WO2021249228A1 - 一种带有高稳定性亲水连接单元的喜树碱类药物及其偶联物 - Google Patents

一种带有高稳定性亲水连接单元的喜树碱类药物及其偶联物 Download PDF

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WO2021249228A1
WO2021249228A1 PCT/CN2021/097302 CN2021097302W WO2021249228A1 WO 2021249228 A1 WO2021249228 A1 WO 2021249228A1 CN 2021097302 W CN2021097302 W CN 2021097302W WO 2021249228 A1 WO2021249228 A1 WO 2021249228A1
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antibody
compound
reaction
add
ligand
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PCT/CN2021/097302
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English (en)
French (fr)
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朱义
万维李
卓识
张勇
张宜英
于天姿
李刚锐
杨秀娟
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四川百利药业有限责任公司
成都多特抗体药物有限责任公司
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Priority to EP21821872.5A priority Critical patent/EP4162954A1/en
Priority to CA3186295A priority patent/CA3186295A1/en
Priority to BR112022024930A priority patent/BR112022024930A2/pt
Priority to JP2022575365A priority patent/JP2023529415A/ja
Priority to IL298787A priority patent/IL298787A/en
Priority to MX2022015695A priority patent/MX2022015695A/es
Priority to US18/008,798 priority patent/US20230226207A1/en
Priority to AU2021289927A priority patent/AU2021289927A1/en
Priority to KR1020237000429A priority patent/KR20230022211A/ko
Publication of WO2021249228A1 publication Critical patent/WO2021249228A1/zh

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    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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Definitions

  • the invention relates to a camptothecin antibody drug conjugate with a high-stability hydrophilic linking structural unit.
  • Antibody-drug conjugates as a new type of targeted drugs, generally consist of three parts: antibodies or antibody-like ligands, small-molecule drugs, and linkers that couple the ligands and drugs.
  • Antibody-drug conjugates use the specific recognition of antibodies to antigens, transport drug molecules to the vicinity of target cells and effectively release drug molecules to achieve the purpose of treatment.
  • FDA U.S. Food and Drug Administration
  • Adcetris TM a new ADC drug developed by Seattle Genetics for the treatment of Hodgkin’s lymphoma and recurrent degenerative large cell lymphoma (ALCL), and its clinical application has been proven The safety and effectiveness of this type of drug are discussed.
  • Camptothecins as small molecule compounds with anti-tumor properties, are known to exhibit anti-tumor effects by inhibiting DNA topoisomerase I, including irinotecan, icinotecan, SN38 and so on. Many camptothecin drugs have been widely used in clinical practice, and the main indications are bone cancer, prostate cancer, breast cancer, pancreatic cancer, etc. Unlike the current clinical use of irinotecan, irinotecan does not need to be activated through the use of enzymes.
  • topoisomerase I has a stronger inhibitory activity and has stronger damage against a variety of cancer cells in vitro. Cell viability.
  • the expression of P-glycoprotein also shows an effect on cancer cells that are resistant to SN-38 and the like. Isinotecan has not been successfully marketed as a single chemotherapeutic drug, which is speculated to be related to its higher cell activity, resulting in a narrow therapeutic window.
  • Antibody-drug conjugate (ADC) drugs have the advantages of increasing water solubility, improving targeting, binding specific antibodies and antigens, carrying drugs around target cells, and effectively killing tumors by releasing drugs near the target cells Cells, reduce toxic side effects. Camptothecin drugs have considerable application prospects in ADC drugs. At present, the antibody conjugate drug trastuzumab deruxtecan (trade name Enhertu) with exenotecan as the toxin has been approved by the U.S. FDA on December 20, 2019. As the first camptothecin ADC drug to be marketed, it has well proved the drug-making ability and application prospects of this type of drug in the ADC field.
  • ADC drug structure includes three key parts: antibody, linker, and toxin. Any part of the defects may affect the overall efficacy of ADC.
  • the structural design defects of Enhertu are obvious: Camptothecin is a class of highly fat-soluble and poorly soluble drugs.
  • the linker-toxin used by Enhertu is designed to be connected to the antibody through a Mc linker, and is connected to a tetrapeptide that can be cleaved.
  • DAR drug-antibody ratio
  • the design of the linker at a high DAR value, will cause the stability of camptothecin ADC drugs to decrease, and the monomer rate will decrease, which will further reduce the efficacy and safety of ADC in vivo.
  • the inventor unexpectedly discovered a series of antibody-drug conjugates of camptothecin derivatives with highly stable hydrophilic polypeptide linking structural units.
  • the ADC molecules of various derivatives of camptothecin carrying the peptide linker show high stability in vivo and in vitro, with a high monomer rate, and have significantly higher pharmacodynamic activity than the control ADC.
  • the inventor created a new deprotection reagent and solvent strategy through the design and innovation of the synthetic route, which can efficiently obtain the complex linker-toxin molecule.
  • One aspect of the present invention provides a ligand-drug conjugate represented by formula I or a pharmaceutically acceptable salt thereof,
  • Ab is a ligand unit, selected from antibodies, antibody fragments or proteins;
  • M is the connection unit connected to Ab
  • D is optional camptothecin drugs
  • the 1-position and 4-position chiral carbon atoms have two chiral configurations: R absolute configuration or S absolute configuration;
  • n is selected from an integer of 1-20.
  • the connecting unit M has a succinimide structure represented by the following formula a or a ring-opened succinimide structure represented by formula b1 and formula b2,
  • the Ac has a structure represented by the following formula c,
  • X is non-limitingly selected from the group consisting of one or more of hydrophilic structure carboxyl group, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid or polyethylene glycol (PEG);
  • Y is an optional scaffold connecting amino group and X;
  • the Ac is non-limitingly selected from glycine, (D/L) alanine, (D/L) leucine, (D/L) isoleucine, (D/L) valine Acid, (D/L) Phenylalanine, (D/L) Proline, (D/L) Tryptophan, (D/L) Serine, (D/L) Tyrosine, (D/L) ) Cysteine, (D/L) Cystine, (D/L) Arginine, (D/L) Histidine, (D/L) Methionine, (D/L) Asparagine, ( D/L) glutamine, (D/L) threonine, (D/L) aspartic acid, (D/L) glutamic acid, natural or unnatural amino acid derivatives or the following structures,
  • the wavy line on the left is connected to the 2nd carbon atom.
  • camptothecin drug having the structure shown in the following formula d,
  • R 1 is selected from hydrogen atom, deuterium atom, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl Or heteroaryl;
  • R 1 and the carbon atom to which it is connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • the chiral carbon atom connected to R 1 has two chirality of R absolute configuration or S absolute configuration;
  • n is selected from 0 or 1;
  • the hydroxyl group of the carbon atom connected to R 1 in the drug d molecule participates in the connection to form the 3-position oxygen atom in formula I.
  • camptothecin drugs provided by the present invention are non-limitingly selected from the following compounds,
  • Another aspect of the present invention provides a linker having the structure shown in the following formula II or a pharmaceutically acceptable salt thereof for coupling with a ligand Ab to form a ligand-drug conjugate shown in formula I -Pharmaceutical compounds,
  • R 1 is selected from hydrogen atom, deuterium atom, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl Or heteroaryl;
  • R 1 and the carbon atom to which it is connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • the chiral carbon atom at position 1 has two chirals: the absolute configuration of R or the absolute configuration of S;
  • n is selected from 0 or 1.
  • the Ac is preferably glycine, phosphoric acid, polyethylene glycol or (D/L) glutamic acid.
  • linker-drug compound provided by the present invention or a pharmaceutically acceptable salt thereof is selected from the following structures without limitation,
  • the 1-position chiral carbon has two configurations of R absolute chirality or S absolute chirality.
  • Another aspect of the present invention provides a structure having the following formula III, formula IV-1 or formula IV-2,
  • the 1-position chiral carbon has two configurations of absolute chirality of R or absolute chirality of S;
  • R 1 , m and n are as described in formula II.
  • 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 murine origin Antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies.
  • the antibody is a monoclonal antibody, which is non-limitingly selected from: 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-AX
  • the antibody or antigen-binding fragment thereof is Trastuzumab, comprising the following sequence:
  • 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 selected from without limitation The following structure or its succinimide ring-opening structure,
  • n is selected from an integer of 1-10.
  • 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:
  • the chiral carbon atom connected to position 1 and R 1 has two chirality of R absolute configuration or S absolute configuration;
  • R 2 is an optional structure that can be converted into Ac
  • R 1 and m are as defined in formula II.
  • 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:
  • Ab is selected from antibody, antibody fragment or protein
  • Ac is selected from hydrophilic structural units
  • the chiral carbon atom connected to position 1 and R 1 has two chirality of R absolute configuration or S absolute configuration;
  • R 1 , m and n are as described in formula II.
  • a pharmaceutical composition which contains 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.
  • the pharmaceutically acceptable salt of the present invention includes sodium salt, potassium salt, calcium salt or magnesium salt formed with the carboxyl functional group in the structural formula, acetate salt, trifluoroethane formed with the nitrogen-containing functional group in the structure Acid salt, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate , Salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate or p-toluenesulfonate.
  • the antibody of the ligand-drug conjugate specifically binds to the target cell of the tumor, autoimmune disease or infectious disease.
  • a ligand-drug conjugate or a pharmaceutically acceptable salt thereof for preparing and treating breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, Bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, Drug use for solid tumors or hematomas such as neuroblastoma, glioma multiforme, sarcoma, lymphoma and leukemia.
  • FIG. 1A SEC-HPLC detection result of Trastuzumab monomer rate disclosed in the present invention.
  • FIG. 1B SEC-HPLC detection result of ADC-2 monomer ratio disclosed in the present invention.
  • FIG. 1C SEC-HPLC detection result of ADC-6 monomer rate disclosed in the present invention.
  • FIG. 1D SEC-HPLC detection result of ADC-10 monomer ratio disclosed in the present invention.
  • FIG. 1E SEC-HPLC detection result of ADC-12 monomer ratio disclosed in the present invention.
  • FIG. 1F The present invention discloses the SEC-HPLC detection result of the ADC-61 monomer ratio of the control group.
  • FIG. 2A RP-HPLC detection result of DAR (drug antibody coupling ratio) value of ADC-02 disclosed in the present invention.
  • FIG. 2B RP-HPLC detection result of DAR (drug antibody coupling ratio) value of ADC-06 disclosed in the present invention.
  • FIG. 2C RP-HPLC detection result of DAR (drug antibody coupling ratio) value of ADC-10 disclosed in the present invention.
  • FIG. 2D RP-HPLC detection result of DAR (drug antibody coupling ratio) value of ADC-12 disclosed in the present invention.
  • FIG. 2E RP-HPLC detection results of the DAR (drug-antibody coupling ratio) value of ADC-61 in the control group disclosed in the present invention.
  • FIG. 3A In vitro drug effects of ADC and naked antibodies disclosed in the present invention on the proliferation of N87 (human gastric cancer cells).
  • Figure 3B The in vitro efficacy of a single agent disclosed in the present invention for inhibiting the proliferation of N87 (human gastric cancer cells).
  • FIG. 4A In vitro drug effect of ADC and naked antibody disclosed in the present invention on the proliferation of SK-BR-3 (human breast adenocarcinoma cells).
  • Figure 4B The in vitro drug effect of the single agent disclosed in the present invention on the proliferation of SK-BR-3 (human breast adenocarcinoma cells).
  • the applicant intends to include the preparation of the brand name product, the generic drug and the active drug portion of the brand name product.
  • the following terms and phrases as used herein are intended to have the following meanings.
  • the brand name includes the product formula, generic drugs, and active pharmaceutical ingredients of the brand name product.
  • ligand is a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell.
  • the role of the ligand is to present the drug to the target cell population that binds to the ligand.
  • ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells.
  • the ligand is expressed as Ab.
  • the ligand can form a linkage with the linking unit through a heteroatom on the ligand, preferably an antibody or an antigen-binding fragment thereof, and the antibody is selected from a chimeric antibody or a human source.
  • Antibodies fully human antibodies or murine antibodies; preferably monoclonal antibodies.
  • the ligand unit is a targeting agent that specifically binds to the target part.
  • the ligand can specifically bind to cellular components or to other target molecules of interest.
  • the target moiety or target is usually on the surface of the cell.
  • 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 (complete) antibodies and antigen-binding fragments thereof.
  • the ligand unit is a non-antibody targeting agent
  • it can be a peptide or polypeptide, or a non-protein molecule.
  • targeting agents include interferons, lymphokines, hormones, growth factors and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell binding molecules or substances.
  • the linker is covalently attached to the sulfur atom of the ligand.
  • the sulfur atom is the sulfur atom of a cysteine residue, which forms an interchain disulfide bond of the antibody.
  • 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.
  • the sulfur atom is the sulfur atom of the cysteine residue that has been introduced into the ligand unit (for example, by site-directed mutagenesis or chemical reaction).
  • the sulfur atom to which the linker binds is selected from the cysteine residues that form the interchain disulfide bond of the antibody or the frontal cysteine residues that have been introduced into the ligand unit (for example, by site-directed mutagenesis or chemical reaction).
  • the EU Kabat ⁇ [Kabat EA et al., (1991)] "Sequences of proteins of Immunological Interest" (Sequences of proteins of Immunological Interest), fifth edition, NIH publication 91-3242 ⁇ Index numbering system.
  • antibody or “antibody unit” includes any part of the structure of an antibody within the scope to which it belongs. This unit can bind, reactively associate, or complex a receptor, antigen, or other receptor unit that the target cell population has.
  • the antibody can be any protein or protein molecule that can bind, complex, or react with a part of the cell population to be treated or biologically modified.
  • the antibody constituting the antibody-drug conjugate in the present invention maintains its original antigen-binding ability in the wild state. Therefore, the antibody of the present invention can specifically bind to the antigen.
  • the antigens involved include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, and molecules related to tissue growth and differentiation (as known or predicted Functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (as known or predicted to be functional).
  • TAA tumor-associated antigens
  • cell survival regulators cell proliferation regulators
  • lymphokines cytokines
  • cytokines molecules involved in cell cycle regulation
  • molecules involved in angiogenesis molecules related to angiogenesis
  • angiogenesis as known or predicted to be functional
  • the tumor-related factor may be a cluster differentiation factor (such as CD protein).
  • Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies directed against cell surface receptors and tumor-associated antigens.
  • tumor-associated antigens are well-known in the industry and can be prepared by antibody preparation methods and information well-known in the industry.
  • researchers are trying to find transmembrane or other tumor-related peptides. These targets can be specifically expressed on the surface of one or more cancer cells, but little or no expression on the surface of one or more non-cancer cells.
  • tumor-associated polypeptides are more overexpressed on the surface of cancer cells compared to the surface of non-cancer cells.
  • tumor-related factors can greatly improve the specific targeting properties of antibody-based treatment of cancer.
  • antigen-related information that is well-known in the industry is marked as follows, including name, other names, and gene bank accession number.
  • Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases, such as Genbank.
  • Antibodies targeting corresponding tumor-associated antigens include all amino acid sequence variants and homologues, and have at least 70%, 80%, 85%, 90% or 95% homology with the sequence confirmed in the reference, or have the same as the cited sequence
  • the tumor-associated antigen sequences in the literature have completely consistent biological properties and characteristics.
  • inhibitor refers to reducing the detectable amount, or preventing it completely.
  • cancer refers to a physiological condition or disease characterized by unregulated cell growth.
  • Tumor includes cancer cells.
  • autoimmune disease is a disease or disorder derived from an individual's own tissues or proteins.
  • drug refers to a cytotoxic drug, which means d, a chemical molecule that can strongly disrupt the normal growth of tumor cells.
  • cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to lack of specificity, while killing tumor cells, it will also cause normal cell apoptosis, leading to serious side effects.
  • the term includes toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g.
  • camptothecin drugs refers to cytotoxic camptothecin and its derivatives, which are non-limitingly selected from 10-hydroxycamptothecin, SN38 (7-ethyl-10-hydroxycamptothecin), topological Tecan, Isinotecan, Irinotecan or 9-nitro-10-hydroxycamptothecin and its derivatives or pharmaceutically acceptable salts.
  • linker or “linking fragment” or “linking unit” refers to a chemical structural fragment or bond with one end connected to a ligand and the other end to a drug. It can also be connected to other linkers before being connected to the drug.
  • Linkers including extensions, spacers and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the linker may be a "cleavable linker" that facilitates the release of the drug in the cell.
  • acid-labile linkers such as hydrazone
  • protease-sensitive such as peptidase-sensitive
  • light-labile linkers dimethyl linkers
  • disulfide-containing linkers Chargei et al. Cancer Research 52:127-131, 1992
  • US Patent No. 5,208,020 US Patent No. 5,208,020.
  • linkers or “linkers of antibody-drug conjugates” can be divided into two categories: non-cleavable linkers and cleavable linkers.
  • the drug release mechanism is as follows: After the conjugate binds to the antigen and is endocytosed by the cell, the antibody is enzymatically digested in the lysosome to release small-molecule drugs. , Linker, and active molecule composed of antibody amino acid residues. The resulting changes in the structure of drug molecules do not reduce their cytotoxicity, but because the active molecules are charged (amino acid residues), they cannot penetrate neighboring cells. Therefore, such active drugs cannot kill adjacent tumor cells that do not express the targeted antigen (antigen-negative cells) (bystander effect) (Ducry et al., 2010, Bioconjugate Chem. 21:5-13).
  • ligand-drug conjugate refers to that the antibody is connected to a biologically active drug through a stable linking unit.
  • ligand-drug conjugate is preferably antibody-drug conjugate (antibody drug conjugate, ADC), which refers to the connection of a monoclonal antibody or antibody fragment to a toxic drug with biological activity through a stable connecting unit .
  • ADC antibody drug conjugate
  • 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 containing 1 to 10 carbons The most preferred is 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-methylhe
  • lower alkyl groups containing 1 to 6 carbon atoms More preferred are lower alkyl groups containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and sec-butyl.
  • Alkyl groups may be substituted or unsubstituted.
  • substituents When substituted, substituents may be substituted at any available attachment point.
  • the substituents are preferably one or more of the following groups, which are independently selected from alkanes Group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane Oxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.
  • substituted alkyl means that the hydrogen in the alkyl group is replaced by a substituent group.
  • R', R" and R"' each independently refers to hydrogen, unsubstituted C 1-8 alkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C 1-8 alkyl, C 1- 8 alkoxy or C 1-8 thioalkoxy, or unsubstituted aryl -C 1-4 alkyl .
  • -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O or S, where the alkyl group is as defined above.
  • alkylene refers to a saturated linear or branched aliphatic hydrocarbon group, which has two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, which is A straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, more preferably an alkylene group containing 1 to 6 carbon atoms.
  • alkylene groups include, but are not limited to, methylene (-CH 2 -, 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene ( -CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and 1,5-butylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -), etc.
  • the alkylene group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is preferably independently optionally selected from alkyl, alkenyl, alkyne Group, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy Substituted by one or more substituents in the group, cycloalkylthio group, heterocycloalkylthio group and oxo group.
  • alkoxy refers to -O-(alkyl) and -O-(cycloalkyl), where alkyl or cycloalkyl is as defined above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • the alkoxy group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent.
  • the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 Carbon atoms, most preferably containing 3 to 8 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Groups, cyclooctyl, etc.; polycyclic cycloalkyls include spiro, fused, and bridged cycloalkyls.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, which contains 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (wherein m is an integer of 0 to 2) heteroatoms, but does not include the ring part of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.
  • cycloalkylalkyl refers to an alkyl group substituted with one or more cycloalkyl groups, preferably with a cycloalkyl group, where alkyl is as defined above and where cycloalkyl is as defined above.
  • haloalkyl refers to an alkyl group substituted with one or more halogens, where the alkyl group is as defined above.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms, where the alkyl group is as defined above.
  • hydroxyl refers to the -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH 2 .
  • nitro refers to -NO 2 .
  • amido refers to -C(O)N(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.
  • carboxylate group refers to -C(O)O(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.
  • aryl refers to a 6 to 14-membered all-carbon monocyclic or fused polycyclic (ie, rings sharing adjacent pairs of carbon atoms) group with a conjugated ⁇ -electron system, preferably 6-10 members, such as phenyl .
  • the aryl group may be substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are not limitedly selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, and alkylthio , Alkylamino, halogen, deuterium atom, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylsulfide Group or heterocycloalkylthio.
  • groups which are not limitedly selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, and alkylthio , Alkylamino, halogen, deuterium atom, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycl
  • the present invention also includes various deuterated forms of Formula I.
  • Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom.
  • Those skilled in the art can refer to relevant literature to synthesize the deuterated form 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, tri-deuterated Borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide, etc.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the antibodies of the present invention are preferably specific antibodies against cell surface antigens on target cells.
  • Non-limiting examples are the following antibodies: anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, and 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-PT
  • solvate or “solvent compound” means that the ligand-drug conjugate of the present invention forms a pharmaceutically acceptable solvate with one or more solvent molecules.
  • solvent molecules include water, ethanol, and acetonitrile. , Isopropanol, DMSO, ethyl acetate.
  • drug loading refers to the average amount of cytotoxic drug loaded on each antibody in Formula I, and can also be expressed as the ratio of drug amount to antibody amount, and the range of drug loading can be connected to each antibody (Ab). -12, preferably 1-10 cytotoxic drugs (D).
  • the drug loading amount is expressed as n, which can be an exemplary average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • Conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test and HPLC characteristics can be used to identify the average number of drug products per ADC molecule after the coupling reaction.
  • the cytotoxic drug is coupled to the interchain cysteine sulfhydryl-SH opened by the antibody and/or to the cysteine sulfhydryl-SH of site-directed mutation through a linking unit, generally, coupled
  • a linking unit generally, coupled
  • the number of drug molecules that can be coupled with the antibody in the reaction will be less than or equal to the theoretical maximum.
  • ligand cytotoxic drug conjugates including:
  • pharmaceutically acceptable salt refers to the salt of the ligand-drug conjugate of the present invention, or the salt of the compound described in the present invention, when such salts are used in mammals. It is safe and effective, and has due biological activity.
  • the ligand-drug coupling compound of the present invention contains at least one carboxyl group, so it can form a salt with a base.
  • pharmaceutically acceptable salts include: sodium Salt, potassium salt, calcium salt or magnesium salt, etc.
  • pharmaceutically acceptable salt refers to the salt of the antibody-drug conjugate of the present invention, or the salt of the compound described in the present invention. Such salts have It is safe and effective, and has due biological activity.
  • the ligand-drug conjugate compound of the present invention contains at least one amino group, so it can form a salt with an acid.
  • Non-limiting examples of pharmaceutically acceptable salts include: hydrochloric acid Salt, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, phosphoric acid Dihydrogen salt, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate , P-toluenesulfonate.
  • Acid amino acid refers to an amino acid whose isoelectric point is less than 7. Acidic amino acid molecules often contain one or more acidic groups such as carboxyl groups, which can be effectively ionized into negative ions in the structure to increase hydrophilicity. Acidic amino acids can be natural or unnatural amino acids.
  • Natural amino acids refer to amino acids synthesized by biology. Natural amino acids are generally L-type, but there are a few exceptions, such as glycine, including natural and biosynthetic.
  • Unnatural amino acid refers to an amino acid obtained by synthetic means.
  • reaction solution1 Add 7a (5g, 9.2mmol) and 15mL DMF to a 50mL single-necked flask, after dissolving it, add DBU (1.68g, 11mmol) in an ice-water bath, and react for 1 hour, which is recorded as reaction solution1;
  • Step 2 Compound 8e-1 and Compound 8e-2
  • reaction solution1 15a (5g, 9.95mmol) and 15mL DMF to a 50mL single-necked flask. After dissolving it, add DBU (1.68g, 11mmol) in an ice-water bath, and react for 1 hour, which is recorded as reaction solution1;
  • reaction solution1 1;
  • reaction solution1 1;
  • reaction solution1 20a (4g, 7.6mmol) and 10mL DMF to a 50mL single-necked flask. After dissolving, add DBU (1.39g, 9.1mmol) in an ice-water bath, and react for 1 hour, which is recorded as reaction solution1;
  • Step 4 Compound 20e-1 and 20e-2
  • Step 2 Compound (R)-2-hydroxy-1,5-glutaric acid tert-butyl ester
  • the antibody molecules whose monomer ratio is greater than 95% are exchanged into a phosphate buffer solution with an ultrafiltration centrifuge tube at a concentration of 10 mg/mL.
  • the linker-drug compound (payload) was added 20 times the number of mole molecules of the antibody, and reacted for 2 hours at room temperature.
  • use an ultrafiltration centrifuge tube with a molecular weight cut-off of 30KDa to exchange the liquid into PBS, and remove uncoupled payload.
  • the ADC sample is filtered with a 0.22 micron sterile filter for use.
  • the sample was centrifuged at 14000 rpm for 5 minutes, and the supernatant was taken for analysis;
  • Mobile phase A: 50mM PB, 300mM NaCl, 200mM Arg, 5% IPA, pH 6.5;
  • the mobile phase A was eluted isocratically for 30 min, flow rate: 0.714 mL/min, column temperature 25°C, detection wavelength: 280 nm.
  • the sample was centrifuged at 14000 rpm for 5 minutes, and the supernatant was taken for analysis;
  • Mobile phase A: 1.5M ammonium sulfate, 0.025M anhydrous sodium phosphate, pH 7.0, B: 0.025M anhydrous sodium phosphate, 25% IPA, pH 7.0;
  • the mobile phase A equilibrates the chromatographic column, the mobile phase A and B are gradient eluted, the flow rate is 0.8mL/min; the column temperature is 25°C, and the detection wavelength is 214nm.
  • ADC-1 was prepared according to the general coupling method.
  • ADC-2 was prepared according to the general coupling method.
  • ADC-3 was prepared according to the general coupling method.
  • ADC-4 was prepared according to the general coupling method.
  • ADC-5 was prepared according to the general coupling method.
  • ADC-6 was prepared according to the general coupling method.
  • ADC-7 was prepared according to the general coupling method.
  • ADC-8 was prepared according to the general coupling method.
  • ADC-9 was prepared according to the general coupling method.
  • ADC-10 was prepared according to the general coupling method.
  • ADC-11 was prepared according to the general coupling method.
  • ADC-12 was prepared according to the general coupling method.
  • ADC-13 was prepared according to the general coupling method.
  • ADC-14 was prepared according to the general coupling method.
  • ADC-15 was prepared according to the general coupling method.
  • ADC-16 was prepared according to the general coupling method.
  • ADC-17 was prepared according to the general coupling method.
  • ADC-18 was prepared according to the general coupling method.
  • ADC-19 was prepared according to the general coupling method.
  • ADC-20 was prepared according to the general coupling method.
  • ADC-21 was prepared according to the general coupling method.
  • ADC-22 was prepared according to the general coupling method.
  • ADC-23 was prepared according to the general coupling method.
  • ADC-24 was prepared according to the general coupling method.
  • ADC-25 was prepared according to the general coupling method.
  • ADC-26 was prepared according to the general coupling method.
  • ADC-27 was prepared according to the general coupling method.
  • ADC-28 was prepared according to the general coupling method.
  • ADC-29 was prepared according to the general coupling method.
  • ADC-30 was prepared according to the general coupling method.
  • ADC-31 was prepared according to the general coupling method.
  • ADC-32 was prepared according to the general coupling method.
  • ADC-33 was prepared according to the general coupling method.
  • ADC-34 was prepared according to the general coupling method.
  • ADC-35 was prepared according to the general coupling method.
  • ADC-36 was prepared according to the general coupling method.
  • ADC-37 was prepared according to the general coupling method.
  • ADC-38 was prepared according to the general coupling method.
  • ADC-39 was prepared according to the general coupling method.
  • ADC-40 was prepared according to the general coupling method.
  • ADC-41 was prepared according to the general coupling method.
  • ADC-42 was prepared according to the general coupling method.
  • ADC-43 was prepared according to the general coupling method.
  • ADC-44 was prepared according to the general coupling method.
  • ADC-45 was prepared according to the general coupling method.
  • ADC-46 was prepared according to the general coupling method.
  • ADC-47 was prepared according to the general coupling method.
  • ADC-48 was prepared according to the general coupling method.
  • ADC-49 was prepared according to the general coupling method.
  • ADC-50 was prepared according to the general coupling method.
  • ADC-51 was prepared according to the general coupling method.
  • ADC-52 was prepared according to the general coupling method.
  • ADC-53 was prepared according to the general coupling method.
  • ADC-54 was prepared according to the general coupling method.
  • ADC-55 was prepared according to the general coupling method.
  • ADC-56 was prepared according to the general coupling method.
  • ADC-57 was prepared according to the general coupling method.
  • ADC-58 was prepared according to the general coupling method.
  • ADC-59 was prepared according to the general coupling method.
  • ADC-60 was prepared according to the general coupling method.
  • Example 108 ADC-61 (control group)
  • ADC-61 was prepared according to the general coupling method.
  • Example 109 Plasma stability
  • ADC samples Take a certain amount of ADC samples and add them to human plasma from which human IgG has been removed. Repeat three tubes of each ADC and place them in a 37°C water bath. After incubating for 72h and 144h respectively, take out the ADC samples and add ProteinA resin(MabSelect) to each tube. SuReTM LX Lot: #10221479GE, 100uL washed with PBS, the vertical mixer is shaken and adsorbed for 2h, after washing and elution steps, the ADC after incubation is obtained. The ADC samples incubated for a specific time were detected by RP-HPLC.
  • the camptothecin ADCs with highly stable hydrophilic linking units disclosed in the present invention have excellent properties of high DAR value (>7.5) and high monomer ratio (>97%), compared to the control ADC-61 has a significantly higher monomer rate.
  • the DAR value can still maintain a higher level compared to the control ADC-61, which proves that the ADC of the present invention has excellent stability in plasma.
  • Example 110 In vitro activity test
  • Tumor cell culture medium Gibco;
  • Detection medium (with 1% FBS, Penicillin/streptomycin (100U/mL);
  • UV lamp in the biological safety cabinet 30 minutes in advance, and ventilate for 3 minutes.
  • the growth medium, detection medium, D-PBS and pancreatin into a 37°C constant temperature water bath to preheat, then disinfect the surface with alcohol and put it in a biological safety cabinet.
  • Select cells with a confluence of ⁇ 80% (logarithmic growth phase) put them in a biological safety cabinet, aspirate the old medium, rinse with D-PBS, aspirate and discard, digest with trypsin for 2 to 3 minutes, and then add to growth Stop trypsin in the medium, and centrifuge at 500 ⁇ g for 5 min.
  • test sample prepare 1.0mL, 2.5 ⁇ M (5 ⁇ Top Dose) test sample with the detection medium, and aliquot it in V Type 96-well plate in the first column, 200 ⁇ L per well; add 180 ⁇ L of detection medium from the second to the eighth column, take 30 ⁇ L from the first column and add to the second column, mix up and down 10 times with a row gun, discard the pipette tip , The remaining detection concentration points are operated in sequence, and a 7-fold gradient concentration dilution is performed. Add the test sample of gradient concentration to the cells in the amount of 20uL per well. At the same time, add only 20uL of detection medium in the 11th column, set 3 replicate wells for each concentration, and then put the 96-well plate into 5% CO 2 , 37 °C cell incubator, culture for 5 days.
  • MTS reagent Take out the MTS reagent after the test sample is exposed for 5 days. After thawing at room temperature and avoiding light, vortex and mix thoroughly. In a biological safety cabinet, add 20 ⁇ L Cell Titer One Solution Reagen MTS reagent for every 100 ⁇ L cell culture volume along the side wall of the well. Gently tap the surface of the plate to mix the MTS solution evenly, and place it in a cell incubator with 5% CO 2 , and incubate at 37°C in the dark for 2 hours. After the reaction, the 96-well plate was taken out, the OD490nm absorbance value was detected in the microplate reader, and the data was recorded, sorted, and stored.
  • Table 3 The IC50 value of the in vitro proliferation inhibition of N87 tumor cells by antibody-drug conjugates and toxins.
  • Table 4 IC50 value of the inhibition of the in vitro proliferation of SK-BR-3 tumor cells by antibody-drug conjugates and toxins.
  • the ligand-drug conjugate of the present invention for HER2 target has obvious in vitro proliferation inhibitory activity on HER2 positive cells N87, which is significantly better than naked antibody (Trastuzumab), control group ADC-61 and toxin single drug .
  • the ADC and the single agent disclosed in the present invention also have obvious in vitro proliferation inhibitory activity on HER2-positive cells SK-BR-3.
  • Example 111 In vivo activity test
  • Tumor cell culture medium Gibco;
  • Balb/c-nu nude mice female, 5-7 weeks (the age of mice at the time of tumor cell inoculation), weighing 18.0-24.0 g, 170 (110 plus 60 surplus mice). Purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.;
  • Test products ADC-61 and ADC-6 were provided by Chengdu Dote Antibody Drug Co., Ltd.
  • Histidine buffer provided by Chengdu Dote Antibody Pharmaceutical Co., Ltd.
  • NCI-H1975 human non-small cell lung cancer adenocarcinoma cells
  • RPMI1640 medium was cultured in RPMI1640 medium.
  • NCI-H1975 cells in the exponential growth phase were collected and resuspended in RPMI1640 medium to a suitable concentration for subcutaneous tumor inoculation in mice.
  • NCI-N87 human gastric cancer cells
  • RPMI1640 medium was resuspended to a suitable concentration for subcutaneous tumor inoculation in mice.
  • mice 85 female nude mice were inoculated subcutaneously on the right shoulder with 5 ⁇ 10 7 NCI-H1975 cells. When the average tumor volume is about 170mm 3 , they are randomly grouped according to the tumor size. Fifty-five tumor-bearing mice with appropriate tumor volume were selected and randomly divided into groups and the administration was started (tail vein injection, the administration volume was 0.1ml/10g). The grouping day is defined as day 0.
  • mice 85 female nude mice were inoculated subcutaneously on the right shoulder with 5 ⁇ 10 7 NCI-N87 cells. When the average tumor volume is about 170mm 3 , they are randomly grouped according to the tumor size. Fifty-five tumor-bearing mice with appropriate tumor volume were selected and randomly divided into groups and the administration was started (tail vein injection, the administration volume was 0.1ml/10g). The grouping day is defined as day 0.
  • tumor inoculation After tumor inoculation, routine monitoring includes tumor growth (the tumor is measured twice a week) and the effect of treatment on the normal behavior of the animal.
  • the specific content includes the activity of the experimental animal, food and drinking status, weight gain or loss (weight is measured weekly 2 times) Conditions, eyes, coat and other abnormal conditions.
  • the 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).
  • Manually recorded data was used in the experiment, including the measurement of the length and short diameter of the tumor and the weighing of the animal's weight.
  • the relative tumor proliferation rate, T/C% is the percentage value of the treatment group and the control group relative to the tumor volume or tumor weight at a certain point in time. Calculated as follows:
  • TGI% (1-T/C) ⁇ 100%.
  • T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment group and the control group at a specific time point, respectively].
  • Table 7 In vivo efficacy of administration of antibody-drug conjugates on NCI-N87 xenografts.
  • Table 8 Effect of administration of antibody drug conjugate (11.25 mg/kg) on the body weight of NCI-H1975 transplanted tumor mice.
  • the ADC-6 disclosed by the present invention in the low-dose control group (3.75mg/Kg) has significantly better in vivo efficacy on tumor-bearing mice NCI-H1975 than the control group ADC-61 and naked antibody; when the dose is increased to At 11.25 mg/Kg, the therapeutic effect of ADC-6 disclosed in the present invention is further improved, and is significantly better than the control ADC-61.
  • the ADC-6 disclosed in the present invention has significantly better in vivo efficacy on tumor-bearing mice NCI-N87 than the control group ADC-61. Compared with the high-dose naked antibody (11.25mg) /Kg) The effect of the medicine in the body is more significant.
  • the present invention discloses that the 11.25mg/Kg of ADC-6 in the high-dose control group has a significantly smaller effect on the body weight of NCI-H1975 tumor-bearing mice than ADC-61, even under the high-dose group , There was no death of mice as shown in the control group, which proves that the ADC drug of the present invention has a significant advantage in terms of safety.

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Abstract

一种带有高稳定性亲水连接单元的喜树碱类药物及其偶联物或其药学上可接受的盐，包括其制备方法和在预防或治疗癌症中的作用。所述偶联物能够特异性地结合肿瘤细胞中高表达的受体。具有良好的水溶性，稳定性及均一性，可用于预防或治疗肿瘤等疾病。

Description

一种带有高稳定性亲水连接单元的喜树碱类药物及其偶联物 技术领域
本发明涉及一种带有高稳定性亲水连接结构单元的喜树碱类抗体药物偶联物。
背景技术
抗体药物偶联物(ADC)作为新型的靶向药物，一般由三部分组成：抗体或抗体类配体，小分子药物以及将配体和药物偶联起来的连接子。抗体药物偶联物利用抗体对抗原的特异性识别，将药物分子运输至靶细胞附近并有效释放药物分子，达到治疗目的。2011年8月，美国食品药品监督管理局(FDA)批准西雅图基因公司研制的用于治疗霍奇金淋巴瘤以及复发性变性大细胞淋巴瘤(ALCL)的ADC新药Adcetris TM上市，临床应用已经证明了此类药物的安全性和有效性。
喜树碱类药物作为抗肿瘤性的小分子化合物，已知作为抑制DNA拓扑异构酶I而呈现抗肿瘤作用，包括伊立替康，依喜替康，SN38等。许多喜树碱类药物已在临床中广泛应用，主要适应症为骨癌、前列腺癌、乳腺癌、胰腺癌等。与目前临床使用的伊立替康不同，依喜替康不需要通过利用酶进行活化。另外，与作为伊立替康的药效本体的SN-38、同在临床中使用的拓扑替康相比，拓扑异构酶I抑制活性更强，在体外针对多种癌细胞具有更强的伤细胞活性。尤其是，对于通过P-糖蛋白的表达面对SN-38等显示耐性的癌细胞也显示效果。依喜替康作为单独化疗药物尚未成功上市，推测与其较高的细胞活性相关，导致治疗窗口窄。
抗体药物偶联物(ADC)类药物的优势在于增加水溶性，提高靶向性，特异性抗体与抗原的结合，将药物携带至靶细胞周围，通过在靶细胞附近释放药物，有效杀灭肿瘤细胞，降低毒副作用。喜树碱类药物在ADC药物中具有可观的应用前景，目前以依喜替康为毒素的抗体偶联药物trastuzumab deruxtecan(商品名为Enhertu)已于2019年12月20日被美国FDA获批上市，作为第一个上市的喜树碱类ADC药物，很好证明了此类药物在ADC领域的成药能力和应用前景。
ADC药物结构中包括了抗体，连接子，及毒素三个关键部分。任何一部分的缺陷都可能影响ADC的整体药效。在本领域，Enhertu的结构设计缺陷是显而易见的：喜树碱为一类高脂溶性，难溶性药物，Enhertu采用的连接子-毒素设计为通过Mc接头与抗体连接，连接可酶切的四肽片段，搭配氨甲氧基自我消除的间隔单元，采用链间半胱氨酸残基以非定点偶联技术实现药物-抗体比例(DAR)为8(参考专利CN104755494)。该连接子的设计，在高DAR值下，将导致喜树碱类ADC药物稳定性降低，单体率降低，进一步降低ADC的体内药效和安全性。
本专利所需要解决的技术问题，就是探索发现更优的抗肿瘤喜树碱类ADC药物，使其具有更高的安全性、有效性，更好满足临床需求。
发明内容
发明人在对ADC类药物综合理解的基础上，意外的发现了一系列带有高稳定性的亲水多肽类连接结构单元的喜树碱类衍生物的抗体药物偶联物，通过实验发现，搭载该多肽类连接子的喜树碱各类衍生物ADC分子在体内外表现出很高的稳定性，单体率高，相比于对照ADC具有明显更高的药效活性。同时，发明人通过合成路线的设计创新，创造的采用了全新的脱保护试剂和溶剂策略，可以高效的获得该复杂连接子-毒素分子。
本发明的一个方面提供一种如式I所示的配体-药物偶联物或其药学上可接受的盐，
Figure PCTCN2021097302-appb-000001
其中：
Ab为配体单元，选自抗体、抗体片段或蛋白；
M为连接单元与Ab连接；
Ac为亲水结构单元；
D为任选的喜树碱类药物；
1位及4位手性碳原子具有R绝对构型或S绝对构型两种手性构型；
n选自1-20的整数。
优选的，所述连接单元M具有如下式a所示的丁二酰亚胺或式b1、式b2所示的开环丁二酰亚胺结构，
Figure PCTCN2021097302-appb-000002
在式a、式b1或式b2中，左侧波浪线表示与Ab连接位点相连，右侧波浪线表示与式I中已标示的1位叔碳原子连接位点相连。
进一步优选的，所述Ac具有如下式c所示结构，
Figure PCTCN2021097302-appb-000003
其中X非限制性地选自亲水结构羧基、磷酸、聚磷酸、亚磷酸、磺酸、亚磺酸或聚乙二醇(PEG)中的一种或多种所组成的组；
Y为任选的连接氨基和X的支架；
Ac通过氨基官能团与结构式I中已标示的2位亚甲基碳相连。
更进一步优选，所述Ac非限制性地选自甘氨酸、(D/L)丙氨酸、(D/L)亮氨酸、(D/L)异亮氨酸、(D/L)缬氨酸、(D/L)苯丙氨酸、(D/L)脯氨酸、(D/L)色氨酸、(D/L)丝氨酸、(D/L)酪氨酸、(D/L)半胱氨酸、(D/L)胱氨酸、(D/L)精氨酸、(D/L)组氨酸、(D/L)蛋氨酸、(D/L)天冬酰胺、(D/L)谷氨酰胺、(D/L)苏氨酸、(D/L)天冬氨酸、(D/L)谷氨酸、天然或非天然氨基酸衍生物或以下结构，
Figure PCTCN2021097302-appb-000004
其中左侧波浪线与2号碳原子相连。
本发明的另一个方面，提供具有如下式d所示结构的喜树碱类药物，
Figure PCTCN2021097302-appb-000005
其中R 1选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R 1及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
R 1相连手性碳原子具有R绝对构型或S绝对构型两种手性；
m选自0或1；
药物d分子中与R 1相连碳原子的羟基参与连接形成式I中的3位氧原子。
优选的，本发明提供的喜树碱类药物非限制性地选自以下化合物，
Figure PCTCN2021097302-appb-000006
本发明的另一个方面提供一种具有如下式II所示的结构或其药学上可接受的盐，用于与配体Ab偶联形成式I所示的配体-药物偶联物的连接子-药物化合物，
Figure PCTCN2021097302-appb-000007
其中R 1选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R 1及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
1位手性碳原子具有R绝对构型或S绝对构型两种手性；
Ac为亲水结构单元；
m选自0或1。
本发明的一个方面，所述Ac优选为甘氨酸、磷酸、聚乙二醇或(D/L)谷氨酸。
更进一步优选的，本发明提供的连接子-药物化合物或其药学上可接受的盐非限制性地选自以下结构，
Figure PCTCN2021097302-appb-000008
Figure PCTCN2021097302-appb-000009
Figure PCTCN2021097302-appb-000010
Figure PCTCN2021097302-appb-000011
Figure PCTCN2021097302-appb-000012
Figure PCTCN2021097302-appb-000013
其中1位手性碳具有R绝对手性或S绝对手性两种构型。
本发明的另一个方面提供一种有如下式Ⅲ、式Ⅳ-1或式Ⅳ-2所示结构，
Figure PCTCN2021097302-appb-000014
Figure PCTCN2021097302-appb-000015
其中Ab为配体单元；
Ac为亲水结构单元；
1位手性碳具有R绝对手性或S绝对手性两种构型；
R 1、m及n如式Ⅱ所述。
优选的，本发明提供的配体-药物偶联物或其药学上可接受的盐，其特征在于：所述配体单元Ab选自抗体、抗体片段或蛋白，其中所述抗体选自鼠源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体。
进一步优选地，所述抗体为单克隆抗体，非限制性地选自：抗EGFRvIII抗体、抗PD-1抗体、抗PD-L1抗体、抗DLL-3抗体、抗PSMA抗体、抗CD70抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM-1抗体、抗LRRC15抗体、抗LIV-1抗体、抗CanAg/AFP抗体、抗cladin 18.2抗体、抗Mesothelin抗体、抗HER2(ErbB2)抗体、抗EGFR抗体、抗c-MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7-H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗TROP-2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P-cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD47抗体、抗CD138抗体、抗CD352抗 体、抗CD25抗体或抗CD123抗体。
更进一步优选的，所述抗体或其抗原结合片段为曲妥珠单抗(Trastuzumab)，包含以下序列：
Light chain
Figure PCTCN2021097302-appb-000016
Heavy chain:
Figure PCTCN2021097302-appb-000017
优选的，本发明提供的配体-药物偶联物或其药学上可接受的盐，其特征在于：所述配体-药物偶联物或其药学上可接受的盐非限制性地选自以下结构或其丁二酰亚胺开环结构，
Figure PCTCN2021097302-appb-000018
Figure PCTCN2021097302-appb-000019
Figure PCTCN2021097302-appb-000020
Figure PCTCN2021097302-appb-000021
Figure PCTCN2021097302-appb-000022
Figure PCTCN2021097302-appb-000023
Figure PCTCN2021097302-appb-000024
其中n选自1-10的整数。
本发明的另一个方面，提供一种制备连接子-药物化合物或其药学上可接受的盐的方法，所述方法包含以下步骤，
Figure PCTCN2021097302-appb-000025
通式L化合物与式d 0依喜替康或其盐在缩合剂存在下，在任选的碱性条件下反应，得到式Ⅳ化合物，进一步转化为式Ⅱ所示结构；
其中1位及R 1相连手性碳原子具有R绝对构型或S绝对构型两种手性；
R 2为任选的可转化为Ac的结构；
Ac、R 1、m如式Ⅱ所定义。
本发明的另一方面提供一种制备配体-药物偶联物或其药学上可接受的盐的方法，所述方法包含以下步骤，
Figure PCTCN2021097302-appb-000026
修饰配体单元Ab后，与式Ⅱ通过偶联反应得到式Ⅲ所示配体-药物偶联物；
其中Ab选自抗体、抗体片段或蛋白；
Ac选自亲水结构单元；
1位及R 1相连手性碳原子具有R绝对构型或S绝对构型两种手性；
R 1、m及n如式Ⅱ所述。
本发明的另一个方面，提供一种药物组合物，其含有治疗有效量的配体-药物偶联物或其药学上可接受的盐或溶剂化物，以及药学上可接受的载体、稀释剂或赋形剂。
本发明药学上可接受的盐，所述的成盐形式包括与结构式中羧基官能团形成的钠盐、钾盐、钙盐或镁盐以及与结构中含氮官能团形成的醋酸盐、三氟乙酸盐、柠檬酸盐、草酸盐、酒石酸盐、苹果酸盐、硝酸盐、氯化物、溴化物、碘化物、硫酸盐、硫酸氢盐、磷酸盐、乳酸盐、油酸盐、抗坏血酸盐、水杨酸盐、甲酸盐、谷氨酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐或对甲苯磺酸盐。
本发明的另一个方面，提供一种配体-药物偶联物或其药学可接受的盐的药物组合物，在制备用于治疗肿瘤、自身免疫疾病或感染性疾病药物中的用途，并且所述的配体-药物偶联物的抗体特异性结合至所述肿瘤、自身免疫疾病或感染性疾病的靶细胞。
本发明的另一个方面，提供一种配体-药物偶联物或其药学可接受的盐在用于制备治疗乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、肺癌、结肠癌、直肠癌、结直肠癌、骨癌、皮肤癌、甲状腺癌、胰腺癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、多形性胶质细胞瘤、肉瘤、淋巴瘤和白血病等实体瘤或血液瘤的药物用途。
附图说明
图1A.本发明公开Trastuzumab单体率SEC-HPLC检测结果。
图1B.本发明公开ADC-2单体率SEC-HPLC检测结果。
图1C.本发明公开ADC-6单体率SEC-HPLC检测结果。
图1D.本发明公开ADC-10单体率SEC-HPLC检测结果。
图1E.本发明公开ADC-12单体率SEC-HPLC检测结果。
图1F.本发明公开对照组ADC-61单体率SEC-HPLC检测结果。
图2A.本发明公开ADC-02的DAR(药物抗体偶联比)值RP-HPLC检测结果。
图2B.本发明公开ADC-06的DAR(药物抗体偶联比)值RP-HPLC检测结果。
图2C.本发明公开ADC-10的DAR(药物抗体偶联比)值RP-HPLC检测结果。
图2D.本发明公开ADC-12的DAR(药物抗体偶联比)值RP-HPLC检测结果。
图2E.本发明公开对照组ADC-61的DAR(药物抗体偶联比)值RP-HPLC检测结果。
图3.本发明公开ADC、单药及裸抗对N87(人胃癌细胞)增殖抑制体外药效。
图3A.本发明公开ADC及裸抗对N87(人胃癌细胞)增殖抑制体外药效。
图3B.本发明公开单药对N87(人胃癌细胞)增殖抑制体外药效。
图4.本发明公开ADC、单药及裸抗对SK-BR-3(人乳腺腺癌细胞)增殖抑制体外药效。
图4A.本发明公开ADC及裸抗对SK-BR-3(人乳腺腺癌细胞)增殖抑制体外药效。
图4B.本发明公开单药对SK-BR-3(人乳腺腺癌细胞)增殖抑制体外药效。
具体实施方式
缩写和定义
除非另有限定，本文所用的所有技术和科学术语均与本发明所属领域普通技术人员的通常理解一致。虽然也可采用与本文所述相似或等同的任何方法和材料实施或测试本发明，但本文描述了优选的方法和材料。描述和要求保护本发明时，依据以下定义使用下列术语。
当本发明中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。
除非另有说明，否则如本文所用的以下术语和短语旨在具有以下含义。当本文中使用商标名称时，除非上下文中另有指明，否则商标名称包括所述商标名称产品的产品配方、通用药物和活性药物成分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“配体”是能识别和结合目标细胞相关的抗原或受体的大分子化合物。配体的作用是将药物呈递给与配体结合的目标细胞群，这些配体包括但不限于蛋白类激素、凝集素、生长因子、抗体或其他能与细胞结合的分子。在本发明实施方式中，配体表示为Ab，配体可通过配体上的杂原子与连接单元形成连接键，优选为抗体或其抗原结合片段，所述抗体选自嵌合抗体、人源化抗体、全人抗体或鼠源抗体；优选为单克隆抗体。
配体单元是与靶标部分特异性结合的靶向剂。所述配体能够特异性结合至细胞组分或结合至其他感兴趣的靶标分子。靶标部分或靶标通常在细胞表面上。在一些方面中，配体单元的作用是将药物单元递送至配体单元与之相互作用的特定靶细胞群。配体包括但不限于蛋白质、多肽和肽，以及非蛋白质如糖。合适的配体单元包括，例如，抗体，例如全长(完整)抗体及其抗原结合片段。在配体单元是非抗体靶向试剂的实施方式中，其可以是肽或多肽，或非蛋白质分子。这类靶向试剂的示例包括干扰素、淋巴因子、激素、生长因子和集落刺激因子、维生素、营养转运分子、或任何其他细胞结合分子或物质。在一些实施方式中，连接子共价连接至配体的硫原子。在一些方面中，硫原子是半胱氨酸残基的硫原子，其形成抗体的链间二硫键。在另一方面中，硫原子是已经导入配体单元的半胱氨酸残基的硫原子，其形成抗体的链间二硫键。在另一方面中，硫原子是已经导入配体单元的半胱氨酸残基的硫原子(例如，通过定点诱变或化学反应)。在其他方面中，连接子结合的硫原子选自形成抗体的链间二硫键的半胱氨酸残基或已经引入配体单元的额半胱氨酸残基(例如，通过定点诱变或化学反应)。在一些实施方式中，按照Kabat{[Kabat E.A等，(1991)]《免疫学感兴趣的蛋白质序列》(Sequences of proteins of Immunological Interest)，第五版，NIH出版物91-3242}中的EU索引编号系统。
如本文所用，“抗体”或“抗体单元”在其所属的范围内，包括抗体结构的任何部分。这一单元可以结合，反应性关联，或者络合一个受体，抗原或者靶向细胞群体具有的其它受体单元。抗体可以是任何蛋白或蛋白类分子，它可以结合、络合或者与待治疗或生物改造的细胞群体的一部分发生反应。本发明中组成抗体药物偶联物的抗体保持其原有野生状态时的抗原结合能力。因此，本发明中的抗体能够专一性地与抗原结合。涉及的抗原包括，例如，肿瘤相关抗原(TAA)，细胞表面受体蛋白和其他细胞表面分子，细胞存活调节因子，细胞增殖调节因子，与组织生长与分化相关的分子(如已知或预知的具有功能性的)，淋巴因子，细胞因子，参与细胞循环调节的分子，参与血管生成的分子，以及与血管生成有关的分子(如已知或预知的具有功能性的)。肿瘤相关因子可以是簇分化因子(如CD蛋白)。
应用在抗体药物偶联物中的抗体包括，但不局限于，针对细胞表面受体和肿瘤相关抗原 的抗体。这样的肿瘤相关抗原是业内所熟知的，可以通过业内熟知的抗体制备方法和信息来制备。为了开发可用于癌症诊断与治疗的有效的细胞水平目标物，研究人员力图找寻跨膜或其他肿瘤相关多肽。这些目标物能够特异性的表达在一种或多种癌细胞表面，而在一种或多种非癌细胞表面表达很少或不表达。通常，相对于非癌细胞表面而言，这样的肿瘤相关多肽在癌细胞表面更加过度表达。确认这样的肿瘤相关因子，可大大提高基于抗体治疗癌症的专一靶向特性。为方便起见，为业内所熟知的抗原相关信息标示如下，包括名称，其他名称，基因库登录号。与肿瘤相关抗原对应的核酸和蛋白序列可参见公开数据库，例如Genbank。抗体靶向对应的肿瘤相关抗原包括所有的氨基酸序列变种和同种，与参考文献中确认的序列具有至少70％,80％,85％,90％或者95％的同源性，或者具备与引用文献中的肿瘤相关抗原序列具有完全一致的生物性质和特征。
术语“抑制”或“的抑制”指，减少了可检测的量，或完全阻止。
术语“癌症”指的是以失调的细胞生长为特征的生理病症或疾病。“肿瘤”包括癌细胞。
术语“自身免疫疾病”是源自针对个体自身的组织或蛋白质的疾病或紊乱。
术语“药物”是指细胞毒性药物，药物表示d，能在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。该术语包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32和Lu 176的放射性同位素)，毒性药物，化疗药物，抗生素和核溶酶，优选为毒性药物。
术语“喜树碱类药物”指具有细胞毒性的喜树碱及其衍生物，非限制性地选自10-羟基喜树碱、SN38(7-乙基-10-羟基喜树碱)、拓扑替康、依喜替康、伊立替康或9-硝基-10-羟基喜树碱及其衍生物或药学上可接受的盐。
术语“连接子”或“连接片段”或“连接单元”是指一端与配体连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。
接头，包括延伸物、间隔物和氨基酸单元，可以通过本领域己知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等Cancer Research 52:127-131,1992)；美国专利No.5,208,020。
按照在细胞内药物释放的机制，如本文所用，“连接子”或“抗体药物偶联物的连接子”可被分为两类：不可断裂连接子和可断裂连接子。对于含有不可断裂连接子的配体-药物偶联物，其药物释放机制为：偶联物与抗原结合并被细胞内吞后，抗体在溶酶体中被酶解，释放出由 小分子药物，连接子，和抗体氨基酸残基共同组成的活性分子。由此带来的药物分子结构改变并不减弱其细胞毒性，但由于活性分子是带电荷的(氨基酸残基)，从而导致其不能渗入邻近细胞。因此，此类活性药物不能杀死邻近不表达靶向抗原(抗原阴性细胞)的肿瘤细胞(旁观者效应，bystander effect)(Ducry等，2010，Bioconjugate Chem.21:5-13)。
术语“配体-药物偶联物”，指抗体通过稳定的连接单元与具有生物活性的药物相连。在本发明中“配体-药物偶联物”优选为抗体-药物偶联物(antibody drug conjugate,ADC)，指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的毒性药物相连。
本公开所用氨基酸三字母代码和单字母代码如J.boil.Chem.1968,243,3558.中所述。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“取代烷基”指烷基中的氢被取代基团取代，除非文中另有说明，烷基的取代基可以是选自下组的多种基团：-卤素、-OR’、-NR’R”、-SR’、-SiR’R”R”’、-OC(O)R’、-C(O)R’、-CO 2R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R”’、-NR”C(O) 2R’、 -NH-C(NH 2)＝NH、-NR’C(NH 2)＝NH、-NH-C(NH 2)＝NR’、-S(O)R’、-S(O) 2R’、-S(O) 2NR’R”、-NR’S(O) 2R”、-CN和-NO 2，取代基数量为0至(2m’+1)，其中m’为该基团中碳原子的总数。R’、R”和R”’各自独立的指代氢、未取代的C 1-8烷基、未取代的芳基、由1-3个卤素取代的芳基、未取代的C 1-8烷基、C 1-8烷氧基或C 1-8硫代烷氧基、或未取代的芳基-C 1-4烷基。R’和R”连接于同一个氮原子时，它们可与该氮原子一起形成3-，4-，5-，6-或7-元环。例如，-NR’R”包括1-吡咯烷基和4-吗啉基。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷基如上所定义。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子，更优选含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH 2-、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)和1,5-亚丁基(-CH 2CH 2CH 2CH 2CH 2-)等。亚烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个取代基所取代。
术语“烷氧基”指-O-(烷基)和-O-(环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O) m(其中 m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子； 更优选环烷基环包含3至10个环原子。单环杂环基的非限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
术语“环烷基烷基”指烷基被一个或多个环烷基取代，优选被一个环烷基取代，其中烷基如上所定义，其中环烷基如上所定义。
术语“卤代烷基”指烷基被一个或多个卤素取代，其中烷基如上所定义。
术语“氘代烷基”指烷基被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH 2。术语“硝基”指-NO 2。
术语“酰胺基"指-C(O)N(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
术语“羧酸酯基"指-C(O)O(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(即共享毗邻碳原子对的环)基团，优选为6-10元，例如苯基。芳基可以是取代的或非取代的，当被取代时，取代基优选为以下一个或多个基团，非限制性地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、氘原子、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基或杂环烷硫基。
本发明还包括各种氘化形式的式Ⅰ。与碳原子连接的各个可用的氢原子可独立地被氘原子替换。本领域技术人员能够参考相关文献合成氘化形式的式Ⅰ。在制备氘代形式的式Ⅰ时可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂的非限制性实例包括:氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。
术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。本发明所述的抗体优选为针对靶细胞上细胞表面抗原的特异性抗体，非限制性实施例为以下抗体：抗EGFRvIII抗体、抗DLL-3抗体、抗PSMA抗体、抗CD70抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM-1抗体、抗LRRC15抗体、抗LIV-1抗体、抗CanAg/AFP抗体、抗cladin 18.2抗体、抗Mesothelin抗体、 抗HER2(ErbB2)抗体、抗EGFR抗体、抗c-MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7-H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗TROP-2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P-cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体或抗CD123抗体中一个或多个；优选为曲妥珠单抗(Trastuzumab,商品名Herceptin)、帕妥珠单抗(Pertuzumab,也被称作2C4，商品名Perjeta)、尼妥珠单抗(Nimotuzumab，商品名泰欣生)、Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、Brentuximab、Gemtuzumab、Bivatuzumab、Lorvotuzumab、cBR96和Glematumamab。
术语“溶剂化物”或“溶剂化合物”指本发明的配体-药物偶联物与一种或多种溶剂分子形成可药用的溶剂化物，溶剂分子的非限制性实例包括水、乙醇、乙腈、异丙醇、DMSO、乙酸乙酯。
术语“载药量”是指式Ⅰ中每个抗体上加载的细胞毒性药物平均数量，也可以表示为药物量和抗体量的比值，药物载量的范围可以是每个抗体(Ab)连接0-12个，优选1-10个细胞毒性药物(D)。在本发明的实施方式中，载药量表示为n,示例性的可以为1,2,3,4,5,6,7,8,9,10的均值。可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的药物品均数量。
本发明的一个实施方式中，细胞毒性药物通过连接单元偶联在抗体打开的链间半胱氨酸巯基-SH上和/或定点突变的半胱氨酸巯基-SH上，一般的，偶联反应中能与抗体偶联的药物分子数将小于或等于理论上的最大值。
可以用以下非限制性方法控制配体细胞毒性药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
常规的药物组合物的制备见中国药典。
术语“药学上可接受的盐”或“可药用盐”是指本发明配体-药物偶联物的盐，或本发明中所 述的化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性，本发明配体-药物偶联化合物至少含有一个羧基，因此可以与碱形成盐，药学上可接受的盐的非限制性实例包括：钠盐、钾盐、钙盐或镁盐等。
术语“药学上可接受的盐”或“可药用盐”是指本发明抗体-药物偶联物的盐，或本发明中所述的化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性，本发明配体-药物偶联化合物至少含有一个氨基，因此可以与酸形成盐，药学上可接受的盐的非限制性实例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
“酸性氨基酸”指氨基酸的等电点小于7，酸性氨基酸分子中往往带有一个或多个羧基等酸性基团，在结构中可有效电离为负离子形式而增加亲水性。酸性氨基酸可以为天然的，也可为非天然的氨基酸。
“天然氨基酸”指由生物合成的氨基酸。天然氨基酸一般情况下是L-型的，但也有少数例外，比如甘氨酸，包括天然的和生物体合成的。
“非天然氨基酸”指通过合成手段所获得的氨基酸。
下面结合具体实施例，进一步阐述本发明，应理解，这些实施例只用于说明本发明，而不用于限制本发明的范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另外说明，否则所有的百分数、比例、比率、或份数按重量计。
除非另行定义，文中所使用的所有专业和科学用于与本领域熟练人员所熟悉的意义相同。
此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1
化合物M1的合成：
Figure PCTCN2021097302-appb-000027
于5000mL单口瓶中加入N-芴甲氧羰基-甘氨酸-甘氨酸(100g,282mmol,1.0eq)，四乙酸铅(175g,553mmol,1.4eq)，2000mL干燥四氢呋喃和670mL甲苯，搅拌均匀，氮气保护，加热至85℃反应2.5h。TLC监控，原料反应完后，冷却至室温，过滤，滤液减压浓缩，残 余物经柱色谱纯化，得化合物M1(87g)；LC-MS：[M+NH 4] +＝386.0。
实施例2
化合物M3的合成：
Figure PCTCN2021097302-appb-000028
于1000mL单口瓶中加入化合物SM-2(按照专利CN108452321A公布的方法合成)(40g,96mmol,1.0eq)，三乙胺(26.7mL,2.0eq)，甲苯(400mL)，升温至120℃下回流反应2h。TLC监测基本完全反应，降温至50℃下减压旋除溶剂。用乙酸乙酯(150mL)，水(40mL)溶解，冰浴搅拌下用1M HCl调pH至2-3，分液。水层用乙酸乙酯再萃取一次，合并有机层，加入无水硫酸钠干燥。过滤后，浓缩得到淡黄色油状粗品，粗品经柱层析纯化(DCM:MeOH＝40:1)，得到化合物M2(26.6g)；LC-MS：[M+H] +＝399.3。
于1000mL单口瓶，加入化合物M2(26.5g，60.5mmol,1.0eq)、五氟苯酚(12.2g,66.5mmol,1.1eq)、DCC(13.7g,66.5mmol,1.1eq)及THF(300mL)，室温反应30min(采用TLC监测)，过滤滤去不溶物。反应液直接经制备纯化，制备液水泵减压水浴35℃浓缩除去乙腈，冻干得到化合物M3(31.5g)，收率64％；LC-MS：[M+H] +＝565.1。
实施例3
化合物ent-M3的合成：
Figure PCTCN2021097302-appb-000029
参照实施例2合成路线，得到化合物ent-M3(27.8g)；LC-MS：[M+H] +＝565.2。
实施例4
化合物1的合成：
Figure PCTCN2021097302-appb-000030
第一步：化合物1a
于250mL单口瓶中，加入M1(6g,16.3mmol)，100mL THF，对甲苯磺酸一水合物(0.31g,1.63mmol)，搅拌冷却至0℃，滴加羟乙酸苄酯(5.4g,32.6mmol)，滴毕自然升温至室温反应(反应约2-4h)，TLC监控。反应结束，加入饱和NaHCO 3溶液，用乙酸乙酯萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，浓缩，残余物经硅胶柱纯化(PE:EA＝10:1-5:1-1:1)得1a(4g)，收率52％；LC-MS：[M+H] +＝475.18。
第二步：化合物1b
于25mL单口瓶，加入1a(2g,4.2mmol)，10mL DMF，0℃搅拌，加入DBU(766mg,5.04mmol)，反应1h，TLC监测Fmoc脱保护完成后，待用；
另取25mL单口瓶中加入M4(参考专利CN111051330 A公布的方法制备)(1.73g,4.2mmol)，PyBOP(2.61g,5.04mmol)，HOBt(680mg,5.04mmol)及10mL DMF，冰水浴下加入DIPEA(830uL,5.04mmol)，继续搅拌30min，将上述反应液加至反应瓶中，升至室温反应。HPLC监测反应结束后，反应液经制备液相纯化，得到产品制备液，制备液经二氯甲烷萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到固体1b(1.7g)，收率63％；LCMS：[M+H] +＝648.26。
第三步：化合物1c
于25mL单口瓶中加入1b(900mg,1.39mmol)，15mL DMF溶清后，加入900mg 5％Pd/C，氢化反应2h，反应完毕，过滤，得滤液，未经纯化直接用于下一步反应。
第四步：化合物1d
将粗产品1c置于冰水浴中，加入DIPEA(235uL,1.39mmol)，再加入化合物M3(784mg,1.39mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到1d(504mg)；LC-MS：[M+H] +＝804.4。
第五步：化合物1e
于50mL单口瓶中加入1d(500mg,0.62mmol)，M5(310mg,0.62mmol)，PyBOP(448mg,0.86mmol)，HOBt(116mg,0.86mmol)及15mL DMF,冰水浴下加入DIPEA(378uL,2.29mmol)，升至室温反应2h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物1e的制备液，制备液冻干得到1e(210mg)；LC-MS：[M+H] +＝1221.6。
第六步：化合物1
于25mL单口瓶中加入1e(100mg,0.081mmol)，溴化锌(368mg,1.63mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体化合物1(60mg)；LC-MS：[M+H] +＝1065.3。
实施例5
化合物2的合成：
Figure PCTCN2021097302-appb-000031
参照实施例4合成路线，得到化合物2(51mg)；LC-MS：[M+H] +＝1065.3。
实施例6
化合物3的合成：
Figure PCTCN2021097302-appb-000032
第一步：化合物3a
于250mL单口瓶中，加入M1(6g,16.3mmol)，100mL THF，对甲苯磺酸一水合物(0.31g,1.63mmol)，搅拌冷却至0℃，滴加2-羟基-2-甲基丙酸苄酯(6.3g,32.6mmol)，滴毕自然升温至室温反应(反应约2-4h)，TLC监控。反应结束，加入饱和NaHCO 3溶液，用乙酸乙酯萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，浓缩，残余物经硅胶柱纯化(PE:EA＝10:1-5:1-2:1)得3a(4.2g)，收率52％；LC-MS：[M+H] +＝503.3。
第二步：化合物3b
于25mL单口瓶，加入3a(2g,4.0mmol)，10mL DMF，0℃搅拌，加入DBU(760mg,5.0mmol)，反应1h，TLC监测Fmoc脱保护完成后，待用；
另取25mL单口瓶中加入M4(1.65g,4.0mmol)，PyBOP(2.59g,5.0mmol)，HOBt(675mg,5.0mmol)及10mL DMF，冰水浴下加入DIPEA(823uL,5.04mmol)，继续搅拌30min，将上述反应液加至反应瓶中，升至室温反应。HPLC监测反应结束后，反应液经制备液相纯化，得到产品制备液，制备液经二氯甲烷萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到固体3b(1.4g)，收率53％；LC-MS：[M+H] +＝676.2。
第三步：化合物3c
于25mL单口瓶中加入3b(700mg,1.04mmol)，10mL DMF溶清后，加入700mg 5％Pd/C，氢化反应1.5h，反应完毕，过滤，得滤液，未经纯化直接用于下一步反应。
第四步：化合物3d
将粗产品3c置于冰水浴中，加入DIPEA(210uL,1.25mmol)，再加入化合物M3(704mg, 1.25mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到3d(486mg)；LC-MS：[M-H] -＝830.5。
第五步：化合物3e
于50mL单口瓶中加入3d(300mg,0.36mmol)，M5(180mg,0.36mmol)，PyBOP(260mg,0.5mmol)，HOBt(67mg,0.5mmol)及10mL DMF,冰水浴下加入DIPEA(219.5uL,1.33mmol)，升至室温反应3h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物3e的制备液，制备液冻干得到3e(157mg)；LC-MS：[M+H] +＝1249.6。
第六步：化合物3
于25mL单口瓶中加入3e(100mg,0.08mmol)，溴化锌(360mg,1.6mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体化合物3(64mg)；LC-MS：[M+H] +＝1093.1。
实施例7
化合物4的合成：
Figure PCTCN2021097302-appb-000033
参照实施例6合成路线，得到化合物4(60mg)；LC-MS：[M+H] +＝1093.2。
实施例8
化合物5A的合成：
Figure PCTCN2021097302-appb-000034
第一步：化合物5a
于25mL单口瓶中加入M1(500mg，1.4mmol，1.0eq),对甲苯磺酸一水合物(26mg，0.1mmol，0.1eq)及10mL THF，搅拌均匀后，降至0℃，再缓慢加入L-乳酸苄酯(1.2g,7.0mmol,5eq)，加完后升至室温反应。TLC监控，反应结束后，加入饱和NaHCO 3溶液，用乙酸乙酯萃取，无水硫酸钠干燥，过滤，浓缩，残余物经反相柱纯化得5a(400mg)；
LC-MS：[M+NH 4] +＝506.2。
1H NMR(400Mz,CDCl 3/CD 3OD):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).。
第二步：化合物5b
于25mL单口瓶中加入化合物5a(400mg,0.8mmol,1.0eq)和4mL DMF，搅拌均匀后，降至0℃，再缓慢加入DBU(137mg,0.9mmol,1.1eq)，加完后升至室温反应。TLC监控，反应结束，记为反应液①；
另取25mL单口瓶中加入M4(372mg,0.9mmol,1.1eq)，PyBOP(852mg,1.6mmol,2.0eq)和3mL DMF，室温搅拌5分钟，加入反应液①，室温反应，HPLC监测。反应完毕，反应液经高效液相纯化得化合物5b(326mg)；LC-MS：[M+NH 4] +＝679.2。
第三步：化合物5c
于100mL单口瓶中加入5b(4.0g,6.05mmol,1.0eq)，DMF(60mL)溶解，再加入5％Pd/C(4g)，室温氢化反应4h(采用HPLC监测反应进程)。过滤Pd/C，滤液未经浓缩，直接置于冰水浴(约0℃)中，待用。
第四步：化合物5d
将粗产品5c置于冰水浴中，加入DIPEA(1.1mL,1.1eq)，再加入化合物M3(3.4g,6.05mmol)，加毕升至室温反应2h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到5d(3.15g)；LC-MS：[M-H] -＝816.3。
第五步：化合物5e
于100mL单口瓶中加入5d(2.07g,2.53mmol,1.0eq)，M5(1.35g,2.53mmol,1.0eq)，PyBOP(1.98g,3.79mmol,1.5eq)，HOBt(0.51g,3.79mmol,1.5eq)及DMF(40mL)，冰水浴下加入DIPEA(1.05mL,1.5eq)，升至室温反应2h(采用HPLC监测)。反应液直接经制备纯化，制备液水泵减压水浴35℃浓缩除去乙腈，冻干得到化合物5e(1.92g)，收率61％；LC-MS：[M+H] +＝1235.4。
第六步：化合物5A
于100mL单口瓶中加入化合物5e(1.0g,0.8mmol,1.0eq)，35mL硝基甲烷，溶解后再加入溴化锌(3.64g,16mmol,20.0eq)，油浴40℃(提前预热稳定)反应30min，水泵减压水浴45℃浓缩除去硝基甲烷，得黄色残余物固体(采用HPLC监测)。经酸法制备，得到化合物5A的制备液，制备液经水泵减压水浴35℃浓缩旋除乙腈，冻干得到化合物5A(786mg)收率90％。
LC-MS：[M+H] +＝1079.4；
1H NMR(400MHz,DMSO-d6)δ9.39–9.02(m,1H),8.70(t,J＝6.5Hz,1H),8.64(t,J＝5.7Hz,1H),8.56(d,J＝8.8Hz,1H),8.34(t,J＝5.7Hz,1H),8.16(d,J＝8.2Hz,1H),8.01(t,J＝5.5Hz,1H),7.71(d,J＝10.9Hz,1H),7.30(s,1H),7.28–7.15(m,4H),7.14(s,2H),5.53(dd,J＝14.5,6.4Hz,1H),5.49–5.34(m,2H),5.22(d,J＝18.8Hz,1H),5.09(d,J＝18.7Hz,1H),5.03(dd,J＝9.6,3.9Hz,1H),4.73(dd,J＝9.9,6.9Hz,1H),4.59(dd,J＝10.1,6.5Hz,1H),4.49(ddd,J＝13.2,8.6,4.4Hz,1H),4.14(dd,J＝13.3,6.6Hz,2H),3.93(s,2H),3.84(dd,J＝16.5,6.3Hz,1H),3.76(dd,J＝16.9,5.7Hz,2H),3.70(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＝12.8,10.1Hz,1H),3.25–3.15(m,1H),3.14–3.05(m,1H),3.01(dd,J＝13.7,4.1Hz,1H),2.73(dd,J＝13.5,9.8Hz,1H),2.54–2.47(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).。
实施例9
化合物5B的合成：
Figure PCTCN2021097302-appb-000035
第一步：化合物5d-1
于25mL单口瓶中加入化合物5b(300mg,0.45mmol,1.0eq)，DMF(3mL)，搅拌溶清，加入5％Pd/C(300mg)，氢气置换三次，氢化反应2h，HPLC监测反应结束。反应结束后，过滤除去Pd/C，将滤液降温至0-5℃，加入DIPEA(65mg,0.5mmol,1.1eq)，再向滤液加入ent-M3(255mg,0.45mmol)，加毕，升至20±5℃反应1h，HPLC监测反应结束。反应结束后， 经HPLC制备纯化，收集产品制备液，冻干得到化合物5d-1(200mg)，收率54％；LC-MS：[M-H] -＝816.3。
第二步：化合物5e-1
于25mL单口瓶中加入化合物5d-1(200mg,0.24mmol,1.0eq)、M5(127mg,0.24mmol，1.0eq)、PyBOP(187mg,0.36mmol,1.2eq)、HOBt(48mg,0.36mmol,1.2eq)及DMF(6mL)，冰水浴降至0-5℃，加入DIPEA(62mg,0.48mmol,2.0eq)，加毕，升至20±5℃反应2h，HPLC监测反应结束。反应液直接HPLC制备纯化，收集产品制备液，冻干得到化合物5e-1(162.8mg)；LC-MS：[M+H] +＝1235.4。
第三步：化合物5B
于25mL单口瓶中依次加入化合物5e-1(110mg,0.089mmol，1.0eq)，ZnBr 2(400mg,1.78mmol,20.0eq)和CH 3NO 2(10mL)，加毕，升至40℃反应0.5h，停止反应，反应液直接45℃减压旋干后得到黄色固体，取样HPLC监测反应。旋干的固体直接HPLC制备纯化，收集产品制备液，冻干得到化合物5B(73.4mg)，收率76.5％；LC-MS：[M+H] +＝1079.4。
实施例10
化合物6A的制备：
Figure PCTCN2021097302-appb-000036
参照实施例8合成路线，得到化合物6A(71mg)；LC-MS：[M+H] +＝1079.4。
实施例11
化合物6B的制备：
Figure PCTCN2021097302-appb-000037
参照实施例9合成路线，得到化合物6B(59mg)；LC-MS：[M+H] +＝1079.4。
实施例12
化合物7A和7B的制备：
Figure PCTCN2021097302-appb-000038
第一步：化合物7a
于250mL单口瓶中加入M1(10g,27.1mmol),3,3,3-三氟乳酸苄酯(参照专利WO2020063673A1公布的方法制备)(12.7g,54.3mmol)，醋酸锌(9.96g,54.3mmol)及100mL甲苯，加热至100℃反应4h。反应完毕，降至室温，过滤除去不溶物，滤液浓缩得粗品。粗品经硅胶柱层析纯化(PE:EA＝10:1-5:1-2:1)得到目标物5.15g，收率35.1％；LC-MS：[M+H] +＝543.17。
第二步：化合物7b
于50mL单口瓶中加入7a(5g,9.2mmol)及15mL DMF，溶清后，冰水浴下，加入DBU(1.68g,11mmol)，反应1h，记为反应液①；
另取50mL单口瓶，加入M4(3.8g,9.2mmol)，PyBOP(5.75g,11mmol)，HOBt(1.49g,11mmol)及10mL DMF，溶清后，冰水浴下，加入DIPEA(1.82mL,11mmol)，继续反应30min，加入反应液①，升至室温反应2h。HPLC监测反应进程，反应完毕后，反应液经高效液相纯化，得制备液。制备液经二氯甲烷萃取、饱和氯化钠溶液洗涤、无水硫酸钠干燥、过滤、浓缩得到固体4.1g，收率62.3％；LC-MS：[M+H] +＝716.25。
第三步：化合物7d
于25mL单口瓶中加入7b(900mg,1.26mmol)，15mL DMF溶清后，加入900mg 5％Pd/C，氢化反应2h，反应完毕，过滤，将滤液置于冰水浴中，加入DIPEA(228uL,1.38mmol)，再加入M3(712mg,1,26mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到产品525mg，收率47.9％；LC-MS：[M-H] -＝870.33。
第四步：化合物7e
于50mL单口瓶中加入7d(500mg,0.57mmol)，M5(305mg,0.57mmol)，PyBOP(448mg,0.86mmol)，HOBt(116mg,0.86mmol)及15mL DMF,冰水浴下加入DIPEA(378uL,2.29mmol)，升至室温反应2h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物7e-1和化合物7e-2的制备液，制备液分别冻干得到150mg化合物7e-1，LC-MS：[M+H] +＝1289.46；220mg化合物7e-2，LC-MS：[M+H] +＝1289.46。
第五步：化合物7A
Figure PCTCN2021097302-appb-000039
于25mL单口瓶中加入7e-1(100mg,0.077mmol)，溴化锌(349mg,1.55mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体52mg；TOF结果：1133.3613。
第六步：化合物7B
Figure PCTCN2021097302-appb-000040
于25mL单口瓶中加入7e-2(100mg,0.077mmol)，溴化锌(349mg,1.55mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体63mg；TOF结果：1133.3668。
实施例13
化合物8A和8B的合成：
Figure PCTCN2021097302-appb-000041
第一步：化合物8d
于25mL单口瓶中加入7c(900mg,1.83mmol)，20mL DMF溶清后，加入DIPEA(303uL,1.83mmol)，再加入ent-M3(1034mg,1.83mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到产品613mg，收率38.5％；LC-MS：[M-H] -＝870.32。
第二步：化合物8e-1和化合物8e-2
于50mL单口瓶中加入8d(500mg,0.57mmol)，M5(305mg,0.57mmol)，PyBOP(448mg,0.86mmol)，HOBt(116mg,0.86mmol)及15mL DMF,冰水浴下加入DIPEA(378uL,2.29mmol)，升至室温反应2h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物8e-1和化合物8e-2的制备液，制备液分别冻干得到140mg化合物8e-1，210mg化合物8e-2。化合物8e-1的LC-MS：[M+H] +＝1289.47；化合物8e-2的LC-MS：[M+H] +＝1289.47。
第三步：化合物8A
Figure PCTCN2021097302-appb-000042
于25mL单口瓶中加入化合物8e-1(100mg,0.077mmol)，溴化锌(349mg,1.55mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体50mg；TOF结果：1133.3623。
第四步：化合物8B
Figure PCTCN2021097302-appb-000043
于25mL单口瓶中加入化合物8e-2(100mg,0.077mmol)，溴化锌(349mg,1.55mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体58mg；TOF结果：1133.3653。
实施例14
化合物9A的合成：
Figure PCTCN2021097302-appb-000044
第一步：化合物9a
于250mL单口瓶中，加入M1(6g,16.3mmol)，100mL THF，对甲苯磺酸一水合物(0.31g,1.63mmol)，搅拌冷却至0℃，滴加2-羟基-2-环丙基乙酸苄酯(参照专利US20050020645A1公布的方法制备)(6.3g,32.6mmol)，滴毕自然升温至室温反应(反应约2-4h)，TLC监控。反应结束，加入饱和NaHCO 3溶液，用乙酸乙酯萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，浓缩，残余物经硅胶柱纯化(PE:EA＝10:1-5:1-2:1)得9a(3.7g)，收率45％；LC-MS：[M+H] +＝501.5。
第二步：化合物9b
于25mL单口瓶，加入9a(2g,4.0mmol)，10mL DMF，0℃搅拌，加入DBU(760mg,5.0mmol)，反应1h，TLC监测Fmoc脱保护完成后，待用；
另取25mL单口瓶中加入M4(1.65g,4.0mmol)，PyBOP(2.59g,5.0mmol)，HOBt(675mg,5.0mmol)及10mL DMF，冰水浴下加入DIPEA(823uL,5.04mmol)，继续搅拌30min，将上述反应液加至反应瓶中，升至室温反应。HPLC监测反应结束后，反应液经制备液相纯化，得到产品制备液，制备液经二氯甲烷萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到固体1.5g，收率56％；LC-MS：[M+H] +＝674.7。
第三步：化合物9c
于25mL单口瓶中加入9b(900mg,1.3mmol)，10mL DMF溶清后，加入900mg 5％Pd/C，氢化反应1.5h，反应完毕，过滤，得滤液，未经纯化直接用于下一步反应。
第四步：化合物9d
将粗产品9c置于冰水浴中，加入DIPEA(223uL,1.3mmol)，再加入化合物M3(750mg,1.3 mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到9d(529mg)；LC-MS：[M-H] -＝828.4。
第五步：化合物9e
于50mL单口瓶中加入9d(500mg,0.6mmol)，M5(300mg,0.6mmol)，PyBOP(416mg,0.8mmol)，HOBt(108mg,0.5mmol)及15mL DMF,冰水浴下加入DIPEA(351uL,2.13mmol)，升至室温反应3h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物9e的制备液，制备液冻干得到9e(257mg)；LC-MS：[M+H] +＝1247.5。
第六步：化合物9A
于25mL单口瓶中加入9e(100mg,0.08mmol)，溴化锌(360mg,1.6mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体化合物9A(55mg)；LC-MS：[M+H] +＝1091.3。
实施例15
化合物9B的合成：
Figure PCTCN2021097302-appb-000045
参照实施例14合成路线，得到化合物9B(44mg)；LC-MS：[M+H] +＝1091.3。
实施例16
化合物10A的合成：
Figure PCTCN2021097302-appb-000046
第一步：化合物10a
于250mL单口瓶中，加入M1(6g,16.3mmol)，100mL THF，对甲苯磺酸一水合物(0.31g,1.63mmol)，搅拌冷却至0℃，滴加3-羟基-2-环丙基丙酸苄酯(参照专利WO2013187496A1公布的方法制备)(6.7g,32.6mmol)，滴毕自然升温至室温反应(反应约2-4h)，TLC监控。反应结束，加入饱和NaHCO 3溶液，用乙酸乙酯萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，浓缩，残余物经硅胶柱纯化(PE:EA＝10:1-5:1-2:1)得10a(4.9g)，收率58％；LC-MS：[M+H] +＝515.4。
第二步：化合物10b
于25mL单口瓶，加入10a(4g,7.8mmol)，10mL DMF，0℃搅拌，加入DBU(1.2g,8.0mmol)，反应1h，TLC监测Fmoc脱保护完成后，待用；
另取25mL单口瓶中加入M4(3.3g,8.0mmol)，PyBOP(5.2g,10.0mmol)，HOBt(1.35g,10.0mmol)及10mL DMF，冰水浴下加入DIPEA(1.65mL,10.1mmol)，继续搅拌50min，将上述反应液加至反应瓶中，升至室温反应。HPLC监测反应结束后，反应液经制备液相纯化，得到产品制备液，制备液经二氯甲烷萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到固体2.3g，收率42％；LC-MS：[M+H] +＝688.8。
第三步：化合物10c
于25mL单口瓶中加入10b(1.0g,1.45mmol)，15mL DMF溶清后，加入1.0g 5％Pd/C，氢化反应1.5h，反应完毕，过滤，得滤液，未经纯化直接用于下一步反应。
第四步：化合物10d
将粗产品10c置于冰水浴中，加入DIPEA(258uL,1.5mmol)，再加入化合物M3(837mg,1.45mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到10d(499mg)；LC-MS：[M-H] -＝842.4。
第五步：化合物10e
于50mL单口瓶中加入10d(400mg,0.48mmol)，M5(240mg,0.48mmol)，PyBOP(250mg,0.48mmol)，HOBt(104mg,0.48mmol)及15mL DMF，冰水浴下加入DIPEA(330uL,2.0mmol)，升至室温反应3h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物10e的制备液，制备液冻干得到10e(188mg)；LC-MS：[M+H] +＝1261.5。
第六步：化合物10A
于25mL单口瓶中加入10e(100mg,0.08mmol)，溴化锌(360mg,1.6mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体化合物10A(61mg)；LC-MS：[M+H] +＝1105.4。
实施例17
化合物10B的合成：
Figure PCTCN2021097302-appb-000047
参照实施例16合成路线，得到化合物10B(75mg)；LC-MS：[M+H] +＝1105.4。
实施例18
化合物11A的合成：
Figure PCTCN2021097302-appb-000048
第一步：化合物11a
于250mL单口瓶中，加入M1(6g,16.3mmol)，100mL THF，对甲苯磺酸一水合物(0.31g,1.63mmol)，搅拌冷却至0℃，滴加2-羟基-2-环丁基乙酸苄酯(参照文献Journal of Medicinal Chemistry,2013,56(13),5541-5552.公布的方法合成)(6.7g,32.6mmol)，滴毕自然升温至室温反应(反应约2-4h)，TLC监控。反应结束，加入饱和NaHCO 3溶液，用乙酸乙酯萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，浓缩，残余物经硅胶柱纯化(PE:EA＝10:1-5:1-2:1)得11a(5.1g)，收率62％；LC-MS：[M+H] +＝515.7。
第二步：化合物11b
于25mL单口瓶，加入11a(4g,7.8mmol)，10mL DMF，0℃搅拌，加入DBU(1.2g,8.0mmol)，反应1h，TLC监测Fmoc脱保护完成后，待用；
另取25mL单口瓶中加入M4(3.3g,8.0mmol)，PyBOP(5.2g,10.0mmol)，HOBt(1.35g, 10.0mmol)及10mL DMF，冰水浴下加入DIPEA(1.63mL,10.0mmol)，继续搅拌40min，将上述反应液加至反应瓶中，升至室温反应。HPLC监测反应结束后，反应液经制备液相纯化，得到产品制备液，制备液经二氯甲烷萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到固体2.3g，收率42％；LC-MS：[M+H] +＝688.3。
第三步：化合物11c
于25mL单口瓶中加入11b(2.0g,2.9mmol)，25mL DMF溶清后，加入2.0g 5％Pd/C，氢化反应3h，反应完毕，过滤，得滤液，未经纯化直接用于下一步反应。
第四步：化合物11d
将粗产品11c置于冰水浴中，加入DIPEA(516uL,3.0mmol)，再加入化合物M3(1.7g,2.9mmol)，加毕升至室温反应2h。HPLC监测反应完毕，反应液经高效液相纯化，得到制备液，制备液冻干得到11d(934mg)；LC-MS：[M-H] -＝842.4。
第五步：化合物11e
于50mL单口瓶中加入11d(800mg,0.96mmol)，M5(480mg,0.96mmol)，PyBOP(500mg,0.96mmol)，HOBt(208mg,0.96mmol)及30mL DMF，冰水浴下加入DIPEA(660uL,4.0mmol)，升至室温反应4h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物11e的制备液，制备液冻干得到11e(401mg)；LC-MS：[M+H] +＝1261.4。
第六步：化合物11A
于25mL单口瓶中加入11e(150mg,0.12mmol)，溴化锌(532mg,2.4mmol)及10mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体化合物11A(86mg)；LC-MS：[M+H] +＝1105.4。
实施例19
化合物11B的合成
Figure PCTCN2021097302-appb-000049
参照实施例18合成路线，得到化合物11B(50mg)。LC-MS：[M+H] +1105.4。
实施例20
化合物12A的合成：
Figure PCTCN2021097302-appb-000050
第一步：化合物12a
于250mL单口瓶中，加入M1(6g,16.3mmol)，100mL THF，对甲苯磺酸一水合物(0.31g,1.63mmol)，搅拌冷却至0℃，滴加3-羟基-2-环丁基丙酸苄酯(参照专利WO2009011285A1公布的方法制备)(7.2g,32.6mmol)，滴毕自然升温至室温反应(反应约2-4h)，TLC监控。反应结束，加入饱和NaHCO 3溶液，用乙酸乙酯萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，浓缩，残余物经硅胶柱纯化(PE:EA＝10:1-5:1-2:1)得12a(4.5g)，收率52％；LC-MS：[M+H] +＝529.4。
第二步：化合物12b
于25mL单口瓶，加入12a(4g,7.6mmol)，10mL DMF，0℃搅拌，加入DBU(1.2g,8.0mmol)，反应1h，TLC监测Fmoc脱保护完成后，待用；
另取25mL单口瓶中加入M4(3.2g,7.6mmol)，PyBOP(4.7g,9.0mmol)，HOBt(1.22g,9.0mmol)及10mL DMF，冰水浴下加入DIPEA(1.49mL,0.9mmol)，继续搅拌30min，将上述反应液加至反应瓶中，升至室温反应。HPLC监测反应结束后，反应液经制备液相纯化，得到产品制备液，制备液经二氯甲烷萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到固体2.0g，收率37％；LC-MS：[M+H] +＝702.8。
第三步：化合物12c
于25mL单口瓶中加入12b(1.0g,1.43mmol)，15mL DMF溶清后，加入1.0g 5％Pd/C，氢化反应1.5h，反应完毕，过滤，得滤液，未经纯化直接用于下一步反应。
第四步：化合物12d
将粗产品12c置于冰水浴中，加入DIPEA(258uL,1.5mmol)，再加入化合物M3(825mg, 1.43mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到12d(522mg)；LC-MS：[M-H] -＝856.4。
第五步：化合物12e
于50mL单口瓶中加入12d(400mg,0.47mmol)，M5(240mg,0.47mmol)，PyBOP(250mg,0.47mmol)，HOBt(101mg,0.47mmol)及15mL DMF，冰水浴下加入DIPEA(330uL,2.0mmol)，升至室温反应3h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物12e的制备液，制备液冻干得到12e(198mg)；LC-MS：[M+H] +＝1275.4。
第六步：化合物12A
于25mL单口瓶中加入12e(100mg,0.08mmol)，溴化锌(360mg,1.6mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体化合物12A(55mg)；LC-MS：[M+H] +＝1119.4。
实施例21
化合物12B的合成：
Figure PCTCN2021097302-appb-000051
参照实施例20合成路线，得到化合物12B(50mg)；LC-MS：[M+H] +＝1119.4。
实施例22
化合物13A的合成：
Figure PCTCN2021097302-appb-000052
第一步：化合物13a
于250mL单口瓶中，加入M1(6g,16.3mmol)，100mL THF，对甲苯磺酸一水合物(0.31g,1.63mmol)，搅拌冷却至0℃，滴加2-羟基-2-环戊基乙酸苄酯(参照文献Journal of Medicinal Chemistry,2013,56(13),5541-5552.公布的方法合成)(7.2g,32.6mmol)，滴毕自然升温至室温反应(反应约2-4h)，TLC监控。反应结束，加入饱和NaHCO 3溶液，用乙酸乙酯萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，浓缩，残余物经硅胶柱纯化(PE:EA＝10:1-5:1-2:1)得13a(4.6g)，收率53％；LC-MS：[M+H] +＝529.5。
第二步：化合物13b
于25mL单口瓶，加入13a(4g,7.6mmol)，10mL DMF，0℃搅拌，加入DBU(1.17g,7.8mmol)，反应1h，TLC监测Fmoc脱保护完成后，待用；
另取25mL单口瓶中加入M4(3.14g,7.6mmol)，PyBOP(4.42g,8.5mmol)，HOBt(1.15g,8.5mmol)及10mL DMF，冰水浴下加入DIPEA(1.39mL,0.85mmol)，继续搅拌30min，将上述反应液加至反应瓶中，升至室温反应。HPLC监测反应结束后，反应液经制备液相纯化，得到产品制备液，制备液经二氯甲烷萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到固体2.1g，收率39％；LC-MS：[M+H] +＝702.8。
第三步：化合物13c
于25mL单口瓶中加入13b(1.5g,1.87mmol)，25mL DMF溶清后，加入1.5g 5％Pd/C，氢化反应3h，反应完毕，过滤，得滤液，未经纯化直接用于下一步反应。
第四步：化合物13d
将粗产品13c置于冰水浴中，加入DIPEA(333uL,1.93mmol)，再加入化合物M3(1.1g,1.87mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得到制备液，制备液冻干得到13d(519mg)；LC-MS：[M-H] -＝856.6。
第五步：化合物13e
于50mL单口瓶中加入13d(400mg,0.47mmol)，M5(240mg,0.48mmol)，PyBOP(250mg,0.48mmol)，HOBt(103mg,48mmol)及15mL DMF，冰水浴下加入DIPEA(330uL,2.0mmol)，升至室温反应4h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物13e的制备液，制备液冻干得到13e(187mg)；LC-MS：[M+H] +＝1275.5。
第六步：化合物13A
于25mL单口瓶中加入13e(100mg,0.08mmol)，溴化锌(355mg,0.16mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体化合物13A(60mg)；LC-MS：[M+H] +＝1119.6。
实施例23
化合物13B的合成：
Figure PCTCN2021097302-appb-000053
参照实施例22合成路线，得到化合物13B(51mg)；LC-MS：[M+H] +＝1119.6。
实施例24
化合物14A的合成：
Figure PCTCN2021097302-appb-000054
第一步：化合物14a
于250mL单口瓶中，加入M1(6g,16.3mmol)，100mL THF，对甲苯磺酸一水合物(0.31g,1.63mmol)，搅拌冷却至0℃，滴加3-羟基-2-环戊基丙酸苄酯(参照专利WO2009011285A1公布的方法合成)(7.6g,32.6mmol)，滴毕自然升温至室温反应(反应约2-4h)，TLC监控。反应结束，加入饱和NaHCO 3溶液，用乙酸乙酯萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，浓缩，残余物经硅胶柱纯化(PE:EA＝10:1-5:1-2:1)得14a(4.4g)，收率49％；LC-MS：[M+H] +＝543.6。
第二步：化合物14b
于25mL单口瓶，加入14a(4g,7.4mmol)，10mL DMF，0℃搅拌，加入DBU(1.2g,8.0mmol)，反应1h，TLC监测Fmoc脱保护完成后，待用；
另取25mL单口瓶中加入M4(3.1g,7.4mmol)，PyBOP(4.6g,8.8mmol)，HOBt(1.19g,8.8mmol)及10mL DMF，冰水浴下加入DIPEA(1.49mL,9.0mmol)，继续搅拌30min，将上述反 应液加至反应瓶中，升至室温反应。HPLC监测反应结束后，反应液经制备液相纯化，得到产品制备液，制备液经二氯甲烷萃取，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到固体2.6g，收率49％；LC-MS：[M+H] +＝716.4。
第三步：化合物14c
于25mL单口瓶中加入14b(1.0g,1.4mmol)，15mL DMF溶清后，加入1.0g 5％Pd/C，氢化反应1.5h，反应完毕，过滤，得滤液，未经纯化直接用于下一步反应。
第四步：化合物14d
将粗产品14c置于冰水浴中，加入DIPEA(248uL,1.5mmol)，再加入化合物M3(808mg,1.4mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到14d(500mg)；LC-MS：[M-H] -＝870.5。
第五步：化合物14e
于50mL单口瓶中加入14d(400mg,0.46mmol)，M5(235mg,0.46mmol)，PyBOP(245mg,0.46mmol)，HOBt(99mg,0.46mmol)及15mL DMF，冰水浴下加入DIPEA(331uL,2.0mmol)，升至室温反应3h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物14e的制备液，制备液冻干得到14e(146mg)；LC-MS：[M+H] +＝1289.5。
第六步：化合物14A
于25mL单口瓶中加入14e(100mg,0.08mmol)，溴化锌(360mg,1.6mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体化合物14A(52mg)；LC-MS：[M+H] +＝1133.4。
实施例25
化合物14B的合成：
Figure PCTCN2021097302-appb-000055
参照实施例24合成路线，得到化合物14B(48mg)；LC-MS：[M+H] +＝1133.4。
实施例26
化合物15A和15B的合成：
Figure PCTCN2021097302-appb-000056
第一步：化合物15a
于250mL单口瓶中加入M1(10g,27.1mmol),2-羟基-丁酸苄酯(参照文献Chemical Communications,2019,55(53),7699-7702.公布的方法制备)(10.5g,54.3mmol)，醋酸锌(9.96g,54.3mmol)及100mL甲苯，加热至100℃反应4h。反应完毕，降至室温，过滤除去不溶物，滤液浓缩得粗品。粗品经硅胶柱层析纯化(PE:EA＝10:1-5:1-2:1)得到目标物5.67g，收率42％；LC-MS：[M+H] +＝503.5。
第二步：化合物15b
于50mL单口瓶中加入15a(5g,9.95mmol)及15mL DMF，溶清后，冰水浴下，加入DBU(1.68g,11mmol),反应1h，记为反应液①；
另取50mL单口瓶，加入M4(4.1g,10.0mmol)，PyBOP(5.75g,11mmol)，HOBt(1.49g,11mmol)及10mL DMF，溶清后，冰水浴下，加入DIPEA(1.82mL,11mmol)，继续反应40min，加入反应液①，升至室温反应2h。HPLC监测反应进程，反应完毕后，反应液经高效液相纯化，得制备液。制备液经二氯甲烷萃取、饱和氯化钠溶液洗涤、无水硫酸钠干燥、过滤、浓缩得到固体4.6g，收率68％；LC-MS：[M+H] +＝676.7。
第三步：化合物15d
于25mL单口瓶中加入15b(2.0g,2.96mmol)，15mL DMF溶清后，加入2.0g 5％Pd/C，氢化反应2h，反应完毕，过滤，将滤液置于冰水浴中，加入DIPEA(496uL,3.0mmol)，再加入M3(1.7g,2.96mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到产品1120.0mg，收率45％；LC-MS：[M-H] -＝830.3。
第四步：化合物15e
于50mL单口瓶中加入15d(500mg,0.60mmol)，M5(321mg,0.60mmol)，PyBOP(469mg,0.90mmol)，HOBt(121mg,0.90mmol)及15mL DMF,冰水浴下加入DIPEA(446uL,2.7mmol)，升至室温反应2h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物15e-1和化合物15e-2的制备液，制备液分别冻干得到138mg化合物15e-1，LC-MS：[M+H] +＝1249.5；140mg化合物15e-2，LC-MS：[M+H] +＝1249.5。
第五步：化合物15A
Figure PCTCN2021097302-appb-000057
于25mL单口瓶中加入15e-1(100mg,0.08mmol)，溴化锌(360mg,1.6mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体59mg；LC-MS：[M+H] +＝1093.4。
第六步：化合物15B
Figure PCTCN2021097302-appb-000058
于25mL单口瓶中加入15e-2(100mg,0.08mmol)，溴化锌(360mg,1.6mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体60mg；LC-MS：[M+H] +＝1093.4。
实施例27
化合物16A和16B的合成：
Figure PCTCN2021097302-appb-000059
参照实施例26合成路线，得到化合物16A(55mg)；LC-MS：[M+H] +＝1093.4。
Figure PCTCN2021097302-appb-000060
参照实施例26合成路线，得到化合物16B(54mg)；LC-MS：[M+H] +＝1093.4。
实施例28
化合物17A和17B的合成：
Figure PCTCN2021097302-appb-000061
第一步：化合物17a
于250mL单口瓶中加入M1(10g,27.1mmol),2-羟基-苯丙酸苄酯(参考文献Nature Communications,2020.11(1),56.公布的方法合成)(14.7g,54.3mmol)，醋酸锌(9.96g,54.3mmol)及100mL甲苯，加热至100℃反应4h。反应完毕，降至室温，过滤除去不溶物，滤液浓缩得粗品。粗品经硅胶柱层析纯化(PE:EA＝10:1-5:1-2:1)得到目标物6.13g，收率40％；LC-MS：[M+H] +＝565.6。
第二步：化合物17b
于50mL单口瓶中加入17a(5g,8.86mmol)及15mL DMF，溶清后，冰水浴下，加入DBU(1.53g,10mmol)，反应1h，记为反应液①；
另取50mL单口瓶，加入M4(3.6g,8.86mmol)，PyBOP(5.23g,10mmol)，HOBt(1.36g,10mmol)及10mL DMF，溶清后，冰水浴下，加入DIPEA(1.65mL,10mmol)，继续反应30min，加入反应液①，升至室温反应2h。HPLC监测反应进程，反应完毕后，反应液经高效液相纯化，得制备液。制备液经二氯甲烷萃取、饱和氯化钠溶液洗涤、无水硫酸钠干燥、过滤、浓 缩得到固体5.0g，收率77％；LC-MS：[M+H] +＝738.3。
第三步：化合物17d
于25mL单口瓶中加入17b(3.0g,4.07mmol)，15mL DMF溶清后，加入3.0g 5％Pd/C，氢化反应2h，反应完毕，过滤，将滤液置于冰水浴中，加入DIPEA(744uL,4.5mmol)，再加入M3(2.34g,4.07mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到产品1.2g，收率33％；LC-MS：[M-H] -＝892.4。
第四步：化合物17e
于50mL单口瓶中加入17d(500mg,0.56mmol)，M5(300mg,0.56mmol)，PyBOP(438mg,0.84mmol)，HOBt(113mg,0.84mmol)及15mL DMF，冰水浴下加入DIPEA(330uL,2.0mmol)，升至室温反应2h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物17e-1和化合物17e-2的制备液，制备液分别冻干得到156mg化合物17e-1，LC-MS：[M+H] +＝1311.4；150mg化合物17e-2，LC-MS：[M+H] +＝1311.7。
第五步：化合物17A
Figure PCTCN2021097302-appb-000062
于25mL单口瓶中加入17e-1(100mg,0.08mmol)，溴化锌(360mg,1.6mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体43mg；LC-MS：[M+H] +＝1155.4。
第六步：化合物17B
Figure PCTCN2021097302-appb-000063
于25mL单口瓶中加入17e-2(100mg,0.08mmol)，溴化锌(360mg,1.6mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体40mg；LC-MS：[M+H] +＝1155.4。
实施例29
化合物18A和18B的合成：
Figure PCTCN2021097302-appb-000064
参照实施例28合成路线，得到化合物18A(54mg)；LC-MS：[M+H] +＝1155.4。
Figure PCTCN2021097302-appb-000065
参照实施例28合成路线，得到化合物18B(55mg)；LC-MS：[M+H] +＝1155.4。
实施例30
化合物19A和19B的合成：
Figure PCTCN2021097302-appb-000066
第一步：化合物19a
于250mL单口瓶中加入M1(10g,27.1mmol),2-环丙基-2-羟基乙酸苄酯(参照专利WO2020244657A1公布的方法制备)(11.2g,54.3mmol)，醋酸锌(9.96g,54.3mmol)及100mL甲苯，加热至100℃反应4h。反应完毕，降至室温，过滤除去不溶物，滤液浓缩得粗品。粗品经硅胶柱层析纯化(PE:EA＝10:1-5:1-2:1)得到目标物4.97g，收率36％；LC-MS：[M+H] +＝515.2。
第二步：化合物19b
于50mL单口瓶中加入19a(4g,7.8mmol)及10mL DMF，溶清后，冰水浴下，加入 DBU(1.42g,9.3mmol),反应1h，记为反应液①；
另取50mL单口瓶，加入M4(3.2g,7.8mmol)，PyBOP(4.5g,8.6mmol)，HOBt(1.16g,8.6mmol)及10mL DMF，溶清后，冰水浴下，加入DIPEA(1.65mL,10mmol)，继续反应30min，加入反应液①，升至室温反应2h。HPLC监测反应进程，反应完毕后，反应液经高效液相纯化，得制备液。制备液经二氯甲烷萃取、饱和氯化钠溶液洗涤、无水硫酸钠干燥、过滤、浓缩得到固体4.2g，收率78％；LC-MS：[M+H] +＝688.3。
第三步：化合物19d
于25mL单口瓶中加入19b(1000mg,1.45mmol)，15mL DMF溶清后，加入1000mg5％Pd/C，氢化反应2h，反应完毕，过滤，将滤液置于冰水浴中，加入DIPEA(248uL,1.5mmol)，再加入M3(720mg,1.45mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到产品503mg，收率41％；LC-MS：[M-H] -＝842.3。
第四步：化合物19e-1和19e-2
于50mL单口瓶中加入19d(500mg,0.59mmol)，M5(317mg,0.59mmol)，PyBOP(339mg,0.65mmol)，HOBt(88mg,0.86mmol)及10mL DMF，冰水浴下加入DIPEA(292uL,1.77mmol)，升至室温反应2h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物19e-1和化合物19e-2的制备液，制备液分别冻干得到112mg化合物19e-1，LC-MS：[M+H] +＝1261.5；131mg化合物19e-2，LC-MS：[M+H] +＝1261.5。
第五步：化合物19A
Figure PCTCN2021097302-appb-000067
于25mL单口瓶中加入19e-1(100mg,0.079mmol)，溴化锌(357mg,1.59mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体55mg；LC-MS：[M+H] +＝1105.4。
第六步：化合物19B
Figure PCTCN2021097302-appb-000068
于25mL单口瓶中加入19e-2(100mg,0.079mmol)，溴化锌(357mg,1.59mmol)及5mL 硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体58mg；LC-MS：[M+H] +＝1105.4。
实施例31
化合物20A和20B的合成：
Figure PCTCN2021097302-appb-000069
第一步：化合物20a
于250mL单口瓶中加入M1(10g,27.1mmol),2-羟基-环丙基丙酸苄酯(参照专利WO2020063676A公布的方法合成)(12.0g,54.3mmol)，醋酸锌(9.96g,54.3mmol)及100mL甲苯，加热至100℃反应4h。反应完毕，降至室温，过滤除去不溶物，滤液浓缩得粗品。粗品经硅胶柱层析纯化(PE:EA＝10:1-5:1-2:1)得到目标物5.09g；LC-MS：[M+H] +＝529.2。
第二步：化合物20b
于50mL单口瓶中加入20a(4g,7.6mmol)及10mL DMF，溶清后，冰水浴下，加入DBU(1.39g,9.1mmol)，反应1h，记为反应液①；
另取50mL单口瓶，加入M4(3.12g,7.6mmol)，PyBOP(4.5g,8.6mmol)，HOBt(1.16g,8.6mmol)及10mL DMF，溶清后，冰水浴下，加入DIPEA(1.65mL,10mmol)，继续反应30min，加入反应液①，升至室温反应2h。HPLC监测反应进程，反应完毕后，反应液经高效液相纯化，得制备液。制备液经二氯甲烷萃取、饱和氯化钠溶液洗涤、无水硫酸钠干燥、过滤、浓缩得到固体4.5g，收率84％；LC-MS：[M+H] +＝702.3。
第三步：化合物20d
于25mL单口瓶中加入20b(1000mg,1.42mmol)，15mL DMF溶清后，加入1000mg5％Pd/C，氢化反应2h，反应完毕，过滤，将滤液置于冰水浴中，加入DIPEA(248uL,1.5mmol)，再加入M5(708mg,1.42mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到产品443mg，收率36％；LC-MS：[M-H] -＝856.4。
第四步：化合物20e-1和20e-2
于50mL单口瓶中加入20d(400mg,0.47mmol)，依喜替康甲磺酸盐(250mg,0.47mmol)，PyBOP(223mg,0.56mmol)，HOBt(83mg,0.56mmol)及10mL DMF，冰水浴下加入DIPEA(248uL,1.5mmol)，升至室温反应2h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物20e-1和化合物20e-2的制备液，制备液分别冻干得到103mg化合物20e-1，LC-MS：[M+H] +＝1275.5；103mg化合物20e-2，LC-MS：[M+H] +＝1275.5。
第五步：化合物20A
Figure PCTCN2021097302-appb-000070
于25mL单口瓶中加入8A(100mg,0.078mmol)，溴化锌(352mg,1.57mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体51mg；LC-MS：[M+H] +＝1119.4。
第六步：化合物20B
Figure PCTCN2021097302-appb-000071
于25mL单口瓶中加入20e-2(100mg,0.079mmol)，溴化锌(357mg,1.59mmol)及5mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体47mg；LC-MS：[M+H] +＝1119.4。
实施例32
化合物21的合成：
Figure PCTCN2021097302-appb-000072
第一步：化合物SM3-1
于2000mL单口瓶中加入77087-60-6(100g,458mmol),马来酸(53.4g,460mmol)，TEA(64mL,460mmol)及1000mL甲苯，加热至100℃反应5h。反应完毕，降至室温，过滤除去不溶物，滤液浓缩得粗品。粗品经硅胶柱层析纯化(PE:EA＝100:1-50:1-20:1)得到目标物75.6g；LC-MS：[M+H] +＝299.1。
第二步：化合物(R)-2-羟基-1,5-戊二酸叔丁酯
于2000mL单口瓶中加入172793-31-6(100g,338mmol)及1000mL水,依次加入亚硝酸钠(35g,507mmol)，浓硫酸(32mL,35mmol)，缓慢升温至室温反应24h。反应完毕，乙酸乙酯500mL萃取三次，有机相用无水硫酸钠干燥，过滤，减压浓缩除去溶剂，得粗品。粗品经硅胶柱层析纯化(PE:EA＝50:1-30:1-2:1)得到目标物91.2g；LC-MS：[M+H] +＝261.4。
第三步：化合物SM3
于2000mL单口瓶中加入(R)-2-羟基-1,5-戊二酸叔丁酯(50g,192mmol)及1000mL无水四氢呋喃，冰水浴降温至0℃，依次加入PPh 3(87.7g,288mmol)，DEAD(50.2g,288mmol)及SM3-1(57.3,192mmol)，缓慢升温至室温反应13h。反应完毕，过滤除去不溶物，滤液浓缩得粗品。粗品经硅胶柱层析纯化(PE:EA＝50:1-30:1-1:1)得到产品68.6g；
将上述产品溶于500mL甲醇中，冰水浴冷却至0℃，于该温度下滴加NaOH(64mL,190mmol,3M/L)，维持该温度下反应12h后，加入HCl(6M/L)调节pH为3，二氯甲烷500mL萃取五次，无水硫酸钠干燥，过滤，滤液减压浓缩，所得粗产品经柱层析(DCM/MeOH＝50/1-20/1-2/1)纯化，得到SM3 50.4g；LC-MS：[M-H] -＝525.5。
第四步：化合物M6
于2000mL单口瓶，加入化合物SM3(50g，95mmol,1.0eq)，五氟苯酚(19.2g,104.5mmol,1.1eq)、DCC(21.5g,104.5mmol,1.1eq)及THF(600mL)，室温反应1h(采用TLC监测)，过滤滤去不溶物。反应液直接经制备纯化，制备液水泵减压水浴35℃浓缩除去乙腈，冻干得到化合物M6(51.9g)，收率79％；LC-MS：[M+H] +＝693.3。
第五步：化合物21a
于25mL单口瓶中加入1c(1g,2.36mmol)，25mL DMF溶清后，加入DIPEA(430uL,2.6mmol)，再加入M6(1177mg,2.36mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到产品555mg；LC-MS：[M-H] -＝931.0。
第六步：化合物21b
于100mL单口瓶中加入21a(500mg,0.54mmol)，依喜替康甲磺酸盐M5(285mg,0.54mmol)，PyBOP(239mg,0.6mmol)，HOBt(239mg,0.6mmol)及10mL DMF，冰水浴下加入DIPEA(248uL,1.5mmol)，升至室温反应2h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物21b的制备液，制备液冻干得到化合物231mg；LC-MS：[M+H] +＝1349.5。
第七步：化合物21
于25mL单口瓶中加入化合物21b(200mg,0.1488mmol)，溴化锌(665mg,2.96mmol)及10mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体103mg；LC-MS：[M+H] +＝1137.5。
实施例33
Figure PCTCN2021097302-appb-000073
化合物22的合成：
以化合物M6及3c为起始原料，参照实施例32的合成路线，得到化合物22(91mg)；LC-MS：[M+H] +＝1165.5。
实施例34
Figure PCTCN2021097302-appb-000074
化合物23及24的合成：
以化合物M6及5c为起始原料，参照实施例32的合成路线，得到102mg化合物23，LC-MS：[M+H] +＝1151.4；得到99mg化合物24，LC-MS：[M+H] +＝1151.4。
实施例35
Figure PCTCN2021097302-appb-000075
化合物25和26的合成：
以化合物M6及7c为起始原料，参照实施例32的合成路线，得到83mg化合物25，LC-MS：[M+H] +＝1205.7；得到80mg化合物26，LC-MS：[M+H] +＝1205.7。
实施例36
Figure PCTCN2021097302-appb-000076
化合物27和28的合成：
以化合物M6及19c为起始原料，参照实施例32的合成路线，得到100mg化合物27，LC-MS：[M+H] +＝1177.5；得到101mg化合物28，LC-MS：[M+H] +＝1177.5。
实施例37
化合物29的合成：
Figure PCTCN2021097302-appb-000077
第一步：化合物SM4-1
于5000mL单口瓶，加入马来酸(50g,431mmol,1.0eq)，114559-25-0(110g,431mmol,1eq)、TEA(263g,2.16mol,5eq)及甲苯(2000mL)，加热至回流反应5h(采用TLC监测)，过滤滤去不溶物。反应液直接减压旋转蒸馏除去溶剂，残余物经硅胶柱层析(PE/EA＝50/1-20/1-1/1)得到SM4-1(64.7g)，收率50％；LC-MS：[M+H] +＝299.2。
第二步：化合物SM4-2
于2000mL单口瓶中加入SM4-1(64g,215mmol)，1000mL DMF溶清后，加入DIPEA(71mL,430mmol)，再加入壬乙二醇单甲醚甲磺酸酯(111.5g,220mmol)，加毕升至室温反应2h。HPLC监测反应完毕，反应液经硅胶柱层析(PE/EA＝50/1-20/1-1/1)纯化，得到产品59.9g；LC-MS：[M+H] +＝709.4。
第三步：化合物SM4
于2000mL单口瓶中加入SM4-2(59g,83mmol)，1000mL MeOH溶清后，加入K 2CO 3(11.75g,85mmol)，加毕于室温反应4h。HPLC监测反应完毕，过滤除去不溶物，反应液直接经制备纯化，制备液水泵减压水浴35℃浓缩除去乙腈，冻干得到化合物SM4(27g)；LC-MS：[M-H] —＝693.5。
第四步：化合物M7
于500mL单口瓶，加入化合物SM4(25g,36mmol,1.0eq)、五氟苯酚(7.3g,40mmol,1.1eq)、DCC(8.2g,40mmol,1.1eq)及THF(200mL)，室温反应1h(采用TLC监测)，过滤滤去不溶物。反应液直接经制备纯化，制备液水泵减压水浴35℃浓缩除去乙腈，冻干得到化合物M7(23.3g)，收率93％；LC-MS：[M+H] +＝695.8。
第五步：化合物29a
于25mL单口瓶中加入1c(1g,2.36mmol)，25mL DMF溶清后，加入DIPEA(430uL,2.6mmol)，再加入M7(1640mg,2.36mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到产品609mg；LC-MS：[M-H] -＝1098.5。
第六步：化合物29b
于100mL单口瓶中加入29a(500mg,0.45mmol)，依喜替康甲磺酸盐M5(240mg,0.45mmol)，PyBOP(215mg,0.54mmol)，HOBt(215mg,0.54mmol)及10mL DMF，冰水浴下加入DIPEA(248uL,1.5mmol)，升至室温反应2h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物29b的制备液，制备液冻干得到化合物187mg；LC-MS：[M+H] +＝1517.6。
第七步：化合物29
于25mL单口瓶中加入化合物29b(150mg,0.988mmol)，溴化锌(223mg,0.988mmol)及10mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体114mg；LC-MS：[M+H] +＝1517.9。
实施例38
Figure PCTCN2021097302-appb-000078
化合物30的合成：
以化合物M7及3c为起始原料，参照实施例37的合成路线，得到化合物30(125mg)；LC-MS：[M+H] +＝1445.6。
实施例39
Figure PCTCN2021097302-appb-000079
化合物31及32的合成：
以化合物M7及5c为起始原料，参照实施例37的合成路线，得到61mg化合物31，LC-MS：[M+H] +＝1431.7；得到63mg化合物32，LC-MS：[M+H] +＝1431.7。
实施例40
Figure PCTCN2021097302-appb-000080
化合物33和34的合成：
以化合物M7及7c为起始原料，参照实施例37的合成路线，得到60mg化合物33，LC-MS：[M+H] +＝1485.6；得到58mg化合物34，LC-MS：[M+H] +＝1485.6。
实施例41
Figure PCTCN2021097302-appb-000081
化合物35和36的合成：
以化合物M7及19c为起始原料，参照实施例37的合成路线，得到102mg化合物35，LC-MS：[M+H] +＝1457.8；得到102mg化合物36，LC-MS：[M+H] +＝1457.8。
实施例42
化合物37的合成：
Figure PCTCN2021097302-appb-000082
第一步：化合物SM5-1
于2000mL单口瓶，加入化合物16947-84-5(100g,295mmol,1.0eq)、DIPEA(50mL,300mmol)，溴化苄(51.3g,300mmol)及THF(1000mL)，室温反应12h(采用TLC监测)，过滤滤 去不溶物。反应液直接减压旋转蒸馏除去溶剂，残余物经硅胶柱层析(PE/EA＝50/1-20/1-2/1)得到SM5-1(110.1g)，收率87％；LC-MS：[M+H] +＝429.2。
第二步：化合物SM5-2
于2000mL单口瓶，加入化合物SM5-1(100g,233.4mmol,1.0eq)及THF(1000mL)，冰水浴冷却至0℃，分批次加入NaH(37.4g,933.5mmol)，MeI(132.5g,933.5mmol)，维持0℃下反应24h(采用TLC监测)，加入饱和NH 4Cl水溶液500mL淬灭反应，乙酸乙酯500mL萃取三次，无水硫酸钠干燥有机相，过滤。滤液直接减压旋转蒸馏除去溶剂，残余物经硅胶柱层析(PE/EA＝100/1-50/1-10/1)得到SM5-2(37.1g)；LC-MS：[M+H] +＝443.3。
第三步：化合物SM5(参照文献Org.Lett.,2006,8,3387-3390.)
于1000mL单口瓶，加入化合物SM5-2(35g,79mmol,1.0eq)及DCE(500mL)，依次加入二乙酸钯(180mg,0.8mmol)，I 2(20g,79mmol)，二乙酸碘苯(40.8g,126.4mmol)，升温至60℃下反应40h(采用TLC监测)，加入饱和硫代硫酸钠水溶液500mL淬灭反应，二氯甲烷500mL萃取三次，无水硫酸钠干燥有机相，过滤。滤液直接减压旋转蒸馏除去溶剂，残余物经硅胶柱层析(PE/EA＝100/1-50/1-10/1)得到SM5(28g)；LC-MS：[M+H] +＝501.3。
第四步：化合物SM6
于500mL单口瓶，加入化合物SM5(25g,50mmol,1.0eq)、磷酸二叔丁酯钾盐(13.66g,55mmol,1.1eq)、一水合对甲苯磺酸(951mg,5mmol,0.1eq)及THF(200mL)，室温反应1h(采用TLC监测)，过滤滤去不溶物。反应液直接经制备纯化，制备液水泵减压水浴35℃浓缩除去乙腈，冻干得到化合物SM6(15.1g)，收率46％；LC-MS：[M+H] +＝651.4。
第五步：化合物SM7
于250mL单口瓶中加入SM6(15g,23mmol)及100mL DMF，溶清后，冰水浴下，加入15g 5％Pd/C，氢气置换体系内的气氛三次，室温下反应12h，过滤除去Pd/C，油泵减压旋蒸除去溶剂，待用；
另取250mL单口瓶，加入上述粗品及100mL甲苯，三乙胺(6.4mL,46mmol)，马来酸酐(2.4g,24mmol)，溶清后，升至100℃反应2h。HPLC监测反应进程，反应完毕后，反应液经高效液相纯化，得制备液。制备液经二氯甲烷萃取、饱和氯化钠溶液洗涤、无水硫酸钠干燥、过滤、浓缩得到固体4.2g，收率36％；LC-MS：[M+H] +＝507.3。
第六步：化合物M8
于100mL单口瓶，加入化合物SM7(4g,7.9mmol,1.0eq)、五氟苯酚(1.6g,8.7mmol,1.1eq)、DCC(1.8g,8.7mmol,1.1eq)及THF(60mL)，室温反应1h(采用TLC监测)，过滤滤去不溶物。反应液直接经制备纯化，制备液水泵减压水浴35℃浓缩除去乙腈，冻干得到化合物 M8(3.7g)，收率70％；LC-MS：[M+H] +＝673.2。
第七步：化合物37a
于25mL单口瓶中加入1c(1g,2.36mmol)，25mL DMF溶清后，加入DIPEA(430uL,2.6mmol)，再加入M8(1.2g,2.36mmol)，加毕升至室温反应1h。HPLC监测反应完毕，反应液经高效液相纯化，得制备液，制备液冻干得到产品488mg；LC-MS：[M-H] -＝911.0。
第八步：化合物37b
于100mL单口瓶中加入37a(400mg,0.44mmol)，依喜替康甲磺酸盐M5(235mg,0.44mmol)，PyBOP(199mg,0.5mmol)，HOBt(69mg,0.5mmol)及10mL DMF，冰水浴下加入DIPEA(218uL,1.32mmol)，升至室温反应2h。HPLC监测反应完毕后，反应液经高效液相纯化，得化合物37b的制备液，制备液冻干得到化合物201mg；LC-MS：[M+H] +＝1329.6。
第九步：化合物37
于25mL单口瓶中加入化合物37b(130mg,0.098mmol)，溴化锌(221mg,0.98mmol)及10mL硝基甲烷，40℃下反应1h。HPLC监测反应完毕后，减压浓缩除去溶剂，得粗品。粗品经高效液相纯化，得到产品制备液，制备液冻干得到固体96mg；LC-MS：[M+H] +＝1117.4。
实施例43
Figure PCTCN2021097302-appb-000083
化合物38的合成：
以化合物M8及3c为起始原料，参照实施例42的合成路线，得到化合物38(51mg)；LC-MS：[M+H] +＝1145.6。
实施例44
Figure PCTCN2021097302-appb-000084
化合物39及40的合成：
以化合物M8及5c为起始原料，参照实施例42的合成路线，得到57mg化合物39，LC-MS：[M+H] +＝1131.4；得到60mg化合物40，LC-MS：[M+H] +＝1131.4。
实施例45
Figure PCTCN2021097302-appb-000085
化合物41和42的合成：
以化合物M7及7c为起始原料，参照实施例42的合成路线，得到44mg化合物41，LC-MS：[M+H] +＝1185.3；得到44mg化合物42，LC-MS：[M+H] +＝1185.3。
实施例46
Figure PCTCN2021097302-appb-000086
化合物43和44的合成：
以化合物M8及19c为起始原料，参照实施例42的合成路线，得到62mg化合物43，LC-MS：[M+H] +＝1157.4；得到59mg化合物44，LC-MS：[M+H] +＝1157.4。
实施例47(对照例)
Figure PCTCN2021097302-appb-000087
化合物45的合成
化合物45参照专利“CN104755494A”的实施例58提供的方法合成。
以下为Trastuzumab的序列：
轻链
Figure PCTCN2021097302-appb-000088
重链
Figure PCTCN2021097302-appb-000089
配体-药物偶联物的制备：
1)通用偶联方法
将通过初步的纯化后单体率大于95％的抗体分子，使用超滤离心管换液至磷酸盐缓冲液中，浓度10mg/mL。加入20倍于抗体摩尔分子数的TCEP，室温下反应4h以打开抗体链间二硫键。加入20倍于抗体摩尔分子数的连接子-药物化合物(payload)，室温下反应2h。反应结束后，使用截留分子量为30KDa的超滤离心管换液至PBS中，并去除未偶联的payload。换液后的ADC样品使用0.22微米除菌过滤器过滤后备用。
2)配体-药物偶联物DAR值的测定
单体率检测条件：
样品14000rpm离心5分钟，取上清液进样分析；
仪器：Waters e2695(2489UV/Vis)；
色谱柱：TSKgel G3000SWXL(7.8×300mm，5μm)；
流动相：A：50mM PB，300mM NaCl，200mM Arg，5％IPA，pH为6.5；
流动相A等度洗脱30min，流速：0.714mL/min，柱温25℃，检测波长：280nm。
DAR检测条件：
样品14000rpm离心5分钟，取上清液进样分析；
仪器：Waters H-class(TUV)；
色谱柱：Proteomix HIC Butyl-NP5(4.6×35mm，5μm)；
流动相：A：1.5M硫酸铵，0.025M无水磷酸钠，pH为7.0，B：0.025M无水磷酸钠，25％IPA，pH为7.0；
流动相A平衡色谱柱，流动相A和B梯度洗脱，流速0.8mL/min；柱温25℃，检测波长：214nm。
实施例48：ADC-1
Figure PCTCN2021097302-appb-000090
按照通用偶联方法制备得到ADC-1。
实施例49：ADC-2
Figure PCTCN2021097302-appb-000091
按照通用偶联方法制备得到ADC-2。
实施例50：ADC-3
Figure PCTCN2021097302-appb-000092
按照通用偶联方法制备得到ADC-3。
实施例51：ADC-4
Figure PCTCN2021097302-appb-000093
按照通用偶联方法制备得到ADC-4。
实施例52：ADC-5
Figure PCTCN2021097302-appb-000094
按照通用偶联方法制备得到ADC-5。
实施例53：ADC-6
Figure PCTCN2021097302-appb-000095
按照通用偶联方法制备得到ADC-6。
实施例54：ADC-7
Figure PCTCN2021097302-appb-000096
按照通用偶联方法制备得到ADC-7。
实施例55：ADC-8
Figure PCTCN2021097302-appb-000097
按照通用偶联方法制备得到ADC-8。
实施例56：ADC-9
Figure PCTCN2021097302-appb-000098
按照通用偶联方法制备得到ADC-9。
实施例57：ADC-10
Figure PCTCN2021097302-appb-000099
按照通用偶联方法制备得到ADC-10。
实施例58：ADC-11
Figure PCTCN2021097302-appb-000100
按照通用偶联方法制备得到ADC-11。
实施例59：ADC-12
Figure PCTCN2021097302-appb-000101
按照通用偶联方法制备得到ADC-12。
实施例60：ADC-13
Figure PCTCN2021097302-appb-000102
按照通用偶联方法制备得到ADC-13。
实施例61：ADC-14
Figure PCTCN2021097302-appb-000103
按照通用偶联方法制备得到ADC-14。
实施例62：ADC-15
Figure PCTCN2021097302-appb-000104
按照通用偶联方法制备得到ADC-15。
实施例63：ADC-16
Figure PCTCN2021097302-appb-000105
按照通用偶联方法制备得到ADC-16。
实施例64：ADC-17
Figure PCTCN2021097302-appb-000106
按照通用偶联方法制备得到ADC-17。
实施例65：ADC-18
Figure PCTCN2021097302-appb-000107
按照通用偶联方法制备得到ADC-18。
实施例66：ADC-19
Figure PCTCN2021097302-appb-000108
按照通用偶联方法制备得到ADC-19。
实施例67：ADC-20
Figure PCTCN2021097302-appb-000109
按照通用偶联方法制备得到ADC-20。
实施例68：ADC-21
Figure PCTCN2021097302-appb-000110
按照通用偶联方法制备得到ADC-21。
实施例69：ADC-22
Figure PCTCN2021097302-appb-000111
按照通用偶联方法制备得到ADC-22。
实施例70：ADC-23
Figure PCTCN2021097302-appb-000112
按照通用偶联方法制备得到ADC-23。
实施例71：ADC-24
Figure PCTCN2021097302-appb-000113
按照通用偶联方法制备得到ADC-24。
实施例72：ADC-25
Figure PCTCN2021097302-appb-000114
按照通用偶联方法制备得到ADC-25。
实施例73：ADC-26
Figure PCTCN2021097302-appb-000115
按照通用偶联方法制备得到ADC-26。
实施例74：ADC-27
Figure PCTCN2021097302-appb-000116
按照通用偶联方法制备得到ADC-27。
实施例75：ADC-28
Figure PCTCN2021097302-appb-000117
按照通用偶联方法制备得到ADC-28。
实施例76：ADC-29
Figure PCTCN2021097302-appb-000118
按照通用偶联方法制备得到ADC-29。
实施例77：ADC-30
Figure PCTCN2021097302-appb-000119
按照通用偶联方法制备得到ADC-30。
实施例78：ADC-31
Figure PCTCN2021097302-appb-000120
按照通用偶联方法制备得到ADC-31。
实施例79：ADC-32
Figure PCTCN2021097302-appb-000121
按照通用偶联方法制备得到ADC-32。
实施例80：ADC-33
Figure PCTCN2021097302-appb-000122
按照通用偶联方法制备得到ADC-33。
实施例81：ADC-34
Figure PCTCN2021097302-appb-000123
按照通用偶联方法制备得到ADC-34。
实施例82：ADC-35
Figure PCTCN2021097302-appb-000124
按照通用偶联方法制备得到ADC-35。
实施例83：ADC-36
Figure PCTCN2021097302-appb-000125
按照通用偶联方法制备得到ADC-36。
实施例84：ADC-37
Figure PCTCN2021097302-appb-000126
按照通用偶联方法制备得到ADC-37。
实施例85：ADC-38
Figure PCTCN2021097302-appb-000127
按照通用偶联方法制备得到ADC-38。
实施例86：ADC-39
Figure PCTCN2021097302-appb-000128
按照通用偶联方法制备得到ADC-39。
实施例87：ADC-40
Figure PCTCN2021097302-appb-000129
按照通用偶联方法制备得到ADC-40。
实施例88：ADC-41
Figure PCTCN2021097302-appb-000130
按照通用偶联方法制备得到ADC-41。
实施例89：ADC-42
Figure PCTCN2021097302-appb-000131
按照通用偶联方法制备得到ADC-42。
实施例90：ADC-43
Figure PCTCN2021097302-appb-000132
按照通用偶联方法制备得到ADC-43。
实施例91：ADC-44
Figure PCTCN2021097302-appb-000133
按照通用偶联方法制备得到ADC-44。
实施例92：ADC-45
Figure PCTCN2021097302-appb-000134
按照通用偶联方法制备得到ADC-45。
实施例93：ADC-46
Figure PCTCN2021097302-appb-000135
按照通用偶联方法制备得到ADC-46。
实施例94：ADC-47
Figure PCTCN2021097302-appb-000136
按照通用偶联方法制备得到ADC-47。
实施例95：ADC-48
Figure PCTCN2021097302-appb-000137
按照通用偶联方法制备得到ADC-48。
实施例96：ADC-49
Figure PCTCN2021097302-appb-000138
按照通用偶联方法制备得到ADC-49。
实施例97：ADC-50
Figure PCTCN2021097302-appb-000139
按照通用偶联方法制备得到ADC-50。
实施例98：ADC-51
Figure PCTCN2021097302-appb-000140
按照通用偶联方法制备得到ADC-51。
实施例99：ADC-52
Figure PCTCN2021097302-appb-000141
按照通用偶联方法制备得到ADC-52。
实施例100：ADC-53
Figure PCTCN2021097302-appb-000142
按照通用偶联方法制备得到ADC-53。
实施例101：ADC-54
Figure PCTCN2021097302-appb-000143
按照通用偶联方法制备得到ADC-54。
实施例102：ADC-55
Figure PCTCN2021097302-appb-000144
按照通用偶联方法制备得到ADC-55。
实施例103：ADC-56
Figure PCTCN2021097302-appb-000145
按照通用偶联方法制备得到ADC-56。
实施例104：ADC-57
Figure PCTCN2021097302-appb-000146
按照通用偶联方法制备得到ADC-57。
实施例105：ADC-58
Figure PCTCN2021097302-appb-000147
按照通用偶联方法制备得到ADC-58。
实施例106：ADC-59
Figure PCTCN2021097302-appb-000148
按照通用偶联方法制备得到ADC-59。
实施例107：ADC-60
Figure PCTCN2021097302-appb-000149
按照通用偶联方法制备得到ADC-60。
实施例108：ADC-61(对照组)
Figure PCTCN2021097302-appb-000150
按照通用偶联方法制备得到ADC-61。
实施例109：血浆稳定性
1)操作
取一定量的ADC样品，加入到已去除人IgG的人血浆中，每种ADC重复三管，放置37℃水浴中孵，分别孵育72h、144h后，取出ADC样品，每管加入ProteinA resin(MabSelect SuReTM  LX Lot:#10221479GE，用取PBS洗涤过的)100uL，垂直混合仪晃动吸附2h,经过洗涤洗脱步骤，获得孵育后的ADC。对孵育特定时间的ADC样品进行RP-HPLC检测。
2)结果
表1.本发明公开配体-药物偶联物(ADC)DAR值及单体率数据。
分子名称 DAR 聚集体％ 单体％
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(对照) 7.59 8.21 91.79
表2.本发明公开配体-药物偶联物(ADC)血浆稳定性数据。
Figure PCTCN2021097302-appb-000151
3)结论
如表1所示，本发明公开的带有高稳定性亲水连接单元的喜树碱类ADC具有DAR值(＞7.5)及单体率(＞97％)高的优异性质，相比于对照ADC-61，具有明显更高的单体率。
如表2所示，本发明公开的ADC血浆中孵育7天后，相比于对照ADC-61，DAR值仍可以保持较高水平，证明本发明的ADC在血浆中具有优异的稳定性。
实施例110：体外活性测试
1)实验材料
细胞：来源于中国科学院细胞库；
肿瘤细胞培养基：Gibco；
FBS：BIOWEST；
2)培养基的配制
生长培养基(with 10％FBS，Penicillin/streptomycin(100U/mL)；
检测培养基(with 1％FBS，Penicillin/streptomycin(100U/mL)；
3)操作
提前30min开启生物安全柜紫外灯照射，后通风3min。将生长培养基、检测培养基、D-PBS和胰酶放入37℃恒温水浴锅预热，之后用酒精对表面进行消毒，放入生物安全柜中。选择汇合度在～80％的细胞(对数生长期)，放于生物安全柜中，吸掉旧培养基，用D-PBS润洗，吸弃，用胰酶消化2～3min，后加入生长培养基终止胰酶，500×g离心5min。吸去离心上清液，用4mL检测培养基混匀，取100uL计数(其中取出50uL细胞液，加入50μL0.4％Trypan Blue Stain并混匀，混匀后计数)。按照之前设置好的细胞数铺板，80uL/孔铺于96孔板中，孔E11、F11、G11只加80uL检测培养基，边缘孔加入200uL的DPBS封边。待铺板细胞完全贴壁后(通常至少需要4小时)，进行受试样品配置与稀释：用检测培养基配置1.0mL,2.5μM(5×Top Dose)的受试样品，分装于V型96孔板第一列，每孔200μL；后面第2至8列分别加入180μL的检测培养基，从第一列中取30μL加入到第二列，用排枪上下混匀10次，弃枪头，剩余检测浓度点依次操作，进行7倍梯度浓度稀释。将梯度浓度的受试样品按照每孔20uL的量加入细胞中，同时第11列只加入20uL的检测培养基，每个浓度设置3个复孔，随后将96孔板放入5％CO 2,37℃细胞培养箱，培养5天。
4)检测
受试样品作用5天后取出MTS试剂，常温避光解冻后，充分涡旋混匀后，在生物安全柜中，沿孔侧壁按每100μL细胞培养体积加入20μL Cell Titer One Solution Reagen MTS试剂，轻轻拍动板面，使MTS溶液混合均匀，放于细胞培养箱中5％CO 2,37℃避光静置孵育2h。反应结束后，取出96孔板，于酶标仪中检测OD490nm吸光值，并进行数据记录、整理、存储。
5)结果
表3：抗体药物偶联物及毒素对N87肿瘤细胞的体外增值抑制的IC50值。
Figure PCTCN2021097302-appb-000152
表4：抗体药物偶联物及毒素对SK-BR-3肿瘤细胞的体外增值抑制的IC50值。
Figure PCTCN2021097302-appb-000153
6)讨论
如表3所示，本发明针对HER2靶标的配体-药物偶联物对HER2阳性细胞N87具有明显的体外增值抑制活性，明显优于裸抗(Trastuzumab)、对照组ADC-61及毒素单药。
如表4所示，与裸抗(Trastuzumab)及对照组ADC对比，本发明公开的ADC及单药对HER2阳性的细胞SK-BR-3也具有明显的体外增值抑制活性。
实施例111：体内活性测试
1)实验材料
细胞：来源于中国科学院细胞库；
肿瘤细胞培养基：Gibco；
Balb/c-nu裸鼠：雌性，5-7周(肿瘤细胞接种时的小鼠周龄)，体重18.0-24.0g，170只(110只加60只富余小鼠)。购自北京维通利华实验动物技术有限公司；
供试品和对照品：
供试品：ADC-61、ADC-6由成都多特抗体药物有限责任公司提供。
Histidine缓冲液，由成都多特抗体药物有限责任公司提供。
0.9％氯化钠注射液：科伦药业有限责任公司。
2)细胞培养
NCI-H1975(人非小细胞肺癌腺癌细胞)培养在RPMI1640培养基中。收集指数生长期的NCI-H1975细胞，RPMI1640培养基重悬至适合浓度后用于小鼠皮下肿瘤接种。
NCI-N87(人胃癌细胞)培养在RPMI1640培养基中。收集指数生长期的NCI-N87细胞，RPMI1640培养基重悬至适合浓度后用于小鼠皮下肿瘤接种。
3)动物造模和随机分组
85只雌性裸鼠右肩侧皮下接种5×10 7个NCI-H1975细胞。待肿瘤平均体积170mm 3左右时，根据肿瘤大小随机分组。选取55只肿瘤体积合适的荷瘤小鼠，随机分组并开始给药(尾静脉注射，给药体积按0.1ml/10g)。分组当天定义为第0天。
85只雌性裸鼠右肩侧皮下接种5×10 7个NCI-N87细胞。待肿瘤平均体积170mm 3左右时，根据肿瘤大小随机分组。选取55只肿瘤体积合适的荷瘤小鼠，随机分组并开始给药(尾静脉注射，给药体积按0.1ml/10g)。分组当天定义为第0天。
4)供试品和对照品的配制
表5.在NCI-H1975(人非小细胞肺癌腺癌细胞)和NCI-N87(人胃癌细胞)裸鼠皮下移植瘤模型中抗肿瘤作用研究的供试品和对照品溶液的配制。
Figure PCTCN2021097302-appb-000154
注：使用前混匀，确保制剂是均一的。
5)实验观察和数据收集
本实验过程中，动物实验操作均根据抗肿瘤药物体内筛选试验标准操作规程的要求。肿瘤接种后，常规监测包括了肿瘤生长(肿瘤每周测量2次)及治疗对动物正常行为的影响，具体内容有实验动物的活动性，摄食和饮水情况，体重增加或降低(体重每周测量2次)情况， 眼睛、被毛及其它异常情况。实验过程中观察到的临床症状均记录在原始数据中。肿瘤体积计算公式：肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。实验中采用人工记录数据，包括肿瘤的长短径的测量和动物体重的称量。
6)疗效评价标准
相对肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式如下：
T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)；或T/C％＝TTW/CTW×100％(TTW：治疗组实验终结时平均瘤重；CTW：溶媒对照组实验终结时平均瘤重)。
相对肿瘤抑制率，TGI(％)，计算公式如下：TGI％＝(1-T/C)×100％。[T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积(RTV)或瘤重(TW)]。
7)结果
表6：给药抗体药物偶联物对NCI-H1975移植瘤的体内疗效。
Figure PCTCN2021097302-appb-000155
表7：给药抗体药物偶联物对NCI-N87移植瘤的体内疗效。
Figure PCTCN2021097302-appb-000156
表8：给药抗体药物偶联物(11.25mg/kg)对NCI-H1975移植瘤小鼠体重影响情况。
NCI-H1975\分组信息\平均体重(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(11.25mg/kg) 19.99 20.66 19.70 20.46 20.65 19.99 20.68 19.89 20.52 20.69
ADC-61(11.25mg/kg)对照 19.78 20.55 19.52 19.32 19.57* 19.78* 19.36* 19.59* 20.14* 18.99*
注：*标识组观察到两只小鼠死亡
8)讨论
如表6所示，本发明公开ADC-6在低剂量对照组(3.75mg/Kg)对荷瘤小鼠NCI-H1975体内药效明显优于对照组ADC-61及裸抗；当剂量增加至11.25mg/Kg时，本发明公开ADC-6的治疗效果进一步提升，且明显优于对照ADC-61。
如表7所示，在相同剂量下(3.75mg/Kg)本发明公开ADC-6对荷瘤小鼠NCI-N87体内药效明显优于对照组ADC-61，对比高剂量裸抗(11.25mg/Kg)体内药效更显著。
如表8所示，本发明公开ADC-6在高剂量对照组11.25mg/Kg对NCI-H1975荷瘤小鼠的体重影响明显相比于ADC-61的影响小，即便在该高剂量组下，也未出现如对照组所示的小鼠死亡，证明本发明所述的ADC药物在安全性方面具有显著的优势。

Claims (20)

  1. 一种如式I所示的带有高稳定性亲水连接单元的配体-药物偶联物或其药学上可接受的盐，
    Figure PCTCN2021097302-appb-100001
    其中：
    Ab为配体单元，选自抗体、抗体片段、靶向蛋白或Fc-融合蛋白；
    M为与Ab连接的连接单元；
    Ac为亲水结构单元；
    D为任选的喜树碱类药物；
    1位及4位手性碳原子具有R绝对构型或S绝对构型两种手性构型；
    n选自1-20的整数。
  2. 如权利要求1所述配体-药物偶联物或其药学上可接受的盐，其特征在于：所述连接单元M具有如下式a所示的丁二酰亚胺或式b1、式b2所示的开环丁二酰亚胺结构，
    Figure PCTCN2021097302-appb-100002
    在式a、式b1或式b2中，左侧波浪线表示与Ab连接位点相连，右侧波浪线表示与式I中已标示的1位叔碳原子连接位点相连。
  3. 如权利要求1中所述配体-药物偶联物或其药学上可接受的盐，其特征在于：所述Ac具有如下式c所示结构，
    Figure PCTCN2021097302-appb-100003
    其中X非限制性地选自亲水结构羧基、磷酸、聚磷酸、亚磷酸、磺酸、亚磺酸或聚乙二醇(PEG)中的一种或多种所组成的组；
    Y为任选的连接氨基和X的支架；
    Ac通过氨基官能团与结构式I中已标示的2位亚甲基碳相连。
  4. 如权利要求1或3中任一项所述的配体-药物偶联物或其药学上可接受的盐，其特征在于：所述Ac非限制性地选自甘氨酸、(D/L)丙氨酸、(D/L)亮氨酸、(D/L)异亮氨酸、(D/L)缬氨酸、(D/L)苯丙氨酸、(D/L)脯氨酸、(D/L)色氨酸、(D/L)丝氨酸、(D/L)酪氨酸、(D/L)半胱氨酸、(D/L)胱氨酸、(D/L)精氨酸、(D/L)组氨酸、(D/L)蛋氨酸、(D/L)天冬酰胺、(D/L)谷氨酰胺、(D/L)苏氨酸、(D/L)天冬氨酸、(D/L)谷氨酸、天然或非天然氨基酸衍生物或以下结构，
    Figure PCTCN2021097302-appb-100004
  5. 如权利要求1中所述配体-药物偶联物或其药学上可接受的盐，其特征在于：所述喜树碱类药物具有如下式d所示的结构；
    Figure PCTCN2021097302-appb-100005
    其中R 1选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R 1及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    R 1相连手性碳原子具有R绝对构型或S绝对构型两种手性；
    m选自0或1；
    药物d分子中与R 1相连碳原子的羟基参与连接形成式I中的3位氧原子。
  6. 如权利要求1或5中任一项所述配体-药物偶联物或其药学上可接受的盐，其特征在于：所述喜树碱类药物非限制性的选自以下化合物；
    Figure PCTCN2021097302-appb-100006
  7. 一种用于与配体Ab偶联形成权利要求1中所述式I配体-药物偶联物的连接子-药物化合物或其药学上可接受的盐，其特征在于：具有如下式II所示的结构，
    Figure PCTCN2021097302-appb-100007
    其中R 1选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、 烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R 1及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    1位手性碳原子具有R绝对构型或S绝对构型两种手性；
    Ac为亲水结构单元；
    m选自0或1。
  8. 如权利要求7所述连接子-药物化合物或其药学上可接受的盐，其特征在于：所述Ac非限制性地选自甘氨酸、磷酸、(D/L)谷氨酸或聚乙二醇亲水结构。
  9. 如权利要求7或8中任一项所述连接子-药物化合物或其药学上可接受的盐，其特征在在于：所述连接子-药物化合物非限制性地选自以下结构，
    Figure PCTCN2021097302-appb-100008
    Figure PCTCN2021097302-appb-100009
    Figure PCTCN2021097302-appb-100010
    Figure PCTCN2021097302-appb-100011
    Figure PCTCN2021097302-appb-100012
    Figure PCTCN2021097302-appb-100013
    其中1位手性碳具有R绝对手性或S绝对手性两种构型。
  10. 如权利要求1-9中任一项所述配体-药物偶联物或其药学上可接受的盐，其特征在于：所述配体-药物偶联物或其药学上可接受的盐具有如下式Ⅲ、式Ⅳ-1或式Ⅳ-2所示结构，
    Figure PCTCN2021097302-appb-100014
    Figure PCTCN2021097302-appb-100015
    其中Ab为配体单元；
    Ac为亲水结构单元；
    1位手性碳具有R绝对手性或S绝对手性两种构型；
    R 1、m及n如式Ⅱ所述。
  11. 如权利要求10所述配体-药物偶联物或其药学上可接受的盐，其特征在于：所述配体单元Ab选自抗体、抗体片段或蛋白，其中所述抗体选自鼠源抗体、兔源抗体、噬菌体展示来源抗体、酵母展示来源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体。
  12. 如权利要求10所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于：所述抗体为单克隆抗体，非限制性地选自：抗EGFRvIII抗体、抗PD-1抗体、抗PD-L1抗体、抗DLL-3抗体、抗PSMA抗体、抗CD70抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM-1抗体、抗LRRC15抗体、抗LIV-1抗体、抗CanAg/AFP抗体、抗cladin 18.2抗体、抗Mesothelin抗体、抗HER2(ErbB2)抗体、抗EGFR抗体、抗c-MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7-H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗TROP-2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P-cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b 抗体、抗CD19抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD47抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体或抗CD123抗体。
  13. 如权利要求10所述配体-药物偶联物或其药学上可接受的盐，其特征在于：所述抗体或其抗原结合片段为曲妥珠单抗(Trastuzumab)，具有以下序列：
    Light chain
    MDMRVPAQLLGLLLLWLRGARC
    DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
    RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*；
                                   SEQ ID NO:1
    Heavy chain:
    MDMRVPAQLLGLLLLWLRGARC
    EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
    ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
                                   SEQ ID NO:2。
  14. 如权利要求10所述配体-药物偶联物或其药学上可接受的盐，其特征在于：所述配体-药物偶联物或其药学上可接受的盐非限制性地选自以下结构或其丁二酰亚胺开环结构，
    Figure PCTCN2021097302-appb-100016
    Figure PCTCN2021097302-appb-100017
    Figure PCTCN2021097302-appb-100018
    Figure PCTCN2021097302-appb-100019
    Figure PCTCN2021097302-appb-100020
    Figure PCTCN2021097302-appb-100021
    其中n选自1-10的整数。
  15. 一种制备权利要求7-9所述连接子-药物化合物或其药学上可接受的盐的方法，其特征在于：所述方法包含以下步骤，
    Figure PCTCN2021097302-appb-100022
    通式L化合物与式d 0依喜替康或其盐在缩合剂存在下，在任选的碱性条件下反应，得到 式Ⅳ化合物，进一步转化为式Ⅱ所示结构；
    其中1位及R 1相连手性碳原子具有R绝对构型或S绝对构型两种手性；
    R 2为任选的可转化为Ac的结构；
    Ac、R 1、m如式Ⅱ所定义。
  16. 一种权利要求15所述制备连接子-药物化合物或其药学上可接受的盐的方法，其特征在于：式Ⅳ转化为式Ⅱ的脱保护试剂为溴化锌，溶剂为硝基甲烷。
  17. 一种制备权利要求1-6所述配体-药物偶联物或其药学上可接受的盐的方法，其特征在于：所述方法包含以下步骤，
    Figure PCTCN2021097302-appb-100023
    修饰配体单元Ab后，与式Ⅱ通过偶联反应得到式Ⅲ所示配体-药物偶联物；
    其中Ab选自抗体、抗体片段或蛋白；
    Ac选自亲水结构单元；
    1位及R 1相连手性碳原子具有R绝对构型或S绝对构型两种手性；
    R 1、m及n如式Ⅱ所述。
  18. 一种药物组合物，其含有治疗有效量的如权利要求1-17中任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，以及药学上可接受的载体、稀释剂或赋形剂。
  19. 一种包括权利要求1-18中任一项所述的配体-药物偶联物或其药学可接受的盐的药物组合物，在制备用于治疗肿瘤、自身免疫疾病或感染性疾病药物中的用途。
  20. 如权利要求19所述的用途，其特征在于：用于制备治疗乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、肺癌、结肠癌、直肠癌、结直肠癌、骨癌、皮肤癌、甲状腺癌、胰腺癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、多形性胶质细胞瘤、肉瘤、淋巴瘤和白血病等实体瘤或血液瘤的药物。
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