DNA toxic dimers and their complexes

Abstract

A DNA toxic dimeric compound, its complex or its pharma- ceutically acceptable salt, its preparation method, and its effect in the prophylaxis or treatment of cancer. The complex can specifically bind to a receptor highly expressed in tumor cells, has good water solubility, stability, and homogeneity, and can be used in the prophylaxis and treatment of diseases such as tumors.

Description

FIELD OF THEINVENTION

[0001] The present invention relates to new cytotoxic compounds and drugs comprising these cytotoxic compounds and cell binding agents. More specifically, the present invention relates to new benzodiazepine dimer compounds, their derivatives, intermediates, pharma- ceutically acceptable salts and complexes, which can be used as drugs, particularly antitumor drugs. [Background technology]

[0002] Ligand-drug conjugates (ADCs) are new targeted drugs that generally consist of three parts: an antibody or antibody-like ligand, a small molecule drug, and a linker that connects the ligand and the drug. Antibody-drug conjugates utilize the specific recognition of antibodies against antigens to deliver drug molecules to the vicinity of target cells and effectively release the drug molecules to achieve the purpose of treatment. In August 2011, the U.S. Food and Drug Administration (FDA) approved the marketing of AdecteisTM, a new ADC drug for the treatment of Hodgkin's lymphoma and recurrent degenerative large cell lymphoma (ALCL) developed by Seattle Genetics. The safety and efficacy of this drug have been clinically proven. With the development of ADC drugs, there is a need for small molecule drugs with more effective and newer mechanisms of action.

[0003] Benzodiazepine derivatives have the ability to recognize and bind to specific DNA sequences, can react with guanine in the minor groove of DNA, and are highly effective interstrand cross-linking agents that form DNA adducts to disrupt DNA processing, and therefore have been used as antitumor drugs (Rahman et al. (2009) Jour. Amer. Chem. Soc. 131(38):13756-13766; Thurston et al. (1994) Chem. Rev., 94:433-465; Bose et al. (1992) J. Am. Chem. Soc. 114:4939-4941; Gregson et al. (2004) Jour. Med. Chem. 47(5):1161-1174).

[0004] The 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) family of DNA minor groove alkylating agents are potent cytotoxins (Atwell et al. (1999) J. Med. Chem., 42:3400) and have been used as effector units in a wide variety of prodrugs designed for cancer therapy, with CBI linked to benzodiazepine derivatives via an alkyl chain (CN105636612A).

[0005] Imidazo[1,2-a]pyridine derivatives are potent DNA binding units and have been used in the synthesis of antitumor antibiotic duocarmycin derivatives, which have shown highly effective cytotoxicity (Ronald C. Elgersma et al. (2015) Mol. Pharmaceutics. 12:1813-1835).

[0006] However, the benzodiazepine derivatives disclosed in the prior art are highly toxic, even at very low doses, and therefore there is a great need for improved benzodiazepine derivatives that have low toxicity but also have therapeutic activity and a wide therapeutic window. Summary of the Invention

[0007] The present invention aims to provide a cytotoxic benzodiazepine dimer derivative and its antibody conjugate with good therapeutic window. The small molecule drug of the newly designed cytotoxic benzodiazepine dimer derivative conjugate has two functional groups, chloromethyl (CBI) and imine (PBD). Chloromethyl is a prodrug structure, which can form a three-membered ring structure after entering the body, and further DNA alkylation can occur. The presence of these two functional groups can strengthen DNA crosslinking. The inventors accidentally discovered that such benzodiazepine-based ADC drugs have good safety and effective antitumor activity.

[0008] The present invention relates to a ligand-drug conjugate represented by formula I, or a pharma- ceutically acceptable salt, deuterated form or solvate thereof. Ab-LD (I) where Ab is a ligand unit selected from an antibody, an antibody fragment, a target protein, or an Fc-fusion protein; L is the binding unit between D and Ab, D is [ka] or a drug unit selected from JPEG0007667870000002.jpg3162; where the wavy lines indicate the binding sites of the drug and L, and only one of the three binding sites is bound to L; R1 is H, deuterium, OH or ether represented by OR3, sulfite ion SO3 - Or OSO3 - and R3 is C1-C 10 is selected from the group consisting of a linear, branched or cyclic alkyl group, an alkenyl group, and an alkynyl group; Double line between N and C [ka] represents a single bond or a double bond. In the case of a double bond, N is not bonded to L and R1 is H. In the case of a single bond, N is bonded to L and R1 is OH or an ether represented by OR3, a sulfite ion SO3 - Or OSO3 - and R3 is selected from C1-C 10 is selected from the group consisting of linear, branched or cyclic alkyl, alkenyl or alkynyl groups; R2 is H or an alkyl substituent; T is C2-C 12a hydrocarbon group, Z, selected from (C1-C6 alkylene group)-Z-(C1-C6 alkylene group), (C1-C6 alkylene group)-Z-(C1-C6 alkylene group)-Z-(C1-C6 alkylene group), (C1-C6 alkenylene group)-Z-(C1-C6 alkenylene group) or (C1-C6 alkynylene group)-Z-(C1-C6 alkynylene group); wherein Z is selected from O, S, NR4, an aryl group or a heteroaryl group; R4 is selected from H, P(O)3H2 or C(O)NR5R6; R5 and R6 are selected from H, a C1-C6 alkyl group, a C1-C6 alkyl group substituted with one or more F, or R5 and R6 comprise a 5- or 6-membered heterocyclyl group; The alkylene, alkenylene, aryl and heteroaryl groups are selected from F, OH, O(C1-C6 alkyl), NH2, NHCH3, N(CH3)2 or substituted with C1-C6 alkyl groups, where the alkyl groups are substituted with one or more F; Y is selected from one or more H or C1-C4 alkyl groups; X is -O-, -N-, -S-, -OC(O)-CR7R8-(CR9R 10 )mO-, -OC(O)-CR7R8-(CR9R 10 )m-NH-, -OC(O)-CR7R8-(CR9R 10 )mS-, -NHC(O)-CR7R8-(CR9R 10 )mO-, -NHC(O)-CR7R8-(CR9R 10 )m-NH- or -NHC(O)-CR7R8-(CR9R 10 ) mS-, wherein R7 and R8 are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a halogenated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, a substituted aryl group, or a heteroaryl group; or R7, R8 and the carbon atom to which they are attached constitute a C3-C6 cycloalkyl group, a cycloalkylalkyl group, or a heterocyclyl group; R9, R 10may be the same or different and each independently represents a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, or a heterocyclyl group; or R, R 10 and the carbon atoms to which they are attached constitute a C3-C6 cycloalkyl group, a cycloalkylalkyl group, or a heterocyclyl group; m is selected from an integer from 0 to 4; X1 is halogen or OSO2R 11 where R 11 is selected from H, a C1-C4 hydrocarbon group, a phenyl group, or a substituted phenyl group.

[0009] Preferably, Ab is an antibody, which forms a bond with the binding unit via a heteroatom thereof, and said antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific or multispecific antibody.

[0010] More preferably, the antibody or antigen-binding fragment thereof includes, but is not limited to, an anti-EGFR VIII antibody, an anti-DLL-3 antibody, an anti-PSMA antibody, an anti-CD70 antibody, an anti-MUC16 antibody, an anti-ENPP3 antibody, an anti-TDGF1 antibody, an anti-ETBR antibody, an anti-MSLN antibody, an anti-TIM-1 antibody, an anti-LRRC15 antibody, an anti-LIV-1 antibody, an anti-CanAg / AFP antibody, an anti-cladin antibody, or an anti-ILV-1 antibody. 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-G PNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibodies, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-Integrin Av antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody or anti-CD123 antibody.

[0011] In the DNA toxic dimeric compounds, their complexes or their pharma- ceutically acceptable salts, deuterated forms and solvates, preferably L is of the cleavable or non-cleavable type.

[0012] In the DNA toxic dimer compounds and their complexes or pharma- ceutically acceptable salts, deuterated salts and solvates thereof, the pharma- ceutically acceptable salts include sodium, potassium, calcium or magnesium salts formed with an acidic functional group in the structural formula; or acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, hydrogen sulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate or p-toluenesulfonate salts formed with a basic functional group in the structural formula.

[0013] Use of a DNA toxic dimeric compound and its complex or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for treating or preventing a tumor.

[0014] Preferably, the tumor is a solid tumor or a hematological tumor, such as breast cancer, 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 or leukemia.

[0015] The present invention further provides a compound of general formula II or III, or a pharma- ceutically acceptable salt or solvate thereof, which is conjugated to Ab. [ka] or JPEG0007667870000005.jpg3262In the formula, R1 is H, OH, or an ether represented by OR3, a sulfite ion SO3 - Or OSO3 - where R3 is C1-C 10 is selected from the group consisting of a linear, branched or cyclic alkyl group, an alkenyl group, and an alkynyl group; Double line between N and C [ka] represents a single bond or a double bond, and when it is a double bond, R 12 When R is absent and R is H and a single bond, R 12 is -C(O)O-L3, where L3 is a bond unit and R1 is OH, an ether represented by OR3, or a sulfite ion represented by SO3 - Or OSO3 - wherein R3 is selected from C1-C 10 is selected from the group consisting of a linear, branched or cyclic alkyl group, an alkenyl group, and an alkynyl group; R2 is H or an alkyl substituent; T is C2-C 12 a hydrocarbon group, Z, selected from (C1-C6 alkylene group)-Z-(C1-C6 alkylene group), (C1-C6 alkylene group)-Z-(C1-C6 alkylene group)-Z-(C1-C6 alkylene group), (C1-C6 alkenylene group)-Z-(C1-C6 alkenylene group) or (C1-C6 alkynylene group)-Z-(C1-C6 alkynylene group); wherein Z is selected from O, S, NR4, an aryl group, and a heteroaryl group; R4 is selected from H, P(O)3H2, C(O)NR5R6; R5 and R6 are selected from H, a C1-C6 alkyl group, a C1-C6 alkyl group substituted with one or more F; or R5 and R6 comprise a 5- or 6-membered heterocyclyl group; The alkylene, alkenylene, aryl and heteroaryl groups are selected from F, OH, O(C1-C6 alkyl), NH2, NHCH3, N(CH3)2 and C1-C6 alkyl substituted groups, the alkyl groups being substituted with one or more F; Y is selected from one or more H or C1-C4 alkyl groups; X is -O-, -N-, -S-, -OC(O)-CR7R8-(CR9R 10 )mO-, -OC(O)-CR7R8-(CR9R 10 )m-NH-, -OC(O)-CR7R8-(CR9R 10 )mS-, -NHC(O)-CR7R8-(CR9R10 )mO-, -NHC(O)-CR7R8-(CR9R 10 )m-NH- or -NHC(O)-CR7R8-(CR9R 10 ) mS-, wherein R7 and R8 are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a halogenated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, a substituted aryl group, or a heteroaryl group; or R7, R8 and the carbon atom to which they are attached constitute a C3-C6 cycloalkyl group, a cycloalkylalkyl group, or a heterocyclyl group; R9, R 10 may be the same or different and each independently represents a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclyl group; or R, R 10 and the carbon atoms to which they are attached constitute a C3-C6 cycloalkyl group, a cycloalkylalkyl group, or a heterocyclyl group; m is selected from an integer from 0 to 4; X1 is halogen or OSO2R 11 where R 11 is selected from H, a C1-C4 hydrocarbon group, a phenyl group, or a substituted phenyl group; L1 and L2 are bonding units or substituents.

[0016] Preferably, T is C2-C 12 The alkylene groups are selected from the following:

[0017] More preferably, T is [ka] It is.

[0018] Preferably, X is -O-, -N- or -NHC(O)-CR7R8-(CR9R10 )mO-.

[0019] Preferably, L3 is [ka] where the wavy line site is bonded to -C(O)O- and L4 is a linking unit that is bonded to the Ligand unit.

[0020] Preferably, L4 is, but not limited to, [ka] , JPEG0007667870000010.jpg1718, JPEG0007667870000011.jpg1718, JPEG0007667870000012.jpg1618, JPEG0007667870000013.jpg1418 or JPEG0007667870000014.jpg1423, where the left side (carbon end) of the wavy line site is bonded to the ligand unit, and the right side (nitrogen end or ester carbonyl group end) is bonded to X2.

[0021] Preferably, Q is [ka] where Q x is an amino acid residue or a peptide residue consisting of amino acids.

[0022] Preferably, X2 is [ka] where a is selected from an integer between 0 and 5, b is selected from an integer between 0 and 16, c is selected from an integer between 0 and 1, and d is selected from an integer between 0 and 5.

[0023] More preferably, L3 is, but not limited to, [ka] or JPEG0007667870000018.jpg19128, where at the site of the wavy line, the left side (succinimide end) is bonded to a Ligand unit and the right side is bonded to -C(O)O-.

[0024] Preferably, in the compound of formula II or III, or a pharma- ceutically acceptable salt or solvate thereof, L1 and L2 are each independently Structure A: a hydrogen atom, C(O)NR'R'', where R' and R'' are selected from H, a C1-C6 alkyl group, a C1-C6 alkyl group substituted with one or more F, or R' and R'' comprise a 5- or 6-membered heterocyclyl group; and Structure B: L4-L5-, L4-L6-, or L4-L7-L8-L9- (wherein L4, L5, L6, L7, L8 and L9 are all binding units, L4 is bonded to the ligand unit, and L5, L6 and L9 are bonded to X). is selected from.

[0025] More preferably, There is a single bond between N and C, i.e., R 12 When present, L1, L2 are each independently selected from Structure A; There is a double bond between N and C, i.e., R 12 is absent, then L1 is structure A or B and L2 is structure B or A.

[0026] More preferably, L5 is -((CH2)sO)r(CH2)sX3L 10 -or-((CH2)sO)r(CH2)sX4L 10 - and L6 is -((CH2)sO)r(CH2)s-, L 10is -(CH2)s- or -((CH2)sNHC(=O)X5X6C(=O)(CH2)s-, where X3 is, but not limited to, [ka] , JPEG0007667870000020.jpg2926 or JPEG0007667870000021.jpg2927, where R 13 are independently selected from a hydrogen atom, a C1-C6 hydrocarbon group, a halogen atom, or a hydroxyl group; X4 is non-restrictive [ka] , JPEG0007667870000023.jpg3630 or JPEG0007667870000024.jpg3428, where R 13 are independently selected from a hydrogen atom, a C1-C6 hydrocarbon group, a halogen atom, or a hydroxyl group; X5 is non-restrictive [ka] , JPEG0007667870000026.jpg1229 or Selected from JPEG0007667870000027.jpg2527, X6 is selected from peptide residues consisting of amino acids, including but not limited to [ka] , JPEG0007667870000029.jpg1248 or JPEG0007667870000030.jpg1225, s is selected from an integer of 1-10, and r is selected from an integer of 1-14.

[0027] More preferably, L7 is -NC(R 14 R 15 )C(O), -NR 16 (CH2) o C(O)-, -NR 16 (CH2CH2O) o , CH2C(O)-, -S(CH2) p C(O)- or a chemical bond, where o is selected from an integer of 0-20 and p is selected from an integer of 0-20; R 14 giving R 15 may be the same or different and are each independently selected from a hydrogen atom, a deuterium atom, an alkyl group, a substituted alkyl group, a deuterated alkyl group, a heteroalkyl group, a carboxyl group, an amino group, and a substituted amino group; R 16 is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a substituted alkyl group, a deuterated alkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, an aryl group, a substituted aryl group, or a heteroaryl group; L8 is selected from peptide residues of an amino acid, preferably one, two or more peptide residues of amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E) or aspartic acid (D); L9, -NR 17 (CR 18 R 19 ) q -, -C(O)NR 17 -, -C(O)NR 17 (CH2) q - or a chemical bond, where q is selected from an integer of 0-6; R 17 , R 18 and R 19 may be the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a substituted alkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, a substituted aryl group, or a heteroaryl group.

[0028] More preferably, L1 and L2 are independently selected from the group consisting of, but not limited to, Structure B. [ka] , JPEG0007667870000032.jpg1877, JPEG0007667870000033.jpg1876JPEG0007667870000034.jpg1665, JPEG0007667870000035.jpg2360, JPEG0007667870000036.jpg2780, JPEG0007667870000037.jpg2962, JPEG0007667870000038.jpg3240 or Selected from JPEG0007667870000039.jpg4648.

[0029] More preferably, the compound of formula II or III, or a pharma- ceutically acceptable salt or solvate thereof is, but not limited to, [ka] , JPEG0007667870000041.jpg5460, JPEG0007667870000042.jpg5272, JPEG0007667870000043.jpg5059, JPEG0007667870000044.jpg3274, JPEG0007667870000045.jpg23128, JPEG0007667870000046.jpg31131, JPEG0007667870000047.jpg23131, JPEG0007667870000048.jpg23129, JPEG0007667870000049.jpg30128, JPEG0007667870000050.jpg31129, JPEG0007667870000051.jpg29128, JPEG0007667870000052.jpg31128, JPEG0007667870000053.jpg32128, JPEG0007667870000054.jpg34128, JPEG0007667870000055.jpg33128, JPEG0007667870000056.jpg31128, JPEG0007667870000057.jpg33128, JPEG0007667870000058.jpg30128, JPEG0007667870000059.jpg33128, JPEG0007667870000060.jpg36128, JPEG0007667870000061.jpg32128, JPEG0007667870000062.jpg48128 or Selected from JPEG0007667870000063.jpg34128.

[0030] Preferably, the antibody-drug conjugate, or a pharma- ceutically acceptable salt, deuterated product, or solvate thereof, includes, but is not limited to, [ka] , JPEG0007667870000065.jpg4666, JPEG0007667870000066.jpg5065, JPEG0007667870000067.jpg5465, JPEG0007667870000068.jpg4777, JPEG0007667870000069.jpg39128, JPEG0007667870000070.jpg36128、 JPEG0007667870000071.jpg30128、 JPEG0007667870000072.jpg30128、 JPEG0007667870000073.jpg28128、 JPEG0007667870000074.jpg27128、 JPEG0007667870000075.jpg34128、 JPEG0007667870000076.jpg29128、 JPEG0007667870000077.jpg29128、 JPEG0007667870000078.jpg34128、 JPEG0007667870000079.jpg34128、 JPEG0007667870000080.jpg33128、 JPEG0007667870000081.jpg33128、 JPEG0007667870000082.jpg34128、 JPEG0007667870000083.jpg32128、 JPEG0007667870000084.jpg33128、 JPEG0007667870000085.jpg35128、 JPEG0007667870000086.jpg36128、 JPEG0007667870000087.jpg37128、 JPEG0007667870000088.jpg36128、 JPEG0007667870000089.jpg34128、 JPEG0007667870000090.jpg35128、 JPEG0007667870000091.jpg35128、 JPEG0007667870000092.jpg36129、 JPEG0007667870000093.jpg46128, JPEG0007667870000094.jpg42128, JPEG0007667870000095.jpg41128, JPEG0007667870000096.jpg39128, JPEG0007667870000097.jpg37128, JPEG0007667870000098.jpg42128, JPEG0007667870000099.jpg43128, JPEG0007667870000100.jpg47128 or JPEG0007667870000101.jpg44128, where u is selected from the integers 1-10. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Abbreviations and Definitions Unless otherwise stated, the following terms and phrases as used herein shall have the following meanings: When trade names are used herein, unless the context dictates otherwise, the trade name includes formulations, generic drugs, and active ingredients of the trade name product.

[0032] Unless stated to the contrary, terms used in the specification and claims have the same meanings as set out below.

[0033] The term "ligand" refers to a macromolecular compound capable of recognizing and binding to an antigen or receptor associated with a target cell. The role of the ligand is to present the drug to the target cell population to which the ligand binds. Such ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules capable of binding to cells. In an embodiment of the invention, the ligand is represented by Ab. The ligand can be bound to the binding unit via a heteroatom on the ligand. It is preferably an antibody or an antigen-binding fragment thereof. The antibody is selected from a chimeric antibody, a humanized antibody, a fully human antibody, and a murine antibody, and is preferably a monoclonal antibody.

[0034] The Ligand unit is a targeting agent that specifically binds to a target moiety. The Ligand can specifically bind to a cellular component or bind to a cellular component or other targeting molecule. The targeting moiety or target is usually on the cell surface. In some embodiments, the function of the Ligand unit is to deliver the Drug unit to a specific target cell population that interacts with the Ligand unit. Ligands include, but are not limited to, non-proteins such as proteins, polypeptides, peptides, and sugars. Suitable Ligand units include, for example, antibodies, such as full length (intact) antibodies and antigen-binding fragments thereof. The Ligand unit may be a peptide or polypeptide, or a non-protein molecule, in embodiments that are non-antibody targeting agents. Examples of such targeting agents include interferons, lymphokines, hormones, growth and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell-binding molecule or substance. In some embodiments, the binding unit is covalently bound 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 an antibody. In another aspect, the sulfur atom is the sulfur atom of a cysteine ​​residue into which the Ligand unit has been introduced, forming an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is the sulfur atom of a cysteine ​​residue into which the Ligand unit has been introduced (e.g., by site-directed mutagenesis or chemical reaction). In another aspect, the sulfur atom attached to the Linking unit is selected from a cysteine ​​residue of an interchain disulfide bond of the antibody and a cysteine ​​residue into which the Ligand unit has been introduced (e.g., by site-directed mutagenesis or chemical reaction). In some embodiments, the EU index numbering system as described in Kabat EA et al., (1991), Sequences of proteins of Immunologic Interest, Fifth Edition, NIH Publication 91-3242.2 is used.

[0035] As used herein, the term "antibody" or "antibody unit" includes within its scope any portion of an antibody structure. This unit can bind, reactively associate, or form a complex with a receptor, antigen, or other receptor unit carried by the target cell population. An antibody may be any protein or protein-like molecule capable of binding, complexing, or reacting with a portion of the cell population to be treated or biologically modified. In the present invention, the antibody conjugate conjugate can maintain its original wild-type antigen-binding ability. Thus, the antibody of the present invention is preferably capable of specifically binding to an antigen. Such antigens include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, regulators of cell survival, regulators of cell proliferation, molecules associated with tissue growth and differentiation (with known or predictable functionality), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (with known or predictable functionality). As described herein, the tumor-associated factor may be a cluster differentiation factor (such as a CD protein).

[0036] Antibodies used in antibody drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art and can be prepared by methods and information well known in the art for preparing antibodies. To develop effective cellular level targets for cancer diagnosis and treatment, researchers have found transmembrane or other tumor-associated polypeptides. These targets can be expressed specifically on the surface of one or more cancer cells, while being little or not expressed on the surface of one or more non-cancer cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells compared to the surface of non-cancer cells. Identifying such tumor-associated factors can greatly improve the specific targeting properties of antibody-based cancer therapy. For convenience, antigen-related information (such as name, other designation, GenBank accession number, etc.) well known in the art is provided below. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases such as Genbank. The tumor-associated antigens corresponding to the antibody targets have at least 70%, 80%, 85%, 90%, or 95% identity to the sequences identified in the references, including all amino acid sequence variants and isotypes, or have biological properties and characteristics that completely match the sequences of the tumor-associated antigens described in the references.

[0037] The terms "inhibit" or "suppress" refer to reducing the detectable amount or preventing it completely.

[0038] The term "cancer" refers to a physiological condition or disease characterized by unregulated cell growth. A "tumor" includes cancer cells.

[0039] The term "autoimmune disease" refers to a disease or disorder that results from the targeting of an individual's own tissues or proteins.

[0040] The term "drug" refers to cytotoxic drugs, which may be denoted as d, and are chemical molecules that can strongly interfere with the normal growth of tumor cells. Cytotoxic drugs can, in principle, kill tumor cells at sufficiently high concentrations, but due to their lack of specificity, while killing tumor cells, they may also cause apoptosis of normal cells, resulting in serious side effects. The term includes toxins, such as small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants or animals, radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , B.I. 212 , P 32 and radioisotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics, and ribozymes, with toxic drugs being preferred.

[0041] The term "linker", "binding fragment" or "binding unit" refers to a chemical structural fragment or bond that is attached to a ligand at one end and to a drug at the other end, and may be attached to another binding unit and then to the drug.

[0042] The linking unit includes a stretcher, a spacer and an amino acid unit, and can be synthesized according to methods known in the art (e.g., the method described in US2005-0238649A1). The linking unit can be a "cleavable linking unit" that facilitates the release of the drug in cells. For example, an acid-labile linking unit (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linking unit, a photolabile linking unit, a dimethyl linking unit, or a disulfide-containing linking unit can be used (CharI et al. Cancer Research 52:127-131,1992; US Patent No.5,208,020).

[0043] According to the mechanism of intracellular drug release, the "binding unit" or "binding unit of antibody-drug conjugate" described herein can be divided into two types: non-cleavable binding unit and cleavable binding unit. The drug release mechanism of antibody-drug conjugates containing non-cleavable binding units is as follows: After the conjugate binds to an antigen and is taken up into a cell, the antibody is enzymatically hydrolyzed in the lysosome to release an active molecule consisting of a small molecule drug, a binding unit, and an antibody amino acid residue. The resulting change in the drug molecule structure does not reduce its cytotoxicity, and since the active molecule is charged (amino acid residue), it does not penetrate into neighboring cells. Therefore, such drugs do not kill neighboring tumor cells that do not express the target antigen (antigen-negative cells) (bystander effect) (Ducry et al., 2010, BIoconjugate Chem. 21:5-13).

[0044] The term "antibody drug conjugate" refers to an antibody conjugated to a biologically active drug via a stable binding unit. In the present invention, "ligand drug conjugate" refers to an antibody drug conjugate (ADC), preferably a monoclonal antibody or an antibody fragment, conjugated to a biologically active toxic drug via a stable binding unit.

[0045] The three-letter and one-letter codes for amino acids used herein are as described in J. Boy. Chem. 1968, 243, 3558.

[0046] The term "alkyl" refers to saturated aliphatic hydrocarbon groups, including straight or branched chain groups having 1 to 20 carbon atoms, preferably alkyl groups having 1 to 12 carbon atoms, more preferably alkyl groups having 1 to 10 carbon atoms, and most preferably alkyl groups having 1 to 6 carbon atoms. 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. , 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and branched isomers thereof.More preferred are lower alkyl groups having 1-6 carbon atoms, non-limiting examples of which 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, etc. The alkyl group may be substituted or unsubstituted. If substituted, the substituent may be substituted at any available point of attachment. The substituents are preferably one or more independently selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen, a mercapto group, a hydroxyl group, a nitro group, a cyano group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocycloalkoxy group, a cycloalkylthio group, a heterocycloalkylthio group, and an oxy group.

[0047] The term "substituted alkyl" refers to a hydrogen atom on an alkyl group being replaced with a substituent. Unless the context dictates otherwise, the substituents on an alkyl group are selected from -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-NO2. The number of substituents is 0-(2m'+1), where m' is the total number of carbon atoms in the group. R', R" and R'" are each independently hydrogen, an unsubstituted C1-8 alkyl group, an unsubstituted aryl group, an aryl group substituted with 1-3 halogens, an unsubstituted C1-8 alkyl group, a C1-8 alkoxy group or a C1-8 thioalkoxy group, or an unsubstituted aryl-C1-4 alkyl group. 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 groups.

[0048] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O and S. Alkyl groups are as defined above.

[0049] The term "alkylene" refers to a saturated linear or branched aliphatic hydrocarbon group having two residues obtained by removing two hydrogen atoms from the same or different carbon atoms of a host alkane, including linear or branched groups having 1 to 20 carbon atoms, preferably alkylene groups having 1 to 12 carbon atoms, and more preferably alkylene groups having 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH-), 1,1-ethylene (-CH(CH)-), 1,2-ethylene (-CHCH)-, 1,1-propylene (-CH(CHCH)-), 1,2-propylene (-CHCH(CH)-), 1,3-propylene (-CHCHCH-), 1,4-butylene (-CHCHCHCHCH-), and 1,5-butylene (-CHCHCHCHCHCH-). The alkylene group may be substituted or unsubstituted. When substituted, the substituents may be substituted at any available attachment point. The substituents are preferably each independently one or more selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogen, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxy groups.

[0050] The term "alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl) groups. Alkyl or cycloalkyl groups are as defined above. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy. Alkoxy groups may be substituted or unsubstituted. If substituted, the substituent may be substituted at any available point of attachment. The substituents are preferably each independently one or more selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio groups.

[0051] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. The cycloalkyl ring contains 3-20, preferably 3-12, more preferably 3-10, and most preferably 3-8 carbon atoms. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. Monocyclic cycloalkyl groups include spiro, fused, or bridged ring cycloalkyl groups.

[0052] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3-20 ring atoms, one or more of which may be nitrogen, oxygen, or S(O). m(m is an integer of 0-2), except for the -OO-, -OS- or -SS- ring moiety, with the remaining ring atoms being carbon. Preferably, the ring contains 3-12 ring atoms, of which 1-4 are heteroatoms. More preferably, the cycloalkyl ring contains 3-10 ring atoms. Examples of monocyclic heterocyclyl groups include, but are not limited to, pyrrolidinylalkyl groups, piperidinyl groups, piperazinyl groups, morpholinyl groups, thiomorpholinyl groups and homopiperazinyl groups. Polycyclic heterocyclyl groups include spiro, fused or bridged ring heterocyclyl groups.

[0053] The term "cycloalkylalkyl" refers to an alkyl group substituted with one or more, preferably one, cycloalkyl. Alkyl and cycloalkyl groups are as defined above.

[0054] The term "halogenated alkyl" refers to an alkyl group substituted with one or more halogens, alkyl being as defined above.

[0055] The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, alkyl group being as defined above.

[0056] The term "hydroxyl" refers to an --OH group.

[0057] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0058] The term "amino" refers to -NH2. The term "nitro" refers to -NO2.

[0059] The term "amido" refers to a -C(O)N-(alkyl) or -(cycloalkyl) group, where alkyl and cycloalkyl are defined above.

[0060] The term "carboxylic acid ester" refers to a -C(O)O-(alkyl) or -(cycloalkyl) group, where alkyl and cycloalkyl are defined above.

[0061] The present invention further includes the deuterated form of formula I. Each hydrogen atom bonded to a carbon atom can be independently replaced with a deuterium atom. Those skilled in the art can synthesize the deuterated form of formula I according to the relevant literature. In synthesizing the deuterated form of formula I, commercially available deuterated starting materials can be used, or deuterated reagents can be used according to conventional methods. Deuterated reagents include, but are not limited to, deuterated borane, trideuterated borane tetrahydrofuran solution, lithium aluminum deuteride, deuterated iodoethane, deuterated iodomethane, etc.

[0062] 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. The antigenicity of immunoglobulins differs due to the different amino acid compositions and sequences in the heavy chain constant regions. This divides immunoglobulins into five types or isotypes: IgM, IgD, IgG, IgA and IgE. The corresponding heavy chains are μ, δ, γ, α and ε chains, respectively. Igs of the same type can be classified into different subclasses depending on the difference in amino acid composition of their hinge regions and the number and position of disulfide bonds in the heavy chains. For example, IgG is divided into IgG1, IgG2, IgG3 and IgG4. Light chains are classified into κ or λ chains depending on the difference in the constant region. Each of the five types of Igs can have κ or λ chains. The antibodies according to the present invention are preferably specific for cell surface antigens on target cells.In non-limiting examples, the antibodies include anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg / AFP antibody, anti-cladIn antibody. 18.2 antibody, anti-MesothelIn antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPI2B / SLC34A2 antibody, anti-G PNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-NectIn 4 antibodies, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-IntegrIn Av antibody, anti-CAIX antibody, anti-P-cadherIn antibody, anti-GD3 antibody, anti-CadherIn 6 antibodies, anti-LAMP1 antibodies, anti-FLT3 antibodies, anti-BCMA antibodies, anti-CD79b antibodies, anti-CD19 antibodies, anti-CD33 antibodies, anti-CD56 antibodies, anti-CD74 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD37 antibodies, anti-CD138 antibodies, anti-CD352 antibodies, anti-CD25 antibodies, or anti-CD123 antibodies.

[0063] The term "solvate" refers to a pharma- ceutically acceptable solvate formed from the Ligand Drug Conjugate of the present invention and one or more solvent molecules. Examples of solvent molecules include, but are not limited to, water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0064] The term "drug loading" refers to the average number of cytotoxic drugs loaded on each antibody in the formula I molecule, and may be expressed as the ratio of drug amount to antibody amount. As a range of drug loading, 0-12, preferably 1-10 cytotoxic drugs (D) can be bound to each antibody (Ab). In an embodiment of the present invention, the drug loading is represented by n, which may be, for example, an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The average number of drugs on each ADC molecule after the coupling reaction can be measured by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA test, and HPLC.

[0065] In one embodiment of the invention, the cytotoxic drug is coupled via a linking unit to the open cysteine ​​thiol-SH of the antibody interchain and / or the thiol-SH of a site-specifically mutated cysteine ​​residue. Generally, the number of drug molecules that can be coupled to the antibody in a coupling reaction is less than or equal to the theoretical maximum.

[0066] The loading of the ligand-cytotoxic drug conjugate can be controlled by the following non-limiting methods. (1) Controlling the molar ratio of binding unit reagent to monoclonal antibody. (2) Controlling reaction time and temperature. (3) Select a different reaction reagent. For the preparation of conventional pharmaceutical compositions, please refer to the Chinese Pharmacopoeia.

[0067] The term "pharmaceutically acceptable salt" or "pharmaceutical acceptable salt" refers to a salt of the ligand drug conjugate of the present invention or a salt of a compound described in the present invention. Such salts have safety and efficacy when used in mammals, and also have appropriate biological activity. The ligand drug conjugate of the present invention has at least one carboxyl, and therefore can form salts with bases. Non-limiting examples of pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts, magnesium salts, etc.

[0068] The term "pharmaceutically acceptable salt" or "pharmaceutical acceptable salt" refers to a salt of the ligand drug conjugate of the present invention or a salt of a compound according to the present invention. Such salts have safety and efficacy when used in mammals and have appropriate biological activity. The antibody drug conjugate compounds of the present invention contain at least one amino group and can therefore form salts with acids. Examples of pharmaceutically acceptable salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0069] "Acidic amino acids" refer to amino acids with an isoelectric point of less than 7. The molecules of acidic amino acids usually have one or more acidic groups such as carboxyl groups, and structurally, they can be effectively ionized to negative ions to improve hydrophilicity. Acidic amino acids can be divided into natural amino acids and unnatural amino acids.

[0070] "Natural amino acid" refers to an amino acid obtained by biosynthesis. Natural amino acids are generally L-type, but there are exceptions, such as glycine, and include both naturally occurring and those synthesized in vivo.

[0071] "Unnatural amino acid" refers to an amino acid obtained synthetically.

[0072] The present invention will be further described below with specific examples, but it should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In the following examples, test methods without specific conditions are usually in accordance with conventional conditions or conditions suggested by manufacturers. Unless otherwise specified, all percentages, proportions, ratios or parts are by weight.

[0073] Example 1 Synthesis of Compound A [ka] Compound A was synthesized according to the method provided in the synthesis of compound 012 in Example 14 of Chinese Patent Application Publication No. CN109180681A.

[0074] Example 2 Synthesis of Compound B and Compounds B1-9 1. Preparation of Compound B [ka] Compound B was synthesized according to the method provided in "General Procedure E: Synthesis Method 1 of CBI-benzodiazepine Derivatives" of Chinese Patent Application Publication No. CN109180681A.

[0075] 2. Preparation of Compound B1 [ka] Compound B (50 mg, 0.072 mmol), hydroxyacetic acid (11 mg, 0.14 mmol), EDCI (20 mg, 0.10 mmol), HOBt (13.5 mg, 0.10 mmol) and 3 mL DMF were placed in a 25 mL one-neck flask and reacted at room temperature for 2 hours. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified directly by high performance liquid chromatography to obtain compound B1 (42 mg). Yield 78%, LC-MS: [M+H] + =747.2.

[0076] 3. Preparation of Compound B2 [ka] Referring to the synthesis method of compound B1, compound B (50 mg, 0.072 mmol), L-lactic acid (12.6 mg, 0.14 mmol), EDCI (20 mg, 0.10 mmol), HOBt (13.5 mg, 0.10 mmol) and 3 mL DMF were placed in a 25 mL one-neck flask and reacted at room temperature for 2 hours, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified directly by high performance liquid chromatography to obtain compound B2 (37 mg). Yield 68%, LC-MS: [M+H] + =761.2.

[0077] 4. Preparation of Compound B3 [ka] Compound B3 was obtained by replacing L-lactic acid with D-lactic acid according to the synthesis method of compound B2. LC-MS: [M+H] + =761.2.

[0078] 5. Preparation of compounds B4 and B5 [ka] Compound B (60 mg, 0.087 mmol), 3,3,3-trifluorolactic acid (25 mg, 0.17 mmol), EDCI (25 mg, 0.13 mmol), HOBt (17.6 mg, 0.13 mmol) and 3 mL DMF were placed in a 25 mL one-neck flask and reacted at room temperature for 1.5 h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified directly by high performance liquid chromatography, and the product fraction was freeze-dried to obtain compound B4 (26 mg) and compound B5 (22 mg), respectively. LC-MS: [M+H] + =815.2.

[0079] 6. Preparation of Compounds B6 and B7 [ka] Compounds B6 and B7 were obtained by purifying the compounds by high performance liquid chromatography according to the synthesis method of compounds B4 and B5. LC-MS: [M+H] +=787.3.

[0080] 7. Preparation of Compounds B8 and B9 [ka] The compounds B8 and B9 were obtained by purifying the compounds by high performance liquid chromatography according to the synthesis method of the compounds B4 and B5. LC-MS: [M+H] + =801.3.

[0081] Example 3 Synthesis of Compounds C and C1-9 1. Preparation of Compound C [ka] Compound C was synthesized according to the method provided in "General Procedure E: Synthesis Method 2 of CBI-benzodiazepine Derivatives" of Chinese Patent Application Publication No. CN109180681A.

[0082] 2. Preparation of Compound C1 [ka] Compound C (50 mg, 0.075 mmol), hydroxyacetic acid (11.4 mg, 0.15 mmol), EDCI (21.6 mg, 0.11 mmol), HOBt (15.2 mg, 0.11 mmol) and 3 mL DMF were placed in a 25 mL one-neck flask and reacted at room temperature for 2 hours, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified directly by high performance liquid chromatography to obtain compound C1 (35 mg). Yield 65%, LC-MS: [M+H] + =723.2.

[0083] 3. Preparation of Compound C2 [ka] Referring to the synthesis method of compound C1, compound C (50 mg, 0.075 mmol), L-lactic acid (13 mg, 0.15 mmol), EDCI (21.6 mg, 0.11 mmol), HOBt (15.2 mg, 0.11 mmol) and 3 mL DMF were placed in a 25 mL one-neck flask and reacted at room temperature for 2 hours, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified directly by high performance liquid chromatography to obtain compound C2 (33.5 mg). Yield 63%, LC-MS: [M+H] + =737.2.

[0084] 4. Preparation of Compound C3 [ka] Compound C3 was obtained by replacing L-lactic acid with D-lactic acid according to the synthesis method of compound C2. LC-MS: [M+H] + =737.2.

[0085] 5. Preparation of Compounds C4 and C5 [ka] Compound C (60 mg, 0.09 mmol), 3,3,3-trifluorolactic acid (26 mg, 0.18 mmol), EDCI (25 mg, 0.13 mmol), HOBt (17.6 mg, 0.13 mmol) and 3 mL DMF were added to a 25 mL one-neck flask, and the reaction was carried out at room temperature for 1.5 h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified directly by high performance liquid chromatography, and the product fraction was freeze-dried to obtain compound C4 (21 mg) and compound C5 (25 mg), respectively. LC-MS: [M+H] + =791.2.

[0086] 6. Preparation of Compounds C6 and C7 [ka] Compounds C6 and C7 were obtained by purifying the compounds by high performance liquid chromatography according to the synthesis method of compounds C4 and C5. LC-MS: [M+H] +=763.3.

[0087] 7. Preparation of Compounds C8 and C9 [ka] The compounds C8 and C9 were obtained by purifying the compounds by high performance liquid chromatography according to the synthesis method of the compounds C4 and C5. LC-MS: [M+H] + =777.3.

[0088] Example 4 Synthesis of Compounds D1 and D1-9 1. Preparation of Compound D [ka] Compound D was synthesized according to the methods provided in "General Procedure A: Synthesis Method 1 for Benzodiazepine Derivatives and General Procedure E: Synthesis Method 2 for CBI-Benzodiazepine Derivatives" of Chinese Patent Application Publication No. CN109180681A.

[0089] 2. Preparation of Compound D1 [ka] Compound D (50 mg, 0.078 mmol), hydroxyacetic acid (12 mg, 0.16 mmol), EDCI (22.4 mg, 0.12 mmol), HOBt (15.8 mg, 0.12 mmol) and 3 mL DMF were placed in a 25 mL one-neck flask and reacted at room temperature for 2 h. The reaction was monitored by HPLC. After completion of the reaction, the reaction solution was purified directly by high performance liquid chromatography to obtain compound D1 (34 mg). Yield 62.5%, LC-MS: [M+H] + =697.2.

[0090] 3. Preparation of Compound D2 [ka] Compound D (50 mg, 0.078 mmol), L-lactic acid (14 mg, 0.16 mmol), EDCI (22.4 mg, 0.12 mmol), HOBt (15.8 mg, 0.12 mmol) and 3 mL DMF were placed in a 25 mL one-neck flask and reacted at room temperature for 2 hours. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified directly by high performance liquid chromatography to obtain compound D2 (33.8 mg). Yield 71%, LC-MS: [M+H] + =711.2.

[0091] 4. Preparation of Compound D3 [ka] Compound D3 was obtained by replacing D-lactic acid with L-lactic acid according to the synthesis method of compound D2. LC-MS: [M+H] + =711.2.

[0092] 4. Preparation of Compounds D4 and D5 [ka] Compound D (60 mg, 0.094 mmol), 3,3,3-trifluorolactic acid (27 mg, 0.187 mmol), EDCI (27 mg, 0.14 mmol), HOBt (19 mg, 0.14 mmol) and 3 mL DMF were placed in a 25 mL one-neck flask and reacted at room temperature for 1.5 h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified directly by high performance liquid chromatography, and the product fraction was freeze-dried to obtain compound D4 (27 mg) and compound D5 (29 mg), respectively. LC-MS: [M+H] + =765.2.

[0093] 6. Preparation of Compounds D6 and D7 [ka] Compounds D6 and D7 were obtained by purifying the compounds by high performance liquid chromatography according to the synthesis method of compounds D4 and D5. LC-MS: [M+H] + =737.3.

[0094] 7. Preparation of Compounds D8 and D9 [ka] Compounds D8 and D9 were obtained by purifying the compounds by high performance liquid chromatography according to the synthesis method of compounds D4 and D5. LC-MS: [M+H] + =751.3.

[0095] Example 5 Synthesis of compound M1 [ka] N-fluorenylmethoxycarbonyl-glycine-glycine (100g, 28mmol, 1.0eq), lead tetraacetate (175g, 395mmol, 1.4eq), 2L tetrahydrofuran and 670mL toluene were added to a 5L one-neck flask, stirred uniformly, heated to 85℃ and reacted for 2.5h. After the raw materials were completely reacted, the mixture was cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (PE:EA=5:1-2:1) to obtain compound M1 (87g). Yield 84.4%, LC-MS: [M+NH4] + =386.0.

[0096] Example 6 Synthesis of compound M3 [ka] Compound SM-2 (synthesized according to the method disclosed in Chinese Patent Application Publication No. CN108452321A) (40 g, 96 mmol, 1.0 eq), triethylamine (26.7 mL, 2.0 eq), and toluene (400 mL) were placed in a 1000 mL one-neck flask, heated to 120 ° C, and refluxed for 2 h. After confirming that the reaction was almost complete by TLC, the temperature was lowered to 50 ° C, and the solvent was removed by rotary evaporation under reduced pressure. The mixture was dissolved in ethyl acetate (150 mL) and water (40 mL), and the pH was adjusted to 2-3 with 1 M HCl while stirring in an ice bath, and the mixture was separated. The aqueous layer was extracted again with ethyl acetate, and the organic layer was combined and dried by adding anhydrous sodium sulfate. After filtration, the mixture was concentrated to obtain a pale yellow oily crude product, which was purified by column chromatography (DCM:MeOH = 40:1) to obtain 26.6 g compound M2. LC-MS: [M + H] + =399.3.

[0097] 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 placed in a 1 L one-neck flask and reacted at room temperature for 30 min (monitored by TLC), and insoluble matter was removed by filtration. The reaction solution was directly separated and purified, and the separated solution was decompressed with a water pump and concentrated in a water bath at 35 °C to remove acetonitrile, and then lyophilized to obtain 31.5 g compound M3. Yield 64%; LC-MS: [M+H] + =565.1.

[0098] Example 7 Synthesis of compound 1 [ka] Step 1: Synthesis of compound 1a M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were placed in a 250 mL one-neck flask, cooled to 0 ° C while stirring, and benzyl hydroxyacetate (5.4 g, 32.6 mmol) was added dropwise. After the addition, the mixture was allowed to naturally warm to room temperature and react (about 2-4 h), and monitored by TLC. After the reaction was completed, 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 obtain compound 1a (4 g). Yield 52%; LC-MS: [M + H] + =475.2.

[0099] Step 2: Synthesis of compound 1b Compound 1a (2 g, 4.2 mmol) and 10 mL DMF were placed in a 25 mL single-neck flask and stirred at 0°C. DBU (766 mg, 5.04 mmol) was added and reacted for 1 h. After completion of Fmoc deprotection was confirmed by TLC, the mixture was stored until use. Compound M4 (prepared according to the method disclosed in Chinese Patent Application Publication No. CN111051330A) (1.73 g, 4.2 mmol), PyBOP (2.61 g, 5.04 mmol), HOBt (680 mg, 5.04 mmol) and 10 mL DMF were added to a new 25 mL one-neck flask, and DIPEA (830 uL, 5.04 mmol) was added under ice water bath, and the mixture was stirred for 30 min. The reaction solution was added to a reaction flask and heated to room temperature to react. After confirming the completion of the reaction by HPLC, the reaction solution was purified by preparative liquid chromatography 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 compound 1b (1.7 g). Yield 63%; LCMS: [M+H] + =648.3.

[0100] Step 3: Synthesis of compound 1c Compound 1b (900 mg, 1.39 mmol) and 15 mL DMF were placed in a 25 mL one-neck flask and dissolved transparently. After that, 900 mg of 5% Pd / C was added and the hydrogenation reaction was carried out for 2 hours. After the reaction was completed, the filtrate was filtered and used in the next reaction without purification.

[0101] Step 4: Synthesis of Compound 1 The DMF solution of compound 1c obtained in the above step was placed in an ice-water bath, DIPEA (235uL, 1.39mmol) was added, and compound M3 (784mg, 1.39mmol) was further added, and then the temperature was raised to room temperature and reacted for 1h. After monitoring the completion of the reaction by HPLC, the reaction solution was purified by high performance liquid chromatography to obtain a fraction, which was then freeze-dried to obtain compound 1 (604mg). Yield 54%; LC-MS: [M+H] + =804.4.

[0102] Example 8 Synthesis of compound 2 [ka] Step 1: Synthesis of compound 1c Compound 1b (900 mg, 1.39 mmol) and 15 mL DMF were placed in a 25 mL one-neck flask and dissolved transparently. After that, 900 mg of 5% Pd / C was added and the hydrogenation reaction was carried out for 2 hours. After the reaction was completed, the filtrate was filtered and used in the next reaction without purification.

[0103] Step 2: Synthesis of compound 2 The DMF solution of compound 1c obtained in the previous step was placed in an ice-water bath, DIPEA (235uL, 1.39mmol) was added, and McOSu (428.5mg, 1.39mmol) was further added, and then the temperature was raised to room temperature and reacted for 1h. After monitoring the completion of the reaction by HPLC, the reaction solution was purified by high performance liquid chromatography to obtain a fraction, which was then freeze-dried to obtain compound 2 (500mg). Yield 58%; LC-MS: [M+H] + =617.3.

[0104] Example 9 Synthesis of Compounds 3A and 3B 1. Preparation of Compound 3A [ka]

[0105] Step 1: Synthesis of compound 3a In a 25mL one-neck flask, M1 (1.5g, 4.0mmol, 1.0eq), p-toluenesulfonic acid monohydrate (77mg, 0.4mmol, 0.1eq) and 15mL THF were added, stirred uniformly, cooled to 0℃, and L-benzyl lactate (2.2g, 12.0mmol, 3eq) was slowly added, and then the temperature was raised to room temperature to react. The reaction was monitored by TLC, and after completion of the reaction, a saturated NaHCO3 solution was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by reverse phase column to obtain compound 3a (1.03g). Yield 52%, LC-MS: [M+NH4] + =506.2.

[0106] Step 2: Synthesis of compound 3b Compound 3a (1 g, 2.04 mmol) and 8 mL DMF were placed in a 25 mL one-neck flask, stirred uniformly, cooled to 0°C, and DBU (373 mg, 2.45 mmol) was slowly added, and then the mixture was allowed to react by heating to room temperature. The reaction was monitored by TLC, and after completion of the reaction, it was designated as reaction solution (1). In a new 25 mL one-neck flask, M4 (846 mg, 2.04 mmol), PyBOP (1.27 g, 2.45 mmol) and 6 mL DMF were added and stirred at room temperature for 5 min. The reaction mixture (1) was added and reacted at room temperature and monitored by HPLC. After completion of the reaction, the reaction mixture was purified by high performance liquid chromatography and freeze-dried to obtain compound 3b (913 mg). Yield 67.6%, LC-MS: [M+NH4] + =679.2.

[0107] Step 3: Synthesis of compound 3c Compound 3b (800 mg, 1.21 mmol, 1.0 eq) was dissolved in DMF (15 mL) in a 100 mL one-neck flask, and 5% Pd / C (800 mg) was added and hydrogenated at room temperature for 2 h (the reaction was monitored by HPLC). The Pd / C was removed by filtration, and the filtrate was used in the next reaction without concentration.

[0108] Step 4: Synthesis of compound 3A The DMF solution of compound 3c obtained in the previous step was placed in an ice-water bath, DIPEA (219uL, 1.21mmol) was added, and compound M3 (683mg, 1.21mmol) was further added, and then the temperature was raised to room temperature and reacted for 1h. After monitoring the completion of the reaction by HPLC, the reaction solution was purified by high performance liquid chromatography to obtain a fraction, which was then freeze-dried to obtain compound 3A (524mg). Yield 53%, LC-MS: [MH] - =816.3.

[0109] 2. Preparation of Compound 3B [ka] Compound 3B was obtained by replacing L-benzyl lactate with D-benzyl lactate in step 1 using the synthesis method for compound 3A described above. LC-MS: [MH] - =816.3.

[0110] Example 10 Synthesis of Compounds 4A and 4B 1. Preparation of Compound 4A [ka] Step 1: Synthesis of compound 3c 3b (800 mg, 1.21 mmol, 1.0 eq) and DMF (15 mL) were added to a 100 mL one-neck flask and dissolved, and 5% Pd / C (800 mg) was added and hydrogenation reaction was carried out at room temperature for 2 h (reaction status was monitored by HPLC). Pd / C was removed by filtration, and the filtrate was used in the next reaction without concentration.

[0111] Step 2: Synthesis of compound 4A The DMF solution of compound 3c obtained in the previous step was placed in an ice-water bath, DIPEA (219 uL, 1.21 mmol) was added, and McOSu (373 mg, 1.21 mmol) was further added, and then the temperature was raised to room temperature and reacted for 1 h. After monitoring the completion of the reaction by HPLC, the reaction solution was purified by high performance liquid chromatography to obtain a fraction, and the fraction was freeze-dried to obtain compound 4A (450 mg). Yield 59%; LC-MS: [M+H] + =631.3.

[0112] 2. Synthesis of compound 4B [ka] Compound 4B was obtained by following the synthesis method of compound 4A. LC-MS: [M+H] + =631.3.

[0113] Example 11 Synthesis of compound 5 [ka] Step 1: Synthesis of compound 5a In a 250 mL one-neck flask, M1 (10 g, 27.1 mmol), 3,3,3-benzyl trifluorolactate (prepared by the method disclosed in WO2020063673A1) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL toluene were placed, heated to 100 ° C and reacted for 4 h. After the reaction was completed, the temperature was lowered to room temperature, insoluble matter was removed by filtration, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain compound 5a (5.15) g. Yield 35.1%; LC-MS: [M + H] + =543.2.

[0114] Step 2: Synthesis of compound 5b Compound 5a (5 g, 9.2 mmol) and 15 mL of DMF were placed in a 50 mL one-neck flask and dissolved transparently. DBU (1.68 g, 11 mmol) was then added in an ice-water bath and the mixture was reacted for 1 hour. This was designated as reaction solution (1). In a new 50 mL one-neck flask, M4 (3.8 g, 9.2 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol) and 10 mL DMF were added and dissolved to a clear solution. Then, DIPEA (1.82 mL, 11 mmol) was added under an ice-water bath, and the mixture was allowed to react for 30 min. The reaction solution (1) was added, and the mixture was heated to room temperature and allowed to react for 2 h. The reaction status 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 compound 5b (4.1) g. Yield 62.3%; LC-MS: [M+H] + =716.3.

[0115] Step 3: Synthesis of compound 5 Compound 5b (900 mg, 1.26 mmol) and 15 mL DMF were placed in a 25 mL one-neck flask, dissolved transparently, then 900 mg of 5% Pd / C was added, and hydrogenation reaction was carried out for 2 h. After the reaction was completed, the mixture was filtered, the filtrate was placed in an ice-water bath, DIPEA (228 uL, 1.38 mmol) was added, and compound M3 (712 mg, 1.26 mmol) was further added, and the mixture was then heated to room temperature and reacted for 1 h. After monitoring the completion of the reaction by HPLC, the reaction solution was purified by high performance liquid chromatography, and the fraction was obtained. The fraction was freeze-dried to obtain the product compound 5 (525) mg. Yield 47.9%; LC-MS: [MH] - =870.3.

[0116] Example 12 Synthesis of compound 6 [ka] Compound 5b (900 mg, 1.26 mmol) and 15 mL DMF were placed in a 25 mL one-neck flask, dissolved transparently, then 900 mg of 5% Pd / C was added and hydrogenated for 2 h. After the reaction was completed, the mixture was filtered, the filtrate was placed in an ice-water bath, DIPEA (228 uL, 1.38 mmol) was added, and McOSu (388 mg, 1.26 mmol) was added. The mixture was then heated to room temperature and reacted for 1 h. After monitoring the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction, which was then freeze-dried to obtain the product compound 6 (450) mg. Yield 52%; LC-MS: [MH] - =683.3.

[0117] Example 13 Synthesis of compound 7 [ka] Step 1: Synthesis of compound 7a In a 250 mL one-neck flask, 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 toluene were added, heated to 100 ° C. and reacted for 4 h. After the reaction was completed, the temperature was lowered to room temperature, insoluble matter was removed by filtration, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain compound 7a (4.97 g). Yield 36%; LC-MS: [M + H] + =515.2.

[0118] Step 2: Synthesis of compound 7b Compound 7a (4 g, 7.8 mmol) and 10 mL of DMF were placed in a 50 mL one-neck flask and dissolved transparently. DBU (1.42 g, 9.3 mmol) was then added in an ice-water bath and the mixture was reacted for 1 hour. This was designated as reaction solution (1). In a new 50mL one-neck flask, M4 (3.2g, 7.8mmol), PyBOP (4.5g, 8.6mmol), HOBt (1.16g, 8.6mmol) and 10mL DMF were added and dissolved transparently. Then, DIPEA (1.65mL, 10mmol) was added under ice water bath, and the mixture was reacted for 30min. The reaction solution (1) was added, and the mixture was heated to room temperature and reacted for 2h. The reaction status 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 compound 7b (4.2g). Yield 78%; LC-MS: [M+H] + =688.3.

[0119] Step 3: Synthesis of compound 7 Compound 7b (1000mg, 1.45mmol) and 15mL DMF were placed in a 25mL one-neck flask, dissolved transparently, then 1000mg of 5% Pd / C was added, and hydrogenation reaction was carried out for 2h. After completion of the reaction, the mixture was filtered, the filtrate was placed in an ice-water bath, DIPEA (248uL, 1.5mmol) was added, and compound M3 (720mg, 1.45mmol) was further added, and the mixture was then heated to room temperature and reacted for 1h. After monitoring the completion of the reaction by HPLC, the reaction solution was purified by high performance liquid chromatography, and the fraction was obtained. The fraction was freeze-dried to obtain the product compound 7 (503mg). Yield 41%; LC-MS: [MH] - =842.3.

[0120] Example 14 Synthesis of compound 8 [ka] Step 1: Synthesis of compound 8a In a 250 mL one-neck flask, M1 (10 g, 27.1 mmol), benzyl 2-hydroxy-3-cyclopropylpropionate (synthesized according to the method disclosed in WO2020063676A) (12.0 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL toluene were added, heated to 100 ° C and reacted for 4 h. After the reaction was completed, the temperature was lowered to room temperature, insoluble matter was removed by filtration, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain the target product 8a (5.09 g). LC-MS: [M + H] + =529.2.

[0121] Step 2: Synthesis of compound 8b Compound 8a (4 g, 7.6 mmol) and 10 mL of DMF were placed in a 50 mL one-neck flask and dissolved transparently. DBU (1.39 g, 9.1 mmol) was then added in an ice-water bath and the mixture was reacted for 1 hour. This was designated as reaction solution (1). In a new 50 mL one-neck flask, M4 (3.12 g, 7.6 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol) and 10 mL DMF were added and dissolved transparently. Then, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath, and the mixture was allowed to react for 30 min. The reaction solution (1) was added, and the mixture was heated to room temperature and allowed to react for 2 h. The reaction status 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 compound 8b (4.5 g). Yield 84%; LC-MS: [M+H] + =702.3. Step 3: Synthesis of compound 8 In a 25mL one-neck flask, 8b (1000mg, 1.42mmol) and 15mL DMF were added, and after dissolving to a clear solution, 1000mg of 5% Pd / C was added and hydrogenated for 2h. After the reaction was completed, the mixture was filtered, the filtrate was placed in an ice-water bath, DIPEA (248uL, 1.5mmol) was added, and compound M3 (708mg, 1.42mmol) was added. The mixture was then heated to room temperature and reacted for 1h. After monitoring the completion of the reaction by HPLC, the reaction solution was purified by high performance liquid chromatography, and the fraction was obtained. The fraction was freeze-dried to obtain the product compound 8 (443mg). Yield 36%; LC-MS: [MH] - =856.4.

[0122] Example 15 Synthesis of compound 9 [ka] Step 1: Synthesis of compound 9a Amino-octapolyethylene glycol-carboxyl (10 g, 22.7 mmol) and 20 mL DMF were added to a 500 mL one-neck flask, dissolved in water, McOSu (8.38 g, 27.2 mmol) and DIEA (5.6 mL, 3.4 mmol) were added, and the mixture was reacted at room temperature for 2 h and monitored by TLC. After the reaction was completed, the reaction solution was added to 100 mL water, extracted three times with dichloromethane, and the organic phases were combined, washed twice with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated under reduced pressure at 45 °C to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM:MeOH = 30:1-10:1), and the product eluate was collected and concentrated to obtain compound 9a (12.1 g). Yield 84.5%, LCMS: [MH] + =633.3.

[0123] Step 2: Synthesis of compound 9b Compound 9a (12.0 g, 18.9 mmol), pentafluorophenol (3.83 g, 20.8 mmol), DCC (4.28 g, 20.8 mmol) and THF (50 mL) were placed in a 500 mL one-neck flask and reacted at room temperature for 1 h and monitored by TLC. After the reaction was completed, insoluble matter was removed by filtration. The filtrate was reduced in pressure with a water pump and concentrated at 45 °C to remove the solvent and obtain a residue. The residue was purified by silica gel column chromatography (PE:EA = 5:1-2:1), and the product eluate was collected and concentrated to obtain compound 9b (13.8 g). Yield 91.5%, LCMS: [M + H] + =801.3.

[0124] Step 3: Synthesis of compound 9 Compound 1c (220 mg, 0.5 mmol) was added with 10 mL DMF, compound 9b (500 mg, 0.6 mmol) was added in an ice-water bath, and the mixture was cooled to 0°C. Compound DIPEA (124 uL, 0.75 mmol) was added, and the mixture was reacted at room temperature for 1 h. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography to obtain a product fraction. After removing acetonitrile from the fraction under reduced pressure, the product was freeze-dried to obtain compound 9 (430 mg). Yield 83%, LCMS: [MH] + =1038.5.

[0125] Example 16 Synthesis of compound 10 [ka] Compound 2 (50 mg, 0.081 mmol), compound B (55.8 mg, 0.081 mmol), PyBOP (50.4 mg, 0.097 mmol), HOBt (13.1 mg, 0.097 mmol) and 5 mL DMF were placed in a 25 mL one-neck flask, DIPEA (15.5 mg, 0.12 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. After confirming the end of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction, and the fraction was freeze-dried to obtain compound 10 (80 mg). Yield 76.7%, LC-MS: [M+H] +=1287.5.

[0126] Example 17 Synthesis of compound 11 [ka] Compound 9 (84.2 mg, 0.081 mmol), compound B (55.8 mg, 0.081 mmol), PyBOP (50.4 mg, 0.097 mmol), HOBt (13.1 mg, 0.097 mmol) and 5 mL DMF were placed in a 25 mL one-neck flask, DIPEA (15.5 mg, 0.12 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a product fraction. The fraction was freeze-dried to obtain compound 11 (100 mg). Yield 70.9%, LC-MS: [M / 2+H] + =855.86.

[0127] Example 18 Synthesis of Compounds 12A and 12B 1. Preparation of Compound 12A [ka] Compound 4A (51 mg, 0.081 mmol), compound B (55.8 mg, 0.081 mmol), PyBOP (50.4 mg, 0.097 mmol), HOBt (13.1 mg, 0.097 mmol) and 5 mL DMF were placed in a 25 mL one-neck flask, DIPEA (15.5 mg, 0.12 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction, which was then freeze-dried to obtain compound 12A (80 mg). Yield 76.2%, LC-MS: [M+H] + =1301.5.

[0128] 2. Preparation of Compound 12B [ka] Compound 12B was obtained by following the synthesis method of compound 12A. LC-MS: [M+H] + =1301.5.

[0129] Example 19 Synthesis of Compounds 13A and 13B [ka] Compound 6 (99.3 mg, 0.145 mmol), compound B (100 mg, 0.145 mmol), PyBOP (90.5 mg, 0.174 mmol), HOBt (23.5 mg, 0.174 mmol) and 10 mL DMF were added to a 50 mL one-neck flask, and DIPEA (28.2 mg, 0.218 mmol) was added under an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 13A and compound 13B. The fractions were each freeze-dried to obtain compound 13A (90 mg) and compound 13B (80 mg). LC-MS: [M+H] + =1355.5.

[0130] Example 20 Synthesis of compound 14 [ka] Step 1: Synthesis of compound 14a Compound 1 (500 mg, 0.62 mmol), compound B (427.3 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 14a. The fraction was freeze-dried to obtain solid compound 14a (550 mg). Yield 60%, LC-MS: [M+H] + =1474.6.

[0131] Step 2: Synthesis of compound 14 Compound 14a (200 mg, 0.136 mmol), zinc bromide (612 mg, 2.72 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 14 (100 mg). Yield 56%, LC-MS: [M+H] + =1318.5.

[0132] Example 21 Synthesis of Compounds 15A and 15B 1. Preparation of Compound 15A [ka] Step 1: Synthesis of compound 15a Compound 3A (507 mg, 0.62 mmol), compound B (427.3 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high performance liquid chromatography to obtain a fraction of compound 15a. The fraction was freeze-dried to obtain solid compound 15a (650 mg). Yield 70%, LC-MS: [M+H] + =1488.6.

[0133] Step 2: Synthesis of compound 15A Compound 15a (200 mg, 0.134 mmol), zinc bromide (603.5 mg, 2.68 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 15A (110 mg). Yield 62%, LC-MS: [M+H] + =1332.5.

[0134] 2. Preparation of Compound 15B [ka] Compound 15B was obtained by following the synthesis method of compound 15A. LC-MS: [M+H] + =1332.5.

[0135] Example 22 Synthesis of Compounds 16A and 16B [ka] Step 1: Synthesis of compounds 16a and 16b Compound 5 (540 mg, 0.62 mmol), compound B (427.3 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were added to a 50 mL one-neck flask, and DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the mixture was allowed to react for 2 h after warming to room temperature. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 16a and compound 16b. The fractions were freeze-dried to obtain compound 16a (350 mg) and compound 16b (300 mg). LC-MS: [M / 2+H] + =771.8.

[0136] Step 2: Synthesis of compound 16A [ka] Compound 16a (200 mg, 0.13 mmol), zinc bromide (585.5 mg, 2.6 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 16A (120 mg). Yield 66%, LC-MS: [M+H] + =1386.4.

[0137] Step 3: Synthesis of compound 16B [ka] Compound 16b (200 mg, 0.13 mmol), zinc bromide (585.5 mg, 2.6 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 16B (115 mg). Yield 64%, LC-MS: [M+H] + =1386.4.

[0138] Example 23 Synthesis of Compounds 17A and 17B [ka] Step 1: Synthesis of compounds 17a and 17b Compound 7 (523 mg, 0.62 mmol), compound B (427.3 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the mixture was allowed to react for 2 h after warming to room temperature. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 17a and compound 17b. The fractions were freeze-dried to obtain compound 17a (320 mg) and compound 17b (280 mg). LC-MS: [M / 2+H] + =757.8.

[0139] Step 2: Synthesis of compound 17A [ka] Compound 17a (200 mg, 0.132 mmol), zinc bromide (594.5 mg, 2.64 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 17A (120 mg). Yield 67%, LC-MS: [M+H] + =1358.5.

[0140] Step 3: Synthesis of compound 17B [ka] Compound 17b (200 mg, 0.132 mmol), zinc bromide (594.5 mg, 2.64 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 17B (116 mg). Yield 65%, LC-MS: [M+H] + =1358.5.

[0141] Example 24 Synthesis of Compounds 18A and 18B [ka] Step 1: Synthesis of compounds 18a and 18b Compound 8 (532 mg, 0.62 mmol), compound B (427.3 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 18a and compound 18b. The fractions were each freeze-dried to obtain compound 18a (300 mg) and compound 18b (280 mg). LC-MS: [M / 2+H] + =764.8.

[0142] Step 2: Synthesis of compound 18A [ka] Compound 18a (200 mg, 0.131 mmol), zinc bromide (590.0 mg, 2.62 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 18A (125 mg). Yield 70%, LC-MS: [M+H] + =1372.5.

[0143] Step 3: Synthesis of compound 18B [ka] Compound 18b (200 mg, 0.131 mmol), zinc bromide (590.0 mg, 2.62 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 18B (120 mg). Yield 67%, LC-MS: [M+H] + =1372.5.

[0144] Example 25 Synthesis of compound 19 [ka] Step 1: Synthesis of compound 19a Compound 1 (500 mg, 0.62 mmol), compound C (412.4 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 19a. The fraction was freeze-dried to obtain solid compound 19a (600 mg). Yield 66%, LC-MS: [M+H] + =1450.6.

[0145] Step 2: Synthesis of compound 19 Compound 19a (200 mg, 0.138 mmol), zinc bromide (621 mg, 2.76 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 19 (120 mg). Yield 67%, LC-MS: [M+H] + =1294.5.

[0146] Example 26 Synthesis of Compound 20A and Compound 20B 1. Preparation of Compound 20A [ka] Step 1: Synthesis of compound 20a Compound 3A (507 mg, 0.62 mmol), compound C (412.4 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, and DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 20a. The fraction was freeze-dried to obtain solid compound 20a (560 mg). Yield 62%, LC-MS: [M+H] + =1464.6.

[0147] Step 2: Synthesis of compound 20A Compound 20a (200 mg, 0.136 mmol), zinc bromide (614.8 mg, 2.73 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 20A (120 mg). Yield 67%, LC-MS: [M+H] + =1308.5.

[0148] 2. Preparation of Compound 20B [ka] Compound 20B was obtained by following the synthesis method of compound 20A. LC-MS: [M+H] + =1308.5.

[0149] Example 27 Synthesis of Compounds 21A and 21B [ka] Step 1: Synthesis of compounds 21a and 21b Compound 5 (540 mg, 0.62 mmol), compound C (412.4 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 21a and compound 21b. The fractions were freeze-dried to obtain compound 21a (330 mg) and compound 21b (300 mg). LC-MS: [M / 2+H] + =759.8.

[0150] Step 2: Synthesis of compound 21A [ka] Compound 21a (200 mg, 0.132 mmol), zinc bromide (594.5 mg, 2.64 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 21A (125 mg). Yield 69%, LC-MS: [M+H] + =1362.4.

[0151] Step 3: Synthesis of compound 21B [ka] Compound 21b (200 mg, 0.132 mmol), zinc bromide (594.5 mg, 2.64 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 21B (112 mg). Yield 62%, LC-MS: [M+H] + =1362.4.

[0152] Example 28 Synthesis of Compounds 22A and 22B [ka] Step 1: Synthesis of compounds 22a and 22b Compound 7 (523 mg, 0.62 mmol), compound C (412.4 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high performance liquid chromatography to obtain fractions of compound 22a and compound 22b. The fractions were freeze-dried to obtain compound 22a (330 mg) and compound 22b (300 mg). LC-MS: [M+H] + =1490.6.

[0153] Step 2: Synthesis of compound 22A [ka] Compound 22a (200 mg, 0.134 mmol), zinc bromide (603.5 mg, 2.68 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction. The fraction was freeze-dried to obtain solid compound 22A (120 mg). Yield 67%, LC-MS: [M+H] + =1334.5.

[0154] Step 3: Synthesis of compound 22B [ka] Compound 22b (200 mg, 0.134 mmol), zinc bromide (603.5 mg, 2.68 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction. The fraction was freeze-dried to obtain solid compound 22B (115 mg). Yield 65%, LC-MS: [M+H]+=1334.5.

[0155] Example 29 Synthesis of Compounds 23A and 23B [ka] Step 1: Synthesis of compounds 23a and 23b Compound 8 (532 mg, 0.62 mmol), compound C (412.4 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 23a and compound 23b. The fractions were each freeze-dried to obtain compound 23a (320 mg) and compound 23b (300 mg). LC-MS: [M / 2+H] + =752.8.

[0156] Step 2: Synthesis of compound 23A [ka] Compound 23a (200 mg, 0.133 mmol), zinc bromide (598.5 mg, 2.66 mmol) and 6 mL of nitromethane were added to a 25 mL one-neck flask. The reaction was allowed to proceed at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 23A (122 mg). Yield 68%, LC-MS: [M+H] + =1348.5.

[0157] Step 3: Synthesis of compound 23B [ka] Compound 23b (200 mg, 0.133 mmol), zinc bromide (598.5 mg, 2.66 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 23B (115 mg). Yield 64%, LC-MS: [M+H] + =1348.5.

[0158] Example 30 Synthesis of compound 24 [ka] Step 1: Synthesis of compound 24a Compound 1 (500 mg, 0.62 mmol), compound D (396.3 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 24a. The fraction was freeze-dried to obtain solid compound 24a (600 mg). Yield 68%, LC-MS: [M+H] +=1424.6.

[0159] Step 2: Synthesis of compound 24 Compound 24a (200 mg, 0.14 mmol), zinc bromide (631 mg, 2.8 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction. The fraction was freeze-dried to obtain solid compound 24 (120 mg). Yield 68%, LC-MS: [M+H] + =1268.4.

[0160] Example 31 Synthesis of Compound 25A and Compound 25B 1. Preparation of Compound 25A [ka] Step 1: Synthesis of compound 25a Compound 3A (507 mg, 0.62 mmol), compound D (396.3 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 25a. The fraction was freeze-dried to obtain solid compound 25a (560 mg). Yield 63%, LC-MS: [M+H] + =1438.6.

[0161] Step 2: Synthesis of compound 25A Compound 25a (200 mg, 0.139 mmol), zinc bromide (626 mg, 2.78 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 25A (115 mg). Yield 65%, LC-MS: [M+H]+=1282.5.

[0162] 2. Preparation of Compound 25B [ka] Compound 26B was obtained by following the synthesis method of compound 25A. LC-MS: [M+H]+=1282.5.

[0163] Example 32 Synthesis of Compounds 26A and 26B [ka] Step 1: Synthesis of compounds 26a and 26b Compound 5 (540 mg, 0.62 mmol), compound D (396.3 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 26a and compound 26b. The fractions were each freeze-dried to obtain compound 26a (330 mg) and compound 26b (300 mg). LC-MS: [M+H] + =1492.5.

[0164] Step 2: Synthesis of compound 26A [ka] Compound 26a (200 mg, 0.134 mmol), zinc bromide (603.5 mg, 2.68 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 26A (125 mg). Yield 70%, LC-MS: [M+H] + =1336.4.

[0165] Step 3: Synthesis of compound 26B [ka] Compound 26b (200 mg, 0.134 mmol), zinc bromide (603.5 mg, 2.68 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction. The fraction was freeze-dried to obtain solid compound 26B (112 mg). Yield 63%, LC-MS: [M+H] + =1336.4.

[0166] Example 33 Synthesis of Compounds 27A and 27B [ka] Step 1: Synthesis of compounds 27a and 27b Compound 7 (523 mg, 0.62 mmol), compound D (396.3 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 27a and compound 27b. The fractions were freeze-dried to obtain compound 27a (330 mg) and compound 27b (300 mg). LC-MS: [M+H] + =1464.6.

[0167] Step 2: Synthesis of compound 27A [ka] Compound 27a (200 mg, 0.136 mmol), zinc bromide (614.8 mg, 2.73 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 27A (125 mg). Yield 70%, LC-MS: [M+H] + =1308.5.

[0168] Step 3: Synthesis of compound 27B [ka] Compound 27b (200 mg, 0.136 mmol), zinc bromide (614.8 mg, 2.73 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 27B (115 mg). Yield 64%, LC-MS: [M+H] + =1308.5.

[0169] Example 34 Synthesis of Compounds 28A and 28B [ka] Step 1: Synthesis of compounds 28a and 28b Compound 8 (532 mg, 0.62 mmol), compound D (396.3 mg, 0.62 mmol), PyBOP (385 mg, 0.74 mmol), HOBt (100 mg, 0.74 mmol) and 15 mL DMF were placed in a 50 mL one-neck flask, DIPEA (120 mg, 0.93 mmol) was added in an ice-water bath, and the temperature was raised to room temperature and reacted for 2 h. The reaction was monitored by HPLC, and after completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain fractions of compound 28a and compound 28b. The fractions were each freeze-dried to obtain compound 28a (320 mg) and compound 28b (300 mg). LC-MS: [M+H] + =1478.6. Step 2: Synthesis of compound 28A [ka] Compound 28a (200 mg, 0.135 mmol), zinc bromide (608 mg, 2.7 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 28A (121 mg). Yield 68%, LC-MS: [M+H] + =1322.5.

[0170] Step 3: Synthesis of compound 28B [ka] Compound 28b (200 mg, 0.135 mmol), zinc bromide (608 mg, 2.7 mmol) and 6 mL of nitromethane were placed in a 25 mL one-neck flask and reacted at room temperature for 1 h. The reaction was monitored by HPLC, and after completion of the reaction, 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. The fraction was freeze-dried to obtain solid compound 28B (115 mg). Yield 64%, LC-MS: [M+H] + =1322.5.

[0171] Example 34 Compound 29 synthesis [ka] Step 1: Synthesis of compound 29d from compound 29a Compound 29d was obtained according to the synthesis of “Compound 26” in CN102933236A.

[0172] Step 2: Synthesis of compound 29e Compound 29d (3.0 g, 5.8 mmol) and 50 mL anhydrous THF were placed in a 250 mL three-neck flask, and kept at 0 ° C in an ice-water bath under nitrogen gas protection. Solid sodium borohydride (329 mg, 8.7 mmol) was added, and the mixture was reacted at 0 ° C for 30 min, then warmed to room temperature and reacted for 2 h. The end point of the reaction was monitored by TLC. After the reaction was completed, the reaction solution was placed in ice water, and 50 mL of water was slowly added to quench the reaction. Further, 1N diluted HCl was added until no bubbles were generated, and ethyl acetate (60 mL * 3) was added for extraction. The organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated crude product was obtained. The crude product was purified by column chromatography (DCM / MeOH = 20: 1-5: 1) to obtain compound 29e (2.1 g, yellow solid). Yield 73.8%, LCMS: [M + H] + =491.2.

[0173] Step 3: Synthesis of compound 29f Compound 29e (2.1 g, 4.3 mmol), TBSCl (1.3 g, 8.6 mmol), and imidazole (580 mg, 8.6 mmol) were placed in a 100 mL one-neck flask, dissolved in 15 mL anhydrous DMF, and reacted at room temperature for 3 h, and the end point of the reaction was monitored by TLC. After the reaction was completed, the reaction solution was added to 30 mL water and extracted with DCM (25 mL * 3), the organic phase was combined, washed with water and brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 5: 1-1: 2) to obtain compound 29f (2.4 g, yellow solid). Yield 89.3%, LCMS: [M + H] + =605.3.

[0174] Step 4: Synthesis of compound 29g Compound 29f (2.4 g, 4.0 mmol) and 30 mL of 5% formic acid / methanol solution were placed in a 100 mL one-neck flask, cooled to below 5 ° C in an ice bath, zinc powder (5.2 g, 80.0 mmol) was slowly added while stirring, and the mixture was allowed to react for 1 h after warming to room temperature. The end point of the reaction was monitored by TLC. After the reaction was completed, the mixture was filtered while still hot, the filter cake was washed with a small amount of methanol, and the filtrate was adjusted to pH 7 with saturated sodium bicarbonate solution, and then concentrated under reduced pressure at 45 ° C to remove the solvent, resulting in a yellow-brown oil. 60 mL DCM was added, the organic layer was washed with saturated saline, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 45 ° C to obtain the crude product. The crude product was purified by column chromatography (PE:EA = 1:1-1:3) to obtain compound 29g (1.9 g, pale yellow solid). Yield 82.5%, LCMS: [M + H] + =575.3.

[0175] Step 5: Synthesis of compound 29h Compound 29g (1.9g, 3.3mmol), Fmoc-VCPABO-PNP (2.5g, 3.3mmol), and 15mL DMF were placed in a 50mL one-neck flask and dissolved to obtain a clear solution. DIPEA (827uL, 5.0mmol) was then added and the reaction was carried out at room temperature for 2h, and the end point of the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was separated and purified by high performance liquid chromatography to obtain a product solution. The product was then lyophilized to obtain compound 29h (3.3g, pale yellow solid). Yield 83.1%, LCMS: [M+H] + =1202.5.

[0176] Step 6: Synthesis of compound 29i Compound 29h (3.3 g, 2.7 mmol), 8 mL THF and 8 mL water were added to a 50 mL one-neck flask and dissolved. 20 mL glacial acetic acid was added and reacted at room temperature. The end point of the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was slowly dripped into 400 mL saturated sodium bicarbonate solution, extracted with ethyl acetate (100 mL * 3), the organic phase was combined, washed with water and brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 10: 1-5: 1) to obtain compound 29i (2.1 g, pale yellow solid). Yield 71.1%, LCMS: [M + H] + =1088.5.

[0177] Step 7: Synthesis of compound 29j Compound 29i (2.1 g, 1.9 mmol) was dissolved in 40 mL of anhydrous DCM in a 100 mL three-neck flask, and Dess-Martin hypervalent iodine reagent (0.88 g, 2.1 mmol) was added under nitrogen gas protection, and the reaction was carried out at room temperature for 4 h, and the reaction end point was monitored by HPLC. After the reaction was completed, the mixture was filtered, and the filtrate was washed with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by high-performance liquid chromatography, and the product fraction was freeze-dried to obtain compound 29j (1.6 g, white solid). Yield 77.6%, LCMS: [M+H] + =1086.5.

[0178] Step 8: Synthesis of compound 29k Compound 29j (1.6 g, 1.5 mmol), 5% palladium barium sulfate (0.29 g), ammonium formate (1.9 g, 30 mmol), and 20 mL of methanol were dissolved in a 50 mL three-neck flask, and reacted at 45 ° C. for 2 h under nitrogen gas protection, and the reaction end point was monitored by HPLC. After the reaction was completed, the mixture was filtered and the filtrate was concentrated, and then 20 mL of DCM and 20 mL of water were added. After homogeneous stirring, the mixture was separated, the aqueous layer was extracted with DCM, and the organic layer was combined, washed with a saturated aqueous sodium chloride solution, separated, dried with anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. Compound 29k (1.3 g, off-white solid) was obtained by lyophilization. Yield 93.3%, LCMS: [M + H] + =996.4.

[0179] Step 9: Synthesis of compound 29l In a 100 mL one-neck flask, 3-((p-toluenesulfonyloxy) methyl)bicyclo[1.1.1]pentane-1-benzyl formate (0.61 g, 1.2 mmol), compound 29k (1.3 g, 1.3 mmol) and 20 mL DMF were added, stirred and dissolved, then cesium carbonate (0.78 g, 2.4 mmol) was added, and the mixture was heated to 60° C. under nitrogen gas protection and reacted for 4 h, and the reaction end point was monitored by TLC. 40 mL of water was added to the reaction solution, and the mixture was extracted twice with 30 mL of EA, and the organic layers were combined and washed with water and saturated saline in that order. The organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 45° C. to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The mixture was lyophilized to obtain compound 29l (1.2 g, off-white solid). Yield 84.0%, LCMS: [M+H] + =1210.5.

[0180] Step 10: Synthesis of compound 29m Compound 29l (1.2 g, 1.0 mmol), 5% palladium barium sulfate (0.24 g), ammonium formate (1.3 g, 20 mmol), and 20 mL of methanol were dissolved in a 50 mL three-neck flask, and reacted at 45 ° C. for 2 h under nitrogen gas protection, and the reaction end point was monitored by HPLC. After the reaction was completed, the mixture was filtered and the filtrate was concentrated, and then 20 mL of DCM and 20 mL of water were added. After homogeneous stirring, the mixture was separated, and the aqueous layer was extracted again with DCM. The organic layers were combined, washed with saturated aqueous sodium chloride solution, separated, dried with anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The mixture was lyophilized to obtain compound 29m (0.92 g, off-white solid). Yield 82.5%, LCMS: [M + H] + =1120.5.

[0181] Step 11: Synthesis of compound 29n Compound 29m (920 mg, 0.82 mmol) and 15 mL DMF were added to a 100 mL one-neck flask, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (496 mg, 2.6 mmol) and DMAP (313 mg, 2.6 mmol) were added in order while stirring at room temperature. The mixture was stirred at room temperature under N2 protection for 1 h, and then compound CBI (CN109180681A) (376 mg, 1.0 mmol) was added and the mixture was reacted at room temperature overnight under N2 protection, and the raw material was almost completely reacted by HPLC. 40 mL of water was added to the reaction solution, and the mixture was extracted twice with 30 mL DCM. The organic layers were combined and washed with water and saturated saline in order, and the organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 40 ° C. to obtain a crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction. The product was freeze-dried to obtain compound 29n (874 mg, white-ish solid). Yield 74.5%, LCMS: [M+H] + =1439.5.

[0182] Step 12: Synthesis of compound 29o Compound 29n (874 mg, 0.60 mmol), 5% palladium barium sulfate (174 mg), ammonium formate (390 mg, 6 mmol), and 20 mL of methanol were dissolved in a 50 mL three-neck flask, and reacted at 45 ° C. for 2 h under nitrogen gas protection, and the reaction end point was monitored by HPLC. After the reaction was completed, the mixture was filtered and the filtrate was concentrated, and then 20 mL of DCM and 20 mL of water were added. After homogeneous stirring, the mixture was separated, and the aqueous layer was extracted again with DCM. The organic layers were combined, washed with saturated aqueous sodium chloride solution, separated, dried with anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The mixture was lyophilized to obtain compound 29o (667 mg, off-white solid). Yield 82.4%, LCMS: [M + H] + =1349.5.

[0183] Step 13: Synthesis of compound 29p Compound 29o (667 mg, 0.49 mmol) and 20% hexahydropyridine DMF solution (15 mL) were placed in a 50 mL one-neck flask and reacted at room temperature for 2 h, and the reaction end point was monitored by HPLC. After the reaction was completed, the mixture was filtered and the filtrate was purified by high performance liquid chromatography to obtain a product fraction. The product was lyophilized to obtain compound 29p (428 mg, off-white solid). Yield 77.5%, LCMS: [M+H] + =1127.5.

[0184] Step 14: Synthesis of compound 29 Compound 29p (428 mg, 0.38 mmol), DIPEA (156 uL, 0.95 mmol), McOSu (166 mg, 0.57 mmol), and 20 mL DMF were dissolved in a 50 mL single-neck flask and reacted at room temperature for 2 h, and the reaction end point was monitored by HPLC. After the reaction was completed, the reaction solution was purified by high-performance liquid chromatography to obtain a product fraction. The product was lyophilized to obtain compound 29 (386 mg, off-white solid). Yield 77.0%, LCMS: [M+H] + =1320.5.

[0185] Example 35 compound 30 synthesis [ka] Step 1: Synthesis of compound 30a Compound 29p (560 mg, 0.50 mmol), DIPEA (161 mg, 1.25 mmol), compound M3 (423 mg, 0.75 mmol), and 10 mL DMF were dissolved in a 50 mL single-neck flask and reacted at room temperature for 2 hours, and the reaction end point was monitored by HPLC. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography to obtain a product fraction. The product was lyophilized to obtain compound 30a (622 mg, off-white solid). Yield 82.6%, LCMS: [M+H] + =1507.6.

[0186] Step 2: Synthesis of compound 30 Compound 30a (622 mg, 0.41 mmol), zinc bromide (922 mg, 4.1 mmol), and 10 mL of nitromethane were dissolved in a 50 mL one-neck flask and reacted at 25°C for 45 min. The end point of the reaction was monitored by HPLC. After the reaction was completed, the solvent was removed by rotary evaporation at reduced pressure at 40°C to obtain a concentrate. The product was purified by high-performance liquid chromatography to obtain a product fraction. The product was freeze-dried to obtain compound 30 (260 mg, off-white solid). Yield 48%, LCMS: [M+H] + =1322.5.

[0187] Example 36 Compound 31 synthesis [ka] JPEG0007667870000181.jpg50128Step 1: Synthesis of compound 31a In a 100mL one-neck flask, 5-bromovalerate benzyl (488mg, 1.8mmol), compound 29k (1.8g, 1.8mmol) and 20mL DMF were placed and dissolved under stirring, followed by the addition of potassium carbonate (372mg, 2.7mmol). The mixture was then heated to 60°C and reacted for 4h, and the end point of the reaction was monitored by HPLC. 40mL of water was added to the reaction solution, and the mixture was extracted twice with 30mL of EA. The organic layers were combined and washed with water and saturated saline in that order, and the organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 45°C to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. The mixture was lyophilized to obtain compound 31a (1.8g, off-white solid). Yield 84.0%, LCMS: [M+H] + =1186.5.

[0188] Step 2: Synthesis of compound 31b Compound 31a (1.8 g, 1.0 mmol), 5% palladium barium sulfate (0.36 g), ammonium formate (0.95 g, 15 mmol), and 20 mL of methanol were placed in a 50 mL three-neck flask, and reacted at 45 ° C. for 2 h under nitrogen gas protection, and the reaction end point was monitored by HPLC. After the reaction was completed, the mixture was filtered and the filtrate was concentrated, and then 20 mL DCM and 20 mL water were added. After homogeneous stirring, the mixture was separated, and the aqueous layer was extracted again with DCM. The organic layers were combined, washed with saturated aqueous sodium chloride solution, separated, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. Compound 31b (0.85 g, off-white solid) was obtained by lyophilization. Yield 78%, LCMS: [M + H] + =1096.5.

[0189] Step 4: Synthesis of compound 31c Compound 31b (850 mg, 0.85 mmol) and 15 mL DMF were placed in a 100 mL one-neck flask, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (514 mg, 2.7 mmol) and DMAP (324 mg, 2.7 mmol) were added in order while stirring at room temperature. After stirring at room temperature for 1 h under N2 protection, compound CBI (CN109180681A) (376 mg, 1.0 mmol) was added and reacted at room temperature overnight under N2 protection, and it was confirmed by HPLC that the raw material had almost completely reacted. 40 mL of water was added to the reaction solution, and the mixture was extracted twice with 30 mL DCM. The organic layers were combined and washed with water and saturated saline in order, and the organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 40 ° C. to obtain a crude product. The crude product was purified by high performance liquid chromatography to obtain a product fraction. The mixture was lyophilized to obtain compound 31c (941 mg, off-white solid). Yield 78.2%, LCMS: [M+H] + =1415.6.

[0190] Step 5: Synthesis of compound 31d Compound 31c (941 mg, 0.66 mmol), 5% palladium barium sulfate (188 mg), ammonium formate (415 mg, 6.6 mmol), and 20 mL of methanol were dissolved in a 50 mL three-neck flask, and reacted at 45 ° C. for 2 h under nitrogen gas protection, and the reaction end point was monitored by HPLC. After the reaction was completed, the mixture was filtered and the filtrate was concentrated, and then 20 mL of DCM and 20 mL of water were added. After homogeneous stirring, the mixture was separated, and the aqueous layer was extracted again with DCM. The organic layers were combined, washed with saturated aqueous sodium chloride solution, separated, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fraction. Compound 31d (735 mg, off-white solid) was obtained by lyophilization. Yield 84.0%, LCMS: [M + H] + =1325.5.

[0191] Step 6: Synthesis of compound 31e Compound 31d (735 mg, 0.55 mmol) and 20% hexahydropyridine DMF solution (15 mL) were placed in a 50 mL one-neck flask and reacted at room temperature for 2 hours, and the reaction end point was monitored by HPLC. After the reaction was completed, the mixture was filtered and the filtrate was purified by high performance liquid chromatography to obtain a product fraction. The product was then lyophilized to obtain compound 31e (428 mg, off-white solid). Yield 86.5%, LCMS: [M+H] + =1103.5.

[0192] Step 7: Synthesis of compound 31f Compound 31e (524 mg, 0.48 mmol), DIPEA (168 mg, 1.30 mmol), compound M3 (441 mg, 0.78 mmol), and 10 mL of DMF were dissolved in a 50 mL one-neck flask and reacted at room temperature for 2 hours, and the reaction end point was monitored by HPLC. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography to obtain a product fraction. The product was freeze-dried to obtain compound 31f (549 mg, off-white solid). Yield 77.2%, LCMS: [M+H] + =1483.6.

[0193] Step 8: Synthesis of compound 31 Compound 31f (549 mg, 0.37 mmol), zinc bromide (832 mg, 3.7 mmol), and 10 mL of nitromethane were dissolved in a 50 mL one-neck flask and reacted at 25°C for 45 min. The end point of the reaction was monitored by HPLC. After the reaction was completed, the solvent was removed by rotary evaporation at 40°C under reduced pressure to obtain a concentrate. The product was purified by high-performance liquid chromatography to obtain a product fraction. The product was freeze-dried to obtain compound 31 (213 mg, off-white solid). Yield 43.5%, LCMS: [M+H] + =1326.5.

[0194] Example 37 Compound 32 synthesis [ka] Step 1: Synthesis of compound 32a Compound N-propargylmaleimide (500mg, 3.7mmol), N3-PEG4-OH (811mg, 3.7mmol), 10mL DMF and 5mL water were added to a 50mL one-neck flask and dissolved. CuSO4·5H2O (1.01g, 4.1mmol) and sodium ascorbate (806mg, 4.1mmol) were added and reacted at room temperature for 1h. The end point 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 product fraction. The fraction was freeze-dried to obtain compound 32a (1.06g). Yield 81%, LCMS: [M+H] + =355.1.

[0195] Step 2: Synthesis of compound 32 Compound 32a (20 mg, 0.056 mmol), compound A (19.8 mg, 0.028 mmol), triphenylphosphine (29.6 mg, 0.11 mmol) and 8 mL DMF were placed in a 50 mL three-neck flask, and diisopropyl azodicarboxylate (28 uL, 0.11 mmol) was added dropwise in an ice-water bath under nitrogen gas protection, and then the temperature was raised to room temperature to react, and the reaction was monitored by HPLC. After the reaction was completed, the product was purified and lyophilized to obtain compound 32 (18 mg). Yield 63%, LC-MS: [M+H] + =1040.4.

[0196] Example 38 Compound 33 synthesis [ka]

[0197] Step 1: Synthesis of compound 33a It was prepared according to the synthesis method of “Compound 22” in Chinese Patent Application Publication No. CN111686259A.

[0198] Step 2: Synthesis of compound 33c Compound 33a (500 mg, 2.0 mmol) was dissolved in 8 mL of DMF in a 50 mL one-neck flask, 300 mg of 5% Pd / C was added, and hydrogenation reaction was carried out for 2 h. The end point of the reaction was monitored by TLC. After the reaction was completed, the mixture was filtered to obtain a DMF solution of compound 32b. The solution was stored until use. Compound Mc-tripeptide (992 mg, 2.0 mmol), PyBop (1.3 g, 2.5 mmol), HOBt (339 mg, 2.5 mmol) and DIEA (415 uL, 2.5 mmol) were added to the above DMF solution, and the reaction was allowed to proceed at room temperature for 2 h. The end point of the reaction was monitored by HPLC. The reaction solution was purified by high performance liquid chromatography to obtain a product fraction. Compound 33c (580 mg) was obtained by lyophilization. Yield 52%, LC-MS: [M+H] + =559.2.

[0199] Step 3: Synthesis of compound 33 Compound 33c (20 mg, 0.035 mmol), compound A (12.6 mg, 0.018 mmol), triphenylphosphine (18.4 mg, 0.07 mmol) and 5 mL DMF were placed in a 50 mL three-neck flask, and diisopropyl azodicarboxylate (14.2 uL, 0.07 mmol) was added dropwise in an ice-water bath under nitrogen gas protection, and then the temperature was raised to room temperature to react, and the reaction was monitored by HPLC. After the reaction was completed, the product was purified and lyophilized to obtain compound 33 (15 mg). Yield 68%, LC-MS: [M+H] + =1244.5.

[0200] Example 39 (Control) Synthesis of compounds 34 and 35 [ka] Compound 34 was synthesized according to the synthesis method of “Compound 56” in Chinese Patent Application Publication No. CN105636612A. [ka] Compound 35 was synthesized according to the synthesis method of “SG3249, tesirine” in “ACS Med Chem Lett. 2016;7:983-987”.

[0201] Example 40 Below is the sequence of Trastuzumab. Light chain MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 1) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 2) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 3) Heavy chain MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 4) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 5) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 6)

[0202] Preparation of ligand-drug conjugates 1) General bonding methods Antibody molecules Ab with a monomer rate of more than 95% after pre-purification were transferred to phosphate buffer (concentration 10 mg / mL) using an ultrafiltration centrifugation tube. TCEP was added in an amount 20 times the antibody mole number and reacted at room temperature for 10 h to cleave the disulfide bonds between the antibody chains. Linker-drug compounds (payload) were added in an amount 20 times the antibody mole number and reacted at room temperature for 2 h. After the reaction was completed, the solution was exchanged with PBS using an ultrafiltration centrifugation tube with a cut-off molecular weight of 30 KDa to remove unbound payload. The ADC sample after the liquid exchange was filtered through a 0.22 micron sterile filter and stored until use. 2) Measurement of DAR value of ligand-drug complex Conditions for detecting monomer ratio Samples were centrifuged at 14000 rpm for 5 min and the supernatant was loaded and analyzed. Equipment:Waters e2695(2489UV / Vis) Column: TSKgel G3000SWXL (7.8 x 300 mm, 5 μm) Mobile phase: A: 50 mM PB, 300 mM NaCl, 200 mM Arg, 5% IPA, pH 6.5 30 min isocratic elution using mobile phase A, flow rate: 0.714 mL / min, column temperature: 25°C, detection wavelength: 280 nm DAR detection conditions Samples were centrifuged at 14000 rpm for 5 min and the supernatant was loaded and analyzed. Equipment: Waters H-class (TUV) Column: Proteomix HIC Butyl-NP5 (4.6 x 35 mm, 5 μm) Mobile phase: A: 1.5 M ammonium sulfate, 0.025 M anhydrous sodium phosphate, pH 7.0; B: 0.025 M anhydrous sodium phosphate, 25% IPA, pH 7.0 The column was equilibrated with mobile phase A, and gradient elution was performed with mobile phases A and B at a flow rate of 0.8 mL / min; column temperature was 25°C, and detection wavelength was 214 nm.

[0203] Example 41 ADC-1 was obtained by preparing the general coupling method of Example 40. [ka] Example 42 ADC-2 was prepared by the general coupling method of Example 40. [ka] Example 43 ADC-3 was prepared by the general coupling method of Example 40. [ka] Example 44 Prepared according to the general coupling method of Example 40 to give ADC-4. [ka] Example 45 Prepared according to the general coupling method of Example 40 to give ADC-5. [ka] Example 46 Prepared according to the general coupling method of Example 40 to give ADC-6. [ka] Example 47 Prepared according to the general coupling method of Example 40 to give ADC-7. [ka] Example 48 Prepared according to the general coupling method of Example 40 to give ADC-8. [ka] Example 49 Prepared according to the general coupling method of Example 40 to give ADC-9. [ka] Example 50 Prepared according to the general coupling method of Example 40 to give ADC-10. [ka] Example 51 Prepared according to the general coupling method of Example 40 to give ADC-11. [ka] Example 52 ADC-12 was prepared by the general coupling method of Example 40. [ka] Example 53 Prepared according to the general coupling method of Example 40 to give ADC-13. [ka] Example 54 Prepared according to the general coupling method of Example 40 to give ADC-14. [ka] Example 55 Prepared according to the general coupling method of Example 40 to give ADC-15. [ka] Example 56 Prepared according to the general coupling method of Example 40 to give ADC-16. [ka] Example 57 Prepared according to the general coupling method of Example 40 to give ADC-17. [ka] Example 58 ADC-18 was prepared by the general coupling method of Example 40. [ka] Example 59 Prepared according to the general coupling method of Example 40 to give ADC-19. [ka] Example 60 Prepared according to the general coupling method of Example 40 to give ADC-20. [ka] Example 61 Prepared according to the general coupling method of Example 40 to give ADC-21. [ka] Example 62 Prepared according to the general coupling method of Example 40 to give ADC-22. [ka] Example 63 Prepared according to the general coupling method of Example 40 to give ADC-23. [ka] Example 64 Prepared according to the general coupling method of Example 40 to give ADC-24. [ka] Example 65 Prepared according to the general coupling method of Example 40 to give ADC-25. [ka] Example 66 Prepared according to the general coupling method of Example 40 to give ADC-26. [ka] Example 67 Prepared according to the general coupling method of Example 40 to give ADC-27. [ka] Example 68 Prepared according to the general coupling method of Example 40 to give ADC-28. [ka] Example 69 Prepared according to the general coupling method of Example 40 to give ADC-29. [ka] Example 70 Prepared according to the general coupling method of Example 40 to give ADC-30. [ka] Example 71 Prepared according to the general coupling method of Example 40 to give ADC-31. [ka] Example 72 Prepared according to the general coupling method of Example 40 to give ADC-32. [ka] Example 73 Prepared according to the general coupling method of Example 40 to give ADC-33. [ka] Example 74 Prepared according to the general coupling method of Example 40 to give ADC-34. [ka] Example 75 Prepared according to the general coupling method of Example 40 to give ADC-35. [ka] Example 76 Prepared according to the general coupling method of Example 40 to give ADC-36. [ka] Example 77 Prepared according to the general coupling method of Example 40 to give ADC-37. [ka] Example 78 Prepared according to the general coupling method of Example 40 to give ADC-38. [ka]

[0204] Example 79 (Control) Prepared according to the general coupling method of Example 40 to give ADC-39. [ka] Example 80 (Control) Prepared according to the general coupling method of Example 40 to give ADC-40. [ka]

[0205] Example 81: Plasma stability study 1.Operation A designated amount of ADC sample is taken and added to human plasma from which human IgG has been removed, and three tubes are prepared for each ADC. Incubate in a water bath at 37°C. After 72 and 144 hours, ADC samples are taken out, and 100ul of ProteinA resIn (MabSelect SuReTM LX Lot: #10221479 GE, washed with PBS) is added to each tube, and the tubes are adsorbed while shaking in a vertical mixer for 2 hours. After washing, the incubated ADC is obtained. The ADC samples incubated for the designated time are detected by RP-HPLC.

[0206] 2.Results Table 1: DAR values ​​and monomer ratio data for ligand drug conjugates (ADCs) of the present invention [Table 1]

[0207] Table 2: Plasma stability data for ligand drug conjugates (ADCs) of the invention. [Table 2]

[0208] 3. Join As shown in Table 1, the ADC disclosed in the present invention has excellent properties, such as a high DAR value (>7.5) and a high monomer ratio (>97%). As shown in Table 2, after the ADC of the present invention was incubated in plasma for 7 days, the DAR value was still at a relatively high level, indicating that the ADC of the present invention has excellent stability in plasma.

[0209] Example 82: In vitro activity test

[0210] 1) Experimental materials Cells: Obtained from the Cell Bank of the Chinese Academy of Sciences. Tumor cell culture medium: GIbco FBS:BIOWEST;

[0211] 2) Preparation of medium Growth medium (containing 10% FBS, penicillin-streptomycin (100 U / mL)) Detection medium (containing 1% FBS, penicillin-streptomycin (100 U / mL))

[0212] 3) Operation The UV light of the safety cabinet was turned on 30 minutes before and ventilated for 3 minutes. Growth medium, detection medium, D-PBS, and trypsin were preheated in a 37 °C constant temperature water bath, then the surfaces were disinfected with alcohol and placed in a biological safety cabinet. Cells with approximately 80% confluence (logarithmic growth phase) were selected, placed in a biological safety cabinet, the old medium was aspirated and removed, rinsed with D-PBS, aspirated and removed, digested with trypsin for 2 to 3 minutes, growth medium was added to stop trypsin, and centrifuged at 500 × g for 5 min. The centrifugation supernatant was aspirated and removed, mixed with 4 mL of detection medium, and 100 uL was taken and counted (50 μL cell solution was taken, 50 μL of 0.4% Trypan Blue StaIn was added and mixed uniformly, and then counted). A given number of cells were plated on a 96-well plate at 80 μl / well, and 80 μL detection medium alone was added to wells E11, F11, and G11, and 200 μL DPBS was added to the edge wells. After the coated cells were completely attached to the wall (usually at least 4 hours), the test samples were prepared and diluted. Specifically, a 1.0 mL, 2.5 μM (5 × Top Dose) test sample was prepared in detection medium and placed in the first row (200 μL / well) of a V-shaped 96-well plate, and 180 μL of detection medium was added to each of the second to eighth rows, and 30 μL was taken out from the first row and placed in the second row, and mixed up and down 10 times with a pipette, and then the pipette tip was discarded, and the remaining detection concentration points were operated in sequence, and the remaining detection concentration points were operated in sequence, and a 7-fold gradient concentration dilution was performed. The gradient concentration test sample was added to the cells at 20 uL / well. Only 20 uL of detection medium was added to the 11th column, and three replicate wells were set up for each concentration. The 96-well plate was then placed in a cell incubator at 5% CO2 and 37°C for 5 days.

[0213] 4) Detection After 4 days, the test samples were removed and thawed at room temperature in the dark, then vortexed to mix thoroughly, then 20 μL of CellTIter One SolutIon Reagen MTS reagent was added per 100 μL of cell culture medium along the sidewall of the well in a biological safety cabinet, and the MTS solution was mixed uniformly by tapping. The wells were then placed in a cell culture incubator at 5% CO2 and 37°C and left in the dark for 2 h. After the reaction was completed, the 96-well plate was removed and the absorbance at OD490nm was measured using a microplate reader, and the data was recorded, organized, and stored.

[0214] 5) Results Table 3: IC50 values ​​of antibody drug conjugates and toxins for inhibition of in vitro proliferation of N87 tumor cells [Table 3]

[0215] Table 4: IC50 values ​​for inhibition of antibody drug conjugates and toxins against in vitro proliferation of SK-BR-3 tumor cells [Table 4]

[0216] 6) Join As shown in Table 3, in the present invention, the ligand-drug conjugate targeting HER2 has a significant in vitro growth inhibitory activity against HER2-positive cells N87, and is clearly superior to the naked antibody (Trastuzumab) and the control group ADC-39. As shown in Table 4, compared with the naked antibody (Trastuzumab) and the control ADC, the ADC and single agent of the present invention have significant in vitro growth inhibitory activity also against HER2-positive cells SK-BR-3.

[0217] Example 83: In vivo safety study 1) Experimental materials Cells: Obtained from the Cell Bank of the Chinese Academy of Sciences. Tumor cell culture medium: GIbco Balb / c-nu nude mice: female, 5-7 weeks old (age of mice at the time of tumor cell inoculation), weighing 18.0-24.0 g, 180 mice (120 mice + 60 supplement mice). Purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Test and control items Test products: ADC-3, ADC-40 (provided by Chengdu Duospecific Antibody Drug Co., Ltd.). Histidine buffer (provided by Chengdu Polyantibody Drug Co., Ltd.). 0.9% Sodium Chloride Injection: Ke-Lun Pharmaceutical Co., Ltd.

[0218] 2) Cell culture NCI-H1975 (human non-small cell lung cancer adenocarcinoma cells) were cultured in RPMI 1640 medium. Exponentially growing NCI-H1975 cells were collected and resuspended in RPMI 1640 medium to an appropriate concentration, and then used for subcutaneous tumor inoculation in mice.

[0219] 3) Animal model construction and random group allocation 120 female nude mice were given 5 × 10 7 NCI-H1975 cells were inoculated subcutaneously. The average tumor volume was approximately 170 mm. 3 After reaching a tumor volume of 100, the mice were randomly divided according to tumor size. 120 mice with tumors of appropriate volume were selected, randomly divided into groups, and treatment was started (tail vein injection, dose 0.1ml / 10g). The day of grouping was defined as day 0.

[0220] 4) Preparation of test and control products Table 5: Preparation of test and control solutions for antitumor activity studies in NCI-H1975 (human non-small cell lung adenocarcinoma cell) transplanted tumor models [Table 5] NOTE: To ensure the formulation is homogenous, mix well before use.

[0221] 5) Experimental observation and data collection In the course of this experiment, the animal experimental operations were carried out in accordance with the requirements of the standard operating procedures for in vivo screening of antitumor drugs. Routine monitoring after tumor inoculation included tumor growth (tumors were measured twice a week) and the effects of treatment on the normal behavior of animals, specifically the activity of the experimental animals, eating and drinking status, weight gain and loss (weight was measured twice a week), eyes, hair coat and other abnormal conditions. Clinical symptoms observed during the experiment were recorded in the original data. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a is the long diameter and b is the short diameter). Manually recorded data such as measurements of the long and short diameters of the tumor and animal weights were used during the experiments.

[0222] 6) Results Table 6: Effect of antibody drug conjugate (11.25 mg / kg) on ​​body weight of NCI-H1975 tumor-implanted mice. [Table 6] Note: Mouse deaths were observed in the groups marked with *.

[0223] 7) Conclusion As shown in Table 8, the effect of ADC-3 of the present invention on the body weight of NCI-H1975 tumor-bearing mice in the high-dose and high-dose control groups was significantly smaller than that of ADC-40, and even in such a high-dose group, there was no mouse death as seen in the control group, indicating that the ADC drugs described in the present invention have a significant advantage in terms of safety.

Claims

1. A ligand-drug conjugate of formula I, or a pharma- ceutically acceptable salt, deuterated salt, or solvate thereof. Ab-L-D (I) where Ab is a ligand unit selected from an antibody, an antibody fragment, a target protein, or an Fc-fusion protein; L is the binding unit between D and Ab, D is a Drug unit; wherein u is selected from an integer of 1 to 10, or a pharma- ceutically acceptable salt, deuterated product, or solvate thereof.

2. The ligand-drug conjugate of claim 1, or a pharma- ceutically acceptable salt, deuterated product, or solvate thereof, wherein Ab is an antibody that forms a bond with the binding unit via a heteroatom thereof, and the antibody is selected from a mouse antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, and a bispecific or multispecific antibody.

3. The antibody or antigen-binding fragment thereof is anti-EGFR VIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg / AFP antibody, anti-cladin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-G PNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibodies, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-Integrin antibody Av antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 3. The ligand-drug conjugate according to claim 1 or 2, characterized in that the ligand-drug conjugate is selected from the group consisting of anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody and anti-CD123 antibody, or a pharma- ceutically acceptable salt, deuterated product or solvate thereof.

4. The ligand-drug conjugate according to claim 1 or 2, or a pharma- ceutically acceptable salt, deuterated product or solvate thereof, wherein L is of a cleavable or non-cleavable type.

5. The ligand-drug conjugate of claim 1 or 2, or a pharma- ceutically acceptable salt, deuterated salt, or solvate thereof, characterized in that the pharma- ceutically acceptable salt comprises a sodium salt, a potassium salt, a calcium salt, or a magnesium salt formed with an acidic functional group in the structural formula; or an acetate, a trifluoroacetate, a citrate, an oxalate, a tartrate, a malate, a nitrate, a chloride, a bromide, an iodide, a sulfate, a hydrogen sulfate, a phosphate, a lactate, an oleate, an ascorbate, a salicylate, a formate, a glutamate, a methanesulfonate, an ethanesulfonate, a benzenesulfonate, or a p-toluenesulfonate formed with a basic functional group in the structural formula.

6. A pharmaceutical composition comprising the ligand-drug conjugate according to any one of claims 1 to 5, or a pharma- ceutically acceptable salt, deuterated product, or solvate thereof, for treating or preventing a tumor.

7. The pharmaceutical composition according to claim 6, wherein the tumor is a solid tumor or a blood tumor.

8. 8. The pharmaceutical composition of claim 7, wherein the tumor is breast cancer, 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 or leukemia.

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