Linker, antibody-drug conjugate containing the linker, and method for producing the same and use thereof

Abstract

The present invention relates to an antibody-drug conjugate comprising a linker, as well as to pharmaceutical combinations comprising the antibody conjugate, and to the use of the linker, and the use of the antibody conjugate in the manufacture of a medicament for the prevention and / or treatment of disease.

Description

[Technical Field]

[0001] The present invention relates to linkers used in antibody-drug conjugates, antibody-drug conjugates produced using such linkers, pharmaceutical compositions containing the antibody-drug conjugates, and the use of these antibody-drug conjugates in the treatment and / or prevention of diseases. [Background technology]

[0002] Currently, conventional chemotherapy remains the first choice for the treatment of malignant tumors. Conventional chemotherapy is primarily based on cytotoxic drugs, but it generally suffers from drawbacks such as poor target specificity, a narrow safety margin, severe toxic side effects, and a tendency toward drug resistance. The emergence of antibody-drug conjugates (ADCs) has provided a new treatment for malignant tumors. ADCs are structured with three components: an antibody, a small molecule cytotoxic drug (cytotoxin), and a linker that organically connects the two. ADCs utilize the targeting specificity of antibodies to accumulate cytotoxic drugs at the target site in tumors, improving efficacy and reducing toxicity. The released cytotoxic drug can further kill surrounding tumor cells through the bystander effect. Combining the dual advantages of the high targeting specificity of monoclonal antibody drugs and the high activity of cytotoxins in tumor tissue, ADCs can efficiently kill tumor cells, resulting in fewer side effects than chemotherapy drugs and superior therapeutic efficacy than conventional antibody-based antitumor drugs. ADC drugs have properties such as high activity, low toxic side effects, and long duration of action, providing a new strategy for "precision therapy" of tumors.

[0003] Abnormal epigenetic regulation is often associated with human diseases, particularly cancer. Abnormal epigenetic regulation in cancer includes DNA methylation, histone methylation, histone acetylation, non-coding RNA, and mRNA methylation. Epigenetic modifications can alter gene expression without altering DNA sequence. Abnormal transcription of oncogenes or tumor suppressor genes mediated by epigenetic enzymes is closely related to tumor development, progression, and prognosis. Based on the reversibility of epigenetic mechanisms, small molecule compounds targeting epigenetic regulation have already become promising therapeutic agents. These compounds target epigenetic regulatory enzymes such as histone modifiers (methylation and acetylation), DNA methyltransferases, enzymes that specifically recognize post-translational modifications, chromatin remodeling enzymes, and post-transcriptional regulators (Jin Y, Liu T, Luo H, Liu Y, Liu D. Targeting Epigenetic Regulatory Enzymes for Cancer Therapeutics: Novel Small-Molecule Epidrug Development. Front Oncol. 2022;12:848221. Published 2022 Mar 28. doi:10.3389 / fonc.2022.848221). Epigenetically targeted drugs have already demonstrated their clinical efficacy in hematopoietic malignancies and therapeutic potential in solid tumors (Jin N, George TL, Otterson GA, et al. Advances in epigenetic therapeutics with focus on solid tumors. Clin Epigenetics. 2021;13(1):83.).Peripheral T-cell lymphoma (PTCL) is a typical epigenetic-related disease that exhibits unique sensitivity to both histone deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors, alone or in combination (Scotto L, Kinahan C, Douglass E, et al. Targeting the T-Cell Lymphoma Epigenome Induces Cell Death, Cancer Testes Antigens, and Immune-Modulatory Signaling Pathways. Mol Cancer Ther. 2021;20(8):1422-1430).

[0004] Although epigenetic drugs have demonstrated clinical efficacy in hematopoietic malignancies, they have not demonstrated significant therapeutic efficacy as monotherapy in solid tumors. Recent studies have demonstrated potential efficacy when combined with chemotherapy and hormonal therapy. Due to their novel mechanisms and high therapeutic potential, it is crucial to reconsider optimal patient selection, drug regimens, study designs, and outcome measures (Juo YY, Gong XJ, Mishra A, et al. Epigenetic therapy for solid tumors: from bench science to clinical trials. Epigenomics. 2015;7(2):215-235. doi:10.2217 / epi.14.73).

[0005] Tucidinostat (Chidamide, Tucidinostat) is a histone deacetylase (HDAC) inhibitor already in clinical use. Studies have shown that treatment of pancreatic cancer cell lines with Tucidinostat significantly reduced the expression of type I HDACs, caspase-3, and p21, while increasing the expression of Bax / Bcl-2. These results suggest that Tucidinostat may inhibit pancreatic tumor cell proliferation by downregulating the expression of type I HDACs and p21, and may promote apoptosis of cells dependent on the mitochondrial apoptosis pathway in a dose-dependent manner (Zhao B, He T. Chidamide, a histone deacetylase inhibitor, functions as a tumor inhibitor by modulating the ratio of Bax / Bcl-2 and p21 in pancreatic cancer. Oncol Rep. 2015;33(1):304-310. doi:10.3892 / or.2014.3595).

[0006] One research team discovered that tucidinostat can increase histone acetylation of the PD-L1 gene by activating the transcription factor STAT1. The HDAC gene family is frequently amplified in patients with soft tissue sarcoma. Based on analysis of drug target gene sets, widespread amplification of the HDAC gene family was observed in 8 of 11 liposarcoma patients (73%). Analysis of the TCGA sarcoma cohort confirmed that amplification of the HDAC gene family was observed in 76.65% (197 / 257) of cases. Class I HDAC expression is associated with poor prognosis in patients with soft tissue sarcoma, and inhibition of its expression can promote cell apoptosis and upregulation of programmed cell death ligand 1 (PD-L1). Tucidinostat, a class I HDAC inhibitor, significantly increased PD-L1 expression in the tumor microenvironment and enhanced CD8 expression. +It can increase T cell infiltration and reduce the number of MDSCs. In mouse models, the combination of tucidinostat with an anti-PD-1 antibody significantly promoted tumor regression and improved survival. Furthermore, the combination of tucidinostat with the anti-PD-1 antibody toripalimab was effective in patients with advanced and metastatic sarcoma, with tolerable side effects (Que Y, Zhang XL, Liu ZX, et al. Frequent amplification of HDAC genes and efficacy of the HDAC inhibitor chidamide and PD-1 blockade combination in soft tissue sarcoma. J Immunother Cancer. 2021;9(2):e001696. doi:10.1136 / jitc-2020-001696).

[0007] To meet the increasing clinical unmet needs in the treatment of solid tumors, antibody-drug conjugates based on histone deacetylase inhibitors need to be developed. Summary of the Invention [Problem to be solved by the invention]

[0008] First, in order to develop a variety of stable ADCs that meet various clinical needs, further development of stable ADCs equipped with new linkers is still necessary. As a result of continuous research, the inventors of the present application have designed a linker having a structure represented by general formula (I).

[0009] Next, the inventors of the present application provide a conjugate represented by the structure of general formula (II) having excellent antitumor effects. [Means for solving the problem]

[0010] As a result of extensive research, the inventors of the present invention have found that a linker having a structure represented by general formula (I) and a conjugate represented by the structure of formula (II) can achieve the desired object, and have thus completed the present invention.

[0011] That is, the present invention relates to the following linkers and conjugates containing the linkers, or stereoisomers thereof, tautomers thereof, pharmaceutically acceptable salts thereof, deuterated compounds thereof, or solvates thereof:

[0012] In a first aspect, the present invention relates to a compound of formula I, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof: [ka] Among them, L1 is [ka] Selected from Preferably, the carbon terminal of L1 is linked to the N of succinimide, and the carbonyl terminal is linked to L2; m and t are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; q is 0, 1, 2 or 3; p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; X is selected from CH2, O and NH; L2 is absent, an amino acid residue, or a peptide residue consisting of 2 to 10 amino acid residues; Preferably, the amino group terminal of L2 is linked to L1, and the carbonyl group terminal is linked to L3; Preferably, the amino acids include, but are not limited to, phenylalanine (F), glycine (G), valine (V), lysine (K), serine (S), glutamic acid (E), asparagine (N), arginine (R), alanine (A), citrulline, and cysteine ​​(C); L3 is not present, or [ka] Selected from Preferably, the amino group terminal of L3 is linked to L2, and the carbonyl group terminal or carbon terminal is linked to D; D is a histone deacetylase inhibitor drug selected from a thiol-based histone deacetylase inhibitor, a hydroxamic acid-based histone deacetylase inhibitor, or a benzamide-based histone deacetylase inhibitor.

[0013] In some embodiments, L1 is [ka] Selected from.

[0014] In some embodiments, L1 is [ka] Selected from.

[0015] In some embodiments, m is selected from 0, 1, 2, 3, 4, and 5. In some embodiments, m is selected from 1, 3, and 4.

[0016] In some embodiments, t is selected from 0, 1, 2, 3, 4, 5, 6, and 7. In some embodiments, t is selected from 1, 2, 3, and 7.

[0017] In some embodiments, q is selected from 0, 1, and 2.

[0018] In some embodiments, p is selected from 10, 11, 12, 13, 14, and 15. In some embodiments, p is selected from 11, 12, and 13. In some embodiments, p is 12.

[0019] In some embodiments, X is selected from O and NH.

[0020] In some embodiments, L1 is [ka] Selected from.

[0021] In some embodiments, L1 is [ka] Selected from.

[0022] In some embodiments, L1 is [ka] Selected from.

[0023] In some embodiments, L1 is [ka] Selected from.

[0024] In some embodiments, L1 is [ka] is.

[0025] In some embodiments, L1 is [ka] is.

[0026] In some embodiments, L1 is [ka] is.

[0027] In some embodiments, L1 is [ka] is.

[0028] In some embodiments, L2 is absent, an amino acid residue, or a peptide residue consisting of 2 to 4 amino acid residues.

[0029] In some embodiments, the amino acids include, but are not limited to, phenylalanine (F), glycine (G), valine (V), alanine (A), citrulline, and cysteine.

[0030] In some embodiments, the amino acids include, but are not limited to, phenylalanine (F), glycine (G), valine (V), alanine (A), and citrulline.

[0031] In some embodiments, L2 is absent, a citrulline residue, or is selected from the following peptide residues: valine-alanine, valine-citrulline, glycine amino group-glycine-phenylalanine-glycine, valine-alanine-phenylalanine-glycine, glycine-glycine-glycine, glycine-phenylalanine-glycine, and valine-cysteine-phenylalanine-glycine.

[0032] In some embodiments, L2 is absent, a citrulline residue, or is selected from the following peptide residues: valine-alanine, valine-citrulline, glycine amino group-glycine-phenylalanine-glycine, and valine-alanine-phenylalanine-glycine.

[0033] In some embodiments, L2 is absent, or [ka] Selected from.

[0034] In some embodiments, L2 is absent, or [ka] Selected from.

[0035] In some embodiments, L2 is absent, or [ka] Selected from.

[0036] In some embodiments, L2 is absent, or [ka] Selected from.

[0037] In some embodiments, L2 is [ka] Selected from.

[0038] In some embodiments, L2 is [ka] is.

[0039] In some embodiments, L3 is [ka] is.

[0040] In some embodiments, D is a benzamide-based histone deacetylase inhibitor.

[0041] In some embodiments, D is [ka] Selected from Among them, A is a phenyl group or a pyridyl group, and the phenyl group or the pyridyl group is each independently optionally substituted with 1 to 4 substituents selected from a halogen, a C1-C4 alkyl group (e.g., a methyl group, an ethyl group), and a C1-C4 haloalkyl group (e.g., a trifluoromethyl group); Preferably, A is a phenyl group or a pyridyl group, and the phenyl group or the pyridyl group is each independently and optionally substituted with 1 to 2 substituents selected from a halogen, a C1-C4 alkyl group (e.g., a methyl group, an ethyl group), and a trifluoromethyl group; More preferably, A is a pyridyl group, and the pyridyl group is optionally substituted with 1 to 2 substituents selected from a C1 to C4 alkyl group; More preferably, A is a pyridyl group, Most preferably, A is [ka] and B is a phenylene group; Preferably, B is [ka] and Y is -CO-NH-CH2-, Preferably, the methylene group terminal of Y is connected to B, and the carbonyl group terminal is connected to an alkenyl carbon or O, R 1 , R 2 are each independently selected from hydrogen and a C1-C4 alkyl group; Preferably, R 1 , R 2 are both hydrogen, X 1 , X 2 , X 3 , X 4 one of which is selected from hydrogen, halogen, and a C1-C4 alkyl group, and the remaining three are hydrogen; Preferably, X 1 , X 2 , X 3 , X4 one of the groups is selected from hydrogen and halogen, and the remaining three are hydrogen; More preferably, X 1 , X 2 , X 3 , X 4 one of which is selected from hydrogen and fluorine, and the remaining three are hydrogen; Most preferably, X 2 is selected from hydrogen and fluorine, and X 1 , X 3 , X 4 is hydrogen.

[0042] In some embodiments, D is [ka] Selected from.

[0043] In some embodiments, D is [ka] is.

[0044] In some embodiments, the compound is [ka] [ka] [ka] [ka] Selected from.

[0045] A second aspect of the present invention relates to the use of the above-mentioned compound or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof in the manufacture of a ligand drug conjugate (e.g., an antibody drug conjugate).

[0046] In a third aspect of the present invention, there is provided a ligand drug conjugate of formula II, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof: [ka] Among them, Ab is a ligand selected from a protein, antibody, polypeptide, enzyme, and small molecule; n is a number between 0.5 and 8.5, for example, n is a number between 0.8 and 5, a number between 1 and 4, a number between 2 and 6, a number between 3 and 7, a number between 4 and 8, a number between 3.5 and 8.5, a number between 3.5 and 4.5, or a number between 6.5 and 8.5, preferably n is about 1, 2, 3, 4, 5, 6, 7, or 8, preferably n is about 2, 3, 4, 5, 6, 7, or 8, preferably n is about 3, 4, 5, 6, 7, or 8, L1 is [ka] Selected from Preferably, the carbon terminal of L1 is linked to the N of succinimide, and the carbonyl terminal is linked to L2; m and t are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; q is 0, 1, 2 or 3; p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; X is selected from CH2, O and NH; L2 is absent, an amino acid residue, or a peptide residue consisting of 2 to 10 amino acid residues; Preferably, the amino group terminal of L2 is linked to L1, and the carbonyl group terminal is linked to L3; Preferably, the amino acids include, but are not limited to, phenylalanine (F), glycine (G), valine (V), lysine (K), serine (S), glutamic acid (E), asparagine (N), arginine (R), alanine (A), citrulline, and cysteine ​​(C); L3 is not present, or [ka] Selected from Preferably, the amino group terminal of L3 is linked to L2, and the carbonyl group terminal or carbon terminal is linked to D; D is a histone deacetylase inhibitor drug selected from a thiol-based histone deacetylase inhibitor, a hydroxamic acid-based histone deacetylase inhibitor, or a benzamide-based histone deacetylase inhibitor.

[0047] In some embodiments, L1 is [ka] Selected from.

[0048] In some embodiments, L1 is [ka] Selected from.

[0049] In some embodiments, m is selected from 0, 1, 2, 3, 4, and 5. In some embodiments, m is selected from 1, 3, and 4.

[0050] In some embodiments, t is selected from 0, 1, 2, 3, 4, 5, 6, and 7. In some embodiments, t is selected from 1, 2, 3, and 7.

[0051] In some embodiments, q is selected from 0, 1, and 2.

[0052] In some embodiments, p is selected from 10, 11, 12, 13, 14, and 15. In some embodiments, p is selected from 11, 12, and 13. In some embodiments, p is 12.

[0053] In some embodiments, X is selected from O and NH.

[0054] In some embodiments, L1 is [ka] Selected from.

[0055] In some embodiments, L1 is [ka] Selected from.

[0056] In some embodiments, L1 is [ka] Selected from.

[0057] In some embodiments, L1 is [ka] Selected from.

[0058] In some embodiments, L1 is [ka]

[0059] In some embodiments, L1 is [ka] is.

[0060] In some embodiments, L1 is [ka] is.

[0061] In some embodiments, L1 is [ka] is.

[0062] In some embodiments, L2 is absent, an amino acid residue, or a peptide residue consisting of 2 to 4 amino acid residues.

[0063] In some embodiments, the amino acids include, but are not limited to, phenylalanine (F), glycine (G), valine (V), alanine (A), citrulline, and cysteine.

[0064] In some embodiments, the amino acids include, but are not limited to, phenylalanine (F), glycine (G), valine (V), alanine (A), and citrulline.

[0065] In some embodiments, L2 is absent, a citrulline residue, or is selected from the following peptide residues: valine-alanine, valine-citrulline, glycine amino group-glycine-phenylalanine-glycine, valine-alanine-phenylalanine-glycine, glycine-glycine-glycine, glycine-phenylalanine-glycine, and valine-cysteine-phenylalanine-glycine.

[0066] In some embodiments, L2 is absent, a citrulline residue, or is selected from the following peptide residues: valine-alanine, valine-citrulline, glycine amino group-glycine-phenylalanine-glycine, and valine-alanine-phenylalanine-glycine.

[0067] In some embodiments, L2 is absent, or [ka] Selected from.

[0068] In some embodiments, L2 is absent, or [ka] Selected from.

[0069] In some embodiments, L2 is absent, or [ka] Selected from.

[0070] In some embodiments, L2 is absent, or [ka] Selected from.

[0071] In some embodiments, L2 is [ka] Selected from.

[0072] In some embodiments, L2 is [ka] is.

[0073] In some embodiments, L3 is [ka] is.

[0074] In some embodiments, D is a benzamide-based histone deacetylase inhibitor.

[0075] In some embodiments, D is [ka] Selected from Among them, A is a phenyl group or a pyridyl group, and the phenyl group or the pyridyl group is each independently optionally substituted with 1 to 4 substituents selected from a halogen, a C1-C4 alkyl group (e.g., a methyl group, an ethyl group), and a C1-C4 haloalkyl group (e.g., a trifluoromethyl group); Preferably, A is a phenyl group or a pyridyl group, and the phenyl group or the pyridyl group is each independently and optionally substituted with 1 to 2 substituents selected from a halogen, a C1-C4 alkyl group (e.g., a methyl group, an ethyl group), and a trifluoromethyl group; More preferably, A is a pyridyl group, and the pyridyl group is optionally substituted with 1 to 2 substituents selected from a C1 to C4 alkyl group; More preferably, A is a pyridyl group, Most preferably, A is [ka] and B is a phenylene group; Preferably, B is [ka] and Y is -CO-NH-CH2-, Preferably, the methylene group terminal of Y is connected to B, and the carbonyl group terminal is connected to an alkenyl carbon or O, R 1 , R 2 are each independently selected from hydrogen and a C1-C4 alkyl group; Preferably, R 1 , R 2 are both hydrogen, X 1 , X 2 , X 3 , X 4 one of which is selected from hydrogen, halogen, and a C1-C4 alkyl group, and the remaining three are hydrogen; Preferably, X 1 , X2 , X 3 , X 4 one of the groups is selected from hydrogen and halogen, and the remaining three are hydrogen; More preferably, X 1 , X 2 , X 3 , X 4 one of which is selected from hydrogen and fluorine, and the remaining three are hydrogen; Most preferably, X 2 is selected from hydrogen and fluorine, and X 1 , X 3 , X 4 is hydrogen, More preferably, D is [ka] Selected from.

[0076] In some embodiments, D is [ka] is.

[0077] In some embodiments, the Ab is an antibody selected from, but not limited to, an anti-EGFR antibody, an anti-CD20 antibody, or an anti-PD-L1 antibody.

[0078] In some embodiments, the Ab is selected from, but not limited to, Cetuximab, Panitumumab, Necitumumab, Rituximab, Tositumomab (Tositumomab + Iodine 131 Tositumomab), Ofatumumab, Obinutuzumab, Ocrelizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab-rwlc.

[0079] In some embodiments, the Ab is atezolizumab, avelumab.

[0080] In some embodiments, the Ligand Drug Conjugate is [ka] [ka] [ka] [ka] Selected from.

[0081] In some embodiments, the Ligand Drug Conjugate is [ka] [ka] Selected from.

[0082] A fourth aspect of the present invention relates to a pharmaceutical composition comprising the above-mentioned compound or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof, or comprising the above-mentioned ligand drug conjugate or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof, optionally further comprising a pharmaceutically acceptable carrier or excipient.

[0083] A fifth aspect of the present invention relates to the use of the above-mentioned compound or a stereoisomer thereof or a tautomer thereof or a pharmaceutically acceptable salt thereof or a deuterated compound thereof or a solvate thereof, or the above-mentioned ligand-drug conjugate or a stereoisomer thereof or a tautomer thereof or a pharmaceutically acceptable salt thereof or a deuterated compound thereof or a solvate thereof, or the above-mentioned pharmaceutical composition, in the manufacture of a medicament for the treatment or prevention of a disease.

[0084] In some embodiments, the disease is a tumor.

[0085] A sixth aspect of the present invention relates to the above-mentioned compound or a stereoisomer thereof or a tautomer thereof or a pharmaceutically acceptable salt thereof or a deuterated compound thereof or a solvate thereof, or the above-mentioned ligand-drug conjugate or a stereoisomer thereof or a tautomer thereof or a pharmaceutically acceptable salt thereof or a deuterated compound thereof or a solvate thereof, or the above-mentioned pharmaceutical composition, for use in the treatment or prevention of a disease.

[0086] In some embodiments, the disease is a tumor.

[0087] A seventh aspect of the present invention relates to a method for treating or preventing a disease, comprising administering to a subject an effective amount of the above-mentioned compound or a stereoisomer thereof or a tautomer thereof or a pharmaceutically acceptable salt thereof or a deuterated compound thereof or a solvate thereof, or the above-mentioned ligand-drug conjugate or a stereoisomer thereof or a tautomer thereof or a pharmaceutically acceptable salt thereof or a deuterated compound thereof or a solvate thereof, or the above-mentioned pharmaceutical composition.

[0088] In some embodiments, the disease is a tumor.

[0089] In an eighth aspect of the present invention, there is provided a method for preparing a compound of formula I, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof, said method comprising: reacting a compound of formula I-1 with D' to obtain a compound of formula I, or reacting a compound of formula I-2 with H-L2-L3-D to obtain a compound of formula I, or reacting a compound of formula I-3 with H-L2-L3-D to obtain a compound of formula I, [ka] [ka] [ka] Preferably, reacting a compound of formula I-1 with D' to obtain a compound of formula I, or reacting a compound of formula I-2 with H-L2-L3-D to obtain a compound of formula I, [ka] [ka] wherein LE is a leaving group, preferably LE is a halogen, [ka] and D' is a histone deacetylase inhibitor drug selected from a thiol-based histone deacetylase inhibitor, a hydroxamic acid-based histone deacetylase inhibitor, or a benzamide-based histone deacetylase inhibitor; Preferably, D' is a benzamide-based histone deacetylase inhibitor; More preferably, D' is [ka] Selected from Among them, A is a phenyl group or a pyridyl group, and the phenyl group or the pyridyl group is each independently optionally substituted with 1 to 4 substituents selected from a halogen, a C1-C4 alkyl group (e.g., a methyl group, an ethyl group), and a C1-C4 haloalkyl group (e.g., a trifluoromethyl group); Preferably, A is a phenyl group or a pyridyl group, and the phenyl group or the pyridyl group is each independently and optionally substituted with 1 to 2 substituents selected from a halogen, a C1-C4 alkyl group (e.g., a methyl group, an ethyl group), and a trifluoromethyl group; More preferably, A is a pyridyl group, and the pyridyl group is optionally substituted with 1 to 2 substituents selected from a C1 to C4 alkyl group; More preferably, A is a pyridyl group, Most preferably, A is [ka] and B is a phenylene group; Preferably, B is [ka] and Y is -CO-NH-CH2-, Preferably, the methylene group terminal of Y is connected to B, and the carbonyl group terminal is connected to an alkenyl carbon or O, R 1 , R 2 are each independently selected from hydrogen and a C1-C4 alkyl group; Preferably, R 1 , R 2 are both hydrogen, X 1 , X 2 , X 3 , X 4 one of which is selected from hydrogen, halogen, and a C1-C4 alkyl group, and the remaining three are hydrogen; Preferably, X 1 , X 2 , X 3 , X4 one of the groups is selected from hydrogen and halogen, and the remaining three are hydrogen; More preferably, X 1 , X 2 , X 3 , X 4 one of which is selected from hydrogen and fluorine, and the remaining three are hydrogen; Most preferably, X 2 is selected from hydrogen and fluorine, and X 1 , X 3 , X 4 is hydrogen, More preferably, D' is [ka] (i.e., tucidinostat, chidamide), [ka] (i.e., entinostat), More preferably, D' is [ka] (i.e., tucidinostat, chidamide), The definitions of L1, L2, L3, and D are as above.

[0090] In a ninth aspect of the present invention, there is provided a method for preparing a ligand drug conjugate of formula II, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof, said method comprising: reacting a compound of formula I or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof with Ab to obtain a ligand drug conjugate of formula II or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof; Preferably, the reaction is carried out under conditions of pH = 5 to 10 and a temperature of 0 to 40°C, [ka] Among them, The definitions of Ab, n, L1, L2, L3, and D are as above.

[0091] In some embodiments, the compound of Formula I is [ka] [ka] [ka] [ka] Selected from.

[0092] In some embodiments, the ligand drug conjugate of Formula II is [ka] [ka] [ka] [ka] Selected from.

[0093] In some embodiments, the ligand drug conjugate of Formula II is [ka] [ka] Selected from. [Effects of the Invention]

[0094] Beneficial effects of the present invention: 1. The present invention provides a novel stable ADC linker and an antibody conjugate with excellent antitumor effects.

[0095] 2. In in vitro tumor cell inhibition tests, the conjugates containing the novel stable linker of the present invention have a significant effect in inhibiting cell growth compared with naked antibodies, small molecule drugs, and combination therapies.

[0096] 3. In in vivo efficacy experiments in mice, the conjugate containing the novel stable linker of the present invention has a significant effect in inhibiting tumor growth compared with the naked antibody and combination therapy.

[0097] 4. The conjugates containing the novel stable linker of the present invention have good plasma stability.

[0098] 5. The complexes containing the novel stable linker of the present invention have excellent endocytosis.

[0099] 6. The conjugate containing the novel stable linker of the present invention has a significant promoting effect on cellular histone acetylation. [Brief explanation of the drawings]

[0100] [Figure 1-1] 1 is a RP-HPLC chromatogram of ADC-1.1. [Figure 1-2] 1 is a RP-HPLC chromatogram of ADC-1.2. [Figure 1-3] 1 is a RP-HPLC chromatogram of ADC-1.3. [Figure 1-4] 1 is a RP-HPLC chromatogram of ADC-1.4. [Figure 1-5] 1 is a RP-HPLC chromatogram of ADC-1.5. [Figure 1-6] 1 is a RP-HPLC chromatogram of ADC-1.6. [Figure 2] 1 is a RP-HPLC chromatogram of ADC-2. [Figure 3-1]1 is a RP-HPLC chromatogram of ADC-3.1. [Figure 3-2] 1 is a RP-HPLC chromatogram of ADC-3.2. [Figure 3-3] 1 is a RP-HPLC chromatogram of ADC-3.3. [Figure 3-4] 1 is a RP-HPLC chromatogram of ADC-3.4. [Figure 4] 1 is a RP-HPLC chromatogram of ADC-4. [Figure 5] 1 is a RP-HPLC chromatogram of ADC-5. [Figure 6] 1 is a RP-HPLC chromatogram of ADC-6. [Figure 7] 1 is a RP-HPLC chromatogram of ADC-7. [Figure 8] Figure 14 shows the results of measuring the affinity of Ate antibody and ADC-3.3 with hPD-L1-his. [Figure 9] Figure 14 shows the results of measuring the affinity of Ate antibody with hPD-L1-his. [Figure 10] Figure 14 shows the results of measuring the affinity between ADC-1.4 and hPD-L1-his. [Figure 11] Figure 14 shows the results of measuring the affinity between ADC-1.5 and hPD-L1-his. [Figure 12] Figure 14 shows the results of measuring the affinity between ADC-1.6 and hPD-L1-his. [Figure 13] Figure 14 shows the results of measuring the affinity between Ate antibody and Cyno PD-L1-his. [Figure 14] Figure 1 shows the results of measuring the affinity between ADC-1.4 and Cyno PD-L1-his. [Figure 15] Figure 1 shows the results of measuring the affinity between ADC-1.5 and Cyno PD-L1-his. [Figure 16] Figure 1 shows the results of measuring the affinity between ADC-1.6 and Cyno PD-L1-his. [Figure 17] Figure 14 shows the results of measuring the affinity between Ate antibody and mPD-L1-his. [Figure 18] FIG. 10 shows the results of measuring the affinity between ADC-1.4 and mPD-L1-his. [Figure 19] FIG. 10 shows the results of measuring the affinity between ADC-1.5 and mPD-L1-his. [Figure 20] FIG. 10 shows the results of measuring the affinity between ADC-1.6 and mPD-L1-his. [Figure 21] Figure 14 shows the results of measuring the affinity between Ate antibody and Rat PD-L1-his. [Figure 22] Figure 14 shows the results of measuring the affinity between ADC-1.4 and Rat PD-L1-his. [Figure 23] Figure 14 shows the results of measuring the affinity between ADC-1.5 and Rat PD-L1-his. [Figure 24] Figure 14 shows the results of measuring the affinity between ADC-1.6 and Rat PD-L1-his. [Figure 25] FIG. 10 is a graph showing the results of measuring the effects of ADC-1.4, ADC-1.5, and ADC-1.6 on histone H4 acetylation levels in NCI-H292 cells. [Figure 26] FIG. 1 shows the results of measuring free toxins from ADC-1.4, ADC-1.5, ADC-1.6, and ADC-3.3. [Figure 27] FIG. 1 shows the results of measuring the stability of ADC-1.6 in human plasma, cynomolgus monkey plasma, SD rat plasma, and C57 mouse plasma. [Figure 28] FIG. 1 shows the results of measuring the endocytosis rate of ADC-1.6 in tumor cells. [Figure 29] Figure 1 shows the results of ADC-3.3 inhibition of cell proliferation of colorectal cancer cell line CT26 hPD-L1-EX. [Figure 30] Figure 1 shows the results of ADC-1.6 inhibition of cell proliferation of colorectal cancer cell line CT26 hPD-L1-EX. [Figure 31] FIG. 10 shows the results of ADC-1.6 inhibition of cell proliferation of human malignant melanoma cell line A375 in a co-culture system of human peripheral blood mononuclear cells (PBMC) and human malignant melanoma cell line A375. [Figure 32]FIG. 10 shows the results of ADC-1.6 inhibition of cell proliferation of human malignant melanoma cell line A375 in a co-culture system of human peripheral blood mononuclear cells (PBMC) and human malignant melanoma cell line A375 (sample concentration: 0.008 μM). [Figure 33] FIG. 1 shows the results of tumor inhibition rates for ADC-1.6 and naked antibody in a C57BL / 6J mouse MC38 tumor model. [Figure 34] FIG. 1 shows the results of tumor inhibition rates for ADC-1.6 and combination therapy in a C57BL / 6J mouse MC38 tumor model. DETAILED DESCRIPTION OF THE INVENTION

[0101] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are merely illustrative of the present invention and do not limit the scope of the present invention.

[0102] Unless otherwise specified, the following terms and phrases used in this disclosure have the following meanings: When a trade name is used in this disclosure, unless the context dictates otherwise, the trade name includes the product formulation, generic drug, and active ingredient of the trademarked product.

[0103] In the present invention, the term "antibody" refers to an immunoglobulin having a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to differences in the amino acid composition and sequence order of the heavy chain constant region. Therefore, immunoglobulins are divided into five types (also called immunoglobulin isotypes): IgM, IgD, IgG, IgA, and IgE, with corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Even within the same type of Ig, subclasses can be further divided based on differences in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ or λ chains based on differences in the constant region. Each of the five types of Ig may have either κ or λ chains.

[0104] The term "drug-antibody ratio (DAR)" refers to the average quantity (e.g., n in Formula II) of cytotoxic drug (i.e., D in Formula I or Formula II) carried by each Ab (e.g., antibody) in Formula II, and may be an integer or a decimal number. The DAR range (e.g., n in general formula II) may be such that an average of 0.5 to 8.5 cytotoxic drugs (i.e., D in formula I or II) are linked to each Ab (e.g., antibody) (i.e., any integer or decimal between 0.5 and 8.5, including the endpoints 0.5 and 8.5). For example, the DAR (e.g., n in formula II) may be 0.5, 1, 1.5, 1.94, 2, 2.5, 3, 3.37, 3.5, 4, 4.08, 4.5, 4.71, 4.74, 4.99, 5, 5.26, 5.43, 5.5, 6, 6.34, 6.46, 6.5, 7, 7.08, 7.5, 7.66, 7.77, 7.80, 7.81, 8, or 8.5.

[0105] The term "pharmaceutically acceptable salt" refers to a salt of a compound or complex of the present invention, which is safe and effective when used in a mammalian body and possesses the desired biological activity. For example, an acidic group in a compound or complex of the present invention may form a salt with a base, non-limiting examples of which include sodium salt, potassium salt, calcium salt, or magnesium salt. Alternatively, a basic group in a compound or complex of the present invention may form a salt with an acid, non-limiting examples of which include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogensulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, phosphate, hydrogenphosphate, dihydrogenphosphate, salicylate, hydrogencitrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0106] The term "solvate" refers to a pharmaceutically acceptable solvate of a compound or complex of the present invention with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0107] The term "stereoisomers" refers to the phenomenon of isomerism in organic chemistry where atoms or substituents in compound molecules having the same molecular formula are bonded in the same order but arranged in different ways in space.

[0108] The term "tautomer" refers to two isomers containing a heteroatom (e.g., nitrogen, oxygen, or sulfur atom), the only structural difference between which is the transition of a double bond corresponding to a proton, and the two isomers coexist in an equilibrium system and are converted into each other at a very high rate, a typical example of which is keto-enol tautomerism.

[0109] The term "deuterated compound" refers to a structure in which one or more hydrogen atoms have been replaced with deuterium atoms in a compound or complex of the present invention.

[0110] The term "amino acid residue" refers to an incomplete amino acid structure remaining after the amino group of an amino acid loses one hydrogen and the carboxy group loses one hydroxyl group, and has a terminal amino group and a terminal carbonyl group. In the present invention, L2 is an amino acid residue or a peptide residue consisting of 2 to 10 (preferably 2 to 4) amino acid residues, of which 2 to 10 (preferably 2 to 4) amino acids may be homologous or different. For example, when L2 is a peptide residue consisting of 4 amino acid residues, and the amino acids are selected from glycine and phenylalanine, L2 may be a peptide residue of glycine residue-glycine residue-phenylalanine residue-glycine residue (Gly-Gly-Phe-Gly), specifically: [ka] may be.

[0111] In the present invention, unless otherwise clearly indicated, the phrase "... are each independently selected..." used throughout this disclosure may mean that specific options represented by the same or different symbols in different groups do not affect each other, or may mean that specific options represented by the same or different symbols in the same group do not affect each other.

[0112] Substituents for the compounds of the invention are disclosed by group type or range. It is intended that the invention encompasses all individual subcombinations of each member of these group types and ranges. For example, the term "C1-C6 alkyl group" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl group, C4 alkyl group, C5 alkyl group, and C6 alkyl group.

[0113] As used herein, "substituted" or "substituted with" means that any one or more hydrogens on the designated atom or group are replaced with one selected from the designated group, but not more than the normal valence of the designated atom.

[0114] In the present invention, "optionally" means that something may or may not be selected. For example, "A is a pyridyl group, and the pyridyl group is optionally substituted with 1 to 2 substituents selected from C1 to C4 alkyl groups" means that A is a pyridyl group, and the pyridyl group may not be substituted with a substituent, or may be substituted with 1 to 2 substituents selected from C1 to C4 alkyl groups.

[0115] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0116] The term "C1-C4 alkyl group" refers to an alkyl group having 1 to 4 carbon atoms, and is preferably a "C1-C3 alkyl group." Illustrative examples of alkyl groups include, but are not limited to, a methyl group, an ethyl group, a propyl group (e.g., n-propyl and isopropyl groups), a butyl group (e.g., n-butyl, isobutyl, tert-butyl groups), and the like.

[0117] The term "C1-C4 haloalkyl group" refers to any of the above C1-C4 alkyl groups in which one or more hydrogen atoms have been substituted with halogen (preferably fluorine), and examples thereof include a monofluoromethyl group, a difluoromethyl group, a difluoroethyl group, a trifluoromethyl group, etc.

[0118] The term "absent" means that the two groups are directly linked. For example, Formula I [ka] In the absence of L2, the structure of formula I is [ka] Therefore, those skilled in the art should understand that when L2 is present, one end of L2 is linked to L1 and the other end is linked to L3, whereas when L2 is absent, L1 is linked to L3. Other similar definitions can be understood by referring to the above content.

[0119] The term "carrier" refers to a system that can change the way a drug enters the human body and its distribution within the body, control the drug release rate, and transport the drug to a target organ. The release and targeting system of a drug carrier can reduce drug degradation and loss, alleviate side effects, and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble into various forms of aggregates due to their unique amphiphilic structure, preferred examples of which include micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules and good membrane permeability, making them excellent drug carriers.

[0120] The term "excipient" refers to additives other than the main drug in a drug formulation, also called auxiliary substances. Examples of auxiliary substances include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerin, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, protamine sulfate, salts or electrolytes such as disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, and lanolin.

[0121] The term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, meaning complete or partial prevention of a disease or its symptoms, and / or therapeutic, meaning partial or complete stabilization or cure of the disease and / or side effects caused by the disease. As used in this disclosure, "treatment" encompasses any treatment of a disease in a patient, including (a) preventing a disease or condition in a patient who is susceptible to, but has not yet been diagnosed with, the disease or condition; (b) inhibiting the symptoms of the disease, i.e., arresting its progression; or (c) alleviating the symptoms of the disease, i.e., causing regression of the disease or condition.

[0122] The term "subject" includes humans or non-human animals. Exemplary human subjects include humans (referred to as patients) suffering from a disease (e.g., a disease described in this disclosure) or normal individuals. The term "non-human animal" in the present invention includes all vertebrates, e.g., non-mammals (e.g., birds, amphibians, reptiles) and mammals (e.g., non-human primates, livestock and / or domesticated animals (sheep, dogs, cats, cows, pigs, etc.)).

[0123] The term "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic effect. A "therapeutically effective amount" of a compound or conjugate of the present invention can vary depending on factors such as the individual's condition, age, sex, and weight, and the ability of the compound or conjugate to elicit a desired response in the individual. A therapeutically effective amount further encompasses an amount in which the therapeutically beneficial effects of the compound or conjugate outweigh any toxic or adverse effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic effect. Typically, a prophylactic dose is administered to a subject before the onset of disease or at an early stage of disease, and therefore the prophylactically effective amount will be lower than the therapeutically effective amount, but this is not necessarily the case. In the case of cancer, administration of a therapeutically effective amount of a drug can reduce the number of cancer cells, shrink tumor volume, inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer.

[0124] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are merely for the purpose of illustrating the present invention and do not limit the scope of the present invention. In the following examples, test methods for which specific conditions are not specified generally follow conventional conditions or conditions suggested by manufacturers.

[0125] Example 1 Preparation of Linker-Toxin (Compound 1) [ka] 1) Preparation of Compound 1c: To a solution of the compound represented by formula 1a (805 mg, 1.25 mmol) in N,N-dimethylformamide (12 mL), 1-hydroxybenzotriazole (506 mg, 3.7 mmol) was added and the mixture was allowed to react at room temperature for 2 hours. After this, entinostat (1b, 470 mg, 1.25 mmol) was added and the mixture was allowed to react at room temperature for 72 hours. After the reaction was complete, the reaction mixture was concentrated and purified by reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0:100% to 50:50%) to obtain the compound represented by formula 1c (200 mg). ESI-MS (m / z): 882.3 [M+H] + .

[0126] 2) Preparation of Compound 1d: In an ice bath, trifluoroacetic acid (2 mL) was slowly added dropwise to a solution of the compound represented by formula 1c (200 mg, 0.23 mmol) in dichloromethane (10 mL), and the reaction was allowed to proceed for 2 hours. The reaction mixture was concentrated and purified by reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0:100% to 40%:60%) to give the compound represented by formula 1d (80 mg). ESI-MS (m / z): 782.4 [M+H] + .

[0127] 3) Preparation of Compound 1: To a solution of the compound represented by formula 1d (80 mg, 0.23 mmol) in N,N-dimethylformamide (10 mL), 1-{15-[(2,5-dioxytrihydro-1H-pyrrol-1-yl)oxy]-15-oxy-3,6,9,12-tetraoxy-1-yl}pyrrole-2,5-dione (1e, 26.5 mg, 0.06 mmol) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated and purified by reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0:100% to 50:50%) to obtain compound 1 represented by formula 1 (27.7 mg). ESI-MS (m / z): 1109.5 [M+H] + .

[0128] Example 2 Preparation of Linker-Toxin (Compound 2) [ka] 4-((17S,20S)-1-(2,5-dioxo-2,5-dioxo-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazacycloalkane-21-amino)benzyl(4-nitrophenyl)carbonate (2a, 252 mg, 0.29 mmol) and DMA (2 mL) were added to a 50 mL round-bottom flask in this order, and the mixture was purged with nitrogen gas three times. HOBT (116 mg, 0.86 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h. Tushidinostat (2b, 112 mg, 0.29 mmol) was added to the reaction mixture, and the reaction mixture was stirred at room temperature for 96 h. The resulting reaction mixture was directly purified by reversed-phase silica gel column chromatography (eluent: acetonitrile / 0.05% formic acid aqueous solution = 0:100% to 35%:65%) to obtain compound 2 (15 mg) represented by formula 2. ESI-MS (m / z): 1123.7 [M+H] + It was. 1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 9.76 (s, 1H), 9.17 (s, 1H), 8.78 - 8.75 (m, 2H), 8.56 (dd, J = 4.8, 1.6 Hz, 1H), 8.10 (d, J = 7.5 Hz, 1H), 8.00 (dt, J = 8.0, 1.9 Hz, 1H), 7.93 (d, J = 8.2 Hz, 2H), 7.85 (d, J = 8.6 Hz, 1H), 7.62 - 7.39 (m, 8H), 7.33 (d, J = 8.6 Hz, 2H), 7.01 - 6.96 (m, 2H), 6.82 (d, J = 15.9 Hz, 1H), 5.98 (t, J = 5.6 Hz, 1H), 5.40 (s, 2H), 5.09 (s, 2H), 4.49 (d, J = 5.9 Hz, 2H), 4.38 (q, J = 8.1 Hz, 1H), 4.23 (dd, J = 8.5, 6.8 Hz, 1H), 3.65 - 3.41 (m, 18H), 3.04 - 2.91 (m, 2H), 2.51 - 2.32 (m, 2H), 1.97 - 1.94 (m, 1H), 1.70 - 1.57 (m, 2H), 1.44 - 1.33 (m, 2H), 0.86 - 0.82 (m, 6H).

[0129] Example 3 Production of Rinotoxin (Compound 3)

change

[0130] Example 4 Synthesis of Linker-Toxin (Compound 4) [ka] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (4a, 200 mg, 0.27 mmol) and anhydrous N,N-dimethylacetamide (1 mL) were added to a 50 mL round-bottom flask in this order, and the flask was purged with nitrogen gas three times. HOBT (109 mg, 0.54 mmol) was added thereto, and the reaction mixture was stirred at room temperature for 2 hours. Tushidinostat (2b, 105 mg, 0.27 mmol) was added thereto, and the reaction mixture was stirred at room temperature for 72 hours. The solvent was evaporated under reduced pressure, and the resulting residue was purified by reverse-phase silica gel column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0:100% to 35%:65%) to obtain compound 4 (18 mg) represented by formula 4. ESI-MS (m / z): 989.5 [M+H] + It was.

[0131] Example 5 Preparation of Linker-Toxin (Compound 5) [ka] By following the procedure of Example 4 and using entinostat (1b) instead of tucidinostat (2b) in Example 4, compound 5 (65 mg) represented by formula 5 was obtained. ESI-MS (m / z): 975.5 [M+H] + It was.

[0132] Example 6 Preparation of Linker-Toxin (Compound 6) [ka] Compound 6 (55 mg) represented by formula 6 was obtained by using the method of Example 4, except that 4-((2S,5S)-19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-4,7,17-trioxo-2-(3-ureidopropyl)-10,13-dioxo-3,6,16-triazaamido)benzyl(4-nitrophenyl)carbonate (6a) was used instead of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (4a) in Example 4. ESI-MS (m / z): 1092.5 [M+H] + It was.

[0133] Example 7: Preparation of Linker-Toxin (Compound 7) [ka] Preparation of compound 7b: To a solution of compound 7a (200 mg, 0.28 mmol) in N,N-dimethylacetamide (0.5 mL), tushidinostat (2b, 107.6 mg, 0.28 mmol) and 1-hydroxybenzotriazole (18.6 mg, 0.14 mmol) were added, and the mixture was allowed to react at 25 °C for 48 h. After the reaction was complete, the reaction mixture was purified by reverse-phase column chromatography (acetonitrile: 0.05% ammonium bicarbonate aqueous solution = 0:100% to 40%:60%) to obtain 7b (94 mg). ESI-MS (m / z): 933.4 [M+H] + It was.

[0134] Preparation of compound 7c: Diethylamine (0.2 mL, 2.76 mmol) was added to a solution of compound 7b (94 mg) in N,N-dimethylformamide (0.8 mL), and the reaction mixture was allowed to react overnight at 25 °C. After completion of the reaction, the reaction mixture was purified by reverse-phase column chromatography (acetonitrile: 0.05% formic acid aqueous solution = 0:100% to 40%:60%) to obtain 7c (51 mg). ESI-MS (m / z): 710.28 [M+H] + It was.

[0135] Preparation of Compound 7: To a solution of compound 7c (29 mg, 0.04 mmol) in N,N-dimethylformamide (0.5 mL), compound 7d (18 mg, 0.04 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (13 mg, 0.04 mmol), and N,N-diisopropylethylamine (5 mg, 0.04 mmol) were added. After addition, the mixture was allowed to react overnight at 25 °C. After completion of the reaction, the reaction mixture was purified by reverse-phase preparative liquid chromatography (acetonitrile: 0.05% formic acid aqueous solution = 0:100% to 40%:60%) to obtain compound 7 (15 mg) represented by formula 7. ESI-MS (m / z): 1213.8 [M+H] + It was.

[0136] Example 8: Preparation of Linker-Toxin (Compound 8) [ka] Compound 7c (29 mg, 0.04 mmol), 1e (18 mg, 0.04 mmol), and N,N-dimethylacetamide (0.5 mL) were added to a 5 mL recovery flask equipped with a stirrer, in that order. N,N-diisopropylethylamine (6 mg, 0.05 mmol) was then added. After addition was complete, the mixture was stirred at room temperature for 3 h. The resulting reaction mixture was purified by reverse-phase column chromatography (eluent: acetonitrile: 0.05% formic acid aqueous solution = 0:100% to 45%:65%) to obtain compound 8 (20 mg, 0.02 mmol, 54.3%) represented by formula 8. ESI-MS (m / z): 1037.5 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 9.96 (s, 1H), 9.79 (s, 1H), 9.21 (s, 1H), 8.82 - 8.76 (m, 2H), 8.58 (dd, J = 4.8, 1.6 Hz, 1H), 8.49 (s, 1H), 8.20 (d, J = 7.2 Hz, 1H), 8.04 - 7.84 (m, 4H), 7.61 - 7.44 (m, 8H), 7.35 (d, J = 8.8 Hz, 2H), 7.03 - 6.98 (m, 2H), 6.84 (d, J = 16 Hz, 1H), 5.11 (s, 2H), 4.51 (d, J = 6 Hz, 2H), 4.44 - 4.36 (m, 1H), 4.22 (dd, J = 8.8, 6.8 Hz, 1H), 3.65 - 3.44 (m, 18H), 2.49 - 2.33 (m, 2H), 2.02 - 1.94 (m, 1H), 1.32 (d, J = 7.2 Hz, 3H), 0.87 (dd, J = 15.2, 6.8 Hz, 6H).

[0137] Example 9: Preparation of Linker-Toxin (Compound 9) [ka] Preparation of compound 9b: To a solution of compound 9a (5000 mg, 6.52 mmol) in N,N-dimethylacetamide (50 mL), tushidinostat (2b, 2545.6 mg, 6.52 mmol) and HOBt (528.6 mg, 3.91 mmol) were added, and the mixture was allowed to react at 25 °C for 72 h. After the reaction was complete, the reaction mixture was purified by reverse-phase column chromatography (acetonitrile: 0.05% ammonium bicarbonate aqueous solution = 0:100% to 40%:60%) to obtain compound 9b (750 mg). ESI-MS (m / z): 509.7 [M / 2+H] + It was.

[0138] Preparation of compound 9c: Diethylamine (0.5 mL) was added to a solution of compound 9b (550 mg, 0.54 mmol) in N,N-dimethylformamide (5 mL), and the reaction mixture was allowed to react overnight at 25 °C. After completion of the reaction, the reaction mixture was purified by reverse-phase preparative liquid chromatography to obtain compound 9c. ESI-MS (m / z): 398.91 [M / 2+H] + .

[0139] Preparation of Compound 9: To a solution of compound 9c (31 mg, 0.04 mmol) in N,N-dimethylacetamide (2 mL), compound 9d (25 mg, 0.04 mmol) and N,N-diisopropylethylamine (0.02 mL, 0.11 mmol) were added, and the mixture was allowed to react at 25 °C for 18 h. After the reaction was complete, the reaction mixture was purified by reverse-phase column chromatography (acetonitrile: 0.05% formic acid aqueous solution = 0:100% to 50%:50%) to obtain compound 9 (3 mg) represented by formula 9. ESI-MS (m / z): 650.33 [M / 2+H] + It was.

[0140] Example 10 Preparation of antibody-drug conjugates 10.1 Production of the anti-PD-L1 antibody Atezolizumab (Ate antibody): The atezolizumab (DrugBank Accession Number: DB11595) antibody gene was CHO codon optimized and synthesized by Shanghai Synchro Bioengineering Co., Ltd., and a plasmid was constructed to obtain the expression plasmid pXC-Atezolizumab.

[0141] The plasmid was introduced into CHO cells by electrotransfection, and the cells were screened, cultured, and expressed. The cell fermentation broth was collected and subjected to affinity chromatography to obtain atezolizumab antibody (referred to as Ate antibody or Ate in this application).

[0142] [Table 1-1]

[0143] 10.2 Manufacturing of the anti-PD-L1 antibody Avelumab (Ave): The antibody gene for Avelumab (DrugBank Accession Number: DB11945) was CHO codon optimized and synthesized by Shanghai Synchro Bioengineering Co., Ltd., and a plasmid was constructed to obtain the expression plasmid pXC-Avelumab.

[0144] The plasmid was introduced into CHO cells by electrotransfection, and the cells were screened, cultured, and expressed. The cell fermentation broth was collected and subjected to affinity chromatography to obtain the Avelumab antibody (abbreviated as Ave antibody or Ave in this application).

[0145] [Table 1-2]

[0146] 10.3 Antibody-Drug Coupling Reactions ADC-1.1 Production: Preparation of buffered salt solutions: Buffer solution-1 (Buffer-1): 4.65 g of L-histidine was dissolved in 1 L of ultrapure water, and the pH was adjusted to approximately 5.50 with 0.1 mol / L of glacial acetic acid. The solution was then filtered through a 0.22 μm filter membrane for sterilization, bottled, and stored at 4°C for a short period of time before use.

[0147] Buffer solution-2: 8.96 g of NaHPO·12H2O and 3.90 g of NaHPO·2H2O were dissolved in 500 mL of ultrapure water, and the pH was adjusted to 8.0 (±0.05). The solution was filtered through a 0.22 μm filter membrane for sterilization, bottled, and stored at 4°C for short-term use.

[0148] Replacement of antibody buffer: The antibody stock solution was slowly dissolved at 4°C and replaced with Buffer-1 by ultrafiltration to a final concentration of >10 mg / mL, and the concentration was measured using an ultraviolet spectrophotometer.

[0149] Antibody reduction: Accurately transfer 1 eq of antibody with a pipette, add a certain amount of Buffer-1 to adjust the final antibody concentration to approximately 5 mg / mL, then add 8 eq of 10 mmol / L TCEP solution, mix uniformly, and then keep at 25°C for 120 min.

[0150] Antibody coupling: The required volume of organic solvent (DMA or DMSO) was accurately transferred to 10% of the total volume. The small molecule (10 eq) of Formula 2 was added to the organic solvent and mixed thoroughly. The resulting mixture was slowly added to the reduced antibody reaction solution. The mixture was then stirred slowly at 25°C for 60 minutes to allow the coupling reaction to proceed.

[0151] Primary purification of the product: After the reaction was completed, the product was replaced with Buffer-1 by ultrafiltration and stored at 4°C for short-term storage or at -80°C for long-term storage until ready for use.

[0152] DAR measurement: DAR was measured using RP-HPLC. Measurement conditions: PLRP-S 8μm 4.6 x 150mm column, column temperature 80°C, flow rate 0.8mL / min, injection volume 20μg, measurement wavelength 214-280nm, gradient elution: 0min 30% B, 5min 35% B, 25min 45% B, 26-30min 90% B, 31-40min 30% B, where B was ACN containing 0.1% TFA. Sample processing: Samples were diluted to 3mg / mL with the corresponding buffer, and DTT was added to a final concentration of 20mM. After uniform mixing, the samples were directly injected for analysis.

[0153] Calculation of the average DAR value: (1) Calculate the peak area percentages of L0 and L1 (the sum of the peak area percentages of L0 + L1 is 100%) and the peak area percentages of H0, H1, H2, and H3 (the sum of the peak area percentages of H0 + H1 + H2 + H3 is 100%); (2) Calculate the weighted percentage of each peak, i.e., peak area percentage × number of drug conjugates; for example, H3 weighted percentage = H3 peak area percentage × 3; (3) Average DAR value = sum of the weighted percentages of each peak × 2 / 100.

[0154] [ka] The measured n was 7.08. The details are shown in Figure 1-1.

[0155] ADC-1.2 Production: The preparation of ADC-1.2 was carried out in accordance with the preparation steps and DAR measurement method of ADC-1.1, with the only difference being that the Ate antibody was replaced with Ave antibody, 4 eq of 10 mmol / L TCEP solution was added during antibody reduction, and 6 eq of small molecule was added during antibody coupling, while the remaining conditions were the same.

[0156] [ka] The measured n was 3.37. The details are shown in Figure 1-2.

[0157] ADC-1.3 Production: The production of ADC-1.3 was based on the production steps and DAR measurement method of ADC-1.1, with the only difference being that the Ate antibody was replaced with the Ave antibody.

[0158] [ka] The measured n was 4.74. The details are shown in Figure 1-3.

[0159] ADC-1.4 Production: The preparation of ADC-1.4 was based on the preparation steps and DAR measurement method of ADC-1.1, with the difference being that 1.2 eq of TCEP was added during antibody reduction and 4 eq of small molecule loading was added during coupling.

[0160] [ka] The measured n was 1.94. The details are shown in Figure 1-4.

[0161] ADC-1.5 Production: The preparation of ADC-1.5 was based on the preparation steps and DAR measurement method of ADC-1.1, with the difference being that 4 eq of TCEP was added during antibody reduction and 6 eq of small molecule loading was added during coupling.

[0162] [ka] The measured n was 4.71. The details are shown in Figure 1-5.

[0163] ADC-1.6 Production: The preparation of ADC-1.6 was based on the preparation steps and DAR measurement method of ADC-1.1, with the exception that 10 eq of TCEP was added during antibody reduction and 14 eq of small molecule loading was added during coupling.

[0164] [ka] The measured n was 7.66. The details are shown in Figure 1-6.

[0165] ADC-2 Production: The preparation of ADC-2 was carried out in accordance with the preparation method and the measurement method of the DAR value of ADC-1.1, with the only difference being that Formula 2 was replaced with the structure shown in Formula 3.

[0166] [ka] The measured n was 6.34. Specifically, this is shown in Figure 2.

[0167] ADC-3.1 Production: The preparation of ADC-3.1 was carried out in accordance with the preparation method and the measurement method of the DAR value of ADC-1.1, with the only difference being that Formula 2 was replaced with the structure shown in Formula 4.

[0168] [ka] The measured n was 6.46. The details are shown in Figure 3-1.

[0169] ADC-3.2 Production: The preparation of ADC-3.2 was based on the preparation steps and DAR measurement of ADC-3.1, with the only difference being that 4 eq of 10 mmol / L TCEP solution was added during antibody reduction, and 6 eq of small molecule was added during antibody coupling.

[0170] [ka] The measured n was 4.99. The details are shown in Figure 3-2.

[0171] ADC-3.3 Production: The preparation of ADC-3.3 was carried out based on the preparation steps and DAR measurement method of ADC-3.1, with the only difference being that 12 eq of 10 mmol / L TCEP solution was added during antibody reduction, and 14 eq of small molecule was added during antibody coupling; the other conditions were the same.

[0172] [ka] The measured n was 7.80. The details are shown in Figure 3-3.

[0173] ADC-3.4 Production: The preparation of ADC-3.4 was based on the preparation steps and DAR measurement method of ADC-3.1, with the only difference being that the Ate antibody was replaced with the Ave antibody, and 4 eq of 10 mmol / L TCEP solution was added during antibody reduction, and 6 eq of small molecule was added during antibody coupling; the rest of the conditions were the same.

[0174] [ka] The measured n was 4.08. The details are shown in Figure 3-4.

[0175] ADC-4 Manufacturing: The preparation of ADC-4 was carried out based on the preparation steps and the measurement method of DAR value of ADC-1.1, with the only difference being that Formula 2 was replaced with the structure shown in Formula 5, and the remaining conditions were the same.

[0176] [ka] The measured n was 5.43. Specifically, this is shown in Figure 4.

[0177] ADC-5 Manufacturing: The preparation of ADC-5 was based on the preparation steps and DAR measurement method of ADC-1.1, with the exception that the small molecule of formula 2 was replaced with the small molecule of formula 7, 12 eq of TCEP was added during antibody reduction, and 10 eq of small molecule loading was added during coupling.

[0178] [ka] The measured n was 7.77. Specifically, this is shown in Figure 5.

[0179] ADC-6 Manufacturing: The preparation of ADC-6 was based on the preparation steps and DAR measurement method of ADC-1.1, with the exception that the small molecule of formula 2 was replaced with the small molecule of formula 8, 12 eq of TCEP was added during antibody reduction, and 10 eq of small molecule loading was added during coupling.

[0180] [ka] The measured n was 7.66. Specifically, this is shown in Figure 6.

[0181] ADC-7 Production: The preparation of ADC-7 was based on the preparation steps and DAR measurement method of ADC-1.1, with the exception that the small molecule of formula 2 was replaced with the small molecule of formula 9, 12 eq of TCEP was added during antibody reduction, and 10 eq of small molecule loading was added during coupling.

[0182] [ka] The measured n was 7.50. Specifically, this is shown in Figure 7.

[0183] Example 11 Measurement of antibody-drug conjugate polymer The ADC measurement instrument was a Thermo Vanquish core, the column was a BioCore SEC-300 5 μm 4.6 × 300 mm, the mobile phase was 1X PBS, the flow rate was 0.3 mL / min, isocratic elution was performed, the analysis time was 20 min, and the measurement wavelength was 280 nm. The results are shown in Table 2.

[0184] [Table 2]

[0185] Among them, / indicates non-existence or not detected.

[0186] The results showed that the purity of the antibody-drug conjugate samples (e.g., ADC-1.4, ADC-1.5, ADC-1.6, ADC-3.3, ADC-5, ADC-6, ADC-7) was comparable to the corresponding naked antibodies.

[0187] Example 12 Measurement of antibody-drug conjugate sample affinity Affinity measurement by SPR method The affinity between antibody-drug conjugate samples (e.g., ADC-1.4, ADC-1.5, ADC-1.6, ADC-3.3) / naked antibodies (e.g., Ate antibody) and Cyno PD-L1-his, hPD-L1-his, mPD-L1-his, and Rat PD-L1-his was measured using a Biacore T200.

[0188] The method is as follows: a CM5 chip was activated with N-hydroxysuccinimide (NHS):1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) = 1v:1v, and then blocked by immobilizing an anti-His antibody.

[0189] 1μg / mL hPD-L1-his was captured on the chip surface, and ADC-3.3 and Ate antibodies were diluted 2-fold in 1x PBST to a total of 9 concentration points, ranging from 25nM to 0.39nM. The program was set up, samples were loaded, and the program was run. Data was kinetically fitted using the 1:1 binding model in Biacore Evaluation Software. The results are shown in Table 3-1 and Figure 8.

[0190] [Table 3-1] 2 μg / mL Cyno PD-L1-his was captured on the chip surface, and the antibody-drug conjugate samples and naked antibody samples were diluted 2-fold with 1x PBST to create a total of seven concentration points, with sample concentrations ranging from 25 nM to 0.39 nM. The program was set up, samples were loaded, and the program was launched. Data was kinetically fitted using the 1:1 binding model in Biacore Evaluation Software. The above steps were repeated to capture 2μg / mL hPD-L1-his, 5μg / mL mPD-L1-his, and 5μg / mL Rat PD-L1-his on the chip surface, and the four samples were diluted 2-fold with 1x PBST. Of these, Ate antibody and ADC-1.4 were set up at eight concentration points, with concentrations ranging from 25nM to 0.39nM, and ADC-1.5 and ADC-1.6 were set up at nine concentration points, with concentrations ranging from 50nM to 0.19nM. The program was set up, samples were loaded, and the program was launched. Data were subjected to kinetic fitting using the 1:1 binding model in Biacore Evaluation Software. The results are shown in Table 3-2 and Figures 9 to 24.

[0191] [Table 3-2]

[0192] The results showed that the affinity between the antibody-drug conjugate samples of the present application (e.g., ADC-1.4, ADC-1.5, ADC-1.6, ADC-3.3) and Cyno PD-L1-his, hPD-L1-his, mPD-L1-his, and Rat PD-L1-his was comparable to that of naked antibodies (e.g., Ate antibody).

[0193] Example 13 Effect of Antibody-Drug Conjugate Samples on Total Cellular Acetylation Levels Measuring the effect of the ADC molecules (e.g., ADC-1.4, ADC-1.5, ADC-1.6) on NCI-H292 cell histone H4 acetylation levels (1) Harvest NCI-H292 cells and count 20 μL of them. (2) Resuspend the cells at 1×10^5 cells / mL. (3) Add 100 μL / well to a 48-well plate and incubate overnight at 37°C and 5% CO2. (4) Dilute the ADC molecules according to the set concentrations to match the tushidinostat concentrations in the added ADC molecules (19 μM in both). Add 100 μL / well to the corresponding wells and incubate at 37°C and 5% CO2 for 48 hours. (5) Aspirate and discard the supernatant. (6) Add 200 μL of fixative (4% paraformaldehyde) to each tube, immediately pipette 2-3 times, and incubate at 4°C for 15 minutes. (7) Add 1 mL of PBS to each tube to wash once. Centrifuge at 700 rcf for 5 minutes, then discard the supernatant. (8) Add 50 μL of membrane disruption solution (1% (9) 0.5 μL / tube of Milli-Mark® Anti-acetyl-Histone H4 Antibody-PE was added, and the mixture was incubated at room temperature for 1 hour away from light. (10) 1 mL of membrane rupture solution was added to each tube, followed by washing once. After centrifugation at 900 rcf for 5 minutes, the supernatant was discarded. (11) 100 μL of membrane rupture solution was added to each tube, and the mixture was pipetted evenly. The mixture was then analyzed by flow cytometry. (12) The data was processed using Graphpad Prism 9.0, and the results are shown in Figure 25.

[0194] The results showed that the antibody-drug conjugate sample ADCs of the present application (e.g., ADC-1.4, ADC-1.5, ADC-1.6) had a clear promoting effect on NCI-H292 histone H4 acetylation.

[0195] Example 14 Detection of free toxin Free linker-toxin was detected in antibody-drug conjugate samples (e.g., ADC-1.4, ADC-1.5, ADC-1.6, ADC-3.3) by RP-HPLC method.

[0196] HPLC analysis conditions: Equipment:Waters e2695 Column: XBridge (registered trademark) C18 3.5 μm, 4.6 × 150 mm column Fluid phase A: 0.1% TFA in water Fluid phase B: 0.1% TFA in ACN Measurement wavelength: 254nm Flow rate: 0.5mL / min Gradient elution: 0-30 min 10%-80% B, 31-35 min 10% B Sample processing: 85 μg of the test sample (e.g., ADC-1.4, ADC-1.5, ADC-1.6, ADC-3.3) was taken, and the sample was mixed with 3 μL of DMSO for 5 minutes. Then, 60 μL of saturated sodium chloride supernatant (dissolved in 30% methanol-acetonitrile) was added to the sample, and the mixture was mixed uniformly for 10 minutes. The mixture was centrifuged at 2000 rpm for 2 minutes, and the supernatant was taken for the test.

[0197] Data analysis: Linker-toxin concentrations of 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 5 μg / mL were injected as standards, and a calibration curve was plotted. If free linker-toxin was present in the sample, its peak area was substituted into a linear equation to calculate the concentration. The results are shown in Figure 26.

[0198] The results showed that no free linker-toxin was detected in the antibody-drug conjugate sample ADCs of the present application (e.g., ADC-1.4, ADC-1.5, ADC-1.6, ADC-3.3).

[0199] Example 15. Plasma stability measurements. Determining the plasma stability of antibody-drug conjugate samples of the present application (e.g., ADC-1.6) in plasma from different species The specific operations are as follows: The antibody-drug conjugate sample was prepared in sterile PBS to a concentration of 1 mg / mL, and was prepared immediately before use. The preparation process was completed in a sterile environment, and all reagent bottles or containers were sterilized.

[0200] Plasma from various genera (human, cynomolgus monkey, SD rat, C57 mouse) was carefully mixed uniformly at room temperature, 3600 μL of plasma from each genera was drawn up, 400 μL of test sample working solution was added, and a sample with a final test sample concentration of 100 μg / mL was prepared. The sample was then gently shaken to mix uniformly and prepared for use.

[0201] The final reaction volume for this incubation test was 200 μL, and samples were collected twice at each time point. Samples were collected after incubation at 37°C under sterile conditions for 0, 4, 8, 24, 48, 72, 120, and 168 hours, respectively. All procedures were performed under sterile and light-protected conditions. Samples at each time point were frozen and stored at -70°C to -90°C.

[0202] After sampling, the samples were measured by RP-HPLC.

[0203] HPLC analysis conditions: Instrument: AB SCIEX TRIPLE QUAD™ 4500 liquid chromatograph mass spectrometer Column: Bridge BEH C18, 2.5 μm, 2.1 × 50 mm, Waters Fluid phase A: 0.1% FA 2mmol / L ammonium formate aqueous solution Fluid phase B: CAN Strong washing solution (SNW): MeOH:ACN:IPA:DMSO = 1:1:1:1 (containing 0.5% FA), v / v / v / v Weak washing solution (WNW): MeOH:H2O = 1:1, v / v Sample processing steps: The samples were vortexed to homogenize, and 20 μL of each sample was transferred to a 96-well plate according to the plate map. 20 μL of ACN:HO (1:1, v / v) was added to the DB and carryover samples. 20 μL of the internal standard working solution (1000,000 ng / mL tolbutamide internal standard working solution) was added to the remaining wells. After vortexing for 1 minute, 160 μL of 0.1% FA in ACN:MeOH (1:1), was added to all wells. The membranes were sealed, and the wells were vortexed at 1000 rpm for 5 minutes to homogenize. The wells were then centrifuged at 4°C and 4700 g for 10 minutes. According to the plate map, 100 μL of the supernatant was transferred to a new 96-well plate, the membranes were sealed, and the samples were injected and analyzed. The calculation results are shown in Table 4 and Figure 27.

[0204] [Table 4]

[0205] Of these, NA stands for not detected. The results showed that the Ate antibody did not detect small molecule drugs in the plasma of different species, and the antibody-drug conjugate sample ADC (e.g., ADC-1.6) of the present application showed a shedding rate of less than 1.0% after incubation at 37°C for 168 hours in the plasma of four species: human, cynomolgus monkey, SD rat, and C57 mouse, and was generally relatively stable.

[0206] Example 16 Measurement of endocytosis of antibody-drug conjugate samples (ADC) in tumor cells Measurement of endocytosis of the antibody-drug conjugate samples (e.g., ADC-1.6) of the present application in human lung cancer cell line NCI-H292 cells (1) NCI-H292 cells were harvested and 20 μL was taken and counted. (2) The cells were resuspended to 6×10^6 cells / mL and added to two 1.5 mL EP tubes at 100 μL per tube. (3) 100 μL per tube of diluted antibody-drug conjugate ADC group and antibody group samples were added. The final concentration of the samples was 150 nM. After uniform mixing, the cells were incubated at 4°C for 1 hour away from light. (4) Pre-cooled PBS was added to wash the cells twice, and the cells were centrifuged at 4°C and 1200 rpm for 5 minutes. The supernatant was discarded. (5) Complete medium was added at 300 μL per tube to resuspend the cells. 150 μL of the cell suspension was aspirated and placed in a new EP tube. One tube was then used for further analysis. The other tube was incubated at 4°C, and the other at 37°C. (6) After 2, 4, and 22 hours, 50 μL of cell suspension was taken from each of the 4°C and 37°C tubes and placed in a new EP tube. 1 mL of pre-chilled PBS was added to each tube, and the tubes were centrifuged at 4°C and 1200 rpm for 5 minutes. The supernatant was discarded. (7) 0.5 μL of secondary antibody was added per tube, mixed evenly, and incubated at 4°C for 30 minutes, away from light. (8) Pre-chilled PBS was added once, and the tubes were centrifuged at 4°C and 1200 rpm for 5 minutes. The supernatant was discarded. (9) 100 μL of pre-chilled PBS was added per tube, and the cells were resuspended. After loading, the cells were measured. (10) The endocytosis rate was calculated using the following formula: Internalization (%) = Positive rate 4℃ (%)-Positive rate 37℃ (%), of which the positive rate 4℃ (%) is the positive rate of cells incubated at 4°C, and the positive rate 37℃ (%) is the positive rate of cells incubated at 37°C, the data was processed by Graphpad Prism 9.0, and the results are shown in Figure 28.

[0207] The results showed that the tumor cell endocytosis rates of the antibody-drug conjugate ADC group (e.g., ADC-1.6) and the antibody group (e.g., Ate antibody) increased over time, and the tumor cell endocytosis rate of the antibody-drug conjugate ADC group was clearly superior to that of the antibody group.

[0208] Example 17 Inhibitory effect of antibody-drug conjugate samples on tumor cells 17.1 Measuring Inhibition of the ADC Molecules of the Invention (e.g., ADC-3.3) on CT26-hPD-L1 Cell Proliferation (1) CT26 hPD-L1-EX cells were harvested and 20 μL was counted. (2) The cells were resuspended at 0.5×10^5 cells / mL and 0.5×10^5 cells / mL in 5 mL each. (3) 50 μL / well was added to two 96-well plates and incubated overnight at 37°C and 5% CO2. (4) ADC samples were diluted according to the set concentrations and added to the wells at 100 μL / well. (5) The plates were incubated at 37°C and 5% CO2 for 48 hours. (6) 50 μL of the culture medium was first aspirated and discarded, and then 20 μL / well of MTS reagent was added. The plates were incubated at 37°C and 5% CO2 for 1 to 4 hours. (7) The readings were taken at 490 nm. The results are shown in Figure 29 and Table 5.

[0209] [Table 5]

[0210] The results showed that the present antibody-drug conjugate sample ADC (e.g., ADC-3.3) had a significant inhibitory effect on CT26-hPD-L1 cell proliferation, and its inhibitory effect was significantly superior to that of small molecule drugs alone (e.g., tushidinostat, Chidamide), naked antibodies alone (e.g., Ate antibody), and the combination of small molecule drugs and naked antibodies (e.g., Chidamide + Ate antibody, i.e., Chi + Ate).

[0211] 17.2 Measuring Inhibition of the Present ADC Molecules (e.g., ADC-1.6) on Colorectal Cancer Cell Line CT26 Cell Proliferation (1) CT26 hPD-L1-EX cells were harvested and counted. (2) The cells were resuspended at 2 x 10^5 cells / mL. (3) 50 μL / well of the cells were seeded into a 96-well plate (1 x 10^4 cells / well) and incubated overnight at 37°C and 5% CO2. (4) The next day, the 96-well plate was removed, and the samples were diluted with complete medium containing 10% FBS. 50 μL / well of the cells were added to the plate to adjust the final concentration of the samples to 10 μM. This day was designated Day 0. The cells were then incubated for two more days at 37°C and 5% CO2. (5) Day 2: After observing the cell growth under a microscope, 10 μL / well of CCK8 was added and the cells were incubated for another 4 hours at 37°C and 5% CO2. (6) The absorbance at 450 nm (OD ) was measured in real time using a microplate reader. 450 ) was read and the results are shown in FIG. 30 and Table 6.

[0212] [Table 6]

[0213] The results showed that the inhibitory effect of antibody-drug conjugate ADC group (e.g., ADC-1.6) on CT26 hPD-L1-EX cell proliferation was superior to that of small molecule drugs (e.g., tucidinostat, Chidamide), antibodies (e.g., Ate antibody), and combination groups (e.g., Chidamide + Ate antibody, i.e., Chi + Ate).

[0214] 17.3 Measurement of the inhibitory effect of the ADC molecule of the present application (e.g., ADC-1.6) on the human melanoma cell line A375 in a co-culture system of human peripheral blood mononuclear cells (PBMC) and the human melanoma cell line A375 The specific operations are as follows: Cryopreserved PBMCs were removed from the liquid nitrogen bath, added to 8 mL of pre-warmed RPMI 1640 medium, and centrifuged at 500 g for 10 minutes. The supernatant was discarded, and the PBMCs were resuspended in a small amount of RPMI 1640 medium and diluted 10-fold. 20 μL of the PBMCs were counted and resuspended at 5E6 cells / mL. The A375-hPDL1-Luciferase cell suspension was collected, counted at 20 μL, and resuspended at 1E6 cells / mL. The PBMCs and A375-hPDL1-Luciferase cell suspension were mixed in equal volumes to produce a cell mixture with an E:T ratio of 5:1. This mixture was then diluted with RPMI 1640 medium. The mixture was supplemented with 1640 complete medium up to 1500 μL, and the Matrigel was placed on ice to dissolve. 1500 μL of the Matrigel and cell mixture was taken and mixed, and added to a black 384-well plate at 20 μL / well. The mixture was incubated at 37°C for 10 minutes, and the ADC-1.6 and Ate antibodies were added at concentrations of 5 μM, 1 μM, 0.2 μM, 0.04 μM, 0.008 μM, 0.0016 μM, and 0 μM. Chidamide was diluted in diluent to 200 μM, 40 μM, 8 μM, 1.6 μM, 0.32 μM, 0.064 μM, 0.0128 μM, and 0 μM. Each sample was diluted in eight concentration gradients, replicated three times, and added at 30 μL / well to a 384-well plate. The plate was then incubated at 37°C and 5% CO2 for 6 days. The 384-well plate was then removed and added at 20 μL / well to the Bio-Lite Luciferase Assay System. The plate was incubated for 5-10 minutes and measured using a multi-mode microplate reader. The results are shown in Figure 31, Figure 32, and Table 7.

[0215] [Table 7] The results showed that the antibody-drug conjugate ADC group (e.g., ADC-1.6) had an IC50 of <0.01 μM, while the Ate antibody group, Chidamide group, and combination group (Chidamide:Ate antibody = 8:1) all had IC50s >0.03 μM. The antibody-drug conjugate ADC group (e.g., ADC-1.6) had a significant growth inhibitory effect on A375-hPDL1-Luciferase cells at lower concentrations than the small molecule group (e.g., Chidamide), antibody group (e.g., Ate antibody), and combination group (e.g., Chidamide + Ate antibody, i.e., Chi + Ate). At a concentration of 0.008 μM, the inhibitory effect of the antibody-drug conjugate ADC group (e.g., ADC-1.6) on A375-hPDL1-Luciferase cell proliferation was superior to that of the small molecule drug group (e.g., Chidamide), antibody group (e.g., Ate antibody), and combination group (e.g., Chidamide + Ate antibody, i.e., Chi + Ate).

[0216] Example 18 Antitumor Effect of Antibody-Drug Conjugate Sample ADC In vivo pharmacodynamic studies of the antibody-drug conjugate ADC (e.g., ADC-1.6) of the present application were conducted using the C57BL / 6J mouse MC38 tumor model. 18.1 The specific method is as follows: On the day of cell inoculation, 3 × 10 cells were injected into each C57BL / 6J mouse. 5 MC38 cells were subcutaneously inoculated (0.1 mL / mouse). The average tumor volume of the mice was approximately 70-80 mm. 3 Once the mice reached 100 mg / kg, they were divided into groups, with 24 mice divided into three groups, and administration began on the day of grouping. The administration cycle was three times a week, and the administration method was intraperitoneal injection. The naked antibody group and the drug conjugate sample ADC group were each intraperitoneally injected with 10 mg / kg of Ate antibody and ADC-1.6, while the solvent control group (Vehicle) was intraperitoneally injected with an equal volume of PBS solution. After the start of administration, body weight and tumor volume were measured three times a week, and the tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2The study was terminated on day 7 after administration, and all mice were euthanized and tumor tissues were collected and weighed. The in vivo antitumor effects of the antibody-drug conjugate sample ADCs are shown in Figure 33.

[0217] The results showed that the tumor volume of the antibody-drug conjugate sample ADC-1.6 of the present application on day 7 after administration was 455.74 mm 3 The tumor volumes in the naked antibody group and the solvent control group were 701.09 mm 3 , 821.79mm 3 The tumor inhibition rate (TGI) (%) of the antibody-drug conjugate sample ADC-1.6 group was 48.97%, which was significantly different from the vehicle control group (P<0.05), and its inhibitory effect was significantly superior to that of the Ate antibody group (TGI=16.26%).

[0218] 18.2 The specific method is as follows: On the day of cell inoculation, 3 × 10 cells were injected into each C57BL / 6J mouse. 5 MC38 cells were subcutaneously inoculated (0.1 mL / mouse). The average tumor volume of the mice was approximately 50-60 mm. 3 Once the mice reached 10 mg / kg, they were divided into three groups, and treatment began on the day of grouping. The treatment cycle was three times a week, and the administration method was intraperitoneal injection. The combination group (Ate antibody + Chidamide) received an intraperitoneal injection of 10 mg / kg of mAb and 0.2 mg / kg of tucidinostat, the drug conjugate sample ADC group received an intraperitoneal injection of 10 mg / kg of ADC-1.6, and the solvent control group (Vehicle) received an intraperitoneal injection of an equal volume of PBS solution. After the start of treatment, body weight and tumor volume were measured three times a week, and the tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The study was terminated on day 7 after administration, and all mice were euthanized and tumor tissues were collected and weighed. The in vivo antitumor effects of the antibody-drug conjugate sample ADCs are shown in Figure 34.

[0219] The results showed that the tumor volume of the antibody-drug conjugate sample ADC-1.6 of the present application on day 7 after administration was 329.63 mm 3The tumor volumes in the combination group and the vehicle control group were 457.35 mm 3 , 499.89mm 3 The TGI (%) of the antibody-drug conjugate sample ADC-1.6 was 38.26%, which was significantly different from the solvent control group (P<0.05), and its inhibitory effect was significantly superior to that of the combination group (TGI=9.63%).

[0220] The present invention has been described by way of example with reference to specific embodiments. However, the present invention is not limited to these specific embodiments. Those skilled in the art can make various modifications and variations within the scope of the present invention, and can combine the technical features described in each part of this specification without departing from the spirit and scope of the present invention. All such modifications and variations are within the scope of the present invention.

Claims

1. A compound of formula I, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof, 【Chemistry 1】 Among them, L １ teeth, 【Chemistry 2】 Selected from Preferably, L １ The carbon end is connected to the N of succinimide, and the carbonyl group end is connected to L ２ Connected to m and t are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; q is 0, 1, 2 or 3; p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; X is CH ２ , O and NH; L ２ is absent, an amino acid residue, or a peptide residue consisting of 2 to 10 amino acid residues, Preferably, L ２ The amino group terminal is L １ The carbonyl group terminal is L ３ Connected to Preferably, the amino acids include, but are not limited to, phenylalanine (F), glycine (G), valine (V), lysine (K), serine (S), glutamic acid (E), asparagine (N), arginine (R), alanine (A), citrulline, and cysteine ​​(C); L ３ does not exist, or 【Transformation 3】 Selected from Preferably, L ３ The amino group terminal is L ２ and the carbonyl group terminal or carbon terminal is connected to D, D is a histone deacetylase inhibitor drug selected from a thiol-based histone deacetylase inhibitor, a hydroxamic acid-based histone deacetylase inhibitor, or a benzamide-based histone deacetylase inhibitor; A compound or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof.

2. Said L １ teeth, 【Chemistry 4】 Selected from Preferably, L １ teeth, 【Transformation 5】 Selected from Preferably, m is selected from 0, 1, 2, 3, 4 and 5, more preferably m is selected from 1, 3 and 4; Preferably, t is selected from 0, 1, 2, 3, 4, 5, 6 and 7, more preferably t is selected from 1, 2, 3 and 7; Preferably, q is selected from 0, 1, and 2; Preferably, p is selected from 10, 11, 12, 13, 14, and 15, more preferably, p is selected from 11, 12, and 13, and even more preferably, p is 12; Preferably, X is selected from O and NH; More preferably, L １ teeth, 【Transformation 6】 Selected from More preferably, L １ teeth, 【Transformation 7】 Selected from More preferably, L １ teeth, 【Transformation 8】 Selected from Even more preferably, L １ teeth, 【Chemistry 9】 and Even more preferably, L １ teeth, 【Chemistry 10】 characterized in that 2. The compound according to claim 1, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof.

3. L ２ is absent, an amino acid residue, or a peptide residue consisting of 2 to 4 amino acid residues, Preferably, the amino acids include, but are not limited to, phenylalanine (F), glycine (G), valine (V), alanine (A), citrulline, and cysteine; More preferably, the amino acids include, but are not limited to, phenylalanine (F), glycine (G), valine (V), alanine (A), citrulline; Preferably, L ２ is absent or is a citrulline residue or is selected from the following peptide residues: valine residue-alanine residue, valine residue-citrulline residue, glycine amino group-glycine residue-phenylalanine residue-glycine residue, valine residue-alanine residue-phenylalanine residue-glycine residue, glycine residue-glycine residue-glycine residue, glycine residue-phenylalanine residue-glycine residue, valine residue-cysteine-phenylalanine residue-glycine residue; More preferably, L ２ is absent, is a citrulline residue, or is selected from the following peptide residues: valine residue-alanine residue, valine residue-citrulline residue, glycine amino group-glycine residue-phenylalanine residue-glycine residue, valine residue-alanine residue-phenylalanine residue-glycine residue; More preferably, the L ２ does not exist, or 【Chemistry 11】 Selected from Even more preferably, the L ２ does not exist, or 【Chemistry 12】 Selected from Or even more preferably, L ２ does not exist, or 【Chemistry 13】 Selected from Most preferably, L ２ does not exist, or 【Chemistry 14】 characterized in that it is selected from 3. The compound according to claim 1, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof.

4. D is a benzamide-based histone deacetylase inhibitor; Preferably, D is 【Chemistry 15】 Selected from Among them, A is a phenyl group or a pyridyl group, and the phenyl group or the pyridyl group each independently optionally contains halogen, C １ ~C ４ Alkyl groups (e.g., methyl groups, ethyl groups), C １ ~C ４ substituted with 1 to 4 substituents selected from haloalkyl groups (e.g., trifluoromethyl groups); Preferably, A is a phenyl group or a pyridyl group, and said phenyl group or pyridyl group each independently optionally contains halogen, C １ ~C ４ substituted with 1 to 2 substituents selected from an alkyl group (e.g., a methyl group, an ethyl group) and a trifluoromethyl group; More preferably, A is a pyridyl group, and said pyridyl group is optionally selected from the group consisting of C １ ~C ４ substituted with 1 to 2 substituents selected from alkyl groups; More preferably, A is a pyridyl group, Most preferably, A is 【Chemistry 16】 and B is a phenylene group; Preferably, B is 【Chemistry 17】 and Y is -CO-NH-CH ２ - and Preferably, the methylene group terminal of Y is connected to B, and the carbonyl group terminal is connected to an alkenyl carbon or O, R １ , R ２ are each independently hydrogen, C １ ~C ４ selected from alkyl groups, Preferably, R １ , R ２ are both hydrogen, X １ , X ２ , X ３ , X ４ Any one of the following is hydrogen, halogen, C １ ~C ４ alkyl groups, and the remaining three are hydrogen; Preferably, X １ , X ２ , X ３ , X ４ any one of the groups is selected from hydrogen and halogen, and the remaining three are hydrogen; More preferably, X １ , X ２ , X ３ , X ４ any one of the groups is selected from hydrogen and fluorine, and the remaining three are hydrogen; Most preferably, X ２ is selected from hydrogen and fluorine, and X １ , X ３ , X ４ is hydrogen, More preferably, D is [Chemistry 18] characterized in that it is selected from 4. The compound according to any one of claims 1 to 3, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof.

5. The compound is 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】 characterized in that it is selected from 5. The compound according to any one of claims 1 to 4, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof.

6. 10. Use of a compound according to any one of claims 1 to 5, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof, in the manufacture of a ligand drug conjugate (e.g., an antibody drug conjugate).

7. A ligand drug conjugate of formula II, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof: 【Chemistry 23】 Among them, Ab is a ligand selected from a protein, antibody, polypeptide, enzyme, and small molecule; n is a number between 0.5 and 8.5, for example, n is a number between 0.8 and 5, a number between 1 and 4, a number between 2 and 6, a number between 3 and 7, a number between 4 and 8, a number between 3.5 and 8.5, a number between 3.5 and 4.5, or a number between 6.5 and 8.5; preferably, n is about 2, 3, 4, 5, 6, 7, or 8; more preferably, n is about 3, 4, 5, 6, 7, or 8; L １ teeth, 【Chemistry 24】 Selected from Preferably, L １ The carbon end is connected to the N of succinimide, and the carbonyl group end is connected to L ２ Connected to m and t are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8; q is 0, 1, 2 or 3; p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; X is CH ２ , O and NH; L ２ is absent, an amino acid residue, or a peptide residue consisting of 2 to 10 amino acid residues, Preferably, L ２ The amino group terminal is L １ The carbonyl group terminal is L ３ Connected to Preferably, the amino acids include, but are not limited to, phenylalanine (F), glycine (G), valine (V), lysine (K), serine (S), glutamic acid (E), asparagine (N), arginine (R), alanine (A), citrulline, and cysteine ​​(C); L ３ does not exist, or 【Chemistry 25】 Selected from Preferably, L ３ The amino group terminal is L ２ and the carbonyl group terminal or carbon terminal is connected to D, D is a histone deacetylase inhibitor drug selected from a thiol-based histone deacetylase inhibitor, a hydroxamic acid-based histone deacetylase inhibitor, or a benzamide-based histone deacetylase inhibitor; A ligand drug conjugate or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof.

8. Said L １ teeth, 【Chemistry 26】 Selected from Preferably, L １ teeth, 【Chemistry 27】 Selected from Preferably, m is selected from 0, 1, 2, 3, 4 and 5, more preferably m is selected from 1, 3 and 4; Preferably, t is selected from 0, 1, 2, 3, 4, 5, 6 and 7, more preferably t is selected from 1, 2, 3 and 7; Preferably, q is selected from 0, 1, and 2; Preferably, p is selected from 10, 11, 12, 13, 14, and 15, more preferably, p is selected from 11, 12, and 13, and even more preferably, p is 12; Preferably, X is selected from O and NH; More preferably, L １ teeth, 【Chemistry 28】 Selected from More preferably, L １ teeth, 【Chemistry 29】 Selected from More preferably, L １ teeth, 【Transformation 30】 Selected from Even more preferably, L １ teeth, 【Chemistry 31】 and Even more preferably, L １ teeth, 【Chemistry 32】 characterized in that 8. The ligand-drug complex of claim 7, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof.

9. L ２ is absent, an amino acid residue, or a peptide residue consisting of 2 to 4 amino acid residues, Preferably, the amino acids include, but are not limited to, phenylalanine (F), glycine (G), valine (V), alanine (A), citrulline, and cysteine; More preferably, the amino acids include, but are not limited to, phenylalanine (F), glycine (G), valine (V), alanine (A), citrulline; Preferably, L ２ is absent or is a citrulline residue or is selected from the following peptide residues: valine residue-alanine residue, valine residue-citrulline residue, glycine amino group-glycine residue-phenylalanine residue-glycine residue, valine residue-alanine residue-phenylalanine residue-glycine residue, glycine residue-glycine residue-glycine residue, glycine residue-phenylalanine residue-glycine residue, valine residue-cysteine-phenylalanine residue-glycine residue; More preferably, L ２ is absent, is a citrulline residue, or is selected from the following peptide residues: valine residue-alanine residue, valine residue-citrulline residue, glycine amino group-glycine residue-phenylalanine residue-glycine residue, valine residue-alanine residue-phenylalanine residue-glycine residue; More preferably, the L ２ does not exist, or 【Transformation 33】 Selected from Even more preferably, the L ２ does not exist, or 【Transformation 34】 Selected from Or even more preferably, L ２ does not exist, or 【Chemistry 35】 Selected from Most preferably, L ２ does not exist, or 【Transformation 36】 characterized in that it is selected from The ligand-drug complex according to any one of claims 7 to 8, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof.

10. D is a benzamide-based histone deacetylase inhibitor; Preferably, D is 【Chemistry 37】 Selected from Among them, A is a phenyl group or a pyridyl group, and the phenyl group or the pyridyl group each independently optionally contains halogen, C １ ~C ４ Alkyl groups (e.g., methyl groups, ethyl groups), C １ ~C ４ substituted with 1 to 4 substituents selected from haloalkyl groups (e.g., trifluoromethyl groups); Preferably, A is a phenyl group or a pyridyl group, and said phenyl group or pyridyl group each independently optionally contains halogen, C １ ~C ４ substituted with 1 to 2 substituents selected from an alkyl group (e.g., a methyl group, an ethyl group) and a trifluoromethyl group; More preferably, A is a pyridyl group, and said pyridyl group is optionally selected from the group consisting of C １ ~C ４ substituted with 1 to 2 substituents selected from alkyl groups; More preferably, A is a pyridyl group, Most preferably, A is 【Transformation 38】 and B is a phenylene group; Preferably, B is 【Chemistry 39】 and Y is -CO-NH-CH ２ - and Preferably, the methylene group terminal of Y is connected to B, and the carbonyl group terminal is connected to an alkenyl carbon or O, R １ , R ２ are each independently hydrogen, C １ ~C ４ alkyl groups, Preferably, R １ , R ２ are both hydrogen, X １ , X ２ , X ３ , X ４ Any one of the following is hydrogen, halogen, or C １ ~C ４ alkyl groups, and the remaining three are hydrogen; Preferably, X １ , X ２ , X ３ , X ４ any one of the groups is selected from hydrogen and halogen, and the remaining three are hydrogen; More preferably, X １ , X ２ , X ３ , X ４ any one of the groups is selected from hydrogen and fluorine, and the remaining three are hydrogen; Most preferably, X ２ is selected from hydrogen and fluorine, and X １ , X ３ , X ４ is hydrogen, More preferably, D is 【Chemistry 40】 characterized in that it is selected from The ligand-drug complex according to any one of claims 7 to 9, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof.

11. The Ab is an antibody selected from, but not limited to, an anti-EGFR antibody, an anti-CD20 antibody, or an anti-PD-L1 antibody; Preferably, the Ab is selected from, but not limited to, Cetuximab, Panitumumab, Necitumumab, Rituximab, Tositumomab (Tositumomab + Iodine 131 Tositumomab), Ofatumumab, Obinutuzumab, Ocrelizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab-rwlc; More preferably, the Ab is atezolizumab or avelumab. The ligand-drug conjugate according to any one of claims 7 to 10, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof.

12. The ligand drug conjugate comprises: 【Chemistry 41】 【Chemistry 42】 【Chemistry 43】 【Chemistry 44】 characterized in that it is selected from The ligand-drug complex according to any one of claims 7 to 11, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof.

13. The ligand drug conjugate comprises: 【Chemistry 45】 【Chemistry 46】 characterized in that it is selected from The ligand-drug complex according to any one of claims 7 to 12, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof.

14. 14. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 5 or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof, or comprising at least one ligand drug conjugate according to any one of claims 7 to 13 or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof, optionally further comprising a pharmaceutically acceptable carrier or excipient.

15. Use of a compound according to any one of claims 1 to 5, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof, or a ligand-drug conjugate according to any one of claims 7 to 13, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a solvate thereof, or a pharmaceutical composition according to claim 14, in the manufacture of a medicament for the treatment or prevention of a disease, Preferably, the disease is a tumor. use.

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