Antibody drug conjugate as well as preparation method and application thereof

Abstract

The invention relates to a novel antibody drug conjugate which is shown in a formula (I) and takes a CBL-b inhibitor as an immune excitation load, a preparation method of the novel antibody drug conjugate, a pharmaceutical composition containing the antibody drug conjugate shown in the formula (I), and medical application of the antibody drug conjugate or the pharmaceutical composition shown in the formula (I).

Description

[0001] This invention claims priority to an earlier application filed on July 25, 2024, with China National Intellectual Property Administration, patent application number 202411004557.2, entitled "Antibody-Drug Conjugates and Preparation Methods Thereof and Uses Thereof". The entire contents of the aforementioned earlier application are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the field of biomedicine, specifically to novel antibody-drug conjugates, their preparation methods, and their uses. Background Technology

[0003] While immunotherapy, primarily based on immune checkpoint inhibitors, has improved the prognosis of patients with various malignant tumors, a significant portion of the population still fails to achieve effective clinical benefits or develops drug resistance. The E3 ligase CBL-b has been shown to play a crucial negative feedback regulatory role in the immune system. CBL-b inhibitors can significantly enhance immune system activity, promote the release of cytokines such as IL-2 and IFN-γ, promote T cell and NK cell proliferation, and enhance the tumor-killing ability of immune cells. Simultaneously, CBL-b inhibitors can restore the function of exhausted T cells and have shown synergistic effects with the immune checkpoint inhibitor anti-PD1 antibody in multiple in vivo models.

[0004] Systemic CBL-b gene knockout in mice resulted in a significant and strong autoimmune response, indicating that systemic CBL-b inhibition carries the risk of inducing a cytokine storm or systemic immune response. Furthermore, due to the high homology (86% amino acid sequence similarity) between the functional regions of CBL-b and c-CBL, small molecule inhibitors struggle to achieve sufficient selectivity. Dual inhibition of CBL-b and c-CBL in dendritic cells (DCs) causes severe hepatotoxicity, leading to liver fibrosis and cirrhosis. Studies have shown that PD-1 is highly expressed in tumor-infiltrating immune cells. Attaching immune activators to PD-1 antibodies can specifically activate immune cells in the tumor microenvironment, reducing the risk of systemic adverse reactions. Therefore, to avoid direct systemic activation of immune cells, this study conjugated a CBL-b small molecule inhibitor as an immune agonist to a PD-1 antibody, constructing a novel antibody-drug conjugate. This aims to enhance the efficacy of PD-1 antibodies while reducing the toxic side effects of systemic administration of the CBL-b small molecule inhibitor. Summary of the Invention

[0005] In a first aspect, this disclosure provides an antibody-drug conjugate of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0006]

[0007] in,

[0008] Ab stands for antibody unit;

[0009] L represents the connecting subunit;

[0010] q is selected from 1 to 10;

[0011] D is a payload having a structure selected from the following, the payload being covalently connected to the connecting subunit.

[0012]

[0013] in,

[0014] Ring A is selected from

[0015] Z 1 Z 2 Z 3 Z 4 Z 5 Z 6 Z 7 Z 8 Z 9 Z 10 and Z 11 Independently selected from CH, CR a Or N;

[0016] R a Selected from halogens, OH, NH2, NO2, CN, C1-C6 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C6 alkylamino, -C1-C6 alkyl-OH, C3-C6 cycloalkyl, C3-C6 cycloalkyl-O-, C3-C6 cycloalkyl-NH-, 4-7 membered heterocyclic, 4-7 membered heterocyclic-O- or 4-7 membered heterocyclic-NH-;

[0017] R 5 Selected from

[0018] R 6 R 7 It is independently selected from H, D, =O, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -C1-C6 alkyl-OH;

[0019] The ring C is selected from 4-10 member nitrogen-containing heterocyclic groups;

[0020] R 8Selected from OH, =O, CN, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, or C3-C6 cycloalkyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, or C3-C6 cycloalkyl is optionally converted to R 8a replace;

[0021] R 8a Selected from OH or C1-C6 alkoxy groups;

[0022] n is an integer selected from 0 to 3;

[0023] R 9 Selected from C1-C6 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic groups, wherein the C1-C6 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic group is optionally R 9a replace;

[0024] R 9a Selected from OH, CN, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -C1-C6 alkyl-OH, -S(O)2-C1-C6 alkyl or C3-C6 cycloalkyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -C1-C6 alkyl-OH, -S(O)2-C1-C6 alkyl or C3-C6 cycloalkyl is optionally R 9b replace;

[0025] R 9b Selected from OH, halogens, or CN;

[0026] Y1, Y2, Y3, and Y4 are independently selected from CR b Or N;

[0027] R b Selected from H, halogen, OH, NH2, CN, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C6 alkylamino, C3-C6 cycloalkyl or 4-7 membered heterocyclic groups;

[0028] k is selected from 0 or 1;

[0029] R 1 R 2 R 3 and R 4 Independently selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic group; or R 1 R 2The atoms bonded to it together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or 4-7 membered heterocyclic group is optionally R 1b Replace; or R 1 R 2 R 3 and R 4 The atoms bonded to it together form a phenyl group, which is optionally R 2b replace;

[0030] R 1b and R 2b Independently selected from halogens, CN, =O, OH, NH2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, or C1-C6 alkylamino, wherein the C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, or C1-C6 alkylamino is optionally R 1c replace;

[0031] R 1c Selected from OH, halogens, or CN;

[0032] Cycle B is selected from a 5-membered heteroaryl group, which is optionally substituted with a C1-C6 alkyl group.

[0033] In some implementation schemes, Z 1 Z 2 Z 3 Z 4 Z 5 Z 6 Z 7 Z 8 Z 9 Z 10 and Z 11 Independently selected from CH or CR a .

[0034] In some implementations, ring A is selected from...

[0035] m is selected from integers from 0 to 3.

[0036] In some implementation schemes, R a Selected from CH3, CF3, F or cyclopropyl.

[0037] In some implementations, k is selected from 0.

[0038] In some implementation schemes, R 1 Selected from H, R 2 Selected from cyclopropyl, or R 1 R 2 The atoms connected to it form together

[0039] In some implementation schemes, R 5 Selected from

[0040] In some implementation schemes, R 5 Selected from

[0041] In some implementation schemes, Selected from

[0042] In some implementation schemes, Selected from

[0043] In some implementation schemes, ring B is selected from... The Optionally substituted with C1-C6 alkyl groups.

[0044] In some implementation schemes, ring B is selected from...

[0045] In some implementation schemes, R 1 R 2 R 3 and R 4 It is independently selected from H, CH3, CF3, F or cyclopropyl.

[0046] In some implementations, D is covalently linked to the linker unit via its nitrogen or oxygen atoms.

[0047] In some implementations, D uses its R 5 The nitrogen atoms contained therein are covalently linked to the linker subunit.

[0048] In some implementations, D is a payload having a structure selected from the following, the payload being covalently connected to the connecting subunit:

[0049]

[0050] Among them, rings C and R 1 R 2 R 3 R 4 R 6 R 7 R 8 R a Y1, Y2, Y3, Y4, n and k are defined in the same way as in formula (A), and m is selected from integers from 0 to 3.

[0051] In some implementations, D is a payload having a structure selected from the following, the payload being covalently connected to the connecting subunit:

[0052]

[0053] Among them, R 1 R 2 R 3 R 4 R 6 R 7 R 9 R a Y1, Y2, Y3, Y4 and k are defined in the same way as in formula (A), and m is selected from integers from 0 to 3.

[0054] In some implementations, D is a payload having a structure selected from the following, the payload being covalently connected to the connecting subunit:

[0055]

[0056] Among them, rings C and R 1 R 2 R 3 R 4 R 6 R 7 R 8 R a Y1, Y2, Y3, Y4, n and k are defined in the same way as in formula (A), and m is selected from integers from 0 to 3.

[0057] In some implementations, D is a payload having a structure selected from the following, the payload being covalently connected to the connecting subunit:

[0058]

[0059] Among them, R 1 R 2 R 3 R 4 R 6 R 7 R 9 R a Y1, Y2, Y3, Y4 and k are defined in the same way as in formula (A), and m is selected from integers from 0 to 3.

[0060] In some implementations, D is a payload having a structure selected from the following, the payload being covalently connected to the connecting subunit:

[0061]

[0062] Among them, rings C and R 1 R2 R 3 R 4 R 6 R 7 R 8 R a Y1, Y2, Y3, Y4, n and k are defined in the same way as in formula (A), and m is selected from integers from 0 to 3.

[0063] In some implementations, D is a payload having a structure selected from the following, the payload being covalently connected to the connecting subunit:

[0064]

[0065] Among them, R 1 R 2 R 3 R 4 R 6 R 7 R 9 R a Y1, Y2, Y3, Y4 and k are defined in the same way as in formula (A), and m is selected from integers from 0 to 3.

[0066] In some implementations, D is a payload having a structure selected from the following, the payload being covalently connected to the connecting subunit:

[0067] In some implementations, D is a payload having a structure selected from the following, the payload being covalently connected to the connecting subunit:

[0068] In some implementations, the Ab is selected from anti-PD-1 antibodies.

[0069] In some implementations, Ab is selected from pembrolizumab.

[0070] In some implementations, L is selected from protease-cleavable linker units.

[0071] In some implementation schemes, L is selected from p is an integer from 2 to 10.

[0072] In some implementation schemes, L is selected from

[0073] In some implementations, q is selected from 2 to 8. In some implementations, q is selected from 4 to 8. In some implementations, q is selected from 6 to 8. In some implementations, q is selected from 8. In some implementations, q is selected from 6.38.

[0074] In some embodiments, the antibody-drug conjugate of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof is selected from the antibody-drug conjugate of formula (II), its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0075]

[0076] Among them, Ab, L, ring C, R 1 R 2 R 3 R 4 R 6 R 7 R 8 R a Y1, Y2, Y3, Y4, n, k and q are defined in the same way as in equation (I), and m is selected from integers from 0 to 3.

[0077] In some embodiments, the antibody-drug conjugate of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof is selected from the antibody-drug conjugate of formula (II-1), its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0078]

[0079] In this context, Ab, L, and q are defined by the same formula (I).

[0080] In some embodiments, the antibody-drug conjugate of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof is selected from the antibody-drug conjugate of formula (II-2), its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0081]

[0082] In this context, Ab, L, and q are defined by the same formula (I).

[0083] In some embodiments, the antibody-drug conjugate represented by formula (I), its stereoisomers, or pharmaceutically acceptable salts thereof are selected from the following antibody-drug conjugates, their stereoisomers, or pharmaceutically acceptable salts thereof.

[0084] q is selected from 1 to 8.

[0085] On the other hand, this disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate or a stereoisomer thereof represented by the formula (I) above, and a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0086] On the other hand, this disclosure provides a method for treating mammalian tumors, comprising administering to a mammal, preferably a human, a therapeutically effective amount of the antibody-drug conjugate of the aforementioned formula (I) or its stereoisomer or its pharmaceutically acceptable salt or a pharmaceutical composition thereof.

[0087] On the other hand, this disclosure provides the use of the antibody-drug conjugate or its stereoisomer or its pharmaceutically acceptable salt or pharmaceutical composition shown in Formula (I) in the preparation of a medicament for treating tumors.

[0088] On the other hand, this disclosure provides the use of the antibody-drug conjugate or its stereoisomer or its pharmaceutically acceptable salt or pharmaceutical composition shown in Formula (I) in the treatment of tumors.

[0089] On the other hand, this disclosure provides antibody-drug conjugates of the aforementioned formula (I) or stereoisomers thereof or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, for the treatment of tumors.

[0090] Terminology Definitions and Explanations

[0091] Unless otherwise stated, the terms used in this disclosure have the following meanings: the definitions of groups and terms recorded in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in the examples, etc., can be arbitrarily combined and combined with each other. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0092] The terms “comprising,” “including,” and “having” are used interchangeably in this document to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” in this document also provides for schemes “consisting of…”.

[0093] The term "linker" or "linker unit" in this article refers to a chemical structural segment that is connected at one end to an antibody unit and at the other end to a drug unit.

[0094] The term "payload" in this article refers to a biologically active substance.

[0095] The term "antibody-drug conjugate" in this article refers to a monoclonal antibody or antibody fragment linked to a biologically active drug via a stable linker unit.

[0096] The term "DAR" or "drug-antibody ratio" in this document refers to the average number of drugs linked to each antibody molecule. In the antibody-drug conjugates disclosed herein, DAR is defined by the variable "q," which can be an integer or a decimal.

[0097] In this article, "antibody" or "antibody unit" refers to an antibody or antigen-binding fragment that can specifically bind to a target antigen, such as monoclonal antibody, polyclonal antibody, monospecific antibody, multispecific antibody (e.g., bispecific antibody), monovalent antibody, multivalent antibody, complete antibody, fragment of complete antibody, naked antibody, conjugated antibody, chimeric antibody, humanized antibody, fully human antibody, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH, and scFv, etc.

[0098] The term "antibody" as used in the broadest sense herein refers to a polypeptide or combination of polypeptides containing sufficient sequence from the variable region of the immunoglobulin heavy chain and / or sufficient sequence from the variable region of the immunoglobulin light chain to specifically bind to an antigen. The term "antibody" as used herein encompasses a variety of forms and structures, as long as they exhibit the desired antigen-binding activity. For example, in some embodiments, the term "antibody," "antibody unit," or "Ab" as used herein refers to an antibody that does not include the N297-linked glycosyl group of the heavy chain. The terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably herein and refer to a structure having a substantially similar structure to that of a natural antibody. The term "antibody" as used herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, complete antibodies, fragments of complete antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0099] The terms "antibody fragment" or "antigen-binding fragment" used herein refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, etc., regardless of its structure. Antibody fragments bind to the same antigen recognized by the intact antibody. "Antibody fragment" or "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions like an antibody by binding to a specific antigen and forming a complex. For example, antibody fragments include isolated fragments consisting of light chain variable regions, "Fv" fragments consisting of heavy and light chain variable regions, recombinant single-chain polypeptide molecules (scFv) in which the light and heavy chain variable regions are linked by peptide linkers, and the smallest recognizing unit consisting of amino acid residues mimicking hypervariable regions. The terms "antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not possess the full structure of a complete antibody, but only contain a portion or a partial variant of the complete antibody capable of binding antigens. "Antigen-binding fragment" or "antibody fragment" as used herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH, and scFv.

[0100] The term "antibody" in this article also includes alternative protein scaffolds or artificial scaffolds having a transplantable complementarity-determining region (CDR) or a CDR derivative. Such scaffolds include antibody-derived scaffolds (which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art for transplanting CDRs, including but not limited to tendinins, fibronectin, peptide aptamers, etc.

[0101] The term "antibody" in this article includes typical "quadruple-chain antibodies," which belong to immunoglobulins composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to-C-terminal direction. Optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to-C-terminal direction. Heavy chains are linked to each other and to each other with disulfide bonds, forming a "Y"-shaped structure. Because the amino acid composition and sequence of the immunoglobulin heavy chain constant region differ, their antigenicity also differs. Based on this, the "immunoglobulins" in this article can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can possess either a κ or λ chain.

[0102] The term "antibody" in this article also includes antibodies that do not contain light chains, such as heavy-chain antibodies (HCAbs) produced by camels such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanacos (Lamaguanicoe), and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (IgNARs) found in cartilaginous fish such as sharks.

[0103] The “antibody” in this article can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0104] In this article Indicates the connection site.

[0105] The diagrammatic representation of racemic or enantiomerically pure compounds in this article is derived from Maehr, J. Chem. Ed. 1985, 62:114-120. Unless otherwise specified, wedge bonds and virtual wedge bonds are used. and ) represents the absolute configuration of a solid center, using black solid bonds and imaginary bonds ( and ) indicates the relative configuration of a stereocenter (such as the cis-trans configuration of alicyclic compounds).

[0106] The term "tautomer" refers to a functional group isomer resulting from the rapid movement of an atom between two positions within a molecule. The compounds disclosed herein can exhibit tautomerism. Tautomers can exist in two or more interconvertible forms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer typically yield a mixture whose physicochemical properties are consistent with those of the mixture of compounds. The equilibrium position depends on the intramolecular chemical characteristics. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the ketone form is dominant; while in phenols, the enol form is dominant. This disclosure encompasses all tautomeric forms of the compounds.

[0107] The term "stereoisomer" refers to isomers that are produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0108] The compounds disclosed herein may have asymmetric atoms such as carbon, sulfur, nitrogen, and phosphorus atoms, or asymmetric double bonds; therefore, the compounds disclosed herein may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E-type and Z-type geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof or other mixtures, such as mixtures enriched with enantiomers or diastereomers. All such isomers and mixtures thereof are within the scope of the definition of the compounds disclosed herein. Structure The double bond can be either E-configuration or Z-configuration. Alkyl groups and other substituents may contain additional asymmetric carbon, sulfur, nitrogen, or phosphorus atoms. All isomers involved in all substituents, as well as mixtures thereof, are included within the scope of the definition of compounds disclosed herein. Compounds containing asymmetric atoms disclosed herein can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0109] The term "substituted" refers to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted; oxo substitution does not occur on aromatic groups.

[0110] The terms “optional” or “optionally” mean that the event or condition subsequently described may or may not occur, including both the occurrence and non-occurrence of said event or condition. For example, “optionally” substituted with a halogen means that the ethyl group can be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0111] When any variable (e.g., R) a R b When a group appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, if a group is surrounded by two R... b Replaced, then each R b Each has its own independent options.

[0112] Unless otherwise specified, the linking direction of the linking groups mentioned in this article is arbitrary.

[0113] C in this article m -C n , refers to having an integer number of carbon atoms in the range mn.

[0114] The term "alkyl" refers to a compound with the general formula C1. n H 2n+1 A monovalent hydrocarbon group, which can be straight-chain or branched. The term "C1-C" is used. 10 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, ... 3-Methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc.; the term "C1-C6 alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms. The "C1-C6 alkyl" used herein... 10"alkyl" can include the range of "C1-C6 alkyl", "C1-C4 alkyl" or "C1-C3 alkyl", and the "C1-C6 alkyl" can further include "C1-C4 alkyl" or "C1-C3 alkyl".

[0115] The term "cycloalkyl" refers to a fully saturated monovalent carbon ring existing in the form of a monocyclic, fused, bridged, or spirocyclic ring. The term "C3-C6 cycloalkyl" should be understood to indicate a saturated monocyclic, fused, spirocyclic, or bridged ring having 3, 4, 5, or 6 carbon atoms, with specific examples including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0116] The term "heterocyclic group" refers to a fully saturated or partially saturated monovalent group existing in the form of a monocyclic, fused, spirocyclic, or bridged ring, whose ring atoms contain 1-5 heteroatoms or heteroatom groups (i.e., atomic groups containing heteroatoms). The "heteroatoms or heteroatom groups" include, but are not limited to, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH-, or -NHC(=O)NH-, etc. The term "4-7 membered heterocyclic group" refers to a heterocyclic group with 4, 5, 6, or 7 ring atoms, and whose ring atoms contain 1-3 independently selected heteroatoms or heteroatom groups as described above. Examples of 4-membered heterocyclic groups include, but are not limited to, azirrobutane and oxobutane; examples of 5-membered heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoalkyl, pyrazolyl, pyrrolinyl, 4,5-dihydrooxazole or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazine, trithiaalkyl, tetrahydropyridinyl or 4H-[1,3,4]thiadiazinyl; examples of 7-membered heterocyclic groups include, but are not limited to, diazacycloheptane. Preferably, "4-7-membered heterocyclic group" can include the ranges of "4-7-membered heterocyclic alkyl", "5-6-membered heterocyclic group", "5-6-membered heterocyclic alkyl", etc.

[0117] The term "halogen" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0118] The term "treatment" refers to surgical or therapeutic treatment aimed at preventing, slowing (reducing) undesirable physiological changes or lesions in the treated individual, such as the progression of cancer, autoimmune diseases, and viral infections. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Individuals requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When terms such as slowing, reducing, weakening, mitigating, or remission are used, they also imply elimination, disappearance, or non-occurrence.

[0119] The term "effective dose" refers to the amount of a therapeutic agent, administered alone or in combination with another therapeutic agent to cells, tissues, or subjects, that is effective in preventing or alleviating symptoms of a disease or the progression of that disease. "Effective dose" also refers to the amount of a compound sufficient to relieve symptoms, such as treating, curing, preventing, or alleviating an associated medical condition, or increasing the rate at which such symptoms are treated, cured, prevented, or alleviated. When an active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined amount of active ingredients that produce the therapeutic effect, regardless of whether they are administered in combination, consecutively, or simultaneously.

[0120] The term "subject" refers to an organism that receives treatment for a specific disease or condition as described in this disclosure. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals that receive treatment for a disease or condition.

[0121] The amount of the disclosed compound constituting a “therapeutic effective amount” varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the content of this disclosure.

[0122] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0123] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of an acid or base, including salts formed by a compound with an inorganic or organic acid, and salts formed by a compound with an inorganic or organic base.

[0124] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or salts thereof with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the disclosed compounds to an organism.

[0125] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0126] The word “comprise” or “include” and its English variants such as comprises or comprising can be understood as having an open, non-exclusive meaning, that is, “including but not limited to”.

[0127] This disclosure also includes compounds of this disclosure that are identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0128] Certain isotope-labeled compounds of this disclosure (e.g., using...) 3 H and 14 C-labeling can be used in the analysis of compound and / or substrate tissue distribution. Tritiumization (i.e., 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes, such as... 15 O、 13 N、 11 C and 18F can be used in positron emission tomography (PET) studies to determine substrate occupancy. The isotopically labeled compounds of this disclosure can typically be prepared by replacing the unlabeled reagent with an isotopically labeled reagent using a procedure similar to those disclosed in the schemes and / or examples below.

[0129] The pharmaceutical compositions disclosed herein are suitable for parenteral administration, such as in suitable unit dosage forms as sterile solutions, suspensions, or lyophilized products. For example, the pharmaceutical compositions disclosed herein may be in the form of sterile aqueous solutions for intramuscular or subcutaneous administration. The pharmaceutical compositions disclosed herein may accept other solvents or media, such as water, Ringer's solution, or isotonic sodium chloride solution, during use.

[0130] In all methods of administration of the compounds described herein, the daily dose is from 0.001 mg / kg to 600 mg / kg body weight, preferably from 0.05 mg / kg to 200 mg / kg body weight, more preferably from 0.1 mg / kg to 100 mg / kg body weight, in the form of single or separate doses.

[0131] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments disclosed herein.

[0132] The chemical reactions in the specific embodiments of this disclosure are carried out in a suitable solvent, which must be suitable for the chemical changes of this disclosure and the reagents and materials required therefor. In order to obtain the compounds of this disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0133] An important consideration in synthetic route planning in this field is the selection of appropriate protecting groups for reactive functional groups (such as amino and carboxyl groups in this disclosure). For example, see Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in this disclosure are incorporated herein by reference in their entirety.

[0134] The following abbreviations are used in this disclosure:

[0135] TFAA: Trifluoroacetic anhydride; DCM: Dichloromethane; Pd(dppf)Cl2: 1,1-bis(diphenylphosphine)ferrocene palladium chloride; Pd(PPh3)4: Tetra(triphenylphosphine)palladium; Pd(Pt-Bu3)2: Bis(tri-tert-butylphosphine)palladium; DIEA: N,N-diisopropylethylamine; DMF: N,N-dimethylformamide; DMF-DMA: N,N-dimethylformamide dimethyl acetal; Con.HCl: Concentrated hydrochloric acid; Xphos Pd G2: Chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II); Xphos: 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl; Py: Pyridine; THF: Tetrahydrofuran; Dess-Martin Reagent: Dys-Martin oxidant; n-BuLi: n-Butyllithium; DME: Dimethyl ethylene glycol ether; AcOH: Acetic acid; SFC: Supercritical fluid chromatography; NaAC-HAC: Sodium acetate-acetic acid; Tris: Tris(hydroxymethyl)aminomethane; DTT: Dithiothreitol.

[0136] Unless otherwise defined in this disclosure, scientific and technical terms used in connection with this disclosure shall have the meanings understood by one of ordinary skill in the art. Attached Figure Description

[0137] Figure 1 This indicates the amount of IL-2 released by ADC-1 in a mixed lymphocyte response.

[0138] Figure 2 This indicates the amount of IFN-γ released by ADC-1 in a mixed lymphocyte response.

[0139] Figure 3 This indicates the amount of IL-2 released in a mixed lymphocyte response stimulated by ADC-2.

[0140] Figure 4 This indicates the amount of IFN-γ released by ADC-2 in a mixed lymphocyte response. Detailed Implementation

[0141] The present disclosure will be further described below with reference to specific embodiments, and the advantages and features of the present disclosure will become clearer with the description. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0142] The embodiments disclosed herein are merely exemplary and do not constitute any limitation on the scope of this disclosure. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions disclosed herein without departing from the spirit and scope of this disclosure, but all such modifications and substitutions fall within the protection scope of this disclosure.

[0143] Example 1: Synthesis of drug-linker compounds

[0144] Example 1.1: Preparation of (3S)-1-((3-(6-cyclopropyl-4-(cis-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-8-methyl-4-carbonyl-4H-chromene-6-yl)methyl)-1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl)-3-methylpiperidin-1-onium 2,2,2-trifluoroacetate (compound 1)

[0145]

[0146] Step 1: Preparation of 2-chloro-4-(3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridine-1-oxy radical (1B)

[0147] Trifluoroacetic anhydride (4.0 mL) was added to a solution of compound 1A (400.0 mg, 1.52 mmol) in dichloromethane (4.0 mL). Hydrogen peroxide (4.0 mL) was slowly added dropwise at room temperature, followed by continuous stirring and monitoring until the starting material disappeared. After the reaction was complete, the reaction solution was concentrated, and the crude product was subjected to reversed-phase column chromatography (column: [column information missing]). Rapid silica column chromatography (mobile phase: acetonitrile / water = 45:55) was used to purify the title compound 1B (300.0 mg).

[0148] MSm / z(ESI): 279.74 [M+H] +

[0149] Step 2: Preparation of 2,6-dichloro-4-(3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridine (1C)

[0150] Compound 1B (300.0 mg, 1.08 mmol) was added to phosphorus oxychloride (3.0 mL), and the reaction mixture was stirred at 70 °C for 1 hour. After the reaction was complete, the reaction mixture was concentrated to dryness, and the crude product was subjected to reversed-phase column chromatography (column: [column information missing]). Rapid silica column chromatography (mobile phase: acetonitrile / water = 35:65) was used to purify the title compound 1C (200.0 mg).

[0151] MSm / z(ESI): 297.06 / 299.06 [M+H] + .

[0152] Step 3: Preparation of cis-2-chloro-6-cyclopropyl-4-(3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridine (1D)

[0153] Compound 1C (200.0 mg, 673.0 μmol), cyclopropylboronic acid (86.7 mg, 1.01 mmol), and potassium phosphate (54.5 mg, 256.9 μmol) were added to a mixture of water (1.0 mL) and 1,4-dioxane (4.0 mL), and nitrogen was introduced. Pd(dppf)Cl2 (49.3 mg, 67.3 μmol) was then added, and the mixture was heated to 100 °C and stirred for 12.0 hours under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature, filtered, and concentrated to obtain the crude product. The crude product was then separated by a preparative high-performance liquid chromatography column (column: Welch Xtimate C18, column length 150 mm, inner diameter 30 mm, particle size 5 μm; mobile phase A: water (containing 0.225% NH3), mobile phase B: acetonitrile; gradient: mobile phase B from 5% to 95% in 24 minutes) to obtain the title compound 1D (142.0 mg).

[0154] MSm / z(ESI): 303.1 / 305.1 [M+H] + .

[0155] Step 4: Preparation of cis-2-cyclopropyl-4-(3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)-6-(trimethyltinyl)pyridine (1E)

[0156] Compound 1D (30.0 mg, 99.1 μmol) and hexamethyldistin (48.7 mg, 148.6 μmol) were added to 1,4-dioxane (2.0 mL), and nitrogen was used to replace the atmosphere. Pd(PPh3)4 (11.5 mg, 9.9 μmol) was added, and the resulting mixture was heated to 100 °C and stirred for 2.0 hours under nitrogen protection. After the reaction was complete, the reaction solution was concentrated under vacuum to obtain crude compound 1E (40.0 mg), which required no further purification.

[0157] MSm / z(ESI):433.1 / 431.1 / 429.1[M+H] + .

[0158] Step 5: Preparation of cis-3-(6-cyclopropyl-4-(3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-8-methyl-6-(((S)-3-methylpiperidin-1-yl)methyl)-4H-chromen-4-one (1G)

[0159] Compounds 1F (40.0 mg, 100.2 μmol), 1E (43.2 mg, 100.2 μmol), and cesium fluoride (33.5 mg, 220.4 μmol) were added to 1,4-dioxane (2.0 mL), and nitrogen was purged. Di(tri-tert-butylphosphine)palladium (10.3 mg, 20.0 μmol) was added, and the resulting mixture was heated to 100 °C under nitrogen protection and stirred for 1.0 h. After the reaction was complete, the crude product was subjected to reversed-phase column chromatography (column: [column name missing]). Purification was performed using a rapid silica column (mobile phase: acetonitrile / water = 80:20) to give 1 G (20.0 mg) of the title compound.

[0160] Step 6: Preparation of (3S)-1-((3-(6-cyclopropyl-4-(cis-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-8-methyl-4-carbonyl-4H-chromene-6-yl)methyl)-1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl)-3-methylpiperidin-1-onium 2,2,2-trifluoroacetate (compound 1)

[0161] Compound 1G (10 mg, 18.60 μmol) and 1H (11.27 mg, 22.32 μmol) were dissolved in DMF (1 mL), followed by the addition of DIEA (4.81 mg, 37.20 μmol). The mixture was heated to 50 °C and reacted for 48 h. LC-MS was used to monitor the completion of the reaction (30% of the target compound). The reaction solution was directly purified using a reverse-phase column chromatography (column: [column information missing]). Rapid silica column chromatography (acetonitrile / water = 5%-60%, time 10 minutes) yielded target compound 1 (4.5 mg).

[0162] MSm / z (ESI): 1006.6 [M] + .

[0163] 1H NMR (400MHz, DMSO-d6) δ10.21(s,1H),9.04(s,1H),8.46(s,1H),8.24(d,J=6.7Hz,1H),8.16(d,J=2.1Hz, 1H),7.93(d,J=1.6Hz,1H),7.85–7.74(m,4H),7.54(d,J=8.3Hz,2H),7.38(d,J=1.7Hz,1H),7.00(s,2H), 4.79(d,J=10.4Hz,2H),4.52–4.12(m,6H),3.36(t,J=7.1Hz,10H),2.86(s,3H),2.59(s,2H),2.36–2.09( m,6H),2.00–1.68(m,4H),1.47(dd,J=10.2,6.0Hz,5H),1.26(dd,J=54.8,7.4Hz,7H),1.10–0.87(m,12H).

[0164] Example 1.2: Preparation of Compound 2

[0165]

[0166] Step 1: Preparation of 1-(5-bromo-2-hydroxy-3-methylphenyl)acetone (compound 2B)

[0167] Compound 2A (1 g, 6.66 mmol) was dissolved in dichloromethane (20 mL) and cooled in an ice bath. A solution of bromine (1.92 g, 11.99 mmol) in dichloromethane (5 mL) was slowly added dropwise to the mixture. The mixture was stirred for 1 hour and heated to room temperature. After the reaction was complete, the solution was quenched with a saturated sodium sulfite aqueous solution (5 mL). The mixture was separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound 2B (1.3 g), which could be used directly in the next step without further purification.

[0168] MS M / z (ESI): 227 / 229 [MH] -

[0169] Step 2: Preparation of 1-(5-bromo-2-hydroxy-3-methylphenyl)-3-(dimethylamino)prop-2-en-1-one (compound 2C)

[0170] Compound 2B (1.3 g, 5.68 mmol) was dissolved in DMF-DMA (5 mL), heated to 100 °C, and stirred for 5 h. After cooling to room temperature, the solution was concentrated to obtain crude compound 2C (1.6 g), which could be used directly in the next step without purification.

[0171] MS M / z (ESI): 284 / 286 [M+H] + ;

[0172] Step 3: Preparation of 6-bromo-8-methyl-chromen-4-one (compound 2D)

[0173] Compound 2C (1.6 g, 5.63 mmol) was dissolved in DCM (10 mL), and concentrated HCl (1 mL) was added under ice bath conditions. The mixture was stirred for 1 h. It was diluted with ethyl acetate (100 mL), washed with saturated NaHCO3 aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (PE:EA = 1:3) to give the title compound 2D (700 mg).

[0174] MS M / z (ESI): 239 / 241 [M+H] + ;

[0175] Step 4: Preparation of (R)-8-methyl-6-((2-methylmorpholino)methyl)-4H-chromen-4-one (2F)

[0176] Compounds 2D (400.0 mg, 1.67 mmol), 2E (369.9 mg, 1.67 mmol), and potassium carbonate (461.8 mg, 3.35 mmol) were added to a mixture of water (2.0 mL) and 1,4-dioxane (10.0 mL), and nitrogen was purged. Under a nitrogen atmosphere, Xphos Pd G2 (131.7 mg, 167.7 μmol) and Xphos (159.62 mg, 334.6 μmol) were added, and the resulting mixture was heated to 100 °C and stirred for 12.0 hours under nitrogen protection. The crude product was analyzed by reversed-phase column chromatography (column: [column name missing]). Purification was performed using a rapid silica column (mobile phase: acetonitrile / water = 80:20) to give the title compound 2F (415.0 mg).

[0177] MSm / z(ESI): 274.14 [M+H] + .

[0178] Step 5: Preparation of (R)-3-iodo-8-methyl-6-((2-methylmorpholino)methyl)-4H-chromen-4-one (2G)

[0179] Compound 2F (200.0 mg, 731.7 μmol) and tetrahydropyrrole (104.1 mg, 1.46 mmol) were added to methanol (4.0 mL), and the reaction mixture was stirred at 60 °C for 1 hour. After the starting material disappeared under TLC monitoring, the reaction mixture was concentrated, and the crude product was added to dichloromethane (4.0 mL), followed by pyridine (0.5 mL) and iodine (371.4 mg, 1.46 mmol). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction mixture was concentrated, and the crude product was purified by reversed-phase column chromatography (column: [column number missing]). Purification was performed using a rapid silica column (mobile phase: acetonitrile / water = 85:15) to give 2 G (210.0 mg) of the title compound.

[0180] MSm / z(ESI): 400.03 [M+H] + .

[0181] Step 6: Preparation of cyclopropyl(2,6-dichloropyridin-4-yl)methanol (compound 2I)

[0182] Compound 2H (2.3 g, 13.07 mmol) was added to tetrahydrofuran (23.0 mL). After cooling the reaction solution to 0 °C, a tetrahydrofuran solution of cyclopropyl magnesium bromide (17.0 mL, 17.0 mmol, 1.0 M) was added dropwise. After the addition was complete, the reaction solution was transferred to room temperature and reacted for 4.0 hours. After the reaction was completed, the reaction solution was cooled to 0 °C in an ice bath, and then quenched by adding a saturated ammonium chloride aqueous solution. The mixture was extracted with ethyl acetate, the organic phase was dried and concentrated, and the crude product was subjected to reversed-phase column chromatography (column: [column number missing]). Rapid silica column chromatography (mobile phase: acetonitrile / water = 60:40) was used to purify the title compound 2I (1.67 g).

[0183] MSm / z(ESI):218.0 / 220.0[M+H] + .

[0184] Step 7: Preparation of (2-chloro-6-cyclopropylpyridin-4-yl)(cyclopropyl)methanol (2J)

[0185] 2I (1.5 g, 6.88 mmol), cyclopropylboronic acid (886.2 mg, 10.3 mmol), and potassium phosphate (2.92 g, 13.76 mmol) were added to a mixture of water (10.0 mL) and 1,4-dioxane (40.0 mL), and nitrogen was introduced to purge the mixture. Pd(dppf)Cl2 (503.5 mg, 687.8 μmol) was added, and the resulting mixture was heated to 100 °C under nitrogen protection and stirred for 12.0 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was then subjected to reversed-phase column chromatography (column: [column information missing]). Rapid silica gel column chromatography; mobile phase: acetonitrile / water = 50:50), yielding the title compound 2J (1.0 g).

[0186] MSm / z(ESI):224.0 / 226.0[M+H] + .

[0187] Step 8: Preparation of (2-chloro-6-cyclopropylpyridin-4-yl)(cyclopropyl)methyl ketone (2K)

[0188] 2 J (1.0 g, 4.47 mmol) was dissolved in anhydrous dichloromethane (20.0 mL). Dysmartin oxidant (2.84 g, 6.71 mmol) was added in portions to the solution under an ice bath at 0 °C. After the addition was complete, the reaction mixture was slowly heated to room temperature and stirred for 2.0 hours. After the reaction was complete, the reaction residue was filtered off, the mixture was washed with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. The solution was then filtered and concentrated to obtain the crude product. The crude product was subjected to reversed-phase column chromatography (column: [column information missing]). Rapid silica column chromatography (mobile phase: acetonitrile / water = 50:50) was used to purify the title compound 2K (800.0 mg).

[0189] MSm / z(ESI):222.0 / 224.0[M+H] + .

[0190] Step 9: Preparation of (2-chloro-6-cyclopropylpyridin-4-yl)(cyclopropyl)(4-methyl-4H-1,2,4-triazol-3-yl)methanol (2 L)

[0191] 4-Methyl-4H-1,2,4-triazole (93.7 mg, 1.13 mmol) was dissolved in DME (10.0 mL). The reaction solution was cooled to -40 °C, and a solution of n-butyllithium in n-hexane (1.6 M, 0.71 mL, 1.13 mmol) was added dropwise. After the addition was complete, the reaction solution was stirred at -40 °C for 1.0 h. Then, a solution of 2K (125.0 mg, 564.0 μmol) in DME (3.0 mL) was added dropwise. After the addition was complete, the reaction solution was transferred to room temperature and reacted for 1.0 h. After the reaction was complete, the reaction solution was cooled to 0 °C in an ice bath, and then a saturated aqueous solution of ammonium chloride was added and stirred for ten minutes. After stirring, the solution was extracted three times with ethyl acetate (20.0 mL). The resulting organic phase was dried and concentrated. The crude product was washed twice with petroleum ether (100.0 mL) to give 2 L (105.0 mg) of the title compound.

[0192] MSm / z(ESI): 305.1 / 307.1 [M+H] + .

[0193] Step 10: Preparation of 2-chloro-4-(chloro(cyclopropyl)(4-methyl-4H-1,2,4-triazol-3-yl)methyl)-6-cyclopropylpyridine (2M)

[0194] Under ice bath conditions, 2 L (70 mg, 229.7 μmol) of compound in 2.0 mL of dichloromethane was added with 273.2 mg (2.3 mmol) of thionyl chloride and DIEA (296.8 mg, 2.3 mmol), and the mixture was stirred at room temperature for 2.0 h. After the reaction was complete, the reaction solution was washed with saturated ammonium chloride aqueous solution, extracted with dichloromethane, and the organic phase was concentrated under vacuum to obtain crude product of the title compound 2M (60.0 mg), which did not require further purification.

[0195] MSm / z(ESI): 323.0 / 325.0 [M+H] + .

[0196] Step 11: Preparation of 2-chloro-6-cyclopropyl-4-(cyclopropyl(4-methyl-4H-1,2,4-triazol-3-yl)methyl)pyridine (2N)

[0197] Zinc powder (241.0 mg, 3.71 mmol) was added to a 2M solution (60.0 mg, 185.6 mmol) of glacial acetic acid (8.0 mL), and stirred at room temperature for 12.0 h. After the reaction was complete, the reaction solution was filtered, and the filtrate was concentrated under vacuum. The crude product was then separated by a reverse-phase column (column: [column name missing]). Rapid silica column chromatography (mobile phase: acetonitrile / water = 50:50) was used to purify the title compound 2N (20.0 mg).

[0198] MSm / z(ESI):289.1 / 291.1[M+H] + .

[0199] Step 12: Preparation of 2-cyclopropyl-4-(cyclopropyl(4-methyl-4H-1,2,4-triazol-3-yl)methyl)-6-(trimethyltinyl)pyridine (2O)

[0200] 2N (30.0 mg, 103.89 μmol) and hexamethyldistin (68.0 mg, 207.8 μmol) were added to 1,4-dioxane (3.0 mL), and nitrogen was purged. Tetra(triphenylphosphine)palladium (24.0 mg, 20.8 μmol) was added, and the resulting mixture was heated to 100 °C and stirred for 18.0 h under nitrogen protection. The mixture was concentrated under vacuum to give crude title compound 2O (25.0 mg), which required no further purification.

[0201] MSm / z(ESI):419.1 / 417.1 / 415.1[M+H] + .

[0202] Step 13: Preparation of 3-(6-cyclopropyl-4-(cyclopropyl(4-methyl-4H-1,2,4-triazol-3-yl)methyl)pyridin-2-yl)-8-methyl-6-(((R)-2-methylmorpholinyl)methyl)-4H-chromen-4-one (compound 2P)

[0203] Compounds 2G (26.3 mg, 65.9 μmol), 2O (25.0 mg, 59.9 μmol), and cesium fluoride (20.0 mg, 131.9 μmol) were added to 1,4-dioxane (2.0 mL), and nitrogen was purged. Di(tri-tert-butylphosphine)palladium (6.2 mg, 12.0 μmol) was added, and the mixture was heated to 100 °C and stirred for 1.0 h under nitrogen protection. After the reaction was complete, the mixture was subjected to reversed-phase column chromatography (column: [column name missing]). Rapid silica column chromatography (mobile phase: acetonitrile / water = 80:20) was used to purify the title compound 2P (6.5 mg).

[0204] MSm / z(ESI): 526.52 [M+H] + .

[0205] 1 H NMR (400MHz, DMSO-d6) δ8.98(s,1H),8.38(s,1H),8.01(d,J=1.5Hz,1H),7.91(d,J=2.1Hz,1H),7.63(d,J=1.3Hz,1H) ,7.23(d,J=1.5Hz,1H),3.73(d,J=10.8Hz,1H),3.61(d,J=9.8Hz,1H),3.56–3.49(m,4H),3.46(s,3H),2.70–2.59(m, 2H),2.09(ddd,J=11.0,7.3,3.9Hz,2H),2.04–1.97(m,2H),1.76(t,J=10.5Hz,1H),1.68–1.63(m,1H),1.48(t,J=7.2 Hz,2H),1.03(dd,J=6.9,3.2Hz,4H),0.95(dd,J=8.1,3.0Hz,2H),0.85(t,J=6.6Hz,2H),0.60(dd,J=14.3,8.8Hz,2H).

[0206] Step 14: Preparation of compound 2Q-1

[0207] Compound 2P was purified by supercritical fluid chromatography (column: REGIS(S,S)WHELK-O1, column length 250 mm, inner diameter 25 mm, particle size 10 μm; mobile phase A: ethanol (containing 0.1% ammonia), mobile phase B: supercritical carbon dioxide; gradient: mobile phase B from 60% to 60%; flow rate: 80 mL / min) to obtain compounds 2Q-1 and 2Q-2. The two title products were then further analyzed using the following chiral HPLC method.

[0208] Chiral HPLC analysis method:

[0209] Chromatographic column: (S,S)Whelk-01, column length 100 mm, inner diameter 4.6 mm, particle size 5 μm; Mobile phase: A: CO2; B: ethanol (containing 0.05% diethylamine); Gradient: mobile phase B from 50% to 50% in 5 minutes; Flow rate 2.5 mL / min; Column temperature: 40℃; ABPR: 100 bar

[0210] 2Q-1:

[0211] The elution time of the chiral HPLC peak was 2.804 min;

[0212] MSm / z(ESI): 526.3 [M+H] +

[0213] 1 H NMR (400MHz, DMSO-d6) δ8.98(s,1H),8.38(s,1H),8.01(d,J=1.5Hz,1H),7.91(d,J=2.1Hz,1H),7.63(d,J=1.3Hz,1H) ,7.23(d,J=1.5Hz,1H),3.73(d,J=10.8Hz,1H),3.61(d,J=9.8Hz,1H),3.56–3.49(m,4H),3.46(s,3H),2.70–2.59(m, 2H),2.09(ddd,J=11.0,7.3,3.9Hz,2H),2.04–1.97(m,2H),1.76(t,J=10.5Hz,1H),1.68–1.63(m,1H),1.48(t,J=7.2 Hz,2H),1.03(dd,J=6.9,3.2Hz,4H),0.95(dd,J=8.1,3.0Hz,2H),0.85(t,J=6.6Hz,2H),0.60(dd,J=14.3,8.8Hz,2H).

[0214] 2Q-2:

[0215] The elution time of the chiral HPLC peak was 3.833 min;

[0216] MSm / z(ESI): 526.3 [M+H] +

[0217] 1 H NMR (400MHz, DMSO-d6) δ8.98(s,1H),8.38(s,1H),8.01(d,J=1.5Hz,1H),7.91(d,J=2.1Hz,1H),7.63(d,J=1.3Hz,1H) ,7.23(d,J=1.5Hz,1H),3.73(d,J=10.8Hz,1H),3.61(d,J=9.8Hz,1H),3.56–3.49(m,4H),3.46(s,3H),2.70–2.59(m, 2H),2.09(ddd,J=11.0,7.3,3.9Hz,2H),2.04–1.97(m,2H),1.76(t,J=10.5Hz,1H),1.68–1.63(m,1H),1.48(t,J=7.2 Hz,2H),1.03(dd,J=6.9,3.2Hz,4H),0.95(dd,J=8.1,3.0Hz,2H),0.85(t,J=6.6Hz,2H),0.60(dd,J=14.3,8.8Hz,2H).

[0218] Step 15: Preparation of Compound 2

[0219] Compound 2Q-1 (50 mg, 95.12 μmol) and 1H (48 mg, 95.12 μmol) were dissolved in DMF (1 mL), followed by the addition of DIEA (37 mg, 285.36 μmol). The mixture was heated to 50 °C and reacted for 48 h. LC-MS was used to monitor the completion of the reaction (30% of the target compound). The reaction solution was directly purified using a reverse-phase column chromatography (column: [column information missing]). Rapid silica column chromatography (acetonitrile / water = 5%-60%, time 10 minutes) yielded the target compound 2 (5 mg).

[0220] m / z (ESI): 994.5 [M] + .

[0221] 1H NMR (400MHz, DMSO-d6) δ10.22(s,1H),9.03(d,J=1.5Hz,1H),8.44(s,1H),8.23(d,J=8.7Hz,2H),8.00–7.93(m,1H),7.80(d, J=8.1Hz,3H),7.56(d,J=8.4Hz,2H),7.27(d,J=1.7Hz,1H),6.99(s,2H),4.99(q,J=14.1Hz,2H),4.68–4.28(m,6H),4.17(t, J=7.7Hz,1H),3.93(d,J=12.9Hz,1H),3.63(d,J=9.7Hz,2H),3.46(d,J=1.3Hz,3H),3.14(d,J=13.1Hz,2H),2.68(s,1H),2.2 4–1.84(m,6H),1.63(s,2H),1.48(p,J=7.3Hz,4H),1.39–0.79(m,19H),0.59(dt,J=17.4,8.9Hz,2H),0.39(d,J=4.7Hz,2H).

[0222] Example 2: Synthesis of antibody-drug conjugates

[0223] 2.1: Antibody Construction and Production

[0224] 2.1.1: Construction and production of anti-human PD-1 monoclonal antibodies

[0225] The heavy and light chain variable region sequences of the anti-human PD-1 antibody are shown in Table 1 below. Both antibodies are hIgG4. The heavy and light chain nucleotide sequences were cloned into the pTT5 vector (purchased from Ubisoft Biotechnology), and plasmids were prepared according to established standard molecular biology methods. For specific methods, please refer to Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). The expression vector and transfection reagent PEI (Polysciences, catalog number: 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021), mixed, and incubated for 15 min. The mixture was then added to Expi293F cells (Thermofisher, catalog number: A14527) and cultured in a shaker at 37°C with 5% CO2 at 120 rpm. On the second day after transfection, OPM-293ProFeed (Shanghai Optima, catalog number: F081918-001) and 6 g / L glucose (manufacturer: Sigma, catalog number: G7528) were added. On the sixth day after transfection, the cell supernatant was collected.

[0226] Table 1. Sequence information of the variable region of the heavy chain of anti-human PD-1 antibody.

[0227]

[0228] 2.1.2: Antibody Purification

[0229] The antibodies were purified from cell culture supernatant using Protein A affinity chromatography (Cytiva, catalog number 17549802). The Protein A affinity column was washed with 3-5 column volumes of 0.5M NaOH, followed by 3-5 column volumes of pure water. The column was equilibrated with 3-5 column volumes of PBS (pH 7.4) as a buffer. Cell supernatant was loaded at a low flow rate for binding, followed by washing the column with 3-5 column volumes of PBS (pH 7.4) until UV absorption returned to baseline. The sample was eluted with 50mM acetate buffer (pH 3.5), and the elution peak was collected based on UV monitoring. The elution product was temporarily stored by rapidly adjusting the pH to 5-6 with 1M Tris-HCl (pH 8.0). The purity of the sample was improved by replacing the buffer system using ultrafiltration, dialysis, or desalting columns, or by using a molecular sieve to remove aggregate components from the elution product. After purification, the protein that meets the purity requirements is aseptically filtered using a 0.22 μm filter (Millipore, catalog number SLGVR13SL). After passing the SEC-HPLC purity test, it is aliquoted and stored at -80℃ for later use.

[0230] 2.2: Antibody-coupled reaction

[0231] The purified antibody was dialyzed to PBS solution (pH 7.2-7.4). 40-80 molar equivalents of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) were added to the antibody solution. The mixture was then incubated on a metal shaker at 25°C for 16-18 hours to reduce the antibody. 25-40 molar equivalents of the drug-linker compound from Example 1 of this disclosure were dissolved in DMSO and added to the reaction system. The reaction mixture was coupled at 25°C for 4 hours. The reaction product was desalted using a G25 column and the buffer was changed to 10 mM NaAC-HAC (containing 9% sucrose, pH 5.5) buffer to remove unreacted free small molecule toxins. The resulting antibody-drug conjugates and their corresponding numbers are shown below:

[0232]

[0233] 2.3: Antibody-Drug Conjugate Purity Assay (SEC-HPLC)

[0234] SEC-HPLC was used to analyze ADC samples, characterize the molecular size uniformity, and determine the purity of the samples. The HPLC system used was an Agilent 1260, with a TSKgel G3000SWXL column from Tosoh Bioscience. The mobile phase was 200 mM phosphate buffer (pH 7.0) / isopropanol (v / v 9:1). The detection temperature was 25℃, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the sample loading was 50 μg, and the analysis time was 40 min. For the SEC-HPLC data, the chromatograms were analyzed using the manual integration method, and the protein purity was calculated using the area normalization method. The main peak was considered the monomer, the peaks before the main peak were called aggregates, and the peaks after the main peak were called fragments. The detection results are shown in Table 2.

[0235] 2.4: Detection of DAR value of antibody-drug conjugate (LC-MS)

[0236] The DAR value of the sample was characterized by analysis using LC-MS.

[0237] Sample preparation: Prepare a 100 mM Tris buffer solution (pH = 8.0) containing 6 M urea. Dilute the sample in half with the buffer solution, add 1 M DTT to a final concentration of 20 mM, and incubate at 37°C for 1 hour. After centrifugation, add formic acid to a final concentration of 0.1%, mix well, and centrifuge again before analysis.

[0238] Liquid chromatography analysis: The HPLC system used in this method was a Thermo Vanquish, with a protein loading amount of 1 μg. The chromatographic column was a Protein BEH SEC column from Waters (specifications: [not specified]). The mobile phase was 25% acetonitrile solution (containing 0.1% formic acid and 0.05% TFA), the flow rate was 0.2 mL / min, the detection wavelengths were 280 nm and 220 nm, and the analysis time was 30 minutes.

[0239] Mass spectrometry analysis: The high-resolution mass spectrometer used in this method was Q Exactive Plus. The mass spectrometry scan time was 4-15 min after sample injection. The mass spectrometry parameters were as follows: Spray Voltage: 3.8 kV; Capillary Temp: 300 °C; S-lens: 50; Sheath Gas: 35 alb; Aux Gas: 10 alb; Probe Heater Temp: 300 °C; Full MS Scan range: 900-4500 m / z; Resolution: 17500; AGC target: 3e6; Maximum IT: 200 ms; Microscans: 1; In-source CID: 0 eV; Spray needle position: C.

[0240] Data Analysis: The raw mass spectrometry data was analyzed using the Biopharma Finder 4.1 mass spectrometry data analysis software. The general Respect algorithm was used for deconvolution processing to calculate the molecular weight information of the light and heavy chain mass spectrometry peaks and the mass spectrometry response signals of each component. The DAR value of the sample was calculated based on the corresponding mass spectrometry response values ​​of each payload. The detection results are shown in Table 2.

[0241] Table 2. ADC, its DAR value, and SEC purity

[0242] ADC number DAR SEC purity (monomer %) ADC-1 6.38 97.20 ADC-2 8.00 93.98

[0243] Example 3: Biological activity and related property testing

[0244] 3.1 Mixed Lymphocyte Reaction (MLR)

[0245] Materials and Instruments: Human PBMCs were purchased from Auscells Biotechnology (Shanghai) Co., Ltd.; RPMI-1640 medium was purchased from BasalMedia (L240KJ); fetal bovine serum was purchased from Gibco (#10091148); human CD14+ monocyte sorting reagent was purchased from Miltenyi (130-096-537); CD4+ T cell sorting reagent was purchased from Miltenyi (130-096-533); human DC differentiation reagent was purchased from R&D Company (CDK004); magnetic sorting rack was purchased from Miltenyi (130-042-303); LS sorting column was purchased from Miltenyi (130-042-401); EasySep... Buffer solutions were purchased from Stemcell (#20144), human IL-2 ELISA reagent from BD (#555190), human IFN-γ ELISA reagent from BD (#555142), and 96-well plates from Corning (#3799). Automated cell counter: Beckman, model Vi-cell XR. Multifunctional microplate reader: PerkinElmer, model Envision2105.

[0246] Mixed lymphocyte reaction: Human primary CD14+ monocytes were sorted from donor 1's PBMCs using a human CD14+ monocyte sorting reagent according to the sorting reagent protocol. Differentiation was performed using a human DC differentiation reagent protocol. Monocytes were cultured for 7 days in RPMI-1640 medium containing 1x GM-CSF, 1x IL-4, and 10% inactivated fetal bovine serum to become immature DCs. On day 7, the immature DCs were cultured for 3 days in RPMI-1640 medium containing 1x TNF-α and 10% inactivated fetal bovine serum to become mature DCs. Mature DCs were used as stimulatory cells for subsequent experiments. CD4+ T cells were sorted from donor 2's PBMCs using a CD4+ T cell sorting reagent. CD4+ T cells were cultured overnight in RPMI-1640 medium containing 10% inactivated fetal bovine serum. On the second day, CD4+ T cells and mature dendritic cells were collected, counted, and the cell density was adjusted. They were co-cultured in 96-well plates at a ratio of 5:1, with 5 x 10⁶ CD4+ T cells per well. 4 1 x 10c DC cells per well 4The ADC obtained in Example 2 and the anti-human PD-1 antibody pembrolizumab produced in Example 2.1 were diluted 20-fold to their final concentrations and then added to each well of a cell culture plate at a volume ratio of 20:1, with a final total volume of 200 μL per well. The control wells contained only CD4+ T cells and mature dendritic cells (DCs). The DC and CD4+ T cell co-culture systems were incubated for 2–7 days under each drug treatment, and the supernatant was collected. The levels of cytokines IL-2 and IFN-γ in the supernatant were detected using an ELISA kit; specific experimental procedures were performed according to the kit's instruction manual.

[0247] Data Analysis: Based on the concentration of the standard samples provided with the kit, the IL-2 or IFN-γ secretion levels in each well were calculated. The amount of IL-2 or IFN-γ secreted represents the degree of T cell activation. Data are presented in a GraphPad Prism bar chart; results are shown below. Figures 1-4 The ADC-1 and ADC-2 obtained in Example 2 of this disclosure can effectively activate CD4+ T cells, with ADC-1 showing stronger activation than pembrolizumab. Figure 1 and Figure 2 ADC-2 showed slightly stronger or comparable activation than pembrolizumab. Figure 3 and Figure 4 ).

Claims

1. An antibody-drug conjugate of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof, in, Ab stands for antibody unit; L represents the connecting subunit; q is selected from 1 to 10; D is a payload having a structure selected from the following, the payload being covalently connected to the connecting subunit. in, Ring A is selected from Z 1 、Z 2 、Z 3 、Z 4 、Z 5 、Z 6 、Z 7 、Z 8 、Z 9 、Z 10 and Z 11 are independently selected from CH, CR a or N; R a Selected from halogens, OH, NH2, NO2, CN, C1-C6 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C6 alkylamino, -C1-C6 alkyl-OH, C3-C6 cycloalkyl, C3-C6 cycloalkyl-O-, C3-C6 cycloalkyl-NH-, 4-7 membered heterocyclic, 4-7 membered heterocyclic-O- or 4-7 membered heterocyclic-NH-; R 5 Selected from R 6 R 7 It is independently selected from H, D, =O, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -C1-C6 alkyl-OH; The ring C is selected from 4-10 member nitrogen-containing heterocyclic groups; R 8 Selected from OH, =O, CN, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, or C3-C6 cycloalkyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, or C3-C6 cycloalkyl is optionally converted to R 8a replace; R 8a Selected from OH or C1-C6 alkoxy groups; n is an integer selected from 0 to 3; R 9 Selected from C1-C6 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic groups, wherein the C1-C6 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic group is optionally R 9a replace; R 9a Selected from OH, CN, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -C1-C6 alkyl-OH, -S(O)2-C1-C6 alkyl or C3-C6 cycloalkyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -C1-C6 alkyl-OH, -S(O)2-C1-C6 alkyl or C3-C6 cycloalkyl is optionally R 9b replace; R 9b Selected from OH, halogens, or CN; Y1, Y2, Y3, and Y4 are independently selected from CR b Or N; R b Selected from H, halogen, OH, NH2, CN, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C6 alkylamino, C3-C6 cycloalkyl or 4-7 membered heterocyclic groups; k is selected from 0 or 1; R 1 R 2 R 3 and R 4 Independently selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic group; or R 1 R 2 The atoms bonded to it together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the C3-C6 cycloalkyl or 4-7 membered heterocyclic group is optionally R 1b Replace; or R 1 R 2 R 3 and R 4 The atoms bonded to it together form a phenyl group, which is optionally R 2b replace; R 1b and R 2b Independently selected from halogens, CN, =O, OH, NH2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, or C1-C6 alkylamino, wherein the C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, or C1-C6 alkylamino is optionally R 1c replace; R 1c Selected from OH, halogens, or CN; Cycle B is selected from a 5-membered heteroaryl group, which is optionally substituted with a C1-C6 alkyl group.

2. The antibody-drug conjugate of formula (I) according to claim 1, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, The ring A is selected from m is selected from integers from 0 to 3.

3. The antibody-drug conjugate of formula (I) according to claim 1 or 2, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, The R a Selected from CH3, CF3, F or cyclopropyl.

4. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 3, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, The k is selected from 0; R 1 Selected from H, R 2 Selected from cyclopropyl, or R 1 R 2 The atoms connected to it form together 5. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 4, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, R 5 Selected from 6. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 5, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, Selected from 7. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 6, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, Ring B is selected from The Optionally substituted with C1-C6 alkyl groups.

8. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 7, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, R 1 R 2 R 3 and R 4 It is independently selected from H, CH3, CF3, F or cyclopropyl.

9. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 8, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, D is covalently connected to the connecting subunit through its nitrogen or oxygen atoms; Alternatively, D is a payload having a structure selected from the following, wherein the payload is covalently connected to the connecting subunit. Among them, rings C and R 1 R 2 R 3 R 4 R 6 R 7 R 8 R a Y1, Y2, Y3, Y4, n and k are defined as in claim 1, and m is selected from integers from 0 to 3; Alternatively, D is a payload having a structure selected from the following, wherein the payload is covalently connected to the connecting subunit.

10. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 9, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, Ab is selected from anti-PD-1 antibody; or, Ab is selected from pembrolizumab.

11. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 10, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, L is selected from protease-cleavable linker units; or, L is selected from... p is an integer from 2 to 10.

12. The antibody-drug conjugate of formula (I) according to any one of claims 1 to 11, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, The antibody-drug conjugate of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof is selected from the antibody-drug conjugate of formula (II), its stereoisomer, or a pharmaceutically acceptable salt thereof. Among them, Ab, L, ring C, R 1 R 2 R 3 R 4 R 6 R 7 R 8 R a Y1, Y2, Y3, Y4, n, k and q are defined as in claim 1, and m is selected from integers from 0 to 3; Alternatively, the antibody-drug conjugate of formula (I), its stereoisomers, or pharmaceutically acceptable salts thereof are selected from the following antibody-drug conjugates, their stereoisomers, or pharmaceutically acceptable salts thereof. q is selected from 1 to 8.

13. A pharmaceutical composition comprising an antibody-drug conjugate of formula (I) according to claims 1 to 12, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

14. Use of the antibody-drug conjugate of formula (I) according to claims 1 to 12, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13, in the preparation of a medicament for treating tumors.

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