Anti-human TROP2 antibody-camptothecin drug conjugate and its medical use

Abstract

Anti-human TROP2 antibody-camptothecin drug conjugates and medical uses thereof are provided. [Solution] A human Trop2-targeting antibody is simultaneously conjugated to a camptothecin drug to form an antibody-drug conjugate with stable treatability and excellent homogeneity, and the drug-antibody ratio (DAR) is 6.0-8.0. The antibody-drug conjugate has a structure shown in general formula I, where Ab refers to a Trop-targeting antibody simultaneously linked to a linker-camptothecin drug. Further disclosed are methods for preparing and purifying the antibody-drug conjugate, and its application in tumor therapy. Further disclosed are linker-drug compounds that can be conjugated to Ab to form the antibody-drug conjugate. TIFF2024541681000450.tif43147

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceuticals, in particular to an antibody-drug conjugate formed by an anti-human Trop2 antibody and a camptothecin drug, as well as a method for preparing and using the antibody-drug conjugate. The present invention also relates to a linker-drug compound that can be bound to an Ab to form an antibody-drug conjugate. [Background technology]

[0002] Human trophoblast cell surface antigen 2 (hTROP2) is a single-pass, type I membrane protein encoded by the TACSTD2 gene. This protein was first discovered by M. Lipinski and colleagues during studies of normal and cancerous human trophoblasts. Subsequent studies have also led to the identification of hTROP2 as a tumor antigen (GA733-1), recognized by the mouse monoclonal antibody GA733 obtained by immunizing a gastric cancer cell line, and as an epidermal glycoprotein (RS7-3G11), recognized by the mouse monoclonal antibody RS7-3G11 obtained by immunizing a non-small cell lung cancer cell line. Following gene cloning in 1995, the identity of these two proteins was confirmed. The hTROP2 protein has a total length of 323 amino acid residues, including an N-terminal 26 amino acid intracellular domain, a C-terminal 248 amino acid extracellular domain, and a 23 amino acid transmembrane domain. hTROP2 has four N-glycosylation sites at amino acid residues 33, 120, 168, and 208 in the extracellular domain. The TROP2 gene belongs to the TACSTD gene family and shares approximately 50% identity with human trophoblast cell surface antigen 1 (hTROP1), another member of this family.

[0003] Although the physiological ligand and molecular function of hTROP2 protein are currently unknown, this protein can regulate calcium signaling in tumor cells. The intracellular domain of hTROP2 contains a PIP2 (phosphatidylinositol 4,5-bisphosphate) binding sequence. Ca 2+ The protein kinase C, a dependent kinase, can phosphorylate serine residue 303. Upon phosphorylation of serine residue 303, PIP2 is hydrolyzed by phospholipase C (PLC) into IP3 (inositol triphosphate) and DAG (diacylglycerol), thereby regulating the process of intracellular calcium signaling. Immunohistochemical analysis of clinical specimens has shown that hTROP2 is expressed exclusively in certain epithelial cells in normal tissues, but is overexpressed in various epithelial cell cancers, including cervical, breast, gastric, lung, prostate, and pancreatic cancers. Several studies have shown that hTROP2 expression levels are closely associated with tumor invasiveness, malignancy, and poor patient prognosis. Meanwhile, hTROP2 can be used as a marker for prostate cancer stem cells during tumor development and progression. Currently, Trop-2 is targeted in tumor therapy, and anti-Trop-2 antibody-drug conjugates (ADCs) are used to treat various metastatic tumors, including triple-negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), and small cell lung cancer (SCLC). In 2016, the American Society of Clinical Oncology (ASCO) published a study on IMMU-132 in the treatment of triple-negative breast cancer. IMMU-132 is an antibody-drug conjugate combining the monoclonal antibody RS7 and the drug SN-38. RS7 can specifically target hTROP2, which has been found to be abundantly expressed in over 80% of triple-negative breast cancer tumor cells.

[0004] Camptothecin (CPT) is a DNA topoisomerase I (TOPI) inhibitor. Camptothecin derivatives can inhibit TOPI and achieve antitumor effects. TOPI catalyzes the cleavage and ligation of single-stranded DNA during DNA translation and transcription, loosening DNA supercoiling and promoting replication and transcription. The TOPI inhibitor SN-38 causes DNA damage by inhibiting DNA strand religation, thereby inhibiting DNA replication and transcription and inducing cell apoptosis. In clinical trials of Trop-2-targeting ADCs using the humanized RS7 antibody, IMMU-132 uses a moderately stable linker. IMMU-132, which releases SN-38, has a half-life in human serum of approximately 24 hours and an elimination half-life of approximately 11 hours in humans and mice. IMMU-132 has a rapid elimination rate and a mean residence time of approximately 15.4 hours, indicating that IMMU-132 can be administered more frequently in clinical treatment. On the other hand, the use of topoisomerase inhibitors such as SN-38, IMMU-132, is often associated with severe side effects such as diarrhea. The use of a more stable linker is expected to extend the mean residence time and reduce drug use. Therefore, further research into safer and more effective Trop-2 targeting technologies is urgently needed. ADCs consist of three components: an antibody, a cytotoxic small molecule drug, and a linker connecting the antibody and drug. The small molecule drug is chemically bonded to the antibody protein. ADCs target cancer cells through antibody-specific recognition, then endocytose the drug, releasing the cytotoxic drug to specifically kill the cancer cells. Clinical studies have shown that ADCs are relatively stable and highly effective in the blood and can effectively reduce the toxicity of cytotoxic small molecule drugs to healthy tissues. ADCs are currently a hot topic in anticancer drug research. Trop-2, a protein specifically and highly expressed in various epithelial cell cancers, is an excellent target candidate for ADCs. Summary of the Invention

[0005] Based on a comprehensive understanding of ADCs, the present inventors disclose anti-human Trop2 antibody-drug conjugates, methods for preparing the conjugates, pharmaceutical compositions containing the conjugates, and uses of the conjugates or pharmaceutical compositions. The present invention further relates to linker-drug compounds that can be bound to anti-human Trop2 antibodies to form antibody-drug conjugates.

[0006] A first aspect of the present invention relates to a compound of general formula I [ka] I is shown by During the ceremony, Ab is a human Trop2-targeting antibody or antigen-binding fragment thereof; L1 may be, but is not limited to: [ka] selected from the group consisting of: Preferably, L1 is [ka] and; Preferably, L1 is [ka] and; L2 has the structure shown in Formula A: [ka] Formula A wherein Y is a scaffold selected from the group consisting of C1-C6 alkyl, substituted C1-C6 alkyl, and C3-C8 cycloalkyl; preferably, Y is C1-C6 alkyl; Ac is a hydrophilic structural unit; the carbon number 2 connected to Y has absolute chiral configuration R or S; L3 is present or absent, and if present, L3 is a PEG hydrophilic unit: [ka] where o is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably from 2 to 8; L4 is an enzyme-cleavable unit; L5 is a bridging unit; In formula I, the chiral carbon atom number 1 linked to N has absolute chiral configuration R or S; R is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl; Preferably, R is selected from the group of a hydrogen atom and a C1-C6 alkyl; R1 is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a carboxyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R1 is selected from the group of a hydrogen atom or a C1-C6 alkyl; More preferably, R1 is selected from C1-C6 alkyl; R2 is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a carboxyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R2 is selected from the group consisting of a hydrogen atom, a halogen, and a C1-C6 alkyl; More preferably, R2 is selected from halogen; X is -C(O)-CR a R b -(CR3R4) m -O-, -C(O)-CR a R b -(CR3R4) m -NH- and -C(O)-CR a R b -(CR3R4) m -S-; Preferably, X is -C(O)-CR a R b -(CR3R4) m -O-; R a and R b are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a deuterated C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C6-C10 arylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R a and R b are each independently selected from the group consisting of a hydrogen atom, a C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, and a C6-C10 arylC1-C6 alkyl; Or R a and R b and R a and R b are linked to form a C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, 3- to 7-membered heterocyclyl, or substituted 3- to 7-membered heterocyclyl; preferably, R a and R b and R a and R b the carbon atoms to which are linked form a C3-C8 cycloalkyl; R3 and R4 are the same or different and each independently represent a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a halogenated C1-C6 alkyl, a deuterated C1-C6 alkyl, a C1-C6 alkoxy, a hydroxyl, an amino, a cyano, a nitro, a hydroxyl C1-C6 alkyl, a C3-C8 cycloalkyl, a 3- to 7-membered heterocyclyl, or a substituted 3- to 7-membered heterocyclyl; Preferably, R3 and R4 are each independently a hydrogen atom or a C1-C6 alkyl; or R3, R4, and the carbon atom to which R3 and R4 are attached form a C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, 3- to 7-membered heterocyclyl, or substituted 3- to 7-membered heterocyclyl; m is selected from integers of 0 to 4 (e.g., 0, 1, 2, 3, or 4), preferably 0 or 1; and n is selected from integers of 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Disclosed is a ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof.

[0007] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, wherein Ab is a TROP2-targeting antibody or antigen-binding fragment thereof. In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, wherein the antibody of Ab comprises two IgG1 heavy chains and two κ light chains. In some embodiments of the first aspect of the present invention, there is disclosed a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody of the Ab has a kappa light chain comprising CDRs as set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and an IgG1 heavy chain comprising CDRs as set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0008] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, wherein the antibody of the Ab comprises a heavy chain and a light chain, the light chain comprising CDRL1, CDRL2, and CDRL3 having nucleic acid coding sequences as set forth in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, and the heavy chain comprising CDRH1, CDRH2, and CDRH3 having nucleic acid coding sequences as set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively. In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, wherein the antibody of Ab comprises a heavy chain and a light chain, the heavy chain comprising a VH having the sequence as set forth in SEQ ID NO: 13, and the light chain comprising a VL having the sequence as set forth in SEQ ID NO: 14. In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, wherein the antibody of the Ab comprises a heavy chain and a light chain, the heavy chain comprising a VH having a nucleic acid coding sequence as set forth in SEQ ID NO: 33, and the light chain comprising a VL having a nucleic acid coding sequence as set forth in SEQ ID NO: 34.

[0009] In some embodiments of the first aspect of the present invention, there is disclosed a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody of Ab has a heavy chain having the amino acid sequence as set forth in SEQ ID NO: 17 and a light chain having the amino acid sequence as set forth in SEQ ID NO: 18. In some embodiments of the first aspect of the present invention, there is disclosed a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody of Ab has a heavy chain having a nucleic acid coding sequence as set forth in SEQ ID NO: 37 and a light chain having a nucleic acid coding sequence as set forth in SEQ ID NO: 38. In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, wherein the antibody of Ab comprises two IgG1 heavy chains and two κ light chains.

[0010] In some embodiments of the first aspect of the present invention, there is disclosed a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody of the Ab has a kappa light chain comprising CDRs as set forth in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, and an IgG1 heavy chain comprising CDRs as set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, wherein the antibody of the Ab comprises a heavy chain and a light chain, the light chain comprising CDRL1, CDRL2, and CDRL3 having nucleic acid coding sequences as set forth in SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, and the heavy chain comprises CDRH1, CDRH2, and CDRH3 having nucleic acid coding sequences as set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, wherein the antibody of Ab comprises a heavy chain and a light chain, the heavy chain comprising a VH having the sequence as set forth in SEQ ID NO: 15, and the light chain comprising a VL having the sequence as set forth in SEQ ID NO: 16.

[0011] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, wherein the antibody of Ab comprises a heavy chain and a light chain, the heavy chain comprising a VH having a nucleic acid coding sequence as set forth in SEQ ID NO: 35, and the light chain comprising a VL having a nucleic acid coding sequence as set forth in SEQ ID NO: 36. In some embodiments of the first aspect of the present invention, there is disclosed a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody of said Ab has a heavy chain having the amino acid sequence as set forth in SEQ ID NO: 19 and a light chain having the amino acid sequence as set forth in SEQ ID NO: 20. In some embodiments of the first aspect of the present invention, there is disclosed a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody of said Ab has a heavy chain having a nucleic acid coding sequence as set forth in SEQ ID NO: 39 and a light chain having a nucleic acid coding sequence as set forth in SEQ ID NO: 40.

[0012] In some embodiments of the first aspect of the present invention, X may be any of the following structures, including but not limited to: [ka] or an isomer thereof, The left wavy line is connected to the camptothecin derivative moiety, and the right wavy line is connected to L5. Disclosed is a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof:

[0013] In some embodiments of the first aspect of the invention, L4 is selected from peptide residues including, but not limited to, the amino acid Optionally, the amino acid may be further substituted with one or more substituents selected from the group consisting of deuterium, halogen, hydroxyl, cyano, amino, nitro, carboxyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, and C3-C8 cycloalkyl or substituted C3-C8 cycloalkyl; Preferably, the peptide residue is a peptide residue consisting of one, two or more amino acids selected from the group consisting of phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline (C), serine (S), glutamic acid (E) or aspartic acid (D); More preferably, the peptide residue is a tetrapeptide residue consisting of glycine (G)-glycine (G)-phenylalanine (F)-glycine (G). Disclosed is a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof: Particularly preferably, the peptide residue is -GGFG-.

[0014] In some embodiments of the first aspect of the present invention, L5 is selected from the group consisting of, but not limited to, -NR5(CR6R7) q - and a chemical bond, and q is selected from an integer from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, or 6), or a pharmaceutically acceptable salt or solvate thereof. R5, R6 and R7 are the same or different and are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R5, R6 and R7 are each independently selected from a hydrogen atom and a C1-C6 alkyl; More preferably, R5, R6 and R7 are each independently selected from a hydrogen atom. In some embodiments, L1 may be any of, but not limited to, [ka] is selected from the group consisting of:

[0015] In some embodiments of the first aspect of the present invention, the bridging unit -L1-L2-L3-L4-L5- has, but is not limited to, the following structure: [ka] Preferably, [ka] is selected from During the ceremony, Ac is a hydrophilic structural unit; R5, R6 and R7 are the same or different and are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R5, R6 and R7 are each independently selected from a hydrogen atom or a C1-C6 alkyl; More preferably, R5, R6 and R7 are each independently selected from a hydrogen atom; The carbon atom number 2 linked to N has absolute chiral configuration R or S; The left wavy line is connected to an antibody or antigen-binding fragment of an antibody, and the right wavy line is connected to an X; o is selected from integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); Disclosed is a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof:

[0016] A second aspect of the present invention relates to compounds of general formula II [ka] II is shown by During the ceremony, Ab is a human Trop2-targeting antibody or antigen-binding fragment thereof; L1 is a bridging unit linked to Ab, including but not limited to: [ka] Selected from; L1 is preferably [ka] and; L1 is preferably [ka] and; L3 is present or absent, and if present, L3 is [ka] where o is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably from 2 to 8; Ac is a hydrophilic structural unit; The chiral carbon atoms at positions 1, 2 and 3 have absolute chiral configuration R or S; R is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl; Preferably, R is selected from the group of a hydrogen atom and a C1-C6 alkyl; R1 is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a carboxyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R1 is selected from the group consisting of a hydrogen atom and a C1-C6 alkyl; More preferably, R1 is selected from C1-C6 alkyl; R2 is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a carboxyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R2 is selected from the group consisting of a hydrogen atom, a halogen, and a C1-C6 alkyl; More preferably, R2 is selected from halogen; X is -C(O)-CR a R b -(CR3R4) m -O-, -C(O)-CR a R b -(CR3R4) m -NH- or -C(O)-CR a R b -(CR3R4) m -S-; Preferably, X is -C(O)-CR a R b -(CR3R4) m -O-; R a and R b are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a deuterated C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R a and R bare each independently selected from the group consisting of a hydrogen atom, a C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, or a C6-C10 arylC1-C6 alkyl; Or R a and R b and R a and R b are linked to form a C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, 3- to 7-membered heterocyclyl, or substituted 3- to 7-membered heterocyclyl; preferably, R a and R b and R a and R b the carbon atoms to which are linked form a C3-C8 cycloalkyl; R3 and R4 are the same or different and each independently represent a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a halogenated C1-C6 alkyl, a deuterated C1-C6 alkyl, a C1-C6 alkoxy, a hydroxyl, an amino, a cyano, a nitro, a hydroxyl C1-C6 alkyl, a C3-C8 cycloalkyl, a 3- to 7-membered heterocyclyl, or a substituted 3- to 7-membered heterocyclyl; Preferably, R3 and R4 are each independently a hydrogen atom or a C1-C6 alkyl; or R3, R4, and the carbon atom to which R3 and R4 are attached form a C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, 3- to 7-membered heterocyclyl, or substituted 3- to 7-membered heterocyclyl; m is selected from integers from 0 to 4 (e.g., 0, 1, 2, 3, or 4), preferably 0 or 1; n is selected from integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); Disclosed is a ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof.

[0017] In some embodiments of the first and second aspects of the invention, Ac is of formula B [ka] B and having a structure represented by During the ceremony, Z is selected from the group consisting of, but not limited to, one or more of the hydrophilic structures carboxyl, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid, and polyethylene glycol (PEG); Preferably, Z is selected from the group consisting of the hydrophilic structures carboxyl, phosphate and PEG; Y' is an optional scaffold connecting -NH- and Z; preferably, Y' is C1-C6 alkylene (e.g., methylene); Ac is linked to the labeled 2-carbon of structural formula I via scaffold Y; Disclosed is a ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof.

[0018] In some embodiments of the first and second aspects of the invention, Ac is selected from the group consisting of, but not limited to, glycine, (D / L)alanine, (D / L)leucine, (D / L)isoleucine, (D / L)valine, (D / L)phenylalanine, (D / L)proline, (D / L)tryptophan, (D / L)serine, (D / L)tyrosine, (D / L)cysteine, (D / L)cystine, (D / L)arginine, (D / L)histidine, (D / L)methionine, (D / L)asparagine, (D / L)glutamine, (D / L)threonine, (D / L)aspartic acid, (D / L)glutamic acid, a natural or unnatural amino acid derivative, or a group having the following structure: [ka] or isomers thereof; Preferably, [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0019] In some embodiments of the first and second aspects of the present invention, a ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, wherein Ac is selected from the group consisting of, but not limited to, glycine, phosphate, (D / L) glutamic acid, and a polyethylene glycol hydrophilic structure.

[0020] In some embodiments of the first and second aspects of the invention, [ka] is the formula d [ka] d having the structure shown in During the ceremony, R is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl; Preferably, R is selected from the group of a hydrogen atom and a C1-C6 alkyl; R1 is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a carboxyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R1 is selected from the group consisting of a hydrogen atom and a C1-C6 alkyl; More preferably, R1 is selected from C1-C6 alkyl; R2 is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a carboxyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R2 is selected from the group consisting of a hydrogen atom, a halogen, and a C1-C6 alkyl; More preferably, R2 is selected from halogen; R a and R b are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a deuterated C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R a and R b are each independently selected from the group consisting of a hydrogen atom, a C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, and a C6-C10 arylC1-C6 alkyl; Preferably, R a and R b are each independently selected from the group consisting of a hydrogen atom, a C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C1-C6 alkyl, and a C6-C10 aryl; preferably, R a and R b are each independently selected from the group consisting of a hydrogen atom, methyl, ethyl, trifluoromethyl, cyclopropylmethyl, and phenyl; Or R a and R b and R a and R bare linked to form a C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, 3- to 7-membered heterocyclyl, or substituted 3- to 7-membered heterocyclyl; preferably, R a and R b and R a and R b the carbon atoms to which are linked form a C3-C8 cycloalkyl (e.g., C3-C5 cycloalkyl); The chiral carbon atom at position 1 has absolute chiral configuration R or S; m is 0 or 1; Disclosed is a ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof.

[0021] In some embodiments of the first and second aspects of the present invention, structural formula d includes, but is not limited to, the following compounds: [ka] TIFF2024541681000023.tif69165, or a pharmaceutically acceptable salt or solvate thereof.

[0022] In certain embodiments of the invention, L1 may contain a succinimide group. In these embodiments, the Ligand-Drug Conjugate may be hydrolyzed under readily hydrolyzable conditions, with hydrolysis occurring at the succinimide group of the bridging unit. When the Ligand contains multiple Linker-Drug units, the extent of hydrolysis may result in the following: The succinimide groups are completely non-hydrolyzable, i.e., all succinimide groups are cyclic. [ka] is; The succinimide group is not completely hydrolyzed, i.e., the succinimide group does not form a ring-closed form. [ka] and open-ring type [ka] There are things like; The succinimide groups are completely hydrolyzed, i.e., all succinimide groups are in the open ring form. [ka] is.

[0023] Thus, when an ADC contains multiple L1s containing succinimide groups (i.e., Ab is linked to multiple drug-linker units containing succinimide groups), these succinimide groups may all be in the closed ring form, some may be in the open ring form, or all may be in the open ring form. The succinimide group shown in the chemical structural formula of the ADC of the present application is in a closed ring form, but it should be understood that it actually includes three cases: completely closed ring, partially opened ring, and completely opened ring.

[0024] A third aspect of the present invention is a compound of formula III [ka] III and having a structure represented by During the ceremony, R is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl; R ais selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a deuterated C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; R b is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a deuterated C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Or R a and R b and R a and R b the carbon atoms to which are linked form a C3-C8 cycloalkyl, a C3-C8 cycloalkyl, a C1-C6 alkyl, a 3- to 7-membered heterocyclyl, and a substituted 3- to 7-membered heterocyclyl; Preferably, R a and R b are each independently selected from the group consisting of a hydrogen atom, a C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C1-C6 alkyl, and a C6-C10 aryl; preferably, R a and R b are each independently selected from the group consisting of a hydrogen atom, methyl, ethyl, trifluoromethyl, cyclopropylmethyl, and phenyl; Or R a and R b and R a and R b are linked to form a C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, 3- to 7-membered heterocyclyl, or substituted 3- to 7-membered heterocyclyl; preferably, R a and Rb and R a and R b the carbon atoms to which are linked form a C3-C8 cycloalkyl (e.g., C3-C5 cycloalkyl); L3 is present or absent, and if present, L3 is [ka] where o is selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); the chiral carbon atom at position 1 or 2 has absolute chiral configuration R or S; Ac is a hydrophilic structural unit; m is 0 or 1; Disclosed is a linker-drug compound, or a pharmaceutically acceptable salt or solvate thereof.

[0025] In some embodiments of the third aspect of the present invention, Ac is of formula B [ka] B and having a structure represented by During the ceremony, Z is composed of one or more hydrophilic structures selected from the group consisting of carboxyl, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid, and polyethylene glycol (PEG); Y' is an optional scaffold connecting -NH- and Z; preferably, Y' is C1-C6 alkylene (e.g., methylene); Ac is linked to the labeled 2-carbon of structural formula I via scaffold Y; Linker-drug compounds, or pharmaceutically acceptable salts or solvates thereof, are disclosed.

[0026] In some embodiments of the third aspect of the present invention, Ac is selected from the group consisting of, but not limited to, glycine, (D / L)alanine, (D / L)leucine, (D / L)isoleucine, (D / L)valine, (D / L)phenylalanine, (D / L)proline, (D / L)tryptophan, (D / L)serine, (D / L)tyrosine, (D / L)cysteine, (D / L)cystine, (D / L)arginine, (D / L)histidine, (D / L)methionine, (D / L)asparagine, (D / L)glutamine, (D / L)threonine, (D / L)aspartic acid, (D / L)glutamic acid, natural or unnatural amino acid derivatives, or amino acids having the following structures: [ka] or a pharmaceutically acceptable salt or solvate thereof.

[0027] In some embodiments of the third aspect of the present invention, a linker-drug compound, or a pharmaceutically acceptable salt or solvate thereof, is disclosed wherein Ac is selected from the group consisting of, but not limited to, glycine, phosphate, (D / L) glutamic acid, and a polyethylene glycol hydrophilic structure.

[0028] In some embodiments of the third aspect of the present invention, the linker-drug compound may have the following structure, including but not limited to: [ka] TIFF2024541681000033.tif250133 TIFF2024541681000034.tif249136 TIFF2024541681000035.tif193106 or an isomer thereof, o is selected from integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); Linker-drug compounds, or pharmaceutically acceptable salts or solvates thereof, are disclosed.

[0029] The linker-drug compound disclosed in the third aspect of the present invention, or a pharmaceutically acceptable salt or solvate thereof, can be used as an intermediate for binding with a ligand Ab to form a ligand-camptothecin derivative conjugate of formula I as described in the first aspect or formula II as described in the second aspect.

[0030] A fourth aspect of the present invention provides a method for preparing a ligand-camptothecin derivative conjugate of general formula I as described in the first aspect or general formula II as described in the second aspect, or a pharmaceutically acceptable salt or solvate thereof, comprising: [ka] coupling the reduced antibody or antigen-binding fragment thereof with a linker-drug compound to obtain a ligand-camptothecin derivative conjugate of general formula I or general formula II; the chiral carbon atom at position 1, 2 or 3 has absolute chiral configuration R or S; Ab, L1, L2, L3, L4, L5, X, R, R1, R2 and n are as defined above. A method is disclosed.

[0031] The present application further relates to the use of a linker-drug compound, or a pharmaceutically acceptable salt or solvate thereof, as disclosed in the third aspect, as an intermediate in the preparation of a ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, is a ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, as described in the first, second, and fourth aspects of the present invention. In certain embodiments, the preparation is carried out according to the preparation method as disclosed in the fourth aspect.

[0032] In some embodiments of the first, second, and fourth aspects of the present invention, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof has the following structure, including but not limited to: [ka] TIFF2024541681000038.tif249148 TIFF2024541681000039.tif245151 TIFF2024541681000040.tif251146 TIFF2024541681000041.tif244146 TIFF2024541681000042.tif250149 TIFF2024541681000043.tif248146 TIFF2024541681000044.tif253150 TIFF2024541681000045.tif239104In formula, hu4D3 is a human Trop2-targeting antibody or antigen-binding fragment thereof; n is selected from integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); Disclosed is a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof selected from derivative structures thereof in which the ring of the succinimide group is open, and isomers thereof.

[0033] In some embodiments of the first, second, and fourth aspects of the present invention, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof has the following structure, including but not limited to: [ka] TIFF2024541681000047.tif251146 TIFF2024541681000048.tif244152 TIFF2024541681000049.tif254151 TIFF2024541681000050.tif246147 TIFF2024541681000051.tif252152 TIFF2024541681000052.tif249139 TIFF2024541681000053.tif250148 TIFF2024541681000054.tif241105In formula, hu7F11 is a human Trop2-targeting antibody or antigen-binding fragment thereof; n is selected from integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); Disclosed is a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof selected from derivative structures thereof in which the succinimide group is in ring-open form, and isomers thereof.

[0034] In some embodiments of the first, second, and fourth aspects of the present invention, a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound or a pharmaceutically acceptable salt or solvate thereof, is disclosed, where the pharmaceutically acceptable salts include sodium, potassium, calcium, or magnesium salts formed at acidic functional groups in the structural formula, and acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate salts formed at basic functional groups in the structural formula.

[0035] A fifth aspect of the present invention discloses a pharmaceutical composition comprising a ligand-camptothecin derivative conjugate as described in the first and second aspects or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound as described in the third aspect or a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutically acceptable carrier.

[0036] A sixth aspect of the present invention discloses a pharmaceutical formulation comprising a ligand-camptothecin derivative conjugate as described in the first and second aspects, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound as described in the third aspect, or a pharmaceutically acceptable salt or solvate thereof.

[0037] A seventh aspect of the present invention relates to the use of a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof as described in the first and second aspects, or a linker-drug compound or a pharmaceutically acceptable salt or solvate thereof as described in the third aspect, a pharmaceutical composition as described in the fifth aspect and / or a pharmaceutical formulation as described in the sixth aspect in the preparation of a medicament for treating or preventing cancer or tumors; or a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof as described in the first and second aspects, or a linker-drug compound or a pharmaceutically acceptable salt or solvate thereof as described in the third aspect, or a pharmaceutical composition as described in the fifth aspect and / or a pharmaceutical formulation as described in the sixth aspect for treating or preventing cancer or tumors. Disclose the Preferably, the cancer or tumor expresses TROP2; More preferably, the cancer or tumor is selected from solid tumors or hematological tumors, such as adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, triple-negative breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia.

[0038] An eighth aspect of the present invention is a method for treating or preventing cancer or tumors, comprising: administering to a subject in need thereof a prophylactically or therapeutically effective amount of a ligand-camptothecin derivative conjugate as described in the first and second aspects, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound as described in the third aspect, or a pharmaceutical composition as described in the fifth aspect and / or a pharmaceutical formulation as described in the sixth aspect; Preferably, the cancer or tumor expresses TROP2; More preferably, the cancer or tumor is selected from a solid tumor or a hematological tumor, such as adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, triple-negative breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia. A method is disclosed.

[0039] In the above aspect of the present invention, "C1-C6 alkyl" and "C1-C6 alkyl" in various hybrid groups including "C1-C6 alkyl" (e.g., "substituted C1-C6 alkyl", "deuterated C1-C6 alkyl") can be substituted with "C1-C20 alkyl", "C1-C12 alkyl" or "C1-C10 alkyl"; "C3-C8 cycloalkyl" and "C3-C8 cycloalkyl" in various hybrid groups containing "C3-C8 cycloalkyl" can be substituted with "C3-C20 cycloalkyl" or "C3-C10 cycloalkyl"; "C1-C6 alkoxy" and "C1-C6 alkoxy" in various hybrid groups containing "C1-C6 alkoxy" can be substituted with "C1-C20 alkoxy", "C1-C12 alkoxy" or "C1-C10 alkoxy"; "C6-C10 aryl" and "C6-C10 aryl" in various hybrid groups containing "C6-C10 aryl" can be substituted with "C6-C12 aryl"; The "3- to 7-membered heterocyclyl" and "3- to 7-membered heterocyclyl" in various hybrid groups containing "3- to 7-membered heterocyclyl" can be substituted with "3- to 20-membered heterocyclyl", "3- to 12-membered heterocyclyl" or "3- to 10-membered heterocyclyl".

[0040] Beneficial effects The anti-human Trop2 antibody-drug conjugate provided by the present invention is an antibody-drug conjugate obtained by conjugating a humanized anti-human Trop2 antibody with a camptothecin drug. Compared to existing drugs of the same type, the anti-human Trop2 antibody-drug conjugate has better molecular stability and preclinical efficacy, and is expected to have excellent clinical therapeutic effects. [Brief explanation of the drawings]

[0041] [Figure 1A] FIG. 1A shows aggregation of DS1062a (DAR=4) detected by SEC-HPLC. [Figure 1B] FIG. 1B shows aggregation of ADC-1 detected by SEC-HPLC. [Figure 1C] FIG. 1C shows aggregation of ADC-2 detected by SEC-HPLC. [Figure 1D] FIG. 1D shows aggregation of ADC-6 detected by SEC-HPLC. [Figure 1E] FIG. 1E shows aggregation of ADC-10 detected by SEC-HPLC. [Figure 2A] FIG. 2A shows the DAR of DS1062a (DAR=4) determined by RP-HPLC. [Figure 2B] FIG. 2B shows the DAR of ADC-1 determined by RP-HPLC. [Figure 2C] FIG. 2C shows the DAR of ADC-2 determined by RP-HPLC. [Figure 2D]FIG. 2D shows the DAR of ADC-12 determined by RP-HPLC. [Figure 2E] FIG. 2E shows the DAR of ADC-6 determined by RP-HPLC. [Figure 2F] FIG. 2F shows the DAR of ADC-10 determined by RP-HPLC. [Figure 3A] FIG. 3A shows that DS1062a retains the same affinity for the antigen TROP2 as antibody TINA1. [Figure 3B] FIG. 3B shows that ADC-1 retains the same affinity for the antigen TROP2 as antibody TINA1. [Figure 3C] Figure 3C shows that ADC-2 retains the same affinity for the antigen TROP2 as antibody hu4D3. [Figure 3D] FIG. 3D shows that ADC-12 retains the same affinity for the antigen TROP2 as antibody hu4D3. [Figure 3E] FIG. 3E shows that ADC-6 retains the same affinity for the antigen TROP2 as antibody hu4D3. [Figure 3F] FIG. 3F shows that ADC-10 retains the same affinity for the antigen TROP2 as antibody hu4D3. [Figure 4] FIG. 4 shows efficacy evaluation of ADCs and corresponding antibodies in BXPC-3 cells. [Figure 5A] FIG. 5A shows that the in vivo efficacy of ADC-6, ADC-10, and ADC-12 is superior to DS1062a and ADC-1 in the BXPC3 mono-tumor model. [Figure 5B] Figure 5B shows the results of in vivo efficacy studies of ADC-1, ADC-6, ADC-12, and DS1062a in A431+SW620 heterogeneous tumors. [Figure 6A] FIG. 6A shows the in vitro tumor inhibitory activity of naked antibodies hu4D3 and hu7F11 and compounds d3, d38, d39, and d44 in a test model of the human tumor cell line N87. [Figure 6B]FIG. 6B shows the in vitro tumor inhibitory activity of naked antibodies hu4D3 and hu7F11 and compounds d3, d38, d39, and d44 in a test model of the human tumor cell line SW620. [Figure 6C] FIG. 6C shows the in vitro tumor inhibitory activity of naked antibodies hu4D3 and hu7F11 and compounds d3, d38, d39, and d44 in a test model of the human tumor cell line HCC827. [Figure 6D] FIG. 6D shows the in vitro tumor inhibitory activity of naked antibodies hu4D3 and hu7F11 and compounds d3, d38, d39, d44 in a test model of the human tumor cell line FaDu. [Figure 6E] FIG. 6E shows the in vitro tumor inhibitory activity of naked antibodies hu4D3 and hu7F11 and compounds d3, d38, d39, and d44 in a test model of the human tumor cell line A431+SW620. [Figure 7A] FIG. 7A shows the in vitro tumor-inhibitory activity of ADC-47, ADC-70, ADC-72, ADC-73, ADC-112, ADC-176, ADC-178, and ADC-179 in a test model of the human tumor cell line N87. [Figure 7B] Figure 7B shows the in vitro tumor-inhibitory activity of ADC-47, ADC-70, ADC-72, ADC-73, ADC-112, ADC-176, ADC-178, and ADC-179 in a test model of the human tumor cell line SW620. [Figure 7C] Figure 7C shows the in vitro tumor-inhibitory activity of ADC-47, ADC-70, ADC-72, ADC-73, ADC-112, ADC-176, ADC-178, and ADC-179 in a test model of the human tumor cell line HCC827. [Figure 7D] FIG. 7D shows the in vitro tumor-inhibitory activity of ADC-47, ADC-70, ADC-72, ADC-73, ADC-112, ADC-176, ADC-178, and ADC-179 in a test model of the human tumor cell line FaDu. [Figure 7E]FIG. 7E shows the in vitro tumor-inhibitory activity of ADC-47, ADC-70, ADC-72, ADC-73, ADC-112, ADC-176, ADC-178, and ADC-179 in a test model of the human tumor cell line A431+SW620. DETAILED DESCRIPTION OF THE INVENTION

[0042] Abbreviations and Definitions Unless otherwise specified, the following terms and phrases, when used herein, are intended to have the following meanings: When trade names are used herein, the trade name includes formulations, generics, and active ingredients of the product bearing that trade name, unless the context indicates otherwise. Unless stated to the contrary, the terms used in the claims and description herein have the following meanings:

[0043] The term "ligand" generally refers to a macromolecular compound capable of recognizing and binding to an antigen or receptor associated with a target cell. The role of the ligand is to deliver a drug to a target cell population that has bound to the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules capable of binding to cells. In an embodiment of the present invention, the ligand is represented as an Ab. The ligand can form a cross-link with the linking unit via a heteroatom on the ligand and is preferably an antibody or an antigen-binding fragment thereof. The antibody is selected from a chimeric antibody, a humanized antibody, a fully human antibody, or a murine antibody. Preferably, the antibody is a monoclonal antibody.

[0044] The Ligand unit is a targeting agent that specifically binds to a targeting moiety. The Ligand can specifically bind to a cellular component, or a cellular component or other target molecule of interest. The targeting moiety or target is usually located on the cell surface. In some embodiments, the Ligand unit serves to deliver the Drug unit to a specific target cell population with which the Ligand unit interacts. Ligands include, but are not limited to, proteins, polypeptides, and peptides, as well as non-proteins such as sugars. Suitable Ligand units include, for example, antibodies, such as full-length (intact) antibodies, and antigen-binding fragments thereof. In embodiments where the Ligand unit is a non-antibody targeting agent, it can be a peptide or polypeptide, or a non-protein molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient transport molecules, or any other cell-binding molecule or substance. In some embodiments, the linker is covalently bonded to a sulfur atom of the Ligand. In some embodiments, the sulfur atom is that of a cysteine ​​residue that forms the interchain disulfide bond of an antibody. In another aspect, the sulfur atom is a sulfur atom introduced into a cysteine ​​residue in the Ligand unit that forms the interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom introduced (e.g., by site-directed mutagenesis or chemical reaction) into a cysteine ​​residue in the Ligand unit. In other aspects, the sulfur atom to which the Linker is attached is selected from cysteine ​​residues that form the interchain disulfide bond of the antibody or cysteine ​​residues introduced (e.g., by site-directed mutagenesis or chemical reaction) into the Ligand unit. In some embodiments, the EU index numbering system is according to Kabat {[Kabat EA et al., (1991)] Sequences of proteins of Immunological Interest, 5th ed., NIH Publication 91-3242"}.

[0045] As used herein, "antibody" or "antibody unit" includes within its scope any portion of an antibody structure. This unit can bind, reactively associate, or complex with a receptor, antigen, or other receptor unit present in a target cell population. An antibody can be any protein or proteinaceous molecule that binds, complexes, or reacts with a portion of a cell population to be treated or biomodified. Antibodies comprising the antibody-drug conjugates of the present invention retain their native antigen-binding ability in the wild. Thus, antibodies of the present invention can specifically bind to antigens. Relevant antigens include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, regulators of cell survival, regulators of cell proliferation, molecules associated with tissue growth and differentiation (such as known or predicted functional molecules), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (such as known or predicted functional molecules). Tumor-associated factors can be cluster differentiation factors (such as CD proteins).

[0046] Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art and can be prepared using antibody production methods and information well known in the art. To develop effective cellular targets for cancer diagnosis and treatment, researchers are striving to find transmembrane peptides or other tumor-associated peptides. These targets can be specifically expressed on the surface of one or more cancer cells and are rarely or not expressed on the surface of one or more non-cancerous cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells than on the surface of non-cancerous cells. Identifying such tumor-associated factors can significantly improve the specific targeting properties of antibody-based cancer therapy. For convenience, antigen-related information known in the art, including names, aliases, and GenBank accession numbers, is presented below. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases such as GenBank. The tumor-associated antigens targeted by the antibody targets include all amino acid sequence variants and isotypes, and have at least 70%, 80%, 85%, 90% or 95% identity to the sequences identified in the references, or have biological properties and characteristics that match exactly the sequences of the tumor-associated antigens in the cited references.

[0047] The term "inhibit" or "inhibition of" means a detectable decrease or complete suppression. The term "cancer" refers to a physiological condition or disease characterized by unregulated cell growth. A "tumor" includes cancerous cells. The term "autoimmune disease" is a disease or disorder caused by and directed against an individual's own tissues or proteins. The term "drug" refers to a cytotoxic drug represented by d, which is a chemical molecule with a strong ability to interfere with the normal growth of tumor cells. In principle, a cytotoxic drug can kill tumor cells at a sufficiently high concentration. However, due to lack of specificity, while killing tumor cells, a cytotoxic drug may also cause apoptosis of normal cells, leading to serious side effects. This term also includes toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 and Lu 176 radioisotopes), toxic drugs, chemotherapeutic drugs, antibiotics and ribozymes, preferably toxic drugs.

[0048] The term "linker" or "bridging fragment" or "bridging unit" refers to a chemical fragment or bond that is linked at one end to a ligand and at the other end to a drug. It may also be linked to another linker and then attached to the drug. Linkers, including extenders, spacers, and amino acid units, can be synthesized according to methods known in the art, such as those described in US2005-0238649A1. The linker can be a "cleavable linker" that facilitates intracellular release of the drug. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al. Cancer Research 52:127-131, 1992; U.S. Patent No. 5,208,020).

[0049] Depending on the intracellular drug release mechanism, "linkers" or "linkers of antibody-drug conjugates," as used herein, can be divided into two types: non-cleavable linkers and cleavable linkers. For antibody-drug conjugates containing non-cleavable linkers, the drug release mechanism is as follows: after the conjugate binds to an antigen and is internalized by the cell, the antibody is enzymatically hydrolyzed in the lysosome, releasing the active molecule consisting of the small molecule drug, the linker, and the antibody amino acid residues. This change in the drug molecule structure does not weaken its cytotoxicity, and the active molecule cannot penetrate neighboring cells because it is charged (amino acid residues). Therefore, such active drugs cannot kill neighboring tumor cells that do not express the target antigen (antigen-negative cells) (Ducry et al., 2010, Bioconjugate Chem. 21: 5-13). For antibody-drug conjugates containing cleavable linkers, the drug release mechanism is as follows: after the conjugate binds to an antigen and is internalized by the cell, it is destroyed within the target cell, releasing the active ingredient (the small molecule drug itself). Cleavable linkers are mainly divided into chemically sensitive linkers and enzyme-sensitive linkers. Chemically sensitive linkers can be selectively cleaved depending on the differences in the characteristics of the plasma, cytoplasm, or tumor microenvironment. These characteristics include pH and glutathione concentration. pH-sensitive linkers, such as hydrazones, carbonates, acetals, and ketals, are relatively stable in the neutral or slightly alkaline environment of blood (pH 7.3-7.5) but are hydrolyzed in the slightly acidic tumor microenvironment (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). Because of the limited plasma stability of acid-cleaved linkers, antibody-drug conjugates based on this type of linker typically have a short half-life (2-3 days). This short half-life limits the application of pH-sensitive linkers to new-generation antibody-drug conjugates. Glutathione-sensitive linkers are also known as disulfide linkers. Drug release is triggered by the difference between high intracellular glutathione concentrations (in the millimolar range) and the relatively low glutathione concentrations in the blood (in the micromolar range). This is particularly true in tumor cells, where low oxygen concentrations lead to increased activity of reductase enzymes, resulting in high glutathione concentrations.Disulfide bonds are thermodynamically stable, resulting in good stability in plasma. Enzyme-labile linkers, such as peptide linkers, allow for better control of drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as cathepsin B. Because proteases are usually inactive outside cells due to inappropriate extracellular pH and serum protease inhibitors, such peptide linkers are considered to be very stable in the plasma circulation. Enzyme-labile linkers are widely used as cleavable linkers in antibody-drug conjugates due to their high plasma stability and good cleavage selectivity and efficacy within cells.

[0050] The term "antibody-drug conjugate" refers to a link between an antibody and a biologically active drug via a stable bridging unit. In the present invention, a "ligand-drug conjugate" refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a stable bridging unit, preferably an antibody-drug conjugate (ADC). The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Boil. Chem. 1968, 243, 3558.

[0051] The term "alkyl" refers to a saturated aliphatic hydrocarbon group that is a straight or branched chain group containing 1 to 20 carbon atoms (i.e., a "C1-C20 alkyl"), preferably an alkyl containing 1 to 12 carbon atoms (i.e., a "C1-C12 alkyl"), more preferably an alkyl containing 1 to 10 carbon atoms (i.e., a "C1-C10 alkyl"), and most preferably an alkyl containing 1 to 6 carbon atoms (i.e., a "C1-C6 alkyl"). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, and 5-methylhexyl. , 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and branched isomers thereof.More preferably, the alkyl group is a lower alkyl having 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl can be substituted or unsubstituted. If substituted, the substituent can be substituted at any available point of attachment. The substituents are preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0052] The term "substituted alkyl" refers to an alkyl group having a hydrogen atom replaced with a substituent. Unless the context indicates otherwise, the alkyl group substituent can be a variety of groups selected from the group consisting of -halogen, -OR', -NR'R'', -SR', -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)R', -NH-C(NH)=NH, -NR'C(NH)=NH, -NH-C(NH)=NR', -S(O)R', -S(O)R', -S(O)NR'R'', -NR'S(O)R'', -CN, and -NO. The number of substituents ranges from 0 to (2m'+1), where m' is the total number of carbon atoms in the group. R', R'', and R''' each independently represent hydrogen, unsubstituted C 1-8Alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted with 1 to 3 halogens, unsubstituted C 1-8 Alkyl, C 1-8 Alkoxy or C 1-8 Thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-C 1-4 When R' and R" are attached to the same nitrogen atom, they can form a 3-, 4-, 5-, 6-, or 7-membered ring together with the nitrogen atom. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

[0053] The term "alkylene" refers to a saturated straight or branched chain aliphatic hydrocarbon group having two residues derived from removing two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. Alkylene is a straight or branched chain group having 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), 1,5-pentylene (-CH2CH2CH2CH2CH2-), and the like. Alkylene may be substituted or unsubstituted. When substituted, the substituents may be substituted at any available point of attachment. The substituents are preferably one or more groups optionally and independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0054] The term "alkoxy" refers to an -O-(alkyl) and -O-(cycloalkyl) group, where alkyl and cycloalkyl are as defined above. Non-limiting examples of C1-C6 alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. An alkoxy can be optionally substituted or unsubstituted. If substituted, the substituents are preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0055] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent having from 3 to 20 carbon atoms (i.e., "C3-C20 cycloalkyl"), preferably from 3 to 12 carbon atoms (i.e., "C3-C12 cycloalkyl"), more preferably from 3 to 10 carbon atoms (i.e., "C3-C10 cycloalkyl"), and most preferably from 3 to 8 carbon atoms (i.e., "C3-C8 cycloalkyl"). Non-limiting examples of monocyclic cycloalkyls (e.g., "C3-C8 cycloalkyl") include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexenyl dialkenyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. Polycyclic cycloalkyls include cycloalkyls having spirocyclic, fused, or bridged rings.

[0056] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon group containing 3 to 20 ring atoms (i.e., "3- to 20-membered heterocyclyl"), in which one or more ring atoms are heteroatoms selected from the group consisting of N, O, and S(O)m (where m is an integer from 0 to 2), excluding -OO-, -OS-, or -SS- in the ring, and the remaining ring atoms are carbon atoms. Preferably, the heterocyclyl contains 3 to 12 ring atoms (i.e., "3- to 12-membered heterocyclyl"), in which 1 to 4 atoms are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms (i.e., "3- to 10-membered heterocyclyl"). Non-limiting examples of monocyclic heterocyclyls (e.g., 3- to 7-membered heterocyclyls) include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyls include heterocyclyls having spiro, fused or bridged rings.

[0057] The term "cycloalkylalkyl" refers to an alkyl group substituted with one or more cycloalkyls, preferably one cycloalkyl, where alkyl and cycloalkyl are as defined above. Examples of cycloalkylalkyl include C3-C8 cycloalkylC1-C6 alkyl. The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, where alkyl is as defined above. Examples of haloalkyl include halogenated C1-C6 alkyls. The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, where alkyl is as defined above. Examples of deuterated alkyls include deuterated C1-C6 alkyls. The term "C6-C12 aryl" refers to a carbocyclic aromatic group having from 6 to 12 carbon atoms. The term "C6-C10 aryl" refers to a carbocyclic aromatic group having 6 to 10 carbon atoms. Examples of C6-C10 aryl include phenyl, naphthyl, and the like.

[0058] The term "5- to 10-membered heteroaryl" refers to an aromatic heterocyclic ring, typically a 5-, 6-, 7-, 8-, 9-, or 10-membered heterocyclic ring, having 1 to 3 heteroatoms selected from N, O, or S. The heteroaryl ring may optionally be further fused or attached to aromatic and non-aromatic carbocyclic and heterocyclic rings. Non-limiting examples of 5-10 membered heteroaryls include, for example, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, isothiazolyl, thioxazolyl, pyrrolyl, phenyl-pyrrolyl, furyl, phenyl-furyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, benzofuranyl, benzothienyl, benzo-1,3-dioxolane (benzodioxin), isoindolyl, benzimidazolyl, indazolyl, quinolyl, isoquinolyl, 1,2,3-triazolyl, 1-phenyl-1,2,3-triazolyl, 2,3-indolinyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, benzopyrine, pyryl, 2,3-dihydrobenzoxazinyl, 2,3-dihydroquinoxalinyl, and the like.

[0059] The term "substituted C6-C10 aryl" or "substituted 5- to 10-membered heteroaryl" or "substituted 3- to 7-membered heterocyclyl" refers to an aryl or heteroaryl or heterocyclyl in which a hydrogen atom is replaced by a substituent, and unless the context indicates otherwise, the substituents on the aryl or heteroaryl or heterocyclyl are -halogen, -OR', -NR'R'', -SR', -SiR'R''R''', -OC(O)R', -C(O )R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NR'S(O)2R'', -CN and -NO2. The number of substituents ranges from 0 to (2m'+1), where m' is the total number of carbon atoms in the group. R', R'' and R''' each independently represent hydrogen, unsubstituted C 1-8 Alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted with 1 to 3 halogens, unsubstituted C 1-8 Alkyl, C 1-8 Alkoxy or C 1-8 Thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-C 1-4 When R' and R" are attached to the same nitrogen atom, they form a 3-, 4-, 5-, 6-, or 7-membered ring together with the nitrogen atom. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

[0060] The term "hydroxy" refers to the group --OH. The term "halogen" refers to fluorine, chlorine, bromine or iodine. The term "amino" refers to the group -NH2. The term "nitro" refers to the group -NO2. The term "amido" refers to a -C(O)N(alkyl) or -C(O)N(cycloalkyl) group, where alkyl and cycloalkyl are as defined above. The term "carboxylic acid group" refers to a -C(O)O(alkyl) or -C(O)O(cycloalkyl) group, where alkyl and cycloalkyl are as defined above.

[0061] The present invention also encompasses various deuterated forms of Formula I. Each available hydrogen atom bonded to a carbon atom can be independently replaced with a deuterium atom. Those skilled in the art can synthesize the deuterated forms of Formula I by referring to relevant literature. The deuterated forms of Formula I can be prepared using commercially available deuterated raw materials or can be synthesized by conventional techniques using deuterated reagents. Non-limiting examples of deuterated reagents include deuterated borane, tritiated borane in tetrahydrofuran, lithium aluminum deuteride, deuterated iodoethane, deuterated iodomethane, etc.

[0062] The term "antibody" refers to an immunoglobulin, a four-peptide chain structure connected by interchain disulfide bonds between two identical heavy chains and two identical light chains. Different immunoglobulin heavy chain constant regions exhibit different amino acid compositions and sequences, and therefore different antigenicity. Therefore, immunoglobulins can be classified into five types, or immunoglobulin isotypes, namely, IgM, IgD, IgG, IgA, and IgE (corresponding to the μ, δ, γ, α, and ε heavy chains, respectively). Depending on the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds, Ig of the same type can be further classified into different subtypes; for example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. Light chains can be classified into kappa or lambda chains based on differences in the constant region. Each of the five types of Ig can have either kappa or lambda chains.The antibodies described in the present invention are preferably specific antibodies against cell surface antigens on target cells, and include, by way of non-limiting example, one or more of the following antibodies: anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg / AFP antibody, anti-cladin antibody. 18.2 antibody, anti-mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (Erb B3) Antibodies, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibody, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody body, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-integrin Av antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-cadherin antibody 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, and anti-CD123 antibody.

[0063] The term "solvate" or "solvate" refers to a pharmaceutically acceptable solvate formed by a Ligand-Drug Conjugate of the present invention and one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate. The term "drug loading" refers to the average number of cytotoxic drugs loaded onto each ligand of Formula I and can also be expressed as a drug-to-antibody ratio (DAR). Drug loading can range from 0 to 12, preferably 1 to 10, cytotoxic drugs (d) per antibody (Ab). In one embodiment of the present invention, drug loading is expressed as n, with exemplary values ​​being an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The average number of drugs per ADC molecule after the conjugation reaction can be determined by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA testing, and HPLC characterization.

[0064] In one embodiment of the present invention, a cytotoxic drug is attached by a bridging unit to the open cysteine ​​thiol-SH between the antibody chains and / or to the thiol-SH of a site-directed mutated cysteine ​​residue. Typically, the number of drug molecules that can be attached to an antibody in a conjugation reaction will be less than the theoretical maximum. The following non-limiting methods can be used to control the addition of the ligand-cytotoxic drug conjugate: (1) Controlling the molar ratio of the cross-linking reagent to the monoclonal antibody; (2) Control of reaction time and temperature, and (3) Selection of various reaction reagents. Methods for preparing conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

[0065] The term "pharmaceutically acceptable salt" or "pharmaceutical salt" generally refers to a salt of a ligand-drug conjugate of the present invention or a salt of a compound of the present invention. Such salts may be safe and / or effective when used in mammals and have corresponding bioactivity. The ligand-drug conjugate compounds of the present invention contain at least one carboxyl and can therefore form salts with bases. Non-limiting examples of pharmaceutically acceptable salts include sodium, potassium, calcium, and magnesium salts. The term "pharmaceutically acceptable salt" or "pharmaceutical salt" generally refers to a salt of an antibody-drug conjugate of the present invention or a salt of a compound of the present invention. Such salts may be safe and / or effective when used in mammals and have corresponding bioactivity. The ligand-drug conjugate compounds of the present invention contain at least one amide and may therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfide, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0066] "Acidic amino acids" refer to amino acids with an isoelectric point of less than 7. Acidic amino acid molecules often contain one or more acidic groups, such as carboxyl, that can be effectively ionized into negative ions in the structure, increasing hydrophilicity. Acidic amino acids can be natural or unnatural amino acids. "Naturally occurring amino acids" refer to amino acids synthesized by living organisms. Naturally occurring amino acids are generally L-configured, with a few exceptions, such as glycine. Naturally occurring amino acids include both naturally occurring and biosynthetic amino acids. "Unnatural amino acid" refers to an amino acid obtained by synthetic means. [Example]

[0067] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In the following examples, experimental methods that do not specify specific conditions are generally carried out under conventional conditions or conditions recommended by manufacturers. Unless otherwise specified, all percentages, proportions, ratios, or parts are calculated by weight.

[0068] Example 1 Synthesis of compound M1: [ka] In a 5000 mL single-neck flask, N-fluorenylmethoxycarbonyl-glycine-glycine (100 g, 282 mmol, 1.0 equiv.), lead tetraacetate (175 g, 395 mmol, 1.4 equiv.), 2000 mL of dry tetrahydrofuran, and 670 mL of toluene were mixed and stirred thoroughly, and then heated to 85 °C under N2 for 2.5 h. After TLC monitoring showed the reaction was complete, the reaction was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give compound M1 (87 g); LC-MS: [M+NH4] + =386.0.

[0069] Example 2 Synthesis of compound M3: [ka] In a 1000 mL single-neck flask, compound SM-2 (prepared according to the synthetic route disclosed in patent application CN108452321A) (40 g, 96 mmol, 1.0 equiv.), triethylamine (26.7 mL, 2.0 equiv.), and toluene (400 mL) were mixed and then heated to 120 °C and refluxed for 2 h. After TLC monitoring showed the reaction was complete, the reaction system was cooled to 50 °C and the solvent was removed by rotary evaporation under reduced pressure. The reaction product was then dissolved in ethyl acetate (150 mL) and water (40 mL). Then, while stirring in a water bath, 1 M HCl was added to adjust the pH to 2-3. The resulting solution was separated, and the aqueous layer was extracted once more with ethyl acetate. The organic layers were combined and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated to give a pale yellow oily crude product. The crude product was purified by column chromatography (DCM:MeOH=40:1) to give compound M2 (26.6 g); LC-MS: [M+H] + =399.3. In a 1000 mL single-neck flask, compound M2 (26.5 g, 60.5 mmol, 1.0 equiv.), pentafluorophenol (12.2 g, 66.5 mmol, 1.1 equiv.), DCC (13.7 g, 66.5 mmol, 1.1 equiv.), and THF (300 mL) were mixed. The reaction was carried out at room temperature for 30 minutes and monitored by TLC. Insoluble materials were filtered off. The reaction solution was directly purified by preparative separation. The preparative solution was concentrated in a vacuum (water pump) water bath at 35°C to remove acetonitrile, and then lyophilized to give compound M3 (31.5 g) in 64% yield; LC-MS: [M+H] + =565.1.

[0070] Example 3 Synthesis of compound ent-M3: [ka] Compound ent-M3 (27.8 g) was obtained by referring to the synthesis route in Example 2; LC-MS: [M+H] + =565.2.

[0071] Example 4 Synthesis of Compound 1: [ka] First step: Compound 1a In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred. After cooling to 0 °C, benzyl glycolate (5.4 g, 32.6 mmol) was added dropwise. The resulting solution was then allowed to warm to room temperature and stirred for approximately 2–4 h. After TLC monitoring showed the reaction was complete, the reaction was quenched with saturated NaHCO3 solution, extracted with ethyl acetate, and the product was washed with saturated NaCl solution, then dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-1:1) to give 1a (4 g) in 52% yield; LC-MS: [M+H] + =475.18.

[0072] Second step: Compound 1b In a 25 mL single-neck flask, compound 1a (2 g, 4.2 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and then DBU (766 mg, 5.04 mmol) was added and reacted for 1 h. After TLC monitoring showed that the Fmoc deprotection was complete, the resulting product was ready for further use. In a separate 25 mL single-neck flask, M4 (prepared according to the synthetic route disclosed in CN111051330 A) (1.73 g, 4.2 mmol), PyBOP (2.61 g, 5.04 mmol), HOBt (680 mg, 5.04 mmol), and 10 mL of DMF were mixed. DIPEA (830 μL, 5.04 mmol) was then added in a water bath. The resulting reaction solution was stirred for 30 minutes and then added to the reaction flask for reaction at room temperature. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by preparative liquid chromatography to obtain a product solution. This solution was extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give the solid product 1b (1.7 g) in 63% yield; LCMS: [M+H] + =648.26.

[0073] Third step: Compound 1c In a 25 mL single-neck flask, compound 1b (900 mg, 1.39 mmol) was completely dissolved in 15 mL of DMF, and then 900 mg of 5% Pd / C was added and the mixture was hydrogenated for 2 hours. After the reaction was determined to be complete, the reaction solution was filtered, and the filtrate was used directly in the next reaction without further purification.

[0074] Fourth step: Compound 1d The crude product 1c was placed in a flask in an ice-water bath, and then DIPEA (235 μL, 1.39 mmol) was added, followed by compound M3 (784 mg, 1.39 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was lyophilized to obtain 1d (504 mg); LC-MS: [M+H] + =804.4.

[0075] 5th step: Compound 1e In a 50 mL single-neck flask, 1d (500 mg, 0.62 mmol), M5 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were mixed, and then DIPEA (378 μL, 2.29 mmol) was added under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 1e. This preparative solution was lyophilized to obtain 1e (210 mg); LC-MS: [M+H] + =1221.6.

[0076] Step 6: Compound 1 In a 25 mL single-neck flask, 1e (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 1 (60 mg); LC-MS: [M+H] + =1065.3.

[0077] Example 5 Synthesis of compound 2: [ka] Compound 2 (51 mg) was obtained by following the synthesis route in Example 4; LC-MS: [M+H] + =1065.3.

[0078] Example 6 Synthesis of compound 3: [ka] First step: Compound 3a In a 250 mL one-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred. After cooling to 0 °C, 2-hydroxy-2-methylpropionic acid benzyl ester (6.3 g, 32.6 mmol) was added dropwise. The resulting solution was then allowed to warm to room temperature and react for approximately 2–4 h. After TLC monitoring showed the reaction was complete, the reaction was quenched with saturated NaHCO3 solution, extracted with ethyl acetate, and the product was washed with saturated NaCl solution, then dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to give 3a (4.2 g) in 52% yield; LC-MS: [M+H] + =503.3.

[0079] Second step: Compound 3b In a 25 mL single-neck flask, compound 3a (2 g, 4.0 mmol) and 10 mL of DMF were mixed and stirred at 0° C. Then, DBU (760 mg, 5.0 mmol) was added and the reaction was continued for 1 h. After TLC monitoring showed that the Fmoc deprotection was complete, the resulting product was ready for further use. In a separate 25 mL single-neck flask, M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol), and 10 mL of DMF were mixed, and then DIPEA (823 μL, 5.04 mmol) was added in an ice-water bath. The resulting reaction solution was stirred for 30 minutes and then added to the reaction flask and allowed to react at room temperature. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by liquid chromatography to obtain a product fraction. This fraction was extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give the solid product 3b (1.4 g) in 53% yield; LC-MS: [M+H] + =676.2.

[0080] Third step: Compound 3c In a 25 mL single-neck flask, compound 3b (700 mg, 1.04 mmol) was completely dissolved in 10 mL of DMF, and then 700 mg of 5% Pd / C was added and the hydrogenation reaction was carried out for 1.5 hours. After the reaction was determined to be complete, the reaction solution was filtered, and the filtrate was used directly in the next reaction without purification.

[0081] Fourth step: Compound 3d The crude product 3c was placed in a flask in an ice-water bath, and then DIPEA (210 μL, 1.25 mmol) was added, followed by compound M3 (704 mg, 1.25 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 3d (486 mg); LC-MS: [M+H] + =830.5.

[0082] 5th step: Compound 3e In a 50 mL single-neck flask, 3d (300 mg, 0.36 mmol), M5 (180 mg, 0.36 mmol), PyBOP (260 mg, 0.5 mmol), HOBt (67 mg, 0.5 mmol), and 10 mL of DMF were mixed, and then DIPEA (219.5 μL, 1.33 mmol) was added under ice-water bath conditions. The reaction was allowed to stand at room temperature for 3 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 3e. This preparative solution was lyophilized to obtain 3e (157 mg); LC-MS: [M+H] + =1249.6.

[0083] Step 6: Compound 3 In a 25 mL single-neck flask, 3e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 3 (64 mg); LC-MS: [M+H] + =1093.1.

[0084] Example 7 Synthesis of compound 4: [ka] Compound 4 (60 mg) was obtained by following the synthesis route in Example 6; LC-MS: [M+H] + =1093.2.

[0085] Example 8 Synthesis of compound 5A: [ka] First step: Compound 5a In a 25 mL one-neck flask, M1 (500 mg, 1.4 mmol, 1.0 equiv.), p-toluenesulfonic acid monohydrate (26 mg, 0.1 mmol, 0.1 equiv.), and 10 mL of THF were mixed and stirred. After cooling to 0 °C, L-lactic acid benzyl ester (1.2 g, 7.0 mmol, 5 equiv.) was slowly added. The resulting solution was stirred and allowed to warm to room temperature. After TLC monitoring showed the reaction was complete, the reaction was quenched with saturated NaHCO3 solution and then extracted with ethyl acetate. The product was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by reverse-phase column to give 5a (400 mg); LC-MS: [M+NH4] + =506.2. 1 H NMR (400 Mz, CDCl3 / CD3OD):1.39 (3H, d, J= 6.8 Hz), 3.78 (2H, t, J= 4.0 Hz), 4.17-4.27 (2H, m), 4.42 (2H, d, J= 4.0 Hz), 4.72-4.85 (2H, m), 5.11-5.58 (2H, m), 5.43 (1H, s), 7.06 (1H, t, J= 8.0 Hz), 7.25-7.33 (6H, m), 7.38 (2H, t, J=8.0 Hz), 7.57 (2H, d, J= 8.0 Hz), 7.75 (2H, d, J= 8.0 Hz).

[0086] Second step: Compound 5b In a 25 mL one-neck flask, compound 5a (400 mg, 0.8 mmol, 1.0 equivalent) and 4 mL of DMF were mixed and stirred, then cooled to 0 °C, and DBU (137 mg, 0.9 mmol, 1.1 equivalent) was slowly added. The resulting solution was then allowed to react at room temperature. After TLC monitoring showed that the reaction was complete, it was designated as reaction solution 1; In another 25 mL single-neck flask, M4 (372 mg, 0.9 mmol, 1.1 equiv.), PyBOP (852 mg, 1.6 mmol, 2.0 equiv.), and 3 mL of DMF were mixed and stirred at room temperature for 5 min. Then, reaction solution 1 was added and the reaction system was allowed to stand at room temperature. After TLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to give compound 5b (326 mg); LC-MS: [M+NH4] + =679.2.

[0087] Third step: Compound 5c In a 100 mL single-neck flask, 5b (4.0 g, 6.05 mmol, 1.0 equiv) was dissolved in DMF (60 mL), and then 5% Pd / C (4 g) was added and the reaction was hydrogenated for 4 h (the progress of the reaction was monitored by HPLC). The Pd / C was filtered, and the filtrate was placed in an ice-water bath (approximately 0 °C) without concentration for further use.

[0088] Fourth step: Compound 5d The crude product 5c was placed in a flask in an ice-water bath, and DIPEA (1.1 mL, 1.1 equivalents) was added, followed by compound M3 (3.4 g, 6.05 mmol). The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 5d (3.15 g); LC-MS: [M-H] - =816.3.

[0089] Fifth step: Compound 5e A 100 mL single-neck flask was charged with 5d (2.07 g, 2.53 mmol, 1.0 equiv.), M5 (1.35 g, 2.53 mmol, 1.0 equiv.), PyBOP (1.98 g, 3.79 mmol, 1.5 equiv.), HOBt (0.51 g, 3.79 mmol, 1.5 equiv.), and DMF (40 mL). DIPEA (1.05 mL, 1.5 equiv.) was added under ice-water bath conditions. The resulting solution was allowed to warm to room temperature and react for 2 h (monitored by HPLC). The reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The reaction solution was directly purified by preparative separation. This preparative solution was concentrated at 35 °C in a vacuum (water pump) water bath to remove acetonitrile, followed by lyophilization to obtain compound 5e (1.92 g) in 61% yield; LC-MS: [M+H] + =1235.4.

[0090] Step 6: Compound 5A In a 100 mL single-neck flask, compound 5e (1.0 g, 0.8 mmol, 1.0 equiv.) and 35 mL of nitromethane were mixed and dissolved, followed by the addition of zinc bromide (3.64 g, 16 mmol, 20.0 equiv.). The reaction was allowed to stand in an oil bath at 40 °C for 30 minutes (the reaction system was preheated and stabilized). The reaction solution was concentrated in a vacuum (water pump) water bath at 45 °C to remove the nitromethane, leaving a yellow solid residue (under HPLC monitoring). A preparative solution of compound 5A was obtained by acid fractionation. This preparative solution was concentrated in a vacuum (water pump) water bath at 35 °C to remove the acetonitrile, followed by lyophilization to give compound 5A (786 mg) in 90% yield. LC-MS: [M+H] + =1079.4; 1H NMR (400 MHz, DMSO-d6) δ9.39 - 9.02 (m, 1H), 8.70 (t, J = 6.5 Hz, 1H), 8.64 (t, J = 5.7 Hz, 1H), 8.56 (d, J = 8.8 Hz, 1H), 8.34 (t, J = 5.7 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H), 8.01 (t, J = 5.5 Hz, 1H), 7.71 (d, J = 10.9 Hz, 1H), 7.30 (s, 1H), 7.28 - 7.15 (m, 4H), 7.14 (s, 2H), 5.53 (dd, J = 14.5, 6.4 Hz, 1H), 5.49 - 5.34 (m, 2H), 5.22 (d, J = 18.8 Hz, 1H), 5.09 (d, J = 18.7 Hz, 1H), 5.03 (dd, J = 9.6, 3.9 Hz, 1H), 4.73 (dd, J = 9.9, 6.9 Hz, 1H), 4.59 (dd, J = 10.1, 6.5 Hz, 1H), 4.49 (ddd, J = 13.2, 8.6, 4.4 Hz, 1H), 4.14 (dd, J = 13.3, 6.6 Hz, 2H), 3.93 (s, 2H), 3.84 (dd, J = 16.5, 6.3 Hz, 1H), 3.76 (dd, J = 16.9, 5.7 Hz, 2H), 3.70 (d, J = 5.2 Hz, 2H), 3.60 (dd, J = 16.7, 5.4 Hz, 1H), 3.52 (dd, J = 16.4, 5.1 Hz, 1H), 3.45 (dd, J = 12.8, 10.1 Hz, 1H), 3.25 - 3.15 (m, 1H), 3.14 - 3.05 (m, 1H), 3.01 (dd, J = 13.7, 4.1 Hz, 1H), 2.73 (dd, J = 13.5, 9.8 Hz, 1H), 2.54 - 2.47 (m, 1H), 2.33 (s, 2H), 2.17 (d, J = 5.5 Hz, 2H), 1.91 - 1.79 (m, 2H), 1.33 (d, J = 6.6 Hz, 2H), 0.87 (t, J = 7.3 Hz, 2H).

[0091] Example 9 Synthesis of compound 5B: [ka] First step: Compound 5d-1 In a 25 mL single-neck flask, compound 5b (300 mg, 0.45 mmol, 1.0 equiv.) was stirred and completely dissolved in DMF (3 mL). 5% Pd / C (300 mg) was added, and the hydrogen was replaced three times. The hydrogenation reaction was carried out for 2 h. After HPLC monitoring showed the reaction was complete, the reaction solution was filtered to remove the Pd / C. The filtrate was cooled to 0-5 °C, and then DIPEA (65 mg, 0.5 mmol, 1.1 equiv.) was added, followed by ent-M3 (255 mg, 0.45 mmol). The resulting solution was heated to 20 ± 5 °C and reacted for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by HPLC, and the product fraction was collected and lyophilized to give compound 5d-1 (200 mg) in 54% yield; LC-MS: [M-H] - =816.3.

[0092] Second step: Compound 5e-1 In a 25 mL single-neck flask, compound 5d-1 (200 mg, 0.24 mmol, 1.0 equiv.), M5 (127 mg, 0.24 mmol, 1.0 equiv.), PyBOP (187 mg, 0.36 mmol, 1.2 equiv.), HOBt (48 mg, 0.36 mmol, 1.2 equiv.), and DMF (6 mL) were mixed and cooled to 0-5 °C in an ice-water bath. DIPEA (62 mg, 0.48 mmol, 2.0 equiv.) was then added. The reaction was allowed to stand at 20 ± 5 °C for 2 h. After HPLC monitoring showed the reaction was complete, the reaction solution was directly purified by preparative HPLC. The product solution was collected and then lyophilized to give compound 5e-1 (162.8 mg); LC-MS: [M+H] + =1235.4.

[0093] Third step: Compound 5B In a 25 mL single-neck flask, compound 5e-1 (110 mg, 0.089 mmol, 1.0 equiv.), ZnBr (400 mg, 1.78 mmol, 20.0 equiv.), and CHNO (10 mL) were added sequentially. The reaction was allowed to stand at 40 °C for 0.5 h before being terminated. Rotary evaporation under reduced pressure at 45 °C gave a yellow solid, which was then sampled and monitored by HPLC. The rotary evaporated solid was directly purified by preparative HPLC. The product solution was collected and then lyophilized to give compound 5B (73.4 mg) in 76.5% yield; LC-MS: [M+H] + =1079.4.

[0094] Example 10 Synthesis of compound 6A: [ka] Compound 6A (71 mg) was obtained by referring to the synthetic route of Example 8; LC-MS: [M+H] + =1079.4.

[0095] Example 11 Synthesis of compound 6B: [ka] Compound 6B (59 mg) was obtained by following the synthesis route of Example 9; LC-MS: [M+H] + =1079.4.

[0096] Example 12 Synthesis of compounds 7A and 7B: [ka]

[0097] First step: Compound 7a In a 250 mL single-neck flask, M1 (10 g, 27.1 mmol), 3,3,3-trifluorolactic acid benzyl ester (prepared according to the synthetic route disclosed in patent application WO2020063673A1) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were mixed and heated to 100 °C for 4 hours. After the reaction was determined to be complete, the reaction solution was cooled to room temperature and then filtered to remove insoluble materials. The filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 5.15 g of the desired product in 35.1% yield; LC-MS: [M+H] + =543.17.

[0098] Second step: Compound 7b In a 50 mL one-neck flask, compound 7a (5 g, 9.2 mmol) was completely dissolved in 15 mL of DMF, and then DBU (1.68 g, 11 mmol) was added in an ice-water bath and reacted for 1 hour to obtain reaction solution 1; In another 50 mL single-neck flask, M4 (3.8 g, 9.2 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol), and 10 mL of DMF were mixed and completely dissolved. Then, DIPEA (1.82 mL, 11 mmol) was added in an ice-water bath and allowed to react for another 30 minutes. Then, reaction solution 1 was added, and the reaction was allowed to stand at room temperature for 2 hours and monitored by HPLC. After the reaction was determined to be complete, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. This fraction was extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated to give 4.1 g of solid product in 62.3% yield; LC-MS: [M+H] + =716.25.

[0099] Third step: Compound 7d In a 25 mL single-neck flask, 7b (900 mg, 1.26 mmol) was completely dissolved in 15 mL of DMF, followed by the addition of 900 mg of 5% Pd / C and hydrogenation for 2 hours. After the reaction was deemed complete, the reaction solution was filtered, and the filtrate was placed in an ice-water bath. DIPEA (228 μL, 1.38 mmol) was added, followed by M3 (712 mg, 1.26 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring indicated the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. This preparative solution was lyophilized to give 525 mg of product in 47.9% yield; LC-MS: [M H] - =870.33.

[0100] Fourth step: Compound 7e In a 50 mL single-neck flask, 7d (500 mg, 0.57 mmol), M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (378 μL, 2.29 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 7e-1 and compound 7e-2. These fractions were separately lyophilized to obtain 150 mg of compound 7e-1. LC-MS: [M+H] + = 1289.46; 220 mg of compound 7e-2, LC-MS: [M+H] + =1289.46 was obtained.

[0101] 5th step: Compound 7A [ka] In a 25 mL single-neck flask, 7e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 52 mg of a solid; TOF result: 1133.3613.

[0102] Sixth Step: Compound 7B [ka] In a 25 mL single-neck flask, 7e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 63 mg of a solid; TOF result: 1133.3668.

[0103] Example 13 Synthesis of compounds 8A and 8B: [ka] First step: Compound 8d In a 25 mL single-neck flask, 7c (900 mg, 1.83 mmol) was completely dissolved in 20 mL of DMF, followed by the addition of DIPEA (303 μL, 1.83 mmol) and then ent-M3 (1034 mg, 1.83 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to give 613 mg of product in 38.5% yield; LC-MS: [M H] - =870.32.

[0104] Second step: Compound 8e-1 and Compound 8e-2 In a 50 mL single-neck flask, 8d (500 mg, 0.57 mmol), M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (378 μL, 2.29 mmol) in an ice-water bath. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 8e-1 and compound 8e-2. These fractions were separately lyophilized to obtain 140 mg of compound 8e-1 and 210 mg of compound 8e-2. LC-MS of compound 8e-1: [M+H] + = 1289.47; LC-MS of compound 8e-2: [M+H] + =1289.47.

[0105] Third step: Compound 8A [ka] In a 25 mL single-neck flask, 8e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. This fraction was lyophilized to obtain 50 mg of a solid; TOF result: 1133.3623.

[0106] Fourth step: Compound 8B [ka] In a 25 mL single-neck flask, compound 8e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. This fraction was lyophilized to obtain 58 mg of a solid; TOF result: 1133.3653.

[0107] Example 14 Synthesis of compound 9A: [ka] First step: Compound 9a In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred, then cooled to 0 °C, and 2-hydroxy-2-cyclopropylacetic acid benzyl ester (prepared according to the synthetic route disclosed in patent application US20050020645 A1) (6.3 g, 32.6 mmol) was added dropwise. The resulting solution was then allowed to warm to room temperature and react for approximately 2 to 4 hours. After TLC monitoring showed the reaction was complete, the reaction was quenched with saturated NaHCO3 solution and then extracted with ethyl acetate, the product was washed with saturated NaCl solution, then dried over anhydrous Na2SO4, filtered and concentrated, and the residue was purified by silica gel column (PE:EA=10:1-5:1-2:1) to give 9a (3.7 g) in 45% yield; LC-MS: [M+H] + =501.5.

[0108] Second step: Compound 9b In a 25 mL single-neck flask, compound 9a (2 g, 4.0 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and then DBU (760 mg, 5.0 mmol) was added and the reaction was continued for 1 h. After TLC monitoring showed that the Fmoc deprotection was complete, the resulting product was ready for further use. In a separate 25 mL single-neck flask, M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol), and 10 mL of DMF were mixed, and then DIPEA (823 μL, 5.04 mmol) was added in an ice-water bath. The resulting reaction solution was stirred for 30 minutes and then placed in a reaction flask and allowed to react at room temperature. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by liquid chromatography to obtain a product fraction. This fraction was extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 1.5 g of solid product in 56% yield; LC-MS: [M+H] + =674.7.

[0109] Third step: Compound 9c In a 25 mL single-neck flask, compound 9b (900 mg, 1.3 mmol) was completely dissolved in 10 mL of DMF, and then 900 mg of 5% Pd / C was added and the hydrogenation reaction was carried out for 1.5 hours. After the reaction was determined to be complete, the reaction solution was filtered, and the filtrate was used directly in the next reaction without purification.

[0110] Fourth step: Compound 9d The crude product 9c was placed in a flask in an ice-water bath, and then DIPEA (223 μL, 1.3 mmol) was added, followed by compound M3 (750 mg, 1.3 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 9d (529 mg); LC-MS: [M+H] + =828.4.

[0111] 5th step: Compound 9e In a 50 mL single-neck flask, 9d (500 mg, 0.6 mmol), M5 (300 mg, 0.6 mmol), PyBOP (416 mg, 0.8 mmol), HOBt (108 mg, 0.5 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (351 μL, 2.13 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 3 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 9e. The preparative solution was lyophilized to obtain 9e (257 mg); LC-MS: [M+H] + =1247.5.

[0112] Step 6: Compound 9A In a 25 mL single-neck flask, 9e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 9A (55 mg); LC-MS: [M+H] + =1091.3.

[0113] Example 15 Synthesis of compound 9B: [ka] Compound 9B (44 mg) was obtained by following the synthesis route of Example 14; LC-MS: [M+H] + =1091.3.

[0114] Example 16 Synthesis of compound 10A: [ka] First step: Compound 10a In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred, then cooled to 0 °C, and then 3-hydroxy-2-cyclopropylpropionic acid benzyl ester (prepared according to the synthetic route disclosed in patent application WO2013187496A1) (6.7 g, 32.6 mmol) was added dropwise. The resulting solution was then allowed to warm to room temperature and react for approximately 2 to 4 hours. After TLC monitoring showed the reaction was complete, saturated NaHCO3 solution was added to the reaction system, then extracted with ethyl acetate, the product was washed with saturated NaCl solution, then dried over anhydrous Na2SO4, filtered and concentrated, and the residue was purified by silica gel column (PE:EA=10:1-5:1-2:1) to give 10a (4.9 g) in 58% yield; LC-MS: [M+H] + =515.4.

[0115] Second step: Compound 10b In a 25 mL single-neck flask, compound 10a (4 g, 7.8 mmol) and 10 mL of DMF were mixed and stirred at 0 °C, and then DBU (1.2 g, 8.0 mmol) was added. The reaction was carried out for 1 h. After TLC monitoring showed that the Fmoc deprotection was complete, the resulting product was ready for further use. In another 25 mL single-neck flask, M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol), and 10 mL of DMF were mixed, and then DIPEA (1.65 mL, 10.1 mmol) was added in an ice-water bath. The resulting reaction solution was stirred for 50 minutes and then added to the reaction flask and reacted at room temperature. After the reaction was completed as monitored by HPLC, the reaction solution was purified by liquid chromatography to obtain a product fraction. This fraction was extracted with dichloromethane and then washed with saturated NaCl solution. The organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give 2.3 g of solid product in 42% yield; LCMS: [M+H] + =688.8.

[0116] Third step: Compound 10c In a 25 mL single-neck flask, compound 10b (1.0 g, 1.45 mmol) was completely dissolved in 15 mL of DMF, and then 1.0 g of 5% Pd / C was added and the hydrogenation reaction was carried out for 1.5 hours. After the reaction was determined to be complete, the reaction solution was filtered, and the filtrate was used directly in the next reaction without purification.

[0117] Fourth step: Compound 10d The crude product 10c was placed in a flask in an ice-water bath, and then DIPEA (258 μL, 1.5 mmol) and compound M3 (837 mg, 1.45 mmol) were added. The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was lyophilized to obtain 10d (499 mg); LC-MS: [M-H] - =842.4.

[0118] Fifth step: Compound 10e In a 50 mL single-neck flask, 10d (400 mg, 0.48 mmol), M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (104 mg, 0.48 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (330 μL, 2.0 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 3 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 10e. This preparative solution was lyophilized to obtain 10e (188 mg); LC-MS: [M+H] + =1261.5.

[0119] Step 6: Compound 10A In a 25 mL single-neck flask, 10e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 10A (61 mg); LC-MS: [M+H] + =1105.4.

[0120] Example 17 Synthesis of compound 10B: [ka] Compound 10B (75 mg) was obtained by following the synthesis route of Example 16; LC-MS: [M+H] + =1105.4.

[0121] Example 18 Synthesis of compound 11A: [ka] First step: Compound 11a In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred, then cooled to 0 °C, and 2-hydroxy-2-cyclobutylacetic acid benzyl ester (prepared according to the synthetic route disclosed in Journal of Medicinal Chemistry, 2013, 56 (13), 5541-5552) (6.7 g, 32.6 mmol) was added dropwise. The resulting solution was then allowed to react at room temperature for approximately 2 to 4 hours. After TLC monitoring showed the reaction was complete, saturated NaHCO3 solution was added, the organic layer was extracted with ethyl acetate, the product was washed with saturated NaCl solution, then dried over anhydrous Na2SO4, filtered and concentrated, and the residue was purified by silica gel column (PE:EA=10:1-5:1-2:1) to give 11a (5.1 g) in 62% yield; LC-MS: [M+H] + =515.7.

[0122] Second step: Compound 11b In a 25 mL single-neck flask, compound 11a (4 g, 7.8 mmol) and 10 mL of DMF were mixed and stirred at 0 °C, and then DBU (1.2 g, 8.0 mmol) was added. The reaction was carried out for 1 h. After TLC monitoring showed that the Fmoc deprotection was complete, the resulting product was ready for further use. In another 25 mL single-neck flask, M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol), and 10 mL of DMF were mixed, and then DIPEA (1.63 mL, 10.0 mmol) was added in an ice-water bath. The resulting reaction solution was stirred for 40 minutes and then added to the reaction flask and reacted at room temperature. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by liquid chromatography to obtain a product fraction. This fraction was extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 2.3 g of solid product in 42% yield; LCMS: [M+H] + =688.3.

[0123] Third step: Compound 11c In a 25 mL single-neck flask, compound 11b (2.0 g, 2.9 mmol) was completely dissolved in 25 mL of DMF, and then 2.0 g of 5% Pd / C was added and the mixture was hydrogenated for 3 hours. After the reaction was determined to be complete, the reaction solution was filtered, and the filtrate was used directly in the next reaction without further purification.

[0124] Fourth step: Compound 11d The crude product 11c was placed in a flask in an ice-water bath, and then DIPEA (516 μL, 3.0 mmol) was added, followed by compound M3 (1.7 g, 2.9 mmol). The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 11d (934 mg); LC-MS: [M-H] - =842.4.

[0125] 5th step: Compound 11e In a 50 mL single-neck flask, 11d (800 mg, 0.96 mmol), M5 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol), and 30 mL of DMF were mixed, followed by the addition of DIPEA (660 μL, 4.0 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 4 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 11e. This preparative solution was lyophilized to obtain 11e (401 mg); LC-MS: [M+H] + =1261.4.

[0126] Step 6: Compound 11A In a 25 mL single-neck flask, 11e (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol), and 10 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 11A (86 mg); LC-MS: [M+H] + =1105.4.

[0127] Example 19 Synthesis of compound 11B: [ka] Compound 11B (50 mg) was obtained by following the synthesis route of Example 18; LC-MS: [M+H] + 1105.4.

[0128] Example 20 Synthesis of compound 12A: [ka] First step: Compound 12a In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred, then cooled to 0 °C, and 3-hydroxy-2-cyclobutylpropionic acid benzyl ester (prepared according to the synthetic route disclosed in patent application WO2009011285A1) (7.2 g, 32.6 mmol) was added dropwise. The resulting solution was then allowed to warm to room temperature and react for approximately 2–4 h. After TLC monitoring showed the reaction was complete, saturated NaHCO3 solution was added, and the organic layer was washed with saturated NaCl solution, extracted with ethyl acetate, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to give 12a (4.5 g) in 52% yield; LC-MS: [M+H] + =529.4.

[0129] Second step: Compound 12b In a 25 mL single-neck flask, compound 12a (4 g, 7.6 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and then DBU (1.2 g, 8.0 mmol) was added and the reaction was continued for 1 h. After TLC monitoring showed that the Fmoc deprotection was complete, the resulting product was ready for further use. In another 25 mL single-neck flask, M4 (3.2 g, 7.6 mmol), PyBOP (4.7 g, 9.0 mmol), HOBt (1.22 g, 9.0 mmol), and 10 mL of DMF were mixed, and then DIPEA (1.49 mL, 0.9 mmol) was added in an ice-water bath. The resulting reaction solution was stirred for 30 minutes and then added to the reaction flask and reacted at room temperature. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by liquid chromatography to obtain a product fraction. This fraction was extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 2.0 g of solid product in 37% yield; LC-MS: [M+H] + =702.8.

[0130] Third step: Compound 12c In a 25 mL single-neck flask, compound 12b (1.0 g, 1.43 mmol) was completely dissolved in 15 mL of DMF, and then 1.0 g of 5% Pd / C was added and the hydrogenation reaction was carried out for 1.5 hours. After the reaction was determined to be complete, the reaction solution was filtered, and the filtrate was used directly in the next reaction without purification.

[0131] Fourth step: Compound 12d The crude product 12c was placed in a flask in an ice-water bath, and then DIPEA (258 μL, 1.5 mmol) was added, followed by compound M3 (825 mg, 1.43 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 12d (522 mg); LC-MS: [M-H] - =856.4.

[0132] Fifth step: Compound 12e In a 50 mL single-neck flask, 12d (400 mg, 0.47 mmol), M5 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (330 μL, 2.0 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 3 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 12e. This preparative solution was lyophilized to obtain 12e (198 mg); LC-MS: [M+H] + =1275.4.

[0133] Step 6: Compound 12A In a 25 mL single-neck flask, 12e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 12A (55 mg); LC-MS: [M+H] + =1119.4.

[0134] Example 21 Synthesis of compound 12B: [ka] Compound 12B (50 mg) was obtained by following the synthesis route of Example 20; LC-MS: [M+H] + =1119.4.

[0135] Example 22 Synthesis of compound 13A: [ka] First step: Compound 13a In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred, then cooled to 0 °C, and 2-hydroxy-2-cyclopentylacetic acid benzyl ester (prepared according to the synthetic route disclosed in Journal of Medicinal Chemistry, 2013, 56 (13), 5541-5552) (7.2 g, 32.6 mmol) was added dropwise. The resulting solution was allowed to warm to room temperature and react for approximately 2 to 4 hours. After TLC monitoring showed the reaction was complete, the reaction was quenched with saturated NaHCO3 solution and then extracted with ethyl acetate, the product was washed with saturated NaCl solution, then dried over anhydrous Na2SO4, filtered and concentrated, and the residue was purified by silica gel column (PE:EA=10:1-5:1-2:1) to give 13a (4.6 g) in 53% yield; LC-MS: [M+H] + =529.5.

[0136] Second step: Compound 13b In a 25 mL single-neck flask, compound 13a (4 g, 7.6 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and then DBU (1.17 g, 7.8 mmol) was added and the reaction was continued for 1 h. After TLC monitoring showed that the Fmoc deprotection was complete, the resulting product was ready for further use. In a separate 25 mL single-neck flask, M4 (3.14 g, 7.6 mmol), PyBOP (4.42 g, 8.5 mmol), HOBt (1.15 g, 8.5 mmol), and 10 mL of DMF were mixed, and then DIPEA (1.39 mL, 0.85 mmol) was added in an ice-water bath. The resulting reaction solution was stirred for 30 minutes and then added to the reaction flask and reacted at room temperature. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by liquid chromatography to obtain a product fraction. This fraction was extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 2.1 g of solid product in 39% yield; LC-MS: [M+H] + =702.8.

[0137] Third step: Compound 13c In a 25 mL single-neck flask, compound 13b (1.5 g, 1.87 mmol) was completely dissolved in 25 mL of DMF, and then 1.5 g of 5% Pd / C was added and the hydrogenation reaction was carried out for 3 hours. After the reaction was determined to be complete, the reaction solution was filtered, and the filtrate was used directly in the next reaction without purification.

[0138] Fourth step: Compound 13d The crude product 13c was placed in a flask in an ice-water bath, and then DIPEA (333 μL, 1.93 mmol) was added, followed by compound M3 (1.1 g, 1.87 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 13d (519 mg); LC-MS: [M-H] - =856.6.

[0139] Fifth step: Compound 13e In a 50 mL single-neck flask, 13d (400 mg, 0.47 mmol), M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (103 mg, 48 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (330 μL, 2.0 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 4 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 13e. The preparative solution was lyophilized to obtain 13e (187 mg); LC-MS: [M+H] + =1275.5.

[0140] Step 6: Compound 13A In a 25 mL single-neck flask, 13e (100 mg, 0.08 mmol), zinc bromide (355 mg, 0.16 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 13A (60 mg); LC-MS: [M+H] + =1119.6.

[0141] Example 23 Synthesis of compound 13B: [ka] Compound 13B (51 mg) was obtained by following the synthesis route of Example 22; LC-MS: [M+H] + =1119.6.

[0142] Example 24 Synthesis of compound 14A: [ka] First step: Compound 14a In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred, then cooled to 0 °C, and 3-hydroxy-2-cyclopentylpropanoic acid benzyl ester (prepared according to the synthetic route disclosed in patent application WO2009011285A1) (7.6 g, 32.6 mmol) was added dropwise. The resulting solution was then allowed to warm to room temperature and react for approximately 2 to 4 hours. After TLC monitoring showed the reaction was complete, the reaction was quenched with saturated NaHCO3 solution, then extracted with ethyl acetate, the product was washed with saturated NaCl solution, then dried over anhydrous Na2SO4, filtered, concentrated, and the residue was purified by silica gel column (PE:EA=10:1-5:1-2:1) to give 14a (4.4 g) in 49% yield; LC-MS: [M+H] + =543.6.

[0143] Second step: Compound 14b In a 25 mL single-neck flask, compound 14a (4 g, 7.4 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and then DBU (1.2 g, 8.0 mmol) was added and the reaction was continued for 1 h. After TLC monitoring showed that the Fmoc deprotection was complete, the resulting product was ready for further use. In another 25 mL single-neck flask, M4 (3.1 g, 7.4 mmol), PyBOP (4.6 g, 8.8 mmol), HOBt (1.19 g, 8.8 mmol), and 10 mL of DMF were mixed, and then DIPEA (1.49 mL, 9.0 mmol) was added in an ice-water bath. The resulting reaction solution was stirred for 30 minutes and then added to the reaction flask and reacted at room temperature. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by liquid chromatography to obtain a product fraction. This fraction was extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give 2.6 g of solid product in 49% yield; LC-MS: [M+H] + =716.4.

[0144] Third step: Compound 14c In a 25 mL single-neck flask, compound 14b (1.0 g, 1.4 mmol) was completely dissolved in 15 mL of DMF, and then 1.0 mL of 5% Pd / C was added and the hydrogenation reaction was carried out for 1.5 hours. After the reaction was determined to be complete, the reaction solution was filtered, and the filtrate was used directly in the next reaction without purification.

[0145] Fourth step: Compound 14d The crude product 14c was placed in a flask in an ice-water bath, and then DIPEA (248 μL, 1.5 mmol) was added, followed by compound M3 (808 mg, 1.4 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. This preparative solution was lyophilized to obtain 14d (500 mg); LC-MS: [M-H] - =870.5.

[0146] Fifth step: Compound 14e In a 50 mL single-neck flask, 14d (400 mg, 0.46 mmol), M5 (235 mg, 0.46 mmol), PyBOP (245 mg, 0.46 mmol), HOBt (99 mg, 0.46 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (331 μL, 2.0 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 3 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 14e. This preparative solution was lyophilized to obtain 14e (146 mg); LC-MS: [M+H] + =1289.5.

[0147] Step 6: Compound 14A In a 25 mL single-neck flask, 14e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 14A (52 mg); LC-MS: [M+H] + =1133.4.

[0148] Example 25 Synthesis of compound 14B: [ka] Compound 14B (48 mg) was obtained by following the synthesis route of Example 24; LC-MS: [M+H] + =1133.4.

[0149] Example 26 Synthesis of compounds 15A and 15B: [ka] First step: Compound 15a In a 250 mL single-neck flask, M1 (10 g, 27.1 mmol), 2-hydroxybutyric acid benzyl ester (prepared according to the synthetic route disclosed in Chemical Communications, 2019, 55 (53), 7699-7702) (10.5 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were mixed and heated to 100 °C for 4 hours. After the reaction was determined to be complete, the reaction solution was cooled to room temperature and then filtered to remove insoluble materials. The filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 5.67 g of the desired product in 42% yield; LC-MS: [M+H] + =503.5.

[0150] Second step: Compound 15b In a 50 mL one-neck flask, compound 15a (5 g, 9.95 mmol) was completely dissolved in 15 mL of DMF, and then DBU (1.68 g, 11 mmol) was added in an ice-water bath and reacted for 1 hour to obtain reaction solution 1; In another 50 mL single-neck flask, M4 (4.1 g, 10.0 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol), and 10 mL DMF were mixed and completely dissolved. Next, DIPEA (1.82 mL, 11 mmol) was added in an ice-water bath and the reaction was continued for another 40 minutes. Then, reaction solution 1 was added, and the reaction was allowed to stand at room temperature for 2 hours and monitored by HPLC. After the reaction was determined to be complete, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. This fraction was extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated to give 4.6 g of solid product in 68% yield; LC-MS: [M+H] + =676.7.

[0151] Third step: Compound 15d In a 25 mL single-neck flask, 15b (2.0 g, 2.96 mmol) was completely dissolved in 15 mL of DMF, followed by the addition of 2.0 g of 5% Pd / C and hydrogenation for 2 hours. After the reaction was deemed complete, the reaction solution was filtered, and the filtrate was placed in an ice-water bath. DIPEA (496 μL, 3.0 mmol) was added, followed by M3 (1.7 g, 2.96 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring indicated the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to give 1120 mg of product in 45% yield; LC-MS: [M H ] - =830.3.

[0152] Fourth step: Compound 15e In a 50 mL single-neck flask, 15d (500 mg, 0.60 mmol), M5 (321 mg, 0.60 mmol), PyBOP (469 mg, 0.90 mmol), HOBt (121 mg, 0.90 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (446 μL, 2.7 mmol) in an ice-water bath. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 15e-1 and compound 15e-2. These fractions were separately lyophilized to obtain 138 mg of compound 15e-1. LC-MS: [M+H] + = 1249.5; 140 mg of compound 15e-2, LC-MS: [M+H] + =1249.5 was obtained.

[0153] Fifth step: Compound 15A [ka] In a 25 mL single-neck flask, 15e-1 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 59 mg of a solid; LC-MS: [M+H] + =1093.4.

[0154] Sixth Step: Compound 15B [ka] In a 25 mL single-neck flask, 15e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 60 mg of a solid; LC-MS: [M+H] + =1093.4.

[0155] Example 27 Synthesis of compounds 16A and 16B: [ka] Compound 16A (55 mg) was obtained by following the synthesis route of Example 26; LC-MS: [M+H] + =1093.4. [ka] Compound 16B (54 mg) was obtained by following the synthesis route of Example 26; LC-MS: [M+H] + =1093.4.

[0156] Example 28 Synthesis of compounds 17A and 17B: [ka] First step: Compound 17a In a 250 mL single-neck flask, M1 (10 g, 27.1 mmol), 2-hydroxy-benzenepropanoic acid benzyl ester (prepared according to the synthetic route disclosed in Nature Communications, 2020. 11 (1), 56.) (14.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were mixed and heated to 100 °C for 4 hours. After the reaction was determined to be complete, the reaction solution was cooled to room temperature and then filtered to remove insoluble materials. The filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 6.13 g of the desired product in 40% yield; LC-MS: [M+H] + =565.6.

[0157] Second step: Compound 17b In a 50 mL one-neck flask, compound 17a (5 g, 8.86 mmol) was completely dissolved in 15 mL of DMF, and then DBU (1.53 g, 10 mmol) was added in an ice-water bath and reacted for 1 hour to obtain reaction solution 1; In another 50 mL single-neck flask, M4 (3.6 g, 8.86 mmol), PyBOP (5.23 g, 10 mmol), HOBt (1.36 g, 10 mmol), and 10 mL of DMF were mixed and completely dissolved. Next, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath and the reaction was continued for another 30 minutes. Then, reaction solution 1 was added, and the reaction was allowed to stand at room temperature for 2 hours and monitored by HPLC. After the reaction was determined to be complete, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. This fraction was extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated to give 5.0 g of solid product in 77% yield; LC-MS: [M+H] + =738.3.

[0158] Third step: Compound 17d In a 25 mL single-neck flask, 17b (3.0 g, 4.07 mmol) was completely dissolved in 15 mL of DMF, followed by the addition of 3.0 g of 5% Pd / C and hydrogenation for 2 hours. After the reaction was deemed complete, the reaction solution was filtered, and the filtrate was placed in an ice-water bath. DIPEA (744 μL, 4.5 mmol) was added, followed by M3 (2.34 g, 4.07 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring indicated the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. This preparative solution was lyophilized to give 1.2 g of product in 33% yield; LC-MS: [M-H] - =892.4.

[0159] Fourth step: Compound 17e In a 50 mL single-neck flask, 17d (500 mg, 0.56 mmol), M5 (300 mg, 0.56 mmol), PyBOP (438 mg, 0.84 mmol), HOBt (113 mg, 0.84 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (330 μL, 2.0 mmol) in an ice-water bath. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 17e-1 and compound 17e-2. These fractions were separately lyophilized to obtain 156 mg of compound 17e-1. LC-MS: [M+H] + = 1311.4; 150 mg of compound 17e-2, LC-MS: [M+H] + =1311.7 was obtained.

[0160] Step 5: Compound 17A [ka] In a 25 mL single-neck flask, 17e-1 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 43 mg of a solid; LC-MS: [M+H] + =1155.4.

[0161] Step 6: Compound 17B [ka] In a 25 mL single-neck flask, 17e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 40 mg of a solid; LC-MS: [M+H] + =1155.4.

[0162] Example 29 Synthesis of compounds 18A and 18B: [ka] Compound 18A (54 mg) was obtained by following the synthetic route of Example 28; LC-MS: [M+H] + =1155.4. [ka] Compound 18B (55 mg) was obtained by following the synthesis route of Example 28; LC-MS: [M+H] + =1155.4.

[0163] Example 30 Synthesis of compounds 19A and 19B: [ka] First step: Compound 19a In a 250 mL single-neck flask, M1 (10 g, 27.1 mmol), 2-cyclopropyl-2-hydroxyacetic acid benzyl ester (prepared according to the synthetic route disclosed in patent application WO2020244657A1) (11.2 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were mixed and heated to 100 °C for 4 hours. After the reaction was determined to be complete, the reaction solution was cooled to room temperature and then filtered to remove insoluble materials. The filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 4.97 g of the desired product in 36% yield; LC-MS: [M+H] + =515.2.

[0164] Second step: Compound 19b In a 50 mL one-neck flask, compound 19a (4 g, 7.8 mmol) was completely dissolved in 10 mL of DMF, and then DBU (1.42 g, 9.3 mmol) was added in an ice-water bath and reacted for 1 hour to obtain reaction solution 1; In another 50 mL single-neck flask, M4 (3.2 g, 7.8 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol), and 10 mL of DMF were mixed and completely dissolved. Next, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath and the reaction was continued for another 30 minutes. Then, reaction solution 1 was added, and the reaction was allowed to stand at room temperature for 2 hours and monitored by HPLC. After the reaction was determined to be complete, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. This fraction was extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated to give 4.2 g of solid product in 78% yield; LC-MS: [M+H] + =688.3.

[0165] Third step: Compound 19d In a 25 mL single-neck flask, 19b (1000 mg, 1.45 mmol) was completely dissolved in 15 mL of DMF and then hydrogenated with 1000 mg of 5% Pd / C for 2 hours. After the reaction was determined to be complete, the reaction solution was filtered, and the filtrate was placed in an ice-water bath. DIPEA (248 μL, 1.5 mmol) was added, followed by M3 (720 mg, 1.45 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring determined the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. This preparative solution was lyophilized to give 503 mg of product in 41% yield; LC-MS: [M-H] - =842.3.

[0166] Fourth step: Compounds 19e-1 and 19e-2 In a 50 mL single-neck flask, 19d (500 mg, 0.59 mmol), M5 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol), and 10 mL of DMF were mixed, followed by the addition of DIPEA (292 μL, 1.77 mmol) in an ice-water bath. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 19e-1 and compound 19e-2. These fractions were separately lyophilized to obtain 112 mg of compound 19e-1. LC-MS: [M+H] + = 1261.5; 131 mg of compound 19e-2, LC-MS: [M+H] + =1261.5 was obtained.

[0167] Step 5: Compound 19A [ka] In a 25 mL single-neck flask, 19e-1 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 55 mg of a solid; LC-MS: [M+H] + =1105.4.

[0168] Step 6: Compound 19B [ka] In a 25 mL single-neck flask, 19e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 58 mg of a solid; LC-MS: [M+H] + =1105.4.

[0169] Example 31 Synthesis of compounds 20A and 20B: [ka] First step: Compound 20a In a 250 mL single-neck flask, M1 (10 g, 27.1 mmol), 2-hydroxycyclopropylpropionic acid benzyl ester (prepared according to the synthetic route disclosed in patent application WO2020063676A) (12.0 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were mixed and heated to 100 °C for 4 hours. After the reaction was determined to be complete, the reaction solution was cooled to room temperature and then filtered to remove insoluble materials. The filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 5.09 g of the desired product; LC-MS: [M+H] + =529.2.

[0170] Second step: Compound 20b In a 50 mL one-neck flask, compound 20a (4 g, 7.6 mmol) was completely dissolved in 10 mL of DMF, and then DBU (1.39 g, 9.1 mmol) was added in an ice-water bath and reacted for 1 hour to obtain reaction solution 1; In another 50 mL single-neck flask, M4 (3.12 g, 7.6 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol), and 10 mL of DMF were mixed and completely dissolved. Next, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath and the reaction was continued for another 30 minutes. Then, reaction solution 1 was added, and the reaction was allowed to stand at room temperature for 2 hours and monitored by HPLC. After the reaction was determined to be complete, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. This fraction was extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated to give 4.5 g of solid product in 84% yield; LC-MS: [M+H] + =702.3.

[0171] Third step: Compound 20d In a 25 mL single-neck flask, 20b (1000 mg, 1.42 mmol) was completely dissolved in 15 mL of DMF, followed by the addition of 1000 mg of 5% Pd / C and hydrogenation for 2 hours. After the reaction was deemed complete, the reaction solution was filtered, and the filtrate was placed in an ice-water bath. DIPEA (248 μL, 1.5 mmol) was added, followed by M5 (708 mg, 1.42 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring indicated the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. This preparative solution was lyophilized to give 443 mg of product in 36% yield; LC-MS: [M-H] - =856.4.

[0172] Fourth step: Compounds 20e-1 and 20e-2 In a 50 mL single-neck flask, 20d (400 mg, 0.47 mmol), ixotecan mesylate (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF were mixed, followed by the addition of DIPEA (248 μL, 1.5 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 20e-1 and compound 20e-2. These fractions were separately lyophilized to obtain 103 mg of compound 20e-1. LC-MS: [M+H] + = 1275.5; 103 mg of compound 20e-2, LC-MS: [M+H] + =1275.5 was obtained.

[0173] Fifth step: Compound 20A [ka] In a 25 mL single-neck flask, 8A (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 51 mg of a solid; LC-MS: [M+H] + =1119.4.

[0174] Sixth Step: Compound 20B [ka] In a 25 mL single-neck flask, 20e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 47 mg of a solid; LC-MS: [M+H] + =1119.4.

[0175] Example 32 Synthesis of compound 21: [ka] First step: Compound SM3-1 In a 2000 mL single-neck flask, 77087-60-6 (100 g, 458 mmol), maleic acid (53.4 g, 460 mmol), TEA (64 mL, 460 mmol), and 1000 mL of toluene were mixed and heated to 100 °C for 5 hours. After the reaction was determined to be complete, the reaction solution was cooled to room temperature and then filtered to remove insoluble materials. The filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 100:1-50:1-20:1) to obtain 75.6 g of the desired product; LC-MS: [M+H] + =299.1.

[0176] Second step: Compound (R)-2-hydroxy-1,5-glutaric acid tert-butyl ester In a 2000 mL single-neck flask, 172793-31-6 (100 g, 338 mmol) and 1000 mL of water were added, followed by sodium nitrite (35 g, 507 mmol) and concentrated sulfuric acid (32 mL, 35 mmol). The resulting solution was allowed to warm to room temperature and react for 24 hours. After the reaction was deemed complete, the reaction solution was extracted three times with 500 mL of ethyl acetate. The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 50:1-30:1-2:1) to obtain 91.2 g of the desired product; LC-MS: [M+H] + =261.4.

[0177] Third step: Compound SM3 In a 2000 mL single-neck flask, (R)-2-hydroxy-1,5-glutaric acid tert-butyl ester (50 g, 192 mmol) and 1000 mL of anhydrous tetrahydrofuran were mixed and cooled to 0 °C in an ice-water bath. PPh3 (87.7 g, 288 mmol), DEAD (50.2 g, 288 mmol), and SM3-1 (57.3 g, 192 mmol) were then added, and the resulting solution was allowed to react at room temperature for 13 hours. After the reaction was determined to be complete, the reaction solution was filtered to remove insoluble materials. The filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 50:1-30:1-1:1) to obtain 68.6 g of product. The above product was dissolved in 500 mL of methanol and then cooled to 0 °C in an ice-water bath. At this temperature, NaOH (64 mL, 190 mmol, 3 M / L) was added dropwise and reacted for 12 hours, and HCl (6 M / L) was added to adjust the pH to 3. The reaction solution was extracted with 500 mL of dichloromethane three times, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (DCM / MeOH = 50 / 1-20 / 1-2 / 1) to give 50.4 g of SM3; LC-MS: [M-H] - =525.5.

[0178] Fourth step: Compound M6 In a 2000 mL single-neck flask, SM3 (50 g, 95 mmol, 1.0 equiv.), pentafluorophenol (19.2 g, 104.5 mmol, 1.1 equiv.), DCC (21.5 g, 104.5 mmol, 1.1 equiv.), and THF (600 mL) were mixed. The reaction was allowed to stand at room temperature for 1 hour and monitored by TLC. Insoluble materials were filtered off. The reaction solution was directly purified by preparative separation. The preparative solution was concentrated in a vacuum (water pump) water bath at 35 °C to remove acetonitrile, followed by lyophilization to give compound M6 (51.9 g) in 79% yield; LC-MS: [M+H]+ = 693.3.

[0179] Fifth step: Compound 21a In a 25 mL single-neck flask, 1c (1 g, 2.36 mmol) was completely dissolved in 25 mL of DMF, followed by the addition of DIPEA (430 μL, 2.6 mmol) and M6 (1177 mg, 2.36 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was lyophilized to give 555 mg of product; LC-MS: [M H] - =931.0.

[0180] Step 6: Compound 21b In a 100 mL single-neck flask, 21a (500 mg, 0.54 mmol), ixotecan mesylate M5 (285 mg, 0.54 mmol), PyBOP (239 mg, 0.6 mmol), HOBt (239 mg, 0.6 mmol), and 10 mL of DMF were mixed, followed by the addition of DIPEA (248 μL, 1.5 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 21b. This preparative solution was lyophilized to obtain 231 mg of compound; LC-MS: [M+H] + =1349.5.

[0181] Seventh step: Compound 21 In a 25 mL single-neck flask, compound 21b (200 mg, 0.1488 mmol), zinc bromide (665 mg, 2.96 mmol), and 10 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 103 mg of a solid; LC-MS: [M+H] + =1137.5.

[0182] Example 33 Synthesis of compound 22: [ka] Using compounds M6 and 3c as starting materials, compound 22 (91 mg) was obtained according to the synthesis route of Example 32; LC-MS: [M+H] + =1165.5.

[0183] Example 34 Synthesis of compounds 23 and 24: [ka] Starting from compounds M6 and 5c, and referring to the synthesis route of Example 32, 102 mg of compound 23 was obtained. LC-MS: [M+H] + = 1151.4; 99 mg of compound 24, LC-MS: [M+H] + =1151.4 was obtained.

[0184] Example 35 Synthesis of compounds 25 and 26: [ka] Starting from compounds M6 and 7c, and referring to the synthesis route of Example 32, 83 mg of compound 25 was obtained. LC-MS: [M+H] + = 1205.7; 80 mg of compound 26, LC-MS: [M+H] + =1205.7 was obtained.

[0185] Example 36 Synthesis of compounds 27 and 28: [ka] Starting from compounds M6 and 19c, and referring to the synthesis route of Example 32, 100 mg of compound 27 was obtained. LC-MS: [M+H] + = 1177.5; 101 mg of compound 28, LC-MS: [M+H] + =1177.5 was obtained.

[0186] Example 37 Synthesis of compound 29: [ka] First step: Compound SM4-1 In a 5000 mL single-neck flask, maleic acid (50 g, 431 mmol, 1.0 equiv.), 114559-25-0 (110 g, 431 mmol, 1 equiv.), TEA (263 g, 2.16 mol, 5 equiv.), and toluene (2000 mL) were mixed and heated to reflux for 5 h. The mixture was monitored by TLC. Insoluble materials were filtered off. The reaction solution was directly subjected to rotary evaporation under reduced pressure to remove the solvent. The residue was subjected to silica gel column chromatography (PE / EA = 50 / 1-20 / 1-1 / 1) to obtain SM4-1 (64.7 g) in 50% yield; LC-MS: [M+H] + =299.2.

[0187] Second step: Compound SM4-2 In a 2000 mL single-neck flask, SM4-1 (64 g, 215 mmol) was completely dissolved in 1000 mL of DMF, and then DIPEA (71 mL, 430 mmol) was added, followed by nonane ethylene glycol monomethyl ether mesylate (111.5 g, 220 mmol). The resulting solution was allowed to react at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by silica gel column chromatography (PE / EA = 50 / 1-20 / 1-1 / 1) to give 59.9 g of product; LC-MS: [M+H] + =709.4.

[0188] Third step: Compound SM4 In a 2000 mL single-neck flask, SM4-2 (59 g, 83 mmol) was completely dissolved in 1000 mL of MeOH, and K2CO3 (11.75 g, 85 mmol) was added. The reaction was left at room temperature for 4 hours and monitored by HPLC. After the reaction was determined to be complete, the insoluble material was filtered off. The reaction solution was directly purified by preparative separation. The preparative solution was concentrated in a vacuum (water pump) water bath at 35 °C to remove acetonitrile, and then lyophilized to obtain compound SM4 (27 g); LC-MS: [MH]- =693.5.

[0189] Fourth step: Compound M7 In a 500 mL single-neck flask, SM4 (25 g, 36 mmol, 1.0 equiv.), pentafluorophenol (7.3 g, 40 mmol, 1.1 equiv.), DCC (8.2 g, 40 mmol, 1.1 equiv.), and THF (200 mL) were mixed, and the reaction was allowed to stand at room temperature for 1 hour and monitored by TLC. Insoluble materials were filtered off. The reaction solution was directly purified by preparative separation. This preparative solution was concentrated in a vacuum (water pump) water bath at 35 °C to remove acetonitrile, and then lyophilized to give compound M7 (23.3 g) in 93% yield; LC-MS: [M+H] + =695.8.

[0190] Fifth step: Compound 29a In a 25 mL single-neck flask, 1c (1 g, 2.36 mmol) was completely dissolved in 25 mL of DMF, followed by the addition of DIPEA (430 μL, 2.6 mmol) and then M7 (1640 mg, 2.36 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 609 mg of product. LC-MS: [M-H] - =1098.5.

[0191] Step 6: Compound 29b In a 100 mL single-neck flask, 29a (500 mg, 0.45 mmol), ixotecan mesylate M5 (240 mg, 0.45 mmol), PyBOP (215 mg, 0.54 mmol), HOBt (215 mg, 0.54 mmol), and 10 mL of DMF were mixed, followed by the addition of DIPEA (248 μL, 1.5 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 29b. This preparative solution was lyophilized to give 187 mg of compound; LC-MS: [M+H] + =1517.6.

[0192] Seventh step: Compound 29 In a 25 mL single-neck flask, compound 29b (150 mg, 0.988 mmol), zinc bromide (223 mg, 0.988 mmol), and 10 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 114 mg of a solid; LC-MS: [M+H] + =1517.9.

[0193] Example 38 Synthesis of compound 30: [ka] Using compounds M7 and 3c as starting materials, compound 30 (125 mg) was obtained according to the synthesis route of Example 37; LC-MS: [M+H] + =1445.6.

[0194] Example 39 Synthesis of compounds 31 and 32: [ka] Starting from compounds M7 and 5c, and referring to the synthesis route of Example 37, 61 mg of compound 31 was obtained. LC-MS: [M+H] + = 1431.7; 63 mg of compound 32, LC-MS: [M+H] + =1431.7 was obtained.

[0195] Example 40 Synthesis of compounds 33 and 34: [ka]

[0196] Starting from compounds M7 and 7c, and referring to the synthesis route of Example 37, 60 mg of compound 33 was obtained. LC-MS: [M+H] + = 1485.6; 58 mg of compound 34, LC-MS: [M+H] + =1485.6 was obtained.

[0197] Example 41 Synthesis of compounds 35 and 36: [ka] Starting from compounds M7 and 19c, and referring to the synthesis route of Example 37, 102 mg of compound 35 was obtained. LC-MS: [M+H] + = 1457.8; 102 mg of compound 36, LC-MS: [M+H] + =1457.8 was obtained.

[0198] Example 42 Synthesis of compound 37: [ka] First step: Compound SM5-1 In a 2000 mL single-neck flask, compound 16947-84-5 (100 g, 295 mmol, 1.0 equiv.), DIPEA (50 mL, 300 mmol), benzyl bromide (51.3 g, 300 mmol), and THF (1000 mL) were mixed. The reaction was allowed to stand at room temperature for 12 hours and monitored by TLC. Insoluble materials were filtered out. The reaction solution was directly subjected to rotary evaporation under reduced pressure to remove the solvent, and the residue was subjected to silica gel column chromatography (PE / EA = 50 / 1-20 / 1-2 / 1) to obtain SM5-1 (110.1 g) in 87% yield; LC-MS: [M+H] + =429.2.

[0199] Second step: Compound SM5-2 In a 2000 mL single-neck flask, compound SM5-1 (100 g, 233.4 mmol, 1.0 equiv.) and THF (1000 mL) were mixed and cooled to 0 °C in an ice-water bath. NaH (37.4 g, 933.5 mmol) and MeI (132.5 g, 933.5 mmol) were then added portionwise. The reaction was allowed to stand at 0 °C for 24 hours and monitored by TLC. 500 mL of saturated NH4Cl in water was added to quench the reaction. The reaction solution was extracted three times with 500 mL of ethyl acetate. The organic phase was dried over anhydrous Na2SO4 and filtered. The filtrate was directly rotary evaporated under reduced pressure to remove the solvent. The residue was subjected to silica gel column chromatography (PE / EA = 100 / 1-50 / 1-10 / 1) to give SM5-2 (37.1 g); LC-MS: [M+H] + =443.3.

[0200] Third step: Compound SM5 (see Org. Lett., 2006, 8, 3387-3390) In a 1000 mL single-neck flask, compound SM5-2 (35 g, 79 mmol, 1.0 equiv.) and DCE (500 mL) were mixed, followed by the sequential addition of palladium diacetate (180 mg, 0.8 mmol), I2 (20 g, 79 mmol), and iodobenzene diacetate (40.8 g, 126.4 mmol). The reaction was allowed to stand at 60 °C for 40 h and monitored by TLC. The reaction was quenched by adding 500 mL of saturated aqueous sodium thiosulfate solution in water. The reaction solution was extracted three times with 500 mL of dichloromethane. The organic phase was dried over anhydrous Na2SO4 and filtered. The filtrate was directly rotary evaporated under reduced pressure to remove the solvent, and the residue was subjected to silica gel column chromatography (PE / EA = 100 / 1-50 / 1-10 / 1) to obtain SM5 (28 g); LC-MS: [M+H] + =501.3.

[0201] Fourth step: Compound SM6 In a 500 mL single-neck flask, compound SM5 (25 g, 50 mmol, 1.0 equiv.), di-tert-butyl phosphate potassium salt (13.66 g, 55 mmol, 1.1 equiv.), p-toluenesulfonic acid monohydrate (951 mg, 5 mmol, 0.1 equiv.), and THF (200 mL) were mixed. The reaction was allowed to stand at room temperature for 1 hour and monitored by TLC. Insoluble materials were filtered off. The reaction solution was directly purified by preparative separation. This preparative solution was concentrated in a vacuum (water pump) water bath at 35°C to remove acetonitrile, and then lyophilized to give compound SM6 (15.1 g) in 46% yield; LC-MS: [M+H] + =651.4.

[0202] 5th step: Compound SM7 In a 250 mL single-neck flask, SM6 (15 g, 23 mmol) was completely dissolved in 100 mL of DMF, and then 15 g of 5% Pd / C was added in a water bath. The atmosphere of the system was replaced with hydrogen three times. The reaction was left at room temperature for 12 hours. The Pd / C was removed, and the solvent was removed by rotary evaporation under reduced pressure (oil pump). The resulting crude product was ready for further use. In another 250 mL single-neck flask, the crude product and 100 mL of toluene, triethylamine (6.4 mL, 46 mmol), and maleic anhydride (2.4 g, 24 mmol) were mixed and dissolved completely. The reaction was allowed to stand at 100 °C for 2 hours and monitored by HPLC. After the reaction was determined to be complete, the reaction solution was purified by liquid chromatography to obtain a fractional solution. This fractional solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated to give 4.2 g of solid product in 36% yield; LCMS: [M+H] + =507.3.

[0203] Sixth step: Compound M8 In a 100 mL single-neck flask, compound SM7 (4 g, 7.9 mmol, 1.0 equiv.), pentafluorophenol (1.6 g, 8.7 mmol, 1.1 equiv.), DCC (1.8 g, 8.7 mmol, 1.1 equiv.), and THF (60 mL) were mixed, and the reaction was allowed to stand at room temperature for 1 hour and monitored by TLC. Insoluble materials were filtered off. The reaction solution was directly purified by preparative separation. This preparative solution was concentrated in a vacuum (water pump) water bath at 35°C to remove acetonitrile, and then lyophilized to give compound M8 (3.7 g) in 70% yield; LC-MS: [M+H] + =673.2.

[0204] Seventh step: Compound 37a In a 25 mL single-neck flask, compound 1c (1 g, 2.36 mmol) was completely dissolved in 25 mL of DMF, followed by the addition of DIPEA (430 μL, 2.6 mmol) and then compound M8 (1.2 g, 2.36 mmol). The reaction was allowed to stand at room temperature for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was lyophilized to obtain 488 mg of product; LC-MS: [M-H] - =911.0.

[0205] Step 8: Compound 37b In a 100 mL single-neck flask, compound 37a (400 mg, 0.44 mmol), ixotecan mesylate M5 (235 mg, 0.44 mmol), PyBOP (199 mg, 0.5 mmol), HOBt (69 mg, 0.5 mmol), and 10 mL of DMF were mixed, followed by the addition of DIPEA (218 μL, 1.32 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 37b. This preparative solution was lyophilized to obtain 201 mg of compound; LC-MS: [M+H] + =1329.6.

[0206] Step 9: Compound 37 In a 25 mL single-neck flask, compound 37b (130 mg, 0.098 mmol), zinc bromide (221 mg, 0.98 mmol), and 10 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 96 mg of a solid; LC-MS: [M+H] + =1117.4.

[0207] Example 43 Synthesis of compound 38: [ka] Using compounds M8 and 3c as starting materials, compound 38 (51 mg) was obtained according to the synthesis route of Example 42; LC-MS: [M+H] + =1145.6.

[0208] Example 44 Synthesis of compounds 39 and 40: [ka] Starting from compounds M8 and 5c, and referring to the synthesis route of Example 42, 57 mg of compound 39 was obtained. LC-MS: [M+H] + = 1131.4; 60 mg of compound 40, LC-MS: [M+H] + =1131.4 was obtained.

[0209] Example 45 Synthesis of compounds 41 and 42: [ka] Starting from compounds M7 and 7c, and referring to the synthesis route of Example 42, 44 mg of compound 41 was obtained. LC-MS: [M+H] + = 1185.3; 44 mg of compound 42, LC-MS: [M+H] + =1185.3 was obtained.

[0210] Example 46 Synthesis of compounds 43 and 44: [ka] Starting from compounds M8 and 19c, and referring to the synthesis route of Example 42, 62 mg of compound 43 was obtained. LC-MS: [M+H] + = 1157.4; 59 mg of compound 44, LC-MS: [M+H] + =1157.4 was obtained.

[0211] Example 47 (Comparative Example) Synthesis of compound 45: [ka] Compound 45 was obtained by referring to the synthetic route of Example 58 of patent application CN104755494A.

[0212] Example 48 Synthesis of compound 46: [ka] First step: Compound 46a In a 50 mL single-neck flask, 1d (500 mg, 0.62 mmol), M9 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (378 μL, 2.29 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 46a (210 mg); LC-MS: [M+H] + =1221.6.

[0213] Second step: Compound 46 In a 25 mL single-neck flask, 46a (200 mg, 0.162 mmol), zinc bromide (736 mg, 3.26 mmol), and 10 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 46 (120 mg); LC-MS: [M+H] + =1065.3.

[0214] Example 49 Synthesis of compound 47: [ka] Compound 47 (81 mg) was obtained by following the synthetic route of Example 48; LC-MS: [M+H] + =1065.3.

[0215] Example 50 Synthesis of compound 48A: [ka] First step: Compound 48a In a 100 mL single-neck flask, 5d (1.66 g, 2.02 mmol, 1.0 equiv.), M9 (1.08 g, 2.02 mmol, 1.0 equiv.), PyBOP (1.58 g, 3.03 mmol, 1.5 equiv.), HOBt (0.41 g, 3.03 mmol, 1.5 equiv.), and DMF (40 mL) were mixed. DIPEA (0.84 mL, 1.5 equiv.) was then added in an ice-water bath. The reaction was allowed to stand at room temperature for 2 h and monitored by HPLC. The reaction solution was directly purified by preparative separation. This solution was concentrated in a vacuum (water pump) water bath at 35 °C to remove acetonitrile, followed by lyophilization to give compound 48a (1.54 g) in 61% yield; LC-MS: [M+H] + =1235.4.

[0216] Step 6: Compound 48A In a 100 mL single-neck flask, compound 48a (1.0 g, 0.8 mmol, 1.0 equiv.) and 35 mL of nitromethane were mixed and dissolved, followed by the addition of zinc bromide (3.64 g, 16 mmol, 20.0 equiv.). The reaction was allowed to stand in an oil bath at 40 °C for 30 minutes (the reaction system was preheated and stabilized). The reaction solution was concentrated in a vacuum (water pump) water bath at 45 °C to remove nitromethane, leaving a yellow solid residue (under HPLC monitoring). A fraction was obtained by acid fractionation. This fraction was concentrated in a vacuum (water pump) water bath at 35 °C to remove acetonitrile, followed by lyophilization to give compound 48A (786 mg) in 90% yield.

[0217] Example 51 Synthesis of compound 48B: [ka] Second step: Compound 48b In a 25 mL single-neck flask, compound 5d-1 (200 mg, 0.24 mmol, 1.0 equiv.), M9 (127 mg, 0.24 mmol, 1.0 equiv.), PyBOP (187 mg, 0.36 mmol, 1.2 equiv.), HOBt (48 mg, 0.36 mmol, 1.2 equiv.), and DMF (6 mL) were mixed and cooled to 0-5 °C in an ice-water bath. DIPEA (62 mg, 0.48 mmol, 2.0 equiv.) was then added. The reaction was allowed to stand at 20 ± 5 °C for 2 h. After HPLC monitoring showed the reaction was complete, the reaction solution was directly purified by preparative HPLC. The product aliquot was collected and then lyophilized to give compound 48b (150.2 mg); LC-MS: [M+H] + =1235.4.

[0218] Second step: Compound 48B In a 25 mL single-neck flask, compound 48b (100 mg, 0.081 mmol, 1.0 equiv.), ZnBr (364 mg, 1.62 mmol, 20.0 equiv.), and CHNO (10 mL) were added sequentially. The reaction was allowed to stand at 40 °C for 0.5 h and then terminated. Rotary evaporation under reduced pressure at 45 °C gave a yellow solid, which was then sampled and monitored by HPLC. The rotary evaporated solid was directly purified by preparative HPLC. The product aliquot was collected and then lyophilized to give compound 48B (70.0 mg); LC-MS: [M+H] + =1079.4.

[0219] Example 52 Synthesis of compound 49A: [ka] Following the route of Example 50, compound 49A (71 mg) was obtained; LC-MS: [M+H] + =1079.4.

[0220] Example 53 Synthesis of compound 49B [ka] Compound 49B (65 mg) was obtained by following the synthesis route of Example 51; LC-MS: [M+H] + =1079.4.

[0221] Example 54 Synthesis of compounds 50A and 50B: [ka] First step: Compounds 50a and 50b In a 50 mL single-neck flask, 7d (500 mg, 0.57 mmol), M9 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (378 μL, 2.29 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 50a and compound 50b. These fractions were separately lyophilized to obtain 170 mg of compound 50a. LC-MS: [M+H] + = 1289.46; 202 mg of compound 50b, LC-MS: [M+H] + =1289.46 was obtained.

[0222] Second step: Compound 50A [ka] In a 25 mL single-neck flask, 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. This fraction was lyophilized to obtain 44 mg of a solid.

[0223] Third step: Compound 50B [ka] In a 25 mL single-neck flask, 50b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. This fraction was lyophilized to obtain 45 mg of a solid.

[0224] Example 55 Synthesis of compounds 51A and 51B: [ka] First step: Compounds 51a and 51b In a 50 mL single-neck flask, 8d (500 mg, 0.57 mmol), M9 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (378 μL, 2.29 mmol) in an ice-water bath. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of Compound 51a and Compound 51b. These fractions were separately lyophilized to obtain 190 mg of Compound 51a and 186 mg of Compound 51b. LC-MS of Compound 51a: [M+H] + = 1289.47; LC-MS of compound 51b: [M+H] + =1289.47.

[0225] Second step: Compound 51A [ka] In a 25 mL single-neck flask, compound 51a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40° C. for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. This fraction was freeze-dried to obtain 39 mg of a solid.

[0226] Third step: Compound 51B [ka] In a 25 mL single-neck flask, compound 51b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. This fraction was lyophilized to obtain 60 mg of a solid.

[0227] Example 56 Synthesis of compound 52A: [ka] First step: Compound 52a In a 50 mL single-neck flask, 11d (800 mg, 0.96 mmol), M9 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol), and 30 mL of DMF were mixed, followed by the addition of DIPEA (660 μL, 4.0 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 4 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 52a. This preparative solution was lyophilized to obtain 52a (388 mg); LC-MS: [M+H] + =1261.4.

[0228] Second step: Compound 52A In a 25 mL single-neck flask, compound 52a (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol), and 10 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 52A (79 mg); LC-MS: [M+H] + =1105.4.

[0229] Example 57 Synthesis of compound 52B: [ka] Compound 52B (50 mg) was obtained by following the synthetic route of Example 56. LC-MS: [M+H] + 1105.4.

[0230] Example 58 Synthesis of compound 53A: [ka] First step: Compound 53a In a 50 mL single-neck flask, 12d (400 mg, 0.47 mmol), M9 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (330 μL, 2.0 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 3 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 53a. The preparative solution was lyophilized to obtain 53a (200 mg); LC-MS: [M+H] + =1275.4.

[0231] Second step: Compound 53A In a 25 mL single-neck flask, 53a (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 53A (51 mg); LC-MS: [M+H] + =1119.4.

[0232] Example 59 Synthesis of compound 53B: [ka] Compound 53B (50 mg) was obtained by following the synthetic route of Example 58; LC-MS: [M+H] + =1119.4.

[0233] Example 60 Synthesis of compounds 54A and 54B: [ka] First step: Compounds 54a and 54b In a 50 mL single-neck flask, 19d (500 mg, 0.59 mmol), M9 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol), and 10 mL of DMF were mixed, followed by the addition of DIPEA (292 μL, 1.77 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compounds 54a and 54b. These fractions were separately lyophilized to obtain 103 mg of compound 54a; LC-MS: [M+H]. + = 1261.5; 111 mg of compound 54b, LC-MS: [M+H] + =1261.5 was obtained.

[0234] Second step: Compound 54A [ka] In a 25 mL single-neck flask, 54a (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 61 mg of a solid; LC-MS: [M+H] + =1105.4.

[0235] Third step: Compound 54B [ka] In a 25 mL single-neck flask, 54b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 57 mg of a solid; LC-MS: [M+H] + =1105.4.

[0236] Example 61 Synthesis of compounds 55A and 55B: [ka] First step: Compounds 55a and 55b In a 50 mL single-neck flask, 20d (400 mg, 0.47 mmol), M9 (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF were mixed, followed by the addition of DIPEA (248 μL, 1.5 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compounds 55a and 55b. These fractions were separately lyophilized to obtain 100 mg of compound 55a. LC-MS: [M+H] + = 1275.5; 101 mg of compound 55b, LC-MS: [M+H] + =1275.5 was obtained.

[0237] Second step: Compound 55A [ka] In a 25 mL single-neck flask, 55a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 42 mg of a solid; LC-MS: [M+H] + =1119.4.

[0238] Third step: Compound 55B [ka] In a 25 mL single-neck flask, 55b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 45 mg of a solid; LC-MS: [M+H] + =1119.4.

[0239] Example 62 Synthesis of compound 56: [ka] Compound 56 (50 mg) was obtained by following the synthesis route of Example 60; LC-MS: [M+H] + =1119.3.

[0240] Example 63 Synthesis of compound 57: [ka] Compound 57 (50 mg) was obtained by following the synthetic route of Example 61; LC-MS: [M+H] + =1119.4.

[0241] Example 64 Synthesis of compound 58: [ka] First step: Synthesis of compound 58a 400 mL of DMF was added to ixotecan mesylate M5 (15 g, 28 mol, obtained by referring to the synthetic route disclosed in patent application EP0737683A1). The resulting solution was then cooled to 0 °C in an ice-water bath, and triethylamine was added dropwise to adjust the pH to 7-8. Benzyl bromide (9.6 g, 56 mmol) was then added dropwise in an ice-water bath, and the reaction was allowed to stand at room temperature (25 °C) for 1 h. After TLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure. The resulting crude product was purified by preparative-grade high-performance liquid chromatography (acetonitrile / pure water system), and the target peak was collected. The acetonitrile was removed under reduced pressure, followed by lyophilization to obtain approximately 11 g of yellow solid compound 58a in 74% yield: MS m / z: [M+H] + 526.3.

[0242] Second step: Synthesis of compound 58b In a 250 mL single-neck flask, compound 58a (11 g, 21 mol) and 120 mL of formic acid were added sequentially and dissolved at room temperature. To the resulting bright yellow solution, 30 mL of formaldehyde (40% in water) was added. The reaction was allowed to stand at 50 °C for 1 hour. After TLC monitoring showed the reaction was complete, the reaction solution was cooled to room temperature and then purified by preparative-grade high-performance liquid chromatography (acetonitrile / pure water system), and the target peak was collected. The acetonitrile was removed under reduced pressure, followed by lyophilization to give approximately 4.5 g of compound 58b as a yellow solid powder in 40% yield; MS m / z: [M+H] + 540.6.

[0243] Third step: Synthesis of compound 58: In a 250 mL single-neck flask, compound 58b (2.3 g, 4.3 mol) was added to 100 mL of DMF and dissolved at room temperature. 2.3 g of 5% Pd / C was added to the resulting bright yellow solution, and the atmosphere in the system was replaced with a hydrogen balloon. The reaction was allowed to stand at room temperature for 1.5 hours. After HPLC monitoring showed the reaction was complete, the Pd / C was filtered off, and the resulting reaction solution was concentrated and then purified by preparative-grade high-performance liquid chromatography (acetonitrile / pure water system) to collect the target peak. The acetonitrile was removed under reduced pressure, followed by lyophilization to obtain approximately 1.0 g of compound 58 as a yellow solid powder in 52% yield; MS m / z: [M+H] + 450.5.

[0244] Example 65 Synthesis of compound 59: [ka] First step: Compound 59a In a 50 mL single-neck flask, 1d (500 mg, 0.62 mmol), 58 (279 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (378 μL, 2.29 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 h. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 59a (166 mg); LC-MS: [M+H] + =1235.6.

[0245] Second step: Compound 59 In a 25 mL single-neck flask, 59a (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol), and 10 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 59 (43 mg); LC-MS: [M+H] + =1079.3.

[0246] Example 66 Synthesis of compound 60: [ka] Compound 60 (40 mg) was obtained by following the synthetic route of Example 65; LC-MS: [M+H] + =1079.3.

[0247] Example 67 Synthesis of compound 61: [ka] First step: Compound 61a In a 100 mL single-neck flask, 5d (1.66 g, 2.02 mmol, 1.0 equiv.), 58 (0.91 g, 2.02 mmol, 1.0 equiv.), PyBOP (1.58 g, 3.03 mmol, 1.5 equiv.), HOBt (0.41 g, 3.03 mmol, 1.5 equiv.), and DMF (40 mL) were mixed, followed by the addition of DIPEA (0.84 mL, 1.5 equiv.) in an ice-water bath. The reaction was allowed to stand at room temperature for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative separation. The preparative solution was concentrated in a vacuum (water pump) water bath at 35°C to remove acetonitrile, and then lyophilized to give Compound 61a (1.21 g); LC-MS: [M+H] + =1249.4.

[0248] Second step: Compound 61 In a 100 mL single-neck flask, compound 61a (1.0 g, 0.8 mmol, 1.0 equiv.) and 35 mL of nitromethane were mixed and dissolved, and then zinc bromide (3.64 g, 16 mmol, 20.0 equiv.) was added. The reaction was allowed to stand in an oil bath at 40 °C for 30 minutes (the reaction system was preheated and stabilized). The reaction solution was concentrated in a vacuum (water pump) water bath at 45 °C to remove nitromethane, yielding a yellow solid residue (under HPLC monitoring). Acid fractionation afforded a fraction. This fraction was concentrated in a vacuum (water pump) water bath at 35 °C to remove acetonitrile, followed by lyophilization to give compound 61 (786 mg); LC-MS: [M+H] + =1093.6.

[0249] Example 68 Synthesis of compound 62: [ka] First step: Compound 62a In a 25 mL single-neck flask, compound 5d-1 (200 mg, 0.24 mmol, 1.0 equiv.), 58 (110.3 mg, 0.24 mmol, 1.0 equiv.), PyBOP (187 mg, 0.36 mmol, 1.2 equiv.), HOBt (48 mg, 0.36 mmol, 1.2 equiv.), and DMF (6 mL) were mixed and cooled to 0-5 °C in an ice-water bath. DIPEA (62 mg, 0.48 mmol, 2.0 equiv.) was then added. The reaction was allowed to stand at 20 ± 5 °C for 2 h. After HPLC monitoring showed the reaction was complete, the reaction solution was directly purified by preparative HPLC. The product aliquot was collected and then lyophilized to give compound 62a (120.9 mg); LC-MS: [M+H] + =1249.4.

[0250] Second step: Compound 62 In a 25 mL single-neck flask, compound 62a (100 mg, 0.081 mmol, 1.0 equiv.), ZnBr (364 mg, 1.62 mmol, 20.0 equiv.), and CHNO (10 mL) were added sequentially. The reaction was allowed to stand at 40 °C for 0.5 h and then terminated. Rotary evaporation under reduced pressure at 45 °C gave a yellow solid, which was then sampled and monitored by HPLC. The rotary evaporated solid was directly purified by preparative HPLC. The product aliquot was collected and then lyophilized to give compound 62 (61 mg); LC-MS: [M+H] + =1093.4.

[0251] Example 69 Synthesis of compound 63: [ka] Following the route of Example 67, compound 63 (60 mg) was obtained; LC-MS: [M+H] + =1093.4.

[0252] Example 70 Synthesis of compound 64: [ka] Compound 64 (65 mg) was obtained by following the synthetic route of Example 68; LC-MS: [M+H] + =1093.4.

[0253] Example 71 Synthesis of compounds 65A and 65B: [ka] First step: Compounds 65a and 65b In a 50 mL single-neck flask, 7d (500 mg, 0.57 mmol), 58 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (378 μL, 2.29 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 65a and compound 65b. These fractions were separately lyophilized to obtain 155 mg of compound 65a. LC-MS: [M+H] + = 1303.4; 158 mg of compound 65b, LC-MS: [M+H] + =1303.6 was obtained.

[0254] Second step: Compound 65A [ka] In a 25 mL single-neck flask, 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 49 mg of a solid.

[0255] Third step: Compound 65B [ka] In a 25 mL single-neck flask, 65b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 47 mg of a solid.

[0256] Example 72 Synthesis of compounds 66A and 66B: [ka] First step: Compounds 66a and 66b In a 50 mL single-neck flask, 8d (500 mg, 0.57 mmol), 58 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (378 μL, 2.29 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to give fractions of compound 66a and compound 66b. These fractions were separately lyophilized to give 160 mg of compound 66a and 160 mg of compound 66b. LC-MS of compound 66a: [M+H] + = 1303.7; LC-MS of compound 66b: [M+H] + =1303.6.

[0257] Second step: Compound 66A [ka] In a 25 mL single-neck flask, compound 66a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 57 mg of a solid; LC-MS: [M+H] + =1147.5.

[0258] Third step: Compound 66B [ka] In a 25 mL single-neck flask, compound 66b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 hour. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 57 mg of a solid; LC-MS: [M+H] + =1147.5.

[0259] Example 73 Synthesis of compound 67A: [ka] First step: Compound 67a In a 50 mL single-neck flask, 11d (800 mg, 0.96 mmol), 58 (432.5 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol), and 30 mL of DMF were mixed, followed by the addition of DIPEA (660 μL, 4.0 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 4 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 67a. The preparative solution was lyophilized to obtain 67a (402 mg); LC-MS: [M+H] + =1275.4.

[0260] Second step: Compound 67A In a 25 mL single-neck flask, 67a (100 mg, 0.78 mmol), zinc bromide (356 mg, 1.57 mmol), and 10 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 67A (47 mg); LC-MS: [M+H] + =1119.5.

[0261] Example 74 Synthesis of compound 67B: [ka] Compound 67B (50 mg) was obtained by following the synthetic route of Example 73; LC-MS: [M+H] + 1119.4.

[0262] Example 75 Synthesis of compound 68A: [ka] First step: Compound 68a In a 50 mL single-neck flask, compound 12d (400 mg, 0.47 mmol), 58 (211.7 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (330 μL, 2.0 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 3 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 68a. This preparative solution was lyophilized to obtain 68a (177 mg); LC-MS: [M+H] + =1289.4.

[0263] Second step: Compound 68A In a 25 mL single-neck flask, 68a (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 68A (45 mg); LC-MS: [M+H] + =1133.4.

[0264] Example 76 Synthesis of compound 68B: [ka] Compound 68B (50 mg) was obtained by following the synthetic route of Example 75; LC-MS: [M+H] + =1133.4.

[0265] Example 77 Synthesis of compounds 69A and 68B: [ka] First step: Compounds 69a and 69b In a 50 mL single-neck flask, 19d (500 mg, 0.59 mmol), 58 (266 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol), and 10 mL of DMF were mixed, followed by the addition of DIPEA (292 μL, 1.77 mmol) in an ice-water bath. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 69a and compound 69b. These fractions were separately lyophilized to obtain 109 mg of compound 69a. LC-MS: [M+H] + = 1275.5; 111 mg of compound 69b, LC-MS: [M+H] + =1275.7 was obtained.

[0266] Second step: Compound 69A [ka] In a 25 mL single-neck flask, 69a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 53 mg of a solid; LC-MS: [M+H] + =1119.4.

[0267] Third step: Compound 69B [ka] In a 25 mL single-neck flask, 69b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 54 mg of a solid; LC-MS: [M+H] + =1119.4.

[0268] Example 78 Synthesis of compounds 70A and 70B: [ka] First step: Compounds 70a and 70b In a 50 mL single-neck flask, 20d (400 mg, 0.47 mmol), 58 (211.7 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF were mixed, followed by the addition of DIPEA (248 μL, 1.5 mmol) in an ice-water bath. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compounds 70a and 70b. These fractions were lyophilized to obtain 106 mg of compound 70a. LC-MS: [M+H] + = 1289.5, and 101 mg of compound 70b, LC-MS: [M+H] + =1289.4 was obtained.

[0269] Second step: Compound 70A [ka] In a 25 mL single-neck flask, 70a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 39 mg of a solid; LC-MS: [M+H] + =1133.4.

[0270] Third step: Compound 70B [ka] In a 25 mL single-neck flask, 70b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol), and 5 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain 35 mg of a solid; LC-MS: [M+H] + =1133.4.

[0271] Example 79 Synthesis of compound 71: [ka] Compound 71 (30 mg) was obtained by following the synthetic route of Example 78; LC-MS: [M+H] + =1133.3.

[0272] Example 80 Synthesis of compound 72: [ka] Compound 72 (33 mg) was obtained by following the synthetic route of Example 78; LC-MS: [M+H] + =1133.4.

[0273] Example 81 [ka] Synthesis of compound M11: In a 100 mL single-neck flask, compound M3 (11.0 g, 19.5 mmol, 1.0 equiv.), DIPEA (2.8 g, 21.4 mmol, 1.1 equiv.), 27-amino-4,7,10,13,16,19,22,25-octaoxaheptacosanoic acid (9.7 g, 20.5 mmol, 1.05 equiv.), and DMF (60 mL) were mixed. The reaction was allowed to stand at room temperature for 20 minutes and monitored by TLC. The reaction solution was directly purified by preparative separation. This solution was concentrated in a vacuum (water pump) water bath at 35 °C to remove acetonitrile, and then lyophilized to give compound M10 (13.2 g) in 78% yield; LC-MS: [M+H] + =866.5. In a 100 mL single-neck flask, M10 (13.0 g, 15 mmol, 1.0 equiv.), pentafluorophenol (3 g, 16.5 mmol, 1.1 equiv.), DCC (3.4 g, 16.5 mmol, 1.1 equiv.), and THF (30 mL) were mixed, and the reaction was allowed to stand at room temperature for 30 minutes and monitored by TLC. Insoluble materials were filtered off. The reaction solution was directly purified by preparative separation. This preparative solution was concentrated in a vacuum (water pump) water bath at 35°C to remove acetonitrile, and then lyophilized to give compound M11 (14.2 g) in 92% yield; LC-MS: [M+H] + =1032.5.

[0274] Example 82 Synthesis of compound 73: [ka] First step: Synthesis of compound 73a 10 mL of DMF was added to M11 (1 g, 0.79 mol). The resulting solution was then cooled to 0 °C in an ice-water bath, after which compound 1c (334 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added. The reaction was allowed to stand under the same conditions for 1 hour. After TLC monitoring showed the reaction was complete, the reaction solution was purified by preparative-grade high-performance liquid chromatography (acetonitrile / pure water system) and the target peak was collected. The acetonitrile was removed under reduced pressure, followed by lyophilization to give approximately 1.2 g of compound 73a; MS m / z: [M+H] + =1271.9.

[0275] Second step: Synthesis of compound 73b In a 25 mL single-neck flask, 73a (1.2 g, 0.94 mmol), M5 (500 mg, 0.94 mmol), PyBOP (625 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol), and 15 mL of DMF were mixed, followed by the addition of DIPEA (310 mg, 2.4 mmol) in an ice-water bath. The reaction was allowed to stand at room temperature for 2 h. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 73b (709 mg); LC-MS: [M+H] + =1720.8.

[0276] Third step: Synthesis of compound 73: In a 25 mL single-neck flask, 73b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol), and 10 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 73 (88 mg); LC-MS: [M+H] + =1532.6.

[0277] Example 83 Synthesis of compound 74: [ka] Compound 74 (90 mg) was obtained by following the synthetic route of Example 82; LC-MS: [M+H] + =1532.6.

[0278] Example 84 Synthesis of compound 75: [ka] First step: synthesis of compound 75a 10 mL of DMF was added to M11 (1 g, 0.79 mol). The resulting solution was then cooled to 0 °C in an ice-water bath, after which compound 5c (345 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added. The reaction was allowed to stand under the same conditions for 1 hour. After TLC monitoring showed the reaction was complete, the reaction solution was purified by preparative-grade high-performance liquid chromatography (acetonitrile / pure water system) and the target peak was collected. The acetonitrile was removed under reduced pressure, followed by lyophilization to give 0.9 g of compound 75a; MS m / z: [M+H] + =1285.6.

[0279] Second step: Synthesis of compound 75b In a 25 mL single-neck flask, 75a (700 mg, 0.54 mmol), M5 (289 mg, 0.54 mmol), PyBOP (313 mg, 0.6 mmol), HOBt (81 mg, 0.6 mmol), and 10 mL of DMF were mixed, followed by the addition of DIPEA (155 mg, 1.2 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 hours. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution of compound 75b (304 mg); LC-MS: [M+H] + =1734.8.

[0280] Third step: Synthesis of compound 75: In a 25 mL single-neck flask, 75b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol), and 10 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 75 (96 mg); LC-MS: [M+H] + =1546.6.

[0281] Example 85 Synthesis of compound 76: [ka] Compound 76 (92 mg) was obtained by following the synthetic route of Example 84; LC-MS: [M+H] + =1546.5.

[0282] Example 86 Synthesis of compound 77: [ka] Compound 77 (87 mg) was obtained by following the synthetic route of Example 84; LC-MS: [M+H] + =1546.5.

[0283] Example 87 Synthesis of compound 78: [ka] Compound 78 (94 mg) was obtained by following the synthetic route of Example 84; LC-MS: [M+H] + =1546.7.

[0284] Example 88 Synthesis of compounds 79 and 80: [ka] First step: Synthesis of compound 79a 10 mL of DMF was added to M11 (1 g, 0.79 mol). The resulting solution was then cooled to 0 °C in an ice-water bath, after which compound 20c (377 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added. The reaction was allowed to stand under the same conditions for 1 hour. After TLC monitoring showed the reaction was complete, the reaction solution was purified by preparative-grade high-performance liquid chromatography (acetonitrile / pure water system) to recover the desired peak. The acetonitrile was removed under reduced pressure, followed by lyophilization to give 783 mg of compound 79a; MS m / z: [M+H] + =1325.8.

[0285] Second step: Synthesis of compounds 79b-1 and 79b-2 In a 25 mL single-neck flask, 79a (600 mg, 0.45 mmol), M5 (240 mg, 0.45 mmol), PyBOP (261 mg, 0.5 mmol), HOBt (68 mg, 0.5 mmol), and 10 mL of DMF were mixed, followed by the addition of DIPEA (130 mg, 1 mmol) under ice-water bath conditions. The reaction was allowed to stand at room temperature for 2 h. After HPLC monitoring showed the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to give fractions of 79b-1 and 79b-2. These fractions were separately lyophilized to give 79b-1 (124 mg); LC-MS: [M+H] + =1743.0;79b-1(122mg);LC-MS:[M+H] + =1743.0 was obtained.

[0286] Third step: Synthesis of compound 79 [ka] In a 25 mL single-neck flask, 79b-1 (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol), and 10 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 79 (30 mg); LC-MS: [M+H] + =1586.9.

[0287] Step 4: Synthesis of Compound 80 [ka] In a 25 mL single-neck flask, 79b-2 (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol), and 10 mL of nitromethane were mixed, and the reaction was allowed to stand at 40 °C for 1 h. After HPLC monitoring showed the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was lyophilized to obtain solid compound 80 (33 mg); LC-MS: [M+H] + =1587.0.

[0288] Example 89 Synthesis of compound 81: [ka] Compound 81 (24 mg) was obtained by following the synthetic route of Example 88; LC-MS: [M+H] + =1586.9.

[0289] Example 90 Synthesis of compound 82: [ka] Compound 82 (29 mg) was obtained by following the synthetic route of Example 88; LC-MS: [M+H] +=1586.9.

[0290] Example 91 1) Expression and purification of antibody hu4D3: The cells were suspended with Expi293 (Shanghai OPM Biosciences Co., Ltd.) to express the antibody hu4D3. One day before transfection, the cells were plated at 0.9 × 10 cells per 1 L shake flask containing 300 mL of OPM-293 CD05 medium (81075-001, Shanghai OPM Biosciences Co., Ltd.). 6 The cells were seeded at a density of 1000 cells / mL and cultured overnight on a cell culture shaker at 37°C, 5% CO2, and 120 rpm. The next day, the antibody expression plasmid was transfected using PEI-MAX at a plasmid:PEI-MAX mass ratio of 1:3. One day after transfection, OPM-293 ProFeed 5% (v / v) was added, and three days after transfection, OPM-293 ProFeed 5% (v / v) was added again. Six days after transfection, the supernatant was collected by centrifugation. The collected cell expression supernatant was loaded onto a Protein A affinity chromatography column (UniMab 50, Suzhou NanoMicro Technology Co., Ltd.) and eluted with 0.05 M sodium acetate (pH 3.6). The captured antibody was adjusted to pH 7.0 with 1 M Tris-HCl (pH 8.8) at a 0.7 / 10 (v / v) ratio. Impurities such as polymers were then removed using a gel filtration chromatography column SEC (Superdex 200, GE Company), while the antibody buffer was replaced with 20 mM PB (pH 6.5).

[0291] Antibody hu4D3: Light chain nucleic acid coding sequence SEQ ID NO: 38 GATATCCAGATGACCCAGTCTCCATCTAGCCTGTCCGCTTCTGTGGGCGATAGAGTGACCATCACATGCAGAGCTTCTCAGGATATCAATAAGTATCTGGCTTGGTATCAGCAGAAGCCTGGAAAGGTGCCTAAGCTGCTGATCTACTCTACATCTACCCTGCAGTCTGGAGTGCCTTCTAGATTTTCTGGATCTGGCTCTGGCACCGATTTTACACTGACAATCTCTTCTCTGCAGCCTGAGGATGTGGCTACATATTATTGTCTGCAGTATGATGATCTGTTCACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG Amino acid sequence of the light chain SEQ ID NO: 18 DIQMTQSPSSLSASVGDRVTITCRASQDINKYLAWYQQKPGKVPKLLIYSTSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLQYDDLFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC In the sequence, the variable region is as follows: SEQ ID NO: 14 DIQMTQSPSSLSASVGDRVTITCRAS QDINKY LAWYQQKPGKVPKLLIY STS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC LQYDDLFT FGQGTKLEIK In the sequence, from left to right, the underlined parts are CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 8), and CDR3 (SEQ ID NO: 9). The nucleic acid coding sequence for the variable region of the light chain is SEQ ID NO:34. GATATCCAGATGACCCAGTCTCCATCTAGCCTGTCCGCTTCTGTGGGCGATAGAGTGACCATCACATGCAGAGCTTCTCAGGATATCAATAAGTATCTGGCTTGGTATCAGCAGAAGCCTGGAAAGGTGCCTAAGCTGCTGATCTACTCTACATCTACC CTGCAGTCTGGAGTGCCTTCTAGATTTTCTGGATCTGGCTCTGGCACCGATTTTACACTGACAATCTCTTCTCTGCAGCCTGAGGATGTGGCTACATATTATTGTCTGCAGTATGATGATCTGTTCACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGC

[0292] In the variable region of the light chain described above, the nucleotide coding sequences of CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 8), and CDR3 (SEQ ID NO: 9) are as follows: SEQ ID NO: 27 CAAGACATTAATAAGTAT SEQ ID NO: 28 TCCACATCT SEQ ID NO: 29 CTGCAGTATGATGATCTATTCACG

[0293] Heavy chain nucleic acid coding sequence SEQ ID NO: 37

[0294] Heavy chain amino acid sequence SEQ ID NO: 17 QVQLVQSGAEVKKPGASVKLSCKASGYTFTSFDINWVRQAPEQRLEWMGWIFPGDGNTKYSQKFQGRATITRDTSASTAYMELSSLRSEDTAVYYCVRGEALYYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG In the sequence, the variable region of the heavy chain is as follows: SEQ ID NO: 13 QVQLVQSGAEVKKPGASVKLSCKAS GYTFTSFD INWVRQAPEQRLEWMGW IFPGDGN TKYSQKFQGRATITRDTSASTAYMELSSLRSEDTAVYYC VRGEALYYFDY WGQGTLVTVSS In the sequence, from left to right, the underlined parts are CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3).

[0295] The nucleic acid coding sequence for the variable region of the heavy chain is SEQ ID NO:33. CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCCTCCGTGAAGCTGAGCTGTAAGGCCTCCGGCTACACCTTCACCTCCTTCGACATTAACTGGGTGCGGCAGGCCCCCGAGCAGCGCCTGGAGTGGATGGGCTGGATCTTCCCCGGCGACGGCAACACCAAG TACTCCCAGAAGTTCCAGGGAAGAGCTACCATCACCAGAGATACATCCGCTTCTACAGCTTACATGGAGCTGTCTAGCCTGAGATCTGAGGATACAGCTGTGTATTACTGTGTGAGAGGAGAGGCTCTGTACTATTTTGATTATTGGGCCAGGGCACCCTGGTGACAGTGTCTTCTG In the variable region of the heavy chain described above, the nucleotide coding sequences of CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3) are as follows: SEQ ID NO: 21 GGCTACACCTTCACAAGCTTTGAT SEQ ID NO: 22 ATTTTTCCTGGAGATGGTAAT SEQ ID NO: 23 GTAAGAGGGGAGGCCCTGTATTACTTTGACTAC

[0296] 2) Expression and purification of antibody hu7F11: Antibody hu7F11 was similarly expressed and purified. Antibody hu7F11: Light chain nucleic acid coding sequence SEQ ID NO: 40 GATATCGTGATGACACAGTCTCCTGATTCTCTGGCCGTGTCTCTGGGCGAAAGAGCTACAATCAACTGTAAGGCTTCTCAGTCTGTGTCTAATGATGTGGTGTGGTACCAGCAGAAGCCTGGGCAGCCCCCCAAGCTGCTGATCTACTACGCCTCCAACAGGTACACCGGCGTGCCCGACAGGTTCTCCGGCTCCGGCTACGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGGACTACTCCTCCCCCTGGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG Amino acid sequence of the light chain SEQ ID NO: 20 DIVMTQSPDSLAVSLGERATINCKASQSVSNDVVWYQQKPGQPPKLLIYYASNRYTGVPDRFSGSGYGTDFTLTISSLQAEDVAVYYCQQDYSSPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC In the sequence, the variable region of the light chain is as follows: SEQ ID NO: 16 DIVMTQSPDSLAVSLGERATINCKAS QSVSND VVWYQQKPGQPPKLLIY YAS NRYTGVPDRFSGSGYGTDFTLTISSLQAEDVAVYYC QQDYSSPWT FGGGTKVEIK In the sequence, from left to right, the underlined parts are CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11), and CDR3 (SEQ ID NO: 12).

[0297] The nucleic acid coding sequence for the variable region of the light chain is SEQ ID NO:36. GATATCGTGATGACACAGTCTCCTGATTCTCTGGCCGTGTCTCTGGGCGAAAGAGCTACAATCAACTGTAAGGCTTCTCAGTCTGTGTCTAATGATGTGGTTGGTACCAGCAGAAGCCTGGGCAGCCCCCCAAGCTGCTGATCTACTACGCCTCCAAACAG GTACACCGGCGTGCCCGACAGGTTCTCCGGCTCCGGCTACGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGGACTACTCCTCCCCTGGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGC In the variable region of the light chain described above, the nucleotide coding sequences of CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11), and CDR3 (SEQ ID NO: 12) are as follows: SEQ ID NO: 30 CAGAGTGTGAGTAATGAT SEQ ID NO: 31 TATGCATCC SEQ ID NO: 32 CAGCAGGATTATTCCTCTCCGTGGACG

[0298] Heavy chain nucleic acid coding sequence SEQ ID NO: 39

[0299] Heavy chain amino acid sequence SEQ ID NO: 19 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHVISWVRQATGQGLEWMGQIYPGSDNSYYAQKFQGRVTLTADKSINTAYMELSSLRSEDTAVYYCAREGYGYGKNGVGYAMD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG In the sequence, the variable region of the heavy chain is as follows: SEQ ID NO: 15 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDHV ISWVRQATGQGLEWMGQ IYPGSDNS YYAQKFQGRVTLTADKSINTAYMELSSLRSEDTAVYYC AREGYGYGKNGVGYAMDY WGQGTLVTVSS In the sequence, from left to right, the underlined parts are CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6).

[0300] The nucleic acid coding sequence for the variable region of the heavy chain is SEQ ID NO:35. CAGGTGCAGCTGGTGCAGTCTGGGGCCGAGGTGAAAAAGCCAGGCGCTTCTGTGAAGGTGTCTTGCAAGGCCTCCGGCTACACCTTCACCGACCACGTGATCTCCTGGGTGCGCCAGGCCACCGGCCAGGGCCTGGAGTGGATGGGCCAGATCTACCCCGGCTCCGACAACTCCTACTACGCCCAGAA GTTCCAGGGCAGGGTGACTCTGACCGCCGACAAGTCCATCAACACCGCCTACATGGAGCTGTCCTCCCTGAGGTCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCTACGGCTACGGCAAGAACGGCGTGGGCTACGCCATGGATTATTGGGCCAGGGCACCCTGGTGACAGTGTCTTCTG In the variable region of the heavy chain described above, the nucleotide coding sequences of CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6) are as follows: SEQ ID NO: 24 GGATACACATTCACTGACCATGTC SEQ ID NO: 25 ATTTATCCTGGAAGTGATAATAGT SEQ ID NO: 26 GCAAGAGAGGGCTATGGTTATGGAAAAAACGGAGTTGGCTATGCTATGGACTAC

[0301] 3) Expression and purification of antibody hTINA1: Antibody hTINA1 was expressed and purified in the same manner. For details, please refer to the "hTINA1-H1L1" section in Table 2 on page 67 / 91 of patent application CN105849126A. Antibody hTINA1: Light chain nucleic acid coding sequence: SEQ ID NO: 43 ATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCTCCGGCGCGTACGGCGACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCCACAGCCGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAGCTGCTGATCTACAGCGCCAGCTACCGGTACACCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCAGCCTGCAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCACTACATCACCCCCCTGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCCCCCTCCGTGTTCATCTTCCCCCCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCCGTGGTGTGCCTGCTGAATAACTTCTACCCCAGAGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGGAACTCCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAAGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCTCCCCCGTCACCAAGAGCTTCAACAGGGGGGAGTGT Amino acid sequence of the light chain: SEQ ID NO: 44 MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The variable regions in the sequence are as follows: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK

[0302] Heavy chain nucleic acid coding sequence: SEQ ID NO: 41

[0303] Heavy chain amino acid sequence: SEQ ID NO: 42 MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCAR SGFGSSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In the sequence, the variable region of the heavy chain is as follows: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS

[0304] Example 92 1) Synthesis of antibody hu4D3-drug conjugate samples by conjugating antibody hu4D3 with a payload: After expression in cells and purification by protein A affinity chromatography and molecular sieve chromatography, the anti-human Trop2 antibody was replaced with 20 mM NaAc-HAc, pH 6.0 buffer, and then concentrated or diluted to a protein concentration of 5 mg / mL. The linker-drug was a white powder and dissolved in DMA to a concentration of 10 mg / mL for further use. To release the interchain disulfide bond of the anti-human Trop2 antibody, 15x TCEP was added based on the molecular ratio and the reaction was allowed to stand at room temperature for 2 hours. Then, 16x linker-drug solution based on the molecular ratio was added based on the molecular ratio and the reaction was allowed to stand at room temperature for 2 hours. After the reaction was determined to be complete, the reaction solution was ultrafiltered using a 30 kDa ultrafiltration centrifuge to remove linker-drug that was not bound to the anti-human Trop2 antibody, and the anti-human Trop2 antibody-drug conjugate sample was obtained. The resulting human Trop2 antibody-drug conjugate samples were examined for the percentage of monomer by SEC-HPLC and for drug loading by RP-HPLC or HIC-HPLC. 2) Similarly, antibody hu7F11 was conjugated to a payload to synthesize an antibody hu7F11-drug conjugate sample.

[0305] Example 93 Synthesis of antibody TINA1-drug conjugate samples by conjugating antibody DS1062a with a payload After expression in cells and purification by protein A affinity chromatography and molecular sieve chromatography, the anti-human Trop2 antibody was substituted into 20 mM NaAc-HAc, pH 6.0 buffer, and then concentrated or diluted to a protein concentration of 5 mg / mL. The linker-drug was a white powder and dissolved in DMA to a concentration of 10 mg / mL for further use. To release the interchain disulfide bond of the anti-human Trop2 antibody, 8-12 times TCEP was added depending on the molecular ratio and the reaction was allowed to stand at room temperature for 2 hours. Next, 5-8 times the linker-drug solution depending on the molecular ratio was added and the reaction was allowed to stand at room temperature for 1-2 hours. After the reaction was determined to be complete, the reaction solution was ultrafiltered using a 30 kDa ultrafiltration centrifuge to remove the linker-drug that was not bound to the anti-human Trop2 antibody, and the anti-human Trop2 antibody-drug conjugate sample was obtained. The resulting human Trop2 antibody-drug conjugate samples were examined for the percentage of monomer by SEC-HPLC and for drug loading by RP-HPLC or HIC-HPLC.

[0306] Example 94 ADC-1 was synthesized according to the general coupling method in Example 92. [ka] Example 95 ADC-2 was synthesized according to the general coupling method in Example 92. [ka] Example 96 ADC-3 was synthesized according to the general coupling method in Example 92. [ka]

[0307] Example 97 ADC-4 was synthesized according to the general coupling method in Example 92. [ka] Example 98 ADC-5 was synthesized according to the general coupling method in Example 92. [ka]

[0308] Example 99 ADC-6 was synthesized according to the general coupling method in Example 92. [ka]

[0309] Example 100 ADC-7 was synthesized according to the general coupling method in Example 92. [ka] Example 101 ADC-8 was synthesized according to the general coupling method in Example 92. [ka]

[0310] Example 102 ADC-9 was synthesized according to the general coupling method in Example 92. [ka] Example 103 ADC-10 was synthesized according to the general coupling method in Example 92. [ka] Example 104 ADC-11 was synthesized according to the general coupling method in Example 92. [ka]

[0311] Example 105 ADC-12 was synthesized according to the general coupling method in Example 92. [ka] Example 106 ADC-13 was synthesized according to the general coupling method in Example 92. [ka] Example 107 ADC-14 was synthesized according to the general coupling method in Example 92. [ka]

[0312] Example 108 ADC-15 was synthesized according to the general coupling method in Example 92. [ka] Example 109 ADC-16 was synthesized according to the general coupling method in Example 92. [ka] Example 110 ADC-17 was synthesized according to the general coupling method in Example 92. [ka]

[0313] Example 111 ADC-18 was synthesized according to the general coupling method in Example 92. [ka] Example 112 ADC-19 was synthesized according to the general coupling method in Example 92. [ka] Example 113 ADC-20 was synthesized according to the general coupling method in Example 92. [ka]

[0314] Example 114 ADC-21 was synthesized according to the general coupling method in Example 92. [ka] Example 115 ADC-22 was synthesized according to the general coupling method in Example 92. [ka] Example 116 ADC-23 was synthesized according to the general coupling method in Example 92. [ka]

[0315] Example 117 ADC-24 was synthesized according to the general coupling method in Example 92. [ka] Example 118 ADC-25 was synthesized according to the general coupling method in Example 92. [ka] Example 119 ADC-26 was synthesized according to the general coupling method in Example 92. [ka]

[0316] Example 120 ADC-27 was synthesized according to the general coupling method in Example 92. [ka] Example 121 ADC-28 was synthesized according to the general coupling method in Example 92. [ka] Example 122 ADC-29 was synthesized according to the general coupling method in Example 92. [ka]

[0317] Example 123 ADC-30 was synthesized according to the general coupling method in Example 92. [ka] Example 124 ADC-31 was synthesized according to the general coupling method in Example 92. [ka] Example 125 ADC-32 was synthesized according to the general coupling method in Example 92. [ka]

[0318] Example 126 ADC-33 was synthesized according to the general coupling method in Example 92. [ka] Example 127 ADC-34 was synthesized according to the general coupling method in Example 92. [ka] Example 128 ADC-35 was synthesized according to the general coupling method in Example 92. [ka]

[0319] Example 129 ADC-36 was synthesized according to the general coupling method in Example 92. [ka] Example 130 ADC-37 was synthesized according to the general coupling method in Example 92. [ka] Example 131 ADC-38 was synthesized according to the general coupling method in Example 92. [ka]

[0320] Example 132 ADC-39 was synthesized according to the general coupling method in Example 92. [ka] Example 133 ADC-40 was synthesized according to the general coupling method in Example 92. [ka] Example 134 ADC-41 was synthesized according to the general coupling method in Example 92. [ka]

[0321] Example 135 ADC-42 was synthesized according to the general coupling method in Example 92. [ka] Example 136 ADC-43 was synthesized according to the general coupling method in Example 92. [ka] Example 137 ADC-44 was synthesized according to the general coupling method in Example 92. [ka]

[0322] Example 138 ADC-45 was synthesized according to the general coupling method in Example 92. [ka] Example 139 ADC-46 was synthesized according to the general coupling method in Example 92. [ka] Example 140 ADC-47 was synthesized according to the general coupling method in Example 92. [ka]

[0323] Example 141 ADC-48 was synthesized according to the general coupling method in Example 92. [ka] Example 142 ADC-49 was synthesized according to the general coupling method in Example 92. [ka] Example 143 ADC-50 was synthesized according to the general coupling method in Example 92. [ka]

[0324] Example 144 ADC-51 was synthesized according to the general coupling method in Example 92. [ka] Example 145 ADC-52 was synthesized according to the general coupling method in Example 92. [ka] Example 146 ADC-53 was synthesized according to the general coupling method in Example 92. [ka]

[0325] Example 147 ADC-54 was synthesized according to the general coupling method in Example 92. [ka] Example 148 ADC-55 was synthesized according to the general coupling method in Example 92. [ka] Example 149 ADC-56 was synthesized according to the general coupling method in Example 92. [ka]

[0326] Example 150 ADC-57 was synthesized according to the general coupling method in Example 92. [ka] Example 151 ADC-58 was synthesized according to the general coupling method in Example 92. [ka] Example 152 ADC-59 was synthesized according to the general coupling method in Example 92. [ka]

[0327] Example 153 ADC-60 was synthesized according to the general coupling method in Example 92. [ka] Example 154 ADC-61 was synthesized according to the general coupling method in Example 92. [ka] Example 155 ADC-62 was synthesized according to the general coupling method in Example 92. [ka]

[0328] Example 156 ADC-63 was synthesized according to the general coupling method in Example 92. [ka] Example 157 ADC-64 was synthesized according to the general coupling method in Example 92. [ka] Example 158 ADC-65 was synthesized according to the general coupling method in Example 92. [ka]

[0329] Example 159 ADC-66 was synthesized according to the general coupling method in Example 92. [ka] Example 160 ADC-67 was synthesized according to the general coupling method in Example 92. [ka] Example 161 ADC-68 was synthesized according to the general coupling method in Example 92. [ka]

[0330] Example 162 ADC-69 was synthesized according to the general coupling method in Example 92. [ka] Example 163 ADC-70 was synthesized according to the general coupling method in Example 92. [ka] Example 164 ADC-71 was synthesized according to the general coupling method in Example 92. [ka]

[0331] Example 165 ADC-72 was synthesized according to the general coupling method in Example 92. [ka] Example 166 ADC-73 was synthesized according to the general coupling method in Example 92. [ka] Example 167 ADC-74 was synthesized according to the general coupling method in Example 92. [ka]

[0332] Example 168 ADC-75 was synthesized according to the general coupling method in Example 92. [ka] Example 169 ADC-76 was synthesized according to the general coupling method in Example 92. [ka] Example 170 ADC-77 was synthesized according to the general coupling method in Example 92. [ka]

[0333] Example 171 ADC-78 was synthesized according to the general coupling method in Example 92. [ka] Example 172 ADC-79 was synthesized according to the general coupling method in Example 92. [ka] Example 173 ADC-80 was synthesized according to the general coupling method in Example 92. [ka]

[0334] Example 174 ADC-81 was synthesized according to the general coupling method in Example 92. [ka] Example 175 ADC-82 was synthesized according to the general coupling method in Example 92. [ka] Example 176 ADC-83 was synthesized according to the general coupling method in Example 92. [ka]

[0335] Example 177 ADC-84 was synthesized according to the general coupling method in Example 92. [ka] Example 178 ADC-85 was synthesized according to the general coupling method in Example 92. [ka] Example 179 ADC-86 was synthesized according to the general coupling method in Example 92. [ka]

[0336] Example 180 ADC-87 was synthesized according to the general coupling method in Example 92. [ka] Example 181 ADC-88 was synthesized according to the general coupling method in Example 92. [ka] Example 182 ADC-89 was synthesized according to the general coupling method in Example 92. [ka]

[0337] Example 183 ADC-90 was synthesized according to the general coupling method in Example 92. [ka] Example 184 ADC-91 was synthesized according to the general coupling method in Example 92. [ka] Example 185 ADC-92 was synthesized according to the general coupling method in Example 92. [ka]

[0338] Example 186 ADC-93 was synthesized according to the general coupling method in Example 92. [ka] Example 187 ADC-94 was synthesized according to the general coupling method in Example 92. [ka] Example 188 ADC-95 was synthesized according to the general coupling method in Example 92. [ka]

[0339] Example 189 ADC-96 was synthesized according to the general coupling method in Example 92. [ka] Example 190 ADC-97 was synthesized according to the general coupling method in Example 92. [ka] Example 191 ADC-98 was synthesized according to the general coupling method in Example 92. [ka]

[0340] Example 192 ADC-99 was synthesized according to the general coupling method in Example 92. [ka] Example 193 ADC-100 was synthesized according to the general coupling method in Example 92. [ka] Example 194 ADC-101 was synthesized according to the general coupling method in Example 92. [ka]

[0341] Example 195 ADC-102 was synthesized according to the general coupling method in Example 92. [ka] Example 196 ADC-103 was synthesized according to the general coupling method in Example 92. [ka] Example 197 ADC-104 was synthesized according to the general coupling method in Example 92. [ka]

[0342] Example 198 ADC-105 was synthesized according to the general coupling method in Example 92. [ka] Example 199 ADC-106 was synthesized according to the general coupling method in Example 92. [ka] Example 200 ADC-DS was synthesized from compound 45 according to the general coupling method in Example 92. [ka]

[0343] Example 201 ADC-107 was synthesized according to the general coupling method in Example 92. [ka] Example 202 ADC-108 was synthesized according to the general coupling method in Example 92. [ka] Example 203 ADC-109 was synthesized according to the general coupling method in Example 92. [ka]

[0344] Example 204 ADC-110 was synthesized according to the general coupling method in Example 92. [ka] Example 205 ADC-111 was synthesized according to the general coupling method in Example 92. [ka] Example 206 ADC-112 was synthesized according to the general coupling method in Example 92. [ka]

[0345] Example 207 ADC-113 was synthesized according to the general coupling method in Example 92. [ka] Example 208 ADC-114 was synthesized according to the general coupling method in Example 92. [ka] Example 209 ADC-115 was synthesized according to the general coupling method in Example 92. [ka]

[0346] Example 210 ADC-116 was synthesized according to the general coupling method in Example 92. [ka] Example 211 ADC-117 was synthesized according to the general coupling method in Example 92. [ka] Example 212 ADC-118 was synthesized according to the general coupling method in Example 92. [ka]

[0347] Example 213 ADC-119 was synthesized according to the general coupling method in Example 92. [ka] Example 214 ADC-120 was synthesized according to the general coupling method in Example 92. [ka] Example 215 ADC-121 was synthesized according to the general coupling method in Example 92. [ka]

[0348] Example 216 ADC-122 was synthesized according to the general coupling method in Example 92. [ka] Example 217 ADC-123 was synthesized according to the general coupling method in Example 92. [ka] Example 218 ADC-124 was synthesized according to the general coupling method in Example 92. [ka]

[0349] Example 219 ADC-125 was synthesized according to the general coupling method in Example 92. [ka] Example 220 ADC-126 was synthesized according to the general coupling method in Example 92. [ka] Example 221 ADC-127 was synthesized according to the general coupling method in Example 92. [ka]

[0350] Example 222 ADC-128 was synthesized according to the general coupling method in Example 92. [ka] Example 223 ADC-129 was synthesized according to the general coupling method in Example 92. [ka] Example 224 ADC-130 was synthesized according to the general coupling method in Example 92. [ka]

[0351] Example 225 ADC-131 was synthesized according to the general coupling method in Example 92. [ka] Example 226 ADC-132 was synthesized according to the general coupling method in Example 92. [ka] Example 227 ADC-133 was synthesized according to the general coupling method in Example 92. [ka]

[0352] Example 228 ADC-134 was synthesized according to the general coupling method in Example 92. [ka] Example 229 ADC-135 was synthesized according to the general coupling method in Example 92. [ka]

[0353] Example 230 ADC-136 was synthesized according to the general coupling method in Example 92. [ka] Example 231 ADC-137 was synthesized according to the general coupling method in Example 92. [ka] Example 232 ADC-138 was synthesized according to the general coupling method in Example 92. [ka]

[0354] Example 233 ADC-139 was synthesized according to the general coupling method in Example 92. [ka] Example 234 ADC-140 was synthesized according to the general coupling method in Example 92. [ka] Example 235 ADC-141 was synthesized according to the general coupling method in Example 92. [ka]

[0355] Example 236 ADC-142 was synthesized according to the general coupling method in Example 92. [ka] Example 237 ADC-143 was synthesized according to the general coupling method in Example 92. [ka] Example 238 ADC-144 was synthesized according to the general coupling method in Example 92. [ka]

[0356] Example 239 ADC-145 was synthesized according to the general coupling method in Example 92. [ka] Example 240 ADC-146 was synthesized according to the general coupling method in Example 92. [ka] Example 241 ADC-147 was synthesized according to the general coupling method in Example 92. [ka]

[0357] Example 242 ADC-148 was synthesized according to the general coupling method in Example 92. [ka] Example 243 ADC-149 was synthesized according to the general coupling method in Example 92. [ka] Example 244 ADC-150 was synthesized according to the general coupling method in Example 92. [ka]

[0358] Example 245 ADC-151 was synthesized according to the general coupling method in Example 92. [ka] Example 246 ADC-152 was synthesized according to the general coupling method in Example 92. [ka] Example 247 ADC-153 was synthesized according to the general coupling method in Example 92. [ka]

[0359] Example 248 ADC-154 was synthesized according to the general coupling method in Example 92. [ka] Example 249 ADC-155 was synthesized according to the general coupling method in Example 92. [ka]

[0360] Example 250 ADC-156 was synthesized according to the general coupling method in Example 92. [ka] Example 251 ADC-157 was synthesized according to the general coupling method in Example 92. [ka] Example 252 ADC-158 was synthesized according to the general coupling method in Example 92. [ka]

[0361] Example 253 ADC-159 was synthesized according to the general coupling method in Example 92. [ka] Example 254 ADC-160 was synthesized according to the general coupling method in Example 92. [ka] Example 255 ADC-161 was synthesized according to the general coupling method in Example 92. [ka]

[0362] Example 256 ADC-162 was synthesized according to the general coupling method in Example 92. [ka] Example 257 ADC-163 was synthesized according to the general coupling method in Example 92. [ka]

[0363] Example 258 ADC-164 was synthesized according to the general coupling method in Example 92. [ka] Example 259 ADC-165 was synthesized according to the general coupling method in Example 92. [ka] Example 260 ADC-166 was synthesized according to the general coupling method in Example 92. [ka]

[0364] Example 261 ADC-167 was synthesized according to the general coupling method in Example 92. [ka] Example 262 ADC-168 was synthesized according to the general coupling method in Example 92. [ka] Example 263 ADC-169 was synthesized according to the general coupling method in Example 92. [ka]

[0365] Example 264 ADC-170 was synthesized according to the general coupling method in Example 92. [ka] Example 265 ADC-171 was synthesized according to the general coupling method in Example 92. [ka] Example 266 ADC-172 was synthesized according to the general coupling method in Example 92. [ka]

[0366] Example 267 ADC-173 was synthesized according to the general coupling method in Example 92. [ka] Example 268 ADC-174 was synthesized according to the general coupling method in Example 92. [ka] Example 269 ADC-175 was synthesized according to the general coupling method in Example 92. [ka]

[0367] Example 270 ADC-176 was synthesized according to the general coupling method in Example 92. [ka] Example 271 ADC-177 was synthesized according to the general coupling method in Example 92. [ka] Example 272 ADC-178 was synthesized according to the general coupling method in Example 92. [ka]

[0368] Example 273 ADC-179 was synthesized according to the general coupling method in Example 92. [ka] Example 274 ADC-180 was synthesized according to the general coupling method in Example 92. [ka] Example 275 ADC-181 was synthesized according to the general coupling method in Example 92. [ka]

[0369] Example 276 ADC-182 was synthesized according to the general coupling method in Example 92. [ka] Example 277 ADC-183 was synthesized according to the general coupling method in Example 92. [ka] Example 278 ADC-184 was synthesized according to the general coupling method in Example 92. [ka]

[0370] Example 279 ADC-185 was synthesized according to the general coupling method in Example 92. [ka] Example 280 ADC-186 was synthesized according to the general coupling method in Example 92. [ka] Example 281 ADC-187 was synthesized according to the general coupling method in Example 92. [ka]

[0371] Example 282 ADC-188 was synthesized according to the general coupling method in Example 92. [ka] Example 283 ADC-189 was synthesized according to the general coupling method in Example 92. [ka] Example 284 ADC-190 was synthesized according to the general coupling method in Example 92. [ka]

[0372] Example 285 ADC-191 was synthesized according to the general coupling method in Example 92. [ka] Example 286 ADC-192 was synthesized according to the general coupling method in Example 92. [ka] Example 287 ADC-193 was synthesized according to the general coupling method in Example 92. [ka]

[0373] Example 288 ADC-194 was synthesized according to the general coupling method in Example 92. [ka] Example 289 ADC-195 was synthesized according to the general coupling method in Example 92. [ka] Example 290 ADC-196 was synthesized according to the general coupling method in Example 92. [ka]

[0374] Example 291 ADC-197 was synthesized according to the general coupling method in Example 92. [ka] Example 292 ADC-198 was synthesized according to the general coupling method in Example 92. [ka] Example 293 ADC-199 was synthesized according to the general coupling method in Example 92. [ka]

[0375] Example 294 ADC-200 was synthesized according to the general coupling method in Example 92. [ka] Example 295 ADC-201 was synthesized according to the general coupling method in Example 92. [ka] Example 296 ADC-202 was synthesized according to the general coupling method in Example 92. [ka]

[0376] Example 297 ADC-203 was synthesized according to the general coupling method in Example 92. [ka] Example 298 ADC-204 was synthesized according to the general coupling method in Example 92. [ka] Example 299 ADC-205 was synthesized according to the general coupling method in Example 92. [ka]

[0377] Example 300 ADC-206 was synthesized according to the general coupling method in Example 92. [ka] Example 301 ADC-207 was synthesized according to the general coupling method in Example 92. [ka] Example 302 ADC-208 was synthesized according to the general coupling method in Example 92. [ka]

[0378] Example 303 ADC-209 was synthesized according to the general coupling method in Example 92. [ka] Example 304 ADC-210 was synthesized according to the general coupling method in Example 92. [ka] Example 305 ADC-211 was synthesized according to the general coupling method in Example 92. [ka]

[0379] Example 306 ADC-212 was synthesized according to the general coupling method in Example 92. [ka] Example 307 ADC-213 was synthesized from compound 45 following the general coupling method of Example 92. [ka] Example 308 DS1062a was synthesized from compound 5A following the general coupling procedure of Example 92. [ka] where the antibody TINA1 is a human Trop2-targeting antibody and n is 4 or 8.

[0380] Example 309 Procedure 1 for determining the percentage of monomer by SEC-HPLC: Chromatography column: Biocore SEC-300 5 μm, 4.6 × 300 mm; Manufacturer: NanoChrom, Product Code: B213-050030-04630S; Mobile phase: 50mM PB+300mM NaCl+200mM Arg+5%IPA, pH=6.5; [Table 1]

[0381] Procedure 2 for determining the percentage of monomer by SEC-HPLC: Chromatography column: ACQUITY UPLC Protein SEC 4.6 x 150 mm 1.7 μm Manufacturer: Waters Product code: 186005225 Mobile phase: 50mM PB+300mM NaCl+200mM Arg+5%IPA, pH=6.5 [Table 2] [Table 3] Conclusion: The ADCs disclosed herein have the excellent properties of low degradation and aggregation characteristics and a high percentage of monomer.

[0382] Example 310 Determination of DAR by RP-HPLC: Chromatography column: Proteomix RP-1000 4.6 × 100 mm 5 μm 1000A; Manufacturer: Sepax; [Table 4] [Table 5] Conclusion: The ADCs disclosed herein have the excellent property of high DAR, which can significantly increase the drug concentration at the target site at the same dose of ADC.

[0383] Example 311 Plasma stability data for ADC A mixture of ADC and IgG-depleted plasma was formulated, with the final ADC concentration at 0.6 mg / mL. The plasma mixture was incubated in a water bath box in a 37°C incubator. The incubation times were set at 0, 3, and 7 days. At the same time, a non-incubated plasma control was set. The incubated sample was purified, extracted, and then tested for DAR to demonstrate the stability of the ADC in plasma. [Table 6] Conclusion: The ADCs disclosed herein have good plasma stability, with no significant changes in DAR during incubation in plasma. However, the control DS-1062a showed a significant decrease in DAR during incubation in plasma, indicating poor plasma stability.

[0384] Example 312 The ADCs maintain the original affinity of the corresponding anti-Trop2 antibodies hu4D3 and hu7F11 for TROP2. Comparison of the relative affinities for TROP2 between hu4D3 and ADC-6 / ADC10 / ADC12 and between TINA1 and ADC-1 / DS1062a was performed using double antigen sandwich ELISA. The specific steps are as follows: Recombinant TROP2-His * Six antigens were coated onto plates, then blocked with 1% bovine serum albumin. Hu4D3 and its corresponding ADC and TINA1 and its corresponding ADC were diluted, starting at 2000 ng / mL, and serially diluted threefold to a total of 11 concentrations. The samples were then incubated on the coated ELISA plate for a set period of time, followed by incubation with a goat anti-human Fc-HRP-conjugated secondary antibody. Finally, TMB was developed and terminated with sulfuric acid solution, and the absorbance at 450 nm was detected on the ELISA plate. The results were plotted against the concentration at OD450 nm.

[0385] Conclusion: As shown in Figures 3A-3F, compared to the corresponding antibody, the conjugated ADC maintained comparable affinity, with no significant difference in EC50, indicating that conjugation of hu4D3 to the toxin does not affect its affinity for the antigen.

[0386] Example 313 In vitro drug efficacy studies: In this study, a human tumor cell line (BxPC-3) was used as a test model to evaluate the in vitro efficacy of ADCs. A fixed amount of tumor cells was seeded into a 96-well plate, and gradient dilutions of the test antibody and the corresponding ADC were added to the cells. The cells were treated for 5 days. Cell viability was detected using MTS, and IC50 values ​​were calculated to evaluate the in vitro inhibitory effects of the test antibody and ADC on the tumor cell line. The cells were treated for 5 days with the antibody drug at an initial concentration of 500 nM, diluted 7-fold for a total of 8 concentrations. Finally, cell viability was calculated as follows: Viability = (Test group - Blank group) / (Control group - Blank group) × 100%. Curve fitting was then performed using GraphPad Prism to calculate the 50% inhibitory concentration (IC50). [Table 7] Conclusion: As shown in Table 7 and Figure 4, in BxPC-3 cells, naked antibodies TINA1 and hu4D3 showed no obvious cytotoxicity against tumor cells; the in vitro cytotoxicity of ADC-2, ADC-6, ADC-10, and ADC-12 of the present invention was significantly higher than that of DS1062a.

[0387] Example 314 In vivo drug efficacy studies: Various human tumor cell lines (BXPC3, A431+SW620) were subcutaneously inoculated into BALB / c nude mice as a test model to evaluate the in vivo efficacy of ADCs. BALB / c nude mice inoculated with a certain number of tumor cell lines were tested for tumor volumes of 150–300 mm. 3 When the tumors reached a critical stage, the antibody or corresponding ADC was injected into the tail vein once a week for a total of four injections, and the tumors were continuously monitored. Tumors were measured twice a week to assess the inhibitory effects of the test antibody and ADC on the tumor cell lines.

[0388] Conclusion: In efficacy studies in tumor-bearing mice inoculated with the cell line BXPC3, which exhibits moderate to high TROP2 expression, the in vivo efficacy results of ADC-6, ADC-10, and ADC-12 were superior to those of DS1062a. In A431+SW620 heterogeneous tumors, the in vivo efficacy results of ADC-6 and ADC-12 were superior to those of DS1062a, and ADC-6 and ADC-12 had a good "bystander effect."

[0389] Example 315 In vivo drug efficacy of linker-drug compound 5A: 1) Test materials: Cells: The cells used in the test were from the Cell Bank of the Chinese Academy of Sciences; Cell culture medium DMEM: Gibco; FBS:BIOWEST 2) Culture medium formulation: Growth medium (containing 10% FBS and penicillin / streptomycin (100 U / mL); Test medium (containing 1% FBS and penicillin / streptomycin (100 U / mL) 3) Operation: The UV light in the biosafety cabinet was turned on 30 minutes before the experiment, and then the ventilation system was turned on for 3 minutes. Growth medium, detection medium, D-PBS, and trypsin were preheated in a 37°C water bath, and the surface was then disinfected with alcohol before being placed in the biosafety cabinet. Cells at approximately 80% confluence were placed in the biosafety cabinet, the old culture medium was removed, and the cells were washed with D-PBS, which was then discarded. The cells were digested with trypsin for 2-3 minutes, neutralized with growth medium, centrifuged at 1200 rpm for 3 minutes, and the supernatant was removed. The cell solution was mixed with 4 mL of test medium, and 100 μL of cells were collected for counting (here, 50 μL of cell solution was mixed thoroughly with 50 μL of trypan blue staining solution before counting). Cells were seeded at 80 μL per well in a 96-well plate according to the previously optimized cell plating density. Wells E11 and F11 received 80 μL of test medium alone, while the outer wells received 150 μL of D-PBS. 24 hours after seeding, 20 μL of diluted antibody was added to each well as a control. 20 μL of test medium alone was added to cells in column 11. Duplicate wells were set up for each concentration. The cells were then mixed thoroughly on a cell vortex shaker at 550 rpm for 3 minutes. Solution dilution: Using test medium, 300 μL of test solution with a starting concentration of 5 μM was prepared in the first column of a V-shaped 96-well plate. 240 μL of test medium was added to the subsequent columns 2 to 10. 60 μL of well-mixed test solution was removed from column 1 and added to column 2. The cell solution was mixed by pipetting up and down 10 times with a pipette, and the pipette tip was discarded. The same procedure was repeated for the other seven concentrations, one by one. 4) Testing: After 4 days, the MTS reagent was removed, thawed at room temperature in the dark, and thoroughly mixed by vortexing. In a biosafety cabinet, 20 μL of CellTiter One Solution Reagen MTS reagent per 100 μL of cell culture volume was added along the sidewall of each well, and the plate surface was gently tapped to thoroughly mix the MTS solution. The cells were incubated in an incubator in the dark for 2 hours. After the reaction, the 96-well plate was removed and the OD490nm absorbance was measured using an ELISA plate. The test data was recorded, organized, analyzed, and stored.

[0390] (5) Results: [Table 8] Table 8 shows that compound 5A (linker-drug) has good inhibitory effects on the above solid tumor cells and blood tumor cells.

[0391] Example 316 Naked antibody and compounds d3, d 38 , d 39 and d 44 In vitro efficacy testing: 1) Test materials: Cells: N87, SW620, SW620+A431, Fadu, and HCC827 were supplied by the Cell Bank of the Chinese Academy of Sciences; Cell culture medium DMEM: Gibco; FBS:BIOWEST 2) Culture medium formulation: Growth medium (containing 10% FBS and penicillin / streptomycin 100 U / mL); Test medium (containing 1% FBS, penicillin / streptomycin 100U / mL) 3) Operation Cells were seeded at an appropriate cell density in a 96-well plate. After 24 hours, drugs were added. After 24 hours, the drugs were diluted with test medium (1 μM starting concentration, 5-fold dilutions, 9 concentrations, test medium was added to column 10 as a blank control). After adding the diluted drugs to the corresponding cell wells, the plates were shaken for 3 minutes at 50 rpm / min using a microplate shaker (model: MX100-4A). After shaking, the cells were incubated in a carbon dioxide incubator for 3 days. 4) Testing After 4 days, the MTS reagent was removed, thawed at room temperature in the dark, and thoroughly mixed by vortexing. In a biosafety cabinet, 20 μL of CellTiter One Solution Reagen MTS reagent per 100 μL of cell culture volume was added along the sidewall of each well, and the plate surface was gently tapped to thoroughly mix the MTS solution. The cells were incubated in an incubator in the dark for 2 hours. After the reaction, the 96-well plate was removed and the OD490nm absorbance was measured using an ELISA plate. The test data was recorded, organized, analyzed, and stored.

[0392] 5) Results [Table 9] Conclusion: As shown in Table 9 and Figures 6A-6E, the small molecule drugs of the present invention exhibit excellent in vitro tumor inhibitory activity in various human tumor cell lines (N87, SW620, Fadu, and HCC827) and the SW620+A431 heterotumor test model.

[0393] Example 317 In vitro drug efficacy testing of ADCs: The in vitro efficacy of the ADC was evaluated using various human tumor cell lines (N87, SW620, Fadu, and HCC827) and a test model of SW620+A431 heterotumor. A fixed number of tumor cells were seeded into a 96-well plate, and gradient dilutions of the test antibody and corresponding ADC were added to the cells. The cells were treated for 5 days. Cell viability was detected using Alamar Blue or MTS, and the IC50 values ​​were calculated to evaluate the inhibitory effect of the test ADC on tumor cell lines. The cells were treated for 5 days with the antibody drug at an initial concentration of 500 nM, diluted 7-fold for a total of 8 concentration points. Finally, cell viability was calculated as follows: Viability = (Test group - Blank group) / (Control group - Blank group) × 100%. Curve fitting was then performed using GraphPad Prism to calculate the 50% inhibitory concentration (IC50). The results are shown in Table 10 and Figures 7A-7E. [Table 10] Conclusion The ADCs prepared according to the present invention exhibit excellent tumor-inhibiting activity in various human tumor cell lines (N87, SW620, Fadu, and HCC827) and in the SW620+A431 heterotumor test model. The test data for the SW620+A431 heterotumor model demonstrate that the ADCs prepared according to the present invention can exert a sufficient bystander effect.

Claims

1. General formula I 【Chemistry 1】 wherein: Ab is a human Trop2-targeting antibody or antigen-binding fragment thereof; L 1 includes, but is not limited to, 【Chemistry 2】 selected from the group consisting of: Preferably, L 1 teeth, 【Transformation 3】 and Preferably, L 1 teeth, 【Chemistry 4】 and L 2 is the formula A 【Transformation 5】 Formula A and having a structure represented by wherein Y is a scaffold selected from the group consisting of C1-C6 alkyl, substituted C1-C6 alkyl, and C3-C8 cycloalkyl; preferably, Y is C1-C6 alkyl; Ac is a hydrophilic structural unit; and the carbon number 2 connected to Y has absolute chiral configuration R or S; L 3 is present or absent, and if present, L 3 is a PEG hydrophilic unit: 【Transformation 6】 where o is selected from an integer from 1 to 10, preferably from 2 to 8; L 4 is an enzyme-cleavable unit; L 5 is a crosslinking unit; In formula I, the chiral carbon atom number 1 linked to N has absolute chiral configuration R or S; R is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl; Preferably, R is selected from the group consisting of a hydrogen atom and a C1-C6 alkyl; R 1 is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a carboxyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R 1 is selected from the group of a hydrogen atom or a C1-C6 alkyl; More preferably, R 1 is selected from C1-C6 alkyl; R 2 is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a carboxyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R 2 is selected from the group consisting of a hydrogen atom, halogen, and C1-C6 alkyl; More preferably, R 2 is selected from halogen; X is -C(O)-CR a R b - (CR 3 R 4 ) m -O-, -C(O)-CR a R b - (CR 3 R 4 ) m —NH— and —C(O)—CR a R b - (CR 3 R 4 ) m -S-; Preferably, X is —C(O)—CR a R b - (CR 3 R 4 ) m -O-; R a and R b are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a deuterated C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C6-C10 arylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R a and R b are each independently selected from the group consisting of a hydrogen atom, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, and C6-C10 aryl C1-C6 alkyl; Or R a and R b and R a and R b are linked to form a C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, 3- to 7-membered heterocyclyl, or substituted 3- to 7-membered heterocyclyl; preferably, R a and R b and R a and R b the carbon atoms to which are linked form a C3-C8 cycloalkyl; R 3 and R 4 are the same or different and each independently represent a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a halogenated C1-C6 alkyl, a deuterated C1-C6 alkyl, a C1-C6 alkoxy, a hydroxyl, an amino, a cyano, a nitro, a hydroxyl C1-C6 alkyl, a C3-C8 cycloalkyl, a 3- to 7-membered heterocyclyl, or a substituted 3- to 7-membered heterocyclyl; Preferably, R 3 and R 4 are each independently a hydrogen atom or a C1-C6 alkyl; Or R 3 and R 4 and R 3 and R 4 the carbon atoms to which are linked form a C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, 3- to 7-membered heterocyclyl, or substituted 3- to 7-membered heterocyclyl; m is selected from integers from 0 to 4, preferably 0 or 1; n is selected from integers from 1 to 10; A ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof.

2. 2. The ligand-camptothecin derivative conjugate of general formula I according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab is an antibody or antigen-binding fragment thereof targeting human Trop2.

3. 2. The ligand-camptothecin derivative conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody of the Ab comprises an IgG1 heavy chain and a κ light chain, and the antibody comprising the IgG1 heavy chain and the κ light chain specifically recognizes human Trop2 protein.

4. 2. The ligand-camptothecin derivative conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein in the antibody of said Ab, the κ light chain comprises CDRs as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, and the IgG1 heavy chain comprises CDRs as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:

3.

5. The ligand-camptothecin derivative conjugate of general formula I according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody of the Ab comprises a heavy chain and a light chain, the heavy chain comprising a VH having the sequence as set forth in SEQ ID NO: 13, and the light chain comprising a VL having the sequence as set forth in SEQ ID NO:

14.

6. The ligand-camptothecin derivative conjugate represented by general formula I according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody of said Ab has a heavy chain having an amino acid sequence as shown in SEQ ID NO: 17 and a light chain having an amino acid sequence as shown in SEQ ID NO:

18.

7. 2. The ligand-camptothecin derivative conjugate of claim 1, wherein the antibody of said Ab has a κ light chain comprising CDRs as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and an IgG1 heavy chain comprising CDRs as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or a pharmaceutically acceptable salt or solvate thereof.

8. The ligand-camptothecin derivative conjugate of general formula I according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody of the Ab comprises a heavy chain and a light chain, the heavy chain comprising a VH having the sequence as shown in SEQ ID NO: 15, and the light chain comprising a VL having the sequence as shown in SEQ ID NO:

16.

9. The ligand-camptothecin derivative conjugate represented by general formula I according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody of said Ab has a heavy chain having an amino acid sequence as shown in SEQ ID NO: 19 and a light chain having an amino acid sequence as shown in SEQ ID NO:

20.

10. X may be, but is not limited to, the following structure: 【Transformation 7】 or an isomer thereof, The left wavy line is connected to the camptothecin derivative moiety, and the right wavy line is L 5 2. The ligand-camptothecin derivative conjugate of claim 1, wherein the ligand is linked to a camptothecin derivative represented by general formula I, or a pharmaceutically acceptable salt or solvate thereof.

11. L 4 is selected from peptide residues including, but not limited to, amino acids, Optionally, the amino acid may be further substituted with one or more substituents selected from the group consisting of deuterium, halogen, hydroxyl, cyano, amino, nitro, carboxyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, and C3-C8 cycloalkyl or substituted C3-C8 cycloalkyl; Preferably, the peptide residue is a peptide residue consisting of one, two or more amino acids selected from the group consisting of phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline (C), serine (S), glutamic acid (E) or aspartic acid (D); More preferably, the peptide residue is a tetrapeptide residue consisting of glycine (G)-glycine (G)-phenylalanine (F)-glycine (G).

2. A ligand-camptothecin derivative complex represented by general formula I according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

12. L 5 but is not limited to -NR 5 (CR 6 R 7 ) q - and a chemical bond, and q is selected from an integer from 0 to 6; R 5 , R 6 and R 7 are the same or different and each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R 5 , R 6 and R 7 are each independently selected from a hydrogen atom and a C1-C6 alkyl; More preferably, R 5 , R 6 and R 7 are each independently selected from a hydrogen atom; 2. A ligand-camptothecin derivative complex represented by general formula I according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

13. The crosslinking unit-L 1 -L 2 -L 3 -L 4 -L 5 - including but not limited to the following structures: 【Transformation 8】 Preferably, 【Chemistry 9】 Selected from: During the ceremony, Ac is a hydrophilic structural unit; R 5 , R 6 and R 7 are the same or different and each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R 5 , R 6 and R 7 are each independently selected from a hydrogen atom or a C1-C6 alkyl; More preferably, R 5 , R 6 and R 7 are each independently selected from a hydrogen atom; The carbon atom number 2 linked to N has absolute chiral configuration R or S; The left wavy line is connected to an antibody or antigen-binding fragment of an antibody, and the right wavy line is connected to an X; o is selected from integers from 1 to 10; 2. A ligand-camptothecin derivative complex represented by general formula I according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

14. General formula II 【Chemistry 10】 II is shown by During the ceremony, Ab is a human Trop2-targeting antibody or antigen-binding fragment thereof; L 1 is a bridging unit linked to Ab, including but not limited to: 【Chemistry 11】 Selected from: L 1 is preferably 【Chemistry 12】 and Preferably, L 1 teeth, 【Chemistry 13】 and L 3 is present or absent, and if present, L 3 teeth, 【Chemistry 14】 where o is selected from an integer from 1 to 10, preferably from 2 to 8; Ac is a hydrophilic structural unit; The chiral carbon atoms at positions 1, 2 and 3 have absolute chiral configuration R or S; R is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl; Preferably, R is selected from the group consisting of a hydrogen atom and a C1-C6 alkyl; R 1 is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a carboxyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R 1 is selected from the group consisting of a hydrogen atom and a C1-C6 alkyl; More preferably, R 1 is selected from C1-C6 alkyl; R 2 is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a carboxyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R 2 is selected from the group consisting of a hydrogen atom, halogen, and C1-C6 alkyl; More preferably, R 2 is selected from halogen; X is -C(O)-CR a R b - (CR 3 R 4 ) m -O-, -C(O)-CR a R b - (CR 3 R 4 ) m —NH— or —C(O)—CR a R b - (CR 3 R 4 ) m -S-; Preferably, X is —C(O)—CR a R b - (CR 3 R 4 ) m -O-; R a and R b are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a deuterated C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R a and R b are each independently selected from the group consisting of a hydrogen atom, a C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl, or a C6-C10 aryl C1-C6 alkyl; Or R a and R b and R a and R b are linked to form a C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, 3- to 7-membered heterocyclyl, or substituted 3- to 7-membered heterocyclyl; preferably, R a and R b and R a and R b the carbon atoms to which are linked form a C3-C8 cycloalkyl; R 3 and R 4 are the same or different and each independently represent a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a halogenated C1-C6 alkyl, a deuterated C1-C6 alkyl, a C1-C6 alkoxy, a hydroxyl, an amino, a cyano, a nitro, a hydroxyl C1-C6 alkyl, a C3-C8 cycloalkyl, a 3- to 7-membered heterocyclyl, or a substituted 3- to 7-membered heterocyclyl; Preferably, R 3 and R 4 are each independently a hydrogen atom or a C1-C6 alkyl; Or R 3 and R 4 and R 3 and R 4 the carbon atoms to which are linked form a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, or a 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl; m is selected from integers from 0 to 4, preferably 0 or 1; n is selected from integers from 1 to 10; A ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof.

15. Ac is formula B 【Chemistry 15】 B and having a structure represented by During the ceremony, Z is selected from the group consisting of, but not limited to, one or more of the hydrophilic structures carboxyl, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid, and polyethylene glycol (PEG); Preferably, Z is selected from the group consisting of the hydrophilic structures carboxyl, phosphate and PEG; Y' is an optional scaffold connecting -NH- and Z; preferably, Y' is C1-C6 alkylene; Ac is linked to the labeled 2-carbon of structural formula I via scaffold Y; The ligand-camptothecin derivative conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

16. Ac is, but is not limited to, glycine, (D / L) alanine, (D / L) leucine, (D / L) isoleucine, (D / L) valine, (D / L) phenylalanine, (D / L) proline, (D / L) tryptophan, (D / L) serine, (D / L) tyrosine, (D / L) cysteine, (D / L) cystine, (D / L) arginine, (D / L) histidine, (D / L) methionine, (D / L) asparagine, (D / L) glutamine, (D / L) threonine, (D / L) aspartic acid, (D / L) glutamic acid, a natural or unnatural amino acid derivative, or a compound having the following structure: 【Chemistry 16】 or isomers thereof; Preferably, 【Chemistry 17】 2. The ligand-camptothecin derivative conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, selected from the group consisting of:

17. 2. The ligand-camptothecin derivative conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein Ac is selected from the group consisting of, but not limited to, glycine, phosphate, (D / L) glutamic acid, and a polyethylene glycol hydrophilic structure. 【Request Item 18】 【Chemistry 18】 is the formula d 【Chemistry 19】 d and having a structure represented by During the ceremony, R is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl; Preferably, R is selected from the group consisting of a hydrogen atom and a C1-C6 alkyl; R 1 is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a carboxyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R 1 is selected from the group consisting of a hydrogen atom and a C1-C6 alkyl; More preferably, R 1 is selected from C1-C6 alkyl; R 2 is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a carboxyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R 2 is selected from the group consisting of a hydrogen atom, halogen, and C1-C6 alkyl; More preferably, R 2 is selected from halogen; R a and R b are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a deuterated C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Preferably, R a and R b are each independently selected from the group consisting of a hydrogen atom, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkyl, and C6-C10 aryl; Or R a and R b and R a and R b are linked to form a C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, 3- to 7-membered heterocyclyl, or substituted 3- to 7-membered heterocyclyl; preferably, R a and R b and R a and R b the carbon atoms to which are linked form a C3-C8 cycloalkyl; the chiral carbon atom at position 1 has absolute chiral configuration R or S; m is 0 or 1; The ligand-camptothecin derivative conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

19. Examples of compounds in which structural formula d is, but is not limited to, the following compounds: 【Chemistry 20】 【change】 The ligand-camptothecin derivative conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, selected from the group consisting of:

20. Formula III 【Chemistry 21】 III and having a structure represented by During the ceremony, R is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl; R a is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a deuterated C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; R b is selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a deuterated C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a C1-C6 alkoxyC1-C6 alkyl, a 3- to 7-membered heterocyclyl, a substituted 3- to 7-membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Or R a and R b and R a and R b the carbon atoms to which are linked form a C3-C8 cycloalkyl, a C3-C8 cycloalkylC1-C6 alkyl, a 3- to 7-membered heterocyclyl, and a substituted 3- to 7-membered heterocyclyl; L 3 is present or absent, and if present, L 3 teeth, 【Chemistry 22】 where o is selected from an integer from 1 to 10; the chiral carbon atom at position 1 or 2 has absolute chiral configuration R or S; Ac is a hydrophilic structural unit; m is 0 or 1; Preferably, it is used to bind with a ligand Ab to form a ligand-camptothecin derivative conjugate of formula I according to claim 1, or to bind with a ligand Ab to form a ligand-camptothecin derivative conjugate of formula II according to claim 14. A linker-drug compound or a pharmaceutically acceptable salt or solvate thereof.

21. Ac is formula B 【Chemistry 23】 B and having a structure represented by During the ceremony, Z is composed of one or more hydrophilic structures selected from the group consisting of carboxyl, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid, and polyethylene glycol (PEG); Y′ is an optional scaffold connecting —NH— and Z; Ac is linked to the labeled 2-carbon of structural formula I via scaffold Y; 21. The linker-drug compound of claim 20, or a pharmaceutically acceptable salt or solvate thereof.

22. Ac may be, but is not limited to, glycine, (D / L) alanine, (D / L) leucine, (D / L) isoleucine, (D / L) valine, (D / L) phenylalanine, (D / L) proline, (D / L) tryptophan, (D / L) serine, (D / L) tyrosine, (D / L) cysteine, (D / L) cystine, (D / L) arginine, (D / L) histidine, (D / L) methionine, (D / L) asparagine, (D / L) glutamine, (D / L) threonine, (D / L) aspartic acid, (D / L) glutamic acid, a natural or unnatural amino acid derivative, or a compound having the following structure: 【Chemistry 24】 21. The linker-drug compound of claim 20, or a pharmaceutically acceptable salt or solvate thereof, selected from:

23. 21. The linker-drug compound of claim 20, or a pharmaceutically acceptable salt or solvate thereof, wherein Ac is selected from the group consisting of, but not limited to, glycine, phosphate, (D / L) glutamic acid, and polyethylene glycol hydrophilic structures.

24. Linker-drug compounds may have, but are not limited to, the following structure: 【Chemistry 25】 【change】 【change】 【change】 or an isomer thereof, wherein o is selected from an integer from 1 to 10.

21. The linker-drug compound of claim 20, or a pharmaceutically acceptable salt or solvate thereof.

25. A method for preparing a ligand-camptothecin derivative conjugate represented by general formula I or general formula II, or a pharmaceutically acceptable salt or solvate thereof, comprising: 【Chemistry 26】 coupling the reduced antibody or antigen-binding fragment thereof with a linker-drug compound to obtain a ligand-camptothecin derivative conjugate of general formula I or general formula II; the chiral carbon atom at position 1, 2 or 3 has absolute chiral configuration R or S; In the step of obtaining a ligand-camptothecin derivative conjugate represented by general formula I, Ab, L 1 , L 2 , L 3 , L 4 , L 5 ,X,R,R 1 , R 2 and n is as defined in claim 1; In the step of obtaining a ligand-camptothecin derivative conjugate represented by general formula II, Ab, L1, L2, L3, L4, L5, X, R, R1, R2 and n are as defined in claim 14; method.

26. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof may have the following structure, but is not limited to: 【Chemistry 27】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 During the ceremony, hu4D3 is a human Trop2-targeting antibody or antigen-binding fragment thereof; n is selected from integers from 1 to 10; 26. The ligand-camptothecin derivative conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, selected from derivative structures thereof in which the ring of the succinimide group is open, and isomers thereof; or the method according to claim 25.

27. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof may have the following structure, but is not limited to: 【Chemistry 28】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 During the ceremony, hu7F11 is a human Trop2-targeting antibody or antigen-binding fragment thereof; n is selected from integers from 1 to 10; 26. The ligand-camptothecin derivative conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, selected from derivative structures thereof in which the ring of the succinimide group is open, and isomers thereof; or the method according to claim 25.

28. 21. The ligand-camptothecin derivative conjugate according to claim 1 or 14, or a pharmaceutically acceptable salt or solvate thereof, wherein the pharmaceutically acceptable salt is a sodium salt, potassium salt, calcium salt, or magnesium salt formed with an acidic functional group in the structural formula, and an acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate salt formed with a basic functional group in the structural formula; or the linker-drug compound according to claim 20, or a pharmaceutically acceptable salt or solvate thereof.

29. A pharmaceutical composition comprising the ligand-camptothecin derivative conjugate of claim 1 or 14, or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound of claim 20, or a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutically acceptable carrier.

30. Use of a pharmaceutical composition comprising the ligand-camptothecin derivative conjugate of claim 1 or 14, or a pharmaceutically acceptable salt or solvate thereof, or the ligand-camptothecin derivative conjugate of claim 1 or 14, or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound of claim 20, or a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutically acceptable carrier, in the preparation of a medicament for treating or preventing cancer or tumors. and Preferably, the cancer or tumor expresses TROP2; More preferably, the cancer or tumor is selected from solid tumors or hematological tumors, such as adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, triple-negative breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia. use.

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