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WO2025171055A1 - Heterocyclic conjugates and uses thereof - Google Patents

Heterocyclic conjugates and uses thereof

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Publication number
WO2025171055A1
WO2025171055A1 PCT/US2025/014660 US2025014660W WO2025171055A1 WO 2025171055 A1 WO2025171055 A1 WO 2025171055A1 US 2025014660 W US2025014660 W US 2025014660W WO 2025171055 A1 WO2025171055 A1 WO 2025171055A1
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WIPO (PCT)
Prior art keywords
alkyl
membered
conjugate
carbocycle
membered heterocycle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/014660
Other languages
French (fr)
Inventor
Yi Liu
Pingda Ren
Baogen Wu
Rasmus Hansen
Matthew R. Janes
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Kumquat Biosciences Inc
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Kumquat Biosciences Inc
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Publication of WO2025171055A1 publication Critical patent/WO2025171055A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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
    • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
    • 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
    • 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
    • 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

Definitions

  • Cancer e.g., tumor, neoplasm, metastases
  • K-Ras Kirsten Ras oncogene
  • PDAC pancreatic ductal adenocarcinoma
  • Ras proteins have long been considered “undruggable,” due to, in part, high affinity to their substrate guanosine-5'-triphosphate (GTP) and/or their smooth surfaces without any obvious targeting region.
  • GTP guanosine-5'-triphosphate
  • the specific G12C Ras gene mutation has been identified as a druggable target to which a number of G12C specific inhibitors have been developed.
  • such therapeutics are still of limited application, as the G12C mutation in Ras exhibits a much lower prevalence rate as compared to other known Ras mutations, such as G12D and G12V. Drug resistance and lack of durability impose further limitations to such therapeutics.
  • KRas inhibitors including pan KRas inhibitors capable of inhibiting two or more KRas mutants and/or wildtype KRas within this given Ras isoform (e.g., inhibitors targeting mutant KRas proteins such as KRas G12D, G12C, G12S, G13D, and/or G12V, for the treatment of Ras-associated diseases (e.g., cancer).
  • compositions and methods can be particularly useful for treating a variety of diseases including, but not limited to, cancers and neoplasia conditions.
  • the present disclosure addresses these needs, and provides additional advantages applicable for diagnosis, prognosis, and/or treatment for a wide diversity of diseases.
  • the present disclosure provides a conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker, and wherein the antigen binding unit and the KRAS inhibitor in the conjugate synergistically inhibits signaling output of the first antigen or KRAS.
  • the conjugate provided herein comprises an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to the small-molecule KRAS inhibitor through a chemical linker.
  • the present disclosure provides a conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker, and wherein the antigen binding unit and the KRAS inhibitor in the conjugate synergistically inhibits signaling output of the first antigen and KRAS.
  • the KRAS inhibitor is characterized by a PAMPA permeability (P e ) less than 1 x 10 -6 cm/s.
  • the conjugate is characterized by an increased concentration of the KRAS inhibitor in tumor tissue relative to plasma as ascertained by the formula: ([KRASi] t-c /[KRASi] p-c ) / ([KRASi] t-k /[KRASi] p-k ) > 1, wherein [KRASi] t-c is concentration of the KRAS inhibitor in tumor tissue at a first time -point following administration of the conjugate; wherein [KRASi] p-c is plasma concentration of the KRAS inhibitor at the same time-point following the administration of the conjugate; wherein [KRASi] t-k is concentration of the KRAS inhibitor in tumor tissue following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point; and wherein [KRASi] p-k is plasma concentration of the KRAS inhibitor at the same time -point following the administration of the KRAS inhibitor alone at the equivalent dose.
  • AgB is an antigen binding unit
  • the linker comprises one or more components selected from Val-Cit, Glu-Val-Cit, Val-Ala, Val- Val, Val-Gly, Gly-Gly, Gly-Cit, Glu-Gly-Cit, Ala-Ala-Asn, Ala-Gly-Ala, Ala-Pro, Ala-Ser, and Phe-Lys.
  • p is selected from 2 to 8.
  • q is selected from 1 to 4.
  • D is a small-molecule KRAS inhibitor.
  • Z is selected from O, N, C(R 5 ) 2 , C(O), S, S(O), and S(O) 2 ;
  • R 13 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, and C 1-6 haloalky I: or R 12 and R 13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R 20 ;
  • R 14 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 14 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), C 3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R 20 ;
  • R 21 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 21 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C 1-3 alkyl, C 1-3 haloalkyl, and -OH;
  • R 23 is independently selected at each occurrence from hydrogen and C 1-6 alkyl; or R 22 and R 23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I) is replaced with a bond to the antigen binding unit or the chemical linker.
  • the compound of Formula (I) is a compound of Formula (I-a): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms
  • R 11 is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle).
  • A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl, such as A is pyridinyl.
  • R 11 is hydrogen.
  • R 10 is selected from hydrogen and halogen; or R 9 and R 10 , together with the atoms to which they are attached, form C 4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted.
  • R 10 is hydrogen.
  • R 9 is optionally substituted
  • C 1-3 alkyl such as R 9 is CH 3 .
  • R 4 is selected from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), each of which is optionally substituted with one or more R 20 .
  • R 4 is selected from C 1-6 alkyl and -C 0-6 alkyl-(3- to 12- membered heterocycle), each of which is optionally substituted with one or more substituents independently selected from halogen, -CH 3 , -NH 2 , -NHCH 3 , and -N(CH 3 ) 2 -
  • R 7 is selected from naphthyl, isoquinolinyl, indazolyl, benzothiazolyl, benzothiophenyl, phenyl, and pyridinyl, each of which is optionally substituted with one, two, three, or four R 20 .
  • R 7 is benzothiophenyl optionally substituted with one, two, three, or four R 20 .
  • X is C(R 6 );
  • A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl;
  • R 3 is independently selected at each occurrence from halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, - C 3-8 carbocycle, 3- to 8-membered heterocycle, -OR 12 , -N(R 12 )(R 13 ), -C(O)OR 12 , -N(R 12 )C(O)N(R 12 )(R 13 ), -C(O)R 12 , -OC(O)R 12 , -C(O)N(R 12 )(R 13 ), and -N(R 12 )C(O)R 12 , wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R 20 ; wherein two R 3 are optionally taken together with the atom or atoms to which they are attached to form C
  • R 7 is benzo [b]thiophen-4-yl optionally substituted with one, two, three, or four R 20 ;
  • R 9 is C 1-3 alkyl optionally substituted with one, two, or three R 20 ;
  • R 10 is selected from hydrogen, halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 carbocycle, 3- to 8- membered heterocycle, -OR 22 , -N(R 22 )(R 23 ), -C(O)OR 22 , -N(R 22 )C(O)N(R 22 )(R 23 ), -C(O)R 22 , -OC(O)R 22 , - C(O)N(R 22 )(R 23 ), and -N(R 22 )C(O)R 22 , wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 3-8 carbocycle, and 3- to 8- membered heterocycle are optionally substituted with one, two, or three substituents independently selected from halogen, oxo, -CN, C 1-6 alkyl, C 1-6 haloalkyl
  • R 2 is selected from hydrogen
  • R 3 is independently selected at each occurrence from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R 20 .
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the conjugate described herein, or a salt thereof, and a pharmaceutically acceptable excipient.
  • the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate described herein, or a salt thereof.
  • the present disclosure provides a method of treating cancer in a subject comprising a Ras mutant protein, the method comprising: inhibiting the Ras mutant protein of said subject by administering to said subject a conjugate described herein, or a salt thereof.
  • the cancer of a method described herein may be a solid tumor or a hematological cancer.
  • the cancer comprises a wildtype K-Ras or a mutant K-Ras including but not limited to K-Ras G12C, G12D, G12S, or G12V mutant protein.
  • the present disclosure provides a method of reducing plasma concentration of a small-molecule KRAS inhibitor, comprising providing a conjugate described herein to a subject, wherein reduced plasma concentration is ascertained by the formula: [KRASi] p-c /[KRASi] p-k ⁇ 1 , wherein [KRASi] p-c is plasma concentration of the KRAS inhibitor at a first time -point following administration of the conjugate; and wherein [KRASi] p-k is plasma concentration of the KRAS inhibitor following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point.
  • the present disclosure provides a method of increasing concentration of a small-molecule KRAS inhibitor in tumor tissue, comprising providing a conjugate described herein to a subject, wherein increased tumor tissue concentration is ascertained by the formula: ([KRASi] t-c /[KRASi] p-c ) / ([KRASi] t .
  • [KRASi] t-c is concentration of the KRAS inhibitor in tumor tissue at a first time-point following administration of the conjugate; wherein [KRASi] p-c is plasma concentration of the KRAS inhibitor at the same time-point following the administration of the conjugate; wherein [KRASi] t-k is concentration of the KRAS inhibitor in tumor tissue following administration of the KRAS inhibitor alone at an equivalent dose at the same time -point; and wherein [KRASi] p-k is plasma concentration of the KRAS inhibitor at the same time -point following the administration of the KRAS inhibitor alone at the equivalent dose.
  • efficacy of the conjugate may be greater than efficacy of a combination of the antigen binding unit and the KRAS inhibitor when each is administered at a comparable concentration. In some embodiments, toxicity of the conjugate is less than toxicity of a combination of the antigen binding unit and the KRAS inhibitor when each is administered at a comparable concentration.
  • FIG. 1 depicts a sequence alignment of various wild type Ras proteins including K-Ras, H-Ras, N-Ras, RalA, and RalB, from top to bottom.
  • C x-y or “C x -C y ” when used in conjunction with a chemical moiety, such as alkyl, alkenyl, or alkynyl, is meant to include groups that contain from x to y carbons in the chain.
  • C x-y alkyl refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched- chain alkyl groups, that contain from x to y carbons in the chain.
  • Alkyl refers to substituted or unsubstituted saturated hydrocarbon groups, including linear and branched alkyl groups.
  • An alkyl group may contain from one to twelve carbon atoms (e.g., C 1-12 alkyl), such as one to eight carbon atoms (C 1-8 alkyl) or one to six carbon atoms (C 1-6 alkyl).
  • alkyl groups include methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl, and decyl.
  • An alkyl group is attached to the rest of the molecule by a single bond. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more substituents such as those substituents described herein.
  • alkenyl refers to substituted or unsubstituted hydrocarbon groups, including linear and branched alkenyl groups, containing at least one double bond.
  • An alkenyl group may contain from two to twelve carbon atoms (e.g., C 2-12 alkenyl), such as two to eight carbon atoms (C 2-8 alkenyl) or two to six carbon atoms (C 2-6 alkenyl).
  • Exemplary alkenyl groups include ethenyl (i.e., vinyl), prop-l -enyl, but-l-enyl, pent-l-enyl, penta- 1,4-dienyl, and the like.
  • an alkenyl group is optionally substituted by one or more substituents such as those substituents described herein.
  • Alkylene or “alkylene chain” refers to substituted or unsubstituted divalent saturated hydrocarbon groups, including linear alkylene and branched alkylene groups, that contain from one to twelve carbon atoms (e.g., C 1-12 alkylene), such as one to eight carbon atoms (C 1-8 alkylene) or one to six carbon atoms (C 1-6 alkylene).
  • Exemplary alkylene groups include methylene, ethylene, propylene, and n-butylene.
  • alkenylene and alkynylene refer to alkylene groups, as defined above, which comprise one or more carbon-carbon double or triple bonds, respectively.
  • alkylene, alkenylene or alkynylene chain can be through one carbon or any two carbons of the chain.
  • an alkylene, alkenylene, or alkynylene group is optionally substituted by one or more substituents such as those substituents described herein.
  • Connection to the rest of the molecule may be through either a heteroatom or a carbon in the heteroalkyl, heteroalkenyl, or heteroalkynyl chain.
  • a heteroalkyl, hetero alkenyl, or heteroalkynyl group is optionally substituted by one or more substituents such as those substituents described herein.
  • Hetero alkylene refers to substituted or unsubstituted alkylene, alkenylene and alkynylene groups, respectively, in which one or more, such as 1, 2 or 3, of the carbon atoms are replaced with a heteroatom, such as O, N, P, Si, S, or combinations thereof. Any nitrogen, phosphorus, and sulfur heteroatoms present in the chain may optionally be oxidized, and any nitrogen heteroatoms may optionally be quatemized. If given, a numerical range refers to the chain length in total. For example, a 3- to 8- membered hetero alkylene group has a chain length of 3 to 8 atoms.
  • the points of attachment of the heteroalkylene, hetero alkenylene or heteroalkynylene chain to the rest of the molecule can be through either one heteroatom or one carbon, or any two heteroatoms, any two carbons, or any one heteroatom and any one carbon in the heteroalkylene, hetero alkenylene or heteroalkynylene chain.
  • a heteroalkylene, heteroalkenylene, or heteroalkynylene group is optionally substituted by one or more substituents such as those substituents described herein.
  • Heterocycle refers to a saturated, unsaturated or aromatic ring comprising one or more heteroatoms, for example 1, 2, 3, or 4 heteroatoms selected from O, S, P, and N. Heterocycle may include 3- to 10-membered monocyclic rings, 5- to 12-membered bicyclic rings, 5- to 18-membered polycyclic rings, 5- to 12-membered spirocyclic rings, and 5- to 12-membered bridged rings. Each ring of a bicyclic or polycyclic heterocycle may be selected from saturated, unsaturated, and aromatic rings.
  • a heterocycle may comprise a fused ring, a bridged ring, a spirocyclic ring, a saturated ring, an unsaturated ring, an aromatic ring, or any combination thereof.
  • a heterocycle e.g., pyridyl
  • a polycyclic heteroaryl contains a number or rings equal to the minimum number of scissions required to convert the heteroaryl into an acyclic skeleton (e.g., bicyclic, tricyclic, tetracyclic, etc.).
  • the heteroatom(s) in the heteroaryl may optionally be oxidized.
  • One or more nitrogen atoms, if present, are optionally quatemized.
  • the heteroaryl may be attached to the rest of the molecule through any atom of the heteroaryl, valence permitting, such as a carbon or nitrogen atom of the heteroaryl.
  • a waved line drawn across or at the end of a bond or a dashed bond are used interchangeably herein to denote where a bond disconnection or attachment occurs.
  • R7 is 2-fluoro-6-hydroxyphenyl as in then R 7 may be depicted as
  • R 21 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, and C 1-6 haloalkyl;
  • “Optional” or “optionally” means that the subsequently described event or circumstances may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.
  • an “optionally substituted” group may be either unsubstituted or substituted.
  • stereoisomers may be obtained by numerous methods that are known in the art, including preparation using chiral synthons or chiral reagents, resolution using chiral chromatography using a suitable chiral stationary phase or support, or by chemically converting them into diastereomers, separating the diastereoisomers by conventional means such as chromatography or recrystallization, then regenerating the original stereoisomer.
  • a mutant Ras may comprise about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more than 50 mutations. In some embodiments, a mutant Ras may comprise up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 mutations.
  • the small molecule has a molecular weight of 100 to 1000 Da, such as 100 to 900 Da, 100 to 850 Da, 100 to 800 Da, 100 to 750 Da, 100 to 700 Da, 100 to 650 Da, 100 to 600 Da, 100 to 550 Da, 100 to 500 Da, 150 to 900 Da, 150 to 850 Da, 150 to 800 Da,
  • ([KRASi] t-c /[KRASi] p-c ) / ([KRASi] t-k /[KRASi] p-k ) is greater than 1.1, such as greater than 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50. In some embodiments, ([KRASi] t-c /[KRASi] p-c ) / ([KRASi] t-k /[KRASi] p-k ) is greater than 2.
  • AgB is an antigen binding unit
  • the conjugate of Formula (A) is selected from:
  • an antigen binding unit is bivalent and mono-specific (i.e., having two binding domains that specifically bind to the same antigen). In embodiments in which an antigen binding unit is trivalent or greater, the antigen binding unit is typically bi-specific or greater. Antigen binding units having a third binding domain attached to the C-terminal end of a light chain and/or the C-terminal end of an Fc domain can be bi-specific, tri-specific or multi-specific.
  • a linker containing such a protease cleavage site may further comprise one or more components selected from pentafluorophenyl, succinimide, maleimide, and para-aminobenzoic acid (PABA).
  • the linker is a compound of the formula: (maleimidocaproyl)-(protease cleavage site)-(para-aminobenzyloxycarbonyl).
  • the linker may comprise a maleimide at one end and a protease cleavage site at the other end.
  • the linker comprises a peptide, such as a dipeptide, tripeptide, or tetrapeptide.
  • the peptide may comprise natural amino acids, unnatural amino acids, or combinations thereof.
  • the peptide comprises L-amino acids.
  • a linker may be polyvalent such that it covalently links more than one KRAS inhibitor to a single site on the antigen binding unit, or monovalent such that it covalently links a single KRAS inhibitor to a single site of the antigen binding unit.
  • Exemplary polyvalent linkers that may be used to attach two or more KRAS inhibitors to an antigen binding unit of the conjugate include Fleximer® linkers.
  • a Fleximer® linker utilizes a solubilizing poly acetal backbone to incorporate two or more KRAS inhibitors (D) via a sequence of ester bonds, for example, utilizing a linker comprising two or more units of the structure shown below. This methodology can render highly- loaded conjugates (e.g., DAR20 — conjugates of Formula (A) wherein q is 20).
  • R 1 is a reactive group
  • L 1 is a bridging spacer
  • Lp is a bivalent peptide spacer
  • G-L 2 -A is a self-immolative spacer
  • R 2 is a hydrophilic moiety
  • L 2 is a bond, a methylene, a neopentylene or a C 2 -C3alkenylene
  • conjugate or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the conjugate includes a structure represented by formula (Xc):
  • the benzene ring of L3 can be substituted with the optionally substituted structural unit -X.
  • the structural unit -X can include optionally substituted wherein X 1 is selected from the group consisting of C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 1 -C 6 cycloalkyl, linear heteroalkyl comprising 1-8 atoms, and linear-cyclic heteroalkyl comprising 1-8 atoms, where the heteroalkyl comprises 1-3 atoms selected from N, O or S, and the C 1 -C 8 alkyl, C 1 -C 8 alkoxy, C 1 -C 6 cycloalkyl, linear heteroalkyl comprising 1-8 atoms, and linear-cyclic heteroalkyl comprising 1 -8 atoms are each independently optionally substituted with one or more substituents selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxy, alkoxy, or cycl
  • the conjugate is a conjugate described in WO2013/173337, which is incorporated herein in its entirety.
  • the conjugate is represented by Formula Xd: or a salt thereof (e.g., pharmaceutically acceptable salts);
  • L is a antigen binding unit;
  • D' is a KRAS inhibitor;
  • L° is the optional secondary linker assembly;
  • L ss is the self-stabilizing linker assembly, wherein M 1 is a succinimide ring or a hydrolyzed succinamide or together with BU forms a dilactam;
  • BU is a Basic unit;
  • HE is a hydrolysis enhancer comprising an electron withdrawing group;
  • the circle represents a scaffold that can be C 1-8 alkylene, Ci.
  • the scaffold is Ci alkylene or C 1 -
  • M 1 represents a succinimide ring or a hydrolyzed succinamide ring or a dilactam formed when the base reacts with the succinimide ring
  • BU is a Basic unit
  • HE is a hydrolysis enhancer comprising an electron withdrawing group
  • the circle represents a scaffold that can be C 1-8 alkylene, C 1-8 heteroalkyiene, C 6-10 arylene, or C 4- 10 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly, D !
  • the scaffold is C 1-3 alkylene or C 1-3 heteroalkyiene.
  • the alkylene is a straight chain or branched alkylene.
  • the circle represents a scaffold that is C1, C2, C3. or C4 straight or branched chain alkylene, r is zero, and the sum of m+q is 1.
  • the circle represents a scaffold that is C1, C2, C3. or C4 straight or branched chain alkylene, and m and r are zero.
  • m is 1 , and q and r are 0; 2) q is 1 , and m and r are 0; 3) r is 1 , and m and q are 0; 4) m is 1 , q and r are 0, and a is 1 ; 5) q is 1 , m and r are 0, and a is 1 ; 6) r is 1 , m and q are 0, and a is 1 ; 7) m is 1 , q and r are 0, and D' is a KRAS inhibitor; 8) q is 1.
  • n and r are 0, and D' is a KRAS inhibitor; 9) r is 1 , m and q are 0, and D' is a KRAS inhibitor, D; 10) m is 3 , q and r are 0, a is 1, and D : is a Drug unit, D: 11) q is 1, m and r are 0, a is 1, and D' is a KRAS inhibitor; or 12) r is 1 , m and q are 0, a is 1 , and D' is a KRAS inhibitor.
  • the Basic unit comprises a primary, a secondary amine, or a tertiary amine.
  • the Basic unit is selected from the group consisting of-(C(R 9 )( R 10 )) x NH 2 , -(C(R 9 )( R 10 )) x NHR a , and -(C(R 9 )( R 10 )) x NR a 2 , wherein x is an integer of from 0-4 (or from 1 to 4) and each R a is independently selected from the group consisting of C 1-6 alkyl and C 1- 6 haloalkyl, or two R a groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group, provided that if x is zero there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide
  • x is an integer of from 1 to 4.
  • the Basic unit is NH 2 -CH 2 NH 2 , -CH 2 CH 2 NH 2 , - CH 2 CH 2 CH 2 NH2, or -CH 2 CH 2 CH 2 CH 2 NH 2 provided that if the Basic unit is -NH 2 , there are no less than 2 intervening atoms between the base and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam.
  • the conjugate is a conjugate described in W02010/093395, which is incorporated herein in its entirety.
  • the conjugate may have the formula Xe, MAb-[L2]-[L l]-[AA] m - [A']-D; where MAb is a disease -targeting antibody; L2 is a component of the cross-linker comprising an antibodycoupling moiety and one or more of acetylene (or azide) groups; L 1 comprises a defined PEG with azide (or acetylene) at one end, complementary to the acetylene (or azide) moiety in L2, and a reactive group such as carboxylic acid or hydroxyl group at the other end; AA is an L-amino acid; m is an integer with values of 0, 1, 2, 3, or 4; and A' is an additional spacer, selected from the group of ethanolamine, 4-hydroxybenzyl alcohol, 4- aminobenzyl alcohol, or substituted or unsubstituted
  • the L amino acids of 'AA' are selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. If the A' group contains hydroxyl, it is linked to the hydroxyl group or amino group of D in the form of a carbonate or carbamate, respectively.
  • A' is a substituted ethanolamine derived from an L-amino acid, wherein the carboxylic acid group of the amino acid is replaced by a hydroxymethyl moiety.
  • A' may be derived from any one of the following L-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • the conjugate is a conjugate described in WO2019081455, which is incorporated herein in its entirety.
  • the conjugate may have the formula Xf wherein D is KRAS inhibitor, X is an optional cleavable moiety for releasing D, Z is an optional spacer, and a is 1 or more, b is 1 or more and m is 1 or more; L is an orthogonal connector that allows for (HP SMW ) to be in an orthogonal orientation with respect to (X-D), HP SMW results from covalent binding to said orthogonal connector L, of a single molecular weight homopolymer having formula (Xfb) wherein n is one or more; R 1 and R 2 are different, and one of R 1 and R 2 is H or an inert group, the other one of R 1 and R 2 being a functionalized reactive group, said group being reactive for covalently binding a bindable group, in such reaction conditions that the inert group is non-reactive, Z 1 and Z 2 ,
  • k is an integer which is at least 2, it is preferably 100 at most, more preferably 50 at most, and specifically 2-30, and more specifically 2-24, 6-24, or 12-24.
  • said functionalized reactive group R 1 or R 2 may be selected from the following groups: carboxylic acid group, amino groups NRR" wherein R and R" are independently selected from H, (C 1 -C 6 ) alkyl optionally interrupted by at least one heteroatom selected among O, N and S, hydroxyl group, halogen atoms, hydrazine (-NH 2 - NH 2 ) group, nitro group, hydroxylamine group, azido group, (C 2 -C 6 ) alkynyl group, (C 2 -C 6 ) alkenyl group, thiol group, activated ester groups such as N-hydroxysuccinimide ester, perfluorinated esters, nitrophenyl esters, azabenzotriazole and benzotriazole activated esters, acylureas, boronic acid B(OR"") 2 groups, wherein R"" is a hydrogen atom or a C 1 -C 6 alkyl group, thio
  • the conjugate of formula Xf is wherein R 6 is —C 1 -C 10 alkylene-, — C 1 -C 10 heteroalkylene-, -C 3 -C 8 carbocyclo-, -O-
  • the linker comprises one or more components independently selected from polyethylene glycol, polysarcosine, a hydrazone, acetal, maleimide, succinimide, asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, valine, alanine, glycine, leucine, isoleucine, methionine, tryptophan, proline, histidine, citrulline, phenylalanine, carboxylate, p-aminobenzyloxy carbonyl, alkyl, alkene, alkyne, aryl, cycloalkyl, heterocycle, glycoside, silyl ether, hydroxy, ether, ketone, ester, carbonate, amide, urea, carbamate, sulfide, disulfide, sulfate, sulfonamide,
  • the linker is a compound of the formula: wherein: indicates an attachment site to the antigen binding unit;
  • Z 1 is a product formed by reaction of the antigen binding unit with a reactive precursor of Z 1 ;
  • Z 2 is absent or an optionally substituted spacer comprising one or more components independently selected from C 1-6 alkyl, (CH 2 CH 2 O) n2 , and -C(O)NH-, or any combination thereof;
  • Z 3 is selected from Val-Cit, Cit-Val, Ala-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu- Val, Ser-Cit, Cit-Ser, Lys-Cit, Cit-Lys, Asp-Cit, Cit-Asp, Val-Ala, Ala-Vai, Val-Val, Val-Gly, Gly-Val, Phe-Lys, Lys-Phe, Val-Lys, Lys-Val, Ala-Lys, Lys-Ala, Phe-Cit, Cit-Phe, Leu-Cit, Cit-Leu, Ile-Cit, Cit-Ile, Phe-Arg, Arg- Phe, Cit-Trp, Trp-Cit, Gly-Gly, Gly-Cit, Cit-Gly, Ala-Pro, Pro-Ala, Ala-Ser, Ser-Ala, Glu- Val-C
  • R z is selected from hydrogen, -C 1-4 alkyl-O n3 -(C 1-4 alkylene) n4 -Z 4 , -C 1-4 alkyl-N-[(C 1-4 alkylene)-Z 4 ] 2 , -C 2-4 alkynyl-C 1-4 alkyl-O n3 -(C 1-4 alkylene) n4 -Z 4 , and -C 2-4 alkynyl-C 1-4 alkyl-N-[(C 1-4 alkylene)-Z 4 ] 2 ;
  • Z 4 is selected from -SO 3 H, -CO 2 H, PEG 4-32, and a sugar moiety; n1, n3, and n4 are each independently 0 or 1 ; n2 is an integer from 1 to 6; and indicates an attachment site to the KRAS inhibitor.
  • the linker is a compound of the formula: wherein: indicates an attachment site to the antigen binding unit;
  • Z 1 is a product formed by reaction of the antigen binding unit with a reactive precursor of Z 1 ;
  • Z 2 is absent or an optionally substituted spacer comprising one or more components independently selected from C 1-6 alkyl, (CH 2 CH 2 O) n2 . -C(O)NH-, -C(O)NCH 3 -, (C(O)CH 2 N(CH 3 )) n2 , or any combination thereof;
  • Z 3 is selected from a bond, Val-Cit, Cit-Val, Ala-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu- Vai, Ser-Cit, Cit-Ser, Lys-Cit, Cit-Lys, Asp-Cit, Cit-Asp, Val-Ala, Ala- Vai, Val-Val, Val-Gly, Gly-Val, Phe- Lys, Lys-Phe, Val-Lys, Lys-Val, Ala-Lys, Lys-Ala, Phe-Cit, Cit-Phe, Leu-Cit, Cit-Leu, Ile-Cit, Cit-Ile, Phe-Arg, Arg-Phe, Cit-Trp, Trp-Cit, Gly-Gly, Gly-Cit, Cit-Gly, Ala-Pro, Pro-Ala, Ala-Ser, Ser-Ala, Glu-
  • R z is selected from hydrogen, Z 4 , -C 1-4 alkyl-O n3 -(C 1-4 alkylene) n4 -Z 4 , -C 1-4 alkyl-N-[(C 1-4 alkylene)-Z 4 ] 2 , - C 2-4 alkynyl-C 1-4 alkyl-O n3 -(C 1-4 alkylene) n4 -Z 4 , and -C 2-4 alkynyl-C 1-4 alkyl-N-[(C 1-4 alkylene)-Z 4 ] 2 ;
  • Z 4 is selected from -SO 3 H, -CO 2 H, PEG 4-32, -(CH 2 N(CH 3 )C(O)) n2 CH 3 , and a sugar moiety; n1, n3, and n4 are each independently 0 or 1 : n2 is an integer from 1 to 20: and indicates an attachment site to the KRAS inhibitor.
  • a conjugate of the present disclosure comprises a linker selected from:
  • a conjugate of the present disclosure comprises a linker selected from:
  • the linker is a compound of the formula: wherein: indicates an attachment site to the antigen binding unit;
  • Z 1 is a product formed by reaction of the antigen binding unit with a reactive precursor of Z 1 ;
  • Z 2 is absent, C 1-6 alkyl, (CH 2 CH 2 O) n2 . -C(O)NH-, -C(O)NCH 3 -, (C(O)CH 2 N(CH 3 )) n2 , - ((CH 2 CH 2 O) n2 (CH 2 CH 2 )C(O))N(CH 2 C(O)N(CH 3 )-(CH 2 CH 2 O) n2 -(CH 2 CH 2 N(CH 3 )-(C(O)CH 2 N(CH 3 )) n2 - C(O)CH 3 )CH 2 -, -(C 1-6 alkyl)C(O)N(CH 2 C(O)N(CH 3 )-(CH 2 CH 2 O) n2 -(CH 2 CH 2 N(CH 3 )-(C(O)CH 2 N(CH 3 )) n2 - C(O)CH 3 )CH 2 -, -(C 1-6 alkyl
  • Z 3 is selected from a bond, Val-Cit, Cit-Val, Ala-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu-Val, Ser-Cit, Cit-Ser, Lys-Cit, Cit-Lys, Asp-Cit, Cit-Asp, Val-Ala, Ala- Vai, Vai- Vai, Val-Gly, Gly-Val, Phe- Lys, Lys-Phe, Val-Lys, Lys-Val, Ala-Lys, Lys-Ala, Phe-Cit, Cit-Phe, Leu-Cit, Cit-Leu, Ile-Cit, Cit-Ile, Phe-Arg, Arg-Phe, Cit-Trp, Trp-Cit, Gly-Gly, Gly-Cit, Cit-Gly, Ala-Pro, Pro-Ala, Ala-Ser, Ser-Ala, Glu
  • R z is selected from hydrogen, -CH 2 N(CH 3 )C(O)-(CH 2 CH 2 O) n2 CH 3 , -CH 2 N(CH 3 )(C(O)CH 2 N(CH 3 )) n2 - C(O)CH 3 , -CH 2 N(CH 3 )C(O)-(CH 2 CH 2 O) n2 -CH 2 CH 2 C(O)-(N(CH 3 )CH 2 C(O)) n2 -N(CH 3 ) 2 , -SO 3 H, -CO 2 H, PEG 4-32, polysarcosine, -(CH 2 N(CH 3 )C(O)) n2 CH 3 , and a sugar moiety; n3, and n4 are each independently 0 or 1 ; n2 is independently an integer from 1 to 20; and indicates an attachment site to the KRAS inhibitor.
  • Z 1a is a moiety capable of forming a covalent bond with an antigen binding unit
  • R z is selected from hydrogen, Z 4 , -C 1-4 alkyl-O n3 -(C 1-4 alkylene) n4 -Z 4 , -C 1-4 alkyl-N-[(C 1-4 alkylene)-Z 4 ] 2 , - C 2-4 alkynyl-C 1-4 alkyl-O n3 -(C 1-4 alkylene) n4 -Z 4 , and -C 2-4 alkynyl-C 1-4 alkyl-N-[(C 1-4 alkylene)-Z 4 ] 2 ;
  • R z is selected from hydrogen, -CH 2 N(CH 3 )C(O)-(CH 2 CH 2 O) n2 CH 3 , -CH 2 N(CH 3 )(C(O)CH 2 N(CH 3 )) n2 - C(O)CH 3 , -CH 2 N(CH 3 )C(O)-(CH 2 CH 2 O) n2 -CH 2 CH 2 C(O)-(N(CH 3 )CH 2 C(O)) n2 -N(CH 3 ) 2 , -SO 3 H, -CO 2 H, PEG 4-32, polysarcosine, -(CH 2 N(CH 3 )C(O)) n2 CH 3 , and a sugar moiety; nl, n3, and n4 are each independently 0 or 1 ; n2 is independently an integer from 1 to 20; and the KRAS inhibitor is optionally a compound of Formula (I), (I-a), (I-b), (I-
  • a KRAS inhibitor for generating a conjugate of the present disclosure is a compound of Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • X is selected from N and C(R 6 );
  • R 2 , R 5 , R 6 , and R 8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3 -i 2 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , - N(R 12 )(R 13 ), -C(O)OR 12 , -OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(
  • R 3 is independently selected at each occurrence from halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , -N(R 12 )(R 13 ), -C(O)OR 12 , - OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(R 12 )(R 13 ), -N(R 12 )C(O
  • R 4 is selected from hydrogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), -C(O)OR 12 , -C(O)R 12 , -C(O)N(R 12 )(R 13 ), -C(O)C(O)N(R 12 )(R 13 ), -S(O) 2 R 12 , - S(O)(NR 12 )R 12 , -S(O) 2 N(R
  • R 7 is selected from C 6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted; m is 0, 1, 2, or 3; n is 1 or 2;
  • R 12 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted;
  • R 13 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, and C 1-6 haloalkyl; or R 12 and R 13 attached to the same nitrogen atom form optionally substituted 3- to 10-membered heterocycle; and
  • R 14 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 14 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), C 3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted; wherein one hydrogen of the compound of Formula (I) is replaced with a bond to the antigen binding unit or the chemical linker.
  • the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
  • the compound of Formula (I) has the formula: wherein all variables are as described for Formula (I).
  • a KRAS inhibitor for generating a conjugate of the present disclosure is a compound of Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • X is selected from N and C(R 6 );
  • Z is selected from O, N, C(R 5 ) 2 , C(O), S, S(O), and S(O) 2 ;
  • R 2 , R 5 , R 6 , and R 8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , - N(R 12 )(R 13 ), -C(O)OR 12 , -OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(R 12
  • R 3 is independently selected at each occurrence from halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , -N(R 12 )(R 13 ), -C(O)OR 12 , - OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(R 12 )(R 13 ), -N(R 12 )C(O
  • R 4 is selected from hydrogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), -C(O)OR 12 , -C(O)R 12 , -C(O)N(R 12 )(R 13 ), -C(O)C(O)N(R 12 )(R 13 ), -S(O) 2 R 12 , - S(O)(NR 12 )R 12 , -S(O) 2 N(R
  • R 7 is selected from C 6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R 20 ; m is 0, 1, 2, or 3; n is 1 or 2;
  • R 12 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R 20 ;
  • R 13 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, and C 1-6 haloalky I: or R 12 and R 13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R 20 ;
  • R 14 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 14 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), C 3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R 20 ;
  • R 21 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 21 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C 1-3 alkyl, C 1-3 haloalkyl, and -OH;
  • R 22 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle); and
  • R 23 is independently selected at each occurrence from hydrogen and C 1-6 alkyl; or R 22 and R 23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I) is replaced with a bond to the antigen binding unit or the chemical linker.
  • the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
  • Formula (I) has the formula: wherein all variables are as described for Formula (I).
  • the KRAS inhibitor for generating a conjugate is a compound of Formula (I-a): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • X is selected from N and C(R 6 );
  • Z is selected from O, N, C(R 5 ) 2 , C(O), S, S(O), and S(O) 2 ;
  • A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms
  • R 2 , R 5 , R 8 , R 9 , and R 10 are each independently selected at each occurrence from hydrogen, halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6- membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -
  • R 3 is independently selected at each occurrence from halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , -N(R 12 )(R 13 ), -C(O)OR 12 , - OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(R 12 )(R 13 ), -N(R 12 )C(O
  • R 6 is selected from hydrogen, halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , -SF 5 , -N(R 12 )(R 13 ), -C(O)OR 12 , -OC(O)N(R 12 )(R 13 ), - N(R 12 )C(O)N(R 12 )(R 13 ), -N(R 12 )
  • R 7 is selected from C 6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R 20 ; m is 0, 1, 2, or 3; n is 1 or 2;
  • R 11 is selected from hydrogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), (5-methyl-2-oxo-l,3-dioxol-4-yl)methyl, -C(O)OR 12 , -C(O)OC(O)R 12 , -C(O)O-(C 1-6 alkyl)-OR 15 , -(C 1-6 alkyl)-OR 15 , -C(O)R 12
  • R 12 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R 20 ;
  • R 13 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, and C 1-6 haloalky I: or R 12 and R 13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R 20 ;
  • R 14 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 14 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), C 3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R 20 ;
  • R 22 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle); and
  • R 23 is independently selected at each occurrence from hydrogen and C 1-6 alkyl; or R 22 and R 23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker.
  • the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
  • Formula (I-a) has the formula: ; wherein all variables are as described for
  • a KRAS inhibitor for generating a conjugate of the present disclosure is a compound of Formula (I-a): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • X is selected from N and C(R 6 );
  • Z is selected from O, N, C(R 5 ) 2 , C(O), S, S(O), and S(O) 2 ;
  • A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms
  • R 2 , R 5 , R 6 , R 8 , R 9 , and R 10 are each independently selected at each occurrence from hydrogen, halogen, - CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6- membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , - SR 12 , -N(R 12 )(R 13 ), -C(O)OR 12 , -OC(O)N(R 12 )(R 13 ), -N(R 12 )
  • R 3 is independently selected at each occurrence from halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , -N(R 12 )(R 13 ), -C(O)OR 12 , - OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(R 12 )(R 13 ), -N(R 12 )C(O
  • R 7 is selected from C 6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R 20 ; m is 0, 1, 2, or 3; n is 1 or 2;
  • R 11 is selected from hydrogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), (5-methyl-2-oxo-l,3-dioxol-4-yl)methyl, -C(O)OR 12 , -C(O)OC(O)R 12 , -C(O)O-(C 1-6 alkyl)-OR 15 , -(C 1-6 alkyl)-OR 15 , -C(O)R 12
  • R 12 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R 20 ;
  • R 13 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, and C 1-6 haloalky I: or R 12 and R 13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R 20 ;
  • R 14 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 14 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), C 3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R 20 ;
  • R 21 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 21 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C 1-3 alkyl, C 1-3 haloalkyl, and -OH;
  • R 22 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle); and
  • R 23 is independently selected at each occurrence from hydrogen and C 1-6 alkyl; or R 22 and R 23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I-a) is replaced with a bond to the antigen binding unit or the chemical linker.
  • the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
  • Formula (I-a) has the formula: ; wherein all variables are as described for
  • a KRAS inhibitor for generating a conjugate of the present disclosure is a compound of Formula (I-a): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • X is selected from N and C(R 6 );
  • Z is selected from O, N, C(R 5 ) 2 , C(O), S, S(O), and S(O) 2 ;
  • A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms
  • R 2 , R 5 , R 6 , and R 8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , - N(R 12 )(R 13 ), -C(O)OR 12 , -OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(R 12
  • R 3 is independently selected at each occurrence from halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , -N(R 12 )(R 13 ), -C(O)OR 12 , - OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(R 12 )(R 13 ), -N(R 12 )C(O
  • R 7 is selected from C 6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R 20 ; m is 0, 1, 2, or 3; n is 1 or 2;
  • R 11 is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle);
  • R 13 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, and C 1-6 haloalkyl; or R 12 and R 13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R 20 ;
  • R 21 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 21 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C 1-3 alkyl, C 1-3 haloalkyl, and -OH;
  • R 22 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle); and
  • R 23 is independently selected at each occurrence from hydrogen and C 1-6 alkyl; or R 22 and R 23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I-a) is replaced with a bond to the antigen binding unit or the chemical linker.
  • Formula (I-a) has the formula: ; wherein all variables are as described for
  • a KRAS inhibitor for generating a conjugate of the present disclosure is a compound of Formula (I-b): or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • R 3 is independently selected at each occurrence from halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , -N(R 12 )(R 13 ), -C(O)OR 12 , - OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(R 12 )(R 13 ), -N(R 12 )C(O
  • R 12 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R 20 ;
  • R 13 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, and C 1-6 haloalky I: or R 12 and R 13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R 20 ;
  • R 14 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 14 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), C 3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R 20 ;
  • R 21 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 21 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C 1-3 alkyl, C 1-3 haloalkyl, and -OH;
  • R 22 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle); and
  • R 23 is independently selected at each occurrence from hydrogen and C 1-6 alkyl; or R 22 and R 23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I-b) is replaced with a bond to the antigen binding unit or the chemical linker.
  • the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
  • the compound of Formula (I-b) is a compound of Formula (I-c): or a pharmaceutically acceptable salt or solvate thereof.
  • (i) X is N; and/or (ii) R 3 is independently selected at each occurrence from C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalkyl)- (C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), wherein C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6- membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6- membered heteroalky l)-(3- to 12-membered heterocycle)
  • the present disclosure provides a compound of Formula (I-d) applicable for generating a subject conjugate: or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • X is selected from C(R 6 ) and N;
  • A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms
  • R 12 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R 20 ;
  • R 14 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 14 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), C 3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R 20 ; R 20 is independently selected at each occurrence from halogen, oxo, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 al
  • R 21 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 21 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C 1-3 alkyl, C 1-3 haloalkyl, and -OH;
  • R 23 is independently selected at each occurrence from hydrogen and C 1-6 alkyl; or R 22 and R 23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker.
  • the present disclosure provides a compound of Formula (I-e) applicable for generating a subject conjugate,: or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms;
  • R 2 , R 8 , and R 10 are each independently selected at each occurrence from hydrogen, halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , - N(R 12 )(R 13 ), -C(O)OR 12 , -OC(O)N(R 12 )
  • R 9 is selected from halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , -N(R 12 )(R 13 ), -C(O)OR 12 , -OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(R 12 )(R 13 ), - N(R 12 )C(O)OR 12 ,
  • R 3 is independently selected at each occurrence from halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , -N(R 12 )(R 13 ), -C(O)OR 12 , - OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(R 12 )(R 13 ), -N(R 12 )C(O
  • R 6 is selected from hydrogen, halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , -SF 5 , -N(R 12 )(R 13 ), -C(O)OR 12 , -OC(O)N(R 12 )(R 13 ), - N(R 12 )C(O)N(R 12 )(R 13 ), -N(R 12 )
  • R 7 is selected from C 6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R 20 ; m is 0, 1, 2, or 3; n is 1 or 2;
  • R 12 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R 20 ;
  • R 10 is halogen.
  • R 10 is -F.
  • R 10 is -Cl.
  • R 10 is -Br.
  • R 10 is C 1-6 alkyl optionally substituted with one, two, or three R 20 .
  • R 10 is C 1-6 alkyl optionally substituted with one, two, or three halogen.
  • compound of Formula (I-e) is selected from
  • X is C(R 6 ), such as C(Cl).
  • X is N.
  • X is C(R 6 ); and R 6 and R 8 are independently selected from hydrogen, halogen, and C 1-3 haloalkyl.
  • X is C(R 6 ); R 6 is selected from chlorine and -CF 3 : and R 8 is fluorine.
  • X is N; and R 8 is selected from hydrogen, halogen, and C 1-3 haloalkyl.
  • X is N; and R 8 is fluorine.
  • X is C(R 6 ); and Z is selected from O and C(R 5 ) 2 . In some embodiments, X is C(R 6 ); and Z is selected from O and CH 2 . In some embodiments, X is C(R 6 ); Z is selected from O and C(R 5 ) 2 : and R 6 and R 8 are independently selected from hydrogen, halogen, and C 1-3 haloalkyl. In some embodiments, X is C(R 6 ); Z is selected from O and C(R 5 ) 2 : R 6 is selected from chlorine and -CF 3 : and R 8 is fluorine.
  • X is C(R 6 ); Z is selected from O and CH 2 : and R 6 and R 8 are independently selected from hydrogen, halogen, and C 1-3 haloalkyl. In some embodiments, X is C(R 6 ); Z is selected from O and CH 2 : R 6 is selected from chlorine and -CF 3 ; and R 8 is fluorine. In some embodiments, X is N; Z is selected from O and CH 2 : and R 8 is selected from hydrogen, halogen, and C 1-3 haloalkyl. In some embodiments, X is N; Z is selected from O and CH 2 : and R 8 is selected from hydrogen, halogen, and C 1-3 haloalkyl. In some embodiments, X is N; Z is selected from O and CH 2 : and R 8 is fluorine. In some embodiments, X is N; Z is selected from O and CH 2 : and R 8 is fluorine. In some embodiments, X is N; Z is selected from O and CH 2 : and
  • R 4 is selected from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), each of which is optionally substituted with one or more R 20 .
  • R 4 is selected from C 1-6 alkyl and -C 0-6 alkyl-(3- to 12-membered heterocycle), each of which is optionally substituted with one or more R 20 .
  • R 4 is -C 1-3 alkyl-(3- to 9-membered heterocycle) optionally substituted with one or more R 20 , and further optionally wherein the 3- to 9-membered heterocycle is selected from azetidinyl, thietanyl,
  • R 4 is -C 1-3 alkyl-(pyridine) optionally substituted with one or more R 20 .
  • R 4 is -C 1-3 alkyl-(3- to 9-membered heterocycle), wherein the 3- to 9- membered heterocycle is substituted with -NH 2 .
  • R 4 is -C 1-3 alkyl-(pyridine), wherein the pyridine is substituted with -NH 2 .
  • R 9 is C 1-3 haloalkyl, such as -CH 2 F, -CHF 2 , -CF 3 , -CH 2 CH 2 F, -CH 2 CHF 2 , or -CH 2 CF 3 .
  • R 9 is selected from -CH 3 , -CHF 2 , - CH 2 CH 3 , and CH 2 CHF 2 .
  • R 9 is C 1-3 alkyl.
  • R 9 is CH 3 . In some
  • R 9 is selected from -C 1-6 alkyl-(C 3-6 carbocycle), -(2- to 6-membered heteroalkyl)-(C 3-6 carbocycle), -C 1-6 alkyl-(3- to 6-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 6-membered heterocycle), wherein -C 1-6 alkyl-(C 3-6 carbocycle), -(2- to 6-membered heteroalkyl)-(C 3-6 carbocycle), -C 1-6 alkyl-(3- to 6-membered heterocycle), and -(2- to 6-membered heteroalkyl)-(3- to 6-membered heterocycle) are each optionally substituted with one, two, or three substituents selected from oxo, -OR 22 , and C 1-6 alkyl optionally substituted with one or more substituents independently selected from oxo, -OR 22 , and -N(
  • R 9 is selected from -C 1-3 alkyl-(C 3-6 carbocycle), -(2- to 3 -membered heteroalky l)-(C 3-6 carbocycle), - C 1-3 alkyl-(4- to 6-membered heterocycle), and -(2- to 3-membered heteroalkyl)-(4- to 6-membered heterocycle), wherein -C 1-3 alkyl-(C 3-6 carbocycle), -(2- to 3-membered heteroalkyl)-(C 3-6 carbocycle), -C 1-3 alkyl-(4- to 6- membered heterocycle), and -(2- to 3 -membered heteroalky l)-(4- to 6-membered heterocycle) are each optionally substituted with one, two, or three substituents selected from oxo, -OCH 3 , and C 1-6 alkyl optionally substituted with one or more substituents independently selected from oxo and -NH 2 .
  • R 9 is selected from -C 1-3 alkyl-(C 3-6 saturated carbocycle), -(2- to 3-membered heteroalkyl)-(C 3-6 saturated carbocycle), -C 1-3 alkyl-(4- to 6- membered saturated heterocycle), -(2- to 3-membered hetero alkyl) -(4- to 6-membered saturated heterocycle), -C 1-3 alkyl-(5- to 6-membered heteroaryl), and -(2- to 3-membered heteroalkyl)-(5- to 6-membered heteroaryl), wherein - C 1-3 alkyl-(C 3-6 saturated carbocycle), -(2- to 3-membered heteroalkyl)-(C 3-6 saturated carbocycle), -C 1-3 alkyl-(4- to 6-membered saturated heterocycle), -(2- to 3 -membered hetero alkyl) -(4- to 6-membered saturated heterocycle), -C 1-3 alkyl-(5
  • R 9 is C 1-6 alkyl optionally substituted with one, two, or three substituents selected from halogen, oxo, -OR 22 , -N(R 22 )(R 23 ), -S(O)R 22 , -C(O)N(R 22 )(R 23 ), -N(R 22 )C(O)R 22 , -S(O) 2 R 22 , -P(O)(R 22 )(R 23 ), and
  • R 9 is C 1-6 alkyl optionally substituted with one, two, or three substituents selected from
  • R 10 is selected from hydrogen, halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, -OR 12 , and -N(R 12 )(R 13 ), wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, and 2- to 6-membered heteroalkyl are optionally substituted with one, two, or three R 20 ; or R 9 and R 10 , together with the atoms to which they are attached, form C 4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R 20 .
  • R 10 is selected from hydrogen, halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6- membered heteroalkyl, -OR 12 , and -N(R 12 )(R 13 ), wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, and 2- to 6-membered heteroalkyl are optionally substituted with one, two, or three R 20 .
  • R 9 and R 10 together with the atoms to which they are attached, form C 4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R 20 .
  • R 11 is selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12- membered heterocycle), -C(O)R 12 , and -C(O)N(R 12 )(R 13 ), wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl- (C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R 20 .
  • R 11 is selected from hydrogen, C 1-6 alkyl, and -C(O)R 12 . In some embodiments, R 11 is hydrogen. In some embodiments, R 11 is C 1-6 alkyl, such as -CH 3 . In some embodiments, R 11 is -C(O)R 12 . In some embodiments, R 11 is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle). In some embodiments, R 11 is a bond to the antigen binding unit or the chemical linker.
  • R 7 is selected from C 6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R 20 . In some embodiments, R 7 is selected from C 10 aryl and 9-membered heteroaryl, each of which is optionally substituted with one or more R 20 . In some embodiments, R 7 is selected from naphthalenyl and benzothiophenyl, each of which is optionally substituted with one or more R 20 .
  • R 7 is selected from bridged bicyclic C 4-10 cycloalkyl, bridged bicyclic 4- to 10-membered heterocycloalkyl, bridged bicyclic C7-10 aryl, and bridged bicyclic 7- to 10-membered heteroaryl, each of which is optionally substituted with one or more R 20 .
  • R 7 is selected from fused bicyclic C 4-10 cycloalkyl, fused bicyclic 4- to 10-membered heterocycloalkyl, fused bicyclic C7-10 aryl, and fused bicyclic 7- to 10-membered heteroaryl, each of which is optionally substituted with one or more R 20 .
  • R 7 is selected from C 6-10 aryl and 5- to 10- membered heteroaryl, each of which is optionally substituted with one, two, three, four, or five R 20 .
  • R 7 is selected from naphthyl, isoquinolinyl, indazolyl, benzothiazolyl, benzothiophenyl, phenyl, and pyridinyl, each of which is optionally substituted with one or more R 20 .
  • R 7 is naphthyl, optionally substituted with one or more R 20 .
  • R 7 is benzothiophenyl, optionally substituted with one or more R 20 .
  • R 7 is selected from: wherein:
  • Q 1 , Q 3 , and Q 5 are independently selected from N and C(R 1a );
  • X 14 , X 15 , X 17 , and X 18 are independently selected from C(O), C(R 1a ), N, C(R 1a ) 2 , and N(R 1b );
  • X 16 is selected from C, N, and C(R 1a ); each R 1a is independently selected from hydrogen, halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , -N(R 12 )(R 13 ), -C(O)OR 12 , - OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(R 12
  • R 7 is selected from
  • R 7 is substituted with -CF 3 , -CH 3 , and -NH 2 . In some embodiments, R 7 is substituted with -CF 3 and -NH 2 . In some embodiments, R 7 is substituted with -CF 3 , -CH 3 , -F, and -NH 2 . In some embodiments, R 7 is substituted with -CF 3 , - F, and -NH 2 .
  • Z is selected from O and C(R 5 ) 2 ;
  • X is selected from C(R 6 ) and N;
  • R 7 is selected from naphthyl, benzothiophenyl, phenyl, and pyridinyl, each of which is optionally substituted with one or more R 20 ; m is 0 or 1 ; and n is 1 or 2.
  • R 11 is hydrogen; m is 0 or 1 ; and n is 1 or 2.
  • X is C(R 6 );
  • A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl;
  • X is N
  • R 11 is hydrogen
  • R 9 is C 1-3 alkyl optionally substituted with one, two, or three R 20 ; m is 0 or 1 ; and n is 1 or 2.
  • A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl;
  • R 3 is independently selected at each occurrence from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R 20 ;
  • R 11 is hydrogen; and m is 0 or 1.
  • R 3 is independently selected at each occurrence from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R 20 ;
  • R 6 and R 8 are independently selected from hydrogen, halogen, and C 1-3 haloalkyl; and m is 0 or 1.
  • R 2 is -OR 12 ;
  • R 3 is independently selected at each occurrence from C 1-6 alkyl optionally substituted with one, two, or three R 20 ; m is 0 or 1 ;
  • R 6 is selected from chlorine and -CF 3 ;
  • R 8 is fluorine
  • R 3 is independently selected at each occurrence from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, -OH, and -OCH 3 ; m is 0 or 1 ;
  • R 6 is selected from chlorine and -CF 3 ;
  • R 8 is fluorine
  • R 3 is independently selected at each occurrence from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, -OH, and -OCH 3 ; m is 0 or 1 ;
  • R 8 is fluorine; and n is 1.
  • X is C(R 6 );
  • Z is O
  • R 6 is selected from chlorine and -CF 3 :
  • R 7 is pyridinyl optionally substituted with one or more R 20 ;
  • R 8 is fluorine; and n is 1.
  • Z is selected from O and C(R 5 ) 2 ;
  • R 3 is independently selected at each occurrence from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, -OH, and -OCH 3 ; m is 0 or 1 ;
  • R 7 is naphthyl optionally substituted with one or more R 20 ;
  • R 8 is fluorine; and n is 1 or 2.
  • the compound of Formula (I) or (I-a) applicable for generating a subject conjugate is selected from:
  • R 2 is selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 3-10 carbocycle, 3- to 10- membered heterocycle, -OR 12 , and -N(R 12 )(R 13 ), wherein C 1-6 alkyl, C 2-6 alkenyl, C 3-10 carbocycle, and 3- to 10- membered heterocycle are optionally substituted with one, two, or three R 20 .
  • R 2 is selected from hydrogen, -(C 0-3 alkylene)-O-(C 0-3 alkylene)-R 20 , C 1-3 alkyl, and 3- to 10-membered heterocycle, wherein each C 0-3 alkylene, C 1-3 alkyl, and 3- to 10-membered heterocycle is optionally substituted with one, two, or three R 20 .
  • R 2 is selected from hydrogen, C 1-3 alkyl, -OR 12 , and 3- to 10-membered heterocycle, wherein C 1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R 20 .
  • R 2 is OR 12 .
  • R 2 is -O(C 1-3 alkylene)(4- to 10-membered heterocycle), wherein
  • R 2 is -OCH 2 (hexahydro-lH-pyrrolizine) optionally substituted with one, two, or three R 20 .
  • R 2 is substituted with halogen, such as fluorine.
  • R 3 is independently selected at each occurrence from halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-6 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-e carbocycle), -C 0-6 alkyl-(3- to 6-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 6-membered heterocycle), -OR 12 , and - N(R 12 )(R 13 ), wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C3-e carbocycle), -(2- to
  • R 8 is selected from hydrogen, halogen, -CN, C 1-6 alkyl, 2- to 6-membered heteroalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -OR 12 , and -N(R 12 )(R 13 ), wherein C 1-6 alkyl, 2- to 6-membered heteroalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one or more R 20 .
  • n is 0, 1, 2, or 3. In some embodiments, n is 1. In some embodiments, n is 2.
  • R 2 is selected from hydrogen, C 1-3 alkyl, -OR 12 , and 3- to 10-membered heterocycle, wherein C 1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R 20 ;
  • R 2 is selected from hydrogen, C 1-3 alkyl, -OR 12 , and 3- to 10-membered heterocycle, wherein C 1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R 20 ;
  • R 6 is selected from chlorine and -CTT and
  • R 3 is independently selected at each occurrence from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R 20 ; m is 0 or 1 ;
  • R 6 is selected from chlorine and -CF 3 :
  • R 8 is fluorine
  • R 3 is independently selected at each occurrence from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, -OH, and -OCH 3 ; m is 0 or 1 ;
  • R 6 is selected from chlorine and -CF 3 :
  • R 8 is fluorine; and n is 1.
  • a compound of Formula (I), (I-a), (I-b), or (I-c) applicable for generating a subject conjugate has the formula: wherein:
  • Ring A is 6-membered heteroaryl comprising one or two ring nitrogen atoms
  • R 9 is selected from C 1-3 alkyl, C 2-4 alkenyl, C 2-3 alkynyl, -C 1-2 alkyl-(C 3-4 saturated carbocycle), and -C 1-2 alkyl-(5- to 6-membered saturated heterocycle); wherein C 1-3 alkyl, C 2-4 alkenyl, C 2-3 alkynyl, -C 1-2 alkyl-(C 3-4 saturated carbocycle), and -C 1-2 alkyl-(5- to 6-membered saturated heterocycle) are each optionally substituted with one or more halogen, R 10 is selected from hydrogen and halogen;
  • R 6 is selected from halogen and -CF 3 ;
  • R 7 is benzothiophenyl optionally substituted with one or more substituents independently selected from -
  • R 8 is halogen; and wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker.
  • the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
  • R 6 is -CF 3 .
  • R 5 is halogen.
  • R 8 is -F.
  • the substituents (for example, R 2 , R 6 , R 7 , R 8 , R 9 , and R 10 ) of formula (I-f) are the same as the corresponding substituents in Formula (I), (I-a), (I-b), and/or (I-c), including in embodiments thereof.
  • a compound of Formula (I-f) is a compound of Formula (I), (I-a), (I-b), and/or (I-c),.
  • a compound of Formula (I), (I-a), (I-b), or (I-c) applicable for generating a subject conjugate has the formula: wherein:
  • R 9 is selected from C 1-3 alkyl, C 2-4 alkenyl, C 2-3 alkynyl, -C 1-2 alkyl-(C 3-4 saturated carbocycle), and -C 1-2 alkyl-(5- to 6-membered saturated heterocycle); wherein C 1-3 alkyl, C 2-4 alkenyl, C 2-3 alkynyl, -C 1-2 alkyl-(C 3-4 saturated carbocycle), and -C 1-2 alkyl-(5- to 6-membered saturated heterocycle) are each optionally substituted with one or more -F,
  • R 6 is selected from -Cl and -CF 3 ;
  • R 7 is benzothiophenyl optionally substituted with one or more substituents independently selected from - NH 2 , -CN, and -F;
  • R 8 is -F ; and wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker.
  • the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
  • R 6 is -CF 3 .
  • the substituents (for example, R 2 , R 6 , R 7 , R 8 , R 9 , and R 10 ) of formula (I-f) are the same as the corresponding substituents in Formula (I), (I-a), (I-b), or (I-c), including in embodiments thereof.
  • a compound of Formula (I-d) applicable for generating a subject conjugate is a compound of Formula (I), (I-a), (I-b), or (I-c).
  • a compound of Formula (I), (I-a), (I-b), or (I-c), applicable for generating a subject conjugate has the formula:
  • R 6 is -CF 3 .
  • R 9 is -CH 3 . In some embodiments of the formulae above, R 9 is -CH 2 CH 3 . In some embodiments of the formulae above, R 6 is -Cl. In some embodiments of the formulae above, R 6 is -CF 3 . In some embodiments of the formulae above, R 7 is embodiments of the formulae above, R 7 is In some embodiments of the formulae above, R 7 is
  • a compound of Formula (I), (I-a), (I-b), or (I-c), applicable for generating a subject conjugate has the formula:
  • a compound of Formula (I-g) applicable for generating a subject conjugate is a compound for generating a subject conjugate, of Formula (I), (I-a), (I-b), and/or (I-c).
  • the compound of Formula (I), (I-a), or (I-b), applicable for generating a subject conjugate is selected from: solvate thereof.
  • the compound of Formula (I), (I-a), (I-b), or (I-c), applicable for generating a subject conjugate is selected from: pharmaceutically acceptable salt or solvate thereof.
  • the compound of Formula (I), (I-a), (I- b), or (I-c), applicable for generating a subject conjugate is selected from:
  • the present disclosure provides a compound selected from:
  • the present disclosure provides a compound for generating a subject conjugate selected from:
  • a KRAS inhibitor for generating a subject conjugate described herein such as a compound of Formula (I), (I-a), (I-b), or (I-c) is provided as a substantially pure stereoisomer.
  • the stereoisomer is provided in at least 80% enantiomeric excess, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% enantiomeric excess.
  • atropisomers refers to conformational stereoisomers which occur when rotation about a single bond in the molecule is prevented, restricted, or greatly slowed as a result of steric interactions with other parts of the molecule and wherein the substituents at both ends of the single bond are asymmetrical (i.e., optical activity arises without requiring an asymmetric carbon center or stereocenter). Where the rotational barrier about the single bond is high enough, and interconversion between conformations is slow enough, separation and isolation of the isomeric species may be permitted.
  • Atropisomers are enantiomers (or epimers) without a single asymmetric atom.
  • compounds for generating conjugates described herein exist as their pharmaceutically acceptable salts.
  • the methods disclosed herein include methods of treating diseases by administering such pharmaceutically acceptable salts.
  • the methods disclosed herein include methods of treating diseases by administering such pharmaceutically acceptable salts as pharmaceutical compositions.
  • a compound of Formula 1g may be prepared according to Scheme 1.
  • heteroaryl amine 1c can be formed from chloride la via a nucleophilic aromatic substitution reaction with amine lb. Ring closure to Id can be followed by an oxidation reaction to provide sulfone le, which can be substituted with R 2 upon addition of a suitable alcohol to afford If.
  • Substitution of the aryl bromide with a suitable boronic ester can provide the corresponding R 7 - substituted compound, which may optionally be subjected to one or more subsequent reactions, such as a deprotection, to provide a compound of Formula 1g.
  • a compound of Formula 2e may be prepared according to Scheme 2.
  • heteroaryl ether 2c can be formed from fluoride 2a via a nucleophilic aromatic substitution reaction with alcohol 2b.
  • Ring closure to 2d can be followed by substitution with a suitable boronic ester to provide the corresponding R 7 - substituted compound, which may optionally be subjected to one or more protecting group manipulations to provide a compound of Formula 2e.
  • a conjugate of Formula 3c may be prepared according to Scheme 3.
  • amine 3a may be coupled to linker L to form intermediate 3b.
  • the antigen binding unit can be coupled to the linker to provide a conjugate of Formula 3c.
  • a Kras inhibitor-chemical linker capable of forming a conjugate of the present disclosure comprises i) a Kras inhibitor for producing a conjugate of the present disclosure of Table 1, wherein one hydrogen bonded to a nitrogen of the Kras inhibitor is replaced with a bond to a chemical linker; and ii) a chemical linker of Table 2, wherein in Table 2 indicates an attachment site to the Kras inhibitor.
  • the nitrogen atom of the Kras inhibitor covalently bonded to chemical linker e.g., chemical linker of Table 2 is a nitrogen atom bonded to a benzothiophenyl of the Kras inhibitor.
  • the nitrogen atom of the Kras inhibitor covalently bonded to chemical linker is in an aminomethyl that is bonded to a pyrrolizidine of the Kras inhibitor.
  • the chemical linkers of Table 2 may each covalently be bonded to the nitrogen atom of the aminomethyl bonded to pyrrolizidine of a compound of Table 1 ; for example, the Kras inhibitor-chemical linker conjugate comprises #377 of Table 1, and 1019 of Table 2.
  • the linker-modified Kras compounds comprising compound 148 of Table 1 and one linker selected from the group of chemical linkers 1001— 1018 and 1020-1057, each results in a linker-modified Kras compound with, respectively, an observed mass of 1794.9, 1703.6, 1075.4 [M/2]+H+ (half-mass), 1305.4, 1825.7, 918.2 [M/2]+H+ (half-mass), 1967.8, 1549.4, 1351.7, 1034.2 [M/2]+H+ (half-mass), 1771.8, 1307.4, 1358.2, 1833.6, 834.9 [M/2]+H+ (half-mass), 1036.9 [M/2]+H+ (half-mass), 1748.8, 1872.9 , 1699.9 , 1779.7 , 1642.5, 1870.9 , 1219.5 , 1776.6 , 1118.7 [M/2]+H+ (halfmass), 1596.4, 1745.7, 1878.
  • Each linker-modified Kras compound formed having the observed mass immediately above comprises a bond between the attachment site of the linker and the nitrogen atom of the amine bonded to the benzothiophenyl of compound 148 of Table 1.
  • the linker-modified Kras compound formed by covalently bonding compound 377 of Table 1 and 1019 of Table 2 (between the aminomethyl nitrogen of 377 and the attachment site of 1019) has an observed mass of 1316.7.
  • linker-modified Kras compounds described herein may be further conjugated to any antigen binding unit disclosed herein including but not limited to Cetuximab to yield a subject conjugate.
  • the linker-attached Kras inhibitors described immediately above each comprise a moiety covalently bonded to cetuximab, which can be produced from the of the linkers of Table 2, wherein indicates the attachment site of th
  • Non-limiting examples of conjugates of the present disclosure include:
  • Ab is an antigen binding unit selected from the group of consisting of AG7, B7-H3, BCMA, CA15-3, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD70, CD71, CD79B, CEA, CLDN18.2, EGFR, FOLR1, GCC, GPC1, HER2, HER3, ICAM1, LeX, LeY, MET, MSLN, MUC1, NECTIN4, SLC44A4, TF, and Trop-2.
  • Ab in the structures shown in this paragraph comprises the amino acid sequence of cetuximab.
  • Ab in the structures shown in this paragraph comprises the amino acid sequence of Sacituzumab.
  • Ab in the structures shown in this paragraph is bonded to the linker through an Ab thiol.
  • m in the structures shown in this paragraph is 2, 4, 6, or 8.
  • m in the structures shown in this paragraph is 4.
  • m in the structures shown in this paragraph is 8.
  • m in the structures shown in this paragraph is 2.
  • Antibody -drug conjugates can also be prepared by methods known in the art and also according to the exemplary procedures provided herein to yield various Kras inhibitor-to-antibody ratio (DAR) ratios, including about 2, 4, 6, 8.
  • DAR Kras inhibitor-to-antibody ratio
  • TCEP tris(2- carboxy ethyljphosphine
  • TCEP tris(2- carboxy ethyljphosphine
  • the reduced antibody is conjugated with excess linker modified Kras inhibitor (5 equiv.) in 10% DMA and the reaction proceeded for 1 h at 22°C.
  • a solution with cysteine (4 equiv.) is added to quench the conjugation reaction by depleting the unconjugated linker-payload.
  • the cell panel include HPAC (KRAS-G12D, pancreatic adenocarcinoma), H1373 (KRAS-G12C, lung adenocarcinoma), H2009 (KRAS-G12A, lung adenocarcinoma), MIA-PaCa2 (KRAS-G12C, pancreatic adenocarcinoma), THP-1 (NRAS-G12D, acute myeloid leukemia), T24 (HRAS-G12V, colorectal adenocarcinoma), and SK -MEL-30 (NRAS-Q61K, melanoma).
  • the present disclosure provides a method of treating a Ras-mediated cancer in a subject in need thereof, comprising administering to the subject a SHP2 inhibitor, a SOS inhibitor, an EGFR inhibitor, a MEK inhibitor, an ERK inhibitor, a CDK4/6 inhibitor, or a BRAF inhibitor and an effective amount of a conjugate described herein.
  • the cancer is a solid tumor.
  • the cancer is a hematological cancer.
  • TI conjugate /Tl KRASi is greater than 1.1, such as greater than 1.2, 1.3, 1.4, 1.5
  • TI conjugate /Tl KRASi is greater than 2. In some embodiments, TI conjugate /Tl KRASi is greater than 5.
  • the present disclosure provides a method of increasing concentration of a small-molecule KRAS inhibitor in tumor tissue, comprising providing a conjugate described herein to a subject, wherein increased tumor tissue concentration is ascertained by the formula: ([KRASi] t-c /[KRASi] p-c ) / ([KRASi] t-k /[KRASi] p-k ) > 1, wherein [KRASi] t-c is concentration of the KRAS inhibitor in tumor tissue at a first time -point following administration of the conjugate; wherein [KRASi] p-c is plasma concentration of the KRAS inhibitor at the same time -point following the administration of the conjugate; wherein [KRASi] t-k is concentration of the KRAS inhibitor in tumor tissue following administration of the KRAS inhibitor alone at an equivalent dose at the same time -point; and wherein [KRASi] p-k is plasma concentration of the KRAS inhibitor
  • ([KRASi] t-c /[KRASi] p-c ) / ([KRASi] t-k /[KRASi] p-k ) is greater than 1.1, such as greater than 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,
  • ([KRASi] t-c /[KRASi] p-c ) / ([KRASi] t-k /[KRASi] p-k ) is greater than 2. In some embodiments, ([KRASi] t-c /[KRASi] p-c ) / ([KRASi] t-k /[KRASi] p-k ) is greater than 5. In some embodiments, the first time-point is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 48 hours after the administration.
  • efficacy of the conjugate may be greater than efficacy of a combination of the antigen binding unit and the KRAS inhibitor when each is administered at a comparable concentration.
  • one or more subject conjugate has been shown to elicit tumor regression more effectively than the antigen binding unit and the Kras inhibitor, when each being administered alone or in combination as separate molecules and not conjugated together to form a cetuximab-Kras ADC-conjugate.
  • cetuximab-Kras conjugates DAR 4 with various linkers elicited tumor regression as early as day 4 after administration, and more than 50% tumor regression on day 22 after administration.
  • toxicity of the conjugate is less than toxicity of a combination of the antigen binding unit and the KRAS inhibitor when each is administered at a comparable concentration.
  • Biological properties that can be determined with SPR include but are not limited to adsorption kinetics, desorption kinetics, antigen binding, affinity, epitope mapping, biomolecular structure, protein interaction, biocompatibility, tissue engineering, lipid biolayers, and any combination thereof.
  • the Ras target to which a subject compound binds can be a Ras mutant (e.g., G12S, G12C, G12D, G12V, G13C, and/or G13D), including a mutant of K-Ras, H-Ras, or N-Ras.
  • the methods of treating cancer can be applied to treat a solid tumor or a hematological cancer.
  • the cancer being treated can be, without limitation, prostate cancer, brain cancer, colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, various lung cancers including non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, non-Hodgkin’s lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of
  • a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, wherein the cancer is a hematological cancer.
  • cancer in some embodiments is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, wherein the cancer is a hematological cancer selected from one or more of chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T- cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma
  • cancer in some embodiments is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, wherein the cancer is one or more cancers selected from the group consisting of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), B cell acute lymphoblastic leukemia (B- ALL), and/or acute lymphoblastic leukemia (ALL).
  • CLL chronic lymphocytic leukemia
  • AML acute myeloid leukemia
  • T-ALL T-cell acute lymphoblastic leukemia
  • B- ALL B cell acute lymphoblastic leukemia
  • ALL acute lymphoblastic leukemia
  • any of the treatment methods disclosed herein can be administered alone or in combination or in conjunction with another therapy or another agent.
  • “combination” it is meant to include (a) formulating a subject composition containing a subject conjugate together with another agent, or (b) using the subject composition separate from the another agent as an overall treatment regimen.
  • “conjunction” it is meant that the another therapy or agent is administered either simultaneously, concurrently or sequentially with a subject composition comprising a conjugate disclosed herein, with no specific time limits, wherein such conjunctive administration provides a therapeutic effect.
  • chemotherapeutic agents include an anthracycline (e.g., doxorubicin (e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosf amide, temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fludarabine)), a TNFR glucocorticoi
  • Anti-cancer agents of particular interest for combinations with a conjugate of the present disclosure include: anthracy clines; alkylating agents; antimetabolites; drugs that inhibit either the calcium dependent phosphatase calcineurin or the p70S6 kinase FK506 or inhibit the p70S6 kinase; mTOR inhibitors; immunomodulators; anthracy clines; vinca alkaloids; proteosome inhibitors; GITR agonists; protein tyrosine phosphatase inhibitors; a CDK4 kinase inhibitor; a BTK inhibitor; a MKN kinase inhibitor; a DGK kinase inhibitor; or an oncolytic virus.
  • compositions provided herein can be administered in combination with radiotherapy, such as radiation.
  • Whole body radiation may be administered at 12 Gy.
  • a radiation dose may comprise a cumulative dose of 12 Gy to the whole body, including healthy tissues.
  • a radiation dose may comprise from 5 Gy to 20 Gy.
  • a radiation dose may be 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 11 Gy, 12, Gy, 13 Gy, 14 Gy, 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, or up to 20 Gy.
  • Radiation may be whole body radiation or partial body radiation. In the case that radiation is whole body radiation it may be uniform or not uniform. For example, when radiation may not be uniform, narrower regions of a body such as the neck may receive a higher dose than broader regions such as the hips.
  • an immunosuppressive agent can be used in conjunction with a subject treatment method.
  • immunosuppressive agents include but are not limited to cyclosporin, azathioprine, methotrexate, my cophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies (e.g., muromonab, otelixizumab) or other antibody therapies, cy toxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation, peptide vaccine, and any combination thereof.
  • the above -described various methods can comprise administering at least one immunomodulatory agent.
  • the at least one immunomodulatory agent is selected from the group consisting of immuno stimulatory agents, checkpoint immune blockade agents (e.g., blockade agents or inhibitors of immune checkpoint genes, such as, for example, PD-1, PD-L1, CTLA-4, IDO, TIM3, LAG3, TIGIT, BTLA, VISTA, ICOS, KIRs and CD39), radiation therapy agents, chemotherapy agents, and combinations thereof.
  • the immuno stimulatory agents are selected from the group consisting of IL-12, an agonist costimulatory monoclonal antibody, and combinations thereof.
  • Immunostimulants can be vaccines, colony stimulating agents, interferons, interleukins, viruses, antigens, costimulatory agents, immunogenicity agents, immunomodulators, or immunotherapeutic agents.
  • An immunostimulant can be a cytokine such as an interleukin.
  • One or more cytokines can be introduced with modified cells provided herein. Cytokines can be utilized to boost function of modified T lymphocytes (including adoptively transferred tumor-specific cytotoxic T lymphocytes) to expand within a tumor microenvironment. In some cases, IL -2 can be used to facilitate expansion of the modified cells described herein. Cytokines such as IL- 15 can also be employed.
  • cytokines in the field of immunotherapy can also be utilized, such as IL-2, IL-7, IL-12, IL-15, IL-21, or any combination thereof.
  • An interleukin can be IL -2, or aldesleukin.
  • Aldesleukin can be administered in low dose or high dose.
  • a high dose aldesleukin regimen can involve administering aldesleukin intravenously every 8 hours, as tolerated, for up to about 14 doses at about 0.037 mg/kg (600,000 lU/kg).
  • An immuno stimulant e.g., aldesleukin
  • a conjugate described herein such as a conjugate, salt, or solvate of Formula (A) is administered in combination or in conjunction with one or more pharmacologically active agents selected from (1) an inhibitor of MEK (e.g., MEK1, MEK2) or of mutants thereof (e.g., trametinib, cobimetinib, binimetinib, selumetinib, refametinib, AZD6244); (2) an inhibitor of epidermal growth factor receptor (EGFR) and/or of mutants thereof (e.g., afatinib, erlotinib, gefitinib, lapatinib, cetuximab panitumumab, osimertinib, olmutinib, EGF-816); (3) an immunotherapeutic agent (e.g., checkpoint immune blockade agents, as disclosed herein); (4) a taxane (e.g., pac), a paclitotin
  • a conjugate described herein such as a conjugate, salt, or solvate of Formula (A) is administered in combination or in conjunction with one or more additional pharmacologically active agents comprising an inhibitor of SOS (e.g., SOS1, SOS2) or of mutants thereof, such as RMC-5845, or BI-1701963.
  • SOS e.g., SOS1, SOS2
  • RMC-5845 e.g., RMC-5845, or BI-1701963.
  • a conjugate described herein such as a conjugate, salt, or solvate of Formula (A) is administered in combination or in conjunction with one or more checkpoint immune blockade agents (e.g., anti-PD- 1 and/or anti-PD-L 1 antibody, anti-CLTA-4 antibody).
  • checkpoint immune blockade agents e.g., anti-PD- 1 and/or anti-PD-L 1 antibody, anti-CLTA-4 antibody.
  • composition comprising at least one conjugate described herein, or a pharmaceutically acceptable salt, together with one or more pharmaceutically acceptable excipients.
  • excipient(s) or carrier(s)
  • the excipient(s) is acceptable or suitable if the excipient is compatible with the other ingredients of the composition and not deleterious to the recipient (i.e., the subject) of the composition.
  • a conjugate described herein is administered either alone or in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition.
  • Administration of a conjugate or composition described herein can be affected by any method that enables delivery of the conjugate to the site of action.
  • enteral routes including oral, gastric or duodenal feeding tube, rectal suppository and rectal enema
  • parenteral routes injection or infusion, including intraarterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and subcutaneous), inhalational, transdermal, transmucosal, sublingual, buccal and topical (including epicutaneous, dermal, enema, eye drops, ear drops, intranasal, vaginal) administration, although the most suitable route may depend upon for example the condition and disorder of the recipient.
  • a conjugate described herein can be administered locally to the area in need of treatment, by, for example, local infusion during surgery, topical application such as creams or ointments, injection, catheter, or implant.
  • the administration can also be by direct injection at the site of a diseased tissue or organ.
  • a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, is administered orally.
  • a pharmaceutical composition suitable for oral administration is presented as a discrete unit such as a capsule, cachet or tablet, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a nonaqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient is presented as a bolus, electuary, or paste.
  • compositions which can be used orally include tablets, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free -flowing form such as a powder or granules, optionally mixed with binders, inert diluents, or lubricating, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered conjugate moistened with an inert liquid diluent.
  • the tablets are coated or scored and are formulated so as to provide slow or controlled release of the active ingredient therein. All formulations for oral administration should be in dosages suitable for such administration.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In some embodiments, stabilizers are added. Dragee cores are provided with suitable coatings.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or Dragee coatings for identification or to characterize different combinations of active conjugate doses.
  • compositions are formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use.
  • sterile liquid carrier for example, saline or sterile pyrogen-free water
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • compositions may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the conjugates may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker, and wherein the KRAS inhibitor selectively inhibits KRAS or a mutant thereof relative to HRAS and NRAS (e.g., 1.1 fold, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, , 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, or 10000 fold).
  • HRAS and NRAS e.g., 1.1 fold, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, , 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700
  • the antigen binding unit is selected from a monoclonal antibody, a Fab, a Fab’, an F(ab’), an Fv, a disulfide linked Fc, an scFv, a single domain antibody, a diabody, a bi-specific antibody, and a multi-specific antibody.
  • the antigen binding unit is a monoclonal antibody.
  • linker comprises one or more components independently selected from alkyl, polyethylene glycol, a hydrazone, maleimide, succinimide, asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, valine, alanine, glycine, leucine, isoleucine, methionine, tryptophan, proline, histidine, citrulline, phenylalanine, carboxylate, and p-aminobenzyloxycarbonyl.
  • the linker comprises one or more components independently selected from alkyl, polyethylene glycol, a hydrazone, maleimide, succinimide, asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, valine, alanine, glycine, leucine,
  • Z 1 is a product formed by reaction of the antigen binding unit with a reactive precursor of Z 1 ;
  • Z 2 is absent, C 1-6 alkyl, (CH 2 CH 2 O) n2 , -C(O)NH-, -C(O)NCH 3 -, (C(O)CH 2 N(CH 3 )) n2 , -
  • Z 3 is selected from a bond, Val-Cit, Cit-Val, Ala-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu- Val, Ser-Cit, Cit-Ser, Lys-Cit, Cit-Lys, Asp-Cit, Cit-Asp, Val-Ala, Ala-Vai, Val-Val, Val-Gly, Gly-Val, Phe-Lys, Lys-Phe, Val-Lys, Lys-Val, Ala-Lys, Lys-Ala, Phe-Cit, Cit-Phe, Leu-Cit, Cit-Leu, Ile-Cit, Cit-Ile, Phe-Arg, Arg- Phe, Cit-Trp, Trp-Cit, Gly-Gly, Gly-Cit, Cit-Gly, Ala-Pro, Pro-Ala, Ala-Ser, Ser-Ala, Glu
  • R z is selected from hydrogen, -CH 2 N(CH 3 )C(O)-(CH 2 CH 2 O) n2 CH 3 , -CH 2 N(CH 3 )(C(O)CH 2 N(CH 3 )) n2 -C(O)CH 3 , -CH 2 N(CH 3 )C(O)-(CH 2 CH 2 O) n2 -CH 2 CH 2 C(O)-(N(CH 3 )CH 2 C(O)) n2 -N(CH 3 ) 2 , -SO 3 H, -CO 2 H, PEG 4-32, polysarcosine, -(CH 2 N(CH 3 )C(O)) n2 CH 3 , and a sugar moiety; nl, n3, and n4 are each independently 0 or 1 ; n2 is independently an integer from 1 to 20; and indicates an attachment site to the KRAS inhibitor.
  • Z 2 is absent, C 1-6 alkyl or -(CH 2 CH 2 O ⁇ CH 2 CH 2 , ;
  • Z 3 is selected from Val-Cit and Val-Ala;
  • R z is selected from hydrogen, -CH 2 N(CH 3 )C(O)-(CH 2 CH 2 O) n2 CH 3 , -CH 2 N(CH 3 )(C(O)CH 2 N(CH 3 )) n2 -C(O)CH 3 , and -CH 2 N(CH 3 )C(O)-(CH 2 CH 2 O) n2 -CH 2 CH 2 C(O)-(N(CH 3 )CH 2 C(O)) n2 -N(CH 3 ) 2 ; n1, n3, and n4 are each independently 0 or 1 ; n2 is independently an integer from 1 to 20; and indicates an attachment site to the KRAS inhibitor.
  • Z is selected from O, N, C(R 5 ) 2 , C(O), S, S(O), and S(O) 2 ;
  • R 3 is independently selected at each occurrence from halogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR 12 , -SR 12 , -N(R 12 )(R 13 ), -C(O)OR 12 , - OC(O)N(R 12 )(R 13 ), -N(R 12 )C(O)N(R 12 )(R 13 ), -N(R 12 )C(O
  • R 4 is selected from hydrogen, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6- membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), -C(O)OR 12 , -C(O)R 12 , -C(O)N(R 12 )(R 13 ), -C(O)C(O)N(R 12 )(R 13 ), -S(O) 2 R 12 , - S(O)(NR 12 )R 12 , -S(O) 2 N(R
  • R 7 is selected from C 6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R 20 ; m is 0, 1, 2, or 3; n is 1 or 2;
  • R 12 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R 20 ;
  • R 13 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, and C 1-6 haloalky I: or R 12 and R 13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R 20 ;
  • R 14 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, - C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 14 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -C 0-6 alkyl-(C 3-12 carbocycle), -C 0-6 alkyl-(3- to 12-membered heterocycle), C 3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R 20 ;
  • R 21 is independently selected at each occurrence from hydrogen, halogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl- (C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle), or two R 21 are taken together with the carbon atom to which they are attached to form C 3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C 1-3 alkyl, C 1-3 haloalkyl, and -OH;
  • R 22 is independently selected at each occurrence from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, -C 0-6 alkyl-(C 3-12 carbocycle), and -C 0-6 alkyl-(3- to 12-membered heterocycle); and
  • R 23 is independently selected at each occurrence from hydrogen and C 1-6 alkyl; or R 22 and R 23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I) is replaced with a bond to the antigen binding unit or the chemical linker.
  • a pharmaceutical composition comprising the conjugate of any one of embodiments 1 to 127, or a salt thereof, and a pharmaceutically acceptable excipient.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a conjugate of any one of embodiments 1 to 127, or a salt thereof. 130.
  • a method of treating cancer in a subject comprising a Ras mutant protein comprising: inhibiting the Ras mutant protein of said subject by administering to said subject a conjugate of any one of embodiments 1 to 127, or a salt thereof.
  • 131. The method of embodiment 129-130, wherein the cancer is a solid tumor or a hematological cancer.
  • 132. The method of any one of embodiments 129-131, wherein the cancer comprises a K-Ras G12C, G12D, G12S, or G12V mutant protein.
  • a method of modulating signaling output of a Ras protein comprising contacting a Ras protein with an effective amount of a conjugate of any one of embodiments 1 to 127, or a salt thereof, thereby modulating the signaling output of the Ras protein.
  • a method of inhibiting cell growth comprising administering an effective amount of a conjugate of any one of embodiments 1 to 127, or a salt thereof, to a cell expressing a Ras protein, thereby inhibiting growth of said cells. 135.
  • the method of any one of embodiments 129-134 comprising administering an additional agent.
  • a method of delivering a small-molecule KRAS inhibitor that exhibits low permeability as characterized by a PAMPA assay comprising contacting a tumor cell with a conjugate of any one of embodiments 1 to 127, or a salt thereof, wherein the KRAS inhibitor exhibits a PAMPA permeability (P e ) value less than 1 x 10 -6 cm/s.
  • a method of enhancing therapeutic efficacy of a small-molecule KRAS inhibitor comprising providing a conjugate of any one of embodiments 1 to 127 to a subject, wherein enhanced therapeutic efficacy is ascertained by the formula:
  • a method of reducing plasma concentration of a small-molecule KRAS inhibitor comprising providing a conjugate of any one of embodiments 1 to 127 to a subject, wherein reduced plasma concentration is ascertained by the formula:
  • a method of increasing concentration of a small-molecule KRAS inhibitor in tumor tissue comprising providing a conjugate of any one of embodiments 1 to 127 to a subject, wherein increased tumor tissue concentration is ascertained by the formula:
  • a method of delivering a small -molecule KRAS inhibitor to the central nervous system of a subject comprising administering a conjugate of any one of embodiments 1 to 127 to the subject, wherein the KRAS inhibitor is released from the conjugate after entering the CNS of the subject.
  • a method of generating a slow-release form of a small-molecule KRAS inhibitor the method comprising conjugating an antigen binding unit to a small-molecule KRAS inhibitor through a chemical linker, wherein the small-molecule inhibitor is released from the conjugate upon introducing the conjugate into a subject or a cell.

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Abstract

The present disclosure provides compounds and pharmaceutically acceptable salts thereof, and methods of using the same. The compounds and methods have a range of utilities as therapeutics, diagnostics, and research tools. In particular, the subject compositions and methods are useful for reducing signaling output of oncogenic protein.

Description

HETEROCYCLIC CONJUGATES AND USES THEREOF
BACKGROUND
CROSS-REFERENCE
[001] This application claims the benefit of U.S. Provisional Application No. 63/550,553, filed February 6, 2024; and U.S. Provisional Application No. 63/700,538, filed September 27, 2024, each incorporated herein by reference in its entirety.
SEQUENCE LISTING
[002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 4, 2025, is named 56690_786_602_SL.xml and is 18,724 bytes in size.
BACKGROUND
[003] Cancer (e.g., tumor, neoplasm, metastases) is the second leading cause of death worldwide estimated to be responsible for about 10 million deaths each year. Many types of cancers are marked with mutations in one or more proteins involved in various signaling pathways leading to unregulated growth of cancerous cells. In some cases, about 25 to 30 percent (%) of tumors are known to harbor Rat sarcoma (Ras) mutations. In particular, mutations in the Kirsten Ras oncogene (K-Ras) are one of the most frequent Ras mutations detected in human cancers, including lung adenocarcinomas (LUADs) and pancreatic ductal adenocarcinoma (PDAC).
[004] Ras proteins have long been considered “undruggable,” due to, in part, high affinity to their substrate guanosine-5'-triphosphate (GTP) and/or their smooth surfaces without any obvious targeting region. The specific G12C Ras gene mutation has been identified as a druggable target to which a number of G12C specific inhibitors have been developed. However, such therapeutics are still of limited application, as the G12C mutation in Ras exhibits a much lower prevalence rate as compared to other known Ras mutations, such as G12D and G12V. Drug resistance and lack of durability impose further limitations to such therapeutics.
SUMMARY
[005] In view of the foregoing, there remains a considerable need for a new design of therapeutics and diagnostics that can specifically target Ras, including wildtype Ras, mutants and/or associated proteins of Ras to reduce Ras signaling output, as well as other signaling pathways in conjunction with the Ras signaling. Of particular interest are KRas inhibitors, including pan KRas inhibitors capable of inhibiting two or more KRas mutants and/or wildtype KRas within this given Ras isoform (e.g., inhibitors targeting mutant KRas proteins such as KRas G12D, G12C, G12S, G13D, and/or G12V, for the treatment of Ras-associated diseases (e.g., cancer). Such compositions and methods can be particularly useful for treating a variety of diseases including, but not limited to, cancers and neoplasia conditions. The present disclosure addresses these needs, and provides additional advantages applicable for diagnosis, prognosis, and/or treatment for a wide diversity of diseases.
[006] In certain aspects, the present disclosure provides a conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker, and wherein the antigen binding unit and the KRAS inhibitor in the conjugate synergistically inhibits signaling output of the first antigen or KRAS. In some embodiments, the conjugate provided herein comprises an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to the small-molecule KRAS inhibitor through a chemical linker. In certain aspects, the present disclosure provides a conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker, and wherein the antigen binding unit and the KRAS inhibitor in the conjugate synergistically inhibits signaling output of the first antigen and KRAS. In some embodiments, the KRAS inhibitor is characterized by a PAMPA permeability (Pe) less than 1 x 10-6 cm/s. In some embodiments, the conjugate is characterized by an enhanced therapeutic efficacy as ascertained by the formula: TIconjugate/TlKRASi > 1 , wherein Tlconjugate = TD50c/ED50c, wherein TD50c is the dose of conjugate required to produce a toxic effect in 50% of test subjects and ED50c is the dose of conjugate required to produce a therapeutic effect in 50% of test subjects; and wherein TIKRASI = TD50k/ED50k, wherein TD50k is the dose of KRAS inhibitor required to produce a toxic effect in 50% of test subjects and ED50k is the dose of KRAS inhibitor required to produce a therapeutic effect in 50% of the test subjects. In some embodiments, the conjugate is characterized by reduced plasma concentration of the KRAS inhibitor as ascertained by the formula: [KRASi]p-c/[KRASi]p-k < 1, wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at a first time-point following administration of the conjugate; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point. In some embodiments, the conjugate is characterized by an increased concentration of the KRAS inhibitor in tumor tissue relative to plasma as ascertained by the formula: ([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) > 1, wherein [KRASi]t-c is concentration of the KRAS inhibitor in tumor tissue at a first time -point following administration of the conjugate; wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at the same time-point following the administration of the conjugate; wherein [KRASi]t-k is concentration of the KRAS inhibitor in tumor tissue following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor at the same time -point following the administration of the KRAS inhibitor alone at the equivalent dose.
[007] In certain aspects, the present disclosure provides a conjugate of Formula (A): wherein:
AgB is an antigen binding unit;
L is a chemical linker;
D is independently selected at each occurrence from a small-molecule KRAS inhibitor, a cytotoxic smallmolecule and a small-molecule agent that selectively modulates a non-KRAS target, wherein at least one D is a KRAS inhibitor; p is selected from 1 to 20; and q is selected from 1 to 20.
[008] In some embodiments, for a conjugate described herein, the antigen binding unit is an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen binding unit is selected from a monoclonal antibody, a Fab, a Fab’, an F(ab’), an Fv, a disulfide linked Fc, an scFv, a single domain antibody, a diabody, a bispecific antibody, and a multi-specific antibody. In some embodiments, the antigen binding unit is a monoclonal antibody. In some embodiments, the antigen binding unit specifically binds a target, such target may include but is not limited to, AG7, B7-H3, BCMA, CA15-3, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD70, CD71, CD79B, CEA, CLDN18.2, EGFR, FOLR1, GCC, GPC1, HER2, HER3, ICAM1, LeX, LeY, MET, MSLN, MUCl, NECTIN4, SLC44A4, TF, or Trop-2. In some embodiments, the antigen binding unit is selected from cetuximab, bevacizumab, paitumumab, ofatumumab, inotuzumab, gemtuzumab, alemtuzumab, and trastuzumab. In some embodiments, the antigen binding unit is an anti-EGFR antibody, such as cetuximab. In some embodiments, the antigen binding unit is an anti-Trop2 antibody, such as sacituzumab.
[009] In some embodiments, for a conjugate described herein, the linker comprises one or more components independently selected from alkyl, alkene, alkyne, aryl, cycloalkyl, heterocycle, glycoside, silyl ether, hydroxy, ether, ketone, ester, carbonate, amide, urea, carbamate, sulfide, disulfide, sulfate, sulfonamide, phosphate, hydrazone, and succinimide. In some embodiments, the linker comprises one or more components independently selected from alkyl, polyethylene glycol, a hydrazone, maleimide, succinimide, asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, valine, alanine, glycine, leucine, isoleucine, methionine, tryptophan, proline, histidine, citrulline, phenylalanine, carboxylate, and p-aminobenzyloxycarbonyl. In some embodiments, the linker comprises one or more components selected from Val-Cit, Glu-Val-Cit, Val-Ala, Val- Val, Val-Gly, Gly-Gly, Gly-Cit, Glu-Gly-Cit, Ala-Ala-Asn, Ala-Gly-Ala, Ala-Pro, Ala-Ser, and Phe-Lys. In some embodiments, p is selected from 2 to 8. In some embodiments, q is selected from 1 to 4. In some embodiments, D is a small-molecule KRAS inhibitor.
[010] In some embodiments, for a conjugate described herein, the KRAS inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is selected from N and C(R6);
Z is selected from O, N, C(R5)2, C(O), S, S(O), and S(O)2;
R2, R5, R6, and R8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, - N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, - C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R5 are taken together with the atom to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein two R5 are taken together to form =O, =NR12, or =C(R14)2;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, - OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -OC(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), - S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R3 are taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one or more R20; optionally wherein two R3 are taken together to form =O, =NR12, or =C(R14)2; and further optionally wherein one R3 and R4 are taken together with the atoms to which they are attached to form 3- to 10-membered heterocycle optionally substituted with one or more R20;
R4 is selected from hydrogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), -C(O)OR12, -C(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), and -S(=O)(=NR12)N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and - (2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20;
R7 is selected from C6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R20; m is 0, 1, 2, or 3; n is 1 or 2;
R12 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20;
R13 is independently selected at each occurrence from hydrogen, C1-6 alkyl, and C1-6 haloalky I: or R12 and R13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R20;
R14 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R14 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R20;
R20 is independently selected at each occurrence from halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23)-, -S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22; wherein two R20 attached to the same or adjacent atoms optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R21 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R21 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and -OH;
R22 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle); and
R23 is independently selected at each occurrence from hydrogen and C1-6 alkyl; or R22 and R23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I) is replaced with a bond to the antigen binding unit or the chemical linker.
[011] In some embodiments, the compound of Formula (I) is a compound of Formula (I-a): or a pharmaceutically acceptable salt or solvate thereof, wherein:
A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms;
R9 and R10 are independently selected from hydrogen, halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23)-, -S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22; wherein R9 and R10 optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, - OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, - C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, - S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23); and
R11 is selected from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle).
[012] In some embodiments, for a compound of Formula (I-a), A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl, such as A is pyridinyl. In some embodiments, R11 is hydrogen. In some embodiments, R10 is selected from hydrogen and halogen; or R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted. In some embodiments, R10 is hydrogen. In some embodiments, In some embodiments, R9 is optionally substituted
C1-3 alkyl, such as R9 is CH3.
[013] In some embodiments, for a compound of Formula (I), R4 is selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), each of which is optionally substituted with one or more R20. In some embodiments, R4 is selected from C1-6 alkyl and -C0-6 alkyl-(3- to 12- membered heterocycle), each of which is optionally substituted with one or more substituents independently selected from halogen, -CH3, -NH2, -NHCH3, and -N(CH3)2-
[014] In some embodiments, for a compound of Formula (I) or (I-a), X is C(R6). In some embodiments, X is N. In some embodiments, Z is O.
[015] In some embodiments, for a compound of Formula (I) or (I-a), R7 is selected from naphthyl, isoquinolinyl, indazolyl, benzothiazolyl, benzothiophenyl, phenyl, and pyridinyl, each of which is optionally substituted with one, two, three, or four R20. In some embodiments, R7 is benzothiophenyl optionally substituted with one, two, three, or four R20. In some embodiments, R7 is substituted with one, two, three, or four substituents independently selected from halogen, -CN, C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, C2-3 alkynyl, -OR22, -N(R22)(R23), and C3-6 cycloalkyl. In some embodiments, R7 is substituted with one, two, three, or four substituents independently selected from halogen, -CN, -CH3, -CH2CH3, -CH=CH2, -CF3, -C=CH, -OH, -NH2, and -cyclopropyl. In some embodiments, R7 is selected
[016] In some embodiments, for a compound of Formula (I-a):
X is C(R6);
A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl;
R2, R6, and R8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), -C(O)OR12, - N(R12)C(O)N(R12)(R13), -C(O)R12, -OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, - C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), -C(O)OR12, -N(R12)C(O)N(R12)(R13), -C(O)R12, -OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20; wherein two R3 are optionally taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one, two, or three R20; and further wherein two R3 are optionally taken together to form =O, =NR12, or =C(R14)2;
R7 is benzo [b]thiophen-4-yl optionally substituted with one, two, three, or four R20;
R9 is C1-3 alkyl optionally substituted with one, two, or three R20;
R10 is selected from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, 3- to 8- membered heterocycle, -OR22, -N(R22)(R23), -C(O)OR22, -N(R22)C(O)N(R22)(R23), -C(O)R22, -OC(O)R22, - C(O)N(R22)(R23), and -N(R22)C(O)R22, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8- membered heterocycle are optionally substituted with one, two, or three substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22,
-S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -
S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R11 is hydrogen; m is 0 or 1 ; and n is 1 or 2.
[017] In some embodiments, the compound of Formula (I) or (I-a) is a compound of Formula (I-b): or a pharmaceutically acceptable salt or solvate thereof.
[018] In some embodiments, the compound of Formula (I) or (I-a) is a compound of Formula (I-c): or a pharmaceutically acceptable salt or solvate thereof.
[019] In some embodiments, for a compound of Formula (I), (I-a), (I-b), or (I-c), R2 is selected from hydrogen,
C1-3 alkyl, -OR12, and 3- to 10-membered heterocycle, wherein C1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R20. In some embodiments, R2 is -OR12. In some embodiments, R2 is -
O(C1-3 alkyl)(4- to 10-membered heterocycle) optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and =C(R21)2, wherein R21 is independently selected at each occurrence from hydrogen, halogen, and C1-3 alkyl. In some embodiments, R2 is selected from
[020] In some embodiments, for a compound of Formula (I), (I-a), (I-b), or (I-c), R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20. In some embodiments, R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, - OH, and -OCH3. In some embodiments, m is 0 or 1, such as m is 0. In some embodiments, R6 and R8 are independently selected from hydrogen, halogen, and C1-3 haloalkyl. In some embodiments, R6 is selected from chlorine and -CF3. In some embodiments, R8 is fluorine. In some embodiments, n is 1.
[021] In certain aspects, the present disclosure provides a pharmaceutical composition comprising the conjugate described herein, or a salt thereof, and a pharmaceutically acceptable excipient.
[022] In certain aspects, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate described herein, or a salt thereof. In certain aspects, the present disclosure provides a method of treating cancer in a subject comprising a Ras mutant protein, the method comprising: inhibiting the Ras mutant protein of said subject by administering to said subject a conjugate described herein, or a salt thereof. The cancer of a method described herein may be a solid tumor or a hematological cancer. In some embodiments, the cancer comprises a wildtype K-Ras or a mutant K-Ras including but not limited to K-Ras G12C, G12D, G12S, or G12V mutant protein.
[023] In certain aspects, the present disclosure provides a method of modulating signaling output of a Ras protein, comprising contacting a Ras protein with an effective amount of a conjugate described herein, or a salt thereof, thereby modulating the signaling output of the Ras protein. In certain aspects, the present disclosure provides a method of inhibiting cell growth, comprising administering an effective amount of a conjugate described herein, or a salt thereof, to a cell expressing a Ras protein, thereby inhibiting growth of said cells. A method described herein may further comprise administering an additional agent.
[024] In certain aspects, the present disclosure provides a method of delivering a small-molecule KRAS inhibitor that exhibits low permeability as characterized by a PAMPA assay, comprising contacting a tumor cell with a conjugate described herein, or a salt thereof, wherein the KRAS inhibitor exhibits a PAMPA permeability (Pe) value less than 1 x 10-6 cm/s. In certain aspects, the present disclosure provides a method of enhancing therapeutic efficacy of a small-molecule KRAS inhibitor, comprising providing a conjugate described herein to a subject, wherein enhanced therapeutic efficacy is ascertained by the formula: TIconjugate/TlKRASi > 1 , wherein TIconjugate = TD50c/ED50c, wherein TD50c is the dose of conjugate required to produce a toxic effect in 50% of test subjects and ED50c is the dose of conjugate required to produce a therapeutic effect in 50% of test subjects; and wherein TIKRASI = TD50k/ED50k, wherein TD50k is the dose of KRAS inhibitor required to produce a toxic effect in 50% of test subjects and ED50k is the dose of KRAS inhibitor required to produce a therapeutic effect in 50% of the test subjects. In certain aspects, the present disclosure provides a method of reducing plasma concentration of a small-molecule KRAS inhibitor, comprising providing a conjugate described herein to a subject, wherein reduced plasma concentration is ascertained by the formula: [KRASi]p-c/[KRASi]p-k < 1 , wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at a first time -point following administration of the conjugate; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point. In certain aspects, the present disclosure provides a method of increasing concentration of a small-molecule KRAS inhibitor in tumor tissue, comprising providing a conjugate described herein to a subject, wherein increased tumor tissue concentration is ascertained by the formula: ([KRASi]t-c/[KRASi]p-c) / ([KRASi]t. k/[KRASi]p-k) > 1, wherein [KRASi]t-c is concentration of the KRAS inhibitor in tumor tissue at a first time-point following administration of the conjugate; wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at the same time-point following the administration of the conjugate; wherein [KRASi]t-k is concentration of the KRAS inhibitor in tumor tissue following administration of the KRAS inhibitor alone at an equivalent dose at the same time -point; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor at the same time -point following the administration of the KRAS inhibitor alone at the equivalent dose.
[025] In certain aspects, the present disclosure provides a method of delivering a small-molecule KRAS inhibitor to the central nervous system of a subject, comprising administering a conjugate described herein to the subject, wherein the KRAS inhibitor is released from the conjugate after entering the CNS of the subject. In certain aspects, the present disclosure provides a method of generating a slow-release form of a small-molecule KRAS inhibitor, the method comprising conjugating an antigen binding unit to a small-molecule KRAS inhibitor through a chemical linker, wherein the small-molecule inhibitor is released from the conjugate upon introducing the conjugate into a subject or a cell. In practicing any of the subject methods, efficacy of the conjugate may be greater than efficacy of a combination of the antigen binding unit and the KRAS inhibitor when each is administered at a comparable concentration. In some embodiments, toxicity of the conjugate is less than toxicity of a combination of the antigen binding unit and the KRAS inhibitor when each is administered at a comparable concentration.
INCORPORATION BY REFERENCE
[026] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[027] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[028] FIG. 1 depicts a sequence alignment of various wild type Ras proteins including K-Ras, H-Ras, N-Ras, RalA, and RalB, from top to bottom.
[029] FIG. 2 shows fluorescence of A-431 cells treated with cetuximab, isotype IgGl, cetuximab-MMAE ADC, Cet-KRAS A, Cet-KRAS B, isotype IgGl KRAS A conjugate, or isotype IgGl KRAS B conjugate, each of which is covalently attached to pH-sensitive pHAB dye. DETAILED DESCRIPTION
[030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs. In the event that there are a plurality of definitions for terms herein, those in this section prevail. All patents, patent applications, publications and published nucleotide and amino acid sequences (e.g., sequences available in GenBank or other databases) referred to herein are incorporated by reference. Chemical structures are named herein according to IUPAC conventions as implemented in ChemDraw® software (Perkin Elmer, Inc., Cambridge, MA). The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes”, and “included”, is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
[031] The term “Cx-y” or “Cx-Cy” when used in conjunction with a chemical moiety, such as alkyl, alkenyl, or alkynyl, is meant to include groups that contain from x to y carbons in the chain. For example, the term “Cx-y alkyl” refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched- chain alkyl groups, that contain from x to y carbons in the chain.
[032] “Alkyl” refers to substituted or unsubstituted saturated hydrocarbon groups, including linear and branched alkyl groups. An alkyl group may contain from one to twelve carbon atoms (e.g., C1-12 alkyl), such as one to eight carbon atoms (C1-8 alkyl) or one to six carbon atoms (C1-6 alkyl). Exemplary alkyl groups include methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl, and decyl. An alkyl group is attached to the rest of the molecule by a single bond. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more substituents such as those substituents described herein.
[033] “Haloalkyl” refers to an alkyl group that is substituted by one or more halogens. Exemplary haloalkyl groups include trifluoromethyl, difluoromethyl, tri chloromethyl, 2,2,2-trifluoroethyl, 1 ,2-difluoroethyl, 3-bromo-2- fluoropropyl, and 1 ,2-dibromoethyl.
[034] “Alkenyl” refers to substituted or unsubstituted hydrocarbon groups, including linear and branched alkenyl groups, containing at least one double bond. An alkenyl group may contain from two to twelve carbon atoms (e.g., C2-12 alkenyl), such as two to eight carbon atoms (C2-8 alkenyl) or two to six carbon atoms (C2-6 alkenyl). Exemplary alkenyl groups include ethenyl (i.e., vinyl), prop-l -enyl, but-l-enyl, pent-l-enyl, penta- 1,4-dienyl, and the like. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted by one or more substituents such as those substituents described herein.
[035] “Alkynyl” refers to substituted or unsubstituted hydrocarbon groups, including linear and branched alkynyl groups, containing at least one triple bond. An alkynyl group may contain from two to twelve carbon atoms (e.g., C2- 12 alkynyl), such as two to eight carbon atoms (C2-8 alkynyl) or two to six carbon atoms (C2-6 alkynyl). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless stated otherwise specifically in the specification, an alkynyl group is optionally substituted by one or more substituents such as those substituents described herein.
[036] “Alkylene” or “alkylene chain” refers to substituted or unsubstituted divalent saturated hydrocarbon groups, including linear alkylene and branched alkylene groups, that contain from one to twelve carbon atoms (e.g., C1-12 alkylene), such as one to eight carbon atoms (C1-8 alkylene) or one to six carbon atoms (C1-6 alkylene). Exemplary alkylene groups include methylene, ethylene, propylene, and n-butylene. Similarly, “alkenylene” and “alkynylene” refer to alkylene groups, as defined above, which comprise one or more carbon-carbon double or triple bonds, respectively. The points of attachment of the alkylene, alkenylene or alkynylene chain to the rest of the molecule can be through one carbon or any two carbons of the chain. Unless stated otherwise specifically in the specification, an alkylene, alkenylene, or alkynylene group is optionally substituted by one or more substituents such as those substituents described herein.
[037] “Heteroalkyl”, “heteroalkenyl” and “heteroalkynyl” refer to substituted or unsubstituted alkyl, alkenyl and alkynyl groups, respectively, in which one or more, such as 1, 2 or 3, of the carbon atoms are replaced with a heteroatom, such as O, N, P, Si, S, or combinations thereof. Any nitrogen, phosphorus, and sulfur heteroatoms present in the chain may optionally be oxidized, and any nitrogen heteroatoms may optionally be quatemized. If given, a numerical range refers to the chain length in total. For example, a 3- to 8-membered heteroalkyl group has a chain length of 3 to 8 atoms. Connection to the rest of the molecule may be through either a heteroatom or a carbon in the heteroalkyl, heteroalkenyl, or heteroalkynyl chain. Unless stated otherwise specifically in the specification, a heteroalkyl, hetero alkenyl, or heteroalkynyl group is optionally substituted by one or more substituents such as those substituents described herein.
[038] “Hetero alkylene”, “hetero alkenylene” and “heteroalkynylene” refer to substituted or unsubstituted alkylene, alkenylene and alkynylene groups, respectively, in which one or more, such as 1, 2 or 3, of the carbon atoms are replaced with a heteroatom, such as O, N, P, Si, S, or combinations thereof. Any nitrogen, phosphorus, and sulfur heteroatoms present in the chain may optionally be oxidized, and any nitrogen heteroatoms may optionally be quatemized. If given, a numerical range refers to the chain length in total. For example, a 3- to 8- membered hetero alkylene group has a chain length of 3 to 8 atoms. The points of attachment of the heteroalkylene, hetero alkenylene or heteroalkynylene chain to the rest of the molecule can be through either one heteroatom or one carbon, or any two heteroatoms, any two carbons, or any one heteroatom and any one carbon in the heteroalkylene, hetero alkenylene or heteroalkynylene chain. Unless stated otherwise specifically in the specification, a heteroalkylene, heteroalkenylene, or heteroalkynylene group is optionally substituted by one or more substituents such as those substituents described herein.
[039] “Carbocycle” refers to a saturated, unsaturated or aromatic ring in which each atom of the ring is a carbon atom. Carbocycle may include C3-10 monocyclic rings, C5-12 bicyclic rings, C5-18 polycyclic rings, C5-12 spirocyclic rings, and C5-12 bridged rings. Each ring of a bicyclic or polycyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. A polycyclic carbocycle contains a number or rings equal to the minimum number of scissions required to convert the carbocycle into an acyclic skeleton (e.g., bicyclic, tricyclic, tetracyclic, etc.). In some embodiments, the carbocycle is a C6-12 aryl group, such as C6-10 aryl. In some embodiments, the carbocycle is a C3-12 cycloalkyl group. In some embodiments, the carbocycle is a C5-12 cycloalkenyl group. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic rings, as valence permits, are included in the definition of carbocycle. A carbocycle may comprise a fused ring, a bridged ring, a spirocyclic ring, a saturated ring, an unsaturated ring, an aromatic ring, or any combination thereof. Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl, adamantly, phenyl, indanyl, and naphthyl. Unless state otherwise specifically in the specification, a carbocycle is optionally substituted by one or more substituents such as those substituents described herein.
[040] “Heterocycle” refers to a saturated, unsaturated or aromatic ring comprising one or more heteroatoms, for example 1, 2, 3, or 4 heteroatoms selected from O, S, P, and N. Heterocycle may include 3- to 10-membered monocyclic rings, 5- to 12-membered bicyclic rings, 5- to 18-membered polycyclic rings, 5- to 12-membered spirocyclic rings, and 5- to 12-membered bridged rings. Each ring of a bicyclic or polycyclic heterocycle may be selected from saturated, unsaturated, and aromatic rings. A polycyclic heterocycle contains a number or rings equal to the minimum number of scissions required to convert the heterocycle into an acyclic skeleton (e.g., bicyclic, tricyclic, tetracyclic, etc.). The heterocycle may be attached to the rest of the molecule through any atom of the heterocycle, valence permitting, such as a carbon or nitrogen atom of the heterocycle. In some embodiments, the heterocycle is a 5- to 10-membered heteroaryl group, such as 5- or 6-membered heteroaryl. In some embodiments, the heterocycle is a 3- to 12-membered heterocycloalkyl group. A heterocycle may comprise a fused ring, a bridged ring, a spirocyclic ring, a saturated ring, an unsaturated ring, an aromatic ring, or any combination thereof. In an exemplary embodiment, a heterocycle, e.g., pyridyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Exemplary heterocycles include pyrrolidinyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, piperidinyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, thiophenyl, oxazolyl, thiazolyl, morpholinyl, indazolyl, indolyl, benzothienyl, benzoxazolyl, and quinolinyl. Unless stated otherwise specifically in the specification, a heterocycle is optionally substituted by one or more substituents such as those substituents described herein.
[041] “Heteroaryl” refers to an aromatic ring that comprises at least one heteroatom, for example 1, 2, 3, or 4 heteroatoms selected from O, S and N. Heteroaryl may include 5- to 10-membered monocyclic rings, 6- to 12- membered bicyclic rings, 6- to 18-membered polycyclic rings, 5- to 12-membered spirocyclic rings, and 6- to 12- membered bridged rings. As used herein, the heteroaryl ring may be selected from monocyclic, bicyclic, or polycyclic — including fused, spirocyclic and bridged ring systems — wherein at least one of the rings in the ring system is aromatic and comprises at least one heteroatom. A polycyclic heteroaryl contains a number or rings equal to the minimum number of scissions required to convert the heteroaryl into an acyclic skeleton (e.g., bicyclic, tricyclic, tetracyclic, etc.). The heteroatom(s) in the heteroaryl may optionally be oxidized. One or more nitrogen atoms, if present, are optionally quatemized. The heteroaryl may be attached to the rest of the molecule through any atom of the heteroaryl, valence permitting, such as a carbon or nitrogen atom of the heteroaryl. Examples of heteroaryl groups include, but are not limited to, azepinyl, benzimidazolyl, benzisothiazolyl, benzisoxazolyl, benzofuranyl, benzothiazolyl, benzothiophenyl, benzoxazolyl, furanyl, imidazolyl, indazolyl, indolyl, isoquinolinyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyridazolyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydroquinolinyl, thiadiazolyl, thiazolyl, and thienyl groups. Unless stated otherwise specifically in the specification, a heteroaryl is optionally substituted by one or more substituents such as those substituents described herein.
[042] Unless stated otherwise, hydrogen atoms are implied in structures depicted herein as necessary to satisfy the valence requirement.
[043] A waved line drawn across or at the end of a bond or a dashed bond are used interchangeably herein to denote where a bond disconnection or attachment occurs. For example, in the structure if R7 is 2-fluoro-6-hydroxyphenyl as in then R7 may be depicted as
[044] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons or heteroatoms of the structure. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms such as nitrogen may have any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
[045] A compound disclosed herein, such as a compound of Formula (I), (I-a), (I-b), or (I-c), is optionally substituted by one or more — such as 1, 2 or 3 — substituents selected from: halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3. 12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), - N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), - C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -S(O)2R22, -S(O)(NR22)R22, -S(O)2N(R22)(R23)-, and - S(=O)(=NR22)N(R22)(R23); wherein two substituents attached to the same or adjacent atoms optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), - N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), - C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -S(O)2R22, -S(O)(NR22)R22, -S(O)2N(R22)(R23), and - S(=O)(=NR22)N(R22)(R23);
R21 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R21 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and -OH;
R22 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein -C0-6 alkyl-(C3-12 carbocycle) and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three groups independently selected from halogen and C1-6 alkyl; and
R23 is independently selected at each occurrence from hydrogen and C1-6 alkyl; or R22 and R23 attached to the same nitrogen atom form 3- to 10 membered heterocycle.
[046] In some embodiments, a compound disclosed herein, such as a compound of Formula (I), (I-a), (I-b), or (I- c), is optionally substituted by one or more — such as 1, 2 or 3 — substituents selected from: halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3. 12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), - N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -OC(O)R22, -C(O)N(R22)(R23), - C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -S(O)2R22, -S(O)(NR22)R22, and -S(O)2N(R22)(R23)-, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, and =C(R21)2;
R21 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, and C1-6 haloalkyl;
R22 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein -C0-6 alkyl-(C3-12 carbocycle) and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three groups independently selected from halogen and C1-6 alkyl;
R23 is independently selected at each occurrence from hydrogen and C1-6 alkyl; or R22 and R23 attached to the same nitrogen atom form 3- to 10 membered heterocycle.
[047] In some embodiments, a compound disclosed herein, such as a compound of Formula (I), (I-a), (I-b), or (I- c), is optionally substituted by one or more — such as 1 , 2 or 3 — substituents selected from halogen, oxo, =NH, -CN, -NO2, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocycle, -CH2-(C3-IO carbocycle), 3- to 10-membered heterocycle, -CH2-(3- to 10-membered heterocycle), -OH, -OCH3, -OCH2CH3, -NH2, -NHCH3, and -NHCH2CH3, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocycle, -CH2-(C3-IO carbocycle), 3- to 10-membered heterocycle, and -CH2-(3- to 10-membered heterocycle) are optionally substituted with one, two, or three groups independently selected from halogen, oxo, =NH, -CN, -NO2, -CH3, -CH2CH3, -CH(CH3)2, -C(CH3)3, -OH, -OCH3, - OCH2CH3, -NH2, -NHCH3, and -NHCH2CH3.
[048] It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as “unsubstituted”, references to chemical moieties herein are understood to include substituted variants. For example, reference to a “heteroaryl” group or moiety implicitly includes both substituted and unsubstituted variants.
[049] Where bivalent substituent groups are specified herein by their conventional chemical formulae, written from left to right, they are intended to encompass the isomer that would result from writing the structure from right to left, e.g., -CH2O- is also intended to encompass -OCH2-.
[050] “Optional” or “optionally” means that the subsequently described event or circumstances may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, an “optionally substituted” group may be either unsubstituted or substituted.
[051] Compounds of the present disclosure also include crystalline and amorphous forms of those compounds, pharmaceutically acceptable salts, and active metabolites having the same type of activity, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, amorphous forms of the compounds, and mixtures thereof.
[052] The compounds described herein may exhibit their natural isotopic abundance, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure. For example, hydrogen has three naturally occurring isotopes, denoted 1H (protium), 2H (deuterium), and 3H (tritium). Protium is the most abundant isotope of hydrogen in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increased in vivo half-life and/or exposure, or may provide a compound useful for investigating in vivo routes of drug elimination and metabolism. Examples of isotopes that may be incorporated into compounds of the present disclosure include, but are not limited to, 2H, 3H, 11C, 14C, 15N, 18O, 17O, 35S, 36Cl, and 18F. Of particular interest are compounds of Formula (I), (I-a), (I-b), or (I-c) enriched in tritium or carbon- 14, which can be used, for example, in tissue distribution studies; compounds of the disclosure enriched in deuterium — especially at a site of metabolism — resulting, for example, in compounds having greater metabolic stability; and compounds of Formula (I), (I-a), (I-b), or (I-c) enriched in a positron emitting isotope, such as 11C, 18F, 15O and 13N, which can be used, for example, in Positron Emission Topography (PET) studies. Isotopically-enriched compounds may be prepared by conventional techniques well known to those skilled in the art.
[053] As used herein, the phrase “of the formula”, “having the formula” or “having the structure” is not intended to be limiting and is used in the same way that the term “comprising” is commonly used. For example, if one structure is depicted, it is understood that all stereoisomer and tautomer forms are encompassed, unless stated otherwise.
[054] Certain compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, the asymmetric centers of which can be defined, in terms of absolute stereochemistry, as (R)- or (S)-. In some embodiments, in order to optimize the therapeutic activity of the compounds of the disclosure, e.g., to treat cancer, it may be desirable that the carbon atoms have a particular configuration (e.g., (R,R), (S,S), (S,R), or (R,S)) or are enriched in a stereoisomeric form having such configuration. The compounds of the disclosure may be provided as racemic mixtures. Accordingly, the disclosure relates to racemic mixtures, pure stereoisomers (e.g., enantiomers and diastereomers), stereoisomer-enriched mixtures, and the like, unless otherwise indicated. When a chemical structure is depicted herein without any stereochemistry, it is understood that all possible stereoisomers are encompassed by such structure. Similarly, when a particular stereoisomer is shown or named herein, it will be understood by those skilled in the art that minor amounts of other stereoisomers may be present in the compositions of the disclosure unless otherwise indicated, provided that the utility of the composition as a whole is not eliminated by the presence of such other isomers. Individual stereoisomers may be obtained by numerous methods that are known in the art, including preparation using chiral synthons or chiral reagents, resolution using chiral chromatography using a suitable chiral stationary phase or support, or by chemically converting them into diastereomers, separating the diastereoisomers by conventional means such as chromatography or recrystallization, then regenerating the original stereoisomer.
[055] Additionally, where applicable, all cis-trans or E/Z isomers (geometric isomers), tautomeric forms and topoisomeric forms of the compounds described herein are included with the scope of the disclosure unless otherwise specified. [056] The term “pharmaceutically acceptable” refers to a material that is not biologically or otherwise unacceptable when used in the subject compositions and methods. For example, the term “pharmaceutically acceptable carrier” refers to a material — such as an adjuvant, excipient, glidant, sweetening agent, diluent, preservative, dye, colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent or emulsifier — that can be incorporated into a composition and administered to a patient without causing unacceptable biological effects or interacting in an unacceptable manner with other components of the composition. Such pharmaceutically acceptable materials typically have met the required standards of toxicological and manufacturing testing, and include those materials identified as suitable inactive ingredients by the U.S. Food and Drug Administration.
[057] The terms “salt” and “pharmaceutically acceptable salt” refer to a salt prepared from a base or an acid. Pharmaceutically acceptable salts are suitable for administration to a patient, such as a mammal (for example, salts having acceptable mammalian safety for a given dosage regime). Salts can be formed from inorganic bases, organic bases, inorganic acids and organic acids. In addition, when a compound contains both a basic moiety, such as an amine, pyridine or imidazole, and an acidic moiety, such as a carboxylic acid or tetrazole, zwitterions may be formed and are included within the term “salt” as used herein. Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
[058] “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxy lie acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc., and include, for example, acetic acid, trifluoro acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S.M. et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Science, 66: 1-19 (1997)). Acid addition salts of basic compounds are, in some embodiments, prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.
[059] “Pharmaceutically acceptable base addition salt” refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts are, in some embodiments, formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, A,A-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, V-methy Iglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, V-ethylpiperidme. poly amine resins and the like. See Berge et al., supra.
[060] The term “effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. An effective amount of an active agent may be administered in a single dose or in multiple doses. A component may be described herein as having at least an effective amount, or at least an amount effective, such as that associated with a particular goal or purpose, such as any described herein. The term “effective amount” also applies to a dose that will provide an image for detection by an appropriate imaging method. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.
[061] As used herein, “treating” or “treatment” refers to an approach for obtaining beneficial or desired results with respect to a disease, disorder, or medical condition (such as cancer) in a subject, including but not limited to the following: (a) ameliorating the disease or medical condition, e.g., eliminating or causing regression of the disease or medical condition in a subject; (b) suppressing the disease or medical condition, e.g., slowing or arresting the development of the disease or medical condition in a subject; or (c) alleviating symptoms of the disease or medical condition in a subject. For example, “treating cancer” would include preventing cancer from reoccurring, ameliorating cancer, suppressing cancer, and alleviating the symptoms of cancer. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
[062] A “therapeutic effect”, as that term is used herein, encompasses a therapeutic benefit and/or prophylactic benefit as described above. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
[063] The terms “antagonist” and “inhibitor” are used interchangeably, and they refer to a compound having the ability to inhibit a biological function (e.g., activity, expression, binding, protein-protein interaction) of a target protein (e.g., K-Ras). Accordingly, the terms “antagonist” and “inhibitor” are defined in the context of the biological role of the target protein. While preferred antagonists herein specifically interact with (e.g., bind to) the target, compounds that inhibit a biological activity of the target protein by interacting with other members of the signal transduction pathway of which the target protein is a member are also specifically included within this definition. [064] The term “selective inhibition” or “selectively inhibit” refers to the ability of a biologically active agent to preferentially reduce the target signaling activity as compared to off-target signaling activity, via direct or indirect interaction with the target.
[065] The terms “subject” and “patient” refer to an animal, such as a mammal, for example a human. The methods described herein can be useful in both human therapeutics and veterinary applications. In some embodiments, the subject is a mammal, such as a human. “Mammal” includes humans and both domestic animals such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and nondomestic animals such as wildlife and the like.
[066] The terms “therapeutic agent”, “therapeutic capable agent” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
[067] The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
[068] The terms “polynucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or noncoding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs, such as peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), 2 ’-fluoro, 2’-OMe, and phosphorothiolated DNA. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component or other conjugation target.
[069] As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
[070] An “antigen” is a moiety or molecule that contains an epitope, and, as such, also specifically binds to an antibody. An “antigen binding unit” may be whole or a fragment (or fragments) of a full-length antibody, a structural variant thereof, a functional variant thereof, or a combination thereof. A full-length antibody may be, for example, a monoclonal, recombinant, chimeric, deimmunized, humanized and human antibody. Examples of a fragment of a full-length antibody may include, but are not limited to, variable heavy (VH), variable light (VL), a heavy chain found in camelids, such as camels, llamas, and alpacas (VHH or VHH), a heavy chain found in sharks (V-NAR domain), a single domain antibody (sdAb, e.g., “nanobody”) that comprises a single antigen-binding domain, Fv, Fd, Fab, Fab', F(ab')2, and “r IgG” (or half antibody). Examples of modified fragments of antibodies may include, but are not limited to scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, minibodies (e.g., (VH-VL- CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2), and multibodies (e.g., tnabodies or tetrabodies).
[071] The terms “antibody” and “antibodies” encompass any antigen binding units, including without limitation: monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, and any other epitope -binding fragments. Antibodies can come in different varieties known as isotypes or classes In humans there are five antibody classes known as IgA, IgD, IgE, IgG, and IgM, which are further subdivided into subclasses such as IgA1, IgA2.
[072] The term “in vivo” refers to an event that takes place in a subject’s body. The term “ex vivo” refers to an event that first takes place outside of the subject’s body for a subsequent in vivo application into a subject’s body. For example, an ex vivo preparation may involve preparation of cells outside of a subject’s body for the purpose of introduction of the prepared cells into the same or a different subject’s body. The term “in vitro” refers to an event that takes place outside of a subject’s body. For example, an in vitro assay encompasses any assay run outside of a subject’s body. In vitro assays encompass cell-based assays in which cells alive or dead are employed. In vitro assays also encompass a cell-free assay in which no intact cells are employed.
[073] The disclosure is also meant to encompass the in vivo metabolic products of the disclosed compounds. Such products may result from, for example, the oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the disclosure includes compounds produced by a process comprising administering a compound disclosed herein to a mammal for a period of time sufficient to yield a metabolic product thereof. Such products are typically identified by administering a radiolabeled compound of the disclosure in a detectable dose to an animal, such as rat, mouse, guinea pig, monkey, or to a human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood or other biological samples.
[074] The term “Ras” or “RAS” refers to a protein in the Rat sarcoma (Ras) superfamily of small GTPases, such as in the Ras subfamily. The Ras superfamily includes, but is not limited to, the Ras subfamily, Rho subfamily, Rab subfamily, Rap subfamily, Arf subfamily, Ran subfamily, Rheb subfamily, RGK subfamily, Rit subfamily, Miro subfamily, and Unclassified subfamily. In some embodiments, a Ras protein is selected from the group consisting of KRAS (also used interchangeably herein as K-Ras, K-ras, or Kras), HRAS (or H-Ras), NRAS (or N-Ras), MRAS (or M-Ras), ERAS (or E-Ras), RRAS2 (or R-Ras2), RALA (or RalA), RALB (or RalB), RIT1, and any combination thereof, such as from KRAS, HRAS, NRAS, RALA, RALB, and any combination thereof.
[075] The terms “mutant Ras” and “Ras mutant”, as used interchangeably herein, refer to a Ras protein with one or more amino acid mutations, such as with respect to a common reference sequence such as a wild-type (WT) sequence. In some embodiments, a mutant Ras is selected from a mutant KRAS, mutant HRAS, mutant NRAS, mutant MRAS, mutant ERAS, mutant RRAS2, mutant RALA, mutant RALB, mutant RIT 1 , and any combination thereof, such as from a mutant KRAS, mutant HRAS, mutant NRAS, mutant RALA, mutant RALB, and any combination thereof. In some embodiments, a mutation can be an introduced mutation, a naturally occurring mutation, or a non-naturally occurring mutation. In some embodiments, a mutation can be a substitution (e.g., a substituted amino acid), insertion (e.g., addition of one or more amino acids), or deletion (e.g., removal of one or more amino acids). In some embodiments, two or more mutations can be consecutive, non-consecutive, or a combination thereof. In some embodiments, a mutation can be present at any position of Ras. In some embodiments, a mutation can be present at position 12, 13, 62, 92, 95, 96 (e.g., Y96D), or any combination thereof of Ras relative to SEQ ID No. 1 when optimally aligned. In some embodiments, a mutant Ras may comprise about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more than 50 mutations. In some embodiments, a mutant Ras may comprise up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 mutations. In some embodiments, the mutant Ras is about or up to about 500, 400, 300, 250, 240, 233, 230, 220, 219, 210, 208, 206, 204, 200, 195, 190, 189, 188, 187, 186, 185, 180, 175, 174, 173, 172, 171, 170, 169, 168, 167, 166, 165, 160, 155, 150, 125, 100, 90, 80, 70, 60, 50, or fewer than 50 ammo acids in length. In some embodiments, an amino acid of a mutation is a proteinogenic, natural, standard, non-standard, non- canonical, essential, non-essential, or non-natural amino acid. In some embodiments, an amino acid of a mutation has a positively charged side chain, a negatively charged side chain, a polar uncharged side chain, a non-polar side chain, a hydrophobic side chain, a hydrophilic side chain, an aliphatic side chain, an aromatic side chain, a cyclic side chain, an acyclic side chain, a basic side chain, or an acidic side chain. In some embodiments, a mutation comprises a reactive moiety. In some embodiments, a substituted amino acid comprises a reactive moiety. In some embodiments, a mutant Ras can be further modified, such as by conjugation with a detectable label. In some embodiments, a mutant Ras is a full-length or truncated polypeptide. For example, a mutant Ras can be a truncated polypeptide comprising residues 1-169 or residues 11-183 (e.g., residues 11-183 of a mutant RALA or mutant RALB).
[076] As used herein, the term “corresponding to” or “corresponds to” as applied to an amino acid residue in a polypeptide sequence refers to the correspondence of such amino acid relative to a reference sequence when optimally aligned (e.g., taking into consideration of gaps, insertions and mismatches; wherein alignment may be primary sequence alignment or three-dimensional structural alignment of the folded proteins). For instance, the serine residue in a K-Ras G12S mutant refers to the serine corresponding to residue 12 of SEQ ID No. 4, which can serve as a reference sequence. For instance, the aspartate residue in a K-Ras G12D mutant refers to the aspartate corresponding to residue 12 of SEQ ID No. 2, which can serve as a reference sequence. When an amino acid of a mutant Ras protein corresponds to an amino acid position in the WT Ras protein, it will be understood that although the mutant Ras protein amino acid may be a different amino acid (e.g., G12D, wherein the wildtype G at position 12 is replaced by an aspartate at position 12 of SEQ ID. No. 1), the mutant amino acid is at the position corresponding to the wildtype amino acid (e.g., of SEQ ID No. 1). In embodiments, a modified Ras mutant protein disclosed herein may comprise truncations at the C-terminus, or truncations at the N-terminal end preceding the serine residue. The serine residue in such N-terminal truncated modified mutant is still considered corresponding to position 12 of SEQ ID No. 1. In addition, an aspartate residue at position 12 of SEQ ID No. 2 finds a corresponding residue in SEQ ID Nos. 6 and 8.
[077] The term “leaving group” is used herein in accordance with its well understood meaning in Chemistry and refers to an atom or group of atoms which breaks away from the rest of the molecule, taking with it the electron pair which used to be the bond between the leaving group and the rest of the molecule.
Conjugates
[078] Disclosed herein are conjugates that comprise an antigen binding unit and a KRAS inhibitor. A subject conjugate exhibits one or more advantageous properties as compared to the corresponding KRAS small molecule inhibitor in the conjugate. For example, a subject conjugate may possess intramolecular synergy mediated by the antigen binding unit targeting a first antigen and a KRAS inhibitor, both together synergistically reduce signaling output of the first antigen or KRAS. In another example, a subject conjugate possesses intramolecular synergy mediated by the antigen binding unit targeting a first antigen, and a KRAS inhibitor, both together synergistically reduce signaling output of the first antigen and KRAS. A conjugate may also improve cell permeability of a small molecule KRAS inhibitor by conjugating to an antibody or fragment thereof that is internalized into the cell upon specific binding to its antigen. A conjugate may also improve cell permeability of an antigen binding unit by conjugating to it a small molecule KRAS inhibitor. A conjugate may also improve tolerability or reduce toxicity of a small molecule KRAS inhibitor by conjugating the small molecule KRAS inhibitor to an antigen binding unit. A conjugate may also provide an enhanced overall therapeutic index via the intramolecular synergy, improved cell permeability of the KRAS inhibitor, reduced off-target toxicity mediated by the KRAS inhibitor, and/or enhance pharmacokinetics profile of the KRAS inhibitor when conjugated to an antigen binding unit. One or more desired advantageous properties of a subject conjugate may be greater than (i) the effect of the KRAS inhibitor alone, (ii) the effect of the antigen binding unit alone, and/or (iii) the sum of the effect the KRAS inhibitor and the antigen binding unit when administered individually (e.g., the sum of the individual effects). The effect of a subject conjugate can be at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1,000%, 5,000%, or more than (i) the effect of the KRAS inhibitor alone, (ii) the effect of the antigen binding unit alone, and/or (iii) the sum of individual effects. The effect can be any measurable effect including but not limited to an enhancement of a therapeutic effect of an individual component within the conjugate (e.g., the KRAS inhibitor or the antigen binding unit) or a reduction in a side effect of an individual component within the conjugate. The present disclosure encompasses conjugates that comprise an antigen binding unit and a KRAS inhibitor (e.g., a smallmolecule KRAS inhibitor), wherein the antigen binding unit targets a first antigen, and the KRAS inhibitor reduces signaling output of the KRAS protein. Where desired, the conjugation of the antigen binding unit and the KRAS inhibitor is constructed to be reversible or cleavable such that the KRAS inhibitor is released from the antigen binding unit inside a cell, following, e.g., internalization of the antigen binding unit by the cell.
[079] In some aspects, the antigen binding unit may be conjugated either directly or through a linker to the KRAS inhibitor to form a conjugate. Preferably, the antigen binding unit is conjugated to the KRAS inhibitor, optionally through a linker, by one or more covalent bonds. In some embodiments, the conjugate may comprise at least two molecules of the KRAS inhibitor per antigen binding unit, such as 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 8, 3 to 6, or 3 to 4 molecules of the KRAS inhibitor per antigen binding unit, as a multi-payload ADC-conjugate.
[080] In certain aspects, the present disclosure provides a conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker, and wherein the antigen binding unit and the KRAS inhibitor in the conjugate synergistically inhibits signaling output of the first antigen or KRAS. In certain aspects, the present disclosure provides a conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker, and wherein the antigen binding unit and the KRAS inhibitor in the conjugate synergistically inhibits signaling output of the first antigen and KRAS. In some aspects, the present disclosure provides a conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker. The antigen binding unit may be conjugated either directly or through a linker to the KRAS inhibitor to form a conjugate. Preferably, the antigen binding unit is conjugated to the KRAS inhibitor by one or more covalent bonds. The conjugate may comprise at least two molecules of the KRAS inhibitor per antigen binding unit, such as 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 8, 3 to 6, or 3 to 4 molecules of the KRAS inhibitor per antigen binding unit.
[081] A small-molecule KRAS inhibitor of the present disclosure typically has a molecular weight of less than 1000 daltons, such as less than 950 Da, less than 900 Da, less than 850 Da, less than 800 Da, less than 790 Da, less than 780 Da, less than 770 Da, less than 760 Da, less than 750 Da, less than 740 Da, less than 730 Da, less than 720 Da, less than 710 Da, less than 700 Da, less than 690 Da, less than 680 Da, less than 670 Da, less than 660 Da, less than 650 Da, less than 600 Da, less than 550 Da, less than 500 Da, less than 450 Da, less than 400 Da, less than 350 Da, less than 300 Da, less than 250 Da, or less than 200 Da. In some embodiments, the small molecule has a molecular weight of 100 to 1000 Da, such as 100 to 900 Da, 100 to 850 Da, 100 to 800 Da, 100 to 750 Da, 100 to 700 Da, 100 to 650 Da, 100 to 600 Da, 100 to 550 Da, 100 to 500 Da, 150 to 900 Da, 150 to 850 Da, 150 to 800 Da,
150 to 750 Da, 150 to 700 Da, 150 to 650 Da, 150 to 600 Da, 150 to 550 Da, 150 to 500 Da, 200 to 900 Da, 200 to
850 Da, 200 to 800 Da, 200 to 750 Da, 200 to 700 Da, 200 to 650 Da, 200 to 600 Da, 200 to 550 Da, 200 to 500 Da,
250 to 900 Da, 250 to 850 Da, 250 to 800 Da, 250 to 790 Da, 250 to 780 Da, 250 to 770 Da, 250 to 760 Da, 250 to
750 Da, 250 to 740 Da, 250 to 730 Da, 250 to 720 Da, 250 to 710 Da, 250 to 700 Da, 250 to 690 Da, 250 to 680 Da,
250 to 670 Da, 250 to 660 Da, 250 to 650 Da, 250 to 600 Da, 250 to 550 Da, or 250 to 500 Da. In some embodiments, a small-molecule KRAS inhibitor has a molecular weight in the range of about 710 Da to about 750 Da. In some embodiments, a small-molecule KRAS inhibitor has a molecular weight in the range of about 550 Da to about 710 Da. In some embodiments, a small-molecule KRAS inhibitor has a molecular weight in the range of about 600 Da to about 700 Da. In some embodiments, a small -molecule KRAS inhibitor has a molecular weight in the range of about 600 Da to about 705 Da. In some embodiments, a small-molecule KRAS inhibitor has a molecular weight in the range of about 600 Da to about 710 Da. In some embodiments, a small -molecule KRAS inhibitor has a molecular weight in the range of about 600 Da to about 715 Da. In some embodiments, a small -molecule KRAS inhibitor has a molecular weight in the range of about 600 Da to about 720 Da. In some embodiments, a smallmolecule KRAS inhibitor has a molecular weight in the range of about 600 Da to about 725 Da. In some embodiments, a small-molecule KRAS inhibitor has a molecular weight in the range of about 600 Da to about 750 Da. In some embodiments, a small-molecule KRAS inhibitor has a molecular weight in the range of about 600 Da to about 800 Da.
[082] In some embodiments, the KRAS inhibitor is characterized by a PAMPA permeability (Pe) less than 1 x 10- 6 cm/s, such as less than 9 x 10-7, 8 x 10-7, 7 x 10-7, 6 x 10-7, 5 x 10-7, 4 x 10-7, 3 x 10-7, 2 x 10-7, 1 x 10-7, 1 x 10-8, or 1 x 10-9 cm/s. In some embodiments, the KRAS inhibitor is characterized by a PAMPA permeability (Pe) greater than 1 x 10-6 cm/s, such as greater than 2 x 10-6, 3 x 10-6, 4 x 10-6, 5 x 10-6, 6 x 10-6, 7 x 10-6, 8 x 10-6, 9 x 10-6, 1 x 10-5, 1 x 10-4, or 1 x 10-3 cm/s.
[083] In some embodiments, the conjugate is characterized by an enhanced therapeutic efficacy. Enhanced therapeutic efficacy may be ascertained by the formula: wherein TIconjugate = TD50c/ED50c, wherein TD50c is the dose of conjugate required to produce a toxic effect in 50% of test subjects and ED50c is the dose of conjugate required to produce a therapeutic effect in 50% of test subjecLs: and wherein TIKRASI = TD50k/ED50k, wherein TD50k is the dose of KRAS inhibitor required to produce a toxic effect in 50% of test subjects and ED50k is the dose of KRAS inhibitor required to produce a therapeutic effect in 50% of the test subjects.
In some embodiments, TIconjugate/TlKRASi is greater than 1.1, such as greater than 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50. In some embodiments, TIconjugate/TlKRASi is greater than 2. In some embodiments, TIconjugate/TlKRASi is greater than 5. In some embodiments, TIconjugate/TlKRASi is between about 2 to about 5. In some embodiments, TIconjugate/TlKRASi is about 5 to about 10. In some embodiments, TIconjugate/TlKRASi is greater than 5.
[084] In some embodiments, the conjugate is characterized by reduced plasma concentration of the KRAS inhibitor. Reduced plasma concentration may be ascertained by the formula:
[KRASi]p-c/[KRASi]p-k < 1 wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at a first time -point following administration of the conjugate; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point.
In some embodiments, [KRASi]p-c/[KRASi]p-k is less than 0.95, such as less than 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, or 0.001. In some embodiments, [KRASi]p-c/[KRASi]p-k is less than 0.5. In some embodiments, [KRASi]p-c/[KRASi]p-k is less than 0.1. In some embodiments, the first time-point is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 48 hours after the administration. As used herein, the term “equivalent dose” refers to an amount of KRAS inhibitor that is approximately the same as the amount of KRAS inhibitor administered as a conjugate in a comparative study. For example, a conjugate comprising two molecules of the KRAS inhibitor per antigen binding unit administered at dose x would typically be compared to a dose of l/2x of free KRAS inhibitor (the equivalent dose).
[085] In some embodiments, the conjugate is characterized by an increased concentration of the KRAS inhibitor in tumor tissue relative to plasma as ascertained by the formula:
([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) > 1 wherein [KRASi]t-c is concentration of the KRAS inhibitor in tumor tissue at a first time -point following administration of the conjugate; wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at the same time -point following the administration of the conjugate; wherein [KRASi]t-k is concentration of the KRAS inhibitor in tumor tissue following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor at the same time-point following the administration of the KRAS inhibitor alone at the equivalent dose.
In some embodiments, ([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) is greater than 1.1, such as greater than 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50. In some embodiments, ([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) is greater than 2. In some embodiments, ([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) is greater than 5. In some embodiments, the first time-point is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 48 hours after the administration.
[086] In some embodiments, the conjugate inhibits signaling output of the first antigen and KRAS in a cell that does not overexpress the first antigen relative to a control cell (e.g., a cancer cell that does not overexpress the first antigen relative to a control cell that is non-cancerous), optionally wherein the first antigen is a tumor antigen. For example, the cell does not overexpress the first antigen, wherein the first antigen is selected from AG7, B7-H3, BCMA, CAI 5-3, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD70, CD71, CD79B, CEA, CLDN18.2, EGFR, F0LR1, GCC, GPC1 , HER2, HER3, ICAM1, LeX, LeY, MET, MSLN, MUCl, NECTIN4, SLC44A4, TF, and Trop-2. In some embodiments, the first antigen is EGFR. In some embodiments, the conjugate inhibits signaling output of the first antigen and KRAS in a cell that overexpress the first antigen relative to a control cell (e.g., a cancer cell that does not overexpress the first antigen relative to a control cell that is non-cancerous), optionally wherein the first antigen is a tumor antigen. For example, a cell of interest overexpress the first antigen, wherein the first antigen is selected from AG7, B7-H3, BCMA, CA15-3, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD70, CD71, CD79B, CEA, CLDN18.2, EGFR, FOLR1, GCC, GPC1, HER2, HER3, ICAM1, LeX, LeY, MET, MSLN, MUCl, NECTIN4, SLC44A4, TF, and Trop-2. In some embodiments, the overexpressed first antigen is EGFR.
[087] In certain aspects, the present disclosure provides a conjugate of Formula (A): wherein:
AgB is an antigen binding unit;
L is a chemical linker;
D is independently selected at each occurrence from a small-molecule KRAS inhibitor, a cytotoxic small-molecule and a small-molecule agent that selectively modulates a non-KRAS target, wherein at least one D is a KRAS inhibitor; p is selected from 1 to 20; and q is selected from 1 to 20. [088] In some embodiments, p is selected from 1 to 10, such as 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 8, 3 to 6, or 3 to 5. In some embodiments, p is 2, 3, 4, 5 or 6, such as p is 2, 3, or 4. In some embodiments, p is about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.
[089] In some embodiments, q is selected from 1 to 10, such as 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 8, 3 to 6, or 3 to 5. In some embodiments, q is 1, 2, 3, 4, 5 or 6, such as q is 1 or 2. In some embodiments, q is 1. In some embodiments, p is 1 to 8 and q is 1 to 5. In some embodiments, p is 1 to 8 and q is 1. In some embodiments, p is 2 to 5 and q is 1.
[090] In some embodiments, D is a small-molecule KRAS inhibitor, such as a KRAS inhibitor described herein. [091] In some embodiments, the conjugate of Formula (A) is selected from:
[092] In some embodiments, the conjugate of Formula (A) is selected from:
[093] In some embodiments, the conjugate of Formula (A) is selected from:
wherein X, Z, A, R2, R3, R4, R6, R7, R8, R9, R10, R11, R22, n, and m are defined in the KRAS Inhibitors section below, for example, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), or (I-g).
Antigen Binding Unit
[094] An antigen binding unit of a conjugate can contain one or more antigen binding domains (also referred to as binding domains). In some embodiments, an antigen binding unit has a first binding domain. In some embodiments, an antigen binding unit has a first and a second binding domain that bind to the same antigen. In some embodiments, an antigen binding unit has a first and a second binding domain that bind to different antigens. In some embodiments, an antigen binding unit has a first and second binding domain, and optionally a third or more binding domain.
[095] A binding domain typically recognizes a single antigen. An antigen binding unit of a conjugate can have binding domains that can recognize, for example, two, three, four, five, six, seven, eight, nine, ten, or more antigens. An antigen binding unit can comprise two binding domains in which each binding domain can recognize the same antigen. An antigen binding unit can comprise two binding domains in which each binding domain can recognize a different antigen. An antigen binding unit can comprise three binding domains in which each binding domain can recognize a different antigen. An antigen binding unit can comprise three binding domains in which two of the binding domains can recognize the same antigen and the third binding domain recognizes a different antigen. In some embodiments, an antigen binding unit is bivalent and mono-specific (i.e., having two binding domains that specifically bind to the same antigen). In embodiments in which an antigen binding unit is trivalent or greater, the antigen binding unit is typically bi-specific or greater. Antigen binding units having a third binding domain attached to the C-terminal end of a light chain and/or the C-terminal end of an Fc domain can be bi-specific, tri-specific or multi-specific.
[096] In some embodiments, an antigen binding unit can be a fusion protein, such as an Fc fusion protein, having a first binding domain and optionally a second binding domain. In some embodiments, two antigen binding domains and an Fc domain can be expressed as a fusion protein, optionally formed by expression of separate polypeptide chains.
[097] A binding domain can specifically bind to an antigen on a cell surface or to a fragment thereof. A binding domain can specifically bind an antigen on a cell surface, for example, a tumor antigen on a tumor cell, on an antigen presenting cell such as a dendritic cell or macrophage, or on another immune cell such as a T cell. In some embodiments, a binding domain can specifically bind to an antigen on a cell surface of a tumor cell or an antigen presenting cell (such as a dendritic cell or macrophage), but not on other immune cells such as T cells. In some embodiments, a binding domain can specifically bind to a tumor antigen. In some embodiments, a binding domain can specifically bind to an antigen on an antigen presenting cell. In some embodiments, a binding domain can be a cell surface receptor agonist, such as an agonistic antibody.
[098] A binding domain can be an antigen-binding portion of an antibody (an antigen binding domain) or an antigen binding antibody fragment. A binding domain can be one or more fragments of an antibody that can retain the ability to specifically bind to an antigen. A binding domain can be in a scaffold, in which a scaffold is a supporting framework for the binding domain. A binding domain, such as an antigen binding fragment of an antibody, can be in an antibody scaffold or antibody-like scaffold. A binding domain can be in a non-antibody scaffold.
[099] Antigen binding units can comprise a binding domain(s) that can specifically bind to a tumor antigen. A tumor antigen can be a tumor specific antigen and/or a tumor associated antigen. As described herein, a “tumor antigen” refers to a molecular marker that can be expressed on a neoplastic tumor cell and/or within a tumor microenvironment. The molecular marker can be a cell surface receptor. For example, a tumor antigen can be an antigen expressed on a cell associated with a tumor, such as a neoplastic cell, stromal cell, endothelial cell, fibroblast, or tumor-infiltrating immune cell. For example, the tumor antigen EGFR can be overexpressed by certain types of head and neck cancer. A tumor antigen can also be ectopically expressed by a tumor and contribute to deregulation of the cell cycle, reduced apoptosis, metastasis, and/or escape from immune surveillance. Tumor antigens are generally proteins or polypeptides derived therefrom, but can be glycans, lipids, or other small organic molecules. Additionally, a tumor antigen can arise through increases or decreases in post-translational processing exhibited by a cancer cell compared to a normal cell, for example, protein glycosylation, protein lipidation, protein phosphorylation, or protein acetylation.
[100] A binding domain of an antigen binding unit can bind to tumor cells, such as an antibody against a cell surface receptor or a tumor antigen. In certain embodiments, a binding domain can specifically bind to a tumor antigen, such as including but not limited to, AG7, CD5, CD19, CD20, CD22, CD25, CD37, CD30, CD33, CD38, CD45, CD52, CD70, CD71, CD79B, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC (PD-L2), HLA-DR, carcinoembryonic antigen (CEA), TAG-72, MUC1, MUC15, MUC16, folate-binding protein (FOLR1), A33, G250, prostate-specific membrane antigen (PSMA), GCC, GD2, GD3, GM2, ICAM1, LeX, LeY, sLe, sLe(a), polysialic acid, fucosyl GM1, GM3, BM3, GloboH, CA15-3, CA-125, CA19-9, epidermal growth factor, HER2, IL -2 receptor, EGFRvIII (de2-7 EGFR), EGFR, fibroblast activation protein (FAP), a tenascin, a metalloproteinase, endosialin, vascular endothelial growth factor, avp3, WT1, LMP2, HPV E6, HPV E7, p53 nonmutant, NY-ESO-1, GLP-3, MelanA/MARTl, Ras mutant, gplOO, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, STn, STN1, TNC, a Sarcoma translocation breakpoint fusion protein, EphA2, EphB2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, mesothehn (MSLN), PSCA, MAGE-A1, MAGE-A3, MET, CYP1B1, PLAV1, BORIS, Tn, TF, CSPG4, ETV6-AML, NY-BR-1, RGS5, SART3, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, MAGE-C2, MAGE-A4, GAGE, TRAIL 1, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumam, Tie 2, Tim 3, PAGE4, VEGFR2, MAD-CT- 1, PDGFR-B, MAD-CT-2, ROR2, CMET, HER3, EpCAM, CA6, NAPI2B, TROP2, Claudm-6 (CLDN6), Claudm-16 (CLDN16), CLDN18.2, RON, LY6E, LY6K, FRA, DLL3, PTK7, Uroplakm-IB (UPK1B), UPK2, LIV1, ROR1, GPC1, GPC3, ADAM12, LRRC15, CDH6, CDH3, TMEFF2, GPNMB, ALPPL2, LAMP-1, STEAP, ENPP3, Nectm4, LYPD3, GPA33, EFNA4, SLITRK6, HAVCR1, SLC44A4, STRA6, TMPRSS3, TMPRSS4, TMEM238, Clorfl86, Fos-related antigen 1, VEGFR1, endoglin, VTCN1 (B7-H4), VISTA, or a fragment thereof. In some embodiments, a binding domain specifically binds a target, such target may include but is not limited to, AG7, B7-H3, BCMA, CA15-3, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD70, CD71, CD79B, CEA, CLDN18.2, EGFR, FOLR1, GCC, GPC1, HER2, HER3, ICAM1, LeX, LeY, MET, MSLN, MUC1, NECTIN4, SLC44A4, TF, or Trop-2. In some embodiments, a binding domain specifically binds EGFR.
[101] EGFR (epidermal growth factor receptor) is a receptor tyrosine kinase that mediates normal cell growth and development. Phosphorylation of EGFR activates downstream signaling pathways, including the PI3K/AKT/mTOR, STAT, and RAS/MAPK pathways, that involve cell proliferation, angiogenesis, apoptosis, and metastasis. Overexpression of EGFR is frequently observed in a variety of tumors, including brain, breast, cervical, colorectal, esophageal, head and neck, kidney, lung, ovarian, and stomach cancers. EGFR mutations that result in constitutive activation of EGFR and downstream signaling pathways are associated with a number of cancers. Examples of antibodies that can target and inhibit EGFR include cetuximab, panitumumab, nimotuzumab, zalutumumab, matuzumab, and amivantamab. In some embodiments, the antigen binding unit is an anti-EGFR antibody, such as cetuximab.
[102] A binding domain of an antigen binding unit can be selected from any domain that specifically binds to an antigen, including a binding domain of an antibody or a non-antibody binding domain. A binding domain of an antibody can be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, or an antigen binding fragment thereof, for example, a heavy chain variable domain (VH) and a light chain variable domain (VL). A binding domain of a non-antibody scaffold can be a lipocalin, an anticalin, ‘T-body ’, a peptide (e.g., a Bicycle™ peptide), an affibody, a peptibody, a DARPin, an affimer, an avimer, a knottin, a monobody, an affinity clamp, an ectodomain, a receptor ectodomain, a receptor, a cytokine, a ligand, an immunocytokine, a centryin, a T-cell receptor, or a recombinant T-cell receptor. In some embodiments, a binding domain of a non-antibody scaffold can be a lipocalin, an anticalin, ‘T- body’, an affibody, a peptide (e.g., a Bicycle™ peptide), a peptibody, a DARPin, an affimer, an avimer, a knottin, a monobody, a centryin or an affinity clamp.
[103] In some embodiments, a binding domain of an antigen binding unit is an antigen binding domain from a monoclonal antibody and can comprise a light chain and a heavy chain. In an embodiment, the monoclonal antibody binds to a tumor antigen and comprises the light chain of a tumor antigen antibody and the heavy chain of a tumor antigen antibody, which specifically bind to the tumor antigen. In another embodiment, the monoclonal antibody binds to an antigen present on the surface of an immune cell (immune cell antigen) and comprises the light chain of an anti-immune cell antigen antibody and the heavy chain of an anti-immune cell antigen antibody, which specifically bind to an immune cell antigen. In another embodiment, the monoclonal antibody specifically binds to an antigen present on the surface of an antigen presenting cell (APC antigen) and comprises the light chain of an anti-APC antigen antibody and the heavy chain of an anti-APC antigen antibody, which bind an APC antigen.
[104] In some embodiments, an antigen binding unit can comprise an antibody, such as a bivalent, mono-specific antibody. An antibody can consist of two identical light protein chains (light chains) and two identical heavy protein chains (heavy chains), all held together covalently by interchain disulfide linkages. The N-terminal regions of the light and heavy chains together can form the antigen recognition site of the antibody. Structurally, various functions of an antibody can be confined to discrete protein domains or regions. The portions that can recognize and can specifically bind to an antigen consist of three complementarity determining regions (CDRs) that he within the variable heavy chain regions and variable light chain regions at the N-terminal ends of the heavy and light chains. The constant domains provide the general framework of the antibody and may not be involved directly in binding the antibody to an antigen, but can be involved in various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).
[105] In some embodiments, an antigen binding unit comprises an antigen binding domain of an antibody that includes the light chain (LC) CDRs (LCDRs) and heavy chain (HC) CDRs (HCDRs) of the antibody. For example, an antigen binding domain of an antibody can comprise one or more of the following: a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2), or a light chain complementary determining region 3 (LCDR3). As another example, an antibody binding domain can comprise one or more of the following: a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), or a heavy chain complementary determining region 3 (HCDR3). In some embodiments an antigen binding domain comprises all of the following: LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3. Unless stated otherwise, the CDRs described herein are defined according to the IMGT (the international ImMunoGeneTics information system) or the Kabat numbering system. In some embodiments, an antigen binding domain can comprise only the heavy chain of an antibody (e.g., does not include the light chain(s)). In some embodiments, an antigen binding domain can comprise only the light chain of an antibody (e.g., does not include the heavy chain(s)). In some embodiments, an antigen binding domain can comprise only the variable region of the heavy chain of an antibody. In some embodiments, an antigen binding domain can comprise only the variable region of the light chain of an antibody.
[106] An antigen binding unit can also comprise any antigen binding fragment of an antibody or recombinant form thereof, including but not limited to an scFv, Fab, Fab’, Fab2, variable Fv fragment (Fv), domain antibody, and any other fragment thereof that can specifically bind to an antigen.
[107] An antibody used herein can be chimeric or “humanized”. Humanized forms of non-human (e.g., murine) antibodies can be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2, scFv, variable Fv fragment (Fv), domain antibody or other antigen-binding subdomains of antibodies), that contain minimal sequences derived from non-human immunoglobulin (e.g., the CDRs). In general, a humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions (FR) are those of a human immunoglobulin sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.
[108] An antibody also can be a human antibody. As used herein, “human antibodies” include antibodies having, for example, the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins that do not express endogenous immunoglobulins. Human antibodies can be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. Completely human antibodies that recognize a selected epitope can be generated using guided selection. In this approach, a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.
[109] An antibody described herein can be a derivatized antibody. For example, derivatized antibodies can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and the like. An antibody can also be modified, such as by defucosylation or deglycosylation.
[HO] An antibody can be a bispecific antibody or a dual variable domain antibody (DVD). Bispecific and DVD antibodies are monoclonal, often human or humanized, antibodies that have binding specificities for at least two different antigens (e.g., bi-specific). An antigen binding unit described herein may comprise a first binding domain and a second binding domain or a third binding domain that specifically binds to a different antigen than the first binding domain. An antigen binding unit may comprise a second binding domain or a third binding domain that specifically binds to a tumor antigen or an immune cell.
[Ill] Conjugates comprising antigen binding units described herein may have a dissociation constant (Kd) that is less than 10 nM for the target of the binding domain. A conjugate comprising a bispecific antigen binding unit may have a dissociation constant (Kd) that is less than 10 nM for the target of the second binding domain. The conjugates or antigen binding units may have a dissociation constant (Kd) that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0. 1 pM.
Linkers [112] The conjugates described herein optionally include a linker or linkers that attach at least one KRAS inhibitor to an antigen binding unit. The linker may be a cleavable linker, such as a peptide linker, or a non- cleavable linker. A conjugate may comprise multiple linkers. These linkers can be the same linker or different linkers. In some embodiments, more than one payload, such as a KRAS inhibitor, is attached to a single linker. The payload may be the same or different.
[113] Typically, linkers of the present disclosure attach a KRAS inhibitor(s) to an antigen binding unit by forming a covalent linkage to the KRAS inhibitor at one location and a covalent linkage to the antigen binding unit of the conjugate at another. Covalent linkages can be formed by reaction between functional groups on the linker and functional groups on the KRAS inhibitor and antigen binding unit of the conjugate. As used herein, the expression “linker” includes linker presented in different context such as (i) unconjugated forms of the linker that comprise a functional group capable of forming a covalent bond with an antigen binding unit; (ii) partially conjugated forms of the linker covalently attached to a KRAS inhibitor, wherein the linker comprises a functional group capable of forming a covalent bond with an antigen binding unit; (iii) partially conjugated forms of the linker covalently attached to an antigen binding unit, wherein the linker comprises a functional group capable of forming a covalent bond with a KRAS inhibitor for generating a conjugate; and (iv) fully conjugated forms of the linker covalently attached to both a KRAS inhibitor and an antigen binding unit.
[114] In some embodiments, the present disclosure provides a conjugate formed by contacting an antigen binding unit with a linker attached to a KRAS inhibitor under conditions in which the linker covalently binds to the antigen binding unit. In some embodiments, the present disclosure provides a method of making a conjugate, comprising contacting an antigen binding unit with a linker attached to a KRAS inhibitor under conditions in which the linker covalently binds to the antigen binding unit, thereby forming the conjugate. In some embodiments, the linker comprises an electrophilic group that is capable of reacting with a nucleophilic group present on the antigen binding unit to form a covalent bond between the linker and the antigen binding unit. In some embodiments, the electrophilic group reacts with a sulfhydryl, hydroxyl, or amino functional group of the antigen binding unit. Suitable electrophilic groups include maleimides, haloacetamides, and NHS esters. In some embodiments, the linker comprises a nucleophilic group that is capable of reacting with an electrophilic group present in the antigen binding unit to form a covalent bond between the linker and the antigen binding unit. In some embodiments, the electrophilic group reacts with a hydrazide, hydroxylamine, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, or arylhydrazide functional group of the linker.
[115] As further described herein, a linker can be short, long, flexible, rigid, hydrophilic, or hydrophobic. A linker may contain segments having different characteristics, such as segments that are flexible or rigid. In some embodiments, a linker is chemically stable in extracellular environments, such as in the blood stream. In some embodiments, a linker or fragment thereof is not stable, such as a linker that undergoes cleavage and/or immolation or otherwise breaks down under certain physiological conditions, such as conditions found inside a tumor cell. In some embodiments, a linker is cleavable by one or more enzymes, such as a protease. A cleavable linker may be cleaved in vitro or in vivo. Cleavable linkers can include chemically or enzymatically unstable or degradable linkages. In some embodiments, cleavable linkers rely on processes inside the cell to liberate a KRAS inhibitor, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by specific proteases or other enzymes within the cell. Cleavable linkers can incorporate one or more chemical bonds that are chemically or enzymatically cleavable, while the remainder of the linker may be non-cleavable.
[116] A linker may contain a chemically labile group such as a hydrazone or disulfide group. Linkers comprising chemically labile groups can exploit differential properties between the plasma and some cytoplasmic compartments. The intracellular conditions that can facilitate KRAS inhibitor release for hydrazine-containing linkers can be the acidic environment of endosomes and lysosomes, while disulfide-containing linkers can be reduced in the cytosol, which can contain high thiol concentrations (e.g., glutathione). The plasma stability of a linker containing a chemically labile group can be increased by introducing steric hindrance using substituents near the chemically labile group.
[117] Acid-labile groups, such as hydrazones, can remain intact during systemic circulation in the neutral pH of the blood (pH 7.3 -7.5) and can undergo hydrolysis and release a KRAS inhibitor once the conjugate is internalized into mildly acidic endosomal (pH 5.0-6.5) or lysosomal (pH 4.5-5.0) compartments of the cell. This pH dependent release mechanism can be associated with nonspecific release of the KRAS inhibitor. To increase the stability of the hydrazone group of the linker, the linker may be varied by chemical modification, e.g., substitution, allowing tuning to achieve more efficient release in the lysosome with minimal loss in circulation.
[118] Hydrazone-containing linkers can contain additional cleavage sites, such as additional acid-labile cleavage sites and/or enzymatically labile cleavage sites. Conjugates including hydrazone-containing linkers may include a
[119] In some embodiments, the linker is cleavable by a protease that is typically present in the disease microenvironment. The linker may contain a protease cleavage site for a protease that is overexpressed in the disease microenvironment. Cleavage of the protease cleavage site allows for the selective release of the KRAS inhibitor in the disease microenvironment while sparing normal cells or tissue from the KRAS inhibitor. In some embodiments, the linker contains a protease cleavage site for a protease selected from legumain, plasmin, TMPRSS3, TMPRSS4, TMPRSS6, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, MT1-MMP, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase- 12, Caspase-13, Caspase-14, TACE, a serine protease, a cysteine protease, human neutrophil elastase, beta- secretase, uPA, fibroblast associated protein, matriptase, PSMA, and PSA.
[120] In some embodiments, the linker is cleavable by a protease found in the extracellular microenvironment of the target cells, whereby cleavage of the protease cleavage site results in release of an active form of the KRAS inhibitor in the extracellular microenvironment. In some embodiments, the linker is cleavable by a protease found in the intracellular microenvironment of the target cells, whereby cleavage of the protease cleavage site results in release of an active form of the KRAS inhibitor in the intracellular microenvironment. In some embodiments, the protease is preferentially localized in the extracellular or intracellular microenvironment of the target cells. Preferential localization of a protease in an extracellular or intracellular microenvironment refers to the increased presence of an active or activatable form of the protease in the vicinity of the target cells associated with a disease to be treated, such that the protease can cleave the protease cleavage site and release an active form of the KRAS inhibitor upon or after antigen binding by the conjugate, as compared to the amount of protease in an extracellular or intracellular environment of normal cells (e.g., cells not associated with the disease). [121] A linker containing such a protease cleavage site may further comprise one or more components selected from pentafluorophenyl, succinimide, maleimide, and para-aminobenzoic acid (PABA). In some embodiments, the linker is a compound of the formula: (maleimidocaproyl)-(protease cleavage site)-(para-aminobenzyloxycarbonyl). The linker may comprise a maleimide at one end and a protease cleavage site at the other end. In some embodiments, the linker comprises a maleimide at one end, a protease cleavage site, and a self-immolative unit on the other end which is activated upon cleavage of the protease cleavage site. In some embodiments, the linker comprises a lysine residue, optionally having an acylated amine, and a protease cleavage site. In some embodiments, the linker is a compound of the formula Rx-SL-Pc-Iu, wherein Rx is a reactive moiety capable of forming a covalent bond with an antigen binding unit; SL is a stretcher unit, such as C1-10 alkyl, PEG 1-5, or a combination thereof; Pc is a protease cleavage site, such as a dipeptide, tripeptide, or tetrapeptide described herein; and Iu is a self-immolative unit, such as p-aniinobenzyI carbamate.
[122] In some embodiments, the linker comprises a peptide, such as a dipeptide, tripeptide, or tetrapeptide. The peptide may comprise natural amino acids, unnatural amino acids, or combinations thereof. In some embodiments, the peptide comprises L-amino acids. In some embodiments, the linker comprises a component selected from Val- Cit, Cit-Val, Ala-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu-Val, Ser-Cit, Cit-Ser, Lys-Cit, Cit- Lys, Asp-Cit, Cit-Asp, Val-Ala, Ala-Vai, Val-Val, Val-Gly, Gly-Val, Phe-Lys, Lys-Phe, Val-Lys, Lys-Val, Ala- Lys, Lys-Ala, Phe-Cit, Cit-Phe, Leu-Cit, Cit-Leu, Ile-Cit, Cit-Ile, Phe-Arg, Arg-Phe, Cit-Trp, Trp-Cit, Gly-Gly, Gly- Cit, Cit-Gly, Ala-Pro, Pro-Ala, Ala-Ser, Ser-Ala, Glu-Val-Cit, Cit- Val-Glu, Glu-Gly-Cit, Cit-Gly-Glu, Asn-Ala-Ala, Ala-Ala-Asn, and Ala-Gly-Ala. In some embodiments, the linker comprises one or more components selected from Val-Cit, Glu-Val-Cit, Val-Ala, Val-Val, Val-Gly, Gly-Gly, Gly-Cit, Glu-Gly-Cit, Ala-Ala-Asn, Ala-Gly-Ala, Ala- Pro, Ala-Ser, and Phe-Lys. In some embodiments, the linker comprises a component selected from Val-Cit and Glu- Val-Cit.
[123] In some embodiments, the linker (L) consists of 10 to 500 atoms, such as 10 to 400 atoms or 10 to 300 atoms. In some embodiments, the linker consists of 30 to 400 atoms, such as 30 to 300 atoms. In some embodiments, the linker comprises one or more components independently selected from alkyl, alkene, alkyne, aryl, cycloalkyl, heterocycle, glycoside, silyl ether, hydroxy, ether, polyether, ketone, ester, polyester, carbonate, amide, polyamide, peptide, polypeptide, urea, carbamate, sulfide, disulfide, sulfate, sulfonamide, phosphate, hydrazone, and succinimide. In some embodiments, the linker comprises one or more components independently selected from alkyl, alkene, alkyne, aryl, cycloalkyl, heterocycle, glycoside, silyl ether, hydroxy, ether, ketone, ester, carbonate, amide, urea, carbamate, sulfide, disulfide, sulfate, sulfonamide, phosphate, hydrazone, and succinimide.
[124] A KRAS inhibitor that is directly attached to a peptide linker can be proteolytically released as an amino acid adduct of the KRAS inhibitor. Such adduct may be active in vivo, or the in vivo activity of the adduct relative to the KRAS inhibitor may be impaired. In some embodiments, a linker comprises a self-immolative spacer to separate the KRAS inhibitor from the site of proteolytic cleavage. Use of a self-immolative spacer can allow for the separation of the fully active, chemically unmodified KRAS inhibitor upon amide bond hydrolysis.
[125] Para-aminobenzyl alcohol is a self-immolative spacer that can be incorporated into a linker of the present disclosure. In particular, the amino group can form an amide bond with an amino acid residue of the linker, while an amine of the KRAS inhibitor can be connected to the benzylic alcohol through a carbamate group. Following cleavage of the amide bond in vivo (e.g., via proteolytic cleavage of a peptide linker), a 1 ,6-elimination reaction releases the KRAS inhibitor (D) as depicted in the following scheme:
Similarly, the same spacer may be used to connect KRAS inhibitors containing a phenol group to the remainder of the linker as a para-aminobenzyl ether (PABE). Self-immolative spacers that comprise a methylene carbamate unit suitable for conjugation with a KRAS inhibitor having a functional group such as a hydroxyl, thiol, amide, or amine are described in WO 2015/095755, which is hereby incorporated by reference in its entirety. Following cleavage of the amide bond in vivo, a 1 ,6-elimination reaction and subsequent decomposition releases the KRAS inhibitor (D) as depicted in the following simplified scheme:
[126] Cleavable linkers described herein may comprise non-cleavable portions or segments. Similarly, an otherwise non-cleavable linker may be modified to include a cleavable portion or segment to render it cleavable. By way of example only, polyethylene glycol (PEG) and related polymers can include cleavable groups in the polymer backbone. For example, a polyethylene glycol or polymer linker may include one or more protease cleavable groups.
[127] Maleimide groups may be used in the preparation of conjugates of the present disclosure due to their specificity for reacting with thiol groups of, for example, cysteine groups of the antigen binding unit of a conjugate. The reaction between a thiol group of an antigen binding unit and a linker comprising a maleimide group may proceed according to the following scheme: wherein indicates an attachment site to the remainder of the linker, optionally wherein the linker comprises the KRAS inhibitor.
[128] The reverse reaction leading to maleimide elimination from a thio-substituted succinimide may also take place. This reverse reaction is undesirable, as the maleimide group may subsequently react with another available thiol group such as other proteins in the body having available cysteine residues. Accordingly, the reverse reaction can undermine the specificity of a conjugate. One method of preventing the reverse reaction is to incorporate a basic group into the linking group as shown in the scheme below. Not wishing to be bound by any particular theory, the presence of the basic group may increase the nucleophilicity of nearby water molecules to promote ring-opening hydrolysis of the succinimide group. The hydrolyzed form of the succinimide is resistant to deconjugation in plasma. So called “self-stabilizing” linkers may provide conjugates with improved stability. A representative example is provided in the following scheme: wherein indicates an attachment site to the remainder of the linker, optionally wherein the linker comprises the KRAS inhibitor. Two possible isomers can result from the hydrolysis reaction even though a mechanism is only depicted for the formation of the first.
[129] The identity of the base as well as the distance between the base and the maleimide group can be modified to tune the rate of hydrolysis of the thio-substituted succinimide group. Bases suitable for inclusion in a linker described herein, e.g., any linker comprising a maleimide group prior to conjugating to an antigen binding unit, may facilitate hydrolysis of a nearby succinimide group formed after conjugation of the antigen binding unit to the linker. Bases may include, for example, amines (e.g., N(R12)(R13)), nitrogen-containing heterocycles (e.g., a 3- to 12- membered heterocycle comprising one or more nitrogen atoms), amidines, guanidines, and carbocycles or heterocycles substituted with one or more amine groups. A basic unit may be separated from a maleimide group by, for example, an alkylene chain, such as C1-10 alkylene, wherein the alkylene is optionally substituted. A linker of the present disclosure that comprises a maleimide group may further comprise an electron withdrawing group, such as - C(O)R12, =O, -CN, -NO2, -CF3, -CBR3, -CC13, -CI3, halogen, -C(O)2R12, -C(O)N(R12)(R13), -C(O)R12, -C(O)F, - C(O)Br, -C(O)C1, -C(O)I, -SO2R12, -SO3R12, -SO2NHR12, -SO2N(R12)(R13), -PO3R12R13, -P(O)(CH3)NHR12, -NO, - N(R12)3 +, -CR12=C(R12)2, and -C=CR12. Such linkers may also comprise an aryl group (e.g., phenyl) or heteroaryl group (e.g., pyridyl), each of which is optionally substituted with one or more electron withdrawing groups such as those described herein. Further examples of self-stabilizing linkers are provided in WO 2013/173337, which is incorporated herein by reference in its entirety. It will be understood that references herein to a linker comprising a maleimide group may equivalently describe a conjugate having a linker comprising a thio-substituted succinimide group or a hydrolyzed, ring-opened thio-substituted succinimide group, and vice versa. In some embodiments, a linker comprises a component selected from , ; wherein indicates an attachment site to the remainder of the linker, optionally wherein the linker comprises the KRAS inhibitor.
[130] A linker may bridge a pair of sulfhydryl groups derived from reduction of a native hinge disulfide bond found in an antigen binding unit. An advantage of this methodology is the ability to synthesize homogenous DAR4 conjugates (e.g., conjugates of Formula (A) wherein p is 4) by full reduction of IgGs to afford 4 pairs of sulfhydryls from interchain disulfide bonds followed by 4 equivalents of the conjugating agent. wherein indicates an attachment site to the remainder of the linker, optionally wherein the linker comprises the KRAS inhibitor. Similarly, a maleimide derivative may be used to bridge a pair of sulfhydryl groups: wherein indicates an attachment site to the remainder of the linker, optionally wherein the linker comprises the KRAS inhibitor.
[131] A linker may be polyvalent such that it covalently links more than one KRAS inhibitor to a single site on the antigen binding unit, or monovalent such that it covalently links a single KRAS inhibitor to a single site of the antigen binding unit. Exemplary polyvalent linkers that may be used to attach two or more KRAS inhibitors to an antigen binding unit of the conjugate include Fleximer® linkers. A Fleximer® linker utilizes a solubilizing poly acetal backbone to incorporate two or more KRAS inhibitors (D) via a sequence of ester bonds, for example, utilizing a linker comprising two or more units of the structure shown below. This methodology can render highly- loaded conjugates (e.g., DAR20 — conjugates of Formula (A) wherein q is 20).
An aliphatic alcohol may be present or introduced into a KRAS inhibitor to utilize this linker. In some embodiments, the alcohol moiety is then attached to an alanine moiety, which is then systematically incorporated into the Fleximer® linker. Liposomal processing of the conjugate in vitro releases the parent alcohol-containing drug. In some embodiment, the linker utilized for producing a subject conjugate may take on different formats. In some instances the linker, linker modified Kras inhibitor, or conjugate may take a form described below in this paragraph. In embodiments, the chemical linker is a chemical linker described in WO2020/236841, which is incorporated herein in its entirety. For example, the Linker- KRAS inhibitor may be a compound having the structure of Formula
(Xa), or a pharmaceutically acceptable salt thereof: wherein: R1 is a reactive group; L1 is a bridging spacer; Lp is a bivalent peptide spacer; G-L2-A is a self-immolative spacer; R2 is a hydrophilic moiety; L2 is a bond, a methylene, a neopentylene or a C2-C3alkenylene; A is a bond, ’-OC(=O)-*, -
OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-* or -OC(=O)N(CH3)C(Ra)2C(Ra)2N(CH3)C(=O)-*, wherein each Ra is independently selected from H, c1-6alkyl or a C3-8cycloalkyl and the * of A indicates the point of attachment to D; L3 is a spacer moiety; and D is a KRAS inhibitor for generating a conjugate of the present disclosure comprising an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the KRAS inhibitor for generating a conjugate of the present disclosure. In embodiments, of the compound of Formula (Xa),Lp is a bivalent peptide spacer comprising one to four amino acid residues. In embodiments, is : wherein the * indicates the point of attachment to an N or a O of the KRAS inhibitor for generating a conjugate of the present disclosure, the *** indicates the point of attachment to Lp. In embodiments, the compound of Formula (Xa), has the formula (Xb): In embodiments, of the compound of Formula (Xa), R1 is
NH2, -SH, -SR3, -SSR4, -S(=O)2(CH=CH2), -
(CH2)2S(=O)2(CH=CH2), -NHS(=O)2(CH=CH2), -NHC(=O)CH2Br, -NHC(=O)CH2I,
C(O)NHNH2,
L1 is *-C(=O)(CH2)mO(CH2)m-**; *-
C(=O)((CH2)mO)t(CH2)n-**; *-C(=O)(CH2)m-**; *-C(=O)NH((CH2)mO)t(CH2)n-**; *-
C(=O)O(CH2)mSSC(R3)2(CH2)mC(=O)NR3(CH2)mNR3C(=O)(CH2)m-**; *-C(=O)O(CH2)mC(=O)NH(CH2)m-**; *- C(=O)(CH2)mNH(CH2)m-**; *-C(=O)(CH2)mNH(CH2)nC(=O)-**; *-C(=O)(CH2)mX1(CH2)m-**; *- C(=O)((CH2)mO)t(CH2)nX1(CH2)n-**; *-C(=O)(CH2)mNHC(=O)(CH2)n-**; *-
C(=O)((CH2)mO)t(CH2)nNHC(=O)(CH2)n-**; *-C(=O)(CH2)mNHC(=O)(CH2)nX1(CH2)n-**; *-
C(=O)((CH2)mO)t(CH2)nNHC(=O)(CH2)nX1(CH2)n-**; *-C(=O)((CH2)mO)t(CH2)nC(=O)NH(CH2)m-**; *- C(=O)(CH2)mC(R3)2-** or *-C(=O)(CH2)mC(=O)NH(CH2)m-**, where the * of L1 indicates the point of attachment to Lp, and the ** of L1 indicates the point of attachment to R1; R2 is a hydrophilic moiety selected from polyethylene glycol, poly alkylene glycol, a sugar, an oligosaccharide, a polypeptide or C2-C6alkyl substituted with 1 to 3 groups; each R3 is independently selected from H and C1-C6alkyl;R4 is 2-pyridyl or 4-pyridyl; each
R5 is independently selected from H, C1-C6alkyl, F, Cl, and-OH; each R6 is independently selected from H, C1- C6alkyl, F, Cl, -NH2, -OCH3, - OCH2CH3, -N(CH3)2, -CN, -NO2 and-OH; each R7 is independently selected from H, C1-6alkyl, fluoro, benzyloxy substituted with-C(=O)OH, benzyl substituted with-C(=O)OH, C1-4alkoxy substituted with- C(=O)OH and C1-4alkyl substituted with-C(=O)OH; X1 is is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each n is independently selected from 1, 2, 3, 4, 5, 6, 7,
8, 9 and 10; each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; Lp is a bivalent peptide spacer comprising an amino acid residue selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine; A is a bond, -OC(=O)-*,
' , -OC(=O)N(CH3)CH2CH2N(CH3)C(=O)-* or -OC(=O)N(CH3)C(Ra)2C(Ra)2N(CH3)C(=O)-*, wherein each Ra is independently selected from H, C1-C6alkyl or a C3-C8cycloalkyl and the * of A indicates the point of attachment to KRAS inhibitor; L3 is a spacer moiety having the structure where (i) W is - CH2O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)C(Rb)2NHC(=O)O-**, - NHC(=O)C(Rb)2NH-**, NHC(=O)C(Rb)2NHC(=O)-**,-CH2N(X-R2)C(=O)O-**, -C(=O)N(X-R2)-**, -CH2N(X-R2)C(=O)-**, -C(=O)NRb- **, -C(=O)NH-**, -CH b 2NRbC(=O)-**, -CH2NR C(=O)NH-**, -CH2NRbC(=O)NRb-**, -NHC(=O)-**, - NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** , -NH-, or - CH2N(Rb)C(=O)CH2-**, wherein each Rb is independently selected from H, C1-C6alkyl or C3-C8cycloalkyl and wherein the ** of W indicates the point of attachment to X; X is a bond, triazolyl or ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R2; or (ii) W is -CH2O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)C(Rb)2NHC(=O)O-**, - NHC(=O)C(Rb)2NH-**, NHC(=O)C(Rb)2NHC(=O)-**, -CH2N(X-R2)C(=O)O-**, -C(=O)N(X-R2)-**, -CH2N(X-R2)C(=O)-**, -C(=O)NRb- **, -C(=O)NH-**, -CH2NRbC(=O)-**, -CH2NRbC(=O)NH-**, -CH2NRbC(=O)NRb-**, -NHC(=O)-**, - NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, -NHS(O)2-**, -C(=O)-, -C(=O)O-** or - NH-, wherein each Rb is independently selected from H, C1-C6alkyl or C3-C8cycloalkyl and wherein the ** of W indicates the point of attachment to X; X is ***-CH2-triazolyl-C1-4 alkylene-OC(O)NHS(O)2NH-*, ***-C4-6 cycloalkylene - OC(O)NHS(O)2NH-*, ***-(CH2CH2O)n- C(O)NHS(O)2NH-*, ***-(CH2CH2O)n-C(O)NHS(O)2NH-(CH2CH2O)n-*, or ***-CH2-triazolyl-C1-4 alkylene-OC(O)NHS(O)2NH-(CH2CH2O)n-*, wherien each n independently is 1, 2, or 3, the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R2; and the * of L3 indicates the point of attachment to R2; and KRAS inhibitor comprising an N or an O, wherein KRAS inhibitor is connected to A via a direct bond from A to the N or the O of the KRAS inhibitor. In embodiments, the conjugate is a conjugate described in WO2023102875, which is incorporated herein in its entirety. For example, the conjugate may have the formula (Xb): Ab-(L-(D)m)n, or a pharmaceutically acceptable salt thereof; wherein Ab is an antibody or antigen binding fragment thereof; L is a linker; D is a KRAS inhibitor moiety; m is an integer from 1 to 8; and n is any number from 1 to 10. In some embodiments, the L is selected from: a cleavable linker and a non-cleavable linker. In some embodiments, the L comprises cleavable peptide. In some embodiments, the cleavable peptide is cleavable by an enzyme. In some embodiments, the enzyme comprises Cathepsin B. In some embodiments, the cleavable peptide or L comprises an amino acid unit. In some embodiments, the amino acid unit comprises a dipeptide, tripeptide, tetrapeptide or pentapeptide. In some embodiments, the amino acid unit is selected from: Val- Cit, Val-Ala(VA), Glu-Val-Cit, Ala-Ala-Asn(AAN), Gly-Val-Cit, Gly-Gly-Gly(GGG)and Gly-Gly-Phe- Gly(GGFG). In some embodiments, the L comprises a spacer. In some embodiments, the spacer comprises self- immolative spacers. In some embodiments, the self-immolative spacer comprises p-aminobenzoxy carbonyl(PABC) or p-aminobenzyl(PAB). In some embodiments, the cleavable peptide is directly spliced to the spacer. In some embodiments, the L comprises: Val-Cit-PABC, Val-Ala-PABC, Glu-Val-Cit-PABC, Ala-Ala-Asn-PABC, Gly-Val- Cit-PABC, Gly-Gly-Gly-PABC, Gly-Gly-Phe-Gly-PABC, Val-Cit-PAB, Val-Ala-PAB, Glu-Val-Cit-PAB, Ala-Ala- Asn-PAB, Gly-Val-Cit-PAB, Gly-Gly-Gly-PAB or Gly-Gly-Phe-Gly-PAB. In some embodiments, the spacer comprises the structure shown in -NH-(CH2)n 1 -La-Lb-Lc-, where La denotes -O-or a single bond; Lb denotes - CR 2(-CR 3)-, or a single bond, where R 2 and R 3 each independently denote C 1-C 6alkyl, -(CH2)n a-NH 2, - (CH2)n b-COOH, or -(CH2)n C-OH, n 1 denotes an integer from 0 to 6, n a, n band n c each independently denote an integer from 1 to 4, but R 2 and R 3 are not the same when n ais 0, and Lc denotes -C(=O)-. In some embodiments, the spacer comprises -NH-(CH2)3-C(=O)-, -NH-CH2-O-CH2-C(=O)-or -NH-(CH2)2-O-CH2-C(=O)-. In some embodiments, the L comprises the structure shown in -L 1-L 2-L 3-, where L 1 denotes -(succinimidyl-3-yl-N)- (CH2)n2 -C(=O)-, -CH2-C(=O)-NH-(CH2)n3 -C(=O)-or -C(=O)-(CH2)n4-C(=O)-, where n denotes an integer from 2 to 8, n 3denotes an integer from 1 to 8, and n 4 denotes an integer from 1 to 8; L 2 denotes amino acid unit; L denotes the self-degradation spacer. In some embodiments, the L is selected from: -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-
GGFG-PABC-; -(succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-PABC-; -(succinimidyl-3-yl-N)- CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-PABC-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O- CH2CH2O-CH2CH2-C(=O)-GGFG-PABC-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O- CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-PABC-; -CH2-C(=O)-NH-CH2CH2-C(=O)-GGFG-PABC-; -C(=O)- CH2CH2CH2CH2CH2CH2-C(=O)-GGFG-PABC-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2- C(=O)-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimidyl-3-yl-N)- CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-; -(succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)- GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-
CH2CH2CH2CH2CH2-C(=O)-; -(succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)- ; -(succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-; -(succinimidyl-3-yl-N)- CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimidyl-3-yl- N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-; -(succinimidyl-3-yl- N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-
C(=O)-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)- GGFG-NH-CH2CH2-C(=O)-; -CH2-C(=O)-NH-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -C(=O)- CH2CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-VA-
PABC-; -(succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-PABC-; -(succinimidyl-3-yl-N)- CH2CH2CH2CH2CH2-C(=O)-VA-NH-PABC-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O- CH2CH2-C(=O)-VA-PABC-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2o-CH2CH2O-CH2CH2O- CH2CH2O-CH2CH2-C(=O)-VA-PABC-; -CH2-C(=O)-NH-CH2CH2-C(=O)-VA-PABC-; -C(=O)- CH2CH2CH2CH2CH2CH2-C(=O)-VA-PABC-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; - (succinimidyl-3-yl-N)-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(succinimidyl-3-yl-N)- CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -(succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA- NH-CH2CH2CH2-C(=O)-; -(succmimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2CH2CH2- C(=O)-; -(succinimidyl-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2-O-CH2-C(=O)-; -(succinimidyl-3-yl-N)- CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2-O-CH2-C(=O)-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH-
CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH- CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -(succinimidyl-3-yl-N)-CH2CH2-C(=O)-NH- CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(succinimidyl-3-yl-N)- CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -CH2- C(=O)-NH-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; and -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-VA-NH- CH2CH2CH2-C(=O)-. In some embodiments, the p-aminobenzoxy carbonyl(PABC) or p-aminobenzyl(PAB) comprises a polysarcosine(poly-N-methylglycine) residue. In some embodiments, the L is selected from the following structure: wherein n 5 denotes an integer from 0 to 20. In some embodiments, the n 5 denotes an integer from 8 to 15. In embodiments, the chemical linker is a chemical linker described in WO2022/228494, which is incorporated herein in its entirety. For example, in one aspect, is provided a conjugate, or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein the conjugate includes a structure represented by formula (Xc):
; wherein, Q1 can be a linking group, L1 can include -L1a-C(=O)-, L1a can be selected from the group consisting of optionally substituted alkylene groups, optionally substituted polyethylene glycol groups, optionally substituted alkenylene groups, optionally substituted alkynylene groups, optionally substituted aliphatic cyclylene groups, optionally substituted aliphatic heterocyclylene groups, optionally substituted arylene groups, and optionally substituted heteroarylene groups; L2 can include an optionally substituted polypeptide residue, L3 can include an optionally substituted spacer group. For example, the spacer group may have self- degrading ability. For example, the spacer group can include optionally substituted optionally substituted ’ wherein, L2 and/or L3 can include optionally substituted polysarcosine residues, T is a KRAS inhibitor, Ab is an antigen binding unit, and m is a number from 1 to 8. In another aspect, the compound, or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof, has the formula Xc; wherein, Q1 can include a linker, L1 can include -L1a-C(=O)-, wherein, L1a can be selected from the group consisting of optionally substituted alkylene groups, optionally substituted polyethylene glycol groups, optionally substituted alkenylene groups, optionally substituted alkynylene groups, optionally substituted aliphatic cyclylene groups, optionally substituted aliphatic heterocyclylene groups, optionally substituted arylene groups, and optionally substituted heteroarylene groups; L2 can include an optionally substituted polypeptide residue, L3 can include an optionally substituted spacer group. For example, wherein, the benzene ring of L3 can be substituted with the optionally substituted structural unit -X. For example, the structural unit -X can be selected from the group consisting of optionally substituted
, wherein X1 is selected from the group consisting of carbonyl, C1-C8 alkyl, C1-C8 alkoxy, C1- C6 cycloalkyl, linear heteroalkyl comprising 1 -8 atoms, and linear-cyclic heteroalkyl comprising 1 -8 atoms, where the heteroalkyl comprises 1 -3 atoms selected from N, O or S; wherein X2 is selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl comprising 1-8 atoms, and linear-cyclic heteroalkyl comprising 1-8 atoms, where the heteroalkyl comprises 1-3 atoms selected from N, O or S; wherein X3 is selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl comprising 1 -8 atoms, and linear-cyclic heteroalkyl comprising 1 -8 atoms, where the heteroalkyl comprises 1 -3 atoms selected from N, O or S; the C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl comprising 1-8 atoms, and linear-cyclic heteroalkyl comprising 1 -8 atoms are each independently optionally substituted with one or more substituents selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxy, alkoxy, or cycloalkyl. For example, wherein, the benzene ring of L3 can be substituted with the optionally substituted structural unit -X. For example, the structural unit -X can include optionally substituted wherein X1 is selected from the group consisting of C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl comprising 1-8 atoms, and linear-cyclic heteroalkyl comprising 1-8 atoms, where the heteroalkyl comprises 1-3 atoms selected from N, O or S, and the C1-C8 alkyl, C1-C8 alkoxy, C1-C6 cycloalkyl, linear heteroalkyl comprising 1-8 atoms, and linear-cyclic heteroalkyl comprising 1 -8 atoms are each independently optionally substituted with one or more substituents selected from deuterium, halogen, cyano, nitro, amino, alkyl, carboxy, alkoxy, or cycloalkyl. In embodiments, the conjugate is a conjugate described in WO2013/173337, which is incorporated herein in its entirety. For example in some embodiments, the conjugate is represented by Formula Xd: or a salt thereof (e.g., pharmaceutically acceptable salts); L is a antigen binding unit; D' is a KRAS inhibitor; L° is the optional secondary linker assembly; and Lss is the self-stabilizing linker assembly, wherein M1 is a succinimide ring or a hydrolyzed succinamide or together with BU forms a dilactam; BU is a Basic unit; HE is a hydrolysis enhancer comprising an electron withdrawing group; the circle represents a scaffold that can be C1-8 alkylene, Ci.
8 heteroalkylene, C6-10 arylene, or C4-10 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or D'; the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C6-10 arylene or C4-10 hetero arylene; the subscript a and b are each 0 or 1 , and the sum of a+b is 1; and the subscript p ranges from 1 to 20. In some aspects, when r is 1, HE does not comprise a carbonyl group, (i.e., C(=O)). In embodiments, r is zero, in some embodiments, a is 1 and b is zero. In other embodiments, a is zero and b is 1. In some embodiments m + q + r is 0. in such embodiments, the scaffold is a C6-10 arylene or C4-10 heteroarylene and acts as the electron withdrawing group. Exemplary aryls and heteroaryls include phenyl and pyridinyl. In some embodiments m + q + r is 1 or 2. In some embodiments, the conjugate is represented by Formula Xd or a salt thereof wherein a is 1 and r is zero. In some embodiments LO is present and is a releasable linker assembly, the circle represents a scaffold that is C1-8 alkylene or C1-8 heteroalkylene (preferably C1-4 alkylene or C1-4 hetero alkylene), a is 1, r is zero, and the sum of m+q is 1. In some such embodiments, the scaffold is C1-3 alkylene or C1-3 hetero alkylene. In some such embodiments, the alkylene is straight chain or branched. In some embodiments, LO is present and is a releasable linker assembly, the circle represents a scaffold that is C1-8 alkylene or C1-8 hetero alkylene (preferably C1-4 alkylene or C1-
4 heteroalkylene), a is 1 , and m and r are zero. In some such embodiments, the scaffold is Ci alkylene or C1 -
3 heteroalkylene, in some such embodiments, the alkylene is straight chain or branched. In embodiments, M1 is preferably a succinimide ring (i.e., non-hydrolyzed) or a hydrolyzed succinimide ring (also referred to herein as hydrolyzed succinimide). In some embodiments, the self-stabilizing linker assembly (Lss) is represented by Formula
Xdb: or a salt thereof (e.g., pharmaceutically acceptable salt) wherein the wavy lines indicate points for attachment of the optional secondary linker assembly to D' or D, and wherein // indicates the point of attachment to a antigen binding unit. In the self-stabilizing linker assembly above, M 1 represents a succinimide ring or a hydrolyzed succinamide ring or a dilactam formed when the base reacts with the succinimide ring, BU is a Basic unit, HE is a hydrolysis enhancer comprising an electron withdrawing group, and the circle represents a scaffold that can be C1-8 alkylene, C1-8 heteroalkyiene, C6-10 arylene, or C4- 10 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly, D!, or D; and the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C6-10 arylene or C4-10 hetero arylene. In some embodiments, when r is 1 , HE does not comprise a carbonyl group, (i.e., C(=O)). In some embodiments, r is zero. In some embodiments m + q + r is 0. In such aspects, the C6-10 arylene or C4-10 heteroarylene act as the electron withdrawing group. Exemplary aryls and heteroaryls include phenyl and pyridinyl. In some embodiments m + q + r is 1 or 2. In some embodiments, the selfstabilizing linker assembly is represented by Formula 11 or a salt thereof wherein the circle represents a scaffold that is C1-8 alkylene or C1-8 heteroalkyiene (preferably C1-4 alkylene or heteroalkyiene), r is zero, and the sum of m+q is 1. In some such aspects, the scaffold is C1-3 alkylene or C1-3 hetero alkylene. In some such aspects, the alkylene is a straight chain or branched alkylene. In some embodiments, the circle represents a scaffold that is C1-8 alkylene or C1- 8 heteroalkyiene (preferably C1-4 alkylene or heteroalkyiene) and m and r are zero. In some such aspects, the scaffold is C1-3 alkylene or C1-3 heteroalkyiene. In some such aspects, the alkylene is a straight chain or branched alkylene. In some embodiments, the circle represents a scaffold that is C1, C2, C3. or C4 straight or branched chain alkylene, r is zero, and the sum of m+q is 1. In some embodiments, the circle represents a scaffold that is C1, C2, C3. or C4 straight or branched chain alkylene, and m and r are zero. In embodiments of the conjugate having the Formula Xd: 1) m is 1 , and q and r are 0; 2) q is 1 , and m and r are 0; 3) r is 1 , and m and q are 0; 4) m is 1 , q and r are 0, and a is 1 ; 5) q is 1 , m and r are 0, and a is 1 ; 6) r is 1 , m and q are 0, and a is 1 ; 7) m is 1 , q and r are 0, and D' is a KRAS inhibitor; 8) q is 1. m and r are 0, and D' is a KRAS inhibitor; 9) r is 1 , m and q are 0, and D' is a KRAS inhibitor, D; 10) m is 3 , q and r are 0, a is 1, and D: is a Drug unit, D: 11) q is 1, m and r are 0, a is 1, and D' is a KRAS inhibitor; or 12) r is 1 , m and q are 0, a is 1 , and D' is a KRAS inhibitor. In embodiments, including those based on each of the selected embodiments of 1), 2) 3), 4), 5), 6), 7), 8), 9), 10), 11), and 12) above , the Basic unit (BU) comprises a primary, a secondary amine, or a tertiary amine. In still other selected embodiments, including those based on each of the selected embodiments of 1), 2) 3), 4), 5), 6), 7), 8), 9), 10), 11), and 12) above, the Basic unit is selected from the group consisting of-(C(R9)( R10 ))xNH2, -(C(R9)( R10))xNHRa, and -(C(R9)( R10))xNRa 2, wherein x is an integer of from 0-4 (or from 1 to 4) and each Ra is independently selected from the group consisting of C1-6 alkyl and C1- 6 haloalkyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group, provided that if x is zero there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam, and each R9and R10 are independently selected from H or C1-3 alkyl. In still other selected embodiments, including those based on each of the selected embodiments of 1), 2) 3), 4), 5), 6), 7), 8), 9), 10), 11), and 12) above, the Basic unit is selected from the group consisting of -(CH2)xNH2, -(CH2)xNHRa, and -(CH2)xNRa 2, wherein x is an integer of from 0 to 6 (preferably 0 to 4, or 1 to 4) provided that if x is zero there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam, and each Ra is independently selected from the group consisting of C1-6 alkyl and C1-6 haloalkyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group. In yet other selected embodiments, x is an integer of from 1 to 4. In even other selected embodiments, including those based on each of the selected embodiments of 1), 2) 3), 4), 5), 6). 7), 8), 9), 10), 11), and 12) above, the Basic unit is NH2 -CH2NH2, -CH2CH2NH2, - CH2CH2CH2NH2, or -CH2CH2CH2CH2NH2 provided that if the Basic unit is -NH2, there are no less than 2 intervening atoms between the base and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam. In embodiments, the conjugate is a conjugate described in W02010/093395, which is incorporated herein in its entirety. For example, the conjugate may have the formula Xe, MAb-[L2]-[L l]-[AA]m- [A']-D; where MAb is a disease -targeting antibody; L2 is a component of the cross-linker comprising an antibodycoupling moiety and one or more of acetylene (or azide) groups; L 1 comprises a defined PEG with azide (or acetylene) at one end, complementary to the acetylene (or azide) moiety in L2, and a reactive group such as carboxylic acid or hydroxyl group at the other end; AA is an L-amino acid; m is an integer with values of 0, 1, 2, 3, or 4; and A' is an additional spacer, selected from the group of ethanolamine, 4-hydroxybenzyl alcohol, 4- aminobenzyl alcohol, or substituted or unsubstituted ethylenediamine. The L amino acids of 'AA' are selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. If the A' group contains hydroxyl, it is linked to the hydroxyl group or amino group of D in the form of a carbonate or carbamate, respectively. In a preferred embodiment of formula Xe, A' is a substituted ethanolamine derived from an L-amino acid, wherein the carboxylic acid group of the amino acid is replaced by a hydroxymethyl moiety. A' may be derived from any one of the following L-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In embodiments, the conjugate is a conjugate described in WO2019081455, which is incorporated herein in its entirety. For example, the conjugate may have the formula Xf wherein D is KRAS inhibitor, X is an optional cleavable moiety for releasing D, Z is an optional spacer, and a is 1 or more, b is 1 or more and m is 1 or more; L is an orthogonal connector that allows for (HPSMW) to be in an orthogonal orientation with respect to (X-D), HPSMW results from covalent binding to said orthogonal connector L, of a single molecular weight homopolymer having formula (Xfb) wherein n is one or more; R1 and R2 are different, and one of R1 and R2 is H or an inert group, the other one of R1 and R2 being a functionalized reactive group, said group being reactive for covalently binding a bindable group, in such reaction conditions that the inert group is non-reactive, Z1 and Z2, identical or different, are optional spacers, and k is 2 or more. Further features of a conjugate of formula Xf, are given below, taken alone or in any combination. In embodiments, k is an integer which is at least 2, it is preferably 100 at most, more preferably 50 at most, and specifically 2-30, and more specifically 2-24, 6-24, or 12-24. In embodiments, said functionalized reactive group R1 or R2 may be selected from the following groups: carboxylic acid group, amino groups NRR" wherein R and R" are independently selected from H, (C1-C6) alkyl optionally interrupted by at least one heteroatom selected among O, N and S, hydroxyl group, halogen atoms, hydrazine (-NH2- NH2) group, nitro group, hydroxylamine group, azido group, (C2-C6) alkynyl group, (C2-C6) alkenyl group, thiol group, activated ester groups such as N-hydroxysuccinimide ester, perfluorinated esters, nitrophenyl esters, azabenzotriazole and benzotriazole activated esters, acylureas, boronic acid B(OR"")2 groups, wherein R"" is a hydrogen atom or a C1-C6 alkyl group, thiol-reactive groups such as maleimide, halomaleimides, haloacetyls, pyridyl disulfides, mesylate group, tosylate group, triflate group, aldehyde group, isocyanate or isothiocyanate group, chlorosulfonyl group, acrylate group. As mentioned above, spacers are optional, both Z1 and Z2 may be present, only one of Z1 and Z2 may be present, they also may not be present. In this latter case and when the homopolymer of the invention is a homopolymer of sarcosine, it has formula Xfc wherein R1, R2 and k are as described above. In embodiments, R1 may be H or an inert group and R2 a functionalized reactive group or R1 may be a functionalized reactive group and R2 is H or an inert group. In embodiments, the functionalized reactive group R1 or R2 is a secondary amine and the inert group R1 or R2 is a carboxylic acid that remains unreacted and unbound on the final conjugate. In embodiments, R1 is selected from OH and NH2, and when R1 is OH, R2 is COCH3 and when R1 is NH2, R2 is CO— G— COOH, G being CH2CH2,
CH2CH2CH2, CH2CH2CH2CH2, CH2OCH2, CH2SCH2, CH2CH(CH3)CH2, CH2C(CH3)2CH2 or CH2N(CH3)CH2. In embodiments, HPSMW wherein the wavy bond represents the attachment point to L or to a spacer Z, if present, k is 2 or more, preferably k is 2 to 50, and R4 is selected from -R', - O , -OR', -SR', -S-, -NR'2, -NR'3 +, =NR', -CX3, -CN, -NRC(=O)R', -C(=O)R', -C(=O)NR'2, -SO3-, -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3 , -PO3H2, -C(=O)X, -C(=S)R', -CO2R', - CO2 , -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR'2, C(=S)NR'2, or C(=NR')NR'2, where each X is independently a halogen: -F, -CI, -Br, or —I, and each R' is independently -H, -C1.20 alkyl, C6-C20 aryl, or C3-14 heterocycle.
Typically, R4 is -OR', -NR'2, or -C(=O)R'. In embodiments, the conjugate of formula Xf is wherein R6 is —C1-C10 alkylene-, — C1-C10 heteroalkylene-, -C3-C8 carbocyclo-, -O-
(C1- C8 alkyl)-, -arylene-, — C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, — C1-C10 alkylene-(C3-
C8 carbocyclo)-, -(C3-C8 carbocyclo)-C1-C10 alkylene-, -C3-C8 heterocyclo-, — C1-C10 alkylene-(C3-C8 heterocyclo)-, -(C3-C8 heterocyclo)— C1-C10 alkylene-, — C1-C10 alkylene-C(=O)-, — C1-C10 heteroalkylene-C(=O)-, -C3-
C8 carbocyclo-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C10 alkylene- arylene-C(=O)-, -arylene-C1- C10 alkylene-C(=O)-, -C1-C10 alkylene-(C3-C8carbocyclo)-C(=O)-, -(C3-C8 carbocyclo)-C1-C10 alkylene-C(=O)-, - C3-C8 heterocyclo-C(=O)-, -C1-C10 alkylene-(C3-C8heterocyclo)-C(=O)-, -(C3-C8 heterocyclo)-C1-C10 alkylene- C(=O)-, -C1-C10 alkylene-NH-, -C1-C10 heteroalkylene-NH-, -C3-C8 carbocyclo-NH-, -O-(C1-C8 alkyl)-NH-, - arylene -NH-, -C1-C10 alkylene-arylene-NH-, -arylene-C1-C10 alkylene-NH-, -C1-C10 alkylene-(C3-C8 carbocyclo)- NH-, -(C3-C8 carbocyclo)-C1-C10 alkylene-NH-, -C3-C8heterocyclo-NH-, -C1-C10 alkylene-(C3-C8 heterocyclo)-NH-, -(C3-C8 heterocyclo)-C1-C10 alkylene-NH-, -C1-C10 alkylene-S-, -C1-C10 heteroalkylene-S -, -C3-C8carbocyclo-S -, - O-(C1-C8 alkyl)-)-S -, -arylene-S-, -C1-C10 alkylene-arylene-S-, -arylene-C1-C10 alkylene-S-, -C1-C10 alkylene-(C3- C8 carbocyclo)-S-, -(C3-C8 carbocyclo)-C1-C10 alkylene-S-, -C3-C8 heterocyclo-S-, -C1-C10 alkylene-(C3-
C8 heterocyclo)-S-, -(C3-C8 heterocyclo)-C1-C10 alkylene-S-, — C1-C10 alkylene-O-C(=O)-, -C3-C8 carbocyclo-O- C(=O)-, -O-(C1-C8 alkyl)-O-C(=O)-, -arylene-O-C(=O)-, -C1-C10 alkylene-arylene-O-C(=O)-, -arylene-C1- C10 alkylene-O-C(=O)-, -C1-Cio alkylene-(C3-C8carbocyclo)-O-C(=O)-,-(C3-C8 carbocyclo)-C1-C10 alkylene-O- C(=O)-, -C3-C8 heterocyclo-O-C(=O)-, -C1-C10 alkylene-(C3-C8heterocyclo)-O-C(=O)-, -(C3-C8 heterocyclo)-C1- C10 alkylene-O-C(=O)-; any of the R6 group is optionally substituted with one or more of the substituents selected from : -X, -R, -O , -OR', =O, -SR', -S-, -NR'2, -NR'3 +, =NR', -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2,
=N2, -N3, -NR'C(=O)R', -C(=O)R', -C(=O)NR'2, -SO3-, -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', - OP(=O)(OR')2, -P(=O)(OR')2, -PO3 , -PO3H2, -C(=O)X, -C(=S)R', -CO2R', -CO2 , -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR'2, C(=S)NR'2, and C(=NR')NR'2, where each X is independently a halogen: -F, -CI, -Br, or -I; and each R' is independently -H, -C1C20 alkyl, -C6-C20 aryl, or -C3-C14 heterocycle; Z is an optional spacer; L is an orthogonal connector; X is an optional cleavable moiety for releasing D; D is KRAS inhibitor, a is 1 or more and b is 0, 1 or more, and HPSMW results from covalent binding to said orthogonal connector L, of a single molecular weight homopolymer having formula (Xfb) above; wherein R1 and R2 are different, and one of R1 and R2 is H or an inert group, the other one of R1 and R2 being a functionalized reactive group, said group being reactive for covalently binding a bindable group, in such reaction conditions that the inert group is non-reactive, Z1 and Z2, identical or different, are optional spacers, and n is 1 or more and k is 2 or more.
[132] In some embodiments, the linker comprises one or more components independently selected from polyethylene glycol, polysarcosine, a hydrazone, acetal, maleimide, succinimide, asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, valine, alanine, glycine, leucine, isoleucine, methionine, tryptophan, proline, histidine, citrulline, phenylalanine, carboxylate, p-aminobenzyloxy carbonyl, alkyl, alkene, alkyne, aryl, cycloalkyl, heterocycle, glycoside, silyl ether, hydroxy, ether, ketone, ester, carbonate, amide, urea, carbamate, sulfide, disulfide, sulfate, sulfonamide, phosphate, phenylboronic ester, phenylboronic acid, thioketal, tartaric acid, 1 ,2-diol acetonide, o-aminoalcohol, selenium, ortho-nitrobenzyl, phenacyl ester, sugar, glucoronide, trioxolane, oxime, acyl hydrazone, cyclobutyl, pyrophosphate, arylsulfate, heptamethine, cyanine fluorophore, o-nitrobenzyl, PC4AP, dsProc, 1,3-dioxane, triazole, piperazine, bis(vinylsulfonyl)piperazine, N- methyl-N-phenylvinylsulfonamide, and Pt.
[133] In some embodiments, the linker is a compound of the formula: wherein: indicates an attachment site to the antigen binding unit;
Z1 is a product formed by reaction of the antigen binding unit with a reactive precursor of Z1 ;
Z2 is absent or an optionally substituted spacer comprising one or more components independently selected from C1-6 alkyl, (CH2CH2O)n2, and -C(O)NH-, or any combination thereof;
Z3 is selected from Val-Cit, Cit-Val, Ala-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu- Val, Ser-Cit, Cit-Ser, Lys-Cit, Cit-Lys, Asp-Cit, Cit-Asp, Val-Ala, Ala-Vai, Val-Val, Val-Gly, Gly-Val, Phe-Lys, Lys-Phe, Val-Lys, Lys-Val, Ala-Lys, Lys-Ala, Phe-Cit, Cit-Phe, Leu-Cit, Cit-Leu, Ile-Cit, Cit-Ile, Phe-Arg, Arg- Phe, Cit-Trp, Trp-Cit, Gly-Gly, Gly-Cit, Cit-Gly, Ala-Pro, Pro-Ala, Ala-Ser, Ser-Ala, Glu- Val-Cit, Cit- Val-Glu, Glu-Gly-Cit, Cit-Gly-Glu, Asn-Ala-Ala, Ala-Ala-Asn, and Ala-Gly-Ala;
Rz is selected from hydrogen, -C1-4 alkyl-On3-(C1-4 alkylene)n4-Z4, -C1-4 alkyl-N-[(C1-4 alkylene)-Z4]2, -C2-4 alkynyl-C1-4 alkyl-On3-(C1-4 alkylene)n4-Z4, and -C2-4 alkynyl-C1-4 alkyl-N-[(C1-4 alkylene)-Z4]2;
Z4 is selected from -SO3H, -CO2H, PEG 4-32, and a sugar moiety; n1, n3, and n4 are each independently 0 or 1 ; n2 is an integer from 1 to 6; and indicates an attachment site to the KRAS inhibitor.
[134] In some embodiments, the linker is a compound of the formula: wherein: indicates an attachment site to the antigen binding unit;
Z1 is a product formed by reaction of the antigen binding unit with a reactive precursor of Z1 ;
Z2 is absent or an optionally substituted spacer comprising one or more components independently selected from C1-6 alkyl, (CH2CH2O)n2. -C(O)NH-, -C(O)NCH3-, (C(O)CH2N(CH3))n2, or any combination thereof;
Z3 is selected from a bond, Val-Cit, Cit-Val, Ala-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu- Vai, Ser-Cit, Cit-Ser, Lys-Cit, Cit-Lys, Asp-Cit, Cit-Asp, Val-Ala, Ala- Vai, Val-Val, Val-Gly, Gly-Val, Phe- Lys, Lys-Phe, Val-Lys, Lys-Val, Ala-Lys, Lys-Ala, Phe-Cit, Cit-Phe, Leu-Cit, Cit-Leu, Ile-Cit, Cit-Ile, Phe-Arg, Arg-Phe, Cit-Trp, Trp-Cit, Gly-Gly, Gly-Cit, Cit-Gly, Ala-Pro, Pro-Ala, Ala-Ser, Ser-Ala, Glu-Val-Cit, Cit-Val- Glu, Glu-Gly-Cit, Cit-Gly-Glu, Asn-Ala-Ala, Ala-Ala-Asn, and Ala-Gly-Ala;
Rz is selected from hydrogen, Z4, -C1-4 alkyl-On3-(C1-4 alkylene)n4-Z4, -C1-4 alkyl-N-[(C1-4 alkylene)-Z4]2, - C2-4 alkynyl-C1-4 alkyl-On3-(C1-4 alkylene)n4-Z4, and -C2-4 alkynyl-C1-4 alkyl-N-[(C1-4 alkylene)-Z4]2;
Z4 is selected from -SO3H, -CO2H, PEG 4-32, -(CH2N(CH3)C(O))n2CH3, and a sugar moiety; n1, n3, and n4 are each independently 0 or 1 : n2 is an integer from 1 to 20: and indicates an attachment site to the KRAS inhibitor.
[135] In some embodiments, a conjugate of the present disclosure comprises a linker selected from:
to the KRAS inhibitor and indicates an attachment site to the antigen binding unit.
[136] In some embodiments, a conjugate of the present disclosure comprises a linker selected from:
wherein indicates an attachment site to the KRAS inhibitor and indicates an attachment site to the antigen binding unit.
[137] In some embodiments, the linker is a compound of the formula: wherein: indicates an attachment site to the antigen binding unit;
Z1 is a product formed by reaction of the antigen binding unit with a reactive precursor of Z1;
Z2 is absent, C1-6 alkyl, (CH2CH2O)n2. -C(O)NH-, -C(O)NCH3-, (C(O)CH2N(CH3))n2, - ((CH2CH2O)n2(CH2CH2)C(O))N(CH2C(O)N(CH3)-(CH2CH2O)n2-(CH2CH2N(CH3)-(C(O)CH2N(CH3))n2- C(O)CH3)CH2-, -(C1-6 alkyl)C(O)N(CH2C(O)N(CH3)-(CH2CH2O)n2-(CH2CH2N(CH3)-(C(O)CH2N(CH3))n2- C(O)CH3)CH2-, -(C1-6 alkyl)C(O)N(CH2C(O)N(CH3)-(CH2CH2O)n2-(CH2CH2N(CH3)-(C(O)CH2N(CH3))n2- C(O)CH2N(H)C(O)CH3)CH2-, -((CH2CH2O)n2(CH2CH2)C(O))N((CH2CH2O)n2CH3)CH2-, -((C1-6 alkyl)C(O))N((CH2CH2O)n2CH3)CH2-, -(C1-6 alkyl)C(O))N((CH2C(O)N(CH3)-(CH2CH2O)n2CH3)CH2-, -((C1-6 alkyl)C(O))N(CH2C(O)N((CH2CH2O)n2CH3)(CH2CH2O)n2CH3)CH2-, - ((CH2CH2O)n2(CH2CH2)C(O))N(CH2C(O)N((CH2CH2O)n2CH3)(CH2CH2O)n2CH3)CH2-, - ((CH2CH2O)n2(CH2CH2)C(O))N(CH2C(O)-(N(CH3)CH2C(O))n2-N(CH3)2)CH2-, -((C1-6 alkyl)C(O))N(CH2C(O)- (N(CH3)CH2C(O))n2-N(CH3)2)CH2-, -(CH2CH2O)n2(CH2CH2)-, -(C1-6 alkyl)-, - ((CH2CH2O)n2(CH2CH2)C(O)NH(CH2CH2)-, -(CH2CH2O)n2(CH2CH2)C(O)-(N(CH3)CH2C(O))n2-N(CH3)CH2)-, - (C1-6 alkyl)C(O)-(N(CH3)CH2C(O))n2-N(CH3)CH2)-, -(Phenyl)-CH2C(O)NH(CH2CH2O)n2(CH2CH2)-, -(C1-6
Z3 is selected from a bond, Val-Cit, Cit-Val, Ala-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu-Val, Ser-Cit, Cit-Ser, Lys-Cit, Cit-Lys, Asp-Cit, Cit-Asp, Val-Ala, Ala- Vai, Vai- Vai, Val-Gly, Gly-Val, Phe- Lys, Lys-Phe, Val-Lys, Lys-Val, Ala-Lys, Lys-Ala, Phe-Cit, Cit-Phe, Leu-Cit, Cit-Leu, Ile-Cit, Cit-Ile, Phe-Arg, Arg-Phe, Cit-Trp, Trp-Cit, Gly-Gly, Gly-Cit, Cit-Gly, Ala-Pro, Pro-Ala, Ala-Ser, Ser-Ala, Glu-Val-Cit, Cit-Val- Glu, Glu-Gly-Cit, Cit-Gly-Glu, Asn-Ala-Ala, Ala-Ala-Asn, and Ala-Gly-Ala;
Rz is selected from hydrogen, -CH2N(CH3)C(O)-(CH2CH2O)n2CH3, -CH2N(CH3)(C(O)CH2N(CH3))n2- C(O)CH3, -CH2N(CH3)C(O)-(CH2CH2O)n2-CH2CH2C(O)-(N(CH3)CH2C(O))n2-N(CH3)2, -SO3H, -CO2H, PEG 4-32, polysarcosine, -(CH2N(CH3)C(O))n2CH3, and a sugar moiety; n3, and n4 are each independently 0 or 1 ; n2 is independently an integer from 1 to 20; and indicates an attachment site to the KRAS inhibitor.
[138] In an aspect is provided a linker-(KRAS inhibitor) of Formula (B): wherein:
Z1a is a moiety capable of forming a covalent bond with an antigen binding unit;
Z2 is absent or an optionally substituted spacer comprising one or more components independently selected from C1-6 alkyl, (CH2CH2O)n2. -C(O)NH-, -C(O)NCH3-, (C(O)CH2N(CH3))n2, or any combination thereof;
Z3 is selected from a bond, Val-Cit, Cit-Val, Ala-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu-Val, Ser-Cit, Cit-Ser, Lys-Cit, Cit-Lys, Asp-Cit, Cit-Asp, Val-Ala, Ala- Vai, Vai- Vai, Val-Gly, Gly-Val, Phe- Lys, Lys-Phe, Val-Lys, Lys-Val, Ala-Lys, Lys-Ala, Phe-Cit, Cit-Phe, Leu-Cit, Cit-Leu, Ile-Cit, Cit-Ile, Phe-Arg, Arg-Phe, Cit-Trp, Trp-Cit, Gly-Gly, Gly-Cit, Cit-Gly, Ala-Pro, Pro-Ala, Ala-Ser, Ser-Ala, Glu-Val-Cit, Cit-Val- Glu, Glu-Gly-Cit, Cit-Gly-Glu, Asn-Ala-Ala, Ala-Ala-Asn, and Ala-Gly-Ala;
Rz is selected from hydrogen, Z4, -C1-4 alkyl-On3-(C1-4 alkylene)n4-Z4, -C1-4 alkyl-N-[(C1-4 alkylene)-Z4]2, - C2-4 alkynyl-C1-4 alkyl-On3-(C1-4 alkylene)n4-Z4, and -C2-4 alkynyl-C1-4 alkyl-N-[(C1-4 alkylene)-Z4]2;
Z4 is selected from -SO3H, -CO2H, PEG 4-32, -(CH2N(CH3)C(O))n2CH3, polysarcosine, and a sugar moiety; n1, n3, and n4 are each independently 0 or 1 ; n2 is independently an integer from 1 to 20; and the KRAS inhibitor is optionally a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), or (I-g).
[139] In an aspect is provided a linker-(KRAS inhibitor) of Formula (B): wherein:
Z1a is a moiety capable of forming a covalent bond with an antigen binding unit (e.g., maleimide);
Z2 is absent, C1-6 alkyl, (CH2CH2O)n2. -C(O)NH-, -C(O)NCH3-, (C(O)CH2N(CH3))n2, - ((CH2CH2O)n2(CH2CH2)C(O))N(CH2C(O)N(CH3)-(CH2CH2O)n2-(CH2CH2N(CH3)-(C(O)CH2N(CH3))n2- C(O)CH3)CH2-, -(C1-6 alkyl)C(O)N(CH2C(O)N(CH3)-(CH2CH2O)n2-(CH2CH2N(CH3)-(C(O)CH2N(CH3))n2- C(O)CH3)CH2-, -(C1-6 alkyl)C(O)N(CH2C(O)N(CH3)-(CH2CH2O)n2-(CH2CH2N(CH3)-(C(O)CH2N(CH3))n2- C(O)CH2N(H)C(O)CH3)CH2-, -((CH2CH2O)n2(CH2CH2)C(O))N((CH2CH2O)n2CH3)CH2-, -((C1-6 alkyl)C(O))N((CH2CH2O)n2CH3)CH2-, -(C1-6 alkyl)C(O))N((CH2C(O)N(CH3)-(CH2CH2O)n2CH3)CH2-, -((C1-6 alkyl)C(O))N(CH2C(O)N((CH2CH2O)n2CH3)(CH2CH2O)n2CH3)CH2-, - ((CH2CH2O)n2(CH2CH2)C(O))N(CH2C(O)N((CH2CH2O)n2CH3)(CH2CH2O)n2CH3)CH2-, - ((CH2CH2O)n2(CH2CH2)C(O))N(CH2C(O)-(N(CH3)CH2C(O))n2-N(CH3)2)CH2-, -((C1-6 alkyl)C(O))N(CH2C(O)- (N(CH3)CH2C(O))n2-N(CH3)2)CH2-, -(CH2CH2O)n2(CH2CH2)-, -(C1-6 alkyl)-, - ((CH2CH2O)n2(CH2CH2)C(O)NH(CH2CH2)-, -(CH2CH2O)n2(CH2CH2)C(O)-(N(CH3)CH2C(O))n2-N(CH3)CH2)-, -
(C1-6 alkyl)C(O)-(N(CH3)CH2C(O))n2-N(CH3)CH2)-, -(Phenyl)-CH2C(O)NH(CH2CH2O)n2(CH2CH2)-, -(C1-6
Z3 is selected from a bond, Val-Cit, Cit-Val, Ala-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu-Val, Ser-Cit, Cit-Ser, Lys-Cit, Cit-Lys, Asp-Cit, Cit-Asp, Val-Ala, Ala- Vai, Vai- Vai, Val-Gly, Gly-Val, Phe- Lys, Lys-Phe, Val-Lys, Lys-Val, Ala-Lys, Lys-Ala, Phe-Cit, Cit-Phe, Leu-Cit, Cit-Leu, Ile-Cit, Cit-Ile, Phe-Arg, Arg-Phe, Cit-Trp, Trp-Cit, Gly-Gly, Gly-Cit, Cit-Gly, Ala-Pro, Pro-Ala, Ala-Ser, Ser-Ala, Glu-Val-Cit, Cit-Val- Glu, Glu-Gly-Cit, Cit-Gly-Glu, Asn-Ala-Ala, Ala-Ala-Asn, and Ala-Gly-Ala:
Rz is selected from hydrogen, -CH2N(CH3)C(O)-(CH2CH2O)n2CH3, -CH2N(CH3)(C(O)CH2N(CH3))n2- C(O)CH3, -CH2N(CH3)C(O)-(CH2CH2O)n2-CH2CH2C(O)-(N(CH3)CH2C(O))n2-N(CH3)2, -SO3H, -CO2H, PEG 4-32, polysarcosine, -(CH2N(CH3)C(O))n2CH3, and a sugar moiety; nl, n3, and n4 are each independently 0 or 1 ; n2 is independently an integer from 1 to 20; and the KRAS inhibitor is optionally a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), or (I-g).
KRAS Inhibitor for Generating Conjugates of the Present Disclosure (e.g., of Formula (A))
[140] In certain aspects, a KRAS inhibitor for generating a conjugate of the present disclosure is a compound of Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is selected from N and C(R6);
Z is selected from O, N, C(R5)2, C(O), S, S(O), and S(O)2;
R2, R5, R6, and R8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-i2 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, - N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, - C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted; optionally wherein two R5 are taken together with the atom to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted; and further optionally wherein two R5 are taken together to form =O, =NR12, or =C(R14)2;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, - OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -0C(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), - S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted; optionally wherein two R3 are taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted; optionally wherein two R3 are taken together to form =O, =NR12, or =C(R14)2; and further optionally wherein one R3 and R4 are taken together with the atoms to which they are attached to form optionally substituted 3- to 10-membered heterocycle;
R4 is selected from hydrogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), -C(O)OR12, -C(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), and -S(=O)(=NR12)N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and - (2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted;
R7 is selected from C6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted; m is 0, 1, 2, or 3; n is 1 or 2;
R12 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted;
R13 is independently selected at each occurrence from hydrogen, C1-6 alkyl, and C1-6 haloalkyl; or R12 and R13 attached to the same nitrogen atom form optionally substituted 3- to 10-membered heterocycle; and
R14 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R14 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted; wherein one hydrogen of the compound of Formula (I) is replaced with a bond to the antigen binding unit or the chemical linker.
[141] In embodiments, the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
[142] In embodiments, the compound of Formula (I) has the formula: wherein all variables are as described for Formula (I).
[143] In certain aspects, a KRAS inhibitor for generating a conjugate of the present disclosure is a compound of Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is selected from N and C(R6);
Z is selected from O, N, C(R5)2, C(O), S, S(O), and S(O)2;
R2, R5, R6, and R8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, - N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, - C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R5 are taken together with the atom to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein two R5 are taken together to form =O, =NR12, or =C(R14)2;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, - OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -0C(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), - S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R3 are taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one or more R20; optionally wherein two R3 are taken together to form =O, =NR12, or =C(R14)2; and further optionally wherein one R3 and R4 are taken together with the atoms to which they are attached to form 3- to 10-membered heterocycle optionally substituted with one or more R20;
R4 is selected from hydrogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), -C(O)OR12, -C(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), and -S(=O)(=NR12)N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and - (2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20;
R7 is selected from C6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R20; m is 0, 1, 2, or 3; n is 1 or 2;
R12 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20;
R13 is independently selected at each occurrence from hydrogen, C1-6 alkyl, and C1-6 haloalky I: or R12 and R13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R20;
R14 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R14 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R20;
R20 is independently selected at each occurrence from halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23)-, -S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22; wherein two R20 attached to the same or adjacent atoms optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R21 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R21 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and -OH;
R22 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle); and
R23 is independently selected at each occurrence from hydrogen and C1-6 alkyl; or R22 and R23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I) is replaced with a bond to the antigen binding unit or the chemical linker.
[144] In embodiments, the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit. In embodiments, the compound of
Formula (I) has the formula: wherein all variables are as described for Formula (I).
[145] In embodiments, the KRAS inhibitor for generating a conjugate is a compound of Formula (I-a): or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is selected from N and C(R6);
Z is selected from O, N, C(R5)2, C(O), S, S(O), and S(O)2;
A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms;
R2, R5, R8, R9, and R10 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6- membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -
SR12, -N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -
C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -
S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R5 are taken together with the atom to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein two R5 are taken together to form =O, =NR12, or =C(R14)2; optionally wherein R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein R3 and R9, together with the atoms to which they are attached, form 4- to 8-membered heterocycle optionally substituted with one or more R20;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, - OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -0C(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), - S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R3 are taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one or more R20; optionally wherein two R3 are taken together to form =O, =NR12, or =C(R14)2;
R6 is selected from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -SF5, -N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), - N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -0C(O)R12, -C(O)N(R12)(R13), - C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6- membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalkyl)- (3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R5 are taken together with the atom to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein two R5 are taken together to form =O, =NR12, or =C(R14)2; optionally wherein R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein R3 and R9, together with the atoms to which they are attached, form 4- to 8-membered heterocycle optionally substituted with one or more R20;
R7 is selected from C6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R20; m is 0, 1, 2, or 3; n is 1 or 2;
R11 is selected from hydrogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), (5-methyl-2-oxo-l,3-dioxol-4-yl)methyl, -C(O)OR12, -C(O)OC(O)R12, -C(O)O-(C1-6 alkyl)-OR15, -(C1-6 alkyl)-OR15, -C(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -S(O)2R12, -S(O)(NR12)R12, - S(O)2N(R12)(R13), and -S(O)(NR12)N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20;
R12 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20;
R13 is independently selected at each occurrence from hydrogen, C1-6 alkyl, and C1-6 haloalky I: or R12 and R13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R20;
R14 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R14 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R20;
R20 is independently selected at each occurrence from halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -SF5, =N-OR22, =N-N(R22)(R23), -P(O)(R22)(R23), -ON=R22, -C(O)OR22, -OC(O)N(R22)(R23), - N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), - C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, -S(O)2N(R22)(R23)-, - S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22: wherein two R20 attached to the same or adjacent atoms optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -SF5, =N-OR22, =N- N(R22)(R23), -P(O)(R22)(R23), -ON=R22, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), - N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), - N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23); R21 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R21 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and -OH;
R22 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle); and
R23 is independently selected at each occurrence from hydrogen and C1-6 alkyl; or R22 and R23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker.
[146] In embodiments, the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit. In embodiments, the compound of
Formula (I-a) has the formula: ; wherein all variables are as described for
Formula (I-a).
[147] In certain aspects, a KRAS inhibitor for generating a conjugate of the present disclosure is a compound of Formula (I-a): or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is selected from N and C(R6);
Z is selected from O, N, C(R5)2, C(O), S, S(O), and S(O)2;
A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms;
R2, R5, R6, R8, R9, and R10 are each independently selected at each occurrence from hydrogen, halogen, - CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6- membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, - SR12, -N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, - C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R5 are taken together with the atom to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein two R5 are taken together to form =O, =NR12, or =C(R14)2; optionally wherein R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein R3 and R9, together with the atoms to which they are attached, form 4- to 8-membered heterocycle optionally substituted with one or more R20;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, - OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -OC(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), - S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R3 are taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one or more R20; optionally wherein two R3 are taken together to form =O, =NR12, or =C(R14)2;
R7 is selected from C6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R20; m is 0, 1, 2, or 3; n is 1 or 2;
R11 is selected from hydrogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), (5-methyl-2-oxo-l,3-dioxol-4-yl)methyl, -C(O)OR12, -C(O)OC(O)R12, -C(O)O-(C1-6 alkyl)-OR15, -(C1-6 alkyl)-OR15, -C(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -S(O)2R12, -S(O)(NR12)R12, - S(O)2N(R12)(R13), and -S(O)(NR12)N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20;
R12 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20;
R13 is independently selected at each occurrence from hydrogen, C1-6 alkyl, and C1-6 haloalky I: or R12 and R13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R20;
R14 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R14 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R20;
R20 is independently selected at each occurrence from halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23)-, -S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22; wherein two R20 attached to the same or adjacent atoms optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R21 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R21 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and -OH;
R22 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle); and
R23 is independently selected at each occurrence from hydrogen and C1-6 alkyl; or R22 and R23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I-a) is replaced with a bond to the antigen binding unit or the chemical linker.
[148] In embodiments, the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit. In embodiments, the compound of
Formula (I-a) has the formula: ; wherein all variables are as described for
Formula (I-a).
[149] In certain aspects, a KRAS inhibitor for generating a conjugate of the present disclosure is a compound of Formula (I-a): or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is selected from N and C(R6);
Z is selected from O, N, C(R5)2, C(O), S, S(O), and S(O)2;
A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms;
R2, R5, R6, and R8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, - N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, - C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R5 are taken together with the atom to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein two R5 are taken together to form =O, =NR12, or =C(R14)2;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, - OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -0C(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), - S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R3 are taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one or more R20; optionally wherein two R3 are taken together to form =O, =NR12, or =C(R14)2;
R7 is selected from C6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R20; m is 0, 1, 2, or 3; n is 1 or 2;
R9 and R10 are independently selected from hydrogen, halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23)-, -S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22; wherein R9 and R10 optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2.6 alkenyl, C2.6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, - OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, - C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, - S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R11 is selected from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle);
R12 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20;
R13 is independently selected at each occurrence from hydrogen, C1-6 alkyl, and C1-6 haloalkyl; or R12 and R13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R20;
R14 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C2.6 alkenyl, C2.6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R14 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R20;
R20 is independently selected at each occurrence from halogen, oxo, -CN, C1-6 alkyl, C2.6 alkenyl, C2.6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23)-, -S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22; wherein two R20 attached to the same or adjacent atoms optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2.6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R21 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R21 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and -OH;
R22 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle); and
R23 is independently selected at each occurrence from hydrogen and C1-6 alkyl; or R22 and R23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I-a) is replaced with a bond to the antigen binding unit or the chemical linker.
[150] In embodiments, the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit. In embodiments, the compound of
Formula (I-a) has the formula: ; wherein all variables are as described for
Formula (I-a).
[151] In certain aspects, a KRAS inhibitor for generating a conjugate of the present disclosure is a compound of Formula (I-b): or a pharmaceutically acceptable salt or solvate thereof, wherein:
R2, R6, and R8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered hetero alky nyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, - N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, - C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, - OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -OC(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), - S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R3 are taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one or more R20; and optionally wherein two R3 are taken together to form =O, =NR12, or =C(R14)2; m is 0, 1, 2, or 3; n is 1 or 2;
R12 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20;
R13 is independently selected at each occurrence from hydrogen, C1-6 alkyl, and C1-6 haloalky I: or R12 and R13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R20;
R14 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R14 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R20;
R20 is independently selected at each occurrence from halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23)-, -S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22; wherein two R20 attached to the same or adjacent atoms optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R21 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R21 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and -OH;
R22 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle); and
R23 is independently selected at each occurrence from hydrogen and C1-6 alkyl; or R22 and R23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I-b) is replaced with a bond to the antigen binding unit or the chemical linker.
[152] In embodiments, the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
[153] In some embodiments, the compound of Formula (I-b), is a compound of Formula (I-c): or a pharmaceutically acceptable salt or solvate thereof.
[154] In some embodiments of Formula (I-a), (i) X is N; and/or (ii) R3 is independently selected at each occurrence from C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)- (C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), wherein C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6- membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6- membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20; wherein two R3 are optionally taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20; and further wherein two R3 are optionally taken together to form =O, =NR12, or =C(R14)2; and/or (iii) R9 is selected from C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl, wherein C1-6 alkyl is substituted with =N-OR22, =N-N(R22)(R23), or -ON=R22, and wherein C2-6 alkenyl and C2-6 alkynyl are optionally substituted with one, two, or three R20; and/or (iv) R6 is selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, - C0-6 alkyl-(C3-12 carbocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), - N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -OC(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), - S(O)(NR12)N(R12)(R13), -OCH2C(O)OR12, and -SF5, wherein C2-6 alkenyl and C2-6 alkynyl are optionally substituted with one, two, or three R20; and wherein C1-6 alkyl and -C0-6 alkyl-(C3-12 carbocycle) are each substituted with one, two, or three R20.
[155] In certain aspects, the present disclosure provides a compound of Formula (I-d) applicable for generating a subject conjugate: or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is selected from C(R6) and N;
A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms;
R2, R8, R9, and R10 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, - N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, - C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R5 are taken together with the atom to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein two R5 are taken together to form =O, =NR12, or =C(R14)2; optionally wherein R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein R3 and R9, together with the atoms to which they are attached, form 4- to 8-membered heterocycle optionally substituted with one or more R20;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, - OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -0C(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), - S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R3 are taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one or more R20; optionally wherein two R3 are taken together to form =O, =NR12, or =C(R14)2;
R6 is selected from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -SF5, -N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), - N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -0C(O)R12, -C(O)N(R12)(R13), - C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6- membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalkyl)- (3- to 12-membered heterocycle) are optionally substituted with one or more R20;
R7 is selected from C6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R20; m is 0, 1, 2, or 3; n is 1 or 2;
R11 is selected from hydrogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), (5-methyl-2-oxo-l,3-dioxol-4-yl)methyl, -C(O)OR12, -C(O)OC(O)R12, -C(O)O-(C1-6 alkyl)-OR15, -(C1-6 alkyl)-OR15, -C(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -S(O)2R12, -S(O)(NR12)R12, - S(O)2N(R12)(R13), and -S(O)(NR12)N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; m is 0, 1, 2, or 3; n is 1 or 2;
R12 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20;
R13 is independently selected at each occurrence from hydrogen, C1-6 alkyl, and C1-6 haloalky I: or R12 and R13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R20;
R14 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R14 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R20; R20 is independently selected at each occurrence from halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -SF5, =N-OR22, =N-N(R22)(R23), -P(O)(R22)(R23), -ON=R22, -C(O)OR22, -OC(O)N(R22)(R23), - N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), - C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, -S(O)2N(R22)(R23)-, - S(O)(NR22)N(R22)(R23), and -OCI PC(O)OR22: wherein two R20 attached to the same or adjacent atoms optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -SF5, =N-OR22, =N- N(R22)(R23), -P(O)(R22)(R23), -ON=R22, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), - N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), - N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R21 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R21 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and -OH;
R22 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle); and
R23 is independently selected at each occurrence from hydrogen and C1-6 alkyl; or R22 and R23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker.
[156] In embodiments, the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
[157] In certain aspects, the present disclosure provides a compound of Formula (I-e) applicable for generating a subject conjugate,: or a pharmaceutically acceptable salt or solvate thereof, wherein:
A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms; R2, R8, and R10 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, - N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, - C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20;
R9 is selected from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), - N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), - N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein R3 and R9, together with the atoms to which they are attached, form 4- to 8-membered heterocycle optionally substituted with one or more R20;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, - OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -OC(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), - S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R3 are taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one or more R20; optionally wherein two R3 are taken together to form =O, =NR12, or =C(R14)2;
R6 is selected from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -SF5, -N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), - N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), - C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6- membered hetero alkenyl, 3- to 6-membered hetero alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalkyl)- (3- to 12-membered heterocycle) are optionally substituted with one or more R20;
R7 is selected from C6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R20; m is 0, 1, 2, or 3; n is 1 or 2;
R11 is selected from hydrogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), (5-methyl-2-oxo-l,3-dioxol-4-yl)methyl, -C(O)OR12, -C(O)OC(O)R12, -C(O)O-(C1-6 alkyl)-OR15, -(C1-6 alkyl)-OR15, -C(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -S(O)2R12, -S(O)(NR12)R12, - S(O)2N(R12)(R13), and -S(O)(NR12)N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; m is 0, 1, 2, or 3; n is 1 or 2;
R12 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20;
R13 is independently selected at each occurrence from hydrogen, C1-6 alkyl, and C1-6 haloalky I: or R12 and R13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R20;
R14 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R14 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R20;
R20 is independently selected at each occurrence from halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -SF5, =N-OR22, =N-N(R22)(R23), -P(O)(R22)(R23), -ON=R22, -C(O)OR22, -OC(O)N(R22)(R23), - N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), - C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, -S(O)2N(R22)(R23)-, - S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22: wherein two R20 attached to the same or adjacent atoms optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -SF5, =N-OR22, =N- N(R22)(R23), -P(O)(R22)(R23), -ON=R22, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), - N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), - N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R21 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R21 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and -OH;
R22 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle); and
R23 is independently selected at each occurrence from hydrogen and C1-6 alkyl; or R22 and R23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker.
[158] In embodiments, the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
[159] In some embodiments, for a compound of Formula (I-e) applicable for generating a subject conjugate, R10 is halogen. In some embodiments, for a compound of Formula (I-e), R10 is -F. In some embodiments, for a compound of Formula (I-e), R10 is -Cl. In some embodiments, for a compound of Formula (I-e), R10 is -Br. In some embodiments, for a compound of Formula (I-e), R10 is C1-6 alkyl optionally substituted with one, two, or three R20. In some embodiments, for a compound of Formula (I-e), R10 is C1-6 alkyl optionally substituted with one, two, or three halogen. In some embodiments, for a compound of Formula (I-e), R10 is C1-6 alkyl optionally substituted with one, two, or three -F. In some embodiments, for a compound of Formula (I-e), R10 is unsubstituted methyl. In some embodiments, for a compound of Formula (I-e), R10 is unsubstituted C1-6 alkyl. In some embodiments, for a compound of Formula (I-e), R10 is -CF3. In some embodiments, for a compound of Formula (I-e), R10 is -CHF2. In some embodiments, for a compound of Formula (I-e), R10 is -OH. In some embodiments, the substituents (for example, R2, R3, R6, R7, R8, R9, and R10) of formula (I-e) are the same as the corresponding substituents in Formula
(I), (I-a), (I-b), (I-c), and/or (I-d). In some embodiments, for a compound of Formula (I-e), is selected compound of Formula (I-e), is selected from
[160] In some embodiments, for a compound of Formula (I) or (I-a) applicable for generating a subject conjugate, X is C(R6), such as C(Cl). In some embodiments, X is N. In some embodiments, X is C(R6); and R6 and R8 are independently selected from hydrogen, halogen, and C1-3 haloalkyl. In some embodiments, X is C(R6); R6 is selected from chlorine and -CF3: and R8 is fluorine. In some embodiments, X is N; and R8 is selected from hydrogen, halogen, and C1-3 haloalkyl. In some embodiments, X is N; and R8 is fluorine. In some embodiments, X is C(R6); and Z is selected from O and C(R5)2. In some embodiments, X is C(R6); and Z is selected from O and CH2. In some embodiments, X is C(R6); Z is selected from O and C(R5)2: and R6 and R8 are independently selected from hydrogen, halogen, and C1-3 haloalkyl. In some embodiments, X is C(R6); Z is selected from O and C(R5)2: R6 is selected from chlorine and -CF3: and R8 is fluorine. In some embodiments, X is C(R6); Z is selected from O and CH2: and R6 and R8 are independently selected from hydrogen, halogen, and C1-3 haloalkyl. In some embodiments, X is C(R6); Z is selected from O and CH2: R6 is selected from chlorine and -CF3; and R8 is fluorine. In some embodiments, X is N; Z is selected from O and CH2: and R8 is selected from hydrogen, halogen, and C1-3 haloalkyl. In some embodiments, X is N; Z is selected from O and CH2: and R8 is selected from hydrogen, halogen, and C1-3 haloalkyl. In some embodiments, X is N; Z is selected from O and CH2: and R8 is fluorine. In some embodiments, X is N; Z is selected from O and CH2: and R8 is fluorine.
[161] In some embodiments, for a compound of Formula (I) applicable for generating a subject conjugate, R4 is selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), each of which is optionally substituted with one or more R20. In some embodiments, R4 is selected from C1-6 alkyl and -C0-6 alkyl-(3- to 12-membered heterocycle), each of which is optionally substituted with one or more R20. In some embodiments, R4 is -C1-3 alkyl-(3- to 9-membered heterocycle) optionally substituted with one or more R20, and further optionally wherein the 3- to 9-membered heterocycle is selected from azetidinyl, thietanyl,
1,1 -dioxide, imidazolyl, thiazolyl, isothiazolyl, triazolyl, pyrazolyl, pyrazinyl, pyidonyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrrolopyridinyl, and pyrazolopyridinyl. In some embodiments, the 3- to 9-membered heterocycle is selected from pyrazolyl, pyridonyl, pyridinyl, pyrimidinyl, and pyridazinyl. In some embodiments, the 3- to 9- membered heterocycle is pyridinyl. In some embodiments, R4 is -C1-3 alkyl-(pyridine) optionally substituted with one or more R20. In some embodiments, R4 is -C1-3 alkyl-(3- to 9-membered heterocycle), wherein the 3- to 9- membered heterocycle is substituted with -NH2. In some embodiments, R4 is -C1-3 alkyl-(pyridine), wherein the pyridine is substituted with -NH2.
[162] In some embodiments, for a compound of Formula (I-a) applicable for generating a subject conjugate, A is 6-membered heteroaryl comprising one or two ring nitrogen atoms. In some embodiments, A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl. In some embodiments, A is selected from pyridin-3-yl, pyridazin- 4-yl, pyrimidin-5-yl, and pyrazin-2-yl. In some embodiments, A is pyridinyl. In some embodiments, A is pyridin-3- yl.
[163] In some embodiments, for a compound of Formula (I-a) applicable for generating a subject conjugate, R9 is selected from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, and -N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky 1)- (C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalkyl)-(3- to 12- membered heterocycle) are optionally substituted with one, two, or three R20; or R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20; or R3 and R9, together with the atoms to which they are attached, form 4- to 8-membered heterocycle optionally substituted with one, two, or three R20. In some embodiments, R9 is selected from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, and -N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky 1)- (C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalkyl)-(3- to 12- membered heterocycle) are optionally substituted with one, two, or three R20. In some embodiments, R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20. In some embodiments, R3 and R9, together with the atoms to which they are attached, form 4- to 8-membered heterocycle optionally substituted with one, two, or three R20. In some embodiments, R9 is C1-3 alkyl optionally substituted with one, two, or three R20; or R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20. In some embodiments, R9 is C1-3 alkyl optionally substituted with one, two, or three R20. In some embodiments, R9 is C1-6 alkyl substituted with -N(R12)C(O)R12, such as -NHC(O)(C2- 6 alkenyl). In some embodiments, R9 is C1-3 alkyl substituted with -N(R12)C(O)R12, such as -NHC(O)(C2-6 alkenyl). In some embodiments, R9 is C1-6 alkyl substituted with -N(R22)C(O)R22, such as -NHC(O)(C2-6 alkenyl). In some embodiments, R9 is C1-3 alkyl substituted with -N(R22)C(O)R22, such as -NHC(O)(C2-6 alkenyl). In some embodiments, R9 is C1-6 alkyl substituted with -NHC(O)CHCH2. In some embodiments, R9 is C1-3 haloalkyl, such as -CH2F, -CHF2, -CF3, -CH2CH2F, -CH2CHF2, or -CH2CF3. In some embodiments, R9 is selected from -CH3, -CHF2, - CH2CH3, and CH2CHF2. In some embodiments, R9 is C1-3 alkyl. In some embodiments, R9 is CH3. In some
[164] In some embodiments, for a compound of Formula (I-a) applicable for generating a subject conjugate, R9 is selected from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, and -N(R22)(R23), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky 1)- (C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalkyl)-(3- to 12- membered heterocycle) are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23). In some embodiments, R9 is C1-3 alkyl optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), - N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), - C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, -S(O)2N(R22)(R23), and - S(O)(NR22)N(R22)(R23).
[165] In embodiments, R9 is selected from C2-6 alkenyl and C2-6 alkynyl, wherein C2-6 alkenyl and C2-6 alkynyl are optionally substituted with one, two, or three substituents selected from halogen, and C3-6 carbocycle. In embodiments, R9 is selected from C2-4 alkenyl and C2-4 alkynyl, wherein C2-4 alkenyl and C2-4 alkynyl are optionally substituted with one, two, or three substituents selected from F and cyclopropyl. In embodiments, R9 is selected
[166] In embodiments, R9 is selected from -C1-6 alkyl-(C3-6 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-6 carbocycle), -C1-6 alkyl-(3- to 6-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 6-membered heterocycle), wherein -C1-6 alkyl-(C3-6 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-6 carbocycle), -C1-6 alkyl-(3- to 6-membered heterocycle), and -(2- to 6-membered heteroalkyl)-(3- to 6-membered heterocycle) are each optionally substituted with one, two, or three substituents selected from oxo, -OR22, and C1-6 alkyl optionally substituted with one or more substituents independently selected from oxo, -OR22, and -N(R22)(R23). In embodiments, R9 is selected from -C1-3 alkyl-(C3-6 carbocycle), -(2- to 3 -membered heteroalky l)-(C3-6 carbocycle), - C1-3 alkyl-(4- to 6-membered heterocycle), and -(2- to 3-membered heteroalkyl)-(4- to 6-membered heterocycle), wherein -C1-3 alkyl-(C3-6 carbocycle), -(2- to 3-membered heteroalkyl)-(C3-6 carbocycle), -C1-3 alkyl-(4- to 6- membered heterocycle), and -(2- to 3 -membered heteroalky l)-(4- to 6-membered heterocycle) are each optionally substituted with one, two, or three substituents selected from oxo, -OCH3, and C1-6 alkyl optionally substituted with one or more substituents independently selected from oxo and -NH2. In embodiments, R9 is selected from -C1-3 alkyl-(C3-6 saturated carbocycle), -(2- to 3-membered heteroalkyl)-(C3-6 saturated carbocycle), -C1-3 alkyl-(4- to 6- membered saturated heterocycle), -(2- to 3-membered hetero alkyl) -(4- to 6-membered saturated heterocycle), -C1-3 alkyl-(5- to 6-membered heteroaryl), and -(2- to 3-membered heteroalkyl)-(5- to 6-membered heteroaryl), wherein - C1-3 alkyl-(C3-6 saturated carbocycle), -(2- to 3-membered heteroalkyl)-(C3-6 saturated carbocycle), -C1-3 alkyl-(4- to 6-membered saturated heterocycle), -(2- to 3 -membered hetero alkyl) -(4- to 6-membered saturated heterocycle), -C1-3 alkyl-(5- to 6-membered heteroaryl), and -(2- to 3-membered heteroalkyl)-(5- to 6-membered heteroaryl) are each optionally substituted with one, two, or three substituents selected from oxo, -OCH3, -CH3, C(O)CH3, C(O)C(NH2)(CH(CH3)2. In embodiments, R9 is selected from
[167] In embodiments, R9 is C1-6 alkyl optionally substituted with one, two, or three substituents selected from halogen, oxo, -OR22, -N(R22)(R23), -S(O)R22, -C(O)N(R22)(R23), -N(R22)C(O)R22, -S(O)2R22, -P(O)(R22)(R23), and
=N-OR22. In embodiments, R9 is C1-6 alkyl optionally substituted with one, two, or three substituents selected from
F, -NH2, -N(CH3)2, -N(CH2CH3)2, OXO, -OH, -OCH3, -OCHF2, -OCF3, -S(O)CH3, -C(O)N(H)(CH3), - N(CH3)C(O)CH3, -S(O)2CH3, -P(O)(CH3)2, and =N-OCH2CH3. In embodiments, R9 is selected from
[168] In some embodiments, for a compound of Formula (I-a) applicable for generating a subject conjugate, R10 is selected from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, -OR12, and -N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, and 2- to 6-membered heteroalkyl are optionally substituted with one, two, or three R20; or R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20. In some embodiments, R10is selected from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6- membered heteroalkyl, -OR12, and -N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, and 2- to 6-membered heteroalkyl are optionally substituted with one, two, or three R20. In some embodiments, R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20. In some embodiments, R10 is selected from hydrogen and halogen; or R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20. In some embodiments, R10 is selected from hydrogen and halogen. In some embodiments, R10 is hydrogen.
[169] In some embodiments, for a compound of Formula (I-a) applicable for generating a subject conjugate, R10 is selected from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, -OR22, and -N(R22)(R23), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, and 2- to 6-membered heteroalkyl are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), - N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), - C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, -S(O)2N(R22)(R23), and - S(O)(NR22)N(R22)(R23). In some embodiments, R10 is selected from hydrogen and halogen.
[170] In some embodiments, for a compound of Formula (I-a) applicable for generating a subject conjugate, R11 is selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12- membered heterocycle), -C(O)R12, and -C(O)N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl- (C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20. In some embodiments, R11 is selected from hydrogen, C1-6 alkyl, and -C(O)R12. In some embodiments, R11 is hydrogen. In some embodiments, R11 is C1-6 alkyl, such as -CH3. In some embodiments, R11 is -C(O)R12. In some embodiments, R11 is selected from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle). In some embodiments, R11 is a bond to the antigen binding unit or the chemical linker.
[171] In some embodiments, for a compound of Formula (I-a) applicable for generating a subject conjugate,
[173] In some embodiments, is selected from ’
[174] In some embodiments, for a compound of Formula (I) or (I-a) applicable for generating a subject conjugate, R7 is selected from C6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R20. In some embodiments, R7 is selected from C10 aryl and 9-membered heteroaryl, each of which is optionally substituted with one or more R20. In some embodiments, R7 is selected from naphthalenyl and benzothiophenyl, each of which is optionally substituted with one or more R20. In some embodiments, R7 is selected from C3-10 cycloalkyl, 3- to 10-membered heterocycloalkyl, C6-10 aryl, and 5- to 10-membered heteroaryl, each of which is optionally substituted with one or more R20. In some embodiments, R7 is selected from bicyclic C4-10 cycloalkyl, bicyclic 4- to 10-membered heterocycloalkyl, bicyclic C7-10 aryl, and bicyclic 7- to 10-membered heteroaryl, each of which is optionally substituted with one or more R20. In some embodiments, R7 is selected from bridged bicyclic C4-10 cycloalkyl, bridged bicyclic 4- to 10-membered heterocycloalkyl, bridged bicyclic C7-10 aryl, and bridged bicyclic 7- to 10-membered heteroaryl, each of which is optionally substituted with one or more R20. In some embodiments, R7 is selected from fused bicyclic C4-10 cycloalkyl, fused bicyclic 4- to 10-membered heterocycloalkyl, fused bicyclic C7-10 aryl, and fused bicyclic 7- to 10-membered heteroaryl, each of which is optionally substituted with one or more R20. In some embodiments, R7 is selected from C6-10 aryl and 5- to 10- membered heteroaryl, each of which is optionally substituted with one, two, three, four, or five R20. In some embodiments, R7 is selected from naphthyl, isoquinolinyl, indazolyl, benzothiazolyl, benzothiophenyl, phenyl, and pyridinyl, each of which is optionally substituted with one or more R20. In some embodiments, R7 is naphthyl, optionally substituted with one or more R20. In some embodiments, R7 is benzothiophenyl, optionally substituted with one or more R20. In some embodiments, R7 is phenyl, optionally substituted with one or more R20. In some embodiments, R7 is pyridinyl, optionally substituted with one or more R20. In some embodiments, R7 is substituted with one, two, three, or four substituents independently selected from halogen, -CN, C1-3 alkyl, C1-3 haloalky 1, C2-3 alkenyl, C2-3 alkynyl, -OR22, -N(R22)(R23), and C3-6 cycloalkyl. In some embodiments, R7 is selected from C6 aryl and 6-membered heteroaryl, each of which is substituted with one, two, three, four, or five R20. In some embodiments, R7 is optionally substituted with one or more R20, such as one, two, three, four, five, six, or seven R20. In some embodiments, R7 is selected from benzothiophenyl, thienopyridinyl, furopyridinyl, and naphthalenyl: wherein each is optionally substituted with one, two, three, four, or five R20.
[175] In some embodiments, for a compound of Formula (I), (I-a), (I-d), or (I-e) applicable for generating a subject conjugate, R7 is selected from: wherein:
Q1, Q3, and Q5 are independently selected from N and C(R1a);
Q4 and Q6 are independently selected from O, S, C(R1a)2, and N(R1b);
X4, X5, X6, X9, and X10 are independently selected from C(R1a) and N;
X7 and X8 are independently selected from C(R1a), C(R1a)2, N, and N(R1b); X13 is selected from a bond, C(R1a), N, C(O), C(R1a)2, C(O)C(R1a)2, C(R1 a)2C(R1a)2, C(R1a)2N(R1b), and
N(R1b);
X14, X15, X17, and X18 are independently selected from C(O), C(R1a), N, C(R1a)2, and N(R1b);
X16 is selected from C, N, and C(R1a); each R1a is independently selected from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, - OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -OC(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), and - S(=O)(=NR12)N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6- membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalkyl)- (3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20; or two R1a bonded to the same carbon are joined to form 3- to 10-membered heterocycle or C3-10 carbocycle, wherein 3- to 10-membered heterocycle and C3-10 carbocycle are optionally substituted with one, two, or three R20; or two R1a bonded to adjacent atoms are joined to form 3- to 10-membered heterocycle or C3-10 carbocycle, wherein 3- to 10-membered heterocycle and C3-10 carbocycle are optionally substituted with one, two, or three R20; or one R1a and one R1b are joined to form 3- to 10-membered heterocycle or C3-10 carbocycle, wherein 3- to 10-membered heterocycle and C3-10 carbocycle are optionally substituted with one, two, or three R20; each R1b is independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 3- to 10-membered heterocycle, and C3-10 carbocycle, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 3- to 10-membered heterocycle, and C3-10 carbocycle are optionally substituted with one, two, or three R20; and indicates a single or double bond such that all valences are satisfied.
[176] In some embodiments, for a compound of Formula (I), (I-a), (I-d), or (I-e) applicable for generating a subject conjugate, R7 is selected from
[177] In some embodiments, for a compound of Formula (I), (I-a), (I-d), or (I-e) applicable for generating a subject conjugate, R7 is benzothiophenyl optionally substituted with one or more R20. In some embodiments, R7 is benzothiophenyl optionally substituted with one, two, three, or four R20. In some embodiments, R7 is benzothiophenyl substituted with three R20. In some embodiments, R7 is benzo [b]thiophen-4-yl optionally substituted with one, two, three, or four R20. In some embodiments, R7 is benzo [b]thiophen-4-yl substituted with three R20. In some embodiments, R7 is selected from
[178] In some embodiments, for a compound of Formula (I), (I-a), (I-d), or (I-e) applicable for generating a subject conjugate, R7 is selected from
[179] In some embodiments, for a compound of Formula (I), (I-a), (I-d), or (I-e) applicable for generating a subject conjugate, R7 is substituted with one, two, three, or four substituents independently selected from halogen, - CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, -OR22, -SR22, and -N(R22)(R23), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, and C3-6 cycloalkyl are optionally substituted with one, two, or three substituents independently selected from halogen, C1-6 alkyl, C1-6 haloalkyl, and -OR22. In some embodiments, R7 is substituted with one, two, three, or four substituents independently selected from halogen, -CN, C1-3 alkyl, C2-3 alkenyl, C2-3 alkynyl, -OR22, and -N(R22)(R23). In some embodiments, R7 is substituted with one, two, three, or four substituents independently selected from halogen, -CN, -CH3, -C=CH, -OH, and -NH2. In some embodiments, R7 is substituted with -F, -CN, and -NH2. In some embodiments, R7 is substituted with -F, -C=CH, and -OH. In some embodiments, R7 is substituted with -CF3, -CH3, and -NH2. In some embodiments, R7 is substituted with -CF3 and -NH2. In some embodiments, R7 is substituted with -CF3, -CH3, -F, and -NH2. In some embodiments, R7 is substituted with -CF3, - F, and -NH2. In some embodiments, R7 is substituted with one, two, three, or four substituents independently selected from halogen, -CN, -CH3, -CH2CH3, -CH=CH2, -CF3, -C=CH, -OH, -NH2, and -cyclopropyl. In some embodiments, R7 is substituted with one, two, or three substituents independently selected from halogen, -CN, and - N(R22)(R23). In some embodiments, R7 is substituted with one, two, or three substituents independently selected from fluorine, chlorine, -CN, and -NH2.
[180] In some embodiments, for a compound of Formula (I) applicable for generating a subject conjugate:
X is selected from C(R6) and N;
Z is selected from O and C(R5)2;
R2, R3, R6, and R8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), -C(O)OR12, - N(R12)C(O)N(R12)(R13), -C(O)R12, -OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20; wherein two R3 are optionally taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20; and further wherein two R3 are optionally taken together to form =O, =NR12, or =C(R14)2;
R7 is selected from naphthyl, benzothiophenyl, phenyl, and pyridinyl, each of which is optionally substituted with one or more R20; m is 0 or 1 ; and n is 1 or 2.
[181] In some embodiments, for a compound of Formula (I) applicable for generating a subject conjugate:
X is selected from C(R6) and N;
Z is selected from O, CH2, and CI I(CI F,): R2 is -OR12;
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20;
R6 and R8 are independently selected from hydrogen, halogen, and C1-3 haloalkyl;
R7 is selected from naphthyl, benzothiophenyl, phenyl, and pyridinyl, each of which is optionally substituted with one or more R20; m is 0 or 1 ; and n is 1 or 2.
[182] In some embodiments, for a compound of Formula (I-a) applicable for generating a subject conjugate:
X is selected from C(R6) and N;
Z is selected from O and C(R5)2;
A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl;
R2, R3, R6, R8, and R10 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), - C(O)OR12, -N(R12)C(O)N(R12)(R13), -C(O)R12, -OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20; wherein two R3 are optionally taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20; and further wherein two R3 are optionally taken together to form =O, =NR12, or =C(R14)2;
R7 is selected from naphthyl, benzothiophenyl, phenyl, and pyridinyl, each of which is optionally substituted with one or more R20;
R9 is C1-3 alkyl optionally substituted with one, two, or three R20;
R11 is hydrogen; m is 0 or 1 ; and n is 1 or 2.
[183] In some embodiments, for a compound of Formula (I-a) applicable for generating a subject conjugate:
X is C(R6);
A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl;
R11 is hydrogen;
R2, R6, R8, and R10 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), -C(O)OR12, - N(R12)C(O)N(R12)(R13), -C(O)R12, -OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, - C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), -C(O)OR12, -N(R12)C(O)N(R12)(R13), -C(O)R12, -OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20; wherein two R3 are optionally taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one, two, or three R20; and further wherein two R3 are optionally taken together to form =O, =NR12, or =C(R14)2;
R7 is benzo [b]thiophen-4-yl optionally substituted with one, two, three, or four R20;
R9 is C1-3 alkyl optionally substituted with one, two, or three R20; m is 0 or 1 ; and n is 1 or 2.
[184] In some embodiments, for a compound of Formula (I-a) applicable for generating a subject conjugate:
X is N;
A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl;
R11 is hydrogen;
R2, R6, R8, and R10 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), -C(O)OR12, - N(R12)C(O)N(R12)(R13), -C(O)R12, -OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, - C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), -C(O)OR12, -N(R12)C(O)N(R12)(R13), -C(O)R12, -OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20; wherein two R3 are optionally taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one, two, or three R20; and further wherein two R3 are optionally taken together to form =O, =NR12, or =C(R14)2;
R7 is selected from naphthyl, phenyl, and pyridinyl, each of which is optionally substituted with one or more R20;
R9 is C1-3 alkyl optionally substituted with one, two, or three R20; m is 0 or 1 ; and n is 1 or 2.
[185] In some embodiments, for a compound of Formula (I-a) applicable for generating a subject conjugate:
A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl;
R2 is selected from hydrogen, C1-3 alkyl, -OR12, and 3- to 10-membered heterocycle, wherein C1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R20;
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20;
R6 and R8 are independently selected from hydrogen, halogen, and C1-3 haloalkyl;
R9 is C1-3 alkyl optionally substituted with one, two, or three R20;
R11 is hydrogen; and m is 0 or 1.
[186] In some embodiments, for a compound of Formula (I), (I-a), (I-b) or (I-c) applicable for generating a subject conjugate:
R2, R3, R6, and R8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), -C(O)OR12, - N(R12)C(O)N(R12)(R13), -C(O)R12, -OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20; wherein two R3 are optionally taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20; and further wherein two R3 are optionally taken together to form =O, =NR12, or =C(R14)2; and m is 0 or 1.
[187] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate:
R2 is selected from hydrogen, C1-3 alkyl, -OR12, and 3- to 10-membered heterocycle, wherein C1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R20;
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20;
R6 and R8 are independently selected from hydrogen, halogen, and C1-3 haloalkyl; and m is 0 or 1.
[188] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate:
R2 is -OR12;
R3 is independently selected at each occurrence from C1-6 alkyl optionally substituted with one, two, or three R20; m is 0 or 1 ;
R6 is selected from chlorine and -CF3; and
R8 is fluorine.
[189] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate:
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, -OH, and -OCH3; m is 0 or 1 ;
R6 is selected from chlorine and -CF3; and
R8 is fluorine.
[190] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate :
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, -OH, and -OCH3; m is 0 or 1 ;
R6 is selected from chlorine and -CF3;
R8 is fluorine; and n is 1. [191] In some embodiments, for a compound of Formula (I) or (I-a) applicable for generating a subject conjugate:
X is C(R6);
Z is O;
R2 is
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, -OH, and -OCH3; m is 0 or 1 ;
R6 is selected from chlorine and -CF3:
R7 is pyridinyl optionally substituted with one or more R20;
R8 is fluorine; and n is 1.
[192] In some embodiments, for a compound of Formula (I) or (I-a) applicable for generating a subject conjugate: X is N;
Z is selected from O and C(R5)2;
R2 is
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, -OH, and -OCH3; m is 0 or 1 ;
R7 is naphthyl optionally substituted with one or more R20;
R8 is fluorine; and n is 1 or 2.
[193] In some embodiments, the compound of Formula (I) or (I-a) applicable for generating a subject conjugate, is selected from:
or a pharmaceutically acceptable salt or solvate thereof.
[194] In some embodiments, the compound of Formula (I) or (I-a) applicable for generating a subject conjugate, is selected from:
[195] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate , R2 is selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C3-10 carbocycle, 3- to 10- membered heterocycle, -OR12, and -N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C3-10 carbocycle, and 3- to 10- membered heterocycle are optionally substituted with one, two, or three R20. In some embodiments, R2 is selected from hydrogen, -(C0-3 alkylene)-O-(C0-3 alkylene)-R20, C1-3 alkyl, and 3- to 10-membered heterocycle, wherein each C0-3 alkylene, C1-3 alkyl, and 3- to 10-membered heterocycle is optionally substituted with one, two, or three R20. In some embodiments, R2 is selected from hydrogen, C1-3 alkyl, -OR12, and 3- to 10-membered heterocycle, wherein C1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R20. In some embodiments, R2 is OR12. In some embodiments, R2 is -O(C1-3 alkylene)(4- to 10-membered heterocycle), wherein
4- to 10-membered heterocycle is optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and =C(R21)2, wherein R21 is independently selected at each occurrence from hydrogen, halogen, and C1-3 alkyl. In some embodiments, R2 is -OCH2(hexahydro-lH-pyrrolizine) optionally substituted with one, two, or three R20. In some embodiments, R2 is -OCH2(hexahydro-lH-pyrrolizine) optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and =C(R21)2, wherein R21 is independently selected at each occurrence from hydrogen, halogen, and C1-3 alkyl.
[196] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for
[197] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate, R2 is substituted with one, two, three, or four substituents independently selected from halogen, oxo, C1-6 alkyl, -OR22, -N(R22)(R23), =C(R21)2, and -OC(O)N(R22)(R23), wherein C1-6 alkyl is optionally substituted with one or more substituents independently selected from halogen, -CN, -OR22, -N(R22)(R23), and -OC(O)N(R22)(R23). In some embodiments, R2 is substituted with one, two, three, or four substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and =C(R21)2, wherein R21 is independently selected at each occurrence from hydrogen, halogen, and C1-3 alkyl. In some embodiments, R2 is substituted with one, two, three, or four substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, =CH2, =CHF, and =CF2. In some embodiments, R2 is substituted with =C(R21)2, such as =CF2. In some embodiments, R2 is substituted with halogen, such as fluorine.
[198] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate, R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, - N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, - C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20; wherein two R3 are optionally taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20; and further wherein two R3 are optionally taken together to form =O, =NR12, or =C(R14)2. In some embodiments, two R3 are taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20. In some embodiments, two R3 are taken together with the atom to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one, two, or three R20. In some embodiments, two R3 attached to adjacent atoms are taken together with the atoms to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20. In some embodiments, two R3 are taken together to form =O, =NR12, or =C(R14)2.
[199] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate, R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-6 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-e carbocycle), -C0-6 alkyl-(3- to 6-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 6-membered heterocycle), -OR12, and - N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-e carbocycle), -(2- to 6-membered heteroalky l)-(C3-6 carbocycle), -C0-6 alkyl-(3- to 6-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 6-membered heterocycle) are optionally substituted with one, two, or three R20; wherein two R3 are optionally taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20; and further wherein two R3 are optionally taken together to form =O, =NR12, or =C(R14)2. In some embodiments, R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20. In some embodiments, R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, - OH, and -OCH3. In some embodiments, R3 is C2-3 alkenyl. In some embodiments, R3 is C2-3 alkynyl. In some embodiments, R3 is C1-3 haloalkyl. In some embodiments, R3 is selected from -CHCH2, -CCH, -CH2CN, and -CHF2.
[200] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate, m is 0, 1, or 2. In some embodiments, m is 0 or 1. In some embodiments, m is 1 or 2. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3.
[201] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate, Z is selected from O, N, C(R5)2, and C(O). In some embodiments, Z is selected from S, S(O), and S(O)2. In some embodiments, Z is selected from O and C(R5)2. In some embodiments, Z is selected from O, CH2, and CH(CH3). In some embodiments, Z is O. In some embodiments, Z is CH2. In some embodiments, Z is CH(CH3).
[202] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate, R6 is selected from hydrogen, halogen, -CN, C1-6 alkyl, 2- to 6-membered heteroalkyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -OR12, and -N(R12)(R13), wherein C1-6 alkyl, 2- to 6-membered heteroalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one or more R20. In some embodiments, R6 is selected from hydrogen, halogen, C1-3 alkyl. In some embodiments, R6 is selected from hydrogen, halogen, and C1-3 haloalkyl. In some embodiments, R6 is selected from halogen and C1-3 haloalkyl. In some embodiments, R6 is selected from hydrogen and halogen. In some embodiments, R6 is hydrogen. In some embodiments, R6 is halogen, such as fluorine. In some embodiments, R6 is chlorine. In some embodiments, R6 is C1-3 haloalkyl, such as -CHF2. In some embodiments, R6 is -CF3. In some embodiments, R6 is -CH2CN. In some embodiments, R6 is -NH2. In some embodiments, R6 is -
N( C1 [3)2. In some embodiments, R6 is -N(CH2CH3)2. In some embodiments, R6 is -SF5.
[203] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate, R8 is selected from hydrogen, halogen, -CN, C1-6 alkyl, 2- to 6-membered heteroalkyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -OR12, and -N(R12)(R13), wherein C1-6 alkyl, 2- to 6-membered heteroalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one or more R20. In some embodiments, R8 is selected from hydrogen, halogen, and C1-3 alkyl. In some embodiments, R8 is selected from hydrogen and halogen. In some embodiments, R8 is hydrogen. In some embodiments, R8 is halogen, such as chlorine. In some embodiments, R8 is fluorine. In some embodiments, R6 and R8 are independently selected from hydrogen, halogen, and C1-3 haloalkyl. In some embodiments, R6 and R8 are independently selected from C1-3 haloalky 1 and halogen. In some embodiments, R6 and R8 are independently selected from hydrogen, halogen, and -CF3. In some embodiments, R6 and R8 are independently selected from -Cl, -F, and -CF3. In some embodiments, R6 and R8 are independently selected from hydrogen and halogen.
[204] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate, n is 0, 1, 2, or 3. In some embodiments, n is 1. In some embodiments, n is 2.
[205] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate:
R2 is selected from hydrogen, C1-3 alkyl, -OR12, and 3- to 10-membered heterocycle, wherein C1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R20;
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20; m is 0 or 1 ; and
R6 and R8 are independently selected from hydrogen, halogen, and CF3.
[206] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate:
R2 is selected from hydrogen, C1-3 alkyl, -OR12, and 3- to 10-membered heterocycle, wherein C1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R20;
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20; m is 0 or 1 ;
R6 and R8 are independently selected from hydrogen, halogen, and -CF3: and n is 1.
[207] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate:
R2 is selected from hydrogen, C1-3 alkyl, -OR12, and 3- to 10-membered heterocycle, wherein C1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R20;
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20; m is 0 or 1 ;
R6 is selected from chlorine and -CTT and
R8 is fluorine.
[208] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate:
R2 is selected from hydrogen, C1-3 alkyl, -OR12, and 3- to 10-membered heterocycle, wherein C1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R20;
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20; m is 0 or 1 ;
R6 is selected from chlorine and -CTT
R8 is fluorine; and n is 1.
[209] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate:
R2 is
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, -OH, and -OCH3; m is 0 or 1 ;
R6 is selected from chlorine and -CF3: and
R8 is fluorine.
[210] In some embodiments, for a compound of Formula (I), (I-a), (I-b), (I-c), (I-d), or (I-e) applicable for generating a subject conjugate:
R2 is
R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, -OH, and -OCH3; m is 0 or 1 ;
R6 is selected from chlorine and -CF3:
R8 is fluorine; and n is 1.
[211] In some embodiments, a compound of Formula (I), (I-a), (I-b), or (I-c) applicable for generating a subject conjugate, has the formula: wherein:
Ring A is 6-membered heteroaryl comprising one or two ring nitrogen atoms;
R2 is selected from -O-CH2-(8- to 10-membered saturated heterocycle) and -O-CH2-(cyclopropylene)-CH2- (5- to 8-membered saturated heterocycle); wherein -O-CH2-(8- to 10-membered saturated heterocycle) and -O-CH2- (cyclopropylene)-CH2-(5- to 8-membered saturated heterocycle) are optionally substituted with one or more substituents independently selected from -F, =CF2, =CH2, and =CHF;
R9 is selected from C1-3 alkyl, C2-4 alkenyl, C2-3 alkynyl, -C1-2 alkyl-(C3-4 saturated carbocycle), and -C1-2 alkyl-(5- to 6-membered saturated heterocycle); wherein C1-3 alkyl, C2-4 alkenyl, C2-3 alkynyl, -C1-2 alkyl-(C3-4 saturated carbocycle), and -C1-2 alkyl-(5- to 6-membered saturated heterocycle) are each optionally substituted with one or more halogen, R10 is selected from hydrogen and halogen;
R6 is selected from halogen and -CF3;
R7 is benzothiophenyl optionally substituted with one or more substituents independently selected from -
NH2, -CN, and -F;
R8 is halogen; and wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker.
[212] In embodiments, the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
[213] In embodiments of a compound of Formula (I-f) applicable for generating a subject conjugate, R6 is -CF3. In embodiments of a compound of Formula (I-f) applicable for generating a subject conjugate, R5 is halogen. In embodiments of a compound of Formula (I-f) applicable for generating a subject conjugate, R8 is -F. In some embodiments, the substituents (for example, R2, R6, R7, R8, R9, and R10) of formula (I-f) are the same as the corresponding substituents in Formula (I), (I-a), (I-b), and/or (I-c), including in embodiments thereof. In embodiments, a compound of Formula (I-f) is a compound of Formula (I), (I-a), (I-b), and/or (I-c),.
[214] In some embodiments, a compound of Formula (I), (I-a), (I-b), or (I-c) applicable for generating a subject conjugate, has the formula: wherein:
R2 is selected from -O-CH2-(8- to 10-membered saturated heterocycle) and -O-CH2-(cyclopropylene)-CH2- (5- to 8-membered saturated heterocycle); wherein -O-CH2-(8- to 10-membered saturated heterocycle) and -O-CH2- (cyclopropylene)-CH2-(5- to 8-membered saturated heterocycle) are optionally substituted with one or more substituents independently selected from -F, =CF2, =CH2, and =CHF;
R9 is selected from C1-3 alkyl, C2-4 alkenyl, C2-3 alkynyl, -C1-2 alkyl-(C3-4 saturated carbocycle), and -C1-2 alkyl-(5- to 6-membered saturated heterocycle); wherein C1-3 alkyl, C2-4 alkenyl, C2-3 alkynyl, -C1-2 alkyl-(C3-4 saturated carbocycle), and -C1-2 alkyl-(5- to 6-membered saturated heterocycle) are each optionally substituted with one or more -F,
R6 is selected from -Cl and -CF3;
R7 is benzothiophenyl optionally substituted with one or more substituents independently selected from - NH2, -CN, and -F;
R8 is -F ; and wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker.
[215] In embodiments, the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
[216] In embodiments of a compound of Formula (I-f) applicable for generating a subject conjugate, R6 is -CF3. In some embodiments, the substituents (for example, R2, R6, R7, R8, R9, and R10) of formula (I-f) are the same as the corresponding substituents in Formula (I), (I-a), (I-b), or (I-c), including in embodiments thereof. In embodiments, a compound of Formula (I-d) applicable for generating a subject conjugate is a compound of Formula (I), (I-a), (I-b), or (I-c).
[217] In some embodiments, a compound of Formula (I), (I-a), (I-b), or (I-c), applicable for generating a subject conjugate, has the formula:
CH3;
R6 is selected from -Cl and -CF3; R7 is selected from
R8 is -F ; and wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker.
[218] In embodiments, the chemical linker is covalently bonded to the antigen binding unit or the chemical linker is capable of covalently conjugating to the antigen binding unit.
[219] In embodiments of a compound of Formula (I-f) applicable for generating a subject conjugate, R6 is -CF3.
In some embodiments, the substituents (for example, R2, R6, R7, R8, R9, and R10) of formula (I-f) are the same as the corresponding substituents in Formula (I), (I-a), (I-b), or (I-c), including in embodiments thereof. In embodiments, a compound of Formula (I-f) applicable for generating a subject conjugate is a compound of Formula (I), (I-a), (I-b), or (I-c). In some embodiments of the formulae above, some embodiments of the formulae above, R2 is embodiments of the formulae above, R2 is In some embodiments of the formulae above, R9 is formulae above, R9 is . In some embodiments of the formulae above, R9 is -CH3. In some embodiments of the formulae above, R9 is -CH2CH3. In some embodiments of the formulae above, R6 is -Cl. In some embodiments of the formulae above, R6 is -CF3. In some embodiments of the formulae above, R7 is embodiments of the formulae above, R7 is In some embodiments of the formulae above, R7 is
In some embodim ents, one embodiment of each of , Ring A, R2, R6, R7, R8, R9, and R10 is combined with a formulae above to generate a single compound.
[220] In some embodiments, a compound of Formula (I), (I-a), (I-b), or (I-c), applicable for generating a subject conjugate, has the formula:
CH3;
R5 is selected from hydrogen and halogen; wherein one hydrogen of the compound of Formula (I-g) is replaced with a bond to the antigen binding unit or the chemical linker. In embodiments, R9 is -CH3 and R10 is hydrogen. In embodiments, R9 is and R10 is hydrogen. In embodiments, R9 is -CH3 and R10 is halogen. In embodiments, R9 is -CH2CH3 and R10 is halogen. In embodiments,
R9 is -CH3 and R10 is -F. In embodiments, R9 is selected from formulae above, R2 is . In some embodiments of the formulae above, R2 is
In some embodiments, the substituents (for example, R2, R6, R7, R8, R9, and R10) of formula (I-g) are the same as the corresponding substituents in Formula (I), (I-a), (I-b), and/or (I-c), including in embodiments thereof. In embodiments, a compound of Formula (I-g) applicable for generating a subject conjugate is a compound for generating a subject conjugate, of Formula (I), (I-a), (I-b), and/or (I-c).
[221] In some embodiments, the compound of Formula (I), (I-a), or (I-b), applicable for generating a subject conjugate, is selected from: solvate thereof.
[222] In some embodiments, the compound of Formula (I), (I-a), (I-b), or (I-c), applicable for generating a subject conjugate, is selected from: pharmaceutically acceptable salt or solvate thereof. In some embodiments, the compound of Formula (I), (I-a), (I- b), or (I-c), applicable for generating a subject conjugate, is selected from:
[223] In some embodiments, a KRAS inhibitor for generating a subject conjugate of the present disclosure, is a compound selected from:
wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker, or a pharmaceutically acceptable salt or solvate thereof. In certain aspects, the present disclosure provides a compound selected from:
wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker, or a pharmaceutically acceptable salt or solvate thereof. In certain aspects, the present disclosure provides a compound for generating a subject conjugate selected from:
wherein one hydrogen of the compound is replaced with a bond to the antigen binding unit or the chemical linker, or a pharmaceutically acceptable salt or solvate thereof.
[224] In some embodiments, a KRAS inhibitor for generating a subject conjugate of the present disclosure is a compound selected from:
replaced with a bond to the antigen binding unit or the chemical linker, or a pharmaceutically acceptable salt or solvate thereof.
[225] In some embodiments, a KRAS inhibitor for generating a subject conjugate described herein, such as a compound of Formula (I), (I-a), (I-b), or (I-c), is provided as a substantially pure stereoisomer. In some embodiments, the stereoisomer is provided in at least 80% enantiomeric excess, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% enantiomeric excess.
[226] In some embodiments, the present disclosure provides an atropisomer of a compound described herein, such as a compound of Formula (I), (I-a), (I-b), or (I-c). In some embodiments, the atropisomer is provided in enantiomeric excess. In some embodiments, the atropisomer is provided in at least 80% enantiomeric excess, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% enantiomeric excess. In some embodiments, the compound of Formula (I), (I-a), (I-b), or (I-c) is used as a non- racemic mixture, wherein one atropisomer is present in excess of its corresponding enantiomer or epimer. Typically, such mixture contains a mixture of the two isomers in a ratio of at least 9: 1, preferably at least 19: 1. In some embodiments, the atropisomer is provided in at least 96% enantiomeric excess, meaning the compound has less than 2% of the corresponding enantiomer. In some embodiments, the atropisomer is provided in at least 96% diastereomeric excess, meaning the compound has less than 2% of the corresponding diastereomer.
[227] The term “atropisomers” refers to conformational stereoisomers which occur when rotation about a single bond in the molecule is prevented, restricted, or greatly slowed as a result of steric interactions with other parts of the molecule and wherein the substituents at both ends of the single bond are asymmetrical (i.e., optical activity arises without requiring an asymmetric carbon center or stereocenter). Where the rotational barrier about the single bond is high enough, and interconversion between conformations is slow enough, separation and isolation of the isomeric species may be permitted. Atropisomers are enantiomers (or epimers) without a single asymmetric atom. Atropisomers are typically considered stable if the barrier to interconversion is high enough to permit the atropisomers to undergo little or no interconversion at room temperature for a least a week, preferably at least a year. In some embodiments, an atropisomeric compound of the disclosure does not undergo more than about 5% interconversion to its opposite atropisomer at room temperature during one week when the atropisomeric compound is in substantially pure form, which is generally a solid state. In some embodiments, an atropisomeric compound of the disclosure does not undergo more than about 5% interconversion to its opposite atropisomer at room temperature (approximately 25 °C) during one year. The present chemical entities, pharmaceutical compositions, and methods are meant to include all such possible atropisomers, including racemic mixtures, diastereomeric mixtures, epimeric mixtures, optically pure forms of single atropisomers, and intermediate mixtures.
[228] In some embodiments, compounds for generating conjugates described herein exist as their pharmaceutically acceptable salts. In some embodiments, the methods disclosed herein include methods of treating diseases by administering such pharmaceutically acceptable salts. In some embodiments, the methods disclosed herein include methods of treating diseases by administering such pharmaceutically acceptable salts as pharmaceutical compositions.
[229] In some embodiments, the compounds for generating conjugates described herein possess acidic or basic groups and therefore react with any of a number of inorganic or organic bases or inorganic or organic acids to form a pharmaceutically acceptable salt. In some embodiments, such salts are prepared in situ during the final isolation and purification of the compounds or conjugates described herein, or by separately reacting a purified compound or conjugate in its free form with a suitable acid or base, and isolating the salt thus formed.
[230] In some embodiments, the compounds for generating conjugates described herein exist as solvates. In some embodiments are methods of treating diseases by administering such solvates. Further described herein are methods of treating diseases by administering such solvates as pharmaceutical compositions.
[231] Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and, in some embodiments, are formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of the compounds or conjugates described herein are conveniently prepared or formed during the processes described herein. By way of example only, hydrates of the compounds or conjugates described herein are conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents including, but not limited to, dioxane, tetrahydrofuran, or MeOH. In addition, the compounds or conjugates provided herein exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the compounds, conjugates, and methods provided herein.
[232] The chemical entities described herein, including KRAS inhibitors for generating a subject conjugate can be synthesized according to one or more illustrative schemes herein and/or techniques known in the art. Materials used herein are either commercially available or prepared by synthetic methods generally known in the art. These schemes are not limited to the compounds listed in the examples or by any particular substituents, which are employed for illustrative purposes. Although various steps are described and depicted in Schemes 1-4, the steps in some cases may be performed in a different order than the order shown in Schemes 1-4. Various modifications to these synthetic reaction schemes may be made and will be suggested to one skilled in the art having referred to the present disclosure. Numberings or R groups in each scheme typically have the same meanings as those defined elsewhere herein unless otherwise indicated. [233] Unless specified to the contrary, the reactions described herein take place at atmospheric pressure, generally within a temperature range from -10 °C to 200 °C. Further, except as otherwise specified, reaction times and conditions are intended to be approximate, e.g., taking place at about atmospheric pressure within a temperature range of about -10 °C to about 110 °C over a period of about 1 to about 24 hours; reactions left to run overnight average a period of about 16 hours.
[234] In general, certain compounds for generating a subject conjugate may be prepared by the following reaction schemes:
Scheme 1
[235] In some embodiments, a compound of Formula 1g may be prepared according to Scheme 1. For example, heteroaryl amine 1c can be formed from chloride la via a nucleophilic aromatic substitution reaction with amine lb. Ring closure to Id can be followed by an oxidation reaction to provide sulfone le, which can be substituted with R2 upon addition of a suitable alcohol to afford If. Substitution of the aryl bromide with a suitable boronic ester can provide the corresponding R7- substituted compound, which may optionally be subjected to one or more subsequent reactions, such as a deprotection, to provide a compound of Formula 1g.
Scheme 2
[236] In some embodiments, a compound of Formula 2e may be prepared according to Scheme 2. For example, heteroaryl ether 2c can be formed from fluoride 2a via a nucleophilic aromatic substitution reaction with alcohol 2b. Ring closure to 2d can be followed by substitution with a suitable boronic ester to provide the corresponding R7- substituted compound, which may optionally be subjected to one or more protecting group manipulations to provide a compound of Formula 2e.
Scheme 3
[237] In some embodiments, a conjugate of Formula 3c may be prepared according to Scheme 3. For example, amine 3a may be coupled to linker L to form intermediate 3b. The antigen binding unit can be coupled to the linker to provide a conjugate of Formula 3c.
Scheme 4
[238] In some embodiments, a conjugate of Formula 4d may be prepared according to Scheme 4. For example, amine 4a may react with the para-nitrophenyl carbonate of linker 4b to form carbamate 4c. A thiol group of the antigen binding unit can then react with the maleimide of 4c to form a conjugate of Formula 4d.
[239] In some embodiments, a compound of Formula 5i applicable for generating a subject conjugate may be prepared according to Scheme 5. For example, 5a can undergo a reductive amination with a suitable R3- substituted amine to provide 5b. Heteroaryl amine 5d can be formed from chloride 7c via a nucleophilic aromatic substitution reaction with amine 5b. Ring closure to 5e can be followed by an oxidation reaction to provide sulfoxide 5f, which can be substituted with -OR12 upon addition of a suitable alcohol to afford 5g. Substitution of the aryl bromide with a suitable boronic ester can provide the corresponding R7- substituted compound, which may optionally be subjected to one or more subsequent reactions, such as a deprotection, to provide a compound for generating a subject conjugate of Formula 5i.
Scheme 6
[240] The Schemes 6 through 12 above may be adapted to incorporate any of the Kras inhibitors for producing a conjugate of the present disclosure, including any compound of Table 1. In the schemes 6 through 12, NH-A- and - N(R2)-A- may be equivalent to Z3 as described herein for a chemical linker; Q may be equivalent to Z2 as described herein for a chemical linker, wherein Q is bonded to maleimide through a carbon atom of Q; R may be equivalent to Rz as described herein for a chemical linker, and R2 may be equivalent to any substituent described herein at the corresponding position of a chemical linker, including hydrogen or R2 may comprise polyethylene glycol, polysarcosine, asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, valine, alanine, glycine, leucine, isoleucine, methionine, tryptophan, proline, histidine, citrulline, phenylalanine, carboxylate, alkyl, alkene, alkyne, aryl, cycloalkyl, heterocycle, glycoside, silyl ether, hydroxy, ether, ketone, ester, carbonate, amide, urea, carbamate, sulfide, disulfide, sulfate, sulfonamide, phosphate, phenylboronic ester, phenylboronic acid, thioketal, tartaric acid, 1,2-diol acetonide, o-aminoalcohol, selenium, ortho-nitrobenzyl, phenacyl ester, sugar, glucoronide, trioxolane, oxime, acyl hydrazone, cyclobutyl, pyrophosphate, arylsulfate, heptamethine, cyanine fluorophore, o-nitrobenzyl, PC4AP, dsProc, 1,3-dioxane, triazole, piperazine, bis(vinylsulfonyl)piperazine, N-methyl-N-phenylvinylsulfonamide, Pt, or any combination thereof. The variables n and m are each an integer of 1 or more, wherein m is equal to or greater than n. Ab comprises an antigen binding unit e.g., cetuximab or any other antigen binding units disclosed herein.
[241] In some embodiments, a Kras inhibitor for producing a conjugate of the present disclosure includes, for example, a compound of a formula given in Table 1, which can be synthesized according to one of the general routes outlined in Schemes 1-6 and 10, or by methods generally known in the art. In some embodiments, exemplary compounds for generating a subject conjugate may include, but are not limited to, a compound selected from Table 1, wherein one hydrogen of the compound is replaced with a bond to an antigen binding unit or a chemical linker disclosed herein.
Table 1
Compounds of Table 1 are depicted with flat, wedged, and/or hashed wedged bonds. It is understood that compounds depicted in Table 1 encompass all possible stereoisomers, including atropisomers, of the compounds of
Table 1. In some instances, the relative stereochemistry at one or more stereocenters of a compound has been determined; in some instances, the absolute stereochemistry has been determined. In some instances, a single compound number represents a mixture of stereoisomers, including atropisomers. In some instances, a single compound number represents a single stereoisomer, such as a single atropisomer. As such, it is understood that if two or more compound numbers in Table 1 are provided with the same depicted structure, then different stereoisomers or mixtures of stereoisomers of the depicted structure are represented by each compound number.
[242] Additional exemplary linkers applicable for conjugation to a Kras inhibitor include any linkers shown below in Table 2. In some embodiments, a Kras inhibitor for producing a conjugate of the present disclosure, as described herein, is covalently bonded to a chemical linker, such as a chemical linker of a formula given in Table 2. in Table 2 indicates an attachment site to the Kras inhibitor for producing a conjugate of the present disclosure, as described herein. The chemical linkers described herein, including in Table 2, can be synthesized according to one of the general routes outlined herein, or by methods generally known in the art. In embodiments a Kras inhibitor-chemical linker capable of forming a conjugate of the present disclosure comprises i) a Kras inhibitor for producing a conjugate of the present disclosure of Table 1, wherein one hydrogen bonded to a nitrogen of the Kras inhibitor is replaced with a bond to a chemical linker; and ii) a chemical linker of Table 2, wherein in Table 2 indicates an attachment site to the Kras inhibitor. In embodiments, the nitrogen atom of the Kras inhibitor covalently bonded to chemical linker (e.g., chemical linker of Table 2) is a nitrogen atom bonded to a benzothiophenyl of the Kras inhibitor. In embodiments, the nitrogen atom of the Kras inhibitor covalently bonded to chemical linker (e.g., chemical linker of Table 2) is in an aminomethyl that is bonded to a pyrrolizidine of the Kras inhibitor.
Table 2
[243] In embodiments, the linker-modified Kras compound comprises i) a Kras inhibitor selected from Table 1, wherein one hydrogen of the compound of Table 1 is replaced with a bond to a chemical linker; and ii) a chemical linker selected from the chemical linkers of Table 2, wherein is the attachment site to the Kras inhibitor of Table 1.
[244] In embodiments, the conjugate comprises i) cetuximab; ii) a Kras inhibitor selected from Table 1, wherein one hydrogen of the compound of Table 1 is replaced with a bond to a chemical linker; and iii) a chemical linker selected from the chemical linkers of Table 2, wherein is the attachment site to the Kras inhibitor of Table 1 and the of the chemical linker is converted to a moiety with indicating the attachment site of the covalently bonded chemical linker to cetuximab of i). In embodiments, the conjugate comprises i) sacituzmab; ii) a Kras inhibitor selected from Table 1, wherein one hydrogen of the compound of Table 1 is replaced with a bond to a chemical linker; and iii) a chemical linker selected from the chemical linkers of Table 2, wherein is the attachment site to the Kras inhibitor of Table 1 and the of the chemical linker is converted to a moiety with indicateing the attachment site of the covalently bonded chemical linker to sacituzmab of i). [245] In some embodiments, each of the exemplified linkers in Table 2 can be used to conjugate to a Kras inhibitor exemplified in Table 1. The chemical linkers of Table 2 may each covalently be bonded to the amine nitrogen bonded to a benzothiophenyl moiety of a compound of Table 1. For example, the Kras inhibitor-chemical linker comprises: a compound selected from compound numbers 101-125 of Table 1 and a chemical linker selected from 1001-1057 of Table 2; a compound selected from compound numbers 126 - 150 of Table 1 and a chemical linker selected from 1001-1057 of Table 2; a compound selected from compound numbers 151 - 175 of Table 1 and a chemical linker selected from 1001-1057 of Table 2; a compound selected from compound numbers 176 - 414 of Table 1 and a chemical linker selected from 1001-1057 of Table 2; a compound selected from compound numbers 101 - 414 of Table 1 and a chemical linker selected from 1001-1025 of Table 2; or a compound selected from compound numbers 101 - 414 of Table 1 and a chemical linker selected from 1026-1057 of Table 2, In some examples, each Kras inhibitor-chemical linker conjugate described above comprises a bond between the linker 1002, 1022, 1031, 1040, 1043, 1046, 1050, or 1054 of Table 2 and amine nitrogen bonded to a benzothiophenyl moiety of any compound in Table 1, e.g., #110, #112, #148, #160, #161, #169, #182, #186, #361, or #372. In some embodiments, the chemical linkers of Table 2 may each covalently be bonded to the nitrogen atom of the aminomethyl bonded to pyrrolizidine of a compound of Table 1 ; for example, the Kras inhibitor-chemical linker conjugate comprises #377 of Table 1, and 1019 of Table 2. In some embodiments, the linker-modified Kras compounds comprising compound 148 of Table 1 and one linker selected from the group of chemical linkers 1001— 1018 and 1020-1057, each results in a linker-modified Kras compound with, respectively, an observed mass of 1794.9, 1703.6, 1075.4 [M/2]+H+ (half-mass), 1305.4, 1825.7, 918.2 [M/2]+H+ (half-mass), 1967.8, 1549.4, 1351.7, 1034.2 [M/2]+H+ (half-mass), 1771.8, 1307.4, 1358.2, 1833.6, 834.9 [M/2]+H+ (half-mass), 1036.9 [M/2]+H+ (half-mass), 1748.8, 1872.9 , 1699.9 , 1779.7 , 1642.5, 1870.9 , 1219.5 , 1776.6 , 1118.7 [M/2]+H+ (halfmass), 1596.4, 1745.7, 1878.7, 1713.4, 1353.6 , 1060.0 [M/2]+H+ (half-mass), 1689.5 , 1703.5, 1446.3, 1617.7, 1857.7 , 1056.9 [M/2]+H+ (half-mass), 1051.7 [M/2]+H+ (half-mass), 1104.3 [M/2]+H+ (half-mass), 961.4 [M/2]+H+ (half-mass), 1439.7 , 1098.3 [M2]+H+ (half-mass), 1312.2, 1656.5, 1791.8, 1903.7, 1623.4 , 1008.6 (M/2]+H+ (half-mass), 1037.4 [M/2]+H+ (half-mass), 1004.5 [M/2]+H+ (half-mass), 1036.9 [M/2]+H+ (half-mass), 1031.4 [M/2]+H+ (half-mass), 1057.2 (M/2]+H+ (half-mass), 1266.6. Each linker-modified Kras compound formed having the observed mass immediately above, comprises a bond between the attachment site of the linker and the nitrogen atom of the amine bonded to the benzothiophenyl of compound 148 of Table 1. The linker-modified Kras compound formed by covalently bonding compound 377 of Table 1 and 1019 of Table 2 (between the aminomethyl nitrogen of 377 and the attachment site of 1019) has an observed mass of 1316.7.
[246] The linker-modified Kras compounds described herein may be further conjugated to any antigen binding unit disclosed herein including but not limited to Cetuximab to yield a subject conjugate. The linker-modified Kras compounds described herein, comprising respectively, the chemical linker No. 1001, 1002, 1005, 1006, 1007, 1008, 1010, 1011, 1013, 1014, 1015, 1016, 1017, 1020, 1021, 1022, 1025, 1027, 1028, 1029, 1030, 1031, 1033, 1034, 1035, 1036, 1038, 1039, 1040, 1041, 1042, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, or 1055, is conjugated to compound 148 of Table 1, each of which was reacted with cetuximab to form a covalently bonded conjugate of the present disclosure. In some embodiments, the linker-attached Kras inhibitors described immediately above, each comprise a moiety covalently bonded to cetuximab, which can be produced from the of the linkers of Table 2, wherein indicates the attachment site of th
[247] e covalently bonded chemical linker to cetuximab.
[248] Non-limiting examples of conjugates of the present disclosure include:
-235-
Ab is an antigen binding unit selected from the group of consisting of AG7, B7-H3, BCMA, CA15-3, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD70, CD71, CD79B, CEA, CLDN18.2, EGFR, FOLR1, GCC, GPC1, HER2, HER3, ICAM1, LeX, LeY, MET, MSLN, MUC1, NECTIN4, SLC44A4, TF, and Trop-2. In some embodiments, Ab in the structures shown in this paragraph comprises the amino acid sequence of cetuximab. In some embodiments, Ab in the structures shown in this paragraph comprises the amino acid sequence of Sacituzumab. In embodiments, Ab in the structures shown in this paragraph is bonded to the linker through an Ab thiol. In embodiments, m in the structures shown in this paragraph is 2, 4, 6, or 8. In embodiments, m in the structures shown in this paragraph is 4. In embodiments, m in the structures shown in this paragraph is 8. In embodiments, m in the structures shown in this paragraph is 2.
[249] Antibody -drug conjugates can also be prepared by methods known in the art and also according to the exemplary procedures provided herein to yield various Kras inhibitor-to-antibody ratio (DAR) ratios, including about 2, 4, 6, 8. To prepare a subject conjugate with DAR being about 2, a solution of about 150 mg antigen binding unit in Tris-HCl buffer adjusted to pH 7.0, is reduced by the addition of an aqueous tris(2- carboxy ethyljphosphine (TCEP) solution (1.35 equiv.) and is incubated for 18 h at 22°C. The reduced antibody is conjugated with excess linker modified Kras inhibitor (5 equiv.) in 10% DMA and the reaction proceeded for 1 h at 22°C. A solution with cysteine (4 equiv.) is added to quench the conjugation reaction by depleting the unconjugated linker-payload.
The reaction mixture is subjected to purification using a desalting column.
[250] To prepare a DAR 4 conjugate, a solution of 76 mg antigen binding unit in Tris-HCl buffer adjusted to pH 7.0, is reduced by the addition of an aqueous tris(2- carboxy ethyl)phosphine (TCEP) solution (2.39 equiv.) and is incubated for 18 h at 22°C. The reduced antibody is conjugated with excess linker modified Kras inhibitor (8 equiv.) in 10% DMA and the reaction proceeded for 1 h at 22°C. A solution with cysteine (4 equiv.) is added to quench the conjugation reaction by depleting the unconjugated linker-payload. The reaction mixture is subjected to purification using a desalting column. [251] After the antigen binding units-chemical linker-Kras inhibitor conjugates are purified, their concentrations are measured using a BCA assay. Antigen binding units-chemical linker-Kras inhibitor conjugates are subjected to additional analyses using various techniques. Hydrophobic interaction chromatography (HIC) and reduced mass spectrometry (MS) are employed to determine the Kras inhibitor-to-antibody ratio (DAR), whereas size exclusion chromatography (SEC) is used to assess monomer purity. To determine the average Kras inhibitor-to-antibody ratio (DAR) of the purified antigen binding units-chemical linker-Kras inhibitor conjugates, the individual contributions of DARO, DAR2, DAR4, DAR6, and DAR8 species are analyzed. This analysis is conducted by integrating the HPLC-HIC chromatograms or MS spectra. The weighted average of each DAR species is used to calculate the average DAR of each antigen binding units-chemical linker-Kras inhibitor conjugate. Based on HIC analyses, subject conjugates of an average DAR from about 2, 4 or higher were generated with a purity of at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.In some embodiments, the KRAS inhibitors of the present disclosure exhibit one or more functional characteristics disclosed herein. For example, a subject KRAS inhibitor binds to a Ras protein, KRAS protein or a mutant form thereof. In some embodiments, a subject KRAS inhibitor binds specifically and also inhibits a Ras protein, KRAS protein or a mutant form thereof. In some embodiments, a subject KRAS inhibitor selectively inhibits a KRAS mutant relative to a wildtype KRAS. In some embodiments, the IC50 of a subject KRAS inhibitor for a KRAS mutant (e.g., including G12C, G12S, G12D, G12V) is less than about 5 μM, less than about 1 μM, less than about 500 nM, less than 250 nM, less than 100 nM, less than 50 nM, or even less, as measured in an in vitro assay known in the art or exemplified herein.
[252] In some embodiments, a KRAS inhibitor of the present disclosure is isotype selective. In some embodiments, a KRAS inhibitor of the present disclosure is not a pan-Ras inhibitor that is promiscuous in inhibiting two or more isotypes of KRAS, HRAS, and NRAS. For example, a subject KRAS inhibitor selectively inhibits KRAS or a mutant thereof relative to HRAS, NRAS, and/or other Ras proteins in the superfamily of small GTPases disclosed herein, including mutants thereof. In some embodiments, a subject KRAS inhibitor exhibits at least 2-fold selectively for a KRAS protein relative to an HRAS and/or NRAS protein, such as at least 3 -fold, at least 4-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 12.5-fold, at least 15-fold, at least 20-fold, or at least 25-fold selectivity for a KRAS protein. In some embodiments, a subject KRAS inhibitor does not substantially inhibit HRAS or NRAS. In an embodiment, a subject KRAS inhibitor selectively inhibits KRAS relative to both HRAS and NRAS. In another embodiment, a subject KRAS inhibitor selectively inhibits KRAS relative to both HRAS and NRAS such that the IC50 value against HRAS and NRAS is at least 2-, 5-, 10-, 100-, 200-, or 500-fold or higher than the IC50 against KRAS when ascertained in an in vitro assay, such as those described In embodiments, a subject KRAS inhibitor selectively inhibits KRAS relative to both HRAS and NRAS such that the IC50 value against HRAS and/or NRAS is greater than 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, or 1000 μM; and the IC50 value against KRAS is less than 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 500 pM, 400 pM, 300 pM, 200 pM, or 100 pM.
[253] In another embodiment, a subject KRAS inhibitor is a KRAS inhibitor that is capable of inhibiting two or more KRAS mutants and/or wildtype KRAS, exhibiting IC50 values against two or more KRAS mutants (including, e.g., KRAS G12C, of less than about 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, or even less. In some embodiments, a subject KRAS inhibitor may be a pan-KRAS inhibitor capable of inhibiting two or more KRAS mutants. In some embodiments, a subject KRAS inhibitor selectively inhibits KRAS G12C relative to wildtype KRAS or another mutant form of KRAS, such as KRAS G12S, KRAS G12D, or KRAS G12V. In some embodiments, a subject KRAS inhibitor exhibits at least 2-fold selectively for KRAS G12C relative to wildtype KRAS or another mutant form of KRAS (e.g., KRAS G12S, KRAS G12D, or KRAS G12V), such as at least 3-fold, at least 4-fold, at least 5-fold, at least 7.5-fold, or at least 10-fold selectivity for KRAS G12C as ascertained in an in vitro assay, such as those described herein. In some embodiments, a subject KRAS inhibitor selectively inhibits KRAS G12S relative to wildtype KRAS or another mutant form of KRAS, such as KRAS G12C, KRAS G12D, or KRAS G12V. In some embodiments, a subject KRAS inhibitor exhibits at least 2- fold selectively for KRAS G12S relative to wildtype KRAS or another mutant form of KRAS (e.g., KRAS G12C, KRAS G12D, or KRAS G12V), such as at least 3-fold, at least 4-fold, at least 5-fold, at least 7.5-fold, or at least 10- fold selectivity for KRAS G12S. In some embodiments, a subject KRAS inhibitor selectively inhibits KRAS G12D relative to wildtype KRAS or another mutant form of KRAS, such as KRAS G12C, KRAS G12S, or KRAS G12V. In some embodiments, a subject KRAS inhibitor exhibits at least 2-fold selectively for KRAS G12D relative to wildtype KRAS or another mutant form of KRAS (e.g., KRAS G12C, KRAS G12S, or KRAS G12V), such as at least 3-fold, at least 4-fold, at least 5-fold, at least 7.5-fold, or at least 10-fold selectivity for KRAS G12D. In some embodiments, a subject KRAS inhibitor selectively inhibits KRAS G12V relative to wildtype KRAS or another mutant form of KRAS, such as KRAS G12C, KRAS G12S, or KRAS G12D. In some embodiments, a subject KRAS inhibitor exhibits at least 2-fold selectively for KRAS G12V relative to wildtype KRAS or another mutant form of KRAS (e.g., KRAS G12C, KRAS G12S, or KRAS G12D), such as at least 3-fold, at least 4-fold, at least 5- fold, at least 7.5-fold, or at least 10-fold selectivity for KRAS G12V.
[254] In some embodiments, a KRAS inhibitor of the present disclosure inhibits both KRAS proteins in the GTP- bound state (KRAS(ON)) and KRAS proteins in the GDP-bound state (KRAS(OFF)). KRAS(ON) and KRAS(OFF) proteins include mutant forms thereof, such as KRAS G12C, KRAS G12S, KRAS G12D, and KRAS G12V in both their GTP-bound and GDP -bound states. In some embodiments, a subject KRAS inhibitor does not exclusively inhibit KRAS(ON). In some embodiments, a subject KRAS inhibitor inhibits KRAS(OFF) with greater potency than KRAS(ON).
[255] In some embodiments, a KRAS inhibitor of the present disclosure is capable of reducing Ras signaling output. Such reduction may be evidenced by one or more of the following: (i) an increase in steady state level of GDP -bound Ras protein; (ii) a reduction in steady state level of GTP-bound Ras protein; (iii) a reduction of phosphorylated AKTs473, (iv) a reduction of phosphorylated ERKT202/y204, (v) a reduction of phosphorylated S6S235/236, and (vi) reduction (e.g., inhibition) of cell growth of Ras-driven tumor cells (e.g., those derived from a tumor cell line disclosed herein). In some cases, the reduction in Ras signaling output can be evidenced by two, three, four, five, or all of (i)-(vi) above.
[256] The inhibitory ability of a candidate Kras inhibitor for producing a conjugate of the present disclosure can be assessed by a variety of methods known in the art. An exemplary method for assessing the Kras inhibition biochemically includes an HTRF (homogenous time-resolved fluorescence) resonance energy transfer assay, which can measure the equilibrium interaction of wildtype KRAS or K-Ras mutant (e.g., wildtype or a mutant thereof) with SOS1 (e.g., hSOSl) Modulation of this interaction may be used as a proxy or an indication for the ability of a subject compound to bind and inhibit Ras protein. The HTRF assay may detect transfer from (i) a fluorescence resonance energy transfer (FRET) donor (e.g., antiGST -Europium) that is bound to GST-tagged K-Ras mutant to (ii) a FRET acceptor (e.g., anti-6His-XL665) bound to a His-tagged hSOSl (see W02022/047260, which is incorporated herein by reference).
[257] Other non-limiting exemplary methods for measuring Kras inhibition biochemically include a GTPase activity assay, which can measure the ability of a compound for producing a conjugate of the present disclosure to inhibit Ras protein signaling through measurement of a reduced GTPase activity in the presence of a candidate compound for producing a conjugate of the present disclosure. This assay can also be used to assess selective inhibition of a mutant Ras protein relative to a wildtype or different mutant Ras protein. In particular, intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activity for a K-Ras construct or a mutant thereof can be measured using EnzCheck phosphate assay system (Life Technologies) (see W02022/047260, which is incorporated herein by reference).
[258] Another non-limiting exemplary method for measuring Kras inhibition biochemically includes a nucleotide exchange assay. The ability of a compound of the present disclosure to inhibit Ras protein signaling can be demonstrated by reduced nucleotide exchange activity. This assay can be also used to assess selective inhibition of a mutant Ras protein relative to a wildtype or different mutant Ras protein. For example, GDP-loaded K-Ras protein (e.g., wildtype or a mutant thereof) is incubated with different concentrations of compounds and SOS1 (catalytic domain) protein is added. The nucleotide exchange reaction is initiated by adding fluorescent labelled GDP (guanosine 5 ’-diphosphate, BODIPY™ FL 2’-(or-3’)-O-(N-(2-aminoethyl)urethane) and fluorescence is measured at 490nm/515nm (excitation/emission). Data is exported and analyzed to calculate an IC50 (see W02022/047260, which is incorporated herein by reference).
[259] The ability of a conjugate of the present disclosure to inhibit the growth of cancer cells (e.g., cancer cells expressing KRAS and/or EGFR mutations or amplifications), can be evaluated using a cell proliferation assay exemplified herein (e.g., Example 11). One or more conjugates of the present disclosure (e.g., Cet-KRAS A and Cet-KRAS B) demonstrate potent growth inhibition of cancer cells such as high EGFR-expressing epidermoid carcinoma cell line A-431, exhibiting an IC50 of less than 50nM, lOnM, or even less. In contrast, cetuximab is approximately 10-fold less potent and isotype IgGl ADC controls (e.g., Isotype-KRAS A and Isotype-KRAS B) are at least 20-fold less potent than the conjugates in the same cell line. Where desired, the inhibitory effect of cetuximab linked to various payload linkers on cell proliferation is evaluated using a range of cell lines with KRAS, NRAS, and HRAS mutations. The cell panel include HPAC (KRAS-G12D, pancreatic adenocarcinoma), H1373 (KRAS-G12C, lung adenocarcinoma), H2009 (KRAS-G12A, lung adenocarcinoma), MIA-PaCa2 (KRAS-G12C, pancreatic adenocarcinoma), THP-1 (NRAS-G12D, acute myeloid leukemia), T24 (HRAS-G12V, colorectal adenocarcinoma), and SK -MEL-30 (NRAS-Q61K, melanoma). A subject conjugate comprising cetuximab conjugated to a Kras inhibitor disclosed herein exhibits at least 30-fold higher potency in inhibiting KRAS driven cell lines relative to NRAS and HRAS cells. When assessed in vivo model (e.g., as described in example 12, Cetuximab (e.g., Cet-KRAS A and Cet-KRAS B) and sacituzumab (e.g., Sac-KRAS A and Sac-KRAS B) KRAS inhibitor conjugates exhibit potent regression of Hl 373 tumors following intravenous (IV) infusion of less than 50 mg/kg on days 1 and 8.
Methods
[260] Conjugates described herein that comprise a KRAS inhibitor have a wide range of applications in therapeutics, diagnostics, and other biomedical research. A subject conjugate exhibits inhibition of a KRAS protein, such as wild-type KRAS or a KRAS mutant protein (e.g., G12S, G12C, G12D, G12V, G13C, and/or G13D).
[261] In certain aspects, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof.
[262] In certain aspects, the present disclosure provides a method of treating a cancer comprising amplified wildtype Ras or a Ras mutant (e.g., G12S, G12C, G12D, G12V, G13C, and/or G13D) protein in a subject, comprising inhibiting amplified wildtype Ras or the Ras mutant protein of said subject by administering to said subject a conjugate described herein, wherein the conjugate releases a KRAS inhibitor that inhibits Ras protein activity or function (e.g., partially or completely), such that said inhibited Ras protein exhibits reduced Ras signaling output (e.g., compared to a corresponding Ras protein not contacted by the conjugate).
[263] In certain aspects, the present disclosure provides a method of modulating activity of a Ras protein (e.g., K- Ras, mutant K-Ras, K-Ras G12S, K-Ras G12C, K-Ras G12D, K-Ras G12V, K-Ras G13C, and/or K-Ras G13D), comprising contacting a Ras protein with an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, thereby modulating the activity of the Ras protein.
[264] In certain aspects, the present disclosure provides a method of inhibiting cell growth, comprising administering an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, to a cell expressing a Ras (e.g., K-Ras) protein, thereby inhibiting growth of said cells. In some embodiments, the subject method comprises administering an additional agent to said cell.
[265] In certain aspects, the present disclosure provides a method of treating a disease mediated at least in part by a Ras protein, such as K-Ras or a mutant thereof, in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate disclosed herein, or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the disease is cancer, such as a solid tumor or a hematological cancer. In some embodiments, the method further comprises administering an additional agent to the subject, such as a SHP2 inhibitor, a SOS inhibitor, an EGFR inhibitor, a MEK inhibitor, an ERK inhibitor, a CDK4/6 inhibitor, a BRAF inhibitor, or a combination thereof.
[266] In certain aspects, the present disclosure provides a method of inhibiting activity of a Ras protein, such as K-Ras or a mutant thereof, comprising contacting the Ras protein with a conjugate disclosed herein, or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the conjugate comprises a Ras inhibitor that exhibits an IC50 against the Ras protein of less than 10 μM, such as less than 5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 50 pM, 10 pM or less.
[267] In certain aspects, the present disclosure provides a method of treating a Ras-mediated cancer in a subject in need thereof, comprising administering to the subject a SHP2 inhibitor, a SOS inhibitor, an EGFR inhibitor, a MEK inhibitor, an ERK inhibitor, a CDK4/6 inhibitor, or a BRAF inhibitor and an effective amount of a conjugate described herein. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer.
[268] In certain aspects, the present disclosure provides a method of delivering a small-molecule KRAS inhibitor that exhibits low permeability as characterized by a PAMPA assay, comprising contacting a tumor cell with a conjugate described herein, or a salt thereof, wherein the KRAS inhibitor exhibits a PAMPA permeability (Pe) value less than 1 x 10-6 cm/s. In some embodiments, the KRAS inhibitor is characterized by a PAMPA permeability (Pe) less than 1 x 10-6 cm/s, such as less than 9 x 10-7, 8 x 10-7, 7 x 10-7, 6 x 10-7, 5 x 10-7, 4 x 10-7, 3 x 10-7, 2 x 10-7, 1 x 10-7, 1 x 10-8, or 1 x 10-9 cm/s.
[269] In certain aspects, the present disclosure provides a method of enhancing therapeutic efficacy of a smallmolecule KRAS inhibitor, comprising providing a conjugate described herein to a subject, wherein enhanced therapeutic efficacy is ascertained by the formula: TIconjugate/TlKRASi > 1 , wherein TIconjugate = TD50c/ED50c, wherein TD50c is the dose of conjugate required to produce a toxic effect in 50% of test subjects and ED50c is the dose of conjugate required to produce a therapeutic effect in 50% of test subjects; and wherein TIKRASI = TD50k/ED50k, wherein TD50k is the dose of KRAS inhibitor required to produce a toxic effect in 50% of test subjects and ED50k is the dose of KRAS inhibitor required to produce a therapeutic effect in 50% of the test subjects. In some embodiments, TIconjugate/TlKRASi is greater than 1.1, such as greater than 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3,
3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50. In some embodiments, TIconjugate/TlKRASi is greater than 2. In some embodiments, TIconjugate/TlKRASi is greater than 5.
[270] In certain aspects, the present disclosure provides a method of reducing plasma concentration of a smallmolecule KRAS inhibitor, comprising providing a conjugate described herein to a subject, wherein reduced plasma concentration is ascertained by the formula: [KRASi]p-c/[KRASi]p-k < 1 , wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at a first time -point following administration of the conjugate; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point. In some embodiments, [KRASi]p-c/[KRASi]p-k is less than 0.95, such as less than 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, or 0.001. In some embodiments, [KRASi]p-c/[KRASi]p-k is less than 0.5. In some embodiments, [KRASi]p-c/[KRASi]p-k is less than 0.1. In some embodiments, the first time-point is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 48 hours after the administration.
[271] In certain aspects, the present disclosure provides a method of increasing concentration of a small-molecule KRAS inhibitor in tumor tissue, comprising providing a conjugate described herein to a subject, wherein increased tumor tissue concentration is ascertained by the formula: ([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) > 1, wherein [KRASi]t-c is concentration of the KRAS inhibitor in tumor tissue at a first time -point following administration of the conjugate; wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at the same time -point following the administration of the conjugate; wherein [KRASi]t-k is concentration of the KRAS inhibitor in tumor tissue following administration of the KRAS inhibitor alone at an equivalent dose at the same time -point; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor at the same time-point following the administration of the KRAS inhibitor alone at the equivalent dose. In some embodiments, ([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) is greater than 1.1, such as greater than 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,
4.5, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50. In some embodiments, ([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) is greater than 2. In some embodiments, ([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) is greater than 5. In some embodiments, the first time-point is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 48 hours after the administration. In some embodiments, the subject conjugates are particularly effective in effectuate tumor growth inhibition and/or regression. Not wishing to be bound by any particular theory to such beneficial effect, the subject conjugates are internalized by tumor cells, and the Kras inhibitor payload is found to be accumulated in the tumor tissue relative to the plasma. A higher concentration of Kras inhibitor is detected in the tumor tissue than that in the plasma after administering a subject conjugate intravenously. For instance, the pharmacokinetics and tumor tissue distribution of the KRAS inhibitor pay load can be evaluated in a mouse model such as an Hl 373 lung adenocarcinoma CDX mouse model using methods known in the art or disclosed in example 15. A subject conjugate comprising an antigen binding unit (e.g., Sacituzumab - an antibody targeting Trop-2) conjugated to a Kras inhibitor disclosed in Table 1 (e.g., cpd# 148) via a linker from Table 2 (e.g., cpd#1032) is found to exhibit such tumor tissue accumulation.
[272] In certain aspects, the present disclosure provides a method of increasing concentration of an antigen binding unit in tumor cells, comprising providing a conjugate described herein to a subject, wherein increased tumor cell concentration is ascertained by the formula: [Conjugate]t-c / [AgB]t-a > 1, wherein [Conjugate]t-c is concentration of the conjugate in tumor cells at a first time-point following administration of the conjugate, and wherein [AgB]t-a is concentration of the antigen binding unit in tumor cells at the same time -point following administration of the antigen binding unit alone at an equivalent dose. Not wishing to be bound by any particular theory, conjugation of a small-molecule KRAS inhibitor to an antigen binding unit, optionally via a chemical linker, may result in greater internalization of the conjugate into cells when compared to an unconjugated antigen binding unit. Accordingly, conjugates of the present disclosure may be used in methods of improving delivery of an antigen binding unit, such as cetuximab, to a cell, such as a tumor cell, even if no other therapeutic benefit is provided by the KRAS inhibitor.
[273] In certain aspects, the present disclosure provides a method of delivering a small-molecule KRAS inhibitor to the central nervous system of a subject, comprising administering a conjugate described herein to the subject, wherein the KRAS inhibitor is released from the conjugate after entering the CNS of the subject.
[274] In certain aspects, the present disclosure provides a method of generating a slow-release form of a smallmolecule KRAS inhibitor, the method comprising conjugating an antigen binding unit to a small-molecule KRAS inhibitor through a chemical linker, wherein the small-molecule inhibitor is released from the conjugate upon introducing the conjugate into a subject or a cell.
[275] In practicing any of the methods disclosed herein, efficacy of the conjugate may be greater than efficacy of a combination of the antigen binding unit and the KRAS inhibitor when each is administered at a comparable concentration. Following the procedure of example 16, one or more subject conjugate has been shown to elicit tumor regression more effectively than the antigen binding unit and the Kras inhibitor, when each being administered alone or in combination as separate molecules and not conjugated together to form a cetuximab-Kras ADC-conjugate. In particular, compared with the vehicle negative control, multiple cetuximab-Kras conjugates (DAR 4) with various linkers elicited tumor regression as early as day 4 after administration, and more than 50% tumor regression on day 22 after administration. By contrast, the mice administered with equivalent amount of unconjugated Kras inhibitors (the equivalent amount is calculated by assuming complete release of Kras as the payload from the cetuximab-Kras conjugates) showed minimal efficacious activity with continue tumor growth on day 11 and were sacrificed owing to tumor burden. Similarly, the group of mice administered with equivalent amount of unconjugated Kras inhibitors together with an equivalent amount of cetuximab (as separate molecules and not in form of an ADC) did not yield tumor regression on 5 or even on day 12. Not wishing to be bound by any particular theory, a subject conjugate provides a tool to synergistically inhibit signaling output of KRAS and the first antigen to which the first antigen binding unit binds.
[276] In some embodiments, toxicity of the conjugate is less than toxicity of a combination of the antigen binding unit and the KRAS inhibitor when each is administered at a comparable concentration.
[277] In practicing any of the methods disclosed herein, specific binding by an antigen binding unit to the bound target can be established by a wide variety of methods and techniques known in the art, including but not limited to direct binding assays, indirect sandwich assays, ligand binding assays, immunoprecipitation, real-time cell-binding assays, imaging analysis, and competition assays. In an aspect, a binding assay can comprise the use of surface plasmon resonance (SPR), bio-layer interference (BLI), scanning probe microscopy, attenuated total reflective infrared spectroscopy, spectral ellipsometry, mass spectrometry, and any combinations thereof. Conversely, the lack of specific binding to the cellular target that is not bound to the exogenous molecule (unbound target) can be established by similar methods. In an aspect, SPR is used to determine affinity of an antigen binding unit to a target or a portion of a target. Additionally, SPR can be used to determine a physical property or biological property of a subject antigen binding unit provided herein. Physical properties include but are not limited to dielectric properties, adsorption processes, surface degradation, hydration, X-ray crystallography, NMR, interferometry, computer modeling and any combination thereof. Biological properties that can be determined with SPR include but are not limited to adsorption kinetics, desorption kinetics, antigen binding, affinity, epitope mapping, biomolecular structure, protein interaction, biocompatibility, tissue engineering, lipid biolayers, and any combination thereof.
[278] In practicing any of the methods disclosed herein, the Ras target to which a subject compound binds, either covalently or reversibly, can be a Ras mutant (e.g., G12S, G12C, G12D, G12V, G13C, and/or G13D), including a mutant of K-Ras, H-Ras, or N-Ras. In some embodiments, the methods of treating cancer can be applied to treat a solid tumor or a hematological cancer. In some embodiments, the cancer being treated can be, without limitation, prostate cancer, brain cancer, colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, various lung cancers including non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, non-Hodgkin’s lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi’s sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers. In some embodiments is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, wherein the cancer is a hematological cancer. In some embodiments is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, wherein the cancer is a hematological cancer selected from one or more of chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T- cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and pre-leukemia. In some embodiments is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, wherein the cancer is one or more cancers selected from the group consisting of chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), B cell acute lymphoblastic leukemia (B- ALL), and/or acute lymphoblastic leukemia (ALL).
[279] Any of the treatment methods disclosed herein can be administered alone or in combination or in conjunction with another therapy or another agent. By “combination” it is meant to include (a) formulating a subject composition containing a subject conjugate together with another agent, or (b) using the subject composition separate from the another agent as an overall treatment regimen. By “conjunction” it is meant that the another therapy or agent is administered either simultaneously, concurrently or sequentially with a subject composition comprising a conjugate disclosed herein, with no specific time limits, wherein such conjunctive administration provides a therapeutic effect.
[280] In some embodiments, a subject treatment method is combined with surgery, cellular therapy, chemotherapy, radiation, and/or immunosuppressive agents. Additionally, compositions of the present disclosure can be combined with other therapeutic agents, such as other anti-cancer agents, anti-allergic agents, anti-nausea agents (or anti-emetics), pain relievers, cytoprotective agents, immunostimulants, and combinations thereof. In one embodiment, a subject treatment method is combined with a chemotherapeutic agent.
[281] Exemplary chemotherapeutic agents include an anthracycline (e.g., doxorubicin (e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosf amide, temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fludarabine)), a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide). Additional chemotherapeutic agents contemplated for use in combination include busulfan (Myleran®), busulfan injection (Busulfex®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), hydroxyurea (Hydrea®), Idarubicin (Idamycin®), mitoxantrone (Novantrone®), Gemtuzumab Ozogamicin (Mylotarg®), anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), dexamethasone, docetaxel (Taxotere®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxy citidine), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (EL SPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6- thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hy camptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).
[282] Anti-cancer agents of particular interest for combinations with a conjugate of the present disclosure include: anthracy clines; alkylating agents; antimetabolites; drugs that inhibit either the calcium dependent phosphatase calcineurin or the p70S6 kinase FK506 or inhibit the p70S6 kinase; mTOR inhibitors; immunomodulators; anthracy clines; vinca alkaloids; proteosome inhibitors; GITR agonists; protein tyrosine phosphatase inhibitors; a CDK4 kinase inhibitor; a BTK inhibitor; a MKN kinase inhibitor; a DGK kinase inhibitor; or an oncolytic virus.
[283] Exemplary antimetabolites include, without limitation, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors: methotrexate (Rheumatrex®, Trexall®), 5-fluorouracil (Adrucil®, Efudex®, Fluoroplex®), floxuridine (FUDF®), cytarabine (Cytosar-U®, Tarabine PFS), 6-mercaptopurine (Puri-Nethol®)), 6-thioguanine (Thioguanine Tabloid®), fludarabine phosphate (Fludara®), pentostatin (Nipent®), pemetrexed (Alimta®), raltitrexed (Tomudex®), cladribine (Leustatin®), clofarabine (Clofarex®, Clolar®), azacitidine (Vidaza®), decitabine and gemcitabine (Gemzar®). Preferred antimetabolites include, cytarabine, clofarabine and fludarabine.
[284] Exemplary alkylating agents include, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes: uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, without limitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®);
Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan (Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (also known as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® and Neosar®); Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine (Matulane®); Mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, Mustargen®); Streptozocin (Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and Bendamustine HC1 (Treanda®).
[285] In certain aspects, compositions provided herein can be administered in combination with radiotherapy, such as radiation. Whole body radiation may be administered at 12 Gy. A radiation dose may comprise a cumulative dose of 12 Gy to the whole body, including healthy tissues. A radiation dose may comprise from 5 Gy to 20 Gy. A radiation dose may be 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 11 Gy, 12, Gy, 13 Gy, 14 Gy, 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, or up to 20 Gy. Radiation may be whole body radiation or partial body radiation. In the case that radiation is whole body radiation it may be uniform or not uniform. For example, when radiation may not be uniform, narrower regions of a body such as the neck may receive a higher dose than broader regions such as the hips.
[286] Where desirable, an immunosuppressive agent can be used in conjunction with a subject treatment method. Exemplary immunosuppressive agents include but are not limited to cyclosporin, azathioprine, methotrexate, my cophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies (e.g., muromonab, otelixizumab) or other antibody therapies, cy toxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation, peptide vaccine, and any combination thereof. In accordance with the presently disclosed subject matter, the above -described various methods can comprise administering at least one immunomodulatory agent. In certain embodiments, the at least one immunomodulatory agent is selected from the group consisting of immuno stimulatory agents, checkpoint immune blockade agents (e.g., blockade agents or inhibitors of immune checkpoint genes, such as, for example, PD-1, PD-L1, CTLA-4, IDO, TIM3, LAG3, TIGIT, BTLA, VISTA, ICOS, KIRs and CD39), radiation therapy agents, chemotherapy agents, and combinations thereof. In some embodiments, the immuno stimulatory agents are selected from the group consisting of IL-12, an agonist costimulatory monoclonal antibody, and combinations thereof. In one embodiment, the immunostimulatory agent is IL- 12. In some embodiments, the agonist costimulatory monoclonal antibody is selected from the group consisting of an anti-4-lBB antibody (e.g., urelumab, PF-05082566), an anti-OX40 antibody (pogalizumab, tavolixizumab, PF-04518600), an anti-ICOS antibody (BMS986226, MEDI-570, GSK3359609, JTX- 2011), and combinations thereof. In one embodiment, the agonist costimulatory monoclonal antibody is an anti-4- 1BB antibody. In some embodiments, the checkpoint immune blockade agents are selected from the group consisting of anti-PD-Ll antibodies (atezolizumab, avelumab, durvalumab, BMS-936559), anti-CTLA-4 antibodies (e.g., tremelimumab, ipilimumab), anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab, cemiplimab), anti-LAG3 antibodies (e.g., C9B7W, 410C9), anti-B7-H3 antibodies (e.g., DS-5573a), anti-TIM3 antibodies (e.g., F38-2E2), and combinations thereof. In one embodiment, the checkpoint immune blockade agent is an anti-PD-Ll antibody. In some cases, a conjugate of the present disclosure can be administered to a subject in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In some cases, expanded cells can be administered before or following surgery. Alternatively, compositions comprising a conjugate described herein can be administered with immunostimulants.
Immunostimulants can be vaccines, colony stimulating agents, interferons, interleukins, viruses, antigens, costimulatory agents, immunogenicity agents, immunomodulators, or immunotherapeutic agents. An immunostimulant can be a cytokine such as an interleukin. One or more cytokines can be introduced with modified cells provided herein. Cytokines can be utilized to boost function of modified T lymphocytes (including adoptively transferred tumor-specific cytotoxic T lymphocytes) to expand within a tumor microenvironment. In some cases, IL -2 can be used to facilitate expansion of the modified cells described herein. Cytokines such as IL- 15 can also be employed. Other relevant cytokines in the field of immunotherapy can also be utilized, such as IL-2, IL-7, IL-12, IL-15, IL-21, or any combination thereof. An interleukin can be IL -2, or aldesleukin. Aldesleukin can be administered in low dose or high dose. A high dose aldesleukin regimen can involve administering aldesleukin intravenously every 8 hours, as tolerated, for up to about 14 doses at about 0.037 mg/kg (600,000 lU/kg). An immuno stimulant (e.g., aldesleukin) can be administered within 24 hours after a cellular administration. An immuno stimulant (e.g., aldesleukin) can be administered in as an infusion over about 15 minutes about every 8 hours for up to about 4 days after a cellular infusion. An immuno stimulant (e.g., aldesleukin) can be administered at a dose from about 100,000 lU/kg, 200,000 lU/kg, 300,000 lU/kg, 400,000 lU/kg, 500,000 lU/kg, 600,000 lU/kg, 700,000 lU/kg, 800,000 lU/kg, 900,000 lU/kg, or up to about 1,000,000 lU/kg. In some cases, aldesleukin can be administered at a dose from about 100,000 lU/kg to 300,000 lU/kg, from 300,000 lU/kg to 500,000 lU/kg, from 500,000 lU/kg to 700,000 lU/kg, from 700,000 lU/kg to about 1,000,000 lU/kg.
[287] In some embodiments, a conjugate described herein, such as a conjugate, salt, or solvate of Formula (A), is administered in combination or in conjunction with one or more pharmacologically active agents selected from (1) an inhibitor of MEK (e.g., MEK1, MEK2) or of mutants thereof (e.g., trametinib, cobimetinib, binimetinib, selumetinib, refametinib, AZD6244); (2) an inhibitor of epidermal growth factor receptor (EGFR) and/or of mutants thereof (e.g., afatinib, erlotinib, gefitinib, lapatinib, cetuximab panitumumab, osimertinib, olmutinib, EGF-816); (3) an immunotherapeutic agent (e.g., checkpoint immune blockade agents, as disclosed herein); (4) a taxane (e.g., paclitaxel, docetaxel); (5) an anti-metabolite (e.g. antifolates such as methotrexate, raltitrexed, pyrimidine analogues such as 5 -fluorouracil (5-FU), ribonucleoside and deoxyribonucleoside analogues, capecitabine and gemcitabine, purine and adenosine analogues such as mercaptopurine, thioguanine, cladribine and pentostatin, cytarabine (ara C), lludarabine): (6) an inhibitor of FGFR1 and/or FGFR2 and/or FGFR3 and/or FGFR4 and/or of mutants thereof (e.g., nintedanib); (7) a mitotic kinase inhibitor (e.g., a CDK4/6 inhibitor, such as, for example, palbociclib, ribociclib, abemaciclib); (8) an anti-angiogenic drug (e.g., an anti-VEGF antibody, such as, for example, bevacizumab); (9) a topoisomerase inhibitor (e.g. epipodophyllotoxins such as for example etoposide and etopophos, teniposide, amsacrin, topotecan, irinotecan, mitoxantrone); (10) a platinum-containing compound (e.g. cisplatin, oxaliplatin, carboplatin); (11) an inhibitor of ALK and/or of mutants thereof (e.g. crizotinib, alectinib, entrectinib, brigatinib); (12) an inhibitor of c-MET and/or of mutants thereof (e.g., K252a, SU11274, PHA665752, PF2341066); (13) an inhibitor of BCR-ABL and/or of mutants thereof (e.g., imatinib, dasatinib, nilotinib); (14) an inhibitor of ErbB2 (Her2) and/or of mutants thereof (e.g., afatinib, lapatinib, trastuzumab, pertuzumab); (15) an inhibitor of AXL and/or of mutants thereof (e.g., R428, amuvatinib, XL -880): (16) an inhibitor of NTRK1 and/or of mutants thereof (e.g., inerestinib): (17) an inhibitor of RET and/or of mutants thereof (e.g., BLU-667, Lenvatinib); (18) an inhibitor of A-Raf and/or B-Raf and/or C-Raf and/or of mutants thereof (RAF-709, L Y-3009120, sorafenib, vemurafenib, dabrafenib, encorafenib, regorafenib, GDC-879); (19) an inhibitor of ERK and/or of mutants thereof (e.g., uhxertmib, MK-8353, LTT462, AZD0364, SCH772984, BIX02189, LY3214996, ravoxertinib); (20) an MDM2 inhibitor (e.g., HDM-201, NVP-CGM097, RG-71 12, MK-8242, RG-7388, SAR405838, AMG-232, DS-3032, RG- 7775, APG-115); (21) an inhibitor of mTOR (e.g., rapamycin, temsirolimus, everolimus, ridaforolimus); (22) an inhibitor of BET (e.g., I-BET 151, 1-BET 762, OTX-015, TEN-010, CPI-203, CPI-0610, olionon, RVX-208, ABBC-744, LY294002, AZD5153, MT-1, MS645); (23) an inhibitor of IGF1/2 and/or of IGF1-R (e.g., xentuzumab, MEDI-573); (24) an inhibitor of CDK9 (e.g., DRB, flavopindol, CR8, AZD 5438, purvalanol B, AT7519, dinaciclib, SNS-032); (25) an inhibitor of famesyl transferase (e.g., tipifamib); (26) an inhibitor of SHIP pathway including SHIP2 inhibitor, as well as SHIP1 inhibitors; (27) an inhibitor of SRC (e.g., dasatinib); (28) an inhibitor of JAK (e.g. tofacitinib); (29) a PARP inhibitor (e.g. Olaparib, Rucaparib, Niraparib, Talazoparib), (30) a BTK inhibitor (e.g. Ibrutinib, Acalabrutinib, Zanubrutinib), (31) a ROS1 inhibitor (e.g., entrectinib), (32) an inhibitor of Src, FLT3, HDAC, VEGFR, PDGFR, LCK, Bcr-Abl or AKT, (33) an inhibitor of KRAS G12C mutant (e.g., including but not limited to AMG510, MRTX849, and any covalent inhibitors binding to the cysteine residue 12 of KRAS, the structures of which are publicly known) (e.g., an inhibitor of Ras G12C as described in US20180334454, US20190144444, US20150239900, US10246424, US20180086753, WO2018143315, WO2018206539, WO20191107519, W02019141250, W02019150305, US9862701, US20170197945, US20180086753, US10144724, US20190055211, US20190092767, US20180127396, US20180273523, US10280172, US20180319775, US20180273515, US20180282307, US20180282308, W02019051291, WO2019213526, WO2019213516, WO2019217691, WO2019241157, WO2019217307, W02020047192, WO2017087528, W02018218070, WO2018218069, W02018218071, W02020027083, W02020027084, WO2019215203, WO2019155399, W02020035031, W02014160200, WO2018195349, WO2018112240, WO2019204442, WO2019204449, W02019104505, WO2016179558, WO2016176338, or related patents and applications, each of which is incorporated by reference in its entirety), (34) an SHC inhibitor (e.g., PP2, AID371185), (35) a GAB inhibitor (e.g., GAB-0001), (36) a GRB inhibitor, (37) a PI-3 kinase inhibitor (e.g., idelalisib, copanlisib, duvelisib, alpelisib, taselisib, perifosine, buparlisib, umbralisib, NVP-BEZ235-AN), (38) a MARPK inhibitor, (39) a CDK4/6 inhibitor (e.g., palbociclib, ribociclib, abemaciclib), (40) a MAPK inhibitor (e.g., VX-745, VX-702, RO-4402257, SCIO-469, BIRB-796, SD-0006, PH-797804, AMG-548, LY2228820, SB-681323, GW-856553, RWJ67657, BCT- 197), or (41) a SHP pathway inhibitor, such as a SHP2 inhibitor (e.g., RMC-4630, ERAS-601,
or a SHP1 inhibitor. In some embodiments, a conjugate described herein, such as a conjugate, salt, or solvate of Formula (A), is administered in combination or in conjunction with one or more checkpoint immune blockade agents (e.g., anti-PD-1 and/or anti-PD-L1 antibody, anti-CLTA-4 antibody). In some embodiments, a conjugate described herein, such as a conjugate, salt, or solvate of Formula (A), is administered in combination or in conjunction with one or more pharmacologically active agents comprising an inhibitor against one or more targets selected from: MEK, epidermal growth factor receptor (EGFR), FGFR1, FGFR2, FGFR3, mitotic kinase, topoisomerase, ALK, ALK5, c-MET, ErbB2, AXL, NTRK1, RET, A-Raf, B-Raf, C-Raf, ERK, MDM2, mTOR, BET, IGF1/2, IGF1-R, CDK9, SHIP1, SHIP2, SHP2, SRC, JAK, PARP,
BTK, FLT3, HDAC, VEGFR, PDGFR, LCK, Bcr-Abl, AKT, KRAS G12C mutant, and ROS1. In some embodiments, a conjugate described herein, such as a conjugate, salt, or solvate of Formula (A), is administered in combination or in conjunction with one or more additional pharmacologically active agents comprising an inhibitor of SOS (e.g., SOS1, SOS2) or of mutants thereof, such as RMC-5845, or BI-1701963. In some embodiments, a conjugate described herein, such as a conjugate, salt, or solvate of Formula (A), is administered in combination or in conjunction with an inhibitor of SOS described in W02021092115, WO2018172250, WO2019201848, WO2019122129, WO2018115380, WO2021127429, W02020180768, or W02020180770, each of which is herein incorporated by reference in its entirety for all purposes.
[288] In some embodiments, a conjugate described herein, such as a conjugate, salt, or solvate of Formula (A), is administered in combination or in conjunction with one or more checkpoint immune blockade agents (e.g., anti-PD- 1 and/or anti-PD-L 1 antibody, anti-CLTA-4 antibody).
[289] In some embodiments, a conjugate described herein, such as a conjugate, salt, or solvate of Formula (A), and one or more pharmacologically active agents are administered either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two or more compounds in the body of the patient.
[290] In some embodiments, a conjugate described herein, such as a conjugate, salt, or solvate of Formula (A), and one or more pharmacologically active agents are administered sequentially in any order by a suitable route, such as infusion or orally. The dosing regimen may vary depending upon the stage of the disease, physical fitness of the patient, safety profiles of the individual drugs, and tolerance of the individual drugs, as well as other criteria known to the attending physician and medical practitioner(s) administering the combination. The conjugate of the present disclosure and other pharmacologically active agent(s) may be administered within minutes of each other, hours, days, or even weeks apart depending upon the particular cycle being used for treatment. In addition, the cycle could include administration of one drug more often than the other during the treatment cycle and at different doses per administration of the drug.
[291] In some cases, a treatment regime may be dosed according to a body weight of a subject. In subjects who are determined obese (BMI > 35) a practical weight may need to be utilized. BMI is calculated by: BMI = weight (kg)/[height (m)]2 Body weight may be calculated for men as 50 kg + 2.3*(number of inches over 60 inches) or for women 45.5 kg + 2.3*(number of inches over 60 inches). An adjusted body weight may be calculated for subjects who are more than 20% of their ideal body weight. An adjusted body weight may be the sum of an ideal body weight + (0.4*(Actual body weight - ideal body weight)). In some cases, a body surface area may be utilized to calculate a dosage. A body surface area (BSA) may be calculated by: BSA (m2) = √Height (cm) *Weight (kg)/3600.
[292] In certain aspects, the present disclosure provides a method of modulating activity of a Ras (e.g., K-Ras) protein, comprising contacting a Ras protein with an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, thereby modulating the activity of the Ras (e.g., K-Ras) protein. In some embodiments, the subject method comprises administering an additional agent or therapy.
[293] In certain aspects, the present disclosure provides a method of modulating activity of a Ras protein, comprising contacting a Ras protein with an effective amount of a conjugate described, or a pharmaceutically acceptable salt or solvate thereof, wherein said modulating comprises inhibiting the Ras (e.g., K-Ras) protein activity. In certain aspects, the present disclosure provides a method of modulating activity of a Ras protein, such as Ras mutant (e.g., G12S, G12C, G12D, G12V, G13C, and/or G13D) proteins of K-Ras, H-Ras, and N-Ras, comprising contacting the Ras protein with an effective amount of a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof.
[294] In certain aspects, the present disclosure provides a method of reducing Ras signaling output in a cell by contacting the cell with a conjugate described herein. A reduction in Ras signaling can be evidenced by one or more of the following: (i) an increase in steady state level of GDP -bound modified protein; (ii) a reduction in steady state level of GTP-bound Ras protein; (iii) a reduction of phosphorylated AKTs473, (iv) a reduction of phosphorylated ERKT202/y204, (v) a reduction of phosphorylated S6S235/236, (vi) a reduction of cell growth of a tumor cell expressing a Ras mutant (e.g., G12S, G12C, G12D, G12V, G13C, and/or G13D) protein, and (vii) a reduction in Ras interaction with a Ras-pathway signaling protein. Non-limiting examples of Ras-pathway signaling proteins include SOS (including SOS1 and SOS2), RAF, SHC, SHP (including SHP1 and SHP2), MEK, MAPK, ERK, GRB, RASA1 , and GNAQ. In some embodiments, the reduction in Ras signaling output can be evidenced by two, three, four, five, six, or all of (i)-(vii) above. In some embodiments, the reduction of any one or more of (i)-(vii) can be 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100- fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or more as compared to a control not treated with a subject conjugate. A reduction in cell growth can be demonstrated with the use of tumor cells or cell lines. A tumor cell line can be derived from a tumor in one or more tissues, e.g., pancreas, lung, ovary, biliary tract, intestine (e.g., small intestine, large intestine, colon), endometrium, stomach, hematopoietic tissue (e.g., lymphoid tissue), etc. Examples of tumor cell lines comprising a K-Ras mutation include, but are not limited to, A549 (e.g., K-Ras G12S), AGS (e.g., K-Ras G12D), ASPC1 (e.g., K-Ras G12D), Calu-6 (e.g., K-Ras Q61K), CFPAC-1 (e.g., K-Ras G12V), CL40 (e.g., K-Ras G12D), COLO678 (e.g., K-Ras G12D), COR-L23 (e.g., K-Ras G12V), DAN-G (e.g., K-Ras G12V), GP2D (e.g., K-Ras G12D), GSU (e.g., K-Ras G12F), HCT116 (e.g., K-Ras G13D), HEC1A (e.g., K-Ras G12D), HEC1B (e.g., K-Ras G12F), HEC50B (e.g., K-Ras G12F), HEYA8 (e.g., K-Ras G12D or G13D), HPAC (e.g., K-Ras G12D), HPAFII (e.g., K- Ras G12D), HUCCT1 (e.g., K-Ras G12D), KARPAS620 (e.g., K-Ras G13D), K0PN8 (e.g., K-Ras G13D), KP-3 (e.g., K-Ras G12V), KP-4 (e.g., K-Ras G12D), L3.3 (e.g., K-Ras G12D), LoVo (e.g., K-Ras G13D), LS180 (e.g., K- Ras G12D), LS513 (e.g., K-Ras G12D), MCAS (e.g., K-Ras G12D), NB4 (e.g., K-Ras A18D), NCI-H1355 (e.g., K- Ras G13C), NCI-H1573 (e.g., K-Ras G12A), NCI-H1944 (e.g., K-Ras G13D), NCI-H2009 (e.g., K-Ras G12A), NCI-H441 (e.g., K-Ras G12V), NCI-H747 (e.g., K-Ras G13D), N0M0-1 (e.g., K-Ras G12D), OV7 (e.g., K-Ras G12D), PANC0203 (e.g., K-Ras G12D), PANC0403 (e.g., K-Ras G12D), PANC0504 (e.g., K-Ras G12D), PANC0813 (e.g., K-Ras G12D), PANCI (e.g., K-Ras G12D), Panc-10.05 (e.g., K-Ras G12D), PaTu-8902 (e.g., K- Ras G12V), PK1 (e.g., K-Ras G12D), PK45H (e.g., K-Ras G12D), PK59 (e.g., K-Ras G12D), SK-CO-1 (e.g., K- Ras G12V), SKLU1 (e.g., K-Ras G12D), SKM-1 (e.g., K-Ras KI 17N), SNU1 (e.g., K-Ras G12D), SNU1033 (e.g., K-Ras G12D), SNU1197 (e.g., K-Ras G12D), SNU407 (e.g., K-Ras G12D), SNU410 (e.g., K-Ras G12D), SNU601 (e.g., K-Ras G12D), SNU61 (e.g., K-Ras G12D), SNU8 (e.g., K-Ras G12D), SNU869 (e.g., K-Ras G12D), SNU- C2A (e.g., K-Ras G12D), SU.86.86 (e.g., K-Ras G12D), SUIT2 (e.g., K-Ras G12D), SW1990 (e.g., K-Ras G12D), SW403 (e.g., K-Ras G12V), SW480 (e.g., K-Ras G12V), SW620 (e.g., K-Ras G12V), SW948 (e.g., K-Ras Q61L), T3M10 (e.g., K-Ras G12D), TCC-PAN2 (e.g., K-Ras G12R), TGBC11TKB (e.g., K-Ras G12D), and MIA Pa-Ca (e.g., MIA Pa-Ca 2 (e.g., K-Ras G12C)).
Pharmaceutical compositions and methods of administration
[295] In an aspect is provided a pharmaceutical composition comprising a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient.
[296] In some embodiments, a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, is administered to a subject in a biologically compatible form suitable for administration to treat or prevent diseases, disorders, or conditions. Administration of a conjugate described herein can be in any pharmacological form including a therapeutically effective amount of a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, alone or in combination with a pharmaceutically acceptable carrier.
[297] In some embodiments, a conjugate described herein is administered as a pure chemical. In some embodiments, the conjugate described herein is combined with a pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier) selected on the basis of a chosen route of administration and standard pharmaceutical practice as described, for example, in Remington: The Science and Practice of Pharmacy (Gennaro, 21st Ed. Mack Pub. Co., Easton, PA (2005)).
[298] Accordingly, provided herein is a pharmaceutical composition comprising at least one conjugate described herein, or a pharmaceutically acceptable salt, together with one or more pharmaceutically acceptable excipients. The excipient(s) (or carrier(s)) is acceptable or suitable if the excipient is compatible with the other ingredients of the composition and not deleterious to the recipient (i.e., the subject) of the composition.
[299] In some embodiments of the methods described herein, a conjugate described herein is administered either alone or in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition. Administration of a conjugate or composition described herein can be affected by any method that enables delivery of the conjugate to the site of action. These methods include, though are not limited to delivery via enteral routes (including oral, gastric or duodenal feeding tube, rectal suppository and rectal enema), parenteral routes (injection or infusion, including intraarterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and subcutaneous), inhalational, transdermal, transmucosal, sublingual, buccal and topical (including epicutaneous, dermal, enema, eye drops, ear drops, intranasal, vaginal) administration, although the most suitable route may depend upon for example the condition and disorder of the recipient. By way of example only, a conjugate described herein can be administered locally to the area in need of treatment, by, for example, local infusion during surgery, topical application such as creams or ointments, injection, catheter, or implant. The administration can also be by direct injection at the site of a diseased tissue or organ. In some embodiments, a conjugate described herein, or a pharmaceutically acceptable salt or solvate thereof, is administered orally.
[300] In some embodiments of the methods described herein, a pharmaceutical composition suitable for oral administration is presented as a discrete unit such as a capsule, cachet or tablet, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a nonaqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. In some embodiments, the active ingredient is presented as a bolus, electuary, or paste.
[301] Pharmaceutical compositions which can be used orally include tablets, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free -flowing form such as a powder or granules, optionally mixed with binders, inert diluents, or lubricating, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered conjugate moistened with an inert liquid diluent. In some embodiments, the tablets are coated or scored and are formulated so as to provide slow or controlled release of the active ingredient therein. All formulations for oral administration should be in dosages suitable for such administration. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In some embodiments, stabilizers are added. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or Dragee coatings for identification or to characterize different combinations of active conjugate doses.
[302] In some embodiments of the methods described herein, pharmaceutical compositions are formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
[303] Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active conjugate which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the conjugates to allow for the preparation of highly concentrated solutions.
[304] Pharmaceutical compositions may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the conjugates may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
[305] ADDITIONAL EMBODIMENTS
[306] 1. A conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker, and wherein the antigen binding unit and the KRAS inhibitor in the conjugate synergistically inhibits signaling output of the first antigen or KRAS.
[307] 2. A conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker, and wherein the antigen binding unit and the KRAS inhibitor in the conjugate synergistically inhibits signaling output of the first antigen and KRAS.
[308] 3. A conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker, and wherein the KRAS inhibitor selectively inhibits KRAS or a mutant thereof relative to HRAS and NRAS (e.g., 1.1 fold, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, , 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, or 10000 fold).
[309] 4. The conjugate of any one of the preceding embodiments, wherein the KRAS inhibitor is characterized by a PAMPA permeability (Pe) less than 1 x 10-6 cm/s.
[310] 5. The conjugate of any one of the preceding embodiments, wherein the conjugate is characterized by an enhanced therapeutic efficacy as ascertained by the formula: wherein TIconjugate = TD50c/ED50c, wherein TD50c is the dose of conjugate required to produce a toxic effect in 50% of test subjects and ED50c is the dose of conjugate required to produce a therapeutic effect in 50% of test subjects; and wherein TIKRASI = TD50k/ED50k, wherein TD50k is the dose of KRAS inhibitor required to produce a toxic effect in 50% of test subjects and ED50k is the dose of KRAS inhibitor required to produce a therapeutic effect in 50% of the test subjects.
[311] 6. The conjugate of any one of the preceding embodiments, wherein the conjugate is characterized by reduced plasma concentration of the KRAS inhibitor as ascertained by the formula:
[KRASi]p-c/[KRASi]p-k < 1 wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at a first time -point following administration of the conjugate; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point.
[312] 7. The conjugate of any one of the preceding embodiments, wherein the conjugate is characterized by an increased concentration of the KRAS inhibitor in tumor tissue relative to plasma as ascertained by the formula:
([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) > 1 wherein [KRASi]t-c is concentration of the KRAS inhibitor in tumor tissue at a first time -point following administration of the conjugate; wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at the same time -point following the administration of the conjugate; wherein [KRASi]t-k is concentration of the KRAS inhibitor in tumor tissue following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor at the same time-point following the administration of the KRAS inhibitor alone at the equivalent dose.
[313] 8. A conjugate of Formula (A): wherein:
AgB is an antigen binding unit;
L is a chemical linker;
D is independently selected at each occurrence from a small-molecule KRAS inhibitor, a cytotoxic smallmolecule and a small-molecule agent that selectively modulates a non-KRAS target, wherein at least one D is a KRAS inhibitor; p is selected from 1 to 20; and q is selected from 1 to 20.
[314] 9. The conjugate of any one of the preceding embodiments, wherein the antigen binding unit is an antibody or an antigen-binding fragment thereof.
[315] 10. The conjugate of any one of the preceding embodiments, wherein the antigen binding unit is selected from a monoclonal antibody, a Fab, a Fab’, an F(ab’), an Fv, a disulfide linked Fc, an scFv, a single domain antibody, a diabody, a bi-specific antibody, and a multi-specific antibody. [316] 11. The conjugate of any one of the preceding embodiments, wherein the antigen binding unit is a monoclonal antibody.
[317] 12. The conjugate of any one of the preceding embodiments, wherein the antigen binding unit specifically binds a target selected from AG7, B7-H3, BCMA, CA15-3, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD70, CD71, CD79B, CEA, CLDN18.2, EGFR, FOLR1, GCC, GPC1, HER2, HER3, ICAM1, LeX, LeY, MET, MSLN, MUC1, NECTIN4, SLC44A4, TF, and Trop-2.
[318] 13. The conjugate of any one of the preceding embodiments, wherein the antigen binding unit is selected from cetuximab, bevacizumab, paitumumab, ofatumumab, inotuzumab, gemtuzumab, alemtuzumab, and trastuzumab.
[319] 14. The conjugate of any one of the preceding embodiments, wherein the antigen binding unit is an anti-EGFR antibody.
[320] 15. The conjugate of any one of the preceding embodiments, wherein the anti-EGFR antibody is cetuximab.
[321] 16. The conjugate of any one of embodiments 1 to 14, wherein the antigen binding unit is sacituzumab.
[322] 17. The conjugate of any one of the preceding embodiments, wherein the linker comprises one or more components independently selected from alkyl, alkene, alkyne, aryl, cycloalkyl, heterocycle, glycoside, silyl ether, hydroxy, ether, ketone, ester, carbonate, amide, urea, carbamate, sulfide, disulfide, sulfate, sulfonamide, phosphate, hydrazone, and succinimide.
[323] 18. The conjugate of any one of the preceding embodiments, wherein the linker comprises one or more components independently selected from alkyl, polyethylene glycol, a hydrazone, maleimide, succinimide, asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, valine, alanine, glycine, leucine, isoleucine, methionine, tryptophan, proline, histidine, citrulline, phenylalanine, carboxylate, and p-aminobenzyloxycarbonyl.
[324] 19. The conjugate of any one of the preceding embodiments, wherein the linker comprises one or more components independently selected from polyethylene glycol, polysarcosine, a hydrazone, acetal, maleimide, succinimide, asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, valine, alanine, glycine, leucine, isoleucine, methionine, tryptophan, proline, histidine, citrulline, phenylalanine, carboxylate, p-aminobenzyloxycarbonyl, alkyl, alkene, alkyne, aryl, cycloalkyl, heterocycle, glycoside, silyl ether, hydroxy, ether, ketone, ester, carbonate, amide, urea, carbamate, sulfide, disulfide, sulfate, sulfonamide, phosphate, phenylboronic ester, phenylboronic acid, thioketal, tartaric acid, 1 ,2-diol acetonide, o-aminoalcohol, selenium, ortho-nitrobenzyl, phenacyl ester, sugar, glucoronide, trioxolane, oxime, acyl hydrazone, cyclobutyl, pyrophosphate, arylsulfate, heptamethine, cyanine fluorophore, o-nitrobenzyl, PC4AP, dsProc, 1,3-dioxane, triazole, piperazine, bis(vinylsulfonyl)piperazine, N-methyl-N-phenylvinylsulfonamide, and Pt.
[325] 20. The conjugate of any one of the preceding embodiments, wherein the linker comprises one or more components selected from Val-Cit, Glu-Val-Cit, Val-Ala, Val-Val, Val-Gly, Gly-Gly, Gly-Cit, Glu- Gly-Cit, Ala-Ala-Asn, Ala-Gly-Ala, Ala-Pro, Ala-Ser, and Phe-Lys.
[326] 21. The conjugate of any one of the preceding embodiments, wherein the linker has the formula: wherein: indicates an attachment site to the antigen binding unit;
Z1 is a product formed by reaction of the antigen binding unit with a reactive precursor of Z1 ;
Z2 is absent, C1-6 alkyl, (CH2CH2O)n2, -C(O)NH-, -C(O)NCH3-, (C(O)CH2N(CH3))n2, -
((CH2CH2O)n2(CH2CH2)C(O))N(CH2C(O)N(CH3)-(CH2CH2O)n2-(CH2CH2N(CH3)-(C(O)CH2N(CH3))n2-
C(O)CH3)CH2-, -(C1-6 alkyl)C(O)N(CH2C(O)N(CH3)-(CH2CH2O)n2-(CH2CH2N(CH3)-(C(O)CH2N(CH3))n2-
C(O)CH3)CH2-, -(C1-6 alkyl)C(O)N(CH2C(O)N(CH3)-(CH2CH2O)n2-(CH2CH2N(CH3)-(C(O)CH2N(CH3))n2-
C(O)CH2N(H)C(O)CH3)CH2-, -((CH2CH2O)n2(CH2CH2)C(O))N((CH2CH2O)n2CH3)CH2-, -((C1-6 alkyl)C(O))N((CH2CH2O)n2CH3)CH2-, -(C1-6 alkyl)C(O))N((CH2C(O)N(CH3)-(CH2CH2O)n2CH3)CH2-, -((C1-6 alkyl)C(O))N(CH2C(O)N((CH2CH2O)n2CH3)(CH2CH2O)n2CH3)CH2-, -
((CH2CH2O)n2(CH2CH2)C(O))N(CH2C(O)N((CH2CH2O)n2CH3)(CH2CH2O)n2CH3)CH2-, -
((CH2CH2O)n2(CH2CH2)C(O))N(CH2C(O)-(N(CH3)CH2C(O))n2-N(CH3)2)CH2-, -((C1-6 alkyl)C(O))N(CH2C(O)-
(N(CH3)CH2C(O))n2-N(CH3)2)CH2-, -(CH2CH2O)n2(CH2CH2)-, -(C1-6 alkyl)-, -
((CH2CH2O)n2(CH2CH2)C(O)NH(CH2CH2)-, -(CH2CH2O)n2(CH2CH2)C(O)-(N(CH3)CH2C(O))n2-N(CH3)CH2)-, -
(C1-6 alkyl)C(O)-(N(CH3)CH2C(O))n2-N(CH3)CH2)-, -(Phenyl)-CH2C(O)NH(CH2CH2O)n2(CH2CH2)-, -(C1-6
Z3 is selected from a bond, Val-Cit, Cit-Val, Ala-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu- Val, Ser-Cit, Cit-Ser, Lys-Cit, Cit-Lys, Asp-Cit, Cit-Asp, Val-Ala, Ala-Vai, Val-Val, Val-Gly, Gly-Val, Phe-Lys, Lys-Phe, Val-Lys, Lys-Val, Ala-Lys, Lys-Ala, Phe-Cit, Cit-Phe, Leu-Cit, Cit-Leu, Ile-Cit, Cit-Ile, Phe-Arg, Arg- Phe, Cit-Trp, Trp-Cit, Gly-Gly, Gly-Cit, Cit-Gly, Ala-Pro, Pro-Ala, Ala-Ser, Ser-Ala, Glu- Val-Cit, Cit- Val-Glu, Glu-Gly-Cit, Cit-Gly-Glu, Asn-Ala-Ala, Ala-Ala-Asn, and Ala-Gly-Ala;
Rz is selected from hydrogen, -CH2N(CH3)C(O)-(CH2CH2O)n2CH3, -CH2N(CH3)(C(O)CH2N(CH3))n2-C(O)CH3, -CH2N(CH3)C(O)-(CH2CH2O)n2-CH2CH2C(O)-(N(CH3)CH2C(O))n2-N(CH3)2, -SO3H, -CO2H, PEG 4-32, polysarcosine, -(CH2N(CH3)C(O))n2CH3, and a sugar moiety; nl, n3, and n4 are each independently 0 or 1 ; n2 is independently an integer from 1 to 20; and indicates an attachment site to the KRAS inhibitor.
[327] 22. The conjugate of any one of the preceding embodiments, wherein the linker has the formula: wherein: indicates an attachment site to the antigen binding unit;
Z1 is
Z2 is absent, C1-6 alkyl or -(CH2CH2O^CH2CH2, ;
Z3 is selected from Val-Cit and Val-Ala;
Rz is selected from hydrogen, -CH2N(CH3)C(O)-(CH2CH2O)n2CH3, -CH2N(CH3)(C(O)CH2N(CH3))n2-C(O)CH3, and -CH2N(CH3)C(O)-(CH2CH2O)n2-CH2CH2C(O)-(N(CH3)CH2C(O))n2-N(CH3)2; n1, n3, and n4 are each independently 0 or 1 ; n2 is independently an integer from 1 to 20; and indicates an attachment site to the KRAS inhibitor.
[328] 23. The conjugate of any one of embodiments 1 to 22, wherein the linker is attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 24.
The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 25. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 26. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 27. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 28.
The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 29. The conjugate of any one of embodiments 1 to 22, wherein the linker is to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 30. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 31. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 32. The conjugate of any one of embodiments 1 to 22, wherein the linker is ; wherein indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit.
33. The conjugate of any one of embodiments 1 to 22, wherein the linker is ; wherein indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 34. The conjugate of any one of embodiments 1 to 22, wherein the linker is wherein indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 35. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 36. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 37. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 38. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 39.
The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 40. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 41. The conjugate of any one of embodiments 1 to 22, wherein the linker is attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 42.
The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 43. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 44. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 45. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 46. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 47.
The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 48. The conjugate of any one of embodiments 1 to 22, wherein the linker is site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 49. The conjugate of any one of embodiments 1 to 22, wherein the linker is attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 50.
The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 51. The conjugate of any one of embodiments 1 to 22, wherein the linker is attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 52.
The conjugate of any one of embodiments 1 to 22, wherein the linker is ; wherein indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit.
53. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 54. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 55. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 56.
The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 57. The conjugate of any one of embodiments 1 to 22, wherein the linker is the Kras inhibitor and indicates the attachment site to the antigen binding unit. 58. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 59.
The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 60. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 61. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 62. The conjugate of any one of embodiments 1 to 22, wherein the linker is ; wherein indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 63. The conjugate of any one of embodiments 1 to 22, wherein the linker is ; wherein indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 64. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the
Kras inhibitor and indicates the attachment site to the antigen binding unit. 65. The conjugate of any one of embodiments 1 to 22, wherein the linker is
; wherein indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 66. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 67. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit.
68. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 69. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 70. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit.
71. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 72. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 73. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 74. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 75. The conjugate of any one of embodiments 1 to 22, wherein the linker is ; wherein indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 76. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 77. The conjugate of any one of embodiments 1 to 22, wherein the linker is indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit. 78. The conjugate of any one of embodiments 1 to 22, wherein the linker is, wherein indicates the attachment site to the Kras inhibitor and indicates the attachment site to the antigen binding unit.
[329] 79. The conjugate of any one of embodiments 8-78, wherein p is selected from 2 to 8. 80. The conjugate of any one of embodiments 8-78, wherein p is 2. 81. The conjugate of any one of embodiments 8-78, wherein p is 4. 82. The conjugate of any one of embodiments 8-78, wherein p is 6. 83. The conjugate of any one of embodiments 8-78, wherein p is 8. 84. The conjugate of any one of embodiments 8-83, wherein q is selected from 1 to 4. 85. The conjugate of any one of embodiments 8- 83, wherein q is 1.
[330] 86. The conjugate of any one of embodiments 8-85, wherein D is a small-molecule KRAS inhibitor. [331] 87. The conjugate of any one of the preceding embodiments, wherein the KRAS inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is selected from N and C(R6);
Z is selected from O, N, C(R5)2, C(O), S, S(O), and S(O)2;
R2, R5, R6, and R8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered hetero alky nyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, - N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, - C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R5 are taken together with the atom to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein two R5 are taken together to form =O, =NR12, or =C(R14)2;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, - OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -0C(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), - S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R3 are taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one or more R20; optionally wherein two R3 are taken together to form =O, =NR12, or =C(R14)2; and further optionally wherein one R3 and R4 are taken together with the atoms to which they are attached to form 3- to 10-membered heterocycle optionally substituted with one or more R20;
R4 is selected from hydrogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6- membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), -C(O)OR12, -C(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), and -S(=O)(=NR12)N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and - (2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20;
R7 is selected from C6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R20; m is 0, 1, 2, or 3; n is 1 or 2;
R12 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20;
R13 is independently selected at each occurrence from hydrogen, C1-6 alkyl, and C1-6 haloalky I: or R12 and R13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R20;
R14 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, - C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R14 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R20;
R20 is independently selected at each occurrence from halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, - C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, - OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, - S(O)2N(R22)(R23)-, -S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22; wherein two R20 attached to the same or adjacent atoms optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12- membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, - C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, - OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, - S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R21 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl- (C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R21 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and -OH;
R22 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle); and
R23 is independently selected at each occurrence from hydrogen and C1-6 alkyl; or R22 and R23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I) is replaced with a bond to the antigen binding unit or the chemical linker.
[332] 88. The conjugate of any one of the preceding embodiments, wherein the KRAS inhibitor is a compound of Formula (I-a): or a pharmaceutically acceptable salt or solvate thereof, wherein:
A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms;
R9 and R10 are independently selected from hydrogen, halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, - C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, - OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, - S(O)2N(R22)(R23)-, -S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22; wherein R9 and R10 optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), - N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), - C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, -S(O)2N(R22)(R23), and - S(O)(NR22)N(R22)(R23); and
R11 is selected from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12- membered heterocycle).
[333] 89. The conjugate of any one of embodiments 87-88, wherein A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl. 90. The conjugate of any one of embodiments 87-88, wherein A is pyridinyl. 91. The conjugate of any one of embodiments 87-90, wherein R11 is hydrogen. 92. The conjugate of any one of embodiments 87-91, wherein R10 is selected from hydrogen and halogen; or R9 and R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted. 93. The conjugate of any one of embodiments 87-91, wherein R10 is hydrogen. 94. The conjugate of any one of embodiments 87-93, wherein 95. The conjugate of any one of embodiments 87-94, wherein R9 is optionally substituted C1-3 alkyl. 96. The conjugate of any one of embodiments 87-94, wherein R9 is CH3. 97. The conjugate of any one of embodiments 87-96, wherein R4 is selected from C1-6 alkyl,
C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), each of which is optionally substituted with one or more R20. 98. The conjugate of any one of embodiments 87-96, wherein R4 is selected from C1-6 alkyl and -C0-6 alkyl-(3- to 12-membered heterocycle), each of which is optionally substituted with one or more substituents independently selected from halogen, -CH3, -NH2, -NHCH3, and -N(CH3)2. 99. The conjugate of any one of embodiments 87-98, wherein X is C(R6). 100. The conjugate of any one of embodiments 87-98, wherein X is N. 101.
The conjugate of any one of embodiments 87-100, wherein Z is O. 102. The conjugate of any one of embodiments 87-101, wherein R7 is selected from naphthyl, isoquinolinyl, indazolyl, benzothiazolyl, benzothiophenyl, phenyl, and pyridinyl, each of which is optionally substituted with one, two, three, or four R20. 103. The conjugate of any one of embodiments 87-101, wherein R7 is benzothiophenyl optionally substituted with one, two, three, or four R20. 104. The conjugate of any one of embodiments 87-103, wherein R7 is substituted with one, two, three, or four substituents independently selected from halogen, - CN, C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, C2-3 alkynyl, -OR22, -N(R22)(R23), and C3-6 cycloalkyl. 105.
The conjugate of any one of embodiments 87-103, wherein R7 is substituted with one, two, three, or four substituents independently selected from halogen, -CN, -CH3, -CH2CH3, -CH=CH2, -CF3, -C=CH, - OH, -NH2, and -cyclopropyl. [334] 106. The conjugate of any one of embodiments 87-101, wherein R7 is selected from 107.The conjugate of any one of embodiments 87-101, wherein R7 is
[335] 108. The conjugate of any one of embodiments 87-107, wherein:
X is C(R6);
A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl;
R2, R6, and R8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), -C(O)OR12, - N(R12)C(O)N(R12)(R13), -C(O)R12, -OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), -C(O)OR12, -N(R12)C(O)N(R12)(R13), -C(O)R12, - OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20; wherein two R3 are optionally taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one, two, or three R20; and further wherein two R3 are optionally taken together to form =O, =NR12, or =C(R14)2;
R7 is benzo [b]thiophen-4-yl optionally substituted with one, two, three, or four R20;
R9 is C1-3 alkyl optionally substituted with one, two, or three R20;
R10 is selected from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, 3- to 8- membered heterocycle, -OR22, -N(R22)(R23), -C(O)OR22, -N(R22)C(O)N(R22)(R23), -C(O)R22, -OC(O)R22, - C(O)N(R22)(R23), and -N(R22)C(O)R22, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8- membered heterocycle are optionally substituted with one, two, or three substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R11 is hydrogen; m is 0 or 1 ; and n is 1 or 2.
[336] 109. The conjugate of any one of embodiments 87-108, wherein the KRAS inhibitor is a compound of or a pharmaceutically acceptable salt or solvate thereof.
[337] 110. The conjugate of any one of embodiments 87-108, wherein the KRAS inhibitor is a compound of or a pharmaceutically acceptable salt or solvate thereof.
[338] 111. The conjugate of any one of embodiments 87-108, wherein the KRAS inhibitor is a compound of formula: or a pharmaceutically acceptable salt or solvate thereof.
[339] 112. The conjugate of any one of embodiments 87-111, wherein R2 is selected from hydrogen, C1-3 alkyl, -OR12, and 3- to 10-membered heterocycle, wherein C1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R20. 113. The conjugate of any one of embodiments 87-111, wherein R2 is -OR12. 114. The conjugate of any one of embodiments 87-111, wherein R2 is -O(C1- 3 alkyl)(4- to 10-membered heterocycle) optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and =C(R21)2, wherein R21 is independently selected at each occurrence from hydrogen, halogen, and C1-3 alkyl. 115. The conjugate of any one of embodiments 87-111, wherein R2 is selected from: [340] 116. The conjugate of any one of embodiments 87-111, wherein R2 is
[341] 117. The conjugate of any one of embodiments 87-116, wherein R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20. 118. The conjugate of any one of embodiments 87-116, wherein R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, -OH, and -OCH3. 119.
The conjugate of any one of embodiments 87-118, wherein m is 0 or 1. 120. The conjugate of any one of embodiments 87-118, wherein m is 0. 121 . The conjugate of any one of embodiments 87-120, wherein R6 and R8 are independently selected from hydrogen, halogen, and C1-3 haloalkyl. 122.
The conjugate of any one of embodiments 87-121, wherein R6 is selected from chlorine and -CF3. 123. The conjugate of any one of embodiments 87-121, wherein R6 is chlorine. 124. The conjugate of any one of embodiments 87-121, wherein R6 is -CF3. 125. The conjugate of any one of embodiments 87-124, wherein R8 is fluorine. 126. The conjugate of any one of embodiments 87-125, wherein n is 1. 127.
The conjugate of any one of embodiments 87-126, wherein R7 is represents the bond to the chemical linker. [342] 128. A pharmaceutical composition comprising the conjugate of any one of embodiments 1 to 127, or a salt thereof, and a pharmaceutically acceptable excipient.
[343] 129. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate of any one of embodiments 1 to 127, or a salt thereof. 130.
A method of treating cancer in a subject comprising a Ras mutant protein, the method comprising: inhibiting the Ras mutant protein of said subject by administering to said subject a conjugate of any one of embodiments 1 to 127, or a salt thereof. 131. The method of embodiment 129-130, wherein the cancer is a solid tumor or a hematological cancer. 132. The method of any one of embodiments 129-131, wherein the cancer comprises a K-Ras G12C, G12D, G12S, or G12V mutant protein. 133. A method of modulating signaling output of a Ras protein, comprising contacting a Ras protein with an effective amount of a conjugate of any one of embodiments 1 to 127, or a salt thereof, thereby modulating the signaling output of the Ras protein. 134. A method of inhibiting cell growth, comprising administering an effective amount of a conjugate of any one of embodiments 1 to 127, or a salt thereof, to a cell expressing a Ras protein, thereby inhibiting growth of said cells. 135. The method of any one of embodiments 129-134, comprising administering an additional agent. 136. A method of delivering a small-molecule KRAS inhibitor that exhibits low permeability as characterized by a PAMPA assay, comprising contacting a tumor cell with a conjugate of any one of embodiments 1 to 127, or a salt thereof, wherein the KRAS inhibitor exhibits a PAMPA permeability (Pe) value less than 1 x 10-6 cm/s.
[344] 137. A method of enhancing therapeutic efficacy of a small-molecule KRAS inhibitor, comprising providing a conjugate of any one of embodiments 1 to 127 to a subject, wherein enhanced therapeutic efficacy is ascertained by the formula:
T Iconjugate/T I KRASi > 1 wherein TIconjugate = TD50c/ED50c, wherein TD50c is the dose of conjugate required to produce a toxic effect in 50% of test subjects and ED50c is the dose of conjugate required to produce a therapeutic effect in 50% of test subjects; and wherein TIKRASI = TD50k/ED50k, wherein TD50k is the dose of KRAS inhibitor required to produce a toxic effect in 50% of test subjects and ED50k is the dose of KRAS inhibitor required to produce a therapeutic effect in 50% of the test subjects.
[345] 138. A method of reducing plasma concentration of a small-molecule KRAS inhibitor, comprising providing a conjugate of any one of embodiments 1 to 127 to a subject, wherein reduced plasma concentration is ascertained by the formula:
[KRASi]p-c/[KRASi]p-k < 1 wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at a first time -point following administration of the conjugate; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point.
[346] 139. A method of increasing concentration of a small-molecule KRAS inhibitor in tumor tissue, comprising providing a conjugate of any one of embodiments 1 to 127 to a subject, wherein increased tumor tissue concentration is ascertained by the formula:
([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) > 1 wherein [KRASi]t-c is concentration of the KRAS inhibitor in tumor tissue at a first time -point following administration of the conjugate; wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at the same time -point following the administration of the conjugate; wherein [KRASi]t-k is concentration of the KRAS inhibitor in tumor tissue following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor at the same time-point following the administration of the KRAS inhibitor alone at the equivalent dose.
[347] 140. A method of delivering a small -molecule KRAS inhibitor to the central nervous system of a subject, comprising administering a conjugate of any one of embodiments 1 to 127 to the subject, wherein the KRAS inhibitor is released from the conjugate after entering the CNS of the subject. 141. A method of generating a slow-release form of a small-molecule KRAS inhibitor, the method comprising conjugating an antigen binding unit to a small-molecule KRAS inhibitor through a chemical linker, wherein the small-molecule inhibitor is released from the conjugate upon introducing the conjugate into a subject or a cell. 142. The method of any one of embodiments 129-141, wherein efficacy of the conjugate is greater than efficacy of a combination of the antigen binding unit and the KRAS inhibitor when each is administered at a comparable concentration. 143. The method of any one of embodiments 129-142, wherein toxicity of the conjugate is less than toxicity of a combination of the antigen binding unit and the KRAS inhibitor when each is administered at a comparable concentration.
EXAMPLES
[348] Example 1: Ras sequences
[349] Human K-Ras Wildtype sequence (SEQ ID NO. 1)
1 MTEYKL VVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET 51 CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI 101 KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ 151 GVDDAFYTLV REIRKHKEKM SKDGKKKKKK SKTKCVIM
[350] Human K-Ras G12D (SEQ ID NO. 2)
1 MTEYKL VVVG ADGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET 51 CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI 101 KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ 151 GVDDAFYTLV REIRKHKEKM SKDGKKKKKK SKTKCVIM
[351] Human K-Ras G12V (SEQ ID NO. 3)
1 MTEYKL VVVG AVGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET 51 CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI 101 KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ 151 GVDDAFYTLV REIRKHKEKM SKDGKKKKKK SKTKCVIM
[352] Human K-Ras G12S (SEQ ID NO. 4):
1 MTEYKL VVVG ASGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET 51 CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI 101 KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ 151 GVDDAFYTLV REIRKHKEKM SKDGKKKKKK SKTKCVIM
[353] Human N-Ras wildtype (SEQ ID NO. 5)
1 MTEYKL VVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET
51 CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNSKSF ADINLYREQI
101 KRVKDSDDVP MVLVGNKCDL PTRTVDTKQA HELAKSYGIP FIETSAKTRQ
151 GVEDAFYTLV REIRQ YRMKK LNSSDDGTQG CMGLPCVVM
[354] H-Ras G12D (SEQ ID NO. 6)
1 MTEYKL VVVG ADGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET
51 CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHQYREQI
101 KRVKDSDDVP MVLVGNKCDL AARTVESRQA QDLARSYGIP YIETSAKTRQ
151 GVEDAFYTLV REIRQHKLRK LNPPDESGPG CMSCKCVLS
[355] H-Ras wildtype (SEQ ID NO. 7)
1 MTEYKL VVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET
51 CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHQYREQI
101 KRVKDSDDVP MVLVGNKCDL AARTVESRQA QDLARSYGIP YIETSAKTRQ
151 GVEDAFYTLV REIRQHKLRK LNPPDESGPG CMSCKCVLS
[356] Human N-Ras G12D (SEQ ID NO. 8)
1 MTEYKL VVVG ADGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET
51 CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNSKSF ADINLYREQI
101 KRVKDSDDVP MVLVGNKCDL PTRTVDTKQA HELAKSYGIP FIETSAKTRQ
151 GVEDAFYTLV REIRQ YRMKK LNSSDDGTQG CMGLPCVVM
[357] Example 2: Exemplary Antigen Binding Units
[358] Cetuximab Sequences
[359] Heavy chain sequence (SEQ ID No. 14)
1 QVQLKQSGPG LVQPSQSLSI TCTVSGFSLT NYGVHWVRQS PGKGLEWLGV IWSGGNTDYN
61 TPFTSRLSIN KDNSKSQVFF KMNSLQSNDT AIYYCARALT YYDYEFAYWG QGTLVTVSAA
121 STKGPSVFPL APSSKSTSGG TAALGCLVKDYFPEPVTVSW NSGALTSGVH TFPAVLQSSG
181 LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPELLGGP
241 SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
301 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM
361 TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ
421 QGNVFSCSVM HEALHNHYTQ KSLSLSPGK
[360] Light chain sequence (SEQ ID No. 15)
1 DILLTQSPVI LSVSPGERVS FSCRASQSIG TNIHWYQQRT NGSPRLLIKY ASESISGIPS
61 RFSGSGSGTD FTLSINSVES EDIADYYCQQ NNNWPTTFGA GTKLELKRTV AAPSVFIFPP
121 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT
181 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
[361] Sacituzumab Sequences
[362] Heavy chain sequence (SEQ ID No. 16)
1 QVQLQQSGSE LKKPGASVKV SCKASGYTFT NYGMNWVKQA PGQGLKWMGW
INTYTGEPTY 61 TDDFKGRFAF SLDTSVSTAY LQISSLKADD TAVYFCARGG FGSSYWYFDV WGQGSLVTVS 121 SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS 181 SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPELLG 241 GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 301NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE 361 EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR 421 WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K
[363] Light chain sequence (SEQ ID No. 17)
1 DIQLTQSPSS LSASVGDRVS ITCKASQDVS IAVAWYQQKP GKAPKLLIYS ASYRYTGVPD 61 RFSGSGSGTD FTLTISSLQP EDFAVYYCQQ HYITPLTFGA GTKVEIKRT V AAPSVFIFPP 121 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 181 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
[364] Example 3: Protein expression
[365] DNA expression constructs encoding one or more protein sequences of interest (e.g., KRAS fragments thereof, mutant variants thereof, etc.) and its corresponding DNA sequences are optimized for expression in E. coli and synthesized by, for example, the GeneArt Technology at Life Technologies. In some cases, the protein sequences of interest are fused with a tag (e.g., glutathione S-transferase (GST), histidine (His), or any other affinity tags) to facilitate recombinant expression and purification of the protein of interest. Such tag can be cleaved subsequent to purification. Alternatively, such tag may remain intact to the protein of interest and may not interfere with activities (e.g., target binding and/or phosphorylation) of the protein of interest
[366] A resulting expression construct is additionally encoded with (i) att-site sequences at the 5’ and 3’ ends for subcloning into various destination vectors using, for example, the Gateway Technology, as well as (ii) a Tobacco Etch Virus (TEV) protease site for proteolytic cleavage of one or more tag sequences. The applied destination vectors can be a pET vector series from Novagen (e.g., with ampicillin resistance gene), which provides an N- terminal fusion of a GST-tag to the integrated gene of interest and/or a pET vector series (e.g., with ampicillin resistance gene), which provides an N-terminal fusion of a HIS-tag to the integrated gene. To generate the final expression vectors, the expression construct of the protein of interest is cloned into any of the applied destination vectors. The expression vectors are transformed into an E. coli strain, e.g., BL21 (DE3). Cultivation of the transformed strains for expression is performed in a 10 L or 1 L fermenter. The cultures are grown, for example, in Terrific Broth media (MP Biomedicals, Kat. #1 13045032) with 200 μg/ml, ampicillin at a temperature of 37 °C to a density of 0.6 (OD600), shifted to a temperature of ~27 °C (for K-Ras expression vectors) induced for expression with 100 mM IPTG, and further cultivated for 24 hours. After cultivation, the transformed E. coli cells are harvested by centrifugation and the resulting pellet is suspended in a lysis buffer, as provided below, and lysed by passing three-times through a high-pressure device. The lysate is centrifuged (49000g, 45 min, 4 °C) and the supernatant is used for further purification.
[367] Example 4: Ras protein purification
[368] A Ras (e.g., K-Ras wildtype or a mutant such as K-Ras G12S, K-Ras G12D, K-Ras G12V or K-Ras G12C) construct or a variant thereof is tagged with GST. E. coli culture from a 10L fermenter is lysed in lysis buffer (50 mM Tris HCI 7.5, 500 mM NaCl, 1 mM DTT, 0.5% CHAPS, Complete Protease Inhibitor Cocktail-(Roche)). As a first chromatography step, the centrifuged lysate is incubated with 50 mL Glutathione Agarose 4B (Macherey- Nagel; 745500.100) in a spinner flask (16 h, 10 °C). The Glutathione Agarose 4B loaded with protein is transferred to a chromatography column connected to a chromatography system, e.g., an Akta chromatography system. The column is washed with wash buffer (50 mM Tris HCI 7.5, 500 mM NaCl, 1 mM DTT) and the bound protein is eluted with elution buffer (50 mM Tris HCI 7.5, 500 mM NaCl, 1 mM DTT, 15 mM glutathione). The main fractions of the elution peak (monitored by OD280) are pooled. For further purification by size-exclusion chromatography, the above eluate volume is applied to a column Superdex 200 HR prep grade (GE Healthcare) and the resulting peak fractions of the eluted fusion protein is collected. Native mass spectrometry analyses of the final purified protein construct can be performed to assess its homogeneous load with GDP.
[369] Example 5: Ras cellular assay
[370] The ability of a compound of the present disclosure to inhibit Ras protein signaling can be demonstrated by inhibiting growth of a given KRAS mutant cell line. For example, this assay can be also used to assess selective growth inhibition of a mutant Ras protein relative to a wildtype or different mutant Ras protein. a. Growth of cells with K-Ras G12C mutation
[371] MIA PaCa-2 (ATCC CRL-1420) and NCI-H1792 (ATCC CRL-5895) cell lines comprise a G12C mutation and can be used to assess Ras cellular signaling in vitro, e.g., in response to an inhibitor compound of the present disclosure. This cellular assay can also be used to discern selective inhibition of a subject compound against certain types of KRAS mutants, e.g., more potent inhibition against KRAS G12C relative to KRAS G12D mutant, by comparing inhibition of MIA PaCa-2 (G12C driven tumor cell line) to inhibition of GP2d (G12D driven tumor cell line). MIA PaCa-2 culture medium is prepared with DMEM/Ham's F12 (e.g., with stable glutamine, 10% FCS, and 2.5% horse serum. NCI-H1792 culture medium is prepared with RPMI 1640 (e.g., with stable glutamine) and 10% FCS.
[372] On a first day (e.g., Day 1), Softagar (Select Agar, Invitrogen, 3% in ddH2O autoclaved) is boiled and tempered at 48 °C. Appropriate culture medium (i.e., medium) is tempered to 37 °C. Agar (3%) is diluted 1 :5 in medium (=0.6%) and plated into 96 well plates (Coming, #3904), then incubated at room temperature for agar solidification. A 3% agar is diluted to 0.25% in medium (1 : 12 dilution) and tempered at 42 °C. Cells are trypsinized, counted, and tempered at 37 °C. The cells (e.g., MIA PaCa-2 at about 125-150 cells, NCI-H1792 at about 1000 cells) are resuspended in 100 mL 0.25% Agar and plated, followed by incubation at room temperature for agar solidification. The wells are overlaid with 50 mL of the medium. Sister wells in a separate plate are plated for time zero determination. All plates are incubated overnight at 37 °C and 5% CO2.
[373] On a second day (e.g., Day 2), time zero values are measured. A 40 mL volume of Cell Titer 96 Aqueous Solution (Promega) is added to each well and incubated in the dark at 37 °C and 5% CO2. Absorption can be measured at 490 nm and reference wavelength 660 nm. DMSO-prediluted test compounds are added to wells of interest, e.g., with HP Dispenser, to one or more desired concentrations (e.g., a final DMSO concentration of 0.3%).
[374] On a tenth day (e.g., Day 10), absorption by wells treated with the test compounds and control wells are measured with, for example, Cell Titer 96 AQueous and analyzed in comparison to the time zero measurements. The IC50 values are determined using the four parameter fit. The resulting IC50 value is a measurement of the ability of the test compound to reduce cell growth of Ras-driven cells (e.g., tumor cell lines) in vitro and/or in vivo. In some embodiments, one or more compounds disclosed herein exhibits an IC50 value less than 1 μM, 100 nM, 10 nM, 1 nM, or even less, against one or more KRAS G12C cell line (such as MIA PaCa-2 or NCI-H1792). b. Growth of cells with K-Ras G12D mutation
[375] ASPC-1 (ATCC CRL-1682), Panc-10.05 (ATCC CRL-2547), A427, and GP2d cell lines, or any other cell lines comprising a G12D mutation, can be used to assess Ras cellular signaling in vitro, e.g., in response to a compound described herein. For example, ASPC-1 culture medium is prepared with RPMI-1640 and 10% heat- inactivated FBS. Panc-10.05 culture medium is prepared with RPMI-1640, 10 units/mL human recombinant insulin, and 10% FBS. A427 cell culture is prepared with RPMI-1640 and 10% heat-inactivated FBS. A CellTiter-Glo (CTG) luminescent based assay (Promega) is used to assess growth of the cells, as a measurement of the ability of the compounds herein to inhibit Ras signaling in the cells. The cells (e.g., 800 per well) are seeded in their respective culture medium in standard tissue culture -treated 384-well format plates (Falcon #08-772-116) or ultralow attachment surface 384-well format plates (S-Bio # MS-9384UZ). The day after plating, cells are treated with a dilution series (e.g., a 9 point, 3-fold dilution series) of the compounds herein (e.g., approximately 40 μL final volume per well). Cell viability can be monitored (e.g., approximately 5 days later) according to the manufacturer’s recommended instructions, where CellTiter-Glo reagent is added (e.g., approximately 10 μL), vigorously mixed, covered, and placed on a plate shaker (e.g., approximately for 20 min) to ensure sufficient cell lysis prior to assessment of luminescent signal. The IC50 values are determined using the four-parameter fit. The resulting IC50 value is a measurement of the ability of the test compound to reduce cell growth of Ras-driven cells (e.g., tumor cell lines) in vitro and/or in vivo. In some embodiments, one or more compounds disclosed herein exhibits an IC50 value less than 1 μM, 100 nM, 10 nM, 1 nM, or even less, against one or more KRAS G12D cell line (such as AsPC-1, Panc-10.05, 1427, or GP2d). c. Growth of cells with K-Ras G12S mutation
[376] A549 (ATCC CRL-185) and LS123 (ATCC CRL-255) cell lines comprise a G12S mutation and can be used to assess Ras cellular signaling in vitro, e.g., in response to treatment with a compound described herein. A549 culture medium is prepared with RPMI-1640 and 10% heat-inactivated FBS. LS123 culture medium is prepared with RPMI-1640 and 10% heat-inactivated FBS. A CellTiter-Glo (CTG) luminescent based assay (Promega) is used to assess growth of the cells, as a measurement of the ability of the compounds herein to inhibit Ras signaling in the cells. The cells (e.g., 800 per well) are seeded in their respective culture medium in standard tissue culture -treated 384-well format plates (Falcon #08-772-116) or ultra-low attachment surface 384-well format plates (S-Bio # MS- 9384WZ). The day after plating, cells are treated with a dilution series (e.g., a 10 point, 3-fold dilution series) of the compounds herein (e.g., approximately 40 μL final volume per well). Cell viability can be monitored (e.g., approximately 6 days later) according to the manufacturer’s recommended instructions, where CellTiter-Glo reagent is added (e.g., approximately 10 μL), vigorously mixed, covered, and placed on a plate shaker (e.g., approximately for 20 min) to ensure sufficient cell lysis prior to assessment of luminescent signal. The IC50 values are determined using the four parameter fit. The resulting IC50 value is a measurement of the ability of the test compound to reduce cell growth of Ras-driven cells (e.g., tumor cell lines) in vitro and/or in vivo. In some embodiments, one or more compounds disclosed herein exhibits an IC50 value less than 1 μM, 100 nM, 10 nM, 1 nM, or even less, against one or more KRAS G12S cell line (such as A549 or LS123).
[377] Example 6: In vivo Ras inhibition
[378] The in vivo reduction in Ras signaling output by a compound of the present disclosure is determined in a mouse tumor xenograft model, particularly by using a mutant K-Ras model including without limitation a K-Ras G12S model, a K-Ras G12C model, a K-Ras G12D model, a K-Ras G13D model, and a K-Ras G13C model. These models can be generated by the methods and procedures described below. In particular, the methods disclosed below involving the use of a K-Ras G12S mutant cell line for generating a K-Ras G12S xenograft model can be applied to other K-Ras mutant animal models using the respective K-Ras mutant cell lines described above.
Xenograft with K-Ras G12D, G12C, or G12S mutation [379] Tumor xenografts are established by administration of tumor cells with a K-Ras G12D mutation (e.g., ASPC-1 cells), a K-Ras G12C mutation (e.g., MIA PaCa-2 cells), or a K-Ras G12S mutation (e.g., A549 or LS123 cells) into mice. Female 6- to 8-week-old athymic BALB/c nude (NCr) nu/nu mice are used for xenografts. The tumor cells (e.g., approximately 5x106) are harvested on the day of use and injected in growth-factor-reduced Matrigel/PBS (e.g., 50% final concentration in 100 μL). One flank is inoculated subcutaneously per mouse. Mice are monitored daily, weighed twice weekly, and caliper measurements begin when tumors become visible. For efficacy studies, animals are randomly assigned to treatment groups by an algorithm that assigns animals to groups to achieve best case distributions of mean tumor size with lowest possible standard deviation. Tumor volume can be calculated by measuring two perpendicular diameters using the following formula: (L x w2) / 2, in which L and w refer to the length and width of the tumor, respectively. Percent tumor volume change can be calculated using the following formula: (Vfinal -Vinitial)/Vinitial x 100. Percent of tumor growth inhibition (%TGI) can be calculated using the following formula: %TGI = 100 x (1 - (average Vfinal -Vinitial of treatment group) / (average Vfinal -Vinitial of control group). When tumors reach a threshold average size (e.g., approximately 200-400 mm3), mice are randomized into 3-10 mice per group and are treated with vehicle (e.g., 100% Labrasol®) or a compound disclosed herein, using, for example, a daily schedule by oral gavage. Results can be expressed as mean and standard deviation of the mean.
[380] Example 7: Conjugate Binding Assay
[381] The ability of a conjugate disclosed herein to specifically bind to an antigen of interest can be determined by any immune-assays, including ELISA, FACS, confocal imaging, immunoprecipitation, real-time cell-binding assays or Westemblots. In particular, a cell expressing the antigen of interest to which the antigen binding unit specifically binds is brought to contact with the conjugate. Any specific labeling of the cell using the conjugate can be determined using FACS or ELISA. Upon internalization of the conjugate, the presence of the conjugate comprising the antigen binding unit can be detected in the cell lysate via Westemblots utilizing a secondary antibody recognizing the antigen binding unit.
[382] Example 8: Characterization of a Subject Conjugate’s Biological Activities
[383] The ability of a conjugate disclosed herein to inhibit signaling output of (i) an antigen to which the antigen binding unit specifically binds and (ii) KRAS, can be demonstrated by a variety of assays disclosed herein. In particular, all of the procedures detailed in Examples 5, 6, 11, 12, 13, and 16 can be performed to detect the KRAS inhibitory activity of the conjugate in vitro or in vivo. Depending on the choice of the antigen binding unit and the corresponding target antigen, a reduction in signaling output of such antigen can be ascertained by the downstream readout associated with this target antigen. For example, if the antigen binding unit specifically binds and inhibits EGFR (e.g., Cetuximab directed to EGFR), then the readout of its inhibitory activity of the conjugate can be inhibition of cancer cell growth (e.g., using MIA PaCa-2 cell) or the reduction of tumor burden in TGI model generated using MIA PaCa-2 cells.
[384] Example 9: Cell Internalization Assay
[385] Internalization of unconjugated cetuximab, a cetuximab conjugate of the present disclosure (Cet-KRAS A), a second cetuximab conjugate of the present disclosure (Cet-KRAS B), cetuximab-MMAE ADC, and corresponding isotype IgGl ADC controls (Isotype-KRAS A and Isotype -KRAS B) was determined by fluorescence detection in the high EGFR-expressing epidermoid carcinoma cell line A-431. A-431 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Cetuximab was purchased from MedChemXpress (Cat. no. HY-P9905, Monmouth Junction, NJ, USA), and cetuximab-MMAE was purchased from Sellick Chem (Cat. no. D4022, Houston, TX, USA). Cetuximab, Cet-KRAS A, Cet-KRAS B, cetuximab -MM AE ADC, Isotype-KRAS A, and Isotype-KRAS B were conjugated with amine -reactive pH-sensitive pHAB dye (Promega, Madison, WI) according to the manufacturer's instructions. The pHAB dye has very low fluorescence at pH > 7 and a dramatic increase in fluorescence as the pH of the solution becomes acidic. Hence, pHAB dye-conjugated antibodies and ADCs become fluorescent upon internalization in cells, as the intracellular pH is < 7.
[386] Briefly, cells were seeded at 50,000 cells/well in RPMI 1640 media (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum in a Coming 3603 clear-bottom black 96 well plate. Dye- conjugated antibodies and ADCs were dosed in the range of 0. 1 nM to 30 nM and incubated for 20 h under standard culture conditions (37 °C/5% CO2). After incubation, the cell media was removed and the cells were washed with PBS three times. PBS buffer (100 μL ) was added to each well. Fluorescence detection (excitation: 532 nm/emission: 560 nm) was performed using a BioTek Synergy Neo2 Hybrid Multimode Reader (Agilent, Santa Clara, CA, USA). Results were plotted using GraphPad Prism Version 10 (GraphPad Software, San Diego, CA, USA). As shown in FIG. 2, conjugates of the present disclosure (Cet-KRAS A and Cet-KRAS B) showed higher levels of internalization than unconjugated cetuximab at all tested concentrations. Cetuximab-MMAE showed lower levels of internalization than unconjugated cetuximab. The isotype IgGl ADCs showed minimal internalization.
[387] Example 10: Cell Payload Release Assay
[388] The internalization and release of the KRAS inhibitor from conjugates disclosed herein were measured using LC-MS/MS detection in the high EGFR-expressing epidermoid carcinoma cell line A-431. Briefly, cells were seeded at one million cells per well in 1.8 mL of RPMI 1640 medium (Thermo Fisher Scientific, W altham, MA, USA) supplemented with 10% fetal bovine serum in a Coming 3516 clear-bottom 6 well plate. Conjugates of the present disclosure were diluted in the RPMI 1640 medium to a concentration of 100 pg/mL. Each conjugate (200 μL) was added to separate wells in duplicate. The cells were incubated with the conjugates for 2 or 20 h under standard culture conditions (37 °C, 5% CO2).
[389] For cell culture media analysis, 100 μL of growth media was collected at both time points, 2 h and 20 h. Samples were aliquoted to 20 μL, then diluted with 20 μL papain activation buffer (1.1 mM EDTA, 0.067 mM mercaptoethanol and 5.5 mM cysteine-HCl) or 20 μL of 1 mg/mL papain in papain activation buffer. Papain obtained from papaya latex was purchased from Sigma-Aldrich (Cat. No. P3125; Atalanta, GA, USA) and was used to completely release the KRAS inhibitor from the conjugates. The samples were incubated for 1 h at 37 °C, then quenched with acetonitrile and the supernatant analyzed for KRAS inhibitor using LC-MS/MS.
[390] For cell analysis, the growth medium was removed and the cells were washed twice with PBS. Trypsin- EDTA solution (200 μL, Thermo Fisher Scientific, Waltham, MA, USA) was added and the cells incubated for 5 min at 37 °C. Cells were collected in clean Eppendorf tubes and washed twice with PBS. The cell volume was estimated to 10 μL. The cells were then lysed by resuspending them in 90 μL of IP lysis buffer. The cell suspension was sonicated (30% amplitude for 10 s) to ensure complete cell lysis. Samples were aliquoted to 20 μL and diluted with 20 μL papain activation buffer (1.1 mM EDTA, 0.067 mM mercaptoethanol and 5.5 mM cysteine-HCl) or 20 μL of 1 mg/mL papain in papain activation buffer. Samples were incubated for 1 h at 37 °C. The samples were quenched with acetonitrile and supernatant was analyzed for KRAS inhibitor using LC-MS/MS. All samples were analyzed using LC-MS/MS (MRM) on a QTRAP 6500 LC-MS/MS System (AB Sciex, Redwood City, CA, USA). A calibration curve was used to calculate KRAS inhibitor concentration in the samples. Conjugates of the present disclosure (e.g., Cet-KRAS A and Cet-KRAS B) demonstrated stability in growth media and exhibited timedependent internalization into A-431 cells. Example 11: Cell Proliferation Assay
[391] The ability of a conjugate of the present disclosure to inhibit the growth of cancer cells, particularly those with KRAS and EGFR mutations or amplifications, is evaluated using a 3D proliferation assay. A-431 cells are obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS. A CellTiter-Glo (CTG) luminescence-based assay is used to assess the growth of cells as a measure of the ability of the conjugates to inhibit cancer cell growth. The cells (e.g., 800-1 ,200 per well) are seeded in culture medium in standard tissue culture-treated 384-well format plates (Coming Costar #3765). The day after plating, the cells are treated with a dilution series (e.g., a 9 point 3-fold dilution series) of each of unconjugated antibodies, conjugates of the present disclosure (e.g., Cet-KRAS A and Cet- KRAS B), and reference compounds (e.g., approximately 40 id, final volume per well). Cell viability is monitored (approximately 3 days later) according to the manufacturer’s instructions, where the CellTiter-Glo reagent (Promega, Madison, WI) is added (approximately 20 μL), vigorously mixed, covered, and placed on a plate shaker (for approximately 30 min) to ensure sufficient cell lysis prior to assessment of the luminescent signal.
Luminescence is detected using a BioTek Synergy Neo2 Hybrid Multimode Reader (Agilent, Santa Clara, CA, USA). Results are plotted using GraphPad Prism Version 10 (GraphPad Software, San Diego, CA, USA). IC50 values are determined using a four-parameter fit. The resulting IC50 value is a measure of the ability of compounds to reduce the growth of EGFR-amplified cells (e.g., tumor cell lines) in vitro and/or in vivo.
[392] The inhibitory effect of cetuximab linked to various payload linkers on 3D cell proliferation can be evaluated using a range of cell lines with KRAS, NRAS, and HRAS mutations. The cell panel can include HPAC (KRAS-G12D, pancreatic adenocarcinoma), H1373 (KRAS-G12C, lung adenocarcinoma), H2009 (KRAS-G12A, lung adenocarcinoma), MIA-PaCa2 (KRAS-G12C, pancreatic adenocarcinoma), THP-1 (NRAS-G12D, acute myeloid leukemia), T24 (HRAS-G12V, colorectal adenocarcinoma), and SK -MEL-30 (NRAS-Q61K, melanoma). Cells are cultured in Ultra-Low Attachment 384-well plates under standard culture conditions. The cell lines are exposed to varying concentrations of antibody drug conjugates prepared in growth medium. After a 3 -day or 5-day incubation period, CellTiter-Glo® 3D reagent (Promega, Madison, WI) is introduced to each well. The plates are then incubated in the dark at room temperature for 60 minutes before measuring luminescence with a BioTek Synergy Neo2 Hybrid Multimode Reader (Agilent Technologies Santa Clara, CA). To determine the IC50, cellular proliferation inhibition is calculated as a percentage and plotted against antibody drug conjugate concentration using GraphPad Prism software (GraphPad Software, San Diego, CA).
[393] Example 12: Tumor Growth Inhibition Model
[394] The tumor growth inhibition of conjugates of the present disclosure is determined in a mouse tumor xenograft model, such as a K-Ras G12C model or an EGFR amplification model. Tumor xenografts are established by administration of tumor cells with a K-Ras G12C mutation (e.g., H1373 cells) into mice. H1373 is a lung adenocarcinoma cell line obtained from American Type Culture Collection (Manassas, VA, USA). Female 6- to 8- week-old athymic BALB/c nude (NCr) nu/nu mice are used for xenografts. Tumor cells (e.g., approximately 5 x
106) are harvested on the day of use and injected in growth-factor-reduced Matrigel/PBS (e.g., 50% final concentration in 100 JJ-L). One flank is inoculated subcutaneously per mouse. Mice are monitored daily, weighed twice weekly, and caliper measurements began when tumors become visible. For efficacy studies, animals are randomly assigned to treatment groups by an algorithm that assigns animals to groups to achieve best case distributions of mean tumor size with lowest possible standard deviation. Tumor volume is calculated by measuring two perpendicular diameters using the following formula: (L x w2)/2, in which L and w refer to the length and width of the tumor, respectively. Percent tumor volume change is calculated using the following formula: (Vfinal - Vinitial)/Vinitial x 100. Percent of tumor growth inhibition (% TGI) is calculated using the following formula: % TGI = 100 x (l-(average Vfinal - Vinitial of treatment group)/(average Vfinal - Vinitial of control group). When tumors reached a threshold average size (e.g., approximately 200-400 mm3), mice are randomized into 3-10 mice per group and are treated with either vehicle (e.g., 20 mM histidine, 8% sucrose, pH 6.0), unconjugated antigen binding units (e.g., cetuximab or sacituzumab), or a conjugate disclosed herein (e.g., Cet-KRAS A, Cet-KRAS B, Sac-KRAS A, or Sac- KRAS B), using, for example, a weekly schedule by intravenous (IV) infusion. Results can be expressed as mean and standard deviation of the mean..
[395] Example 13: Therapeutic Index of Antibody-drug Conjugates Relative to Kras Inhibitor in Murine Tolerability Study
[396] The tolerability of antibody -drug conjugates (ADCs) consisting of cetuximab linked to various pay load linkers is evaluated in mice using single doses of 30, 100, and 200 mg/kg. Female CD-I mice, aged 6-8 weeks and weighing approximately 20-30 g, received intravenous injections of ADCs at these doses. Six mice from each dose group are monitored for two weeks after administration. The KRAS inhibitor payload alone is also tested using the same protocol, with six mice per dose group observed for two weeks post-injection. Mice are euthanized if their body weight dropped more than 20% below pre -dose levels. Blood samples are scheduled for collection from all mice at 24 hours and 7 days after dosing for pharmacokinetic analysis. The investigation determined the dose of ADC or payload required to induce toxicity in 50% of the mice (TD50). Tumor growth inhibition (TGI) studies are conducted using the Hl 373 KRAS-mutant model to establish the dose needed to produce a therapeutic effect in 50% of the mice (ED50) for both ADCs and payload separately. The therapeutic index (TI) is calculated as the ratio of TD50 to ED50. TI for one or more subject conjugate is expected to exceed that of the KRAS inhibitor payload alone (TIconjugate/TIpayload >1).
[397] Example 14: Plasma Antibody-Drug Conjugates Concentration Relative to that of Unconjugated Kras Inhibitor
[398] An assessment of the KRAS inhibitor pay load's pharmacokinetics is conducted in CD1 mice using the following protocol. The KRAS inhibitor unbound to an antigen binding unit is introduced intravenously at a dosage of 2 mg/kg. Blood samples are obtained from three mice at 10 min, 30 min, 1, 2, 4, 8, and 24 h after administration. The blood is converted to plasma and stored at -80°C until pharmacokinetic analysis. In a separate study, a subject conjugate comprising an antigen binding unit (e.g., cetuximab) conjugated to a Kras inhibitor in Table 1 via a linker in T able 2 can be examined once administered intravenously in CD 1 mice over various time points from 20 min till 168 hours. The plasma is prepared and frozen at -80°C for LC-MS analysis. The KRAS inhibitor concentration in plasma can be determined using LC-MS analysis with a standard calibration curve, employing a QTRAP 6500+ mass spectrometer (AB Sciex, Framingham, MA, USA). The plasma sample from mice administered with the subject conjugate does not yield any detectable amount of the Kras inhibitor (falling below the lower limit of quantification (0.02 ng/mL) at all measured time points), whereas Kras inhibitor unbound to an antigen binding unit exhibits a curve showing a rise of the concentration in plasma followed by a decline over the time course investigated.
[399] Example 15: Concentration of Antibody-Drug Conjugates in Tumor and in Plasma
[400] The pharmacokinetics and tumor tissue distribution of the KRAS inhibitor pay load can be evaluated in an Hl 373 lung adenocarcinoma CDX mouse model using the following protocol. A subject conjugate comprising an antigen binding unit is administered intravenously to six mice at a dose of ~40 mg/kg when the tumors reached an average size of approximately 250 mm3. Samples of plasma and tumor tissue are obtained from the six mice, with three specimens collected at the 4-hour mark and another three at the 24-hour point. Plasma and tumor tissues are frozen at -80°C for LC-MS analysis. LC-MS analysis with a standard calibration curve, employing a QTRAP 6500+ mass spectrometer (AB Sciex, Framingham, MA, USA) is utilized to assess the concentration of KRAS inhibitor in the plasma or in the tumor tissue.
[401] Example 16: In Vivo Efficacy Assessment In H1373 Lung Adenocarcinoma Mouse Model
[402] The anti-tumor efficacy of a subject conjugate (e.g., cetuximab-Kras conjugate with various DAR) can be evaluated in vivo using Hl 373 cell-line derived xenograft (CDX) models, as outlined below. H1373 tumor cells (ATCC, Manassas, VA, USA) are maintained in an incubator at 37C with 5% CO2. The cells are passaged three times prior to implantation. To enhance tumor growth in immunodeficient athymic nude mice, cells are harvested when they reach 70-80% confluence in the culture medium. At the time of harvesting, the cell viability exceeds 90%, and implantation is performed within one hour of cell collection. Approximately 100 μL of the tumor cell suspension is injected with a tuberculin syringe and a 25-gauge 0.5-inch needle into the mouse subcutaneous space on the flank in the right axillary region. The tumor growth is monitored daily. Tumor volume is measured using calipers, and animal body weight is measured once or twice weekly based on the tumor growth rate. In one study, cetuximab-Kras conjugate is administered intravenously (with a single dose) when the tumor size reached ~240 mm .
[403] It shall be understood that different aspects of the disclosure can be appreciated individually, collectively, or in combination with each other. Various aspects described herein may be applied to any of the particular applications disclosed herein. The compositions of matter, including conjugates and KRAS inhibitors of any formulae disclosed in the conjugate or KRAS inhibitor sections, of the present disclosure may be utilized in the method section, including methods of use and production disclosed herein, or vice versa.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker, and wherein the antigen binding unit and the KRAS inhibitor in the conjugate synergistically inhibits signaling output of the first antigen and KRAS.
2. A conjugate comprising an antigen binding unit exhibiting binding specificity for at least a first antigen that is not KRAS, wherein the antigen binding unit is covalently attached to a small-molecule KRAS inhibitor, optionally through a chemical linker, and wherein the KRAS inhibitor selectively inhibits KRAS or a mutant thereof relative to HRAS and NRAS.
3. The conjugate of claim 1 or 2, wherein the KRAS inhibitor is characterized by a PAMPA permeability (Pe) less than 1 x 10-6 cm/s.
4. The conjugate of any one of the preceding claims, wherein the conjugate is characterized by an enhanced therapeutic efficacy as ascertained by the formula: wherein TIconjugate = TD50c/ED50c, wherein TD50c is the dose of conjugate required to produce a toxic effect in 50% of test subjects and ED50c is the dose of conjugate required to produce a therapeutic effect in 50% of test subjects; and wherein TIKRASI = TD50k/ED50k, wherein TD50k is the dose of KRAS inhibitor required to produce a toxic effect in 50% of test subjects and ED50k is the dose of KRAS inhibitor required to produce a therapeutic effect in 50% of the test subjects.
5. The conjugate of any one of the preceding claims, wherein the conjugate is characterized by reduced plasma concentration of the KRAS inhibitor as ascertained by the formula:
[KRASi]p-c/[KRASi]p-k < 1 wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at a first time -point following administration of the conjugate; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point.
6. The conjugate of any one of the preceding claims, wherein the conjugate is characterized by an increased concentration of the KRAS inhibitor in tumor tissue relative to plasma as ascertained by the formula:
([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) > 1 wherein [KRASi]t-c is concentration of the KRAS inhibitor in tumor tissue at a first time -point following administration of the conjugate; wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at the same time -point following the administration of the conjugate; wherein [KRASi]t-k is concentration of the KRAS inhibitor in tumor tissue following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor at the same time-point following the administration of the KRAS inhibitor alone at the equivalent dose.
7. A conjugate of Formula (A): wherein:
AgB is an antigen binding unit;
L is a chemical linker;
D is independently selected at each occurrence from a small-molecule KRAS inhibitor, a cytotoxic smallmolecule and a small-molecule agent that selectively modulates a non-KRAS target, wherein at least one D is a KRAS inhibitor; p is selected from 1 to 20; and q is selected from 1 to 20.
8. The conjugate of any one of the preceding claims, wherein the antigen binding unit is an antibody or an antigen-binding fragment thereof.
9. The conjugate of any one of the preceding claims, wherein the antigen binding unit is selected from a monoclonal antibody, a Fab, a Fab’, an F(ab’), an Fv, a disulfide linked Fc, an scFv, a single domain antibody, a diabody, a bi-specific antibody, and a multi-specific antibody.
10. The conjugate of any one of the preceding claims, wherein the antigen binding unit is a monoclonal antibody.
11. The conjugate of any one of the preceding claims, wherein the antigen binding unit specifically binds a target selected from AG7, B7-H3, BCMA, CA15-3, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD70, CD71, CD79B, CEA, CLDN18.2, EGFR, FOLR1, GCC, GPC1, HER2, HER3, ICAM1, LeX, LeY, MET, MSLN, MUCl, NECTIN4, SLC44A4, TF, and Trop-2.
12. The conjugate of any one of the preceding claims, wherein the antigen binding unit is selected from cetuximab, bevacizumab, paitumumab, ofatumumab, inotuzumab, gemtuzumab, alemtuzumab, and trastuzumab.
13. The conjugate of any one of claims 1 to 11, wherein the antigen binding unit is an anti -EGFR antibody.
14. The conjugate of claim 13, wherein the anti-EGFR antibody is cetuximab.
15. The conjugate of any one of the preceding claims, wherein the linker comprises one or more components independently selected from alkyl, alkene, alkyne, aryl, cycloalkyl, heterocycle, glycoside, silyl ether, hydroxy, ether, ketone, ester, carbonate, amide, urea, carbamate, sulfide, disulfide, sulfate, sulfonamide, phosphate, hydrazone, and succinimide.
16. The conjugate of any one of the preceding claims, wherein the linker comprises one or more components independently selected from alkyl, polyethylene glycol, a hydrazone, maleimide, succinimide, asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, valine, alanine, glycine, leucine, isoleucine, methionine, tryptophan, proline, histidine, citrulline, phenylalanine, carboxylate, and p- aminobenzyloxy carbonyl.
17. The conjugate of any one of the preceding claims, wherein the linker comprises one or more components selected from Val-Cit, Glu-Val-Cit, Val-Ala, Val-Val, Val-Gly, Gly-Gly, Gly-Cit, Glu-Gly-Cit, Ala-Ala-Asn, Ala- Gly-Ala, Ala-Pro, Ala-Ser, and Phe-Lys.
18. The conjugate of any one of claims 7 to 17, wherein p is selected from 2 to 8.
19. The conjugate of any one of claims 7 to 18, wherein q is selected from 1 to 4.
20. The conjugate of any one of claims 7 to 19, wherein D is a small-molecule KRAS inhibitor.
21. The conjugate of any one of the preceding claims, wherein the KRAS inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt or solvate thereof, wherein:
X is selected from N and C(R6);
Z is selected from O, N, C(R5)2, C(O), S, S(O), and S(O)2;
R2, R5, R6, and R8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alky nyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, - N(R12)(R13), -C(O)OR12, -OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, - C(O)R12, -S(O)R12, -OC(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), -S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered hetero alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R5 are taken together with the atom to which they are attached to form C3-8 carbocycle or 3- to 8-membered heterocycle, each of which is optionally substituted with one or more R20; and further optionally wherein two R5 are taken together to form =O, =NR12, or =C(R14)2;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR12, -SR12, -N(R12)(R13), -C(O)OR12, - OC(O)N(R12)(R13), -N(R12)C(O)N(R12)(R13), -N(R12)C(O)OR12, -N(R12)S(O)2R12, -C(O)R12, -S(O)R12, -0C(O)R12, - C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -N(R12)C(O)R12, -S(O)2R12, -S(O)(NR12)R12, -S(O)2N(R12)(R13), - S(O)(NR12)N(R12)(R13), and -OCH2C(O)OR12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20; optionally wherein two R3 are taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one or more R20; optionally wherein two R3 are taken together to form =O, =NR12, or =C(R14)2; and further optionally wherein one R3 and R4 are taken together with the atoms to which they are attached to form 3- to 10-membered heterocycle optionally substituted with one or more R20;
R4 is selected from hydrogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalkyl)-(3- to 12-membered heterocycle), -C(O)OR12, -C(O)R12, -C(O)N(R12)(R13), -C(O)C(O)N(R12)(R13), -S(O)2R12, - S(O)(NR12)R12, -S(O)2N(R12)(R13), and -S(=O)(=NR12)N(R12)(R13), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), and - (2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle) are optionally substituted with one or more R20;
R7 is selected from C6-12 aryl and 5- to 12-membered heteroaryl, each of which is optionally substituted with one or more R20; m is 0, 1, 2, or 3; n is 1 or 2;
R12 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle) are optionally substituted with one, two, or three R20;
R13 is independently selected at each occurrence from hydrogen, C1-6 alkyl, and C1-6 haloalky I: or R12 and R13 attached to the same nitrogen atom form 3- to 10-membered heterocycle optionally substituted with one, two, or three R20;
R14 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R14 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one, two, or three R20;
R20 is independently selected at each occurrence from halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23)-, -S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22; wherein two R20 attached to the same or adjacent atoms optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalky l)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R21 is independently selected at each occurrence from hydrogen, halogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), or two R21 are taken together with the carbon atom to which they are attached to form C3-12 carbocycle or 3- to 12-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and -OH;
R22 is independently selected at each occurrence from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle); and
R23 is independently selected at each occurrence from hydrogen and C1-6 alkyl; or R22 and R23 attached to the same nitrogen atom form 3- to 10 membered heterocycle; wherein one hydrogen of the compound of Formula (I) is replaced with a bond to the antigen binding unit or the chemical linker.
22. The conjugate of claim 21, wherein the KRAS inhibitor is a compound of Formula (I-a): or a pharmaceutically acceptable salt or solvate thereof, wherein:
A is 6-membered heteroaryl comprising one, two, or three ring nitrogen atoms;
R9 and R10 are independently selected from hydrogen, halogen, oxo, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered heteroalkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23)-, -S(O)(NR22)N(R22)(R23), and -OCH2C(O)OR22; wherein R9 and R10 optionally join to form C3-12 carbocycle or 3- to 12-membered heterocycle; wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 2- to 6-membered heteroalkyl, 3- to 6-membered hetero alkenyl, 3- to 6-membered heteroalkynyl, -C0-6 alkyl-(C3-12 carbocycle), -(2- to 6-membered heteroalkyl)-(C3-12 carbocycle), -C0-6 alkyl-(3- to 12-membered heterocycle), -(2- to 6-membered heteroalky l)-(3- to 12-membered heterocycle), C3-12 carbocycle, and 3- to 12-membered heterocycle are optionally substituted with one or more substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, - OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, - C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, -S(O)(NR22)R22, - S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23); and
R11 is selected from hydrogen, C1-6 alkyl, C1-6 haloalkyl, -C0-6 alkyl-(C3-12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle).
23. The conjugate of claim 22, wherein A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl.
24. The conjugate of claim 22 or 23, wherein A is pyridinyl.
25. The conjugate of any one of claims 22 to 24, wherein R11 is hydrogen.
26. The conjugate of any one of claims 22 to 25, wherein R10 is selected from hydrogen and halogen; or R9 and
R10, together with the atoms to which they are attached, form C4-8 carbocycle or 4- to 8-membered heterocycle, each of which is optionally substituted.
27. The conjugate of any one of claims 22 to 26, wherein R10 is hydrogen.
28. The conjugate of any one of claims 22 to 27, wherein
29. The conjugate of any one of claims 22 to 28, wherein R9 is optionally substituted C1-3 alkyl.
30. The conjugate of any one of claims 22 to 29, wherein R9 is CH3.
31. The conjugate of claim 21, wherein R4 is selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C0-6 alkyl-(C3- 12 carbocycle), and -C0-6 alkyl-(3- to 12-membered heterocycle), each of which is optionally substituted with one or more R20.
32. The conjugate of claim 31, wherein R4 is selected from C1-6 alkyl and -C0-6 alkyl-(3- to 12-membered heterocycle), each of which is optionally substituted with one or more substituents independently selected from halogen, -CH3, -NH2, -NHCH3, and -N(CH3)2.
33. The conjugate of any one of claims 21 to 32, wherein X is C(R6).
34. The conjugate of any one of claims 21 to 32, wherein X is N.
35. The conjugate of any one of claims 21 to 34, wherein Z is O.
36. The conjugate of any one of claims 21 to 35, wherein R7 is selected from naphthyl, isoquinolinyl, indazolyl, benzothiazolyl, benzothiophenyl, phenyl, and pyridinyl, each of which is optionally substituted with one, two, three, or four R20.
37. The conjugate of any one of claims 21 to 36, wherein R7 is benzothiophenyl optionally substituted with one, two, three, or four R20.
38. The conjugate of any one of claims 21 to 37, wherein R7 is substituted with one, two, three, or four substituents independently selected from halogen, -CN, C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, C2-3 alkynyl, -OR22, - N(R22)(R23), and C3-6 cycloalkyl.
39. The conjugate of any one of claims 21 to 38, wherein R7 is substituted with one, two, three, or four substituents independently selected from halogen, -CN, -CH3, -CH2CH3, -CH=CH2, -CF3, -C=CH, -OH, -NH2, and - cyclopropyl.
40. The conjugate of any one of claims 21 to 39, wherein R7 is selected from
41. The conjugate of any one of claims 21 to 40, wherein R7 is
42. The conjugate of claim 22, wherein:
X is C(R6);
A is selected from pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl;
R2, R6, and R8 are each independently selected at each occurrence from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), -C(O)OR12, - N(R12)C(O)N(R12)(R13), -C(O)R12, -OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20;
R3 is independently selected at each occurrence from halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, - C3-8 carbocycle, 3- to 8-membered heterocycle, -OR12, -N(R12)(R13), -C(O)OR12, -N(R12)C(O)N(R12)(R13), -C(O)R12, -OC(O)R12, -C(O)N(R12)(R13), and -N(R12)C(O)R12, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8-membered heterocycle are optionally substituted with one, two, or three R20; wherein two R3 are optionally taken together with the atom or atoms to which they are attached to form C3-8 carbocycle or 3- to 8- membered heterocycle, each of which is optionally substituted with one, two, or three R20; and further wherein two R3 are optionally taken together to form =O, =NR12, or =C(R14)2;
R7 is benzo [b]thiophen-4-yl optionally substituted with one, two, three, or four R20;
R9 is C1-3 alkyl optionally substituted with one, two, or three R20;
R10 is selected from hydrogen, halogen, -CN, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, 3- to 8- membered heterocycle, -OR22, -N(R22)(R23), -C(O)OR22, -N(R22)C(O)N(R22)(R23), -C(O)R22, -OC(O)R22, - C(O)N(R22)(R23), and -N(R22)C(O)R22, wherein C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C3-8 carbocycle, and 3- to 8- membered heterocycle are optionally substituted with one, two, or three substituents independently selected from halogen, oxo, -CN, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, -OR22, -SR22, -N(R22)(R23), =NR22, =C(R21)2, -C(O)OR22, -OC(O)N(R22)(R23), -N(R22)C(O)N(R22)(R23), -N(R22)C(O)OR22, -N(R22)S(O)2R22, -C(O)R22, -S(O)R22, -OC(O)R22, -C(O)N(R22)(R23), -C(O)C(O)N(R22)(R23), -N(R22)C(O)R22, -OS(O)2R22, -S(O)2R22, - S(O)(NR22)R22, -S(O)2N(R22)(R23), and -S(O)(NR22)N(R22)(R23);
R11 is hydrogen; m is 0 or 1 ; and n is 1 or 2.
43. The conjugate of claim 22, wherein the KRAS inhibitor is a compound of Formula (I-b): or a pharmaceutically acceptable salt or solvate thereof.
44. The conjugate of claim 43, wherein the KRAS inhibitor is a compound of Formula (I-c): or a pharmaceutically acceptable salt or solvate thereof.
45. The conjugate of any one of claims 21 to 44, wherein R2 is selected from hydrogen, C1-3 alkyl, -OR12, and 3- to 10-membered heterocycle, wherein C1-3 alkyl and 3- to 10-membered heterocycle are optionally substituted with one, two, or three R20.
46. The conjugate of any one of claims 21 to 45, wherein R2 is -OR12.
47. The conjugate of any one of claims 21 to 46, wherein R2 is -O(C1-3 alkyl)(4- to 10-membered heterocycle) optionally substituted with one, two, or three substituents independently selected from halogen, C1-3 alkyl, C1-3 haloalkyl, and =C(R21)2, wherein R21 is independently selected at each occurrence from hydrogen, halogen, and C1-3 alkyl.
48. The conjugate of any one of claims 21 to 47, wherein R2 is selected from:
49. The conjugate of any one of claims 21 to 48, wherein R2 is
50. The conjugate of any one of claims 21 to 49, wherein R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three R20.
51. The conjugate of any one of claims 21 to 50, wherein R3 is independently selected at each occurrence from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 carbocycle, and 3- to 8-membered heterocycle, each of which is optionally substituted with one, two, or three substituents independently selected from halogen, -CN, -OH, and -OCH3.
52. The conjugate of any one of claims 21 to 51, wherein m is 0 or 1.
53. The conjugate of any one of claims 21 to 52, wherein m is 0.
54. The conjugate of any one of claims 21 to 53, wherein R6 and R8 are independently selected from hydrogen, halogen, and C1-3 haloalkyl.
55. The conjugate of any one of claims 21 to 54, wherein R6 is selected from chlorine and -CF3.
56. The conjugate of any one of claims 21 to 55, wherein R8 is fluorine.
57. The conjugate of any one of claims 21 to 56, wherein n is 1.
58. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 57, or a salt thereof, and a pharmaceutically acceptable excipient.
59. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate of any one of claims 1 to 57, or a salt thereof.
60. A method of treating cancer in a subject comprising a Ras mutant protein, the method comprising: inhibiting the Ras mutant protein of said subject by administering to said subject a conjugate of any one of claims 1 to 57, or a salt thereof.
61. The method of claim 59 or 60, wherein the cancer is a solid tumor or a hematological cancer.
62. The method of any one of claims 59 to 61, wherein the cancer comprises a K-Ras G12C, G12D, G12S, or G12V mutant protein.
63. A method of modulating signaling output of a Ras protein, comprising contacting a Ras protein with an effective amount of a conjugate of any one of claims 1 to 57, or a salt thereof, thereby modulating the signaling output of the Ras protein.
64. A method of inhibiting cell growth, comprising administering an effective amount of a conjugate of any one of claims 1 to 57, or a salt thereof, to a cell expressing a Ras protein, thereby inhibiting growth of said cells.
65. The method of any one of claims 59 to 64, comprising administering an additional agent.
66. A method of delivering a small-molecule KRAS inhibitor that exhibits low permeability as characterized by a PAMPA assay, comprising contacting a tumor cell with a conjugate of any one of claims 1 to 57, or a salt thereof, wherein the KRAS inhibitor exhibits a PAMPA permeability (Pe) value less than 1 x 10-6 cm/s.
67. A method of enhancing therapeutic efficacy of a small -molecule KRAS inhibitor, comprising providing a conjugate of any one of claims 1 to 57 to a subject, wherein enhanced therapeutic efficacy is ascertained by the formula: wherein TIconjugate = TD50c/ED50c, wherein TD50c is the dose of conjugate required to produce a toxic effect in 50% of test subjects and ED50c is the dose of conjugate required to produce a therapeutic effect in 50% of test subjects; and wherein TIKRASI = TD50k/ED50k, wherein TD50k is the dose of KRAS inhibitor required to produce a toxic effect in 50% of test subjects and ED50k is the dose of KRAS inhibitor required to produce a therapeutic effect in 50% of the test subjects.
68. A method of reducing plasma concentration of a small-molecule KRAS inhibitor, comprising providing a conjugate of any one of claims 1 to 57 to a subject, wherein reduced plasma concentration is ascertained by the formula:
[KRASi]p-c/[KRASi]p-k < 1 wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at a first time -point following administration of the conjugate; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point.
69. A method of increasing concentration of a small-molecule KRAS inhibitor in tumor tissue, comprising providing a conjugate of any one of claims 1 to 57 to a subject, wherein increased tumor tissue concentration is ascertained by the formula:
([KRASi]t-c/[KRASi]p-c) / ([KRASi]t-k/[KRASi]p-k) > 1 wherein [KRASi]t-c is concentration of the KRAS inhibitor in tumor tissue at a first time -point following administration of the conjugate; wherein [KRASi]p-c is plasma concentration of the KRAS inhibitor at the same time -point following the administration of the conjugate; wherein [KRASi]t.k is concentration of the KRAS inhibitor in tumor tissue following administration of the KRAS inhibitor alone at an equivalent dose at the same time-point; and wherein [KRASi]p-k is plasma concentration of the KRAS inhibitor at the same time-point following the administration of the KRAS inhibitor alone at the equivalent dose.
70. A method of delivering a small-molecule KRAS inhibitor to the central nervous system of a subject, comprising administering a conjugate of any one of claims 1 to 57 to the subject, wherein the KRAS inhibitor is released from the conjugate after entering the CNS of the subject.
71. A method of generating a slow-release form of a small-molecule KRAS inhibitor, the method comprising conjugating an antigen binding unit to a small-molecule KRAS inhibitor through a chemical linker, wherein the small-molecule inhibitor is released from the conjugate upon introducing the conjugate into a subject or a cell.
72. The method of any one of claims 59 to 71, wherein efficacy of the conjugate is greater than efficacy of a combination of the antigen binding unit and the KRAS inhibitor when each is administered at a comparable concentration.
73. The method of any one of claims 59 to 72, wherein toxicity of the conjugate is less than toxicity of a combination of the antigen binding unit and the KRAS inhibitor when each is administered at a comparable concentration.
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