WO2024007908A1 - 特异性拓扑异构酶抑制剂和可用于抗体药物偶联物及其制备方法 - Google Patents

特异性拓扑异构酶抑制剂和可用于抗体药物偶联物及其制备方法 Download PDF

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WO2024007908A1
WO2024007908A1 PCT/CN2023/103058 CN2023103058W WO2024007908A1 WO 2024007908 A1 WO2024007908 A1 WO 2024007908A1 CN 2023103058 W CN2023103058 W CN 2023103058W WO 2024007908 A1 WO2024007908 A1 WO 2024007908A1
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antibody
add
drug conjugate
pharmaceutically acceptable
racemate
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French (fr)
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王方道
王东
王猛
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博石丰生命科技(南通)有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • 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
    • A61K47/54—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 organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • 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
    • A61K47/6889—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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala

Definitions

  • the present invention relates to a conjugate containing a specific topoisomerase inhibitor and a drug linker that can be used in an antibody-drug conjugate, as well as a method for preparing the compound and related antibody-drug conjugate and in the preparation of a drug for treating cancer.
  • the use belongs to the field of medicinal chemistry technology.
  • the basic modules of antibody-conjugated drugs include antibodies, linkers, and toxin molecules.
  • Antibodies are used to transport toxin molecules to the tumor where they are enriched, thereby killing tumor cells.
  • Traditional toxin molecules are mostly highly active tubulin inhibitors or directly targeting DNA cytotoxic drugs, which usually have major toxic side effects, limiting the application of ADCs.
  • Immunomedics has developed a new ADC drug IMMU-132 using a camptothecin compound as a warhead molecule, which has shown good anti-tumor effects.
  • Daiichi Sankyo has developed another camptothecin compound as a warhead molecule ADC drug DS-8201a. , also showed good anti-tumor effect.
  • camptothecin compounds and antibodies are mainly connected by modifying the existing linker technology.
  • the ideal linker in ADC needs to meet the following requirements: First, ensure that small molecule drugs are not present in the plasma. After detaching from the antibody and entering the cell, the linker breaks under appropriate conditions and quickly releases the active small molecule drug; secondly, the linker must have good physical and chemical properties so that it can be connected with the antibody to form a conjugate; thirdly, the connection It should be easy to prepare to lay the foundation for large-scale production of ADC.
  • IMMU-132 uses a pH-sensitive linker, which has poor stability.
  • DS-8201a uses a glycine-glycine-phenylalanine-glycine (GGFG) tetrapeptide structure, which has better stability.
  • the toxins released by the above-mentioned ADC drugs are SN38 and Dxd, both of which are Pgp substrates. There are still problems such as multi-drug resistance in tumors.
  • camptothecin derivatives and ADC drugs with better efficacy and/or safety.
  • the present invention provides an inhibitor compound or its tautomer, meso, racemate, enantiomer, diastereomer, or mixture thereof. , or a pharmaceutically acceptable salt thereof, the compound comprising the structure represented by formula (A):
  • the Q is a group that can be coupled to the sulfhydryl group on the antibody, and is selected from maleimide.
  • the L1 is an amino linking group between the linker and the drug, which is selected from the group consisting of: L2 is optionally substituted C3-C7 alkylene, C3-C8 cycloalkyl, optionally substituted diethylene glycol to octaethylene glycol acyl, AA is a peptide segment composed of 2 to 4 amino acids, M is methylene , C1-C6 alkyl or cycloalkyl substituted methylene, trifluoromethyl substituted methylene, C3-C6 cycloalkyl.
  • the L1 is an amino linking group between the linker and the drug, selected from the group consisting of: L2 is optionally substituted C3-C7 alkylene, C3-C8 cycloalkyl, optionally substituted diethylene glycol to octaethylene glycol acyl.
  • the invention also provides an antibody-drug conjugate or its tautomer, meso, racemate, enantiomer, diastereomer, or mixture thereof, or its mixture.
  • Pharmaceutically acceptable salts the compound comprising the structure represented by formula (B):
  • the Q includes a linker coupled with a thiol group, selected from the group consisting of
  • the L1 is an amino linking group between the linker and the drug, which is selected from the group consisting of: L2 is optionally substituted C3-C7 alkylene, C3-C8 cycloalkyl, optionally substituted diethylene glycol to octaethylene glycol acyl, AA is a peptide segment composed of 2 to 4 amino acids, M is methylene , C1-C6 alkyl or cycloalkyl substituted methylene, trifluoromethyl substituted methylene, C3-C6 cycloalkyl.
  • the L1 is an amino linking group between the linker and the drug, selected from the group consisting of: L2 is optionally substituted C 3 -C 7 alkylene, C 3 -C 8 cycloalkyl, optionally substituted diethylene glycol to octaethylene glycol acyl.
  • the structure of formula B specifically includes the following:
  • the Ab is selected from the group consisting of murine antibodies, chimeric antibodies, humanized antibodies or fully human antibodies.
  • the antibody includes a monoclonal antibody.
  • the antibody includes a bispecific antibody.
  • the antibody can interact with HER2, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC -3, c-KIT, ROR1, PD-L1, CD27L, 5T4, Mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3 or Claudin 18.2 tumors Antibodies that bind to relevant antigens.
  • the present invention also provides a pharmaceutical composition, including: (a) the above-mentioned antibody drug conjugate; and (b) a pharmaceutically acceptable diluent, carrier or excipient.
  • the present invention also provides the use of the above-mentioned antibody-drug conjugate in preparing drugs for treating tumors.
  • the invention also provides a method for preparing the antibody drug conjugate, which includes the following steps:
  • step b Use the linker-drug conjugate (A) to cross-link the reduced antibody obtained in step a in a mixture of buffer and organic solvent to obtain an antibody-drug conjugate.
  • the term "pharmaceutically acceptable” ingredients refers to substances that are suitable for humans and/or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, have a reasonable benefit/risk ratio.
  • the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or that exhibits a detectable therapeutic or preventive effect.
  • the precise effective amount for a given subject will depend on the size and health of the subject, the nature and extent of the condition, and the therapeutic agent and/or combination of therapeutic agents chosen to be administered. Therefore, it is useless to pre-specify the exact effective amount. However, routine experimentation can be used to determine the effective amount for a given condition and the clinician will be able to judge this.
  • each chiral carbon atom may optionally be in R configuration or S configuration, or a mixture of R configuration and S configuration.
  • the term "compound of the invention” refers to a compound of Formula I.
  • the term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula I.
  • salts refers to salts of compounds of the invention with acids or bases suitable for use as pharmaceuticals.
  • Pharmaceutically acceptable salts include inorganic salts and organic salts.
  • One preferred class of salts are the salts of the compounds of the invention with acids.
  • Acids suitable for forming salts include, but are not limited to: hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and other inorganic acids, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, benzenesulfonic acid and other organic acids; as well as aspartic acid, glutamic acid and other acidic amino acids.
  • amino acid as used herein is intended to include any conventional amino acid, such as aspartic acid, glutamic acid, cysteine, asparagine, phenylalanine, glutamine, tyrosine , serine, methionine (methionine), tryptophan, glycine, valine, leucine, alanine, isoleucine, proline, threonine, histidine, lysine, Arginine.
  • amino acid as used herein is intended to include any conventional amino acid, such as aspartic acid, glutamic acid, cysteine, asparagine, phenylalanine, glutamine, tyrosine , serine, methionine (methionine), tryptophan, glycine, valine, leucine, alanine, isoleucine, proline, threonine, histidine, lysine, Arginine.
  • the trade name is intended to include the formulation of the trade name product, its corresponding generic version, and the active pharmaceutical ingredient of the trade name product.
  • antibody is used herein in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, clonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired biological activity (Miller et al. (2003) Journal of Immunology 170:4854-4861) .
  • Antibodies can be murine, human, humanized, chimeric, or derived from other species.
  • Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York).
  • the target antigen generally has a large number of binding sites recognized by the CDRs of multiple antibodies, also called epitopes. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen can have more than one corresponding antibody.
  • Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen or portion thereof that specifically binds to a target of interest, including, but not limited to, cancer cells or cells producing Cells of autoimmune antibodies associated with autoimmune diseases.
  • the immunoglobulins disclosed herein may be of any type (eg, IgG, IgE, IgM, IgD, and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass of immunoglobulin molecules.
  • Immunoglobulins can be derived from any species. However, in one aspect, the immunoglobulin is derived from human, mouse or rabbit.
  • antibody fragment includes a portion of a full-length antibody, typically its antigen-binding or variable region.
  • antibody fragments include: Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4):315- 323); Fragments prepared from Fab expression libraries; anti-idiotypic (anti-Id) antibodies; CDRs (complementarity determining regions); and any of the above epitopes that bind cancer cell antigens, viral antigens or microbial antigens in an immunospecific manner - binding fragments; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • the antibodies constituting the antibody-drug conjugates of the present invention should preferably maintain their original antigen-binding ability in the wild state. Therefore, the antibodies of the invention are capable of binding, preferably specifically, to the antigen.
  • Antigens involved include, for example, tumor associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (such as known or predicted Functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as known antibody-bound antigens can be one or a subset of the above classifications , while other subsets contain other molecules/antigens with special properties (compared to the target antigen).
  • 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 Known antibody preparation methods and information to prepare.
  • researchers strive to find transmembrane or other tumor-associated peptides. These targets can be specifically expressed on the surface of one or more cancer cells but have little or no expression on the surface of one or more non-cancer cells.
  • tumor-associated polypeptides are more overexpressed on cancer cell surfaces relative to non-cancer cell surfaces. Identifying such tumor-associated factors could greatly improve the specific targeting properties of antibody-based cancer treatments.
  • the term "ligand” generally refers to a macromolecular compound capable of recognizing and binding to an antigen or receptor associated with a target cell.
  • the role of the ligand can be to present the drug to the target cell population that binds to the ligand.
  • These ligands include but are not limited to protein hormones, lectins, growth factors, antibodies or others that can bind to cells, receptors and/or antigens. of molecules.
  • the ligand can be expressed as Ab, and the ligand antigen forms a connection bond with the connecting unit through the heteroatom on the ligand, and can be an antibody or an antigen-binding fragment thereof.
  • the antibody can be selected from chimeric antibodies, human EL antibody, fully human antibody or murine antibody; the antibody can be a monoclonal antibody.
  • the antibody can be an antibody targeting the following targets: HER2, HER3, B7H, TROP2, Claudin 18.2, CD30, CD33, CD70, EGFR, 5T4, AGS-16, ANGPTL4, ApoE, CD19, CTGF, CXCR5, FGF2,MCPT8,MF12,MS4A7,NCA,Sema5b,SLITRK6,STC2,TGF,0772P,ST4,ACTA2,ADGRE1,AG-7,AIF1,AKRIC1,AKR1C2,ASLG659,Axl,B7H3,BAFF-R,BCMA,BMPRIB, BNIP3,C1QA,C1QB,CA6,CADM1,CCD79b,CCL5,CCR5,CCR7,CD11c,CD123,CD138,CD142,CD147,CD166,CD19,CD22,CD21,
  • Enzyme-labile linkers such as peptide linkers, enable better control of drug release.
  • Peptide linkers can be effectively cleaved by intralysosomal proteases, such as cathepsin (CathepsinB) or plasmin (the content of these enzymes is increased in some tumor tissues). This peptide linkage is believed to be very stable in the plasma circulation, where proteases are normally inactive due to inappropriate extracellular pH and serum protease inhibitors.
  • Enzyme-labile linkers are widely used as cleavable linkers for antibody-drug conjugates due to their high plasma stability and good intracellular cleavage selectivity and effectiveness.
  • Typical enzyme-labile linkers include Val-Cit (VC), Phe-Lys, etc.
  • Self-releasing linkers are generally embedded between the cleavable linker and the active drug, or themselves are part of the cleavable linker.
  • the mechanism of action of the self-releasing linker is: when the cleavable linker is broken under appropriate conditions, the self-releasing linker can spontaneously undergo structural rearrangement, thereby releasing the active drug connected to it.
  • Common suicide linkers include para-aminobenzyl alcohols (PAB) and ⁇ -glucuronides ( ⁇ -Glucuronide).
  • the disulfide bonds between the antibody chains are reduced to generate a total of 8 sulfhydryl groups, which replace the maleamide linker drug conjugate and cross-link with the reduced antibody sulfhydryl groups to generate the corresponding antibody drug conjugate.
  • maleimide compounds can be added directly without purification (preliminarily 10 mg/mL dissolved in dimethyl sulfoxide (DMSO), dimethylformamide (DMF) or two Ethyl acetamide (DMA)), and ensure that the volume proportion of organic solvent in the reaction solution does not exceed 15%.
  • DMSO dimethyl sulfoxide
  • DMF dimethylformamide
  • DMA Ethyl acetamide
  • the coupling reaction was stirred at 10-25°C for 2 hours. If DTT is used for reduction, it is necessary to remove excess DTT through a desalting column or ultrafiltration after the reduction reaction is completed, and then add substituted maleamide compounds for coupling.
  • the drug-to-antibody conjugation ratio (DAR8) of the obtained antibody-drug conjugate is relatively uniform.
  • the drug linker compound described in this patent can also be used to produce antibody-drug conjugates with certain differences in product homogeneity. If it is necessary to obtain more homogeneous Good samples can be further separated and purified by but not limited to the following methods: hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), and ion exchange chromatography (IEC).
  • HIC hydrophobic interaction chromatography
  • SEC size exclusion chromatography
  • IEC ion exchange chromatography
  • compositions and methods of administration are provided.
  • the antibody-drug conjugate provided by the present invention can target a special cell population and bind to a specific protein (antigen) on the cell surface, the drug can be released into the cell in an active form through endocytosis of the conjugate or drug penetration. Therefore, the antibody-drug conjugate of the present invention can be used to treat target diseases, and the above-mentioned antibody-drug conjugate can be administered to a subject (such as a human) through a suitable route in a therapeutically effective amount.
  • a subject in need of treatment may be a patient who is at risk for, or suspected of having, a condition related to the activity or expression of a particular antigen. Such patients can be identified through routine physical examination.
  • compositions may be administered by other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally or by implantation.
  • parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. Additionally, it may be administered to the subject via an injectable depot route of administration, for example using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
  • Injectable compositions may contain various carriers such as vegetable oil, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerin, propylene glycol, liquid polyethylene glycol, etc.).
  • the water-soluble antibody can be administered by drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is administered by infusion.
  • Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients.
  • Intramuscular preparations e.g., sterile preparations of the antibody in the form of a suitable soluble salt
  • delivery can be performed by conventional methods in the art. For example, it can be introduced into cells through the use of liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres.
  • the nucleic acid or vector may be delivered locally by direct injection or by use of an infusion pump.
  • Other approaches include the use of various delivery and carrier systems through the use of conjugates and biodegradable polymers.
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of a solution, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other auxiliaries.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the active ingredients are administered in amounts that are therapeutically effective.
  • the effective amount of the antibody-drug conjugate of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: pharmacokinetic parameters of the bifunctional antibody conjugate such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, and the patient's immunity. conditions, route of administration, etc. Generally, when the antibody-drug conjugate of the present invention is administered at a dose of about 0.0001 mg-50 mg/kg animal body weight (preferably 0.001 mg-10 mg/kg animal body weight) every day, satisfactory effects can be obtained. For example, several divided doses may be administered daily, or the dosage may be proportionally reduced as dictated by the exigencies of the treatment situation.
  • Dosage forms for topical administration of the compounds of this invention include ointments, powders, patches, sprays and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.
  • the compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.
  • a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, and the dosage when administered is a pharmaceutically effective dosage.
  • a mammal such as a human
  • the daily dose is usually 1-2000 mg, preferably 5-500 mg.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.
  • A1-7 (1.6g, 2.2mmol), N,N-dimethylformamide (11mL), and DBU (0.3g, 2mmol) were added, and the reaction was stirred at room temperature under nitrogen replacement protection for 1.5h.
  • the sampled raw materials reacted completely.
  • 10% Pd/C (0.2g), replace with hydrogen at normal pressure, stir and react at room temperature for 2 hours, filter through diatomaceous earth, add 20mL of process purified water, add 10mL of methylene chloride, stir, separate layers, and decompress the resulting water phase Concentrate to dryness to obtain target compound A1-8 (0.9 g, yield: 98.0%).
  • A1-9X-1X (642g, 4200mmol), triphenylphosphine (1155g, 4410mmol), and acetonitrile (6L), and heat to reflux and insulate for 24h. Cool to room temperature, concentrate the reaction solution to dryness under reduced pressure, add 6L of methyl tert-butyl ether, filter, soak the filter cake in methyl tert-butyl ether, and dry the filter cake under reduced pressure to obtain A1-9X-2X (1500g, yield: 86.0%).
  • A1-9X-2 (594.0, 3000mmol) and ethyl acetate (2500mL) to dissolve, then add 10% Pd/C (50% water, 75g), then replace with nitrogen and hydrogen respectively, and hydrogenate at normal pressure at 25-35°C. React for 16 hours, filter through diatomaceous earth, and concentrate the filtrate to dryness under reduced pressure to obtain A1-9X-3 (600.0 g, yield 100%).
  • A1-9X-4 (125g, 686.8mmol) to concentrated sulfuric acid (1200g) while controlling the temperature below 5°C. Then add sodium nitrate (70g, 823.5mmol) in batches and complete the reaction at room temperature for 2-3 hours.
  • the reaction solution was slowly poured into 6.5kg of ice water, and extracted with a mixed solution of 1.5L methyl tert-butyl ether and 150mL ethyl acetate.
  • the organic layers were combined, washed with saturated sodium bicarbonate and brine in sequence, and the organic phase was dried and concentrated under reduced pressure to obtain a crude product. After column purification, A1-9X-5 (71.0 g, yield: 45.5%) was obtained.
  • A1-9X-7 (9.0g, 37.6mmol) and dimethyl sulfoxide (150mL), slowly add 25% ammonia water (250g) while stirring, transfer the reaction solution to an autoclave, and raise the temperature to 65-75°C for 16 hours. . Cool down, add an appropriate amount of water, extract with ethyl acetate several times, combine the organic layers, wash with water and saturated brine in sequence, dry the organic layer, and concentrate to dryness under reduced pressure. The crude product was purified by column to obtain A1-9X-8 (4.5g, yield: 50.6%).
  • A1-10X-4 80g, 320mmol
  • concentrated hydrochloric acid 500mL
  • A1-10X-5 (66g, 320mmol) and dichloromethane (1.5L), stir to dissolve, add pyridine (52mL, 650mmol), cool to 0°C, and add trifluoroacetic anhydride (94mL, 676mmol) dropwise within 40min. , continue the reaction for 20 minutes after dripping, concentrate the reaction solution to dryness under reduced pressure, then add 1L dichloromethane to dissolve, wash with 1N hydrochloric acid until there is no pyridine, then wash with saturated sodium bicarbonate solution until it is alkaline, and wash with saturated brine. The organic phase was dried and concentrated under reduced pressure to obtain A1-10X-6 (62g, yield: 64.1%).
  • A1-10X-6 (54g, 180mmol) in dichloromethane (100mL) and add it to the system. Cool to 0°C and add in batches within 50 minutes. Zinc powder (150g, 2290mmol), continue the reaction for 1.5h. Filter with suction, separate the filtrate, wash the organic phase with saturated sodium carbonate solution and saturated brine, dry the organic phase and concentrate under reduced pressure to obtain A1-10X-7 (42g, yield: 85.7%).
  • A1-10X-9 (6g, 27.5mmol), A1-10X-8B (8g, 30.3mmol), PPTS (6.9g, 27.5mmol), toluene (1L), under nitrogen protection, raise the temperature to 120°C and keep the reaction for 4 hours. . Lower to 0-5°C, suction filter, dissolve the filter cake with dichloromethane and methanol, and concentrate to dryness under reduced pressure. The concentrate was slurried with 50 mL of acetone at 0-5°C, and filtered with suction to obtain A1-10X-10 (12.2 g, yield: 100%).
  • A1-10X-10 (12.2g, 27.5mmol), concentrated hydrochloric acid (80mL), and water (80mL), raise the temperature to 80°C, and keep the reaction for 2.5h. Cool to room temperature, add a small amount of methanol, and concentrate the reaction solution to dryness under reduced pressure. The concentrate was slurried with 30 mL acetone at room temperature, filtered with suction, and the filter cake was dried under reduced pressure to obtain A1-10X (11.1 g, yield: 100%).
  • A1-8A (2.5g, 11.8mmol), N,N-dimethylformamide (12.5mL), N,N-diisopropylethylamine (2.2g, 17.0mmol), and cool to -5 ⁇ 0°C, add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (5.2g, 13.7mmol), keep warm and react 0.5 ⁇ 1h. Add the entire batch of A10-9 reaction solution prepared in step nine. Return to room temperature and stir the reaction until the raw materials react completely.
  • A15-3X (10g, 41.3mmol), acetonitrile (100mL), water (100mL), triethylamine (41.8g, 41.3mmol), A14-3 (17.4g, 41.3mmol), nitrogen replacement protection, and stir at room temperature for 2 hours. .
  • 6-aminocaproic acid is replaced by 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propionic acid:
  • A20-1 (10.0g, 67.5mmol) and dichloromethane (500mL), cool to 0 ⁇ 5°C, add Boc2O (7.4g, 33.9mmol) dropwise, keep stirring for 5h, raise the temperature to room temperature and stir the reaction overnight.
  • Boc2O 7.4g, 33.9mmol
  • A20-2 (3.0g, 12.1mmol) and saturated aqueous sodium bicarbonate solution (90mL), and stir at room temperature for 15min. Filter, cool the filtrate to 0 ⁇ 5°C, add A20-4 (2.0g, 11.8mmol), and stir at room temperature for 16h. The aqueous solution was concentrated to dryness under reduced pressure, and water was added to toluene. To the concentrate, sodium acetate (10g, 121.9mmol) and ethanol were added. Acid anhydride (20 mL, 220.0 mmol), heated to 120°C and kept for 0.5 h. Cool the temperature, concentrate the reaction solution to dryness under reduced pressure, add 50 mL of water, and extract with 50 mL of dichloromethane*2. The organic phase is dried and concentrated under reduced pressure to obtain A20-5 (5.7 g, yield: 100%).
  • A20-5 (5.7g, 17.4mmol), dichloromethane (50mL), and trifluoroacetic acid (10g, 87.7mmol), stir at room temperature for 2 to 3 hours, solid precipitates, filter, and the filter cake is dried under reduced pressure to obtain A20-6 ( 5.3g, yield: 89.1%).
  • A19-1X (10.0g, 166.4mmol) and dichloromethane (200mL), cool to 0 ⁇ 5°C, add Boc2O (18.1g, 83.2mmol) dropwise, keep stirring for 3 hours, raise the temperature to room temperature and stir until the raw material Boc2O reacts After completion, 100 mL of water was added to wash the organic phase, and the aqueous phase was extracted with 50 mL of DCM. The combined organic phases were dried, filtered, and concentrated to dryness under reduced pressure to obtain A19-2X (12.1 g, yield: 90.8%).
  • A20-1 was replaced with amino-monoethylene glycol-carboxylic acid.
  • A20-1 was replaced with 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propionic acid.
  • A1-10X-5 (66g, 320mmol) and dichloromethane (1.5L), stir to dissolve, add pyridine (52mL, 650mmol), cool to 0°C, add trifluoroacetic anhydride (94mL, 676mmol) dropwise within 40min. After the dripping, the reaction was continued for 20 minutes. The reaction solution was concentrated to dryness under reduced pressure, then 1 L of dichloromethane was added to dissolve, washed with 1N hydrochloric acid until there was no pyridine, washed with saturated sodium bicarbonate solution until alkaline, and washed with saturated brine. The organic phase was dried and concentrated under reduced pressure to obtain A1-10X-6 (62g, yield: 64.1%).
  • A1-10X-6 54g, 180mmol dichloromethane (100mL) into the system, cool to 0°C, and add zinc powder in batches within 50 minutes (150g, 2290mmol), continue the reaction for 1.5h. Filter with suction, separate the filtrate, wash the organic phase with saturated sodium carbonate solution and saturated brine, dry the organic phase and concentrate under reduced pressure to obtain A1-10X-7 (42g, yield: 85.7%).
  • A1-9X-2 (594.0, 3000mmol) and ethyl acetate (2500mL) to dissolve, then add 10% Pd/C (50% water, 75g), then replace with nitrogen and hydrogen respectively, and hydrogenate under normal pressure at 25-35°C. React for 16 hours, filter through diatomaceous earth, and concentrate the filtrate to dryness under reduced pressure to obtain A1-9X-3 (600.0 g, yield 100%).
  • A1-9X-3 600.0g, 3000mmol
  • concentrated sulfuric acid 3000g
  • the temperature is controlled below 35°C
  • the reaction is completed at room temperature for 2-3 hours.
  • Slowly pour the reaction solution into 6.5kg of ice water add 3.5L of methyl tert-butyl ether and 0.5L of ethyl acetate for extraction and separation, combine the organic layers with saturated sodium bicarbonate and wash with brine, dry the organic phase and concentrate to dryness under reduced pressure.
  • the crude product was purified by column to obtain A1-9X-4 (230.0 g, yield: 42.1%).
  • A1-9X-4 (125g, 686.8mmol) to concentrated sulfuric acid (1200g) while controlling the temperature below 5°C. Then add sodium nitrate (70g, 823.5mmol) in batches and complete the reaction at room temperature for 2-3 hours.
  • the reaction solution was slowly poured into 6.5kg of ice water, extracted with a mixed solution of 1.5L methyl tert-butyl ether and 150mL ethyl acetate, the organic layers were combined, washed with saturated sodium bicarbonate and brine, and the organic phase was dried under reduced pressure and concentrated to obtain a crude product that was passed through a column. After purification, A1-9X-5 (71.0 g, yield: 45.5%) was obtained.
  • A1-9X-7 (9.0g, 37.6mmol) and dimethyl sulfoxide (150mL), slowly add 25% ammonia water (250g) while stirring, transfer the reaction solution to an autoclave, and raise the temperature to 65-75°C for 16 hours. . Cool down, add an appropriate amount of water, extract with ethyl acetate several times, combine the organic layers, wash with water and saturated brine in sequence, dry the organic layer, and concentrate to dryness under reduced pressure. The crude product was purified by column to obtain A1-9X-8 (4.5g, yield: 50.6%).
  • the DAR values were measured using HIC-HPLC, RP-HPLC or LCMS, and the conjugates were detected by SEC-HPLC.
  • the polymer ratio of the conjugate is within the normal range, indicating that the antibody-drug conjugate of the present invention has good solubility and drug-forming properties, and no precipitation occurs during the conjugation process.
  • the stably transfected SK-BR-3 and BT-474 human breast cancer cells with high expression of Her2, NCI-N87 human gastric cancer cells and human non-small cell lung cancer cell A549 were selected as the cell lines used for in vitro activity detection in this experiment. Observation The dose and effect of different antibody conjugated drugs on cell killing. Preliminarily select the density of the seed plate for each cell: 2 ⁇ 10 3 cells/well, and measure the cytotoxic activity after 16-24 hours; secondly, test the antibody-conjugated drug prepared in Example 2 and add the final concentration to 5000nM.
  • the linkers in the present invention are all cleavable linkers, so the released camptothecin analogs are first tested for in vitro cytotoxic activity, and the results are as shown in the table below. The test results show that the cell killing activity of most compounds is better than that of the control compound Dxd.
  • the conjugated ADC drugs were further tested for in vitro cytotoxic activity. Judging from the activity test results, the ADCs prepared from the compounds of the present application all showed certain anti-tumor activity, with IC 50 reaching 10 -6 ⁇ 10 - 10 M, showing significantly stronger anti-tumor activity compared with the control sample.
  • the efficacy of the combinations of the invention is measured in vivo by implanting allografts or xenografts of cancer cells in rodents and treating tumors with the combination.
  • Test mice are treated with drug or control and monitored for weeks or longer to measure time to tumor doubling, log cell killing, and tumor inhibition.
  • mice BALB/cA-nude nude mice, 6-7 weeks old, female mice, were purchased from Shanghai Lingchang Biotechnology Co., Ltd.
  • T/C (%) (TT 0 )/(CC 0 ) ⁇ 100%, where T and C are the tumor volumes at the end of the experiment in the test group and the control group respectively; T 0 and C 0 are respectively the test group and the control group. Tumor volume at the beginning of the experiment.
  • the ADCs prepared from the compounds of the present application all show certain anti-tumor activity in vivo, and can show significantly stronger anti-tumor activity compared with the control sample.
  • the tumor-bearing mice tolerated the above drugs well, and no symptoms such as weight loss occurred.
  • the inhibitor of the present invention is Compound A or its tautomer, meso, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof , the antibody drug conjugate can be obtained through further preparation; the antibody drug conjugate of the present invention has good solubility and druggability, and no precipitation occurs during the coupling process; the ADC exhibits obvious anti-tumor activity in vivo, and can be displayed compared with the control sample showed significantly stronger anti-tumor activity.

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Abstract

一种特异性拓扑异构酶抑制剂和可用于抗体药物偶联物及其制备方法，属于药物化学技术领域。所述抑制剂为化合物A或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐，化合物A结构为该化合物还可进一步制备得到抗体药物偶联物；该抗体药物偶联物具有很好溶解性和成药性，偶联过程不发生沉淀；ADC表现出明显体内抗肿瘤活性，与对照样品相比可以显示出显著更强抗肿瘤活性。

Description

特异性拓扑异构酶抑制剂和可用于抗体药物偶联物及其制备方法 技术领域
本发明涉及包含特异性拓扑异构酶抑制剂和可用于抗体药物偶联物的药物连接子缀合物，以及所述化合物及相关的抗体偶联药物的制备方法和在制备治疗癌症的药物中的用途，属于药物化学技术领域。
背景技术
抗体偶联药物的基本模块包含抗体、连接子、毒素分子，利用抗体将毒素分子传输到肿瘤部分富集，从而杀死肿瘤细胞。传统的毒素分子大都是高活性的微管蛋白抑制剂或直接靶向DNA细胞毒药物，通常有较大毒副作用，限制了ADC应用。
近来Immunomedics公司开发了以喜树碱化合物作为弹头分子新型ADC药物IMMU-132，表现出了较好抗肿瘤效果，第一三共开发了另一种喜树碱化合物作为弹头分子ADC药物DS-8201a，同样表现出来良好抗肿瘤效果。
现有ADC技术中，主要是通过改造现有连接子技术将喜树碱化合物与抗体的进行连接，一般来讲，ADC中的理想连接子需满足以下要求：首先，血浆中保证小分子药物不与抗体脱离，进入细胞后，连接子在适当条件下断裂，迅速释放出活性小分子药物；其次，连接子还要具有较好的理化性质，以便能与抗体连接形成偶联物；再次，连接子要易于制备从而为ADC规模化生产奠定基础。IMMU-132采用pH敏感型连接子，稳定性较差，DS-8201a使用一种含有甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(GGFG)四肽结构，具有较好稳定性。上述ADC药物所释放毒素为SN38和Dxd，其均为Pgp底物，仍然存在有肿瘤多药耐药等问题。
因此，仍需进一步开发疗效和/或安全性更好的喜树碱衍生物以及ADC药物。
发明内容
为了克服上述技术缺陷，本发明提供了一种抑制剂化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，所述化合物包含式(A)所示结构：
其中：X为氢、氟；Q为可与抗体偶联的接头或氢，L1是接头或氢与药物氨基连接基团。
进一步地，在上述技术方案中，所述Q为可与抗体上巯基偶联的基团，选自马来酰亚胺。
进一步地，在上述技术方案中，所述L1为接头与药物氨基连接基团，其选自L2为任选取代的C3-C7亚烷基、C3-C8环烷基、任选取代的二甘醇至八甘醇酰基，AA为2至4个氨基酸组成的肽段，M为亚甲基、C1-C6烷基或环烷基取代亚甲基、三氟甲基取代亚甲基、C3-C6环烷基。
进一步地，在上述技术方案中，所述L1为接头与药物氨基连接基团，其选自AA多肽残基选自：NH-Phe-Lys-C＝O、NH-Val-Cit-C＝O、NH-Val-Ala-C＝O、NH-Phe-Cit-C＝O、NH-Gly-Val-C＝O、NH-Ala-Lys-C＝O、NH-Ala-Ala-Ala-C＝O、NH-Glu-Val-Ala-C＝O、NH-Glu-Val-Cit-C＝O、NH-Gly-Gly-Phe-Gly-C＝O。
进一步地，在上述技术方案中，所述L1为接头与药物氨基连接基团，选自L2为任选取代的C3-C7亚烷基、C3-C8环烷基、任选取代的二甘醇至八甘醇酰基。
进一步地，在上述技术方案中，所述式A具体分子结构如下：



本发明还提供了一种抗体药物偶联物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，所述化合物包含式(B)所示结构：
其中：X为氢、氟；Q为可与巯基偶联的接头，L1是接头与药物氨基连接基团，Ab为配体，n＝1-8。
进一步地，在上述技术方案中，所述Q包含与巯基偶联后的连接体，选自
进一步地，在上述技术方案中，所述L1为接头与药物氨基连接基团，其选自L2为任选取代的C3-C7亚烷基、C3-C8环烷基、任选取代的二甘醇至八甘醇酰基，AA为2至4个氨基酸组成的肽段，M为亚甲基、C1-C6烷基或环烷基取代亚甲基、三氟甲基取代亚甲基、C3-C6环烷基。
进一步地，在上述技术方案中，所述L1为接头与药物氨基连接基团，选自AA多肽残基选自：NH-Phe-Lys-C＝O、NH-Val-Cit-C＝O、NH-Val-Ala-C＝O、NH-Phe-Cit-C＝O、NH-Gly-Val-C＝O、NH-Ala-Lys-C＝O、NH-Ala-Ala-Ala-C＝O、NH-Glu-Val-Ala-C＝O、NH-Glu-Val-Cit-C＝O、NH-Gly-Gly-Phe-Gly-C＝O。
进一步地，在上述技术方案中，所述L1为接头与药物氨基连接基团，选自L2为任选取代的C3-C7亚烷基、C3-C8环烷基、任选取代的二甘醇至八甘醇酰基。
进一步地，在上述技术方案中，所述式B结构具体包括如下：




其中：Ab为配体，n＝1-8。
进一步地，在上述技术方案中，所述所述Ab选自鼠源抗体、嵌合抗体、人源化抗体或全人源抗体。
进一步地，在上述技术方案中，所述所述抗体包含单克隆抗体。
进一步地，在上述技术方案中，所述所述抗体包含双特异性抗体。
进一步地，在上述技术方案中，所述所述抗体能够与HER2、HER3、CD19、CD20、CD22、CD30、CD33、CD37、CD45、CD56、CD66e、CD70、CD74、CD79b、CD138、CD147、CD223、EpCAM、Mucin 1、STEAP1、GPNMB、FGF2、FOLR1、EGFR、EGFRvIII、Tissue factor、c-MET、FGFR、Nectin 4、AGS-16、Guanylyl cyclase C、Mesothelin、SLC44A4、PSMA、EphA2、AGS-5、GPC-3、c-KIT、ROR1、PD-L1、CD27L、5T4、Mucin 16、NaPi2b、STEAP、SLITRK6、ETBR、BCMA、Trop-2、CEACAM5、SC-16、SLC39A6、Delta-like protein3或Claudin 18.2肿瘤相关抗原结合的抗体。
本发明还提供了一种药物组合物，包括：(a)上述抗体药物偶联物；和(b)药学上可接受的稀释剂，载剂或赋形剂。
本发明还提供了上述抗体药物偶联物在制备治疗肿瘤药物中的用途。
本发明还提供了所述抗体药物偶联物的制备方法，包括如下步骤：
a、采用抗体与还原试剂在缓冲液中反应，得到经还原后的抗体；
b、采用连接子-药物缀合物(A)与步骤a得到经还原后的抗体在缓冲液与有机溶剂混合液中进行交联，得到抗体药物偶联物。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
本发明中，术语“药学上可接受的”成分是指适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)，即有合理的效益/风险比物质。
本发明中，术语“有效量”指治疗剂治疗、缓解或预防目标疾病或状况的量，或是表现出可检测治疗或预防效果量。对于某一对象精确有效量取决于该对象的体型和健康状况、病症性质和程度、以及选择给予的治疗剂和/或治疗剂组合。因此，预先指定准确有效量是没用的。然而，对于某给定状况而言，可以用常规实验来确定该有效量，临床医师是能够判断出来的。
除非特别说明，本发明中，所有出现的化合物均意在包括所有可能的光学异构体，如单一手性的化合物，或各种不同手性化合物的混合物(即外消旋体)。本发明的所有化合物之中，各手性碳原子可以任选地为R构型或S构型，或R构型和S构型的混合物。
如本文所用，术语“本发明化合物”指式I所示的化合物。该术语还包括及式I化合物的各种晶型形式、药学上可接受的盐、水合物或溶剂合物。
如本文所用，术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。
除非特别说明，本文中所使用的“氨基酸”意在包括任何常规氨基酸，如天冬氨酸、谷氨酸、半胱氨酸、天冬酰胺、苯丙氨酸、谷氨酰胺、酪氨酸、丝氨酸、甲硫氨酸(蛋氨酸)、色氨酸、甘氨酸、缬氨酸、亮氨酸、丙氨酸、异亮氨酸、脯氨酸、苏氨酸、组氨酸、赖氨酸、精氨酸。
当本文中使用商品名时，该商品名意在包括商品名产品制剂、其相应的仿制药，以及商品名产品的活性药物组分。
本文的术语“抗体”以其最广泛的含义使用并且特别覆盖单克隆抗体、多克 隆抗体、二聚体、多聚体、多特异性抗体(例如双特异性抗体)和抗体片段，只要它们表现出所需的生物活性(Miller等(2003)Journal of Immunology 170:4854-4861)。抗体可以为鼠、人、人源化、嵌合的抗体或来源于其它物种。抗体为由能够识别和结合特异性抗原的免疫系统产生的蛋白质(Janeway,C.,Travers,P.,Walport,M.,Shlomchik(2001)ImmunoBiology,5thEd.,Garland Publishing,NewYork)。靶抗原一般具有由多种抗体的CDRs识别的大量结合位点，也称作表位。特异性结合不同表位的各抗体具有不同的结构。因此，一种抗原可以具有一种以上相应的抗体。抗体包括全-长免疫球蛋白分子或全-长免疫球蛋白分子的免疫活性部分，即含有特异性结合所关注靶标的抗原或其部分的分子，这类靶标包括，但不限于癌细胞或产生与自身免疫性疾病相关的自身免疫抗体的细胞。本文披露的免疫球蛋白可以具有免疫球蛋白分子的任意类型(例如IgG、IgE、IgM、IgD和IgA)、类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类。免疫球蛋白可以来源于任意的物种。然而，在一个方面中，免疫球蛋白来源于人、鼠或兔。
“抗体片段”包含全长抗体的一部分，一般为其抗原结合区或可变区。抗体片段的实例包括：Fab、Fab’、F(ab’)2和Fv片段；双抗体；线性抗体；微抗体(minibody)(Olafsen等(2004)Protein Eng.Design&Sel.17(4):315-323)；Fab表达文库制备的片段；抗-独特型(抗-Id)抗体；CDR(互补决定区)；和以免疫特异性方式结合癌细胞抗原、病毒抗原或微生物抗原的上述任意的表位-结合片段；单-链抗体分子；和由抗体片段形成的多特异性抗体。
本发明中组成抗体药物偶联物的抗体最好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，最好专一性地，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(如已知抗体结合的抗原可以是上述分类中一个或一个子集，而其它的子集则包含其它的具有特殊性质的分子/抗原(与目标抗原相比)。
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地表达在一种或多种癌症细胞表面，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。
在本申请中，术语“配体”通常指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。配体的作用可以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、受体和/或抗原结合的分子。在本申请中，配体可以表示为Ab，配体抗原通过配体上的杂原子与连接单元形成连接键，可以为抗体或其抗原结合片段，所述抗体可以选自嵌合抗体、人源化抗体、全人抗体或鼠源抗体；所述抗体可以是单克隆抗体。例如所述抗体可以是，靶向为如下靶点抗体：HER2,HER3,B7H,TROP2,Claudin 18.2,CD30,CD33,CD70,EGFR,5T4,AGS-16,ANGPTL4,ApoE,CD19,CTGF,CXCR5,FGF2,MCPT8,MF12,MS4A7,NCA,Sema5b,SLITRK6,STC2,TGF,0772P,ST4,ACTA2,ADGRE1,AG-7,AIF1,AKRIC1,AKR1C2,ASLG659,Axl,B7H3,BAFF-R,BCMA,BMPRIB,BNIP3,C1QA,C1QB,CA6,CADM1,CCD79b,CCL5,CCR5,CCR7,CD11c,CD123,CD138,CD142,CD147,CD166,CD19,CD22,CD21,CD20,CD205,CD22,CD223,CD228,CD25,CD30,CD33,CD37,CD38,CD40,CD45,CD45(PTPRC),CD46,CD47,CD49D(ITGA4),CD56,CD66e,CD70,CD71,CD72,CD74,CD79a,CD79b,CDS0,CDCP1,CDH11,CD11b,CEA,CEACAMS,c-Met,COL6A3,COL7A1,CRIPTO,CSF1R,CTSD,CTSS,CXCL11,CXCL10,DDIT4,DLL3,DLL4,DR5,E16,EFNA4,EGFR,EGFRVIII,EGLN,EGLN3,EMR2,ENPP3,EpCAM,EphA2,EphB2R,ETBR,FcRH2,FcRHI,FGFR2,FGFR3,FLT3,FOLR-ALPHA,GD2,GEDA,GPC-1,GPNMB,GPR20,GZMB,HER2,HER3,HLA-DOB,HMOX1,IFI6,IFNG,IGF-1R,IGFBP3,IL-13R,IL-2,IL20Ra,IL-3,IL-4,IL-6,IRTA2,KISS1R,KRT33A,LIV-1,LOX,LRP-1,LRRC15,LUM,LY64,LY6E,Ly86,LYPD3,MDP,MMP10,MMP14,MMP16,MPF,MSG783,MSLN,MUC-1,NaPi2b,Napi3b,Nectin-4,NOG,P2X5,pAD,P-Cadherin,PDGFRA,PDK1,PD-LI,PFKFB3,PGF,PGK1,PIK3AP1,PIK3CD,PLOD2,PSCA,PSCAh1g,PSMA,PTK7,P-钙黏着蛋白,RNF43,NaPi2b,ROR1,ROR2,SERPINE1,SLC39A6,SLTRK6,STAT1,STEAP1,STEAP2,TCF4, TENB2,TGFB1,TGFB2,TGFBR1,TNFRSF21,TNFSF9,Trop-2,TrpM4,Tyro7,UPK1B,VEGFA,WNTSA,表皮生长因子，短蛋白柔糖，间皮素，磷酸钠协同转运蛋白2B，密封蛋白18.2，内皮肤受体，黏蛋白(如黏蛋白1和黏蛋白16)，鸟苷酸环化酶C，整合素a4p7，整合素a5p6，滋养层细胞糖蛋白或组织因子。
酶不稳定连接子，如肽连接子，能够更好地控制药物释放。肽连接子能够被溶酶体内蛋白酶，如组织蛋白酶(CathepsinB)或纤溶酶(在一些肿瘤组织中此类酶含量增加)，有效地切断。这种肽连接被认为在血浆循环中非常稳定，这是因为细胞外不合宜的pH值及血清蛋白酶抑制剂导致蛋白酶通常在不具备活性。鉴于较高的血浆稳定性和良好的细胞内断裂选择性和有效性，酶不稳定连接子被广泛用做抗体药物偶联物的可断裂连接子。典型的酶不稳定连接子包括Val-Cit(VC),Phe-Lys等。
自释放连接子一般嵌合在可断裂连接子与活性药物之间，或者本身就是可断裂连接子的一部分。自释放连接子的作用机制是：当可断裂连接子在合宜的条件下断裂后，自释放连接子能够自发地进行结构重排，进而释放与之连接的活性药物。常见的自杀式连接子包括对氨基苄醇类(PAB)和β-葡萄糖醛酸苷类(β-Glucuronide)等。
抗体-药物偶联物的制备方法
抗体链间二硫键被还原产生共8个巯基基团骤，取代马来酰胺类连接子药物缀合物与还原后的抗体巯基交联，生成相应的抗体药物偶联物。
将抗体原液用反应缓冲液稀释至2-10mg/mL，加入6.0-20倍过量摩尔比三(2-羧乙基)膦盐酸盐(TCEP)，或加入140-200倍过量摩尔比二硫苏糖醇(DTT)，反应液于25℃搅动2-4小时；在此所述反应缓冲液为50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH＝6-9；50mM磷酸氢二钠-柠檬酸/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH＝6-9；50mM硼酸-硼砂/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH＝6-9；50mM组氨酸-氢氧化钠/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9和PBS//1mM二乙基三胺五乙酸(DTPA)，pH＝6-9。
将上述反应液冷至0-10℃，若采用TCEP还原可不经纯化直接加入马来酰亚胺类化合物(预先10mg/mL溶在二甲亚砜(DMSO)，二甲基甲酰胺(DMF)或二 乙基乙酰胺(DMA)中)，并保证反应液中有机溶剂的体积占比不超过15％。偶联反应于10-25℃搅动2小时。若采用DTT还原，需在还原反应完成后过脱盐柱或超滤除去过量DTT，再加入取代马来酰胺类化合物进行偶联。
采用脱盐柱将偶联反应混合物用琥珀酸钠/150mM NaCl缓冲液或组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。或超滤数遍。然后经由0.22微米孔径的过滤装置除菌，-80℃保存。
所得抗体药物偶联物的药物抗体偶联比(DAR8)较为均一，采用本专利中所述的药物连接子化合物也将产物均一性有一定差别的抗体药物偶联物，如需要获得均一性更好的样品，可进一步利用但不限于以下方法进行分离纯化：疏水作用层析方法(HIC)、分子排阻色谱法(SEC)、离子交换层析(IEC)。
药物组合物和施用方法
由于本发明提供的抗体-药物偶联物，可以靶向瞄准特殊的细胞群体，与细胞表面特异蛋白(抗原)结合，从而通过结合物内吞或药物渗入使得药物以活性形式释放到细胞内，因此，本发明的抗体-药物偶联物可以用于治疗目标疾病，上面提到的抗体-药物偶联物可以以治疗有效量，通过合适途径给予受试者(例如人)。需要治疗受试者可以是有风险，或怀疑患有与特定抗原的活性或表达量有关病症的患者。这样的患者可以通过常规体检来鉴定。
常规方法，已知的医学领域的普通技术人员，可以用于施用药物组合物给受试者，这取决于疾病的要治疗的类型或疾病的部位。此组合物还可以通过其它常规途径，例如，口服，肠胃外给药，通过吸入喷雾，局部，直肠，经鼻，口腔，阴道或通过植入进行给药。本文所用的术语“肠胃外”包括皮下，皮内，静脉内，肌内，关节内，动脉内，滑膜内，胸骨内，鞘内，病灶内和颅内注射或输注技术。此外，它可以施用到通过施用可注射的贮库途径，例如使用1-，3-，或6个月的贮库可注射或可生物降解的材料和方法的主题。
注射组合物可以含有各种载体如植物油，二甲基乙酰胺(dimethylactamide)，二甲基甲酰胺，乳酸乙酯，碳酸乙酯，肉豆蔻酸异丙酯，乙醇，多元醇(甘油，丙二醇，液体聚乙二醇，等等)。对于静脉内注射，水溶性抗体可以通过点滴方法，由此含有抗体和生理上可接受的赋形剂的药物制剂输注给药。生理上可接受的赋形剂可以包括，例如，5％葡萄糖，0.9％盐水，林格溶液或其它合适的赋形剂。肌内制剂，例如，抗体的一个合适的可溶盐形式的无菌制剂，可以溶解 和施用的药用赋形剂诸如水换注射液，0.9％盐水，或5％葡萄糖溶液。
当用本发明抗体-药物偶联物治疗时，可以通过本领域常规方法进行递送。例如，它可以通过使用脂质体，水凝胶，环糊精，生物可降解的纳米胶囊，或生物粘附性微球被引入到细胞中。或者，所述核酸或载体可在本地通过直接注射或通过使用输注泵递送。其它方法包括通过使用缀合物和生物可降解的聚合物的使用各种运输和载体系统。
本发明药物组合物含有安全有效量的本发明抗体-药物偶联物以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物可以被制成溶液剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。
本发明所述抗体-药物偶联物的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的双功能抗体偶联物的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的抗体-药物偶联物每天以约0.0001mg-50mg/kg动物体重(较佳的0.001mg-10mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的治疗剂联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1-2000mg，优选5-500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅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实验方 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，否则百分比和份数按重量计算。
实施例1、化合物的合成与制备
1、化合物A1的合成
第一步
加入双甘肽A1-1(2.0g，15.2mmol)、芴甲氧羰酰氯(4.7g，18.2mmol)、二氧六环(20mL)，降温到0～5℃，滴加1N碳酸钠水溶液(18mL)。滴完恢复到室温搅拌反应约2h。降温到0～5℃，以1M HCl约30mL调pH＝2，水相以乙酸乙酯100mL*3提取，合并有机相，减压浓缩干，得类白色固体6.0g。加入甲基叔丁基醚19mL，室温打浆20min，过滤，滤饼减压浓缩干，得A1-2(4.3g，收率：81.3％)。MS(ESI)(m/z)：355([M+H]+)。
第二步
加入四氢呋喃(450mL)、A1-2(29.0g，81.9mmol)、乙酸(90mL)、氮气置换保护，升温到40℃，加入四乙酸铅(60.0g，135.4mmol)，升温到回流且保温反应16h。降温到室温，过硅藻土饼，滤饼以乙酸乙酯泡洗，滤液减压浓缩干得油状物粗品，接着过柱纯化得到A1-3(25.0g，收率：82.8％)。MS(ESI)(m/z)：391([M+Na]+)。
第三步
加入二氯甲烷(112mL)、A1-3(9.8g，26.6mmol)、A1-3X(17.6g，106.0mmol)、PPTS(1.3g，5.2mmol)，氮气置换保护，升温到45℃回流反应2h。反应液减压浓缩，过柱纯化得到A1-4(5.4g，收率：43.2％)，MS(ESI)(m/z)：497([M+Na]+)。
第四步
加入A1-4(2.8g，5.9mmol)、加入N,N-二甲基甲酰胺(14mL)、加入DBU(0.9g，5.9mmol)，氮气置换保护后室温搅拌3～4h，待原料反应完全后，得到粗品A1-5，没有进一步处理，直接进行下一步反应。
第五步
加入冰醋酸(39mL)、马来酸酐(3.5g，36mmol)、6-氨基己酸(3.9g，30mmol)，升温到120℃且保温搅拌4h，反应完全后降温。反应液减压浓缩干。浓缩物中加水，以乙酸乙酯提取，有机相依次水洗、饱和食盐水洗，有机相减压浓缩干。加水50mL，室温搅拌，过滤，50℃减压干燥得到A1-8A(4.5g，收率：71.0％)。
第六步
加入A1-8A(4.5g，21.3mmol)、乙腈(45mL)、N-羟基丁二酰亚胺(2.7g，23.4mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(4.9g，25.6mmol)，室温搅拌反应。将体系减压浓缩干，加入饱和碳酸氢钠水溶液50mL，二氯甲烷提取，有机相干燥，减压浓缩，粗品过柱纯化，收集A1-8X(5.0g，收率：79.8％)。MS(ESI)(m/z)：315.3([M+Na]+)。
第七步
加入L-苯丙氨酸(5.2g，31.5mmol)、乙腈(56mL)、纯化水(56mL)、三乙胺(3.7g，36.5mmol)、A1-2(15.8g，44.6mmol)，室温搅拌16h直至原料反应完全，减压浓缩，过柱纯化得到目标物A1-6(12.0g，收率：76.9％)。MS(ESI)(m/z)：502([M+H]+)。
第八步：
加入A1-6(3.0g，6.0mmol)、N,N-二甲基甲酰胺(15mL)、N,N-二异丙基乙基胺(1.1g)，氮气保护下降温到-5～0℃，加入2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(2.62g，6.9mmol)，保温反应0.5～1h。加入第四步备用A1-5粗品。恢复到室温搅拌反应直至原料反应完全。加水100mL淬灭反应，乙酸乙酯提取，合并有机相再以pH＝3～5的稀酸水洗涤、饱和食盐水洗涤，有机相干燥、减压浓缩得到粗品过柱纯化，得到目标物A1-7(1.6g，两步收率：37.5％)。MS(ESI)(m/z)：758([M+Na]+)。
第九步：
加入A1-7(1.6g，2.2mmol)、N,N-二甲基甲酰胺(11mL)、DBU(0.3g，2mmol)，氮气置换保护室温搅拌反应1.5h。取样原料反应完全。直接加入10％Pd/C(0.2g)，氢气常压置换，室温搅拌反应2h，硅藻土过滤，加入20mL工艺纯化水，加入10mL‘二氯甲烷，搅拌，分层，所得水相减压浓缩干得到目标物A1-8(0.9g，收率：98.0％)。
第十步
加入A1-8(0.9g，2mmol)、乙腈(6.3mL)、水(6.3mL)、三乙胺(0.2g，2mmol)、A1-8X(0.5g，1.7mmol)，氮气置换保护，室温搅拌2h。加水20mL，以乙酸乙酯20mL*2洗涤，水相以乙酸调pH到4～5，水相减压浓缩干得到A1-9(1.0g，收率95.2％)。MS(ESI)(m/z)：639([M+Na]+)。
第十一步
加入A1-9(1.0g，1.6mmol)、二氯甲烷(16mL)、4-二甲氨基吡啶(0.26g)，室温搅拌下加入A1-9X(0.68g,1.57mmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.4g)，室温搅拌16h。待原料反应完全后，反应液依次以10％柠檬酸水溶液洗涤，水洗，饱和碳酸钠水溶液洗、饱和食盐水洗，有机相干燥减压浓缩干，粗品过柱纯化，得到目标物A1(1.2g,收率73.0％)。
1H-NMR(400MHz,DMSO-d6):10.05(s,1H),9.00(s,2H),8.2(br,1H),7.85(s,2H),7.44(m,1H),7.10–7.25(m,5H),6.74(s,1H),4.70–4.80(m,3H),4.32(s,2H),4.25(s,2H),4.15(s,2H),4.10–3.55(m,15H),3.40–3.05(m,6H),3.00–1.15(m,10H),0.99(t,3H).MS(ESI)(m/z)1043([M+Na]+)。
2、化合物A1片段A1-9X的合成
第一步
加入A1-9X-1X(642g，4200mmol)、三苯基膦(1155g，4410mmol)、乙腈(6L)，升温到回流保温反应24h。降温到室温，反应液减压浓缩干，加入甲基叔丁基醚6L，过滤，滤饼甲基叔丁基醚泡洗，滤饼减压干燥得A1-9X-2X(1500g，收率：86.0％)。
第二步
加入四氢呋喃(2.4L)、A1-9X-1(426g，3000mmol)、A1-9X-2X(1500g，3600mmol)，氮气保护下降温至-5℃，再滴加叔丁醇钠(840g，7500mmol)/四氢呋喃(2.4L)溶液，保温反应5-8h。将体系倒入7.5L冰水中，40-45℃减压浓去大部分THF，再用5.0L MTBE分三次萃取副产物，水相用6.0N盐酸调pH＝4-5，再用4.0L EA分三次萃取产物，合并有机层，分别用水、盐洗、无水硫酸钠干燥、过滤浓干得A1-9X-2(594.0g，收率100％)。
第三步
加入A1-9X-2(594.0，3000mmol)、乙酸乙酯(2500mL)溶解后加入10％Pd/C(含水50％，75g)，再分别用氮气和氢气置换，25-35℃常压加氢反应16h，硅藻土过滤，滤液减压浓缩干得A1-9X-3(600.0g，收率100％)。
第四步
将A1-9X-3(600.0g，3000mmol)加入到浓硫酸(3000g)中，期间温度控制在35℃以下，加完室温反应2-3h。将反应液缓慢倒入6.5kg冰水中，加入甲基叔丁基醚3.5L及乙酸乙酯0.5L萃取分液，合并有机层依次饱和碳酸氢钠和盐水洗涤，有机相干燥减压浓缩，粗品过柱纯化得A1-9X-4(230.0g，收率：42.1％)。1H-NMR(400MHz,CDCl3):6.79-6.69(m,2H),2.96(t,J＝6.0Hz,2H),2.64(t,J＝6.0Hz,2H),2.11(m,2H).
第五步
将A1-9X-4(125g，686.8mmol)加入浓硫酸(1200g)中，期间温度控制在5℃以下，再分批加入硝酸钠(70g，823.5mmol)，加完室温反应2-3h。反应液缓慢倒入6.5kg冰水中，以1.5L甲基叔丁基醚及150mL乙酸乙酯混合溶液萃取，合并有机层，依次用饱和碳酸氢钠，盐水洗涤，有机相干燥减压浓缩所得粗品过柱纯化，得到A1-9X-5(71.0g，收率：45.5％)。
第六步
加入乙醇(2.1L)、水(0.3L)、氯化铵(70g，1308mmol)、A1-9X-5(100g，440mmol)，搅拌溶解后，缓慢加入铁粉(200g，3571mmol)，升温到80℃反应1-2h。降温过滤，滤液减压浓去大部分乙醇，再加入水和2.6L二氯甲烷萃取，合并有机层，再分别用饱和碳酸氢钠，盐水洗涤，干燥后得到A1-9X-6萃取液，直接进行下一步。
第七步
向A1-9X-6萃取液中加入吡啶(150g，1900mmol)、4-二甲氨基吡啶(1.0g)控制温度在15℃以下缓慢滴加醋酸酐(99g，968mmol)，室温反应2-3h。将反应液缓慢倒入1.5kg冰水中，有机层再用1.0N稀盐酸洗至酸性，再分别用饱和碳酸氢钠，盐水洗涤，有机相减压浓缩干。浓缩物中加入1.0L甲醇溶解，用25.0％NaOH调pH＝11-12，35-45℃水解1-3小时，减压浓缩掉有机溶剂。加入二氯甲烷和水，萃取，分层，有机层减压浓缩干，浓缩物甲基叔丁基醚打浆，过滤，滤饼干燥得到A1-9X-7(55.0g，收率：52.3％)。1H-NMR(400MHz,CDCl3):6.83 (m,1H),2.86(t,J＝6.0Hz,2H),2.64(t,J＝6.0Hz，2H),2.25(s,3H),2.06(m,2H).
第八步
加入A1-9X-7(9.0g，37.6mmol)、二甲亚砜(150mL)，搅拌下缓慢加入25％氨水(250g)，将反应液转移至高压釜中，升温到65-75℃反应16h。降温，加入适量的水，用乙酸乙酯多次萃取，合并有机层，依次用水、饱和食盐水洗涤，有机层干燥，减压浓缩干。粗品过柱纯化，得到A1-9X-8(4.5g，收率：50.6％)。1H-NMR(400MHz,DMSO-d6):9.09(s,1H),6.43(d,J＝12.4Hz,1H),2.66(t,J＝6.0Hz,2H),2.50(m,2H),2.00(s,3H),1.87(t,J＝6.0Hz,2H).LC-MS:(m/z):237.1[M+H]+。
第九步
加入A1-9X-8(4g，17mmol)、A1-10X-8B(4.9g，18.6mmol)、PPTS(4.8g，17mmol)、甲苯(800mL)，氮气保护，升温到120℃且保温反应8h。降温，反应液减压浓缩，浓缩物用丙酮50mL打浆，降至0-5℃，抽滤，滤饼减压干燥得A1-9X-9(6.5g，收率：82.5％)。LC-MS:(m/z):464.2[M+H]+。
第十步
加入A1-9X-9(5.0g，10.8mmol)，浓盐酸(25mL)，水(25mL)，升温到80℃，保温反应1h。反应液减压浓缩干，加30mL丙酮打浆，抽滤得A1-9X(3.1g，收率：68.1％)。LC-MS:(m/z):422.1[M+H]+。
3、化合物A2～A6的合成
参照化合物A1的合成，分别将A1-3X替换为：
4、化合物A7、A8的合成
参照化合物A1、A5的合成，将A1-9X替换为A1-10X:
5、化合物A7、A8片段A1-10X的合成
第一步
加入二氯甲烷(6L)，A1-10X-1(914g，6220mmol),三乙胺(754g，7464mmol)，降温至0℃。滴加醋酸酐(698g，6842mmol)，约1h滴完。继续反应30min。向体系中加入水1L，静置分液，水层用二氯甲烷800mL萃取一遍，合并有机相。有机相用1N盐酸调PH为2左右，分出有机相，水层用二氯甲烷萃取一遍，合并有机相。有机相用饱和碳酸氢钠溶液1L洗，滤液减压浓缩干得A1-10X-2(1150g，收率：97.8％)。
第二步
加入硫酸(2L)、A1-10X-2(300g，1580mmol),降温至0℃，于2h内分批加入硝酸钠(134.3g，1580mmol)，反应30min。搅拌状态下将反应液缓慢倒入8L冰 水中，抽滤，滤饼用冰水淋洗两次。滤饼用1L二氯甲烷溶解，用饱和碳酸氢钠溶液洗至碱性，有机相干燥，减压浓缩干。加400mL甲基叔丁基醚打浆，降温0-5℃，抽滤，滤饼减压干燥得A1-10X-3(264g，收率：71.3％)。
第三步
加入丙酮(5L)、A1-10X-3(160g，680mmol),再将硫酸镁(111.6g，930mmol)溶解于水(620mL)中，加入体系。降温至0℃，于3h分批加入高锰酸钾(323g，2050mmol)。加完自然升温。反应1.5h。将配置好的40％硫代硫酸钠溶液(2L)滴加至反应体系，至检测无氧化性。抽滤，滤液浓缩，加入二氯甲烷6L,有机相以水洗，用饱和碳酸氢钠溶液洗，饱和食盐水洗，干燥，减压浓缩干得粗品。过柱纯化得到A1-10X-4(100g，收率：59.2％)。
第四步
加入A1-10X-4(80g，320mmol)、浓盐酸(500mL)，加热90-100℃且保温反应2h，降温0-10℃，将反应液倒入2L冰水中，过滤，用冰水淋洗滤饼，得A1-10X-5(66g，收率：100％)。
第五步
加入A1-10X-5(66g，320mmol)、二氯甲烷(1.5L)，搅拌溶解，加入吡啶(52mL，650mmol)，降温至0℃，于40min内滴加三氟乙酸酐(94mL，676mmol)，滴完继续反应20min，反应液减压浓缩干，再加入1L二氯甲烷溶解，用1N盐酸洗至无吡啶，再用饱和碳酸氢钠溶液洗至碱性，用饱和食盐水洗。有机相干燥，减压浓缩干得A1-10X-6(62g，收率：64.1％)。
第六步
加入甲醇(1L)、甲酸(50mL)、水(50mL)，将A1-10X-6(54g，180mmol)用二氯甲烷(100mL)溶解加入体系中，降温至0℃，于50min内分批加入锌粉(150g，2290mmol)，继续反应1.5h。抽滤，滤液分液，有机相用饱和碳酸氛钠溶液洗，饱和食盐水洗，有机相干燥，减压浓缩得A1-10X-7(42g，收率：85.7％)。
第七步
加入二氯甲烷(1.6L)、A1-10X-7(40g，147.0mmol)、三乙胺(49mL，352.5mmol)，降温0-5℃，滴加乙酰氯(27mL，380.0mmol)，继续反应50min，加600mL水。抽滤，滤饼用水打浆，得A1-10X-8(46.2g，收率：100％)。
第八步
加入甲醇(2L)，A1-10X-8(30g，96mmol)，水(150mL)，升温到50℃，加入碳酸钾(50g，360mmol)，继续反应20min。降至室温，反应液减压浓缩干，加入400mL水打浆，抽滤，滤饼再用300mL水打浆，抽滤得A1-10X-9(7.8g，收率：37.3％)。
第九步
加入A1-10X-9(6g，27.5mmol)、A1-10X-8B(8g，30.3mmol)、PPTS(6.9g，27.5mmol)，甲苯(1L)，氮气保护，升温到120℃且保温反应4h。降至0-5℃，抽滤，滤饼用二氯甲烷和甲醇溶解，减压浓缩干。浓缩物以丙酮50mL于0-5℃打浆，抽滤，得A1-10X-10(12.2g，收率：100％)。
第十步
加入A1-10X-10(12.2g，27.5mmol)、浓盐酸(80mL)、水(80mL)，升温到80℃且保温反应2.5h。降温到室温，加少量甲醇，反应液减压浓缩干。浓缩物用30mL丙酮室温打浆，抽滤，滤饼减压干燥得A1-10X(11.1g，收率：100％)。1H-NMR(400MHz,DMSO-d6):8.28(d,J＝7.2Hz,1H),7.45(br,2H),7.25(d,J＝7.2Hz,1H),6.70(s,1H),4.65(m,2H),4.20(s,2H),3.50(m,2H),2.73(m,2H),2.20(m,2H),1.81(m,1H),0.91(t,J＝5.0Hz,2H)。LC-MS:(m/z):404.1[M+H]+
6、化合物A10的合成

第一步
加入A10-1(5.0g，66.6mmol)、芴甲氧羰酰氯(20.6g，79.6mmol)、二氧六环(50mL)，降温到0～5℃，滴加1N碳酸钠水溶液(60mL)。滴完恢复到室温搅拌反应约2h。降温到0～5℃，以1M HCl约60mL调pH＝2，水相乙酸乙酯100mL*3提取，合并有机相减压浓缩，得到固体15g。加入甲基叔丁基醚50mL，室温打浆30min，过滤，滤饼减压浓缩，得到A10-2(17.9g，收率：90.3％)。MS(ESI)(m/z)：298([M+H]+)。
第二步
加入A10-2(17.9g，60.2mmol)、乙腈(180mL)、N-羟基丁二酰亚胺(7.7g，66.9mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(13.8g，72.0mmol)，室温搅拌反应。将体系减压浓缩干，加入饱和碳酸氢钠水溶液270mL，二氯甲烷提取，有机相干燥后减压浓缩干，粗品过柱纯化，收集A10-3(19.2g，收率：84.3％)。MS(ESI)(m/z)：401([M+Na]+)。
第三步
加入A10-4(5.0g，43.4mmol)、甲醇(75mL)，降温到-5～0℃，缓慢滴加氯化 亚砜(10.3g，86.6mmol)，滴完恢复到室温搅拌1h，升温到回流且保温反应1h。降温，反应液减压浓缩干，过柱纯化得到A10-5(5.16g，收率：92.0％)。
第四步
加入A10-5(5.0g，38.7mmol)、乙腈(50mL)、水(50mL)、三乙胺(8.2g，81.0mmol)、A10-3(17.6g，46.5mmol)，氮气置换保护，室温搅拌2h。补加水80mL，乙酸乙酯50mL*2洗涤，水相以乙酸调pH＝4-5，水相减压浓缩干得A10-6(11.9g，收率75.3％)。MS(ESI)(m/z)：431([M+Na]+)。
第五步
加入A10-6(11g，26.9mmol)、加入N,N-二甲基甲酰胺(55mL)、DBU(4.1g，26.9mmol)，氮气置换保护后室温搅拌3～4h。原料反应完全未作处理，A10-7粗品直接投料下一步。
第六步
加入A1-6(13.5g，26.9mmol)、N,N-二甲基甲酰胺(55mL)、N,N-二异丙基乙基胺(4.9g，37.9mmol)，氮气保护下降温到-5～0℃，加入2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(11.8g，31.0mmol)，保温反应0.5～1h。加入第五步所制备的整批A10-7反应液。恢复到室温搅拌反应直至原料反应完全。加水200mL淬灭反应，以乙酸乙酯提取，合并有机相再以pH＝3～5稀酸水洗涤、饱和食盐水洗涤，有机相干燥、减压浓缩得到的粗品过柱纯化，得A10-8(8.1g，两步收率：45.0％)。MS(ESI)(m/z)：693([M+Na]+)。
第七步
加入A10-8(8.0g，11.9mmol)、加入N,N-二甲基甲酰胺(40mL)、DBU(1.8g，11.8mmol)，氮气置换保护后室温搅拌3～4h。原料反应完后，A10-9粗品未做处理直接投料下一步。
第八步
加入A1-8A(2.5g，11.8mmol)、N,N-二甲基甲酰胺(12.5mL)、N,N-二异丙基乙基胺(2.2g，17.0mmol)，氮气保护下降温到-5～0℃，加入2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(5.2g，13.7mmol)，保温反应0.5～1h。加入第九步所制备的整批A10-9反应液。恢复到室温搅拌反应直至原料反应完全。加水100mL淬灭反应，以乙酸乙酯提取，合并有机相再以pH＝3～5稀酸水洗涤、饱和食盐水洗涤，有机相干燥、减压浓缩得到粗品过柱纯化，得A10-10(4.78g， 两步收率：62.8％)。MS(ESI)(m/z)：663([M+Na]+)。
第九步
氮气保护下，加入A10-10(4.7g，7.3mmol)、干燥甲醇(50mL)，室温搅拌溶清。加入无水氢氧化锂(0.63g，26.3mmol)，室温继续搅拌反应2h，加入乙酸调pH＝4.5，减压浓缩干得到A10-11(4.1g，收率：89.1％)。
第十步
加入A10-11(4.0g，6.4mmol)、二氯甲烷(50mL)、4-二甲氨基吡啶(1.1g，9.0mmol)，室温搅拌下加入A1-9X(2.7g，6.4mmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1.5g)，室温搅拌16h。待原料反应完全，反应液依次以10％柠檬酸水溶液洗涤，水洗，饱和碳酸钠水溶液洗、饱和食盐水洗，有机相干燥减压浓缩，粗品过柱纯化，得A10(4.0g，收率60.6％)。1H-NMR(400MHz,DMSO-d6):δ10.05(s,1H),9.00(s,2H),8.2(br,1H),7.85(s,2H),7.44(m,1H),7.10–7.25(m,5H),6.74(s,1H),4.70–4.80(m,3H),4.32(s,2H),4.25(s,2H),4.15(s,2H),4.10–3.55(m,15H),3.40–3.05(m,6H),3.00–1.15(m,10H),0.99(t,3H).MS(ESI)(m/z)：1053([M+Na]+)。
7、化合物A9、A11、A12的合成
参照化合物A10的合成，分别将A10-4替换为：
8、化合物A13的合成
参照化合物A11的合成，将A1-9X替换为A1-10X:
9、化合物A15的合成
第一步
加入A15-3X-2(50g，342mmol)、二氯甲烷(500mL)，搅拌溶解，加入吡啶(13.5g，171mmol)，降温至0℃，于40min内滴加三氟乙酸酐(35.9g，171mmol)，滴完继续反应20min，反应液减压浓缩干，再加入1L二氯甲烷溶解，用1N盐酸洗至无吡啶，再用饱和碳酸氢钠溶液洗至碱性，饱和食盐水洗。有机相干燥，减压浓缩干过柱纯化得到A15-3X(13.2g，收率：32.0％)。
第二步
加入A15-3X(10g，41.3mmol)、乙腈(100mL)、水(100mL)、三乙胺(41.8g，41.3mmol)、A14-3(17.4g，41.3mmol)，氮气置换保护，室温搅拌2h。加水200mL，以乙酸乙酯200mL*2洗涤，水相以乙酸调pH＝4～5，水相减压浓缩干得A15-4(17.7g，收率76.3％)。
第三步
加入A15-4(17.7g，31.4mmol)、乙腈(180mL)、N-羟基丁二酰亚胺(4.0g，34.5mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(7.2g，37.7mmol)，室温搅拌反应。将体系减压浓缩干，加入饱和碳酸氢钠水溶液100mL，以二氯甲烷提取，有机相干燥后减压浓缩干，粗品过柱纯化，收集A15-5(16.4g，收率：81.3％)。
第四步
加入甘氨酸(1.9g，24.8mmol)、乙腈(50mL)、水(50mL)、三乙胺(2.5g，24.8mmol)、A15-5(16.0g，24.8mmol)，氮气置换保护，室温搅拌1.5h。加水80mL，乙酸乙酯50mL*2洗涤，水相以乙酸调pH＝4～5，水相减压浓缩干得到A15-6(13.2g，收率85.6％)。
第五步
加入四氢呋喃(90mL)、A14-6(13.0g，20.9mmol)、乙酸(55mL)、氮气置换保护，升温到40℃，加入四乙酸铅(37.1g，83.6mmol)，升温到回流且保温反应16h。降温到室温，过硅藻土饼，滤饼以乙酸乙酯泡洗，滤液减压浓缩干，过柱纯化得到A15-7(13.2g，收率：100％)。
第六步
加入二氯甲烷(100mL)、A15-7(13.0g，20.5mmol)、羟基乙酸(6.2g，82mmol)、PPTS(1.0g，4.1mmol)，氮气置换保护，升温到45℃回流反应2h。反应液减压浓缩，过柱纯化得到A15-8(6.8g，收率：51.1％)。
第七步
加入A14-8(6.5g，10.0mmol)、加入N,N-二甲基甲酰胺(32.5mL)、DBU(1.5g，10.0mmol)，氮气置换保护后室温搅拌3～4h，原料反应完后未后处理，A15-9粗品直接投料下一步。
第八步
加入A1-8X(2.9g，10.0mmol)、N,N-二甲基甲酰胺(45mL)、N,N-二异丙基乙基胺(1.8g，14.0mmol)，氮气保护下降温到-5～0℃，加入2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(4.4g，11.5mmol)，保温反应0.5～1h。加入A15-9的整批反应液。恢复到室温搅拌反应直至原料反应完全。加水100mL淬灭反应，乙酸乙酯提取，合并有机相再以pH＝3～5的稀酸水洗涤、饱和食盐水洗涤，有机相干燥、减压浓缩得到的粗品过柱纯化，得到A15-10(3.1g，两步收率：49.2％)。
第九步
氮气保护下，加入A15-10(3.0g，4.7mmol)、干燥甲醇(20mL)，室温搅拌溶清后加入无水氢氧化锂(0.45g，18.8mmol)，于室温继续搅拌反应3h，加入乙酸调pH＝4.5，减压浓缩干得到A15-11(2.7g，收率：93.4％)。
第十步
加入A15-11(2.5g，4.0mmol)、二氯甲烷(20mL)、4-二甲氨基吡啶(0.67g，5.5mmol)，室温搅拌下加入A1-9X(1.7g，4.0mmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.95g，5.0mmol)，室温搅拌16h。待原料反应完全后，反应液依次以10％柠檬酸水溶液洗涤，水洗，饱和碳酸钠水溶液洗、饱和食盐水洗，有机相干燥后减压浓缩干，粗品过柱纯化，得到A15-12(2.8g，收率：68.7％)。
第十一步
加入甲醇(25mL)、A15-12(2.5g，2.4mmol)、水(12.5mL)，升温到50℃，加入碳酸钾(1.3g，9.6mmol)，继续反应20min。降至室温，反应液减压浓缩干，加入40mL水打浆，抽滤，滤饼再用30mL水打浆，抽滤得到A15(0.83g，收率：37.3％)。
10、化合物A14的合成
参照化合物A15的合成，将A15-3X替换为D-丙氨酸:
11、化合物A16的合成
参照化合物A15的合成，将A15-3X替换为D-丙氨酸，A1-9X替换为A1-10X:
12、化合物A17的合成
参照化合物A1的合成，将6-氨基己酸替换为3-[2-[2-(2-氨基乙氧基)乙氧基]乙氧基]丙酸：
13、化合物A18的合成
参照化合物A15的合成，将A15-3X替换为D-丙氨酸，A1-9X替换为A1-10X，
将6-氨基己酸替换为3-[2-[2-(2-氨基乙氧基)乙氧基]乙氧基]丙酸：
14、化合物A20的合成
第一步
加入A20-1(10.0g，67.5mmol)、二氯甲烷(500mL)，降温至0～5℃，滴加Boc2O(7.4g，33.9mmol)，保温搅拌5h，升温到室温搅拌反应过夜。待原料Boc2O反应完全后加水200mL洗涤有机相，水相再以DCM 50mL提取，合并有机相干燥过滤，减压浓缩干得A20-2(8.4g，收率：100％)。
第二步
加入A20-3(9.7g，100mmol)、乙酸乙酯(50mL)、N-甲基吗啉(11mL，100mmol)，降温到0～5℃，缓慢加入氯甲酸乙酯(7.7mL，80.5mmol)，期间放热不明显。加完后恢复到室温搅拌反应1h。反应液加水50mL，分液，水相再以乙酸乙酯30mL*2提取，合并有机相减压浓缩干，拌样过柱纯化，收集A20-4(7.3g，收率：54.0％)。
第三步
加入A20-2(3.0g，12.1mmol)、饱和碳酸氢钠水溶液(90mL)、室温搅拌15min。过滤，滤液降温到0～5℃，加入A20-4(2.0g，11.8mmol)，室温搅拌16h。水溶液减压浓缩干，以甲苯带水。浓缩物中加入乙酸钠(10g，121.9mmol)、乙 酸酐(20mL，220.0mmol),加热升温到120℃且保温反应0.5h。降温，将反应液减压浓缩干，加水50mL，二氯甲烷50mL*2提取，有机相干燥，减压浓缩，得A20-5(5.7g，收率：100％)。
第四步
加入A20-5(5.7g，17.4mmol)、二氯甲烷(50mL)、三氟乙酸(10g，87.7mmol)，室温搅拌2～3h，固体析出，过滤，滤饼减压干燥得A20-6(5.3g，收率：89.1％)。
第五步
氮气保护下，加入A20-7(15.0g，90.3mmol)、DMF(240mL)、碳酸氢钾(9.1g，90.9mmol)，降温到0～5℃，滴加溴化苄(10.8mL，90.9mmol)，滴完恢复到室温搅拌反应16h，原料反应完全后将反应液倒入冰水中，搅拌1h，过滤，滤饼乙酸乙酯50mL溶解，有机相干燥，过滤，减压浓缩干，得A20-8(20.4g，收率：88.3％)。
第六步
加入40％溴化氢/乙酸溶液(25mL)、降温到0～5℃，缓慢分批加入A20-9(5.0g，13.3mmol)。加完恢复到室温，搅拌反应2h，加甲苯50mL，混合物减压浓缩干，浓缩物中加入乙酸乙酯100mL，有机相饱和碳酸氢钠水溶液100mL洗涤，洗完水相pH＝8。有机相无水硫酸钠干燥，过滤，减压浓缩干得A20-10(5.1g，收率：96.6％)。
第七步
加入A20-8(2.69g，10.5mmol)、A20-10(5.0g，12.6mmol)、乙腈(50mL)、活化分子筛(5.0g)、氧化银(9.3g，40.1mmol)，氮气置换保护，室温搅拌2h，取样TLC(PE/EA＝10/1，UV)，原料反应完全。加水100mL，搅拌硅藻土过滤，滤液乙酸乙酯150mL*3提取，有机相合并无水硫酸钠干燥，过滤，滤液减压浓缩干，得A20-11(6.0g，收率：100％)。MS(ESI)(m/z)：595([M+Na]+)。
第八步
加入A20-11(6.0g，10.5mmol)、异丙醇(18mL)/二氯甲烷(90mL)的混合溶液，200～300目硅胶(6.0g)，降温到0～5℃，缓慢分批加入硼氢化钠(0.8g，21.1mmol)，加完恢复室温搅拌反应2h。加水100mL，以乙酸乙酯提取，有机相干燥，过滤减压浓缩。浓缩物加入无水乙醇(275mL)、10％Pd/C(0.55g)，氢气置换常压常温 反应10～20min。过硅藻土饼，滤饼以乙醇洗涤，滤液减压浓缩干得A20-12(4.2g，两步收率：81.9％)。
第九步
加入A20-12(2.8g，5.8mmol)、N,N-二甲基甲酰胺(20mL)，搅拌降温到0～5℃，加入三乙胺(1.7g，16.8mmol)、4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(3.5g，11.9mmol)、A20-6(2.0g，5.8mmol)。室温搅拌反应1h，加水50mL，二氯甲烷50mL*3提取，有机相干燥浓缩所得粗品过柱纯化得A20-13(1.4g，收率：32.2％)。
第十步
加入A1-9X(0.7g，1.7mmol)、乙腈(20mL)、4-二甲氨基吡啶(0.26g，2.1mmol)，室温搅拌下加入三光气(0.5g，1.7mmol)，室温搅拌2h。加入A20-13(1.2g，1.7mmol)，室温搅拌16h，反应液减压浓缩干，粗品过柱纯化，得到A20(1.15g，收率：68.8％)。
15、化合物A19片段A19-4的合成
第一步
加入A19-1X(10.0g，166.4mmol)、二氯甲烷(200mL)，降温至0～5℃，滴加Boc2O(18.1g，83.2mmol)，保温搅拌3h，升温到室温搅拌,待原料Boc2O反应完全后加水100mL洗涤有机相，水相再以DCM 50mL提取，合并有机相干燥过滤，减压浓缩干得到A19-2X(12.1g，收率：90.8％)。
第二步
加入A19-2X(12.0g，74.9mmol)、N,N-二甲基甲酰胺(100mL)、N,N-二异丙基乙胺(48.5g，374.9mmol)、A1-8A(15.8g，74.9mmol)、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(34.3g，90.1mmol)，反应于室温搅拌15min，反 应液减压浓缩干，加入二氯甲烷100mL，有机相水洗，饱和食盐水洗，有机相干燥减压浓缩干得到A19-3X(16.3g，收率：61.6％)。MS(ESI)(m/z)：354([M+H]+)。
第三步
加入A19-3X(16.0g，45.3mmol)、二氯甲烷(160mL)，缓慢加入三氟乙酸(32g，280.7mmol)，室温搅拌1h。将反应液减压浓缩干得A19-4X(10.9g，收率：95.6％)。
16、化合物A19的合成
参照化合物A20的合成，将A1-9X替换为A1-10X，将A20-6替换为A19-4X。
17、化合物A21的合成
参照化合物A20的合成，将A20-6替换为A19-4X。
18、化合物A22的合成
参照化合物A20的合成，将A20-1替换为氨基-单乙二醇-羧酸。
19、化合物A23的合成
参照化合物A20的合成，将A1-9X替换为A1-10X，将A20-1替换为氨基-单乙二醇-羧酸。
20、化合物A24的合成
参照化合物A20的合成，将A20-1替换为3-[2-[2-(2-氨基乙氧基)乙氧基]乙氧基]丙酸。
21、化合物A25的合成
参照化合物A20的合成，将A20-1替换为3-[2-[2-(2-氨基乙氧基)乙氧基]乙氧基]丙酸，将A1-9X替换为A1-10X。
22、中间体A1-10X的合成
第一步
加入二氯甲烷(6L)，A1-10X-1(914g，6220mmol),三乙胺(754g，7464mmol)，降温至0℃。滴加醋酸酐(698g，6842mmol)，约1h滴完。继续反应30min。向体系中加入水1L，静置分液，水层二氯甲烷800mL萃取，合并有机相。有机相用1N盐酸调pH＝2，分出有机相，水层二氯甲烷萃取，合并有机相。有机相饱和碳酸氢钠溶液1L洗，滤液减压浓缩干得A1-10X-2(1150g，收率：97.8％)。
第二步
加入硫酸(2L)、A1-10X-2(300g，1580mmol),降温至0℃，于2h内分批加入硝酸钠(134.3g，1580mmol)，反应30min。搅拌状态下将反应液缓慢倒入8L冰水中，抽滤，滤饼用冰水淋洗两次。滤饼1L二氯甲烷溶解，饱和碳酸氢钠溶液洗至碱性，有机相干燥，减压浓缩干。400mL甲基叔丁基醚打浆，降温0-5℃，抽滤，滤饼减压干燥得到A1-10X-3(264g，收率：71.3％)。
第三步
加入丙酮(5L)、A1-10X-3(160g，680mmol)，将硫酸镁(111.6g，930mmol)溶于水(620mL)中，加入体系。降温至0℃，3h分批加入高锰酸钾(323g，2050mmol)。加完自然升温。反应1.5h。将配置好40％硫代硫酸钠溶液(2L)滴加至反 应体系，至检测无氧化性。抽滤，滤液浓缩，加入二氯甲烷6L,有机相以水洗，用饱和碳酸氢钠溶液洗，饱和食盐水洗，干燥，减压浓缩干得粗品。过柱纯化得到A1-10X-4(100g，收率：59.2％)。LC-MS:(m/z):274.3[M+Na]+。
第四步
加入A1-10X-4(80g，320mmol)、浓盐酸(500mL)，加热90-100℃且保温反应2h，降温0-10℃，将反应液倒入2L冰水中，过滤，冰水淋洗滤饼，得A1-10X-5(66g，收率：100％)。
第五步
加入A1-10X-5(66g，320mmol)、二氯甲烷(1.5L)，搅拌溶解，加入吡啶(52mL，650mmol)，降温至0℃，40min内滴加三氟乙酸酐(94mL，676mmol)，滴完继续反应20min，反应液减压浓缩干，再加入1L二氯甲烷溶解，1N盐酸洗至无吡啶，饱和碳酸氢钠溶液洗至碱性，饱和食盐水洗。有机相干燥，减压浓缩干得A1-10X-6(62g，收率：64.1％)。
第六步
加入甲醇(1L)、甲酸(50mL)、水(50mL)，将A1-10X-6(54g，180mmol)二氯甲烷(100mL)溶解加入体系中，降温至0℃，50min内分批加入锌粉(150g，2290mmol)，继续反应1.5h。抽滤，滤液分液，有机相饱和碳酸氛钠溶液洗，饱和食盐水洗，有机相干燥，减压浓缩得到A1-10X-7(42g，收率：85.7％)。
第七步
加入二氯甲烷(1.6L)、A1-10X-7(40g，147.0mmol)、三乙胺(49mL，352.5mmol)，降温0-5℃，滴加乙酰氯(27mL，380.0mmol)，继续反应50min，加600mL水。抽滤，滤饼用水打浆，得到A1-10X-8(46.2g，收率：100％)。1H-NMR(400MHz,DMSO-d6):13.30(s,1H),9.60(s,1H),8.31(d,J＝9.2Hz，1H),7.72(d,J＝9.2Hz,1H),2.86(t,J＝7.0Hz,1H),2.74(t,J＝1.6Hz,1H),2.00(m,1H).LC-MS:(m/z):315.1[M+H]+。
第八步
加入甲醇(2L)，A1-10X-8(30g，96mmol)，水(150mL)，升温到50℃，加入碳酸钾(50g，360mmol)，继续反应20min。降至室温，反应液减压浓缩干，加入400mL水打浆，抽滤，滤饼再用300mL水打浆，抽滤得A1-10X-9(7.8g，收率：37.3％)。LC-MS:(m/z):219.1[M+H]+。
第九步
加入A1-10X-9(6g，27.5mmol)、A1-10X-8B(8g，30.3mmol)、PPTS(6.9g，27.5mmol)，甲苯(1L)，氮气保护，升温到120℃且保温反应4h。降至0-5℃，抽滤，滤饼二氯甲烷和甲醇溶解，减压浓缩干。浓缩物丙酮50mL 0-5℃打浆，抽滤，得A1-10X-10(12.2g，收率：100％)。LC-MS:(m/z):446.2[M+H]+。
第十步
加入A1-10X-10(12.2g，27.5mmol)、浓盐酸(80mL)、水(80mL)，升温到80℃且保温反应2.5h。降温到室温，加少量甲醇，反应液减压浓缩干。浓缩物用30mL丙酮室温打浆，抽滤滤饼减压干燥得到A1-10X(11.1g，收率：100％)。LC-MS:(m/z):404.1[M+H]+。
23、中间体A1-9X的合成
第一步
加入四氢呋喃(2.4L)、A1-9X-1(426g，3000mmol)、A1-9X-1X(1500g，3600mmol)，氮气保护下降温至-5℃，再滴加叔丁醇钠(840g，7500mmol)/四氢呋喃(2.4L)溶液，保温反应5-8h。将体系倒入7.5L的冰水中，40-45℃减压浓去大部分THF，再用5.0L的MTBE分三次萃取副产物，水相用6.0N盐酸调pH＝4-5，再用4.0LEA分三次萃取产物，合并有机层，分别用水、盐洗、无水硫酸钠干燥、过滤浓干得到A1-9X-2(594.0g，收率100％)。
第二步
加入A1-9X-2(594.0，3000mmol)、乙酸乙酯(2500mL)溶解后加入10％Pd/C(含水50％，75g)，再分别用氮气、氢气置换，25-35℃常压加氢反应16h，硅藻土过滤，滤液减压浓缩干得A1-9X-3(600.0g，收率100％)。
第三步
将A1-9X-3(600.0g，3000mmol)加入到浓硫酸(3000g)中，期间温度控制在35℃以下，加完室温反应2-3h。将反应液缓慢倒入6.5kg冰水中，加入甲基叔丁基醚3.5L及乙酸乙酯0.5L萃取分液，合并有机层依次饱和碳酸氢钠，盐水洗涤，有机相干燥减压浓缩干，粗品过柱纯化得到A1-9X-4(230.0g，收率：42.1％)。1H-NMR(400MHz,CDCl3):6.79-6.69(m,2H),2.96(t,J＝6.0Hz,2H),2.64(t,J＝6.0Hz，2H),2.11(m,2H).
第四步
将A1-9X-4(125g，686.8mmol)加入浓硫酸(1200g)中，期间温度控制在5℃以下，再分批加入硝酸钠(70g，823.5mmol)，加完室温反应2-3h。反应液缓慢倒入6.5kg冰水中，1.5L甲基叔丁基醚及150mL乙酸乙酯混合溶液萃取，合并有机层，依次饱和碳酸氢钠，盐水洗涤，有机相干燥减压浓缩所得粗品过柱纯化，得到A1-9X-5(71.0g，收率：45.5％)。
第五步
加入乙醇(2.1L)、水(0.3L)、氯化铵(70g，1308mmol)、A1-9X-5(100g，440mmol)，搅拌溶解后，缓慢加入铁粉(200g，3571mmol)，升温到80℃反应1-2h。降温过滤，滤液减压浓去大部分乙醇，再加入水和2.6L二氯甲烷萃取，合并有机层，再分别饱和碳酸氢钠，盐水洗涤，干燥后得A1-9X-6萃取液，未作处理直接进行下一步。LC-MS:(m/z):198.1[M+H]+。
第六步
往A1-9X-6萃取液中加入吡啶(150g，1900mmol)、4-二甲氨基吡啶(1.0g)控制温度在15℃以下缓慢滴加醋酸酐(99g，968mmol)，室温反应2-3h。将反应液缓慢倒入1.5kg冰水中，有机层再用1.0N稀盐酸洗至酸性，再分别用饱和碳酸氢钠，盐水洗涤有机相减压浓缩干。浓缩物中加入1.0L甲醇溶解，用25.0％NaOH调pH＝11-12，35-45℃水解1-3小时，减压浓缩掉有机溶剂。加入二氯甲烷和水，萃取，分层，有机层减压浓缩干，浓缩物用甲基叔丁基醚打浆，过滤， 滤饼干燥得A1-9X-7(55.0g，收率：52.3％)。1H-NMR(400MHz,CDCl3):6.83(m,1H),2.86(t,J＝6.0Hz,2H),2.64(t,J＝6.0Hz，2H),2.25(s,3H),2.06(m,2H).
第七步
加入A1-9X-7(9.0g，37.6mmol)、二甲亚砜(150mL)，搅拌下缓慢加入25％氨水(250g)，将反应液转移至高压釜中，升温到65-75℃反应16h。降温，加入适量的水，用乙酸乙酯多次萃取，合并有机层，依次用水、饱和食盐水洗涤，有机层干燥，减压浓缩干。粗品过柱纯化，得到A1-9X-8(4.5g，收率：50.6％)。1H-NMR(400MHz,DMSO-d6):9.09(s,1H),6.43(d,J＝12.4Hz,1H),2.66(t,J＝6.0Hz,2H),2.50(m,2H),2.00(s,3H),1.87(t,J＝6.0Hz,2H).LC-MS:(m/z):237.1[M+H]+。
第八步
加入A1-9X-8(4g，17mmol)、A1-10X-8B(4.9g，18.6mmol)、PPTS(4.8g，17mmol)、甲苯(800mL)，氮气保护，升温到120℃且保温反应8h。降温，反应液减压浓缩，浓缩物丙酮50mL打浆，降至0-5℃，抽滤滤饼减压干燥得A1-9X-9(6.5g，收率：82.5％)。LC-MS:(m/z):464.2[M+H]+。
第九步
加入A1-9X-9(5.0g，10.8mmol)，浓盐酸(25mL)，水(25mL)，升温到80℃，保温反应1h。反应液减压浓缩干，加30mL丙酮打浆，抽滤得A1-9X(3.1g，收率：68.1％)。LC-MS:(m/z):422.1[M+H]+。
实施例2、抗体偶联物的制备
将靶向HER2抗体Trastuzumab使用G25脱盐柱将其置换至50mM PB/1.0mM EDTA缓冲液申(pH7.0)，加入8当量TECP，37℃搅拌2小时，以使抗体链间二硫键完全打开，随后使用磷酸将还原后的抗体溶液pH调至6.0，并将水浴温度降至25℃，以备偶联反应。将按照上述实施例一方法制备得到连接子-药物偶联物和GGFG-Dxd(对照化合物)分别用DMA溶解，从中吸取12当量连接子-药物偶联物逐滴加至还原后的抗体溶液中，并补加DMA至其终浓度为10％(V/V)，25℃搅拌反应0.5小时，反应完成，采用0.22um膜过滤样品。使用切问流超滤系统纯化去除过量偶联小分子，缓冲液为50mM PB/1.0mM EDTA溶液(pH＝6.0)，纯化后添加终浓度6％蔗糖放置于-20℃冰箱中保存。使用UV法分别在280nm和370nm测定其吸光度值，计算DAR值。本技术方案 中大部分连接子-药物偶联物偶联过程中未产生沉淀，偶联物DAR值均为6-8，DAR值采用HIC-HPLC,RP-HPLC或LCMS进行测定，通过SEC-HPLC检测偶联物的聚体比例在正常范围，表明本发明抗体药物偶联物具有很好溶解性和成药性，偶联过程不发生沉淀。
实施例3体外细胞毒活性测试
选择稳定转染高表达Her2高表达SK-BR-3和BT-474人乳腺癌细胞、NCI-N87人胃癌细胞作和人非小细胞肺癌细胞A549为本次实验体外活性检测用细胞株，观察不同抗体偶联药物对细胞杀伤量效情况。初步选择每种细胞的种板密度：2×103细胞数/孔，16-24小时后进行细胞毒活性测定；其次测试实施例2制备得到抗体偶联药物加样后终浓度设定5000nM为起始浓度，5000-0.006nM设计系列10个浓度(4-10倍比稀释)，观察120小时杀伤(或抑制)变化，化学发光染色(Luminescent Cell Viability Assay)，读取荧光数据后计算IC50。
本发明中的连接子均为可裂解型连接子，因此对于所释放出的喜树碱类似物首先进行体外细胞毒活性测试，结果如下表所示。测试结果显示大部分化合物细胞杀伤活性优于对照化合物Dxd。

进一步对于偶联后的ADC药物进行体外细胞毒活性测试。从活性测试结果看，本申请化合物制备得到ADC均表现出一定抗肿瘤活性，IC50达到10-6～10- 10M，与对照样品相比显示出显著更强抗肿瘤活性。
实施例4体内抗肿瘤功效测定
在体内测量本发明组合的功效，即在啮齿类动物中植入癌细胞的同种异体移植物或异种移植物，并用所述组合处理肿瘤。将受试小鼠用药物或对照处理，并监测数周或更长时间以测量到达肿瘤倍增的时间、对数细胞杀伤和肿瘤抑制。
1)实验动物
BALB/cA-nude裸小鼠，6-7周，雌鼠，购自上海灵畅生物科技有限公司。
2)实验步骤
裸小鼠皮下接种人胃癌NCI-N87细胞，待肿瘤生长至100-250mm3后，将动物随机分组(D0)。每周测2-3次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：V＝1/2×a×b2其中a、b分别表示长、宽。T/C(％)＝(T-T0)/(C-C0)×100％，其中T、C分别为试验组和对照组实验结束时的肿瘤体积；T0、C0分别为试验组和对照组实验开始时肿瘤体积。
从活性测试结果看，本申请的化合物制备得ADC均表现出一定的体内抗肿瘤活性，与对照样品相比可以显示出显著更强抗肿瘤活性。荷瘤小鼠对以上药物均能很好耐受，没有体重减轻等症状发生。
工业实用性
本发明抑制剂为化合物A或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式、或其可药用的盐，进一步制备可得到抗体药物偶联物；本发明抗体药物偶联物具有很好溶解性和成药性，偶联过程不发生沉淀；ADC表现出明显体内抗肿瘤活性，与对照样品相比可以显示出显著更强抗肿瘤活性。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (19)

  1. 一种抑制剂化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：所述化合物包含式(A)所示结构：
    其中：X为氢、氟；Q为可与抗体偶联的接头或氢，L1是接头或氢与药物氨基连接基团。
  2. 根据权利要求1所述化合物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：Q为可与抗体上巯基偶联的基团，选自马来酰亚胺。
  3. 根据权利要求1所述化合物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：L1为接头与药物氨基连接基团，其选自L2为任选取代的C3-C7亚烷基、C3-C8环烷基、任选取代的二甘醇至八甘醇酰基，AA为2至4个氨基酸组成的肽段，M为亚甲基、C1-C6烷基或环烷基取代亚甲基、三氟甲基取代亚甲基、C3-C6环烷基。
  4. 根据权利要求3所述化合物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：L1为接头与药物氨基连接基团，其选自AA多肽残基选自：NH-Phe-Lys-C＝O、NH-Val-Cit-C＝O、NH-Val-Ala-C＝O、NH-Phe-Cit-C＝O、NH-Gly-Val-C＝O、NH-Ala-Lys-C＝O、NH-Ala-Ala-Ala-C＝O、NH-Glu-Val-Ala-C＝O、NH-Glu-Val-Cit-C＝O、NH-Gly-Gly-Phe-Gly-C＝O。
  5. 根据权利要求1所述化合物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：L1 为接头与药物氨基连接基团，选自L2为任选取代的C3-C7亚烷基、C3-C8环烷基、任选取代的二甘醇至八甘醇酰基。
  6. 如权利要求1-5所述化合物，其特征在于：式A具体分子结构如下：



  7. 一种抗体药物偶联物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：所述化合物包含式(B)所示结构：
    其中：X为氢、氟；Q为可与巯基偶联的接头，L1是接头与药物氨基连接基团，Ab为配体，n＝1-8。
  8. 根据权利要求7所述抗体药物偶联物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：Q包含与巯基偶联后的连接体，选自
  9. 根据权利要求7所述抗体药物偶联物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：L1为接头与药物氨基连接基团，其选自L2为任选取代的C3-C7亚烷基、C3-C8环烷基、任选取代的二甘醇至八甘醇酰基，AA为2至4个氨基酸组成的肽段，M为亚甲基、C1-C6烷基或环烷基取代亚甲基、三氟甲基取代亚甲基、C3-C6环烷基。
  10. 根据权利要求9所述抗体药物偶联物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：L1为接头与药物氨基连接基团，选自AA多肽残基选自：NH-Phe-Lys-C＝O、NH-Val-Cit-C＝O、NH-Val-Ala-C＝O、NH-Phe-Cit-C＝O、NH-Gly-Val-C＝O、NH-Ala-Lys-C＝O、NH-Ala-Ala-Ala-C＝O、NH-Glu-Val-Ala-C＝O、NH-Glu-Val-Cit-C＝O、NH-Gly-Gly-Phe-Gly-C＝O。
  11. 根据权利要求7所述抗体药物偶联物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：L1为接头与药物氨基连接基团，选自L2为任选取代的C3-C7亚烷基、C3-C8环烷基、任选取代的二甘醇至八甘醇酰基。
  12. 如权利要求7-11所述抗体药物偶联物，其特征在于：式B结构包括如下：



    其中：Ab为配体，n＝1-8。
  13. 根据权利要求12所述抗体药物偶联物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：所述Ab选自鼠源抗体、嵌合抗体、人源化抗体或全人源抗体。
  14. 根据权利要求13所述抗体药物偶联物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：所述抗体包含单克隆抗体。
  15. 根据权利要求14所述抗体药物偶联物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其特征在于：所述抗体包含双特异性抗体。
  16. 如权利要求15所述抗体药物偶联物，其特征在于：所述抗体能够与HER2、HER3、CD19、CD20、CD22、CD30、CD33、CD37、CD45、CD56、CD66e、CD70、CD74、CD79b、CD138、CD147、CD223、EpCAM、Mucin 1、STEAP1、GPNMB、FGF2、FOLR1、EGFR、EGFRvIII、Tissue factor、c-MET、FGFR、Nectin 4、AGS-16、Guanylyl cyclase C、Mesothelin、SLC44A4、PSMA、EphA2、AGS-5、GPC-3、c-KIT、ROR1、PD-L1、CD27L、5T4、Mucin 16、NaPi2b、STEAP、SLITRK6、ETBR、BCMA、Trop-2、CEACAM5、SC-16、SLC39A6、Delta-like protein3或Claudin 18.2肿瘤相关抗原结合的抗体。
  17. 一种药物组合物，其特征在于，包括：(a)如权利要求7-16任意一项所述抗体药物偶联物；和(b)药学上可接受的稀释剂，载剂或赋形剂。
  18. 如权利要求7-17任意一项所述抗体药物偶联物在制备治疗肿瘤药物中的用途。
  19. 如权利要求7-17任意一项所述抗体药物偶联物的制备方法，其特征在于，包括如下步骤：
    a.采用抗体与还原试剂在缓冲液中反应，得到经还原后的抗体；
    b.采用连接子-药物缀合物(A)与步骤a得到经还原后的抗体在缓冲液与有机溶剂混合液中进行交联，得到抗体药物偶联物。
PCT/CN2023/103058 2022-07-05 2023-06-28 特异性拓扑异构酶抑制剂和可用于抗体药物偶联物及其制备方法 WO2024007908A1 (zh)

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