Bispecific antibody-camptothecin drug conjugates and pharmaceutical uses thereof

Abstract

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 has a drug-antibody ratio (DAR) of 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, which is coupled with a linker-camptothecin drug. Furthermore, the present invention further relates to a method for producing and purifying the antibody-toxin conjugate, and its application in tumor therapy. Furthermore, the present invention further relates to a linker-drug compound that can be coupled with Ab to form an antibody-toxin conjugate. TIFF2024543508000518.tif44161

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 the preparation and application of 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 include EGFR (ErbB-1), HER2 / c-neu (ErbB-2), HER3 (ErbB-3) and HER4 (ErbB-4).

[0003] Epidermal growth factor receptor is a glycoprotein with a molecular weight of 170 kDa that crosses the cell membrane and is activated by binding to a ligand. Upon activation, EGFR is converted from a monomer to an auto-dimer or forms a heterodimer with other HER family members. The formation of the dimer activates intracellular kinase pathways and directs phosphorylation of downstream pathways such as MAPK, Akt, and JNK pathways to induce cell proliferation. Studies have shown that high expression of EGFR is associated with tumor cell proliferation, angiogenesis, tumor invasion, and the like. EGFR-related signaling pathways play a very important role in the maintenance and proliferation of epithelial tissues. In particular, in breast cancer, malignant glioma, and lung cancer, EGFR can promote tumor formation. In lung cancer tissues, the EGFR signaling pathway shows a stimulated state, and there is a positive correlation between the expression level of EGFR and the stage of cancer development. Meanwhile, due to the discovery of secondary mutations of EGFR under drug stress, EGFR is being used in more and more studies as a biomarker of tumor drug resistance.

[0004] Epidermal growth factor receptor 3 (HER3 or ErbB3) also has the structure of a general epidermal growth factor receptor, but HER3 lacks the structural domain of intracellular protein tyrosine kinase and therefore cannot autophosphorylate. HER3 binds to ligand proteins and promotes its heterodimerization with other human epidermal growth factor receptor family members to activate receptor-mediated signaling pathways, acting not only as a means of signal diversification but also as a signal amplifier, promoting tumor progression. Heregulin (glial cell growth factor, neu differentiation factor) can activate intracellular kinase-dependent multistep signaling pathways when bound to the transmembrane receptors HER3 and HER4. Downregulation of this signaling pathway leads to Alzheimer's disease, heart failure, atherosclerosis, cancer, etc. 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 has the potential to regulate EGFR / HER2 signaling pathway-mediated drug resistance in cancer cells. Studies have also shown that HER3 renders cancer treatment 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 found to drive tumor progression in solid tumors, such as breast, lung, gastric, and pancreatic cancers. 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 practice.

[0006] Monoclonal antibodies have been widely used in antitumor treatment in recent years, but their efficacy has left much to be desired. Many tumor patients have poor clinical responses, and some patients with clinical responses develop drug resistance after a period of monoclonal antibody treatment, causing 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, thus achieving various new and unique antitumor 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 mainly due to the fact that multiple mediators are involved in the pathogenesis of tumors through specific or overlapping mechanisms, and when multiple targets are blocked simultaneously, this will bring about better therapeutic effects than the inhibition of a single target, and at the same time, the action of multiple targets greatly reduces the possibility of drug resistance occurring. Currently, two bispecific antibodies, catumaxomab and blinatumomab, are approved for sale in the United States, and there are over 50 bispecific antibody molecules in clinical trials.

[0007] Antibody-drug conjugates (ADCs) are molecules with specific targeted killing effects, obtained by binding small molecule toxins with cell death effects 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, and therefore have tumor specificity and potential that cannot be achieved 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 practice are directed at a single target, and the synergistic benefits of dual-targeting therapy cannot be achieved. Summary of the Invention [Means for solving the problem]

[0008] Based on a comprehensive understanding of the ADC class of drugs, the inventors disclose bispecific antibody drug conjugates, methods for producing the same, pharmaceutical compositions comprising said conjugates, and uses of said 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 pharma- ceutically 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 one of the following groups: [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 of number symbol 2 bonded to Y has absolute chirality in the R or S configuration; L3 is 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 an enzyme cleavage unit: L5 is a linking unit; In formula I, the chiral carbon atom numbered 1 attached to N has absolute chirality in the R or S configuration; R is selected from hydrogen atoms, deuterium atoms, halogens, C1-C6 alkyls, substituted C1-C6 alkyls, deuterated C1-C6 alkyls, C3-C8 cycloalkyl C1-C6 alkyls, C1-C6 alkoxy C1-C6 alkyls, C6-C10 aryls, substituted C6-C10 aryls, 5-10 membered heteroaryls, and substituted 5-10 membered heteroaryls; 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, 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 3-7 membered heterocyclyl, a substituted 3-7 membered heterocyclyl, a C6-C10 aryl, a substituted C6-C10 aryl, a 5-10 membered heteroaryl, or a substituted 5-10 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 hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, substituted 5-10 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- is selected; 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 cycloalkyl C1-C6 alkyl, a C6-C10 aryl C1-C6 alkyl, a C1-C6 alkoxy C1-C6 alkyl, a 3-7 membered heterocyclic group, a substituted 3-7 membered heterocyclic group, a C6-C10 aryl, a substituted C6-C10 aryl, a 5-10 membered heteroaryl, and a substituted 5-10 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 atom to which they are attached constitute a C3-C8 cycloalkyl group; R3 and R4 are the same or different, and each independently is a hydrogen atom, a deuterium atom, a halogen, 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 from 0 to 4 (eg, 0, 1, 2, 3, or 4), preferably 0, 1; and n is selected from integers from 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, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, is disclosed, 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.

[0011] In some embodiments of the first aspect of the present invention, the disclosure of a ligand-camptothecin derivative conjugate of general formula I, or a pharma- ceutically acceptable salt or solvate thereof, comprises an Ab antibody comprising: an IgG1 heavy chain, a κ light chain, and a single chain Fv (scFv) structural domain; said single chain Fv (scFv) structural domain forming a construct with an IgG1 heavy chain or a κ light chain; said IgG1 heavy chain and κ light chain forming an IgG moiety having binding specificity for EGFR; said scFv structural domain having binding specificity for HER3, and said scFv structural domain comprising a linker (e.g., (gly-gly-gly-gly- the single-chain Fv structural domain has an amino acid sequence of (gly-gly-gly-gly-ser)m, where m is an integer of at least 3, and preferably n is an integer of 1 to 10), which is linked to the C-terminus or N-terminus of the IgG1 heavy chain or the C-terminus or N-terminus of the κ light chain; and the single-chain Fv structural domain has a structural order of 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 has an amino acid sequence of (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 of a ligand-camptothecin derivative conjugate of general formula I, or a pharma- ceutically acceptable salt or solvate thereof, is characterized in that the Ab antibody's kappa light chain comprises the CDRs shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, the IgG1 heavy chain comprises the CDRs shown 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 shown in SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, and the light chain variable region CDRs shown 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 as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, characterized in that the Ab antibody light chain comprises a variable region as shown in SEQ ID NO:28, the IgG1 heavy chain comprises a variable region as shown in SEQ ID NO:38, and the single-chain Fv (scFv) structural domain comprises a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:40.

[0014] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, is disclosed, 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, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, is disclosed, 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 single-chain Fv (scFv) structural domain construct 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 as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, characterized in that the Ab antibody kappa light chain comprises the CDRs shown in SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, the IgG1 heavy chain comprises the CDRs shown 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 shown in SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, and the light chain variable region CDRs shown 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, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, is disclosed, characterized in that the Ab antibody light chain comprises a variable region as shown in SEQ ID NO: 44, the IgG1 heavy chain comprises a variable region as shown in SEQ ID NO: 48, and the single-chain Fv (scFv) structural domain comprises a heavy chain variable region as shown in SEQ ID NO: 39 and a light chain variable region as shown in SEQ ID NO: 40.

[0018] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, is disclosed, 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, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, is disclosed, characterized in that the nucleic acid coding sequence of the light chain of the Ab antibody is SEQ ID NO:5 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:7.

[0020] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, is disclosed, characterized in that the Ab antibody light chain comprises a variable region as shown in SEQ ID NO: 49, and the IgG1 heavy chain comprises a variable region as shown in SEQ ID NO: 52, and the single-chain Fv (scFv) structural domain comprises a heavy chain variable region as shown in SEQ ID NO: 50, and a light chain variable region as shown in SEQ ID NO: 51.

[0021] In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharma- ceutically 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 pharma- ceutically 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 light chain and single chain Fv (scFv) structural domains of the antibody has a 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 as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, is disclosed, characterized in that the Ab antibody has a 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 pharma- ceutically 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 as shown in general formula I, or a pharma- ceutically 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 pharma- ceutically 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 light chain and single chain Fv (scFv) structural domains of the antibody has a 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 as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, characterized in that the Ab antibody light chain comprises a variable region as shown in SEQ ID NO: 53, the IgG1 heavy chain comprises a variable region as shown in SEQ ID NO: 54, and the single-chain Fv (scFv) structural domain comprises a heavy chain variable region as shown in SEQ ID NO: 50 and a light chain variable region as shown 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 pharma- ceutically 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 pharma- ceutically 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 a 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 pharma- ceutically 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 invention, X has the following structure or an isomer thereof: [ka] The present invention discloses a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, characterized in that it is selected without limitation from 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, a ligand-camptothecin derivative conjugate as shown in general formula I, or a pharma- ceutically acceptable salt or solvate thereof, is disclosed, characterized in that L4 is selected, without limitation, from a peptide residue formed of an amino acid, In the above formula, optionally, the amino acid is 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, said peptide residue is a peptide residue 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, said 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, 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 3-7 membered heterocyclyl group, a substituted 3-7 membered heterocyclyl group, a C6-C10 aryl group, a substituted C6-C10 aryl, a 5-10 membered heteroaryl, and a substituted 5-10 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 as shown in general formula I, or a pharma- ceutically 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] are selected without restriction.

[0036] In some embodiments of the first aspect of the invention, the linking unit -L1-L2-L3-L4-L5- has the following structure: [ka] Preferably [ka] are selected without restriction from In the above formula, Ac is a hydrophilic structural unit; R5, R6 and R7 are the same or different, and each is independently selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 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 an integer 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 pharma- ceutically acceptable salt or solvate thereof; [ka] Disclose the following: 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] where o is selected from an integer in the range of 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably an integer in the range of 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 cycloalkyl C1-C6 alkyl, a C1-C6 alkoxy C1-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 hydrogen atoms, deuterium atoms, halogens, C1-C6 alkyls, substituted C1-C6 alkyls, deuterated C1-C6 alkyls, C3-C8 cycloalkyl C1-C6 alkyls, C1-C6 alkoxy C1-C6 alkyls, carboxyl, 3-7 membered heterocyclyls, substituted 3-7 membered heterocyclyls, C6-C10 aryls, substituted C6-C10 aryls, 5-10 membered heteroaryls, and substituted 5-10 membered heteroaryls; 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 hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, substituted 5-10 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- is selected; 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 3-7 membered heterocyclic group, a substituted 3-7 membered heterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 membered heteroaryl, and a substituted 5-10 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 atom to which they are attached constitutes a C3-C8 cycloalkyl group; R3 and R4 are the same or different and each independently represent a hydrogen atom, a deuterium atom, a halogen, 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 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 an integer selected from 0 to 4 (i.e., 0, 1, 2, 3, or 4), preferably 0 or 1; n is selected from an integer 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 pharma- ceutically acceptable salt or solvate thereof, may be such that Ac is of formula B: [ka] It is disclosed that the compound has a structure as shown in In the above formula, Z is selected from the group consisting of one or more of the hydrophilic structures carboxyl group, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid, or polyethylene glycol (PEG), without limitation; Preferably, Z is selected from the hydrophilic structures carboxyl, phosphoric acid or 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 2-position carbon labeled in structural formula I by scaffold Y.

[0039] In some embodiments of the first and second aspects of the invention, the ligand-camptothecin derivative conjugate, or a pharma- ceutically acceptable salt or solvate thereof, is characterized in that 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 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 pharma- ceutically 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 pharma- ceutically acceptable salt or solvate thereof comprises [ka] is the following formula d: [ka] It has a 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 5-10 membered heteroaryl, and a substituted 5-10 membered heteroaryl; Preferably, R is selected from a hydrogen atom or a C1 to C6 alkyl group; R1 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, carboxyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, substituted 5-10 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 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, carboxyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, substituted 5-10 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 cycloalkyl C1-C6 alkyl, a C1-C6 alkoxy C1-C6 alkyl, a 3-7 membered heterocyclic group, a substituted 3-7 membered heterocyclic group, a C6-C10 aryl, a substituted C6-C10 aryl, a 5-10 membered heteroaryl, and a substituted 5-10 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; 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 atom to which they are attached constitutes 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 a pharma- ceutically acceptable salt or solvate thereof further comprises the step of: [ka] [ka] The present invention is characterized in that the above-mentioned is selected without limitation from the above.

[0043] In some embodiments of the invention, L1 may contain a succinimide group. In these embodiments, the Ligand-Drug conjugate may undergo hydrolysis under readily hydrolyzable conditions, and the site of hydrolysis is 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 ring-closed form [ka] and a part of the succinimide groups is in a ring-open form. [ka] and; Complete hydrolysis of succinimide groups, i.e., all succinimide groups are ring-open [ka] It 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), the succinimide groups can all be ring-closed, some can be ring-open, or all can be ring-open.

[0045] Although the succinimide group appears in the chemical structure of the ADC in closed form, it will be understood that the present application actually encompasses three scenarios: all closed, some open, and all open succinimides. A third aspect of the invention is a compound represented by formula III: [ka] The present invention discloses a linker-drug compound, or a pharma- ceutically acceptable salt or solvate thereof, characterized in that it has the structure: 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 5-10 membered heteroaryl, and a substituted 5-10 membered heteroaryl; R a is selected from hydrogen atoms, deuterium atoms, halogens, C1-C6 alkyls, deuterated C1-C6 alkyls, halogenated C1-C6 alkyls, C3-C8 cycloalkyls, C3-C8 cycloalkylC1-C6 alkyls, C1-C6 alkoxyC1-C6 alkyls, 3-7 membered heterocyclic groups, substituted 3-7 membered heterocyclic groups, C6-C10 aryls, substituted C6-C10 aryls, 5-10 membered heteroaryls, and substituted 5-10 membered heteroaryls; R bis selected from hydrogen atoms, deuterium atoms, halogens, C1-C6 alkyls, deuterated C1-C6 alkyls, halogenated C1-C6 alkyls, C3-C8 cycloalkyls, C3-C8 cycloalkylC1-C6 alkyls, C1-C6 alkoxyC1-C6 alkyls, 3-7 membered heterocyclic groups, substituted 3-7 membered heterocyclic groups, C6-C10 aryls, substituted C6-C10 aryls, 5-10 membered heteroaryls, and substituted 5-10 membered heteroaryls; 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 atom 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 invention, the linker-drug compound, or a pharma- ceutically acceptable salt or solvate thereof, is a compound in which Ac is a group of the following formula B: [ka] It has a structure shown in In the above formula, Z is selected from the group consisting of one or more of the hydrophilic structures carboxyl group, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid, or polyethylene glycol (PEG), without limitation; Y' is any scaffold linking 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 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 derivative of the following structure: [ka] Disclosed is a linker-drug compound, 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 pharma- ceutically 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 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, 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 pharma- ceutically 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 as described in the first and second aspects.

[0051] In a fourth aspect of the present invention, there is provided a method for preparing a ligand-camptothecin derivative conjugate of general formula I or general formula II according to the first and second aspects, or a pharma- ceutical acceptable salt or solvate thereof, comprising the steps of: [ka] Including, A ligand-camptothecin derivative conjugate as 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 pharma- ceutically acceptable salt or solvate thereof, as an intermediate in the preparation of a ligand-camptothecin derivative conjugate or a pharma- ceutically acceptable salt or solvate thereof. In certain embodiments, said ligand-camptothecin derivative conjugate or a pharma- ceutically acceptable salt or solvate thereof is a ligand-camptothecin derivative conjugate or a pharma- ceutically acceptable salt or solvate thereof as disclosed in the first, second and fourth aspects of the invention. In certain embodiments, said 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 pharma- ceutically acceptable salt or solvate thereof, further comprises the ligand-camptothecin derivative conjugate, or a pharma- ceutically acceptable salt or solvate thereof, having the following structure: or a succinimide ring-open structure thereof or an isomer thereof: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] is 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 pharma- ceutically acceptable salt or solvate thereof, further comprises the ligand-camptothecin derivative conjugate, or a pharma- ceutically acceptable salt or solvate thereof, having the following structure: or a succinimide ring-open structure thereof or an isomer thereof: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] is 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 pharma- ceutically acceptable salt or solvate thereof, further comprises the ligand-camptothecin derivative conjugate, or a pharma- ceutically acceptable salt or solvate thereof, having the following structure: or a succinimide ring-open structure thereof or an isomer thereof: [ka] is 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 pharma- ceutically acceptable salt or solvate thereof, further comprises the ligand-camptothecin derivative conjugate, or a pharma- ceutically acceptable salt or solvate thereof, having the following structure: or a succinimide ring-open structure thereof or an isomer thereof: [ka] is 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 pharma- ceutically acceptable salt or solvate thereof, further comprises the ligand-camptothecin derivative conjugate, or a pharma- ceutically acceptable salt or solvate thereof, having the following structure: or a succinimide ring-open structure thereof or an isomer thereof: [ka] is 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 pharma- ceutically acceptable salt or solvate thereof, further comprises the ligand-camptothecin derivative conjugate, or a pharma- ceutically acceptable salt or solvate thereof, having the following structure: or a succinimide ring-open structure thereof or an isomer thereof: [ka] is 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 pharma- ceutically acceptable salt or solvate thereof, or the linker-drug compound or a pharma- ceutically acceptable salt or solvate thereof, is characterized in that the pharma- ceutically 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, a pharmaceutical composition is disclosed comprising a ligand-camptothecin derivative conjugate as described in the first and second aspects, or a pharma- ceutically acceptable salt or solvate thereof, or a linker-drug compound as described in the third aspect, or a pharma- ceutically acceptable salt or solvate thereof, and optionally a pharma- ceutically acceptable carrier.

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

[0062] In a seventh aspect of the invention there is provided the use of: a ligand-camptothecin derivative conjugate as defined in the first and second aspects, or a pharma- ceutically acceptable salt or solvate thereof, or a linker-drug compound as defined in the third aspect, or a pharmaceutical composition as defined in the fifth aspect and / or a pharmaceutical formulation as defined in the sixth aspect, in the manufacture of a medicament for treating or preventing cancer or tumours; Alternatively, the present invention discloses the use of a ligand-camptothecin derivative conjugate according to the first and second aspects, or a pharma- ceutically acceptable salt or solvate thereof, or a linker-drug compound according to the third aspect, 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, gastric 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 invention there is provided a method for treating or preventing cancer or tumours comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a ligand-camptothecin derivative conjugate as defined in the first and second aspects, or a pharma- ceutically acceptable salt or solvate thereof, or a linker-drug compound as defined in the third aspect, or a pharmaceutical composition as defined in the fifth aspect and / or a pharmaceutical formulation as defined in 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, gastric 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 hematological 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 and good preclinical efficacy, as well as excellent clinical therapeutic effects. [Brief description 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] FIG. 1 shows RP-HPLC detection / measurement of ADC-6 drug-antibody conjugation ratio (DAR). [Figure 2C] FIG. 1 shows 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] RP-HPLC detection / measurement of ADC-219 drug-antibody conjugation ratio (DAR). [Figure 2J] 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 in vitro efficacy of six naked antibodies as well as six ADCs in A431. [Figure 4B] Shows in vitro efficacy of six naked antibodies as well as six ADCs in BXPC-3. [Figure 4C] The in vitro efficacy of six naked antibodies as well as six ADCs in FaDu is shown. [Figure 4D] Shows in vitro efficacy of six naked antibodies as well as six ADCs in HARA-B. [Figure 4E] Shows in vitro efficacy of six naked antibodies as well as six ADCs in HCC827. [Figure 4F] Shows in vitro efficacy of six naked antibodies as well as six ADCs in SW620. [Figure 5A]1 shows the results of in vivo efficacy experiments of ADC-6 and SI-1×6.4 naked antibodies in the A431 single tumor model. [Figure 5B] 1 shows the results of in vivo efficacy studies of ADC-6, SI-1×6.4 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 the BXPC-3 single tumor model. [Figure 7A] Figure 2 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 PREFERRED EMBODIMENTS

[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 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 that can bind to cells. In an embodiment of the invention, the ligand is designated Ab, the ligand can form a linkage bond with a linkage unit through a heteroatom on the ligand, and is preferably an antibody or an antigen-binding fragment thereof, the antibody being selected from a chimeric, humanized, fully human, or murine antibody; preferably a monoclonal antibody.

[0070] The Ligand unit is a targeting agent that specifically binds to a target moiety. The Ligand can specifically bind to or bind to a cellular component or other target molecule of interest. The target moiety or target is generally on the surface of a cell. In some aspects, the Ligand unit serves to deliver the Drug unit to a specific target cell population with which the Ligand unit interacts. Ligands include, but are not limited to, proteins, polypeptides and peptides, 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 a sulfur atom of the Ligand. In some aspects, the sulfur atom is a sulfur atom of a cysteine ​​residue, forming an interchain disulfide bond of the antibody. In another embodiment, the sulfur atom is a sulfur atom of a cysteine ​​residue that has been introduced into the Ligand unit and forms an interchain disulfide bond of the antibody. In another embodiment, the sulfur atom is a 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 embodiments, the linker bond sulfur atom is selected from a cysteine ​​residue that forms an interchain disulfide bond of the antibody or a cysteine ​​residue that has 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 the target cell population. The antibody may be any protein or protein-like molecule capable of binding, forming a complex, or reacting with a portion of the cell population to be treated or biologically modified. The antibody forming the antibody-drug conjugate in the present invention maintains its original antigen-binding ability from the wild state. Thus, the antibody of the present invention may exclusively bind to the antigen. The antigens involved include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation 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). The tumor-associated factor may be a cluster differentiation factor (e.g., CD protein).

[0072] Antibodies applied 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 by methods and information for antibody production well known in the art. To develop effective cellular level targets that can be used in cancer diagnosis and treatment, researchers are trying to discover transmembrane or other tumor-associated peptides. These targets can be specifically expressed on the surface of one or more cancer cells, and are expressed little or not at all on the surface of one or more non-cancer cells. In general, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells than on the surface of non-cancer cells. Identification of 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 gene bank 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 relevant 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 cited.

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

[0074] The term "cancer" refers to a physiological condition or disease characterized by dysregulated cell proliferation. 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 cytotoxic drugs, denoted by d, which are chemical molecules with a strong ability to disrupt normal proliferation in tumor cells. Cytotoxic drugs in principle kill tumor cells at sufficiently high concentrations, but due to lack of specificity, they may also cause apoptosis of normal cells at the same time as killing tumor cells, thereby causing severe side effects. The term 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" refers to a fragment or bond of a chemical structure 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 including extensions, spacers, and amino acid units can be synthesized by methods known in the art, such as those described in US Patent Publication No. 2005-0238649A1. The connectors can be "cleavable connectors" that facilitate the release of drugs 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] According to the mechanism of intracellular drug release, as used herein, the "linker" or "linker of antibody-drug conjugate" can be classified into two types: non-cleavable linker and cleavable linker. For antibody-drug conjugates containing non-cleavable linkers, the mechanism of drug release is as follows: after the conjugate binds to the antigen and is taken up by the 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. Although the resulting change in the structure of the drug molecule does not reduce its cytotoxicity, it cannot penetrate adjacent cells because the active molecule is charged (amino acid residues). Therefore, such an agent cannot kill adjacent tumor cells (antigen-negative cells) that do not express the targeted antigen (bystander effect) (Ducry et al., 2010, Bioconjugate Chem. 21:5-13). For antibody-drug conjugates containing cleavable linkers, the mechanism of drug release is as follows: after the conjugate binds to the antigen and is taken up by the cell, the conjugate is cleaved, releasing the active component (the small molecule drug itself) in the target cell. Cleavable linkers are mainly classified into chemically sensitive linkers and enzyme sensitive linkers. Chemically sensitive linkers are selectively cleaved due to the difference in the properties of plasma and cytoplasm or tumor microenvironment. Such properties include pH, glutathione concentration, etc. However, pH sensitive linkers, which are relatively stable in the neutral or weakly alkaline environment of blood (pH 7.3-7.5), are believed to be hydrolyzed in the weakly acidic tumor microenvironment (pH 5.0-6.5) and lysosomes (pH 4.5-5.0), such as hydrazones, carbonates, acetals, and ketals. Due to the limited plasma stability of acid cleavable linkers, antibody drug conjugates based on such linkers have a short half-life (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 concentration. 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 plasma circulation due to unfavorable extracellular pH and serum protease inhibitors that render the proteases normally inactive outside cells. Considering the high plasma stability and excellent intracellular cleavage selectivity and efficacy, enzyme-labile linkers are widely used as cleavable linkers for 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 symbols for amino acids used in this disclosure are as described 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 1 to 20 carbon atoms (i.e., a "C1-C20 alkyl"), preferably an alkyl group containing 1 to 12 carbon atoms (i.e., a "C1-12 alkyl"), more preferably an alkyl group containing 1 to 10 carbon atoms (i.e., a "C1-C10 alkyl"), and most preferably an alkyl group containing 1 to 6 carbon atoms (i.e., a "C1-C6 alkyl"). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, Examples of dimethylpentyl 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. An alkyl group may be substituted or unsubstituted, and when substituted, the substituents may be substituted at available points of attachment, and said substituents are preferably one or more of 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 is replaced by a substituent. Unless otherwise specified in the text, the substituents of 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', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NR'S(O)2R'', -CN and -NO2, 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" refers to a saturated straight or branched chain aliphatic hydrocarbon group having two residues derived by the removal of two hydrogen atoms from the same carbon atom or from two different carbon atoms of a parent alkane, and is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkylene group containing from 1 to 12 carbon atoms, and more preferably from 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 optionally substituted. or unsubstituted, and when substituted, the substituents may be substituted at available points of attachment, said substituents being preferably 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, cyclohexyloxy. An alkoxy group may be optionally substituted or unsubstituted, and if substituted, the substituents are preferably one or more of the 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" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic 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 which is nitrogen, oxygen, or S(O). m(m is an integer from 0 to 2), except for the -OO-, -OS-, or -SS- ring moieties, the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms (i.e., a "3- to 12-membered heterocyclic group"), of which 1 to 4 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, where 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, the alkyl group being as defined above, for example, a deutero C1-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 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 in an aryl or heteroaryl or heterocyclic group is replaced by a substituent. Unless otherwise specified in the text, the substituents on the 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)2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NR'S(O)2R'', -CN and -NO2, 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 defined above.

[0100] The present invention also includes 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 the preparation of the deuterated forms of formula I, commercially available deuterated starting materials can be used, or deuterated reagents can be synthesized by conventional techniques, including non-limiting examples of deuterated reagents such as deuteroborane, trideuteroborane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide, and deuterated methyl iodide.

[0101] The term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in the composition and order of amino acids in the constant region of the heavy chains and therefore in their antigenicity. Thus, immunoglobulins can be classified into five classes, or isoforms, of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. The same class of Ig can be further divided into different subclasses depending on the difference in the amino acid composition of its hinge region and the number and position of the disulfide bonds in the heavy chains, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are divided into kappa or lambda chains depending on the difference in the constant region. Each of the five classes of Ig 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-glinAv antibody, an anti-CAIX antibody, an anti-P-cadherin antibody, an anti-GD3 antibody, an anti-cadherin 6 antibody, an anti-LAMP1 antibody, an anti-FLT3 antibody, an anti-BCMA antibody, an anti-CD79b antibody, an anti-CD19 antibody, an anti-CD33 antibody, an anti-CD56 antibody, an anti-CD74 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD37 antibody, an anti-CD138 antibody, an anti-CD352 antibody, an anti-CD25 antibody, or an anti-CD123 antibody.

[0102] The term "solvate" or "solvate" refers to the formation of a pharma- ceutically usable solvate from the ligand-drug complex 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 an embodiment of the present invention, the drug load is indicated as n, which can illustratively be an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The average number of drugs per ADC molecule after the coupling reaction can be identified by conventional methods such as UV-visible spectroscopy, mass spectrometry, ELISA test and HPLC characterization.

[0104] In one embodiment of the invention, a cytotoxic drug is coupled to the open cysteine ​​sulfhydryl-SH and / or sulfhydryl-SH of site-directed mutagenesis cysteine ​​residues between antibody chains by a linker unit, and generally the number of drug molecules that can be coupled to an 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 and 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 salts" or "pharmaceutically usable salts" refers to salts of the ligand-drug conjugates of the present invention or compounds 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, etc.

[0108] The term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" refers to a salt of the ligand-drug conjugate of the present invention or a salt of 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 that has an isoelectric point of less than 7. Acidic amino acid molecules tend to have one or more acidic groups, such as carboxyl groups, that can effectively ionize into a negative ionic form in their structure, making them more hydrophilic. Acidic amino acids can be natural or unnatural.

[0110] The term "natural amino acid" refers to biologically synthesized amino acids. Natural amino acids are generally L-configured, with a few exceptions, such as glycine, that include both natural and biologically synthesized amino acids.

[0111] By "unnatural amino acid" is meant 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 shown 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. EXAMPLES

[0113] Example 1 Synthesis of compound M1: [ka] In a 5000mL one-neck flask, N-fluorenylmethoxycarbonyl-glycine-glycine (100g, 282mmol, 1.0 equivalent), lead tetraacetate (175g, 395mmol, 1.4 equivalent), 2000mL of anhydrous tetrahydrofuran and 670mL of toluene were added, stirred uniformly, protected by nitrogen, heated to 85℃, and reacted for 2.5 hours. The reaction was monitored by TLC, and 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 (87g); LC-MS: [M+NH4] + =386.0.

[0114] Example 2 Synthesis of compound M3: [ka] In a 1000 mL single-neck flask, compound SM-2 (synthesized according to the method disclosed in China Patent No. 108452321A) (40 g, 96 mmol, 1.0 equiv.), triethylamine (26.7 mL, 2.0 equiv.), and toluene (400 mL) were added, and the reaction was carried out by heating to 120° C. and refluxing for 2 h. When TLC monitoring showed that the reaction was essentially complete, cooling to 50° C. was carried out, and the solvent was removed by rotation under reduced pressure. Dissolution with ethyl acetate (150 mL) and water (40 mL) was carried out. The pH was adjusted to 2-3 with 1 M HCl under stirring in an ice bath, and liquid separation was carried out. The aqueous layer was extracted once more with ethyl acetate, and the organic layers were combined and dried by adding 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] In a 1000 mL single-neck flask, 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 and reacted at room temperature for 30 min (monitored by TLC). Insoluble materials were filtered off. The reaction solution was directly purified by preparative LC, and 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 obtain 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 of Example 2; LC-MS: [M+H] + =565.2.

[0117] Example 4 Synthesis of compound 1: [ka] Step 1: Compound 1a In a 250mL one-neck flask, M1 (6g, 16.3mmol), 100mL THF, p-toluenesulfonic acid monohydrate (0.31g, 1.63mmol) were added, stirred, cooled to 0℃, benzyl hydroxyacetate (5.4g, 32.6mmol) was added dropwise, and then allowed to warm to room temperature naturally for reaction with TLC monitoring (the reaction lasts for about 2-4 hours). 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, concentrated, and the residue was purified by silica gel column (PE:EA=10:1-5:1-1:1) to give 1a (4g) in 52% yield: LC-MS: [M+H] + =475.18.

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

[0119] M4 (prepared according to the method disclosed in China Patent No. 111051330A) (1.73g, 4.2mmol), PyBOP (2.61g, 5.04mmol), HOBt (680mg, 5.04mmol) and DMF 10mL were added to another 25mL one-neck flask, DIPEA (830uL, 5.04mmol) was added to 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 the product preparation liquid, the preparation liquid was extracted by dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain solid 1b (1.7g) 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 one-neck flask and dissolved in 15 mL of DMF, followed by addition of 900 mg of 5% Pd / C, and the hydrogenation reaction was carried out for 2 hours. After the reaction was completed, filtration was carried out to obtain the filtrate, which 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 uL, 1.39 mmol) was added, compound M3 (784 mg, 1.39 mmol) was added, and then the temperature was 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, DIPEA (378 uL, 2.29 mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted for 2 hours. After the completion of the reaction was shown by monitoring with HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 1e, which was freeze-dried to obtain 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 h. After the completion of the reaction was shown by monitoring with HPLC, concentration under reduced pressure was carried out to remove the solvent and obtain the crude product. The crude product was purified by HPLC to obtain a product fraction, and the fraction was freeze-dried 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 referring to the synthetic route of Example 4; LC-MS: [M+H] + =1065.3.

[0125] Example 6 Synthesis of compound 3: [ka] Step 1: Compound 3a In a 250mL one-neck flask, M1 (6g, 16.3mmol), 100mL THF, p-toluenesulfonic acid monohydrate (0.31g, 1.63mmol) were added, stirred, cooled to 0℃, 2-hydroxy-2-methylpropionate benzyl (6.3g, 32.6mmol) was added dropwise, and then allowed to warm to room temperature naturally for reaction while monitoring by TLC (the reaction lasts for about 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 with anhydrous sodium sulfate, filtering, concentrating, and the residue was purified by silica gel column (PE:EA=10:1-5:1-2:1) to give 3a (4.2g) in 52% yield; LC-MS: [M+H] + =503.3.

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

[0127] M4 (1.65g, 4.0mmol), PyBOP (2.59g, 5.0mmol), HOBt (675mg, 5.0mmol) and DMF 10mL were added to another 25mL one-neck flask, DIPEA (823uL, 5.04mmol) was added in 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 shown by monitoring with 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, filtered, and the filtrate was concentrated under reduced pressure to obtain solid 3b (1.4g) 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 one-neck flask and dissolved in 10 mL of DMF, and 5% Pd / C 700 mg was added, and the hydrogenation reaction was carried out for 1.5 h. 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 Crude product 3c was placed in an ice-water bath, DIPEA (210 uL, 1.25 mmol) was added, then compound M3 (704 mg, 1.25 mmol) was added, and then the reaction was allowed to warm to room temperature for 1 hour. After completion of the reaction as shown by HPLC monitoring, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to obtain 3d (486 mg); LC-MS: [MH] - =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, DIPEA (219.5 uL, 1.33 mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted for 3 hours. After completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 3e, which was freeze-dried to obtain 3e (157 mg); LC-MS: [M+H] + =1249.6.

[0131] Step 6: Compound 3 In a 25 mL one-neck flask, 3e (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 mixture was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was freeze-dried 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 referring to the synthetic route of Example 6; LC-MS: [M+H] + =1093.2.

[0133] Example 8 Synthesis of compound 5A: [ka] Step 1: Compound 5a In a 25mL one-neck flask, M1 (500mg, 1.4mmol, 1.0eq), p-toluenesulfonic acid monohydrate (26mg, 0.1mmol, 0.1eq) and 10mL of THF were added, stirred well, then cooled to 0°C, then L-lactic acid benzyl ester (1.2g, 7.0mmol, 5eq) was added slowly, 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, concentrating, and the residue was purified by reverse phase column to give 5a (400mg); 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 In a 25 mL single-neck flask, compound 5a (400 mg, 0.8 mmol, 1.0 equiv.) and 4 mL of DMF were added, stirred well, and then cooled to 0° C. before slowly adding DBU (137 mg, 0.9 mmol, 1.1 equiv.). After the addition was complete, the temperature was raised to room temperature for reaction. 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, and reaction solution (1) was added. 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 obtain 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 one-neck flask and dissolved in DMF (60 mL), then 5% Pd / C (4 g) was added and the hydrogenation reaction was carried out at room temperature for 4 h (the reaction progress was monitored using HPLC). The Pd / C was filtered and without concentration, the filtrate was placed directly into an ice-water bath (approximately 0° C.) and was ready for use.

[0136] Step 4: Compound 5d Crude product 5c was placed in an ice-water bath, DIPEA (1.1 mL, 1.1 eq.) was added, followed by compound M3 (3.4 g, 6.05 mmol), and then warmed to room temperature and reacted 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 obtain 5d (3.15 g); LC-MS: [MH] - =816.3.

[0137] Step 5: Compound 5e In a 100 mL one-neck flask, 5d (2.07 g, 2.53 mmol, 1.0 eq.), M5 (1.35 g, 2.53 mmol, 1.0 eq.), PyBOP (1.98 g, 3.79 mmol, 1.5 eq.), HOBt (0.51 g, 3.79 mmol, 1.5 eq.) and DMF (40 mL) were added, and DIPEA (1.05 mL, 1.5 eq.) was added in an ice-water bath, warmed to room temperature, and reacted for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative LC, and the fraction was concentrated by water pump at 35 °C under reduced pressure in a water bath to remove acetonitrile and freeze-dried 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 eq.) and 35 mL of nitromethane were added to a 100 mL one-neck flask, dissolved, and then zinc bromide (3.64 g, 16 mmol, 20.0 eq.) was added, and the mixture was reacted for 30 minutes in a 40° C. oil bath (stabilized by preheating in advance), and concentrated under reduced pressure at 45° C. in a water bath by a water pump to remove nitromethane, and a yellow residual solid was formed (monitored by HPLC). After acid preparation, a fraction of compound 5A was obtained, concentrated under reduced pressure at 35° C. in a water bath by a water pump, acetonitrile was removed by rotation, and lyophilized to obtain compound 5A (786 mg) in a yield of 90%. 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) are added to a 25 mL one-neck flask, the temperature of the ice-water bath is lowered to 0-5 °C, and DIPEA (62 mg, 0.48 mmol, 2.0 equiv) is added. After the addition is complete, the temperature is raised to 20 ± 5 °C, and the reaction is allowed to proceed for 2 h. HPLC is used to monitor the completion of the reaction. The reaction solution is subjected to preparative purification by HPLC, and the product aliquots are 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 eq.), ZnBr2 (400 mg, 1.78 mmol, 20.0 eq.) and CH3NO2 (10 mL) are added successively to a 25 mL single-neck flask. After addition, the temperature is raised to 40° C., and the reaction is allowed to proceed for 0.5 h, and then the reaction is stopped. The reaction solution is directly dried at 45° C. by rotary evaporation under reduced pressure to obtain a yellow solid. A sample is taken and the reaction is monitored by HPLC. The rotary dried solid is directly purified by HPLC preparative, and the product fraction is collected and freeze-dried to obtain compound 5B (73.4 mg) in 76.5% yield; LC-MS: [M+H] + =1079.4.

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

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

[0144] Example 12 Preparation of Compounds 7A and 7B: [ka] Step 1: Compound 7a In a 250 mL one-neck flask, M1 (10 g, 27.1 mmol), benzyl 3,3,3-trifluorolactate (prepared according to the method disclosed in WO2020063673A1) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added, 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 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 5.15 g of the desired material in 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 one-neck flask and dissolve until clear, then add DBU (1.68 g, 11 mmol) in an ice-water bath and react for 1 h, and record the reaction solution as reaction solution (1).

[0146] In another 50 mL one-neck flask, add M4 (3.8 g, 9.2 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol) and 10 mL of DMF. When dissolved, add DIPEA (1.82 mL, 11 mmol) in ice-water bath and continue the reaction for 30 minutes, then add reaction solution (1), warm to room temperature and react for 2 hours. The progress of the reaction was monitored by HPLC. After the reaction was completed, 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 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 one-neck flask, dissolved in 15 mL of DMF, and then 900 mg of 5% Pd / C was added, and the hydrogenation reaction was carried out for 2 hours. After the reaction was completed, filtration was performed, the filtrate was placed in an ice-water bath, DIPEA (228 uL, 1.38 mmol) was added, and then M3 (712 mg, 1.26 mmol) was added, and then the temperature was raised to room temperature and reacted for 1 hour. When the reaction was completed as detected by HPLC, the reaction solution was purified by high performance liquid chromatography to obtain a fraction, which was freeze-dried to obtain 525 mg of product in 47.9% yield; LC-MS: [MH] - =870.33.

[0148] Step 4: Compound 7e In a 50mL one-neck flask, 7d (500mg, 0.57mmol), M5 (305mg, 0.57mmol), PyBOP (448mg, 0.86mmol), HOBt (116mg, 0.86mmol) and DMF 15mL were added, and DIPEA (378uL, 2.29mmol) was added to an ice-water bath, the room temperature was raised, and the reaction was carried out for 2 hours. After the completion of the reaction detected by HPLC, the reaction solution was purified by high performance liquid chromatography to obtain a fraction of compound 7e-1 and compound 7e-2, and the fraction was freeze-dried to obtain compound 7e-1 150mg; LC-MS: [M+H] + =1289.46, and compound 7e-2 220mg;LC-MS:[M+H] + =1289.46 respectively.

[0149] Step 5: Compound 7A [ka] In a 25 mL single-neck flask, 7e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were 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, and the fraction was freeze-dried to obtain 52 mg of solid; TOF result: 1133.3613.

[0150] Step 6: Compound 7B [ka] In a 25 mL single-neck flask, 7e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were 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, and the fraction was freeze-dried to obtain 63 mg of solid; TOF result: 1133.3668.

[0151] Example 13 Synthesis of compounds 8A and 8B: [ka] Step 1: Compound 8d In a 25mL one-neck flask, 7c (900mg, 1.83mmol) was added and dissolved using 20mL of DMF, then DIPEA (303uL, 1.83mmol) was added, ent-M3 (1034mg, 1.83mmol) was added, the temperature was raised to room temperature, and reacted for 1 hour. After the completion of the reaction detected by HPLC, the reaction solution was purified by high performance liquid chromatography to obtain a fraction, which was freeze-dried to obtain 613mg of product with a yield of 38.5%; LC-MS: [MH] - =870.32.

[0152] Step 2: Compound 8e-1 and Compound 8e-2 In a 50mL one-neck flask, 8d (500mg, 0.57mmol), M5 (305mg, 0.57mmol), PyBOP (448mg, 0.86mmol), HOBt (116mg, 0.86mmol) and 15mL of DMF were added, and DIPEA (378uL, 2.29mmol) was added in an ice-water bath, and the mixture was heated to room temperature and reacted for 2 hours. After the completion of the reaction detected by HPLC, the reaction solution was purified by high performance liquid chromatography to obtain fractions of compound 8e-1 and compound 8e-2, and the fractions were freeze-dried to obtain 140mg of compound 8e-1 and 210mg 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 nitromethane (5 mL) 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 freeze-dried to obtain 50 mg of 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 nitromethane (5 mL) 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 freeze-dried to obtain 58 mg of solid; TOF result: 1133.3653.

[0155] Example 14 Synthesis of compound 9A: [ka] Step 1: Compound 9a 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, 2-hydroxy-2-cyclopropyl benzyl acetate (prepared according to the method published in US Patent Publication No. 20050020645A1) (6.3 g, 32.6 mmol) was added dropwise, allowed to warm to room temperature naturally, and reacted while monitoring by TLC (the reaction lasts about 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 with anhydrous sodium sulfate, filtering, concentrating, and purifying the residue 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 one-neck flask, 9a (2 g, 4.0 mmol) and 10 mL of DMF were added and stirred at 0° C., and DBU (760 mg, 5.0 mmol) was added and reacted for 1 h. After completion of Fmoc deprotection monitored by TLC, the reaction solution was set aside for ready use.

[0157] In another 25mL one-neck flask, M4 (1.65g, 4.0mmol), PyBOP (2.59g, 5.0mmol), HOBt (675mg, 5.0mmol) and DMF 10mL were added, DIPEA (823uL, 5.04mmol) was added in 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 to react. 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 a solid 1.5g 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 one-neck flask and dissolved in 10 mL of DMF, and then 900 mg of 5% Pd / C was added and the hydrogenation reaction was carried out for 1.5 h. 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 Crude product 9c was placed in an ice-water bath, DIPEA (223uL, 1.3mmol) was added, then compound M3 (750mg, 1.3mmol) was added, and then the temperature was raised to room temperature and reacted for 1 hour. After the completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to obtain 9d (529mg); LC-MS: [MH] - =828.4.

[0160] Step 5: Compound 9e In a 50mL one-neck flask, 9d (500mg, 0.6mmol), M5 (300mg, 0.6mmol), PyBOP (416mg, 0.8mmol), HOBt (108mg, 0.5mmol) and 15mL of DMF were added, and DIPEA (351uL, 2.13mmol) in an ice-water bath was added, and the mixture was warmed to room temperature and reacted for 3 hours. After the completion of the reaction detected by HPLC, the reaction solution was purified by high performance liquid chromatography to obtain a preparation of compound 9e, which was freeze-dried to obtain 9e (257mg); LC-MS: [M+H] + =1247.5.

[0161] Step 6: Compound 9A In a 25 mL one-neck flask, 9e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and nitromethane 5 mL were added, and the reaction was carried out at 40° C. for 1 h. After the completion of the reaction 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, and the fraction was freeze-dried 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 referring to the synthetic route of Example 14; LC-MS: [M+H] + =1091.3.

[0163] Example 16 Synthesis of compound 10A: [ka] Step 1: Compound 10a 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, cooled to 0 °C, 3-hydroxy-2-cyclopropylpropionate benzyl (prepared with reference to the method disclosed in WO2013187496A1) (6.7 g, 32.6 mmol) was added dropwise, and then allowed to warm to room temperature and react while monitoring by TLC (about 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 with anhydrous sodium sulfate, filtering, concentrating, and purifying the residue by silica gel column (PE:EA=10:1-5:1-2:1) to obtain 10a (4.9 g) in 58% yield; LC-MS: [M+H] + =515.4.

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

[0165] M4 (3.3g, 8.0mmol), PyBOP (5.2g, 10.0mmol), HOBt (1.35g, 10.0mmol) and DMF 10mL were added to another 25mL one-neck flask, DIPEA (1.65mL, 10.1mmol) was added in 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 a solid 2.3g 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 one-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 h. After the reaction was completed, filtration was carried out to obtain the filtrate, which was directly used 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, DIPEA (258 uL, 1.5 mmol) was added, then compound M3 (837 mg, 1.45 mmol) was added, and the temperature was then raised to room temperature and reacted 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 obtain 10d (499 mg); LC-MS: [MH] - =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, DIPEA (330 uL, 2.0 mmol) was added in an ice-water bath, and the temperature was raised to room temperature for 3 hours. After completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 10e, which was lyophilized to obtain 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 freeze-dried 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 referring to the synthetic route of Example 16; LC-MS: [M+H] + =1105.4.

[0171] Example 18 Synthesis of compound 11A: [ka] Step 1: Compound 11a 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, cooled to 0°C, and 2-hydroxy-2-cyclobutyl benzyl acetate (synthesized by the method published in the literature Journal of Medicinal Chemistry, 2013, 56(13), 5541-5552) (6.7 g, 32.6 mmol) was added dropwise, then allowed to warm to room temperature and react while monitoring with TLC (reaction time of about 2 to 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 purifying the residue 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 one-neck flask, 11a (4 g, 7.8 mmol) and 10 mL of DMF were added and stirred at 0° C., and DBU (1.2 g, 8.0 mmol) was added and reacted for 1 h. After completion of Fmoc deprotection monitored by TLC, the reaction solution was set aside for ready use.

[0173] M4 (3.3g, 8.0mmol), PyBOP (5.2g, 10.0mmol), HOBt (1.35g, 10.0mmol) and DMF 10mL were added to another 25mL one-neck flask, DIPEA (1.63mL, 10.0mmol) was added in 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 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 a solid 2.3g 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 one-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, DIPEA (516uL, 3.0mmol) was added, then compound M3 (1.7g, 2.9mmol) was added, and then the temperature was raised to room temperature and reacted for 2 hours. After the completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was freeze-dried to obtain 11d (934mg); LC-MS: [MH] - =842.4.

[0176] Step 5: Compound 11e In a 50mL one-neck flask, 11d (800mg, 0.96mmol), M5 (480mg, 0.96mmol), PyBOP (500mg, 0.96mmol), HOBt (208mg, 0.96mmol) and 30mL of DMF were added, and DIPEA (660uL, 4.0mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted for 4 hours. After the completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 11e, which was freeze-dried to obtain 11e (401mg); 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 freeze-dried 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 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, cooled to 0 °C, and 3-hydroxy-2-cyclobutylpropionate benzyl (prepared according to the method published in WO2009011285A1) (7.2 g, 32.6 mmol) was added dropwise. Then, it was allowed to warm to room temperature and reacted while monitoring by TLC (about 2-4 hours of reaction). At the end of the reaction, saturated NaHCO3 solution was added, followed by extraction with ethyl acetate, washing with saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, concentrating, and purifying the residue by silica gel column (PE:EA=10:1-5:1-2:1) to give 12a (4.5 g) in 52% yield; LC-MS: [M+H] + =529.4.

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

[0181] M4 (3.2g, 7.6mmol), PyBOP (4.7g, 9.0mmol), HOBt (1.22g, 9.0mmol) and 10mL DMF were added to another 25mL one-neck flask, DIPEA (1.49mL, 0.9mmol) was added in 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 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 a solid 2.0g 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 one-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 h. 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, DIPEA (258uL, 1.5mmol) was added, then compound M3 (825mg, 1.43mmol) was added, and then the temperature was raised to room temperature and reacted for 1 hour. After the completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was freeze-dried to obtain 12d (522mg); LC-MS: [MH] - =856.4.

[0184] Step 5: Compound 12e In a 50mL one-neck flask, 12d (400mg, 0.47mmol), M5 (240mg, 0.47mmol), PyBOP (250mg, 0.47mmol), HOBt (101mg, 0.47mmol) and 15mL of DMF were added, and DIPEA (330uL, 2.0mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted for 3 hours. After the completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 12e, which was freeze-dried to obtain 12e (198mg); 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 freeze-dried 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 referring to the synthetic 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 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, cooled to 0°C, and 2-hydroxy-2-cyclopentyl benzyl acetate (synthesized by the method published in the literature Journal of Medicinal Chemistry, 2013, 56(13), 5541-5552) (7.2 g, 32.6 mmol) was added dropwise, and then allowed to warm naturally to room temperature and react while monitoring with TLC (reaction time of about 2 to 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 purifying the residue 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 one-neck flask, 13a (4 g, 7.6 mmol) and 10 mL of DMF were added and stirred at 0° C., and DBU (1.17 g, 7.8 mmol) was added and reacted for 1 h. After completion of Fmoc deprotection monitored by TLC, the reaction solution was set aside for ready use.

[0189] M4 (3.14g, 7.6mmol), PyBOP (4.42g, 8.5mmol), HOBt (1.15g, 8.5mmol) and 10mL DMF were added to another 25mL one-neck flask, DIPEA (1.39mL, 0.85mmol) was added in 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 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 a solid 2.1g 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 one-neck flask and dissolved in 25 mL of DMF, and then 1.5 g of 5% Pd / C was added and the hydrogenation reaction was carried out for 3 hours. After the reaction was completed, filtration was carried out to obtain the filtrate, which was directly used in the next step of the reaction without purification.

[0191] Step 4: Compound 13d The crude product 13c was placed in an ice-water bath and DIPEA (333 uL, 1.93 mmol) was added, the temperature was raised to room temperature and reacted for 1 h, then 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 obtain 13d (519 mg); LC-MS: [MH] - =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 uL, 2.0 mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted for 4 hours. After the reaction was completed as detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 13e, which was lyophilized to obtain 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 obtain the crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction, which was freeze-dried to obtain 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 referring to the synthetic 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, cooled to 0 °C, 3-hydroxy-2-cyclopentylpropionate benzyl (synthesized by the method disclosed in WO2009011285A1) (7.6 g, 32.6 mmol) was added dropwise, and then allowed to warm to room temperature naturally and react while monitoring by TLC (about 2-4 hours of reaction). At the end of the reaction, saturated NaHCO3 solution was added, followed by extraction with ethyl acetate, washing with saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, concentrating, and purifying the residue by silica gel column (PE:EA=10:1-5:1-2:1) to give 14a (4.4 g) in 49% yield; LC-MS: [M+H] + =543.6.

[0196] Step 2: Compound 14b In a 25 mL one-neck flask, 14a (4 g, 7.4 mmol) and 10 mL of DMF were added and stirred at 0° C., and DBU (1.2 g, 8.0 mmol) was added and reacted for 1 h. After completion of Fmoc deprotection 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 another 25 mL one-neck flask, DIPEA (1.49 mL, 9.0 mmol) was added in 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 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 2.6 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 one-neck flask and dissolved in 15 mL of DMF, and then 1.0 g of 5% Pd / C was added and the hydrogenation reaction was carried out for 1.5 hours. After the reaction was completed, filtration was carried out to obtain the filtrate, which was directly used in the next step of the reaction without purification.

[0199] Step 4: Compound 14d The crude product 14c was placed in an ice-water bath, DIPEA (248 uL, 1.5 mmol) was added, then compound M3 (808 mg, 1.4 mmol) was added, and the temperature was then raised to room temperature and reacted 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 obtain 14d (500 mg); LC-MS: [MH] - =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 uL, 2.0 mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted for 3 hours. After completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 14e, which was freeze-dried to obtain 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 freeze-dried 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 referring to the synthetic route of Example 24; LC-MS: [M+H] + =1133.4.

[0203] Example 26 Synthesis of compounds 15A and 15B: [ka] Step 1: Compound 15a In a 250 mL one-neck flask, M1 (10 g, 27.1 mmol), 2-hydroxy-benzyl butyrate (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 were added and heated to 100 °C for 4 hours. After the reaction was completed, the temperature was lowered to room temperature, filtration was performed 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 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, and after dissolving, 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 one-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 after dissolving, DIPEA (1.82 mL, 11 mmol) was added in an ice-water bath, and the reaction was continued for 40 minutes, and then reaction solution (1) was added, and the temperature was raised to room temperature and reacted for 2 hours. The progress of the reaction was monitored by HPLC. After the reaction was completed, 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 solid in 68% yield; LC-MS: [M+H] + =676.7.

[0206] Step 3: Compound 15d 15b (2.0g, 2.96mmol) was added to a 25mL single-neck flask and dissolved in 15mL of DMF, then 2.0g of 5% Pd / C was added, and the hydrogenation reaction was carried out for 2 hours. After the reaction was completed, filtration was carried out, the filtrate was placed in an ice-water bath, DIPEA (496uL, 3.0mmol) was added, followed by M3 (1.7g, 2.96mmol), and then the temperature was raised to room temperature and reacted for 1 hour. Once the reaction was completed, as detected by HPLC, the reaction solution was purified by high performance liquid chromatography to obtain a fraction, which was freeze-dried to obtain 1120.0mg of product in 45% yield; LC-MS: [MH] - =830.3.

[0207] Step 4: Compound 15e In a 50mL one-neck flask, 15d (500mg, 0.60mmol), M5 (321mg, 0.60mmol), PyBOP (469mg, 0.90mmol), HOBt (121mg, 0.90mmol) and DMF 15mL were added, DIPEA (446uL, 2.7mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted 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 15e-1 and compound 15e-2, which was freeze-dried to give compound 15e-1 138mg; 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 one-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 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 freeze-dried to obtain 59 mg of solid; LC-MS: [M+H] + =1093.4.

[0209] Step 6: Compound 15B [ka] In a 25 mL one-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 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 freeze-dried to obtain 60 mg of solid; LC-MS: [M+H] + =1093.4.

[0210] Example 27 Synthesis of compounds 16A and 16B: [ka] Compound 16A (55 mg) was obtained by referring to the synthetic route of Example 26; LC-MS: [M+H] + =1093.4. [ka]

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

[0212] Example 28 Synthesis of compounds 17A and 17B: [ka] Step 1: Compound 17a In a 250 mL one-neck flask, M1 (10 g, 27.1 mmol), benzyl 2-hydroxy-phenylpropionate (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 were added, 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 yield of 40%; LC-MS: [M + H] + =565.6.

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

[0214] In another 50 mL one-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 after dissolving, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath, and the reaction was continued for 30 minutes, and then reaction solution (1) was added, and the temperature was raised to room temperature and reacted for 2 hours. The progress of the reaction was monitored by HPLC. After the reaction was completed, 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 solid in 77% yield; LC-MS: [M+H] + =738.3.

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

[0216] Step 4: Compound 17e In a 50mL one-neck flask, 17d (500mg, 0.56mmol), M5 (300mg, 0.56mmol), PyBOP (438mg, 0.84mmol), HOBt (113mg, 0.84mmol) and 15mL of DMF were added, and DIPEA (330uL, 2.0mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted 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 17e-1 and compound 17e-2, which was freeze-dried to give compound 17e-1 156mg; LC-MS: [M+H] +=1311.4 and compound 17e-2 150mg;,LC-MS:[M+H] + =1311.7, respectively.

[0217] Step 5: Compound 17A [ka] In a 25 mL one-neck flask, 17e-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 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 freeze-dried to obtain 43 mg of solid; LC-MS: [M+H] + =1155.4.

[0218] Step 6: Compound 17B [ka] In a 25 mL one-neck flask, 17e-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 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 freeze-dried to obtain 40 mg of solid; LC-MS: [M+H] + =1155.4.

[0219] Example 29 Synthesis of compounds 18A and 18B: [ka] Compound 18A (54 mg) was obtained by referring to 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 WO2020244657A1) (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. After the reaction was completed, the temperature was lowered to room temperature, filtration was performed 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 4.97 g of the desired material in 36% yield; LC-MS: [M + H] + =515.2.

[0222] Step 2: Compound 19b In a 50 mL one-neck flask, 19a (4 g, 7.8 mmol) and 10 mL of DMF were added, and after dissolution, DBU (1.42 g, 9.3 mmol) was then added in an ice-water bath and reacted for 1 h. The reaction solution was recorded as reaction solution (1).

[0223] In another 50 mL one-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 after dissolving, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath, and the reaction was continued for 30 minutes, and then reaction solution (1) was added, and the reaction was allowed to warm to room temperature and react for 2 hours. The progress of the reaction was monitored by HPLC, and after the reaction was completed, 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 solid in 78% yield; LC-MS: [M+H] + =688.3.

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

[0225] Step 4: Compounds 19e-1 and 19e-2 In a 50mL single-neck flask, 19d (500mg, 0.59mmol), M5 (317mg, 0.59mmol), PyBOP (339mg, 0.65mmol), HOBt (88mg, 0.86mmol) and 10mL of DMF were added, and (292uL, 1.77mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted 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 19e-1 and compound 19e-2, which was freeze-dried to obtain compound 19e-1 112mg; 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] In a 25 mL one-neck flask, 19e-1 (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 high performance liquid chromatography to obtain a product fraction, which was freeze-dried to obtain 55 mg of solid; LC-MS: [M+H] + =1105.4.

[0227] Step 6: Compound 19B [ka] In a 25 mL one-neck flask, 19e-2 (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 high performance liquid chromatography to obtain a product fraction, which was freeze-dried to obtain 58 mg of solid; LC-MS: [M+H] + =1105.4.

[0228] Example 31 Synthesis of compounds 20A and 20B: [ka] Step 1: Compound 20a In a 250 mL one-neck flask, M1 (10 g, 27.1 mmol), benzyl 2-hydroxy-cyclopropylpropionate (prepared according to the method published in WO2020063676A) (12.0 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added and heated to 100 °C and reacted for 4 h. After the reaction was completed, 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 In a 50 mL one-neck flask, 20a (4 g, 7.6 mmol) and 10 mL of DMF were added, and after dissolution, DBU (1.39 g, 9.1 mmol) was then added in an ice-water bath and reacted for 1 hour. The reaction solution was recorded as reaction solution (1).

[0230] In another 50mL one-neck flask, M4 (3.12g, 7.6mmol), PyBOP (4.5g, 8.6mmol), HOBt (1.16g, 8.6mmol) and 10mL of DMF were added, and after dissolving, DIPEA (1.65mL, 10mmol) was added in ice-water bath, and the reaction was continued for 30 minutes, and then reaction solution (1) was added, and the reaction was allowed to warm to room temperature and react for 2 hours. HPLC was carried out to monitor the progress of the reaction, and when the reaction was completed, 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 4.5g of solid in 84% yield; LC-MS: [M+H] + =702.3.

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

[0232] Step 4: Compounds 20e-1 and 20e-2 In a 50mL single-neck flask, 20d (400mg, 0.47mmol), exatecan mesylate (250mg, 0.47mmol), PyBOP (223mg, 0.56mmol), HOBt (83mg, 0.56mmol) and DMF 10mL were added, DIPEA (248uL, 1.5mmol) was added in ice-water bath, the temperature was raised to room temperature and reacted 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 20e-1 and compound 20e-2, which was lyophilized to obtain compound 20e-1 103mg; LC-MS: [M+H] + =1275.5 and compound 20e-2 103mg;LC-MS:[M+H] + =1275.5, respectively.

[0233] Step 5: Compound 20A [ka] In a 25 mL one-neck flask, 8A (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 high performance liquid chromatography to obtain a product fraction, which was freeze-dried to obtain 51 mg of solid; LC-MS: [M+H] + =1119.4.

[0234] Step 6: Compound 20B [ka] In a 25 mL one-neck flask, 20e-2 (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 high performance liquid chromatography to obtain a product fraction, which was freeze-dried to obtain 47 mg of solid; LC-MS: [M+H] + =1119.4.

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

[0236] Step 2: Compound (R)-tert-butyl 2-hydroxy-1,5-glutarate In a 2000mL single-neck flask, 172793-31-6 (100g, 338mmol) and 1000mL of water were added, then sodium nitrite (35g, 507mmol) and concentrated sulfuric acid (32mL, 35mmol) were added successively, the temperature was slowly raised to room temperature, and the reaction was carried out for 24 hours. After the reaction was completed, extraction was carried out three times with 500mL of ethyl acetate, the organic phase was dried with 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 91.2g of the desired material; LC-MS: [M+H] + =261.4.

[0237] Step 3: Compound SM3 In a 2000mL one-neck flask, (R)-tert-butyl 2-hydroxy-1,5-glutarate (50g, 192mmol) and 1000mL of anhydrous tetrahydrofuran were added, and the temperature was cooled to 0°C in an ice-water bath, and then PPh3 (87.7g, 288mmol), DEAD (50.2g, 288mmol) and SM3-1 (57.3, 192mmol) were added in order. The temperature was slowly raised to room temperature and reacted 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 68.6g of product.

[0238] The above product was dissolved in 500mL of ethanol, cooled to 0°C in an ice-water bath, NaOH (64mL, 190mmol, 3M / L) was added dropwise at this temperature, and the reaction was carried out for 12 hours while maintaining this temperature. Then, the pH was adjusted to 3 by adding HCl (6M / L), and extracted five times with 500mL of dichloromethane, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained crude product was purified by column chromatography (DCM / MeOH=50 / 1-20 / 1-2 / 1) to obtain 50.4g of SM3; LC-MS: [MH] - =525.5.

[0239] Step 4: Compound M6 Compound SM3 (50 g, 95 mmol, 1.0 eq.), pentafluorophenol (19.2 g, 104.5 mmol, 1.1 eq.), DCC (21.5 g, 104.5 mmol, 1.1 eq.) and THF (600 mL) were added to a 2000 mL single-neck flask, the reaction was carried out at room temperature for 1 h (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly 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 freeze-dried to obtain compound M6 (51.9 g) in 79% yield; LC-MS: [M+H] + =693.3.

[0240] Step 5: Compound 21a In a 25mL one-neck flask, 1c (1g, 2.36mmol) was added and dissolved in 25mL of DMF, then DIPEA (430uL, 2.6mmol) was added, then M6 (1177mg, 2.36mmol) was added, then the temperature was raised to room temperature and reacted 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 freeze-dried to obtain 555mg of product; LC-MS: [MH] - =931.0.

[0241] Step 6: Compound 21b In a 100mL one-neck flask, 21a (500mg, 0.54mmol), exatecan mesylate M5 (285mg, 0.54mmol), PyBOP (239mg, 0.6mmol), HOBt (239mg, 0.6mmol) and 10mL of DMF were added, DIPEA (248uL, 1.5mmol) 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 compound 231mg; 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 concentrating 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 freeze-dried to obtain 103 mg of 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 100mg; 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 In a 5000 mL one-neck flask, maleic acid (50 g, 431 mmol, 1.0 eq.), 114559-25-0 (110 g, 431 mmol, 1 eq.), TEA (263 g, 2.16 mol, 5 eq.) and toluene (2000 mL) were added, heated and reacted under reflux for 5 h (monitored by TLC), and insoluble materials were filtered off. The reaction solution was directly subjected to rotary evaporation under reduced pressure to remove the solvent, and the residue was subjected to silica gel column chromatography (PE / EA=50 / 1-20 / 1-1 / 1) to obtain SM4-1 (64.7 g) in 50% yield; LC-MS: [M+H] + =299.2.

[0248] Step 2: Compound SM4-2 In a 2000mL one-neck flask, SM4-1 (64g, 215mmol) was added, dissolved in 1000mL of DMF, then DIPEA (71mL, 430mmol) was added, then nonaethylene glycol monomethyl ether methanesulfonate (111.5g, 220mmol) was added, then the temperature was raised to room temperature and reacted 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 59.9g 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, dissolved in 1000 mL of MeOH, and then K2CO3 (11.75 g, 85 mmol) was added, and the reaction was carried out at room temperature for 4 hours. When the reaction was completed as detected by HPLC, the insoluble material was removed by filtration, and the reaction solution was directly purified by preparative LC, and the preparative solution was concentrated under reduced pressure in a water bath at 35°C with a water pump to remove acetonitrile, and then freeze-dried to obtain compound SM4 (27 g); LC-MS: [MH] - =693.5.

[0250] Step 4: Compound M7 Compound SM4 (25 g, 36 mmol, 1.0 eq.), pentafluorophenol (7.3 g, 40 mmol, 1.1 eq.), DCC (8.2 g, 40 mmol, 1.1 eq.) and THF (200 mL) were added to a 500 mL single-neck flask, the reaction was carried out at room temperature for 1 h (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly 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 freeze-dried to give compound M7 (23.3 g) in 93% yield; LC-MS: [M+H] + =695.8.

[0251] Step 5: Compound 29a 1c (1g, 2.36mmol) was added to a 25mL one-neck flask and dissolved in 25mL of DMF, then DIPEA (430uL, 2.6mmol) was added, then M7 (1640mg, 2.36mmol) was added, then the temperature was raised to room temperature and reacted 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 freeze-dried to obtain 609mg of product; LC-MS: [MH] - =1098.5.

[0252] Step 6: Compound 29b In a 100mL single-neck flask, 29a (500mg, 0.45mmol), exatecan mesylate M5 (240mg, 0.45mmol), PyBOP (215mg, 0.54mmol), HOBt (215mg, 0.54mmol) and 10mL of DMF were added, DIPEA (248uL, 1.5mmol) was added in an ice-water bath, the temperature was raised to room temperature and reacted 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 29b, which was freeze-dried to obtain 187mg of compound; 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 nitromethane (10 mL) were added to a 25 mL single-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 freeze-dried to obtain 114 mg of 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 synthesis 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 synthesis 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 synthesis route of Example 37, compound 33 60mg; 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 synthesis 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 eq.), 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 h (monitored by TLC), and the insoluble material was filtered off. The solvent was removed directly from the reaction solution by rotary evaporation under reduced pressure, and the residue was subjected to silica gel column chromatography (PE / EA=50 / 1-20 / 1-2 / 1) to give 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 one-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, and extraction was performed three times with 500 mL of ethyl acetate, followed by drying the organic phase with anhydrous sodium sulfate and filtering. The solvent was removed directly 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, palladium(II) acetate (180 mg, 0.8 mmol), I2 (20 g, 79 mmol) and iodobenzene diacetate (40.8 g, 126.4 mmol) were added successively, and the temperature was raised to 60 °C for 40 h reaction (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, followed by drying the organic phase with anhydrous sodium sulfate and filtering. The solvent was removed directly 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 eq.), di-tert-butyl potassium phosphate (13.66 g, 55 mmol, 1.1 eq.), p-toluenesulfonic acid monohydrate (951 mg, 5 mmol, 0.1 eq.) and THF (200 mL) were added to a 500 mL single-neck flask, the reaction was carried out at room temperature for 1 h (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly 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 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 one-neck flask, and after dissolving, 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, and the crude product was set aside for ready use.

[0263] In another 250 mL one-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, and the temperature was raised to 100° C. for 2 hours. The progress of the reaction was monitored using HPLC, and when the reaction was completed, 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 solid in 36% yield; LC-MS: [M+H] + =507.3.

[0264] Step 6: Compound M8 Compound SM7 (4 g, 7.9 mmol, 1.0 eq.), pentafluorophenol (1.6 g, 8.7 mmol, 1.1 eq.), DCC (1.8 g, 8.7 mmol, 1.1 eq.) and THF (60 mL) were added to a 100 mL single-neck flask, and the reaction was carried out at room temperature for 1 h (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly 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 freeze-dried to give compound M8 (3.7 g) in 70% yield; LC-MS: [M+H] + =673.2.

[0265] Step 7: Compound 37a In a 25mL one-neck flask, 1c (1g, 2.36mmol) was added, dissolved in 25mL of DMF, then DIPEA (430uL, 2.6mmol) was added, then M8 (1.2g, 2.36mmol) was added, then the temperature was raised to room temperature and reacted 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 freeze-dried to obtain 488mg of product; LC-MS: [MH] - =911.0.

[0266] Step 8: Compound 37b 37a (400mg, 0.44mmol), exatecan mesylate M5 (235mg, 0.44mmol), PyBOP (199mg, 0.5mmol), HOBt (69mg, 0.5mmol) and 10mL DMF were added to a 100mL one-neck flask, DIPEA (218uL, 1.32mmol) was added in an ice-water bath, the temperature was 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 of compound 37b, which was lyophilized to obtain compound 201mg; 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 h. After the reaction was completed as detected by HPLC, the solvent was removed by concentrating 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 freeze-dried to obtain 96 mg of 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 synthesis 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, 62mg 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 "China 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, DIPEA (378 uL, 2.29 mmol) was added in an ice-water bath, the temperature was raised to room temperature and reacted for 2 h. 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 freeze-dried 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 concentrating 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 freeze-dried 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 synthesis 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 eq.), M9 (1.08 g, 2.02 mmol, 1.0 eq.), PyBOP (1.58 g, 3.03 mmol, 1.5 eq.), HOBt (0.41 g, 3.03 mmol, 1.5 eq.) and DMF (40 mL) were added to a 100 mL one-neck flask, DIPEA (0.84 mL, 1.5 eq.) 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, and the fraction 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 48a (1.54 g) in 61% yield; LC-MS: [M+H] + =1235.4.

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

[0278] Example 51 Synthesis of compound 48B: [ka] Step 1: Compound 48b In a 25 mL one-neck flask, compound 5d-1 (200 mg, 0.24 mmol, 1.0 equiv), M9 (127 mg, 0.24 mmol, 1.0 equiv), PyBOP (187 mg, 0.36 mmol, 1.2 equiv), HOBt (48 mg, 0.36 mmol, 1.2 equiv), and DMF (6 mL) were added. The temperature was lowered to 0-5°C in an ice-water bath, DIPEA (62 mg, 0.48 mmol, 2.0 equiv) was added, and then the temperature was raised to 20±5°C and reacted for 2 h. The completion of the reaction was detected by HPLC. The reaction solution was directly purified by HPLC fractionation, and the product fraction 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), ZnBr2 (364 mg, 1.62 mmol, 20.0 equiv) and CH3NO2 (10 mL) were added in order to a 25 mL one-neck flask, then the temperature was raised to 40 °C and reacted for 0.5 h. The reaction was stopped and the reaction solution was directly rotary dried at 45 °C under reduced pressure to obtain a yellow solid, and a sample was taken to monitor the reaction by HPLC. The rotary dried solid was directly purified by HPLC preparative, 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 procedure 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 procedure 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 In a 50mL one-neck flask, 7d (500mg, 0.57mmol), M9 (305mg, 0.57mmol), PyBOP (448mg, 0.86mmol), HOBt (116mg, 0.86mmol) and 15mL of DMF were added, and DIPEA (378uL, 2.29mmol) was added in an ice-water bath, and the mixture was allowed to react for 2 hours after warming to room temperature. 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, which were each freeze-dried to give compound 50a 170mg; LC-MS: [M+H] + =1289.46 and compound 50b 202mg;LC-MS:[M+H] + =1289.46 was obtained.

[0283] Step 2: Compound 50A [ka] In a 25 mL single-neck flask, 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40° C. for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 44 mg of solid.

[0284] Step 3: Compound 50B [ka] In a 25 mL single-neck flask, 50b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40° C. for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 solid.

[0285] Example 55 Synthesis of compounds 51A and 51B: [ka] Step 1: Compound 51a and Compound 51b In a 50mL one-neck flask, 8d (500mg, 0.57mmol), M9 (305mg, 0.57mmol), PyBOP (448mg, 0.86mmol), HOBt (116mg, 0.86mmol) and 15mL of DMF were added, and DIPEA (378uL, 2.29mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted for 2 hours. After the completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of compound 51a and compound 51b, which were lyophilized to obtain 190mg of compound 51a and 186mg 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 nitromethane (5 mL) 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 concentrating 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 solid.

[0287] Step 3: Compound 51B [ka] Compound 51b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and nitromethane (5 mL) 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 concentrating 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 solid.

[0288] Example 56 Synthesis of compound 52A: [ka] Step 1: Compound 52a In a 50 mL one-neck flask, 11d (800 mg, 0.96 mmol), M9 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol) and 30 mL of DMF were added, and DIPEA (660 uL, 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 obtain 52a (388 mg); LC-MS: [M+H] + =1261.4.

[0289] Step 2: Compound 52A In a 25 mL one-neck flask, 52a (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol) and nitromethane (10 mL) were added and reacted at 40° C. for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 uL, 2.0 mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted for 3 hours. After completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 53a, which was lyophilized to obtain 53a (200 mg); LC-MS: [M+H] + =1275.4.

[0292] Step 2: Compound 53A In a 25 mL one-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 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 referring to 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 In a 50 mL single-neck flask, 19d (500 mg, 0.59 mmol), M9 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol) and 10 mL of DMF were added, and DIPEA (292 uL, 1.77 mmol) was added in an ice-water bath, and the mixture was 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 54a and 54b. The fractions were lyophilized separately 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 concentrating 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 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 concentrating 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 solid; LC-MS: [M+H] + =1105.4.

[0297] Example 61 Synthesis of compounds 55A and 55B: [ka] Step 1: Compounds 55a and 55b In a 50mL single-neck flask, 20d (400mg, 0.47mmol), M9 (250mg, 0.47mmol), PyBOP (223mg, 0.56mmol), HOBt (83mg, 0.56mmol) and DMF 10mL were added, DIPEA (248uL, 1.5mmol) was added in ice-water bath, and the mixture was 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 fractions of compounds 55a and 55b. The fractions were lyophilized separately to obtain compound 55a 100mg; 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 one-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 concentrating 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 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 concentrating 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 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 synthesis 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 the patent application "EP 0737683A1") was added with 400 mL of DMF, cooled to 0 ° C in an ice-water bath, triethylamine was added dropwise, and the pH was adjusted to 7-8. Benzyl bromide (9.6 g, 56 mmol) was added dropwise in an ice-water bath, the temperature was raised to room temperature (25 ° C), and the reaction was carried out for 1 hour, and the completion of the reaction was detected by TLC. The reaction solution was concentrated under reduced pressure, and the obtained crude product was purified by preparative high performance liquid chromatography (acetonitrile / purified water system), the target peak was collected, acetonitrile was removed under reduced pressure, and then lyophilization was carried out to obtain compound 58a, about 11 g of a yellow solid, with a yield of about 74%; MS m / z: [M + H] + 526.3.

[0303] Step 2: Synthesis of compound 58b At room temperature, compound 58a (11 g, 21 mol) and 120 mL of formic acid for dissolution were successively added to a 250 mL one-neck flask, and 30 mL of formaldehyde (40% aqueous solution) was added to the resulting light yellow solution, and the temperature was raised to 50° C. and reacted for 1 h. After the reaction was completed 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) to collect the target peak. The acetonitrile was removed under reduced pressure, followed by lyophilization to obtain compound 58b, about 4.5 g of yellow powdery solid, with a yield of about 40%; MS m / z: [M+H] + 540.6.

[0304] Step 3: Synthesis of compound 58: At room temperature, compound 58b (2.3 g, 4.3 mol) was added to a 250 mL one-neck flask, and 100 mL of DMF was added to dissolve it. 2.3 g of 5% Pd / C was added to the resulting bright yellow solution, and a hydrogen balloon was used to replace the atmosphere in the system, and the reaction was maintained at room temperature for 1.5 hours. Then, the completion of the reaction was detected by HPLC, 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, acetonitrile was removed under reduced pressure, and lyophilization was performed to obtain compound 58, about 1.0 g of a yellow powdery solid, with a yield of about 52%; MS m / z: [M+H] + 450.5.

[0305] Example 65 Synthesis of compound 59: [ka] Step 1: Compound 59a In a 50 mL one-neck flask, 1d (500 mg, 0.62 mmol), 58 (279 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, and DIPEA (378 uL, 2.29 mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted for 2 h. After completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to obtain 59a (166 mg); LC-MS: [M+H] + =1235.6.

[0306] Step 2: Compound 59 In a 25 mL one-neck flask, 59a (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol) and nitromethane (10 mL) were added and reacted at 40° C. for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 freeze-dried 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 referring to the synthetic route of Example 65; LC-MS: [M+H] + =1079.3.

[0308] Example 67 Synthesis of compound 61: [ka] Step 1: Compound 61a In a 100 mL one-neck flask, 5d (1.66 g, 2.02 mmol, 1.0 equiv), 58 (0.91 g, 2.02 mmol, 1.0 equiv), PyBOP (1.58 g, 3.03 mmol, 1.5 equiv), HOBt (0.41 g, 3.03 mmol, 1.5 equiv), and DMF (40 mL) were added, and DIPEA (0.84 mL, 1.5 equiv) was added in an ice-water bath, and the temperature was raised to room temperature and reacted 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 freeze-dried 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 eq.) and 35 mL of nitromethane were added to a 100 mL one-neck flask, and after dissolving, zinc bromide (3.64 g, 16 mmol, 20.0 eq.) was added, and the mixture was reacted in a 40° C. oil bath (preheated to stabilize) for 30 minutes, and concentrated at 45° C. in a water bath with a water pump under reduced pressure to remove nitromethane and obtain a yellow residue solid (monitored by HPLC). After acid preparation, a fraction was obtained. The fraction was concentrated at 35° C. in a water bath with a water pump under reduced pressure, acetonitrile was removed by rotation, and lyophilized to obtain 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 one-neck flask, 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 reacted for 2 h. HPLC was used to monitor the completion of the reaction. The reaction solution was directly purified by HPLC fractionation, and the product fraction 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.), ZnBr2 (364 mg, 1.62 mmol, 20.0 equiv.) and CH3NO2 (10 mL) were added successively to a 25 mL one-neck flask. After the addition was completed, the temperature was raised to 40° C. and reacted 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 the reaction was monitored by HPLC. The rotary evaporated solid was directly purified by HPLC preparative, 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 referring to 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 referring to 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 In a 50 mL one-neck flask, 7d (500 mg, 0.57 mmol), 58 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, and DIPEA (378 uL, 2.29 mmol) was added in an ice-water bath, and the temperature was raised to room temperature for 2 hours. After the completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of compounds 65a and 65b. The fractions were lyophilized separately 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 one-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 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a fraction, which was lyophilized to obtain 49 mg of solid.

[0316] Step 3: Compound 65B [ka] In a 25 mL one-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 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a fraction, which was lyophilized to obtain 47 mg of solid.

[0317] Example 72 Synthesis of compounds 66A and 66B: [ka] Step 1: Compound 66a and Compound 66b In a 50mL one-neck flask, 8d (500mg, 0.57mmol), 58 (256.8mg, 0.57mmol), PyBOP (448mg, 0.86mmol), HOBt (116mg, 0.86mmol) and 15mL of DMF were added, and DIPEA (378uL, 2.29mmol) was added in an ice-water bath, and the mixture was allowed to react for 2 hours after warming to room temperature. After the reaction was completed as detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of compound 66a and compound 66b, which were separately freeze-dried to obtain 160mg of compound 66a and 160mg 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 one-neck flask and reacted at 40° C. for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 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 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 solid: [M+H] + =1147.5.

[0320] Example 73 Synthesis of compound 67A: [ka] Step 1: Compound 67a In a 50 mL one-neck flask, 11d (800 mg, 0.96 mmol), 58 (432.5 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol) and 30 mL of DMF were added, and DIPEA (660 uL, 4.0 mmol) was added in an ice-water bath, and the mixture was allowed to react for 4 hours at room temperature. After completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction of compound 67a, which was lyophilized to obtain 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 nitromethane (10 mL) were added and reacted at 40° C. for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 synthesis route of Example 73. LC-MS: [M+H] + 1119.4.

[0323] Example 75 Synthesis of compound 68A: [ka] Step 1: Compound 68a In a 50 mL one-neck flask, 12d (400 mg, 0.47 mmol), 58 (211.7 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol) and 15 mL of DMF were added, and DIPEA (330 uL, 2.0 mmol) was added in an ice-water bath, and the mixture was allowed to react for 3 hours at room temperature. After completion of the reaction 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 one-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 concentrating 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 referring to 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 In a 50 mL one-neck flask, 19d (500 mg, 0.59 mmol), 58 (266 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol) and 10 mL of DMF were added, and DIPEA (292 uL, 1.77 mmol) was added in an ice-water bath, and the mixture was allowed to react for 2 hours at room temperature. After the reaction was completed 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.

[0327] Step 2: Compound 69A [ka] In a 25 mL one-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 concentrating 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 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 concentrating 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 solid; LC-MS: [M+H] + =1119.4.

[0329] Example 78 Synthesis of compounds 70A and 70B: [ka] Step 1: Compounds 70a and 70b In a 50 mL single-neck flask, 20d (400 mg, 0.47 mmol), 58 (211.7 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol) and 10 mL of DMF were added, and then (248 uL, 1.5 mmol) was added in an ice-water bath, and the mixture was allowed to react for 2 hours at room temperature. 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 compound 70a 106 mg; 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 one-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 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 39 mg of solid; LC-MS: [M+H] + =1133.4.

[0331] Step 3: Compound 70B [ka] In a 25 mL one-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 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 35 mg of solid; LC-MS: [M+H] + =1133.4.

[0332] Example 79 Synthesis of compound 71: [ka] Compound 71 (30 mg) was obtained by referring to 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 referring to 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 eq.), DIPEA (2.8 g, 21.4 mmol, 1.1 eq.), 27-amino-4,7,10,13,16,19,22,25-octaoxaheptacosanoic acid (9.7 g, 20.5 mmol, 1.05 eq.) 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, and the fraction was concentrated under reduced pressure in a water bath at 35° C. with a water pump to remove acetonitrile and freeze-dried to obtain compound M10 (13.2 g) in 78% yield; LC-MS: [M+H] + =866.5.

[0335] Compound M10 (13.0 g, 15 mmol, 1.0 eq.), pentafluorophenol (3 g, 16.5 mmol, 1.1 eq.), DCC (3.4 g, 16.5 mmol, 1.1 eq.) and THF (30 mL) were added to a 100 mL single-neck flask, reacted at room temperature for 30 min (monitored by TLC), and insoluble materials were filtered off. The reaction solution was directly 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 freeze-dried to obtain 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 M11 (1g, 0.79mol) was added with 10mL of DMF, cooled to 0°C in an ice-water bath, compound 1c (334mg, 0.79mol) and DIPEA (154mg, 1.19mol) were added, and the reaction was carried out for 1 hour while maintaining the 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 carried out to obtain about 1.2g of compound 73a; MS m / z: [M+H] + =1271.9.

[0337] Step 2: Synthesis of compound 73b In a 25 mL one-neck flask, 73a (1.2 g, 0.94 mmol), M5 (500 mg, 0.94 mmol), PyBOP (625 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol) and 15 mL of DMF were added, and DIPEA (310 mg, 2.4 mmol) was added in an ice-water bath, and the mixture was allowed to react for 2 h at room temperature. After completion of the reaction 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 nitromethane (10 mL) were added and reacted at 40° C. for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 referring to 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 M11 (1 g, 0.79 mol) was added with 10 mL of DMF, 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, and the reaction was carried out for 1 hour while maintaining the 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 carried out to obtain 0.9 g of compound 75a; MS m / z: [M+H] + =1285.6.

[0341] Step 2: Synthesis of compound 75b In a 25 mL one-neck flask, 75a (700 mg, 0.54 mmol), M5 (289 mg, 0.54 mmol), PyBOP (313 mg, 0.6 mmol), HOBt (81 mg, 0.6 mmol) and 10 mL of DMF were added, and DIPEA (155 mg, 1.2 mmol) was added in an ice-water bath, and the mixture was allowed to react for 2 h at room temperature. After completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain a fraction, which was lyophilized to obtain 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 nitromethane (10 mL) were added and reacted at 40° C. for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 referring to 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 synthesis route of Example 84; LC-MS: [M+H] + =1546.5.

[0345] Example 87 Synthesis of compound 78: [ka] Following the synthesis route of Example 84, compound 78 (94 mg) was obtained; LC-MS: [M+H] + =1546.7.

[0346] Example 88 Synthesis of compounds 79 and 80: [ka] Step 1: Synthesis of compound 79a M11 (1 g, 0.79 mol) was added with 10 mL of DMF, 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, and the reaction was carried out for 1 hour while maintaining the 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 carried out to obtain 783 mg of compound 79a; MS m / z: [M+H] + =1325.8.

[0347] Step 2: Synthesis of compounds 79b-1 and 79b-2 In a 25 mL one-neck flask, 79a (600 mg, 0.45 mmol), M5 (240 mg, 0.45 mmol), PyBOP (261 mg, 0.5 mmol), HOBt (68 mg, 0.5 mmol) and 10 mL of DMF were added, and DIPEA (130 mg, 1 mmol) was added in an ice-water bath, and the mixture was allowed to react for 2 hours at room temperature. After completion of the reaction detected by HPLC, the reaction solution was purified by HPLC to obtain fractions of compounds 79b-1 and 79b-2, which were freeze-dried 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 nitromethane (10 mL) were added and reacted at 40° C. for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 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 nitromethane (10 mL) were added and reacted at 40° C. for 1 h. After completion of the reaction as detected by HPLC, the solvent was removed by concentrating 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 referring to 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 referring to the synthetic route of Example 88; LC-MS: [M+H] + =1586.9.

[0352] Example 91 1) Expression and purification of SI-1×6.4 antibody: Expi293 (Shanghai OPM Biosciences Co., Ltd.) suspension cells were used to express the SI-1x6.4 antibody. The day before transfection, cells were cultured at a density of 0.9x10 in a 1L shake flask containing 300mL 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, with a mass ratio of plasmid:PEI-MAX of 1:3. On the first day after transfection, OPM-293 ProFeed supplement was added at 5% (v / v), then on the third day after transfection, it was added again at 5% (v / v), and on the sixth day after transfection, centrifugation was performed to collect the supernatant.

[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.), and the captured antibody was adjusted to pH 7.0 with 1 M Tris-HCl (pH 8.8) at 0.7 / 10 (v / v), and 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, from left to right, are CDR1 (SEQ ID NO:25), CDR2 (SEQ ID NO:26), and CDR3 (SEQ ID NO:27), respectively.

[0355] Nucleic acid coding sequences for constructs 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, from left to right, are 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, from left to right, are 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 the 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, from left to right, are 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, from left to right, are 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] where the underlined portions, from left to right, are 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, from left to right, are CDR1 (SEQ ID NO:25), CDR2 (SEQ ID NO:26), and CDR3 (SEQ ID NO:27), respectively.

[0368] Nucleic acid coding sequences for constructs 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 the single chain Fv (scFv) is: SEQ ID NO:39 [ka] and the underlined portions, from left to right, are 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, from left to right, are 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 cultured at a density of 0.9x10 in a 1L shake flask containing 300mL 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, with a mass ratio of plasmid:PEI-MAX of 1:3. On the first day after transfection, OPM-293 ProFeed supplement was added at 5% (v / v), then on the third day after transfection, it was added again at 5% (v / v), and on the sixth day after transfection, centrifugation was performed to collect the supernatant.

[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.), and the captured antibody was adjusted to pH 7.0 with 1 M Tris-HCl (pH 8.8) at 0.7 / 10 (v / v), and 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, from left to right, are CDR1 (SEQ ID NO:41), CDR2 (SEQ ID NO:42), and CDR3 (SEQ ID NO:43), respectively.

[0376] Nucleic acid coding sequences for constructs 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, from left to right, are CDR1 (SEQ ID NO:45), CDR2 (SEQ ID NO:46), and CDR3 (SEQ ID NO:47), respectively.

[0378] Variable regions of heavy chains in the structural domains of single chain Fv (scFv) SEQ ID NO:39 [ka] and the underlined portions, from left to right, are 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, from left to right, are 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: A similar method was 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 the 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, from left to right, are 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, from left to right, are 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, from left to right, are 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: A similar method was 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 the 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, from left to right, are 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, from left to right, are CDR1 (SEQ ID NO:32), CDR2 (SEQ ID NO:33), and CDR3 (SEQ ID NO:34), respectively.

[0391] The variable region of the light chain in the structural domain of the single chain Fv (scFv) is: SEQ ID NO:51 [ka] and the underlined portions, from left to right, are 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, from left to right, are CDR1 (SEQ ID NO:45), CDR2 (SEQ ID NO:46), and CDR3 (SEQ ID NO:47), respectively.

[0394] Example 93 1) A sample of SI-1x6.4 antibody drug conjugate was prepared by coupling SI-1x6.4 antibody with a payload: After cell expression and purification by protein A affinity chromatography and molecular sieve chromatography, SI-1x6.4 antibody was substituted in 20mM PB, pH6.5 buffer, and SI-1x6.4 antibody was concentrated or diluted to a protein concentration of 3mg / mL. The payload was a white powder, which was dissolved to 20mg / mL using DMA and set aside. To open the interchain disulfide bond of SI-1x6.4 antibody, 20x TECP was first added according to the molecular ratio and reacted at room temperature for 3 hours. Then, 20x payload solution was added according to the molecular ratio and reacted at room temperature for 1 hour. After the reaction was completed, the payload that was not coupled to SI-1x6.4 was removed by ultrafiltration using a 30KDa ultrafiltration centrifuge tube, and the SI-1x6.4 antibody drug conjugate sample was thus obtained.

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

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

[0397] Example 94 1) A sample of SI-1x4 antibody-drug conjugate was prepared by coupling SI-1x4 antibody with payload: After cell expression and purification by protein A affinity chromatography and molecular sieve chromatography, SI-1x4 antibody was substituted in 20mM PB, pH6.5 buffer, and SI-1x4 antibody was concentrated or diluted to a protein concentration of 3mg / mL. The payload was a white powder, which was dissolved to 20mg / mL using DMA and set aside. To open the interchain disulfide bond of SI-1x4 antibody, 20x TECP was first added according to the molecular ratio and reacted at room temperature for 3 hours. Then, 20x payload solution was added according to the molecular ratio and reacted at room temperature for 1 hour. When the reaction was completed, the payload that was not coupled to SI-1x4 was removed by ultrafiltration using a 30KDa ultrafiltration centrifuge tube, and a SI-1x4 antibody-drug conjugate sample was obtained.

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

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

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

[0401] Example 96 ADC-2 was prepared following the general coupling procedure in Example 93; [ka]

[0402] Example 97 ADC-3 was prepared following the general coupling procedure in Example 93; [ka]

[0403] Example 98 ADC-4 was prepared following the general coupling procedure in Example 93; [ka]

[0404] Example 99 ADC-5 was prepared following the general coupling procedure in Example 93; [ka]

[0405] Example 100 ADC-6 was prepared following the general coupling procedure in Example 93; [ka]

[0406] Example 101 ADC-7 was prepared following the general coupling procedure in Example 93; [ka]

[0407] Example 102 ADC-8 was prepared following the general coupling procedure in Example 93; [ka]

[0408] Example 103 ADC-9 was prepared following the general coupling procedure in Example 93; [ka]

[0409] Example 104 ADC-10 was prepared following the general coupling procedure in Example 93; [ka]

[0410] Example 105 ADC-11 was prepared following the general coupling procedure in Example 93; [ka]

[0411] Example 106 ADC-12 was prepared following the general coupling procedure in Example 93; [ka]

[0412] Example 107 ADC-13 was prepared following the general coupling procedure in Example 93; [ka]

[0413] Example 108 ADC-14 was prepared following the general coupling procedure in Example 93; [ka]

[0414] Example 109 ADC-15 was prepared following the general coupling procedure in Example 93; [ka]

[0415] Example 110 ADC-16 was prepared following the general coupling procedure in Example 93; [ka]

[0416] Example 111 ADC-17 was prepared following the general coupling procedure in Example 93; [ka]

[0417] Example 112 ADC-18 was prepared following the general coupling procedure in Example 93; [ka]

[0418] Example 113 ADC-19 was prepared following the general coupling procedure in Example 93; [ka]

[0419] Example 114 ADC-20 was prepared following the general coupling procedure in Example 93; [ka]

[0420] Example 115 ADC-21 was prepared following the general coupling procedure in Example 93; [ka]

[0421] Example 116 ADC-22 was prepared following the general coupling procedure in Example 93; [ka]

[0422] Example 117 ADC-23 was prepared following the general coupling procedure in Example 93; [ka]

[0423] Example 118 ADC-24 was prepared following the general coupling procedure in Example 93; [ka]

[0424] Example 119 ADC-25 was prepared following the general coupling procedure in Example 93; [ka]

[0425] Example 120 ADC-26 was prepared following the general coupling procedure in Example 93; [ka]

[0426] Example 121 ADC-27 was prepared following the general coupling procedure in Example 93; [ka]

[0427] Example 122 ADC-28 was prepared following the general coupling procedure in Example 93; [ka]

[0428] Example 123 ADC-29 was prepared following the general coupling procedure in Example 93; [ka]

[0429] Example 124 ADC-30 was prepared following the general coupling procedure in Example 93; [ka]

[0430] Example 125 ADC-31 was prepared following the general coupling procedure in Example 93; [ka]

[0431] Example 126 ADC-32 was prepared following the general coupling procedure in Example 93; [ka]

[0432] Example 127 ADC-33 was prepared following the general coupling procedure in Example 93; [ka]

[0433] Example 128 ADC-34 was prepared following the general coupling procedure in Example 93; [ka]

[0434] Example 129 ADC-35 was prepared following the general coupling procedure in Example 93; [ka]

[0435] Example 130 ADC-36 was prepared following the general coupling procedure in Example 93; [ka]

[0436] Example 131 ADC-37 was prepared following the general coupling procedure in Example 93; [ka]

[0437] Example 132 ADC-38 was prepared following the general coupling procedure in Example 93; [ka]

[0438] Example 133 ADC-39 was prepared following the general coupling procedure in Example 93; [ka]

[0439] Example 134 ADC-40 was prepared following the general coupling procedure in Example 93; [ka]

[0440] Example 135 ADC-41 was prepared following the general coupling procedure in Example 93; [ka]

[0441] Example 136 ADC-42 was prepared following the general coupling procedure in Example 93; [ka]

[0442] Example 137 ADC-43 was prepared following the general coupling procedure in Example 93; [ka]

[0443] Example 138 ADC-44 was prepared following the general coupling procedure in Example 93; [ka]

[0444] Example 139 ADC-45 was prepared following the general coupling procedure in Example 93; [ka]

[0445] Example 140 ADC-46 was prepared following the general coupling procedure in Example 93; [ka]

[0446] Example 141 ADC-47 was prepared following the general coupling procedure in Example 93; [ka]

[0447] Example 142 ADC-48 was prepared following the general coupling procedure in Example 93; [ka]

[0448] Example 143 ADC-49 was prepared following the general coupling procedure in Example 93; [ka]

[0449] Example 144 ADC-50 was prepared following the general coupling procedure in Example 93; [ka]

[0450] Example 145 ADC-51 was prepared following the general coupling procedure in Example 93; [ka]

[0451] Example 146 ADC-52 was prepared following the general coupling procedure in Example 93; [ka]

[0452] Example 147 ADC-53 was prepared following the general coupling procedure in Example 93; [ka]

[0453] Example 148 ADC-54 was prepared following the general coupling procedure in Example 93; [ka]

[0454] Example 149 ADC-55 was prepared following the general coupling procedure in Example 93; [ka]

[0455] Example 150 ADC-56 was prepared following the general coupling procedure in Example 93; [ka]

[0456] Example 151 ADC-57 was prepared following the general coupling procedure in Example 93; [ka]

[0457] Example 152 ADC-58 was prepared following the general coupling procedure in Example 93; [ka]

[0458] Example 153 ADC-59 was prepared following the general coupling procedure in Example 93; [ka]

[0459] Example 154 ADC-60 was prepared following the general coupling procedure in Example 93; [ka]

[0460] Example 155 ADC-61 was prepared following the general coupling procedure in Example 93; [ka]

[0461] Example 156 ADC-62 was prepared following the general coupling procedure in Example 93; [ka]

[0462] Example 157 ADC-63 was prepared following the general coupling procedure in Example 93; [ka]

[0463] Example 158 ADC-64 was prepared following the general coupling procedure in Example 93; [ka]

[0464] Example 159 ADC-65 was prepared following the general coupling procedure in Example 93; [ka]

[0465] Example 160 ADC-66 was prepared following the general coupling procedure in Example 93; [ka]

[0466] Example 161 ADC-67 was prepared following the general coupling procedure in Example 93; [ka]

[0467] Example 162 ADC-68 was prepared following the general coupling procedure in Example 93; [ka]

[0468] Example 163 ADC-69 was prepared following the general coupling procedure in Example 93; [ka]

[0469] Example 164 ADC-70 was prepared following the general coupling procedure in Example 93; [ka]

[0470] Example 165 ADC-71 was prepared following the general coupling procedure in Example 93; [ka]

[0471] Example 166 ADC-72 was prepared following the general coupling procedure in Example 93; [ka]

[0472] Example 167 ADC-73 was prepared following the general coupling procedure in Example 93; [ka]

[0473] Example 168 ADC-74 was prepared following the general coupling procedure in Example 93; [ka]

[0474] Example 169 ADC-75 was prepared following the general coupling procedure in Example 93; [ka]

[0475] Example 170 ADC-76 was prepared following the general coupling procedure in Example 93; [ka]

[0476] Example 171 ADC-77 was prepared following the general coupling procedure in Example 93; [ka]

[0477] Example 172 ADC-78 was prepared following the general coupling procedure in Example 93; [ka]

[0478] Example 173 ADC-79 was prepared following the general coupling procedure in Example 93; [ka]

[0479] Example 174 ADC-80 was prepared following the general coupling procedure in Example 93; [ka]

[0480] Example 175 ADC-81 was prepared following the general coupling procedure in Example 93; [ka]

[0481] Example 176 ADC-82 was prepared following the general coupling procedure in Example 93; [ka]

[0482] Example 177 ADC-83 was prepared following the general coupling procedure in Example 93; [ka]

[0483] Example 178 ADC-84 was prepared following the general coupling procedure in Example 93; [ka]

[0484] Example 179 ADC-85 was prepared following the general coupling procedure in Example 93; [ka]

[0485] Example 180 ADC-86 was prepared following the general coupling procedure in Example 93; [ka]

[0486] Example 181 ADC-87 was prepared following the general coupling procedure in Example 93; [ka]

[0487] Example 182 ADC-88 was prepared following the general coupling procedure in Example 93; [ka]

[0488] Example 183 ADC-89 was prepared following the general coupling procedure in Example 93; [ka]

[0489] Example 184 ADC-90 was prepared following the general coupling procedure in Example 93; [ka]

[0490] Example 185 ADC-91 was prepared following the general coupling procedure in Example 93; [ka]

[0491] Example 186 ADC-92 was prepared following the general coupling procedure in Example 93; [ka]

[0492] Example 187 ADC-93 was prepared following the general coupling procedure in Example 93; [ka]

[0493] Example 188 ADC-94 was prepared following the general coupling procedure in Example 93; [ka]

[0494] Example 189 ADC-95 was prepared following the general coupling procedure in Example 93; [ka]

[0495] Example 190 ADC-96 was prepared following the general coupling procedure in Example 93; [ka]

[0496] Example 191 ADC-97 was prepared following the general coupling procedure in Example 93; [ka]

[0497] Example 192 ADC-98 was prepared following the general coupling procedure in Example 93; [ka]

[0498] Example 193 ADC-99 was prepared following the general coupling procedure in Example 93; [ka]

[0499] Example 194 ADC-100 was prepared following the general coupling procedure in Example 93; [ka]

[0500] Example 195 ADC-101 was prepared following the general coupling procedure in Example 93; [ka]

[0501] Example 196 ADC-102 was prepared following the general coupling procedure in Example 93; [ka]

[0502] Example 197 ADC-103 was prepared following the general coupling procedure in Example 93; [ka]

[0503] Example 198 ADC-104 was prepared following the general coupling procedure in Example 93; [ka]

[0504] Example 199 ADC-105 was prepared following the general coupling procedure in Example 93; [ka]

[0505] Example 200 ADC-106 was prepared following the general coupling procedure in Example 93; [ka]

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

[0507] Example 202 ADC-107 was prepared following the general coupling procedure in Example 94; [ka]

[0508] Example 203 ADC-108 was prepared following the general coupling procedure in Example 94; [ka]

[0509] Example 204 ADC-109 was prepared following the general coupling procedure in Example 94; [ka]

[0510] Example 205 ADC-110 was prepared following the general coupling procedure in Example 94; [ka]

[0511] Example 206 ADC-111 was prepared following the general coupling procedure in Example 94; [ka]

[0512] Example 207 ADC-112 was prepared following the general coupling procedure in Example 94; [ka]

[0513] Example 208 ADC-113 was prepared following the general coupling procedure in Example 94; [ka]

[0514] Example 209 ADC-114 was prepared following the general coupling procedure in Example 94; [ka]

[0515] Example 210 ADC-115 was prepared following the general coupling procedure in Example 94; [ka]

[0516] Example 211 ADC-116 was prepared following the general coupling procedure in Example 94; [ka]

[0517] Example 212 ADC-117 was prepared following the general coupling procedure in Example 94; [ka]

[0518] Example 213 ADC-118 was prepared following the general coupling procedure in Example 94; [ka]

[0519] Example 214 ADC-119 was prepared following the general coupling procedure in Example 94; [ka]

[0520] Example 215 ADC-120 was prepared following the general coupling procedure in Example 94; [ka]

[0521] Example 216 ADC-121 was prepared following the general coupling procedure in Example 94; [ka]

[0522] Example 217 ADC-122 was prepared following the general coupling procedure in Example 94; [ka]

[0523] Example 218 ADC-123 was prepared following the general coupling procedure in Example 94; [ka]

[0524] Example 219 ADC-124 was prepared following the general coupling procedure in Example 94; [ka]

[0525] Example 220 ADC-125 was prepared following the general coupling procedure in Example 94; [ka]

[0526] Example 221 ADC-126 was prepared following the general coupling procedure in Example 94; [ka]

[0527] Example 222 ADC-127 was prepared following the general coupling procedure in Example 94; [ka]

[0528] Example 223 ADC-128 was prepared following the general coupling procedure in Example 94; [ka]

[0529] Example 224 ADC-129 was prepared following the general coupling procedure in Example 94; [ka]

[0530] Example 225 ADC-130 was prepared following the general coupling procedure in Example 94; [ka]

[0531] Example 226 ADC-131 was prepared following the general coupling procedure in Example 94; [ka]

[0532] Example 227 ADC-132 was prepared following the general coupling procedure in Example 94; [ka]

[0533] Example 228 ADC-133 was prepared following the general coupling procedure in Example 94; [ka]

[0534] Example 229 ADC-134 was prepared following the general coupling procedure in Example 94; [ka]

[0535] Example 230 ADC-135 was prepared following the general coupling procedure in Example 94; [ka]

[0536] Example 231 ADC-136 was prepared following the general coupling procedure in Example 94; [ka]

[0537] Example 232 ADC-137 was prepared following the general coupling procedure in Example 94; [ka]

[0538] Example 233 ADC-138 was prepared following the general coupling procedure in Example 94; [ka]

[0539] Example 234 ADC-139 was prepared following the general coupling procedure in Example 94; [ka]

[0540] Example 235 ADC-140 was prepared following the general coupling procedure in Example 94; [ka]

[0541] Example 236 ADC-141 was prepared following the general coupling procedure in Example 94; [ka]

[0542] Example 237 ADC-142 was prepared following the general coupling procedure in Example 94; [ka]

[0543] Example 238 ADC-143 was prepared following the general coupling procedure in Example 94; [ka]

[0544] Example 239 ADC-144 was prepared following the general coupling procedure in Example 94; [ka]

[0545] Example 240 ADC-145 was prepared following the general coupling procedure in Example 94; [ka]

[0546] Example 241 ADC-146 was prepared following the general coupling procedure in Example 94; [ka]

[0547] Example 242 ADC-147 was prepared following the general coupling procedure in Example 94; [ka]

[0548] Example 243 ADC-148 was prepared following the general coupling procedure in Example 94; [ka]

[0549] Example 244 ADC-149 was prepared following the general coupling procedure in Example 94; [ka]

[0550] Example 245 ADC-150 was prepared following the general coupling procedure in Example 94; [ka]

[0551] Example 246 ADC-151 was prepared following the general coupling procedure in Example 94; [ka]

[0552] Example 247 ADC-152 was prepared following the general coupling procedure in Example 94; [ka]

[0553] Example 248 ADC-153 was prepared following the general coupling procedure in Example 94; [ka]

[0554] Example 249 ADC-154 was prepared following the general coupling procedure in Example 94; [ka]

[0555] Example 250 ADC-155 was prepared following the general coupling procedure in Example 94; [ka]

[0556] Example 251 ADC-156 was prepared following the general coupling procedure in Example 94; [ka]

[0557] Example 252 ADC-157 was prepared following the general coupling procedure in Example 94; [ka]

[0558] Example 253 ADC-158 was prepared following the general coupling procedure in Example 94; [ka]

[0559] Example 254 ADC-159 was prepared following the general coupling procedure in Example 94; [ka]

[0560] Example 255 ADC-160 was prepared following the general coupling procedure in Example 94; [ka]

[0561] Example 256 ADC-161 was prepared following the general coupling procedure in Example 94; [ka]

[0562] Example 257 ADC-162 was prepared following the general coupling procedure in Example 94; [ka]

[0563] Example 258 ADC-163 was prepared following the general coupling procedure in Example 94; [ka]

[0564] Example 259 ADC-164 was prepared following the general coupling procedure in Example 94; [ka]

[0565] Example 260 ADC-165 was prepared following the general coupling procedure in Example 94; [ka]

[0566] Example 261 ADC-166 was prepared following the general coupling procedure in Example 94; [ka]

[0567] Example 262 ADC-167 was prepared following the general coupling procedure in Example 94; [ka]

[0568] Example 263 ADC-168 was prepared following the general coupling procedure in Example 94; [ka]

[0569] Example 264 ADC-169 was prepared following the general coupling procedure in Example 94; [ka]

[0570] Example 265 ADC-170 was prepared following the general coupling procedure in Example 94; [ka]

[0571] Example 266 ADC-171 was prepared following the general coupling procedure in Example 94; [ka]

[0572] Example 267 ADC-172 was prepared following the general coupling procedure in Example 94; [ka]

[0573] Example 268 ADC-173 was prepared following the general coupling procedure in Example 94; [ka]

[0574] Example 269 ADC-174 was prepared following the general coupling procedure in Example 94; [ka]

[0575] Example 270 ADC-175 was prepared following the general coupling procedure in Example 94; [ka]

[0576] Example 271 ADC-176 was prepared following the general coupling procedure in Example 94; [ka]

[0577] Example 272 ADC-177 was prepared following the general coupling procedure in Example 94; [ka]

[0578] Example 273 ADC-178 was prepared following the general coupling procedure in Example 94; [ka]

[0579] Example 274 ADC-179 was prepared following the general coupling procedure in Example 94; [ka]

[0580] Example 275 ADC-180 was prepared following the general coupling procedure in Example 94; [ka]

[0581] Example 276 ADC-181 was prepared following the general coupling procedure in Example 94; [ka]

[0582] Example 277 ADC-182 was prepared following the general coupling procedure in Example 94; [ka]

[0583] Example 278 ADC-183 was prepared following the general coupling procedure in Example 94; [ka]

[0584] Example 279 ADC-184 was prepared following the general coupling procedure in Example 94; [ka]

[0585] Example 280 ADC-185 was prepared following the general coupling procedure in Example 94; [ka]

[0586] Example 281 ADC-186 was prepared following the general coupling procedure in Example 94; [ka]

[0587] Example 282 ADC-187 was prepared following the general coupling procedure in Example 94; [ka]

[0588] Example 283 ADC-188 was prepared following the general coupling procedure in Example 94; [ka]

[0589] Example 284 ADC-189 was prepared following the general coupling procedure in Example 94; [ka]

[0590] Example 285 ADC-190 was prepared following the general coupling procedure in Example 94; [ka]

[0591] Example 286 ADC-191 was prepared following the general coupling procedure in Example 94; [ka]

[0592] Example 287 ADC-192 was prepared following the general coupling procedure in Example 94; [ka]

[0593] Example 288 ADC-193 was prepared following the general coupling procedure in Example 94; [ka]

[0594] Example 289 ADC-194 was prepared following the general coupling procedure in Example 94; [ka]

[0595] Example 290 ADC-195 was prepared following the general coupling procedure in Example 94; [ka]

[0596] Example 291 ADC-196 was prepared following the general coupling procedure in Example 94; [ka]

[0597] Example 292 ADC-197 was prepared following the general coupling procedure in Example 94; [ka]

[0598] Example 293 ADC-198 was prepared following the general coupling procedure in Example 94; [ka]

[0599] Example 294 ADC-199 was prepared following the general coupling procedure 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 following the general coupling procedure in Example 94; [ka]

[0602] Example 297 ADC-202 was prepared following the general coupling procedure in Example 94; [ka]

[0603] Example 298 ADC-203 was prepared following the general coupling procedure in Example 94; [ka]

[0604] Example 299 ADC-204 was prepared following the general coupling procedure in Example 94; [ka]

[0605] Example 300 ADC-205 was prepared following the general coupling procedure in Example 94; [ka]

[0606] Example 301 ADC-206 was prepared following the general coupling procedure in Example 94; [ka]

[0607] Example 302 ADC-207 was prepared following the general coupling procedure in Example 94; [ka]

[0608] Example 303 ADC-208 was prepared following the general coupling procedure in Example 94; [ka]

[0609] Example 304 ADC-209 was prepared following the general coupling procedure in Example 94; [ka]

[0610] Example 305 ADC-210 was prepared following the general coupling procedure in Example 94; [ka]

[0611] Example 306 ADC-211 was prepared following the general coupling procedure in Example 94; [ka]

[0612] Example 307 ADC-212 was prepared following the general coupling procedure in Example 94; [ka]

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

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

[0615] Example 310 ADC-215 for HER3 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 following the general coupling procedure of Example 93; [ka]

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

[0618] Example 313 ADC-239 was prepared from ADC-232 following the general coupling procedure of Example 94; [ka]

[0619] Example 314 ADC-247 was prepared from ADC-240 following the general coupling procedure of Example 94; [ka]

[0620] Example 315 The SEC-HPLC method was used to determine the monomer percentage: Chromatography column: Biocore SEC-300 5μm, 4.6×300mm 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 to antibody ratio DAR: Chromatography column name: Proteomix RP-1000 4.6×100mm 5μm 1000A Manufacturer: Sepax

[0625] [Table 3]

[0626] [Table 4]

[0627] Conclusion: The ADC disclosed in this invention has the excellent property of high DAR value, which can significantly increase the concentration of the drug 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 6 antigen-coated plates were closed with 1% bovine serum protein; then SI-1x6.4, ADC-6, SI-1x4, and ADC-112 were diluted respectively, then diluted in a continuous 3-fold gradient with a starting concentration of 5,000ng / mL for a total of 11 concentrations; the samples were incubated on the coated ELISA plates for a certain period of time, followed by biotin-labeled HER3-Fc antigen incubation, followed by streptavidin-HRP label incubation; finally, TMB color development was performed, stopped with sulfuric acid solution, and the absorbance value at 450nm was detected with a microplate reader. The results of the assay were plotted against the concentration of OD450nm.

[0629] Conclusion: As shown in 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 the 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 similar affinities 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 cancer cells SW620) were used as experimental models to evaluate the in vitro efficacy of the ADC-conjugated drugs in the present invention. A certain 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; Alamar Blue or MTS was used to evaluate the inhibitory effect of the test antibodies and ADCs on the tumor cell lines by measuring cell viability and calculating IC50. The starting concentration of the antibody drug was 500 nM, the dilution was 7-fold, totaling 8 concentration points, and the treatment was performed for 5 days. The final calculation method was survival rate = (experimental group - blank) / (control group - blank group) x 100%, and then curve fitting was performed using Graph Pad Prism to calculate the 50% inhibitory concentration (IC50) as well as efficacy (%).

[0632] [Table 5]

[0633] [Table 6]

[0634] [Table 7]

[0635] [Table 8]

[0636] [Table 9]

[0637] [Table 10]

[0638] Conclusion: 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, 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, SI-1x26) (Figures 4A-4F and Tables 5-10).

[0639] Example 319 In vivo efficacy testing: In the present invention, 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, and tumors with a volume of 150-300 mm were cultured. 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 monitored continuously with tumor measurements taken twice a week to assess the inhibitory effect of the test antibodies and ADCs on the tumor cell lines.

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

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

[0642] In A431+SW620 xenogeneic tumors, ADC-6 at 5, 15, and 30 mg / kg similarly demonstrated robust tumor inhibition relative to SI-1×6.4 naked antibody at 30 mg / kg ( FIG. 5C ).

[0643] Example 320 In vitro efficacy testing: In the present invention, human epithelial cancer cells A431 and human pancreatic cancer cells BxPC3 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 the right shoulder of BALB / c nude mice, and tumors with a volume of 180 to 250 mm were cultured. 3 When the tumors grew to a saturation level, the corresponding ADC drugs were injected into the tail vein once a week for four consecutive doses under continuous observation, and the tumor volumes were measured twice a week to evaluate the inhibitory effects of the test ADCs 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-high A431 single-body tumor model, 42 days after the first administration, each of the tested ADC drugs caused significant tumor growth inhibition (P<0.05) in the subcutaneous transplantation of human epithelial carcinoma cell A431 tumors in BALB / c nude mice, and ADC-6, ADC-219, ADC-235, ADC-227, and ADC-112 had comparable tumor-inhibitory effects, all of which showed strong tumor-inhibitory effects (Figure 6A).

[0646] In the BxPC3 monoclonal tumor model with moderate EGFR expression, 35 days after the first administration, each of the tested ADC drugs caused significant tumor growth inhibition (P<0.05) in the human epithelial carcinoma cell BxPC3 BALB / c nude mouse subcutaneously xenografted tumor model, with ADC-6, ADC-219, ADC-235, ADC-227 and ADC-112 having comparable tumor-inhibitory effects, all of which showed strong tumor-inhibitory 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: In advance, the ultraviolet light of the biosafety cabinet was turned on for 30 minutes, and then the ventilation was turned on for 3 minutes. The growth medium, detection medium, D-PBS and pancreatin were placed in a 37 °C constant temperature water bath and preheated, then the surface was sterilized with alcohol and placed in the biosafety cabinet. The cells were placed in the biosafety cabinet at about 80% confluence, the old medium was removed, rinsed with D-PBS, removed and discarded, digested with pancreatin for 2-3 minutes, then the growth medium was neutralized and centrifuged at 1200 rpm for 3 minutes. The centrifuged supernatant was removed and mixed uniformly with 4 mL of detection medium, and 100 uL was collected for counting (if 50 uL of cell solution was removed, 50 uL of trypan blue stain was added, mixed uniformly, and then counted). Plate spreading was performed according to the previously optimized cell spreading density; 80ul / well was spread on a 96-well plate, 80uL of detection medium was added to wells E11 and F11 only, and 150uL of DPBS was added to the edge wells. 24 hours after spreading the plate, 20uL / well of diluted antibody was added to set up the control; 20uL of detection medium was added to the 11th column only, and duplicate wells were set up for each concentration; after addition, uniform mixing was performed on a cell vortex mixer at 550rpm 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 was mixed evenly, add it to the second column, mix evenly by moving up and down 10 times with a multichannel pipette, discard the pipette tip, and perform the operation for the following 7 concentrations in sequence.

[0651] 4) Detection: After 4 days, the MTS reagent was removed and thawed at room temperature in the dark, then mixed thoroughly and uniformly using a vortex mixer, then 20 μL of CellTiter One Solution Reagen MTS reagent was added per 100 μL of cell culture liquid volume along the sidewall of the well in a biosafety cabinet, the plate surface was gently tapped to mix the MTS solution uniformly, and 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 value was measured in a microplate reader, recorded, collated, analyzed and stored.

[0652] 5) Results: Table 11 illustrates that compound 5A (payload) showed 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 cells SK-MES-1).

[0655] A certain number of tumor cells were inoculated in a 96-well plate, gradient-diluted test antibodies and corresponding ADC drugs were added to the cells, and the cells were treated for 5 days, and the inhibitory effects of the test antibodies, ADCs, and small molecule drugs d3 on tumor cell lines were evaluated by measuring cell viability using Alamar Blue or MTS, and calculating IC50. The starting concentration of the antibody drug was 500nM, the dilution was 7-fold, totaling 7 concentration points, and the treatment was performed for 5 days. The final calculation method was viability = (experimental group-blank) / (control group-blank group) x 100%, and then curve fitting was performed using Graph Pad 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. formula: 【Chemistry 1】 [In the formula, The following groups: 【Chemistry 2】 are each independently optionally 【Chemistry 3】 and SI-1x24 is a tetravalent bispecific antibody comprising an IgG that binds to EGFR and a scFV that binds to HER3, wherein the IgG heavy chain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:29, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:31; the IgG kappa light chain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:25, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:26, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:27; and the scFV comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:32, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:33, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:34, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:35, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

37. or a pharma- ceutical acceptable salt thereof.

2. The antibody-drug conjugate of claim 1, wherein the IgG heavy chain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38, the IgG kappa light chain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28, and the scFv comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

40.

3. 2. The antibody drug conjugate of claim 1, wherein SI-1x24 comprises a first IgG1 heavy chain-connector-scFv domain polypeptide comprising the amino acid sequence of SEQ ID NO:4; a second IgG1 heavy chain-connector-scFv domain polypeptide comprising the amino acid sequence of SEQ ID NO:4; a first kappa light chain comprising the amino acid sequence of SEQ ID NO:2; and a second kappa light chain comprising the amino acid sequence of SEQ ID NO:

2.

4. formula: 【Chemistry 4】 [wherein the following groups: 【Chemistry 5】 are each independently optionally 【Chemistry 6】 may be substituted with The antibody-drug conjugate according to any one of claims 1 to 3,

5. formula: 【Chemistry 7】 [wherein the following groups: 【Chemistry 8】 are each independently optionally 【Chemistry 9】 may be substituted with A pharma- ceutically acceptable salt of the antibody-drug conjugate according to any one of claims 1 to 3,

6. formula: 【Chemistry 9】 is covalently bonded to one or more sulfur atoms of one or more cysteine ​​residues of SI-1x24 and has the following group: 【Chemistry 10】 However, each independently, optionally 【Chemistry 11】 The antibody-drug conjugate according to any one of claims 1 to 3, which is optionally substituted with:

7. The antibody-drug conjugate of claim 6, wherein the one or more cysteine ​​residues result from cleavage of one or more interchain disulfide bonds of SI-1x24.

8. Each light chain of SI-1x24 has 0 to 1 amino acid sequence of the formula: 【Chemistry 12】 and each heavy chain of SI-1x24 is linked to a moiety of the formula: 【Chemistry 13】 and a group represented by the following formula: 【Chemistry 14】 However, each independently, optionally 【Chemistry 15】 The antibody-drug conjugate according to any one of claims 1 to 3, which is optionally substituted with:

9. formula: 【Chemistry 16】 The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 3,

10. formula: 【Chemistry 17】 The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 3,

11. formula: 【Chemistry 18】 The antibody-drug conjugate or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 3,

12. The antibody-drug conjugate of any one of claims 1 to 3, wherein n is 6, 7 or 8.

13. The antibody-drug conjugate of claim 12 , wherein n is 8.

14. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 3 and a pharma- ceutical acceptable carrier.

15. 4. A pharmaceutical composition comprising a plurality of the antibody-drug conjugates of any one of claims 1 to 3, and optionally a pharma- ceutical acceptable carrier, wherein the pharmaceutical composition has an average drug-antibody binding ratio (DAR) of 6 to 8.

16. 16. The pharmaceutical composition of claim 15, wherein the average DAR of the pharmaceutical composition is about 6.

99.

17. 16. The pharmaceutical composition of claim 15, wherein the average DAR of the pharmaceutical composition is about 8.

18. Use of the antibody-drug conjugate of any one of claims 1 to 3 in the manufacture of a medicament for treating or preventing cancer in a subject in need of such treatment or prevention.

19. 19. The use of claim 18, wherein the cancer is selected from the group consisting of adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia.

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