WO2022056696A1 - 一种喜树碱类药物及其抗体偶联物 - Google Patents

一种喜树碱类药物及其抗体偶联物 Download PDF

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WO2022056696A1
WO2022056696A1 PCT/CN2020/115429 CN2020115429W WO2022056696A1 WO 2022056696 A1 WO2022056696 A1 WO 2022056696A1 CN 2020115429 W CN2020115429 W CN 2020115429W WO 2022056696 A1 WO2022056696 A1 WO 2022056696A1
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cancer
drug
antibody
aryl
compound
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PCT/CN2020/115429
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English (en)
French (fr)
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朱义
万维李
卓识
秦文芳
张勇
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四川百利药业有限责任公司
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Priority to BR112022002353A priority Critical patent/BR112022002353A2/pt
Priority to KR1020217042066A priority patent/KR20220038601A/ko
Priority to JP2021570456A priority patent/JP2022552757A/ja
Priority to CA3170019A priority patent/CA3170019A1/en
Priority to MX2022003397A priority patent/MX2022003397A/es
Priority to PCT/CN2020/115429 priority patent/WO2022056696A1/zh
Priority to AU2020442003A priority patent/AU2020442003B2/en
Priority to US17/601,055 priority patent/US20220378928A1/en
Application filed by 四川百利药业有限责任公司 filed Critical 四川百利药业有限责任公司
Priority to EP20928032.0A priority patent/EP3995496A4/en
Priority to TW110133499A priority patent/TW202216724A/zh
Priority to IL288215A priority patent/IL288215A/en
Publication of WO2022056696A1 publication Critical patent/WO2022056696A1/zh
Priority to AU2023285718A priority patent/AU2023285718A1/en
Priority to JP2024002606A priority patent/JP2024050606A/ja

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    • 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
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • 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
    • 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
    • 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
    • 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
    • A61K47/6835—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
    • A61K47/6849—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 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
    • A61K47/6835—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
    • A61K47/6851—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 the antibody targeting a determinant of a tumour cell
    • 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
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • 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

Definitions

  • the present invention relates to camptothecin drugs used as antitumor drugs and their antibody drug conjugates.
  • Antibody-drug conjugates as new 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 utilize the specific recognition of antigens by antibodies to transport drug molecules to the vicinity of target cells and effectively release drug molecules to achieve therapeutic purposes.
  • FDA U.S. Food and Drug Administration
  • Adecteis TM developed by Seattle Genetics for the treatment of Hodgkin lymphoma and relapsed anaplastic large cell lymphoma (ALCL), and its clinical application has been proven. safety and efficacy of these drugs.
  • Camptothecins as small molecule compounds with anti-tumor properties, are known to exhibit anti-tumor effects by inhibiting DNA topoisomerase I, including irinotecan, ixitecan, SN38, etc.
  • Many camptothecin drugs have been widely used in clinic, the main indications are bone cancer, prostate cancer, breast cancer, pancreatic cancer and so on.
  • ixitecan does not require activation by the use of enzymes.
  • SN-38 which is the main body of irinotecan, and topotecan used in the clinic, topoisomerase I inhibitory activity is stronger, and it has stronger damage to various cancer cells in vitro. cell activity.
  • ADC Antibody Drug Conjugates
  • the technical problem to be solved by this patent is to explore and discover better anti-tumor camptothecin compounds, improve the safety and effectiveness of anti-tumor small molecule compounds in ADC drug application, and obtain anti-tumor drugs with excellent curative effect.
  • the inventors Based on the comprehensive understanding of ADC drugs, the inventors have designed a series of anti-tumor camptothecin derivatives. Through experiments, it is found that the anti-tumor small molecule compounds show higher anti-tumor activity in cell experiments.
  • the present invention aims to provide a camptothecin derivative with more excellent antitumor effect and an antibody drug conjugate thereof, an antitumor camptothecin compound as shown in formula I or a pharmaceutically acceptable salt thereof :
  • R 1 and R 2 are independently selected from C 1 -C 3 alkyl or substituted alkyl, -H, -CF 3 , aryl or substituted aryl; or R 1 , R 2 together with the carbon atom to which they are attached Form cyclobutane, cyclopentane or cyclohexane; R 1 and R 2 are not hydrogen at the same time.
  • R 1 is hydrogen
  • R 2 is C 1 -C 3 alkyl, -CF 3 , aryl, heteroaryl, mono-fluoro substituted aryl or di-fluoro substituted aryl
  • R 1 and R 2 is a C 1 -C 3 alkyl group, -CF 3 , an aryl group, a heteroaryl group, a mono-fluorine-substituted aryl group or a di-fluorine-substituted aryl group
  • R 1 , R 2 together with the carbon atoms to which they are attached form a cyclobutane alkane, cyclopentane or cyclohexane:
  • R 1 is hydrogen
  • the carbon attached to R 2 has two configurations, R and S.
  • camptothecin compound or a pharmaceutically acceptable salt thereof includes the following structure:
  • the camptothecin compound or a pharmaceutically acceptable salt thereof is an antitumor drug, which is used for lung cancer, kidney cancer, urethral cancer, colon cancer, rectal cancer, prostate cancer, and glioblastoma multiforme , ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, lung cancer or esophageal cancer and other solid tumors and hematological tumors.
  • Another aspect of the present invention includes the antibody-drug conjugate shown in formula II, which exerts drug effect by releasing drug D after the conjugate reaches the target cell.
  • Ab is an antibody, antibody fragment or protein
  • L is an optional linking unit
  • D is selected from any of the above-mentioned camptothecin compounds or pharmaceutically acceptable salts thereof, which are linked to L through a hydroxyl group in the molecule.
  • n is selected from an integer of 1-20.
  • the linking unit L comprises a chemical bond selected from the group consisting of -O-, -N(R)n 1 -, -CH 2 -, -CH(R)n 1 -, amide, ester bond, -S-, -(PEG)n 2 -, the group consisting of; n 1 is selected from 1-3 integers, and n 2 is selected from 1-20 integers.
  • Another aspect of the invention includes a method of treating a patient in need thereof, comprising administering to the patient the antibody drug conjugate of any preceding claim, wherein the patient has a tumor, autologous Immune disease or infectious disease, and the antibody of the drug-ligand conjugate specifically binds to the target cell of the cancer, autoimmune disease.
  • the antibody conjugated drug or its salt is an antitumor drug or an anticancer drug, which is used for lung cancer, kidney cancer, urethral cancer, colon cancer, rectal cancer, prostate cancer, and glioma multiforme.
  • trade name includes the product formulation, generic drug and active pharmaceutical ingredient of the trade name product.
  • alkylene refers to a divalent straight chain saturated hydrocarbon group having from 1 to 20 carbon atoms, including groups from 1 to 10 carbon atoms.
  • alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2-CH2-), n-propylene, n-butylene, n-pentylene, and n-hexylene .
  • aryl refers to a polyunsaturated, generally aromatic, hydroxyl group which may be monocyclic or polycyclic (up to three rings) fused or covalently linked.
  • heteroaryl refers to an aryl group (or ring) containing 1-5 heteroatoms selected from N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternary Ammonized. Heteroaryl groups can be attached to the remainder of the molecule through a heteroatom.
  • Non-limiting examples of aryl groups include: phenyl, naphthyl, and diphenyl
  • non-limiting examples of heteroaryl groups include: pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, Triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnoline, phthalaziniyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzene Triazolyl, benzisoxazolyl, isobenzofuranyl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridyl, thienopyrimidinyl, pyridopyrimidinyl, imidazopyridine , benzothiaxolyl, benzofuranyl, benzothienyl, indolyl
  • R', R" and R"' each independently refer to hydrogen, unsubstituted C 1-8 alkanes group, unsubstituted aryl, aryl substituted by 1-3 halogens, unsubstituted C 1-8 alkyl, C 1-8 alkoxy or C 1-8 thioalkoxy, or unsubstituted aryl-C 1-4 alkyl.
  • R' and R" When R' and R" are attached to the same nitrogen atom, they can form a 3-, 4-, 5-, 6- or 7-membered ring together with the nitrogen atom.
  • -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.
  • a “derivative" of a compound refers to a substance that has a similar chemical structure to the compound but also contains at least one chemical group not present in the compound and/or lacks at least one chemical group present in the compound.
  • the compound to which the derivative is compared is referred to as the "parent” compound.
  • “derivatives” can be produced from the parent compound in one or more chemical reaction steps.
  • Ligand units are targeting agents that specifically bind to the target moiety.
  • the ligands are capable of specifically binding to cellular components or to cellular components or to other target molecules of interest.
  • the target moiety or target is usually on the cell surface.
  • the function of the Ligand unit is to deliver the Drug unit to a specific target cell population with which the Ligand unit interacts.
  • Ligands include, but are not limited to, proteins, polypeptides and peptides, as well as non-proteins such as sugars.
  • Suitable Ligand units include, for example, antibodies, such as full-length (intact) antibodies and antigen-binding fragments thereof.
  • the Ligand unit is a non-antibody targeting agent
  • it may be a peptide or polypeptide, or a non-proteinaceous molecule.
  • targeting agents include interferons, lymphokines, hormones, growth and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell binding molecule or substance.
  • the linker is covalently attached to the sulfur atom of the ligand.
  • the sulfur atom is that of a cysteine residue, which forms an interchain disulfide bond of the antibody.
  • the sulfur atom is one that has been introduced into a cysteine residue of the Ligand unit, which forms an interchain disulfide bond of the antibody.
  • the sulfur atom is one that has been introduced into a cysteine residue of the Ligand unit (eg, by site-directed mutagenesis or chemical reaction).
  • the sulfur atom to which the linker binds is selected from cysteine residues that form interchain disulfide bonds of the antibody or frontal cysteine residues that have been introduced into the Ligand unit (eg, by site-directed mutagenesis or chemical reaction).
  • the EU index in Kabat Kabat (Kabat EA et al., (1991) Sequences of proteins of Immunological Interest, Fifth Edition, NIH publication 91-3242) numbering system.
  • an “antibody” or “antibody unit” is within its scope to include any portion of an antibody structure. This unit may bind, reactively associate, or complex a receptor, antigen, or other receptor unit possessed by the targeted cell population.
  • An antibody can be any protein or protein-like molecule that can bind, complex, or otherwise react with a portion of the cell population to be treated or bioengineered.
  • the antibody constituting the antibody-drug conjugate preferably maintains the antigen-binding ability of the original wild state. Therefore, the antibody of the present invention can, preferably specifically, bind to the antigen.
  • Antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, regulators of cell survival, regulators of cell proliferation, molecules associated with tissue growth and differentiation (as known or predicted) functional), lymphokines, cytokines, molecules involved in the regulation of the cell cycle, molecules involved in angiogenesis, and molecules involved in angiogenesis (as known or predicted to be functional).
  • TAAs tumor-associated antigens
  • cell surface receptor proteins and other cell surface molecules regulators of cell survival, regulators of cell proliferation, molecules associated with tissue growth and differentiation (as known or predicted) functional
  • lymphokines cytokines
  • molecules involved in the regulation of the cell cycle molecules involved in angiogenesis, and molecules involved in angiogenesis (as known or predicted to be functional.
  • the tumor-associated factor can be
  • Antibodies for use in antibody drug conjugates include, but are not limited to, antibodies directed against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art and can be prepared by methods and information well known in the art for preparing antibodies.
  • tumor-associated antigens are well known in the art and can be prepared by methods and information well known in the art for preparing antibodies.
  • To develop effective cellular-level targets for cancer diagnosis and therapy researchers seek to find transmembrane or other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells, with little or no expression on the surface of one or more non-cancer cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. Identifying such tumor-associated factors could greatly improve the specific targeting properties of antibody-based cancer therapy.
  • Tumor-associated antigens include, but are not limited to, tumor-associated antigens (1)-(36) listed below. For convenience, antigen-related information that is well known in the industry is indicated below, including name, other names, and GenBank accession numbers. 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 isotypes that are at least 70%, 80%, 85%, 90%, or 95% homologous to sequences identified in references, or have The tumor-associated antigen sequences in the cited literature have completely identical biological properties and characteristics.
  • inhibitor refers to reducing by a detectable amount, or preventing 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 that results from targeting an individual's own tissues or proteins.
  • pharmaceutically acceptable salt refers to a pharmaceutically acceptable organic or inorganic salt of a compound (eg, drug, drug-linker or ligand-linker-drug conjugate).
  • the compounds may contain at least one amino or carboxyl group and thus may form addition salts with the corresponding acids or bases.
  • Exemplary salts include, but are not limited to: sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid Phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannin, pantothenate, bitartrate, ascorbate, salicylate, Formate, this formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, potassium salt, sodium salt, etc.
  • pharmaceutically acceptable salts have more than one point atom in the structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counter-examples. For example, a pharmaceutically acceptable salt has one or more charged atoms and/or one or more counter atoms.
  • linkers or “linkers of antibody drug conjugates” can be divided into two categories: non-cleavable linkers and cleavable linkers.
  • the drug release mechanism is: after the conjugate binds to the antigen and is endocytosed by the cell, the antibody is enzymatically hydrolyzed in the lysosome to release the small molecule drug, which is linked to the It is an active molecule composed of amino acid residues of antibodies.
  • the resulting changes in the molecular structure of the drug do not reduce its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into adjacent cells.
  • active drugs cannot kill adjacent tumor cells that do not express the target antigen (antigen-negative cells) (bystander effect) (Ducry et al., 2010, Bioconjugate Chem. 21:5-13).
  • Cleavable linkers can be cleaved within the target cell and release the active drug (the small molecule drug itself).
  • Cleavable linkers can be divided into two main categories: chemically labile linkers and enzymatically labile linkers.
  • Chemically labile linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties. Such properties include pH, glutathione concentration, etc.
  • pH-sensitive linkers are also commonly referred to as acid-cleavable linkers. Such linkers are relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be blocked in the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). hydrolysis. Most of the first-generation antibody-drug conjugates use such linkers, such as hydrazones, carbonates, acetals, and ketals. Antibody drug conjugates based on such linkers typically have short half-lives (2-3 days) due to the limited plasma stability of acid-cleavable linkers. This short half-life limits the application of pH-sensitive linkers in new-generation antibody-drug conjugates to a certain extent.
  • glutathione-sensitive linkers also known as disulfide linkers.
  • Drug release is based on the difference between high intracellular glutathione concentrations (in the millimolar range) and relatively low glutathione concentrations in the blood (in the micromolar range). This is especially true for tumor cells, whose low oxygen content results in enhanced reductase activity and thus higher glutathione concentrations.
  • Disulfide bonds are thermodynamically stable and therefore have good stability in plasma.
  • Enzyme-labile linkers can better control drug release.
  • Peptide linkers can be efficiently cleaved by lysosomal proteases, such as cathepsin (Cathepsin B) or plasmin (which are increased in some tumor tissues). This peptide linkage is believed to be very stable in the plasma circulation because proteases are generally inactive due to unfavorable 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 and the like.
  • Suicide linkers are typically chimeric between the cleavable linker and the active drug, or are themselves part of the cleavable linker.
  • the mechanism of action of the suicide linker is that when the cleavable linker is broken under suitable conditions, the suicide linker can spontaneously rearrange its structure, thereby releasing the active drug linked to it.
  • Common suicide linkers include p-aminobenzyl alcohols (PAB) and ⁇ -glucuronide ( ⁇ -Glucuronide).
  • Trifluorolactic acid (3.5g, 24.3mmol , 1.0eq) and K2CO3 (5.0g, 36.5mmol , 1.5eq) were dissolved in dry 35mL DMF, and benzyl bromide (5.0g, 29.2mmol, 1.2g) was added dropwise under an ice-water bath. eq), nitrogen protection, after adding, the reaction was raised to room temperature for 5 hours, TLC detected that the reaction was complete, quenched by adding water, extracted with dichloromethane (100 mL ⁇ 3), the organic phases were combined, washed with saturated brine successively, and dried over anhydrous sodium sulfate.
  • reaction solution of compound 18 was filtered into a 25mL single-neck flask, and SMCC (80mg, 0.24mmol, 2.0eq), DIEA (62mg, 0.48mmol, 4.0eq) were sequentially added under an ice-water bath, under nitrogen protection, after adding, the reaction was raised to room temperature for 1h , monitored by HPLC, purified by preparation, lyophilized to give compound 19 (66 mg, 78%), MS: [MH] 709.2.
  • Embodiment 24 Camptothecin drug cell activity test
  • camptothecin drugs were added to human tumors expressing A431, Fadu, Bxpc-3 (EGFR positive expressing cells) and U87-MG, SW620 (negative control cells), respectively In cell culture medium, cell viability was determined after 72 hours of cell culture.
  • Cell-based in vitro assays were used to determine cell viability, cytotoxicity, and apoptosis induced by the camptothecin drugs of the present invention.
  • CellTiter The AqueousOne Solution Cell Proliferation Assay is commercially available (Promega Corp., Madison, WI).
  • CellTiter AQueous One Solution Cell Proliferation Assay(a) is a colorimetric assay for the detection of viable cell numbers in cell proliferation and cytotoxicity assays.
  • This reagent contains a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and An electron coupling agent (phenazine ethosulfate; PES).
  • PES has enhanced chemical stability, which allows it to be mixed with MTS to form stable solutions. This convenient "single-solution" mode was introduced in the first generation CellTiter Improvements based on AQueous Assay, CellTiter The electron coupling agent PMS used in the AQueous Assay is provided separately from the MTS solution.
  • MTS Oleen's reagent
  • Figure 1 MTS (Owen's reagent) is reduced by cellular organisms to a colored formazan product, which can be directly dissolved in the culture medium ( Figure 1). This conversion is most likely accomplished by the action of NADPH or NADH produced by dehydrogenases in metabolically active cells.
  • NADPH NADPH
  • NADH NADH produced by dehydrogenases
  • the amount of formazan product detected at 490 nm is proportional to the number of viable cells in culture. Since the formazan product of MTS is soluble in tissue culture medium, CellTiter AQueous One Solution Assay has fewer steps than MTT or INT methods.
  • A431, Fadu, Bxpc-3 (EGFR positive expression cells) and U87-MG, SW620 (negative control cells) are used as research systems for in vitro drug efficacy detection. Plate in 96-well plates using the appropriate cell density, and 24 hours later, dosing with camptothecin drug. After 24 hours, dilute the camptothecin drug with the detection medium (1uM initial, 5-fold dilution, 9 concentrations, add the detection medium in the tenth column as a blank control), and add the diluted camptothecin drug to the corresponding The cells were then shaken with a microplate shaker (model: MX100-4A) for 3 min at a shaking speed of 550 rpm/min.
  • a microplate shaker model: MX100-4A
  • SN38 is a classic high-activity camptothecin drug and has been clinically proven in IMMU-132ADC.
  • camptothecin derivatives described in the present invention are comparable to or higher than SN38 in representative tumor cells Fadu, BXPC-3, A431, U87-MG and SW620. active.
  • the antibody molecule C with a monomer rate greater than 95% after the preliminary purification was exchanged into a phosphate buffer using an ultrafiltration centrifuge tube at a concentration of 10 mg/ml.
  • use an ultrafiltration centrifuge tube with a molecular weight cut-off of 30KDa to change the medium to PBS, and remove the uncoupled payload.
  • the ADC samples after the liquid change were filtered through a 0.22-micron sterile filter before use.
  • the coupling compounds 11, 20, 21, 23, and 24 were coupled with the antibody molecule C using the general coupling method described in Example 25.
  • Compound number The ADC number obtained by coupling 11 C-11 20 C-20 twenty one C-21 twenty three C-23 twenty four C-24
  • A431, Fadu, Bxpc-3 (antigen positive expressing cells) and SW620 (antigen negative control cells) are used as the research system for in vitro drug efficacy detection in the present invention. Plated in 96-well plates using the appropriate cell density and 24 hours later, ADC drug dosing was performed. After 24 hours, the ADC drug was diluted with detection medium (1uM initial, 5-fold dilution, 9 concentrations, the tenth column was added with detection medium as a blank control), and the diluted ADC drug was added to the corresponding cell wells and then Shake with a microplate shaker (model: MX100-4A) for 3 min at a shaking speed of 550 rpm/min.
  • the camptothecin drug of the present invention showed good antitumor activity in multiple antigen-positive tumor cell lines after coupling with the antibody through the linking unit L, and has great clinical Value.
  • an A431 tumor-bearing mouse model is established to evaluate the in vivo efficacy of the toxin ADC conjugated drug. That is, 3 ⁇ 10 6 A431 cells were subcutaneously injected into the right side of 4-6 weeks old BALB/c nude mice, and when the average tumor size of the mice grew to 140-150 mm3, they were randomly divided into groups, 5 in each group. On days 7, 14, and 21, blank control (blank buffer solution) and antibody-drug conjugate C-11 were administered respectively, and they were administered intravenously at a dose of 10 mg/kg.
  • the tumor volume measurement data is shown as the mean tumor volume ⁇ SE at the time of measurement, and the changes in the body weight of the mice were recorded to observe the preliminary toxicity of ADC drugs in vivo.
  • camptothecin drug of the present invention after coupling with the antibody through the linking unit L, showed a clear anti-tumor activity in tumor-bearing mice, and the average tumor size was significantly lower than the blank control.
  • the body weight of the mice did not change significantly during the administration period, and no mice died in the group.
  • the camptothecin drug of the present invention has good safety.

Abstract

一种喜树碱类药物及其抗体偶联物，发明人在对ADC类药物综合理解的基础上，设计出一系列抗肿瘤依喜替康活性衍生物，通过实验发现，新设计的抗肿瘤的分子化合物在实验中表现出很好的抗肿瘤活性。

Description

一种喜树碱类药物及其抗体偶联物 技术领域
本发明涉及作为抗肿瘤药用的喜树碱类药物及其抗体药物偶联物。
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Adecteis 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。
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化合物，提高对抗肿瘤的小分子化合物在ADC药物应用中的安全性、有效性，得到具有优异疗效的抗肿瘤药。
本发明人在对ADC类药物综合理解的基础上，设计出一系列抗肿瘤喜树碱活性衍生物，通过实验发现，抗肿瘤的小分子化合物在细胞实验中表现出更高的抗肿瘤活性。
发明内容
本发明旨在提供一种具有更优异抗肿瘤效果的喜树碱衍生物及其抗体药物偶联物，一种如式I所示的抗肿瘤喜树碱类化合物或其药学上可接受的盐：
Figure PCTCN2020115429-appb-000001
其中R 1及R 2分别独立的选自C 1-C 3烷基或取代烷基、-H、-CF 3、芳基或取代的芳基；或R 1、R 2连同其所连接碳原子构成环丁烷、环戊烷或环己烷；R 1、R 2不同时为氢。
作为优选方式，R 1为氢，R 2为C 1-C 3烷基、-CF 3、芳基、杂芳基、单氟取代的芳基或者双氟取代的芳基；或R 1及R 2为C 1-C 3烷基、-CF 3、芳基、杂芳基、单氟取代的芳基或者双氟取代的芳基；或R 1、R 2连同其所连接碳原子构成环丁烷、环戊烷或环己烷：
Figure PCTCN2020115429-appb-000002
在结构式(a)中，R 2独立地为-(CH 2)n 1-CH 3、-CF 3、芳基、杂芳基、单氟取代的芳基或者双氟取代的芳基，其中n 1＝0、1或2；
在结构式(b)中，R 1及R 2独立地为-(CH 2)n 1-CH 3、-CF 3、芳基、杂芳基、单氟取代的芳基或者双氟取代的芳基，其中n 1＝0、1或2；
在结构式(c)中，R 1、R 2连同其所连接碳原子构成环丁烷、环戊烷、环己烷,m＝1、2或3。
更加优选的，R 1为氢，R 2独立地为-(CH 2)n 1-CH 3、-CF 3、芳基、杂芳基、单氟取代的芳基或者双氟取代的芳基，其中n 1＝0、1或2：
Figure PCTCN2020115429-appb-000003
R 2所连接碳有R、S两种构型，
在结构式(a-1)中，R 2所连接碳为R构型，
在结构式(a-2)中，R 2所连接碳为S构型。
作为优选例，所述的喜树碱化合物或其药学上可接受的盐，包括以下结构：
Figure PCTCN2020115429-appb-000004
作为优选例，所述的喜树碱化合物或其药学上可接受的盐为抗肿瘤药，其用于肺癌、肾癌、尿道癌、结肠癌、直肠癌、前列腺癌、多形成胶质细胞瘤、卵巢癌、胰腺癌、乳腺癌、黑色素瘤、肝癌、膀胱癌、胃癌、肺癌或食道癌等实体瘤及血液肿瘤。
本发明的另一方面，包括如式II所示的抗体药物偶联物，该偶联物到达靶细胞后通过释放药物D发挥药效。
Figure PCTCN2020115429-appb-000005
其中Ab是抗体，抗体片段或蛋白；
L是任选的连接单元；
D选自上述述的任一喜树碱化合物或其药学上可接受的盐，其通过分子中的羟基与L相连。
m选自1-20的整数。
作为优选例，所述的抗体药物偶联物，连接单元L包含选自化学键-O-，-N(R)n 1-，-CH 2-，-CH(R)n 1-，酰胺，酯键，-S-，—(PEG)n 2—，组成的组；n 1选自1-3整数，n 2选自1-20整数。
本发明的另一方面，包括一种治疗有此需要的患者的方法，包括向所述患者给予前述权利要求中任一项所述的抗体药物偶联物，其中所述患者患有肿瘤、自身免疫疾病或感染性疾病，并且所述的药物-配体偶联物的抗体特异性结合至所述癌症、自身免疫疾病的靶细胞。
优选的，所述的抗体偶联药物或其盐，为抗肿瘤药或抗癌药，其用于肺癌、肾癌、尿道癌、结肠癌、直肠癌、前列腺癌、多形性胶质细胞瘤、卵巢癌、胰腺癌、乳腺癌、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌等实体瘤及血液肿瘤。
具体实施方式
缩写和定义
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。
术语“亚烷基”是指具有1-20个碳原子的二价直链饱和烃基团，包括从1至10碳原子的基团。亚烷基基团的实施例包括但不限于亚甲基(-CH2-),亚乙基(-CH2-CH2-),亚正丙基，亚正丁基，亚正戊基和亚正己基。除非另有说明，术语“芳基”指多不饱和、一般是芳族的羟基团，它可以是单环或者稠合或共价连接的多环(至多三个环)。术语“芳杂基”指含有1-5个选自N、O或S的杂原子的芳基(或环)，其中所述氮和硫原子任选被氧化，所述氮原子任选被季铵化。杂芳基团可通过杂原子连接于分子的其余部分。芳基基团的非限制性例子包括：苯基、萘基和二苯基，而杂芳基团的非限制性例子包括：吡啶基、哒嗪基、吡嗪基、嘧啶基(pyrimindinyl)、三嗪基、喹啉基、喹喔啉基、喹唑啉基、噌啉基、酞嗪基(phthalaziniyl)、苯并三嗪基、嘌呤基、苯并咪唑基、苯并吡唑基、苯并三唑基、苯并异唑基、异苯并呋喃基、异吲哚基、吲嗪基、苯并三嗪基、噻吩并吡啶基、噻吩并嘧啶基、吡啶并嘧啶基、咪唑并吡啶、苯并噻唑基(benzothiaxolyl)、苯并呋喃基、苯并噻吩基、吲哚基、喹啉基、异喹啉基、异噻唑基、吡唑基、吲唑基、蝶啶基、咪唑基、三唑基、四唑基、噁唑基、异噁唑基、噻二唑基、吡咯基、噻唑基、呋喃基、以及噻吩基等。当描述为“取代的”时，上述芳环和杂芳环系统的取代基选自下述可接受的取代基。
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。
本文中所用的化合物的“衍生物”是指具有与化合物相似的化学结构但还含有至少一个化合物中不存在的化学基团和/或缺少至少一个化合物中存在的化学基团的物质。衍生物所比较的化合物被称为“母体”化合物。通常，“衍生物”可在一个或多个化学反应步骤中由母体 化合物产生。
L-配体
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，最好专一性的与抗原结合。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)。与本发明中所述
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。
肿瘤相关抗原包括，但不局限于以下列出的肿瘤相关抗原(1)-(36)。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。
本文中所用的短语“药学上可接受的盐”指的是，化合物(例如，药物，药物-接头或配体-接头-药物偶联物)的药学上可接收到有机或无机盐。该化合物可含有至少一个氨基或羧基，并且因此可与相应的酸或碱形成加成盐。示例性的盐包括但不限于：硫酸盐、三氟乙酸盐、柠檬酸盐、乙酸盐、草酸盐、氯化物、溴化物、碘化物、硝酸盐、硫酸氢盐、磷酸盐、酸性磷酸盐、异烟酸盐、乳酸盐、水杨酸盐、酸性柠檬酸盐、酒石酸盐、油酸盐、单宁酸盐、泛酸盐、酒石酸氢盐、抗坏血酸盐、水杨酸盐、甲酸盐、本甲酸盐、谷氨酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐，钾盐、钠盐等。另外，药学上可接受的盐在结构中具有超过一个的带点原子。其中多个带电原子是药学上可接受的盐的一部分的示例能有多个抗衡例子。例如，药学上可接受的盐具有一个或多个带电原子和/或一个或多个抗衡原子。
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抗体药物偶联物，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)。
可断裂连接子，顾名思义，可以在目标细胞内断裂并释放出活性药物(小分子药物本身)。可断裂连接子可分为两个主要的类别：化学不稳定连接子和酶不稳定连接子。
化学不稳定连接子可以由于血浆和细胞质性质的不同而选择性的断裂。这样的性质包括pH值，谷胱甘肽浓度等。
对pH值敏感的连接子，通常又称为酸断裂连接子。这样的连接子在血液的中性环境下相对稳定(pH7.3-7.5)，但是在弱酸性的内涵体(pH5.0-6.5)和溶酶体(pH4.5-5.0)内将会被水解。第一代的抗体药物偶联物大多应用这类连接子，例如腙，碳酸酯，缩醛，缩酮类。由于酸断裂连接子有限的血浆稳定性，基于此类连接子的抗体药物偶联物通常具有较短的半衰期(2-3天)。这种较短的半衰期在一定程度上限制了pH敏感连接子在新一代抗体药物偶联物中的应用。
对于谷胱甘肽敏感的连接子，又称二硫键连接子。药物释放是基于细胞内谷胱甘肽的高浓度(毫摩尔范围)与血液中相对较低的谷胱甘肽浓度(微摩尔范围)差异引起的。对于肿瘤细胞而言尤其如此，其低含氧量导致还原酶的活性增强，因而导致更高的谷胱甘肽浓度。二硫键具有热力学稳定性，因此在血浆中具有较好的稳定性。
酶不稳定连接子，如肽连接子，能够更好的控制药物释放。肽连接子能够被溶酶体内蛋白酶，如组织蛋白酶(Cathepsin B)或纤溶酶(在一些肿瘤组织中此类酶含量增加)，有效的切断。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。典型的酶不稳定性连接子包括Val-Cit(vc)，Phe-Lys等。
自杀式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。自杀式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，自杀式连接子能够自发的进行结构重排，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。
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2的合成
Figure PCTCN2020115429-appb-000006
将化合物1(依喜替康甲磺酸盐，外购)(40mg,75.3mmol,1.0eq)和L-乳酸(10mg,113.0mmol,1.5eq)溶于干燥5mL DMF，再加入PyBop(58.8mg,113.0mmol,1.5eq)和DIEA(15.7uL,113.0mmol,1.5eq)。室温搅拌3小时后，TLC检测反应完全，加水淬灭，二氯甲烷萃取(10mL×3)，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩，残余物经柱色谱纯化，得化合物2(30.9mg，81.1％)。LC-MS：[M+H]+:508.2。1H NMR(400Mz,CDCl3/CD3OD):0.91-0.94(3H,m),1.32-1.39(3H,m),1.71-1.83(2H,m),2.31(3H,s),2.78-3.02(2H,m),3.16-3.26(2H,m),4.27-4.35(1H,m),4.81-4.92(1H,m),5.15-5.24(2H,m),5.49-5.76(2H,m),7.52(1H,d,J＝12.0Hz),7.58(1H,s),7.75(1H,d,J＝12.0Hz)。
实施例2化合物4的合成
Figure PCTCN2020115429-appb-000007
于500mL单口瓶中加入化合物3：N-芴甲氧羰基-甘氨酰-甘氨酸(10g,28.2mmol,1.0eq)，四乙酸铅(17.5g,55.3mmol,1.4eq)，200mL干燥四氢呋喃和67mL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4(8.7g，83.7％))。
实施例3化合物5的合成
Figure PCTCN2020115429-appb-000008
于25mL单口瓶中加入化合物3(500mg，1.4mmol，1.0eq),对甲苯磺酸一水合物(26mg，0.1mmol，0.1eq)及10mLTHF，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NaHCO3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化合物5(400mg，60.3％)。1H NMR(400Mz,CDCl3):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)。
实施例4化合物6的合成
Figure PCTCN2020115429-appb-000009
于25mL单口瓶中加入化合物5(400mg，0.8mmol，1.0eq)和10mL 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，反应结束后，浓缩，得化合物6粗品(550mg)，不经纯化，直接投下一步。
实施例5化合物7的合成
Figure PCTCN2020115429-appb-000010
于25mL单口瓶中加入Z-Gly-Gly-Phe-OH(372mg，0.9mmol，1.1eq)，PyBOP(852mg，1.6mmol，2.0eq)和3mL DMF，室温搅拌5分钟，再加入化合物6粗品(550mg)，室温反应，HPLC监测。反应完毕，加入水，乙酸乙酯萃取，无水硫酸钠干燥，过滤，浓缩，残余物经反相柱柱纯化得化合物7(326mg，59.2％)。
实施例6化合物8的合成
Figure PCTCN2020115429-appb-000011
于25mL单口瓶中加入化合物7(50mg，1.0eq，0.08mmol)，5％Pd/C(50mg)，3mLDMF，室温氢化反应。HPLC监控，反应结束后加水过滤，滤液浓缩得化合物8粗品(52mg)，不经纯化，直接投下一步。
实施例7化合物9的合成
Figure PCTCN2020115429-appb-000012
于25mL单口瓶中加入化合物8(52mg)，SMCC(23mg，0.07mmol，1.0eq)，DIEA(22.2mg，0.24mmol,2.5eq)及3mLDMF，室温反应，HPLC监控，制备纯化，冻干得化合物9(9.0mg，18.1％)。MS：[M-H]655.1。
实施例8化合物11的合成
Figure PCTCN2020115429-appb-000013
于25mL单口瓶中加入化合物9(9.0mg，0.014mmol，1.0eq)，依喜替康甲磺酸盐(6.6mg，0.014mmol，1.0eq)，PyBOP(14.3mg，0.028mmol,2.0eq)，DIEA(6.2mg，0.048mmol,3.5eq)及0.5mLDMF，室温反应，HPLC监控，制备纯化，冻干得化合物11(7.0mg，48.3％)。TOF：[M+Na]+1096.42。
实施例9化合物12、13的合成
Figure PCTCN2020115429-appb-000014
将化合物1(依喜替康甲磺酸盐)(40mg,75.3mmol,1.0eq)和三氟乳酸(16.3mg,113.0mmol,1.5eq)溶于干燥5mL DMF，再加入PyBop(58.8mg,113.0mmol,1.5eq)和DIEA(15.7uL,113.0mmol,1.5eq)。室温搅拌3小时后，TLC检测反应完全，加水淬灭，二氯甲烷萃取(10mL×3)，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩，残余物经柱色谱纯化， 得化合物12(13.5mg，32％)。LC-MS：[M+H]+:562.2。1H NMR(400Mz,CDCl3/CD3OD):0.91-0.95(3H,m),1.78-1.84(2H,m),2.34(3H,s),3.04-3.14(2H,m),3.27-3.32(2H,m),4.42-4.47(1H,m),5.08-5.20(3H,m),5.41-5.58(2H,m),7.23-7.25(1H,m)，7.52-7.55(1H,m)；化合物13(15.5mg，36.7％)。LC-MS：[M+H]+:562.2。1H NMR(400Mz,CDCl3/CD3OD):0.90-1.00(3H,m),1.74-1.89(2H,m),2.34(3H,s),3.01-3.09(2H,m),3.32-3.38(2H,m),4.65-4.71(1H,m),4.89-4.96(1H,m),5.17-5.30(2H,m),5.55-5.65(2H,m),7.53-7.61(2H,m)。
实施例10化合物14的合成
Figure PCTCN2020115429-appb-000015
将三氟乳酸(3.5g,24.3mmol,1.0eq)和K 2CO 3(5.0g,36.5mmol,1.5eq)溶于干燥35mLDMF，冰水浴下滴加溴化苄(5.0g,29.2mmol,1.2eq)，氮气保护，加完后升至室温反应5小时，TLC检测反应完全，加水淬灭，二氯甲烷萃取(100mL×3)，合并有机相，依次用饱和食盐水洗，无水硫酸钠干燥，过滤，滤液减压浓缩，残余物经柱色谱纯化，得化合物14(3.14g，55％)。1H NMR(400Mz,DMSO):4.91-4.94(1H,m),5.25(2H,s),7.17-7.19(1H,d),7.39(5H,s)；
实施例11化合物15的合成
Figure PCTCN2020115429-appb-000016
于50mL单口瓶中加入化合物4(1.45g，3.9mmol，1.0eq),化合物14(1.84g，7.8mmol，2.0eq),Zn(OAc) 2(1.44g，7.86mmol，2.0eq)及25mLTol，氮气保护，搅拌均匀后，升至100℃反应5.5h。TLC监控，产品点明显，过滤，滤液浓缩得黄色油状物(4.0g)，粗品经柱色谱纯化，得化合物15(0.99g，46％)。1H NMR(400Mz,CDCl3):3.68-3.83(2H,m),4.20-4.23(1H,m),4.49(2H,d,J＝8.0Hz),4.73-4.78(1H,m),4.89-5.00(2H,m),5.19(1H,s),5.25(2H,s),7.11(1H,s,),7.29-7.35(7H,m),7.43(2H,t,J＝8.0Hz),7.59(2H,d,J＝8.0Hz),7.79(2H,d,J＝8.0Hz)。
实施例12化合物16的合成
Figure PCTCN2020115429-appb-000017
于25mL单口瓶中加入化合物15(990mg，1.8mmol，1.0eq)和10mL DMF，搅拌均匀后，降至0℃，再缓慢加入DBU(335mg，2.2mmol，1.2eq)，氮气保护，加完后继续0℃反应30min。TLC监控，原料反应完，反应液直接下一步反应。
实施例13化合物17的合成
Figure PCTCN2020115429-appb-000018
于50mL单口瓶中加入Z-Gly-Gly-Phe-OH(909mg，2.2mmol，1.2eq)，PyBOP(1.4g，2.7mmol，1.5eq)和10mL DMF，冰水浴下滴加DIEA，氮气保护，继续反应10min，将化合物16反应液冰水浴下缓慢滴加至此反应液中，加完后升至室温反应1.5h，HPLC监测，反应完毕，制备纯化，冻干得化合物17(0.91g，71％)。
实施例14化合物18的合成
Figure PCTCN2020115429-appb-000019
于25mL单口瓶中加入化合物17(85mg，1.0eq，0.12mmol)，5％Pd/C(85mg)，6mLDMF，室温氢化反应1h。HPLC监控，原料反应完，反应液过滤，滤液直接投下一步反应。
实施例15化合物19的合成
Figure PCTCN2020115429-appb-000020
将化合物18反应液过滤至25mL单口瓶中，冰水浴下依次加入SMCC(80mg，0.24mmol，2.0eq)，DIEA(62mg，0.48mmol,4.0eq)，氮气保护，加完后升至室温反应1h，HPLC监控，制备纯化，冻干得化合物19(66m g，78％)，MS：[M-H]709.2。
实施例16化合物20、21的合成
Figure PCTCN2020115429-appb-000021
将化合物19(10mg,14umol,1.0eq)、化合物1(9mg,21umol,1.5eq)和PyBop(14.6mg,28mmol,2.0eq)溶于干燥DMF(0.5mL)，冰水浴下加入DIEA(5uL,28umol,2.0eq)，氮气保护，加后升至室温反应1h，HPLC监控，原料化合物19反应完，反应液直接HPLC纯水制备后分别得到化合物20(2.77mg，17.5％)，LC-MS：[M+H]+:1128.0；化合物21(3.92mg，24.8％)，LC-MS：[M+H]+:1128.0。
实施例17化合物22的合成
Figure PCTCN2020115429-appb-000022
将化合物18反应液(0.15mmol,1.0eq)过滤至25mL单口瓶中，冰水浴下依次加入MC (93mg，0.3mmol，2.0eq)，DIEA(78mg，0.6mmol,4.0eq)，氮气保护，加完后升至室温反应1h，HPLC监控，纯水制备纯化，冻干得化合物22(90m g，86％)，MS：[M-H]683.2。
实施例18化合物23、24的合成
Figure PCTCN2020115429-appb-000023
将化合物22(15mg,21.9umol,1.0eq)、化合物1(14.3mg,32.8umol,1.5eq)和PyBop(22.8mg,43.8mmol,2.0eq)溶于干燥DMF(0.8mL)，冰水浴下加入DIEA(7.3uL,43.8umol,2.0eq)，氮气保护，加后升至室温反应1h，HPLC监控，原料化合物22反应完，反应液直接HPLC纯水制备后分别得到化合物23(6.01mg，25％)，LC-MS：[M+H]+:1102.0；化合物24(5.57mg，23.2％)，LC-MS：[M+H]+:1102.0。
实施例18化合物25的合成
Figure PCTCN2020115429-appb-000024
于5mL单口瓶中加入扁桃酸(42mg，0.09mmol，1.1eq)，依喜替康(35mg，0.08mmol，1.0eq)，PyBOP(84mg，0.16mmol,2.0eq)，DIEA(36.4mg，0.28mmol,3.5eq)及1mLDMF，室温反应，HPLC监控，制备纯化，冻干得化合物25(15.0mg，32.6％)。1H NMR(CDCl 3，400Mz)δ7.70(d，1H，J＝8.0Hz)，7.64(s，1H)，5.64-5.75(m，2H)，5.48-5.38(m，1H)，5.29-5.21(m，1H)，5.19-5.11(m，1H)，3.37-3.11(m，2H)，2.55-2.38(m，4H)，2.32-2.15(m，2H)，2.08-1.99(m，1H)，1.94-1.85(m，4H)，1.33-1.24(m，4H)，1.05(t，3H，J＝7.2Hz)；LC-MS：[M+H]548.4。
实施例19化合物26的合成
Figure PCTCN2020115429-appb-000025
于5mL单口瓶中加入D-乳酸(11.2mg，0.08mmol，1.1eq)，依喜替康(30.0mg，0.07mmol，1.0eq)，PyBOP(119.5mg，0.14mmol,2.0eq)，DIEA(31.2mg，0.25mmol,3.5eq)及1mLDMF，室温反应，HPLC监控，制备纯化，冻干得化合物26(7.2mg，20.6％)。1H NMR(CDCl 3，400Mz)δ7.75(d，1H，J＝10.4Hz)，7.70(s，1H)，5.75-5.63(m，2H)，5.46-5.38(m，1H)，5.30-5.16(m，2H)，4.50-4.40(m，1H)，3.34-3.13(m，2H)，2.50-2.36(m，3H)，2.34-2.21(m，1H)，2.05-2.02(s，1H)，1.96-1.84(m，2H)，1.35-1.23(m，3H)，1.06(t，3H，J＝4.0Hz)；LC-MS：[M+H]508.3。
实施例20化合物27的合成
Figure PCTCN2020115429-appb-000026
于5mL单口瓶中加入2-甲基乳酸(10.5mg，0.10mmol，1.1eq)，依喜替康(40mg，0.09mmol，1.0eq)，PyBOP(95.6mg，0.18mmol,2.0eq)，DIEA(41.4mg，0.32mmol,3.5eq)及1mLDMF，室温反应，HPLC监控，制备纯化，冻干得化合物27(10.0mg，20.8％)。1H NMR(CDCl 3，400Mz)δ7.68(d，1H，J＝24Hz)，7.63(s，1H)，5.77-5.59(m，2H)，5.48-5.39(m，1H)，5.30-5.22(m，1H)，5.19-5.11(m，1H)，3.33-3.10(m，2H)，2.24(s，3H)，1.71-1.63(m，2H)，1.58-1.52(m，2H)，1.40-1.20(m，6H)，1.05(t，3H，J＝7.2Hz)；LC-MS：[M+H]522.2。
实施例4化合物28的合成
Figure PCTCN2020115429-appb-000027
于5mL单口瓶中加入R)-(-)-扁桃酸(15.2mg，0.10mmol，1.1eq)，依喜替康(40mg，0.09mmol，1.0eq)，PyBOP(95.6mg，0.18mmol,2.0eq)，DIEA(41.4mg，0.32mmol,3.5eq)及1mLDMF，室温反应，HPLC监控，制备纯化，冻干得化合物28(12.2mg，23.3％)。1H NMR(CDCl 3，400Mz)δ7.76(d，1H，J＝8.0Hz)，7.69(s，1H)，7.53-7.35(m，5H)，5.77-5.70(m，1H)，5.65-5.55(m，1H)，5.34-5.20(m，4H)，3.32-3.31(m，2H)，2.47-2.40(m，3H)，2.30-2.27(m，1H)，2.05-2.02(s，1H)，1.93-1.89(m，3H)，1.07(t，3H，J＝8.0Hz)；LC-MS：[M+H]570.2。
实施例21化合物29的合成
Figure PCTCN2020115429-appb-000028
于5mL单口瓶中加入3,5-二氟扁桃酸(23.7mg，0.13mmol，1.1eq)，依喜替康(50mg，0.11mmol，1.0eq)，PyBOP(119.5mg，0.23mmol,2.0eq)，DIEA(37.1mg，0.29mmol,2.5eq)及1mLDMF，室温反应，HPLC监控，制备纯化，冻干得化合物29(13.5mg，19.4％)。1H NMR(DMSO，400Mz)δ8.76(d，1H，J＝8.4Hz)，7.81(d，1H，J＝10.8Hz)，7.31(s，1H)，7.27-7.07(m，4H)，5.54-5.47(m，1H)，5.43(s，2H)，5.20-5.03(m，4H)，3.20-3.09(m，2H)，2.43-2.38(m，3H)，2.17-2.09(m，1H)，1.96-1.79(m，3H)，0.88(t，3H，J＝7.2Hz)；LC-MS：[M+H]606.2。
实施例22化合物30的合成
Figure PCTCN2020115429-appb-000029
于5mL单口瓶中加入3,5-二氟扁桃酸(22.5mg，0.13mmol，1.1eq)，依喜替康(50mg，0.11mmol，1.0eq)，PyBOP(119.5mg，0.23mmol,2.0eq)，DIEA(37.1mg，0.29mmol,2.5eq)及1mLDMF，室温反应，HPLC监控，制备纯化，冻干得化合物30(8.2mg，11.7％)。1H NMR(DMSO，400Mz)δ8.60(d，1H，J＝8.4Hz)，7.79(d，1H，J＝11.2Hz)，7.31(s，1H)，7.02-6.90(m，2H)，6.90-6.72(m，1H)，6.05-5.93(m，2H)，5.52-5.40(m，2H)，5.19-5. 09(m，1H)，5.09-4.92(m，2H)，2.98-2.85(m，2H)，2.22-2.14(m，3H)，1.94-1.83(m，1H)，1.75-1.59(m，1H)，1.53-1.45(m，2H)，0.66(t，3H，J＝7.2Hz)；LC-MS：[M+H]614.2。
实施例23化合物31的合成
Figure PCTCN2020115429-appb-000030
于5mL单口瓶中加入S-2-羟基丁酸(16.3mg，0.16mmol，1.1eq)，依喜替康(68.0mg，0.16mmol，1.0eq)，HATU(59.4mg，0.16mmol,1.0eq)，DIEA(50.5mg，0.39mmol,2.5eq)及1mLDMF，室温反应，HPLC监控，制备纯化，冻干得化合物31(16.3mg，20.1％)。1H NMR(DMSO，400Mz)δ8.36(d，1H，J＝8.8Hz)，7.79(d，1H，J＝11.2Hz)，7.31(s，1H)，6.53(s，1H)，5.60-5.52(m，1H)，5.46-5.40(m，3H)，5.24-5.17(m，2H)，3.23-3.09(m，2H)，2.45-2.38(m，3H)，2.28-2.08(m，2H)，1.94-1.80(m，2H)，1.79-1.66(m，1H)，1.66-1.55(m，1H)，1.05-0.84(m，6H)；LC-MS：[M+H]522.3。
实施例24喜树碱药物细胞活性测试
通过下列实验过程测定喜树碱药物细胞毒性活性：将喜树碱药物分别加入到A431、Fadu、Bxpc-3(EGFR阳性表达细胞)和U87-MG、SW620(阴性对照细胞)表达的人的肿瘤细胞培养基中，细胞培养72小时后测定细胞存活率。基于细胞的体外实验用于测定细胞存活率、细胞毒性和本发明喜树碱药物诱导的细胞程序性死亡。
通过细胞增殖试验测定喜树碱药物的体外药效。CellTiter
Figure PCTCN2020115429-appb-000031
AqueousOne Solution Cell Proliferation Assay为商购的(Promega Corp.，Madison,WI)。CellTiter
Figure PCTCN2020115429-appb-000032
AQueous One Solution Cell Proliferation Assay(a)是一种用比色法来检测细胞增殖和细胞毒性实验中的活细胞数量的检测试剂。此试剂含有一个新型的四唑化合物[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt；MTS]和一种电子偶联剂(phenazine ethosulfate；PES)。PES具有增强的化学稳定性，这使它可与MTS混合形成稳定的溶液。这种方便的“单溶液”模式，是在第一代CellTiter
Figure PCTCN2020115429-appb-000033
AQueous Assay的基础上的改进，CellTiter
Figure PCTCN2020115429-appb-000034
AQueous Assay中使用的电子偶联剂PMS与MTS溶液是分开提供的。MTS(Owen’s reagent)被细胞生物还原成为一种有色的甲臜产物，可直接溶解于培养基中(图1)。这 种转化很可能是在代谢活跃的细胞中的脱氢酶产生的NADPH或NADH的作用下完成的。检测时，只需将少量的CellTiter
Figure PCTCN2020115429-appb-000035
AQueous One Solution Reagent直接加入培养板孔的培养基中，孵育1–4小时，然后以酶标仪读取490nm的吸光度值。
Figure PCTCN2020115429-appb-000036
在490nm处检测到的甲臜产物的量与培养中的活细胞数成正比。由于MTS的甲臜产物在组织培养基中可溶，CellTiter
Figure PCTCN2020115429-appb-000037
AQueous One Solution Assay与MTT或INT法相比操作步骤更少。
本发明中采用A431、Fadu、Bxpc-3(EGFR阳性表达细胞)和U87-MG、SW620(阴性对照细胞)作为体外药效检测的研究体系。在96孔板中，使用适当的细胞密度进行铺板，24小时后，进行喜树碱药物加药。24小时后用检测培养基稀释喜树碱药物(1uM起始，5倍稀释，9个浓度，第十列添加检测培养基用来作为空白对照)，将稀释好的喜树碱药物加入对应的细胞孔中然后用微孔板震荡器(型号：MX100-4A)震荡3min，震速550rpm/min,震荡完放入二氧化碳培养箱孵育3天。3天后每孔加入20ul MTS(Promega，G3581)反应2小时，酶标仪(Molecular Device，型号：SpectraMAX190)490nM读数。通过检测线粒体内的脱氢酶的活性，评价喜树碱药物对细胞的增殖抑制作用。
Figure PCTCN2020115429-appb-000038
Figure PCTCN2020115429-appb-000039
SN38为经典的高活性喜树碱药物，并已经在IMMU-132ADC中获得临床证明。发明人通过细胞活性实验，证明本发明所述的喜树碱衍生物在代表性的肿瘤细胞Fadu、BXPC-3、A431、U87-MG、SW620中均表现出了与SN38相当或更高的细胞活性。
实施例25偶联制备ADC通法
将通过初步的纯化后单体率大于95％的抗体分子C，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。将偶联用化合物11、20、21、23、24采用本实施例25所述偶联通法，与抗体分子C偶联。
化合物编号 偶联所得ADC编号
11 C-11
20 C-20
21 C-21
23 C-23
24 C-24
实施例26 ADC抗肿瘤细胞活性测试
与喜树碱药物细胞活性测试方法相似，本发明中采用A431、Fadu、Bxpc-3(抗原阳性表达细胞)、SW620(抗原阴性对照细胞)作为体外药效检测的研究体系。在96孔板中，使用适当的细胞密度进行铺板，24小时后，进行ADC药物加药。24小时后用检测培养基稀释ADC药物(1uM起始，5倍稀释，9个浓度，第十列添加检测培养基用来作为空白对照)，将稀释好的ADC药物加入对应的细胞孔中然后用微孔板震荡器(型号：MX100-4A)震荡3min，震速550rpm/min,震荡完放入二氧化碳培养箱孵育3天。3天后每孔加入20ul MTS(Promega，G3581)反应2小时，酶标仪(Molecular Device，型号：SpectraMAX190)490nM读数。通过检测线粒体内的脱氢酶的活性，评价ADC药物对细胞的增殖抑制作用。
Figure PCTCN2020115429-appb-000040
Figure PCTCN2020115429-appb-000041
经过以上ADC细胞活性测试，本发明所述的喜树碱药物在通过连接单元L与抗体偶联后，在多个抗原阳性肿瘤细胞系中均表现出良好的抗肿瘤活性，具有极大的临床应用价值。
实施例27 ADC体内药效测试
本发明中建立了A431荷瘤小鼠模型，以评价毒素ADC偶联药物的体内药效。即以3×10 6A431细胞通过皮下注射到4～6周鼠龄的BALB/c裸鼠右侧，待小鼠肿瘤平均大小生长至140～150mm3，随机分组，每组5只，在第0,7,14,21天分别给与空白对照(缓冲溶液空白)、抗体药物偶联物C-11，均以10mg/kg剂量进行静脉给药。肿瘤体积测量数据显示为测量时肿瘤平均体积±SE，同时记录小鼠体重变化情况，用以观察ADC药物的体内初步毒性。
Figure PCTCN2020115429-appb-000042
Figure PCTCN2020115429-appb-000043
经过以上ADC小鼠体内药效实验，证明本发明所述的喜树碱药物在通过连接单元L与抗体偶联后，在荷瘤小鼠中表现出了明确的抗肿瘤活性，平均瘤体明显低于空白对照。小鼠体重在给药期间无明显变化，组内无小鼠死亡，本发明所述的喜树碱药物具有良好的安全性。

Claims (10)

  1. 一种如式I所示的喜树碱类化合物或其药学上可接受的盐：
    Figure PCTCN2020115429-appb-100001
    其中R 1及R 2分别独立的选自C 1-C 3烷基或取代烷基、-H、-CF 3、芳基、取代的芳基或杂芳基；或R 1、R 2连同其所连接碳原子构成环丁烷、环戊烷或环己烷；R 1、R 2不同时为氢。
  2. 如权利要求1所述的喜树碱类化合物或其药学上可接受的盐，其特征在于：R 1为氢，R 2为C 1-C 3烷基、-CF 3、芳基、取代的芳基或杂芳基；或R 1及R 2为C 1-C 3烷基、-CF 3、芳基、杂芳基或取代的芳基；或R 1、R 2连同其所连接碳原子构成环丁烷、环戊烷或环己烷：
    Figure PCTCN2020115429-appb-100002
    在结构式(a)中，R 2独立地为-(CH 2)n 1-CH 3、-CF 3、芳基、杂芳基、取代的芳基，其中n 1＝0、1或2；
    在结构式(b)中，R 1及R 2独立地为-(CH 2)n 1-CH 3、-CF 3、芳基、杂芳基、取代的芳基，其中n 1＝0、1或2；
    在结构式(c)中，R 1、R 2连同其所连接碳原子构成环丁烷、环戊烷、环己烷,n 2＝1、2或3。
  3. 如权利要求2所述的喜树碱类化合物或其药学上可接受的盐，其特征在于：R 1为氢，R 2独立地为-(CH 2)n 1-CH 3、-CF 3、芳基、杂芳基或取代的芳基，其中n 1＝0、1或2：
    Figure PCTCN2020115429-appb-100003
    R 2所连接碳有R、S两种构型，
    在结构式(a-1)中，R 2所连接碳为R构型，
    在结构式(a-2)中，R 2所连接碳为S构型。
  4. 如权利要求1-3任一所述的喜树碱类化合物或其药学上可接受的盐，其特征在于：芳基取代基选自卤素、烃基、烷氧基、羟基、硝基、氨基、羟基或氰基。
  5. 如权利要求1所述的喜树碱类化合物或其药学上可接受的盐，其特征在于：，选自以下结构：
    Figure PCTCN2020115429-appb-100004
  6. 包括权利要求1～5任一所述的喜树碱类化合物或其药学上可接受的盐的抗肿瘤药，其特征在于：用于肺癌、肾癌、尿道癌、结肠癌、直肠癌、前列腺癌、多形成胶质细胞瘤、卵巢癌、胰腺癌、乳腺癌、黑色素瘤、肝癌、膀胱癌、胃癌、肺癌或食道癌等实体瘤或血液肿瘤。
  7. 一种如式II所示的抗体药物偶联物，该偶联物到达靶细胞后通过释放药物D发挥药效：
    Figure PCTCN2020115429-appb-100005
    其中Ab是抗体，抗体片段或蛋白；
    L是任选的连接单元，一端与Ab连接，一端与药物D连接；
    D选自权利要求1～6任一所述的喜树碱类化合物或其药学上可接受的盐，其通过分子中的羟基与L相连；
    m选自1-20整数；
  8. 如权利要求7所述的抗体药物偶联物，其特征在于：连接单元L结构中包含选自化学键-O-，-N(R)n 1-，-CH 2-，-CH(R)n 1-，酰胺，酯键，-S-，—(PEG)n 2—，组成的组；n 1选自1-3整数，n 2选自1-20整数。
  9. 如权利要求7或8中所述的抗体药物偶联物，其特征在于：所述的药物-配体偶联物的抗体特异性结合至癌症、自身免疫疾病的靶细胞。
  10. 包括权利要求7～9中任一所述的抗体偶联药物的药物，其特征在于：用于肺癌、肾癌、尿道癌、结肠癌、直肠癌、前列腺癌、多形性胶质细胞瘤、卵巢癌、胰腺癌、乳腺癌、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌等实体瘤或血液肿瘤。
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