Protease / enzyme cleavable linker-payloads and protein conjugates

Abstract

The present invention provides compounds, conjugate products thereof, methods and pharmaceutical compositions for treatment and diagnosis.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims and enjoys the benefit of U.S. Provisional Application No. 63 / 389,666, filed on July 15, 2022, the entire contents of which are incorporated herein for all purposes. Field of the Invention

[0002] The present invention relates to linker-payload compounds and macromolecular conjugates thereof; pharmaceutical compositions comprising linker-payload compounds and / or conjugates; methods for preparing linker-payload compounds and / or conjugates; and methods of using the linker-payload compounds, conjugates and compositions for treating, for example, cell proliferative diseases (including but not limited to cancer). Background of the Invention

[0003] Biotherapeutics offer a wealth of therapeutic and diagnostic potential for patients worldwide. However, many drugs based on large molecules, such as proteins, peptides, and antibodies, have limitations in their effective use, including limitations in bioavailability, absorption, distribution, metabolism, and excretion (ADME). Some of these limitations affect drug dosing, half-life, side effects, and toxicity. Strategies to improve the effectiveness of biotherapeutics continue to be needed. Summary of the Invention

[0004] In one aspect, the present invention provides a compound represented by formula (I): or a pharmaceutically acceptable salt and / or regioisomer thereof; in, L 1 Selected from Ring A is an optionally substituted bridged, fused, or spiro bicyclic carbocycle, or an optionally substituted bridged, fused, or spiro bicyclic heterocycle, wherein the carbocycle or the heterocycle of Ring A is optionally substituted by one or more selected from alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents; Ring B is an optionally substituted N-linked bridged, fused, or spiro bicyclic heterocycle, wherein Ring B is optionally substituted by one or more selected from alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents; R a and R b are independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; a is an integer independently selected from 0, 1, 2, 3, 4, 5 and 6; b is an integer selected from 0 and 1; R 1 is H or alkyl, the alkyl being optionally substituted by one or more selected from cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aryl and heteroaryl substituents; R 2 and R 3 are each independently selected from H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; Y is *-C(O)-(CR a R b ) c -NH-, or *-C(O)-(CR a R b ) c -, where * indicates the position where Y is attached to RG; c is an integer selected from 1, 2, 3, 4, 5 and 6; RG is the reactive group; L 2 Does not exist, or L 2 is a linker comprising a hydrophilic polymer residue; L 3 Does not exist, or L 3 It is -C(O)-AA-, -C(O)-AA-Z-(CR a Rb ) a -Z-(CR a R b ) a -C(O)-, -C(O)-Z-(CR a R b ) a -C(O)-ZL 4 -OC(O)-, -Z-AA-, -AA-, -C(O)-, -C(O)-AA-Z-(CR a R b ) a -、-AA-C(O)-、-C(O)-(CR a R b ) a -Z-(CR a R b ) a -Z-AA-C(O)-, -C(O)OL 4 -ZC(O)-(CR a R b ) a -ZC(O)-, -AA-Z-, or -(CR a R b ) a -Z-AA-C(O)-; Z is selected from -NR 2 - and -O-; AA is an amino acid residue or a peptide residue; L 4 yes Where Su is the hexose form of a monosaccharide; d is an integer independently selected from 1, 2 and 3; D is a cytotoxic payload; and represents the point of attachment to the rest of the compound.

[0005] In some embodiments, the compound represented by formula (I) is a compound represented by formula (IA): or a pharmaceutically acceptable salt and / or regioisomer thereof; where the integers c, RG, R a , R b , Ring B, L 2 , L 3 and D are as defined in the present invention.

[0006] In certain embodiments, the compound of formula (I) is a compound of formula (IB): or a pharmaceutically acceptable salt and / or regioisomer thereof; where the integers c, RG, R a , R b , Ring B, L 2 , L 3 and D are as defined in the present invention.

[0007] In certain embodiments, the compound of formula (I) is a compound of formula (IC): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring A, L 2 , L 3 and D are as defined in the present invention.

[0008] In certain embodiments, the compound of formula (I) is a compound of formula (ID): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring A, L 2 , L 3 and D are as defined in the present invention.

[0009] In certain embodiments, the compound of formula (I) is a compound of formula (IE): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring B, L 3 POLY 2 and D are as defined in the present invention.

[0010] In certain embodiments, the compound of formula (I) is a compound of formula (IF): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring B, L 3 POLY 2 and D are as defined in the present invention.

[0011] In certain embodiments, the compound of formula (I) is a compound of formula (IG): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring A, L 3 POLY 2 and D are as defined in the present invention.

[0012] In certain embodiments, the compound of formula (I) is a compound of formula (IH): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring A, L 3 POLY 2 and D are as defined in the present invention.

[0013] In one aspect, the present invention provides a conjugate represented by formula (II): or a pharmaceutically acceptable salt and / or regioisomer thereof; in, L 1 Selected from Ring A is an optionally substituted bridged, fused, or spiro bicyclic carbocycle, or an optionally substituted bridged, fused, or spiro bicyclic heterocycle, wherein the carbocycle or the heterocycle of Ring A is optionally substituted by one or more selected from alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents; Ring B is an optionally substituted N-linked bridged, fused, or spiro bicyclic heterocycle, wherein Ring B is optionally substituted by one or more selected from alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3)2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents; R a and R b are independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; a is an integer independently selected from 0, 1, 2, 3, 4, 5 and 6; b is an integer selected from 0 and 1; R 1 is H or alkyl, the alkyl being optionally substituted by one or more selected from cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aryl and heteroaryl substituents; R 2 and R 3 are each independently selected from H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; Y is *-C(O)-(CR a R b ) c -NH-, or *-C(O)-(CR a R b ) c -, where * indicates the position where Y is attached to RL; c is an integer selected from 1, 2, 3, 4, 5 and 6; RL is the reactive residue; L 2 Does not exist, or L 2 is a linker comprising a hydrophilic polymer residue; L 3 Does not exist, or L 3 It is -C(O)-AA-, -C(O)-AA-Z-(CR a R b ) a -Z-(CR a Rb ) a -C(O)-, -C(O)-Z-(CR a R b ) a -C(O)-ZL 4 -OC(O)-, -Z-AA-, -AA-, -C(O)-, -C(O)-AA-Z-(CR a R b ) a -、-AA-C(O)-、-C(O)-(CR a R b ) a -Z-(CR a R b ) a -Z-AA-C(O)-, -C(O)OL 4 -ZC(O)-(CR a R b ) a -ZC(O)-, -AA-Z-, or -(CR a R b ) a -Z-AA-C(O)-; Z is selected from -NR 2 - and -O-; AA is an amino acid residue or a peptide residue; L 4 yes Where Su is the hexose form of a monosaccharide; d is an integer independently selected from 1, 2 and 3; D is the cytotoxic payload; COMP is the residue of a second compound; and represents the point of attachment to the rest of the compound.

[0014] In certain embodiments, the compound represented by formula (II) is a compound represented by formula (IIA): or a pharmaceutically acceptable salt and / or regioisomer thereof; where the integers c, RL, and R a , R b , Ring B, L 2 , L 3 , D and COMP are as defined in the present invention.

[0015] In certain embodiments, the compound of formula (II) is a compound of formula (IIB): or a pharmaceutically acceptable salt and / or regioisomer thereof; where the integers c, RL, and R a , R b , Ring B, L 2 , L 3 , D and COMP are as defined in the present invention.

[0016] In certain embodiments, the compound of formula (II) is a compound of formula (IIC): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RL, R a , R b , Ring A, L 2 , L 3 , D and COMP are as defined in the present invention.

[0017] In certain embodiments, the compound of formula (II) is a compound of formula (IID): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RL, R a , R b , Ring A, L 2 , L 3 , D and COMP are as defined in the present invention.

[0018] In certain embodiments, the compound of formula (II) is a compound of formula (IIE): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RL, R a , R b , Ring B, L 3 POLY 2 , D and COMP are as defined in the present invention.

[0019] In certain embodiments, the compound of formula (II) is a compound of formula (IIF): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RL, R a , R b , Ring B, L 3 POLY 2, D and COMP are as defined in the present invention.

[0020] In certain embodiments, the compound of formula (II) is a compound of formula (IIG): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring A, L 3 POLY 2 and D are as defined in the present invention.

[0021] In certain embodiments, the compound of formula (II) is a compound of formula (IIH): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RL, R a , R b , Ring A, L 3 POLY 2 , D and COMP are as defined in the present invention.

[0022] In one aspect, the present invention provides a conjugate represented by formula (III): or a pharmaceutically acceptable salt and / or regioisomer thereof; in, L 5 is a linker comprising an unnatural amino acid; and RG, Y, L 2 , L 3 and D are as defined in the present invention.

[0023] Non-limiting unnatural amino acids include cysteic acid, hydroxyproline (Hyp), β-alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3-nitrotyrosine, nitroarginine, pyroglutamate (Pyr), naphthylalanine (Nal), 2,4-diaminobutyric acid (DAB), methionine sulfoxide, and methionine sulfone.

[0024] In certain embodiments, the compound represented by formula (III) is a compound represented by formula (IIIA): or a pharmaceutically acceptable salt and / or regioisomer thereof; Among them, RG, Y, L 2 , L 3 and D are as defined in the present invention.

[0025] In certain embodiments, the compound of formula (III) is a compound of formula (IIIB): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b POLY 1 , AA and D are as defined in the present invention.

[0026] In certain embodiments, the compound represented by formula (IIIB) is a compound represented by the following formula: or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b POLY 1 , AA and D are as defined in the present invention.

[0027] In one aspect, the present invention provides a conjugate represented by formula (IV): or a pharmaceutically acceptable salt and / or regioisomer thereof; in, L 5 is a linker comprising an unnatural amino acid; and RL, COMP, Y, L 2 , L 3 and D are as defined in the present invention.

[0028] In certain embodiments, the compound of formula (IV) is a compound of formula (IVA): or a pharmaceutically acceptable salt and / or regioisomer thereof; Among them, RL, COMP, Y, L 2 , L 3 and D are as defined in the present invention.

[0029] In certain embodiments, the compound of formula (IV) is a compound of formula (IVB): or a pharmaceutically acceptable salt and / or regioisomer thereof; Among them, integer a, integer c, RL, COMP, R a , R b POLY 1, AA and D are as defined in the present invention.

[0030] In certain embodiments, the compound represented by formula (IVB) is a compound represented by the following formula: or a pharmaceutically acceptable salt and / or regioisomer thereof; Among them, integer a, integer c, RL, COMP, R a , R b POLY 1 , AA and D are as defined in the present invention.

[0031] The present invention provides at least the following embodiments: a) a compound represented by Formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIIA), or (IIIB), or a pharmaceutically acceptable salt, solvate, regioisomer, and / or stereoisomer thereof; b) a compound selected from Compound 1 to Compound 25 or a pharmaceutically acceptable salt, solvate, regioisomer, and / or stereoisomer thereof; c) a pharmaceutical composition comprising the compound described in (a) or (b) and a pharmaceutically acceptable excipient, diluent or carrier; d) Formula (II), Formula (IIA), Formula (IIB), Formula (IIC), Formula (IID), Formula (IIE), Formula (IIF), Formula (IIG), Formula (IIH), Formula (IVA), or Formula (IVB) or a pharmaceutically acceptable salt, solvate, regioisomer, and / or stereoisomer thereof; e) a compound selected from Compound 101A to Compound 125B or a pharmaceutically acceptable salt, solvate, regioisomer, and / or or stereoisomers; f) a pharmaceutical composition comprising the compound described in (d) or (e) and a pharmaceutically acceptable excipient, diluent or carrier; g) a method for treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the compound of (a) or (b) or (c) The pharmaceutical composition; h) a method for treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the compound of (d) or (e) or (f) The pharmaceutical composition; i) A method for inhibiting tubulin polymerization in a subject in need thereof, comprising administering a therapeutically effective amount of (a), (b), (d) or (e) the compound or the pharmaceutical composition of (c) or (f); j) The method of (g) or (h), wherein the disease or condition is abnormal cell proliferation; k) The method as described in (j), wherein the abnormal cell proliferation is cancer; l)(k) The method of claim 1, wherein the cancer is small cell lung cancer, non-small cell lung cancer, ovarian cancer, platinum-resistant ovarian cancer, ovarian adenocarcinoma, endometrial cancer, breast cancer, breast cancer overexpressing HER2, triple-negative breast cancer, lymphoma, large cell lymphoma, diffuse mixed histiocytic and lymphocytic lymphoma, follicular B-cell lymphoma, colon cancer, colon carcinoma (colon carcinoma), colon adenocarcinoma, colorectal adenocarcinoma, melanoma, prostate cancer, or multiple myeloma; m) use of a therapeutically effective amount of the compound (a) or (b) or the pharmaceutical composition (c) for treating a disease or condition in a subject in need thereof; n) use of a therapeutically effective amount of the compound (d) or (e) or the pharmaceutical composition (f) for treating a disease or condition in a subject in need thereof; o) use of a therapeutically effective amount of the compound (a) or (b) or the pharmaceutical composition (c) in the preparation of a medicament for treating a disease or condition in a subject in need thereof; p) use of a therapeutically effective amount of the compound of (d) or (e) or the pharmaceutical composition of (f) in the preparation of a medicament for treating a disease or condition in a subject in need thereof; q) use of a therapeutically effective amount of the compound of (a), (b), (d) or (e) or the pharmaceutical composition of (d) or (f) in the preparation of a medicament for inhibiting tubulin polymerization in a subject in need thereof; r) The use as described in (m)-(p), wherein the disease or disorder is abnormal cell proliferation; s)(r) The use as described in claim 1, wherein the abnormal cell proliferation is cancer; The use of (s), wherein the cancer is small cell lung cancer, non-small cell lung cancer, ovarian cancer, platinum-resistant ovarian cancer, ovarian adenocarcinoma, endometrial cancer, breast cancer, breast cancer overexpressing HER2, triple-negative breast cancer, lymphoma, large cell lymphoma, diffuse mixed histiocytic and lymphocytic lymphoma, follicular B-cell lymphoma, colon cancer, colon carcinoma (colon carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, melanoma, prostate cancer, or multiple myeloma; and u) A method for preparing a conjugate of formula (II), formula (IIA), formula (IIB), formula (IIC), formula (IID), formula (IIE), formula (IIF), formula (IIG), formula (IIH), formula (IVA), or formula (IVB) or a pharmaceutically acceptable salt thereof, which comprises reacting a conjugate of formula (I), formula (IA), formula (IB), or formula (IVB) with a molecule of formula (I) or (IIB) wherein the conjugate is Under conditions where the compound represented by formula (I), formula (IA), formula (IB), formula (IC), formula (ID), formula (IE), formula (IF), formula (IG), formula (IH), formula (IIIA), or formula (IIIB) or a pharmaceutically acceptable salt thereof is contacted with the second compound, wherein the second compound comprises an alkyne, a cyclooctyne, a strained olefin, a tetrazine, a methylcyclopropene, a thiol, a maleimide, a carbonyl, an amine, an oxyamine, or an azide. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1A : is a graph showing the killing activity of the anti-FolRα ADCs conjugated with Compound 1, Compound 2 and Compound 3 described in Example 18 on FolRα-positive Igrov1 cells.

[0033] Figure 1B is a graph showing that the anti-FolRα ADCs conjugated with Compound 1, Compound 2, and Compound 3 described in Example 18 have no killing activity against FolRα-negative A549 cells.

[0034] Figure 2A : is a graph showing the killing activity of the anti-FolRα ADCs conjugated with Compound 6, Compound 7, Compound 8 and Compound 9 described in Example 18 on FolRα-positive Igrov1 cells.

[0035] Figure 2B is a graph showing that the anti-FolRα ADCs conjugated with Compound 6, Compound 7, Compound 8 and Compound 9 described in Example 18 have no killing activity against hFolRα-negative A549 cells.

[0036] Figure 3A is a graph showing the killing activity of the anti-FolRα ADCs conjugated with Compound 13, Compound 14, and Compound 15 described in Example 19 against FolRα-positive Igrov1 cells.

[0037] Figure 3Bis a graph showing that the anti-FolRα ADCs conjugated with Compound 13, Compound 14, and Compound 15 described in Example 19 have no killing activity against FolRα-negative A549 cells. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention describes compounds of formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), or (IIB), which can be used to regulate, for example, the bioavailability and ADME of macromolecular conjugate compounds. In some embodiments, the compounds of the present invention can be used to prepare conjugates, such as conjugates of formula (II), (IIA), (IIB), (IIC), (IID), (IIE), (IIF), (IIG), (IIH), (IVA), or (IVB) for in vivo use. In certain embodiments, the compounds and conjugates have the function of being easily enzymatically cleaved to release the payload compound for use in vivo or elsewhere. The compounds can be altered to adjust the physiochemical properties and plasma stability of the conjugates. This provides a platform for regulating the bioavailability and ADME of macromolecules in vivo. definition

[0039] Unless otherwise defined, all technical terms, symbol descriptions, and other scientific terms used in the present invention are intended to have the meanings commonly understood by those having ordinary knowledge in the art to which the present invention belongs. In many cases, terms with commonly understood meanings are defined in the present invention for clarity and / or ease of reference. The techniques and procedures described or referred to in the present invention are generally widely understood by those having ordinary knowledge in the art and are frequently used using conventional methods, such as, for example, the widely used Green & Sambrook, Molecular Cloning: A Laboratory Manual 4 th Molecular cloning methods described in ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons. Where appropriate, methods involving the use of commercially available kits and reagents were generally performed according to manufacturer-defined protocols and conditions / parameters unless otherwise specified.

[0040] As used herein, the singular forms "a", "an", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0041] The term "about" means and encompasses the indicated value and the range above and below this value. In certain embodiments, the term "about" means a specified value ± 10%, ± 5%, or ± 1%. In certain embodiments, the term "about" means a specified value ± one standard deviation of this value. In certain embodiments, for example, for a logarithmic scale (e.g., pH), the term "about" means a specified value ± 0.3, ± 0.2, or ± 0.1.

[0042] When referring to the compounds provided by the present invention, unless otherwise stated, the following terms have the following meanings. Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those of ordinary skill in the art are generally understood. If a term of the present invention has multiple definitions, unless otherwise stated, the definitions in this section shall prevail.

[0043] As used herein, the terms "alkoxy" and "alkoxy group" refer to the group -OR", where R" is an alkyl or cycloalkyl group. In certain embodiments, the alkoxy group includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

[0044] The term "alkoxyamine" as used herein refers to the group -alkylene-O-NH2, wherein alkylene is as defined herein. In some embodiments, the alkoxyamine group can react with an aldehyde to form an oxime residue. Examples of alkoxyamine groups include -CH2CH2-O-NH2, -CH2-O-NH2, and -O-NH2.

[0045] The term "alkyl" as used herein, unless otherwise specified, refers to a saturated straight or branched chain hydrocarbon. In certain embodiments, the alkyl group is a primary, secondary or tertiary hydrocarbon. In certain embodiments, the alkyl group comprises 1-10 carbon atoms (i.e., C1-C 10 In certain embodiments, the alkyl group is a lower alkyl group, such as C 1-6Alkyl etc. In certain embodiments, the alkyl group is selected from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl and 2,3-dimethylbutyl. In certain embodiments, "substituted alkyl" refers to alkyl being substituted by, for example, 1, 2 or 3 groups independently selected from halogen (e.g., fluorine (F), chlorine (Cl), bromine (Br) or iodine (I)), alkyl, -CN, -NO2, amide, -C (O) -, -C (S) -, ester, carbamate, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, dialkylamino, haloalkyl, hydroxyl, amino, alkylamino and alkoxy groups. In some embodiments, alkyl is unsubstituted.

[0046] As used herein, the term "alkylene", unless otherwise indicated, refers to a divalent alkyl group, the alkyl being as defined herein. "Substituted alkylene" refers to an alkylene substituted as described herein for an alkyl. In some embodiments, the alkylene is unsubstituted.

[0047] As used herein, the term "alkenyl" refers to an ethylenically unsaturated hydrocarbon radical, in certain embodiments, having up to about 11 carbon atoms or 2 to 6 carbon atoms (e.g., "lower alkenyl"), which may be straight chain or branched, and having at least 1 or 1-2 sites of ethylenic unsaturation. "Substituted alkenyl" refers to an alkenyl group substituted as described herein for alkyl.

[0048] The term "alkenylene" as used herein refers to a divalent alkenyl group as defined herein. Lower alkenylene is, for example, C2-C6-alkenylene.

[0049] The term "alkynyl" as used herein refers to an acetylenically unsaturated hydrocarbon radical, in certain embodiments, having up to about 11 carbon atoms or 2-6 carbon atoms (e.g., "lower alkynyl"), which may be straight or branched, and having at least 1 or 1-2 acetylenically unsaturated sites. Non-limiting examples of alkynyl groups include acetylenic ethynyl (-C≡CH), propargyl (-CH2C≡CH), and the like. "Substituted alkynyl" refers to an alkynyl group substituted as described herein for alkyl.

[0050] The term "alkynylene" as used herein refers to a divalent alkynyl group as defined herein. Lower alkynylene is, for example, C2-C6-alkynylene.

[0051] The term "amino" as used herein refers to -NH2.

[0052] The term "alkylamino" as used herein, unless otherwise indicated, refers to the group -NHR", wherein R" is, for example, C 1-10 Alkyl, C 2-10Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, C 1-10 In certain embodiments, alkylamino is C 1-6 Alkylamino.

[0053] The term "dialkylamino" as used herein, unless otherwise specified, refers to the group -NR"R", wherein each R" is independently C 1-10 In certain embodiments, dialkylamino is, for example, di-C 1-6 Alkylamino, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, C 1-10 Haloalkyl, etc.

[0054] The term "aryl" used in the present invention refers to phenyl, biphenyl or naphthyl, unless otherwise indicated. The term includes substituted and unsubstituted moieties. The aryl group can be substituted by any described moiety, including but not limited to one or more moieties selected from the group consisting of the following (for example, in some embodiments, 1, 2 or 3 moieties): halogen (fluorine (F), chlorine (Cl), bromine (Br) or iodine (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate (phosphate) and phosphonate (phosphonate), wherein each moiety is independently unprotected, or protected as required, as known to those skilled in the art (see, for example, Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991); and wherein the aryl in the arylamino and aryloxy substituents is not further substituted.

[0055] The term "arylamino", as used herein, and unless otherwise indicated, refers to a -NR'R" group, wherein R' is H or C1-C6-alkyl; and R" is aryl, as defined herein.

[0056] The term "arylene" as used herein, unless otherwise indicated, refers to a divalent aryl group, wherein aryl is as defined herein.

[0057] The term "aryloxy" as used herein, unless otherwise indicated, refers to a -OR group, wherein R is an aryl group, as defined herein.

[0058] "Alkarylene" refers to an arylene group, as defined herein, wherein the aryl ring is substituted with 1 or 2 alkyl groups. "Substituted alkarylene" refers to an alkylarylene group, as defined herein, wherein the arylene group is further substituted as defined herein for aryl.

[0059] "Aralkylene" refers to a -CH2-arylene-, -arylene-CH2-, or -CH2-arylene-CH2- group, wherein arylene is as defined herein. "Substituted aralkylene" refers to arylenealkyl, as defined herein, wherein the aralkylene group is substituted as defined herein for aryl.

[0060] As used herein, the term "carboxyl" or "carboxyl group" refers to -C(O)OH or -COOH.

[0061] The term "cycloalkyl" or "carbocycle" used in the present invention, unless otherwise specified, refers to a saturated, unsaturated or aromatic ring in which all atoms on the ring are C atoms. In certain embodiments, the "cycloalkyl" or "carbocycle" group can be a saturated, and / or bridged, and / or non-bridged, and / or fused bicyclic group, and / or a spirocyclic bicyclic group. In certain embodiments, the "cycloalkyl" or "carbocycle" group contains 3-10 carbon atoms (i.e., C3-C 10 In some embodiments, the "cycloalkyl" or "carbocycle" has 3-15 carbon atoms (C 3-15 ), 3-10 carbon atoms (C 3-10 ), 3-7 carbon atoms (C 3-7), or 3-6 carbon atoms (C3-C6) (i.e., "low cycloalkyl"). In certain embodiments, the "cycloalkyl" or "carbocycle" group is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, cycloheptyl, bicyclo [2.1.1] hexyl, bicyclo [2.2.1] heptyl, decalinyl or adamantyl. Exemplary "cycloalkyl" or "carbocycle" include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl and naphthyl. "Cycloalkyl" or "carbocycle" includes a monocycle composed of 3 to 10 atoms, a bicycle composed of 6 to 12 atoms and a bridged ring composed of 6 to 12 atoms. Each ring of the bicyclic cycloalkyl or carbocycle can be selected from saturated rings, unsaturated rings and aromatic rings. Bicyclic cycloalkyl or bicyclic carbocycle includes any combination of saturated bicyclic, unsaturated bicyclic and aromatic bicyclic, as long as the valence allows. Bicyclic cycloalkyl or bicyclic carbocycle includes any combination of ring sizes, such as 4-5 fused ring system, 5-5 fused ring system, 5-6 fused ring system, 6-6 fused ring system, 5-7 fused ring system, 6-7 fused ring system, 5-8 fused ring system and 6-8 fused ring system. Non-limiting examples of bridged bicyclic cycloalkyl or bridged bicyclic carbocycle groups include, but are not limited to, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.1.1]hexyl, bicyclo[3.1.1]heptyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2]decyl, and 2-oxabicyclo[2.2.2]octyl. Non-limiting examples of spirocyclic cycloalkyl or spirocyclic carbocyclic groups include, but are not limited to, spiro[3.3]heptyl, spiro[3.4]octyl, spiro[3.5]nonyl, spiro[3.6]decyl, spiro[4.4]nonyl, spiro[4.5]decyl, spiro[5.5]undecyl, spiro[5.6]dodecyl, and spiro[5.7]tridecyl.

[0062] The term "bicyclic ring system" includes structures consisting of 6-12 (e.g., 8-12 or 9, 10 or 11) atoms forming two rings, wherein the two rings have at least one common atom (e.g., two common atoms). The bicyclic rings can be fused, bridged or spirocyclic. Bicyclic ring systems include bicyclic aliphatics (e.g., bicyclic alkyls or bicyclic alkenyls), bicyclic heteroaliphatics, bicyclic aryls and bicyclic heteroaryls.

[0063] The term "bridged bicyclic ring system" refers to a bicyclic heteroalicyclic ring system or a bicyclic alicyclic ring system with a bridge between the rings. Examples of bridged bicyclic ring systems include, but are not limited to, adamantyl, norbornyl, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.1.1]hexyl, bicyclo[3.1.1]heptyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2]decyl, 2-oxabicyclo[2.2.2] octyl, 6-azabicyclo[3.1.1]heptyl, 6-azabicyclo[3.1.1]heptyl, 1-azabicyclo[2.2.1]heptyl, 2-azabicyclo[2.2.1]heptyl, 7-azabicyclo[2.2.1]heptyl, 1-azabicyclo[2.2.2]octyl, 3-azabicyclo[3.2.1]octyl, 2-oxabicyclo[3.1.1]heptyl, and 2,6-dioxatricyclo[3.3.1.0 3,7 The bridged bicyclic ring system may be optionally substituted with one or more substituents such as alkyl (including carboxyalkyl, hydroxyalkyl, and haloalkyl such as trifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxyl, alkoxycarbonyl, alkylcarbonyl, alkylcarbonyl, alkyloxy ... oxo, aminocarbonyl, alkylcarbonylamino, cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halogen, hydroxy, acyl, thiol, alkylsulfanyl, sulfinyloxy, urea, thiourea, sulfamoyl, sulfonamido, oxo, or carbamoyl.

[0064] The term "spiro bicyclic ring system" refers to a bicyclic heteroalicyclic ring system or a bicyclic alicyclic ring system in which two or three rings are linked together by a common atom. Spiro compounds depicted with overlapping rings indicate that the rings may be linked at any vertex. For example, in the spiro group , the two rings may be connected at any of the three available vertex atoms in either ring.

[0065] The term "cycloalkylene" as used herein refers to a divalent cycloalkyl group, wherein the cycloalkyl group is as defined herein. In certain embodiments, the cycloalkylene group is cyclopropylene. Cyclobutylene Cyclopentylene Cyclohexylene Cycloheptylene etc. Lower cycloalkylene refers to C3-C6-cycloalkylene.

[0066] The term "cycloalkylalkyl" as used herein, unless otherwise indicated, refers to an alkyl group as defined herein substituted with 1 or 2 cycloalkyl groups as defined herein.

[0067] The term "ester" as used herein refers to -C(O)OR or -COOR, wherein R is an alkyl group as defined herein.

[0068] The term "fluorene" used in the present invention refers to Any one or more C atoms carrying one or more H atoms may be replaced by the chemical functional groups described in the present invention.

[0069] The term "haloalkyl" refers to an alkyl group, as defined herein, substituted with one or more independently selected halogen atoms (eg, in some embodiments 1, 2, 3, 4, or 5).

[0070] The term "heteroalkyl" refers to an alkyl group defined in the present invention, wherein one or more carbon atoms are replaced by heteroatoms. "Heteroalkenyl" used in the present invention refers to an alkenyl group defined in the present invention, wherein one or more carbon atoms are replaced by heteroatoms. "Heteroalkynyl" used in the present invention refers to an alkynyl group defined in the present invention, wherein one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen (N), oxygen (O) and sulfur (S) atoms. Heteroalkyl, heteroalkenyl and heteroalkynyl groups may all be optionally substituted. Examples of heteroalkyl group moieties include, but are not limited to, aminoalkyl, sulfonylalkyl and sulfinylalkyl. Examples of heteroalkyl group moieties also include, but are not limited to, methylamino, methylsulfonyl and methylsulfinyl. "Substituted heteroalkyl" refers to heteroalkyl substituted by 1, 2 or 3 groups independently selected from halogen (e.g., fluorine (F), chlorine (Cl), bromine (Br) or iodine (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino and alkoxy groups. In some embodiments, the heteroalkyl group may contain 1, 2, 3 or 4 heteroatoms. Those skilled in the art will recognize that heteroalkyl groups consisting of 4 atoms may typically contain 1 or 2 heteroatoms, heteroalkyl groups consisting of 5 or 6 atoms may typically contain 1, 2 or 3 heteroatoms, and heteroalkyl groups consisting of 7 to 10 atoms may typically contain 1, 2, 3 or 4 heteroatoms.

[0071] As used herein, the term "heteroalkylene" refers to a divalent heteroalkyl group, which is as defined herein. "Substituted heteroalkylene" refers to a divalent heteroalkyl group, as defined herein, which is substituted as described herein for heteroalkyl groups.

[0072] The term "heterocycloalkyl" or "heterocycle" refers to a saturated, unsaturated or aromatic ring comprising one or more heteroatoms. Exemplary heteroatoms include N, O, Si, P, B and S atoms, wherein N or S atoms may be optionally oxidized, and N atoms may be optionally quaternized, and the remaining ring atoms of the non-aromatic rings are all carbon atoms."Heterocycloalkyl" or "heterocycle" includes a monocycle composed of 3 to 10 atoms, a bicyclic ring composed of 6 to 12 atoms, and a bridged ring composed of 6 to 12 atoms. In certain embodiments, "heterocycloalkyl" or "heterocycle" is a monovalent monocyclic or polycyclic fully saturated ring system. In certain embodiments, the "heterocycloalkyl" or "heterocycle" group may be unsaturated, and / or bridged, and / or non-bridged, and / or fused bicyclic groups, and / or spirocyclic bicyclic groups. Bicyclic "heterocycloalkyl" or "heterocycle" includes any combination of ring sizes, such as 4-5 fused ring systems, 5-5 fused ring systems, 5-6 fused ring systems, 6-6 fused ring systems, 5-7 fused ring systems, 6-7 fused ring systems, 5-8 fused ring systems, and 6-8 fused ring systems. In certain embodiments, the "heterocycloalkyl" or "heterocycle" group has 3 to 20, 3 to 15, 3 to 10, 3 to 8, 4 to 7, 4 to 11, or 5 to 6 ring atoms. The "heterocycloalkyl" or "heterocycle" can be connected to the parent core structure at any heteroatom or carbon atom thereof to form a stable compound. In certain embodiments, the "heterocycloalkyl" or "heterocycle" is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a fused or bridged or spirocyclic ring system, and wherein the N or S atom may be optionally oxidized, and / or the N atom may be optionally quaternized. In some embodiments, "heterocycloalkyl" or "heterocyclic" groups include, but are not limited to, 2,5-diazabicyclo[2.2.2]octyl, decahydroisoquinolinyl, dihydrobenzoisoxazinyl, dihydrofuranyl, dihydroisoindolyl, dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, 1,4-dithianyl, furanonyl, imidazolidinyl, imidazolinyl, Indolinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinone, oxazolidinyl, oxiranyl, piperazinyl, piperidinyl, 4-piperidone, pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydrothiophenyl, thiamorpholinyl, thiazolidinyl, tetrahydroquinolinyl, and 1,3,5-trithianyl.Non-limiting examples of bridged heterocycloalkyl or bridged heterocyclic groups include, but are not limited to, 6-azabicyclo[3.1.1]heptyl, 6-azabicyclo[3.1.1]heptyl, 1-azabicyclo[2.2.1]heptyl, 2-azabicyclo[2.2.1]heptyl, 7-azabicyclo[2.2.1]heptyl, 1-azabicyclo[2.2.2]octyl, 3-azabicyclo[3.2.1]octyl, 2-oxabicyclo[3.1.1]heptyl, and 2,6-dioxa-tricyclo[3.3.1.03,7]nonyl. Non-limiting examples of spirocyclic heterocycloalkyl or spirocyclic heterocyclic groups include, but are not limited to, 2,8-diazaspiro[4.5]decyl; 2,7-diazaspiro[3.5]nonyl; 3,9-diazaspiro[5.5]undecyl; 3-azaspiro[5.5]undecyl; 2-oxa-6-azaspiro[3.4]octyl; 2-oxa-9-azaspiro[5.5]undecyl; 3-oxa-9-azaspiro[5.5]undecyl; 7-azaspiro[3.5]nonyl; 2-azaspiro[3.5]nonyl; 7-oxaspiro[3.5]nonyl; and 2-oxaspiro[3.5]nonyl.

[0073] In certain embodiments, "heterocycloalkyl" or "heterocycle" may also be optionally substituted as described herein. In certain embodiments, "heterocycloalkyl" or "heterocycle" is substituted by 1, 2 or 3 groups independently selected from halogen (e.g., fluorine (F), chlorine (Cl), bromine (Br) or iodine (I)), alkyl, haloalkyl, hydroxy, amino, alkylamino, and alkoxy. In some embodiments, the heterocycloalkyl or "heterocycle" group may contain 1, 2, 3 or 4 heteroatoms. Those skilled in the art will recognize that "heterocycloalkyl" or "heterocycle" composed of 4 atoms may generally contain 1 or 2 heteroatoms, "heterocycloalkyl" or "heterocycle" composed of 5 or 6 atoms may generally contain 1, 2 or 3 heteroatoms, and "heterocycloalkyl" or "heterocycle" composed of 7 to 10 atoms may generally contain 1, 2, 3 or 4 heteroatoms.

[0074] "Heterocycloalkylene" refers to a divalent heterocycloalkyl group, wherein the heterocycloalkyl group is as defined herein.

[0075] The term "heteroaryl" refers to a monovalent monocyclic aromatic group and / or a polycyclic aromatic group, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S and N in the ring. Each ring of the heteroaryl group may contain 1 or 2 O atoms, 1 or 2 S atoms and / or 1-4 N atoms, provided that the total number of heteroatoms in each ring is 4 or less, and each ring contains at least one carbon atom. In certain embodiments, the heteroaryl group has 5-20, 5-15 or 5-10 ring atoms. The heteroaryl group may be connected to the rest of the molecule by a nitrogen or carbon atom. In some embodiments, the monocyclic heteroaryl group includes, but is not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, triazolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothiophenyl, benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothiophenyl, isoindolyl, isoquinolyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridinyl, quinolyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidinyl, and thienopyridinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthroline, phenanthridinyl, phenanthroline, phenanthroline, phenanthroline, phenanthroline and xanthenyl. In certain embodiments, heteroaryl can also be optionally substituted as described herein. "Substituted heteroaryl" is a heteroaryl substituted as defined by the present invention for aryl.

[0076] The term "heteroarylene" refers to a divalent heteroaryl radical, wherein heteroaryl is as defined herein. "Substituted heteroarylene" is a heteroarylene that is substituted as defined herein for aryl.

[0077] The term "protecting group" used in the present invention, unless otherwise specified, refers to a group added to an oxygen, nitrogen or phosphorus atom to prevent (protected) oxygen, nitrogen or phosphorus from further reaction or for other purposes. Various oxygen protecting groups and nitrogen protecting groups are known to those skilled in the art of organic synthesis (see, for example, Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Fourth Edition, 2006, which is incorporated herein by reference in its entirety).

[0078] "Pharmaceutically acceptable salt" refers to any salt of a compound provided herein which retains its biological properties and is non-toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from various organic and inorganic counterions known in the art. Such salts include, but are not limited to: (1) with organic or inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, caproic acid, cyclopentylpropionic acid, glycolic acid, glutaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, picric acid , cinnamic acid, mandelic acid, phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphoric acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthalene or (2) when an acidic proton is present in the parent compound, (a) it is replaced by a metal ion such as an alkali metal ion, an alkaline earth metal ion or an aluminum ion, or an alkali metal or alkaline earth metal hydroxide such as sodium, potassium, calcium, magnesium, aluminum, lithium, zinc and barium, or ammonia, or (b) it is coordinated with an organic base such as an aliphatic, alicyclic or aromatic organic amine, including but not limited to ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, procaine, N-benzylphenethylamine, N-methylglucamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, etc. to form a base addition salt.

[0079] By way of example only and not limitation, pharmaceutically acceptable salts further include sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts, and tetraalkylammonium salts and the like; when the compound contains a basic functional group, it is a salt of a non-toxic organic or inorganic acid, such as a hydrohalide such as hydrochloride and hydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate, tartrate, citrate, benzoate, 3-(4-hydroxybenzoyl) Benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate), ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (benzenesulfonate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylaminosulfonate, quinate, muconate, etc.

[0080] In terms of composition, the term "substantially free of" or "substantially absent" refers to a composition comprising at least 85% by weight or 90% by weight, in certain embodiments, comprising 95%, 98%, 99% by weight or 100% by weight, or in certain embodiments, comprising 95%, 98%, 99% or 100% of the specified enantiomer or diastereomer of the compound. In certain embodiments, in the methods and compounds provided by the invention, the compound is substantially free of one of two enantiomers. In certain embodiments, in the methods and compounds provided by the invention, the compound is substantially free of one of two diastereomers. In certain embodiments, in the methods and compounds provided by the invention, the compound is substantially free of enantiomers (that is, the compound is not a racemic compound or a mixture of 50:50 compounds).

[0081] Similarly, with respect to a composition, the term "isolated" means that the composition contains at least 85%, 90%, 95%, 98%, 99% to 100% by weight of the compound, with the remainder comprising other chemical species, enantiomers, or diastereomers.

[0082] "Solvate" means that the compound or salt thereof provided by the present invention further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0083] The term "substituted" refers to a moiety with a substituent that replaces one or more carbons or H on a replaceable heteroatom (e.g., NH or NH2 of a compound). It should be understood that "substituted" or "substituted by..." includes implicit conditions, i.e., such substitutions meet the allowed valences of the substituted atoms and substituents, and such substitutions can produce stable compounds, i.e., compounds that do not spontaneously undergo transformations (e.g., by rearrangement, cyclization, elimination, etc.). In certain embodiments, substituted refers to a moiety with a substituent that replaces two H atoms on the same carbon atom, such as replacing two H atoms on a single carbon with an oxo, imino, or thio group. The term "substituted" used in the present invention is intended to include all allowed substituents of organic compounds. In a broad sense, allowed substituents in organic compounds include non-cyclic and cyclic, branched and straight-chain, carbocyclic and heterocyclic, aromatic and non-aromatic substituents. For appropriate organic compounds, the allowed substituents may be one or more, and may be the same or different.

[0084] In some embodiments, the substituents may include any of the substituents described herein, for example, halogen, hydroxyl, oxo (=O), thio (=S), cyano (-CN), nitro (-NO2), imino (=NH), oxime (=N-OH), hydrazine (=N-NH2), -R b1 -OR a1 , -R b1 -OC(O)-R a1 , -R b1 -OC(O)-OR a1 , -R b1 -OC(O)-N(R a1 )2. -R b1 -N(R a )2. -R b1 -C(O)R a1 , -R b1 -C(O)OR a1 , -R b1 -C(O)N(R a1 )2. -R b1 -OR c1 -C(O)N(R a1 )2. -R b1 -N(R a1 )C(O)OR a1 , -R b1 -N(R a )C(O)R a1 , -R b1 -N(R a1 )S(O) t R a1 (where t is 1 or 2), -Rb1 -S(O) t R a1 (where t is 1 or 2), -R b1 -S(O) t OR a1 (where t is 1 or 2), and -R b1 -S(O) t N(R a1 )2 (wherein t is 1 or 2); and alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl and heteroarylalkyl, any of which may be optionally substituted by alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thio (=S), cyano (-CN), nitro (-NO2), imino (=NH), oxime (=N-OH), hydrazine (=N-NH2), -R b1 -OR a1 , -R b1 -OC(O)-R a1 , -R b1 -OC(O)-OR a1 , -R b1 -OC(O)-N(R a1 )2. -R b1 -N(R a )2. -R b1 -C(O)R a1 , -R b1 -C(O)OR a1 , -R b1 -C(O)N(R a1 )2. -R b1 -OR c1 -C(O)N(R a1 )2. -R b1 -N(R a1 )C(O)OR a1 , -R b1 -N(R a1 )C(O)R a1 , -R b1 -N(R a1 )S(O) t R a1 (where t is 1 or 2), -R b1 -S(O) t R a1 (where t is 1 or 2), -R b1 -S(O) t OR a1 (where t is 1 or 2) and -R b1 -S(O)t N(R a1 )2 (where t is 1 or 2); where each R a1 Each R is independently selected from H, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, wherein each R a1 , if valence permits, may be optionally substituted by the following groups: alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thio (=S), cyano (-CN), nitro (-NO2), imino (=NH), oxime (=N-OH), hydrazine (=N-NH2), -R b1 -OR a1 , -R b1 -OC(O)-R a1 , -R b1 -OC(O)-OR a1 , -R b1 -OC(O)-N(R a1 )2. -R b1 -N(R a1 )2. -R b1 -C(O)R a1 , -R b1 -C(O)OR a1 , -R b1 -C(O)N(R a1 )2. -R b1 -OR c1 -C(O)N(R a1 )2. -R b1 -N(R a1 )C(O)OR a1 , -R b1 -N(R a1 )C(O)R a1 , -R b1 -N(R a )S(O) t R a1 (where t is 1 or 2), -R b1 -S(O) t R a1 (where t is 1 or 2), -R b1 -S(O) t OR a1 (where t is 1 or 2) and -R b1 -S(O) t N(R a1 )2 (wherein t is 1 or 2); and wherein each R b1 are independently selected from a direct bond or a straight or branched alkylene, alkenylene or alkynylene chain, and each R cA straight or branched alkylene, alkenylene or alkynylene chain, respectively.

[0085] Those skilled in the art will appreciate that substituents themselves may be substituted if appropriate. Unless expressly stated as "unsubstituted", references to chemical moieties in the present invention should be understood to include substituted variants. For example, references to "heteroaryl" groups or moieties implicitly include both substituted and unsubstituted variants unless otherwise stated.

[0086] The term "amino acid" or "amino acid residue" refers to a D- or L-natural or non-natural amino acid. Representative amino acids include, but are not limited to, alanine, β-alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, proline, serine, threonine, valine, tryptophan, or tyrosine, etc. "Non-natural amino acids" are naturally occurring or chemically synthesized non-proteinogenic amino acids. Non-limiting examples of non-natural amino acids include cysteic acid, hydroxyproline (Hyp), β-alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3-nitrotyrosine, nitroarginine, pyroglutamic acid (Pyr), naphthylalanine (Nal), 2,4-diaminobutyric acid (DAB), methionine sulfoxide, and methionine sulfone.

[0087] "Isotopic composition" refers to the amount of each isotope present for a given atom, and "natural isotopic composition" refers to the naturally occurring isotopic composition or abundance of a given atom. Atoms having their natural isotopic composition may also be referred to as "non-enriched" atoms in the present invention. Unless otherwise indicated, atoms of the compounds described herein are intended to represent any stable isotopes of the atom. For example, unless otherwise indicated, when a position is specifically designated as "hydrogen" (H), the position is understood to be hydrogen having its natural isotopic composition.

[0088] "Isotopic enrichment" refers to the percentage of incorporation of a specific isotope at a given atom in a molecule in place of the natural isotopic abundance of the atom. For example, a 1% deuterium (D) enrichment at a given position means that 1% of the molecules in a given sample contain deuterium at the specified position. Since the natural distribution of deuterium is about 0.0156%, the deuterium enrichment at any position in a compound synthesized using a non-enriched starting material is about 0.0156%. The isotopic enrichment of the compounds provided by the present invention can be measured using conventional analytical methods known to those of ordinary skill in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy.

[0089] "Isotopically enriched" refers to atoms having an isotopic composition other than the natural isotopic composition of said atoms. "Isotopically enriched" may also refer to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of said atoms.

[0090] As used herein, “alkyl,” “alkylene,” “alkylamino,” “dialkylamino,” “cycloalkyl,” “aryl,” “arylene,” “alkoxy,” “amino,” “carboxy,” “heterocycloalkyl,” “heteroaryl,” “heteroarylene,” “carboxy,” and “amino acid” groups optionally contain deuterium (D) at one or more positions where there is a hydrogen atom (H), and wherein the deuterium composition of said one or more atoms is not the natural isotopic composition.

[0091] Likewise, the "alkyl", "alkylene", "alkylamino", "dialkylamino", "cycloalkyl", "aryl", "arylene", "alkoxy", "amino", "carboxyl", "heterocycloalkyl", "heteroaryl", "heteroarylene", "carboxyl" and "amino acid" groups used herein optionally contain C-13 ( 13 C).

[0092] The term "macromolecule" or "macromolecule moiety" refers to a protein, peptide, antibody, nucleic acid, carbohydrate, or other macromolecule composed of polymerized monomers. It includes peptides of two or more residues or ten or more residues. In certain embodiments, the mass of the macromolecule is at least 1000 Da. In certain embodiments, the macromolecule has at least 1000 atoms. In certain embodiments, the macromolecule can be modified. For example, the protein, peptide or antibody can be modified with one or more carbohydrates and / or small molecule therapeutic compounds.

[0093] The term "immunoglobulin" refers to a class of structurally related proteins that generally comprise two pairs of polypeptide chains: a pair of light (L) chains and a pair of heavy (H) chains. In a "complete immunoglobulin," all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been characterized in detail. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. In brief, each heavy chain generally comprises a heavy chain variable region (V H or VH) and the heavy chain constant region (C H The heavy chain constant region generally contains three domains, abbreviated as C H1 (or CH1), C H2 (or CH2) and C H3(or CH3). Each light chain generally comprises a light chain variable region (V L or VL) and a light chain constant region. The light chain constant region generally comprises a domain, abbreviated as C L or CL.

[0094] The term "antibody" is used in the broadest sense in the present invention. Antibodies include complete antibodies (e.g., complete immunoglobulins) and antibody fragments (e.g., antigen-binding fragments or antigen-binding fragments of antibodies). Antibodies contain at least one antigen-binding domain. An example of an antigen-binding domain is a V H -V L Dimerized antigen-binding domain.

[0095] The term "amino acid" refers to the 20 common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V), as well as the less common pyrrolysine and selenocysteine. Natural amino acids also include citrulline. Naturally encoded amino acids include post-translational variants of 22 naturally occurring amino acids, such as prenylated amino acids, isoprenylated amino acids, myristoylated amino acids, palmitoylated amino acids, N-linked glycosylated amino acids, O-linked glycosylated amino acids, phosphorylated amino acids, and acylated amino acids. The term "amino acid" also includes non-natural (or unnatural) or synthetic α-, β-, γ-, or δ-amino acids, and includes, but is not limited to, amino acids found in proteins, i.e., glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine. In certain embodiments, the amino acid is in the L configuration. In certain embodiments, the amino acid is in the D configuration. Alternatively, the amino acid may be a derivative of alanyl, valyl, leucyl, isoleucyl, prolyl, phenylalanyl, tryptophanyl, methionyl, glycyl, seryl, threonyl, cysteinyl, tyrosyl, asparaginyl, glutaminyl, aspartyl, glutaryl, lysyl, arginyl, histidyl, β-alanyl, β-valyl, β-leucyl, β-isoleucyl, β-prolyl, β-phenylalanyl, β-tryptophanyl, β-methionyl, β-glycyl, β-seryl, β-threonyl, β-cysteinyl, β-tyrosyl, β-asparaginyl, β-glutaminyl, β-aspartyl, β-glutaryl, β-lysyl, β-arginyl, or β-histidyl. Unnatural amino acids are not proteinogenic amino acids, nor are they post-translationally modified variants thereof. Specifically, the term unnatural amino acid refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine, nor a post-translationally modified variant thereof.

[0096] The term "conjugate" refers to a compound or drug moiety of the present invention connected to one or more macromolecular moieties. The macromolecular moiety is as defined herein, or is any macromolecule deemed suitable by a person skilled in the art. The compound or drug moiety can be any compound or drug moiety described herein. The compound or drug moiety can be directly connected to the macromolecular moiety via a covalent bond, or the compound or drug moiety can be indirectly connected to the macromolecular moiety via a linker. Typically, the linker is covalently linked to the macromolecular moiety and is also covalently linked to the compound or drug moiety.

[0097] "pAMF," "pAMF residue," or "pAMF mutation" refers to a variant phenylalanine residue (ie, p-azidomethyl-L-phenylalanine) that is added to or substituted into a polypeptide.

[0098] The term "connector" refers to a molecular group portion that can form at least two covalent bonds. Typically, a connector can form at least one covalent bond with a macromolecular group portion, and form at least another covalent bond with a compound or drug group portion. In certain embodiments, a connector can form more than one covalent bond with a macromolecular group portion. In certain embodiments, a connector can form more than one covalent bond with a compound or drug group portion, or can form multiple covalent bonds with more than one compound or drug group portion. After a connector forms a bond with a macromolecular group portion, or a compound or drug group portion, or both, the remaining structure (i.e., the residue of the connector after forming one or more covalent bonds ("connector residue")) can still be referred to as a "connector" in the present invention. The term "connector precursor" refers to a connector having one or more reactive groups that can form covalent bonds with a macromolecule, or a compound or drug group portion, or both. A person of ordinary skill in the art, after understanding the context in which the term "linker" is used, will understand whether "linker" refers to a linker precursor having one active group, a linker precursor having more than one active group, a linker residue covalently linked to a macromolecule, a linker residue covalently linked to a compound or drug group portion, and / or a linker residue covalently linked to a macromolecule and covalently linked to a compound or drug group portion. In some embodiments, the linker is a cleavable linker. For example, a cleavable linker can be a linker that is released by biological instability or enzymatic function, which may or may not be engineered. In some embodiments, the linker is a non-cleavable linker. For example, a non-cleavable linker can be a linker that is released after the macromolecular group portion is degraded.

[0099] The "EC 50” refers to the dose, concentration or amount of a specific test compound that elicits a dose-dependent response at 50% of the maximal expression of a specific response induced, elicited or potentiated by the specific test compound.

[0100] Unless otherwise specified, the term "IC 50 ” refers to the amount, concentration or dose of a particular test compound that achieves a 50% inhibition of the maximal response in an assay measuring such response.

[0101] The terms "subject" and "patient" used in the present invention are used interchangeably herein. The term "subject" refers to an animal, such as a mammal including non-primates (e.g., cattle, pigs, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys such as cynomolgus monkeys, chimpanzees, and humans), and in certain embodiments is a human. In certain embodiments, the subject is a farm animal (e.g., horse, cattle, pig, etc.) or a pet (e.g., dog or cat). In certain embodiments, the subject is a human.

[0102] As used herein, the terms "therapeutic agent" and "therapeutic drug / agent" refer to any drug / agent that can be used to treat or prevent a disease / disorder or one or more symptoms thereof. In certain embodiments, the term "therapeutic agent" includes the compounds and / or conjugates provided herein. In certain embodiments, a therapeutic agent is a drug / agent that is known to be useful or has been used or is currently being used to treat or prevent a disease / disorder or one or more symptoms thereof.

[0103] "Therapeutically effective amount" refers to the amount of a compound or composition that is sufficient to achieve treatment of the disease or condition when administered to a subject. A "therapeutically effective amount" may vary depending on, inter alia, the compound, the disease or condition and its severity, and the age, weight, etc., of the subject to be treated.

[0104] In certain embodiments, "treating" or "managing" any disease or condition refers to improving the disease or condition present in a subject. In another embodiment, "treating" or "managing" includes improving at least one physical parameter, which may not be perceived by the subject. In yet another embodiment, "treating" or "managing" includes regulating the disease or condition, whether physically (e.g., stabilizing perceptible symptoms) or physiologically (e.g., stabilizing physiological parameters) or both. In another embodiment, "treating" or "managing" includes postponing or preventing the onset of the disease or condition, or postponing or preventing the recurrence of the disease or condition. In yet another embodiment, "treating" or "managing" includes reducing or eliminating the disease or condition, or delaying the progression of the disease or condition or one or more symptoms of the disease or condition, or reducing the severity of the disease or condition or one or more symptoms of the disease or condition.

[0105] As used herein, the term "inhibit growth" (e.g., in reference to cells, such as tumor cells) is intended to include any measurable reduction in cell growth (e.g., tumor cell growth) when contacted with a compound, drug moiety, or conjugate of the invention, compared to the growth of the same cells not contacted with the compound, drug moiety, or conjugate of the invention. In some embodiments, growth may be inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Reduction in cell growth may occur by a variety of mechanisms, including, but not limited to, conjugate, compound, or drug moiety internalization, apoptosis, necrosis, and / or effector function-mediated activity.

[0106] The term "prophylactic agent" as used herein and the term "prophylactic drug / agent" as used herein refer to any drug / agent that can be used to prevent a disease / disorder or one or more symptoms thereof. In certain embodiments, the term "prophylactic agent" includes a compound, drug group portion, or conjugate provided herein. In certain other embodiments, the term "prophylactic agent" does not refer to a compound, drug group portion, or conjugate provided herein. For example, a prophylactic agent is a drug / agent that is known to be useful or has been used or is being used to prevent or stop the onset, development, progression, and / or severity of a disease / disorder.

[0107] The phrase "prophylactically effective amount" as used herein refers to an amount of a treatment / therapy (e.g., a prophylactic agent) sufficient to result in the prevention or reduction of the development, recurrence or onset of one or more symptoms associated with a disease / disorder, or to enhance or improve the prophylactic effect of another treatment / therapy (e.g., another prophylactic agent).

[0108] In some chemical structures shown in the present invention, certain substituents, chemical groups and atoms are represented by curved / wavy / bent lines (e.g., ) depicts a curve / wavy line / curved line intersecting one or more bonds to represent the atoms through which the substituents, chemical groups, and atoms are connected. For example, in certain structures, such as but not limited to The curved / wavy / curved lines represent atoms in the backbone of the conjugate, compound or drug group portion of the structure to which the chemical entity is attached. In some structures, for example but not limited to The curved / wavy / curved lines represent atoms in a macromolecule to which the indicated chemical entity is attached and atoms in the backbone of a conjugate, compound or drug group moiety structure to which the indicated chemical entity is attached.

[0109] As used herein, diagrams showing substituents attached to cyclic groups (e.g., aromatic, heteroaromatic, fused rings, and saturated or unsaturated cycloalkyl or heterocycloalkyl) through bonds between ring atoms are intended to indicate that, unless otherwise indicated, the cyclic group may be substituted with the substituent at any ring position in the cyclic group or on any ring in a fused ring group, according to the techniques described herein or known in the art to which the invention pertains. For example, the group The positions of the substituents O-Su are generally described, i.e., not directly connected to any vertex of the bond line structure, i.e., a specific ring carbon atom, including the following non-limiting examples of groups in which the substituents O-Su are connected to specific ring carbon atoms:

[0110] The term "site-specific" refers to the modification of a polypeptide at a predetermined sequence position in the polypeptide. The modification is at a single predictable residue of the polypeptide with little or no change. In certain embodiments, the modified amino acid is introduced at the sequence position, for example, by recombination or synthesis. Similarly, a group moiety can be "site-specifically" attached to a residue at a specific sequence position in a polypeptide. In certain embodiments, a polypeptide may contain more than one site-specific modification. Compounds represented by formula (I) and (III) and conjugates represented by formula (II) and (IV)

[0111] In one aspect, the compound is a compound represented by formula (I): or a pharmaceutically acceptable salt and / or regioisomer thereof; in, L 1 Selected from Ring A is an optionally substituted bridged, fused, or spiro bicyclic carbocycle, or an optionally substituted bridged, fused, or spiro bicyclic heterocycle, wherein the carbocycle or the heterocycle of Ring A is optionally substituted by one or more selected from alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl substituents; Ring B is an optionally substituted N-linked bridged, fused, or spiro bicyclic heterocycle, wherein Ring B is optionally substituted by one or more selected from alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl substituents; R a and R b are independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; a is an integer independently selected from 0, 1, 2, 3, 4, 5 and 6; b is an integer selected from 0 and 1; R 1 is H or alkyl, the alkyl being optionally substituted by one or more selected from cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aryl and heteroaryl substituents; R 2 and R 3 are each independently selected from H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; Y is *-C(O)-(CR a R b ) c -NH-, or *-C(O)-(CR a R b ) c -, where * indicates the position where Y is attached to RG; c is an integer selected from 1, 2, 3, 4, 5 and 6; RG is the reactive group; L 2 Does not exist, or L 2 is a linker comprising a hydrophilic polymer residue; L 3 Does not exist, or L 3 It is -C(O)-AA-, -C(O)-AA-Z-(CR a Rb ) a -Z-(CR a R b ) a -C(O)-, -C(O)-Z-(CR a R b ) a -C(O)-ZL 4 -OC(O)-, -Z-AA-, -AA-, -C(O)-, -C(O)-AA-Z-(CR a R b ) a -、-AA-C(O)-、-C(O)-(CR a R b ) a -Z-(CR a R b ) a -Z-AA-C(O)-, -C(O)OL 4 -ZC(O)-(CR a R b ) a -ZC(O)-, -AA-Z-, or -(CR a R b ) a -Z-AA-C(O)-; Z is selected from -NR 2 - and -O-; AA is an amino acid residue or a peptide residue; L 4 yes Where Su is the hexose form of a monosaccharide; d is an integer independently selected from 1, 2 and 3; D is a cytotoxic payload; and represents the point of attachment to the rest of the compound.

[0112] In certain embodiments, the compound of formula (I) is a compound of formula (IA): or a pharmaceutically acceptable salt and / or regioisomer thereof; where the integers c, RG, R a , R b , Ring B, L 2 , L 3 and D are as defined in the present invention.

[0113] In certain embodiments, the compound represented by formula (IA) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0114] In certain embodiments, the compound of formula (I) is a compound of formula (IB): or a pharmaceutically acceptable salt and / or regioisomer thereof; where the integers c, RG, R a , R b , Ring B, L 2 , L 3 and D are as defined in the present invention.

[0115] In certain embodiments, the compound represented by formula (IB) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0116] In certain embodiments, the compound of formula (I) is a compound of formula (IC): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring A, L 2 , L 3 and D are as defined in the present invention.

[0117] In certain embodiments, the compound represented by formula (IC) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0118] In certain embodiments, the compound of formula (I) is a compound of formula (ID): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring A, L 2 , L 3 and D are as defined in the present invention.

[0119] In certain embodiments, the compound represented by formula (ID) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0120] In certain embodiments, the compound of formula (I) is a compound of formula (IE): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring B, L 3 POLY 2 and D are as defined in the present invention.

[0121] In certain embodiments, the compound represented by formula (IE) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0122] In certain embodiments, the compound of formula (I) is a compound of formula (IF): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring B, L 3 POLY 2 and D are as defined in the present invention.

[0123] In certain embodiments, the compound represented by formula (IF) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0124] In certain embodiments, the compound of formula (I) is a compound of formula (IG): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring A, L 3 POLY 2 and D are as defined in the present invention.

[0125] In certain embodiments, the compound represented by formula (IG) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0126] In certain embodiments, the compound of formula (I) is a compound of formula (IH): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b , Ring A, L 3 POLY 2 and D are as defined in the present invention.

[0127] In certain embodiments, the compound represented by formula (IH) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0128] In one aspect, the present invention provides a conjugate represented by formula (II): or a pharmaceutically acceptable salt and / or regioisomer thereof; in, L 1 Selected from Ring A is an optionally substituted bridged, fused, or spiro bicyclic carbocycle, or an optionally substituted bridged, fused, or spiro bicyclic heterocycle, wherein the carbocycle or the heterocycle of Ring A is optionally substituted by one or more selected from alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents; Ring B is an optionally substituted N-linked bridged, fused, or spiro bicyclic heterocycle, wherein Ring B is optionally substituted by one or more selected from alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents; R a and R b are independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; a is an integer independently selected from 0, 1, 2, 3, 4, 5 and 6; R 1 is H or alkyl, the alkyl being optionally substituted by one or more selected from cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aryl and heteroaryl substituents; R 2 and R 3 are each independently selected from H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; Y is *-C(O)-(CR a R b ) c -NH-, or *-C(O)-(CR a R b ) c -, where * indicates the position where Y is attached to RL; c is an integer selected from 1, 2, 3, 4, 5 and 6; RL is the reactive residue; L 2 Does not exist, or L 2 is a linker comprising a hydrophilic polymer residue; L 3 Does not exist, or L 3 It is -C(O)-AA-, -C(O)-AA-Z-(CR a R b ) a -Z-(CR a R b ) a -C(O)-, -C(O)-Z-(CRa R b ) a -C(O)-ZL 4 -OC(O)-, -Z-AA-, -AA-, -C(O)-, -C(O)-AA-Z-(CR a R b ) a -、-AA-C(O)-、-C(O)-(CR a R b ) a -Z-(CR a R b ) a -Z-AA-C(O)-, -C(O)OL 4 -ZC(O)-(CR a R b ) a -ZC(O)-, -AA-Z-, or -(CR a R b ) a -Z-AA-C(O)-; Z is selected from -NR 2 - and -O-; AA is an amino acid residue or a peptide residue; L 4 yes Where Su is the hexose form of a monosaccharide; d is an integer independently selected from 1, 2 and 3; D is the cytotoxic payload; COMP is the residue of a second compound; and represents the point of attachment to the rest of the compound.

[0129] In certain embodiments, the compound represented by formula (II) is a compound represented by formula (IIA): or a pharmaceutically acceptable salt and / or regioisomer thereof; where the integers c, RL, and R a , R b , Ring B, L 2 , L 3 , D and COMP are as defined in the present invention.

[0130] In certain embodiments, the compound represented by formula (IIA) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0131] In certain embodiments, the compound of formula (II) is a compound of formula (IIB): or a pharmaceutically acceptable salt and / or regioisomer thereof; where the integers c, RL, and R a , R b , Ring B, L 2 , L 3 , D and COMP are as defined in the present invention.

[0132] In certain embodiments, the compound represented by formula (IIB) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0133] In certain embodiments, the compound of formula (II) is a compound of formula (IIC): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RL, R a , R b , Ring A, L 2 , L 3 , D and COMP are as defined in the present invention.

[0134] In certain embodiments, the compound represented by formula (IIC) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0135] In certain embodiments, the compound of formula (II) is a compound of formula (IID): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RL, R a , R b , Ring A, L 2 , L 3 , D and COMP are as defined in the present invention.

[0136] In certain embodiments, the compound represented by formula (IID) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0137] In certain embodiments, the compound of formula (II) is a compound of formula (IIE): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RL, R a , R b , Ring B, L 3 POLY 2 , D and COMP are as defined in the present invention.

[0138] In certain embodiments, the compound represented by formula (IIE) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0139] In certain embodiments, the compound of formula (II) is a compound of formula (IIF): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RL, R a , R b , Ring B, L 3 POLY 2 , D and COMP are as defined in the present invention.

[0140] In certain embodiments, the compound represented by formula (IIF) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0141] In certain embodiments, the compound of formula (II) is a compound of formula (IIG): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RL, R a , R b , Ring A, L 3 POLY 2 , D and COMP are as defined in the present invention.

[0142] In certain embodiments, the compound represented by formula (IIG) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0143] In certain embodiments, the compound of formula (II) is a compound of formula (IIH): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RL, R a , R b , Ring A, L 3 POLY 2 , D and COMP are as defined in the present invention.

[0144] In certain embodiments, the compound represented by formula (IIH) is selected from the following compounds: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0145] In one aspect, the present invention provides a conjugate represented by formula (III): or a pharmaceutically acceptable salt and / or regioisomer thereof; in, L 5 is a linker comprising an unnatural amino acid; and RG, Y, L 2 , L 3 and D are as defined in the present invention.

[0146] Non-limiting unnatural amino acids include cysteic acid, hydroxyproline (Hyp), β-alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3-nitrotyrosine, nitroarginine, pyroglutamate (Pyr), naphthylalanine (Nal), 2,4-diaminobutyric acid (DAB), methionine sulfoxide, and methionine sulfone.

[0147] In certain embodiments, the compound represented by formula (III) is a compound represented by formula (IIIA): or a pharmaceutically acceptable salt and / or regioisomer thereof; Among them, RG, Y, L 2 , L 3 and D are as defined in the present invention.

[0148] In certain embodiments, the compound of formula (III) is a compound of formula (IIIB): or a pharmaceutically acceptable salt and / or regioisomer thereof; where integer a, integer c, RG, R a , R b POLY 1 , AA and D are as defined in the present invention.

[0149] In certain embodiments, the compound represented by formula (IIIB) is selected from the compounds represented by the following formulae: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0150] In one aspect, the present invention provides a conjugate represented by formula (IV): or a pharmaceutically acceptable salt and / or regioisomer thereof; in, L 5 is a linker comprising an unnatural amino acid; and RL, COMP, Y, L 2 , L 3 and D are as defined in the present invention.

[0151] In certain embodiments, the compound of formula (IV) is a compound of formula (IVA): or a pharmaceutically acceptable salt and / or regioisomer thereof; Among them, RL, COMP, Y, L 2 , L 3 and D are as defined in the present invention.

[0152] In certain embodiments, the compound of formula (IV) is a compound of formula (IVB): or a pharmaceutically acceptable salt and / or regioisomer thereof; Among them, integer a, integer c, RL, COMP, R a , R b POLY 1 , AA and D are as defined in the present invention.

[0153] In certain embodiments, the compound represented by formula (IVB) is selected from the compounds represented by the following formulae: or a pharmaceutically acceptable salt and / or regioisomer thereof.

[0154] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes

[0155] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes

[0156] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring B is a 5 to 12-atom N-linked bridged, fused, or spiro bicyclic heterocyclic ring, comprising 1, 2, or 3 heteroatoms independently selected from N, O, and S, including the N atom to which the ring is attached. In certain embodiments of Formulae (I)-(IIH), including any of the foregoing embodiments, L 1 Ring B of is a 5 to 12-atom N-linked spiro bicyclic heterocyclic ring containing 1, 2 or 3 heteroatoms independently selected from N, O and S, including the N atom to which the ring is attached.

[0157] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring B is an optionally substituted 5 to 12-atom N-linked bridged, fused, or spiro bicyclic heterocyclic ring, comprising 1, 2 or 3 heteroatoms independently selected from N, O and S, including the N atom to which the ring is attached, wherein the heterocyclic ring of Ring B is optionally substituted by one or more selected from C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents. In certain embodiments of formula (I)-(IIH), including any of the foregoing embodiments, L 1 Ring B is an optionally substituted 5 to 12-atom N-attached spiro bicyclic heterocycle, the 5 to 12-atom N-attached spiro bicyclic heterocycle containing 1, 2 or 3 heteroatoms independently selected from N, O and S, including the N atom to which the ring is attached, wherein the heterocycle of Ring B is optionally substituted by one or more selected from C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents.

[0158] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring B is selected from wherein m is an integer selected from 1, 2, 3, 4 and 5; and n and o are each an integer independently selected from 1, 2 and 3.

[0159] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring B is In certain embodiments of Formula (I)-(IIH), Selected from In certain embodiments of Formula (I)-(IIH), Selected from In certain embodiments of Formula (I)-(IIH), L 1 Ring B is In certain embodiments, L 1 Ring B is

[0160] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring B is In certain embodiments of Formula (I)-(IIH), Selected from In certain embodiments of Formula (I)-(IIH), Selected from In certain embodiments of Formula (I)-(IIH), L 1 Ring B is In certain embodiments of Formula (I)-(IIH), L 1 Ring B is

[0161] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring B is In certain embodiments of Formula (I)-(IIH), yes wherein m is 1, 2, or 3. In certain embodiments of Formula (I)-(IIH), yes wherein m is 1, 2, or 3. In certain embodiments of Formula (I)-(IIH), yes wherein m is 1, 2, or 3. In certain embodiments of Formula (I)-(IIH), yes Wherein m is 1, 2, or 3.

[0162] In certain embodiments of Formula (I)-(IIH), yes In certain embodiments of Formula (I)-(IIH), yes In certain embodiments of Formula (I)-(IIH), yes In certain embodiments of Formula (I)-(IIH), yes In certain embodiments of Formula (I)-(IIH), yes

[0163] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring B is In certain embodiments of Formula (I)-(IIH), yes wherein m is 1, 2, or 3. In certain embodiments of Formula (I)-(IIH), yes wherein m is 1, 2, or 3. In certain embodiments of Formula (I)-(IIH), yes wherein m is 1, 2, or 3. In certain embodiments of Formula (I)-(IIH), yes Wherein m is 1, 2, or 3.

[0164] In certain embodiments of Formula (I)-(IIH), yes In certain embodiments of Formula (I)-(IIH), yes In certain embodiments of Formula (I)-(IIH), yes In certain embodiments, yes In certain embodiments of Formula (I)-(IIH), yes

[0165] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring B is

[0166] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring B is selected from

[0167] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring B is selected from

[0168] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Selected from wherein m is an integer selected from 1, 2, 3, 4 and 5; and n and o are each an integer independently selected from 1, 2 and 3.

[0169] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Selected from

[0170] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring B is selected from Where X 1 , X 2 , X 3 and X 4 are independently selected from -C(R 4 )2-, -NH-, -O-, and -S-, where X 1 , X 2 and X 3 When both exist, X 1 -X 3 At least one of them is -C(R 4 )2-, and when X 1 , X 2 , X 3 and X 4When both exist, X 1 -X 4 At least two of them are -C(R 4 )2-; and R 4 independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; or two R on the same carbon 4 The groups together form an oxo group. 4 Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl; or two R on the same carbon 4 The groups are taken together to form an oxo group.

[0171] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring B is selected from Where X 1 , X 2 , X 3 and X 4 are independently selected from -C(R 4 )2-, -NH-, -O-, and -S-, where X 1 , X 2 and X 3 When both exist, X 1 -X 3 At least one of them is -C(R 4 )2-, and when X 1 , X 2 , X 3 and X 4 When both exist, X 1 -X 4 At least two of them are -C(R4 )2-; and R 4 independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; or two R on the same carbon 4 The groups together form an oxo group. 4 Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl; or two R on the same carbon 4 The groups are taken together to form an oxo group.

[0172] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring A is a bridged, fused, or spiro bicyclic carbocyclic ring. In certain embodiments, including any of the foregoing embodiments, L 1 Ring A is C 4-12 In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, L 1 Ring A is C 4-12 In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, L 1 Ring A is C 4-8 Bridged bicyclic carbocycles.

[0173] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring A is an optionally substituted bridged, fused, or spiro bicyclic carbocyclic ring, wherein the carbocyclic ring of Ring A is optionally substituted by one or more selected from C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents. In certain embodiments, including any of the foregoing embodiments, L 1 Ring A is optionally substituted C 4-12 In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, L 1 Ring A is optionally substituted C 4-12 In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, L 1 Ring A is optionally substituted C 4-8 Bridged bicyclic carbocycles.

[0174] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring A is selected from Where X 1 , X 2 , X 3 and X 4 are independently selected from -C(R 4 )2-, -NH-, -O-, and -S-, where X 1 , X 2 and X 3 When both exist, X 1 -X 3 At least one of them is -C(R 4 )2-, and when X 1 , X 2 , X 3 and X 4 When both exist, X 1 -X 4 At least two of them are -C(R 4 )2-; X 5 Yes CR 4 or N; and R 4 independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; or two R on the same carbon 4 The groups together form an oxo group. 4 Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl; or two R on the same carbon 4 The groups are taken together to form an oxo group.

[0175] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring A is selected from

[0176] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring A is selected from

[0177] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring A is selected from

[0178] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring A is selected from

[0179] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring A is In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 Ring A is

[0180] In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, X 1 , X2 , X 3 , and / or X 4 Yes-C(R 4 In certain embodiments of Formula (I)-(IIH), including any of the foregoing embodiments, X 1 and X 2 -C(R 4 In certain embodiments of Formula (I)-(IIH), including any of the foregoing embodiments, X 1 , X 2 and X 3 -C(R 4 In certain embodiments of Formula (I)-(IIH), including any of the foregoing embodiments, X 1 , X 2 , X 3 and X 4 -C(R 4 In certain embodiments of Formula (I)-(IIH), including any of the foregoing embodiments, X 1 In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, X 2 In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, X 3 In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, X 4 In certain embodiments, including any of the foregoing embodiments, X 1 In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, X 2 In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, X 3 In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, X 4 Yes -O-.

[0181] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, L 1 yes In certain embodiments, L 1 yes

[0182] In certain embodiments, including any of the foregoing embodiments, a is 0. In certain embodiments, including any of the foregoing embodiments, a is 1. In certain embodiments, including any of the foregoing embodiments, a is 2. In certain embodiments, including any of the foregoing embodiments, a is 3. In certain embodiments, including any of the foregoing embodiments, a is 4. In certain embodiments, including any of the foregoing embodiments, a is 5. In certain embodiments, including any of the foregoing embodiments, a is 6.

[0183] In certain embodiments of formula (I)-(IIH), b is 0. In certain embodiments of formula (I)-(IIH), b is 1.

[0184] In certain embodiments of formula (I)-(IIH), b is 0, and a is 0. In certain embodiments of formula (I)-(IIH), b is 0, and a is 1. In certain embodiments of formula (I)-(IIH), b is 0, and a is 2. In certain embodiments of formula (I)-(IIH), b is 0, and a is 3. In certain embodiments of formula (I)-(IIH), b is 0, and a is 4. In certain embodiments of formula (I)-(IIH), b is 0, and a is 5. In certain embodiments of formula (I)-(IIH), b is 0, and a is 6. In certain embodiments of formula (I)-(IIH), b is 1, and a is 1. In certain embodiments of formula (I)-(IIH), b is 1, and a is 2. In certain embodiments of formula (I)-(IIH), b is 1, and a is 3. In certain embodiments of formula (I)-(IIH), b is 1, and a is 4. In certain embodiments of formula (I)-(IIH), b is 1, and a is 5. In certain embodiments of formula (I)-(IIH), b is 1, and a is 6.

[0185] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, R 1 is H. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R 1 is an unsubstituted alkyl group. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R 1 In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R 1 is an alkyl group optionally substituted with one or more substituents selected from cycloalkyl, halogen, alkoxy, -CN, -NO2 and -OH.

[0186] In certain embodiments of Formulas (III)-(IVB), L 5is a linker comprising at least one amino acid selected from cysteic acid, hydroxyproline (Hyp), β-alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3-nitrotyrosine, nitroarginine, pyroglutamic acid (Pyr), naphthylalanine (Nal), 2,4-diaminobutyric acid (DAB), methionine sulfoxide, and methionine sulfone. In certain embodiments of formula (III)-(IVB), L 5 is included In certain embodiments of Formulae (III)-(IVB), L 5 is included In certain embodiments of Formulae (III)-(IVB), L 5 yes In certain embodiments of Formulas (III)-(IVB), L 5 yes

[0187] In certain embodiments, including any of the foregoing embodiments, R a is H, and R b is selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl. In certain embodiments, including any of the foregoing embodiments, R a is H, and R b is selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, -C(O)OH, aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl. In certain embodiments, including any of the foregoing embodiments, R a is H, and R b is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH. In certain embodiments, including any of the foregoing embodiments, R a and R b Both are H.

[0188] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, R a It is H; R bR is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH; 1 is H; a is 1; and b is 1. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a It is H; R b is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH; R 1 is H; a is 2; and b is 1. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a It is H; R b R is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH; 1 is H; a is 3; and b is 1.

[0189] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, R a It is H; R b is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH; a is 1; and b is 0. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a It is H; R b is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH; a is 2; and b is 0. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a It is H; R b is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH; a is 3; and b is 0.

[0190] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, R a It is H; R b is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH; R 1 is methyl; a is 1; and b is 1. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a It is H; R bR is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH; 1 is methyl; a is 2; and b is 1. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a It is H; R b R is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH; 1 is methyl; a is 3; and b is 1.

[0191] In certain embodiments, including any of the foregoing formulae (I)-(IIH), R a and R b Both are H; R 1 is H; a is 1, and b is 1. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b Both are H; R 1 is H; a is 2, and b is 1. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b Both are H; R 1 is H; a is 3, and b is 1. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b Both are H; R 1 is H; a is 4, and b is 1. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b Both are H; R 1 is H; a is 5, and b is 1. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b Both are H; R 1 is H; a is 6, and b is 1.

[0192] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, R a and R b are H; a is 1; and b is 0. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b are H; a is 2; and b is 0. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and Rb are H; a is 3; and b is 0. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b are H; a is 4; and b is 0. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b are H; a is 5; and b is 0. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b are all H; a is 6; and b is 0.

[0193] In certain embodiments of Formulas (I)-(IIH), including any of the preceding embodiments, R a and R b Both are H; R 1 is methyl; a is 1; and b is 0. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b Both are H; R 1 is methyl; a is 2; and b is 0. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b Both are H; R 1 is methyl; a is 3; and b is 0. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b Both are H; R 1 is methyl; a is 4; and b is 0. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b Both are H; R 1 is methyl; a is 5; and b is 0. In certain embodiments of Formulas (I)-(IIH), including any of the foregoing embodiments, R a and R b Both are H; R 1 is methyl; a is 6; and b is 0.

[0194] In certain embodiments of Formulae (III)-(IVB), including any of the preceding embodiments, R a It is H; R b is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH; a is 1; and c is 1. In certain embodiments of Formulas (III)-(IVB), including any of the foregoing embodiments, Ra It is H; R b is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH; a is 2; and c is 1. In certain embodiments of Formulas (III)-(IVB), including any of the foregoing embodiments, R a It is H; R b is selected from the group consisting of H, alkyl, halogen, alkoxy, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)OH; a is 3; and c is 1.

[0195] In certain embodiments, including any of the preceding embodiments, Y is *-C(O)-(CR a R b ) c -NH-, wherein * represents the position at which Y is attached to RG in formulas (I)-(IH) and (III)-(IIIB) and RL in formulas (II)-(IIH) and (IV)-(IVB). In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2) c -NH-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)-NH-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)2-NH-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)3-NH-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)4-NH-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)5-NH-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)6-NH-.

[0196] In certain embodiments, including any of the preceding embodiments, Y is *-C(O)-(CR a R b )-NH-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CR a R b )2-NH-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CR a R b )3-NH-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CR a R b In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CR a Rb )5-NH-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CR a R b )6-NH-.

[0197] In certain embodiments, including any of the preceding embodiments, Y is *-C(O)-(CR a R b ) c -NH-, where R a is H, and R b is selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CR a R b )2-NH-, *-C(O)-(CR a R b )3-NH-, or *-C(O)-(CR a R b )4-NH-, where R a is H, and R b is selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl.

[0198] In certain embodiments, including any of the preceding embodiments, Y is *-C(O)-(CR a R b ) c -, wherein * represents the position at which Y is attached to RG in formulas (I)-(IH) and (III)-(IIIB) and RL in formulas (II)-(IIH) and (IV)-(IVB). In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2) c-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)2-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)3-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)4-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)5-. In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)6-.

[0199] In certain embodiments, including any of the preceding embodiments, Y is *-C(O)-(CR a R b In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CR a R b In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CR a R b In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CR a R b In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CR a R b In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CR a R b )6-.

[0200] In certain embodiments, including any of the preceding embodiments, Y is *-C(O)-(CR a R b ) c -, where R a is H, and R b is selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CR a R b )2-, *-C(O)-(CR a Rb )3-, or *-C(O)-(CR a R b )4-, where R a is H, and R b is selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl.

[0201] In certain embodiments, including any of the foregoing embodiments, Y is *-C(O)-(CH2)2-NH- or *-C(O)-(CH2)4-.

[0202] In certain embodiments, including any of the foregoing embodiments, L 2 In certain embodiments, including any of the foregoing embodiments, L 2 is a linker comprising a hydrophilic polymer residue.

[0203] In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -. In certain embodiments, including any of the foregoing embodiments, L 2 Yes-CH2-POLY 1 -. In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -. In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)3-POLY 1 -. In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)4-POLY 1 -. In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)5-POLY 1 -. In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)6-POLY 1 -. In certain embodiments, including any of the foregoing embodiments, L 2 Yes-CR a R b-POLY 1 -. In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b )2-POLY 1 -. In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b )3-POLY 1 -. In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b )4-POLY 1 -.

[0204] In certain embodiments, including any of the foregoing embodiments, L 2 Yes-POLY 1 -.

[0205] In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -(CR a R b ) a -. In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -(CR a R b ) a -, wherein a is independently selected from 0, 1, 2, 3, 4, 5, or 6. In certain embodiments, including any of the foregoing embodiments, L 2 Yes - (CH2) a -POLY 1 -(CH2) a -, wherein a is independently selected from 0, 1, 2, 3, 4, 5, or 6. In certain embodiments, including any of the foregoing embodiments, L 2 Yes - (CH2) a -POLY 1 -(CR a R b ) a -, wherein a is independently selected from 0, 1, 2, 3, 4, 5, or 6. In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CRa R b ) a -POLY 1 -(CH2) a -, wherein a is independently selected from 0, 1, 2, 3, 4, 5, or 6.

[0206] In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of a non-peptide hydrophilic polymer. 1 is a divalent residue of polyethylene glycol (PEG), polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(enols), polyvinyl pyrrolidone, poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), polysaccharides, poly(α-hydroxy acids), polyvinyl alcohol, polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), polysarcosine, or a combination thereof. In certain embodiments, including any of the foregoing embodiments, POLY 1 It is POLY 1 It is a divalent residue of polyethylene glycol (PEG), polypropylene glycol (PPG), or a copolymer of ethylene glycol and propylene glycol.

[0207] In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of polyethylene glycol (PEG). In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of polypropylene glycol (PPG). In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of a copolymer of ethylene glycol and propylene glycol. In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of a poly(oxyethylated polyol). In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of a poly(enol). In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of polyvinylpyrrolidone. In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of poly(hydroxyalkylmethacrylamide). In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of poly(hydroxyalkyl methacrylate). In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of a polysaccharide. In certain embodiments, including any of the foregoing embodiments, POLY 1is a divalent residue of poly(α-hydroxy acid). In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of polyvinyl alcohol. In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of polyphosphazene. In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of a polyoxazoline (POZ). In certain embodiments, including any of the foregoing embodiments, POLY 1 is a divalent residue of poly(N-acryloylmorpholine). In certain embodiments, including any of the foregoing embodiments, POLY 1 It is a divalent residue of polysarcosine.

[0208] In certain embodiments, including any of the foregoing embodiments, POLY 1 yes Where R 5 is H or methyl, x is an integer from 1 to 100, inclusive, and represents the point of attachment to the rest of the compound or conjugate. In certain embodiments, including any of the foregoing embodiments, x is an integer between 1 and 25. In certain embodiments, including any of the foregoing embodiments, x is an integer between 5 and 15. In certain embodiments, including any of the foregoing embodiments, x is 1. In certain embodiments, including any of the foregoing embodiments, x is 2. In certain embodiments, including any of the foregoing embodiments, x is 3. In certain embodiments, including any of the foregoing embodiments, x is 4. In certain embodiments, including any of the foregoing embodiments, x is 5. In certain embodiments, including any of the foregoing embodiments, x is 6. In certain embodiments, including any of the foregoing embodiments, x is 7. In certain embodiments, including any of the foregoing embodiments, x is 8. In certain embodiments, including any of the foregoing embodiments, x is 9. In certain embodiments, including any of the foregoing embodiments, x is 10. In certain embodiments, including any of the foregoing embodiments, x is 11. In certain embodiments, including any of the foregoing embodiments, x is 12. In certain embodiments, including any of the foregoing embodiments, x is 13. In certain embodiments, including any of the foregoing embodiments, x is 14. In certain embodiments, including any of the foregoing embodiments, x is 15. In certain embodiments, including any of the foregoing embodiments, x is 16. In certain embodiments, including any of the foregoing embodiments, x is 17. In certain embodiments, including any of the foregoing embodiments, x is 18. In certain embodiments, including any of the foregoing embodiments, x is 19. In certain embodiments, including any of the foregoing embodiments, x is 20. In certain embodiments, including any of the foregoing embodiments, x is an integer between 25 and 50. In certain embodiments, including any of the foregoing embodiments, x is an integer between 35 and 45. In certain embodiments, including any of the foregoing embodiments, x is an integer between 50 and 75. In certain embodiments, including any of the foregoing embodiments, x is an integer between 55 and 65. In certain embodiments, including any of the foregoing embodiments, x is an integer between 75 and 100. In certain embodiments, including any of the foregoing embodiments, x is an integer between 85 and 95. In certain embodiments, including any of the preceding embodiments, x is an integer ranging from 1 to 25, 20 to 45, 40 to 65, 60 to 85, 70 to 95, or 75 to 100.

[0209] In certain embodiments, including any of the foregoing embodiments, R 5 is H. In certain embodiments, including any of the foregoing embodiments, R 5 It's methyl.

[0210] In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b) a -POLY 1 -, where POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -, where POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes - (CH2) a -POLY 1 -, where POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -, where POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -, where POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -, where POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -, where POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -, where POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -, where POLY 1 yes

[0211] In certain embodiments, including any of the foregoing embodiments, L2 yes In certain embodiments, including any of the foregoing embodiments, L 2 yes In certain embodiments, including any of the foregoing embodiments, L 2 yes In certain embodiments, including any of the foregoing embodiments, L 2 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -(CR a R b ) a -, where POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -(CR a R b ) a -, where POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -(CR a R b ) a -, where POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes – (CR a R b ) a -POLY 1 -(CR a R b ) a -, where POLY 1 yes

[0212] In certain embodiments, including any of the foregoing embodiments, L 2 Selected from In certain embodiments, including any of the foregoing embodiments, L 2 yes In certain embodiments, including any of the foregoing embodiments, L 2 yes

[0213] In certain embodiments, including any of the foregoing embodiments, L 2 yes In certain embodiments, including any of the foregoing embodiments, L 2 yes In certain embodiments, including any of the foregoing embodiments, L 2 yes In certain embodiments, including any of the foregoing embodiments, L 2 yes In certain embodiments, including any of the foregoing embodiments, L 2 yes

[0214] In certain embodiments, including any of the foregoing embodiments, POLY 2 is the residue of a non-peptide hydrophilic polymer. 2 is a residue of polyethylene glycol (PEG), methoxypolyethylene glycol (mPEG), polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(enols), polyvinyl pyrrolidone, poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), polysaccharides, poly(α-hydroxy acids), polyvinyl alcohol, polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), polysarcosine, or a combination thereof. In certain embodiments, including any of the foregoing embodiments, POLY 2 is a residue of polyethylene glycol (PEG), methoxypolyethylene glycol (mPEG), polypropylene glycol (PPG), or a copolymer of ethylene glycol and propylene glycol. In certain embodiments, including any of the foregoing embodiments, POLY 2 is the residue of methoxypolyethylene glycol (mPEG).

[0215] In certain embodiments, including any of the foregoing embodiments, POLY 2 is a residue of polyethylene glycol (PEG). In certain embodiments, including any of the foregoing embodiments, POLY 2 is a residue of polypropylene glycol (PPG). In certain embodiments, including any of the foregoing embodiments, POLY 2 is the residue of a copolymer of ethylene glycol and propylene glycol. In certain embodiments, including any of the foregoing embodiments, POLY 2 is the residue of a poly(oxyethylated polyol). In certain embodiments, including any of the foregoing embodiments, POLY 2is the residue of a poly(enol). In certain embodiments, including any of the foregoing embodiments, POLY 2 is the residue of polyvinylpyrrolidone. In certain embodiments, including any of the foregoing embodiments, POLY 2 is the residue of poly(hydroxyalkylmethacrylamide). In certain embodiments, including any of the foregoing embodiments, POLY 2 is the residue of poly(hydroxyalkyl methacrylate). In certain embodiments, including any of the foregoing embodiments, POLY 2 is a polysaccharide residue. In certain embodiments, including any of the foregoing embodiments, POLY 2 is a residue of poly(α-hydroxy acid). In certain embodiments, including any of the foregoing embodiments, POLY 2 is the residue of polyvinyl alcohol. In certain embodiments, including any of the foregoing embodiments, POLY 2 is the residue of a polyphosphazene. In certain embodiments, including any of the foregoing embodiments, POLY 2 is a polyoxazoline (POZ) residue. In certain embodiments, including any of the foregoing embodiments, POLY 2 is the residue of poly(N-acryloylmorpholine). In certain embodiments, including any of the foregoing embodiments, POLY 2 It is a polysarcosine residue.

[0216] In certain embodiments, including any of the foregoing embodiments, POLY 2 yes Where R 5 is H or methyl, x is an integer from 1 to 100, inclusive, and represents the point of attachment to the rest of the compound or conjugate. In certain embodiments, including any of the foregoing embodiments, x is an integer between 1 and 25. In certain embodiments, including any of the foregoing embodiments, x is an integer between 5 and 15. In certain embodiments, including any of the foregoing embodiments, x is 1. In certain embodiments, including any of the foregoing embodiments, x is 2. In certain embodiments, including any of the foregoing embodiments, x is 3. In certain embodiments, including any of the foregoing embodiments, x is 4. In certain embodiments, including any of the foregoing embodiments, x is 5. In certain embodiments, including any of the foregoing embodiments, x is 6. In certain embodiments, including any of the foregoing embodiments, x is 7. In certain embodiments, including any of the foregoing embodiments, x is 8. In certain embodiments, including any of the foregoing embodiments, x is 9. In certain embodiments, including any of the foregoing embodiments, x is 10. In certain embodiments, including any of the foregoing embodiments, x is 11. In certain embodiments, including any of the foregoing embodiments, x is 12. In certain embodiments, including any of the foregoing embodiments, x is 13. In certain embodiments, including any of the foregoing embodiments, x is 14. In certain embodiments, including any of the foregoing embodiments, x is 15. In certain embodiments, including any of the foregoing embodiments, x is 16. In certain embodiments, including any of the foregoing embodiments, x is 17. In certain embodiments, including any of the foregoing embodiments, x is 18. In certain embodiments, including any of the foregoing embodiments, x is 19. In certain embodiments, including any of the foregoing embodiments, x is 20. In certain embodiments, including any of the foregoing embodiments, x is an integer between 25 and 50. In certain embodiments, including any of the foregoing embodiments, x is an integer between 35 and 45. In certain embodiments, including any of the foregoing embodiments, x is an integer between 50 and 75. In certain embodiments, including any of the foregoing embodiments, x is an integer between 55 and 65. In certain embodiments, including any of the foregoing embodiments, x is an integer between 75 and 100. In certain embodiments, including any of the foregoing embodiments, x is an integer between 85 and 95. In certain embodiments, including any of the preceding embodiments, x is an integer ranging from 1 to 25, 20 to 45, 40 to 65, 60 to 85, 70 to 95, or 75 to 100.

[0217] In certain embodiments, including any of the foregoing embodiments, R 5 is H. In certain embodiments, including any of the foregoing embodiments, R 5 It's methyl.

[0218] In certain embodiments, including any of the foregoing embodiments, L 2 Selected from the group consisting of:

[0219] In certain embodiments, including any of the foregoing embodiments, L 2 Selected from the group consisting of:

[0220] In certain embodiments, including any of the foregoing embodiments, L 2 yes In certain embodiments, including any of the foregoing embodiments, L 2 yes

[0221] In certain embodiments, including any of the foregoing embodiments, L 2 yes In certain embodiments, including any of the foregoing embodiments, L 2 yes

[0222] In certain embodiments, including any of the foregoing embodiments, L 3 It is -C(O)-AA-.

[0223] In certain embodiments, including any of the foregoing embodiments, L 3 Yes-C(O)-AA-Z-(CR a R b ) a -Z-(CR a R b ) a -C(O)-. In certain embodiments, including any of the foregoing embodiments, L 3 Yes-C(O)-AA-NR 2 -(CR a R b ) a -NR 2 -(CR a R b ) a -C(O)-. In certain embodiments, including any of the foregoing embodiments, L 3 Yes -C(O)-AA-NH-(CR a R b ) a -NH-(CR a R b ) a -C(O)-. In certain embodiments, including any of the foregoing embodiments, L 3 Yes -C(O)-AA-NH-(CH2) a -NH-(CH2) a-C(O)-. In certain embodiments, including any of the foregoing embodiments, L 3 Yes -C(O)-AA-NH-(CH2) a -NH-(CH2) a -C(O)-, wherein a is selected from 1, 2, and 3. In certain embodiments, including any of the foregoing embodiments, L 3 It is -C(O)-AA-NH-CH2-NH-CH2-C(O)-.

[0224] In certain embodiments, including any of the foregoing embodiments, L 3 Yes-C(O)-AA-Z-(CR a R b ) a In certain embodiments, including any of the foregoing embodiments, L 3 Yes-C(O)-AA-NR 2 -(CH2) a In certain embodiments, including any of the foregoing embodiments, L 3 It is -C(O)-AA-NH-(CH2)2.

[0225] In certain embodiments, including any of the foregoing embodiments, L 3 In certain embodiments, including any of the foregoing embodiments, L 3 yes

[0226] In certain embodiments, including any of the foregoing embodiments, -AA- is an amino acid residue. In certain embodiments, including any of the foregoing embodiments, -AA- is a peptide residue. In certain embodiments, including any of the foregoing embodiments, -AA- is a dipeptide residue, a tripeptide residue, a tetrapeptide residue, or a pentapeptide residue. In certain embodiments, including any of the foregoing embodiments, -AA- comprises at least one amino acid residue selected from alanine, glycine, valine, and asparagine. In certain embodiments, including any of the foregoing embodiments, -AA- comprises at least one amino acid residue selected from alanine and glycine. In certain embodiments, including any of the foregoing embodiments, -AA- is selected from the group consisting of:

[0227] In certain embodiments, including any of the foregoing embodiments, -AA- is selected from the group consisting of:

[0228] In certain embodiments, including any of the foregoing embodiments, L 3 It is -C(O)-.

[0229] In certain embodiments, including any of the foregoing embodiments, L 3 Yes-C(O)-Z-(CR a R b ) a -C(O)-ZL 4 -OC(O)-, where L 4 yes Su is a hexose form of a monosaccharide, and d is an integer independently selected from 1, 2, and 3. In certain embodiments, including any of the foregoing embodiments, L 3 Yes-C(O)-NR 2 -(CR a R b ) a -C(O)-NR 2 -L 4 -OC(O)-. In certain embodiments, including any of the foregoing embodiments, L 3 Yes-C(O)-NR 2 -(CH2)2-C(O)-NR 2 -L 4 -OC(O)-. In certain embodiments, including any of the foregoing embodiments, L 3 It is -C(O)-NH-(CR a R b ) a -C(O)-NH-L 4 -OC(O)-. In certain embodiments, including any of the foregoing embodiments, L 3 Yes -C(O)-NH-(CH2)2-C(O)-NH-L 4 -OC(O)-.

[0230] In certain embodiments, including any of the foregoing embodiments, L 4 yes In certain embodiments, including any of the foregoing embodiments, L 4 yes In certain embodiments, including any of the foregoing embodiments, L 4 yes

[0231] In some embodiments, including any of the foregoing embodiments, Su is a sugar moiety. In some embodiments, Su is a hexose form of a monosaccharide. Su can be a glucuronic acid or mannose residue. In certain embodiments, including any of the foregoing embodiments, Su is in In certain embodiments, including any of the foregoing embodiments, Su is in represents the point of attachment to the rest of the compound.

[0232] In certain embodiments, including any of the foregoing embodiments, L 4 yes

[0233] In certain embodiments, including any of the foregoing embodiments, L 3 Yes -C(O)-NH-(CH2)2-C(O)-NH-L 4 -OC(O)-, where L 4 yes In certain embodiments, including any of the foregoing embodiments, L 3 Yes -C(O)-NH-(CH2)2-C(O)-NH-L 4 -OC(O)-, where L 4 yes

[0234] In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -, and L 3 In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -;L 3 is -C(O)-AA-; and POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes - (CH2) a -POLY 1 -;L 3 is-C(O)-AA-; POLY 1 yes and x is an integer between 10 and 15. In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -;L 3 is -C(O)-AA-; and POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -;L 3is-C(O)-AA-; POLY 1 yes and AA is a dipeptide residue, a tripeptide residue, a tetrapeptide residue, or a pentapeptide residue.

[0235] In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -, and L 3 Yes-C(O)-AA-Z-(CR a R b ) a -Z-(CR a R b ) a -C(O)-. In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -;L 3 Yes-C(O)-AA-Z-(CR a R b ) a -Z-(CR a R b ) a -C(O)-;POLY 1 yes and Z is -NH-. In certain embodiments, including any of the foregoing embodiments, L 2 Yes - (CH2) a -POLY 1 -;L 3 Yes -C(O)-AA-NH-(CH2) a -NH-(CH2) a -C(O)-;POLY 1 yes and x is an integer between 10 and 15. In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -;L 3 is -C(O)-AA-NH-CH2-NH-CH2-C(O)-; and POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -;L 3Yes -C(O)-AA-NH-CH2-NH-CH2-C(O)-; POLY 1 yes and AA is a dipeptide residue, a tripeptide residue, a tetrapeptide residue, or a pentapeptide residue.

[0236] In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -, and L 3 is -C(O). In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -;L 3 is -C(O); and POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes - (CH2) a -POLY 1 -;L 3 Yes -C(O); POLY 1 yes and x is an integer between 10 and 15. In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -;L 3 is -C(O); and POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -;L 3 Yes -C(O); POLY 1 yes and AA is a dipeptide residue, a tripeptide residue, a tetrapeptide residue, or a pentapeptide residue.

[0237] In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -, and L 3 In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a-POLY 1 -;L 3 does not exist; and POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes - (CH2) a -POLY 1 -;L 3 Does not exist; POLY 1 yes and x is an integer between 10 and 15. In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -;L 3 does not exist; and POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -;L 3 Does not exist; POLY 1 yes and AA is a dipeptide residue, a tripeptide residue, a tetrapeptide residue, or a pentapeptide residue.

[0238] In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -, and L 3 Yes-C(O)-AA-Z-(CR a R b ) a -. In certain embodiments, including any of the foregoing embodiments, L 2 Yes-(CR a R b ) a -POLY 1 -;L 3 Yes-C(O)-AA-Z-(CR a R b ) a -; and POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes - (CH2) a -POLY 1 -;L 3 Yes-C(O)-AA-NR 2 -(CR a R b )a - POLY 1 yes and x is an integer between 10 and 15. In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -;L 3 Yes -C(O)-AA-NH-(CR a R b ) a -; and POLY 1 yes In certain embodiments, including any of the foregoing embodiments, L 2 Yes -(CH2)2-POLY 1 -;L 3 Yes -C(O)-AA-NH-(CR a R b ) a - POLY 1 yes and AA is a dipeptide residue, a tripeptide residue, a tetrapeptide residue, or a pentapeptide residue.

[0239] -L 2 -L 3 Non-limiting examples of - include:

[0240] -L 2 -L 3 Additional non-limiting examples of - include:

[0241] In certain embodiments, including any of the foregoing embodiments, L 2 yes and L 3 Yes-C(O)-Z-(CR a R b ) a -C(O)-ZL 4 -OC(O)-, where L 4 yes Su is a hexose form of a monosaccharide, and d is an integer independently selected from 1, 2, and 3. In certain embodiments, including any of the foregoing embodiments, L 2 yes and L 3 Yes-C(O)-Z-(CR a R b ) a-C(O)-ZL 4 -OC(O)-.

[0242] In certain embodiments, including any of the foregoing embodiments, L 2 yes and L 3 In certain embodiments, including any of the foregoing embodiments, L 2 yes and L 3 Does not exist.

[0243] In certain embodiments, including any of the foregoing embodiments, L 2 Selected from the group consisting of: and L 3 It is -C(O)-NH-(CR a R b ) a -C(O)-NH-L 4 -OC(O)-.

[0244] In certain embodiments, including any of the foregoing embodiments, L 2 Selected from the group consisting of: and L 3 It is -C(O)-NH-(CR a R b ) a -C(O)-NH-L 4 -OC(O)-.

[0245] In certain embodiments, including any of the foregoing embodiments, L 2 Selected from the group consisting of: and L 3 Yes -C(O)-NH-(CH2)2-C(O)-NH-L 4 -OC(O)-, where L 4 yes

[0246] In certain embodiments, including any of the foregoing embodiments, L 2 Selected from the group consisting of: and L 3 It is -C(O).

[0247] -L2 -L 3 Non-limiting examples of - include:

[0248] In-L 2 -L 3 In any embodiment of -, D can be selected from

[0249] -L 2 -L 3 Non-limiting examples of -D include:

[0250] In certain embodiments of Formula (I)-(IH), yes In certain embodiments of Formula (I)-(IH), yes In certain embodiments of Formula (I)-(IH), yes In certain embodiments of Formula (I)-(IH), yes

[0251] In certain embodiments of Formula (I)-(IH), yes Where R a and R b In certain embodiments of Formulas (I)-(IH), yes Where R a and R b H, and c is 2. In certain embodiments of Formula (I)-(IH), yes Where R a and R b are H, c is 2, and RG is

[0252] In certain embodiments of Formula (I)-(IH), yes Where R a , R b and R 1 In certain embodiments of Formulas (I)-(IH), yes Where R a , R b and R 1 are H, respectively; c is 2; and a is 1. In certain embodiments of Formulas (I)-(IH), yes Where R a , R b and R 1 are H; c is 2; a is 1; and RG is

[0253] In certain embodiments of Formula (I)-(IH), yes Where R a and R b In certain embodiments of Formulas (I)-(IH), yes Where R a and R b H, and c is 4, respectively. In certain embodiments of Formula (I)-(IH), yes Where R a and R b are H, c is 4, and RG is

[0254] In certain embodiments of Formula (I)-(IH), yes Where R a and R b In certain embodiments of Formulas (I)-(IH), yes Where R a and R b H, and c is 4, respectively. In certain embodiments of Formula (I)-(IH), yes Where R a and R b are H, c is 4, and RG is

[0255] Non-limiting examples include:

[0256] In certain embodiments of Formula (III), (IIIA) or (IIIB), yes In certain embodiments of Formula (III), (IIIA) or (IIIB), yes In certain embodiments of Formula (III), (IIIA) or (IIIB), yes In certain embodiments of Formula (III), (IIIA) or (IIIB), yes In certain embodiments of Formula (III), (IIIA) or (IIIB), yes

[0257] In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG is a group comprising an alkyne, a cyclooctyne, a strained olefin, a tetrazine, an amine, a methylcyclopropene, a thiol, a p-acetylphenylalanine residue, an oxyamine, a maleimide, or an azide. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG comprises an alkyne. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG comprises a cyclooctyne. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG comprises a strained olefin. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG comprises a tetrazine. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG comprises an amine. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG comprises a methylcyclopropene. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG comprises a thiol. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG comprises an acetylphenylalanine residue. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG comprises an oxamine. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG comprises a maleimide. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG comprises an azide. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG is selected from the group consisting of: –N3, –NH2, methylcyclopropene, and –SH; where R T It is C 1-6 Alkyl; and In certain embodiments of Formula (I)-(IH) or (III)-(IIIB), RG is and In certain embodiments of Formula (I)-(IH) or (III)-(IIIB), RG is and In certain embodiments of Formula (I)-(IH) or (III)-(IIIB), RG is In certain embodiments of Formula (I)-(IH) or (III)-(IIIB), RG is In certain embodiments of Formula (I)-(IH) or (III)-(IIIB), RG is In certain embodiments of Formula (I)-(IH) or (III)-(IIIB), RG is Where R T It is C 1-6 Alkyl, and In certain embodiments, R T is methyl, ethyl, or propyl. In certain embodiments, R T In certain embodiments, R T In certain embodiments, R T In certain embodiments, R T In certain embodiments, R T In certain embodiments, R T In certain embodiments of Formula (I)-(IH) or (III)-(IIIB), RG is In certain embodiments of Formula (I)-(IH) or (III)-(IIIB), RG is In certain embodiments of Formula (I)-(IH) or (III)-(IIIB), RG is In certain embodiments of Formula (I)-(IH) or (III)-(IIIB), RG is In certain embodiments of Formula (I)-(IH) or (III)-(IIIB), RG is In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG is -N3. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG is -NH2. In certain embodiments of formula (I)-(IIIB), RG is methylcyclopropene. In certain embodiments of formula (I)-(IH) or (III)-(IIIB), RG is -SH.

[0258] In certain embodiments of Formula (II)-(IIH), yes In certain embodiments of Formula (II)-(IIH), yes In certain embodiments of Formula (II)-(IIH), yes In certain embodiments of Formula (II)-(IIH), yes

[0259] In certain embodiments of Formula (II)-(IIH), yes Where R a and R b In certain embodiments of Formula (II)-(IIH), yes Where R a and R b H, and c is 2. In certain embodiments of Formula (II)-(IIH), yes Where R a and R b are H, c is 2, and RL is

[0260] In certain embodiments of Formula (II)-(IIH), yes Where R a , R b and R 1 In certain embodiments of Formula (II)-(IIH), yes Where R a , R b and R 1 are H, respectively; c is 2; and a is 1. In certain embodiments of Formulas (II)-(IIH), yes Where R a , R band R 1 are H; c is 2; a is 1; and RL is

[0261] In certain embodiments of Formula (II)-(IIH), yes Where R a and R b In certain embodiments of Formula (II)-(IIH), yes Where R a and R b H, and c is 4, respectively. In certain embodiments of Formula (II)-(IIH), yes Where R a and R b are H, c is 4, and RL is

[0262] In certain embodiments of Formula (II)-(IIH), yes Where R a and R b They are H respectively.

[0263] In certain embodiments of Formula (II)-(IIH), yes Where R a and R b H, and c is 4, respectively. In certain embodiments of Formula (II)-(IIH), yes Where R a and R b are H, c is 4, and RL is

[0264] In certain embodiments of Formulae (IV)-(IVB), yes In certain embodiments of Formulae (IV)-(IVB), yes In certain embodiments of Formulae (IV)-(IVB), yes In certain embodiments of Formulae (IV)-(IVB), yes In certain embodiments of Formulae (IV)-(IVB), yes

[0265] In certain embodiments of formula (II)-(IIH), (IVA) or (IVB), RL is a group comprising a triazole, a pyridazine, a thiol or an oxime. In certain embodiments of formula (II)-(IIH), (IV), (IVA) or (IVB), RL is a group comprising a triazole. In certain embodiments of formula (II)-(IIH), (IV), (IVA) or (IVB), RL is a group comprising a pyridazine. In certain embodiments of formula (II)-(IIH), (IV), (IVA) or (IVB), RL is a group comprising a thiol. In certain embodiments of formula (II)-(IIH), (IV), (IVA) or (IVB), RL is a group comprising an oxime.

[0266] In certain embodiments of Formula (II)-(IIH), (IV), (IVA), or (IVB), RL is selected from the group consisting of: In certain embodiments of Formula (II)-(IIH), (IV), (IVA) or (IVB), RL is In certain embodiments of Formula (II)-(IIH), (IV), (IVA) or (IVB), RL is In certain embodiments of Formula (II)-(IIH), (IV), (IVA) or (IVB), RL is In certain embodiments of Formula (II)-(IIH), (IV), (IVA) or (IVB), RL is In certain embodiments of Formula (II)-(IIH), (IV), (IVA) or (IVB), RL is represents the point of attachment to the rest of the compound.

[0267] In certain embodiments, including any of the foregoing embodiments, D is a cytotoxic payload selected from a tubulin inhibitor, a DNA topoisomerase I inhibitor, and a DNA topoisomerase II inhibitor, or a derivative thereof. In certain embodiments, including any of the foregoing embodiments, D is a tubulin inhibitor or a derivative thereof. In certain embodiments, including any of the foregoing embodiments, D is a DNA topoisomerase I inhibitor or a derivative thereof. In certain embodiments, including any of the foregoing embodiments, D is a DNA topoisomerase I inhibitor selected from the group consisting of: irinotecan, SN-38, topotecan, exatecan; or a derivative thereof. In certain embodiments, including any of the foregoing embodiments, D is irinotecan or a derivative thereof. In certain embodiments, including any of the foregoing embodiments, D is SN-38 or a derivative thereof. In certain embodiments, including any of the foregoing embodiments, D is topotecan or a derivative thereof. In certain embodiments, including any of the foregoing embodiments, D is exatecan or a derivative thereof. In certain embodiments, including any of the foregoing embodiments, D is a DNA topoisomerase II inhibitor or a derivative thereof. In certain embodiments, including any of the foregoing embodiments, D is a DNA topoisomerase II inhibitor selected from the group consisting of: etoposide, teniposide, and tafluposide; or a derivative thereof. In certain embodiments, including any of the foregoing embodiments, D is etoposide or a derivative thereof. In certain embodiments, including any of the foregoing embodiments, D is teniposide or a derivative thereof. In certain embodiments, including any of the foregoing embodiments, D is tafluposide or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a payload selected from the group consisting of: hemiasterlins, camptothecins, and anthracyclines; or a derivative thereof. Anthracyclines may include PNU-159682 and EDA PNU-159682 derivatives. In some embodiments, including any of the foregoing embodiments, D is an anthracycline selected from the group consisting of: daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin; or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is daunorubicin or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is doxorubicin or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is epirubicin or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is idarubicin or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is mitoxantrone or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is valrubicin or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is hemicycline or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is camptothecin or a derivative thereof.In some embodiments, including any of the foregoing embodiments, D is an anthracycline or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is PNU-159682 or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is an EDA PNU compound or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is an EDA PNU-159682 derivative. In some embodiments, including any of the foregoing embodiments, D is hemicycline, exitecan, PNU-159682, or an EDA PNU-159682 derivative. In some embodiments, including any of the foregoing embodiments, D is hemicycline or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is exitecan or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is PNU-159682 or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is an EDA PNU-159682 compound or derivative. In some embodiments, including any of the foregoing embodiments, D is not an immunostimulatory compound.

[0268] In some embodiments, including any of the foregoing embodiments, D is an alkylating agent or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a bifunctional alkylating agent or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a bifunctional alkylating agent selected from the group consisting of cyclophosphamide, mechlorethamine, chlorambucil, and melphalan; or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is cyclophosphamide or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is mechlorethamine or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is chlorambucil or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is melphalan or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a monofunctional alkylating agent or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a monofunctional alkylating agent selected from the group consisting of dacarbazine, nitrosourea, and temozolomide; or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is dacarbazine or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a nitrosourea or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is temozolomide or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a cytoskeletal disruptor (e.g., a taxane) or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a cytoskeletal disruptor selected from the group consisting of paclitaxel, docetaxel, albumin-bound paclitaxel (abraxane), and taxotere; or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is paclitaxel or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is docetaxel or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is albumin-bound paclitaxel (abraxane) or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is taxotere or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is epothilone or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is an epothilone selected from the group consisting of: epothilone A, epothilone B, epothilone C, epothilone D, and ixabepilone; or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is epothilone A or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is epothilone B or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is epothilone C or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is epothilone D or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is ixabepilone or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a histone deacetylase inhibitor or a derivative thereof.In some embodiments, including any of the preceding embodiments, D is a histone deacetylase inhibitor selected from the group consisting of: vorinostat and romidepsin; or a derivative thereof. In some embodiments, including any of the preceding embodiments, D is vorinostat or a derivative thereof. In some embodiments, including any of the preceding embodiments, D is romidepsin or a derivative thereof. In some embodiments, including any of the preceding embodiments, D is a kinase inhibitor or a derivative thereof. In some embodiments, including any of the preceding embodiments, D is a kinase inhibitor selected from the group consisting of: bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vemurafenib; or a derivative thereof. In some embodiments, including any of the preceding embodiments, D is bortezomib or a derivative thereof. In some embodiments, including any of the preceding embodiments, D is erlotinib or a derivative thereof. In some embodiments, including any of the preceding embodiments, D is gefitinib or a derivative thereof. In some embodiments, including any of the preceding embodiments, D is imatinib or a derivative thereof. In some embodiments, including any of the preceding embodiments, D is vemurafenib or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is vismodegib or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a nucleotide analog and / or precursor analog or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a nucleotide analog and / or precursor analog selected from the group consisting of azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine (formerly known as mercaptoguanine); or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is azacitidine or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is azathioprine or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is capecitabine or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is cytarabine or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is doxifluridine or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is fluorouracil or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is gemcitabine or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is hydroxyurea or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is mercaptopurine or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is methotrexate or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is thioguanine (formerly known as mercaptoguanine) or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a peptide antibiotic or a derivative thereof.In some embodiments, including any of the foregoing embodiments, D is a peptide antibiotic selected from the group consisting of: bleomycin and actinomycin; or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is bleomycin or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is actinomycin or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a platinum-based payload or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a platinum-based payload selected from the group consisting of: carboplatin, cisplatin, and oxaliplatin; or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is carboplatin or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is cisplatin or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is oxaliplatin or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a retinoid or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a retinoid selected from the group consisting of: tretinoin, alitretinoin, and bexarotene; or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is tretinoin or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is alitretinoin or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is bexarotene or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a vinca alkaloid or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is a vinca alkaloid or a derivative thereof selected from the group consisting of vinblastine, vincristine, vindesine, and vinorelbine. In some embodiments, including any of the foregoing embodiments, D is vinblastine or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is vincristine or a derivative thereof. In some embodiments, including any of the foregoing embodiments, D is vindesine or a derivative thereof.

[0269] In some embodiments, including any of the preceding embodiments, D is selected from or its derivatives.

[0270] In some embodiments, including any of the preceding embodiments, D is selected from or its derivatives.

[0271] Non-limiting examples of compounds of the present invention include:

[0272] Representative compounds of the present invention are shown in Table A. Table A

[0273] Non-limiting examples of conjugates of the present invention include:

[0274] Representative conjugates of the present invention are shown in Table B. Table B Optically active compounds

[0275] In certain embodiments, compound provided by the invention can have several chiral centers, and can exist and be separated in optically active form and racemic form. In certain embodiments, some compounds can show polymorphism. It will be appreciated by those skilled in the art that compound provided by the invention can exist in any racemic form, optical active form, diastereoisomer, polymorph, regioisomer and / or stereoisomer form and / or its mixture.

[0276] Those skilled in the art will also recognize that the compounds of the present invention having the useful properties of the present invention are within the scope of the present invention. Those skilled in the art will also recognize how to prepare optically active forms of the compounds of the present invention, for example, by resolving racemic forms by recrystallization techniques, by synthesizing from optically active starting materials, by chiral synthesis, or by chromatographic separation using chiral stationary phases. In addition, most amino acids are chiral (i.e., designated as L or D, wherein the L enantiomer is the naturally occurring configuration) and can exist as separate enantiomers.

[0277] Examples of methods for obtaining optically active substances are all known in the art and include at least the following: i) Physical separation of crystals - a technique by which macroscopic crystals of individual enantiomers are separated manually. This technique can be used if crystals of the individual enantiomers exist (ie the material is an aggregate and the crystals are visually distinct). ii) simultaneous crystallization - a technique by which the individual enantiomers are crystallized separately from a solution of the racemate, provided that the latter is an aggregate in the solid state; iii) Enzymatic resolution - a technique that partially or completely separates racemates by taking advantage of the different reaction rates of enantiomers in the presence of enzymes; iv) Enzymatic asymmetric synthesis - a synthetic technique that uses an enantiomerically pure or enriched synthetic precursor of the desired enantiomer by at least one step of the synthesis; v) Chemical asymmetric synthesis - a synthetic technique in which the desired enantiomer is synthesized from achiral precursors under conditions that result in asymmetry (i.e., chirality) of the product, which can be achieved using chiral catalysts or chiral auxiliaries; vi) Diastereoisomer separation - a technique in which a racemic compound is reacted with an enantiomerically pure reagent (chiral auxiliary) to convert the individual enantiomers into diastereomers. The resulting diastereomers are then separated by chromatography or crystallization, by virtue of their now more pronounced diastereomeric differences, and the chiral auxiliary is subsequently removed to obtain the individual enantiomers; vii) primary and secondary asymmetric transformations - techniques whereby the diastereomers of a racemate are allowed to equilibrate in solution to produce a predominance of a diastereomer over the desired enantiomer in solution, or a diastereomer preferentially crystallizes over the desired enantiomer to disturb the equilibrium so that ultimately in principle all of the material is converted from the desired enantiomer to the crystalline diastereomer. The desired enantiomer is then derived from the diastereomer; viii) Kinetic resolution - the technique is the partial or complete resolution of a racemate (or further resolution of a partially resolved compound) due to the unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions; ix) Stereospecific synthesis from non-racemic precursors - synthetic techniques by which the desired enantiomer is obtained from chiral starting materials and wherein the stereochemical integrity is not or only minimally compromised during the synthetic process; x) Chiral liquid chromatography - a technique that allows the enantiomers of a racemate to be separated in a liquid mobile phase by virtue of their different interactions with the stationary phase. The stationary phase may be made of a chiral material, or the mobile phase may contain additional chiral materials to induce different interactions; xi) Chiral gas chromatography – a technique whereby the racemate is volatilized and the enantiomers are separated by their differential interaction in a gaseous mobile phase with a chromatographic column containing a stationary non-racemic adsorbent phase; xii) Chiral solvent extraction - a technique that allows the separation of enantiomers by kinetically or thermodynamically dissolving one enantiomer in a specific chiral solvent; xiii) Transport across chiral membranes - a technique where the racemate is brought into contact with a thin membrane barrier. The barrier typically separates two miscible fluids, one of which contains the racemate, and a driving force such as concentration or pressure differential allows preferential transport across the membrane barrier. The separation occurs due to the non-racemic chiral nature of the membrane, which allows only one enantiomer of the racemate to pass.

[0278] In some embodiments, the present invention provides a composition of the compounds of the present invention, comprising compounds of formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (III), (IIIA), and (IIIB) and conjugates of formula (II), (IIA), (IIB), (IIC), (IID), (IIE), (IIF), (IIG), (IIH), (IV), (IVA), and (IVB), wherein the compounds or conjugates are substantially free of a specified stereoisomer of the compound or conjugate. In certain embodiments, in the methods, compounds, and conjugates of the present invention, the compounds or conjugates are substantially free of other stereoisomers. In some embodiments, the composition comprises at least 85%, 90%, 95%, 98%, or 99% to 100% by weight of the compound or conjugate, with the remainder comprising other chemical substances or enantiomers. In some embodiments, the present invention provides a composition of a compound of formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (III), (IIIA) and (IIIB) and a conjugate of formula (II), (IIA), (IIB), (IIC), (IID), (IIE), (IIF), (IIG), (IIH), (IV), (IVA) and (IVB), wherein the compound or conjugate is substantially free of a specified enantiomer of the compound or conjugate. In certain embodiments, in the methods, compounds and conjugates of the present invention, the compound or conjugate is substantially free of other enantiomers. In some embodiments, the composition comprises at least 85%, 90%, 95%, 98%, or 99% to 100% by weight of the compound or conjugate, with the remainder comprising other chemical substances or enantiomers. Isotopically enriched compounds

[0279] The present invention also provides isotopically enriched compounds and conjugates, including but not limited to isotopically enriched compounds of formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (III), (IIIA) and (IIIB) and conjugates of formula (II), (IIA), (IIB), (IIC), (IID), (IIE), (IIF), (IIG), (IIH), (IV), (IVA) and (IVB).

[0280] It has been previously demonstrated with certain classes of drugs that isotopic enrichment (eg, deuteration) of drugs can improve pharmacokinetic ("PK"), pharmacodynamic ("PD"), and / or toxicity profiles. See, for example, Lijinsky et.al., Food Cosmet. Toxicol., 20:393 (1982); Lijinsky et.al., J. Nat. Cancer Inst., 69:1127 (1982); Mangold et.al., Mutation Res. 308:33 (1994); Gordon et.al., Drug Metab. Dispos., 15:589 (1987); Zello et.al., Metabolism, 43:487 (1994); Gately et.al., J. Nucl. Med., 27:388 (1986); Wade D, Chem. Biol. Interact. 117:191 (1999).

[0281] Isotopic enrichment of a drug can be used, for example, to (1) reduce or eliminate unwanted metabolites, (2) increase the half-life of the parent drug, (3) reduce the number of doses required to achieve a desired effect, (4) reduce the amount of dose necessary to achieve a desired effect, (5) increase the formation of active metabolites (if any are formed), and / or (6) reduce the production of deleterious metabolites in specific tissues. Isotopic enrichment of a drug can also be used to prepare drugs for more effective and / or safer combination therapies, whether the combination therapy is intentional or unintentional.

[0282] Replacing an atom with one of its isotopes usually causes a change in the reaction rate of a chemical reaction. This phenomenon is called the kinetic isotope effect ("KIE"). For example, if a C-H bond breaks during the rate-determining step (i.e., the step with the highest transition state energy) in a chemical reaction, then replacing this active hydrogen with a (heavier) isotope will result in a decrease in the reaction rate. The deuterium kinetic isotope effect ("DKIE") is the most common form of KIE. (See, e.g., Foster et al., Adv. Drug Res., vol. 14, pp. 1-36 (1985); Kushner et al., Can. J. Physiol. Pharmacol., vol. 77, pp. 79-88 (1999)).

[0283] The magnitude of the DKIE can be expressed as the ratio between the rates of a given reaction in which a C—H bond is broken and the same reaction in which deuterium replaces hydrogen and the C—D bond is broken. The DKIE can range from about 1 (no isotope effect) to very large values, such as 50 or more, meaning that the reaction can be 50 or more times slower when deuterium replaces hydrogen.

[0284] Substitution of hydrogen with tritium ("T") produces a stronger bond than deuterium and a numerically larger isotope effect. Similarly, isotope substitution of other elements, including but not limited to, 13 C or 14 C replaces carbon, 33 S. 34 S, or 36 S replaces sulfur, 15 N replaces nitrogen, and 17 O or 18 O can replace oxygen and produce similar kinetic isotope effects.

[0285] Animals express various enzymes to remove foreign substances, such as therapeutic agents, from their circulatory systems. Examples of such enzymes include cytochrome P450 enzymes ("CYPs"), esterases, proteases, reductases, dehydrogenases, and monoamine oxidases to react with and convert these foreign substances into more polar intermediates or metabolites for renal excretion. Some of the most common metabolic reactions of drug compounds involve oxidation of carbon-hydrogen (CH) bonds to carbon-oxygen (CO) or carbon-carbon (C=C) π bonds. The resulting metabolites may be stable or unstable under physiological conditions and may have significantly different PK / PD and acute and long-term toxicity characteristics relative to the parent compound. For many drugs, such oxidation reactions are rapid. Therefore, these drugs typically require multiple or high-dose daily dosing.

[0286] Therefore, isotopic enrichment at certain positions of the compounds provided herein will produce a detectable KIE compared to similar compounds with natural isotopic compositions, which will affect the pharmacological, PK, PD, and / or toxicological characteristics of the compounds provided herein. Conjugates represented by formula (II), (IIA), (IIB), (IIC), (IID), (IIE), (IIF), (IIG), (IIH), (III), (IIIA) and (IIIB)

[0287] The present invention provides a conjugate of a macromolecule and one of the compounds shown in formula (I)-(IH), (III), (IIIA) and (IIIB) of the present invention. The conjugate is directly or indirectly covalently linked via a linker. In certain embodiments, the conjugate comprises a macromolecule coupled to one or more compounds shown in formula (I)-(IH), (III), (IIIA) and (IIIB) of the present invention. In certain embodiments, the conjugate comprises more than one macromolecule. In certain embodiments, the macromolecule is connected to 1, 2, 3, 4, 5, 6, 7, 8 or more compounds shown in formula (I)-(IH), (III), (IIIA) and (IIIB).

[0288] The linker can be any linker capable of forming at least one bond with the macromolecule and forming at least one bond with the compound represented by formula (I)-(IH), (III), (IIIA) and (IIIB). Useful linkers are described in the present invention section and in the examples, in particular the linkers described below.

[0289] Macromolecule can be any macromolecule that a person skilled in the art finds suitable. In certain embodiments, the macromolecule is a second compound. In certain embodiments, COMP is a residue of the second compound. In certain embodiments, the macromolecule is a protein, a peptide, an antibody or its antigen-binding fragment, a nucleic acid, a carbohydrate or other macromolecules composed of polymerized monomers. In certain embodiments, the macromolecule is a peptide with two or more residues. In certain embodiments, the macromolecule is a peptide with 10 or more residues. In certain embodiments, the mass of the macromolecule is at least 1000Da. In certain embodiments, the macromolecule comprises at least 1000 atoms. Useful macromolecules are described in the following sections. Macromolecules (COMP)

[0290] Macromolecule (COMP) can be any macromolecule that one skilled in the art considers suitable. In certain embodiments, the macromolecule is a protein, a peptide, an antibody or its antigen-binding fragment, a nucleic acid, a carbohydrate, or other macromolecules composed of polymerized monomers. In certain embodiments, the macromolecule is a protein. In certain embodiments, the macromolecule is an antibody or its antigen-binding fragment. In some embodiments, COMP is a residue of a polypeptide. In some embodiments, COMP is a residue of an antibody. In some embodiments, COMP is a residue of an antibody chain.

[0291] In some embodiments, the macromolecule is an antibody or an antigen-binding fragment thereof. In some embodiments, the macromolecule is a known antibody. Useful antibodies include, but are not limited to, rituximab ( IDEC / Genentech / Roche) (see, e.g., U.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody approved for the treatment of non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently being developed by Genmab, an anti-CD20 antibody described in U.S. Pat. No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 (PCT Application No. PCT / US2003 / 040426), trastuzumab ( Genentech (see, e.g., U.S. Pat. No. 5,677,171), a humanized anti-Her2 / neu antibody approved for the treatment of breast cancer; Pertuzumab (rhuMab-2C4, ), currently under development by Genentech; anti-Her2 antibody (U.S. Patent No. 4,753,894); cetuximab ( Imclone) (U.S. Pat. No. 4,943,533; PCT Publication No. WO 96 / 40210), a chimeric anti-EGFR antibody in clinical trials for a variety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883), currently being developed by Abgenix-Immunex-Amgen; HuMax-EGFR (U.S. Pat. No. 7,247,301), currently being developed by Genmab; 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864; Murthy, et al. (1987) Arch. Biochem. Biophys. 252(2):549-60; Rodeck, et al. (1987) J. Cell. Biochem. 35(4):315-20; Kettleborough, et al. (1991) Protein Eng.4(7):773-83); ICR62 (Institute of Cancer Research) (PCT Publication No. WO 95 / 20045; Modjtahedi, et al. (1993) J. Cell. Biophys. 22 (I-3): 129-46; Modjtahedi, et al. (1993) Br.J.Cancer 67(2):247-53; Modjtahedi, et al. (1996) Br. J. Cancer 73(2):228-35; Modjtahedi, et al. (2003) Int. J. Cancer 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada; and Centrode Immunologia Molecular, Cuba) (U.S. Patent No. 5,891,996; U.S. Patent No. 6,506,883; Mateo, et al. (1997) Immunotechnol. 3 (1): 71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth, et al. (2003) Proc. Natl. Acad. Sci. USA.100(2):639-44)); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT Publication No. WO 01 / 62931A2); and SC100 (Scancell) (PCT Publication No. WO 01 / 88138); alemtuzumab (. Millenium), a humanized mAb currently approved for the treatment of B-cell chronic lymphocytic leukemia; muromonab CD3 (Orthoclone ), an anti-CD3 antibody developed by OrthoBiotech / Johnson & Johnson; Ibritumomab It is an anti-CD20 antibody developed by IDEC / Schering AG; gemtuzumab ozogamicin It is an anti-CD33 (P67 protein) antibody developed by Celltech / Wyeth; Afacept Anti-LFA-3 Fc fusion protein developed by Biogen; Abciximab Developed by Centocor / Eli Lilly; basiliximab Developed by Novartis; Palivizumab Developed by Medimmune; infliximab An anti-TNFα antibody developed by Centocor; adalimumab Anti-TNFα antibody developed by Abbott; Anti-TNFα antibody developed by Celltech; golimumab (CNTO-148), a fully human TNF antibody developed by Centocor; etanercept p75 TNF receptor Fc fusion protein being developed by Immunex / Amgen; Ienercept, a p55 TNF receptor Fc fusion protein previously developed by Roche; ABX-CBL, an anti-CD147 antibody being developed by Abgenix; ABX-IL8, an anti-IL8 antibody being developed by Abgenix; ABX-MA1, an anti-MUC18 antibody being developed by Abgenix; Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 antibody being developed by Antisoma; Therex (R1550), an anti-MUC1 antibody being developed by Antisoma; AngioMab (AS1405), being developed by Antisoma; HuBC-1, being developed by Antisoma; Thioplatin (AS1407), being developed by Antisoma; (natalizumab), an anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody being developed by Biogen; VLA-1 mAb, an anti-VLA-1 integrin antibody being developed by Biogen; LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody being developed by Biogen; CAT-152, an anti-TGF-beta antibody being developed by Cambridge Antibody Technology; ABT 874 (J695), an anti-IL-12p40 antibody being developed by Abbott; CAT-192, an anti-TGFβ1 antibody being developed by Cambridge Antibody Technology and Genzyme; CAT-213, an anti-Eotaxin1 antibody being developed by Cambridge Antibody Technology; Anti-Blys antibody being developed by Cambridge Antibody Technology and Human Genome Sciences Inc.; TRAIL-R1 mAb, anti-TRAIL-R1 antibody being developed by Cambridge Antibody Technology and Human GenomeSciences Inc.; Bevacizumab, rhuMAb-VEGF, an anti-VEGF antibody being developed by Genentech; an anti-HER receptor family antibody being developed by Genentech; anti-tissue factor (ATF), an anti-tissue factor antibody being developed by Genentech; (omalizumab), an anti-IgE antibody being developed by Genentech; (efalizumab), an anti-CD11a antibody being developed by Genentech and Xoma; the MLN-02 antibody (formerly known as LDP-02), being developed by Genentech and Millenium Pharmaceuticals; HuMax CD4, an anti-CD4 antibody being developed by Genmab; HuMax-IL15, an anti-IL15 antibody being developed by Genmab and Amgen; HuMax-Inflam, being developed by Genmab and Medarex; HuMax-Cancer, an anti-heparanase I antibody being developed by Genmab, Medarex and Oxford GcoSciences; HuMax-Lymphoma, being developed by Genmab and Amgen; HuMax-TAC, being developed by Genmab; IDEC-131, an anti-CD40L antibody being developed by IDEC Pharmaceuticals; IDEC-151 (cleliximab), an anti-CD40L antibody being developed by IDEC Pharmaceuticals; and an anti-CD4 antibody being developed by IDEC Pharmaceuticals; IDEC-114, an anti-CD80 antibody being developed by IDEC Pharmaceuticals; IDEC-152, an anti-CD23 antibody being developed by IDEC Pharmaceuticals; an anti-macrophage migration factor (MIF) antibody being developed by IDEC Pharmaceuticals; BEC2, an anti-idiotypic antibody being developed by Imclone; IMC-1C11, an anti-KDR antibody being developed by Imclone; DC101, an anti-flk-1 antibody being developed by Imclone; an anti-VE-cadherin antibody being developed by Imclone; (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibody being developed by Immunomedics; (epratuzumab), an anti-CD22 antibody being developed by Immunomedics; AFP-Cide being developed by Immunomedics; MyelomaCide being developed by Immunomedics; LkoCide being developed by Immunomedics; ProstaCide being developed by Immunomedics; MDX-010, an anti-CTLA4 antibody being developed by Medarex; MDX-060, an anti-CD30 antibody being developed by Medarex; MDX-070 being developed by Medarex; MDX-018 being developed by Medarex; and Immuno-Designed Molecules being developed by Medarex and Medarex. (IDM-1) and anti-Her2 antibody; -CD4, which is an anti-CD4 antibody being developed by Medarex and Genmab; HuMax-IL15, which is an anti-IL15 antibody being developed by Medarex and Genmab; CNTO 148, which is an anti-TNFα antibody being developed by Medarex and Centocor / J&J; CNTO 1275, which is an anti-cytokine antibody being developed by Centocor / J&J; MOR101 and MOR102, which are anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies being developed by MorphoSys; MOR201, which is an anti-fibroblast growth factor receptor 3 (FGFR-3) antibody being developed by MorphoSys; (Visilizumab), an anti-CD3 antibody being developed by Protein Design Labs; which is an anti-gamma interferon antibody being developed by Protein Design Labs; anti-alpha5beta1 interferon antibody being developed by Protein Design Labs; anti-IL-12 being developed by Protein Design Labs; ING-1, which is an anti-Ep-CAM antibody being developed by Xoma; (omalizumab), a humanized anti-IgE antibody being developed by Genentech and Novartis; and MLN01, a humanized anti-β2 integrin antibody being developed by Xoma.

[0292] In another embodiment, the therapeutic agent includes KRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for Cancer Research); CNTO 95 (αV integrin, Centocor); MEDI-522 (αVβ3 integrin, Medimmune); volociximab (αVβ1 integrin, Biogen / PDL); human mAb 216 (B cell glycosylation antigen epitope, NCL); BiTE MT103 (bispecific CD19×CD3, MedImmune); 4G7×H22 (bispecific B cell×FcγR1, Medarex / Merck KGa); rM28 (bispecific CD28×MAPG, European Patent No. EP1444268); MDX447 (EMD 82633) (bispecific CD64 × EGFR, Medarex); catumaxomab (removab) (bispecific EpCAM × anti-CD3, Trion / Fres); ertuinomab (bispecific HER2 / CD3, Fresenius Biotech); oregovomab (OvaRex) (CA-125, ViRexx); (WX G250) (Carbonic Anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507) (CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituxan (CD20, Genentech); veltuzumab (hA20) (CD20, Immunomedics); Epratuzumab (CD22, Amgen); Ruximab (IDEC 152)(CD23, Biogen); muromonab-CD3(CD3, Ortho); HuM291(CD3 fc receptor, PDL Biopharma); HeFi-1(CD30, NCl); MDX-060(CD30, Medarex); MDX-1401(CD30, Medarex); SGN-30(CD30, Seattle Genentics); SGN-33(lintuzumab)(CD33, Seattle Genentics); zanolimumab (HUMAX-CD4) (CD4, Genmab); HCD122 (CD40, Novartis); SGN-40 (CD40, Seattle Genentics); MabCampath (alemtuzumab) (CD52, Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38, Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293 (TRC093 / D93) (cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma); ipilimumab (MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab (Ticilimumab, CP-675,2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab) (DR4TRAIL-R1 agonist, Human Genome Science / Glaxo Smith Kline); AMG-655 (DR5, Amgen);Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-ETR2 (lexalimumab) (DR5TRAIL-R2 agonist, HGS); Cetuximab (Erbitux) (EGFR, Imclone); IMC-11F8, (EGFR, Imclone); Nimotuzumab (EGFR, YM Bio); Panitumumab (Vectabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANT Immunotherapeutics; adecatumumab (MT201) (Epcam, Merck); edrecolomab (Panorex, 17-1A) (Epcam, Glaxo / Centocor); MORAb-003 (folate receptor alpha, Morphotech); KW-2871 (ganglioside GD3, Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); trastuzumab (Herceptin) (HER2, Celldex); pertuzumab (rhuMAb 2C4) (HER2 (DI), Genentech); apolizumab (HLA-DR β chain, PDL Pharma); AMG-479 (IGF-1R, Amgen); anti-IGF-1R R1507 (IGF1-R, Roche); CP751871 (IGF1-R, Pfizer); IMC-A12 (IGF1-R, Imclone); BIIB022 (IGF-1R, Biogen); MIK-β-1 (IL-2Rb (CD122), Hoffman LaRoche); CNTO 328 (IL6, Centocor); anti-KIR (1-7F9) (Killer cell Ig-like receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute for Cancer Research); hCBE-11 (LTβR, Biogen); HuHMFG1 (MUC1, Antisoma / NCL); RAV12 (N-linked carbohydrate epitope, Raven); CAL (parathyroid hormone-related protein (PTH-RP), University of California); CT-011 (PD1, CureTech); MDX-1106 (ONO-4538) (PD1, Medarex / Ono); MAb CT-011 (PD1, Curetech);IMC-3G3 (PDGFRa, Imclone); bavituximab (phosphatidylserine, Peregrine); huJ591 (PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research Foundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); infliximab (Remicade) (TNFa, Centocor); A27.15 (transferrin receptor, Salk Institute, INSERN WO 2005 / 111082); E2.3 (transferrin receptor, Salk Institute); bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab, PCT Publication No. WO / 2000 / 034337, University of Texas); IMC-18F1 (VEGFR1, Imclone); IMC-1121 (VEGFR2, Imclone). ;

[0293] Examples of useful bispecific antibodies include, but are not limited to, those having one antibody directed against a tumor cell antigen and another directed against a cytotoxic triggering molecule, such as anti-FcγRI / anti-CD15, anti-p185 HER2 / FcγRIII(CD16), anti-CD3 / anti-malignant B cells(1D10), anti-CD3 / anti-p185 HER2, anti-CD3 / anti-p97, anti-CD3 / anti-renal cell carcinoma, anti-CD3 / anti-OVCAR-3, anti-CD3 / L-D1 (anti-colon cancer), anti-CD3 / anti-melanocyte stimulating hormone analog, anti-EGF receptor / anti-CD3, anti-CD3 / anti-CAMA1, anti-CD3 / anti-CD19, anti-CD3 / MoV18, anti-neural cell adhesion molecule (NCAM) / anti-CD3, anti-folate binding protein (FBP) / anti-CD3, anti-pan-cancer associated antigen (AMOC-31) / anti-CD3; Bispecific antibodies with one antibody that specifically binds to a tumor antigen and another antibody that binds to a toxin, such as anti-saporin / anti-Id-1, anti-CD22 / anti-saporin, anti-CD7 / anti-saporin, anti-CD38 / anti-saporin, anti-CEA / anti-ricin A chain, anti-interferon alpha (IFN-α) / anti-hybridoma idiotype, anti-CEA / anti-vinca alkaloid; bispecific antibodies for converting enzyme-activated prodrugs, such as anti-CD30 / anti-alkaline phosphatase (which converts mitomycin phosphate to phosphate) prodrug catalytic conversion into mitomycin alcohol); bispecific antibodies that can be used as fibrinolytic agents, such as anti-fibrinogen / anti-tissue plasminogen activator (tPA), anti-fibrinogen / anti-urokinase-type plasminogen activator (uPA); immune complex bispecific antibodies for targeting cell surface receptors, such as anti-low-density lipoprotein (LDL) / anti-Fc receptor (e.g., FcγRI, FcγRII or FcγRIII); bispecific antibodies for treating infectious diseases, such as anti-CD3 / anti-herpes simplex virus (HSV), anti-T cell receptor:CD3 complex / anti-influenza, anti-FcγR / anti-HIV; bispecific antibodies for in vitro or in vivo tumor detection, such as anti-CEA / anti-EOTUBE, anti-CEA / anti-DPTA, anti-p185 HER2 / anti-half antibody; bispecific antibodies as vaccine adjuvants (see Fanger, MW et al., Crit Rev Immunol. 1992; 12 (34): 101-24, incorporated herein by reference); and bispecific antibodies as diagnostic tools, such as anti-rabbit IgG / anti-ferritin, anti-horseradish peroxidase (HRP) / anti-hormone, anti-somatostatin / anti-substance P, anti-HRP / anti-FITC, anti-CEA / anti-β-galactosidase (see Nolan, O et R. O'Kennedy, Biochim Biophys Acta. 1990 Aug. 1; 1040 (1): 1-11, incorporated herein by reference). Examples of trispecific antibodies include anti-CD3 / anti-CD4 / anti-CD37, anti-CD3 / anti-CD5 / anti-CD37, and anti-CD3 / anti-CD8 / anti-CD37. Coupling

[0294] In certain embodiments, the conjugate may be formed by a macromolecule comprising one or more active (reactive) groups. In certain embodiments, the conjugate may be formed by a macromolecule comprising all naturally encoded amino acids. Those skilled in the art will recognize that several naturally encoded amino acids include active groups that can be coupled to compounds or connectors shown in formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (III), (IIIA) and (IIIB). These active groups include cysteine ​​side chains, lysine side chains, and amino terminal groups. In these embodiments, the conjugate may include compounds or connectors shown in formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (III), (IIIA) and (IIIB) connected to antibody active group residues. In these embodiments, the compound precursor or linker precursor shown in formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (III), (IIIA) and (IIIB) comprises an active group capable of forming a bond with an antibody or antigen-binding fragment thereof active group. Typical active groups include maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl esters), activated esters (including but not limited to, N-hydroxysuccinimide, p-nitrophenyl esters and aldehydes). Particularly useful active groups include maleimide and succinimide, such as N-hydroxysuccinimide, for forming bonds with cysteine ​​and lysine side chains. Further active groups will be described in the following sections and examples. Active Group

[0295] The reactive group can promote the coupling of the compound of formula (I)-(IH), (III), (IIIA) or (IIIB) described in the present invention with a second compound (e.g., a macromolecule described in the present invention (i.e., COMP)) to form a conjugate of formula (II)-(IIH), (III), (IIIA) or (IIIB) described in the present invention. In certain embodiments, the reactive group is designated as RG in the present invention. The reactive group can react by any suitable reaction mechanism known to those skilled in the art. In certain embodiments, the reactive group (RG) reacts by a [3+2] alkyne-azide cycloaddition reaction, a reverse electron demand Diels-Alder ligation reaction, a thiol-electrophile reaction, or a carbonyl-oxamine reaction, as described in detail herein. In certain embodiments, the reactive group (RG) comprises an alkyne, a strained alkyne, a tetrazine, a thiol, a para-acetylphenylalanine residue, an oxamine, a maleimide, or an azide. In certain embodiments, the reactive group is –N3, or –SH; where R T In certain embodiments, R T is methyl, ethyl or propyl. In some embodiments, R T In some embodiments, R T In some embodiments, R T Other active groups are described in, for example, U.S. Patent Application Publication No. US2014 / 0356385, U.S. Patent Application Publication No. US2013 / 0189287, U.S. Patent Application Publication No. US2013 / 0251783, U.S. Patent No. US8,703,936, U.S. Patent No. US9,145,361, U.S. Patent No. US9,222,940 and U.S. Patent No. US8,431,558.

[0296] After coupling, a divalent residue of the active group (referred to as RL in the present invention) is formed and connected to the residue of the second compound (eg, COMP). The structure of the divalent residue is determined by the type of coupling reaction used to form the conjugate.

[0297] [3+2] Alkyne-Azide Cycloaddition

[0298] Advantageously, the compounds of the present invention comprising a coupled alkyne or azide group are conducive to selective and efficient reaction with a second compound comprising a complementary azide or alkyne group. It is believed that the azide group and the alkyne group react in a 1,3-dipolar cycloaddition reaction to form a 1,2,3-triazolyl radical moiety, which connects the compounds of the present invention comprising an alkyne or azide group to the second compound. This reaction between the azide group and the alkyne group to form a triazole is generally referred to by those skilled in the art as a Huisgen cycloaddition reaction or a [3+2] alkyne-azide cycloaddition reaction.

[0299] The unique reactivity of azide and alkyne functional groups makes them useful for selective modification of peptides and other biomolecules. Organic azides (particularly aliphatic azides) and alkynes are generally stable to common reactive chemical conditions. In particular, both azide and alkyne functional groups are inert to the side chains of the 20 common amino acids found in naturally occurring peptides. It is believed that when azide and alkyne groups are brought into close proximity, their "spring-loaded" nature is revealed, and the azide and alkyne groups react selectively and efficiently via a [3+2] alkyne-azide cycloaddition reaction to generate the corresponding triazoles. See, e.g., Chin J., et al., Science 301:964-7 (2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin, JW, et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

[0300] Since the [3+2] alkyne-azide cycloaddition reaction involves a selective cycloaddition reaction [see, e.g., Padwa, A., in COMPREHENSIVE ORGANIC SYNTHESIS, Vol. 4, (ed. Trost, BM, 1991), pp. 1069-1109; Huisgen, R. in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), pp. 1-176] rather than a nucleophilic substitution reaction, the introduction of non-naturally encoded amino acids with azide and alkyne-containing side chains allows the resulting polypeptide to be selectively modified at the position of the non-naturally encoded amino acid. Cycloaddition reactions involving azide or alkyne-containing compounds can be carried out at room temperature under aqueous conditions by adding Cu(II) (including but not limited to a catalytic amount of CuSO4) and in the presence of a reducing agent to reduce Cu(II) in situ to Cu(I) in a catalytic amount. See, e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe, CW, et al., J. Org. Chem. 67: 3057-3064 (2002); Rostovtsev, et al., Angew. Chem. Int. Ed. 41: 2596-2599 (2002). Exemplary reducing agents include, but are not limited to, ascorbic acid, metallic copper, quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe 2+ 、Co 2+ , and the applied potential.

[0301] In certain embodiments, when the conjugate is formed by a [3+2] alkyne-azide cycloaddition reaction, the divalent residue of the reactive group (e.g., RL) comprises a triazole ring or a fused cyclic group containing a triazole ring. In certain embodiments, when the conjugate is formed by a strain-promoted [3+2] alkyne-azide cycloaddition (SPAAC) reaction, the divalent residue of the reactive group (e.g., RL) is

[0302] If the conjugate of formula (II)-(IIH), (IV), (IVA) or (IVB) is formed by [3+2] alkyne-azide cycloaddition, the conjugate comprises two regioisomers. In certain embodiments, the conjugate of formula (II)-(IIH), (IV), (IVA) or (IVB) is a mixture of regioisomers formed by [3+2] alkyne-azide cycloaddition.

[0303] Reverse electron demand ligation

[0304] Advantageously, compounds comprising a terminal tetrazine or strained olefin group can promote selective and efficient reaction with a second compound comprising a strained olefin or tetrazine group. It is believed that the tetrazine and strained olefin react in a reverse electron demand Diels-Alder reaction followed by a reverse Diels-Alder reaction, which connects the compound comprising a terminal tetrazine or strained olefin group to the second compound. It is believed that the reaction is specific and has little cross-reactivity with functional groups in biomolecules. The reaction can be carried out under mild conditions, such as at room temperature without a catalyst. This reaction between a tetrazine and a strained olefin is generally referred to as a tetrazine ligation reaction by those skilled in the art.

[0305] In certain embodiments, when the conjugate is formed by tetrazine reverse electron demand Diels-Alder ligation reaction, the divalent residue of the active group (e.g., RL) comprises a fused bicyclic ring having at least two adjacent nitrogen atoms in the ring. In certain embodiments, when the conjugate is formed by tetrazine reverse electron demand Diels-Alder ligation reaction, the divalent residue of the active group (e.g., RL) is

[0306] If the conjugate shown in formula (II)-(IIH), (IV), (IVA) or (IVB) is formed by a reverse electron demand ligation reaction, the conjugate comprises two regioisomers. In certain embodiments, the conjugate shown in formula (II)-(IIH), (IV), (IVA) or (IVB) is a mixture of regioisomers formed by a reverse electron demand ligation reaction.

[0307] Thiol reaction

[0308] Advantageously, compounds containing a terminal thiol group or a suitable electrophilic group or disulfide forming group are favorable for selective and efficient reaction with a second compound containing a complementary electrophilic group or disulfide forming group or thiol group. It is believed that these reactions are selective with little cross-reactivity with functional groups within biomolecules. In some embodiments, the thiol reaction does not include the reaction of the maleimide group.

[0309] In certain embodiments, when the conjugate is formed by a thiol-maleimide reaction, the divalent residue of the reactive group comprises In certain embodiments, when the conjugate of formula (II)-(IIH), (IV), (IVA) or (IVB) is formed by a thiol-maleimide reaction, The divalent residue of the active group (e.g., RL) is In other embodiments, when the conjugate of formula (II)-(IIH), (IV), (IVA) or (IVB) is formed by a thiol-maleimide reaction, The divalent residue of the active group (e.g., RL) is

[0310] Carbonyl-oxyamine reaction

[0311] Advantageously, compounds containing a terminal carbonyl or oxamine group are favorable for selective and efficient reaction with a second compound containing an oxamine or carbonyl group. It is believed that the carbonyl group reacts with the oxamine to form an oxime bond. It is believed that the reaction is specific with little cross-reactivity with functional groups within biomolecules.

[0312] In certain embodiments, when the conjugate of formula (II)-(IIH), (IV), (IVA) or (IVB) is formed by an oxime coupling reaction, the divalent residue of the reactive group comprises a divalent residue of a non-natural amino acid. In certain embodiments, when the conjugate of formula (II)-(IIH), (IV), (IVA) or (IVB) is formed by an oxime coupling reaction, the divalent residue of the reactive group (e.g., RL) is In certain embodiments, when the conjugate of formula (II)-(IIH), (IV), (IVA) or (IVB) is formed by an oxime coupling reaction, the divalent residue of the reactive group comprises an oxime bond. In certain embodiments, when the conjugate of formula (II)-(IIH), (IV), (IVA) or (IVB) is formed by an oxime coupling reaction, the divalent residue of the reactive group (e.g., RL) is

[0313] Thiol-N-hydroxysuccinimide reaction

[0314] Advantageously, compounds containing a terminal thiol-N-hydroxysuccinimide group favor reaction with a second compound containing an amine group (eg, glutamine) to form an amide.

[0315] In certain embodiments, when the conjugate of formula (II)-(IIH), (IV), (IVA) or (IVB) is formed by a thiol-N-hydroxysuccinimide coupling reaction, the divalent residue of the reactive group comprises a divalent residue of an amino acid (e.g., glutamine). In certain embodiments, when the conjugate of formula (II)-(IIH), (IV), (IVA) or (IVB) is formed by a thiol-N-hydroxysuccinimide coupling reaction, the divalent residue of the reactive group (e.g., RL) is In certain embodiments, when the conjugate of formula (II)-(IIH), (IV), (IVA) or (IVB) is formed by a thiol-N-hydroxysuccinimide coupling reaction, the divalent residue of the reactive group comprises an amide bond.

[0316] Other reactions

[0317] Other suitable coupling reactions are described in the literature. See, for example, Lang, K. and Chin, J. 2014, Bioorthogonal Reactions for Labeling Proteins, ACS Chem Biol 9, 16-20; Paterson, DM et al. 2014, Finding the Right (Bioorthogonal) Chemistry, ACS Chem Biol 9, 592-605; King, M. and Wagner, A. 2014, Developments in the Field of Bioorthogonal Bond Forming Reactions–Past and Present Trends, Bioconjugate Chem., 2014, 25(5), pp825-839; and Ramil, CP and Lin, Q., 2013, Bioorthogonal chemistry: strategies and recent developments, Chem Commun 49, 11007-11022.

[0318] Release reaction

[0319] The release reaction is a reaction for releasing a biologically active portion from a compound or conjugate of the present invention in vivo and / or in vitro. In certain embodiments, the released biologically active portion is a compound (e.g., a cytotoxic agent) or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof as described elsewhere in the present invention. An example of a release reaction is an intramolecular reaction between an eliminator group and a release trigger group of a compound or conjugate of the present invention to release the biologically active portion of the compound or conjugate of the present invention. The elimination group itself can be decomposed into two reactive components, as shown in these reactions, where X is a drug having a heteroatom of N or O for connection. An exemplary release reaction is shown in the following scheme: Water soluble polymer

[0320] In certain embodiments, the compound or conjugate of the present invention comprises one or more water-soluble polymers. Various macromolecular polymers and other molecules can be connected to the polypeptide of the present invention to regulate the biological properties of the polypeptide, and / or provide new biological properties for the polypeptide. These macromolecular polymers can be connected to the polypeptide via naturally encoded amino acids, via non-naturally encoded amino acids or any functional substituents of natural or modified amino acids, or added to any substituents or functional groups of natural or modified amino acids. The molecular weight of the polymer may include a wide range, including but not limited to about 100Da to about 100,000Da, or larger.

[0321] The selected polymer may be water soluble so that the protein to which it is attached will not precipitate in an aqueous environment such as a physiological environment. The polymer may be branched or unbranched. In certain embodiments, the polymer will be pharmaceutically acceptable for therapeutic use of the final product formulation.

[0322] In certain embodiments, the ratio of polyethylene glycol molecules to polypeptide molecules will vary, and their concentrations in the reaction mixture will also vary. Generally, the optimal ratio (in terms of reaction efficiency, there is a minimum excess of unreacted protein or polymer) can be determined by the molecular weight of the selected polyethylene glycol and the number of available reactive groups. In terms of molecular weight, generally the higher the molecular weight of the polymer, the fewer the number of polymer molecules that can be attached to the protein. Similarly, when optimizing these parameters, the branching of the polymer should be considered. Generally, the higher the molecular weight (or the more branches), the higher the polymer: protein ratio.

[0323] The water-soluble polymer can be in any structural form, including but not limited to straight chain, forked or branched. Typically, the water-soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water-soluble polymers may also be used. For example, PEG is used to describe certain embodiments.

[0324] PEG is a well-known water-soluble polymer that can be purchased commercially or prepared by ring-opening polymerization of ethylene glycol using methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). The term "PEG" is used broadly to include any polyethylene glycol molecule, regardless of the size or modification of the PEG terminus, and can be represented as being attached to a polypeptide by the formula: X'O-(CH2CH2O) n –CH2CH2–Y′, wherein n is an integer selected from 2 to 10,000, and X′ is H or a terminal modification, including but not limited to C 1-4alkyl, and Y' is the point of attachment to the polypeptide.

[0325] In some cases, PEG is terminated at one end with a hydroxyl or methoxy group, ie, X' is H or CH3 (also known as "methoxy PEG"). Alternatively, the PEG can be terminated with an active (reactive) group to form a bifunctional polymer. Typical PEG reactive groups may include those reactive groups that are commonly used to react with the following functional groups: functional groups present in the 20 common amino acids (including but not limited to maleimide groups, activated carbonates (including but not limited to p-nitrophenyl esters), activated esters (including but not limited to N-hydroxysuccinimide, p-nitrophenyl esters and aldehydes); and functional groups that are inert to the 20 common amino acids but specifically react with complementary functional groups present in non-naturally encoded amino acids (including but not limited to azide groups, alkyne groups). It should be noted that the other end of the PEG, represented by Y' in the above formula, will be directly or indirectly linked to the polypeptide via a naturally occurring or non-naturally encoded amino acid. For example, Y' can be an amide, carbamate or urea bond of an amine group of a polypeptide (including but not limited to the epsilon amine or N-terminus of lysine). Alternatively, Y' can be a thiol group (including but not limited to the thiol of cysteine). Alcohol group). Alternatively, Y' can be a bond of a residue that is not usually available via 20 common amino acids. For example, the azide group on PEG can react with the alkyne group on the polypeptide to form a Huisgen [3 + 2] cycloaddition product. Alternatively, the alkyne group on PEG can react with the azide group present in non-naturally encoded amino acids (e.g., modified amino acids described in the present invention) to form similar products. In some embodiments, as applicable, strong nucleophiles (including but not limited to hydrazine, hydrazide, hydroxylamine, semicarbazide) can react with aldehyde or ketone groups present in non-naturally encoded amino acids to form hydrazones, oximes or semicarbazones, which can be further reduced by treatment with an appropriate reducing agent in some cases. Alternatively, the strong nucleophile can be incorporated into the polypeptide via non-naturally encoded amino acids and used to preferentially react with ketone or aldehyde groups present in water-soluble polymers.

[0326] Depending on the actual needs, any molecular weight PEG can be used, including but not limited to, from about 100 Daltons (Da) to 100,000 Da or greater (including but not limited to, in certain embodiments, 0.1-50 kDa or 10-40 kDa) as required. Branched PEGs include, but are not limited to, PEG molecules where each chain has a molecular weight (MW) ranging from 1-100 kDa (including but not limited to, 1-50 kDa or 5-20 kDa). A wide range of PEG molecules are described in the product catalog of Shearwater Polymers and the product catalog of Nektar Therapeutics, both of which are incorporated herein by reference.

[0327] Typically, at least one end of the PEG molecule can be used to react with the rest of the compound shown in formula (I)-(IVB). For example, PEG derivatives with alkynes and azide groups for reacting with amino acid side chains can be used to attach PEG to non-naturally encoded amino acids as described herein. If the non-naturally encoded amino acid comprises an azide, PEG will typically contain either an alkyne group portion to achieve formation of a [3+2] cycloaddition product, or an activated PEG species (i.e., ester, carbonate) containing a phosphine group to achieve formation of an amide bond. Alternatively, if the non-naturally encoded amino acid comprises an alkyne, PEG will typically contain an azide group portion to achieve formation of a [3+2] Huisgen cycloaddition product. If the non-naturally encoded amino acid comprises a carbonyl group, the PEG will typically contain a nucleophile (including but not limited to, hydrazide, hydrazine, hydroxylamine, or semicarbazide functional group) to achieve formation of corresponding hydrazone, oxime, and semicarbazide bonds, respectively. In other alternatives, the reverse orientation of the active (reactive) groups described herein can be used (ie, the azide portion of the non-naturally encoded amino acid can be reacted with the alkyne-containing PEG derivative).

[0328] In some embodiments, the polypeptide variant with a PEG derivative comprises a chemical functionality that reacts with a chemical functionality present on the side chain of the non-naturally encoded amino acid.

[0329] In certain embodiments, the water-soluble polymer is a polymer containing an azide or acetylene, comprising a water-soluble polymer backbone, having an average molecular weight from about 800Da to about 100,000Da. The polymer backbone of the water-soluble polymer can be polyethylene glycol. However, it should be understood that a variety of water-soluble polymers including but not limited to polyethylene glycol and other related polymers (including poly (dextran)) and polypropylene glycol) are also suitable for use, and the use of the term "PEG" or "polyethylene glycol" is intended to cover and include all such molecules. The term "PEG" further includes but is not limited to any form of polyethylene glycol, including bifunctional PEG, multi-arm PEG, derivatized PEG, forked PEG, branched PEG, pendant PEG (i.e., PEG or related polymers having one or more functional groups pendant to the polymer backbone), or PEG having a degradable bond therein.

[0330] The polymer backbone can be linear or branched. Branched polymer backbones are generally known in the art. Typically, branched polymers have a central branching core group portion and multiple linear polymer chains attached to the central branching core. PEG is typically used in a branched form that can be prepared by adding ethylene oxide to various polyols, such as glycerol, glycerol oligomers, pentaerythritol, and sorbitol. The central branching group portion can also be derived from several amino acids, such as lysine. The branched polyethylene glycol can be represented in the general form as R-(-PEG-OH) m , wherein R is derived from a core group portion, such as glycerol, glycerol oligomers, or pentaerythritol, and m represents the number of arms. Multi-arm PEG molecules, such as those described in the following documents, can also be used as the polymer backbone: U.S. Patent Nos. 5,932,462, 5,643,575, 5,229,490 and 4,289,872; U.S. Patent Application No. US2003 / 0143596; and WO96 / 21469; and WO 93 / 21259, each of which is incorporated into the present invention in its entirety by reference.

[0331] Branched PEG can also be PEG(-Y″CHZ2) n The forked PEG form represented by PEG is a linking group, and Z is an activated terminal group connected to CH with a chain of atoms of defined length.

[0332] Another branching form, pendant PEG, has PEG active (reactive) groups, such as carboxyl groups, along the PEG backbone rather than at the termini of the PEG chains.

[0333] In addition to these forms of PEG, the polymers can also be prepared using weak or degradable bonds in the backbone. For example, PEG can be prepared using ester bonds in the polymer backbone that are subject to hydrolysis. As shown in the present invention, the hydrolysis allows the polymer to be cut into lower molecular weight fragments: -PEG-CO2-PEG-+H2O→PEG-CO2H+HO-PEG-. It will be understood by those skilled in the art that the term "polyethylene glycol" or PEG represents or includes all forms known in the art, including but not limited to those disclosed in the present invention.

[0334] Many other polymers are also suitable for use. In some embodiments, a water-soluble polymer backbone having from 2 to about 300 terminals is particularly suitable. Examples of suitable polymers include, but are not limited to, other poly(alkylene glycols) (e.g., polypropylene glycol ("PPG")), copolymers thereof (including but not limited to copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like. Although the molecular weight of each chain of the polymer backbone can vary, it is generally in the range of about 800 Da to about 100,000 Da, typically about 6,000 Da to about 80,000 Da.

[0335] Those of ordinary skill in the art will recognize that the foregoing list of substantially water-soluble backbones is by no means exhaustive, but is merely exemplary, and all polymeric materials having the properties described herein are considered suitable for use.

[0336] In some embodiments, the polymer derivative is "multifunctional", meaning that the polymer backbone has at least two termini, and potentially up to about 300 termini, functionalized or activated with functional groups. Multifunctional polymer derivatives include, but are not limited to, linear polymers having two termini, wherein each terminus is attached to a functional group that may be the same or different. Composition and use Pharmaceutical composition and administration method

[0337] The conjugates provided by the present invention can be formulated into pharmaceutical compositions using methods available in the art and those disclosed in the present invention. Any conjugate provided by the present invention can be provided in a suitable pharmaceutical composition and administered by a suitable route of administration.

[0338] The method provided by the present invention includes administering a pharmaceutical composition comprising at least one conjugate provided by the present invention and one or more compatible and pharmaceutically acceptable carriers. In this case, the term "pharmaceutically acceptable" refers to those approved by the regulatory agency of the federal or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for animals, and in certain embodiments for humans. The term "carrier" includes a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), auxiliary material or vehicle used together with the therapeutic agent. Such drug carriers can be sterile liquids, such as water and oil, including oils from petroleum, animals, plants or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water can be used as a carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Examples of suitable pharmaceutical carriers are described in Martin, EW, Remington's Pharmaceutical Sciences.

[0339] In clinical practice, the pharmaceutical composition or conjugate provided by the present invention can be administered by any route known in the art. Exemplary routes of administration include, but are not limited to, oral, inhaled, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes. In some embodiments, the pharmaceutical composition or conjugate provided by the present invention is administered orally. In some embodiments, the pharmaceutical composition or conjugate provided by the present invention is administered parenterally.

[0340] Compositions for parenteral administration can be emulsions or sterile solutions. Parenteral compositions can include, for example, propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters (e.g., ethyl oleate). These compositions can also include wetting agents, isotonic agents, emulsifiers, dispersants and stabilizers. Sterilization can be carried out in several ways, for example, using a bacterial filter, by irradiation or by heating. Parenteral compositions can also be prepared in the form of sterile solid compositions, which can be dissolved in sterile water or any other injectable sterile medium when used.

[0341] In some embodiments, the compositions provided herein are pharmaceutical compositions or single unit dosage forms. The pharmaceutical compositions and single unit dosage forms provided herein contain a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic conjugates.

[0342] The pharmaceutical composition may include one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, wherein a person of ordinary skill in the art is able to select a suitable pharmaceutical excipient. Non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art, including but not limited to the manner in which the dosage form is applied to the subject and the specific conjugate in the dosage form. If desired, the composition or single unit dosage form may also include a small amount of a wetting agent or emulsifier, or a pH buffer. Therefore, the pharmaceutical excipients provided below are exemplary only and are not intended to be limiting. Other pharmaceutical excipients include, for example, those described in Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), which are incorporated herein by reference in their entirety.

[0343] In some embodiments, the pharmaceutical composition comprises a defoamer. Any suitable defoamer can be used. In some aspects, the defoamer is selected from alcohol, ether, oil, wax, polysiloxane, surfactant, and a combination thereof. In some aspects, the defoamer is selected from mineral oil, vegetable oil, vinyl bis stearamide, paraffin, ester wax, fatty alcohol wax, long-chain fatty alcohol, fatty acid soap, fatty acid ester, silicon glycol, fluorosilicone, polyethylene glycol-polypropylene glycol copolymer, polydimethylsiloxane-silicon dioxide, ether, octanol, caprylic alcohol, sorbitan trioleate, ethanol, 2-ethyl-hexanol, dimethicone, oleyl alcohol, dimethicone, and a combination thereof.

[0344] In some embodiments, the pharmaceutical composition comprises a co-solvent. Illustrative examples of co-solvents include ethanol, poly(ethylene) glycol, butylene glycol, dimethylacetamide, glycerol, and propylene glycol.

[0345] In some embodiments, the pharmaceutical composition comprises a buffer. Illustrative examples of buffers include acetate, borate, carbonate, lactate, malate, phosphate, citrate, hydroxide, diethanolamine, monoethanolamine, glycine, methionine, guar gum, and monosodium glutamate.

[0346] In some embodiments, the pharmaceutical composition comprises a carrier or filler. Illustrative examples of carriers or fillers include lactose, maltodextrin, mannitol, sorbitol, chitosan, stearic acid, xanthan gum, and guar gum.

[0347] In some embodiments, the pharmaceutical composition comprises a surfactant. Illustrative examples of surfactants include d-alpha tocopherol, benzalkonium chloride, benzethonium chloride, cetrimonium bromide, cetylpyridinium chloride, docusate sodium, behenyl glyceryl, monooleyl glyceryl, lauric acid, macrogol 15hydroxystearate, myristyl alcohol, phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxyglycerol esters, sodium lauryl sulfate, sorbitan esters, and vitamin E polyethylene glycol succinate.

[0348] In some embodiments, the pharmaceutical composition comprises an anti-caking agent. Illustrative examples of anti-caking agents include calcium phosphate (trivalent), hydroxymethylcellulose, hydroxypropylcellulose, and magnesium oxide.

[0349] Other excipients that can be used together with the pharmaceutical composition include, for example, albumin, antioxidants, antibacterial agents, antifungal agents, bioabsorbable polymers, chelating agents, controlled release agents, diluents, dispersants, dissolution enhancers, emulsifiers, gelling agents, ointment bases, penetration enhancers, preservatives, solubilizers, solvents, stabilizers, and sugars. Specific examples of each of these agents are described in, for example, Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), The Pharmaceutical Press, which is incorporated herein by reference in its entirety.

[0350] In some embodiments, the pharmaceutical composition comprises a solvent. In some aspects, the solvent is a saline solution, such as a sterile isotonic saline solution or a glucose solution. In some aspects, the solvent is water for injection.

[0351] In some embodiments, the pharmaceutical composition is in the form of particles, such as microparticles or nanoparticles. Microparticles and nanoparticles can be formed from any suitable material, such as a polymer or lipid. In some aspects, the microparticles or nanoparticles are micelles, liposomes, or polymer vesicles.

[0352] The present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the conjugates, since, in some embodiments, water can cause degradation of certain antibodies or antigen-binding fragments thereof.

[0353] The anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture content ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms comprising lactose and at least one active ingredient comprising a primary or secondary amine can be anhydrous if significant contact with moisture and / or humidity is expected during manufacture, packaging and / or storage.

[0354] Anhydrous pharmaceutical compositions should be prepared and stored in a manner that maintains their anhydrous nature. Thus, anhydrous compositions may be packaged using materials known to prevent exposure to water so that they can be included in appropriately formulated kits. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0355] The lactose-free compositions provided by the present invention may include excipients well known in the art, and include, for example, those listed in U.S. Pharmacopoeia (USP) SP (XXI) / NF (XVI). Typically, the lactose-free compositions include pharmaceutically compatible and pharmaceutically acceptable amounts of active ingredients, binders / fillers, and lubricants. Exemplary lactose-free dosage forms include active ingredients, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

[0356] The present invention also provides pharmaceutical compositions and dosage forms, which contain one or more excipients that can reduce the decomposition rate of the conjugate. Such excipients are referred to as "stabilizers" in the present invention, including but not limited to antioxidants such as ascorbic acid, pH buffers, or salt buffers. Parenteral Dosage Forms

[0357] In certain embodiments, the invention provides parenteral dosage forms. Parenteral dosage forms can be administered to subjects by various routes, including but not limited to subcutaneous, intravenous (including bolus injection), intramuscular and intra-arterial administration. Because its administration usually bypasses the natural defense of the subject against pollutants, parenteral dosage forms are usually sterile or can be sterilized before being applied to the subject. Examples of parenteral dosage forms include but are not limited to ready-to-use injection solutions, dry products that can be immediately dissolved or suspended in pharmaceutically acceptable injection vehicles, ready-to-use injection suspensions and emulsions.

[0358] Suitable vehicles that can be used to provide parenteral dosage forms are known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0359] Excipients that increase the solubility of one or more of the antibodies disclosed herein may also be included in the parenteral dosage forms. Dosage and unit dosage form

[0360] In the treatment of humans, the physician will determine the dosing which he considers most appropriate according to the prophylactic or therapeutic treatment and according to the age, weight, condition and other particular factors of the subject to be treated.

[0361] In certain embodiments, the compositions provided herein are pharmaceutical compositions or single unit dosage forms. The pharmaceutical compositions and single unit dosage forms provided herein contain a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic antibodies or antigen-binding fragments thereof.

[0362] The amount of the conjugate or composition that is effective in preventing or treating a disorder or one or more symptoms of the disorder will vary with the nature and severity of the disease or disorder, and with the route of administration of the conjugate. The frequency and dosage will also vary according to factors specific to each subject, which depend on the specific therapy (e.g., therapeutic or prophylactic agent) administered, the severity of the disorder, disease or condition, the route of administration, and the age, body, weight, response and previous medical history of the subject. The effective dose can be extrapolated from a dose-response curve derived from an in vitro or animal model test system.

[0363] In certain embodiments, exemplary dosages of the conjugate or composition include milligram or microgram amounts of the antibody per kilogram of subject or sample body weight (e.g., about 10 micrograms per kilogram to about 50 milligrams per kilogram, about 100 micrograms per kilogram to about 25 milligrams per kilogram, or about 100 micrograms per kilogram to about 10 milligrams per kilogram).

[0364] The dosage can be administered according to an appropriate schedule. In some cases, it may be necessary to use an antibody conjugate dosage outside the scope of the present disclosure, which is obvious to those of ordinary skill in the art. In addition, it should be noted that the clinician or attending physician will understand how and when to interrupt, adjust or terminate treatment in conjunction with the subject's response.

[0365] Different therapeutically effective amounts may be applicable to different diseases and conditions, which are obvious to those of ordinary skill in the art. Similarly, amounts sufficient to prevent, treat, treat or improve such conditions, but insufficient to cause or sufficient to reduce adverse reactions associated with the antibodies or antigen-binding fragments thereof provided by the present invention are also encompassed within the dosage and dosage frequency time course described herein. In addition, when multiple doses of the compositions provided by the present invention are administered to a subject, not all doses need to be the same. For example, the dose administered to a subject may be increased to improve the preventive or therapeutic effect of the compositions, or it may be reduced to reduce one or more adverse reactions experienced by a particular subject.

[0366] In certain embodiments, treatment or prevention can be initiated with one or more loading doses of a conjugate or composition provided herein and then maintained with one or more maintenance doses.

[0367] In certain embodiments, the dosage of the conjugate or composition provided by the invention can be administered so that the conjugate reaches a steady-state concentration in the subject's blood or serum. The steady-state concentration can be determined by the assay techniques available to the technician, or can be determined based on the subject's physical characteristics such as height, weight and age. Therapeutic applications

[0368] For therapeutic applications, the conjugates of the invention are administered to mammals (in certain embodiments, humans) in pharmaceutically acceptable dosage forms (such as those known in the art and as discussed herein). For example, the conjugates of the invention may be administered to humans by intravenous bolus or by continuous infusion for a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, or intratumoral routes. The conjugates may also be appropriately administered by peri-tumor, intralesional, or perilesional routes to exert local as well as systemic therapeutic effects. The intraperitoneal route may be particularly useful, for example, in the treatment of ovarian tumors.

[0369] The conjugates provided by the present invention can be used to treat any disease or condition described herein (e.g., inflammatory and / or proliferative diseases or conditions). In some embodiments, the disease or condition is a disease or condition that can be diagnosed by overexpression of an antigen. In some embodiments, the disease or condition is a disease or condition that can benefit from macromolecular therapy. In some embodiments, the disease or condition is abnormal cell proliferation.

[0370] In some embodiments, the disease or condition is cancer. In certain embodiments, the cancer is small cell lung cancer, non-small cell lung cancer, ovarian cancer, platinum-resistant ovarian cancer, ovarian adenocarcinoma, endometrial cancer, breast cancer, breast cancer overexpressing HER2, triple-negative breast cancer, lymphoma, large cell lymphoma, diffuse mixed histiocytic and lymphocytic lymphoma, follicular B-cell lymphoma, colon cancer, colon carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, melanoma, prostate cancer, or multiple myeloma. Diagnostic Applications

[0371] In some embodiments, the conjugates provided herein are used for diagnostic applications. These assays can be used, for example, to diagnose and / or prognose diseases such as cancer.

[0372] In some diagnostic and prognostic applications or embodiments, the conjugate may be labeled with a detectable moiety. Suitable detectable moieties include, but are not limited to, radioisotopes, fluorescent labels, and enzyme-substrate labels. In another embodiment, the conjugate need not be labeled, and the presence of the conjugate may be detected using a labeled antibody or antigen-binding fragment thereof that specifically binds to the conjugate. Reagent test kit

[0373] In some embodiments, the conjugates provided herein are provided in the form of a kit (i.e., a packaged combination of reagents in predetermined amounts with instructions for use to perform a procedure). In some embodiments, the procedure is a diagnostic assay. In other embodiments, the procedure is a therapeutic procedure.

[0374] In some embodiments, the kit further comprises a solvent for reconstituting the conjugate. In some embodiments, the conjugate is provided in the form of a pharmaceutical composition.

[0375] In some embodiments, the kit may include a conjugate or composition provided by the present invention, an optional second agent or composition, and instructions for providing information about the usage of the treatment of a disease to a medical service staff. Instructions may be provided in printed form or in the form of an electronic medium (e.g., a floppy disk, a CD, or a DVD), or in the form of a website address from which such instructions may be obtained. The unit dose of a conjugate or composition provided by the present invention or a second agent or composition may include such a dose, i.e., when applied to a subject, the treatment or prevention effective plasma level of the compound or composition may be maintained in the subject for at least 1 day. In some embodiments, the compound or composition may be included as a sterile aqueous pharmaceutical composition or a dry powder (e.g., lyophilized) composition.

[0376] In some embodiments, the invention provides suitable packaging. As used in the present invention, "packaging" includes solid substrates or materials that are generally used in systems, and can accommodate compounds provided by the invention and / or be suitable for being applied to a second medicament for a subject within fixed limits. Such materials include glass and plastic (e.g., polyethylene, polypropylene and polycarbonate) bottles, vials, paper, plastic and plastic foil laminated envelopes, etc. If electron beam sterilization technology is used, packaging should have a sufficiently low density to allow contents to be sterilized. Preparation and Synthesis Procedure Coupling

[0377] The conjugate can be prepared by standard techniques. In certain embodiments, the macromolecule is contacted with a compound shown in formula (I)-(IH), (III), (IIIA) or (IIIB) under conditions suitable for the macromolecule to form a bond with the compound shown in formula (I)-(IH), (III), (IIIA) or (IIIB) to form a conjugate, such as a conjugate shown in formula (II)-(IIH), (IV), (IVA) or (IVB). In certain embodiments, the macromolecule is contacted with a linker precursor under conditions suitable for the macromolecule to form a bond with the linker. The resulting macromolecule-linker is contacted with a compound or drug group portion under conditions suitable for the macromolecule-linker to form a bond with the compound or drug group portion to form a conjugate. In certain embodiments, the compound or drug group portion is contacted with a linker precursor under conditions suitable for the compound or drug group portion to form a bond with the linker. The resulting compound-linker or drug moiety-linker and macromolecule are contacted under conditions suitable for forming a bond between the compound-linker or drug moiety-linker and macromolecule to form a conjugate. For example, in certain embodiments, the second compound comprises a tetrazine; and RG comprises a strained olefin. In some embodiments, RG is In certain embodiments, the second compound comprises an azide; and RG comprises an alkyne. In certain embodiments, RG is In certain embodiments, the second compound comprises an alkyne; and RG comprises an azide. In certain embodiments, the second compound comprises a strained olefin; and RG comprises a tetrazine. In certain embodiments, the second compound comprises a thiol; and RG comprises a maleimide. In some embodiments, RG is In certain embodiments, the second compound comprises maleimide; and RG comprises a thiol. In some embodiments, the second compound comprises In certain embodiments, the second compound comprises a carbonyl group; and RG comprises an oxamine. In certain embodiments, RG is In some embodiments, the second compound comprises In certain embodiments, the second compound comprises an oxamine; and RG comprises a carbonyl group. In certain embodiments, RG is In some embodiments, RG is In some embodiments, RG is In certain embodiments, the second compound comprises In certain embodiments, the second compound is a polypeptide. In certain embodiments, the second compound is an antibody. In certain embodiments, the second compound is an antibody chain. Suitable linkers for preparing conjugates are disclosed herein, and exemplary conditions for conjugation are described in the Examples below. Example

[0378] The compounds provided by the present invention can be prepared, isolated or obtained by any method obvious to those skilled in the art. The compounds provided by the present invention can be prepared according to the exemplary preparation schemes provided below. The reaction conditions, steps and reactants not provided in the exemplary preparation schemes are obvious to those skilled in the art and are known. The symbols and conventions used in the present invention for the process methods, schemes and embodiments, whether or not specific abbreviations are specifically defined, are consistent with those used in contemporary scientific literature, such as the Journal of the American Chemical Society or the Journal of Biological Chemistry. Specifically, but not limited to, the following abbreviations can be used in the embodiments and throughout the specification: g (gram); mg (milligram); mL (milliliter); μL (microliter); mM (millimolar concentration); μM (micromolar concentration); Hz (hertz); MHz (megahertz); mmol (millimole); h, hr or hrs (hours); min (minutes); MS (mass spectrometry); ESI (electrospray ionization); LCMS (liquid chromatography-mass spectrometry); TLC (thin layer chromatography); HPLC (high performance liquid chromatography); rt (room temperature); atm (atmospheric pressure); calc d (calculated); equiv (equivalent); CDCl3 (deuterated chloroform); DBCO (dibenzocyclooctynamine); DCE (dichloroethane); DCM (dichloromethane); DIPEA (diisopropylethylamine); DMSO (dimethyl sulfoxide); DMSO-d6 (deuterated dimethyl sulfoxide); EtOAc (ethyl acetate); EtOH (ethanol); MeCN (acetonitrile); MeOH (methanol); RB (round-bottom flask); TFA (trifluoroacetic acid); THF (tetrahydrofuran); DMF (dimethylformamide); and BOC (tert-butyloxycarbonyl).

[0379] For all of the following examples, standard post-treatment and purification methods known to those skilled in the art may be used. Unless otherwise stated, all temperatures are expressed in ° C (Celsius). Unless otherwise stated, all reactions are carried out at room temperature. The synthetic methodology shown in the present invention is intended to illustrate applicable chemistry by using specific examples, rather than to indicate the scope of the present invention disclosed.

[0380] Unless otherwise stated, all anhydrous solvents were commercially available and stored in Sure-Seal bottles under nitrogen. All other reagents and solvents were purchased of the highest grade and used without further purification. NMR spectra were recorded on an Avance II HD (500 MHz) spectrometer equipped with a 5 mm Prodiogy H / F-BBO cryogenic probe and a BCU-I temperature controller. Chemical shifts (δ) are reported in parts per million (ppm) with reference to tetramethylsilane of δ 0.00, and coupling constants (J) are reported in Hz. Low-resolution mass spectral data were acquired by LC-MS on an Agilent G6125B spectrometer connected to an Agilent 1260 high-performance liquid chromatograph. The product was purified by RP-HPLC method, system: Shimadzu LC equipped with CTC IFC, Phenomenex Gemini NX 5μ, C18, 150 x 50 mm reverse phase column, using mobile phase B (CH3CN) / A (water containing 0.1% TFA) linear gradient, flow rate of 50 mL / min. Analytical HPLC was performed on a Waters 2695 instrument. For analytical HPLC, the stationary phases used were Phenomenex Gemini NX 5μ, C18, 150x 4.6mm RP column. The product was eluted on an acidic linear gradient (designated Gradient A) of mobile phase B (CH3CN with 0.05% TFA; 5% to 95% in 20 minutes) / mobile phase A (0.05% TFA / water) at a flow rate of 1.0 mL / min. Preparative HPLC purifications were performed on a Shimadzu LC equipped with a CTC IFC. All other preparative normal phase purifications were performed by standard flash silica gel chromatography using an ISCO fast liquid system.

[0381] Example 1. Synthesis of DBCO-nnAA-PEG13-AAN-exitecan (Compound 1):

[0382] Scheme 1: Synthesis of AAN-Ixitectin (3):

[0383] Fmoc-AAN(Trt)-OH 2 (250 mg, 0.33 mmol), EDC (65 mg, 0.34 mmol), HOAt (46 mg, 0.34 mmol) and DIPEA (53 μL) were added to a suspension of isotecan mesylate 1 (MsOH salt, 150 mg, 0.28 mmol) in anhydrous DMF (3 mL) at room temperature. The reaction was stirred at room temperature for 1 hour, LCMS showed the desired product, and then 0.3 mL of piperidine was added. The mixture was stirred for 5 minutes, and then a 1 / 1 hexane / ether mixture (45 mL) was added. The precipitate was collected by centrifugation and the solvent was removed by decantation to give compound 2b. The residue 2b was dissolved in 3 mL of TFA, and the mixture was stirred at room temperature for 10 minutes. TFA was then removed under reduced pressure, and the crude mixture was purified by reverse phase HPLC. The pure fractions were lyophilized to give compound 3 as a TFA salt (63 mg); LCMS m / z (ESI + ): Calculated value C 34 H 38 FN7O8, 691.28; found 692.4 (M+H).

[0384] Scheme 2: Synthesis of DBCO-nnAA-PEG13-PFP linker (4):

[0385] Synthesis of compound 7: Compound 5 (1.4 g, 3.34 mmol) was dissolved in DMF (10 mL). HATU (1.2 g, 3.34 mmol) and DIPEA (861 mg, 6.68 mmol) were added to the clear solution. The resulting solution was stirred at room temperature for about 30 seconds, and a solution of DBCO-amine 7 (922 mg, 3.34 mmol) in DMF (2 mL) was added. After the mixture was stirred at room temperature for 20 minutes, diethylamine (2 mL) was added and stirring was continued for 30 minutes. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse phase HPLC to obtain compound 7 (790 mg); LCMS m / z (ESI + ): Calculated value C 29 H 33 N3O2, 455.26; found 456.4 (M+H).

[0386] Synthesis of compound 4: A solution of compound 8 (3 g, 2.9 mmol), compound 7 (780 mg, 1.37 mmol) and DIPEA (353 mg, 2.74 mmol) in DMF (20 mL) was stirred for 20 minutes. LCMS showed that the reaction was complete. The reaction mixture was directly purified by reverse phase HPLC to give compound 4 (1.52 g); LCMS m / z (ESI + ): Calculated value C65 H 88 F5N3O 18 ,1293.60; Found 1293.7(M+H).

[0387] Scheme 3: Final coupling for the synthesis of compound 1:

[0388] Compound 3 (TFA salt, 478 mg, 0.6 mmol) and DIPEA (206 μL) were added to a solution of compound 4 (770 mg, 0.6 mmol) in anhydrous DMF (3 mL). The mixture was stirred at room temperature for 10 minutes, and LCMS showed that the reaction was complete. The reaction mixture was then directly purified by reverse phase HPLC to give compound 1 as a light yellow solid (710 mg); 1 H NMR(500MHz,DMSO-d6)δ11.41(s,1H),8.25(d,J=8.5Hz,1H),8.07–7.91(m,3H),7 .77(d,J=10.9Hz,1H),7.68–7.54(m,2H),7.47(dddd,J=13.4,7.6,4.9,2.8Hz,4H ),7.43–7.22(m,5H),6.98–6.83(m,1H),5.51(dt,J=8.8,4.5Hz,1H),5.43(s,2H) ,5.22(s,2H),5.04(d,J=14.0Hz,1H),4.47(q,J=6.8Hz,1H),4.09(dp,J=18.0,7.1 Hz,3H),3.73(s,10H),3.67–3.27(m,61H),3.22–3.01(m,3H),2.92(dq,J=13.3,6 .7Hz,1H),2.68–2.45(m,8H),2.45–2.28(m,6H),2.19(dq,J=9.3,4.8Hz,1H),1.8 7(qd,J=13.8,7.1Hz,4H),1.61(d,J=13.1Hz,2H),1.49–1.21(m,7H),1.13(dd,J= 26.8,7.1Hz,7H),0.97(ddd,J=16.1,11.6,6.4Hz,2H),0.88(t,J=7.3Hz,3H); LCMS m / z(ESI + ): Calculated value C 93 H 125 FN 10 O 25 ,1801.9; Measured value 1802.9[M+H] + .

[0389] Example 2: Synthesis of DBCO-nnAA-PEG13-VKG-exitecan (Compound 2):

[0390] Scheme 1: Synthesis of VK(Fmoc)G-exitecan (10):

[0391] To a solution of compound Boc-VK(Fmoc)-G-OH 9 (562 mg, 0.9 mmol) in anhydrous DMF (10 mL) was added isotecan mesylate (1) (478 mg, 0.9 mmol) and DIPEA (470 μL). After all components were dissolved, HATU (342 mg, 0.9 mmol) was added and the mixture was stirred at room temperature for 10 minutes. The reaction was then diluted with water (80 mL) and extracted with EtOAc (150 mL). The organic layer was washed with hydrochloric acid (0.2 M, 50 mL) and brine (50 mL), dried over Na2SO4, and evaporated to dryness under reduced pressure. The resulting residue was treated with TFA / DCM (1 / 4, 20 mL) at room temperature for 30 minutes, and the reactant was evaporated to dryness under reduced pressure. The crude mixture was dissolved in 5 mL DMF and purified by reverse phase HPLC to obtain compound 10 as a light yellow solid (771 mg). MS calculated value C 52 H 56 FN7O9,941.4; Found 942.9 [M+H] + .

[0392] Scheme 2: Final coupling and deprotection of the synthesis of compound 2:

[0393] Compound 10 (TFA salt, 347 mg, 0.33 mmol) and DIPEA (130 μL) were added to a solution of compound 4 (426 mg, 0.33 mmol) in anhydrous DMF (3 mL). The mixture was stirred at room temperature for 15 minutes, and LCMS showed the formation of the desired product. DBU (366 μL) was then added dropwise, and the mixture was stirred at room temperature for another 10 minutes. LCMS showed that the reaction was complete. The mixture was then directly purified by reverse phase HPLC to give compound 2 as a light yellow solid (350 mg); HRMS m / z (ESI + ): Calculated value C 96 H 133 FN 10 O 24 ,1828.94; Found 1829.95[M+H] + ; 1H NMR (500MHz, DMSO) δ8.43(d,J=8.5Hz,1H),8.14(t,J=5.6Hz,1H),7.98(d,J =7.4Hz,1H),7.81(d,J=10.9Hz,1H),7.77–7.53(m,7H),7.54–7.23(m,10H) ,5.57(dt,J=8.7,4.4Hz,1H),5.47–5.38(m,2H),5.25(d,J=3.3Hz,2H),5.0 4(d,J=14.1Hz,2H),4.16(td,J=8.1,5.9Hz,3H),4.08(dd,J=8.5,6.8Hz,3H ),3.88–3.68(m,13H),3.50(d,J=3.6Hz,71H),3.25–3.13(m,4H),3.13–3.0 3(m,2H),2.92(dq,J=13.4,6.8Hz,2H),2.78(q,J=6.8Hz,3H),2.60–2.26(m ,18H),2.26–2.03(m,3H),1.98–1.74(m,6H),1.74–1.46(m,8H),1.46–1.09 (m,12H),0.97(s,2H),0.88(t,J=7.3Hz,3H),0.78(dd,J=14.1,6.7Hz,7H).

[0394] Example 3: Synthesis of DBCO-nnAA-PEG13-AAA-exitecan (Compound 3):

[0395] Scheme 1: Synthesis of AAA-exitecan (13):

[0396] Scheme 2: Synthesis of DBCO-nnAA-PEG13-AAA-exitecan (Compound 3):

[0397] Compound 3 was synthesized in a similar manner using the same method as above. LCMS m / z (ESI + ): Calculated value C 92 H 124 FN9O 24 ,1757.87; Found 1759.1[M+H] + .

[0398] Example 4: Synthesis of DBCO-nnAA-PEG13-VK-exitecan (Compound 4):

[0399] Scheme 1: Synthesis of VK(Fmoc)-exitecan (15):

[0400] Scheme 2: Final coupling and deprotection of DBCO-PEG13-VK-Ixitectin (Compound 4):

[0401] Compound 4 was synthesized in a similar manner using the same method as above. LCMS m / z (ESI + ): Calculated value C 94 H 130 FN9O 23 ,1771.93; Measured value 1773.1[M+H] + .

[0402] Example 5: Synthesis of DBCO-nnAA-PEG13-NN-exitecan (Compound 5):

[0403] Scheme 1: Synthesis of Asn-Asn-exitecan (18):

[0404] PyAOP (110 mg) was added to a solution of Fmoc-Asn(Trt)-OH 16 (120 mg, 0.2 mmol) and isotecan mesylate 1 (106 mg, 0.2 mmol) in anhydrous DMF (4 mL), followed by DIPEA (0.14 mL). The reaction was stirred at room temperature for 1 hour, and LCMS showed that the desired product was formed. Hydrochloric acid (0.5 M, 30 mL) was added to the reaction. The mixture was extracted with EtOAc (60 mL), the organic layer was dried over Na2SO4, and evaporated to dryness under reduced pressure. The resulting residue was dissolved in DMF (3 mL), and piperidine (0.4 mL) was added. The mixture was stirred at room temperature for reaction. After 20 minutes, the mixture was concentrated to about 2 mL under reduced pressure, and the residue was ground with ether / hexane (1 / 1, 50 mL) to obtain compound 17. Compound 17 was diluted with DMF (5 mL), and Fmoc-Asn(Trt)-OH 16 (360 mg, 0.6 mmol) was added to the resulting mixture, followed by PyAOP (330 mg) and DIPEA (0.21 mL). The mixture was stirred at room temperature for 10 minutes and then diluted with EtOAc (100 mL). The mixture was washed with 0.5 M hydrochloric acid (50 mL) and water (50 mL). The organic layer was dried over Na2SO4 and evaporated to dryness under reduced pressure. The resulting gum was treated with TFA / TIS (9 / 1, 5 mL) at room temperature for 20 minutes. 100 mL of ether was added and the precipitated solid was collected by filtration to obtain a crude product, which was treated with piperidine (20% in DMF, 3 mL) at room temperature for 10 minutes. LCMS showed that the reaction was complete. The mixture was purified by RP-HPLC and lyophilized to give compound 18 (120 mg) as a light yellow solid; LCMS m / z (ESI + ): Calculated value C 32 H 34 FN7O8, 663.25; Found 664.3 [M+H] + .

[0405] Scheme 2. Synthesis of compound 5:

[0406] Compound 4 and compound 18 were coupled using the same method as above to obtain compound 5 as a yellow solid. LCMS m / z (ESI + ): Calculated value C 91 H 121 FN 10 O 25 ,1772.85; Measured value 1773.91[M+H] + .

[0407] Example 6. Synthesis of Compound 6, Compound 7, Compound 8 and Compound 9:

[0408] Compound 6, Compound 7, Compound 8 and Compound 9 were synthesized, purified and characterized in a similar manner from the common intermediate Compound 10 using the same method as described above.

[0409] Example 7. Synthesis of Compound 10:

[0410] Scheme 1: Synthesis of DBCO linker (Compound 21)

[0411] Scheme 2: Synthesis of Fmoc mPEG12 linker (24):

[0412] Scheme 3: Synthesis of DBCO-mPEG12-Pfp ester linker (27):

[0413] Compound 27 was synthesized on a 2-chlorotrityl chloride resin solid support using the general method: 2-chlorotrityl chloride resin loading, Fmoc removal with DMF / piperidine, chain extension and amide coupling with HATU, DMF, DIPEA, and the final resin was cleaved by treatment with 2 mL TFA / DCM (4%). The crude carboxylic acid was dissolved in DMF (4 mL), Pftu (86 mg, 0.2 mmol) was added, and then DIEA (70 μL) was added. The mixture was stirred at room temperature for 10 minutes and directly purified by reverse phase HPLC to give compound 27 (185 mg) after lyophilization as a viscous syrup.

[0414] Scheme 4: Synthesis of β-Glu-exitecan benzylamine (29)

[0415] Scheme 5: The final coupling synthesis of compound 10:

[0416] DIPEA (38 μL) was added to a 2 mL anhydrous DMF solution of compound 29 (TFA salt, 40 mg, 44 μmol) and compound 27 (55 mg, 40 μmol), and the mixture was stirred at room temperature for 30 minutes. LCMS showed that the reaction was complete. The mixture was then purified by reverse phase HPLC to give compound 10 (52.9 mg) as a yellow solid. LCMS m / z (ESI + ): Calculated value C 103 H 134 FN9O 31 ,2011.92; Measured value 2013.1[M+H] + .

[0417] Example 8. Synthesis of Compound 11 and Compound 12:

[0418] Compound 11 and Compound 12 were synthesized, purified and characterized from the common intermediate Compound 29 in the same manner as the above Example Compound 10.

[0419] Example 9. Synthesis of Compound 13:

[0420] Compound 13 was synthesized in a similar manner using the same method as compound 2 described in Example 2.

[0421] Example 10. Synthesis of Compound 14:

[0422] Compound 14 was synthesized in a similar manner using the same method as compound 1 described in Example 1.

[0423] Example 11. Synthesis of Compound 15:

[0424] Compound 15 was synthesized in a similar manner using the same method as compound 10 described in Example 7.

[0425] Example 12. Synthesis of Compound 16:

[0426] Compound 4 (66 mg, 51 μmol) and DIPEA (18 μL, 156 μmol) were added to a solution of compound 28 (44 mg, 51 μmol) in anhydrous DMF (2 mL). The mixture was stirred at room temperature for 20 minutes, and LCMS showed that the reaction was complete. The mixture was then directly purified by reverse phase HPLC to give compound 16 as a yellow solid (61 mg). LCMS m / z (ESI + ): Calculated value C 98 H 127 FN 10 O 25 ,1862.90; Measured value 1864.1[M+H] + .

[0427] Example 13: Synthesis of Compound 17:

[0428] Compound 17 was synthesized in a similar manner using the same method as compound 16 described in Example 12.

[0429] Example 14: Synthesis of Compound 18:

[0430] Compound 18 was synthesized in a similar manner using the same method as compound 16 described in Example 12.

[0431] Example 15: Synthesis of Compound 19, Compound 20, Compound 21, Compound 22 and Compound 23:

[0432] Compound 19, Compound 20, Compound 21, Compound 22 and Compound 23 were all synthesized using the same method as above.

[0433] Example 16. Linker-Payload Compound Coupling

[0434] The small molecule drug linker was dissolved in DMSO to a final concentration of 5 mM. The conjugation was performed in 1xPBS with an antibody concentration of 1 mg / mL, a drug linker to pAMF ratio of 3, and 15% DMSO. The reaction mixture was incubated overnight at 30°C. The conjugation efficiency was determined by MALDI. Unconjugated drug linker was removed by desalting. The conjugate purity was determined by Sepax SEC-300. The conjugate was formulated in 1xPBS.

[0435] The linker drug was conjugated to aFolR mAb, where 4 pAMF sites were incorporated at the Y180F404 site of the heavy chain and 8 pAMF sites at the K42E161 site of the light chain, respectively. According to the above conjugation conditions, the conjugation efficiency of all linker payloads exceeded 94%. Analytical SEC results showed that all conjugates were of high purity with a monomer content of >99% (Table 1). Table 1. Selected linker-payload conjugation results

[0436] Example 17: Free drug cell killing activity

[0437] The in vitro cytotoxic activity of exitecan, glycine-exitecan, and hemicaterin was evaluated in a panel of breast and lung cancer cell lines.

[0438] MDA-MB-361, SKBR3, BT20, HCC38, HCC1143, HCC1937, JIMT1, MCF-7, A549, NCI-H441, NCI-H520, NCI-H1703, NCI-H1975, NCI-H2110, NCI-H226, NCI-H292, NCI-H358 cells were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). All cell lines were maintained in DMEM / F12 (1:1), high glucose (Corning, Corning, New York), supplemented with 10% heat-inactivated fetal bovine serum (ThermoScientific, Grand Island, NY), 2mM glutamax (Thermo Scientific, GrandIsland, NY) and 1x penicillin / streptomycin (Corning, Corning, NY). The cytotoxic effect of Exatecan warhead was determined using a cell proliferation assay. One day before the actual assay, cells were seeded in 384-well flat-bottom white polystyrene plates at a concentration of 625 cells / 25 μL. Exatecan was prepared in cell culture medium at 2 times the starting concentration, serially diluted (1:3) under sterile conditions, and added to the cells in triplicate. The plates were incubated in a CO2 incubator at 37°C for 120 h. To determine cell viability, 30 μL Cell Reagent (Promega Corp, Madison, WI) was added to each well, and the plates were processed according to the manufacturer's instructions. Relative luminescence was measured on a plate reader (Perkin-Elmer; Waltham, MA). Untreated cells were used as controls and relative luminescence readings were converted to percent viability (%). Data were analyzed by nonlinear regression using GraphPad Prism using log(inhibitor) vs. response, variable slope, 4-parameter fit equation.

[0439] The warheads without ixotecan showed potent cell killing effects against all tested cell lines, with an EC 50 The values ​​ranged from 0.32 nM to 4.2 nM (Table 2), which was similar to that of hemipteratin. Glycine-exitecan showed slightly lower cell killing activity, with an EC 50 The values ​​ranged from 3.8 nM to 44 nM (Table 2). Table 2. Cytotoxic activity of free drugs

[0440] Example 18: In vitro cell killing activity of exitecan ADC:

[0441] The anti-FolRa ADCs were all generated by conjugating the anti-FolRa antibody 1848-H01 with the exotecan linker warhead at DAR = 4 and DAR = 8. The anti-FolRa antibody 1848-H01 was conjugated with compound 27 and compound 26 at DAR = 4 and DAR = 8 and used as positive controls.

[0442] FolRα-positive Igrov1 cells were licensed from NCI (National Cancer Institute, Frederick, Maryland). FolRα-negative A549 cells were purchased from ATCC (American Type Culture Collection). Both cell lines were maintained in DMEM / F12 (1:1), high glucose (Corning), supplemented with 10% heat-inactivated fetal bovine serum (Thermo Scientific), 2 mM glutamine (Thermo Scientific), and 1× penicillin / streptomycin (Corning). The cytotoxic effects of the ADCs were determined using a cell proliferation assay. One day before the assay, Igrov1 and A549 cells were seeded in 384-well flat-bottom white polystyrene plates at a concentration of 625 cells / 25 μL. The ADCs were prepared in cell culture medium at 2 times the starting concentration and sterile filtered through SpinX 0.22 μm filter centrifuge tubes (Corning Costar). The filter-sterilized samples were serially diluted (1:3) under sterile conditions and added to the cells in triplicate. The plates were incubated in a CO2 incubator at 37°C for 120 hours. To determine cell viability, 30 μL Cell Reagent (Promega Corp, Madison, WI) was added to each well, and the plates were processed according to the manufacturer's instructions. Relative luminescence was measured on a plate reader (Perkin-Elmer; Waltham, MA). Untreated cells were used as controls and relative luminescence readings were converted to percent viability (%). Data were analyzed by nonlinear regression using GraphPad Prism using log(inhibitor) vs. response, variable slope, 4-parameter fit equation.

[0443] like Figure 1A and Figure 1B As shown, at DAR = 4 and DAR = 8, the anti-FolRα ADCs coupled with compounds 1, 2, and 3, respectively, showed potent cell killing effects on FolRα-positive Igrov1 cells, while no cell killing effects were observed on FolRα-negative A549 cells. The results showed that there was no nonspecific release of free warheads to kill target-negative cells, which means that all tested linkers were stable in the cell culture medium for 5 days. Compared with the ADC of DAR4, the ADC of DAR8 showed stronger cell killing effects and lower EC 50 And a larger kill span.

[0444] like Figure 2A and Figure 2BAs shown, at DAR = 8, the anti-FolRα ADCs coupled with compounds 6, 7, 8, and 9 respectively showed potent cell killing effects on FolRα-positive Igrov1 cells, while no cell killing effects were observed on hFolRα-negative A549 cells. The anti-FolRα ADCs coupled with compound 24 had no cell killing effects on FolRα-positive Igrov1 cells and FolRα-negative A549 cells.

[0445] The cell killing activities of exitecan ADC are summarized in Tables 3 and 4. Table 3. Cytotoxic activity of exitecan ADC Table 4. Cytotoxic activity of exitecan ADC

[0446] Example 19: In vitro cell killing activity of hemiesterin ADC

[0447] At DAR = 4, anti-FolRa ADCs conjugated to hemiglutinin with different cathepsin-cleavable linkers were also generated and the cell killing activity was evaluated against FolRα-positive Igrov1 cells and FolRα-negative A549 cells. Figure 3A , Figure 3B As shown in Table 5, similar to the anti-FolRα ADC conjugate 127-3, the anti-FolRα ADCs conjugated with DAR4 to compounds 13, 14 and 15 respectively showed potent cell killing effects on FolRα-positive Igrov1 cells, while no cell killing effects were observed on FolRα-negative A549 cells, indicating that all tested linkers were stably present in the cell culture medium for 5 days. Table 5. Cell killing activity of hemipteratin ADC

[0448] The content of the present invention described above may include multiple different embodiments with independent practicality. Although each of these embodiments is disclosed, the specific embodiments disclosed and shown in the present invention should not be regarded as limiting, because various variations are possible. The subject matter of the embodiments of the present invention includes all novel and non-obvious combinations and sub-combinations of various elements / elements, features, functions and / or properties disclosed in the present invention. The following claims specifically point out certain combinations and sub-combinations that are considered novel and non-obvious. Inventions embodied in other combinations and sub-combinations of features, functions, elements and / or properties may be claimed in this application, an application claiming priority to this application, or a related application. This claim, whether it is about different embodiments or about the same embodiment and whether the scope is broader, narrower, equal or different than the original claim, is also considered to be included in the subject matter of the invention of the present invention.

[0449] One or more features of any embodiment described herein or in the accompanying drawings may be combined with one or more features of any other embodiment described herein or in the accompanying drawings without departing from the scope of the invention.

[0450] All publications, patents and patent applications cited in this specification are incorporated herein by reference, just as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the above invention has been described in detail by way of illustration and example for the purpose of clear understanding, it is obvious to those skilled in the art based on the teachings of the present invention that certain changes / variations and modifications / modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

1. Compound represented by formula (I): or a pharmaceutically acceptable salt and / or regioisomer thereof; in, L 1 Selected from Ring A is an optionally substituted bridged, fused, or spiro bicyclic carbocycle, or an optionally substituted bridged, fused, or spiro bicyclic heterocycle, wherein the carbocycle or the heterocycle of Ring A is optionally substituted by one or more selected from alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents; Ring B is an optionally substituted N-linked bridged, fused, or spiro bicyclic heterocycle, wherein Ring B is optionally substituted by one or more selected from alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents; R a and R b are independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; a is an integer independently selected from 0, 1, 2, 3, 4, 5 and 6; b is an integer selected from 0 and 1; R 1 is H or alkyl, the alkyl being optionally substituted by one or more selected from cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aryl and heteroaryl substituents; R 2 and R 3 are each independently selected from H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; Y is *-C(O)-(CR a R b ) c -NH-, or *-C(O)-(CR a R b ) c -, where * indicates the position where Y is attached to RG; c is an integer selected from 1, 2, 3, 4, 5 and 6; RG is the reactive group; L 2 Does not exist, or L 2 is a linker comprising a hydrophilic polymer residue; L 3 Absent, or L 3 is -C(O)-AA-, -C(O)-AA-Z-(CR a R b ) a -Z-(CR a R b ) a -C(O)-, -C(O)-Z-(CR a R b ) a -C(O)-Z-L 4 -OC(O)-, -Z-AA-, -AA-, -C(O)-, -C(O)-AA-Z-(CR a R b ) a -, -AA-C(O)-, -C(O)-(CR a R b ) a -Z-(CR a R b ) a -Z-AA-C(O)-, -C(O)O-L 4 -Z-C(O)-(CR a R b ) a -Z-C(O)-, -AA-Z-, or -(CR a R b ) a -Z-AA-C(O)-; Z is selected from -NR 2 - and -O-; AA is an amino acid residue or a peptide residue; L 4 yes Where Su is the hexose form of a monosaccharide; d is an integer independently selected from 1, 2 and 3; D is the cytotoxic payload; and represents the point of attachment to the rest of the compound.

2. The compound according to claim 1, wherein L 1 yes 3. The compound according to claim 2, wherein L 1 yes 4. The compound according to claim 1, wherein L 1 yes 5. The compound according to any one of claims 1 to 4, wherein L 1 Ring A is an optionally substituted bridged, fused, or spirocyclic bicyclic carbocyclic ring, wherein the carbocyclic ring of Ring A is optionally substituted with one or more substituents selected from the group consisting of: 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl.

6. The compound according to claim 5, wherein L 1 Ring A is optionally substituted C 4-12 Bridged, fused, or spiro bicyclic carbocycles.

7. The compound according to claim 5, wherein L 1 Ring A is optionally substituted C 4-12 Bridged bicyclic carbocycles.

8. The compound according to claim 5, wherein L 1 Ring A is optionally substituted C 4-8 Bridged bicyclic carbocycles.

9. The compound according to claim 1 or 4, wherein L 1 yes 10. The compound according to claim 9, wherein L 1 yes 11. The compound according to claim 9, wherein L 1 yes 12. The compound according to claim 1, wherein L 1 yes 13. The compound according to claim 1 or 12, wherein L 1 Ring B is an optionally substituted 5 to 12-atom N-linked bridged, fused, or spiro bicyclic heterocyclic ring, comprising 1, 2 or 3 heteroatoms independently selected from N, O and S, including the N atom to which the ring is attached, wherein the heterocyclic ring of Ring B is optionally substituted by one or more selected from C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents.

14. The compound according to claim 13, wherein L 1 Ring B is an optionally substituted 5 to 12-atom N-attached spiro bicyclic heterocycle containing 1, 2 or 3 heteroatoms independently selected from N, O and S, including the N atom to which the ring is attached.

15. The compound according to claim 1 or 12, wherein L 1 Selected from wherein m is an integer selected from 1, 2, 3, 4 and 5; and n and o are each an integer independently selected from 1, 2 and 3.

16. The compound according to claim 1 or 12, wherein L 1 Selected from wherein m is an integer selected from 1, 2, 3, 4 and 5; and n and o are each an integer independently selected from 1, 2 and 3.

17. The compound according to claim 1 or 12, wherein L 1 Ring B is selected from Where X 1 , X 2 , X 3 and X 4 are independently selected from -C(R 4 )2-, -NH-, -O-, and -S-, where X 1 , X 2 and X 3 When both exist, X 1 -X 3 At least one of them is -C(R 4 )2-, and when X 1 , X 2 , X 3 and X 4 When both exist, X 1 -X 4 At least two of them are -C(R 4 )2-; and R 4 Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; or Two R's on the same carbon 4 The groups are taken together to form an oxo group.

18. The compound according to claim 17, wherein Ring B is selected from 19. The compound according to claim 1 or 12, wherein Ring B is selected from Where X 1 , X 2 , X 3 and X 4 are independently selected from -C(R 4 )2-, -NH-, -O-, and -S-, where X 1 , X 2 and X 3 When both exist, X 1 -X 3 At least one of them is -C(R 4 )2-, and when X 1 , X 2 , X 3 and X 4 When both exist, X 1 -X 4 At least two of them are -C(R 4 )2-; and R 4 Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; or Two R's on the same carbon 4 The groups are taken together to form an oxo group.

20. The compound according to claim 19, wherein Ring B is selected from 21. The compound according to any one of claims 1 to 4, wherein Ring A is selected from Where X 1 , X 2 , X 3 and X 4 are independently selected from -C(R 4 )2-, -NH-, -O-, and -S-, where X 1 , X 2 and X 3 When both exist, X 1 -X 3 At least one of them is -C(R 4 )2-, and when X 1 , X 2 , X 3 and X 4 When both exist, X 1 -X 4 At least two of them are -C(R 4 )2-; X 5 Yes-CR 4 -or-N-; and R 4 Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; or Two R's on the same carbon 4 The groups are taken together to form an oxo group.

22. Compound shown in (III): or a pharmaceutically acceptable salt and / or regioisomer thereof; in, L 5 is a linker comprising an unnatural amino acid; Y is *-C(O)-(CR a R b ) c -NH-, or *-C(O)-(CR a R b ) c -, where * indicates the position where Y is attached to RG; c is an integer selected from 1, 2, 3, 4, 5 and 6; RG is the reactive group; L 2 Does not exist, or L 2 is a linker comprising a hydrophilic polymer residue; L 3 Absent, or L 3 is -C(O)-AA-, -C(O)-AA-Z-(CR a R b ) a -Z-(CR a R b ) a -C(O)-, -C(O)-Z-(CR a R b ) a -C(O)-Z-L 4 -OC(O)-, -Z-AA-, -AA-, -C(O)-, -C(O)-AA-Z-(CR a R b ) a -, -AA-C(O)-, -C(O)-(CR a R b ) a -Z-(CR a R b ) a -Z-AA-C(O)-, -C(O)O-L 4 -Z-C(O)-(CR a R b ) a -Z-C(O)-, -AA-Z-, or -(CR a R b ) a -Z-AA-C(O)-; R a and R b are independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; a is an integer independently selected from 1, 2, 3, 4, 5 and 6; Z is selected from -NR 2 - and -O-; AA is an amino acid residue or a peptide residue; L 4 yes Where Su is the hexose form of a monosaccharide; d is an integer independently selected from 1, 2 and 3; D is the cytotoxic payload; and represents the point of attachment to the rest of the compound.

23. The compound according to claim 22, wherein L 5 is a linker comprising at least one amino acid selected from cysteic acid, hydroxyproline (Hyp), β-alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3-nitrotyrosine, nitroarginine, pyroglutamate (Pyr), naphthylalanine (Nal), 2,4-diaminobutyric acid (DAB), methionine sulfoxide, and methionine sulfone.

24. The compound according to claim 22, wherein L 5 is included connector.

25. The compound according to claim 22, wherein L 5 yes 26. The compound according to any one of claims 1 to 25, wherein L 2 Selected from -(CR a R b ) a -POLY 1 -、-POLY 1 -、-(CR a R b ) a -POLY 1 -(CR a R b ) a -、 POLY 1 Is a divalent hydrophilic polymer residue; POLY 2 is a residue of a hydrophilic polymer; and represents the point of attachment to the rest of the compound.

27. The compound according to any one of claims 1 to 26, wherein Y is -C(O)-(CR a R b ) c -NH-.

28. The compound according to claim 27, wherein Y is -C(O)-CH2CH2-NH-.

29. The compound according to any one of claims 1 to 26, wherein Y is -C(O)-(CR a R b ) c -.

30. The compound according to claim 29, wherein Y is -C(O)-(CH2)4-.

31. The compound according to any one of claims 1 to 30, wherein RG comprises alkyne, cyclooctyne, strained olefin, tetrazine, methylcyclopropene (Mecyp), thiol, p-acetylphenylalanine residue, oxyamine, maleimide, or azide.

32. The compound according to claim 31, wherein RG is selected from –N3, –NH2, and –SH; among which R T It is C 1-6 Alkyl; and represents the point of attachment to the rest of the compound.

33. The compound according to claim 32, wherein RG is selected from 34. The compound according to claim 32, wherein RG is 35. A compound according to any one of claims 1-25 and 31-34, wherein L 2 Yes-(CR a R b ) a -POLY 1 -.

36. The compound according to claim 35, wherein -POLY 1 - is a divalent residue of polyethylene glycol (PEG), polypropylene glycol (PPG), or a copolymer of ethylene glycol and propylene glycol.

37. The compound according to claim 36, wherein The divalent hydrophilic polymer residue has a structure shown in the following formula: Where R 5 is H or methyl, and x is an integer between 1 and 100, inclusive.

38. The compound according to claim 37, wherein yes 39. The compound according to claim 36 or 37, wherein x is an integer between 1 and 25, inclusive.

40. The compound according to claim 36 or 37, wherein x is an integer between 1 and 15, inclusive.

41. The compound according to claim 36 or 37, wherein x is 13.

42. A compound according to any one of claims 1 to 25, wherein L 2 yes 43. The compound according to claim 42, wherein POLY 2 It is a residue of polyethylene glycol (PEG), methoxypolyethylene glycol (mPEG), polypropylene glycol (PPG), a copolymer of ethylene glycol and propylene glycol, or polysarcosine.

44. The compound according to claim 43, wherein POLY 2 yes Where R 5 is H or methyl, and x is an integer between 1 and 100, inclusive.

45. The compound according to claim 44, wherein yes 46. ​​The compound according to claim 43 or 44, wherein x is an integer between 1 and 25, inclusive.

47. The compound according to claim 43 or 44, wherein x is an integer between 1 and 15, inclusive.

48. The compound according to claim 43 or 44, wherein x is 11.

49. A compound according to any one of claims 1 to 48, wherein L 3 Does not exist, or L 3 It is -C(O)-AA-, -C(O)-AA-Z-(CR a R b ) a -Z-(CR a R b ) a -C(O)-, -C(O)-Z-(CR a R b ) a -C(O)-ZL 4 -OC(O)-, -Z-AA-, -AA-, or -C(O)-.

50. The compound according to any one of claims 1 to 48, wherein L 3 It is -C(O)-AA-.

51. A compound according to any one of claims 1 to 48, wherein L 3 is -C(O)-AA-Z-(CR a R b ) a -Z-(CR a R b ) a -C(O)-, -Z-AA-, or -AA-.

52. The compound according to claim 51, wherein Z is -NH-.

53. A compound according to any one of claims 1 to 48, wherein L 3 It is -AA-.

54. The compound according to claim 53, wherein -AA-Yes 55. A compound according to any one of claims 50 to 53, wherein AA is a dipeptide residue, a tripeptide residue, a tetrapeptide residue, or a pentapeptide residue.

56. The compound according to claim 55, wherein AA is a tripeptide residue.

57. A compound according to any one of claims 50-53 and 55-56, wherein AA contains at least one amino acid residue selected from the group consisting of alanine, glycine, valine, and asparagine.

58. A compound according to any one of claims 50-53 and 55-56, wherein AA contains at least one amino acid residue selected from alanine and glycine.

59. A compound according to any one of claims 50 to 53, wherein AA is selected from the group consisting of:

60. The compound according to any one of claims 1 to 48, wherein L 3 is -C(O)-Z-(CR a R b ) a -C(O)-Z-L 4 -OC(O)-; L 4 yes and Su is the hexose form of a monosaccharide.

61. The compound according to claim 60, wherein Su is 62. The compound according to claim 61, wherein Su is 63. A compound according to any one of claims 1 to 62, wherein D is a cytotoxic payload selected from the group consisting of a tubulin inhibitor, a DNA damaging agent, a DNA topoisomerase I inhibitor, a DNA topoisomerase II inhibitor, and an RNA polymerase II inhibitor.

64. The compound according to claim 63, wherein D is a cytotoxic payload selected from the group consisting of: hemiasterlin or a derivative thereof, camptothecin or a derivative thereof, anthracycline or a derivative thereof, PNU-159682 or a derivative thereof, PBD or a derivative thereof, and duocarmycin or a derivative thereof.

65. The compound according to claim 63, wherein D is selected from: or its derivatives.

66. The compound according to claim 63, wherein D is the exatecan payload.

67. The compound according to claim 1, wherein The compound is represented by the following formula: or a pharmaceutically acceptable salt thereof.

68. The compound according to claim 67, wherein The compound is represented by the following formula: or a pharmaceutically acceptable salt thereof.

69. The compound according to claim 67, wherein The compound is represented by the following formula: or a pharmaceutically acceptable salt thereof.

70. The compound according to claim 1, wherein The compound is represented by the following formula: or a pharmaceutically acceptable salt thereof.

71. The compound according to claim 70, wherein The compound is represented by the following formula: or a pharmaceutically acceptable salt thereof.

72. The compound according to claim 71, wherein The compound is represented by the following formula: or a pharmaceutically acceptable salt thereof.

73. The compound according to claim 1, wherein The compound is selected from: or a pharmaceutically acceptable salt thereof.

74. The compound according to claim 73, wherein The compound is selected from: or a pharmaceutically acceptable salt thereof.

75. The compound according to claim 72, wherein The compound is selected from: or a pharmaceutically acceptable salt thereof.

76. The compound according to claim 22, having the structure shown in the following formula: or a pharmaceutically acceptable salt thereof.

77. The compound according to claim 76, having the structure shown in the following formula: or a pharmaceutically acceptable salt thereof.

78. A conjugate comprising a compound according to any one of claims 1 to 77 or a pharmaceutically acceptable salt thereof linked to a second compound.

79. The conjugate according to claim 78, having a structure shown in Formula II: or a pharmaceutically acceptable salt thereof; in, L 1 Selected from Ring A is an optionally substituted bridged, fused, or spiro bicyclic carbocycle, or an optionally substituted bridged, fused, or spiro bicyclic heterocycle, wherein the carbocycle or the heterocycle of Ring A is optionally substituted by one or more selected from alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents; Ring B is an optionally substituted N-linked bridged, fused, or spiro bicyclic heterocycle, wherein Ring B is optionally substituted by one or more selected from alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents; R a and R b are independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; a is an integer independently selected from 0, 1, 2, 3, 4, 5 and 6; b is an integer selected from 0 and 1; R 1 is H or alkyl, the alkyl being optionally substituted by one or more selected from cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aryl and heteroaryl substituents; R 2 and R 3 are each independently selected from H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; Y is *-C(O)-(CR a R b ) c -NH-, or *-C(O)-(CR a R b ) c -, where * indicates the position where Y is attached to RL; c is an integer selected from 1, 2, 3, 4, 5 and 6; RL is the reactive residue; L 2 Does not exist, or L 2 is a linker comprising a hydrophilic polymer residue; L 3 is absent, or L 3 is -C(O)-AA-, -C(O)-AA-Z-(CR a R b ) a -Z-(CR a R b ) a -C(O)-, -C(O)-Z-(CR a R b ) a -C(O)-Z-L 4 -OC(O)-, -Z-AA-, -AA-, -C(O)-, -C(O)-AA-Z-(CR a R b ) a -, -AA-C(O)-, -C(O)-(CR a R b ) a -Z-(CR a R b ) a -Z-AA-C(O)-, -C(O)O-L 4 -Z-C(O)-(CR a R b ) a -Z-C(O)-, -AA-Z-, or -(CR a R b ) a -Z-AA-C(O)-; Z is selected from -NR 2 - and -O-; AA is an amino acid residue or a peptide residue; L 4 yes Where Su is the hexose form of a monosaccharide; d is an integer independently selected from 1, 2 and 3; D is the cytotoxic payload; COMP is the residue of a second compound; and represents the point of attachment to the rest of the compound.

80. The conjugate according to claim 78, having a structure shown in formula (IV): or a pharmaceutically acceptable salt and / or regioisomer thereof; in, L 5 is a linker comprising an unnatural amino acid; Y is *-C(O)-(CR a R b ) c -NH-, or *-C(O)-(CR a R b ) c -, where * indicates the position where Y is attached to RL; c is an integer selected from 1, 2, 3, 4, 5 and 6; RL is the reactive residue; L 2 Does not exist, or L 2 is a linker comprising a hydrophilic polymer residue; L 3 is absent, or L 3 is -C(O)-AA-, -C(O)-AA-Z-(CR a R b ) a -Z-(CR a R b ) a -C(O)-, -C(O)-Z-(CR a R b ) a -C(O)-Z-L 4 -OC(O)-, -Z-AA-, -AA-, -C(O)-, -C(O)-AA-Z-(CR a R b ) a -, -AA-C(O)-, -C(O)-(CR a R b ) a -Z-(CR a R b ) a -Z-AA-C(O)-, -C(O)O-L 4 -Z-C(O)-(CR a R b ) a -Z-C(O)-, -AA-Z-, or -(CR a R b ) a -Z-AA-C(O)-; R a and R b are independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; a is an integer independently selected from 1, 2, 3, 4, 5 and 6; Z is selected from -NR 2 - and -O-; AA is an amino acid residue or a peptide residue; L 4 yes Where Su is the hexose form of a monosaccharide; d is an integer independently selected from 1, 2 and 3; D is the cytotoxic payload; COMP is the residue of a second compound; and represents the point of attachment to the rest of the compound.

81. The conjugate according to claim 79 or 80, wherein COMP is a residue of a polypeptide.

82. The conjugate according to claim 79 or 80, wherein COMP is the residue of antibody.

83. The conjugate according to claim 79 or 80, wherein COMP is the residue of the antibody chain.

84. The conjugate according to any one of claims 79 and 81-83, wherein L 1 yes 85. The conjugate according to any one of claims 79 and 81-83, wherein L 1 Ring A is an optionally substituted bridged, fused, or spirocyclic bicyclic carbocyclic ring, wherein the carbocyclic ring of Ring A is optionally substituted with one or more substituents selected from the group consisting of: 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl.

86. The conjugate according to claim 85, wherein L 1 Ring A is optionally substituted C 4-12 Bridged, fused, or spiro bicyclic carbocycles.

87. The conjugate according to claim 86, wherein L 1 Ring A is optionally substituted C 4-12 Bridged bicyclic carbocycles.

88. The conjugate according to claim 87, wherein L 1 Ring A is optionally substituted C 4-8 Bridged bicyclic carbocycles.

89. The conjugate according to any one of claims 79 and 81-83, wherein L 1 yes 90. The conjugate according to any one of claims 79 and 81-83, wherein L 1 Ring B is an optionally substituted 5 to 12-atom N-linked bridged, fused, or spiro bicyclic heterocyclic ring, comprising 1, 2 or 3 heteroatoms independently selected from N, O and S, including the N atom to which the ring is attached, wherein the heterocyclic ring of Ring B is optionally substituted by one or more selected from C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)-、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl and heteroarylalkyl substituents.

91. The conjugate according to any one of claims 79 and 81-83, wherein L 1 Ring B is an optionally substituted 5 to 12-atom N-attached spiro bicyclic heterocycle containing 1, 2 or 3 heteroatoms independently selected from N, O and S, including the N atom to which the ring is attached.

92. The conjugate according to any one of claims 79 and 81-83, wherein L 1 Selected from wherein m is an integer selected from 1, 2, 3, 4 and 5; and n and o are each an integer independently selected from 1, 2 and 3.

93. The conjugate according to any one of claims 79 and 81-83, wherein L 1 Selected from wherein m is an integer selected from 1, 2, 3, 4 and 5; and n and o are each an integer independently selected from 1, 2 and 3.

94. The conjugate according to any one of claims 79 and 81-83, wherein Ring B is selected from Where X 1 , X 2 , X 3 and X 4 are independently selected from -C(R 4 )2-, -NH-, -O-, and -S-, where X 1 , X 2 and X 3 When both exist, X 1 -X 3 At least one of them is -C(R 4 )2-, and when X 1 , X 2 , X 3 and X 4 When both exist, X 1 -X 4 At least two of them are -C(R 4 )2-; and R 4 Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; or two R on the same carbon 4 The groups are taken together to form an oxo group.

95. The conjugate according to claim 94, wherein Ring B is selected from 96. The conjugate according to any one of claims 78 and 81-83, wherein Ring B is selected from Where X 1 , X 2 , X 3 and X 4 are independently selected from -C(R 4 )2-, -NH-, -O-, and -S-, where X 1 , X 2 and X 3 When both exist, X 1 -X 3 At least one of them is -C(R 4 )2-, and when X 1 , X 2 , X 3 and X 4 When both exist, X 1 -X 4 At least two of them are -C(R 4 )2-; and R 4 Independently selected from H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; or two R on the same carbon 4 The groups are taken together to form an oxo group.

97. The conjugate according to claim 96, wherein Ring B is selected from 98. The conjugate according to any one of claims 79 and 81-83, wherein Ring A is selected from Where X 1 , X 2 , X 3 and X 4 are independently selected from -C(R 4 )2-, -NH-, -O-, and -S-, where X 1 , X 2 and X 3 When both exist, X 1 -X 3 At least one of them is -C(R 4 )2-, and when X 1 , X 2 , X 3 and X 4 When both exist, X 1 -X 4 At least two of them are -C(R 4 )2-; X 5 Yes-CR 4 -or-N-; and R 4 Independently selected from H, C 2-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-12 Cycloalkyl, halogen, alkoxy, -CN, -NO2, -OH, -N(R 2 R 3 )2、-C(O)N(R 2 R 3 )2. -C(O)OR 2 , aminoalkyl, hydroxyalkyl, haloalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; or two R on the same carbon 4 The groups are taken together to form an oxo group.

99. The conjugate according to any one of claims 79 and 81-83, wherein L 1 yes 100. The conjugate according to claim 99, wherein L 1 yes 101. The conjugate according to claim 80, wherein L 5 is a linker comprising at least one amino acid selected from cysteic acid, hydroxyproline (Hyp), β-alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3-nitrotyrosine, nitroarginine, pyroglutamate (Pyr), naphthylalanine (Nal), 2,4-diaminobutyric acid (DAB), methionine sulfoxide, and methionine sulfone.

102. The conjugate according to claim 101, wherein L 5 is included connector.

103. The conjugate according to claim 102, wherein L 5 yes 104. The conjugate according to any one of claims 73 to 103, wherein L 2 Selected from -(CR a R b ) a -POLY 1 -、-POLY 1 -、-(CR a R b ) a -POLY 1 -(CR a R b ) a -、 POLY 1 Is a divalent hydrophilic polymer residue; POLY 2 is a residue of a hydrophilic polymer; and represents the point of attachment to the rest of the compound.

105. The conjugate according to any one of claims 79 to 104, wherein Y is -C(O)-(CR a R b ) c -NH-.

106. The conjugate according to claim 105, wherein Y is -C(O)-CH2CH2-NH-.

107. The conjugate according to any one of claims 79 to 104, wherein Y is -C(O)-(CR a R b ) c -.

108. The conjugate according to claim 107, wherein Y is -C(O)-(CH2)4-.

109. The conjugate according to any one of claims 79 to 108, wherein RL contains a triazole.

110. The conjugate according to any one of claims 79 to 108, wherein The RL is selected from the group consisting of:

111. The conjugate according to claim 110, wherein RL is and represents the point of attachment to the rest of the compound.

112. The conjugate according to any one of claims 79-103 and 109-111, wherein L 2 Yes-(CR a R b ) a -POLY 1 -.

113. The conjugate according to claim 112, wherein -POLY 1 - is a divalent residue of polyethylene glycol (PEG), polypropylene glycol (PPG), or a copolymer of ethylene glycol and propylene glycol.

114. The conjugate according to claim 113, wherein The divalent hydrophilic polymer residue has a structure shown in the following formula: Where R 5 is H or methyl, and x is an integer between 1 and 100, inclusive.

115. The conjugate according to claim 114, wherein x is 1-25, inclusive.

116. The conjugate according to claim 115, wherein x is 13.

117. The conjugate according to any one of claims 79-103 and 109-111, wherein L 2 yes in represents the point of attachment to the rest of the compound.

118. The conjugate according to claim 117, wherein POLY 2 It is polyethylene glycol (PEG), methoxypolyethylene glycol (mPEG), polypropylene glycol (PPG), or a copolymer of ethylene glycol and propylene glycol.

119. The conjugate according to claim 118, wherein POLY 2 yes Where R 5 is H or methyl, and x is an integer between 1 and 100, inclusive.

120. The conjugate according to claim 119, wherein x is 1 to 25, inclusive.

121. The conjugate according to claim 120, wherein x is 11.

122. The conjugate according to any one of claims 79 to 121, wherein L 3 It is -C(O)-AA-.

123. The conjugate according to any one of claims 79 to 121, wherein L 3 is -C(O)-AA-Z-(CR a R b ) a -Z-(CR a R b ) a -C(O)-, -Z-AA-, or -AA-.

124. The conjugate according to any one of claims 79 to 121, wherein L 3 It is -AA-.

125. The conjugate according to claim 124, wherein -AA-Yes 126. The conjugate according to any one of claims 122 to 124, wherein AA is a dipeptide residue, a tripeptide residue, a tetrapeptide residue, or a pentapeptide residue.

127. The conjugate according to claim 126, wherein AA is a tripeptide residue.

128. The conjugate according to any one of claims 122-124 and 126-127, wherein AA contains at least one amino acid residue selected from the group consisting of alanine, glycine, valine, and asparagine.

129. The conjugate according to any one of claims 122-124 and 126-127, wherein AA contains at least one amino acid residue selected from alanine and glycine.

130. The conjugate according to any one of claims 122-124 and 126-127, wherein AA is selected from the group consisting of:

131. The conjugate according to any one of claims 79 to 121, wherein L 3 is -C(O)-Z-(CR a R b ) a -C(O)-Z-L 4 -OC(O)-; L 4 yes and Su is the hexose form of a monosaccharide.

132. The conjugate according to claim 131, wherein Su is 133. The conjugate according to claim 132, wherein Su is 134. The conjugate according to any one of claims 79 to 133, wherein The D is a cytotoxic payload selected from the group consisting of a tubulin inhibitor, a DNA damaging agent, a DNA topoisomerase I inhibitor, a DNA topoisomerase II inhibitor, and an RNA polymerase II inhibitor.

135. The conjugate according to claim 134, wherein D is a cytotoxic payload selected from the group consisting of: hemiasterlin or a derivative thereof, camptothecin or a derivative thereof, camptothecin, anthracycline or a derivative thereof, PNU-159682 or a derivative thereof, PBD or a derivative thereof, and duocarmycin or a derivative thereof.

136. The conjugate according to claim 134, wherein D is selected from in represents the point of attachment to the rest of the compound.

137. The conjugate according to claim 134, wherein D is the exatecan payload.

138. The conjugate of claim 79, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

139. The conjugate according to claim 138, having a structure shown in the following formula: or a pharmaceutically acceptable salt thereof.

140. The conjugate according to claim 80, having a structure shown in the following formula: or a pharmaceutically acceptable salt thereof.

141. A pharmaceutical composition comprising a compound according to any one of claims 1 to 77 or a pharmaceutically acceptable salt thereof, or a conjugate according to any one of claims 78 to 140 or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient, carrier, or diluent.

142. A method for treating a disease or condition in a subject in need thereof, comprising administering a compound or pharmaceutically acceptable salt thereof as described in any one of claims 1-77, a conjugate or pharmaceutically acceptable salt thereof as described in any one of claims 78-140, or a pharmaceutical composition as described in claim 141.

143. A method for inhibiting tubulin polymerization in a subject in need thereof, comprising administering a compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-77, a conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 78-140, or a pharmaceutical composition as described in claim 141.

144. The method of claim 142, wherein: The disease or disorder is abnormal cell proliferation.

145. The method of claim 144, wherein: Such abnormal cell proliferation is cancer.

146. The method of claim 145, wherein: The cancer is small cell lung cancer, non-small cell lung cancer, ovarian cancer, platinum-resistant ovarian cancer, ovarian adenocarcinoma, endometrial cancer, breast cancer, breast cancer overexpressing HER2, triple-negative breast cancer, lymphoma, large cell lymphoma, diffuse mixed histiocytic and lymphocytic lymphoma, follicular B-cell lymphoma, colon cancer, colon carcinoma (colon carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, melanoma, prostate cancer, or multiple myeloma.

147. Use of a compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-77, a conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 78-140, or a pharmaceutical composition as described in claim 141 for treating a disease or condition in a subject in need thereof.

148. Use of a compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-77, a conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 78-140, or a pharmaceutical composition as described in claim 141 in the preparation of a medicament for treating a disease or condition in a subject in need thereof.

149. The use according to claim 147 or 148, wherein The treatment is the inhibition of tubulin polymerization. Use of the compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 77, the conjugate or pharmaceutically acceptable salt thereof according to any one of claims 78 to 140, or the pharmaceutical composition according to claim 141 in the preparation of a medicament for inhibiting tubulin polymerization in a subject in need thereof.

150. The use according to claim 147 or 148, wherein The disease or condition is abnormal cell proliferation, or cancer.

151. A method of reducing cell proliferation in a subject in need thereof, comprising administering a compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-77, a conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 78-140, or a pharmaceutical composition as described in claim 141.

152. A method for preparing a conjugate, comprising contacting a compound as described in any one of claims 1-77 with a second compound under conditions suitable for coupling the compound as described in any one of claims 1-77 with the second compound; wherein the second compound comprises an alkyne, a cyclooctyne, a strained olefin, a tetrazine, a methylcyclopropene, a thiol, a maleimide, a carbonyl, an amine, an oxyamine, or an azide.

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