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WO2024155747A2 - Asgpr binding compounds and conjugates - Google Patents

Asgpr binding compounds and conjugates Download PDF

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Publication number
WO2024155747A2
WO2024155747A2 PCT/US2024/011898 US2024011898W WO2024155747A2 WO 2024155747 A2 WO2024155747 A2 WO 2024155747A2 US 2024011898 W US2024011898 W US 2024011898W WO 2024155747 A2 WO2024155747 A2 WO 2024155747A2
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Prior art keywords
optionally substituted
certain embodiments
alkyl
alkylene
formula
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PCT/US2024/011898
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French (fr)
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WO2024155747A3 (en
Inventor
Jason G. Lewis
Tao Chen
Darin Hildebrandt
Steven W. RANK
Eric D. Turtle
Shuai ZHENG
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Lycia Therapeutics Inc
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Lycia Therapeutics Inc
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Priority to KR1020257026847A priority Critical patent/KR20250144397A/en
Priority to AU2024208960A priority patent/AU2024208960A1/en
Priority to CN202480015602.6A priority patent/CN120813382A/en
Priority to EP24705914.0A priority patent/EP4651902A2/en
Publication of WO2024155747A2 publication Critical patent/WO2024155747A2/en
Publication of WO2024155747A3 publication Critical patent/WO2024155747A3/en
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/02—Heterocyclic radicals containing only nitrogen as ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/056—Triazole or tetrazole radicals

Definitions

  • the asialoglycoprotein receptor also known as the Ashwell Morell receptor, is the transmembrane glycoprotein receptor found primarily in hepatocytes which plays an important role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine (GalNAc) residues.
  • ASGPR cycles between endosomes and the cell surface.
  • the present disclosure provides a class of compounds including a ligand moiety that specifically binds to a cell surface asialoglycoprotein receptor (ASGPR).
  • ASGPR asialoglycoprotein receptor
  • the cell surface ASGPR binding compounds can trigger the receptor to internalize into the cell a bound compound.
  • the ligand moieties of this disclosure can be linked to a variety of moieties of interest without impacting the specific binding to, and function of, the cell surface receptor ASGPR.
  • conjugates of the ligand moieties linked to a biomolecule such as an antibody
  • conjugates can harness cellular pathways to remove specific proteins of interest from the cell surface or from the extracellular milieu.
  • the conjugates described herein may sequester and/or degrade a target molecule of interest in a cell’s lysosome.
  • compositions comprising such conjugates and methods of using the conjugates to target a polypeptide of interest for sequestration and/or lysosomal degradation, and methods of using the conjugates to treat disorders or disease.
  • FIG.1 shows a graph of cell fluorescence (MFI) versus antibody conjugate concentration ([Ab]) indicating that various antibody conjugates of exemplary ASGPR binding compounds, and an example M6PR binding compound (520) exhibited comparable robust uptake into HepG2 cells after one hour incubation.
  • FIG.2A-2D shows graphs of cell fluorescence versus antibody conjugate concentration indicating that various antibody conjugates of exemplary ASGPR binding compounds exhibited robust uptake into HepG2 cells after one hour incubation.
  • FIG.3 illustrates the fluorescence polarization screening results for example trivalent compounds (1901 (I-171), 1902 (I-172), XB32 and 2101).
  • FIG.4 illustrates the binding of example monovalent compounds (591, XB20, XB23, XB21, 592 and 593) as a percentage of the activity of reference compound XB149.
  • FIG.5 illustrates the fluorescence polarization screening results for example monovalent compounds (XB20, XB21, 592, XB23, 591).
  • FIG.6 shows a graph of cellular uptake of various conjugates of OMA (anti-IgE) with example compounds I-160 to I-163 and I-141 bound to Alexa488 labeled-target IgE in HepG2 cells.
  • FIG.7 illustrates affinity-dependent clearance of OMA-example compounds (I-160 to I-163) as compared to OMA (reference).
  • FIG.8 illustrates dose titration OMA-I-163 IgE clearance.
  • FIG.9 illustrates affinity-dependent clearance of OMA-example compounds (I-160 to I-163) as compared to hIgE (reference).
  • this disclosure provides classes of compounds including a ligand moiety that specifically binds an ASGPR of a cell of interest.
  • This disclosure includes compounds of formula (I): X n L Y (I) or a prodrug thereof, or a salt thereof, wherein: X is a moiety that binds to a ASGPR cell surface receptor (e.g., as described herein); n is 1 to 500; L is a linker (e.g., monovalent or multivalent, as described herein, of defined length); and Y is a moiety of interest (e.g., as described herein).
  • conjugates that comprise a moiety, X, that binds to such an ASGPR internalizing cell surface receptor, for example, for sequestration and/or lysosomal degradation.
  • this disclosure includes target binding conjugate of formula (I): or a prodrug thereof, or a pharmaceutically acceptable salt thereof, wherein: X is a moiety that binds to an ASGPR cell surface receptor (e.g., as described herein); n is 1 to 500; L is a linker (e.g., monovalent or multivalent, as described herein); m is 1 to 20; Y is a biomolecule that specifically binds an extracellular target molecule.
  • target binding conjugate is of formula (II’): (II’) or a prodrug thereof, or a salt thereof, wherein: n is 1 to 3; m is 1 to 3; X and Y are each independently as defined herein; each L 1 to L 6 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; and a, b, c, d, and e are each independently 1, 2, 3, 4, or 5. [0020]
  • the ASGPR binding compounds and conjugates and methods of this disclosure are described in greater detail below. Linkers (L) and moieties of interest (Y) which find use in the ASGPR binding compounds, and the biomolecule conjugates are also described.
  • ASGPR Ligands [0021] As summarized above, this disclosure provides a class of compounds including a ligand moiety that specifically binds to a cell surface ASGPR.
  • the ASGPR ligand moieties of this disclosure can be linked to a variety of moieties of interest without impacting the specific binding to, and function of, the cell surface ASGPR.
  • compounds of this disclosure can utilize the functions of cell surface ASGPRs in a biological system, e.g., for internalization and sequestration of a compound to the lysosome of a cell, and in certain embodiments subsequent lysosomal degradation.
  • the compounds of this disclosure find use in a variety of applications.
  • asialoglycoprotein receptor also known as the Ashwell Morell receptor, means the transmembrane glycoprotein receptor found primarily in hepatocytes which plays an important role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine (GalNAc) residues.
  • ASGPR cycles between endosomes and the cell surface.
  • the ASGPR is Homo sapiens asialoglycoprotein receptor 1 (ASGR1) (see, e.g., NCBI Reference Sequence: NM_001197216).
  • a compound comprising such ASGPR binding moiety may bind to other receptors, for example, may bind with lower affinity as determined by, e.g., immunoassays or other assays known in the art.
  • X, or a compound as described herein comprising such X specifically binds to the cell surface ASGPR with an affinity that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the affinity when X or the compound or the conjugate bind to another cell surface receptor.
  • X or a compound as described herein comprising X specifically binds to ASGPR with an affinity (K d ) 20 mM or less.
  • affinity (K d ) is 10 mM or less, 1 mM or less, 100 uM or less, 10 uM or less, 1 uM or less, 100 nM or less, 10 nM or less, or 1 nM or less.
  • the terms “binds,” “binds to,” “specifically binds” or “specifically binds to” in this context are used interchangeably.
  • the ASGPR binding compounds of this disclosure include a moiety (X) that specifically binds to the cell surface receptor ASGPR.
  • the ASGPR binding compounds can be monovalent or multivalent (e.g., bivalent or trivalent or of higher valency), where a monovalent compound includes a single ASGPR ligand moiety, and a multivalent compound includes two or more such moieties.
  • the ASGPR binding moiety X is able to bind to a specific cell surface ASGPR, and direct (or target) the molecule to this receptor.
  • the ASGPR binding moiety X is capable of binding to the ASGPR and directing (or targeting) a compound or conjugate described herein for internalization and sequestration to the lysosome, and/or subsequent lysosomal degradation.
  • the ASGPR binding moiety X includes an amino sugar ring derivative of galactose (e.g., N-acetylgalactosamine, and analogs thereof), that is linked via a linking moiety to the 1, 6 or 2-position of the sugar ring.
  • the linking moiety can be of 1-10 atoms in length, such as 1-6, or 1- 5, 1-4, or 1-3 atoms in length.
  • the amino sugar ring derivative of galactose is linked via a linking moiety to an oxygen, sulfur, nitrogen or carbon atom at the 1-position of the ring.
  • the amino sugar ring derivative of galactose is linked via a linking moiety to an oxygen, sulfur, nitrogen or carbon atom the 6-position of the ring. In some embodiments, the amino sugar ring derivative of galactose is linked via a linking moiety to an oxygen, sulfur, nitrogen or carbon atom the 2-position of the ring. In certain embodiments, the amino sugar derivative of galactose is linked via a linking moiety to a heteroaryl group at the 1, 6 or 2 position of the ring. In certain embodiments, the amino sugar derivative of galactose is a bicyclic structure.
  • the ASGPR binding compounds is monovalent (e.g., in Formula (I), n is 1), such that the ASGPR binding compound includes a single ASGPR ligand moiety (X) that is linked to a moiety of interest (Y) via a linking moiety at the 1, 6, or 2-position of (X).
  • n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking the ASGPR ligand (X) to Y via a linking moiety at any of the 1, 2 or 6-positions of X.
  • L is 20 to 100 consecutive atoms, such as 25 to 80, 25 to 60, or 25 to 50.
  • n is 1, and L comprises a backbone of 25 or more consecutive atoms covalently linking the ASGPR ligand (X) to Y.
  • the ASGPR binding compounds are multivalent (e.g., in Formula (I), n is 2 or more, such that the ASGPR binding compound includes two or more ASGPR ligand binding moieties (X) that are each covalently linked to a moiety of interest (Y) via a branched linker (e.g., L is a branched linker).
  • the ASGPR binding compound is divalent (e.g., n is 2 in Formula I).
  • each branch of the branched linker comprises a liner linker of 14 or more consecutive atoms to covalently link a linking moiety of each X to a branching point in the linker.
  • each branch of the linker includes 14 to 50 consecutive atoms, such as 14 to 40, 14 to 30, or 14 to 20 atoms.
  • each branch of the linker includes a linear linker of 20 or more consecutive atoms.
  • the linker comprises a linear linker of 12 or more consecutive atoms to covalently link the branching point of L to a moiety of interest (Y), such as 15 or more, 20 or more, 30 or more, or even more consecutive atoms to covalently link the branching point of L to Y.
  • Y moiety of interest
  • the ASGPR ligand moieties e.g., X n -L or (X-L) n of formula (I), and other formulae described herein
  • the bifunctional molecule specifically bind to ASGPR with an affinity (K d ) of 300 nM or less, such as 100 nM or less, 30 nM or less, 10 nM or less, 3 nM or less, or 1 nM or less.
  • K d affinity
  • the terms “binds,” “binds to,” “specifically binds” or “specifically binds to” in this context are used interchangeably.
  • a compound of formula (I): or a prodrug thereof, or a salt thereof, wherein: n is 1 to 500; m is 1 to 20; Y is a moiety of interest; L is a linker; and X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (II): wherein: R 1 is selected from –Z 1 –*, –H, –OH, optionally substituted (C 1 -C 6 )alkyl, –OCH 3 ,–OCH 2 CH CH, optionally substituted -S-(C 1 -C 6 )alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, and optionally substituted -S-heteroaryl; R 2 is selected from –Z 1 –*, –NHCOCH 3 , –NHCOCF 3 , –NHCOCH 2 CF 3 ,
  • Z 1 is a linking moiety selected from -Z 11 -A 1 - and -A 2 -; and -A 1 - and - A 2 - are optionally substituted heterocyclylene; or -A 2 - is optionally substituted isoxazolyl.
  • R 6 is -OR, optionally substituted (C 1 -C 6 )alkyl, –OC(O)-optionally substituted heteroaryl, -C(O)NH-optionally substituted heteroaryl, -NR xx R yy , optionally substituted aryl, optionally substituted heteroaryl–NHCOR, or –NRCOR, provided the heteroaryl is other than triazole; where each R is independently optionally substituted (C 1 -C 6 )alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R xx and R yy are independently H, optionally substituted (C 1 -C 6 )alkyl, or R xx and R yy can cyclize to form an optionally substituted heterocyclyl.
  • R 1 is optionally substituted C 2-6 alkyl, optionally substituted -S-(C 1 - C 6 )alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, or optionally substituted -S-heteroaryl.
  • at least one R 21 is -COR or optionally substituted heteroaryl.
  • n is 1, 2, or 3; and m is 1-3.
  • R 6 is -OR, optionally substituted (C 1 -C 6 )alkyl, –OC(O)-optionally substituted heteroaryl, - C(O)NH-optionally substituted heteroaryl, -NR xx R yy , optionally substituted aryl, optionally substituted heteroaryl–NHCOR, or –NRCOR, provided the heteroaryl is other than triazole; where each R is independently optionally substituted (C 1 -C 6 )alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R xx and R yy are independently H, optionally substituted (C 1 -C 6 )alkyl, or R xx and R yy can cyclize to form an optionally substituted heterocyclyl; D) R 1 is optionally substituted C 2-6 alkyl, optionally substituted -S-(C 1 -C 6 )alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substitute
  • n is 1, 2, or 3; and m is 1-3.
  • X is of formula (a-II): (a-II).
  • X is of formula (a-II), R 1 is n-propyl; and R 2 is -Z 1 -*.
  • Z 1 is a linking moiety selected from -Z 11 -A 1 - and -A 2 -; and -A 1 - and - A 2 - are optionally substituted heterocyclylene.
  • -L-Y comprises: monocyclic heteroaryl.
  • -L-Y comprises:
  • R 6 is -OR, optionally substituted (C 1 -C 6 )alkyl, –OC(O)-optionally substituted heteroaryl, -C(O)NH-optionally substituted heteroaryl, -NR xx R yy , optionally substituted aryl, or optionally substituted heteroaryl, provided the heteroaryl is other than triazole; where R is optionally substituted (C 1 -C 6 )alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R xx and R yy are independently H, optionally substituted (C 1 -C 6 )alkyl, or R xx and R yy can cyclize to form an optionally substituted heterocyclyl.
  • R 6 is -O-(C 1 -C 6 )alkyl, optionally substituted heterocyclyl, or -O-aryl.
  • R 1 is optionally substituted C 2-6 alkyl, optionally substituted -S-(C 1 - C 6 )alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, or optionally substituted -S-heteroaryl
  • R 1 is optionally substituted C 2-6 alkyl.
  • at least one R 21 is -COR or optionally substituted heteroaryl.
  • Y is an antibody or antibody fragment.
  • n is 1, 2, or 3; and m is 1-3.
  • the chemoselective ligation group comprises a carboxylic acid or active ester, maleimide, isocyanate or isothiocyanate, alkyl halide, alkyl tosylate, aldehyde, haloacetamide or alpha-leaving group acetamide, 2-sulfonylpyridine, diazirine, sulfonyl halide or vinyl sulfone, hydrazide, hydrazino, hydroxylamino, pyridyl disulfide, (HIPS) hydrazinyl-indolyl group, or (aza-HIPS) hydrazinyl-pyrrolo-pyridinyl group, alkyne or cyclooctyne, azide, or amine.
  • the chemoselective ligation group comprises a carboxylic acid or active ester, maleimide, iso
  • A) Z 1 is a linking moiety selected from -Z 11 -A 1 - and -A 2 -; and -A 1 - and -A 2 - are optionally substituted heterocyclylene; or -A 2 - is optionally substituted isoxazolyl;
  • B) -L-Y comprises: C) R 6 is -OR, optionally substituted (C 1 -C 6 )alkyl, –OC(O)-optionally substituted heteroaryl, - C(O)NH-optionally substituted heteroaryl, -NR xx R yy , optionally substituted aryl, optionally substituted heteroaryl–NHCOR, or –NRCOR, provided the heteroaryl is other than triazole; where each R is independently optionally substituted (C 1 -C 6 )alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R xx and R yyy
  • the compound of formula (II) is represented by formula (a-II): (a-II).
  • R 1 is –Z 1 –*, –H, or (C 1 -C 6 )alkyl.
  • R 2 is –Z 1 –* or –NHCOCH 3 .
  • R 3 and R 4 are each –H.
  • R 6 is –OH, –OC(O)R, -NR xx R yy , or aryl; R is (C 1 -C 6 )alkyl; and R xx and R yy cyclize to form an optionally substituted heterocyclyl.
  • * represents a point of connection of Z 1 to the linker (L).
  • R 1 is –Z 1 –*.
  • R 2 is –Z 1 –*.
  • L comprises of 10 to 60 consecutive linear or branched chain atoms.
  • L is of formula (IIb’): wherein: each L 1 to L 6 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y.
  • n is 1-3. In some embodiments of formula (IIb’), n is 1. In some embodiments of formula (IIb’), n is 2. In some embodiments of formula (IIb’), n is 3.
  • L is of formula (IIb’): wherein: n is 1, 2, or 3; each L 1 to L 6 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y.
  • a compound of formula (II’): or a prodrug thereof, or a salt thereof, wherein: n is 1 to 3; m is 1 to 20; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; each L 1 to L 6 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; Y is as defined herein; each X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (a-II): (a-II) wherein: R 1 is selected from –Z 1 –*, –H, –OH, optionally substituted (C 1 -C 6 )alkyl, –OCH 3 , –OCH 2 CH CH, optionally substituted -S-(C 1 -C 6 )alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-
  • R 1 is selected from –Z 1
  • each L 1 to L 5 independently comprises one or more linking moieties independently selected from –C 1-20 -alkylene–, –NHC(O)-C 1-6 -alkylene–, –C(O)NH-C 1-6 -alkylene–, –NH- C 1-6 -alkylene–, –NHC(O)NH-C 1-6 -alkylene–, –NHC(S)NH-C 1-6 -alkylene–, –C 1-6 -alkylene–NHC(O)-, – C 1-6 -alkylene–C(O)NH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NHC(O)NH-, –C 1-6 -alkylene–NHC(S)NH- , -O(CH 2 ) p –, –(OCH 2 CH 2 ) p –, –NHC(O)–, –, –NHC(S)
  • each R 16 is independently (C 1 -C 6 )alkyl or monocyclic heteroaryl. In some embodiments, each R 16 is independently R”. [0074] In some embodiments, each L 1 to L 5 is independently selected from –C 1-20 -alkylene–, –NHC(O)-C 1-6 -alkylene–, –C(O)NH-C 1-6 -alkylene–, –NH-C 1-6 -alkylene–, –NHC(O)NH-C 1-6 -alkylene–, –NHC(S)NH-C 1-6 -alkylene–, –C 1-6 -alkylene–NHC(O)-, –C 1-6 -alkylene–C(O)NH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NHC(O)NH-, –C 1-6 -alkylene–NH-,
  • each L 1 to L 5 is independently selected from –C 1-20 -alkylene–, –NHC(O)-C 1-6 -alkylene–, –C(O)NH-C 1-6 -alkylene–, –NH-C 1-6 -alkylene–, –NHC(O)NH-C 1-6 -alkylene–, –NHC(S)NH-C 1-6 -alkylene–, –C 1-6 -alkylene–NHC(O)-, –C 1-6 -alkylene–C(O)NH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NHC(O)NH-, –C 1-6 -alkylene–NHC(S)NH-, -O(CH 2 ) p –, –(OCH 2 CH 2 ) p –, –NHC(O)–, –C(O)NH– –NH—NHC(S)NH-
  • n is 1. [0078] In some embodiments of formula (II’), n is 2. [0079] In some embodiments of formula (II’), n is 3. [0080] In some embodiments, at least one L 1 is –C 1-20 -alkylene– optionally substituted with one to five halo. [0081] In some embodiments, at least one L 1 is -CF 2 CH 2 -. [0082] In some embodiments, at least one L 2 is –(OCH 2 CH 2 ) p –. [0083] In some embodiments, p is 2-3.
  • At least one L 3 is NHCONH-C 1-6 -alkylene–.
  • at least one L 4 is –C 1-6 -alkylene–NHCONH-.
  • at least one L 5 is –(OCH 2 CH 2 ) p –.
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Iia): (Iia) wherein R 2 , R 3 , R 4 , R 6 and Z 1 are as defined herein.
  • R 6 is selected from –OH, –OC(O)R, and -C(O)NHR; and R 2 is selected from –NHCOCH 3 , –NHCOCF 3 , and –NHCOCH 2 CF 3 .
  • Z 1 is in a beta configuration, and can be described by formula (Iia-1): (Iia-1).
  • Z 1 is in an alpha configuration, and can be described by formula (Iia-2) (Iia-2). [0092] In certain embodiments of formula (Iia), (Iia-1) or (Iia-2), Z 1 is -Z 11 -A 1 -, wherein A 1 - is optionally substituted arylene or optionally substituted heteroarylene. In certain embodiments, A 1 is an optionally substituted heteroarylene. In certain embodiments, the heteroarylene is a 5 or 6-membered heteroarylene. In certain embodiments, the heteroarylene is a 5-membered heteroarylene. In certain embodiments, the 5-membered heteroarylene is a triazole.
  • the triazole is a 1,2,3- triazole moiety.
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (IIIa) or (IIIb): (IIIa) (IIIb) wherein: -Z 11 - is -O-, -S-, -N(R 21 )-, or -C(R 22 ) 2 -, where each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 -C 6 )alkyl, and R 21 is H or optionally substituted (C 1 -C 6 )alkyl; and -A 1 - is arylene, substituted arylene, heteroarylene, or substituted heteroarylene.
  • Z 11 is -S-.
  • Z 11 is -C(R 22 ) 2 -.
  • Z 11 is -CH 2 -.
  • Z 11 is -C(R 22 ) 2 , where at least one R 22 is H. In certain embodiments, both R 22 are H.
  • Z 11 is -O-.
  • Z 11 is -S-.
  • Z 11 is -N(R 21 ), where R 21 is H or (C 1- C 3 )alkyl.
  • -A 1 - is triazole.
  • Z 1 is -C(R 22 ) 2 -triazole-. In certain embodiments, Z 1 is: * * . In certain embodiments, Z 1 is: . [0099] In certain embodiments of formula (Iia), (Iia-1) or (Iia-2), Z 1 is Z 11 . In certain embodiments, Z 11 is -C(R 22 ) 2 . In certain embodiments, at least one R 22 is H. In certain embodiments, both R 22 are H, and Z 11 is -CH 2 -. In certain cases Z 11 is -O-. In certain embodiments, Z 11 is -S-.
  • Z 11 is -N(R 21 ), where R 21 is H or (C 1 -C 3 )alkyl. [0100] In certain embodiments of formula (Iia), (Iia-1) or (Iia-2), Z 1 is monocyclic 5 or 6-membered heteroaryl or aryl. In certain embodiments, Z 1 is . In certain embodiments, Z 1 .
  • Z 1 is selected from -O-, -S-, wherein: X 1 is O or S; t is 0 or 1; R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
  • X 1 is O or S
  • t is 0 or 1
  • R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl)
  • each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
  • Z 1 is optionally substituted (C 1 - C 6 )alkyl.
  • the alkyl is methyl.
  • the alkyl is ethyl.
  • the alkyl is propyl.
  • the alkyl is butyl.
  • the alkyl is pentyl.
  • the alkyl is hexyl.
  • the ASGPR binding moiety (X) of formula (Iia-1) is selected from one of the following structures:
  • Z 1 is in a beta configuration and X is of formula (IIIb-2): (IIIb-2) wherein: -A 1 - is arylene, substituted arylene, heteroarylene, or substituted heteroarylene.
  • a 1 is a triazole.
  • X is of formula (X A -4).
  • Z 1 is in a alpha configuration at the 1-position carbon of the galactosamine ring.
  • Z 1 is S, and each X is of formula (X A -1).
  • each X is of formula (X A -2). In some embodiments of formula (Iia-1), each X is of formula (X A -3). In some embodiments of formula (Iia-1), each X is of formula (X A -4). In some embodiments of formula (Iia-1), each X is of formula (X A -5). [0107] In certain embodiments, the compound of formula (Iia-2) is selected from one of the following structures: [0108] In some embodiments of formula (Iia-2), each X is of formula (X B -1). [0109] In some embodiments of formula (Iia-2), each X is of formula (X B -2).
  • each X is of formula (X B -3). [0111] In some embodiments of formula (Iia-2), each X is of formula (X B -4). [0112] In some embodiments of formula (Iia-2), Z 1 is in an alpha configuration and X is of formula (IIIb-1): wherein -A 1 - is arylene, substituted arylene, heteroarylene, or substituted heteroarylene. [0113] In certain embodiments of formula (IIIb-1), A 1 is an optionally substituted heteroarylene. In certain embodiments, the heteroarylene is a 5 or 6-membered heteroarylene. In certain embodiments, the heteroarylene is a 5-membered heteroarylene.
  • the 5-membered heteroarylene is a triazole. In certain embodiments, the triazole is a 1,2,3-triazole moiety.
  • the X of formula (IIIb-1) is selected from one of the following structures: [0115] In some embodiments of formula (IIIb-1), each X is of formula (X C -1). [0116] In some embodiments of formula (IIIb-1), each X is of formula (X C -2). [0117] Exemplary ligand moieties that bind ASGPR, and synthons thereof, which can be utilized in the compounds of this disclosure are shown in Tables 1-5. In certain embodiments, the compound of formula (Iia) is a compound shown in Table 1:
  • Z 1 is in the alpha configuration such that the ASGPR binding moiety X1-X5.1 is derived from formula (Iia-2): 2-linked ASGPR ligand moieties [0119]
  • the ASGPR binding moiety (X) is linked via the 2-postion of the sugar analog.
  • the ASGPR binding moiety (X) has a reduced ring carbon at the 1- position relative to a galactosamine derived sugar.
  • the ASGPR binding moiety (X) of the bifunctional molecules of this disclosure is described by formula (Iib): wherein R 1 , R 3 , R 4 , R 6 , R 11 , and Z 1 are as defined herein.
  • the ASGPR binding moiety (X) of the compounds of this disclosure are described by formula (Iib’): wherein R 3 -R 4 , R 6 , and Z 1 are as defined herein.
  • the ASGPR binding moiety (X) of the compounds of this disclosure are described by formula (Iva): wherein R 1 , R 11 , and Z 1 are as defined herein.
  • Z1 is selected from optionally substituted –(C(R 22 ) 2 ) q -heteroarylene, , wherein q is 0 or 1.
  • Z 1 is optionally substituted – (C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
  • R is H, or C (1-3) -alkyl.
  • Z 1 is -NR 23 CO-, wherein R 23 is H or C (1-3) -alkyl.
  • R 23 is H or C (1-3) -alkyl.
  • Z 1 is selected from optionally substituted –(C(R 22 ) 2 ) q -heteroaryl, , wherein q is 0 or 1.
  • Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
  • Z 1 is selected from -O-, - wherein: X 1 is O or S; t is 0 or 1; R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
  • X 1 is O or S
  • t is 0 or 1
  • R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl)
  • each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
  • the compound of formula of formulae (Iib), (Iib’) or (Iva) is selected from one of the following structures: , wherein R 1A is independently H or (C 1 - 3 )alkyl.
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ivb) or (Ivc): OH OH R 11 R 11 HO O HO O HO R 1 HO R 1 Z 11 A 1 A 2 * * (Ivb) (Ivc), wherein: -Z 11 - is -O-, -S-, -N(R 21 )-, or -C(R 22 ) 2 ; -A 1 - and -A 2 - are optionally substituted arylene or optionally substituted heteroarylene; each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
  • halogen e.g., F
  • R 1 is H.
  • R 2 is –Z 1 –*
  • R 11 is a group of the formula -CH 2 O- that forms a bridge (i.e., is cyclically linked) to the 1-position carbon atom on the sugar ring.
  • –Z 1 –* or –Z 1 –L- comprises [0137]
  • R 11 is H and the compound is of Table 2:
  • the compound of formula (Iib) is a compound shown in Table 3:
  • the compound of formula (Iib), the configuration at C1 (i.e., R 1 ) is alpha.
  • the compound of formula (Iib), the configuration at C1 (i.e., R 1 ) is beta.
  • the compound of formula (Id’) is a compound shown in Table 4: [0140]
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ivb-1) or (Ivc-1): (Ivb-1) (Ivc-1), wherein R 11 is the bridging moiety that connects the 5-position carbon to the 1-position carbon.
  • Z 11 is -C(R 22 ) 2 .
  • at least one R 22 is H.
  • both R 22 are H.
  • Z 11 is -O-.
  • Z 11 is -S-. In certain embodiments, Z 11 is -N(R 21 ), where R 21 is H or (C 1 -C 3 )alkyl. [0142] In certain embodiments of formulae (Ivb), (Ivc), (Ivb-1) or (Ivc-1), -A 1 - and -A 2 - are each independently an optionally substituted heteroarylene.
  • the heteroarylene is a 5 or 6-membered heteroarylene. In certain embodiments, the heteroarylene is a 5-membered heteroarylene. In certain embodiments, the heteroarylene is a 6-membered heteroarylene.
  • the A 1 ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan.
  • the A 1 ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine.
  • the A 1 ring is triazole.
  • the A 1 ring is pyridine.
  • the A 1 ring is pyrimidine.
  • the A 1 ring is thiadiazole. In certain embodiments, the A 1 ring is a 5 or 6- membered arylene or heteroarylene that is further substituted with one or more substituents. In certain embodiments, the A 1 ring is further substituted with one or more substituents selected from halogen, (C 1- C 6 )alkyl and substituted (C 1- C 6 )alkyl (e.g., CF 3 ).
  • the A 2 ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan.
  • the A 2 ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine.
  • the A 2 ring is triazole.
  • the A 2 ring is pyridine.
  • the A 2 ring is pyrimidine.
  • the A 2 ring is thiadiazole. In certain embodiments, the A 2 ring is a 5 or 6- membered arylene or heteroarylene that is further substituted with one or more substituents. In certain embodiments, the A 2 ring is further substituted with one or more substituents selected from halogen, (C 1- C 6 )alkyl and substituted (C 1- C 6 )alkyl (e.g., CF 3 ).
  • -Z 11 -A 1 - is a monocyclic 5 or 6- memebered heteroarylene of one of the following structures: [0146] In certain embodiments of formulae (Ivc) or (Ivc-1), -A 2 - is a monocyclic 5 or 6-membered heteroarylene of the following structure: . [0147] It is understood that a variety of substituents can be utilized to connect a particular -Z 11 -A 1 - group to an adjacent linker.
  • -Z 11 -A 1 - is a monocyclic 5 or 6-membered heteroarylene that is attached to a linking moiety as shown in one of the following structures: [0148] In certain embodiments of formulae (Ivc) or (Ivc-1), -Z 11 -A 1 - is a monocyclic 5 or 6- membered heteroarylene that is attached to a linking moiety as shown in one of the following structures: .
  • R 1 is H, such that the compound of formula of formulae (Iib), or (Iva)-(Ivc) has no non-hydrogen substituents at the 1-position of the sugar ring.
  • the compound of formula (Iib) is of any one of formulae (Ivd)-(Ivg): (Ivf), and (Ivg), wherein the A 1 and A 2 rings, R 6 , R 4 , R 3 , R 11 , and R 21 are as defined herein.
  • the A 1 ring is a 5 or 6-membered arylene or heteroarylene.
  • the A 1 ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, imidazole, and furan.
  • the A 1 ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine.
  • the A 1 ring is triazole.
  • the A 1 ring is pyridine In certain embodiments the A 1 ring is pyrimidine In certain embodiments, the A 1 ring is thiadiazole. In certain embodiments, the A 1 ring is pyrazine. In certain embodiments, the A 1 ring is a 5 or 6-membered arylene or heteroarylene that is further substituted with one or more substituents. In certain embodiments, the A 1 ring is further substituted with one or more substituents selected from halogen, (C 1- C 6 )alkyl and substituted (C 1- C 6 )alkyl (e.g., CF 3 ).
  • the A 2 ring is a 5 or 6-membered arylene or heteroarylene.
  • the A 2 ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan.
  • the A 2 ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine.
  • the A 2 ring is triazole.
  • the A 2 ring is pyridine. In certain embodiments, the A 2 ring is pyrimidine. In certain embodiments, the A 2 ring is thiadiazole. In certain embodiments, the A 2 ring is a 5 or 6-membered arylene or heteroarylene that is further substituted with one or more substituents. In certain embodiments, the A 2 ring is further substituted with one or more substituents selected from halogen, (C 1- C 6 )alkyl and substituted (C 1- C 6 )alkyl (e.g., CF 3 ). [0153] In some embodiments of any one of formulae (Ivd)-(Ivg),the A 1 or A 2 ring is absent.
  • the A 1 or A 2 ring is phenylene or substituted phenylene.
  • the A 2 ring is a 5 or 6-membered heteroarylene. In certain cases of formula (Ivd), the A 2 ring is a 5-membered heteroarylene. In certain embodiments of formula (Ivd), the A 2 ring is triazole. In certain embodiments of (Ivd), the A 2 ring is absent.
  • the A 1 ring is a 5 or 6-membered heteroarylene and R 21 is H.
  • the A ring is triazole. In certain cases of formula (Ive), the A 1 ring is pyridine. In certain cases of formula (Ive), the A 1 ring is pyrimidine. In certain cases of formula (Ive), the A 1 ring is thiadiazole. In some embodiments of formula (Ive), the A 1 ring is absent and R 21 is H or optionally substituted acyl. In certain embodiments, R 21 is -COCH 3 . In certain embodiments, R 21 is H. [0157] In some embodiments of formula (Ivf), the A 1 ring is a 5 or 6-membered heteroarylene.
  • the A 1 ring is a 5-membered heteroarylene. In certain embodiments of formula (Ivf), the A 1 ring is triazole. In certain embodiments of (Ivf), the A 1 ring is absent. [0158] In some embodiments of formula (Ivg), the A 2 ring is a 5 or 6-membered heteroarylene. In certain cases of formula (Ivg), the A 2 ring is a 5-membered heteroarylene. In certain embodiments of formula (Ivg), the A 2 ring is triazole. In certain embodiments of (Ivg), the A 2 ring is absent. [0159] In certain embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by any one of formulae (Ivh)-(Ivk):
  • R 6 , R 4 , R 3 , and R 21 are as defined herein; Y 1 -Y 3 are each independently N or CR 25 ; and R 24 and R 25 are each independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen.
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by any one of formulae (Ivl)-(Ivm): wherein: R 6 , R 4 , R 3 , and R 21 are as defined herein; Y 1 -Y 3 are each independently N or CR 25 ; Y 4 is N or CR 24 ; Y 5 is S, O, or NH; and R 24 and R 25 are each independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen. [0161] In some embodiments of formula (Ivi) at least one of Y 1 to Y 3 is N.
  • Y 1 to Y 3 are N.
  • Y 1 and Y 4 are N.
  • Y 1 and Y 3 are N and Y 2 is CR 25 .
  • Y 1 and Y 2 are N and Y 3 is CR 25 .
  • Y 1 and Y 2 are CR 25 and Y 3 is N.
  • R 6 is H.
  • R 4 and R 3 are each H. In certain embodiments, at least one of R 4 -R 3 is a promoiety. In certain embodiments, R 4 and R 3 are cyclically linked to form a promoiety (e.g., as described herein).
  • the compound of formula (Ivi) is of formula (Ivi-1): [0170] wherein R 24 and R 25 are independently selected from H, halogen, (C 1- C 6 )alkyl and substituted (C 1- C 6 )alkyl (e.g., CF 3 ).
  • R 25 is H.
  • R 25 is C (1-3) -alkyl, or C (1-3) -fluoroalkyl.
  • the fluoroalkyl is CF 3 .
  • R 24 is H.
  • R 24 is C (1-3) -alkyl, or C (1-3) - fluoroalkyl. In certain embodiments, the fluoroalkyl is CF 3 .
  • the compound of formula (Ivi-1) is of formula (X D ): [0172]
  • the compound of formula (Ivk-1) is of formula (X E ): [0173]
  • the compound of formula (Ivl) is of formula (Ivl-1): wherein: R 6 , R 4 , R 3 , and R 21 are as defined herein; Y 1 -Y 4 are each independently N or CR 25 ; Y 5 is S, O, or NH; and each R 25 is independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen.
  • each R 25 is H.
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by one of the following structures: .
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by one of the following structures: [0177]
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by one of the following structures: * [0178]
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by one of the following structures: * [0179]
  • R 1 R 3 , R 4 , and R 11 are H
  • R 6 is OH: wherein Z 1 is -NH-, -CH 2 -, -S- or -O-.
  • R 3 , R 4 are H, and R 6 is OH: wherein Z 1 is -NH-, -CH 2 -, -S-, -O-, triazole, 6-linked ASGPR ligand moieties
  • the ASGPR binding moiety (X) is linked via the 6-postion of the sugar analog.
  • the ASGPR binding moiety (X) has a reduced ring carbon at the 1- position relative to a galactosamine derived sugar.
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Iic): * wherein R 1 -R 4 and Z 1 are as defined herein.
  • Z 1 is selected from -O-, -S-, -CONR 21 -, and optionally substituted –(C(R 22 ) 2 ) q - heteroarylene, wherein q is 0 or 1.
  • Z 1 is -O-.
  • Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
  • Z 1 is -Z 11 -A 1 -, wherein -A 1 - is or optionally substituted -A 1 - or optionally substituted arylene. In certain embodiments, -A 1 - is an optionally substituted heteroarylene. In certain embodiments, the heteroarylene is a 5 or 6-membered heteroarylene. In certain embodiments, the heteroarylene is a 5-membered heteroarylene. In certain embodiments, the 5-membered heteroarylene is a triazole. In certain embodiments, the triazole is a 1,2,3-triazole moiety. In certain embodiments, Z 11 is -C(R 22 ) 2 .
  • At least one R 22 is H. In certain embodiments, both R 22 are H. In certain cases Z 11 is -O-. In certain embodiments, Z 11 is -S-. In certain other cases, Z 11 is -N(R 21 ), where R 21 is H or (C 1 -C 3 )alkyl. In certain embodiments, Z 1 is -C(R 22 ) 2 - * triazole-. In certain embodiments, Z 1 is: . [0186] In certain embodiments of formula (Iic), Z 1 is Z 11 . In certain embodiments, Z 11 is -C(R 22 ) 2 . In certain embodiments, at least one R 22 is H.
  • both R 22 are H, and Z 11 is -CH 2 -. In certain cases Z 11 is -O-. In certain embodiments, Z 11 is -S-. In certain other cases, Z 11 is -N(R 21 ), where R 21 is H or (C 1- C 3 )alkyl. [0187] In certain embodiments of formula (Iic), Z 1 is monocyclic 5 or 6-membered heteroarylene or arylene.
  • Z1 is selected from -O-, -S-, -C(R22)2-, -N(R21) - wherein: X 1 is O or S; t is 0 or 1; R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
  • the compound of formula (Iic) is the following structure: .
  • the compound of formula (Iic) is the following structure: .
  • R 11 is H and the compound is of Table 5:
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Iid): wherein: R 6 , R 4 , R 3 and Z 1 are as defined herein; Y 6 and Y 5 are each independently selected from -O-, -S-, NR 21 -, and -C(R 22 ) 2 ; R 21 is selected from H, optionally substituted (C 1 -C 6 )alkyl, and -C(O)R 22 ; each R 22 is independently selected from H, halogen and optionally substituted (C 1 -C 6 )alkyl; and ring B is a 5 or 6-membered optionally substituted cyclic group.
  • Y 5 is connected to the sugar ring via an alpha configuration. In some embodiments of formula (Iid), Y 5 is connected to the sugar ring via a beta configuration.
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Iid’): wherein: R 6 , R 4 , R 3 and Z 1 are as defined herein; Y 5 and Y 6 are each independently selected from -O-, -S-, NR 21 -, and -C(R 22 ) 2 ; R 21 is selected from H, optionally substituted (C 1 -C 6 )alkyl, and -C(O)R 22 ; each R 22 is independently selected from H, halogen and optionally substituted (C 1 -C 6 )alkyl; and ring B is a 5 or 6-membered optionally substituted cyclic group.
  • Y 5 is O. In certain embodiments, Y 5 is S. In certain embodiments, Y 5 is -NR 21 -. In certain embodiments, Y 5 is -C(R 22 ) 2 and each R 22 is H. [0195] In some embodiments of formula (Iid)-(Iid’) Y 6 is -NR 21 - where R 21 is H. In certain embodiments, Y 6 is -NR 21 - where R 21 is -C(O)R 22 . In certain embodiments, R 22 is methyl.
  • the B ring is a 5 or 6-membered heterocycle. In certain embodiments, the B ring is a 5-membered heterocycle. In certain embodiments, the B ring is a 6- membered heterocycle.
  • Z 1 is Z 11 , where Z 11 is selected from -O-, -S-, NR 21 -, and -C(R 22 ) 2 . In certain embodiments, Z 1 is -O-. In certain embodiments, Z 1 is -S-. In certain embodiments, Z 1 is NR 21 where R 21 is H.
  • Z 1 is -C(R 22 ) 2 where each R 22 is H. [0198] In some embodiments of formula (Iid)-(Iid’) Z 1 is optionally substituted Z 11 -heteroarylene or optionally substituted Z 11 -arylene. In some embodiments, Z 1 is CH 2 -heteroarylene or CH 2 -arylene. In some embodiments of formula (Iid)-(Iid’) Z 1 is optionally substituted amide. In some embodiments of formula (Iid)-(Iid’) Z 1 is optionally substituted sulfonamide.
  • Z 1 is optionally substituted urea or optionally substituted thiourea.
  • the compound of formula (Iid)-(Iid’) has one of the following structures: , . [0200]
  • R 6 is OH.
  • R 6 is -OC(O)R.
  • R 6 is -C(O)NHR, where R is an optionally substituted alkyl.
  • R terminates in an alkenyl or an alkynyl group.
  • R 6 is optionally substituted triazole.
  • the triazole is of the following structure: .
  • R 2 is -NHCOCH 3 .
  • R 2 is –NHCOCF 3 .
  • R 2 is –NHCOCH 2 CF 3 .
  • R 2 is – OH.
  • R 2 is an optionally substituted triazole.
  • the triazole in of the following structure: .
  • R 6 or R 2 when R 6 or R 2 is a substituted triazole, the triazole is a 1,2,3-trizole, and the substituent is at the 4 or 5-position.
  • the substituent on the triazole moiety includes but is not limited to, an optionally substituted (C 1 - 6 )alkyl, optionally substituted (C 1 - 6 )alkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkaryl, and an optionally substituted alkyheteroaryl. It will be understood that any convenient substituent can be included in the triazole moiety, see, e.g., triazole moieties disclosed in Mamidayala et al, J.
  • Z 1 , Z 11 , and Z 11 -Ar linking moieties can be considered part of the X group of formula (I).
  • -Z 1 - can be linked to an - L 1 - moiety (e.g., of the linker as described herein) via a variety of bonds and linking moieties, depending on the method of preparation.
  • the subject compounds comprise a -Z 1 -L 1 - moiety selected from: wherein each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 -C 6 )alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 0 to 6. [0204] In some embodiments, the subject compounds comprise a -Z 1 -L 1 - moiety selected from:
  • each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 -C 6 )alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6.
  • the Z 1 -L 1 - group is , and o is 1 or 2.
  • the Z 1 -L 1 - group 22 each R is H, and p is 1 or 2.
  • the Z 1 -L 1 - group is . [0210] In certain embodiments, the Z 1 -L 1 - group are each independently 1-3. [0211] In certain embodiments, the Z 1 -L 1 - group i [0212] In certain embodiments, the Z 1 -L 1 - group i are each independently is 1-3. [0213] In certain embodiments, the Z 1 -L 1 - group is , where x is 0-3. [0214] In certain embodiments, the Z 1 -L 1 - group [0215] In certain embodiments, the Z 1 -L 1 - group is , where R 21 is H, and z is 1-4.
  • the Z 1 -L 1 - group is , where R 21 is H, and z1 is 1-4. [0217] In certain embodiments, the Z 1 -L 1 - group is , where each R 22 is H, and q is 0-3. In certain embodiments, the Z 1 -L 1 - group is , where each R 22 is H, and q is 1-3. [0218] In certain embodiments, the Z 1 -L 1 - group is , where q is 1-3.
  • the subject compounds comprise a -Z 1 -L- group selected from: [0220]
  • the Z 1 -L 1 - group is q O , where q is 1-3. In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. [0221] In certain embodiments, the Z 1 -L 1 - group i . [0222] In certain embodiments, the Z 1 -L 1 - group i . [0223] In certain embodiments, the Z 1 -L 1 - group is . [0224] In certain embodiments, -Z 1 -L 1 - comprises an optionally substituted -NH-heteroarylene-.
  • the heteroarylene is a triazole. In certain embodiments, the heteroarylene is pyridine. In certain embodiments, the heteroarylene is pyrimidine. In certain embodiments, the heteroarylene is thiadiazole. [0225] In certain embodiments, the -Z 1 -L 1 - comprises a group selected from: wherein each R 21 is independently selected from H, optionally substituted (C 1 -C 6 )alkyl, and optionally substituted acyl; and R 24 and R 25 are each independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen.
  • the -Z 1 -L 1 - comprises a group selected from: wherein R 24 and R 25 are each independently selected from H, optionally substituted C (1-6) - alkyl, optionally substituted fluoroalkyl, and halogen; and each R 21 is independently selected from H, optionally substituted (C 1 -C 6 )alkyl, and optionally substituted acyl.
  • R 21 is H.
  • R 24 is C (1-3) -alkyl, or C (1-3) - fluoroalkyl.
  • the fluoroalkyl is CF 3 .
  • R 25 is C (1-3) -alkyl, or C (1-3) -fluoroalkyl.
  • the fluoroalkyl is CF 3 .
  • - It is understood that a variety of substituents and chemistries can be utilized to connect a particular X ligand moiety (e.g., as described herein) to an adjacent linker.
  • a linking moiety of the linker comprises a triazole that derives from a Click chemistry conjugation.
  • the ASGPR ligand moiety (X) is attached to a linking moiety as shown in one of the following structures: , , [0230]
  • R 1 R 3 , R 4 , and R 11 are H, and R 6 is OH: wherein Z 1 is triazole, -NH-heteroaryl (e.g., -NH- attached to pyridine, pyrazine, or pyrimidine) , -NH-, - O-, or -CH 2 - , and/or Z 1 is attached to a linking moiety as shown in one of the following structures: [0231]
  • R 1 R 3 , R 4 , and R 11 are H, and R 6 is OH: wherein Z 1 is attached to a linking moiety as shown in one of the following structures: Additional Exemplary ASGPR Binding Moieties [0232]
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ib): wherein R 1 , R 2 , R 4 , R 5 and Z 1 are as defined herein.
  • R 1 is selected from –OH, –OC(O)R, and -C(O)NHR; and R 2 is selected from –NHCOCH 3 , –NHCOCF 3 , and – NHCOCH 2 CF 3
  • Z 1 is in an alpha configuration .
  • Z 1 is in an alpha configuration and X is of the following formula: .
  • Z 1 is in an alpha configuration and X is of the following formula: .
  • Z 1 is in an alpha configuration and X is of the following formula: .
  • Z 1 is in an alpha configuration and X is of the following formula: .
  • Z 1 is in an alpha configuration and X is of the following formula: .
  • Z 1 is in a beta configuration .
  • Z 1 is in a beta configuration and X is of the following formula: .
  • Z 1 is in a beta configuration and X is of the following formula: .
  • Z 1 is in a beta configuration and X is of the following formula: .
  • Z 1 is in a beta configuration and X is of the following formula: .
  • Z 1 is in a beta configuration and X is of the following formula: .
  • Z 1 is in a beta configuration and X is of the following formula: .
  • Z 1 is Z 11 -Ar , wherein Ar is or optionally substituted heteroaryl or optionally substituted aryl.
  • Ar is an optionally substituted heteroaryl.
  • the heteroaryl is a 5 or 6-membered heteroaryl.
  • the heteroaryl is a 5-membered heteroaryl.
  • the 5-membered heteroaryl is a triazole.
  • the triazole is a 1,2,3-triazole moiety.
  • Z 11 is -C(R 22 ) 2 .
  • at least one R 22 is H.
  • both R 22 are H.
  • Z 11 is -O-.
  • Z 11 is -S-.
  • Z 11 is -NR 21 , where R 21 is H or (C 1-3 )alkyl.
  • Z 1 is -C(R 22 ) 2 -triazole-. In certain * * embodiments, Z 1 is: . In certain embodiments, Z 1 is: . [0250] In certain embodiments of formula (Ib), Z 1 is Z 11 . In certain embodiments, Z 11 is -C(R 22 ) 2 . In certain embodiments, at least one R 22 is H. In certain embodiments, both R 22 are H, and Z 11 is -CH 2 -. In certain cases Z 11 is -O-. In certain embodiments, Z 11 is -S-.
  • Z 11 is -NR 21 , where R 21 is H or (C1-3)alkyl.
  • R 21 is H or (C1-3)alkyl.
  • Z 1 is monocyclic 5 or 6-membered heteroaryl or aryl. In certain embodiments, Z 1 is .
  • Z 1 is selected from -O-, -S-, -C(R 22 ) 2 -, -NR 21 -, - wherein: X 1 is O or S; t is 0 or 1; R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
  • X 1 is O or S
  • t is 0 or 1
  • R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl)
  • each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
  • Z 1 is optionally substituted (C 1 -C 6 )alkyl.
  • the alkyl is methyl.
  • the alkyl is ethyl.
  • the alkyl is propyl.
  • the alkyl is butyl.
  • the alkyl is pentyl.
  • the alkyl is hexyl.
  • the compound of formula (Ib) is selected from one of the following structures: wherein R 5 is independently H or a promoiety.
  • the compound of formula (Ib) is selected from one of the following structures: wherein R 5 and R 4 independently H or a promoiety, or R 5 and R 4 are cyclically linked to form a promoiety; and n1 is an integer from 1 to 6. [0256] In certain embodiments, the compound of formula (Ib) is selected from one of the following structures: . [0257] In some embodiments, at least one of R 4 -R 5 is of the formula -COCH 3 , -COCH(CH 3 ) 2 or - COC(CH 3 ) 3 . In certain embodiments, at least one of R 4 -R 5 is of the formula -CH 2 OCOC(CH 3 ) 3 .
  • R 4 -R 5 is of the formula -COC(CH 3 ) 3 or -CH 2 OCOC(CH 3 ) 3 .
  • R 4 is H and R 5 is selected from -COCH 3 , -COCH(CH 3 ) 2 , -COC(CH 3 ) 3 and - CH 2 OCOC(CH 3 ) 3 .
  • R 4 is H and R 5 is -COC(CH 3 ) 3 .
  • R 4 is H and R 5 is -CH 2 OCOC(CH 3 ) 3 .
  • the compound of formula (Ib) is selected from one of the following structures: wherein n2 is an integer from 1 to 6.
  • R 5 and R 4 are cyclically linked to form a promoiety.
  • the compound of formula (Ib) is selected from one of the following structures: wherein n2 is an integer from 1 to 6; and Y 4 is a suitable counterion.
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ic): wherein R 2 -R 5 and Z 1 are as defined herein.
  • Z 1 is selected from -O-, -S-, -CONR 21 -, and optionally substituted –(C(R 22 ) 2 ) q -heteroaryl, wherein q is 0 or 1.
  • Z 1 is -O-.
  • Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
  • Z 1 is Z 11 -Ar , wherein Ar is or optionally substituted heteroaryl or optionally substituted aryl.
  • Ar is an optionally substituted heteroaryl.
  • the heteroaryl is a 5 or 6-membered heteroaryl.
  • the heteroaryl is a 5-membered heteroaryl.
  • the 5-membered heteroaryl is a triazole.
  • the triazole is a 1,2,3-triazole moiety.
  • Z 11 is -C(R 22 ) 2 .
  • at least one R 22 is H.
  • both R 22 are H.
  • Z 11 is -O-.
  • Z 11 is -S-.
  • Z 11 is -NR 21 , where R 21 is H or (C1-3)alkyl.
  • Z 1 is -C(R 22 ) 2 -triazole-.
  • Z 1 is: .
  • Z 1 is Z 11 .
  • Z 11 is -C(R 22 ) 2 .
  • at least one R 22 is H.
  • both R 22 are H, and Z 11 is -CH 2 -.
  • Z 11 is -O-.
  • Z 11 is -S-.
  • Z 11 is -NR 21 , where R 21 is H or (C 1-3 )alkyl.
  • Z 1 is monocyclic 5 or 6-membered heteroaryl or aryl. In certain embodiments, [0264] In certain embodiments of formula (Ic), Z 1 is selected from -O-, -S-, -C(R 22 ) 2 -, -NR 21 -, - wherein: X 1 is O or S; t is 0 or 1; R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
  • X 1 is O or S
  • t is 0 or 1
  • R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl)
  • the compound of formula (Ic) is the following structure: .
  • the compound of formula (Ic) is the following structure: .
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Id): wherein R 1 , R 3 -R 5 and Z 1 are as defined herein.
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Id’): [0269] In some embodiments, Z 1 is selected from optionally substituted –(C(R 22 ) 2 ) q -heteroaryl, and [0270] In some embodiments, Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1. [0271] In some embodiments, Z 1 is . In some embodiments, Z 1 . [0272] In some embodiments, wherein R 23 is H, or C(1-3)-alkyl.
  • Z 1 is -NR 23 CO-, wherein R 23 is H or C (1-3) -alkyl.
  • Z 1 is selected from optionally substituted – (C(R 22 ) 2 ) q -heteroaryl, , wherein q is 0 or 1.
  • Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
  • R 23 is H, or C(1-3)-alkyl.
  • Z 1 is -NR 23 CO-, wherein R 23 is H or C (1-3) -alkyl.
  • Z 1 is monocyclic 5 or 6-membered heteroaryl or aryl.
  • Z 1 is a monocyclic 5 or 6-memebered heteroaryl of one of the following structures: [0280] In certain embodiments of formula (Id), Z 1 is of one of the following structures: [0281] In certain embodiments of formula (Id), Z 1 is selected from -O-, -S-, -C(R 22 ) 2 -, -NR 21 -, - wherein: X 1 is O or S; t is 0 or 1; R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
  • X 1 is O or S
  • t is 0 or 1
  • R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )al
  • the compound of formula (Id) is selected from one of the following structures: wherein R 6 is independently H or (C 1 - 3 )alkyl. [0283] In some embodiments of the compound of formula (Id) R 3 is H, such that the compound of formula (Id) has no non-hydrogen substituents at the 1-position of the sugar ring.
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ie’): wherein: R 1 , R 4 , R 5 and R 11 are as defined herein; Z 2 is absent or selected from -O-, -S-, NR 25 -, and -C(R 22 ) 2 , and optionally substituted Z 12 -alkyl; ring A is absent or selected from a 5 or 6-membered optionally substituted aryl and a 5 or 6- membered optionally substituted heteroaryl; Z 3 is a linking moiety selected from Z 12 , optionally substituted alkyl, optionally substituted Z 12 - alkyl, optionally substituted Z 12 -heteroaryl, optionally substituted Z 12 -aryl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiour
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ie’’): [0286] In some embodiments of formula (Ie’), the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ie): wherein: R 1 , R 4 , R 5 and R 11 are as defined herein; Z 2 is absent or selected from -O-, -S-, NR 25 -, and -C(R 22 ) 2 , and optionally substituted Z 12 -alkyl; ring A is absent or selected from a 5 or 6-membered optionally substituted aryl and a 5 or 6- membered optionally substituted heteroaryl; Z 3 is a linking moiety selected from Z 12 , optionally substituted alkyl, optionally substituted Z 12 - alkyl, optionally substituted Z 12 -heteroaryl, optionally substituted Z 12 -aryl, optionally
  • Z 2 is absent.
  • Z 2 is C(R 22 ) 2 where R 22 is H or optionally substituted (C 1 -C 3 )alkyl.
  • Z 2 is -CH 2 -.
  • Z 2 is NR 25 where R 25 is selected from H, optionally substituted (C 1 -C 3 )alkyl and optionally substituted acyl.
  • Z 2 is -N(COCH 3 )-.
  • Z 2 is -NH-.
  • Z 2 is - S-.
  • Z 2 is O.
  • the compound of formula (Ie) is of any one of formulae (If)-(Ii): wherein the A ring, R 1 , R 4 , R 5 , R 11 , Z 3 and R 25 are as defined herein.
  • the A ring is a 5 or 6-membered aryl or heteroaryl.
  • the A ring is a 5-membered heteroaryl selected from selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan.
  • the A ring is a 6-membered heteroaryl selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine. In certain embodiments, the A ring is triazole. In certain embodiments, the A ring is pyridine. In certain embodiments, the A ring is pyrimidine. In certain embodiments, the A ring is thiadiazole. [0290] In some embodiments of any one of formulae (Ie)-(Ii), the A ring is absent. [0291] In some embodiments of any one of formulae (Ie)-(Ii), the A ring is phenyl or substituted phenyl.
  • the A ring is a 5 or 6-membered heteroaryl. In certain cases of formula (If), the A ring is a 5-membered heteroaryl. In certain embodiments of formula (If), the A ring is triazole. In certain embodiments of (If), the A ring is absent. [0293] In some embodiments of formula (Ig), the A ring is a 5 or 6-membered heteroaryl and R 25 is H. In certain embodiments of formula (Ig), the A ring is triazole. In certain embodiments of formula (Ig), the A ring is pyridine. In certain cases of formula (Ig), the A ring is pyrimidine.
  • the A ring is thiadiazole. In some embodiments of formula (Ig), the A ring is absent and R 25 is H or optionally substituted acyl. In certain embodiments, R 25 is -COCH 3 . In certain embodiments, R 25 is H. [0294] In some embodiments of formula (Ih), the A ring is a 5 or 6-membered heteroaryl. In certain cases of formula (Ih), the A ring is a 5-membered heteroaryl. In certain embodiments of formula (Ih), the A ring is triazole. In certain embodiments of (Ih), the A ring is absent.
  • the A ring is a 5 or 6-membered heteroaryl. In certain cases of formula (Ii), the A ring is a 5-membered heteroaryl. In certain embodiments of formula (Ii), the A ring is triazole. In certain embodiments of (Ii), the A ring is absent. [0296] In certain embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by any one of formulae (Ij)-(Im): (Il), and (Im) wherein: R 1 , R 4 , R 5 , R 11 , Z 3 and R 25 are as defined herein.
  • Y 1 -Y 3 are each independently N or CR 27 ; and R 24 and R 27 are each independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen.
  • Z 3 is selected from -O-, -CH 2 O-, - OCH 2 -, optionally substituted -OCH 2 -heteroaryl, optionally substituted -OCH 2 -aryl, optionally substituted -CH 2 O-heteroaryl, and optionally substituted -CH 2 O-aryl.
  • Z 3 is selected from: [0299] In some embodiments of any one of formulae (Ie)-(Im), Z 3 is selected from -C(R 22 ) 2 -, optionally substituted alkyl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiourea. In some embodiments, Z 3 is -CH 2 -. In some embodiments, Z 3 is -CH 2 CH 2 -. In some embodiments, Z 3 is -CH 2 CH 2 CH 2 -. In some embodiments, Z 3 is -NHSO 2 -(C 1-3 -alkyl).
  • Z 3 is -N(Ac)-(C 1-3 -alkyl). [0300] In some embodiments of any one of formulae (Ie)-(Im), Z 3 is selected from -S- and -NR 26 -, where R 26 is selected from H and optionally substituted (C 1 -C 3 )alkyl. [0301] In some embodiments of formula (Ik) at least one of Y 1 to Y 3 is N. In certain embodiments, at least two of Y 1 to Y 3 are N. In certain embodiments, Y 1 and Y 3 are N and Y 2 is CR 25 . In certain embodiments, Y 1 and Y 2 are N and Y 3 is CR 25 .
  • Y 1 and Y 2 are CR 25 and Y 3 is N.
  • R 25 is H.
  • R 24 is C (1-3) -alkyl, or C (1-3) -fluoroalkyl.
  • the fluoroalkyl is CF 3 .
  • R 1 is OH.
  • R 4 and R 5 are each H.
  • at least one of R 4 -R 5 is a promoiety.
  • R 4 and R 5 are cyclically linked to form a promoiety (e.g., as described herein).
  • the compound of formula (Ie) is selected from one of the following structures:
  • the compound of formula (Ie) is selected from one of the following structures: .
  • the compound of formula (Ie) is: .
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (In’): wherein: R 1 , R 4 , R 5 and Z 1 are as defined herein; Y 1 and Y 2 are each independently selected from -O-, -S-, NR 28 -, and -C(R 22 ) 2 ; R 28 is selected from H, optionally substituted (C 1 -C 6 )alkyl, and -C(O)R 22 ; each R 22 is independently selected from H, halogen and optionally substituted (C 1 -C 6 )alkyl; and ring B is a 5 or 6-membered optionally substituted cyclic group.
  • Y 1 is connected to the sugar ring via an alpha configuration. In some embodiments of formula (In’), Y 1 is connected to the sugar ring via a beta configuration.
  • the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (In): wherein: R 1 , R 4 , R 5 and Z 1 are as defined herein; Y 1 and Y 2 are each independently selected from -O-, -S-, NR 28 -, and -C(R 22 ) 2 ; R 28 is selected from H, optionally substituted (C 1 -C 6 )alkyl, and -C(O)R 22 ; each R 22 is independently selected from H, halogen and optionally substituted (C 1 -C 6 )alkyl; and ring B is a 5 or 6-membered optionally substituted cyclic group.
  • Y 1 is O. In certain embodiments, Y 1 is S. In certain embodiments, Y 1 is -NR 28 -. In certain embodiments, Y 1 is -C(R 22 ) 2 and each R 22 is H. [0310] In some embodiments of formula (In)-(In’) Y 2 is -NR 28 - where R 28 is H. In certain embodiments, Y 2 is -NR 28 - where R 28 is -C(O)R 22 . In certain embodiments, R 22 is methyl. [0311] In some embodiments of formula (In)-(In’) the B ring is a 5 or 6-membered heterocycle.
  • the B ring is a 5-membered heterocycle. In certain embodiments, the B ring is a 6- membered heterocycle. [0312] In some embodiments of formula (In)-(In’) Z 1 is Z 11 , where Z 11 is selected from -O-, -S-, NR 21 -, and -C(R 22 ) 2 . In certain embodiments, Z 1 is -O-. In certain embodiments, Z 1 is -S-. In certain embodiments, Z 1 is NR 21 where R 21 is H. In certain embodiments, Z 1 is -C(R 22 ) 2 where each R 22 is H.
  • Z 1 is optionally substituted Z 11 -heteroaryl or optionally substituted Z 11 -aryl.
  • Z 1 is CH 2 -heteroaryl or CH 2 -aryl.
  • Z 1 is optionally substituted amide.
  • Z 1 is optionally substituted sulfonamide.
  • Z 1 is optionally substituted urea or optionally substituted thiourea.
  • the compound of formula (In)-(In’) has one of the following structures: .
  • n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking X to Y via Z 1 , such as a backbone of 25 or more consecutive atoms, or 30 or more consecutive atoms, and in certain embodiments, up to 100 consecutive atoms.
  • linker L separates X and Y (or Z 1 ) by a chain of 20 to 50 consecutive atoms.
  • linker L separates X and Y (or Z 1 ) by a chain of 21 to 50 consecutive atoms, by a chain of 22 to 50 consecutive atoms, by a chain of 23 to 50 consecutive atoms, by a chain of 24 to 50 consecutive atoms, by a chain of 25 to 50 consecutive atoms, by a chain of 26 to 50 consecutive atoms, by a chain of 27 to 50 consecutive atoms, by a chain of 28 to 50 consecutive atoms, or by a chain of 29 to 50 consecutive atoms.
  • linker L separates X and Y (or Z 1 ) by a chain of 30 to 60 consecutive atoms.
  • linker L separates X and Y (or Z 1 ) by a chain of 31 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 32 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 33 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 34 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 35 to 50 consecutive atoms.
  • linker L separates X and Y (or Z 1 ) by a chain of 36 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 41 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 46 to 50 consecutive atoms. [0316] In certain embodiments of any one of formulae (Ia)-(In), n is 2 or more, and L is a branched linker that covalently links 2 or more X moieties to Y via the linking moiety Z 1 .
  • n is 2 or more and each branch of L comprises a linear linker of 14 or more consecutive atoms to covalently link via Z 1 each X moiety to a branching point of the linker L, such as 15 or more consecutive atoms, 16 or more consecutive atoms, or 17 or more consecutive atoms, and in certain embodiments, up to 50 consecutive atoms.
  • each branch of L comprises a linear linker of 14 to 50 consecutive atoms, such as 14 to 45, 14 to 40, 14 to 35 or 14 to 30 consecutive atoms.
  • each branch of L comprises 14 to 30 consecutive atoms, such as 14 to 29, 14 to 28, 14 to 27, 14 to 26, 14 to 25, 14 to 24, 14 to 23, 14 to 22, 14 to 21, or 14 to 20 consecutive atoms.
  • L comprises more than 14 consecutive atoms covalently linking each X moiety (via each Z 1 group) to a branching point of the linker.
  • L comprises 15 consecutive atoms separating each Z 1 group from a branching point of L.
  • L comprises 16 consecutive atoms separating each Z 1 group from a branching point of L.
  • L comprises 17 consecutive atoms separating each Z 1 group from a branching point of L.
  • L comprises 18 consecutive atoms separating each Z 1 group from a branching point of L. In certain embodiments, L comprises 19 consecutive atoms separating each Z 1 group from a branching point of L. In certain embodiments, L comprises 20 consecutive atoms separating each Z 1 group from a branching point of L. In certain other cases, L comprises a liner linker of 20 or more consecutive atoms separating each Z 1 group from a branching point L. [0318] In certain embodiments of any one of formulae (Ia)-(In), n is 2, and L comprises a branched linker having 14 or more consecutive atoms separating each Z 1 group of X from a branching point of L.
  • n is 3, and L comprises a branched linker having 14 or more consecutive atoms separating each Z 1 group of X from a branching point of L.
  • the linker L is of the formula (II) (e.g., as described herein).
  • R 1 is OH. In certain other cases, R 1 is -OC(O)R. In certain embodiments, R 1 is -C(O)NHR, where R is an optionally substituted alkyl.
  • R terminates in an alkenyl or an alkynyl group.
  • R 1 is optionally substituted triazole.
  • the triazole is of the following structure: .
  • R 2 is -NHCOCH 3 .
  • R 2 is — NHCOCF 3 .
  • R 2 is –NHCOCH 2 CF 3 .
  • R 2 is –OH.
  • R 2 is an optionally substituted triazole.
  • the triazole in of the following structure: . [0323] In certain embodiments when R 1 or R 2 is a substituted triazole.
  • the triazole is a 1,2,3-trizole, and the substituent is at the 4 or 5-position.
  • the substituent on the triazole moiety includes but is not limited to, an optionally substituted (C 1 - 6 )alkyl, optionally substituted (C 1 - 6 )alkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkaryl, and an optionally substituted alkyheteroaryl. It will be understood that any convenient substituent can be included in the triazole moiety, see, e.g., triazole moieties disclosed in Mamidayala et al, J. Am. Chem. Soc.2012, 134, 1978-1981.
  • At least one of R 4 -R 5 is a promoiety.
  • the promoiety is an ester.
  • at least one of R 4 -R 5 is of the formula -COCH 3 , -COCH(CH 3 ) 2 or -COC(CH 3 ) 3 .
  • at least one of R 4 -R 5 is of the formula -CH 2 OCOC(CH 3 ) 3
  • R 4 is a promoiety and R 5 is H.
  • R 5 is H and R 4 is a promoiety. In certain embodiments, both R 4 and R 5 are both promoieties. In certain embodiments, R 4 and R 5 are cyclically linked to form a promoiety. In certain embodiments, R 4 and R 5 are cyclically linked to form a promoiety of formulae (Io) or (Ip): wherein R 1 -R 3 and Y 4 are as defined herein. [0325] In certain embodiments of any one of formulae (Ia)-(In), both R 4 and R 5 are H. [0326] In certain embodiments of formula (I), n is 2 or 3, and X is selected from one of the wherein R 5 and R 23 are independently H or (C 1 - 3 )alkyl.
  • n is 1, 2 or 3, and X is selected from one of the following structures: [0328] wherein R 5 and R 4 independently H or a promoiety, or R 5 and R 4 are cyclically linked to form a promoiety; n1 and n2 are each independently an integer from 1 to 6; and Y 4 is a suitable counterion. In some embodiments, Y 4 is sodium. [0329] In certain embodiments of formula (I), n is 1, 2 or 3, and X is selected from one of the following structures: . [0330] In certain embodiments of formula (I), n is 1, 2 or 3, and X is selected from one of the following structures:
  • n is 1, 2 or 3, and X is selected from one of the following structures: .
  • n is 1, 2 or 3, and X is the following structure: .
  • n is 1, 2 or 3, and X is the following structure: .
  • X is the following structure: .
  • n is 1 and X is .
  • -Z 1 - is linked to an -L 1 - moiety (e.g., of the linker of any of formulae (II), (IIa) or (IIb) described herein).
  • the subject compounds comprise a -Z 1 -L 1 - group selected from: wherein each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 -C 6 )alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6. [0336] In certain embodiments, the Z 1 -L 1 - group is , and o is 1 or 2.
  • the Z 1 -L 1 - group each R 22 is H, and p is 1 or 2. [0340] In certain embodiments, the Z 1 -L 1 - group is , where r is 1-3. [0341] In certain embodiments, the Z 1 -L 1 - group are each independently 1-3. [0342] In certain embodiments, the Z 1 -L 1 - group [0343] In certain embodiments, the Z 1 -L 1 - group i are each independently is 1-3. [0344] In certain embodiments, the Z 1 -L 1 - group is , where x is 0-3. [0345] In certain embodiments, the Z 1 -L 1 - group -3.
  • the Z 1 -L 1 - group i where R 21 is H, and z is 1-4.
  • the Z 1 -L 1 - group is , where R 21 is H, and z1 is 1-4.
  • the Z 1 -L 1 - group i is 1-3.
  • the Z 1 -L 1 - group i where q is 1-3.
  • the subject compounds comprise a -Z 1 -L- group selected from: [0351] In certain embodiments, the Z 1 -L 1 - group is , where q is 1-3.
  • q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. [0352] In certain embodiments, the Z 1 -L 1 - group i . [0353] In certain embodiments, the Z 1 -L 1 - group i . [0354] In certain embodiments, the Z 1 -L 1 - group is . [0355] In certain embodiments, -Z 1 -L 1 - comprises an optionally substituted -NH-heteroaryl-. In certain embodiments the heteroaryl is a triazole. In certain embodiments, the heteroaryl is pyridine.
  • the heteroaryl is pyrimidine In certain cases the heteroaryl is thiadiazole [0356]
  • the -Z 1 -L 1 - comprises a group selected from: wherein each R 24 is independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen; and each R 25 is independently selected from H, optionally substituted (C 1 -C 6 )alkyl, and optionally substituted acyl.
  • R 25 is H.
  • R 24 is C (1-3) -alkyl, or C (1-3) -fluoroalkyl.
  • the fluoroalkyl is CF 3 .
  • Exemplary ASGPR Ligands Exemplary moieties that bind ASGPR, and synthons which can be utilized in the preparation of compounds of this disclosure that include the ASGPR ligand of interest are shown in Tables 1-5. [0358] In certain embodiments, the compound of formula (Ib) is a compound shown in Table 1: [0359] In certain embodiments, the compound of formula (Ib) is a compound shown in Table 1A:
  • the compound of formula (Ib) is a compound shown in Table 1B: [0361] In certain embodiments, the compound of formula (Ic) is a compound shown in Table 2a:
  • the compound of formula (Id) is a compound shown in Table 3a: [0363] In certain embodiments, the compound of formula (Id) is a compound shown in Table 4:
  • the compound of formula (Id’) is a compound shown in Table 5: [0365] Additional exemplary moieties that bind ASGPR, and synthons which can be utilized in the preparation of compounds of this disclosure that include the ASGPR ligand of interest are shown in the tables below.
  • the building blocks described herein can be used to prepare the compounds disclosed herein. As is appreciated by one of skill in the art, reactive functional groups present on the building blocks described herein can be reacted with complimentary functional groups on a linker moiety to bond the ASGPR binding compound X to Y. [0366] For example, compounds of this disclosure can be prepared using the building blocks described herein as exemplified in Scheme 1.
  • the compounds of this disclosure have various L moieties which may be constructed by coupling X to one or more first portions of the linker L (e.g., an -L 1 - moiety) via Z 1 to provide exemplary ASGPR binding compound X building blocks.
  • R M1 and R M2 are each independently reactive functional groups for coupling reactions (e.g., alkyne, -N 3 , -C(O)OH, -NH 2 , etc.); and Y’ is Y or a chemoselective a chemoselective ligation group capable of conjugating to an amino acid residue(s) of Y.
  • R 3 is H such that the ASGPR ligand (X) includes CH 2 at the 1-position, and R 2 is a linking moiety, Z 1 .
  • Exemplary building blocks that can be used in the preparation of compounds of this disclosure that include ASGPR ligands (X) of interest are shown in Table 7.
  • the ASGPR ligand (X) building blocks that can be used in the preparation of compounds of this disclosure is a bicyclic structure. Exemplary building blocks that can be used in the preparation of compounds of this disclosure that include ASGPR ligands (X) of interest are shown in Table 8.
  • Prodrugs [0375] Aspects of this disclosure include prodrugs of any of the ASGPR binding moieties described herein that are incorporated into the compounds and conjugates of this disclosure.
  • the term “prodrug” refers to an agent which is converted into the drug in vivo by some physiological or chemical process (e.g., a prodrug on being brought to the physiological pH is converted to the desired drug form).
  • Prodrugs forms of any of the ASGPR binding moieties described herein can be useful because, for example, can lead to particular therapeutic benefits as a consequence of an extension of the half-life of the resulting compound or conjugate in the body or a reduction in the active dose required.
  • Pro-drugs can also be useful in some situations, as they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The pro-drug may also have improved solubility in pharmacological compositions over the parent drug.
  • Prodrug derivative of a ASGPR binding moiety generally includes a promoiety substituent at a suitable labile site of the compound. The promoiety refers to the group that is removed by enzymatic or chemical reactions, when a prodrug is converted to the drug in vivo.
  • the promoiety is a group attached via an ester linkage to a hydroxyl group of the compound or drug.
  • a prodrug derivative of one or more of the hydroxyl groups of the sugar ring may be incorporated into the compounds.
  • an ester promoiety can be incorporated at one or more of the hydroxyl groups at the 3 and/or 4 positions of the sugar (e.g., as described herein).
  • the hydroxyl groups at the 3 and 4 positions of the sugar are cyclically linked to form a promoiety (e.g., as described herein).
  • Linker Valency [0382]
  • the ASGPR ligand moieties (X) can be used in a monovalent or multivalent configuration with respect to the binding to ASGPR of the “n” X groups that are displayed on the linker scaffold.
  • a monovalent configuration includes a single ASGPR ligand moiety (X) per linker of the bifunctional molecule, where it is understood that one or more linkers may be connected to Y.
  • a multivalent configuration includes two or more such ASGPR ligand moieties per linker (e.g., bivalent or trivalent or of higher valency linker). This disclosure provides particular linker scaffolds and linker valencies that display preferred ASGPR ligand moieties in the bifunctional molecules of this disclosure.
  • the linked ASGPR ligand moiety (X) of the bifunctional molecule is monovalent (e.g., in Formula (I), n is 1), such that a linker covalently links a single ASGPR ligand moiety (X) via a linking moiety at the 1, 6, or 2-position of the sugar ring analog to a biomolecule (Y).
  • n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms (e.g., 25 or more) covalently linking the ASGPR ligand X to Y via a linking moiety at any of the 1-, 2- or 6-positions of X.
  • the linker L includes a backbone of 20 to 100 consecutive atoms linking the ASGPR ligand (X) to Y, such as 25 to 80, 25 to 60, or 25 to 50 consecutive atoms.
  • the bifunctional molecule is multivalent with respect to X, where in Formula (I), n is 2 or more, such that the conjugate includes two or more ASGPR ligand binding moieties (X) per multivalent linker which connects to Y.
  • the multivalent linker (L) is a branched linker or a dendrimer linker.
  • the bifunctional molecule has one or more divalent linkers (e.g., n is 2 in Formula (I)).
  • each branch of a branched linker includes a linear linker portion covalently connecting each X moiety (via the linking moiety described herein) to a branching point in the branched linker or dendrimer linker.
  • each branch of the linker includes a linear linker portion having a backbone of 8 or more consecutive atoms, such as 10 or more, 12 or more, 14 or more, 16 or more, 18 or more or 20 or more consecutive atoms between the X ligand moiety and the branching point in the linker.
  • each branch of the linker includes a linear linker portion having a backbone of 8 to 50 consecutive atoms, such as 10 to 50, 12 to 50, 14 to 50, or 14 to 40, 14 to 30, or 14 to 20 consecutive atoms.
  • Linkers [0386]
  • the terms “linker”, “linking moiety” and “linking group” are used interchangeably and refer to a linking moiety that covalently connects two or more moieties, compounds or other biomolecules, such as ligands and proteins of interest.
  • the linker is divalent and connects two moieties.
  • the linker is a branched linking group that is trivalent or of a higher multivalency.
  • the linker that connects the two or more moieties has a linear or branched backbone of 500 atoms or less (such as 400 atoms or less, 300 atoms or less, 200 atoms or less, 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or even 20 atoms or less) in length, e.g., as measured between the two or more moieties.
  • 500 atoms or less such as 400 atoms or less, 300 atoms or less, 200 atoms or less, 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or even 20 atoms or less
  • a linking moiety may be a covalent bond that connects two groups or a linear or branched chain of between 1 and 500 atoms in length, for example of about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100, 150, 200, 300, 400 or 500 carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom.
  • one, two, three, four, five or more, ten or more, or even more carbon atoms of a linker backbone may be optionally substituted with heteroatoms, e.g., sulfur, nitrogen or oxygen heteroatom.
  • linker when the linker includes an ethylene glycol, or longer polyethylene glycol (PEG) linking group, e.g., where every third atom of that segment of the linker backbone is substituted with an oxygen.
  • PEG polyethylene glycol
  • the bonds between backbone atoms of a linker may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone.
  • the linker may include one or more substituent groups, for example an alkyl, aryl or alkenyl group.
  • a linker may include, without limitations, one or more of the following: oligo(ethylene glycol) (also referred to as PEG), ether, thioether, disulfide, amide, carbonate, carbamate, urea, sulfonamide, thiourea, tertiary amine, alkyl which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1- methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like.
  • oligo(ethylene glycol) also referred to as PEG
  • ether also referred to as PEG
  • thioether disulfide
  • amide carbonate
  • carbamate urea
  • sulfonamide thiourea
  • tertiary amine alkyl which may be straight or branched, e
  • the linker backbone may include a cyclic group, for example, an aryl, a heterocycle, a cycloalkyl group or a heterocycle group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone.
  • a “linker” or linking moiety is derived from a molecule with a reactive terminus, e.g., suitable for conjugation to a protein of interest.
  • the reactive terminus of the linker precursor includes a chemoselective ligation group capable of conjugating to amino acid residue(s) of a polypeptide.
  • the chemoselective ligation group conjugates to a cysteine thiol group, or a lysine sidechain amine group of the polypeptide that is accessible.
  • a variety of conjugation chemistries can be utilized in the conjugtaes of this disclosure (e.g., as described herein).
  • the chemoselective ligation group is a thiol-reactive group such as maleimide or dibromomaleimide.
  • the chemoselective ligation group is an amine- reactive group such as an active ester, e.g., perfluorophenyl ester or tetrafluorophenyl ester, or N- hydroxysuccinimidyl ester (NHS) or sulfo-NHS, or as defined herein.
  • the linker L includes one or more straight or branched-chain carbon moieties and/or polyether (e.g., ethylene glycol) moieties (e.g., repeating units of -CH 2 CH 2 O-), and combinations thereof.
  • these linkers optionally have amide linkages, urea or thiourea linkages, carbamate linkages, ester linkages, amino linkages, ether linkages, thioether linkages, sulfhydryl linkages, heteroaryl linkages, or other hetero functional linkages.
  • the linker backbone includes one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof.
  • the linker includes one or more of an ether bond, thioether bond, amine bond, amide bond, carbon-carbon bond, carbon-nitrogen bond, carbon-oxygen bond, carbon-sulfur bond, and combinations thereof.
  • the linker includes a linear structure.
  • the linker includes a branched structure. In certain embodiments, the linker includes a cyclic structure. In certain embodiments, the linker includes one or more heteroaryl cyclic structures, e.g., a triazole, such as a 1,2,3- traizole.
  • L is a linker between about 5 ⁇ and about 500 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 400 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 300 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 200 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 100 ⁇ .
  • linker L separates X (or Z 1 ) and Y by a chain of 10 to 100 consecutive atoms. In certain embodiments, linker L separates X (or Z 1 ) and Y by a chain of 10 to 60 consecutive atoms, by a chain of 12 to 60 consecutive atoms, by a chain of 16 to 50 consecutive atoms, by a chain of 20 to 50 consecutive atoms, by a chain of 30 to 50 consecutive atoms, by a chain of 40 to 50 consecutive atoms. [0391] It is understood that the linker may be considered as connecting directly to a Z 1 group of a ASGPR ligand moiety (X) (e.g., as described herein).
  • X ASGPR ligand moiety
  • the linker may be considered as connecting directly to the Z 1 group.
  • the -Z 1 -L 1 - group e.g., as described herein
  • the disclosure is meant to include all such configurations of ASGPR ligand moiety (X) and linker (L).
  • L is a linker of formula (XI): wherein each L 1 and L 3 are independently a linear linking moiety, and L 2 is a branched linking moiety, wherein L 1 to L 3 together provide a linear or branched linker between X and Y; a, b and c are independently 0 or 1; * represents the point of attachment of L 1 to X via Z 1 ; and ** represents the point of conjugation of the linker L to Y; wherein: when n is 1, b is 0 and at least one of a and c is 1; and when n is 2 or 3, a, b and c are each 1.
  • the linear linker of formula (Xia) has a backbone of 10 or more consecutive atoms covalently linking X to Y via Z 1 , such as a backbone of 12 or more consecutive atoms, 14 or more consecutive atoms, or 16 or more consecutive atoms, and in certain embodiments, up to 100 consecutive atoms.
  • the linear linker separates X (or Z 1 ) and Y by a chain of 20 to 50 consecutive atoms. In certain embodiments of formula (Xa), the linear linker separates X (or Z 1 ) and Y by a chain of 30 to 60 consecutive atoms.
  • each L 1 is of formula (XII): wherein: L 10 is a linking moiety, and * represents the point of attachment of L 1 to X via Z 1 ; and L 11 to L 19 are independently absent or a linking moiety, wherein L 10 to L 19 of each L 1 is independently selected from–C 1-6 -alkylene–,-–C 1-12 -alkylene–, – C 1-20 -alkylene–,–NHCO-C 1-6 -alkylene–,
  • each L 1 is of formula (XII): wherein: L 10 is a linking moiety, and * represents the point of attachment of L 1 to X via Z 1 ; and L 11 to L 19 are independently absent or a linking moiety, wherein L 10 to L 19 of each L 1 is independently selected from –C 1-6 -alkylene–,-CF 2 -, –C 1-12 - alkylene–, –C 1-20 -alkylene–,–NHCO-C 1-6 -alkylene–, –CONH-C 1-6 -alkylene–, –NH-C 1-6 -alkylene–, – NHCONH-C 1-6 -alkylene–, –NHCSNH-C 1-6 -alkylene–, –C 1-6 -alkylene–NHCO-, –C 1-6 -alkylene–CONH-, —C 1-6 -alkylene–CONH-, —C 1-6 -alkylene–CONH-, —C
  • the linking moiety L 1 includes a linear backbone of 6 to 40 consecutive atoms, such as 10 to 40, 10 to 30, 16 to 30, or 20 to 30 consecutive atoms.
  • the linking moiety L 1 includes a linear backbone of each L 1 comprises a linear backbone of 6 to 20 consecutive atoms, such as 6 to 16 consecutive atoms, such as 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive atoms.
  • the linking moiety of formula (XII) includes one or repeating ethylene glycol moieties (e.g., -CH 2 CH 2 O- or -OCH 2 CH 2 -).
  • the linking moiety of formula (XII) includes 1 to 10 ethylene glycol moieties, such as 1, 2, 3, 4, 5 or 6 ethylene glycol moieties.
  • the linking moiety of formula (XII) includes one or more triazole (e.g., 1,2,3-triazole) containing linking moieties. It is understood that the triazole may be derived from an azido-alkyne click chemistry and thus have two possible orientations depending on the method of synthesis: [0403]
  • the triazole containing linking moiety is : wherein w1 and u1 are independently 0 to 12, such as 0, 1, 2, 3, 4, 5 or 6.
  • L 20 is a branched linking moiety including one or more linking moieties independently selected from amino acid residue (e.g., a residue such as Gly, Ala, beta-Al Glu, Ser, Cys, or a derivative thereof), –NH-CH[(CH 2 ) q ] 2 O– or –NH-C[(CH 2 ) q ] 3 O–, alkylene–, –NHCO-, –CONH–, –NHSO 2 –, –SO 2 NH–, –CO–, –SO 2 –, –O–, –S–, pyrrolidine-2,5-dione, 1,2,3-triazole, –NH–, and –Nme–, –NHC(O)NH–, – NHC(S)
  • b is 1 and the linking moiety L 2 is selected from one of (L2A)-(L2D): wherein: each Z 2 and Z 3 is independently absent or selected from –NHCO-, –CONH–, –CO–, –O–, –NH–, and –Nme–; x is 1 to 12 (e.g., 1 to 6, or 1 to 3); and y is 0 to 12 (e.g., 1 to 6, or 1 to 3). [0406] In some embodiments of any one of L2A-L2D, Z 2 is –NHCO-. In some embodiments of any one of L2A-L2D, Z 2 is –CONH–.
  • Z 2 is –CO–. In some embodiments of any one of L2A-L2D, Z 2 is –O–. In some embodiments of any one of L2A-L2D, Z 2 is –NH–. In some embodiments of any one of L2A-L2D, Z 2 is –Nme–. In some embodiments of any one of L2A-L2D, Z 2 is absent. [0407] In some embodiments of any one of L2A-L2D, Z 3 is –NHCO-. In some embodiments of any one of L2A-L2D, Z 3 is –CONH–.
  • Z 3 is –CO–. In some embodiments of any one of L2A-L2D, Z 3 is –O–. In some embodiments of any one of L2A-L2D, Z 3 is –NH–. In some embodiments of any one of L2A-L2D, Z 3 is –Nme–. In some embodiments of any one of L2A-L2D, Z 3 is absent.
  • L2A In some embodiments of L2A, Z 2 is –O–, y is 0 and the linking moiety is of the structure L2Ai: [0409] In some embodiments of L2B, Z 2 is –O– or –CO—, and the linking moiety is of the structure L2Bi or L2Bii: [0410] In some embod the linking moiety is of the structure L2Ci, L2Cii, L2Ciii, or L2Civ: [0411] In some embodiments of L2D, Z 2 is absent and the linking moiety is of the structure L2Di: [0412] In some embodiments, of any one of formulae L2A-L2Di, x is 1 to 6.
  • x is 1 to 3. In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, x is 3. [0413] In some embodiments of any one of formulae L2A-L2Di, y is 0 to 6. In certain embodiments, y is 0 to 3. In certain embodiments, y is 0. In certain embodiments, y is 1. In certain embodiments, y is 2. In certain embodiments, y is 3.
  • b is 1 and the linking moiety L 2 is selected from: [0415] In some embodiments of the linker of formula (XI), b is 1 and the linking moiety L 2 is of the formula (XIV): wherein: r is 1 or 2; and when n is 2, r is 1, when n is 3, r is 2. [0416] In some embodiments of the linker of formula (XI), b is 1 and the linking moiety L 2 is of the formula (Xva) or (XVb): wherein: r is 1 or 2; and when n is 2, r is 1, when n is 3, r is 2.
  • L 2 is of formula (XIIIa) or (XIIIb) and L 2 includes two 2 or more amino acid residues (e.g., 3 or more, or 4 or more amino acid residues, linear or dendrimer).
  • L 2 includes 4 or more amino acid residues that are branched linking moieties selected from Lys, Orn, Asp, Glu, Ser, and Cys (e.g., where the sidechain, amino and carboxylic acid are each linked to an adjacent moiety).
  • each L 3 is of the formulae (XVI): wherein: L 30 to L 39 are independently absent or a linking moiety; and Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group of the linker to a compatible group of Y; wherein L 30 to L 39 are each independently selected from –C 1-20 -alkylene–, –NHCO-C 1-6 - alkylene–, –CONH-C 1-6 -alkylene–, –NH C 1-6 -alkylene–, –NHCONH-C 1-6 -alkylene–, – NHCSNH-C 1-6 - alkylene–, –C 1-6 -alkylene–NHCO-, –C 1-6 -alkylene–CONH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–CONH-, –C 1-6 -alkylene–NH-, –C 1-6 -al
  • the linking moiety of formula (XVI) includes a linear backbone of 6 to 40 consecutive atoms, such as 10 to 40, 10 to 30, or 20 to 30 consecutive atoms.
  • the linking moiety of formula (XVI) includes repeating ethylene glycol moieties (e.g., -CH 2 CH 2 O- or -OCH 2 CH 2 -).
  • the linking moiety of formula (XVI) includes 2 to 20 ethylene glycol moieties, such as 2 to 15, 2 to 10, 3 to 20, 3 to 15, 3 to 10, 4 to 15, 5 to 15 or 5 to 10 ethylene glycol moieties.
  • the linking moiety of formula (XVI) includes 2 or more ethylene glycol moieties, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or even more ethylene glycol moieties.
  • the linking moiety of formula (XVI) includes one or more triazole linking moieties.
  • the linker includes one or more 1,2,3-triazole linking moieties.
  • the one or more 1,2,3-triazoel moieties is selected from one of the following structures: , wherein w1, u1 and q1 are independently 1 to 25 (e.g., 1 to 12, such as 1 to 6).
  • the linking moiety L 3 includes (C 10 -C 20 -alkylene (e.g., C 12 -alkylene), or –(OCH 2 CH 2 ) p –, where p is 1 to 25, such as 3 to 25, 5 to 24, 7 to 25, 10 to 25, 15 to 25 or 20 to 24.
  • the linker L is of formula XVII: wherein: a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); f is 1 to 6 (e.g., 1, 2, or 3); Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group (e.g., as described herein) of a linker precursor to a compatible group of Y.
  • a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to
  • Z is a residual moiety resulting from the covalent linkage (e.g., via a thioether bond) of a thiol-reactive chemoselective ligation group to one or more cysteine residue(s) of Y.
  • the thiol-reactive chemoselective ligation group includes maleimide, bromomaleimide, haloacetamide, vinyl sulfone, or thiolactone.
  • the thiol-reactive group is selected from one of the following structures: wherein: u is 1 to 11 (e.g., 1 to 5); v is 1 to 11 (e.g., 1 to 5); and X is H or Br. [0425] In some embodiments, the thiol-reactive group comprises:
  • Z is a residual moiety resulting from the covalent linkage (e.g., via an amide bond) of an amine-reactive chemoselective ligation group to one or more lysine residue(s) of Y.
  • the amine-reactive chemoselective ligation group includes an active ester (e.g., N-hydroxysuccinimidyl (NHS) ester, sulfo-NHS ester, pentafluorophenyl (PFP) ester, tetrafluorophenyl (TFP) ester, or the like).
  • the linker L includes one of (XVIIIa)-(XVIIIc): wherein: a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); and f is 1 to 6 (e.g., 1, 2, or 3).
  • a is 2 to 6, such as 2 to 3. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5. In some embodiments a is 6. [0430] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), b is 1 to 4, such as 1 to 3. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3.
  • c is 1 to 4, such as 1 to 3. In some embodiments, c is 1. In some embodiments, c is 2. In some embodiments, c is 3. [0432] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), r is 1. In some embodiments, r is 2. [0433] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), d is 1 to 4, such as 1 to 3. In some embodiments, d is 1. In some embodiments, d is 2. In some embodiments, d is 3.
  • e is 1 to 5, such as 1 to 3. In some embodiments, e is 1. In some embodiments, e is 2. In some embodiments, e is 3. In some embodiments, e is 4. In some embodiments, e is 5. [0435] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), f is 1 to 4, such as 1 to 3. In some embodiments, f is 1. In some embodiments, f is 2. In some embodiments, f is 3.
  • a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3.
  • a is 1-4; b is 1-4; c is 1-3; r is 2; d is 1-3; e is 1-6; and f is 1-3.
  • a is 2; b is 1; c is 2; r is 1; d is 2; e is 3; and f is 2.
  • a is 2; b is 1; c is 2; r is 2; d is 2; e is 3; and f is 2.
  • a is 4; b is 1; c is 2; r is 1; d is 2; e is 3; and f is 2.
  • a is 4; b is 1; c is 2; r is 2; d is 2; e is 3; and f is 2.
  • a is 2; b is 2; c is 2; r is 1; d is 2; e is 3; and f is 2.
  • a is 2; b is 2; c is 2; r is 2; d is 2; e is 3; and f is 2.
  • a is 0; b is 3; c is 2; r is 2; d is 2; e is 3; and f is 2.
  • a is 2; b is 4; c is 2; r is 2; d is 2; e is 3; and f is 2.
  • a is 2; b is 4; c is 2; r is 1; d is 2; e is 3; and f is 2.
  • the linker L includes L A : (L A ), wherein: Z 4 is selected from -NHC(O)NH-, -NHC(O)-, -C(O)NH-, -O-, -NH-; a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); and f is 1 to 6 (e.g., 1, 2, or 3).
  • Z 4 is -NHC(O)NH-. In certain embodiments, Z 4 is -NHC(O)-. In certain embodiments, Z 4 is -C(O)NH-. In certain embodiments, Z 4 is -O-. In certain embodiments, Z 4 is -NH-.
  • a is 1-4; b is 1-4; c is 1-3; d is 1-3; e is 1-6; and f is 1-3. In some embodiments, a is 4; b is 1; c is 2; d is 2; e is 5; and f is 2.
  • Z 4 is -NHC(O)NH- and a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3.
  • Z 4 is -NHC(O)- and a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3.
  • the linker L includes L B : wherein: a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); and f is 1 to 6 (e.g., 1, 2, or 3).
  • a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3.
  • a is 4; b is 1; c is 2; r is 1; d is 2; e is 5; and f is 2.
  • a is 2; b is 1; c is 2; r is 1; d is 2; e is 3; and f is 2.
  • a is 4; b is 1; c is 2; r is 1; d is 2; e is 3; and f is 2.
  • a is 1; b is 2; c is 2; r is 1; d is 2; e is 3; and f is 2.
  • a is 0; b is 3; c is 2; r is 1; d is 2; e is 3; and f is 2.
  • L B a is 1-4; b is 1-4; c is 1-3; r is 2; d is 1-3; e is 1-6; and f is 1-3.
  • a is 2; b is 1; c is 2; r is 2; d is 2; e is 3; and f is 2.
  • a is 4; b is 1; c is 2; r is 2; d is 2; e is 3; and f is 2.
  • a is 1; b is 2; c is 2; r is 2; d is 2; e is 3; and f is 2.
  • a is 0; b is 3; c is 2; r is 2; d is 2; e is 3; and f is 2.
  • the linker L includes L C : wherein: a is 0 to 12 (e.g., 1 to 6, 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1 to 4, such as 1, 2, or 3); c is 1 to 6 (e.g., 1 to 3, such as 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1 to 3, such as 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); and f is 1 to 6 (e.g., 1 to 3, such as 1, 2, or 3).
  • a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3.
  • a is 2; b is 4; c is 2; r is 1; d is 2; e is 5; and f is 2.
  • a is 1-4; b is 1-4; c is 1-3; r is 2; d is 1-3; e is 1-6; and f is 1-3.
  • a is 2; b is 4; c is 2; r is 2; d is 2; e is 5; and f is 2.
  • -Z 1 - is linked to an -L 1 - moiety (e.g., of the linker as described herein).
  • the subject compounds comprise a -Z 1 -L 1 - moiety comprising a linking moiety selected from: wherein each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 -C 6 )alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6.
  • the Z 1 -L 1 - group is , and o is 1 or 2.
  • the Z 1 -L 1 - group 22 each R is H, and p is 1 or 2.
  • the Z 1 -L 1 - group is , where r is 1-3.
  • the Z 1 -L 1 - group are each independently 1-3.
  • the Z 1 -L 1 - group i are each independently is 1-3.
  • the Z -L - group is N N N 1 1 , where x is 0-3.
  • the Z 1 -L 1 - group R 21 [0468] In certain embodiments, the Z 1 -L 1 - group is , where R 21 is H, and z is 1-4.
  • R 21 [0469] In certain embodiments, the Z 1 -L 1 - group is , where R 21 is H, and z1 is 1-4.
  • the Z 1 -L 1 - group is , where each R 22 is H, and q is 1-3.
  • the Z 1 -L 1 - group i where q is 1-3.
  • the subject compounds comprise a -Z 1 -L- group comprising a linking moiety selected from: where R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 -C 6 )alkyl (e.g., methyl).
  • R 21 is H.
  • each R 22 is H.
  • the -Z 1 -L 1 - group is , where q is 1-3. In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. [0474] In certain embodiments, the -Z 1 -L 1 - group is H . [0475] In certain embodiments, -Z 1 -L 1 - includes an optionally substituted -NH-heteroarylene-. In certain embodiments, the heteroarylene is a triazole. In certain embodiments, the heteroarylene is pyridine. In certain embodiments, the heteroarylene is pyrimidine. In certain embodiments, the heteroarylene is thiadiazole.
  • the -Z 1 -L 1 - includes a group selected from: wherein R 24 and R 25 are each independently selected from H, optionally substituted C (1-6) - alkyl, optionally substituted fluoroalkyl, and halogen; and each R 21 is independently selected from H, optionally substituted (C 1 -C 6 )alkyl, and optionally substituted alkanoyl.
  • R 21 is H.
  • R 24 is C (1-3) -alkyl, or C (1-3) -fluoroalkyl.
  • the fluoroalkyl is CF 3 .
  • R 25 is C (1-3) -alkyl, or C (1-3) -fluoroalkyl.
  • the fluoroalkyl is CF 3 .
  • the linker includes a polypeptide scaffold where some or all of the sidechain groups of the amino acid residues of such a polypeptide scaffold have been modified to attach a X binding moiety (e.g., as described herein). It is understood that X binding moieties (e.g., as described herein) can be conjugated to amino acid residues, such as Asp, Lys, Orn, Glu, and Ser, of a polypeptide containing linker via a convenient conjugation chemistry.
  • the linker contains a polylysine polypeptide. In some embodiments, the linker contains a polyornithine polypeptide. In some embodiments, the linker contains a polyserine polypeptide. In some embodiments, the linker contains a polyaspartate polypeptide.
  • the polypeptide backbone of such a linker can be a randomly polymerized polymer having an average length, or a polymer of defined length prepared e.g., in a controlled stepwise fashion. In certain embodiments, the polypeptide linker has a length of 10-100 amino acid residues, such as 20-90, or 20-50 amino acid residues.
  • the N-terminal or C-terminal of the polypeptide linker is modified to include a linking moiety to an additional X binding moiety (e.g., as described herein).
  • the N-terminal or C-terminal of the polypeptide linker segment is modified with one or more linking moieties (e.g., as described herein) suitable for attachment to a protein construct (Y) including a polypeptide that specifically binds an autoantibody [0478]
  • a “linker” or linking moiety is derived from a molecule with two reactive termini, one for conjugation to a moiety of interest (Y), e.g., a biomolecule (e.g., an antibody) and the other for conjugation to a moiety (noted as X) that binds to a ASGPR cell surface receptor.
  • the polypeptide conjugation reactive terminus of the linker is in some cases a site that is capable of conjugation to the polypeptide through a cysteine thiol or lysine amine group on the polypeptide, and so is can be a thiol-reactive group such as a maleimide or a dibromomaleimide, or as defined herein, or an amine-reactive group such as an active ester (e.g., perfluorophenyl ester or tetrafluorophenyl ester), or as defined herein.
  • a thiol-reactive group such as a maleimide or a dibromomaleimide, or as defined herein
  • an amine-reactive group such as an active ester (e.g., perfluorophenyl ester or tetrafluorophenyl ester), or as defined herein.
  • the linker L comprises one or more straight or branched-chain carbon moieties and/or polyether (e.g., ethylene glycol) moieties (e.g., repeating units of -CH 2 CH 2 O-), and combinations thereof.
  • these linkers optionally have amide linkages, urea or thiourea linkages, carbamate linkages, ester linkages, amino linkages, ether linkages, thioether linkages, sulfhydryl linkages, heteroaryl linkages, or other hetero functional linkages.
  • the linker comprises one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof.
  • the linker comprises one or more of an ether bond, thioether bond, amine bond, amide bond, carbon-carbon bond, carbon-nitrogen bond, carbon-oxygen bond, carbon-sulfur bond, and combinations thereof.
  • the linker comprises a linear structure.
  • the linker comprises a branched structure.
  • the linker comprises a cyclic structure.
  • the linker comprises one or more heteroaryl cyclic structures, e.g., a triazole, such as a 1,2,3-traizole.
  • L is between about 10 ⁇ and about 20 ⁇ in length.
  • L is between about 15 ⁇ and about 20 ⁇ in length. In certain embodiments, L is about 15 ⁇ in length. In certain embodiments, L is about 16 ⁇ in length. In certain embodiments, L is about 17 ⁇ in length. [0481] In certain embodiments, L is a linker between about 5 ⁇ and about 500 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 400 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 300 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 200 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 100 ⁇ .
  • L is between about 10 ⁇ and about 20 ⁇ , between about 20 ⁇ and about 30 ⁇ , between about 30 ⁇ and about 40 ⁇ , between about 40 ⁇ and about 50 ⁇ , between about 50 ⁇ and about 60 ⁇ , between about 60 ⁇ and about 70 ⁇ , between about 70 ⁇ and about 80 ⁇ , between about 80 ⁇ and about 90 ⁇ , or between about 90 ⁇ and about 100 ⁇ .
  • L is a linker between about 5 ⁇ and about 500 ⁇ , which comprises an optionally substituted arylene linked to X, an optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X.
  • L is a linker between about 10 ⁇ and about 500 ⁇ , which comprises an optionally substituted arylene linked to X, or optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X. In certain embodiments, L is a linker between about 10 ⁇ and about 400 ⁇ , which comprises an optionally substituted arylene linked to X, or optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X.
  • L is a linker between about 10 ⁇ and about 200 ⁇ , which comprises an optionally substituted arylene linked to X, or optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X.
  • linker L separates X and Y (or Z 1 ) by a chain of 4 to 500 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 4 to 50 consecutive atoms.
  • linker L separates X and Y (or Z 1 ) by a chain of 6 to 50 consecutive atoms, by a chain of 11 to 50 consecutive atoms, by a chain of 16 to 50 consecutive atoms, by a chain of 21 to 50 consecutive atoms, by a chain of 26 to 50 consecutive atoms, by a chain of 31 to 50 consecutive atoms, by a chain of 36 to 50 consecutive atoms, by a chain of 41 to 50 consecutive atoms, or by a chain of 46 to 50 consecutive atoms.
  • linker L separates X and Y (or Z 1 ) by a chain of 6 to 50 consecutive atoms.
  • linker L separates X and Y (or Z 1 ) by a chain of 11 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 16 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 21 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 26 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 31 to 50 consecutive atoms.
  • linker L separates X and Y (or Z 1 ) by a chain of 36 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 41 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 46 to 50 consecutive atoms.
  • linker L separates X and Y (or Z 1 ) by a chain of 4 or 5 consecutive atoms, by a chain of 6 to 10 consecutive atoms, by a chain of 11 to 15 consecutive atoms, by a chain of 16 to 20 consecutive atoms, by a chain of 21 to 25 consecutive atoms, by a chain of 26 to 30 consecutive atoms, by a chain of 31 to 35 consecutive atoms, by a chain of 36 to 40 consecutive atoms, by a chain of 41 to 45 consecutive atoms, or by a chain of 46 to 50 consecutive atoms.
  • linker L is a chain of 5 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or heteroatom linked to X.
  • linker L is a chain of 7 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or heteroatom linked to X.
  • linker L is a chain of 10 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or heteroatom linked to X. In certain embodiments, linker L is a chain of 15 to 400 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or heteroatom linked to X.
  • linker L is a chain of 5 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an alkylene, a heteroatom, or optionally substituted heteroarylene linked to X. In certain embodiments, linker L is a chain of 7 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an alkylene, a heteroatom, or optionally substituted heteroarylene linked to X. In certain embodiments, linker L is a chain of 10 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an comprises an alkylene, a heteroatom, or optionally substituted heteroarylene linked to X.
  • linker L is a chain of 15 to 400 consecutive atoms separating X and Y (or Z 1 ) and which comprises an alkylene, a heteroatom, or optionally substituted heteroarylene linked to X. [0486] In certain embodiments, linker L is a chain of 5 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted triazole linked to X. In certain embodiments, linker L is a chain of 7 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted triazole linked to X.
  • linker L is a chain of 10 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted triazole linked to X. In certain embodiments, linker L is a chain of 15 to 400 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted triazole linked to X. [0487] In certain embodiments, linker L is a chain of 16 to 400 consecutive atoms separating X and Y (or Z) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or a heteroatom linked to X.
  • the linker may be considered as connecting directly to a Z 1 group of a ASGPR binding moiety (X) (e.g., as described herein). In some embodiments of any of formulae (Ia)- (Ip), the linker may be considered as connecting directly to the Z 1 group.
  • the -Z 1 -L 1 - group e.g., as described herein
  • the disclosure is meant to include all such configurations of ASGPR binding moiety (X) and linker (L).
  • L is a linker of formula (II): wherein L 1 and L 3 are independently a linker, and L 2 is a branched linking moiety, wherein L 1 to L 3 together provide a linear or branched linker between X and Y; a, b and c are independently 0 or 1; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y; wherein: when n is 1, a is 1, and b is 0; when n is >1, a is 1, and b is 1.
  • L 1 to L 3 each independently comprise one or more linking moieties independently selected from –C 1-20 -alkylene–, –NHCO-C 1-6 -alkylene–, – CONH-C 1-6 -alkylene–, –NH C 1-6 -alkylene–, –NHCONH-C 1-6 -alkylene–, – NHCSNH-C 1-6 -alkylene–, – C 1-6 -alkylene–NHCO-, –C 1-6 -alkylene–CONH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NHCONH-, –C 1-6 - alkylene–NHCSNH-, -O(CH 2 ) p –, –(OCH 2 CH 2 ) p –, –NHCO—, —CONH–, –NHSO 2 –, –SO 2 NH–
  • any of L 1 -L 3 comprises repeating ethylene glycol moieties (e.g., -CH 2 CH 2 O- or -OCH 2 CH 2 -).
  • the linker of formula (II) comprises 1 to 25 ethylene glycol moieties, such as 3 to 25, 5 to 25, 7 to 25, 10 to 25, 15 to 25, 17 to 25, 20 to 25 or 22 to 25 ethylene glycol moieties.
  • the linker of formulae (II) comprises 3 or more ethylene glycol moieties, such as 5 or more, 7 or more, 10 or more, 15 or more, 20 or more, or even more ethylene glycol moieties.
  • any of L 1 -L 3 comprises one or more triazole linking moieties.
  • the linker comprises one or more 1,2,3-triazole linking moieties.
  • the one or more 1,2,3-triazole moieties is selected from one of the following structures: , wherein w1, u1 and q1 are independently 1 to 25 (e.g., 1 to 12, such as 1 to 6).
  • n is 1, such that b is 0, and the linker is of the formula (IIa): wherein L 1 and L 3 are independently a linker (e.g., as described herein), wherein L 1 to L 3 together provide a linear linker between X and Y; a is 1; c is 0 or 1; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y.
  • L 1 and L 3 are independently a linker (e.g., as described herein), wherein L 1 to L 3 together provide a linear linker between X and Y; a is 1; c is 0 or 1; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y.
  • the linear linker has a backbone of 20 or more consecutive atoms covalently linking X to Y via Z 1 , such as a backbone of 25 or more consecutive atoms, or 30 or more consecutive atoms, and in certain embodiments, up to 100 consecutive atoms.
  • the linear linker separates X and Y (or Z 1 ) by a chain of 20 to 50 consecutive atoms.
  • the linear linker separates X and Y (or Z 1 ) by a chain of 21 to 50 consecutive atoms, by a chain of 22 to 50 consecutive atoms, by a chain of 23 to 50 consecutive atoms, by a chain of 24 to 50 consecutive atoms, by a chain of 25 to 50 consecutive atoms, by a chain of 26 to 50 consecutive atoms, by a chain of 27 to 50 consecutive atoms, by a chain of 28 to 50 consecutive atoms, or by a chain of 29 to 50 consecutive atoms.
  • the linear linker separates X and Y (or Z 1 ) by a chain of 30 to 60 consecutive atoms.
  • the linear linker separates X and Y (or Z 1 ) by a chain of 31 to 60 consecutive atoms. In certain embodiments, the linear linker separates X and Y (or Z 1 ) by a chain of 32 to 60 consecutive atoms. In certain embodiments, the linear linker separates X and Y (or Z 1 ) by a chain of 33 to 60 consecutive atoms. In certain embodiments, the linear linker separates X and Y (or Z 1 ) by a chain of 34 to 60 consecutive atoms. In certain embodiments, the linear linker L separates X and Y (or Z 1 ) by a chain of 35 to 50 consecutive atoms.
  • the linear linker L separates X and Y (or Z 1 ) by a chain of 36 to 50 consecutive atoms. In certain embodiments, the linear linker L separates X and Y (or Z 1 ) by a chain of 41 to 50 consecutive atoms. In certain embodiments, the linear linker L separates X and Y (or Z 1 ) by a chain of 46 to 50 consecutive atoms. [0495] In certain other embodiments of formula (II), n is 2 or more, such that L 1 to L 3 together provide a branched linker between X and Y.
  • n is 2 or more, and L 2 is selected from: wherein each x and y are independently 1 to 10.
  • L 1 -L 2 comprises a backbone of 14 or more consecutive atoms between X and the branching atom, such as 14 to 50, 14 to 40, 14 to 35 or 14 to 30 consecutive atoms between X and the branching atom.
  • L 3 comprises a backbone of 10 to 80 consecutive atoms, such as 12 to 70, 12 to 60, or 12 to 50 consecutive atoms.
  • L comprises of 12 to 70, 12 to 60, 12 to 50, or 10 to 60 consecutive linear or branched chain atoms.
  • L 3 comprises a linking moiety selected from (C 10 -C 20 -alkylene (e.g., C 12 -alkylene), or –(OCH 2 CH 2 ) p –, where p is 1 to 25, such as 3 to 25, 5 to 24, 7 to 25, 10 to 25, 15 to 25 or 20 to 24.
  • L is of formula (Iib): wherein each L 1 to L 5 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 0, 1, or 2; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y; wherein: when n is 1, a is 1, and c is 0; and when n is >1, a is 1, and c is 1.
  • L is of formula (IIb’): wherein: each L 1 to L 6 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y.
  • L is of formula (IIb’): wherein: each L 1 to L 6 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y.
  • each L 1 to L 5 independently comprises one or more linking moieties independently selected from –C 1-20 -alkylene–, –NHC(O)-C 1-6 -alkylene–, –C(O)NH-C 1-6 -alkylene–, —NH- C 1-6 -alkylene–, –NHC(O)NH-C 1-6 -alkylene–, –NHC(S)NH-C 1-6 -alkylene–, –C 1-6 -alkylene–NHC(O)-, – C 1-6 -alkylene–C(O)NH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NHC(O)NH-, –C 1-6 -alkylene–NHC(S)NH- , -O(CH 2 ) p –, –(OCH 2 CH 2 ) p –, –NHC(O)–, –, –NHC(S)
  • each L 1 to L 5 is independently selected from –C 1-20 -alkylene–, – NHC(O)-C 1-6 -alkylene–, –C(O)NH-C 1-6 -alkylene–, –NH-C 1-6 -alkylene–, –NHC(O)NH-C 1-6 -alkylene–, – NHC(S)NH-C 1-6 -alkylene–, –C 1-6 -alkylene–NHC(O)-, –C 1-6 -alkylene–C(O)NH-, –C 1-6 -alkylene–NH-, – C 1-6 -alkylene–NHC(O)NH-, –C 1-6 -alkylene–NHC(S)NH-, -O(CH 2 ) p –, –(OCH 2 CH 2 ) p –, –NHC(O)–, – C(O)NH–, – C(O)NH–,
  • -(L 1 ) a - comprises an optionally substituted alkyl or ethylene glycol linking moiety.
  • L 1 comprises an optionally substituted -C 1-6 -alkylene–.
  • L 1 comprises an ethylene glycol linking moiety.
  • L 1 is independently selected from: -C 1-6 -alkylene–, –(CH 2 CH 2 O) t –, –-C 1-6 -alkylene-NR 4 CO–, –C 1-6 -alkyleneCONH–,or OCH 2 , wherein t is 1 to 20; and R 4 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl.
  • L 1 is -C 1-6 -alkylene–, such as -C 1-3 -alkylene–.
  • L 1 is – (CH 2 CH 2 O) t –, where t is 1 to 20, such as 1 to 15, 1 to 10, 1 to 8, 1 to 6, or 1 to 4.
  • L 1 is –-C 1-6 -alkylene-NR 4 CO–.
  • L 1 is –C 1-6 -alkyleneCONH–.
  • L 1 is or OCH 2 .
  • one or more L 1 is independently –CH 2 O–; – wherein: R 13 is selected from H, halogen, OH, optionally substituted (C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkoxy, COOH, NO 2 , CN, NH 2 , -N(R 21 ) 2 , -OCOR 21 , -COOR 21 , -CONHR 21 , and -NHCOR 21 ; each r independently 0 to 20, and any of the L 1 moieties are optionally further substituted.
  • L 2 is independently selected from: is 1 to 10, u is 0 to 10, w is 1 to 10, and R 4’ is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl.
  • L 2 is –NR 4’ CO-C 1-6 -alkylene–.
  • L 2 is – CONR 4’ -C 1-6 -alkylene.
  • [0510] In certain embodiments, [0511] In certain embodiments, [0512] In certain embodiments, is 1. [0513] In certain embodiments, [0514] In certain embodiments, [0515] In certain embodiments, L 2 is -OCH 2 -.
  • L 2 is (OCH 2 CH 2 ) q –, and q is 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2. In certain embodiments, q is 2 to 8, such as 2 to 6 , 4 to 6, or 2 to 4. [0516] In certain embodiments of formula (Iib), L 4 is absent or independently selected from -C 1-6 -alkylene–, –(CH 2 CH 2 O) t –, –-C 1-6 -alkylene-NHCO–, –C 1-6 -alkyleneCONH–,or OCH 2 , wherein t is 1 to 20. In certain embodiments, L 4 is absent.
  • L 4 is -C 1-6 -alkylene–. In certain embodiments, L 4 is –(CH 2 CH 2 O) t –, where t is 1 to 20, such as 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 3. In certain embodiments, L 4 is –-C 1-6 -alkylene-NHCO–. In certain embodiments, L 4 is –C 1-6 -alkyleneCONH–. In certain embodiments, L 4 is OCH 2 . [0518] In some embodiments of the subject compounds, n is 1 and L 3 in formula (Iib) is absent.
  • L 3 of formula (Iib) is a branched linking moiety.
  • L 3 is a branched linking moiety, e.g., a divalent, or a trivalent linking moiety.
  • an L 3 linking moiety can be of the one of the following general formula: .
  • the branched linking moiety can be of higher valency and be described by one of the one of the following general formula: where any two L 3 groups can be directed linked or connected via optional linear linking moieties (e.g., as described herein).
  • the branched linking moiety can include one, two or more L 3 linking moieties, each being trivalent moieties, which when linked together can provide for multiple branching points for covalent attachment of the ligands and be described by the following general formula: where t is 0 to 500, such as 0 to 100, 0 to 20, or 0 to 10.
  • the branched linking moiety comprises one or more of: an amino acid residue (e.g., Asp, Lys, Orn, Glu), N-substituted amido (-N(-)C(O)-), tertiary amino, polyol (e.g., O-substituted glycerol), and the like.
  • an amino acid residue e.g., Asp, Lys, Orn, Glu
  • N-substituted amido e.g., Asp, Lys, Orn, Glu
  • N-substituted amido e.g., Asp, Lys, Orn, Glu
  • N-substituted amido e.g., Asp, Lys, Orn, Glu
  • N-substituted amido e.g., Asp, Lys, Orn, Glu
  • N-substituted amido e.g., Asp, Lys, Orn, Glu
  • each x and y are each independently 1 to 10, such as 1-6, 1-3, e.g., 1 or 2. In certain embodiments, each x is 1, 2 or 3, e.g., 2. [0525]
  • L 5 is selected from –CH 2 O–; –(CH 2 CH 2 O) t –, wherein: R 13 is selected from H, halogen, OH, optionally substituted (C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkoxy, COOH, NO 2 , CN, NH 2 , -N(R 21 ) 2 , -OCOR 21 , -COOR 21 , -CONHR 21 , and - NHCOR 21 ; and each r independently 0 to 20, and any of the L 5 moieties are optionally further substituted.
  • L 5 is –CH 2 O–. In certain embodiments, L 5 is –(CH 2 CH 2 O) t –, where t is 1 to 20, such as 1-15, 1-12, 1-10, 1-8, 1-6, or 1 to 4. In certain embodiments, L 5 is –NR 4 CO–, where R 4 is H, or optionally substituted (C 1 -C 6 )alkyl. In certain embodiments, L 5 is -C 1-6 -alkylene–. [0527] In certain embodiments, L 5 is , where r is 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5.
  • each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5 and R 13 is H, or optionally substituted (C 1 -C 6 )alkyl.
  • 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5 and R 13 is H, or optionally substituted (C 1 -C 6 )alkyl.
  • 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5, and R 13 is H, or optionally substituted (C 1 -C 6 )alkyl.
  • each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5, and R 13 is H, or optionally substituted (C 1 -C 6 )alkyl.
  • each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5.
  • L 5 is , where each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5.
  • L 5 is , where each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5.
  • L 5 is , where each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5. [0536] In certain embodiments, L 5 is , where r is 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5.
  • L 5 comprises one or more of: an amino acid residue (e.g., Asp, Lys, Orn, Glu), an amino acid analogue, N-substituted amido (-N(-)C(O)-), tertiary amino, polyol (e.g., O-substituted glycerol), and the like.
  • an amino acid residue e.g., Asp, Lys, Orn, Glu
  • an amino acid analogue e.g., N-substituted amido (-N(-)C(O)-), tertiary amino, polyol (e.g., O-substituted glycerol), and the like.
  • Analogs of an amino acid include but not limited to, unnatural amino acids, as well as other modifications known in the art.
  • the amino acid includes L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art
  • L 1 -L 5 comprises one or more of the following units: R a , where R a is (C 1 -C 6 )alkyl or substituted (C 1 -C 6 )alkyl, e.g., a (C 1 -C 6 )alkyl optionally substituted with amine, a tertiary amine, optionally substituted alkoxy, optionally substituted carboxyl, optionally substituted aryl, or optionally substituted heteroaryl. It is understood that R a can be linked to a M6PR binding moiety.
  • the linker includes a polypeptide scaffold where some or all of the sidechain groups of the amino acid residues have been modified to attach a ASGPR binding moiety (e.g., as described herein). It is understood that ASGPR binding moieties (e.g., as described herein) can be conjugated to amino acid residues, such as Asp, Lys, Orn, Glu, and Ser, of a polypeptide containing linker via a convenient conjugation chemistry.
  • the linker contains a polylysine polypeptide.
  • the linker contains a polyornithine polypeptide.
  • the linker contains a polyserine polypeptide.
  • the linker contains a polyaspartate polypeptide.
  • the polypeptide can be a randomly polymerized polymer having an average length, or a polymer of defined length prepared e.g., in a controlled stepwise fashion.
  • the polypeptide linker segment has a length of 10-100 amino acid residues, such as 20-90, or 20-50 amino acid residues.
  • the N-terminal or C-terminal of the polypeptide linker segment is modified to include a linking unit to an additional M6PR binding moiety (e.g., as described herein).
  • the N-terminal or C-terminal of the polypeptide linker segment is modified with one or more linking units (e.g., as described herein) suitable for attachment to a Y moiety of interest.
  • a is 1.
  • at least one of b, c, d, and e is not 0.
  • b is 1 or 2.
  • c is 1 or 2.
  • e is 1 or 2.
  • b, d and e are independently 1 or 2.
  • a, b, d, and e are each 1, and c is 0.
  • the linker comprises 20 to 100 consecutive atoms, such as 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40 or 20 to 30 consecutive atoms.
  • the linker comprises 25 to 100 consecutive atoms, such as 30 to 100, 35 to 100, 40 to 100, 45 to 100, 50 to 100, 55 to 100, 60 to 100, 65 to 100, 70 to 100, 75 to 100, 80 to 100, 85 to 100, 90 to 100, or 95 to 100 consecutive atoms.
  • the linker comprises 25 or more consecutive atoms, such as 26 or more, 27 or more, 28 or more, 29 or more or 30 or more consecutive atoms.
  • the linker comprises 30 or more consecutive atoms, such as 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37, or more, 38 or more, 39 or more, 40 or even more consecutive atoms.
  • ASGPR binding compounds of this disclosure having a particular configuration with a linker of desired valency and length can specifically bind with high affinity to both the ASGPR and a target simultaneously, and exhibit high uptake activity of a target.
  • the conjugates of this disclosure can thus provide for sequestering of a target protein in the cell’s lysosome and degrading of the target protein.
  • conjugates of trivalent ASGPR binding compounds with 14 or more atoms between the ASGPR binding moiety (e.g., Z 1 group) and the branching point of the linker can exhibit superior uptake of cells as compared to conjugates of trivalent ASGPR binding compounds with shorter linkers (e.g., linkers less than 14 atoms) between the ASGPR binding compound (e.g., Z 1 group) and the branching point.
  • linkers e.g., linkers less than 14 atoms
  • a conjugate having a 1-triazole moiety and a short linkage (e.g., 6 atoms) from the ASGPR ligand to the branching point of the ligand (I-157, linker length of 6 atoms to branching point) exhibited less uptake activity in HepG2 cells than the conjugate having a 1-triazole moiety and a longer linkage (e.g., 14 atoms) from the ASGPR ligand to the branching point (I-143, length of 14 atoms) (see, e.g., FIG.2A).
  • multivalent ASGPR binding compounds having a certain linker length range between the ASGPR binding moiety and the linker branching point which provides desirable binding and cellular uptake of a bound target.
  • conjugates of multivalent ASGPR binding compounds with 12 or more atoms between the branching point of the linker and the Y moiety of interest can exhibit superior uptake of cells as compared to conjugates of multivalent ASGPR binding compounds with shorter linkers (e.g., linkers less than 12 atoms) between the branching point of the linker and the Y moiety of interest.
  • conjugates of ASGPR binding compounds having more than 12 atoms between the branching point of the linker and Y exhibit comparable uptake activity.
  • conjugates having longer linkers between the ASGPR linker and Y e.g., conjugates of compounds I- 137, having 81 atoms between the branching point and Y; and I-129, having 33 atoms between the branching point and Y
  • a reference conjugate e.g., conjugate of compound I-124, having 12 atoms between the branching point and Y
  • each branch of the linker comprises a linear linker of 14 or more consecutive atoms to covalently link via Z 1 each X moiety to a branching point of the linker.
  • each branch of the linker comprises a linear linker of 15 or more consecutive atoms to the branching point.
  • each branch of the linker comprises a linear linker of 16 or more consecutive atoms to the branching point.
  • each branch of the linker comprises a linear linker of 17 or more consecutive atoms to the branching point.
  • each branch of the linker comprises a linear linker of 18 or more consecutive atoms to the branching point. In certain embodiments, each branch of the linker comprises a linear linker of 19 or more consecutive atoms to the branching point.
  • the linker is a branched linker comprising branches covalently linking via Z 1 each X moiety to a branching point of the linker, and a linear linker covalently linking the branching point to Y. In certain embodiments, the linear linker covalently linking the branching point to Y is 12 or more consecutive atoms.
  • the linear linker covalently linking the branching point to Y is 15 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 20 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 25 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 30 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 40 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 50 or more consecutive atoms.
  • the linear linker covalently linking the branching point to Y is 60 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 70 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 80 or more consecutive atoms.
  • Exemplary linkers and linking moieties [0548] Exemplary linkers and linking moieties that can be utilized in the preparation of compounds of this disclosure (e.g., that link the ASGPR ligand (X) to the moiety of interest (Y)) are shown in Tables 9-11. [0549] In certain embodiments, the linker is a linear linker or linking moiety as shown in Table 9.
  • Table 10 includes various linker component synthetic precursors (e.g., linear and branched linker precursors) that can be utilized in the preparation of the subject compounds.
  • linker component synthetic precursors e.g., linear and branched linker precursors
  • the linker is a branched linker or linking moiety as shown in Table 11.
  • Y is a chemoselective ligation group, or a precursor thereof.
  • a chemoselective ligation group is a group having a reactive functionality or function group capable of conjugation to a compatible group of a second moiety.
  • chemoselective ligation groups may be one of a pair of groups associated with a conjugation chemistry such as azido-alkyne click chemistry, copper free click chemistry, Staudinger ligation, tetrazine ligation, hydrazine-iso-Pictet-Spengler (HIPS) ligation, cysteine-reactive ligation chemistry (e.g., thiol-maleimide, thiol-haloacetamide or alkyne hydrothiolation), amine-active ester coupling, tyrosine specific conjugation chemistry (e.g., e-Y-CLICK), methionine specific conjugation chemistry (e.g., oxaziridine-based or ReACT chemistry), reductive amination, dialkyl squarate chemistry, etc.
  • a conjugation chemistry such as azido-alkyne click chemistry, copper free click chemistry, Staudinger ligation, t
  • Chemoselective ligation groups that may be utilized in linking two moieties, include, but are not limited to, amino (e.g., a N-terminal amino or a lysine sidechain group of a polypeptide), azido, aryl azide, alkynyl (e.g., ethynyl or cyclooctyne or derivative), active ester (e.g., N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester or PFP ester or thioester), haloacetamide (e.g., iodoacetamide or bromoacetamide), chloroacetyl, bromoacetyl, bromomethyl-aryl, chloromethyl-aryl, bromomethyl- heteroaryl, chloromethyl-heteroaryl, hydrazide, maleimide, vinyl sulfone, 2-sulfonyl pyridine,
  • chemoselective ligation group is capable of spontaneous conjugation to a compatible chemical group when the two groups come into contact under suitable conditions (e.g., copper free Click chemistry conditions). In some instances, the chemoselective ligation group is capable of conjugation to a compatible chemical group when the two groups come into contact in the presence of a catalyst or other reagent (e.g., copper catalyzed Click chemistry conditions).
  • the chemoselective ligation group is a photoactive ligation group.
  • a diazirine group can form reactive carbenes, which can insert into C-H, N-H, and O-H bonds of a second moiety.
  • Y is a precursor of the reactive functionality or function group capable of conjugation to a compatible group of a second moiety.
  • a carboxylic acid is a precursor of an active ester chemoselective ligation group.
  • Y is a reactive moiety capable forming a covalent bond to a polypeptide (e.g., with an amino acid sidechain of a polypeptide having a compatible reactive group).
  • Y is a thio-reactive chemoselective ligation group (e.g., as described in Table 12). In certain embodiments, Y can produce a residual moiety Z resulting from the covalent linkage of a thiol-reactive chemoselective ligation group to one or more cysteine residue(s) of a protein, e.g., Ab.
  • Y is a Cys-reactive chemoselective ligation group (e.g., a maleimide derivative as described in table 12).
  • the Cys-reactive chemoselective ligation group includes a maleimide group.
  • the chemoselective ligation group includes a maleimide group of Table 12, e.g., mal-1 to mal-7.
  • Y is an amino-reactive chemoselective ligation group (e.g., as described in Table 12).
  • Y can produce a residual moiety Z resulting from the covalent linkage of an amine-reactive chemoselective ligation group to one or more lysine residue(s) a protein, e.g., Ab.
  • Y is a Lys-reactive chemoselective ligation group (e.g., an active ester as described in table 12).
  • the Lys-reactive chemoselective ligation group is a PFP ester.
  • Exemplary chemoselective ligation groups, and synthetic precursors thereof, which may be adapted for use in the compounds of this disclosure are shown in Table 12.
  • Table 12 the can represent a point of attachment of Y to a linking moiety or a linked X moiety.
  • Table 12a shows exemplary residual moieties, wherein the “***” indicates the point of attachment of Y.
  • This disclosure includes compounds of formula (I) which can include: (1) one or more particular ASGPR ligand (X) (e.g., as described herein, such as ligands X1-X20 of Tables 1-4) or a particular ASGPR ligand (X) (e.g., as described herein), (2) a linker including one or more linking moieties (e.g., as described herein, such as any one or more of the linking moieties of Tables 8 to 10); and (3) a chemoselective ligation group (Y) e.g., as described herein, such as any one of the groups of Table 12).
  • ASGPR ligand e.g., as described herein, such as ligands X1-X20 of Tables 1-4
  • a linker including one or more linking moieties e.g., as described herein, such as any one or more of the linking moieties of Tables 8 to 10
  • Y chemoselective ligation
  • the chemoselective ligation group can be tailored to provide linkages which confer additional benefits, such as, but not limited to, stability of the conjugate.
  • the chemoselective ligation group comprises:
  • Table 13 illustrates various monovalent ligand-linker compounds for use in conjugates of the disclosure.
  • Tables 14 illustrates various multivalent ligand-linker compounds for use in conjugates of the disclosure.
  • the compound of formula (I) is an ASGPR binding compound as described in International Application No. WO/2023288033, filed July 14, 2022, and the disclosure of which is herein incorporated by reference in its entirety.
  • the following Tables illustrate several exemplary ASGPR binding compounds of this disclosure that include a chemoselective ligation group, or a precursor thereof. It is understood that this disclosure includes Y (e.g., as described herein) conjugates of each of the exemplary compounds of Tables 13-23. For example, conjugates where the chemoselective ligation group has been conjugated to a different Y, such as a biomolecule or a small molecule ligand for a target protein.
  • the chemoselective ligation group of such compounds can be utilized to connect to another Y moiety of interest (e.g., as described below). It is understood that any of these compounds can also be prepared de novo to include an alternative Y moiety of interest (e.g., as described below) rather than the chemoselective ligation group.
  • such compounds are referred to as a conjugate, e.g., a biomolecule conjugate that specifically binds a target protein.
  • the present disclosure is meant to encompass stereoisomers of any one of the compounds described herein.
  • the compound includes an enantiomer of the D- N- acetylgalactosamine (GalNAc), or an analog or derivative of GalNAc.
  • GalNAc D- N- acetylgalactosamine
  • Table 19 illustrates exemplary ASGPR binding compounds of this disclosure that include a binding moiety, or a precursor thereof.
  • Table 20 illustrates exemplary trivalent ASGPR binding intermediate compounds of this disclosure including X groups of formula (Ie).
  • Table 21 illustrates exemplary monovalent ASGPR binding intermediate compounds of this disclosure that include a promoiety and X groups that are of formula (Ib).
  • Table 22 illustrates exemplary ASGPR binding intermediate compounds of this disclosure that include X groups that are of formula (In).
  • Table 23 illustrates exemplary ASGPR binding intermediate compounds.
  • the present disclosure is meant to encompass stereoisomers of any one of the compounds described herein.
  • the compound includes an enantiomer of the D-N- acetylgalactosamine (GalNAc), or an analog or derivative of GalNAc.
  • Conjugates with Moiety of Interest [0580]
  • the compounds of this disclosure can be referred to as a conjugate, e.g., when the moiety of interest (Y) is a molecule (e.g., as described herein).
  • conjugates can be prepared by conjugation of a chemoselective ligation group of any one of the compounds described herein with a compatible reactive group of a molecule Y.
  • the compatible group of the molecule Y can be introduced by modification prior to conjugation, or can be a group present in the molecule.
  • conjugates can be prepared de novo, e.g., via modification of a Y molecule of interest starting material to introduce a linker, e.g., to which a ligand X can be attached.
  • the moiety of interest to which the ASGPR binding moiety is linked is a biomolecule.
  • the moiety of interest is a biomolecule.
  • the biomolecule is selected from peptide, protein, polynucleotide, polysaccharide, glycan, glycoprotein, lipid, enzyme, antibody, and antibody fragment.
  • the moiety of interest Y is selected from small molecule, small molecule drug, chemotherapeutic agent, cytotoxic agent, diagnostic agent, dye, fluorophore, and the like.
  • the moiety of interest is a molecule that specifically binds to a target of interest, i.e., a target-binding moiety.
  • the conjugates of this disclosure can provide for cellular uptake of the target after it non-covalently binds to the conjugate, and/or degradation.
  • conjugates of this disclosure having a particular configuration of ASGPR binding moiety of a desired affinity, with a linker of desired valency and length can specifically bind with high affinity to both the ASGPR and the target simultaneously.
  • the conjugates of this disclosure can thus provide for sequestering of a target protein in the cell’s lysosome and degrading of the target protein.
  • the moiety of interest is a molecule that does not bind to an extracellular target, but rather is a molecule that is itself desirable to deliver intracellularly.
  • the moiety of interest is selected from enzymes (e.g., lysosomal enzyme), a nanoparticle, a viral composition (e.g., viral particle), therapeutic protein, therapeutic antibodies and cytotoxic agents.
  • enzymes e.g., lysosomal enzyme
  • a nanoparticle e.g., a viral composition
  • therapeutic protein e.g., therapeutic antibodies and cytotoxic agents.
  • the moiety of interest is a lysosomal enzyme for delivery to a cell for use in enzyme replacement therapy, such as acid alpha-glucosidase (GAA).
  • GAA acid alpha-glucosidase
  • Lysosomal enzymes of interest that may be adapted for use in conjugates of this disclosure include, but are not limited to, acid alpha-glucosidase, acid beta-galactosidase-1, acid sphingomyelinase, alpha-D-mannosidase, alpha- fucosidase, alpha-galactosidase A, alpha-glucosaminide acetyltransferase, alpha-glucosidase, alpha-L- iduronidase, alpha-N-acetylgalactosaminidase, alpha-acetylglucosaminidase, alpha-D-neuraminidase, arylsulfatase A, arylsulfatase B, beta-galactosidase, beta-glucuronidase, beta-mannosidase, cathepsin D, cathepsin K, ceramidase, cysti
  • aspects of this disclosure include compounds of formula (I) where the moiety of interest Y is a selected from small molecule, dye, fluorophore, monosaccharide, disaccharide, trisaccharide, and biomolecule.
  • Y is a small molecule that specifically binds to a target molecule, such as a target protein.
  • Y is a biomolecule.
  • the biomolecule is selected from protein, polynucleotide, polysaccharide, peptide, glycoprotein, lipid, enzyme, antibody, and antibody fragment.
  • Y is a biomolecule that specifically binds to a target molecule, such as a target protein.
  • the compounds of this disclosure can, in certain embodiments, be referred to as a conjugate, e.g., when the moiety of interest (Y) is a molecule such as a biomolecule, where the conjugate can be derived from a conjugation or coupling reaction between a chemoselective ligation group and a compatible group on the biomolecule.
  • the biomolecule is conjugated via a naturally occurring group of the biomolecule.
  • the biomolecule is conjugated via a compatible functional group that is introduced into the biomolecule prior to chemoselective conjugation.
  • the linking moiety between X and Y incorporates the residual group (e.g., Z) that is the product of the chemoselective ligation chemistry.
  • Z residual group
  • aspects of this disclosure include compounds of formula (I) where the moiety of interest Y is a moiety that specifically binds to a target molecule, such as a target protein.
  • the target protein can be the target protein is a membrane bound protein or an extracellular protein.
  • Y is a biomolecule that specifically binds to a target protein.
  • the conjugate includes a moiety of interest Y that specifically binds a target protein, and can find use in methods of cell uptake or internalization of the target protein via binding to the cell surface receptor, and eventual degradation of the target protein.
  • Y is an aptamer that specifically binds to a target molecule, such as a target protein.
  • Y is a peptide or protein (e.g., peptidic binding motif, protein domain, engineered polypeptide, or glycoprotein) that specifically binds to a target molecule, such as a target protein.
  • Y is an antibody or antibody fragment that specifically binds to a target molecule, such as a target protein.
  • Y is a polynucleotide or oligonucleotide that specifically binds to a target molecule, such as a target protein or a target nucleic acid.
  • one Y biomolecule is conjugated to a single moiety (X) that specifically binds to the cell surface receptor (e.g., ASGPR) via a linker L.
  • Y can be conjugated to two or more (Xn-L)- groups, wherein each (Xn-L)- group may itself be monovalent or multivalent (e.g., bivalent, trivalent, etc.).
  • the ratio of linked (Xn-L)- groups to biomolecule can be referred to as 2 or more.
  • Y is a moiety that specifically binds the target protein and the compound is a conjugate of formula (III’): wherein: n is 1 to 20; m is an average loading of 1 to 80; each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group to a compatible group of Y; and Y is a moiety of interest binds the target protein.
  • Y is an antibody or an antibody fragment.
  • Y is an antibody or antibody fragment that specifically binds the target protein and the compound is a conjugate of formula (III): wherein: n is 1 to 20; m is an average loading of 1 to 80; each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group to a compatible group of Ab; and Ab is the antibody or antibody fragment that specifically binds the target protein.
  • n is 1 to 6.
  • n is 1, such that the antibody is conjugated to a monovalent ligand and the linker is of the formula (IIa) (e.g., as described herein). In certain cases, n is at least 2, such that the antibody is conjugated to a multivalent ligand. In certain cases, n is 2. In certain cases n is 3. [0595] In certain embodiments of the conjugate of formula (III), Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation moiety of Table 12 (e.g., Table 12a). [0596] In certain embodiments of formula (III) or (III’), each X is independently of formula (Ib) (e.g., as described herein). In certain embodiments, each X is independently selected from a compound of Table 1. In certain embodiments, each X is independently selected from one of the following compounds:
  • each X is independently selected from one of the following compounds: wherein R 5 and R 4 independently H or a promoiety, or R 5 and R 4 are cyclically linked to form a promoiety; n1 and n2 are each independently an integer from 1 to 6; and Y 4 is a suitable counterion. In some embodiments, Y 4 is sodium.
  • n is 1 and X is: .
  • each X is independently of the formula (Ic) (e.g., as described herein).
  • each X is independently selected from a compound of Table 2 or 2a.
  • each X is independently of formula (Id) (e.g., as described herein).
  • each X is independently selected from a compound of Table 3 or 3a.
  • each X is independently selected from a compound of Table 4.
  • each X is a compound of Table 5.
  • each X is independently selected from one of the following compounds: [0602]
  • L is a linker of formula (II) (e.g., as described herein).
  • n is 1 to 6. In certain embodiments, n is 1, such that the antibody is conjugated to a monovalent ASGPR ligand and the linker is of the formula (IIa) (e.g., as described herein). In certain embodiments, n is at least 2, such that the antibody is conjugated to a multivalent ASGPR ligand. In certain embodiments, n is 2. In certain cases n is 3. [0604] In certain embodiments of the conjugate of formula (III) or (III’), Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation moiety of Table 9.
  • Z is a residual moiety resulting from the covalent linkage of a thiol reactive chemoselective ligation group to one or more cysteine residue(s) of Ab.
  • the thiol-reactive chemoselective ligation group is a maleimide derivative.
  • Z is a residual moiety resulting from the covalent linkage of an amine-reactive chemoselective ligation group to one or more lysine residue(s) of Ab.
  • the amine-reactive chemoselective ligation group is an active ester.
  • the active ester is a PFP ester.
  • the conjugates with their linker structures described herein have weaker binding affinity to cell surface receptors. Without being bound to any particular mechanism or theory, such weaker binding affinity may be corrected to longer half-life of the conjugates, and may be useful for tuning (e.g., modifying) the pharmacokinetic properties of the conjugates described herein. In certain embodiments, such weaker binding conjugates still have sufficiently robust uptake.
  • Conjugates of a polypeptide may be made using a variety of bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC- SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo- KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4- vinylsulfone)benzoate).
  • bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC- SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo- KMUS,
  • the conjugates described herein may be prepared using any suitable methods as disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2d ed.2008)).
  • L is bonded through an amide bond to a lysine residue of the polypeptide.
  • L is bonded through a thioether bond to a cysteine residue of the polypeptide.
  • L is bonded through an amide bond to a lysine residue of Ab.
  • L is bonded through a thioether bond to a cysteine residue of Ab. In certain embodiments of the conjugates described herein, L is bonded through two thioether bonds to two cysteine residues of Ab, wherein the two cysteine residues are from an opened cysteine-cysteine disulfide bond in Ab. In certain embodiments, the opened cysteine-cysteine disulfide bond is an interchain disulfide bond. [0610] In certain embodiments of the conjugates described herein, when L is bonded through an amide bond to a lysine residue of a polypeptide (e.g., an antibody), m is an integer from 1 to 80.
  • m is an integer from 1 to 80.
  • conjugation to the polypeptide, or the antibody Ab may be via site- specific conjugation. Site-specific conjugation may, for example, result in homogeneous loading and minimization of conjugate subpopulations with potentially altered antigen-binding or pharmacokinetics.
  • conjugation may comprise engineering of cysteine substitutions at positions on the polypeptide or antibody, e.g., on the heavy and/or light chains of an antibody that provide reactive thiol groups and do not disrupt polypeptide or antibody folding and assembly or alter polypeptide or antigen binding (see, e.g., Junutula et al., J. Immunol. Meth.2008; 332: 41-52; and Junutula et al., Nature Biotechnol.2008; 26: 925-32; see also WO2006/034488 (herein incorporated by reference in its entirety)).
  • selenocysteine is cotranslationally inserted into a polypeptide or antibody sequence by recoding the stop codon UGA from termination to selenocysteine insertion, allowing site specific covalent conjugation at the nucleophilic selenol group of selenocysteine in the presence of the other natural amino acids (see, e.g., Hofer et al., Proc. Natl. Acad. Sci. USA 2008; 105: 12451-56; and Hofer et al., Biochemistry 2009; 48(50): 12047-57).
  • Non-limiting techniques that allow for site-specific conjugation to polypeptides or antibodies include engineering of non-natural amino acids, including, e.g., p-acetylphenylalanine (p-acetyl-Phe), p- azidomethyl-N-phenylalanine (p-azidomethyl-Phe), and azidolysine (azido-Lys) at specific linkage sites, and can further include engineering unique functional tags, including, e.g., LPXTG, LLQGA, sialic acid, and GlcNac, for enzyme mediated conjugation. See Jackson, Org. Process Res.
  • the term “DAR” refers to the average value of “m” or the loading of the conjugate.
  • the number of “X” moieties (e.g., folate moieties) per each unit of “Xn-L-” or “Xn-” is represented by “n” in formula (III).
  • the term “valency” or “valencies” refers to the number of “X” moieties per unit (“n”). It will be understood that loading, or DAR, is not necessarily equivalent to the number of “X” moieties per conjugate molecule.
  • total valency refers to the total number of “X” moieties per conjugate molecule (n x m; total valency).
  • DAR loading
  • the conjugates provided herein may include collections of polypeptides, antibodies or antigen binding fragments conjugated with a range of units, e.g., from 1 to 80.
  • the average number of units per polypeptide or antibody in preparations of the conjugate from conjugation reactions may be characterized by conventional means such as mass spectroscopy.
  • the quantitative distribution of DAR (loading) in terms of m may also be determined.
  • the DAR for a conjugate provided herein ranges from 1 to 80. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 70. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 60. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 50. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 40. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 35.
  • the DAR for a conjugate provided herein ranges from 1 to 30. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 25. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 20. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 18. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 15. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 12. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 10. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 9.
  • the DAR for a conjugate provided herein ranges from 1 to 8. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 7. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 6. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 5. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 4. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 3. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 12. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 10.
  • the DAR for a conjugate provided herein ranges from 2 to 9. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 8. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 7. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 6. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 5. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 4. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 12. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 10.
  • the DAR for a conjugate provided herein ranges from 3 to 9. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 8. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 7. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 6. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 5. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 4.
  • the DAR for a conjugate provided herein ranges from 1 to about 8; from about 2 to about 6; from about 3 to about 5; from about 3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about 3.7.
  • the DAR for a conjugate provided herein is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or more.
  • the DAR for a conjugate provided herein is about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. [0617] In some embodiments, the DAR for a conjugate provided herein ranges from 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, or 2 to 13. In some embodiments, the DAR for a conjugate provided herein ranges from 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, or 3 to 13. In some embodiments, the DAR for a conjugate provided herein is about 1. In some embodiments, the DAR for a conjugate provided herein is about 2.
  • the DAR for a conjugate provided herein is about 3. In some embodiments, the DAR for a conjugate provided herein is about 4. In some embodiments, the DAR for a conjugate provided herein is about 3.8. In some embodiments, the DAR for a conjugate provided herein is about 5. In some embodiments, the DAR for a conjugate provided herein is about 6. In some embodiments, the DAR for a conjugate provided herein is about 7. In some embodiments, the DAR for a conjugate provided herein is about 8. In some embodiments, the DAR for a conjugate provided herein is about 9. In some embodiments, the DAR for a conjugate provided herein is about 10. In some embodiments, the DAR for a conjugate provided herein is about 11.
  • the DAR for a conjugate provided herein is about 12. In some embodiments, the DAR for a conjugate provided herein is about 13. In some embodiments, the DAR for a conjugate provided herein is about 14. In some embodiments, the DAR for a conjugate provided herein is about 15. In some embodiments, the DAR for a conjugate provided herein is about 16. In some embodiments, the DAR for a conjugate provided herein is about 17. In some embodiments, the DAR for a conjugate provided herein is about 18. In some embodiments, the DAR for a conjugate provided herein is about 19. In some embodiments, the DAR for a conjugate provided herein is about 20.
  • the DAR for a conjugate provided herein is about 25. In some embodiments, the DAR for a conjugate provided herein is about 30. In some embodiments, the DAR for a conjugate provided herein is about 35. In some embodiments, the DAR for a conjugate provided herein is about 40. In some embodiments, the DAR for a conjugate provided herein is about 50. In some embodiments, the DAR for a conjugate provided herein is about 60. In some embodiments, the DAR for a conjugate provided herein is about 70. In some embodiments, the DAR for a conjugate provided herein is about 80.
  • a polypeptide may contain, for example, lysine residues that do not react with the compound or linker reagent.
  • antibodies do not contain many free and reactive cysteine thiol groups which may be linked to a drug unit; indeed most cysteine thiol residues in antibodies exist as disulfide bridges.
  • an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups.
  • DTT dithiothreitol
  • TCEP tricarbonylethylphosphine
  • an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.
  • the compound is conjugated via a lysine residue on the antibody.
  • the linker unit or a drug unit is conjugated via a cysteine residue on the antibody.
  • the amino acid that attaches to a unit is in the heavy chain of an antibody.
  • the amino acid that attaches to a unit is in the light chain of an antibody.
  • the amino acid that attaches to a unit is in the hinge region of an antibody.
  • the amino acid that attaches to a unit is in the Fc region of an antibody.
  • the amino acid that attaches to a unit is in the constant region (e.g., CH1, CH2, or CH3 of a heavy chain, or CH1 of a light chain) of an antibody.
  • the amino acid that attaches to a unit or a drug unit is in the VH framework regions of an antibody.
  • the amino acid that attaches to unit is in the VL framework regions of an antibody.
  • the DAR (loading) of a conjugate may be controlled in different ways, e.g., by: (i) limiting the molar excess of compound or conjugation reagent relative to polypeptide, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the polypeptide, such that the number and position of cysteine residues is modified for control of the number and/or position of linker- drug attachments (such as for thiomabs prepared as disclosed in WO2006/034488 (herein incorporated by reference in its entirety)).
  • m is 1 to 20, such as 2 to 10, 2 to 8, or 2 to 6. In certain embodiments, m is 10 or less.
  • m is 2 to 8. In certain embodiments, m is 2 to 6. In certain embodiments, m is an average loading of about 4. [0624] It is to be understood that the preparation of the conjugates described herein may result in a mixture of conjugates with a distribution of one or more units attached to a polypeptide, for example, an antibody. Individual conjugate molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography, including such methods known in the art. In certain embodiments, a homogeneous conjugate with a single DAR (loading) value may be isolated from the conjugation mixture by electrophoresis or chromatography.
  • the target-binding moiety can be any moiety that has an affinity for the target of less than 1 ⁇ M, such as 300nM or less, 100nM or less, 30nM or less, 10nM or less, 3nM or less, or 1nM or less, e.g., as measured in an in vitro binding assay.
  • the target-binding moiety is a biomolecule.
  • the target-binding moiety is a biomolecule that specifically binds to a target protein.
  • the biomolecule is selected from peptide, protein, polynucleotide, polysaccharide, glycan, glycoprotein, lipid, enzyme, antibody, and antibody fragment.
  • the target-binding moiety is a polypeptide (e.g., peptide or protein binding motif, protein domain, engineered polypeptide, or glycoprotein) that specifically binds to a target molecule, such as a target protein.
  • the target-binding moiety of the bifunctional compound includes a polypeptide that binds to a soluble (e.g., secreted) target protein of interest.
  • the target-binding is a polypeptide ligand that includes a receptor ligand, or a receptor-binding portion or fragment of the receptor ligand, that binds a target cell surface receptor.
  • Target-binding polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of naturally occurring amino acids, non-naturally occurring amino acids, and/or amino acid modifications or analogs known in the art. Useful modifications include, e.g., N-terminal acetylation, amidation, methylation, etc.
  • the target-binding moiety is a polynucleotide that specifically binds to a target molecule, such as a target protein or a target nucleic acid.
  • a target molecule such as a target protein or a target nucleic acid.
  • the terms polynucleotide and nucleic acid can be used interchangeably.
  • the target-binding moiety is a nucleic acid aptamer that specifically binds to a target molecule, such as a target protein.
  • the target-binding moiety is a glycan.
  • the target-binding moiety is a glycan epitope for an autoantibody.
  • the target-binding moiety is an antibody or antibody fragment that specifically binds to a target moiety, such as a target protein.
  • the ASGPR binding moiety can be site-specifically covalently linked to the antibody or antibody fragment, via an optional linking moiety.
  • ASGPR binding moiety can be covalently linked to the antibody or antibody fragment via a site-specific cysteine modification on the antibody or antibody fragment (e.g., L443C) and a thiol-reactive chemoselective ligation group.
  • the bifunctional conjugate of this disclosure includes an antibody (Ab).
  • Ab is a monoclonal antibody.
  • Ab is a human antibody.
  • Ab is a humanized antibody.
  • Ab is a chimeric antibody.
  • Ab is a full-length antibody that includes two heavy chains and two light chains.
  • Ab is an IgG antibody, e.g., is an IgG1, IgG2, IgG3 or IgG4 antibody.
  • Ab is a single chain antibody.
  • the target- binding moiety is an antigen-binding fragment of an antibody, e.g., a Fab fragment.
  • the antibody or antibody fragment specifically binds to a cancer antigen.
  • the antibody or antibody fragment specifically binds to a hepatocyte antigen.
  • the antibody or antibody fragment specifically binds to an antigen presented on a macrophage.
  • the antibody or antibody fragment specifically binds to an intact complement or a fragment thereof.
  • the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within intact complement or a fragment thereof. [0637] In some embodiments, the antibody or antibody fragment specifically binds to a cell surface receptor. In some embodiments, the antibody or antibody fragment specifically binds to a cell surface receptor ligand. [0638] In some embodiments, the antibody or antibody fragment specifically binds to an epidermal growth factor (EGF) protein, e.g., a human EGF. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within an EGF protein.
  • EGF epidermal growth factor
  • the antibody or antibody fragment specifically binds to an epidermal growth factor receptor (EGFR) protein, e.g., a human EGFR. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within an EGFR protein. In some embodiments, the antibody or antibody fragment comprises the CDRs present in cetuximab. In some embodiments, the antibody or antibody fragment includes the variable light chain and variable heavy chain present in cetuximab. In some embodiments, the antibody is cetuximab. In some embodiments, the antibody or antibody fragment includes the CDRs present in matuzumab.
  • EGFR epidermal growth factor receptor
  • the antibody or antibody fragment includes the variable light chain and variable heavy chain present in matuzumab. In some embodiments, the antibody is matuzumab. [0640] In some embodiments, the antibody or antibody fragment specifically binds to vascular endothelial growth factor (VEGF) protein, e.g., human VEGF protein. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within a VEGF protein. [0641] In some embodiments, the antibody or antibody fragment specifically binds to a vascular endothelial growth factor receptor (VEGFR) protein, e.g., human VEGFR protein.
  • VEGF vascular endothelial growth factor
  • VEGFR vascular endothelial growth factor receptor
  • the antibody or antibody fragment specifically binds vascular endothelial growth factor receptor 2 (VEGFR2) protein, e.g., a human VEGFR2 protein.
  • the antibody or antibody fragment specifically binds a vascular endothelial growth factor receptor 3 (VEGFR3) protein, e.g., a human VEGFR3 protein.
  • the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within a VEGFR protein, a VEGFR2 protein or a VEGFR3 protein.
  • the antibody or antibody fragment specifically binds to a fibroblast growth factor (FGF), e.g., a human FGF.
  • FGF fibroblast growth factor
  • the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within a FGF protein.
  • the antibody or antibody fragment specifically binds to a fibroblast growth factor receptor (FGFR), e.g., a human FGFR.
  • FGFR2 fibroblast growth factor receptor 2
  • FGFR3 fibroblast growth factor receptor 3
  • the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within a FGFR protein, a FGFR2 protein or a FGFR3 protein.
  • the antibody specifically binds to a receptor tyrosine kinase cMET protein.
  • the antibody specifically binds to one or more immunodominant epitope(s) within a receptor tyrosine kinase cMET protein.
  • the antibody specifically binds to a CD47 protein, e.g., a human CD47 protein.
  • the antibody specifically binds to one or more immunodominant epitope(s) within a CD47 protein. [0646] In some embodiments, the antibody specifically binds to an immune checkpoint inhibitor. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within an immune checkpoint inhibitor. In some embodiments, the antibody specifically binds to a programmed death protein, e.g., a human PD-1. In some embodiments, the antibody specifically binds to one or more immunodominant epitope(s) within PD-1 protein.
  • the antibody specifically binds to a programmed death ligand-1 (PD- L1) protein, e.g., a human PD-L1. In some embodiments, the antibody specifically binds to one or more immunodominant epitope(s) within PD-L1 protein. [0648] In some embodiments, the antibody binds to TIM3. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within TIM3. [0649] In some embodiments, the antibody specifically binds to a lectin. In some embodiments, the antibody specifically binds to one or more immunodominant epitope(s) within a lectin.
  • PD- L1 programmed death ligand-1
  • the antibody binds to SIGLEC. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within SIGLEC. In some embodiments, the antibody binds to a cytokine receptor. In some embodiments, the antibody binds to a one or more immunodominant epitope(s) within cytokine receptor. In some embodiments, the antibody binds to sIL6R. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within sIL6R. In some embodiments, the antibody binds to a cytokine.
  • the antibody binds to one or more immunodominant epitope(s) within a cytokine. In some embodiments, the antibody binds to MCP-1, TNF (e.g., a TNF- alpha), IL1a, IL1b, IL4, IL5, IL6, IL12/IL23, IL13, IL17 or p40. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within MCP-1, TNF (e.g., a TNF-alpha), IL1a, IL1b, IL4, IL5, IL6, IL12/IL23, IL13, IL17 or p40.
  • TNF e.g., a TNF-alpha
  • the antibody binds to a major histocompatibility protein (e.g., a MHC class I or class II molecule). In some embodiments, the antibody binds to one or more immunodominant epitope(s) within a major histocompatibility protein (e.g., a MHC class I or class II molecule). In some embodiments, the antibody binds to beta 2 microglobulin. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within beta 2 microglobulin.
  • Y is a viral particle, viral capsid, a viral envelope or a viral protein.
  • the viral composition is a viral particle that comprises a transgene.
  • the viral protein is a viral capsid protein or a viral envelope protein.
  • modified viral compositions comprising a viral composition, for example, a virus particle, a virus capsid or a viral protein (e.g., a viral capsid protein or an envelope protein) attached to (e.g., conjugated to, directly or indirectly, for example via an intervening linker sequence) an ASGPR binding moiety that binds to a cell surface receptor.
  • a modified viral composition comprises a virus particle that comprises a polynucleotide that optionally comprises a transgene, e.g., a transgene useful for therapeutic applications.
  • the modified viral compositions, e.g., viral conjugates, presented herein may comprise any viral composition described herein e.g., any virus particle, capsid or viral protein, for example capsid protein or envelope protein, or fragment thereof, as described herein.
  • a viral composition described herein may comprise a virus particle.
  • the terms “virus particle,” “viral particle,” “virus vector” or “viral vector” are used interchangeably herein.
  • a “virus particle” refers to a virus capsid and a polynucleotide (DNA or RNA), which may comprise a viral genome, a portion of a viral genome, or a polynucleotide derived from a viral genome (e.g., one or more ITRs), which polynucleotide optionally comprises a transgene.
  • a virus particle further comprises an envelope (which generally comprises lipid moieties and envelope proteins), surrounding or partially surrounding the capsid.
  • a viral particle may be referred to as a “recombinant viral particle,” or “recombinant virus particle,” which terms as used herein refer to a virus particle that has been genetically altered, e.g., by the deletion or other mutation of an endogenous viral gene and/or the addition or insertion of a heterologous nucleic acid construct into the polynucleotide of the virus particle.
  • a recombinant virus particle generally refers to a virus particle comprising a capsid coat or shell (and an optional outer envelope) within which is packaged a polynucleotide sequence that comprises sequences of viral origin and sequences not of viral origin (i.e., a polynucleotide heterologous to the virus).
  • a viral composition described herein may comprise an “viral capsid,” “empty viral particle,” “empty virus particle,” or “capsid,” or “empty particle” when referred to herein in the context of the virus, which terms as used herein refer to a three-dimensional shell or coat comprising a viral capsid protein, optionally surrounded or partially surrounded by an outer envelope.
  • the viral composition is a virus particle or a fragment thereof, virus capsid or fragment thereof, a viral protein, for example, a virus capsid protein or fragment thereof or envelope protein, or fragment thereof.
  • the virus used in a modified viral composition provided herein is adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), rhabdoviruses, murine leukemia virus); herpes simplex virus, coronavirus, reovirus, and the like.
  • the viral vector, viral particle or viral protein used in the present disclosure is derived from a non- enveloped virus, e.g., an adeno-associated virus (AAV).
  • lentiviral vectors can be used for CAR-T gene delivery, vaccines, or research tools, e.g., to introduce genes into mature T cells to generate immunity to cancer through the delivery of chimeric antigen receptors (CARs) or cloned T-cell receptors.
  • CARs chimeric antigen receptors
  • Naturally occurring AAV forms a virus particle that comprises a three-dimensional capsid coat or shell (a “capsid”) made up of capsid proteins (VP1, VP2 and VP3) and, contained within the capsid, an AAV viral genome.
  • the modified AAV compositions may comprise any AAV composition described herein, e.g., any AAV particle, capsid or capsid protein, or fragment thereof, as described herein.
  • AAV capsid protein or “AAV cap protein” refers to a protein encoded by an AAV capsid (cap) gene (e.g., VP1, VP2, and VP3) or a variant or fragment thereof.
  • the term includes a capsid protein expressed by or derived from an AAV, e.g., a recombinant AAV, such as a chimeric AAV.
  • the term includes but not limited to a capsid protein derived from any AAV serotype such as AAV1, AAV2, AAV2i8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV rh10, AAV11, AAV12, AAV13, AAV-DJ, AAV3b, AAV LK03, AAV rh74, AAV Anc81, Anc82, Anc83, Anc84, Anc110, Anc113, Anc126, or Anc127, AAV_go.1, AAV hu.37, or AAV rh.8 or a variant thereof.
  • AAV serotype such as AAV1, AAV2, AAV2i8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV rh10, AAV11, AAV12, AAV13, AAV-DJ, AAV3b, AAV LK03, AAV
  • Y is a bridging moiety that specifically binds to a viral composition, for example, a viral particle, viral capsid, viral envelope or viral protein (e.g., a viral capsid protein or envelope protein), wherein the binding is not via a covalent linkage.
  • a viral particle, viral capsid, viral envelope or viral protein e.g., a viral capsid protein or envelope protein
  • Any suitable moiety that binds a viral particle, viral capsid, viral envelope or viral protein e.g., a viral capsid protein or envelope protein
  • a bridging moiety is a polypeptide that specifically binds a viral composition.
  • the bridging moiety is a polypeptide that binds to a viral composition, e.g., a virus particle, virus capsid, virus envelope, or a viral protein, for example, a viral capsid protein or viral envelope protein.
  • the bridging composition binds the viral capsid protein or a viral envelope protein, when the viral protein is part of a virus particle.
  • a bridging moiety is an antibody or antibody fragment (e.g., an antigen binding fragment of an antibody) that specifically binds a viral composition.
  • a bridging moiety that binds a viral protein may also bind a viral particle, for example, via binding to the viral protein incorporated in a viral particle.
  • a bridging moiety that binds a viral particle may also bind a viral protein even if the viral protein is not incorporated in a viral particle.
  • the viral particle can be an AAV virus particle.
  • the viral protein can be a AAV capsid protein.
  • the bridging moieties of this disclosure specifically bind to an AAV composition, e.g., an AAV particle, AAV capsid, or AAV viral protein (e.g., an AAV capsid protein, for example, a VP1, VP2 or VP3 protein).
  • AAV composition e.g., an AAV particle, AAV capsid, or AAV viral protein (e.g., an AAV capsid protein, for example, a VP1, VP2 or VP3 protein).
  • An antibody or antigen binding fragment that may be utilized in connection with the modified viral compositions provided herein, e.g., in connection with the bridging compositions and bridging moieties presented herein, includes, without limitation, monoclonal antibodies, antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain antibodies, and fragments thereof (e.g., domain antibodies).
  • the target-binding moiety of the bifunctional compound of this disclosure is a small molecule that specifically binds to a target molecule, such as a target protein.
  • the bifunctional compound includes a small molecule inhibitor or ligand of a target protein.
  • a small molecule target-binding moiety can be covalently linked to one or more ASGPR binding moieties via a linker.
  • the linker can be covalently attached to the small molecule via substitution at any suitable site of the small molecule such that binding to the target protein is substantially retained.
  • the target-binding moiety is a small molecule inhibitor or antagonist of a target protein (e.g., as described herein).
  • the target-binding moiety is a small molecule inhibitor or antagonist of VEGF.
  • the target-binding moiety is a small molecule inhibitor or antagonist of PD-L1.
  • the target-binding moiety is a small molecule inhibitor or antagonist of EGFR protein, a VEGFR protein, a FGFR2 protein or a FGFR3 protein.
  • the target-binding moiety is a small molecule inhibitor or antagonist of TNF protein (e.g., TNF-alpha).
  • TNF-alpha is a soluble cytokine produced by monocytes and macrophages as part of immune and inflammatory processes and is involved in a diverse range of cellular responses including differentiation, proliferation, inflammation, and cell death.
  • TNF ⁇ is a type II transmembrane protein that can be cleaved and secreted as a soluble form. Both the transmembrane and soluble biologically active forms of TNF ⁇ are homotrimeric complexes that can signal through TNF receptors 1 and 2 (TNF-R1 and TNF-R2).
  • TNF ⁇ is directly involved in systemic inflammation through the regulation of the intracellular NF- ⁇ B, JNK and p38-MAPK signaling pathways.
  • the TNF ⁇ binding moiety can be a TNF ⁇ inhibitor, such as a competitive inhibitor of TNF receptor binding or an allosteric inhibitor of TNF signaling.
  • the compounds of this disclosure can include a potent TNF ⁇ inhibitor, e.g., an inhibitor having sub-micromolar inhibitory activity.
  • the TNF ⁇ inhibitor is an allosteric inhibitor.
  • the TNF ⁇ binding moiety is an allosteric desymmetrization TNF ⁇ inhibitor.
  • An allosteric desymmetrization TNF ⁇ inhibitor refers to a compound that binds to an allosteric site within TNF ⁇ and stabilizes the trimeric unit in a nonsymmetrical conformation that allows the TNF ⁇ trimer to recruit only two out of the three copies of TNF Receptor (TNFR, e.g., TNFR1), leading to an incompetent TNF ⁇ -TNFR signaling complex.
  • TNFR TNF Receptor
  • An allosteric desymmetrization TNF ⁇ inhibitor can act via a particular mechanism of action to provide potent inhibitory activity.
  • the TNF ⁇ inhibitor binding site is a cavity within the TNF ⁇ trimer created via movement of monomer A
  • the inhibitor stabilizes the TNF ⁇ trimer in an inactive conformation by forming key ⁇ and hydrogen bonding interactions
  • an allosteric desymmetrization TNF ⁇ inhibitor binds to TNF ⁇ trimer leading to major disruption of one TNFR binding site and minor disruption of a second site, while the third site remains unchanged
  • the allosteric desymmetrization TNF ⁇ inhibitor modulates TNF-R activity through an allosteric mechanism rather than direct competition with TNFR.
  • the bifunctional compounds of this disclosure can include a moiety of interest (Y) that specifically binds a target molecule.
  • the target molecule can be a cell surface molecule or an extracellular molecule.
  • the target molecule is a cell surface molecule.
  • cell surface molecule is meant a target molecule associated with a cell membrane, e.g., because the molecule has a domain that inserts into or spans a cell membrane, e.g., a cell membrane- tethering domain or a transmembrane domain.
  • the cell surface molecule may be any cell surface molecule which is desired for targeted degradation via the endosomal/lysosomal pathway.
  • the cell surface molecule is a cell surface receptor.
  • Cell surface receptors of interest include, but are not limited to, stem cell receptors, immune cell receptors, growth factor receptors, cytokine receptors, hormone receptors, receptor tyrosine kinases, a receptor in the epidermal growth factor receptor (EGFR) family (e.g., HER2 (human epidermal growth factor receptor 2), etc.), a receptor in the fibroblast growth factor receptor (FGFR) family, a receptor in the vascular endothelial growth factor receptor (VEGFR) family, a receptor in the platelet derived growth factor receptor (PDGFR) family, a receptor in the rearranged during transfection (RET) receptor family, a receptor in the Eph receptor family, a receptor in the discoidin domain receptor (DDR) family, and a mucin protein (e.g., MUC1 ).
  • EGFR epidermal growth factor receptor
  • HER2 human epidermal growth factor receptor 2
  • FGFR fibroblast growth factor receptor
  • VEGFR vascular endot
  • the cell surface molecule is CD71 (transferrin receptor).
  • the cell surface receptor is an immune cell receptor selected from a T cell receptor, a B cell receptor, a natural killer (NK) cell receptor, a macrophage receptor, a monocyte receptor, a neutrophil receptor, a dendritic cell receptor, a mast cell receptor, a basophil receptor, and an eosinophil receptor.
  • the moiety of interest (Y) specifically binds a cell surface molecule which mediates its effect not through a specific molecular interaction (and therefore is not susceptible to blocking), but rather through bulk biophysical or aggregate effects.
  • a non-limiting example of such a cell surface molecule is a mucin.
  • mucins include, but are not limited to, MUC1 , MUC16, MUC2, MUC5AC, MUC4, CD43, CD45, GPIb, and the like.
  • cancer cell is meant a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage- independent growth potential, ability to promote tumor growth and/or development in an immunocompromised non-human animal model, and/or any appropriate indicator of cellular transformation.
  • Cancer cell may be used interchangeably herein with “tumor cell”, “malignant cell” or “cancerous cell”, and encompasses cancer cells of a solid tumor, a semi-solid tumor, a hematological malignancy (e.g., a leukemia cell, a lymphoma cell, a myeloma cell, etc.), a primary tumor, a metastatic tumor, and the like.
  • the cell surface molecule present on the cancer cell is a tumor-associated antigen or a tumor-specific antigen.
  • the moiety of interest (Y) specifically binds a cell surface molecule, the cell surface molecule is present on an immune cell.
  • the cell surface molecule is present on an immune cell selected from a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mast cell, a basophil, and an eosinophil.
  • the cell surface molecule present on the immune cell is an inhibitory immune receptor.
  • an “inhibitory immune receptor” is a receptor present on an immune cell that negatively regulates an immune response.
  • Ig superfamily including but not limited to: CD200R, CD300a (IRp60; mouse MAIR-I), CD300f (IREM-1 ), CEACAM1 (CD66a), FcyRIIb, ILT-2 (LIR-1 ; LILRB1 ;
  • Siglec sialic acid-binding Ig-like lectin
  • C- type lectins including but not limited to: CLEC4A (DCIR), Ly49Q and MICL. Details regarding inhibitory immune receptors may be found, e.g., in Steevels et al. (2011 ) Eur. J. Immunol.
  • the cell surface molecule present on the immune cell is a ligand of an inhibitory immune receptor.
  • the cell surface molecule present on the immune cell is an immune checkpoint molecule.
  • immune checkpoint molecules to which the moiety of interest (Y) may specifically bind include PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT, and a member of the B7 family.
  • the target molecule is an extracellular molecule.
  • extracellular molecule is meant a soluble molecule external to the cell membranes of any cells in the vicinity of the soluble molecule.
  • the extracellular molecule may be any extracellular molecule which is desired for targeted degradation via the endosomal/lysosomal pathway.
  • the extracellular molecule is a soluble target protein.
  • the extracellular molecule is a secreted protein that accumulates in disease (e.g., alpha- synuclein), a cholesterol carrier (e.g., ApoB), an infectious disease toxin (e.g., AB toxins, ESAT-6), an infectious particle (e.g., a whole virus, a whole bacterium, etc.), a clotting factor (e.g., Factor IX), the target of any FDA approved antibody that binds to an extracellular molecule (e.g., TNFalpha), any chemokine or cytokine (e.g., mediators of sepsis or chronic inflammation such at IL-1 ), a proteinaceous hormone (e.g., insulin, ACTH, etc.), a proteinaceous mediator of
  • the target molecule is an extracellular molecule that is an antibody, e.g., an antibody that specifically binds a cell surface molecule or different extracellular molecule.
  • the antibody is an autoantibody.
  • the target is a human immunoglobulin A(IgA).
  • the IgA is a particular antibody that plays a crucial role in the immune function of mucous membranes. In the blood, IgA interacts with an Fc receptor called CD89 expressed on immune effector cells, to initiate inflammatory reactions. Aberrant IgA expression has been implicated in a number of autoimmune and immune-mediated disorders.
  • the target is a human immunoglobulin G (IgG).
  • the Fc regions of IgGs include a conserved N- glycosylation site at asparagine 297 in the constant region of the heavy chain.
  • Various N-glycans can b eattached to this site.
  • the N-glycan IgG composition has been linked to several autoimmune, infectious and metabolic diseases.
  • overexpression of IgG4 has been associated with IG4-related diseases.
  • the target is human immunoglobulin E (IgE).
  • IgE is a type of immunoglobulin that plays an essential role in type I hypersensitivity, which can manifest into various allergic diseases and conditions.
  • the extracellular molecule is a ligand for a cell surface receptor.
  • Cell surface receptor ligands of interest include, but are not limited to, growth factors (e.g., epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and the like), cytokines (e.g., an interleukin, an interferon, a tumor necrosis factor (TNF), a transforming growth factor b (TGF-b), including any particular subtypes of such cytokines), hormones, and the like.
  • the moiety of interest (Y) specifically binds apolipoprotein E4 (ApoE4).
  • Pharmaceutical Compositions comprising one or more conjugates disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions provided herein contain therapeutically effective amounts of one or more of the conjugates provided herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier.
  • Pharmaceutical carriers suitable for administration of the conjugates provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the conjugates described herein can be formulated as the sole pharmaceutically active ingredient in the composition or can be combined with other active ingredients.
  • the conjugate is formulated into one or more suitable pharmaceutical preparations, such as solutions, suspensions, powders, sustained release formulations or elixirs in sterile solutions or suspensions for parenteral administration, or as transdermal patch preparation and dry powder inhalers.
  • a conjugate described herein may be mixed with a suitable pharmaceutical carrier.
  • the concentration of the conjugate in the compositions can, for example, be effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates a condition or disorder described herein or a symptom thereof.
  • the pharmaceutical compositions provided herein are formulated for single dosage administration.
  • compositions described herein are provided for administration to a subject, for example, humans or animals (e.g., mammals) in unit dosage forms, such as sterile parenteral (e.g., intravenous) solutions or suspensions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.
  • compositions are also provided for administration to humans and animals in unit dosage form, including oral or nasal solutions or suspensions and oil-water emulsions containing suitable quantities of a conjugate or pharmaceutically acceptable derivatives thereof.
  • the conjugate is, in certain embodiments, formulated and administered in unit-dosage forms or multiple-dosage forms.
  • Unit-dose forms as used herein refers to physically discrete units suitable for human or animal (e.g., mammal) subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of a conjugate sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged capsules.
  • Unit-dose forms can be administered in fractions or multiples thereof.
  • a multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of capsules or bottles. Hence, in specific aspects, multiple dose form is a multiple of unit-doses which are not segregated in packaging.
  • the conjugates herein are in a liquid pharmaceutical formulation.
  • Liquid pharmaceutically administrable formulations can, for example, be prepared by dissolving, dispersing, or otherwise mixing a conjugate and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, and the like, to thereby form a solution or suspension.
  • a pharmaceutical composition provided herein to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, and pH buffering agents and the like.
  • the injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol.
  • Other routes of administration may include, enteric administration, intracerebral administration, nasal administration, intraarterial administration, intracardiac administration, intraosseous infusion, intrathecal administration, and intraperitoneal administration.
  • Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions.
  • the solutions can be either aqueous or nonaqueous.
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.
  • Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.
  • intravenous or intraarterial infusion of a sterile aqueous solution containing a conjugate described herein is an effective mode of administration.
  • the pharmaceutical formulations are lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They can also be reconstituted and formulated as solids or gels.
  • the lyophilized powder is prepared by dissolving a conjugate provided herein, in a suitable solvent.
  • the lyophilized powder is sterile. Suitable solvents can contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder.
  • Excipients that can be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent.
  • a suitable solvent can also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in certain embodiments, about neutral pH.
  • Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides an example of a formulation.
  • the resulting solution will be apportioned into vials for lyophilization. Lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
  • the conjugates provided herein can be formulated for local administration or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.
  • provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from a cell’s surface. In one aspect, provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the extracellular milieu. For example, in one embodiment, provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the surface of a cell by sequestering the target protein in the cell’s lysosome.
  • provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the extracellular space (the extracellular milieu) of a cell by sequestering the target protein in the cell’s lysosome.
  • provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the extracellular space (the extracellular milieu) of a cell by sequestering the target protein in the cell’s lysosome and degrading the target protein.
  • Removal of a target protein may refer to reduction, or depletion, of the target protein from the cell surface or from the extracellular space, or the extracellular milieu, that is, a reduction, or depletion, of the amount of the target protein on the cell surface or in the extracellular milieu.
  • provided herein are methods of using the conjugates described herein to sequester a polypeptide of interest (a target protein) in a cell’s lysosome.
  • provided herein are methods of using the conjugates described herein to sequester a polypeptide of interest (a target protein) in a cell’s lysosome and to degrade the polypeptide of interest. [0708] In one aspect, provided herein are methods of using the conjugates described herein to degrade a polypeptide of interest (a target protein). [0709] In one aspect, provided herein are methods of depleting a polypeptide of interest (a target protein) described herein by degradation through a cell’s lysosomal pathway.
  • a polypeptide of interest a target protein described herein by administering to a subject in need thereof an effective amount of a conjugate or pharmaceutically acceptable salt described herein, or a pharmaceutical composition described herein.
  • the subject is a mammal (e.g., human).
  • the target protein is a VEGF protein, an EGFR protein, a VEGFR protein, a PD-L1 protein, an FGFR2 protein or an FGFR3 protein.
  • administering refers to the act of injecting or otherwise physically delivering a substance (e.g., a conjugate or pharmaceutical composition provided herein) to a subject or a patient (e.g., human), such as by mucosal, topical, intradermal, parenteral, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
  • administering is by intravenous infusion.
  • a therapeutic e.g., a conjugate or pharmaceutical composition provided herein
  • administration is by intravenous infusion.
  • a therapeutic e.g., a conjugate or pharmaceutical composition provided herein
  • a therapeutic e.g., a conjugate or pharmaceutical composition provided herein
  • a therapeutic e.g., a conjugate or pharmaceutical composition provided herein
  • these terms also encompass an amount necessary for the reduction, slowing, or amelioration of the advancement or progression of a given disease, reduction, slowing, or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy or to serve as a bridge to another therapy.
  • “effective amount” as used herein also refers to the amount of a conjugate described herein to achieve a specified result.
  • “effective amount” or “therapeutically effective amount” mean that amount of a conjugate or pharmaceutical composition provided herein which, when administered to a human suffering from a cancer, is sufficient to effect treatment for the cancer.
  • “Treating” or “treatment” of the cancer includes one or more of: (1) limiting/inhibiting growth of the cancer, e.g. limiting its development; (2) reducing/preventing spread of the cancer, e.g. reducing/preventing metastases; (3) relieving the cancer, e.g.
  • a subject can be a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkey and human), for example a human.
  • a mammal e.g., a human, diagnosed with a disease or disorder provided herein.
  • the subject is a mammal, e.g., a human, at risk of developing a disease or disorder provided herein.
  • the subject is human.
  • therapies can refer to any protocol(s), method(s), compositions, formulations, and/or agent(s) that can be used in the prevention, treatment, management, or amelioration of a disease or disorder or symptom thereof (e.g., a disease or disorder provided herein or one or more symptoms or condition associated therewith).
  • the terms “therapies” and “therapy” refer to drug therapy, adjuvant therapy, radiation, surgery, biological therapy, supportive therapy, and/or other therapies useful in treatment, management, prevention, or amelioration of a disease or disorder or one or more symptoms thereof.
  • the term “therapy” refers to a therapy other than a conjugate described herein or pharmaceutical composition thereof.
  • the disease or disorder is treated by depletion of the target protein by degradation through the lysosomal pathway.
  • the disease or disorder is treated by depletion of certain proteins, for example, soluble proteins, e.g., secreted proteins, cell surface proteins (for example, cell surface receptor proteins, e.g., tyrosine kinase receptors, soluble cytokine receptors, and immune checkpoint receptors, e.g., EGFR, VEGFR, FGFR, and PD-L1), lectins, complements, lipoproteins, transport proteins, MHC class I and class II molecules, cytokines, chemokines, and/or receptors , or fragments or subunits of any of the foregoing.
  • the disease or disorder is a cancer.
  • the cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, endometrial cancer, hepatocellular carcinoma, kidney cancer, melanoma, myeloid neoplasms, non-small cell lung cancer (NSCLC), Ewing’s sarcoma, and Hodgkin’s Lymphoma.
  • the cancer is a solid tumor.
  • the disease or disorder is an inflammatory or autoimmune disease.
  • the disease or disorder is an inflammatory disease.
  • the disease or disorder is an autoimmune disease.
  • the disease or disorder is a viral disease.
  • the viral disease is hepatitis B. Definitions [0726] It is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. [0727] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of embodiments of the present disclosure.
  • Proteins may include moieties other than amino acids (e.g., may be glycoproteins, etc.) and/or may be otherwise processed or modified.
  • a “protein” can be a complete protein chain as produced by a cell (with or without a signal sequence), or can be a protein portion thereof.
  • a protein can sometimes include more than one protein chain, for example non-covalently or covalently attached, e.g., linked by one or more disulfide bonds or associated by other means.
  • a polypeptide can occur as a single chain or as two or more associated chains, e.g., may be present as a multimer, e.g., dimer, a trimer.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.
  • polypeptides containing one or more analogs of an amino acid including but not limited to, unnatural amino acids, as well as other modifications known in the art.
  • Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc.
  • proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.
  • proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
  • antibody and “immunoglobulin” are terms of art and can be used interchangeably herein in their broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope.
  • an isolated antibody e.g., monoclonal antibody described herein, or an antigen-binding fragment thereof, which specifically binds to a protein of interest, for example, EGFR, is conjugated to one or more lysosomal targeting moieties, for example, via a linker.
  • an “antigen” is a moiety or molecule that contains an epitope to which an antibody can specifically bind. As such, an antigen is also is specifically bound by an antibody.
  • the antigen, to which an antibody described herein binds is a protein of interest, for example, EGFR (e.g., human EGFR), or a fragment thereof, or for example, an extracellular domain of EGFR (e.g., human EGFR).
  • EGFR e.g., human EGFR
  • An “epitope” is a term known in the art and refers to a localized region of an antigen to which an antibody can specifically bind.
  • An epitope can be a linear epitope of contiguous amino acids or can comprise amino acids from two or more non-contiguous regions of the antigen.
  • binds refers to antibody binding to an antigen (e.g., epitope) as such binding is understood by one skilled in the art.
  • an antigen e.g., epitope
  • a molecule that specifically binds to an antigen may bind to other polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BiacoreTM, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art.
  • molecules that specifically bind to an antigen bind to the antigen with an affinity (Kd ) that is at least 2 logs, 2.5 logs, 3 logs, 4 logs lower (higher affinity) than the K d when the molecules bind to another antigen.
  • Kd affinity
  • molecules that specifically bind to an antigen do not cross react with other proteins.
  • EGFR is the protein of interest, molecules that specifically bind to an antigen do not cross react with other non-EGFR proteins.
  • An antibody specifically includes, but is not limited to, full length antibodies (e.g., intact immunoglobulins), antibody fragments, monoclonal antibodies, polyclonal antibodies,, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain/antibody heavy chain pair, an antibody with two light chain/heavy chain pairs (e.g., identical pairs), intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, bivalent antibodies (including monospecific or bispecific bivalent antibodies), single chain antibodies, or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’) fragments, F(ab’) 2 fragments, disulfide-linked Fvs (scF
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule.
  • antibodies described herein are IgG antibodies (e.g., human IgG), or a class (e.g., human IgG1, IgG2, IgG3 or IgG4) or subclass thereof.
  • an antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs, wherein the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical.
  • the H and L chains comprise constant regions, for example, human constant regions.
  • the L chain constant region of such antibodies is a kappa or lambda light chain constant region, for example, a human kappa or lambda light chain constant region.
  • the H chain constant region of such antibodies comprise a gamma heavy chain constant region, for example, a human gamma heavy chain constant region.
  • such antibodies comprise IgG constant regions, for example, human IgG constant regions.
  • the term “constant region” or “constant domain” is a well-known antibody term of art (sometimes referred to as “Fc”), and refers to an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor.
  • the terms refer to a portion of an immunoglobulin molecule having a generally more conserved amino acid sequence relative to an immunoglobulin variable domain.
  • the term “heavy chain” when used in reference to an antibody can refer to any distinct types, e.g., alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu ( ⁇ ), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG 1 , IgG 2 , IgG 3 and IgG 4 .
  • the term “light chain” when used in reference to an antibody can refer to any distinct types, e.g., kappa ( ⁇ ) of lambda ( ⁇ ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.
  • the term “monoclonal antibody” is a well-known term of art that refers to an antibody obtained from a population of homogenous or substantially homogeneous antibodies. The term “monoclonal” is not limited to any particular method for making the antibody. Generally, a population of monoclonal antibodies can be generated by cells, a population of cells, or a cell line.
  • a “monoclonal antibody,” as used herein, is an antibody produced by a single cell (e.g., hybridoma or host cell producing a recombinant antibody), wherein the antibody specifically binds to an epitope as determined, e.g., by ELISA or other antigen-binding or competitive binding assay known in the art or in the Examples provided herein.
  • a monoclonal antibody can be a chimeric antibody or a humanized antibody.
  • a monoclonal antibody is a monovalent antibody or multivalent (e.g., bivalent) antibody.
  • a monoclonal antibody is a monospecific or multispecific antibody (e.g., bispecific antibody).
  • variable region refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 100 amino acids in the mature light chain.
  • Variable regions comprise complementarity determining regions (CDRs) flanked by framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the spatial orientation of CDRs and FRs are as follows, in an N-terminal to C-terminal direction: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen and for the specificity of the antibody for an epitope.
  • numbering of amino acid positions of antibodies described herein is according to the EU Index, as in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242.
  • the variable region is a human variable region.
  • the CDRs of an antibody can be determined according to (i) the Kabat numbering system (Kabat et al. (1971) Ann. NY Acad.
  • full length antibody “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, and are not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain the Fc region.
  • Antibody fragments comprise only a portion of an intact antibody, wherein the portion retains at least one, two, three and as many as most or all of the functions normally associated with that portion when present in an intact antibody. In one aspect, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen.
  • an antibody fragment such as an antibody fragment that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody. Such functions may include FcRn binding, antibody half life modulation, conjugate function and complement binding.
  • an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody.
  • such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.
  • Antibody fragments suitable for use in the compounds of this disclosure include, for example, Fv fragments, Fab fragments, F(ab’) 2 fragments, Fab’ fragments, scFv (sFv) fragments, and scFv-Fc fragments.
  • Polynucleotide or “nucleic acid,” as used interchangeably herein, and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers.
  • the nucleic acid molecule may be linear or circular.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • the nucleic acid molecule may be an aptamer.
  • purified refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance of interest comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises 50%, 80%-85%, 90-99%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the sample.
  • Techniques for purifying polynucleotides, polypeptides and virus particles of interest are well- known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • treatment “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect, such as reduction of tumor burden.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease (as in liver fibrosis that can result in the context of chronic HCV infection); (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease (e.g., reduction in of tumor burden).
  • the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to an animal, including, but not limited to, human and non-human primates, including simians and humans; rodents, including rats and mice; bovines; equines; ovines; felines; canines; and the like.
  • "Mammal” means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, e.g., non-human primates, and humans.
  • Non-human animal models e.g., mammals, e.g.
  • a “therapeutically effective amount” or “efficacious amount” means the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to effect such treatment for the disease, condition, or disorder.
  • the “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • all alternative isomers are intended to be encompassed within the scope of the claimed subject matter.
  • the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures.
  • the compounds provided herein may contain chiral centers. Such chiral centers may be of either the (R) or (S) configurations, or may be a mixture thereof. The chiral centers of the compounds provided herein may undergo epimerization in vivo.
  • the present disclosure also encompasses all suitable isotopic variants of the compounds according to the present disclosure, whether radioactive or not.
  • An isotopic variant of a compound according to the present disclosure is understood to mean a compound in which at least one atom within the compound according to the present disclosure has been exchanged for another atom of the same atomic number, but with a different atomic mass than the atomic mass which usually or predominantly occurs in nature.
  • isotopes which can be incorporated into a compound according to the present disclosure are those of hydrogen, carbon, nitrogen, oxygen, fluorine, chlorine, bromine and iodine, such as 2 H (deuterium), 3 H (tritium), 13 C, 14 C, 15 N, 17 O, 18 O, 18 F, 36 Cl, 82 Br, 123 I, 124 I, 125 I, 129 I and 131 I.
  • Particular isotopic variants of a compound according to the present disclosure especially those in which one or more radioactive isotopes have been incorporated, may be beneficial, for example, for the examination of the mechanism of action or of the active compound distribution in the body.
  • Isotopic variants of the compounds according to the present disclosure can be prepared by various, including, for example, the methods described below and in the working examples, by using corresponding isotopic modifications of the particular reagents and/or starting compounds therein.
  • any of the embodiments described herein are meant to include a salt, a single stereoisomer, a mixture of stereoisomers and/or an isotopic form of the compounds.
  • a "pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” and “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use.
  • “A pharmaceutically acceptable excipient, diluent, carrier and adjuvant” as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.
  • a "pharmaceutical composition” is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human.
  • a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade).
  • Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like.
  • pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and, more particularly in humans.
  • pharmaceutically acceptable salt refers to those salts which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).
  • salts can be prepared in situ during the final isolation and purification of the conjugate compounds, or separately by reacting the free base function or group of a compound with a suitable organic acid.
  • pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, or salts of an amino group formed with inorganic acids [0761] “Acyl” refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)- , hetero
  • acyl includes the “acetyl” group CH 3 C(O)- [0762]
  • alkyl refers to a branched or unbranched saturated hydrocarbon group (i.e., a mono-radical) typically although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like.
  • alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms.
  • the term "lower alkyl” intends an alkyl group of 1 to 6 carbon atoms.
  • “Substituted alkyl” refers to alkyl substituted with one or more substituent groups, and this includes instances wherein two hydrogen atoms from the same carbon atom in an alkyl substituent are replaced, such as in a carbonyl group (i.e., a substituted alkyl group may include a -C(O)- moiety).
  • heteroatom-containing alkyl and “heteroalkyl” refer to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom, as described in further detail infra. If not otherwise indicated, the terms “alkyl” and “lower alkyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl or lower alkyl, respectively.
  • substituted alkyl is meant to include an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as -O- , -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thi
  • alkenyl refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like.
  • alkenyl groups herein may contain 2 to about 18 carbon atoms, and for example may contain 2 to 12 carbon atoms.
  • lower alkenyl intends an alkenyl group of 2 to 6 carbon atoms.
  • substituted alkenyl refers to alkenyl substituted with one or more substituent groups
  • heteroatom-containing alkenyl and “heteroalkenyl” refer to alkenyl in which at least one carbon atom is replaced with a heteroatom.
  • alkenyl and “lower alkenyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkenyl and lower alkenyl, respectively.
  • alkynyl refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups herein may contain 2 to about 18 carbon atoms, and such groups may further contain 2 to 12 carbon atoms. The term “lower alkynyl” intends an alkynyl group of 2 to 6 carbon atoms.
  • substituted alkynyl refers to alkynyl substituted with one or more substituent groups
  • heteroatom-containing alkynyl and “heteroalkynyl” refer to alkynyl in which at least one carbon atom is replaced with a heteroatom.
  • alkynyl and “lower alkynyl” include linear, branched, unsubstituted, substituted, and/or heteroatom-containing alkynyl and lower alkynyl, respectively.
  • alkoxy refers to an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy” group may be represented as -O-alkyl where alkyl is as defined above.
  • a "lower alkoxy” group refers to an alkoxy group containing 1 to 6 carbon atoms, and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc.
  • Substituents identified as "C1-C6 alkoxy” or “lower alkoxy” herein may, for example, may contain 1 to 3 carbon atoms, and as a further example, such substituents may contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).
  • substituted alkoxy refers to the groups substituted alkyl-O-, substituted alkenyl- O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.
  • aryl refers to an aromatic substituent generally, although not necessarily, containing 5 to 30 carbon atoms and containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety).
  • Aryl groups may, for example, contain 5 to 20 carbon atoms, and as a further example, aryl groups may contain 5 to 12 carbon atoms.
  • aryl groups may contain one aromatic ring or two or more fused or linked aromatic rings (i.e., biaryl, aryl-substituted aryl, etc.).
  • substituted aryl refers to an aryl moiety substituted with one or more substituent groups
  • heteroatom-containing aryl and “heteroaryl” refer to aryl substituent, in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra.
  • Aryl is intended to include stable cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated C 3 -C 14 moieties, exemplified but not limited to phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazoyl; which may further be substituted with one to five members selected from the group consisting of hydroxy, C 1 -C 8 alkoxy, C 1 -C 8 branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (see e.g.
  • aryl includes unsubstituted, substituted, and/or heteroatom-containing aromatic substituents.
  • aralkyl refers to an alkyl group with an aryl substituent
  • alkaryl refers to an aryl group with an alkyl substituent, wherein “alkyl” and “aryl” are as defined above.
  • aralkyl and alkaryl groups herein contain 6 to 30 carbon atoms.
  • Aralkyl and alkaryl groups may, for example, contain 6 to 20 carbon atoms, and as a further example, such groups may contain 6 to 12 carbon atoms.
  • alkylene refers to a multi-valent (e.g., di-radical alkyl group, tri-radical alkyl group, tetra-radical alkyl group, etc.) . Unless otherwise indicated, such groups include saturated hydrocarbon chains containing from 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may be heteroatom-containing. "Lower alkylene” refers to alkylene linkages containing from 1 to 6 carbon atoms.
  • alkenylene alkynylene
  • arylene aralkylene
  • alkarylene refer to di-radical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.
  • the "alkylene” refers to a multi-valent (e.g., di-valent alkyl group, tri-valent alkyl group, tetra-valent alkyl group, etc.).
  • the terms “alkenylene,” “alkynylene,” “arylene,” “aralkylene,” and “alkarylene” can refer to multi-valent alkenyl, multi-valent alkynyl, multi-valent aryl, multi-valent aralkyl, and multi-valent alkaryl groups, respectively.
  • amino refers to the group -NRR’ wherein R and R’ are independently hydrogen or nonhydrogen substituents, with nonhydrogen substituents including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and/or heteroatom-containing variants thereof.
  • halo and “halogen” are used in the conventional sense to refer to a chloro, bromo, fluoro or iodo substituent.
  • Carboxyl,” “carboxy” or “carboxylate” refers to –C(O)OH or salts thereof.
  • Cycloalkyl refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems.
  • suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like.
  • Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
  • substituted cycloalkyl refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,
  • heteroatom-containing refers to a molecule, linkage or substituent in which one or more carbon atoms are replaced with an atom other than carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur.
  • heteroalkyl refers to an alkyl substituent that is heteroatom-containing
  • heterocycloalkyl refers to a cycloalkyl substituent that is heteroatom-containing
  • heterocyclic or “heterocycle” refer to a cyclic substituent that is heteroatom-containing
  • heteroaryl and “heteroaromatic” respectively refer to “aryl” and “aromatic” substituents that are heteroatom-containing, and the like.
  • heteroalkyl groups include alkoxyaryl, alkylsulfanyl- substituted alkyl, N-alkylated amino alkyl, and the like.
  • heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, furyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl, etc. [0779] “Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring.
  • heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic, provided that the point of attachment is through an atom of an aromatic ring.
  • the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N ⁇ O), sulfinyl, or sulfonyl moieties.
  • heteroaryl substituent can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclo
  • heterocycle refers to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 15 ring atoms, including 1 to 4 hetero atoms. These ring heteroatoms are selected from nitrogen, sulfur and oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring.
  • the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or –SO 2 - moieties.
  • heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenox
  • heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,
  • Hydrocarbyl refers to univalent hydrocarbyl radicals containing 1 to about 30 carbon atoms, including 1 to about 24 carbon atoms, further including 1 to about 18 carbon atoms, and further including about 1 to 12 carbon atoms, including linear, branched, cyclic, saturated and unsaturated species, such as alkyl groups, alkenyl groups, aryl groups, and the like.
  • a hydrocarbyl may be substituted with one or more substituent groups.
  • heteroatom-containing hydrocarbyl refers to hydrocarbyl in which at least one carbon atom is replaced with a heteroatom.
  • hydrocarbyl is to be interpreted as including substituted and/or heteroatom-containing hydrocarbyl moieties.
  • substituted as in “substituted hydrocarbyl,” “substituted alkyl,” “substituted aryl,” and the like, as alluded to in some of the aforementioned definitions, is meant that in the hydrocarbyl, alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents.
  • substituents include, without limitation, functional groups, and the hydrocarbyl moieties C 1 -C 24 alkyl (including C 1 -C 18 alkyl, further including C 1 -C 12 alkyl, and further including C 1 -C 6 alkyl), C 2 -C 24 alkenyl (including C 2 -C 18 alkenyl, further including C 2 -C 12 alkenyl, and further including C 2 -C 6 alkenyl), C 2 -C 24 alkynyl (including C 2 -C 18 alkynyl, further including C 2 -C 12 alkynyl, and further including C 2 -C 6 alkynyl), C 5 -C 30 aryl (including C 5 -C 20 aryl, and further including C 5 -C 12 aryl), and C 6 -C 30 aralkyl (including C 6 -C 20 aralkyl, and further including C 6 -C 12 aralkyl).
  • hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any of the groups described herein are to be interpreted as including substituted and/or heteroatom-containing moieties, in addition to unsubstituted groups.
  • “Sulfonyl” refers to the group SO 2 -alkyl, SO 2 -substituted alkyl, SO 2 -alkenyl, SO 2 -substituted alkenyl, SO 2 -cycloalkyl, SO 2 -substituted cylcoalkyl, SO 2 -cycloalkenyl, SO 2 -substituted cylcoalkenyl, SO 2 -aryl, SO 2 -substituted aryl, SO 2 -heteroaryl, SO 2 -substituted heteroaryl, SO 2 -heterocyclic, and SO 2 - substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, wherein al
  • Sulfonyl includes, by way of example, methyl-SO 2 -, phenyl-SO 2 -, and 4-methylphenyl-SO 2 -.
  • functional groups chemical groups such as halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-
  • linking or "linker” as in “linking group,” “linker moiety,” etc., is meant a linking moiety that connects two groups via covalent bonds.
  • the linker may be linear, branched, cyclic or a single atom.
  • linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups including, without limitation: amido (-NH-CO-), ureylene (-NH-CO-NH-), imide (-CO-NH-CO-) , epoxy (-O-), epithio (-S-), epidioxy (-O-O-), carbonyldioxy (-O-CO-O-), alkyldioxy (-O-(CH2)n-O-), epoxyimino (-O-NH-), epimino (-NH-), carbonyl (-CO-), etc.
  • one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom.
  • the bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone.
  • the linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group.
  • a linker may include, without limitations, poly(ethylene glycol) unit(s) (e.g., -(CH 2 -CH 2 -O)-); ethers, thioethers, amines, alkyls (e.g., (C 1 -C 12 )alkyl) , which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n- pentyl, 1,1-dimethylethyl (t-butyl), and the like.
  • poly(ethylene glycol) unit(s) e.g., -(CH 2 -CH 2 -O)-
  • ethers e.g., -(CH 2 -CH 2 -O)-
  • ethers e.g., -(CH 2 -CH 2 -O)-
  • ethers e.g.,
  • the linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone.
  • a linker may be cleavable or non-cleavable. Any convenient orientation and/or connections of the linkers to the linked groups may be used. [0788] When the term "substituted" appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group.
  • substituted alkyl and aryl is to be interpreted as “substituted alkyl and substituted aryl.”
  • substituted when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.
  • Each M + may independently be, for example, an alkali ion, such as K + , Na + , Li + ; an ammonium ion, such as + N(R 60 ) 4 ; or an alkaline earth ion, such as [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5 (“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound as disclosed herein, and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound can serve as the counter ion for such divalent alkali earth ions).
  • an alkali ion such as K + , Na + , Li +
  • an ammonium ion such as + N(R 60 ) 4
  • -NR 80 R 80 is meant to include -NH 2 , -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N- methyl-piperazin-1-yl and N-morpholinyl.
  • substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R 60 , halo, -O-M + , -OR 70 , -SR 70 , -S – M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , -N 3 , -SO 2 R 70 , -SO 3 – M + , -SO 3 R 70 , -OSO 2 R 70 , -OSO 3 – M + , -OSO 3 R 70 , -PO 3 -2 (M + ) 2 , -P(O)(OR 70 )O – M + , -P(O)(OR 70 ) 2 , -C(O)R 70
  • substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O-M + , -OR 70 , -SR 70 , -S-M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -NO, -NO 2 , -S(O) 2 R 70 , -S(O) 2 O-M + , -S(O) 2 OR 70 , -OS(O) 2 R 70 , -OS(O) 2 O-M + , -O S(O) 2 OR 70 , -P(O)(O-) 2 (M + ) 2 , -P(O)(OR 70 )O-M + , -P(O)(OR 70 )(OR 70 ), -C(O
  • a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
  • substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment.
  • substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O-C(O)-.
  • any of the groups disclosed herein which contain one or more substituents it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
  • the subject compounds include all stereochemical isomers arising from the substitution of these compounds.
  • a substituent may contribute to optical isomerism and/or stereo isomerism of a compound. Salts, solvates, hydrates, and prodrug forms of a compound are also of interest. All such forms are embraced by the present disclosure.
  • a compound may be a metabolized into a pharmaceutically active derivative.
  • reference to an atom is meant to include isotopes of that atom.
  • reference to H is meant to include 1 H, 2 H (i.e., D) and 3 H (i.e., T)
  • reference to C is meant to include 12 C and all isotopes of carbon (such as 13 C).
  • the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, or 3 standard deviations. In certain embodiments, the term “about” or “approximately” means within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.25%, 0.2%, 0.1% or 0.05% of a given value or range.
  • Embodiment A-1 A cell surface ASGPR binding compound of formula (I): X n L Y ( I) or a prodrug thereof, or a salt thereof, wherein: Y is a moiety of interest; n is 1 to 500; L is a linker; and X is a moiety that binds to a cell surface asialoglycoprotein receptor (ASGPR) of formula (Ia): wherein: R 1 is selected from –OH, –OC(O)R, -C(O)NHR, –Z 1 –*, and optionally substituted triazole, where R is optionally substituted C 1-6 alkyl or optionally substituted aryl; R 2 is selected from–NHCOCH 3 , –NHCOCF 3, –NHCOCH 2 CF 3 , –OH, optionally substituted triazole, and –Z 1 –*; R 3 is selected from –H, –OH, –
  • Embodiment A-2 The compound of embodiment A-1, wherein each X is independently of formula (Ib): wherein: R 1 is selected from –OH, –OC(O)R, and -C(O)NHR; and R 2 is selected from –NHCOCH 3 , –NHCOCF 3 , and –NHCOCH 2 CF 3 .
  • Embodiment A-3 The compound of embodiment A-3, wherein Z 1 is selected from -O-, -S-, and -C(R 22 ) 2 -.
  • Embodiment A-4 The compound of embodiment A-3, wherein Z 1 is Z 11 -Ar , wherein Ar is optionally substituted heteroaryl or optionally substituted aryl.
  • Embodiment A-5 The compound of embodiment A-4, wherein: Z 11 is O, S, or C(R 22 ) 2 ; and Ar is a monocyclic 5 or 6-membered heteroaryl or aryl.
  • Embodiment A-6 The compound of embodiment A-5, wherein Z 1 is -C(R 22 ) 2 -triazole-. * [0808] Embodiment A-7: The compound of embodiment A-6, wherein Z 1 is or .
  • Embodiment A-8 The compound of embodiment A-3, wherein Z 1 is monocyclic 5 or 6- membered heteroaryl or aryl.
  • Embodiment A-9 The compound of embodiment A-8, wherein .
  • Embodiment A-10 The compound of embodiment A-2, wherein each X is independently of the formula: wherein R 4 and R 5 are each H.
  • Embodiment A-11 The compound of embodiment A-10, wherein each X is independently of formula: [0813]
  • Embodiment A-12 The compound of embodiment A-10, wherein Z 1 is selected from monocyclic 5 or 6-membered heteroaryl, monocyclic 5 or 6-membered aryl and Z 11 -Ar, wherein Ar is optionally substituted heteroaryl or optionally substituted aryl.
  • Embodiment A-13 The compound of embodiment A-12, wherein each X is [0815]
  • Embodiment A-14 The compound of embodiment A-2, wherein each X is independently of the formula: wherein R 4 and R 5 are each H.
  • Embodiment A-19 The compound of embodiment A-18, wherein each X is independently of formula (Ie): wherein: Z 2 is absent or selected from -O-, -S-, NR 25 -, -C(R 22 ) 2 -, and optionally substituted Z 12 -alkyl; ring A is absent or selected from a 5 or 6-membered optionally substituted aryl and a 5 or 6- membered optionally substituted heteroaryl; Z 3 is a linking moiety selected from Z 12 , optionally substituted alkyl, optionally substituted Z 12 - alkyl, optionally substituted Z 12 -heteroaryl, optionally substituted Z 12 -aryl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiourea; and Z 12 is selected from -CH 2 O-, -O-, -
  • Embodiment A-20 The compound of embodiment A-17, wherein each X is independently of one of formula (If)-(Ii): [0822]
  • Embodiment A-21 The compound of embodiment A-20, wherein each X is independently of one of formula (Ij)-(Im) wherein: Y 1 -Y 3 are each independently N or CR 27 ; and R 24 and R 27 are each independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen.
  • Embodiment A-22 The compound of embodiment A-21, wherein Z 3 is selected from -O-, - CH 2 O-, -OCH 2 -, optionally substituted -OCH 2 -heteroaryl, optionally substituted -OCH 2 -aryl, optionally substituted -CH 2 O-heteroaryl, and optionally substituted -CH 2 O-aryl.
  • Embodiment A-23 The compound of embodiment A-21 or A-22, wherein X is independently one of the following structures:
  • Embodiment A-24 The compound of embodiment A-18, wherein X is: .
  • Embodiment A-26 The compound of embodiment A-25, wherein Z 1 is selected from -O-, - S-, -CONR 21 -, and optionally substituted –(C(R 22 ) 2 ) q -heteroaryl, wherein q is 0 or 1.
  • Embodiment A-27 The compound of embodiment A-26, wherein Z 1 is -O-.
  • Embodiment A-28 The compound of embodiment A-36, wherein Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
  • Embodiment A-29 The compound of embodiment A-28, wherein [0832] Embodiment A-30: The compound of any one of embodiments A-1 to A-20, wherein n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking X to Y via Z 1 .
  • Embodiment A-31 The compound of any one of embodiments A-1 to A-20, wherein n is 2 or more, and L is a branched linker that covalently links 2 or more X moieties to Y via the linking moiety Z 1 .
  • Embodiment A-32 The compound of any one of embodiment A-1 to A-31, wherein L is of formula (IIb): IIb) wherein each L 1 to L 5 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 0, 1, or 2; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y, wherein when n is >1, d is 1 or 2 and L4 is a branching moiety.
  • L is of formula (IIb): IIb) wherein each L 1 to L 5 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 0, 1, or 2; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y, wherein when n is
  • Embodiment A-33 The compound of claim 32, wherein L 1 to L 5 each independently comprise one or more linking moieties independently selected from –C 1-20 -alkylene–, –NHCO-C 1-6 - alkylene–, –CONH-C 1-6 -alkylene–, –NH-C 1-6 -alkylene–, —NHCONH-C 1-6 -alkylene–, – NHCSNH-C 1-6 - alkylene–, –C 1-6 -alkylene–NHCO-, –C 1-6 -alkylene–CONH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene– NHCONH-, –C 1-6 -alkylene–NHCSNH-, -O(CH 2 ) p –, –(OCH 2 CH 2 ) p –, –NHCO—, –CONH–, –NHSO 2 –, – SO
  • Embodiment A-34 The compound of embodiment A-32 or A-33, wherein L comprises repeating ethylene glycol moieties.
  • Embodiment A-35 The compound of embodiment A-34, wherein L comprises 1 to 25 ethylene glycol moieties.
  • Embodiment A-36 The compound of any one of embodiments A-32 to A-35, wherein L comprises one or more 1,2,3-triazole linking moieties.
  • Embodiment A-37 The compound of any one of embodiments A-32 to A-36, wherein n is 1.
  • Embodiment A-38 The compound of any one of embodiments A-32 to A-36, wherein n is 2 or more.
  • Embodiment A-39 The compound of embodiment A-38, wherein L 4 is a branching moiety [0842] wherein each x and y are each independently 1 to 10.
  • Embodiment A-40 The compound of any one of embodiments A-32 to A-39, wherein L 1 -L 4 comprises a backbone of 14 or more consecutive atoms between X and the branching atom.
  • Embodiment A-41 The compound of any one of embodiments A-32 to A-40, wherein L 5 comprises a backbone of 10 to 80 consecutive atoms.
  • Embodiment A-42 The compound of embodiment A-33, wherein L 5 comprises a linking moiety selected from (C 10 -C 20 -alkylene, or –(OCH 2 CH 2 ) p –, where p is 1 to 25.
  • Embodiment A-43 The compound of any one of embodiments A-32 to A-42, wherein the linker of formula (IIb) comprises a backbone of 20 to 100 consecutive atoms.
  • Embodiment A-44 The compound of embodiment A-43, wherein the linker of formula (IIb) comprises a backbone of 25 or more consecutive atoms.
  • Embodiment A-45 The compound of embodiment A-44, wherein the linker of formula (IIb) comprises a backbone of 30 or more consecutive atoms.
  • Embodiment A-46 The compound of any one of embodiments A-1 to A-45, wherein -Z 1 -L 1 - comprises a group selected from: wherein: each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6.
  • Embodiment A-47 The compound of embodiment A-46, wherein -Z 1 -L 1 - comprises a group selected from: wherein q is 1 to 3.
  • Embodiment A-48 The compound of any one of embodiments A-1 to A-45, wherein -Z 1 -L 1 - comprises an optionally substituted -NH-heteroaryl-.
  • Embodiment A-49 The compound of embodiment A-48, wherein -Z 1 -L 1 - comprises a group selected from: wherein: each R 24 is independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen; and each R 25 is independently selected from H, optionally substituted (C 1 -C 6 )alkyl, and optionally substituted acyl.
  • Embodiment A-50 The compound of any one of embodiment A-1 to A-49, wherein Y is selected from small molecule, dye, fluorophore, monosaccharide, polysaccharide, lipid, protein, polynucleotide, enzyme, enzyme substrate, polymer, and chemoselective ligation group or precursor thereof.
  • Embodiment A-51 The compound of any one of embodiment A-1 to A-49, wherein Y is a moiety that specifically binds an extracellular target protein.
  • Embodiment A-52 The compound of embodiment A-51, wherein the target protein is a membrane bound protein.
  • Embodiment A-53 The compound of embodiment A-51, wherein the target protein is a soluble extracellular protein.
  • Embodiment A-54 The compound of any one of embodiments A-51 to A-53, wherein Y is a target-binding small molecule.
  • Embodiment A-55 The compound of any one of embodiments A-51 to A-53, wherein Y is a target-binding biomolecule.
  • Embodiment A-56 The compound of embodiment A-55, wherein the biomolecule is selected from peptide, protein, glycoprotein, polynucleotide, aptamer, and antibody or antibody fragment.
  • Embodiment A-57 The compound of embodiment A-56, wherein Y is selected from antibody, antibody fragment, chimeric fusion protein, an engineered protein domain, and D-protein binder of target protein.
  • Embodiment A-58 The compound of embodiment A-55, wherein Y is a protein, n is 1 to 6, and m is 1 to 20.
  • Embodiment A-59 The compound of any one of embodiment A-51 to A-57, wherein Y is a moiety that specifically binds the target protein and the compound is a conjugate of formula (III’): wherein: n is 1 to 20; m is 1 to 80 (e.g., an average loading or a discrete loading); each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group to a compatible group of Y; and Y is a moiety of interest binds the target protein.
  • formula (III’) wherein: n is 1 to 20; m is 1 to 80 (e.g., an average loading or a discrete loading); each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage
  • Embodiment A-60 The compound of embodiment A-59, wherein Y is an antibody or an antibody fragment.
  • Embodiment A-61 The compound of embodiment A-59, wherein Y is selected from chimeric fusion protein, and engineered protein domain.
  • Embodiment A-62 The compound of any one of embodiments A-59 to A-61, wherein n is 1 to 6.
  • Embodiment A-63 The compound of any one of embodiments A-59 to A-61, wherein n is 1 to 4.
  • Embodiment A-64 The compound of any one of embodiments A-59 to A-61, wherein n is 3.
  • Embodiment A-65 The compound of any one of embodiments A-59 to A-61, wherein n is 2.
  • Embodiment A-66 The compound of any one of embodiments A-59 to A-61, wherein n is 1.
  • Embodiment A-67 The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 20.
  • Embodiment A-68 The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 10.
  • Embodiment A-69 The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 6.
  • Embodiment A-70 The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 4.
  • Embodiment A-71 The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 2.
  • Embodiment A-72 The compound of any one of embodiments A-59 to A-66, wherein m is 2.
  • Embodiment A-73 The compound of any one of embodiments A-59 to A-66, wherein m is 1.
  • Embodiment A-74 The compound of any one of embodiments A-59 to A-73, wherein Z is a residual moiety resulting from the covalent linkage of a thiol-reactive chemoselective ligation group to one or more cysteine residue(s) of Ab.
  • Embodiment A-75 The compound of embodiment A-74, wherein the thiol-reactive chemoselective ligation group comprises a maleimide.
  • Embodiment A-76 The compound of any one of embodiments A-59 to A-73, wherein Z is a residual moiety resulting from the covalent linkage of an amine-reactive chemoselective ligation group to one or more lysine residue(s) of Ab.
  • Embodiment A-77 The compound of embodiment A-76, wherein the amine-reactive chemoselective ligation group comprises a pentafluorophenyl (PFP) active ester.
  • PFP pentafluorophenyl
  • Embodiment A-78 A method of internalizing a target protein in a cell comprising a cell surface asialoglycoprotein receptor (ASGPR), the method comprising: contacting a cellular sample comprising the cell and the target protein with an effective amount of a compound according to any one of embodiments A-1 to A-77, wherein the compound specifically binds the target protein and specifically binds the ASGPR to facilitate cellular uptake of the target protein.
  • ASSGPR cell surface asialoglycoprotein receptor
  • Embodiment A-79 The method of embodiment A-78, wherein the target protein is a membrane bound protein.
  • Embodiment A-80 The method of embodiment A-78, wherein the target protein is an extracellular protein.
  • Embodiment A-81 A method of reducing levels of a target protein in a biological system, the method comprising: contacting the biological system with an effective amount of a compound according to any one of embodiments A-1 to A-77, wherein the compound specifically binds the target protein and specifically binds a ASGPR of cells in the biological system to facilitate cellular uptake and degradation of the target protein.
  • Embodiment A-82 The method of embodiment A-81, wherein the biological system is a human subject.
  • Embodiment A-83 The method of any one of embodiments A-81 to A-82, wherein the biological system is an in vitro cellular sample.
  • Embodiment A-84 The method of any one of embodiments A-81 to A-83, wherein the target protein is a membrane bound protein.
  • Embodiment A-85 The method of any one of embodiments A-81 to 83, wherein the target protein is an extracellular protein.
  • Embodiment B-2 The compound of embodiment B-1, wherein each X is independently of formula (IIa): wherein: R 6 is selected from –OH, –OC(O)R, and -C(O)NHR; and R 2 is selected from –NHCOCH 3 , –NHCOCF 3 , and –NHCOCH 2 CF 3 .
  • Embodiment B-3 The compound of embodiment B-3, wherein Z 1 is selected from -O-, -S-, and -C(R 22 ) 2 -.
  • Embodiment B-4 The compound of embodiment B-3, wherein Z 1 is Z 11 -Ar , wherein Ar is optionally substituted heteroaryl or optionally substituted aryl.
  • Embodiment B-5 The compound of embodiment B-4, wherein: Z 11 is O, S, or C(R 22 ) 2 ; and Ar is a monocyclic 5 or 6-membered heteroaryl or aryl.
  • Embodiment B-6 The compound of embodiment B-5, wherein Z 1 is -C(R 22 ) 2 -triazole-.
  • Embodiment B-7 The compound of embodiment B-6, wherein Z 1 is .
  • Embodiment B-8 The compound of embodiment B-3, wherein Z 1 is monocyclic 5 or 6- membered heteroaryl or aryl.
  • Embodiment B-9 The compound of embodiment B-8, wherein .
  • Embodiment B-10 The compound of embodiment B-2, wherein each X is independently of the formula: wherein R 3 and R 4 are each H.
  • Embodiment B-11 The compound of embodiment B-10, wherein each X is independently of formula: [0900]
  • Embodiment B-12 The compound of embodiment B-10, wherein Z 1 is selected from monocyclic 5 or 6-membered heteroaryl, monocyclic 5 or 6-membered aryl and Z 11 -Ar, wherein Ar is optionally substituted heteroaryl or optionally substituted aryl.
  • Embodiment B-13 The compound of embodiment B-12, wherein each X is [0902]
  • Embodiment B-14 The compound of embodiment B-2, wherein each X is independently of the formula: wherein R 3 and R 4 are each H.
  • Embodiment B-19 The compound of embodiment B-18, wherein each X is independently of formula (IVb) or (IVc): wherein -Z 11 - is -O-, -S-, -N(R 21 )-, or -C(R 22 ) 2 .
  • Embodiment B-20 The compound of embodiment B-17, wherein each X is independently of formula (IVb-1) or (IVc-1): (IVb-1) (IVc-1) wherein R 11 is the bridging moiety that connects the 5-position carbon to the 1-position carbon.
  • Embodiment B-21 The compound of embodiment B-19 or B-20, wherein each X is independently of formula ( wherein R 21 and R 22 are independently selected from H, halogen, (C 1- C 6 )alkyl and substituted (C 1- C 6 )alkyl (e.g., CF 3 ).
  • Embodiment B-22 The compound of embodiment B-21, wherein Z 3 R 21 and R 22 are independently H, or CF 3 .
  • Embodiment B-23 The compound of embodiment B-21 or B-22, wherein X is independently one of the following structures:
  • Embodiment B-26 The compound of embodiment B-25, wherein Z 1 is selected from -O-, -S- , -CONR 21 -, and optionally substituted –(C(R 22 ) 2 ) q -heteroaryl, wherein q is 0 or 1.
  • Embodiment B-27 The compound of embodiment B-26, wherein Z 1 is -O-.
  • Embodiment B-28 The compound of embodiment B-36, wherein Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
  • Embodiment B-29 The compound of embodiment B-28, wherein [0918] Embodiment B-30: The compound of any one of embodiments B-1 to B-20, wherein n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking X to Y via Z 1 .
  • Embodiment B-31 The compound of any one of embodiments B-1 to B-20, wherein n is 2 or more, and L is a branched linker that covalently links 2 or more X moieties to Y via the linking moiety Z 1 .
  • Embodiment B-32 The compound of any one of embodiments B-1 to B-31, wherein L is of formula (XI): wherein each L 1 and L 3 are independently a linear linking moiety, and L 2 is a branched linking moiety, wherein L 1 to L 3 together provide a linear or branched linker between X and Y; a, b and c are independently 0 or 1, wherein: when n is 1, b is 0 and at least one of a and c is 1; and when n is 2 or 3, a, b and c are each 1; * represents the point of connection of L 1 to X via Z 1 ; and ** represents a point of conjugation of the linker L to Y.
  • L is of formula (XI): wherein each L 1 and L 3 are independently a linear linking moiety, and L 2 is a branched linking moiety, wherein L 1 to L 3 together provide a linear or branched linker between X and Y; a, b
  • Embodiment B-33 The compound of embodiment B-32, wherein: n is 1; and a is 1, b is 0 and c is 1, whereby L is of formula (XIa): * L1 L3 ** (XIa).
  • Embodiment B-34 The compound of embodiment B-32, wherein: n is 2; and a is 1, b is 1, and c is 1, whereby L is of formula (XIb): (XIb).
  • Embodiment B-35 The compound of embodiment B-32, wherein: n is 3; and a is 1, b is 1, and c is 1, whereby L is of formula (XIc): (XIc).
  • Embodiment B-36 The compound of any one of embodiments B-32 to B-35, wherein each L 1 is of the formula (XII) wherein: L 10 is a linking moiety; and L 11 to L 19 are independently absent or a linking moiety, wherein L 10 to L 19 of each L 1 is each independently selected from –C 1-20 -alkylene–, –NHCO-C 1-6 - alkylene–, –CONH-C 1-6 -alkylene–, –NH-C 1-6 -alkylene–, —NHCONH-C 1-6 -alkylene–, – NHCSNH-C 1-6 - alkylene–, –C 1-6 -alkylene–NHCO-, –C 1-6 -alkylene–CONH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NHCONH-, –C 1-6 -alkylene–NHCSNH-, -O
  • Embodiment B-37 The compound of any one of embodiments B-32 to B-36, wherein each L 1 comprises a linear backbone of 6 to 20 consecutive atoms (e.g., 6 to 16 consecutive atoms, such as 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive atoms).
  • Embodiment B-38 The compound of any one of embodiments B-32, and B-34 to B-37, wherein L 2 is of formula (XIIIa) or (XIIIb): (XIIIa) (XIIIb) wherein: L 20 is a branched linking moiety comprising: a carbon atom or nitrogen atom that is the branching point of the branched linking moiety; and one or more (e.g., 1 to 20, 1 to 10, or 1 to 6) linking moieties independently selected from amino acid residue (e.g., a residue such a s, or a derivative thereof), –NH-CH[(CH 2 ) q ] 2 O– alkylene–, – NHCO-, –CONH–, –NHSO 2 –, –SO 2 NH–, –CO–, –SO 2 –, –O–, –S–, pyrrolidine-2,5-dione, 1,2,3- triazole, –NH–, and
  • Embodiment B-39 The compound of any one of embodiments B-32, and B-34 to B-38, wherein L 2 comprises a linking moiety selected from: wherein: Z 2 is connected to L 1 , and Z 3 is connected to L 3 ; each Z 2 and Z 3 is independently selected from –NHCO-, –CONH–, –CO–, –O–, –NH–, and – N(CH 3 )–; x is 1 to 12 (e.g., 1 to 6, or 1 to 3); and y is 0 to 12 (e.g., 1 to 6, or 1 to 3).
  • Embodiment B-40 The compound of embodiment B-39, wherein L 2 comprises a linking moiety selected from: [0929]
  • Embodiment B-41 The compound of embodiment B-40, wherein L 2 comprises a linking moiety of formula (XIV): wherein: r is 1 or 2; and when n is 2, r is 1, when n is 3, r is 2.
  • Embodiment B-42 The compound of embodiment B-41, wherein L 2 is of formula (XVa) or (XVb): (XVa) (XVb).
  • Embodiment B-43 The compound of embodiment B-41 or B-42, wherein L 2 is of formula (XVc) or (XVd): (XVc) (XVc) wherein r is 1 or 2.
  • Embodiment B-44 The compound of embodiment B-38, wherein L 2 comprises two 2 or more amino acid residues (e.g., 3 or more, or 4 or more amino acid residues, linear or dendrimer).
  • Embodiment B-45 The compound of embodiment B-38, wherein L 2 comprises 4 or more amino acid residues that are branched linking moieties selected from Lys, Orn, Asp, Glu, Ser, and Cys (e.g., where the sidechain, amino and carboxylic acid are each linked to an adjacent moiety).
  • Embodiment B-46 The compound of any one of embodiments B-32 to B-45, wherein each L 3 is of the formula (XVI): wherein: L 30 to L 39 are independently absent or a linking moiety; and Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group of the linker to a compatible group of Y; wherein L 30 to L 39 are each independently selected from –C 1-20 -alkylene–, –NHCO-C 1-6 -alkylene–, –CONH-C 1-6 -alkylene–, –NH C 1-6 -alkylene–, –NHCONH-C 1-6 -alkylene–, – NHCSNH-C 1-6 -alkylene–, – C 1-6 -alkylene–NHCO-, –C 1-6 -alkylene–CONH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–CONH
  • Embodiment B-47 The compound of embodiment B-46, wherein L 3 comprises a linear backbone of 6 to 40 consecutive atoms (e.g., 10 to 30 consecutive atoms, or 20 to 30 consecutive atoms).
  • Embodiment B-48 The compound of embodiment B-46 or B-47, wherein the linker L has one of the following structures: , , wherein: a is 1 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); f is 1 to 6 (e.g., 1, 2, or 3); g is 1 to 20 (e.g., 1 to 12, or 6 to 20 or 6 to 12); Z is a residual moiety resulting from the
  • Embodiment B-50 The compound of embodiment B-32, wherein L comprises repeating ethylene glycol moieties.
  • Embodiment B-51 The compound of embodiment B-32, wherein L comprises 1 to 25 ethylene glycol moieties.
  • Embodiment B-52 The compound of embodiment B-32, wherein L comprises one or more 1,2,3-triazole linking moieties.
  • Embodiment B-53 The compound of embodiment B-38, wherein L 2 is a branching moiety selected from: wherein each x and y are each independently 1 to 10.
  • Embodiment B-54 The compound of any one of embodiments B-32 to B-54, wherein the linker comprises a backbone of 14 or more consecutive atoms between each X and the branching atom.
  • Embodiment B-55 The compound of any one of embodiments B-32 to B-54, wherein L 3 comprises a backbone of 10 to 80 consecutive atoms.
  • Embodiment B-56 The compound of embodiment B-55, wherein L 3 comprises a linking moiety selected from (C 10 -C 20 -alkylene, or –(OCH 2 CH 2 ) p –, where p is 1 to 25.
  • Embodiment B-57 The compound of any one of embodiment B-32 to B-56, wherein the linker of formula (XI) comprises a backbone of 20 to 100 consecutive atoms.
  • Embodiment B-58 The compound of embodiment B-57, wherein the linker of formula (XI) comprises a backbone of 25 or more consecutive atoms.
  • Embodiment B-59 The compound of embodiment B-58, wherein the linker of formula (XI) comprises a backbone of 30 or more consecutive atoms.
  • Embodiment B-60 The compound of any one of embodiments B-1 to B-59, wherein -Z 1 -L 1 - comprises a group selected from: wherein: each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6.
  • each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl
  • each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl
  • Embodiment B-61 The compound of embodiment B-60, wherein -Z 1 -L 1 - comprises a group selected from: wherein q is 1 to 3.
  • Embodiment B-62 The compound of any one of embodiments B-1 to B-61, wherein -Z 1 -L 1 - comprises an optionally substituted -NH-heteroaryl-.
  • Embodiment B-63 The compound of embodiment B-62, wherein -Z 1 -L 1 - comprises a group selected from: wherein: each R 24 is independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen; and each R 25 is independently selected from H, optionally substituted (C 1 -C 6 )alkyl, and optionally substituted acyl.
  • Embodiment B-64 The compound of any one of embodiments B-1 to B-63, wherein Y is selected from small molecule, dye, fluorophore, monosaccharide, polysaccharide, lipid, protein, polynucleotide, enzyme, enzyme substrate, polymer, and chemoselective ligation group or precursor thereof.
  • Embodiment B-65 The compound of any one of embodiments B-1 to B-64, wherein Y is a moiety that specifically binds an extracellular target protein.
  • Embodiment B-66 The compound of embodiment B-65, wherein the target protein is a membrane bound protein.
  • Embodiment B-67 The compound of embodiment B-65, wherein the target protein is a soluble extracellular protein.
  • Embodiment B-68 The compound of any one of embodiments B-65 to B-67, wherein Y is a target-binding small molecule.
  • Embodiment B-69 The compound of any one of embodiments B-65 to B-67, wherein Y is a target-binding biomolecule.
  • Embodiment B-70 The compound of embodiment B-69, wherein the biomolecule is selected from peptide, protein, glycoprotein, polynucleotide, aptamer, and antibody or antibody fragment.
  • Embodiment B-71 The compound of embodiment B-69, wherein Y is selected from antibody, antibody fragment, chimeric fusion protein, an engineered protein domain, and D-protein binder of target protein.
  • Embodiment B-72 The compound of embodiment B-69, wherein Y is a protein, n is 1 to 10 (e.g., 1 to 8, 1 to 6, such as 1, 2, 3, 4-5, 5-6, or 6-7), and m is 1 to 20.
  • Embodiment B-73 The compound of any one of embodiments B-51 to B-57, wherein Y is a moiety that specifically binds the target protein and the compound is a conjugate of formula (III’): wherein: n is 1 to 20; m is 1 to 80 (e.g., an average loading or a discrete loading); each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group to a compatible group of Y; and Y is a moiety of interest (e.g., a protein, antibody, aptamer, peptide) binds the target protein.
  • formula (III’) wherein: n is 1 to 20; m is 1 to 80 (e.g., an average loading or a discrete loading); each X is a moiety that binds to a cell surface ASGPR; each L
  • Embodiment B-74 The compound of embodiment B-59, wherein Y is an antibody or an antibody fragment.
  • Embodiment B-75 The compound of embodiment B-59, wherein Y is selected from chimeric fusion protein, and engineered protein domain.
  • Embodiment B-76 The compound of any one of embodiments B-73 to B-75, wherein n is to 10 (e.g., 1 to 8, or 1 to 6).
  • Embodiment B-77 The compound of any one of embodiments B-73 to B-75, wherein n is 1 to 4.
  • Embodiment B-78 The compound of any one of embodiments B-73 to B-75, wherein n is 3.
  • Embodiment B-79 The compound of any one of embodiments B-73 to B-75, wherein n is 2.
  • Embodiment B-80 The compound of any one of embodiments B-73 to B-75, wherein n is 1.
  • Embodiment B-81 The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 20.
  • Embodiment B-82 The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 10.
  • Embodiment B-83 The compound of any one of embodiments B-73 to B-80, wherein m is 4 to 8 (e.g., 4-6, 4-5, 5-6, or 6-7).
  • Embodiment B-84 The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 6.
  • Embodiment B-85 The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 4.
  • Embodiment B-86 The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 3 (e.g., 1, 2, or 3).
  • Embodiment B-87 The compound of any one of embodiments B-73 to B-80, wherein m is 2.
  • Embodiment B-88 The compound of any one of embodiments B-73 to B-80, wherein m is 1.
  • Embodiment B-89 The compound of any one of embodiments B-73 to B-88, wherein Z is a residual moiety resulting from the covalent linkage of a thiol-reactive chemoselective ligation group to one or more cysteine residue(s) of Ab.
  • Embodiment B-90 The compound of embodiment B-89, wherein the thiol-reactive chemoselective ligation group comprises a maleimide or phenylene-maleimide.
  • Embodiment B-91 The compound of any one of embodiments B-73 to B-88, wherein Z is a residual moiety resulting from the covalent linkage of an amine-reactive chemoselective ligation group to one or more lysine residue(s) of Ab.
  • Embodiment B-92 The compound of embodiment B-91, wherein the amine-reactive chemoselective ligation group comprises a pentafluorophenyl (PFP) active ester.
  • PFP pentafluorophenyl
  • Embodiment B-93 A method of internalizing a target protein in a cell comprising a cell surface asialoglycoprotein receptor (ASGPR), the method comprising: contacting a cellular sample comprising the cell and the target protein with an effective amount of a compound according to any one of embodiments B-1 to B-92, wherein the compound specifically binds the target protein and specifically binds the ASGPR to facilitate cellular uptake of the target protein.
  • Embodiment B-94 The method of embodiment B-93, wherein the target protein is a membrane bound protein.
  • Embodiment B-95 The method of embodiment B-93, wherein the target protein is an extracellular protein.
  • Embodiment B-96 A method of reducing levels of a target protein in a biological system, the method comprising: contacting the biological system with an effective amount of a compound according to any one of embodiments B-1 to B-92, wherein the compound specifically binds the target protein and specifically binds a ASGPR of cells in the biological system to facilitate cellular uptake and degradation of the target protein.
  • Embodiment B-97 The method of embodiment B-96, wherein the biological system is a human subject.
  • Embodiment B-98 The method of any one of embodiments B-96 to B-97, wherein the biological system is an in vitro cellular sample.
  • Embodiment B-99 The method of any one of embodiments B-96 to B-98, wherein the target protein is a membrane bound protein.
  • Embodiment B-100 The method of any one of embodiments B-96 to B-98, wherein the target protein is an extracellular protein.
  • EXAMPLES [0989] The examples in this section are offered by way of illustration, and not by way of limitation.
  • Example 1 Preparation of Compounds [0990] The following are illustrative schemes and examples of how the compounds described herein can be prepared and tested. Although the examples can represent only some embodiments, it should be understood that the following examples are illustrative and not limiting. All substituents, unless otherwise specified, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art. The specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare the compounds described herein. Synthesis of compound XC28:
  • reaction mixture was concentrated, washed with diethyl ether and dried to afford 2-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)isoindoline-1,3-dione (48-6) as a light purple solid which was used as such for next reaction. Yield: 7.0 g (Crude).
  • reaction mixture was neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated, washed with diethyl ether and dried to afford 2-((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)isoindoline-1,3-dione (48-8) as an off white solid. Yield: 0.640 g, 83.2 %; ELSD m/z 294.15 [M+1] + .
  • reaction mixture was cooled, water was added and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-25 % ethyl acetate in hexane to afford 2- ((3S,4R,5R,6R)-4,5-bis(methoxymethoxy)-6-((methoxymethoxy)methyl)tetrahydro-2H-pyran-3- yl)isoindoline-1,3-dione (48-9) as colourless viscous liquid.
  • reaction mixture was cooled and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford (3S,4R,5R,6R)-4,5-bis(methoxymethoxy)-6- ((methoxymethoxy)methyl)tetrahydro-2H-pyran-3-amine (48-10) as colourless viscous liquid. Yield: 0.310 g, 76.99 %; ELSD m/z 296.20 [M+1] + .
  • reaction mixture was concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford 2,5-dioxopyrrolidin-1-yl 3,3,3-trifluoropropanoate (2) as colorless viscous liquid. Yield: 0.050 g, 28.57 %; LCMS m/z No ionization; 1 H NMR (400 MHz, CDCl 3 ) ⁇ 3.54-3.47 (m, 2H), 2.87 (s, 4H).
  • reaction mixture was concentrated to get crude which was purified by prep HPLC (25-33 % acetonitrile in water with 0.1 % trifluoroacetic acid). Fractions containing the desired product were combined and lyophilized to dryness to afford N-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)-3,3,3- trifluoropropanamide (XC28) as a cream solid.
  • reaction mixture was concentrated under reduce pressure to get crude which was purified by flash column chromatography on silica gel using 0-50% ethyl acetate in hexane to afford a (S)-3-((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)-5-(methoxymethyl)oxazolidin-2-one (3) as colorless liquid. Yield: 0.2 g; 51.49 %, LCMS m/z 548.0 [M+1] + .
  • tert-butyl (4-aminobutyl)carbamate (1.1 eq.0.20 g, 1.16 mmol) was added, and stirred the reaction mixture at room temperature for 12 h. After completion (monitored by TLC), water was added, and extracted with dichloromethane.
  • reaction mixture was filtered on celite pad and washed with methanol. The filtrate was evaporated under reduced pressure to give crude which was again treated with a mixture trifluoroacetic acid and dichloromethane (10 ml, 1:1, v/v) and stirred for 1h at room temperature.
  • reaction mixture was purified by prep-HPLC (20-30% acetonitrile in water with 0.1% TFA) to give N-(4-aminobutyl)-2- ((3S,4aS,6R,7R,8R,8aR)-7,8-dihydroxy-6-(hydroxymethyl)-2-oxohexahydro-1H,6H-pyrano[2,3- b][1,4]oxazin-3-yl)acetamide (XC25) as colorless semi solid.
  • reaction mixture was concentrated under vacuum to get crude product which was re-dissolved in ethyl acetate, washed with ice cold water. The organic layer was dried over anhydrous sodium sulfate, concentrated to get crude which was purified by silica gel flash column chromatography using 2-5% methanol in dichloromethane to afford 2-(((3S,4R,5R,6R)-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)-N-(2-methoxyethyl)acetamide (3) as green oily liquid.
  • reaction mixture was quenched by 15 % sodium hydroxide solution, brine solution was added, then extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to give crude N1-((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro- 2H-pyran-3-yl)-N2-(2-methoxyethyl)ethane-1,2-diamine (4) as brown syrup which was used for next reaction without further purification.
  • reaction mixture was concentrated to give crude residue which was purified by flash column chromatography on silica gel using 50% ethyl acetate in hexane to afford a 1-((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H- pyran-3-yl)-3-(2-methoxyethyl)imidazolidin-2-one (5) as colorless liquid. Yield: 0.080 g, 69.36 %; LCMS m/z 561.15 [M+1] + .
  • reaction mixture was concentrated to get crude. which was purified by column chromatography using silica gel (100-200 mesh) and 0-10 % methanol in DCM to afford N-((3aR,4S,7S,8R,8aR)-4-(methoxymethyl)-2,2-dimethylhexahydro-4,7- epoxy[1,3]dioxolo[4,5-d]oxepin-8-yl)-4-(prop-2-yn-1-yloxy)-6-(trifluoromethyl)pyrimidin-2-amine (4) as a white solid. Yield: 0.070 g, 55.07%; LCMS m/z 446.05 [M+1] + .
  • reaction mixture was purified by prep HPLC (45-55% ACN in H 2 O with 0.1% TFA). Fractions containing desired compound were collected and lyophilized to afford (1S,2R,3R,4R,5S)-1-(methoxymethyl)-4-((4-(prop-2- yn-1-yloxy)-6-(trifluoromethyl)pyrimidin-2-yl)amino)-6,8-dioxabicyclo[3.2.1]octane-2,3-diol (XC11) as an off white solid. Yield: 0.0013 g, 1.37 %; m/z 406.0 [M+1] + .
  • N-((3aR,4R,7S,7aR)-4-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran- 7-yl)acetamide (XC17-A, 1.00 eq, 17.0 mg, 0.0693 mmol) was added and after another 10 minutes, 4- methoxyphenol (1.5 eq, 12.9 mg, 0.104 mmol) was added and the reaction was stirred at room temperature for 1 hour then at 50 °C for 18 h.
  • reaction was purified directly by reversed-phase HPLC (10-50% acetonitrile in water w/0.1% FA) to give N-((3aR,4R,7S,7aR)-4-((4- methoxyphenoxy)methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)acetamide.
  • LCMS m/z 351.5 [M+H] + .
  • reaction mixture was stirred for 16h at room temperature. After completion (monitored by TLC), the reaction mixture was poured into cold 1N HCl, and extracted with dichloromethane. The organic part was then washed with saturated bicarbonate followed by brine, and dried over anhydrous sodium sulfate, filtered, and concentrated to give crude residue which was purified by silica gel flash column chromatography using 0-30% ethyl acetate in hexane to afford 6- ((2R,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2- yl)hexa-3,5-diyn-1-yl 4-methylbenzenesulfonate (3) as white solid.
  • reaction mixture was extracted with ethyl acetate, and the collected ethyl acetate was dried over anhydrous sodium sulphate, filtered, and concentrated to give crude (2R,3R,4R,5S,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-oxoethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (4) as colorless syrup. This was immediately used for next step without purification. Yield: 1.4 g (Crude); ELSD-MS m/z 374.19 [M+H] + .
  • N,N-dibenzyl-2-(2-prop-2- ynoxyethoxy)ethanamine 2 was dissolved in 10 mL toluene then concentrated to dryness and left under high vacuum.
  • the reaction was filtered then re-cooled in an ice bath before it was treated with 5M aq. sodium hydroxide (350 eq, 214 mL, 1070 mmol) at such a rate as to keep the internal temperature below 24 °C.
  • 5M aq. sodium hydroxide 350 eq, 214 mL, 1070 mmol
  • the layers were partitioned then the aqueous layer was washed with DCM (80 mL).
  • the aqueous layer was washed with DCM (50 mL) then the combined organic layer was washed with brine then dried over Na2SO4, filtered, concentrated under reduced pressure and left under high vacuum.
  • reaction mixture was cooled, water was added, neutralized with solid sodium bicarbonate and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-20 % ethyl acetate in dichloromethane to afford (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a- tetrahydro-5H-pyrano[3,2-d]thiazole-6,7-diyl diacetate (2) as light yellow viscous liquid.
  • reaction mixture was concentrated, azeotroped with toluene (2-3 times) and dried to get afford (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6- mercaptotetrahydro-2H-pyran-3,4-diyl diacetate (3) as a light yellow viscous liquid. Yield: 1.6 g (Crude); LCMS m/z 364.10 [M+H].
  • reaction mixture was concentrated to get crude which was first purified by column chromatography using silica gel (100-200 mesh) and 0-10 % methanol in dichloromethane and then by prep HPLC (10-25 % acetonitrile in water with 0.1 % trifluoroacetic acid).
  • reaction mixture was concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((2,2- dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-yl)thio)tetrahydro-2H-pyran-3,4-diyl diacetate (2) as a colorless semi solid.
  • reaction mixture was neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated, washed with diethyl ether and dried to afford tert-butyl (2-(2-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)ethoxy)ethoxy)ethoxy)ethyl)carbamate (3) as an off white solid. Yield: 0.320 g, 88.64 %; LCMS m/z 513.10 [M+H].
  • reaction mixture stirred at ambient temperature for 30 minutes, at which time 8 mL of 1N aqueous hydrochloric acid was added slowly to achieve approximate final pH of 1-2.
  • the reaction mixture was concentrated to approximately 1 ⁇ 4 volume and purified by preparatory HPLC, eluting with 1-30% acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford Compound 3 as white solid. Yield: 244 mg (64%); LCMS m/z 423.06 [M+H].
  • reaction was monitored by ELSD. After completion, reaction mixture was cooled, diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution and water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford (2R,3R,4R,5R,6S)-5-acetamido-2-(acetoxymethyl)-6-((3-methoxy-3-oxopropyl)thio)tetrahydro-2H- pyran-3,4-diyl diacetate (2) as a colorless viscous liquid.
  • reaction mixture was neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated and dried to afford methyl 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoate (3) as an off white solid. Yield: 1.7 g, 59.0 %; LCMS m/z 324.0 [M+H].
  • reaction mixture was concentrated, methanol was added, neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated and dried to afford 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoic acid (4) as an off white sticky solid. Yield: 2.4 g (Crude); LCMS m/z 310.0 [M+H].
  • reaction mixture was concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-7 % methanol in dichloromethane to afford 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoic acid (XB5) as a colorless viscous liquid.
  • reaction mixture was quenched with triethyl amine and concentrated under reduced pressure to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 2-5% methanol in dichloromethane to afford (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((3-oxo-1-phenyl-2,7,10-trioxa-4- azatridecan-13-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (3) as colorless viscous liquid.
  • the mixture was stirred under nitrogen atmosphere at ambient temperature for approximately 15 minutes until completion.
  • the reaction mixture was diluted with water, which formed a precipitate, then 2 drops of trifluoroacetic acid was added to clear the solution.
  • the product was isolated from the diluted mixture by preparatory HPLC, eluting with 1-20% acetonitrile in water with 0.1% trifluoroacetic acid.
  • tert-butyl (2-(2-(2- bromoethoxy)ethoxy)ethyl)carbamate (4a, 0.195 g, 1 eq., 0.625 mmol) was added.
  • the reaction mixture was stirred at room temperature for 1h. After completion (monitored by TLC), ice-cold water (10 mL) was added to the reaction mixture. Organic part was extracted with ethyl acetate (3 ⁇ 10 mL), combined and dried over anhydrous sodium sulphate.
  • reaction mixture was cooled down to 0 °C, quenched with the addition of an aqueous sodium thiosulfate solution (1M) and saturated sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate.
  • reaction mixture was stirred at room temperature for 1h. Progress of the reaction was monitored by LCMS. After completion of reaction, solvent was evaporated under reduced pressure to obtain crude residue. Crude was purified by RP prep-HPLC (60% acetonitrile in water with 0.1% trifluoroacetic acid). Fractions containing desired product were combined and lyophilized to afford (2R,3R,4R,5S)-5-((4-((2-(2-(2- aminoethoxy)ethoxy)ethoxy)methyl)thiazol-2-yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB82) as a white sticky solid.
  • reaction mixture was stirred at room temperature for 12h. After completion the reaction mixture was concentrated under reduced pressure to get a crude which was diluted with water and extracted with ethyl acetate (5 X 120 mL). Organic part was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to afford tert-butyl ((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-propyltetrahydro-2H-pyran-3-yl)carbamate (1a) as off white solid. Yield: 5.0 g, crude; LCMS m/z 306.1[M + H] + .
  • reaction mixture was neutralized using triethyl amine and concentrated under reduced pressure to afford crude which was purified by silica gel flash column chromatography using 20-50 % ethyl acetate/hexane as eluent to afford tert-butyl ((3aR,4R,6R,7S,7aR)-4-(hydroxymethyl)-2,2-dimethyl-6- propyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (1) as off white solid.
  • reaction mixture was stirred at room temperature for 4h. After completion (monitored by TLC), reaction mixture was concentrated under reduce pressure and co-evaporated with dichloromethane three times to obtain crude residue, which was further lyophilized to afford (2R,3R,4R,5R,6R)-5-amino-2-(methoxymethyl)-6- propyltetrahydro-2H-pyran-3,4-diol (3) as light yellow syrup. The crude residue was directly used for next step. Yield: 0.6 g (Crude). LCMS m/z 220.1 [M+H] + .
  • reaction mixture was concentrated under reduced pressure.
  • the crude residue was purified by silica gel flash column chromatography using 2-3% methanol in dichloromethane as the eluent to afford tert-butyl (2-(2-((2-(((2R,3R,4R,5R,6R)- 4,5-dihydroxy-6-(methoxymethyl)-2-propyltetrahydro-2H-pyran-3-yl)amino)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4) as brown gummy liquid. Yield: 0.64 g, 67.1%.
  • reaction mixture was concentrated under reduced pressure to get crude which was purified by RP prep HPLC (20% acetonitrile in water with 0.1 % acetic acid). Fractions containing desired product were combined and lyophilized to get (2,2-dimethyl- 4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)glycine (3) as a white solid. Yield: 2.5 g, 59.3%.
  • reaction mixture was concentrated under reduced pressure to afford crude which was again dissolved in tetrahydrofuran (20 mL) and acetic anhydride (2.32 mL, 3 eq., 24.5 mmol) was added at 0 °C and reaction mixture was allowed to stir at room temperature for 36h. After completion, reaction mixture was concentrated to afford crude which was purified by silica gel column chromatography using 2-3% methanol/dichloromethane as eluent to afford 3-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)- 1,2,3-oxadiazol-3-ium-5-olate (4) as yellow liquid.
  • reaction mixture was quenched with methanol and concentrated under reduced pressure to afford crude which was purified by RP prep-HPLC (40% of acetonitrile in water with 0.1% TFA).
  • Fractions containing desired product were combined and lyophilized to afford N-((2S,3R,4R,5R,6R)-2-(1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-pyrazol-3-yl)- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB84) as colorless foamy solid.
  • reaction mixture was filtered through syringe filter and washed with methanol. The filtrate was concentrated and dried to obtained crude. Crude was purified by silica gel chromatography using 10% methanol in dichloromethane to afford tert-butyl (2-(2-(3-(2-bromopyrimidin-4- yl)propoxy)ethoxy)ethyl)carbamate (3) as colourless liquid. Yield: 0.11 g, 38.5%; LCMS: m/z 403.9 [M+H].
  • reaction was monitored by TLC. After completion of reaction, reaction mixture was diluted with ice cold water and extracted with ethyl acetate (3 ⁇ 20 mL). Then, organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford crude tert-butyl (Z)-(2-(2-(2-chloro-2- (hydroxyimino)ethoxy)ethoxy)ethyl)carbamate (4, 1.6 g) which was used for the next step as such.
  • reaction mixture was concentrated under reduced pressure to afford crude which was purified by silica gel flash column chromatography using 70-80% ethyl acetae- heptane as eluent to afford tert-butyl (2-(2-((5-((2S,3R,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)isoxazol-3-yl)methoxy)ethoxy)ethyl)carbamate (5) as yellowish sticky liquid. Yield: 0.27 g, 65.7%.
  • reaction mixture was filtered through syringe filter and washed with methanol. The filtrate was concentrated and dried to afford crude tert-butyl (2-(2-(3-(6-(((3S,4R,5R,6R)-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)propoxy)ethoxy)ethyl)carbamate (5) as colour less liquid. Yield: 0.25 g, crude; LCMS: m/z 487.00 [M+H].
  • reaction mixture was quenched with water (10 mL) and extracted with dichloromethane to obtain crude, which was purified by flash column chromatography (using 10% ethyl acetate/heptane as eluent) to afford tert-butyl(dec-9-yn-1-yloxy)dimethylsilane (2) as colourless liquid.
  • Methyl iodide (0.566 mL, 1.20 eq., 9.10 mmol) was added and the reaction mixture was allowed to warm to room temperature and stirred for 4h. After completion, the mixture was dried, diluted with water and extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulphate, concentrated under reduced pressure to get crude, which was purified by silica gel flash column chromatography using 20-40% ethyl acetate in heptane as eluent to afford benzyl methyl(2-(2-(prop-2-yn-1- yloxy)ethoxy)ethyl)carbamate (3) as a brown liquid.
  • reaction mixture was stirred at room temperature under hydrogen for 16 h. Reaction was monitored by ELSD. After completion, reaction mixture was filtered through syringe filter and filtrate was concentrated to get crude which was purified by prep HPLC (14-28 % acetonitrile in water with 0.1 % trifluoroacetic acid) to afford (R)-5-((2-(2-(2 aminoethoxy)ethoxy)ethoxy)methyl)-3- ((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxazolidin-2-one (XB91) as colorless viscous syrup.
  • reaction mixture was stirred at room temperature for 2h. After completion, the reaction mixture was diluted with water and extracted with dichloromethane. The combined organic layer was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using 10-25% ethyl acetate/heptane as eluent to afford ((3aR,4R,7S,7aR)-7-((tert-butoxycarbonyl)amino)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran- 4-yl)methyl 4-methylbenzenesulfonate (2) as off-white solid.

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Abstract

The present disclosure provides a class of compounds including a ligand moiety that specifically binds to a cell surface asialoglycoprotein receptor (ASGPR). The cell surface ASGPR binding compounds can trigger the receptor to internalize into the cell a bound compound. The ligand moieties of this disclosure can be linked to a variety of moieties of interest without impacting the specific binding to, and function of, the cell surface receptor ASGPR. Also provided are compounds that are conjugates of the ligand moieties linked to a biomolecule, such as an antibody, which conjugates can harness cellular pathways to remove specific proteins of interest from the cell surface or from the extracellular milieu. Also provided herein methods of using the conjugates to target a polypeptide of interest for sequestration and/or lysosomal degradation.

Description

ASGPR BINDING COMPOUNDS AND CONJUGATES CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application Numbers 63/439,811, filed January 18, 2023, and 63/518,532, filed August 9, 2023, each of which is hereby incorporated by reference in its entirety. BACKGROUND [0002] Many therapeutics act by binding a functionally important site on a target protein, thereby modulating the activity of that protein, or by recruiting immune effectors, as with many monoclonal antibody drugs, to act upon the target protein. However, there is an untapped reservoir of medically important human proteins that are considered to be “undruggable” because these proteins are not readily amenable to currently available therapeutic targeting approaches. Thus, there is a need for therapies that can target a wider range of proteins. [0003] The asialoglycoprotein receptor (ASGPR), also known as the Ashwell Morell receptor, is the transmembrane glycoprotein receptor found primarily in hepatocytes which plays an important role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine (GalNAc) residues. ASGPR cycles between endosomes and the cell surface. [0004] Alternative ligands that provide for binding to cell surface ASGPRs followed by transport across cell membranes are of great interest. SUMMARY [0005] The present disclosure provides a class of compounds including a ligand moiety that specifically binds to a cell surface asialoglycoprotein receptor (ASGPR). The cell surface ASGPR binding compounds can trigger the receptor to internalize into the cell a bound compound. The ligand moieties of this disclosure can be linked to a variety of moieties of interest without impacting the specific binding to, and function of, the cell surface receptor ASGPR. Also provided are compounds that are conjugates of the ligand moieties linked to a biomolecule, such as an antibody, which conjugates can harness cellular pathways to remove specific proteins of interest from the cell surface or from the extracellular milieu. For example, the conjugates described herein may sequester and/or degrade a target molecule of interest in a cell’s lysosome. Also provided herein are compositions comprising such conjugates and methods of using the conjugates to target a polypeptide of interest for sequestration and/or lysosomal degradation, and methods of using the conjugates to treat disorders or disease. BRIEF DESCRIPTION OF THE DRAWINGS [0006] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where: [0007] FIG.1 shows a graph of cell fluorescence (MFI) versus antibody conjugate concentration ([Ab]) indicating that various antibody conjugates of exemplary ASGPR binding compounds, and an example M6PR binding compound (520) exhibited comparable robust uptake into HepG2 cells after one hour incubation. [0008] FIG.2A-2D shows graphs of cell fluorescence versus antibody conjugate concentration indicating that various antibody conjugates of exemplary ASGPR binding compounds exhibited robust uptake into HepG2 cells after one hour incubation. [0009] FIG.3 illustrates the fluorescence polarization screening results for example trivalent compounds (1901 (I-171), 1902 (I-172), XB32 and 2101). [0010] FIG.4 illustrates the binding of example monovalent compounds (591, XB20, XB23, XB21, 592 and 593) as a percentage of the activity of reference compound XB149. [0011] FIG.5 illustrates the fluorescence polarization screening results for example monovalent compounds (XB20, XB21, 592, XB23, 591). [0012] FIG.6 shows a graph of cellular uptake of various conjugates of OMA (anti-IgE) with example compounds I-160 to I-163 and I-141 bound to Alexa488 labeled-target IgE in HepG2 cells. [0013] FIG.7 illustrates affinity-dependent clearance of OMA-example compounds (I-160 to I-163) as compared to OMA (reference). [0014] FIG.8 illustrates dose titration OMA-I-163 IgE clearance. [0015] FIG.9 illustrates affinity-dependent clearance of OMA-example compounds (I-160 to I-163) as compared to hIgE (reference). DETAILED DESCRIPTION [0016] As summarized above, this disclosure provides classes of compounds including a ligand moiety that specifically binds an ASGPR of a cell of interest. [0017] This disclosure includes compounds of formula (I): Xn L Y (I) or a prodrug thereof, or a salt thereof, wherein: X is a moiety that binds to a ASGPR cell surface receptor (e.g., as described herein); n is 1 to 500; L is a linker (e.g., monovalent or multivalent, as described herein, of defined length); and Y is a moiety of interest (e.g., as described herein). [0018] Also provided herein are conjugates that comprise a moiety, X, that binds to such an ASGPR internalizing cell surface receptor, for example, for sequestration and/or lysosomal degradation. Accordingly, this disclosure includes target binding conjugate of formula (I):
Figure imgf000004_0001
or a prodrug thereof, or a pharmaceutically acceptable salt thereof, wherein: X is a moiety that binds to an ASGPR cell surface receptor (e.g., as described herein); n is 1 to 500; L is a linker (e.g., monovalent or multivalent, as described herein); m is 1 to 20; Y is a biomolecule that specifically binds an extracellular target molecule. [0019] In some embodiments target binding conjugate is of formula (II’):
Figure imgf000005_0001
(II’) or a prodrug thereof, or a salt thereof, wherein: n is 1 to 3; m is 1 to 3; X and Y are each independently as defined herein; each L1 to L6 is independently a linking moiety which together provide a linear or branched linker between Z1 and Y; and a, b, c, d, and e are each independently 1, 2, 3, 4, or 5. [0020] The ASGPR binding compounds and conjugates and methods of this disclosure are described in greater detail below. Linkers (L) and moieties of interest (Y) which find use in the ASGPR binding compounds, and the biomolecule conjugates are also described. Methods in which the compounds and conjugates of this disclosure find use are also described. ASGPR Ligands [0021] As summarized above, this disclosure provides a class of compounds including a ligand moiety that specifically binds to a cell surface ASGPR. The ASGPR ligand moieties of this disclosure can be linked to a variety of moieties of interest without impacting the specific binding to, and function of, the cell surface ASGPR. In certain embodiments, compounds of this disclosure can utilize the functions of cell surface ASGPRs in a biological system, e.g., for internalization and sequestration of a compound to the lysosome of a cell, and in certain embodiments subsequent lysosomal degradation. The compounds of this disclosure find use in a variety of applications. [0022] The term “asialoglycoprotein receptor” (ASGPR), also known as the Ashwell Morell receptor, means the transmembrane glycoprotein receptor found primarily in hepatocytes which plays an important role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine (GalNAc) residues. ASGPR cycles between endosomes and the cell surface. In particular embodiments, the ASGPR is Homo sapiens asialoglycoprotein receptor 1 (ASGR1) (see, e.g., NCBI Reference Sequence: NM_001197216). [0023] A compound comprising such ASGPR binding moiety (X) (e.g., as described herein), may bind to other receptors, for example, may bind with lower affinity as determined by, e.g., immunoassays or other assays known in the art. In a specific embodiment, X, or a compound as described herein comprising such X specifically binds to the cell surface ASGPR with an affinity that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the affinity when X or the compound or the conjugate bind to another cell surface receptor. In a specific embodiment, X or a compound as described herein comprising X, specifically binds to ASGPR with an affinity (Kd) 20 mM or less. In particular embodiments, such binding is with an affinity (Kd) is 10 mM or less, 1 mM or less, 100 uM or less, 10 uM or less, 1 uM or less, 100 nM or less, 10 nM or less, or 1 nM or less. The terms “binds,” “binds to,” “specifically binds” or “specifically binds to” in this context are used interchangeably. [0024] The ASGPR binding compounds of this disclosure include a moiety (X) that specifically binds to the cell surface receptor ASGPR. The ASGPR binding compounds can be monovalent or multivalent (e.g., bivalent or trivalent or of higher valency), where a monovalent compound includes a single ASGPR ligand moiety, and a multivalent compound includes two or more such moieties. [0025] In certain embodiments, the ASGPR binding moiety X is able to bind to a specific cell surface ASGPR, and direct (or target) the molecule to this receptor. In certain embodiments, the ASGPR binding moiety X is capable of binding to the ASGPR and directing (or targeting) a compound or conjugate described herein for internalization and sequestration to the lysosome, and/or subsequent lysosomal degradation. [0026] In some embodiments, the ASGPR binding moiety X includes an amino sugar ring derivative of galactose (e.g., N-acetylgalactosamine, and analogs thereof), that is linked via a linking moiety to the 1, 6 or 2-position of the sugar ring. The linking moiety can be of 1-10 atoms in length, such as 1-6, or 1- 5, 1-4, or 1-3 atoms in length. In some embodiments, the amino sugar ring derivative of galactose is linked via a linking moiety to an oxygen, sulfur, nitrogen or carbon atom at the 1-position of the ring. In some embodiments, the amino sugar ring derivative of galactose is linked via a linking moiety to an oxygen, sulfur, nitrogen or carbon atom the 6-position of the ring. In some embodiments, the amino sugar ring derivative of galactose is linked via a linking moiety to an oxygen, sulfur, nitrogen or carbon atom the 2-position of the ring. In certain embodiments, the amino sugar derivative of galactose is linked via a linking moiety to a heteroaryl group at the 1, 6 or 2 position of the ring. In certain embodiments, the amino sugar derivative of galactose is a bicyclic structure. [0027] In some embodiments, the ASGPR binding compounds is monovalent (e.g., in Formula (I), n is 1), such that the ASGPR binding compound includes a single ASGPR ligand moiety (X) that is linked to a moiety of interest (Y) via a linking moiety at the 1, 6, or 2-position of (X). In certain embodiments of formula (I), n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking the ASGPR ligand (X) to Y via a linking moiety at any of the 1, 2 or 6-positions of X. In certain embodiments, L is 20 to 100 consecutive atoms, such as 25 to 80, 25 to 60, or 25 to 50. In certain embodiments of formula (I), n is 1, and L comprises a backbone of 25 or more consecutive atoms covalently linking the ASGPR ligand (X) to Y. [0028] In some embodiments, the ASGPR binding compounds are multivalent (e.g., in Formula (I), n is 2 or more, such that the ASGPR binding compound includes two or more ASGPR ligand binding moieties (X) that are each covalently linked to a moiety of interest (Y) via a branched linker (e.g., L is a branched linker). In certain embodiments, the ASGPR binding compound is divalent (e.g., n is 2 in Formula I). In certain other cases, the ASGPR binding compound is trivalent (e.g., n is 3 in Formula I). In certain embodiments, each branch of the branched linker comprises a liner linker of 14 or more consecutive atoms to covalently link a linking moiety of each X to a branching point in the linker. In certain embodiments, each branch of the linker includes 14 to 50 consecutive atoms, such as 14 to 40, 14 to 30, or 14 to 20 atoms. In certain embodiments, each branch of the linker includes a linear linker of 20 or more consecutive atoms. In certain embodiments, the linker comprises a linear linker of 12 or more consecutive atoms to covalently link the branching point of L to a moiety of interest (Y), such as 15 or more, 20 or more, 30 or more, or even more consecutive atoms to covalently link the branching point of L to Y. [0029] Exemplary ASGPR ligand moieties which can be adapted for use in the conjugates of this disclosure are described in WO/2023288033, filed July 14, 2022, the disclosure of which is herein incorporated by reference in its entirety. [0030] Certain ASGPR ligand moieties are described below. [0031] In some embodiments, the ASGPR ligand moieties (e.g., Xn-L or (X-L)n of formula (I), and other formulae described herein), of the bifunctional molecule specifically bind to ASGPR with an affinity (Kd) of 300 nM or less, such as 100 nM or less, 30 nM or less, 10 nM or less, 3 nM or less, or 1 nM or less. The terms “binds,” “binds to,” “specifically binds” or “specifically binds to” in this context are used interchangeably. [0032] In some embodiments, provided is a compound of formula (I):
Figure imgf000007_0001
or a prodrug thereof, or a salt thereof, wherein: n is 1 to 500; m is 1 to 20; Y is a moiety of interest; L is a linker; and X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (II):
Figure imgf000007_0002
wherein: R1 is selected from –Z1–*, –H, –OH, optionally substituted (C1-C6)alkyl, –OCH3,–OCH2CH=CH, optionally substituted -S-(C1-C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, and optionally substituted -S-heteroaryl; R2 is selected from –Z1–*, –NHCOCH3, –NHCOCF3, –NHCOCH2CF3, –OH, –NHR, and optionally substituted triazole; R6 is selected from –Z1–*, –OH, -OR, optionally substituted (C1-C6)alkyl, –OC(O)R, -C(O)NHR, -NRxxRyy, optionally substituted aryl, optionally substituted heteroaryl, –NHCOR, and –NRCOR; each R is independently optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl; wherein one of R1, R2, and R6 is –Z1–*, and “ * ” represents a point of connection of Z1 to the linker (L); R3 and R4 are each independently H, or a promoiety, or R3 and R4 are cyclically linked to form a promoiety; R11 is H, or a bridging moiety that connects the 5-position carbon to the 1-position carbon of the ring; Z1 is a linking moiety selected from -Z11-, -Z11-A1-, -A2-, -NR21CO-, - CONR21-, -NR21SO2-, - SO2NR21-, -NR21C(=O)NR21-, and -NR21C(=S)NR21-; -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2; -A1- and -A2- are optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene; each R21 is independently selected from H, optionally substituted (C1-C6)alkyl, -COR, and optionally substituted heteroaryl; and each R22 is independently selected from H, halogen, and optionally substituted (C1-C6)alkyl. [0033] In some embodiments, Z1 is a linking moiety selected from -Z11-A1- and -A2-; and -A1- and - A2- are optionally substituted heterocyclylene; or -A2- is optionally substituted isoxazolyl.
Figure imgf000008_0001
Figure imgf000008_0002
Figure imgf000009_0001
[0035] In some embodiments, R6 is -OR, optionally substituted (C1-C6)alkyl, –OC(O)-optionally substituted heteroaryl, -C(O)NH-optionally substituted heteroaryl, -NRxxRyy, optionally substituted aryl, optionally substituted heteroaryl–NHCOR, or –NRCOR, provided the heteroaryl is other than triazole; where each R is independently optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl. [0036] In some embodiments, R1 is optionally substituted C2-6 alkyl, optionally substituted -S-(C1- C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, or optionally substituted -S-heteroaryl. [0037] In some embodiments, at least one R21 is -COR or optionally substituted heteroaryl. [0038] In some embodiments, n is 1, 2, or 3; and m is 1-3. [0039] In some embodiments, provided is a compound of formula (I):
Figure imgf000009_0002
or a prodrug thereof, or a salt thereof, wherein: n is 1 to 500; m is 1 to 20; Y is a moiety of interest; L is a linker; and X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (II):
Figure imgf000009_0003
(II) wherein: R1 is selected from –Z1–*, –H, –OH, optionally substituted (C1-C6)alkyl, –OCH3,–OCH2CH=CH, optionally substituted -S-(C1-C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, and optionally substituted -S-heteroaryl; R2 is selected from –Z1–*, –NHCOCH3, –NHCOCF3, –NHCOCH2CF3, –OH, –NHR, and optionally substituted triazole; R6 is selected from –Z1–*, –OH, -OR, optionally substituted (C1-C6)alkyl, –OC(O)R, -C(O)NHR, -NRxxRyy, optionally substituted aryl, optionally substituted heteroaryl, –NHCOR, and –NRCOR; each R is independently optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl; wherein one of R1, R2, and R6 is –Z1–*, and “ * ” represents a point of connection of Z1 to the linker (L); R3 and R4 are each independently H, or a promoiety, or R3 and R4 are cyclically linked to form a promoiety; R11 is H, or a bridging moiety that connects the 5-position carbon to the 1-position carbon of the ring; Z1 is a linking moiety selected from -Z11-, -Z11-A1-, -A2-, -NR21CO-, - CONR21-, -NR21SO2-, - SO2NR21-, -NR21C(=O)NR21-, and -NR21C(=S)NR21-; -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2; -A1- and -A2- are optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene; each R21 is independently selected from H, optionally substituted (C1-C6)alkyl, -COR, and optionally substituted heteroaryl; and each R22 is independently selected from H, halogen, and optionally substituted (C1-C6)alkyl; provided at least one of the following occurs: A) Z1 is a linking moiety selected from -Z11-A1- and -A2-; and -A1- and -A2- are optionally substituted heterocyclylene; or -A2- is optionally substituted isoxazolyl; B) -L-Y comprises:
Figure imgf000010_0001
Figure imgf000011_0001
C) R6 is -OR, optionally substituted (C1-C6)alkyl, –OC(O)-optionally substituted heteroaryl, - C(O)NH-optionally substituted heteroaryl, -NRxxRyy, optionally substituted aryl, optionally substituted heteroaryl–NHCOR, or –NRCOR, provided the heteroaryl is other than triazole; where each R is independently optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl; D) R1 is optionally substituted C2-6 alkyl, optionally substituted -S-(C1-C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, or optionally substituted -S-heteroaryl; or E) at least one R21 is -COR or optionally substituted heteroaryl. [0040] In some embodiments, n is 1, 2, or 3; and m is 1-3. [0041] In some embodiments, X is of formula (a-II):
Figure imgf000011_0002
(a-II). [0042] In some embodiments, X is of formula (a-II), R1 is n-propyl; and R2 is -Z1-*. [0043] In some embodiments, Z1 is a linking moiety selected from -Z11-A1- and -A2-; and -A1- and - A2- are optionally substituted heterocyclylene. [0044] In some embodiments,
Figure imgf000012_0001
[0045] In some embodiments, -L-Y comprises:
Figure imgf000012_0002
monocyclic heteroaryl. [0046] In some embodiments, -L-Y comprises:
Figure imgf000012_0003
Figure imgf000013_0001
[0047] In some embodiments, R6 is -OR, optionally substituted (C1-C6)alkyl, –OC(O)-optionally substituted heteroaryl, -C(O)NH-optionally substituted heteroaryl, -NRxxRyy, optionally substituted aryl, or optionally substituted heteroaryl, provided the heteroaryl is other than triazole; where R is optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl. [0048] In some embodiments, R6 is -O-(C1-C6)alkyl, optionally substituted heterocyclyl, or -O-aryl. [0049] In some embodiments, R1 is optionally substituted C2-6 alkyl, optionally substituted -S-(C1- C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, or optionally substituted -S-heteroaryl [0050] In some embodiments, R1 is optionally substituted C2-6 alkyl. [0051] In some embodiments, at least one R21 is -COR or optionally substituted heteroaryl. [0052] In some embodiments, Y is an antibody or antibody fragment. [0053] In some embodiments, provided is a compound of formula (I):
Figure imgf000013_0002
or a prodrug thereof, or a salt thereof, wherein: n is 1 to 500; m is 1 to 20; L is a linker; and X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (II):
Figure imgf000014_0001
wherein: R1 is selected from –Z1–*, –H, –OH, optionally substituted (C1-C6)alkyl, –OCH3,–OCH2CH=CH, optionally substituted -S-(C1-C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, and optionally substituted -S-heteroaryl; R2 is selected from –Z1–*, –NHCOCH3, –NHCOCF3, –NHCOCH2CF3, –OH, and optionally substituted triazole; R6 is selected from –Z1–*, –OH, -OR, optionally substituted (C1-C6)alkyl, –OC(O)R, -C(O)NHR, -NRxxRyy, optionally substituted aryl, and optionally substituted heteroaryl, R is optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl; wherein one of R1, R2, and R6 is –Z1–*, and “ * ” represents a point of connection of Z1 to the linker (L); R3 and R4 are each independently H, or a promoiety, or R3 and R4 are cyclically linked to form a promoiety; R11 is H, or a bridging moiety that connects the 5-position carbon to the 1-position carbon of the ring; Z1 is a linking moiety selected from -Z11-, -Z11-A1-, -A2-, -NR21CO-, - CONR21-, -NR21SO2-, - SO2NR21-, -NR21C(=O)NR21-, and -NR21C(=S)NR21-; -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2; -A1- and -A2- are optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene; each R21 is independently selected from H, optionally substituted (C1-C6)alkyl, -COR, and optionally substituted heteroaryl; and each R22 is independently selected from H, halogen, and optionally substituted (C1-C6)alkyl; R3 and R4 are each independently H, or a promoiety, or R3 and R4 are cyclically linked to form a promoiety; R11 is H, or a bridging moiety that connects the 5-position carbon to the 1-position carbon of the ring; Z1 is a linking moiety selected from -Z11-, -Z11-A1-, -A2-, -NR21CO-, -CONR21-, -NR21SO2-, - SO2NR21-, -NR21C(=O)NR21-, and -NR21C(=S)NR21-; -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2; -A1- and -A2- are optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene; each R21 is independently selected from H, optionally substituted (C1-C6)alkyl, -COR, and optionally substituted heteroaryl; and each R22 is independently selected from H, halogen, and optionally substituted (C1-C6)alkyl; and Y is a chemoselective ligation group. [0054] In some embodiments, n is 1, 2, or 3; and m is 1-3. [0055] In some embodiments, the chemoselective ligation group comprises a carboxylic acid or active ester, maleimide, isocyanate or isothiocyanate, alkyl halide, alkyl tosylate, aldehyde, haloacetamide or alpha-leaving group acetamide, 2-sulfonylpyridine, diazirine, sulfonyl halide or vinyl sulfone, hydrazide, hydrazino, hydroxylamino, pyridyl disulfide, (HIPS) hydrazinyl-indolyl group, or (aza-HIPS) hydrazinyl-pyrrolo-pyridinyl group, alkyne or cyclooctyne, azide, or amine. [0056] In some embodiments, the chemoselective ligation group is selected from:
Figure imgf000015_0001
Figure imgf000016_0001
. [0057] In some embodiments, at least one of the following occurs: A) Z1 is a linking moiety selected from -Z11-A1- and -A2-; and -A1- and -A2- are optionally substituted heterocyclylene; or -A2- is optionally substituted isoxazolyl; B) -L-Y comprises:
Figure imgf000016_0002
Figure imgf000017_0001
C) R6 is -OR, optionally substituted (C1-C6)alkyl, –OC(O)-optionally substituted heteroaryl, - C(O)NH-optionally substituted heteroaryl, -NRxxRyy, optionally substituted aryl, optionally substituted heteroaryl–NHCOR, or –NRCOR, provided the heteroaryl is other than triazole; where each R is independently optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl; D) R1 is optionally substituted C2-6 alkyl, optionally substituted -S-(C1-C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, or optionally substituted -S-heteroaryl; or E) at least one R21 is -COR or optionally substituted heteroaryl. [0058] In some embodiments, the compound of formula (II) is represented by formula (a-II):
Figure imgf000017_0002
(a-II). [0059] In some embodiments, R1 is –Z1–*, –H, or (C1-C6)alkyl. [0060] In some embodiments, R2 is –Z1–* or –NHCOCH3. [0061] In some embodiments, R3 and R4 are each –H. [0062] In some embodiments, R6 is –OH, –OC(O)R, -NRxxRyy, or aryl; R is (C1-C6)alkyl; and Rxx and Ryy cyclize to form an optionally substituted heterocyclyl.
Figure imgf000017_0003
wherein “ * ” represents a point of connection of Z1 to the linker (L). [0064] In some embodiments, R1 is –Z1–*. [0065] In some embodiments, R2 is –Z1–*. [0066] In some embodiments, L comprises of 10 to 60 consecutive linear or branched chain atoms. [0067] In some embodiments, L is of formula (IIb’):
Figure imgf000018_0001
wherein: each L1 to L6 is independently a linking moiety which together provide a linear or branched linker between Z1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L1 of X via Z1; and *** represents the point of attachment to Y. [0068] In some embodiments of formula (IIb’), n is 1-3. In some embodiments of formula (IIb’), n is 1. In some embodiments of formula (IIb’), n is 2. In some embodiments of formula (IIb’), n is 3. [0069] In some embodiments, L is of formula (IIb’):
Figure imgf000018_0002
wherein: n is 1, 2, or 3; each L1 to L6 is independently a linking moiety which together provide a linear or branched linker between Z1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L1 of X via Z1; and *** represents the point of attachment to Y. [0070] In some embodiments, provided is a compound of formula (II’):
Figure imgf000018_0003
(II’) or a prodrug thereof, or a salt thereof, wherein: n is 1 to 3; m is 1 to 20; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; each L1 to L6 is independently a linking moiety which together provide a linear or branched linker between Z1 and Y; X and Y are each independently as defined herein. [0071] In some embodiments, provided is a compound of formula (II’):
Figure imgf000019_0001
or a prodrug thereof, or a salt thereof, wherein: n is 1 to 3; m is 1 to 20; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; each L1 to L6 is independently a linking moiety which together provide a linear or branched linker between Z1 and Y; Y is as defined herein; each X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (a-II):
Figure imgf000019_0002
(a-II) wherein: R1 is selected from –Z1–*, –H, –OH, optionally substituted (C1-C6)alkyl, –OCH3, –OCH2CH=CH, optionally substituted -S-(C1-C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, and optionally substituted -S-heteroaryl; R2 is selected from –Z1–*, –NHCOCH3, –NHCOCF3, –NHCOCH2CF3, –OH, and optionally substituted triazole; R6 is selected from –Z1–*, –OH, -OR, optionally substituted (C1-C6)alkyl, –OC(O)R, -C(O)NHR, -NRxxRyy, optionally substituted aryl, and optionally substituted heteroaryl, R is optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl; wherein one of R1, R2, and R6 is –Z1–*, and “ * ” represents a point of connection of Z1 to the linker (L); R3 and R4 are each independently H, or a promoiety, or R3 and R4 are cyclically linked to form a promoiety; R11 is H, or a bridging moiety that connects the 5-position carbon to the 1-position carbon of the ring; Z1 is a linking moiety selected from -Z11-, -Z11-A1-, -A2-, -NR21CO-, - CONR21-, -NR21SO2-, - SO2NR21-, -NR21C(=O)NR21-, and -NR21C(=S)NR21-; -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2; -A1- and -A2- are optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene; each R21 is independently selected from H, optionally substituted (C1-C6)alkyl, -COR, and optionally substituted heteroaryl; and each R22 is independently selected from H, halogen, and optionally substituted (C1-C6)alkyl; R3 and R4 are each independently H, or a promoiety, or R3 and R4 are cyclically linked to form a promoiety; R11 is H, or a bridging moiety that connects the 5-position carbon to the 1-position carbon of the ring; Z1 is a linking moiety selected from -Z11-, -Z11-A1-, -A2-, -NR21CO-, - CONR21-, -NR21SO2-, - SO2NR21-, -NR21C(=O)NR21-, and -NR21C(=S)NR21-; -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2; -A1- and -A2- are optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene; each R21 is independently selected from H, optionally substituted (C1-C6)alkyl, -COR, and optionally substituted heteroaryl; and each R22 is independently selected from H, halogen, and optionally substituted (C1-C6)alkyl. [0072] In some embodiments, each L1 to L5 independently comprises one or more linking moieties independently selected from –C1-20-alkylene–, –NHC(O)-C1-6-alkylene–, –C(O)NH-C1-6-alkylene–, –NH- C1-6-alkylene–, –NHC(O)NH-C1-6-alkylene–, –NHC(S)NH-C1-6-alkylene–, –C1-6-alkylene–NHC(O)-, – C1-6-alkylene–C(O)NH-, –C1-6-alkylene–NH-, –C1-6-alkylene–NHC(O)NH-, –C1-6-alkylene–NHC(S)NH- , -O(CH2)p–, –(OCH2CH2)p–, –NHC(O)–, –C(O)NH–, –NHS(O)2–, –S(O)2NH–, –C(O)–, –S(O)2–, –O–, – S–, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, amino acid residue, –NH–, and – NMe–; wherein each L1 to L5 is independently optionally substituted with one to five halo; each p is independently1 to 50; L6 is a linking group comprising one or more linking moieties independently selected from –C1-20-alkylene–, –NR16C(O)-C1-6-alkylene–, –C(O)NR16-C1-6-alkylene–, –NR16-C1-6-alkylene–, –NR16C(O)NR16-C1-6-alkylene–, –NR16C(S)NR16-C1-6-alkylene–, –C1-6-alkylene–NR16C(O)-, –C1-6-alkylene–C(O)NR16-, –C1-6-alkylene–NR16-, –C1-6-alkylene–NR16C(O)N R16-, –C1-6-alkylene– NR16C(S)NR16-, -O(CH2)p–, –(OCH2CH2)p–, –NR16C(O)–, –C(O)NR16–, –NHS(O)2–, –S(O)2NH–, –C(O)–, –S(O)2–, –O–, –S–, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, amino acid residue, or –NR16–; and each R16 is independently –H, (C1-C6)alkyl, or monocyclic heteroaryl. [0073] In some embodiments, each R16 is independently (C1-C6)alkyl or monocyclic heteroaryl. In some embodiments, each R16 is independently R”. [0074] In some embodiments, each L1 to L5 is independently selected from –C1-20-alkylene–, –NHC(O)-C1-6-alkylene–, –C(O)NH-C1-6-alkylene–, –NH-C1-6-alkylene–, –NHC(O)NH-C1-6-alkylene–, –NHC(S)NH-C1-6-alkylene–, –C1-6-alkylene–NHC(O)-, –C1-6-alkylene–C(O)NH-, –C1-6-alkylene–NH-, –C1-6-alkylene–NHC(O)NH-, –C1-6-alkylene–NHC(S)NH-, -O(CH2)p–, –(OCH2CH2)p–, –NHC(O)–, –C(O)NH–, –NHS(O)2–, –S(O)2NH–, –C(O)–, –S(O)2–, –O–, –S–, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, monocyclic cycloalkyl, amino acid residue, –NH–, and –NMe–; wherein each L1 to L5 is independently optionally substituted with one to five halo; each p is independently1 to 50; and
Figure imgf000021_0001
; and R16 is (C1-C6)alkyl or monocyclic heteroaryl. [0075] In some embodiments, each L1 to L5 is independently selected from –C1-20-alkylene–, –NHC(O)-C1-6-alkylene–, –C(O)NH-C1-6-alkylene–, –NH-C1-6-alkylene–, –NHC(O)NH-C1-6-alkylene–, –NHC(S)NH-C1-6-alkylene–, –C1-6-alkylene–NHC(O)-, –C1-6-alkylene–C(O)NH-, –C1-6-alkylene–NH-, –C1-6-alkylene–NHC(O)NH-, –C1-6-alkylene–NHC(S)NH-, -O(CH2)p–, –(OCH2CH2)p–, –NHC(O)–, –C(O)NH– –NHS(O)2– –S(O)2NH– –C(O)– –S(O)2– –O– –S– monocyclic heteroaryl monocyclic aryl, monocyclic heterocycle, monocyclic cycloalkyl, amino acid residue, –NH–, and –NMe–; wherein each L1 to L5 is independently optionally substituted with one to five halo;
Figure imgf000022_0001
[0076] In some embodiments, each L1 to L5 is independently selected from –C1-20-alkylene–, – NHC(O)-C1-6-alkylene–, –C(O)NH-C1-6-alkylene–, –NH-C1-6-alkylene–, –NHC(O)NH-C1-6-alkylene–, – NHC(S)NH-C1-6-alkylene–, –C1-6-alkylene–NHC(O)-, –C1-6-alkylene–C(O)NH-, –C1-6-alkylene–NH-, – C1-6-alkylene–NHC(O)NH-, –C1-6-alkylene–NHC(S)NH-, -O(CH2)p–, –(OCH2CH2)p–, –NHC(O)–, – C(O)NH–, –NHS(O)2–, –S(O)2NH–, –C(O)–, –S(O)2–, –O–, –S–, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, amino acid residue, –NH–, and –NMe–; wherein each L1 to L5 is independently optionally substituted with one to five halo; each p is independently1 to 50; and
Figure imgf000023_0001
[0077] In some embodiments of formula (II’), n is 1. [0078] In some embodiments of formula (II’), n is 2. [0079] In some embodiments of formula (II’), n is 3. [0080] In some embodiments, at least one L1 is –C1-20-alkylene– optionally substituted with one to five halo. [0081] In some embodiments, at least one L1 is -CF2CH2-. [0082] In some embodiments, at least one L2 is –(OCH2CH2)p–. [0083] In some embodiments, p is 2-3. [0084] In some embodiments, at least one L3 is NHCONH-C1-6-alkylene–. [0085] In some embodiments, at least one L4 is –C1-6-alkylene–NHCONH-. [0086] In some embodiments, at least one L5 is –(OCH2CH2)p–. [0087] In some embodiments, the lysosomal targeting bifunctional molecules of this disclosure (e.g., of formula (I)-(Ia)) include an ASGPR ligand moiety of formula (II):
Figure imgf000023_0002
(II) wherein: R1 is selected from –Z1–*, –H, –OH, –CH3, –OCH3, and –OCH2CH=CH; R2 is selected from –Z1–*, –NHCOCH3, –NHCOCF3, –NHCOCH2CF3, –OH, and optionally substituted triazole; R6 is selected from –Z1–*, –OH, –OC(O)R, -C(O)NHR, and optionally substituted triazole, where R is optionally substituted (C1-C6)alkyl or optionally substituted aryl; wherein one of R1, R2, and R6 is –Z1–*, and “ * ” represents a point of connection of Z1 to the linker (L); R3 and R4 are each independently H, or a promoiety, or R3 and R4 are cyclically linked to form a promoiety; R11 is H, or a bridging moiety that connects the 5-position carbon to the 1-position carbon of the ring; Z1 is a linking moiety selected from -Z11-, -Z11-A1-, -A2-, -NR21CO-, - CONR21-, -NR21SO2-, -SO2NR21-, -NR21C(=O)NR21-, and -NR21C(=S)NR21-; -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2; -A1- and -A2- are optionally substituted arylene or optionally substituted heteroarylene; each R21 is independently selected from H, and optionally substituted (C1-C6)alkyl; and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1-C6)alkyl. [0088] In some embodiments of formula (II): when n is 3, R6 is OH, R2 is –NHCOCH3, R3-R4 are H, and R1 is Z1, then Z1 is not O; ii) when n is 2 or 3, R6 is OAc, R2 is –NHCOCH3, R3-R4 are Ac, and R1 is Z1, then Z1 is not O; iii) when n is 2 or 3, R6 is -OBz, R2 is –NHCOCH3, R3-R4 are Bz, and R1 is Z1, then Z1 is not O; iv) when n is 3, R6 is OH, R2 is –NHCOCH3, R3-R4 are H, and R1 is Z1, and Z11 is O, then L comprises a backbone of at least 16 consecutive atoms to a branching point; v) when n is 3, R6 is Z1, where Z1 is O, and R3-R4 are H, then R1 is not -CH3 –OCH3,or –OCH2CH=CH; and vi) when R11 is a group of the formula -CH2O- that forms a bridge (i.e., is cyclically linked) to the 1-position carbon atom on the sugar ring, R2 is –NHCOCH3, R3-R4 are H, then R1 and R3 are not Z1. 1-linked ASGPR ligand moieties [0089] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Iia):
Figure imgf000024_0001
(Iia) wherein R2, R3, R4, R6 and Z1 are as defined herein. In some embodiments of formula (Iia), R6 is selected from –OH, –OC(O)R, and -C(O)NHR; and R2 is selected from –NHCOCH3, –NHCOCF3, and –NHCOCH2CF3. [0090] In some embodiments of formula (II), Z1 is in a beta configuration, and can be described by formula (Iia-1):
Figure imgf000025_0001
(Iia-1). [0091] In some embodiments of formula (II), Z1 is in an alpha configuration, and can be described by formula (Iia-2)
Figure imgf000025_0002
(Iia-2). [0092] In certain embodiments of formula (Iia), (Iia-1) or (Iia-2), Z1 is -Z11-A1-, wherein A1- is optionally substituted arylene or optionally substituted heteroarylene. In certain embodiments, A1 is an optionally substituted heteroarylene. In certain embodiments, the heteroarylene is a 5 or 6-membered heteroarylene. In certain embodiments, the heteroarylene is a 5-membered heteroarylene. In certain embodiments, the 5-membered heteroarylene is a triazole. In certain embodiments, the triazole is a 1,2,3- triazole moiety. [0093] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (IIIa) or (IIIb):
Figure imgf000025_0003
(IIIa) (IIIb) wherein: -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2-, where each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1-C6)alkyl, and R21 is H or optionally substituted (C1-C6)alkyl; and -A1- is arylene, substituted arylene, heteroarylene, or substituted heteroarylene. [0094] In some embodiments of formula (IIIa) or (IIIb), Z11 is -S-. [0095] In some embodiments, Z11 is -C(R22)2-. In some embodiments, Z11 is -CH2-. [0096] In certain embodiments, Z11 is -C(R22)2, where at least one R22 is H. In certain embodiments, both R22 are H. In certain embodiments Z11 is -O-. In certain embodiments, Z11 is -S-. In certain embodiments cases, Z11 is -N(R21), where R21 is H or (C1-C3)alkyl. [0097] In certain embodiments, -A1- is triazole. [0098] In certain embodiments, Z1 is -C(R22)2-triazole-. In certain embodiments, Z1 is: * *
Figure imgf000026_0001
. In certain embodiments, Z1 is:
Figure imgf000026_0002
. [0099] In certain embodiments of formula (Iia), (Iia-1) or (Iia-2), Z1 is Z11. In certain embodiments, Z11 is -C(R22)2. In certain embodiments, at least one R22 is H. In certain embodiments, both R22 are H, and Z11 is -CH2-. In certain cases Z11 is -O-. In certain embodiments, Z11 is -S-. In certain other cases, Z11 is -N(R21), where R21 is H or (C1-C3)alkyl. [0100] In certain embodiments of formula (Iia), (Iia-1) or (Iia-2), Z1 is monocyclic 5 or 6-membered heteroaryl or aryl. In certain embodiments, Z1 is
Figure imgf000026_0003
. In certain embodiments, Z1
Figure imgf000026_0004
. [0101] In certain embodiments of formula (Iia), (Iia-1) or (Iia-2), Z1 is selected from -O-, -S-,
Figure imgf000026_0005
wherein: X1 is O or S; t is 0 or 1; R21 and each R23 is independently selected from H, and optionally substituted (C1-C6)alkyl (e.g., C(1-3)-alkyl, such as methyl); and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl. [0102] In certain embodiments of formula (Iia), (Iia-1), or (Iia-2), Z1 is optionally substituted (C1- C6)alkyl. In certain embodiments, of Z1 the alkyl is methyl. In certain embodiments, of Z1, the alkyl is ethyl. In certain embodiments, of Z1, the alkyl is propyl. In certain embodiments, of Z1, the alkyl is butyl. In certain embodiments, of Z1, the alkyl is pentyl. In certain embodiments, of Z1, the alkyl is hexyl. [0103] In certain embodiments, the ASGPR binding moiety (X) of formula (Iia-1) is selected from one of the following structures:
Figure imgf000027_0001
[0104] In some embodiments of formula (Iia-2), Z1 is in a beta configuration and X is of formula (IIIb-2):
Figure imgf000027_0002
(IIIb-2) wherein: -A1- is arylene, substituted arylene, heteroarylene, or substituted heteroarylene. [0105] In some embodiments of formula (IIIb-2), A1 is a triazole. In some embodiments of formula (IIIb-2), X is of formula (XA-4). [0106] In some embodiments of formula (Iia-1), Z1 is in a alpha configuration at the 1-position carbon of the galactosamine ring. In some embodiments of formula (Iia-1), Z1 is S, and each X is of formula (XA-1). In some embodiments of formula (Iia-1), each X is of formula (XA-2). In some embodiments of formula (Iia-1), each X is of formula (XA-3). In some embodiments of formula (Iia-1), each X is of formula (XA-4). In some embodiments of formula (Iia-1), each X is of formula (XA-5). [0107] In certain embodiments, the compound of formula (Iia-2) is selected from one of the following structures:
Figure imgf000027_0003
Figure imgf000028_0001
[0108] In some embodiments of formula (Iia-2), each X is of formula (XB-1). [0109] In some embodiments of formula (Iia-2), each X is of formula (XB-2). [0110] In some embodiments of formula (Iia-2), each X is of formula (XB-3). [0111] In some embodiments of formula (Iia-2), each X is of formula (XB-4). [0112] In some embodiments of formula (Iia-2), Z1 is in an alpha configuration and X is of formula (IIIb-1):
Figure imgf000028_0002
wherein -A1- is arylene, substituted arylene, heteroarylene, or substituted heteroarylene. [0113] In certain embodiments of formula (IIIb-1), A1 is an optionally substituted heteroarylene. In certain embodiments, the heteroarylene is a 5 or 6-membered heteroarylene. In certain embodiments, the heteroarylene is a 5-membered heteroarylene. In certain embodiments, the 5-membered heteroarylene is a triazole. In certain embodiments, the triazole is a 1,2,3-triazole moiety. [0114] In certain embodiments, the X of formula (IIIb-1) is selected from one of the following structures:
Figure imgf000028_0003
[0115] In some embodiments of formula (IIIb-1), each X is of formula (XC-1). [0116] In some embodiments of formula (IIIb-1), each X is of formula (XC-2). [0117] Exemplary ligand moieties that bind ASGPR, and synthons thereof, which can be utilized in the compounds of this disclosure are shown in Tables 1-5. In certain embodiments, the compound of formula (Iia) is a compound shown in Table 1:
Figure imgf000029_0003
[0118] In some embodiments of any one of X1-X5.1, Z1 is in the alpha configuration such that the ASGPR binding moiety X1-X5.1 is derived from formula (Iia-2):
Figure imgf000029_0001
2-linked ASGPR ligand moieties [0119] In some embodiments, the ASGPR binding moiety (X) is linked via the 2-postion of the sugar analog. In some embodiments, the ASGPR binding moiety (X) has a reduced ring carbon at the 1- position relative to a galactosamine derived sugar. In some embodiments, the ASGPR binding moiety (X) of the bifunctional molecules of this disclosure is described by formula (Iib):
Figure imgf000029_0002
wherein R1, R3, R4, R6, R11, and Z1 are as defined herein. [0120] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure are described by formula (Iib’):
Figure imgf000030_0001
wherein R3-R4, R6, and Z1 are as defined herein. [0121] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure are described by formula (Iva):
Figure imgf000030_0002
wherein R1, R11, and Z1 are as defined herein. [0122] In some embodiments of formulae (Iib), (Iib’) or (Iva), Z1 is selected from optionally substituted –(C(R22)2)q-heteroarylene,
Figure imgf000030_0003
, wherein q is 0 or 1. [0123] In some embodiments of formulae (Iib), (Iib’) or (Iva), Z1 is optionally substituted – (C(R22)2)q-triazole wherein q is 0 or 1. [0124] In some embodiments of formulae (Iib),
Figure imgf000030_0004
some embodiments,
Figure imgf000030_0005
[0125] In some embodiments of formulae (Iib), 23
Figure imgf000030_0006
, wherein R is H, or C(1-3)-alkyl. [0126] In some embodiments of formulae (Iib), (Iib’) or (Iva), Z1 is -NR23CO-, wherein R23 is H or C(1-3)-alkyl. [0127] In certain embodiments of formula of formulae (Iib), (Iib’) or (Iva), Z1 is selected from optionally substituted –(C(R22)2)q-heteroaryl,
Figure imgf000031_0001
, wherein q is 0 or 1. [0128] In certain embodiments of formula of formulae (Iib), (Iib’) or (Iva), Z1 is optionally substituted –(C(R22)2)q-triazole wherein q is 0 or 1. In certain embodiments,
Figure imgf000031_0002
[0129] In certain cases of formulae (Iib), , wh 23
Figure imgf000031_0003
erein R is H, or C(1-3)-alkyl. [0130] In certain cases of formulae (Iib), (Iib’) or (Iva), Z1 is -NR23CO-, wherein R23 is H or C(1-3)- alkyl. [0131] In certain embodiments of formula of formulae (Iib), (Iib’) or (Iva), Z1 is monocyclic 5 or 6- membered heteroarylene or arylene. In certain embodiments,
Figure imgf000031_0004
[0132] In certain embodiments of formula of formulae (Iib), (Iib’) or (Iva), Z1 is selected from -O-, -
Figure imgf000031_0005
wherein: X1 is O or S; t is 0 or 1; R21 and each R23 is independently selected from H, and optionally substituted (C1-C6)alkyl (e.g., C(1-3)-alkyl, such as methyl); and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl. [0133] In certain embodiments, the compound of formula of formulae (Iib), (Iib’) or (Iva) is selected from one of the following structures:
Figure imgf000031_0006
, , wherein R1A is independently H or (C1-3)alkyl. [0134] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ivb) or (Ivc): OH OH R11 R11 HO O HO O HO R1 HO R1 Z11 A1 A2 * * (Ivb) (Ivc), wherein: -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2; -A1- and -A2- are optionally substituted arylene or optionally substituted heteroarylene; each R21 is independently selected from H, and optionally substituted (C1-C6)alkyl; and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl. In some embodiments of formula (Ivb) or (Ivc), R1 is H. [0135] In some embodiments, R2 is –Z1–*, and R11 is a group of the formula -CH2O- that forms a bridge (i.e., is cyclically linked) to the 1-position carbon atom on the sugar ring. [0136] In some embodiments, –Z1–* or –Z1–L- comprises
Figure imgf000032_0001
[0137] In certain embodiments of formula (Iib), R11 is H and the compound is of Table 2:
Figure imgf000032_0002
[0138] In certain embodiments, the compound of formula (Iib) is a compound shown in Table 3: In certain embodiments, the compound of formula (Iib), the configuration at C1 (i.e., R1) is alpha. In certain embodiments, the compound of formula (Iib), the configuration at C1 (i.e., R1) is beta.
Figure imgf000033_0001
Figure imgf000034_0002
[0139] In certain embodiments, the compound of formula (Id’) is a compound shown in Table 4:
Figure imgf000034_0003
[0140] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ivb-1) or (Ivc-1):
Figure imgf000034_0001
(Ivb-1) (Ivc-1), wherein R11 is the bridging moiety that connects the 5-position carbon to the 1-position carbon. [0141] In some embodiments of formulae (Ivb), or (Ivb-1), Z11 is -C(R22)2. In certain embodiments, at least one R22 is H. In certain embodiments, both R22 are H. In certain embodiments, Z11 is -O-. In certain embodiments, Z11 is -S-. In certain embodiments, Z11 is -N(R21), where R21 is H or (C1-C3)alkyl. [0142] In certain embodiments of formulae (Ivb), (Ivc), (Ivb-1) or (Ivc-1), -A1- and -A2- are each independently an optionally substituted heteroarylene. In certain embodiments, the heteroarylene is a 5 or 6-membered heteroarylene. In certain embodiments, the heteroarylene is a 5-membered heteroarylene. In certain embodiments, the heteroarylene is a 6-membered heteroarylene. [0143] In some embodiments of formulae (Ivb), or (Ivb-1), the A1 ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan. In certain embodiments, the A1 ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine. In certain embodiments, the A1 ring is triazole. In certain embodiments, the A1 ring is pyridine. In certain embodiments, the A1 ring is pyrimidine. In certain embodiments, the A1 ring is thiadiazole. In certain embodiments, the A1 ring is a 5 or 6- membered arylene or heteroarylene that is further substituted with one or more substituents. In certain embodiments, the A1 ring is further substituted with one or more substituents selected from halogen, (C1- C6)alkyl and substituted (C1-C6)alkyl (e.g., CF3). [0144] In some embodiments of any one of formulae (Ivc), or (Ivc-1), the A2 ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan. In certain embodiments, the A2 ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine. In certain embodiments, the A2 ring is triazole. In certain embodiments, the A2 ring is pyridine. In certain embodiments, the A2 ring is pyrimidine. In certain embodiments, the A2 ring is thiadiazole. In certain embodiments, the A2 ring is a 5 or 6- membered arylene or heteroarylene that is further substituted with one or more substituents. In certain embodiments, the A2 ring is further substituted with one or more substituents selected from halogen, (C1- C6)alkyl and substituted (C1-C6)alkyl (e.g., CF3). [0145] In certain embodiments of formulae (Ivb) or (Ivb-1), -Z11-A1- is a monocyclic 5 or 6- memebered heteroarylene of one of the following structures:
Figure imgf000035_0001
[0146] In certain embodiments of formulae (Ivc) or (Ivc-1), -A2- is a monocyclic 5 or 6-membered heteroarylene of the following structure:
Figure imgf000035_0002
. [0147] It is understood that a variety of substituents can be utilized to connect a particular -Z11-A1- group to an adjacent linker. In certain embodiments of formulae (Ivb) or (Ivb-1), -Z11-A1- is a monocyclic 5 or 6-membered heteroarylene that is attached to a linking moiety as shown in one of the following structures:
Figure imgf000036_0001
[0148] In certain embodiments of formulae (Ivc) or (Ivc-1), -Z11-A1- is a monocyclic 5 or 6- membered heteroarylene that is attached to a linking moiety as shown in one of the following structures:
Figure imgf000036_0002
. [0149] In some embodiments of the compound of formula of formulae (Iib), or (Iva)-(Ivc), R1 is H, such that the compound of formula of formulae (Iib), or (Iva)-(Ivc) has no non-hydrogen substituents at the 1-position of the sugar ring. [0150] In some embodiments, the compound of formula (Iib) is of any one of formulae (Ivd)-(Ivg):
Figure imgf000036_0003
(Ivf), and (Ivg), wherein the A1 and A2 rings, R6, R4, R3, R11, and R21 are as defined herein. [0151] In some embodiments of any one of formulae (Ivd)-(Ivg), the A1 ring is a 5 or 6-membered arylene or heteroarylene. In certain embodiments, the A1 ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, imidazole, and furan. In certain embodiments, the A1 ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine. In certain embodiments, the A1 ring is triazole. In certain embodiments the A1 ring is pyridine In certain embodiments the A1 ring is pyrimidine In certain embodiments, the A1 ring is thiadiazole. In certain embodiments, the A1 ring is pyrazine. In certain embodiments, the A1 ring is a 5 or 6-membered arylene or heteroarylene that is further substituted with one or more substituents. In certain embodiments, the A1 ring is further substituted with one or more substituents selected from halogen, (C1-C6)alkyl and substituted (C1-C6)alkyl (e.g., CF3). [0152] In some embodiments of any one of formulae (Ivd)-(Ivg), the A2 ring is a 5 or 6-membered arylene or heteroarylene. In certain embodiments, the A2 ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan. In certain embodiments, the A2 ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine. In certain embodiments, the A2 ring is triazole. In certain embodiments, the A2 ring is pyridine. In certain embodiments, the A2 ring is pyrimidine. In certain embodiments, the A2 ring is thiadiazole. In certain embodiments, the A2 ring is a 5 or 6-membered arylene or heteroarylene that is further substituted with one or more substituents. In certain embodiments, the A2 ring is further substituted with one or more substituents selected from halogen, (C1- C6)alkyl and substituted (C1-C6)alkyl (e.g., CF3). [0153] In some embodiments of any one of formulae (Ivd)-(Ivg),the A1 or A2 ring is absent. [0154] In some embodiments of any one of formulae (Ivd)-(Ivg),the A1 or A2 ring is phenylene or substituted phenylene. [0155] In some embodiments of formula (Ivd), the A2 ring is a 5 or 6-membered heteroarylene. In certain cases of formula (Ivd), the A2 ring is a 5-membered heteroarylene. In certain embodiments of formula (Ivd), the A2 ring is triazole. In certain embodiments of (Ivd), the A2 ring is absent. [0156] In some embodiments of formula (Ive), the A1 ring is a 5 or 6-membered heteroarylene and R21 is H. In certain embodiments of formula (Ive), the A ring is triazole. In certain cases of formula (Ive), the A1 ring is pyridine. In certain cases of formula (Ive), the A1 ring is pyrimidine. In certain cases of formula (Ive), the A1 ring is thiadiazole. In some embodiments of formula (Ive), the A1 ring is absent and R21 is H or optionally substituted acyl. In certain embodiments, R21 is -COCH3. In certain embodiments, R21 is H. [0157] In some embodiments of formula (Ivf), the A1 ring is a 5 or 6-membered heteroarylene. In certain cases of formula (Ivf), the A1 ring is a 5-membered heteroarylene. In certain embodiments of formula (Ivf), the A1 ring is triazole. In certain embodiments of (Ivf), the A1 ring is absent. [0158] In some embodiments of formula (Ivg), the A2 ring is a 5 or 6-membered heteroarylene. In certain cases of formula (Ivg), the A2 ring is a 5-membered heteroarylene. In certain embodiments of formula (Ivg), the A2 ring is triazole. In certain embodiments of (Ivg), the A2 ring is absent. [0159] In certain embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by any one of formulae (Ivh)-(Ivk):
Figure imgf000038_0001
(Ivj), and (Ivk) wherein: R6, R4, R3, and R21 are as defined herein; Y1-Y3 are each independently N or CR25; and R24 and R25 are each independently selected from H, optionally substituted C(1-6)-alkyl, optionally substituted fluoroalkyl, and halogen. [0160] In certain embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by any one of formulae (Ivl)-(Ivm):
Figure imgf000038_0002
wherein: R6, R4, R3, and R21 are as defined herein; Y1-Y3 are each independently N or CR25; Y4 is N or CR24; Y5 is S, O, or NH; and R24 and R25 are each independently selected from H, optionally substituted C(1-6)-alkyl, optionally substituted fluoroalkyl, and halogen. [0161] In some embodiments of formula (Ivi) at least one of Y1 to Y3 is N. [0162] In certain embodiments, at least two of Y1 to Y3 are N. [0163] In certain embodiments, Y1 and Y4 are N. [0164] In certain embodiments, Y1 and Y3 are N and Y2 is CR25. [0165] In certain embodiments, Y1 and Y2 are N and Y3 is CR25. [0166] In certain embodiments, Y1 and Y2 are CR25 and Y3 is N. [0167] In certain embodiments of any one of formulae (Ivd)-(Ivk), or (Ivd)-(Ivm), R6 is H. [0168] In some embodiments of any one of formulae (Ivd)-(Ivk), or (Ivd)-(Ivm), R4 and R3 are each H. In certain embodiments, at least one of R4-R3 is a promoiety. In certain embodiments, R4 and R3 are cyclically linked to form a promoiety (e.g., as described herein). [0169] In some embodiments, the compound of formula (Ivi) is of formula (Ivi-1):
Figure imgf000039_0001
[0170] wherein R24 and R25 are independently selected from H, halogen, (C1-C6)alkyl and substituted (C1-C6)alkyl (e.g., CF3).In some embodiments of formula (Ivi)-(Ivi-1), R25 is H. In certain embodiments, R25 is C(1-3)-alkyl, or C(1-3)-fluoroalkyl. In certain embodiments, the fluoroalkyl is CF3. In some embodiments of formula (Ivi) or (Ivi-1), R24 is H. In certain embodiments, R24 is C(1-3)-alkyl, or C(1-3)- fluoroalkyl. In certain embodiments,, the fluoroalkyl is CF3. [0171] In some embodiments, the compound of formula (Ivi-1) is of formula (XD):
Figure imgf000039_0002
[0172] In some embodiments, the compound of formula (Ivk-1) is of formula (XE):
Figure imgf000039_0003
[0173] In certain embodiments, the compound of formula (Ivl) is of formula (Ivl-1):
Figure imgf000039_0004
wherein: R6, R4, R3, and R21 are as defined herein; Y1-Y4 are each independently N or CR25; Y5 is S, O, or NH; and each R25 is independently selected from H, optionally substituted C(1-6)-alkyl, optionally substituted fluoroalkyl, and halogen. [0174] In certain embodiments, each R25 is H. [0175] In certain embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by one of the following structures:
Figure imgf000040_0001
. [0176] In certain embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by one of the following structures:
Figure imgf000040_0002
[0177] In certain embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by one of the following structures: *
Figure imgf000040_0003
[0178] In certain embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by one of the following structures: *
Figure imgf000040_0004
[0179] In certain embodiments of formula (Iib), R1 R3, R4, and R11 are H, and R6 is OH:
Figure imgf000041_0001
wherein Z1 is -NH-, -CH2-, -S- or -O-. [0180] In certain embodiments of formula (Iib’), R3, R4 are H, and R6 is OH:
Figure imgf000041_0002
wherein Z1 is -NH-, -CH2-, -S-, -O-, triazole,
Figure imgf000041_0003
6-linked ASGPR ligand moieties [0181] In some embodiments, the ASGPR binding moiety (X) is linked via the 6-postion of the sugar analog. In some embodiments, the ASGPR binding moiety (X) has a reduced ring carbon at the 1- position relative to a galactosamine derived sugar. [0182] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Iic): *
Figure imgf000041_0004
wherein R1-R4 and Z1 are as defined herein. [0183] In certain embodiments of formula (Iic), Z1 is selected from -O-, -S-, -CONR21-, and optionally substituted –(C(R22)2)q- heteroarylene, wherein q is 0 or 1. In certain embodiments, Z1 is -O-. In certain other cases, Z1 is optionally substituted –(C(R22)2)q-triazole wherein q is 0 or 1. [0184] In certain embodiments,
Figure imgf000041_0005
[0185] In certain embodiments of formula (Iic), Z1 is -Z11-A1-, wherein -A1- is or optionally substituted -A1- or optionally substituted arylene. In certain embodiments, -A1- is an optionally substituted heteroarylene. In certain embodiments, the heteroarylene is a 5 or 6-membered heteroarylene. In certain embodiments, the heteroarylene is a 5-membered heteroarylene. In certain embodiments, the 5-membered heteroarylene is a triazole. In certain embodiments, the triazole is a 1,2,3-triazole moiety. In certain embodiments, Z11 is -C(R22)2. In certain embodiments, at least one R22 is H. In certain embodiments, both R22 are H. In certain cases Z11 is -O-. In certain embodiments, Z11 is -S-. In certain other cases, Z11 is -N(R21), where R21 is H or (C1-C3)alkyl. In certain embodiments, Z1 is -C(R22)2- * triazole-. In certain embodiments, Z1 is:
Figure imgf000042_0001
. [0186] In certain embodiments of formula (Iic), Z1 is Z11. In certain embodiments, Z11 is -C(R22)2. In certain embodiments, at least one R22 is H. In certain embodiments, both R22 are H, and Z11 is -CH2-. In certain cases Z11 is -O-. In certain embodiments, Z11 is -S-. In certain other cases, Z11 is -N(R21), where R21 is H or (C1-C3)alkyl. [0187] In certain embodiments of formula (Iic), Z1 is monocyclic 5 or 6-membered heteroarylene or arylene. In certain embodiments,
Figure imgf000042_0002
[0188] In certain embodiments of formula (Iic), Z1 is selected from -O-, -S-, -C(R22)2-, -N(R21) -
Figure imgf000042_0003
wherein: X1 is O or S; t is 0 or 1; R21 and each R23 is independently selected from H, and optionally substituted (C1-C6)alkyl (e.g., C(1-3)-alkyl, such as methyl); and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl. [0189] In certain embodiments, the compound of formula (Iic) is the following structure:
Figure imgf000042_0004
. [0190] In certain embodiments, the compound of formula (Iic) is the following structure:
Figure imgf000043_0001
. [0191] In certain embodiments of formula (Iic), R11 is H and the compound is of Table 5:
Figure imgf000043_0003
[0192] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Iid):
Figure imgf000043_0002
wherein: R6, R4, R3 and Z1 are as defined herein; Y6 and Y5 are each independently selected from -O-, -S-, NR21-, and -C(R22)2; R21 is selected from H, optionally substituted (C1-C6)alkyl, and -C(O)R22; each R22 is independently selected from H, halogen and optionally substituted (C1-C6)alkyl; and ring B is a 5 or 6-membered optionally substituted cyclic group. In some embodiments of formula (Iid), Y5 is connected to the sugar ring via an alpha configuration. In some embodiments of formula (Iid), Y5 is connected to the sugar ring via a beta configuration. [0193] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Iid’):
Figure imgf000044_0001
wherein: R6, R4, R3 and Z1 are as defined herein; Y5 and Y6 are each independently selected from -O-, -S-, NR21-, and -C(R22)2; R21 is selected from H, optionally substituted (C1-C6)alkyl, and -C(O)R22; each R22 is independently selected from H, halogen and optionally substituted (C1-C6)alkyl; and ring B is a 5 or 6-membered optionally substituted cyclic group. [0194] In some embodiments of formula (Iid)-(Iid’) Y5 is O. In certain embodiments, Y5 is S. In certain embodiments, Y5 is -NR21-. In certain embodiments, Y5 is -C(R22)2 and each R22 is H. [0195] In some embodiments of formula (Iid)-(Iid’) Y6 is -NR21- where R21 is H. In certain embodiments, Y6 is -NR21- where R21 is -C(O)R22. In certain embodiments, R22 is methyl. [0196] In some embodiments of formula (Iid)-(Iid’) the B ring is a 5 or 6-membered heterocycle. In certain embodiments, the B ring is a 5-membered heterocycle. In certain embodiments, the B ring is a 6- membered heterocycle. [0197] In some embodiments of formula (Iid)-(Iid’) Z1 is Z11, where Z11 is selected from -O-, -S-, NR21-, and -C(R22)2. In certain embodiments, Z1 is -O-. In certain embodiments, Z1 is -S-. In certain embodiments, Z1 is NR21 where R21 is H. In certain embodiments, Z1 is -C(R22)2 where each R22 is H. [0198] In some embodiments of formula (Iid)-(Iid’) Z1 is optionally substituted Z11-heteroarylene or optionally substituted Z11-arylene. In some embodiments, Z1 is CH2-heteroarylene or CH2-arylene. In some embodiments of formula (Iid)-(Iid’) Z1 is optionally substituted amide. In some embodiments of formula (Iid)-(Iid’) Z1 is optionally substituted sulfonamide. In some embodiments of formula (Iid)- (Iid’) Z1 is optionally substituted urea or optionally substituted thiourea. [0199] In some embodiments, the compound of formula (Iid)-(Iid’) has one of the following structures:
Figure imgf000044_0002
, . [0200] In certain embodiments of any one of formulae (Iia), (Iib) or (Iid), R6 is OH. In certain other cases, R6 is -OC(O)R. In certain embodiments, R6 is -C(O)NHR, where R is an optionally substituted alkyl. In certain embodiments, R terminates in an alkenyl or an alkynyl group. In certain other cases R6 is optionally substituted triazole. In certain embodiments, the triazole is of the following structure:
Figure imgf000045_0001
. [0201] In certain embodiments of (Iia), and (Iic), R2 is -NHCOCH3. In certain other embodiments, R2 is –NHCOCF3. In certain other embodiments, R2 is –NHCOCH2CF3. In certain embodiments, R2 is – OH. In certain other cases, R2 is an optionally substituted triazole. In certain embodiments, the triazole in of the following structure:
Figure imgf000045_0002
. [0202] In certain embodiments when R6 or R2 is a substituted triazole, the triazole is a 1,2,3-trizole, and the substituent is at the 4 or 5-position. In certain embodiments, the substituent on the triazole moiety includes but is not limited to, an optionally substituted (C1-6)alkyl, optionally substituted (C1- 6)alkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkaryl, and an optionally substituted alkyheteroaryl. It will be understood that any convenient substituent can be included in the triazole moiety, see, e.g., triazole moieties disclosed in Mamidayala et al, J. Am. Chem. Soc.2012, 134, 1978-1981. [0203] It is understood that the Z1, Z11, and Z11-Ar linking moieties can be considered part of the X group of formula (I). In the ASGPR binding moieties (X) as described herein, -Z1- can be linked to an - L1- moiety (e.g., of the linker as described herein) via a variety of bonds and linking moieties, depending on the method of preparation. In some embodiments, the subject compounds comprise a -Z1-L1- moiety selected from:
Figure imgf000045_0003
wherein each R21 is independently selected from H, and optionally substituted (C1-C6)alkyl; each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1-C6)alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 0 to 6. [0204] In some embodiments, the subject compounds comprise a -Z1-L1- moiety selected from:
Figure imgf000046_0001
wherein each R21 is independently selected from H, and optionally substituted (C1-C6)alkyl; each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1-C6)alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6. [0205] In certain embodiments, the Z1-L1- group is
Figure imgf000046_0002
, and o is 1 or 2. [0206] In certain embodiments, the Z1-L1- group 22
Figure imgf000046_0003
each R is H, and p is 1 or 2.
Figure imgf000046_0007
[0209] In certain embodiments, the Z1-L1- group is
Figure imgf000046_0004
. [0210] In certain embodiments, the Z1-L1- group
Figure imgf000046_0005
are each independently 1-3. [0211] In certain embodiments, the Z1-L1- group i
Figure imgf000046_0006
[0212] In certain embodiments, the Z1-L1- group i
Figure imgf000047_0001
are each independently is 1-3. [0213] In certain embodiments, the Z1-L1- group is
Figure imgf000047_0002
, where x is 0-3. [0214] In certain embodiments, the Z1-L1- group
Figure imgf000047_0003
Figure imgf000047_0004
[0215] In certain embodiments, the Z1-L1- group is
Figure imgf000047_0005
, where R21 is H, and z is 1-4. R21 [0216] In certain embodiments, the Z1-L1- group is
Figure imgf000047_0006
, where R21 is H, and z1 is 1-4. [0217] In certain embodiments, the Z 1 -L 1 - group is
Figure imgf000047_0007
, where each R22 is H, and q is 0-3. In certain embodiments, the Z 1 -L 1 - group is
Figure imgf000047_0008
, where each R22 is H, and q is 1-3. [0218] In certain embodiments, the Z1-L1- group is
Figure imgf000047_0009
, where q is 1-3. [0219] In certain embodiments, the subject compounds comprise a -Z1-L- group selected from:
Figure imgf000047_0010
[0220] In certain embodiments, the Z 1 -L 1 - group is q O , where q is 1-3. In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. [0221] In certain embodiments, the Z1-L1- group i
Figure imgf000048_0001
. [0222] In certain embodiments, the Z1-L1- group i
Figure imgf000048_0002
. [0223] In certain embodiments, the Z1-L1- group is
Figure imgf000048_0003
. [0224] In certain embodiments, -Z1-L1- comprises an optionally substituted -NH-heteroarylene-. In certain embodiments the heteroarylene is a triazole. In certain embodiments, the heteroarylene is pyridine. In certain embodiments, the heteroarylene is pyrimidine. In certain embodiments, the heteroarylene is thiadiazole. [0225] In certain embodiments, the -Z1-L1- comprises a group selected from:
Figure imgf000048_0004
wherein each R21 is independently selected from H, optionally substituted (C1-C6)alkyl, and optionally substituted acyl; and R24 and R25 are each independently selected from H, optionally substituted C(1-6)-alkyl, optionally substituted fluoroalkyl, and halogen. [0226] In certain embodiments, the -Z1-L1- comprises a group selected from:
Figure imgf000048_0005
wherein R24 and R25 are each independently selected from H, optionally substituted C(1-6)- alkyl, optionally substituted fluoroalkyl, and halogen; and each R21 is independently selected from H, optionally substituted (C1-C6)alkyl, and optionally substituted acyl. [0227] In certain embodiments, R21 is H. In certain embodiments, R24 is C(1-3)-alkyl, or C(1-3)- fluoroalkyl. In certain embodiments, the fluoroalkyl is CF3. In certain embodiments, R25 is C(1-3)-alkyl, or C(1-3)-fluoroalkyl. In certain embodiments, the fluoroalkyl is CF3. [0228] In certain embodiments, -
Figure imgf000049_0001
[0229] It is understood that a variety of substituents and chemistries can be utilized to connect a particular X ligand moiety (e.g., as described herein) to an adjacent linker. In some embodiments, a linking moiety of the linker comprises a triazole that derives from a Click chemistry conjugation. In certain embodiments, the ASGPR ligand moiety (X) is attached to a linking moiety as shown in one of the following structures: , ,
Figure imgf000049_0002
[0230] In certain embodiments of formula (Iib), R1 R3, R4, and R11 are H, and R6 is OH:
Figure imgf000050_0001
wherein Z1 is triazole, -NH-heteroaryl (e.g., -NH- attached to pyridine, pyrazine, or pyrimidine) , -NH-, - O-, or -CH2- , and/or Z1 is attached to a linking moiety as shown in one of the following structures:
Figure imgf000050_0002
[0231] In certain embodiments, of formula (Iib), R1 R3, R4, and R11 are H, and R6 is OH:
Figure imgf000050_0003
wherein Z1 is attached to a linking moiety as shown in one of the following structures:
Figure imgf000051_0001
Additional Exemplary ASGPR Binding Moieties [0232] The ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ia):
Figure imgf000051_0002
wherein: R1 is selected from –OH, –OC(O)R, -C(O)NHR, –Z1–*, and optionally substituted triazole, where R is optionally substituted C1-6 alkyl or optionally substituted aryl; R2 is selected from–NHCOCH3, –NHCOCF3, –NHCOCH2CF3, –OH, optionally substituted triazole, and –Z1–*; R3 is selected from –H, –OH, –CH3, –OCH3, –OCH2CH=CH and –Z1–*; one of R1 to R3 is –Z1–*, wherein “ * ” represents a point of attachment of Z1 to the linker (L); R4 and R5 are each independently selected from H, and a promoiety; or R4 and R5 are cyclically linked to form a promoiety; R11 is H, or a group that forms a bridge (e.g., a 2 atom bridge cyclically linked) to the 1-position carbon atom; Z1 is a linking moiety selected from Z11, optionally substituted Z11-heteroaryl, optionally substituted Z11-aryl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted alkyl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiourea; Z11 is selected from -O-, -S-, NR21-, and -C(R22)2, each R21 is independently selected from H, and optionally substituted (C1-C6)alkyl; and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl. [0233] In some embodiments of formula (Ia): i) when n is 3, R1 is OH, R2 is –NHCOCH3, R4-R5 are H, and R3 is Z1, then Z1 is not O; ii) when n is 2 or 3, R1 is OAc, R2 is –NHCOCH3, R4-R5 are Ac, and R3 is Z1, then Z1 is not O; iii) when n is 2 or 3, R1 is OBz, R2 is –NHCOCH3, R4-R5 are Bz, and R3 is Z1, then Z1 is not O; iv) when n is 3, R1 is OH, R2 is –NHCOCH3, R4-R5 are H, and R3 is Z1, and Z11 is O, then L comprises a backbone of at least 16 consecutive atoms to a branching point; v) when n is 3, R1 is Z1, where Z1 is O, and R4-R5 are H, then R3 is not -CH3 –OCH3,or – OCH2CH=CH; and vi) when R11 is a group of the formula -CH2O- that forms a bridge (i.e., is cyclically linked) to the 1-position carbon atom on the sugar ring, R2 is –NHCOCH3, R4-R5 are H, then R1 and R3 are not Z1. [0234] In some embodiments the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ia-1):
Figure imgf000052_0001
(Ia-1) wherein: R1 is selected from –OH, –OC(O)R, -C(O)NHR, –Z1–*, and optionally substituted triazole, where R is optionally substituted C1-6 alkyl or optionally substituted aryl; R2 is selected from–NHCOCH3, –NHCOCF3, –NHCOCH2CF3, –OH, optionally substituted triazole, and –Z1–*; R3 is selected from –H, –OH, –CH3, –OCH3, –OCH2CH=CH and –Z1–*; one of R1 to R3 is –Z1–*, wherein “ * ” represents a point of attachment of Z1 to the linker (L); R4 and R5 are each independently selected from H, and a promoiety (e.g., an ester promoiety); Z1 is a linking moiety selected from Z11, optionally substituted Z11-heteroaryl, optionally substituted Z11-aryl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiourea; Z11 is selected from -O-, -S-, NR21-, and -C(R22)2, each R21 is independently selected from H, and optionally substituted (C1-C6)alkyl; and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1-C6)alkyl. [0235] In some embodiments of formula (Ia-1): i) when n is 3, R1 is OH, R2 is –NHCOCH3, R4-R5 are H, and R3 is Z1, then Z1 is not O; ii) when n is 2 or 3, R1 is OAc, R2 is –NHCOCH3, R4-R5 are Ac, and R3 is Z1, then Z1 is not O; iii) when n is 2 or 3, R1 is OBz, R2 is –NHCOCH3, R4-R5 are Bz, and R3 is Z1, then Z1 is not O; iv) when n is 3, R1 is OH, R2 is –NHCOCH3, R4-R5 are H, and R3 is Z1, and Z1 is O, then L comprises a backbone of at least 16 consecutive atoms to a branching point; and/or v) when n is 3, R1 is Z1, where Z1 is O, and R4-R5 are H, then R3 is not -CH3. [0236] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ib):
Figure imgf000053_0001
wherein R1, R2, R4, R5 and Z1 are as defined herein. In some embodiments of formula (Ib), R1 is selected from –OH, –OC(O)R, and -C(O)NHR; and R2 is selected from –NHCOCH3, –NHCOCF3, and – NHCOCH2CF3 [0237] In some embodiments of Formula (Ib), Z1 is in an alpha configuration
Figure imgf000053_0002
. [0238] In some embodiments of Formula (Ib), Z1 is in an alpha configuration and X is of the following formula:
Figure imgf000053_0003
. [0239] In some embodiments of Formula (Ib), Z1 is in an alpha configuration and X is of the following formula:
Figure imgf000053_0004
. [0240] In some embodiments of Formula (Ib), Z1 is in an alpha configuration and X is of the following formula:
Figure imgf000054_0001
. [0241] In some embodiments of Formula (Ib), Z1 is in an alpha configuration and X is of the following formula:
Figure imgf000054_0002
. [0242] In some embodiments of Formula (Ib), Z1 is in an alpha configuration and X is of the following formula:
Figure imgf000054_0003
. [0243] In some embodiments of Formula (Ib), Z1 is in a beta configuration
Figure imgf000054_0004
. [0244] In some embodiments of Formula (Ib), Z1 is in a beta configuration and X is of the following formula:
Figure imgf000054_0005
. [0245] In some embodiments of Formula (Ib), Z1 is in a beta configuration and X is of the following formula:
Figure imgf000055_0001
. [0246] In some embodiments of Formula (Ib), Z1 is in a beta configuration and X is of the following formula:
Figure imgf000055_0002
. [0247] In some embodiments of Formula (Ib), Z1 is in a beta configuration and X is of the following formula:
Figure imgf000055_0003
. [0248] In some embodiments of Formula (Ib), Z1 is in a beta configuration and X is of the following formula:
Figure imgf000055_0004
. [0249] In certain embodiments of formula (Ib), Z1 is Z11-Ar , wherein Ar is or optionally substituted heteroaryl or optionally substituted aryl. In certain embodiments, Ar is an optionally substituted heteroaryl. In certain embodiments, the heteroaryl is a 5 or 6-membered heteroaryl. In certain embodiments, the heteroaryl is a 5-membered heteroaryl. In certain embodiments, the 5-membered heteroaryl is a triazole. In certain embodiments, the triazole is a 1,2,3-triazole moiety. In certain embodiments, Z11 is -C(R22)2. In certain embodiments, at least one R22 is H. In certain embodiments, both R22 are H. In certain cases Z11 is -O-. In certain embodiments, Z11 is -S-. In certain other cases, Z11 is -NR21, where R21 is H or (C1-3)alkyl. In certain embodiments, Z1 is -C(R22)2-triazole-. In certain * * embodiments, Z1 is:
Figure imgf000056_0001
. In certain embodiments, Z1 is:
Figure imgf000056_0002
. [0250] In certain embodiments of formula (Ib), Z1 is Z11. In certain embodiments, Z11 is -C(R22)2. In certain embodiments, at least one R22 is H. In certain embodiments, both R22 are H, and Z11 is -CH2-. In certain cases Z11 is -O-. In certain embodiments, Z11 is -S-. In certain other cases, Z11 is -NR21, where R21 is H or (C1-3)alkyl. [0251] In certain embodiments of formula (Ib), Z1 is monocyclic 5 or 6-membered heteroaryl or aryl. In certain embodiments, Z1 is
Figure imgf000056_0003
. In certain embodiments, Z1
Figure imgf000056_0004
[0252] In certain embodiments of formula (Ib), Z1 is selected from -O-, -S-, -C(R22)2-, -NR21-, -
Figure imgf000056_0005
wherein: X1 is O or S; t is 0 or 1; R21 and each R23 is independently selected from H, and optionally substituted (C1-C6)alkyl (e.g., C(1-3)-alkyl, such as methyl); and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl. [0253] In certain embodiments of formula (Ib), Z1 is optionally substituted (C1-C6)alkyl. In certain embodiments of Z1 the alkyl is methyl. In certain embodiments of Z1, the alkyl is ethyl. In certain embodiments of Z1, the alkyl is propyl. In certain embodiments of Z1, the alkyl is butyl. In certain embodiments of Z1, the alkyl is pentyl. In certain embodiments of Z1, the alkyl is hexyl. [0254] In certain embodiments, the compound of formula (Ib) is selected from one of the following structures:
Figure imgf000056_0006
wherein R5 is independently H or a promoiety. [0255] In some embodiments, the compound of formula (Ib) is selected from one of the following structures:
Figure imgf000057_0001
wherein R5 and R4 independently H or a promoiety, or R5 and R4 are cyclically linked to form a promoiety; and n1 is an integer from 1 to 6. [0256] In certain embodiments, the compound of formula (Ib) is selected from one of the following structures:
Figure imgf000057_0002
. [0257] In some embodiments, at least one of R4-R5 is of the formula -COCH3, -COCH(CH3)2 or - COC(CH3)3. In certain embodiments, at least one of R4-R5 is of the formula -CH2OCOC(CH3)3. In certain embodiments, at least one of R4-R5 is of the formula -COC(CH3)3 or -CH2OCOC(CH3)3. In certain embodiments, R4 is H and R5 is selected from -COCH3, -COCH(CH3)2, -COC(CH3)3 and - CH2OCOC(CH3)3. In certain embodiments, R4 is H and R5 is -COC(CH3)3. In certain embodiments, R4 is H and R5 is -CH2OCOC(CH3)3. In some embodiments, the compound of formula (Ib) is selected from one of the following structures:
Figure imgf000057_0003
wherein n2 is an integer from 1 to 6. [0258] In some embodiments of the compound of formula (Ib), R5 and R4 are cyclically linked to form a promoiety. In certain embodiments , the compound of formula (Ib) is selected from one of the following structures:
Figure imgf000058_0001
wherein n2 is an integer from 1 to 6; and Y4 is a suitable counterion. [0259] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ic):
Figure imgf000058_0002
wherein R2-R5 and Z1 are as defined herein. [0260] In certain embodiments of formula (Ic), Z1 is selected from -O-, -S-, -CONR21-, and optionally substituted –(C(R22)2)q-heteroaryl, wherein q is 0 or 1. In certain embodiments, Z1 is -O-. In certain other cases, Z1 is optionally substituted –(C(R22)2)q-triazole wherein q is 0 or 1. In certain embodiments,
Figure imgf000058_0003
[0261] In certain embodiments of formula (Ic), Z1 is Z11-Ar , wherein Ar is or optionally substituted heteroaryl or optionally substituted aryl. In certain embodiments, Ar is an optionally substituted heteroaryl. In certain embodiments, the heteroaryl is a 5 or 6-membered heteroaryl. In certain embodiments, the heteroaryl is a 5-membered heteroaryl. In certain embodiments, the 5-membered heteroaryl is a triazole. In certain embodiments, the triazole is a 1,2,3-triazole moiety. In certain embodiments, Z11 is -C(R22)2. In certain embodiments, at least one R22 is H. In certain embodiments, both R22 are H. In certain cases Z11 is -O-. In certain embodiments, Z11 is -S-. In certain other cases, Z11 is -NR21, where R21 is H or (C1-3)alkyl. In certain embodiments, Z1 is -C(R22)2-triazole-. In certain embodiments, Z1 is:
Figure imgf000058_0004
. [0262] In certain embodiments of formula (Ic), Z1 is Z11. In certain embodiments, Z11 is -C(R22)2. In certain embodiments, at least one R22 is H. In certain embodiments, both R22 are H, and Z11 is -CH2-. In certain cases Z11 is -O-. In certain embodiments, Z11 is -S-. In certain other cases, Z11 is -NR21, where R21 is H or (C1-3)alkyl. [0263] In certain embodiments of formula (Ic), Z1 is monocyclic 5 or 6-membered heteroaryl or aryl. In certain embodiments,
Figure imgf000059_0001
[0264] In certain embodiments of formula (Ic), Z1 is selected from -O-, -S-, -C(R22)2-, -NR21-, -
Figure imgf000059_0002
wherein: X1 is O or S; t is 0 or 1; R21 and each R23 is independently selected from H, and optionally substituted (C1-C6)alkyl (e.g., C(1-3)-alkyl, such as methyl); and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl. [0265] In certain embodiments, the compound of formula (Ic) is the following structure:
Figure imgf000059_0003
. [0266] In certain embodiments, the compound of formula (Ic) is the following structure:
Figure imgf000059_0004
. [0267] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Id):
Figure imgf000059_0005
wherein R1, R3-R5 and Z1 are as defined herein. [0268] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Id’):
Figure imgf000060_0001
[0269] In some embodiments, Z1 is selected from optionally substituted –(C(R22)2)q-heteroaryl, and
Figure imgf000060_0002
[0270] In some embodiments, Z1 is optionally substituted –(C(R22)2)q-triazole wherein q is 0 or 1. [0271] In some embodiments, Z1 is
Figure imgf000060_0003
. In some embodiments, Z1
Figure imgf000060_0004
. [0272] In some embodiments,
Figure imgf000060_0005
, wherein R 23 is H, or C(1-3)-alkyl. [0273] In some embodiments, Z1 is -NR23CO-, wherein R23 is H or C(1-3)-alkyl. [0274] In certain embodiments of formula (Id), Z1 is selected from optionally substituted – (C(R22)2)q-heteroaryl,
Figure imgf000060_0006
, wherein q is 0 or 1. [0275] In certain embodiments of formula (Id), Z1 is optionally substituted –(C(R22)2)q-triazole wherein q is 0 or 1. In certain embodiments,
Figure imgf000060_0007
[0276] In certain embodiments,
Figure imgf000060_0008
, wherein R 23 is H, or C(1-3)-alkyl. [0277] In certain embodiments, Z1 is -NR23CO-, wherein R23 is H or C(1-3)-alkyl. [0278] In certain embodiments of formula (Id), Z1 is monocyclic 5 or 6-membered heteroaryl or aryl. In certain embodiments,
Figure imgf000061_0001
[0279] In certain embodiments of formula (Id), Z1 is a monocyclic 5 or 6-memebered heteroaryl of one of the following structures:
Figure imgf000061_0002
[0280] In certain embodiments of formula (Id), Z1 is of one of the following structures:
Figure imgf000061_0003
[0281] In certain embodiments of formula (Id), Z1 is selected from -O-, -S-, -C(R22)2-, -NR21-, -
Figure imgf000061_0004
wherein: X1 is O or S; t is 0 or 1; R21 and each R23 is independently selected from H, and optionally substituted (C1-C6)alkyl (e.g., C(1-3)-alkyl, such as methyl); and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl. [0282] In certain embodiments, the compound of formula (Id) is selected from one of the following structures:
Figure imgf000061_0005
wherein R6 is independently H or (C1-3)alkyl. [0283] In some embodiments of the compound of formula (Id) R3 is H, such that the compound of formula (Id) has no non-hydrogen substituents at the 1-position of the sugar ring. [0284] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ie’):
Figure imgf000062_0001
wherein: R1, R4, R5 and R11 are as defined herein; Z2 is absent or selected from -O-, -S-, NR25-, and -C(R22)2, and optionally substituted Z12-alkyl; ring A is absent or selected from a 5 or 6-membered optionally substituted aryl and a 5 or 6- membered optionally substituted heteroaryl; Z3 is a linking moiety selected from Z12, optionally substituted alkyl, optionally substituted Z12- alkyl, optionally substituted Z12-heteroaryl, optionally substituted Z12-aryl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiourea; and Z12 is selected from -CH2O-, -O-, -S-, -NR26-, and -C(R22)2-; R25 and R26 are each independently selected from H, optionally substituted (C1-C6)alkyl (e.g., C(1- 3)-alkyl, such as methyl), and optionally substituted acyl; and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl. [0285] In some embodiments of formula (Ie’), the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ie’’):
Figure imgf000062_0002
[0286] In some embodiments of formula (Ie’), the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ie):
Figure imgf000062_0003
wherein: R1, R4, R5 and R11 are as defined herein; Z2 is absent or selected from -O-, -S-, NR25-, and -C(R22)2, and optionally substituted Z12-alkyl; ring A is absent or selected from a 5 or 6-membered optionally substituted aryl and a 5 or 6- membered optionally substituted heteroaryl; Z3 is a linking moiety selected from Z12, optionally substituted alkyl, optionally substituted Z12- alkyl, optionally substituted Z12-heteroaryl, optionally substituted Z12-aryl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiourea; and Z12 is selected from -CH2O-, -O-, -S-, -NR26-, and -C(R22)2-; R25 and R26 are each independently selected from H, optionally substituted (C1-C6)alkyl (e.g., C(1- 3)-alkyl, such as methyl), and optionally substituted acyl; and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl. [0287] In some embodiments Z2 is absent. In some embodiments, Z2 is C(R22)2 where R22 is H or optionally substituted (C1-C3)alkyl. In some embodiments, Z2 is -CH2-. In some embodiments, Z2 is NR25 where R25 is selected from H, optionally substituted (C1-C3)alkyl and optionally substituted acyl. In some embodiments, Z2 is -N(COCH3)-. In some embodiments, Z2 is -NH-. In some embodiments, Z2 is - S-. In some embodiments, Z2 is O. [0288] In some embodiments, the compound of formula (Ie) is of any one of formulae (If)-(Ii):
Figure imgf000063_0001
wherein the A ring, R1, R4, R5, R11, Z3 and R25 are as defined herein. [0289] In some embodiments of any one of formulae (Ie)-(Ii), the A ring is a 5 or 6-membered aryl or heteroaryl. In certain embodiments, the A ring is a 5-membered heteroaryl selected from selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan. In certain embodiments, the A ring is a 6-membered heteroaryl selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine. In certain embodiments, the A ring is triazole. In certain embodiments, the A ring is pyridine. In certain embodiments, the A ring is pyrimidine. In certain embodiments, the A ring is thiadiazole. [0290] In some embodiments of any one of formulae (Ie)-(Ii), the A ring is absent. [0291] In some embodiments of any one of formulae (Ie)-(Ii), the A ring is phenyl or substituted phenyl. [0292] In some embodiments of formula (If), the A ring is a 5 or 6-membered heteroaryl. In certain cases of formula (If), the A ring is a 5-membered heteroaryl. In certain embodiments of formula (If), the A ring is triazole. In certain embodiments of (If), the A ring is absent. [0293] In some embodiments of formula (Ig), the A ring is a 5 or 6-membered heteroaryl and R25 is H. In certain embodiments of formula (Ig), the A ring is triazole. In certain embodiments of formula (Ig), the A ring is pyridine. In certain cases of formula (Ig), the A ring is pyrimidine. In certain cases of formula (Ig), the A ring is thiadiazole. In some embodiments of formula (Ig), the A ring is absent and R25 is H or optionally substituted acyl. In certain embodiments, R25 is -COCH3. In certain embodiments, R25 is H. [0294] In some embodiments of formula (Ih), the A ring is a 5 or 6-membered heteroaryl. In certain cases of formula (Ih), the A ring is a 5-membered heteroaryl. In certain embodiments of formula (Ih), the A ring is triazole. In certain embodiments of (Ih), the A ring is absent. [0295] In some embodiments of formula (Ii), the A ring is a 5 or 6-membered heteroaryl. In certain cases of formula (Ii), the A ring is a 5-membered heteroaryl. In certain embodiments of formula (Ii), the A ring is triazole. In certain embodiments of (Ii), the A ring is absent. [0296] In certain embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by any one of formulae (Ij)-(Im):
Figure imgf000064_0001
(Il), and (Im) wherein: R1, R4, R5, R11, Z3 and R25 are as defined herein. Y1-Y3 are each independently N or CR27; and R24 and R27 are each independently selected from H, optionally substituted C(1-6)-alkyl, optionally substituted fluoroalkyl, and halogen. [0297] In some embodiments of any one of formulae (Ie)-(Im), Z3 is selected from -O-, -CH2O-, - OCH2-, optionally substituted -OCH2-heteroaryl, optionally substituted -OCH2-aryl, optionally substituted -CH2O-heteroaryl, and optionally substituted -CH2O-aryl. [0298] In some embodiments, Z3 is selected from:
Figure imgf000065_0001
[0299] In some embodiments of any one of formulae (Ie)-(Im), Z3 is selected from -C(R22)2-, optionally substituted alkyl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiourea. In some embodiments, Z3 is -CH2-. In some embodiments, Z3 is -CH2CH2-. In some embodiments, Z3 is -CH2CH2CH2-. In some embodiments, Z3 is -NHSO2-(C1-3-alkyl). In some embodiments, Z3 is -N(Ac)-(C1-3-alkyl). [0300] In some embodiments of any one of formulae (Ie)-(Im), Z3 is selected from -S- and -NR26-, where R26 is selected from H and optionally substituted (C1-C3)alkyl. [0301] In some embodiments of formula (Ik) at least one of Y1 to Y3 is N. In certain embodiments, at least two of Y1 to Y3 are N. In certain embodiments, Y1 and Y3 are N and Y2 is CR25. In certain embodiments, Y1 and Y2 are N and Y3 is CR25. In certain embodiments, Y1 and Y2 are CR25 and Y3 is N. In certain embodiments, R25 is H. In certain embodiments, R24 is C(1-3)-alkyl, or C(1-3)-fluoroalkyl. In certain embodiments, the fluoroalkyl is CF3. [0302] In certain embodiments of any one of formulae (Ie)-(Im) R1 is OH. [0303] In some embodiments of any one of formulae (Ie)-(Im), R4 and R5 are each H. In certain embodiments, at least one of R4-R5 is a promoiety. In certain embodiments, R4 and R5 are cyclically linked to form a promoiety (e.g., as described herein). [0304] In certain embodiments, the compound of formula (Ie) is selected from one of the following structures:
Figure imgf000065_0002
Figure imgf000066_0001
[0305] In certain embodiments, the compound of formula (Ie) is selected from one of the following structures:
Figure imgf000066_0002
. [0306] In certain embodiments, the compound of formula (Ie) is:
Figure imgf000067_0001
. [0307] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (In’):
Figure imgf000067_0002
wherein: R1, R4, R5 and Z1 are as defined herein; Y1 and Y2 are each independently selected from -O-, -S-, NR28-, and -C(R22)2; R28 is selected from H, optionally substituted (C1-C6)alkyl, and -C(O)R22; each R22 is independently selected from H, halogen and optionally substituted (C1-C6)alkyl; and ring B is a 5 or 6-membered optionally substituted cyclic group. In some embodiments of formula (In’), Y1 is connected to the sugar ring via an alpha configuration. In some embodiments of formula (In’), Y1 is connected to the sugar ring via a beta configuration. [0308] In some embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (In):
Figure imgf000067_0003
wherein: R1, R4, R5 and Z1 are as defined herein; Y1 and Y2 are each independently selected from -O-, -S-, NR28-, and -C(R22)2; R28 is selected from H, optionally substituted (C1-C6)alkyl, and -C(O)R22; each R22 is independently selected from H, halogen and optionally substituted (C1-C6)alkyl; and ring B is a 5 or 6-membered optionally substituted cyclic group. [0309] In some embodiments of formula (In)-(In’) Y1 is O. In certain embodiments, Y1 is S. In certain embodiments, Y1 is -NR28-. In certain embodiments, Y1 is -C(R22)2 and each R22 is H. [0310] In some embodiments of formula (In)-(In’) Y2 is -NR28- where R28 is H. In certain embodiments, Y2 is -NR28- where R28 is -C(O)R22. In certain embodiments, R22 is methyl. [0311] In some embodiments of formula (In)-(In’) the B ring is a 5 or 6-membered heterocycle. In certain embodiments, the B ring is a 5-membered heterocycle. In certain embodiments, the B ring is a 6- membered heterocycle. [0312] In some embodiments of formula (In)-(In’) Z1 is Z11, where Z11 is selected from -O-, -S-, NR21-, and -C(R22)2. In certain embodiments, Z1 is -O-. In certain embodiments, Z1 is -S-. In certain embodiments, Z1 is NR21 where R21 is H. In certain embodiments, Z1 is -C(R22)2 where each R22 is H. [0313] In some embodiments of formula (In)-(In’) Z1 is optionally substituted Z11-heteroaryl or optionally substituted Z11-aryl. In some embodiments, Z1 is CH2-heteroaryl or CH2-aryl. In some embodiments of formula (In)-(In’) Z1 is optionally substituted amide. In some embodiments of formula (In)-(In’) Z1 is optionally substituted sulfonamide. In some embodiments of formula (In)-(In’) Z1 is optionally substituted urea or optionally substituted thiourea. [0314] In some embodiments, the compound of formula (In)-(In’) has one of the following structures:
Figure imgf000068_0001
. [0315] In certain embodiments of any one of formulae (Ia)-(In), n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking X to Y via Z1, such as a backbone of 25 or more consecutive atoms, or 30 or more consecutive atoms, and in certain embodiments, up to 100 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 20 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 21 to 50 consecutive atoms, by a chain of 22 to 50 consecutive atoms, by a chain of 23 to 50 consecutive atoms, by a chain of 24 to 50 consecutive atoms, by a chain of 25 to 50 consecutive atoms, by a chain of 26 to 50 consecutive atoms, by a chain of 27 to 50 consecutive atoms, by a chain of 28 to 50 consecutive atoms, or by a chain of 29 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 30 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 31 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 32 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 33 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 34 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 35 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 36 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 41 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 46 to 50 consecutive atoms. [0316] In certain embodiments of any one of formulae (Ia)-(In), n is 2 or more, and L is a branched linker that covalently links 2 or more X moieties to Y via the linking moiety Z1. [0317] In certain embodiments of any one of formulae (Ia)-(In), n is 2 or more and each branch of L comprises a linear linker of 14 or more consecutive atoms to covalently link via Z1 each X moiety to a branching point of the linker L, such as 15 or more consecutive atoms, 16 or more consecutive atoms, or 17 or more consecutive atoms, and in certain embodiments, up to 50 consecutive atoms. In certain embodiments, each branch of L comprises a linear linker of 14 to 50 consecutive atoms, such as 14 to 45, 14 to 40, 14 to 35 or 14 to 30 consecutive atoms. In certain embodiments, each branch of L comprises 14 to 30 consecutive atoms, such as 14 to 29, 14 to 28, 14 to 27, 14 to 26, 14 to 25, 14 to 24, 14 to 23, 14 to 22, 14 to 21, or 14 to 20 consecutive atoms. In certain embodiments, L comprises more than 14 consecutive atoms covalently linking each X moiety (via each Z1 group) to a branching point of the linker. In certain embodiments, L comprises 15 consecutive atoms separating each Z1 group from a branching point of L. In certain embodiments, L comprises 16 consecutive atoms separating each Z1 group from a branching point of L. In certain embodiments, L comprises 17 consecutive atoms separating each Z1 group from a branching point of L. In certain embodiments, L comprises 18 consecutive atoms separating each Z1 group from a branching point of L. In certain embodiments, L comprises 19 consecutive atoms separating each Z1 group from a branching point of L. In certain embodiments, L comprises 20 consecutive atoms separating each Z1 group from a branching point of L. In certain other cases, L comprises a liner linker of 20 or more consecutive atoms separating each Z1 group from a branching point L. [0318] In certain embodiments of any one of formulae (Ia)-(In), n is 2, and L comprises a branched linker having 14 or more consecutive atoms separating each Z1 group of X from a branching point of L. [0319] In certain embodiments of any one of formulae (Ia)-(In), n is 3, and L comprises a branched linker having 14 or more consecutive atoms separating each Z1 group of X from a branching point of L. [0320] In certain embodiments of any one of formulae (Ia)-(In), the linker L is of the formula (II) (e.g., as described herein). [0321] In certain embodiments of any one of formulae (Ia), (Ib) or (Id)-(In), R1 is OH. In certain other cases, R1 is -OC(O)R. In certain embodiments, R1 is -C(O)NHR, where R is an optionally substituted alkyl. In certain embodiments, R terminates in an alkenyl or an alkynyl group. In certain other cases R1 is optionally substituted triazole. In certain embodiments, the triazole is of the following structure:
Figure imgf000069_0001
. [0322] In certain embodiments of (Ia)-(Ic), R2 is -NHCOCH3. In certain other embodiments, R2 is – NHCOCF3. In certain other embodiments, R2 is –NHCOCH2CF3. In certain embodiments, R2 is –OH. In certain other cases, R2 is an optionally substituted triazole. In certain embodiments, the triazole in of the following structure:
Figure imgf000070_0001
. [0323] In certain embodiments when R1 or R2 is a substituted triazole. The triazole is a 1,2,3-trizole, and the substituent is at the 4 or 5-position. In certain embodiments, the substituent on the triazole moiety includes but is not limited to, an optionally substituted (C1-6)alkyl, optionally substituted (C1- 6)alkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkaryl, and an optionally substituted alkyheteroaryl. It will be understood that any convenient substituent can be included in the triazole moiety, see, e.g., triazole moieties disclosed in Mamidayala et al, J. Am. Chem. Soc.2012, 134, 1978-1981. [0324] In certain embodiments of any one of formulae (Ia)-(In), at least one of R4-R5 is a promoiety. In certain embodiments, the promoiety is an ester. In certain embodiments the ester of the formula - OCOCH3, -OCOCH(CH3)2 or -OCOC(CH3)3. In certain embodiments, at least one of R4-R5 is of the formula -COCH3, -COCH(CH3)2 or -COC(CH3)3. In certain embodiments, at least one of R4-R5 is of the formula -CH2OCOC(CH3)3 In certain embodiments, R4 is a promoiety and R5 is H. In certain other cases, R5 is H and R4 is a promoiety. In certain embodiments, both R4 and R5 are both promoieties. In certain embodiments, R4 and R5 are cyclically linked to form a promoiety. In certain embodiments, R4 and R5 are cyclically linked to form a promoiety of formulae (Io) or (Ip):
Figure imgf000070_0002
wherein R1-R3 and Y4 are as defined herein. [0325] In certain embodiments of any one of formulae (Ia)-(In), both R4 and R5 are H. [0326] In certain embodiments of formula (I), n is 2 or 3, and X is selected from one of the
Figure imgf000070_0003
Figure imgf000071_0001
wherein R5 and R23 are independently H or (C1-3)alkyl. [0327] In certain embodiments of formula (I), n is 1, 2 or 3, and X is selected from one of the following structures:
Figure imgf000071_0002
[0328] wherein R5 and R4 independently H or a promoiety, or R5 and R4 are cyclically linked to form a promoiety; n1 and n2 are each independently an integer from 1 to 6; and Y4 is a suitable counterion. In some embodiments, Y4 is sodium. [0329] In certain embodiments of formula (I), n is 1, 2 or 3, and X is selected from one of the following structures:
Figure imgf000071_0003
. [0330] In certain embodiments of formula (I), n is 1, 2 or 3, and X is selected from one of the following structures:
Figure imgf000072_0001
[0331] In certain embodiments of formula (I), n is 1, 2 or 3, and X is selected from one of the following structures:
Figure imgf000072_0002
Figure imgf000073_0001
. [0332] In certain embodiments of formula (I), n is 1, 2 or 3, and X is the following structure:
Figure imgf000073_0002
. [0333] In certain embodiments of formula (I), n is 1, 2 or 3, and X is the following structure:
Figure imgf000073_0003
. [0334] In certain embodiments of formula (I), n is 1 and X is
Figure imgf000073_0004
. [0335] In certain embodiments of any one of formulae (Ia)-(Ip), -Z1- is linked to an -L1- moiety (e.g., of the linker of any of formulae (II), (IIa) or (IIb) described herein). In some embodiments, the subject compounds comprise a -Z1-L1- group selected from:
Figure imgf000073_0005
wherein each R21 is independently selected from H, and optionally substituted (C1-C6)alkyl; each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1-C6)alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6. [0336] In certain embodiments, the Z1-L1- group is
Figure imgf000074_0001
, and o is 1 or 2. [0337] In certain embodiments, the Z1-L1- group
Figure imgf000074_0002
each R22 is H, and p is 1 or 2.
Figure imgf000074_0009
[0340] In certain embodiments, the Z1-L1- group is
Figure imgf000074_0003
, where r is 1-3. [0341] In certain embodiments, the Z1-L1- group
Figure imgf000074_0004
are each independently 1-3. [0342] In certain embodiments, the Z1-L1- group
Figure imgf000074_0005
[0343] In certain embodiments, the Z1-L1- group i
Figure imgf000074_0006
are each independently is 1-3. [0344] In certain embodiments, the Z1-L1- group is
Figure imgf000074_0007
, where x is 0-3. [0345] In certain embodiments, the Z1-L1- group
Figure imgf000074_0008
-3. R21 [0346] In certain embodiments, the Z1-L1- group i
Figure imgf000075_0001
, where R21 is H, and z is 1-4. R21 [0347] In certain embodiments, the Z1-L1- group is
Figure imgf000075_0002
, where R21 is H, and z1 is 1-4. [0348] In certain embodiments, the Z 1 -L 1 - group i
Figure imgf000075_0003
is 1-3. [0349] In certain embodiments, the Z1-L1- group i
Figure imgf000075_0004
, where q is 1-3. [0350] In certain embodiments, the subject compounds comprise a -Z1-L- group selected from:
Figure imgf000075_0005
[0351] In certain embodiments, the Z 1 -L 1 - group is
Figure imgf000075_0006
, where q is 1-3. In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. [0352] In certain embodiments, the Z1-L1- group i
Figure imgf000075_0007
. [0353] In certain embodiments, the Z1-L1- group i
Figure imgf000075_0008
. [0354] In certain embodiments, the Z1-L1- group is
Figure imgf000075_0009
. [0355] In certain embodiments, -Z1-L1- comprises an optionally substituted -NH-heteroaryl-. In certain embodiments the heteroaryl is a triazole. In certain embodiments, the heteroaryl is pyridine. In certain embodiments the heteroaryl is pyrimidine In certain cases the heteroaryl is thiadiazole [0356] In certain embodiments, the -Z1-L1- comprises a group selected from:
Figure imgf000076_0001
wherein each R24 is independently selected from H, optionally substituted C(1-6)-alkyl, optionally substituted fluoroalkyl, and halogen; and each R25 is independently selected from H, optionally substituted (C1-C6)alkyl, and optionally substituted acyl. In certain embodiments, R25 is H. In certain embodiments, R24 is C(1-3)-alkyl, or C(1-3)-fluoroalkyl. In certain embodiments, the fluoroalkyl is CF3. Exemplary ASGPR Ligands [0357] Exemplary moieties that bind ASGPR, and synthons which can be utilized in the preparation of compounds of this disclosure that include the ASGPR ligand of interest are shown in Tables 1-5. [0358] In certain embodiments, the compound of formula (Ib) is a compound shown in Table 1:
Figure imgf000076_0002
[0359] In certain embodiments, the compound of formula (Ib) is a compound shown in Table 1A:
Figure imgf000076_0003
Figure imgf000077_0001
[0360] In certain embodiments, the compound of formula (Ib) is a compound shown in Table 1B:
Figure imgf000077_0002
[0361] In certain embodiments, the compound of formula (Ic) is a compound shown in Table 2a:
Figure imgf000077_0003
Figure imgf000078_0001
[0362] In certain embodiments, the compound of formula (Id) is a compound shown in Table 3a:
Figure imgf000078_0002
[0363] In certain embodiments, the compound of formula (Id) is a compound shown in Table 4:
Figure imgf000078_0003
Figure imgf000079_0001
Figure imgf000080_0001
[0364] In certain embodiments, the compound of formula (Id’) is a compound shown in Table 5:
Figure imgf000080_0002
[0365] Additional exemplary moieties that bind ASGPR, and synthons which can be utilized in the preparation of compounds of this disclosure that include the ASGPR ligand of interest are shown in the tables below. The building blocks described herein, in some embodiments, can be used to prepare the compounds disclosed herein. As is appreciated by one of skill in the art, reactive functional groups present on the building blocks described herein can be reacted with complimentary functional groups on a linker moiety to bond the ASGPR binding compound X to Y. [0366] For example, compounds of this disclosure can be prepared using the building blocks described herein as exemplified in Scheme 1. In Scheme 1, compounds of formula (I): Xn L m Y (I) is represented by formula (II’):
Figure imgf000081_0001
(II’) wherein: n is 1 to 3; m is 1 to 20; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; each L1 to L6 is independently a linking moiety which together provide a linear or branched linker between Z1 and Y; and X and Y are as defined herein. [0367] Scheme I is intended to be exemplary and in no way is intended to limit the scope of the disclosure. However, as can be appreciated by one of skill in the art, the compounds of this disclosure have various L moieties which may be constructed by coupling X to one or more first portions of the linker L (e.g., an -L1- moiety) via Z1 to provide exemplary ASGPR binding compound X building blocks. In Scheme 1, RM1 and RM2 are each independently reactive functional groups for coupling reactions (e.g., alkyne, -N3, -C(O)OH, -NH2, etc.); and Y’ is Y or a chemoselective a chemoselective ligation group capable of conjugating to an amino acid residue(s) of Y. Scheme 1
Figure imgf000081_0002
[0368] Methods for the steps and exemplary reagents and starting materials (i.e., compounds of Formula 1-1, 1-2, 1-3) are described throughout or can be derived from the art. [0369] Exemplary building blocks (e.g., compounds of formula 1-1, 1-2, or 1-3 in Scheme 1) are shown in the tables below. [0370] Exemplary building blocks (e.g., compounds of formula 1-1, 1-2, or 1-3 in Scheme 1) that can be used in the preparation of compounds of this disclosure that include ASGPR ligands (X) of interest are shown in Table 6.
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
[0371] In some embodiments of the ASGPR ligand (X) building blocks that can be used in the preparation of compounds of this disclosure, R3 is H such that the ASGPR ligand (X) includes CH2 at the 1-position, and R2 is a linking moiety, Z1. Exemplary building blocks that can be used in the preparation of compounds of this disclosure that include ASGPR ligands (X) of interest are shown in Table 7.
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
[0372] In some embodiments, the ASGPR ligand (X) building blocks that can be used in the preparation of compounds of this disclosure is a bicyclic structure. Exemplary building blocks that can be used in the preparation of compounds of this disclosure that include ASGPR ligands (X) of interest are shown in Table 8.
Figure imgf000114_0001
Figure imgf000115_0001
[0373] Other building blocks that can be used and/or modified to assemble ASGPR ligands of this disclosure are shown in Tables 8A-8B.
Figure imgf000115_0002
Figure imgf000116_0001
[0374] Table 8B: Monovalent binding examples, 2R or 6R modifications
Figure imgf000116_0002
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Prodrugs [0375] Aspects of this disclosure include prodrugs of any of the ASGPR binding moieties described herein that are incorporated into the compounds and conjugates of this disclosure. [0376] The term “prodrug” refers to an agent which is converted into the drug in vivo by some physiological or chemical process (e.g., a prodrug on being brought to the physiological pH is converted to the desired drug form). [0377] Prodrugs forms of any of the ASGPR binding moieties described herein can be useful because, for example, can lead to particular therapeutic benefits as a consequence of an extension of the half-life of the resulting compound or conjugate in the body or a reduction in the active dose required. [0378] Pro-drugs can also be useful in some situations, as they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The pro-drug may also have improved solubility in pharmacological compositions over the parent drug. [0379] Prodrug derivative of a ASGPR binding moiety generally includes a promoiety substituent at a suitable labile site of the compound. The promoiety refers to the group that is removed by enzymatic or chemical reactions, when a prodrug is converted to the drug in vivo. [0380] In some embodiments, the promoiety is a group attached via an ester linkage to a hydroxyl group of the compound or drug. [0381] In some embodiments, a prodrug derivative of one or more of the hydroxyl groups of the sugar ring may be incorporated into the compounds. For example, an ester promoiety can be incorporated at one or more of the hydroxyl groups at the 3 and/or 4 positions of the sugar (e.g., as described herein). In some embodiments, the hydroxyl groups at the 3 and 4 positions of the sugar are cyclically linked to form a promoiety (e.g., as described herein). Linker Valency [0382] The ASGPR ligand moieties (X) can be used in a monovalent or multivalent configuration with respect to the binding to ASGPR of the “n” X groups that are displayed on the linker scaffold. A monovalent configuration includes a single ASGPR ligand moiety (X) per linker of the bifunctional molecule, where it is understood that one or more linkers may be connected to Y. A multivalent configuration includes two or more such ASGPR ligand moieties per linker (e.g., bivalent or trivalent or of higher valency linker). This disclosure provides particular linker scaffolds and linker valencies that display preferred ASGPR ligand moieties in the bifunctional molecules of this disclosure. [0383] In some embodiments, the linked ASGPR ligand moiety (X) of the bifunctional molecule is monovalent (e.g., in Formula (I), n is 1), such that a linker covalently links a single ASGPR ligand moiety (X) via a linking moiety at the 1, 6, or 2-position of the sugar ring analog to a biomolecule (Y). In certain embodiments of formula (I), n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms (e.g., 25 or more) covalently linking the ASGPR ligand X to Y via a linking moiety at any of the 1-, 2- or 6-positions of X. In certain embodiments, the linker L includes a backbone of 20 to 100 consecutive atoms linking the ASGPR ligand (X) to Y, such as 25 to 80, 25 to 60, or 25 to 50 consecutive atoms. [0384] In some embodiments, the bifunctional molecule is multivalent with respect to X, where in Formula (I), n is 2 or more, such that the conjugate includes two or more ASGPR ligand binding moieties (X) per multivalent linker which connects to Y. In such cases, the multivalent linker (L) is a branched linker or a dendrimer linker. In certain embodiments, the bifunctional molecule has one or more divalent linkers (e.g., n is 2 in Formula (I)). In certain embodiments, the bifunctional molecule has one or more trivalent linkers (e.g., n is 3 in Formula (I)). [0385] In certain embodiments, each branch of a branched linker includes a linear linker portion covalently connecting each X moiety (via the linking moiety described herein) to a branching point in the branched linker or dendrimer linker. In certain embodiments, each branch of the linker includes a linear linker portion having a backbone of 8 or more consecutive atoms, such as 10 or more, 12 or more, 14 or more, 16 or more, 18 or more or 20 or more consecutive atoms between the X ligand moiety and the branching point in the linker. In certain embodiments, each branch of the linker includes a linear linker portion having a backbone of 8 to 50 consecutive atoms, such as 10 to 50, 12 to 50, 14 to 50, or 14 to 40, 14 to 30, or 14 to 20 consecutive atoms. Linkers [0386] The terms “linker”, “linking moiety” and “linking group” are used interchangeably and refer to a linking moiety that covalently connects two or more moieties, compounds or other biomolecules, such as ligands and proteins of interest. In certain embodiments, the linker is divalent and connects two moieties. In certain embodiments, the linker is a branched linking group that is trivalent or of a higher multivalency. In certain embodiments, the linker that connects the two or more moieties has a linear or branched backbone of 500 atoms or less (such as 400 atoms or less, 300 atoms or less, 200 atoms or less, 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or even 20 atoms or less) in length, e.g., as measured between the two or more moieties. A linking moiety may be a covalent bond that connects two groups or a linear or branched chain of between 1 and 500 atoms in length, for example of about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100, 150, 200, 300, 400 or 500 carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom. In certain embodiments, one, two, three, four, five or more, ten or more, or even more carbon atoms of a linker backbone may be optionally substituted with heteroatoms, e.g., sulfur, nitrogen or oxygen heteroatom. In certain instances, when the linker includes an ethylene glycol, or longer polyethylene glycol (PEG) linking group, e.g., where every third atom of that segment of the linker backbone is substituted with an oxygen. The bonds between backbone atoms of a linker may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone. The linker may include one or more substituent groups, for example an alkyl, aryl or alkenyl group. A linker may include, without limitations, one or more of the following: oligo(ethylene glycol) (also referred to as PEG), ether, thioether, disulfide, amide, carbonate, carbamate, urea, sulfonamide, thiourea, tertiary amine, alkyl which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1- methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle, a cycloalkyl group or a heterocycle group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone. [0387] In some embodiments, a “linker” or linking moiety is derived from a molecule with a reactive terminus, e.g., suitable for conjugation to a protein of interest. In some instances, the reactive terminus of the linker precursor includes a chemoselective ligation group capable of conjugating to amino acid residue(s) of a polypeptide. In certain instances, the chemoselective ligation group conjugates to a cysteine thiol group, or a lysine sidechain amine group of the polypeptide that is accessible. A variety of conjugation chemistries can be utilized in the conjugtaes of this disclosure (e.g., as described herein). In some embodiments, the chemoselective ligation group is a thiol-reactive group such as maleimide or dibromomaleimide. In some embodiments, the chemoselective ligation group is an amine- reactive group such as an active ester, e.g., perfluorophenyl ester or tetrafluorophenyl ester, or N- hydroxysuccinimidyl ester (NHS) or sulfo-NHS, or as defined herein. [0388] In certain embodiments of the formula described herein, the linker L includes one or more straight or branched-chain carbon moieties and/or polyether (e.g., ethylene glycol) moieties (e.g., repeating units of -CH2CH2O-), and combinations thereof. In certain embodiments, these linkers optionally have amide linkages, urea or thiourea linkages, carbamate linkages, ester linkages, amino linkages, ether linkages, thioether linkages, sulfhydryl linkages, heteroaryl linkages, or other hetero functional linkages. In certain embodiments, the linker backbone includes one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof. In certain embodiments, the linker includes one or more of an ether bond, thioether bond, amine bond, amide bond, carbon-carbon bond, carbon-nitrogen bond, carbon-oxygen bond, carbon-sulfur bond, and combinations thereof. In certain embodiments, the linker includes a linear structure. In certain embodiments, the linker includes a branched structure. In certain embodiments, the linker includes a cyclic structure. In certain embodiments, the linker includes one or more heteroaryl cyclic structures, e.g., a triazole, such as a 1,2,3- traizole. [0389] In certain embodiments, L is a linker between about 5 Å and about 500 Å. In certain embodiments, L is between about 10 Å and about 400 Å. In certain embodiments, L is between about 10 Å and about 300 Å. In certain embodiments, L is between about 10 Å and about 200 Å. In certain embodiments, L is between about 10 Å and about 100 Å. [0390] In certain embodiments, linker L separates X (or Z1) and Y by a chain of 10 to 100 consecutive atoms. In certain embodiments, linker L separates X (or Z1) and Y by a chain of 10 to 60 consecutive atoms, by a chain of 12 to 60 consecutive atoms, by a chain of 16 to 50 consecutive atoms, by a chain of 20 to 50 consecutive atoms, by a chain of 30 to 50 consecutive atoms, by a chain of 40 to 50 consecutive atoms. [0391] It is understood that the linker may be considered as connecting directly to a Z1 group of a ASGPR ligand moiety (X) (e.g., as described herein). In some embodiments of formula II (or any formulae described herein for the ASGPR ligand moiety (X)), the linker may be considered as connecting directly to the Z1 group. Alternatively, the -Z1-L1- group (e.g., as described herein) can be considered part of a linking moiety that connects L to Y. The disclosure is meant to include all such configurations of ASGPR ligand moiety (X) and linker (L). [0392] In some embodiments of formula (I), L is a linker of formula (XI):
Figure imgf000122_0001
wherein each L1 and L3 are independently a linear linking moiety, and L2 is a branched linking moiety, wherein L1 to L3 together provide a linear or branched linker between X and Y; a, b and c are independently 0 or 1; * represents the point of attachment of L1 to X via Z1; and ** represents the point of conjugation of the linker L to Y; wherein: when n is 1, b is 0 and at least one of a and c is 1; and when n is 2 or 3, a, b and c are each 1. [0393] In some embodiments of the linker of formula (XI), n is 1, a is 1, b is 0, and c is 1, such that the linker L is of formula (Xia):
Figure imgf000123_0001
[0394] In some embodiments of the linker of formula (XI), n is 1, a is 1, b is 0, and c is 0, such that the linker L is of formula:
Figure imgf000123_0002
. [0395] In certain embodiments, the linear linker of formula (Xia) has a backbone of 10 or more consecutive atoms covalently linking X to Y via Z1, such as a backbone of 12 or more consecutive atoms, 14 or more consecutive atoms, or 16 or more consecutive atoms, and in certain embodiments, up to 100 consecutive atoms. In certain embodiments of formula (Xia), the linear linker separates X (or Z1) and Y by a chain of 20 to 50 consecutive atoms. In certain embodiments of formula (Xa), the linear linker separates X (or Z1) and Y by a chain of 30 to 60 consecutive atoms. [0396] In some embodiments of the linker of formula (XI), n is 2, a is 1, b is 1, and c is 1, such that the linker L is of formula (Xib):
Figure imgf000123_0003
[0397] In some embodiments of the linker of formula (XI), n is 3, a is 1, b is 1, and c is 1, such that the linker L is of formula (Xic):
Figure imgf000123_0004
[0398] In some embodiments of the linker of any one of formulae (XI) or (Xia)-(Xic), each L1 is of formula (XII):
Figure imgf000123_0005
wherein: L10 is a linking moiety, and * represents the point of attachment of L1 to X via Z1; and L11 to L19 are independently absent or a linking moiety, wherein L10 to L19 of each L1 is independently selected from–C1-6-alkylene–,-–C1-12-alkylene–, – C1-20-alkylene–,–NHCO-C1-6-alkylene–, –CONH-C1-6-alkylene–, –NH-C1-6-alkylene–, –NHCONH-C1-6- alkylene–, –NHCSNH-C1-6-alkylene–, –C1-6-alkylene–NHCO-, –C1-6-alkylene–CONH-, –C1-6-alkylene– NH-, –C1-6-alkylene–NHCONH-, –C1-6-alkylene–NHCSNH-, -O(CH2)p–, –(OCH2CH2)p–, –NHCO–, – CONH–, –NHSO2–, –SO2NH–, –NHCONH-,–NHCSNH-, –CO–, –SO2–, –O–, –S–, arylene, heteroarylene, heteroalkylene, cycloalkylene, –NH–, –N(C1-6-alkyl)–, and –N(CH3)–, wherein each L10 to L19 of each L1 is independently optionally substituted with one or more halo (e.g., 1 to 3, or 1 to 5); and p is independently 1 to 50, such as 1 to 20, 1 to 12, 1 to 10, 1 to 8, or 1 to 6, e.g., 1, 2, 3, 4, 5 or 6. [0399] In some embodiments of the linker of any one of formulae (XI) or (Xia)-(Xic), each L1 is of formula (XII):
Figure imgf000124_0001
wherein: L10 is a linking moiety, and * represents the point of attachment of L1 to X via Z1; and L11 to L19 are independently absent or a linking moiety, wherein L10 to L19 of each L1 is independently selected from –C1-6-alkylene–,-CF2-, –C1-12- alkylene–, –C1-20-alkylene–,–NHCO-C1-6-alkylene–, –CONH-C1-6-alkylene–, –NH-C1-6-alkylene–, – NHCONH-C1-6-alkylene–, –NHCSNH-C1-6-alkylene–, –C1-6-alkylene–NHCO-, –C1-6-alkylene–CONH-, –C1-6-alkylene–NH-, –C1-6-alkylene–NHCONH-, –C1-6-alkylene–NHCSNH-, -O(CH2)p–, –(OCH2CH2)p– , –NHCO–, –CONH–, –NHSO2–, –SO2NH–, –NHCONH-,–NHCSNH-, –CO–, –SO2–, –O–, –S–, pyrrolidine-2,5-dione, 1,2,3-triazole, –NH–, –N(C1-6-alkyl)–, and –N(CH3)–, wherein each p is independently 1 to 50, such as 1 to 20, 1 to 12, 1 to 10, 1 to 8, or 1 to 6, e.g., 1, 2, 3, 4, 5 or 6. [0400] In certain embodiments of formula (XII), the linking moiety L1 includes a linear backbone of 6 to 40 consecutive atoms, such as 10 to 40, 10 to 30, 16 to 30, or 20 to 30 consecutive atoms. In certain embodiments of formula (XII), the linking moiety L1 includes a linear backbone of each L1 comprises a linear backbone of 6 to 20 consecutive atoms, such as 6 to 16 consecutive atoms, such as 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive atoms. [0401] In certain embodiments, the linking moiety of formula (XII) includes one or repeating ethylene glycol moieties (e.g., -CH2CH2O- or -OCH2CH2-). In certain embodiments, the linking moiety of formula (XII) includes 1 to 10 ethylene glycol moieties, such as 1, 2, 3, 4, 5 or 6 ethylene glycol moieties. [0402] In certain embodiments, the linking moiety of formula (XII) includes one or more triazole (e.g., 1,2,3-triazole) containing linking moieties. It is understood that the triazole may be derived from an azido-alkyne click chemistry and thus have two possible orientations depending on the method of synthesis:
Figure imgf000124_0002
[0403] In certain embodiments, the triazole containing linking moiety is :
Figure imgf000125_0001
wherein w1 and u1 are independently 0 to 12, such as 0, 1, 2, 3, 4, 5 or 6. [0404] In some embodiments of the linker of formula (XI), b is 1 and L2 is of the formula (XIIIa) or (XIIIb):
Figure imgf000125_0002
(XIIIa) (XIIIb) wherein: L20 is a branched linking moiety including one or more linking moieties independently selected from amino acid residue (e.g., a residue such as Gly, Ala, beta-Al Glu, Ser, Cys, or a derivative thereof), –NH-CH[(CH2)q]2O– or –NH-C[(CH2)q]3O–,
Figure imgf000125_0003
alkylene–, –NHCO-, –CONH–, –NHSO2–, –SO2NH–, –CO–, –SO2–, –O–, –S–, pyrrolidine-2,5-dione, 1,2,3-triazole, –NH–, and –Nme–, –NHC(O)NH–, – NHC(S)NH–, –O(CH2)p–, and –(OCH2CH2)p–; wherein each p is independently 1 to 50, and q is 1-6. [0405] In some embodiments of the linker of formula (XI), b is 1 and the linking moiety L2 is selected from one of (L2A)-(L2D):
Figure imgf000125_0004
wherein: each Z2 and Z3 is independently absent or selected from –NHCO-, –CONH–, –CO–, –O–, –NH–, and –Nme–; x is 1 to 12 (e.g., 1 to 6, or 1 to 3); and y is 0 to 12 (e.g., 1 to 6, or 1 to 3). [0406] In some embodiments of any one of L2A-L2D, Z2 is –NHCO-. In some embodiments of any one of L2A-L2D, Z2 is –CONH–. In some embodiments of any one of L2A-L2D, Z2 is –CO–. In some embodiments of any one of L2A-L2D, Z2 is –O–. In some embodiments of any one of L2A-L2D, Z2 is –NH–. In some embodiments of any one of L2A-L2D, Z2 is –Nme–. In some embodiments of any one of L2A-L2D, Z2 is absent. [0407] In some embodiments of any one of L2A-L2D, Z3 is –NHCO-. In some embodiments of any one of L2A-L2D, Z3 is –CONH–. In some embodiments of any one of L2A-L2D, Z3 is –CO–. In some embodiments of any one of L2A-L2D, Z3 is –O–. In some embodiments of any one of L2A-L2D, Z3 is –NH–. In some embodiments of any one of L2A-L2D, Z3 is –Nme–. In some embodiments of any one of L2A-L2D, Z3 is absent. [0408] In some embodiments of L2A, Z2 is –O–, y is 0 and the linking moiety is of the structure L2Ai:
Figure imgf000126_0001
[0409] In some embodiments of L2B, Z2 is –O– or –CO–, and the linking moiety is of the structure L2Bi or L2Bii: [0410] In some embod
Figure imgf000126_0004
the linking moiety is of the structure L2Ci, L2Cii, L2Ciii, or L2Civ:
Figure imgf000126_0002
[0411] In some embodiments of L2D, Z2 is absent and the linking moiety is of the structure L2Di:
Figure imgf000126_0003
[0412] In some embodiments, of any one of formulae L2A-L2Di, x is 1 to 6. In certain embodiments, x is 1 to 3. In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, x is 3. [0413] In some embodiments of any one of formulae L2A-L2Di, y is 0 to 6. In certain embodiments, y is 0 to 3. In certain embodiments, y is 0. In certain embodiments, y is 1. In certain embodiments, y is 2. In certain embodiments, y is 3. [0414] In certain embodiments of formula (XI), b is 1 and the linking moiety L2 is selected from:
Figure imgf000127_0001
[0415] In some embodiments of the linker of formula (XI), b is 1 and the linking moiety L2 is of the formula (XIV):
Figure imgf000127_0002
wherein: r is 1 or 2; and when n is 2, r is 1, when n is 3, r is 2. [0416] In some embodiments of the linker of formula (XI), b is 1 and the linking moiety L2 is of the formula (Xva) or (XVb):
Figure imgf000127_0003
wherein: r is 1 or 2; and when n is 2, r is 1, when n is 3, r is 2. [0417] In some embodiments L2 is of formula (XIIIa) or (XIIIb) and L2 includes two 2 or more amino acid residues (e.g., 3 or more, or 4 or more amino acid residues, linear or dendrimer). In some embodiments, L2 includes 4 or more amino acid residues that are branched linking moieties selected from Lys, Orn, Asp, Glu, Ser, and Cys (e.g., where the sidechain, amino and carboxylic acid are each linked to an adjacent moiety). [0418] In some embodiments of the linker of any one of formulae (XI) or (Xa)-(Xc), each L3 is of the formulae (XVI):
Figure imgf000128_0001
wherein: L30 to L39 are independently absent or a linking moiety; and Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group of the linker to a compatible group of Y; wherein L30 to L39 are each independently selected from –C1-20-alkylene–, –NHCO-C1-6- alkylene–, –CONH-C1-6-alkylene–, –NH C1-6-alkylene–, –NHCONH-C1-6-alkylene–, – NHCSNH-C1-6- alkylene–, –C1-6-alkylene–NHCO-, –C1-6-alkylene–CONH-, –C1-6-alkylene–NH-, –C1-6-alkylene– NHCONH-, –C1-6-alkylene–NHCSNH-, -O(CH2)p–, –(OCH2CH2)p–, –NHCO–, –CONH–, –NHSO2–, – SO2NH–, –NHCONH-, –NHCSNH-, –CO–, –SO2–, –O–, –S–, pyrrolidine-2,5-dione, 1,2,3-triazole, – NH–, and –NMe–, wherein each p is independently 1 to 50. [0419] In certain embodiments, the linking moiety of formula (XVI) includes a linear backbone of 6 to 40 consecutive atoms, such as 10 to 40, 10 to 30, or 20 to 30 consecutive atoms. [0420] In certain embodiments, the linking moiety of formula (XVI) includes repeating ethylene glycol moieties (e.g., -CH2CH2O- or -OCH2CH2-). In certain embodiments, the linking moiety of formula (XVI) includes 2 to 20 ethylene glycol moieties, such as 2 to 15, 2 to 10, 3 to 20, 3 to 15, 3 to 10, 4 to 15, 5 to 15 or 5 to 10 ethylene glycol moieties. In some instances, the linking moiety of formula (XVI) includes 2 or more ethylene glycol moieties, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or even more ethylene glycol moieties. [0421] In certain embodiments, the linking moiety of formula (XVI) includes one or more triazole linking moieties. In some instances, the linker includes one or more 1,2,3-triazole linking moieties. In certain embodiments, the one or more 1,2,3-triazoel moieties is selected from one of the following structures:
Figure imgf000128_0002
Figure imgf000128_0003
, wherein w1, u1 and q1 are independently 1 to 25 (e.g., 1 to 12, such as 1 to 6). [0422] In certain embodiments, the linking moiety L3 includes (C10-C20-alkylene (e.g., C12-alkylene), or –(OCH2CH2)p–, where p is 1 to 25, such as 3 to 25, 5 to 24, 7 to 25, 10 to 25, 15 to 25 or 20 to 24. [0423] In some embodiments, the linker L is of formula XVII:
Figure imgf000129_0001
wherein: a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); f is 1 to 6 (e.g., 1, 2, or 3); Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group (e.g., as described herein) of a linker precursor to a compatible group of Y. [0424] In some embodiments of the formula XVII, Z is a residual moiety resulting from the covalent linkage (e.g., via a thioether bond) of a thiol-reactive chemoselective ligation group to one or more cysteine residue(s) of Y. In some embodiments, the thiol-reactive chemoselective ligation group includes maleimide, bromomaleimide, haloacetamide, vinyl sulfone, or thiolactone. In some embodiments, the thiol-reactive group is selected from one of the following structures:
Figure imgf000129_0002
wherein: u is 1 to 11 (e.g., 1 to 5); v is 1 to 11 (e.g., 1 to 5); and X is H or Br. [0425] In some embodiments, the thiol-reactive group comprises:
Figure imgf000129_0003
Figure imgf000130_0001
[0427] In some embodiments of formula XVII, Z is a residual moiety resulting from the covalent linkage (e.g., via an amide bond) of an amine-reactive chemoselective ligation group to one or more lysine residue(s) of Y. In some embodiments, the amine-reactive chemoselective ligation group includes an active ester (e.g., N-hydroxysuccinimidyl (NHS) ester, sulfo-NHS ester, pentafluorophenyl (PFP) ester, tetrafluorophenyl (TFP) ester, or the like). [0428] In some embodiments, the linker L includes one of (XVIIIa)-(XVIIIc):
Figure imgf000130_0002
wherein: a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); and f is 1 to 6 (e.g., 1, 2, or 3). [0429] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), a is 2 to 6, such as 2 to 3. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5. In some embodiments a is 6. [0430] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), b is 1 to 4, such as 1 to 3. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3. [0431] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), c is 1 to 4, such as 1 to 3. In some embodiments, c is 1. In some embodiments, c is 2. In some embodiments, c is 3. [0432] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), r is 1. In some embodiments, r is 2. [0433] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), d is 1 to 4, such as 1 to 3. In some embodiments, d is 1. In some embodiments, d is 2. In some embodiments, d is 3. [0434] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), e is 1 to 5, such as 1 to 3. In some embodiments, e is 1. In some embodiments, e is 2. In some embodiments, e is 3. In some embodiments, e is 4. In some embodiments, e is 5. [0435] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), f is 1 to 4, such as 1 to 3. In some embodiments, f is 1. In some embodiments, f is 2. In some embodiments, f is 3. [0436] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3. [0437] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), a is 1-4; b is 1-4; c is 1-3; r is 2; d is 1-3; e is 1-6; and f is 1-3. [0438] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), a is 2; b is 1; c is 2; r is 1; d is 2; e is 3; and f is 2. [0439] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), a is 2; b is 1; c is 2; r is 2; d is 2; e is 3; and f is 2. [0440] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), a is 4; b is 1; c is 2; r is 1; d is 2; e is 3; and f is 2. [0441] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), a is 4; b is 1; c is 2; r is 2; d is 2; e is 3; and f is 2. [0442] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), a is 2; b is 2; c is 2; r is 1; d is 2; e is 3; and f is 2. [0443] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), a is 2; b is 2; c is 2; r is 2; d is 2; e is 3; and f is 2. [0444] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), a is 0; b is 3; c is 2; r is 2; d is 2; e is 3; and f is 2. [0445] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), a is 2; b is 4; c is 2; r is 2; d is 2; e is 3; and f is 2. [0446] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), a is 2; b is 4; c is 2; r is 1; d is 2; e is 3; and f is 2. [0447] In some embodiments, the linker L includes LA:
Figure imgf000132_0001
(LA), wherein: Z4 is selected from -NHC(O)NH-, -NHC(O)-, -C(O)NH-, -O-, -NH-; a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); and f is 1 to 6 (e.g., 1, 2, or 3). [0448] In some embodiments of LA, Z4 is -NHC(O)NH-. In certain embodiments, Z4 is -NHC(O)-. In certain embodiments, Z4 is -C(O)NH-. In certain embodiments, Z4 is -O-. In certain embodiments, Z4 is -NH-. [0449] In some embodiments of LA, a is 1-4; b is 1-4; c is 1-3; d is 1-3; e is 1-6; and f is 1-3. In some embodiments, a is 4; b is 1; c is 2; d is 2; e is 5; and f is 2. [0450] In some embodiments, Z4 is -NHC(O)NH- and a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3. In some embodiments, Z4 is -NHC(O)- and a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3. [0451] In some embodiments, the linker L includes LB:
Figure imgf000132_0002
wherein: a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); and f is 1 to 6 (e.g., 1, 2, or 3). [0452] In some embodiments of LB, a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3. In some embodiments, a is 4; b is 1; c is 2; r is 1; d is 2; e is 5; and f is 2. In some embodiments, a is 2; b is 1; c is 2; r is 1; d is 2; e is 3; and f is 2. In some embodiments, a is 4; b is 1; c is 2; r is 1; d is 2; e is 3; and f is 2. In some embodiments, a is 1; b is 2; c is 2; r is 1; d is 2; e is 3; and f is 2. In some embodiments, a is 0; b is 3; c is 2; r is 1; d is 2; e is 3; and f is 2. [0453] In some embodiments of LB, a is 1-4; b is 1-4; c is 1-3; r is 2; d is 1-3; e is 1-6; and f is 1-3. In some embodiments, a is 2; b is 1; c is 2; r is 2; d is 2; e is 3; and f is 2. In some embodiments, a is 4; b is 1; c is 2; r is 2; d is 2; e is 3; and f is 2. In some embodiments, a is 1; b is 2; c is 2; r is 2; d is 2; e is 3; and f is 2. In some embodiments, a is 0; b is 3; c is 2; r is 2; d is 2; e is 3; and f is 2. [0454] In some embodiments, the linker L includes LC:
Figure imgf000133_0001
wherein: a is 0 to 12 (e.g., 1 to 6, 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1 to 4, such as 1, 2, or 3); c is 1 to 6 (e.g., 1 to 3, such as 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1 to 3, such as 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); and f is 1 to 6 (e.g., 1 to 3, such as 1, 2, or 3). [0455] In some embodiments of Lc, a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3. In some embodiments, a is 2; b is 4; c is 2; r is 1; d is 2; e is 5; and f is 2. [0456] In some embodiments of Lc, a is 1-4; b is 1-4; c is 1-3; r is 2; d is 1-3; e is 1-6; and f is 1-3. In some embodiments, a is 2; b is 4; c is 2; r is 2; d is 2; e is 5; and f is 2. [0457] In certain embodiments of the ASGPR binding moiety (X) as described herein, -Z1- is linked to an -L1- moiety (e.g., of the linker as described herein). In some embodiments, the subject compounds comprise a -Z1-L1- moiety comprising a linking moiety selected from:
Figure imgf000133_0002
Figure imgf000134_0001
wherein each R21 is independently selected from H, and optionally substituted (C1-C6)alkyl; each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1-C6)alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6. [0458] In certain embodiments, the Z1-L1- group is
Figure imgf000134_0002
, and o is 1 or 2. [0459] In certain embodiments, the Z1-L1- group 22
Figure imgf000134_0003
each R is H, and p is 1 or 2.
Figure imgf000134_0008
[0462] In certain embodiments, the Z1-L1- group is
Figure imgf000134_0004
, where r is 1-3. [0463] In certain embodiments, the Z1-L1- group
Figure imgf000134_0005
are each independently 1-3. [0464] In certain embodiments, the Z1-L1- group i
Figure imgf000134_0006
[0465] In certain embodiments, the Z1-L1- group i
Figure imgf000134_0007
are each independently is 1-3. O [0466] In certain embodiments, the Z -L - group is N N N
Figure imgf000135_0001
1 1 , where x is 0-3. [0467] In certain embodiments, the Z1-L1- group
Figure imgf000135_0002
R21 [0468] In certain embodiments, the Z1-L1- group is
Figure imgf000135_0003
, where R21 is H, and z is 1-4. R21 [0469] In certain embodiments, the Z1-L1- group is
Figure imgf000135_0004
, where R21 is H, and z1 is 1-4. [0470] In certain embodiments, the Z 1 -L 1 - group is
Figure imgf000135_0005
, where each R22 is H, and q is 1-3. [0471] In certain embodiments, the Z1-L1- group i
Figure imgf000135_0006
, where q is 1-3. [0472] In certain embodiments, the subject compounds comprise a -Z1-L- group comprising a linking moiety selected from:
Figure imgf000135_0007
where R21 is independently selected from H, and optionally substituted (C1-C6)alkyl (e.g., methyl); and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1-C6)alkyl (e.g., methyl). In certain embodiments, R21 is H. In certain embodiments, each R22 is H. [0473] In certain embodiments, the -Z 1 -L 1 - group is
Figure imgf000135_0008
, where q is 1-3. In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3.
Figure imgf000136_0001
[0474] In certain embodiments, the -Z1-L1- group is H . [0475] In certain embodiments, -Z1-L1- includes an optionally substituted -NH-heteroarylene-. In certain embodiments, the heteroarylene is a triazole. In certain embodiments, the heteroarylene is pyridine. In certain embodiments, the heteroarylene is pyrimidine. In certain embodiments, the heteroarylene is thiadiazole. [0476] In certain embodiments, the -Z1-L1- includes a group selected from:
Figure imgf000136_0002
wherein R24 and R25 are each independently selected from H, optionally substituted C(1-6)- alkyl, optionally substituted fluoroalkyl, and halogen; and each R21 is independently selected from H, optionally substituted (C1-C6)alkyl, and optionally substituted alkanoyl. In certain embodiments, R21 is H. In certain embodiments, R24 is C(1-3)-alkyl, or C(1-3)-fluoroalkyl. In certain embodiments, the fluoroalkyl is CF3. In certain embodiments, R25 is C(1-3)-alkyl, or C(1-3)-fluoroalkyl. In certain embodiments, the fluoroalkyl is CF3. [0477] In some embodiments, the linker includes a polypeptide scaffold where some or all of the sidechain groups of the amino acid residues of such a polypeptide scaffold have been modified to attach a X binding moiety (e.g., as described herein). It is understood that X binding moieties (e.g., as described herein) can be conjugated to amino acid residues, such as Asp, Lys, Orn, Glu, and Ser, of a polypeptide containing linker via a convenient conjugation chemistry. In some embodiments, the linker contains a polylysine polypeptide. In some embodiments, the linker contains a polyornithine polypeptide. In some embodiments, the linker contains a polyserine polypeptide. In some embodiments, the linker contains a polyaspartate polypeptide. The polypeptide backbone of such a linker can be a randomly polymerized polymer having an average length, or a polymer of defined length prepared e.g., in a controlled stepwise fashion. In certain embodiments, the polypeptide linker has a length of 10-100 amino acid residues, such as 20-90, or 20-50 amino acid residues. In some embodiments, the N-terminal or C-terminal of the polypeptide linker is modified to include a linking moiety to an additional X binding moiety (e.g., as described herein). In some embodiments, the N-terminal or C-terminal of the polypeptide linker segment is modified with one or more linking moieties (e.g., as described herein) suitable for attachment to a protein construct (Y) including a polypeptide that specifically binds an autoantibody [0478] In some embodiments, a “linker” or linking moiety is derived from a molecule with two reactive termini, one for conjugation to a moiety of interest (Y), e.g., a biomolecule (e.g., an antibody) and the other for conjugation to a moiety (noted as X) that binds to a ASGPR cell surface receptor. When Y is a polypeptide, the polypeptide conjugation reactive terminus of the linker is in some cases a site that is capable of conjugation to the polypeptide through a cysteine thiol or lysine amine group on the polypeptide, and so is can be a thiol-reactive group such as a maleimide or a dibromomaleimide, or as defined herein, or an amine-reactive group such as an active ester (e.g., perfluorophenyl ester or tetrafluorophenyl ester), or as defined herein. [0479] In certain embodiments of the formula described herein, the linker L comprises one or more straight or branched-chain carbon moieties and/or polyether (e.g., ethylene glycol) moieties (e.g., repeating units of -CH2CH2O-), and combinations thereof. In certain embodiments, these linkers optionally have amide linkages, urea or thiourea linkages, carbamate linkages, ester linkages, amino linkages, ether linkages, thioether linkages, sulfhydryl linkages, heteroaryl linkages, or other hetero functional linkages. In certain embodiments, the linker comprises one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof. In certain embodiments, the linker comprises one or more of an ether bond, thioether bond, amine bond, amide bond, carbon-carbon bond, carbon-nitrogen bond, carbon-oxygen bond, carbon-sulfur bond, and combinations thereof. In certain embodiments, the linker comprises a linear structure. In certain embodiments, the linker comprises a branched structure. In certain embodiments, the linker comprises a cyclic structure. In certain embodiments, the linker comprises one or more heteroaryl cyclic structures, e.g., a triazole, such as a 1,2,3-traizole. [0480] In certain embodiments, L is between about 10 Å and about 20 Å in length. In certain embodiments, L is between about 15 Å and about 20 Å in length. In certain embodiments, L is about 15 Å in length. In certain embodiments, L is about 16 Å in length. In certain embodiments, L is about 17 Å in length. [0481] In certain embodiments, L is a linker between about 5 Å and about 500 Å. In certain embodiments, L is between about 10 Å and about 400 Å. In certain embodiments, L is between about 10 Å and about 300 Å. In certain embodiments, L is between about 10 Å and about 200 Å. In certain embodiments, L is between about 10 Å and about 100 Å. In certain embodiments, L is between about 10 Å and about 20 Å, between about 20 Å and about 30 Å, between about 30 Å and about 40 Å, between about 40 Å and about 50 Å, between about 50 Å and about 60 Å, between about 60 Å and about 70 Å, between about 70 Å and about 80 Å, between about 80 Å and about 90 Å, or between about 90 Å and about 100 Å. In certain embodiments, L is a linker between about 5 Å and about 500 Å, which comprises an optionally substituted arylene linked to X, an optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X. In certain embodiments, L is a linker between about 10 Å and about 500 Å, which comprises an optionally substituted arylene linked to X, or optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X. In certain embodiments, L is a linker between about 10 Å and about 400 Å, which comprises an optionally substituted arylene linked to X, or optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X. In certain embodiments, L is a linker between about 10 Å and about 200 Å, which comprises an optionally substituted arylene linked to X, or optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X. [0482] In certain embodiments, linker L separates X and Y (or Z1) by a chain of 4 to 500 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 4 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 6 to 50 consecutive atoms, by a chain of 11 to 50 consecutive atoms, by a chain of 16 to 50 consecutive atoms, by a chain of 21 to 50 consecutive atoms, by a chain of 26 to 50 consecutive atoms, by a chain of 31 to 50 consecutive atoms, by a chain of 36 to 50 consecutive atoms, by a chain of 41 to 50 consecutive atoms, or by a chain of 46 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 6 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 11 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 16 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 21 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 26 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 31 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 36 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 41 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z1) by a chain of 46 to 50 consecutive atoms. [0483] In certain embodiments, linker L separates X and Y (or Z1) by a chain of 4 or 5 consecutive atoms, by a chain of 6 to 10 consecutive atoms, by a chain of 11 to 15 consecutive atoms, by a chain of 16 to 20 consecutive atoms, by a chain of 21 to 25 consecutive atoms, by a chain of 26 to 30 consecutive atoms, by a chain of 31 to 35 consecutive atoms, by a chain of 36 to 40 consecutive atoms, by a chain of 41 to 45 consecutive atoms, or by a chain of 46 to 50 consecutive atoms. [0484] In certain embodiments, linker L is a chain of 5 to 500 consecutive atoms separating X and Y (or Z1) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or heteroatom linked to X. In certain embodiments, linker L is a chain of 7 to 500 consecutive atoms separating X and Y (or Z1) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or heteroatom linked to X. In certain embodiments, linker L is a chain of 10 to 500 consecutive atoms separating X and Y (or Z1) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or heteroatom linked to X. In certain embodiments, linker L is a chain of 15 to 400 consecutive atoms separating X and Y (or Z1) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or heteroatom linked to X. [0485] In certain embodiments, linker L is a chain of 5 to 500 consecutive atoms separating X and Y (or Z1) and which comprises an alkylene, a heteroatom, or optionally substituted heteroarylene linked to X. In certain embodiments, linker L is a chain of 7 to 500 consecutive atoms separating X and Y (or Z1) and which comprises an alkylene, a heteroatom, or optionally substituted heteroarylene linked to X. In certain embodiments, linker L is a chain of 10 to 500 consecutive atoms separating X and Y (or Z1) and which comprises an comprises an alkylene, a heteroatom, or optionally substituted heteroarylene linked to X. In certain embodiments, linker L is a chain of 15 to 400 consecutive atoms separating X and Y (or Z1) and which comprises an alkylene, a heteroatom, or optionally substituted heteroarylene linked to X. [0486] In certain embodiments, linker L is a chain of 5 to 500 consecutive atoms separating X and Y (or Z1) and which comprises an optionally substituted triazole linked to X. In certain embodiments, linker L is a chain of 7 to 500 consecutive atoms separating X and Y (or Z1) and which comprises an optionally substituted triazole linked to X. In certain embodiments, linker L is a chain of 10 to 500 consecutive atoms separating X and Y (or Z1) and which comprises an optionally substituted triazole linked to X. In certain embodiments, linker L is a chain of 15 to 400 consecutive atoms separating X and Y (or Z1) and which comprises an optionally substituted triazole linked to X. [0487] In certain embodiments, linker L is a chain of 16 to 400 consecutive atoms separating X and Y (or Z) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or a heteroatom linked to X. [0488] It is understood that the linker may be considered as connecting directly to a Z1 group of a ASGPR binding moiety (X) (e.g., as described herein). In some embodiments of any of formulae (Ia)- (Ip), the linker may be considered as connecting directly to the Z1 group. Alternatively, the -Z1-L1- group (e.g., as described herein) can be considered part of a linking moiety that connects L to Y. The disclosure is meant to include all such configurations of ASGPR binding moiety (X) and linker (L). [0489] In some embodiments of formula (I)-(Ia), L is a linker of formula (II):
Figure imgf000139_0001
wherein L1 and L3 are independently a linker, and L2 is a branched linking moiety, wherein L1 to L3 together provide a linear or branched linker between X and Y; a, b and c are independently 0 or 1; ** represents the point of attachment to L1 of X via Z1; and *** represents the point of attachment to Y; wherein: when n is 1, a is 1, and b is 0; when n is >1, a is 1, and b is 1. [0490] In certain embodiments of the linker of formula (II), L1 to L3 each independently comprise one or more linking moieties independently selected from –C1-20-alkylene–, –NHCO-C1-6-alkylene–, – CONH-C1-6-alkylene–, –NH C1-6-alkylene–, –NHCONH-C1-6-alkylene–, – NHCSNH-C1-6-alkylene–, – C1-6-alkylene–NHCO-, –C1-6-alkylene–CONH-, –C1-6-alkylene–NH-, –C1-6-alkylene–NHCONH-, –C1-6- alkylene–NHCSNH-, -O(CH2)p–, –(OCH2CH2)p–, –NHCO–, –CONH–, –NHSO2–, –SO2NH–, –CO–, – SO2–, –O–, –S–, monocyclic heteroaryl (e.g., 1,2,3-triazole), monocyclic aryl (e.g., phenyl, e.g., 1,4- linked phenyl or 1,3-linked phenyl), monocyclic heterocycle (e.g., pyrrolidine-2,5-dione, piperazine or piperidine ring as described herein), amino acid residue (naturally or non- naturally occurring amino acid residue), –NH–, and –Nme–, wherein each p is independently1 to 50. [0491] In certain embodiments of the linker of formula (II), any of L1-L3 comprises repeating ethylene glycol moieties (e.g., -CH2CH2O- or -OCH2CH2-). In certain embodiments, the linker of formula (II) comprises 1 to 25 ethylene glycol moieties, such as 3 to 25, 5 to 25, 7 to 25, 10 to 25, 15 to 25, 17 to 25, 20 to 25 or 22 to 25 ethylene glycol moieties. In some instances, the linker of formulae (II) comprises 3 or more ethylene glycol moieties, such as 5 or more, 7 or more, 10 or more, 15 or more, 20 or more, or even more ethylene glycol moieties. [0492] In certain embodiments of the linker of formula (II), any of L1-L3 comprises one or more triazole linking moieties. In some instances, the linker comprises one or more 1,2,3-triazole linking moieties. In certain embodiments, the one or more 1,2,3-triazole moieties is selected from one of the following structures:
Figure imgf000140_0001
Figure imgf000140_0002
, wherein w1, u1 and q1 are independently 1 to 25 (e.g., 1 to 12, such as 1 to 6). [0493] In certain embodiments of the linker of formula (II), n is 1, such that b is 0, and the linker is of the formula (IIa):
Figure imgf000140_0003
wherein L1 and L3 are independently a linker (e.g., as described herein), wherein L1 to L3 together provide a linear linker between X and Y; a is 1; c is 0 or 1; ** represents the point of attachment to L1 of X via Z1; and *** represents the point of attachment to Y. [0494] In certain embodiments of the linker of formula (IIa), the linear linker has a backbone of 20 or more consecutive atoms covalently linking X to Y via Z1, such as a backbone of 25 or more consecutive atoms, or 30 or more consecutive atoms, and in certain embodiments, up to 100 consecutive atoms. In certain embodiments of formula (IIa), the linear linker separates X and Y (or Z1) by a chain of 20 to 50 consecutive atoms. In certain embodiments, the linear linker separates X and Y (or Z1) by a chain of 21 to 50 consecutive atoms, by a chain of 22 to 50 consecutive atoms, by a chain of 23 to 50 consecutive atoms, by a chain of 24 to 50 consecutive atoms, by a chain of 25 to 50 consecutive atoms, by a chain of 26 to 50 consecutive atoms, by a chain of 27 to 50 consecutive atoms, by a chain of 28 to 50 consecutive atoms, or by a chain of 29 to 50 consecutive atoms. In certain embodiments of formula (IIa), the linear linker separates X and Y (or Z1) by a chain of 30 to 60 consecutive atoms. In certain embodiments, the linear linker separates X and Y (or Z1) by a chain of 31 to 60 consecutive atoms. In certain embodiments, the linear linker separates X and Y (or Z1) by a chain of 32 to 60 consecutive atoms. In certain embodiments, the linear linker separates X and Y (or Z1) by a chain of 33 to 60 consecutive atoms. In certain embodiments, the linear linker separates X and Y (or Z1) by a chain of 34 to 60 consecutive atoms. In certain embodiments, the linear linker L separates X and Y (or Z1) by a chain of 35 to 50 consecutive atoms. In certain embodiments, the linear linker L separates X and Y (or Z1) by a chain of 36 to 50 consecutive atoms. In certain embodiments, the linear linker L separates X and Y (or Z1) by a chain of 41 to 50 consecutive atoms. In certain embodiments, the linear linker L separates X and Y (or Z1) by a chain of 46 to 50 consecutive atoms. [0495] In certain other embodiments of formula (II), n is 2 or more, such that L1 to L3 together provide a branched linker between X and Y. [0496] In certain embodiments of formula (II), n is 2 or more, and L2 is selected from:
Figure imgf000141_0001
wherein each x and y are independently 1 to 10. [0497] In certain embodiments of formula (II), L1-L2 comprises a backbone of 14 or more consecutive atoms between X and the branching atom, such as 14 to 50, 14 to 40, 14 to 35 or 14 to 30 consecutive atoms between X and the branching atom. [0498] In certain embodiments of formula (II) or (IIa), L3 comprises a backbone of 10 to 80 consecutive atoms, such as 12 to 70, 12 to 60, or 12 to 50 consecutive atoms. In some embodiments, L comprises of 12 to 70, 12 to 60, 12 to 50, or 10 to 60 consecutive linear or branched chain atoms. [0499] In certain embodiments of formula (II) or (IIa), wherein L3 comprises a linking moiety selected from (C10-C20-alkylene (e.g., C12-alkylene), or –(OCH2CH2)p–, where p is 1 to 25, such as 3 to 25, 5 to 24, 7 to 25, 10 to 25, 15 to 25 or 20 to 24. [0500] In certain embodiments, L is of formula (Iib):
Figure imgf000142_0001
wherein each L1 to L5 is independently a linking moiety which together provide a linear or branched linker between Z1 and Y; a, b, c, d, and e are each independently 0, 1, or 2; ** represents the point of attachment to L1 of X via Z1; and *** represents the point of attachment to Y; wherein: when n is 1, a is 1, and c is 0; and when n is >1, a is 1, and c is 1. [0501] In some embodiments, L is of formula (IIb’):
Figure imgf000142_0002
wherein: each L1 to L6 is independently a linking moiety which together provide a linear or branched linker between Z1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L1 of X via Z1; and *** represents the point of attachment to Y. [0502] In certain embodiments, L is of formula (IIb’):
Figure imgf000142_0003
wherein: each L1 to L6 is independently a linking moiety which together provide a linear or branched linker between Z1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L1 of X via Z1; and *** represents the point of attachment to Y. [0503] In certain embodiments, each L1 to L5 independently comprises one or more linking moieties independently selected from –C1-20-alkylene–, –NHC(O)-C1-6-alkylene–, –C(O)NH-C1-6-alkylene–, –NH- C1-6-alkylene–, –NHC(O)NH-C1-6-alkylene–, –NHC(S)NH-C1-6-alkylene–, –C1-6-alkylene–NHC(O)-, – C1-6-alkylene–C(O)NH-, –C1-6-alkylene–NH-, –C1-6-alkylene–NHC(O)NH-, –C1-6-alkylene–NHC(S)NH- , -O(CH2)p–, –(OCH2CH2)p–, –NHC(O)–, –C(O)NH–, –NHS(O)2–, –S(O)2NH–, –C(O)–, –S(O)2–, –O–, – S–, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, monocyclic carbocycle, amino acid residue, –NH–, and –NMe–; wherein each L1 to L5 is independently optionally substituted with one to five halo; each p is independently1 to 50; L6 is a linking group comprising one or more linking moieties independently selected from –C1- 20-alkylene–, –NR16C(O)-C1-6-alkylene–, –C(O)NR16-C1-6-alkylene–, –NR16-C1-6-alkylene–, – NR16C(O)NR16-C1-6-alkylene–, –NR16C(S)NR16-C1-6-alkylene–, –C1-6-alkylene–NR16C(O)-, –C1-6- alkylene–C(O)NR16-, –C1-6-alkylene–NR16-, –C1-6-alkylene–NR16C(O)N R16-, –C1-6-alkylene– NR16C(S)NR16-, -O(CH2)p–, –(OCH2CH2)p–, –NR16C(O)–, –C(O)NR16–, –NHS(O)2–, –S(O)2NH–, – C(O)–, –S(O)2–, –O–, –S–, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, amino acid residue, or –NR16–; and each R16 is independently –H, (C1-C6)alkyl, or monocyclic heteroaryl. [0504] In certain embodiments, each L1 to L5 is independently selected from –C1-20-alkylene–, – NHC(O)-C1-6-alkylene–, –C(O)NH-C1-6-alkylene–, –NH-C1-6-alkylene–, –NHC(O)NH-C1-6-alkylene–, – NHC(S)NH-C1-6-alkylene–, –C1-6-alkylene–NHC(O)-, –C1-6-alkylene–C(O)NH-, –C1-6-alkylene–NH-, – C1-6-alkylene–NHC(O)NH-, –C1-6-alkylene–NHC(S)NH-, -O(CH2)p–, –(OCH2CH2)p–, –NHC(O)–, – C(O)NH–, –NHS(O)2–, –S(O)2NH–, –C(O)–, –S(O)2–, –O–, –S–, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, monocyclic carbocycle, amino acid residue, –NH–, and –NMe–; wherein each L1 to L5 is independently optionally substituted with one to five halo;
Figure imgf000143_0001
[0505] In certain embodiments of the linker of formula (Iib) or (IIb’), L1 to L5 each independently comprise one or more linking moieties independently selected from –C1-20-alkylene–, –NHCO-C1-6- alkylene–, –CONH-C1-6-alkylene–, –NH C1-6-alkylene–, –NHCONH-C1-6-alkylene–, – NHCSNH-C1-6- alkylene–, –C1-6-alkylene–NHCO-, –C1-6-alkylene–CONH-, –C1-6-alkylene–NH-, –C1-6-alkylene– NHCONH-, –C1-6-alkylene–NHCSNH-, -O(CH2)p–, –(OCH2CH2)p–, –NHCO–, –CONH–, –NHSO2–, – SO2NH–, –CO–, –SO2–, –O–, –S–, monocyclic heteroaryl (e.g., 1,2,3-triazole), monocyclic aryl (e.g., phenyl, e.g., 1,4-linked phenyl or 1,3-linked phenyl), monocyclic heterocycle (e.g., pyrrolidine-2,5-dione, piperazine or piperidine ring as described herein), amino acid residue (naturally or non- naturally occurring amino acid residue), –NH–, and –Nme–, wherein each p is independently1 to 50. [0506] In certain embodiments of formula (Iib) or (IIb’), -(L1)a- comprises an optionally substituted alkyl or ethylene glycol linking moiety. In certain embodiments, L1 comprises an optionally substituted -C1-6-alkylene–. In certain embodiments, L1 comprises an ethylene glycol linking moiety. [0507] In certain embodiments of formula (Iib), L1 is independently selected from: -C1-6-alkylene–, –(CH2CH2O)t–, –-C1-6-alkylene-NR4CO–, –C1-6-alkyleneCONH–,or OCH2, wherein t is 1 to 20; and R4 is independently selected from H, and optionally substituted (C1-C6)alkyl. In certain embodiments, L1 is -C1-6-alkylene–, such as -C1-3-alkylene–. In certain embodiments, L1 is – (CH2CH2O)t–, where t is 1 to 20, such as 1 to 15, 1 to 10, 1 to 8, 1 to 6, or 1 to 4. In certain embodiments, L1 is –-C1-6-alkylene-NR4CO–. In certain embodiments, L1 is –C1-6-alkyleneCONH–. In certain embodiments, L1 is or OCH2. [0508] In some embodiments of formula (Iib) or (IIb’), one or more L1 is independently –CH2O–; –
Figure imgf000144_0001
wherein: R13 is selected from H, halogen, OH, optionally substituted (C1-C6)alkyl, optionally substituted (C1-C6)alkoxy, COOH, NO2, CN, NH2, -N(R21)2, -OCOR21, -COOR21, -CONHR21, and -NHCOR21; each r independently 0 to 20, and any of the L1 moieties are optionally further substituted. [0509] In certain embodiments of formula (Iib) or (IIb’), L2 is independently selected from:
Figure imgf000145_0006
is 1 to 10, u is 0 to 10, w is 1 to 10, and R4’ is independently selected from H, and optionally substituted (C1-C6)alkyl. In certain embodiments, L2 is –NR4’CO-C1-6-alkylene–. In certain embodiments, L2 is – CONR4’-C1-6-alkylene. [0510] In certain embodiments,
Figure imgf000145_0001
[0511] In certain embodiments,
Figure imgf000145_0002
[0512] In certain embodiments,
Figure imgf000145_0003
is 1. [0513] In certain embodiments,
Figure imgf000145_0004
[0514] In certain embodiments,
Figure imgf000145_0005
[0515] In certain embodiments, L2 is -OCH2-. In certain other embodiments, L2 is (OCH2CH2)q–, and q is 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2. In certain embodiments, q is 2 to 8, such as 2 to 6 , 4 to 6, or 2 to 4. [0516] In certain embodiments of formula (Iib), L4 is absent or independently selected from -C1-6-alkylene–, –(CH2CH2O)t–, –-C1-6-alkylene-NHCO–, –C1-6-alkyleneCONH–,or OCH2, wherein t is 1 to 20. In certain embodiments, L4 is absent. [0517] In certain embodiments, L4 is -C1-6-alkylene–. In certain embodiments, L4 is –(CH2CH2O)t–, where t is 1 to 20, such as 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 3. In certain embodiments, L4 is –-C1-6-alkylene-NHCO–. In certain embodiments, L4 is –C1-6-alkyleneCONH–. In certain embodiments, L4 is OCH2. [0518] In some embodiments of the subject compounds, n is 1 and L3 in formula (Iib) is absent. [0519] In certain embodiments of the subject compounds, n is 2 or more, and L3 of formula (Iib) is a branched linking moiety. [0520] Accordingly, in some embodiments of formula (Iib) or (IIb’), L3 is a branched linking moiety, e.g., a divalent, or a trivalent linking moiety. For example, an L3 linking moiety can be of the one of the following general formula:
Figure imgf000146_0001
. [0521] In some embodiments of formula (Iib) or (IIb’), the branched linking moiety can be of higher valency and be described by one of the one of the following general formula:
Figure imgf000146_0002
where any two L3 groups can be directed linked or connected via optional linear linking moieties (e.g., as described herein). [0522] In some embodiments of formula (Iib) or (IIb’), the branched linking moiety can include one, two or more L3 linking moieties, each being trivalent moieties, which when linked together can provide for multiple branching points for covalent attachment of the ligands and be described by the following general formula:
Figure imgf000146_0003
where t is 0 to 500, such as 0 to 100, 0 to 20, or 0 to 10. [0523] In some embodiments, the branched linking moiety (e.g., L3) comprises one or more of: an amino acid residue (e.g., Asp, Lys, Orn, Glu), N-substituted amido (-N(-)C(O)-), tertiary amino, polyol (e.g., O-substituted glycerol), and the like. [0524] In some embodiments of formula (Iib) or (IIb’), one or more L4 is a branching moiety selected from:
Figure imgf000147_0001
wherein each x and y are each independently 1 to 10, such as 1-6, 1-3, e.g., 1 or 2. In certain embodiments, each x is 1, 2 or 3, e.g., 2. [0525] In some embodiments of formula (Iib) or (IIb’), L5 is selected from –CH2O–; –(CH2CH2O)t–,
Figure imgf000147_0002
wherein: R13 is selected from H, halogen, OH, optionally substituted (C1-C6)alkyl, optionally substituted (C1-C6)alkoxy, COOH, NO2, CN, NH2, -N(R21)2, -OCOR21, -COOR21, -CONHR21, and - NHCOR21; and each r independently 0 to 20, and any of the L5 moieties are optionally further substituted. [0526] In certain embodiments, L5 is –CH2O–. In certain embodiments, L5 is –(CH2CH2O)t–, where t is 1 to 20, such as 1-15, 1-12, 1-10, 1-8, 1-6, or 1 to 4. In certain embodiments, L5 is –NR4CO–, where R4 is H, or optionally substituted (C1-C6)alkyl. In certain embodiments, L5 is -C1-6-alkylene–. [0527] In certain embodiments, L5 is
Figure imgf000147_0003
, where r is 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5. [0528] In certain embodiments,
Figure imgf000148_0001
each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5 and R13 is H, or optionally substituted (C1-C6)alkyl. [0529] In certain embodiments,
Figure imgf000148_0002
20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5 and R13 is H, or optionally substituted (C1-C6)alkyl. [0530] In certain embodiments,
Figure imgf000148_0003
20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5, and R13 is H, or optionally substituted (C1-C6)alkyl. [0531] In certain embodiments,
Figure imgf000148_0004
20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5, and R13 is H, or optionally substituted (C1-C6)alkyl. [0532] In certain embodiments,
Figure imgf000148_0005
each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5.
Figure imgf000148_0006
[0533] In certain embodiments, L5 is , where each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5. [0534] In certain embodiments, L5 is
Figure imgf000148_0007
, where each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5. [0535] In certain embodiments, L5 is
Figure imgf000148_0008
, where each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5. [0536] In certain embodiments, L5 is
Figure imgf000149_0001
, where r is 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5. [0537] In some embodiments of formula (Iib) or (IIb’), L5 comprises one or more of: an amino acid residue (e.g., Asp, Lys, Orn, Glu), an amino acid analogue, N-substituted amido (-N(-)C(O)-), tertiary amino, polyol (e.g., O-substituted glycerol), and the like. Analogs of an amino acid, include but not limited to, unnatural amino acids, as well as other modifications known in the art. The amino acid includes L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. [0538] In some embodiments of formula (Iib) or (IIb’), L1-L5 comprises one or more of the following units: Ra
Figure imgf000149_0002
, where Ra is (C1-C6)alkyl or substituted (C1-C6)alkyl, e.g., a (C1-C6)alkyl optionally substituted with amine, a tertiary amine, optionally substituted alkoxy, optionally substituted carboxyl, optionally substituted aryl, or optionally substituted heteroaryl. It is understood that Ra can be linked to a M6PR binding moiety. [0539] In some embodiments, the linker includes a polypeptide scaffold where some or all of the sidechain groups of the amino acid residues have been modified to attach a ASGPR binding moiety (e.g., as described herein). It is understood that ASGPR binding moieties (e.g., as described herein) can be conjugated to amino acid residues, such as Asp, Lys, Orn, Glu, and Ser, of a polypeptide containing linker via a convenient conjugation chemistry. In some embodiments, the linker contains a polylysine polypeptide. In some embodiments, the linker contains a polyornithine polypeptide. In some embodiments, the linker contains a polyserine polypeptide. In some embodiments, the linker contains a polyaspartate polypeptide. The polypeptide can be a randomly polymerized polymer having an average length, or a polymer of defined length prepared e.g., in a controlled stepwise fashion. In certain embodiments, the polypeptide linker segment has a length of 10-100 amino acid residues, such as 20-90, or 20-50 amino acid residues. In some embodiments, the N-terminal or C-terminal of the polypeptide linker segment is modified to include a linking unit to an additional M6PR binding moiety (e.g., as described herein). In some embodiments, the N-terminal or C-terminal of the polypeptide linker segment is modified with one or more linking units (e.g., as described herein) suitable for attachment to a Y moiety of interest. [0540] In certain embodiments of formula (Iib) or (IIb’), a is 1. In certain embodiments, at least one of b, c, d, and e is not 0. In certain embodiments, b is 1 or 2. In certain embodiments, c is 1 or 2. In certain embodiments, e is 1 or 2. In certain embodiments, b, d and e are independently 1 or 2. In certain embodiments, a, b, d, and e are each 1, and c is 0. [0541] In certain embodiments of formula (II), (IIa) or (Iib), the linker comprises 20 to 100 consecutive atoms, such as 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40 or 20 to 30 consecutive atoms. In certain embodiments, the linker comprises 25 to 100 consecutive atoms, such as 30 to 100, 35 to 100, 40 to 100, 45 to 100, 50 to 100, 55 to 100, 60 to 100, 65 to 100, 70 to 100, 75 to 100, 80 to 100, 85 to 100, 90 to 100, or 95 to 100 consecutive atoms. [0542] In certain embodiments of formula (II), (IIa) or (Iib), the linker comprises 25 or more consecutive atoms, such as 26 or more, 27 or more, 28 or more, 29 or more or 30 or more consecutive atoms. In certain embodiments of formula (II), (IIa) or (Iib), the linker comprises 30 or more consecutive atoms, such as 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37, or more, 38 or more, 39 or more, 40 or even more consecutive atoms. [0543] In certain embodiments, ASGPR binding compounds of this disclosure having a particular configuration with a linker of desired valency and length can specifically bind with high affinity to both the ASGPR and a target simultaneously, and exhibit high uptake activity of a target. The conjugates of this disclosure can thus provide for sequestering of a target protein in the cell’s lysosome and degrading of the target protein. For example, conjugates of trivalent ASGPR binding compounds with 14 or more atoms between the ASGPR binding moiety (e.g., Z1 group) and the branching point of the linker can exhibit superior uptake of cells as compared to conjugates of trivalent ASGPR binding compounds with shorter linkers (e.g., linkers less than 14 atoms) between the ASGPR binding compound (e.g., Z1 group) and the branching point. For example, In certain embodiments, a conjugate having a 1-triazole moiety and a short linkage (e.g., 6 atoms) from the ASGPR ligand to the branching point of the ligand (I-157, linker length of 6 atoms to branching point) exhibited less uptake activity in HepG2 cells than the conjugate having a 1-triazole moiety and a longer linkage (e.g., 14 atoms) from the ASGPR ligand to the branching point (I-143, length of 14 atoms) (see, e.g., FIG.2A). Based on this discovery, described herein are multivalent ASGPR binding compounds having a certain linker length range between the ASGPR binding moiety and the linker branching point which provides desirable binding and cellular uptake of a bound target. [0544] Further, conjugates of trivalent ASGPR binding compounds (e.g., compounds of formula (I) where n = 3) can exhibit superior uptake activity in cells as compared to conjugates of divalent or monovalent ASGPR binding compounds (e.g., compounds of formula (I) where n = 2 or 1). In certain embodiments, conjugate (I-124, n = 3) showed superior uptake activity in HepG2 cells as compared to the divalent conjugate (I-144, n = 2) (see, e.g., FIG.2B). [0545] Still further, conjugates of multivalent ASGPR binding compounds with 12 or more atoms between the branching point of the linker and the Y moiety of interest can exhibit superior uptake of cells as compared to conjugates of multivalent ASGPR binding compounds with shorter linkers (e.g., linkers less than 12 atoms) between the branching point of the linker and the Y moiety of interest. In certain embodiments, conjugates of ASGPR binding compounds having more than 12 atoms between the branching point of the linker and Y exhibit comparable uptake activity. For example, it was observed that conjugates having longer linkers between the ASGPR linker and Y (e.g., conjugates of compounds I- 137, having 81 atoms between the branching point and Y; and I-129, having 33 atoms between the branching point and Y) exhibit comparable activity to a reference conjugate (e.g., conjugate of compound I-124, having 12 atoms between the branching point and Y) (see, e.g., FIG.2B). [0546] As such, in certain embodiments where the linker of formula (II), or (IIb’), or (Iib) is a branched linker, each branch of the linker comprises a linear linker of 14 or more consecutive atoms to covalently link via Z1 each X moiety to a branching point of the linker. In certain embodiments, each branch of the linker comprises a linear linker of 15 or more consecutive atoms to the branching point. In certain embodiments, each branch of the linker comprises a linear linker of 16 or more consecutive atoms to the branching point. In certain embodiments, each branch of the linker comprises a linear linker of 17 or more consecutive atoms to the branching point. In certain embodiments, each branch of the linker comprises a linear linker of 18 or more consecutive atoms to the branching point. In certain embodiments, each branch of the linker comprises a linear linker of 19 or more consecutive atoms to the branching point. [0547] In certain embodiments of formula (II) , or (IIb’), or (Iib), the linker is a branched linker comprising branches covalently linking via Z1 each X moiety to a branching point of the linker, and a linear linker covalently linking the branching point to Y. In certain embodiments, the linear linker covalently linking the branching point to Y is 12 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 15 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 20 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 25 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 30 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 40 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 50 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 60 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 70 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 80 or more consecutive atoms. Exemplary linkers and linking moieties [0548] Exemplary linkers and linking moieties that can be utilized in the preparation of compounds of this disclosure (e.g., that link the ASGPR ligand (X) to the moiety of interest (Y)) are shown in Tables 9-11. [0549] In certain embodiments, the linker is a linear linker or linking moiety as shown in Table 9.
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
[0550] Table 10 includes various linker component synthetic precursors (e.g., linear and branched linker precursors) that can be utilized in the preparation of the subject compounds.
Figure imgf000154_0002
Figure imgf000155_0001
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
Figure imgf000161_0001
Figure imgf000162_0001
Figure imgf000163_0001
Figure imgf000164_0001
[0551] In certain embodiments, the linker is a branched linker or linking moiety as shown in Table 11.
Figure imgf000164_0002
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
Chemoselective ligation group [0552] In certain embodiments of formula (I), Y is a chemoselective ligation group, or a precursor thereof. A chemoselective ligation group is a group having a reactive functionality or function group capable of conjugation to a compatible group of a second moiety. For example, chemoselective ligation groups (or a precursor thereof) may be one of a pair of groups associated with a conjugation chemistry such as azido-alkyne click chemistry, copper free click chemistry, Staudinger ligation, tetrazine ligation, hydrazine-iso-Pictet-Spengler (HIPS) ligation, cysteine-reactive ligation chemistry (e.g., thiol-maleimide, thiol-haloacetamide or alkyne hydrothiolation), amine-active ester coupling, tyrosine specific conjugation chemistry (e.g., e-Y-CLICK), methionine specific conjugation chemistry (e.g., oxaziridine-based or ReACT chemistry), reductive amination, dialkyl squarate chemistry, etc. [0553] Chemoselective ligation groups that may be utilized in linking two moieties, include, but are not limited to, amino (e.g., a N-terminal amino or a lysine sidechain group of a polypeptide), azido, aryl azide, alkynyl (e.g., ethynyl or cyclooctyne or derivative), active ester (e.g., N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester or PFP ester or thioester), haloacetamide (e.g., iodoacetamide or bromoacetamide), chloroacetyl, bromoacetyl, bromomethyl-aryl, chloromethyl-aryl, bromomethyl- heteroaryl, chloromethyl-heteroaryl, hydrazide, maleimide, vinyl sulfone, 2-sulfonyl pyridine, cyano- alkyne, thiol (e.g., a cysteine residue), disulfide or protected thiol, isocyanate, isothiocyanate, aldehyde, ketone, alkoxyamine, hydrazide, aminooxy, phosphine, HIPS hydrazinyl-indolyl group, or aza-HIPS hydrazinyl-pyrrolo-pyridinyl group, tetrazine, cyclooctene, squarate, and the like. [0554] In some instances, chemoselective ligation group is capable of spontaneous conjugation to a compatible chemical group when the two groups come into contact under suitable conditions (e.g., copper free Click chemistry conditions). In some instances, the chemoselective ligation group is capable of conjugation to a compatible chemical group when the two groups come into contact in the presence of a catalyst or other reagent (e.g., copper catalyzed Click chemistry conditions). [0555] In some embodiments, the chemoselective ligation group is a photoactive ligation group. For example, upon irradiation with ultraviolet light, a diazirine group can form reactive carbenes, which can insert into C-H, N-H, and O-H bonds of a second moiety. [0556] In some instances, Y is a precursor of the reactive functionality or function group capable of conjugation to a compatible group of a second moiety. For example, a carboxylic acid is a precursor of an active ester chemoselective ligation group. [0557] In certain embodiments of formula (I), Y is a reactive moiety capable forming a covalent bond to a polypeptide (e.g., with an amino acid sidechain of a polypeptide having a compatible reactive group). The reactive moiety can be referred to as a chemoselective ligation group. [0558] In certain embodiments of formula (I), Y is a thio-reactive chemoselective ligation group (e.g., as described in Table 12). In certain embodiments, Y can produce a residual moiety Z resulting from the covalent linkage of a thiol-reactive chemoselective ligation group to one or more cysteine residue(s) of a protein, e.g., Ab. [0559] In certain embodiments of formula (I), Y is a Cys-reactive chemoselective ligation group (e.g., a maleimide derivative as described in table 12). In certain embodiments, the Cys-reactive chemoselective ligation group includes a maleimide group. In some embodiments, the chemoselective ligation group includes a maleimide group of Table 12, e.g., mal-1 to mal-7. [0560] In certain embodiments of formula (I), Y is an amino-reactive chemoselective ligation group (e.g., as described in Table 12). In certain embodiments, Y can produce a residual moiety Z resulting from the covalent linkage of an amine-reactive chemoselective ligation group to one or more lysine residue(s) a protein, e.g., Ab. [0561] In certain embodiments of formula (I), Y is a Lys-reactive chemoselective ligation group (e.g., an active ester as described in table 12). In some embodiments the Lys-reactive chemoselective ligation group is a PFP ester. [0562] Exemplary chemoselective ligation groups, and synthetic precursors thereof, which may be adapted for use in the compounds of this disclosure are shown in Table 12.
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000174_0001
[0563] In Table 12, the can represent a point of attachment of Y to a linking moiety or a linked X moiety. [0564] Table 12a shows exemplary residual moieties, wherein the “***” indicates the point of attachment of Y.
Figure imgf000175_0001
Exemplary Compounds with Chemoselective Ligation Group [0565] This disclosure includes compounds of formula (I) which can include: (1) one or more particular ASGPR ligand (X) (e.g., as described herein, such as ligands X1-X20 of Tables 1-4) or a particular ASGPR ligand (X) (e.g., as described herein), (2) a linker including one or more linking moieties (e.g., as described herein, such as any one or more of the linking moieties of Tables 8 to 10); and (3) a chemoselective ligation group (Y) e.g., as described herein, such as any one of the groups of Table 12). [0566] In some embodiments, the chemoselective ligation group can be tailored to provide linkages which confer additional benefits, such as, but not limited to, stability of the conjugate. [0567] In some embodiments, the chemoselective ligation group comprises:
Figure imgf000176_0001
[0568] Table 13 illustrates various monovalent ligand-linker compounds for use in conjugates of the disclosure.
Figure imgf000176_0002
Figure imgf000177_0001
[0569] Tables 14 illustrates various multivalent ligand-linker compounds for use in conjugates of the disclosure.
Figure imgf000177_0002
Figure imgf000178_0001
[0570] In some embodiments, the compound of formula (I) is an ASGPR binding compound as described in International Application No. WO/2023288033, filed July 14, 2022, and the disclosure of which is herein incorporated by reference in its entirety. [0571] The following Tables illustrate several exemplary ASGPR binding compounds of this disclosure that include a chemoselective ligation group, or a precursor thereof. It is understood that this disclosure includes Y (e.g., as described herein) conjugates of each of the exemplary compounds of Tables 13-23. For example, conjugates where the chemoselective ligation group has been conjugated to a different Y, such as a biomolecule or a small molecule ligand for a target protein. [0572] The chemoselective ligation group of such compounds can be utilized to connect to another Y moiety of interest (e.g., as described below). It is understood that any of these compounds can also be prepared de novo to include an alternative Y moiety of interest (e.g., as described below) rather than the chemoselective ligation group. In some embodiments, such compounds are referred to as a conjugate, e.g., a biomolecule conjugate that specifically binds a target protein.
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
Figure imgf000191_0001
Figure imgf000192_0001
Figure imgf000193_0001
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
Figure imgf000199_0001
Figure imgf000200_0001
Figure imgf000201_0001
Figure imgf000202_0001
Figure imgf000203_0001
Figure imgf000204_0001
Figure imgf000205_0001
Figure imgf000206_0001
Figure imgf000207_0001
Figure imgf000208_0001
Figure imgf000209_0001
Figure imgf000210_0002
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000212_0001
[0573] The present disclosure is meant to encompass stereoisomers of any one of the compounds described herein. In some instance, the compound includes an enantiomer of the D- N- acetylgalactosamine (GalNAc), or an analog or derivative of GalNAc. Other Exemplary Compounds [0574] Table 19 illustrates exemplary ASGPR binding compounds of this disclosure that include a binding moiety, or a precursor thereof.
Figure imgf000213_0001
Figure imgf000214_0001
[0575] Table 20 illustrates exemplary trivalent ASGPR binding intermediate compounds of this disclosure including X groups of formula (Ie).
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000218_0001
Figure imgf000219_0001
Figure imgf000220_0001
Figure imgf000221_0001
Figure imgf000222_0001
Figure imgf000223_0001
Figure imgf000224_0001
Figure imgf000225_0001
Figure imgf000226_0001
[0576] Table 21 illustrates exemplary monovalent ASGPR binding intermediate compounds of this disclosure that include a promoiety and X groups that are of formula (Ib).
Figure imgf000226_0002
[0577] Table 22 illustrates exemplary ASGPR binding intermediate compounds of this disclosure that include X groups that are of formula (In).
Figure imgf000227_0002
[0578] Table 23 illustrates exemplary ASGPR binding intermediate compounds.
Figure imgf000227_0001
Figure imgf000228_0001
Figure imgf000229_0001
Figure imgf000230_0001
Figure imgf000231_0001
Figure imgf000232_0001
Figure imgf000233_0001
Figure imgf000234_0001
Figure imgf000235_0001
Figure imgf000236_0001
Figure imgf000237_0001
Figure imgf000238_0001
Figure imgf000239_0001
Figure imgf000240_0001
Figure imgf000241_0001
Figure imgf000242_0001
Figure imgf000243_0001
Figure imgf000244_0001
Figure imgf000245_0001
Figure imgf000246_0001
Figure imgf000247_0001
Figure imgf000248_0001
Figure imgf000249_0001
Figure imgf000250_0001
Figure imgf000251_0001
Figure imgf000252_0001
Figure imgf000253_0001
Figure imgf000254_0001
Figure imgf000255_0001
[0579] The present disclosure is meant to encompass stereoisomers of any one of the compounds described herein. In some instance, the compound includes an enantiomer of the D-N- acetylgalactosamine (GalNAc), or an analog or derivative of GalNAc. Conjugates with Moiety of Interest [0580] The compounds of this disclosure can be referred to as a conjugate, e.g., when the moiety of interest (Y) is a molecule (e.g., as described herein). Such conjugates can be prepared by conjugation of a chemoselective ligation group of any one of the compounds described herein with a compatible reactive group of a molecule Y. The compatible group of the molecule Y can be introduced by modification prior to conjugation, or can be a group present in the molecule. Alternatively, such conjugates can be prepared de novo, e.g., via modification of a Y molecule of interest starting material to introduce a linker, e.g., to which a ligand X can be attached. [0581] In some embodiments, the moiety of interest to which the ASGPR binding moiety is linked is a biomolecule. In some embodiments, the moiety of interest is a biomolecule. In some embodiments, the biomolecule is selected from peptide, protein, polynucleotide, polysaccharide, glycan, glycoprotein, lipid, enzyme, antibody, and antibody fragment. In some embodiments, the moiety of interest Y is selected from small molecule, small molecule drug, chemotherapeutic agent, cytotoxic agent, diagnostic agent, dye, fluorophore, and the like. [0582] In preferred embodiments, the moiety of interest is a molecule that specifically binds to a target of interest, i.e., a target-binding moiety. In such embodiments, the conjugates of this disclosure can provide for cellular uptake of the target after it non-covalently binds to the conjugate, and/or degradation. In certain embodiments, conjugates of this disclosure having a particular configuration of ASGPR binding moiety of a desired affinity, with a linker of desired valency and length can specifically bind with high affinity to both the ASGPR and the target simultaneously. The conjugates of this disclosure can thus provide for sequestering of a target protein in the cell’s lysosome and degrading of the target protein. [0583] In some embodiments, the moiety of interest is a molecule that does not bind to an extracellular target, but rather is a molecule that is itself desirable to deliver intracellularly. In some embodiments, the moiety of interest is selected from enzymes (e.g., lysosomal enzyme), a nanoparticle, a viral composition (e.g., viral particle), therapeutic protein, therapeutic antibodies and cytotoxic agents. [0584] In some embodiments, the moiety of interest is a lysosomal enzyme for delivery to a cell for use in enzyme replacement therapy, such as acid alpha-glucosidase (GAA). Lysosomal enzymes of interest that may be adapted for use in conjugates of this disclosure include, but are not limited to, acid alpha-glucosidase, acid beta-galactosidase-1, acid sphingomyelinase, alpha-D-mannosidase, alpha- fucosidase, alpha-galactosidase A, alpha-glucosaminide acetyltransferase, alpha-glucosidase, alpha-L- iduronidase, alpha-N-acetylgalactosaminidase, alpha-acetylglucosaminidase, alpha-D-neuraminidase, arylsulfatase A, arylsulfatase B, beta-galactosidase, beta-glucuronidase, beta-mannosidase, cathepsin D, cathepsin K, ceramidase, cystinosine, ganglioside activator GM2, galactocerebrosidase, glucocerebrosidase, heparan sulfatase, hexosaminidase A, hexosaminidase B, hyaluronidase, iduronate-2- sulfatase, LAMP2, lysosomal acid lipase, N-acetylglucosamine-1-phosphotransferase, N- acetylgalactosamine 6-sulfatase, N-acetylglucosamine-1-phosphotransferase, N-acetylglucosamine-6- sulfate sulfatase, N-aspartyl-beta-glucosaminidase, palmitoyl-thioesterase-1, acid phosphatase, protected protein/cathepsin A (PPCA), sialin, tripeptidyl-peptidase 1. [0585] Aspects of this disclosure include compounds of formula (I) where the moiety of interest Y is a selected from small molecule, dye, fluorophore, monosaccharide, disaccharide, trisaccharide, and biomolecule. In some embodiments, Y is a small molecule that specifically binds to a target molecule, such as a target protein. [0586] In some embodiments of the compounds of this disclosure, Y is a biomolecule. In some embodiments, the biomolecule is selected from protein, polynucleotide, polysaccharide, peptide, glycoprotein, lipid, enzyme, antibody, and antibody fragment. In some embodiments, Y is a biomolecule that specifically binds to a target molecule, such as a target protein. [0587] The compounds of this disclosure can, in certain embodiments, be referred to as a conjugate, e.g., when the moiety of interest (Y) is a molecule such as a biomolecule, where the conjugate can be derived from a conjugation or coupling reaction between a chemoselective ligation group and a compatible group on the biomolecule. In some embodiments, the biomolecule is conjugated via a naturally occurring group of the biomolecule. In some embodiments, the biomolecule is conjugated via a compatible functional group that is introduced into the biomolecule prior to chemoselective conjugation. In such embodiments, the linking moiety between X and Y incorporates the residual group (e.g., Z) that is the product of the chemoselective ligation chemistry. [0588] Aspects of this disclosure include compounds of formula (I) where the moiety of interest Y is a moiety that specifically binds to a target molecule, such as a target protein. The target protein can be the target protein is a membrane bound protein or an extracellular protein. In some embodiments of the compounds of this disclosure, Y is a biomolecule that specifically binds to a target protein. This disclosure provides conjugates of the particular ASGPR binding compounds and conjugates. In some embodiments, the conjugate includes a moiety of interest Y that specifically binds a target protein, and can find use in methods of cell uptake or internalization of the target protein via binding to the cell surface receptor, and eventual degradation of the target protein. [0589] In some embodiments, Y is an aptamer that specifically binds to a target molecule, such as a target protein. In some embodiments, Y is a peptide or protein (e.g., peptidic binding motif, protein domain, engineered polypeptide, or glycoprotein) that specifically binds to a target molecule, such as a target protein. In some embodiments, Y is an antibody or antibody fragment that specifically binds to a target molecule, such as a target protein. In some embodiments, Y is a polynucleotide or oligonucleotide that specifically binds to a target molecule, such as a target protein or a target nucleic acid. [0590] In some embodiments, one Y biomolecule is conjugated to a single moiety (X) that specifically binds to the cell surface receptor (e.g., ASGPR) via a linker L. In some embodiments, one Y biomolecule is conjugated to one (Xn-L)- group, wherein when n =1 the (Xn-L)- group is referred to as monovalent, and when n > 1 the (Xn-L)- group is referred to as multivalent (e.g., bivalent, trivalent, etc.). It is understood that in some embodiments of formula (I), where Y is a biomolecule, Y can be conjugated to two or more (Xn-L)- groups, wherein each (Xn-L)- group may itself be monovalent or multivalent (e.g., bivalent, trivalent, etc.). In such embodiments, the ratio of linked (Xn-L)- groups to biomolecule can be referred to as 2 or more. [0591] In some embodiments, Y is a moiety that specifically binds the target protein and the compound is a conjugate of formula (III’):
Figure imgf000258_0001
wherein: n is 1 to 20; m is an average loading of 1 to 80; each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group to a compatible group of Y; and Y is a moiety of interest binds the target protein. [0592] In some embodiments of formula (III’), Y is an antibody or an antibody fragment. [0593] In some embodiments, Y is an antibody or antibody fragment that specifically binds the target protein and the compound is a conjugate of formula (III):
Figure imgf000258_0002
wherein: n is 1 to 20; m is an average loading of 1 to 80; each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group to a compatible group of Ab; and Ab is the antibody or antibody fragment that specifically binds the target protein. [0594] In certain embodiments of the conjugate of formula (III), n is 1 to 6. In certain cases, n is 1, such that the antibody is conjugated to a monovalent ligand and the linker is of the formula (IIa) (e.g., as described herein). In certain cases, n is at least 2, such that the antibody is conjugated to a multivalent ligand. In certain cases, n is 2. In certain cases n is 3. [0595] In certain embodiments of the conjugate of formula (III), Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation moiety of Table 12 (e.g., Table 12a). [0596] In certain embodiments of formula (III) or (III’), each X is independently of formula (Ib) (e.g., as described herein). In certain embodiments, each X is independently selected from a compound of Table 1. In certain embodiments, each X is independently selected from one of the following compounds:
Figure imgf000259_0001
. [0597] In certain embodiments of formula (III) or (III’), each X is independently selected from one of the following compounds:
Figure imgf000259_0002
wherein R5 and R4 independently H or a promoiety, or R5 and R4 are cyclically linked to form a promoiety; n1 and n2 are each independently an integer from 1 to 6; and Y4 is a suitable counterion. In some embodiments, Y4 is sodium. [0598] In certain embodiments of formula (III) or (III’), n is 1 and X is:
Figure imgf000259_0003
. [0599] In certain other embodiments of formula (III) or (III’), each X is independently of the formula (Ic) (e.g., as described herein). In certain embodiments, each X is independently selected from a compound of Table 2 or 2a. [0600] In certain other embodiments of formula (III) or (III’), each X is independently of formula (Id) (e.g., as described herein). In certain embodiments, each X is independently selected from a compound of Table 3 or 3a. In certain embodiments, each X is independently selected from a compound of Table 4. In certain embodiments, each X is a compound of Table 5. [0601] In certain embodiments of formula (III), each X is independently selected from one of the following compounds:
Figure imgf000260_0001
[0602] In certain embodiments of the conjugate of formula (III) or (III’), L is a linker of formula (II) (e.g., as described herein). [0603] In certain embodiments of the conjugate of formula (III) or (III’), n is 1 to 6. In certain embodiments, n is 1, such that the antibody is conjugated to a monovalent ASGPR ligand and the linker is of the formula (IIa) (e.g., as described herein). In certain embodiments, n is at least 2, such that the antibody is conjugated to a multivalent ASGPR ligand. In certain embodiments, n is 2. In certain cases n is 3. [0604] In certain embodiments of the conjugate of formula (III) or (III’), Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation moiety of Table 9. [0605] In certain embodiments of the conjugate of formula (III) or (III’), Z is a residual moiety resulting from the covalent linkage of a thiol reactive chemoselective ligation group to one or more cysteine residue(s) of Ab. In certain embodiments, the thiol-reactive chemoselective ligation group is a maleimide derivative. [0606] In certain other embodiments of the conjugate of formula (III), Z is a residual moiety resulting from the covalent linkage of an amine-reactive chemoselective ligation group to one or more lysine residue(s) of Ab. In certain embodiments, the amine-reactive chemoselective ligation group is an active ester. In certain embodiments, the active ester is a PFP ester. [0607] In certain embodiments, the conjugates with their linker structures described herein have weaker binding affinity to cell surface receptors. Without being bound to any particular mechanism or theory, such weaker binding affinity may be corrected to longer half-life of the conjugates, and may be useful for tuning (e.g., modifying) the pharmacokinetic properties of the conjugates described herein. In certain embodiments, such weaker binding conjugates still have sufficiently robust uptake. [0608] Conjugates of a polypeptide, e.g., an antibody (Ab) and compound (Xn-L-Y) may be made using a variety of bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC- SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo- KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4- vinylsulfone)benzoate). The present disclosure further contemplates that the conjugates described herein may be prepared using any suitable methods as disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2d ed.2008)). [0609] In certain embodiments of the conjugates described herein, L is bonded through an amide bond to a lysine residue of the polypeptide. In certain embodiments of the conjugates described herein, L is bonded through a thioether bond to a cysteine residue of the polypeptide. In certain embodiments of the conjugates described herein, L is bonded through an amide bond to a lysine residue of Ab. In certain embodiments of the conjugates described herein, L is bonded through a thioether bond to a cysteine residue of Ab. In certain embodiments of the conjugates described herein, L is bonded through two thioether bonds to two cysteine residues of Ab, wherein the two cysteine residues are from an opened cysteine-cysteine disulfide bond in Ab. In certain embodiments, the opened cysteine-cysteine disulfide bond is an interchain disulfide bond. [0610] In certain embodiments of the conjugates described herein, when L is bonded through an amide bond to a lysine residue of a polypeptide (e.g., an antibody), m is an integer from 1 to 80. In certain embodiments of the conjugates described herein, when L is bonded through a thioether bond to a cysteine residue of P, m is an integer from 1 to 8. [0611] In certain embodiments, conjugation to the polypeptide, or the antibody Ab may be via site- specific conjugation. Site-specific conjugation may, for example, result in homogeneous loading and minimization of conjugate subpopulations with potentially altered antigen-binding or pharmacokinetics. In certain embodiments, for example, conjugation may comprise engineering of cysteine substitutions at positions on the polypeptide or antibody, e.g., on the heavy and/or light chains of an antibody that provide reactive thiol groups and do not disrupt polypeptide or antibody folding and assembly or alter polypeptide or antigen binding (see, e.g., Junutula et al., J. Immunol. Meth.2008; 332: 41-52; and Junutula et al., Nature Biotechnol.2008; 26: 925-32; see also WO2006/034488 (herein incorporated by reference in its entirety)). In another non-limiting approach, selenocysteine is cotranslationally inserted into a polypeptide or antibody sequence by recoding the stop codon UGA from termination to selenocysteine insertion, allowing site specific covalent conjugation at the nucleophilic selenol group of selenocysteine in the presence of the other natural amino acids (see, e.g., Hofer et al., Proc. Natl. Acad. Sci. USA 2008; 105: 12451-56; and Hofer et al., Biochemistry 2009; 48(50): 12047-57). Yet other non- limiting techniques that allow for site-specific conjugation to polypeptides or antibodies include engineering of non-natural amino acids, including, e.g., p-acetylphenylalanine (p-acetyl-Phe), p- azidomethyl-N-phenylalanine (p-azidomethyl-Phe), and azidolysine (azido-Lys) at specific linkage sites, and can further include engineering unique functional tags, including, e.g., LPXTG, LLQGA, sialic acid, and GlcNac, for enzyme mediated conjugation. See Jackson, Org. Process Res. Dev.2016; 20: 852-866; and Tsuchikama and An, Protein Cell 2018; 9(1):33-46, the contents of each of which is incorporated by reference in its entirety. See also US 2019/0060481 A1 & US 2016/0060354 A1, the contents of each of which is incorporated by reference in its entirety. All such methodologies are contemplated for use in connection with making the conjugates described herein. [0612] Loading of the compounds of formula (I) to the polypeptides (e.g., antibodies) described herein is represented by “m” in formula (III), and is the average number of units of “Xn-L-” or “Xn-” per conjugate molecule. As used herein, the term “DAR” refers to the average value of “m” or the loading of the conjugate. The number of “X” moieties (e.g., folate moieties) per each unit of “Xn-L-” or “Xn-” is represented by “n” in formula (III). As used herein, the term “valency” or “valencies” refers to the number of “X” moieties per unit (“n”). It will be understood that loading, or DAR, is not necessarily equivalent to the number of “X” moieties per conjugate molecule. By means of example, where there is one “X” moiety per unit (n = 1; valency is “1”), and one “Xn-L-” unit per conjugate (m = 1), there will be 1 x 1 = 1 “X” moiety per conjugate. However, where there are two “X” moieties per unit (n = 2; valency is “2”), and four “Xn-L-” units per conjugate (m = 4), there will be 2 x 4 = 8 “X” moieties per conjugate. Accordingly, for the conjugates described herein, the total number of “X” moieties per conjugate molecule will be n x m. As used herein, the term “total valency” or “total valencies” refers to the total number of “X” moieties per conjugate molecule (n x m; total valency). [0613] DAR (loading) may range from 1 to 80 units per conjugate. The conjugates provided herein may include collections of polypeptides, antibodies or antigen binding fragments conjugated with a range of units, e.g., from 1 to 80. The average number of units per polypeptide or antibody in preparations of the conjugate from conjugation reactions may be characterized by conventional means such as mass spectroscopy. The quantitative distribution of DAR (loading) in terms of m may also be determined. In some instances, separation, purification, and characterization of homogeneous conjugate where m is a certain value may be achieved by means such as electrophoresis. [0614] In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 80. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 70. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 60. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 50. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 40. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 35. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 30. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 25. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 20. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 18. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 15. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 12. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 10. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 9. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 8. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 7. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 6. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 5. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 4. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 3. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 12. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 10. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 9. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 8. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 7. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 6. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 5. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 4. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 12. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 10. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 9. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 8. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 7. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 6. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 5. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 4. [0615] In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to about 8; from about 2 to about 6; from about 3 to about 5; from about 3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about 3.7. [0616] In certain embodiments, the DAR for a conjugate provided herein is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or more. In some embodiments, the DAR for a conjugate provided herein is about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. [0617] In some embodiments, the DAR for a conjugate provided herein ranges from 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, or 2 to 13. In some embodiments, the DAR for a conjugate provided herein ranges from 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, or 3 to 13. In some embodiments, the DAR for a conjugate provided herein is about 1. In some embodiments, the DAR for a conjugate provided herein is about 2. In some embodiments, the DAR for a conjugate provided herein is about 3. In some embodiments, the DAR for a conjugate provided herein is about 4. In some embodiments, the DAR for a conjugate provided herein is about 3.8. In some embodiments, the DAR for a conjugate provided herein is about 5. In some embodiments, the DAR for a conjugate provided herein is about 6. In some embodiments, the DAR for a conjugate provided herein is about 7. In some embodiments, the DAR for a conjugate provided herein is about 8. In some embodiments, the DAR for a conjugate provided herein is about 9. In some embodiments, the DAR for a conjugate provided herein is about 10. In some embodiments, the DAR for a conjugate provided herein is about 11. In some embodiments, the DAR for a conjugate provided herein is about 12. In some embodiments, the DAR for a conjugate provided herein is about 13. In some embodiments, the DAR for a conjugate provided herein is about 14. In some embodiments, the DAR for a conjugate provided herein is about 15. In some embodiments, the DAR for a conjugate provided herein is about 16. In some embodiments, the DAR for a conjugate provided herein is about 17. In some embodiments, the DAR for a conjugate provided herein is about 18. In some embodiments, the DAR for a conjugate provided herein is about 19. In some embodiments, the DAR for a conjugate provided herein is about 20. [0618] In some embodiments, the DAR for a conjugate provided herein is about 25. In some embodiments, the DAR for a conjugate provided herein is about 30. In some embodiments, the DAR for a conjugate provided herein is about 35. In some embodiments, the DAR for a conjugate provided herein is about 40. In some embodiments, the DAR for a conjugate provided herein is about 50. In some embodiments, the DAR for a conjugate provided herein is about 60. In some embodiments, the DAR for a conjugate provided herein is about 70. In some embodiments, the DAR for a conjugate provided herein is about 80. [0619] In certain embodiments, fewer than the theoretical maximum of units are conjugated to the polypeptide, e.g., antibody, during a conjugation reaction. A polypeptide may contain, for example, lysine residues that do not react with the compound or linker reagent. Generally, for example, antibodies do not contain many free and reactive cysteine thiol groups which may be linked to a drug unit; indeed most cysteine thiol residues in antibodies exist as disulfide bridges. In certain embodiments, an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups. In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine. In some embodiments, the compound is conjugated via a lysine residue on the antibody. In some embodiments, the linker unit or a drug unit is conjugated via a cysteine residue on the antibody. [0620] In certain embodiments, the amino acid that attaches to a unit is in the heavy chain of an antibody. In certain embodiments, the amino acid that attaches to a unit is in the light chain of an antibody. In certain embodiments, the amino acid that attaches to a unit is in the hinge region of an antibody. In certain embodiments, the amino acid that attaches to a unit is in the Fc region of an antibody. In certain embodiments, the amino acid that attaches to a unit is in the constant region (e.g., CH1, CH2, or CH3 of a heavy chain, or CH1 of a light chain) of an antibody. In yet other embodiments, the amino acid that attaches to a unit or a drug unit is in the VH framework regions of an antibody. In yet other embodiments, the amino acid that attaches to unit is in the VL framework regions of an antibody. [0621] The DAR (loading) of a conjugate may be controlled in different ways, e.g., by: (i) limiting the molar excess of compound or conjugation reagent relative to polypeptide, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the polypeptide, such that the number and position of cysteine residues is modified for control of the number and/or position of linker- drug attachments (such as for thiomabs prepared as disclosed in WO2006/034488 (herein incorporated by reference in its entirety)). [0622] It is to be understood that the preparation of the conjugates described herein may result in a mixture of conjugates with a distribution of one or more units attached to a polypeptide, for example, an antibody. Individual conjugate molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography, including such methods known in the art. In certain embodiments, a homogeneous conjugate with a single DAR (loading) value may be isolated from the conjugation mixture by electrophoresis or chromatography. [0623] In certain embodiments of the conjugate of formula (III) m is 1 to 20, such as 2 to 10, 2 to 8, or 2 to 6. In certain embodiments, m is 10 or less. In certain embodiments, m is 2 to 8. In certain embodiments, m is 2 to 6. In certain embodiments, m is an average loading of about 4. [0624] It is to be understood that the preparation of the conjugates described herein may result in a mixture of conjugates with a distribution of one or more units attached to a polypeptide, for example, an antibody. Individual conjugate molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography, including such methods known in the art. In certain embodiments, a homogeneous conjugate with a single DAR (loading) value may be isolated from the conjugation mixture by electrophoresis or chromatography. Target-binding Moieties [0625] The target-binding moiety can be any moiety that has an affinity for the target of less than 1 µM, such as 300nM or less, 100nM or less, 30nM or less, 10nM or less, 3nM or less, or 1nM or less, e.g., as measured in an in vitro binding assay. [0626] In some embodiments, the target-binding moiety is a biomolecule. In some embodiments, the target-binding moiety is a biomolecule that specifically binds to a target protein. In some embodiments, the biomolecule is selected from peptide, protein, polynucleotide, polysaccharide, glycan, glycoprotein, lipid, enzyme, antibody, and antibody fragment. [0627] In some embodiments, the target-binding moiety is a polypeptide (e.g., peptide or protein binding motif, protein domain, engineered polypeptide, or glycoprotein) that specifically binds to a target molecule, such as a target protein. In some embodiments, the target-binding moiety of the bifunctional compound includes a polypeptide that binds to a soluble (e.g., secreted) target protein of interest. In some embodiments, the target-binding is a polypeptide ligand that includes a receptor ligand, or a receptor-binding portion or fragment of the receptor ligand, that binds a target cell surface receptor. Target-binding polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of naturally occurring amino acids, non-naturally occurring amino acids, and/or amino acid modifications or analogs known in the art. Useful modifications include, e.g., N-terminal acetylation, amidation, methylation, etc. [0628] In some embodiments, the target-binding moiety is a polynucleotide that specifically binds to a target molecule, such as a target protein or a target nucleic acid. The terms polynucleotide and nucleic acid can be used interchangeably. In some embodiments, the target-binding moiety is a nucleic acid aptamer that specifically binds to a target molecule, such as a target protein. [0629] In some embodiments, the target-binding moiety is a glycan. In some embodiments, the target-binding moiety is a glycan epitope for an autoantibody. Antibodies [0630] In some embodiments, the target-binding moiety is an antibody or antibody fragment that specifically binds to a target moiety, such as a target protein. [0631] The ASGPR binding moiety can be site-specifically covalently linked to the antibody or antibody fragment, via an optional linking moiety. ASGPR binding moiety can be covalently linked to the antibody or antibody fragment via a site-specific cysteine modification on the antibody or antibody fragment (e.g., L443C) and a thiol-reactive chemoselective ligation group. ASGPR binding moiety can be covalently linked to the antibody or antibody fragment via one or more lysine residues of the antibody or antibody fragment and an amine-reactive chemoselective ligation group. [0632] In some embodiments, the bifunctional conjugate of this disclosure includes an antibody (Ab). In some embodiments, Ab is a monoclonal antibody. In some embodiments, Ab is a human antibody. In some embodiments, Ab is a humanized antibody. In some embodiments, Ab is a chimeric antibody. In some embodiments, Ab is a full-length antibody that includes two heavy chains and two light chains. In some embodiments, Ab is an IgG antibody, e.g., is an IgG1, IgG2, IgG3 or IgG4 antibody. In some embodiments, Ab is a single chain antibody. In some embodiments, the target- binding moiety is an antigen-binding fragment of an antibody, e.g., a Fab fragment. [0633] In some embodiments, the antibody or antibody fragment specifically binds to a cancer antigen. [0634] In some embodiments, the antibody or antibody fragment specifically binds to a hepatocyte antigen. [0635] In some embodiments, the antibody or antibody fragment specifically binds to an antigen presented on a macrophage. [0636] In some embodiments, the antibody or antibody fragment specifically binds to an intact complement or a fragment thereof. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within intact complement or a fragment thereof. [0637] In some embodiments, the antibody or antibody fragment specifically binds to a cell surface receptor. In some embodiments, the antibody or antibody fragment specifically binds to a cell surface receptor ligand. [0638] In some embodiments, the antibody or antibody fragment specifically binds to an epidermal growth factor (EGF) protein, e.g., a human EGF. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within an EGF protein. [0639] In some embodiments, the antibody or antibody fragment specifically binds to an epidermal growth factor receptor (EGFR) protein, e.g., a human EGFR. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within an EGFR protein. In some embodiments, the antibody or antibody fragment comprises the CDRs present in cetuximab. In some embodiments, the antibody or antibody fragment includes the variable light chain and variable heavy chain present in cetuximab. In some embodiments, the antibody is cetuximab. In some embodiments, the antibody or antibody fragment includes the CDRs present in matuzumab. In some embodiments, the antibody or antibody fragment includes the variable light chain and variable heavy chain present in matuzumab. In some embodiments, the antibody is matuzumab. [0640] In some embodiments, the antibody or antibody fragment specifically binds to vascular endothelial growth factor (VEGF) protein, e.g., human VEGF protein. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within a VEGF protein. [0641] In some embodiments, the antibody or antibody fragment specifically binds to a vascular endothelial growth factor receptor (VEGFR) protein, e.g., human VEGFR protein. In some embodiments, the antibody or antibody fragment specifically binds vascular endothelial growth factor receptor 2 (VEGFR2) protein, e.g., a human VEGFR2 protein. In some embodiments, the antibody or antibody fragment specifically binds a vascular endothelial growth factor receptor 3 (VEGFR3) protein, e.g., a human VEGFR3 protein. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within a VEGFR protein, a VEGFR2 protein or a VEGFR3 protein. [0642] In some embodiments, the antibody or antibody fragment specifically binds to a fibroblast growth factor (FGF), e.g., a human FGF. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within a FGF protein. [0643] In some embodiments, the antibody or antibody fragment specifically binds to a fibroblast growth factor receptor (FGFR), e.g., a human FGFR. In some embodiments, the antibody or antibody fragment specifically binds fibroblast growth factor receptor 2 (FGFR2) protein, e.g., a human FGFR2 protein, for example, a FGFR2b protein. In some embodiments, the antibody or antibody fragment specifically binds a fibroblast growth factor receptor 3 (FGFR3) protein, e.g., a human FGFR3 protein. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within a FGFR protein, a FGFR2 protein or a FGFR3 protein. [0644] In some embodiments, the antibody specifically binds to a receptor tyrosine kinase cMET protein. In some embodiments, the antibody specifically binds to one or more immunodominant epitope(s) within a receptor tyrosine kinase cMET protein. [0645] In some embodiments, the antibody specifically binds to a CD47 protein, e.g., a human CD47 protein. In some embodiments, the antibody specifically binds to one or more immunodominant epitope(s) within a CD47 protein. [0646] In some embodiments, the antibody specifically binds to an immune checkpoint inhibitor. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within an immune checkpoint inhibitor. In some embodiments, the antibody specifically binds to a programmed death protein, e.g., a human PD-1. In some embodiments, the antibody specifically binds to one or more immunodominant epitope(s) within PD-1 protein. [0647] In some embodiments, the antibody specifically binds to a programmed death ligand-1 (PD- L1) protein, e.g., a human PD-L1. In some embodiments, the antibody specifically binds to one or more immunodominant epitope(s) within PD-L1 protein. [0648] In some embodiments, the antibody binds to TIM3. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within TIM3. [0649] In some embodiments, the antibody specifically binds to a lectin. In some embodiments, the antibody specifically binds to one or more immunodominant epitope(s) within a lectin. In some embodiments, the antibody binds to SIGLEC. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within SIGLEC. In some embodiments, the antibody binds to a cytokine receptor. In some embodiments, the antibody binds to a one or more immunodominant epitope(s) within cytokine receptor. In some embodiments, the antibody binds to sIL6R. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within sIL6R. In some embodiments, the antibody binds to a cytokine. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within a cytokine. In some embodiments, the antibody binds to MCP-1, TNF (e.g., a TNF- alpha), IL1a, IL1b, IL4, IL5, IL6, IL12/IL23, IL13, IL17 or p40. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within MCP-1, TNF (e.g., a TNF-alpha), IL1a, IL1b, IL4, IL5, IL6, IL12/IL23, IL13, IL17 or p40. [0650] In some embodiments, the antibody binds to a major histocompatibility protein (e.g., a MHC class I or class II molecule). In some embodiments, the antibody binds to one or more immunodominant epitope(s) within a major histocompatibility protein (e.g., a MHC class I or class II molecule). In some embodiments, the antibody binds to beta 2 microglobulin. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within beta 2 microglobulin. Modified Viral Compositions [0651] In specific embodiments, Y is a viral particle, viral capsid, a viral envelope or a viral protein. In some embodiments, the viral composition is a viral particle that comprises a transgene. In some embodiments, the viral protein is a viral capsid protein or a viral envelope protein. [0652] In certain aspects, provided herein are modified viral compositions comprising a viral composition, for example, a virus particle, a virus capsid or a viral protein (e.g., a viral capsid protein or an envelope protein) attached to (e.g., conjugated to, directly or indirectly, for example via an intervening linker sequence) an ASGPR binding moiety that binds to a cell surface receptor. In certain embodiments, a modified viral composition comprises a virus particle that comprises a polynucleotide that optionally comprises a transgene, e.g., a transgene useful for therapeutic applications. [0653] The modified viral compositions, e.g., viral conjugates, presented herein may comprise any viral composition described herein e.g., any virus particle, capsid or viral protein, for example capsid protein or envelope protein, or fragment thereof, as described herein. [0654] In certain aspects, a viral composition described herein may comprise a virus particle. The terms “virus particle,” “viral particle,” “virus vector” or “viral vector” are used interchangeably herein. A “virus particle” refers to a virus capsid and a polynucleotide (DNA or RNA), which may comprise a viral genome, a portion of a viral genome, or a polynucleotide derived from a viral genome (e.g., one or more ITRs), which polynucleotide optionally comprises a transgene. In certain instances, a virus particle further comprises an envelope (which generally comprises lipid moieties and envelope proteins), surrounding or partially surrounding the capsid. [0655] A viral particle may be referred to as a “recombinant viral particle,” or “recombinant virus particle,” which terms as used herein refer to a virus particle that has been genetically altered, e.g., by the deletion or other mutation of an endogenous viral gene and/or the addition or insertion of a heterologous nucleic acid construct into the polynucleotide of the virus particle. Thus, a recombinant virus particle generally refers to a virus particle comprising a capsid coat or shell (and an optional outer envelope) within which is packaged a polynucleotide sequence that comprises sequences of viral origin and sequences not of viral origin (i.e., a polynucleotide heterologous to the virus). This polynucleotide sequence is typically a sequence of interest for the genetic alteration of a cell. [0656] In certain aspects, a viral composition described herein may comprise an “viral capsid,” “empty viral particle,” “empty virus particle,” or “capsid,” or “empty particle” when referred to herein in the context of the virus, which terms as used herein refer to a three-dimensional shell or coat comprising a viral capsid protein, optionally surrounded or partially surrounded by an outer envelope. In particular embodiments, the viral composition is a virus particle or a fragment thereof, virus capsid or fragment thereof, a viral protein, for example, a virus capsid protein or fragment thereof or envelope protein, or fragment thereof. [0657] In some embodiments, the virus used in a modified viral composition provided herein is adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), rhabdoviruses, murine leukemia virus); herpes simplex virus, coronavirus, reovirus, and the like. In some embodiments, the viral vector, viral particle or viral protein used in the present disclosure is derived from a non- enveloped virus, e.g., an adeno-associated virus (AAV). [0658] In some embodiments, lentiviral vectors can be used for CAR-T gene delivery, vaccines, or research tools, e.g., to introduce genes into mature T cells to generate immunity to cancer through the delivery of chimeric antigen receptors (CARs) or cloned T-cell receptors. [0659] Naturally occurring AAV forms a virus particle that comprises a three-dimensional capsid coat or shell (a “capsid”) made up of capsid proteins (VP1, VP2 and VP3) and, contained within the capsid, an AAV viral genome. [0660] The modified AAV compositions, e.g., AAV conjugates or fusions, presented herein may comprise any AAV composition described herein, e.g., any AAV particle, capsid or capsid protein, or fragment thereof, as described herein. The term “AAV capsid protein” or “AAV cap protein” refers to a protein encoded by an AAV capsid (cap) gene (e.g., VP1, VP2, and VP3) or a variant or fragment thereof. The term includes a capsid protein expressed by or derived from an AAV, e.g., a recombinant AAV, such as a chimeric AAV. For example, the term includes but not limited to a capsid protein derived from any AAV serotype such as AAV1, AAV2, AAV2i8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV rh10, AAV11, AAV12, AAV13, AAV-DJ, AAV3b, AAV LK03, AAV rh74, AAV Anc81, Anc82, Anc83, Anc84, Anc110, Anc113, Anc126, or Anc127, AAV_go.1, AAV hu.37, or AAV rh.8 or a variant thereof. Bridging Moieties that Bind Virus Composition [0661] In some embodiments, Y is a bridging moiety that specifically binds to a viral composition, for example, a viral particle, viral capsid, viral envelope or viral protein (e.g., a viral capsid protein or envelope protein), wherein the binding is not via a covalent linkage. [0662] Any suitable moiety that binds a viral particle, viral capsid, viral envelope or viral protein (e.g., a viral capsid protein or envelope protein) can be adapted for use in the conjugates of this disclosure. [0663] In certain embodiments, a bridging moiety is a polypeptide that specifically binds a viral composition. In some embodiments, the bridging moiety is a polypeptide that binds to a viral composition, e.g., a virus particle, virus capsid, virus envelope, or a viral protein, for example, a viral capsid protein or viral envelope protein. In certain aspects, the bridging composition binds the viral capsid protein or a viral envelope protein, when the viral protein is part of a virus particle. [0664] In certain embodiments, a bridging moiety is an antibody or antibody fragment (e.g., an antigen binding fragment of an antibody) that specifically binds a viral composition. In certain embodiments, a bridging moiety that binds a viral protein may also bind a viral particle, for example, via binding to the viral protein incorporated in a viral particle. Likewise, in certain embodiments, a bridging moiety that binds a viral particle may also bind a viral protein even if the viral protein is not incorporated in a viral particle. The viral particle can be an AAV virus particle. The viral protein can be a AAV capsid protein. [0665] In some embodiments, the bridging moieties of this disclosure specifically bind to an AAV composition, e.g., an AAV particle, AAV capsid, or AAV viral protein (e.g., an AAV capsid protein, for example, a VP1, VP2 or VP3 protein). [0666] An antibody or antigen binding fragment that may be utilized in connection with the modified viral compositions provided herein, e.g., in connection with the bridging compositions and bridging moieties presented herein, includes, without limitation, monoclonal antibodies, antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain antibodies, and fragments thereof (e.g., domain antibodies). Small Molecules [0667] In some embodiments, the target-binding moiety of the bifunctional compound of this disclosure is a small molecule that specifically binds to a target molecule, such as a target protein. In some embodiments, the bifunctional compound includes a small molecule inhibitor or ligand of a target protein. A small molecule target-binding moiety can be covalently linked to one or more ASGPR binding moieties via a linker. The linker can be covalently attached to the small molecule via substitution at any suitable site of the small molecule such that binding to the target protein is substantially retained. [0668] In some embodiments, the target-binding moiety is a small molecule inhibitor or antagonist of a target protein (e.g., as described herein). Any convenient small molecules known to bind a target of interest can be adapted for use in the subject compounds and conjugates. [0669] In some embodiments, the target-binding moiety is a small molecule inhibitor or antagonist of VEGF. [0670] In some embodiments, the target-binding moiety is a small molecule inhibitor or antagonist of PD-L1. [0671] In some embodiments, the target-binding moiety is a small molecule inhibitor or antagonist of EGFR protein, a VEGFR protein, a FGFR2 protein or a FGFR3 protein. [0672] In some embodiments, the target-binding moiety is a small molecule inhibitor or antagonist of TNF protein (e.g., TNF-alpha). TNF-alpha (TNFα) is a soluble cytokine produced by monocytes and macrophages as part of immune and inflammatory processes and is involved in a diverse range of cellular responses including differentiation, proliferation, inflammation, and cell death. TNFα is a type II transmembrane protein that can be cleaved and secreted as a soluble form. Both the transmembrane and soluble biologically active forms of TNFα are homotrimeric complexes that can signal through TNF receptors 1 and 2 (TNF-R1 and TNF-R2). TNFα is directly involved in systemic inflammation through the regulation of the intracellular NF-κB, JNK and p38-MAPK signaling pathways. [0673] The TNFα binding moiety can be a TNFα inhibitor, such as a competitive inhibitor of TNF receptor binding or an allosteric inhibitor of TNF signaling. The compounds of this disclosure can include a potent TNFα inhibitor, e.g., an inhibitor having sub-micromolar inhibitory activity. In some embodiments, the TNFα inhibitor is an allosteric inhibitor. In some embodiments, the TNFα binding moiety is an allosteric desymmetrization TNFα inhibitor. An allosteric desymmetrization TNFα inhibitor refers to a compound that binds to an allosteric site within TNFα and stabilizes the trimeric unit in a nonsymmetrical conformation that allows the TNFα trimer to recruit only two out of the three copies of TNF Receptor (TNFR, e.g., TNFR1), leading to an incompetent TNFα-TNFR signaling complex. [0674] See e.g., Xiao et al. in Journal of Medicinal Chemistry 202063 (23), 15050-15071, and McMillan et al. in Nature Communications (2021) 12:582, which discloses an analysis of the X-ray co- crystal structure of exemplary inhibitors bound to TNFα. An allosteric desymmetrization TNFα inhibitor can act via a particular mechanism of action to provide potent inhibitory activity. For example, (a) the TNFα inhibitor binding site is a cavity within the TNFα trimer created via movement of monomer A, (b) the inhibitor stabilizes the TNFα trimer in an inactive conformation by forming key π−π and hydrogen bonding interactions, (c) an allosteric desymmetrization TNFα inhibitor binds to TNFα trimer leading to major disruption of one TNFR binding site and minor disruption of a second site, while the third site remains unchanged, and (d) the allosteric desymmetrization TNFα inhibitor modulates TNF-R activity through an allosteric mechanism rather than direct competition with TNFR. Thus, the binding of an allosteric desymmetrization TNFα inhibitor to the symmetric TNFα trimer can lead to the formation of an asymmetric trimer which prevents the recruitment of three TNF receptor molecules that are necessary for signaling. Targets [0675] As summarized above, the bifunctional compounds of this disclosure can include a moiety of interest (Y) that specifically binds a target molecule. The target molecule can be a cell surface molecule or an extracellular molecule. [0676] In some embodiments of the compounds and methods of this disclosure, the target molecule is a cell surface molecule. By “cell surface molecule” is meant a target molecule associated with a cell membrane, e.g., because the molecule has a domain that inserts into or spans a cell membrane, e.g., a cell membrane- tethering domain or a transmembrane domain. The cell surface molecule may be any cell surface molecule which is desired for targeted degradation via the endosomal/lysosomal pathway. In some embodiments, the cell surface molecule is a cell surface receptor. [0677] Cell surface receptors of interest include, but are not limited to, stem cell receptors, immune cell receptors, growth factor receptors, cytokine receptors, hormone receptors, receptor tyrosine kinases, a receptor in the epidermal growth factor receptor (EGFR) family (e.g., HER2 (human epidermal growth factor receptor 2), etc.), a receptor in the fibroblast growth factor receptor (FGFR) family, a receptor in the vascular endothelial growth factor receptor (VEGFR) family, a receptor in the platelet derived growth factor receptor (PDGFR) family, a receptor in the rearranged during transfection (RET) receptor family, a receptor in the Eph receptor family, a receptor in the discoidin domain receptor (DDR) family, and a mucin protein (e.g., MUC1 ). In some embodiments, the cell surface molecule is CD71 (transferrin receptor). In certain aspects, the cell surface receptor is an immune cell receptor selected from a T cell receptor, a B cell receptor, a natural killer (NK) cell receptor, a macrophage receptor, a monocyte receptor, a neutrophil receptor, a dendritic cell receptor, a mast cell receptor, a basophil receptor, and an eosinophil receptor. [0678] In some embodiments, the moiety of interest (Y) specifically binds a cell surface molecule which mediates its effect not through a specific molecular interaction (and therefore is not susceptible to blocking), but rather through bulk biophysical or aggregate effects. A non-limiting example of such a cell surface molecule is a mucin. Examples of mucins include, but are not limited to, MUC1 , MUC16, MUC2, MUC5AC, MUC4, CD43, CD45, GPIb, and the like. [0679] In some embodiments, when the moiety of interest specifically binds a cell surface molecule, the cell surface molecule is present on a cancer cell. By “cancer cell” is meant a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage- independent growth potential, ability to promote tumor growth and/or development in an immunocompromised non-human animal model, and/or any appropriate indicator of cellular transformation. “Cancer cell” may be used interchangeably herein with “tumor cell”, “malignant cell” or “cancerous cell”, and encompasses cancer cells of a solid tumor, a semi-solid tumor, a hematological malignancy (e.g., a leukemia cell, a lymphoma cell, a myeloma cell, etc.), a primary tumor, a metastatic tumor, and the like. In some embodiments, the cell surface molecule present on the cancer cell is a tumor-associated antigen or a tumor-specific antigen. In certain aspects, when the moiety of interest (Y) specifically binds a cell surface molecule, the cell surface molecule is present on an immune cell. In some embodiments, the cell surface molecule is present on an immune cell selected from a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mast cell, a basophil, and an eosinophil. In certain aspects, the cell surface molecule present on the immune cell is an inhibitory immune receptor. As used herein, an “inhibitory immune receptor” is a receptor present on an immune cell that negatively regulates an immune response. Examples of inhibitory immune receptors which may be inhibited according to the methods of the present disclosure include inhibitory immune receptors of the Ig superfamily, including but not limited to: CD200R, CD300a (IRp60; mouse MAIR-I), CD300f (IREM-1 ), CEACAM1 (CD66a), FcyRIIb, ILT-2 (LIR-1 ; LILRB1 ; CD85j), ILT-3 (LIR-5; CD85k; LILRB4), ILT-4 (LIR-2; LILRB2), ILT-5 (LIR-3; LILRB3; mouse PIR-B); LAIR-1 , PECAM-1 (CD31 ), PILR-a (FDF03), SIRL-1 , and SIRP-a. Further examples of inhibitory immune receptors which may be inhibited according to the methods of the present disclosure include sialic acid-binding Ig-like lectin (Siglec) receptors, e.g., Siglec 7, Siglec 9, and/or the like. Additional examples of inhibitory immune receptors which may be inhibited according to the methods of the present disclosure include C- type lectins, including but not limited to: CLEC4A (DCIR), Ly49Q and MICL. Details regarding inhibitory immune receptors may be found, e.g., in Steevels et al. (2011 ) Eur. J. Immunol.41 (3):575- 587. In some embodiments, the cell surface molecule present on the immune cell is a ligand of an inhibitory immune receptor. In certain aspects, the cell surface molecule present on the immune cell is an immune checkpoint molecule. Non-limiting examples of immune checkpoint molecules to which the moiety of interest (Y) may specifically bind include PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT, and a member of the B7 family. [0680] In some embodiments of the compounds and methods of this disclosure, the target molecule is an extracellular molecule. By “extracellular molecule” is meant a soluble molecule external to the cell membranes of any cells in the vicinity of the soluble molecule. The extracellular molecule may be any extracellular molecule which is desired for targeted degradation via the endosomal/lysosomal pathway. [0681] In some embodiments, the extracellular molecule is a soluble target protein. In some embodiments, the extracellular molecule is a secreted protein that accumulates in disease (e.g., alpha- synuclein), a cholesterol carrier (e.g., ApoB), an infectious disease toxin (e.g., AB toxins, ESAT-6), an infectious particle (e.g., a whole virus, a whole bacterium, etc.), a clotting factor (e.g., Factor IX), the target of any FDA approved antibody that binds to an extracellular molecule (e.g., TNFalpha), any chemokine or cytokine (e.g., mediators of sepsis or chronic inflammation such at IL-1 ), a proteinaceous hormone (e.g., insulin, ACTH, etc.), a proteinaceous mediator of a mood disorder, a proteinaceous mediator of energy homeostasis (e.g., leptin, ghrelin, etc.), a proteinaceous allergen present in the bloodstream or an antibody against such an allergen (e.g., for peanut allergies), a proteinaceous toxin (e.g., snake venom hyaluronidase, etc.), an autoantibody, etc. [0682] In some embodiments, the target molecule is an extracellular molecule that is an antibody, e.g., an antibody that specifically binds a cell surface molecule or different extracellular molecule. In some embodiments, the antibody is an autoantibody. In some embodiments, the target is a human immunoglobulin A(IgA). In some embodiments, the IgA is a particular antibody that plays a crucial role in the immune function of mucous membranes. In the blood, IgA interacts with an Fc receptor called CD89 expressed on immune effector cells, to initiate inflammatory reactions. Aberrant IgA expression has been implicated in a number of autoimmune and immune-mediated disorders. In some embodiments, the target is a human immunoglobulin G (IgG). The Fc regions of IgGs include a conserved N- glycosylation site at asparagine 297 in the constant region of the heavy chain. Various N-glycans can b eattached to this site. The N-glycan IgG composition has been linked to several autoimmune, infectious and metabolic diseases. In addition, overexpression of IgG4 has been associated with IG4-related diseases. In some embodiments, the target is human immunoglobulin E (IgE). IgE is a type of immunoglobulin that plays an essential role in type I hypersensitivity, which can manifest into various allergic diseases and conditions. [0683] In some embodiments, the extracellular molecule is a ligand for a cell surface receptor. Cell surface receptor ligands of interest include, but are not limited to, growth factors (e.g., epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and the like), cytokines (e.g., an interleukin, an interferon, a tumor necrosis factor (TNF), a transforming growth factor b (TGF-b), including any particular subtypes of such cytokines), hormones, and the like. In certain aspects, the moiety of interest (Y) specifically binds apolipoprotein E4 (ApoE4). Pharmaceutical Compositions [0684] In another embodiment, provided herein are pharmaceutical compositions comprising one or more conjugates disclosed herein and a pharmaceutically acceptable carrier. [0685] In certain embodiments, the pharmaceutical compositions provided herein contain therapeutically effective amounts of one or more of the conjugates provided herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. [0686] Pharmaceutical carriers suitable for administration of the conjugates provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. [0687] The conjugates described herein can be formulated as the sole pharmaceutically active ingredient in the composition or can be combined with other active ingredients. [0688] In certain embodiments, the conjugate is formulated into one or more suitable pharmaceutical preparations, such as solutions, suspensions, powders, sustained release formulations or elixirs in sterile solutions or suspensions for parenteral administration, or as transdermal patch preparation and dry powder inhalers. [0689] In compositions provided herein, a conjugate described herein may be mixed with a suitable pharmaceutical carrier. The concentration of the conjugate in the compositions can, for example, be effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates a condition or disorder described herein or a symptom thereof. [0690] In certain embodiments, the pharmaceutical compositions provided herein are formulated for single dosage administration. To formulate a composition, the weight fraction of conjugate is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration such that the treated condition is relieved, prevented, or one or more symptoms are ameliorated. [0691] Concentrations of the conjugate in a pharmaceutical composition provided herein will depend on, e.g., the physicochemical characteristics of the conjugate, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. [0692] Pharmaceutical compositions described herein are provided for administration to a subject, for example, humans or animals (e.g., mammals) in unit dosage forms, such as sterile parenteral (e.g., intravenous) solutions or suspensions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof. Pharmaceutical compositions are also provided for administration to humans and animals in unit dosage form, including oral or nasal solutions or suspensions and oil-water emulsions containing suitable quantities of a conjugate or pharmaceutically acceptable derivatives thereof. The conjugate is, in certain embodiments, formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms as used herein refers to physically discrete units suitable for human or animal (e.g., mammal) subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of a conjugate sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged capsules. Unit-dose forms can be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of capsules or bottles. Hence, in specific aspects, multiple dose form is a multiple of unit-doses which are not segregated in packaging. [0693] In certain embodiments, the conjugates herein are in a liquid pharmaceutical formulation. Liquid pharmaceutically administrable formulations can, for example, be prepared by dissolving, dispersing, or otherwise mixing a conjugate and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, and the like, to thereby form a solution or suspension. In certain embodiments, a pharmaceutical composition provided herein to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, and pH buffering agents and the like. [0694] Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see, e.g., Remington: The Science and Practice of Pharmacy (2012) 22nd ed., Pharmaceutical Press, Philadelphia, PA Dosage forms or compositions containing antibody in the range of 0.005% to 100% with the balance made up from non-toxic carrier can be prepared. [0695] Parenteral administration, in certain embodiments, is characterized by injection, either subcutaneously, intramuscularly or intravenously is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. The injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. Other routes of administration may include, enteric administration, intracerebral administration, nasal administration, intraarterial administration, intracardiac administration, intraosseous infusion, intrathecal administration, and intraperitoneal administration. [0696] Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions can be either aqueous or nonaqueous. [0697] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof. [0698] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances. [0699] Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment. [0700] In certain embodiments, intravenous or intraarterial infusion of a sterile aqueous solution containing a conjugate described herein is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing a conjugate described herein injected as necessary to produce the desired pharmacological effect. [0701] In certain embodiments, the pharmaceutical formulations are lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They can also be reconstituted and formulated as solids or gels. [0702] The lyophilized powder is prepared by dissolving a conjugate provided herein, in a suitable solvent. In some embodiments, the lyophilized powder is sterile. Suitable solvents can contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. A suitable solvent can also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in certain embodiments, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides an example of a formulation. In certain embodiments, the resulting solution will be apportioned into vials for lyophilization. Lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature. [0703] Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. [0704] In certain embodiments, the conjugates provided herein can be formulated for local administration or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered. Uses and Methods [0705] In one aspect, provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from a cell’s surface. In one aspect, provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the extracellular milieu. For example, in one embodiment, provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the surface of a cell by sequestering the target protein in the cell’s lysosome. In another embodiment, provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the extracellular space (the extracellular milieu) of a cell by sequestering the target protein in the cell’s lysosome. In another embodiment, provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the surface of a cell by sequestering the target protein in the cell’s lysosome and degrading the target protein. In another embodiment, provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the extracellular space (the extracellular milieu) of a cell by sequestering the target protein in the cell’s lysosome and degrading the target protein. [0706] Removal of a target protein may refer to reduction, or depletion, of the target protein from the cell surface or from the extracellular space, or the extracellular milieu, that is, a reduction, or depletion, of the amount of the target protein on the cell surface or in the extracellular milieu. [0707] In one aspect, provided herein are methods of using the conjugates described herein to sequester a polypeptide of interest (a target protein) in a cell’s lysosome. In one aspect, provided herein are methods of using the conjugates described herein to sequester a polypeptide of interest (a target protein) in a cell’s lysosome and to degrade the polypeptide of interest. [0708] In one aspect, provided herein are methods of using the conjugates described herein to degrade a polypeptide of interest (a target protein). [0709] In one aspect, provided herein are methods of depleting a polypeptide of interest (a target protein) described herein by degradation through a cell’s lysosomal pathway. [0710] In another aspect, provided herein are methods of depleting a polypeptide of interest (a target protein) described herein by administering to a subject in need thereof an effective amount of a conjugate or pharmaceutically acceptable salt described herein, or a pharmaceutical composition described herein. In certain embodiments, the subject is a mammal (e.g., human). [0711] In certain embodiments, the target protein is a VEGF protein, an EGFR protein, a VEGFR protein, a PD-L1 protein, an FGFR2 protein or an FGFR3 protein. [0712] In another aspect, provided herein are methods of treating a disease or disorder by administering to a subject, e.g., a human, in need thereof an effective amount of a conjugate or pharmaceutically acceptable salt described herein, or a pharmaceutical composition described herein. [0713] The terms “administer”, “administration”, or "administering" refer to the act of injecting or otherwise physically delivering a substance (e.g., a conjugate or pharmaceutical composition provided herein) to a subject or a patient (e.g., human), such as by mucosal, topical, intradermal, parenteral, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. In a particular embodiment, administration is by intravenous infusion. [0714] The terms “effective amount” or “therapeutically effective amount” refer to an amount of a therapeutic (e.g., a conjugate or pharmaceutical composition provided herein) which is sufficient to treat, diagnose, prevent, delay the onset of, reduce and/or ameliorate the severity and/or duration of a given condition, disorder or disease and/or a symptom related thereto. These terms also encompass an amount necessary for the reduction, slowing, or amelioration of the advancement or progression of a given disease, reduction, slowing, or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy or to serve as a bridge to another therapy. In some embodiments, “effective amount” as used herein also refers to the amount of a conjugate described herein to achieve a specified result. [0715] In certain embodiments, when the disorder or disease is cancer, “effective amount” or “therapeutically effective amount” mean that amount of a conjugate or pharmaceutical composition provided herein which, when administered to a human suffering from a cancer, is sufficient to effect treatment for the cancer. “Treating” or “treatment” of the cancer includes one or more of: (1) limiting/inhibiting growth of the cancer, e.g. limiting its development; (2) reducing/preventing spread of the cancer, e.g. reducing/preventing metastases; (3) relieving the cancer, e.g. causing regression of the cancer, (4) reducing/preventing recurrence of the cancer; and (5) palliating symptoms of the cancer. [0716] The terms “subject” and “patient” are used interchangeably. A subject can be a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkey and human), for example a human. In certain embodiments, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder provided herein. In another embodiment, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder provided herein. In a specific embodiment, the subject is human. [0717] The terms “therapies” and “therapy” can refer to any protocol(s), method(s), compositions, formulations, and/or agent(s) that can be used in the prevention, treatment, management, or amelioration of a disease or disorder or symptom thereof (e.g., a disease or disorder provided herein or one or more symptoms or condition associated therewith). In certain embodiments, the terms “therapies” and “therapy” refer to drug therapy, adjuvant therapy, radiation, surgery, biological therapy, supportive therapy, and/or other therapies useful in treatment, management, prevention, or amelioration of a disease or disorder or one or more symptoms thereof. In certain embodiments, the term “therapy” refers to a therapy other than a conjugate described herein or pharmaceutical composition thereof. [0718] In certain embodiments, the disease or disorder is treated by depletion of the target protein by degradation through the lysosomal pathway. [0719] In certain embodiments, the disease or disorder is treated by depletion of certain proteins, for example, soluble proteins, e.g., secreted proteins, cell surface proteins (for example, cell surface receptor proteins, e.g., tyrosine kinase receptors, soluble cytokine receptors, and immune checkpoint receptors, e.g., EGFR, VEGFR, FGFR, and PD-L1), lectins, complements, lipoproteins, transport proteins, MHC class I and class II molecules, cytokines, chemokines, and/or receptors , or fragments or subunits of any of the foregoing. [0720] In certain embodiments, the disease or disorder is a cancer. [0721] In certain embodiments, the cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, endometrial cancer, hepatocellular carcinoma, kidney cancer, melanoma, myeloid neoplasms, non-small cell lung cancer (NSCLC), Ewing’s sarcoma, and Hodgkin’s Lymphoma. [0722] In certain embodiments, the cancer is a solid tumor. [0723] In certain embodiments, the disease or disorder is an inflammatory or autoimmune disease. [0724] In certain embodiments, the disease or disorder is an inflammatory disease. [0725] In certain embodiments, the disease or disorder is an autoimmune disease. In certain embodiments, the disease or disorder is a viral disease. In certain embodiments, the viral disease is hepatitis B. Definitions [0726] It is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. [0727] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of embodiments of the present disclosure. [0728] It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes not only a single compound but also a combination of two or more compounds, reference to "a substituent" includes a single substituent as well as two or more substituents, and the like. [0729] Certain terminology will be used in accordance with the definitions set out below. It will be appreciated that the definitions provided herein are not intended to be mutually exclusive. Accordingly, some chemical moieties may fall within the definition of more than one term. [0730] As used herein, the phrases “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify more general subject matter. These examples are provided only as an aid for understanding the disclosure, and are not meant to be limiting in any fashion. [0731] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. [0732] The terms “protein” and “polypeptide” are used interchangeably. Proteins may include moieties other than amino acids (e.g., may be glycoproteins, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete protein chain as produced by a cell (with or without a signal sequence), or can be a protein portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one protein chain, for example non-covalently or covalently attached, e.g., linked by one or more disulfide bonds or associated by other means. In certain embodiments, a polypeptide can occur as a single chain or as two or more associated chains, e.g., may be present as a multimer, e.g., dimer, a trimer. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof. [0733] The terms “antibody” and “immunoglobulin” are terms of art and can be used interchangeably herein in their broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. [0734] In a certain embodiments, an isolated antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to a protein of interest, for example, EGFR, is conjugated to one or more lysosomal targeting moieties, for example, via a linker. [0735] An “antigen” is a moiety or molecule that contains an epitope to which an antibody can specifically bind. As such, an antigen is also is specifically bound by an antibody. In a specific embodiment, the antigen, to which an antibody described herein binds, is a protein of interest, for example, EGFR (e.g., human EGFR), or a fragment thereof, or for example, an extracellular domain of EGFR (e.g., human EGFR). [0736] An “epitope” is a term known in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be a linear epitope of contiguous amino acids or can comprise amino acids from two or more non-contiguous regions of the antigen. [0737] The terms “binds,” “binds to,” “specifically binds” or “specifically binds to” in the context of antibody binding refer to antibody binding to an antigen (e.g., epitope) as such binding is understood by one skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other polypeptides, generally with lower affinity as determined by, e.g., immunoassays, Biacore™, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In a specific embodiment, molecules that specifically bind to an antigen bind to the antigen with an affinity (Kd ) that is at least 2 logs, 2.5 logs, 3 logs, 4 logs lower (higher affinity) than the Kd when the molecules bind to another antigen. In another specific embodiment, molecules that specifically bind to an antigen do not cross react with other proteins. In another specific embodiment, where EGFR is the protein of interest, molecules that specifically bind to an antigen do not cross react with other non-EGFR proteins. [0738] An antibody specifically includes, but is not limited to, full length antibodies (e.g., intact immunoglobulins), antibody fragments, monoclonal antibodies, polyclonal antibodies,, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain/antibody heavy chain pair, an antibody with two light chain/heavy chain pairs (e.g., identical pairs), intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, bivalent antibodies (including monospecific or bispecific bivalent antibodies), single chain antibodies, or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and epitope-binding fragments of any of the above. [0739] Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, antibodies described herein are IgG antibodies (e.g., human IgG), or a class (e.g., human IgG1, IgG2, IgG3 or IgG4) or subclass thereof. [0740] In a particular embodiment, an antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs, wherein the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical. In a specific embodiment, the H and L chains comprise constant regions, for example, human constant regions. In a yet more specific embodiment, the L chain constant region of such antibodies is a kappa or lambda light chain constant region, for example, a human kappa or lambda light chain constant region. In another specific embodiment, the H chain constant region of such antibodies comprise a gamma heavy chain constant region, for example, a human gamma heavy chain constant region. In a particular embodiment, such antibodies comprise IgG constant regions, for example, human IgG constant regions. [0741] The term “constant region” or “constant domain” is a well-known antibody term of art (sometimes referred to as “Fc”), and refers to an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor. The terms refer to a portion of an immunoglobulin molecule having a generally more conserved amino acid sequence relative to an immunoglobulin variable domain. [0742] The term “heavy chain” when used in reference to an antibody can refer to any distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (µ), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3 and IgG4. [0743] The term “light chain” when used in reference to an antibody can refer to any distinct types, e.g., kappa (κ) of lambda (λ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain. [0744] The term “monoclonal antibody” is a well-known term of art that refers to an antibody obtained from a population of homogenous or substantially homogeneous antibodies. The term “monoclonal” is not limited to any particular method for making the antibody. Generally, a population of monoclonal antibodies can be generated by cells, a population of cells, or a cell line. In specific embodiments, a “monoclonal antibody,” as used herein, is an antibody produced by a single cell (e.g., hybridoma or host cell producing a recombinant antibody), wherein the antibody specifically binds to an epitope as determined, e.g., by ELISA or other antigen-binding or competitive binding assay known in the art or in the Examples provided herein. In particular embodiments, a monoclonal antibody can be a chimeric antibody or a humanized antibody. In certain embodiments, a monoclonal antibody is a monovalent antibody or multivalent (e.g., bivalent) antibody. In particular embodiments, a monoclonal antibody is a monospecific or multispecific antibody (e.g., bispecific antibody). [0745] The terms “variable region” or “variable domain” refer to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 100 amino acids in the mature light chain. Variable regions comprise complementarity determining regions (CDRs) flanked by framework regions (FRs). Generally, the spatial orientation of CDRs and FRs are as follows, in an N-terminal to C-terminal direction: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4. Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen and for the specificity of the antibody for an epitope. In a specific embodiment, numbering of amino acid positions of antibodies described herein is according to the EU Index, as in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242. In certain embodiments, the variable region is a human variable region. [0746] In certain aspects, the CDRs of an antibody can be determined according to (i) the Kabat numbering system (Kabat et al. (1971) Ann. NY Acad. Sci.190:382-391 and, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242); or (ii) the Chothia numbering scheme, which will be referred to herein as the “Chothia CDRs” (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196: 901-917; Al- Lazikani et al., 1997, J. Mol. Biol., 273: 927-948; Chothia et al., 1992, J. Mol. Biol., 227: 799-817; Tramontano et al., 1990, J. Mol. Biol.215(1):175-82; U.S. Patent No.7,709,226; and Martin, A., “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp.422-439, Springer-Verlag, Berlin (2001)); or (iii) the ImMunoGeneTics (IMGT) numbering system, for example, as described in Lefranc, 1999, The Immunologist, 7: 132-136 and Lefranc et al., 1999, Nucleic Acids Res., 27: 209-212 (“IMGT CDRs”); or (iv) the AbM numbering system, which will be referred to herein as the “AbM CDRs”, for example as described in MacCallum et al., 1996, J. Mol. Biol., 262: 732-745. See also, e.g., Martin, A., “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp.422-439, Springer-Verlag, Berlin (2001); or (v) the Contact numbering system, which will be referred to herein as the “Contact CDRs” (the Contact definition is based on analysis of the available complex crystal structures (bioinf.org.uk/abs) (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 262:732-745)). [0747] The terms “full length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, and are not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain the Fc region. [0748] “Antibody fragments” comprise only a portion of an intact antibody, wherein the portion retains at least one, two, three and as many as most or all of the functions normally associated with that portion when present in an intact antibody. In one aspect, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another aspect, an antibody fragment, such as an antibody fragment that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody. Such functions may include FcRn binding, antibody half life modulation, conjugate function and complement binding. In another aspect, an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody. For example, such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment. Antibody fragments suitable for use in the compounds of this disclosure include, for example, Fv fragments, Fab fragments, F(ab’)2 fragments, Fab’ fragments, scFv (sFv) fragments, and scFv-Fc fragments. [0749] “Polynucleotide” or “nucleic acid,” as used interchangeably herein, and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. The nucleic acid molecule may be an aptamer. [0750] The term “purified” refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance of interest comprises the majority percent of the sample in which it resides. Typically in a sample a substantially purified component comprises 50%, 80%-85%, 90-99%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the sample. Techniques for purifying polynucleotides, polypeptides and virus particles of interest are well- known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density. [0751] The terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect, such as reduction of tumor burden. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease (as in liver fibrosis that can result in the context of chronic HCV infection); (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease (e.g., reduction in of tumor burden). [0752] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to an animal, including, but not limited to, human and non-human primates, including simians and humans; rodents, including rats and mice; bovines; equines; ovines; felines; canines; and the like. "Mammal" means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, e.g., non-human primates, and humans. Non-human animal models, e.g., mammals, e.g. non-human primates, murines, lagomorpha, etc. may be used for experimental investigations. [0753] A "therapeutically effective amount" or "efficacious amount" means the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to effect such treatment for the disease, condition, or disorder. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated. [0754] Unless specifically stated otherwise, where a compound may assume alternative tautomeric, regioisomeric and/or stereoisomeric forms, all alternative isomers, are intended to be encompassed within the scope of the claimed subject matter. For example, when a compound is described as a particular optical isomer D- or L-, it is intended that both optical isomers be encompassed herein. For example, where a compound is described as having one of two tautomeric forms, it is intended that both tautomers be encompassed herein. Thus, the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures. The compounds provided herein may contain chiral centers. Such chiral centers may be of either the (R) or (S) configurations, or may be a mixture thereof. The chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form. [0755] The present disclosure also encompasses all suitable isotopic variants of the compounds according to the present disclosure, whether radioactive or not. An isotopic variant of a compound according to the present disclosure is understood to mean a compound in which at least one atom within the compound according to the present disclosure has been exchanged for another atom of the same atomic number, but with a different atomic mass than the atomic mass which usually or predominantly occurs in nature. Examples of isotopes which can be incorporated into a compound according to the present disclosure are those of hydrogen, carbon, nitrogen, oxygen, fluorine, chlorine, bromine and iodine, such as 2H (deuterium), 3H (tritium), 13C, 14C, 15N, 17O, 18O, 18F, 36Cl, 82Br, 123I, 124I, 125I, 129I and 131I. Particular isotopic variants of a compound according to the present disclosure, especially those in which one or more radioactive isotopes have been incorporated, may be beneficial, for example, for the examination of the mechanism of action or of the active compound distribution in the body. Compounds labelled with 3H, 14C and/or 18F isotopes are suitable for this purpose. In addition, the incorporation of isotopes, for example of deuterium, can lead to particular therapeutic benefits as a consequence of greater metabolic stability of the compound, for example an extension of the half-life in the body or a reduction in the active dose required. In some embodiments, hydrogen atoms of the compounds described herein may be replaced with deuterium atoms. In certain embodiments, “deuterated” as applied to a chemical group and unless otherwise indicated, refers to a chemical group that is isotopically enriched with deuterium in an amount substantially greater than its natural abundance. Isotopic variants of the compounds according to the present disclosure can be prepared by various, including, for example, the methods described below and in the working examples, by using corresponding isotopic modifications of the particular reagents and/or starting compounds therein. [0756] Thus, any of the embodiments described herein are meant to include a salt, a single stereoisomer, a mixture of stereoisomers and/or an isotopic form of the compounds. [0757] A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use. "A pharmaceutically acceptable excipient, diluent, carrier and adjuvant" as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant. [0758] A "pharmaceutical composition" is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like. [0759] The term “pharmaceutically acceptable” means being approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and, more particularly in humans. [0760] The term "pharmaceutically acceptable salt" refers to those salts which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in situ during the final isolation and purification of the conjugate compounds, or separately by reacting the free base function or group of a compound with a suitable organic acid. Examples of pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, or salts of an amino group formed with inorganic acids [0761] “Acyl” refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)- , heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the “acetyl” group CH3C(O)- [0762] The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group (i.e., a mono-radical) typically although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Generally, although not necessarily, alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms. The term "lower alkyl" intends an alkyl group of 1 to 6 carbon atoms. "Substituted alkyl" refers to alkyl substituted with one or more substituent groups, and this includes instances wherein two hydrogen atoms from the same carbon atom in an alkyl substituent are replaced, such as in a carbonyl group (i.e., a substituted alkyl group may include a -C(O)- moiety). The terms "heteroatom-containing alkyl" and "heteroalkyl" refer to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom, as described in further detail infra. If not otherwise indicated, the terms "alkyl" and "lower alkyl" include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl or lower alkyl, respectively. [0763] The term “substituted alkyl” is meant to include an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as -O- , -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, - SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NRaRb, wherein R’ and R” may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic. [0764] The term "alkenyl" refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like. Generally, although again not necessarily, alkenyl groups herein may contain 2 to about 18 carbon atoms, and for example may contain 2 to 12 carbon atoms. The term "lower alkenyl" intends an alkenyl group of 2 to 6 carbon atoms. The term "substituted alkenyl" refers to alkenyl substituted with one or more substituent groups, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to alkenyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms "alkenyl" and "lower alkenyl" include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkenyl and lower alkenyl, respectively. [0765] The term "alkynyl" refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups herein may contain 2 to about 18 carbon atoms, and such groups may further contain 2 to 12 carbon atoms. The term "lower alkynyl" intends an alkynyl group of 2 to 6 carbon atoms. The term "substituted alkynyl" refers to alkynyl substituted with one or more substituent groups, and the terms "heteroatom-containing alkynyl" and "heteroalkynyl" refer to alkynyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms "alkynyl" and "lower alkynyl" include linear, branched, unsubstituted, substituted, and/or heteroatom-containing alkynyl and lower alkynyl, respectively. [0766] The term "alkoxy" refers to an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy" group may be represented as -O-alkyl where alkyl is as defined above. A "lower alkoxy" group refers to an alkoxy group containing 1 to 6 carbon atoms, and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc. Substituents identified as "C1-C6 alkoxy" or "lower alkoxy" herein may, for example, may contain 1 to 3 carbon atoms, and as a further example, such substituents may contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy). [0767] The term “substituted alkoxy” refers to the groups substituted alkyl-O-, substituted alkenyl- O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein. [0768] The term "aryl", unless otherwise specified, refers to an aromatic substituent generally, although not necessarily, containing 5 to 30 carbon atoms and containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Aryl groups may, for example, contain 5 to 20 carbon atoms, and as a further example, aryl groups may contain 5 to 12 carbon atoms. For example, aryl groups may contain one aromatic ring or two or more fused or linked aromatic rings (i.e., biaryl, aryl-substituted aryl, etc.). Examples include phenyl, naphthyl, biphenyl, diphenylether, diphenylamine, benzophenone, and the like. "Substituted aryl" refers to an aryl moiety substituted with one or more substituent groups, and the terms "heteroatom-containing aryl" and "heteroaryl" refer to aryl substituent, in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra. Aryl is intended to include stable cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated C3-C14 moieties, exemplified but not limited to phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazoyl; which may further be substituted with one to five members selected from the group consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (see e.g. Katritzky, Handbook of Heterocyclic Chemistry). If not otherwise indicated, the term "aryl" includes unsubstituted, substituted, and/or heteroatom-containing aromatic substituents. [0769] The term "aralkyl" refers to an alkyl group with an aryl substituent, and the term "alkaryl" refers to an aryl group with an alkyl substituent, wherein "alkyl" and "aryl" are as defined above. In general, aralkyl and alkaryl groups herein contain 6 to 30 carbon atoms. Aralkyl and alkaryl groups may, for example, contain 6 to 20 carbon atoms, and as a further example, such groups may contain 6 to 12 carbon atoms. [0770] The term "alkylene" refers to a multi-valent (e.g., di-radical alkyl group, tri-radical alkyl group, tetra-radical alkyl group, etc.) . Unless otherwise indicated, such groups include saturated hydrocarbon chains containing from 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may be heteroatom-containing. "Lower alkylene" refers to alkylene linkages containing from 1 to 6 carbon atoms. Examples include, methylene (--CH2--), ethylene (--CH2CH2--), propylene (--CH2CH2CH2--), 2-methylpropylene (--CH2--CH(CH3)--CH2--), hexylene (-- (CH2)6--) and the like. [0771] Similarly, the terms "alkenylene," "alkynylene," "arylene," "aralkylene," and "alkarylene" refer to di-radical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively. [0772] In some embodiments, such as in branched constructs, the "alkylene" refers to a multi-valent (e.g., di-valent alkyl group, tri-valent alkyl group, tetra-valent alkyl group, etc.). Similarly, the terms "alkenylene," "alkynylene," "arylene," "aralkylene," and "alkarylene" can refer to multi-valent alkenyl, multi-valent alkynyl, multi-valent aryl, multi-valent aralkyl, and multi-valent alkaryl groups, respectively. [0773] The term "amino" refers to the group -NRR’ wherein R and R’ are independently hydrogen or nonhydrogen substituents, with nonhydrogen substituents including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and/or heteroatom-containing variants thereof. [0774] The terms "halo" and "halogen" are used in the conventional sense to refer to a chloro, bromo, fluoro or iodo substituent. [0775] “Carboxyl,” “carboxy” or “carboxylate” refers to –C(O)OH or salts thereof. [0776] “Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like. [0777] The term “substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -S(O)-alkyl, -S(O)-substituted alkyl, -S(O)-aryl, -S(O)-heteroaryl, - S(O)2-alkyl, -S(O)2-substituted alkyl, -S(O)2-aryl and -S(O)2-heteroaryl. [0778] The term "heteroatom-containing" as in a "heteroatom-containing alkyl group" (also termed a "heteroalkyl" group) or a "heteroatom-containing aryl group" (also termed a "heteroaryl" group) refers to a molecule, linkage or substituent in which one or more carbon atoms are replaced with an atom other than carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur. Similarly, the term "heteroalkyl" refers to an alkyl substituent that is heteroatom-containing, the term "heterocycloalkyl" refers to a cycloalkyl substituent that is heteroatom-containing, the terms "heterocyclic" or “heterocycle” refer to a cyclic substituent that is heteroatom-containing, the terms "heteroaryl" and "heteroaromatic" respectively refer to "aryl" and "aromatic" substituents that are heteroatom-containing, and the like. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl- substituted alkyl, N-alkylated amino alkyl, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, furyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl, etc. [0779] “Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic, provided that the point of attachment is through an atom of an aromatic ring. In certain embodiments, the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N→O), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, - SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl, and trihalomethyl. [0780] The terms “heterocycle,” “heterocyclic” and “heterocyclyl” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 15 ring atoms, including 1 to 4 hetero atoms. These ring heteroatoms are selected from nitrogen, sulfur and oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or –SO2- moieties. [0781] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7- tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like. [0782] Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO- aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle. [0783] "Hydrocarbyl" refers to univalent hydrocarbyl radicals containing 1 to about 30 carbon atoms, including 1 to about 24 carbon atoms, further including 1 to about 18 carbon atoms, and further including about 1 to 12 carbon atoms, including linear, branched, cyclic, saturated and unsaturated species, such as alkyl groups, alkenyl groups, aryl groups, and the like. A hydrocarbyl may be substituted with one or more substituent groups. The term "heteroatom-containing hydrocarbyl" refers to hydrocarbyl in which at least one carbon atom is replaced with a heteroatom. Unless otherwise indicated, the term "hydrocarbyl" is to be interpreted as including substituted and/or heteroatom-containing hydrocarbyl moieties. [0784] By "substituted" as in "substituted hydrocarbyl," "substituted alkyl," "substituted aryl," and the like, as alluded to in some of the aforementioned definitions, is meant that in the hydrocarbyl, alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation, functional groups, and the hydrocarbyl moieties C1-C24 alkyl (including C1-C18 alkyl, further including C1-C12 alkyl, and further including C1-C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, and further including C2-C6 alkynyl), C5-C30 aryl (including C5-C20 aryl, and further including C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, and further including C6-C12 aralkyl). The above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any of the groups described herein are to be interpreted as including substituted and/or heteroatom-containing moieties, in addition to unsubstituted groups. [0785] “Sulfonyl” refers to the group SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-cycloalkyl, SO2-substituted cylcoalkyl, SO2-cycloalkenyl, SO2-substituted cylcoalkenyl, SO2-aryl, SO2-substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2-heterocyclic, and SO2- substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-. [0786] By the term “functional groups” is meant chemical groups such as halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO- ), carbamoyl (-(CO)-NH2), mono-substituted C1-C24 alkylcarbamoyl (- (CO)-NH(C1-C24 alkyl)), di-substituted alkylcarbamoyl (-(CO)-N(C1-C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano (- C≡N), isocyano (-N+≡C-), cyanato (-O-C≡N), isocyanato (-O-N+≡C-), isothiocyanato (-S-C≡N), azido (- N=N+=N-), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)- substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2-C24 alkylamido (-NH-(CO)- alkyl), C5-C20 arylamido (-NH-(CO)-aryl), imino (-CR=NH where R = hydrogen, C1-C24 alkyl, C5- C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R = hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), where R = hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl; also termed "alkylthio"), arylsulfanyl (-S-aryl; also termed "arylthio"), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P(O)(O-)), phospho (-PO2), and phosphino (-PH2), mono- and di-(C1-C24 alkyl)-substituted phosphino, mono- and di-(C5-C20 aryl)- substituted phosphine. In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above. [0787] By "linking" or "linker" as in "linking group," "linker moiety," etc., is meant a linking moiety that connects two groups via covalent bonds. The linker may be linear, branched, cyclic or a single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups including, without limitation: amido (-NH-CO-), ureylene (-NH-CO-NH-), imide (-CO-NH-CO-) , epoxy (-O-), epithio (-S-), epidioxy (-O-O-), carbonyldioxy (-O-CO-O-), alkyldioxy (-O-(CH2)n-O-), epoxyimino (-O-NH-), epimino (-NH-), carbonyl (-CO-), etc. In certain embodiments, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. The bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone. The linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group. A linker may include, without limitations, poly(ethylene glycol) unit(s) (e.g., -(CH2-CH2-O)-); ethers, thioethers, amines, alkyls (e.g., (C1-C12)alkyl) , which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n- pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone. A linker may be cleavable or non-cleavable. Any convenient orientation and/or connections of the linkers to the linked groups may be used. [0788] When the term "substituted" appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group. For example, the phrase "substituted alkyl and aryl" is to be interpreted as "substituted alkyl and substituted aryl." [0789] In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below. [0790] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =O, =NR70, =N-OR70, =N2 or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R60, halo, =O, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R70, -SO2O–
Figure imgf000293_0001
where R60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70 is independently hydrogen or R60; each R80 is independently R70 or alternatively, two R80’s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3 alkyl substitution; and each M+ is a counter ion with a net single positive charge. Each M+ may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as +N(R60)4; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]0.5, or [Ba2+]0.5 (“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound as disclosed herein, and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR80R80 is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N- methyl-piperazin-1-yl and N-morpholinyl. [0791] In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R60, halo, -O-M+, -OR70, -SR70, -S–M+, -NR80R80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R70, -SO3 –M+, -SO3R70, -OSO2R70, -OSO3 – M+, -OSO3R70, -PO3 -2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2 – M+, -CO2R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OCO2 – M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2 – M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80, where R60, R70, R80 and M+ are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O-M+, -OR70, -SR70, or -S–M+. [0792] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R60, -O-M+, -OR70, -SR70, -S-M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R70, -S(O)2O-M+, -S(O)2OR70, -OS(O)2R70, -OS(O)2O-M+, -O S(O)2OR70, -P(O)(O-)2(M+)2, -P(O)(OR70)O-M+, -P(O)(OR70)(OR70), -C(O)R70, -C(S)R70, -C(NR70)R70, -C (O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)OR70, -OC(S)OR 70, -NR70C(O)R70, -NR70C(S)R70, -NR70C(O)OR70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R7 0 and -NR70C(NR70)NR80R80, where R60, R70, R80 and M+ are as previously defined. [0793] In some embodiments, the term “optionally substituted” means that a group has 0-5, or 0-3, or 1, or 2, or 3, substituents independently selected from -R60, halo, =O, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R70, -SO2OR70, -OSO2R70, -OSO2OR70, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -C (O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)OR70, -OC(S)OR 70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80, where R60 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70 is independently hydrogen or R60; each R80 is independently R70 or alternatively, two R80’s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3 alkyl substitution. [0794] In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent. [0795] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O-C(O)-. [0796] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds. [0797] In certain embodiments, a substituent may contribute to optical isomerism and/or stereo isomerism of a compound. Salts, solvates, hydrates, and prodrug forms of a compound are also of interest. All such forms are embraced by the present disclosure. Thus the compounds described herein include salts, solvates, hydrates, prodrug and isomer forms thereof, including the pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof. In certain embodiments, a compound may be a metabolized into a pharmaceutically active derivative. [0798] Unless otherwise specified, reference to an atom is meant to include isotopes of that atom. For example, reference to H is meant to include 1H, 2H (i.e., D) and 3H (i.e., T), and reference to C is meant to include 12C and all isotopes of carbon (such as 13C). [0799] Unless otherwise indicated, the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, or 3 standard deviations. In certain embodiments, the term “about” or “approximately” means within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.25%, 0.2%, 0.1% or 0.05% of a given value or range. In certain embodiments, where an integer is required, the term “about” means within plus or minus 10% of a given value or range, rounded either up or down to the nearest integer. [0800] In the description herein, if there is any discrepancy between a chemical name and chemical structure, the chemical structure shall prevail. [0801] Definitions of other terms and concepts appear throughout the detailed description. NUMBERED EMBODIMENTS [0802] Embodiment A-1: A cell surface ASGPR binding compound of formula (I): Xn L Y (I) or a prodrug thereof, or a salt thereof, wherein: Y is a moiety of interest; n is 1 to 500; L is a linker; and X is a moiety that binds to a cell surface asialoglycoprotein receptor (ASGPR) of formula (Ia):
Figure imgf000296_0001
wherein: R1 is selected from –OH, –OC(O)R, -C(O)NHR, –Z1–*, and optionally substituted triazole, where R is optionally substituted C1-6 alkyl or optionally substituted aryl; R2 is selected from–NHCOCH3, –NHCOCF3, –NHCOCH2CF3, –OH, optionally substituted triazole, and –Z1–*; R3 is selected from –H, –OH, –CH3, –OCH3, –OCH2CH=CH and –Z1–*; one of R1 to R3 is –Z1–*, wherein “ * ” represents a point of attachment of Z1 to the linker R4 and R5 are each independently selected from H, and a promoiety, or R4 and R5 are cyclically linked to form a promoiety; R11 is H, or a group that forms a bridge to the 1-position carbon atom; Z1 is a linking moiety selected from Z11, optionally substituted Z11-heteroaryl, optionally substituted Z11-aryl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted alkyl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiourea; Z11 is selected from -O-, -S-, NR21-, and -C(R22)2, each R21 is independently selected from H, and optionally substituted (C1-C6)alkyl; and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1-C6)alkyl; wherein: i) when n is 3, R1 is OH, R2 is –NHCOCH3, R4-R5 are H, and R3 is Z1, then Z1 is not O; ii) when n is 2 or 3, R1 is OAc, R2 is –NHCOCH3, R4-R5 are Ac, and R3 is Z1, then Z1 is not O; iii) when n is 2 or 3, R1 is OBz, R2 is –NHCOCH3, R4-R5 are Bz, and R3 is Z1, then Z1 is not O; iv) when n is 3, R1 is OH, R2 is –NHCOCH3, R4-R5 are H, and R3 is Z1, and Z11 is O, then L comprises a backbone of at least 16 consecutive atoms to a branching point; v) when n is 3, R1 is Z1, where Z1 is O, and R4-R5 are H, then R3 is not -CH3 –OCH3,or – OCH2CH=CH; and vi) when R11 is a group of the formula -CH2O- that forms a bridge to the 1-position carbon atom, R2 is –NHCOCH3, R4-R5 are H, then R1 and R3 are not Z1. [0803] Embodiment A-2: The compound of embodiment A-1, wherein each X is independently of formula (Ib):
Figure imgf000297_0001
wherein: R1 is selected from –OH, –OC(O)R, and -C(O)NHR; and R2 is selected from –NHCOCH3, –NHCOCF3, and –NHCOCH2CF3. [0804] Embodiment A-3: The compound of embodiment A-3, wherein Z1 is selected from -O-, -S-, and -C(R22)2-. [0805] Embodiment A-4: The compound of embodiment A-3, wherein Z1 is Z11-Ar , wherein Ar is optionally substituted heteroaryl or optionally substituted aryl. [0806] Embodiment A-5: The compound of embodiment A-4, wherein: Z11 is O, S, or C(R22)2; and Ar is a monocyclic 5 or 6-membered heteroaryl or aryl. [0807] Embodiment A-6: The compound of embodiment A-5, wherein Z1 is -C(R22)2-triazole-. * [0808] Embodiment A-7: The compound of embodiment A-6, wherein Z1 is
Figure imgf000297_0002
or
Figure imgf000297_0003
. [0809] Embodiment A-8: The compound of embodiment A-3, wherein Z1 is monocyclic 5 or 6- membered heteroaryl or aryl. [0810] Embodiment A-9: The compound of embodiment A-8, wherein
Figure imgf000297_0004
Figure imgf000297_0005
. [0811] Embodiment A-10: The compound of embodiment A-2, wherein each X is independently of the formula:
Figure imgf000297_0006
wherein R4 and R5 are each H. [0812] Embodiment A-11: The compound of embodiment A-10, wherein each X is independently of formula:
Figure imgf000298_0001
[0813] Embodiment A-12: The compound of embodiment A-10, wherein Z1 is selected from monocyclic 5 or 6-membered heteroaryl, monocyclic 5 or 6-membered aryl and Z11-Ar, wherein Ar is optionally substituted heteroaryl or optionally substituted aryl. [0814] Embodiment A-13: The compound of embodiment A-12, wherein each X is
Figure imgf000298_0002
[0815] Embodiment A-14: The compound of embodiment A-2, wherein each X is independently of the formula:
Figure imgf000298_0003
wherein R4 and R5 are each H. [0816] Embodiment A-15: The compound of embodiment A-14, wherein each X is selected from the following structures: ,
Figure imgf000298_0004
[0817] Embodiment A-16: The compound of embodiment A-14, wherein n is 1 and X is
Figure imgf000299_0001
. [0818] Embodiment A-17: The compound of embodiment A-1, wherein each X is independently of formula (Id):
Figure imgf000299_0002
wherein: R1 is selected from –OH, –OC(O)R, and -C(O)NHR; and R3 is selected from –H, –OH, –CH3, –OCH3, and –OCH2CH=CH. [0819] Embodiment A-18: The compound of embodiment A-17, wherein R3 is H. [0820] Embodiment A-19: The compound of embodiment A-18, wherein each X is independently of formula (Ie):
Figure imgf000299_0003
wherein: Z2 is absent or selected from -O-, -S-, NR25-, -C(R22)2-, and optionally substituted Z12-alkyl; ring A is absent or selected from a 5 or 6-membered optionally substituted aryl and a 5 or 6- membered optionally substituted heteroaryl; Z3 is a linking moiety selected from Z12, optionally substituted alkyl, optionally substituted Z12- alkyl, optionally substituted Z12-heteroaryl, optionally substituted Z12-aryl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiourea; and Z12 is selected from -CH2O-, -O-, -S-, -NR26-, and -C(R22)2-; R25 and R26 are each independently selected from H, optionally substituted (C1-C6)alkyl (e.g., C(1- 3)-alkyl, such as methyl), and optionally substituted acyl; and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl. [0821] Embodiment A-20: The compound of embodiment A-17, wherein each X is independently of one of formula (If)-(Ii):
Figure imgf000300_0001
[0822] Embodiment A-21: The compound of embodiment A-20, wherein each X is independently of one of formula (Ij)-(Im)
Figure imgf000300_0002
wherein: Y1-Y3 are each independently N or CR27; and R24 and R27 are each independently selected from H, optionally substituted C(1-6)-alkyl, optionally substituted fluoroalkyl, and halogen. [0823] Embodiment A-22: The compound of embodiment A-21, wherein Z3 is selected from -O-, - CH2O-, -OCH2-, optionally substituted -OCH2-heteroaryl, optionally substituted -OCH2-aryl, optionally substituted -CH2O-heteroaryl, and optionally substituted -CH2O-aryl. [0824] Embodiment A-23: The compound of embodiment A-21 or A-22, wherein X is independently one of the following structures:
Figure imgf000301_0001
[0826] Embodiment A-24: The compound of embodiment A-18, wherein X is:
Figure imgf000301_0002
. [0827] Embodiment A-25: The compound of embodiment A-1, wherein each X is independently of formula (Ic): *
Figure imgf000301_0003
wherein: R2 is selected from –NHCOCH3, –NHCOCF3, and –NHCOCH2CF3; and R3 is selected from –H, –OH, –CH3, –OCH3, and –OCH2CH=CH. [0828] Embodiment A-26: The compound of embodiment A-25, wherein Z1 is selected from -O-, - S-, -CONR21-, and optionally substituted –(C(R22)2)q-heteroaryl, wherein q is 0 or 1. [0829] Embodiment A-27: The compound of embodiment A-26, wherein Z1 is -O-. [0830] Embodiment A-28: The compound of embodiment A-36, wherein Z1 is optionally substituted –(C(R22)2)q-triazole wherein q is 0 or 1. [0831] Embodiment A-29: The compound of embodiment A-28, wherein
Figure imgf000302_0001
[0832] Embodiment A-30: The compound of any one of embodiments A-1 to A-20, wherein n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking X to Y via Z1. [0833] Embodiment A-31: The compound of any one of embodiments A-1 to A-20, wherein n is 2 or more, and L is a branched linker that covalently links 2 or more X moieties to Y via the linking moiety Z1. [0834] Embodiment A-32: The compound of any one of embodiment A-1 to A-31, wherein L is of formula (IIb):
Figure imgf000302_0002
IIb) wherein each L1 to L5 is independently a linking moiety which together provide a linear or branched linker between Z1 and Y; a, b, c, d, and e are each independently 0, 1, or 2; ** represents the point of attachment to L1 of X via Z1; and *** represents the point of attachment to Y, wherein when n is >1, d is 1 or 2 and L4 is a branching moiety. [0835] Embodiment A-33: The compound of claim 32, wherein L1 to L5 each independently comprise one or more linking moieties independently selected from –C1-20-alkylene–, –NHCO-C1-6- alkylene–, –CONH-C1-6-alkylene–, –NH-C1-6-alkylene–, –NHCONH-C1-6-alkylene–, – NHCSNH-C1-6- alkylene–, –C1-6-alkylene–NHCO-, –C1-6-alkylene–CONH-, –C1-6-alkylene–NH-, –C1-6-alkylene– NHCONH-, –C1-6-alkylene–NHCSNH-, -O(CH2)p–, –(OCH2CH2)p–, –NHCO–, –CONH–, –NHSO2–, – SO2NH–, –CO–, –SO2–, –O–, –S–, pyrrolidine-2,5-dione, 1,2,3-triazole, –NH–, and –NMe–, wherein each p is independently 1 to 50. [0836] Embodiment A-34: The compound of embodiment A-32 or A-33, wherein L comprises repeating ethylene glycol moieties. [0837] Embodiment A-35: The compound of embodiment A-34, wherein L comprises 1 to 25 ethylene glycol moieties. [0838] Embodiment A-36: The compound of any one of embodiments A-32 to A-35, wherein L comprises one or more 1,2,3-triazole linking moieties. [0839] Embodiment A-37: The compound of any one of embodiments A-32 to A-36, wherein n is 1. [0840] Embodiment A-38: The compound of any one of embodiments A-32 to A-36, wherein n is 2 or more. [0841] Embodiment A-39: The compound of embodiment A-38, wherein L4 is a branching moiety
Figure imgf000303_0001
[0842] wherein each x and y are each independently 1 to 10. [0843] Embodiment A-40: The compound of any one of embodiments A-32 to A-39, wherein L1-L4 comprises a backbone of 14 or more consecutive atoms between X and the branching atom. [0844] Embodiment A-41: The compound of any one of embodiments A-32 to A-40, wherein L5 comprises a backbone of 10 to 80 consecutive atoms. [0845] Embodiment A-42: The compound of embodiment A-33, wherein L5 comprises a linking moiety selected from (C10-C20-alkylene, or –(OCH2CH2)p–, where p is 1 to 25. [0846] Embodiment A-43: The compound of any one of embodiments A-32 to A-42, wherein the linker of formula (IIb) comprises a backbone of 20 to 100 consecutive atoms. [0847] Embodiment A-44: The compound of embodiment A-43, wherein the linker of formula (IIb) comprises a backbone of 25 or more consecutive atoms. [0848] Embodiment A-45: The compound of embodiment A-44, wherein the linker of formula (IIb) comprises a backbone of 30 or more consecutive atoms. [0849] Embodiment A-46: The compound of any one of embodiments A-1 to A-45, wherein -Z1-L1- comprises a group selected from:
Figure imgf000303_0002
Figure imgf000304_0001
wherein: each R21 is independently selected from H, and optionally substituted (C1-C6)alkyl; each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6. [0850] Embodiment A-47: The compound of embodiment A-46, wherein -Z1-L1- comprises a group selected from:
Figure imgf000304_0002
wherein q is 1 to 3. [0851] Embodiment A-48: The compound of any one of embodiments A-1 to A-45, wherein -Z1-L1- comprises an optionally substituted -NH-heteroaryl-. [0852] Embodiment A-49: The compound of embodiment A-48, wherein -Z1-L1- comprises a group selected from:
Figure imgf000304_0003
wherein: each R24 is independently selected from H, optionally substituted C(1-6)-alkyl, optionally substituted fluoroalkyl, and halogen; and each R25 is independently selected from H, optionally substituted (C1-C6)alkyl, and optionally substituted acyl. [0853] Embodiment A-50: The compound of any one of embodiment A-1 to A-49, wherein Y is selected from small molecule, dye, fluorophore, monosaccharide, polysaccharide, lipid, protein, polynucleotide, enzyme, enzyme substrate, polymer, and chemoselective ligation group or precursor thereof. [0854] Embodiment A-51: The compound of any one of embodiment A-1 to A-49, wherein Y is a moiety that specifically binds an extracellular target protein. [0855] Embodiment A-52: The compound of embodiment A-51, wherein the target protein is a membrane bound protein. [0856] Embodiment A-53: The compound of embodiment A-51, wherein the target protein is a soluble extracellular protein. [0857] Embodiment A-54: The compound of any one of embodiments A-51 to A-53, wherein Y is a target-binding small molecule. [0858] Embodiment A-55: The compound of any one of embodiments A-51 to A-53, wherein Y is a target-binding biomolecule. [0859] Embodiment A-56: The compound of embodiment A-55, wherein the biomolecule is selected from peptide, protein, glycoprotein, polynucleotide, aptamer, and antibody or antibody fragment. [0860] Embodiment A-57: The compound of embodiment A-56, wherein Y is selected from antibody, antibody fragment, chimeric fusion protein, an engineered protein domain, and D-protein binder of target protein. [0861] Embodiment A-58: The compound of embodiment A-55, wherein Y is a protein, n is 1 to 6, and m is 1 to 20. [0862] Embodiment A-59: The compound of any one of embodiment A-51 to A-57, wherein Y is a moiety that specifically binds the target protein and the compound is a conjugate of formula (III’):
Figure imgf000305_0001
wherein: n is 1 to 20; m is 1 to 80 (e.g., an average loading or a discrete loading); each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group to a compatible group of Y; and Y is a moiety of interest binds the target protein. [0863] Embodiment A-60: The compound of embodiment A-59, wherein Y is an antibody or an antibody fragment. [0864] Embodiment A-61: The compound of embodiment A-59, wherein Y is selected from chimeric fusion protein, and engineered protein domain. [0865] Embodiment A-62: The compound of any one of embodiments A-59 to A-61, wherein n is 1 to 6. [0866] Embodiment A-63: The compound of any one of embodiments A-59 to A-61, wherein n is 1 to 4. [0867] Embodiment A-64: The compound of any one of embodiments A-59 to A-61, wherein n is 3. [0868] Embodiment A-65: The compound of any one of embodiments A-59 to A-61, wherein n is 2. [0869] Embodiment A-66: The compound of any one of embodiments A-59 to A-61, wherein n is 1. [0870] Embodiment A-67: The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 20. [0871] Embodiment A-68: The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 10. [0872] Embodiment A-69: The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 6. [0873] Embodiment A-70: The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 4. [0874] Embodiment A-71: The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 2. [0875] Embodiment A-72: The compound of any one of embodiments A-59 to A-66, wherein m is 2. [0876] Embodiment A-73: The compound of any one of embodiments A-59 to A-66, wherein m is 1. [0877] Embodiment A-74: The compound of any one of embodiments A-59 to A-73, wherein Z is a residual moiety resulting from the covalent linkage of a thiol-reactive chemoselective ligation group to one or more cysteine residue(s) of Ab. [0878] Embodiment A-75: The compound of embodiment A-74, wherein the thiol-reactive chemoselective ligation group comprises a maleimide. [0879] Embodiment A-76: The compound of any one of embodiments A-59 to A-73, wherein Z is a residual moiety resulting from the covalent linkage of an amine-reactive chemoselective ligation group to one or more lysine residue(s) of Ab. [0880] Embodiment A-77: The compound of embodiment A-76, wherein the amine-reactive chemoselective ligation group comprises a pentafluorophenyl (PFP) active ester. [0881] Embodiment A-78: A method of internalizing a target protein in a cell comprising a cell surface asialoglycoprotein receptor (ASGPR), the method comprising: contacting a cellular sample comprising the cell and the target protein with an effective amount of a compound according to any one of embodiments A-1 to A-77, wherein the compound specifically binds the target protein and specifically binds the ASGPR to facilitate cellular uptake of the target protein. [0882] Embodiment A-79: The method of embodiment A-78, wherein the target protein is a membrane bound protein. [0883] Embodiment A-80: The method of embodiment A-78, wherein the target protein is an extracellular protein. [0884] Embodiment A-81: A method of reducing levels of a target protein in a biological system, the method comprising: contacting the biological system with an effective amount of a compound according to any one of embodiments A-1 to A-77, wherein the compound specifically binds the target protein and specifically binds a ASGPR of cells in the biological system to facilitate cellular uptake and degradation of the target protein. [0885] Embodiment A-82: The method of embodiment A-81, wherein the biological system is a human subject. [0886] Embodiment A-83: The method of any one of embodiments A-81 to A-82, wherein the biological system is an in vitro cellular sample. [0887] Embodiment A-84: The method of any one of embodiments A-81 to A-83, wherein the target protein is a membrane bound protein. [0888] Embodiment A-85: The method of any one of embodiments A-81 to 83, wherein the target protein is an extracellular protein. [0889] Embodiment B-1l: A cell surface ASGPR binding compound of formula (I): Xn L Y (I) or a prodrug thereof, or a salt thereof, wherein: Y is a moiety of interest; n is 1 to 500; L is a linker; and X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (II):
Figure imgf000307_0001
wherein: R1 is selected from –Z1–*, –H, –OH, –CH3, –OCH3, and –OCH2CH=CH; R2 is selected from –Z1–*, –NHCOCH3, –NHCOCF3, –NHCOCH2CF3, –OH, and optionally substituted triazole; R6 is selected from –Z1–*, –OH, –OC(O)R, -C(O)NHR, and optionally substituted triazole, where R is optionally substituted (C1-C6)alkyl or optionally substituted aryl; wherein one of R1, R2, and R6 is –Z1–*, and “ * ” represents a point of connection of Z1 to the linker (L); R3 and R4 are each independently H, or a promoiety, or R3 and R4 are cyclically linked to form a promoiety; R11 is H, or a bridging moiety that connects the 5-position carbon to the 1-position carbon of the ring; Z1 is a linking moiety selected from -Z11- -Z11-A1- -A2- -NR21CO- - CONR21- -NR21SO2- - SO2NR21-, -NR21C(O)NR21-, and -NR21C(S)NR21-; -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2; -A1- and -A2- are optionally substituted arylene or optionally substituted heteroarylene; each R21 is independently selected from H, and optionally substituted (C1-C6)alkyl; and each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl; wherein: i) when n is 3, R6 is OH, R2 is –NHCOCH3, R3-R4 are H, and R1 is Z1, then Z1 is not O; ii) when n is 2 or 3, R6 is OAc, R2 is –NHCOCH3, R3-R4 are Ac, and R1 is Z1, then Z1 is not O; iii) when n is 2 or 3, R6 is Obz, R2 is –NHCOCH3, R3-R4 are Bz, and R1 is Z1, then Z1 is not O; iv) when n is 3, R6 is OH, R2 is –NHCOCH3, R3-R4 are H, and R1 is Z1, and Z11 is O, then L comprises a backbone of at least 16 consecutive atoms to a branching point; v) when n is 3, R6 is Z1, where Z1 is O, and R3-R4 are H, then R1 is not -CH3 –OCH3,or – OCH2CH=CH; and vi) when R11 is a group of the formula -CH2O- that forms a bridge to the 1-position carbon atom, R2 is –NHCOCH3, R3-R4 are H, then R6 and R1 are not Z1. [0890] Embodiment B-2: The compound of embodiment B-1, wherein each X is independently of formula (IIa):
Figure imgf000308_0001
wherein: R6 is selected from –OH, –OC(O)R, and -C(O)NHR; and R2 is selected from –NHCOCH3, –NHCOCF3, and –NHCOCH2CF3. [0891] Embodiment B-3: The compound of embodiment B-3, wherein Z1 is selected from -O-, -S-, and -C(R22)2-. [0892] Embodiment B-4: The compound of embodiment B-3, wherein Z1 is Z11-Ar , wherein Ar is optionally substituted heteroaryl or optionally substituted aryl. [0893] Embodiment B-5: The compound of embodiment B-4, wherein: Z11 is O, S, or C(R22)2; and Ar is a monocyclic 5 or 6-membered heteroaryl or aryl. [0894] Embodiment B-6: The compound of embodiment B-5, wherein Z1 is -C(R22)2-triazole-. [0895] Embodiment B-7: The compound of embodiment B-6, wherein Z1 is
Figure imgf000309_0001
Figure imgf000309_0002
. [0896] Embodiment B-8: The compound of embodiment B-3, wherein Z1 is monocyclic 5 or 6- membered heteroaryl or aryl. [0897] Embodiment B-9: The compound of embodiment B-8, wherein
Figure imgf000309_0003
Figure imgf000309_0004
. [0898] Embodiment B-10: The compound of embodiment B-2, wherein each X is independently of the formula:
Figure imgf000309_0005
wherein R3 and R4 are each H. [0899] Embodiment B-11: The compound of embodiment B-10, wherein each X is independently of formula:
Figure imgf000309_0006
[0900] Embodiment B-12: The compound of embodiment B-10, wherein Z1 is selected from monocyclic 5 or 6-membered heteroaryl, monocyclic 5 or 6-membered aryl and Z11-Ar, wherein Ar is optionally substituted heteroaryl or optionally substituted aryl. [0901] Embodiment B-13: The compound of embodiment B-12, wherein each X is
Figure imgf000309_0007
[0902] Embodiment B-14: The compound of embodiment B-2, wherein each X is independently of the formula:
Figure imgf000310_0001
wherein R3 and R4 are each H. [0903] Embodiment B-15: The compound of embodiment B-14, wherein each X is selected from the following structures:
Figure imgf000310_0002
[0904] Embodiment B-16: The compound of embodiment B-14, wherein n is 1 and X is
Figure imgf000310_0003
. [0905] Embodiment B-17: The compound of embodiment B-1, wherein each X is independently of formula (IIb):
Figure imgf000310_0004
wherein: R6 is selected from –OH, –OC(O)R, and -C(O)NHR; and R1 is selected from –H, –OH, –CH3, –OCH3, and –OCH2CH=CH. [0906] Embodiment B-18: The compound of embodiment B-17, wherein R1 is H. [0907] Embodiment B-19: The compound of embodiment B-18, wherein each X is independently of formula (IVb) or (IVc):
Figure imgf000311_0001
wherein -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2. [0908] Embodiment B-20: The compound of embodiment B-17, wherein each X is independently of formula (IVb-1) or (IVc-1):
Figure imgf000311_0002
(IVb-1) (IVc-1) wherein R11 is the bridging moiety that connects the 5-position carbon to the 1-position carbon. [0909] Embodiment B-21: The compound of embodiment B-19 or B-20, wherein each X is independently of formula (
Figure imgf000311_0003
wherein R21 and R22 are independently selected from H, halogen, (C1-C6)alkyl and substituted (C1- C6)alkyl (e.g., CF3). [0910] Embodiment B-22: The compound of embodiment B-21, wherein Z3R21 and R22 are independently H, or CF3. [0911] Embodiment B-23: The compound of embodiment B-21 or B-22, wherein X is independently one of the following structures:
Figure imgf000312_0001
. [0913] Embodiment B-25: The compound of embodiment B-1, wherein each X is independently of formula (Ic):
Figure imgf000312_0002
wherein: R2 is selected from –NHCOCH3, –NHCOCF3, and –NHCOCH2CF3; and R1 is selected from –H, –OH, –CH3, –OCH3, and –OCH2CH=CH. [0914] Embodiment B-26: The compound of embodiment B-25, wherein Z1 is selected from -O-, -S- , -CONR21-, and optionally substituted –(C(R22)2)q-heteroaryl, wherein q is 0 or 1. [0915] Embodiment B-27: The compound of embodiment B-26, wherein Z1 is -O-. [0916] Embodiment B-28: The compound of embodiment B-36, wherein Z1 is optionally substituted –(C(R22)2)q-triazole wherein q is 0 or 1. [0917] Embodiment B-29: The compound of embodiment B-28, wherein
Figure imgf000313_0001
[0918] Embodiment B-30: The compound of any one of embodiments B-1 to B-20, wherein n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking X to Y via Z1. [0919] Embodiment B-31: The compound of any one of embodiments B-1 to B-20, wherein n is 2 or more, and L is a branched linker that covalently links 2 or more X moieties to Y via the linking moiety Z1. [0920] Embodiment B-32: The compound of any one of embodiments B-1 to B-31, wherein L is of formula (XI):
Figure imgf000313_0002
wherein each L1 and L3 are independently a linear linking moiety, and L2 is a branched linking moiety, wherein L1 to L3 together provide a linear or branched linker between X and Y; a, b and c are independently 0 or 1, wherein: when n is 1, b is 0 and at least one of a and c is 1; and when n is 2 or 3, a, b and c are each 1; * represents the point of connection of L1 to X via Z1; and ** represents a point of conjugation of the linker L to Y. [0921] Embodiment B-33: The compound of embodiment B-32, wherein: n is 1; and a is 1, b is 0 and c is 1, whereby L is of formula (XIa): * L1 L3 ** (XIa). [0922] Embodiment B-34: The compound of embodiment B-32, wherein: n is 2; and a is 1, b is 1, and c is 1, whereby L is of formula (XIb):
Figure imgf000313_0003
(XIb). [0923] Embodiment B-35: The compound of embodiment B-32, wherein: n is 3; and a is 1, b is 1, and c is 1, whereby L is of formula (XIc):
Figure imgf000314_0004
(XIc). [0924] Embodiment B-36: The compound of any one of embodiments B-32 to B-35, wherein each L1 is of the formula (XII)
Figure imgf000314_0001
wherein: L10 is a linking moiety; and L11 to L19 are independently absent or a linking moiety, wherein L10 to L19 of each L1 is each independently selected from –C1-20-alkylene–, –NHCO-C1-6- alkylene–, –CONH-C1-6-alkylene–, –NH-C1-6-alkylene–, –NHCONH-C1-6-alkylene–, – NHCSNH-C1-6- alkylene–, –C1-6-alkylene–NHCO-, –C1-6-alkylene–CONH-, –C1-6-alkylene–NH-, –C1-6-alkylene– NHCONH-, –C1-6-alkylene–NHCSNH-, -O(CH2)p–, –(OCH2CH2)p–, –NHCO–, –CONH–, –NHSO2–, – SO2NH–, –NHCONH–, –NHCSNH–, –CO–, –SO2–, –O–, –S–, pyrrolidine-2,5-dione, 1,2,3-triazole, – NH–, and –NMe–, wherein each p is independently 1 to 50. [0925] Embodiment B-37: The compound of any one of embodiments B-32 to B-36, wherein each L1 comprises a linear backbone of 6 to 20 consecutive atoms (e.g., 6 to 16 consecutive atoms, such as 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive atoms). [0926] Embodiment B-38: The compound of any one of embodiments B-32, and B-34 to B-37, wherein L2 is of formula (XIIIa) or (XIIIb):
Figure imgf000314_0002
(XIIIa) (XIIIb) wherein: L20 is a branched linking moiety comprising: a carbon atom or nitrogen atom that is the branching point of the branched linking moiety; and one or more (e.g., 1 to 20, 1 to 10, or 1 to 6) linking moieties independently selected from amino acid residue (e.g., a residue such a s, or a derivative thereof), –NH-CH[(CH2)q]2O–
Figure imgf000314_0003
alkylene–, – NHCO-, –CONH–, –NHSO2–, –SO2NH–, –CO–, –SO2–, –O–, –S–, pyrrolidine-2,5-dione, 1,2,3- triazole, –NH–, and –N(CH3)–, –NHC(O)NH–, – NHC(S)NH–, –O(CH2)p–, and –(OCH2CH2)p–, wherein each p is independently 1 to 50, and q is 1-6. [0927] Embodiment B-39: The compound of any one of embodiments B-32, and B-34 to B-38, wherein L2 comprises a linking moiety selected from:
Figure imgf000315_0001
wherein: Z2 is connected to L1, and Z3 is connected to L3; each Z2 and Z3 is independently selected from –NHCO-, –CONH–, –CO–, –O–, –NH–, and – N(CH3)–; x is 1 to 12 (e.g., 1 to 6, or 1 to 3); and y is 0 to 12 (e.g., 1 to 6, or 1 to 3). [0928] Embodiment B-40: The compound of embodiment B-39, wherein L2 comprises a linking moiety selected from:
Figure imgf000315_0002
[0929] Embodiment B-41: The compound of embodiment B-40, wherein L2 comprises a linking moiety of formula (XIV):
Figure imgf000315_0003
wherein: r is 1 or 2; and when n is 2, r is 1, when n is 3, r is 2. [0930] Embodiment B-42: The compound of embodiment B-41, wherein L2 is of formula (XVa) or (XVb):
Figure imgf000316_0001
(XVa) (XVb). [0931] Embodiment B-43: The compound of embodiment B-41 or B-42, wherein L2 is of formula (XVc) or (XVd):
Figure imgf000316_0002
(XVc) (XVc) wherein r is 1 or 2. [0932] Embodiment B-44: The compound of embodiment B-38, wherein L2 comprises two 2 or more amino acid residues (e.g., 3 or more, or 4 or more amino acid residues, linear or dendrimer). [0933] Embodiment B-45: The compound of embodiment B-38, wherein L2 comprises 4 or more amino acid residues that are branched linking moieties selected from Lys, Orn, Asp, Glu, Ser, and Cys (e.g., where the sidechain, amino and carboxylic acid are each linked to an adjacent moiety). [0934] Embodiment B-46: The compound of any one of embodiments B-32 to B-45, wherein each L3 is of the formula (XVI):
Figure imgf000316_0003
wherein: L30 to L39 are independently absent or a linking moiety; and Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group of the linker to a compatible group of Y; wherein L30 to L39 are each independently selected from –C1-20-alkylene–, –NHCO-C1-6-alkylene–, –CONH-C1-6-alkylene–, –NH C1-6-alkylene–, –NHCONH-C1-6-alkylene–, – NHCSNH-C1-6-alkylene–, – C1-6-alkylene–NHCO-, –C1-6-alkylene–CONH-, –C1-6-alkylene–NH-, –C1-6-alkylene–NHCONH-, –C1-6- alkylene–NHCSNH-, -O(CH2)p–, –(OCH2CH2)p–, –NHCO–, –CONH–, –NHSO2–, –SO2NH–, – NHCONH-, –NHCSNH-, –CO–, –SO2–, –O–, –S–, pyrrolidine-2,5-dione, 1,2,3-triazole, –NH–, and – NMe–, wherein each p is independently 1 to 50. [0935] Embodiment B-47: The compound of embodiment B-46, wherein L3 comprises a linear backbone of 6 to 40 consecutive atoms (e.g., 10 to 30 consecutive atoms, or 20 to 30 consecutive atoms). [0936] Embodiment B-48: The compound of embodiment B-46 or B-47, wherein the linker L has one of the following structures: , ,
Figure imgf000317_0001
wherein: a is 1 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); f is 1 to 6 (e.g., 1, 2, or 3); g is 1 to 20 (e.g., 1 to 12, or 6 to 20 or 6 to 12); Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group of the linker to a compatible group of Y. [0937] Embodiment B-49: The conjugate of embodiment B-32, wherein the linker L comprises one of the following structures:
Figure imgf000318_0001
. [0938] Embodiment B-50: The compound of embodiment B-32, wherein L comprises repeating ethylene glycol moieties. [0939] Embodiment B-51: The compound of embodiment B-32, wherein L comprises 1 to 25 ethylene glycol moieties. [0940] Embodiment B-52: The compound of embodiment B-32, wherein L comprises one or more 1,2,3-triazole linking moieties. [0941] Embodiment B-53: The compound of embodiment B-38, wherein L2 is a branching moiety selected from:
Figure imgf000318_0002
wherein each x and y are each independently 1 to 10. [0942] Embodiment B-54: The compound of any one of embodiments B-32 to B-54, wherein the linker comprises a backbone of 14 or more consecutive atoms between each X and the branching atom. [0943] Embodiment B-55: The compound of any one of embodiments B-32 to B-54, wherein L3 comprises a backbone of 10 to 80 consecutive atoms. [0944] Embodiment B-56: The compound of embodiment B-55, wherein L3 comprises a linking moiety selected from (C10-C20-alkylene, or –(OCH2CH2)p–, where p is 1 to 25. [0945] Embodiment B-57: The compound of any one of embodiment B-32 to B-56, wherein the linker of formula (XI) comprises a backbone of 20 to 100 consecutive atoms. [0946] Embodiment B-58: The compound of embodiment B-57, wherein the linker of formula (XI) comprises a backbone of 25 or more consecutive atoms. [0947] Embodiment B-59: The compound of embodiment B-58, wherein the linker of formula (XI) comprises a backbone of 30 or more consecutive atoms. [0948] Embodiment B-60: The compound of any one of embodiments B-1 to B-59, wherein -Z1-L1- comprises a group selected from:
Figure imgf000319_0001
wherein: each R21 is independently selected from H, and optionally substituted (C1-C6)alkyl; each R22 is independently selected from H, halogen (e.g., F) and optionally substituted (C1- C6)alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6. [0949] Embodiment B-61: The compound of embodiment B-60, wherein -Z1-L1- comprises a group selected from:
Figure imgf000319_0002
wherein q is 1 to 3. [0950] Embodiment B-62: The compound of any one of embodiments B-1 to B-61, wherein -Z1-L1- comprises an optionally substituted -NH-heteroaryl-. [0951] Embodiment B-63: The compound of embodiment B-62, wherein -Z1-L1- comprises a group selected from:
Figure imgf000319_0003
wherein: each R24 is independently selected from H, optionally substituted C(1-6)-alkyl, optionally substituted fluoroalkyl, and halogen; and each R25 is independently selected from H, optionally substituted (C1-C6)alkyl, and optionally substituted acyl. [0952] Embodiment B-64: The compound of any one of embodiments B-1 to B-63, wherein Y is selected from small molecule, dye, fluorophore, monosaccharide, polysaccharide, lipid, protein, polynucleotide, enzyme, enzyme substrate, polymer, and chemoselective ligation group or precursor thereof. [0953] Embodiment B-65: The compound of any one of embodiments B-1 to B-64, wherein Y is a moiety that specifically binds an extracellular target protein. [0954] Embodiment B-66: The compound of embodiment B-65, wherein the target protein is a membrane bound protein. [0955] Embodiment B-67: The compound of embodiment B-65, wherein the target protein is a soluble extracellular protein. [0956] Embodiment B-68: The compound of any one of embodiments B-65 to B-67, wherein Y is a target-binding small molecule. [0957] Embodiment B-69: The compound of any one of embodiments B-65 to B-67, wherein Y is a target-binding biomolecule. [0958] Embodiment B-70: The compound of embodiment B-69, wherein the biomolecule is selected from peptide, protein, glycoprotein, polynucleotide, aptamer, and antibody or antibody fragment. [0959] Embodiment B-71: The compound of embodiment B-69, wherein Y is selected from antibody, antibody fragment, chimeric fusion protein, an engineered protein domain, and D-protein binder of target protein. [0960] Embodiment B-72: The compound of embodiment B-69, wherein Y is a protein, n is 1 to 10 (e.g., 1 to 8, 1 to 6, such as 1, 2, 3, 4-5, 5-6, or 6-7), and m is 1 to 20. [0961] Embodiment B-73: The compound of any one of embodiments B-51 to B-57, wherein Y is a moiety that specifically binds the target protein and the compound is a conjugate of formula (III’):
Figure imgf000320_0001
wherein: n is 1 to 20; m is 1 to 80 (e.g., an average loading or a discrete loading); each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group to a compatible group of Y; and Y is a moiety of interest (e.g., a protein, antibody, aptamer, peptide) binds the target protein. [0962] Embodiment B-74: The compound of embodiment B-59, wherein Y is an antibody or an antibody fragment. [0963] Embodiment B-75: The compound of embodiment B-59, wherein Y is selected from chimeric fusion protein, and engineered protein domain. [0964] Embodiment B-76: The compound of any one of embodiments B-73 to B-75, wherein n is to 10 (e.g., 1 to 8, or 1 to 6). [0965] Embodiment B-77: The compound of any one of embodiments B-73 to B-75, wherein n is 1 to 4. [0966] Embodiment B-78: The compound of any one of embodiments B-73 to B-75, wherein n is 3. [0967] Embodiment B-79: The compound of any one of embodiments B-73 to B-75, wherein n is 2. [0968] Embodiment B-80: The compound of any one of embodiments B-73 to B-75, wherein n is 1. [0969] Embodiment B-81: The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 20. [0970] Embodiment B-82: The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 10. [0971] Embodiment B-83: The compound of any one of embodiments B-73 to B-80, wherein m is 4 to 8 (e.g., 4-6, 4-5, 5-6, or 6-7). [0972] Embodiment B-84: The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 6. [0973] Embodiment B-85: The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 4. [0974] Embodiment B-86: The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 3 (e.g., 1, 2, or 3). [0975] Embodiment B-87: The compound of any one of embodiments B-73 to B-80, wherein m is 2. [0976] Embodiment B-88: The compound of any one of embodiments B-73 to B-80, wherein m is 1. [0977] Embodiment B-89: The compound of any one of embodiments B-73 to B-88, wherein Z is a residual moiety resulting from the covalent linkage of a thiol-reactive chemoselective ligation group to one or more cysteine residue(s) of Ab. [0978] Embodiment B-90: The compound of embodiment B-89, wherein the thiol-reactive chemoselective ligation group comprises a maleimide or phenylene-maleimide. [0979] Embodiment B-91: The compound of any one of embodiments B-73 to B-88, wherein Z is a residual moiety resulting from the covalent linkage of an amine-reactive chemoselective ligation group to one or more lysine residue(s) of Ab. [0980] Embodiment B-92: The compound of embodiment B-91, wherein the amine-reactive chemoselective ligation group comprises a pentafluorophenyl (PFP) active ester. [0981] Embodiment B-93: A method of internalizing a target protein in a cell comprising a cell surface asialoglycoprotein receptor (ASGPR), the method comprising: contacting a cellular sample comprising the cell and the target protein with an effective amount of a compound according to any one of embodiments B-1 to B-92, wherein the compound specifically binds the target protein and specifically binds the ASGPR to facilitate cellular uptake of the target protein. [0982] Embodiment B-94: The method of embodiment B-93, wherein the target protein is a membrane bound protein. [0983] Embodiment B-95: The method of embodiment B-93, wherein the target protein is an extracellular protein. [0984] Embodiment B-96: A method of reducing levels of a target protein in a biological system, the method comprising: contacting the biological system with an effective amount of a compound according to any one of embodiments B-1 to B-92, wherein the compound specifically binds the target protein and specifically binds a ASGPR of cells in the biological system to facilitate cellular uptake and degradation of the target protein. [0985] Embodiment B-97: The method of embodiment B-96, wherein the biological system is a human subject. [0986] Embodiment B-98: The method of any one of embodiments B-96 to B-97, wherein the biological system is an in vitro cellular sample. [0987] Embodiment B-99: The method of any one of embodiments B-96 to B-98, wherein the target protein is a membrane bound protein. [0988] Embodiment B-100: The method of any one of embodiments B-96 to B-98, wherein the target protein is an extracellular protein. EXAMPLES [0989] The examples in this section are offered by way of illustration, and not by way of limitation. Example 1: Preparation of Compounds [0990] The following are illustrative schemes and examples of how the compounds described herein can be prepared and tested. Although the examples can represent only some embodiments, it should be understood that the following examples are illustrative and not limiting. All substituents, unless otherwise specified, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art. The specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare the compounds described herein. Synthesis of compound XC28:
Figure imgf000322_0001
Figure imgf000323_0001
3 XC28 [0991] To a solution of (2R,3R,4R,5S)-5-amino-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol hydrochloride (which was prepared by literature routes described in WO2022035997A1) (48-4, 1.0 eq, 2.0 g, 10.0 mmol) in methanol (40 mL), sodium methoxide (25% in methanol) (1.2 eq, 2.89 mL, 12.0 mmol) was added and reaction mixture was stirred at room temperature for 30 minutes. Then, phthalic anhydride (48-1a, 1.2 eq, 1.78 g, 12.0 mmol) was added and reaction mixture was stirred at room temperature for 30 minutes. Then, triethylamine (1.4 eq, 2.03 mL, 14.0 mmol) and a second portion of phthalic anhydride (48-1a, 1.2 eq, 1.78 g, 12.0 mmol) was added and reaction mixture was stirred at room temperature for 16 h. After that, reaction mixture was concentrated, washed with diethyl ether and dried to afford 2-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)isoindoline-1,3-dione (48-6) as a light purple solid which was used as such for next reaction. Yield: 7.0 g (Crude). [0992] A solution of 2-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)isoindoline-1,3-dione (48-6, 1.0 eq, 7.0 g, 23.9 mmol), in pyridine (70 mL) was cooled at 0 °C, acetic anhydride (10.0 eq, 24.4 mL, 239.0 mmol) was added and reaction mixture was stirred at room temperature for 16 h. After completion, reaction mixture was diluted with 1N hydrochloric acid and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-50 % ethyl acetate in hexane to afford (2R,3R,4R,5S)-2-(acetoxymethyl)-5-(1,3-dioxoisoindolin-2-yl)tetrahydro-2H-pyran-3,4-diyl diacetate (48-7) as an off white sticky solid. Yield: 1.1 g, 10.99 %; ELSD m/z 437.45 [M+18]+. [0993] A solution of (2R,3R,4R,5S)-2-(acetoxymethyl)-5-(1,3-dioxoisoindolin-2-yl)tetrahydro-2H- pyran-3,4-diyl diacetate (48-7, 1.0 eq, 1.1 g, 2.62 mmol) in methanol (11 mL) was cooled at 0 °C, sodium methoxide (25 % solution in methanol) (0.1 eq, 0.06 mL, 0.262 mmol) was added and reaction mixture was stirred at room temperature for 3 h. After completion, reaction mixture was neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated, washed with diethyl ether and dried to afford 2-((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)isoindoline-1,3-dione (48-8) as an off white solid. Yield: 0.640 g, 83.2 %; ELSD m/z 294.15 [M+1]+. [0994] A solution of 2-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)isoindoline-1,3-dione (48-8, 1.0 eq, 0.900 g, 3.07 mmol) in dichloroethane (9 mL) was cooled at 0 °C, N,N-disopropylethylamine (8.0 eq, 4.53 mL, 24.6 mmol) and chloromethyl methyl ether (6.0 eq, 1.48 mL, 18.4 mmol) were added and reaction mixture was heated at 80 °C for 16 h. After completion, reaction mixture was cooled, water was added and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-25 % ethyl acetate in hexane to afford 2- ((3S,4R,5R,6R)-4,5-bis(methoxymethoxy)-6-((methoxymethoxy)methyl)tetrahydro-2H-pyran-3- yl)isoindoline-1,3-dione (48-9) as colourless viscous liquid. Yield: 0.600 g, 45.96 %; ELSD m/z 443.25 [M+18]+. [0995] To a solution of 2-((3S,4R,5R,6R)-4,5-bis(methoxymethoxy)-6- ((methoxymethoxy)methyl)tetrahydro-2H-pyran-3-yl)isoindoline-1,3-dione (48-9, 1.0 eq, 0.580 g, 1.36 mmol) in ethanol (6 mL), ethylene diamine (10.0 eq, 0.91 mL, 13.6 mmol) was added and reaction mixture was heated at 80 °C for 16 h. After completion, reaction mixture was cooled and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford (3S,4R,5R,6R)-4,5-bis(methoxymethoxy)-6- ((methoxymethoxy)methyl)tetrahydro-2H-pyran-3-amine (48-10) as colourless viscous liquid. Yield: 0.310 g, 76.99 %; ELSD m/z 296.20 [M+1]+. [0996] To a solution of 3,3,3-trifluoropropanoic acid (1, 1.0 eq, 0.100 g, 0.781 mmol) in dichloromethane (2 mL), N-hydroxysuccinimide (1a, 1.5 eq, 0.134 g, 1.17 mmol) and N,N'- dicyclohexylcarbodiimide (2.0 eq, 0.321 mL, 1.56 mmol) were added and reaction mixture was stirred at room temperature for 16 h. After completion, reaction mixture was concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford 2,5-dioxopyrrolidin-1-yl 3,3,3-trifluoropropanoate (2) as colorless viscous liquid. Yield: 0.050 g, 28.57 %; LCMS m/z No ionization; 1H NMR (400 MHz, CDCl3) δ 3.54-3.47 (m, 2H), 2.87 (s, 4H). [0997] To a solution of (3S,4R,5R,6R)-4,5-bis(methoxymethoxy)-6- ((methoxymethoxy)methyl)tetrahydro-2H-pyran-3-amine (48-10, 1.0 eq, 0.070 g, 0.237 mmol) in N,N- dimethylformamide (1 mL), 2,5-dioxopyrrolidin-1-yl 3,3,3-trifluoropropanoate (2, 1.5 eq, 0.080 g, 0.356 mmol) and N,N-diisopropylethylamine (3.0 eq, 0.12 mL, 0.711 mmol) were added and reaction mixture was stirred at room temperature for 16 h. After completion, water was added to the reaction mixture and extracted with ethyl acetate. The organic layer was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-20 % ethyl acetate in hexane to afford N-((3S,4R,5R,6R)-4,5- bis(methoxymethoxy)-6-((methoxymethoxy)methyl)tetrahydro-2H-pyran-3-yl)-3,3,3- trifluoropropanamide (3) as light yellow viscous liquid. Yield: 0.030 g, 31.22 %; LCMS m/z 423.20 [M+18]+. [0998] To a solution of N-((3S,4R,5R,6R)-4,5-bis(methoxymethoxy)-6- ((methoxymethoxy)methyl)tetrahydro-2H-pyran-3-yl)-3,3,3-trifluoropropanamide (3, 1.0 eq, 0.035 g, 0.086 mmol) in tetrahydrofuran (0.5 mL) was cooled at 0 °C, 6N hydrochloric acid (0.5 mL) was added and reaction mixture was stirred at room temperature for 16 h. After completion, reaction mixture was concentrated to get crude which was purified by prep HPLC (25-33 % acetonitrile in water with 0.1 % trifluoroacetic acid). Fractions containing the desired product were combined and lyophilized to dryness to afford N-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)-3,3,3- trifluoropropanamide (XC28) as a cream solid. Yield: 0.008 g, 33.91 %; LCMS m/z 274.10 [M+1]+; 1H NMR (400 MHz,CD3OD) δ 4.18-4.12 (m, 1H), 3.98-3.93 (m, 1H), 3.86 (d, J = 3.2 Hz, 1H), 3.75-3.70 (m, 1H), 3.67-3.63 (m, 1H), 3.52-3.49 (m, 1H), 3.41-3.38 (m, 1H), 3.21-3.13 (m, 2H), 3.08 (t, J = 10.8 Hz, 1H). Synthesis of Compound XC10:
Figure imgf000325_0001
[0999] To a stirred solution of (3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro- 2H-pyran-3-amine (which can be prepared by literature routes described in WO2022035997A1) (1a, 1.0 eq.,0.50 g.,4.11 mmol.) and (S)-2-(methoxymethyl)oxirane (1, 1.0 eq., 0.0914 g, 1.04 mmol.) in ethanol (5.0 mL) was added N,N-Diisopropylethylamine (2.0 eq,.0.403 mL, 2.31 mmol.), and reaction mixture was refluxed for 12 h. After completion reaction (monitored by LCMS.), ethanol was evaporated under reduce pressure to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-10 % methanol in dichloromethane to afford (S)-1-(((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)-3-methoxypropan-2-ol (2) as an off white solid. Yield: 0.47 g, 78.12 %; LCMS m/z 522.15 [M+1]+. [1000] To a stirred solution of (S)-1-(((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)-3-methoxypropan-2-ol (2, 1.0 eq., 0.37 g., 0.709 mmol) in acetonitrile (4.0 mL), 1,1'-carbonyldiimidazole (CDI) (1.5 eq., 0.173 g., 1.06 mmol) and N,N- dimethylpyridin-4-amine (, 0.1 eq., 8.67 mg 0.070 mmol) were added and reaction mixture was stirred at room temperature for 16h. After completion (monitored by LCMS), reaction mixture was concentrated under reduce pressure to get crude which was purified by flash column chromatography on silica gel using 0-50% ethyl acetate in hexane to afford a (S)-3-((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)-5-(methoxymethyl)oxazolidin-2-one (3) as colorless liquid. Yield: 0.2 g; 51.49 %, LCMS m/z 548.0 [M+1]+. [1001] To a solution of (S)-3-((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro- 2H-pyran-3-yl)-5-(methoxymethyl)oxazolidin-2-one (3, 1.0 eq., 0.2 g, 0.365 mmol) in methanol (2.0 mL) and tetrahydrofuran (2.0 mL) was added 10% palladium on carbon (0.20 g). Thereafter, reaction mixture was stirred under hydrogen atmosphere at room temperature for 24h. After completion (monitored by LCMS), catalyst was removed by filtration on celite bed and rinsed with methanol, solvents were evaporated under reduced pressure and directly purified by prep HPLC (25-35% acetonitrile in water with 0.1% TFA) Fractions containing desired product were combined and lyophilized to dryness to afford (S)-3-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)-5- (methoxymethyl)oxazolidin-2-one (XC10) as an off white solid. Yield: 0.070 g, 73.08 %; LCMS m/z 278.10 [M+1]+.1H NMR (400 MHz, DMSO-d6 with D2O exchange) δ 4.65-4.59 (m,1H), 3.80-3.73 (m, 2H), 3.62-3.56 (m, 4H), 3.48-3.40 (m, 4H), 3.27 (s, 3H), 3.26-3.20 (m, 2H). Synthesis of Compound XC25:
Figure imgf000326_0001
Figure imgf000327_0001
[1002] To a stirred solution of (2R,3R,4R)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-5-nitro-3,4- dihydro-2H-pyran (3, 1.0 eq., 4.5 g, 9.75 mmol) and dimethyl (S)-2-hydroxysuccinate (2.0 eq., 3.16 g, 19.5 mmol) in anhydrous toluene (30 mL) under Ar, was added activated molecular sieves (3 Å, 1.50 g) and stirred for 1h at room temperature. Then, potassium tert-butoxide (1 M solution in THF, 4.88 mmol, 4.88 mL, 0.5 eq.,) was added at 0 °C and stirred for 4h at room temperature. Thereafter, acetic acid (0.5 mL) was added to quench the reaction, and molecular sieves were filtered off. The filtrate was concentrated under reduced pressure to get residue which was purified by silica gel flash column chromatography using 10% ethyl acetate in hexane to furnish dimethyl (2S)-2-(((3R,4R,5R,6R)-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)-3-nitrotetrahydro-2H-pyran-2-yl)oxy)succinate (4) as colorless oil. Yield 3.1 g, 50.98%. LCMS m/z 641.32 [M+18]+. [1003] To a stirred solution of dimethyl (2S)-2-(((3R,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)-3-nitrotetrahydro-2H-pyran-2-yl)oxy)succinate (4, 1.0 eq., 3.1 g, 3.48 mmol) in glacial acetic acid (15 mL), zinc (12.0 eq., 2.73g, 41.8 mmol) was added at room temperature and then refluxed for 6h. After completion (monitored by TLC), the reaction mixture was diluted with methanol and filtered over celite pad. The filtrate was evaporated to get crude dimethyl (2S)-2-(((3R,4R,5R,6R)-3- amino-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)succinate (5) as brown syrup.. Yield: 2.0 g, crude. LCMS m/z 594.12 [M+H]+. [1004] The solution of dimethyl (2S)-2-(((4R,5R,6R)-3-amino-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)succinate (5, 1.0 eq.2.0 g, 3.37 mmol) in methanol (20 mL) was refluxed for 6h. The volatile was evaporated to get crude which was purified by silica gel flash column chromatography using 60-70% ethyl acetate in hexane to furnish methyl 2- ((3S,6R,7R,8R,8aR)-7,8-bis(benzyloxy)-6-((benzyloxy)methyl)-2-oxohexahydro-1H,6H-pyrano[2,3- b][1,4]oxazin-3-yl)acetate (6) as yellow sticky solid. Yield: 0.80 g, 42.28%; LCMS m/z 560.12 [M-H]. [1005] To a stirred solution of methyl 2-((3S,6R,7R,8R,8aR)-7,8-bis(benzyloxy)-6- ((benzyloxy)methyl)-2-oxohexahydro-1H,6H-pyrano[2,3-b][1,4]oxazin-3-yl)acetate (6, 1.0 eq., 0.78 g, 1.39 mmol) in mixture of tetrahydrofuran:methanol:water (7 mL, 4:2:1,v/v/v), litium hydroxide monohydrate (1.1 eq., 0.064 g, 1.53 mmol) was added at 0 °C and stirred at room temperature for 3h. After completion, the reaction mixture was acidified with 1N HCl and extracted by dichloromethane (3x100 mL). Then organic part was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to give crude 2-((3S,6R,7R,8R,8aR)-7,8-bis(benzyloxy)-6-((benzyloxy)methyl)-2- oxohexahydro-1H,6H-pyrano[2,3-b][1,4]oxazin-3-yl)acetic acid (7) which was used for next step without further purification. Yield: 0.530 g, crude; LCMS m/z 546.12 [M-H] +. [1006] To a stirred solution 2-((3S,6R,7R,8R,8aR)-7,8-bis(benzyloxy)-6-((benzyloxy)methyl)-2- oxohexahydro-1H,6H-pyrano[2,3-b][1,4]oxazin-3-yl)acetic acid (7, 1.0 eq.0.530 g, 0.968 mmol) in dry N,N-dimethylformamide (9 mL), 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (1.2 eq., 0.442 g, 1.16 mmol), N,N-diisopropylethylamine, (3.0 eq., 0.521 mL, 2.9 mmol) were added at 0 °C. Then, tert-butyl (4-aminobutyl)carbamate (1.1 eq.0.20 g, 1.16 mmol) was added, and stirred the reaction mixture at room temperature for 12 h. After completion (monitored by TLC), water was added, and extracted with dichloromethane. The organic part was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated to give crude which was purified by pre-HPLC (60% acetonitrile in water with 0.1% acetic acid) to give tert-butyl (4-(2- ((3S,6R,7R,8R,8aR)-7,8-bis(benzyloxy)-6-((benzyloxy)methyl)-2-oxohexahydro-1H,6H-pyrano[2,3- b][1,4]oxazin-3-yl)acetamido)butyl)carbamate (mixture of both isomer) which was further passed through Chiral SFC to give tert-butyl (4-(2-((3S,4aR,6R,7R,8R,8aR)-7,8-bis(benzyloxy)-6- ((benzyloxy)methyl)-2-oxohexahydro-1H,6H-pyrano[2,3-b][1,4]oxazin-3-yl)acetamido)butyl)carbamate (8α, 0.120 g, 18%) and tert-butyl (4-(2-((3S,4aS,6R,7R,8R,8aR)-7,8-bis(benzyloxy)-6- ((benzyloxy)methyl)-2-oxohexahydro-1H,6H-pyrano[2,3-b][1,4]oxazin-3-yl)acetamido)butyl)carbamate (8β, 0.015 [1007]
Figure imgf000328_0001
8.44 (d, J = 4.8 Hz, 1H), 7.88 (t, J = 4.8 Hz, 1H), 7.44 (d, J = 7.2 Hz, 2H), 7.36-7.22 (m, 13H), 6.75 (t, J = 5.6 Hz, 1H), 5.29 (d, J = 2.4 Hz, 1H), 4.79- 4.70 (m, 2H), 4.65-4.62 (m, 1H), 4.60-4.57 (m, 1H), 4.51-4.41 (m, 3H), 4.01 (s, 2H), 3.81 (d, J = 10 Hz, 1H), 3.56-3.48 (m, 3H), 3.05-2.99 (m, 2H), 2.88-2.87 (m, 2H), 2.68-2.62 (m, 1H), 2.43-2.32 (m, 1H), 1.36 (s, 13H). [1008] (8β) 1H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 4.0 Hz, 1H), 7.88 (t, J = 6.0 Hz, 1H), 7.53 (d, J = 9.2 Hz, 2H), 7.49-7.20 (m, 13H), 6.77 (s, 1H), 5.25 (brs, 1H), 4.81-4.78 (m, 1H), 4.73-4.65 (m, 2H), 4.54-4.40 (m, 4H), 4.03-3.97 (m, 3H), 3.57-3.44 (m,, 3H), 3.16-3.02 (m, 2H), 2.90 (s, 2H), 2.66- 2.51 (m, 2H), 1.36 (s, 14H), 1.21 (s, 1H). [1009] Method for Chiral Separation: Column Name: Chiralpak IB-N5 (250*4.6)mm, 5um; Mobile Phase : CO2:0.1%IPamine in IPA 60:40; Flow Rate : 3.0 mL/min Column; Temperature : 40°C. [1010] To a solution of tert-butyl (4-(2-((3S,4aS,6R,7R,8R,8aR)-7,8-bis(benzyloxy)-6- ((benzyloxy)methyl)-2-oxohexahydro-1H,6H-pyrano[2,3-b][1,4]oxazin-3-yl)acetamido)butyl)carbamate (8β, 0.500 g, 0.697 mmol, 1.0 eq.) in methanol (10 mL), 10% Pd/C (0.900 g) was added and stirred at room temperature under H2 gas (balloon pressure) for 48h. After completion (monitor by LCMS), the reaction mixture was filtered on celite pad and washed with methanol. The filtrate was evaporated under reduced pressure to give crude which was again treated with a mixture trifluoroacetic acid and dichloromethane (10 ml, 1:1, v/v) and stirred for 1h at room temperature. Thereafter, the reaction mixture was purified by prep-HPLC (20-30% acetonitrile in water with 0.1% TFA) to give N-(4-aminobutyl)-2- ((3S,4aS,6R,7R,8R,8aR)-7,8-dihydroxy-6-(hydroxymethyl)-2-oxohexahydro-1H,6H-pyrano[2,3- b][1,4]oxazin-3-yl)acetamide (XC25) as colorless semi solid. Yield: 0.060 g, 35%; LCMS 348.30 [M+H] +; 1H NMR (400 MHz, DMSO-d6 with D2O exchange) δ 5.17 (d, J = 2.8 Hz, 1H), 4.48-4.46 (m, 1H), 3.76 (t, J = 6.4 Hz, 1H), 3.72-3.67 (m, 2H), 3.52-3.43 (m, 2H), 3.24-3.09 (m, 2H), 3.27 (dd, J = 9.6, 2.8 Hz, 1H), 3.05 (t, J = 6.8 Hz, 1H), 2.75 (t, J = 7.2 Hz, 1H), 2.61(dd, J = 14.8, 3.2 Hz, 1H), 1.52-1.46 (m, 2H), 1.44-1.39 (m, 2H). Synthesis of XC9:
Figure imgf000329_0001
XC9 [1011] To a stirred solution of 2-methoxyethan-1-amine (1.0 eq., 1.16 mL, 13.3 mmol) in dichloromethane (50 mL), 2-bromoacetyl chloride (0.7 eq,.0.772 mL, 9.32 mmol) was added dropwise at 0°C under N2 and then allowed to stir at room temperature for 12h. After completion (monitored by TLC), reaction mixture was quenched by addition of ice cold water followed by extracted with dichloromethane. Organic layer was then washed with brine, dried over anhydrous sodium sulfate and concentrated under vacuum to get 2-bromo-N-(2-methoxyethyl)acetamide (1.0 g, 5.1 mmol) as colorless liquid. Yield: 1.0 g (crude), 38.31 %; 1H NMR (400 MHz, DMSO-d6): δ 8.30 (bs, 1H), 4.05 (d, J = 7.20 Hz, 1H), 3.85 (s, 1H), 3.36-3.52 (m, 2H), 3.21-3.35 (m, 5H). [1012] A solution of 2-bromo-N-(2-methoxyethyl)acetamide (2.0 eq., 0.181 g 0.923 mmol) and (3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-amine hydrochloride (2a, 1.0 eq., 0.2 g 0.461 mmol) in acetonitrile (5.0 mL) was cooled at 0 °C, N,N-diisopropylethylamine (0.403 mL, 50 eq., 1.02 mmol) was added and the reaction mixture was stirred at room temperature for 16h. After completion (monitored by LCMS), reaction mixture was concentrated under vacuum to get crude product which was re-dissolved in ethyl acetate, washed with ice cold water. The organic layer was dried over anhydrous sodium sulfate, concentrated to get crude which was purified by silica gel flash column chromatography using 2-5% methanol in dichloromethane to afford 2-(((3S,4R,5R,6R)-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)-N-(2-methoxyethyl)acetamide (3) as green oily liquid. Yield: 0.120 g, 47.41 %; LCMS m/z 549.10 [M+1]+. [1013] A solution of 2-(((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H- pyran-3-yl)amino)-N-(2-methoxyethyl)acetamide (3, 1.0 eq, 0.220 g, 0.401mmol) in tetrahydrofuran (4.0 mL) was cooled at 0 °C, lithium aluminum hydride (40 eq., 0.656 mL, 1.31 mmol, 1M in THF) was added and reaction mixture was stirred at room temperature for 12 h. After completion (monitored by LCMS), reaction mixture was quenched by 15 % sodium hydroxide solution, brine solution was added, then extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to give crude N1-((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro- 2H-pyran-3-yl)-N2-(2-methoxyethyl)ethane-1,2-diamine (4) as brown syrup which was used for next reaction without further purification.. Yield: 0.170 g (Crude); LCMS m/z 535.10 [M+1]+. [1014] To a solution of N1-((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro- 2H-pyran-3-yl)-N2-(2-methoxyethyl)ethane-1,2-diamine (4, 1.0 eq., 0.11 g, 0.206 mmol) in acetonitrile (2.75 mL), 1-(1H-imidazole-1-carbonyl)-1H-imidazole (0.05 g, 1.5 eq., 0.309 mmol) and N,N- dimethylpyridin-4-amine (0.1 eq., 0.0025 g 0.0020 mmol) were added and reaction mixture was stirred at room temperature for 16h. After completion (monitored by LCMS), reaction mixture was concentrated to give crude residue which was purified by flash column chromatography on silica gel using 50% ethyl acetate in hexane to afford a 1-((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H- pyran-3-yl)-3-(2-methoxyethyl)imidazolidin-2-one (5) as colorless liquid. Yield: 0.080 g, 69.36 %; LCMS m/z 561.15 [M+1]+. [1015] To a stirred solution of 1-((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)-3-(2-methoxyethyl)imidazolidin-2-one (5, 1 eq, 0.047 g 0.0838 mmol) in methanol (3 mL) were added 20% Palladium hydroxide on carbon (0.05 g) and 10% palladium on carbon (0.5 g). Then reaction mixture was purged with hydrogen gas and stirred under hydrogen atmosphere. After completion, the reaction mixture was filtered through celite bed and rinsed with methanol The filtrate was concentrated under reduce pressure to afford a crude which was purified by prep HPLC (20-30% acetonitrile in water with 0.1% TFA) to afford 1-((3S,4R,5R,6R)-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)-3-(2-methoxyethyl)imidazolidin-2-one (XC9) as a colorless semi solid. Yield: 0.003 g, 12.5 %; LCMS m/z 291.15 [M+1]+; 1H NMR (400 MHz, DMSO-d6 with D2O exchange): δ 3.85-3.75 (m, 1H), 3.71 (bs, 1H), 3.61-3.53 (m, 1H), 3.45 (d, J = 6.0 Hz, 1H), 3.38 (t, J = 5.6 Hz, 1H), 3.30-3.16 (m, 7H). Synthesis of XC11:
Figure imgf000330_0001
Figure imgf000331_0001
[1016] To a stirred solution of ((3aR,4S,7S,8R,8aR)-8-azido-2,2-dimethyltetrahydro-4,7- epoxy[1,3]dioxolo[4,5-d]oxepin-4(5H)-yl)methanol (which can be prepared by literature routes described in WO 2017083368A1) (1, 1.0 eq., 0.430 g., 1.67 mmol.) in tetrahydrofuran (5 mL) was add sodium hydride (1.5 eq., 0.1 g, 2.51 mmol.) at 0° C and stirred for 30 minutes, then methyl iodide (5.0 eq., 0.520 ml.,8.36 mmol) was added and the mixture was stirred at room temperature for 2 h. After completion (monitored by TLC), the reaction mixture was quenched with cold water and extracted with dichloromethane (2-3 times). The organic layer was dried over anhydrous sodium sulphate, filtered, and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-60 % ethyl acetate in hexane to afford (3aR,4S,7S,8R,8aR)-8-azido-4-(methoxymethyl)-2,2- dimethylhexahydro-4,7-epoxy[1,3]dioxolo[4,5-d]oxepine (2) as colorless liquid. Yield: 0.30 g, 66.16%; 1H NMR (400 MHz, DMSO-d6): δ 5.43 (d, J = 2.0 Hz, 1H), 4.29 (d, J = 4.8 Hz, 2H), 3.91 (d, J = 7.6 Hz, 1H), 3.77 (d, J = 10.0 Hz, 1H), 3.66 (d, J = 7.6 Hz, 1H), 3.58 (d, J = 10.0 Hz, 1H), 3.39 (t, J = 4.8 Hz, 1H), 3.32 (d, J = 6.0 Hz, 3H), 1.45 (s, 3H), 1.31 (s, 3H). [1017] To a stirred solution of (3aR,4S,7S,8R,8aR)-8-azido-4-(methoxymethyl)-2,2- dimethylhexahydro-4,7-epoxy[1,3]dioxolo[4,5-d]oxepine (2, 1.0 eq., 0.3 g., 1.11 mmol) in methanol (5.0 mL) was added 10% palladium on carbon (0.12 g) and stirred under hydrogen atmosphere. After completion (monitored by TLC and LCMS), the reaction mixture was filtered through celite bed and rinsed with methanol. The filtrate was concentrated under vacuum to afford (3aR,4S,7S,8R,8aR)-4- (methoxymethyl)-2,2-dimethylhexahydro-4,7-epoxy[1,3]dioxolo[4,5-d]oxepin-8-amine (3) as a light yellow viscous liquid. Yield: 0.070 g, 25.81 %; LCMS m/z 246.10 [M+1]+. [1018] To a stirred solution of (3aR,4S,7S,8R,8aR)-4-(methoxymethyl)-2,2-dimethylhexahydro-4,7- epoxy[1,3]dioxolo[4,5-d]oxepin-8-amine (3, 1.0 eq., 0.070 g.,0.285 mmol) and 2-chloro-4-(prop-2-yn-1- yloxy)-6-(trifluoromethyl)pyrimidine (3a, 1.0 eq., 0.0675 g 0.285 mmol) in acetonitrile (1.0 mL) was added N,N-diisopropylethylamine (5.0 eq., 0.249 mL 1.43 mmol) at room temperature, and then heated at 70°C for 16h. After completion (monitored by LCMS), reaction mixture was concentrated to get crude. which was purified by column chromatography using silica gel (100-200 mesh) and 0-10 % methanol in DCM to afford N-((3aR,4S,7S,8R,8aR)-4-(methoxymethyl)-2,2-dimethylhexahydro-4,7- epoxy[1,3]dioxolo[4,5-d]oxepin-8-yl)-4-(prop-2-yn-1-yloxy)-6-(trifluoromethyl)pyrimidin-2-amine (4) as a white solid. Yield: 0.070 g, 55.07%; LCMS m/z 446.05 [M+1]+. [1019] A solution of N-((3aR,4S,7S,8R,8aR)-4-(methoxymethyl)-2,2-dimethylhexahydro-4,7- epoxy[1,3]dioxolo[4,5-d]oxepin-8-yl)-4-(prop-2-yn-1-yloxy)-6-(trifluoromethyl)pyrimidin-2-amine (4, 1.0 eq., 0.080 g, 0.180 mmol) in dichloromethane (1.0 mL) was cooled at 0 °C, trifluoroacetic acid (1.0 mL) was added and reaction mixture was stirred at room temperature for 1 h. After completion, reaction mixture was purified by prep HPLC (45-55% ACN in H2O with 0.1% TFA). Fractions containing desired compound were collected and lyophilized to afford (1S,2R,3R,4R,5S)-1-(methoxymethyl)-4-((4-(prop-2- yn-1-yloxy)-6-(trifluoromethyl)pyrimidin-2-yl)amino)-6,8-dioxabicyclo[3.2.1]octane-2,3-diol (XC11) as an off white solid. Yield: 0.0013 g, 1.37 %; m/z 406.0 [M+1]+. Synthesis of XC17:
Figure imgf000332_0001
[1020] A solution of XC-17A which can be prepared by literature routes described in WO2022235699A2 (XC17-A, 1.0 eq, 80.0 mg, 0.326 mmol) in THF (0.815 mL) was cooled in an ice bath then triethylamine (2.2 eq, 100 mL, 0.718 mmol) was added followed by p-toluenesulfonyl chloride (2.0 eq, 124 mg, 0.652 mmol). The reaction was stirred at room temperature for 1 hour then 50 °C for 18 h. The crude reaction was adsorbed to celite then purified by silica gel chromatography (0-20% MeOH in DCM) to give ((3aR,4R,7S,7aR)-7-acetamido-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4- yl)methyl 4-methylbenzenesulfonate. Yield: 90 mg, 69%. LCMS m/z 400.1 [M+H]+. [1021] A mixture of sodium hydride (3.0 eq, 4.5 mg, 0.113 mmol) and 1H-pyrazole (3.2 eq, 8.2 mg, 0.120 mmol) under nitrogen was dissolved in DMF (0.188 mL) then ((3aR,4R,7S,7aR)-7-acetamido-2,2- dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl 4-methylbenzenesulfonate (1.00 eq, 15.0 mg, 0.0376 mmol) was added and the reaction was stirred at room temperature for 3 h then 19 h at 80 °C. The reaction was diluted with DCM (2 mL) and water (3 mL). The organic layer was washed with water (5 mL) then dried over Na2SO4, filtered, and concentrated to give N-((3aS,4R,7S,7aR)-4-((1H- pyrazol-1-yl)methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)acetamide as a solid. The residue was used in the next step without further purification. LCMS m/z 296.2 [M+H]+. [1022] N-((3aS,4R,7S,7aR)-4-((1H-pyrazol-1-yl)methyl)-2,2-dimethyltetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-7-yl)acetamide (1.0 eq, 7.0 mg, 0.0237 mmol) was dissolved in water (150 mL) and trifluoroacetic acid (88.8 eq, 150 mL, 2.10 mmol). After 90 minutes, the reaction was concentrated under vacuum then diluted further with water and MeOH before being purified by reversed- phase HPLC (5-30% ACN in water with 0.1% FA) to give N-((3S,4R,5R,6R)-6-((1H-pyrazol-1- yl)methyl)-4,5-dihydroxytetrahydro-2H-pyran-3-yl)acetamide (XC17). Yield: 1.0 mg, 16.5%. LCMS m/z 312.1 [M+H]+. Synthesis of XC12:
Figure imgf000333_0001
[1023] A solution of triphenylphosphine (1.5 eq, 27.3 mg, 0.104 mmol) in THF (0.693 mL) was cooled in an ice bath before adding diisopropyl azodicarboxylate (1.2 eq, 16 uL, 0.0832 mmol). After 5 minutes, N-((3aR,4R,7S,7aR)-4-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran- 7-yl)acetamide (XC17-A, 1.00 eq, 17.0 mg, 0.0693 mmol) was added and after another 10 minutes, 4- methoxyphenol (1.5 eq, 12.9 mg, 0.104 mmol) was added and the reaction was stirred at room temperature for 1 hour then at 50 °C for 18 h. The reaction was purified directly by reversed-phase HPLC (10-50% acetonitrile in water w/0.1% FA) to give N-((3aR,4R,7S,7aR)-4-((4- methoxyphenoxy)methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)acetamide. LCMS m/z 351.5 [M+H]+. [1024] A solution of N-((3aR,4R,7S,7aR)-4-((4-methoxyphenoxy)methyl)-2,2-dimethyltetrahydro- 4H-[1,3]dioxolo[4,5-c]pyran-7-yl)acetamide in a solution of TFA/water (1:1) was stirred at room temperature for 1 hour. The reaction was purified by reversed-phase HPLC (5-50% ACN in water with 0.1% FA) to give N-((3S,4R,5R,6R)-4,5-dihydroxy-6-((4-methoxyphenoxy)methyl)tetrahydro-2H-pyran- 3-yl)acetamide, XC12, as a white solid. Yield: 1.1 mg, 5%. LCMS m/z 312.1 [M+H]+. Synthesis of XC15:
Figure imgf000333_0002
48-4 XC15 [1025] A solution of (2R,3R,4R,5S)-5-amino-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4- diol;hydrochloride (48-4, 1.00 eq, 10.0 mg, 0.0501 mmol), 2-chloropyrimidine (3.00 eq, 17.2 mg, 0.150 mmol) and DIPEA (4.00 eq, 35 mL, 0.200 mmol) in IPA (0.251 mL) and NMP (0.251 mL) was heated to 120 °C for 18 h. The reaction was diluted with water then purified by reversed-phase HPLC (4-100% acetonitrile in water w 200 mM NH4OH) to give (2R,3R,4R,5S)-2-(hydroxymethyl)-5-(pyrimidin-2- ylamino)tetrahydro-2H-pyran-3,4-diol (XC15). Yield: 1.2 mg, 10%. LCMS m/z 242.1 [M+H]+. Synthesis of XC22 and XC23:
Figure imgf000334_0001
XC22 XC23 [1026] A solution of N-((3aR,4R,7S,7aR)-4-(hydroxymethyl)-2,2-dimethyltetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-7-yl)acetamide (XC17-A, 1.0 eq, 30.0 mg, 0.122 mmol) and sodium hydride (2.0 eq, 9.8 mg, 0.245 mmol) in DMF (0.612 mL) was cooled in an ice bath before adding iodomethane (1.5 eq, 11 mL, 0.183 mmol) then removing the ice bath. After 1 hour, the reaction was filtered then purified by RPHPLC (10-50% ACN in water) to give N-((3aR,4R,7S,7aR)-4-(methoxymethyl)-2,2- dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)acetamide (22 mg, 0.0848 mmol, 69 % yield) and N-((3aR,4R,7S,7aR)-4-(methoxymethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5- c]pyran-7-yl)-N-methylacetamide. Yield: 7.0 mg, 21% and 22 mg, 69% respectively. LCMS m/z 274.0 [M+H]+. [1027] A solution of N-((3aR,4R,7S,7aR)-4-(methoxymethyl)-2,2-dimethyltetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-7-yl)-N-methylacetamide (1.0 eq, 7.0 mg, 0.0256 mmol) in water (300 mL) was treated with trifluoroacetic acid (10.0 eq, 0.018 mL, 0.256 mmol). The reaction was filtered then purified by reversed-phase HPLC (5-50% ACN in water) to give N-((3S,4R,5R,6R)-4,5-dihydroxy-6- (methoxymethyl)tetrahydro-2H-pyran-3-yl)-N-methylacetamide (XC22). Yield: 3.4 mg, 57 %. LCMS m/z 234.0 [M+H]+. [1028] A solution of N-((3aR,4R,7S,7aR)-4-(methoxymethyl)-2,2-dimethyltetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-7-yl)acetamide (1.0 eq, 22.0 mg, 0.0848 mmol) in water (300 mL) was treated with formic acid (16.0 eq, 51 mL, 1.36 mmol) and after 1h, some starting material was remaining so trifluoroacetic acid (8.4 eq, 51 mL, 0.716 mmol) was added. The reaction was filtered then purified by reversed-phase HPLC (5-50% ACN in water) to give N-((3S,4R,5R,6R)-4,5-dihydroxy-6- (methoxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XC23). Yield: 6.6 mg, 35%. LCMS m/z 234.0 [M+H]+. Synthesis of XC18:
Figure imgf000335_0001
[1029] A vial containing (2R,3R,4R,5S)-5-amino-2-(hydroxymethyl)tetrahydropyran-3,4- diol;hydrochloride (48-4, 1.5 eq, 53.1 mg, 0.266 mmol), 6-chloro-4-(trifluoromethyl)picolinic acid (1.0 eq, 40.0 mg, 0.177 mmol) and BrettPhos Pd G3 (0.2 eq, 32.2 mg, 0.0355 mmol) was placed under nitrogen in a dry box then anhydrous DMA (1.773 mL) was added followed by an anhydrous solution of sodium tert-butoxide solution (2M in THF, 4.0 eq, 355 mL, 0.709 mmol) and the reaction was sealed then heated to 100 °C for 2h. The reaction was diluted with water and DMSO then purified by reversed- phase HPLC (3-70% acetonitrile in water w/0.1% TFA) to give 6-[[(3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]amino]-4-(trifluoromethyl)pyridine-2-carboxylic acid as a mixture of regio-isomers. Yield: 15 mg, 24%. LCMS m/z 353.1 [M+H]+. [1030] A solution of the mixture from the synthesis of A (15 mg), propargylamine (3.00 eq, 5.5 mL, 0.0852 mmol), and DIPEA (1.0 eq, 2.9 mg, 0.0284 mmol) in DMSO (0.600 mL) was treated with HATU (1.2 eq, 13.0 mg, 0.0341 mmol) at room temperature. After 2 h, the solution was purified by reversed- phase HPLC (10-50% acetonitrile in water w/0.1% TFA) to give 6-(((2R,3R,4R,5S)-5-amino-3,4- dihydroxytetrahydro-2H-pyran-2-yl)methoxy)-N-(prop-2-yn-1-yl)-4-(trifluoromethyl)picolinamide. Yield: 3.3 mg, 30%. LCMS m/z 390.0 [M+H]+. [1031] A solution of 6-[[(2R,3R,4R,5S)-5-amino-3,4-dihydroxy-tetrahydropyran-2-yl]methoxy]-N- prop-2-ynyl-4-(trifluoromethyl)pyridine-2-carboxamide (1.0 eq, 3.3 mg, 0.00848 mmol) in DMSO (0.500 mL) was treated with triethylamine (3.0 eq, 3.5 mL, 0.0254 mmol) followed by acetic anhydride (1.0 eq, 0.80 mL, 0.00848 mmol) and the reaction was stirred at room temperature. After 2 h, the reaction was purified by reversed-phase HPLC (10-50% acetonitrile in water w/0.1% TFA) to give 6- [[(2R,3R,4R,5S)-5-acetamido-3,4-dihydroxy-tetrahydropyran-2-yl]methoxy]-N-prop-2-ynyl-4- (trifluoromethyl)pyridine-2-carboxamide (XC18). Yield: 1.1 mg, 31%. LCMS m/z 432.2 [M+H]+. Synthesis of XC19:
Figure imgf000336_0001
[1032] A vial containing (2R,3R,4R,5S)-5-amino-2-(hydroxymethyl)tetrahydropyran-3,4- diol;hydrochloride (1.5 eq, 53.1 mg, 0.266 mmol), 6-chloro-4-(trifluoromethyl)picolinic acid (1.0 eq, 40.0 mg, 0.177 mmol) and BrettPhos Pd G3 (0.2 eq, 32.2 mg, 0.0355 mmol) was placed under nitrogen in a dry box then anhydrous DMA (1.773 mL) was added followed by an anhydrous solution of sodium tert-butoxide (2M in THF, 4.0 eq, 355 mL, 0.709 mmol) and the reaction was sealed then heated to 100 °C for 2h. The reaction was diluted with water and DMSO then purified by reversed-phase HPLC (3- 70% acetonitrile in water w/0.1% TFA) to give 6-[[(3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]amino]-4-(trifluoromethyl)pyridine-2-carboxylic acid as a mixture of regio-isomers. Yield: 15 mg, 24%. LCMS m/z 353.1 [M+H]+. [1033] A solution of propargylamine (3.00 eq, 5.5 mL, 0.0852 mmol), 6-[[(3S,4R,5R,6R)-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]amino]-4-(trifluoromethyl)pyridine-2-carboxylic acid (1.0 eq, 10.0 mg, 0.0284 mmol) and DIPEA (1.0 eq, 2.9 mg, 0.0284 mmol) in DMSO (0.600 mL) was treated with HATU (1.2 eq, 13.0 mg, 0.0341 mmol) at room temperature. After 2 h, the solution was purified by reversed-phase HPLC (10-50% acetonitrile in water w/0.1% TFA) to give 6-[[(3S,4R,5R,6R)- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]amino]-N-prop-2-ynyl-4- (trifluoromethyl)pyridine-2-carboxamide (XC19). Yield: 1.1 mg, 10%. LCMS m/z 390.0 [M+H]+. Synthesis of N-((2R,3R,4R,5R,6R)-2-(6-aminohexyl)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-3-yl)acetamide (XB45)
Figure imgf000336_0002
4 XB45 [1034] Synthesis of N-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-2-(6- hydroxyhexa-1,3-diyn-1-yl)tetrahydro-2H-pyran-3-yl)acetamide (2) [1035] To a stirred solution of N-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-2- ethynyltetrahydro-2H-pyran-3-yl)acetamide (XB147, 2.0 g, 1.0 eq., 4.0 mmol), piperidine (0.852 g, 2.5 eq., 10.0 mmol), copper(I) bromide (0.056 g, 0.1 eq., 0.4 mmol), and hydroxylamine hydrochloride (0.055 g, 0.2 eq., 0.4 mmol) in methanol (8.0 mL) under nitrogen atmosphere at room temperature, was added a degassed solution of 4-bromobut-3-yn-1-ol (1, 0.656 g, 1.1 eq., 2.2 mmol) in methanol (2 mL) over a period of 1.5 hrs. The mixture was stirred for 2 h. After completion (monitored by LCMS & TLC), solvent was removed under reduced pressure to get crude residue which was dissolved again in ethyl acetate and washed with ice cold water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to give crude product which was purified by silica gel flash column chromatography using 0-50% ethyl acetate in hexane to afford N-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)- 6-((benzyloxy)methyl)-2-(6-hydroxyhexa-1,3-diyn-1-yl)tetrahydro-2H-pyran-3-yl)acetamide (2) as an off white solid. Yield: 1.9 g, 83.0%. LCMS m/z 568.13 [M+H]. [1036] Synthesis of 6-((2R,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)hexa-3,5-diyn-1-yl 4-methylbenzenesulfonate (3) [1037] To a stirred solution of N-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-2- (6-hydroxyhexa-1,3-diyn-1-yl)tetrahydro-2H-pyran-3-yl)acetamide (2, 0.80 g, 1.0 eq., 1.41 mmol) in dichloromethane (10 mL), pyridine (0.341 mL, 3.0 eq., 4.23 mmol), 4-dimethylaminopyridine (0.08 g, 0.3 eq., 0.50 mmol) and 4-methylbenzene-1-sulfonyl chloride (0.80 g, 3.0 eq., 4.23 mmol) were added sequentially at 0 °C. Then reaction mixture was stirred for 16h at room temperature. After completion (monitored by TLC), the reaction mixture was poured into cold 1N HCl, and extracted with dichloromethane. The organic part was then washed with saturated bicarbonate followed by brine, and dried over anhydrous sodium sulfate, filtered, and concentrated to give crude residue which was purified by silica gel flash column chromatography using 0-30% ethyl acetate in hexane to afford 6- ((2R,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2- yl)hexa-3,5-diyn-1-yl 4-methylbenzenesulfonate (3) as white solid. Yield: 0.81 g, 80.0%; LCMS, m/z 722.06 [M+H]. [1038] Synthesis of N-((2R,3S,4R,5R,6R)-2-(6-azidohexa-1,3-diyn-1-yl)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide (4) [1039] A solution of 6-((2R,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)hexa-3,5-diyn-1-yl 4-methylbenzenesulfonate (3, 0.715 g, 1 eq., 0.990 mmol) in N, N-dimethylformamide (5 mL) was treated with sodium azide (0.322 mg, 5 eq., 4.95 mmol). The suspension was then heated at 80°C for 6h. After completion (monitored by TLC), the reaction mixture was allowed to come to room temperature, and poured it to water, and extracted with ethyl acetate. The organic part was dried over anhydrous sodium sulfate, filtered, and concentrated to give crude which was purified by silica gel flash column chromatography using 0-30% ethyl acetate in hexane to afford N-((2R,3S,4R,5R,6R)-2-(6-azidohexa-1,3-diyn-1-yl)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide (4) as an off white solid. Yield: 0.330 g, 56.0%; LCMS m/z 593.20 [M+H]. [1040] Synthesis of N-((2R,3R,4R,5R,6R)-2-(6-aminohexyl)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB45) [1041] To a stirred solution of N-((2R,3S,4R,5R,6R)-2-(6-azidohexa-1,3-diyn-1-yl)-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide (4, 1.0 eq.,0.2 g, 0.337 mmol) in a mixture of methanol (5 mL), tetrahydrofuran (2 mL), acetic acid (0.20 mL) and water (0.20 mL) was added 10% palladium on carbon (0.30 g) and the reaction mixture was stirred at room temperature under H2 gas balloon pressure. After completion of reaction, the reaction mixture was filtered through celite bed and rinsed with methanol. The filtrate was concentrated under vacuum and purified by prep HPLC (30% acetonitrile in water with 0.1 % TFA) to afford N-((2R,3R,4R,5R,6R)-2-(6-aminohexyl)-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide. Yield: 0.051 g, 49.6%; LCMS m/z 305.15 [M+H].1H NMR (400 MHz, DMSO-d6 with D2O exchange) δ 4.02-3.94 (m, 1H), 3.83-3.80 (m, 1H), 3.70 (s, 1H), 3.56-3.46 (m, 4H), 2.73 (t, J = 7.6 Hz, 2H), 1.82 (s, 3H), 1.52-1.47 (m, 3H), 1.31-1.12 (m, 7H). Synthesis of N-((2R,3R,4R,5R,6R)-2-((1-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3- triazol-4-yl)methyl)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide trifluoroacetic acid (XB46) 2a
Figure imgf000338_0001
1 (0.1 eq), i) NMO (1.5 eq), OsO4 Bestmann reagent, (CH3)2 CO:H 2 O (5:1), 0 °C-Rt K2CO3 (2.0 eq), ii) NaIO4 MeOH, Rt (CH3)2 CO:H 2 O (2:1), Rt
Figure imgf000338_0002
Figure imgf000338_0003
Figure imgf000338_0004
[1042] A solution of (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5- triyl triacetate (1, 1.0 eq, 20.0 g, 51.4 mmol) in acetyl chloride (60 mL) was cooled at 0 °C, methanol (2.4 eq, 4.98 mL, 123.3 mmol) was added and reaction mixture was stirred at room temperature for 48 h. After completion, the reaction mixture was concentrated, washed with 10 % diethyl ether in ethyl acetate and dried to afford (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-chlorotetrahydro-2H-pyran-3,4-diyl diacetate (2) as a white solid. Yield: 16.0 g, 85.16 %; 1H NMR (400 MHz, CDCl3) δ 6.08 (d, J = 2.0 Hz, 1H), 5.36-5.31 (m, 2H), 5.26-5.25 (m, 1H), 5.29-5.25 (m, 1H), 4.15-4.02 (m, 3H), 2.16 (d, J = 3.2 Hz, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.00 (s, 3H). [1043] To a stirred solution of (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-chlorotetrahydro- 2H-pyran-3,4-diyl diacetate (2, 5.0 g, 1.0 eq., 13.7 mmol) in dry tetrahydrofuran (50 mL) was added allyltributylstannane (2a, 42.7 mL, 10.0 eq., 137 mmol) and azobisisobutyronitrile (AIBN) (0.673 g, 0.3 eq., 4.1 mmol). The reaction mixture was then purged with argon and refluxed for 12 h. Thereafter, volatiles were removed in vacuo, and the remaining residue was partitioned between acetonitrile and pentane. Acetonitrile layer was extracted with pentane (3 times) to remove the remaining organotin compounds and then concentrated to dryness to get crude which was purified by silica gel flash column chromatography eluting with 45-50% ethyl acetate in hexane to afford (2R,3R,4R,5S,6R)-5-acetamido-2- (acetoxymethyl)-6-allyltetrahydro-2H-pyran-3,4-diyl diacetate as sticky colorless syrup. Yield: 2.4 g, 47.0 %; ELSD-MS m/z 372.21 [M+H]+. [1044] To a stirred solution of (2R,3R,4R,5S,6R)-5-acetamido-2-(acetoxymethyl)-6-allyltetrahydro- 2H-pyran-3,4-diyl diacetate (1.5 g, 1 eq.4.03 mmol) in acetone:water (5:1) (15 mL) was added N- methylmorpholine-N-oxide (0.708 g, 1.5 eq, 6.04 mmol) followed by osmium tetraoxide (4.0 wt % in water, 0.274 mL, 0.1 eq, 0.0403 mmol) at 0°C, and stirred for 2 h at room temperature. After completion, the reaction mixture was extracted with ethyl acetate. The organic part was then dried over anhydrous sodium sulphate, filtered, and concentrated to give crude product which was again dissolved in acetone:water (2:1) (15 mL), and added sodium periodate (1.55 g, 2.0 eq., 8.06 mmol), stirred for another 3 h at room temperature. Thereafter, reaction mixture was extracted with ethyl acetate, and the collected ethyl acetate was dried over anhydrous sodium sulphate, filtered, and concentrated to give crude (2R,3R,4R,5S,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-oxoethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (4) as colorless syrup. This was immediately used for next step without purification. Yield: 1.4 g (Crude); ELSD-MS m/z 374.19 [M+H]+. [1045] To a stirred solution of (2R,3R,4R,5S,6R)-5-acetamido-2-(acetoxymethyl)-6-(2- oxoethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (1.0 g, 1.00 eq, 2.68 mmol) in methanol (25.0 mL) at 0 °C, were added potassium carbonate (1.11 g, 3.0 eq., 8.04 mmol), dimethyl (1-diazo-2- oxopropyl)phosphonate (1.03 g, 2.0 eq., 5.36 mmol) and stirred at room temperature for 5 h. Thereafter, volatiles were evaporated in vacuo to get crude which was purified by prep-HPLC (70 % acetonitrile in water with 0.1 % trifluoroacetic acid). Fractions containing the desired product were combined and lyophilized to dryness to afford N-((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-(prop-2-yn-1- yl)tetrahydro-2H-pyran-3-yl)acetamide (XB4B) as an off-white solid.. Yield: 0.112 g, 17.04 %. LCMS m/z 244.11 [M+H]+.1H NMR (400 MHz, DMSO-d6 with D2O exchange) δ 4.05-4.00 (m, 2H), 3.73 (bs, 1H), 3.58-3.45 (m, 4H), 2.60 (t, J = 2.4 Hz, 1H), 2.46-2.41 (m, 1H), 2.33-2.27 (m, 1H), 1.82 (s, 3H). [1046] Synthesis of N-((2R,3R,4R,5R,6R)-2-((1-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-1H- 1,2,3-triazol-4-yl)methyl)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide trifluoroacetic acid (XB46) [1047] To a solution of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine (5) (101.3 mg, 0.464 mmol, 1.00 eq) and N-((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-(prop-2-yn-1- yl)tetrahydro-2H-pyran-3-yl)acetamide (XB4B) (116.1 mg, 0.477 mmol, 1.03 eq) in 2 mL dimethyl sulfoxide was added tetrakis(acetonitrile)copper(I) hexafluorophosphate(187.3 mg, 0.403 mmol, 1.1 eq) as a solid in one portion. The mixture stirred under nitrogen atmosphere at ambient temperature for approximately 30 minutes, then directly purified by preparatory HPLC, eluting with 1-30% acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford trifluoroacetic acid salt of Compound XB46, as a white foam. Yield: 217 mg (81%); LCMS m/z 462.4 [M+H]. Synthesis of XB48 °
Figure imgf000340_0001
XB48 [1048] Synthesis of N,N-dibenzyl-2-(2-((3-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)-3-nitrotetrahydro-2H-pyran-2-yl)prop-2-yn-1-yl)oxy)ethoxy)ethan-1-amine (2) [1049] A mixture of 2-[2-(2-propynyloxy)ethoxy]ethylamine (1, 1.00 eq, 9.75 g, 68.1 mmol), anhydrous potassium carbonate (2.32 eq, 21.82 g, 158 mmol) and acetonitrile (306.46 mL) was treated with benzyl bromide (2.10 eq, 17 mL, 143 mmol) then heated to 50 °C for 2h. The reaction was filtered through celite and the filtrate concentrated under vacuum. The residue was adsorbed to silica then purified by column chromatography (5-100% EtOAc in hexanes) to give N,N-dibenzyl-2-(2-prop-2- ynoxyethoxy)ethanamine 2 as a clear oil. Yield: 18.4 g, 83%. LCMS m/z 324.22 [M+H]. [1050] N,N-dibenzyl-2-(2-prop-2-ynoxyethoxy)ethanamine (2, 1.43 eq, 3.70 g, 11.4 mmol) was dissolved in 10 mL toluene then concentrated to dryness and left under high vacuum. Next, (2R,3R,4R)- 3,4-dibenzyloxy-2-(benzyloxymethyl)-5-nitro-3,4-dihydro-2H-pyran (3, 1.00 eq, 3.70 g, 8.02 mmol) was dissolved in 10 mL toluene and concentrated under high vacuum. In an oven dried flask, a solution of N,N-dibenzyl-2-(2-prop-2-ynoxyethoxy)ethanamine (2, 1.43 eq, 3.70 g, 11.4 mmol) in anhydrous THF (31.8 mL) under nitrogen via balloon was cooled to -50 °C (dry-ice bath - 1:1 MeOH/water) then treated with the slow addition of butyl lithium, 2.5M in hexanes (1.20 eq, 3.8 mL, 9.62 mmol) and the reaction was stirred @ -50 °C for 60 minutes. Next, a solution of (2R,3R,4R)-3,4-dibenzyloxy-2- (benzyloxymethyl)-5-nitro-3,4-dihydro-2H-pyran (3, 1.00 eq, 3.70 g, 8.02 mmol) in dry THF (12.7 mL) was added dropwise over 5 minutes while maintaining -50 °C externally. After 60 minutes, the reaction was quenched with the addition of 7.1M aq. ammonium chloride (32.8 eq, 37 mL, 263 mmol) until a reaction pH of 9-10 was reached, then the cold bath was removed and the slurry was warmed to room temperature. The desired product was extracted with EtOAc (100 mL, 50 mL) and the combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give crude oil. The oil was adsorbed to silica gel then purified by silica gel chromatography (10% then 20% 2- MeTHF in hexanes) to give: N,N-dibenzyl-2-[2-[3-[(2R,3S,4R,5R,6R)-4,5-dibenzyloxy-6- (benzyloxymethyl)-3-nitro-tetrahydropyran-2-yl]prop-2-ynoxy]ethoxy]ethanamine (4, 2.19 g, 2.79 mmol, 35 % yield). LCMS m/z 785.34 [M+H]. and N,N-dibenzyl-2-(2-((3-((2S,3S,4R,5R,6R)-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)-3-nitrotetrahydro-2H-pyran-2-yl)prop-2-yn-1- yl)oxy)ethoxy)ethan-1-amine (3.11g, 3.9 mmol, 49% yield) [1051] A solution of N,N-dibenzyl-2-[2-[3-[(2R,3S,4R,5R,6R)-4,5-dibenzyloxy-6- (benzyloxymethyl)-3-nitro-tetrahydropyran-2-yl]prop-2-ynoxy]ethoxy]ethanamine (4, 1.00 eq, 2.40 g, 3.06 mmol) in THF (182 mL), water (77.6 mL) and acetic acid (46.1 mL) was cooled over ice then treated with zinc dust (18.8 eq, 3.76 g, 57.5 mmol) followed by 12M HCl (44.3 eq, 11 mL, 135 mmol). After 90m, the reaction was filtered then re-cooled in an ice bath before it was treated with 5M aq. sodium hydroxide (350 eq, 214 mL, 1070 mmol) at such a rate as to keep the internal temperature below 24 °C. Next, the layers were partitioned then the aqueous layer was washed with DCM (80 mL). The aqueous layer was washed with DCM (50 mL) then the combined organic layer was washed with brine then dried over Na2SO4, filtered, concentrated under reduced pressure and left under high vacuum. [1052] The crude amine from step 3 (2.3 g) was dissolved in DCM (20 mL) before adding triethylamine (6.00 eq, 2.6 mL, 18.3 mmol), 4-(dimethylamino)pyridine (0.050 eq, 18.7 mg, 0.153 mmol) then acetic anhydride (9.80 eq, 2.8 mL, 30.0 mmol). After several hours, the reaction was quenched with water (20 mL) for several minutes. The organic layer was collected, and the aqueous layer was washed with DCM (2x10 mL). The organic layer was dried over Na2SO4, filtered and concentrated in the presence of silica gel for purification by silica gel chromatography (0-15% 2-MeTHF in DCM) to give N-[(2R,3S,4R,5R,6R)-4,5-dibenzyloxy-6-(benzyloxymethyl)-2-[3-[2-[2- (dibenzylamino)ethoxy]ethoxy]prop-1-ynyl]tetrahydropyran-3-yl]acetamide (5). Yield: 1.86 g, 76%. LCMS m/z 797.5 [M+H]. [1053] A mixture of N-[(2R,3S,4R,5R,6R)-4,5-dibenzyloxy-6-(benzyloxymethyl)-2-[3-[2-[2- (dibenzylamino)ethoxy]ethoxy]prop-1-ynyl]tetrahydropyran-3-yl]acetamide (5, 1.00 eq, 2.02 g, 2.53 mmol), and 10% Pd/C w/w (dry basis) (0.040 eq, 0.22 g, 0.101 mmol) in acetic acid (86 mL) under nitrogen was evacuated then back-filled with hydrogen gas via balloon - a process that was repeated 3 times before leaving under an atmosphere of hydrogen. After 1h, 10% Pd/C w/w (dry basis) (0.200 eq, 1.08 g, 0.507 mmol) and 20% w/w (dry basis) palladium hydroxide (0.300 eq, 1.07 g, 0.760 mmol) were added and the reaction was again placed under hydrogen. After 3 more hours, the reaction was filtered over a pad of celite then the filter cake was rinsed while stirring with methanol (100 mL). Solvents were removed under reduced pressure and the residue was concentrated from toluene then left under high vacuum. The residue was purified by RPHPLC (5-20% ACN in water w/ 20mM NH4OH) and fractions lyophilized to give N,N-dibenzyl-2-(2-((3-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)-3-nitrotetrahydro-2H-pyran-2-yl)prop-2-yn-1-yl)oxy)ethoxy)ethan-1-amine, XB48, as a white solid. Yield: 562 mg, 63%. LCMS m/z 351.4 [M+H]. Synthesis of 3-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H- pyran-2-yl)thio)-N-(5-aminopentyl)propanamide (XB49)
Figure imgf000342_0001
[1054] Synthesis of (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H- pyrano[3,2-d]thiazole-6,7-diyl diacetate (2) [1055] To a solution of (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran- 2,4,5-triyl triacetate (1, 1.0 eq, 4.0 g, 10.3 mmol) in toluene (33.2 mL), Lawesson's reagent (0.85 eq, 3.53 g, 8.73 mmol) was added and reaction mixture was heated at 100 °C for 1.5 h. After completion, reaction mixture was cooled, water was added, neutralized with solid sodium bicarbonate and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-20 % ethyl acetate in dichloromethane to afford (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a- tetrahydro-5H-pyrano[3,2-d]thiazole-6,7-diyl diacetate (2) as light yellow viscous liquid. Yield: 2.0 g, 56.37 %; LCMS m/z 346.10 [M+18]+. [1056] Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-mercaptotetrahydro-2H- pyran-3,4-diyl diacetate (3) [1057] A solution of (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H- pyrano[3,2-d]thiazole-6,7-diyl diacetate (2, 1.0 eq, 1.6 g, 4.63 mmol) in methanol (16 mL) and water (0.16 mL) was cooled at 0 °C, trifluoroacetic acid (0.16 mL) was added and reaction mixture was stirred at room temperature for 16 h. After completion, reaction mixture was concentrated, azeotroped with toluene (2-3 times) and dried to get afford (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6- mercaptotetrahydro-2H-pyran-3,4-diyl diacetate (3) as a light yellow viscous liquid. Yield: 1.6 g (Crude); LCMS m/z 364.10 [M+H]. [1058] Synthesis of 3-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro- 2H-pyran-2-yl)thio)propanoic acid (XB5B) [1059] A solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-mercaptotetrahydro-2H- pyran-3,4-diyl diacetate (3, 1.0 eq, 2.4 g, 6.6 mmol) in N,N-dimethylformamide (24 mL) was cooled at - 78 °C, Lithium bis(trimethylsilyl)amide (LiHMDS, 1M in tetrahydrofuran) (1.0 eq, 6.6 mL, 6.6 mmol) was added and reaction mixture was stirred at the same temperature for 1 h. Then, oxetan-2-one (3a, 1.3 eq, 0.617 g, 8.58 mmol) was added and reaction mixture stirred at room temperature for 16 h. After completion, reaction mixture was concentrated to get crude which was first purified by column chromatography using silica gel (100-200 mesh) and 0-10 % methanol in dichloromethane and then by prep HPLC (10-25 % acetonitrile in water with 0.1 % trifluoroacetic acid). Fractions containing the desired product were combined and lyophilized to dryness to afford 3-(((2R,3R,4R,5R,6R)-3-acetamido- 4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoic acid (XB5B) as a white solid. Yield: 0.732 g, 25.68 %; LCMS m/z 436.05 [M+H]; 1H NMR (400 MHz, DMSO-d6) δ 12.26 (bs, 1H), 8.15 (d, J = 7.2 Hz, 1H), 5.60 (d, J = 5.2 Hz, 1H), 5.34 (d, J = 2.4 Hz, 1H), 4.88 (dd, J = 12.0 Hz & 3.2 Hz, 1H), 4.45 (t, J = 6.8 Hz, 1H), 4.40-4.34 (m, 1H), 4.09-4.00 (m, 2H), 2.76-2.64 (m, 2H), 2.54 (d, J = 7.2 Hz, 2H), 2.10 (s, 3H), 2.10 (s, 3H), 1.90 (s, 3H), 1.80 (s, 3H). [1060] Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((3-((5-((tert- butoxycarbonyl)amino)pentyl)amino)-3-oxopropyl)thio)tetrahydro-2H-pyran-3,4-diyl diacetate (4) [1061] A solution of 3-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoic acid (XB5B, 1.00 eq, 100 mg, 0.230 mmol) and tert-butyl (5-aminopentyl)carbamate (1.1 eq, 51.1 mg, 0.253 mmol) and Diisopropylethylamine (DIPEA) (3.0 eq, 0.12 mL, 0.689 mmol) in DMF (1.15 mL) was cooled in an ice bath before adding HATU (1.2 eq, 105 mg, 0.276 mmol). After 45 minutes, the reaction was diluted with water (5 mL) and brine (5 mL) and the products were extracted with EtOAc (2x5 mL). The partitioned aqueous layer was washed with EtOAc (5 mL) and the combined organic layer was washed with citric acid then with sodium bicarbonate before being dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give crude material that was used in the next step without further purification. LCMS m/z 620.0 [M+H]. [1062] Synthesis of tert-butyl (5-(3-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanamido)pentyl)carbamate (5) [1063] A solution of 25% w/w sodium methoxide in methanol (6.0 eq, 0.29 mL, 1.26 mmol) in methanol (0.839 mL) was treated with (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6- ((3-((5-((tert-butoxycarbonyl)amino)pentyl)amino)-3-oxopropyl)thio)tetrahydro-2H-pyran-3,4-diyl diacetate (4, 1.0 eq, 130 mg, 0.210 mmol). After 30 minutes, the reaction was cooled in an ice bath and the reaction solution was neutralized with 12M HCl (5.9 eq, 103 uL, 1.24 mmol) then diluted with DCM (7mL) and filtered. The filtrate was concentrated under reduced pressure to give 177 mg of crude material that was used in the next step without further purification. LCMS m/z 494.0 [M+H]. [1064] Synthesis of 3-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)-N-(5-aminopentyl)propanamide (XB49) [1065] A mixture of tert-butyl (5-(3-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanamido)pentyl)carbamate (1.0 eq, 103 mg, 0.209 mmol) and trifluoroacetic acid (40.0 eq, 595 uL, 8.35 mmol) in DCM (1.04 mL) was stirred at room temp for 2 h. The reaction was concentrated under reduced pressure then dissolved in water and ammonium hydroxide for purification by reversed-phase HPLC (3-50% acetonitrile in water with 200 mM NH4OH) to give 3-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-2-yl)thio)-N-(5-aminopentyl)propenamide, XB49, as a white solid. Yield: 46 mg, 56 % (over 3 steps). LCMS m/z 394.2 [M+H]. Synthesis of N-((2R,3R,4R,5R,6R)-2-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)thio)-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB50) 1a
Figure imgf000344_0001
[1066] Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((2,2-dimethyl-4-oxo- 3,8,11,14-tetraoxa-5-azahexadecan-16-yl)thio)tetrahydro-2H-pyran-3,4-diyl diacetate (2) [1067] A solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-mercaptotetrahydro-2H- pyran-3,4-diyl diacetate (1, 1.0 eq, 0.800 g, 2.2 mmol) in N,N-dimethylformamide (8 mL) was cooled at - 78 °C, Lithium bis(trimethylsilyl)amide (LiHMDS, 1M in tetrahydrofuran) (1.0 eq, 2.2 mL, 2.2 mmol) was added and reaction mixture was stirred at the same temperature for 1 h. Then, tert-butyl (2-(2-(2-(2- bromoethoxy)ethoxy)ethoxy)ethyl)carbamate (1a, 1.2 eq, 0.940 g, 2.64 mmol) was added and reaction mixture stirred at room temperature for 16 h. After completion, reaction mixture was concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((2,2- dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-yl)thio)tetrahydro-2H-pyran-3,4-diyl diacetate (2) as a colorless semi solid. Yield: 0.600 g, 44.11 %; LCMS m/z 639.05 [M+H]; 1H NMR (400 MHz, DMSO-d6 with D2O) δ 5.57 (d, J = 5.2 Hz, 1H), 5.31-5.30 (m, 1H), 4.89-4.85 (m, 1H), 4.43 (t, J = 6.0 Hz, 1H), 4.36-4.32 (m, 1H), 4.06-3.97 (m, 2H), 3.60-3.56 (m, 1H), 3.54-3.51 (m, 4H), 3.36 (t, J = 6.0 Hz, 2H), 3.03 (t, J = 5.6 Hz, 2H), 2.92 (s, 2H), 2.76 (s, 1H), 2.73-2.69 (m, 1H), 2.64-2.58 (m, 1H), 2.06 (s, 3H), 1.96-1.94 (m, 5H), 1.87 (s, 3H), 1.79 (s, 3H), 1.33 (s, 9H). [1068] Synthesis of tert-butyl (2-(2-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)ethoxy)ethoxy)ethoxy)ethyl)carbamate (3) [1069] A solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((2,2-dimethyl-4-oxo- 3,8,11,14-tetraoxa-5-azahexadecan-16-yl)thio)tetrahydro-2H-pyran-3,4-diyl diacetate (2, 1.0 eq, 0.450 g, 0.705 mmol) in methanol (5 mL) was cooled at 0 °C, sodium methoxide (25 % solution in methanol) (2.0 eq, 0.33 mL, 1.41 mmol) was added and reaction mixture was stirred at room temperature for 3 h. After completion, reaction mixture was neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated, washed with diethyl ether and dried to afford tert-butyl (2-(2-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)ethoxy)ethoxy)ethoxy)ethyl)carbamate (3) as an off white solid. Yield: 0.320 g, 88.64 %; LCMS m/z 513.10 [M+H]. [1070] Synthesis of N-((2R,3R,4R,5R,6R)-2-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)thio)- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB50) [1071] A solution of tert-butyl (2-(2-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)ethoxy)ethoxy)ethoxy)ethyl)carbamate (3, 1.0 eq, 0.320 g, 0.624 mmol) in dichloromethane (1.6 mL) was cooled at 0 °C, trifluoroacetic acid (1.6 mL) was added and reaction mixture was stirred at room temperature for 1 h. After completion, the reaction mixture was concentrated, azeotroped with dichloromethane (2-3 times), washed with diethyl ether (2-3 times) and purified by prep HPLC (32-50 % acetonitrile in water with 0.1 % trifluoroacetic acid). Fractions containing the desired product were combined and lyophilized to dryness to afford N- ((2R,3R,4R,5R,6R)-2-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)thio)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide, XB50, as a light yellow viscous liquid. Yield: 0.220 g, 85.33 %; LCMS m/z 413.10 [M+H]; 1H NMR (400 MHz, DMSO-d6) δ 7.79-7.73 (m, 3H), 5.43 (d, J = 5.6 Hz, 1H), 4.67-4.60 (m, 3H), 4.18-4.12 (m, 1H), 3.88 (t, J = 5.6 Hz, 1H), 3.73 (bs, 1H), 3.60- 3.45 (m, 15H), 2.98-2.97 (m, 2H), 2.70-2.64 (m, 1H), 2.57-2.54 (m, 1H), 1.81 (s, 3H). Synthesis of (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (trifluoromethyl)pyrimidin-2-yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB51)
Figure imgf000346_0001
[1072] Synthesis of tert-butyl (2-(2-(2-((2-chloro-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (2) [1073] To stirred solution of tert-butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (1, 1.15 g, 1.0 eq, 4.61 mmol) in tetrahydrofuran (10 mL), sodium hydride (0.277 g, 1.5 eq., 6.91 mmol, 60% in oil) was added portion wise at 0°C under nitrogen and allowed to stir at 0 °C for 30 minutes. Then the resultant solution was added dropwise to a solution of 2,4-dichloro-6-(trifluoromethyl)pyrimidine (1.0 g, 1.0 eq, 4.61 mmol) in tetrahydrofuran (10 mL) under nitrogen atmosphere at 0°C and allowed to stir for 10 minutes. After completion (monitored by TLC), the reaction was quenched by the addition of ice, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate and concentrated to get crude which was passed through silica gel (silica gel 100-200 mesh, eluent: 10% ethyl acetate in hexane) to get crude product. (1.57 g, 3.65 mmol) as light yellow liquid which was further purified by SFC to get tert-butyl (2-(2-(2-((2-chloro-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (2) as colorless oil. Yield: 0.60 g, 35%; LCMS m/z 430.3 [M+H]; 1H NMR (400 MHz, DMSO-d6): δ 7.64 (s, 1H), 6.75 (t, J = 4.8 Hz, 1H), 4.56-4.54 (m, 2H), 3.78-3.76 (m, 2H), 3.58-3.56 (m, 2H), 3.51-3.48 (m, 2H), 3.38 (t, J = 6.0 Hz, 2H), 3.06-3.02 (m, 2H), 1.35 (s, 9H). [1074] SFC Purification Method: [1075] Column: AMYLOSE-1 (250*4.6)mm, 5um; Mobile Phase: CO2:0.1% IPAmine in HEXANE:IPA 50:50 (20:80); Flow Rate: 3.0 mL/min; Column Temperature: 40 °C [1076] A suspension of tert-butyl N-[2-[2-[2-[2-chloro-6-(trifluoromethyl)pyrimidin-4- yl]oxyethoxy]ethoxy]ethyl]carbamate (2, 1.00 eq, 51.0 mg, 0.119 mmol), (2R,3R,4R,5S)-5-amino-2- (hydroxymethyl)tetrahydropyran-3,4-diol;hydrochloride (48-4, 2.00 eq, 47.4 mg, 0.237 mmol), diisopropylethylamine (DIPEA) (4.00 eq, 0.083 mL, 0.475 mmol), in 420 µL of anhydrous acetonitrile was heated to 75 °C under nitrogen in a sealed vial. The mixture was heated for 1 hr, added 1 eq of DIEA (20 µL) and heated overnight at 80 °C, then heated at 95 °C for 24 hours. The mixture was cooled, diluted with water and formic acid, and the crude material was purified by Prep HPLC, eluting from a C18 column with a gradient of 10-100% CH3CN:water + 0.1% FA to give 51 mg of a white powder [77% yield]; LCMS: 557.1 [M+H] [1077] A solution of tert-butyl N-[2-[2-[2-[2-[[(3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]amino]-6-(trifluoromethyl)pyrimidin-4- yl]oxyethoxy]ethoxy]ethyl]carbamate (3, 1.00 eq, 31.0 mg, 0.0557 mmol) in 6 mL DCM was cooled to 0 °C and 2 mL of TFA was added. The solution was stirred for 1 hr at room temperature, at which time the solution was concentrated to a crude residue which was subsequently lyophilized from water to give (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6-(trifluoromethyl)pyrimidin-2-yl)amino)-2- (hydroxymethyl)tetrahydro-2H-pyran-3,4-diol, XB51: 37.3 mg of a white solid (crude, TFA salt); LCMS 457.2 [M+H] Synthesis of N-((2S,3R,4R,5R,6R)-2-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethoxy)-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB52)
Figure imgf000347_0001
[1078] Synthesis of tert-butyl (2-(2-(2-(2-(((2S,3R,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)-3-nitrotetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (2) [1079] To a stirred solution of (2R,3R,4R)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-5-nitro-3,4- dihydro-2H-pyran (1, 5.0 g, 1.0 eq., 10.8 mmol) and tert-butyl (2-(2-(2-(2- hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (1a, 4.77 g, 1.5 eq., 16.3 mmol) in anhydrous toluene (40 mL) under Ar, activated molecular sieves (3 Å, 1.50 g) were added and the mixture stirred for 1 h at room temperature. Thereafter, t-BuOK (0.608 g, 0.5 eq., 5.42 mmol, 1 M solution in THF) was added at 0°C, and stirred for 12 h at room temperature. After completion (monitored by LCMS, & TLC), acetic acid (0.05 mL) was added to quench the reaction. Molecular sieves were filtered off and the filtrate was removed under reduced pressure to afford crude which was purified by silica gel flash column chromatography to (using 0-70% ethyl acetate in hexane) to afford tert-butyl (2-(2-(2-(2- (((2S,3R,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3-nitrotetrahydro-2H-pyran-2- yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (2) as colorless syrup. Yield: 2.5 g, 30.0%; LCMS m/z 755.37 [M+H] +. [1080] Synthesis of tert-butyl (2-(2-(2-(2-(((2S,3R,4R,5R,6R)-3-amino-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (3) [1081] To a solution of tert-butyl (2-(2-(2-(2-(((2S,3R,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)-3-nitrotetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (2, 2.5 g, 3.31 mmol, 1.0 eq.) in glacial acetic acid (30 mL); zinc (2.6 g, 12.0 eq., 39.7 mmol) was added and then heated at 40 °C for 3 h. After completion (monitored by TLC), the reaction mixture was diluted with methanol and was filtered through celite pad. The volatiles were evaporated out on high vacuum to yield crude tert-butyl (2-(2-(2-(2-(((2S,3R,4R,5R,6R)-3-amino-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (3) as syrup which was used for next step without further purification. Yield: 2.5 g (crude); LCMS m/z 725.65 [M+H]. [1082] Synthesis of tert-butyl (2-(2-(2-(2-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (4) [1083] To a solution of tert-butyl (2-(2-(2-(2-(((2S,3R,4R,5R,6R)-3-amino-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (3, 2.5 g, 3.45 mmol, 1.0 eq.) in pyridine (20 mL), acetic anhydride (10 mL) was added at 0 °C and stirred the reaction mixture for 16 h at room temperature. After completion, the volatiles were evaporated under reduced pressure to get crude which was purified by silica gel flash column chromatography (40-60% ethyl acetate/hexane) to afford tert-butyl (2-(2-(2-(2-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (4) as off- white semi solid Yield: 2.5 g, 94.52%.; LCMS m/z 767.50 [M+H].1H NMR (400 MHz, DMSO-d6): δ 7.82 (d, J = 8.8 Hz, 1H), 7.35-7.23 (m, 15H), 6.74 (t, J = 5.6 Hz, 1H), 5.75 (s, 1H), 4.76-4.70 (m, 3H), 4.59-4.43 (m, 4H), 4.29-4.23 (m, 1H), 4.04 (bs, 1H), 3.94 (t, J = 6.4 Hz, 1H), 3.75 (dd, J = 11.2, 2.4 Hz 1H), 3.68-3.63 (m, 1H), 3.58-3.54 (m, 4H), 3.53-3.47 (m, 9H), 3.37-3.34 (m, 2H), 3.05 (q, J = 6.0 Hz, 2H), 1.83 (s, 3H), 1.36 (s, 9H). [1084] Synthesis of N-((2S,3R,4R,5R,6R)-2-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethoxy)-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB52) [1085] To a solution of tert-butyl (2-(2-(2-(2-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)- 6-((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (4, 1.0 g, 1.0 eq., 1.3 mmol) in methanol (15 mL), 10% Pd/C (1.0 g), and concentrated HCl (0.083 mL, 2.0 eq., 2.61 mmol) were added. Then reaction mixture was stirred at room temperature under H2 gas balloon pressure for 48h. After completion, the reaction mixture was filtered on celite pad and washed the pad with methanol. The volatiles were evaporated in high vacuum to afford crude which was purified by prep-HPLC (25% acetonitrile in water with 0.1 % TFA ) to afford N-((2S,3R,4R,5R,6R)-2-(2-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)ethoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)acetamide (XB52) as light brown semi solid Yield: 0.109 g., 21.09%; LCMS m/z 397.15 [M+H] 1H NMR (400 MHz, DMSO-d6 with D2O exchange) δ 4.70 (d, J = 2.0 Hz, 1H), 4.01-3.98 (m, 1H), 3.77-3.72 (m, 2H), 3.63-3.44 (m, 16H), 2.95 (t, J = 4.8 Hz, 2H), 1.84 (s, 3H). Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-(2- azidoethoxy)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (XB53)
Figure imgf000349_0001
[1086] To a solution of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol (1) (246 mg, 1.12 mmol, 0.9eq.) and (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (1a) (486 mg, 1.24 mmol, 1.0 eq.) in 5 mL of 1,2-dicholomethane, stirring at ambient temperature under nitrogen atmosphere, was slowly added trimethylsilyl trifluoromethanesulfonate (45 mL, 0.25 mmol, 0.2 eq.). The mixture stirred at ambient temperature for approximately 5 minutes, then was heated to 60C. After 3 hrs., the mixture was cooled to ambient temperature and quenched with addition of triethylamine (70 mL, 0.51 mmol, 0.4 eq.). The reaction mixture was diluted further with 1,2-dicholomethane, evaporated onto silica, and purified by flash column chromatography column, eluting with 0-100% ethyl acetate/dichloromethane to afford Compound 2 as a clear thick syrup. Yield: 520 mg (75%); LCMS m/z 548.97 [M+H]. [1087] Synthesis of N-((2R,3R,4R,5R,6R)-2-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (3) [1088] To a solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-(2- azidoethoxy)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (2) (492 mg, 0.897 mmol) in 6 mL of methanol, stirring under nitrogen atmosphere at 0-5C, was added sodium methoxide (25% w/w in methanol) (1359 mg, 6.29 mmol, 7.0 eq) diluted in 3 mL of methanol. The reaction mixture stirred at ambient temperature for 30 minutes, at which time 8 mL of 1N aqueous hydrochloric acid was added slowly to achieve approximate final pH of 1-2. The reaction mixture was concentrated to approximately ¼ volume and purified by preparatory HPLC, eluting with 1-30% acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford Compound 3 as white solid. Yield: 244 mg (64%); LCMS m/z 423.06 [M+H]. [1089] Synthesis of N-((2R,3R,4R,5R,6R)-2-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethoxy)-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (4) [1090] To a solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-(2- azidoethoxy)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (3) (239 mg, 0.566 mmol) in 15 mL methanol was added 10% w/w palladium on carbon (128 mg). The mixture was degassed under vacuum then stirred under hydrogen atmosphere via balloon. After approximately 15 minutes, the solution was filtered over Celite and washed with methanol. The filtrate was concentrated to residue, then co-evaporated from acetonitrile to afford Compound 4 (XB53), as a clear oil. Yield: 243 mg (108%); LCMS m/z 397.25 [M+H]. Synthesis of 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H- pyran-2-yl)thio)-N-(5-aminopentyl)propanamide (XB54)
Figure imgf000350_0001
LiOH.H2O Ac2O THF, MeOH, Pyridine, rt H2O, rt
Figure imgf000350_0002
Figure imgf000350_0003
Figure imgf000350_0004
[1091] Synthesis of (2R,3R,4R,5R,6S)-5-acetamido-2-(acetoxymethyl)-6-((3-methoxy-3- oxopropyl)thio)tetrahydro-2H-pyran-3,4-diyl diacetate (2) [1092] A solution of (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5- triyl triacetate (1, 1.0 eq, 5.0 g, 12.8 mmol) and methyl 3-mercaptopropanoate (1a, 2.0 eq, 3.09 mL, 25.7 mmol) in dichloromethane (50 mL) was cooled at 0 °C, boron trifluoride diethyl etherate (5.0 eq, 8.28 mL, 64.2 mmol) was added dropwise and reaction mixture was heated at 40 °C for 16 h. Reaction was monitored by ELSD. After completion, reaction mixture was cooled, diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution and water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford (2R,3R,4R,5R,6S)-5-acetamido-2-(acetoxymethyl)-6-((3-methoxy-3-oxopropyl)thio)tetrahydro-2H- pyran-3,4-diyl diacetate (2) as a colorless viscous liquid. Yield: 5.2 g, 87.39 %; LCMS m/z 450.1 [M+H]. [1093] Synthesis of methyl 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoate (3) [1094] To a solution of (2R,3R,4R,5R,6S)-5-acetamido-2-(acetoxymethyl)-6-((3-methoxy-3- oxopropyl)thio)tetrahydro-2H-pyran-3,4-diyl diacetate (2, 1.0 eq, 4.0 g, 8.9 mmol) in methanol (40 mL), sodium methoxide (25 % solution in methanol) (0.1 eq, 0.21 mL, 0.89 mmol) was added and reaction mixture was stirred at room temperature for 3 h. After completion, reaction mixture was neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated and dried to afford methyl 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoate (3) as an off white solid. Yield: 1.7 g, 59.0 %; LCMS m/z 324.0 [M+H]. [1095] Synthesis of 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoic acid (4) [1096] To a solution of methyl 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoate (3, 1.0 eq, 2.0 g, 6.19 mmol) in tetrahydrofuran (18 mL), methanol (12 mL) and water (6 mL), lithium hydroxide monohydrate (2.0 eq, 0.519 g, 12.4 mmol) was added and reaction mixture was stirred at room temperature for 2 h. After completion, reaction mixture was concentrated, methanol was added, neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated and dried to afford 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoic acid (4) as an off white sticky solid. Yield: 2.4 g (Crude); LCMS m/z 310.0 [M+H]. [1097] Synthesis of 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro- 2H-pyran-2-yl)thio)propanoic acid (XB5) [1098] To a solution of 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoic acid (4, 1.0 eq, 1.1 g, 3.56 mmol) in pyridine (11 mL), acetic anhydride (10.0 eq, 3.36 mL, 35.6 mmol) was added and reaction mixture was stirred at room temperature for 16 h. After completion, reaction mixture was concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-7 % methanol in dichloromethane to afford 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoic acid (XB5) as a colorless viscous liquid. [1099] Yield: 1.25 g, 76.74 %; LCMS m/z 436.0 [M+H]; 1H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 7.88 (d, J = 9.6 Hz, 1H), 5.27 (d, J = 3.2 Hz, 1H), 4.95 (dd, J = 2.4, 10.8 Hz, 1H), 4.66 (d, J = 10.4 Hz, 1H), 4.10-3.96 (m, 4H), 2.85-2.79 (m, 1H), 2.74-2.69 (m, 1H), 2.58 (t, J = 7.2 Hz, 2H), 2.11(s, 3H), 2.00 (s, 3H), 1.90 (s, 3H), 1.77 (s, 3H). [1100] A solution of tert-butyl (5-aminopentyl)carbamate (1.2 eq, 166 mg, 0.821 mmol) and 3- (((2S,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2- yl)thio)propanoic acid (XB5, 1.0 eq, 302 mg, 0.694 mmol) and DIPEA (3.0 eq, 0.36 mL, 2.08 mmol) in DMF (3.5 mL) was cooled in an ice bath before adding HATU (1.2 eq, 316 mg, 0.832 mmol) then removing the ice bath. After 45 minutes, the reaction was diluted with water (10 mL) and brine (10 mL) and the products were extracted with EtOAc (2x10 mL). The partitioned aqueous layer was washed with EtOAc (5 mL) and the combined organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give crude material that was adsorbed to silica gel for purification by column chromatography (50-100% EtOAc in hexanes) to give (2R,3R,4R,5R,6S)-5-acetamido-2-(acetoxymethyl)-6-((3-((5-((tert- butoxycarbonyl)amino)pentyl)amino)-3-oxopropyl)thio)tetrahydro-2H-pyran-3,4-diyl diacetate. Yield: 319 mg, 74.2 %. LCMS m/z 619.95 [M+H]. [1101] A solution of [(2R,3R,4R,5R,6S)-5-acetamido-3,4-diacetoxy-6-[3-[5-(tert- butoxycarbonylamino)pentylamino]-3-oxo-propyl]sulfanyl-tetrahydropyran-2-yl]methyl acetate (1.0 eq, 188 mg, 0.303 mmol) in methanol (1.5 mL) was treated with 25% w/w sodium methoxide in methanol (4.0 eq, 0.28 mL, 1.21 mmol). After 1 h, the reaction was cooled in an ice bath, neutralized with 4M HCl in dioxane (4.0 eq, 0.303 mL, 1.21 mmol) then concentrated under reduced pressure to give crude material. The residue was dissolved in 1:1 MeOH/DCM (3 mL) then treated with 4M HCl in dioxane (4.0 eq, 0.303 mL, 1.21 mmol). After 1h, the reaction was concentrated under reduced pressure. The residue was dissolved in concentrated NH4OH then purified by reversed-phase HPLC (3-30% acetonitrile in water w/ 10 mM NH4OH) then lyophilized to give 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)-N-(5-aminopentyl)propenamide (XB54). Yield: 102 mg, 85.5 %. LCMS m/z 394.2 [M+H]. Synthesis of N-((2R,3R,4R,5R,6R)-2-(3-(2-(2-aminoethoxy)ethoxy)propoxy)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB55)
Figure imgf000352_0001
[1102] Synthesis of benzyl (2-(2-(3-hydroxypropoxy)ethoxy)ethyl)carbamate (2) [1103] A solution of 3-oxo-1-phenyl-2,7,10-trioxa-4-azatridecan-13-oic acid (1, 7.0 g, 1.0 eq., 22.5 mmol) in dry tetrahydrofuran (70 mL) was cooled to 0° C. To this, borane tetrahydrofuran complex (1M in THF, 112.5 mL, 5.0 eq., 112.5 mmol) was added slowly, and the resulting reaction mixture was stirred at room temperature for 21h. After completion, conc. HCl was added dropwise to quench excess borane complex, and the resulting mixture was stirred for another 30 minutes. Thereafter, the reaction mixture was concentrated under reduced pressure to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 2-5% methanol in dichloromethane to afford benzyl (2-(2-(3- hydroxypropoxy)ethoxy)ethyl)carbamate (2) as colorless viscous liquid. Yield: 2.8 g, 41.88%; LCMS: m/z 298.1 [M+H] [1104] Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((3-oxo-1-phenyl-2,7,10- trioxa-4-azatridecan-13-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (3) [1105] To a stirred solution of (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H- pyran-2,4,5-triyl triacetate (2a, 1.45 g, 1.0 eq., 3.72 mmol), and benzyl (2-(2-(3- hydroxypropoxy)ethoxy)ethyl)carbamate (2, 1.11 g, 1 eq., 3.72 mmol) in 1,2-dichloroethane (15 mL) at room temperature, was added trimethylsilyl trifluoromethanesulfonate (67.6 µL, 0.1 eq., 372 µmol) drop- wise and the reaction mixture was heated at 65° C for 6h. After completion, the reaction mixture was quenched with triethyl amine and concentrated under reduced pressure to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 2-5% methanol in dichloromethane to afford (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((3-oxo-1-phenyl-2,7,10-trioxa-4- azatridecan-13-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (3) as colorless viscous liquid. Yield: 2.08 g, 89.13%; LCMS: m/z 627.0 [M+H] [1106] Synthesis of benzyl (2-(2-(3-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propoxy)ethoxy)ethyl)carbamate (4) [1107] A solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((3-oxo-1-phenyl-2,7,10- trioxa-4-azatridecan-13-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (3, 0.350 g, 0.559 mmol) in methanol (4 mL) was cooled to 0° C. To this, sodium methoxide (25% solution in methanol) (0.02 mL, 0.4 eq., 0.223 mmol) was added, and the resulting reaction mixture was stirred at room temperature for 3h. After completion, the reaction mixture was neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated to afford benzyl (2-(2-(3-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)propoxy)ethoxy)ethyl)carbamate (4) as colorless viscous liquid. Yield: 0.26 g, 93.0%; LCMS: m/z 501.1 [M+H] [1108] Synthesis of N-((2R,3R,4R,5R,6R)-2-(3-(2-(2-aminoethoxy)ethoxy)propoxy)-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB55) [1109] To a stirred solution of benzyl (2-(2-(3-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propoxy)ethoxy)ethyl)carbamate (4, 0.26 g, 0.519 mmol) in methanol (4 mL), 10% palladium on carbon (0.166 g) and conc. HCl (20 µL, 1.0 eq., 0.519 mmol) were added and the reaction mixture was stirred at room temperature under hydrogen gas balloon pressure for 2h. After completion, the reaction mixture was filtered through syringe filter and washed with methanol. The filtrate was concentrated and purified by prep-HPLC (30-50% acetonitrile in water with 0.1 % trifluoroacetic acid). Fractions containing the desired product were combined and lyophilized to dryness to afford N-((2R,3R,4R,5R,6R)-2-(3-(2-(2-aminoethoxy)ethoxy)propoxy)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB55) as a colorless semi-solid. Yield: 0.071 g, 37.3%; LCMS: m/z 367.1 [M+H]; 1H NMR (400 MHz, DMSO-d6 with D2O exchange): δ 4.20 (d, J = 8.4 Hz, 1H), 3.76-3.64 (m, 3H), 3.59-3.55 (m, 4H), 3.58-3.48 (m, 4H), 3.47-3.39 (m, 4H), 3.31-3.28 (t, J = 6.0 Hz, 1H), 2.95 (t, J = 5.2 Hz, 2H), 1.86 (s, 3H), 1.66 (t, J = 6.8, 2H). Synthesis of (1S,2R,3R,4R,5S)-4-(4-((2-(2-(2-aminoethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol- 1-yl)-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane-2,3-diol TFA salt (XB57)
Figure imgf000354_0001
XB57 [1110] Synthesis of (1S,2R,3R,4R,5S)-4-azido-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane- 2,3-diol (2) [1111] To a solution of ((3aR,4S,7S,8R,8aR)-8-azido-2,2-dimethyltetrahydro-4,7- epoxy[1,3]dioxolo[4,5-d]oxepin-4(5H)-yl)methanol (1) (58.2 mg, 0.226 mmol) in 1.2 mL DCM was added 0.3 mL of trifluoroacetic acid. The reaction was monitored by TLC until completion, then solvent evaporated under a stream of nitrogen. The residue was dissolved in a solution of acetonitrile and water, then frozen and lyophilized to dryness to afford (1S,2R,3R,4R,5S)-4-azido-1-(hydroxymethyl)-6,8- dioxabicyclo[3.2.1]octane-2,3-diol as a tan color solid. Yield: 31.0 mg (106%); LCMS m/z 219.8 [M+H]. [1112] Synthesis of (1S,2R,3R,4R,5S)-4-(4-((2-(2-(2-aminoethoxy)ethoxy)ethoxy)methyl)-1H- 1,2,3-triazol-1-yl)-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane-2,3-diol 2,2,2-trifluoroacetic acid (XB57) [1113] To a solution of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-amine (2a) (39.7 mg, 0.183 mmol, 1.0 eq.) and (1S,2R,3R,4R,5S)-4-azido-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane- 2,3-diol (2) (34.4 mg, 0.184 mmol, 1.0 eq.) in 0.4 mL of dimethyl sulfoxide was added cuprous;acetonitrile;hexafluorophosphate (67.4 mg, 0.181 mmol, 0.99 eq) as a solid in one portion. The mixture was stirred under nitrogen atmosphere at ambient temperature for approximately 15 minutes until completion. The reaction mixture was diluted with water, which formed a precipitate, then 2 drops of trifluoroacetic acid was added to clear the solution. The product was isolated from the diluted mixture by preparatory HPLC, eluting with 1-20% acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford (1S,2R,3R,4R,5S)-4- (4-((2-(2-(2-aminoethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)-1-(hydroxymethyl)-6,8- dioxabicyclo[3.2.1]octane-2,3-diol 2,2,2-trifluoroacetic acid (XB57) as a clear oil. Yield: 47.9 mg (50%); LCMS m/z 405.3 [M+H]. Synthesis of (2R,3R,4R,5S)-5-((6-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-1,1- difluoroethyl)pyrazin-2-yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB80)
Figure imgf000355_0001
[1114] Synthesis of methyl 2-(6-chloropyrazin-2-yl)-2,2-difluoroacetate (2) [1115] To a stirred suspension of copper (1.97 g, 3 eq., 31 mmol) in dimethyl sulfoxide (8 mL), methyl 2-bromo-2,2-difluoroacetate (1a, 3.7 mL, 3 eq., 31 mmol) was added at room temperature under inert atmosphere. After 30 minutes, 2-bromo-6-chloropyrazine (1, 2 g, 1 eq., 10.3 mmol) was added and heated at 90 °C for 3 h. After completion of reaction (monitored by TLC), the reaction mixture was quenched with saturated aqueous ammonium chloride solution and extracted with ethyl acetate (3 × 25 mL). The combined organic phase was washed with brine, dried with anhydrous sodium sulphate, filtered, and concentrated under reduced pressure to afford methyl 2-(6-chloropyrazin-2-yl)-2,2- difluoroacetate (2.5 g, crude) as dark brown liquid.1H NMR (400 MHz, DMSO-d6) for crude compound: δ 9.15 (s, 1H), 9.11 (s, 1H), 3.89 (s, 3H). [1116] Synthesis of 2-(6-chloropyrazin-2-yl)-2,2-difluoroethan-1-ol (3) [1117] To a stirred solution of methyl 2-(6-chloropyrazin-2-yl)-2,2-difluoroacetate (2, 2.5 g, crude) in methanol (20 mL), sodium borohydride (1.2 g, 3 eq., 33 mmol) was added portion wise for 10 min at 0 °C. After stirring 30 minutes at room temperature, the reaction mixture was quenched in ice cold water and organic part was extracted with dichloromethane (3 × 20 mL). Combined dichloromethane layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude was purified by combi flash column chromatography using ethyl acetate/ heptane (0-30% gradient) as eluent to afford 2-(6-chloropyrazin-2-yl)-2,2-difluoroethan-1-ol as colourless liquid. Yield: 0.8 g, 39% over two steps.1H NMR (400 MHz, DMSO-d6): δ 9.02 (s, 1H), 8.95 (s, 1H), 5.73 (t, J = 6.4 Hz, 1H), 4.03-3.94 (m, 2H). [1118] Synthesis of 2-(6-(((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H- pyran-3-yl)amino)pyrazin-2-yl)-2,2-difluoroethan-1-ol (4) [1119] To a solution of (3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H- pyran-3-amine-hydrochloride (3a, 0.550 g, 1 eq., 1.17 mmol) in 1-methyl-2-pyrrolidinone (4 mL) was added N,N-Diisopropylethylamine (2.16 mL, 10 eq., 11.7 mmol) and stirred at room temperature for 10 minutes. After that, 2-(6-chloropyrazin-2-yl)-2,2-difluoroethan-1-ol (3, 0.228 g, 1 eq., 1.17 mmol) was added and heated the reaction mixture at 150 °C for 48 h. After completion (monitored by LCMS), reaction mixture was concentrated under reduced pressure to give crude which was purified by combi flash column chromatography using ethyl acetate/heptane as eluting system to afford 2-(6- (((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2- yl)-2,2-difluoroethan-1-ol (4) as yellow dense liquid. Yield: 0.37 g, 53%. LCMS: m/z 592.44 [M+H]. [1120] Synthesis of tert-butyl (2-(2-(2-(2-(6-(((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)-2,2- difluoroethoxy)ethoxy)ethoxy)ethyl)carbamate (5) [1121] To a stirred solution of 2-(6-(((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)-2,2-difluoroethan-1-ol (4, 0.37 g, 1 eq., 0.625 mmol) in N,N-dimethylformamide (3 mL), sodium hydride (0.025 g, 1 eq., 625 µmol) was added and stirred for 5 minutes at 0 °C temperature. After that, tert-butyl (2-(2-(2- bromoethoxy)ethoxy)ethyl)carbamate (4a, 0.195 g, 1 eq., 0.625 mmol) was added. The reaction mixture was stirred at room temperature for 1h. After completion (monitored by TLC), ice-cold water (10 mL) was added to the reaction mixture. Organic part was extracted with ethyl acetate (3 × 10 mL), combined and dried over anhydrous sodium sulphate. Then, solvent was evaporated under reduced pressure to give crude which was purified by combi flash column chromatography using 60% ethyl acetate/heptane as eluting system to afford tert-butyl (2-(2-(2-(2-(6-(((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)-2,2- difluoroethoxy)ethoxy)ethoxy)ethyl)carbamate (5) as brown dense liquid. Yield: 0.23 g, 45.0%. LCMS: m/z 823.05 [M+H]. [1122] Synthesis of (2R,3R,4R,5S)-5-((6-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-1,1- difluoroethyl)pyrazin-2-yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB80) [1123] To a stirred solution of tert-butyl (2-(2-(2-(2-(6-(((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)-2,2- difluoroethoxy)ethoxy)ethoxy)ethyl)carbamate (5, 0.43 g, 1 eq., 0.522 mmol) in dichloromethane ( 4 mL ) was added trichloroborane (7.4 mL, 20 eq., 7.31 mmol, 1M solution) solution in dichloromethane at - 78 °C dropwise and stirred at the same temperature for 3 h. After completion (monitored by LCMS), reaction mixture was quenched with methanol and concentrated under reduced pressure to afford crude which was purified by RP prep-HPLC (15-20% acetonitrile in water with 0.1% TFA). Fractions containing desire product were combined and lyophilized to afford (2R,3R,4R,5S)-5-((6-(2-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)-1,1-difluoroethyl)pyrazin-2-yl)amino)-2-(hydroxymethyl)tetrahydro-2H- pyran-3,4-diol (XB80) as brown sticky solid. Yield: 0.112 g, 47%.1H NMR (400 MHz, DMSO-d6): δ 8.07 (s, 1H), 7.90 (s, 1H), 7.72 (brs, 3H), 7.35 (d, J = 7.6 Hz, 1H), 7.78 (d, J = 6.4 Hz, 1H), 4.63-4.58 (m, 2H), 4.08-3.98 (m, 3H), 3.94-3.90 (m, 1H), 3.75 (brs, 1H), 3.64-3.58 (m, 2H), 3.57-3.49 (m, 11H), 3.31- 3.28 (m, 1H), 2.97-2.89 (m, 3H). Synthesis of (2R,3R,4R,5S)-5-((6-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)pyrazin-2-yl)amino)-2- (hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB81)
Figure imgf000357_0001
[1124] Synthesis of 2-bromo-6-fluoropyrazine (2) [1125] To a solution of 6-bromopyrazin-2-amine (1, 1.0 g, 1.0 eq.5.75 mmol) in HBF4 (3 mL) was added sodium nitrite (0.793 g, 2 eq., 11.5 mmol) in portions at 0 °C. The mixture was stirred at 20 °C for 2h. After completion, the reaction mixture was quenched with water (10 mL) and extracted with pentane (3×20 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated via distillation to remove pentane. After distillation, 2-bromo-6-fluoropyrazine (2) was obtained as brown oil. Yield: 0.5 g, 49.16%. [1126] Synthesis of tert-butyl (2-(2-(2-((6-bromopyrazin-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (3) [1127] To a stirred solution of tert-butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (2a, 0.5 g, 0.8 eq, 2.0 mmol) in tetrahydrofuran (5 mL), sodium hydride (0.12 g, 1.2 eq., 3.0 mmol) was added in portion at 0 °C under N2 atmosphere. The resultant reaction mixture was allowed to stirred at 0 °C for 30 minutes. To this, a solution of 2-bromo-6-fluoropyrazine (2, 0.44 g, 1.0 eq, 2.5 mmol) in tetrahydrofuran (1 mL,) was added drop-wise under N2-atmosphere and allowed to stir for another 30 minutes. After completion, the resulting reaction mixture was quenched by the addition of ice, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulphate and concentrated to obtained crude which was purified by column chromatography (using 20-30% ethylacetate-heptane as eluent) to afford tert-butyl (2-(2-(2-((6-bromopyrazin-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (3) as sticky off brown liquid. Yield: 0.55 g, 54.0%; LCMS: m/z 406 [M+H] [1128] Synthesis of tert-butyl (2-(2-(2-((6-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4) [1129] A solution (A) of tert-butyl (2-(2-(2-((6-bromopyrazin-2- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (3, 0.4 g, 1.0 eq, 1.0 mmol), (2R,3R,4R,5S)-5-amino-2- (hydroxymethyl)tetrahydro-2H-pyran-3,4-diol hydrochloride (48-4, 0.25 g, 1.3 eq., 1.28 mmol) and potassium tert-butoxide (0.33 g, 3 eq., 2.95 mmol) in dry 1,4-dioxane (4 mL) were taken in a sealed tube and purged with N2 gas for 60 minutes. Another solution (B) of Tris(dibenzylideneacetone)dipalladium(0) (0.18 g, 0.2 eq., 0.197 mmol) and xanthphos (0.228 g, 0.4 eq., 0.394 mol) in dry 1,4-dioxane (1 mL) were taken in another sealed tube and purged with nitrogen for 60 minutes. Thereafter, solution (B) was added to solution (A) through syringe, and the reaction mixture was heated at 90 °C for 12h. After completion, the reaction mixture was diluted with 20% methanol- dichloromethane and filtered through syringe filter. Filtrate was concentrated under reduced pressure, and the crude mixture was purified by column chromatography (using 5-10% methanol-dichloromethane as eluent) to afford tert-butyl (2-(2-(2-((6-(((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H- pyran-3-yl)amino)pyrazin-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4) as light brown viscous liquid. Yield: 0.085 g, 17.6%; LCMS: m/z 489.9 [M+H] [1130] Synthesis of (2R,3R,4R,5S)-5-((6-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)pyrazin-2-yl)amino)- 2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB81) [1131] To a stirred solution of tert-butyl (2-(2-(2-((6-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4, 0.135 g, 0.102 mol) in dichloromethane (2 mL) was added trifluoroacetic acid (0.6 mL) at 0 °C. The reaction mixture was then stirred at room temperature for 3h. Progress of the reaction was monitored by LCMS. After completion, the reaction mixture was concentrated under vacuum to get crude which was purified by RP prep-HPLC (50-70% acetonitrile in water with 0.1% trifluoroacetic acid). Fractions containing the desired product were combined and lyophilized to dryness to afford (2R,3R,4R,5S)-5-((6- (2-(2-(2-aminoethoxy)ethoxy)ethoxy)pyrazin-2-yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4- diol (XB81) as brown viscous liquid. Yield: 0.037 g, 34.4%; LCMS m/z 389.15 [M+H]; 1H NMR (400 MHz, DMSO-d6---D2O exchange): δ 7.44 (s, 1H), 7.24 (s, 1H), 4.30 (d, J = 4.80 Hz, 2H), 4.03-3.96 (m, 1H), 3.72 (t, J = 4.40 Hz, 3H), 3.58-3.56 (m, 7H), 3.47 (t, J = 6.00 Hz, 3H), 3.29 (t, J = 5.60 Hz, 1H), 2.97-2.92 (m, 3H). Synthesis of (2R,3R,4R,5S)-5-((4-((2-(2-(2-aminoethoxy)ethoxy)ethoxy)methyl)thiazol-2-yl)amino)- 2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB82)
Figure imgf000359_0001
[1132] Synthesis of (2R,3R,4R,5S)-2-(hydroxymethyl)-5-isothiocyanatotetrahydro-2H-pyran-3,4- diol (5c) [1133] To a suspension of (2R,3R,4R,5S)-5-amino-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol hydrochloride (48-4, 0.180 g, 0.902 mmol) in water (1.6 mL) and acetone (1.6 mL) were sequentially added sodium hydrogen carbonate (0.227 g, 3 eq., 2.7 mmol) and thiophosgene (0.082 mL, 1.2 eq., 1.08 mmol). The mixture was stirred at room temperature for 2 h. Progress of the reaction was monitored by TLC and LCMS. After completion, reaction mixture was concentrated to dryness. The residue was co- evaporated with toluene (2 X 5 mL) and purified by flash column chromatography using 0-4% methanol/ethyl acetate as eluting solvent to afford (2R,3R,4R,5S)-2-(hydroxymethyl)-5- isothiocyanatotetrahydro-2H-pyran-3,4-diol (5c) as a white solid. Yield: 0.130 g, 70.25%; LCMS: m/z 206.0 [M+H]. [1134] Synthesis of 1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)thiourea (5a) [1135] A solution of (2R,3R,4R,5S)-2-(hydroxymethyl)-5-isothiocyanatotetrahydro-2H-pyran-3,4- diol (5c, 0.130 g, 0.633 mmol) and 7M ammonia in methanol (5 mL) was stirred at room temperature for 2h. Progress of the reaction was monitored by TLC and LCMS. After completion, the reaction mixture was concentrated under reduced pressure to obtain crude residue which was washed with diethyl ether (2- 3 times) and dried under vacuum to afford 1-((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)thiourea (5a) as a white solid which was directly used for the next step without further purification. Yield: 0.10 g, Crude; LCMS: m/z 223.0 [M+H]. [1136] Synthesis of tert-butyl (2-(2-(2-(allyloxy)ethoxy)ethoxy)ethyl)carbamate (2) [1137] To a solution of tert-butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (1, 1.00 g, 4.01 mmol) in anhydrous tetrahydrofuran (10 mL), sodium hydride (0.176 g, 1.1 eq., 4.41 mmol) was added portion-wise at 0 °C. After being stirred for 20 min, 3-bromopropene (0.381 mL, 1.1 eq., 4.41 mmol) was added and the reaction mixture was stirred for 2 h at 0 °C. Then it was brought to room temperature and stirred for another 1h. Progress of the reaction was monitored by TLC and LCMS. After completion, the reaction mixture was quenched with ice-cold water (10 mL) and organic part was extracted with dichloromethane (3 X 20 mL). Combined organic layer was dried over anhydrous sodium sulphate, filtered and the filtrate was concentrated under reduced pressure to obtain crude residue. The crude was purified by flash column chromatography using 0-40% ethyl acetate/heptane as eluting solvent to afford tert-butyl (2-(2-(2-(allyloxy)ethoxy)ethoxy)ethyl)carbamate (2) as a colourless viscous liquid. Yield: 0.750 g, 62.03%; LCMS-MS: m/z 290.1 [M+H]. [1138] Synthesis of tert-butyl (2-(2-(2-(oxiran-2-ylmethoxy)ethoxy)ethoxy)ethyl)carbamate (3) [1139] To a solution of tert-butyl (2-(2-(2-(allyloxy)ethoxy)ethoxy)ethyl)carbamate (2, 0.6 g, 2.07 mmol) in anhydrous dichloromethane (10 mL) under argon atmosphere, a solution of m- chloroperbenzoic acid (m-CPBA, 1.43 g, 4.0 eq., 8.29 mmol) in dichloromethane (4 mL) was added dropwise at 0°C. The solution was allowed to reach room temperature and stirred for 20h. Progress of the reaction was monitored by TLC and LCMS. After completion, saturated aqueous sodium bicarbonate solution was added to the reaction mixture and the aqueous layer was extracted with ethyl acetate for three times. Combined organic extracts were dried over anhydrous sodium sulfate, filtered and filtrate was concentrated in vacuo to give crude which was purified by flash column chromatography using 40% ethyl acetate in heptane as eluent to afford tert-butyl (2-(2-(2-(oxiran-2- ylmethoxy)ethoxy)ethoxy)ethyl)carbamate (3) as a light yellow oil. Yield: 0.250 g, 39.48%; LCMS: m/z 306.0 [M+H]. [1140] Synthesis of tert-butyl (2-(2-(2-(3-bromo-2-hydroxypropoxy)ethoxy)ethoxy)ethyl)carbamate (4) [1141] To a stirred solution of tert-butyl (2-(2-(2-(oxiran-2- ylmethoxy)ethoxy)ethoxy)ethyl)carbamate (3, 1.25 g, 4.09 mmol) in anhydrous tetrahydrofuran (50 mL), lithium bromide (1.42 g, 4 eq., 16.4 mmol) and acetic acid (0.709 mL, 3.0 eq., 12.3 mmol) was added at 0 °C and reaction mixture was stirred at room temperature for 1h. Progress of the reaction was monitored by TLC & LCMS. After completion, volatiles were removed under vacuum to obtain crude tert-butyl (2- (2-(2-(3-bromo-2-hydroxypropoxy)ethoxy)ethoxy)ethyl)carbamate (4, 1.35 g) which was directly used in the next step without further purification. Yield: 1.35 g, Crude; LCMS: m/z 387.75 [M+H] [1142] Synthesis of tert-butyl (2-(2-(2-(3-bromo-2-oxopropoxy)ethoxy)ethoxy)ethyl)carbamate (5) [1143] To a solution of tert-butyl (2-(2-(2-(3-bromo-2- hydroxypropoxy)ethoxy)ethoxy)ethyl)carbamate (4, 1.35 g, 3.49 mmol) in dry dichloromethane (60 mL) at room temperature and under argon was added Dess-Martin periodinane (DMP, 4.45 g, 3.0 eq., 10.5 mmol). After 3 h, the reaction mixture was cooled down to 0 °C, quenched with the addition of an aqueous sodium thiosulfate solution (1M) and saturated sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate. Combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuo to give residue which was purified by flash column chromatography 40% using ethyl acetate in heptane as eluent to afford tert-butyl (2-(2-(2-(3-bromo-2-oxopropoxy)ethoxy)ethoxy)ethyl)carbamate (5) as a colorless oil. Yield: 0.750 g, 47.47%; LCMS: m/z 386.0 [M+H] [1144] Synthesis of tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)thiazol-4-yl)methoxy)ethoxy)ethoxy)ethyl)carbamate (6) [1145] A solution of tert-butyl (2-(2-(2-(3-bromo-2-oxopropoxy)ethoxy)ethoxy)ethyl)carbamate (5, 0.520 g, 1.0 eq, 1.15 mmol) and 1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H- pyran-3-yl)thiourea (5a, 0.284 g, 1.15 mmol) in acetone (5 mL) was heated at 50 °C and the reaction mixture was stirred at same temperature for 3h. Progress of the reaction was monitored by TLC and LCMS. After completion of reaction, solvent was evaporated under reduced pressure and dried to obtain crude tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)amino)thiazol-4-yl)methoxy)ethoxy)ethoxy)ethyl)carbamate (6, 1.2 g) which was directly used in the next step without further purification. LCMS: m/z 508.12 [M+H]. [1146] Synthesis of (2R,3R,4R,5S)-5-((4-((2-(2-(2-aminoethoxy)ethoxy)ethoxy)methyl)thiazol-2- yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB82) [1147] To a stirred solution of tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)thiazol-4-yl)methoxy)ethoxy)ethoxy)ethyl)carbamate (6, 1.2 g) in dichloromethane (10 mL), trifluoroacetic acid (10 mL) was added at 0 °C. Then, reaction mixture was stirred at room temperature for 1h. Progress of the reaction was monitored by LCMS. After completion of reaction, solvent was evaporated under reduced pressure to obtain crude residue. Crude was purified by RP prep-HPLC (60% acetonitrile in water with 0.1% trifluoroacetic acid). Fractions containing desired product were combined and lyophilized to afford (2R,3R,4R,5S)-5-((4-((2-(2-(2- aminoethoxy)ethoxy)ethoxy)methyl)thiazol-2-yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB82) as a white sticky solid. Yield: 0.376 g, 48.79%; LCMS m/z 408.25 [M+H]; 1H-NMR (400 MHz, DMSO-d6): δ 7.73 (brs, 3H), 6.58 (s, 1H), 4.28 (s, 2H), 3.94-3.91 (m, 1H), 3.83-3.82 (m, 1H), 3.74 (d, J = 6.4 Hz, 1H), 3.59-3.52 (m, 10H), 3.51-3.42 (m, 3H), 3.28 (t, J = 6.0 Hz, 1H), 3.00-2.95 (m, 3H). Synthesis of (2R,3R,4R,5R,6R)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)pyrimidin-2-yl)amino)-2- (methoxymethyl)-6-propyltetrahydro-2H-pyran-3,4-diol (XB83)
Figure imgf000362_0001
XB83 [1148] tert-butyl ((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-propyltetrahydro-2H- pyran-3-yl)carbamate (1a) [1149] To a stirred solution of (2R,3R,4R,5R,6R)-5-amino-2-(hydroxymethyl)-6-propyltetrahydro- 2H-pyran-3,4-diol hydrochloride (1b, 5.3 g, 21.9 mmol) in methanol (45 mL), were added triethylamine (3 eq., 9 mL, 65.8 mmol) and di-tert-butyl dicarbonate (1.2 eq., 6.04 mL, 26.3 mmol) gradually at 0° C under nitrogen atmosphere. Then reaction mixture was stirred at room temperature for 12h. After completion the reaction mixture was concentrated under reduced pressure to get a crude which was diluted with water and extracted with ethyl acetate (5 X 120 mL). Organic part was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to afford tert-butyl ((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-propyltetrahydro-2H-pyran-3-yl)carbamate (1a) as off white solid. Yield: 5.0 g, crude; LCMS m/z 306.1[M + H]+. [1150] tert-butyl ((3aR,4R,6R,7S,7aR)-4-(hydroxymethyl)-2,2-dimethyl-6-propyltetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (1) [1151] To a stirred solution of tert-butyl ((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2- propyltetrahydro-2H-pyran-3-yl)carbamate (1a, 1.0 eq., 3.0 g, 9.82 mmol) in 2,2-dimethoxypropane (15.0 mL, 22 eq., 218 mmol) and acetone (15.0 mL) was added camphor sulfonic acid (0.2 eq., 0.454 g, 1.96 mmol) at 0°C and reaction mixture was sonicated for 30 min. Then 0.5 mL of methanol was added to the reaction mixture and reaction was stirred at room temperature for 10 min. After completion of reaction, reaction mixture was neutralized using triethyl amine and concentrated under reduced pressure to afford crude which was purified by silica gel flash column chromatography using 20-50 % ethyl acetate/hexane as eluent to afford tert-butyl ((3aR,4R,6R,7S,7aR)-4-(hydroxymethyl)-2,2-dimethyl-6- propyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (1) as off white solid. Yield: 1.6 g, 47%; ELSD-MS m/z 346.2 [M + H]+ . [1152] Synthesis of tert-butyl ((3aR,4R,6R,7S,7aR)-4-(methoxymethyl)-2,2-dimethyl-6- propyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (2) [1153] To a stirred solution of tert-butyl ((3aR,4R,6R,7S,7aR)-4-(hydroxymethyl)-2,2-dimethyl-6- propyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (1, 1.0 g, 1 eq., 2.89 mmol) in dry tetrahydrofuran (15 mL), sodium hydride (127 mg, 1.1 eq., 3.18 mmol) was added portion-wise at 0 °C under nitrogen atmosphere. The reaction was allowed to stir at room temperature for 30 min. After that, methyl iodide (0.360 mL, 2 eq., 5.79 mmol) was added slowly at 0 °C, and stirred at room temperature for another 30 min. After completion (monitored by TLC), ice-cold water was added to the reaction mixture and extracted with dichloromethane (3 × 10 mL). Combined organic portions were dried over anhydrous sodium sulphate and concentrated under reduced pressure to give crude residue which was purified by silica gel flash column chromatography using 40% ethyl acetate in heptane as the eluent to afford tert-butyl ((3aR,4R,6R,7S,7aR)-4-(methoxymethyl)-2,2-dimethyl-6-propyltetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (2) as yellow sticky liquid. Yield: 0.75 g, 72.0%. LCMS m/z 360.20 [M+H]. [1154] Synthesis of (2R,3R,4R,5R,6R)-5-amino-2-(methoxymethyl)-6-propyltetrahydro-2H-pyran- 3,4-diol (3) [1155] To a stirred solution of ((3aR,4R,6R,7S,7aR)-4-(methoxymethyl)-2,2-dimethyl-6- propyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (2, 1.1 g, 1.0 eq., 3.06 mmol ) in dichloromethane (15 mL), trifluoroacetic acid (15 mL) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 4h. After completion (monitored by TLC), reaction mixture was concentrated under reduce pressure and co-evaporated with dichloromethane three times to obtain crude residue, which was further lyophilized to afford (2R,3R,4R,5R,6R)-5-amino-2-(methoxymethyl)-6- propyltetrahydro-2H-pyran-3,4-diol (3) as light yellow syrup. The crude residue was directly used for next step. Yield: 0.6 g (Crude). LCMS m/z 220.1 [M+H] +. [1156] Synthesis of tert-butyl (2-(2-(2-((2-(((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(methoxymethyl)- 2-propyltetrahydro-2H-pyran-3-yl)amino)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4) [1157] To a stirred solution of (2R,3R,4R,5R,6R)-5-amino-2-(methoxymethyl)-6-propyltetrahydro- 2H-pyran-3,4-diol (XB96, intermediate 3, 0.7 g, 1.2 eq., 2.1 mmol) in dry 1-methyl-2-pyrrolidinone (2 mL), N,N-Diisopropylethylamine (3.66 mL, 12 eq., 21 mmol) was added at room temperature and then heated at 140 °C for 16h. After completion (monitored by ELSD-MS), reaction mixture was concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography using 2-3% methanol in dichloromethane as the eluent to afford tert-butyl (2-(2-(2-((2-(((2R,3R,4R,5R,6R)- 4,5-dihydroxy-6-(methoxymethyl)-2-propyltetrahydro-2H-pyran-3-yl)amino)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4) as brown gummy liquid. Yield: 0.64 g, 67.1%. LCMS m/z 545.90 [M+H] +. [1158] Synthesis of (2R,3R,4R,5R,6R)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)pyrimidin-2- yl)amino)-2-(methoxymethyl)-6-propyltetrahydro-2H-pyran-3,4-diol (XB83) [1159] A solution of tert-butyl (2-(2-(2-((2-(((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(methoxymethyl)- 2-propyltetrahydro-2H-pyran-3-yl)amino)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4, 0.140 g, 1 eq., 0.257 mmol) in dichloromethane (1 mL) was cooled at 0 °C and trifluoroacetic acid (1 mL) was added and the reaction mixture was stirred at room temperature for 3h. After completion, the reaction mixture was concentrated under reduce pressure to get crude residue which was purified by RP prep HPLC (25% acetonitrile in water with 0.1% TFA) to afford ((2R,3R,4R,5R,6R)-5-((4-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)pyrimidin-2-yl)amino)-2-(methoxymethyl)-6-propyltetrahydro-2H-pyran- 3,4-diol (XB83) as colorless sticky solid. Yield: 0.058 g, 50.8%. LCMS m/z 445.3 [M+H] +.1H NMR (400 MHz, DMSO-D6 with D2O): δ 7.99 (d, J = 6.4 Hz, 1H), 6.18 (d, J = 6.0 Hz, 1H), 4.44-4.43 (m, 2H), 4.18-4.14 (m, 1H), 4.10-4.06 (m, 1H), 3.75-3.70 (m, 5H), 3.60-3.58 (m, 6H), 3.50 (d, J = 4.4 Hz, 1H), 3.25 (s, 3H), 2.97 (t, J = 5.2 Hz, 2H), 1.61-1.55 (m, 1H), 1.33-1.18 (m, 4H), 0.81 (t, J = 6.8 Hz, 3H). Synthesis of N-((2S,3R,4R,5R,6R)-2-(1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-pyrazol-3-yl)-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB84)
Figure imgf000364_0001
XB84 [1160] Synthesis of ethyl (2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)glycinate (2) [1161] To a stirred solution of tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (1, 5.0 g, 20.1 mmol) in dry tetrahydrofuran (50 mL), were added triethylamine (8.49 mL, 3 eq., 60.4 mmol) and ethyl 2-bromoacetate (1.78 mL, 0.8 eq., 16.1 mmol) in dry tetrahydrofuran (5.0 mL) over a period of 30 min. The above suspension was stirred at room temperature for 8h. After completion, reaction mixture was concentrated to give crude which was purified by silica gel flash column chromatography using 0- 5% methanol-dichloromethane to afford ethyl (2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13- yl)glycinate(2) as white solid. Yield: 4.6 g, 68.3%, LCMS m/z 335.2 [M+H]. [1162] Synthesis of (2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)glycine (3) [1163] To a stirred solution of ethyl (2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13- yl)glycinate(2, 4.6 g, 1.0 eq.,13.8 mmol) in tetrahydrofuran (35 mL) and water (15 mL) was added lithium hydroxide monohydrate (0.659 g, 2 eq., 27.5 mmol) at room temperature. Then reaction mixture was stirred for 3h at room temperature. After completion, reaction mixture was concentrated under reduced pressure to get crude which was purified by RP prep HPLC (20% acetonitrile in water with 0.1 % acetic acid). Fractions containing desired product were combined and lyophilized to get (2,2-dimethyl- 4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)glycine (3) as a white solid. Yield: 2.5 g, 59.3%. LCMS m/z 307.1 [M+H]. [1164] Synthesis of 3-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)-1,2,3-oxadiazol-3- ium-5-olate (4) [1165] To a stirred solution of (2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)glycine (3, 2.5 g, 8.16 mmol) in dichloromethane (15 mL) was added tert-butyl nitrite (1.26 mL, 1.1 eq., 8.98 mmol) at 0 °C. Reaction mixture was then stirred at room temperature for another 1h. After completion, reaction mixture was concentrated under reduced pressure to afford crude which was again dissolved in tetrahydrofuran (20 mL) and acetic anhydride (2.32 mL, 3 eq., 24.5 mmol) was added at 0 °C and reaction mixture was allowed to stir at room temperature for 36h. After completion, reaction mixture was concentrated to afford crude which was purified by silica gel column chromatography using 2-3% methanol/dichloromethane as eluent to afford 3-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)- 1,2,3-oxadiazol-3-ium-5-olate (4) as yellow liquid. Yield: 2.0 g, 77.0 % LCMS m/z 317.1 [M+H]. [1166] Synthesis of tert-butyl (2-(2-(2-(3-((2S,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)-1H-pyrazol-1-yl)ethoxy)ethoxy)ethyl)carbamate (5) [1167] Compound 3-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)-1,2,3-oxadiazol-3-ium- 5-olate (4, 2.54 g, 5 eq., 8.01 mmol) and N-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)-2-ethynyltetrahydro-2H-pyran-3-yl)acetamide (XB147, 0.8 g, 1.6 mmol) were taken in 1,4-dioxane (3.5 mL) and reaction mixture was stirred at 140°C temperature for 72h. After completion (monitored by LCMS), reaction mixture was concentrated to get crude which was purified by silica gel flash chromatography using 5% methanol/dichloromethane to afford tert-butyl (2-(2-(2-(3- ((2S,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)- 1H-pyrazol-1-yl)ethoxy)ethoxy)ethyl)carbamate (5) as sticky yellow solid. Yield: 0.48 g, 38.8% LCMS m/z 773.4 [M+H]. [1168] Synthesis of N-((2S,3R,4R,5R,6R)-2-(1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-pyrazol-3- yl)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB84) [1169] To a stirred solution of tert-butyl (2-(2-(2-(3-((2S,3S,4R,5R,6R)-3-acetamido-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)-1H-pyrazol-1- yl)ethoxy)ethoxy)ethyl)carbamate (5, 0.17 g, 0.220 mmol) in dichloromethane ( 4.25 mL ) was added trichloroborane (0.309 g, 12 eq., 2.64 mmol, 1M in dichloromethane) at -78°C dropwise and stirred at the same temperature for 3h. After completion of reaction, reaction mixture was quenched with methanol and concentrated under reduced pressure to afford crude which was purified by RP prep-HPLC (40% of acetonitrile in water with 0.1% TFA). Fractions containing desired product were combined and lyophilized to afford N-((2S,3R,4R,5R,6R)-2-(1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-pyrazol-3-yl)- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB84) as colorless foamy solid. Yield: 0.047 g, 53.1%; LCMS m/403.00 [M+H]. z 1H NMR (400 MHz, DMSO-d6): δ 7.86 (s, 3H), 7.65 (d, J = 2.40 Hz, 1H), 7.43 (d, J = 8.00 Hz, 1H), 6.20 (d, J = 2.00 Hz, 1H), 5.00 (d, J = 5.20 Hz, 1H), 4.26- 4.20 (m, 3H), 3.98 (dd, J = 2.8, 9.6 Hz, 1H),), 3.98-3.96 (m, 7H), 3.61 (t, J = 5.6 Hz, 1H), 3.55-3.48 (m, 9H), 2.95-2.89 (m, 2H), 1.73 (s, 3H). Synthesis of (2R,3R,4R,5S)-5-((4-(3-(2-(2-aminoethoxy)ethoxy)propyl)pyrimidin-2-yl)amino)-2- (hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB85)
Figure imgf000366_0001
[1170] Synthesis of tert-butyl (2-(2-((3-(2-bromopyrimidin-4-yl)prop-2-yn-1- yl)oxy)ethoxy)ethyl)carbamate (2) [1171] A solution of 2,4-dibromopyrimidine (1, 0.5 g, 1.0 eq., 2.1 mmol) and tert-butyl-2-[2-(2- propynyloxy)ethoxy]ethylaminoformylate (2a, 0.511 g, 1.0 eq., 2.1 mmol) in tetrahydrofuran (5 mL) was purged with N2 gas for 10 min. To this, triethylamine (0.880 mL, 3 eq., 6.31 mmol), Bis(triphenylphosphine)palladium(II) dichloride (0.0738 g, 0.05 eq., 0.105 mmol) and copper (I) iodide (0.04 g, 0.1 eq., 0.210 mmol) were added, and the reaction mixture was purged with N2 for another 30 min. Then the resultant reaction mixture was stirred at 50 °C for 12h. After completion, the reaction mixture was filtered using sintered funnel, and the filtrate was concentrated to obtained crude, which was purified by silica gel column chromatography eluting with 30% ethyl acetate-heptane to afford tert-butyl (2-(2-((3-(2-bromopyrimidin-4-yl)prop-2-yn-1-yl)oxy)ethoxy)ethyl)carbamate (2) as colorless viscous liquid. Yield: 0.45 g, 53.40%; LCMS: m/z 400.0 [M+H]. [1172] Synthesis of tert-butyl (2-(2-(3-(2-bromopyrimidin-4-yl)propoxy)ethoxy)ethyl)carbamate (3) [1173] To a solution of tert-butyl (2-(2-((3-(2-bromopyrimidin-4-yl)prop-2-yn-1- yl)oxy)ethoxy)ethyl)carbamate (2, 0.28 g, 0.7 mmol) in tetrahydrofuran (6.0 mL), 10% Pd/C (0.28 g) was added and the reaction mixture was stirred at room temperature under hydrogen gas balloon pressure for 4h. After completion, reaction mixture was filtered through syringe filter and washed with methanol. The filtrate was concentrated and dried to obtained crude. Crude was purified by silica gel chromatography using 10% methanol in dichloromethane to afford tert-butyl (2-(2-(3-(2-bromopyrimidin-4- yl)propoxy)ethoxy)ethyl)carbamate (3) as colourless liquid. Yield: 0.11 g, 38.5%; LCMS: m/z 403.9 [M+H]. [1174] Synthesis of tert-butyl (2-(2-(3-(2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrimidin-4-yl)propoxy)ethoxy)ethyl)carbamate (4) [1175] To a stirred solution of tert-butyl (2-(2-(3-(2-bromopyrimidin-4- yl)propoxy)ethoxy)ethyl)carbamate (3, 0.3 g, 1.0 eq., 742 µmol) and (2R,3R,4R,5S)-5-amino-2- (hydroxymethyl)tetrahydro-2H-pyran-3,4-diol hydrochloride salt (48-4, 0.207 g, 1.4 eq., 1.04 mmol) in N-Methyl-2-pyrrolidone (3 mL), N,N-Diisopropylethylamine (1.29 mL, 10 eq., 7.42 mmol) was added, and the resulting reaction mixture was allowed to stirred at 150° C for 48h. After completion, volatiles were removed under reduced pressure to obtain the crude which was purified by silica gel column chromatography using 5-20% methanol in dichloromethane to afford tert-butyl (2-(2-(3-(2- (((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrimidin-4- yl)propoxy)ethoxy)ethyl)carbamate (4) as viscous oil. Yield: 0.17 g, 44.73%; LCMS: m/z 487.0 [M+H]. [1176] Synthesis of (2R,3R,4R,5S)-5-((4-(3-(2-(2-aminoethoxy)ethoxy)propyl)pyrimidin-2- yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB85) [1177] To a stirred solution of tert-butyl (2-(2-(3-(2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrimidin-4-yl)propoxy)ethoxy)ethyl)carbamate (4, 0.119 g, 1.0 eq., 0.245 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (0.8 mL) at 0 °C. The reaction mixture was stirred at room temperature for 2h. After completion, the reaction mixture was concentrated under vacuum to give crude which was purified by RP prep-HPLC (20-30% acetonitrile in water with 0.1% trifluoroacetic acid). Fractions containing the desired product were combined and lyophilized to dryness to afford (2R,3R,4R,5S)-5-((4-(3-(2-(2-aminoethoxy)ethoxy)propyl)pyrimidin-2- yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB85) as white solid. Yield: 0.064 g, 67.71%; LCMS m/z 387.15 [M+H]; 1H NMR (400 MHz, DMSO-d6-D2O exchange): δ 8.12 (d, J = 5.20 Hz, 1H), 6.62 (d, J = 5.60 Hz, 1H), 4.08 (m, 1H), 3.87-3.83 (m, 1H), 3.73 (d, J = 3.20 Hz, 1H), 3.57-3.47 (m, 9H), 3.41 (t, J = 6.40 Hz, 2H), 3.29 (t, J = 6.00 Hz, 1H), 3.02 (t, J = 10.80 Hz, 1H), 2.94 (t, J = 5.20 Hz, 2H), 2.60 (t, J = 7.20 Hz, 2H), 1.88-1.81 (m, 2H). Synthesis of N-((2S,3R,4R,5R,6R)-2-(3-((2-(2-aminoethoxy)ethoxy)methyl)isoxazol-5-yl)-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB86)
Figure imgf000368_0001
[1178] Synthesis of tert-butyl (2-(2-(2-oxoethoxy)ethoxy)ethyl)carbamate (2) [1179] To a stirred solution of oxalic dichloride (1.38 mL, 2 eq., 16 mmol) in dichloromethane (20 mL) at –78 °C was added dropwise a solution of dimethyl sulfoxide (1.28 mL, 2.25 eq,, 18.1 mmol). After being stirred at –78 °C for 20 minutes, a solution of tert-butyl-2-[2-(2- hydroxyethoxy)ethoxy]ethylaminoformylate (1, 2 g, 1 eq., 8.02 mmol) in dichloromethane (20 mL) was added to the mixture. The reaction mixture was further stirred at –78 °C for 90 minutes, followed by addition of triethylamine (9.02 mL, 8.0 eq, 64.2 mmol). The resulting mixture was allowed to reach at room temperature over 1h. The reaction mixture was diluted with dichloromethane and washed with water followed by brine solution. The organic layer was dried over anhydrous sodium sulphate, filtered, and concentrated under reduced pressure to afford tert-butyl (2-(2-(2-oxoethoxy)ethoxy)ethyl)carbamate (1.9 g, crude) as sticky liquid. The crude was forwarded as such for next step. [1180] Synthesis of tert-butyl (E)-(2-(2-(2-(hydroxyimino)ethoxy)ethoxy)ethyl)carbamate (3) [1181] To a stirred solution of tert-butyl-2-[2-(formylmethoxy)ethoxy]ethylaminoformylate (2, 1.98 g, 1.0 eq., 8.01 mmol) in ethanol (12 mL), sodium acetate (0.985 g, 1.5 eq., 12 mmol) was added. After being stirred at 0 °C, solution of hydroxylamine hydrochloride (0.835 g, 1.5 eq., 12 mmol) in water (4 mL) was added dropwise over 10 min. The reaction mixture was further stirred at room temperature for 12h, After completion (monitored by LCMS), the reaction mixture was diluted with ice cold water and extracted with dichloromethane (3 × 30 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford crude residue. The crude was purified by silica gel flash column chromatography using 20% ethyl acetate in hexane as eluent to afford tert-butyl (E)-(2-(2-(2-(hydroxyimino)ethoxy)ethoxy)ethyl)carbamate as a yellowish liquid. Yield: 1.75 g, 83.3%. LCMS: m/z 263.25 [M+H]. [1182] Synthesis of tert-butyl (Z)-(2-(2-(2-chloro-2-(hydroxyimino)ethoxy)ethoxy)ethyl)carbamate (4) [1183] To a stirred solution of tert-butyl (E)-(2-(2-(2-(hydroxyimino)ethoxy)ethoxy)ethyl)carbamate (3, 1.5 g, 1 eq., 5.72 mmol) in N, N-dimethylformaide (10 ml), was added n-chlorosuccinimide ( 0.764 g., 1 eq., 5.72 mmol ) in one portion and stirred at 40 °C for 2h. Reaction was monitored by TLC. After completion of reaction, reaction mixture was diluted with ice cold water and extracted with ethyl acetate (3 × 20 mL). Then, organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford crude tert-butyl (Z)-(2-(2-(2-chloro-2- (hydroxyimino)ethoxy)ethoxy)ethyl)carbamate (4, 1.6 g) which was used for the next step as such. [1184] Synthesis of tert-butyl (2-(2-((5-((2S,3R,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)isoxazol-3-yl)methoxy)ethoxy)ethyl)carbamate (5) [1185] Compound N-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-2- ethynyltetrahydro-2H-pyran-3-yl)acetamide (XB147, 0.27 g, 1 eq., 0.54 mmol) and tert-butyl (Z)-(2-(2- (2-chloro-2-(hydroxyimino)ethoxy)ethoxy)ethyl)carbamate (4, 1.6 g, 5 eq, 2.7 mmol) were taken in 1,4- dioxane (10 mL) and reaction mixture was stirred under 120 °C for 3 days. Progress of reaction was monitored by LCMS. After completion, reaction mixture was concentrated under reduced pressure to afford crude which was purified by silica gel flash column chromatography using 70-80% ethyl acetae- heptane as eluent to afford tert-butyl (2-(2-((5-((2S,3R,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)isoxazol-3-yl)methoxy)ethoxy)ethyl)carbamate (5) as yellowish sticky liquid. Yield: 0.27 g, 65.7%. LCMS: m/z 760.00 [M+H]. [1186] Synthesis of N-((2S,3R,4R,5R,6R)-2-(3-((2-(2-aminoethoxy)ethoxy)methyl)isoxazol-5-yl)- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB86) [1187] To a stirred solution of tert-butyl (2-(2-((5-((2S,3R,4R,5R,6R)-3-acetamido-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)isoxazol-3- yl)methoxy)ethoxy)ethyl)carbamate (5, 0.27 g, 1.0 eq., 0.93 mmol) in dry dichloromethane (10 mL), trichloroborane (4.34 ml, 12.0 eq., 4.34 mmol 1M solution in dichloromethane) was added dropwise and stirred at -78 °C for 3h. After completion (monitored by LCMS), reaction mixture was quenched with methanol and concentrated under reduced pressure to afford crude which was purified by RP prep-HPLC (40% acetonitrile in water with 0.1% TFA) to afford N-((2S,3R,4R,5R,6R)-2-(3-((2-(2- aminoethoxy)ethoxy)methyl)isoxazol-5-yl)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)acetamide (XB86) as colorless foamy solid. Yield: 0.072 g, 51%. LCMS: m/z 390.00 [M+H]. 1H NMR (400 MHz, DMSO-d6 with D2O): δ 6.53 (s, 1H), 5.24 (d, J = 6.00 Hz, 1H), 4.54 (s, 2H), 4.24-4.20 (m, 1H), 3.83-3.80 (m, 2H), 3.59-3.55 (m, 7H), 3.49 (d, J = 6.00 Hz, 2H), 2.94 (t, J = 5.20 Hz, 2H), 1.73 (s, 3H). Synthesis of (2R,3R,4R,5S)-5-((6-(3-(2-(2-aminoethoxy)ethoxy)propyl)pyrazin-2-yl)amino)-2- (hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB87)
Figure imgf000370_0001
XB87 [1188] Synthesis of 2-bromo-6-fluoropyrazine (2) [1189] To a solution of 6-bromopyrazin-2-amine (1, 1 g, 1.0 eq.5.75 mmol) in HBF4 (3 mL) was added sodium nitrite (0.793 g, 2 eq., 11.5 mmol) in portions at 0 °C. The mixture was stirred at 20 °C for 2h. After completion, the reaction mixture was quenched with water (10 mL) and extracted with pentane (3×20 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated via distillation to remove pentane. Desired compound 2-bromo-6-fluoropyrazine (2) was obtained as brown oil. Yield: 0.5 g, 49.16% [1190] Synthesis of (2R,3R,4R,5S)-5-((6-bromopyrazin-2-yl)amino)-2-(hydroxymethyl)tetrahydro- 2H-pyran-3,4-diol (3) [1191] To a stirred solution of 2-bromo-6-fluoropyrazine (2, 0.365 g, 1.0 eq., 2.06 mmol) and (2R,3R,4R,5S)-5-amino-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol hydrochloride (48-4, 0.494 g, 1.2 eq., 2.47 mmol) in N-Methyl-2-pyrrolidone (4 mL), N,N-Diisopropylethylamine (3.59 mL, 10 eq., 20.6 mmol) was added, and the resultant reaction mixture was allowed to stir at 100 °C for 12h. After completion, volatiles were removed under reduced pressure to obtain crude which was purified by silica gel column chromatography using 5-20% methanol in dichloromethane to afford (2R,3R,4R,5S)-5-((6- bromopyrazin-2-yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (3) as white semi-solid. Yield: 0.450 g, 66.79%; LCMS: m/z 320.0 [M+H]. [1192] Synthesis of tert-butyl (2-(2-((3-(6-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)prop-2-yn-1- yl)oxy)ethoxy)ethyl)carbamate (4) [1193] A stirred solution of (2R,3R,4R,5S)-5-((6-bromopyrazin-2-yl)amino)-2- (hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (3, 0.05 mg, 1.0 eq., 0.156 mmol) in tetrahydrofuran (0.5 mL) was purged with N2-gas for 30 min. To this, triethylamine (0.0652 mL, 3 eq., 0.469 mmol), tert- butyl (2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)carbamate (0.038 g, 1.0 eq., 0.156 mmol), copper iodide (0.00297 g, 0.1 eq., 0.0156 mmol) and Bis(triphenylphosphine)palladium(II) dichloride (0.0055 g, 0.05 eq., 0.0078 mmol) were added and the mixture was purged with N2-gas for another 30 minutes. The reaction mixture was stirred at 100 °C for 24h. After completion, the reaction mixture was filtered through celite bed and rinsed with 20% methanol-dichloromethane. The filtrate was dried to obtained crude, which was purified by silica gel chromatography using 5% methanol in dichloromethane to afford tert-butyl (2-(2-((3-(6-(((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)amino)pyrazin-2-yl)prop-2-yn-1-yl)oxy)ethoxy)ethyl)carbamate (4) as a colourless liquid. Yield: 0.011 g, 14.6%; LCMS: m/z 483.5 [M+H] [1194] Synthesis of tert-butyl (2-(2-(3-(6-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)propoxy)ethoxy)ethyl)carbamate (5) [1195] To a solution of tert-butyl (2-(2-((3-(6-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)prop-2-yn-1- yl)oxy)ethoxy)ethyl)carbamate (4, 0.28 g, 0.580 mmol) in methanol (5 mL), 10% Pd/C (0.28 g) was added and the reaction mixture was stirred at room temperature under hydrogen gas balloon pressure for 1h. After completion, reaction mixture was filtered through syringe filter and washed with methanol. The filtrate was concentrated and dried to afford crude tert-butyl (2-(2-(3-(6-(((3S,4R,5R,6R)-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)propoxy)ethoxy)ethyl)carbamate (5) as colour less liquid. Yield: 0.25 g, crude; LCMS: m/z 487.00 [M+H]. [1196] Synthesis of (2R,3R,4R,5S)-5-((6-(3-(2-(2-aminoethoxy)ethoxy)propyl)pyrazin-2-yl)amino)- 2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB87) [1197] To a stirred solution of tert-butyl (2-(2-(3-(6-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)propoxy)ethoxy)ethyl)carbamate (5, 0.28 g, 0.575 mol) in dichloromethane (6 mL) was added trifluoroacetic acid (1.5 mL) at 0 °C. The reaction mixture was stirred at room temperature for 2h. After completion, the reaction mixture was concentrated under vacuum to get crude which was purified by RP prep-HPLC (20-30% acetonitrile in water with 0.1% trifluoroacetic acid). Fractions containing the desired product were combined and lyophilized to dryness to afford (2R,3R,4R,5S)-5-((6-(3-(2-(2-aminoethoxy)ethoxy)propyl)pyrazin-2-yl)amino)-2- (hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB87) as colourless semi solid. Yield: 0.107 g, 48.11%; LCMS m/z 387.10 [M+H] ; 1H NMR (400 MHz, DMSO-d6, D2O exchange): δ 7.70 (s, 1H), 7.51 (s, 1H), 4.06-4.00 (m, 1H), 3.90-3.86 (m, 1H), 3.73-3.72 (d, J = 2.8 Hz, 1H), 3.57-3.46 (m, 9H), 3.42-3.39 (t, J = 6.4 Hz, 2H), 3.31-3.28 (t, J = 6.0 Hz, 1H), 2.95-2.90 (m, 3H), 2.56-2.50 (m, 2H), 1.85-1.78 (m, 2H) Synthesis of N-((2R,3R,4R,5R,6R)-2-(5-(8-aminooctyl)pyridazin-4-yl)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB88)
Figure imgf000372_0001
Figure imgf000373_0001
XB88 [1198] Synthesis of tert-butyl(dec-9-yn-1-yloxy)dimethylsilane (2) [1199] To a solution of dec-9-yn-1-ol (1, 1.0 g, 1.0 eq., 6.48 mmol) in dichloromethane (20 mL) was added imidazole (1.32 g, 3 eq., 19.4 mmol) followed by tert-butyldimethylsilyl chloride (1.47 g, 1.5 eq., 9.72 mmol) at 0 °C under nitrogen atmosphere and the resultant reaction mixture was stirred at room temperature for 12h. After completion, the reaction mixture was quenched with water (10 mL) and extracted with dichloromethane to obtain crude, which was purified by flash column chromatography (using 10% ethyl acetate/heptane as eluent) to afford tert-butyl(dec-9-yn-1-yloxy)dimethylsilane (2) as colourless liquid. Yield: 1.50 g, 86.17%; 1H NMR (400 MHz, CDCl3): δ 3.59 (t, J = 6.4 Hz, 2H), 2.20- 2.16 (m, 2H), 1.93 (t, J = 2.8 Hz, 1H), 1.56-1.52 (m, 4H), 1.30-1.26 (m, 8H), 0.87-0.83 (m, 10H), 0.05 (s, 6H). [1200] Synthesis of ((10-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3- nitrotetrahydro-2H-pyran-2-yl)dec-9-yn-1-yl)oxy)(tert-butyl)dimethylsilane (3) [1201] To a stirred solution of tert-butyl(dec-9-yn-1-yloxy)dimethylsilane (2, 0.698 g, 1.2 eq., 2.6 mmol) in dry tetrahydrofuran (3 ml) was added n-butyllithium (1.30 mL, 1.5 eq., 3.25 mmol, 2.5 M solution in hexane) at -40 °C under nitrogen atmosphere. The resulting reaction mixture was stirred at -40 °C for 1h. To this, a solution of (2R,3R,4R)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-5-nitro-3,4- dihydro-2H-pyran (2a, 1.0 g, 1.0 e.q., 2.17 mmol) in dry tetrahydrofuran (2 ml) was added dropwise at - 50 °C and the reaction mixture was stirred for another 30 minutes. After completion, the reaction mixture was quenched with saturated aqueous ammonium chloride solution and was allowed to stir at room temperature for 10 min. The resulting solution was extracted with ethyl acetate (3×10 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain crude which was purified by column chromatography using 10-15% ethyl acetate/heptane as eluent to afford ((10-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3-nitrotetrahydro- 2H-pyran-2-yl)dec-9-yn-1-yl)oxy)(tert-butyl)dimethylsilane (3) as pale yellow semi-solid. Yield: 0.56 g, 35.4%; LCMS: m/z 730.4 [M+ H]. [1202] Synthesis of 10-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3- nitrotetrahydro-2H-pyran-2-yl)dec-9-yn-1-ol (4) [1203] To a stirred solution of ((10-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)- 3-nitrotetrahydro-2H-pyran-2-yl)dec-9-yn-1-yl)oxy)(tert-butyl)dimethylsilane (3, 3.0 g, 1.0 eq., 4.11 mmol) in dry tetrahydrofuran (25.0 mL) was added tetrabutylammonium fluoride (1.78 mL, 1.5 eq., 6.16 mmol, 1M solution tetrahydrofuran) at 0 °C. The reaction was stirred for 2h at room temperature. After completion, the reaction mixture was diluted with water and extracted with ethyl acetate. Organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated to obtain crude, which was purified by column chromatography using 60-70% ethyl acetate/heptane to afford 10-((2R,3S,4R,5R,6R)- 4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3-nitrotetrahydro-2H-pyran-2-yl)dec-9-yn-1-ol (4) as sticky liquid. Yield: 1.30 g, 51.3%; LCMS m/z 616.3 [M+H]. [1204] Synthesis of 2-(10-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3- nitrotetrahydro-2H-pyran-2-yl)dec-9-yn-1-yl)isoindoline-1,3-dione (5) [1205] To a stirred solution of 10-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3- nitrotetrahydro-2H-pyran-2-yl)dec-9-yn-1-ol (4, 1.3 g, 1 eq, 2.11 mmol) in dry tetrahydrofuran (10 mL) were added phthalimide (0.311 g, 1.0 eq, 2.11 mmol) and triphenylphosphine (0.664 g, 1.2 eq., 2.53 mmol) at room temperature and was stirred for 20 min. To this, diisopropyl azodicarboxylate (0.622 mL, 1.5 eq., 3.17 mmol) was added under nitrogen atmosphere and the resulting reaction mixture was stirred at room temperature for 16h. After completion, the reaction mixture was concentrated under reduced pressure to obtain crude, which was purified by silica gel flash chromatography using 50-60% ethyl acetate/heptane to afford 2-(10-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3- nitrotetrahydro-2H-pyran-2-yl)dec-9-yn-1-yl)isoindoline-1,3-dione (5) as a pale yellow semi-solid. Yield: 1.27 g, 80.76%; LCMS: m/z 340.22 [M+H]. [1206] Synthesis of 2-(10-((2R,3S,4R,5R,6R)-3-amino-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)dec-9-yn-1-yl)isoindoline-1,3-dione (6) [1207] To a stirred solution of 2-(10-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)- 3-nitrotetrahydro-2H-pyran-2-yl)dec-9-yn-1-yl)isoindoline-1,3-dione (5, 2.7 g, 1.0 eq., 3.62 mmol) in ethanol (22 ml) was added iron (1.01 g, 5.0 eq., 18.1 mmol) and a aqueous solution of ammonium chloride (0.969 g, 5.0 eq., 18.1 mmol, dissolved in 7 ml of water). The resultant reaction mixture was stirred at 65 °C for 2h. After completion, the reaction mixture was concentrated under reduce pressure, diluted with dichloromethane, filtered through celite-bed, and filtrate was concentrated to afford crude 2- (10-((2R,3S,4R,5R,6R)-3-amino-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2- yl)dec-9-yn-1-yl)isoindoline-1,3-dione (6) as a semi-solid. Yield: 2.50 g, Crude; LCMS: m/z 715.3 [M+H]. [1208] Synthesis of N-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-2-(10-(1,3- dioxoisoindolin-2-yl)dec-1-yn-1-yl)tetrahydro-2H-pyran-3-yl)acetamide (7) [1209] To a solution of 2-(10-((2R,3S,4R,5R,6R)-3-amino-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)dec-9-yn-1-yl)isoindoline-1,3-dione (6, 1.44 g, 2.01 mmol) in dichloromethane (10 mL), triethylamine (5.66 mL, 20 eq., 40.3 mmol), N,N-dimethyl-4- pyridylamine (24.6 mg, 0.1 eq., 0.201 mmol) and acetic anhydride (1.9 mL, 10 eq., 20.1 mmol) were added at 0 °C and the resultant reaction mixture was stirred at room temperature for 12h. After completion, water was added to the reaction mixture and the organic layer was extracted with dichloromethane. The combined organic extract was dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using 40-55 % ethyl acetate/heptane to afford N-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-2-(10-(1,3- dioxoisoindolin-2-yl)dec-1-yn-1-yl)tetrahydro-2H-pyran-3-yl)acetamide (7) as off white solid. Yield: 0.63 g, 41.33%, LCMS: m/z 757.3 [M+H]. [1210] Synthesis of N-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-2-(5-(9-(1,3- dioxoisoindolin-2-yl)nonyl)pyridazin-4-yl)tetrahydro-2H-pyran-3-yl)acetamide (8) [1211] To a stirred solution of N-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-2- (10-(1,3-dioxoisoindolin-2-yl)dec-1-yn-1-yl)tetrahydro-2H-pyran-3-yl)acetamide (7, 1.0 g,1.32 mmol) and 1,2,4,5-tetraazine (0.217 g, 2 eq., 2.64 mmol) in dry 1,4-dioxane (10 mL) were heated at 140 °C for 24h. After completion, the reaction mixture was concentrated under reduced pressure to obtain crude which was purified by silica gel column chromatography using 40-55 % ethyl acetate in hexane to afford N-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-2-(5-(9-(1,3-dioxoisoindolin-2- yl)nonyl)pyridazin-4-yl)tetrahydro-2H-pyran-3-yl)acetamide (8) yellow sticky liquid. Yield: 0.67 g, 62.53%, LCMS: m/z 825.4 [M+H]. [1212] Synthesis of N-((2R,3R,4R,5R,6R)-2-(5-(8-(1,3-dioxoisoindolin-2-yl)octyl)pyridazin-4-yl)- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (9) [1213] To a solution of N-((2R,3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-2-(5-(9- (1,3-dioxoisoindolin-2-yl)nonyl)pyridazin-4-yl)tetrahydro-2H-pyran-3-yl)acetamide (8, 0.05 g, 1.0 eq., 0.061mmol) in dichloromethane (3 mL), was added boron trichloride (0.74 mL, 12 eq., 6.95 mmol, 1M in dichloromethane) dropwise at -78 °C and stirred at the same temperature for 2h. After completion, the reaction mixture was quenched with methanol and concentrated under reduced pressure to afford crude N-((2R,3R,4R,5R,6R)-2-(5-(8-(1,3-dioxoisoindolin-2-yl)octyl)pyridazin-4-yl)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (9) as a brown gum. Yield: 0.03 g, Crude; LCMS m/z 541.26 [M+H]. [1214] Synthesis of N-((2R,3R,4R,5R,6R)-2-(5-(8-aminooctyl)pyridazin-4-yl)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB88) [1215] To a solution of N-((2R,3R,4R,5R,6R)-2-(5-(8-(1,3-dioxoisoindolin-2-yl)octyl)pyridazin-4- yl)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (9, 0.35 g, 1.0 q., 0.647 mmol) in methanol (3.0 ml) was added hydrazine hydrate (50%, 0.047 mL, 1.5 eq., 0.971 mmol) at 0 °C and the resultant reaction mixture was allowed to attain room temperature and stirred for 2h. After completion, the reaction mixture was concentrated under reduced pressure to obtain crude which was purified by RP prep-HPLC (40% acetonitrile in water with 0.1% TFA). Fractions containing desire product were collected and lyophilized to afford of N-((2R,3R,4R,5R,6R)-2-(5-(8-aminooctyl)pyridazin-4-yl)-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB88) as off-white solid. Yield: 0.052 g, 19.5%, LCMS m/z 411.15 [M+H].1H NMR (400 MHz, DMSO-d6, D2O exchange): δ 9.17 (s, 1H), 9.03 (s, 1H), 5.38 (s, 2H), 4.11 (s, 1H), 4.04 (s, 1H), 3.65-3.58 (m, 2H), 2.74 (t, J = 7.6 Hz, 1H), 2.67-2.56 (m, 2H), 1.60-1.49 (m, 7H), 1.30-1.27 (m, 9H). Synthesis of N-((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-(5-(2-(2- (methylamino)ethoxy)ethoxy)pentyl)tetrahydro-2H-pyran-3-yl)acetamide (XB89)
Figure imgf000376_0001
[1216] Synthesis of benzyl (2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)carbamate (2) [1217] To a solution of 2-(2-(prop-2-yn-1-yloxy)ethoxy)ethan-1-amine (1, 1.50 g, 1.0 eq., 10.5 mmol) in mixture of 1,4-dioxane:water (4:1 v/v; 10 mL) was added sodium carbonate (1.67 g, 1.5 eq., 15.7 mmol) and the mixture was cooled to 0 °C. To this, benzyl chloroformate (7.66 g, 1.5 eq., 15.7 mmol) was added and the reaction mixture was stirred at room temperature for 2h. After completion, the reaction mixture was diluted by adding water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to afford crude. Crude was triturated with pentane to afford benzyl (2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)carbamate (2) as colourless liquid. Yield: 2.10 g, 72.28%; LCMS: m/z 278.0 [M+H]. [1218] Synthesis of benzyl methyl(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)carbamate (3) [1219] A solution of benzyl (2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)carbamate (2, 2.10 g, 1.0 eq., 7.57 mmol) in tetrahydrofuran (20 mL) was cooled to 0 °C. Then sodium hydride (0.363 g, 1.2 eq., 9.10 mmol, 60% in oil) was added and the reaction mixture was stirred at 0 °C for 20 mins. Methyl iodide (0.566 mL, 1.20 eq., 9.10 mmol) was added and the reaction mixture was allowed to warm to room temperature and stirred for 4h. After completion, the mixture was dried, diluted with water and extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulphate, concentrated under reduced pressure to get crude, which was purified by silica gel flash column chromatography using 20-40% ethyl acetate in heptane as eluent to afford benzyl methyl(2-(2-(prop-2-yn-1- yloxy)ethoxy)ethyl)carbamate (3) as a brown liquid. Yield: 1.50 g, 68.0%; LCMS: m/z 292.00 [M+H]. [1220] Synthesis of benzyl (2-(2-((3-bromoprop-2-yn-1-yl)oxy)ethoxy)ethyl)(methyl)carbamate (4) [1221] Benzyl methyl(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)carbamate (3, 1.50 g, 1.0 eq., 5.15 mmol) was dissolved in acetone (20 mL). To this, 1-bromo-2,5-pyrrolidinedione (1.01 g, 1.1 eq., 5.66 mmol) and silver nitrate (87.5 mg, 0.1 eq., 0.515 mmol) were added. The resultant reaction mixture was stirred at room temperature for 12h. After completion, the reaction mixture was concentrated to remove acetone; and then diluted with ethyl acetate and water. The aqueous layer was extracted with ethyl acetate and the combined organic layer was dried over anhydrous sodium sulphate, filtered, and concentrated to obtained crude which was purified by silica gel flash column chromatography using 30- 40% ethyl acetate in heptane as eluent to afford benzyl (2-(2-((3-bromoprop-2-yn-1- yl)oxy)ethoxy)ethyl)(methyl)carbamate (4) as viscous oil. Yield: 0.740 g, 38.88%; LCMS: m/z 369.95 [M+H]. [1222] Synthesis of benzyl (2-(2-((5-((2R,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)penta-2,4-diyn-1-yl)oxy)ethoxy)ethyl)(methyl)ca-- rbamate (5) [1223] A solution containing N-((2R,3S,4R,5S,6R)-4,5-bis(benzyloxy)-6-ethyl-2-ethynyltetrahydro- 2H-pyran-3-yl)acetamide (XB147, 0.80 g, 1.6 mmol), piperidine (0.341 g, 2.5 eq., 4 mmol) , copper(I)bromide (0.023 g, 0.1 eq., 160 µmol) and hydroxylamine hydrochloride (0.0223 g, 0.2 eq., 0.320 mmol) in methanol (9 mL) was degassed under N2 atmosphere at room temperature. To this, a degassed solution of benzyl (2-(2-((3-bromoprop-2-yn-1-yl)oxy)ethoxy)ethyl)(methyl)carbamate (4, 0.711 g, 1.20 eq., 1.92 mmol) in methanol (9 mL) was added in portion over a period of 15 mins. The resultant reaction mixture was stirred at room temperature for 12h. After completion, methanol was removed under reduced pressure and the crude product obtained was poured into ice cold water (20 mL) and extracted with ethyl acetate. The organic layer was washed with saturated brine solution (20 mL) and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtained crude which was purified by silica gel flash column chromatography using 10-40% ethyl acetate in heptane as an eluent to afford benzyl (2-(2-((5-((2R,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)penta-2,4-diyn-1-yl)oxy)ethoxy)ethyl)(methyl)carbamate (5) as an off-white solid. Yield: 0.700 g, 63.30%; LCMS: m/z 789.05 [M+H]. [1224] Synthesis of N-((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-(5-(2-(2- (methylamino)ethoxy)ethoxy)pentyl)tetrahydro-2H-pyran-3-yl)acetamide (XB89) [1225] To a stirred solution of benzyl (2-(2-((5-((2R,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)- 6-((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)penta-2,4-diyn-1- yl)oxy)ethoxy)ethyl)(methyl)carbamate (5, 0.570 g, 1.0 eq., 0.722 mmol) in methanol (10 mL) and acetic acid (1.5 mL), 10% Pd/C (0.275 g) and 20% Pd(OH)2/C (0.275 g) were added, and the resultant reaction mixture was stirred under H2-balloon pressure at room temperature for 6h. After completion, the mixture was filtered through syringe filter and concentrated under reduced pressure to get crude. Crude was triturated with diethyl ether and pentane several times. Then crude was dissolved in water and lyophilized to afford N-((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-(5-(2-(2- (methylamino)ethoxy)ethoxy)pentyl)tetrahydro-2H-pyran-3-yl)acetamide (XB89) as colourless solid. Yield: 0.153 g, 53.6%, LCMS-MS m/z 393.15 [M+H]. [1226] 1H NMR (400 MHz, DMSO-d6-D2O) δ 3.97-3.94 (m, 1H), 3.82-3.79 (m, 1H), 3.71-3.70 (m, 2H), 3.53-3.46 (m, 10H), 3.34 (t, J = 6.40 Hz, 2H), 2.77 (t, J = 5.60 Hz, 2H), 2.36 (t, J = 15.20 Hz, 3H), 1.81 (s, 1H), 1.48-1.43 (m, 3H), 1.31-0.16 (m, 6H). Synthesis of (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (trifluoromethyl)pyrimidin-2-yl)amino)-2-((4-((1-phenylpiperidin-4-yl)methyl)piperazin-1- yl)methyl)tetrahydro-2H-pyran-3,4-diol (XB90)
Figure imgf000378_0001
XB90 [1227] Synthesis of (1-phenylpiperidin-4-yl)methyl methanesulfonate (2) [1228] To a solution of (1-phenylpiperidin-4-yl)methanol (1, 0.8 g, 4.18 mmol) in dichloromethane (10 mL) were added triethylamine (1.76 mL, 3 eq., 12.5 mmol) and methanesulfonyl chloride (0.971 mL, 3 eq., 12.5 mmol) at 0 °C, and the resultant reaction mixture was stirred at room temperature for 3h. After completion, the reaction mixture was quenched with water (20 mL), and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated to get crude (1- phenylpiperidin-4-yl)methyl methanesulfonate (2) as pale yellow semi-solid. Yield: 1.10 g, Crude; LCMS: m/z 270.1 [M+H]. [1229] Synthesis of tert-butyl 4-((1-phenylpiperidin-4-yl)methyl)piperazine-1-carboxylate (3) [1230] To a stirred solution of (1-phenylpiperidin-4-yl)methyl methanesulfonate (2, 1.2 g, 1.0 eq., 4.45 mmol) in tetrahydrofuran (10 mL) were added tert-butyl piperazine-1-carboxylate (2a, 1.66 g, 2 eq., 8.91 mmol) and triethylamine (1.88 ml, 3.0 eq., 13.4 mmol) under nitrogen atmosphere. Then the reaction mixture was stirred at 80 °C for 12h. After completion, the reaction mixture was concentrated under reduced pressure. The crude was purified by column chromatography on silica gel using 40% ethyl acetate/hexane as eluent to afford tert-butyl 4-((1-phenylpiperidin-4-yl)methyl)piperazine-1-carboxylate (3) as yellow solid. Yield: 1.50 g, 93.6%; LCMS: m/z 360.2 [M+H]. [1231] Synthesis of 1-((1-phenylpiperidin-4-yl)methyl)piperazine hydrochloride (4) [1232] To a solution of tert-butyl 4-((1-phenylpiperidin-4-yl)methyl)piperazine-1-carboxylate (3, 1.0 g.,1.0 eq., 2.78 mmol) in dichloromethane (5.0 mL) was added 4M HCl in 1,4-dioxane (5.0 mL) at 0 °C and reaction mixture was stirred at room temperature for 3h. After completion, the reaction mixture was concentrated under reduced pressure and washed with diethyl ether to afford 1-((1-phenylpiperidin- 4-yl)methyl)piperazine hydrochloride (4) as viscous brown liquid. Yield: 0.7 g (Crude), LCMS: m/z 260.2 [M+H]. [1233] Synthesis of tert-butyl ((3aS,4R,7S,7aR)-2,2-dimethyl-4-((4-((1-phenylpiperidin-4- yl)methyl)piperazin-1-yl)methyl)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (5) [1234] To a stirred solution of 1-((1-phenylpiperidin-4-yl)methyl)piperazine hydrochloride (4, 0.369 g, 1.3 eq., 1.42 mmol) in tetrahydrofuran (3 mL) was added triethylamine (1.54 mL, 10 eq., 10.9 mmol) under nitrogen atmosphere at room temperature. Subsequently, ((3aR,4R,7S,7aR)-7-((tert- butoxycarbonyl)amino)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl 4- methylbenzenesulfonate (4a, 0.5 g, 1 eq., 1.09 mmol) and potassium iodide (0.181 g, 1 eq., 1.09 mmol) were added, and reaction mixture was stirred at 100 ℃ for 48h. After completion, the reaction mixture was concentrated under reduced pressure to afford crude which was purified by silica gel column chromatography using 15-20% methanol/dichloromethane to afford of tert-butyl ((3aS,4R,7S,7aR)-2,2- dimethyl-4-((4-((1-phenylpiperidin-4-yl)methyl)piperazin-1-yl)methyl)tetrahydro-4H-[1,3]dioxolo[4,5- c]pyran-7-yl)carbamate (5) as pale-yellow semi-solid. Yield: 0.55 g, 92.4%, LCMS: m/z 545.3[M+H]. [1235] Synthesis of (2R,3R,4R,5S)-5-amino-2-((4-((1-phenylpiperidin-4-yl)methyl)piperazin-1- yl)methyl)tetrahydro-2H-pyran-3,4-diol (6) [1236] To a stirred solution of tert-butyl ((3aS,4R,7S,7aR)-2,2-dimethyl-4-((4-((1-phenylpiperidin- 4-yl)methyl)piperazin-1-yl)methyl)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (5, 0.550 g, 1.0 eq., 1.01 mmol) in dichloromethane (3 mL), trifluoroacetic acid (3 mL) was added at 0 °C, and the resultant reaction mixture was stirred at room temperature for 4h. After completion, the reaction mixture was concentrated under reduce pressure co-evaporated with dichloromethane to afford crude (2R,3R,4R,5S)-5-amino-2-((4-((1-phenylpiperidin-4-yl)methyl)piperazin-1-yl)methyl)tetrahydro-2H- pyran-3,4-diol (6) as colorless gum. Yield: 0.33 g, Crude, LCMS: m/z 405.2 [M+H]. [1237] Synthesis of tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6-((4-((1-phenylpiperidin- 4-yl)methyl)piperazin-1-yl)methyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (7) [1238] To a stirred solution of (2R,3R,4R,5S)-5-amino-2-((4-((1-phenylpiperidin-4- yl)methyl)piperazin-1-yl)methyl)tetrahydro-2H-pyran-3,4-diol (6, 0.476 g, 1.5 eq., 0.919 mmol) and tert- butyl (2-(2-(2-((2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (6a (XB95-4A), 0.290 g, 0.612 mmol) in dry N,N- dimethylformamide (3 mL,), N,N-diisopropylethylamine (1.28 mL, 12 eq., 7.35 mmol) was added at 0 °C and allowed to stir at 50 °C for 3h. After completion, the reaction mixture was concentrated under reduced pressure to obtain crude tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6-((4-((1- phenylpiperidin-4-yl)methyl)piperazin-1-yl)methyl)tetrahydro-2H-pyran-3-yl)amino)-6- (trifluoromethyl)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (7) as brown gum which was used for next step without purification. Yield: 0.50 g, Crude; LCMS: m/z 798.4 [M+H]. [1239] Synthesis of (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (trifluoromethyl)pyrimidin-2-yl)amino)-2-((4-((1-phenylpiperidin-4-yl)methyl)piperazin-1- yl)methyl)tetrahydro-2H-pyran-3,4-diol (XB90) [1240] To a stirred solution of tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6-((4-((1- phenylpiperidin-4-yl)methyl)piperazin-1-yl)methyl)tetrahydro-2H-pyran-3-yl)amino)-6- (trifluoromethyl)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (7, 0.489 g, 1.0 eq., 0.612 mmol) in dichloromethane (3 mL), was added trifluoroacetic acid (3 mL) at 0 °C. The resultant reaction was stirred at room temperature for 4h. After completion, the reaction mixture was concentrated under reduced pressure and co-evaporated with dichloromethane to get crude, which was purified by RP prep-HPLC (75-82% acetonitrile in water with 0.1% TFA). Fractions containing desired product were lyophilized to afford (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6-(trifluoromethyl)pyrimidin-2- yl)amino)-2-((4-((1-phenylpiperidin-4-yl)methyl)piperazin-1-yl)methyl)tetrahydro-2H-pyran-3,4-diol (XB90) as white solid. Yield: 0.14 g, 32.7%, LCMS: m/z 698.3 [M+H]. 1H NMR (400 MHz, DMSO-d6): δ 7.74 (m, 3H), 7.15 (t, J = 8.0 Hz, 2H), 6.58 (t, J = 7.2 Hz, 1H), 6.51 (d, J = 8.0 Hz, 2H), 6.45 (d, J = 14.4 Hz, 1H), 4.85 (bs, 2H), 4.42-4.41 (m, 2H), 4.11-4.10 (m, 2H), 3.89-3.88 (m, 3H), 3.75 (t, J = 4.0 Hz, 3H), 3.67 (s, 3H), 3.60-3.57 (m, 8H), 3.44 (t, J = 10.0 Hz, 2H), 3.41-3.39 (m, 2H), 3.32-3.27 (m, 2H), 3.06-3.03 (m, 2H), 2.99-2.98 (m, 3H), 2.85 (m, 2H), 2.27 (m, 1H), 2.07 (m, 1H), 1.74 (m, 2H), 1.64 (m, 1H). Synthesis of (R)-5-((2-(2-(2 aminoethoxy)ethoxy)ethoxy)methyl)-3-((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxazolidin-2-one (XB91) 1a'
Figure imgf000381_0001
3 XB91 [1241] Synthesis of (R)-2-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)methyl)oxirane (1a) [1242] A solution of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (1a’, 1.0 eq, 0.947 g, 5.4 mmol) in tetrahydrofuran (5 mL) was cooled at 0 °C, sodium hydride (60 % suspension in mineral oil) (1.1 eq, 0.238 g, 5.94 mmol) was added and reaction mixture was stirred at 0° C for 30 minutes. Then, a solution of (R)-2-(chloromethyl)oxirane (1’, 1.0 eq, 0.500 g, 5.4 mmol) in tetrahydrofuran (5 mL) was added and reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was poured into ice cold water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to get crude residue which was purified by column chromatography using silica gel (100-200 mesh, 0-100 % ethyl acetate in hexane) to afford (R)-2-((2-(2- (2-azidoethoxy)ethoxy)ethoxy)methyl)oxirane (1a) as colorless viscous liquid. Yield: 0.350 g, 28.0 %; LCMS m/z 249.1 [M+18]+. [1243] Synthesis of (R)-1-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-3-(((3S,4R,5R,6R)-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)propan-2-ol (2) [1244] To a solution of (3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H- pyran-3-amine (1, 1.0 eq, 0.450 g, 1.04 mmol) and (R)-2-((2-(2-(2- azidoethoxy)ethoxy)ethoxy)methyl)oxirane (1a, 0.9 eq, 0.216 g, 0.93 mmol) in ethanol (9 mL) N,N- diisopropylethylamine (2.0 eq, 0.38 mL, 2.08 mmol) was added and reaction mixture was heated for at 80 °C for 16 h. After completion, the reaction mixture was concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh, 0-3 % methanol in dichloromethane) to afford (R)-1-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-3-(((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)propan-2-ol (2) as colorless viscous liquid. Yield: 0.420 g, 60.9 %; LCMS m/z 665.7 [M+H] +. [1245] Synthesis of (R)-5-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)methyl)-3-((3S,4R,5R,6R)-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxazolidin-2-one (3) [1246] To a solution of (R)-1-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-3-(((3S,4R,5R,6R)-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)propan-2-ol (2, 1.0 eq, 0.420 g, 0.63 mmol) in acetonitrile (10 mL), 1,1'-carbonyldiimidazole (CDI) (1.5 eq, 0.154 g, 0.95 mmol) and 4- dimethylaminopyridine (DMAP) (0.1 eq, 0.007 g, 0.063 mmol) were added and the reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh, 0-50 % ethyl acetate in hexane) to afford (R)-5-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)methyl)-3-((3S,4R,5R,6R)-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxazolidin-2-one (3) as colorless viscous liquid. Yield: 0.310 g, 71.0 %; LCMS m/z 691.7 [M+H] +. [1247] Synthesis of (R)-5-((2-(2-(2 aminoethoxy)ethoxy)ethoxy)methyl)-3-((3S,4R,5R,6R)-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxazolidin-2-one (XB91) [1248] To a solution of (R)-5-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)methyl)-3-((3S,4R,5R,6R)-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxazolidin-2-one (3, 1.0 eq, 0.310 g, 0.45 mmol) in methanol (6 mL), conc. hydrochloric acid (0.31 mL) and 10 % Palladium on carbon (0.310 g) were added and the reaction mixture was stirred at room temperature under hydrogen for 16 h. Reaction was monitored by ELSD. After completion, reaction mixture was filtered through syringe filter and filtrate was concentrated to get crude which was purified by prep HPLC (14-28 % acetonitrile in water with 0.1 % trifluoroacetic acid) to afford (R)-5-((2-(2-(2 aminoethoxy)ethoxy)ethoxy)methyl)-3- ((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxazolidin-2-one (XB91) as colorless viscous syrup. Yield: 0.064 g, 36.1 %; LCMS m/z 395.1 [M+H] +; 1H NMR (400 MHz, DMSO-d6 with D2O) δ 4.63-4.61 (m, 1H), 3.76-3.72 (m, 2H), 3.62-3.47 (m, 15H), 3.45 (d, J = 5.2 Hz, 2H), 3.27-3.22 (m, 3H), 2.94 (t, J = 4.8 Hz, 2H). Synthesis of (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (trifluoromethyl)pyrimidin-2-yl)amino)-2-((4-methylpiperazin-1-yl)methyl)tetrahydro-2H-pyran-
Figure imgf000382_0001
4
Figure imgf000383_0002
Figure imgf000383_0001
[1249] Synthesis of ((3aR,4R,7S,7aR)-7-((tert-butoxycarbonyl)amino)-2,2-dimethyltetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-4-yl)methyl 4-methylbenzenesulfonate (2) [1250] To a solution of tert-butyl ((3aR,4R,7S,7aR)-4-(hydroxymethyl)-2,2-dimethyltetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (1, 0.5 g, 1.65 mmol) in dichloromethane (8 mL), triethylamine (0.92 mL, 4 eq., 6.59 mmol) and 4-methylbenzenesulfonyl chloride (0.377 g, 1.2 eq., 1.98 mmol) were added at 0 °C. The reaction mixture was stirred at room temperature for 2h. After completion, the reaction mixture was diluted with water and extracted with dichloromethane. The combined organic layer was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using 10-25% ethyl acetate/heptane as eluent to afford ((3aR,4R,7S,7aR)-7-((tert-butoxycarbonyl)amino)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran- 4-yl)methyl 4-methylbenzenesulfonate (2) as off-white solid. Yield: 0.50 g, 66.3 % LCMS: m/z 458.1 [M+H] [1251] Synthesis of tert-butyl ((3aS,4R,7S,7aR)-2,2-dimethyl-4-((4-methylpiperazin-1- yl)methyl)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (3) [1252] To a stirred solution of ((3aR,4R,7S,7aR)-7-((tert-butoxycarbonyl)amino)-2,2- dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl 4-methylbenzenesulfonate (2, 0.413 g, 1.0 eq., 0.903 mmol) in tetrahydrofuran (6 mL) was added triethylamine (0.381 mL, 3 eq., 2.71 mmol) and 1- methylpiperazine (2a, 0.181 g, 2 eq., 1.81 mmol) at 0 °C. The resultant reaction mixture was stirred at 80 °C for 12h. After completion, the reaction mixture was poured into water, and extracted with dichloromethane. The combined organic layer was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude which was purified by column chromatography using 0- 5% methanol in dichloromethane to afford tert-butyl ((3aS,4R,7S,7aR)-2,2-dimethyl-4-((4- methylpiperazin-1-yl)methyl)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (3) as viscous brown liquid. Yield: 0.20 g, 57.4 % LCMS: m/z 386.2 [M+H]. [1253] Synthesis (2R,3R,4R,5S)-5-amino-2-((4-methylpiperazin-1-yl)methyl)tetrahydro-2H-pyran- 3,4-diol (4) [1254] To a solution of tert-butyl ((3aS,4R,7S,7aR)-2,2-dimethyl-4-((4-methylpiperazin-1- yl)methyl)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (3, 0.3 g,1.0 eq., 0.778 mmol) in dichloromethane (2.0 mL) was added trifluoroacetic acid (2 mL) at 0 °C and reaction mixture was stirred at room temperature for 3h. After completion, the reaction mixture was concentrated under reduced pressure and co-evaporated with dichloromethane to afford (2R,3R,4R,5S)-5-amino-2-((4- methylpiperazin-1-yl)methyl)tetrahydro-2H-pyran-3,4-diol (4) as brown semi-solid. Yield: 0.20 g, LCMS: m/z 246.1 [M+H]. [1255] Synthesis of tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6-((4-methylpiperazin-1- yl)methyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (5) [1256] To a stirred solution of (2R,3R,4R,5S)-5-amino-2-((4-methylpiperazin-1- yl)methyl)tetrahydro-2H-pyran-3,4-diol (4, 0.364 g, 1.2 eq., 0.845 mmol) and tert-butyl (2-(2-(2-((2- (methylsulfonyl)-6-(trifluoromethyl)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4a (XB95- 4A)0.4 g, 1 eq., 0.845 mmol) in dry N,N-dimethylformamide (4 mL) was added N,N- diisopropylethylamine (1.77 mL, 12 eq.) at 0 °C. The reaction was stirred at 80 °C for 5h. After completion, the reaction mixture was concentrated under reduced pressure to afford crude tert-butyl (2- (2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6-((4-methylpiperazin-1-yl)methyl)tetrahydro-2H-pyran-3- yl)amino)-6-(trifluoromethyl)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (5) as brown semi- solid. Yield: 0.50 g, LCMS: m/z 639.3 [M+H]. The crude material was used for the next step without purification. [1257] Synthesis of (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (trifluoromethyl)pyrimidin-2-yl)amino)-2-((4-methylpiperazin-1-yl)methyl)tetrahydro-2H-pyran-3,4-diol (XB92) [1258] To a stirred solution of tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6-((4- methylpiperazin-1-yl)methyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (5, 0.27 g, 1.0 eq.,0.42 mmol) in dichloromethane (0.2 mL) was added trifluoroacetic acid (0.2 mL) at 0 °C and reaction was stirred at room temperature for 4h. After completion of reaction as indicated by LCMS, reaction mixture was concentrated under reduce pressure to get crude. Crude was purified by RP prep HPLC (80% acetonitrile in water with 0.1% TFA) to afford (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6-(trifluoromethyl)pyrimidin-2-yl)amino)-2- ((4-methylpiperazin-1-yl)methyl)tetrahydro-2H-pyran-3,4-diol (XB92) as white sticky solid. Yield: 0.094 g; 22.3 %, LCMS: m/z 539.2 [M+H]; 1H-NMR (400 MHz, DMSO-d6-D2O exchange, high temperature): δ 6.37 (s, 1H), 4.43 (brs, 2H), 4.12-4.07 (m, 1H), 3.90-3.86 (m, 1H), 3.74 (t, J = 4.4 Hz, 2H), 3.71 (brs, 1H), 3.61-3.57 (m, 9H), 3.08-3.03 (m, 6H), 2.97 (t, J = 4.8 Hz, 2H), 2.91-2.80 (m, 4H), 2.78-2.75 (m, 2H), 2.68-2.67 (m, 3H). Synthesis of (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (trifluoromethyl)pyrimidin-2-yl)amino)-2-(phenoxymethyl)tetrahydro-2H-pyran-3,4-diol (XB93)
Figure imgf000384_0001
Figure imgf000385_0001
[1259] Synthesis of tert-butyl ((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H- pyran-3-yl)carbamate (2) [1260] To a stirred solution of (2R,3R,4R,5S)-5-amino-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4- diol hydrochloride (84-4, 1.0 eq., 2.0 g, 10.0 mmol) in methanol (20.0 mL) were added triethylamine (3 eq., 4.11 mL, 30.1 mmol) and di-tert-butyl dicarbonate (1.2 eq., 2.76 mL, 12.0 mmol) gradually at 0° C under a nitrogen atmosphere. Then reaction mixture was stirred at room temperature for 12h. After completion reaction mixture was concentrated under reduced pressure to get crude tert-butyl ((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)carbamate (2) as brown solid. Yield: 2.5 g, Crude; LCMS m/z 264.1 [M+H]. [1261] Synthesis of tert-butyl ((3aR,4R,7S,7aR)-4-(hydroxymethyl)-2,2-dimethyltetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (3) [1262] To a stirred solution of tert-butyl ((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)carbamate (2, 1 eq , 2.0 g, 7.6 mmol) in acetone ( 15 mL) 2,2- dimethoxypropane (2a, 4.0 eq., 3.6 g, 30.4 mmol) and (7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1- yl)methanesulfonic acid (CSA) (0.2 eq., 0.351 g, 1.5 mmol) were added. Then reaction mixture was sonicated for 30 min. After completion, the reaction mixture was neutralized using aqueous sodium bicarbonate solution and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product obtained was purified by column chromatography on silica gel with 10-25% Ethyl acetate/heptane as eluent to afford tert-butyl ((3aR,4R,7S,7aR)-4-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7- yl)carbamate (3) as white solid. Yield: 2.0 g, 86.8%; LCMS m/z 304.1 [M+H] . [1263] Synthesis of tert-butyl ((3aR,4R,7S,7aR)-2,2-dimethyl-4-(phenoxymethyl)tetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (4) [1264] To a stirred solution of tert-butyl ((3aR,4R,7S,7aR)-4-(hydroxymethyl)-2,2- dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (3, 1 eq., 1 g, 3.3 mmol) in 1,2- dichloroethane (15.0 mL) were added phenylboronic acid (1.5 eq , 0.603 g, 4.94 mmol), copper bis(acetate) (1 eq , 0.599 g, 3.3 mmol,) and triethylamine (3eq., 1.38 mL, 9.89 mmol) and the reaction was stirred under oxygen atmosphere at room temperature for 12h. After 12h reaction mixture was filtered through bed of celite, washed with 1,2-dichloroethane and concentrated under reduce pressure to get crude which was purified by flash column using 10% ethyl acetate/heptane as eluent to afford tert- butyl ((3aR,4R,7S,7aR)-2,2-dimethyl-4-(phenoxymethyl)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7- yl)carbamate (4) as yellow sticky liquid. Yield: 0.3 g.23.9% LCMS m/z 380.2 [M+H]. [1265] Synthesis of (2R,3R,4R,5S)-5-amino-2-(phenoxymethyl)tetrahydro-2H-pyran-3,4-diol (5) [1266] A solution of tert-butyl ((3aR,4R,7S,7aR)-2,2-dimethyl-4-(phenoxymethyl)tetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (4, 1.0 eq., 0.25 g, 0.659 mmol) in dichloromethane (2.0 mL) was added trifluoroacetic acid (2.0 mL) dropwise at 0°C. Then the reaction mixture was stirred at room temperature for 3h. After completion, the reaction mixture was co-distilled with dichloromethane and diethyl ether to obtain crude (2R,3R,4R,5S)-5-amino-2-(phenoxymethyl)tetrahydro-2H-pyran-3,4-diol (5) as viscous brown liquid. Yield: 0.15 g, Crude; LCMS m/z 240.1 [M+H]. [1267] Synthesis of tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (phenoxymethyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (6) [1268] To a stirred solution of (2R,3R,4R,5S)-5-amino-2-(phenoxymethyl)tetrahydro-2H-pyran-3,4- diol (5, 0.328 g, 1.1 eq., 0.929 mmol) and tert-butyl (2-(2-(2-((2-(methylsulfonyl)-6- (trifluoromethyl)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (5a (XB95-4A), 1eq., 0.4 g, 0.845 mmol) in dry N,N-dimethylformamide (3 mL) was added N,N-diisopropyl ethylamine (1.77 mL, 12 eq., 10.1 mmol) at 0°C and allowed to stir at 80°C for 8h. After completion reaction mixture was concentrated under reduced pressure to get crude tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy- 6-(phenoxymethyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (6) as brown gum which was used as such for next step. Yield: 0.45 g, Crude; LCMS m/z 633.2 [M+H]. [1269] Synthesis of (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (trifluoromethyl)pyrimidin-2-yl)amino)-2-(phenoxymethyl)tetrahydro-2H-pyran-3,4-diol (XB93) [1270] To a stirred solution of tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (phenoxymethyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (6, 0.534 g, 1.0 eq.,0.845 mmol) in dichloromethane (4.0 mL) was added trifluoroacetic acid (4.0 mL) at 0°C and reaction was stirred at room temperature for 2h. After completion, reaction mixture was concentrated under reduce pressure to get crude which was purified by reverse phase prep HPLC (25% acetonitrile in water with 0.1% TFA). Fractions containing desired compound were combined and lyophilized to afford (2R,3R,4R,5S)-5-((4-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)-6-(trifluoromethyl)pyrimidin-2-yl)amino)-2-(phenoxymethyl)tetrahydro- 2H-pyran-3,4-diol (XB93) as white solid. Yield: 0.139 g, 30.9% LCMS m/z 533.21[M+H]. 1H NMR (400 MHz, DMSO-d6): δ 7.67 (d, J = 8.00 Hz, 1H), 7.42 (d, J = 8.00 Hz, 1H), 7.29 (t, J = 7.20 Hz, 3H), 6.92 (d, J = 8.40 Hz, 3H), 6.44 (d, J = 11.20 Hz, 1H), 4.90 (d, J = 4.80 Hz, 1H), 4.47-4.44 (m, 2H), 4.11- 4.03 (m, 3H), 3.95-3.93 (m, 1H), 3.84 (s, 1H), 3.77-3.76 (m, 2H), 3.68-3.60 (m, 8H), 3.08-3.01 (m, 1H), 2.91 (t, J = 4.80 Hz, 2H), 2.07 (s, 1H). Synthesis of (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (trifluoromethyl)pyrimidin-2-yl)amino)-2-(methoxymethyl)tetrahydro-2H-pyran-3,4-diol (XB94)
Figure imgf000387_0001
[1271] Synthesis of tert-butyl ((3aR,4R,7S,7aR)-4-(methoxymethyl)-2,2-dimethyltetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (2) [1272] To a stirred solution of tert-butyl ((3aR,4R,7S,7aR)-4-(hydroxymethyl)-2,2- dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (1, 0.3 g, 1 eq., 0.989 mmol) in dry tetrahydrofuran (5 mL), sodium hydride (47.5 mg, 2 eq., 1.98 mmol) was added portion-wise at 0 °C. After that, reaction mixture was stirred at room temperature for 30 minutes. Then, iodomethane (0.092 mL, 1.5 eq., 1.48 mmol) was added to the reaction mixture at 0 °C and stirred at room temperature for 30 minutes. After completion (monitored by ELSD-MS), reaction mixture was quenched with ice-cold water followed by concentration gave crude residue which was re-dissolved in ethyl acetate and poured in water, then extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulphate, filtered, and concentrated to give crude residue which was purified by silica gel column chromatography using 40% ethyl acetate in heptane as eluent to afford tert-butyl (3aR,4R,7S,7aR)-4-(methoxymethyl)- 2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate as off-white solid. Yield: 0.28 g, 89.0 %. LCMS: m/z 318.10 [M+H]. [1273] Synthesis of (2R,3R,4R,5S)-5-amino-2-(methoxymethyl)tetrahydro-2H-pyran-3,4-diol (3) [1274] To a stirred solution of (3aR,4R,7S,7aR)-4-(methoxymethyl)-2,2-dimethyltetrahydro-4H- [1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (0.28 g, 1 eq., 0.88 mmol) in dichloromethane (5 mL), trifluoroacetic acid (5 mL) was added to the reaction mixture at 0 °C. Then, reaction mixture was stirred at room temperature for 2h. After completion (monitored by ELSD-MS), reaction mixture was directly concentrated and washed with diethyl ether to get crude (2R,3R,4R,5S)-5-amino-2- (methoxymethyl)tetrahydro-2H-pyran-3,4-diol (3) as brown solid. Yield: 0.18 g, Crude. LCMS: m/z 178.2 [M+H]. [1275] Synthesis of tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (methoxymethyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4) [1276] To a stirred solution of (2R,3R,4R,5S)-5-amino-2-(methoxymethyl)tetrahydro-2H-pyran-3,4- diol (3, 0.185 g, 1 eq., 0.634 mmol) and tert-butyl (2-(2-(2-((2-(methylsulfonyl)-6- (trifluoromethyl)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (3a (XB95-4A), 0.3 g, 1 eq., 0.634 mmol) in dry N,N-dimethylformamide (3 mL), was added N,N-Diisopropylethylamine (1.32 mL, 12 eq., 7.6 mmol) at 0 °C and allowed to stir at 120 °C for 6h. After completion (monitored by LCMS), reaction mixture was concentrated to afford crude tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (methoxymethyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4) as brown liquid which was used for the next step without purification. Yield: 0.317 g, Crude. LCMS: m/z 571.15 [M+H]. [1277] Synthesis of (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (trifluoromethyl)pyrimidin-2-yl)amino)-2-(methoxymethyl)tetrahydro-2H-pyran-3,4-diol (XB94) [1278] A solution of tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (methoxymethyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4, 0.362 g, 1 eq., 0.634 mmol) in dichloromethane (3 mL) was cooled at 0 °C. Then, trifluoroacetic acid (3 mL) was added to it and reaction mixture was stirred at room temperature for 3h. After completion (monitored by LCMS), reaction mixture was concentrated under reduced pressure to afford crude which was purified by RP prep-HPLC (14% acetonitrile in water with 0.1%TFA). Fractions containing the desired product were collected and lyophilized to afford (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6-(trifluoromethyl)pyrimidin-2-yl)amino)-2- (methoxymethyl)tetrahydro-2H-pyran-3,4-diol (XB94) as a white solid. Yield: 0.078 g, 30.19 %. LCMS: m/z 471.30 [M+H].1H NMR (400 MHz, DMSO-d6, high temperature): δ 7.63 (brs, 3H), 7.20 (brs, 1H), 6.37 (brs, 1H), 4.47-4.48 (t, J = 4.8 Hz, 2H), 4.14-4.06 (m, 1H), 3.94-3.89 (m, 1H), 3.77 (t, J = 4.8 Hz, 2H), 3.73 (brs, 1H), 3.66-3.59 (m, 7H), 3.54-3.43 (m, 3H), 3.28 (s, 3H), 2.98 (t, J = 5.2 Hz, 3H). Synthesis of (2R,3R,4R,5R,6R)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (trifluoromethyl)pyrimidin-2-yl)amino)-2-(hydroxymethyl)-6-propyltetrahydro-2H-pyran-3,4-diol
Figure imgf000389_0001
XB95 Synthesis of intermediate 4a
Figure imgf000389_0002
[1279] Synthesis of (2R,3R,4R,5S,6R)-5-acetamido-2-(acetoxymethyl)-6-allyltetrahydro-2H-pyran- 3,4-diyl diacetate (2) [1280] To a stirred solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6- chlorotetrahydro-2H-pyran-3,4-diyl diacetate (1, 20 g, 1 eq., 54.7 mmol), and allyl tributylstannane (171 mL, 10 eq., 547 mmol)) in tetrahydrofuran (120 mL) was added azobisisobutyronitrile (2.69 g, 0.3 eq., 16.4 mmol). The solution was purged with nitrogen gas and then heated at 80 °C for 8 h. After that, reaction mixture was cooled to room temperature and solvent was removed under reduced pressure. The residue was partitioned between 100 mL of acetonitrile and 400 mL of pentane. The acetonitrile layer was extracted with additional pentane (5 × 100 mL) to remove the remaining organotin compounds (soluble in pentane). Then, acetonitrile layer was concentrated to give crude which was purified by column chromatography using 10 % methanol in dichloromethane as eluting system to afford (2R,3R,4R,5S,6R)-5-acetamido-2-(acetoxymethyl)-6-allyltetrahydro-2H-pyran-3,4-diyl diacetate (2) as a sticky solid. Yield: 12.0 g, 59.1%. LCMS: m/z 372.05 [M+H]. [1281] Synthesis of (2R,3R,4R,5S,6R)-5-acetamido-2-(acetoxymethyl)-6-propyltetrahydro-2H- pyran-3,4-diyl diacetate (3) [1282] To a solution of (2R,3R,4R,5S,6R)-5-acetamido-2-(acetoxymethyl)-6-allyltetrahydro-2H- pyran-3,4-diyl diacetate (2, 3.0 g, 1.0 eq., 8.08 mmol) in methanol (10.0 mL), purged with hydrogen gas and maintained hydrogen atmosphere using hydrogen gas balloon. Then, the reaction mixture was stirred for 12 h at room temperature. After completion, the reaction mixture was filtered over celite pad, the pad was then washed with methanol. The collected solvent was evaporated under reduced pressure to afforded (2R,3R,4R,5S,6R)-5-acetamido-2-(acetoxymethyl)-6-propyltetrahydro-2H-pyran-3,4-diyl diacetate (3) as colorless gummy liquid. Yield: 2.8 g, 92.0%, crude. LCMS: m/z 374.40 [M+H]. [1283] Synthesis of (2R,3R,4R,5R,6R)-5-amino-2-(hydroxymethyl)-6-propyltetrahydro-2H-pyran- 3,4-diol hydrochloride (4) [1284] (2R,3R,4R,5S,6R)-5-acetamido-2-(acetoxymethyl)-6-propyltetrahydro-2H-pyran-3,4-diyl diacetate (3, 2.6 g,1.0 eq., 6.96 mmol) was taken in 2.5 M HCl solution (45 mL) and was refluxed at 100 °C for 16 h. After completion of reaction (monitored by LCMS), reaction mixture was concentrated under reduced pressure and wash with diethyl ether twice and finally dried under high vacuum to afford (2R,3R,4R,5R,6R)-5-amino-2-(hydroxymethyl)-6-propyltetrahydro-2H-pyran-3,4-diol hydrochloride as light gray solid. Yield: 1.6 g, 96.0%. LCMS: m/z 206.15 [M+H]. [1285] Synthesis of tert-butyl (2-(2-(2-((2-(methylthio)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4d) [1286] To a stirred solution of tert-butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (4c, 1.09 g, 1eq., 4.37 mmol) in dry tetrahydrofuran (10 mL) was added sodium hydride (262 mg, 1.5 eq., 6.56 mmol) portion-wise at 0 °C under inert atmosphere. The resulting suspension was stirred at ambient temperature for 0.5 h. To the resulting reaction mixture a pre-prepared solution of 4-chloro-2- (methylthio)-6-(trifluoromethyl)pyrimidine (4b, 1 g, 1 eq., 4.37 mmol) in dry tetrahydrofuran (4 mL) was added at 0 °C under inert atmosphere. The reaction mixture was stirred at ambient temperature for 10 min. After completion (monitored by TLC), reaction mixture was quenched with ice-cold water (10 mL) and extracted with ethyl acetate (3 × 50 mL). Combined organic phases were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to get crude compound. Then, crude was purified by flash column chromatography using 30-35% ethyl acetate/heptane as eluent to afford tert-butyl(2-(2-(2-((2-(methylthio)-6-(trifluoromethyl)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4d) as colorless gummy liquid. Yield: 1.3 g, 67.3%. LCMS: m/z 442.0 [M+H]. [1287] Synthesis of tert-butyl (2-(2-(2-((2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (XB95-4a) [1288] To a solution of tert-butyl(2-(2-(2-((2-(methylthio)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4d, 1.3 g, 1 eq., 2.94 mmol) in dry dichloromethane (13 mL) was added meta-chloroperbenzoic acid (2.03 g, 3 eq., 8.83 mmol) portion-wise at 0°C under nitrogen atmosphere. The resulting suspension was stirred at ambient temperature under nitrogen atmosphere for 8h. After completion (monitored by TLC and LCMS), reaction mixture was quenched with saturated aqueous solution of sodium hydrogen carbonate (20 mL) and extracted with ethyl acetate (3 × 30 mL). Combined organic phases were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to get crude residue. Then, the crude was purified by combi-flash column chromatography using 45-47% ethyl acetae/heptane as eluent to afford tert-butyl (2-(2-(2-((2- (methylsulfonyl)-6-(trifluoromethyl)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (XB95-4a) as colorless gummy liquid. Yield: 0.8 g, 57.4%. LCMS: m/z 474.05 [M+H].1H-NMR (400 MHz, CDCl3): δ 7.28 (brs, 1H), 4.95 (s, 1H), 4.77-4.75 (m, 2H), 3.95-3.93 (m, 2H), 3.70-3.66 (m, 2H), 3.63-3.61 (m, 2H), 3.53 (t, J = 6.4 Hz, 2H), 3.39 (s, 3H), 3.31 (m, 2H), 1.43 (s, 9H). [1289] Synthesis of tert-butyl (2-(2-(2-((2-(((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)- 2-propyltetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (5) [1290] To a stirred solution of tert-butyl (2-(2-(2-((2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidin- 4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (XB95-4a, 0.553 g, 1 eq., 1.17 mmol) and (2R,3R,4R,5R,6R)-5- amino-2-(hydroxymethyl)-6-propyloxane-3,4-diol hydrochloride (4, 0.311 g, 1.1 eq., 1.28 mmol) in dimethylformamide (5 mL), was added N,N-Diisopropylethylamine (2.44 mL, 12 eq., 14 mmol) at 0 °C and allowed to stir at 120 °C for 16h. After completion (monitored by LMCS), reaction mixture was concentrated under reduced pressure to get crude tert-butyl (2-(2-(2-((2-(((2R,3R,4R,5R,6R)-4,5- dihydroxy-6-(hydroxymethyl)-2-propyltetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (5) (0.9 g) which was directly forwarded to next step. [1291] Synthesis of (2R,3R,4R,5R,6R)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (trifluoromethyl)pyrimidin-2-yl)amino)-2-(hydroxymethyl)-6-propyltetrahydro-2H-pyran-3,4-diol (XB95) [1292] To a suspension of tert-butyl (2-(2-(2-((2-(((2R,3R,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)-2-propyltetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (5) (1.2 g., 1 eq., 2 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (5 ml) at 0 °C and stirred the reaction mixture for 2h at room temperature. After completion of reaction (monitored by LCMS), reaction mixture was concentrated under reduced pressure to get crude which was purified by RP prep-HPLC (20-25% acetonitrile in water with 0.1 TFA) to afford (2R,3R,4R,5R,6R)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6-(trifluoromethyl)pyrimidin-2- yl)amino)-2-(hydroxymethyl)-6-propyltetrahydro-2H-pyran-3,4-diol (XB95) as white solid. Yield: 0.322 g, 32.2 %. LCMS: m/z 499.40 [M+H].1H NMR (400 MHz, DMSO-d6, High temperature): δ 7.65 (brs, 3H), 7.18 (brs, 1H), 6.37 (s, 1H), 4.49-4.47 (m, 2H), 4.35 (brs, 1H), 4.24-4.12 (m, 2H), 3.82-3.73 (m, 4H), 3.64-3.50 (m, 9H), 2.99 (t, J = 5.2 Hz, 2H), 1.68-1.60 (m, 1H), 1.42-1.19 (m, 3H), 0.85 (t, J = 7.2 Hz, 3H). Synthesis of (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)pyrimidin-2-yl)amino)-2- (hydroxymethyl)tetrahydro-2H-pyran-3,4-diol) (XB96)
Figure imgf000392_0001
[1293] Synthesis of tert-butyl (2-(2-(2-((2-(methylthio)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (2) [1294] To a stirred solution of tert-butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (1a, 0.706 g, 1 eq., 2.83 mmol) in tetrahydrofuran (5 mL), sodium hydride (170 mg, 1.5 eq., 4.25 mmol) was added portion wise at 0 °C under N2 atmosphere and allowed to stir at 0 °C for 30 minutes. Then, the resultant solution was added dropwise to a solution of 4-chloro-2-(methylthio)pyrimidine (1, 0.5 g, 1 eq., 2.83 mmol) in tetrahydrofuran (2.5 mL) under N2 atmosphere and allowed to stir for 20 minutes at 0 °C. After completion (monitored by TLC), the reaction was quenched by the addition of ice-cold water, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate and concentrated to get crude. Crude residue was purified by silica gel flash chromatography (eluent: 30% ethyl acetate in hexane) to afforded tert-butyl (2-(2-(2-((2-(methylthio)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (2) as sticky liquid. Yield: 1.0 g, 85.5%. LCMS: m/z 374.05 [M+H]. [1295] Synthesis of tert-butyl (2-(2-(2-((2-(methylsulfonyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (3) [1296] To a stirred solution of tert-butyl (2-(2-(2-((2-(methylthio)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (2, 1.3 g, 1 eq., 3.48 mmol) in dichloromethane (15 mL), meta- chloroperbenzoic acid (1.8 g, 2.3 eq., 7.83 mmol) was added portion wise at 0 °C under N2 atmosphere and allowed to stir at room temperature for 12h. After completion (monitored by LCMS), water (20 mL) was added and extracted with dichloromethane (3 × 15 mL), washed with sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to get crude residue which was purified by silica gel flash chromatography (eluent: 40% ethyl acetate in hexane) to give tert-butyl (2-(2-(2-((2-(methylsulfonyl)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (3) as white solid. Yield: 0.80 g, 56.68%. LCMS: m/z 406.6 [M+H]. [1297] Synthesis of tert-butyl (2-(2-(2-((2-(methylsulfonyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4) [1298] To a stirred solution of tert-butyl (2-(2-(2-((2-(methylsulfonyl)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (3, 0.4 g, 1 eq., 0.987 mmol) and (2R,3R,4R,5S)-5-amino-2- (hydroxymethyl)tetrahydro-2H-pyran-3,4-diol hydrochloride (3a, 0.236 g, 1.2 eq., 1.18 mmol) in N- Methyl-2-pyrrolidone (4 mL), N,N-Diisopropylethylamine (1.72 mL, 10 eq., 9.87 mmol) was added at 0 °C and heated at 150 °C for 12h. After completion (monitored by LCMS), reaction mixture was poured into water and washed with dichloromethane. The aqueous part was lyophilized to give crude tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)amino)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate, which was directly forwarded to next step. Yield: 0.60 g (Crude). LCMS: m/z 489.10 [M+H]. [1299] Synthesis of (2R,3R,4R,5S)-5-((4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)pyrimidin-2- yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB96) [1300] To a stirred solution of tert-butyl (2-(2-(2-((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrimidin-4-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (0.460 g, 1 eq., 941.58 mmol) in dichloromethane (3 mL), trifluoroacetic acid (3 mL) was added at 0 °C and reaction mixture was stirred at room temperature for 3h. Progress of reaction was monitored by LCMS. After completion, reaction mixture was concentrated under reduced pressure to give crude compound was purified by RP prep-HPLC (20-25% acetonitrile in water with 0.1% TFA). Fractions containing the desired product were combined and lyophilized to afford (2R,3R,4R,5S)-5-((4-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)pyrimidin-2-yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB96) as white solid. Yield: 0.15 g, 33.0%; LCMS-MS: m/z 489.20 [M+H].1H NMR (400 MHz, DMSO-d6 with D2O exchange): δ 7.99 (brs, 1H), 6.31 (brs, 1H), 4.47 (brs, 2H), 4.13 (s, 1H), 3.74 (d, J = 2.8 Hz, 3H), 3.66-3.47 (m, 10H), 3.30 (t, J = 5.6 Hz, 1H), 3.09-3.04 (m, 1H), 2.93 (t, J = 4.8 Hz, 2H), 1.23-1.17 (m, 2H). Synthesis of N-((2S,3R,4R,5R,6R)-2-(1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB97)
Figure imgf000393_0001
[1301] Synthesis of N-((2S,3S,4R,5R,6R)-2-(1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-1,2,3- triazol-4-yl)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide (1) [1302] To a solution of N-[(2R,3S,4R,5R,6R)-4,5-dibenzyloxy-6-(benzyloxymethyl)-2-ethynyl- tetrahydropyran-3-yl]acetamide (XB147, 1.00 eq, 46.0 mg, 0.092 mmol) in DMSO (0.25 mL) was added azido-PEG2-amine (1.20 eq, 19.2 mg, 0.11 mmol) and tetrakis(acetonitrile)copper(I) hexafluorophosphate (1.50 eq, 51.5 mg, 0.14 mmol) . The mixture was stirred at rt for 1h and purified by prep. HPLC (5 - 70 % MeCN/water with 0.1% TFA) to give 1 as a white solid (54 mg, yield: 87%). LCMS m/z 674.4 [M+H]. [1303] Synthesis of N-((2S,3R,4R,5R,6R)-2-(1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-1,2,3- triazol-4-yl)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB97) [1304] To a mixture of N-((2S,3S,4R,5R,6R)-2-(1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-1,2,3- triazol-4-yl)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide (1.00 eq, 54.0 mg, 0.080 mmol) in MeOH (4 mL) was added 10% Pd/C (170 mg). The mixture was stirred at rt under hydrogen for 7h, filtered, concentrated, and purified by prep. HPLC (3 - 25% MeCN/ 20 mM NH4OH solution) to give XB97 as a white solid (19.6 mg, yield: 61%). LCMS: 404.3 [M+H]. Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((3-aminopropyl)amino)-5- oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (XB148)
Figure imgf000394_0001
[1305] To the solution of (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran- 2,4,5-triyl triacetate (1.0 eq, 5.05 g, 13.0 mmol) and benzyl N-[3-(5-hydroxypentanamido) propyl]carbamate (CAS: 1637413-93-4, 1.0 eq, 4.00 g, 13.0 mmol) in dichloromethane (50.0 mL), trimethylsilyl trifluoromethanesulfonate (1.1 eq, 2.52 mL, 14.3 mmol) was added dropwise at room temperature. The reaction mixture was stirred at 40 °C for 5 h. After completion, the reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered, and concentrated under high vacuum to get crude. The crude was purified by reverse phase chromatography using 0-30% acetonitrile in water to afford Compound 1 as yellow viscous liquid, Yield: (5.80 g, 70.12%); LCMS m/z 638.2 [M+1]+ [1306] To a solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((3- (((benzyloxy)carbonyl)amino)propyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (1, 1.0 eq, 4.80 g, 7.53 mmol) in methanol (40.0 mL), 10 % Palladium on carbon (1.60 g) was added and stirred at room temperature under hydrogen atmosphere for 4 h. After completion, the reaction mixture was filtered through syringe filter, filtrate was concentrated and dried to get crude. The crude was triturated with diethyl ether to afford Compound XB148 as a pale yellow viscous liquid. Yield: (3.4 g, 80.73%); LCMS m/z 504.37 [M+1]+. [1307] The following compounds were prepared according to the procedures described above using the appropriate starting material.
Figure imgf000395_0001
[1308] The following compounds were prepared according to the procedures described above using the appropriate starting material.
Figure imgf000395_0002
Figure imgf000396_0001
Figure imgf000397_0001
Figure imgf000398_0001
Figure imgf000399_0001
Figure imgf000400_0001
CAS: 190714-38-6
Figure imgf000401_0001
[1309] Synthesis of perfluorophenyl 16-(4-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethyl)piperazin-1-yl)-16-oxo-4,7,10,13-tetraoxahexadecanoate, TFA salt (Int1) [1310] To a suspension of 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione;dihydrochloride (1.00 eq, 202 mg, 0.716 mmol) in 1.5mL of DMF was added Bis-PEG4-PFP ester (1.50 eq, 673 mg, 1.07 mmol) in 1 mL DMF, the mixture was cooled to 0°C and triethylamine (3.00 eq, 0.30 mL, 2.15 mmol) was added dropwise - mixture remains cloudy, partial slurry - stirred at 0°C for 20 min and then let warm to room temp and stirred for 30 min. The mixture was cooled to 0°C, acidified with 165uL TFA (3 eq) in approx. 0.50 mL water, diluted with CH3CN, filtered and purified by preparative HPLC on C18 column (30x250mm) eluting with a gradient of 15-75-100% CH3CN/water + 0.1 %TFA. Collected 343 mg of the desired product as a clear oil after lyophilization of fractions, 63% yield. HPLC: 100% by ELSD, >99% @ 254nm [ 5-99 CH3CN/water+0.1% TFA over 8 min., 0.9 mL/min, Agilent-poroshell 120, C18, 2.7 um, 50x3 mm] LCMS 652.5 [M+H], [10-100 CH3CN/water+0.1% FA over 7 min., 0.9 ml/min, Agilent, poroshell120,C18, 2.1 mmx50mm, 2.7um]; 1H NMR (400 MHz, CDCl3) δ 6.75 (s, 2H), 3.91 (m, 6H), 3.79 (t, J = 5.8 Hz, 2H), 3.71 – 3.56 (m, 14H), 3.39 (t, J = 5.6 Hz, 2H), 3.39 (brs, 4H), 2.97 (t, J = 6.1 Hz, 2H), 2.67 (br s, 2H). LCMS: 652.5 [M+H]
Figure imgf000401_0002
[1311] Synthesis of bis(perfluorophenyl) 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((3-oxo-3- (perfluorophenoxy)propoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate (Int2) [1312] A solution of 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((2-carboxyethoxy)methyl)propane- 1,3-diyl)bis(oxy))dipropionic acid (1.0 eq, 10.0 g, 21.2 mmol) in ethyl acetate (100 mL) was cooled at 0 °C, 2,3,4,5,6-pentafluorophenol (3.0 eq, 11.7 g, 63.6 mmol) and N,N'-diisopropylcarbodiimide (4.0 eq, 13.3 mL, 84.8 mmol) were added and reaction mixture was stirred at room temperature for 16 h. After completion, reaction mixture was filtered through sintered funnel (without celite) and filtrate was concentrated to a crude residue, which was purified by column chromatography using silica gel (100-200 mesh) and 0-15 % ethyl acetate in hexane to afford bis(perfluorophenyl) 3,3'-((2- (((benzyloxy)carbonyl)amino)-2-((3-oxo-3-(perfluorophenoxy)propoxy)methyl)propane-1,3- diyl)bis(oxy))dipropionate (Int2) as an off white solid. Yield: 17.0 g, 82.66 %; LCMS m/z 970.39 [M+H]; 1H NMR (400 MHz, DMSO-d6) δ 7.34-7.26 (m, 5H), 6.58 (s, 1H), 4.95 (s, 2H), 3.75 (t, J = 5.6 Hz, 6H), 3.59 (s, 6H), 2.98 (t, J = 5.6 Hz, 6H). [1313] Synthesis of benzyl (1,31-bis((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-16-(15-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-5-oxo-2,9,12-trioxa-6-azapentadecyl)-11,21-dioxo- 4,7,14,18,25,28-hexaoxa-10,22-diazahentriacontan-16-yl)carbamate [1314] A solution of N-[(2R,3R,4R,5R,6R)-2-[3-[2-(2-aminoethoxy)ethoxy]propyl]-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide (Compound XB48, 3.30 eq, 346 mg, 0.987 mmol) and (2,3,4,5,6-pentafluorophenyl) 3-[2-(benzyloxycarbonylamino)-3-[3-oxo-3-(2,3,4,5,6- pentafluorophenoxy)propoxy]-2-[[3-oxo-3-(2,3,4,5,6- pentafluorophenoxy)propoxy]methyl]propoxy]propanoate (1.00 eq, 290 mg, 0.299 mmol) in DMSO (1.4955 mL) was treated with diisopropylethylamine (DIPEA) (3.00 eq, 156 uL, 0.897 mmol). After 90 minutes, the reaction was diluted with water (500 uL), formic acid (150 uL) and methanol (350 uL) and the reaction was purified by RPHPLC (3-50% ACN in water w/0.1% FA) to give benzyl N-[2-[3-[2-[2- [3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-1,1-bis[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]methyl]ethyl]carbamate as a white solid. Yield: 405 mg, 92%. LCMS m/z 1469.1 [M+H] [1315] Synthesis of N-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethyl]-3-[3-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]-2-[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]-2-amino- propoxy]propenamide [1316] A mixture of benzyl N-[2-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-1,1-bis[[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]ethyl]carbamate (1.00 eq, 390 mg, 0.266 mmol) and 10% Pd/C, Evonik Noblyst (0.700 eq, 396 mg, 0.186 mmol) in methanol (5.3 mL) was stirred vigorously under an atmosphere of nitrogen gas. After 1h, the reaction was filtered through celite, the filter cake was washed with methanol (50mL) and the filtrate was concentrated under reduced pressure to a syrup. The syrup (~1.8mL) was filtered (some darkness of carbon observed) then dripped into EtOAc (8 mL) but a sticky ppt was formed so the slurry was concentrated under reduced pressure to give N-[2- [2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethyl]-3-[3-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-2-[[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]-2-amino-propoxy]propenamide as a white solid. The crude material was used in the next step without further purification. Yield: 326 mg, 92%. LCMS m/z 1335.1 [M+H] [1317] Synthesis of 4-(33-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-18,18-bis(15-((2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-5-oxo-2,9,12-trioxa-6-azapentadecyl)-16,23- dioxo-4,7,10,13,20,27,30-heptaoxa-17,24-diazatritriacontanoyl)-1-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol- 1-yl)ethyl)piperazin-1-ium (Compound 1248) [1318] A mixture of N-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethyl]-3-[3-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]-2-[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]-2-amino- propoxy]propanamide (1.00 eq, 250 mg, 0.187 mmol) and (2,3,4,5,6-pentafluorophenyl) 3-[2-[2-[2-[3-[4- [2-(2,5-dioxopyrrol-1-yl)ethyl]piperazin-1-yl]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]propanoate;2,2,2- trifluoroacetic acid (1.20 eq, 172 mg, 0.225 mmol) in DMF (1.8734 mL) was treated with diisopropylethylamine (4.00 eq, 131 uL, 0.749 mmol) and the reaction was stirred at room temperature. After 19h, another charge of PFP-ester was added (0.24 eq, 34 mg). After 50h, another charge of PFP-ester (0.26 eq, 40mg) was added. After 68h, the reaction was diluted with DMSO (500uL) then purified by RPHLC (2% then 10% then 10-20% ACN in water w/0.1% TFA) to give Compound 1248 as a white solid. Yield: 245mg, 68%. LCMS m/z 902.3 [M+2H]++. HPLC: 99% based on ELSD,
Figure imgf000404_0001
Figure imgf000405_0001
[1319] Synthesis of (Cmpd A) [1320] A solution of perfluorophenyl 1-azido-15-oxo-17,17-bis((3-oxo-3- (perfluorophenoxy)propoxy)methyl)-3,6,9,12,19-pentaoxa-16-azadocosan-22-oate (Int3, 1.0 eq, 72.0 mg, 0.0649 mmol) and DIPEA (9.0 eq, 102 mL, 0.584 mmol) in DMSO (649 mL) was treated with 3- [(2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]sulfanyl-N-(5- aminopentyl)propanamide (XB54, 3.3 eq, 84.3 mg, 0.214 mmol). After 2 h, the reaction was purified by reversed-phase HPLC (10-50% acetonitrile in water w/0.1% TFA) to give (Cmpd A) N-[2-[3-[5-[3- [(2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]sulfanylpropanoylamino]pentylamino]-3-oxo-propoxy]-1,1-bis[[3-[5-[3-[(2S,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]sulfanylpropanoylamino]pentylamino]-3-oxo-propoxy]methyl]ethyl]-3-[2-[2-[2-(2- azidoethoxy)ethoxy]ethoxy]ethoxy]propanamide. Yield: 81 mg, 72%. LCMS m/z 1737.4 [M+H]. [1321] Synthesis of (Cmpd B) [1322] A mixture of Cmpd A (1.0 eq, 42.0 mg, 0.0242 mmol), 10% Pd/C, Evonik Noblyst (0.84 eq, 43.4 mg, 0.0204 mmol) and acetic acid (1.67 mL) was placed under an hydrogen atmosphere via balloon for 1 h. The reaction was filtered through celite then a 0.2 mm syringe filter then concentrated under reduced pressure. The residue was concentrated from water then from MeCN before leaving under high vacuum to give Cmpd B. Yield: 40 mg, 93%. LCMS m/z 1710.8 [M+H]. [1323] Synthesis of (Compound 1255) [1324] A cold solution of perfluorophenyl 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoate(1.2 eq, 5.2 mg, 0.0136 mmol) in DMA (100 mL) was added to a cold solution of Cmpd B (1.0 eq, 20.0 mg, 0.0113 mmol) and DIPEA (4.00 eq, 7.9 mL, 0.0452 mmol) in DMA (100 mL) being cooled in an ice bath. After 90 minutes, the reaction was diluted with DMSO, water and some methanol for rinsing then the crude solution was purified by reversed-phase HPLC (5-50% MeCN in water w/0.1% formic acid) to give Compound 1255. Yield: 10 mg, 48%. LCMS m/z 1910.6 [M+H]. Synthesis of Int3
Figure imgf000406_0001
[1325] Synthesis of tert-butyl 1-azido-17,17-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-15-oxo- 3,6,9,12,19-pentaoxa-16-azadocosan-22-oate (2) [1326] A solution of di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane- 1,3-diyl)bis(oxy))dipropionate (1, CAS: 175724-30-8) (452 mg, 0.894 mmol, 1.00 eq.) and 2,5- dioxopyrrolidin-1-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (1a) (356 mg, 0.916 mmol, 1.02 eq.) in 1 mL acetonitrile was stirred under nitrogen atmosphere and heated at 45C for 2 days until minimal Compound 1 remained. The reaction mixture was diluted with dichloromethane and evaporated onto silica, then purified by flash column chromatography column, eluting with 0-100% ethyl acetate/dichloromethane. Fractions containing product were concentrate and dried further under high vacuum at ambient temperature to afford Compound 2 as a clear thick syrup. Yield: 559 mg (78%); LCMS m/z 779.2 [M+H]. [1327] Synthesis of 1-azido-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16- azadocosan-22-oic acid (3) [1328] To tert-butyl 1-azido-17,17-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-15-oxo-3,6,9,12,19- pentaoxa-16-azadocosan-22-oate (2) (509 mg, 0.654 mmol) was added 10 mL of a pre-mixed solution of 30% trifluoroacetic acid in dichloromethane. The mixture stirred at ambient temperature for 3.5 hrs., until consumption of Compound 2. The solvent was evaporated to residue under a stream of nitrogen, dried further under high vacuum at ambient temperature, then dissolved in 20% water/acetonitrile and lyophilized to dryness to afford Compound 3 as a thick dark yellow color syrup. Yield: 424 mg (99%); LCMS m/z 611.3 [M+H], 609.4 [M-1]-. [1329] perfluorophenyl 1-azido-15-oxo-17,17-bis((3-oxo-3-(perfluorophenoxy)propoxy)methyl)- 3,6,9,12,19-pentaoxa-16-azadocosan-22-oate (Int3) [1330] A solution of 1-azido-17,17-bis((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16- azadocosan-22-oic acid (3) (81.3 mg, 0.133 mmol, 1.0 eq.) and 2,3,4,5,6-pentafluorophenol (3a) (91.8 mg, 0.499 mmol, 3.7 eq.) in 1.5 mL of dichloromethane was added N,N′-diisopropylcarbodiimide (90 mL, 0.581 mmol, 4.4 eq.). The mixture stirred at ambient temperature for 1 hr., until consumption of Compound 3. The reaction mixture was diluted with dichloromethane, evaporated onto silica, then purified by flash column chromatography column, eluting with 0-100% ethyl acetate/hexanes. Fractions containing product were concentrated and dried further under high vacuum at ambient temperature to afford Int3 as a clear thick syrup. Yield: 104 mg (67%); LCMS m/z 1109.1 [M+H].
Synthesis of Compound 1251: General Synthesis, Method C:
Figure imgf000408_0001
Figure imgf000409_0001
[1331] To a mixture of 12-tert-butoxy-12-oxo-dodecanoic acid (1.00 eq, 553 mg, 1.93 mmol) in DMF (9.4 mL) were added Diisopropylethylamine (DIPEA) (4.00 eq, 1.3 mL, 7.72 mmol) and HATU (1.20 eq, 881 mg, 2.32 mmol), followed by addition of 1-(2-aminoethyl)pyrrole-2,5-dione;hydrochloride (1.00 eq, 341 mg, 1.93 mmol). The mixture was stirred at room temperature for 1h, diluted with EtOAc, washed with water (2x) and brine (1x), dried, concentrated, and purified by column (0 - 90% EtOAc/hexane) to give 00A as a white solid (644 mg, yield: 82%). LCMS m/z 431.2 [M + Na]+. [1332] To a mixture of tert-butyl 12-[2-(2,5-dioxopyrrol-1-yl)ethylamino]-12-oxo-dodecanoate (00A, 1.00 eq, 581 mg, 1.42 mmol) in DCM (2 mL) was added TFA (6 mL). The mixture was stirred at room temperature for 2h, concentrated, and lyophilized to give 00B as a white solid (502 mg, yield: 100%). LCMS m/z 353.2 [M+H] [1333] To a mixture of 12-[2-(2,5-dioxopyrrol-1-yl)ethylamino]-12-oxo-dodecanoic acid (00B, 1.00 eq, 502 mg, 1.42 mmol) and2,3,4,5,6-Pentafluorophenol (1.20 eq, 315 mg, 1.71 mmol) in DCM (6 mL) was added 1,3-diisopropylcarbodiimide (1.50 eq, 0.33 mL, 2.14 mmol). The mixture was stirred at room temperature for 2h, filtered, concentrated, purified by column (0 -90% EtOAc/hexane) to give 00C as a white solid (728.5 mg, yield: 99%). LCMS m/z 519.1 [M+H]. [1334] To a mixture of (2,3,4,5,6-pentafluorophenyl) 3-[2-(benzyloxycarbonylamino)-3-[3-oxo-3- (2,3,4,5,6-pentafluorophenoxy)propoxy]-2-[[3-oxo-3-(2,3,4,5,6- pentafluorophenoxy)propoxy]methyl]propoxy]propanoate (Int2, 1.00 eq, 29.0 mg, 0.0299 mmol) and N- [(2R,3R,4R,5R,6R)-2-[5-[2-(2-aminoethoxy)ethoxy]pentyl]-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide;2,2,2-trifluoroacetic acid (XB47, 3.10 eq, 45.7 mg, 0.0927 mmol) in DMSO (0.7 mL) was added DIPEA (10.0 eq, 0.052 mL, 0.299 mmol). The mixture was stirred at room temperature for 1h and was purified by prep. HPLC (2 - 40% MeCN/water with 0.1% TFA) to give 00D as a white solid (42.2 mg, yield: 91%). LCMS m/z 1552.6 [M+H]. [1335] To a mixture of 00D (1.00 eq, 42.2 mg, 0.0272 mmol) in MeOH (4 mL) was added 10% Pd/C (14mg). The mixture was stirred at room temperature under hydrogen for 1h, filtered, concentrated to give 00E as a white solid. (38.8 mg, yield: 100%) LCMS m/z 1418.8 [M+H] [1336] To a mixture of (2,3,4,5,6-pentafluorophenyl) 12-[2-(2,5-dioxopyrrol-1-yl)ethylamino]-12- oxo-dodecanoate (00C, 1.50 eq, 10.1 mg, 0.0196 mmol) in DMF (0.7 mL) were added N-[2-[2-[5- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]pentoxy]ethoxy]ethyl]-3-[3-[3-[2-[2-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]pentoxy]ethoxy]ethylamino]-3-oxo-propoxy]-2-[[3-[2-[2-[5- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]pentoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]-2-amino-propoxy]propanamide (00E, 1.00 eq, 18.5 mg, 0.0130 mmol) and Diisopropylethylamine (DIPEA) (4.00 eq, 0.0091 mL, 0.0522 mmol). The mixture was stirred at room temperature for 5h and more DIPEA (2.5 mL) was added. The mixture was stirred at room temperature overnight. More DIEA (2 mL) was added, and the mixture was stirred at room temperature for 5h and purified by prep. HPLC (2 - 50%MeCN/water with 0.1%TFA) to give Compound 1251 as a white solid (12.8 mg, yield: 56%). LCMS m/z 1753.7 [M+H]. Method D: Synthesis of Compound 2313 from XB48
Figure imgf000410_0001
Figure imgf000411_0001
[1337] Synthesis of tert-butyl 3-(2-(2-(2-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl)sulfonamido)ethoxy)ethoxy)ethoxy)propanoate (1) [1338] To a mixture of 4-(2,5-dioxopyrrol-1-yl)benzenesulfonyl chloride (1.02 eq, 160 mg, 0.59 mmol) in DMF (1.6 mL) at 0 °C was added DIPEA (1.30 eq, 0.13 mL, 0.75 mmol) and a solution of tert- butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate (1.00 eq, 161 mg, 0.58 mmol) in DMF (1.7 mL). The mixture was stirred at 0 °C for 1h then purified by prep HPLC (10 - 80% MeCN/water with 0.1% TFA) to give 1 as a clear syrup. Yield: 174.4 mg, 62%. LCMS m/z 457.0 [M -Boc + H]+. [1339] Synthesis of 3-(2-(2-(2-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl)sulfonamido)ethoxy)ethoxy)ethoxy)propanoic acid (2) [1340] To a mixture of tert-butyl 3-(2-(2-(2-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl)sulfonamido)ethoxy)ethoxy)ethoxy)propanoate (1, 1.00 eq, 90.0 mg, 0.18 mmol) in DCM (1 mL) was added TFA (1 mL). The mixture was stirred at rt for 30 minutes then concentrated and purified by prep HPLC (5 - 70% MeCN/water) to give 2 as clear syrup. Yield: 66 mg, 82%. LCMS m/z 457.1 [M+H]. [1341] Synthesis of benzyl (4-(3-(1H-imidazole-1-carboxamido)propyl)-1,7-bis(1H-imidazole-1- carboxamido)heptan-4-yl)carbamate (3) [1342] To a mixture of carbonyldiimidazole (7.00 eq, 162 mg, 1.00 mmol) in DMSO (0.5 mL) was added a solution of benzyl N-[4-amino-1,1-bis(3-aminopropyl)butyl]carbamate trifluoroacetate salt (3a, 1.00 eq, 97.0 mg, 0.14 mmol) in DMSO (1.3 mL) dropwise. The mixture was stirred at rt for 1h then diluted with EtOAc, washed with water (1x) and the aqueous layer was extracted with EtOAc. The combined organic layer was washed with water (1x) and brine (1x), dried, concentrated, and purified by column (0 -10% MeOH/DCM) to give 3 as clear syrup. Yield: 62.7 mg, 71%. LCMS m/z 619.1 [M+H]. [1343] Synthesis of benzyl (1,31-bis((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-16-(15-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-5-oxo-9,12-dioxa-4,6-diazapentadecyl)-11,21-dioxo- 4,7,25,28-tetraoxa-10,12,20,22-tetraazahentriacontan-16-yl)carbamate (4) [1344] To a mixture of benzyl (4-(3-(1H-imidazole-1-carboxamido)propyl)-1,7-bis(1H-imidazole-1- carboxamido)heptan-4-yl)carbamate (3, 1.00 eq, 93.4 mg, 0.15 mmol) in DMSO (0.8 mL) was added a solution of N-[(2R,3R,4R,5R,6R)-2-[3-[2-(2-aminoethoxy)ethoxy]propyl]-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB48, 3.00 eq, 159 mg, 0.45 mmol) in DMSO (2.2 mL). The mixture was stirred at 45 °C overnight then purified by prep HPLC (5-12-30% MeCN/water with 0.1% formic acid) to give 4 as a white solid. Yield: 129 mg, 58%. LCMS m/z 1465.7 [M+H]. [1345] Synthesis of N-[(2R,3R,4R,5R,6R)-2-[3-[2-[2-[[7-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-4-[3-[2- [2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]-4-amino- heptyl]carbamoylamino]ethoxy]ethoxy]propyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3- yl]acetamide (XB133) [1346] To a mixture of benzyl (1,31-bis((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-16-(15-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-5-oxo-9,12-dioxa-4,6-diazapentadecyl)-11,21-dioxo- 4,7,25,28-tetraoxa-10,12,20,22-tetraazahentriacontan-16-yl)carbamate (4, 1.00 eq, 129 mg, 0.088 mmol) in MeOH (6 mL) was added 10% Pd/C (39 mg). The mixture was stirred at rt under hydrogen for 1h, filtered, concentrated, and purified by prep HPLC (3 - 50% MeCN/20 mM NH4OH aqueous solution) to give XB133 as a white solid. Yield: 104.2 mg, 89%. LCMS m/z 1331.7 [M+H]. [1347] Synthesis of Compound 2313 [1348] To a mixture of 3-[2-[2-[2-[[4-(2,5-dioxopyrrol-1- yl)phenyl]sulfonylamino]ethoxy]ethoxy]ethoxy]propanoic acid (2, 1.10 eq, 3.7 mg, 0.0082 mmol) in DMF (0.25 mL) were added DIEA (3.00 eq, 0.0039 mL, 0.022 mmol) , HATU (1.10 eq, 3.1 mg, 0.0082 mmol) and N-[(2R,3R,4R,5R,6R)-2-[3-[2-[2-[[7-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-4-[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]-4-amino- heptyl]carbamoylamino]ethoxy]ethoxy]propyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3- yl]acetamide (Compound 2313, 1.00 eq, 9.9 mg, 0.0074 mmol) . The mixture was stirred at rt for 30 minutes. To a solution of 3-[2-[2-[2-[[4-(2,5-dioxopyrrol-1- yl)phenyl]sulfonylamino]ethoxy]ethoxy]ethoxy]propanoic acid (0.68 mg) in DMF (0.1 mL) were added DIEA (1 uL) and HATU (0.6 mg). This mixture was added to the above reaction mixture. The mixture was stirred at rt for 30 minutes and purified by prep. HPLC (5 - 13 -25% MeCN/water with 0.1% TFA) to give Compound 2313 as a white solid. Yield: 8.2 mg, 62%. LCMS m/z 1771.1 [M+H]. Synthesis of 3a
Figure imgf000413_0001
[1349] Synthesis of benzyl di-tert-butyl (4-(3-((tert-butoxycarbonyl)amino)propyl)heptane-1,4,7- triyl)tricarbamate (2a) [1350] A solution of di-tert-butyl (4-amino-4-(3-((tert-butoxycarbonyl)amino)propyl)heptane-1,7- diyl)dicarbamate (1a, 1.0 eq, 1.1 g, 2.19 mmol) in 1,4-dioxane (9 mL) and water (3 mL) was cooled at 0 °C. Sodium carbonate (3.0 eq, 0.70 g, 6.56 mmol) and benzyl chloroformate (50 % solution in toluene) (1.5 eq, 1.12 mL, 3.28 mmol) were added and reaction mixture was stirred at room temperature for 16 h. After completion, water was added to the reaction mixture and product extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh, 0-15 % ethyl acetate in hexane) to afford benzyl di-tert-butyl (4-(3-((tert-butoxycarbonyl)amino)propyl)heptane-1,4,7-triyl)tricarbamate (2a) as a colorless viscous liquid. Yield: 1.3 g, 93.3 %; LCMS m/z 637.55 [M+1]+. [1351] Synthesis of benzyl (1,7-diamino-4-(3-aminopropyl)heptan-4-yl)carbamate (3a) [1352] A solution of benzyl di-tert-butyl (4-(3-((tert-butoxycarbonyl)amino)propyl)heptane-1,4,7- triyl)tricarbamate (1, 1.0 eq, 1.3 g, 2.04 mmol) in dichloromethane (13 mL) was cooled at 0 °C. Trifluoroacetic acid (13 mL) was added and reaction mixture was stirred at room temperature for 1 h. After completion, the reaction mixture was concentrated, azeotroped with dichloromethane (2-3 times), dissolved in minimum amount of water, neutralized with ammonia solution (pH~8) and concentrated to get crude which was purified by prep HPLC (15-25 % acetonitrile in water with 0.1 % ammonium hydroxide) to afford 3a as an off white solid. Yield: 0.495 g, 72.1 %; LCMS m/z 337.20 [M+1]+; 1H NMR (400 MHz, DMSO-d6 with D2O & d-TFA) δ 7.34-7.26 (m, 5H), 4.96 (s, 2H), 2.72 (t, J = 6.8 Hz, 6H), 1.62-1.52 (m, 6H), 1.51-1.41 (m, 6H).
Figure imgf000414_0001
[1353] Synthesis of N,N'-(4-(3-(1H-imidazole-1-carboxamido)propyl)-4-(2,2,2- trifluoroacetamido)heptane-1,7-diyl)bis(1H-imidazole-1-carboxamide) (2) [1354] To a solution of N-[4-amino-1,1-bis(3-aminopropyl)butyl]-2,2,2-trifluoro-acetamide;2,2,2- trifluoroacetic acid (1) (57.0 mg, 0.0890 mmol, 1.0 eq.) in 0.6 mL DMSO was added carbonyldiimidazole (108.2 mg, 0.668 mmol, 7.5 eq). The reaction solution was stirred under nitrogen atmosphere and ambient temperature for 1 hr. The reaction mixture was diluted with 1 mL of DMSO and purified by reverse phase HPLC (5-100% acetonitrile in water). Fractions containing desired product were combined and lyophilized to dryness to afford N-[7-(imidazole-1-carbonylamino)-4-[3-(imidazole- 1-carbonylamino)propyl]-4-[(2,2,2-trifluoroacetyl)amino]heptyl]imidazole-1-carboxamide (2) as a white solid. Yield: 55 mg, 106%; LCMS m/z 581.04 [M+H]. [1355] Synthesis of N-[4-[2-[2-[2-[3-[(2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]pyrazol-1-yl]ethoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2- [2-[2-[3-[(2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]pyrazol-1-yl]ethoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]-2,2,2-trifluoro-acetamide (3) [1356] To a stirring solution of N-[(2S,3R,4R,5R,6R)-2-[1-[2-[2-(2- aminoethoxy)ethoxy]ethyl]pyrazol-3-yl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3- yl]acetamide;2,2,2-trifluoroacetic acid (XB84) (21.7 mg, 0.0419 mmol, 3.1 eq.) in 0.2 mL of DMSO and diisopropylethylamine (DIPEA) (14.3 mL, 0.0821 mmol, 6.0 eq.) was added a solution of N-[7- (imidazole-1-carbonylamino)-4-[3-(imidazole-1-carbonylamino)propyl]-4-[(2,2,2- trifluoroacetyl)amino]heptyl]imidazole-1-carboxamide (2) (7.9 mg, 0.014 mmol, 1.0 eq) in 0.1 mL of DMSO. The reaction solution was stirred under a nitrogen atmosphere at 40 °C overnight, then was cooled to ambient temperature, diluted with 1 mL of DMSO, and purified by reverse phase HPLC (1- 40% acetonitrile in water with 0.1% TFA modifier). Fractions containing desired product were combined and lyophilized to dryness to afford N-[4-[2-[2-[2-[3-[(2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]pyrazol-1-yl]ethoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2- [2-[2-[3-[(2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]pyrazol-1-yl]ethoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]-2,2,2-trifluoro-acetamide (3) as a white solid. Yield: 8.7 mg, 40%; LCMS m/z 1583.6 [M+H]. [1357] Synthesis of N-[(2S,3R,4R,5R,6R)-2-[1-[2-[2-[2-[[7-[2-[2-[2-[3-[(2S,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]pyrazol-1- yl]ethoxy]ethoxy]ethylcarbamoylamino]-4-[3-[2-[2-[2-[3-[(2S,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]pyrazol-1- yl]ethoxy]ethoxy]ethylcarbamoylamino]propyl]-4-amino- heptyl]carbamoylamino]ethoxy]ethoxy]ethyl]pyrazol-3-yl]-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide (4) [1358] To a solution of N-[4-[2-[2-[2-[3-[(2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]pyrazol-1-yl]ethoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2- [2-[2-[3-[(2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]pyrazol-1-yl]ethoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]-2,2,2-trifluoro-acetamide (3) (8.7 mg, 0.0055 mmol, 1.0 eq.) in 0.9 mL of methanol was added 0.3 mL of aqueous 5N sodium hydroxide. The reaction stirred at 40 °C overnight, then the solution was concentrated to ~1/4 original volume under a stream of nitrogen. The concentrated crude mixture was diluted with 2 mL of H2O and purified by reverse phase HPLC (2-40% acetonitrile in water with 20 mM ammonium hydroxide modifier). Fractions containing desired product were combined and lyophilized to dryness to afford N-[(2S,3R,4R,5R,6R)-2- [1-[2-[2-[2-[[7-[2-[2-[2-[3-[(2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]pyrazol-1-yl]ethoxy]ethoxy]ethylcarbamoylamino]-4-[3-[2-[2-[2- [3-[(2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]pyrazol-1- yl]ethoxy]ethoxy]ethylcarbamoylamino]propyl]-4-amino- heptyl]carbamoylamino]ethoxy]ethoxy]ethyl]pyrazol-3-yl]-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide (4) as a white solid. Yield: 5.1 mg, 63%; LCMS m/z 1488.4 [M+H]. [1359] Synthesis of Compound 2305 [1360] A solution of 3-[2-[2-[2-[[4-(2,5-dioxopyrrol-1- yl)phenyl]sulfonylamino]ethoxy]ethoxy]ethoxy]propanoic acid (4a) (2.3 mg, 0.0051 mmol, 1.5 eq.) in 0.1 mL of DMF was added HATU (2.1 mg, 0.0056 mmol, 1.7 eq.) and DIPEA (4.0 mL, 0.023 mmol, 7.0 eq.). The solution stirred under nitrogen atmosphere at ambient temperature for ~5 min, then was added to a stirring solution of N-[(2S,3R,4R,5R,6R)-2-[1-[2-[2-[2-[[7-[2-[2-[2-[3-[(2S,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]pyrazol-1- yl]ethoxy]ethoxy]ethylcarbamoylamino]-4-[3-[2-[2-[2-[3-[(2S,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]pyrazol-1- yl]ethoxy]ethoxy]ethylcarbamoylamino]propyl]-4-amino- heptyl]carbamoylamino]ethoxy]ethoxy]ethyl]pyrazol-3-yl]-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide (4) (4.9 mg, 0.0033 mmol, 1.0 eq) dissolved in 1.0 mL of DMF. The reaction solution stirred under nitrogen atmosphere at ambient temperature for 1 hr., then was diluted with 1 mL of DMSO and purified by reverse phase HPLC (5-100% acetonitrile in water with 0.1% TFA modifier). Fractions containing desired product were combined and lyophilized to dryness to afford Compound 2305 as a white solid. Yield: 4.3 mg, 67%; LCMS m/z 1926.5 [M+H]. Synthesis of Compound 2378: General Synthesis Method F:
Figure imgf000417_0001
Figure imgf000418_0001
[1361] Synthesis of benzyl (1,31-bis((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-16-(15-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-5-oxo-2,9,12-trioxa-6-azapentadecyl)-11,21-dioxo- 4,7,14,18,25,28-hexaoxa-10,22-diazahentriacontan-16-yl)carbamate (2) [1362] A mixture of N-((2R,3R,4R,5R,6R)-2-(3-(2-(2-aminoethoxy)ethoxy)propyl)-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB48, 3.24 eq, 1.72 g, 4.91 mmol) and bis(perfluorophenyl) 3,3'-((2-(((benzyloxy)carbonyl)amino)-2-((3-oxo-3- (perfluorophenoxy)propoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate (1.00 eq, 1.47 g, 1.52 mmol) was treated with diisopropylethylamine (1, 3.00 eq, 862 mL, 4.95 mmol) then dissolved in DMSO (6.89 mL) with sonication. After 90 minutes the reaction was purified by reversed-phase HPLC (5% then 15- 50% acetonitrile in water w/0.1% FA) to give 2. Yield: 2.21 g, 91%. LCMS 1468.6 [M+H]. [1363] Synthesis of N-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethyl]-3-[3-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]-2-[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]-2-amino- propoxy]propenamide (XB68A) [1364] A solution of 2 (1.00 eq, 400 mg, 0.272 mmol) in methanol (20 mL) was purged with nitrogen then treated with 10% Pd/C (0.400 eq, 232 mg, 0.109 mmol) before being evacuated then back- filled with hydrogen via balloon. After 2h, the reaction was treated with 500 mg celite then filtered over a pad of celite. The filter cake was rinsed with methanol and the filtrate was concentrated under reduced pressure. The residue was dissolved in water with minimal DMSO then purified by reversed-phase HPLC (5-50% acetonitrile in water w/ 0.2 mM NH4OH) to give XB68A. Yield: 315 mg, 86.7%. LCMS 1335.6 [M+H]. [1365] Synthesis of perfluorophenyl 33-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-18,18-bis(15-((2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-5-oxo-2,9,12-trioxa-6-azapentadecyl)-16,23- dioxo-4,7,10,13,20,27,30-heptaoxa-17,24-diazatritriacontanoate (Compound 2378) [1366] A solution of XB68A (1.00 eq, 1.10 g, 0.824 mmol) in DMF (13.7 mL) was added to a solution of bis(perfluorophenyl) 4,7,10,13-tetraoxahexadecanedioate (3, 3.00 eq, 1.55g, 2.47 mmol) in DMF (5.6 mL) drop-wise via syringe over 16 minutes. Next, DMF (2 mL) was added to the amine flask for rinsing then this solution was added to the reaction dropwise via syringe. The reaction was stirred at room temperature overnight. After 26h, the reaction was purified directly by reversed-phase HPLC (5- 15-40% acetonitrile in water w/0.1% TFA) to give solids that were not easily manipulated. The residue was treated with acetonitrile to remove water via azeotropic distillation. The residue was then dissolved in MeOH (1 volume) then diluted with DCM (95 volumes) then concentrated under high vacuum for 18h to give Compound 2378 as a nice powder. Yield: 1050 mg, 71%. LCMS 1777.58 [M+H]. [1367] The following compounds were prepared according to the procedures disclosed above using the starting amines disclosed above via the general methods indicated, A-F.
Figure imgf000419_0001
[1368] The following compounds were prepared according to the procedures disclosed above using the starting amines disclosed above via the general methods indicated, A-F.
Figure imgf000420_0001
Figure imgf000421_0001
Figure imgf000422_0001
Figure imgf000423_0001
Figure imgf000424_0001
Figure imgf000425_0001
Figure imgf000426_0001
Figure imgf000427_0001
Figure imgf000428_0001
Figure imgf000429_0001
Figure imgf000430_0001
Figure imgf000431_0001
Figure imgf000432_0001
Figure imgf000433_0001
Figure imgf000434_0001
Figure imgf000435_0001
Figure imgf000436_0001
Figure imgf000437_0001
Figure imgf000438_0001
Figure imgf000439_0001
[1369] Compound 1 was synthesized from 6-((2R,3S,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)hexanoic acid, which was prepared by literature routes described in WO 2015/006740A2. [1370] Synthesis of (2) [1371] To a solution of (1) (75 mg, 0.039 mmol) in 5 mL of methanol was added 10% w/w palladium on carbon (24 mg). The mixture was degassed under vacuum, then stirred under hydrogen atmosphere via balloon. After 2.5 hrs., the solution was filtered over Celite and washed with methanol. The filtrate was concentrated to residue and dried further under high vacuum at ambient temperature to afford compound 2 as a white residue. Yield: 65 mg, 93%; LCMS m/z 1787.6 [M+H]. [1372] Synthesis of (3) [1373] To a solution of (2) (36 mg, 0.020 mmol) in 1 mL of methanol, stirring under nitrogen atmosphere at 0-5C, was added sodium methoxide (25% w/w in methanol) (85.5 mg, 0.396 mmol, 19 eq) diluted in 02 mL of methanol Following addition the reaction mixture stirred at ambient temperature for 30 minutes until completion, at which time 0.4 mL of 1N aqueous hydrochloric acid was added slowly to achieve approximate final pH of 1-2. The reaction mixture was diluted further with water and purified by preparatory HPLC, eluting with 1-40% acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford compound 3 as white solid. Yield: 19.2 mg, 68%; LCMS m/z 1409.75 [M+H]. [1374] Synthesis of tert-butyl 1-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-17,17-bis((3-((3-(6-((2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)hexanamido)propyl)amino)-3- oxopropoxy)methyl)-6,12,19-trioxo-15-oxa-7,11,18-triazatriacontan-30-oate (4) [1375] To a solution of 12-(tert-butoxy)-12-oxododecanoic acid (3a) (6.8 mg, 0.024 mmol, 1.8 eq.) in 0.1 mL of dimethylformamide was added N,N-diisopropylethylamine (12 mL, 0.069 mmol, 5.1 eq.) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (9.4 mg, 0.025 mmol, 1.8 eq.). The acid activation reaction mixture stirred at ambient temperature for approximately 10 min, followed by addition of (3) (19.0 mg, 0.0135 mmol, 1.0 eq.) dissolved in 0.3 mL of dimethylformamide. The reaction continued to stir at ambient temperature until completion, at which time the mixture was diluted further with dimethyl sulfoxide and purified by preparatory HPLC, eluting with acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford compound 4 as white solid. Yield: 13.9 mg, 61%; LCMS m/z 1678.7 [M+H]. [1376] Synthesis of 1-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-2-yl)-17,17-bis((3-((3-(6-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)hexanamido)propyl)amino)-3-oxopropoxy)methyl)-6,12,19- trioxo-15-oxa-7,11,18-triazatriacontan-30-oic acid (Compound 1232A) [1377] To tert-butyl 1-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-17,17-bis((3-((3-(6-((2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)hexanamido)propyl)amino)-3- oxopropoxy)methyl)-6,12,19-trioxo-15-oxa-7,11,18-triazatriacontan-30-oate (4) (14 mg, 0.0083 mmol) was added 2 mL of a pre-mixed solution of 30% trifluoroacetic acid in dichloromethane. The mixture stirred at ambient temperature for 1 hr., then solvent was evaporated to residue under a stream of nitrogen. The crude residue was diluted with dimethyl sulfoxide and purified by preparatory HPLC, eluting with acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford compound 1232A, as a residue. Yield: 6.38 mg, 39%; LCMS m/z 1622.7 [M+H]. [1378] Synthesis of N1-(1,33-bis((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-17-((3-((3-(6-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)hexanamido)propyl)amino)-3-oxopropoxy)methyl)- 6,12,22,28-tetraoxo-15,19-dioxa-7,11,23,27-tetraazatritriacontan-17-yl)-N12-(2-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)ethyl)dodecanediamide (Compound 1232) [1379] To a solution of 1-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-17,17-bis((3-((3-(6-((2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)hexanamido)propyl)amino)-3- oxopropoxy)methyl)-6,12,19-trioxo-15-oxa-7,11,18-triazatriacontan-30-oic acid (5) (6.5 mg, 0.0040 mmol, 1.0 eq.) in 0.3 mL of dimethylformamide was added N,N-diisopropylethylamine (5 mL, 0.029 mmol, 7 eq.) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (2.3 mg, 0.0060 mmol, 1.5 eq.). The acid activation reaction mixture stirred at ambient temperature for approximately 10 min, followed by addition of 2-maleimidoethylamine hydrochloride (5a) (0.92 mg, 0.0052 mmol, 1.3 eq.) in one portion as a solid. The mixture stirred at ambient temperature for approximately 20 minutes until completion, then was diluted with dimethyl sulfoxide and acidified with 2 drops of trifluoroacetic acid. The product was isolated by preparatory HPLC, eluting with acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford compound 1232, as a white solid. Yield: 3.3 mg, 65%; LCMS m/z 1745.0 [M+H]. Synthesis of Compound 1915A:
Figure imgf000441_0001
Figure imgf000442_0001
[1380] Synthesis of perfluorophenyl 14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadec-15- yn-1-yl)-4,7,10,22,25,28-hexaoxa-13,18-diazahentriacont-1-yn-31-oate (2) [1381] To a stirred solution of bis(perfluorophenyl) 3,3'-((oxybis(ethane-2,1- diyl))bis(oxy))dipropionate (1, 3.0 eq, 1.16 g, 1.99 mmol) and 4-amino-4-(3-oxo-7,10,13-trioxa-4- azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)heptanediamide (35- 3, 1.0 eq, 0.5 g, 0.662 mmol) in acetonitrile (5.0 mL), N,N-diisopropylethylamine (5.0 eq, 0.578 mL, 3.31 mmol) was added at 0 °C and reaction mixture was stirred at room temperature for 12 h. After completion (monitored by LCMS), the reaction mixture was concentrated under reduced pressure to get crude which was purified by prep HPLC (using 30-45% ACN in H2O with 0.1% TFA) to yield perfluorophenyl 14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-4,7,10,22,25,28- hexaoxa-13,18-diazahentriacont-1-yn-31-oate (2) as a colorless, sticky solid. Yield: 0.450 g, 58.9 %; LCMS m/z 1153.35 [M+H]. [1382] Synthesis of 4-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-oxo-7,10,13-trioxa-3- azahexadecan-16-amido)-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2- yn-1-yloxy)ethoxy)ethoxy)ethyl)heptanediamide (3) [1383] To a stirred solution of perfluorophenyl 14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4- azahexadec-15-yn-1-yl)-4,7,10,22,25,28-hexaoxa-13,18-diazahentriacont-1-yn-31-oate (2, 1.0 eq., 0.028 g 0.0243 mmol) and 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (2a, 1.5 eq., 0.0063 g 0.0364 mmol) in acetonitrile (2 mL) was added N,N-diisopropylethylamine (6.0 eq., 0.00250 mL 0.146 mmol) at 0 °C and then stirred at room temperature for 1.5h After completion (monitored by LCMS), the reaction mixture was concentrated under reduced pressure to get crude which was purified by prep HPLC (5-55% acetonitrile in water with 0.1 % TFA). Fractions containing the desired product were combined and lyophilized to dryness to afford 4-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-oxo-7,10,13-trioxa-3- azahexadecan-16-amido)-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2- yn-1-yloxy)ethoxy)ethoxy)ethyl)heptanediamide (3) as an off white solid. Yield: 0.008 g, 29.7 %; LCMS m/z 1109.25 [M+H]; 1H-NMR (400 MHz, DMSO-d6 with D2O exchange): δ 6.87 (s, 2H), 4.09 (d, J = 2.0 Hz, 6H), 3.56-3.42 (m, 39H), 3.38 (t, J = 5.6 Hz, 6H), 3.25 (t, J = 2.0 Hz, 2H), 3.18-3.14 (m, 8H), 2.28 (t, J = 6.0 Hz, 2H), 2.20 (t, J = 6.4 Hz, 2H), 2.01-1.97 (m, 6H), 1.77-1.74 (m, 6H). [1384] Synthesis of 4-(1-(1-((1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8- dioxabicyclo[3.2.1]octan-4-yl)-1H-1,2,3-triazol-4-yl)-12-oxo-2,5,8-trioxa-11-azatetradecan-14-yl)- N1,N7-bis(2-(2-(2-((1-((1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8- dioxabicyclo[3.2.1]octan-4-yl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethyl)-4-(1-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-4-oxo-7,10,13-trioxa-3-azahexadecan-16-amido)heptanediamide (Compound 1915A) [1385] To a vial was added 8.9 mg (0.0080 mmol) of 4-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- 4-oxo-7,10,13-trioxa-3-azahexadecan-16-amido)-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)- N1,N7-bis(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)heptanediamide, 6.6 mg (3, 0.029 mmol, 3.8 eq) of (1S,2R,3R,4R,5S)-4-azido-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane-2,3-diol (XB39, prepared as in Sanhueza, J. Am. Chem. Soc.2017, 139, 3528−3536) and 0.3 mL DMSO. The mixture stirred at RT until all solids dissolved, then 14.4 mg (0.0386 mmol, 3.9 eq) of tetrakis(acetonitrile)copper(I) hexafluorophosphatewas added. The vial was flushed with nitrogen, and reaction stirred at ambient temperature. After 30 min, the reaction mixture was diluted with DMSO and injected onto prep-LC, eluting with MeCN/H2O (0.1% TFA). Fractions containing desired product were frozen and lyophilized to a white solid to give Compound 1915A. Yield 8.1 mg, 57%. LCMS m/z 1761.3 [M+H].
Figure imgf000444_0001
Figure imgf000445_0001
[1386] Synthesis of (9H-fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4-azahexadec- 15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazatritriaconta-1,32-diyn-17-yl)carbamate (2) [1387] A solution of bis(perfluorophenyl) 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3- oxo-3-(perfluorophenoxy)propyl)heptanedioate (1, 1.0 eq, 1.87 g, 1.93 mmol) and 2-(2-(2-(prop-2-yn-1- yloxy)ethoxy)ethoxy)ethan-1-amine (1a, 3.0 eq, 1.09 g, 5.80 mmol) in N,N-dimethylformamide (18 mL) was stirred at room temperature for 16 h. After completion, water was added to the reaction mixture and the product was extracted with ethyl acetate. The organic layer was washed with water then dried over anhydrous sodium sulfate, filtered and concentrated to give crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford (9H- fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-4,7,10,24,27,30- hexaoxa-13,21-diazatritriaconta-1,32-diyn-17-yl)carbamate (2) as a colorless, viscous liquid. Yield: 1.67 g, 88.4 %; LCMS m/z 977.36 [M+1]+. [1388] To a solution of (9H-fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4- azahexadec-15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazatritriaconta-1,32-diyn-17-yl)carbamate (2, 1.0 eq, 1.57 g, 1.61 mmol) in methanol (20 mL), diethylamine (20.0 eq, 3.36 mL, 32.1 mmol) was added and the reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was concentrated to get crude which was triturated with diethyl ether (2-3 times) to afford 4-amino-4-(3- oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-yn-1- yloxy)ethoxy)ethoxy)ethyl)heptanediamide (35-3) as a light yellow, viscous liquid. Yield: 1.2 g, 98.9 %; LCMS m/z 755.2 [M+1]+. [1389] Synthesis of 4-amino-4-(1-(1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-3-yl)-1H-1,2,3-triazol-4-yl)-12-oxo-2,5,8-trioxa-11-azatetradecan-14-yl)-N1,N7-bis(2-(2-(2- ((1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)-1H-1,2,3-triazol-4- yl)methoxy)ethoxy)ethoxy)ethyl)heptanediamide (XB32) [1390] To a stirred solution of 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7- bis(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)heptanediamide (35-3, 0.3 g, 1.0 eq., 0.397 mmol) and (2R,3R,4R,5S)-5-azido-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB29 (prepared as Stokmaier et al, 2009, Bioorg Med. Chem., pg 7254-7264), 0.271 g, 3.6 eq., 1.43 mmol) in dimethyl sulfoxide (3 mL), tetrakis(acetonitrile)copper(I) hexafluorophosphate (1.11 g, 7.5 eq., 2.98 mmol) was added and the reaction mixture was stirred at room temperature for 30 minutes. After completion (monitored by TLC), acetic acid (0.5 mL) was added and the crude reaction mixture was purified by prep HPLC (10-15 % acetonitrile in water with 0.1 % TFA). Fractions containing the desired product were combined and lyophilized to afford 4-amino-4-(1-(1-((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)-1H-1,2,3-triazol-4-yl)-12-oxo-2,5,8-trioxa-11-azatetradecan- 14-yl)-N1,N7-bis(2-(2-(2-((1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethyl)heptanediamide (XB32) as an off-white solid. Yield: 0.184 g, 35.0%. LCMS m/z 1322.80 [M+H]. 1H NMR (400 MHz, DMSO-d6 with D2O exchange) δ 8.07 (m,3H), 4.70-4.63 (m, 3H), 4.50 (s, 6H), 4.00-3.94 (m, 6H), 3.82 (d, J = 2.8 Hz, 3H), 3.62 (t, J = 10.8 Hz, 3H), 3.54-3.48 (m, 33H), 3.39 (t, J = 6.0 Hz, 6H), 3.18 (bs, 6H), 2.14 (bs, 6H), 1.72 (bs, 6H), [1391] Synthesis of N1-(1,29-bis(1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-3-yl)-1H-1,2,3-triazol-4-yl)-15-(1-(1-((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)-1H-1,2,3-triazol-4-yl)-12-oxo-2,5,8-trioxa-11-azatetradecan- 14-yl)-12,18-dioxo-2,5,8,22,25,28-hexaoxa-11,19-diazanonacosan-15-yl)-N12-(2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)ethyl)dodecanediamide (Compound 1908) [1392] To a solution of 4-amino-4-(1-(1-((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)-1H-1,2,3-triazol-4-yl)-12-oxo-2,5,8-trioxa-11-azatetradecan- 14-yl)-N1,N7-bis(2-(2-(2-((1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethyl)heptanediamide (XB32, 0.210 g, 1.0 eq, 0.159 mmol) and perfluorophenyl 12-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-12- oxododecanoate (2a, 0.124 g, 1.5 eq, 0.238 mmol) in dimethyl sulfoxide (3.0 mL), N,N- diisopropylethylamine (5.0 eq, 0.138 mL, 794 mmol) was added and the reaction mixture was stirred at room temperature for 16h. After completion (monitored by LCMS), reaction mixture was concentrated to afford crude which was purified by prep HPLC (35-65 % acetonitrile in water with 0.05% TFA). Fractions containing the desired compound were combined and lyophilized to afford N1-(1,29-bis(1- ((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)-1H-1,2,3-triazol-4-yl)-15- (1-(1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)-1H-1,2,3-triazol-4- yl)-12-oxo-2,5,8-trioxa-11-azatetradecan-14-yl)-12,18-dioxo-2,5,8,22,25,28-hexaoxa-11,19- diazanonacosan-15-yl)-N12-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)dodecanediamide (Compound 1908) as a white solid. Yield: 0.052 g, 19.7%. LCMS m/z 1654.80 [M-H]; 1H-NMR (400 MHz, DMSO-d6 with D2O): δ 8.06 (s, 3H), 6.87 (s, 2H), 4.66-4.62 (m, 3H), 4.49 (s, 6H), 4.00-3.93 (m, 7H), 3.63 (t, J = 11.6 Hz, 5H), 3.53-3.47 (m, 35H), 3.42 (t, J = 6.4 Hz, 3H), 3.37 (t, J = 6.0 Hz, 6H), 3.18- 3.13 (m, 9H), 3.15-3.13 (m, 6H), 2.01-1.91 (m, 11H), 1.78-1.76 (m, 6H), 1.41-1.37 (m, 5H), 1.21-1.11 (m, 14H). Synthesis of Compound 1905A
Figure imgf000447_0001
Figure imgf000448_0001
[1393] Synthesis of 2-chloro-4-(prop-2-yn-1-yloxy)-6-(trifluoromethyl)pyrimidine (2’) and 4- chloro-2-(prop-2-yn-1-yloxy)-6-(trifluoromethyl)pyrimidine (2’a) [1394] To a stirred solution of prop-2-yn-1-ol (1’a, 0.775 g, 1.0 eq., 13.8 mmol) in diethyl ether (30 mL) and acetonitrile (30 mL) was added sodium hydride (0.398 g, 1.2 eq.,16.56 mmol) portion-wise at 0 °C, and stirred for 30 min at the same temperature. Thereafter, 2,4-dichloro-6- (trifluoromethyl)pyrimidine (1’, 3.0 g, 1.0 eq.,13.8 mmol) was added and allowed to come the temperature to room temperature during 1 h. The reaction mixture was quenched with ice cold water, and extracted with ethyl acetate (3 times). The combined organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain crude which was purified by silica gel flash chromatography using 0-5-% ethyl acetate in hexane to afford an inseparable mixture of 2-chloro-4-(prop-2-yn-1-yloxy)-6-(trifluoromethyl)pyrimidine (2’) and 4-chloro-2-(prop-2-yn-1-yloxy)- 6-(trifluoromethyl)pyrimidine (2’a) as a white solid. Yield: 2.9 g, 89%. 1H NMR (400 MHz, CDCl3) δ 7.35 (s, 0.2 H), 7.06 (s, 1H), 5.09 (d, J = 1.6 Hz, 2H), 5.08 (d, J = 1.6 Hz, 0.40 H), 2.58 (t, J = 1.6 Hz, 1H), 2.52 (t, J = 1.6 Hz, 0.2 H). [1395] Synthesis of (2R,3R,4R,5S)-2-(hydroxymethyl)-5-((4-(prop-2-yn-1-yloxy)-6- (trifluoromethyl) pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3,4-diol (4’) and (2R,3R,4R,5S)-2- (hydroxymethyl)-5-((2-(prop-2-yn-1-yloxy)-6-(trifluoromethyl)pyrimidin-4-yl)amino)tetrahydro-2H- pyran-3,4-diol (5’) [1396] A mixture of 2-chloro-4-(prop-2-yn-1-yloxy)-6-(trifluoromethyl)pyrimidine (2’) and 4- chloro-2-(prop-2-yn-1-yloxy)-6-(trifluoromethyl)pyrimidine (2’a) (1.73 g, 1.2 eq., 7.33 mmol) was dissolved in acetonitrile (25 mL), and then (2R,3R,4R,5S)-5-amino-2-(hydroxymethyl)oxane-3,4-diol hydrochloride (3’, 1.22 g, 1.0 eq., 6.11 mmol) was added followed by N,N-diisopropylethylamine (5.49 mL, 30.6 mmol) and stirred at 70°C for 16 h. Thereafter, the reaction mixture was concentrated under reduced pressure to give crude which was purified by prep-HPLC (37% acetonitrile in water with 0.1% TFA) to afford (2R,3R,4R,5S)-2-(hydroxymethyl)-5-((4-(prop-2-yn-1-yloxy)-6-(trifluoromethyl) pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3,4-diol (4’) as an off-white solid. Yield: 0.193 g, 9%. LCMS m/z 364 [M+H]+; 1HNMR (400 MHz, DMSO-d6 with D2O exchange) δ 6.45 (d, J = 8.8 Hz, 1H), 5.01-4.98 (m, 2H), 4.09 (td, J = 3.2 Hz, 1H), 3.81 (dd, J = 10.8, 5.21H), 3.77 (d, J = 2.4 Hz, 1H), 3.54 (dd, J = 10.8, 3.2 Hz, 1H), 3.48 (d, J = 6.0 Hz, 2H), 3.41 (s, 1H), 3.27 (t, J = 10.8 Hz, 2H), 3.04-2.94 (m, 1H), and (2R,3R,4R,5S)-2-(hydroxymethyl)-5-((2-(prop-2-yn-1-yloxy)-6-(trifluoromethyl)pyrimidin-4- yl)amino)tetrahydro-2H-pyran-3,4-diol (5’), as a white solid. Yield: 0.056 g, 3%. LCMS m/z 364 [M+1]+; 1HNMR (400 MHz, DMSO with D2O exchange) δ:6.57 (s, 1H), 4.96-4.86 (m, 2H), 4.21 (td, J = 10.4, 5.2 Hz, 1H), 3.93 (d, J = 2.4 Hz, 1H), 3.72 (d, J = 2.4 Hz, 1H), 3.53-3.47 (m, 3H), 3.34 (t, J = 2.0 Hz, 1H), 3.29 (t, J = 6 Hz, 1H), 2.97 (t, J = 10.8 Hz, 1H). [1397] Synthesis of perfluorophenyl 1-(4-(((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4-yl)oxy)methyl)-1H- 1,2,3-triazol-1-yl)-13,13-bis(3-((2-(2-(2-(4-(((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4-yl)oxy)methyl)-1H- 1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)amino)-3-oxopropyl)-10,15-dioxo-3,6-dioxa-9,14- diazahexacosan-26-oate (2) [1398] To a solution of perfluorophenyl 1-azido-13,13-bis(3-((2-(2-(2- azidoethoxy)ethoxy)ethyl)amino)-3-oxopropyl)-10,15-dioxo-3,6-dioxa-9,14-diazahexacosan-26-oate (1) (25.2 mg, 0.023 mmol, 1.0 eq.) and (2R,3R,4R,5S)-2-(hydroxymethyl)-5-((4-(prop-2-yn-1-yloxy)-6- (trifluoromethyl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3,4-diol trifluoroacetic acid (4’) (36.3 mg, 0.0761 mmol, 3.3 eq.) in 0.5 mL of dimethyl sulfoxide was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (34.5 mg, 0.0926 mmol, 4.0 eq.) in one portion as a solid. The mixture stirred under a nitrogen atmosphere at ambient temperature for approximately 50 minutes until consumption of (1). The reaction mixture was diluted further with dimethyl sulfoxide and purified by preparatory HPLC, eluting with acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford compound 2 as a white solid. Yield: 35.3 mg, 70%. LCMS m/z 1092.2 [M+2H]++. [1399] Synthesis of N1-(1,25-bis(4-(((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4-yl)oxy)methyl)-1H- 1,2,3-triazol-1-yl)-13-(3-((2-(2-(2-(4-(((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4-yl)oxy)methyl)-1H-1,2,3-triazol-1- yl)ethoxy)ethoxy)ethyl)amino)-3-oxopropyl)-10,16-dioxo-3,6,20,23-tetraoxa-9,17-diazapentacosan-13- yl)-N12-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)dodecanediamide (Compound 1905A) [1400] To a solution of perfluorophenyl 1-(4-(((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4-yl)oxy)methyl)-1H- 1,2,3-triazol-1-yl)-13,13-bis(3-((2-(2-(2-(4-(((2-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)-6-(trifluoromethyl)pyrimidin-4-yl)oxy)methyl)-1H- 1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)amino)-3-oxopropyl)-10,15-dioxo-3,6-dioxa-9,14- diazahexacosan-26-oate (2) (10.2 mg, 0.0047 mmol, 1.0 eq.) and 2-maleimidoethylamine hydrochloride (2a) (1.41 mg, 0.0080 mmol, 1.7 eq.) in 0.4 mL of dimethylformamide was added triethylamine (3.5 mL, 0.025 mmol, 5.3 eq.). The mixture stirred under a nitrogen atmosphere at ambient temperature for approximately 30 minutes until consumption of (2). The reaction mixture was diluted further with dimethyl sulfoxide, acidified by addition of 2 drops of trifluoroacetic acid, and purified by preparatory HPLC, eluting with acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford Compound 1905A as a white solid. Yield: 2.6 mg, 25%. LCMS m/z 1070.8 [M+2H]++. Synthesis of Compound 1234
Figure imgf000450_0001
Figure imgf000451_0001
[1401] Synthesis of tert-butyl 12-chloro-12-oxododecanoate (3) [1402] A solution of 12-(tert-butoxy)-12-oxododecanoic acid (3a, 1.0 eq, 0.200 g, 0.698 mmol) in dichloromethane (2 mL) was cooled at 0 °C, then oxalyl chloride (1.5 eq, 0.032 mL, 1.05 mmol) and DIPEA (0.032 mL) were added and reaction mixture was stirred at room temperature for 1 h. After completion, the reaction mixture was concentrated under nitrogen gas atmosphere and dried to afford tert-butyl 12-chloro-12-oxododecanoate (3) as a light brown, viscous liquid which was directly used as such for the next reaction. Yield: 0.23 g (crude). [1403] Synthesis of (9H-fluoren-9-yl)methyl (1,31-diazido-16-(1-azido-13-oxo-3,6,9-trioxa-12- azapentadecan-15-yl)-13,19-dioxo-3,6,9,23,26,29-hexaoxa-12,20-diazahentriacontan-16-yl)carbamate (2) [1404] To a solution of bis(perfluorophenyl) 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3- oxo-3-(perfluorophenoxy)propyl)heptanedioate (1, 1.0 eq, 1.9 g, 1.96 mmol) in acetonitrile (20.0 mL) was added 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine (1a, 4.0 eq, 1.71 g, 7.84 mmol) followed by N,N-diisopropylethylamine (6.0 eq, 2.08 mL, 11.8 mmol) at 0 °C. The reaction mixture was then stirred at room temperature for 12 h. After completion, reaction mixture was concentrated under reduced pressure to obtain crude which was purified by silica gel flash chromatography using 3-15 % methanol in dichloromethane to afford (9H-fluoren-9-yl)methyl (1,31-diazido-16-(1-azido-13-oxo-3,6,9- trioxa-12-azapentadecan-15-yl)-13,19-dioxo-3,6,9,23,26,29-hexaoxa-12,20-diazahentriacontan-16- yl)carbamate (2) as colorless, sticky solid. Yield: 1.4 g, 66.7 %. LCMS m/z: 1070.51 [M+H]. [1405] Synthesis of 4-amino-4-(1-azido-13-oxo-3,6,9-trioxa-12-azapentadecan-15-yl)-N1,N7-bis(2- (2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)heptanediamide (3b) [1406] To a solution of (9H-fluoren-9-yl)methyl (1,31-diazido-16-(1-azido-13-oxo-3,6,9-trioxa-12- azapentadecan-15-yl)-13,19-dioxo-3,6,9,23,26,29-hexaoxa-12,20-diazahentriacontan-16-yl)carbamate (2, 1.0 eq, 1.4 g, 1.31 mmol) in methanol (10 mL), diethylamine (20.0 eq, 5.0 mL, 47.9 mmol) was added and reaction mixture was stirred at room temperature for 16 h. After completion, reaction mixture was concentrated to get crude which was purified by silica gel column chromatography using 0-15 % methanol in dichloromethane to afford 4-amino-4-(1-azido-13-oxo-3,6,9-trioxa-12-azapentadecan-15-yl)- N1,N7-bis(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)heptanediamide (3b) as a light yellow, viscous liquid. Yield: 0.920 g, 82.9 %. LCMS m/z 848.35 [M+H]. [1407] Synthesis of tert-butyl 1-azido-16,16-bis(1-azido-13-oxo-3,6,9-trioxa-12-azapentadecan-15- yl)-13,18-dioxo-3,6,9-trioxa-12,17-diazanonacosan-29-oate (4) [1408] To a stirred solution of tert-butyl 12-chloro-12-oxododecanoate (3, 2.0 eq, 0.144 g, 0.472 mmol) in dichloromethane, a solution of 4-amino-4-(1-azido-13-oxo-3,6,9-trioxa-12-azapentadecan-15- yl)-N1,N7-bis(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)heptanediamide (3b, 1.0 eq, 0.200 g, 0.236 mmol) and N,N-diisopropylethylamine (5.0 eq, 0.218 mL, 1.18 mmol) in dichloromethane (3.0 mL) was added dropwise at 0 °C and the reaction mixture was stirred at room temperature for 1 h. After completion, water was added to the reaction mixture and product extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to give crude which was purified by silica gel column chromatography using 0-8 % methanol in dichloromethane to afford tert- butyl 1-azido-16,16-bis(1-azido-13-oxo-3,6,9-trioxa-12-azapentadecan-15-yl)-13,18-dioxo-3,6,9-trioxa- 12,17-diazanonacosan-29-oate (4) as a light brown, viscous liquid. Yield: 0.190 g, 72.2 %; LCMS m/z 1116.40 [M+H]. [1409] Synthesis of 1-azido-16,16-bis(1-azido-13-oxo-3,6,9-trioxa-12-azapentadecan-15-yl)-13,18- dioxo-3,6,9-trioxa-12,17-diazanonacosan-29-oic acid (LC44) [1410] To a solution of tert-butyl 1-azido-16,16-bis(1-azido-13-oxo-3,6,9-trioxa-12-azapentadecan- 15-yl)-13,18-dioxo-3,6,9-trioxa-12,17-diazanonacosan-29-oate (4, 1.0 eq, 0.16 g, 0.163 mmol) in dichloromethane (3 mL), trifluoroacetic acid (1.5 mL) was added and reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was concentrated to give crude which was triturated with dichloromethane (2-3 times) to afford 1-azido-16,16-bis(1-azido-13-oxo-3,6,9-trioxa- 12-azapentadecan-15-yl)-13,18-dioxo-3,6,9-trioxa-12,17-diazanonacosan-29-oic acid (5) as a light yellow, viscous liquid. Yield: 0.148 g, 98.0 %. LCMS m/z 1060.35 [M+H].1H NMR (400 MHz, DMSO-d6 with D2O exchange) δ 3.59-3.48 (m, 23H), 3.48 (t, J = 5.6 Hz, 4H), 3.33 (t, J = 4.8 Hz, 4H), 3.16 (t, J = 6.0 Hz, 4H), 3.06 (q, J = 7.6 Hz, 2H), 2.54 (s, 1H), 2.16 (t, J = 6.8 Hz, 1H), 2.03-1.96 (m, 6H), 1.81-1.76 (m, 4H), 1.48-1.39 (m, 4H), 1.23-1.19 (m, 27H). [1411] Synthesis of N1-(1,31-bis(4-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-1-yl)-16-(1-(4-((2R,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-1-yl)-13-oxo- 3,6,9-trioxa-12-azapentadecan-15-yl)-13,19-dioxo-3,6,9,23,26,29-hexaoxa-12,20-diazahentriacontan-16- yl)-N12-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)dodecanediamide (Compound 1234) [1412] To a mixture of 12-[[4-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethylamino]-1,1-bis[3-[2-[2- [2-(2-azidoethoxy)ethoxy]ethoxy]ethylamino]-3-oxo-propyl]-4-oxo-butyl]amino]-12-oxo-dodecanoic acid (5, 1.00 eq, 12.9 mg, 0.0122 mmol) in DMSO (0.3 mL) were added diisopropylethylamine (4.00 eq, 0.0085 mL, 0.0487 mmol) and HATU (1.10 eq, 5.1 mg, 0.0134 mmol), followed by the addition of 2- maleimidoethylamine hydrochloride (1.00 eq, 2.1 mg, 0.0122 mmol). The mixture was stirred at room temperature for 30 minutes and then N-[(2S,3R,4R,5R,6R)-2-ethynyl-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB145, 3.30 eq, 9.2 mg, 0.0402 mmol) was added. The mixture was purged with nitrogen and tetrakis(acetonitrile)copper(I) hexafluorophosphate (6.00 eq, 27.5 mg, 0.0730 mmol) was added. The mixture was stirred at room temperature for 3h then purified by prep HPLC (10- 30% MeCN/water with 0.1%TFA) to give Compound 1234 as a white solid. Yield: 5.5 mg, 24%. LCMS m/z 1870.9 [M+H]. [1413] The following compounds were prepared according to the procedures described above using the appropriate starting material.
Figure imgf000453_0001
[1414] The following compounds were prepared according to the procedures described above using the appropriate starting material.
Figure imgf000453_0002
Synthesis of Compound 1217
Figure imgf000454_0001
[1415] Compound 1217 is synthesized by employing the procedures described for Compound I-168 using LC43 and XB4. LCMS m/z 1396.2 [M+H]. Synthesis of XB4
Figure imgf000454_0002
NaIO i) OsO4 4 Bestmann's reagent acetone: H 2O, RT K CO , MeOH ii) NaIO 2 3 4, acetone: H2O, RT 0 °C to RT
Figure imgf000454_0003
Figure imgf000454_0004
Figure imgf000454_0005
[1416] Synthesis of 2 [1417] To a solution of 0.25 M sodium methoxide (4.95 mL) in methanol (90.0 mL) was added D- galactosamine hydrochloride (1, 1.0 eq, 5.0 g, 23.2 mmol) in small portions. After stirring for 15 minutes, 3,4,5,6-tetrachloropthalic anhydride (1a, 0.6 eq, 4.0 g, 14.0 mmol) was added and the reaction mixture was vigorously stirred for 20 minutes at room temperature. Triethylamine (1.4 eq, 4.5 mL, 32.5 mmol) and a second lot of 3,4,5,6-tetrachloropthalic anhydride (1a, 0.6 eq, 4.0 g, 14.0 mmol) was added and the reaction mixture was stirred at room temperature for 24 h. After completion (monitored by LCMS), methanol was concentrated under reduced pressure to obtain 4,5,6,7-tetrachloro-2-((3R,4R,5R,6R)-2,4,5- trihydroxy-6 (hydroxymethyl)tetrahydro-2H-pyran-3-yl)isoindoline-1,3-dione (2) as orange syrup which was used as such for next step without any further purification. Yield: 10.0 g (crude); LC-MS m/z 447.8 [M+1]+. [1418] Synthesis of 3 [1419] To a solution of 4,5,6,7-tetrachloro-2-((3R,4R,5R,6R)-2,4,5-trihydroxy-6 (hydroxymethyl)tetrahydro-2H-pyran-3-yl)isoindoline-1,3-dione (2, 1.0 eq, 10.0 g, 22.4 mmol) in pyridine (100.0 mL) was added acetic anhydride (15.0 eq, 31.7 mL, 335.5 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. After completion (monitored by TLC), the reaction mixture was poured into ice water and product extracted with dichloromethane. The organic layer was washed subsequently with 5% aqueous hydrochloric acid solution, saturated solution of sodium bicarbonate and water. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain crude. The crude was purified by column chromatography (silica mesh: 100- 200; elution: 30-40% ethyl acetate in hexane) to afford (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(4,5,6,7- tetrachloro-1,3-dioxoisoindolin-2-yl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (3) as white a fluffy solid as anomeric mixture. Yield: 6.8 g, 49.4 %; LC-MS m/z 631.31 [M+17]+. [1420] Synthesis of 4 [1421] To a solution of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(4,5,6,7-tetrachloro-1,3- dioxoisoindolin-2-yl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (3, 1.0 eq., 6.0 g, 9.76 mmol) in acetonitrile (40.0 mL) were added allyltrimethylsilane (4.0 eq, 3.1 mL, 19.5 mmol) followed by boron trifluoride diethyl etherate (4.0 eq, 2.4 mL, 19.5 mmol) and trimethylsilyl trifluoromethanesulfonate (0.3 eq, 0.26 mL, 1.46 mmol) sequentially at 0 °C under nitrogen. The reaction mixture was stirred at room temperature for 16 h. After completion (monitored by TLC), the reaction mixture was poured into ice- cold saturated sodium bicarbonate solution and product extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain crude. The crude was purified by column chromatography (silica mesh: 100-200; elution: 30-40% ethyl acetate in hexane) to afford (2R,3R,4R,5S,6S)-2-(acetoxymethyl)-6-allyl-5-(4,5,6,7-tetrachloro-1,3- dioxoisoindolin-2-yl)tetrahydro-2H-pyran-3,4-diyl diacetate (4) as a yellow liquid. Yield: 4.3 g, 73.8%; LC-MS m/z 598.2 [M+H]+. [1422] Synthesis of 6 [1423] To a solution of (2R,3R,4R,5S,6S)-2-(acetoxymethyl)-6-allyl-5-(4,5,6,7-tetrachloro-1,3- dioxoisoindolin-2-yl)tetrahydro-2H-pyran-3,4-diyl diacetate (4, 1.0 eq, 4.3 g, 7.2 mmol) in ethanol (4.0 mL) was added ethane-1,2-diamine (10.0 eq, 4.8 mL, 72 mmol). The reaction mixture was heated at 80 °C for 8 h. After completion, the reaction mixture was concentrated under reduced pressure and dried to afford (2R,3R,4R,5R,6S)-6-allyl-5-amino-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (5) as crude (3.0 g). To a solution of (2R,3R,4R,5S,6S)-2-(acetoxymethyl)-6-allyl-5-aminotetrahydro-2H-pyran-3,4- diyl diacetate (1.0 eq, 3.0g (crude), 9.1 mmol) in pyridine (30.0 mL) was acetic anhydride (3.0 eq, 2.6 mL, 27.0 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. After completion (monitored by TLC), the reaction mixture was poured onto ice water and extracted with dichloromethane. The organic layer was washed sequentially with 5% aqueous hydrochloric acid solution, a saturated solution of sodium bicarbonate and water. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain crude. The crude was purified by column chromatography (silica mesh: 100-200; elution: 50-60% ethyl acetate in hexane) to afford (2R,3R,4R,5S,6S)-5-acetamido- 2-(acetoxymethyl)-6-allyltetrahydro-2H-pyran-3,4-diyl diacetate (6) as viscous liquid. Yield: 0.84 g (Peak-1) and 0.24 g (Peak-2), 42%; LC-MS m/z 372.1 [M+H]+. [1424] Synthesis of 7 [1425] To a solution of (2R,3R,4R,5S,6S)-5-acetamido-2-(acetoxymethyl)-6-allyltetrahydro-2H- pyran-3,4-diyl diacetate (6, Peak-1, 1.0 eq, 0.78 g, 2.09 mmol) in acetone: water (5:1) (10.0 mL) were added N-methylmorpholine N-oxide (1.5 eq, 0.4 mL, 3.1 mmol) and osmium tetraoxide (4% in water) (0.1 eq, 1.33 mL, 0.210 mmol). The reaction mixture was stirred at room temperature for 2 h. After completion (monitored by TLC), the reaction mixture was diluted with ethyl acetate and the layers were separated. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain crude. The crude was dissolved in acetone: water (2:1) (20 mL) and sodium meta periodate (2.0 eq, 0.9 g, 4.2 mmol) was added. After completion (monitored by TLC), the reaction mixture was diluted with ethyl acetate and the layers were separated. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to afford (2R,3R,4R,5S,6S)-5- acetamido-2-(acetoxymethyl)-6-(2-oxoethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (7) as sticky solid which was used as such for next step without any further purification. Yield: 0.85 g (crude); LCMS: 374.0 [M+1]+. [1426] Synthesis of XB4 [1427] To a solution of (2R,3R,4R,5S,6S)-5-acetamido-2-(acetoxymethyl)-6-(2- oxoethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (0.85 g, 1.0 eq, 2.30 mmol) in methanol (20.0 mL) at 0 °C, were added potassium carbonate (0.950 g, 3.0 eq, 6.89 mmol) and dimethyl (1-diazo-2- oxopropyl)phosphonate (0.88 g, 2.0 eq, 4.59 mmol). The reaction mixture was stirred at room temperature for 3 h. After completion (monitored by TLC), the reaction mixture was concentrated under reduced pressure to obtain crude. The crude was purified by reverse phase preparative HPLC, fractions containing desired product were lyophilized to afford N-((2S,3R,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)-2-(prop-2-yn-1-yl)tetrahydro-2H-pyran-3-yl)acetamide (XB4) as white solid. Yield: 0.13 g, 20.89%; LCMS: 244.0 [M+1]+. Synthesis of Compound 1117 (I-168)
Figure imgf000457_0001
[1428] To a mixture of compound 1-azido-13,18-dioxo-3,6,9-trioxa-12,17-diazanonacosan-29-oic acid (1, 1.00 eq, 14.7 mg, 0.0285 mmol) in DMSO (0.5 mL) were added DIEA (4.00 eq, 0.020 mL, 0.114 mmol) and HATU (1.10 eq, 11.9 mg, 0.0314 mmol), followed by addition of 2-maleimidoethylamine hydrochloride (1.00 eq, 5.0 mg, 0.0285 mmol). The mixture was stirred at room temperature for 30 minutes and to this mixture was added compound XB4B (1.00 eq, 6.9 mg, 0.0285 mmol). The mixture was purged with nitrogen and tetrakis(acetonitrile)copper(I) hexafluorophosphate (2.00 eq, 21.5 mg, 0.0570 mmol) was added. The mixture was stirred at room temperature for 3h. The mixture was purified by prep. HPLC (10 - 40% MeCN/water with 0.1% TFA) to give N1-(1-(4-(((2R,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-1-yl)- 13-oxo-3,6,9-trioxa-12-azahexadecan-16-yl)-N12-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethyl)dodecanediamide (I-168) as a white solid. Yield: 11.8 mg, 47%. LCMS m/z 881.2 [M + H]+.
Synthesis of Compound 1235
Figure imgf000458_0001
[1429] Synthesis of 46A [1430] To a mixture of 12-[[4-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethylamino]-1-[3-[2-[2-[2-(2- azidoethoxy)ethoxy]ethoxy]ethylamino]-3-oxo-propyl]-4-oxo-butyl]amino]-12-oxo-dodecanoic acid (LC43, 1.00 eq, 60.7 mg, 0.0770 mmol) in DCM (0.5 mL) were added 2,3,4,5,6-pentafluorophenol (1.20 eq, 17.0 mg, 0.0924 mmol) and 1,3-diisopropylcarbodiimide (1.50 eq, 0.018 mL, 0.116 mmol). The mixture was stirred at room temperature for 2h, concentrated, and purified by prep HPLC (20 - 80% MeCN/water with 0.1% TFA) to give 46A as a clear solid. Yield: 33 mg, 45%. LCMS m/z 954.4 [M+H]. [1431] Synthesis of Compound 1235 [1432] To a mixture of (2,3,4,5,6-pentafluorophenyl) 12-[[4-[2-[2-[2-(2- azidoethoxy)ethoxy]ethoxy]ethylamino]-1-[3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethylamino]-3-oxo- propyl]-4-oxo-butyl]amino]-12-oxo-dodecanoate (46A, 1.00 eq, 15.6 mg, 0.0164 mmol) and N- [(2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-prop-2-ynyl-tetrahydropyran-3-yl]acetamide (XB4B, 2.30 eq, 9.1 mg, 0.0376 mmol) in NMP (0.25 mL) was added Tetrakis(acetonitrile)copper(I) hexafluorophosphate (4.00 eq, 24.6 mg, 0.0654 mmol). The mixture was stirred at room temperature for 1.5h. The mixture was purified by prep. HPLC (20 - 60% MeCN/water with 0.1% TFA) to give Compound 1235 as a white solid (12.7 mg, Yield: 54%). LCMS m/z 1440.3 [M+H]. Synthesis of Compound 1119 and Compound 2384
Figure imgf000459_0001
[1433] Synthesis of 14A [1434] To a mixture of carbonyldiimidazole (1.30 eq, 18.1 mg, 0.112 mmol) in DMF (0.4 mL) was added a solution of amino-PEG6-t-butyl ester (1.30 eq, 45.7 mg, 0.112 mmol) in DMF (0.4 mL). The mixture was stirred at room temperature for 1h, then N-[(2R,3R,4R,5R,6R)-2-[5-[2-(2- aminoethoxy)ethoxy]pentyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB47, 1.00 eq, 32.5 mg, 0.0859 mmol) was added and more DMF (0.6 mL) was added. The mixture was stirred at room temperature for 5h then purified by prep HPLC (9 - 50% MeCN/water with 0.1%FA) to give 14A, as a white solid. Yield: 55 mg, 79%. LCMS m/z 814.3 [M+H]. [1435] Synthesis of Compound 2384 [1436] To a mixture of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]pentoxy]ethoxy]ethylcarbamoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (14A, 1.00 eq, 55.0 mg, 0.0676 mmol) in DCM (1 mL) was added a solution of TFA (1 mL) and water (100 uL). The mixture was stirred at rt for 30 minutes, concentrated, co-evaporated with water (2x), and lyophilized to give Compound 2384 as a clear syrup. Yield: 54.6 mg, 107%. LCMS: 758.4 [M+H]. [1437] To a mixture of 3-[2-[2-[2-[2-[2-[2-[2-[2-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]pentoxy]ethoxy]ethylcarbamoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (14B, 1.00 eq, 25.5 mg, 0.0336 mmol) in DMF (0.6 mL) at 0 °C was added bis(pentafluorophenyl) carbonate (1.00 eq, 13.3 mg, 0.0336 mmol), followed by the addition of a solution of 4- methylmorpholine (1.00 eq, 0.0037 mL, 0.0336 mmol) in DMF (30 uL). The mixture was stirred at 0 °C for 30 minutes and purified by prep HPLC (10 - 70% MeCN/water with 0.1% TFA) to give Compound 1119 as a white solid. Yield: 17.3 mg, 56%. LCMS m/z 924.4 [M+H]. [1438] The following compounds were prepared according to the procedures described above using the appropriate starting material.
Figure imgf000460_0002
Synthesis of Compound 1120 and Compound 2385
Figure imgf000460_0001
[1439] To a mixture of carbonyldiimidazole (1.40 eq, 10.8 mg, 0.0666 mmol) in DMF (0.2 mL) was added a solution of 2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethanamine (1.40 eq, 20.4 mg, 0.0666 mmol) in DMF (0.3 mL). The mixture was stirred at room temperature for 2h, then N- [(2R,3R,4R,5R,6R)-2-[5-[2-(2-aminoethoxy)ethoxy]pentyl]-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB47, 1.00 eq, 18.0 mg, 0.0476 mmol) was added. The mixture was stirred at room temperature for 5h and was purified by prep HPLC (9 - 50% MeCN/water with 0.1% TFA) to give 05D as a white solid. Yield: 26.8 mg, 79%. LCMS m/z 711.3 [M+H]. [1440] To a mixture of N-[(2R,3R,4R,5R,6R)-2-[5-[2-[2-[2-[2-[2-[2-[2-(2- azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoylamino]ethoxy]ethoxy]pentyl]-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide (05D, 1.00 eq, 30.6 mg, 0.0430 mmol) in MeOH (4 mL) was added 10% Pd/C (13 mg). The mixture was stirred at room temperature under hydrogen for 30 minutes, filtered, concentrated, and purified by prep HPLC (2- 50% MeCN/20mM NH4OH aqueous solution) to give Compound 2385 as a white solid. Yield: 14.9 mg, 66%. LCMS m/z 685.4 [M+H]. [1441] To a mixture of 2,3,4,5,6-Pentafluorophenol (1.20 eq, 300 mg, 1.63 mmol) and 4- Maleimidobenzoic acid (1.00 eq, 295 mg, 1.36 mmol) in DCM (6 mL) was added 1,3- diisopropylcarbodiimide (1.50 eq, 0.32 mL, 2.04 mmol). The mixture was stirred at room temperature for 2h and filtered. The filtrate was concentrated and purified by column (0- 25% EtOAc/hexane) to give 05F as a white solid. Yield: 449 mg, 86%.1H NMR (400 MHz, CDCl3) δ 8.37 – 8.28 (m, 2H), 7.72 – 7.62 (m, 2H), 6.95 (s, 2H). [1442] To a mixture of (2,3,4,5,6-pentafluorophenyl) 4-(2,5-dioxopyrrol-1-yl)benzoate (05F, 1.10 eq, 11.9 mg, 0.0312 mmol) in DMA (0.1 mL) at 0 °C was added a solution of N-[(2R,3R,4R,5R,6R)-2- [5-[2-[2-[2-[2-[2-[2-[2-(2- aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoylamino]ethoxy]ethoxy]pentyl]-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide (05E, 1.00 eq, 19.4 mg, 0.0283 mmol) in DMA (0.3 mL) dropwise. The mixture was stirred at 0 °C for 10 minutes. DIPEA (1.00 eq, 0.0049 mL, 0.0283 mmol) was added. The mixture was stirred at 0 °C for 20 minutes. The mixture was purified by prep HPLC (9 - 40 % MeCN/water with 0.1%formic acid) to give Compound 1120 as a white solid. Yield: 15.5 mg, 62%. LCMS: 884.3. [M+H]. [1443] The following compounds were prepared according to the procedures described above using the appropriate starting material.
Figure imgf000461_0001
Synthesis of XB118
Figure imgf000462_0001
[1444] Synthesis of bis(perfluorophenyl) 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3-oxo- 3-(perfluorophenoxy)propyl)heptanedioate (4) [1445] A solution of 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(2- carboxyethyl)heptanedioic acid (3, 1.0 eq, 8.6 g, 18.3 mmol) in ethyl acetate (80 mL) was cooled at 0 °C, 2,3,4,5,6-pentafluorophenol (3a, 3.0 eq, 10.1 g, 55.0 mmol) and N,N'-diisopropylcarbodiimide (4.0 eq, 11.6 mL, 73.3 mmol) were added and reaction mixture was stirred at room temperature for 16 h. After completion, reaction mixture was filtered through celite bed and celite bed was washed with ethyl acetate. The filtrate was concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-10 % ethyl acetate in hexane to afford bis(perfluorophenyl) 4-((((9H- fluoren-9-yl)methoxy)carbonyl)amino)-4-(3-oxo-3-(perfluorophenoxy)propyl)heptanedioate (4) as a colorless gel. Yield: 9.8 g, 52.3 %; LCMS m/z 968.59 [M+H]. [1446] Synthesis of (9H-fluoren-9-yl)methyl (1,25-diazido-13-(3-((2-(2-(2- azidoethoxy)ethoxy)ethyl)amino)-3-oxopropyl)-10,16-dioxo-3,6,20,23-tetraoxa-9,17-diazapentacosan- 13-yl)carbamate (5) [1447] A solution of bis(perfluorophenyl) 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3- oxo-3-(perfluorophenoxy)propyl)heptanedioate (4, 1.0 eq, 3.70 g, 3.82 mmol) and 2-(2-(2- azidoethoxy)ethoxy)ethan-1-amine (4a, 3.0 eq, 2.0 g, 11.5 mmol) in N,N-dimethylformamide (37 mL) was stirred at room temperature for 16 h. After completion, water was added to the reaction mixture and extracted with ethyl acetate. The organic layer was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford (9H-fluoren-9-yl)methyl (1,25- diazido-13-(3-((2-(2-(2-azidoethoxy)ethoxy)ethyl)amino)-3-oxopropyl)-10,16-dioxo-3,6,20,23-tetraoxa- 9,17-diazapentacosan-13-yl)carbamate (5) as a colorless viscous liquid. Yield: 2.6 g, 72.5 %; LCMS m/z 938.41 [M+H]. [1448] Synthesis of 4-amino-N1,N7-bis(2-(2-(2-azidoethoxy)ethoxy)ethyl)-4-(3-((2-(2-(2 azidoethoxy)ethoxy)ethyl)amino)-3-oxopropyl)heptanediamide (6) [1449] To a solution of (9H-fluoren-9-yl)methyl (1,25-diazido-13-(3-((2-(2-(2- azidoethoxy)ethoxy)ethyl)amino)-3-oxopropyl)-10,16-dioxo-3,6,20,23-tetraoxa-9,17-diazapentacosan- 13-yl)carbamate (5, 1.0 eq, 2.6 g, 2.77 mmol) in methanol (26 mL), diethylamine (20.0 eq, 5.79 mL, 55.4 mmol) was added and reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was concentrated to give crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-15 % methanol in dichloromethane to afford 4-amino-N1,N7-bis(2-(2-(2- azidoethoxy)ethoxy)ethyl)-4-(3-((2-(2-(2 azidoethoxy)ethoxy)ethyl)amino)-3-oxopropyl)heptanediamide (6) as a light yellow, viscous liquid. Yield: 1.7 g, 85.4 %. LCMS m/z 716.2 [M+H]. [1450] Synthesis of N1,N7-bis(2-(2-(2-(4-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)-4-(3-((2- (2-(2-(4-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)amino)-3-oxopropyl)-4-aminoheptanediamide trifluoroacetic acid (XB118) [1451] To a solution of 4-amino-N1,N7-bis(2-(2-(2-azidoethoxy)ethoxy)ethyl)-4-(3-((2-(2-(2- azidoethoxy)ethoxy)ethyl)amino)-3-oxopropyl)heptanediamide (1) (8.7 mg, 0.012 mmol, 1.0 eq.) and N- ((2S,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-(prop-2-yn-1-yl)tetrahydro-2H-pyran-3- yl)acetamide (XB4) (10.7 mg, 0.044 mmol, 3.6 eq.) in 0.2 mL of dimethyl sulfoxide was added tetrakis(acetonitrile)copper(I) hexafluorophosphate(18.6 mg, 0.050, 4.1 eq.) in one portion as a solid. The mixture stirred under nitrogen atmosphere at ambient temperature until completion. The reaction was then diluted with dimethyl sulfoxide and acidified with 2 drops of trifluoroacetic acid. The diluted mixture was purified by preparatory HPLC, eluting with acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford the trifluoroacetic acid salt of XB118 as a white solid. Yield: 10.8 mg, 61%; LCMS m/z 1445.6 [M+H]. Synthesis of Compound 1923 and Compound 1924
Figure imgf000464_0001
Figure imgf000465_0001
[1452] Synthesis of tert-butyl 1-(1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H- pyran-3-yl)-1H-1,2,3-triazol-4-yl)-15,15-bis(1-(1-((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)-1H-1,2,3-triazol-4-yl)-12-oxo-2,5,8-trioxa-11-azatetradecan- 14-yl)-12,17-dioxo-2,5,8,20,23,26,29-heptaoxa-11,16-diazadotriacontan-32-oate (Compound 1924) [1453] To a solution of 2,2-dimethyl-4-oxo-3,7,10,13,16-pentaoxanonadecan-19-oic acid (1a) (13.8 mg, 0.0394 mmol, 2.0 eq) in 0.5 mL of dimethylformamide was added N,N-diisopropylethylamine (20 mL, 0.012 mmol, 5.8 eq.) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (18.4 mg, 0.0484 mmol, 2.4 eq.). The acid activation reaction mixture stirred at ambient temperature for approximately 10 min, followed by addition of a solution containing 4-amino-4- (1-(1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)-1H-1,2,3-triazol-4- yl)-12-oxo-2,5,8-trioxa-11-azatetradecan-14-yl)-N1-(2-(2-(2-((1-((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethyl)-N7-(2- (2-(2-((1-((3S,4S,6S)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)-1H-1,2,3-triazol-4- yl)methoxy)ethoxy)ethoxy)ethyl)heptanediamide (XB32) (26 mg, 0.020 mmol, 1.0 eq.) dissolved in 0.5 mL of dimethylformamide. The reaction continued to stir at ambient temperature for 1 hour until completion, at which time the mixture was diluted further with dimethyl sulfoxide and purified by preparatory HPLC, eluting with acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford Compound 1924 as white solid. Yield: 18.1 mg, 55%; LCMS m/z 1654.5 [M+H]. [1454] Synthesis of 1-(1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)-1H-1,2,3-triazol-4-yl)-15,15-bis(1-(1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-3-yl)-1H-1,2,3-triazol-4-yl)-12-oxo-2,5,8-trioxa-11-azatetradecan-14-yl)-12,17-dioxo- 2,5,8,20,23,26,29-heptaoxa-11,16-diazadotriacontan-32-oic acid (Compound 1923) [1455] To tert-butyl 1-(1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)-1H-1,2,3-triazol-4-yl)-15,15-bis(1-(1-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-3-yl)-1H-1,2,3-triazol-4-yl)-12-oxo-2,5,8-trioxa-11-azatetradecan-14-yl)-12,17-dioxo- 2,5,8,20,23,26,29-heptaoxa-11,16-diazadotriacontan-32-oate (2) (12.4 mg, 0.0075 mmol) was added 2 mL of a pre-mixed solution of 30% trifluoroacetic acid in dichloromethane. The mixture stirred at ambient temperature for 1 hr., then solvent was evaporated to residue under a stream of nitrogen. The crude residue was diluted with dimethyl sulfoxide and purified by preparatory HPLC, eluting with acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford Compound 1923 as a white residue. Yield: 2.37 mg, 20%; LCMS m/z 1598.6 [M+H]. Synthesis of XB34
Figure imgf000466_0001
[1456] Synthesis of (2R,3R,4R)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-3,4-dihydro-2H-pyran- 5-carbaldehyde (2) [1457] To a stirred solution of ((2R,3R,4R)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-3,4-dihydro- 2H-pyran (1, 10 g, 1.0 eq., 24.0 mmol) in dimethylformamide (80 mL) was added phosphorus oxychloride (6.73 mL, 3.0 eq., 72.0 mmol) dropwise at 0 °C and stirred for 5h at room temperature. After completion, the reaction mixture was diluted with cold water and extracted with diethyl ether. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain crude which was purified by silica gel flash chromatography (using 10 % ethyl acetate in hexane) to afford (2R,3R,4R)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-3,4-dihydro-2H-pyran-5-carbaldehyde (2) as a colorless, gummy liquid. Yield: 7.7 g, 58.4 %; LCMS m/z 445.19. [1458] Synthesis of ((4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3- yl)methanol (3) [1459] To a stirred solution of (2R,3R,4R)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-3,4-dihydro- 2H-pyran-5-carbaldehyde (2, 7.7 g, 1.0 eq., 17.3 mmol) in methanol (190 mL) was added nickel dichloride hexahydrate (20.6 g, 5 eq., 86.6 mmol) followed by sodium borohydride (6.55 g, 10 eq., 173.0 mmol) at -78 °C and stirred for 2h. The reaction mixture was then filtered through celite pad and the filtrate was concentrated under reduced pressure to get crude which was purified by silica gel flash chromatography (using 30 % ethyl acetate in hexane) to afford ((4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)methanol (3) as a colorless oil. Yield: 2.5 g, 32.2%. LCMS m/z 449.15 [M+H]. [1460] Synthesis of ((4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3- yl)methyl 4-methylbenzenesulfonate (4) [1461] To a stirred solution of ((4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro- 2H-pyran-3-yl)methanol( 3, 2.5 g, 1 eq., 5.57 mmol) in dichloromethane (15 mL) were added triethylamine (0.874 mL, 2.0 eq., 11.1 mmol) and 4-methylbenzene-1-sulfonyl chloride (1.28 mg, 6.69 mmol, 1.2 eq.) at 0 °C and stirred for 16 h. The reaction mixture was concentrated under reduced pressure to obtain crude which was purified by flash chromatography (silica mesh: 100-200; elution: 50 % ethyl acetate in hexane) to afford ((4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro- 2H-pyran-3-yl)methyl 4-methylbenzenesulfonate (4) as white a solid.. Yield: 1.8 g, 53.6%. LCMS, m/z 603.21 [M+H]. [1462] Synthesis of (2R,3R,4R,5S)-5-(azidomethyl)-3,4-bis(benzyloxy)-2- ((benzyloxy)methyl)tetrahydro-2H-pyran (5) and (2R,3R,4R,5R)-5-(azidomethyl)-3,4-bis(benzyloxy)-2- ((benzyloxy)methyl)tetrahydro-2H-pyran [1463] A solution of ((4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3- yl)methyl 4-methylbenzenesulfonate (4, 1.8 g, 1.0 eq., 2.99 mmol) in N,N-dimethylformamide (15 mL) was treated with sodium azide (0.582 g, 3.0 eq., 8.96 mmol). The suspension was heated at 80°C for 6h. After the completion (monitored by the TLC), the reaction mixture diluted with water and extracted with ethyl acetate. Then organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to give crude which was purified by silica gel flash column chromatography (using 0-30% ethyl acetate in hexane) to afford mixture of (2R,3R,4R,5S)-5-(azidomethyl)-3,4-bis(benzyloxy)-2- ((benzyloxy)methyl)tetrahydro-2H-pyran (5, Peak 1) and (2R,3R,4R,5R)-5-(azidomethyl)-3,4- bis(benzyloxy)-2-((benzyloxy)methyl)tetrahydro-2H-pyran (5, Peak 2). Both isomers were then separated by prep-HPLC using (20% acetonitrile in water with 0.1% TFA). Peak-1 was lyophilized to afford (2R,3R,4R,5S)-5-(azidomethyl)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)tetrahydro-2H-pyran (5, Peak 1) as a sticky liquid. Yield: 0.180 g, 12.85%; LCMS m/z 474.16 [M+H], 1H NMR (400 MHz, DMSO-d6) δ 7.40-7.24 (m, 15H), 4.75 (t, J = 11.2 Hz, 2H), 4.65-4.42 (m, 4H), 4.03 (s, 1H), 3.85-3.82 (m, 1H), 3.57-3.49 (m, 5H), 3.41-3.37 (m, 1H), 3.20 (t, J = 11.2 Hz, 1H), 2.22-2.19 (m, 1H). Similarly, Peak-2 was lyophilized to afford 2R,3R,4R,5R)-5-(azidomethyl)-3,4-bis(benzyloxy)-2- ((benzyloxy)methyl)tetrahydro-2H-pyran (5, Peak 2) as an oily liquid. Yield: 0.210 g 15.00%. LCMS m/z 474.18 [M+H]. 1H NMR (400 MHz, DMSO-d6) δ 7.39-7.26 (m, 15H), 4.87 (d, J = 11.6 Hz, 1H), 4.63 (q, J = 12.0 Hz, 2H), 4.55-4.51 (m, 2H), 4.43 (d, J = 12.0 Hz, 1H), 4.09-4.05 (m, 1H), 3.87-3.81 (m, 1H), 3.79 (s, 1H), 3.76-3.72 (m, 1H), 3.67-3.62 (m, 2H), 3.55-3.51 (m, 2H), 3.43 (d, J = 12 Hz, 1H), 2.10-2.08 (m, 1H). [1464] Synthesis of (2R,3R,4R,5S)-5-(azidomethyl)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4- diol (6) [1465] To a solution of (2R,3R,4R,5S)-5-(azidomethyl)-3,4-bis(benzyloxy)-2- ((benzyloxy)methyl)tetrahydro-2H-pyran (5, Peak 1, 0.140 g, 0.296 mmol, 1.0 eq.) in ethyl acetate (3.0 mL) was added a solution of sodium bromate (NaBrO3) (0.357 g, 2.36 mmol., 8.0 eq.) dissolved in 1 mL water. Thereafter, a solution of sodium dithionite (Na2S2O4) (0.463 g, 2.66 mmol, 7.0 eq.) in water (1.5 mL) was added over 5 minutes and the reaction mixture was vigorously stirred for 12h at room temperature. After completion, volatiles were evaporated to get crude which was purified by prep-HPLC (20-30% acetonitrile in water with 0.1% TFA) to afford (2R,3R,4R,5S)-5-(azidomethyl)-2- (hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (6, Yield: 0.016 g, 26.6%. LCMS m/z 202.1 [M-H] -; and 1H NMR (400 MHz, DMSO-d6 with D2O exchange) δ 3.80 (dd, J = 11.6, 4.4 Hz, 1H), 3.58-3.57 (m, 1H), 3.54 (d, J = 3.2 Hz, 1H), 3.43 (d, J = 6.0 Hz, 2H), 3.29-3.22 (m, 2H), 3.19 (t, J = 6.0 Hz, 1H), 3.07 (t, J = 11.2 Hz, 1H), 2.05-1.98 (m, 1H). [1466] Synthesis of 4-amino-4-(1-(1-(((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-3-yl)methyl)-1H-1,2,3-triazol-4-yl)-12-oxo-2,5,8-trioxa-11-azatetradecan-14-yl)-N1,N7-bis(2- (2-(2-((1-(((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)methyl)-1H-1,2,3- triazol-4-yl)methoxy)ethoxy)ethoxy)ethyl)heptanediamide (XB34) [1467] To a stirred solution of 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7- bis(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)heptanediamide (35-3, 0.030 g,.1 eq,.0.039 mmol) and (2R,3R,4R,5S)-5-(azidomethyl)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol added (6, 3.6 eq, 0.029 g, 0.143 mmol) in dimethyl sulfoxide (1 mL), was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (7.5 eq, 0.11 g, 0.293 mmol) was added and reaction mixture was stirred at room temperature for 30 minutes. After completion (monitored by LCMS) the reaction mixture was purified by prep-HPLC (20% acetonitrile in water, 0.01% TFA). Fractions containing the desired compound were combined and lyophilized to afford 4-amino-4-(1-(1-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)methyl)-1H-1,2,3-triazol-4-yl)-12-oxo-2,5,8-trioxa-11- azatetradecan-14-yl)-N1,N7-bis(2-(2-(2-((1-(((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)methyl)-1H-1,2,3-triazol-4- yl)methoxy)ethoxy)ethoxy)ethyl)heptanediamide (XB34) as colorless, sticky liquid. Yield: 0.02 g, 36.9%; LCMS m/z 1364.65 [M+H].1H NMR (400 MHz, DMSO-d6 with D2O exchange) δ 7.96 (s, 3H), 4.54 (dd, J = 11.2, 3.2 Hz, 3H), 4.49 (s, 6H), 4.21 (dd, J = 14.0, 9.6 Hz, 3H), 3.59 (d, J = 2.4 Hz, 3H), 3.51-3.37 (m, 39H), 3.26 (dd, J = 11.2, 3.2 Hz, 3H), 3.17 (t, J = 5.6 Hz, 9H), 3.05 (t, J = 11.6 Hz, 3H), 2.33-2.13 (m, 9H), 1.77-1.72 (m, 6H). [1468] The following compounds were prepared according to the procedures described above using the appropriate starting material.
Figure imgf000469_0001
Figure imgf000470_0001
Figure imgf000471_0001
Figure imgf000472_0001
Figure imgf000473_0001
Figure imgf000474_0001
Figure imgf000475_0001
Figure imgf000476_0001
Figure imgf000477_0001
Figure imgf000478_0001
Figure imgf000478_0002
Figure imgf000479_0001
Figure imgf000480_0001
Figure imgf000481_0001
Figure imgf000482_0002
Synthesis of Compound 1239
Figure imgf000482_0001
Figure imgf000483_0001
[1470] Synthesis of tert-butyl (1,31-diazido-13,19-dioxo-3,6,9,23,26,29-hexaoxa-12,20- diazahentriacontan-16-yl)carbamate (2) [1471] To a stirred solution of 4-((tert-butoxycarbonyl)amino)heptanedioic acid (1, 0.25 g, 1.0 eq, 0.908 mmol) in N,N-dimethylformamide (4 mL), N,N-diisopropylethylamine (0.79 mL, 5.0 eq, 4.54 mmol) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (1.04 g, 3.0 eq, 2.72 mmol) were added at 0 °C and stirred for 10 minutes. Then 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine (0.47 g, 2.4 eq, 2.18 mmol) in N,N- dimethylformamide (4 mL) was added and reaction mixture was stirred at room temperature for 16 h. After completion of reaction, ice cold water was added to the reaction mixture, and product extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to get crude which was purified by silica gel flash column chromatography using 1-4% methanol in dichloromethane to afford tert-butyl (1,31-diazido-13,19-dioxo- 3,6,9,23,26,29-hexaoxa-12,20-diazahentriacontan-16-yl)carbamate (2) as brown, viscous liquid. Yield: 0.45 g, 73.3%; LCMS m/z 676.15 [M+H]. [1472] Synthesis of 4-amino-N1,N7-bis(2-(2-(2-(2- azidoethoxy)ethoxy)ethoxy)ethyl)heptanediamide (3) [1473] To a stirred solution of tert-butyl (1,31-diazido-13,19-dioxo-3,6,9,23,26,29-hexaoxa-12,20- diazahentriacontan-16-yl)carbamate (2, 0.45 g, 1.0 eq, 0.666 mmol) in dichloromethane (10 mL), trifluroacetic acid (5 mL) was added at 0°C, and the reaction mixture was stirred at room temperature for 2 h. After completion, the reaction mixture was concentrated under reduced pressure to get crude which was triturated thrice with dichloromethane to get 4-amino-N1,N7-bis(2-(2-(2-(2- azidoethoxy)ethoxy)ethoxy)ethyl)heptanediamide (3) as yellow syrup. Yield: 0.40 g, crude; LCMS m/z 576.16 [M+H]. [1474] Synthesis of tert-butyl 1-azido-16-(1-azido-13-oxo-3,6,9-trioxa-12-azapentadecan-15-yl)- 13,18-dioxo-3,6,9-trioxa-12,17-diazanonacosan-29-oate (4) [1475] To a stirred solution of tert-butyl 12-chloro-12-oxododecanoate (0.35 g, 1.50 eq, 1.17 mmol) in dichloromethane (10 mL), a mixture of 4-amino-N1,N7-bis(2-(2-(2-(2- azidoethoxy)ethoxy)ethoxy)ethyl)heptanediamide (3, 0.45 g, 1.0 eq, 0.782 mmol) and N,N- diisopropylethylamine (2.05 mL, 15.0 eq,11.7 mmol) in dichloromethane (5 mL) was added at 0°C and reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was concentrated under reduced pressure to get crude which was purified by silica gel flash column chromatography using 0-10% methanol in dichloromethane to afford tert-butyl 1-azido-16-(1-azido-13- oxo-3,6,9-trioxa-12-azapentadecan-15-yl)-13,18-dioxo-3,6,9-trioxa-12,17-diazanonacosan-29-oate (4) as brown, viscous liquid. Yield: 0.50 g, 85.3%; LCMS m/z 845.15 [M+H]. [1476] Synthesis of 1-azido-16-(1-azido-13-oxo-3,6,9-trioxa-12-azapentadecan-15-yl)-13,18-dioxo- 3,6,9-trioxa-12,17-diazanonacosan-29-oic acid (LC43) [1477] To a stirred solution of tert-butyl 1-azido-16-(1-azido-13-oxo-3,6,9-trioxa-12-azapentadecan- 15-yl)-13,18-dioxo-3,6,9-trioxa-12,17-diazanonacosan-29-oate (4, 0.650 g, 0.770 mmol) in dichloromethane (10 mL), trifluoroacetic acid (3 mL) was added at 0°C and reaction mixture was stirred at room temperature for 2 h. After the completion, the reaction mixture was concentrated under reduced pressure to get crude which was triturated thrice with dichloromethane to afford 1-azido-16-(1-azido-13- oxo-3,6,9-trioxa-12-azapentadecan-15-yl)-13,18-dioxo-3,6,9-trioxa-12,17-diazanonacosan-29-oic acid (LC43) as brownish semi-solid. Yield: 0.22 g, 36.7%; LCMS m/z 788.50 [M+H]; 1H-NMR (400 MHz, DMSO-d6 with D2O) δ 3.69-3.48 (m, 21H), 3.38-3.32 (m, 8H), 3.15 (d, J = 5.6 Hz, 4H), 2.15 (t, J = 7.2 Hz, 2H), 2.03-1.99 (m, 6H), 1.61-.158 (m, 2H), 1.50-1.44 (m, 6H), 1.19 (s, 12H). [1478] Synthesis of 1-(4-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-1-yl)-16-(1-(4-(((2R,3R,4R,5R,6R)- 3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-1-yl)- 13-oxo-3,6,9-trioxa-12-azapentadecan-15-yl)-13,18-dioxo-3,6,9-trioxa-12,17-diazanonacosan-29-oic acid (Compound 1239): [1479] To a mixture of 12-[[4-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethylamino]-1-[3-[2-[2-[2-(2- azidoethoxy)ethoxy]ethoxy]ethylamino]-3-oxo-propyl]-4-oxo-butyl]amino]-12-oxo-dodecanoic acid (LC43, 1.00 eq, 6.3 mg, 0.00800 mmol) and N-[(2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2- prop-2-ynyl-tetrahydropyran-3-yl]acetamide (XB4B, 2.20 eq, 4.3 mg, 0.0176 mmol) in NMP (0.2 mL) was added tetrakis(acetonitrile)copper(I) hexafluorophosphate (4.00 eq, 12.0 mg, 0.0320 mmol). The mixture was stirred at room temperature for 1h. The mixture was purified by prep. HPLC (10 - 50% MeCN/water with 0.1% TFA) to give Compound 1239 as a white solid (8.5 mg, yield: 83%). LCMS m/z 1274.7 [M+H]. Synthesis of Compound 1243
Figure imgf000485_0001
[1480] Compound 1243 is synthesized by employing the procedures described for compound 1239 using LC44 in lieu of LC43 and XB145 in lieu of compound XB4B LCMS m/z 1748.9 [M+H].
Figure imgf000485_0002
[1481] Compound XB75 is synthesized by employing the procedures described for compound XB74B using 1-azido-17-((2-carboxyethoxy)methyl)-15-oxo-3,6,9,12,19-pentaoxa-16-azadocosan-22- oic acid in lieu of azido-PEG4-amido-tri-(carboxyethoxymethyl)-methane. LCMS m/z 1203.8 [M+H]. Synthesis of XB67
Figure imgf000485_0003
[1482] Compound XB67 is synthesized by employing the procedures described for compound 00E (in Compound 1251, General Method C) using compound XB48 in lieu of compound XB47. LCMS m/z 900.9 [M+H]. [1483] The following compounds were prepared according to the procedures described above using the appropriate starting material.
Figure imgf000486_0001
Figure imgf000487_0002
[1484] The following compounds were prepared according to the procedures described above using the appropriate starting material.
Figure imgf000487_0001
Synthesis of XB10A and XB144
Figure imgf000488_0001
[1485] To a mixture of tert-butyl N-[2-(2-prop-2-ynoxyethoxy)ethyl]carbamate (1.00 eq, 204 mg, 0.838 mmol) in acetone (10 mL) were added NBS (1.34 eq, 200 mg, 1.13 mmol) and silver nitrate (0.144 eq, 20.5 mg, 0.121 mmol). The mixture was stirred at room temperature for 1h and concentrated. The residue was diluted with EtOAc and washed with water. The aqueous layer was extracted with EtOAc (2x). The combined organic layer was washed with brine, dried, concentrated, and purified by column (0 - 50% EtOAc/hexane) to give 36A as a clear oil. Yield: 220 mg, 82%. LCMS m/z 344.0 [M + Na]+, 222 [M -Boc + H]+. [1486] To a mixture of N-[(2R,3R,4R,5R,6R)-2-ethynyl-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB44A, 1.00 eq, 12.0 mg, 0.0523 mmol) in water (1 mL) were added tert-butyl N-[2-[2-(3-bromoprop-2-ynoxy)ethoxy]ethyl]carbamate (36A, 1.10 eq, 18.6 mg, 0.0576 mmol) and piperidine (5.00 eq, 0.026 mL, 0.262 mmol). The mixture was purged with nitrogen, cooled to 0 ˚C and CuCl (0.1000 eq, 1.0 mg, 0.00523 mmol) was added. The mixture was stirred at 0 ˚C for 1h and at room temperature for 2h. MeCN (0.5 mL) was added then the mixture was purged with nitrogen and cooled to 0 ˚C. More tert-butyl N-[2-[2-(3-bromoprop-2- ynoxy)ethoxy]ethyl]carbamate (14.7 mg) and CuCl (1.5 mg) were added. The mixture was stirred at 0 ˚C for 1h and at room temperature overnight. The mixture was purified by prep HPLC (2 - 50% MeCN/water with 0.1%TFA) to give XB10A as a white solid. Yield: 12 mg, 49%. LCMS: 470.9 [M+H]. [1487] To a mixture of tert-butyl N-[2-[2-[5-[(2R,3S,4R,5R,6R)-3-acetamido-4,5-dibenzyloxy-6- (benzyloxymethyl)tetrahydropyran-2-yl]penta-2,4-diynoxy]ethoxy]ethyl]carbamate (XB10A, 1.00 eq, 8.7 mg, 0.0117 mmol) in MeOH (4mL) was added 10% Pd/C (5 mg). The mixture was stirred at rt under hydrogen for 2h. More 10% Pd/C (5 mg) was added and the mixture was stirred at rt under hydrogen for 1h. The mixture was filtered, and the filtrate was concentrated and purified by prep HPLC (5 -50% MeCN/water with 0.1% TFA) to give XB144 as a white solid. Yield: 3.3 mg, 59%. LCMS: 479.0 [M+H]. Synthesis of XB44A
Figure imgf000489_0001
[1488] Synthesis of N-((2R,3R,4R,5R,6R)-2-ethynyl-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-3-yl)acetamide (XB44A) [1489] To a mixture of N-[(2R,3S,4R,5R,6R)-4,5-dibenzyloxy-6-(benzyloxymethyl)-2-ethynyl- tetrahydropyran-3-yl]acetamide which was prepared by literature routes described in Rouzier, et al, Synthesis 2019, 51, A–E, (1.00 eq, 55.4 mg, 0.111 mmol) in DCM (2.9 mL) at -10 ˚C was added boron trichloride solution in DCM (10.5 eq, 1.2 mL, 1.16 mmol) dropwise. The mixture was stirred at rt for 45 minutes and cooled to 0 ˚C. The reaction was quenched with sat. NaHCO3. The mixture was concentrated to remove organic solvent. The aqueous solution was purified by prep. HPLC (2 -10 % MeCN/water with 0.1% TFA) to give XB44A as a white solid. Yield: 25 mg, 98%. LCMS m/z 230.2 [M+H]. Synthesis of XB145
Figure imgf000489_0002
[1490] N-((2S,3R,4R,5R,6R)-2-ethynyl-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)acetamide was synthesized analogously to example XB44A from intermediate 4a of that example. LCMS: 230.0 [M+H] [1491] The following compounds were prepared according to the procedures described above using the appropriate starting material.
Figure imgf000489_0003
Synthesis of Compound 2307
Figure imgf000490_0001
Synthesis of N-(1,7-diamino-4-(3-aminopropyl)heptan-4-yl)-2,2,2-trifluoroacetamide (6a)
Figure imgf000490_0002
[1492] Synthesis of 4-(3-aminopropyl)-4-nitroheptane-1,7-diamine (2a) [1493] A solution of 4-(2-cyanoethyl)-4-nitroheptanedinitrile (1a, 5.0 g, 22.7 mmol) in dry tetrahydrofuran (25 mL) was cooled to 0 °C. To this, borane-tetrahydrofuran complex (1M in THF, 114 mL, 5 eq., 114 mmol) was added slowly and the resulting reaction mixture was heated at 80 °C for 24h. After completion, the reaction mixture was allowed to attain room temperature and conc. hydrochloric acid solution was added dropwise to quench excess borane complex. The resultant reaction mixture was heated to 50 °C for 30 min and then concentrated under reduced pressure. The residue was neutralized by adding 40% aqueous sodium hydroxide solution (80 mL) and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulphate, filtered and the filtrate was concentrated under reduced pressure to obtain 4-(3-aminopropyl)-4-nitroheptane-1,7-diamine (2a) as an off-white solid. Yield: 5.0 g, crude. LCMS: m/z 233.2 [M+H]. [1494] Synthesis of di-tert-butyl (4-(3-((tert-butoxycarbonyl)amino)propyl)-4-nitroheptane-1,7- diyl)dicarbamate (3a) [1495] To a solution of 4-(3-aminopropyl)-4-nitroheptane-1,7-diamine (2a, 8 g, 34.4 mmol) in methanol (150 mL), were added di-tert-butyl dicarbonate (26.1 mL, 3.3 eq., 114 mmol) and triethylamine (20.2 mL, 4.2 eq., 145 mmol) at 0 °C and the resultant reaction mixture was refluxed for 6 h. Progress of the reaction was monitored by TLC and LCMS. After completion, the reaction mixture was concentrated under reduced pressure to get crude which was purified by flash chromatography using silica gel (100- 200 mesh; 20-40% ethyl acetate-heptane) to afford di-tert-butyl (4-(3-((tert- butoxycarbonyl)amino)propyl)-4-nitroheptane-1,7-diyl)dicarbamate (3a) as a colourless oil (which turns glassy solid when cooled at -20 °C). Yield: 9.0 g, 49.1%; LCMS: m/z 533.2 [M+H]. [1496] Synthesis of di-tert-butyl (4-amino-4-(3-((tert-butoxycarbonyl)amino)propyl)heptane-1,7- diyl)dicarbamate (4a) [1497] A suspension of nickel dichloride hexahydrate (7.14 g, 2 eq., 30 mmol) in methanol (80 mL) was cooled to 0 °C. To this, sodium borohydride (1.55 g, 2.6 eq., 5 mmol) was added and the resulting black suspension was stirred for 30 min. Thereafter, the black suspension was further diluted with methanol (30 mL) followed by addition of di-tert-butyl (4-(3-((tert-butoxycarbonyl)amino)propyl)-4- nitroheptane-1,7-diyl)dicarbamate (3a, 8.0 g, 15 mmol) and sodium borohydride (1.55 g, 2.6 eq., 5 mmol). The resulting black suspension was stirred for 60 min. Subsequently, a last portion of sodium borohydride (1.55 g, 2.6 eq., 5 mmol) was added and the reaction mixture was stirred at room temperature for 4 h. After completion, the black suspension was filtrated through a celite bed, and the filtrate was concentrated under reduced pressure. The residue was diluted by adding water and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulphate, filtrated,filtered and the filtrate was concentrated under reduced pressure to afford di-tert-butyl (4-amino-4-(3-((tert- butoxycarbonyl)amino)propyl)heptane-1,7-diyl)dicarbamate (4a) as a white solid. Yield: 6.0 g, 79.5%; LCMS: m/z 503.2 [M+H]. 1H NMR (400 MHz, DMSO-d6 with D2O): δ 2.83 (t, J = 6.80 Hz, 6H), 1.34 (s, 27H), 1.32-1.28 (m, 6H), 1.14-1.12 (m, 6H). [1498] Synthesis of di-tert-butyl (4-(3-((tert-butoxycarbonyl)amino)propyl)-4-(2,2,2- trifluoroacetamido)heptane-1,7-diyl)dicarbamate (5a) [1499] A solution of di-tert-butyl (4-amino-4-(3-((tert-butoxycarbonyl)amino)propyl)heptane-1,7- diyl)dicarbamate (4a, 1.0 g, 1.99 mmol) and triethylamine (1.39 mL, 5.0 eq., 9.95 mmol) in dry dichloromethane (10 mL) was cooled to 0 °C. To this, trifluoroacetic anhydride (1.25 mL, 3.0 eq., 5.97 mmol) was added slowly, and the resulting reaction mixture was stirred at room temperature for 12 h. Progress of the reaction was monitored by TLC and LCMS. After completion, the reaction mixture was concentrated under reduced pressure to obtain crude. The crude was purified by flash chromatography using silica gel (100-200 mesh, eluent: 10-20% ethyl acetate-heptane) to afford di-tert-butyl (4-(3-((tert- butoxycarbonyl)amino)propyl)-4-(2,2,2-trifluoroacetamido)heptane-1,7-diyl)dicarbamate (5a) as a colorless, viscous liquid. Yield: 0.75 g, 63.0%; LCMS: m/z 599.45 [M+H]. [1500] Synthesis of N-(1,7-diamino-4-(3-aminopropyl)heptan-4-yl)-2,2,2-trifluoroacetamide (6a) [1501] To a stirred solution of di-tert-butyl (4-(3-((tert-butoxycarbonyl)amino)propyl)-4-(2,2,2- trifluoroacetamido)heptane-1,7-diyl)dicarbamate (5a, 0.75 g, 1.25 mmol) in dichloromethane (8 mL), was added trifluoroacetic acid (3 mL) at 0 °C. The resultant reaction mixture was stirred at room temperature for 3 h. Progress of the reaction was monitored by TLC and LCMS. After completion, the reaction mixture was concentrated under reduced pressure to obtain crude. The was purified by reverse phase prep-HPLC (30-40 % acetonitrile in water with 0.1 % ammonium hydroxide). Fractions containing the desired product were combined and lyophilized to dryness to afford N-(1,7-diamino-4-(3- aminopropyl)heptan-4-yl)-2,2,2-trifluoroacetamide (6a) as an off-white solid. Yield: 0.070 g, 18.7%; LCMS m/z 299.25 [M+H]. 1H-NMR (400 MHz, DMSO-d6 with D2O): δ 3.13 (t, J = 6.72 Hz, 1H), 2.85 (bs, 2H), 1.70-1.53 (m, 6H), 1.49-1.40 (m, 1H), 1.27-1.21 (m , 10H). Synthesis of 14-((4-nitro-N-(tetrahydro-2H-pyran-4-yl)phenyl)sulfonamido)-3,6,9,12- tetraoxatetradecanoic acid (4b)
Figure imgf000492_0001
[1502] Synthesis of tert-butyl 14-((tetrahydro-2H-pyran-4-yl)amino)-3,6,9,12- tetraoxatetradecanoate (2b) [1503] To a stirred solution of tert-butyl 14-amino-3,6,9,12-tetraoxatetradecanoate (1b, 2 g, 6.51 mmol) in methanol (16 mL) and dichloromethane (4 mL) were added tetrahydro-4H-pyran-4-one (1b’, 0.717 g, 1.1 eq., 7.16 mmol), acetic acid (0.0372 mL, 0.1 eq., 0.651 mmol) and stirred for 1h at room temperature. Then sodium cyanoborohydride (0.467 g, 1.2 eq., 7.81 mmol) was added and stirred for another 4h. After completion, that reaction mixture was concentrated and portioned between water and ethyl acetate. The ethyl acetate part was dried over anhydrous sodium sulphate, filtered, and concentrated to give crude which was purified by reverse phase prep HPLC (70-75 % acetonitrile in water with 0.1 %TFA). Fractions containing desired product were combined and lyophilized to dryness to afford tert- butyl 14-((tetrahydro-2H-pyran-4-yl)amino)-3,6,9,12-tetraoxatetradecanoate (2b) as sticky solid. Yield: 0.8 g, 32%; LCMS m/z 392.85 [M+H]. [1504] Synthesis of tert-butyl 14-((4-nitro-N-(tetrahydro-2H-pyran-4-yl)phenyl)sulfonamido)- 3,6,9,12-tetraoxatetradecanoate (3b) [1505] To a stirred solution of tert-butyl 14-((tetrahydro-2H-pyran-4-yl)amino)-3,6,9,12- tetraoxatetradecanoate (2b, 0.85 g, 1 eq., 2.17 mmol ) in tetrahydrofuran (9.0 mL) was added triethylamine (3.05 mL, 10 eq., 21.7 mmol) and 4-nitrobenzenesulfonyl chloride (2.41 g, 5.0 eq., 10.9 mmol) at 0°C and stirred the reaction mixture at 30°C for 3 h. Progress of reaction was monitored by TLC After completion reaction mixture was diluted with water and extracted with dichloromethane to afford crude which was purified by silica gel column chromatography (60% ethyl acetate/hexane) to afford tert-butyl 14-((4-nitro-N-(tetrahydro-2H-pyran-4-yl)phenyl)sulfonamido)-3,6,9,12- tetraoxatetradecanoate (3b) as yellow sticky liquid. Yield: 0.66 g, 52.72%; LCMS m/z 577.05 [M+H]. [1506] Synthesis of 14-((4-nitro-N-(tetrahydro-2H-pyran-4-yl)phenyl)sulfonamido)-3,6,9,12- tetraoxatetradecanoic acid (4b) [1507] To a solution of tert-butyl 14-((4-nitro-N-(tetrahydro-2H-pyran-4-yl)phenyl)sulfonamido)- 3,6,9,12-tetraoxatetradecanoate (3b, 0.660 g, 1.14 mmol) in dichloromethane (3 mL) was added hydrogen chloride solution (3 ml, 4M in 1,4-dioxane) dropwise at 0 °C and reaction was stirred at same temperature for 3h. After completion, reaction mixture was concentrated and co-distilled with dichloromethane to afford crude which was purified by reverse phase prep HPLC (80% acetonitrile in water with 0.1% TFA). Fractions containing desired product were combined and lyophilized to dryness to afford 14-((4-nitro-N-(tetrahydro-2H-pyran-4-yl)phenyl)sulfonamido)-3,6,9,12-tetraoxatetradecanoic acid (4b) as colorless viscous liquid. Yield: 0.475 g, 80%. LCMS m/z 519.4 [M-1].1H NMR (400 MHz, DMSO-d6) δ 12.57 (s, 1H), 8.36 (d, J = 8.8 Hz, 2H), 8.14 (d, J = 8.8 Hz, 2H), 3.99 (s, 2H), 3.88-3.84 (m, 1H), 3.79 (dd, J = 4.0, 11.2 Hz, 2H), 3.57-3.48 (m, 15H), 3.29-3.26 (m, 3H), 1.74-1.64 (m, 2H), 1.36- 1.34 (m, 2H).
Figure imgf000493_0001
Figure imgf000494_0001
[1508] Synthesis of N,N'-(4-(3-(1H-imidazole-1-carboxamido)propyl)-4-(2,2,2- trifluoroacetamido)heptane-1,7-diyl)bis(1H-imidazole-1-carboxamide) (2) [1509] A solution of N-[4-amino-1,1-bis(3-aminopropyl)butyl]-2,2,2-trifluoro-acetamide;2,2,2- trifluoroacetic acid (6a) (57.0 mg, 0.0890 mmol, 1.0 eq.) in 0.6 mL DMSO was added carbonyldiimidazole (108.2 mg, 0.668 mmol, 7.5 eq). The reaction solution stirred under nitrogen atmosphere and ambient temperature for 1 hr. The reaction mixture was diluted with 1 mL of DMSO and purified by reverse phase HPLC (5-100% acetonitrile in water). Fractions containing desired product were combined and lyophilized to dryness to afford N-[7-(imidazole-1-carbonylamino)-4-[3-(imidazole- 1-carbonylamino)propyl]-4-[(2,2,2-trifluoroacetyl)amino]heptyl]imidazole-1-carboxamide (2) as a white solid. Yield: 55 mg (106%); LCMS m/z 581.04 [M+H]. [1510] Synthesis of N-[4-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]-2,2,2-trifluoro-acetamide (3) [1511] To a stirring solution of N-[(2R,3R,4R,5R,6R)-2-[3-[2-(2-aminoethoxy)ethoxy]propyl]-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB48) (67.3 mg, 0.192 mmol, 3.4 eq.) in 0.6 mL of DMSO was added a solution of N-[7-(imidazole-1-carbonylamino)-4-[3-(imidazole-1- carbonylamino)propyl]-4-[(2,2,2-trifluoroacetyl)amino]heptyl]imidazole-1-carboxamide (2) (32.7 mg, 0.0564 mmol, 1.0 eq) in 0.3 mL of DMSO. The reaction solution stirred under nitrogen atmosphere at 40C for 3 hrs. The reaction mixture was diluted with 1 mL of DMSO and purified by reverse phase HPLC (5-40% acetonitrile in water with 0.1% TFA modifier). Fractions containing desired product were combined and lyophilized to dryness to afford N-[4-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2- [2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]-2,2,2-trifluoro-acetamide (3) as a white solid. Yield: 65.3 mg (81%); LCMS m/z 1427.4 [M+H]. [1512] Synthesis of N-[(2R,3R,4R,5R,6R)-2-[3-[2-[2-[[7-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-4-[3-[2- [2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]-4-amino- heptyl]carbamoylamino]ethoxy]ethoxy]propyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3- yl]acetamide (4) [1513] To a solution of N-[4-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]-2,2,2-trifluoro-acetamide (3) (53.9 mg, 0.0378 mmol, 1.0 eq.) in 0.9 mL of methanol was added 0.4 mL of aqueous 5N sodium hydroxide. The reaction stirred at 40C overnight, then the solution was concentrated to ~1/4 original volume under a stream of nitrogen. The concentrated crude mixture was diluted with 2 mL of H2O and purified by reverse phase HPLC (2-40% acetonitrile in water with 20mM ammonium hydroxide modifier). Fractions containing desired product were combined and lyophilized to dryness to afford N-[(2R,3R,4R,5R,6R)-2-[3-[2-[2- [[7-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]-4-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]-4-amino- heptyl]carbamoylamino]ethoxy]ethoxy]propyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3- yl]acetamide (4) as a white solid. Yield: 40.3 mg (75%); LCMS m/z 1331.6 [M+H]. [1514] Synthesis of N-[4-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]-2-[2-[2-[2-[2-[(2-nitrophenyl)sulfonyl- tetrahydropyran-4-yl-amino]ethoxy]ethoxy]ethoxy]ethoxy]acetamide (5) [1515] A solution of 2-[2-[2-[2-[2-[(4-nitrophenyl)sulfonyl-tetrahydropyran-4-yl- amino]ethoxy]ethoxy]ethoxy]ethoxy]acetic acid (4b) (19.7 mg, 0.0378 mmol, 1.2 eq.) in 0.2 mL of DMF was added [dimethylamino(triazolo[4,5-b]pyridin-3-yloxy)methylene]-dimethyl- ammonium;hexafluorophosphate (HATU) (15.1 mg, 0.0397 mmol, 1.3 eq.) and diisopropylethylamine (DIPEA) (16.1 mL, 0.0924 mmol, 3.0 eq.). The solution stirred under nitrogen atmosphere at ambient temperature for ~5 min, then was added to a stirring solution of N-[(2R,3R,4R,5R,6R)-2-[3-[2-[2-[[7-[2- [2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]-4-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]-4-amino- heptyl]carbamoylamino]ethoxy]ethoxy]propyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3- yl]acetamide (4) (40.3 mg, 0.0303 mmol, 1.0 eq) dissolved in 0.7 mL of DMF. The reaction solution stirred under nitrogen atmosphere at ambient temperature for 1 hr. The reaction mixture was diluted with 1 mL of DMSO and purified by reverse phase HPLC (5-100% acetonitrile in water with 0.1% TFA modifier). Fractions containing desired product were combined and lyophilized to dryness to afford N-[4- [2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]- 2-[2-[2-[2-[2-[(2-nitrophenyl)sulfonyl-tetrahydropyran-4-yl- amino]ethoxy]ethoxy]ethoxy]ethoxy]acetamide (5) as a white solid. Yield: 38.3 mg (69%); LCMS m/z 1834.5 [M+H]. [1516] Synthesis of N-[4-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]-2-[2-[2-[2-[2-(tetrahydropyran-4- ylamino)ethoxy]ethoxy]ethoxy]ethoxy]acetamide (6) [1517] To a solution of N-[4-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]-2-[2-[2-[2-[2-[(2-nitrophenyl)sulfonyl- tetrahydropyran-4-yl-amino]ethoxy]ethoxy]ethoxy]ethoxy]acetamide (5) (36.6 mg, 0.0200 mmol, 1.0 eq.) in 1.5 mL of methanol was added K2CO3 (42.1 mg, 0.305 mmol, 15.3 eq.) and 2-thioglycolic acid (7 mL, 0.100 mmol, 5.0 eq.). The reaction solution stirred under nitrogen atmosphere at ambient temperature for 4 hrs. The reaction solution was concentrated under a stream of nitrogen to yellow solid. The concentrated crude solid was diluted with 2 mL of H2O and purified by reverse phase HPLC (2-40% acetonitrile in water with 20mM ammonium hydroxide modifier). Fractions containing desired product were combined and lyophilized to dryness to afford N-[4-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2- [2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]-2-[2-[2-[2-[2-(tetrahydropyran-4- ylamino)ethoxy]ethoxy]ethoxy]ethoxy]acetamide (6) as a white solid. Yield: 26.9 mg (82%); LCMS m/z 1649.6 [M+H]. [1518] Synthesis of N-[4-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]-2-[2-[2-[2-[2-[(2-bromoacetyl)-tetrahydropyran- 4-yl-amino]ethoxy]ethoxy]ethoxy]ethoxy]acetamide (Compound 2307) [1519] A solution of N-[4-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]-2-[2-[2-[2-[2-(tetrahydropyran-4- ylamino)ethoxy]ethoxy]ethoxy]ethoxy]acetamide (6) (11.3 mg, 0.0068 mmol, 1.0 eq.) in 0.3 mL of dimethylacetamide (DMA) was stirred under nitrogen atmosphere at ~-20C to form a white slurry, then a solution of 2-bromoacetic anhydride (2.4 mg, 0.0092 mmol, 1.3 eq.), dissolved in 0.2 mL of DMA, was added dropwise over 2 min. The reaction slurry continued to stir cold while warming to ambient temperature to form a clear solution. After 40 min., the reaction mixture was diluted with 1 mL of DMA and purified by reverse phase HPLC (2-40% acetonitrile in water with 0.1% TFA modifier). Fractions containing desired product were combined and lyophilized to dryness to afford N-[4-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]-1,1-bis[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]butyl]- 2-[2-[2-[2-[2-[(2-bromoacetyl)-tetrahydropyran-4-yl-amino]ethoxy]ethoxy]ethoxy]ethoxy]acetamide (Compound 2307) as a white solid. Yield: 5.6 mg (46%); LCMS m/z 1770.4 [M+H].
Figure imgf000498_0001
Figure imgf000499_0001
[1520] Synthesis of benzyl N-[2-[2-[2-[2-(imidazole-1- carbonylamino)ethoxy]ethoxy]ethoxy]ethyl]carbamate (Int3) [1521] To a mixture of Carbonyldiimidazole (1.10 eq, 115 mg, 0.71 mmol) in DMSO (0.4 mL) was added a solution of CbzNH-PEG3-CH2CH2NH2 (1.00 eq, 210 mg, 0.64 mmol) in DMSO (1.2 mL) dropwise. The mixture was stirred at rt for 2h, diluted with EtOAc, washed with water (1x), and the aqueous layer was extracted with EtOAc. The combined organic layer was washed with water (1x) and brine (1x), dried, and concentrated, and purified by column (0 -10% MeOH/DCM) to give Int3 as clear syrup (179 mg, yield: 66%). LCMS m/z 420.9 [M+H]. [1522] Synthesis of benzyl (30-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-15,15-bis(15-((2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-5-oxo-9,12-dioxa-4,6-diazapentadecyl)-13,20- dioxo-3,6,9,24,27-pentaoxa-12,14,19,21-tetraazatriacontyl)carbamate (2) [1523] To N-[(2R,3R,4R,5R,6R)-2-[3-[2-[2-[[7-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-4-[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]-4-amino- heptyl]carbamoylamino]ethoxy]ethoxy]propyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3- yl]acetamide (XB133, 1.00 eq, 50.0 mg, 0.038 mmol) was added a solution of benzyl N-[2-[2-[2-[2- (imidazole-1-carbonylamino)ethoxy]ethoxy]ethoxy]ethyl]carbamate (Int3, 1.80 eq, 28.4 mg, 0.068 mmol) in DMSO (0.7 mL). The mixture was stirred at 45 °C overnight and purified by prep. HPLC (5 - 12 - 30% MeCN/water with 0.1% formic acid), and re-purified by prep. HPLC (5 - 12 - 30% MeCN/water with 0.1% TFA) to give 2 as a white solid (45 mg, yield: 71%). LCMS m/z 1683.8 [M+H]. [1524] Synthesis of intermediate Int1 [1525] To a mixture of benzyl (30-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-15,15-bis(15-((2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-5-oxo-9,12-dioxa-4,6-diazapentadecyl)-13,20- dioxo-3,6,9,24,27-pentaoxa-12,14,19,21-tetraazatriacontyl)carbamate (2, 1.00 eq, 45.0 mg, 0.027 mmol) in MeOH (4 mL) was added 10% Pd/C (14 mg). The mixture was stirred at rt under hydrogen for 1h, filtered, concentrated, and purified by prep. HPLC (3 - 50% MeCN/20 mM NH4OH solution) to give Int1 as a white solid (31.9 mg, yield: 77%). LCMS m/z 1549.8 [M+H]. [1526] Synthesis of Compound 2314 [1527] To a mixture of 4-Maleimidobenzoic acid (1.15 eq, 2.2 mg, 0.010 mmol) in DMF (0.1 mL) were added DIPEA (3.00 eq, 0.0046 mL, 0.026 mmol) and HATU (1.15 eq, 3.8 mg, 0.010 mmol), and a solution of intermediate Int1 (1.00 eq, 13.6 mg, 0.0088 mmol) in DMF (0.2 mL). The mixture was stirred at rt for 30 minutes and purified by prep. HPLC (3 - 14 - 27% MeCN/water with 0.1% TFA) to give Compound 2314 as a white solid (12.8 mg, yield: 83%). LCMS m/z 1749.5 [M+H]. Synthesis of Compound 2315
Figure imgf000500_0001
[1528] Synthesis of Compound 2315 [1529] To a mixture of 4-(2,5-dioxopyrrol-1-yl)benzenesulfonyl chloride (1.05 eq, 1.9 mg, 0.0068 mmol) in DMF (0.05 mL) at 0 °C were added DIPEA (1.40 eq, 0.0016 mL, 0.0091 mmol) and a mixture of Int1 (1.00 eq, 10.1 mg, 0.0065 mmol) in DMF (0.2 mL). The mixture was stirred at 0 °C for 20 minutes and purified by prep. HPLC (3-13-25% MeCN/water with 0.1% formic acid) to give Compound 2315 as a white solid (7.9 mg, yield: 68%). LCMS 1785.8 [M+H]. Synthesis of Compound 2321
Figure imgf000501_0001
[1530] Synthesis of Compound 2321 [1531] To a mixture of 2,5-dioxypyrrolidin-1-ul 2-bromoacetate (1.49 eq, 2.3 mg, 0.0098 mmol) in DMF (0.2 mL) at 0 °C was added a mixture of Int1 (1.00 eq, 10.2 mg, 0.0066 mmol) in DMF (0.3 mL). The mixture was stirred at 0 °C for 30 minutes and purified by prep. HPLC (3-10-24% with 0.1% formic acid) and re-purified by prep. HPLC (3-10-24% MeCN/water with 0.1%TFA) to give Compound 2321 as a white solid (5.5 mg, yield: 50%). LCMS: 1671.7 [M+H]. Synthesis of Compound 2316
Figure imgf000502_0001
[1532] Synthesis of benzyl N-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethyl]- N-methyl-carbamate (1) [1533] To a mixture of tert-butyl N-[2-[2-[2-[2- (methylamino)ethoxy]ethoxy]ethoxy]ethyl]carbamate (1.00 eq, 198 mg, 0.65 mmol) in DMF (0.4 mL) was added a solution of benzyl 2,5-dioxopyrrolidin-1-yl carbonate (1.30 eq, 210 mg, 0.84 mmol) in DMF (1 mL). The mixture was stirred at rt for 2h and purified by prep. HPLC (20-100% MeCN/water with 0.1% formic acid) to give 1 as clear syrup (192 mg, yield: 67%). LCMS 341.3 [M -Boc + H]+. [1534] Synthesis of benzyl N-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]-N-methyl-carbamate (2) [1535] To a mixture of benzyl N-[2-[2-[2-[2-(tert- butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethyl]-N-methyl-carbamate (1, 1.00 eq, 282 mg, 0.64 mmol) in DCM (1 mL) was added TFA (1 mL). The mixture was stirred at rt for 1h and concentrated. The residue was basified with sat. NaHCO3 and extracted with DCM (3x). The combined organic layer was dried, concentrated to give 2 as yellow syrup (230 mg, yield: 105%). LCMS m/z 341.2 [M+H]. [1536] Synthesis of intermediate Int2 [1537] Intermediate Int2 was synthesized by employing the procedures described for intermediate Compound 2321-Int1 using benzyl N-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]-N-methyl- carbamate (2) in lieu of CbzNH-PEG3-CH2CH2NH2. LCMS m/z 1564.8 [M+H]. [1538] Synthesis of Compound 2316 [1539] Compound 2316 was synthesized by employing the procedures described for Compound 2321 using intermediate Int2 in lieu of intermediate Compound 2321-Int1. LCMS m/z 1685.7 [M+H]. Synthesis of Compound 2317
Figure imgf000503_0001
[1540] Synthesis of tert-butyl 3-[2-[2-[2-[[4-(2,5-dioxopyrrol-1- yl)benzoyl]amino]ethoxy]ethoxy]ethoxy]propanoate (1) [1541] To a mixture of 4-Maleimidobenzoic acid (1.00 eq, 102 mg, 0.47 mmol) in DMF (0.6 mL) at 0 °C were added HATU (1.10 eq, 196 mg, 0.52 mmol) and Diisopropylethylamine (DIPEA) (2.00 eq, 0.16 mL, 0.94 mmol) and a solution of tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate (1.00 eq, 130 mg, 0.47 mmol) in DMF (0.4 mL). The mixture was stirred at 0 °C for 20 minutes and was purified by prep. HPLC (10 - 60% MeCN/water with 0.1% TFA) to give 1 as yellow syrup (211 mg, yield: 95%). LCMS 477.6 [M+H]. [1542] Synthesis of 3-[2-[2-[2-[[4-(2,5-dioxopyrrol-1- yl)benzoyl]amino]ethoxy]ethoxy]ethoxy]propanoic acid (2) [1543] To a mixture of tert-butyl 3-[2-[2-[2-[[4-(2,5-dioxopyrrol-1- yl)benzoyl]amino]ethoxy]ethoxy]ethoxy]propanoate (1, 1.00 eq, 211 mg, 0.44 mmol) in DCM (1 mL) was added TFA (1mL). The mixture was stirred at rt for 30 minutes, concentrated, and purified by prep. HPLC (10 - 70 % MeCN/water) to give 2 as clear syrup (103 mg, yield: 55%). LCMS: 421.5 [M+H]. [1544] Synthesis of N-[(2R,3R,4R,5R,6R)-2-[5-[2-[2-[3-[3-[3-[2-[2-[5-[(2R,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]pentoxy]ethoxy]ethylcarbamoylamino]propoxy]-2-[3-[2-[2-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]pentoxy]ethoxy]ethylcarbamoylamino]propoxymethyl]-2-amino- propoxy]propylcarbamoylamino]ethoxy]ethoxy]pentyl]-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB130) [1545] Intermediate XB130 was synthesized by employing the procedures described for intermediate XB133 using benzyl (1,3-bis(3-aminopropoxy)-2-((3-aminopropoxy)methyl)propan-2- yl)carbamate; 3TFA salt in lieu of benzyl N-[4-amino-1,1-bis(3-aminopropyl)butyl]carbamate;3TFA salt and using N-((2R,3R,4R,5R,6R)-2-(5-(2-(2-aminoethoxy)ethoxy)pentyl)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB47) in lieu of N-[(2R,3R,4R,5R,6R)-2-[3-[2- (2-aminoethoxy)ethoxy]propyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB48). LCMS m/z 1505.5 [M+H]. [1546] Synthesis of Compound 2317 [1547] Compound 2317 was synthesized by employing the procedures described for Compound 2313 using 3-[2-[2-[2-[[4-(2,5-dioxopyrrol-1-yl)benzoyl]amino]ethoxy]ethoxy]ethoxy]propanoic acid (2) in lieu of 3-(2-(2-(2-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl)sulfonamido)ethoxy)ethoxy)ethoxy)propanoic acid and using N-[(2R,3R,4R,5R,6R)-2-[5-[2- [2-[3-[3-[3-[2-[2-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran- 2-yl]pentoxy]ethoxy]ethylcarbamoylamino]propoxy]-2-[3-[2-[2-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]pentoxy]ethoxy]ethylcarbamoylamino]propoxymethyl]-2-amino- propoxy]propylcarbamoylamino]ethoxy]ethoxy]pentyl]-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB130) in lieu of N-[(2R,3R,4R,5R,6R)-2-[3-[2-[2- [[7-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]-4-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]-4-amino- heptyl]carbamoylamino]ethoxy]ethoxy]propyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3- yl]acetamide (XB133). LCMS m/z 1908.5 [M+H]. Synthesis of Compound 2318
Figure imgf000505_0001
Figure imgf000506_0001
Compound 2318 [1548] Synthesis of tert-butyl N-[4-[2-[2-[2-[2- (benzyloxycarbonylamino)ethoxy]ethoxy]ethoxy]ethylcarbamoylamino]-7-(tert-butoxycarbonylamino)- 4-methyl-heptyl]carbamate (1) [1549] To a mixture of benzyl N-[2-[2-[2-[2-(imidazole-1- carbonylamino)ethoxy]ethoxy]ethoxy]ethyl]carbamate (Int3, 1.46 eq, 51.2 mg, 0.12 mmol) in DMSO (0.25 mL) was added tert-butyl N-[4-amino-7-(tert-butoxycarbonylamino)-4-methyl-heptyl]carbamate (LC42, 1.00 eq, 30.0 mg, 0.083 mmol) and DIPEA (1.00 eq, 0.015 mL, 0.083 mmol). The mixture was stirred at 50 °C overnight and at 60 °C for 5h and purified by prep. HPLC (20 - 80% MeCN/water with 0.1% formic acid) to give 1 as clear syrup (41.6 mg, yield: 70%). LCMS m/z 712.2 [M+H]. [1550] Synthesis of benzyl N-[2-[2-[2-[2-[[4-amino-1-(3-aminopropyl)-1-methyl- butyl]carbamoylamino]ethoxy]ethoxy]ethoxy]ethyl]carbamate;2,2,2-trifluoroacetic acid (2) [1551] To a mixture of tert-butyl N-[4-[2-[2-[2-[2- (benzyloxycarbonylamino)ethoxy]ethoxy]ethoxy]ethylcarbamoylamino]-7-(tert-butoxycarbonylamino)- 4-methyl-heptyl]carbamate (1, 1.00 eq, 63.7 mg, 0.089 mmol) in DCM (1 mL) was added TFA (1 mL). The mixture was stirred at rt for 1h, concentrated, and lyophilized to give 2 as clear syrup (83 mg, yield: 108%). LCMS m/z 512.2 [M+H]. [1552] Synthesis of benzyl N-[2-[2-[2-[2-[[4-(imidazole-1-carbonylamino)-1-[3-(imidazole-1- carbonylamino)propyl]-1-methyl-butyl]carbamoylamino]ethoxy]ethoxy]ethoxy]ethyl]carbamate (3) [1553] To a mixture of carbonyldiimidazole (5.80 eq, 91.4 mg, 0.56 mmol) in DMSO (0.2 mL) was added a solution of benzyl N-[2-[2-[2-[2-[[4-amino-1-(3-aminopropyl)-1-methyl- butyl]carbamoylamino]ethoxy]ethoxy]ethoxy]ethyl]carbamate;2,2,2-trifluoroacetic acid (2, 1.00 eq, 83.0 mg, 0.097 mmol) in DMSO (0.8 mL). The mixture was stirred at rt for 2h, diluted with EtOAc, washed with water (1x), and the aqueous layer was extracted with EtOAc (1x). The combined organic layer was washed with water (1x) and brine (1x), dried, and concentrated to give 3 as clear syrup (59.5 mg, yield: 87%). LCMS m/z 700.2 [M+H]. [1554] Synthesis of benzyl N-[2-[2-[2-[2-[[4-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-1-[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]-1-methyl- butyl]carbamoylamino]ethoxy]ethoxy]ethoxy]ethyl]carbamate (4) [1555] To a mixture of benzyl N-[2-[2-[2-[2-[[4-(imidazole-1-carbonylamino)-1-[3-(imidazole-1- carbonylamino)propyl]-1-methyl-butyl]carbamoylamino]ethoxy]ethoxy]ethoxy]ethyl]carbamate (3, 1.00 eq, 32.4 mg, 0.046 mmol) in DMSO (0.5 mL) was addedN-[(2R,3R,4R,5R,6R)-2-[3-[2-(2- aminoethoxy)ethoxy]propyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB48 2.10 eq, 34.1 mg, 0.097 mmol) . The mixture was stirred at rt over weekend, purified by prep. HPLC (5 - 35% MeCN/water with 0.1% formic acid) to give 4 as a white solid (39.5 mg, yield: 67%). LCMS 1264.5 [M+H]. [1556] Synthesis of N-[(2R,3R,4R,5R,6R)-2-[3-[2-[2-[[7-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-4-[2-[2- [2-(2-aminoethoxy)ethoxy]ethoxy]ethylcarbamoylamino]-4-methyl- heptyl]carbamoylamino]ethoxy]ethoxy]propyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3- yl]acetamide (5) [1557] To a mixture of benzyl N-[2-[2-[2-[2-[[4-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-1-[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]propyl]-1-methyl- butyl]carbamoylamino]ethoxy]ethoxy]ethoxy]ethyl]carbamate (4, 1.00 eq, 39.5 mg, 0.031 mmol) in MeOH (3 mL) was added 10% Pd/C (12 mg). The mixture was stirred at rt under hydrogen for 1h, filtered, concentrated, purified by prep. HPLC (5 - 50% MeCN/20 mM NH4OH solution) to give 5 as a white solid (24.6 mg, yield: 70%). LCMS 1130.6 [M+H]. [1558] Synthesis of N,N'-((2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(16-(3-(1-(4-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-yl)ureido)-16-methyl-11,21-dioxo- 4,7,25,28-tetraoxa-10,12,20,22-tetraazahentriacontane-1,31-diyl)bis(4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2,3-diyl))diacetamide (Compound 2318) [1559] To a mixture of 4-Maleimidobenzoic acid (1.10 eq, 1.8 mg, 0.0081 mmol) in DMF (0.3 mL) were added DIPEA (3.00 eq, 0.0038 mL, 0.022 mmol) , HATU (1.10 eq, 3.1 mg, 0.0081 mmol) , and N- [(2R,3R,4R,5R,6R)-2-[3-[2-[2-[[7-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylcarbamoylamino]-4-[2-[2-[2-(2- aminoethoxy)ethoxy]ethoxy]ethylcarbamoylamino]-4-methyl- heptyl]carbamoylamino]ethoxy]ethoxy]propyl]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3- yl]acetamide (5, 1.00 eq, 8.3 mg, 0.0073 mmol) . The mixture was stirred at rt for 1h and purified by prep. HPLC (10 - 25% MeCN/water with 0.1% formic acid) to give Compound 2318 as a white solid (5.9 mg, yield: 60%). LCMS 1329.5 [M+H]. Synthesis of Compound 2319
Figure imgf000508_0001
[1560] Synthesis of N-(29-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-19-oxo-3,6,9,12,15,23,26-heptaoxa-18,20-diazanonacosyl)- 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamide (Compound 2319) [1561] To a mixture of 4-maleimidobenzoic acid (1.10 eq, 3.6 mg, 0.0165 mmol) in DMF (0.1 mL) were added DIPEA (3.00 eq, 0.0078 mL, 0.0450 mmol) , HATU (1.10 eq, 6.3 mg, 0.0165 mmol) , and a solution of N-[(2R,3R,4R,5R,6R)-2-[3-[2-[2-[2-[2-[2-[2-[2-(2- aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoylamino]ethoxy]ethoxy]propyl]-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide (Compound 2386, 1.00 eq, 9.9 mg, 0.0150 mmol) in DMF (0.2 mL). The mixture was stirred at rt for 1h and purified by prep. HPLC (10 - 25% MeCN/water with 0.1% formic acid) to give Compound 2319 as a white solid (9 mg, yield: 70%). LCMS m/z 856.4 [M+H].
Figure imgf000509_0001
[1562] Synthesis of benzyl (1,27-bis(5-((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)isoxazol-3-yl)-14-(1-(5-((2S,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)isoxazol-3-yl)-9-oxo-2,5-dioxa-8,10- diazatridecan-13-yl)-9,19-dioxo-2,5,23,26-tetraoxa-8,10,18,20-tetraazaheptacosan-14-yl)carbamate (1) [1563] Compound 1 was synthesized according to General Method D using N-((2S,3R,4R,5R,6R)- 2-(3-((2-(2-aminoethoxy)ethoxy)methyl)isoxazol-5-yl)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H- pyran-3-yl)acetamide (XB86) in lieu of (XB48) to give intermediate (1). LCMS 1583.97 [M+H]. [1564] Synthesis of N-[(2S,3R,4R,5R,6R)-2-[3-[2-[2-[[7-[2-[2-[[5-[(2S,3R,4R,5R,6R)-3-acetamido- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]isoxazol-3- yl]methoxy]ethoxy]ethylcarbamoylamino]-4-[3-[2-[2-[[5-[(2S,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]isoxazol-3- yl]methoxy]ethoxy]ethylcarbamoylamino]propyl]-4-amino- heptyl]carbamoylamino]ethoxy]ethoxymethyl]isoxazol-5-yl]-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide (2) [1565] Using air-free techniques, a mixture of 1 (1.00 eq, 9.0 mg, 0.00569 mmol) and MeCN (0.057 mL) was treated with iodo(trimethyl)silane (12.0 eq, 9.8 uL, 0.0682 mmol) . More MeCN was added (0.200 mL) to enable stirring when a precipitate formed, then the reaction was heated to 30 ˚C. After 1h, the reaction was cooled to room temperature then diluted with concentrated ammonium hydroxide (0.050 mL) and DMSO (1 mL), then purified by reversed-phase HPLC (10-50% acetonitrile in water with 0.2 mM NH4OH modifer) to give (2) (5.0 mg, 0.00345 mmol, 60.7 % yield). LCMS 1448.35 [M+H]. [1566] Synthesis of Compound 2325 [1567] Following the HATU coupling method outlined in General Method D, 2 was converted to Compound 2325. LCMS 1888.25 [M+H]. Synthesis of Compound 2336
Figure imgf000510_0001
[1568] Synthesis of tert-butyl (2-(4-((17-azido-3,6,9,12,15- pentaoxaheptadecyl)carbamoyl)piperazin-1-yl)ethyl)carbamate (1) [1569] To a mixture of Carbonyldiimidazole (1.10 eq, 77.8 mg, 0.48 mmol) in DMF (0.4 mL) was added a solution of Azido-PEG5-amine (1.10 eq, 147 mg, 0.48 mmol) in DMF (0.6 mL). The mixture was stirred at ambient temperature for 1h, then a solution of tert-butyl N-(2-piperazin-1- ylethyl)carbamate (1.00 eq, 100 mg, 0.44 mmol) in DMF (0.4 mL) was added. The mixture was stirred at ambient temperature overnight, then 60 oC for 3hr. The reaction mixture was then concentrated, diluted with EtOAc, washed with water (2x), brine, dried, concentrated, and purified by column (0 - 10% MeOH/DCM) to give 1 as clear syrup (139.1 mg, yield: 57%). LCMS 562.3 [M+H]. [1570] Synthesis of tert-butyl (2-(4-((17-amino-3,6,9,12,15- pentaoxaheptadecyl)carbamoyl)piperazin-1-yl)ethyl)carbamate (2) [1571] To a mixture of tert-butyl (2-(4-((17-azido-3,6,9,12,15- pentaoxaheptadecyl)carbamoyl)piperazin-1-yl)ethyl)carbamate (1, 1.00 eq, 139 mg, 0.25 mmol) in MeOH (4 mL) was added 10% Pd/C (45 mg). The mixture was stirred at ambient temperature under hydrogen atmosphere for 1hr, filtered to remove solids, then concentrated to give 2 as clear syrup (104.1 mg, yield 78%). LCMS 536.4 [M+H]. [1572] Synthesis of tert-butyl (2-(4-((31-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-19-oxo-3,6,9,12,15,23,26-heptaoxa-18,20- diazahentriacontyl)carbamoyl)piperazin-1-yl)ethyl)carbamate; TFA salt (3) [1573] To a mixture of carbonyldiimidazole (1.17 eq, 12.5 mg, 0.0773 mmol) in DMF (0.1 mL) was added a solution of tert-butyl (2-(4-((17-amino-3,6,9,12,15-pentaoxaheptadecyl)carbamoyl)piperazin-1- yl)ethyl)carbamate (2, 1.17 eq, 41.4 mg, 0.077 mmol) in DMF (0.5 mL). The mixture was stirred at ambient temperature for 1hr, then N-[(2R,3R,4R,5R,6R)-2-[5-[2-(2-aminoethoxy)ethoxy]pentyl]-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB47, 1.00 eq, 25.0 mg, 0.066 mmol) was added. The mixture was stirred at ambient temperature overnight, then DIPEA (1.00 eq, 0.012 mL, 0.066 mmol) was added. The mixture then stirred at 60 oC for 5h, followed by purification by prep. HPLC (5 - 35%MeCN/water with 0.1%TFA) to give 3 as a white solid (TFA salt, 53 mg, yield: 76%). LCMS 940.5 [M+H]. [1574] Synthesis of N-(31-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-19-oxo-3,6,9,12,15,23,26-heptaoxa-18,20- diazahentriacontyl)-4-(2-aminoethyl)piperazine-1-carboxamide (4) [1575] To a mixture of tert-butyl (2-(4-((31-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-19-oxo-3,6,9,12,15,23,26-heptaoxa-18,20- diazahentriacontyl)carbamoyl)piperazin-1-yl)ethyl)carbamate; TFA salt (3, 1.00 eq, 53.0 mg, 0.050 mmol) in DCM (0.5 mL) was added TFA (0.5 mL). The mixture was stirred at ambient temperature for 1hr, concentrated, and purified by prep. HPLC (5 - 50% MeCN/20 mM NH4OH solution) to give 4 as a white solid (31 mg, yield: 73%). LCMS 840.6 [M+H]. [1576] Synthesis of N-(31-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)-19-oxo-3,6,9,12,15,23,26-heptaoxa-18,20- diazahentriacontyl)-4-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)piperazine-1-carboxamide (Compound 2336) [1577] To a mixture of N-[2-[2-[2-[2-[2-[2-[2-[2-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]pentoxy]ethoxy]ethylcarbamoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]-4-(2- aminoethyl)piperazine-1-carboxamide (1.00 eq, 13.3 mg, 0.0158 mmol) in acetic acid (0.45 mL) was added Maleic anhydride (1.00 eq, 1.5 mg, 0.0158 mmol) . The mixture was stirred at ambient temperature for 1hr, then stirred at 100 oC for 20 minutes, followed by purification by prep. HPLC (5 - 20% MeCN/water with 0.1%formic acid) to give Compound 2336 as a white solid (7.1 mg, yield:47%). LCMS 920.7 [M+H]. Synthesis of Compound 2337
Figure imgf000512_0001
[1578] Synthesis of tert-butyl (17-((5-nitropyridin-2-yl)oxy)-3,6,9,12,15- pentaoxaheptadecyl)carbamate (1) [1579] To a mixture of tert-butyl N-[2-[2-[2-[2-[2-(2- hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (1.10 eq, 331 mg, 0.87 mmol) and 2- chloro-5-nitro-pyridine (1.00 eq, 125 mg, 0.79 mmol) in DMF (0.3 mL) was added 1M Potassium tert- butoxide solution in THF (1.20 eq, 0.95 mL, 0.95 mmol) . The mixture was stirred at ambient temperture for 2h, then diluted with EtOAc, washed with water (2x), brine (1x), dried, concentrated, and purified by silica column (0 -100% EtOAc/hexane) to give 1 as yellow syrup (244.3 mg, yield: 62%). LCMS m/z 404.2 [M-Boc+H]+. [1580] Synthesis of 17-((5-nitropyridin-2-yl)oxy)-3,6,9,12,15-pentaoxaheptadecan-1-amine (2) [1581] To a mixture of tert-butyl (17-((5-nitropyridin-2-yl)oxy)-3,6,9,12,15- pentaoxaheptadecyl)carbamate (1, 1.00 eq, 244 mg, 0.485 mmol) in DCM (1 mL) was added TFA (1 mL), The mixture was stirred at ambient temperature for 1hr, concentrated, and purified by prep. HPLC (10 - 70% MeCN/20mM NH4OH) to give 2 as yellow syrup (187 mg, yield: 96%). LCMS m/z 404.2 [M+H]. [1582] Synthesis of N-((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-(1-((5-nitropyridin- 2-yl)oxy)-19-oxo-3,6,9,12,15,23,26-heptaoxa-18,20-diazahentriacontan-31-yl)tetrahydro-2H-pyran-3- yl)acetamide (3) [1583] To a mixture of carbonyldiimidazole (1.15 eq, 13.2 mg, 0.081 mmol) in DMF (0.1 mL) was added a solution of 17-((5-nitropyridin-2-yl)oxy)-3,6,9,12,15-pentaoxaheptadecan-1-amine (2, 1.15 eq, 32.9 mg, 0.081 mmol) in DMF (0.4 mL). The mixture was stirred at ambient temperature for 1hr, then N- [(2R,3R,4R,5R,6R)-2-[5-[2-(2-aminoethoxy)ethoxy]pentyl]-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide (XB47, 1.00 eq, 26.8 mg, 0.0708 mmol) was added. The mixture continued to stir at ambient temperature overnight, then DIPEA (1.00 eq, 0.012 mL, 0.071 mmol) was added, and the mixture was stirred at 60 oC for 5hr. The product was isolated by prep. HPLC (5 - 50%MeCN/water with 0.1%TFA) to give 3 as a white solid (19.3 mg, yield: 34%). LCMS m/z 808.3 [M+H]. [1584] Synthesis of N-((2R,3R,4R,5R,6R)-2-(1-((5-aminopyridin-2-yl)oxy)-19-oxo- 3,6,9,12,15,23,26-heptaoxa-18,20-diazahentriacontan-31-yl)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (4) [1585] To a mixture of N-((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-(1-((5- nitropyridin-2-yl)oxy)-19-oxo-3,6,9,12,15,23,26-heptaoxa-18,20-diazahentriacontan-31-yl)tetrahydro- 2H-pyran-3-yl)acetamide (3, 1.00 eq, 19.3 mg, 0.024 mmol) in MeOH (3 mL) was added 10% Pd/C (6 mg). The mixture was stirred at ambient temperature under hydrogen atmosphere for 1hr, filtered, concentrated, and purified by prep. HPLC (5 - 50% MeCN/20 mM NH4OH solution) to give 4 as a yellow solid (10.6 mg, yield: 57%). LCMS m/z 778.4 [M+H]. [1586] Synthesis of N-((2R,3R,4R,5R,6R)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pyridin- 2-yl)oxy)-19-oxo-3,6,9,12,15,23,26-heptaoxa-18,20-diazahentriacontan-31-yl)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide; formate salt (Compound 2337) [1587] To a mixture of N-((2R,3R,4R,5R,6R)-2-(1-((5-aminopyridin-2-yl)oxy)-19-oxo- 3,6,9,12,15,23,26-heptaoxa-18,20-diazahentriacontan-31-yl)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (4, 1.00 eq, 10.6 mg, 0.014 mmol) in Acetic acid (0.45 mL) was added Maleic anhydride (1.00 eq, 1.3 mg, 0.014 mmol) . The mixture was stirred at ambient temperature for 1hr, stirred at 100 oC for 50 minutes, then concentrated and purified by prep. HPLC (5 - 50% MeCN/water with 0.1%TFA) to give a white solid (7 mg), followed by repurification prep. HPLC (5 - 50% MeCN/water with 0.1%formic acid) to give Compound 2337 as a white solid (formate salt, 4.8 mg, yield: 39%). LCMS m/z 858.6 [M+H]. Synthesis of Compound 2339
Figure imgf000514_0001
[1588] Synthesis of tert-butyl (17-((4-nitrophenyl)sulfonamido)-3,6,9,12,15- pentaoxaheptadecyl)carbamate (1) [1589] To a mixture of nosyl chloride (1.00 eq, 86.8 mg, 0.39 mmol) in THF (1.5 mL) at 0 oC were added DIPEA (1.30 eq, 0.089 mL, 0.51 mmol) and a solution of t-Boc-N-amido-PEG5-amine (1.00 eq, 149 mg, 0.39 mmol) in THF (1 mL). The mixture was stirred at ambient temperature overnight, concentrated, and purified by silica column (0 - 100% EtOAc/hexane) to give 1 as clear syrup (174.4 mg, yield:79%). LCMS m/z 466.2 [M-Boc+H]+. [1590] Synthesis of N-((2R,3R,4R,5R,6R)-2-(1-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl)sulfonamido)-19-oxo-3,6,9,12,15,23,26-heptaoxa-18,20-diazahentriacontan-31-yl)-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (Compound 2339) [1591] Compound 2339 was synthesized by employing the procedures described for Compound 2337 using tert-butyl (17-((4-nitrophenyl)sulfonamido)-3,6,9,12,15-pentaoxaheptadecyl)carbamate (1) in lieu of tert-butyl (17-((5-nitropyridin-2-yl)oxy)-3,6,9,12,15-pentaoxaheptadecyl)carbamate. LCMS m/z 920.4 [M+H]. Synthesis of Compound 2340
Figure imgf000514_0002
[1592] Synthesis of N-((2R,3R,4R,5R,6R)-2-(1-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl)amino)-1,21-dioxo-5,8,11,14,17,25,28-heptaoxa-2,20,22-triazatritriacontan-33-yl)-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (Compound 2340) [1593] To a mixture of 4-(maleinimdo)phenyl isocyanate (1.00 eq, 3.2 mg, 0.015 mmol) in DMSO (0.05 mL) at 0 oC was added a solution of N-[(2R,3R,4R,5R,6R)-2-[5-[2-[2-[2-[2-[2-[2-[2-(2- aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethylcarbamoylamino]ethoxy]ethoxy]pentyl]-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide (Compound 2385, 1.00 eq, 10.1 mg, 0.015 mmol) in DMSO (0.15 mL). The mixture was stirred at ambient temperature for 30 minutes and purified by prep. HPLC (10 - 40% MeCN/water with 0.1% formic acid) to give a yellow solid (4.1 mg, yield: 31%). LCMS m/z 899.4 [M+H]. Synthesis of Compound 2341
Figure imgf000515_0001
[1594] Synthesis of tert-butyl (1-((1-(2-(((benzyloxy)carbonyl)amino)ethyl)piperidin-4-yl)amino)-1- oxo-5,8,11,14,17-pentaoxa-2-azanonadecan-19-yl)carbamate (1) [1595] To a mixture of carbonyldiimidazole (1.10 eq, 42.5 mg, 0.26 mmol) in DMF ( 0.5 mL) was added a solution of t-Boc-N-amido-PEG5-amine (1.10 eq, 99.8 mg, 0.26 mmol) in DMF (0.5 mL). The mixture was stirred at rt for 1h and DIPEA (3.00 eq, 0.12 mL, 0.72 mmol) and benzyl N-[2-(4-amino-1- piperidyl)ethyl]carbamate;trihydrochloride (1.00 eq, 92.2 mg, 0.24 mmol) were added. The mixture was stirred at ambient temperature overnight, 60 oC for 2hr, then purified by silica column (0 - 15% MeOH/DCM) to give 1 as yellow syrup (120.7 mg, yield: 74%). LCMS m/z 684.5 [M+H]. [1596] Synthesis of N-((2R,3R,4R,5R,6R)-2-(1-((1-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethyl)piperidin-4-yl)amino)-1,21-dioxo-5,8,11,14,17,25,28-heptaoxa-2,20,22-triazatritriacontan-33- yl)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (Compound 2341) [1597] Compound 2341 was synthesized by employing the procedures described for Compound 2336 using tert-butyl (1-((1-(2-(((benzyloxy)carbonyl)amino)ethyl)piperidin-4-yl)amino)-1-oxo- 5,8,11,14,17-pentaoxa-2-azanonadecan-19-yl)carbamate (1) in lieu of tert-butyl (17-((5-nitropyridin-2- yl)oxy)-3,6,9,12,15-pentaoxaheptadecyl)carbamate. LCMS m/z 934.5 [M+H]. Synthesis of Compound 2347
Figure imgf000516_0001
[1598] Synthesis of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[3-[(2R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (1) [1599] A solution of carbonyldiimidazole (1.40 eq, 11.7 mg, 0.0719 mmol) in DMF (60 uL) was added to a solution of amino-PEG6-t-butyl ester (1.40 eq, 29.5 mg, 0.0719 mmol) in DMF (130 uL), then after 1 hour, N-[(2R,4R,5R,6R)-2-[3-[2-(2-aminoethoxy)ethoxy]propyl]-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-3-yl]acetamide (1.00 eq, 18.0 mg, 0.0514 mmol) was added. The reaction was diluted with water and DMSO then purified by reversed-phase HPLC (5-50% acetonitrile in water w/0.1% FA) to give tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[3-[(2R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (1). Yield: 14 mg, 34 %. LCMS 786.20 [M+H]. [1600] Synthesis of 3-[2-[2-[2-[2-[2-[2-[2-[2-[3-[(2R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (2) [1601] A mixture of 1 (1.00 eq, 13.8 mg, 0.0176 mmol) in water (0.100 mL) and DCM (0.060 mL) was treated with 4M HCl Dioxane (25.0 eq, 0.11 mL, 0.439 mmol). After 5 hr., the reaction was complete and volatile components were removed under reduced pressure, then the residue dried further under high vacuum overnight to give 3-[2-[2-[2-[2-[2-[2-[2-[2-[3-[(2R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylcarbamoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (2). Yield: 18 mg, 136 %. LCMS 730.27 [M+H]. [1602] Synthesis of N-((2R,3R,4R,5R,6R)-2-(33-(4-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethyl)piperazin-1-yl)-11,33-dioxo-4,7,15,18,21,24,27,30-octaoxa-10,12-diazatritriacontyl)-4,5- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (Compound 2347) [1603] A solution of 2 (1.00 eq, 17.0 mg, 0.0233 mmol) in DMF (0.1165 mL) was added to a cold solution of 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione;dihydrochloride (1.00 eq, 6.6 mg, 0.0233 mmol), HATU (1.10 eq, 9.7 mg, 0.0256 mmol), and DIPEA (3.00 eq, 12 uL, 0.0699 mmol) in DMF (0.116 mL). After 3hrs., the reaction was diluted with DMSO then purified by reversed-phase HPLC (3-50% acetonitrile in water w/0.1% TFA) to give N-((2R,3R,4R,5R,6R)-2-(33-(4-(2-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)ethyl)piperazin-1-yl)-11,33-dioxo-4,7,15,18,21,24,27,30-octaoxa-10,12-diazatritriacontyl)- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (Compound 2347). Yield: 2.5 mg, 11.6 %. LCMS 921.33 [M+H].
Figure imgf000517_0001
[1604] 1 was made following General Method B using XB48 in lieu of XB54. Compound 2356 was made following General Method B using 4-(chloromethyl)benzene-1-sulfonyl chloride in lieu of Int3 to give Compound 2356. LCMS 1771.5 [M+H]. [1605] Synthesis of Compound 2356 [1606] A suspension of 1 (1.00 eq, 7.0 mg, 0.00443 mmol) and DIPEA (3.00 eq, 2.3 uL, 0.0133 mmol) in DMF (44 uL) was treated with 4-(chloromethyl)benzene-1-sulfonyl chloride (1.00 eq, 1.0 mg, 0.00443 mmol) and the reaction was stored at -20C for 12h. The reaction was diluted with DMSO then purified by reversed-phase HPLC (5-40% acetonitrile in water w/0.1% TFA) to give Compound 2356. Yield: 3.5 mg, 44 %. LCMS 1771.5 [M+H]. Synthesis of Compound 2357
Figure imgf000518_0001
[1607] Synthesis of methyl 2-(bromomethyl)isonicotinate (2) [1608] To a solution of methyl 2-methylisonicotinate (1) (292.8 mg, 1.937 mmol) in 4.8 mL of CCl4 was added N-bromosuccinimide (439.2 mg, 2.468 mmol, 1.3 eq.) and azo-isobutyronitrile (280.3 mg, 1.707 mmol, 0.9 eq.). The mixture was stirred at reflux (80C) for 4 hours, cooled to ambient temperature, then evaporated onto silica. The product was isolated via silica chromatography, eluting with isocratic 15% ethyl acetate in hexanes. Fractions containing product were concentrated and dried further under high-vacuum at ambient temperature to afford methyl 2-(bromomethyl)isonicotinate (2) as a dark blue color solid. Yield: 88.0 mg (20%); LCMS m/z 231.9 [M+H]. [1609] Synthesis of 2-(bromomethyl)isonicotinic acid (3) [1610] To a solution of methyl 2-(bromomethyl)isonicotinate (2) (80.8 mg, 0.351 mmol) in 3.3 mL THF was added lithium hydroxide monohydrate (36.9 mg, 0.879 mmol, 2.5 eq.). The reaction stirred for 20 minutes until hydrolysis complete. The reaction mixture was diluted with 1 mL H2O and acidified to pH 5 with 0.4 mL of aqueous 1N HCl. While stirring, the majority of THF solvent was evaporated via stream of N2 to ~1/3 original volume. The concentrate was diluted with DMA solvent and product isolated by preparatory HPLC, eluting with 1-20% acetonitrile in water. Fractions containing the desired product were combined and lyophilized to dryness to afford 2-(bromomethyl)isonicotinic acid (3) as a dark blue color solid. Yield: 31.8 mg (42%); LCMS m/z 217.7 [M+H]. [1611] Synthesis of N-[2-[2-[2-[2-[3-[[2-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-1,1- bis[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]ethyl]amino]-3-oxo- propoxy]ethoxy]ethoxy]ethoxy]ethyl]-2-(bromomethyl)pyridine-4-carboxamide (Compound 2357) [1612] A solution of -(bromomethyl)isonicotinic acid (3) (2.64 mg, 0.0122 mmol, 1.6 eq.), [dimethylamino(triazolo[4,5-b]pyridin-3-yloxy)methylene]-dimethyl-ammonium;hexafluorophosphate (HATU) (4.88 mg, 0.0128 mmol, 1.7 eq.) and 2,6-lutidine (4.0 uL, 0.028 mmol, 3.0 eq.) in 0.1 mL DMF was stirred at ambient temperature for 10 minutes, then a solution of N-[2-[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-1,1-bis[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]methyl]ethyl]-3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propenamide (2356, intermediate 1) (11.9 mg, 0.0076 mmol, 1.0 eq.), dissolved in 0.1 mL DMF, was added. The reaction solution stirred for 35 minutes at ambient temperature until consumption of amine by LCMS. The reaction solution was injected directly onto preparatory HPLC, eluting with 1-40% acetonitrile in water. Fractions containing the desired product were combined and lyophilized to dryness to afford N-[2-[2-[2- [2-[3-[[2-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran- 2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-1,1-bis[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]methyl]ethyl]amino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethyl]-2-(bromomethyl)pyridine-4- carboxamide (Compound 2357) as a white/slight purple color solid. Yield: 2.6 mg (18%); LCMS m/z 1781.6 [M+H].
Figure imgf000520_0001
[1613] Synthesis of N-[2-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-1,1-bis[[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]ethyl]-3-[2-[2-[2-[2-[(2- chloroacetyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]propanamide (Compound 2358) [1614] To a solution of chloroacetic acid (1.23 mg, 0.0130 mmol, 1.7 eq.) in 50 uL DMF was added N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (3.53 mg, 0.0143 mmol, 1.9 eq.). The acid activation reaction stirred under nitrogen atmosphere at ambient temperature for approximately 90 minutes, then a solution of N-[2-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-1,1-bis[[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]ethyl]-3-[2-[2-[2-(2- aminoethoxy)ethoxy]ethoxy]ethoxy]propenamide (2356-1) (11.77 mg, 0.0074 mmol, 1.0 eq.) dissolved in 100 uL DMF was added. The reaction continued to stir under nitrogen atmosphere at ambient temperature for an additional 2 hours, at which time the reaction was diluted with DMF and product isolated by preparatory HPLC, eluting with 1-40% acetonitrile in water (0.1% trifluoracetic acid modifier). Fractions containing the desired product were combined and lyophilized to dryness to afford N-[2-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-1,1-bis[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]methyl]ethyl]-3-[2-[2-[2-[2-[(2-chloroacetyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]propenamide (Compound 2358) as a white solid. Yield: 3.79 mg (31%); LCMS m/z 1657.9 [M+H]. Synthesis of Compound 2352
Figure imgf000521_0001
[1615] Synthesis of tert-butyl 3-[2-[2-[2-[2-[(2-bromoacetyl)-methyl- amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (2) [1616] To a solution of methylamino-PEG4-t-butyl ester (34.4 mg, 0.103 mmol, 1.0 eq.) in 0.2 mL of DCM was added diisopropylethylamine (DIPEA) (36 uL, 0.20 mmol, 2.0 eq.). The solution stirred at ~-78C (CO2/acetone bath) under nitrogen atmosphere, then a solution of bromoacetyl bromide (22.4 mg, 0.111 mmol, 1.1 eq.) dissolved in 0.1 mL of DCM was added dropwise over 5 minutes. The reaction solution continued to stir while warming to ambient temperature. The reaction progress was monitored by LCMS. After 90 minutes, the reaction mixture was diluted with DCM and evaporated onto silica. The product was isolated by silica chromatography, eluting with EtOAc/DCM. Fractions containing desired product were combined, concentrated, and dried under high-vacuum at ambient temperature to afford tert-butyl 3-[2-[2-[2-[2-[(2-bromoacetyl)-methyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (2) as slight yellow color syrup. Yield: 25.1 mg (54%); LCMS m/z 456.3 [M+H]. [1617] Synthesis of 3-[2-[2-[2-[2-[(2-bromoacetyl)-methyl- amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (3) [1618] To a solution of tert-butyl 3-[2-[2-[2-[2-[(2-bromoacetyl)-methyl- amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (2) (20.9 mg, 0.0458 mmol) in 0.9 mL of DCM was added 0.1 mL of trifluoroacetic acid. The reaction stirred at ambient temperature for 2 hours, then was concentrated to residue under a stream of nitrogen. The residue was dissolved in acetonitrile/water, frozen, and lyophilized to dryness to afford 3-[2-[2-[2-[2-[(2-bromoacetyl)-methyl- amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (3) as a dark yellow color syrup. Yield: 12.9 mg (70%); LCMS m/z 400.3 [M+H]. [1619] Synthesis of N-[2-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-1,1-bis[[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]ethyl]-3-[2-[2-[2-[2-[(2-bromoacetyl)-methyl- amino]ethoxy]ethoxy]ethoxy]ethoxy]propenamide (Compound 2352) [1620] To a solution of 3-[2-[2-[2-[2-[(2-bromoacetyl)-methyl- amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (3) (5.42 mg, 0.0135 mmol, 1.9 eq.) in 0.1 mL DCM was added N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (3.66 mg, 0.0148 mmol, 2.1 eq.). The acid activation reaction stirred under nitrogen atmosphere at ambient temperature for approximately 2 hours, then a solution of N-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethyl]-3-[3-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]-2-[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]-2-amino- propoxy]propenamide (XB68A), dissolved in 0.1 mL of DMF, was added. The reaction mixture stirred overnight under nitrogen atmosphere, then was diluted with 1 mL DMF, and purified by reverse phase HPLC (2-40% acetonitrile in water with 0.1% trifluoroacetic acid modifier). Fractions containing desired product were combined and lyophilized to dryness to afford N-[2-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]-1,1-bis[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]ethyl]-3-[2- [2-[2-[2-[(2-bromoacetyl)-methyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]propenamide (Compound 2352) as a white solid. Yield: 0.7 mg (5%); LCMS m/z 1717.5 [M+H]. Synthesis of Compound 2372
Figure imgf000523_0001
[1621] Synthesis of 1-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)piperidine-4-carboxylic acid (1) [1622] To a mixture of 1-(2-aminoethyl)piperidine-4-carboxylic acid (1.00 eq, 81.4 mg, 0.47 mmol) and maleic anhydride (1.00 eq, 46.3 mg, 0.47 mmol) was added HOAc (1.5 mL). The mixture was stirred at rt for 1h, concentrated, and co-evaporated with toluene (2x). To the residue was added toluene (1.5 mL). The mixture was stirred at 110 oC for 1hr and concentrated to give 1 as a pink solid which was used without purification. LCMS m/z 253.1 [M+H] [1623] Synthesis of tert-butyl 1-(1-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)piperidin-4-yl)- 1-oxo-5,8,11,14-tetraoxa-2-azaheptadecan-17-oate (2) [1624] To a mixture of crude 1-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)piperidine-4- carboxylic acid (0.47 mmol) in DMF (1.2 mL) at 0 oC were added HATU (1.30 eq, 234 mg, 0.62 mmol) and diisopropylethylamine (DIPEA) (2.50 eq, 0.21 mL, 1.18 mmol) , followed by addition of amino-PEG4-t-butyl ester (0.900 eq, 137 mg, 0.43 mmol) . The mixture was stirred at ambient temperature for 1hr and purified by prep. HPLC (5 - 40% MeCN/water with 0.1%TFA) to give 2 as clear syrup (51 mg, yield: 19% two steps). LCMS m/z 556.2 [M+H]. [1625] Synthesis of Compound 2372 [1626] Compound 2372 was synthesized by employing the procedures described for Compound 1251 using tert-butyl 1-(1-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)piperidin-4-yl)-1-oxo- 5,8,11,14-tetraoxa-2-azaheptadecan-17-oate (2) in lieu of tert-butyl 12-((2-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)ethyl)amino)-12-oxododecanoate (Compound 1251, 00A) and using XB48 in place of XB47. LCMS m/z 1816.5 [M+H]. Synthesis of Compound 2376
Figure imgf000524_0001
Figure imgf000525_0001
[1627] Synthesis of tert-butyl 1-((4-nitrophenyl)sulfonamido)-3,6,9,12-tetraoxapentadecan-15-oate (1) [1628] A solution of 4-nitrobenzenesulfonyl chloride (1.00 eq, 0.93 g, 4.21 mmol) in THF (18.4 mL) was treated with amino-PEG4-t-butyl ester (1.15 eq, 1.56 g, 4.84 mmol), followed by diisopropylethylamine (DIPEA) (2.00 eq, 1.5 mL, 8.42 mmol) . The reaction was stirred at room temperature for 15hr before adsorbing onto silica gel for purification by silica gel chromatography (100% DCM then 2% MeOH in DCM) to give tert-butyl 3-[2-[2-[2-[2-[(4- nitrophenyl)sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate. Yield: 1.73 g, 81 %. LCMS 505.3 [M-H]. [1629] Synthesis of tert-butyl 1-((4-aminophenyl)sulfonamido)-3,6,9,12-tetraoxapentadecan-15-oate (2) [1630] A solution of tert-butyl 3-[2-[2-[2-[2-[(4- nitrophenyl)sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (1.00 eq, 1.70 g, 3.36 mmol) and palladium hydroxide (1.00 eq, 471 mg, 3.36 mmol) on carbon in methanol (67.1 mL) was placed under an atmosphere of hydrogen gas via balloon. After 1hr, the reaction was filtered and the filtrate concentrated under reduced pressure. The resulting residue was used directly in the next step. Yield: 1.58 g, 98 %. LCMS 476.87 [M+H]. [1631] Synthesis of 1-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)sulfonamido)-3,6,9,12- tetraoxapentadecan-15-oic acid (3) [1632] A solution of tert-butyl 3-[2-[2-[2-[2-[(4- aminophenyl)sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (1.00 eq, 200 mg, 0.420 mmol) in acetic acid (0.6994 mL) was treated with maleic anhydride (1.62 eq, 66.7 mg, 0.680 mmol) before heating to 150 C. After 1h, the reaction was diluted with DMSO then purified by RPHPLC (5-50% MeCN in water w0.1% TFA) to give 1-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)sulfonamido)- 3,6,9,12-tetraoxapentadecan-15-oic acid (3). Yield: 140 mg, 66.6 %. LCMS 501.5 [M+H]. [1633] Synthesis of (Compound 2376) [1634] Following the HATU coupling method outlined in General Method D, XB68A was converted to Compound 2376 using 3 in lieu of 3-(2-(2-(2-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl)sulfonamido)ethoxy)ethoxy)ethoxy)propanoic acid to give Final Product (Compound 2376). LCMS 1817.1 [M+H]. Synthesis of Compound 2379
Figure imgf000526_0001
[1635] Synthesis of perfluorophenyl 16-(4-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethyl)piperazin-1-yl)-16-oxo-4,7,10,13-tetraoxahexadecanoate (1) [1636] To a suspension of 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione;dihydrochloride (1.00 eq, 202 mg, 0.716 mmol) in 1.5 mL of DMF was added Bis-PEG4-PFP ester (1.50 eq, 673 mg, 1.07 mmol) in 1 mL DMF, the mixture was cooled to 0˚C and DIPEA (3.00 eq, 0.30 mL, 2.15 mmol) was added dropwise - mixture remains a slurry - stirred at 0˚C for 20 min, and warmed to room temp and stirred for 30 min. The mixture was cooled to 0˚C, acidified with 165 uL TFA in approximately 0.5 mL water, diluted with CH3CN, filtered, and purified on C18, eluting with a gradient of 15-75-100% CH3CN/water + 0.1 %TFA to give 1. Yield: 343 mg, 62%. LCMS 652.5 [M+H]. [1637] Synthesis of N-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethyl]-3-[2-[2-[2-[3-[4-[2-(2,5-dioxopyrrol-1- yl)ethyl]piperazin-1-yl]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]propanamide;2,2,2-trifluoroacetic acid (Compound 2379) [1638] A mixture of 1 (1.00 eq, 10.0 mg, 0.0285 mmol) and N-((2R,3R,4R,5R,6R)-2-(3-(2-(2- aminoethoxy)ethoxy)propyl)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)acetamide (XB48, 1.00 eq, 10.0 mg, 0.0285 mmol) were dissolved in DMF (0.342 mL) then diisopropylethylamine (DIPEA) (2.00 eq, 9.9 uL, 0.0571 mmol) was added. After 2h, the reaction was purified by reversed-phase HPLC (5-40% acetonitrile in water w/0.1% TFA) then lyophilized to give (Compound 2379). Yield: 20 mg, 75 %. Synthesis of Compound 2350
Figure imgf000527_0001
Figure imgf000528_0001
[1639] Synthesis of benzyl 4-(N-(16,16-dimethyl-14-oxo-3,6,9,12,15- pentaoxaheptadecyl)sulfamoyl)piperidine-1-carboxylate (1a) [1640] To a solution of tert-butyl 14-amino-3,6,9,12-tetraoxatetradecanoate (0.7 g, 2.28 mmol, 1.0 eq.) in acetonitrile (10 mL) was added triethylamine (0.96 mL, 6.83 mmol, 3.0 eq.) and benzyl 4- (chlorosulfonyl)piperidine-1-carboxylate (0.724 g, 2.28 mmol, 1.0 eq.) at 0 °C and the reaction mixture was stirred at room temperature for 6hr. After completion, as indicated by TLC and LCMS, the reaction mixture was concentrated to get crude, which was purified by silica gel flash column chromatography using 40-50% ethyl acetate in hexane to afford benzyl 4-(N-(16,16-dimethyl-14-oxo-3,6,9,12,15- pentaoxaheptadecyl)sulfamoyl)piperidine-1-carboxylate (1) as colorless gum. Yield: 0.9 g, 67.1%; LCMS m/z 606.3 [M+18]+ [1641] Synthesis of 14-((1-((benzyloxy)carbonyl)piperidine)-4-sulfonamido)-3,6,9,12- tetraoxatetradecanoic acid (1b) [1642] To a solution of benzyl 4-(N-(16,16-dimethyl-14-oxo-3,6,9,12,15- pentaoxaheptadecyl)sulfamoyl)piperidine-1-carboxylate (1) (0.7 g, 1.19 mmol) in dichloromethane (7 mL) was added 4M hydrogen chloride in 1,4-dioxane (7 mL) dropwise at 0 °C and reaction was stirred at same temperature for 3hr. After completion, the reaction was concentrated and co-distilled with dichloromethane to afford crude, which was purified by reverse phase prep HPLC (80% acetonitrile in water with 0.1% TFA). Pure fractions were collected and lyophilized to afford 14-((1- ((benzyloxy)carbonyl)piperidine)-4-sulfonamido)-3,6,9,12-tetraoxatetradecanoic acid (1b) as colourless viscous liquid. Yield: 0.563 g, 88.9%; LCMS m/z 531.4 [M - H]-.1H NMR (400 MHz, DMSO-d6-D2O exchange): δ 7.37-7.30 (m, 5H), 5.04 (s, 2H), 4.06 (d, J = 13.2 Hz, 2H), 3.99 (s, 2H), 3.55-3.52 (m, 2H), 3.50-3.48 (m, 10H), 3.41 (t, J = 5.2 Hz, 2H), 3.26-3.20 (m, 1H), 3.09-3.06 (m, 2H), 2.82 (s, 2H), 1.96 (d, J = 11.6 Hz, 2H), 1.45-1.35 (m, 2H). [1643] Synthesis of benzyl 4-[2-[2-[2-[2-[2-[[2-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-1,1- bis[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]ethyl]amino]-2-oxo- ethoxy]ethoxy]ethoxy]ethoxy]ethylsulfamoyl]piperidine-1-carboxylate (1) [1644] A solution of 14-((1-((benzyloxy)carbonyl)piperidine)-4-sulfonamido)-3,6,9,12- tetraoxatetradecanoic acid (1b) (25.8 mg, 0.0485 mmol, 2.4 eq.) in 0.2 mL of DMF was added [dimethylamino(triazolo[4,5-b]pyridin-3-yloxy)methylene]-dimethyl-ammonium;hexafluorophosphate (HATU) (19.4 mg, 0.0510 mmol, 2.6 eq.) and diisopropylethylamine (DIPEA) (13.6 mL, 0.0781 mmol, 4.0 eq.). The solution stirred under nitrogen atmosphere at ambient temperature for ~5 min, then was added to a stirring solution of N-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethyl]-3-[3-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]-2-[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]-2-amino- propoxy]propanamide (XB68A) (26.1 mg, 0.0196 mmol, 1.0 eq) dissolved in 0.2 mL of DMF. The reaction solution stirred under nitrogen atmosphere at ambient temperature for 15 minutes then was diluted with DMSO and purified by reverse phase HPLC (5-50% acetonitrile in water with 0.1% TFA modifier). Fractions containing desired product were combined and lyophilized to dryness to afford benzyl 4-[2-[2-[2-[2-[2-[[2-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-1,1-bis[[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]ethyl]amino]-2-oxo- ethoxy]ethoxy]ethoxy]ethoxy]ethylsulfamoyl]piperidine-1-carboxylate (1) as a white solid. Yield: 22.3 mg (61%); LCMS m/z 1849.6 [M+H]. [1645] Synthesis of N-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethyl]-3-[3-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]-2-[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]-2-[[2-[2-[2- [2-[2-(4-piperidylsulfonylamino)ethoxy]ethoxy]ethoxy]ethoxy]acetyl]amino]propoxy]propanamide (2) [1646] To a solution of benzyl 4-[2-[2-[2-[2-[2-[[2-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-1,1- bis[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]ethyl]amino]-2-oxo- ethoxy]ethoxy]ethoxy]ethoxy]ethylsulfamoyl]piperidine-1-carboxylate (1) (21.9 mg, 0.0118 mmol) in 5 mL of methanol was added 17.1 mg of 10% w/w palladium on carbon. The solution stirred under hydrogen atmosphere at ambient temperature for 20 minutes then was filtered over diatomaceous earth and concentrated to crude film. The crude material was dissolved in 2 mL of H2O and purified by reverse phase HPLC (1-40% acetonitrile in water with 20 mM ammonium hydroxide modifier). Fractions containing desired product were combined and lyophilized to dryness to afford N-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethyl]-3-[3-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-2-[[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]-2-[[2-[2-[2-[2-[2-(4- piperidylsulfonylamino)ethoxy]ethoxy]ethoxy]ethoxy]acetyl]amino]propoxy]propenamide (2) as a white solid. Yield: 14.6 mg (72%); LCMS m/z 1715.8 [M+H]. [1647] Synthesis of N-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethyl]-3-[3-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]-2-[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]-2-[[2-[2-[2- [2-[2-[[1-(2-bromoacetyl)-4- piperidyl]sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]acetyl]amino]propoxy]propanamide (Compound 2350) [1648] To a solution of N-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethyl]-3-[3-[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3- acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]-2-[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]methyl]-2-[[2-[2-[2- [2-[2-(4-piperidylsulfonylamino)ethoxy]ethoxy]ethoxy]ethoxy]acetyl]amino]propoxy]propanamide (2) (13.3 mg, 0.0078 mmol, 1.0 eq.) in 0.5 mL of DMF was added 2,5-dioxopyrrolidin-1-yl 2-bromoacetate (6.3 mg, 0.0266 mmol, 3.4 eq.) dissolved in 0.1 mL of DMF. The reaction solution stirred under nitrogen atmosphere at ambient temperature for 90 minutes then was diluted with DMF and purified by reverse phase HPLC (2-40% acetonitrile in water with 0.1% TFA modifier). Fractions containing desired product were combined and lyophilized to dryness to afford N-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethyl]-3-[3-[3-[2-[2-[3- [(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2- yl]propoxy]ethoxy]ethylamino]-3-oxo-propoxy]-2-[[3-[2-[2-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]propoxy]ethoxy]ethylamino]-3-oxo- propoxy]methyl]-2-[[2-[2-[2-[2-[2-[[1-(2-bromoacetyl)-4- piperidyl]sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]acetyl]amino]propoxy]propenamide (Compound 2350) as a white solid. Yield: 6.8 mg (48%); LCMS m/z 1836.5 [M+H]. Synthesis of LC38
Figure imgf000531_0001
[1649] Synthesis of tert-butyl 1-((5-nitropyridin-2-yl)amino)-3,6,9,12-tetraoxapentadecan-15-oate (2) [1650] To a solution of 2-chloro-5-nitropyridine (1, 1.0 eq., 0.48 g, 3.03 mmol) and tert-butyl 1- amino-3,6,9,12-tetraoxapentadecan-15-oate (1a, 1.0 eq., 0.97 g, 3.03 mmol) in acetonitrile (10 mL) was added and N,N-diisopropylethyl amine (5.0 eq., 2.64 mL, 15.1 mmol) and the reaction mixture was stirred at 70 °C for 16hrs. The reaction mixture was concentrated under reduced pressure to afford crude which was purified by silica gel flash column chromatography (45-55% ethyl acetate/hexane) to afford 2 as yellow gum. Yield: 1.2 g, 89.4%, LCMS m/z 444.15 [M +H]+ . [1651] Synthesis of tert-butyl 1-((5-aminopyridin-2-yl)amino)-3,6,9,12-tetraoxapentadecan-15-oate (3) [1652] To a solution of tert-butyl 1-((5-nitropyridin-2-yl)amino)-3,6,9,12-tetraoxapentadecan-15- oate (2, 1.2 g, 2.71 mmol) in methanol (16 mL), 10% palladium on carbon (1.3 g) was added and the reaction mixture was stirred at room temperature under hydrogen atmosphere (balloon pressure) for 6hrs. The reaction mixture was filtered through sintered funnel and the filtrate was concentrated under reduced pressure to afford 3 as a dark brown sticky solid. Yield: 1.0 g, LCMS m/z 414.2 [M + H]+ . [1653] Synthesis of 1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pyridin-2-yl)amino)-3,6,9,12- tetraoxapentadecan-15-oic acid (LC38) [1654] To a solution of tert-butyl 1-((5-aminopyridin-2-yl)amino)-3,6,9,12-tetraoxapentadecan-15- oate (3, 0.2 g, 0.48 mmol) in glacial acetic acid (2 mL) was added maleic anhydride (3a, 1.2 eq., 0.56 g, 0.58 mmol) and the reaction mixture was stirred at 120 °C under microwave irradiation for 10 mins. The reaction mixture was cooled to 0 °C and trifluoroacetic acid (1.5 mL) was added. The resulting reaction mixture was further stirred at room temperature for 3hrs. The reaction mixture was concentrated and purified with prep-HPLC (15% acetonitrile in water with 0.1% acetic acid) to afford LC38 as brown gum. Yield: 0.161 g, 76.1%, LCMS m/z 438.3 [M + H]+ .1H-NMR (400 MHz, DMSO-d6 D2O exchange): δ 7.86 (d, J = 2.40 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.16 (s, 2H), 6.63 (d, J = 8.4 Hz, 1H), 3.59 (t, J = 6.0 Hz, 2H), 3.54 (d, J = 4.4 Hz, 6H), 3.49 (d, J = 6.4 Hz, 8H), 3.44 (t, J = 4.4 Hz, 2H), 2.43 (t, J = 6.4 Hz, 2H), 2.07 (s, 1H). Synthesis of LC39 ll 2
Figure imgf000532_0001
[1655] Synthesis of tert-butyl 3-(2-(2-(2-(piperazin-1-yl)ethoxy)ethoxy)ethoxy)propanoate (1) [1656] To a solution of benzyl 4-(14,14-dimethyl-12-oxo-3,6,9,13-tetraoxapentadecyl)piperazine-1- carboxylate (0.62 g, 1.29 mmol) in methanol (10 mL), 20% palladium hydroxide on carbon (0.51 g) was added and the resultant reaction mixture was stirred at room temperature under hydrogen gas balloon pressure for 3h. The reaction mixture was filtered through syringe filter and concentrated under reduced pressure to get crude. The crude was triturated with diethyl ether and pentane to afford 1 as brown viscous liquid. Yield: 0.40 g (Crude); 77.9%; LCMS m/z 347.2 [M+H]+. [1657] Synthesis of tert-butyl 3-(2-(2-(2-(4-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)benzoyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)propanoate (2) [1658] To a solution of tert-butyl 3-(2-(2-(2-(piperazin-1-yl)ethoxy)ethoxy)ethoxy)propanoate (1, 0.40 g, 1.15 mmol) in dimethylformamide (8 mL), perfluorophenyl 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol- 1-yl)benzoate (0.487 g, 1.1 eq., 1.27 mmol) was added, and reaction mixture was stirred at room temperature for 1hr. The reaction mixture was concentrated under reduced pressure to give 2 as brown color crude. The crude was used for the next step without purification. Yield: 0.80 g (Crude); LCMS m/z 546.1 [M+H]+. [1659] Synthesis of 3-(2-(2-(2-(4-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoyl)piperazin-1- yl)ethoxy)ethoxy)ethoxy)propanoic acid (LC39) [1660] To a solution of tert-butyl 3-(2-(2-(2-(4-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)benzoyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)propanoate (2, 1.20 g, 2.2 mmol) in dichloromethane (12 mL) at 0 °C was added trifluoroacetic acid (12 mL) and the reaction mixture was stirred at room temperature for 3hrs, concentrated under reduced pressure, and triturated thrice with dichloromethane to afford crude which was purified by prep-HPLC (30-50% acetonitrile in water with 0.1% acetic acid) to afford LC39 as colorless semi-solid. Yield: 0.56 g, 52.0%, LCMS m/z 490.4 [M+H]+ ; 1H NMR (400 MHz, DMSO-d6-D2O) δ 7.55 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.09 (s, 2H), 3.70 (t, J = 5.6 Hz, 2H), 3.54-3.44 (m, 14H), 3.15 (m, 6H), 2.39 (t, J = 6.0 Hz, 2H). Synthesis of LC40
Figure imgf000533_0001
[1661] Synthesis of tert-butyl 1-((5-nitropyridin-2-yl)oxy)-3,6,9,12-tetraoxapentadecan-15-oate (1) [1662] To a stirred solution of 2-chloro-5-nitropyridine (2.0 eq., 0.197 g, 1.24 mmol) and tert-butyl 1-hydroxy-3,6,9,12-tetraoxapentadecan-15-oate (1.0 eq., 0.200 g, 0.620 mmol) in N,N- dimethylformamide (2 mL) was added potassium carbonate (4.0 eq., 0.343 g, 2.48 mmol) at room temperature and then the reaction mixture was heated at 70 °C for 16hrs. The reaction mixture was concentrated under reduced pressure to afford crude which was purified by silica gel flash column chromatography using 50-55 % ethyl acetate/heptane as eluent to afford 1 as a yellow sticky compound. Yield: 0.084g, 30.5%; LCMS m/z 445.0 [M + H]+ . [1663] Synthesis of tert-butyl 1-((5-aminopyridin-2-yl)oxy)-3,6,9,12-tetraoxapentadecan-15-oate (2) [1664] To a stirred solution of tert-butyl 1-((5-nitropyridin-2-yl)oxy)-3,6,9,12-tetraoxapentadecan- 15-oate (1, 0.4 g, 0.9 mmol) in methanol (5 mL) was added 10% palladium on carbon (0.4 g) and the reaction mixture was stirred under hydrogen gas atmosphere (balloon pressure) at room temperature for 4hrs. The reaction mixture was filtered through pad of celite and the filtrate was concentrated under reduced pressure to afford 2 as brown sticky liquid. Yield: 0.33 g. lCMS m/z 415.1 [M + H]+. [1665] Synthesis of 1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pyridin-2-yl)oxy)-3,6,9,12- tetraoxapentadecan-15-oic acid (LC40) [1666] To a solution of tert-butyl 1-((5-aminopyridin-2-yl)oxy)-3,6,9,12-tetraoxapentadecan-15-oate (2, 1.0 eq., 0.220 g, 0.531 mmol) in glacial acetic acid (2 mL) was added maleic anhydride (1.2 eq., 0.0625 g, 0.637 mmol) and the reaction mixture was stirred at 120 °C under microwave irradiation for 10 mins. Then reaction mixture was cooled to 0 °C and trifluoroacetic acid (1.5 mL) was added. The resulting reaction mixture was further stirred at room temperature for 3hrs. The reaction mixture was concentrated and purified with reverse phase prep-HPLC (35% acetonitrile in water with 0.1% acetic acid) to afford LC40 as an off white solid. Yield: 0.15 g, 64.46%, LCMS m/z 439.3 [M + H]+.1H-NMR (400 MHz, DMSO-d6 D2O exchange): δ 8.09 (d, J = 2.8 Hz, 1H), 7.67 (dd, J = 8.8, 2.4 Hz, 1H), 7.15 (s, 2H), 6.95 (d, J = 8.8 Hz, 1H), 4.38 (t, J = 4.4 Hz, 2H), 3.74 (t, J = 4.4 Hz, 2H), 3.59-3.55 (m, 4H), 3.52- 3.50 (m, 2H), 3.47 (d, J = 7.2 Hz, 8H), 2.42 (t, J = 6.0 Hz, 2H).
Figure imgf000534_0001
[1667] Synthesis of 4-methyl-4-nitroheptane-1,7-diamine (1) [1668] To a solution of 4-methyl-4-nitroheptanedinitrile (5.0 g, 27.6 mmol) in dry tetrahydrofuran (25 mL) at 0 °C was added slowly borane tetrahydrofuran complex (1M in tetrahydrofuran, 9.49 g, 5.0 eq., 110 mmol) and the resulting reaction mixture was heated at 80 °C for 24hrs. The reaction mixture was allowed to cool to room temperature and concentrated hydrochloric acid solution was added dropwise to acidify (pH~1). The resultant reaction mixture was stirred at 50 °C for 30 mins and concentrated under reduced pressure. The residue was neutralized by adding sodium hydroxide solution (40%, 80 mL) and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulphate, filtered, and concentrated under reduced pressure to obtain 1 as colourless oil. Yield: 3.80 g (Crude), 84.7%; LCMS m/z 190.2 [M+H]+ [1669] Synthesis of di-tert-butyl (4-methyl-4-nitroheptane-1,7-diyl)dicarbamate (2) [1670] A solution of 4-methyl-4-nitroheptane-1,7-diamine (1, 4.0 g, 21.1 mmol) in methanol (25 mL) was cooled to 0 °C. Then di-tert-butyl dicarbonate (15.2 g, 3.3 eq., 69.7 mmol) and triethylamine (12.4 mL, 4.2 eq., 88.8 mmol) were added and the reaction mixture was reflux for 6hrs. After completion, the reaction mixture was concentrated under reduced pressure to get crude, which was purified by silica gel flash chromatography using 30-40% ethyl acetate-heptane as eluent to afford 2 as colourless viscous oil. Yield: 3.8 g, 44.8%; LCMS: m/z 390.15 [M+H]+ [1671] Synthesis of di-tert-butyl (4-amino-4-methylheptane-1,7-diyl)dicarbamate (LC42) [1672] A suspension of nickel dichloride hexahydrate (1.83 g, 1.0 eq., 7.7 mmol) in methanol (60 mL) was cooled to 0 °C. Then sodium borohydride (0.874 g 3.0 eq., 23.1 mmol) was added and stirred for 30 mins. Thereafter, reaction mixture was further diluted with methanol (20 mL), and di-tert-butyl (4- methyl-4-nitroheptane-1,7-diyl)dicarbamate (2, 3.0 g, 7.7 mmol) was added followed by another portion of sodium borohydride (0.874 g 3.0 eq., 23.1 mmol). The resulting black suspension was stirred at room temperature for 4hrs. After completion, the black suspension was filtrated through celite bed, and the filtrate was concentrated under reduced pressure to get crude. The crude was purified by RP prep-HPLC (25-30% acetonitrile in water with 0.1% acetic acid) to afford LC42 as a white crystalline solid. Yield: 0.775 g, 27.9%; LCMS: m/z 360.2 [M+H]+; 1H NMR (400 MHz, DMSO-d6): δ 6.83 (t, J = 5.60 Hz, 2H), 2.86 (d, J = 6.00 Hz, 4H), 1.86 (s, 2H), 1.37-1.32 (m, 26H), 1.00 (s, 3H). Synthesis of LC45
Figure imgf000535_0001
[1673] Synthesis of 3,3'-((2-aminopropane-1,3-diyl)bis(oxy))dipropanenitrile (2) [1674] To a solution of tert-butyl N-[1,3-bis(2-cyanoethoxy)propan-2-yl]carbamate (1, 1.0 g, 3.36 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (10 mL) at 0 °C, and the reaction mixture was stirred at room temperature for 3hrs. The reaction mixture was concentrated under reduced pressure to give crude which was further triturated with ether to afford 2 as yellow oil. Yield: 0.5 g, 65.3 %; LCMS m/z 198.1 [M + H]+. [1675] Synthesis of benzyl (1,3-bis(2-cyanoethoxy)propan-2-yl)carbamate (3) [1676] To a solution of 3,3'-((2-aminopropane-1,3-diyl)bis(oxy))dipropanenitrile (2, 6.0 g, 30.4 mmol) in a mixture of 1,4-dioxane (20 mL) and water (5 mL) was added sodium carbonate (12.9 g, 4 eq., 122 mmol) and the reaction mixture was cooled to 0 °C. Then benzyl chloroformate (17.2 g, 1.3 eq., 39.5 mmol) was added to the reaction mixture and the reaction mixture was stirred at room temperature for 24 hrs. The reaction mixture was diluted with water and product extracted with ethyl acetate. Ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to get crude which was purified by silica gel flash column chromatography using 30-40% ethyl acetate in heptane as eluent to obtain 3 as a white solid. Yield: 3.0 g, 47%; LCMS m/z 332.1 [M + H]+. [1677] Synthesis of benzyl (2,2,20,20-tetramethyl-4,18-dioxo-3,9,13,19-tetraoxa-5,17- diazahenicosan-11-yl)carbamate (4) [1678] To a solution of nickel (II) chloride hexahydrate (2.66 g, 2 eq., 11.2 mmol) in methanol (80 mL) was added sodium borohydride (0.424 g, 2 eq., 11.2 mmol) and the reaction mixture was sonicated for 30 mins. Then benzyl (1,3-bis(2-cyanoethoxy)propan-2-yl)carbamate (3, 1.8 g, 5.6 mmol) in methanol (10 mL) was added along with di-tert-butyl dicarbonate (4.89 g, 4 eq., 22.4 mmol) to the reaction mixture and the reaction mixture was sonicated for 4 hrs, with the addition of sodium borohydride (0.21 g) after every 30 mins. The progress of the reaction was monitored by LCMS. After completion of reaction, the reaction mixture was filtered through celite bed and the filtrate was concentrated under reduced pressure to get crude which was purified by silica gel flash column chromatography using 30% ethyl acetate in heptane as an eluent to give 4 as colorless oil. Yield: 2.50 g, 81.9%. LCMS m/z 540.1 [M+1]+. [1679] Synthesis of benzyl (1,3-bis(3-aminopropoxy)propan-2-yl)carbamate (LC45) [1680] To a solution of benzyl (2,2,20,20-tetramethyl-4,18-dioxo-3,9,13,19-tetraoxa-5,17- diazahenicosan-11-yl)carbamate (4, 0.55 g, 1.02 mmol) in dichloromethane (5 mL) was added 4M HCl in 1,4-dioxane (5 mL) at 0°C, then reaction was stirred at room temperature for 12 hrs. The reaction mixture was then concentrated and purified by reverse phase prep HPLC (10-18 % acetonitrile in water with 0.1% trifluoroacetic acid) to afford LC45 as colorless gum. Yield: 0.267 g, 77.2% LCMS m/z 340.2 [M + H]+.1H NMR (400 MHz, DMSO-d6 with D2O) δ 7.39-7.30 (m, 5H), 7.28 (d, J = 8.0 Hz, 1H), 5.02 (s, 2H), 3.81-3.76 (m, 1H), 3.45-3.34 (m, 8H), 2.86-2.79 (m, 4H), 1.78-1.71 (m, 4H). [1681] The following compounds were prepared according to the procedures described herein using the appropriate starting materials.
Figure imgf000536_0001
Preparation of Conjugates Example 2: Conjugation of isothiocyanate-based ligand-linker compounds with anti-EGFR and anti-PD-L1 antibodies. [1682] This example provides a general protocol for the conjugation of the isothiocyanate-based ligand-linker compounds with the primary amines on lysine residues of anti-EGFR antibodies (e.g., matuzumab, cetuximab) and anti-PD-L1 antibodies (e.g., atezolizumab, anti-PD-L1(29E.2A3)). [1683] The antibody is buffer exchanged into 100 mM sodium bicarbonate buffer pH 9.0 at 5 mg/mL concentration, after which about 30 equivalents of the isothiocyanate-based ligand-linker compound (e.g., freshly prepared as 20 mM stock solution in DMSO) is added and incubated overnight at ambient temperature in a tube revolver at 10 rpm. [1684] The conjugates are purified using a PD-10 desalting column (GE Healthcare) and followed by formulating the final conjugate into PBS pH 7.4 with Amicon Ultra 15 mL Centrifugal Filters with 30 kDa molecular weight cutoff. Example 3: Conjugation of perfluorophenoxy-based ligand-linker compounds with anti-EGFR and IgG antibodies. [1685] This example provides a general protocol for the conjugation of the perfluorophenoxy-based ligand-linker compounds (e.g., Compound I-141) with the primary amines on lysine residues of anti- EGFR antibodies (e.g., matuzumab, cetuximab) and IgG antibodies (e.g., IgG2a-UNLB). [1686] The antibody is buffer exchanged into 50 mM sodium phosphate buffer pH 8.0 at 5 mg/mL concentration, after which about 22 equivalents of perfluorophenoxy-based ligand-linker compound (e.g., Compound I-141; freshly prepared as 20 mM stock solution in DMSO) is added and incubated for 3 hours at ambient temperature in a tube revolver at 10 rpm. [1687] The conjugates containing on average four ligand-linker moieties per antibody are purified using a PD-10 desalting column (GE Healthcare) and followed with formulating the final conjugate into PBS pH 7.4 with Amicon Ultra 15 mL Centrifugal Filters with 30 kDa molecular weight cutoff. Example 4: Determination of LPR values by mass spectrometry. [1688] This example provides the method for determining LPR (ligand-linker to protein ratio), also referred to as DAR values for the conjugates prepared as described in Examples 55 and 56. To determine the DAR value, 10 μg of the antibody (unconjugated or conjugated) was treated 2 μL of non-reducing denaturing buffer (10X, New England Biolabs) for 10 minutes at 75 °C. The denatured antibody solution was then deglycosylated by adding 1.5 μL of Rapid-PNGase F (New England Biolabs) and incubated for 10 minutes at 50 °C. Deglycosylated samples were diluted 50-fold in water and analyzed on a Waters ACQUITY UPLC interfaced to Xevo G2-S QToF mass spectrometer. Deconvoluted masses were obtained using Waters MassLynx 4.2 Software. DAR values were calculated using a weighted average of the peak intensities corresponding to each loading species using the formula below:
Figure imgf000537_0001
[1689] DAR values for the conjugates prepared are shown in Tables 15 to 18. Example 5: Determination of purity of conjugates by SEC method. [1690] Purity of the conjugates prepared as described in Examples 55 and 56 was determined through size exclusion high performance liquid chromatography (SEC-HPLC) using a 20 minute isocratic method with a mobile phase of 0.2 M sodium phosphate, 0.2 M potassium chloride, 15 w/v isopropanol, pH 6.8. An injection volume of 10 μL was loaded to a TSKgel SuperSW3000 column, at a constant flow rate of 0.35 mL/min. Chromatographs were integrated based on elution time to calculate the purity of monomeric conjugate species. Example 6: Antibody disulfide reduction and thiol-reactive ligand-linker conjugation to antibody. [1691] This example provides an exemplary protocol for reduction of the disulfides of the antibodies described herein, and conjugation of the reduced antibodies to thiol-reactive ligand-linker compounds described herein, e.g., containing a maleimide chemoselective ligation group. [1692] Protocol: Antibody disulfide reduction A) Dilute antibody to 15 mg/mL (0.1 mM IgG) in PBS, pH 7.4. B) Prepare a fresh 20 mM (5.7 mg/mL) stock solution of tris(2 carboxyethyl)phosphine (TCEP) in H2O. C) Add 25 µL of TCEP stock solution from step B) above to 1 mL of antibody from step A) above (0.5 mM final concentration TCEP). D) Incubate at 37 °C for 2 hours (check for free thiols using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) test). E) Aliquot the reduced antibody into 4 tubes (250 µL each). [1693] Ligand-linker conjugation to antibody A) Prepare 10 mM stock solution of thiol-reactive ligand-linker compound in DMSO (DMA, DMF or CH3CN are also acceptable). B) Add 5 equivalents of 12.5 µL stock solution from step A) above to each tube of reduced antibody (0.5 mM final concentration ligand-linker compound stock solution). C) Incubate overnight at 4 °C for 4 hours at room temperature; check for free thiols using DTNB test. D) Run analytical hydrophobic interaction chromatography (HIC) to determine DAR and homogeneity. Example 6: Preparation of Omalizumab Conjugates [1694] A series of conjugates of the exemplary antibody omalizumab (an anti-IgE antibody) with a series of perfluorophenyl ester containing ligand-linker compounds were prepared and characterized using methods similar to those described in Examples 62 to 65. [1695] These conjugates were assessed for cell uptake activity as described in Example 7.
Figure imgf000538_0001
Figure imgf000538_0002
Figure imgf000538_0003
Figure imgf000539_0001
Assessment of Activity of Compounds and Conjugates [1696] Example 8: Alexa Fluor labelling. [1697] Cetuximab, matuzumab and human IgG isotype antibodies are conjugated to Alexa Fluor 647 using Alexa Fluor™ Protein Labeling Kit (Invitrogen) per the manufacturer’s protocol. In brief, antibodies to be labeled are diluted to 2 mg/mL in PBS to a total volume of 500 μL. A 15 DOL (degree of labeling) is used for the conjugation with the fluorophore. Free dye is removed by pre-wetting an Amicon 30 kDa filter with PBS. After incubation, the conjugation reaction is then added to the filter and spun at high speed for 10 minutes. Retained solution is then resuspended in PBS to a final volume of 1 mL and stored at 4 °C indefinitely. [1698] The above procedure can be adapted to fluorescently label a variety of antibodies or target proteins of interest with alternative dyes such as Alexa Fluor™ 488 using e.g., NHS – lysine conjugation chemistry. Example 9: Measurement of EGFR/IgG levels by surface staining. [1699] This example provides a protocol for the measurement of the time course activity of cetuximab, and matuzumab conjugates on surface EGFR and IgG levels in Hela parental cells measured by surface staining. [1700] Day -1 [1701] 1e6 Hela parental cells are plated in 2 mL of media in 6 well plates. [1702] Day 0 [1703] Media are replaced with 1.5 mL of fresh media. [1704] PBS, unconjugated antibodies and conjugated antibodies are added to respective wells at a final concentration of 20 nM. [1705] Day 1/2/3 [1706] Media is aspirated from wells and washed thrice with PBS.750 μL of Enzyme-Free Dissociation buffer is added and cells are allowed to detach on ice. [1707] Cell are collected in tubes and spun down at 300xg for 5 mins @ 4 °C. [1708] Cells are resuspended in PBS and volume is split equally into two tubes. [1709] All tubes are spun at 300xg for 5 mins at 4 °C. One set, the PBS is aspirated and pellets are frozen at -80 °C. [1710] The other set, the PBS is aspirated and washed 2x with cold FACS buffer. [1711] After final wash, pellets are resuspended in 300 μL FACS buffer. [1712] The 300 μL suspension is split into three wells (100 μL each) of a 96 well plate. Set 1: Ctx::AF647 at 1:100 dilution and incubated on ice in the dark for 1 h. Set 2: Mtz::AF647 at 1:100 dilution and incubated on ice in the dark for 1 h. Set 3: Goat anti-human IgG PE at 2 μg/mL and incubated on ice in the dark for 1 h. [1713] Cells are spun down at 1000xg at 4 °C for 3 minutes and liquid is decanted. Cell pellets are resuspended in 200 μL of cold FACS buffer. Repeated 3x total. [1714] After final wash and decant, cells are resuspended in 100 μL cold FACS buffer with DAPI (25 ug/mL final). [1715] Stained cells are then analyzed on Biorad ZE5. Example 10: Live-Cell EGFR Surface Staining by Flow Cytometry. [1716] This example provides an alternate protocol for the determination of the effect of matuzumab conjugates on surface EGFR levels measured by surface staining using flow cytometry. [1717] Hela parental cells are plated in 6 well plates and treated with vehicle (PBS), unconjugated anti-EGFR antibody (matuzumab, Mtz), or matuzumab conjugates for the indicated period of time. [1718] After incubation, media is aspirated and cells are washed three times with PBS, lifted using Accutase and pelleted by centrifugation at 300xg for 5 minutes. Cells are resuspended in cold FACS buffer and kept cold for the remainder of the staining procedure. A portion of cells are excluded from staining procedure as an unstained control. Cells are stained with either human IgG Isotype-AF647 or cetuximab-AF647 conjugates for 1 h on ice in the dark. Cells are then spun at 300xg for 5 min at 4 °C and washed with cold FACS buffer for a total of three washes. After the final wash, cells are resuspended in 100 μL of FACS buffer with DAPI added at a final concentration of 5 μg/mL. Cells are analyzed using a BioRad ZE5 flow cytometer and data is analyzed using FlowJo software. Cells are first gated to remove debris, doublets and dead cells (DAPI negative). EGFR cell surface levels are determined based on AF647 mean fluorescence intensity (MFI). [1719] In parental Hela cells, treatment with the conjugated antibodies can result in reduced cell surface levels of EGFR compared to cells treated with unconjugated antibodies (Ctx or Mtz). [1720] Treatment of cells with the conjugates described herein can induce reduction in targeted cell surface receptors. Example 11: Measurement of total EGFR levels by Western blotting. [1721] This example provides the protocol for the measurement of the time course activity of cetuximab, and matuzumab conjugates on total EGFR levels in Hela parental cells measured by traditional Western blotting. [1722] Once all time-points from Example above are collected, all cell pellets are resuspended in 50 μL of radioimmunoprecipitation assay (RIPA) buffer (+protease/phosphatase inhibitor +nuclease). [1723] Lysates are incubated on ice for 1 h. [1724] Lysates are then spun at high-speed for 10 min at 4 °C [1725] 40 μL of cleared lysate is transferred to a 96 well plate. [1726] All lysate concentrations are calculated using BCA assay (1:3 dilution). [1727] All lysates are equalized to 2 mg/mL using RIPA as diluent. [1728] Equal volumes (15 μL) of lysate are then mixed with LDS sample buffer (3x LDS + 2.5x reducing agent). [1729] Samples are incubated at 98 °C for mins and allowed to cool. [1730] Samples are vortexed and spun down. [1731] 15 µL of sample is loaded onto a 26-well bis-tris 4-12% midi-gel. [1732] Gel is allowed to run at 180V for 20 mins. [1733] Gels are transferred to nitrocellulose membrane using iBlot 2 (20V constant, 7 mins). [1734] Membranes are washed 1x in PBS and then placed in Odyssey blocking buffer for 1h RT with shaking. [1735] Primary antibodies mouse anti-β-actin (SCB) and rabbit anti-EGFR (CST) are diluted 1:1000 in blocking buffer and allowed to incubate overnight at 4 °C with shaking. [1736] Membranes are washed thrice with PBS-T (Tween200.1%), at least 5 mins each wash. [1737] Secondary antibodies anti-mouse 680rd and anti-rabbit 800cw are diluted 1:5000 in blocking buffer and allowed to incubate for 1 h at RT with shaking. [1738] Membranes are washed thrice with PBS-T (Tween200.1%), at least 5 mins each wash. [1739] Membranes are imaged using licor odyssey scanner. Example 11: Measurement of total EGFR levels by in-cell Western blotting. [1740] This example provides a protocol for the measurement of the dose response of cetuximab and matuzumab conjugates on total EGFR levels in Hela parental measured by in-cell Western blotting. [1741] Day -1 [1742] 3e4 Hela parental cells are plated 100 μL per well in a clear bottom black walled 96 well plate (Costar 3603) [1743] Day 0 [1744] Media is decanted and 100 μL of fresh media is added back to wells. [1745] 50 μL of a 3x dose response of unconjugated and conjugated antibodies are added to respective wells. [1746] 80 nM final starting concentration, 1:2 dilution. EGF is added in 3 wells at a concentration of 50 ng/mL final. [1747] Day 2 [1748] Media is decanted and wells are washed thrice with PBS. [1749] Wells are fixed with 4% PFA in PBS for 15 minutes at RT. [1750] Wells are washed thrice with PBS. [1751] Cells are permeabilized with 0.2% triton-x100 in PBS for 15 mins. Repeated 3x total. [1752] Cells are blocked in Odyssey blocking buffer with 0.2% triton-x100 for 1 h at RT. [1753] Cells are stained with goat anti-EGFR (AF231, R&D, 1 μg/mL final) in block buffer overnight at 4 °C. [1754] Cells are washed 3x with PBS-T (Tween200.1%). [1755] Cells are stained with donkey anti-goat 800CW secondary (1:200) and CellTag700 (1:500) in blocking buffer for 1 h at RT in dark. [1756] Cells are washed 3x with PBS-T (Tween200.1%). [1757] Last wash is decanted and plates are blotted on paper towel to remove residual liquid. [1758] Plates are imaged on Licor scanner (3 mm offset). Example 12: Conjugates of M6PR or ASGPR binding compounds mediate uptake of IgG2a into human liver cancer cells [1759] The uptake of antibody conjugates of exemplary M6PR or ASGPR binding compounds was assessed in Hep G2 cells, using a method similar to that described in Example above. FIG.1 shows a graph of cell fluorescence versus antibody conjugate concentration indicating that various antibody conjugates of exemplary ASGPR binding compounds, and an example M6PR binding compound (see, e.g., PCT US21/12846, herein incorporated by reference) exhibited robust uptake into HepG2 cells after one hour incubation. Conjugates of compound 1209 (ASGPR binding compound I-124) (average loading DAR6), compound 1210 (ASGPR binding compound I-123) (average loading DAR4) and M6PR binding compound 520 (average loading DAR4) exhibited comparable HepG2 cellular uptake.
Figure imgf000542_0001
Figure imgf000543_0001
Example 13: Uptake of target protein in HepG2 Cells [1760] The omalizumab conjugates were bound to IgE-Alexa488 (prepared according to Example 2), as follows: equal molar ratios of omalizumab (anti-IgE) conjugate and IgE-Alexa488 were added in tissue culture media for 30 minutes at room temperature. The resulting anti-IgE conjugate:IgE antibody- Alexa488 compositions were added to HepG2 cells (100k cells/50ul per well, n=2), and Alexa488 fluorescence levels in the cells were measured at 1 hour by flow cytometry. As the fluorescently labelled target (IgE antibody) accumulates in cells, the fluorescence presents a way to measure total intracellular uptake by cells over time. [1761] FIG.2A shows a graph of cellular uptake of various conjugates of omalizumab (anti-IgE) with exemplary ASGPR ligand-linkers of Table 26, bound to Alexa488 labeled-target IgE in HepG2 cells. The conjugates are ordered in Table 26 according to the relative uptake activity as shown in FIG. 2A.
Figure imgf000543_0002
[1762] As seen in FIG.2A all of the conjugates include multivalent compounds (e.g., n = 3). The conjugate having a 1-triazole moiety (I-143, ASGPR ligand X4) has superior activity to the reference conjugate having a 1-oxygen moiety (I-124, ASGPR ligand X1). [1763] The conjugate having a 6-position oxygen linkage (I-136, ASGPR ligand X8) has superior activity to the reference conjugate I-124. [1764] The conjugate having a 1-triazole moiety and a shorter linkage from the ASGPR ligand to the branching point of the ligand (I-157, linker length of 6 atoms to branching point) exhibits less activity than the conjugate having a 1-triazole moiety and a longer linkage from the ASGPR ligand to the branching point (I-143, length of 14 atoms). [1765] FIG.2B shows a graph of cellular uptake of various conjugates of omalizumab (anti-IgE) with exemplary ASGPR ligand-linkers of Table 27, bound to Alexa488 labeled-target IgE in HepG2 cells. The conjugates are ordered in Table 27 according to the relative uptake activity as shown in FIG. 2B.
Figure imgf000544_0001
[1766] As seen in FIG.2B all of the conjugates include the ASGPR ligand X1 and branched trivalent linker L29, or its divalent equivalent L30. The conjugates having longer linkers between the ASGPR linker and Y (e.g., conjugates of compounds I-137 and I-129) exhibit comparable activity to the reference conjugate (e.g., conjugate of compound I-124). [1767] The reference conjugate (I-124, n = 3) showed superior activity to the divalent conjugate (I- 144, n = 2). [1768] FIG.2C shows a graph of cellular uptake of various conjugates of omalizumab (anti-IgE) with exemplary ASGPR ligand-linkers of Table 28, bound to Alexa488 labeled-target IgE in HepG2 cells. The conjugates are ordered in Table 28 according to the relative uptake activity as shown in FIG. 2C.
Figure imgf000544_0002
[1769] As seen in FIG.2C the conjugate having an S-glycoside (I-141, ASGPR ligand X2) shows superior uptake to that of the O-glycoside (I-145, ASGPR ligand X1). [1770] The conjugates having a 1-methylene moiety (I-140, ASGPR ligand X3) has comparable activity to the conjugate having the O-glycoside (I-145, ASGPR ligand X1). [1771] FIG.2D shows a graph of cellular uptake of various conjugates of omalizumab (anti-IgE) with exemplary ASGPR ligand-linkers of Table 29, bound to Alexa488 labeled-target IgE in HepG2 cells. The conjugates are ordered in Table 29 according to the relative uptake activity as shown in FIG. 2D.
Figure imgf000545_0002
[1772] As seen in FIG.2D the conjugate having a valency of 3 (I-145, n = 3) shows superior uptake to conjugates having a valency of 2 or 1 (I-111, n =2; and I-146, n=1). [1773] The conjugates having the linker attached to the ASGPR ligand at the 2-position (e.g., I-153, ASGPR ligand X10; I-154, ASGPR ligand X11; and I-155, ASGPR ligand X12) also exhibit activity in this assay, however, are less active than the 1-position linked conjugate (e.g., I-145, ASGPR ligand X1). Example 14: Binding Affinity Assessment of Trivalent compounds by Fluorescence Polarization Assay [1774] Example trivalent compounds (1901 (I-171), 1902 (I-172), XB32 and 2101) were assessed for binding affinity to ASGPR by fluorescence polarization assay as compared to a reference compound XB149 (structure below) as a positive control, and the corresponding carboxylic acid of compound I-124 (I-124acid, structure below) as a second reference compound.
Figure imgf000545_0001
Figure imgf000546_0001
[1775] Assay Procedure: ASGPR binding was measured in black 96-well plates using a fluorescence polarization assay. A fluorescent probe consisting of a tri-GalNAc linked to Cy5 (GalNac- Cy5, depicted below) was custom synthesized. Example compounds were resuspended in DMSO and 3- fold serial dilutions were made at 100x final concentrations. Binding reactions were conducted in 100 µl final volume in 20 mM Hepes (pH 7.5) 50 mM NaCl 5 mM CaCl20.015% Triton X-1001% DMSO with 80 nM ASGPR (Acro Biosystems) and 1 nM probe. Fluorescence polarization was measured using λex = 620 nm, λem = 688 nm on an Envision plate reader (Perkin Elmer) after 2 hr incubation time. Dose responses were conducted in duplicate and normalized to the response with DMSO (high) and 1 µM reference compound XB149 (low) on each plate. IC50 values were determined by fitting to 4-parameter curves in GraphPad Prism.
Figure imgf000547_0001
[1776] Dose response curves were measured for example compounds. It was observed that Compounds 1901 (I-171), 1902 (I-172) and XB32 bind ASGPR with comparable affinity as reference compound XB149. The other reference compound I-124acid had reduced ASGPR binding affinity as compared to other trivalent compounds assayed. [1777] FIG.3 illustrates the fluorescence polarization screening results for example trivalent compounds (1901 (I-171), 1902 (I-172), XB32 and 2101). [1778] The IC50 ranges for the compounds assayed are presented in Table 30. In Table 30, A: IC50 ≤ 50 nM; B: 50 nM < IC50 ≤100 nM; and C: IC50 > 100 nM
Figure imgf000547_0003
Figure imgf000547_0002
Example 15: Binding Affinity Assessment of Monomer compounds by Fluorescence Polarization Assay [1779] Example monovalent compounds (depicted below) were assessed at 100 µM as compared to a reference compound XB149 as a positive control, following the assay procedure outlined in Example 8.
Figure imgf000548_0001
[1780] It was observed that all monovalent compounds screened, with the exception of compound 593, bind with at least 50% of the activity of reference compound XB149. [1781] FIG.4 illustrates the binding of example monovalent compounds (591, XB20, XB23, XB21, 592 and 593) as a percentage of the activity of reference compound XB149. [1782] Dose response curves were measured for example compounds. It was observed that Compound 591 binds ASGPR with the highest affinity of the tested monomers. [1783] FIG.5 illustrates the fluorescence polarization screening results for example monovalent compounds (XB20, XB21, 592, XB23, 591). [1784] The IC50 ranges for the monovalent compounds assayed are presented in Table 31. In Table 31, A: IC50 ≤ 1 µM; B: 1 µM < IC50 ≤50 µM; and C: IC50 > 50 µM
Figure imgf000548_0002
Example 16: Binding Affinity Assessment of Exemplary ligands [1785] The binding affinity of example compounds were assessed. In Table 31B and reported as follows: A: 1nM < Kd ≤ 10 nM; B: 10 nM < Kd ≤100 nM; C: 100 nM < Kd ≤1 uM; D: 1 uM < KD ≤10 uM; E: Kd > 10 uM.
Figure imgf000549_0001
Figure imgf000549_0002
Figure imgf000550_0002
Figure imgf000550_0001
Example 17: Comparison of trivalent compounds of formula (Ib) with atom variations at the anomeric position (1-position, Z1 group) [1786] To investigate the merits of alternative glycoside linkages at the anomeric position of the ASGPR ligand (e.g., S-glycoside linkage and C-glycoside linkages), compounds having various alternative linkages at the anomeric position (1-position) of the ASGPR ligand were compared to reference compound having a 1-position O-glycoside linkage (compound I-163). [1787] OMA Ab conjugates of example compounds I-160-I-163 and I-141 were bound to IgE- Alexa488 (prepared by adapting literature procedure), as follows: equal molar ratios of OMA(anti-IgE) conjugate and IgE-Alexa488 were added in tissue culture media for 30 minutes at room temperature. The resulting anti-IgE conjugate:IgE antibody-Alexa488 compositions were added to HepG2 cells (100k cells/50ul per well, n=2), and Alexa488 fluorescence levels in the cells were measured at 1 hour by flow cytometry. As the fluorescently labelled target (IgE antibody) accumulates in cells, the fluorescence presents a way to measure total intracellular uptake by cells over time. [1788] FIG.6 shows a graph of cellular uptake of various conjugates of OMA (anti-IgE) with example compounds of table 32 bound to Alexa488 labeled-target IgE in HepG2 cells. The conjugates are ordered in Table 32 according to the relative uptake activity as shown in FIG.6.
Figure imgf000551_0001
[1789] As seen in FIG.6 all of the conjugates include multivalent compounds (e.g., n = 3) linked to the linker through the anomeric position (1-position of the ASGPR ligand). The conjugate having a 1- triazole moiety (I-161) has essentially identical activity to the reference conjugate having a 1-oxygen moiety (I-163). The conjugate having a 1-position -S-glycoside linkage has similar activity to compounds I-163 and I-161. [1790] In vivo comparison of example compounds having atom variations at the anomeric position [1791] The three most active anomeric position variants (I-161, I-162 and I-160) identified in the HepG2 uptake assay were evaluated in comparison to the O-glycoside linked compound I-163 in vivo. Pharmacokinetic studies [1792] Pharmacokinetic (PK) studies were undertaken to determine the pharmacokinetic profile of various OMA linked example compounds (I-161-I-163). [1793] Protocol: Female C57BL/6 mice are dosed via IV with 5 mg/kg of OMA-example compounds, and OMA-reference. Serum samples are collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 72, and 168 hours after dosing with 3 animals per timepoint. [1794] Serum hIgG concentrations were measured by ELISA. [1795] FIG.7 illustrates affinity-dependent clearance of OMA-linked example compounds (I-160 to I-163) as compared to OMA IgG antibody alone (reference). [1796] These results demonstrate that compounds with alternative anomeric position glycoside linkages to oxygen (e.g., 1-position -S- (I-162), -CH2- (I-160) and -triazole- (I-161)) have distinct PK profiles from reference compound I-163 (1-position -O- linkage), with near complete depletion of particular OMA linked compounds to lower limit of detection (LLOD) by day 7. Pharmacodynamic Studies [1797] A dose titration of the effect of OMA-linked compound I-163 on target IgE clearance was undertaken to determine the pharmacodynamic (PD) parameters of IgE and OMA-I-163 in mice. [1798] Protocol: Female C57BL/6 mice were IV dosed 1 mg/kg of IgE followed by 0.1, 0.3, 1, or 3 mg/kg of OMA-I-16316 hours later. Serum samples were collected at 0 (pre-bleed), 1, 4, and 24 hours after OMA-I-163 dose with 3 animals per timepoint. [1799] Serum hIgE and hIgG concentrations were measured by ELISA. [1800] FIG.8 illustrates dose titration OMA-I-163 IgE clearance. [1801] These results demonstrate that compound OMA-I-163 at 0.1 mg/kg gave partial clearance of IgE dosed at 1 mg/kg at 2 and 4 h, and complete clearance at 24 h. From these results the limiting dose of IgE and OMA-I-163 in mice was determined to be 0.1 mg/kg. [1802] Next, the effect of example compound conjugate on IgE clearance was investigated at the determined limiting dose to compare the PD activity of OMA-I-163 (reference compound) to OMA- linked compounds I-160, I-161 and I-162. [1803] Protocol: Female C57BL/6 mice were IV dosed 1 mg/kg of IgE followed by 0.1 mg/kg of OMA-I-163, OMA-I-162, OMA-I-161, and OMA-I-16016 hours later. Serum samples were collected at 0 (pre-bleed), 1, 4, and 24 hours after LYTAC dose with 3 animals per timepoint. [1804] Serum concentrations were measured by Elisa. [1805] FIG.9 illustrates the clearance of target IgE via OMA-example compounds (I-160 to I-163) as compared to hIgE (reference) was affinity-dependent. [1806] These results demonstrate that at low dose (0.1 mg/kg of the example OMA conjugate) OMA example compounds with alternative anomeric position glycoside linkages to oxygen (e.g., 1-position -S- (I-162), -CH2- (I-160), and -triazole- (I-161)) clear IgE more efficiently than the reference compound with an oxygen linkage at the anomeric position (OMA-I-163). All of compounds OMA-I-60, OMA-I- 161, and OMA-I-162 cleared IgE completely within 4 hours. [1807] In summary, in vivo and in vitro data set for ASGPR compounds with alternative anomeric position glycoside linkages to oxygen indicates that increased target clearance can be attained by replacing the 1-position oxygen atom, e.g., by -S-, -CH2-, or -triazole-. Example 18: Internalization of Conjugates in Cells (Uptake Assay) [1808] HepG2 cells (25,000) were plated in 96-well plates 48 hours before the experiment was performed. On the day of the experiment, indicated the conjugate was mixed with an equimolar stock of Alexa Fluor647 labelled human IgE (Enzo Life Sciences, BPD-DIA-HE1) in fresh EMEM as a 600 nM stock. The precomplexed conjugate:IgE mixture was incubated at room temperature for 30 minutes. During the precomplexing step, media from HepG2 was removed, and 40 ul of EMEM with 1/100 Fc Receptor Blocking Solution (Biolegend Human TruStain FcX, 422302) was added. Cells incubated in the receptor blocking solution for 10 minutes while making serial dilutions (1:3) of conjugate:IgE stock solution in EMEM.40 ul of the conjugate:IgE mixture in EMEM was then added to cells (six dilutions, with final concentrations ranging from 300 nM to 1.23 nM) for 2 hours before washing with DPBS and detaching with 0.25% Trypsin/EDTA. Cells were pelleted in 96-well conical bottom plates, washed once with FACS Buffer with BSA (Rockland Immunochemicals), and resuspended in FACS Buffer with BSA. Uptake of fluorescently labelled IgE was measured by analyzing median cellular AF647 intensity by flow cytometry. [1809] Table 38 shows uptake (cellular internalization) of Alexa Fluor 647 labelled IgE in wild type and ASGPR1/2 KO HepG2 cells treated with the conjugate. Cells were incubated with equimolar ratio of conjugate and AF647 IgE at indicated concentrations, with data normalized to unconjugated parent protein (Oma S35H/Y57H-LC). No significant increase in uptake was observed in ASGPR1/2 KO cells versus the unconjugated control.
Figure imgf000553_0001
Example 19: Lysosomal Degradation of Target [1810] HepG2 cells (15,000) were plated in 96-well plates 72 hours before performing the experiment. On the day of the experiment, indicated the conjugate was diluted in fresh EMEM and mixed with an equimolar stock of LysoLight-labelled human IgE (Enzo Life Sciences, BPD-DIA-HE1), at 200 nM of each compound. The precomplexed conjugate:IgE mixture was incubated at room temperature for 30 minutes. During the precomplexing step, media from HepG2 was removed, and 40 ul of EMEM with 1/100 Fc Receptor Blocking Solution (Biolegend Human TruStain FcX, 422302) was added. Cells incubated in the receptor blocking solution for 10 minutes while making serial dilutions (1:3) of conjugate:IgE stock solution in EMEM.40 ul of the conjugate:IgE mixture in EMEM was then added to cells (five dilutions, with final concentrations ranging from 100 nM to 1.23 nM). Cells were placed in an IncuCyte Live-Cell Analysis System (Sartorius) for long term live cell imaging analysis (10x magnification, 3 images per well). One hour after addition of compound, image acquisition to measure red fluorescence from the LysoLight probe was started, with further Images collected every hour for 72 hours. Fluorescence of the LysoLight probe only occurs when cleaved by lysosomal cathepsin proteases, confirming lysosomal delivery and degradation of target. After 72 hours, mean red fluorescence was measured for each time point, and the area under the curve of fluorescence over time was used to measure total degradation over the 3-day time period. [1811] Table 39 shows lysosomal degradation of target in the conjugate treated cells tracked with IgE conjugated to a quenched cathepsin-cleavage fluorescence probe. HepG2 cells were incubated with equimolar conjugate:LysoLight IgE (Thermo Fisher LysoLight Deep Red) at indicated concentrations; deep red fluorescence observed from cleavage of IgE in the lysosome was tracked using live cell imaging over three days and quantified as the area under the curve of fluorescence signal over time, normalized to signal from unconjugated parent protein (Oma S35H/Y57H-LC). Background degradation of unconjugated control was significantly higher at 100 nM than at 3.7 nM due to increased nonspecific internalization from pinocytosis, leading to smaller fold changes of conjugates versus control.
Figure imgf000554_0001
Figure imgf000555_0001
EQUIVALENTS AND INCORPORATION BY REFERENCE [1812] While the disclosure has been particularly shown and described with reference to certain embodiments and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the disclosure. [1813] All publications, patents, patent applications, including International Application No. PCT/US2022/037227, filed July 14, 2022, and U.S. Provisional Application No.63/439,811, filed January 18, 2023, and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

Claims

WHAT IS CLAIMED IS: 1. A compound of formula (I):
Figure imgf000556_0001
or a prodrug thereof, or a salt thereof, wherein: n is 1 to 500; m is 1 to 20; Y is a moiety of interest; L is a linker; and X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (II):
Figure imgf000556_0002
wherein: R1 is selected from –Z1–*, –H, –OH, optionally substituted (C1-C6)alkyl, –OCH3,–OCH2CH=CH, optionally substituted -S-(C1-C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, and optionally substituted -S-heteroaryl; R2 is selected from –Z1–*, –NHCOCH3, –NHCOCF3, –NHCOCH2CF3, –OH, –NHR, and optionally substituted triazole; R6 is selected from –Z1–*, –OH, -OR, optionally substituted (C1-C6)alkyl, –OC(O)R, -C(O)NHR, -NRxxRyy, optionally substituted aryl, optionally substituted heteroaryl, –NHCOR, and –NRCOR; each R is independently optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl; wherein one of R1, R2, and R6 is –Z1–*, and “ * ” represents a point of connection of Z1 to the linker (L); R3 and R4 are each independently H, or a promoiety, or R3 and R4 are cyclically linked to form a promoiety; R11 is H, or a bridging moiety that connects the 5-position carbon to the 1-position carbon of the ring; Z1 is a linking moiety selected from -Z11-, -Z11-A1-, -A2-, -NR21CO-, - CONR21-, -NR21SO2-, - SO2NR21-, -NR21C(=O)NR21-, and -NR21C(=S)NR21-; -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2; -A1- and -A2- are optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene; each R21 is independently selected from H, optionally substituted (C1-C6)alkyl, -COR, and optionally substituted heteroaryl; and each R22 is independently selected from H, halogen, and optionally substituted (C1-C6)alkyl; provided at least one of the following occurs: A) Z1 is a linking moiety selected from -Z11-A1- and -A2-; and -A1- and -A2- are optionally substituted heterocyclylene; or -A2- is optionally substituted isoxazolyl; B) -L-Y comprises:
Figure imgf000557_0001
C) R6 is -OR, optionally substituted (C1-C6)alkyl, –OC(O)-optionally substituted heteroaryl, - C(O)NH-optionally substituted heteroaryl, -NRxxRyy, optionally substituted aryl, optionally substituted heteroaryl–NHCOR, or –NRCOR, provided the heteroaryl is other than triazole; where each R is independently optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl; D) R1 is optionally substituted C2-6 alkyl, optionally substituted -S-(C1-C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, or optionally substituted -S-heteroaryl; or E) at least one R21 is -COR or optionally substituted heteroaryl.
2. The compound of claim 1, wherein X is of formula (a-II):
Figure imgf000558_0001
(a-II).
3. The compound of claim 1 or 2, wherein Z1 is a linking moiety selected from -Z11-A1- and -A2-; and -A1- and -A2- are optionally substituted heterocyclylene.
4. The compound of any preceding claim, wherein
Figure imgf000558_0002
5. The compound of any preceding claim, wherein -L-Y comprises:
Figure imgf000558_0003
Figure imgf000559_0001
6. The compound of any preceding claim, wherein R6 is -OR, optionally substituted (C1-C6)alkyl, – OC(O)-optionally substituted heteroaryl, -C(O)NH-optionally substituted heteroaryl, -NRxxRyy, optionally substituted aryl, or optionally substituted heteroaryl, provided the heteroaryl is other than triazole; where R is optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl.
7. The compound of any preceding claim, wherein R6 is -O-(C1-C6)alkyl, optionally substituted heterocyclyl, or -O-aryl.
8. The compound of any preceding claim, wherein R1 is optionally substituted C2-6 alkyl, optionally substituted -S-(C1-C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, or optionally substituted -S-heteroaryl
9. The compound of any preceding claim, wherein R1 is optionally substituted C2-6 alkyl.
10. The compound of any preceding claim, wherein at least one R21 is -COR or optionally substituted heteroaryl.
11. The compound of any preceding claim, wherein Y is an antibody or antibody fragment.
12. A compound of formula (I):
Figure imgf000559_0002
or a prodrug thereof, or a salt thereof, wherein: n is 1 to 500; m is 1 to 20; L is a linker; and X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (II):
Figure imgf000560_0001
wherein: R1 is selected from –Z1–*, –H, –OH, optionally substituted (C1-C6)alkyl, –OCH3,–OCH2CH=CH, optionally substituted -S-(C1-C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, and optionally substituted -S-heteroaryl; R2 is selected from –Z1–*, –NHCOCH3, –NHCOCF3, –NHCOCH2CF3, –OH, and optionally substituted triazole; R6 is selected from –Z1–*, –OH, -OR, optionally substituted (C1-C6)alkyl, –OC(O)R, -C(O)NHR, -NRxxRyy, optionally substituted aryl, and optionally substituted heteroaryl, R is optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl; wherein one of R1, R2, and R6 is –Z1–*, and “ * ” represents a point of connection of Z1 to the linker (L); R3 and R4 are each independently H, or a promoiety, or R3 and R4 are cyclically linked to form a promoiety; R11 is H, or a bridging moiety that connects the 5-position carbon to the 1-position carbon of the ring; Z1 is a linking moiety selected from -Z11-, -Z11-A1-, -A2-, -NR21CO-, - CONR21-, -NR21SO2-, - SO2NR21-, -NR21C(=O)NR21-, and -NR21C(=S)NR21-; -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2; -A1- and -A2- are optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene; each R21 is independently selected from H, optionally substituted (C1-C6)alkyl, -COR, and optionally substituted heteroaryl; and each R22 is independently selected from H, halogen, and optionally substituted (C1-C6)alkyl; R3 and R4 are each independently H, or a promoiety, or R3 and R4 are cyclically linked to form a promoiety; R11 is H, or a bridging moiety that connects the 5-position carbon to the 1-position carbon of the ring; Z1 is a linking moiety selected from -Z11-, -Z11-A1-, -A2-, -NR21CO-, -CONR21-, -NR21SO2-, - SO2NR21-, -NR21C(=O)NR21-, and -NR21C(=S)NR21-; -Z11- is -O-, -S-, -N(R21)-, or -C(R22)2; -A1- and -A2- are optionally substituted heterocyclylene, optionally substituted arylene, or optionally substituted heteroarylene; each R21 is independently selected from H, optionally substituted (C1-C6)alkyl, -COR, and optionally substituted heteroaryl; and each R22 is independently selected from H, halogen, and optionally substituted (C1-C6)alkyl; and Y is a chemoselective ligation group; wherein at least one of the following occurs: A) Z1 is a linking moiety selected from -Z11-A1- and -A2-; and -A1- and -A2- are optionally substituted heterocyclylene; or -A2- is optionally substituted isoxazolyl; B) -L-Y comprises:
Figure imgf000561_0001
C) R6 is -OR, optionally substituted (C1-C6)alkyl, –OC(O)-optionally substituted heteroaryl, - C(O)NH-optionally substituted heteroaryl, -NRxxRyy, optionally substituted aryl, optionally substituted heteroaryl–NHCOR, or –NRCOR, provided the heteroaryl is other than triazole; where each R is independently optionally substituted (C1-C6)alkyl, optionally substituted aryl, or optionally substituted heteroaryl; Rxx and Ryy are independently H, optionally substituted (C1-C6)alkyl, or Rxx and Ryy can cyclize to form an optionally substituted heterocyclyl; D) R1 is optionally substituted C2-6 alkyl, optionally substituted -S-(C1-C6)alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, or optionally substituted -S-heteroaryl; or E) at least one R21 is -COR or optionally substituted heteroaryl.
13. The compound of claim 12, wherein the compound of formula (II) is represented by formula (a- II):
Figure imgf000562_0001
(a-II).
14. The compound of any one of claims 1-13, wherein R1 is –Z1–*, –H, or (C1-C6)alkyl.
15. The compound of any one of claims 1-13, wherein R2 is –Z1–* or –NHCOCH3.
16. The compound of any preceding claim, wherein R3 and R4 are each –H.
17. The compound of any preceding claim, wherein R6 is –OH, –OC(O)R, -NRxxRyy, or aryl; R is (C1-C6)alkyl; and Rxx and Ryy cyclize to form an optionally substituted heterocyclyl.
18. The compound of any preceding claim, wherein –Z1–* is -S-, -CH2-,
Figure imgf000562_0002
, ,
Figure imgf000562_0003
19. The compound of claim 18, wherein R1 is –Z1–*.
20. The compound of claim 18, wherein R2 is –Z1–*.
21. The compound of any preceding claim, wherein L comprises of 10 to 60 consecutive chain atoms.
22. The compound of any preceding claim, wherein L is of formula (IIb’):
Figure imgf000563_0001
wherein: n is 1, 2, or 3; each L1 to L6 is independently a linking moiety which together provide a linear or branched linker between Z1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L1 of X via Z1; and *** represents the point of attachment to Y.
23. The compound of claim 22, wherein each L1 to L5 independently comprises one or more linking moieties independently selected from –C1-20-alkylene–, –NHC(O)-C1-6-alkylene–, –C(O)NH-C1-6- alkylene–, –NH-C1-6-alkylene–, –NHC(O)NH-C1-6-alkylene–, –NHC(S)NH-C1-6-alkylene–, –C1-6- alkylene–NHC(O)-, –C1-6-alkylene–C(O)NH-, –C1-6-alkylene–NH-, –C1-6-alkylene–NHC(O)NH-, –C1-6- alkylene–NHC(S)NH-, -O(CH2)p–, –(OCH2CH2)p–, –NHC(O)–, –C(O)NH–, –NHS(O)2–, –S(O)2NH–, – C(O)–, –S(O)2–, –O–, –S–, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, monocyclic cycloalkyl, amino acid residue, –NH–, and –NMe–; wherein each L1 to L5 is independently optionally substituted with one to five halo; each p is independently1 to 50; L6 is a linking group comprising one or more linking moieties independently selected from –C1- 20-alkylene–, –NR16C(O)-C1-6-alkylene–, –C(O)NR16-C1-6-alkylene–, –NR16-C1-6-alkylene–, – NR16C(O)NR16-C1-6-alkylene–, –NR16C(S)NR16-C1-6-alkylene–, –C1-6-alkylene–NR16C(O)-, –C1-6- alkylene–C(O)NR16-, –C1-6-alkylene–NR16-, –C1-6-alkylene–NR16C(O)N R16-, –C1-6-alkylene– NR16C(S)NR16-, -O(CH2)p–, –(OCH2CH2)p–, –NR16C(O)–, –C(O)NR16–, –NHS(O)2–, –S(O)2NH–, – C(O)–, –S(O)2–, –O–, –S–, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, amino acid residue, or –NR16–; and each R16 is independently –H, (C1-C6)alkyl, or monocyclic heteroaryl.
24. The compound of claim 22, wherein each L1 to L5 is independently selected from –C1-20- alkylene–, –NHC(O)-C1-6-alkylene–, –C(O)NH-C1-6-alkylene–, –NH-C1-6-alkylene–, –NHC(O)NH-C1-6- alkylene–, –NHC(S)NH-C1-6-alkylene–, –C1-6-alkylene–NHC(O)-, –C1-6-alkylene–C(O)NH-, –C1-6- alkylene–NH-, –C1-6-alkylene–NHC(O)NH-, –C1-6-alkylene–NHC(S)NH-, -O(CH2)p–, –(OCH2CH2)p–, – NHC(O)–, –C(O)NH–, –NHS(O)2–, –S(O)2NH–, –C(O)–, –S(O)2–, –O–, –S–, monocyclic heteroaryl, monocyclic aryl, monocyclic heterocycle, monocyclic cycloalkyl, amino acid residue, –NH–, and –NMe– ; wherein each L1 to L5 is independently optionally substituted with one to five halo; each p is independently1 to 50; and L6 is
Figure imgf000564_0001
25. The compound of any one of claims 22-24, wherein n is 1.
26. The compound of any one of claims 22-24, wherein n is 2.
27. The compound of any one of claims 22-24, wherein n is 3.
28. The compound of any one of claims 22-27, wherein at least one L1 is –C1-20-alkylene– optionally substituted with one to five halo.
29. The compound of any one of claims 22-28, wherein at least one L1 is -CF2CH2-.
30. The compound of any one of claims 22-29, wherein at least one L2 is –(OCH2CH2)p–.
31. The compound of claim 30, wherein p is 2-3.
32. The compound of any one of claims 22-31, wherein at least one L3 is NHCONH-C1-6-alkylene–.
33. The compound of any one of claims 22-32, wherein at least one L4 is –C1-6-alkylene–NHCONH-.
34. The compound of any one of claims 22-33, wherein at least one L5 is –(OCH2CH2)p–.
35. A pharmaceutical composition comprising the compound of any one of claims 1-34 and one or more pharmaceutically acceptable excipients.
36. A method of internalizing a target molecule in a cell comprising a cell surface asialoglycoprotein receptor (ASGPR), the method comprising: contacting a cellular sample comprising the cell and the target molecule with an effective amount of a compound according to any one of claims 1 to 34, wherein the compound specifically binds the target protein and specifically binds the ASGPR to facilitate cellular uptake of the target protein.
37. The method of claim 36, wherein the target molecule is a membrane bound protein.
38. The method of claim 37, wherein the target molecule is an extracellular protein.
39. A method of reducing levels of a target molecule in a biological system, the method comprising: contacting the biological system with an effective amount of a compound according to any one of claims 1 to 34, wherein the compound specifically binds the target protein and specifically binds a ASGPR of cells in the biological system to facilitate cellular uptake and degradation of the target protein.
40. The method of claim 39 wherein the biological system is a human subject. 41 The method of claim 39, wherein the biological system is an in vitro cellular sample. 42. The method of any one of claims 36 to 41, wherein the target molecule is a membrane bound protein. 43. The method of any one of claims 36 to 41, wherein the target molecule is an extracellular protein.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025067423A1 (en) * 2023-09-27 2025-04-03 上海拓界生物医药科技有限公司 Triazole-containing compound, and preparation method and use therefor
WO2025143096A1 (en) * 2023-12-29 2025-07-03 キッセイ薬品工業株式会社 Nitrogen-containing condensed ring compound

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006034488A2 (en) 2004-09-23 2006-03-30 Genentech, Inc. Cysteine engineered antibodies and conjugates
US7709226B2 (en) 2001-07-12 2010-05-04 Arrowsmith Technology Licensing Llc Method of humanizing antibodies by matching canonical structure types CDRs
US20160060354A1 (en) 2014-03-19 2016-03-03 Genzyme Corporation Site-specific glycoengineering of targeting moieties
US20190060481A1 (en) 2014-10-09 2019-02-28 Genzyme Corporation Glycoengineered antibody drug conjugates
WO2023288033A1 (en) 2021-07-14 2023-01-19 Lycia Therapeutics, Inc. Asgpr cell surface receptor binding compounds and conjugates

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3167272A1 (en) * 2020-01-10 2021-07-15 Lycia Therapeutics, Inc. Cell surface receptor binding compounds and conjugates
AU2021213822A1 (en) * 2020-01-31 2022-09-22 Avilar Therapeutics, Inc. ASGPR-binding compounds for the degradation of extracellular proteins

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7709226B2 (en) 2001-07-12 2010-05-04 Arrowsmith Technology Licensing Llc Method of humanizing antibodies by matching canonical structure types CDRs
WO2006034488A2 (en) 2004-09-23 2006-03-30 Genentech, Inc. Cysteine engineered antibodies and conjugates
US20160060354A1 (en) 2014-03-19 2016-03-03 Genzyme Corporation Site-specific glycoengineering of targeting moieties
US20190060481A1 (en) 2014-10-09 2019-02-28 Genzyme Corporation Glycoengineered antibody drug conjugates
WO2023288033A1 (en) 2021-07-14 2023-01-19 Lycia Therapeutics, Inc. Asgpr cell surface receptor binding compounds and conjugates

Non-Patent Citations (23)

* Cited by examiner, † Cited by third party
Title
"NCBI", Database accession no. NM_001197216
"Remington: The Science and Practice of Pharmacy", 2012, PHARMACEUTICAL PRESS
AL-LAZIKANI ET AL., J. MOL. BIOL., vol. 273, 1997, pages 927 - 948
CHOTHIA ET AL., J. MOL. BIOL., vol. 227, 1992, pages 799 - 817
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
HOFER ET AL., BIOCHEMISTRY, vol. 48, no. 50, 2009, pages 12047 - 57
HOFER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 105, 2008, pages 12451 - 56
JACKSON, ORG. PROCESS RES. DEV., vol. 20, 2016, pages 852 - 866
JUNUTULA ET AL., J. IMMUNOL. METH., vol. 332, 2008, pages 41 - 52
JUNUTULA ET AL., NATURE BIOTECHNOL., vol. 26, 2008, pages 925 - 32
KABAT ET AL., ANN. NY ACAD. SCI., vol. 190, 1971, pages 382 - 391
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
LEFRANC ET AL., NUCLEIC ACIDS RES., vol. 27, 1999, pages 209 - 212
LEFRANC, THE IMMUNOLOGIST, vol. 7, 1999, pages 132 - 136
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745
MAMIDAYALA ET AL., J. AM. CHEM. SOC., vol. 134, 2012, pages 1978 - 1981
MARTIN, A.: "Antibody Engineering", vol. 31, 2001, SPRINGER-VERLAG, article "Protein Sequence and Structure Analysis of Antibody Variable Domains", pages: 422 - 439
MCMILLAN ET AL., NATURE COMMUNICATIONS, vol. 12, 2021, pages 582
S. M. BERGE ET AL., J. PHARMACEUTICAL SCIENCES, vol. 66, 1977, pages 1 - 19
STEEVELS ET AL., EUR. J. IMMUNOL., vol. 41, no. 3, 2011, pages 575 - 587
TRAMONTANO ET AL., J. MOL. BIOL., vol. 215, no. 1, 1990, pages 175 - 82
TSUCHIKAMAAN, PROTEIN CELL, vol. 9, no. 1, 2018, pages 33 - 46
XIAO ET AL., JOURNAL OF MEDICINAL CHEMISTRY, vol. 63, no. 23, 2020, pages 15050 - 15071

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WO2025143096A1 (en) * 2023-12-29 2025-07-03 キッセイ薬品工業株式会社 Nitrogen-containing condensed ring compound

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