A DNA toxic dimer compound and its conjugate

Abstract

A DNA toxic dimer compound and its conjugate or a pharmaceutically acceptable salt thereof, including its preparation method and its role in preventing or treating cancer. The conjugate can specifically bind to receptors highly expressed in tumor cells. It has good water solubility, stability and homogeneity, and can be used to prevent or treat diseases such as tumors.

Description

Field of the Invention

[0001] The present invention relates to novel cytotoxic compounds and drugs comprising these cytotoxic compounds and cell binding agents. More specifically, the present invention relates to novel benzodiazepine dimer compounds, their derivatives, their intermediates, their pharmaceutically acceptable salts and conjugates thereof, which can be used as drugs, especially as anti-tumor drugs. Background of the Invention

[0002] As a new type of targeted drug, ligand-drug conjugate (ADC) is generally composed of three parts: antibody or antibody-like ligand, small molecule drug and linker that couples ligand and drug. Antibody-drug conjugate uses the antibody's specific recognition of antigen to transport drug molecules to the vicinity of target cells and effectively release drug molecules to achieve the purpose of treatment. In August 2011, the U.S. Food and Drug Administration (FDA) approved Adecteis, a new ADC drug developed by Seattle Genetics for the treatment of Hodgkin's lymphoma and relapsed anaplastic large cell lymphoma (ALCL). TM The safety and effectiveness of such drugs have been proven in clinical applications. With the development of ADC drugs, more effective small molecule drugs with newer mechanisms of action are needed.

[0003] Benzodiazepine derivatives have the ability to recognize and bind to specific DNA sequences and are highly effective interchain crosslinkers that react with guanine in the minor groove of DNA to form DNA adducts, interfering with DNA processing, and are therefore used as anti-tumor 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] DNA minor groove alkylators of the 1-(chloromethyl)-2,3-dihydro-1H-benz[e]indole (CBI) class are potent cytotoxics (Atwell et al. (1999) J. Med. Chem., 42:3400) and have been used as effector units in a variety of prodrugs designed for cancer therapy. CBI and benzodiazepine derivatives have been linked together via alkyl chains (CN105636612 A).

[0005] Imidazolo[1,2-a]pyridine derivatives are powerful DNA binding units that have been used in the synthesis of antitumor antibiotic duocarmycins derivatives and exhibit very potent cytotoxicity (Ronald C. Elgersma et al. (2015) Mol. Pharmaceutics. 12: 1813-1835).

[0006] The benzodiazepine derivatives disclosed in the prior art are extremely toxic, and are toxic even at extremely low doses. Therefore, improved benzodiazepine derivatives with lower toxicity, still therapeutic activity, and a high therapeutic window are very necessary. Summary of the invention

[0007] The present invention aims to provide a cytotoxic benzodiazepine dimer derivative with a good therapeutic window and an antibody conjugate thereof. The small molecule drug in the conjugate of the newly designed cytotoxic benzodiazepine dimer derivative has two functional groups: chloromethyl (CBI) and imine (PBD). The chloromethyl group 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 enhance DNA cross-linking. The inventor unexpectedly found that this type of benzodiazepine ADC drug has good safety and efficient anti-tumor activity.

[0008] The present invention discloses a ligand-drug conjugate as shown in Formula I, or a pharmaceutically acceptable salt, deuterated substance, and solvate thereof:

[0009] Ab-LD

[0010] (I)

[0011] in:

[0012] Ab is a ligand unit selected from an antibody, an antibody fragment, a targeting protein or an Fc-fusion protein;

[0013] L is the connecting unit between D and Ab;

[0014] D is a drug unit selected from the following structures:

[0015]

[0016] in:

[0017] The wavy line indicates the site where the drug is attached to L, and only one of the three sites is attached to L;

[0018] R1 is H, deuterium, OH, or ether represented by OR3, sulfite SO3 - or OSO3 - , wherein R3 is selected from C1-C10 a straight chain, branched chain or cyclic alkyl, alkenyl or alkynyl group;

[0019] Double wire between N and C represents a single bond or a double bond, provided that when it is a double bond, N is not connected to L and R1 is H; when it is a single bond, N is connected to L and R1 is selected from OH or an ether represented by OR3, a sulfite SO3 - or OSO3 - , wherein R3 is selected from C1-C 10 a straight chain, branched chain or cyclic alkyl, alkenyl or alkynyl group;

[0020] R2 is H or an alkyl substituent;

[0021] T is selected from C2-C 12 Hydrocarbyl, Z, (C1-C6 alkylene)-Z-(C1-C6 alkylene), (C1-C6 alkylene)-Z-(C1-C6 alkylene)-Z-(C1-C6 alkylene), (C1-C6 alkenylene)-Z-(C1-C6 alkenylene), or (C1-C6 alkynylene)-Z-(C1-C6 alkynylene);

[0022] in:

[0023] Z is independently selected from O, S, NR4, aryl or heteroaryl; wherein R4 is selected from H, P(O)3H2 or C(O)NR5R6; wherein R5 and R6 are selected from H, C1-C6 alkyl, C1-C6 alkyl substituted with one or more F, or R5 and R6 form a five-membered or six-membered heterocyclyl group;

[0024] Alkylene, alkenylene, aryl and heteroaryl are independently and optionally substituted with F, OH, O(C1-C6 alkyl), NH2, NHCH3, N(CH3)2 or C1-C6 alkyl, wherein alkyl is optionally substituted with one or more F;

[0025] Y is selected from one or more H or C1-C4 alkyl;

[0026] X is independently selected from -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-etc.;

[0027] in:

[0028] R7 and R8 are independently hydrogen, deuterium, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl, substituted aryl or heteroaryl; or, R7, R8 and the carbon atom to which they are connected constitute a C3-C6 cycloalkyl, cycloalkylalkyl or heterocyclic group;

[0029] R9, R 10 are the same or different and are independently hydrogen, deuterium, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl or heterocyclic; or, R9, R 10 and the carbon atoms to which they are connected constitute a C3-C6 cycloalkyl group, cycloalkylalkyl group or heterocyclic group;

[0030] m is an integer selected from 0 to 4;

[0031] X1 is selected from halogen or OSO2R 11 , where R 11 It can be independently selected from H, C1-C4 hydrocarbon group, phenyl or substituted phenyl.

[0032] As a preferred embodiment, Ab is an antibody, which can form a connecting bond with a connecting unit through its heteroatom, and the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody or a multispecific antibody.

[0033] Further preferably, the antibody or antigen-binding fragment thereof is selected from, but not limited to, 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-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD27 6) Antibodies, anti-PTK7 / CCK4 antibodies, anti-PRLR antibodies, anti-EFNA4 antibodies, anti-5T4 antibodies, anti-NOTCH3 antibodies, anti-Nectin4 antibodies, anti-TROP-2 antibodies, anti-CD142 antibodies, anti-CA6 antibodies, anti-GPR20 antibodies, anti-CD174 antibodies, anti-CD71 antibodies, anti-EphA2 antibodies, anti-LYPD3 antibodies, anti-FGFR2 antibodies, anti-FGFR3 antibodies, anti-FRα antibodies, anti-CEACAMs antibodies, anti-GCC antibodies, 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.

[0034] A DNA toxic dimer compound and its conjugate or pharmaceutically acceptable salt, deuterated substance and solvate thereof, preferably: L is cleavable or non-cleavable.

[0035] A DNA toxic dimer compound and its conjugate or its pharmaceutically acceptable salt, deuterated substance and solvate, wherein the pharmaceutically acceptable salt comprises sodium salt, potassium salt, calcium salt or magnesium salt formed with the acidic functional group in the structural formula; or acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate or p-toluenesulfonate formed with the basic functional group in the structure.

[0036] A DNA toxic dimer compound and a conjugate thereof or a pharmaceutically acceptable salt thereof, and use of the compound in preparing a drug for treating or preventing tumors.

[0037] Preferably, the tumor is a solid tumor or a blood 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 and leukemia.

[0038] The present invention also discloses a compound of the general formula II or III connected to Ab, or a pharmaceutically acceptable salt or solvate thereof:

[0039]

[0040] in:

[0041] R1 is H, OH or ether represented by OR3, sulfite SO3 - or OSO3 - , wherein R3 is selected from C1-C 10 a straight chain, branched chain or cyclic alkyl, alkenyl or alkynyl group;

[0042] Double wire between N and C represents a single bond or a double bond, provided that, in the case of a double bond, R 12 When it is not present and R1 is H; when it is a single bond, R 12 -C(O)O-L3, wherein L3 is a linking unit; R1 is selected from OH, ether represented by OR3, sulfite SO3 - or OSO3 - , wherein R3 is selected from C1-C 10 a straight chain, branched chain or cyclic alkyl, alkenyl or alkynyl group;

[0043] R2 is H or an alkyl substituent;

[0044] T is selected from C2-C12 Hydrocarbyl, Z, (C1-C6 alkylene)-Z-(C1-C6 alkylene), (C1-C6 alkylene)-Z-(C1-C6 alkylene)-Z-(C1-C6 alkylene), (C1-C6 alkenylene)-Z-(C1-C6 alkenylene), or (C1-C6 alkynylene)-Z-(C1-C6 alkynylene);

[0045] in:

[0046] Z is independently selected from O, S, NR4, aryl and heteroaryl; wherein R4 is selected from H, P(O)3H2, C(O)NR5R6, wherein R5 and R6 are selected from H, C1-C6 alkyl, one or more F-substituted C1-C6 alkyl, or R5 and R6 form a five-membered or six-membered heterocyclyl group;

[0047] Alkylene, alkenylene, aryl and heteroaryl are independently and optionally substituted with F, OH, O(C1-C6 alkyl), NH2, NHCH3, N(CH3)2 and C1-C6 alkyl, wherein alkyl is optionally substituted with one or more F;

[0048] Y is selected from one or more H or C1-C4 alkyl;

[0049] X is independently selected from -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-;

[0050] in:

[0051] R7 and R8 are independently hydrogen, deuterium, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl, substituted aryl or heteroaryl; or, R7, R8 and the carbon atom to which they are connected constitute a C3-C6 cycloalkyl, cycloalkylalkyl or heterocyclic group;

[0052] R9, R 10 are the same or different and are independently hydrogen, deuterium, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl or heterocyclic; or, R9, R 10and the carbon atoms to which they are connected constitute a C3-C6 cycloalkyl group, cycloalkylalkyl group or heterocyclic group;

[0053] m is an integer selected from 0 to 4;

[0054] X1 is selected from halogen or OSO2R 11 , where R 11 Can be independently selected from H, C1-C4 hydrocarbon group, phenyl or substituted phenyl;

[0055] L1 and L2 are linking units or substituents.

[0056] As a preferred embodiment, T is selected from C2-C 12 of alkylene.

[0057] More preferably, T is

[0058] As a preferred embodiment, X is -O-, -N- or -NHC(O)-CR7R8-(CR9R 10 )mO-.

[0059] As a preferred embodiment, L3 is:

[0060]

[0061] Among them: the wavy line is connected to -C(O)O-; L4 is a connecting unit, which is connected to the ligand unit.

[0062] As a preferred embodiment, L4 is selected from the group consisting of:

[0063]

[0064] Among them: at the wavy line, the left side (carbon end) is connected to the ligand unit, and the right side (nitrogen end or ester carbonyl end) is connected to X2.

[0065] As a preferred embodiment, Q is:

[0066]

[0067] Where Q x It is an amino acid residue or a peptide residue composed of amino acids.

[0068] As a preferred embodiment, X2 is:

[0069]

[0070] wherein a is selected from an integer of 0-5, b is selected from an integer of 0-16, c is selected from an integer of 0-1, and d is selected from an integer of 0-5.

[0071] More preferably, L3 is selected from the group consisting of:

[0072]

[0073] Among them: at the wavy line, the left side (succinimide end) is connected to the ligand unit, and the right side is connected to -C(O)O-.

[0074] As a preferred embodiment, in the compound of formula II or III or a pharmaceutically acceptable salt or solvate thereof, L1 and L2 are independently selected from:

[0075] Structure A: hydrogen atom, C(O)NR′R″, wherein R′ and R″ are selected from H, C1-C6 alkyl, C1-C6 alkyl substituted with one or more F, or R′ and R″ form a five-membered or six-membered heterocyclyl group;

[0076] Structure B: L4-L5-, L4-L6- or L4-L7-L8-L9-, wherein L4, L5, L6, L7, L8 and L9 are all connecting units, L4 is connected to the ligand unit, and L5, L6, L9 are connected to X.

[0077] Further preferably,

[0078] When there is a single bond between N and C, R 12 When present, L1 and L2 are independently selected from structure A;

[0079] When there is a double bond between N and C, R 12 When not present, L1 is structure A or B, and L2 is structure B or A.

[0080] More preferably, L5 is -((CH2)sO)r(CH2)sX3L 10 -or-((CH2)sO)r(CH2)sX4L 10 -;

[0081] L6 is -((CH2)sO)r(CH2)s-;

[0082] L 10 is -(CH2)s- or -((CH2)sNHC(=O)X5X6C(=O)(CH2)s-;

[0083] in:

[0084] X3 is selected from, but not limited to:

[0085] Where R 13 Independently selected from a hydrogen atom, a C1-C6 hydrocarbon group, a halogen atom or a hydroxyl group;

[0086] X4 is selected from, but not limited to:

[0087] Where R 13 Independently selected from a hydrogen atom, a C1-C6 hydrocarbon group, a halogen atom or a hydroxyl group;

[0088] X5 is selected from, but not limited to:

[0089]

[0090] X6 is selected from a peptide residue consisting of amino acids, non-limitingly selected from:

[0091]

[0092] s is selected from an integer of 1-10, and r is selected from an integer of 1-14.

[0093] 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 chemical bond, wherein o is selected from an integer of 0-20; p is selected from an integer of 0-20; R 14 With R 15 are 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;

[0094] L8 is selected from a peptide residue consisting of amino acids, preferably a peptide residue consisting of one, two or more amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E) or aspartic acid (D);

[0095] L9-NR 17 (CR 18 R 19 ) q -、-C(O)NR 17 -、-C(O)NR 17 (CH2) q - or a chemical bond, wherein q is selected from an integer of 0-6; R 17 , R 18 and R 19are 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 heterocyclic group, an aryl group, a substituted aryl group or a heteroaryl group.

[0096] Further preferably, L1 and L2 are independently selected from the following structures without limitation from structure B:

[0097]

[0098]

[0099] More preferably, the compound of formula II or III or a pharmaceutically acceptable salt or solvate thereof is selected from the following structures without limitation:

[0100]

[0101]

[0102]

[0103]

[0104] Preferably, the antibody-drug conjugate or its pharmaceutically acceptable salt, deuterated substance and solvate is selected from the following structures without limitation:

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] wherein u is selected from an integer of 1-10. DETAILED DESCRIPTION

[0114] Abbreviations and definitions

[0115] Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings.When a trade name is used herein, unless the context indicates otherwise, the trade name includes the product formulation, generic drug, and active ingredient of the trade name product.

[0116] Unless stated otherwise, the terms used in the claims and specification herein have the following meanings.

[0117] The term "ligand" is a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell. The function of a ligand is to present the drug to a target cell population that binds to the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells. In an embodiment of the present invention, the ligand is represented by Ab, and the ligand can form a linking bond with a linking unit through a heteroatom on the ligand, preferably an antibody or an antigen-binding fragment thereof, wherein the antibody is selected from a chimeric antibody, a humanized antibody, a fully human antibody, or a mouse antibody; preferably a monoclonal antibody.

[0118] The ligand unit is a targeting agent that specifically binds to the target portion. The ligand can specifically bind to a cell component or bind to a cell component or bind to other target molecules of interest. The target portion or target is usually on the cell surface. In some aspects, the role of the ligand unit is to deliver the drug unit to a specific target cell population with which the ligand unit interacts. The ligand includes, but is not limited to, proteins, polypeptides and peptides, and non-proteins such as sugars. Suitable ligand units include, for example, antibodies, such as full-length (complete) antibodies and antigen-binding fragments thereof. In the embodiment where the ligand unit is a non-antibody targeting agent, it can be a peptide or polypeptide, or a non-protein molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth factors and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell binding molecules or substances. In some embodiments, the linker is covalently attached to the sulfur atom of the ligand. In some aspects, the sulfur atom is the sulfur atom of a cysteine ​​residue, which forms the interchain disulfide bond of the antibody. On the other hand, the sulfur atom is the sulfur atom of a cysteine ​​residue that has been introduced into the ligand unit, which forms the interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine ​​residue that has been introduced into the Ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In other aspects, the sulfur atom to which the linker is bound is selected from a cysteine ​​residue that forms an interchain disulfide bond of an antibody or an additional cysteine ​​residue that has been introduced into the Ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In some embodiments, the EU index numbering system is according to Kabat {[Kabat EA et al., (1991)] Sequences of proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242}.

[0119] As used herein, "antibody" or "antibody unit" includes any part of the antibody structure within the scope to which it belongs. This unit can bind, reactively associate, or complex a receptor, antigen or other receptor unit possessed by the targeted cell population. The antibody can be any protein or protein-like molecule that can bind, complex or react with a part of the cell population to be treated or biomodified. The antibodies that constitute the antibody drug conjugates in the present invention retain their antigen binding ability in their original wild state. Therefore, the antibodies in the present invention can bind specifically to the antigen. The antigens involved include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulatory factors, cell proliferation regulatory factors, molecules related to tissue growth and differentiation (such as known or predicted functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as known or predicted functional). Tumor-related factors can be cluster differentiation factors (such as CD proteins).

[0120] 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 industry and can be prepared by antibody preparation methods and information well known in the industry. In order to develop effective cellular level targets that can be used for cancer diagnosis and treatment, researchers strive to find transmembrane or other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells, and rarely or not expressed on the surface of one or more non-cancerous cells. Generally, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancerous cells. Confirming such tumor-associated factors can greatly improve the specific targeting characteristics of antibody-based cancer treatment. For convenience, antigen-related information well known in the industry is marked as follows, including name, other names, and gene bank accession number. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases, such as Genbank. The antibody targets the corresponding tumor-associated antigen, including all amino acid sequence variants and isotypes, which have at least 70%, 80%, 85%, 90% or 95% homology with the sequence confirmed in the reference, or have biological properties and characteristics that are completely consistent with the tumor-associated antigen sequence in the cited literature.

[0121] The term "inhibit" or "inhibition of" means reducing by a detectable amount, or completely preventing.

[0122] The term "cancer" refers to the physiological condition or disease characterized by unregulated cell growth. A "tumor" includes cancer cells.

[0123] The term "autoimmune disease" is a disease or disorder that arises from a host directed against an individual's own tissues or proteins.

[0124] The term "drug" refers to cytotoxic drugs, which are chemical molecules that have a strong ability to disrupt the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells in principle at sufficiently high concentrations, but due to the lack of specificity, while killing tumor cells, they can also cause apoptosis of normal cells, leading to serious side effects. The term includes toxins, such as small molecule toxins or enzyme-active toxins from bacteria, fungi, plants or animals, radioactive isotopes (such as At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 He Lu 176 radioactive isotopes), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.

[0125] The term "linker" or "linking fragment" or "linking unit" refers to a chemical structure fragment or bond that is connected to a ligand at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug.

[0126] Linkers, including extensions, spacers, and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1. Linkers can be "cleavable linkers" that facilitate release of the drug in cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al. Cancer Research 52: 127-131, 1992); U.S. Pat. No. 5,208,020.

[0127] According to the mechanism of drug release in cells, as used herein, "linkers" or "linkers of antibody drug conjugates" can be divided into two categories: non-cleavable linkers and cleavable linkers. For antibody-drug conjugates containing non-cleavable linkers, the drug release mechanism is: after the conjugate binds to the antigen and is endocytosed by the cell, the antibody is enzymatically hydrolyzed in the lysosome, releasing the active molecule composed of small molecule drugs, linkers, and antibody amino acid residues. The resulting changes in the molecular structure of the drug do not weaken its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into neighboring cells. Therefore, such active drugs cannot kill neighboring tumor cells that do not express the targeted antigen (antigen-negative cells) (bystander effect) (Ducry et al., 2010, Bioconjugate Chem. 21: 5-13).

[0128] The term "antibody-drug conjugate" refers to an antibody connected to a biologically active drug via a stable linker. In the present invention, "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment connected to a biologically active toxic drug via a stable linker.

[0129] The three letter codes and one letter codes for amino acids used in this disclosure are as described in J. boil. Chem. 1968, 243, 3558.

[0130] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 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 various branched chain isomers thereof. More preferred are lower alkyl groups having 1 to 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, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0131] The term "substituted alkyl" refers to an alkyl group in which a hydrogen atom is replaced by a substituent group. Unless otherwise specified herein, the substituent group of the alkyl group may be a variety of groups selected from the group consisting of -halogen, -OR', -NR'R", -SR', -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR "C(O)2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R", -NR'S(O)2R", -CN and -NO2, the number of substituents is 0 to (2m'+1), where m' is the total number of carbon atoms in the group. R', R" and R'" each independently refer to hydrogen, unsubstituted C 1-8 Alkyl, unsubstituted aryl, aryl substituted by 1-3 halogens, unsubstituted C 1-8 Alkyl, C 1-8 Alkoxy or C 1-8 Thioalkoxy, or unsubstituted aryl-C 1-4 Alkyl. When R' and R" are attached to the same nitrogen atom, they may form a 3-, 4-, 5-, 6- or 7-membered ring together with the nitrogen atom. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

[0132] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.

[0133] The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of an alkane radical by removing two hydrogen atoms, and is a straight or branched group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-, 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH 2CH2CH2CH2CH2-) etc. Alkylene may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available attachment point, and the substituent is preferably independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0134] The term "alkoxy" refers to-O-(alkyl) and-O-(cycloalkyl), wherein the definition of alkyl or cycloalkyl is as described above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0135] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyls include spirocyclic, fused and bridged cycloalkyls.

[0136] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O). m (in mis an integer from 0 to 2) of heteroatoms, but does not include -OO-, -OS- or -SS- ring moieties, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups.

[0137] The term "cycloalkylalkyl" refers to an alkyl group substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein alkyl is as defined above, and wherein cycloalkyl is as defined above.

[0138] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0139] The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

[0140] The term "hydroxy" refers to an -OH group.

[0141] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0142] The term "amino" refers to -NH2. The term "nitro" refers to -NO2.

[0143] The term "amido" refers to -C(O)N(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0144] The term "carboxylate" refers to -C(O)O(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0145] The present invention also includes various deuterated forms of formula I. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of formula I with reference to the relevant literature. Commercially available deuterated starting materials can be used when preparing deuterated forms of formula I, or they can use conventional techniques to adopt deuterated reagents to synthesize, and the non-limiting examples of deuterated reagents include: deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane etc.

[0146] The term "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain respectively. The same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains or λ chains according to the difference in constant regions. Each of the five classes of Ig can have κ chains or λ chains. The antibody of the present invention is preferably a specific antibody against a cell surface antigen on a target cell, and non-limiting examples are the following antibodies: anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg / AFP antibody, anti-cladin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (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 antibody, 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-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody or anti-CD123 antibody.

[0147] The term "solvate" or "solvate compound" refers to a pharmaceutically acceptable solvate formed between the ligand-drug conjugate of the present invention and one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0148] The term "drug loading" refers to the average number of cytotoxic drugs loaded on each antibody in Formula I, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The range of drug loading can be 0-12, preferably 1-10 cytotoxic drugs (D) connected to each antibody (Ab). In an embodiment of the present invention, the drug loading is expressed as n, which can be exemplified as an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The average number of drugs per ADC molecule after the coupling reaction can be identified by conventional methods such as UV / visible light spectroscopy, mass spectrometry, ELISA test and HPLC characteristics.

[0149] In one embodiment of the present invention, the cytotoxic drug is coupled to the thiol-SH of the opened cysteine ​​residues between antibody chains and / or the thiol-SH of the site-directed mutated cysteine ​​residues through a linker unit. Generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than or equal to the theoretical maximum value.

[0150] The loading of the ligand cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:

[0151] (1) Control the molar ratio of the linking reagent and the monoclonal antibody,

[0152] (2) Control the reaction time and temperature,

[0153] (3) Select different reaction reagents.

[0154] The preparation of conventional pharmaceutical compositions is described in the Chinese Pharmacopoeia.

[0155] The term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" refers to a salt of the ligand-drug conjugate of the present invention, or a salt of the compound described in the present invention. Such salts are safe and effective when used in mammals and have the desired biological activity. The ligand-drug conjugate compound of the present invention contains at least one carboxyl group and can therefore form salts with bases. Non-limiting examples of pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts or magnesium salts, etc.

[0156] The term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" refers to a salt of the antibody-drug conjugate of the present invention, or a salt of the compound described in the present invention. Such salts are safe and effective when used in mammals and have the desired biological activity. The ligand-drug conjugate compound of the present invention contains at least one amino group and can therefore form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0157] "Acidic amino acids" refer to amino acids with an isoelectric point of less than 7. Acidic amino acids often have one or more acidic groups such as carboxyl groups in their molecules, which can be effectively ionized into negative ion forms in the structure to increase hydrophilicity. Acidic amino acids can be natural or non-natural amino acids.

[0158] "Natural amino acids" refer to amino acids synthesized by organisms. Natural amino acids are generally L-type, but there are a few exceptions, such as glycine, including natural and biologically synthesized amino acids.

[0159] "Unnatural amino acid" refers to an amino acid obtained by synthetic means.

[0160] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, ratios, or parts are by weight.

[0161] Embodiment 1:

[0162] Synthesis of compound A:

[0163]

[0164] Compound A was synthesized according to the method provided in Example 14 "Synthesis of Compound 012" of patent "CN109180681A".

[0165] Embodiment 2:

[0166] Synthesis of Compound B and Compound B1-9:

[0167] 1. Preparation of Compound B

[0168]

[0169] Compound B was synthesized according to the method provided in "General Step E: Synthesis Method 1 of CBI-Benzodiazepine Derivatives" of patent "CN109180681A".

[0170] 2. Preparation of Compound B1

[0171]

[0172] Compound B (50 mg, 0.072 mmol), glycolic acid (11 mg, 0.14 mmol), EDCI (20 mg, 0.10 mmol), HOBt (13.5 mg, 0.10 mmol) and 3 mL DMF were added to a 25 mL single-mouth bottle, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly purified by high-performance liquid chromatography to obtain compound B1 (42 mg) with a yield of 78%. LC-MS: [M+H] + =747.2.

[0173] 3. Preparation of Compound B2

[0174]

[0175] 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 added to a 25 mL single-mouth bottle, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly purified by high-performance liquid chromatography to obtain compound B2 (37 mg) with a yield of 68%. LC-MS: [M+H] + =761.2.

[0176] 4. Preparation of Compound B3

[0177]

[0178] Referring to the synthesis method of compound B2, L-lactic acid was replaced with D-lactic acid to obtain compound B3. LC-MS: [M+H] + =761.2.

[0179] 5. Preparation of Compounds B4 and B5

[0180]

[0181] 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 added to a 25 mL single-mouth bottle, and the reaction was carried out at room temperature for 1.5 h. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly purified by high-performance liquid chromatography, and the product preparation solution was lyophilized to obtain compound B4 (26 mg) and compound B5 (22 mg), respectively. LC-MS: [M+H] + =815.2.

[0182] 6. Preparation of Compounds B6 and B7

[0183]

[0184] Referring to the synthesis method of compounds B4 and B5, compounds B6 and B7 were obtained by high performance liquid chromatography purification. LC-MS: [M+H] + =787.3.

[0185] 7. Preparation of Compounds B8 and B9

[0186]

[0187] Referring to the synthesis method of compounds B4 and B5, compounds B8 and B9 were obtained by high performance liquid chromatography purification. LC-MS: [M+H] + =801.3.

[0188] Embodiment 3:

[0189] Synthesis of Compound C and C1-9:

[0190] 1. Preparation of Compound C

[0191]

[0192] Compound C was synthesized according to the method provided in "General Step E: Synthesis Method 2 of CBI-Benzodiazepine Derivatives" of patent "CN109180681A".

[0193] 2. Preparation of Compound C1

[0194]

[0195] Compound C (50 mg, 0.075 mmol), glycolic 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 added to a 25 mL single-mouth bottle, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly purified by high-performance liquid chromatography to obtain compound C1 (35 mg) with a yield of 65%. LC-MS: [M+H] + =723.2.

[0196] 3. Preparation of Compound C2

[0197]

[0198] 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 added to a 25 mL single-mouth bottle, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly purified by high-performance liquid chromatography to obtain compound C2 (33.5 mg) with a yield of 63%. LC-MS: [M+H] + =737.2.

[0199] 4. Preparation of compound C3:

[0200]

[0201] Referring to the synthesis method of compound C2, L-lactic acid was replaced with D-lactic acid to obtain compound C3. LC-MS: [M+H] + =737.2.

[0202] 5. Preparation of compounds C4 and C5:

[0203]

[0204] 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 single-mouth bottle, and the reaction was carried out at room temperature for 1.5 h. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly purified by high-performance liquid chromatography, and the product preparation solution was lyophilized to obtain compound C4 (21 mg) and compound C5 (25 mg), respectively. LC-MS: [M+H] + =791.2.

[0205] 6. Preparation of compounds C6 and C7:

[0206]

[0207] Referring to the synthesis method of compounds C4 and C5, compounds C6 and C7 were obtained by high performance liquid chromatography purification. LC-MS: [M+H] + =763.3.

[0208] 7. Preparation of compounds C8 and C9:

[0209]

[0210] Referring to the synthesis method of compounds C4 and C5, compounds C8 and C9 were obtained by high performance liquid chromatography purification. LC-MS: [M+H] + =777.3.

[0211] Embodiment 4:

[0212] Synthesis of compounds D1 and D1-9:

[0213] 1. Preparation of Compound D

[0214]

[0215] Compound D was synthesized with reference to the method provided in "General Step A: Synthesis of Benzodiazepine Derivatives I and General Step General Step E: Synthesis Method II of CBI-Benzodiazepine Derivatives" of patent "CN109180681A".

[0216] 2. Preparation of Compound D1

[0217]

[0218] Compound D (50 mg, 0.078 mmol), glycolic 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 added to a 25 mL single-mouth bottle, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly purified by high-performance liquid chromatography to obtain compound D1 (34 mg) with a yield of 62.5%. LC-MS: [M+H] + =697.2.

[0219] 3. Preparation of Compound D2

[0220]

[0221] 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 added to a 25 mL single-mouth bottle, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly purified by high-performance liquid chromatography to obtain compound D2 (33.8 mg) with a yield of 71%. LC-MS: [M+H] + =711.2.

[0222] 4. Preparation of Compound D3

[0223]

[0224] Referring to the synthesis method of compound D2, D-lactic acid was substituted for L-lactic acid to obtain compound D3. LC-MS: [M+H] + =711.2.

[0225] 4. Preparation of Compounds D4 and D5

[0226]

[0227] 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 added to a 25 mL single-mouth bottle, and the reaction was carried out at room temperature for 1.5 h. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly purified by high-performance liquid chromatography, and the product preparation solution was lyophilized to obtain compound D4 (27 mg) and compound D5 (29 mg), respectively. LC-MS: [M+H] + =765.2.

[0228] 6. Preparation of Compounds D6 and D7

[0229]

[0230] Referring to the synthesis method of compounds D4 and D5, compounds D6 and D7 were obtained by high performance liquid chromatography purification. LC-MS: [M+H] + =737.3.

[0231] 7. Preparation of Compounds D8 and D9

[0232]

[0233] Referring to the synthesis method of compounds D4 and D5, compounds D8 and D9 were obtained by high performance liquid chromatography purification. LC-MS: [M+H]+ =751.3.

[0234] Embodiment 5:

[0235] Synthesis of compound M1:

[0236]

[0237] In a 5L single-mouth bottle, add N-Fmoc-glycine-glycine (100g, 28mmol, 1.0eq), lead tetraacetate (175g, 395mmol, 1.4eq), 2L tetrahydrofuran and 670mL toluene, stir evenly, and heat to 85℃ for 2.5h. After the reaction of the raw materials, cool to room temperature, filter, and concentrate the filtrate under reduced pressure. The residue is purified by column chromatography (PE:EA=5:1-2:1) to obtain compound M1 (87g), with a yield of 84.4%, LC-MS: [M+NH4] + =386.0.

[0238] Embodiment 6:

[0239] Synthesis of compound M3:

[0240]

[0241] Compound SM-2 (synthesized according to the method disclosed in patent CN108452321A) (40 g, 96 mmol, 1.0 eq), triethylamine (26.7 mL, 2.0 eq), and toluene (400 mL) were added to a 1000 mL single-mouth bottle, and the temperature was raised to 120 ° C and refluxed for 2 h. TLC monitored the reaction to be basically complete, and the temperature was reduced to 50 ° C and the solvent was removed by vacuum rotation. Dissolved with ethyl acetate (150 mL) and water (40 mL), adjusted to pH 2-3 with 1M HCl under ice bath stirring, and separated. The aqueous layer was extracted once more with ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. After filtration, it 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 of compound M2; LC-MS: [M+H] + =399.3.

[0242] Compound M2 (26.5 g, 60.5 mmol, 1.0 eq), pentafluorophenol (12.2 g, 66.5 mmol, 1.1 eq), DCC (13.7 g, 66.5 mmol, 1.1 eq) and THF (300 mL) were added to a 1 L single-mouth bottle, reacted at room temperature for 30 min (monitored by TLC), and filtered to remove insoluble matter. The reaction solution was directly purified by preparative purification, and the preparative liquid was concentrated by a water pump under reduced pressure in a water bath at 35 ° C to remove acetonitrile, and lyophilized to obtain 31.5 g of compound M3, with a yield of 64%; LC-MS: [M+H]+ =565.1.

[0243] Embodiment 7:

[0244] Synthesis of compound 1:

[0245]

[0246] Step 1: Synthesis of compound 1a

[0247] In a 250mL single-mouth bottle, add M1 (6g, 16.3mmol), 100mL THF, p-toluenesulfonic acid monohydrate (0.31g, 1.63mmol), stir and cool to 0°C, add benzyl glycolate (5.4g, 32.6mmol) dropwise, and naturally warm to room temperature for reaction (reaction for about 2-4h), monitored by TLC. After the reaction is completed, add saturated NaHCO3 solution, extract with ethyl acetate, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, filter, concentrate, and the residue is purified by silica gel column (PE:EA=10:1-5:1-1:1) to obtain compound 1a (4g), with a yield of 52%; LC-MS: [M+H] + =475.2.

[0248] Step 2: Synthesis of compound 1b

[0249] In a 25 mL single-necked bottle, compound 1a (2 g, 4.2 mmol) and 10 mL DMF were added, stirred at 0°C, and DBU (766 mg, 5.04 mmol) was added. The reaction was continued for 1 h. After the Fmoc deprotection was completed, the mixture was monitored by TLC and then set aside.

[0250] In another 25mL single-mouth bottle, compound M4 (prepared by the method disclosed in reference patent CN111051330 A) (1.73g, 4.2mmol), PyBOP (2.61g, 5.04mmol), HOBt (680mg, 5.04mmol) and 10mL DMF were added, and DIPEA (830uL, 5.04mmol) was added under ice-water bath, and stirring was continued for 30min. The above reaction solution was added to the reaction bottle and warmed to room temperature for reaction. After the reaction was completed by HPLC monitoring, the reaction solution was purified by preparative liquid phase to obtain the product preparation solution, 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 (1.7g), with a yield of 63%; LCMS: [M+H] + =648.3.

[0251] Step 3: Synthesis of compound 1c

[0252] Compound 1b (900 mg, 1.39 mmol) was added to a 25 mL single-necked bottle, dissolved in 15 mL DMF, and then 900 mg 5% Pd / C was added. The hydrogenation reaction was carried out for 2 h. After the reaction was completed, the filtrate was filtered and used directly in the next step without purification.

[0253] Step 4: Synthesis of Compound 1

[0254] The DMF solution of compound 1c obtained in the previous step was placed in an ice-water bath, and DIPEA (235uL, 1.39mmol) was added, followed by compound M3 (784mg, 1.39mmol). After the addition, the mixture was heated to room temperature and reacted for 1h. The reaction was monitored by HPLC, and the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain compound 1 (604mg), with a yield of 54%; LC-MS: [M+H] + =804.4.

[0255] Embodiment 8:

[0256] Synthesis of compound 2:

[0257]

[0258] Step 1: Synthesis of compound 1c

[0259] Compound 1b (900 mg, 1.39 mmol) was added to a 25 mL single-necked bottle, dissolved in 15 mL DMF, and then 900 mg 5% Pd / C was added. The hydrogenation reaction was carried out for 2 h. After the reaction was completed, the filtrate was filtered and used directly in the next step without purification.

[0260] Step 2: Synthesis of Compound 2

[0261] The DMF solution of compound 1c obtained in the previous step was placed in an ice-water bath, and DIPEA (235uL, 1.39mmol) was added, followed by McOSu (428.5mg, 1.39mmol). After the addition, the mixture was heated to room temperature and reacted for 1h. The reaction was monitored by HPLC. The reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution, which was lyophilized to obtain compound 2 (500mg) with a yield of 58%; LC-MS: [M+H] + =617.3.

[0262] Embodiment 9:

[0263] Synthesis of compounds 3A and 3B:

[0264] 1. Preparation of Compound 3A

[0265]

[0266] Step 1: Synthesis of compound 3a

[0267] M1 (1.5 g, 4.0 mmol, 1.0 eq), p-toluenesulfonic acid monohydrate (77 mg, 0.4 mmol, 0.1 eq) and 15 mL THF were added to a 25 mL single-mouth bottle, stirred evenly, cooled to 0 ° C, and then L-benzyl lactate (2.2 g, 12.0 mmol, 3 eq) was slowly added. After the addition, the temperature was raised to room temperature for reaction. TLC monitoring, after the reaction was completed, 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 (1.03 g), with a yield of 52%, LC-MS: [M+NH4] + =506.2.

[0268] Step 2: Synthesis of compound 3b

[0269] Add compound 3a (1 g, 2.04 mmol) and 8 mL DMF to a 25 mL single-necked bottle, stir evenly, cool to 0 °C, slowly add DBU (373 mg, 2.45 mmol), and warm to room temperature to react. Monitor by TLC, the reaction is complete, and is recorded as reaction solution ①;

[0270] In another 25mL single-mouth bottle, add M4 (846mg, 2.04mmol), PyBOP (1.27g, 2.45mmol) and 6mL DMF, stir at room temperature for 5min, add reaction solution ①, react at room temperature, and monitor by HPLC. After the reaction is completed, the reaction solution is purified by high-performance liquid chromatography and freeze-dried to obtain compound 3b (913mg), with a yield of 67.6%, LC-MS: [M+NH4] + =679.2.

[0271] Step 3: Synthesis of compound 3c

[0272] Compound 3b (800 mg, 1.21 mmol, 1.0 eq) was added to a 100 mL single-necked bottle, dissolved in DMF (15 mL), and then 5% Pd / C (800 mg) was added, and hydrogenation was carried out at room temperature for 2 h (HPLC was used to monitor the reaction progress). Pd / C was filtered, and the filtrate was used directly in the next step without concentration.

[0273] Step 4: Synthesis of compound 3A

[0274] The DMF solution of compound 3c obtained in the previous step was placed in an ice-water bath, and DIPEA (219uL, 1.21mmol) was added, followed by compound M3 (683mg, 1.21mmol). After the addition, the mixture was heated to room temperature and reacted for 1h. The reaction was monitored by HPLC and the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain compound 3A (524mg), with a yield of 53%. LC-MS: [MH]- =816.3.

[0275] 2. Preparation of Compound 3B

[0276]

[0277] Referring to the synthesis method of compound 3A, L-benzyl lactate in the first step was replaced with D-benzyl lactate to obtain compound 3B. LC-MS: [MH] - =816.3.

[0278] Embodiment 10:

[0279] Synthesis of compounds 4A and 4B:

[0280] 1. Preparation of Compound 4A

[0281]

[0282] Step 1: Synthesis of compound 3c

[0283] 3b (800 mg, 1.21 mmol, 1.0 eq) was added to a 100 mL single-mouth bottle, dissolved in DMF (15 mL), and then 5% Pd / C (800 mg) was added, and hydrogenation was carried out at room temperature for 2 h (HPLC was used to monitor the reaction progress). Pd / C was filtered, and the filtrate was used directly in the next step without concentration.

[0284] Step 2: Synthesis of compound 4A

[0285] The DMF solution of compound 3c obtained in the previous step was placed in an ice-water bath, and DIPEA (219uL, 1.21mmol) was added, followed by McOSu (373mg, 1.21mmol). After the addition, the mixture was heated to room temperature and reacted for 1h. The reaction was monitored by HPLC and the reaction solution was purified by high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain compound 4A (450mg) with a yield of 59%; LC-MS: [M+H] + =631.3.

[0286] 2. Synthesis of Compound 4B

[0287]

[0288] Referring to the synthesis method of compound 4A, compound 4B was obtained, LC-MS: [M+H] + =631.3.

[0289] Embodiment 11:

[0290] Synthesis of compound 5:

[0291]

[0292] Step 1: Synthesis of compound 5a

[0293] M1 (10 g, 27.1 mmol), 3,3,3-trifluorobenzyl lactate (prepared according to the method disclosed in patent WO2020063673A1) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added to a 250 mL single-mouth bottle and heated to 100 ° C for 4 h. After the reaction was completed, the mixture was cooled to room temperature, the insoluble matter was filtered out, 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, with a yield of 35.1%; LC-MS: [M+H] + =543.2.

[0294] Step 2: Synthesis of compound 5b

[0295] Compound 5a (5 g, 9.2 mmol) and 15 mL DMF were added to a 50 mL single-necked bottle. After dissolving, DBU (1.68 g, 11 mmol) was added under ice-water bath and reacted for 1 h. This was recorded as reaction solution ①.

[0296] Take another 50mL single-mouth bottle, add M4 (3.8g, 9.2mmol), PyBOP (5.75g, 11mmol), HOBt (1.49g, 11mmol) and 10mL DMF, after dissolving, add DIPEA (1.82mL, 11mmol) in an ice-water bath, continue to react for 30min, add reaction solution ①, warm to room temperature and react for 2h. HPLC monitors the reaction progress. After the reaction is completed, the reaction solution is purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 5b (4.1) g, with a yield of 62.3%; LC-MS: [M+H] + =716.3.

[0297] Step 3: Synthesis of Compound 5

[0298] Compound 5b (900 mg, 1.26 mmol) was added to a 25 mL single-mouth bottle, 15 mL DMF was used to dissolve it, and then 900 mg 5% Pd / C was added. The hydrogenation reaction was continued for 2 h. After the reaction was completed, the mixture was filtered and the filtrate was placed in an ice-water bath. DIPEA (228 uL, 1.38 mmol) was added, and then compound M3 (712 mg, 1.26 mmol) was added. After the addition, the mixture was heated to room temperature and reacted for 1 h. The reaction was completed by HPLC monitoring. The reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain the product compound 5 (525) mg, with a yield of 47.9%; LC-MS: [MH]- =870.3.

[0299] Embodiment 12:

[0300] Synthesis of compound 6:

[0301]

[0302] Compound 5b (900 mg, 1.26 mmol) was added to a 25 mL single-mouth bottle, 15 mL DMF was added to dissolve, and 900 mg 5% Pd / C was added. The hydrogenation reaction was continued for 2 h. After the reaction was completed, the filtrate was filtered and placed in an ice-water bath. DIPEA (228 uL, 1.38 mmol) was added, and then McOSu (388 mg, 1.26 mmol) was added. After the addition, the temperature was raised to room temperature and the reaction was continued for 1 h. The reaction was completed by HPLC monitoring. The reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain the product compound 6 (450) mg, with a yield of 52%; LC-MS: [MH] - =683.3.

[0303] Embodiment 13:

[0304] Synthesis of compound 7:

[0305]

[0306] Step 1: Synthesis of compound 7a

[0307] M1 (10 g, 27.1 mmol), 2-cyclopropyl-2-hydroxybenzyl acetate (prepared according to the method disclosed in patent WO2020244657A1) (11.2 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added to a 250 mL single-mouth bottle and heated to 100 ° C for 4 hours. After the reaction was completed, the temperature was cooled to room temperature, the insoluble matter was filtered out, 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) with a yield of 36%; LC-MS: [M+H] + =515.2.

[0308] Step 2: Synthesis of compound 7b

[0309] Compound 7a (4 g, 7.8 mmol) and 10 mL DMF were added to a 50 mL single-necked bottle. After dissolving, DBU (1.42 g, 9.3 mmol) was added under ice-water bath and reacted for 1 h. This was recorded as reaction solution ①.

[0310] Take another 50mL single-mouth bottle, add M4 (3.2g, 7.8mmol), PyBOP (4.5g, 8.6mmol), HOBt (1.16g, 8.6mmol) and 10mL DMF, after dissolving, add DIPEA (1.65mL, 10mmol) in an ice-water bath, continue to react for 30min, add reaction solution ①, warm to room temperature and react for 2h. HPLC monitors the reaction progress. After the reaction is completed, the reaction solution is purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 7b (4.2g), with a yield of 78%; LC-MS: [M+H] + =688.3.

[0311] Step 3: Synthesis of Compound 7

[0312] Compound 7b (1000 mg, 1.45 mmol) was added to a 25 mL single-mouth bottle, 15 mL DMF was added to dissolve, 1000 mg 5% Pd / C was added, hydrogenation reaction was carried out for 2 h, the reaction was completed, filtered, the filtrate was placed in an ice-water bath, DIPEA (248 uL, 1.5 mmol) was added, and then compound M3 (720 mg, 1.45 mmol) was added, and the temperature was raised to room temperature for 1 h. The reaction was monitored by HPLC, and the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution, which was lyophilized to obtain the product compound 7 (503 mg), with a yield of 41%; LC-MS: [MH] - =842.3.

[0313] Embodiment 14:

[0314] Synthesis of compound 8:

[0315]

[0316] Step 1: Synthesis of compound 8a

[0317] M1 (10 g, 27.1 mmol), 2-hydroxy-3-cyclopropylpropionic acid benzyl ester (synthesized according to the method disclosed in patent WO2020063676A) (12.0 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added to a 250 mL single-mouth bottle and heated to 100 ° C for 4 h. After the reaction was completed, the mixture was cooled to room temperature, the insoluble matter was filtered out, 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.

[0318] Step 2: Synthesis of compound 8b

[0319] Compound 8a (4 g, 7.6 mmol) and 10 mL DMF were added to a 50 mL single-necked bottle. After dissolution, DBU (1.39 g, 9.1 mmol) was added under ice-water bath and reacted for 1 h. This was recorded as reaction solution ①.

[0320] Take another 50mL single-mouth bottle, add M4 (3.12g, 7.6mmol), PyBOP (4.5g, 8.6mmol), HOBt (1.16g, 8.6mmol) and 10mL DMF, after dissolving, add DIPEA (1.65mL, 10mmol) in an ice-water bath, continue to react for 30min, add reaction solution ①, warm to room temperature and react for 2h. HPLC monitors the reaction progress. After the reaction is completed, the reaction solution is purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 8b (4.5g), with a yield of 84%; LC-MS: [M+H] + =702.3.

[0321] Step 3: Synthesis of Compound 8

[0322] 8b (1000 mg, 1.42 mmol) was added to a 25 mL single-mouth bottle, and 15 mL DMF was added to dissolve it. Then 1000 mg 5% Pd / C was added. The hydrogenation reaction was carried out for 2 h. After the reaction was completed, the filtrate was filtered and placed in an ice-water bath. DIPEA (248 uL, 1.5 mmol) was added, and then compound M3 (708 mg, 1.42 mmol) was added. After the addition, the temperature was raised to room temperature and the reaction was carried out for 1 h. The reaction was monitored by HPLC. The reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain the product compound 8 (443 mg), with a yield of 36%; LC-MS: [MH] - =856.4.

[0323] Embodiment 15:

[0324] Synthesis of compound 9:

[0325]

[0326] Step 1: Synthesis of compound 9a

[0327] Add amino-octapolyethylene glycol-carboxyl (10 g, 22.7 mmol) and 20 mL DMF to a 500 mL single-mouth bottle, add McOSu (8.38 g, 27.2 mmol), DIEA (5.6 mL, 3.4 mmol), react at room temperature for 2 h, and monitor by TLC. After the reaction, pour the reaction solution into 100 mL of water, extract three times with dichloromethane, combine the organic phases, wash twice with saturated sodium chloride solution, and dry over anhydrous sodium sulfate. Filter and concentrate under reduced pressure at 45 ° C to obtain a crude product. The crude product is purified by silica gel column chromatography (DCM: MeOH = 30: 1-10: 1), collect the product eluate, and concentrate to obtain compound 9a (12.1 g), with a yield of 84.5%, LCMS: [MH] + =633.3.

[0328] Step 2: Synthesis of compound 9b

[0329] 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 added to a 500 mL single-mouth bottle, and the mixture was reacted at room temperature for 1 h and monitored by TLC. After the reaction was completed, the insoluble matter was filtered out. The filtrate was concentrated at 45 °C under reduced pressure by a water pump to remove the solvent to 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) with a yield of 91.5%, LCMS: [M+H] + =801.3.

[0330] Step 3: Synthesis of compound 9

[0331] 10 mL of DMF was added to compound 1c (220 mg, 0.5 mmol), compound 9b (500 mg, 0.6 mmol) was added in an ice-water bath and cooled to 0°C, compound DIPEA (124 uL, 0.75 mmol) was added, and the reaction was allowed to react at room temperature for 1 h, and 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 preparation solution, and the preparation solution was decompressed to remove acetonitrile, and then lyophilized to obtain compound 9 (430 mg), with a yield of 83%, LCMS: [MH] + =1038.5.

[0332] Embodiment 16:

[0333] Synthesis of compound 10:

[0334]

[0335] 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 added to a 25 mL single-mouth bottle, and DIPEA (15.5 mg, 0.12 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution, which was lyophilized to obtain compound 10 (80 mg) with a yield of 76.7%, LC-MS: [M+H] + =1287.5.

[0336] Embodiment 17:

[0337] Synthesis of compound 11:

[0338]

[0339] 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 added to a 25 mL single-mouth bottle, and DIPEA (15.5 mg, 0.12 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain a product preparation solution, which was lyophilized to obtain compound 11 (100 mg) with a yield of 70.9%, LC-MS: [M / 2+H] + =855.86.

[0340] Embodiment 18:

[0341] Synthesis of compounds 12A and 12B:

[0342] 1. Preparation of Compound 12A

[0343]

[0344] 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 added to a 25 mL single-mouth bottle, and DIPEA (15.5 mg, 0.12 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution, which was lyophilized to obtain compound 12A (80 mg) with a yield of 76.2%, LC-MS: [M+H]+ =1301.5.

[0345] 2. Preparation of Compound 12B

[0346]

[0347] Referring to the synthetic method of compound 12A, compound 12B was obtained. LC-MS: [M+H] + =1301.5.

[0348] Embodiment 19:

[0349] Synthesis of compounds 13A and 13B:

[0350]

[0351] 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 single-mouth bottle, and DIPEA (28.2 mg, 0.218 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain the preparation solutions of compound 13A and compound 13B, which were lyophilized to obtain compound 13A (90 mg) and compound 13B (80 mg). LC-MS: [M+H] + =1355.5.

[0352] Embodiment 20:

[0353] Synthesis of compound 14:

[0354]

[0355] Step 1: Synthesis of compound 14a

[0356] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of compound 14a, which was lyophilized to obtain solid compound 14a (550 mg) with a yield of 60%, LC-MS: [M+H] + =1474.6.

[0357] Step 2: Synthesis of compound 14

[0358] Compound 14a (200 mg, 0.136 mmol), zinc bromide (612 mg, 2.72 mmol) and 6 mL nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, 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 preparation solution, which was lyophilized to obtain solid compound 14 (100 mg) with a yield of 56%. LC-MS: [M+H] + =1318.5.

[0359] Embodiment 21:

[0360] Synthesis of compounds 15A and 15B:

[0361] 1. Preparation of Compound 15A

[0362]

[0363] Step 1: Synthesis of compound 15a

[0364] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of compound 15a, which was lyophilized to obtain solid compound 15a (650 mg) with a yield of 70%, LC-MS: [M+H] + =1488.6.

[0365] Step 2: Synthesis of compound 15A

[0366] Compound 15a (200 mg, 0.134 mmol), zinc bromide (603.5 mg, 2.68 mmol) and 6 mL of nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, 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 preparation solution, which was lyophilized to obtain solid compound 15A (110 mg) with a yield of 62%. LC-MS: [M+H] + =1332.5.

[0367] 2. Preparation of Compound 15B

[0368]

[0369] Referring to the synthetic method of compound 15A, compound 15B was obtained. LC-MS: [M+H] + =1332.5.

[0370] Embodiment 22:

[0371] Synthesis of compounds 16A and 16B:

[0372]

[0373] Step 1: Synthesis of compounds 16a and 16b

[0374] 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 single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain the preparation solutions of compound 16a and compound 16b, which were lyophilized to obtain compound 16a (350 mg) and compound 16b (300 mg), respectively. LC-MS: [M / 2+H] + =771.8.

[0375] Step 2: Synthesis of compound 16A

[0376]

[0377] Compound 16a (200 mg, 0.13 mmol), zinc bromide (585.5 mg, 2.6 mmol) and 6 mL nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 16A (120 mg) with a yield of 66%. LC-MS: [M+H] + =1386.4.

[0378] Step 3: Synthesis of compound 16B

[0379]

[0380] Compound 16b (200 mg, 0.13 mmol), zinc bromide (585.5 mg, 2.6 mmol) and 6 mL nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 16B (115 mg) with a yield of 64%. LC-MS: [M+H] + =1386.4.

[0381] Embodiment 23:

[0382] Synthesis of compounds 17A and 17B:

[0383]

[0384] Step 1: Synthesis of compounds 17a and 17b

[0385] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain the preparation solutions of compound 17a and compound 17b, which were lyophilized to obtain compound 17a (320 mg) and compound 17b (280 mg), respectively. LC-MS: [M / 2+H] + =757.8.

[0386] Step 2: Synthesis of compound 17A

[0387]

[0388] Compound 17a (200 mg, 0.132 mmol), zinc bromide (594.5 mg, 2.64 mmol) and 6 mL of nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, 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 preparation solution, which was lyophilized to obtain solid compound 17A (120 mg) with a yield of 67%. LC-MS: [M+H] + =1358.5.

[0389] Step 3: Synthesis of compound 17B

[0390]

[0391] Compound 17b (200 mg, 0.132 mmol), zinc bromide (594.5 mg, 2.64 mmol) and 6 mL of nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, 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 preparation solution, which was lyophilized to obtain solid compound 17B (116 mg) with a yield of 65%. LC-MS: [M+H] + =1358.5.

[0392] Embodiment 24:

[0393] Synthesis of compounds 18A and 18B:

[0394]

[0395] Step 1: Synthesis of compounds 18a and 18b

[0396] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain the preparation solutions of compound 18a and compound 18b, which were lyophilized to obtain compound 18a (300 mg) and compound 18b (280 mg), respectively. LC-MS: [M / 2+H] + =764.8.

[0397] Step 2: Synthesis of compound 18A

[0398]

[0399] Compound 18a (200 mg, 0.131 mmol), zinc bromide (590.0 mg, 2.62 mmol) and 6 mL of nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, 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 preparation solution, which was lyophilized to obtain solid compound 18A (125 mg) with a yield of 70%. LC-MS: [M+H] + =1372.5.

[0400] Step 3: Synthesis of compound 18B

[0401]

[0402] Compound 18b (200 mg, 0.131 mmol), zinc bromide (590.0 mg, 2.62 mmol) and 6 mL of nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 18B (120 mg) with a yield of 67%. LC-MS: [M+H] + =1372.5.

[0403] Embodiment 25:

[0404] Synthesis of compound 19:

[0405]

[0406] Step 1: Synthesis of compound 19a

[0407] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of compound 19a, and the preparation solution was lyophilized to obtain solid compound 19a (600 mg), with a yield of 66%, LC-MS: [M+H] + =1450.6.

[0408] Step 2: Synthesis of compound 19

[0409] Compound 19a (200 mg, 0.138 mmol), zinc bromide (621 mg, 2.76 mmol) and 6 mL nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, 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 preparation solution, which was lyophilized to obtain solid compound 19 (120 mg) with a yield of 67%. LC-MS: [M+H] + =1294.5.

[0410] Embodiment 26:

[0411] Synthesis of Compound 20A and Compound 20B:

[0412] 1. Preparation of Compound 20A

[0413]

[0414] Step 1: Synthesis of compound 20a

[0415] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of compound 20a, which was lyophilized to obtain solid compound 20a (560 mg) with a yield of 62%, LC-MS: [M+H] + =1464.6.

[0416] Step 2: Synthesis of compound 20A

[0417] Compound 20a (200 mg, 0.136 mmol), zinc bromide (614.8 mg, 2.73 mmol) and 6 mL of nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, 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 preparation solution, which was lyophilized to obtain solid compound 20A (120 mg) with a yield of 67%. LC-MS: [M+H] + =1308.5.

[0418] 2. Preparation of Compound 20B

[0419]

[0420] Referring to the synthetic method of compound 20A, compound 20B was obtained. LC-MS: [M+H] + =1308.5.

[0421] Embodiment 27:

[0422] Synthesis of compounds 21A and 21B:

[0423]

[0424] Step 1: Synthesis of compounds 21a and 21b

[0425] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain the preparation solutions of compound 21a and compound 21b, which were lyophilized to obtain compound 21a (330 mg) and compound 21b (300 mg), respectively. LC-MS: [M / 2+H] + =759.8.

[0426] Step 2: Synthesis of compound 21A

[0427]

[0428] Compound 21a (200 mg, 0.132 mmol), zinc bromide (594.5 mg, 2.64 mmol) and 6 mL of nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 21A (125 mg) with a yield of 69%. LC-MS: [M+H] + =1362.4.

[0429] Step 3: Synthesis of compound 21B

[0430]

[0431] Compound 21b (200 mg, 0.132 mmol), zinc bromide (594.5 mg, 2.64 mmol) and 6 mL of nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 21B (112 mg) with a yield of 62%. LC-MS: [M+H] + =1362.4.

[0432] Embodiment 28:

[0433] Synthesis of compounds 22A and 22B:

[0434]

[0435] Step 1: Synthesis of compounds 22a and 22b

[0436] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain the preparation solutions of compound 22a and compound 22b, which were lyophilized to obtain compound 22a (330 mg) and compound 22b (300 mg), respectively. LC-MS: [M+H] + =1490.6.

[0437] Step 2: Synthesis of compound 22A

[0438]

[0439] Compound 22a (200 mg, 0.134 mmol), zinc bromide (603.5 mg, 2.68 mmol) and 6 mL nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 22A (120 mg) with a yield of 67%. LC-MS: [M+H] + =1334.5.

[0440] Step 3: Synthesis of compound 22B

[0441]

[0442] Compound 22b (200 mg, 0.134 mmol), zinc bromide (603.5 mg, 2.68 mmol) and 6 mL of nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 22B (115 mg) with a yield of 65%, LC-MS: [M+H]+=1334.5.

[0443] Embodiment 29:

[0444] Synthesis of compounds 23A and 23B:

[0445]

[0446] Step 1: Synthesis of compounds 23a and 23b

[0447] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain the preparation solutions of compound 23a and compound 23b, which were lyophilized to obtain compound 23a (320 mg) and compound 23b (300 mg), respectively. LC-MS: [M / 2+H] + =752.8.

[0448] Step 2: Synthesis of compound 23A

[0449]

[0450] 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 single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, 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 preparation solution, which was lyophilized to obtain solid compound 23A (122 mg) with a yield of 68%. LC-MS: [M+H] + =1348.5.

[0451] Step 3: Synthesis of compound 23B

[0452]

[0453] Compound 23b (200 mg, 0.133 mmol), zinc bromide (598.5 mg, 2.66 mmol) and 6 mL of nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 23B (115 mg) with a yield of 64%. LC-MS: [M+H] + =1348.5.

[0454] Embodiment 30:

[0455] Synthesis of compound 24:

[0456]

[0457] Step 1: Synthesis of compound 24a

[0458] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of compound 24a, and the preparation solution was lyophilized to obtain solid compound 24a (600 mg), with a yield of 68%, LC-MS: [M+H] + =1424.6.

[0459] Step 2: Synthesis of compound 24

[0460] Compound 24a (200 mg, 0.14 mmol), zinc bromide (631 mg, 2.8 mmol) and 6 mL nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, 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 preparation solution, which was lyophilized to obtain solid compound 24 (120 mg) with a yield of 68%. LC-MS: [M+H] + =1268.4.

[0461] Embodiment 31:

[0462] Synthesis of Compound 25A and Compound 25B:

[0463] 1. Preparation of Compound 25A

[0464]

[0465] Step 1: Synthesis of compound 25a

[0466] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of compound 25a, which was lyophilized to obtain solid compound 25a (560 mg) with a yield of 63%, LC-MS: [M+H] + =1438.6.

[0467] Step 2: Synthesis of compound 25A

[0468] Compound 25a (200 mg, 0.139 mmol), zinc bromide (626 mg, 2.78 mmol) and 6 mL nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 25A (115 mg) with a yield of 65%, LC-MS: [M+H]+=1282.5.

[0469] 2. Preparation of Compound 25B

[0470]

[0471] Referring to the synthesis method of compound 25A, compound 26B was obtained, LC-MS: [M+H]+=1282.5.

[0472] Embodiment 32:

[0473] Synthesis of compounds 26A and 26B:

[0474]

[0475] Step 1: Synthesis of compounds 26a and 26b

[0476] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain the preparation solutions of compound 26a and compound 26b, which were lyophilized to obtain compound 26a (330 mg) and compound 26b (300 mg), respectively. LC-MS: [M+H] + =1492.5.

[0477] Step 2: Synthesis of compound 26A

[0478]

[0479] Compound 26a (200 mg, 0.134 mmol), zinc bromide (603.5 mg, 2.68 mmol) and 6 mL nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 26A (125 mg) with a yield of 70%. LC-MS: [M+H]+ =1336.4.

[0480] Step 3: Synthesis of compound 26B

[0481]

[0482] Compound 26b (200 mg, 0.134 mmol), zinc bromide (603.5 mg, 2.68 mmol) and 6 mL nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 26B (112 mg) with a yield of 63%. LC-MS: [M+H] + =1336.4.

[0483] Embodiment 33:

[0484] Synthesis of compounds 27A and 27B:

[0485]

[0486] Step 1: Synthesis of compounds 27a and 27b

[0487] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain the preparation solutions of compound 27a and compound 27b, which were lyophilized to obtain compound 27a (330 mg) and compound 27b (300 mg), respectively. LC-MS: [M+H] + =1464.6.

[0488] Step 2: Synthesis of compound 27A

[0489]

[0490] Compound 27a (200 mg, 0.136 mmol), zinc bromide (614.8 mg, 2.73 mmol) and 6 mL of nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, 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 preparation solution, which was lyophilized to obtain solid compound 27A (125 mg) with a yield of 70%. LC-MS: [M+H]+ =1308.5.

[0491] Step 3: Synthesis of compound 27B

[0492]

[0493] Compound 27b (200 mg, 0.136 mmol), zinc bromide (614.8 mg, 2.73 mmol) and 6 mL of nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 27B (115 mg) with a yield of 64%. LC-MS: [M+H] + =1308.5.

[0494] Embodiment 34:

[0495] Synthesis of compounds 28A and 28B:

[0496]

[0497] Step 1: Synthesis of compounds 28a and 28b

[0498] 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 added to a 50 mL single-mouth bottle, and DIPEA (120 mg, 0.93 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high-performance liquid chromatography to obtain the preparation solutions of compound 28a and compound 28b, which were lyophilized to obtain compound 28a (320 mg) and compound 28b (300 mg), respectively. LC-MS: [M+H] + =1478.6.

[0499] Step 2: Synthesis of compound 28A

[0500]

[0501] Compound 28a (200 mg, 0.135 mmol), zinc bromide (608 mg, 2.7 mmol) and 6 mL nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, 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 preparation solution, which was lyophilized to obtain solid compound 28A (121 mg) with a yield of 68%. LC-MS: [M+H] +=1322.5.

[0502] Step 3: Synthesis of compound 28B

[0503]

[0504] Compound 28b (200 mg, 0.135 mmol), zinc bromide (608 mg, 2.7 mmol) and 6 mL nitromethane were added to a 25 mL single-mouth bottle and reacted at room temperature for 1 h. After the reaction was completed by HPLC monitoring, the solvent was removed by vacuum concentration to obtain a crude product. The crude product was purified by HPLC to obtain a product preparation solution, which was lyophilized to obtain solid compound 28B (115 mg) with a yield of 64%. LC-MS: [M+H] + =1322.5.

[0505] Embodiment 34:

[0506] Synthesis of compound 29:

[0507]

[0508] Step 1: Synthesis of Compounds 29a to 29d

[0509] Compound 29d was obtained by referring to the synthesis of "Compound 26" in patent CN102933236A.

[0510] Step 2: Synthesis of compound 29e

[0511] In a 250mL three-necked flask, compound 29d (3.0g, 5.8mmol) and 50mL anhydrous THF were added. Under nitrogen protection, an ice-water bath was maintained at 0°C, and sodium borohydride solid (329mg, 8.7mmol) was added. The mixture was reacted at 0°C for 30min, and then heated to room temperature for 2h. The reaction end point was monitored by TLC. After the reaction, the reaction solution was placed in ice water, 50mL of water was slowly added to quench the reaction, and 1N dilute HCl was added until no bubbles appeared. Ethyl acetate was added for extraction (60mL*3), and the organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (DCM / MeOH=20:1-5:1) to obtain compound 29e (2.1g, yellow solid) with a yield of 73.8%, LCMS: [M+H] + =491.2.

[0512] Step 3: Synthesis of compound 29f

[0513] Compound 29e (2.1 g, 4.3 mmol), TBSCl (1.3 g, 8.6 mmol), imidazole (580 mg, 8.6 mmol) were added to a 100 mL single-mouth bottle and dissolved in 15 mL of anhydrous DMF. The mixture was reacted at room temperature for 3 h and the reaction end point was monitored by TLC. After the reaction was completed, the reaction mixture was poured into 30 mL of water and extracted with DCM (25 mL*3). The organic phases were combined, washed with water and brine, dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain a 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) with a yield of 89.3%. LCMS: [M+H] + =605.3.

[0514] Step 4: Synthesis of compound 29g

[0515] Compound 29f (2.4 g, 4.0 mmol) and 30 mL of 5% formic acid / methanol solution were added to a 100 mL single-mouth bottle, cooled to below 5°C in an ice bath, and zinc powder (5.2 g, 80.0 mmol) was slowly added under stirring, and then the temperature was raised to room temperature for reaction for 1 h. The end point of the reaction was monitored by TLC. After the reaction was completed, the mixture was filtered while hot, the filter cake was washed with a small amount of methanol, 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 to obtain a brown-yellow oil. 60 mL of DCM was added, the organic layer was separated, washed once with saturated brine, 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 column chromatography (PE:EA=1:1-1:3) to obtain compound 29g (1.9 g, light yellow solid), with a yield of 82.5%, LCMS: [M+H] + =575.3.

[0516] Step 5: Synthesis of compound 29h

[0517] Compound 29g (1.9 g, 3.3 mmol), Fmoc-VCPABO-PNP (2.5 g, 3.3 mmol), 15 mL DMF were added to a 50 mL single-mouth bottle, and DIPEA (827 uL, 5.0 mmol) was added. The reaction was allowed to react at room temperature for 2 h, and the reaction endpoint was monitored by HPLC. After the reaction was completed, the reaction solution was purified by high-performance liquid chromatography to obtain a product preparation solution, which was then lyophilized to obtain compound 29h (3.3 g, light yellow solid), with a yield of 83.1%, LCMS: [M+H] + =1202.5.

[0518] Step 6: Synthesis of compound 29i

[0519] Compound 29h (3.3 g, 2.7 mmol) was added to a 50 mL single-mouth bottle, dissolved in 8 mL THF and 8 mL water, and then 20 mL glacial acetic acid was added. The reaction was reacted at room temperature and the reaction end point 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 phases were combined, washed with water and brine, dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (DCM / MeOH=10:1-5:1) to obtain compound 29i (2.1 g, light yellow solid), with a yield of 71.1%, LCMS: [M+H] + =1088.5.

[0520] Step 7: Synthesis of compound 29j

[0521] Compound 29i (2.1 g, 1.9 mmol) was added to a 100 mL three-necked flask and dissolved in 40 mL of anhydrous DCM. Under nitrogen protection, Dess-Martin periodic reagent (0.88 g, 2.1 mmol) was added and reacted at room temperature for 4 h. The reaction end point was monitored by HPLC. After the reaction was completed, the filtrate 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 preparation solution was lyophilized to obtain compound 29j (1.6 g, white solid), with a yield of 77.6%, LCMS: [M+H] + =1086.5.

[0522] Step 8: Synthesis of compound 29k

[0523] Compound 29j (1.6 g, 1.5 mmol), 5% palladium barium sulfate (0.29 g), ammonium formate (1.9 g, 30 mmol) were added to a 50 mL three-necked flask and dissolved in 20 mL methanol. The mixture was reacted at 45 °C for 2 h under nitrogen protection. The reaction endpoint was monitored by HPLC. After the reaction was completed, the mixture was filtered and the filtrate was concentrated. 20 mL DCM and 20 mL water were added and stirred evenly. The aqueous layer was extracted once with DCM. The organic layers were combined and washed once with saturated sodium chloride aqueous solution. The mixture was 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 preparation solution, which was lyophilized to obtain compound 29k (1.3 g, off-white solid) with a yield of 93.3%. LCMS: [M+H] + =996.4.

[0524] Step 9: Synthesis of compound 29l

[0525] 3-((p-Toluenesulfonyloxy)methyl)bicyclo[1.1.1]pentane-1-carboxylic acid benzyl ester (0.61 g, 1.2 mmol), compound 29k (1.3 g, 1.3 mmol) and 20 mL of DMF were added to a 100 mL single-mouth bottle, and cesium carbonate (0.78 g, 2.4 mmol) was added after stirring and dissolving. After the addition was completed, the temperature was raised to 60°C under nitrogen protection for 4 h, and the reaction end point was monitored by TLC. 40 mL of water was added to the reaction solution, and 30 mL of EA was used for extraction twice. The organic layers were combined, washed with water and saturated brine respectively, separated, 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 preparation solution, which was lyophilized to obtain compound 29l (1.2 g, off-white solid), with a yield of 84.0%, LCMS: [M+H] + =1210.5.

[0526] Step 10: Synthesis of compound 29m

[0527] Compound 29l (1.2 g, 1.0 mmol), 5% palladium barium sulfate (0.24 g), ammonium formate (1.3 g, 20 mmol) were added to a 50 mL three-necked flask and dissolved in 20 mL methanol. The mixture was reacted at 45 °C for 2 h under nitrogen protection. The reaction endpoint was monitored by HPLC. After the reaction was completed, the mixture was filtered and the filtrate was concentrated. 20 mL DCM and 20 mL water were added and stirred evenly. The aqueous layer was extracted once with DCM. The organic layers were combined and washed once with saturated sodium chloride aqueous solution. The mixture was 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 preparation solution, which was lyophilized to obtain compound 29m (0.92 g, off-white solid) with a yield of 82.5%. LCMS: [M+H] + =1120.5.

[0528] Step 11: Synthesis of compound 29n

[0529] Compound 29m (920 mg, 0.82 mmol) and 15 mL DMF were added to a 100 mL single-mouth bottle, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (496 mg, 2.6 mmol) and DMAP (313 mg, 2.6 mmol) were added successively under stirring at room temperature, and stirred at room temperature for 1 h under N2 protection, and then compound CBI (refer to patent CN109180681A) (376 mg, 1.0 mmol) was added, and the reaction was carried out overnight under N2 protection at room temperature. HPLC showed that the raw materials were basically reacted completely. 40 mL of water was added to the reaction solution, and extracted twice with 30 mL of DCM, the organic layers were combined, and washed once with water and saturated brine in turn, the organic layer was separated, 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 HPLC to obtain a product preparation solution, which was lyophilized to obtain compound 29n (874 mg, off-white solid) with a yield of 74.5%. LCMS: [M+H] + =1439.5.

[0530] Step 12: Synthesis of compound 29o

[0531] Compound 29n (874 mg, 0.60 mmol), 5% palladium barium sulfate (174 mg), ammonium formate (390 mg, 6 mmol) were added to a 50 mL three-necked flask and dissolved in 20 mL methanol. The mixture was reacted at 45 °C for 2 h under nitrogen protection. The reaction endpoint was monitored by HPLC. After the reaction was completed, the mixture was filtered and the filtrate was concentrated. 20 mL DCM and 20 mL water were added and stirred evenly. The aqueous layer was extracted once with DCM, the organic layers were combined, washed once with saturated sodium chloride aqueous 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 preparation solution, which was lyophilized to obtain compound 29o (667 mg, off-white solid) with a yield of 82.4%. LCMS: [M+H] + =1349.5.

[0532] Step 13: Synthesis of compound 29p

[0533] Compound 29o (667 mg, 0.49 mmol) and 20% hexahydropyridine DMF solution (15 mL) were added to a 50 mL single-mouth bottle and reacted at room temperature for 2 h. The reaction endpoint 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 preparation solution, which was lyophilized to obtain compound 29p (428 mg, off-white solid) with a yield of 77.5%. LCMS: [M+H] + =1127.5.

[0534] Step 14: Synthesis of compound 29

[0535] Compound 29p (428 mg, 0.38 mmol), DIPEA (156 uL, 0.95 mmol), McOSu (166 mg, 0.57 mmol) were added to a 50 mL single-mouth bottle and dissolved in 20 mL DMF. The mixture was reacted at room temperature for 2 h and the reaction endpoint was monitored by HPLC. After the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a product preparation solution, which was lyophilized to obtain compound 29 (386 mg, off-white solid) with a yield of 77.0%. LCMS: [M+H] + =1320.5.

[0536] Embodiment 35:

[0537] Synthesis of compound 30:

[0538]

[0539] Step 1: Synthesis of compound 30a

[0540] Compound 29p (560 mg, 0.50 mmol), DIPEA (161 mg, 1.25 mmol), compound M3 (423 mg, 0.75 mmol) were added to a 50 mL single-mouth bottle, dissolved in 10 mL DMF, and reacted at room temperature for 2 h. The reaction endpoint was monitored by HPLC. After the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a product preparation solution, which was lyophilized to obtain compound 30a (622 mg, off-white solid), with a yield of 82.6%, LCMS: [M+H] + =1507.6.

[0541] Step 2: Synthesis of compound 30

[0542] Compound 30a (622 mg, 0.41 mmol) and zinc bromide (922 mg, 4.1 mmol) were added to a 50 mL single-mouth bottle and dissolved in 10 mL nitromethane. The mixture was reacted at 25 °C for 45 min. The reaction endpoint was monitored by HPLC. After the reaction, the solvent was removed under reduced pressure at 40 °C to obtain a concentrate. The concentrate was purified by high performance liquid chromatography to obtain a product preparation solution. The product was lyophilized to obtain compound 30 (260 mg, off-white solid). The yield was 48%. LCMS: [M+H] + =1322.5.

[0543] Embodiment 36:

[0544] Synthesis of compound 31:

[0545]

[0546]

[0547] Step 1: Synthesis of compound 31a

[0548] Benzyl 5-bromovalerate (488 mg, 1.8 mmol), compound 29k (1.8 g, 1.8 mmol) and 20 mL DMF were added to a 100 mL single-mouth bottle, and potassium carbonate (372 mg, 2.7 mmol) was added after stirring and dissolving. After the addition was completed, the temperature was raised to 60°C for reaction for 4 h, and the reaction end point was monitored by HPLC. 40 mL of water was added to the reaction solution, and 30 mL of EA was used for extraction twice. The organic layers were combined, washed with water and saturated brine respectively, separated, 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 preparation solution, which was lyophilized to obtain compound 31a (1.8 g, off-white solid) with a yield of 84.0%, LCMS: [M+H] + =1186.5.

[0549] Step 2: Synthesis of compound 31b

[0550] Compound 31a (1.8 g, 1.0 mmol), 5% palladium barium sulfate (0.36 g), ammonium formate (0.95 g, 15 mmol) were added to a 50 mL three-necked flask and dissolved in 20 mL methanol. The mixture was reacted at 45 °C for 2 h under nitrogen protection. The reaction endpoint was monitored by HPLC. After the reaction was completed, the mixture was filtered and the filtrate was concentrated. 20 mL DCM and 20 mL water were added and stirred evenly. The aqueous layer was extracted once with DCM. The organic layers were combined and washed once with saturated sodium chloride aqueous solution. The mixture was separated and 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 preparation solution, which was lyophilized to obtain compound 31b (0.85 g, off-white solid) with a yield of 78%. LCMS: [M+H] + =1096.5.

[0551] Step 4: Synthesis of compound 31c

[0552] Compound 31b (850 mg, 0.85 mmol) and 15 mL DMF were added to a 100 mL single-mouth bottle, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (514 mg, 2.7 mmol) and DMAP (324 mg, 2.7 mmol) were added in sequence under stirring at room temperature, and stirred at room temperature for 1 h under N2 protection, and then compound CBI (refer to patent CN109180681A) (376 mg, 1.0 mmol) was added, and the reaction was allowed to proceed overnight under N2 protection at room temperature. HPLC showed that the raw materials were basically reacted completely. 40 mL of water was added to the reaction solution, and the mixture was extracted twice with 30 mL of DCM, the organic layers were combined, and washed once with water and saturated brine in sequence, the organic layer was separated, 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 HPLC to obtain a product preparation solution, which was lyophilized to obtain compound 31c (941 mg, off-white solid) with a yield of 78.2%. LCMS: [M+H] + =1415.6.

[0553] Step 5: Synthesis of compound 31d

[0554] Compound 31c (941 mg, 0.66 mmol), 5% palladium barium sulfate (188 mg), ammonium formate (415 mg, 6.6 mmol) were added to a 50 mL three-necked flask and dissolved in 20 mL methanol. The mixture was reacted at 45 °C for 2 h under nitrogen protection. The reaction endpoint was monitored by HPLC. After the reaction was completed, the mixture was filtered and the filtrate was concentrated. 20 mL DCM and 20 mL water were added and stirred evenly. The aqueous layer was extracted once with DCM, the organic layers were combined, washed once with saturated sodium chloride aqueous 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 preparation solution, which was lyophilized to obtain compound 31d (735 mg, off-white solid) with a yield of 84.0%. LCMS: [M+H] + =1325.5.

[0555] Step 6: Synthesis of compound 31e

[0556] Compound 31d (735 mg, 0.55 mmol) and 20% hexahydropyridine DMF solution (15 mL) were added to a 50 mL single-mouth bottle and reacted at room temperature for 2 h. The reaction endpoint 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 preparation solution, which was lyophilized to obtain compound 31e (428 mg, off-white solid) with a yield of 86.5%. LCMS: [M+H] + =1103.5.

[0557] Step 7: Synthesis of compound 31f

[0558] Compound 31e (524 mg, 0.48 mmol), DIPEA (168 mg, 1.30 mmol), compound M3 (441 mg, 0.78 mmol) were added to a 50 mL single-mouth bottle, dissolved in 10 mL DMF, and reacted at room temperature for 2 h. The reaction endpoint was monitored by HPLC. After the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a product preparation solution, which was lyophilized to obtain compound 31f (549 mg, off-white solid) with a yield of 77.2%. LCMS: [M+H] + =1483.6.

[0559] Step 8: Synthesis of compound 31

[0560] Compound 31f (549 mg, 0.37 mmol) and zinc bromide (832 mg, 3.7 mmol) were added to a 50 mL single-mouth bottle and dissolved in 10 mL nitromethane. The mixture was reacted at 25 °C for 45 min. The reaction endpoint was monitored by HPLC. After the reaction, the solvent was removed under reduced pressure at 40 °C to obtain a concentrate. The concentrate was purified by high performance liquid chromatography to obtain a product preparation solution, which was lyophilized to obtain compound 31 (213 mg, off-white solid). The yield was 43.5%. LCMS: [M+H] + =1326.5.

[0561] Embodiment 37:

[0562] Synthesis of compound 32:

[0563]

[0564] Step 1: Synthesis of compound 32a

[0565] In a 50mL single-mouth bottle, add compound N-propargylmaleimide (500mg, 3.7mmol), N3-PEG4-OH (811mg, 3.7mmol), 10mL DMF and 5mL water to dissolve, then add CuSO4·5H2O (1.01g, 4.1mmol), sodium ascorbate (806mg, 4.1mmol), react at room temperature for 1h, and monitor the reaction end point by HPLC. After the reaction, the reaction solution is purified by high-performance liquid chromatography to obtain a product preparation solution, which is lyophilized to obtain compound 32a (1.06g), with a yield of 81%, LCMS: [M+H] + =355.1.

[0566] Step 2: Synthesis of compound 32

[0567] 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 added to a 50 mL three-necked flask, and diisopropyl azodicarboxylate (28 uL, 0.11 mmol) was added dropwise in an ice-water bath under nitrogen protection. The temperature was raised to room temperature for reaction and monitored by HPLC. After the reaction, the product was purified and freeze-dried to obtain compound 32 (18 mg) with a yield of 63%. LC-MS: [M+H] + =1040.4.

[0568] Embodiment 38:

[0569] Synthesis of compound 33:

[0570]

[0571] Step 1: Synthesis of compound 33a

[0572] Prepared according to the synthesis method of "Compound 22" in patent CN111686259A.

[0573] Step 2: Synthesis of compound 33c

[0574] Compound 33a (500 mg, 2.0 mmol) was added to a 50 mL single-mouth bottle, dissolved in 8 mL DMF, and 300 mg 5% Pd / C was added. The hydrogenation reaction was carried out for 2 h, and the reaction endpoint was monitored by TLC. After the reaction was completed, the DMF solution of compound 32b was filtered and used for later use.

[0575] 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 carried out at room temperature for 2 h. The reaction end point was monitored by HPLC. The reaction solution was purified by high performance liquid chromatography to obtain a product preparation solution, which was lyophilized to obtain compound 33c (580 mg) with a yield of 52%. LC-MS: [M+H] + =559.2.

[0576] Step 3: Synthesis of compound 33

[0577] 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 added to a 50 mL three-necked flask, and diisopropyl azodicarboxylate (14.2 uL, 0.07 mmol) was added dropwise in an ice-water bath under nitrogen protection. The temperature was raised to room temperature for reaction and monitored by HPLC. After the reaction, the product was purified and freeze-dried to obtain compound 33 (15 mg) with a yield of 68%. LC-MS: [M+H] + =1244.5.

[0578] Embodiment 39 (comparative example):

[0579] Synthesis of compounds 34 and 35:

[0580]

[0581] Compound 34 was synthesized according to the synthesis method of “Compound 56” in patent CN105636612 A.

[0582]

[0583] Compound 35 was synthesized according to the synthesis method of “SG3249, tesirine” in the literature “ACS Med Chem Lett. 2016; 7:983–987”.

[0584] Embodiment 40:

[0585] The following is the sequence of Trastuzumab:

[0586] Light chain

[0587] MDMRVPAQLLGLLLLWLRGARC

[0588] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK

[0589] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

[0590] Heavy chain

[0591] MDMRVPAQLLGLLLLWLRGARC

[0592] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

[0593] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0594] Preparation of ligand-drug conjugates:

[0595] 1) General coupling method

[0596] After the preliminary purification, the antibody molecules with a monomer rate greater than 95% are replaced with an ultrafiltration centrifuge tube to a phosphate buffer solution at a concentration of 10 mg / mL. TCEP 20 times the number of antibody moles is added and reacted at room temperature for 4 hours to open the disulfide bonds between the antibody chains. A linker-drug compound (payload) 20 times the number of antibody moles is added and reacted at room temperature for 2 hours. After the reaction is completed, an ultrafiltration centrifuge tube with a molecular weight cutoff of 30KDa is used to replace the solution in PBS and remove the uncoupled payload. The ADC sample after the solution change is filtered with a 0.22 micron sterilizing filter for standby use.

[0597] 2) Determination of DAR value of ligand-drug conjugate

[0598] Monomer rate detection conditions:

[0599] The sample was centrifuged at 14000 rpm for 5 min, and the supernatant was sampled and analyzed;

[0600] Instrument: Waters e2695 (2489 UV / Vis);

[0601] Chromatographic column: TSKgel G3000SWXL (7.8×300mm, 5μm);

[0602] Mobile phase: A: 50 mM PB, 300 mM NaCl, 200 mM Arg, 5% IPA, pH 6.5;

[0603] The mobile phase A was used for isocratic elution for 30 min, the flow rate was 0.714 mL / min, the column temperature was 25 °C, and the detection wavelength was 280 nm.

[0604] DAR detection conditions:

[0605] The sample was centrifuged at 14000 rpm for 5 min, and the supernatant was sampled and analyzed;

[0606] Instrument: Waters H-class (TUV);

[0607] Chromatographic column: Proteomix HIC Butyl-NP5 (4.6×35 mm, 5 μm);

[0608] 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;

[0609] The chromatographic column was balanced with mobile phase A, and mobile phases A and B were used for gradient elution at a flow rate of 0.8 mL / min; the column temperature was 25°C, and the detection wavelength was 214 nm.

[0610] Embodiment 41:

[0611] ADC-1 was prepared according to the general coupling method of Example 40:

[0612]

[0613] Embodiment 42:

[0614] ADC-2 was prepared according to the general coupling method of Example 40:

[0615]

[0616] Embodiment 43:

[0617] ADC-3 was prepared according to the general coupling method of Example 40:

[0618]

[0619] Embodiment 44:

[0620] ADC-4 was prepared according to the general coupling method of Example 40:

[0621]

[0622] Embodiment 45:

[0623] ADC-5 was prepared according to the general coupling method of Example 40:

[0624]

[0625] Embodiment 46:

[0626] ADC-6 was prepared according to the general coupling method of Example 40:

[0627]

[0628] Embodiment 47:

[0629] ADC-7 was prepared according to the general coupling method of Example 40:

[0630]

[0631] Embodiment 48:

[0632] ADC-8 was prepared according to the general coupling method of Example 40:

[0633]

[0634] Embodiment 49:

[0635] ADC-9 was prepared according to the general coupling method of Example 40:

[0636]

[0637] Embodiment 50:

[0638] ADC-10 was prepared according to the general coupling method of Example 40:

[0639]

[0640] Embodiment 51:

[0641] ADC-11 was prepared according to the general coupling method of Example 40:

[0642]

[0643] Embodiment 52:

[0644] ADC-12 was prepared according to the general coupling method of Example 40:

[0645]

[0646] Embodiment 53:

[0647] ADC-13 was prepared according to the general coupling method of Example 40:

[0648]

[0649] Embodiment 54:

[0650] ADC-14 was prepared according to the general coupling method of Example 40:

[0651]

[0652] Embodiment 55:

[0653] ADC-15 was prepared according to the general coupling method of Example 40:

[0654]

[0655] Embodiment 56:

[0656] ADC-16 was prepared according to the general coupling method of Example 40:

[0657]

[0658] Embodiment 57:

[0659] ADC-17 was prepared according to the general coupling method of Example 40:

[0660]

[0661] Embodiment 58:

[0662] ADC-18 was prepared according to the general coupling method of Example 40:

[0663]

[0664] Embodiment 59:

[0665] ADC-19 was prepared according to the general coupling method of Example 40:

[0666]

[0667] Embodiment 60:

[0668] ADC-20 was prepared according to the general coupling method of Example 40:

[0669]

[0670] Example 61:

[0671] ADC-21 was prepared according to the general coupling method of Example 40:

[0672]

[0673] Embodiment 62:

[0674] ADC-22 was prepared according to the general coupling method of Example 40:

[0675]

[0676] Embodiment 63:

[0677] ADC-23 was prepared according to the general coupling method of Example 40:

[0678]

[0679] Embodiment 64:

[0680] ADC-24 was prepared according to the general coupling method of Example 40:

[0681]

[0682] Embodiment 65:

[0683] ADC-25 was prepared according to the general coupling method of Example 40:

[0684]

[0685] Embodiment 66:

[0686] ADC-26 was prepared according to the general coupling method of Example 40:

[0687]

[0688] Embodiment 67:

[0689] ADC-27 was prepared according to the general coupling method of Example 40:

[0690]

[0691] Embodiment 68:

[0692] ADC-28 was prepared according to the general coupling method of Example 40:

[0693]

[0694] Embodiment 69:

[0695] ADC-29 was prepared according to the general coupling method of Example 40:

[0696]

[0697] Embodiment 70:

[0698] ADC-30 was prepared according to the general coupling method of Example 40:

[0699]

[0700] Embodiment 71:

[0701] ADC-31 was prepared according to the general coupling method of Example 40:

[0702]

[0703] Embodiment 72:

[0704] ADC-32 was prepared according to the general coupling method of Example 40:

[0705]

[0706] Embodiment 73:

[0707] ADC-33 was prepared according to the general coupling method of Example 40:

[0708]

[0709] Embodiment 74:

[0710] ADC-34 was prepared according to the general coupling method of Example 40:

[0711]

[0712] Embodiment 75:

[0713] ADC-35 was prepared according to the general coupling method of Example 40:

[0714]

[0715] Embodiment 76:

[0716] ADC-36 was prepared according to the general coupling method of Example 40:

[0717]

[0718] Embodiment 77:

[0719] ADC-37 was prepared according to the general coupling method of Example 40:

[0720]

[0721] Embodiment 78:

[0722] ADC-38 was prepared according to the general coupling method of Example 40:

[0723]

[0724] Example 79 (control):

[0725] ADC-39 was prepared according to the general coupling method of Example 40:

[0726]

[0727] Example 80 (control):

[0728] ADC-40 was prepared according to the general coupling method of Example 40:

[0729]

[0730] Example 81: Plasma stability experiment

[0731] 1. Operation

[0732] Take a certain amount of ADC sample and add it to human plasma from which human IgG has been removed. Repeat three tubes for each ADC and place them in a 37°C water bath for incubation. After incubation for 72 hours and 144 hours, take out the ADC sample and add 100uL of ProteinA resin (MabSelectSuReTM LX Lot: #10221479GE, washed with PBS) to each tube. Shake and adsorb for 2 hours with a vertical mixer. After washing and elution steps, obtain the incubated ADC. Perform RP-HPLC detection on the ADC samples incubated for a specific time.

[0733] 2. Results

[0734] Table 1. DAR values ​​and monomer rate data of ligand-drug conjugates (ADCs) disclosed in the present invention.

[0735]

[0736]

[0737] Table 2. Plasma stability data of ligand-drug conjugates (ADCs) disclosed in the present invention.

[0738]

[0739] 3) Conclusion

[0740] As shown in Table 1, the ADC disclosed in the present invention has excellent properties of high DAR value (>7.5) and monomer rate (>97%).

[0741] As shown in Table 2, the DAR value of the ADC disclosed in the present invention can still be maintained at a high level after incubation in plasma for 7 days, which proves that the ADC of the present invention has excellent stability in plasma.

[0742] Example 82: In vitro activity test

[0743] 1) Experimental Materials

[0744] Cells: Derived from the Cell Bank of the Chinese Academy of Sciences;

[0745] Tumor cell culture medium: Gibco;

[0746] FBS: BIOWEST;

[0747] 2) Preparation of culture medium

[0748] Growth medium (with 10% FBS, Penicillin / streptomycin (100 U / mL);

[0749] Assay medium (with 1% FBS, Penicillin / streptomycin (100 U / mL);

[0750] 3) Operation

[0751] Turn on the ultraviolet light of the biosafety cabinet 30 minutes in advance, and then ventilate for 3 minutes. Preheat the growth medium, detection medium, D-PBS and trypsin in a 37°C constant temperature water bath, then disinfect the surface with alcohol and put it in the biosafety cabinet. Select cells with a confluence of ~80% (logarithmic growth phase), place them in the biosafety cabinet, remove the old culture medium, rinse with D-PBS, discard, digest with trypsin for 2-3 minutes, then add growth medium to terminate trypsin, and centrifuge at 500×g for 5 minutes. Aspirate the centrifugal supernatant, mix with 4mL detection medium, take 100uL for counting (take out 50uL of cell fluid, add 50μL0.4% TrypanBlue Stain and mix, and count after mixing). Plate according to the previously set cell number, 80uL / well plate in a 96-well plate, add only 80uL detection medium to wells E11, F11, and G11, and add 200uL DPBS to the edge wells to seal the edges. After the plated cells are completely attached to the wall (usually at least 4 hours), prepare and dilute the test samples: prepare 1.0mL, 2.5μM (5×Top Dose) test samples with detection medium, and dispense them into the first column of the V-shaped 96-well plate, 200μL per well; add 180μL of detection medium to the second to eighth columns, take 30μL from the first column and add it to the second column, mix up and down 10 times with a gun, discard the gun tip, and operate the remaining detection concentration points in sequence, and perform 7-fold gradient concentration dilution. Add 20uL of the gradient concentration test samples to the cells per well, and only add 20uL of detection medium to the 11th column. Set 3 replicates for each concentration, then put the 96-well plate into a 5% CO2, 37℃ cell culture incubator and culture for 5 days.

[0752] 4) Detection

[0753] After 5 days of exposure to the test sample, the MTS reagent was removed, thawed at room temperature and away from light, and fully vortexed to mix. In the biosafety cabinet, 20 μL of Cell Titer One Solution Reagen MTS reagent was added along the side wall of the well for every 100 μL of cell culture volume. The plate surface was gently tapped to mix the MTS solution evenly, and placed in a cell culture incubator with 5% CO2, 37°C and away from light for 2 hours. After the reaction was completed, the 96-well plate was removed, the OD490 nm absorbance value was detected in an ELISA instrument, and the data was recorded, sorted, and stored.

[0754] 5) Results

[0755] Table 3: IC50 values ​​of antibody drug conjugates and toxins for inhibiting the proliferation of N87 tumor cells in vitro.

[0756]

[0757]

[0758] Table 4: IC50 values ​​of antibody drug conjugates and toxins for inhibiting the proliferation of SK-BR-3 tumor cells in vitro.

[0759] Sample IC50(nM) Compound A 0.52 Compound B1 4.23 Compound C1 8.67 Compound D1 10.33 ADC-3 0.45 ADC-5 0.60 ADC-7 4.55 ADC-12 5.82 ADC-21 6.11 ADC-30 9.05 ADC-39 (control) 26.12

[0760] 6) Conclusion

[0761] As shown in Table 3, the ligand-drug conjugate targeting HER2 of the present invention has significant in vitro proliferation inhibition activity on HER2-positive cells N87, which is significantly better than naked antibody (Trastuzumab) and control group ADC-39.

[0762] As shown in Table 4, compared with naked antibody (Trastuzumab) and control group ADC, the ADC and single drug disclosed in the present invention also have obvious in vitro proliferation inhibition activity on HER2-positive cells SK-BR-3.

[0763] Example 83: In vivo safety testing

[0764] 1) Experimental Materials

[0765] Cells: Derived from the Cell Bank of the Chinese Academy of Sciences;

[0766] Tumor cell culture medium: Gibco;

[0767] Balb / c-nu nude mice: female, 5-7 weeks (age of mice at the time of tumor cell inoculation), weight 18.0-24.0 g, 180 mice (120 plus 60 surplus mice), purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.;

[0768] Test and control articles:

[0769] Test samples: ADC-3 and ADC-40 were provided by Chengdu Duote Antibody Drug Co., Ltd.

[0770] Histidine buffer was provided by Chengdu Duote Antibody Drug Co., Ltd.

[0771] 0.9% Sodium Chloride Injection: Kelun Pharmaceutical Co., Ltd.

[0772] 2) Cell culture

[0773] NCI-H1975 (human non-small cell lung cancer adenocarcinoma cells) were cultured in RPMI 1640 medium. NCI-H1975 cells in the exponential growth phase were collected, resuspended in RPMI 1640 medium to a suitable concentration, and then used for subcutaneous tumor inoculation in mice.

[0774] 3) Animal modeling and random grouping

[0775] 120 female nude mice were subcutaneously inoculated with 5×10 7 NCI-H1975 cells. Average tumor volume 170mm 3 Around 1:00 pm, the mice were randomly divided into groups according to the tumor size. 120 tumor-bearing mice with appropriate tumor volume were selected, randomly divided into groups, and drug administration (tail vein injection, drug volume of 0.1 mL / 10 g) was started. The day of grouping was defined as day 0.

[0776] 4) Preparation of test and reference substances

[0777] Table 5. Preparation of test article and control article solutions for the anti-tumor effect study in the NCI-H1975 (human non-small cell lung cancer adenocarcinoma cell) xenograft tumor model.

[0778]

[0779]

[0780] Note: Mix well before use to ensure the preparation is homogeneous.

[0781] 5) Experimental observation and data collection

[0782] During this experiment, animal experimental operations were carried out in accordance with the requirements of the standard operating procedures for in vivo screening tests of anti-tumor drugs. After tumor inoculation, routine monitoring included tumor growth (tumor was measured twice a week) and the effect of treatment on the normal behavior of the animals. Specific contents included the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, hair and other abnormalities. 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 represents the long diameter and b represents the short diameter). The data were recorded manually in the experiment, including the measurement of the long and short diameters of the tumor and the weighing of the animal body weight.

[0783] 6) Results

[0784] Table 6: Effects of administration of antibody drug conjugates (11.25 mg / kg) on ​​body weight of mice transplanted with NCI-H1975 tumors.

[0785]

[0786]

[0787] Note: * indicates the group in which mice died

[0788] 7) Conclusion

[0789] As shown in Table 6, the effects of ADC-3 disclosed in the present invention on the body weight of NCI-H1975 tumor-bearing mice at low and high doses were significantly smaller than those of ADC-40. Even in the high-dose group, there was no mouse death as shown in the control group, demonstrating that the ADC drug disclosed in the present invention has significant advantages in terms of safety.

Claims

1. A ligand-drug conjugate as shown in Formula I, or a pharmaceutically acceptable salt, deuterated substance, or solvate thereof: Ab-LD (I) in: Ab is a ligand unit selected from an antibody, an antibody fragment, a targeting protein or an Fc-fusion protein; L is the connecting unit between D and Ab; D is the drug unit; The ligand-drug conjugate is selected from the following structures: Wherein, u is selected from an integer of 1-10.

2. The ligand-drug conjugate or a pharmaceutically acceptable salt, deuterated substance or solvate thereof according to claim 1, characterized in that: Ab is an antibody and can form a connecting bond with a connecting unit through its heteroatom. The antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody or a multispecific antibody.

3. The ligand-drug conjugate or a pharmaceutically acceptable salt, deuterated substance or solvate thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof is selected from: 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-GPNMB 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 antibody, 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.

4. The ligand-drug conjugate or a pharmaceutically acceptable salt, deuterated substance or solvate thereof as claimed in claim 1 or 2, characterized in that: The pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts or magnesium salts formed with acidic functional groups in the structural formula; or acetates, trifluoroacetates, citrates, oxalates, tartrates, malates, nitrates, chlorides, bromides, iodides, sulfates, bisulfates, phosphates, lactates, oleates, ascorbic acid salts, salicylates, formates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates or p-toluenesulfonates formed with basic functional groups in the structure.

5. Use of the ligand-drug conjugate according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, deuterated substance or solvate thereof in the preparation of a drug for treating tumors.

6. The use according to claim 5, characterized in that: The tumor is gastric cancer, breast cancer or lung cancer.

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