Camptothecin drug with highly stable hydrophilic linker unit and conjugate thereof

Abstract

A camptothecin-based drug with a highly stable hydrophilic linker and its conjugate, or a pharmaceutically acceptable salt thereof, including its preparation method and its use in preventing or treating cancer. The conjugate can specifically bind to receptors highly expressed in tumor cells. It has good water solubility, stability, and homogeneity, and can be used to prevent or treat diseases such as tumors.

Description

Technical Field

[0001] The present invention relates to a camptothecin antibody-drug conjugate with a highly stable hydrophilic connecting structural unit. Background Art

[0002] Antibody-drug conjugates (ADCs), as a new type of targeted drug, are generally composed of three parts: an antibody or antibody-like ligand, a small molecule drug, and a linker that couples the ligand and drug. Antibody-drug conjugates utilize the antibody's specific recognition of the antigen to transport the drug molecule to the vicinity of the target cell and effectively release the drug molecule to achieve the therapeutic purpose. In August 2011, the U.S. Food and Drug Administration (FDA) approved Adcetris, a new ADC developed by Seattle Genetics for the treatment of Hodgkin's lymphoma and relapsed anaplastic large cell lymphoma (ALCL). TM The safety and effectiveness of these drugs have been demonstrated in clinical applications.

[0003] Camptothecins, small molecule anti-tumor compounds, are known to exhibit anti-tumor effects by inhibiting DNA topoisomerase I, including irinotecan, exotecan, and SN38. Many camptothecins have been widely used clinically, primarily for bone cancer, prostate cancer, breast cancer, and pancreatic cancer. Unlike irinotecan, currently used clinically, exotecan does not require enzyme activation. Furthermore, compared to SN-38, the active ingredient in irinotecan, and topotecan, also used clinically, exotecan exhibits stronger topoisomerase I inhibitory activity and exhibits enhanced cytotoxicity against various cancer cells in vitro. In particular, it is effective against cancer cells that exhibit resistance to SN-38, such as those resistant to SN-38, as demonstrated by P-glycoprotein expression. Exotecan has not yet been successfully marketed as a standalone chemotherapy agent, presumably due to its high cellular activity, resulting in a narrow therapeutic window.

[0004] The advantages of antibody-drug conjugates (ADCs) lie in their increased water solubility, enhanced targeting, and specific antibody-antigen binding, which delivers the drug to target cells. By releasing the drug near target cells, it effectively kills tumor cells and reduces toxic side effects. Camptothecins hold considerable promise for application in ADCs. Trastuzumab deruxtecan (trade name Enhertu), an antibody-drug conjugate containing deruxtecan as a toxin, was approved for marketing by the US FDA on December 20, 2019. As the first marketed camptothecin ADC, it demonstrates the druggability and application prospects of this class of drugs in the ADC field.

[0005] The ADC drug structure includes three key parts: antibody, linker, and toxin. Defects in any part may affect the overall efficacy of the ADC. In this field, the structural design defects of Enhertu are obvious: camptothecin is a class of highly lipid-soluble and poorly soluble drugs. The linker-toxin used by Enhertu is designed to be connected to the antibody through an Mc linker, connecting an enzymatically cleavable tetrapeptide fragment, with an aminomethoxy self-eliminating spacer unit, and using interchain cysteine ​​residues to achieve a drug-antibody ratio (DAR) of 8 using non-site-specific coupling technology (reference patent CN104755494). The design of this linker, at a high DAR value, will lead to reduced stability of camptothecin ADC drugs and reduced monomer rate, further reducing the in vivo efficacy and safety of ADC.

[0006] The technical problem that this patent needs to solve is to explore and discover better anti-tumor camptothecin ADC drugs so that they have higher safety and effectiveness and better meet clinical needs. Summary of the Invention

[0007] Based on a comprehensive understanding of ADC drugs, the inventors unexpectedly discovered a series of camptothecin derivative antibody-drug conjugates (ADCs) with highly stable hydrophilic peptide linkers. Experiments revealed that the camptothecin derivative ADCs equipped with these peptide linkers exhibited high stability in vitro and in vivo, high monomer yields, and significantly higher pharmacodynamic activity than control ADCs. Furthermore, through innovative synthetic route design, the inventors developed a novel deprotection reagent and solvent strategy that efficiently yielded these complex linker-toxin molecules.

[0008] One aspect of the present invention provides a ligand-drug conjugate as shown in Formula I or a pharmaceutically acceptable salt thereof,

[0009]

[0010] in:

[0011] Ab is a ligand unit selected from an antibody, an antibody fragment or a protein;

[0012] M is a linker unit connected to Ab;

[0013] Ac is a hydrophilic structural unit;

[0014] D is an optional camptothecin drug;

[0015] The chiral carbon atoms at positions 1 and 4 have two chiral configurations: R absolute configuration or S absolute configuration;

[0016] n is selected from integers of 1-20.

[0017] Preferably, the linking unit M has a succinimide structure as shown in formula a or a ring-opened succinimide structure as shown in formula b1 or formula b2.

[0018]

[0019] In Formula a, Formula b1 or Formula b2, the wavy line on the left indicates connection to the Ab connection site, and the wavy line on the right indicates connection to the marked tertiary carbon atom connection site at position 1 in Formula I.

[0020] Further preferably, the Ac has the structure shown in the following formula c:

[0021]

[0022] wherein X is selected from, but not limited to, the group consisting of one or more of a hydrophilic structure carboxyl group, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid, or polyethylene glycol (PEG);

[0023] Y is an optional scaffold connecting the amino group and X;

[0024] Ac is connected to the methylene carbon at position 2 indicated in structural formula I via the amino functional group.

[0025] Still further preferably, the Ac is selected from, but not limited to, 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 or the following structure:

[0026]

[0027] The wavy line on the left is connected to carbon atom 2.

[0028] Another aspect of the present invention provides a camptothecin drug having a structure as shown in the following formula d:

[0029]

[0030] wherein R1 is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a haloalkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, a substituted aryl group, or a heteroaryl group;

[0031] Alternatively, R1 and the carbon atom to which it is attached constitute C 3-6cycloalkyl, cycloalkylalkyl, or heterocyclyl;

[0032] The chiral carbon atom connected to R1 has two chirality: R absolute configuration or S absolute configuration;

[0033] m is selected from 0 or 1;

[0034] The hydroxyl group of the carbon atom connected to R1 in the drug d molecule participates in the connection to form the oxygen atom at position 3 in formula I.

[0035] Preferably, the camptothecin drugs provided by the present invention are non-limitingly selected from the following compounds,

[0036]

[0037] Another aspect of the present invention provides a linker-drug compound having a structure shown in Formula II or a pharmaceutically acceptable salt thereof, for coupling with a ligand Ab to form a ligand-drug conjugate shown in Formula I,

[0038]

[0039] wherein R1 is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a haloalkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, a substituted aryl group, or a heteroaryl group;

[0040] Alternatively, R1 and the carbon atom to which it is attached constitute C 3-6 cycloalkyl, cycloalkylalkyl, or heterocyclyl;

[0041] The chiral carbon atom at position 1 has two chirality: R absolute configuration or S absolute configuration;

[0042] Ac is a hydrophilic structural unit;

[0043] m is selected from 0 or 1.

[0044] In one aspect of the present invention, the Ac is preferably glycine, phosphoric acid, polyethylene glycol or (D / L) glutamic acid.

[0045] More preferably, the linker-drug compound provided by the present invention or a pharmaceutically acceptable salt thereof is selected from the following structures without limitation,

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] The chiral carbon at position 1 has two configurations: R absolute chirality or S absolute chirality.

[0053] Another aspect of the present invention provides a compound having a structure shown in Formula III, Formula IV-1 or Formula IV-2,

[0054]

[0055]

[0056] Where Ab is the ligand unit;

[0057] Ac is a hydrophilic structural unit;

[0058] The chiral carbon at position 1 has two configurations: R absolute chirality or S absolute chirality;

[0059] R1, m and n are as described in Formula II.

[0060] Preferably, the ligand-drug conjugate provided by the present invention or a pharmaceutically acceptable salt thereof is characterized in that: the ligand unit Ab is selected from an antibody, an antibody fragment or a protein, wherein the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody and a multispecific antibody.

[0061] Further preferably, the antibody is a monoclonal antibody, selected from, but not limited to, 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-cladin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibody, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-Integrin Av antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, or anti-CD123 antibody.

[0062] More preferably, the antibody or antigen-binding fragment thereof is trastuzumab, comprising the following sequence:

[0063] Light chain

[0064] MDMRVPAQLLGLLLLWLRGARC

[0065] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK

[0066] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*;

[0067] SEQ ID NO:1

[0068] Heavy chain:

[0069] MDMRVPAQLLGLLLLWLRGARC

[0070] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

[0071] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

[0072] SEQ ID NO:2

[0073] Preferably, the ligand-drug conjugate or a pharmaceutically acceptable salt thereof provided by the present invention is characterized in that: the ligand-drug conjugate or a pharmaceutically acceptable salt thereof is non-restrictively selected from the following structures or their succinimide ring-opening structures,

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] wherein n is selected from an integer of 1-10.

[0082] Another aspect of the present invention provides a method for preparing a linker-drug compound or a pharmaceutically acceptable salt thereof, the method comprising the following steps:

[0083]

[0084] The compound of formula L reacts with ixetec or its salt of formula d0 in the presence of a condensing agent under optional alkaline conditions to obtain a compound of formula IV, which is further converted into the structure shown in formula II;

[0085] The chiral carbon atoms at position 1 and connected to R1 have two chiralities: R absolute configuration or S absolute configuration;

[0086] R2 is an optional structure that can be converted into Ac;

[0087] Ac, R1, and m are as defined in formula II.

[0088] Another aspect of the present invention provides a method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, the method comprising the following steps:

[0089]

[0090] After modifying the ligand unit Ab, a coupling reaction is performed with Formula II to obtain a ligand-drug conjugate of Formula III;

[0091] wherein Ab is selected from an antibody, an antibody fragment or a protein;

[0092] Ac is selected from hydrophilic structural units;

[0093] The chiral carbon atom at position 1 and connected to R1 has two chirality: R absolute configuration or S absolute configuration;

[0094] R1, m and n are as described in Formula II.

[0095] Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0096] The pharmaceutically acceptable salts of the present invention include sodium salts, potassium salts, calcium salts or magnesium salts formed with the carboxyl functional group in the structural formula, and acetates, trifluoroacetates, citrates, oxalates, tartrates, malates, nitrates, chlorides, bromides, iodides, sulfates, bisulfates, phosphates, lactates, oleates, ascorbic acid salts, salicylates, formates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates or p-toluenesulfonates formed with the nitrogen-containing functional group in the structure.

[0097] Another aspect of the present invention provides a pharmaceutical composition of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, and its use in preparing a drug for treating tumors, autoimmune diseases or infectious diseases, wherein the antibody of the ligand-drug conjugate specifically binds to the target cells of the tumor, autoimmune disease or infectious disease.

[0098] Another aspect of the present invention provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof for use in preparing a medicament for treating solid tumors or hematological tumors such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1A .The present invention discloses the SEC-HPLC detection results of Trastuzumab monomer rate.

[0100] Figure 1B .The present invention discloses the SEC-HPLC detection results of ADC-2 monomer rate.

[0101] Figure 1C .The present invention discloses the SEC-HPLC detection results of ADC-6 monomer rate.

[0102] Figure 1D .The present invention discloses the SEC-HPLC detection results of ADC-10 monomer rate.

[0103] Figure 1E .The present invention discloses the SEC-HPLC detection results of ADC-12 monomer rate.

[0104] Figure 1F .The present invention discloses the SEC-HPLC detection results of ADC-61 monomer rate in the control group.

[0105] Figure 2A The present invention discloses the RP-HPLC test results of the DAR (drug-antibody conjugation ratio) value of ADC-02.

[0106] Figure 2B The present invention discloses the RP-HPLC test results of the DAR (drug-antibody conjugation ratio) value of ADC-06.

[0107] Figure 2C The present invention discloses the RP-HPLC test results of the DAR (drug-antibody conjugation ratio) value of ADC-10.

[0108] Figure 2D The present invention discloses the RP-HPLC test results of the DAR (drug-antibody conjugation ratio) value of ADC-12.

[0109] Figure 2E .The present invention discloses the RP-HPLC test results of the DAR (drug-antibody coupling ratio) value of the control group ADC-61.

[0110] Figure 3A .The present invention discloses the in vitro efficacy of ADC, single drug and naked antibody in inhibiting the proliferation of N87 (human gastric cancer cells).

[0111] Figure 3B .The present invention discloses the in vitro efficacy of ADC and naked antibody in inhibiting the proliferation of N87 (human gastric cancer cells).

[0112] Figure 3C .The present invention discloses the in vitro efficacy of a single drug in inhibiting the proliferation of N87 (human gastric cancer cells).

[0113] Figure 4A The present invention discloses the in vitro efficacy of ADC, single drug and naked antibody in inhibiting the proliferation of SK-BR-3 (human breast adenocarcinoma cells).

[0114] Figure 4B The present invention discloses the in vitro efficacy of ADC and naked antibody in inhibiting the proliferation of SK-BR-3 (human breast cancer cells).

[0115] Figure 4C The present invention discloses the in vitro efficacy of a single drug in inhibiting the proliferation of SK-BR-3 (human breast adenocarcinoma cells). DETAILED DESCRIPTION

[0116] Abbreviations and definitions

[0117] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, the preferred methods and materials are described herein. In describing and claiming the present invention, the following terms are used in accordance with the following definitions.

[0118] When a trade name is used herein, applicants intend to include formulations of the trade name product, generic versions of the trade name product, and the active drug portion of the trade name product.

[0119] Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings. When a trade name is used herein, unless the context indicates otherwise, the trade name includes the product formulation, generic drug and active pharmaceutical ingredient of the trade name product.

[0120] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0121] The term "ligand" refers to a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell. The function of a ligand is to present a drug to the target cell population bound to the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules capable of binding to cells. In embodiments of the present invention, the ligand is represented by Ab, and the ligand can form a bond with a linker via a heteroatom on the ligand. The ligand is preferably an antibody or an antigen-binding fragment thereof, selected from a chimeric antibody, a humanized antibody, a fully human antibody, or a murine antibody; preferably, a monoclonal antibody.

[0122] The Ligand unit is a targeting agent that specifically binds to a target moiety. The ligand can specifically bind to a cellular component or to other target molecules of interest. The target moiety or target is typically on the cell surface. In some aspects, the role of the Ligand unit is to deliver the Drug unit to a specific target cell population with which the Ligand unit interacts. Ligands include, but are not limited to, proteins, polypeptides, and peptides, as well as non-proteins such as sugars. Suitable Ligand units include, for example, antibodies, such as full-length (intact) antibodies and antigen-binding fragments thereof. In embodiments where the Ligand unit is a non-antibody targeting agent, it can be a peptide or polypeptide, or a non-protein molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth factors, colony-stimulating factors, vitamins, nutrient transport molecules, or any other cell-binding molecule or substance. In some embodiments, a linker is covalently linked to a sulfur atom of the ligand. In some aspects, the sulfur atom is a sulfur atom of a cysteine ​​residue, which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine ​​residue that has been introduced into the Ligand unit, which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine ​​residue that has been introduced into the Ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In other aspects, the sulfur atom to which the linker is bound is selected from a cysteine ​​residue that forms an interchain disulfide bond of the antibody or an additional cysteine ​​residue that has been introduced into the Ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In some embodiments, the numbering system is based on the EU index as in Kabat {[Kabat EA et al., (1991)] Sequences of proteins of Immunological Interest, 5th ed., NIH publication 91-3242}.

[0123] As used herein, "antibody" or "antibody unit" includes any part of the antibody structure within the scope thereof. This unit can bind to, reactively associate with, or complex with a receptor, antigen or other receptor unit possessed by the target cell population. An antibody can be any protein or protein-like molecule that can bind to, complex with or react with a portion of the cell population to be treated or biomodified. The antibodies that constitute the antibody drug conjugates of the present invention retain their original antigen binding ability in the wild state. Therefore, the antibodies of the present invention can specifically bind to antigens. The antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulatory factors, cell proliferation regulatory factors, molecules related to tissue growth and differentiation (such as known or predicted functional ones), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as known or predicted functional ones). Tumor-associated factors can be cluster differentiation factors (such as CD proteins).

[0124] Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the industry and can be prepared using methods and information for antibody preparation that are well known in the industry. In order to develop effective cellular targets that can be used for cancer diagnosis and treatment, researchers strive to find transmembrane or other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells, while being rarely or not expressed on the surface of one or more non-cancerous cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells than on the surface of non-cancerous cells. Identifying such tumor-associated factors can greatly improve the specific targeting properties of antibody-based cancer treatment. For convenience, information related to antigens well known in the industry is marked below, including name, other names, and gene bank accession number. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases, such as Genbank. The antibody targets the corresponding tumor-associated antigen, including all amino acid sequence variants and isotypes, and has at least 70%, 80%, 85%, 90% or 95% homology with the sequence confirmed in the reference, or has biological properties and characteristics that are completely consistent with the tumor-associated antigen sequence in the cited reference.

[0125] The term "inhibit" or "inhibition of" refers to reducing by a detectable amount, or completely preventing.

[0126] The term "cancer" refers to the physiological condition or disease characterized by unregulated cell growth. A "tumor" includes cancer cells.

[0127] The term "autoimmune disease" is a disease or disorder that arises from a host directed against an individual's own tissues or proteins.

[0128] The term "drug" refers to cytotoxic drugs, which are chemical molecules that can strongly disrupt the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells in principle at sufficiently high concentrations, but due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. The term includes toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 He Lu 176 radioactive isotopes), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.

[0129] The term "camptothecin drugs" refers to cytotoxic camptothecin and its derivatives, selected from, but not limited to, 10-hydroxycamptothecin, SN38 (7-ethyl-10-hydroxycamptothecin), topotecan, exitecan, irinotecan or 9-nitro-10-hydroxycamptothecin and its derivatives or pharmaceutically acceptable salts.

[0130] The term "linker" or "linker fragment" or "linker unit" refers to a chemical structure fragment or bond that is connected to a ligand at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug.

[0131] Linkers, including extenders, spacers, and amino acid units, can be synthesized by methods known in the art, such as those described in US 2005-0238649 A1. The linker can be a "cleavable linker" that facilitates release of the drug in the cell. For example, an acid-labile linker (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al. Cancer Research 52:127-131, 1992); U.S. Patent No. 5,208,020.

[0132] According to the mechanism of drug release in cells, as used herein, "linkers" or "linkers of antibody drug conjugates" can be divided into two categories: non-cleavable linkers and cleavable linkers. For ligand-drug conjugates containing non-cleavable linkers, the drug release mechanism is as follows: after the conjugate binds to the antigen and is internalized by the cell, the antibody is enzymatically hydrolyzed in the lysosome, releasing the active molecule composed of the small molecule drug, the linker, and the antibody amino acid residues. The resulting change in the drug molecular structure does not weaken its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into neighboring cells. Therefore, this type of active drug cannot kill neighboring tumor cells that do not express the target antigen (antigen-negative cells) (bystander effect) (Ducry et al., 2010, Bioconjugate Chem. 21: 5-13).

[0133] The term "ligand-drug conjugate" refers to an antibody linked to a biologically active drug via a stable linker. In the present invention, the "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a stable linker.

[0134] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. boil. Chem. 1968, 243, 3558.

[0135] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0136] The term "substituted alkyl" refers to an alkyl group in which a hydrogen atom is replaced by a substituent group. Unless otherwise specified herein, the substituent group of the alkyl group may be a variety of groups selected from the group consisting of -halogen, -OR', -NR'R", -SR', -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR "C(O)2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R", -NR'S(O)2R", -CN and -NO2, the number of substituents is 0 to (2m'+1), where m' is the total number of carbon atoms in the group. R', R" and R'' independently refer to hydrogen, unsubstituted C 1-8 Alkyl, unsubstituted aryl, aryl substituted by 1-3 halogens, unsubstituted C 1-8 Alkyl, C 1-8 Alkoxy or C 1-8 Thioalkoxy, or unsubstituted aryl-C 1-4 Alkyl. When R' and R" are attached to the same nitrogen atom, they may form a 3-, 4-, 5-, 6-, or 7-membered ring together with the nitrogen atom. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

[0137] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.

[0138] The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of a parent alkane, and is a straight or branched group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-, 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH 2CH2CH2CH2CH2-) etc. Alkylene may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available attachment point. The substituent is preferably independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0139] The term "alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl), wherein the definition of alkyl or cycloalkyl is as described above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0140] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.

[0141] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m(wherein m is an integer from 0 to 2) heteroatoms, excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, the ring contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyls include spirocyclic, fused, and bridged heterocyclyls.

[0142] The term "cycloalkylalkyl" refers to an alkyl group substituted with one or more cycloalkyl groups, preferably with one cycloalkyl group, wherein alkyl is as defined above and wherein cycloalkyl is as defined above.

[0143] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0144] The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

[0145] The term "hydroxy" refers to an -OH group.

[0146] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0147] The term "amino" refers to -NH2. The term "nitro" refers to -NO2.

[0148] The term "amido" refers to -C(O)N(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0149] The term "carboxylate" refers to -C(O)O(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0150] The term "aryl" refers to a 6- to 14-membered, all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6-10 members, such as phenyl. The aryl group may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, including, but not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, deuterium atom, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, or heterocycloalkylthio.

[0151] The present invention also includes various deuterated forms of Formula I. Each available hydrogen atom attached to a carbon atom can be independently replaced with a deuterium atom. Those skilled in the art will be able to synthesize deuterated forms of Formula I by referring to relevant literature. When preparing deuterated forms of Formula I, commercially available deuterated starting materials can be used, or they can be synthesized using conventional techniques using deuterated reagents. Non-limiting examples of deuterated reagents include: deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated ethyl iodide, and deuterated methyl iodide.

[0152] The term "antibody" refers to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and order of the constant region of immunoglobulins' heavy chains vary, resulting in different antigenicity. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, corresponding to the μ, δ, γ, α, and ε heavy chains, respectively. Within the same class, Ig can be further divided into subclasses based on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on differences in the constant region. Each of the five Ig classes can have either kappa or lambda chains.The antibody of the present invention is preferably a specific antibody against a cell surface antigen on a target cell, and non-limiting examples thereof are the following antibodies: anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg / AFP antibody, anti-cladin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibodies, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-Integrin Av antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody or anti-CD123 antibody or more; preferably trastuzumab (Trastuzumab, trade name Herceptin), pertuzumab (Pertuzumab, also known as 2C4, trade name Perjeta), nimotuzumab (Nimotuzumab, trade name Taixinsheng), enoblituzumab, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96 and glematumamab.

[0153] The term "solvate" or "solvate compound" refers to a pharmaceutically acceptable solvate formed between the ligand-drug conjugate of the present invention and one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0154] The term "drug loading" refers to the average amount of cytotoxic drug attached to each antibody in Formula I. It can also be expressed as the ratio of the amount of drug to the amount of antibody. The drug loading can range from 0-12, preferably 1-10, cytotoxic drugs (D) attached to each antibody (Ab). In embodiments of the present invention, the drug loading is expressed as n, which can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The average amount of drug per ADC molecule after the conjugation reaction can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, and HPLC characterization.

[0155] In one embodiment of the present invention, the cytotoxic drug is coupled to the opened interchain cysteine ​​thiol-SH group of the antibody and / or the site-directed mutated cysteine ​​thiol-SH group via a linker. Generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than or equal to the theoretical maximum value.

[0156] The loading capacity of the ligand cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:

[0157] (1) Control the molar ratio of the linker and the monoclonal antibody,

[0158] (2) Control reaction time and temperature,

[0159] (3) Select different reaction reagents.

[0160] The preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

[0161] The term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" refers to a salt of the ligand-drug conjugate of the present invention, or a salt of the compound described in the present invention. Such salts are safe and effective when used in mammals and have the desired biological activity. The ligand-drug conjugate compound of the present invention contains at least one carboxyl group and can therefore form salts with bases. Non-limiting examples of pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts, or magnesium salts.

[0162] The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of the antibody-drug conjugate of the present invention, or a salt of the compound described herein. Such salts are safe and effective when used in mammals and have the desired biological activity. The ligand-drug conjugate compound of the present invention contains at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include 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.

[0163] "Acidic amino acids" refer to amino acids with an isoelectric point of less than 7. Acidic amino acids often contain one or more acidic groups, such as carboxyl groups, within their molecules. These groups can effectively dissociate into negative ions within their structure, increasing their hydrophilicity. Acidic amino acids can be either natural or unnatural.

[0164] "Natural amino acids" refer to amino acids synthesized by organisms. Natural amino acids are generally L-type, but there are a few exceptions, such as glycine, including those that are naturally occurring and those that are synthesized by organisms.

[0165] "Unnatural amino acid" refers to an amino acid obtained by synthetic means.

[0166] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The test methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0167] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those commonly understood by one skilled in the art.

[0168] In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0169] Example 1

[0170] Synthesis of compound M1:

[0171]

[0172] To a 5000 mL single-necked flask, add N-Fmoc-glycine-glycine (100 g, 282 mmol, 1.0 eq), lead tetraacetate (175 g, 553 mmol, 1.4 eq), 2000 mL of dry tetrahydrofuran, and 670 mL of toluene. Stir thoroughly, protect with nitrogen, and heat to 85°C for 2.5 h. Monitor by TLC. After the reaction is complete, cool to room temperature, filter, and concentrate the filtrate under reduced pressure. The residue is purified by column chromatography to obtain compound M1 (87 g); LC-MS: [M+NH4] + =386.0.

[0173] Example 2

[0174] Synthesis of compound M3:

[0175]

[0176] Compound SM-2 (synthesized according to the method disclosed in patent CN108452321A) (40 g, 96 mmol, 1.0 eq), triethylamine (26.7 mL, 2.0 eq), and toluene (400 mL) were added to a 1000 mL single-necked flask and refluxed at 120°C for 2 h. TLC monitoring indicated that the reaction was essentially complete, and the temperature was lowered to 50°C and the solvent was removed under reduced pressure. The mixture was dissolved with 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. The layers were separated. The aqueous layer was extracted once more with ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated to obtain a pale yellow oily crude product, which was purified by column chromatography (DCM:MeOH=40:1) to obtain compound M2 (26.6 g); LC-MS: [M+H] + =399.3.

[0177] Compound M2 (26.5 g, 60.5 mmol, 1.0 eq), pentafluorophenol (12.2 g, 66.5 mmol, 1.1 eq), DCC (13.7 g, 66.5 mmol, 1.1 eq), and THF (300 mL) were added to a 1000 mL single-necked flask and reacted at room temperature for 30 min (monitored by TLC). The insoluble material was then filtered off. The reaction solution was directly purified by preparative purification. The preparative solution was concentrated in a water bath at 35°C under reduced pressure to remove acetonitrile and lyophilized to obtain compound M3 (31.5 g) in a 64% yield. LC-MS: [M+H] + =565.1.

[0178] Example 3

[0179] Synthesis of compound ent-M3:

[0180]

[0181] Referring to the synthetic route of Example 2, compound ent-M3 (27.8 g) was obtained; LC-MS: [M+H] + =565.2.

[0182] Example 4

[0183] Synthesis of compound 1:

[0184]

[0185] Step 1: Compound 1a

[0186] In a 250 mL single-necked flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added. The mixture was stirred and cooled to 0°C. Benzyl glycolate (5.4 g, 32.6 mmol) was added dropwise. The temperature was naturally raised to room temperature for reaction (approximately 2-4 h) and monitored by TLC. After the reaction, saturated NaHCO₃ solution was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-1:1) to afford 1a (4 g) in a 52% yield; LC-MS: [M+H] + =475.18.

[0187] Step 2: Compound 1b

[0188] In a 25 mL single-necked bottle, add 1a (2 g, 4.2 mmol) and 10 mL of DMF, stir at 0°C, add DBU (766 mg, 5.04 mmol), react for 1 h, monitor the Fmoc deprotection by TLC, and set aside.

[0189] In a separate 25 mL single-necked flask, M4 (prepared by the method disclosed in patent CN111051330 A) (1.73 g, 4.2 mmol), PyBOP (2.61 g, 5.04 mmol), HOBt (680 mg, 5.04 mmol), and 10 mL of DMF were added. DIPEA (830 μL, 5.04 mmol) was added under ice-water bath and stirring was continued for 30 min. The above reaction solution was added to the reaction flask and the mixture was allowed to warm to room temperature for reaction. After completion of the reaction, the reaction solution 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) in a 63% yield; LCMS: [M+H] + =648.26.

[0190] Step 3: Compound 1c

[0191] 1b (900 mg, 1.39 mmol) was added to a 25 mL single-necked bottle and dissolved in 15 mL DMF. 900 mg of 5% Pd / C was then added and hydrogenated for 2 h. After completion of the reaction, the filtrate was filtered and used directly in the next step without purification.

[0192] Step 4: Compound 1d

[0193] The crude product 1c was placed in an ice-water bath, and DIPEA (235 μL, 1.39 mmol) was added, followed by compound M3 (784 mg, 1.39 mmol). After addition, the mixture was warmed to room temperature and reacted for 1 h. The reaction was monitored for completion by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 1d (504 mg); LC-MS: [M+H] + =804.4.

[0194] Step 5: Compound 1e

[0195] To a 50 mL single-necked vial were added 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. DIPEA (378 μL, 2.29 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 2 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain a preparation of compound 1e. The preparation was lyophilized to yield 1e (210 mg); LC-MS: [M+H] + =1221.6.

[0196] Step 6: Compound 1

[0197] 1e (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield solid compound 1 (60 mg); LC-MS: [M+H] + =1065.3.

[0198] Example 5

[0199] Synthesis of compound 2:

[0200]

[0201] Referring to the synthetic route of Example 4, compound 2 (51 mg) was obtained; LC-MS: [M+H] + =1065.3.

[0202] Example 6

[0203] Synthesis of compound 3:

[0204]

[0205] Step 1: Compound 3a

[0206] In a 250 mL single-necked flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added. The mixture was stirred and cooled to 0°C. Benzyl 2-hydroxy-2-methylpropanoate (6.3 g, 32.6 mmol) was added dropwise. The temperature was naturally raised to room temperature for reaction (approximately 2-4 h) and monitored by TLC. After the reaction, saturated NaHCO₃ solution was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to afford 3a (4.2 g) in a 52% yield; LC-MS: [M+H] + =503.3.

[0207] Step 2: Compound 3b

[0208] In a 25 mL single-necked bottle, 3a (2 g, 4.0 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (760 mg, 5.0 mmol) was added. The reaction was allowed to proceed for 1 h. After the Fmoc deprotection was completed, the product was set aside for use after monitoring by TLC.

[0209] In a separate 25 mL single-necked flask, M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol), and 10 mL of DMF were added. DIPEA (823 μL, 5.04 mmol) was added under ice-water bath and stirring continued for 30 min. The reaction mixture was added to the reaction flask and allowed to warm to room temperature. After completion of the reaction, the reaction solution was purified by preparative liquid chromatography to obtain a preparative solution of the product. 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 a solid 3b (1.4 g) in a 53% yield; LC-MS: [M+H] + =676.2.

[0210] Step 3: Compound 3c

[0211] 3b (700 mg, 1.04 mmol) was added to a 25 mL single-necked bottle and dissolved in 10 mL DMF. 700 mg of 5% Pd / C was then added and hydrogenated for 1.5 h. After completion of the reaction, the filtrate was filtered and used directly in the next step without purification.

[0212] Step 4: Compound 3d

[0213] The crude product 3c was placed in an ice-water bath, and DIPEA (210 μL, 1.25 mmol) was added, followed by compound M3 (704 mg, 1.25 mmol). After addition, the mixture was warmed to room temperature and reacted for 1 h. The reaction was monitored for completion by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 3d (486 mg); LC-MS: [MH] - =830.5.

[0214] Step 5: Compound 3e

[0215] To a 50 mL single-necked vial were added 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. DIPEA (219.5 μL, 1.33 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 3 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain a preparation of compound 3e. The preparation was lyophilized to yield 3e (157 mg); LC-MS: [M+H] + =1249.6.

[0216] Step 6: Compound 3

[0217] 3e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield solid compound 3 (64 mg); LC-MS: [M+H] + =1093.1.

[0218] Example 7

[0219] Synthesis of compound 4:

[0220]

[0221] Referring to the synthetic route of Example 6, compound 4 (60 mg) was obtained; LC-MS: [M+H] + =1093.2.

[0222] Example 8

[0223] Synthesis of compound 5A:

[0224]

[0225] Step 1: Compound 5a

[0226] To a 25 mL single-necked flask, add 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. After stirring, cool the mixture to 0°C and slowly add benzyl L-lactate (1.2 g, 7.0 mmol, 5 eq). After complete addition, warm the mixture to room temperature for reaction. Monitor the reaction by TLC. After completion of the reaction, add saturated NaHCO₃ solution, extract with ethyl acetate, dry over anhydrous sodium sulfate, filter, and concentrate. The residue is purified by reverse-phase column chromatography to afford 5a (400 mg).

[0227] LC-MS: [M+NH4] + =506.2.

[0228] 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), 5.11 -5.58(2H,m),5.43(1H,s),7.06(1H,t,J=8.0Hz),7.25-7.33(6H,m),7.38(2H,t,J=8.0Hz),7.57(2H,d,J=8.0Hz),7.75(2H,d,J=8.0Hz).

[0229] Step 2: Compound 5b

[0230] Compound 5a (400 mg, 0.8 mmol, 1.0 eq) and 4 mL of DMF were added to a 25 mL single-necked vial. After stirring, the temperature was lowered to 0°C and DBU (137 mg, 0.9 mmol, 1.1 eq) was slowly added. After the addition was complete, the temperature was raised to room temperature. The reaction was completed by TLC monitoring and was designated as reaction solution ①.

[0231] In another 25 mL single-necked flask, M4 (372 mg, 0.9 mmol, 1.1 eq), PyBOP (852 mg, 1.6 mmol, 2.0 eq), and 3 mL of DMF were added and stirred at room temperature for 5 minutes. Then, reaction solution ① was added and the mixture was allowed to react at room temperature under HPLC monitoring. After completion of the reaction, the reaction solution was purified by HPLC to obtain compound 5b (326 mg); LC-MS: [M+NH4] + =679.2.

[0232] Step 3: Compound 5c

[0233] To a 100 mL single-necked flask, add 5b (4.0 g, 6.05 mmol, 1.0 eq) and dissolve in DMF (60 mL). Then add 5% Pd / C (4 g) and hydrogenate at room temperature for 4 h (reaction progress monitored by HPLC). Filter the Pd / C and place the filtrate in an ice-water bath (approximately 0°C) without concentration until ready for use.

[0234] Step 4: Compound 5d

[0235] The crude product 5c was placed in an ice-water bath, and DIPEA (1.1 mL, 1.1 eq) was added, followed by compound M3 (3.4 g, 6.05 mmol). After addition, the mixture was warmed to room temperature and reacted for 2 h. HPLC monitoring of the reaction was performed, and the reaction solution was purified by HPLC to obtain a preparative solution. The preparative solution was lyophilized to obtain 5d (3.15 g); LC-MS: [MH] - =816.3.

[0236] Step 5: Compound 5e

[0237] 5d (2.07 g, 2.53 mmol, 1.0 eq), M5 (1.35 g, 2.53 mmol, 1.0 eq), PyBOP (1.98 g, 3.79 mmol, 1.5 eq), HOBt (0.51 g, 3.79 mmol, 1.5 eq), and DMF (40 mL) were added to a 100 mL single-necked vial. DIPEA (1.05 mL, 1.5 eq) was added under ice-water bath conditions, and the mixture was allowed to warm to room temperature for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative purification and concentrated under reduced pressure in a water bath at 35°C to remove acetonitrile. The product was then lyophilized to afford compound 5e (1.92 g) in a 61% yield. LC-MS: [M+H] + =1235.4.

[0238] Step 6: Compound 5A

[0239] Compound 5e (1.0 g, 0.8 mmol, 1.0 eq) and 35 mL of nitromethane were added to a 100 mL single-necked flask. After dissolution, zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added. The mixture was reacted in an oil bath at 40°C (preheated for stabilization) for 30 min. The nitromethane was removed by concentrating the mixture in a water bath at 45°C with a water pump to obtain a yellow solid residue (monitored by HPLC). A solution of compound 5A was prepared by an acid method. The solution was concentrated in a water bath at 35°C with a water pump to remove acetonitrile and then lyophilized to obtain compound 5A (786 mg) in a 90% yield.

[0240] LC-MS: [M+H] + =1079.4;

[0241] 1 H 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).

[0242] Example 9

[0243] Synthesis of compound 5B:

[0244]

[0245] Step 1: Compound 5d-1

[0246] Compound 5b (300 mg, 0.45 mmol, 1.0 eq) and DMF (3 mL) were added to a 25 mL single-necked vial and stirred to dissolve. 5% Pd / C (300 mg) was added and the atmosphere was replaced with hydrogen three times. The hydrogenation reaction was carried out for 2 h and the reaction was monitored by HPLC. After the reaction, the Pd / C was removed by filtration, the filtrate was cooled to 0-5°C, DIPEA (65 mg, 0.5 mmol, 1.1 eq) was added, and ent-M3 (255 mg, 0.45 mmol) was added to the filtrate. After the addition was complete, the temperature was raised to 20±5°C and the reaction was allowed to react for 1 h. The reaction was monitored by HPLC. After the reaction was completed, the product was purified by HPLC and the prepared solution was collected and lyophilized to obtain compound 5d-1 (200 mg) in a 54% yield; LC-MS: [MH] - =816.3.

[0247] Step 2: Compound 5e-1

[0248] 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 added to a 25 mL single-necked vial. The mixture was cooled to 0-5°C in an ice-water bath and DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added. After addition, the temperature was raised to 20±5°C and the reaction was allowed to react for 2 h. The reaction was monitored by HPLC. The reaction mixture was directly purified by HPLC. The product preparation solution was collected and lyophilized to give compound 5e-1 (162.8 mg); LC-MS: [M+H] + =1235.4.

[0249] Step 3: Compound 5B

[0250] 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 single-necked vial. After addition, the mixture was heated to 40°C for 0.5 h, after which the reaction was terminated. The reaction solution was directly dried under reduced pressure at 45°C to afford a yellow solid. Samples were taken for HPLC monitoring. The dried solid was directly purified by HPLC. The product solution was collected and lyophilized to afford compound 5B (73.4 mg) in a 76.5% yield. LC-MS: [M+H] + =1079.4.

[0251] Example 10

[0252] Preparation of compound 6A:

[0253]

[0254] Referring to the synthetic route of Example 8, compound 6A (71 mg) was obtained; LC-MS: [M+H] + =1079.4.

[0255] Example 11

[0256] Preparation of compound 6B:

[0257]

[0258] Referring to the synthetic route of Example 9, compound 6B (59 mg) was obtained; LC-MS: [M+H] + =1079.4.

[0259] Example 12

[0260] Preparation of compounds 7A and 7B:

[0261]

[0262] Step 1: Compound 7a

[0263] M1 (10 g, 27.1 mmol), benzyl 3,3,3-trifluorolactate (prepared according to the method disclosed in patent WO2020063673A1) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were added to a 250 mL single-necked flask and heated to 100°C for 4 h. After the reaction was completed, the mixture was cooled to room temperature, the insoluble matter was filtered out, and the filtrate was concentrated to obtain 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 compound, with a yield of 35.1%; LC-MS: [M+H] + =543.17.

[0264] Step 2: Compound 7b

[0265] 7a (5 g, 9.2 mmol) and 15 mL of DMF were added to a 50 mL single-necked bottle. After dissolution, DBU (1.68 g, 11 mmol) was added under ice-water bath and reacted for 1 h. This was recorded as reaction solution ①.

[0266] In a separate 50 mL single-necked flask, add M4 (3.8 g, 9.2 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol), and 10 mL of DMF. After dissolution, add DIPEA (1.82 mL, 11 mmol) in an ice-water bath and continue the reaction for 30 min. Then add reaction solution ① and warm to room temperature for 2 h. The reaction progress is monitored by HPLC. After completion, the reaction solution is purified by HPLC to obtain a preparative solution. The preparative solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to yield 4.1 g of a solid (yield: 62.3%); LC-MS: [M+H] + =716.25.

[0267] Step 3: Compound 7d

[0268] To a 25mL single-necked vial, 7b (900mg, 1.26mmol) was added. Dissolved in 15mL of DMF, 900mg of 5% Pd / C was added, and hydrogenation was carried out for 2h. After completion, the reaction was filtered, and the filtrate was placed in an ice-water bath. DIPEA (228uL, 1.38mmol) was added, followed by M3 (712mg, 1.26mmol). After addition, the mixture was warmed to room temperature and reacted for 1h. Completion of the reaction was monitored by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution. The preparative solution was lyophilized to yield 525mg of the product, with a yield of 47.9%; LC-MS: [MH] - =870.33.

[0269] Step 4: Compound 7e

[0270] To a 50 mL single-necked vial were added 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. DIPEA (378 μL, 2.29 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 2 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain preparations of compound 7e-1 and compound 7e-2. The preparations were lyophilized to yield 150 mg of compound 7e-1. LC-MS: [M+H] + =1289.46; 220 mg of compound 7e-2, LC-MS: [M+H] + =1289.46.

[0271] Step 5: Compound 7A

[0272]

[0273] 7e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and allowed to react at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to yield the crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 52 mg of a solid; TOF result: 1133.3613.

[0274] Step 6: Compound 7B

[0275]

[0276] 7e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and allowed to react at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 63 mg of a solid; TOF result: 1133.3668.

[0277] Example 13

[0278] Synthesis of compounds 8A and 8B:

[0279]

[0280] Step 1: Compound 8d

[0281] To a 25 mL single-necked vial, 7c (900 mg, 1.83 mmol) was added. Dissolved in 20 mL of DMF, followed by DIPEA (303 μL, 1.83 mmol) and ent-M3 (1034 mg, 1.83 mmol). The mixture was allowed to warm to room temperature for 1 h. Completion of the reaction was monitored by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution. The preparative solution was lyophilized to yield 613 mg of the product, with a yield of 38.5%. LC-MS: [MH] - =870.32.

[0282] Step 2: Compound 8e-1 and Compound 8e-2

[0283] To a 50 mL single-necked vial were added 8d (500 mg, 0.57 mmol), M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF. DIPEA (378 μL, 2.29 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 2 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain preparations of compounds 8e-1 and 8e-2. The preparations were lyophilized to yield 140 mg of compound 8e-1 and 210 mg of compound 8e-2, respectively. LC-MS of compound 8e-1: [M+H] + =1289.47; LC-MS of compound 8e-2: [M+H] + =1289.47.

[0284] Step 3: Compound 8A

[0285]

[0286] Compound 8e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and allowed to react at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 50 mg of a solid; TOF result: 1133.3623.

[0287] Step 4: Compound 8B

[0288]

[0289] Compound 8e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and allowed to react at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 58 mg of a solid; TOF result: 1133.3653.

[0290] Example 14

[0291] Synthesis of compound 9A:

[0292]

[0293] Step 1: Compound 9a

[0294] To a 250 mL single-necked flask, add M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). Stir and cool to 0°C. Add 2-hydroxy-2-cyclopropylacetic acid benzyl ester (prepared according to the method disclosed in US Patent No. 20050020645 A1) (6.3 g, 32.6 mmol) dropwise. Allow to warm to room temperature for approximately 2-4 hours, monitored by TLC. Upon completion of the reaction, add saturated NaHCO₃ solution, extract with ethyl acetate, wash with saturated sodium chloride solution, dry over anhydrous sodium sulfate, filter, and concentrate. The residue is purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to afford 9a (3.7 g) in a 45% yield. LC-MS: [M+H] + =501.5.

[0295] Step 2: Compound 9b

[0296] In a 25 mL single-necked bottle, 9a (2 g, 4.0 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (760 mg, 5.0 mmol) was added. The reaction was allowed to proceed for 1 h. After the Fmoc deprotection was completed, the product was set aside for use after monitoring by TLC.

[0297] In a separate 25 mL single-necked flask, M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol), and 10 mL of DMF were added. DIPEA (823 μL, 5.04 mmol) was added under ice-water bath and stirring continued for 30 min. The reaction mixture was added to the reaction flask and allowed to warm to room temperature for reaction. After completion of the reaction, monitored by HPLC, the reaction solution 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 a solid, with a yield of 56%. LC-MS: [M+H] + =674.7.

[0298] Step 3: Compound 9c

[0299] 9b (900 mg, 1.3 mmol) was added to a 25 mL single-necked bottle and dissolved in 10 mL DMF. 900 mg of 5% Pd / C was then added and hydrogenated for 1.5 h. After completion of the reaction, the filtrate was filtered and used directly in the next step without purification.

[0300] Step 4: Compound 9d

[0301] The crude product 9c was placed in an ice-water bath, and DIPEA (223 μL, 1.3 mmol) was added, followed by compound M3 (750 mg, 1.3 mmol). After addition, the mixture was warmed to room temperature and reacted for 1 h. The reaction was monitored for completion by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 9d (529 mg); LC-MS: [MH] - =828.4.

[0302] Step 5: Compound 9e

[0303] To a 50 mL single-necked vial were added 9d (500 mg, 0.6 mmol), M5 (300 mg, 0.6 mmol), PyBOP (416 mg, 0.8 mmol), HOBt (108 mg, 0.5 mmol), and 15 mL of DMF. DIPEA (351 μL, 2.13 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 3 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain a preparation of compound 9e. The preparation was lyophilized to yield 9e (257 mg); LC-MS: [M+H] + =1247.5.

[0304] Step 6: Compound 9A

[0305] 9e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield solid compound 9A (55 mg); LC-MS: [M+H] + =1091.3.

[0306] Example 15

[0307] Synthesis of compound 9B:

[0308]

[0309] Referring to the synthetic route of Example 14, compound 9B (44 mg) was obtained; LC-MS: [M+H] + =1091.3.

[0310] Example 16

[0311] Synthesis of compound 10A:

[0312]

[0313] Step 1: Compound 10a

[0314] To a 250 mL single-necked flask, add M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). Stir and cool to 0°C. Add benzyl 3-hydroxy-2-cyclopropylpropionate (prepared according to the method disclosed in patent WO2013187496A1) (6.7 g, 32.6 mmol) dropwise. Allow to warm to room temperature for approximately 2-4 hours, monitored by TLC. Upon completion of the reaction, add saturated NaHCO₃ solution, extract with ethyl acetate, wash with saturated sodium chloride solution, dry over anhydrous sodium sulfate, filter, and concentrate. The residue is purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to afford 10a (4.9 g) in a 58% yield. LC-MS: [M+H] + =515.4.

[0315] Step 2: Compound 10b

[0316] In a 25 mL single-necked bottle, 10a (4 g, 7.8 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (1.2 g, 8.0 mmol) was added. The reaction was continued for 1 h. After the Fmoc deprotection was completed, the product was set aside for use after monitoring by TLC.

[0317] In a separate 25 mL single-necked flask, M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol), and 10 mL of DMF were added. DIPEA (1.65 mL, 10.1 mmol) was added under ice-water bath and stirring continued for 50 min. The above reaction solution was added to the reaction flask and the reaction was allowed to warm to room temperature. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography to obtain the product preparation solution. The preparation 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, with a yield of 42%; LC-MS: [M+H] + =688.8.

[0318] Step 3: Compound 10c

[0319] 10b (1.0 g, 1.45 mmol) was added to a 25 mL single-necked bottle and dissolved in 15 mL DMF. 1.0 g of 5% Pd / C was then added and hydrogenated for 1.5 h. After completion of the reaction, the filtrate was filtered and used directly in the next step without purification.

[0320] Step 4: Compound 10d

[0321] The crude product 10c was placed in an ice-water bath, and DIPEA (258 μL, 1.5 mmol) was added, followed by compound M3 (837 mg, 1.45 mmol). After addition, the mixture was warmed to room temperature and reacted for 1 h. The reaction was monitored for completion by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 10d (499 mg); LC-MS: [MH] - =842.4.

[0322] Step 5: Compound 10e

[0323] To a 50 mL single-necked vial were added 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. DIPEA (330 μL, 2.0 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 3 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain a preparative solution of compound 10e. The preparative solution was lyophilized to yield 10e (188 mg); LC-MS: [M+H] + =1261.5.

[0324] Step 6: Compound 10A

[0325] 10e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield solid compound 10A (61 mg); LC-MS: [M+H] + =1105.4.

[0326] Example 17

[0327] Synthesis of compound 10B:

[0328]

[0329] Referring to the synthetic route of Example 16, compound 10B (75 mg) was obtained; LC-MS: [M+H] + =1105.4.

[0330] Example 18

[0331] Synthesis of compound 11A:

[0332]

[0333] Step 1: Compound 11a

[0334] In a 250 mL single-necked bottle, M1 (6 g, 16.3 mmol), 100 mL THF, p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred, cooled to 0°C, and 2-hydroxy-2-cyclobutylacetic acid benzyl ester (synthesized according to the method published in Journal of Medicinal Chemistry, 2013, 56 (13), 5541-5552) (6.7 g, 32.6 mmol) was added dropwise. After the addition, the temperature was naturally raised to room temperature for reaction (reaction time of about 2-4 h) and monitored by TLC. After the reaction was completed, saturated NaHCO3 solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA = 10:1-5:1-2:1) to obtain 11a (5.1 g), with a yield of 62%; LC-MS: [M+H] + =515.7.

[0335] Step 2: Compound 11b

[0336] In a 25 mL single-necked bottle, 11a (4 g, 7.8 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (1.2 g, 8.0 mmol) was added. The reaction was continued for 1 h. After the Fmoc deprotection was completed, the product was set aside after monitoring by TLC.

[0337] In a separate 25 mL single-necked flask, M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol), and 10 mL of DMF were added. DIPEA (1.63 mL, 10.0 mmol) was added under ice-water bath and stirring continued for 40 min. The above reaction solution was added to the reaction flask and the reaction was allowed to warm to room temperature. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography to obtain the product preparation solution. The preparation 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, with a yield of 42%; LC-MS: [M+H] + =688.3.

[0338] Step 3: Compound 11c

[0339] 11b (2.0 g, 2.9 mmol) was added to a 25 mL single-necked bottle and dissolved in 25 mL DMF. 2.0 g of 5% Pd / C was then added and hydrogenated for 3 h. After completion of the reaction, the filtrate was filtered and used directly in the next step without purification.

[0340] Step 4: Compound 11d

[0341] The crude product 11c was placed in an ice-water bath, and DIPEA (516 μL, 3.0 mmol) was added, followed by compound M3 (1.7 g, 2.9 mmol). After addition, the mixture was warmed to room temperature and reacted for 2 h. The reaction was monitored for completion by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to obtain 11d (934 mg); LC-MS: [MH] - =842.4.

[0342] Step 5: Compound 11e

[0343] To a 50 mL single-necked vial were added 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. DIPEA (660 μL, 4.0 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 4 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain a preparative solution of compound 11e. The preparative solution was lyophilized to yield 11e (401 mg); LC-MS: [M+H] + =1261.4.

[0344] Step 6: Compound 11A

[0345] 11e (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield solid compound 11A (86 mg); LC-MS: [M+H] + =1105.4.

[0346] Example 19

[0347] Synthesis of compound 11B

[0348]

[0349] Referring to the synthetic route of Example 18, compound 11B (50 mg) was obtained. LC-MS: [M+H] + 1105.4.

[0350] Example 20

[0351] Synthesis of compound 12A:

[0352]

[0353] Step 1: Compound 12a

[0354] To a 250 mL single-necked flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added. The mixture was stirred and cooled to 0°C. Benzyl 3-hydroxy-2-cyclobutylpropionate (prepared according to the method disclosed in patent WO2009011285A1) (7.2 g, 32.6 mmol) was added dropwise. The temperature was naturally raised to room temperature for reaction (approximately 2-4 hours) and monitored by TLC. After the reaction, saturated NaHCO₃ solution was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to afford 12a (4.5 g) in a 52% yield; LC-MS: [M+H] + =529.4.

[0355] Step 2: Compound 12b

[0356] In a 25 mL single-necked bottle, 12a (4 g, 7.6 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (1.2 g, 8.0 mmol) was added. The reaction was continued for 1 h. After the Fmoc deprotection was completed, the product was set aside after monitoring by TLC.

[0357] In a separate 25 mL single-necked flask, M4 (3.2 g, 7.6 mmol), PyBOP (4.7 g, 9.0 mmol), HOBt (1.22 g, 9.0 mmol), and 10 mL of DMF were added. DIPEA (1.49 mL, 0.9 mmol) was added under ice-water bath and stirring continued for 30 min. The above reaction solution was added to the reaction flask and the reaction was allowed to warm to room temperature. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography to obtain the product preparation solution. The preparation 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, with a yield of 37%; LC-MS: [M+H] + =702.8.

[0358] Step 3: Compound 12c

[0359] 12b (1.0 g, 1.43 mmol) was added to a 25 mL single-necked bottle and dissolved in 15 mL DMF. 1.0 g of 5% Pd / C was then added and hydrogenated for 1.5 h. After completion of the reaction, the filtrate was filtered and used directly in the next step without purification.

[0360] Step 4: Compound 12d

[0361] The crude product 12c was placed in an ice-water bath, and DIPEA (258 μL, 1.5 mmol) was added, followed by compound M3 (825 mg, 1.43 mmol). After addition, the mixture was warmed to room temperature and reacted for 1 h. The reaction was monitored for completion by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 12d (522 mg); LC-MS: [MH] - =856.4.

[0362] Step 5: Compound 12e

[0363] To a 50 mL single-necked vial were added 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. DIPEA (330 μL, 2.0 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 3 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain a preparation of compound 12e. The preparation was lyophilized to yield 12e (198 mg); LC-MS: [M+H] + =1275.4.

[0364] Step 6: Compound 12A

[0365] 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-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield solid compound 12A (55 mg); LC-MS: [M+H] + =1119.4.

[0366] Example 21

[0367] Synthesis of compound 12B:

[0368]

[0369] Referring to the synthetic route of Example 20, compound 12B (50 mg) was obtained; LC-MS: [M+H] + =1119.4.

[0370] Example 22

[0371] Synthesis of compound 13A:

[0372]

[0373] Step 1: Compound 13a

[0374] In a 250 mL single-necked bottle, M1 (6 g, 16.3 mmol), 100 mL THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred, and cooled to 0°C. Benzyl 2-hydroxy-2-cyclopentyl acetate (synthesized according to the method published in Journal of Medicinal Chemistry, 2013, 56(13), 5541-5552) (7.2 g, 32.6 mmol) was added dropwise. After addition, the temperature was naturally raised to room temperature for reaction (reaction time of about 2-4 h) and monitored by TLC. After the reaction was completed, saturated NaHCO3 solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA=10:1-5:1-2:1) to obtain 13a (4.6 g) with a yield of 53%; LC-MS: [M+H] + =529.5.

[0375] Step 2: Compound 13b

[0376] In a 25 mL single-necked bottle, 13a (4 g, 7.6 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (1.17 g, 7.8 mmol) was added. The reaction was continued for 1 h. After the Fmoc deprotection was completed, the product was set aside by TLC.

[0377] In a separate 25 mL single-necked flask, M4 (3.14 g, 7.6 mmol), PyBOP (4.42 g, 8.5 mmol), HOBt (1.15 g, 8.5 mmol), and 10 mL of DMF were added. DIPEA (1.39 mL, 0.85 mmol) was added under ice-water bath and stirring continued for 30 min. The above reaction solution was added to the reaction flask and the reaction was allowed to warm to room temperature. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography to obtain the product preparation solution. The preparation 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, with a yield of 39%; LC-MS: [M+H] + =702.8.

[0378] Step 3: Compound 13c

[0379] 13b (1.5 g, 1.87 mmol) was added to a 25 mL single-necked bottle and dissolved in 25 mL DMF. 1.5 g of 5% Pd / C was then added and hydrogenated for 3 h. After completion of the reaction, the filtrate was filtered and used directly in the next step without purification.

[0380] Step 4: Compound 13d

[0381] The crude product 13c was placed in an ice-water bath, and DIPEA (333 μL, 1.93 mmol) was added, followed by compound M3 (1.1 g, 1.87 mmol). After addition, the mixture was warmed to room temperature and reacted for 1 h. The reaction was monitored for completion by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 13d (519 mg); LC-MS: [MH] - =856.6.

[0382] Step 5: Compound 13e

[0383] To a 50 mL single-necked vial were added 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. DIPEA (330 μL, 2.0 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 4 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain a preparation of compound 13e. The preparation was lyophilized to yield 13e (187 mg); LC-MS: [M+H] + =1275.5.

[0384] Step 6: Compound 13A

[0385] 13e (100 mg, 0.08 mmol), zinc bromide (355 mg, 0.16 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield solid compound 13A (60 mg); LC-MS: [M+H] + =1119.6.

[0386] Example 23

[0387] Synthesis of compound 13B:

[0388]

[0389] Referring to the synthetic route of Example 22, compound 13B (51 mg) was obtained; LC-MS: [M+H] + =1119.6.

[0390] Example 24

[0391] Synthesis of compound 14A:

[0392]

[0393] Step 1: Compound 14a

[0394] To a 250 mL single-necked flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added. The mixture was stirred and cooled to 0°C. Benzyl 3-hydroxy-2-cyclopentylpropionate (synthesized according to the method disclosed in patent WO2009011285A1) (7.6 g, 32.6 mmol) was added dropwise. The temperature was naturally raised to room temperature for the reaction (approximately 2-4 hours) and monitored by TLC. After the reaction, saturated NaHCO₃ solution was added, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to afford 14a (4.4 g) in a 49% yield; LC-MS: [M+H] + =543.6.

[0395] Step 2: Compound 14b

[0396] In a 25 mL single-necked bottle, 14a (4 g, 7.4 mmol) and 10 mL of DMF were added, stirred at 0°C, and DBU (1.2 g, 8.0 mmol) was added. The reaction was continued for 1 h. After the Fmoc deprotection was completed, the product was set aside by TLC.

[0397] In a separate 25 mL single-necked flask, M4 (3.1 g, 7.4 mmol), PyBOP (4.6 g, 8.8 mmol), HOBt (1.19 g, 8.8 mmol), and 10 mL of DMF were added. DIPEA (1.49 mL, 9.0 mmol) was added under ice-water bath and stirring continued for 30 min. The above reaction solution was added to the reaction flask and the reaction was allowed to warm to room temperature. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography to obtain the product preparation solution. The preparation 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, with a yield of 49%; LC-MS: [M+H] + =716.4.

[0398] Step 3: Compound 14c

[0399] 14b (1.0 g, 1.4 mmol) was added to a 25 mL single-necked bottle and dissolved in 15 mL DMF. 1.0 g of 5% Pd / C was then added and hydrogenated for 1.5 h. After completion of the reaction, the filtrate was filtered and used directly in the next step without purification.

[0400] Step 4: Compound 14d

[0401] The crude product 14c was placed in an ice-water bath, and DIPEA (248 μL, 1.5 mmol) was added, followed by compound M3 (808 mg, 1.4 mmol). After addition, the mixture was warmed to room temperature and reacted for 1 h. The reaction was monitored for completion by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 14d (500 mg); LC-MS: [MH] - =870.5.

[0402] Step 5: Compound 14e

[0403] To a 50 mL single-necked vial were added 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. DIPEA (331 μL, 2.0 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 3 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain a preparation of compound 14e. The preparation was lyophilized to yield 14e (146 mg); LC-MS: [M+H] + =1289.5.

[0404] Step 6: Compound 14A

[0405] 14e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield solid compound 14A (52 mg); LC-MS: [M+H] + =1133.4.

[0406] Example 25

[0407] Synthesis of compound 14B:

[0408]

[0409] Referring to the synthetic route of Example 24, compound 14B (48 mg) was obtained; LC-MS: [M+H] + =1133.4.

[0410] Example 26

[0411] Synthesis of compounds 15A and 15B:

[0412]

[0413] Step 1: Compound 15a

[0414] M1 (10 g, 27.1 mmol), 2-hydroxy-butyric acid benzyl ester (prepared according to the method published in Chemical Communications, 2019, 55 (53), 7699-7702) (10.5 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added to a 250 mL single-necked bottle and heated to 100 ° C for 4 h. After the reaction was completed, the mixture was cooled to room temperature, the insoluble matter was removed by filtration, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 5.67 g of the target product with a yield of 42%; LC-MS: [M+H] + =503.5.

[0415] Step 2: Compound 15b

[0416] 15a (5 g, 9.95 mmol) and 15 mL of DMF were added to a 50 mL single-necked flask. After dissolution, DBU (1.68 g, 11 mmol) was added under ice-water bath and reacted for 1 h. This was recorded as reaction solution ①.

[0417] In a separate 50 mL single-necked flask, add M4 (4.1 g, 10.0 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol), and 10 mL of DMF. After dissolution, add DIPEA (1.82 mL, 11 mmol) in an ice-water bath and continue the reaction for 40 min. Then add reaction solution ① and warm to room temperature for 2 h. The reaction progress is monitored by HPLC. After completion, the reaction solution is purified by HPLC to obtain a preparative solution. The preparative solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to yield 4.6 g of a solid (68% yield); LC-MS: [M+H] + =676.7.

[0418] Step 3: Compound 15d

[0419] To a 25 mL single-necked vial, 15b (2.0 g, 2.96 mmol) was added. Dissolved in 15 mL of DMF, 2.0 g of 5% Pd / C was added, and hydrogenation was carried out for 2 h. Upon completion, the reaction was filtered, and the filtrate was placed in an ice-water bath. DIPEA (496 μL, 3.0 mmol) was added, followed by M3 (1.7 g, 2.96 mmol). After addition, the mixture was warmed to room temperature and reacted for 1 h. Completion of the reaction was monitored by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution. The preparative solution was lyophilized to yield 1120.0 mg of the product, with a yield of 45%. LC-MS: [MH] - =830.3.

[0420] Step 4: Compound 15e

[0421] To a 50 mL single-necked vial were added 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. DIPEA (446 μL, 2.7 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 2 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain preparations of compound 15e-1 and compound 15e-2. The preparations were lyophilized to yield 138 mg of compound 15e-1. LC-MS: [M+H] + =1249.5; 140 mg of compound 15e-2, LC-MS: [M+H] + =1249.5.

[0422] Step 5: Compound 15A

[0423]

[0424] 15e-1 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 59 mg of a solid; LC-MS: [M+H] + =1093.4.

[0425] Step 6: Compound 15B

[0426]

[0427] 15e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 60 mg of a solid; LC-MS: [M+H] + =1093.4.

[0428] Example 27

[0429] Synthesis of compounds 16A and 16B:

[0430]

[0431] Referring to the synthetic route of Example 26, compound 16A (55 mg) was obtained; LC-MS: [M+H] + =1093.4.

[0432]

[0433] Referring to the synthetic route of Example 26, compound 16B (54 mg) was obtained; LC-MS: [M+H] + =1093.4.

[0434] Example 28

[0435] Synthesis of compounds 17A and 17B:

[0436]

[0437] Step 1: Compound 17a

[0438] M1 (10 g, 27.1 mmol), 2-hydroxy-phenylpropionic acid benzyl ester (synthesized by the method published in Nature Communications, 2020.11(1), 56.) (14.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added to a 250 mL single-necked bottle and heated to 100 ° C for 4 h. After the reaction was completed, the mixture was cooled to room temperature, the insoluble matter was removed by filtration, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA=10:1-5:1-2:1) to obtain 6.13 g of the target product with a yield of 40%; LC-MS: [M+H] + =565.6.

[0439] Step 2: Compound 17b

[0440] 17a (5 g, 8.86 mmol) and 15 mL of DMF were added to a 50 mL single-necked flask. After dissolution, DBU (1.53 g, 10 mmol) was added under ice-water bath and reacted for 1 h. This was recorded as reaction solution ①.

[0441] In a separate 50 mL single-necked flask, add M4 (3.6 g, 8.86 mmol), PyBOP (5.23 g, 10 mmol), HOBt (1.36 g, 10 mmol), and 10 mL of DMF. After dissolution, add DIPEA (1.65 mL, 10 mmol) in an ice-water bath and continue the reaction for 30 min. Then add reaction solution ① and warm to room temperature for 2 h. The reaction progress is monitored by HPLC. After completion, the reaction solution is purified by HPLC to obtain a preparative solution. The preparative solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 5.0 g of a solid (77% yield); LC-MS: [M+H]+ =738.3.

[0442] Step 3: Compound 17d

[0443] To a 25 mL single-necked vial, 17b (3.0 g, 4.07 mmol) was added. Dissolved in 15 mL of DMF, 3.0 g of 5% Pd / C was added, and hydrogenation was carried out for 2 h. Upon completion, the reaction was filtered, and the filtrate was placed in an ice-water bath. DIPEA (744 μL, 4.5 mmol) was added, followed by M3 (2.34 g, 4.07 mmol). After addition, the mixture was warmed to room temperature and reacted for 1 h. Completion of the reaction was monitored by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution. The preparative solution was lyophilized to yield 1.2 g of the product, with a yield of 33%. LC-MS: [MH] - =892.4.

[0444] Step 4: Compound 17e

[0445] To a 50 mL single-necked vial were added 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. DIPEA (330 μL, 2.0 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 2 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain preparations of compound 17e-1 and compound 17e-2. The preparations were lyophilized to yield 156 mg of compound 17e-1. LC-MS: [M+H] + =1311.4; 150 mg of compound 17e-2, LC-MS: [M+H] + =1311.7.

[0446] Step 5: Compound 17A

[0447]

[0448] 17e-1 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 43 mg of a solid; LC-MS: [M+H] + =1155.4.

[0449] Step 6: Compound 17B

[0450]

[0451] 17e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 40 mg of a solid; LC-MS: [M+H] + =1155.4.

[0452] Example 29

[0453] Synthesis of compounds 18A and 18B:

[0454]

[0455] Referring to the synthetic route of Example 28, compound 18A (54 mg) was obtained; LC-MS: [M+H] + =1155.4.

[0456]

[0457] Referring to the synthetic route of Example 28, compound 18B (55 mg) was obtained; LC-MS: [M+H] + =1155.4.

[0458] Example 30

[0459] Synthesis of compounds 19A and 19B:

[0460]

[0461] Step 1: Compound 19a

[0462] M1 (10 g, 27.1 mmol), 2-cyclopropyl-2-hydroxybenzyl acetate (prepared according to the method disclosed in patent WO2020244657A1) (11.2 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were added to a 250 mL single-necked flask and heated to 100°C for 4 h. After the reaction was complete, the mixture was cooled to room temperature, the insoluble matter was filtered out, 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 compound, with a yield of 36%; LC-MS: [M+H] + =515.2.

[0463] Step 2: Compound 19b

[0464] 19a (4 g, 7.8 mmol) and 10 mL of DMF were added to a 50 mL single-necked flask. After dissolution, DBU (1.42 g, 9.3 mmol) was added under ice-water bath and reacted for 1 h. This was recorded as reaction solution ①.

[0465] In a separate 50 mL single-necked flask, add M4 (3.2 g, 7.8 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol), and 10 mL of DMF. After dissolution, add DIPEA (1.65 mL, 10 mmol) in an ice-water bath and continue the reaction for 30 min. Then add reaction solution ① and warm to room temperature for 2 h. The reaction progress is monitored by HPLC. After completion, the reaction solution is purified by HPLC to obtain a preparative solution. The preparative solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.2 g of a solid (78% yield); LC-MS: [M+H] + =688.3.

[0466] Step 3: Compound 19d

[0467] To a 25 mL single-necked vial, 19b (1000 mg, 1.45 mmol) was added. Dissolved in 15 mL of DMF, 1000 mg of 5% Pd / C was added, and hydrogenation was carried out for 2 h. Upon completion, the reaction was filtered, and the filtrate was placed in an ice-water bath. DIPEA (248 μL, 1.5 mmol) was added, followed by M3 (720 mg, 1.45 mmol). After addition, the mixture was warmed to room temperature and reacted for 1 h. Completion of the reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain a preparative solution. The preparative solution was lyophilized to yield 503 mg of the product, with a yield of 41%; LC-MS: [MH] - =842.3.

[0468] Step 4: Compounds 19e-1 and 19e-2

[0469] To a 50 mL single-necked vial were added 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. DIPEA (292 μL, 1.77 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 2 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain preparations of compound 19e-1 and compound 19e-2. The preparations were lyophilized to yield 112 mg of compound 19e-1. LC-MS: [M+H] + =1261.5; 131 mg of compound 19e-2, LC-MS: [M+H] + =1261.5.

[0470] Step 5: Compound 19A

[0471]

[0472] 19e-1 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 55 mg of a solid; LC-MS: [M+H] + =1105.4.

[0473] Step 6: Compound 19B

[0474]

[0475] 19e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 58 mg of a solid; LC-MS: [M+H] + =1105.4.

[0476] Example 31

[0477] Synthesis of compounds 20A and 20B:

[0478]

[0479] Step 1: Compound 20a

[0480] M1 (10 g, 27.1 mmol), benzyl 2-hydroxycyclopropylpropionate (synthesized according to the method disclosed in patent WO2020063676A) (12.0 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were added to a 250 mL single-necked flask and heated to 100°C for 4 h. After the reaction was complete, the mixture was cooled to room temperature, the insoluble matter was filtered out, 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 compound; LC-MS: [M+H] + =529.2.

[0481] Step 2: Compound 20b

[0482] 20a (4 g, 7.6 mmol) and 10 mL of DMF were added to a 50 mL single-necked bottle. After dissolution, DBU (1.39 g, 9.1 mmol) was added under ice-water bath and reacted for 1 h. This was recorded as reaction solution ①.

[0483] In a separate 50 mL single-necked flask, add M4 (3.12 g, 7.6 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol), and 10 mL of DMF. After dissolution, add DIPEA (1.65 mL, 10 mmol) in an ice-water bath and continue the reaction for 30 min. Then add reaction solution ① and warm to room temperature for 2 h. The reaction progress is monitored by HPLC. After completion, the reaction solution is purified by HPLC to obtain a preparative solution. The preparative solution is extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.5 g of a solid (84% yield); LC-MS: [M+H] + =702.3.

[0484] Step 3: Compound 20d

[0485] To a 25mL single-necked vial, 20b (1000mg, 1.42mmol) was added. Dissolved in 15mL of DMF, followed by the addition of 1000mg of 5% Pd / C. Hydrogenation was carried out for 2h. Upon completion, the reaction was filtered, and the filtrate was placed in an ice-water bath. DIPEA (248uL, 1.5mmol) was added, followed by M5 (708mg, 1.42mmol). After addition, the mixture was warmed to room temperature and reacted for 1h. Completion of the reaction was monitored by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution. The preparative solution was lyophilized to yield 443mg of the product, with a yield of 36%; LC-MS: [MH] - =856.4.

[0486] Step 4: Compounds 20e-1 and 20e-2

[0487] To a 50 mL single-necked vial were added 20d (400 mg, 0.47 mmol), exitecan mesylate (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF. DIPEA (248 uL, 1.5 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 2 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain preparations of compound 20e-1 and compound 20e-2. The preparations were lyophilized to yield 103 mg of compound 20e-1. LC-MS: [M+H] + =1275.5; 103 mg of compound 20e-2, LC-MS: [M+H] + =1275.5.

[0488] Step 5: Compound 20A

[0489]

[0490] 8A (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 51 mg of a solid; LC-MS: [M+H] + =1119.4.

[0491] Step 6: Compound 20B

[0492]

[0493] 20e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 47 mg of a solid; LC-MS: [M+H] + =1119.4.

[0494] Example 32

[0495] Synthesis of compound 21:

[0496]

[0497] Step 1: Compound SM3-1

[0498] To a 2000 mL single-necked flask, add 77087-60-6 (100 g, 458 mmol), maleic acid (53.4 g, 460 mmol), TEA (64 mL, 460 mmol), and 1000 mL of toluene. Heat to 100°C and react for 5 h. After the reaction is complete, cool to room temperature, filter out insoluble matter, and concentrate the filtrate to obtain the crude product. The crude product is purified by silica gel column chromatography (PE:EA = 100:1-50:1-20:1) to obtain 75.6 g of the desired product; LC-MS: [M+H] + =299.1.

[0499] Step 2: Compound (R)-2-hydroxy-1,5-pentanedioic acid tert-butyl ester

[0500] To a 2000mL single-necked flask, add 172793-31-6 (100g, 338mmol) and 1000mL of water. Then, add sodium nitrite (35g, 507mmol) and concentrated sulfuric acid (32mL, 35mmol). Slowly warm the mixture to room temperature and allow to react for 24 hours. After completion, extract the mixture three times with 500mL of ethyl acetate. The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain the crude product. The crude product is purified by silica gel column chromatography (PE:EA = 50:1-30:1-2:1) to yield 91.2g of the desired product; LC-MS: [M+H] + =261.4.

[0501] Step 3: Compound SM3

[0502] To a 2000mL single-necked flask, add (R)-tert-butyl 2-hydroxy-1,5-pentanedioate (50g, 192mmol) and 1000mL of anhydrous tetrahydrofuran. Cool to 0°C in an ice-water bath, then add PPh3 (87.7g, 288mmol), DEAD (50.2g, 288mmol), and SM3-1 (57.3, 192mmol) in sequence. Slowly warm to room temperature and react for 13h. After the reaction is complete, filter to remove insoluble matter, and concentrate the filtrate to obtain the crude product. The crude product is purified by silica gel column chromatography (PE:EA = 50:1-30:1-1:1) to obtain 68.6g of the product.

[0503] The above product was dissolved in 500 mL of methanol and cooled to 0°C in an ice-water bath. NaOH (64 mL, 190 mmol, 3 M / L) was added dropwise at this temperature. The reaction was maintained at this temperature for 12 h, and then HCl (6 M / L) was added to adjust the pH to 3. The product was extracted five times with 500 mL of dichloromethane, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (DCM / MeOH = 50 / 1-20 / 1-2 / 1) to obtain 50.4 g of SM3; LC-MS: [MH] - =525.5.

[0504] Step 4: Compound M6

[0505] Compound SM3 (50 g, 95 mmol, 1.0 eq), pentafluorophenol (19.2 g, 104.5 mmol, 1.1 eq), DCC (21.5 g, 104.5 mmol, 1.1 eq), and THF (600 mL) were added to a 2000 mL single-necked flask and reacted at room temperature for 1 h (monitored by TLC). The insoluble material was then filtered off. The reaction solution was directly purified by preparative purification. The preparative solution was concentrated in a water bath at 35°C under reduced pressure to remove acetonitrile and lyophilized to obtain compound M6 (51.9 g) in a 79% yield. LC-MS: [M+H] + =693.3.

[0506] Step 5: Compound 21a

[0507] To a 25 mL single-necked vial, add 1c (1 g, 2.36 mmol). Dissolve in 25 mL of DMF, then add DIPEA (430 μL, 2.6 mmol) and M6 (1177 mg, 2.36 mmol). After addition, warm to room temperature and react for 1 h. HPLC monitoring of the reaction revealed completion. The reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 555 mg of the product; LC-MS: [MH] - =931.0.

[0508] Step 6: Compound 21b

[0509] To a 100 mL single-necked vial were added 21a (500 mg, 0.54 mmol), exitecan mesylate M5 (285 mg, 0.54 mmol), PyBOP (239 mg, 0.6 mmol), HOBt (239 mg, 0.6 mmol), and 10 mL of DMF. DIPEA (248 μL, 1.5 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 2 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain a preparation of compound 21b. The preparation was lyophilized to yield compound 231 mg; LC-MS: [M+H] + =1349.5.

[0510] Step 7: Compound 21

[0511] Compound 21b (200 mg, 0.1488 mmol), zinc bromide (665 mg, 2.96 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked vial and allowed to react at 40°C for 1 h. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 103 mg of a solid; LC-MS: [M+H] + =1137.5.

[0512] Example 33

[0513]

[0514] Synthesis of compound 22:

[0515] Compound M6 and 3c were used as starting materials and the synthetic route of Example 32 was followed to obtain compound 22 (91 mg); LC-MS: [M+H] + =1165.5.

[0516] Example 34

[0517]

[0518] Synthesis of compounds 23 and 24:

[0519] Using compounds M6 and 5c as starting materials, and referring to the synthetic route of Example 32, 102 mg of compound 23 was obtained. LC-MS: [M+H] + =1151.4; 99 mg of compound 24 was obtained, LC-MS: [M+H] + =1151.4.

[0520] Example 35

[0521]

[0522] Synthesis of compounds 25 and 26:

[0523] Using compounds M6 and 7c as starting materials, and referring to the synthetic route of Example 32, 83 mg of compound 25 was obtained. LC-MS: [M+H] + =1205.7; 80 mg of compound 26 was obtained, LC-MS: [M+H] + =1205.7.

[0524] Example 36

[0525]

[0526] Synthesis of compounds 27 and 28:

[0527] Using compounds M6 and 19c as starting materials, the synthetic route of Example 32 was followed to obtain 100 mg of compound 27. LC-MS: [M+H] + =1177.5; 101 mg of compound 28 was obtained, LC-MS: [M+H] + =1177.5.

[0528] Example 37

[0529] Synthesis of compound 29:

[0530]

[0531] Step 1: Compound SM4-1

[0532] To a 5000 mL single-necked 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 to reflux for 5 h (monitored by TLC), and the insoluble matter was removed by filtration. The reaction solution was directly subjected to rotary distillation under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (PE / EA = 50 / 1-20 / 1-1 / 1) to afford SM4-1 (64.7 g) in a 50% yield. LC-MS: [M+H] + =299.2.

[0533] Step 2: Compound SM4-2

[0534] SM4-1 (64 g, 215 mmol) was added to a 2000 mL single-necked flask. Dissolved in 1000 mL of DMF, DIPEA (71 mL, 430 mmol) was added, followed by nonanediol monomethyl ether methanesulfonate (111.5 g, 220 mmol). After addition, the mixture was allowed to warm to room temperature and react for 2 h. The reaction was monitored 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.

[0535] Step 3: Compound SM4

[0536] SM4-2 (59 g, 83 mmol) was added to a 2000 mL single-necked flask. Dissolved in 1000 mL of MeOH, K2CO3 (11.75 g, 85 mmol) was added, and the mixture was allowed to react at room temperature for 4 h. The reaction was monitored by HPLC for completion, and the insoluble matter was removed by filtration. The reaction solution was directly purified by preparative purification. The preparative solution was concentrated in a water bath at 35°C under reduced pressure to remove acetonitrile, and lyophilized to obtain compound SM4 (27 g); LC-MS: [MH] — =693.5.

[0537] Step 4: Compound M7

[0538] Compound SM4 (25 g, 36 mmol, 1.0 eq), pentafluorophenol (7.3 g, 40 mmol, 1.1 eq), DCC (8.2 g, 40 mmol, 1.1 eq), and THF (200 mL) were added to a 500 mL single-necked flask and reacted at room temperature for 1 h (monitored by TLC). The insoluble material was then filtered off. The reaction solution was directly purified by preparative purification. The preparative solution was concentrated in a water bath at 35°C under reduced pressure to remove acetonitrile and lyophilized to obtain compound M7 (23.3 g) in a 93% yield. LC-MS: [M+H] + =695.8.

[0539] Step 5: Compound 29a

[0540] To a 25 mL single-necked vial, add 1c (1 g, 2.36 mmol). Dissolve in 25 mL of DMF, then add DIPEA (430 μL, 2.6 mmol) and M7 (1640 mg, 2.36 mmol). After addition, warm to room temperature and react for 1 h. The reaction was monitored by HPLC. The reaction solution was purified by HPLC to obtain a preparative solution. The preparative solution was lyophilized to yield 609 mg of the product; LC-MS: [MH] - =1098.5.

[0541] Step 6: Compound 29b

[0542] To a 100 mL single-necked vial were added 29a (500 mg, 0.45 mmol), exitecan mesylate M5 (240 mg, 0.45 mmol), PyBOP (215 mg, 0.54 mmol), HOBt (215 mg, 0.54 mmol), and 10 mL of DMF. DIPEA (248 μL, 1.5 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 2 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain a preparation of compound 29b. The preparation was lyophilized to yield 187 mg of compound 29b; LC-MS: [M+H] + =1517.6.

[0543] Step 7: Compound 29

[0544] Compound 29b (150 mg, 0.988 mmol), zinc bromide (223 mg, 0.988 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked vial and allowed to react at 40°C for 1 h. After completion of the reaction, the solvent was removed by HPLC and concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 114 mg of a solid; LC-MS: [M+H] + =1517.9.

[0545] Example 38

[0546]

[0547] Synthesis of compound 30:

[0548] Compound M7 and 3c were used as starting materials and the synthetic route of Example 37 was followed to obtain compound 30 (125 mg); LC-MS: [M+H] + =1445.6.

[0549] Example 39

[0550]

[0551] Synthesis of compounds 31 and 32:

[0552] Using compounds M7 and 5c as starting materials, and referring to the synthetic route of Example 37, 61 mg of compound 31 was obtained. LC-MS: [M+H] + =1431.7; 63 mg of compound 32 was obtained, LC-MS: [M+H] + =1431.7.

[0553] Example 40

[0554]

[0555] Synthesis of compounds 33 and 34:

[0556] Using compounds M7 and 7c as starting materials, and referring to the synthetic route of Example 37, 60 mg of compound 33 was obtained. LC-MS: [M+H] + =1485.6; 58 mg of compound 34 was obtained, LC-MS: [M+H] + =1485.6.

[0557] Example 41

[0558]

[0559] Synthesis of compounds 35 and 36:

[0560] Using compounds M7 and 19c as starting materials, and referring to the synthetic route of Example 37, 102 mg of compound 35 was obtained. LC-MS: [M+H] + =1457.8; 102 mg of compound 36 was obtained, LC-MS: [M+H] + =1457.8.

[0561] Example 42

[0562] Synthesis of compound 37:

[0563]

[0564] Step 1: Compound SM5-1

[0565] Compound 16947-84-5 (100 g, 295 mmol, 1.0 eq), DIPEA (50 mL, 300 mmol), benzyl bromide (51.3 g, 300 mmol), and THF (1000 mL) were added to a 2000 mL single-necked flask. The mixture was reacted at room temperature for 12 h (monitored by TLC), and the insoluble material was removed by filtration. The reaction solution was directly subjected to rotary distillation under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (PE / EA = 50 / 1-20 / 1-2 / 1) to afford SM5-1 (110.1 g) in an 87% yield. LC-MS: [M+H] + =429.2.

[0566] Step 2: Compound SM5-2

[0567] Compound SM5-1 (100 g, 233.4 mmol, 1.0 eq) and THF (1000 mL) were added to a 2000 mL single-necked flask and cooled to 0°C in an ice-water bath. NaH (37.4 g, 933.5 mmol) and MeI (132.5 g, 933.5 mmol) were added portionwise. The reaction was maintained at 0°C for 24 h (monitored by TLC). The reaction was quenched by adding 500 mL of saturated aqueous NH4Cl solution. The mixture was extracted three times with 500 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was directly subjected to rotary distillation under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (PE / EA = 100 / 1-50 / 1-10 / 1) to obtain SM5-2 (37.1 g); LC-MS: [M+H] + =443.3.

[0568] Step 3: Compound SM5 (refer to Org. Lett., 2006, 8, 3387-3390)

[0569] Compound SM5-2 (35 g, 79 mmol, 1.0 eq) and DCE (500 mL) were added to a 1000 mL single-necked flask. Palladium diacetate (180 mg, 0.8 mmol), I2 (20 g, 79 mmol), and iodobenzene diacetate (40.8 g, 126.4 mmol) were then added sequentially. The mixture was heated to 60°C and reacted for 40 h (monitored by TLC). The reaction was quenched by the addition of 500 mL of saturated aqueous sodium thiosulfate. The mixture was extracted three times with 500 mL of dichloromethane. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was directly subjected to rotary evaporation under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (PE / EA = 100 / 1-50 / 1-10 / 1) to afford SM5 (28 g); LC-MS: [M+H] + =501.3.

[0570] Step 4: Compound SM6

[0571] To a 500 mL single-necked flask, compound SM5 (25 g, 50 mmol, 1.0 eq), potassium di-tert-butyl phosphate (13.66 g, 55 mmol, 1.1 eq), p-toluenesulfonic acid monohydrate (951 mg, 5 mmol, 0.1 eq), and THF (200 mL) were added. The mixture was reacted at room temperature for 1 h (monitored by TLC), and the insoluble material was removed by filtration. The reaction solution was directly purified by preparative purification. The preparative solution was concentrated in a water bath at 35°C under reduced pressure to remove acetonitrile and lyophilized to obtain compound SM6 (15.1 g) in a 46% yield. LC-MS: [M+H] + =651.4.

[0572] Step 5: Compound SM7

[0573] SM6 (15 g, 23 mmol) and 100 mL of DMF were added to a 250 mL single-necked bottle. After dissolving, 15 g of 5% Pd / C was added under an ice-water bath. The atmosphere in the system was replaced with hydrogen three times. The reaction was carried out at room temperature for 12 h. The Pd / C was removed by filtration, and the solvent was removed by vacuum rotary evaporation using an oil pump. The mixture was set aside.

[0574] In another 250 mL single-necked flask, add the crude product, 100 mL of toluene, triethylamine (6.4 mL, 46 mmol), and maleic anhydride (2.4 g, 24 mmol). After dissolving, heat to 100°C and react for 2 h. Monitor the reaction progress by HPLC. After completion, purify the solution by HPLC to obtain a preparative solution. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to yield 4.2 g of a solid (36% yield). LC-MS: [M+H] + =507.3.

[0575] Step 6: Compound M8

[0576] Compound SM7 (4 g, 7.9 mmol, 1.0 eq), pentafluorophenol (1.6 g, 8.7 mmol, 1.1 eq), DCC (1.8 g, 8.7 mmol, 1.1 eq), and THF (60 mL) were added to a 100 mL single-necked flask and reacted at room temperature for 1 h (monitored by TLC). The insoluble material was then filtered off. The reaction solution was directly purified by preparative purification. The preparative solution was concentrated in a water bath at 35°C under reduced pressure to remove acetonitrile and lyophilized to obtain compound M8 (3.7 g) in a 70% yield. LC-MS: [M+H] + =673.2.

[0577] Step 7: Compound 37a

[0578] To a 25mL single-necked vial, add 1c (1g, 2.36mmol). Dissolve in 25mL of DMF, then add DIPEA (430uL, 2.6mmol) and M8 (1.2g, 2.36mmol). After addition, warm to room temperature and react for 1h. HPLC monitoring of the reaction completion was performed. The reaction solution was purified by HPLC to obtain a preparative solution. The preparative solution was lyophilized to yield 488mg of the product; LC-MS: [MH] - =911.0.

[0579] Step 8: Compound 37b

[0580] To a 100 mL single-necked vial were added 37a (400 mg, 0.44 mmol), exitecan mesylate M5 (235 mg, 0.44 mmol), PyBOP (199 mg, 0.5 mmol), HOBt (69 mg, 0.5 mmol), and 10 mL of DMF. DIPEA (218 μL, 1.32 mmol) was added under an ice-water bath and the mixture was allowed to warm to room temperature for 2 h. After completion of the reaction, the reaction solution was purified by HPLC to obtain a preparation of compound 37b. The preparation was lyophilized to yield 201 mg of compound 37b; LC-MS: [M+H] + =1329.6.

[0581] Step 9: Compound 37

[0582] Compound 37b (130 mg, 0.098 mmol), zinc bromide (221 mg, 0.98 mmol), and 10 mL of nitromethane were added to a 25 mL single-necked vial and allowed to react at 40°C for 1 h. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain a preparative solution, which was lyophilized to yield 96 mg of a solid; LC-MS: [M+H] + =1117.4.

[0583] Example 43

[0584]

[0585] Synthesis of compound 38:

[0586] Compound M8 and 3c were used as starting materials and the synthetic route of Example 42 was followed to obtain compound 38 (51 mg); LC-MS: [M+H] + =1145.6.

[0587] Example 44

[0588]

[0589] Synthesis of compounds 39 and 40:

[0590] Using compounds M8 and 5c as starting materials and referring to the synthetic route of Example 42, 57 mg of compound 39 was obtained. LC-MS: [M+H] + =1131.4; 60 mg of compound 40 was obtained, LC-MS: [M+H] + =1131.4.

[0591] Example 45

[0592]

[0593] Synthesis of compounds 41 and 42:

[0594] Using compounds M7 and 7c as starting materials and referring to the synthetic route of Example 42, 44 mg of compound 41 was obtained. LC-MS: [M+H] + =1185.3; 44 mg of compound 42 was obtained, LC-MS: [M+H] + =1185.3.

[0595] Example 46

[0596]

[0597] Synthesis of compounds 43 and 44:

[0598] Using compounds M8 and 19c as starting materials and referring to the synthetic route of Example 42, 62 mg of compound 43 was obtained. LC-MS: [M+H] + =1157.4; 59 mg of compound 44 was obtained, LC-MS: [M+H] + =1157.4.

[0599] Example 47 (Comparative Example)

[0600]

[0601] Synthesis of compound 45

[0602] Compound 45 was synthesized according to the method provided in Example 58 of patent “CN104755494A”.

[0603] The following is the sequence of Trastuzumab:

[0604] light chain

[0605] MDMRVPAQLLGLLLLWLRGARC

[0606] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVP

[0607] SRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK

[0608] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

[0609] Heavy chain

[0610] MDMRVPAQLLGLLLLWLRGARC

[0611] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

[0612] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

[0613] Preparation of ligand-drug conjugate:

[0614] 1) General conjugation method

[0615] After preliminary purification, antibody molecules with a monomer rate greater than 95% were exchanged into phosphate buffered saline (PBS) using an ultrafiltration centrifuge tube at a concentration of 10 mg / mL. TCEP (20 times the number of moles of antibody) was added and allowed to react at room temperature for 4 hours to break the disulfide bonds between the antibody chains. A linker-drug compound (payload) (20 times the number of moles of antibody) was added and allowed to react at room temperature for 2 hours. After the reaction, the solution was exchanged into PBS using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa, and the uncoupled payload was removed. The ADC sample after the exchange was filtered through a 0.22 micron sterilizing filter and set aside.

[0616] 2) Determination of DAR value of ligand-drug conjugate

[0617] Monomer rate detection conditions:

[0618] The sample was centrifuged at 14000 rpm for 5 min, and the supernatant was injected into the sample for analysis;

[0619] Instrument: Waters e2695 (2489 UV / Vis);

[0620] Chromatographic column: TSKgel G3000SWXL (7.8 × 300 mm, 5 μm);

[0621] Mobile phase: A: 50 mM PB, 300 mM NaCl, 200 mM Arg, 5% IPA, pH 6.5;

[0622] The mobile phase A was used for isocratic elution for 30 min, the flow rate was 0.714 mL / min, the column temperature was 25°C, and the detection wavelength was 280 nm.

[0623] DAR detection conditions:

[0624] The sample was centrifuged at 14000 rpm for 5 min, and the supernatant was injected into the sample for analysis;

[0625] Instrument: Waters H-class (TUV);

[0626] Chromatographic column: Proteomix HIC Butyl-NP5 (4.6×35 mm, 5 μm);

[0627] Mobile phase: A: 1.5 M ammonium sulfate, 0.025 M anhydrous sodium phosphate, pH 7.0; B: 0.025 M anhydrous sodium phosphate, 25% IPA, pH 7.0;

[0628] The column was equilibrated with mobile phase A, and gradient elution with mobile phases A and B was performed at a flow rate of 0.8 mL / min; the column temperature was 25°C, and the detection wavelength was 214 nm.

[0629] Example 48: ADC-1

[0630]

[0631] ADC-1 was prepared according to the general coupling method.

[0632] Example 49: ADC-2

[0633]

[0634] ADC-2 was prepared according to the general coupling method.

[0635] Example 50: ADC-3

[0636]

[0637] ADC-3 was prepared according to the general coupling method.

[0638] Example 51: ADC-4

[0639]

[0640] ADC-4 was prepared according to the general coupling method.

[0641] Example 52: ADC-5

[0642]

[0643] ADC-5 was prepared according to the general coupling method.

[0644] Example 53: ADC-6

[0645]

[0646] ADC-6 was prepared according to the general coupling method.

[0647] Example 54: ADC-7

[0648]

[0649] ADC-7 was prepared according to the general coupling method.

[0650] Example 55: ADC-8

[0651]

[0652] ADC-8 was prepared according to the general coupling method.

[0653] Example 56: ADC-9

[0654]

[0655] ADC-9 was prepared according to the general coupling method.

[0656] Example 57: ADC-10

[0657]

[0658] ADC-10 was prepared according to the general coupling method.

[0659] Example 58: ADC-11

[0660]

[0661] ADC-11 was prepared according to the general coupling method.

[0662] Example 59: ADC-12

[0663]

[0664] ADC-12 was prepared according to the general coupling method.

[0665] Example 60: ADC-13

[0666]

[0667] ADC-13 was prepared according to the general coupling method.

[0668] Example 61: ADC-14

[0669]

[0670] ADC-14 was prepared according to the general coupling method.

[0671] Example 62: ADC-15

[0672]

[0673] ADC-15 was prepared according to the general coupling method.

[0674] Example 63: ADC-16

[0675]

[0676] ADC-16 was prepared according to the general coupling method.

[0677] Example 64: ADC-17

[0678]

[0679] ADC-17 was prepared according to the general coupling method.

[0680] Example 65: ADC-18

[0681]

[0682] ADC-18 was prepared according to the general coupling method.

[0683] Example 66: ADC-19

[0684]

[0685] ADC-19 was prepared according to the general coupling method.

[0686] Example 67: ADC-20

[0687]

[0688] ADC-20 was prepared according to the general coupling method.

[0689] Example 68: ADC-21

[0690]

[0691] ADC-21 was prepared according to the general coupling method.

[0692] Example 69: ADC-22

[0693]

[0694] ADC-22 was prepared according to the general coupling method.

[0695] Example 70: ADC-23

[0696]

[0697] ADC-23 was prepared according to the general coupling method.

[0698] Example 71: ADC-24

[0699]

[0700] ADC-24 was prepared according to the general coupling method.

[0701] Example 72: ADC-25

[0702]

[0703] ADC-25 was prepared according to the general coupling method.

[0704] Example 73: ADC-26

[0705]

[0706] ADC-26 was prepared according to the general coupling method.

[0707] Example 74: ADC-27

[0708]

[0709] ADC-27 was prepared according to the general coupling method.

[0710] Example 75: ADC-28

[0711]

[0712] ADC-28 was prepared according to the general coupling method.

[0713] Example 76: ADC-29

[0714]

[0715] ADC-29 was prepared according to the general coupling method.

[0716] Example 77: ADC-30

[0717]

[0718] ADC-30 was prepared according to the general coupling method.

[0719] Example 78: ADC-31

[0720]

[0721] ADC-31 was prepared according to the general coupling method.

[0722] Example 79: ADC-32

[0723]

[0724] ADC-32 was prepared according to the general coupling method.

[0725] Example 80: ADC-33

[0726]

[0727] ADC-33 was prepared according to the general coupling method.

[0728] Example 81: ADC-34

[0729]

[0730] ADC-34 was prepared according to the general coupling method.

[0731] Example 82: ADC-35

[0732]

[0733] ADC-35 was prepared according to the general coupling method.

[0734] Example 83: ADC-36

[0735]

[0736] ADC-36 was prepared according to the general coupling method.

[0737] Example 84: ADC-37

[0738]

[0739] ADC-37 was prepared according to the general coupling method.

[0740] Example 85: ADC-38

[0741]

[0742] ADC-38 was prepared according to the general coupling method.

[0743] Example 86: ADC-39

[0744]

[0745] ADC-39 was prepared according to the general coupling method.

[0746] Example 87: ADC-40

[0747]

[0748] ADC-40 was prepared according to the general coupling method.

[0749] Example 88: ADC-41

[0750]

[0751] ADC-41 was prepared according to the general coupling method.

[0752] Example 89: ADC-42

[0753]

[0754] ADC-42 was prepared according to the general coupling method.

[0755] Example 90: ADC-43

[0756]

[0757] ADC-43 was prepared according to the general coupling method.

[0758] Example 91: ADC-44

[0759]

[0760] ADC-44 was prepared according to the general coupling method.

[0761] Example 92: ADC-45

[0762]

[0763] ADC-45 was prepared according to the general coupling method.

[0764] Example 93: ADC-46

[0765]

[0766] ADC-46 was prepared according to the general coupling method.

[0767] Example 94: ADC-47

[0768]

[0769] ADC-47 was prepared according to the general coupling method.

[0770] Example 95: ADC-48

[0771]

[0772] ADC-48 was prepared according to the general coupling method.

[0773] Example 96: ADC-49

[0774]

[0775] ADC-49 was prepared according to the general coupling method.

[0776] Example 97: ADC-50

[0777]

[0778] ADC-50 was prepared according to the general coupling method.

[0779] Example 98: ADC-51

[0780]

[0781] ADC-51 was prepared according to the general coupling method.

[0782] Example 99: ADC-52

[0783]

[0784] ADC-52 was prepared according to the general coupling method.

[0785] Example 100: ADC-53

[0786]

[0787] ADC-53 was prepared according to the general coupling method.

[0788] Example 101: ADC-54

[0789]

[0790] ADC-54 was prepared according to the general coupling method.

[0791] Example 102: ADC-55

[0792]

[0793] ADC-55 was prepared according to the general coupling method.

[0794] Example 103: ADC-56

[0795]

[0796] ADC-56 was prepared according to the general coupling method.

[0797] Example 104: ADC-57

[0798]

[0799] ADC-57 was prepared according to the general coupling method.

[0800] Example 105: ADC-58

[0801]

[0802] ADC-58 was prepared according to the general coupling method.

[0803] Example 106: ADC-59

[0804]

[0805] ADC-59 was prepared according to the general coupling method.

[0806] Example 107: ADC-60

[0807]

[0808] ADC-60 was prepared according to the general coupling method.

[0809] Example 108: ADC-61 (control group)

[0810]

[0811] ADC-61 was prepared according to the general coupling method.

[0812] Example 109: Plasma Stability

[0813] 1) Operation

[0814] A certain amount of ADC sample was added to IgG-depleted human plasma. Three tubes of each ADC were incubated in a 37°C water bath for 72 and 144 hours, respectively. ADC samples were removed and 100 μL of Protein A resin (MabSelectSuRe™ LX Lot: #10221479GE, washed with PBS) was added to each tube. The ADCs were adsorbed using a vertical mixer for 2 hours. After washing and elution steps, the ADCs were obtained. ADC samples incubated for the specified time were analyzed by RP-HPLC.

[0815] 2) Results

[0816] Table 1. DAR values ​​and monomer rate data of ligand-drug conjugates (ADCs) disclosed in the present invention.

[0817] Molecular name DAR Aggregate % monomer% Trastuzumab NA 1.61 98.39 ADC-2 7.67 1.51 98.49 ADC-6 7.55 1.61 98.39 ADC-10 7.66 1.45 98.55 ADC-12 7.64 2.28 97.72 ADC-15 7.63 1.44 98.56 ADC-20 7.60 1.40 98.60 ADC-29 7.66 1.62 98.38 ADC-35 7.59 1.67 98.33 ADC-36 7.68 1.38 98.62 ADC-41 7.64 1.51 98.49 ADC-48 7.67 1.77 98.23 ADC-52 7.58 1.61 98.39 ADC-56 7.60 1.61 98.39 ADC-61 (control) 7.59 8.21 91.79

[0818] Table 2. Plasma stability data of ligand-drug conjugates (ADCs) disclosed in the present invention.

[0819]

[0820] 3) Conclusion

[0821] As shown in Table 1, the camptothecin ADC with a highly stable hydrophilic linker disclosed in the present invention has excellent properties of high DAR value (>7.5) and monomer rate (>97%), and has a significantly higher monomer rate than the control ADC-61.

[0822] As shown in Table 2, after 7 days of incubation in plasma, the DAR value of the ADC disclosed in the present invention can still be maintained at a higher level compared with the control ADC-61, demonstrating that the ADC of the present invention has excellent stability in plasma.

[0823] Example 110: In vitro activity test

[0824] 1) Experimental Materials

[0825] Cells: From the Cell Bank of the Chinese Academy of Sciences;

[0826] Tumor cell culture medium: Gibco;

[0827] FBS: BIOWEST;

[0828] 2) Preparation of culture medium

[0829] Growth medium (with 10% FBS, Penicillin / streptomycin (100 U / mL);

[0830] Assay medium (with 1% FBS, Penicillin / streptomycin (100 U / mL);

[0831] 3) Operation

[0832] Turn on the UV light of the biosafety cabinet 30 minutes in advance, and then ventilate for 3 minutes. Preheat the growth medium, detection medium, D-PBS and trypsin in a 37°C constant temperature water bath, then disinfect the surface with alcohol and place it in the biosafety cabinet. Select cells with a confluence of ~80% (logarithmic growth phase), place them in the biosafety cabinet, remove the old culture medium, rinse with D-PBS, discard, digest with trypsin for 2-3 minutes, then add growth medium to stop trypsin, and centrifuge at 500×g for 5 minutes. Aspirate the centrifugal supernatant, mix with 4mL detection medium, and take 100uL for counting (take out 50uL of cell fluid, add 50μL0.4% TrypanBlue Stain and mix, and count after mixing). Plate according to the previously set cell number, 80uL / well in a 96-well plate, add only 80uL detection medium to wells E11, F11, and G11, and add 200uL of DPBS to the edge wells to seal. After the plated cells have completely adhered (usually requiring at least 4 hours), prepare and dilute the test sample: Prepare 1.0mL of 2.5μM (5× Top Dose) test sample with assay medium and dispense it into the first column of a V-shaped 96-well plate, 200μL per well. Add 180μL of assay medium to each of columns 2 to 8. Take 30μL from the first column and add it to the second column. Mix up and down 10 times with a pipette, discard the pipette tip, and repeat the same process for the remaining test concentration points, performing a 7-fold gradient dilution. Add 20uL of the gradient concentration test sample to the cells per well. At the same time, add only 20uL of assay medium to column 11. Set up three replicates for each concentration. Then, place the 96-well plate in a 5% CO2, 37°C cell culture incubator and incubate for 5 days.

[0833] 4) Detection

[0834] After 5 days of exposure to the test sample, remove the MTS reagent, thaw at room temperature in the dark, and thoroughly vortex to mix. In a biosafety cabinet, add 20 μL of Cell Titer One Solution Reagen MTS reagent per 100 μL of cell culture volume along the side of the wells. Gently tap the plate to mix the MTS solution evenly. Incubate in a cell culture incubator in 5% CO2, 37°C in the dark for 2 hours. After the reaction is complete, remove the 96-well plate and measure the OD490 nm absorbance using a microplate reader. Record, organize, and store the data.

[0835] 5) Results

[0836] Table 3: IC50 values ​​of antibody drug conjugates and toxins for inhibition of N87 tumor cell proliferation in vitro.

[0837]

[0838] Table 4: IC50 values ​​of antibody drug conjugates and toxins for inhibition of SK-BR-3 tumor cell proliferation in vitro.

[0839]

[0840] 6) Discussion

[0841] As shown in Table 3, the ligand-drug conjugate targeting HER2 of the present invention has significant in vitro proliferation inhibitory activity against HER2-positive cells N87, which is significantly better than naked antibody (Trastuzumab), control group ADC-61 and toxin alone.

[0842] As shown in Table 4, compared with naked antibody (Trastuzumab) and control ADC, the ADC and single drug disclosed in the present invention also have significant in vitro proliferation inhibition activity against HER2-positive cells SK-BR-3.

[0843] Example 111: In vivo activity test

[0844] 1) Experimental Materials

[0845] Cells: From the Cell Bank of the Chinese Academy of Sciences;

[0846] Tumor cell culture medium: Gibco;

[0847] Balb / c-nu nude mice: female, 5-7 weeks (age of mice at the time of tumor cell inoculation), weighing 18.0-24.0 g, 170 mice (110 plus 60 surplus mice), purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0848] Test and control articles:

[0849] Test samples: ADC-61 and ADC-6 were provided by Chengdu Duote Antibody Drug Co., Ltd.

[0850] Histidine buffer was provided by Chengdu Duote Antibody Drug Co., Ltd.

[0851] 0.9% sodium chloride injection: Kelun Pharmaceutical Co., Ltd.

[0852] 2) Cell culture

[0853] NCI-H1975 (human non-small cell lung cancer adenocarcinoma cells) were cultured in RPMI 1640 medium. NCI-H1975 cells were collected during the exponential growth phase, resuspended in RPMI 1640 medium to an appropriate concentration, and then used for subcutaneous tumor inoculation in mice.

[0854] NCI-N87 (human gastric cancer cells) were cultured in RPMI 1640 medium. NCI-N87 cells were collected during the exponential growth phase, resuspended in RPMI 1640 medium to a suitable concentration, and then used for subcutaneous tumor inoculation in mice.

[0855] 3) Animal modeling and random grouping

[0856] 85 female nude mice were subcutaneously inoculated with 5×10 7 NCI-H1975 cells. The average tumor volume is 170 mm 3 Around day 0, the mice were randomly divided into groups according to tumor size. Fifty-five tumor-bearing mice with appropriate tumor volumes were randomly divided into groups and drug administration was initiated (injected into the tail vein at a volume of 0.1 ml / 10 g). The day of grouping was defined as day 0.

[0857] 85 female nude mice were subcutaneously inoculated with 5×10 7 NCI-N87 cells. The average tumor volume is 170 mm 3 Around day 0, the mice were randomly divided into groups according to tumor size. Fifty-five tumor-bearing mice with appropriate tumor volumes were randomly divided into groups and drug administration was initiated (injected into the tail vein at a volume of 0.1 ml / 10 g). The day of grouping was defined as day 0.

[0858] 4) Preparation of test and reference substances

[0859] Table 5. Preparation of test article and control solution for anti-tumor effect study in NCI-H1975 (human non-small cell lung cancer cell) and NCI-N87 (human gastric cancer cell) nude mouse subcutaneous xenograft tumor models.

[0860]

[0861] Note: Mix well before use to ensure the preparation is homogeneous.

[0862] 5) Experimental observation and data collection

[0863] During this experiment, animal experimental operations were carried out in accordance with the requirements of the standard operating procedures for in vivo screening tests of anti-tumor drugs. After tumor inoculation, routine monitoring included tumor growth (tumor was measured twice a week) and the effect of treatment on the normal behavior of the animals. Specifically, the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eyes, fur and other abnormalities. Clinical symptoms observed during the experiment were recorded in the original data. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the long diameter and b represents the short diameter). The data were recorded manually, including the measurement of the long and short diameters of the tumor and the weighing of the animal body weight.

[0864] 6) Efficacy evaluation criteria

[0865] The relative tumor growth rate, T / C%, is the percentage of the tumor volume or weight of the treatment group and the control group at a certain time point. The calculation formula is as follows:

[0866] T / C% = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt / V0, V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment); or T / C% = TTW / CTW × 100% (TTW: average tumor weight of the treatment group at the end of the experiment; CTW: average tumor weight of the vehicle control group at the end of the experiment).

[0867] The relative tumor inhibition rate (TGI) was calculated as follows: TGI% = (1-T / C) × 100% [T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment and control groups at a specific time point, respectively].

[0868] 7) Results

[0869] Table 6: In vivo efficacy of administered antibody drug conjugates on NCI-H1975 xenograft tumors.

[0870]

[0871] Table 7: In vivo efficacy of administered antibody drug conjugates on NCI-N87 xenograft tumors.

[0872]

[0873] Table 8: Effects of administration of antibody drug conjugates (11.25 mg / kg) on ​​body weight of mice bearing NCI-H1975 tumor xenografts.

[0874] NCI-H1975\Group Information\Average Weight (g) D0 D3 D7 D10 D14 D17 D21 D24 D28 D31 Vehicle 19.83 20.52 20.13 19.79 20.42 19.83 19.57 20.45 18.21 19.79 ADC-6 (11.25 mg / kg) 19.99 20.66 19.70 20.46 20.65 19.99 20.68 19.89 20.52 20.69 ADC-61 (11.25 mg / kg) control 19.78 20.55 19.52 19.32 19.57* 19.78* 19.36* 19.59* 20.14* 18.99*

[0875] Note: Two mice died in the group marked with *

[0876] 8) Discussion

[0877] As shown in Table 6, the in vivo efficacy of ADC-6 disclosed in the present invention in the low-dose control group (3.75 mg / Kg) in NCI-H1975 tumor-bearing mice was significantly better than that of the control group ADC-61 and naked antibody. When the dose was increased to 11.25 mg / Kg, the therapeutic effect of ADC-6 disclosed in the present invention was further enhanced and was significantly better than that of the control ADC-61.

[0878] As shown in Table 7, at the same dose (3.75 mg / Kg), the ADC-6 disclosed in the present invention has a significantly better in vivo efficacy on tumor-bearing mice NCI-N87 than the control group ADC-61, and is more significant than the high-dose naked antibody (11.25 mg / Kg).

[0879] As shown in Table 8, the effect of ADC-6 disclosed in the present invention on the body weight of NCI-H1975 tumor-bearing mice in the high-dose control group of 11.25 mg / Kg was significantly smaller than that of ADC-61. Even in this high-dose group, no mouse deaths occurred as shown in the control group, demonstrating that the ADC drug disclosed in the present invention has significant advantages in terms of safety.

Claims

1. A ligand-drug conjugate with a highly stable hydrophilic linker as shown in Formula I, or a pharmaceutically acceptable salt thereof, in: Ab is a ligand unit selected from an antibody, an antibody fragment, a targeting protein or an Fc-fusion protein; M is a linker unit connected to Ab, and the linker unit M has a succinimide structure as shown in formula a or a ring-opened succinimide structure as shown in formula b1 or formula b2, In Formula a, Formula b1, or Formula b2, the wavy line on the left indicates connection to the Ab attachment site, and the wavy line on the right indicates connection to the marked tertiary carbon atom attachment site at position 1 in Formula I; Ac is a hydrophilic structural unit, and Ac is connected to the methylene carbon at position 2 indicated in structural formula I through an amino functional group, and Ac is selected from glycine, D / L alanine, D / L serine, D / L cysteine, D / L cystine, D / L arginine, D / L asparagine, D / L glutamine, D / L threonine, D / L aspartic acid, D / L glutamic acid or the following structure: D is a camptothecin drug having a structure as shown in the following formula d; The chiral carbon atom connected to R1 has two absolute configurations: R configuration or S configuration; The hydroxyl group of the carbon atom connected to R1 in the molecule of formula d participates in the connection to form the oxygen atom at position 3 in formula I; The chiral carbon atoms at positions 1 and 4 have two absolute configurations: R configuration or S configuration; n is an integer selected from 1 to 20; The camptothecin drug as a whole is selected from the following structures:

2. A linker-drug compound or a pharmaceutically acceptable salt thereof, characterized in that: Having the structure shown in the following formula II, The chiral carbon atom at position 1 has two absolute configurations: R configuration or S configuration; The linker-drug compound as a whole is selected from the following structures, 3. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that: The ligand-drug conjugate or a pharmaceutically acceptable salt thereof has a structure as shown in Formula III, Formula IV-1 or Formula IV-2 below: Where Ab is the ligand unit; Ac is a hydrophilic structural unit, Ac is as defined in claim 2; The chiral carbon at position 1 has two absolute configurations: R configuration or S configuration; R1, m as defined in claim 2; n is selected from integers of 1-20.

4. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 3, wherein: The ligand unit Ab is selected from an antibody, an antibody fragment or a protein, wherein the antibody is selected from a monoclonal antibody, a mouse antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, a fully human antibody or a bispecific antibody.

5. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 3, wherein: The ligand unit Ab is selected from an antibody, an antibody fragment or a protein, wherein the antibody is a multispecific antibody.

6. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 4, wherein: The antibody is a monoclonal antibody selected from the group consisting of: 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-cladin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 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 antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 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-IntegrinαV antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, or anti-CD123 antibody.

7. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: The antibody is trastuzumab, which has a light chain as shown in SEQ ID NO: 1 and a heavy chain as shown in SEQ ID NO:

2.

8. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 3, wherein: The ligand-drug conjugate or a pharmaceutically acceptable salt thereof is selected from one or more of the following structures or succinimide ring-opening structures thereof: wherein n is selected from an integer of 1-10.

9. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 3, wherein: The ligand-drug conjugate or a pharmaceutically acceptable salt thereof is selected from one or more of the following structures:

10. A method for preparing the linker-drug compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The method comprises the following steps, The compound of formula L reacts with ixetem or its salt of formula d0 in the presence of a condensing agent under alkaline conditions to obtain a compound of formula IV, which is further converted into the structure shown in formula II; The chiral carbon atoms at position 1 and connected to R1 have two absolute configurations: R configuration or S configuration; R2 is a structure that can be converted into Ac; Ac, R1, and m are as defined in claim 2.

11. The method according to claim 10, wherein: The deprotection reagent for converting Formula IV into Formula II is zinc bromide, and the solvent is nitromethane.

12. A method for preparing the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: The method comprises the following steps, After modifying the ligand unit Ab, a coupling reaction is performed with Formula II to obtain a ligand-drug conjugate of Formula III; wherein Ab is selected from an antibody, an antibody fragment or a protein; Ac is selected from a hydrophilic structural unit, Ac is as defined in claim 2; The chiral carbon atom at position 1 and connected to R1 has two absolute configurations: R configuration or S configuration; R1, m and n are as defined in claim 2.

13. A pharmaceutical composition comprising a therapeutically effective amount of the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 and 3-9, or the linker-drug compound or a pharmaceutically acceptable salt thereof according to claim 2, and a pharmaceutically acceptable carrier or excipient.

14. The pharmaceutical composition according to claim 13, wherein The excipient is a diluent.

15. Use of the pharmaceutical composition according to claim 13 or 14 in the preparation of a medicament for treating tumors, autoimmune diseases or infectious diseases.

16. Use of the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 and 3-9, or the linker-drug compound or a pharmaceutically acceptable salt thereof according to claim 2, in the preparation of a medicament for treating tumors, autoimmune diseases or infectious diseases.

17. The use according to claim 15 or 16, characterized in that: The drug is used to treat breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, salivary gland cancer, esophageal cancer, lung cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma or leukemia.

18. The use according to claim 15 or 16, characterized in that: The drug is used to treat endometrial cancer, colon cancer or rectal cancer.

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