Bispecific antibody-camptothecin drug conjugates and pharmaceutical uses thereof

Abstract

To provide a bispecific antibody-drug conjugate that exhibits the synergistic benefits of dual-targeted therapy. [Solution] A bispecific antibody that simultaneously targets two different epitopes or targets is coupled with a camptothecin drug to form a bispecific antibody-toxin conjugate, which is stable in therapy, has excellent homogeneity, and its drug-antibody ratio (DAR) is 6.0-8.0. The antibody-toxin conjugate has a structure represented by general formula (I), where Ab represents a bispecific antibody that simultaneously targets two different epitopes or targets and is coupled with a linker-camptothecin drug. Furthermore, methods for producing and purifying the antibody-toxin conjugate and its application in tumor therapy are provided. Furthermore, a linker-drug compound that can be coupled with Ab to form the antibody-toxin conjugate is provided. JPEG2026035591000517.jpg44161

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceuticals, specifically to antibody-drug conjugates formed by bispecific antibodies and camptothecin drugs, as well as methods for producing and applying the antibody-drug conjugates.The present invention also relates to linker-drug compounds that can be coupled with Abs to form antibody-toxin conjugates. [Background technology]

[0002] The epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (also known as HER3 and ErbB3) are receptor protein tyrosine kinases that belong to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases, which includes EGFR (ErbB-1), HER2 / c-neu (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4).

[0003] The epidermal growth factor receptor (EGFR) is a glycoprotein with a molecular weight of 170 kDa that spans the cell membrane and is activated upon ligand binding. Upon activation, EGFR converts from a monomer to an autodimer or forms a heterodimer with other HER family members. Dimerization activates intracellular kinase pathways, directing phosphorylation of downstream pathways such as MAPK, Akt, and JNK pathways, thereby inducing cell proliferation. Studies have shown that high EGFR expression is associated with tumor cell proliferation, angiogenesis, tumor invasion, and other conditions. EGFR-related signaling pathways play a crucial role in the maintenance and proliferation of epithelial tissues. EGFR may promote tumorigenesis, particularly in breast cancer, malignant glioma, and lung cancer. In lung cancer tissue, the EGFR signaling pathway exhibits a stimulated state, and there is a positive correlation between EGFR expression levels and the stage of cancer development. Meanwhile, due to the discovery of secondary EGFR mutations under drug stress, EGFR is increasingly being used as a biomarker for tumor drug resistance.

[0004] Epidermal growth factor receptor 3 (HER3 or ErbB3) also shares the structure of a typical epidermal growth factor receptor; however, HER3 lacks the structural domain of an intracellular protein tyrosine kinase and therefore cannot autophosphorylate. HER3 binds to ligand proteins and promotes their heterodimerization with other members of the human epidermal growth factor receptor family, activating receptor-mediated signaling pathways. This not only acts as a signal diversifier but also plays a role in signal amplification, promoting tumor progression. Heregulin (glial cell growth factor, neu differentiation factor) can activate intracellular kinase-dependent multistep signaling pathways upon binding to the transmembrane receptors HER3 and HER4. Downregulation of this signaling pathway can lead to Alzheimer's disease, heart failure, atherosclerosis, and cancer. Upregulation of HER3 expression levels can promote tumor formation and growth by interacting with receptor tyrosine kinases (RTKs). HER3 is also a heterodimeric molecular chaperone for other EGFR family members, and thus may modulate drug resistance mediated by the EGFR / HER2 signaling pathway in cancer cells. Studies have also shown that HER3 renders cancer treatments ineffective by activating the PI3K / AKT, MAPK / ERK, and JAK / STAT signaling pathways.

[0005] Overexpression / dysregulation of EGFR and HER3 are closely related to the development of various tumors. EGFR and HER3 have been shown to drive tumor progression in solid tumors such as breast cancer, lung cancer, gastric cancer, and pancreatic cancer. Several studies have shown that high expression of HER3 is associated with the clinical failure of EGFR antibodies and inhibitors. Several combination drug therapies targeting EGFR and HER3 are already underway in clinical settings.

[0006] Monoclonal antibodies have been widely used in anti-tumor treatment in recent years, but their efficacy leaves much to be desired. Many tumor patients experience poor clinical responses, and some patients who experience clinical responses develop drug resistance after a period of monoclonal antibody treatment, leading to tumor recurrence. Bispecific monoclonal antibodies are monoclonal antibody molecules with two different antigen recognition sequences and can bind to protein molecules of two antigen epitopes. Therefore, they can achieve various new and unique anti-tumor mechanisms, such as mediating tumor cell death by immune cells, mediating tumor cell death by toxic small molecules, or blocking signaling pathways that promote tumor growth. The development of bispecific monoclonal antibodies is primarily driven by the fact that multiple mediators are involved in tumor pathogenesis through specific or overlapping mechanisms. When multiple targets are blocked simultaneously, this results in a superior therapeutic effect than the inhibition of a single target. At the same time, the action of multiple targets significantly reduces the likelihood of drug resistance. Currently, two bispecific antibodies, catumaxomab and blinatumomab, are approved for sale in the United States, and more than 50 bispecific antibody molecules are in clinical trials.

[0007] Antibody-drug conjugates (ADCs) are molecules with specifically targeted killing properties. They are obtained by attaching small-molecule toxins with cell-killing properties to antibodies and are widely used in the treatment of tumors and other diseases. Antibody-drug coupled drugs use antibodies that can specifically bind to proteins on the surface of tumor cells, thus providing tumor specificity and potential unattainable by conventional drugs. Currently, 12 ADC drugs have been approved for sale worldwide, and hundreds of programs are in clinical trials. However, most ADC programs currently on the market or in clinical settings are directed at a single target, preventing the synergistic benefits of dual-targeted therapy. Summary of the Invention [Means for solving the problem]

[0008] Based on a comprehensive understanding of the ADC class of drugs, the present inventors disclose bispecific antibody-drug conjugates, methods for producing the same, pharmaceutical compositions containing the conjugates, and uses of the conjugates or pharmaceutical compositions. The present invention also relates to linker-drug compounds that can be coupled to bispecific antibodies to form antibody-toxin conjugates.

[0009] A first aspect of the present invention discloses a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharmaceutically acceptable salt or solvate thereof; [ka] In the above formula: Ab is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets two different epitopes or targets; L1 is a member of the following group: [ka] Selected without limitation from; Preferably, L1 is [ka] and; Preferably, L1 is [ka] and; L2 has the structure shown in Formula A below: [ka] In the above formula, Y is a scaffold selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl; preferably, Y is C1-C6 alkyl; Ac is a hydrophilic structural unit; and the carbon atom of number 2 bonded to Y has absolute chirality in the R or S configuration; L3 may be present or absent, and if present, L3 is a PEG hydrophilic unit: [ka] where o is selected from an integer ranging from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably an integer ranging from 2 to 8; L4 is the enzyme cleavage unit: L5 is the linking unit; In formula I, the chiral carbon atom of number 1 attached to N has absolute chirality in the R or S configuration; R is selected from a hydrogen atom, a deuterium atom, a halogen, 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 C6-C10 aryl, a substituted C6-C10 aryl, a 5-10 membered heteroaryl, and a substituted 5-10 membered heteroaryl; Preferably, R is selected from a hydrogen atom or a C1-C6 alkyl group; R1 is selected from 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, or a substituted 5- to 10-membered heteroaryl; Preferably, R1 is selected from a hydrogen atom or a C1-C6 alkyl group; More preferably, R1 is selected from C1-C6 alkyl; R2 is selected from 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 a hydrogen atom, a halogen, or a C1-C6 alkyl group; 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- selected from; Preferably, X is -C(O)-CR a R b -(CR3R4) m -O-; R a and R b are each independently selected from 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 heterocyclic group, a substituted 3- to 7-membered heterocyclic group, 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 a hydrogen atom, a C1-C6 alkyl, a halo-C1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, or a C6-C10 arylC1-C6 alkyl; Instead, R a , R b and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclyl group, or a substituted 3- to 7-membered heterocyclyl group; preferably R a , R b and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group; R3 and R4 are the same or different, and each independently represents 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 hydroxy 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 group; Alternatively, R3, R4 and the carbon atoms attached thereto comprise a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclic group, or a substituted 3- to 7-membered heterocyclic group; m is selected from integers of 0 to 4 (eg, 0, 1, 2, 3, or 4), preferably 0 and 1; and n is selected from integers of 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0010] In some embodiments of the first aspect of the present invention, there is disclosed a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharmaceutically acceptable salt or solvate thereof, characterized in that the Ab is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets two different epitopes or targets, preferably a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3.

[0011] In some embodiments of the first aspect of the present invention, the disclosure provides a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab antibody comprises: an IgG1 heavy chain, a κ light chain, and a single-chain Fv (scFv) structural domain; the single-chain Fv (scFv) structural domain forms a construct with an IgG1 heavy chain or a κ light chain; the IgG1 heavy chain and the κ light chain form an IgG moiety having binding specificity for EGFR; the scFv structural domain has binding specificity for HER3, and the scFv structural domain is linked to a linker (e.g., (gly-gly-gly-gly- the single-chain Fv structural domain has the structural order N-terminus-heavy chain variable region-joint-light chain variable region-C-terminus, or N-terminus-light chain variable region-joint-heavy chain variable region-C-terminus (for example, the joint consists of the amino acid sequence (gly-gly-gly-gly-ser)m, where m is an integer of at least 3, and preferably m is 3, 4, 5, or 6).

[0012] In some embodiments of the first aspect of the present invention, the disclosure provides a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, characterized in that the Ab antibody's kappa light chain comprises the CDRs set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, the IgG1 heavy chain comprises the CDRs set forth in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, and the single-chain Fv (scFv) structural domain comprises the heavy chain variable region CDRs set forth in SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, and the light chain variable region CDRs set forth in SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37.

[0013] 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, characterized in that the Ab antibody light chain comprises a variable region set forth in SEQ ID NO: 28, the IgG1 heavy chain comprises a variable region set forth in SEQ ID NO: 38, and the single-chain Fv (scFv) structural domain comprises a heavy chain variable region set forth in SEQ ID NO: 39 and a light chain variable region set forth in SEQ ID NO: 40.

[0014] 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, characterized in that the amino acid sequence of the Ab antibody light chain is SEQ ID NO: 2 and the amino acid sequence of the construct of the antibody heavy chain and single-chain Fv (scFv) structural domain is SEQ ID NO: 4.

[0015] In some embodiments of the first aspect of the present invention, there is disclosed a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharmaceutically acceptable salt or solvate thereof, characterized in that the nucleic acid coding sequence of the Ab antibody light chain is SEQ ID NO: 1 and the nucleic acid coding sequence of the construct of the antibody heavy chain and single-chain Fv (scFv) structural domain is SEQ ID NO: 3.

[0016] 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, characterized in that the Ab antibody's kappa light chain comprises the CDRs set forth in SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, the IgG1 heavy chain comprises the CDRs set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, and the single-chain Fv (scFv) structural domain comprises the heavy chain variable region CDRs set forth in SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, and the light chain variable region CDRs set forth in SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37.

[0017] 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, characterized in that the Ab antibody light chain comprises a variable region set forth in SEQ ID NO: 44, the IgG1 heavy chain comprises a variable region set forth in SEQ ID NO: 48, and the single-chain Fv (scFv) structural domain comprises a heavy chain variable region set forth in SEQ ID NO: 39 and a light chain variable region set forth in SEQ ID NO: 40.

[0018] 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, characterized in that the amino acid sequence of the Ab antibody light chain is SEQ ID NO: 6 and the amino acid sequence of the construct of the antibody heavy chain and single-chain Fv (scFv) structural domain is SEQ ID NO: 8.

[0019] 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, characterized in that the nucleic acid coding sequence of the Ab antibody light chain is SEQ ID NO: 5 and the nucleic acid coding sequence of the antibody heavy chain and single-chain Fv (scFv) structural domain construct is SEQ ID NO: 7.

[0020] 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, characterized in that the Ab antibody light chain comprises a variable region set forth in SEQ ID NO: 49, the IgG1 heavy chain comprises a variable region set forth in SEQ ID NO: 52, and the single-chain Fv (scFv) structural domain comprises a heavy chain variable region set forth in SEQ ID NO: 50 and a light chain variable region set forth in SEQ ID NO: 51.

[0021] 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, characterized in that the Ab antibody has the heavy chain amino acid sequence of SEQ ID NO: 12, and the antibody light chain and single-chain Fv (scFv) structural domain construct has the amino acid sequence of SEQ ID NO: 10.

[0022] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the Ab antibody has a heavy chain nucleic acid coding sequence of SEQ ID NO: 11, and the construct of the antibody light chain and single-chain Fv (scFv) structural domains has the nucleic acid coding sequence of SEQ ID NO: 9.

[0023] 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, characterized in that the Ab antibody has the light chain amino acid sequence of SEQ ID NO: 14, and the antibody heavy chain and single-chain Fv (scFv) structural domain construct has the amino acid sequence of SEQ ID NO: 16.

[0024] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the Ab antibody has a light chain nucleic acid coding sequence of SEQ ID NO: 13, and the antibody heavy chain and single-chain Fv (scFv) structural domain construct has a nucleic acid coding sequence of SEQ ID NO: 15.

[0025] 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, characterized in that the Ab antibody has the heavy chain amino acid sequence of SEQ ID NO: 20, and the antibody light chain and single-chain Fv (scFv) structural domain construct has the amino acid sequence of SEQ ID NO: 18.

[0026] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the Ab antibody has a heavy chain nucleic acid coding sequence of SEQ ID NO: 19, and the construct of the antibody light chain and single-chain Fv (scFv) structural domains has the nucleic acid coding sequence of SEQ ID NO: 17.

[0027] 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, characterized in that the Ab antibody light chain comprises a variable region set forth in SEQ ID NO: 53, the IgG1 heavy chain comprises a variable region set forth in SEQ ID NO: 54, and the single-chain Fv (scFv) structural domain comprises a heavy chain variable region set forth in SEQ ID NO: 50 and a light chain variable region set forth in SEQ ID NO: 51.

[0028] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the Ab antibody has a heavy chain amino acid sequence of SEQ ID NO: 24, and the amino acid sequence of the construct of the antibody light chain and single-chain Fv (scFv) structural domain is SEQ ID NO: 22.

[0029] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the Ab antibody has a heavy chain nucleic acid coding sequence of SEQ ID NO: 23, and the antibody light chain and single-chain Fv (scFv) structural domain construct has the nucleic acid coding sequence of SEQ ID NO: 21.

[0030] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharmaceutically acceptable salt or solvate thereof, is disclosed, characterized in that the Ab antibody comprises: two IgG1 heavy chains; two κ light chains; and two single-chain Fv (scFv) structural domains.

[0031] In some embodiments of the first aspect of the present invention, X has the following structure or an isomer thereof: [ka] The present invention provides a ligand-camptothecin derivative conjugate of general formula I, or a pharmaceutically acceptable salt or solvate thereof, characterized in that the ligand-camptothecin derivative conjugate is selected without limitation from the group consisting of: In the above formula, the left wavy line is connected to the camptothecin derivative moiety, and the right wavy line is connected to L5.

[0032] In some embodiments of the first aspect of the present invention, there is disclosed a ligand-camptothecin derivative conjugate according to general formula I, or a pharmaceutically acceptable salt or solvate thereof, characterized in that L4 is selected without limitation from a peptide residue formed of an amino acid: wherein the amino acid is optionally further substituted with one or more substituents selected from deuterium atom, 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 formed from one, two or more amino acids selected from 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).

[0033] Particularly preferably, the peptide residue is -GGFG-.

[0034] In some embodiments of the first aspect of the invention, as follows: L5 is -NR5(CR6R7) q- or a chemical bond, where q is selected from an integer from 0 to 6 (e.g., 0, 1, 2, 3, 4, 5, or 6); R5, R6 and R7 are the same or different, and each is independently selected from a hydrogen atom, a deuterium atom, a halogen atom, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a 3- to 7-membered heterocyclyl group, a substituted 3- to 7-membered heterocyclyl group, a C6-C10 aryl group, 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 group; More preferably, there is disclosed a ligand-camptothecin derivative conjugate according to general formula I, or a pharmaceutically acceptable salt or solvate thereof, characterized in that R5, R6 and R7 are each independently selected from a hydrogen atom.

[0035] In certain embodiments, L1 is: [ka] can be selected without restriction.

[0036] In some embodiments of the first aspect of the present invention, the linking unit -L1-L2-L3-L4-L5- has the following structure: [ka] Preferably [ka] can be selected without restriction from In the above formula, Ac is a hydrophilic structural unit; R5, R6 and R7 are the same or different and are each independently selected from hydrogen atoms, deuterium atoms, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl; Preferably, R5, R6 and R7 are each independently selected from a hydrogen atom or a C1-C6 alkyl group; More preferably, R5, R6 and R7 are each independently selected from a hydrogen atom; The carbon atom numbered 2 attached to N has absolute chirality in the R or S configuration; The left wavy line is connected to an antibody or antigen-binding fragment thereof portion, and the right wavy line is connected to an X; o is selected from integers from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0037] A second aspect of the present invention relates to a ligand-camptothecin derivative conjugate of general formula II, or a pharmaceutically acceptable salt or solvate thereof; [ka] Disclose the In the above formula, Ab is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3; L1 is [ka] a linking unit linked to the Ab, selected without limitation from: Preferably, L1 is [ka] and; Preferably, L1 is [ka] and L3 is either present or absent, and if present, L3 is [ka] wherein o is selected from an integer ranging from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably an integer ranging from 2 to 8; Ac is a hydrophilic structural unit; The chiral carbon atoms at positions 1, 2, and 3 have two chiral configurations, namely, the R absolute configuration or the S absolute configuration; R is selected from a hydrogen atom, a deuterium atom, a halogen, 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 C6-C10 aryl, a substituted C6-C10 aryl, a 5-10 membered heteroaryl, and a substituted 5-10 membered heteroaryl; Preferably, R is selected from a hydrogen atom or a C1-C6 alkyl group; R1 is selected from 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 a hydrogen atom or a C1-C6 alkyl group; More preferably, R1 is selected from C1-C6 alkyl; R2 is selected from 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 a hydrogen atom, a halogen, or a C1-C6 alkyl group; 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- selected from; Preferably, X is —C(O)—CR a R b -(CR3R4) m -O-; R a and R b are each independently selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a 3- to 7-membered heterocyclic group, a substituted 3- to 7-membered heterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, 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 a hydrogen atom, a C1-C6 alkyl, a haloC1-C6 alkyl, a C3-C8 cycloalkylC1-C6 alkyl, or a C6-C10 arylC1-C6 alkyl; Instead, R a , R band the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclyl group, or a substituted 3- to 7-membered heterocyclyl group; preferably R a , R b and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group; R3 and R4 are the same or different and 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 hydroxy 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 independently a hydrogen atom or a C1-C6 alkyl group; Alternatively, R3, R4 and the carbon atoms bonded thereto form a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclic group, or a substituted 3- to 7-membered heterocyclic group; m is an integer selected from 0 to 4 (i.e., 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).

[0038] In some embodiments of the first and second aspects of the present invention, the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, is such that Ac is a compound of formula B: [ka] It is disclosed that the compound has the structure shown in In the above formula, Z is selected from the group consisting of one or more of a hydrophilic structure, a carboxyl group, a phosphoric acid, a polyphosphoric acid, a phosphorous acid, a sulfonic acid, a sulfinic acid, or a polyethylene glycol (PEG), without limitation; Preferably, Z is selected from the hydrophilic structure of a carboxyl group, a phosphate group, or a polyethylene glycol (PEG); Y' is optionally a scaffold linking an amino group to Z; preferably, Y' is a C1-C6 alkylene group (e.g., methylene); Ac is linked to the carbon at position 2, which is labeled in structural formula I, by scaffold Y.

[0039] In some embodiments of the first and second aspects of the present invention, the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, is characterized in that Ac is 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 the following structures or isomers thereof: [ka] Preferably, [ka] It is disclosed that the present invention is characterized by being selected without limitation from:

[0040] In some embodiments of the first and second aspects of the present invention, the disclosure of the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is characterized in that Ac is selected from, without limitation, glycine, phosphate, (D / L) glutamic acid, or a polyethylene glycol hydrophilic structure.

[0041] In some embodiments of the first and second aspects of the present invention, the disclosure of the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof comprises [ka] is the following formula d: [ka] It has the structure shown in In the above formula, R is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl, a C1-C6 alkoxy C1-C6 alkyl, 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 is selected from a hydrogen atom or a C1-C6 alkyl group; R1 is selected from 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 a hydrogen atom or a C1-C6 alkyl group; More preferably, R1 is selected from C1-C6 alkyl; R2 is selected from 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 a hydrogen atom, a halogen, or a C1-C6 alkyl group; More preferably, R2 is selected from halogen; R a and R bare each independently selected from 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 heterocyclic group, a substituted 3- to 7-membered heterocyclic group, 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 a hydrogen atom, C1-C6 alkyl, halo-C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or C6-C10 arylC1-C6 alkyl; Preferably R a and R b are each independently selected from a hydrogen atom, C1-C6 alkyl, halo-C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkyl, and C6-C10 aryl; preferably R a and R b are each independently selected from a hydrogen atom, a methyl group, an ethyl group, a trifluoromethyl group, a cyclopropylmethyl group, and a phenyl group; Instead, R a , R b and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclyl group, or a substituted 3- to 7-membered heterocyclyl group; preferably, R a , R b and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group (e.g., a C3-C5 cycloalkyl group); The chiral carbon atom at position 1 has two chiral configurations, namely, the R absolute configuration or the S absolute configuration; m is selected from 0 or 1.

[0042] In some embodiments of the first and second aspects of the present invention, the disclosure of the ligand-camptothecin derivative conjugate or pharmaceutically acceptable salt or solvate thereof further comprises the structure d being the following compound: [ka] [ka] It is characterized in that it is selected without limitation from:

[0043] In some embodiments of the present invention, L1 may contain a succinimide group. In these embodiments, the Ligand-Drug Conjugate may undergo hydrolysis under readily hydrolyzable conditions, with the site of hydrolysis being the succinimide group of the Linker unit. When the Ligand contains multiple Linker-Drugs, the following scenarios may occur with varying degrees of hydrolysis: The succinimide groups are completely non-hydrolyzable, i.e., all succinimide groups are in the closed ring form. [ka] and; Incomplete hydrolysis of the succinimide group, i.e., some of the succinimide groups are in the cyclic form [ka] and a part of the succinimide groups is in a ring-opened form. [ka] and; Complete hydrolysis of succinimide groups, i.e., all succinimide groups are ring-opened [ka] is.

[0044] Thus, when multiple L1s containing succinimide groups are simultaneously present in an ADC (i.e., Ab is linked to multiple drug-linkers containing succinimide groups), these succinimide groups can all be ring-closed, some may be ring-open, or all may be ring-open.

[0045] It will be understood that even though the succinimide group appearing in the chemical structure of the ADC is in closed ring form, the present application actually encompasses three scenarios: all closed, some open, and all open succinimides. A third aspect of the present invention is a compound of formula III: [ka] The present invention discloses a linker-drug compound, or a pharmaceutically acceptable salt or solvate thereof, characterized in that it has the structure shown in In the above formula, R is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl, a C1-C6 alkoxy C1-C6 alkyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; R a is selected from 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 heterocyclic group, a substituted 3- to 7-membered heterocyclic group, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; R bis selected from 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 heterocyclic group, a substituted 3- to 7-membered heterocyclic group, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Instead, R a , R b and the carbon atoms bonded thereto constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclic group, or a substituted 3- to 7-membered heterocyclic group; Preferably, R a and R b are each independently selected from a hydrogen atom, C1-C6 alkyl, halo-C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 alkyl, and C6-C10 aryl; preferably, R a and R b are each independently selected from a hydrogen atom, a methyl group, an ethyl group, a trifluoromethyl group, a cyclopropylmethyl group, and a phenyl group; Instead, R a , R b and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclyl group, or a substituted 3- to 7-membered heterocyclyl group; preferably, R a , R b and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group (e.g., a C3-C5 cycloalkyl group); L3 is either 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 two chiralities, i.e., R or S absolute configuration; Ac is a hydrophilic structural unit; m is selected from 0 or 1.

[0046] In some embodiments of the third aspect of the present invention, the linker-drug compound, or a pharmaceutically acceptable salt or solvate thereof, is a compound wherein Ac is a group of formula B: [ka] It has the structure shown in In the above formula, Z is selected from the group consisting of one or more of a hydrophilic structure, a carboxyl group, a phosphoric acid, a polyphosphoric acid, a phosphorous acid, a sulfonic acid, a sulfinic acid, or a polyethylene glycol (PEG), without limitation; Y' is any scaffold that links an amino acid to Z; preferably, Y' is a C1-C6 alkylene group (e.g., methylene); It is disclosed that Ac is characterized in that it is linked to the 2-carbon labeled in structural formula I by a scaffold Y.

[0047] In some embodiments of the third aspect of the present invention, Ac is selected from the group consisting of 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] Disclosed is a linker-drug compound, or a pharmaceutically acceptable salt or solvate thereof, characterized in that it is selected without limitation from:

[0048] In some embodiments of the third aspect of the present invention, there is disclosed a linker-drug compound, or a pharmaceutically acceptable salt or solvate thereof, characterized in that Ac is selected, without limitation, from glycine, phosphate, (D / L) glutamic acid, or a polyethylene glycol hydrophilic structure.

[0049] In some embodiments of the third aspect of the invention, the linker-drug compound has the following structure: [ka] [ka] [ka] [ka] [ka] (wherein o is selected from an integer of 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)) Disclosed is a linker-drug compound, or a pharmaceutically acceptable salt or solvate thereof, characterized in that it is selected without limitation from:

[0050] The linker-drug compound disclosed in the third aspect of the present invention, or a pharmaceutically acceptable salt or solvate thereof, may be used as an intermediate for coupling with a ligand Ab to form a ligand-camptothecin derivative conjugate of formula I or formula II described in the first and second aspects.

[0051] In a fourth aspect of the present invention, there is provided a method for producing a ligand-camptothecin derivative conjugate of general formula I or general formula II according to the first and second aspects, or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of: [ka] Including, A ligand-camptothecin derivative conjugate shown in general formula I or general formula II is obtained by a coupling reaction between a linker-drug compound and a reduced antibody or an antigen-binding fragment thereof; the chiral carbon atom at position 1, 2, or 3 has absolute chirality in the R or S configuration; wherein Ab, L1, L2, L3, L4, L5, X, R, R1, R2 and n are as defined above.

[0052] The present application also relates to the use of a linker-drug compound as disclosed and described in the third aspect, or a pharmaceutically acceptable salt or solvate thereof, 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 disclosed in the first, second, and fourth aspects of the present invention. In certain embodiments, the preparation is carried out according to the preparation method disclosed in the fourth aspect.

[0053] In some embodiments of the first, second, and fourth aspects of the present invention, the disclosure of the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, further provides that the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] are selected without limitation from In the above formula, SI-1×6.4 is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3; n is selected from an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0054] In some embodiments of the first, second, and fourth aspects of the present invention, the disclosure of the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, further provides that the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] are selected without limitation from In the above formula, SI-1×4 is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3; n is selected from an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0055] In some embodiments of the first, second, and fourth aspects of the present invention, the disclosure of the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, further provides that the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, has the following structure: [ka] are selected without limitation from In the above formula, SI-1×22 is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3; n is selected from an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0056] In some embodiments of the first, second, and fourth aspects of the present invention, the disclosure of the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, further provides that the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, has the following structure: [ka] are selected without limitation from In the above formula, SI-1×24 is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3; n is selected from an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0057] In some embodiments of the first, second, and fourth aspects of the present invention, the disclosure of the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, further provides that the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, has the following structure: [ka] are selected without limitation from In the above formula, SI-1×25 is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3; n is selected from an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0058] In some embodiments of the first, second, and fourth aspects of the present invention, the disclosure of the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, further provides that the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, has the following structure: [ka] are selected without limitation from In the above formula, SI-1×26 is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3; n is selected from an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0059] In some embodiments of the first, second, and third aspects of the present invention, the disclosure of the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof, is characterized in that the pharmaceutically acceptable salt comprises a sodium, potassium, calcium, or magnesium salt formed with an acidic functional group in the structure, 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 structure.

[0060] In a fifth aspect of the present invention, there is disclosed a pharmaceutical composition comprising a ligand-camptothecin derivative conjugate according to the first and second aspects, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound according to the third aspect, or a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutically acceptable carrier.

[0061] In a sixth aspect of the present invention, there is disclosed a pharmaceutical composition comprising a ligand-camptothecin derivative conjugate according to the first and second aspects, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound according to the third aspect, or a pharmaceutically acceptable salt or solvate thereof.

[0062] In a seventh aspect of the present invention, there is provided use of: a ligand-camptothecin derivative conjugate according to the first and second aspects, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound according to the third aspect, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to the fifth aspect and / or a pharmaceutical formulation according to the sixth aspect, in the manufacture of a medicament for treating or preventing cancer or tumors; Alternatively, the present invention discloses the use of a ligand-camptothecin derivative conjugate according to the first and second aspects, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound according to the third aspect, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to the fifth aspect and / or a pharmaceutical formulation according to the sixth aspect, in treating or preventing cancer or tumors, Preferably, the cancer or tumor expresses EGFR and / or HER3; More preferably, the cancer or tumor is selected from 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, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia, and other solid tumors or blood cancers.

[0063] In an eighth aspect of the present invention, there is provided a method for treating or preventing cancer or tumors, comprising administering to a subject in need thereof a prophylactically effective or therapeutically effective amount of a ligand-camptothecin derivative conjugate according to the first and second aspects, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound according to the third aspect, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to the fifth aspect and / or a pharmaceutical formulation according to the sixth aspect; Preferably, the cancer or tumor expresses EGFR and / or HER3; More preferably, the cancer or tumor is selected from 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, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia, and other solid tumors or blood cancers.

[0064] In the above aspects and embodiments of the present invention, "C1-C6 alkyl" and "C1-C6 alkyl" in various composite groups containing "C1-C6 alkyl" (e.g., "substituted C1-C6 alkyl", "deuterated C1-C6 alkyl") may be substituted with "C1-C20 alkyl", "C1-C12 alkyl", or "C1-C10 alkyl"; "C3-C8 cycloalkyl" and "C3-C8 cycloalkyl" in various composite 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 composite groups containing it can be substituted with "C1-C20 alkoxy", "C1-C12 alkoxy" or "C1-C10 alkoxy"; "C6-C10 aryl" and "C6-C10 aryl" in various composite groups containing it can be substituted with "C6-C12 aryl"; The "3- to 7-membered heterocyclic group" and the "3- to 7-membered heterocyclic group" in various composite groups containing it can be substituted with a "3- to 20-membered heterocyclic group", a "3- to 12-membered heterocyclic group" or a "3- to 10-membered heterocyclic group".

[0065] Beneficial effects The EGFR / HER3 bispecific antibody-drug conjugate provided by the present invention is a bispecific antibody ADC that targets both EGFR and HER3, and is expected to have good molecular stability, good preclinical efficacy, and excellent clinical therapeutic effects. [Brief explanation of the drawings]

[0066] [Figure 1A] SEC-HPLC detection / measurement of ADC-5 aggregation. [Figure 1B] SEC-HPLC detection / measurement of ADC-6 aggregation. [Figure 1C] SEC-HPLC detection / measurement of ADC-64 aggregation. [Figure 1D] SEC-HPLC detection / measurement of ADC-DS aggregation. [Figure 1E]SEC-HPLC detection / measurement of ADC-108 aggregation. [Figure 1F] SEC-HPLC detection / measurement of ADC-112 aggregation. [Figure 1G] SEC-HPLC detection / measurement of ADC-215 aggregation. [Figure 1H] SEC-HPLC detection / measurement of ADC-219 aggregation. [Figure 1I] SEC-HPLC detection / measurement of ADC-227 aggregation. [Figure 1J] SEC-HPLC detection / measurement of ADC-235 aggregation. [Figure 2A] RP-HPLC detection / measurement of ADC-5 drug-antibody conjugation ratio (DAR). [Figure 2B] RP-HPLC detection / measurement of ADC-6 drug-antibody conjugation ratio (DAR). [Figure 2C] RP-HPLC detection / measurement of ADC-10 drug-antibody conjugation ratio (DAR). [Figure 2D] RP-HPLC detection / measurement of ADC-12 drug-antibody conjugation ratio (DAR). [Figure 2E] RP-HPLC detection / measurement of ADC-64 drug-antibody conjugation ratio (DAR). [Figure 2F] Figure 1 shows RP-HPLC detection / measurement of ADC-108 drug-antibody conjugation ratio (DAR). [Figure 2G] RP-HPLC detection / measurement of ADC-112 drug-antibody conjugation ratio (DAR). [Figure 2H] RP-HPLC detection / measurement of ADC-215 drug-antibody conjugation ratio (DAR). [Figure 2I] Figure 1 shows RP-HPLC detection / measurement of ADC-219 drug-antibody conjugation ratio (DAR). [Figure 2J] Figure 1 shows RP-HPLC detection / measurement of ADC-227 drug-antibody conjugation ratio (DAR). [Figure 2K] RP-HPLC detection / measurement of ADC-235 drug-antibody conjugation ratio (DAR). [Figure 2L]RP-HPLC detection / measurement of ADC-243 drug-antibody conjugation ratio (DAR). [Figure 3A] 1 shows that the ADC-112 and SI-1x4 antibodies maintain affinity for both antigens EGFR and HER3-Fc. [Figure 3B] 1 shows that the ADC-6 and SI-1x6.4 antibodies maintain affinity for both antigens EGFR and HER3-Fc. [Figure 3C] 1 shows that the ADC-219 and SI-1x22 antibodies maintain affinity for both antigens EGFR and HER3-Fc. [Figure 3D] 1 shows that the ADC-227 and SI-1x24 antibodies maintain affinity for both antigens EGFR and HER3-Fc. [Figure 3E] 1 shows that the ADC-235 and SI-1x25 antibodies maintain affinity for both antigens EGFR and HER3-Fc. [Figure 3F] 1 shows that the ADC-243 and SI-1x26 antibodies maintain affinity for both antigens EGFR and HER3-Fc. [Figure 4A] Shows the in vitro efficacy of six naked antibodies as well as six ADCs in A431. [Figure 4B] Figure 1 shows the in vitro efficacy of six naked antibodies as well as six ADCs on BXPC-3. [Figure 4C] The in vitro efficacy of six naked antibodies as well as six ADCs in FaDu is shown. [Figure 4D] Shows the in vitro efficacy of six naked antibodies as well as six ADCs in HARA-B. [Figure 4E] Shows the in vitro efficacy of six naked antibodies as well as six ADCs in HCC827. [Figure 4F] Figure 1 shows the in vitro efficacy of six naked antibodies as well as six ADCs in SW620. [Figure 5A]1 shows the results of in vivo efficacy studies of ADC-6 and SI-1x6.4 naked antibodies in the A431 single tumor model. [Figure 5B] 1 shows the results of in vivo efficacy experiments of ADC-6, SI-1x6.4 naked antibody, cetuximab (Cet), and ADC-214 in the SW620 single tumor model. [Figure 5C] 1 shows the results of in vivo efficacy experiments of ADC-6 and SI-1×6.4 naked antibodies in A431+SW620 xenogeneic tumors. [Figure 6A] 1 shows the results of in vivo efficacy studies of ADC-6, ADC-219, ADC-235, ADC-227, and ADC-112 in the A431 single tumor model. [Figure 6B] 1 shows the results of in vivo efficacy studies of ADC-6, ADC-219, ADC-235, ADC-227, and ADC-112 in a BXPC-3 single tumor model. [Figure 7A] Figure 1 shows the in vitro efficacy of SI-1x6.4, cetuximab, ADC-6, ADC-214 and d3 against the human poorly differentiated lung squamous cell carcinoma cell line Oka-c-1. [Figure 7B] 1 shows the in vitro efficacy of SI-1×6.4, cetuximab, ADC-6, ADC-214, and d3 against human lung squamous cell carcinoma cells SK-MES-1. DETAILED DESCRIPTION OF THE INVENTION

[0067] Abbreviations and Definitions Unless otherwise specified, the following terms and phrases as used herein are intended to have the meanings set forth below: When trade names are used herein, unless the context indicates otherwise, the trade name includes the product ingredients, generic drugs, and active ingredients of said trade name.

[0068] Unless specified to the contrary, terms used in the claims and this specification have the meanings set forth below.

[0069] The term "ligand" refers to a macromolecular compound capable of recognizing and binding to an antigen or receptor associated with a target cell. Ligands serve to present drugs to a target cell population that is bound to the ligand; these ligands include, but are not limited to, proteinaceous hormones, lectins, growth factors, antibodies, or other molecules capable of binding to cells. In an embodiment of the present invention, the ligand is designated as Ab, and the ligand can form a linkage bond with a linkage unit via a heteroatom on the ligand, and is preferably an antibody or an antigen-binding fragment thereof, wherein the antibody is selected from chimeric, humanized, fully human, or murine antibodies; preferably, a monoclonal antibody.

[0070] The Ligand unit is a targeting agent that specifically binds to a targeting moiety. The Ligand can specifically bind to a cellular component, or can bind to a cellular component, or can bind to other target molecules of interest. The targeting moiety or target is generally located on the surface of a cell. 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, peptides, and 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 transporter molecules, or any other cell-binding molecule or substance. In some embodiments, the linker is covalently bonded to the sulfur atom of the Ligand. In some embodiments, the sulfur atom is the sulfur atom of a cysteine ​​residue, forming an interchain disulfide bond of an antibody. In another aspect, the sulfur atom is the sulfur atom of a cysteine ​​residue that has been introduced into the Ligand unit and forms the interchain disulfide bond of the antibody. In another aspect, the sulfur atom is the sulfur atom of a cysteine ​​residue that has been introduced into the Ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In other aspects, the linker-bonded sulfur atom is selected from cysteine ​​residues that form the interchain disulfide bond of the antibody or cysteine ​​residues that have been introduced into the Ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In some embodiments, the system is numbered according to the EU index in Kabat {[Kabat EA et al, (1991)] "Sequences of proteins of Immunological Interest", Fifth Edition, NIH Publication 91-3242}.

[0071] As used herein, "antibody" or "antibody unit" includes, to that extent, any portion of the antibody structure. This unit may bind, reactively associate, or form a complex with a receptor, antigen, or other receptor unit possessed by a target cell population. An antibody may be any protein or protein-like molecule capable of binding, forming a complex, or reacting with a portion of a cell population to be treated or biologically modified. Antibodies forming antibody-drug conjugates of the present invention maintain their original antigen-binding ability from their wild-type state. Thus, antibodies of the present invention can exclusively bind to antigens. Relevant antigens include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell growth regulators, molecules associated with tissue growth and differentiation (e.g., known or predicted to be functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules involved in angiogenesis (e.g., known or predicted to be functional). Tumor-associated factors may be cluster differentiation factors (e.g., CD proteins).

[0072] 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 produced using antibody production methods and information well known in the art. To develop effective cellular targets that can be used in cancer diagnosis and treatment, researchers are seeking to discover transmembrane or other tumor-associated peptides. These targets can be specifically expressed on the surface of one or more cancer cells, with little or no expression on the surface of one or more non-cancerous cells. Generally, 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 greatly improve the specific targeting properties of antibody-based cancer therapy. For convenience, information related to antigens known in the art, such as names, other names, and GenBank accession numbers, is listed below. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in publicly available databases such as GenBank. Tumor-associated antigens suitable for antibody targeting include all amino acid sequence variants and isoforms that have at least 70%, 80%, 85%, 90%, or 95% homology with the sequence identified in the reference sequence or that possess biological properties and characteristics identical to the tumor-associated antigen sequence in the literature.

[0073] The terms "inhibit" or "inhibition" mean that the detectable amount is reduced or prevented completely.

[0074] The term "cancer" means a physiological condition or disease characterized by dysregulated cell growth. A "tumor" includes cancer cells.

[0075] The term "autoimmune disease" refers to a disease or disorder that originates in the tissues or proteins of an individual's own body, targeting it.

[0076] The term "drug" refers to a cytotoxic drug, designated by d, which is a chemical molecule with a strong ability to disrupt normal growth in tumor cells. Cytotoxic drugs, in principle, kill tumor cells at sufficiently high concentrations, but due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, thereby causing severe side effects. The term also refers to toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (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 nucleases, preferably toxic drugs.

[0077] The term "linker" or "linker fragment" or "linker unit" means a chemical structure fragment or bond that can be attached to a ligand on one end and to a drug on the other end, or to another connector and then to a drug.

[0078] Connectors containing extensions, spacers, and amino acid units can be synthesized by methods known in the art, such as those described in U.S. Patent Application Publication No. 2005-0238649A1. The connectors can be "cleavable connectors" that facilitate drug release in cells. For example, acid-labile connectors (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) connectors, photolabile connectors, dimethyl connectors, or disulfide-containing connectors can be used (Chari et al. Cancer Research 52:127-131, 1992); U.S. Patent No. 5,208,020.

[0079] As used herein, "linkers" or "linkers of antibody-drug conjugates" can be classified into two types according to the mechanism of intracellular drug release: 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 a cell, the antibody is enzymatically cleaved in the lysosome, releasing the active molecule consisting of the small molecule drug, the linker, and the amino acid residues of the antibody. The resulting structural change in the drug molecule does not reduce its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate neighboring cells. Therefore, such drugs cannot kill neighboring tumor cells that do not express the targeted antigen (antigen-negative cells) (bystander effect) (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 a cell, the conjugate is cleaved, releasing the active component (the small molecule drug itself) into the target cell. Cleavable linkers are mainly classified into chemically sensitive linkers and enzyme-sensitive linkers. Chemically sensitive linkers are selectively cleaved due to differences in the properties of plasma and the cytoplasm or tumor microenvironment. Such properties include pH and glutathione concentration. However, pH-sensitive linkers, which are relatively stable in the neutral or slightly alkaline environment of blood (pH 7.3-7.5), are thought to be hydrolyzed in the slightly acidic tumor microenvironment (pH 5.0-6.5) and lysosomes (pH 4.5-5.0), e.g., hydrazones, carbonates, acetals, and ketals. Due to the limited plasma stability of acid-cleavable linkers, antibody-drug conjugates based on such linkers have short half-lives (2-3 days). This short half-life somewhat limits the use of pH-sensitive linkers in the new generation of antibody-drug conjugates. Glutathione-sensitive linkers are also known as disulfide bond linkers. Drug release is based on the difference between the high intracellular glutathione concentration (millimolar range) and the relatively low glutathione concentration in the blood (micromolar range).This is especially true for tumor cells, whose low oxygen content leads to enhanced reductase activity and thus higher glutathione concentrations. Disulfide bonds are thermodynamically stable and therefore have better stability in plasma. Enzyme-labile linkers, such as peptides, provide better control of drug release. Peptide linkers can be efficiently cleaved by lysosomal proteases, such as cathepsin (cathepsin B). This peptide bond is considered to be very stable in the plasma circulation due to unfavorable extracellular pH and serum protease inhibitors, which render the protease normally inactive outside the cell. Considering their high plasma stability and excellent intracellular cleavage selectivity and efficacy, enzyme-labile linkers are widely used as cleavable linkers in antibody-drug conjugates.

[0080] The term "antibody drug conjugate" refers to the attachment of an antibody to a biologically active drug by a stable linking unit. In the context of the present invention, a "ligand-drug conjugate", preferably an antibody drug conjugate (ADC), refers to the attachment of a monoclonal antibody or antibody fragment to a biologically active toxic drug by a stable linking unit.

[0081] The three-letter and one-letter codes for amino acids used in this disclosure are as set forth in J. Boil. Chem. 1968, 243, 3558.

[0082] The term "alkyl" refers to a saturated aliphatic hydrocarbon group that is a straight or branched chain group containing from 1 to 20 carbon atoms (i.e., a "C1-C20 alkyl"), preferably an alkyl group containing from 1 to 12 carbon atoms (i.e., a "C1-12 alkyl"), more preferably an alkyl group containing from 1 to 10 carbon atoms (i.e., a "C1-C10 alkyl"), and most preferably an alkyl group containing from 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, 5-methylhexyl, 2, Examples include 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 various branched chain isomers thereof.Lower alkyl groups containing 1 to 6 carbon atoms are more preferred, and non-limiting embodiments 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. Alkyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at available points of attachment and are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.

[0083] The term "substituted alkyl" means that a hydrogen in an alkyl group has been replaced by a substituent. Unless otherwise specified in the text, the substituents on the alkyl group can be various groups selected from the following groups: -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, where 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''' are each independently hydrogen, unsubstituted C 1~8Alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C substituted with 1-3 halogens 12 Aryl (or C6-C10 aryl), 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 be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

[0084] The term "alkylene" means a saturated straight-chain or branched-chain aliphatic hydrocarbon group having two residues derived by removing two hydrogen atoms from the same carbon atom or from two different carbon atoms of a parent alkane, and is a straight-chain or branched-chain group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH-), 1,1-ethylidene (-CH(CH)-), 1,2-ethylidene (-CHCH)-, 1,1-propylidene (-CH(CHCH)-), 1,2-propylidene (-CHCH(CH)-), 1,3-propylidene (-CHCHCHCH-), 1,4-butylidene (-CHCHCHCHCH), and 1,5-butylidene (-CHCHCHCHCHCH-), among others. Alkylene groups may be substituted or unsubstituted. or unsubstituted, and when substituted, the substituents may be substituted at available points of attachment, said substituents preferably being independently and optionally substituted with one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halo, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclo, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0085] The term "alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl), where alkyl or cycloalkyl are as defined above. Non-limiting examples of C1-C6 alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy. Alkoxy groups can be optionally substituted or unsubstituted, and if substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0086] The term "cycloalkyl" means a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, in which the cycloalkyl ring contains 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 cycloalkyl groups (e.g., "C3-C8 cycloalkyl") include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0087] The term "heterocyclyl" means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms (i.e., a "3- to 20-membered heterocyclyl"), one or more of whose ring atoms is nitrogen, oxygen, or S(O). m(m is an integer of 0 to 2), except for the -OO-, -OS-, or -SS- ring moiety, the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms (i.e., a "3- to 12-membered heterocyclic group"), 1 to 4 of which are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms (i.e., a "3- to 10-membered heterocyclic group"). Non-limiting examples of monocyclic heterocyclic groups (e.g., 3- to 7-membered heterocyclic groups) include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.

[0088] The term "cycloalkylalkyl" refers to an alkyl group substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, where alkyl is defined as above and cycloalkyl is defined as above, for example, C3-C8 cycloalkyl, C1-C6 alkyl.

[0089] The term "haloalkyl" means an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above, for example, haloC1-C6 alkyl.

[0090] The term "deuteroalkyl" means an alkyl group substituted with one or more deuterium atoms, wherein the alkyl group is as defined above, for example, deuteroC1-C6 alkyl.

[0091] The term "C6-C12 aryl" means a carbocyclic aromatic group having from 6 to 12 carbon atoms.

[0092] The term "C6-C10 aryl" refers to a carbocyclic aromatic group having 6 to 10 carbon atoms, such as phenyl, naphthyl, and the like.

[0093] The term "5- to 10-membered heteroaryl" refers to aromatic heterocyclic rings, typically 5-, 6-, 7-, 8-, 9-, and 10-membered heterocyclic rings having 1 to 3 heteroatoms selected from N, O, or S; heteroaryl rings can optionally be further fused to or attached to aromatic and non-aromatic carbocyclic and heterocyclic rings. Non-limiting examples of the 5- to 10-membered heteroaryl ring include pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, isothiazolyl, thioxazolyl, pyrrolyl, phenyl-pyrrolyl, furanyl, phenyl-furanyl, oxazolyl, isoxazolyl, pyrazolyl, thiophenyl, benzofuranyl, benzothiophenyl, benzo-1,3-dioxolane (benzodioxole), isodihydroindolyl, benzimidazolyl, indazolyl, quinolinyl, isoquinolinyl, 1,2,3-triazolyl, 1-phenyl-1,2,3-triazolyl, 2,3-dihydroindolyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothiophenyl, benzopyranyl, 2,3-dihydrobenzoxazinyl, and 2,3-dihydroquinoxalinyl.

[0094] The term "substituted C6-C10 aryl" or "substituted 5- to 10-membered heteroaryl" or "substituted 3- to 7-membered heterocyclyl" means that a hydrogen atom in an aryl or heteroaryl or heterocyclic group is replaced by a substituent. Unless otherwise specified in the text, substituents on an aryl or heteroaryl or heterocyclyl group can be various groups selected from the following groups: -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, where the number of substituents ranges from 0 to (2m'+1), where m' is the total number of carbons in the group. R', R'' and R''' are each independently hydrogen, unsubstituted C 1~8 Alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C substituted with 1-3 halogens 12 Aryl (or C6-C10 aryl), 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 be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

[0095] The term "hydroxyl" refers to an --OH group.

[0096] The term "halogen" means fluorine, chlorine, bromine or iodine.

[0097] The term "amino" means -NH2. The term "nitro" means -NO2.

[0098] The term "amide group" means -C(O)N(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0099] The term "carboxylate group" means -C(O)O(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0100] The present invention also encompasses various deuterated forms of Formula I. Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of Formula I by referring to relevant literature. In preparing the deuterated forms of Formula I, commercially available deuterated starting materials may be used, or the compounds may be synthesized by conventional techniques using deuterated reagents, non-limiting examples of which include deuterated deuteroborane, trideuteroborane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide, and deuterated methyl iodide.

[0101] The term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in the amino acid composition and order in the constant region of their heavy chains, and therefore in their antigenicity. Therefore, immunoglobulins can be classified into five immunoglobulin classes or isoforms: IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. The same Ig class can be further divided into different subclasses depending on the amino acid composition of their hinge regions and the number and position of disulfide bonds in the heavy chains; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into kappa or lambda chains depending on the difference in the constant region. Each of the five Ig classes can have kappa or lambda chains.The antibodies described herein are preferably specific for cell surface antigens on target cells, and non-limiting embodiments include 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 cladin18.2 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-SL AMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD 276) Antibodies, anti-PTK7 / CCK4 antibodies, anti-PRLR antibodies, anti-EFNA4 antibodies, anti-5T4 antibodies, anti-NOTCH3 antibodies, anti-Nectin4 antibodies, anti-TROP-2 antibodies, anti-CD142 antibodies, anti-CA6 antibodies, 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-INTE and one or more of an anti-CD30 antibody, an anti-CD37 antibody, an anti-CD38 antibody, an anti-CD39 antibody, an anti-CD40 antibody, an anti-CD41 antibody, an anti-CD42 antibody, an anti-CD43 antibody, an anti-CD44 antibody, an anti-CD45 antibody, an anti-CD46 antibody, an anti-CD47 antibody, an anti-CD48 antibody, an anti-CD49 antibody, an anti-CD50 antibody, an anti-CD51 antibody, an anti-CD52 antibody, an anti-CD53 antibody, an anti-CD54 antibody, an anti-CD55 antibody, an anti-CD60 antibody, an anti-CD61 antibody, an anti-CD62 antibody, an anti-CD63 antibody, an anti-CD70 antibody, an anti-CD71 antibody, an anti-CD72 antibody, an anti-CD73 antibody, an anti-CD74 antibody, an anti-CD80 antibody, an anti-CD81 antibody, an anti-CD82 antibody, an anti-CD83 antibody, an anti-CD84 antibody, an anti-CD85 antibody, an anti-CD86 antibody, an anti-CD87 antibody, an anti-CD88 antibody, an anti-CD90 antibody, an anti-CD91 antibody, an anti-CD92 antibody, an anti-CD93 antibody, an anti-CD94 antibody, an anti-CD95 antibody, an anti-CD96 antibody, an anti-CD97 antibody, an anti-CD98 antibody, an anti-CD9 ...9 antibody, an anti-CD90 antibody, an anti-CD91 antibody, an anti-CD92 antibody, an anti-CD93 antibody, an anti

[0102] The term "solvate" or "solvate" refers to the formation of a pharmaceutically usable solvate from a Ligand-Drug Conjugate of the present invention with one or more solvating molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0103] The term "drug load" refers to the average number of cytotoxic drugs loaded onto each antibody in Formula I. It can also be expressed as the ratio of drug amount to antibody amount, and the drug load can range from 0 to 12, preferably 1 to 10, cytotoxic drugs (D) linked to each antibody (Ab). In embodiments of the present invention, the drug load is represented as n, which can illustratively be an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The average number of drugs per ADC molecule after the coupling reaction can be determined by conventional methods such as UV-visible spectroscopy, mass spectrometry, ELISA testing, and HPLC characterization.

[0104] In one embodiment of the present invention, a cytotoxic drug is coupled by a linker unit to the open cysteine ​​sulfhydryl-SH and / or sulfhydryl-SH of the site-directed mutagenesized cysteine ​​residues between the antibody chains; generally, the number of drug molecules that can be coupled to the antibody in a coupling reaction will be less than or equal to the theoretical maximum.

[0105] Loading of the ligand-cytotoxic drug conjugate can be accomplished by the following non-limiting methods: (1) Control the molar ratio of the linking reagent to the monoclonal antibody; (2) control of reaction time and temperature; (3) It can be controlled by the selection of different reaction reagents.

[0106] For the preparation of conventional pharmaceutical compositions, refer to the Chinese Pharmacopoeia.

[0107] The term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" refers to a salt of a ligand-drug conjugate of the present invention or a salt of a compound described herein; salts of this type are safe and effective when used in mammals, and have the desired biological activity. The ligand-drug conjugates of the present invention contain at least one carboxyl group and are therefore capable of forming salts with bases; non-limiting examples of pharmaceutically acceptable salts include sodium, potassium, calcium, or magnesium salts.

[0108] The term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" refers to a salt of a ligand-drug conjugate of the present invention or a compound described herein; this type of salt is safe and effective when used in mammals, and has the desired biological activity. The ligand-drug conjugates of the present invention contain at least one amino group and therefore can form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodate, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, phosphate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0109] "Acidic amino acid" refers to an amino acid having an isoelectric point of less than 7. Acidic amino acid molecules tend to have one or more acidic groups, such as carboxyl groups, in their structure that can effectively ionize to a negative ionic form, increasing their hydrophilicity. Acidic amino acids can be natural or unnatural.

[0110] The term "natural amino acid" refers to a biologically synthesized amino acid. Natural amino acids are generally L-configured, with a few exceptions, such as glycine, which include naturally occurring amino acids and amino acids synthesized by living organisms.

[0111] "Unnatural amino acid" means an amino acid obtained by synthetic means.

[0112] The present invention will be further described in detail below in connection with specific embodiments, and it should be understood that the embodiments are intended to illustrate the present invention only and are not intended to limit the present invention. Test methods for which specific conditions are not given in the following embodiments generally follow conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all percentages, proportions, ratios, or parts are by weight. [Example]

[0113] Example 1 Synthesis of compound M1: [ka] In a 5000 mL single-neck flask, N-fluorenylmethoxycarbonyl-glycine-glycine (100 g, 282 mmol, 1.0 equivalent), lead tetraacetate (175 g, 395 mmol, 1.4 equivalent), 2000 mL of anhydrous tetrahydrofuran, and 670 mL of toluene were added, stirred evenly, protected with nitrogen, heated to 85 °C, and reacted for 2.5 hours. The reaction was monitored by TLC. After the starting material had reacted, it was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain compound M1 (87 g); LC-MS: [M+NH4] + =386.0.

[0114] Example 2 Synthesis of compound M3: [ka] Compound SM-2 (synthesized according to the method disclosed in Chinese Patent No. 108452321A) (40 g, 96 mmol, 1.0 equiv.), triethylamine (26.7 mL, 2.0 equiv.), and toluene (400 mL) were added to a 1000 mL single-neck flask and heated to 120 °C and refluxed for 2 h. When TLC monitoring showed the reaction was essentially complete, the mixture was cooled to 50 °C and the solvent was removed by rotation under reduced pressure. Dissolution was carried out with ethyl acetate (150 mL) and water (40 mL). The pH was adjusted to 2-3 with 1 M HCl under stirring in an ice bath, and the liquid was separated. The aqueous layer was extracted once more with ethyl acetate, and the combined organic layers were dried over anhydrous sodium sulfate. After filtration, concentration gave a pale yellow oily crude product, which was purified by column chromatography (DCM:MeOH=40:1) to give compound M2 (26.6 g); LC-MS: [M+H] + =399.3.

[0115] Compound M2 (26.5 g, 60.5 mmol, 1.0 eq.), pentafluorophenol (12.2 g, 66.5 mmol, 1.1 eq.), DCC (13.7 g, 66.5 mmol, 1.1 eq.), and THF (300 mL) were added to a 1000 mL single-neck flask and the reaction was carried out at room temperature for 30 minutes (monitored by TLC). Insoluble materials were filtered off. The reaction solution was directly purified by preparative LC. The prepared solution was concentrated by pumping water under reduced pressure in a water bath at 35 °C to remove acetonitrile and freeze-dried to give compound M3 (31.5 g) in 64% yield; LC-MS: [M+H] + =565.1.

[0116] Example 3 Synthesis of compound ent-M3: [ka] Compound ent-M3 (27.8 g) was obtained by referring to the synthetic route in Example 2; LC-MS: [M+H] + =565.2.

[0117] Example 4 Synthesis of Compound 1: [ka] Step 1: Compound 1a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol), stirred, and cooled to 0 °C. Benzyl hydroxyacetate (5.4 g, 32.6 mmol) was added dropwise, and then the mixture was allowed to warm to room temperature under TLC monitoring (the reaction lasted approximately 2-4 h). At the end of the reaction, saturated NaHCO3 solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by 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.

[0118] Step 2: Compound 1b In a 25 mL single-neck flask, 1a (2 g, 4.2 mmol) and 10 mL of DMF were added and stirred at 0° C. DBU (766 mg, 5.04 mmol) was added and reacted for 1 h. After completion of Fmoc deprotection as detected by TLC, the reaction solution was set aside for ready use.

[0119] M4 (prepared according to the method disclosed in Chinese Patent No. 111051330A) (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 added to another 25 mL one-neck flask. DIPEA (830 μL, 5.04 mmol) was added to an ice-water bath. Stirring was continued for 30 minutes. The above reaction solution was added to the reaction flask, and the temperature was raised to room temperature for reaction. When HPLC monitoring showed the completion of the reaction, the reaction solution was purified by preparative LC to obtain a preparation liquid of the product. The preparation liquid was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain solid 1b (1.7 g) in 63% yield; LCMS: [M+H] + =648.26.

[0120] Step 3: Compound 1c 1b (900 mg, 1.39 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 900 mg of 5% Pd / C was added, and the hydrogenation reaction was carried out for 2 hours. After the reaction was completed, the filtrate was filtered, and the filtrate was used directly in the next step of the reaction without purification.

[0121] Step 4: Compound 1d The crude product 1c was placed in an ice-water bath, DIPEA (235 μL, 1.39 mmol) was added, and compound M3 (784 mg, 1.39 mmol) was added. The temperature was then raised to room temperature and reacted for 1 hour. When the reaction was complete, as shown by HPLC monitoring, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to obtain 1d (504 mg); LC-MS: [M+H] + =804.4.

[0122] Step 5: Compound 1e 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 added to a 50 mL one-neck flask, and DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath. The mixture was then warmed to room temperature and reacted for 2 hours. After completion of the reaction was confirmed by HPLC monitoring, the reaction solution was purified by HPLC to obtain a fraction of compound 1e, which was lyophilized to give 1e (210 mg); LC-MS: [M+H] + =1221.6.

[0123] Step 6: Compound 1 1e (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask, and the reaction was carried out at 40° C. for 1 hour. After the completion of the reaction was shown by HPLC monitoring, the mixture was concentrated under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain solid compound 1 (60 mg); LC-MS: [M+H] + =1065.3.

[0124] 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.

[0125] Example 6 Synthesis of compound 3: [ka] Step 1: Compound 3a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The flask was stirred and cooled to 0 °C. Benzyl 2-hydroxy-2-methylpropionate (6.3 g, 32.6 mmol) was added dropwise, and the mixture was then allowed to warm to room temperature while being monitored by TLC (the reaction lasted approximately 2-4 h). At the end of the reaction, saturated NaHCO3 solution was added, followed by extraction with ethyl acetate, washing with saturated sodium chloride solution, drying over anhydrous sodium sulfate, filtering, and concentration. The residue was purified by 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.

[0126] Step 2: Compound 3b In a 25 mL single-neck flask, 3a (2 g, 4.0 mmol) and 10 mL of DMF were added and stirred at 0° C. DBU (760 mg, 5.0 mmol) was added and reacted for 1 h. After completion of Fmoc deprotection, as detected by TLC, the reaction solution was set aside for ready use.

[0127] 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 added to a 25 mL single-neck flask, and DIPEA (823 μL, 5.04 mmol) was added in an ice-water bath. Stirring was continued for 30 minutes, and then the above reaction solution was added to the reaction flask and warmed to room temperature for reaction. After the completion of the reaction was confirmed by HPLC monitoring, the reaction solution was purified by preparative LC to obtain a product fraction, which was extracted with dichloromethane, washed with saturated sodium chloride solution, filtered, and the filtrate was concentrated under reduced pressure to give solid 3b (1.4 g) in 53% yield; LC-MS: [M+H] + =676.2.

[0128] Step 3: Compound 3c 3b (700 mg, 1.04 mmol) was added to a 25 mL single-neck flask and dissolved in 10 mL of DMF. 700 mg of 5% Pd / C was added, and the hydrogenation reaction was carried out for 1.5 hours. After the reaction was completed, filtration was performed to obtain the filtrate, which was directly used in the next step of the reaction without purification.

[0129] Step 4: Compound 3d The crude product 3c was placed in an ice-water bath, DIPEA (210 μL, 1.25 mmol) was added, and then compound M3 (704 mg, 1.25 mmol) was added. The mixture was then warmed to room temperature and reacted for 1 hour. After the reaction was completed as indicated by HPLC monitoring, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to give 3d (486 mg); LC-MS: [M−H] - =830.5.

[0130] Step 5: Compound 3e 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 added to a 50 mL one-neck flask, and DIPEA (219.5 μL, 1.33 mmol) was added in an ice-water bath. The mixture was then warmed to room temperature and reacted for 3 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 3e, which was lyophilized to give 3e (157 mg); LC-MS: [M+H] + =1249.6.

[0131] Step 6: Compound 3 A 25 mL single-neck flask was charged with 3e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane, and the mixture was reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the mixture 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, which was lyophilized to obtain solid compound 3 (64 mg); LC-MS: [M+H] + =1093.1.

[0132] 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.

[0133] Example 8 Synthesis of compound 5A: [ka] Step 1: Compound 5a In a 25 mL single-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 added, stirred well, then cooled to 0 °C, and then L-lactic acid benzyl ester (1.2 g, 7.0 mmol, 5 equiv.) was slowly added, and then warmed to room temperature for reaction. TLC monitoring was performed, and at the end of the reaction, saturated NaHCO3 solution was added, followed by extraction with ethyl acetate, drying with anhydrous sodium sulfate, filtering, and concentrating. 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=6.8Hz),3.78(2H,t,J=4.0Hz),4.17-4.27(2H,m),4.42(2H,d,J=4.0Hz),4.72-4.85(2H,m),5.11-5. 58(2H,m),5.43(1H,s),7.06(1H,t,J=8.0Hz),7.25-7.33(6H,m),7.38(2H,t,J=8.0Hz),7.57(2H,d,J=8.0Hz),7.75(2H,d,J=8.0Hz).

[0134] Step 2: Compound 5b Compound 5a (400 mg, 0.8 mmol, 1.0 eq) and 4 mL of DMF were added to a 25 mL single-neck flask, stirred well, and then cooled to 0°C before slowly adding DBU (137 mg, 0.9 mmol, 1.1 eq). After the addition was completed, the reaction was allowed to warm to room temperature. The reaction was monitored by TLC, and at the end of the reaction, the reaction solution was recorded as reaction solution (1); In another 25 mL one-neck flask, M4 (372 mg, 0.9 mmol, 1.1 eq.), PyBOP (852 mg, 1.6 mmol, 2.0 eq.), and 3 mL of DMF were added and stirred at room temperature for 5 minutes, followed by addition of reaction solution (1). The reaction was carried out at room temperature and monitored by HPLC. Upon completion of the reaction, the reaction solution was purified by HPLC to give compound 5b (326 mg); LC-MS: [M+NH4] + =679.2.

[0135] Step 3: Compound 5c 5b (4.0 g, 6.05 mmol, 1.0 equiv) was added to a 100 mL single-neck flask and dissolved in DMF (60 mL). 5% Pd / C (4 g) was then added, and the hydrogenation reaction was carried out at room temperature for 4 h (the progress of the reaction was monitored using HPLC). The Pd / C was filtered off, and the filtrate was placed directly in an ice-water bath (approximately 0 °C) without concentration, and was ready for use.

[0136] Step 4: Compound 5d The crude product 5c was placed in an ice-water bath, DIPEA (1.1 mL, 1.1 equivalents) was added, and then compound M3 (3.4 g, 6.05 mmol) was added. The mixture was then warmed to room temperature and reacted for 2 hours. After the reaction was completed as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to give 5d (3.15 g); LC-MS: [M H] - =816.3.

[0137] Step 5: 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 in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative LC. The aliquot was concentrated under reduced pressure in a water bath with a water pump at 35 °C to remove acetonitrile, and lyophilized to give compound 5e (1.92 g) in 61% yield; LC-MS: [M+H] + =1235.4.

[0138] Step 6: Compound 5A Compound 5e (1.0 g, 0.8 mmol, 1.0 equivalent) and 35 mL of nitromethane were added to a 100 mL single-neck flask. After dissolution, zinc bromide (3.64 g, 16 mmol, 20.0 equivalents) was added and the mixture was allowed to react for 30 minutes in a 40 °C oil bath (preheated and stabilized). The mixture was then concentrated under reduced pressure at 45 °C in a water bath with a water pump to remove the nitromethane, resulting in the formation of a yellow residual solid (monitored by HPLC). After acid preparation, a fraction of compound 5A was obtained, which was concentrated under reduced pressure at 35 °C in a water bath with a water pump. The acetonitrile was removed by rotation and lyophilized to give compound 5A (786 mg) in a 90% yield. LC-MS: [M+H] + =1079.4; 1H NMR(400MHz,DMSO-d6)δ 9.39-9.02(m,1H),8.70(t,J=6.5Hz,1H),8.64(t,J=5.7Hz,1H),8.56(d,J=8.8Hz,1H),8.34(t,J=5.7Hz,1H), 8.16(d,J=8.2Hz,1H),8.01(t,J=5.5Hz,1H),7.71(d,J=10.9Hz,1H),7.30(s,1H),7.28-7.15(m,4H),7.14(s, 2H),5.53(dd,J=14.5,6.4Hz,1H),5.49-5.34(m,2H),5.22(d,J=18.8Hz,1H),5.09(d,J=18.7Hz,1H),5.03(dd ,J=9.6,3.9Hz,1H),4.73(dd,J=9.9,6.9Hz,1H),4.59(dd,J=10.1,6.5Hz,1H),4.49(ddd,J=13.2,8.6,4.4Hz, 1H),4.14(dd,J=13.3,6.6Hz,2H),3.93(s,2H),3.84(dd,J=16.5,6.3Hz,1H),3.76(dd,J=16.9,5.7Hz,2H),3. 70(dd,J=5.2Hz,2H),3.60(dd,J=16.7,5.4Hz,1H),3.52(dd,J=16.4,5.1Hz,1H),3.45(dd,J=12.8,10.1Hz,1H ),3.25-3.15(m,1H),3.14-3.05(m,1H),3.01(dd,J=13.7,4.1Hz,1H),2.73(dd,J=13.5,9.8Hz,1H),2.54-2.4 7(m,1H),2.33(s,2H),2.17(d,J=5.5Hz,2H),1.91-1.79(m,2H),1.33(d,J=6.6Hz,2H),0.87(t,J=7.3Hz,2H).

[0139] Example 9 Synthesis of compound 5B:

change

[0140] Step 2: Compound 5e-1 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 added to a 25 mL one-neck flask. The temperature of the ice-water bath was lowered to 0-5 °C, and DIPEA (62 mg, 0.48 mmol, 2.0 equiv.) was added. After the addition was complete, the temperature was raised to 20 ± 5 °C and the reaction was allowed to proceed for 2 h. The reaction was monitored for completion using HPLC. The reaction solution was purified by HPLC, and the product aliquot was collected and lyophilized to give compound 5e-1 (162.8 mg). LC-MS: [M+H] + =1235.4.

[0141] Step 3: Compound 5B 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 to a 25 mL single-neck flask. After the addition, the temperature was raised to 40 °C, and the reaction was allowed to proceed for 0.5 h, after which the reaction was stopped. The reaction solution was directly dried at 45 °C by rotary evaporation under reduced pressure to obtain a yellow solid. A sample was taken, and the reaction was monitored by HPLC. The rotary-dried solid was directly purified by HPLC preparative HPLC, and the product fraction was collected and lyophilized to obtain compound 5B (73.4 mg) in a 76.5% yield; LC-MS: [M+H] + =1079.4.

[0142] Example 10 Preparation of Compound 6A: [ka] Compound 6A (71 mg) was obtained by following the synthesis route of Example 8; LC-MS: [M+H] + =1079.4.

[0143] Example 11 Preparation of Compound 6B: [ka] Compound 6B (59 mg) was obtained by following the synthesis route of Example 9; LC-MS: [M+H] + =1079.4.

[0144] Example 12 Preparation of Compounds 7A and 7B: [ka] Step 1: Compound 7a A 250 mL single-neck flask was charged with M1 (10 g, 27.1 mmol), benzyl 3,3,3-trifluorolactate (prepared according to the method disclosed in WO 2020063673 A1) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene. The mixture was heated to 100 °C and reacted for 4 hours. Upon completion of the reaction, the mixture was cooled to room temperature, filtered to remove insoluble material, and 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 material in a 35.1% yield; LC-MS: [M+H] + =543.17.

[0145] Step 2: Compound 7b Add 7a (5 g, 9.2 mmol) and 15 mL of DMF to a 50 mL single-neck flask and dissolve until clear. Then add DBU (1.68 g, 11 mmol) in an ice-water bath and react for 1 hour. Record the reaction solution as reaction solution (1).

[0146] Into 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 added. When dissolved, DIPEA (1.82 mL, 11 mmol) was added in an ice-water bath and the reaction was continued for 30 minutes. Then, reaction solution (1) was added, and the mixture was warmed to room temperature and allowed to react for 2 hours. The progress of the reaction was monitored by HPLC. After completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.1 g of a solid in 62.3% yield; LC-MS: [M+H] + =716.25.

[0147] Step 3: Compound 7d 7b (900 mg, 1.26 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 900 mg of 5% Pd / C was added, and the hydrogenation reaction was carried out for 2 hours. After completion of the reaction, the mixture was filtered, placed in an ice-water bath, and DIPEA (228 μL, 1.38 mmol) was added, followed by M3 (712 mg, 1.26 mmol). The mixture was then warmed to room temperature and reacted for 1 hour. When the reaction was complete as detected by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction, which was lyophilized to give 525 mg of product in 47.9% yield; LC-MS: [M H] - =870.33.

[0148] Step 4: Compound 7e A 50 mL single-neck flask was charged with 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. The reaction mixture was then cooled to room temperature and heated to 378 μL (2.29 mmol) of DIPEA in an ice-water bath. The reaction mixture was then allowed to react for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 7e-1 and compound 7e-2. The fractions were lyophilized to give 150 mg of compound 7e-1; LC-MS: [M+H] + =1289.46, and compound 7e-2 220mg;LC-MS:[M+H] + =1289.46 respectively.

[0149] Step 5: Compound 7A [ka] 7e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-neck flask and reacted at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 52 mg of a solid; TOF result: 1133.3613.

[0150] Step 6: Compound 7B [ka] A 25 mL single-neck flask was charged with 7e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane, and the mixture was reacted at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 63 mg of a solid; TOF result: 1133.3668.

[0151] Example 13 Synthesis of compounds 8A and 8B: [ka] Step 1: Compound 8d 7c (900 mg, 1.83 mmol) was added to a 25 mL single-neck flask and dissolved in 20 mL of DMF. Then, DIPEA (303 μL, 1.83 mmol) and ent-M3 (1034 mg, 1.83 mmol) were added. The temperature was raised to room temperature and reacted for 1 hour. After the reaction was completed as detected by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction, which was lyophilized to give 613 mg of product in 38.5% yield; LC-MS: [M H] - =870.32.

[0152] Step 2: Compound 8e-1 and Compound 8e-2 A 50 mL single-neck flask was charged with 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. DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 8e-1 and compound 8e-2. The fractions were lyophilized to obtain 140 mg of compound 8e-1 and 210 mg of compound 8e-2, respectively. LC-MS of compound 8e-1: [M+H] + = 1289.47; LC-MS of compound 8e-2: [M+H] + =1289.47.

[0153] Step 3: Compound 8A [ka] Compound 8e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-neck flask and reacted at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 50 mg of a solid; TOF result: 1133.3623.

[0154] Step 4: Compound 8B [ka] Compound 8e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-neck flask and reacted at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 58 mg of a solid; TOF result: 1133.3653.

[0155] Example 14 Synthesis of compound 9A: [ka] Step 1: Compound 9a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was stirred and cooled to 0 °C. 2-Hydroxy-2-cyclopropylbenzyl acetate (prepared according to the method disclosed in U.S. Patent Application Publication No. 20050020645 A1) (6.3 g, 32.6 mmol) was added dropwise and allowed to warm to room temperature. The reaction was monitored by TLC (the reaction lasted approximately 2-4 h). At the end of the reaction, saturated NaHCO3 solution was added, followed by extraction with ethyl acetate, washing with saturated sodium chloride solution, drying over anhydrous sodium sulfate, filtering, and concentrating. 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.

[0156] Step 2: Compound 9b In a 25 mL single-neck flask, 9a (2 g, 4.0 mmol) and 10 mL of DMF were added and stirred at 0° C. DBU (760 mg, 5.0 mmol) was added and reacted for 1 h. After completion of Fmoc deprotection as monitored by TLC, the reaction solution was set aside for ready use.

[0157] In another 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 added, and DIPEA (823 μL, 5.04 mmol) was added in an ice-water bath. Stirring was continued for 30 minutes, and then the above reaction solution was added to the reaction flask and allowed to warm to room temperature. After completion of the reaction as detected by HPLC, the reaction solution was purified by preparative LC to obtain a product fraction, which was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1.5 g of solid in 56% yield; LC-MS: [M+H] + =674.7.

[0158] Step 3: Compound 9c 9b (900 mg, 1.3 mmol) was added to a 25 mL single-neck flask and dissolved in 10 mL of DMF. 900 mg of 5% Pd / C was added, and the hydrogenation reaction was carried out for 1.5 hours. After completion of the reaction, filtration was performed to obtain the filtrate, which was used directly in the next step without purification.

[0159] Step 4: Compound 9d The crude product 9c was placed in an ice-water bath, DIPEA (223 μL, 1.3 mmol) was added, and then compound M3 (750 mg, 1.3 mmol) was added. The mixture was then warmed to room temperature and reacted for 1 hour. After the reaction was completed as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to give 9d (529 mg); LC-MS: [M−H] - =828.4.

[0160] Step 5: Compound 9e A 50 mL single-neck flask was charged with 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. DIPEA (351 μL, 2.13 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 3 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation of compound 9e, which was lyophilized to give 9e (257 mg); LC-MS: [M+H] + =1247.5.

[0161] 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 added, and the reaction was carried out at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain solid compound 9A (55 mg); LC-MS: [M+H] + =1091.3.

[0162] 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.

[0163] Example 16 Synthesis of compound 10A: [ka] Step 1: Compound 10a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was stirred and cooled to 0 °C. Benzyl 3-hydroxy-2-cyclopropylpropionate (prepared according to the method disclosed in WO 2013187496 A1) (6.7 g, 32.6 mmol) was added dropwise. The mixture was then allowed to warm to room temperature and reacted for approximately 2-4 h while being monitored by TLC. At the end of the reaction, saturated NaHCO3 solution was added, followed by extraction with ethyl acetate, washing with saturated sodium chloride solution, drying over anhydrous sodium sulfate, filtering, and concentrating. The residue was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give 10a (4.9 g) in 58% yield; LC-MS: [M+H] + =515.4.

[0164] Step 2: Compound 10b In a 25 mL single-neck flask, 10a (4 g, 7.8 mmol) and 10 mL of DMF were added and stirred at 0° C. DBU (1.2 g, 8.0 mmol) was added and the reaction was allowed to proceed for 1 h. After completion of Fmoc deprotection as monitored by TLC, the reaction solution was set aside for ready use.

[0165] 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 added to another 25 mL one-neck flask, and DIPEA (1.65 mL, 10.1 mmol) was added in an ice-water bath. Stirring was continued for 50 minutes, and then the above reaction solution was added to the reaction flask, warmed to room temperature, and reacted. After the completion of the reaction detected by HPLC, the reaction solution was purified by preparative LC to obtain a product fraction, which was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.3 g of solid in 42% yield; LC-MS: [M+H] + =688.8.

[0166] Step 3: Compound 10c 10b (1.0 g, 1.45 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF until clear. 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 completed, the filtrate was filtered and used directly in the next step of the reaction without purification.

[0167] Step 4: Compound 10d The crude product 10c was placed in an ice-water bath, and DIPEA (258 μL, 1.5 mmol) was added, followed by compound M3 (837 mg, 1.45 mmol). The temperature was then raised to room temperature and allowed to react for 1 hour. Upon completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to give 10d (499 mg); LC-MS: [M H] - =842.4.

[0168] Step 5: Compound 10e 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 added to a 50 mL one-neck flask, and DIPEA (330 μL, 2.0 mmol) was added in an ice-water bath. The temperature was raised to room temperature and the reaction was carried out for 3 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 10e, which was lyophilized to give 10e (188 mg); LC-MS: [M+H] + =1261.5.

[0169] 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 added, and the reaction was carried out at 40° C. for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain solid compound 10A (61 mg); LC-MS: [M+H] +=1105.4.

[0170] 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.

[0171] Example 18 Synthesis of compound 11A: [ka] Step 1: Compound 11a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol), and cooled to 0°C. 2-Hydroxy-2-cyclobutylbenzyl acetate (synthesized by the method published in Journal of Medicinal Chemistry, 2013, 56(13), 5541-5552) (6.7 g, 32.6 mmol) was added dropwise, and the mixture was allowed to warm to room temperature and react while monitoring by TLC (approximately 2-4 hours). At the end of the reaction, saturated NaHCO3 solution was added, followed by extraction with ethyl acetate, washing with saturated sodium chloride solution, drying over anhydrous sodium sulfate, filtering, concentrating, 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.

[0172] Step 2: Compound 11b In a 25 mL single-neck flask, 11a (4 g, 7.8 mmol) and 10 mL of DMF were added and stirred at 0° C. DBU (1.2 g, 8.0 mmol) was added and the reaction was allowed to proceed for 1 h. After completion of Fmoc deprotection as monitored by TLC, the reaction solution was set aside for ready use.

[0173] 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 added to another 25 mL one-neck flask, and DIPEA (1.63 mL, 10.0 mmol) was added in an ice-water bath. Stirring was continued for 40 minutes, and then the above reaction solution was added to the reaction flask, warmed to room temperature, and reacted. After completion of the reaction detected by HPLC, the reaction solution was purified by preparative LC to obtain a product fraction, which was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.3 g of solid in 42% yield; LC-MS: [M+H] + =688.3.

[0174] Step 3: Compound 11c 11b (2.0 g, 2.9 mmol) was added to a 25 mL single-neck flask and dissolved in 25 mL of DMF. Then, 2.0 g of 5% Pd / C was added, and the hydrogenation reaction was carried out for 3 hours. After the reaction was completed, it was filtered to obtain the filtrate, which was directly used in the next step of the reaction without purification.

[0175] Step 4: Compound 11d The crude product 11c was placed in an ice-water bath, and DIPEA (516 μL, 3.0 mmol) was added, followed by compound M3 (1.7 g, 2.9 mmol). The temperature was then raised to room temperature and reacted for 2 hours. After the reaction was completed as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to obtain 11d (934 mg); LC-MS: [M−H] - =842.4.

[0176] Step 5: 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 added, and DIPEA (660 μL, 4.0 mmol) was added in an ice-water bath. The mixture was then warmed to room temperature and reacted for 4 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 11e, which was lyophilized to give 11e (401 mg); LC-MS: [M+H] + =1261.4.

[0177] Step 6: Compound 11A 11e (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol), and 10 mL of nitromethane were added to a 25 mL one-neck flask, and the reaction was carried out at 40 °C for 1 h. After the reaction was completed as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain solid compound 11A (86 mg); LC-MS: [M+H] + =1105.4.

[0178] 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.

[0179] Example 20 Synthesis of compound 12A: [ka] Step 1: Compound 12a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was stirred and cooled to 0 °C. Benzyl 3-hydroxy-2-cyclobutylpropionate (7.2 g, 32.6 mmol) (prepared according to the method disclosed in WO 2009011285 A1) was added dropwise. The mixture was then allowed to warm to room temperature and reacted while being monitored by TLC (approximately 2-4 h). At the end of the reaction, saturated NaHCO3 solution was added, followed by extraction with ethyl acetate, washing with saturated sodium chloride solution, drying over anhydrous sodium sulfate, filtration, and concentration. The residue was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give 12a (4.5 g) in 52% yield; LC-MS: [M+H] + =529.4.

[0180] Step 2: Compound 12b In a 25 mL single-neck flask, 12a (4 g, 7.6 mmol) and 10 mL of DMF were added and stirred at 0° C. DBU (1.2 g, 8.0 mmol) was added and the reaction was allowed to proceed for 1 h. After completion of Fmoc deprotection as monitored by TLC, the reaction solution was set aside for ready use.

[0181] 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 added to another 25 mL one-neck flask, and DIPEA (1.49 mL, 0.9 mmol) was added in an ice-water bath. Stirring was continued for 30 minutes, and then the above reaction solution was added to the reaction flask, warmed to room temperature, and reacted. After completion of the reaction as detected by HPLC, the reaction solution was purified by preparative LC to obtain a product fraction, which was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.0 g of solid in 37% yield; LC-MS: [M+H] + =702.8.

[0182] Step 3: Compound 12c 12b (1.0 g, 1.43 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF until clear, 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 completed, it was filtered to obtain the filtrate, which was directly used in the next step of the reaction without purification.

[0183] Step 4: Compound 12d The crude product 12c was placed in an ice-water bath, and DIPEA (258 μL, 1.5 mmol) was added, followed by compound M3 (825 mg, 1.43 mmol). The temperature was then raised to room temperature and reacted for 1 hour. After the reaction was completed as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to obtain 12d (522 mg); LC-MS: [M−H] - =856.4.

[0184] Step 5: 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 added, and DIPEA (330 μL, 2.0 mmol) was added in an ice-water bath. The mixture was then warmed to room temperature and reacted for 3 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 12e, which was lyophilized to give 12e (198 mg); LC-MS: [M+H] + =1275.4.

[0185] Step 6: Compound 12A 12e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask, and the reaction was carried out at 40 °C for 1 h. After the reaction was completed as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain solid compound 12A (55 mg); LC-MS: [M+H] +=1119.4.

[0186] 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.

[0187] Example 22 Synthesis of compound 13A: [ka] Step 1: 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 added, stirred, and cooled to 0°C. 2-hydroxy-2-cyclopentylbenzyl acetate (synthesized by the method published in Journal of Medicinal Chemistry, 2013, 56(13), 5541-5552) (7.2 g, 32.6 mmol) was added dropwise, and then the mixture was allowed to warm to room temperature and reacted while being monitored by TLC (approximately 2-4 hours). At the end of the reaction, saturated NaHCO3 solution was added, followed by extraction with ethyl acetate, washing with saturated sodium chloride solution, drying over anhydrous sodium sulfate, filtering, concentrating, 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.

[0188] Step 2: Compound 13b In a 25 mL single-neck flask, 13a (4 g, 7.6 mmol) and 10 mL of DMF were added and stirred at 0° C. DBU (1.17 g, 7.8 mmol) was added and the reaction was allowed to proceed for 1 h. After completion of Fmoc deprotection as monitored by TLC, the reaction solution was set aside for ready use.

[0189] 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 added to another 25 mL one-neck flask, and DIPEA (1.39 mL, 0.85 mmol) was added in an ice-water bath. Stirring was continued for 30 minutes, and then the above reaction solution was added to the reaction flask, warmed to room temperature, and reacted. After completion of the reaction detected by HPLC, the reaction solution was purified by preparative LC to obtain a product fraction, which was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.1 g of solid in 39% yield; LC-MS: [M+H] + =702.8.

[0190] Step 3: Compound 13c 13b (1.5 g, 1.87 mmol) was added to a 25 mL single-neck flask and dissolved in 25 mL of DMF. 1.5 g of 5% Pd / C was added and the hydrogenation reaction was carried out for 3 hours. After the reaction was completed, the filtrate was filtered and used directly in the next step of the reaction without further purification.

[0191] Step 4: Compound 13d The crude product 13c was placed in an ice-water bath, DIPEA (333 μL, 1.93 mmol) was added, and the temperature was raised to room temperature and allowed to react for 1 h before compound M3 (1.1 g, 1.87 mmol) was added. Upon completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to give 13d (519 mg); LC-MS: [M−H] - =856.6.

[0192] Step 5: 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 added, and DIPEA (330 μL, 2.0 mmol) was added in an ice-water bath. The mixture was then warmed to room temperature and reacted for 4 hours. Upon completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 13e, which was lyophilized to give 13e (187 mg); LC-MS: [M+H] + =1275.5.

[0193] Step 6: Compound 13A 13e (100 mg, 0.08 mmol), zinc bromide (355 mg, 0.16 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask, and the reaction was carried out at 40 °C for 1 h. Upon completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to give the crude product. The crude product was purified by high performance liquid chromatography to give a product fraction, which was lyophilized to give solid compound 13A (60 mg); LC-MS: [M+H] + =1119.6.

[0194] 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.

[0195] Example 24 Synthesis of compound 14A: [ka] Step 1: Compound 14a 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 added, stirred, and cooled to 0 °C. Benzyl 3-hydroxy-2-cyclopentylpropionate (synthesized by the method disclosed in WO 2009011285 A1) (7.6 g, 32.6 mmol) was added dropwise, and the mixture was allowed to warm to room temperature. The reaction was monitored by TLC (approximately 2-4 h). At the end of the reaction, saturated NaHCO3 solution was added, followed by extraction with ethyl acetate, washing with saturated sodium chloride solution, drying over anhydrous sodium sulfate, filtering, and concentrating. The residue was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to give 14a (4.4 g) in 49% yield; LC-MS: [M+H] + =543.6.

[0196] Step 2: Compound 14b In a 25 mL single-neck flask, 14a (4 g, 7.4 mmol) and 10 mL of DMF were added and stirred at 0° C. DBU (1.2 g, 8.0 mmol) was added and the reaction was allowed to proceed for 1 h. After completion of Fmoc deprotection as monitored by TLC, the reaction solution was set aside for ready use.

[0197] 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 added to a separate 25 mL one-neck flask, and DIPEA (1.49 mL, 9.0 mmol) was added in an ice-water bath. Stirring was continued for 30 minutes, and then the above reaction solution was added to the reaction flask, warmed to room temperature, and reacted. After completion of the reaction as detected by HPLC, the reaction solution was purified by preparative LC to obtain a product fraction, which was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.6 g of solid in 49% yield; LC-MS: [M+H] + =716.4.

[0198] Step 3: Compound 14c 14b (1.0 g, 1.4 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 1.0 g of 5% Pd / C was added and the hydrogenation reaction was carried out for 1.5 hours. After the reaction was completed, the filtrate was filtered and used directly in the next step of the reaction without further purification.

[0199] Step 4: Compound 14d The crude product 14c was placed in an ice-water bath, and DIPEA (248 μL, 1.5 mmol) was added, followed by compound M3 (808 mg, 1.4 mmol). The temperature was then raised to room temperature and allowed to react for 1 hour. Upon completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to give 14d (500 mg); LC-MS: [M−H] - =870.5.

[0200] Step 5: 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 added, and DIPEA (331 μL, 2.0 mmol) was added in an ice-water bath. The mixture was then warmed to room temperature and reacted for 3 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 14e, which was lyophilized to give 14e (146 mg); LC-MS: [M+H] + =1289.5.

[0201] Step 6: Compound 14A 14e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask, and the reaction was carried out at 40 °C for 1 h. After the reaction was completed as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain solid compound 14A (52 mg); LC-MS: [M+H] + =1133.4.

[0202] 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.

[0203] Example 26 Synthesis of compounds 15A and 15B: [ka] Step 1: Compound 15a A 250 mL single-neck flask was charged with M1 (10 g, 27.1 mmol), benzyl 2-hydroxybutyrate (prepared by the method published 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. The mixture was heated to 100 °C and reacted for 4 hours. After the reaction was completed, the temperature was lowered to room temperature, filtered to remove insoluble materials, and 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 material in a 42% yield; LC-MS: [M+H] + =503.5.

[0204] Step 2: Compound 15b 15a (5 g, 9.95 mmol) and 15 mL of DMF were added to a 50 mL single-neck flask, dissolved, and then DBU (1.68 g, 11 mmol) was added in an ice-water bath and reacted for 1 hour. The reaction solution was recorded as reaction solution (1).

[0205] 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 of DMF were added and dissolved. After dissolving, DIPEA (1.82 mL, 11 mmol) was added in an ice-water bath. The reaction was continued for 40 minutes, and then reaction solution (1) was added. The temperature was raised to room temperature, and the reaction was continued for 2 hours. The progress of the reaction was monitored by HPLC. After completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.6 g of a solid in 68% yield; LC-MS: [M+H] + =676.7.

[0206] Step 3: Compound 15d 15b (2.0 g, 2.96 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 2.0 g of 5% Pd / C was then added, and the hydrogenation reaction was carried out for 2 h. After completion of the reaction, it 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 temperature was then raised to room temperature and the reaction was carried out for 1 h. Upon completion of the reaction, as detected by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction, which was lyophilized to give 1120 mg of product in 45% yield; LC-MS: [MH] - =830.3.

[0207] Step 4: 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 added, and DIPEA (446 μL, 2.7 mmol) was added in an ice-water bath. The mixture was then warmed to room temperature and reacted for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 15e-1 and compound 15e-2, which was lyophilized to give 138 mg of compound 15e-1; LC-MS: [M+H]+ =1249.5 and compound 15e-2 140mg;LC-MS:[M+H] + =1249.5, respectively.

[0208] Step 5: 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 added and reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 59 mg of a solid; LC-MS: [M+H] + =1093.4.

[0209] Step 6: 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 added and reacted at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 60 mg of a solid; LC-MS: [M+H] + =1093.4.

[0210] 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]

[0211] Compound 16B (54 mg) was obtained by following the synthesis route of Example 26; LC-MS: [M+H] + =1093.4.

[0212] Example 28 Synthesis of compounds 17A and 17B: [ka] Step 1: Compound 17a A 250 mL single-neck flask was charged with M1 (10 g, 27.1 mmol), benzyl 2-hydroxyphenylpropionate (prepared by the method published 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. The mixture was heated to 100 °C and reacted for 4 hours. After the reaction was completed, the temperature was lowered to room temperature, filtered to remove insoluble materials, and 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 material in a 40% yield; LC-MS: [M+H] + =565.6.

[0213] Step 2: Compound 17b In a 50 mL single-neck flask, 17a (5 g, 8.86 mmol) and 15 mL of DMF were added and dissolved, followed by the addition of DBU (1.53 g, 10 mmol) in an ice-water bath, followed by reaction for 1 hour, and the reaction solution was recorded as reaction solution (1).

[0214] 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 added and dissolved. After dissolving, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath. The reaction was continued for 30 minutes, and then reaction solution (1) was added. The temperature was raised to room temperature, and the reaction was continued for 2 hours. The progress of the reaction was monitored by HPLC. After completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 5.0 g of a solid in 77% yield; LC-MS: [M+H] + =738.3.

[0215] Step 3: Compound 17d 17b (3.0 g, 4.07 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 3.0 g of 5% Pd / C was added and the hydrogenation reaction was carried out for 2 h. After the reaction was completed, it was filtered. The filtrate was placed in an ice-water bath and DIPEA (744 μL, 4.5 mmol) was added, followed by M3 (2.34 g, 4.07 mmol). The temperature was then raised to room temperature and the reaction was carried out for 1 h. Upon completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to give 1.2 g of product in 33% yield; LC-MS: [M-H] - =892.4.

[0216] Step 4: 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 added. DIPEA (330 μL, 2.0 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 17e-1 and compound 17e-2, which was lyophilized to give 156 mg of compound 17e-1; LC-MS: [M+H] +=1311.4 and compound 17e-2 150mg;,LC-MS:[M+H] + =1311.7, respectively.

[0217] Step 5: Compound 17A [ka] A 25 mL single-neck flask was charged with 17e-1 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane, and the reaction was carried out at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 43 mg of a solid; LC-MS: [M+H] + =1155.4.

[0218] Step 6: Compound 17B [ka] A 25 mL single-neck flask was charged with 17e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane, and the reaction was carried out at 40 °C for 1 hour. After the reaction was completed as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 40 mg of a solid; LC-MS: [M+H] + =1155.4.

[0219] 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]

[0220] Compound 18B (55 mg) was obtained by following the synthesis route of Example 28; LC-MS: [M+H] + =1155.4.

[0221] Example 30 Synthesis of compounds 19A and 19B: [ka] Step 1: Compound 19a M1 (10 g, 27.1 mmol), 2-cyclopropyl-2-hydroxybenzyl acetate (prepared according to the method disclosed in WO 2020244657 A1) (11.2 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were added and heated to 100 °C for 4 hours. Upon completion of the reaction, the temperature was lowered to room temperature, filtered to remove insoluble material, and 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 material in 36% yield; LC-MS: [M+H] + =515.2.

[0222] Step 2: Compound 19b 19a (4 g, 7.8 mmol) and 10 mL of DMF were added to a 50 mL single-neck flask. After dissolving, DBU (1.42 g, 9.3 mmol) was added in an ice-water bath and the reaction was continued for 1 hour. The reaction solution was recorded as reaction solution (1).

[0223] 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 added and dissolved. After dissolving, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath. The reaction was continued for 30 minutes, followed by the addition of reaction solution (1), which was then warmed to room temperature and reacted for 2 hours. The reaction progress was monitored by HPLC. Upon completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.2 g of a solid in 78% yield; LC-MS: [M+H] + =688.3.

[0224] Step 3: Compound 19d 19b (1000 mg, 1.45 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 1000 mg of 5% Pd / C was added and the hydrogenation reaction was carried out for 2 h. After the reaction was completed, it was filtered. The filtrate was placed in an ice-water bath and DIPEA (248 μL, 4.5 mmol) was added, followed by M3 (720 mg, 1.45 mmol). The temperature was then raised to room temperature and the reaction was carried out for 1 h. Upon completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to give 503 mg of product in 41% yield; LC-MS: [M-H] - =842.3.

[0225] Step 4: Compounds 19e-1 and 19e-2 A 50 mL single-neck flask was charged with 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. The resulting mixture was heated to room temperature in an ice-water bath and reacted for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of compound 19e-1 and compound 19e-2. The fractions were lyophilized to give 112 mg of compound 19e-1; LC-MS: [M+H]+ = 1261.5 and compound 19e-2 131 mg; LC-MS: [M+H] + =1261.5, respectively.

[0226] Step 5: Compound 19A [ka] A 25 mL single-neck flask was charged with 19e-1 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane, and the reaction was carried out at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 55 mg of a solid; LC-MS: [M+H] + =1105.4.

[0227] Step 6: Compound 19B [ka] A 25 mL single-neck flask was charged with 19e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane, and the reaction was carried out at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 58 mg of a solid; LC-MS: [M+H] + =1105.4.

[0228] Example 31 Synthesis of compounds 20A and 20B: [ka] Step 1: Compound 20a A 250 mL single-neck flask was charged with M1 (10 g, 27.1 mmol), benzyl 2-hydroxycyclopropylpropionate (prepared according to the method disclosed in WO 2020063676A) (12.0 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene. The mixture was heated to 100 °C and reacted for 4 hours. Upon completion of the reaction, the temperature was lowered to room temperature. The insoluble material was removed by filtration, and 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 material; LC-MS: [M+H] + =529.2.

[0229] Step 2: Compound 20b 20a (4 g, 7.6 mmol) and 10 mL of DMF were added to a 50 mL single-neck flask. After dissolution, DBU (1.39 g, 9.1 mmol) was added in an ice-water bath and the reaction was continued for 1 hour. The reaction solution was recorded as reaction solution (1).

[0230] 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 added and dissolved. After dissolving, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath. The reaction was continued for 30 minutes, and then reaction solution (1) was added, warmed to room temperature, and reacted for 2 hours. HPLC was performed to monitor the progress of the reaction. Upon completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.5 g of a solid in 84% yield; LC-MS: [M+H] + =702.3.

[0231] Step 3: Compound 20d 20b (1000 mg, 1.42 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 1000 mg of 5% Pd / C was added and the hydrogenation reaction was carried out for 2 hours. After the reaction was completed, it was filtered. The filtrate was placed in an ice-water bath and DIPEA (248 μL, 1.5 mmol) was added, followed by M5 (708 mg, 1.42 mmol). The temperature was then raised to room temperature and the reaction was carried out for 1 hour. Upon completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to give 443 mg of product in 36% yield; LC-MS: [M-H] - =856.4.

[0232] Step 4: Compounds 20e-1 and 20e-2 In a 50 mL single-neck flask, 20d (400 mg, 0.47 mmol), exatecan mesylate (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF were added, and DIPEA (248 μL, 1.5 mmol) was added in an ice-water bath. The temperature was raised to room temperature and reacted for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 20e-1 and compound 20e-2, which was lyophilized to give 103 mg of compound 20e-1; LC-MS: [M+H] + =1275.5 and compound 20e-2 103mg;LC-MS:[M+H] + =1275.5, respectively.

[0233] Step 5: Compound 20A [ka] A 25 mL single-neck flask was charged with 8A (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol), and 5 mL of nitromethane, and the reaction was carried out at 40 °C for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 51 mg of a solid; LC-MS: [M+H] + =1119.4.

[0234] Step 6: Compound 20B [ka] A 25 mL single-neck flask was charged with 20e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane, and the reaction was carried out at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 47 mg of a solid; LC-MS: [M+H] + =1119.4.

[0235] Example 32 Synthesis of compound 21: [ka] Step 1: Compound SM3-1 A 2000 mL single-neck flask was charged with 77087-60-6 (100 g, 458 mmol), maleic acid (53.4 g, 460 mmol), TEA (64 mL, 460 mmol), and 1000 mL of toluene. The mixture was heated to 100 °C and reacted for 5 hours. After the reaction was completed, the temperature was lowered to room temperature, and the mixture 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 = 100:1-50:1-20:1) to obtain 75.6 g of the desired material; LC-MS: [M+H] + =299.1.

[0236] Step 2: Compound (R)-tert-butyl 2-hydroxy-1,5-glutarate A 2000 mL single-neck flask was charged with 172793-31-6 (100 g, 338 mmol) and 1000 mL of water, followed by the sequential addition of sodium nitrite (35 g, 507 mmol) and concentrated sulfuric acid (32 mL, 35 mmol). The temperature was slowly raised to room temperature and the reaction was allowed to proceed for 24 hours. After the reaction was completed, the mixture was extracted three times with 500 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, 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 material; LC-MS: [M+H] + =261.4.

[0237] Step 3: Compound SM3 A 2000 mL single-neck flask was charged with (R)-tert-butyl 2-hydroxy-1,5-glutarate (50 g, 192 mmol) and 1000 mL of anhydrous tetrahydrofuran. The mixture was cooled to 0 °C in an ice-water bath, and then PPh3 (87.7 g, 288 mmol), DEAD (50.2 g, 288 mmol), and SM3-1 (57.3 g, 192 mmol) were added in that order. The temperature was slowly raised to room temperature, and the reaction was continued for 13 hours. After the reaction was completed, the insoluble material was removed by filtration, and 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.

[0238] The above product was dissolved in 500 mL of ethanol and cooled to 0°C in an ice-water bath. NaOH (64 mL, 190 mmol, 3 M / L) was added dropwise at this temperature, and the reaction was carried out for 12 hours while maintaining the temperature. Then, the pH was adjusted to 3 by adding HCl (6 M / L), and the mixture was extracted five times with 500 mL of dichloromethane. The mixture was dried over anhydrous sodium sulfate, filtered, and 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: [MH] - =525.5.

[0239] Step 4: Compound M6 Compound 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 added to a 2000 mL single-neck flask, and the reaction was carried out at room temperature for 1 hour (monitored by TLC). Insoluble materials were filtered off. The reaction solution was directly purified by preparative LC. The fraction was concentrated under reduced pressure in a water bath at 35°C with a water pump to remove acetonitrile, and lyophilized to give compound M6 (51.9 g) in 79% yield; LC-MS: [M+H] + =693.3.

[0240] Step 5: Compound 21a 1c (1 g, 2.36 mmol) was added to a 25 mL single-neck flask and dissolved in 25 mL of DMF. DIPEA (430 μL, 2.6 mmol) was added, followed by M6 (1177 mg, 2.36 mmol). The temperature was then raised to room temperature and the reaction was allowed to proceed for 1 hour. The completion of the reaction was confirmed by HPLC. The reaction solution was purified by high-performance liquid chromatography to obtain a fraction, which was lyophilized to give 555 mg of product; LC-MS: [M-H] - =931.0.

[0241] Step 6: Compound 21b In a 100 mL single-neck flask, 21a (500 mg, 0.54 mmol), exatecan 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 added, and DIPEA (248 μL, 1.5 mmol) was added in an ice-water bath. The temperature was raised to room temperature and the reaction was carried out for 2 hours. After the completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 21b, which was lyophilized to obtain 231 mg of compound 21b; LC-MS: [M+H] + =1349.5.

[0242] Step 7: Compound 21 Compound 21b (200 mg, 0.1488 mmol), zinc bromide (665 mg, 2.96 mmol), and 10 mL of nitromethane were added to a 25 mL one-neck flask, and the reaction was carried out at 40 °C for 1 h. After the reaction was completed as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 103 mg of a solid; LC-MS: [M+H] + =1137.5.

[0243] Example 33 Synthesis of compound 22: [ka] Using compounds M6 and 3c as starting materials and referring to the synthetic route of Example 32, compound 22 (91 mg) was obtained; LC-MS: [M+H] + =1165.5.

[0244] Example 34 Synthesis of compounds 23 and 24: [ka] Using compounds M6 and 5c as starting materials and referring to the synthetic route of Example 32, compound 23 102 mg; LC-MS: [M+H] +=1151.4;Compound 24 99mg;LC-MS:[M+H] + =1151.4 was obtained.

[0245] Example 35 Synthesis of compounds 25 and 26: [ka] Using compounds M6 and 7c as starting materials and referring to the synthetic route of Example 32, compound 25 83 mg; LC-MS: [M+H] + =1205.7;Compound 26 80mg;LC-MS:[M+H] + =1205.7 was obtained.

[0246] Example 36 Synthesis of compounds 27 and 28: [ka] Using compounds M6 and 19c as starting materials and referring to the synthetic route of Example 32, compound 27 100 mg; LC-MS: [M+H] + =1177.5;Compound 28 101mg;LC-MS:[M+H] + =1177.5 was obtained.

[0247] Example 37 Synthesis of compound 29: [ka] Step 1: Compound SM4-1 A 5000 mL single-neck flask was charged with 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). The mixture was heated and refluxed for 5 hours (monitored by TLC). The insoluble material was 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 give SM4-1 (64.7 g) in 50% yield; LC-MS: [M+H] + =299.2.

[0248] Step 2: Compound SM4-2 SM4-1 (64 g, 215 mmol) was added to a 2000 mL single-neck flask and dissolved in 1000 mL of DMF. DIPEA (71 mL, 430 mmol) was then added, followed by nonaethylene glycol monomethyl ether methanesulfonate (111.5 g, 220 mmol). The temperature was then raised to room temperature and the reaction was carried out for 2 hours. The completion of the reaction was detected by HPLC, and the reaction solution was purified by silica gel column chromatography (PE / EA = 50 / 1-20 / 1-1 / 1) to obtain 59.9 g of product; LC-MS: [M+H] + =709.4.

[0249] Step 3: Compound SM4 SM4-2 (59 g, 83 mmol) was added to a 2000 mL single-neck flask and dissolved in 1000 mL of MeOH. K2CO3 (11.75 g, 85 mmol) was then added, and the reaction was carried out at room temperature for 4 hours. Upon completion of the reaction as detected by HPLC, the insoluble material was removed by filtration, and the reaction solution was directly purified by preparative LC. The fraction was concentrated under reduced pressure in a water bath at 35 °C with a water pump to remove acetonitrile, and then lyophilized to obtain compound SM4 (27 g); LC-MS: [M-H] - =693.5.

[0250] Step 4: Compound M7 Compound 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 added to a 500 mL single-neck flask. The reaction was carried out at room temperature for 1 hour (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly purified by preparative LC. The fraction was concentrated under reduced pressure in a water bath at 35°C with a water pump to remove acetonitrile, and lyophilized to give compound M7 (23.3 g) in 93% yield; LC-MS: [M+H] + =695.8.

[0251] Step 5: Compound 29a 1c (1 g, 2.36 mmol) was added to a 25 mL single-neck flask and dissolved in 25 mL of DMF. DIPEA (430 μL, 2.6 mmol) was added, followed by M7 (1640 mg, 2.36 mmol). The temperature was then raised to room temperature and the reaction was allowed to proceed for 1 hour. The completion of the reaction was detected by HPLC, and the reaction solution was purified by high-performance liquid chromatography to obtain a fraction, which was lyophilized to give 609 mg of product; LC-MS: [M H] - =1098.5.

[0252] Step 6: Compound 29b In a 100 mL single-neck flask, 29a (500 mg, 0.45 mmol), exatecan 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 added, and DIPEA (248 μL, 1.5 mmol) was added in an ice-water bath. The temperature was raised to room temperature and the reaction was carried out for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 29b, which was lyophilized to obtain 187 mg of compound 29b; LC-MS: [M+H] + =1517.6.

[0253] Step 7: Compound 29 Compound 29b (150 mg, 0.988 mmol), zinc bromide (223 mg, 0.988 mmol), and 10 mL of nitromethane were added to a 25 mL single-neck flask, and the reaction was carried out at 40° C. for 1 hour. After the reaction was completed as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 114 mg of a solid; LC-MS: [M+H] + =1517.9.

[0254] Example 38 Synthesis of compound 30: [ka] Using compounds M7 and 3c as starting materials and referring to the synthetic route of example 37, compound 30 (125 mg) was obtained; LC-MS: [M+H] + =1445.6.

[0255] Example 39 Synthesis of compounds 31 and 32: [ka] Using compounds M7 and 5c as starting materials and referring to the synthetic route of Example 37, compound 31 61 mg; LC-MS: [M+H] + =1431.7;Compound 32 63mg;LC-MS:[M+H] + =1431.7 was obtained.

[0256] Example 40 Synthesis of compounds 33 and 34: [ka] Using compounds M7 and 7c as starting materials and referring to the synthetic route of Example 37, compound 33 60 mg; LC-MS: [M+H] + =1485.6;Compound 34 58mg;LC-MS:[M+H] + =1485.6 was obtained.

[0257] Example 41 Synthesis of compounds 35 and 36: [ka] Using compounds M7 and 19c as starting materials and referring to the synthetic route of Example 37, compound 35 102 mg; LC-MS: [M+H] + =1457.8;Compound 36 102mg;LC-MS:[M+H] + =1457.8 was obtained.

[0258] Example 42 Synthesis of compound 37: [ka] Step 1: Compound SM5-1 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 added to a 2000 mL single-neck flask. The reaction was carried out at room temperature for 12 hours (monitored by TLC). The insoluble material was filtered off. The solvent was directly removed from the reaction solution by rotary evaporation under reduced pressure. 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.

[0259] Step 2: Compound SM5-2 Compound SM5-1 (100 g, 233.4 mmol, 1.0 equiv.) and THF (1000 mL) were added to a 2000 mL single-neck flask 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 added in batches, and the reaction was maintained at 0 °C for 24 h (monitored by TLC). 500 mL of saturated aqueous NH4Cl was added to quench the reaction, followed by extraction with 500 mL of ethyl acetate three times. The organic phase was then dried with anhydrous sodium sulfate and filtered. The solvent was directly removed from the filtrate by rotary evaporation under reduced pressure, and 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.

[0260] Step 3: Compound SM5 (see Org. Lett., 2006, 8, 3387-3390) Compound SM5-2 (35 g, 79 mmol, 1.0 equiv.) and DCE (500 mL) were added to a 1000 mL single-neck flask, followed by the sequential addition of palladium(II) acetate (180 mg, 0.8 mmol), I2 (20 g, 79 mmol), and iodobenzene diacetate (40.8 g, 126.4 mmol). The temperature was raised to 60 °C and the reaction was continued for 40 h (monitored by TLC). The reaction was quenched by adding 500 mL of saturated aqueous sodium thiosulfate solution, and extracted three times with 500 mL of dichloromethane. The organic phase was then dried with anhydrous sodium sulfate and filtered. The solvent was directly removed from the filtrate by rotary evaporation under reduced pressure, and the residue was subjected to silica gel column chromatography (PE / EA = 100 / 1-50 / 1-10 / 1) to give SM5 (28 g); LC-MS: [M+H] + =501.3.

[0261] Step 4: Compound SM6 Compound SM5 (25 g, 50 mmol, 1.0 equiv.), di-tert-butyl potassium phosphate (13.66 g, 55 mmol, 1.1 equiv.), p-toluenesulfonic acid monohydrate (951 mg, 5 mmol, 0.1 equiv.), and THF (200 mL) were added to a 500 mL single-neck flask. The reaction was carried out at room temperature for 1 hour (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly purified by preparative LC. The aliquot was concentrated under reduced pressure in a water bath at 35°C with a water pump to remove acetonitrile and freeze-dried to give compound SM6 (15.1 g) in 46% yield; LC-MS: [M+H] + =651.4.

[0262] Step 5: Compound SM7 SM6 (15 g, 23 mmol) and 100 mL of DMF were added to a 250 mL single-neck flask. After dissolution, 15 g of 5% Pd / C was added in an ice-water bath. The atmosphere in the system was replaced with hydrogen three times, and the reaction was carried out at room temperature for 12 h. The Pd / C was removed by filtration, and then the solvent was removed by rotary evaporation under reduced pressure using an oil pump. The crude product was set aside for immediate use.

[0263] Into another 250 mL single-neck flask, the above crude product, 100 mL of toluene, triethylamine (6.4 mL, 46 mmol), and maleic anhydride (2.4 g, 24 mmol) were added and dissolved. After dissolving, the temperature was raised to 100°C and the reaction was carried out for 2 hours. The progress of the reaction was monitored using HPLC. Upon completion of the reaction, the reaction solution was purified by HPLC to obtain a fraction. The fraction was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.2 g of a solid in 36% yield; LC-MS: [M+H] + =507.3.

[0264] Step 6: Compound M8 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 added to a 100 mL single-neck flask, and the reaction was carried out at room temperature for 1 hour (monitored by TLC). Insoluble materials were filtered off. The reaction solution was directly purified by preparative LC. The fraction was concentrated under reduced pressure in a water bath at 35 °C with a water pump to remove acetonitrile, and lyophilized to give compound M8 (3.7 g) in 70% yield; LC-MS: [M+H] + =673.2.

[0265] Step 7: Compound 37a In a 25 mL single-neck flask, 1c (1 g, 2.36 mmol) was added and dissolved in 25 mL of DMF. DIPEA (430 μL, 2.6 mmol) was added, followed by M8 (1.2 g, 2.36 mmol). The temperature was then raised to room temperature and the reaction was continued for 1 hour. The completion of the reaction was detected by HPLC, and the reaction solution was purified by high-performance liquid chromatography to obtain a fraction, which was lyophilized to give 488 mg of product; LC-MS: [M H] - =911.0.

[0266] Step 8: Compound 37b 37a (400 mg, 0.44 mmol), exatecan 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 added to a 100 mL single-neck flask, and DIPEA (218 μL, 1.32 mmol) was added in an ice-water bath. The temperature was raised to room temperature and the reaction was continued for 2 hours. After the reaction was completed as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 37b, which was lyophilized to give 201 mg of compound 37b; LC-MS: [M+H] + =1329.6.

[0267] Step 9: Compound 37 Compound 37b (130 mg, 0.098 mmol), zinc bromide (221 mg, 0.98 mmol), and 10 mL of nitromethane were added to a 25 mL one-neck flask, and the reaction was carried out at 40 °C for 1 hour. After the reaction was completed as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain 96 mg of a solid; LC-MS: [M+H] + =1117.4.

[0268] Example 43 Synthesis of compound 38: [ka] Using compounds M8 and 3c as starting materials and referring to the synthetic route of example 42, compound 38 (51 mg) was obtained; LC-MS: [M+H] + =1145.6.

[0269] Example 44 Synthesis of compounds 39 and 40: [ka] Using compounds M8 and 5c as starting materials and referring to the synthetic route of Example 42, compound 39 57 mg; LC-MS: [M+H] + =1131.4;Compound 40 60mg;LC-MS:[M+H] + =1131.4 was obtained.

[0270] Example 45 Synthesis of compounds 41 and 42: [ka] Using compounds M7 and 7c as starting materials 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 was obtained; LC-MS: [M+H] + =1185.3.

[0271] Example 46 Synthesis of compounds 43 and 44: [ka] Using compounds M8 and 19c as starting materials 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 was obtained; LC-MS: [M+H] + =1157.4.

[0272] Example 47 (Comparative Example) Synthesis of compound 45: [ka] Compound 45 was synthesized according to the method provided in Example 58 of the patent "CN Patent No. 104755494A".

[0273] Example 48 Synthesis of compound 46: [ka] Step 1: Compound 46a 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 added to a 50 mL one-neck flask, and DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath. The temperature was raised to room temperature and the reaction was continued for 2 h. After the reaction was complete as detected by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction, which was lyophilized to give 46a (210 mg); LC-MS: [M+H] + =1221.6.

[0274] Step 2: Compound 46 46a (200 mg, 0.162 mmol), zinc bromide (736 mg, 3.26 mmol), and 10 mL of nitromethane were added to a 25 mL one-neck flask, and the reaction was carried out at 40 °C for 1 h. After the reaction was completed as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain solid compound 46 (120 mg); LC-MS: [M+H] + =1065.3.

[0275] 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.

[0276] Example 50 Synthesis of compound 48A: [ka] Step 1: Compound 48a 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 added to a 100 mL one-neck flask, and DIPEA (0.84 mL, 1.5 equiv.) was added in an ice-water bath. The temperature was raised to room temperature and the reaction was allowed to proceed for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative LC. The fraction was concentrated under reduced pressure in a water bath at 35 °C with a water pump to remove acetonitrile and lyophilized to give compound 48a (1.54 g) in 61% yield; LC-MS: [M+H] + =1235.4.

[0277] Step 6: Compound 48A Compound 48a (1.0 g, 0.8 mmol, 1.0 equiv.) and 35 mL of nitromethane were added to a 100 mL single-neck flask. After dissolution, zinc bromide (3.64 g, 16 mmol, 20.0 equiv.) was added and the mixture was allowed to react for 30 minutes in a 40 °C oil bath (preheated to stabilize). The mixture was then concentrated under reduced pressure at 45 °C in a water bath with a water pump to remove the nitromethane, yielding a yellow residual solid (monitored by HPLC). A aliquot was obtained by acid preparation. The aliquot was concentrated under reduced pressure at 35 °C in a water bath with a water pump, the acetonitrile was removed by rotation, and the mixture was lyophilized to give compound 48A (786 mg) in a 90% yield.

[0278] Example 51 Synthesis of compound 48B: [ka] Step 1: Compound 48b 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 added to a 25 mL single-neck flask. The temperature was lowered to 0-5 °C in an ice-water bath, and DIPEA (62 mg, 0.48 mmol, 2.0 equiv.) was added. The temperature was then raised to 20 ± 5 °C and the reaction was allowed to proceed for 2 h. The completion of the reaction was detected by HPLC. The reaction solution was directly purified by HPLC preparative HPLC, and the product aliquot was collected and lyophilized to give compound 48b (150.2 mg); LC-MS: [M+H] + =1235.4.

[0279] Step 2: Compound 48B 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 to a 25 mL one-neck flask, and the temperature was then raised to 40 °C for 0.5 h. The reaction was stopped, and the reaction solution was directly rotary evaporated under reduced pressure at 45 °C to obtain a yellow solid. A sample was taken and the reaction was monitored by HPLC. The rotary dried solid was directly purified by HPLC preparative HPLC, and the product aliquot was collected and lyophilized to obtain compound 48B (70.0 mg); LC-MS: [M+H] + =1079.4.

[0280] 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.

[0281] Example 53 Preparation of compound 49B: [ka] Following the route of Example 51, compound 49B (65 mg) was obtained; LC-MS: [M+H] + =1079.4.

[0282] Example 54 Synthesis of compounds 50A and 50B: [ka] Step 1: Compounds 50a and 50b A 50 mL single-neck flask was charged with 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. DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of compound 50a and compound 50b. The fractions were lyophilized to give 170 mg of compound 50a; LC-MS: [M+H] + =1289.46 and compound 50b 202mg;LC-MS:[M+H] + =1289.46 was obtained.

[0283] Step 2: Compound 50A [ka] A 25 mL single-neck flask was charged with 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane, and the mixture was reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 44 mg of a solid.

[0284] Step 3: Compound 50B [ka] A 25 mL single-neck flask was charged with 50b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane, and the mixture was reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 45 mg of a solid.

[0285] Example 55 Synthesis of compounds 51A and 51B: [ka] Step 1: Compound 51a and Compound 51b A 50 mL single-neck flask was charged with 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. DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of Compound 51a and Compound 51b. These fractions were lyophilized to give 190 mg of Compound 51a and 186 mg of Compound 51b, respectively. LC-MS of Compound 51a: [M+H] + = 1289.47; LC-MS of compound 51b: [M+H] + =1289.47.

[0286] Step 2: Compound 51A [ka] Compound 51a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-neck flask and reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 39 mg of a solid.

[0287] Step 3: Compound 51B [ka] Compound 51b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-neck flask and reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 60 mg of a solid.

[0288] Example 56 Synthesis of compound 52A: [ka] Step 1: Compound 52a A 50 mL single-neck flask was charged with 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. DIPEA (660 μL, 4.0 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 4 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 52a, which was lyophilized to give 52a (388 mg); LC-MS: [M+H] + =1261.4.

[0289] Step 2: Compound 52A In a 25 mL single-neck flask, 52a (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol), and 10 mL of nitromethane were added and reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain solid compound 52A (79 mg); LC-MS: [M+H] + =1105.4.

[0290] 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.

[0291] Example 58 Synthesis of compound 53A: [ka] Step 1: 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 added, and DIPEA (330 μL, 2.0 mmol) was added in an ice-water bath. The mixture was then warmed to room temperature and reacted for 3 h. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 53a, which was lyophilized to give 53a (200 mg); LC-MS: [M+H] + =1275.4.

[0292] Step 2: 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 added and reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain solid compound 53A (51 mg); LC-MS: [M+H] + =1119.4.

[0293] 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.

[0294] Example 60 Synthesis of compounds 54A and 54B: [ka] Step 1: Compounds 54a and 54b A 50 mL single-neck flask was charged with 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. DIPEA (292 μL, 1.77 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of compounds 54a and 54b. The fractions were separately lyophilized to give 103 mg of compound 54a; LC-MS: [M+H]. + =1261.5, and compound 54b 111mg;LC-MS:[M+H] + =1261.5 was obtained.

[0295] Step 2: Compound 54A [ka] In a 25 mL one-neck flask, 54a (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added and reacted at 40 °C for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 61 mg of a solid; LC-MS: [M+H] + =1105.4.

[0296] Step 3: Compound 54B [ka] In a 25 mL one-neck flask, 54b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added and reacted at 40 °C for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 57 mg of a solid; LC-MS: [M+H] + =1105.4.

[0297] Example 61 Synthesis of compounds 55A and 55B: [ka] Step 1: Compounds 55a and 55b A 50 mL single-neck flask was charged with 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. DIPEA (248 μL, 1.5 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of compounds 55a and 55b. The fractions were separately lyophilized to give 100 mg of compound 55a; LC-MS: [M+H] + =1275.5 and compound 55b 101mg;LC-MS:[M+H] + =1275.5 was obtained.

[0298] Step 2: 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 added and reacted at 40 °C for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 42 mg of a solid; LC-MS: [M+H] + =1119.4.

[0299] Step 3: Compound 55B [ka] In a 25 mL one-neck flask, 55b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added and reacted at 40 °C for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 45 mg of a solid; LC-MS: [M+H] + =1119.4.

[0300] 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.

[0301] 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.

[0302] Example 64 Synthesis of compound 58: [ka] Step 1: Synthesis of Compound 58a Exatecan mesylate M5 (15 g, 28 mol, prepared by the method disclosed in European Patent Application Publication No. 0737683A1) was added to 400 mL of DMF and cooled to 0 °C in an ice-water bath. Triethylamine was added dropwise to adjust the pH to 7-8. Benzyl bromide (9.6 g, 56 mmol) was added dropwise in an ice-water bath, and the temperature was raised to room temperature (25 °C). The reaction was allowed to proceed for 1 hour. After completion of the reaction was detected by TLC, the reaction solution was concentrated under reduced pressure. The resulting crude product was purified by preparative high-performance liquid chromatography (acetonitrile / purified water system). The target peak was collected, and the acetonitrile was removed under reduced pressure. The crude product was then lyophilized to give compound 58a, a yellow solid (approximately 11 g) in a yield of approximately 74%; MS m / z: [M+H]. + 526.3.

[0303] Step 2: Synthesis of Compound 58b Compound 58a (11 g, 21 mol) and 120 mL of formic acid for dissolution were added sequentially to a 250 mL single-neck flask at room temperature. 30 mL of formaldehyde (40% aqueous solution) was added to the resulting bright yellow solution, and the temperature was raised to 50 °C for 1 h. Upon completion of the reaction as detected by TLC, the temperature was cooled to room temperature, and the reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile / purified water system) and the target peak was collected. The acetonitrile was removed under reduced pressure, followed by lyophilization to obtain compound 58b, approximately 4.5 g of a yellow powdery solid, in approximately 40% yield; MS m / z: [M+H] + 540.6.

[0304] Step 3: Synthesis of compound 58: Compound 58b (2.3 g, 4.3 mol) was added to a 250 mL single-neck flask at room temperature and dissolved in 100 mL of DMF. 2.3 g of 5% Pd / C was added to the resulting bright yellow solution. The atmosphere in the system was replaced using a hydrogen balloon, and the reaction was maintained at room temperature for 1.5 hours. The completion of the reaction was then detected by HPLC. The Pd / C was removed by filtration, and the resulting reaction solution was concentrated and then purified by preparative high-performance liquid chromatography (acetonitrile / purified water system). The target peak was collected, the acetonitrile was removed under reduced pressure, and the mixture was lyophilized to obtain compound 58, approximately 1.0 g of a yellow powdery solid, in approximately 52% yield; MS m / z: [M+H] + 450.5.

[0305] Example 65 Synthesis of compound 59: [ka] Step 1: Compound 59a A 50 mL single-neck flask was charged with 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. DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 2 h. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to give 59a (166 mg); LC-MS: [M+H] + =1235.6.

[0306] Step 2: 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 added and reacted at 40 °C for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was lyophilized to obtain solid compound 59 (43 mg); LC-MS: [M+H] + =1079.3.

[0307] 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.

[0308] Example 67 Synthesis of compound 61: [ka] Step 1: Compound 61a A 100 mL single-neck flask was charged with 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). The reaction mixture was added with DIPEA (0.84 mL, 1.5 equiv.) in an ice-water bath, and the temperature was raised to room temperature for 2 h (monitored by HPLC). The reaction solution was purified by preparative LC, and the fraction was concentrated under reduced pressure in a water bath at 35 °C with a water pump to remove acetonitrile and lyophilized to give compound 61a (1.21 g); LC-MS: [M+H] + =1249.4.

[0309] Step 2: Compound 61 Compound 61a (1.0 g, 0.8 mmol, 1.0 equivalent) and 35 mL of nitromethane were added to a 100 mL single-neck flask. After dissolution, zinc bromide (3.64 g, 16 mmol, 20.0 equivalents) was added and the mixture was allowed to react in a 40 °C oil bath (preheated to stabilize) for 30 minutes. The mixture was then concentrated under reduced pressure at 45 °C in a water bath using a water pump to remove the nitromethane, resulting in a yellow solid residue (monitored by HPLC). After acid preparation, a fraction was obtained. The fraction was concentrated under reduced pressure at 35 °C in a water bath using a water pump, the acetonitrile was removed by rotation, and the mixture was lyophilized to give compound 61 (786 mg). LC-MS: [M+H] + =1093.6.

[0310] Example 68 Synthesis of compound 62: [ka] Step 1: Compound 62a 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 added to a 25 mL single-neck flask. The temperature was cooled to 0-5 °C in an ice-water bath, and DIPEA (62 mg, 0.48 mmol, 2.0 equiv.) was added. The temperature was then raised to 20 ± 5 °C and the reaction was allowed to proceed for 2 h. The reaction was monitored for completion by HPLC. The reaction solution was directly purified by HPLC preparative HPLC, and the product aliquot was collected and lyophilized to give compound 62a (120.9 mg). LC-MS: [M+H] + =1249.4.

[0311] Step 2: Compound 62 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 to a 25 mL single-neck flask. After the addition was complete, the temperature was raised to 40 °C and the reaction was allowed to proceed for 0.5 h. The reaction was stopped and the reaction solution was directly rotary evaporated under reduced pressure at 45 °C to obtain a yellow solid, which was sampled and used for HPLC monitoring. The rotary dried solid was directly purified by HPLC preparative HPLC, and the product aliquot was collected and lyophilized to obtain compound 62 (61 mg); LC-MS: [M+H] + =1093.4.

[0312] Example 69 Preparation of Compound 63: [ka] Compound 63 (60 mg) was obtained by following the synthetic route of Example 67; LC-MS: [M+H] + =1093.4.

[0313] Example 70 Preparation of Compound 64: [ka] Compound 64 (65 mg) was obtained by following the synthetic route of Example 68; LC-MS: [M+H] + =1093.4.

[0314] Example 71 Preparation of Compounds 65A and 65B: [ka] Step 1: Compounds 65a and 65b A 50 mL single-neck flask was charged with 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. DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath, and the temperature was raised to room temperature for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of compounds 65a and 65b. The fractions were separately lyophilized to give 155 mg of compound 65a; LC-MS: [M+H]. + =1303.4 and compound 65b 158mg;LC-MS:[M+H] + =1303.6 was obtained.

[0315] Step 2: 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 added and reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a fraction, which was lyophilized to obtain 49 mg of a solid.

[0316] Step 3: 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 added and reacted at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a fraction, which was lyophilized to obtain 47 mg of a solid.

[0317] Example 72 Synthesis of compounds 66A and 66B: [ka] Step 1: Compound 66a and Compound 66b A 50 mL single-neck flask was charged with 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. DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of compound 66a and compound 66b. The 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.

[0318] Step 2: Compound 66A [ka] Compound 66a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-neck flask and reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 57 mg of a solid; LC-MS: [M+H] + =1147.5.

[0319] Step 3: Compound 66B [ka] Compound 66b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-neck flask and reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 57 mg of a solid: [M+H] + =1147.5.

[0320] Example 73 Synthesis of compound 67A: [ka] Step 1: Compound 67a A 50 mL single-neck flask was charged with 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. The flask was then cooled to room temperature and reacted for 4 hours in an ice-water bath. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 67a, which was lyophilized to give 67a (402 mg); LC-MS: [M+H] + =1275.4.

[0321] Step 2: Compound 67A In a 25 mL one-neck flask, 67a (100 mg, 0.78 mmol), zinc bromide (356 mg, 1.57 mmol), and 10 mL of nitromethane were added and reacted at 40 °C for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain solid compound 67A (47 mg); LC-MS: [M+H] + =1119.5.

[0322] 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.

[0323] Example 75 Synthesis of compound 68A: [ka] Step 1: Compound 68a A 50 mL single-neck flask was charged with 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. The resulting mixture was added with DIPEA (330 μL, 2.0 mmol) in an ice-water bath, and the mixture was allowed to warm to room temperature for 3 h. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 68a, which was lyophilized to give 68a (177 mg); LC-MS: [M+H] + =1289.4.

[0324] Step 2: 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 added and reacted at 40 °C for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain solid compound 68A (45 mg); LC-MS: [M+H] + =1133.4.

[0325] 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.

[0326] Example 77 Synthesis of compounds 69A and 68B: [ka] Step 1: Compounds 69a and 69b A 50 mL single-neck flask was charged with 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. The flask was then cooled to room temperature and reacted for 2 hours with DIPEA (292 μL, 1.77 mmol). After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compounds 69a and 69b. The fraction was lyophilized to give 109 mg of compound 69a; LC-MS: [M+H]. + =1275.5, and compound 69b 111 mg; LC-MS:[M+H] + =1275.7 was obtained.

[0327] Step 2: 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 added and reacted at 40 °C for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 53 mg of a solid; LC-MS: [M+H] + =1119.4.

[0328] Step 3: Compound 69B [ka] In a 25 mL one-neck flask, 69b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol), and 5 mL of nitromethane were added and reacted at 40 °C for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 54 mg of a solid; LC-MS: [M+H] + =1119.4.

[0329] Example 78 Synthesis of compounds 70A and 70B: [ka] Step 1: Compounds 70a and 70b A 50 mL single-neck flask was charged with 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 (248 μL, 1.5 mmol) in an ice-water bath. The mixture was then warmed to room temperature and reacted for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of compounds 70a and 70b. The fractions were separately lyophilized to give 106 mg of compound 70a; LC-MS: [M+H] + =1289.5 and compound 70b 101mg;LC-MS:[M+H] + =1289.4 was obtained.

[0330] Step 2: 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 added and reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 39 mg of a solid; LC-MS: [M+H] + =1133.4.

[0331] Step 3: 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 added and reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain 35 mg of a solid; LC-MS: [M+H] + =1133.4.

[0332] 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.

[0333] 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.

[0334] Example 81 [ka] Synthesis of compound M11: 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 added to a 100 mL single-neck flask and reacted at room temperature for 20 min (monitored by TLC). The reaction solution was directly purified by preparative LC. The aliquot was concentrated under reduced pressure in a water bath at 35 °C with a water pump to remove acetonitrile and lyophilized to give compound M10 (13.2 g) in 78% yield; LC-MS: [M+H] + =866.5.

[0335] Compound 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 added to a 100 mL single-neck flask and reacted at room temperature for 30 minutes (monitored by TLC). The insoluble material was filtered off. The reaction solution was directly purified by preparative LC. The fraction was concentrated under reduced pressure in a water bath at 35 °C with a water pump to remove acetonitrile, and lyophilized to give compound M11 (14.2 g) in 92% yield; LC-MS: [M+H] + =1032.5.

[0336] Example 82 Synthesis of compound 73: [ka] Step 1: Synthesis of Compound 73a 10 mL of DMF was added to M11 (1 g, 0.79 mol), cooled to 0 °C in an ice-water bath, and compound 1c (334 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added. The reaction was continued for 1 hour under the same conditions. The completion of the reaction was monitored by TLC. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile / purified water system), and the target peak was collected. After removing acetonitrile under reduced pressure, lyophilization was performed to obtain approximately 1.2 g of compound 73a; MS m / z: [M+H] + =1271.9.

[0337] Step 2: Synthesis of Compound 73b A 25 mL single-neck flask was charged with 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. DIPEA (310 mg, 2.4 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 2 h. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC and lyophilized to give 73b (709 mg); LC-MS: [M+H] + =1720.8.

[0338] Step 3: Synthesis of compound 73: In a 25 mL one-neck flask, 73b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol), and 10 mL of nitromethane were added and reacted at 40° C. for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain solid compound 73 (88 mg); LC-MS: [M+H] + =1532.6.

[0339] 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.

[0340] Example 84 Synthesis of compound 75: [ka] Step 1: Synthesis of Compound 75a 10 mL of DMF was added to M11 (1 g, 0.79 mol), cooled to 0 °C in an ice-water bath, and compound 5c (345 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added. The reaction was continued for 1 hour under the same conditions. The completion of the reaction was monitored by TLC. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile / purified water system), and the target peak was collected. After removing acetonitrile under reduced pressure, the mixture was lyophilized to obtain 0.9 g of compound 75a; MS m / z: [M+H] + =1285.6.

[0341] Step 2: Synthesis of Compound 75b A 25 mL single-neck flask was charged with 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. DIPEA (155 mg, 1.2 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature for 2 h. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to give 75b (304 mg); LC-MS: [M+H] + =1734.8.

[0342] Step 3: Synthesis of compound 75: In a 25 mL one-neck flask, 75b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol), and 10 mL of nitromethane were added and reacted at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain solid compound 75 (96 mg); LC-MS: [M+H] + =1546.6.

[0343] 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.

[0344] 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.

[0345] 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.

[0346] Example 88 Synthesis of compounds 79 and 80: [ka] Step 1: Synthesis of compound 79a 10 mL of DMF was added to M11 (1 g, 0.79 mol), cooled to 0 °C in an ice-water bath, and compound 20c (377 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added. The reaction was continued for 1 hour under the same conditions. The completion of the reaction was monitored by TLC. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile / purified water system), and the target peak was collected. After removing acetonitrile under reduced pressure, the mixture was lyophilized to give 783 mg of compound 79a; MS m / z: [M+H] + =1325.8.

[0347] Step 2: Synthesis of Compounds 79b-1 and 79b-2 A 25 mL single-neck flask was charged with 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. The flask was then cooled to room temperature and reacted for 2 hours. After completion of the reaction as detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of compounds 79b-1 and 79b-2. The fractions were lyophilized to give 79b-1 (124 mg); LC-MS: [M+H] + = 1743.0; and 79b-1 (122 mg); LC-MS: [M+H] + =1743.0 was obtained.

[0348] Step 3: Synthesis of Compound 79 [ka] In a 25 mL one-neck flask, 79b-1 (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol), and 10 mL of nitromethane were added and reacted at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain solid compound 79 (30 mg); LC-MS: [M+H] + =1586.9.

[0349] Step 4: Synthesis of Compound 80 [ka] In a 25 mL one-neck flask, 79b-2 (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol), and 10 mL of nitromethane were added and reacted at 40 °C for 1 hour. After completion of the reaction as detected by HPLC, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, which was lyophilized to obtain solid compound 80 (33 mg); LC-MS: [M+H] + =1587.0.

[0350] 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.

[0351] 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.

[0352] Example 91 1) Expression and purification of SI-1x6.4 antibody: Expi293 (Shanghai OPM Biosciences Co., Ltd.) suspension cells were used to express the SI-1x6.4 antibody. The day before transfection, cells were grown at a density of 0.9x10 in a 1 L shake flask containing 300 mL of OPM-293 CD05 medium (81075-001, Shanghai OPM Biosciences Co., Ltd.). 6Cells were seeded at 1000 cells / mL and cultured overnight at 37°C, 5% CO2, and 120 rpm in a cell culture shaker. The next day, the antibody expression plasmid was transfected with PEI-MAX at a mass ratio of 1:3. OPM-293 ProFeed supplement was added at 5% (v / v) on day 1 posttransfection, and again at 5% (v / v) on day 3 posttransfection. Centrifugation was performed on day 6 posttransfection, and the supernatant was collected.

[0353] The collected cell expression supernatant was eluted with 0.05 M sodium acetate (pH 3.6) on a protein A affinity chromatography column (UniMab 50, Suzhou Nanomicro Technology Co., Ltd.). The captured antibody was adjusted to pH 7.0 with 1 M Tris-HCl (pH 8.8) at a ratio of 0.7 / 10 (v / v). It was then passed through a gel filtration chromatography column SEC (Superdex 200, GE) to remove impurities such as polymers, and the antibody buffer was replaced with 20 mM PB (pH 6.5).

[0354] Antibody SI-1×6.4: Light chain nucleic acid coding sequence SEQ ID NO: 1 [ka] Light chain amino acid sequence SEQ ID NO: 2 [ka] In the above formula, the variable region is: SEQ ID NO: 28 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 25), CDR2 (SEQ ID NO: 26), and CDR3 (SEQ ID NO: 27), respectively.

[0355] Nucleic acid coding sequences for the construction of heavy chain and single-chain Fv (scFv) structural domains SEQ ID NO: 3 [ka]

[0356] Amino acid sequences of heavy chain and single-chain Fv (scFv) structural domain constructs SEQ ID NO:4 [ka] In the above formula, the variable region of the heavy chain is: SEQ ID NO: 38 [ka] and the underlined portions, from left to right, are CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 30), and CDR3 (SEQ ID NO: 31), respectively.

[0357] The variable region of the heavy chain in the structural domain of the single chain Fv (scFv) is: SEQ ID NO: 39 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 32), CDR2 (SEQ ID NO: 33), and CDR3 (SEQ ID NO: 34), respectively.

[0358] The variable region of the light chain in the single-chain Fv (scFv) structural domain is: SEQ ID NO: 40 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 35), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 37), respectively.

[0359] 2) Expression and purification of SI-1x22 antibody: Similar methods were followed for expression and purification of SI-1x22 antibody.

[0360] Antibody SI-1×22: Nucleic acid coding sequences for constructs of light chain and single-chain Fv (scFv) structural domains SEQ ID NO:9 [ka]

[0361] Amino acid sequences of light chain and single-chain Fv (scFv) structural domain constructs SEQ ID NO: 10 [ka] In the above formula, the light chain variable region is: SEQ ID NO: 49 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 25), CDR2 (SEQ ID NO: 26), and CDR3 (SEQ ID NO: 27), respectively.

[0362] The variable region of the heavy chain in the structural domain of the single chain Fv (scFv) is: SEQ ID NO:50 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 32), CDR2 (SEQ ID NO: 33), and CDR3 (SEQ ID NO: 34), respectively.

[0363] The variable region of the light chain of the single-chain Fv (scFv) structural domain is: SEQ ID NO:51 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 35), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 37), respectively.

[0364] Heavy chain nucleic acid coding sequence SEQ ID NO: 11 [ka] Heavy chain amino acid sequence SEQ ID NO: 12 [ka] In the above formula, the variable region of the heavy chain is: SEQ ID NO:52 [ka] and the underlined portions, from left to right, are CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 30), and CDR3 (SEQ ID NO: 31), respectively.

[0365] (3) Expression and purification of SI-1x24 antibody: Similar methods were followed for expression and purification of SI-1x24 antibody.

[0366] Antibody SI-1×24: Light chain nucleic acid coding sequence SEQ ID NO: 13 [ka]

[0367] Light chain amino acid sequence SEQ ID NO: 14 [ka] In the above formula, the variable region is: SEQ ID NO: 28 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 25), CDR2 (SEQ ID NO: 26), and CDR3 (SEQ ID NO: 27), respectively.

[0368] Nucleic acid coding sequences for the construction of heavy chain and single-chain Fv (scFv) structural domains SEQ ID NO: 15 [ka]

[0369] Amino acid sequences of heavy chain and single-chain Fv (scFv) structural domain constructs SEQ ID NO: 16 [ka] In the above formula, the variable region of the heavy chain is: SEQ ID NO: 38 [ka] and the underlined portions, from left to right, are CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 30), and CDR3 (SEQ ID NO: 31), respectively.

[0370] The variable region of the heavy chain of the structural domain of a single chain Fv (scFv) is: SEQ ID NO: 39 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 32), CDR2 (SEQ ID NO: 33), and CDR3 (SEQ ID NO: 34), respectively.

[0371] The variable region of the light chain of the single-chain Fv (scFv) structural domain is: SEQ ID NO: 40 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 35), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 37), respectively.

[0372] Example 92 1) Expression and purification of SI-1x4 antibody: (Shanghai OPM Biosciences Co., Ltd.) suspension cells were used to express the SI-1x4 antibody. The day before transfection, cells were grown at a density of 0.9x10 in a 1 L shake flask containing 300 mL of OPM-293 CD05 medium (81075-001, Shanghai OPM Biosciences Co., Ltd.). 6 Cells were seeded at 1000 cells / mL and cultured overnight at 37°C, 5% CO2, and 120 rpm in a cell culture shaker. The next day, the antibody expression plasmid was transfected with PEI-MAX at a mass ratio of 1:3. OPM-293 ProFeed supplement was added at 5% (v / v) on day 1 posttransfection, and again at 5% (v / v) on day 3 posttransfection. Centrifugation was performed on day 6 posttransfection, and the supernatant was collected.

[0373] The collected cell expression supernatant was eluted with 0.05 M sodium acetate (pH 3.6) on a protein A affinity chromatography column (UniMab 50, Suzhou Nanomicro Technology Co., Ltd.). The captured antibody was adjusted to pH 7.0 with 1 M Tris-HCl (pH 8.8) at a ratio of 0.7 / 10 (v / v). It was then passed through a gel filtration chromatography column SEC (Superdex 200, GE) to remove impurities such as polymers, and the antibody buffer was replaced with 20 mM PB (pH 6.5).

[0374] Antibody SI-1×4: Light chain nucleic acid coding sequence SEQ ID NO:5 [ka]

[0375] Light chain amino acid sequence SEQ ID NO:6 [ka] In the above formula, the variable region is: SEQ ID NO: 44 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 41), CDR2 (SEQ ID NO: 42), and CDR3 (SEQ ID NO: 43), respectively.

[0376] Nucleic acid coding sequences for the construction of heavy chain and single-chain Fv (scFv) structural domains SEQ ID NO:7 [ka]

[0377] Amino acid sequences of heavy chain and single-chain Fv (scFv) structural domain constructs SEQ ID NO:8 [ka] In the above formula, the variable region of the heavy chain is: SEQ ID NO: 48 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 46), and CDR3 (SEQ ID NO: 47), respectively.

[0378] Heavy chain variable region in the structural domain of single chain Fv (scFv) SEQ ID NO: 39 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 32), CDR2 (SEQ ID NO: 33), and CDR3 (SEQ ID NO: 34), respectively.

[0379] The variable region of the light chain of the single-chain Fv (scFv) structural domain is: SEQ ID NO: 40 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 35), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 37), respectively.

[0380] 2) Expression and purification of SI-1x25 antibody: Similar methods were followed for expression and purification of SI-1x25 antibody.

[0381] Antibody SI-1×25: Nucleic acid coding sequences for constructs of light chain and single-chain Fv (scFv) structural domains SEQ ID NO: 17 [ka]

[0382] Amino acid sequences of light chain and single-chain Fv (scFv) structural domain constructs SEQ ID NO: 18 [ka] In the above formula, the light chain variable region is: SEQ ID NO: 49 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 25), CDR2 (SEQ ID NO: 26), and CDR3 (SEQ ID NO: 27), respectively.

[0383] The variable region of the heavy chain in the structural domain of the single chain Fv (scFv) is: SEQ ID NO:50 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 32), CDR2 (SEQ ID NO: 33), and CDR3 (SEQ ID NO: 34), respectively.

[0384] The variable region of the light chain in the single-chain Fv (scFv) structural domain is: SEQ ID NO:51 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 35), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 37), respectively.

[0385] Heavy chain nucleic acid coding sequence SEQ ID NO: 19 [ka]

[0386] Heavy chain amino acid sequence SEQ ID NO: 20 [ka] In the above formula, the variable region of the heavy chain is: SEQ ID NO:52 [ka] and the underlined portions, from left to right, are CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 30), and CDR3 (SEQ ID NO: 31), respectively.

[0387] (3) Expression and purification of SI-1x26 antibody: Similar methods were followed for expression and purification of SI-1x26 antibody.

[0388] Antibody SI-1×26: Nucleic acid coding sequences for constructs of light chain and single-chain Fv (scFv) structural domains SEQ ID NO: 21 [ka]

[0389] Amino acid sequences of light chain and single-chain Fv (scFv) structural domain constructs SEQ ID NO: 22 [ka] In the above formula, the light chain variable region is: SEQ ID NO:53 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 41), CDR2 (SEQ ID NO: 42), and CDR3 (SEQ ID NO: 43), respectively.

[0390] The variable region of the heavy chain in the structural domain of the single chain Fv (scFv) is: SEQ ID NO:50 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 32), CDR2 (SEQ ID NO: 33), and CDR3 (SEQ ID NO: 34), respectively.

[0391] The structural domain of the light chain variable region of the single chain Fv (scFv) is: SEQ ID NO:51 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 35), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 37), respectively.

[0392] Heavy chain nucleic acid coding sequence SEQ ID NO: 23 [ka]

[0393] Heavy chain amino acid sequence SEQ ID NO: 24 [ka] In the above formula, the variable region of the heavy chain is: SEQ ID NO:54 [ka] and the underlined portions are, from left to right, CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 46), and CDR3 (SEQ ID NO: 47), respectively.

[0394] Example 93 1) Samples of SI-1x6.4 antibody-drug conjugates were prepared by coupling the SI-1x6.4 antibody with a payload: After cell expression and purification by protein A affinity chromatography and molecular sieve chromatography, the SI-1x6.4 antibody was substituted into 20 mM PB, pH 6.5 buffer, and concentrated or diluted to a protein concentration of 3 mg / mL. The payload was a white powder, which was dissolved to 20 mg / mL using DMA and set aside. To open the interchain disulfide bonds of the SI-1x6.4 antibody, 20x TECP was first added according to the molecular ratio and the reaction was allowed to proceed at room temperature for 3 hours. Next, 20x payload solution was added according to the molecular ratio and the reaction was allowed to proceed at room temperature for 1 hour. After the reaction was complete, the payload that was not coupled to SI-1x6.4 was removed by ultrafiltration using a 30 kDa ultrafiltration centrifuge tube, and the SI-1x6.4 antibody-drug conjugate sample was thus obtained.

[0395] 2) Following a similar method, SI-1x22 antibody-drug conjugate samples were prepared by coupling SI-1x22 antibody with a payload.

[0396] 3) Following a similar method, SI-1x24 antibody-drug conjugate samples were prepared by coupling SI-1x24 antibody with payload.

[0397] Example 94 1) SI-1x4 antibody-drug conjugate samples were prepared by coupling the SI-1x4 antibody with a payload. After cell expression and purification by protein A affinity chromatography and molecular sieve chromatography, the SI-1x4 antibody was substituted into 20 mM PB, pH 6.5 buffer, and concentrated or diluted to a protein concentration of 3 mg / mL. The payload was a white powder, which was dissolved to 20 mg / mL using DMA and set aside. To open the interchain disulfide bond of the SI-1x4 antibody, 20x TECP was first added according to the molecular ratio, and the reaction was allowed to proceed at room temperature for 3 hours. Next, 20x payload solution was added according to the molecular ratio, and the reaction was allowed to proceed at room temperature for 1 hour. Upon completion of the reaction, the payload not coupled to SI-1x4 was removed by ultrafiltration using a 30 kDa ultrafiltration centrifuge tube, yielding the SI-1x4 antibody-drug conjugate sample.

[0398] 2) Following a similar method, SI-1x25 antibody-drug conjugate samples were prepared by coupling SI-1x25 antibody with payload.

[0399] 3) Following a similar method, SI-1x26 antibody-drug conjugate samples were prepared by coupling SI-1x26 antibody with a payload.

[0400] Example 95 ADC-1 was prepared according to the general coupling method in Example 93; [ka]

[0401] Example 96 ADC-2 was prepared according to the general coupling method in Example 93; [ka]

[0402] Example 97 ADC-3 was prepared according to the general coupling method in Example 93; [ka]

[0403] Example 98 ADC-4 was prepared according to the general coupling method in Example 93; [ka]

[0404] Example 99 ADC-5 was prepared according to the general coupling method in Example 93; [ka]

[0405] Example 100 ADC-6 was prepared according to the general coupling method in Example 93; [ka]

[0406] Example 101 ADC-7 was prepared according to the general coupling method in Example 93; [ka]

[0407] Example 102 ADC-8 was prepared according to the general coupling method in Example 93; [ka]

[0408] Example 103 ADC-9 was prepared according to the general coupling method in Example 93; [ka]

[0409] Example 104 ADC-10 was prepared according to the general coupling method of Example 93; [ka]

[0410] Example 105 ADC-11 was prepared according to the general coupling method in Example 93; [ka]

[0411] Example 106 ADC-12 was prepared according to the general coupling method in Example 93; [ka]

[0412] Example 107 ADC-13 was prepared according to the general coupling method in Example 93; [ka]

[0413] Example 108 ADC-14 was prepared according to the general coupling method in Example 93; [ka]

[0414] Example 109 ADC-15 was prepared according to the general coupling method in Example 93; [ka]

[0415] Example 110 ADC-16 was prepared according to the general coupling method in Example 93; [ka]

[0416] Example 111 ADC-17 was prepared according to the general coupling method in Example 93; [ka]

[0417] Example 112 ADC-18 was prepared according to the general coupling method in Example 93; [ka]

[0418] Example 113 ADC-19 was prepared according to the general coupling method in Example 93; [ka]

[0419] Example 114 ADC-20 was prepared according to the general coupling method in Example 93; [ka]

[0420] Example 115 ADC-21 was prepared according to the general coupling method in Example 93; [ka]

[0421] Example 116 ADC-22 was prepared according to the general coupling method in Example 93; [ka]

[0422] Example 117 ADC-23 was prepared according to the general coupling method in Example 93; [ka]

[0423] Example 118 ADC-24 was prepared according to the general coupling method in Example 93; [ka]

[0424] Example 119 ADC-25 was prepared according to the general coupling method in Example 93; [ka]

[0425] Example 120 ADC-26 was prepared according to the general coupling method in Example 93; [ka]

[0426] Example 121 ADC-27 was prepared according to the general coupling method in Example 93; [ka]

[0427] Example 122 ADC-28 was prepared according to the general coupling method in Example 93; [ka]

[0428] Example 123 ADC-29 was prepared according to the general coupling method in Example 93; [ka]

[0429] Example 124 ADC-30 was prepared according to the general coupling method in Example 93; [ka]

[0430] Example 125 ADC-31 was prepared according to the general coupling method in Example 93; [ka]

[0431] Example 126 ADC-32 was prepared according to the general coupling method in Example 93; [ka]

[0432] Example 127 ADC-33 was prepared according to the general coupling method in Example 93; [ka]

[0433] Example 128 ADC-34 was prepared according to the general coupling method in Example 93; [ka]

[0434] Example 129 ADC-35 was prepared according to the general coupling method in Example 93; [ka]

[0435] Example 130 ADC-36 was prepared according to the general coupling method in Example 93; [ka]

[0436] Example 131 ADC-37 was prepared according to the general coupling method in Example 93; [ka]

[0437] Example 132 ADC-38 was prepared according to the general coupling method in Example 93; [ka]

[0438] Example 133 ADC-39 was prepared according to the general coupling method in Example 93; [ka]

[0439] Example 134 ADC-40 was prepared according to the general coupling method in Example 93; [ka]

[0440] Example 135 ADC-41 was prepared according to the general coupling method in Example 93; [ka]

[0441] Example 136 ADC-42 was prepared according to the general coupling method in Example 93; [ka]

[0442] Example 137 ADC-43 was prepared according to the general coupling method in Example 93; [ka]

[0443] Example 138 ADC-44 was prepared according to the general coupling method in Example 93; [ka]

[0444] Example 139 ADC-45 was prepared according to the general coupling method in Example 93; [ka]

[0445] Example 140 ADC-46 was prepared according to the general coupling method in Example 93; [ka]

[0446] Example 141 ADC-47 was prepared according to the general coupling method in Example 93; [ka]

[0447] Example 142 ADC-48 was prepared according to the general coupling method in Example 93; [ka]

[0448] Example 143 ADC-49 was prepared according to the general coupling method in Example 93; [ka]

[0449] Example 144 ADC-50 was prepared according to the general coupling method in Example 93; [ka]

[0450] Example 145 ADC-51 was prepared according to the general coupling method in Example 93; [ka]

[0451] Example 146 ADC-52 was prepared according to the general coupling method of Example 93; [ka]

[0452] Example 147 ADC-53 was prepared according to the general coupling method in Example 93; [ka]

[0453] Example 148 ADC-54 was prepared according to the general coupling method in Example 93; [ka]

[0454] Example 149 ADC-55 was prepared according to the general coupling method in Example 93; [ka]

[0455] Example 150 ADC-56 was prepared according to the general coupling method in Example 93; [ka]

[0456] Example 151 ADC-57 was prepared according to the general coupling method in Example 93; [ka]

[0457] Example 152 ADC-58 was prepared according to the general coupling method in Example 93; [ka]

[0458] Example 153 ADC-59 was prepared according to the general coupling method in Example 93; [ka]

[0459] Example 154 ADC-60 was prepared according to the general coupling method in Example 93; [ka]

[0460] Example 155 ADC-61 was prepared according to the general coupling method in Example 93; [ka]

[0461] Example 156 ADC-62 was prepared according to the general coupling method of Example 93; [ka]

[0462] Example 157 ADC-63 was prepared according to the general coupling method in Example 93; [ka]

[0463] Example 158 ADC-64 was prepared according to the general coupling method of Example 93; [ka]

[0464] Example 159 ADC-65 was prepared according to the general coupling method in Example 93; [ka]

[0465] Example 160 ADC-66 was prepared according to the general coupling method in Example 93; [ka]

[0466] Example 161 ADC-67 was prepared according to the general coupling method in Example 93; [ka]

[0467] Example 162 ADC-68 was prepared according to the general coupling method in Example 93; [ka]

[0468] Example 163 ADC-69 was prepared according to the general coupling method in Example 93; [ka]

[0469] Example 164 ADC-70 was prepared according to the general coupling method in Example 93; [ka]

[0470] Example 165 ADC-71 was prepared according to the general coupling method in Example 93; [ka]

[0471] Example 166 ADC-72 was prepared according to the general coupling method of Example 93; [ka]

[0472] Example 167 ADC-73 was prepared according to the general coupling method in Example 93; [ka]

[0473] Example 168 ADC-74 was prepared according to the general coupling method of Example 93; [ka]

[0474] Example 169 ADC-75 was prepared according to the general coupling method in Example 93; [ka]

[0475] Example 170 ADC-76 was prepared according to the general coupling method in Example 93; [ka]

[0476] Example 171 ADC-77 was prepared according to the general coupling method in Example 93; [ka]

[0477] Example 172 ADC-78 was prepared according to the general coupling method in Example 93; [ka]

[0478] Example 173 ADC-79 was prepared according to the general coupling method in Example 93; [ka]

[0479] Example 174 ADC-80 was prepared according to the general coupling method of Example 93; [ka]

[0480] Example 175 ADC-81 was prepared according to the general coupling method in Example 93; [ka]

[0481] Example 176 ADC-82 was prepared according to the general coupling method in Example 93; [ka]

[0482] Example 177 ADC-83 was prepared according to the general coupling method in Example 93; [ka]

[0483] Example 178 ADC-84 was prepared according to the general coupling method of Example 93; [ka]

[0484] Example 179 ADC-85 was prepared according to the general coupling method in Example 93; [ka]

[0485] Example 180 ADC-86 was prepared according to the general coupling method in Example 93; [ka]

[0486] Example 181 ADC-87 was prepared according to the general coupling method in Example 93; [ka]

[0487] Example 182 ADC-88 was prepared according to the general coupling method in Example 93; [ka]

[0488] Example 183 ADC-89 was prepared according to the general coupling method in Example 93; [ka]

[0489] Example 184 ADC-90 was prepared according to the general coupling method of Example 93; [ka]

[0490] Example 185 ADC-91 was prepared according to the general coupling method of Example 93; [ka]

[0491] Example 186 ADC-92 was prepared according to the general coupling method of Example 93; [ka]

[0492] Example 187 ADC-93 was prepared according to the general coupling method in Example 93; [ka]

[0493] Example 188 ADC-94 was prepared according to the general coupling method of Example 93; [ka]

[0494] Example 189 ADC-95 was prepared according to the general coupling method of Example 93; [ka]

[0495] Example 190 ADC-96 was prepared according to the general coupling method of Example 93; [ka]

[0496] Example 191 ADC-97 was prepared according to the general coupling method of Example 93; [ka]

[0497] Example 192 ADC-98 was prepared according to the general coupling method of Example 93; [ka]

[0498] Example 193 ADC-99 was prepared according to the general coupling method in Example 93; [ka]

[0499] Example 194 ADC-100 was prepared according to the general coupling method of Example 93; [ka]

[0500] Example 195 ADC-101 was prepared according to the general coupling method in Example 93; [ka]

[0501] Example 196 ADC-102 was prepared according to the general coupling method in Example 93; [ka]

[0502] Example 197 ADC-103 was prepared according to the general coupling method in Example 93; [ka]

[0503] Example 198 ADC-104 was prepared according to the general coupling method in Example 93; [ka]

[0504] Example 199 ADC-105 was prepared according to the general coupling method in Example 93; [ka]

[0505] Example 200 ADC-106 was prepared according to the general coupling method in Example 93; [ka]

[0506] Example 201 ADC-DS was prepared from compound 45 according to the general coupling method in Example 93; [ka]

[0507] Example 202 ADC-107 was prepared according to the general coupling method in Example 94; [ka]

[0508] Example 203 ADC-108 was prepared according to the general coupling method of Example 94; [ka]

[0509] Example 204 ADC-109 was prepared according to the general coupling method in Example 94; [ka]

[0510] Example 205 ADC-110 was prepared according to the general coupling method of Example 94; [ka]

[0511] Example 206 ADC-111 was prepared according to the general coupling method of Example 94; [ka]

[0512] Example 207 ADC-112 was prepared according to the general coupling method of Example 94; [ka]

[0513] Example 208 ADC-113 was prepared according to the general coupling method in Example 94; [ka]

[0514] Example 209 ADC-114 was prepared according to the general coupling method of Example 94; [ka]

[0515] Example 210 ADC-115 was prepared according to the general coupling method of Example 94; [ka]

[0516] Example 211 ADC-116 was prepared according to the general coupling method in Example 94; [ka]

[0517] Example 212 ADC-117 was prepared according to the general coupling method in Example 94; [ka]

[0518] Example 213 ADC-118 was prepared according to the general coupling method of Example 94; [ka]

[0519] Example 214 ADC-119 was prepared according to the general coupling method in Example 94; [ka]

[0520] Example 215 ADC-120 was prepared according to the general coupling method of Example 94; [ka]

[0521] Example 216 ADC-121 was prepared according to the general coupling method in Example 94; [ka]

[0522] Example 217 ADC-122 was prepared according to the general coupling method in Example 94; [ka]

[0523] Example 218 ADC-123 was prepared according to the general coupling method in Example 94; [ka]

[0524] Example 219 ADC-124 was prepared according to the general coupling method in Example 94; [ka]

[0525] Example 220 ADC-125 was prepared according to the general coupling method of Example 94; [ka]

[0526] Example 221 ADC-126 was prepared according to the general coupling method in Example 94; [ka]

[0527] Example 222 ADC-127 was prepared according to the general coupling method in Example 94; [ka]

[0528] Example 223 ADC-128 was prepared according to the general coupling method of Example 94; [ka]

[0529] Example 224 ADC-129 was prepared according to the general coupling method in Example 94; [ka]

[0530] Example 225 ADC-130 was prepared according to the general coupling method of Example 94; [ka]

[0531] Example 226 ADC-131 was prepared according to the general coupling method in Example 94; [ka]

[0532] Example 227 ADC-132 was prepared according to the general coupling method of Example 94; [ka]

[0533] Example 228 ADC-133 was prepared according to the general coupling method in Example 94; [ka]

[0534] Example 229 ADC-134 was prepared according to the general coupling method in Example 94; [ka]

[0535] Example 230 ADC-135 was prepared according to the general coupling method in Example 94; [ka]

[0536] Example 231 ADC-136 was prepared according to the general coupling method in Example 94; [ka]

[0537] Example 232 ADC-137 was prepared according to the general coupling method in Example 94; [ka]

[0538] Example 233 ADC-138 was prepared according to the general coupling method in Example 94; [ka]

[0539] Example 234 ADC-139 was prepared according to the general coupling method in Example 94; [ka]

[0540] Example 235 ADC-140 was prepared according to the general coupling method of Example 94; [ka]

[0541] Example 236 ADC-141 was prepared according to the general coupling method in Example 94; [ka]

[0542] Example 237 ADC-142 was prepared according to the general coupling method of Example 94; [ka]

[0543] Example 238 ADC-143 was prepared according to the general coupling method in Example 94; [ka]

[0544] Example 239 ADC-144 was prepared according to the general coupling method in Example 94; [ka]

[0545] Example 240 ADC-145 was prepared according to the general coupling method in Example 94; [ka]

[0546] Example 241 ADC-146 was prepared according to the general coupling method in Example 94; [ka]

[0547] Example 242 ADC-147 was prepared according to the general coupling method in Example 94; [ka]

[0548] Example 243 ADC-148 was prepared according to the general coupling method of Example 94; [ka]

[0549] Example 244 ADC-149 was prepared according to the general coupling method in Example 94; [ka]

[0550] Example 245 ADC-150 was prepared according to the general coupling method of Example 94; [ka]

[0551] Example 246 ADC-151 was prepared according to the general coupling method in Example 94; [ka]

[0552] Example 247 ADC-152 was prepared according to the general coupling method of Example 94; [ka]

[0553] Example 248 ADC-153 was prepared according to the general coupling method in Example 94; [ka]

[0554] Example 249 ADC-154 was prepared according to the general coupling method in Example 94; [ka]

[0555] Example 250 ADC-155 was prepared according to the general coupling method in Example 94; [ka]

[0556] Example 251 ADC-156 was prepared according to the general coupling method in Example 94; [ka]

[0557] Example 252 ADC-157 was prepared according to the general coupling method in Example 94; [ka]

[0558] Example 253 ADC-158 was prepared according to the general coupling method in Example 94; [ka]

[0559] Example 254 ADC-159 was prepared according to the general coupling method in Example 94; [ka]

[0560] Example 255 ADC-160 was prepared according to the general coupling method of Example 94; [ka]

[0561] Example 256 ADC-161 was prepared according to the general coupling method in Example 94; [ka]

[0562] Example 257 ADC-162 was prepared according to the general coupling method in Example 94; [ka]

[0563] Example 258 ADC-163 was prepared according to the general coupling method in Example 94; [ka]

[0564] Example 259 ADC-164 was prepared according to the general coupling method in Example 94; [ka]

[0565] Example 260 ADC-165 was prepared according to the general coupling method in Example 94; [ka]

[0566] Example 261 ADC-166 was prepared according to the general coupling method in Example 94; [ka]

[0567] Example 262 ADC-167 was prepared according to the general coupling method in Example 94; [ka]

[0568] Example 263 ADC-168 was prepared according to the general coupling method in Example 94; [ka]

[0569] Example 264 ADC-169 was prepared according to the general coupling method in Example 94; [ka]

[0570] Example 265 ADC-170 was prepared according to the general coupling method in Example 94; [ka]

[0571] Example 266 ADC-171 was prepared according to the general coupling method in Example 94; [ka]

[0572] Example 267 ADC-172 was prepared according to the general coupling method of Example 94; [ka]

[0573] Example 268 ADC-173 was prepared according to the general coupling method in Example 94; [ka]

[0574] Example 269 ADC-174 was prepared according to the general coupling method in Example 94; [ka]

[0575] Example 270 ADC-175 was prepared according to the general coupling method in Example 94; [ka]

[0576] Example 271 ADC-176 was prepared according to the general coupling method in Example 94; [ka]

[0577] Example 272 ADC-177 was prepared according to the general coupling method in Example 94; [ka]

[0578] Example 273 ADC-178 was prepared according to the general coupling method in Example 94; [ka]

[0579] Example 274 ADC-179 was prepared according to the general coupling method in Example 94; [ka]

[0580] Example 275 ADC-180 was prepared according to the general coupling method in Example 94; [ka]

[0581] Example 276 ADC-181 was prepared according to the general coupling method in Example 94; [ka]

[0582] Example 277 ADC-182 was prepared according to the general coupling method in Example 94; [ka]

[0583] Example 278 ADC-183 was prepared according to the general coupling method in Example 94; [ka]

[0584] Example 279 ADC-184 was prepared according to the general coupling method in Example 94; [ka]

[0585] Example 280 ADC-185 was prepared according to the general coupling method in Example 94; [ka]

[0586] Example 281 ADC-186 was prepared according to the general coupling method in Example 94; [ka]

[0587] Example 282 ADC-187 was prepared according to the general coupling method in Example 94; [ka]

[0588] Example 283 ADC-188 was prepared according to the general coupling method in Example 94; [ka]

[0589] Example 284 ADC-189 was prepared according to the general coupling method in Example 94; [ka]

[0590] Example 285 ADC-190 was prepared according to the general coupling method in Example 94; [ka]

[0591] Example 286 ADC-191 was prepared according to the general coupling method in Example 94; [ka]

[0592] Example 287 ADC-192 was prepared according to the general coupling method in Example 94; [ka]

[0593] Example 288 ADC-193 was prepared according to the general coupling method in Example 94; [ka]

[0594] Example 289 ADC-194 was prepared according to the general coupling method in Example 94; [ka]

[0595] Example 290 ADC-195 was prepared according to the general coupling method in Example 94; [ka]

[0596] Example 291 ADC-196 was prepared according to the general coupling method in Example 94; [ka]

[0597] Example 292 ADC-197 was prepared according to the general coupling method in Example 94; [ka]

[0598] Example 293 ADC-198 was prepared according to the general coupling method in Example 94; [ka]

[0599] Example 294 ADC-199 was prepared according to the general coupling method in Example 94; [ka]

[0600] Example 295 ADC-200 was prepared according to the general coupling method of Example 94; [ka]

[0601] Example 296 ADC-201 was prepared according to the general coupling method in Example 94; [ka]

[0602] Example 297 ADC-202 was prepared according to the general coupling method of Example 94; [ka]

[0603] Example 298 ADC-203 was prepared according to the general coupling method in Example 94; [ka]

[0604] Example 299 ADC-204 was prepared according to the general coupling method of Example 94; [ka]

[0605] Example 300 ADC-205 was prepared according to the general coupling method of Example 94; [ka]

[0606] Example 301 ADC-206 was prepared according to the general coupling method in Example 94; [ka]

[0607] Example 302 ADC-207 was prepared according to the general coupling method in Example 94; [ka]

[0608] Example 303 ADC-208 was prepared according to the general coupling method of Example 94; [ka]

[0609] Example 304 ADC-209 was prepared according to the general coupling method of Example 94; [ka]

[0610] Example 305 ADC-210 was prepared according to the general coupling method of Example 94; [ka]

[0611] Example 306 ADC-211 was prepared according to the general coupling method in Example 94; [ka]

[0612] Example 307 ADC-212 was prepared according to the general coupling method of Example 94; [ka]

[0613] Example 308 ADC-213 was prepared from compound 45 according to the general coupling method of Example 94; [ka]

[0614] Example 309 ADC-214 was prepared from compound 5A according to the general coupling method of Example 93; [ka]

[0615] Example 310 HER3 ADC-215 was prepared from compound 5A according to the general coupling method of Example 93. [ka] In the above formula, the H3 antibody is part of the SI-1x6.4 antibody knockout that targets EGFR.

[0616] Example 311 ADC-223 was prepared from ADC-216 according to the general coupling method of Example 93; [ka]

[0617] Example 312 ADC-231 was prepared from ADC-224 according to the general coupling method of Example 93; [ka]

[0618] Example 313 ADC-239 was prepared from ADC-232 according to the general coupling method of Example 94; [ka]

[0619] Example 314 ADC-247 was prepared from ADC-240 according to the general coupling method of Example 94; [ka]

[0620] Example 315 The SEC-HPLC method was used to determine the monomer percentage: Chromatography column: Biocore SEC-300 5 μm, 4.6 × 300 mm Manufacturer: NanChrom, Product Number: B213-050030-04630S Mobile phase: 50mM PB+300mM NaCl+200mM Arg+5%IPA, pH=6.5

[0621] [Table 1]

[0622] [Table 2]

[0623] Conclusion: The ADC disclosed in this invention is characterized by low degradation rate and low aggregation rate, and has excellent properties of high monomer rate.

[0624] Example 316 The RP-HPLC method was used to determine the drug-antibody ratio DAR: Chromatography column name: Proteomix RP-1000 4.6 x 100 mm 5 μm 1000A Manufacturer: Sepax

[0625] [Table 3]

[0626] [Table 4]

[0627] Conclusion: The ADC disclosed in the present invention has the excellent property of a high DAR value, which can significantly increase the drug concentration at the target site with the same dose of the ADC drug administered.

[0628] Example 317 The ADCs maintained the affinity of the corresponding original bispecific antibodies SI-1x6.4, SI-1x4, SI-1x22, SI-1x24, SI-1x25, and SI-1x26 for EGFR and HER3: The relative affinities of SI-1x6.4 vs. ADC-6 and SI-1x4 vs. ADC-112 for EGFR and HER3 were compared by double antigen sandwich ELISA. The specific steps are as follows: Recombinant EGFR-His * The six antigen-coated plates were sealed with 1% bovine serum protein; SI-1x6.4, ADC-6, SI-1x4, and ADC-112 were then diluted, starting at 5,000 ng / mL, in a continuous 3-fold gradient for a total of 11 concentrations; the samples were incubated on the coated ELISA plate for a certain period, followed by biotin-labeled HER3-Fc antigen incubation and then streptavidin-HRP-labeled incubation; finally, TMB color development was performed, stopped with sulfuric acid solution, and the absorbance value at 450 nm was detected using a microplate reader. The assay results were plotted against the concentration at OD450 nm.

[0629] Conclusion: As shown in the attached Figures 3A and 3B, after coupling, ADC-6 and ADC-112 maintained similar affinities to those of SI-1x6.4 and SI-1x4, respectively, with no significant differences in EC50 values; indicating that coupling of toxins to SI-1x6.4 and SI-1x4 did not affect their affinity for antigen.

[0630] Similarly, using a test method similar to that described above, the results are shown in Figures 3C, 3D, 3E, and 3F; after coupling, ADC-219, ADC-227, ADC-235, and ADC-243 maintained affinities similar to those of SI-1x22, SI-1x24, SI-1x25, and SI-1x26, respectively, with no significant differences in EC50 values, indicating that coupling of toxins to SI-1x22, SI-1x24, SI-1x25, and SI-1x26 did not affect their affinity for the antigen.

[0631] Example 318 In vitro pharmacodynamic assay: Various human-derived tumor cell lines (human epithelial carcinoma cells A431, human in situ pancreatic adenocarcinoma cells BXPC-3, human pharyngeal squamous cell carcinoma cells FaDu, human lung cancer squamous cell carcinoma cell line HARA-B, human non-small cell lung cancer cells HCC827, and human colon carcinoma cells SW620) were used as experimental models to evaluate the in vitro efficacy of the ADC-conjugated drugs of the present invention. A fixed number of tumor cells were seeded into a 96-well plate, and gradient-diluted test antibodies and corresponding ADC drugs were added to the cells followed by treatment for 5 days. Cell viability was measured using Alamar Blue or MTS, and the inhibitory effects of the test antibodies and ADC drugs on the tumor cell lines were evaluated by calculating the IC50. The starting concentration of the antibody drug was 500 nM, and the dilutions were 7-fold, for a total of 8 concentration points, and the treatment was continued for 5 days. The final calculation method was survival rate = (experimental group - blank) / (control group - blank group) × 100%, and then curve fitting was performed using Graph Pad Prism to calculate the 50% inhibitory concentration (IC50) and efficacy (%).

[0632] [Table 5]

[0633] [Table 6]

[0634] [Table 7]

[0635] [Table 8]

[0636] [Table 9]

[0637] [Table 10]

[0638] Conclusions: In human epithelial carcinoma cells A431, human in situ pancreatic adenocarcinoma cells BXPC-3, human pharyngeal squamous cell carcinoma cells FaDu, human lung cancer squamous cell carcinoma cell line HARA-B, human non-small cell lung cancer cells HCC827, and human colon carcinoma cells SW620, all six ADCs (ADC-112, ADC-6, ADC-219, ADC-227, ADC-235, and ADC-243) showed more sensitive efficacy and stronger tumor cell growth inhibition than their corresponding naked antibodies (SI-1x4, SI-1x6.4, SI-1x22, SI-1x24, SI-1x25, and SI-1x26) (Figures 4A-4F and Tables 5-10).

[0639] Example 319 In vivo efficacy testing: In this study, various human tumor cell lines (A431, SW620, A431+SW620) were subcutaneously inoculated into BALB / c nude mice as an experimental model to evaluate the in vivo efficacy of ADC conjugates. A certain number of tumor cell lines were subcutaneously inoculated into BALB / c nude mice until the tumor volume reached 150-300 mm. 3 When the tumor cell lines reached maturity, the antibodies and corresponding ADC drugs were injected into the tail vein; the drugs were administered once a week for four days, and the tumors were continuously monitored and measured twice a week to evaluate the inhibitory effects of the test antibodies and ADCs on the tumor cell lines.

[0640] Conclusion: In the A431 single tumor model with high EGFR expression, ADC-6 (DAR=8) 10 mg / kg showed strong tumor inhibition relative to SI-1x6.4 naked antibody (Figure 5A).

[0641] In the SW620 single tumor model with low EGFR expression, ADC-6 at 10 and 15 mg / kg also exhibited strong tumor inhibition relative to SI-1x6.4 naked antibody at 15 mg / kg; and at equivalent molar doses, it was even stronger than that of Cet-ADC (ADC-214, DAR=8) (Figure 5B).

[0642] In A431+SW620 xenogeneic tumors, ADC-6 at 5, 15, and 30 mg / kg demonstrated similarly strong tumor inhibition relative to SI-1x6.4 naked antibody at 30 mg / kg (Figure 5C).

[0643] Example 320 In vitro efficacy testing: In this study, human epithelial cancer cell line A431 and human pancreatic cancer cell line BxPC3 were subcutaneously inoculated into BALB / c nude mice as experimental models to evaluate the in vivo efficacy of ADC conjugates. A certain number of tumor cell lines were subcutaneously inoculated into the right shoulder of BALB / c nude mice, and tumors with a volume of 180 to 250 mm were observed. 3 When the tumors grew to a normal size, the corresponding ADC drug was injected into the tail vein once a week for four consecutive doses under continuous observation, and the tumor volume was measured twice a week to evaluate the inhibitory effect of the test ADC on tumor growth.

[0644] % change relative to D0 = (Dn-D0) / D0 * 100; TGI%=1-[change in tumor volume in treated group / change in tumor volume in control group]×100.

[0645] In the EGFR-highly expressing A431 single-body tumor model, 42 days after the first administration, each of the tested ADC drugs significantly inhibited tumor growth (P<0.05) in the subcutaneously transplanted human epithelial carcinoma A431 tumor model in BALB / c nude mice. ADC-6, ADC-219, ADC-235, ADC-227, and ADC-112 all had comparable tumor-suppressing effects, and all of them demonstrated strong tumor-suppressing effects (Figure 6A).

[0646] In a BxPC3 monoclonal tumor model with moderate EGFR expression, 35 days after the first administration, each of the tested ADC drugs significantly inhibited tumor growth (P<0.05) in a subcutaneously implanted BxPC3 human epithelial carcinoma cell line in BALB / c nude mice. ADC-6, ADC-219, ADC-235, ADC-227, and ADC-112 all exhibited comparable tumor-suppressing effects, all of which demonstrated potent tumor-suppressing effects (Figure 6B).

[0647] Example 321 In vitro efficacy of payload compound 5A: 1) Experimental materials: Cells: Test cells were obtained from the Cell Bank of the Chinese Academy of Sciences; Cell medium DMEM: Gibco; FBS:BIOWEST.

[0648] 2) Preparation of medium: Growth medium (containing 10% FBS, penicillin / streptomycin (100 U / mL)); Detection medium (containing 1% FBS, penicillin / streptomycin (100 U / mL)).

[0649] 3) Operation: The UV light in the biosafety cabinet was turned on for 30 minutes, followed by 3 minutes of ventilation. Growth medium, detection medium, D-PBS, and pancreatin were preheated in a 37°C constant-temperature water bath. The surface was then sterilized with alcohol and placed in the biosafety cabinet. Cells were placed in the biosafety cabinet at approximately 80% confluence, the old medium was removed, rinsed with D-PBS, and discarded. The cells were then digested with pancreatin for 2-3 minutes, followed by neutralization of the growth medium and centrifugation at 1200 rpm for 3 minutes. The supernatant was removed and mixed evenly with 4 mL of detection medium. 100 μL was collected for counting. (If 50 μL of cell solution was removed, 50 μL of trypan blue stain was added, mixed evenly, and then counted.) Plate spreading was performed according to the previously optimized cell spreading density; 80 μL / well was spread on a 96-well plate, and 80 μL of detection medium alone was added to wells E11 and F11, and 150 μL of DPBS was added to the edge wells. 24 hours after plate spreading, 20 μL / well of diluted antibody was added to set up the control; 20 μL of detection medium alone was added to the 11th column, and duplicate wells were set up for each concentration. After addition, the cells were mixed uniformly using a cell vortex mixer at 550 rpm for 3 minutes.

[0650] Dilution of the solution: Using detection medium, prepare 300uL of test sample solution with a starting concentration of 5uM in the first column of a V-shaped 96-well plate, add 240uL of detection medium to each of the second to tenth columns, take 60uL from the first column that has been uniformly mixed, add it to the second column, mix it up and down 10 times with a multi-channel pipette, discard the pipette tip, and perform the operation sequentially for the next seven concentrations.

[0651] 4) Detection: After 4 days, the MTS reagent was removed and thawed at room temperature in the dark. Then, using a vortex mixer, 20 μL of CellTiter One Solution Reagen MTS reagent was added per 100 μL of cell culture liquid along the sidewall of the well in a biosafety cabinet. The plate surface was gently tapped to mix the MTS solution uniformly. The plate was then placed in a cell culture incubator and incubated in the dark for 2 hours. At the end of the reaction, the 96-well plate was removed, and the OD490nm absorbance values ​​were measured in a microplate reader. The data was then recorded, analyzed, and stored.

[0652] 5) Results: Table 11 illustrates that compound 5A (payload) exhibited excellent inhibition of the following solid tumor cells and hematoma cells:

[0653] [Table 11]

[0654] Example 322: In vitro efficacy data for ADC-6 and ADC-214: The in vitro efficacy of ADC-conjugated drugs was evaluated using experimental models of two human tumor cell lines (human poorly differentiated lung squamous cell carcinoma cell line Oka-c-1 and human lung squamous cell carcinoma cell line SK-MES-1).

[0655] A fixed number of tumor cells were seeded in a 96-well plate, and gradient-diluted test antibodies and corresponding ADC drugs were added to the cells. The cells were treated for 5 days. Cell viability was measured using Alamar Blue or MTS, and the IC50 values ​​were calculated to evaluate the inhibitory effects of the test antibodies, ADCs, and small molecule drug d3 on tumor cell lines. The starting concentration of the antibody drug was 500 nM, and the dilutions were 7-fold, resulting in a total of 7 concentration points. The treatment was continued for 5 days. The final calculation method was viability = (experimental group - blank) / (control group - blank group) × 100%, and the curve was then fitted using GraphPad Prism to calculate the 50% inhibitory concentration (IC50). The results are shown in Figures 7A and 7B and Table 12.

[0656] [Table 12]

Claims

1. General formula I: 【Chemistry 1】 (wherein Ab is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets two different epitopes or targets; L １ teeth, 【Chemistry 2】 Selected without limitation from: Preferably, L １ teeth, 【Transformation 3】 and Preferably, L １ teeth, 【Chemistry 4】 and L ２ is represented by the following formula A: 【Transformation 5】 wherein Y is a scaffold selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl; preferably, Y is C1-C6 alkyl; Ac is a hydrophilic structural unit; and the carbon atom numbered 2 attached to Y has absolute chirality in the R or S configuration; L ３ is present or absent, and if present, L ３ is a PEG hydrophilic unit: 【Transformation 6】 where o is selected from integers from 1 to 10, preferably from 2 to 8; L ４ is the enzyme cleavage unit: L ５ is a concatenation unit; The chiral carbon atom number 1 attached to N in formula I has absolute chirality in the R or S configuration; R is selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl; Preferably, R is selected from a hydrogen atom or a C1-C6 alkyl group; R １ is selected from 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 a hydrogen atom or a C1 to C6 alkyl group; More preferably, R １ is selected from C1-C6 alkyl; R ２ is selected from 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 a hydrogen atom, a halogen, or a C1-C6 alkyl group; 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 ａｂ - (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 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 heterocyclic group, a substituted 3- to 7-membered heterocyclic group, 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 a hydrogen atom, C1-C6 alkyl, halo-C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or C6-C10 arylC1-C6 alkyl; Instead, R ａ , R ｂ and the carbon atoms to which they are attached form a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclyl group, or a substituted 3- to 7-membered heterocyclyl group; preferably, R ａ , R ｂ and the carbon atoms to which they are attached form a C3-C8 cycloalkyl group; R ３ , R ４ are the same or different and each independently represents 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 hydroxy C1-C6 alkyl, a C3-C8 cycloalkyl, a 3- to 7-membered heterocyclyl, or a substituted 3- to 7-membered heterocyclyl; Preferably, R ３ , R ４ are each independently a hydrogen atom or a C1 to C6 alkyl group; Instead, R ３ , R ４ and the carbon atoms attached thereto form a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclic group, or a substituted 3- to 7-membered heterocyclic group; m is an integer selected from 0 to 4, preferably 0 or 1; and n is an integer selected from 1 to 10), 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, characterized in that Ab is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets two different epitopes or targets, preferably a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3.

3. The Ab antibody comprises: an IgG1 heavy chain, a kappa light chain, and a single-chain Fv (scFv) structural domain; the single-chain Fv (scFv) structural domain forms a construct with the IgG1 heavy chain or the kappa light chain; the IgG1 heavy chain and the kappa light chain form an IgG moiety having binding specificity for EGFR; the scFv structural domain has binding specificity for HER3, and the scFv structural domain is linked to a linker (e.g., (gly-gly-gly-gly-ser) ｎ wherein n is an integer of at least 1, preferably n is an integer of 1 to 10), and the single-chain Fv structural domain has a structural order of N-terminus-heavy chain variable region-junction-light chain variable region-C-terminus, or N-terminus-light chain variable region-junction-heavy chain variable region-C-terminus (for example, the linking site is (gly-gly-gly-gly-ser) ｍ wherein m is an integer of at least 3, preferably 3, 4, 5, or 6.

4. 4. The ligand-camptothecin derivative conjugate of general formula I according to claim 1, wherein the κ light chain of the Ab antibody comprises the CDRs set forth in SEQ ID NOs:25, 26, and 27, the IgG1 heavy chain comprises the CDRs set forth in SEQ ID NOs:29, 30, and 31, and the single-chain Fv (scFv) structural domain comprises the heavy chain variable region CDRs set forth in SEQ ID NOs:32, 33, and 34, and the light chain variable region CDRs set forth in SEQ ID NOs:35, 36, and 37, or a pharmaceutically acceptable salt or solvate thereof.

5. The ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 4, characterized in that the light chain of the Ab antibody comprises a variable region set forth in SEQ ID NO: 28, the IgG1 heavy chain comprises a variable region set forth in SEQ ID NO: 38, and the single-chain Fv (scFv) structural domain comprises a heavy chain variable region set forth in SEQ ID NO: 39 and a light chain variable region set forth in SEQ ID NO: 40, or a pharmaceutically acceptable salt or solvate thereof.

6. 6. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 5, characterized in that the amino acid sequence of the Ab antibody light chain is SEQ ID NO: 2, and the amino acid sequence of the antibody heavy chain and the single-chain Fv (scFv) structural domain construct is SEQ ID NO: 4, or a pharmaceutically acceptable salt or solvate thereof.

7. 7. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 6, characterized in that the nucleic acid coding sequence of the Ab antibody light chain is SEQ ID NO: 1, and the nucleic acid coding sequence of the antibody heavy chain and the construct of the single-chain Fv (scFv) structural domain is SEQ ID NO: 3, or a pharmaceutically acceptable salt or solvate thereof.

8. The ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, characterized in that the κ light chain of the Ab antibody comprises the CDRs set forth in SEQ ID NOs: 41, 42, and 43, the IgG1 heavy chain comprises the CDRs set forth in SEQ ID NOs: 45, 46, and 47, and the single-chain Fv (scFv) structural domain comprises the heavy chain variable region CDRs set forth in SEQ ID NOs: 32, 33, and 34, and the light chain variable region CDRs set forth in SEQ ID NOs: 35, 36, and 37.

9. 9. The ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3 and 8, characterized in that the light chain of the Ab antibody comprises a variable region set forth in SEQ ID NO: 44, the IgG1 heavy chain comprises a variable region set forth in SEQ ID NO: 48, and the single-chain Fv (scFv) structural domain comprises a heavy chain variable region set forth in SEQ ID NO: 39 and a light chain variable region set forth in SEQ ID NO: 40, or a pharmaceutically acceptable salt or solvate thereof.

10. 10. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3 and 8 to 9, characterized in that the amino acid sequence of the Ab antibody light chain is SEQ ID NO: 6, and the amino acid sequence of the antibody heavy chain and the single-chain Fv (scFv) structural domain construct is SEQ ID NO: 8, or a pharmaceutically acceptable salt or solvate thereof.

11. 11. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3 and 8 to 10, characterized in that the nucleic acid coding sequence of the Ab antibody light chain is SEQ ID NO: 5, and the nucleic acid coding sequence of the antibody heavy chain and the construct of the single-chain Fv (scFv) structural domain is SEQ ID NO: 7, or a pharmaceutically acceptable salt or solvate thereof.

12. 5. The ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, characterized in that the light chain of the Ab antibody comprises a variable region set forth in SEQ ID NO: 49, the IgG1 heavy chain comprises a variable region set forth in SEQ ID NO: 52, and the single-chain Fv (scFv) structural domain comprises a heavy chain variable region set forth in SEQ ID NO: 50 and a light chain variable region set forth in SEQ ID NO:

51.

13. 13. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3, 4 and 12, characterized in that the heavy chain amino acid sequence of the Ab antibody is SEQ ID NO: 12, and the construct of the antibody light chain and the single-chain Fv (scFv) structural domain is SEQ ID NO: 10, or a pharmaceutically acceptable salt or solvate thereof.

14. 14. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3, 4 and 12 to 13, characterized in that the nucleic acid coding sequence of the heavy chain of the Ab antibody is SEQ ID NO: 11, and the nucleic acid coding sequence of the construct of the antibody light chain and the single-chain Fv (scFv) structural domain is SEQ ID NO: 9, or a pharmaceutically acceptable salt or solvate thereof.

15. 6. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3, 4 and 5, characterized in that the amino acid sequence of the Ab antibody light chain is SEQ ID NO: 14, and the amino acid sequence of the antibody heavy chain and the single-chain Fv (scFv) structural domain construct is SEQ ID NO: 16, or a pharmaceutically acceptable salt or solvate thereof.

16. 16. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3, 4, 5 and 15, characterized in that the nucleic acid coding sequence of the Ab antibody light chain is SEQ ID NO: 13, and the nucleic acid coding sequence of the antibody heavy chain and the construct of the single-chain Fv (scFv) structural domain is SEQ ID NO: 15, or a pharmaceutically acceptable salt or solvate thereof.

17. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3, 4 and 12, characterized in that the heavy chain amino acid sequence of the Ab antibody is SEQ ID NO: 20, and the construct of the antibody light chain and the single-chain Fv (scFv) structural domain is SEQ ID NO: 18, or a pharmaceutically acceptable salt or solvate thereof.

18. 18. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3, 4, 12 and 17, characterized in that the nucleic acid coding sequence of the heavy chain of the Ab antibody is SEQ ID NO: 19, and the nucleic acid coding sequence of the construct of the antibody light chain and the single-chain Fv (scFv) structural domain is SEQ ID NO: 17, or a pharmaceutically acceptable salt or solvate thereof.

19. 9. The ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3 and 8, characterized in that the light chain of the Ab antibody comprises a variable region set forth in SEQ ID NO: 53, the IgG1 heavy chain comprises a variable region set forth in SEQ ID NO: 54, and the single-chain Fv (scFv) structural domain comprises a heavy chain variable region set forth in SEQ ID NO: 50 and a light chain variable region set forth in SEQ ID NO: 51, or a pharmaceutically acceptable salt or solvate thereof.

20. 20. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3, 8 and 19, characterized in that the heavy chain amino acid sequence of the Ab antibody is SEQ ID NO: 24, and the construct of the antibody light chain and the single-chain Fv (scFv) structural domain is SEQ ID NO: 22, or a pharmaceutically acceptable salt or solvate thereof.

21. 21. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 3, 8 and 19 to 20, or a pharmaceutically acceptable salt or solvate thereof, characterized in that the nucleic acid coding sequence of the heavy chain of the Ab antibody is SEQ ID NO: 23, and the nucleic acid coding sequence of the construct of the antibody light chain and the single-chain Fv (scFv) structural domain is SEQ ID NO:

21.

22. 22. The ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 21, characterized in that the Ab antibody comprises two IgG1 heavy chains; two κ light chains; and two single-chain Fv (scFv) structural domains, or a pharmaceutically acceptable salt or solvate thereof.

23. The X is the following structure or an isomer thereof: 【Transformation 7】 (wherein the left wavy line is linked to the camptothecin derivative moiety and the right wavy line is L ５ 23. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 22, or a pharmaceutically acceptable salt or solvate thereof, characterized in that:

24. L ４ is selected without limitation from peptide residues formed from amino acids, Optionally, the amino acid is further substituted with one or more substituents selected from deuterium atoms, 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 formed from one, two or more amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline (C), serine (S), glutamic acid (E) or aspartic acid (D); More preferably, the ligand-camptothecin derivative conjugate represented by general formula I according to any one of claims 1 to 23, or a pharmaceutically acceptable salt or solvate thereof, is characterized in that the peptide residue is a tetrapeptide residue consisting of glycine (G)-glycine (G)-phenylalanine (F)-glycine (G).

25. L ５ is -NR ５ (CR ６ R ７ ) ｑ - or a chemical bond, and q is selected from integers 0 to 6; R ５ , R ６ and R ７ are the same or different, and each is independently selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a 3- to 7-membered heterocyclic group, a substituted 3- to 7-membered heterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5- to 10-membered heteroaryl group, and a substituted 5- to 10-membered heteroaryl group; Preferably, R ５ , R ６ and R ７ are each independently selected from a hydrogen atom or a C1 to C6 alkyl group; More preferably, R ５ , R ６ and R ７ are each independently selected from a hydrogen atom, or a pharmaceutically acceptable salt or solvate thereof.

26. Consolidated unit-L １ -L ２ -L ３ -L ４ -L ５ - has the following structure: 【Transformation 8】 Preferably, 【Chemistry 9】 are selected without restriction from In the above formula, Ac is a hydrophilic structural unit; R ５ , R ６ and R ７ are the same or different and each independently selected from 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 to C6 alkyl group; More preferably, R ５ , R ６ and R ７ are each independently selected from a hydrogen atom; The carbon atom numbered 2 attached to N has absolute chirality in the R or S configuration; the left wavy line is connected to the antibody or antigen-binding fragment thereof portion and the right wavy line is connected to the X; 26. A ligand-camptothecin derivative conjugate of general formula I according to any one of claims 1 to 25, or a pharmaceutically acceptable salt or solvate thereof, wherein o is selected from the integers 1 to 10.

27. General formula II: 【Chemistry 10】 (Ab is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3; L １ is the linking unit linked to Ab, and 【Chemistry 11】 Selected without limitation from: L １ is preferably 【Chemistry 12】 and Preferably, L １ teeth, 【Chemistry 13】 and L ３ is present or absent, and if L3 is 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 two chiral configurations, the R absolute configuration or the S absolute configuration; R is selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl; Preferably, R is selected from a hydrogen atom or a C1-C6 alkyl group; R １ is selected from 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 a hydrogen atom or a C1 to C6 alkyl group; More preferably, R １ is selected from C1-C6 alkyl; R ２ is selected from 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 a hydrogen atom, a halogen, or a C1-C6 alkyl group; More preferably, R ２ is selected from halogens; 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 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 heterocyclic group, a substituted 3- to 7-membered heterocyclic group, 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 a hydrogen atom, C1-C6 alkyl, haloC1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, or C6-C10 arylC1-C6 alkyl; Instead, R ａ , R ｂ and the carbon atoms to which they are attached form a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclic group, or a substituted 3- to 7-membered heterocyclic group; preferably, R ａ , R ｂ and the carbon atoms to which they are attached form a C3-C8 cycloalkyl group; 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 hydroxy 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 to C6 alkyl group; Instead, R ３ , R ４ and the carbon atoms attached thereto form a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclic group, or a substituted 3- to 7-membered heterocyclic group; m is an integer selected from 0 to 4, preferably 0 and 1; and n is selected from an integer of 1 to 10), or a pharmaceutically acceptable salt or solvate thereof.

28. The Ac is represented by the following formula B: 【Chemistry 15】 It has the structure shown in wherein Z is selected, without limitation, from the group consisting of one or more of a hydrophilic structure: a carboxyl group, a phosphoric acid, a polyphosphoric acid, a phosphorous acid, a sulfonic acid, a sulfinic acid, or a polyethylene glycol (PEG); Preferably, Z is selected from the hydrophilic structure of a carboxyl group, a phosphoric acid or a polyethylene glycol (PEG); Y' is any scaffold that links an amino group to Z; preferably, Y' is a C1-C6 alkyl; 28. The ligand-camptothecin derivative conjugate of any one of claims 1 to 27, or a pharmaceutically acceptable salt or solvate thereof, characterized in that Ac is linked to the 2-carbon marked in structural formula I by a scaffold Y.

29. The Ac is selected from the group consisting of 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, and the following structures or isomers thereof: 【Chemistry 16】 Preferably, 【Chemistry 17】 29. The ligand-camptothecin derivative conjugate according to any one of claims 1 to 28, or a pharmaceutically acceptable salt or solvate thereof, characterized in that it is selected without limitation from the following:

30. 30. The ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, according to any one of claims 1 to 29, wherein Ac is selected from, without limitation, glycine, phosphate, (D / L) glutamic acid, or a polyethylene glycol hydrophilic structure. 【Request Item 31】 【Chemistry 18】 is expressed by the following formula d: 【Chemistry 19】 It has the structure shown in In the above formula, R is selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl, a C1-C6 alkoxy C1-C6 alkyl, 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 is selected from a hydrogen atom or a C1-C6 alkyl group; R １ is selected from 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 C1-C6 alkyl, a C1-C6 alkoxy C1-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 a hydrogen atom or a C1 to C6 alkyl group; More preferably, R １ is selected from C1-C6 alkyl; R ２ is selected from 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 C1-C6 alkyl, a C1-C6 alkoxy C1-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 a hydrogen atom, a halogen, or a C1-C6 alkyl group; More preferably, R ２ is selected from halogens; R ａ and R ｂ are each independently selected from 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 heterocyclic group, a substituted 3- to 7-membered heterocyclic group, 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 a hydrogen atom, a C1-C6 alkyl group, a halo-C1-C6 alkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, and a C6-C10 aryl group; Instead, R ａ , R ｂ and the carbon atoms to which they are attached form a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclic group, or a substituted 3- to 7-membered heterocyclic group; preferably, R ａ , R ｂ and the carbon atoms to which they are attached form a C3-C8 cycloalkyl group; The chiral carbon atom at position 1 has two chiral configurations, the R absolute configuration or the S absolute configuration; 31. The ligand-camptothecin derivative conjugate according to any one of claims 1 to 30, or a pharmaceutically acceptable salt or solvate thereof, characterized in that m is selected from 0 or 1.

32. The structural formula d is a compound as follows: 【Chemistry 20】 【Chemistry 21】 32. The ligand-camptothecin derivative conjugate according to any one of claims 1 to 31, or a pharmaceutically acceptable salt or solvate thereof, characterized in that it is selected without limitation from the following:

33. The following formula III: 【Chemistry 22】 wherein R is selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl; R ａ is selected from 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 heterocyclic group, a substituted 3- to 7-membered heterocyclic group, 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 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 heterocyclic group, a substituted 3- to 7-membered heterocyclic group, a C6-C10 aryl, a substituted C6-C10 aryl, a 5- to 10-membered heteroaryl, and a substituted 5- to 10-membered heteroaryl; Instead, R ａ , R ｂ and the carbon atoms to which they are linked form a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3- to 7-membered heterocyclic group, or a substituted 3- to 7-membered heterocyclic group; L ３ is present or absent, L ３ If exists, 【Chemistry 23】 where o is selected from an integer from 1 to 10; The chiral carbon atom at position 1 or 2 has two chiralities, R or S absolute configuration; Ac is a hydrophilic structural unit; wherein m is selected from 0 or 1), or a pharmaceutically acceptable salt or solvate thereof.

34. The Ac is represented by the following formula B: 【Chemistry 24】 wherein Z is selected, without limitation, from the group consisting of one or more of a hydrophilic structure: a carboxyl group, a phosphoric acid, a polyphosphoric acid, a phosphorous acid, a sulfonic acid, a sulfinic acid, or a polyethylene glycol (PEG); Y′ is an optional scaffold connecting the amino group and Z; Ac is linked to the 2-carbon atom labeled in structural formula I by scaffold Y; Preferably, the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is used to couple with a ligand Ab to form a ligand-camptothecin derivative conjugate of formula I or formula II according to any one of claims 1 to 32.

35. The Ac may be 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 the following structure: 【Chemistry 25】 35. The linker-drug compound of claim 33 or 34, or a pharmaceutically acceptable salt or solvate thereof, selected without limitation from:

36. 36. The linker-drug compound of any one of claims 33 to 35, or a pharmaceutically acceptable salt or solvate thereof, wherein Ac is selected without limitation from glycine, phosphate, (D / L) glutamic acid, or polyethylene glycol hydrophilic structures.

37. The linker-drug compound has the following structure or an isomer thereof: 【Chemistry 26】 【Chemistry 27】 【Chemistry 28】 【Chemistry 29】 wherein o is selected from an integer of 1 to 10.

37. The linker-drug compound of any one of claims 33 to 36, or a pharmaceutically acceptable salt or solvate thereof, selected without limitation from:

38. The following steps: 【Transformation 30】 The invention is characterized in that it comprises A ligand-camptothecin derivative conjugate shown in general formula I or general formula II is obtained by a coupling reaction between a linker-drug compound and a reduced antibody or an antigen-binding fragment thereof; the 1-, 2-, or 3-position chiral carbon atom has absolute chirality in the R or S configuration; Ab, L １ , L ２ , L ３ , L ４ , L ５ ,X,R,R １ , R ２ and n is as defined in any one of claims 1 to 37, or a pharmaceutically acceptable salt or solvate thereof.

39. The ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, has the following structure: 【Chemistry 31】 【Chemistry 32】 【Transformation 33】 【Transformation 34】 【Chemistry 35】 【Transformation 36】 【Chemistry 37】 【Transformation 38】 【Chemistry 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 wherein SI-1x6.4 is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3, preferably as described in any one of claims 2 to 7 and 22; and n is selected from the group consisting of an integer of 1 to 10, without limitation.

40. The ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, has the following structure: 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】 【Chemistry 46】 【Chemistry 47】 【Chemistry 48】 【Chemistry 49】 [Transformation 50] 【Chemistry 51】 【Chemistry 52】 wherein SI-1x4 is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3, preferably as described in any one of claims 2 to 3, 8 to 11 and 22; and n is selected from the group consisting of an integer of 1 to 10, without limitation.

41. The ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, has the following structure: 【Chemistry 53】 wherein SI-1x22 is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3, preferably as described in any one of claims 2 to 3, 4, 12 to 14 and 22; and n is selected from the group consisting of an integer of 1 to 10, without limitation.

42. The ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, has the following structure: 【Chemistry 54】 wherein SI-1x24 is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3, preferably as described in any one of claims 2 to 5, 15, 16 and 22; and n is selected from the group consisting of an integer of 1 to 10, without limitation.

43. The ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, has the following structure: 【Transformation 55】 wherein SI-1x25 is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3, preferably as described in any one of claims 2 to 3, 4, 12, 17, 18 and 22; and n is selected from the group consisting of an integer of 1 to 10, without limitation.

44. The ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, has the following structure: 【Transformation 56】 wherein SI-1x26 is a bispecific antibody or antigen-binding fragment thereof that simultaneously targets EGFR and HER3, preferably as described in any one of claims 2 to 3, 8, 19 to 21 and 22; and n is selected without limitation from the group consisting of an integer of 1 to 10, and n is selected from the group consisting of an integer of 1 to 10.

45. 45. The ligand-camptothecin derivative conjugate of any one of claims 1 to 32 or 39 to 44, or a pharmaceutically acceptable salt or solvate thereof; or the linker-drug compound of any one of claims 33 to 37, or a pharmaceutically acceptable salt or solvate thereof, characterized in that the pharmaceutically acceptable salts include sodium, potassium, calcium, or magnesium salts formed with 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 with basic functional groups in the structural formula.

46. 46. ​​A pharmaceutical composition comprising a ligand-camptothecin derivative conjugate according to any one of claims 1 to 32 or 39 to 44, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound according to any one of claims 33 to 37, or a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutically acceptable carrier.

47. 46. ​​A pharmaceutical formulation comprising a ligand-camptothecin derivative conjugate according to any one of claims 1 to 32 or 39 to 44, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound according to any one of claims 33 to 37, or a pharmaceutically acceptable salt or solvate thereof.

48. Use of a ligand-camptothecin derivative conjugate according to any one of claims 1 to 32 or 39 to 44, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound according to any one of claims 33 to 37, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to claim 46 and / or a pharmaceutical formulation according to claim 47, in the manufacture of a medicament for the treatment or prevention of cancer or tumor; or 10. Use of a ligand-camptothecin derivative conjugate according to any one of claims 1 to 32 or 39 to 44, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound according to any one of claims 33 to 37, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to claim 46 and / or a pharmaceutical formulation according to claim 47, for the treatment or prevention of cancer or tumors, comprising: Preferably, the cancer or tumor expresses EGFR and / or HER3; More preferably, the cancer or tumor is selected from 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, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia, and other solid tumors or blood cancers.

49. 1. 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 according to any one of claims 1 to 32 or 39 to 44, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound according to any one of claims 33 to 37, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to claim 46 and / or a pharmaceutical formulation according to claim 47, Preferably, the cancer or tumor expresses EGFR and / or HER3; More preferably, the method wherein the cancer or tumor is selected from 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, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia, and other solid tumors or blood cancers.

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