Camptothecin drugs having a highly stable hydrophilic binding unit and complexes thereof

Abstract

A camptothecin-class drug having a highly stable hydrophilic binding unit, its conjugate, or its pharmaceutically acceptable salt, a method for preparing the same, and its effect in the prevention or treatment of cancer. The conjugate can specifically bind to a receptor highly expressed in tumor cells, has good water solubility, stability, and homogeneity, and is applicable to the prevention or treatment of diseases such as tumors.

Description

[Technical Field]

[0001] The present invention relates to a camptothecin antibody-drug conjugate having a highly stable hydrophilic binding structure unit. [Background technology]

[0002] As a new form of targeted therapy, antibody-drug conjugates (ADCs) typically consist of three parts: an antibody or antibody-based ligand, a small molecule drug, and a linker that binds the ligand to the drug. Antibody-drug conjugates utilize the antibody's specific recognition of an antigen to deliver the drug molecule to target cells, effectively releasing the drug molecule and achieving therapeutic objectives. In August 2011, the U.S. Food and Drug Administration (FDA) approved AdcetrIs, a new ADC drug developed by Seattle Genetic Ics for the treatment of Hodgkin lymphoma and relapsing large cell lymphoma (ALCL). TM The drug has been approved for market release. Its safety and efficacy have been demonstrated through clinical use.

[0003] Camptothecines (including irinotecan, exatecan, and SN38), small molecule compounds with antitumor properties, exert their antitumor effects by inhibiting DNA topoisomerase I. Many camptothecin drugs are widely used in clinical practice, with their main indications being bone cancer, prostate cancer, breast cancer, and pancreatic cancer. Unlike irinotecan, which is currently in clinical use, exatecan does not require enzymatic activation. Furthermore, compared to SN-38, the active ingredient in irinotecan, and topothecan, which is used clinically, exatecan has stronger inhibitory activity against topoisomerase I, causing stronger damage to various cancer cells in vitro. In particular, the expression of P-glycoprotein has shown efficacy even in cancer cells resistant to SN-38 and other agents. Exatecan has not been successfully marketed as a monotherapy drug, and it is speculated that its high cellular activity limits its therapeutic range.

[0004] The advantages of antibody-drug conjugates (ADCs) lie in increased water solubility, improved targeting, specific binding to antigens, drug transport to the vicinity of target cells, tumor cell death through drug release near target cells, and reduced toxic side effects. Camptothecin-type drugs show considerable potential in the field of ADCs. Currently, trastuzumab deruxtecan (trade name: Enhertu), an antibody-conjugated drug with exatecan as its toxin, was approved for market by the US FDA on December 20, 2019. As the first commercially available camptothecin-type ADC, it has fully demonstrated the drug discovery capabilities and application prospects of such drugs in the ADC field.

[0005] The ADC drug structure consists of three key components: the antibody, the linker, and the toxin. A defect in any of these components can affect the overall efficacy of the ADC. In the art, a structural design defect in Enhertu is evident. Camptothecin is a highly lipophilic and poorly soluble drug. The linker toxin used by Enhertu is designed to bind to the antibody via a Mc linker, and the fragment, which is linked to a cleavable tetrapeptide and an aminomethoxy autoeliminating spacer unit, achieves a drug-antibody ratio (DAR) of 8 by non-site-directed binding using interchain cysteine ​​residues (see patent CN104755494). This linker design leads to reduced stability of the camptothecin ADC drug, decreased monomer ratio, and further reduced efficacy and safety of the ADC in vivo at high DAR values.

[0006] The problem that this invention aims to solve is to provide a superior antitumor camptothecin-based ADC drug that has higher safety and efficacy, and better meets clinical needs. [Overview of the project]

[0007] Based on a comprehensive understanding of ADC drugs, the inventors unexpectedly discovered a series of antibody-drug conjugates containing camptothecin derivatives with highly stable hydrophilic polypeptide-binding structural units. Experiments revealed that ADC molecules containing each of these polypeptide-linked camptothecin derivatives exhibit high stability both in vitro and in vivo, high monomer content, and significantly higher pharmacokinetic activity compared to control ADCs. Furthermore, the inventors demonstrated that complex linker-toxin molecules can be efficiently generated by creatively employing novel deprotection reagents and solvent strategies through the design and innovation of synthetic pathways.

[0008] In one aspect of the present invention, a ligand-drug conjugate represented by formula I or a pharmaceutically acceptable salt thereof is provided. [ka] In the formula, Ab is a ligand unit selected from an antibody, antibody fragment, or protein. M is a binding unit that binds to Ab, Ac is a hydrophilic structural unit, D is any camptothecin-type drug, The chiral carbon atoms at positions 1 and 4 have either an R absolute configuration or an S absolute configuration. n is an integer selected from 1 to 20.

[0009] Preferably, the bonding unit M has a succinimide structure represented by formula a below or an open-ring succinimide structure represented by formulas b1 and b2. [ka] In formulas a, b1, or b2, the wavy line on the left indicates a bond with the Ab bonding site, and the wavy line on the right indicates a bond with the tertiary carbon atom at position 1 in formula I.

[0010] More preferably, the Ac has a structure represented by the following formula c, [ka] In the formula, X is one or more substances non-limitingly selected from the group consisting of hydrophilic structures: carboxyl, phosphoric acid, polyphosphate, phosphorous acid, sulfonic acid, sulfinic acid, and polyethylene glycol (PEG). Y is any scaffold that binds the amino group and X, Ac is bonded to the methylene carbon at position 2 in structural formula I via an amino group.

[0011] More preferably, Ac is glycine, (D / L)alanine, (D / L)leucine, (D / L)isoleucine, (D / L)valine, (D / L)phenylalanine, (D / L)proline, (D / L)tryptophan, (D / L)serine, (D / L)tyrosine, (D / L)cysteine, (D / L)cystine, (D / L)arginine, (D / L)histidine, (D / L)methionine, (D / L)asparagine, (D / L)glutamine, (D / L)threonine, (D / L)aspartic acid, (D / L)glutamic acid, natural or non-natural amino acid derivatives, and the following structures: [ka] Selected non-restrictively from the group consisting of, Here, the wavy line on the left represents a bond to the carbon atom at position 2.

[0012] In another aspect of the present invention, a camptothecin-type drug having a structure represented by the following formula d is provided. [ka] In the formula, R1 is selected from hydrogen, deuterium, halogen, alkyl, deuterated alkyl, halogenated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl and heteroaryl, or R1 and the carbon atom bonded to R1 are C 3-6 Constituting a cycloalkyl, cycloalkylalkyl, or heterocycline, The chiral carbon atom bonded to R1 has either an R absolute configuration or an S absolute configuration. m is either 0 or 1. The hydroxyl group of the carbon atom bonded to R1 in the drug molecule d is involved in the formation of the oxygen atom at position 3 in formula I.

[0013] Preferably, the camptothecin-type drugs of the present invention are [ka] Selected without restriction from the given range.

[0014] In another aspect of the present invention, a linker-drug compound having the structure represented by the following formula II, or a pharmaceutically acceptable salt thereof, is provided for binding with ligand Ab to form a ligand-drug complex represented by formula I as described in claim 1. [ka] In the formula, R1 is selected from hydrogen, deuterium, halogen, alkyl, deuterated alkyl, halogenated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or heteroaryl, or R1 and the carbon atom bonded to R1 are C 3-6 Constituting a cycloalkyl, cycloalkylalkyl, or heterocycline, The chiral carbon atom at position 1 has either an R absolute configuration or an S absolute configuration. Ac is a hydrophilic structural unit, m is either 0 or 1.

[0015] In one aspect of the present invention, Ac is preferably glycine, phosphoric acid, polyethylene glycol, or (D / L) glutamic acid.

[0016] More preferably, the linker-drug compound of the present invention or a pharmaceutically acceptable salt thereof is [ka] Selected non-restrictively from JPEG2023529415000010.jpg211140JPEG2023529415000011.jpg195140JPEG2023529415000012.jpg190140JPEG2023529415000013.jpg191140JPEG2023529415000014.jpg82140, Here, the chiral carbon at position 1 has either an R absolute configuration or an S absolute configuration.

[0017] In another aspect of the present invention, a structure represented by formula III, formula IV-1, or formula IV-2 is provided. [ka] In Equation III, Equation IV-1, or Equation IV-2, Ab is a ligand unit, Ac is a hydrophilic structural unit, The chiral carbon at position 1 has either an R absolute configuration or an S absolute configuration. R1, m, and n are expressed by Equation II.

[0018] Preferably, in the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt thereof, the ligand unit Ab is selected from an antibody, an antibody fragment, or a protein, and the antibody is selected from a mouse antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody, and a multispecific antibody.

[0019] More preferably, the antibody is a monoclonal antibody selected from, without limitation, anti-EGFRvIII antibody, anti-PD-1 antibody, anti-PD-L1 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-claudin 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 αv 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, and anti-CD123 antibody.

[0020] More preferably, the antibody or its antigen-binding fragment is MDMRVPAQLLGLLLLWLRGARC DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(Sequence ID 1) A light chain containing, MDMRVPAQLLGLLLLWLRGARC EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 2) A heavy chain containing, Trastuzumab is a drug that contains [unclear / unclear].

[0021] Preferably, the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt thereof is selected from the following structures or its succinimide ring-opening structures. [ka] JPEG2023529415000017.jpg184141JPEG2023529415000018.jpg207141JPEG2023529415000019.jpg210140JPEG2023529415000020.jpg208140JPEG2023529415000021.jpg29136 Here, n is an integer selected from 1 to 10.

[0022] Another aspect of the present invention provides a method for preparing a linker-drug compound or a pharmaceutically acceptable salt thereof. The method comprises the steps shown in the following formula. [ka] In the formula, a compound of general formula L is reacted with exatecane of formula d0 or a salt thereof under arbitrary alkaline conditions in the presence of a condensing agent to obtain a compound of formula IV, which is then converted to the structure represented by formula II. In the formula, the chiral carbon atom at position 1 and the chiral carbon atom bonded to R1 have either an R absolute configuration or an S absolute configuration. R2 is any structure that can be converted to Ac, Ac, R1, and m are as defined in Equation II.

[0023] Another aspect of the present invention provides a method for preparing ligand-drug conjugates or pharmaceutically acceptable salts thereof. The method comprises the steps shown in the following formula. [ka] In the formula, after modifying the ligand unit Ab, it is coupled with the compound of formula II to obtain the ligand-drug conjugate of formula III. Ab is selected from antibodies, antibody fragments, and proteins. Ac is a hydrophilic structural unit, The chiral carbon atom at position 1 and the chiral carbon atom bonded to R1 have either an R absolute configuration or an S absolute configuration. R1, m, and n are as defined in Equation II.

[0024] In another aspect of the present invention, a pharmaceutical composition is provided comprising a therapeutically effective amount of a ligand-drug conjugate, a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0025] The pharmaceutically acceptable salts of the present invention include, in terms of the manner of salt formation, sodium salts, potassium salts, calcium salts or magnesium salts formed with carboxyl group functional groups in the structural formula; and acetates, trifluoroacetates, citrates, oxalates, tartrates, malates, nitrates, chlorides, bromides, iodides, sulfates, bisulfates, phosphates, lactates, oleates, ascorbicates, salicylates, formic acid, glutamates, mesylates, ethanesulfons, benzenesulfons, or p-toluenesulfons formed with nitrogen-containing functional groups in the structure.

[0026] Another aspect of the present invention provides the use of a pharmaceutical composition of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof in the preparation of a therapeutic agent for tumors, autoimmune diseases, or infectious diseases. The antibody of the ligand-drug conjugate specifically binds to target cells of the tumor, autoimmune disease, or infectious disease.

[0027] Another aspect of the present invention provides the use of ligand-drug conjugates or pharmaceutically acceptable salts thereof in the preparation of therapeutic agents for solid tumors or hematological malignancies, including breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma, and leukemia. [Brief explanation of the drawing]

[0028] [Figure 1A] This is the SEC-HPLC detection result of the trastuzumab monomer ratio in the present invention. [Figure 1B] This is the SEC-HPLC detection result of the ADC-2 monomer ratio according to the present invention. [Figure 1C]This is the SEC-HPLC detection result of the ADC-6 monomer ratio according to the present invention. [Figure 1D] This is the SEC-HPLC detection result of the ADC-10 monomer ratio in the present invention. [Figure 1E] This is the SEC-HPLC detection result of the ADC-12 monomer ratio according to the present invention. [Figure 1F] This is the SEC-HPLC detection result of the ADC-61 monomer rate in the control group of the present invention. [Figure 2A] This is the RP-HPLC detection result of the DAR (drug-antibody binding ratio) value of ADC-02 of the present invention. [Figure 2B] This is the RP-HPLC detection result of the DAR (drug-antibody binding ratio) value of ADC-06 of the present invention. [Figure 2C] This is the RP-HPLC detection result of the DAR (drug-antibody binding ratio) value of ADC-10 of the present invention. [Figure 2D] This is the RP-HPLC detection result of the DAR (drug-antibody binding ratio) value of ADC-12 of the present invention. [Figure 2E] This is the RP-HPLC detection result of the DAR (drug-antibody binding ratio) value of ADC-61, the control group of the present invention. [Figure 3] This describes the in vitro effects of the ADC, monotherapy, and naked antibody of the present invention on the proliferation of N87 (human gastric cancer cells). [Figure 3A] This describes the in vitro effect of the ADC and naked antibody of the present invention on the proliferation of N87 (human gastric cancer cells). [Figure 3B] This describes the in vitro effect of the present invention's monotherapy on the proliferation of N87 (human gastric cancer cells). [Figure 4] This describes the in vitro effects of the ADC, monotherapy, and naked antibody of the present invention on inhibiting the proliferation of SK-BR-3 (human mammary gland cancer cells). [Figure 4A] This describes the in vitro effect of the ADC and naked antibody of the present invention on inhibiting the proliferation of SK-BR-3 (human mammary gland cancer cells). [Figure 4B] This describes the in vitro effect of the present invention as a monotherapy agent on inhibiting the proliferation of SK-BR-3 (human mammary gland cancer cells). [Modes for carrying out the invention]

[0029] Abbreviations and Definitions Unless otherwise defined, all technical and scientific terms used herein are the same as those commonly understood by an ordinary person skilled in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure, but exemplary methods and materials are described herein. In describing and asserting the invention, the following terms are used according to the following definitions.

[0030] When a product name is used in this specification, it is intended to include the formulation, generic drug, and active ingredient of the product to which the product name pertains.

[0031] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings: Where a trade name is used herein, unless the context indicates otherwise, the trade name includes the trade formula, generic drugs, and the active ingredients of the trade name product.

[0032] Unless otherwise stated, terms used herein and in the claims have the same meanings as set forth below.

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

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

[0035] In this specification, the terms “antibody” or “antibody unit” include, to the extent that they apply, any part of an antibody structure. This unit can bind to, reactively interact with, or form complexes with receptors, antigens, or other receptor units possessed by a target cell population. The antibody may be any protein or protein-like molecule that can bind to, form complexes with, or react with a portion of a cell population being treated or biologically modified. In the present invention, the antibody constituting the antibody-drug conjugate may maintain its original wild-state antigen-binding ability. Therefore, the antibody of the present invention may preferably bind specifically to an antigen. Such antigens include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, regulators of cell survival, regulators of cell proliferation, molecules associated with tissue growth and differentiation (with known or predictable functionality), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (with known or predictable functionality). As described herein, tumor-associated factors may also be cluster differentiation factors (such as CD proteins).

[0036] Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art and can be prepared by methods and information well known in the art for antibody preparation. To develop effective cellular-level targets for cancer diagnosis and treatment, researchers are finding transmembrane or other tumor-associated polypeptides. These targets are little to no expression on the surface of one or more non-cancer cells but can be specifically expressed on the surface of one or more cancer cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells compared to the surface of non-cancer cells. Identifying such tumor-associated factors can significantly improve the specific targeting characteristics of antibody-based cancer treatment. For convenience, antigen-related information (names, other designations, GenBank accession numbers, etc.) that is well known in the art is listed below. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases such as GenBank. The tumor-associated antigens corresponding to the antibody targets include all amino acid sequence variants and isotypes, and have at least 70%, 80%, 85%, 90%, or 95% identity with the sequences identified in the references, or possess biological properties and characteristics that are completely identical to the sequences of tumor-associated antigens described in the references.

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

[0038] The term "cancer" refers to a physiological condition or disease characterized by uncontrolled cell proliferation. "Tumor" includes cancer cells.

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

[0040] The term "drug" refers to a cytotoxic drug that can be denoted as d, which is a chemical molecule that can strongly interfere with the normal growth of tumor cells. Cytotoxic drugs can, in principle, kill tumor cells at a sufficiently high concentration, but due to lack of specificity, while killing tumor cells, they may cause apoptosis of normal cells and serious side effects. This term includes toxins such as small molecule toxins or enzyme active toxins derived from bacteria, fungi, plants or animals, radioisotopes (e.g., At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、BI 212 、P 32 and radioisotopes of Lu176), toxic drugs, chemotherapeutic drugs, antibiotics, and ribolysin, and is preferably a toxic drug.

[0041] The term "camptothecin drugs" refers to camptothecin having cytotoxicity and its derivatives, and is non - restrictively selected from 10 - hydroxycamptothecin, SN38 (7 - ethyl - 10 - hydroxycamptothecin), topotecan, exatecan, irinotecan or 9 - nitro - 10 - hydroxycamptothecin, its derivatives, and pharmaceutically acceptable salts thereof.

[0042] The term "linker unit", "linker fragment" or "linker" is a chemical structure fragment or bond where one end is bound to a ligand and the other end is bound to a drug, and may be bound to another linker and then to the drug.

[0043] The linker comprises a stretcher, a spacer, and an amino acid unit, and can be synthesized according to methods known in the art (e.g., the method described in US2005-0238649A1). The linker may be a “cleavable linker” that facilitates the release of a drug into a cell. For example, an acid-unstable linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a light-unstable linker, a dimethyl linker, or a disulfide-containing linker can be used (Char I et al. Cancer Research 52: 127-131, 1992; US Patent No. 5,208,020).

[0044] Based on the mechanism of intracellular drug release, the "linkers" or "antibody-drug conjugate linkers" described herein are divided into two types: non-cleavable linkers and cleavable linkers. The drug release mechanism of ligand-drug conjugates containing non-cleavable linkers is as follows: After the conjugate binds to an antigen and is taken up by the cell, the antibody is enzymatically hydrolyzed within the lysosome, releasing an active molecule consisting of a small molecule drug, a linker, and antibody amino acid residues. This change in the drug molecule structure does not reduce its cytotoxicity, and because the active molecule is charged (amino acid residues), it does not penetrate neighboring cells. Therefore, such drugs do not kill adjacent tumor cells that do not express the target antigen (antigen-negative cells) (bystander effect) (Ducry et al., 2010, BIoconjugate Chem. 21:5-13).

[0045] The term "ligand-drug conjugate" refers to an antibody binding to a biologically active drug via a stable linker. In the present invention, "ligand-drug conjugate" refers to an antibody-drug conjugate (ADC), preferably a monoclonal antibody or antibody fragment, binding to a biologically active toxic drug via a stable linker.

[0046] The three-letter and one-letter amino acid codes used herein are as described in J.boy.Chem.1968,243,3558.

[0047] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group, and includes linear or branched groups having 1 to 20 carbon atoms, preferably alkyl groups having 1 to 12 carbon atoms, more preferably alkyl groups having 1 to 10 carbon atoms, and most preferably alkyl groups having 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, and 5-methylhexyl. Examples include 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 their branched isomers.More preferably, the alkyl group is a lower alkyl group having 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and 2,3-dimethylbutyl. The alkyl group may be substituted or unsubstituted. If substituted, the substituent may be substituted at any available bond point. The substituents are preferably one or more independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxy groups.

[0048] The term "substituted alkyl group" refers to an alkyl group in which hydrogen atoms are replaced by substituents. Unless otherwise specified in the context, substituents on an alkyl group are selected from -halogen, -OR', -NR'R'', -SR', -SIR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NR'S(O)2R'', -CN and -NO2. The number of substituents is 0-(2m'+1), where m' is the total number of carbon atoms in the group. R', R'', and R''' are each independently hydrogen and unsubstituted carbon. 1-8Alkyl groups, unsubstituted aryl groups, aryl groups substituted with 1-3 halogens, unsubstituted C 1-8 Alkyl alkyl group, C 1-8 Alkoxy group or C 1-8 Thioalkoxy group, or unsubstituted aryl group -C 1-4 It is an alkyl group. When R' and R'' are bonded to the same nitrogen atom, they can form a 3-, 4-, 5-, 6-, or 7-membered ring together with this nitrogen atom. For example, -NR'R'' contains a 1-pyrrolidinyl group and a 4-morpholinyl group.

[0049] The term "heteroalkyl group" refers to an alkyl group containing one or more heteroatoms selected from N, O, and S. Alkyl group is synonymous with the above.

[0050] The term "alkylene group" refers to a saturated linear or branched aliphatic hydrocarbon group having two residues obtained by removing two hydrogen atoms from the same or different carbon atoms of the main alkane, and includes linear or branched groups having 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene 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 (-CH2CH2CH2CH2CH2-). The alkylene group may be substituted or unsubstituted. If substituted, the substituent may be substituted at any available bond point. The substituent is preferably one or more independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxy groups.

[0051] The term "alkoxy group" refers to -O-(alkyl) and -O-(cycloalkyl) groups. Alkyl group or cycloalkyl group is synonymous with the above. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy. Alkoxy groups may be substituted or unsubstituted. If substituted, the substituent may be substituted at any available bond point. The substituent is preferably one or more independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0052] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent. A cycloalkyl ring contains 3-20, preferably 3-12, more preferably 3-10, and most preferably 3-8 carbon atoms. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl. Monocyclic cycloalkyl groups include spiro, condensed, or bridged cycloalkyl groups.

[0053] The term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring-forming atoms, one or more of which are nitrogen, oxygen, and S(O). mThe heteroatom is selected from (where m is an integer between 0 and 2), but the -OO-, -OS-, or -SS- ring portion is excluded, and the remaining ring constituent atoms are carbon. Preferably, it contains 3 to 12 ring constituent atoms, of which 1 to 4 are heteroatoms. More preferably, the cycloalkyl ring contains 3 to 10 ring constituent atoms. Examples of monocyclic heterocyclyl groups include, but are not limited to, pyrrolidinyl alkyl groups, piperidinyl groups, piperazinyl groups, morpholinyl groups, thiomorpholinyl groups, and homopiperazinyl groups. Polycyclic heterocyclyl groups include spiro, condensed, or bridge ring heterocyclyl groups.

[0054] The term "cycloalkylalkyl group" refers to a group in which one or more alkyl groups, preferably one cycloalkyl group, are substituted. Alkyl group and cycloalkyl group are synonymous with the terms used above.

[0055] The term "halogenated alkyl group" refers to a group in which one or more halogens are substituted for an alkyl group. The term "alkyl group" is synonymous with the above.

[0056] The term "deuterated alkyl group" refers to a group in which one or more deuterium atoms are substituted for an alkyl group. The term "alkyl group" is synonymous with the above.

[0057] The term "hydroxyl group" refers to the -OH group.

[0058] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0059] The term "amino group" refers to -NH2. The term "nitro group" refers to -NO2.

[0060] The term "amide group" refers to a -C(O)N-(alkyl) or (cycloalkyl) group, and alkyl and cycloalkyl are synonymous with the above.

[0061] The term "carboxylic acid ester group" refers to a -C(O)O-(alkyl) or (cycloalkyl) group, and alkyl and cycloalkyl are synonymous with the above.

[0062] The term "aryl group" refers to a 6-14 member, preferably 6-10 member, all-carbon monocyclic or fused polycyclic (i.e., a ring sharing pairs of adjacent carbon atoms) group having a conjugated electron system, such as a phenyl group. The aryl group may be substituted or unsubstituted. If substituted, the substituents are preferably one or more independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, deuterium atoms, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0063] The present invention further includes deuterated formula (I). Each hydrogen atom bonded to a carbon atom can be independently substituted with a deuterium atom. Those skilled in the art can synthesize deuterated formula (I) compounds according to relevant literature. When synthesizing deuterated formula (I) compounds, commercially available deuterated starting materials may be used, or they may be synthesized using deuterated reagents by conventional methods. Deuterated reagents include, but are not limited to, borane deuterated, trideuterated boranetetrahydrofuran solution, lithium aluminum deuterated, deuterated iodoethane, and deuterated iodomethane.

[0064] The term "antibody" refers to immunoglobulins, which are tetrapeptide chain structures consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Because the amino acid composition and sequence in the constant region of the heavy chain of immunoglobulins differ, their antigenicity also differs. This allows immunoglobulins to be classified into five types or isotypes: IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are the μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be classified into different subclasses depending on the differences in the amino acid composition of their hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified into κ or λ chains depending on the differences in the constant region. Each of the five types of Ig can have either a κ or λ chain. The antibodies described in this invention are preferably specific antibodies against cell surface antigens on target cells.In non-limiting embodiments, the antibody is one or more of an anti-EGFRvIII antibody, an anti-DLL-3 antibody, an anti-PSMA antibody, an anti-CD70 antibody, an anti-MUC16 antibody, an anti-ENPP3 antibody, an anti-TDGF1 antibody, an anti-ETBR antibody, an anti-MSLN antibody, an anti-TIM-1 antibody, an anti-LRRC15 antibody, an anti-LIV-1 antibody, an anti-CanAg / AFP antibody, an anti-claudin 18.2 antibody, an anti-Mesothelin antibody, an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-c-MET antibody, an anti-SLITRK6 antibody, an anti-KIT / CD117 antibody, an anti-STEAP1 antibody, an anti-SLAMF7 / CS1 antibody, an anti-NaPi2B / SLC34A2 antibody, an anti-GPNMB antibody, an anti-HER3 (ErbB3) antibody, an anti-MUC1 / CD227 antibody, an anti-AXL antibody, an anti-CD166 antibody, an anti-B7-H3 (CD276) antibody, an anti-PTK7 / CCK4 antibody, an anti-PRLR antibody, an anti-EFNA4 antibody, an anti-5T4 antibody, an anti-NOTCH3 antibody, an anti-NectIn 4 antibody, an anti-TROP-2 antibody, an anti-CD142 antibody, an anti-CA6 antibody, an anti-GPR20 antibody, an anti-CD174 antibody, an anti-CD71 antibody, an anti-EphA2 antibody, an anti-LYPD3 antibody, an anti-FGFR2 antibody, an anti-FGFR3 antibody, an anti-FRα antibody, an anti-CEACAMs antibody, an anti-GCC antibody, an anti-Integrin αv antibody, an anti-CAIX antibody, an anti-P-cadherin antibody, an anti-GD3 antibody, an anti-Cadherin 6 antibody, an anti-LAMP1 antibody, an anti-FLT3 antibody, an anti-BCMA antibody, an anti-CD79b antibody, an anti-CD19 antibody, an anti-CD33 antibody, an anti-CD56 antibody, an anti-CD74 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD37 antibody, an anti-CD138 antibody, an anti-CD352 antibody, an anti-CD25 antibody or an anti-CD123 antibody. Preferably, it is Trastuzumab (trade name Herceptin), Pertuzumab (also called 2C4; trade name Perjeta), Nimotuzumab (trade name: Taishengxing), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 and Glematumamab.

[0065] The term "solvate" refers to a pharmaceutically acceptable solvate formed from the ligand-drug conjugate of the present invention and one or more solvent molecules. Examples of solvent molecules include, but are not limited to, water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0066] The term "drug load" refers to the average number of cytotoxic agents loaded onto each antibody in Formula I, and may be expressed as a ratio of drug amount to antibody amount. Within the drug load range, 0-12, preferably 1-10 cytotoxic agents (D) can be bound to each antibody (Ab). In embodiments of the present invention, the drug load is denoted by n, and may be an average value such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The average number of drugs on each ADC molecule after the coupling reaction can be measured by conventional methods such as UV / visible light spectroscopy, mass spectrometry, ELISA, and HPLC.

[0067] In one embodiment of the present invention, the cytotoxic drug is bound to the ring-opened interchain cysteinethiol-SH and / or site-directly mutated cysteinethiol-SH of the antibody via a binding unit, and generally, in the coupling reaction, the number of drug molecules that can bind to the antibody is less than or equal to the theoretical maximum value.

[0068] The amount of ligand-cytotoxic drug conjugates can be controlled by the following non-restrictive methods. (1) Control the molar ratio of the linker reagent to the monoclonal antibody. (2) Control the reaction time and temperature. (3) Select different reaction reagents. For the manufacture of typical pharmaceutical compositions, please refer to the Chinese Pharmacopoeia.

[0069] The terms “pharmaceutically acceptable salt” or “pharmaceutically acceptable salt” refer to a salt of the ligand-drug conjugate of the present invention or a salt of a compound described in the present invention. Such salts are safe and effective when used in mammals and possess appropriate biological activity. The ligand-drug conjugate of the present invention has at least one carboxyl and can therefore form salts with bases. Non-restrictive examples of pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts, magnesium salts, and the like.

[0070] The terms “pharmaceutically acceptable salt” or “pharmaceutically acceptable salt” refer to a salt of the ligand-drug conjugate of the present invention or a salt of a compound described in the present invention. Such salts are safe and effective when used in mammals and have appropriate biological activity. The antibody-drug conjugate compounds of the present invention contain at least one amino group and can therefore form salts with acids. Examples of pharmaceutically acceptable salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0071] "Acidic amino acids" refer to amino acids with an isoelectric point of less than 7. Acidic amino acid molecules typically have one or more acidic groups, such as carboxyl groups, and structurally, they can be effectively ionized to become negative ions, thereby improving their hydrophilicity. Acidic amino acids can be divided into natural and unnatural amino acids.

[0072] "Natural amino acids" refer to amino acids obtained through biosynthesis. Natural amino acids are generally L-type, but there are exceptions, such as glycine, which includes both naturally occurring forms and those synthesized within the body.

[0073] "Non-natural amino acids" refer to amino acids obtained through synthesis.

[0074] The present invention will be further described below with reference to specific examples, but please understand that these examples are used solely to illustrate the present invention and are not intended to limit the scope of the invention. In the following examples, test methods for which specific conditions are not indicated generally follow conventional conditions or conditions proposed by the manufacturer. Unless otherwise specified, all percentages, proportions, ratios or parts are by weight.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly known in the art. Furthermore, any methods and materials similar or equivalent to those described herein are applicable to the methods of the present invention. The preferred methods and materials described herein are illustrative only.

[0076] Example 1 Synthesis of compound M1 [ka] In a 5000 mL single-neck flask, N-fluorenemethoxycarbonyl-glycine-glycine (100 g, 282 mmol, 1.0 eq), lead tetraacetate (175 g, 553 mmol, 1.4 eq), 2000 mL dry tetrahydrofuran, and 670 mL toluene were placed and mixed uniformly. The mixture was then protected with nitrogen gas and heated to 85 °C for 2.5 hours. After confirming that the starting materials had reacted completely by TLC, the mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound M1 (87 g). LC-MS: [M + NH4] + =386.0.

[0077] Example 2 Synthesis of compound M3 [ka] 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 placed in a 1000 mL necked flask. The mixture was heated to 120 °C and refluxed for 2 hours. After confirming that the reaction was almost complete by TLC, the temperature was lowered to 50 °C, and the solvent was removed by rotating evaporation under reduced pressure. The compound was dissolved in ethyl acetate (150 mL) and water (40 mL), and the pH was adjusted to 2-3 with 1 M HCl while stirring in an ice bath, followed by liquid-liquid extraction. The aqueous layer was further extracted with ethyl acetate, and the organic layers were combined. Anhydrous sodium sulfate was added and the mixture was dried. After filtration, the mixture was concentrated to obtain a pale yellow oily crude product. The crude product was purified by column chromatography (DCM:MeOH=40:1) to obtain compound M2 (26.6 g). LC-MS:[M+H] + = 399.3.

[0078] Compound M2 (26.5g, 60.5 mmol, 1.0eq), pentafluorophenol (12.2g, 66.5 mmol, 1.1eq), DCC (13.7g, 66.5 mmol, 1.1eq), and THF (300mL) were placed in a 1000mL neck flask and reacted at room temperature for 30mIn (monitored by TLC). Insoluble matter was removed by filtration. The reaction mixture was then preparatively purified, and the preparative solution was concentrated under reduced pressure with a water pump, in a water bath, and at 35°C to remove acetonitrile. The mixture was then freeze-dried to obtain compound M3 (31.5g). Yield: 64%; LC-MS: [M+H] + = 565.1.

[0079] Example 3 Synthesis of compound ent-M3 [ka] Compound ent-M3 (27.8 g) was obtained according to the synthesis route of Example 2. LC-MS: [M+H] + = 565.2.

[0080] Example 4 Synthesis of Compound 1 [ka]

[0081] Step 1: Compound 1a In a 250 mL neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were placed. The mixture was cooled to 0°C while stirring, and benzyl hydroxyacetate (5.4 g, 32.6 mmol) was added dropwise. After the addition was complete, the mixture was allowed to rise naturally to room temperature for approximately 2-4 hours, and the reaction was monitored by TLC. After the reaction was complete, saturated NaHCO3 solution was added, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA = 10:1-5:1-1:1) to obtain 1a (4 g). Yield 52%; LC-MS: [M+H] + = 475.18.

[0082] Step 2: Compound 1b 1a (2g, 4.2 mmol) and 10 mL of DMF were placed in a 25 mL neck flask, stirred at 0°C, and DBU (766 mg, 5.04 mmol) was added. The reaction was allowed to proceed for 1 hour, and after confirming that Fmoc deprotection was complete by TLC, the flask was stored until use. A new 25 mL neck flask was taken and M4 (prepared according to the method disclosed in patent CN111051330A) (1.73 g, 4.2 mmol), PyBOP (2.61 g, 5.04 mmol), HOBt (680 mg, 5.04 mmol), and 10 mL DMF were added. DIPEA (830 μL, 5.04 mmol) was added under an ice bath, and the mixture was stirred for 30 mIn. The reaction mixture was then transferred to a reaction flask and allowed to react at room temperature. After confirmation of the completion of the reaction by HPLC, the reaction mixture was purified by preparative liquid chromatography to obtain the product preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain solid 1b (1.7 g). Yield 63%; LCMS: [M+H] + = 648.26.

[0083] Step 3: Compound 1c In a 25 mL neck flask, 1b (900 mg, 1.39 mmol) and 15 mL of DMF were placed and dissolved uniformly. Then, 900 mg of 5% Pd / C was added, and the hydrogenation reaction was carried out for 2 hours. After the reaction was complete, the filtrate was filtered and used directly in the next reaction without purification.

[0084] Step 4: Compound 1d Crude product 1c was placed in an ice bath, DIPEA (235 μL, 1.39 mmol) was added, followed by compound M3 (784 mg, 1.39 mmol). The mixture was then heated to room temperature and reacted for 1 hour. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 1d (504 mg). LC-MS: [M+H] + = 804.4.

[0085] Step 5: Compound 1e 1d (500 mg, 0.62 mmol), M5 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were placed in a 50 mL neck flask. DIPEA (378 μL, 2.29 mmol) was added under ice water bath, and the mixture was heated to room temperature and reacted for 2 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative of compound 1e. The preparative was freeze-dried to obtain 1e (210 mg). LC-MS: [M+H] + = 1221.6.

[0086] Step 6: Compound 1 1e (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain solid compound 1 (60 mg). LC-MS: [M+H] + = 1065.3.

[0087] Example 5 Synthesis of Compound 2 [ka] Compound 2 (51 mg) was obtained according to the synthesis route of Example 4. LC-MS: [M+H] + = 1065.3.

[0088] Example 6 Synthesis of Compound 3 [ka]

[0089] Step 1: Compound 3a In a 250 mL neck flask, M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were placed and cooled to 0°C with stirring. Benzyl 2-hydroxy-2-methylpropionate (6.3 g, 32.6 mmol) was added dropwise. After the addition was complete, the mixture was allowed to rise naturally to room temperature for approximately 2-4 hours, and the reaction was monitored by TLC. After the reaction was complete, saturated NaHCO3 solution was added, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was analyzed by silica gel column (PE:EA = 10:1-5:1-2:1) to obtain 3a (4.2 g). Yield 52%; LC-MS: [M+H] + = 503.3.

[0090] Step 2: Compound 3b 3a (2g, 4.0 mmol) and 10 mL of DMF were placed in a 25 mL neck flask, stirred at 0°C, and DBU (760 mg, 5.0 mmol) was added. The reaction was allowed to proceed for 1 hour, and after confirming that Fmoc deprotection was complete by TLC, the solution was stored until use.

[0091] A new 25 mL neck flask was taken, and M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol), and 10 mL DMF were added. DIPEA (823 μL, 5.04 mmol) was added under an ice bath, and the mixture was stirred for 30 mIn. The reaction mixture was then transferred to a reaction flask and allowed to react at room temperature. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by preparative liquid chromatography to obtain the product fraction. The fraction was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain solid 3b (1.4 g). Yield 53%; LC-MS: [M+H] + = 676.2.

[0092] Step 3: Compound 3c 3b (700 mg, 1.04 mmol) and 10 mL of DMF were placed in a 25 mL neck flask, dissolved, and then 700 mg of 5% Pd / C was added. The hydrogenation reaction was carried out for 1.5 hours, and after the reaction was complete, the filtrate was filtered and used directly in the next reaction without purification.

[0093] Step 4: Compound 3D Crude product 3c was placed in an ice bath, DIPEA (210 μL, 1.25 mmol) was added, followed by compound M3 (704 mg, 1.25 mmol). The mixture was then heated to room temperature and reacted for 1 hour. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 3d (486 mg). LC-MS: [MH] - = 830.5.

[0094] Step 5: Compound 3e 3d (300 mg, 0.36 mmol), M5 (180 mg, 0.36 mmol), PyBOP (260 mg, 0.5 mmol), HOBt (67 mg, 0.5 mmol), and 10 mL of DMF were placed in a 50 mL neck flask. DIPEA (219.5 μL, 1.33 mmol) was added under an ice bath, and the mixture was heated to room temperature and reacted for 3 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative of compound 3e. The preparative was freeze-dried to obtain 3e (157 mg). LC-MS: [M+H] + = 1249.6.

[0095] Step 6: Compound 3 3e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain solid compound 3 (64 mg). LC-MS: [M+H] + = 1093.1.

[0096] Example 7 Synthesis of Compound 4 [ka] Compound 4 (60 mg) was obtained according to the synthesis route of Example 6. LC-MS: [M+H] + = 1093.2.

[0097] Example 8 Synthesis of compound 5A [ka]

[0098] Step 1: Compound 5a In a 25 mL neck flask, M1 (500 mg, 1.4 mmol, 1.0 eq), p-toluenesulfonic acid monohydrate (26 mg, 0.1 mmol, 0.1 eq), and 10 mL of THF were placed and mixed uniformly. After cooling to 0°C, L-benzyl lactate (1.2 g, 7.0 mmol, 5 eq) was slowly added, and the mixture was then heated to room temperature to allow the reaction to proceed. The reaction was monitored by TLC, and after completion, saturated NaHCO3 solution was added, the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by reverse-phase column chromatography to obtain 5a (400 mg).

[0099] LC-MS:[M+NH4] + = 506.2. 1 H NMR(400Mz,CDCl3 / CD3OD):1.39(3H,d,J=6.8Hz),3.78(2H,t,J=4.0Hz),4.17-4.27(2H,m),4.42(2H,d,J=4.0Hz),4.72-4.85(2H,m),5.1 1-5.58(2H,m),5.43(1H,s),7.06(1H,t,J=8.0Hz),7.25-7.33(6H,m),7.38(2H,t,J=8.0Hz),7.57(2H,d,J=8.0Hz),7.75(2H,d,J=8.0Hz)

[0100] Step 2: Compound 5b Compound 5a (400 mg, 0.8 mmol, 1.0 eq) and 4 mL of DMF were placed in a 25 mL neck flask, mixed uniformly, cooled to 0°C, and then DBU (137 mg, 0.9 mmol, 1.1 eq) was slowly added. The reaction was then allowed to proceed by raising the temperature to room temperature. The reaction was monitored by TLC, and after completion, it was recorded as reaction solution (1). A new 25 mL neck flask was placed in it and M4 (372 mg, 0.9 mmol, 1.1 eq), PyBOP (852 mg, 1.6 mmol, 2.0 eq), and 3 mL DMF. The mixture was stirred at room temperature for 5 minutes, and reaction solution (1) was added. The reaction was allowed to proceed at room temperature and monitored by TLC. After the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain compound 5b (326 mg). LC-MS: [M + NH4] + = 679.2.

[0101] Step 3: Compound 5c Dissolve 5b (4.0g, 6.05 mmol, 1.0eq) in DMF (60mL) in a 100mL neck flask, then add 5% Pd / C (4g) and allow the hydrogenation reaction to proceed at room temperature for 4 hours (the reaction was monitored by HPLC). Filter out the Pd / C, and store the filtrate in an ice bath (approximately 0°C) without concentration until use.

[0102] Step 4: Compound 5d Crude product 5c was placed in an ice bath, DIPEA (1.1 mL, 1.1 eq) was added, followed by compound M3 (3.4 g, 6.05 mmol). The mixture was then heated to room temperature and reacted for 2 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 5d (3.15 g). LC-MS: [MH] - = 816.3.

[0103] Step 5: Compound 5e In a 100 mL neck flask, 5d (2.07 g, 2.53 mmol, 1.0 eq), M5 (1.35 g, 2.53 mmol, 1.0 eq), PyBOP (1.98 g, 3.79 mmol, 1.5 eq), HOBt (0.51 g, 3.79 mmol, 1.5 eq), and DMF (40 mL) were placed. DIPEA (1.05 mL, 1.5 eq) was added under an ice bath, and the mixture was heated to room temperature and reacted for 2 hours (monitored by HPLC). The reaction mixture was then preparatively purified, and the preparative solution was concentrated under reduced pressure with a water pump, in a water bath, and at 35°C to remove acetonitrile. The mixture was then freeze-dried to obtain compound 5e (1.92 g). Yield: 61%; LC-MS: [M+H] + = 1235.4.

[0104] Step 6: Compound 5A Compound 5e (1.0 g, 0.8 mmol, 1.0 eq) and 35 mL of nitromethane were placed in a 100 mL neck flask. After dissolution, zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added, and the mixture was reacted for 30 mIn in an oil bath at 40°C (preheated and stabilized beforehand). The mixture was concentrated under reduced pressure with a water pump in a water bath at 45°C to remove the nitromethane, yielding a yellow solid residue (monitored by HPLC). Compound 5A was prepared by the acid method to obtain a preparative solution. The preparative solution was concentrated under reduced pressure with a water pump in a water bath at 35°C, acetonitrile was removed by rotary evaporation, and the solution was freeze-dried to obtain compound 5A (786 mg). Yield: 90%. LC-MS:[M+H] + =1079.4 1H NMR(400MHz,DMSO-d6)δ9.39-9.02(m,1H),8.70(t,J=6.5Hz,1H),8.64(t,J=5.7Hz,1H),8.56(d,J=8.8Hz,1H),8 .34(t,J=5.7Hz,1H),8.16(d,J=8.2Hz,1H),8.01(t,J=5.5Hz,1H),7.71(d,J=10.9Hz,1H),7.30(s,1H),7.28-7.1 5(m,4H),7.14(s,2H),5.53(dd,J=14.5,6.4Hz,1H),5.49-5.34(m,2H),5.22(d,J=18.8Hz,1H),5.09(d,J=18.7H z,1H),5.03(dd,J=9.6,3.9Hz,1H),4.73(dd,J=9.9,6.9Hz,1H),4.59(dd,J=10.1,6.5Hz,1H),4.49(ddd,J=13.2, 8.6,4.4Hz,1H),4.14(dd,J=13.3,6.6Hz,2H),3.93(s,2H),3.84(dd,J=16.5,6.3Hz,1H),3.76(dd,J=16.9,5.7H z,2H),3.70(d,J=5.2Hz,2H),3.60(dd,J=16.7,5.4Hz,1H),3.52(dd,J=16.4,5.1Hz,1H),3.45(dd,J=12.8,10.1H z,1H),3.25-3.15(m,1H),3.14-3.05(m,1H),3.01(dd,J=13.7,4.1Hz,1H),2.73(dd,J=13.5,9.8Hz,1H),2.54-2 .47(m,1H),2.33(s,2H),2.17(d,J=5.5Hz,2H),1.91-1.79(m,2H),1.33(d,J=6.6Hz,2H),0.87(t,J=7.3Hz,2H).

[0105] Example 9 Synthesis of Compound 5B

change

[0106] ステップ1: Compound 5d-1 Compound 5b (300 mg, 0.45 mmol, 1.0 eq) and DMF (3 mL) were placed in a 25 mL neck flask, stirred to dissolve, and 5% Pd / C (300 mg) was added. The mixture was purged three times with hydrogen gas and the hydrogenation reaction was carried out for 2 hours. The completion of the reaction was confirmed by HPLC. After the reaction was complete, the Pd / C was removed by filtration, and the filtrate was cooled to 0-5°C. DIPEA (65 mg, 0.5 mmol, 1.1 eq) was added, and then ent-M3 (255 mg, 0.45 mmol) was added to the filtrate. The temperature was then raised to 20 ± 5°C and the reaction was carried out for 1 hour. The completion of the reaction was confirmed by HPLC. After the reaction was complete, the product was separated and purified by HPLC, and the product was collected and freeze-dried to obtain compound 5d-1 (200 mg). Yield 54%; LC-MS: [MH] - = 816.3.

[0107] Step 2: Compound 5e-1 Compound 5d-1 (200 mg, 0.24 mmol, 1.0 eq), M5 (127 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2 eq), HOBt (48 mg, 0.36 mmol, 1.2 eq), and DMF (6 mL) were placed in a 25 mL neck flask. The mixture was cooled to 0–5°C in an ice bath, DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added, and the temperature was raised to 20±5°C for 2 hours. The reaction was confirmed to be complete by HPLC. The reaction mixture was then preparatively purified by HPLC, and the product fraction was collected and freeze-dried to obtain compound 5e-1 (162.8 mg). LC-MS: [M+H] + = 1235.4.

[0108] Step 3: Compound 5B Compound 5e-1 (110 mg, 0.089 mmol, 1.0 eq), ZnBr2 (400 mg, 1.78 mmol, 20.0 eq), and CH3NO2 (10 mL) were added sequentially to a 25 mL neck flask. The mixture was then heated to 40°C and reacted for 0.5 hours. After stopping the reaction, the reaction solution was dried at 45°C under reduced pressure by rotary evaporation to obtain a yellow solid. The reaction was monitored by HPLC after sampling. The solid dried by rotary evaporation was then preparatively purified by HPLC, and the product fraction was collected and freeze-dried to obtain compound 5B (73.4 mg). Yield: 76.5%; LC-MS: [M+H] + = 1079.4.

[0109] Example 10 Preparation of compound 6A [ka] Compound 6A (71 mg) was obtained according to the synthesis route of Example 8. LC-MS: [M+H] + = 1079.4.

[0110] Example 11 Preparation of compound 6B [ka] Compound 6B (59 mg) was obtained according to the synthesis route of Example 9. LC-MS: [M+H] + = 1079.4.

[0111] Example 12 Preparation of compounds 7A and 7B [ka]

[0112] Step 1: Compound 7a In a 250 mL neck flask, M1 (10 g, 27.1 mmol), benzyl trifluorolactic acid (prepared by 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 placed and heated to 100 °C for 4 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 5.15 g of the target product. Yield 35.1%; LC-MS: [M + H] + = 543.17.

[0113] Step 2: Compound 7b 7a (5g, 9.2 mmol) and 15 mL of DMF were placed in a 50 mL neck flask, dissolved, and then DBU (1.68 g, 11 mmol) was added under ice water bath. The mixture was reacted for 1 hour and recorded as reaction solution (1). A new 50 mL neck flask was taken, and M4 (3.8 g, 9.2 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol), and 10 mL DMF were added and dissolved. Then, DIPEA (1.82 mL, 11 mmol) was added under an ice bath, and the reaction was continued for 30 mIn. Reaction solution (1) was added, and the mixture was heated to room temperature and reacted for 2 hours. The reaction was monitored by HPLC, and after the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative. The preparative was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.1 g of solid. Yield 62.3%; LC-MS: [M+H] + = 716.25.

[0114] Step 3: Compound 7d In a 25 mL neck flask, 7b (900 mg, 1.26 mmol) and 15 mL of DMF were added and dissolved. Then, 900 mg of 5% Pd / C was added, and the hydrogenation reaction was carried out for 2 hours. After the reaction was complete, the mixture was filtered, and the filtrate was placed in an ice bath. DIPEA (228 μL, 1.38 mmol) was added, followed by M3 (712 mg, 1.26 mmol). The mixture was then heated to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 525 mg of the product. Yield 47.9%; LC-MS: [MH] - = 870.33.

[0115] Step 4: Compound 7e 7d (500 mg, 0.57 mmol), M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were placed in a 50 mL neck flask. DIPEA (378 μL, 2.29 mmol) was added under ice water bath, and the mixture was heated to room temperature and reacted for 2 hours. After confirmation of the completion of the reaction by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain preparative solutions of compound 7e-1 and compound 7e-2. Each preparative solution was freeze-dried, and 150 mg of compound 7e-1 (LC-MS: [M+H]) was obtained. + =1289.46), 220 mg compound 7e-2 (LC-MS:[M+H] + We obtained (=1289.46).

[0116] Step 5: Compound 7A [ka]

[0117] 7e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 52 mg of solid. TOF result: 1133.3613.

[0118] Step 6: Compound 7B [ka] 7e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 63 mg of solid. TOF result: 1133.3668.

[0119] Example 13 Synthesis of compounds 8A and 8B [ka]

[0120] Step 1: Compound 8d In a 25 mL neck flask, 7c (900 mg, 1.83 mmol) and 20 mL of DMF were dissolved. Then, DIPEA (303 μL, 1.83 mmol) was added, followed by ent-M3 (1034 mg, 1.83 mmol). The mixture was then heated to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 613 mg of the product. Yield: 38.5%; LC-MS: [MH] - = 870.32.

[0121] Step 2: Compound 8e-1 and Compound 8e-2 8d (500 mg, 0.57 mmol), M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were placed in a 50 mL neck flask. DIPEA (378 μL, 2.29 mmol) was added under ice water bath, and the mixture was heated to room temperature and reacted for 2 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain preparative solutions of compound 8e-1 and compound 8e-2. The preparative solutions were freeze-dried to obtain 140 mg of compound 8e-1 and 210 mg of compound 8e-2. LC-MS of compound 8e-1: [M+H] + =1289.47; LC-MS of compound 8e-2:[M+H] + = 1289.47.

[0122] Step 3: Compound 8A [ka] Compound 8e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 50 mg of solid. TOF result: 1133.3623.

[0123] Step 4: Compound 8B [ka] Compound 8e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 58 mg of solid. TOF result: 1133.3653.

[0124] Example 14 Synthesis of compound 9A [ka]

[0125] Step 1: Compound 9a In a 250 mL neck flask, M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were placed and cooled to 0°C with stirring. 2-hydroxy-2-cyclopropylbenzyl acetate (prepared according to the method disclosed in patent US20050020645A1) (6.3 g, 32.6 mmol) was added dropwise. After the addition was complete, the mixture was allowed to rise naturally to room temperature for approximately 2-4 hours, and monitored by TLC. After the reaction was complete, saturated NaHCO3 solution was added, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA = 10:1-5:1-2:1) to obtain 9a (3.7 g). Yield 45%; LC-MS: [M+H] + = 501.5.

[0126] Step 2: Compound 9b 9a (2g, 4.0 mmol) and 10 mL of DMF were placed in a 25 mL neck flask, stirred at 0°C, and DBU (760 mg, 5.0 mmol) was added. The reaction was allowed to proceed for 1 hour, and after confirming that Fmoc deprotection was complete by TLC, the solution was stored until use. A new 25 mL neck flask was taken, and M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol), and 10 mL DMF were added. DIPEA (823 μL, 5.04 mmol) was added under an ice bath, and the mixture was stirred for 30 mIn. The reaction mixture was then transferred to a reaction flask and the mixture was heated to room temperature to allow the reaction to proceed. After confirmation of the completion of the reaction by HPLC, the reaction mixture was purified by preparative liquid chromatography to obtain the product preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1.5 g of solid. Yield 56%; LC-MS: [M+H] + = 674.7.

[0127] Step 3: Compound 9c Dissolve 9b (900 mg, 1.3 mmol) in 10 mL of DMF in a 25 mL neck flask, then add 900 mg of 5% Pd / C and allow the hydrogenation reaction to proceed for 1.5 hours. After the reaction is complete, filter to obtain the filtrate. This filtrate was used directly in the next reaction without purification.

[0128] Step 4: Compound 9d Crude product 9c was placed in an ice bath, DIPEA (223 μL, 1.3 mmol) was added, followed by compound M3 (750 mg, 1.3 mmol). The mixture was then heated to room temperature and reacted for 1 hour. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 9d (529 mg). LC-MS: [MH] - = 828.4.

[0129] Step 5: Compound 9e 9d (500 mg, 0.6 mmol), M5 (300 mg, 0.6 mmol), PyBOP (416 mg, 0.8 mmol), HOBt (108 mg, 0.5 mmol), and 15 mL of DMF were placed in a 50 mL neck flask. DIPEA (351 μL, 2.13 mmol) was added under ice water bath, and the mixture was heated to room temperature and reacted for 3 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative of compound 9e. The preparative was freeze-dried to obtain 9e (257 mg). LC-MS: [M+H] + = 1247.5.

[0130] Step 6: Compound 9A 9e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain solid compound 9A (55 mg). LC-MS: [M+H] + = 1091.3.

[0131] Example 15 Synthesis of compound 9B [ka] Compound 9B (44 mg) was obtained according to the synthesis route of Example 14. LC-MS: [M+H] + = 1091.3.

[0132] Example 16 Synthesis of compound 10A [ka]

[0133] Step 1: Compound 10a In a 250 mL neck flask, M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were placed and cooled to 0°C with stirring. Benzyl 3-hydroxy-2-cyclopropylpropionate (prepared according to the method disclosed in patent WO2013187496A1) (6.7 g, 32.6 mmol) was added dropwise. After the addition was complete, the mixture was allowed to rise naturally to room temperature for approximately 2-4 hours, and monitored by TLC. After the reaction was complete, saturated NaHCO3 solution was added, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA = 10:1-5:1-2:1) to obtain 10a (4.9 g). Yield 58%; LC-MS: [M+H] + = 515.4.

[0134] Step 2: Compound 10b 10a (4g, 7.8 mmol) and 10 mL of DMF were placed in a 25 mL neck flask, stirred at 0°C, and DBU (1.2 g, 8.0 mmol) was added. The reaction was allowed to proceed for 1 hour, and after confirming that Fmoc deprotection was complete by TLC, the solution was stored until use. A new 25 mL neck flask was taken, and M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol), and 10 mL DMF were added. DIPEA (1.65 mL, 10.1 mmol) was added under an ice bath, and the mixture was stirred for 50 mL. The reaction mixture was then transferred to a reaction flask and allowed to react at room temperature. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by preparative liquid chromatography to obtain the product preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.3 g of solid. Yield 42%; LC-MS: [M+H] + = 688.8.

[0135] Step 3: Compound 10c Dissolve 10b (1.0g, 1.45 mmol) in 15mL DMF in a 25mL neck flask, then add 1.0g of 5% Pd / C and allow the hydrogenation reaction to proceed for 1.5 hours. After the reaction is complete, filter to obtain the filtrate. This filtrate was used directly in the next reaction without purification.

[0136] Step 4: Compound 10d Crude product 10c was placed in an ice bath, DIPEA (258 μL, 1.5 mmol) was added, followed by the addition of compound M3 (837 mg, 1.45 mmol). The mixture was then heated to room temperature and reacted for 1 hour. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 10d (499 mg). LC-MS: [MH] - = 842.4.

[0137] Step 5: Compound 10e In a 50 mL neck flask, 10d (400 mg, 0.48 mmol), M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (104 mg, 0.48 mmol), and 15 mL of DMF were placed. Under an ice bath, DIPEA (330 μL, 2.0 mmol) was added, and the mixture was heated to room temperature for 3 hours. After confirming the completion of the reaction by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a fraction of compound 10e. The fraction was freeze-dried to obtain 10e (188 mg). LC-MS: [M+H] + = 1261.5.

[0138] Step 6: Compound 10A 10e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain solid compound 10A (61 mg). LC-MS: [M+H] + = 1105.4.

[0139] Example 17 Synthesis of Compound 10B

Chemical formula

[0140] Example 18 Synthesis of Compound 11A

Chemical formula

[0141] Step 1: Compound 11a M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were placed in a 250 mL single-neck flask, cooled to 0 °C with stirring, and benzyl 2-hydroxy-2-cyclobutylacetate (synthesized by the method disclosed in Journal of Medicinal Chemistry, 2013, 56(13), 5541 - 5552) (6.7 g, 32.6 mmol) was added dropwise. After the addition was complete, the temperature was allowed to rise to room temperature naturally and the reaction was carried out (about 2 - 4 h), monitored by TLC. After the reaction was completed, saturated NaHCO3 solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA = 10:1 - 5:1 - 2:1) to obtain 11a (5.1 g). Yield 62%; LC-MS: [M+H] + = 515.7

[0142] Step 2: Compound 11b 11a (4 g, 7.8 mmol) and 10 mL of DMF were placed in a 25 mL single-neck flask, stirred at 0 °C, DBU (1.2 g, 8.0 mmol) was added, and the reaction was carried out for 1 h. After it was confirmed by TLC that the Fmoc deprotection was complete, it was stored until use. A new 25 mL neck flask was taken, and M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol), and 10 mL DMF were added. DIPEA (1.63 mL, 10.0 mmol) was added under an ice bath, and the mixture was stirred for 40 mIn. The reaction mixture was then transferred to a reaction flask and the mixture was heated to room temperature to allow the reaction to proceed. After confirmation of the completion of the reaction by HPLC, the reaction mixture was purified by preparative liquid chromatography to obtain the product preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.3 g of solid. Yield 42%; LC-MS: [M+H] + = 688.3.

[0143] Step 3: Compound 11c Dissolve 11b (2.0 g, 2.9 mmol) in 25 mL of DMF in a 25 mL neck flask, then add 2.0 g of 5% Pd / C and allow the hydrogenation reaction to proceed for 3 hours. After the reaction is complete, filter to obtain the filtrate. This filtrate was used directly in the next reaction without purification.

[0144] Step 4: Compound 11d Crude product 11c was placed in an ice bath, DIPEA (516 μL, 3.0 mmol) was added, followed by compound M3 (1.7 g, 2.9 mmol). The mixture was then heated to room temperature and reacted for 2 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 11d (934 mg). LC-MS: [MH] - = 842.4.

[0145] Step 5: Compound 11e 11d (800 mg, 0.96 mmol), M5 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol), and 30 mL of DMF were placed in a 50 mL neck flask. DIPEA (660 μL, 4.0 mmol) was added under an ice bath, and the mixture was heated to room temperature and reacted for 4 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative of compound 11e. The preparative was freeze-dried to obtain 11e (401 mg). LC-MS: [M+H] + = 1261.4.

[0146] Step 6: Compound 11A 11e (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol), and 10 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain solid compound 11A (86 mg). LC-MS: [M+H] + = 1105.4.

[0147] Example 19 Synthesis of compound 11B [ka] Compound 11B (50 mg) was obtained according to the synthesis route of Example 18. LC-MS: [M+H] + 1105.4.

[0148] Example 20 Synthesis of compound 12A [ka]

[0149] Step 1: Compound 12a In a 250 mL neck flask, M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were placed and cooled to 0°C with stirring. 3-hydroxy-2-cyclobutylpropionate benzyl (prepared by the method disclosed in patent WO2009011285A1) (7.2 g, 32.6 mmol) was added dropwise. After the addition was complete, the mixture was allowed to rise naturally to room temperature for approximately 2-4 hours, and monitored by TLC. After the reaction was complete, saturated NaHCO3 solution was added, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA = 10:1-5:1-2:1) to obtain 12a (4.5 g). Yield 52%; LC-MS: [M+H] + = 529.4.

[0150] Step 2: Compound 12b 12a (4g, 7.6 mmol) and 10 mL of DMF were mixed in a 25 mL neck flask at 0°C and stirred. DBU (1.2 g, 8.0 mmol) was added and the mixture was allowed to react for 1 hour. After confirming that Fmoc deprotection was complete by TLC, the mixture was stored until use. A new 25 mL neck flask was taken, and M4 (3.2 g, 7.6 mmol), PyBOP (4.7 g, 9.0 mmol), HOBt (1.22 g, 9.0 mmol) and 10 mL DMF were added. DIPEA (1.49 mL, 0.9 mmol) was added under an ice bath, and the mixture was stirred for 30 mL. The reaction mixture was then transferred to a reaction flask and allowed to react at room temperature. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by preparative liquid chromatography to obtain the product preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.0 g of solid. Yield 37%; LC-MS: [M+H] + = 702.8.

[0151] Step 3: Compound 12c 12b (1.0 g, 1.43 mmol) and 15 mL of DMF were added to a 25 mL single-neck flask and dissolved. Then, 1.0 g of 5% Pd / C was added, and the mixture was subjected to a hydrogenation reaction for 1.5 h. After the reaction was completed, it was filtered to obtain a filtrate, which was used directly in the next reaction without purification.

[0152] Step 4: Compound 12d The crude product 12c was placed in an ice-water bath, DIPEA (258 μL, 1.5 mmol) was added, and then compound M3 (825 mg, 1.43 mmol) was added. Subsequently, the temperature was raised to room temperature and the reaction was carried out for 1 h. After the completion of the reaction was confirmed by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fractionation solution. The fractionation solution was lyophilized to obtain 12d (522 mg). LC-MS: [M-H] - = 856.4.

[0153] Step 5: Compound 12e 12d (400 mg, 0.47 mmol), M5 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol) and 15 mL of DMF were added to a 50 mL single-neck flask. Under an ice-water bath, DIPEA (330 μL, 2.0 mmol) was added, and the temperature was raised to room temperature and the reaction was carried out for 3 h. After the completion of the reaction was confirmed by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fractionation solution of compound 12e. The fractionation solution was lyophilized to obtain 12e (198 mg). LC-MS: [M+H] + = 1275.4.

[0154] Step 6: Compound 12A 12e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL single-neck flask, and the reaction was carried out at 40 °C for 1 h. After the completion of the reaction was confirmed by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a product fractionation solution. The fractionation solution was lyophilized to obtain solid compound 12A (55 mg). LC-MS: [M+H] + = 1119.4.

[0155] Example 21 Synthesis of compound 12B [ka] Compound 12B (50 mg) was obtained according to the synthesis route of Example 20. LC-MS: [M+H] + = 1119.4.

[0156] Example 22 Synthesis of compound 13A [ka]

[0157] Step 1: Compound 13a In a 250 mL neck flask, M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were placed, and the mixture was cooled to 0°C while stirring. 2-hydroxy-2-cyclopentyl acetate benzyl (synthesized by the method disclosed in the Journal of Medical Inal Chemistry, 2013, 56(13), 5541-5552) (7.2 g, 32.6 mmol) was added, and after the addition was complete, the mixture was allowed to rise naturally to room temperature for the reaction (approximately 2-4 hours), and monitored by TLC. After the reaction was complete, saturated NaHCO3 solution was added, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA = 10:1-5:1-2:1) to obtain 13a (4.6 g). Yield 53%;LC-MS:[M+H] + = 529.5.

[0158] Step 2: Compound 13b 13a (4g, 7.6 mmol) and 10 mL of DMF were placed in a 25 mL neck flask, stirred at 0°C, and DBU (1.17 g, 7.8 mmol) was added. The reaction was allowed to proceed for 1 hour, and after confirming that Fmoc deprotection was complete by TLC, the solution was stored until use. A new 25 mL neck flask was taken, and M4 (3.14 g, 7.6 mmol), PyBOP (4.42 g, 8.5 mmol), HOBt (1.15 g, 8.5 mmol) and 10 mL DMF were added. DIPEA (1.39 mL, 0.85 mmol) was added in an ice bath, and the mixture was stirred for 30 mL. The reaction mixture was then transferred to a reaction flask and allowed to react at room temperature. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by preparative liquid chromatography to obtain the product preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.1 g of solid. Yield 39%; LC-MS: [M+H] + = 702.8.

[0159] Step 3: Compound 13c Dissolve 13b (1.5g, 1.87 mmol) in 25mL DMF in a 25mL neck flask, then add 1.5g of 5% Pd / C and allow the hydrogenation reaction to proceed for 3 hours. After the reaction is complete, filter to obtain the filtrate. This filtrate was used directly in the next reaction without purification.

[0160] Step 4: Compound 13d Crude product 13c was placed in an ice bath, DIPEA (333 μL, 1.93 mmol) was added, followed by compound M3 (1.1 g, 1.87 mmol). The mixture was then heated to room temperature and reacted for 1 hour. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 13d (519 mg). LC-MS: [MH] - = 856.6.

[0161] Step 5: Compound 13e In a 50 mL neck flask, 13d (400 mg, 0.47 mmol), M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (103 mg, 48 mmol), and 15 mL of DMF were placed. Under an ice bath, DIPEA (330 μL, 2.0 mmol) was added, and the mixture was heated to room temperature and reacted for 4 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a fraction of compound 13e. The fraction was freeze-dried to obtain 13e (187 mg). LC-MS: [M+H] + = 1275.5.

[0162] Step 6: Compound 13A 13e (100 mg, 0.08 mmol), zinc bromide (355 mg, 0.16 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain solid compound 13A (60 mg). LC-MS: [M+H] + = 1119.6.

[0163] Example 23 Synthesis of compound 13B [ka] Compound 13B (51 mg) was obtained according to the synthesis route of Example 22. LC-MS: [M+H] + = 1119.6.

[0164] Example 24 Synthesis of compound 14A [ka]

[0165] Step 1: Compound 14a In a 250 mL neck flask, M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were placed and cooled to 0°C with stirring. 3-hydroxy-2-cyclopentylpropionate benzyl (synthesized by the method disclosed in patent WO2009011285A1) (7.6 g, 32.6 mmol) was added dropwise. After the addition was complete, the mixture was allowed to rise naturally to room temperature for approximately 2-4 hours, and monitored by TLC. After the reaction was complete, saturated NaHCO3 solution was added, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA = 10:1-5:1-2:1) to obtain 14a (4.4 g). Yield 49%; LC-MS: [M+H] + = 543.6.

[0166] Step 2: Compound 14b 14a (4g, 7.4 mmol) and 10 mL of DMF were placed in a 25 mL neck flask, stirred at 0°C, and DBU (1.2 g, 8.0 mmol) was added. The reaction was allowed to proceed for 1 hour, and after confirming that Fmoc deprotection was complete by TLC, the solution was stored until use. A new 25 mL neck flask was taken, and M4 (3.1 g, 7.4 mmol), PyBOP (4.6 g, 8.8 mmol), HOBt (1.19 g, 8.8 mmol) and 10 mL DMF were added. DIPEA (1.49 mL, 9.0 mmol) was added under an ice bath, and the mixture was stirred for 30 mL. The reaction mixture was then transferred to a reaction flask and allowed to react at room temperature. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by preparative liquid chromatography to obtain the product preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.6 g of solid. Yield 49%; LC-MS: [M+H] + = 716.4.

[0167] Step 3: Compound 14c Dissolve 14b (1.0 g, 1.4 mmol) in 15 mL of DMF in a 25 mL neck flask, then add 1.0 g of 5% Pd / C and allow the hydrogenation reaction to proceed for 1.5 hours. After the reaction is complete, filter to obtain the filtrate. This filtrate was used directly in the next reaction without purification.

[0168] Step 4: Compound 14d Crude product 14c was placed in an ice bath, DIPEA (248 μL, 1.5 mmol) was added, followed by compound M3 (808 mg, 1.4 mmol). The mixture was then heated to room temperature and reacted for 1 hour. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 14d (500 mg). LC-MS: [MH] - = 870.5.

[0169] Step 5: Compound 14e In a 50 mL neck flask, 14d (400 mg, 0.46 mmol), M5 (235 mg, 0.46 mmol), PyBOP (245 mg, 0.46 mmol), HOBt (99 mg, 0.46 mmol), and 15 mL of DMF were placed. Under an ice bath, DIPEA (331 μL, 2.0 mmol) was added, and the mixture was heated to room temperature and reacted for 3 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a fraction of compound 14e. The fraction was freeze-dried to obtain 14e (146 mg). LC-MS: [M+H] + = 1289.5.

[0170] Step 6: Compound 14A 14e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain solid compound 14A (52 mg). LC-MS: [M+H] + = 1133.4.

[0171] Example 25 Synthesis of Compound 14B

Chemical Structure

[0172] Example 26 Synthesis of Compounds 15A and 15B

Chemical Structure

[0173] Step 1: Compound 15a M1 (10 g, 27.1 mmol), benzyl 2-hydroxy-butyrate (prepared by the method disclosed in Chemical Communications, 2019, 55(53), 7699 - 7702.) (10.5 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added to a 250 mL one-neck flask, and the mixture was heated to 100 °C and reacted for 4 h. After the reaction was completed, it was cooled to room temperature, and the insoluble matter removed by filtration. 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 5.67 g of the target product. Yield 42%; LC-MS: [M+H] + = 503.5

[0174] Step 2: Compound 15b 15a (5 g, 9.95 mmol) and 15 mL of DMF were added to a 50 mL one-neck flask. After dissolution, DBU (1.68 g, 11 mmol) was added under an ice-water bath, and the mixture was reacted for 1 h, and the reaction solution was recorded as (1). A new 50 mL neck flask was taken, and M4 (4.1 g, 10.0 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol), and 10 mL DMF were added and dissolved. Then, DIPEA (1.82 mL, 11 mmol) was added under an ice bath, and the reaction was continued for 40 mIn. Reaction solution (1) was added, and the mixture was heated to room temperature and reacted for 2 hours. The reaction was monitored by HPLC, and after the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative. The preparative was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.6 g of solid. Yield 68%; LC-MS: [M+H] + = 676.7.

[0175] Step 3: Compound 15d Dissolve 15b (2.0 g, 2.96 mmol) and 15 mL of DMF in a 25 mL neck flask. Then add 2.0 g of 5% Pd / C and allow hydrogenation reaction for 2 hours. After the reaction is complete, filter the solution and place the filtrate in an ice bath. Add DIPEA (496 μL, 3.0 mmol) and then M3 (1.7 g, 2.96 mmol). The mixture is then heated to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction mixture is purified by high-performance liquid chromatography to obtain a preparative. The preparative is freeze-dried to obtain 1120.0 mg of the product. Yield 45%; LC-MS: [MH] - = 830.3.

[0176] Step 4: Compound 15e 15d (500 mg, 0.60 mmol), M5 (321 mg, 0.60 mmol), PyBOP (469 mg, 0.90 mmol), HOBt (121 mg, 0.90 mmol), and 15 mL of DMF were placed in a 50 mL neck flask. DIPEA (446 μL, 2.7 mmol) was added under ice water bath, and the mixture was heated to room temperature and reacted for 2 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain fractions of compound 15e-1 and compound 15e-2. Each fraction was freeze-dried, and 138 mg of compound 15e-1 (LC-MS: [M+H]) was obtained. += 1249.5) and 140 mg of Compound 15e-2 (LC-MS: [M+H] + = 1249.5) was obtained.

[0177] Step 5: Compound 15A

Chemical formula

[0178] Step 6: Compound 15B

Chemical formula

[0179] Example 27 Synthesis of Compounds 16A and 16B

Chemical formula

Chemical formula

[0180] Compound 16B (54 mg) was obtained according to the synthesis route of Example 26. LC-MS: [M+H] + = 1093.4. Example 28 Synthesis of compounds 17A and 17B [ka]

[0181] Step 1: Compound 17a In a 250 mL neck flask, M1 (10 g, 27.1 mmol), benzyl 2-hydroxyphenylpropionate (synthesized by the method disclosed in Nature Communiquéns, 2020. 11(1), 56.) (14.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were placed and heated to 100 °C, where the mixture was reacted for 4 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 6.13 g of the target product. Yield 40%; LC-MS: [M + H] + = 565.6.

[0182] Step 2: Compound 17b 17a (5g, 8.86 mmol) and 15 mL of DMF were dissolved in a 50 mL neck flask, then DBU (1.53 g, 10 mmol) was added under ice water bath, and the mixture was reacted for 1 hour. The reaction solution was recorded as (1). A new 50 mL neck flask was taken, and M4 (3.6 g, 8.86 mmol), PyBOP (5.23 g, 10 mmol), HOBt (1.36 g, 10 mmol) and 10 mL DMF were added and dissolved. Then, DIPEA (1.65 mL, 10 mmol) was added under an ice bath, and the reaction was continued for 30 mIn. Reaction solution (1) was added, and the mixture was heated to room temperature and reacted for 2 hours. The reaction was monitored by HPLC, and after the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative. The preparative was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 5.0 g of solid. Yield 77%; LC-MS: [M+H] + = 738.3.

[0183] Step 3: Compound 17d Dissolve 17b (3.0 g, 4.07 mmol) in 15 mL of DMF in a 25 mL neck flask, then add 3.0 g of 5% Pd / C and allow hydrogenation reaction for 2 hours. After the reaction is complete, filter the solution, place the filtrate in an ice bath, add DIPEA (744 μL, 4.5 mmol), then add M3 (2.34 g, 4.07 mmol), and allow the reaction to proceed at room temperature for 1 hour. After confirming the completion of the reaction by HPLC, purify the reaction mixture by high-performance liquid chromatography to obtain a preparative. Freeze-dry the preparative to obtain 1.2 g of the product. Yield 33%; LC-MS: [MH] - = 892.4.

[0184] Step 4: Compound 17e 17d (500 mg, 0.56 mmol), M5 (300 mg, 0.56 mmol), PyBOP (438 mg, 0.84 mmol), HOBt (113 mg, 0.84 mmol), and 15 mL of DMF were placed in a 50 mL neck flask. DIPEA (330 μL, 2.0 mmol) was added under ice water bath, and the mixture was heated to room temperature and reacted for 2 hours. After confirmation of the completion of the reaction by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain fractions of compound 17e-1 and compound 17e-2. Each fraction was freeze-dried, and 156 mg of compound 17e-1 (LC-MS: [M+H]) was obtained. +=1311.4) and 150 mg compound 17e-2 (LC-MS:[M+H] + We obtained (=1311.7).

[0185] Step 5: Compound 17A [ka] 17e-1 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 43 mg of solid. LC-MS: [M+H] + = 1155.4.

[0186] Step 6: Compound 17B [ka] 17e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 40 mg of solid. LC-MS: [M+H] + = 1155.4.

[0187] Example 29 Synthesis of compounds 18A and 18B [ka] Compound 18A (54 mg) was obtained according to the synthesis route of Example 28. LC-MS: [M+H] + = 1155.4. [ka] Compound 18B (55 mg) was obtained according to the synthesis route of Example 28. LC-MS: [M+H] + = 1155.4.

[0188] Example 30 Synthesis of compounds 19A and 19B [ka]

[0189] Step 1: Compound 19a In a 250 mL neck flask, M1 (10 g, 27.1 mmol), 2-cyclopropyl-2-hydroxybenzyl acetate (prepared by 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 placed and heated to 100 °C for 4 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 4.97 g of the target product. Yield 36%; LC-MS: [M + H] + = 515.2.

[0190] Step 2: Compound 19b 19a (4g, 7.8 mmol) and 10 mL of DMF were dissolved in a 50 mL neck flask, then DBU (1.42 g, 9.3 mmol) was added under ice water bath, and the reaction was allowed to proceed for 1 hour. The reaction was recorded as reaction solution (1). A new 50 mL neck flask was taken, and M4 (3.2 g, 7.8 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol) and 10 mL DMF were added and dissolved. Then, DIPEA (1.65 mL, 10 mmol) was added under an ice bath, and the reaction was continued for 30 mIn. Reaction solution (1) was added, and the mixture was heated to room temperature and reacted for 2 hours. The reaction was monitored by HPLC, and after the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative. The preparative was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.2 g of solid. Yield 78%; LC-MS: [M+H] + = 688.3.

[0191] Step 3: Compound 19d Dissolve 19b (1000 mg, 1.45 mmol) in 15 mL of DMF in a 25 mL neck flask. Then add 1000 mg of 5% Pd / C and allow hydrogenation reaction for 2 hours. After the reaction is complete, filter the solution, place the filtrate in an ice bath, add DIPEA (248 μL, 1.5 mmol), then add M3 (720 mg, 1.45 mmol), and allow the reaction to proceed at room temperature for 1 hour. After confirming the completion of the reaction by HPLC, purify the reaction mixture by high-performance liquid chromatography to obtain a preparative. Freeze-dry the preparative to obtain 503 mg of the product. Yield 41%; LC-MS: [MH] - = 842.3.

[0192] Step 4: Compounds 19e-1 and 19e-2 19d (500 mg, 0.59 mmol), M5 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol), and 10 mL of DMF were placed in a 50 mL neck flask. DIPEA (292 μL, 1.77 mmol) was added under ice water bath, and the mixture was heated to room temperature and reacted for 2 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain fractions of compound 19e-1 and compound 19e-2. Each fraction was freeze-dried, and 112 mg of compound 19e-1 (LC-MS: [M+H]) was obtained. +=1261.5) and 131 mg compound 19e-2 (LC-MS:[M+H] + We obtained (=1261.5).

[0193] Step 5: Compound 19A [ka] 19e-1 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 55 mg of solid. LC-MS: [M+H] + = 1105.4.

[0194] Step 6: Compound 19B [ka] 19e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 58 mg of solid. LC-MS: [M+H] + = 1105.4.

[0195] Example 31 Synthesis of compounds 20A and 20B [ka]

[0196] Step 1: Compound 20a In a 250 mL neck flask, M1 (10 g, 27.1 mmol), benzyl 2-hydroxycyclopropylpropionate (synthesized by 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 placed and heated to 100 °C for 4 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 5.09 g of the target product; LC-MS: [M + H] + = 529.2 was obtained.

[0197] Step 2: Compound 20b 20a (4g, 7.6 mmol) and 10 mL of DMF were dissolved in a 50 mL neck flask, then DBU (1.39 g, 9.1 mmol) was added under ice water bath, and the reaction was allowed to proceed for 1 hour. The reaction was recorded as reaction solution (1). A new 50 mL neck flask was taken, and M4 (3.12 g, 7.6 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol) and 10 mL DMF were added and dissolved. Then, DIPEA (1.65 mL, 10 mmol) was added under an ice bath, and the reaction was continued for 30 mIn. Reaction solution (1) was added, and the mixture was heated to room temperature and reacted for 2 hours. The reaction was monitored by HPLC, and after the reaction was complete, the reaction solution was purified by high-performance liquid chromatography to obtain a preparative. The preparative was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.5 g of solid. Yield 84%; LC-MS: [M+H] + = 702.3.

[0198] Step 3: Compound 20d Dissolve 20b (1000 mg, 1.42 mmol) in 15 mL of DMF in a 25 mL neck flask. Then add 1000 mg of 5% Pd / C and allow hydrogenation reaction for 2 hours. After the reaction is complete, filter the solution and place the filtrate in an ice bath. Add DIPEA (248 μL, 1.5 mmol), then M5 (708 mg, 1.42 mmol), and allow the reaction to proceed at room temperature for 1 hour. After confirming the completion of the reaction by HPLC, purify the reaction mixture by high-performance liquid chromatography to obtain a preparative. Freeze-dry the preparative to obtain 443 mg of the product. Yield 36%; LC-MS: [MH] - = 856.4.

[0199] Step 4: Compounds 20e-1 and 20e-2 20d (400 mg, 0.47 mmol), exatecan mesylate (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF were placed in a 50 mL neck flask. DIPEA (248 μL, 1.5 mmol) was added under an ice bath, and the mixture was heated to room temperature and reacted for 2 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain fractions of compound 20e-1 and compound 20e-2. Each fraction was freeze-dried, and 103 mg of compound 20e-1 (LC-MS: [M+H]) was obtained. + =1275.5) and 103 mg compound 20e-2 (LC-MS:[M+H] + We obtained (=1275.5).

[0200] Step 5: Compound 20A [ka] 8A (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 51 mg of solid. LC-MS: [M+H]+ = 1119.4.

[0201] Step 6: Compound 20B [ka] 20 e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 47 mg of solid. LC-MS: [M+H] + = 1119.4.

[0202] Example 32 Synthesis of Compound 21 [ka]

[0203] Step 1: Compound SM3-1 77087-60-6 (100g, 458 mmol), maleic acid (53.4g, 460 mmol), TEA (64mL, 460 mmol), and 1000 mL of toluene were placed in a 2000 mL necked flask and heated to 100°C for 5 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 100:1-50:1-20:1) to obtain 75.6 g of the target product. LC-MS: [M+H] + =299.1.

[0204] Step 2: Compound (R)-2-hydroxy-1,5-glutarate tert-butyl 100 g, 338 mmol of 172793-31-6 and 1000 mL of water were placed in a 2000 mL necked flask. Sodium nitrite (35 g, 507 mmol) and concentrated sulfuric acid (32 mL, 35 mmol) were added in sequence, and the mixture was slowly heated to room temperature and reacted for 24 hours. After the reaction was complete, the mixture was extracted three times with 500 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 50:1-30:1-2:1) to obtain 91.2 g of the target product. LC-MS: [M+H] + = 261.4.

[0205] Step 3: Compound SM3 50 g, 192 mmol of (R)-2-hydroxy-1,5-glutarate tert-butyl and 1000 mL of anhydrous tetrahydrofuran were placed in a 2000 mL necked flask, cooled to 0°C in an ice bath, and PPh3 (87.7 g, 288 mmol), DEAD (50.2 g, 288 mmol), and SM3-1 (57.3 g, 192 mmol) were added in sequence. The mixture was slowly heated to room temperature and reacted for 13 hours. After the reaction was complete, the mixture was filtered to remove insoluble matter, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 50:1-30:1-1:1) to obtain 68.6 g of the product. The above product was dissolved in 500 mL of methanol, cooled to 0°C in an ice bath, and NaOH (64 mL, 190 mmol, 3 M / L) was added dropwise at this temperature. The reaction was carried out for 12 hours while maintaining this temperature, then HCl (6 M / L) was added to adjust the pH to 3. The mixture was extracted five times with 500 mL of dichloromethane, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting crude product was purified by column chromatography (DCM / MeOH = 50 / 1-20 / 1-2 / 1) to obtain 50.4 g of SM3. LC-MS: [MH] - = 525.5.

[0206] Step 4: Compound M6 Compound SM3 (50 g, 95 mmol, 1.0 eq), pentafluorophenol (19.2 g, 104.5 mmol, 1.1 eq), DCC (21.5 g, 104.5 mmol, 1.1 eq), and THF (600 mL) were placed in a 2000 mL neck flask and reacted at room temperature for 1 hour (monitored by TLC). The mixture was filtered to remove insoluble matter. The reaction mixture was then preparatively purified, and the preparative solution was concentrated under reduced pressure using a water pump, in a water bath, and at 35°C to remove acetonitrile. The mixture was then freeze-dried to obtain compound M6 (51.9 g). Yield: 79%; LC-MS: [M+H] + = 693.3.

[0207] Step 5: Compound 21a In a 25 mL neck flask, 1 c (1 g, 2.36 mmol) and 25 mL of DMF were dissolved. Then, DIPEA (430 μL, 2.6 mmol) was added, followed by M6 (1177 mg, 2.36 mmol). The mixture was then heated to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 555 mg of the product. LC-MS: [MH] - =931.0.

[0208] Step 6: Compound 21b 21a (500 mg, 0.54 mmol), exatecan mesylate M5 (285 mg, 0.54 mmol), PyBOP (239 mg, 0.6 mmol), HOBt (239 mg, 0.6 mmol), and 10 mL of DMF were placed in a 100 mL neck flask. DIPEA (248 μL, 1.5 mmol) was added under an ice bath, and the mixture was heated to room temperature and reacted for 2 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a fraction of compound 21b. The fraction was freeze-dried to obtain 231 mg of compound 21b. LC-MS: [M+H] + = 1349.5.

[0209] Step 7: Compound 21 Compound 21b (200 mg, 0.1488 mmol), zinc bromide (665 mg, 2.96 mmol), and 10 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 103 mg of solid. LC-MS: [M+H] + = 1137.5.

[0210] Example 33 [ka]

[0211] Synthesis of compound 22 Compound 22 (91 mg) was obtained using compounds M6 and 3c as starting materials, following the synthesis route of Example 32. LC-MS: [M+H] + = 1165.5.

[0212] Example 34 [ka]

[0213] Synthesis of compounds 23 and 24 Starting with compounds M6 and 5c, 102 mg of compound 23 (LC-MS: [M+H]) was synthesized according to the synthesis route of Example 32. + =1151.4) and 99 mg compound 24 (LC-MS:[M+H] + We obtained (=1151.4).

[0214] Example 35 [ka]

[0215] Synthesis of compounds 25 and 26 Starting with compounds M6 and 7c, 83 mg of compound 25 (LC-MS: [M+H]) was synthesized according to the synthesis route of Example 32. +=1205.7) and 80mg compound 26 (LC-MS:[M+H] + We obtained (=1205.7).

[0216] Example 36 [ka]

[0217] Synthesis of compounds 27 and 28 Starting with compounds M6 and 19c, 100 mg of compound 27 (LC-MS: [M+H]) was synthesized according to the synthesis route of Example 32. + =1177.5) and 101mg compound 28 (LC-MS:[M+H] + We obtained (=1177.5).

[0218] Example 37 Synthesis of compound 29 [ka]

[0219] Step 1: Compound SM4-1 In a 5000 mL neck flask, maleic acid (50 g, 431 mmol, 1.0 eq), 114559-25-0 (110 g, 431 mmol, 1 eq), TEA (263 g, 2.16 mol, 5 eq), and toluene (2000 mL) were added. The mixture was heated under reflux and reacted for 5 hours (monitored by TLC), and the insoluble matter was removed by filtration. The reaction mixture was then evaporated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (PE / EA = 50 / 1-20 / 1-1 / 1) to obtain SM4-1 (64.7 g). Yield 50%; LC-MS: [M+H] + = 299.2.

[0220] Step 2: Compound SM4-2 In a 2000 mL neck flask, SM4-1 (64 g, 215 mmol) and 1000 mL of DMF were dissolved. Then, DIPEA (71 mL, 430 mmol) was added, followed by nonaethylene glycol monomethyl ether mesylate (111.5 g, 220 mmol). The mixture was then heated to room temperature and reacted for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by silica gel column chromatography (PE / EA = 50 / 1-20 / 1-1 / 1) to obtain 59.9 g of the product. LC-MS: [M+H] + = 709.4.

[0221] Step 3: Compound SM4 SM4-2 (59g, 83 mmol) and 1000 mL of MeOH were dissolved in a 2000 mL necked flask, then K2CO3 (11.75 g, 85 mmol) was added, and the reaction was allowed to proceed at room temperature for 4 hours. After confirming the completion of the reaction by HPLC, the mixture was filtered to remove insoluble matter, and the reaction solution was preparatively purified. The preparative solution was concentrated under reduced pressure with a water pump, in a water bath, and at 35°C to remove acetonitrile, and then freeze-dried to obtain compound SM4 (27 g). LC-MS: [MH] - = 693.5.

[0222] Step 4: Compound M7 Compound SM4 (25g, 36 mmol, 1.0eq), pentafluorophenol (7.3g, 40 mmol, 1.1eq), DCC (8.2g, 40 mmol, 1.1eq), and THF (200mL) were placed in a 500mL neck flask and reacted at room temperature for 1 hour (monitored by TLC). The mixture was filtered to remove insoluble matter. The reaction mixture was then preparatively purified, and the preparative solution was concentrated under reduced pressure using a water pump, in a water bath, and at 35°C to remove acetonitrile. The mixture was then freeze-dried to obtain compound M7 (23.3g). Yield: 93%; LC-MS: [M+H] + = 695.8.

[0223] Step 5: Compound 29a In a 25 mL neck flask, 1 c (1 g, 2.36 mmol) and 25 mL of DMF were dissolved. Then, DIPEA (430 μL, 2.6 mmol) was added, followed by M7 (1640 mg, 2.36 mmol). The mixture was then heated to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 609 mg of the product. LC-MS: [MH] - = 1098.5.

[0224] Step 6: Compound 29b In a 100 mL neck flask, 29a (500 mg, 0.45 mmol), exatecan mesylate M5 (240 mg, 0.45 mmol), PyBOP (215 mg, 0.54 mmol), HOBt (215 mg, 0.54 mmol), and 10 mL of DMF were placed. Under an ice bath, DIPEA (248 μL, 1.5 mmol) was added, and the mixture was heated to room temperature and reacted for 2 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a fraction of compound 29b. The fraction was freeze-dried to obtain 187 mg of the compound. LC-MS: [M+H] + = 1517.6.

[0225] Step 7: Compound 29 Compound 29b (150 mg, 0.988 mmol), zinc bromide (223 mg, 0.988 mmol), and 10 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 114 mg of solid. LC-MS: [M+H] + = 1517.9.

[0226] Example 38 [ka]

[0227] Synthesis of compound 30 Compound 30 (125 mg) was obtained using compounds M7 and 3c as starting materials, following the synthesis route of Example 37. LC-MS: [M+H] + = 1445.6.

[0228] Example 39 [ka]

[0229] Synthesis of compounds 31 and 32 Starting with compounds M7 and 5c, 61 mg of compound 31 (LC-MS: [M+H]) was synthesized according to the synthesis route of Example 37. + =1431.7) and 63mg compound 32 (LC-MS:[M+H] + We obtained (=1431.7).

[0230] Example 40 [ka]

[0231] Synthesis of compounds 33 and 34 Starting with compounds M7 and 7c, 60 mg of compound 33 (LC-MS: [M+H]) was synthesized according to the synthesis route of Example 37. + =1485.6) and 58 mg compound 34 (LC-MS:[M+H] + We obtained (=1485.6).

[0232] Example 41 [ka]

[0233] Synthesis of compounds 35 and 36 Starting with compounds M7 and 19c, 102 mg of compound 35 (LC-MS: [M+H]) was synthesized according to the synthesis route of Example 37. + =1457.8) and 102 mg compound 36 (LC-MS:[M+H] + We obtained (=1457.8).

[0234] Example 42 Synthesis of Compound 37 [ka]

[0235] Step 1: Compound SM5-1 Compound 16947-84-5 (100g, 295 mmol, 1.0eq), DIPEA (50mL, 300 mmol), benzyl bromide (51.3g, 300 mmol), and THF (1000mL) were placed in a 2000mL neck flask. The reaction was allowed to proceed at room temperature for 12 hours (monitored by TLC), and the mixture was filtered to remove insoluble matter. The reaction mixture was then evaporated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (PE / EA = 50 / 1-20 / 1-2 / 1) to obtain SM5-1 (110.1g). Yield 87%; LC-MS: [M+H] + = 429.2.

[0236] Step 2: Compound SM5-2 Compound SM5-1 (100g, 233.4 mmol, 1.0eq) and THF (1000mL) were placed in a 2000mL neck flask. The mixture was cooled to 0°C in an ice bath. NaH (37.4g, 933.5 mmol) and MeI (132.5g, 933.5 mmol) were added in batches, and the reaction was allowed to proceed for 24 hours while maintaining 0°C (monitored by TLC). The reaction was quenched by adding 500mL of saturated NH4Cl aqueous solution, and the mixture was extracted three times with 500mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was rotated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (PE / EA = 100 / 1-50 / 1-10 / 1) to obtain SM5-2 (37.1g). LC-MS: [M+H] + = 443.3.

[0237] Step 3: Compound SM5 (See reference Org. Lett., 2006, 8, 3387-3390.) Compound SM5-2 (35g, 79 mmol, 1.0eq) and DCE (500mL) were placed in a 1000mL neck flask. Palladium diacetate (180mg, 0.8 mmol), I2 (20g, 79 mmol), and iodobenzene diacetate (40.8g, 126.4 mmol) were added in sequence. The mixture was heated to 60°C and reacted for 40 hours (monitored by TLC). 500mL of saturated sodium thiosulfate aqueous solution was added to quench the reaction. The mixture was extracted three times with 500mL of dichloromethane, the organic phase was dried over anhydrous sodium sulfate, and the solution was filtered. The filtrate was rotated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (PE / EA = 100 / 1-50 / 1-10 / 1) to obtain SM5 (28g). LC-MS: [M+H] + = 501.3.

[0238] Step 4: Compound SM6 Compound SM5 (25g, 50 mmol, 1.0eq), di-tert-butyl potassium phosphate (13.66g, 55 mmol, 1.1eq), monohydrate p-toluenesulfonic acid (951mg, 5 mmol, 0.1eq), and THF (200mL) were placed in a 500mL neck flask. The reaction was allowed to proceed at room temperature for 1 hour (monitored by TLC), and the mixture was filtered to remove insoluble matter. The reaction mixture was then preparatively purified, and the preparative solution was concentrated under reduced pressure using a water pump, in a water bath, and at 35°C to remove acetonitrile. The mixture was then freeze-dried to obtain compound SM6 (15.1g). Yield: 46%; LC-MS: [M+H] + = 651.4.

[0239] Step 5: Compound SM7 In a 250 mL neck flask, SM6 (15 g, 23 mmol) and 100 mL of DMF were placed and dissolved. Then, 15 g of 5% Pd / C was added under ice water bath, the atmosphere in the reaction system was replaced three times with hydrogen gas, and the reaction was carried out at room temperature for 12 hours. The Pd / C was removed by filtration, and the solvent was removed by rotary evaporation under reduced pressure with an oil pump. The solution was then stored until use. A new 250 mL necked flask was taken, and the crude product described above, along with 100 mL of toluene, triethylamine (6.4 mL, 46 mmol), and maleic anhydride (2.4 g, 24 mmol) were added and dissolved. The mixture was then heated to 100 °C and reacted for 2 hours. The reaction was monitored by HPLC, and after completion, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.2 g of solid. Yield 36%; LC-MS: [M+H] + = 507.3.

[0240] Step 6: Compound M8 Compound SM7 (4g, 7.9 mmol, 1.0eq), pentafluorophenol (1.6g, 8.7 mmol, 1.1eq), DCC (1.8g, 8.7 mmol, 1.1eq), and THF (60mL) were placed in a 100mL neck flask and reacted at room temperature for 1 hour (monitored by TLC). The mixture was filtered to remove insoluble matter. The reaction mixture was then preparatively purified, and the preparative solution was concentrated under reduced pressure using a water pump, in a water bath, and at 35°C to remove acetonitrile. The mixture was then freeze-dried to obtain compound M8 (3.7g). Yield: 70%; LC-MS: [M+H] + = 673.2.

[0241] Step 7: Compound 37a In a 25 mL neck flask, 1 c (1 g, 2.36 mmol) and 25 mL of DMF were dissolved. Then, DIPEA (430 μL, 2.6 mmol) was added, followed by M8 (1.2 g, 2.36 mmol). The mixture was then heated to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a preparative. The preparative was freeze-dried to obtain 488 mg of the product. LC-MS: [MH] - =911.0.

[0242] Step 8: Compound 37b 37a (400 mg, 0.44 mmol), exatecan mesylate M5 (235 mg, 0.44 mmol), PyBOP (199 mg, 0.5 mmol), HOBt (69 mg, 0.5 mmol), and 10 mL of DMF were placed in a 100 mL neck flask. DIPEA (218 μL, 1.32 mmol) was added under an ice bath, and the mixture was heated to room temperature and reacted for 2 hours. After confirmation of the reaction completion by HPLC, the reaction mixture was purified by high-performance liquid chromatography to obtain a fraction of compound 37b. The fraction was freeze-dried to obtain 201 mg of the compound. LC-MS: [M+H] + = 1329.6.

[0243] Step 9: Compound 37 Compound 37b (130 mg, 0.098 mmol), zinc bromide (221 mg, 0.98 mmol), and 10 mL of nitromethane were placed in a 25 mL neck flask and reacted at 40°C for 1 hour. After confirmation of the reaction completion by HPLC, the solvent was removed by vacuum concentration to obtain the crude product. The crude product was purified by high-performance liquid chromatography to obtain the product fraction. The fraction was freeze-dried to obtain 96 mg of solid. LC-MS: [M+H] + = 1117.4.

[0244] Example 43 [ka] Synthesis of compound 38 Compound 38 (51 mg) was obtained using compounds M8 and 3c as starting materials, following the synthesis route of Example 42. LC-MS: [M+H] + = 1145.6.

[0245] Example 44 [ka] Synthesis of compounds 39 and 40 Starting with compounds M8 and 5c, 57 mg of compound 39 (LC-MS: [M+H]) was synthesized according to the synthesis route of Example 42. + =1131.4) and 60 mg compound 40 (LC-MS:[M+H] +We obtained =1131.4).

[0246] Example 45 [ka] Synthesis of compounds 41 and 42 Starting with compounds M7 and 7c, 44 mg of compound 41 (LC-MS: [M+H]) was synthesized according to the synthesis route of Example 42. + =1185.3) and 44mg compound 42 (LC-MS:[M+H] + =1185.3) was obtained.

[0247] Example 46 [ka] Synthesis of compounds 43 and 44 Starting with compounds M8 and 19c, 62 mg of compound 43 (LC-MS: [M+H]) was synthesized according to the synthesis route of Example 42. + =1157.4) and 59mg compound 44 (LC-MS:[M+H] + We obtained (=1157.4).

[0248] Example 47 (Comparative Example) [ka] Synthesis of compound 45 Compound 45 was synthesized by the method described in Example 58 of Patent CN104755494A.

[0249] The following is the sequence of Trastuzumab. Light chain MDMRVPAQLLGLLLLWLRGARC DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* Heavy chain MDMRVPAQLLGLLLLWLRGARC EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0250] Preparation of ligand-drug conjugates 1) General coupling methods Antibody molecules with a monomer content exceeding 95% after preliminary purification were transferred to phosphate buffer (10 mg / mL concentration) using an ultrafiltration centrifuge tube. 20 times the amount of antibody moles in TCEP were added, and the mixture was reacted at room temperature for 4 hours to break the disulfide bonds between antibody chains. 20 times the amount of antibody moles in linker-drug compound (payload) was added, and the mixture was reacted at room temperature for 2 hours. After the reaction was complete, the solution was replaced with PBS using an ultrafiltration centrifuge tube with a cutoff molecular weight of 30 kDa to remove the unbound payload. The ADC sample after the solution change was filtered through a 0.22 micron sterile filter and stored until use. 2) Measurement of ligand-drug complex DAR value Monomer concentration detection conditions The sample was centrifuged at 14,000 rpm for 5 minutes, and the supernatant was collected and analyzed. Equipment:Waters e2695(2489UV / VIs) Column: TSKgel G3000SWXL (7.8 × 300 mm, 5 μm) Mobile phase: A: 50mM PB, 300mM NaCl, 200mM Arg, 5%IPA, pH6.5 30 mIn isocratic elution was performed with mobile phase A. Flow rate: 0.714 mL / mIn, column temperature: 25°C, detection wavelength: 280 nm.

[0251] Monomer concentration detection conditions The sample was centrifuged at 14,000 rpm for 5 minutes, and the supernatant was collected and analyzed. Equipment: Waters H-class (TUV); Column: ProteomIx HICButyl-NP5 (4.6 × 35 mm, 5 μm); Mobile phase: A: 1.5M ammonium sulfate, 0.025M anhydrous sodium phosphate, pH 7.0; B: 0.025M anhydrous sodium phosphate, 25% IPA, pH 7.0 The column was equilibrated with mobile phase A, and gradient elution was performed with mobile phases A and B. Flow rate: 0.8 mL / mIn, column temperature: 25°C, detection wavelength: 214 nm.

[0252] Example 48: ADC-1 [ka] ADC-1 was obtained by preparation using a general coupling method.

[0253] Example 49: ADC-2 [ka] ADC-2 was obtained by preparation using a general coupling method.

[0254] Example 50: ADC-3 [ka] ADC-3 was obtained by preparation using a general coupling method.

[0255] Example 51: ADC-4 [ka] ADC-4 was obtained by preparation using a general coupling method.

[0256] Example 52: ADC-5 [ka] ADC-5 was obtained by preparation using a general coupling method.

[0257] Example 53: ADC-6 [ka] ADC-6 was obtained by preparation using a general coupling method.

[0258] Example 54: ADC-7 [ka] ADC-7 was obtained by preparation using a general coupling method.

[0259] Example 55: ADC-8 [ka] ADC-8 was obtained by preparation using a general coupling method.

[0260] Example 56: ADC-9 [ka] ADC-9 was obtained by preparation using a general coupling method.

[0261] Example 57: ADC-10 [ka] ADC-10 was obtained by preparation using a general coupling method.

[0262] Example 58: ADC-11 [ka] ADC-11 was obtained by preparation using a general coupling method.

[0263] Example 59: ADC-12 [ka] ADC-12 was obtained by preparation using a general coupling method.

[0264] Example 60: ADC-13 [ka] ADC-13 was obtained by preparation using a general coupling method.

[0265] Example 61: ADC-14 [ka] ADC-14 was obtained by preparation using a general coupling method.

[0266] Example 62: ADC-15 [ka] ADC-15 was obtained by preparation using a general coupling method.

[0267] Example 63: ADC-16 [ka] ADC-16 was obtained by preparation using a general coupling method.

[0268] Example 64: ADC-17 [ka] ADC-17 was obtained by preparation using a general coupling method.

[0269] Example 65: ADC-18 [ka] ADC-18 was obtained by preparation using a general coupling method.

[0270] Example 66: ADC-19 [ka] ADC-19 was obtained by preparation using a general coupling method.

[0271] Example 67: ADC-20 [ka] ADC-20 was obtained by preparation using a general coupling method.

[0272] Example 68: ADC-21 [ka] ADC-21 was obtained by preparation using a general coupling method.

[0273] Example 69: ADC-22 [ka] ADC-22 was obtained by preparation using a general coupling method.

[0274] Example 70: ADC-23 [ka] ADC-23 was obtained by preparation using a general coupling method.

[0275] Example 71: ADC-24 [ka] ADC-24 was obtained by preparation using a general coupling method.

[0276] Example 72: ADC-25 [ka] ADC-25 was obtained by preparation using a general coupling method.

[0277] Example 73: ADC-26 [ka] ADC-26 was obtained by preparation using a general coupling method.

[0278] Example 74: ADC-27 [ka] ADC-27 was obtained by preparation using a general coupling method.

[0279] Example 75: ADC-28 [ka] ADC-28 was obtained by preparation using a general coupling method.

[0280] Example 76: ADC-29 [ka] ADC-29 was obtained by preparation using a general coupling method.

[0281] Example 77: ADC-30 [ka] ADC-30 was obtained by preparation using a general coupling method.

[0282] Example 78: ADC-31 [ka] ADC-31 was obtained by preparation using a general coupling method.

[0283] Example 79: ADC-32 [ka] ADC-32 was obtained by preparation using a general coupling method.

[0284] Example 80: ADC-33 [ka] ADC-33 was obtained by preparation using a general coupling method.

[0285] Example 81: ADC-34 [ka] ADC-34 was obtained by preparation using a general coupling method.

[0286] Example 82: ADC-35 [ka] ADC-35 obtained by preparation using a general coupling method.

[0287] Example 83: ADC-36 [ka] ADC-36 was obtained by preparation using a general coupling method.

[0288] Example 84: ADC-37 [ka] ADC-37 was obtained by preparation using a general coupling method.

[0289] Example 85: ADC-38 [ka] ADC-38 was obtained by preparation using a general coupling method.

[0290] Example 86: ADC-39 [ka] ADC-39 was obtained by preparation using a general coupling method.

[0291] Example 87: ADC-40 [ka] ADC-40 was obtained by preparation using a general coupling method.

[0292] Example 88: ADC-41 [ka] ADC-41 was obtained by preparation using a general coupling method.

[0293] Example 89: ADC-42 [ka] ADC-42 was obtained by preparation using a general coupling method.

[0294] Example 90: ADC-43 [ka] ADC-43 was obtained by preparation using a general coupling method.

[0295] Example 91: ADC-44 [ka] ADC-44 was obtained by preparation using a general coupling method.

[0296] Example 92: ADC-45 [ka] ADC-45 was obtained by preparation using a general coupling method.

[0297] Example 93: ADC-46 [ka] ADC-46 was obtained by preparation using a general coupling method.

[0298] Example 94: ADC-47 [ka] ADC-47 was obtained by preparation using a general coupling method.

[0299] Example 95: ADC-48 [ka] ADC-48 was obtained by preparation using a general coupling method.

[0300] Example 96: ADC-49 [ka] ADC-49 was obtained by preparation using a general coupling method.

[0301] Example 97: ADC-50 [ka] ADC-50 was obtained by preparation using a general coupling method.

[0302] Example 98: ADC-51 [ka] ADC-51 was obtained by preparation using a general coupling method.

[0303] Example 99: ADC-52 [ka] ADC-52 was obtained by preparation using a general coupling method.

[0304] Example 100: ADC-53 [ka] ADC-53 was obtained by preparation using a general coupling method.

[0305] Example 101: ADC-54 [ka] ADC-54 was obtained by preparation using a general coupling method.

[0306] Example 102: ADC-55 [ka] ADC-55 was obtained by preparation using a general coupling method.

[0307] Example 103: ADC-56 [ka] ADC-56 was obtained by preparation using a general coupling method.

[0308] Example 104: ADC-57 [ka] ADC-57 was obtained by preparation using a general coupling method.

[0309] Example 105: ADC-58 [ka] ADC-58 was obtained by preparation using a general coupling method.

[0310] Example 106: ADC-59 [ka] ADC-59 was obtained by preparation using a general coupling method.

[0311] Example 107: ADC-60 [ka] ADC-60 was obtained by preparation using a general coupling method.

[0312] Example 108: ADC-61 (control group) [ka] ADC-61 was obtained by preparation using a general coupling method.

[0313] Example 109: Plasma Stability 1) Operation A predetermined amount of ADC sample is taken and added to human plasma from which human IgG has been removed. Three tubes are prepared for each ADC. The tubes are incubated in a 37°C water bath. After 72 hours and 144 hours, the ADC samples are removed, 100 µl of ProteInA resIn (MabSelect SuRe™ LX Lot:#10221479 GE, washed with PBS) is added to each tube, and the mixture is adsorbed by shaking in a vertical mixer for 2 hours. After washing, the incubated ADCs are obtained. The ADC samples incubated for the predetermined time are detected by RP-HPLC. 2) Results

[0314] Table 1: Data on DAR values ​​and monomer ratios of the ligand-drug conjugate (ADC) of the present invention [Table 1]

[0315] Table 2: Plasma stability data of the ligand-drug conjugate (ADC) of the present invention [Table 2]

[0316] 3) Conclusion As shown in Table 1, the camptothecin ADCs disclosed in this invention, which have a highly stable hydrophilic binding unit, exhibit excellent properties such as a high DAR value (>7.5) and a high monomer content (>97%), and have a significantly higher monomer content than the control ADC-61. As shown in Table 2, after incubation of the ADC of the present invention in plasma for 7 days, the DAR value remained at a relatively high level compared to the control ADC-61, indicating that the ADC of the present invention has excellent stability in plasma.

[0317] Example 110: In vitro activity test

[0318] 1) Experimental materials Cells: Obtained from the Chinese Academy of Sciences Cell Bank Tumor cell culture medium: GIBCO FBS:BIOWEST;

[0319] 2) Preparation of culture medium Growth medium (containing 10% FBS and penicillin-streptomycin (100 U / mL)) Detection medium (containing 1% FBS and penicillin-streptomycin (100 U / mL))

[0320] 3) Operation The UV light in the safety cabinet was turned on 30 minutes prior to the procedure, and the cabinet was ventilated for 3 minutes. Growth medium, detection medium, D-PBS, and trypsin were preheated in a 37°C constant temperature water bath, the surface was disinfected with alcohol, and the cabinet was placed in the biological safety cabinet. Cells with approximately 80% confluence (logarithmic growth phase) were selected and placed in the biological safety cabinet. Old medium was aspirated and removed, the cells were rinsed with D-PBS, aspirated and removed, digested with trypsin for 2-3 minutes, growth medium was added to stop the trypsin reaction, and the cells were centrifuged at 500×g for 5mIn. The supernatant was aspirated and removed, mixed with 4mL of detection medium, and then 100μL was taken and counted (50μL of cell solution was taken, 50μL of 0.4% Trypan Blue Stain was added and mixed uniformly, and then counted). Cells were plated in a 96-well plate at 80 μL / well with the specified number of cells. 80 μL of detection medium was added to wells E11, F11, and G11, and 200 μL of DPBS was added to the edge wells. After the coated cells had completely adhered to the wall (usually at least 4 hours), the test samples were prepared and diluted. Specifically, a 1.0 mL, 2.5 μM (5 × Top Dose) test sample was prepared in detection medium and placed in the first row (200 μL / well) of a V-type 96-well plate. 180 μL of detection medium was added to each of the following rows 2 through 8. 30 μL was taken from row 1 and placed in row 2. After mixing 10 times up and down with a pipette, the pipette tip was discarded, and the remaining detection concentration points were processed sequentially to perform a 7-fold gradient dilution. The gradient concentration test samples were added to the cells at 20 μL / well. Only 20 μL of detection medium was added to the 11th row, and three replication wells were set up for each concentration. The 96-well plate was placed in a cell incubator at 5% CO2 and 37°C and cultured for 5 days.

[0321] 4) Detection Four days later, the test samples were removed, thawed in the dark at room temperature, and then vortexed to mix thoroughly. Subsequently, 20 μL of CellTIter One Solution Reagen MTS reagent was added along the side wall of each well in a biological safety cabinet, and the MTS solution was uniformly mixed by gently tapping. The mixture was then placed in a cell culture incubator at 5% CO2 and 37°C, and left to stand in the dark for 2 hours. After the reaction was complete, the 96-well plate was removed, the absorbance at OD490 nm was measured using a microplate reader, and the data was recorded, organized, and stored.

[0322] 5) Results Table 3: IC50 values ​​of antibody-drug conjugates and toxins inhibiting in vitro proliferation of N87 tumor cells [Table 3]

[0323] Table 4: IC50 values ​​of antibody-drug conjugates and toxins inhibiting in vitro proliferation of SK-BR-3 tumor cells [Table 4]

[0324] 6) Discussion As shown in Table 3, in the present invention, the ligand-drug conjugate targeting HER2 exhibits remarkable in vitro growth inhibitory activity against HER2-positive cell N87, and is clearly superior to the naked antibody (Trastuzumab), the control group ADC-61, and the toxin monotherapy. As shown in Table 4, the ADC and monotherapy agents of the present invention exhibit significant in vitro growth inhibitory activity against HER2-positive SK-BR-3 cells compared to the naked antibody (Trastuzumab) and the control group ADC.

[0325] Example 111: In vivo activity test

[0326] 1) Experimental materials Cells: Obtained from the Chinese Academy of Sciences Cell Bank Tumor cell culture medium: GIBCO Balb / c-nu nude mice: female, 5-7 weeks old (age of the mouse at the time of tumor cell inoculation), body weight 18.0-24.0g, 170 mice (110 mice + 60 supplement mice). Purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Test specimens and control specimens Test products: ADC-61, ADC-6 (provided by Chengdu DuoSpecial Antibody Drug Co., Ltd.). Histidine buffer (provided by Chengdu Duote Antibody Drug Co., Ltd.). 0.9% Sodium Chloride Injection: Ke-Lun Pharmaceutical Co., Ltd.

[0327] 2)Cell culture NCI-H1975 (human non-small cell lung cancer adenocarcinoma cells) were cultured in RPMI1640 medium. NCI-H1975 cells in the exponential growth phase were collected, resuspended in RPMI1640 medium at an appropriate concentration, and then used for subcutaneous tumor inoculation in mice. NCI-N87 (human gastric cancer cells) were cultured in RPMI1640 medium. NCI-N87 cells in the exponential growth phase were collected, resuspended in RPMI1640 medium at an appropriate concentration, and then used for subcutaneous tumor inoculation in mice.

[0328] 3) Construction of animal models and random group assignment 85 female nude mice with 5 x 10 7 NCI-H1975 cells were subcutaneously inoculated. The average tumor volume was approximately 170 mm². 3 After this stage, the mice were randomly divided according to tumor size. 55 tumor-bearing mice with appropriate tumor volume were selected and randomly divided into groups for administration (tail vein injection, dose 0.1 ml / 10 g). The day of group division was defined as day 0. 85 female nude mice with 5 x 10 7 NCI-N87 cells were subcutaneously inoculated. The average tumor volume was approximately 170 mm². 3 After this stage, the mice were randomly divided according to tumor size. 55 tumor-bearing mice with appropriate tumor volume were selected and randomly divided into groups for administration (tail vein injection, dose 0.1 ml / 10 g). The day of group division was defined as day 0.

[0329] 4) Preparation of test samples and control samples Table 5: Preparation of test and control solutions for studying antitumor effects in nude mouse subcutaneous tumor models using NCI-H1975 (human non-small cell lung cancer adenocarcinoma cells) and NCI-N87 (human gastric cancer cells). [Table 5] Note: Mix thoroughly before use to ensure the formulation is uniform.

[0330] 5) Experimental observation and data collection In this experiment, animal experiments were conducted in accordance with the requirements of the standard operating procedures for in vivo screening of antitumor drugs. Routine monitoring after tumor inoculation included the effects of treatment on tumor growth (tumors measured twice weekly) and the normal behavior of the animals, specifically, the activity, feeding, weight changes (weight measured twice weekly), eyes, coat, and other abnormal conditions of the experimental animals. 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 (Here, a represents the major axis and b represents the minor axis). During the experiment, manually recorded data such as measurements of the major and minor axes of the tumor and measurements of the animal's body weight were used.

[0331] 6) Standards for evaluating treatment effectiveness The relative tumor growth rate (T / C%) is the percentage of tumor volume or tumor weight at a specific time point compared to the treatment group and the control group, and is calculated as follows: T / C% = TRTV / CRTV × 100% (TRTV: mean RTV of the treatment group; CRTV: mean RTV of the solvent control group; RTV = Vt / V0, where V0 is the tumor volume of the animal at the time of group division and Vt is the tumor volume of the animal after treatment). Alternatively, T / C% = TTW / CTW × 100% (TTW: mean tumor weight at the end of the experiment in the treatment group; CTW: mean tumor weight at the end of the experiment in the solvent control group). The formula for calculating the relative tumor inhibition rate (TGI) (%) is as follows: TGI% = (1 - T / C) × 100%. [T and C are the relative tumor volume (RTV) or tumor weight (TW) at a specific time point for the treatment group and the control group, respectively].

[0332] 7) Results Table 6: In vivo effects of administered antibody-drug conjugates against transplanted tumors (NCI-H1975) [Table 6]

[0333] Table 7: In vivo effects of administered antibody-drug conjugates against NCI-N87 transplanted tumors [Table 7]

[0334] Table 8: Effect of administered antibody-drug conjugate (11.25 mg / kg) on ​​body weight in NCI-H1975 transplanted tumor mice [Table 8] Note: *Two mice died in the identification group.

[0335] 8) Discussion As shown in Table 6, the ADC-6 of the present invention exhibits significantly superior in vivo efficacy against tumor-bearing mice NCI-H1975 in the low-dose control group (3.75 mg / kg) compared to the control group ADC-61 and the naked antibody. When the dose is increased to 11.25 mg / kg, the therapeutic effect of the ADC-6 of the present invention is further improved and significantly superior to that of the control ADC-61. As shown in Table 7, at the same dose (3.75 mg / kg), the ADC-6 of the present invention showed significantly higher in vivo efficacy against tumor-bearing mice NCI-N87 than the control group ADC-61, and its in vivo efficacy was even more pronounced compared to the high-dose naked antibody (11.25 mg / kg). As shown in Table 8, the effect of the present invention on body weight in NCI-H1975 tumor-bearing mice at a high dose of 11.25 mg / kg was significantly smaller than that of ADC-61. Even at such a high dose, there were no mouse deaths as observed in the control group, indicating that the ADC drug described in the present invention has a significant safety advantage.

Claims

1. A ligand-drug conjugate having a highly stable hydrophilic binding unit or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate is represented by Formula I below: 【Chemistry 1】 wherein Ab is a ligand unit selected from an antibody, an antibody fragment, a target protein, or an Fc-fusion protein; M is a binding unit that binds to Ab, Ac is a hydrophilic structural unit, D is any camptothecin class drug; the asymmetric carbon atoms at positions 1 and 4 have the R or S absolute configuration; n is an integer selected from 1 to 20.

2. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, characterized in that the linking unit M has a succinimide structure represented by the following formula a, or an open-ring succinimide structure represented by formula b1 or formula b2: 【Chemistry 2】 (In Formula a, Formula b1, or Formula b2, the wavy line on the left side represents a bond to the binding site of Ab, and the wavy line on the right side represents a bond to the binding site of the tertiary carbon atom at position 1 in Formula I.)

3. The Ac has a structure represented by the following formula c: 【Transformation 3】 (In the formula, X represents one or more hydrophilic structures selected from the group consisting of carboxyl, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid, and polyethylene glycol (PEG), but not limited thereto; Y is an optional scaffold connecting the amino group and X.

2. The ligand-drug conjugate or pharmaceutically acceptable salt thereof of claim 1, wherein Ac is attached to the methylene carbon at position 2 in structural formula I via an amino group.

4. The Ac may be selected from the group consisting of glycine, (D / L) alanine, (D / L) leucine, (D / L) isoleucine, (D / L) valine, (D / L) phenylalanine, (D / L) proline, (D / L) tryptophan, (D / L) serine, (D / L) tyrosine, (D / L) cysteine, (D / L) cystine, (D / L) arginine, (D / L) histidine, (D / L) methionine, (D / L) asparagine, (D / L) glutamine, (D / L) threonine, (D / L) aspartic acid, (D / L) glutamic acid, natural or unnatural amino acid derivatives, and amino acids having the following structure: 【Chemistry 4】 4. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 3, characterized in that it is selected from the group consisting of, but not limited to:

5. The camptothecin-class drug has a structure represented by the following formula d: 【Transformation 5】 In the formula, R １ is selected from hydrogen, deuterium, halogen, alkyl, deuterated alkyl, halogenated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl, and heteroaryl; or R １ and R １ The carbon atom bonded to C ３－６ cycloalkyl, cycloalkylalkyl or heterocyclyl, R １ has the R or S absolute configuration; m is 0 or 1; R in the drug molecule １ 2. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, characterized in that the hydroxyl group of the carbon atom bonded to is involved in the formation of the oxygen atom at position 3 in formula I.

6. The camptothecin-class drug is 【Transformation 6】 【change】 6. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 5, characterized in that it is selected from, but not limited to:

7. A linker-drug compound or a pharmaceutically acceptable salt thereof for binding with a ligand Ab to form a ligand-drug conjugate of formula I according to claim 1, A linker-drug compound or a pharmaceutically acceptable salt thereof, characterized in that it has a structure represented by Formula II below: 【Transformation 7】 (In the formula, R １ is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, a deuterated alkyl, a halogenated alkyl, a cycloalkyl, a cycloalkylalkyl, an alkoxyalkyl, a heterocyclyl, an aryl, a substituted aryl, or a heteroaryl; R １ and R １ The carbon atom bonded to C ３－６ cycloalkyl, cycloalkylalkyl or heterocyclyl, the asymmetric carbon atom at position 1 has the R or S absolute configuration; Ac is a hydrophilic structural unit, m is 0 or 1.

8. The linker-drug compound or a pharmaceutically acceptable salt thereof according to claim 7, wherein Ac is selected from, but not limited to, glycine, phosphate, (D / L) glutamic acid, and polyethylene glycol hydrophilic structures.

9. The linker-drug compound comprises: 【Transformation 8】 【change】 【change】 【change】 【change】 【change】 selected, but not limited to, from 9. The linker-drug compound or pharmaceutically acceptable salt thereof according to claim 7 or 8, wherein the asymmetric carbon at position 1 has an R or S absolute configuration.

10. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, characterized in that it has a structure represented by the following Formula III, Formula IV-1 or Formula IV-2: 【Chemistry 9】 (In Formula III, Formula IV-1 or Formula IV-2, Ab is a ligand unit; Ac is a hydrophilic structural unit, the asymmetric carbon at position 1 has an R or S absolute configuration; R １ , m and n are represented by Formula II.

11. the ligand unit Ab is selected from an antibody, an antibody fragment, or a protein; The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 10, wherein the antibody is selected from a mouse antibody, a rabbit antibody, a phage-displayed antibody, a yeast display-derived antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody, and a multispecific antibody.

12. The antibodies include anti-EGFRvIII antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, and anti-ENPP3 antibody. Antibodies, anti-TDGF1 antibodies, anti-ETBR antibodies, anti-MSLN antibodies, anti-TIM-1 antibodies, anti-LRRC15 antibodies, anti-LIV-1 antibodies, anti-CanAg / AFP antibodies, anti-cladIn 18.2 antibody, anti-MesothelIn antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPI2B / SLC34A2 antibody, anti-G PNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-NectIn 4 antibodies, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-IntegrIn Av antibody, anti-CAIX antibody, anti-P-cadherIn antibody, anti-GD3 antibody, anti-CadherIn 11. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 10, wherein the monoclonal antibody is selected from the group consisting of anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, and anti-CD123 antibody, but not limited to.

13. The antibody or antigen-binding fragment thereof MDMRVPAQLLGLLLWLRGARC DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 1) and a light chain comprising MDMRVPAQLLGLLLWLRGARC EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2) and a heavy chain comprising The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 10, characterized in that it is trastuzumab having the formula:

14. The ligand-drug conjugate or its pharmaceutically acceptable salt according to claim 10, characterized in that the ligand-drug conjugate or its pharmaceutically acceptable salt is selected from the following structures or their succinimide ring-open structures: 【Chemistry 10】 【change】 【change】 【change】 【change】 (wherein n is an integer selected from 1 to 10).

15. A method for preparing a linker-drug compound or a pharmaceutically acceptable salt thereof according to any one of claims 7 to 9, comprising: The method is characterized in that it comprises the steps shown in the following formula: 【Chemistry 11】 (Wherein, a compound of general formula L and a compound of formula d ０ and exatecan or a salt thereof in the presence of a condensing agent under any alkaline condition to obtain a compound of formula IV, which is then converted into a structure represented by formula II; In the formula, the asymmetric carbon atom at the 1-position and R １ has the R or S absolute configuration; R ２ is any structure that can be converted to Ac, Ac, R １ , m is as defined in Formula II.

16. 16. The method of claim 15, wherein the deprotecting agent used in converting formula IV to formula II is zinc bromide and the solvent is nitromethane.

17. 7. A method for preparing the ligand-drug conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 6, comprising: The method is characterized in that it comprises the steps shown in the following formula: 【Chemistry 12】 (wherein the ligand unit Ab is modified and then coupled with a compound of formula II to obtain a ligand-drug conjugate of formula III; Ab is selected from an antibody, an antibody fragment, and a protein; Ac is a hydrophilic structural unit, The asymmetric carbon atom at position 1 and R １ has the R or S absolute configuration; R １ , m and n are as defined in Formula II.

18. 20. A pharmaceutical composition comprising a therapeutically effective amount of a ligand-drug conjugate of any one of claims 1 to 17, a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

19. 20. Use of a pharmaceutical composition comprising the ligand-drug conjugate of any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of a tumor, an autoimmune disease or an infectious disease.

20. 20. The use according to claim 19, characterized in that it is used for the preparation of a medicament for treating solid tumors or hematological tumors, including breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma, leukemia.

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