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WO2024243706A1 - Analogues d'amatoxine et leurs utilisations - Google Patents

Analogues d'amatoxine et leurs utilisations Download PDF

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WO2024243706A1
WO2024243706A1 PCT/CA2024/050737 CA2024050737W WO2024243706A1 WO 2024243706 A1 WO2024243706 A1 WO 2024243706A1 CA 2024050737 W CA2024050737 W CA 2024050737W WO 2024243706 A1 WO2024243706 A1 WO 2024243706A1
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formula
amatoxin
analog
target
halo
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David M. Perrin
Shambhu Deo CHANDRA
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University of British Columbia
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University of British Columbia
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6831—Fungal toxins, e.g. alpha sarcine, mitogillin, zinniol or restrictocin
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/22—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated the carbon skeleton being further substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
    • C07C251/02—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups
    • C07C251/04—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups having carbon atoms of imino groups bound to hydrogen atoms or to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • AMATOXIN ANALOGS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [001] The present application claims the benefit of priority from co-pending U.S. provisional application no. 63/470,542 filed on June 2, 2023, the contents of which are incorporated herein by reference in their entirety.
  • FIELD [002] The present disclosure relates, for example, to amatoxin analogs, methods for their preparation, conjugates comprising such amatoxin analogs and their use, for example, in the treatment of cancer.
  • BACKGROUND [003] ⁇ -Amanitin is an extremely toxic bicyclic octapeptide extracted from the death- cap mushroom, Amanita phalloides.
  • RNA polymerase II the enzyme indispensable for cellular function and homeostasis. Since RNA polymerase II catalyzed transcription is obligatory for cellular viability, it empowers ⁇ - amanitin to target actively dividing and quiescent cells. With the appealing properties of stability, potency, and a unique mechanism of action, it has shown daunting promise as a payload in antibody-drug conjugates (ADCs) for cancer treatment.
  • ADCs antibody-drug conjugates
  • the present disclosure includes an amatoxin analog comprising a DHIle residue with a sidechain of the formula: , wherein R 1 is H, OR ', N(R ') 2 , SR ', halo, -N 3 , -S-S-R ', imidazole, -CN, -S(O)R ',- ON(R ') 2 , -N(R ')OR ', -S(O) 2 R ', -SeR ' or -N(R ')N(R) 2 ; and R 2 is H, OR ', NHR ', SR ' or halo; or R 1 and R 2 , together with the carbon atoms to which they are attached, form epoxy; each R ' is independently H, C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycl
  • the amatoxin analog is a compound of Formula I: , wherein A is S, S(O), SO 2 , Se, Se(O) or SeO 2 ; R 1 is H, OR ', NR 'R 2 , SR ', halo, -N 3 , -S-S-R ', imidazole, -CN, -S(O)R ', - ON(R ') 2 , -N(R ')OR ', -S(O) 2 R ', -SeR ' or -N(R ')N(R) 2 ; and R 2 is H, OR ', NHR ', SR ' or halo; or R 1 and R 2 , together with the carbon atoms to which they are attached, form epoxy; R 3 is OR ', NH(OR '), NH-NHR ' or N(R ') 2 ; R 4 is OR ',
  • the present disclosure also includes an amatoxin-linker construct comprising an amatoxin analog as described herein (which may include pharmaceutically acceptable salts thereof) coupled to a linker, wherein the linker comprises a reactive group R 6 for conjugating the amatoxin-linker construct to a target-binding moiety.
  • the present disclosure also includes a conjugate comprising a target-binding moiety conjugated to an amatoxin analog or an amatoxin-linker construct as described herein.
  • the present disclosure also includes a pharmaceutical composition comprising an amatoxin analog or conjugate of the present disclosure and a pharmaceutically acceptable carrier.
  • the present disclosure also includes a use of an amatoxin analog of the present disclosure for treatment of cancer in a subject in need thereof.
  • the present disclosure also includes a use of an amatoxin analog or a conjugate of the present disclosure for preparation of a medicament for treatment of cancer in a subject in need thereof.
  • the present disclosure also includes a dihydroxyisoleucine (DHIle) analog of the Formula II: , wherein R 1a is H, OR ', N(R ') 2 , SR ', halo, -N 3 , -S-S-R ', imidazole, -CN, -S(O)R ', - ON(R ') 2 , -N(R ')OR ', -S(O) 2 R ', -SeR ' or -N(R ')N(R) 2 , a protected form thereof or a precursor thereto; and PG 1 and PG 2 are each independently protecting groups.
  • DHIle dihydroxyisoleucine
  • the present disclosure also includes a dihydroxyisoleucine (DHIle) analog of the Formula III: , wherein R 1 is OR ', N(R ') 2 , SR ', halo, -N 3 , -S-S-R ', imidazole, -CN, -S(O)R ', -ON(R ') 2 , -N(R ')OR ', -S(O) 2 R ', -SeR ' or -N(R ')N(R) 2 ; and each R ' is independently H, C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl or heteroaryl, wherein one or more available carbon atoms in the C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl is optionally replaced by a heteroatom,
  • an amatoxin compound of Formula I where R 1 is selected from -H, -OH, -F, -Cl, -Br, -N 3 , -S-S- t Bu, - SH, -NH 2 , epoxy, imidazole, -CN, -SOR, -ONH 2 , -NHOH, -SO 2 R, -SeR, and -NHNH 2 (where R is selected from H, Me, Et, iPr or another optionally substituted linear or branched 1-10 carbon alkyl group) and R 2 is either -CH 2 CH 2 N 3 or H.
  • the amatoxin analog is selected from the following compounds: ; ; ; ; ;
  • DHIle analogs according to the following formula: where R 1 is selected from -H, -OH, -F, -Cl, -Br, -N 3 , -S-S- t Bu, -SH, -NH 2 , epoxy, imidazole, -CN, -SOR, -ONH 2 , -NHOH, -SO 2 R, -SeR, and -NHNH 2 (where R is selected from H, Me, Et, iPr or another optionally substituted linear or branched 1-10 carbon alkyl group).
  • the DHIle analog is selected from the following compounds: .
  • a method for synthesizing (2S,3R,4R/S)-DHIle analogs from a ⁇ -keto-L-isoleucine precursor wherein a synthetic step involving Rubottom oxidation of a silyl enol ether isoleucine intermediate to form ⁇ -OH substituted keto-isoleucine intermediates (see also Scheme 2): .
  • a compound-linker construct comprising an amatoxin analog and a linker for conjugating the construct to a target-binding moiety.
  • the target-binding moiety of the conjugate is an antibody, an antigen-binding fragment thereof or an antibody-like protein.
  • the use of an effective amount of the compound or conjugate for treatment of cancer in a subject is the use of an effective amount of the compound or conjugate for treatment of cancer in a subject.
  • FIG.1 shows a nuclear magnetic resonance spectroscopy (NMR) COSY spectrum of ((3S,4R,5R)-5-(hydroxymethyl)-4-methyl-2-oxotetrahydrofuran-3-yl)carbamate ( 1 H, 1 H) 400 MHz, CD 2 Cl 2 (upper) and a zoomed in image of the COSY spectrum (lower).
  • NMR nuclear magnetic resonance spectroscopy
  • FIG. 2 shows a NMR NOESY spectrum of ((3S,4R,5R)-5-(hydroxymethyl)-4- methyl-2-oxotetrahydrofuran-3-yl)carbamate ( 1 H, 1 H) 400 MHz, CD 2 Cl 2 (upper) and a zoomed in image of the NOESY spectrum (lower).
  • FIG. 3 shows the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) viability assay on human embryonic kidney (HEK) 293 cells for (2S,3R,4S)-Mono-OH-Ama (A1), (2S,3R,4R)-F-OH-Ama (A2), (2S,3R,4S)-F-OH- Ama (A3) and (2S,3R,4R)-Azido-OH-Ama (A4) in comparison to ⁇ -amanitin.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide
  • FIG.4 shows the results of an MTT viability assay on Chinese hamster ovary (CHO) cells for A1, A2, A3 and A4 in comparison to ⁇ -amanitin.
  • FIG.5 shows the results of an MTT viability assay on HeLa cells for A1, A2, A3 and A4 in comparison to ⁇ -amanitin.
  • FIG.6 shows the results of an MTT viability assay on HepG2 cells for A1, A2, A3 and A4 in comparison to ⁇ -amanitin.
  • FIG. 7 shows the results of an MTT viability assay on HEK 293 cells for (2S,3R,4R)-tBu-S-S-OH-Ama (A6) in comparison to ⁇ -amanitin.
  • FIG. 8 shows the results of an MTT viability assay on CHO cells for A6 in comparison to ⁇ -amanitin.
  • FIG. 9 shows the results of an MTT viability assay on HeLa cells for A6 in comparison to ⁇ -amanitin.
  • FIG.10 shows the results of an MTT viability assay on HepG2 cells for A6 in comparison to ⁇ -amanitin. [0041] FIG.
  • FIG. 11 shows the results of an MTT viability assay on HEK 293 cells for (2S,3R,4R)-NH 2 -OH-Ama (A7) in comparison to ⁇ -amanitin.
  • FIG.12 shows the results of an MTT viability assay on HepG2 cells for A7 and (2S,3R,4R)-epoxy-Ama (A8) in comparison to ⁇ -amanitin.
  • FIG.13 shows the results of an MTT viability assay on HEK 293 cells for A8 in comparison to ⁇ -amanitin.
  • FIG.14 shows the results of an MTT viability assay on CHO cells for A8 in comparison to ⁇ -amanitin.
  • FIG.15 shows the results of an MTT viability assay on HeLa cells for A7 and A8 in comparison to ⁇ -amanitin.
  • FIG.16 shows the results of an MTT viability assay on CHO cells for A7 in comparison to ⁇ -amanitin.
  • FIG.17 shows the results of an MTT viability assay on HEK 293 cells for (2S,3R,4R)- SH-OH-Ama (A9) and (2S,3R,4R)-S-S-OH-Ama (A10) in comparison to ⁇ -amanitin.
  • FIG.18 shows the results of an MTT viability assay on CHO cells for A9 in comparison to ⁇ -amanitin.
  • FIG.19 shows the results of an MTT viability assay on CHO cells for A10 in comparison to ⁇ -amanitin.
  • FIG.20 shows the results of an MTT viability assay on HeLa cells for A9 in comparison to ⁇ -amanitin.
  • FIG.21 shows the results of an MTT viability assay on HeLa cells for A10 in comparison to ⁇ -amanitin.
  • FIG.22 shows the results of an MTT viability assay on HepG2 cells for A10 in comparison to ⁇ -amanitin.
  • FIG.23 shows the results of an MTT viability assay on HepG2 cells for A9 in comparison to ⁇ -amanitin.
  • FIG.24 shows the results of an MTT viability assay on SK-BR-3 cells for A9 ADC in comparison to ⁇ -amanitin, A9, A10 and Kadcyla TM .
  • the words “comprising” (and any form thereof, such as “comprise” and “comprises”), “having” (and any form thereof, such as “have” and “has”), “including” (and any form thereof, such as “include” and “includes”) or “containing” (and any form thereof, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process/method steps.
  • the word “consisting” and its derivatives are intended to be close-ended terms that specify the presence of the stated features, elements, components, groups, integers and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • the term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of these features, elements, components, groups, integers and/or steps.
  • alkyl as used herein, whether it is used alone or as part of another group, means straight or branched chain, saturated alkyl groups. The number of carbon atoms that are possible in the referenced alkyl group are indicated by the numerical prefix “Cn1-n2”.
  • Cn1-n2 the number of carbon atoms that are possible in the referenced alkyl group are indicated by the numerical prefix “Cn1-n2”.
  • alkenyl as used herein, whether it is used alone or as part of another group, means straight or branched chain, unsaturated alkenyl groups. The number of carbon atoms that are possible in the referenced alkenyl group are indicated by the numerical prefix “Cn1-n2”.
  • C 2-20 alkenyl means an alkenyl group having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms and at least one double bond, for example 1-3, 1-2 or 1 double bond.
  • alkynyl as used herein, whether it is used alone or as part of another group, means straight or branched chain, unsaturated alkynyl groups. The number of carbon atoms that are possible in the referenced alkynyl group are indicated by the numerical prefix “Cn1-n2”.
  • C 2-20 alkynyl means an alkenyl group having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms and at least one triple bond, for example 1-3, 1-2 or 1 triple bond.
  • cycloalkyl as used herein, whether it is used alone or as part of another group, means a mono- or bicyclic, saturated cycloalkyl group. The number of carbon atoms that are possible in the referenced cycloalkyl group are indicated by the numerical prefix “C n1-n2 ”.
  • C3-10cycloalkyl means a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
  • cyclic structures When a cycloalkyl group contains more than one cyclic structure or rings, the cyclic structures may be fused, bridged, spiro connected or linked by a single bond.
  • fused as used herein in reference to a first cyclic structure being “fused” with a second cyclic structure means the first cyclic structure and the second cyclic structure share at least two adjacent atoms therebetween.
  • bridged as used herein in reference to a first cyclic structure being “bridged” with a second cyclic structure means the first cyclic structure and the second cyclic structure share at least two non-adjacent atoms therebetween.
  • spiro-connected in reference to a first cyclic structure being “spiro connected” with a second cyclic structure means the first cyclic structure and the second cyclic structure share one atom therebetween.
  • aryl as used herein, whether it is used alone or as part of another group, refers to cyclic groups that contain at least one aromatic ring. The number of carbon atoms that are possible in the referenced aryl group are optionally indicated by the numerical prefix “Cn1-n2”. For example, the term C 6-30 aryl means an aryl group having 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms.
  • the aryl group contains from 6, 9, 10 or 14 carbon atoms, such as phenyl, naphthyl, indanyl or anthracenyl.
  • heterocycloalkyl refers to a non-aromatic, ring-containing group having one or more multivalent heteroatoms, for example, independently selected from O, S and Se, as a part of the ring structure and including at least 3 and up to 20 atoms in the ring(s).
  • Heterocycloalkyl groups are either saturated or unsaturated (i.e. contain one or more double bonds) and may contain more than one ring.
  • heteroaryl refers to an aromatic, ring-containing group having one or more multivalent heteroatoms, for example, independently selected from O, S, Se and N, as a part of the ring structure and including at least 5 and up to 20 atoms in the ring(s). Heteroaryl groups may contain more than one ring.
  • halo refers to a halogen atom and includes F, Cl and Br.
  • the term “available”, as used herein in reference to “available hydrogens”, “available carbon atoms”, “available atoms” and the like refers to atoms that would be known to a person skilled in the art to be capable of modification and/or replacement by another atom or substituent.
  • the term “replaced with its radioactive isotope” as used herein in reference to one or more available atoms in the amatoxin analogs of the present disclosure means that a non- radioactive isotope of the atom is replaced with the corresponding radioactive isotope.
  • a non-radioactive isotope of hydrogen is replaced with 3 H (tritium).
  • a non-radioactive isotope of carbon is replaced with 11 C or 14 C.
  • a non-radioactive isotope of iodine is replaced with 123 I, 125 I or 131 I.
  • a non-radioactive isotope of sulfur is replaced with 35 S. It will be appreciated by a person skilled in the art that depending, for example, on the radioactive isotope, such radiolabelled compounds may, for example, be useful as radiopharmaceuticals for treatment and/or diagnostic applications. The selection of a suitable radioactive isotope and methods for the preparation of radiolabelled compounds can be made by a person skilled in the art.
  • protecting group or “protecting group” or “PG” or the like as used herein refer to a chemical moiety which protects or masks a reactive portion of a molecule to prevent side reactions in those reactive portions of the molecule, while manipulating or reacting a different portion of the molecule. After the manipulation or reaction is complete, the protecting group is removed under conditions that do not degrade or decompose the remaining portions of the molecule.
  • PG protecting group
  • the selection of a suitable protecting group can be made by a person skilled in the art. Many conventional protecting groups are known in the art, for example as described in “Protective Groups in Organic Chemistry” McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W.
  • protecting groups include, but are not limited to Boc, Ac, Ts, Ms, silyl ethers such as TMS, TBS, TBDMS, TBDPS, Tf, Ns, Bn, Fmoc, dimethoxytrityl, methoxyethoxymethyl ether, methoxymethyl ether, pivaloyl, p-methoxyphenyl p-methyoxybenzyl ether, tetrahydropyranyl, trityl, ethoxyethyl ethers, carbobenzyloxy, benzoyl and the like.
  • the term “subject” as used herein includes all members of the animal kingdom including mammals. In an embodiment, the subject is a human.
  • pharmaceutically acceptable means compatible with the treatment of subjects, for example, mammals such as humans.
  • pharmaceutically acceptable salt means either an acid addition salt or a base addition salt that is compatible with the treatment of subjects.
  • An “acid addition salt that is compatible with the treatment of subjects” is any suitable non-toxic organic or inorganic salt of any basic compound.
  • Basic compounds that form an acid addition salt include, for example, compounds comprising an amine group susceptible to protonation.
  • Illustrative inorganic acids which form suitable salts include but are not limited to hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as but not limited to sodium monohydrogen orthophosphate and potassium hydrogen sulfate.
  • Illustrative organic acids that may form suitable salts include but are not limited to mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as but not limited to p-toluene sulfonic and methanesulfonic acids.
  • the selection of a suitable salt can be made by a person skilled in the art.
  • the formation of a desired acid addition salt is, for example, achieved using standard techniques.
  • a “base addition salt that is compatible with the treatment of subjects” is any suitable nontoxic inorganic or organic salt of any acidic compound.
  • Acidic compounds that form a base addition salt include, for example, compounds comprising a sulfonic acid group.
  • Inorganic bases that may form suitable salts include, without limitation, lithium, sodium, potassium, calcium, magnesium or barium hydroxide.
  • Organic bases that may form suitable salts include, without limitation, aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia.
  • a suitable salt can be made by a person skilled in the art.
  • the formation of a desired base addition salt is, for example, achieved using standard techniques.
  • the neutral compound is treated with the desired base in a suitable solvent and the salt which is thereby formed then isolated by filtration, extraction and/or any other suitable method.
  • beneficial or desired clinical results include but are not limited to alleviation or amelioration of one or more symptoms of a disease (e.g. cancer), diminishment of the extent of the disease, stabilization (i.e.
  • the term “effective amount” means an amount effective, at dosages and for periods of time necessary to achieve a desired result.
  • an effective amount of an amatoxin analog or conjugate of the present disclosure is an amount that, for example, reduces the cancer compared to the cancer without administration or use of the amatoxin analog or conjugate, respectively.
  • Effective amounts may vary according to factors such as the disease state, age, sex and/or weight of the subject.
  • the amount of a given amatoxin analogue or conjugate, as the case may be, that will correspond to such an amount will vary depending upon various factors, such as the given amatoxin analog or conjugate, the pharmaceutical formulation, the type of disease being treated, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.
  • suitable means that the selection of the particular compound and/or conditions would depend on the specific synthetic manipulation to be performed, and/or the identity of the compound(s) to be transformed, but the selection would be well within the skill of a person skilled in the art. All synthetic method steps described herein are to be conducted under conditions sufficient to provide the product shown. A person skilled in the art would understand that all reaction conditions, including, for example, reaction solvent, reaction time, reaction temperature, reaction pressure, reactant ratio and whether or not the reaction should be performed under an anhydrous or inert atmosphere, can be varied to optimize the yield of the desired product and it is within their skill to do so. II.
  • the present disclosure includes an amatoxin analog comprising a DHIle residue with a sidechain of the formula: , wherein R 1 is H, OR ', N(R ') 2 , SR ', halo, -N 3 , -S-S-R ', imidazole, -CN, -S(O)R ', - ON(R ') 2 , -N(R ')OR ', -S(O) 2 R ', -SeR ' or -N(R ')N(R) 2 ; and R 2 is H, OR ', NHR ', SR ' or halo; or R 1 and R 2 , together with the carbon atoms to which they are attached, form epoxy; each R ' is independently H, C 1-20 alkyl, C 2-20 alkenyl, C 2-20 al
  • the amatoxin can be any suitable amatoxin.
  • amatoxin as used herein includes all cyclic peptides composed of 8 amino acids as isolated from the genus Amanita and described, for example, by Wieland, T. and Faulstich, H. in CRC Critical Reviews in Biochemistry 1978, 5 (3), 185-260 as well as all suitable chemical derivatives, semisynthetic analogs and synthetic analogs thereof built from building blocks according to the master structure of the natural compounds (cyclic, 8 amino acids).
  • amatoxin as used herein also includes all suitable synthetic or semisynthetic analogs of amatoxins containing non- hydroxylated amino acids instead of the hydroxylated amino acids and/or amino acids comprising beta-alkyl groups such as beta-methyl groups and/or in which the sulfoxide moiety is replaced by a sulfone, or thioether, or by atoms different from sulfur, e.g., a carbon atom as in a carbanalog of amanitin.
  • the amatoxin has a functional group (e.g.
  • the amatoxin is an ⁇ -amanitin, ⁇ -amanitin, ⁇ -amanitin, ⁇ -amanitin, amanullin, amanullinic acid, amanin, amaninamide, ⁇ -amanin or ⁇ -amaninamide or a derivative thereof.
  • the amatoxin analog is a compound of Formula I: , wherein A is S, S(O), SO 2 , Se, Se(O) or SeO 2 ;
  • R 1 is H, OR ', NR 'R2, SR ', halo, -N 3 , -S-S-R ', imidazole, -CN, -S(O)R ', - ON(R ') 2 , -N(R ')OR ', -S(O) 2 R ', -SeR ' or -N(R ')N(R) 2 ; and
  • R 2 is H, OR ', NHR ', SR ' or halo; or R 1 and R 2 , together with the carbon atoms to which they are attached, form epoxy;
  • R 3 is OR ', NH(OR '), NH-NHR ' or N(R ') 2 ;
  • R 4 is OR ', NH
  • the amatoxin analog of Formula I has the following stereochemistry: .
  • R 4 is OR ' or N(R ') 2 .
  • R 4 is OR ' or N(R ') 2 , wherein R ' is H.
  • R 4 is NH 2 .
  • R 4 is -N(R ') 2 , wherein one R ' is H and the other R ' is C 1-20 alkyl substituted with -N 3 .
  • R 4 is -N(R ') 2 , wherein one R ' is H and the other R ' is C1-6alkyl substituted with -N 3 .
  • R 4 is -N(H)CH 2 CH 2 N 3 .
  • R 5 is H. In another embodiment, R 5 is OH.
  • R 2 is H or OR '. In another embodiment, R 2 is OH. In an embodiment, R 2 is -OH (S). In another embodiment, R 2 is -OH (R). [0086] In an embodiment, n is an integer from 0 to 3. In another embodiment, n is 0, 1 or 2. In another embodiment, n is 0 or 1. In an embodiment, n is 0. In another embodiment, n is 1. [0087] In an embodiment, each R 3 is independently OR '. In another embodiment, R 3 is OH.
  • R 3 is 4-OH, 5-OH, 6-OH or 7-OH. In an embodiment, R 3 is 5-OH.
  • A is S, (R)-S(O), Se or (R)-Se(O). In an embodiment, A is S or (R)-S(O). In another embodiment, A is S. In a further embodiment, A is (R)-S(O).
  • R 1 is selected from -H, -OH, -F, -Cl, -Br, -N 3 , -S-S- t Bu, - SH, -NH 2 , epoxy, imidazole, -CN, -SOR, -ONH 2 , -NHOH, -SO 2 R, -SeR, and -NHNH 2 (where R is selected from H, Me, Et, iPr or another optionally substituted C1-10alkyl group).
  • R 1 is H, OR ', N(R ') 2 , SR ', halo, -N 3 , or -S-S-R '.
  • R 1 is H, OR ', N(R ') 2 , SR ', halo, -N 3 , or -S-S-R '.
  • R 1 is H, N(R ') 2 , SR ', halo, -N 3 , or -S-S-R ', wherein R ' is H or C1-6alkyl, or R 1 and R 2 , together with the carbon atoms to which they are attached, form epoxy.
  • R 1 is F, Br, -N 3 , -S-S- t Bu, NH 2 , SH or -S-S-CH(CH 3 )CH(CH 3 ) 2 .
  • R 1 is F, Br, -N 3 , -S-S- t Bu, or NH 2 .
  • R 1 is F.
  • R 1 is Br.
  • R 1 is -N 3 .
  • R 1 is -S-S- t Bu.
  • R 1 is NH 2 .
  • R 1 and R 2 together with the carbon atoms to which they are attached, form epoxy.
  • R 1 is SH.
  • R 1 is -S-S-CH(CH 3 )CH(CH 3 ) 2 .
  • R ' is H, C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl or heteroaryl, wherein one or more available carbon atoms in the C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl is optionally replaced by a heteroatom, and wherein the C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl and heteroaryl is unsubstituted.
  • R ' is H, C 1-20 alkyl, C 2-20 alkenyl, C 2- 20alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl or heteroaryl, wherein the C 1-20 alkyl, C 2- 20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl and heteroaryl is further substituted with one or more groups selected from OR ' ', SR ' ', halo, -N3 and nitrile, wherein R ' ' is independently H, C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl or heteroaryl.
  • each R ' is independently H or C1-20alkyl. In an embodiment, R ' is C1-6alkyl. In another embodiment, R ' is H.
  • the amatoxin analog is: (A9); or (A10) or a pharmaceutically acceptable salt thereof. [0093] In an embodiment, the amatoxin analog is: (A1). [0094] In an embodiment, the amatoxin analog is: (A2). [0095] In an embodiment, the amatoxin analog is: (A3). [0096] In an embodiment, the amatoxin analog is: (A4). [0097] In an embodiment, the amatoxin analog is: (A5).
  • the amatoxin analog is: (A6). [0099] In an embodiment, the amatoxin analog is: (A7). [00100] In an embodiment, the amatoxin analog is: (A8). [00101] In an embodiment, the amatoxin analog is: (A9). [00102] In an embodiment, the amatoxin analog is: (A10). [00103] In an embodiment, the amatoxin analog is not in the form of a salt. In an alternative embodiment, the amatoxin analog is in the form of a pharmaceutically acceptable salt.
  • amatoxin analogs of the present disclosure such as the compounds of Formula I, and salts thereof, as described herein may, for example, be coupled to a linker to provide an amatoxin-linker construct that may, for example, be used in the manufacture of conjugates which may, for example, be useful in the treatment of diseases such as cancer.
  • the present disclosure also includes an amatoxin-linker construct comprising an amatoxin analog as described herein (which may include pharmaceutically acceptable salts thereof) coupled to a linker, wherein the linker comprises a reactive group R 6 for conjugating the amatoxin-linker construct to a target-binding moiety.
  • amatoxin analogs of the present disclosure are coupled to the linker in any suitable configuration, the selection of which can be made by a person skilled in the art.
  • the location of the coupling may depend, for example, on the identity of the amatoxin analog and/or the linker.
  • the linker is coupled to the amatoxin analog or the salt thereof at a site indicated by R 1 , R 2 , R 3 , R 4 and/or R 5 in an amatoxin analog as described herein.
  • the linker is coupled to the amatoxin analog or the salt thereof through a moiety obtained from the reaction of R 1 , R 2 , R 3 , R 4 and/or R 5 in an amatoxin analog as described herein with a complementary functional group on the linker.
  • the linker can be any suitable linker.
  • amatoxins are relatively non-toxic when coupled to a biomolecule carrier such as a target-binding moiety, and advantageously only exert their cytotoxic activity after internalization in the target cells.
  • a conjugate comprising the target-binding moiety conjugated to the compound coupled to the linker is advantageously substantially stable in the plasma after administration and a suitable linker may desirably allow, for example, for substantially releasing the compound subsequent to internalization in the target cells.
  • a suitable linker may desirably allow, for example, for substantially releasing the compound subsequent to internalization in the target cells.
  • linkers suitable for use in amatoxin-linker constructs are known in the art, for example, suitable linkers for some embodiments of the present disclosure are described in PCT Application Publication No. WO/2023/097407 entitled “Modified Amatoxins and Uses Thereof”. [00108]
  • the linker is stable linker.
  • stable linker refers to a linker that typically releases the compound after the target-binding moiety to which it is conjugated is degraded intracellularly, for example, in the lysosomes.
  • the linker is substantially stable in an intracellular reducing environment and in the presence of enzymes such as lysosomal peptidases (e.g. Cathepsin B).
  • the stable linker is devoid of an enzyme-cleavable structure (e.g. a dipeptide sequence cleavable by Cathepsin B) and/or a disulfide group.
  • the stable linker comprises an –[CH 2 -X 1 -CH 2 ] s –CH 2 – moiety, wherein X 1 is O, CH 2 or S, and s is an integer from 1 to 8.
  • the linker is a cleavable linker.
  • cleavable linker refers to a linker that is cleavable by an enzyme and/or in a reducing environment. In an embodiment of the present disclosure, the cleavable linker is cleavable by an intracellular protease.
  • the linker is cleavable by at least one agent selected from the group consisting of cysteine protease, metalloproteinase, serine protease, threonine protease, aspartic protease, glycosidase, phosphodiesterase, and reductase.
  • Such linkers may, for example, comprise a peptide motif cleavable by such an enzyme.
  • the linker comprises a motif selected from the group consisting of: Val-Ala, Val-Cit, Val- Lys, Val-Arg, Phe-Lys-Gly-Pro-Leu-Gly, Ala-Ala-Pro-Val, ⁇ -glucuronide and ⁇ -galactoside.
  • the linker is a stable linker, but the linker is coupled to the amatoxin analog or the salt thereof through a moiety cleavable by an enzyme and/or in a reducing environment.
  • the linker is coupled to the amatoxin analog or the salt thereof through a moiety cleavable in a reducing environment.
  • the moiety cleavable in a reducing environment is a disulfide.
  • the linker further comprises a self-immolating moiety.
  • self-immolating moiety refers to a moiety, which, after enzymatic cleavage of the linker, spontaneously cleaves from the remainder of the amatoxin-linker construct, thereby releasing the compound.
  • the self-immolating moiety is a p-aminobenzyl alcohol (PAB) moiety.
  • PAB is conjugated to a peptide (e.g. a dipeptide) portion of the linker via the aromatic amine group of the PAB.
  • the PAB is conjugated to the compound via a carbamate group coupled to a primary or secondary amine or in an alternative embodiment, the PAB is coupled directly to the amatoxin analogue.
  • the reactive group R 6 is any reactive group suitable for conjugating the amatoxin- linker construct to a target-binding moiety and may depend, for example, on the identity of the target-binding moiety and/or a complimentary reactive group thereon.
  • R 6 is selected from: ; ; ; ; ; ; ; ; and , wherein represents the site of attachment of R 6 to the remainder of the linker; or R 6 comprises an azide, thiol, tetrazine, trans-cyclooctene or acrylamide functional group.
  • R 6 is .
  • R 1 and/or R 4 comprise an N 3 moiety, the complementary functional group on the linker is an alkyne, and the linker is coupled to the amatoxin analog or the salt thereof through a 1,2,3-triazole.
  • R 4 comprises an N 3 moiety, the complementary functional group on the linker is an alkyne, and the linker is coupled to the amatoxin analog or the salt thereof through a 1,2,3-triazole.
  • R 4 comprises -CH 2 CH 2 N 3 , the complementary functional group on the linker is an alkyne, and the linker is coupled to the amatoxin analog or the salt thereof through a 1,2,3-triazole.
  • R 1 comprises an SH moiety, the complementary functional group comprises an orthopyridyl disulfide, and the linker is coupled to the amatoxin analog or the salt thereof through a disulfide.
  • the present disclosure also includes a conjugate comprising a target-binding moiety conjugated to an amatoxin analog or an amatoxin-linker construct as described herein.
  • the conjugate comprises the target-binding moiety conjugated to the amatoxin analog.
  • the conjugate comprises the target-binding moiety conjugated to the amatoxin-linker construct.
  • target-binding moiety refers to any suitable moiety that can specifically bind to a target molecule (e.g. protein) or epitope.
  • the target-binding moiety is a peptide, an oligonucleotide, a lipid, a lipid carrier, a nanoparticle or combinations thereof.
  • the target-binding moiety is an antibody, an antigen-binding fragment thereof, an antibody-like protein, an aptamer, a vitamin, an oligonucleotide, an affibody, a minibody, a lipid, a lipid carrier, a nanoparticle or combinations thereof.
  • the target-binding moiety is an antibody, an antigen-binding fragment thereof, an antibody-like protein, an aptamer, a vitamin, an oligonucleotide, an affibody, a minibody, a lipid, a lipid carrier or a nanoparticle.
  • the target-binding moiety is an antibody, antigen-binding fragment thereof or an antibody-like protein.
  • the target-binding moiety is an antibody.
  • the conjugate has a drug-to-target-binding moiety ratio e.g., a drug-to- antibody ratio (DAR) of about 0.1 to about 10, about 0.5 to about 5 or about 1 to about 3.
  • DAR drug-to- antibody ratio
  • the conjugate has a drug-to-target-binding moiety ratio e.g., a drug-to- antibody ratio (DAR) of about 2.
  • DAR drug-to- antibody ratio
  • the term “antigen-binding fragment thereof” as used herein means that the fragment of the antibody comprises at least a functional antigen-binding domain.
  • antibody-like protein refers to a protein that is not strictly an antibody but has the capability of binding to a target molecule (e.g. protein) or epitope.
  • the “antibody-like protein” is a protein that has been engineered (e.g. by mutagenesis of Ig loops) to specifically bind to the target molecule.
  • such an antibody-like protein comprises at least one variable peptide loop attached at both ends to a protein scaffold.
  • This double structural constraint greatly increases the binding affinity of the antibody-like protein to levels comparable to that of an antibody.
  • the length of the variable peptide loop typically consists of 10 to 20 amino acids.
  • the scaffold protein may be any protein having good solubility properties. In an embodiment, the scaffold protein is a small globular protein.
  • the scaffold of antibody-like proteins can be based on, for example, without limitation, affilin proteins, affibodies, anti-calins, lipocalins, ubiquitin, leucine-rich repeat proteins, and designed ankyrin repeat proteins (see, for example: Binz et al., “Engineering novel binding proteins from nonimmunoglobulin domains” Nat Biotechnol. 2005, 23:10, 1257-68).
  • the antibody or antigen-binding fragment thereof can be from any suitable immunoglobulin type (e.g.
  • Suitable antibodies and/or antigen-binding fragments thereof may include but are not limited to polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized (e.g. CDR-grafted), deimmunized, and/or chimeric antibodies, single chain antibodies (e.g.
  • the antigen-binding fragments are human antigen-binding antibody fragments and include, but are not limited to, Fab, Fab' and F(ab') 2 , Fd, single- chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (dsFv) and fragments comprising either a VL or VH domain.
  • Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable domain(s) alone or in combination with the entirety or a portion of the following: hinge region, CL, CH1, CH2, and CH3 domains.
  • antigen-binding fragments also comprising any combination of variable domain(s) with a hinge region, CL, CH1, CH2, and CH3 domains.
  • the antibody, antigen- binding fragment thereof or antibody-like protein may be from any animal origin including birds and mammals.
  • the antibody, antigen-binding fragment thereof or antibody-like protein is from human, rodent (e.g. mouse, rat, guinea pig, or rabbit), chicken, pig, sheep, goat, camel, cow, horse, donkey, cat, or dog origin.
  • the antibody, antigen-binding fragment thereof or antibody-like protein is of human or murine origin.
  • human as used herein in reference to antibodies includes antibodies having the amino acid sequence of a human immunoglobulin and also includes antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described for example in U.S. Patent No.5,939,598.
  • specifically bind as used herein in reference to a target-binding moiety specifically binding to a target molecule or epitope means, for example, that it has a dissociation constant K D to the target molecule or epitope of at most about 100 ⁇ M.
  • KD is about 100 ⁇ M or lower, about 50 ⁇ M or lower, about 30 ⁇ M or lower, about 20 ⁇ M or lower, about 10 ⁇ M or lower, about 5 ⁇ M or lower, about 1 ⁇ M or lower, about 900 nM or lower, about 800 nM or lower, about 700 nM or lower, about 600 nM or lower, about 500 nM or lower, about 400 nM or lower, about 300 nM or lower, about 200 nM or lower, about 100 nM or lower, about 90 nM or lower, about 80 nM or lower, about 70 nM or lower, about 60 nM or lower, about 50 nM or lower, about 40 nM or lower, about 30 nM or lower, about 20 nM or lower or about 10 nM or lower, about 1 nM or lower, about 900 pM or lower, about 800 pM or lower, about 700 pM or lower, about 600 pM or lower, about 500 pM or or
  • the target molecule or epitope associated with cancer or non-cancerous neoplasms is present on the surface of one or more tumor cell types or tumor-associated cells in an increased concentration and/or in a different steric configuration as compared to the surface of non- tumor cells.
  • the target molecule or epitope associated with cancer is an epitope of human epidermal growth receptor 2 (HER2), prostate-specific membrane antigen (PSMA), CD20, CD269, sialyl Lewis X , HER-2/neu or epithelial cell adhesion molecule (EpCAM).
  • the target molecule or epitope associated with cancer is an epitope of human epidermal growth receptor 2 (HER2).
  • the target- binding moiety is an anti-HER2 antibody.
  • the anti-HER2 antibody is trastuzumab.
  • the target molecule or target epitope is associated with autoimmune disease.
  • the target molecule or epitope associated with autoimmune disease is preferentially expressed on cells involved in an autoimmune disease.
  • the target molecule or target epitope is associated with a viral infection.
  • the target molecule or epitope associated with a viral infection is preferentially expressed on cells involved in a viral infection.
  • the conjugate comprises the amatoxin-linker construct
  • the target-binding moiety comprises an engineered acceptor residue
  • R 6 is a reactive group to a moiety comprised in the engineered acceptor residue
  • the amatoxin-linker construct is conjugated to the target-binding moiety via a moiety resulting from the reaction of the moiety comprised in the engineered acceptor residue with R 6 .
  • engineered acceptor residue refers to a residue that is introduced into a peptide sequence of a suitable target-binding moiety that is generally not present in the native peptide sequence of the target-binding moiety.
  • engineered acceptor residue is an engineered cystine residue and R 6 is a thiol-reactive group.
  • engineered cysteine residue refers to a cysteine residue that is introduced into the peptide sequence of a suitable target-binding moiety that is generally not present in the native peptide sequence of the target-binding moiety. Such cysteine residues are available for conjugation but desirably do not substantially affect, for example, immunoglobulin folding, antibody assembly, antigen binding and/or Fc domain effector functions.
  • the engineered cysteine residue can take the place of the amino acid that naturally occurs at a given position in the peptide sequence; i.e. in an embodiment of the present disclosure, the engineered cysteine residue is a cysteine substitution.
  • Engineered cysteine residues can be introduced into the peptide sequence of the target-binding moiety through suitable techniques such as site-directed mutagenesis, the selection of which can be made by a person skilled in the art.
  • the target-binding moiety comprises one engineered cysteine residue.
  • the target-binding moiety comprises greater than one engineered cysteine residue, for example, two engineered cysteine residues.
  • the engineered cysteine residue is selected from the group consisting of heavy chain 118Cys, heavy chain 239Cys, and heavy chain 265Cys. In another embodiment, the engineered cysteine residue is heavy chain 265Cys.
  • the target-binding moiety comprises a biorthogonal functional group
  • R 6 is a complementary biorthogonal functional group
  • the amatoxin-linker construct is conjugated to the target-binding moiety via a moiety resulting from the reaction of the biorthogonal functional group on the target-binding moiety with R 6 .
  • biorthogonal refers to a chemical reaction capable of occurring inside of a living system without interfering with native biochemical processes.
  • Biorthogonal reactions are well known in the art and include but are not limited to reaction between an azide and a soft nucleophile such as a phosphine; reaction between an azide and a suitable acetylene, and 1,3-dipolar cycloaddition between an azide and a suitable cyclooctyne or azacyclooctyne (copper-free click chemistry).
  • the target-binding moiety comprises a primary amino group
  • R 6 is a reactive group to the primary amino group
  • the amatoxin-linker construct is conjugated to the target-binding moiety via a moiety resulting from the reaction of the primary amino group with R 6 .
  • the present disclosure also includes a composition comprising an amatoxin analog or conjugate of the present disclosure and a carrier.
  • the composition comprises the amatoxin analog of the present disclosure and the carrier.
  • the composition comprises the conjugate of the present disclosure and the carrier.
  • the amatoxin analogs and/or conjugates of the present disclosure are optionally formulated into pharmaceutical compositions for administration or use in a biologically compatible form, for example, a form suitable for administration to or for use in subjects in vivo.
  • the present disclosure also includes a pharmaceutical composition comprising an amatoxin analog or conjugate of the present disclosure and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises the amatoxin analog of the present disclosure and the pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises the conjugate of the present disclosure and the pharmaceutically acceptable carrier.
  • the amatoxin analog or conjugate of the present disclosure can be administered to a subject or used in a variety of forms depending on the selected route of administration or use, as will be understood by a person skilled in the art.
  • the amatoxin analog or conjugate of the present disclosure is suitably administered to the subject or for use parenterally; i.e. taken into the body or administered or used in a manner other than through the gastrointestinal tract.
  • the amatoxin analog or conjugate of the present disclosure is administered or for use as an injectable or infusion.
  • Injectables can be formulated in the form of ampules and/or as a ready-for-use injectable such as a ready-to-use syringe, a single-use syringe and/or in a puncturable flask for multiple withdrawal.
  • the injectable is administered or for use in the form of a subcutaneous (s.c.), intramuscular (i.m.), intravenous (i.v.) or intracutaneous (i.c.) injection.
  • the infusion is in the form of an isotonic solution, fatty emulsion, liposomal formulation or micro-emulsion. A person skilled in the art would know how to prepare suitable formulations.
  • the injectable or infusion formulation is in the form of a concentrate which can be dissolved or dispersed with aqueous isotonic diluents. Injectable formulations can also be administered or for use in the form of a permanent infusion e.g. via a mini-pump.
  • the parenteral formulation further comprises albumin, plasma, expander, surface-active substances, organic diluents, pH-influencing substances, complexing substances, polymeric substances or combinations thereof, for example to influence the adsorption of the amatoxin analog or conjugate of the present disclosure to proteins or polymers and/or to reduce the adsorption of the amatoxin analog or conjugate of the present disclosure to materials like injection instruments or packaging-materials, for example, plastic or glass.
  • adjuvants and carriers in the pharmaceutical compositions formulated as parenterals are one or more of aqua sterilisata (sterilized water), pH value influencing substances (for example, suitable organic or inorganic acids or bases and salts thereof or suitable combinations thereof), buffering substances for adjusting pH values, substances for isotonization (for example, sodium chloride, sodium hydrogen carbonate, glucose, fructose or combinations thereof), surfactants (for example, partial esters of fatty acids of polyoxyethylene sorbitans such as a Tween TM surfactant or fatty acid esters of polyoxyethylenes such as a Cremophor TM surfactant or combinations thereof), fatty oils (for example, soybean oil, castor oil or combinations thereof), synthetic esters of fatty acids (for example, ethyl oleate, isopropyl myristate or combinations thereof), polymeric adjuvants (for example, gelatine, dextran, polyvinylpyrrolidone or combinations thereof), additive
  • modified-release formulations include, for example, sustained-release (SR), extended-release (ER, XR, or XL), time-release or timed-release, controlled-release (CR), or continuous-release (CR or Contin), employed, for example, in the form of a coated tablet, an osmotic delivery device, a coated capsule, a microencapsulated microsphere, an agglomerated particle, e.g., as molecular sieving type particles, or, a fine hollow permeable fiber bundle, or chopped hollow permeable fibers, agglomerated or held in a fibrous packet.
  • Timed-release compositions can be formulated, e.g.
  • the present disclosure also includes all uses for the amatoxin analogs, amatoxin- linker constructs and conjugates of the present disclosure, including, for example, use in therapeutic methods, diagnostic assays and as research tools whether alone or in combination with another active pharmaceutical ingredient.
  • the synthesis and biochemical evaluation of new amatoxin analogs comprising modifications of the DHIle residue is disclosed herein. Such amatoxin analogs and conjugates of the present disclosure may be useful as medicaments. Accordingly, the present disclosure also includes an amatoxin analog or conjugate of the present disclosure for use as a medicament.
  • the amatoxin analog of the present disclosure is for use as a medicament.
  • the conjugate of the present disclosure is for use as a medicament.
  • the present disclosure also includes a method of treating a disease associated with cells presenting a target in a subject in need thereof, the method comprising administering an effective amount of an amatoxin analog or a conjugate of the present disclosure to the subject, wherein the target-binding moiety is specific for the target.
  • the present disclosure also includes a method of treating a disease associated with cells presenting a target in a subject in need thereof, the method comprising administering an effective amount of an amatoxin analog of the present disclosure to the subject, wherein the target-binding moiety is specific for the target.
  • the present disclosure also includes a method of treating a disease associated with cells presenting a target in a subject in need thereof, the method comprising administering an effective amount of a conjugate of the present disclosure to the subject, wherein the target-binding moiety is specific for the target.
  • the present disclosure also includes a use of an amatoxin analog or a conjugate of the present disclosure for treatment of a disease associated with cells presenting a target in a subject in need thereof, wherein the target-binding moiety is specific for the target.
  • the present disclosure also includes a use of an amatoxin analog of the present disclosure for treatment of a disease associated with cells presenting a target in a subject in need thereof, wherein the target- binding moiety is specific for the target.
  • the present disclosure also includes a use of a conjugate of the present disclosure for treatment of a disease associated with cells presenting a target in a subject in need thereof, wherein the target-binding moiety is specific for the target.
  • the present disclosure also includes a use of an amatoxin analog or a conjugate of the present disclosure for preparation of a medicament for treatment of a disease associated with cells presenting a target in a subject in need thereof, wherein the target-binding moiety is specific for the target.
  • the present disclosure also includes a use of an amatoxin analog of the present disclosure for preparation of a medicament for treatment of a disease associated with cells presenting a target in a subject in need thereof, wherein the target-binding moiety is specific for the target.
  • the present disclosure also includes a use of a conjugate of the present disclosure for preparation of a medicament for treatment of a disease associated with cells presenting a target in a subject in need thereof, wherein the target-binding moiety is specific for the target.
  • the present disclosure also includes an amatoxin analog or a conjugate of the present disclosure for use to treat a disease associated with cells presenting a target in a subject, wherein the target-binding moiety is specific for the target.
  • the present disclosure also includes an amatoxin analog of the present disclosure for use to treat a disease associated with cells presenting a target in a subject, wherein the target-binding moiety is specific for the target.
  • the present disclosure also includes a conjugate of the present disclosure for use to treat a disease associated with cells presenting a target in a subject, wherein the target-binding moiety is specific for the target.
  • the term “disease associated with cells presenting a target” as used herein refers, for example, to a pathological state characterized by a cellular marker such as but not limited to a receptor and/or enzyme which can be used in a targeted fashion.
  • the disease associated with cells presenting a target is cancer, a disease associated with non- cancerous neoplasms, an autoimmune disease, an inflammatory disease or a viral infection.
  • the disease associated with cells presenting a target is cancer, an autoimmune disease or a viral infection.
  • the term “cancer” as used herein refers to diseases caused by uncontrolled cell division and/or the ability of cells to metastasize, or to establish new growth in additional sites.
  • the cancer is a skin cancer (e.g., melanoma), a connective tissue cancer (e.g., sarcomas), an adipose cancer, a breast cancer, a head and neck cancer, a lung cancer (e.g., mesothelioma), a stomach cancer, a pancreatic cancer, an ovarian cancer, a cervical cancer, a uterine cancer, an anogenital cancer (e.g., testicular cancer), a kidney cancer, a bladder cancer, a colon cancer, a prostate cancer, a central nervous system (CNS) cancer, retinal cancer, a blood cancer, a neuroblastoma, multiple myeloma, or a lymphoid cancer (e.g., Hodgkin’s and non-Hodgkin’s lymph
  • the cancer is breast cancer. In a further embodiment, the cancer is HER2- positive breast cancer.
  • autoimmune disease can be used interchangeably with the term “autoimmune disorder” and refers to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunologic reaction of the subject to its own cells, tissues and/or organs.
  • inflammatory disease can be used interchangeably with the term “inflammatory disorder” and refers to a condition in a subject characterized by inflammation, such as chronic inflammation. Autoimmune disorders may or may not be associated with inflammation. Moreover, inflammation may or may not be caused by an autoimmune disorder. Thus, the skilled person would appreciate that certain disorders may e.g. be characterized as both autoimmune and inflammatory disorders.
  • the inflammatory disease is systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), Autoimmune Hemolytic Anaemia (AIHA), or Sjögren’s syndrome.
  • the disease associated with cells presenting a target is a viral infection
  • the cells presenting a target are virally infected cells.
  • the viral infection is human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • the present disclosure also includes a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of an amatoxin analog of the present disclosure to the subject.
  • the present disclosure also includes a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of a conjugate of the present disclosure to the subject.
  • the present disclosure also includes a use of an amatoxin analog or a conjugate of the present disclosure for treatment of cancer in a subject in need thereof.
  • the present disclosure also includes a use of an amatoxin analog of the present disclosure for treatment of cancer in a subject in need thereof.
  • the present disclosure also includes a use of a conjugate of the present disclosure for treatment of cancer in a subject in need thereof.
  • the present disclosure also includes a use of an amatoxin analog or a conjugate of the present disclosure for preparation of a medicament for treatment of cancer in a subject in need thereof.
  • the present disclosure also includes a use of an amatoxin analog of the present disclosure for preparation of a medicament for treatment of cancer in a subject in need thereof.
  • the present disclosure also includes a use of a conjugate of the present disclosure for preparation of a medicament for treatment of cancer in a subject in need thereof.
  • the present disclosure also includes an amatoxin analog or a conjugate of the present disclosure for use to treat cancer in a subject.
  • the present disclosure also includes an amatoxin analog of the present disclosure for use to treat cancer in a subject.
  • the present disclosure also includes a conjugate of the present disclosure for use to treat cancer in a subject.
  • Treatment methods or uses comprise administering to a subject or use of an effective amount of an amatoxin analog or conjugate of the present disclosure, optionally consisting of a single administration or use, or alternatively comprising a series of administrations or uses.
  • the amatoxin analogs or conjugates of the present disclosure are administered or used at least once a week.
  • the amatoxin analog or conjugate is administered to the subject or for use from one time per three weeks, or one time per week to once daily for a given treatment or use.
  • the length of the treatment period or use depends on a variety of factors, such as the severity of the disease, the age of the subject, the activity of the amatoxin analog or conjugate of the present disclosure and/or a combination thereof.
  • amatoxin analog or conjugate used for the treatment or use may increase or decrease over the course of a particular treatment regime or use. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration or use is required.
  • the amatoxin analog or conjugate of the present disclosure is administered or for use in an amount and for a duration suitable to treat the subject.
  • the amatoxin analog or conjugate of the present disclosure may be administered or used alone or in combination with other therapeutic agents useful for treating a disease (e.g. cancer).
  • the amatoxin analog or conjugate of the present disclosure is administered or for use contemporaneously with those therapeutic agents.
  • the term “contemporaneous” in reference to administration of two substances to a subject or use means providing each of the two substances so that they are both biologically active in the individual at the same time. The exact details of the administration or use will depend on the pharmacokinetics of the two substances in the presence of each other, and can include administration or use of the two substances within a few hours of each other, or even administration or use of one substance within 24 hours of administration or use of the other, if the pharmacokinetics are suitable. Design of suitable dosing regimens is routine for a person skilled in the art.
  • two substances will be administered or for use substantially simultaneously, i.e. within minutes of each other, or in a single composition that includes both substances. It is a further embodiment of the present disclosure that a combination of the two substances is administered to a subject or for use in a non-contemporaneous fashion.
  • the dosage of the amatoxin analog or conjugate of the disclosure can vary depending on many factors such as the pharmacodynamic properties of the amatoxin analog or conjugate, the age, health and/or weight of the subject, the nature and/or extent of the symptoms of the disease, the frequency of the treatment or use and the type of concurrent treatment or use, if any, and the clearance rate of the amatoxin analog or conjugate in the subject.
  • the amatoxin analog or conjugate of the present disclosure is administered or for use initially in a suitable dosage that is optionally adjusted as required, depending on the clinical response.
  • the present disclosure also includes a kit comprising an amatoxin analog as described herein, a linker as described herein and optionally instructions for preparing a amatoxin-linker construct as described herein from the amatoxin analog and the linker.
  • the present disclosure also includes a kit comprising an amatoxin analog as described herein, a linker as described herein, a target-binding moiety as described herein and optionally instructions for preparing a conjugate as described herein from the amatoxin analog, the linker and the target-binding moiety.
  • the present disclosure also includes a kit comprising an amatoxin-linker construct as described herein, a target-binding moiety and optionally instructions for preparing a conjugate as described herein from the amatoxin-linker construct and the target-binding moiety. IV.
  • the present disclosure includes a method of preparing a dihydroxyisoleucine (DHIle) analog, the method comprising: (i) saponification of a lactone of Formula IV to provide a diol of Formula V: , wherein: R 1a is H, OR ', N(R ') 2 , SR ', halo, -N 3 , -S-S-R ', imidazole, -CN, -S(O)R ', - ON(R ') 2 , -N(R ')OR ', -S(O) 2 R ', -SeR ' or -N(R ')N(R) 2 , a protected form thereof or a precursor thereto; and PG 1 is a protecting group; and optionally further comprising: (ii) protection of the diol of Formula V to provide a compound of Formula VI: , wherein: R 1a is H, OR ', N(R ') 2
  • the method comprises the saponification of the lactone of Formula IV to provide the diol of Formula V, protection of the diol of Formula V to provide the compound of Formula VI, and conversion of the compound of Formula VI to the NHS ester of Formula II.
  • the conditions for saponification of the lactone of Formula IV to provide the diol of Formula V can be any suitable conditions.
  • the conditions comprise reacting the lactone of Formula IV with a suitable base (for example, an alkali metal hydroxide such as lithium hydroxide) to convert the lactone of Formula IV to the corresponding carboxylate salt followed by neutralization of the carboxylate salt with a suitable acid (for example, HCl) to obtain the diol of Formula V.
  • a suitable base for example, an alkali metal hydroxide such as lithium hydroxide
  • the conditions for protection of the diol of Formula V to provide the compound of Formula VI can be any suitable conditions, the selection of which may depend, for example, on the identity of R 1a and/or PG 2 .
  • PG 2 is a silyl ether.
  • PG 2 is tert-butyldimethylsilyl.
  • the conditions comprise treatment of the diol of Formula V with tert-butyldimethylsilyl chloride.
  • the conditions for conversion of the compound of Formula VI to the NHS ester of Formula II can be any suitable conditions.
  • the conditions comprise reaction of the compound of Formula VI with N,N’-disuccinimidyl carbonate.
  • R 1a is other t R is -SRi7R O 8 , w HhNer PeGin1han H, an each R 8 is indepe1d na the lactone of Formula IV is prepared by a method comprising: (i) conversion of the silyl enol ether of Formula VII to t de Ohe ⁇ ntly H-k CNe 1 Pto -6G-i al1soleucine of Formula VIII: rein: PG 1 is a pro (VteIcIt)ing g OrEotup; 7 R (VIII) O OkyElt , whe O or aryl; and R 1a is H, OR ', N(R ') 2 , SR ', halo, -N 3 , -S-S-R ', imidazole, -CN, -S(O)R ', - ON(R ') 2 , -N(R ')OR '
  • the present disclosure also includes a method of preparing a ⁇ -keto-is PG 1 is 2R a7 p Orote HcNtin P( Og OG g ) Or 2REotup; R 7 is -SiR 8 , wherein each R 8 is indep ', -SeR ' or -N Re (R1nade ')N Ontly (R) HI C 2)N1 , a P- O6 pGal r Oky otEl etoleucine of Formula VIII, the method comprising conversion of the silyl enol ether of Formula VII to the ⁇ -keto-isoleucine of Formula VIII:1 1 , wherein: (VII) (VII or aryl; and R 1a is H, OR ', N(R ') 2 , SR ', halo, -N 3 , -S-S-R ', imidazole, -CN,
  • R 1a is OH and the conversion of the silyl enol ether of Formula VII to the ⁇ -keto-isoleucine of Formula VIII comprises Rubottom oxidation.
  • R 1a is Br, and the conversion of the silyl enol ether of Formula VII to the ⁇ -keto-isoleucine of Formula VIII comprises electrophilic bromination of the silyl enol ether of Formula VII to provide a ⁇ -bromo- ⁇ -keto-isoleucine of Formula VIII.
  • the method further comprises nucleophilic substitution of R 1a in the ⁇ -bromo- ⁇ -keto-isoleucine of Formula VIII.
  • the nucleophilic substitution provides a ⁇ -fluoro- ⁇ -keto-isoleucine of Formula VIII.
  • the conditions for reduction of the ⁇ -keto-isoleucine of Formula VIII to obtain the lactone of Formula IV can be any suitable conditions.
  • the conditions comprise borane-THF reduction with the 2-methyl-CBS-oxazaborolidine catalyst.
  • the method further comprises nucleophilic substitution of R 1a in the lactone of Formula IV.
  • the nucleophilic substitution provides a lactone of Formula IV, wherein R 1a is N 3 , -SH, -S-S- t Bu or -S-S-CH(CH 3 )CH(CH 3 ) 2 .
  • R 1a is -SH and the method comprises: nucleophilic displacement of bromine with a suitable source of thioacetate such as an alkali metal ethanethioate (e.g., potassium ethanethioate) followed by treatment with a suitable reagent (e.g., hydrazine) to obtain the lactone of Formula IV, wherein R 1a is SH.
  • a suitable source of thioacetate such as an alkali metal ethanethioate (e.g., potassium ethanethioate) followed by treatment with a suitable reagent (e.g., hydrazine) to obtain the lactone of Formula IV, wherein R 1a is SH.
  • R 1a is -S-S-R '.
  • R 1a is -S-S- t Bu or -S- S-CH(CH 3 3)CH(CH H 3 3S).
  • the method erein R is SH w such as CsCOR)' ca ith to Sn H o S further S-R ' u btain th O comprise: reacting the lactone of For HmNula IV, whS R' 1a Hn eNder lac O suitable conditions (e.g., in the presence of a suitPaGbl1e base 2 3 Boctone of Formula IV, wherein R 1a is -S-S-R ' such as wherein R 1a is -S-S- t Bu or -S-S-CH(CH 3 3)CH(CH 3 3).
  • the present disclosure also includes a method of preparing a (2S,3R,4S) dihydroxyisoleucine (DHIle) analog, wherein the reduction of the ⁇ -keto-isoleucine of Formula VIII as described herein further or alternatively provides a compound of Formula IX: , wherein: PG 1 is a protecting group; and R 1a is OR ', N(R ') 2 , SR ', halo, -N 3 , -S-S-R ', imidazole, -CN, -S(O)R ', -ON(R ') 2 , - N(R ')OR ', -S(O) 2 R ', -SeR ' or -N(R ')N(R) 2 , a protected form thereof or a precursor thereto, the method comprising: (i) removal of the ethyl group to provide a diol of Formula V ': , wherein:
  • the method comprises the protection of the diol of Formula V ' to provide the compound of Formula VI '; and the conversion of the compound of Formula VI ' to the NHS ester of Formula II '.
  • R 1a is F.
  • the conditions for removal of the ethyl group to provide the diol of Formula V ' can comprise any suitable conditions.
  • the conditions comprise reaction of the diol of Formula V ' with a suitable base such as an alkali metal hydroxide (e.g., LiOH).
  • the conditions for protection of the diol of Formula V ' to provide a compound of Formula VI ' can be any suitable conditions, the selection of which may depend, for example, on the identity of R 1a and/or PG 2 .
  • PG 2 is a silyl ether.
  • PG 2 is tert-butyldimethylsilyl.
  • the conditions comprise treatment of the diol of Formula V ' with tert-butyldimethylsilyl chloride.
  • the conditions for conversion of the compound of Formula VI ' to the NHS ester of Formula II ' can be any suitable conditions.
  • the conditions comprise reaction of the compound of Formula VI ' with N,N'-disuccinimidyl carbonate.
  • the silyl enol ether of Formula VII is prepared by a method comprising reacting a ⁇ -keto-isoleucine of Formula X with a silane of Formula XI in the presence of a base: wherein: PG 1 is a protecting group; each R 8 is independently C1-6alkyl or aryl; and R 9 is a hydrolysable group.
  • the hydrolysable group can be any suitable hydrolysable group, the selection of which can be made by a person skilled in the art.
  • R 9 is chloro.
  • each R 8 is independently C 1-6 alkyl. In an embodiment each R 8 is methyl.
  • the compound of Formula X is prepared by a method comprising: removal of the p-methoxyphenyl (PMP) group from a ⁇ -keto-L-isoleucine of Formula XII followed by protection of the amino group to provide the compound of Formula X: .
  • the conditions for removal of the PMP can be any suitable conditions. In an embodiment, the conditions comprise treatment with trichloroisocyanuric acid.
  • the conditions for protection of the amino group to provide the compound of Formula X can be any suitable conditions, the selection of which may depend, for example, on the identity of PG 1 .
  • PG 1 is Boc.
  • the conditions comprise reaction with (Boc) 2 O in the presence of a suitable base.
  • R 1 is H
  • the lactone of Formula IV is prepared by a method comprising: (i) reduction of a ⁇ -keto-isoleucine of Formula XII to provide a lactone of Formula IV(a): and protection of the lactone of Formula XV(a) to provide the lactone of Formula IV.
  • the conditions for reduction of the ⁇ -keto-isoleucine of Formula XII to provide the lactone of Formula IV(a) can comprise any suitable conditions.
  • the conditions comprise treatment with trichloroisocyanuric acid.
  • the conditions for protection of the lactone of Formula XV(a) to provide the lactone of Formula IV can comprise any suitable conditions, the selection of which may depend, for example, on the identity of PG 1 .
  • PG 1 is Boc.
  • the conditions comprise reaction with (Boc) 2 O in the presence of a suitable base.
  • the ⁇ -keto-L-isoleucine of Formula XII is prepared by a method comprising: (i) L-proline-catalyzed Mannich reaction of p-methoxyphenyl-iminoester of Formula XIII with butanone to provide ⁇ -keto-L-allo-isoleucine of Formula XIV: ; and (ii) catalytic epimerization of the ⁇ -keto-L-allo-isoleucine of Formula XIV to provide the ⁇ -keto-L-isoleucine of Formula XII: .
  • the conditions for the catalytic epimerization of the ⁇ -keto-L-allo-isoleucine of Formula XIV to provide the ⁇ -keto-L-isoleucine of Formula XII can comprise any suitable conditions.
  • the conditions comprise reaction of the ⁇ -keto-L-allo- isoleucine of Formula XIV with 1,5-diazabicyclo[4.3.0]non-5-ene (DBN).
  • the p-methoxyphenyl-iminoester of Formula XIII is prepared by reaction of p-anisidine with ethyl glyoxalate.
  • the method is for preparing a dihydroxyisoleucine (DHIle) analog of Formula III(a): , the method comprising deprotection of the diol of Formula V or Formula V ' to provide the compound of Formula III.
  • the conditions for deprotection can be any suitable conditions, which may depend, for example, on the identity of R 1a and/or R 1 as well as the identity of the protecting groups. A person skilled in the art could readily select such conditions.
  • the present disclosure also includes use of a compound of Formula II as described herein or an NHS ester of Formula II or Formula II ' prepared by a method as described herein in the preparation of an amatoxin or an amatoxin analog.
  • the present disclosure also includes a method of preparing an ⁇ -amanitin or an ⁇ -amanitin analog, the method comprising coupling a compound of Formula II as described herein or an NHS ester of Formula II or Formula II ' prepared by a method as described herein to a monocyclic heptapeptide ⁇ -amanitin or ⁇ -amanitin analog precursor, followed by deprotection and macrolactamization.
  • the monocyclic heptapeptide ⁇ -amanitin or ⁇ - amanitin analog precursor can be prepared by any suitable method.
  • the preparation can comprise a method in line with that reported Matinkhoo et al.
  • PG is tert-butyloxycarbonyl and the conditions for deprotection comprise HCl having a concentration of about 1 M in a solvent comprising Et 2 O.
  • R 1 is N 3 , and the method further comprises reduction of the ⁇ -amanitin analog to convert R 1 into an NH 2 and/or an epoxide.
  • the present disclosure also includes a dihydroxyisoleucine (DHIle) analog of the Formula II: , wherein R 1a is H, OR ', N(R ') 2 , SR ', halo, -N 3 , -S-S-R ', imidazole, -CN, -S(O)R ', - ON(R ') 2 , -N(R ')OR ', -S(O) 2 R ', -SeR ' or -N(R ')N(R) 2 , a protected form thereof or a precursor thereto; and PG 1 and PG 2 are each independently protecting groups.
  • DHIle dihydroxyisoleucine
  • R 1 is H, O-PG 3 , N(R ') 2 , SR ', halo, -N 3 or -S-S-R '.
  • R 1 is H, O-PG 3 , N(R ') 2 , SR ', halo, -N 3 or -S-S-R ', wherein R ' is H or C 1-6 alkyl.
  • R 1 is H, O-PG 3 , F, Br, -N 3 , -S-S- t Bu, SH or -S-S- CH(CH 3 )CH(CH 3 ) 2 .
  • R 1 is H, O-PG 3 , F, Br, -N 3 , -S-S- t Bu or NH 2 .
  • R 1 is F, Br, -N 3 , -S-S- t Bu, SH or -S-S-CH(CH 3 )CH(CH 3 ) 2 .
  • R 1 is H.
  • R 1 is O-PG 3 .
  • R 1 is F.
  • R 1 is Br.
  • R 1 is -N 3 .
  • R 1 is -S-S- t Bu.
  • R 1 is NH 2 .
  • R 1 is SH.
  • R 1 is -S-S-CH(CH 3 )CH(CH 3 ) 2 .
  • PG 1 , PG 2 and PG 3 can be any suitable protecting groups, the selection of which can be made by a person skilled in the art.
  • PG 1 , PG 2 and PG 3 are capable of use in solid phase peptide synthesis (SPPS).
  • SPPS solid phase peptide synthesis
  • PG 1 is tert- butyloxycarbonyl.
  • PG 2 is tert-butyldimethylsilyl.
  • O-PG 3 is a silyl ether.
  • PG 3 is tert-butyldimethylsilyl.
  • the present disclosure also includes a dihydroxyisoleucine (DHIle) analog of the Formula III: , wherein R 1 is OR ', N(R ') 2 , SR ', halo, -N 3 , -S-S-R ', imidazole, -CN, -S(O)R ', -ON(R ') 2 , -N(R ')OR ', -S(O) 2 R ', -SeR ' or -N(R ')N(R) 2 ; and each R ' is independently H, C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl or heteroaryl, wherein one or more available carbon atoms in the C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl is optionally replaced by a heteroatom,
  • the compound of Formula III has the following stereochemistry: .
  • the compound of Formula III has the following stereochemistry: .
  • R 1 is OR ', N(R ') 2 , SR ', halo, -N 3 or -S-S-R '.
  • R 1 is N(R ') 2 , SR ', halo, -N 3 or -S-S-R ', wherein R ' is H or C1-6alkyl.
  • R 1 is F, Br, -N 3 , -S-S- t Bu, SH or -S-S-CH(CH 3 )CH(CH 3 ) 2 .
  • R 1 is F, Br, -N 3 , or -S-S- t Bu.
  • R 1 is F.
  • R 1 is Br.
  • R 1 is -N 3 .
  • R 1 is -S-S- t Bu.
  • R 1 is SH.
  • R 1 is -S-S-CH(CH 3 )CH(CH 3 ) 2 .
  • R ' is H, C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl or heteroaryl, wherein one or more available carbon atoms in the C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl is optionally replaced by a heteroatom, and wherein the C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl and heteroaryl is unsubstituted.
  • R ' is H, C 1-20 alkyl, C 2-20 alkenyl, C 2- 20alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl or heteroaryl, wherein the C 1-20 alkyl, C 2- 20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl and heteroaryl is further substituted with one or more groups selected from OR ' ', SR ' ', halo, -N 3 and nitrile, wherein R ' ' is independently H, C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl or heteroaryl.
  • each R ' is independently H or C 1-20 alkyl. In an embodiment, R ' is C1-6alkyl. In another embodiment, R ' is H.
  • the present disclosure also includes a compound of Formula VII: , wherein: PG 1 is a protecting group; and R 7 is -SiR 8 , wherein each R 8 is independently C1-6alkyl or aryl. [00191] In an embodiment, PG 1 is Boc. In an embodiment, each R 8 is independently C 1- 6alkyl. In an embodiment each R 8 is methyl.
  • the present disclosure also includes a compound of Formula VIII: , wherein: PG 1 is a protecting group; and R 1a is OH or a protected form thereof or halo.
  • PG 1 is Boc.
  • R 1a is OH.
  • R 1a is bromo.
  • R 1a is fluoro.
  • the present disclosure also includes a compound of Formula IV(b): , wherein: PG 1 is a protecting group; R 1a is SR ' or -S-S-R '; and each R ' is independently H, C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocycloalkyl or heteroaryl, wherein one or more available carbon atoms in the C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl is optionally replaced by a heteroatom, and wherein the C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, heterocyclo
  • PG 1 is Boc.
  • R 1a is SR '.
  • R 1a is SH.
  • R 1a is -S-S-R '.
  • R 1a is -S-S- t Bu.
  • R 1a is -S-S-CH(CH 3 )CH(CH 3 ) 2 .
  • Controlled temperature reactions were performed using a mineral oil bath and a temperature-controlled hot plate (IKA Ceramag Midi). Temperatures below room temperature were achieved in an ice/water bath (0 °C), and dry ice/acetone bath (-78 °C). Solvents were removed under reduced pressure using a Büchi rotary evaporator. Anhydrous solvents were prepared by distillation under a nitrogen atmosphere. DMSO, THF and DMF were dried over 4 ⁇ molecular sieves under argon atmosphere. n-Butyl lithium solutions in tetrahydrofuran were titrated with di- phenylacetic acid (DPAA) to the yellow endpoint.
  • DPAA di- phenylacetic acid
  • TLC Thin-layer chromatography
  • EM Science EM Science
  • Detection of TLC spots was performed using an ultraviolet (UV) lamp at 254 nm, p-anisaldehyde, potassium permanganate, ninhydrin, 2,4-dinitrophenylhydrazine and bromo cresol green prepared according to literature procedures.
  • Flash column chromatography purifications were performed using silica gel 60 (230-400 mesh, Silicycle, Quebec).
  • ⁇ -keto-L-allo-Isoleucine 2 (5.9 g, 21.12 mmol) was added followed by 1,5- diazabicyclo(4.3.0)non-5-ene (DBN, 0.2 ml, 1.68 mmol).
  • DBN 1,5- diazabicyclo(4.3.0)non-5-ene
  • the resulting solution was stirred under argon for 2 hours at room temperature. After that, it was stirred open to the atmosphere at room temperature for 24 hours.
  • the solvent was removed under reduced pressure to obtain the crude product.
  • the crude was dissolved in a minimum amount of boiling ethanol and kept at -20° C. A white crystalline solid was obtained which was filtered and washed with cold ethanol to give the pure product. Yield: 60%.
  • reaction mixture was quenched saturated solution of sodium thiosulphate (10 ml) and extracted with DCM (3 ⁇ 10 ml). The organic layer was washed with brine and dried over anhydrous sodium sulphate. The solvent was evaporated under reduced pressure to give crude, which was immediately dissolved in 20 ml THF and was stirred at 0 °C.1 M solution of tetra-n-butylammonium fluoride (TBAF) in THF (1.81 ml.1.81 mmol) was added and the mixture was stirred at 0 °C for 4 hours. The reaction mixture was washed with brine (15 ml) and extracted with ethyl acetate (3 ⁇ 10 ml).
  • TBAF tetra-n-butylammonium fluoride
  • the yellowish crude (42 mg, 0.16 mmol) was dissolved in 3.3 ml dimethylacetamide (DMA) followed by the addition of imidazole (108 mg, 1.6 mmol) and tert-butyldimethylsilyl chloride (TBSCl, 192 mg, 1.28 mmol).
  • Another 60 mg of imidazole was added to adjust the pH to 8 and the reaction mixture was stirred for 24 hours.
  • pH was adjusted to 2-3 using 0.1 M HCl and extracted with EtOAc (4 ⁇ 5 ml). The organic layer was washed with water, and brine and was dried over anhydrous sodium sulphate.
  • the organic layer was washed with brine and dried over anhydrous sodium sulfate.
  • the yellowish crude was dissolved in 25 mL DMA followed by the addition of imidazole (0.56 g, 8.16 mmol) and TBS-Cl (0.98 g, 6.53 mmol). Another 0.2 g of imidazole was added to adjust the pH to 8 and the reaction mixture was stirred for 24 hours. After the completion of the reaction, pH was adjusted to 2 using 0.1 M HCl and extracted with EtOAc (4 ⁇ 10 ml). The organic layer was washed with water, and brine and dried over anhydrous sodium sulphate.
  • N,N'-Disuccinimidyl carbonate (DSC, 26.08 mg, 0.10 mmol) and collidine (0.019 ml, 0.14 mmol) were added subsequently and the reaction mixture was warmed to room temperature over 24 hours.
  • the reaction mixture was diluted with ethyl acetate (30 ml); and the organic layer was washed with saturated KH 2 PO 4 , saturated NaHCO 3 , and brine and dried over anhydrous sodium sulphate.
  • the crude was purified on silica gel using flash column chromatography (EtOAc:Hexane 25:75 v/v) to afford the product.
  • Ester 6 was subjected to borane-THF reduction using the R-CBS catalyst (Corey et al., 1987; Lohray et al., 1992); and the resultant diol spontaneously formed lactone 7 with good diastereoselectivity. Boc removal provided lactone 7a (not shown) that was crystallized for x- ray diffraction to confirm the absolute stereochemical configuration of 7a and therefore that of 7. Saponification of lactone 7 gave diol 8, which was protected by TBS-Cl treatment, resulting in 9. Subsequent conversion to the NHS ester provided a SPPS-compatible NHS-ester of DHIle 10.
  • This method for obtaining Boc-protected DHIle 10 obviates the use of transition metals, highly reactive pyrophoric reagents, intricate transformations, and safety concerns associated with the use of cyanide in large-scale reactions.
  • This synthetic pathway entails conventional, cost-effective steps minimizing the purification requirements at each stage, enhancing scalability, and making it more accessible for large-scale production.
  • ⁇ -proline may alternatively be used for the direct production of the anti-Mannich product (Zhang et al., 2006; Mitsumori et al., 2006; Garg & Tanaka, 2020).
  • Silyl-enol ether 5 provides an especially attractive node for generating ⁇ -halogenated DHIle analogs that may be transposed further.
  • ketones 4 (or 6) provide an attractive handle for substitution.
  • the crude reaction mixture was dried under reduced pressure and analyzed by reverse-phase HPLC.
  • A integral area of the peak of interest determined using standard Agilent data analysis software.
  • amanitin A0 When tested against HEK-293 cells, amanitin A0 showed an IC 50 (0.31 ⁇ M), a value comparable to that of ⁇ - amanitin IC 50 (0.39 ⁇ M). Due to its high acute toxicity, only milligram quantities of the final toxin were synthesized. Accordingly, an optimized method for incorporating N-Boc-DHIle into the amanitin core is reported, which has not been previously documented, opening the opportunity for incorporating other Boc-protected amino acids e.g. homoserine and its analogs into peptides, as they are highly susceptible to undergoing N-to-O acyl transfer.
  • Boc-protected amino acids e.g. homoserine and its analogs into peptides
  • the synthetic route starts with a condensation reaction between p-anisidine and ethyl glyoxalate to give the (PMP)-protected imine of glycine ethyl ester.
  • This undergoes L- proline catalyzed Mannich condensation with 2-butanone that results in ⁇ -keto L-allo- isoleucine C1.
  • Scheme 8 shows the coupling reaction between heptapeptide monocycle and dihydroxy-isoleucine analogs for incorporation into ⁇ -amanitin where X, Y and Z are as defined in Table 4.
  • X, Y and Z are as defined in Table 4.
  • Scheme 9 shows a synthesis of a heptapeptide monocyclic precursor with the azide handle.
  • Scheme 9 Synthesis of heptapeptide monocyclic precursor with azide handle.
  • TCEP tris(2-carboxyethyl)phosphine
  • Scheme 10 Synthesis of (2S,3R,4R)-NH 2 -OH-Ama and (2S,3R,4R)-Epoxy-Ama analogs.
  • Additional ⁇ -amanitin analogs shown in Table 5 were prepared by similar methods as those described herein. Table 5 III.
  • FIG.3 shows the results of an MTT viability assay on HEK 293 cells
  • FIG. 4 shows the results of an MTT viability assay on CHO cells
  • FIG.5 shows the results of an MTT viability assay on HeLa cells
  • FIG.6 shows the results of an MTT viability assay on HepG2 cells for A1, A2, A3 and A4 in comparison to ⁇ -amanitin.
  • FIG.7 shows the results of an MTT viability assay on HEK 293 cells
  • FIG.8 shows the results of an MTT viability assay on CHO cells
  • FIG.9 shows the results of an MTT viability assay on HeLa cells
  • FIG.10 shows the results of an MTT viability assay on HepG2 cells for A6 in comparison to ⁇ - amanitin
  • FIG.11 shows the results of an MTT viability assay on HEK 293 cells for A7 in comparison to ⁇ -amanitin
  • FIG.12 shows the results of an MTT viability assay on HepG2 cells for A7 and A8 in comparison to ⁇ -amanitin.
  • FIG.13 shows the results of an MTT viability assay on HEK 293 cells
  • FIG.14 shows the results of an MTT viability assay on CHO cells for A8 in comparison to ⁇ -amanitin
  • FIG.15 shows the results of an MTT viability assay on HeLa cells for A7 and A8 in comparison to ⁇ -amanitin
  • FIG.16 shows the results of an MTT viability assay on CHO cells for A7 in comparison to ⁇ -amanitin.
  • FIG.17 shows the results of an MTT viability assay on HEK 293 cells for (2S,3R,4R)-SH-OH-Ama (A9) and (2S,3R,4R)-S-S-OH-Ama (A10) in comparison to ⁇ -amanitin;
  • FIG.18 shows the results of an MTT viability assay on CHO cells for A9 in comparison to ⁇ -amanitin;
  • FIG.19 shows the results of an MTT viability assay on CHO cells for A10 in comparison to ⁇ -amanitin;
  • FIG.20 shows the results of an MTT viability assay on HeLa cells for A9 in comparison to ⁇ -amanitin;
  • FIG.21 shows the results of an MTT viability assay on HeLa cells for A10 in comparison to ⁇ -amanitin-;
  • FIG.22 shows the results of an MTT viability assay on HepG2 cells for A10 in comparison to ⁇ -amanitin;
  • FIG.23 shows the
  • Table 6 includes a summary of the results.
  • Table 6 mantn [00223] Curiously, the synthesized amatoxins exhibited differential toxicity, hence these analogs could prove to be useful in the selective diagnosis of specific cancers.
  • disulfide derivative of amanitin A6 exhibited less than 3-fold lower toxicity as compared to ⁇ - amanitin in HEK293 but in liver-derived HepG2 cells, it was completely inactive.
  • fluoro-derivative A2 exhibited almost identical toxicity as ⁇ -amanitin in HEK293 however, it was 13-fold less toxic on HepG2 cells.
  • HPLC Purification Agilent 5 Prep C-18250 ⁇ 10 mm (L ⁇ ID). Solvent A was 0.1% formic acid H 2 O (filtered through a 0.2 ⁇ m PALL filter). Solvent B was 0.1% formic acid MeCN (purchased at HPLC grade, formic acid was then added). Absorbance was monitored simultaneously at 230 nm, 260 nm, and 290 nm.
  • the drug-antibody ratio was determined to be 2.0 by MALDI and protein quantification was carried out using Bradford-Lowry to give a recovery of 77%.
  • the toxicity of the A9 ADC was evaluated on SK-BR-3 cells in comparison to ⁇ - amanitin, amatoxin analogs A9 and A10, and ado-trastuzumab emtansine (Kadcyla TM ) (FIG.24).
  • Kadcyla TM ado-trastuzumab emtansine

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Abstract

La présente divulgation concerne, par exemple, des analogues d'amatoxine contenant des analogues de di-hydroxyisoleucine (DHIle) et, éventuellement, des éléments fonctionnels supplémentaires pour la bioconjugaison, ainsi que des procédés pour leur préparation, des constructions comprenant de tels analogues d'amatoxine couplés à un lieur et des conjugués comprenant de tels analogues d'amatoxine ou constructions de lieur d'amatoxine. La divulgation concerne également des utilisations de tels analogues d'amatoxine, par exemple, dans le traitement du cancer. Par exemple, l'analogue d'amatoxine peut être un composé de formule I.
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Citations (6)

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WO2012041504A1 (fr) * 2010-09-30 2012-04-05 Heidelberg Pharma Gmbh Conjugués d'amatoxine à liants améliorés
WO2014135282A1 (fr) * 2013-03-04 2014-09-12 Heidelberg Pharma Gmbh Dérivés d'amatoxine
WO2017210288A1 (fr) * 2016-05-31 2017-12-07 Sorrento Therapeutics, Inc. Conjugués anticorps-médicaments ayant des dérivés d'amatoxine en tant que médicament
WO2020216947A1 (fr) * 2019-04-24 2020-10-29 Heidelberg Pharma Research Gmbh Conjugués anticorps-médicaments d'amatoxine et leurs utilisations
US20210030887A1 (en) * 2017-08-18 2021-02-04 Sichuan Baili Pharmaceutical Co. Ltd. Non-natural amatoxin-type antibody conjugate
WO2022120476A1 (fr) * 2020-12-08 2022-06-16 The University Of British Columbia Analogues d'amatoxine et leurs utilisations

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WO2012041504A1 (fr) * 2010-09-30 2012-04-05 Heidelberg Pharma Gmbh Conjugués d'amatoxine à liants améliorés
WO2014135282A1 (fr) * 2013-03-04 2014-09-12 Heidelberg Pharma Gmbh Dérivés d'amatoxine
WO2017210288A1 (fr) * 2016-05-31 2017-12-07 Sorrento Therapeutics, Inc. Conjugués anticorps-médicaments ayant des dérivés d'amatoxine en tant que médicament
US20210030887A1 (en) * 2017-08-18 2021-02-04 Sichuan Baili Pharmaceutical Co. Ltd. Non-natural amatoxin-type antibody conjugate
WO2020216947A1 (fr) * 2019-04-24 2020-10-29 Heidelberg Pharma Research Gmbh Conjugués anticorps-médicaments d'amatoxine et leurs utilisations
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MATINKHOO, K. ET AL.: "Design, Synthesis, and Biochemical Evaluation of Alpha- Amanitin Derivatives Containing Analogs of the trans-Hydroxyproline Residue for Potential Use in Antibody-Drug Conjugates", CHEMISTRY - A EUROPEAN JOURNAL, vol. 27, no. 40, 31 May 2021 (2021-05-31), pages 10282 - 10292, XP055947821, ISSN: 0947-6539, Retrieved from the Internet <URL:https://doi.org/10.1002/chem.202101373> DOI: 10.1002/chem.202101373 *

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