WO2021052402A1 - 一种喜树碱衍生物及其偶联物 - Google Patents

一种喜树碱衍生物及其偶联物 Download PDF

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WO2021052402A1
WO2021052402A1 PCT/CN2020/115807 CN2020115807W WO2021052402A1 WO 2021052402 A1 WO2021052402 A1 WO 2021052402A1 CN 2020115807 W CN2020115807 W CN 2020115807W WO 2021052402 A1 WO2021052402 A1 WO 2021052402A1
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group
antibody
alkyl
cycloalkyl
compound
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PCT/CN2020/115807
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English (en)
French (fr)
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朱义
万维李
卓识
张伟
张宜英
胥涛
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四川百利药业有限责任公司
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Priority to EP20864446.8A priority Critical patent/EP4032892A4/en
Priority to JP2022517395A priority patent/JP7467610B2/ja
Priority to BR112022004913A priority patent/BR112022004913A2/pt
Priority to US17/761,417 priority patent/US20220411436A1/en
Priority to AU2020347715A priority patent/AU2020347715A1/en
Publication of WO2021052402A1 publication Critical patent/WO2021052402A1/zh
Priority to IL291337A priority patent/IL291337A/en

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    • 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
    • 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
    • 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
    • A61K47/6851—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
    • A61K47/6855—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 the tumour determinant being from breast cancer 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
    • 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

Definitions

  • the present invention relates to camptothecin derivatives and their antibody-drug conjugates used as anti-tumor drugs. Specifically, the present invention relates to obtaining a better camptothecin anti-tumor drug through a series of molecular structure modification, so as to be more suitable as a drug for antibody coupling.
  • Antibody-drug conjugate as a new type of targeted drug, generally consists of three parts: antibody or antibody-like ligand, small molecule drug base, and linker that couples the ligand to the drug.
  • the antibody-drug conjugate uses the specific recognition of the antibody to the antigen, transports the drug molecule to the vicinity of the target cell and effectively releases the drug molecule to achieve the purpose of treatment.
  • antibody-drug conjugates can accurately bind to tumor cells and reduce the impact on normal cells [Mullard A, (2013) Nature Reviews Drug Discovery, 12: 329-332; DiJoseph JF, Armellino DC , (2004) Blood, 103: 1807-1814].
  • Kadcyla ado-trastezumab emtansine, T-DM1
  • T-DM1 trastuzumab
  • Herceptin Herceptin
  • paclitaxel resistant advanced or metastatic breast cancer patients WO2005037992; US8088387
  • ADC commonly used toxins include MMAE, T-DM1, PBD and other highly toxic toxins. Such toxins are highly toxic, and even after the formation of ADC drugs, the therapeutic window is narrow.
  • Camptothecin derivatives that have anti-tumor effects by inhibiting topoisomerase I are a class of small molecules used in antibody-drug conjugates.
  • Isinotecan was developed by Daiichi Sankyo. The drug was used as a single chemotherapeutic agent to advance to phase III clinical. The main indications are bone cancer, prostate cancer, breast cancer, pancreatic cancer, etc., because of side effects. The treatment window is large and the treatment window is narrow. In the end, the direct administration of exenotecan failed to be marketed.
  • Daiichi Sankyo has developed exenotecan as an ADC toxin and has been successfully marketed.
  • the reduction in the number of conjugated drugs will reduce the therapeutic index of ADC drugs.
  • the technical problem to be solved by the present invention is to explore and find better anti-tumor camptothecin derivatives, reduce the toxicity of the compound, improve its safety and effectiveness in the application of ADC drugs, and obtain an anti-tumor ADC with excellent curative effect. drug.
  • the present invention designs and synthesizes a series of camptothecin derivatives and antibody conjugates with significant anti-tumor activity, and the resulting camptothecin derivatives The toxicity of the base derivatives is significantly reduced, and finally the anti-tumor activity in vivo is tested to prove its good anti-tumor activity.
  • the present invention aims to provide a kind of camptothecin derivatives and their conjugates or tautomers, mesosomes, racemates, enantiomers, non- Enantiomers or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof, wherein the antibody-drug conjugate comprises an anti-tumor camptothecin compound represented by formula D.
  • R is selected from deuterium atom, C 1-6 alkyl, deuterated alkyl, substituted alkyl, aryl, substituted aryl or heteroaryl, preferably C 1-6 alkyl or substituted alkyl;
  • R 1 is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a haloalkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a carboxyl group, a carboxyl group, a heterocyclic group, an aryl group, Substituted aryl or heteroaryl;
  • R 2 is selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, carboxyl, carboxyl, heterocyclic, aryl, Substituted aryl or heteroaryl;
  • X is selected from -C(O)-CR a R b -O-(CR 3 R 4 ) n -, -C(O)-CR a R b -NH-(CR 3 R 4 ) n -or -C( O)-CR a R b -(CR 3 R 4 ) n -S-;
  • R a is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, an aryl group, a substituted aryl group or Heteroaryl
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl
  • R a , R b together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • R 3 and R 4 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group , Cycloalkyl or heterocyclic group;
  • R 3 and R 4 together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • n is selected from an integer from 0 to 4.
  • camptothecin derivatives or tautomers mesoisomers, racemates, enantiomers, diastereomers or mixtures thereof are provided , Or a pharmaceutically acceptable salt or solvate, including a compound having the general formula D 1 or a pharmaceutically acceptable salt or solvate thereof;
  • R is selected from deuterium atom, C 1-6 alkyl or substituted alkyl, aryl, substituted aryl or heteroaryl;
  • X is selected from -C(O)-CR a R b -(CR 3 R 4 ) n -O-, -C(O)-CR a R b -(CR 3 R 4 ) n -NH- or -C( O)-CR a R b -(CR 3 R 4 ) n -S-;
  • R a is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, an aryl group, a substituted aryl group or Heteroaryl
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl
  • R a , R b together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • R 3 and R 4 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a hydroxyl group, an amino group, a cyano group, a nitro group, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, and a hydroxyalkyl group. , Cycloalkyl or heterocyclic group;
  • R 3 and R 4 together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • n is selected from an integer from 0 to 4.
  • the wavy line in formula D 1 represents a hydrogen atom, or is covalently connected to a linker unit or an antibody that binds to an antigen expressed by a tumor target cell.
  • the structural unit -X- is -C(O)-CR a R b -(CR 3 R 4 ) n -O- or the structural unit -X- is -C(O)-CR a R b -(CH 2 ) n -O-;
  • R a is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, an aryl group, a substituted aryl group or Heteroaryl
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl
  • R a , R b together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • R 3 and R 4 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group , Cycloalkyl or heterocyclic group;
  • R 3 and R 4 together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • n 0 or 1.
  • a camptothecin derivative or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the O end of the structural unit -X- is connected to the linking unit L, and the structural unit -X- is selected from , But not limited to:
  • One aspect of the present invention provides a compound represented by the general formula (D 2 ) or its tautomer, meso, racemate, enantiomer, diastereomer Or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof,
  • R is selected from deuterium atom, C 1-6 alkyl or substituted alkyl, aryl, substituted aryl or heteroaryl, preferably C 1-6 alkyl or substituted alkyl;
  • R a is selected from cycloalkyl or cycloalkylalkyl, preferably C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl;
  • R b is selected from a hydrogen atom, a deuterium atom, an alkyl group, a haloalkyl group, a cycloalkyl group or a cycloalkylalkyl group, preferably a hydrogen atom or a deuterium atom;
  • R a , R b together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • n 0 or 1
  • the wavy line in formula D 2 represents a hydrogen atom, or is covalently connected to a linker unit or an antibody that binds to an antigen expressed by a target cell.
  • a preferred solution in one aspect of the present invention provides a compound represented by general formula (D) or its tautomer, meso, racemate, enantiomer, diastereomer Or its mixture form, or its pharmaceutically acceptable salt or solvate, including the compound represented by the general formula (D 3 ) or its tautomer, meso, racemate, enantiomer Forms, diastereomers or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof:
  • R is selected from deuterium atom, C 1-6 alkyl or substituted alkyl, aryl, substituted aryl or heteroaryl;
  • R a is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, an aryl group, a substituted aryl group or Heteroaryl; preferably C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl;
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl; preferably C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl;
  • R a , R b together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • n 0 or 1
  • the compounds shown in the general formula (D) shown in the present invention include but are not limited to:
  • Camptothecin derivatives of the compound are used in the preparation of lung cancer, kidney cancer, urethral cancer, colon cancer, rectal cancer, prostate cancer, multiform glioblastoma, ovarian cancer, pancreatic cancer, and breast cancer without limitation. Cancer, melanoma, liver cancer, bladder cancer, stomach cancer, lung cancer or esophageal cancer and other solid tumors or blood tumor drugs.
  • the antibody-drug conjugate or pharmaceutically acceptable salt or solvent compound thereof according to the present invention, wherein the linker unit -L- is -L 1 -L 2- L 3 -L 4 -, the L 1 end is connected to the antibody, and the L 4 end is connected to X;
  • L 1 is selected from, but not limited to: -YC(O)-, -CH 2 -C(O)-NR 5 -YC(O)- or -C(O)-YC(O)-, wherein Y is non-limitingly selected from a C 1-8 alkyl group, a C 1-8 alkyl-cycloalkyl group, or a linear or linear-cyclic heteroalkyl group of 1-8 atoms, and the heteroalkyl group includes the group consisting of: From 1-3 atoms of N, O or S, the C 1-8 alkyl, cycloalkyl, linear or linear cyclic heteroalkyl are each independently selected from deuterated atoms, halogens, and hydroxyl groups. , Cyano, nitro, amino, alkyl, heteroalkyl, substituted alkyl, carboxy, alkoxy or cycloalkyl substituted by one or more substituents;
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof wherein the linking unit L 1 is selected from, but not limited to: -(succinimid-3-yl -N-)-(CH 2 ) o -C(O)-, -(succinimide-3-yl-N)-CH 2 -cyclohexyl-C(O)-, -(succinimide- 3-base-N-)-(CH 2 CH 2 O) o -C(O)-, -CH 2 -C(O)-NR 5 -(CH 2 ) o -C(O)- or -C( O)-(CH 2 ) o -C(O)-, wherein o is selected from an integer of 2-8, preferably 5;
  • L 2 is selected from -NR 6 (CH 2 CH 2 O) p CH 2 CH 2 C(O)-, -NR 6 (CH 2 CH 2 O) p CH 2 C(O)-, -S(CH 2 ) p C(O)- or chemical bond, where p is selected from an integer of 0-20;
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the linking unit L 2 is selected from -NR 6 (CH 2 CH 2 O) p CH 2 C( O)-or a chemical bond, where p is selected from an integer of 0-12.
  • L 3 is non-limitingly selected from peptide residues composed of 2-7 amino acids, wherein the optional amino acid is further selected from deuterium atom, halogen, hydroxyl, cyano, amino, nitro, alkyl, substituted alkyl, alkane One or more substituents in oxy and cycloalkyl or substituted cycloalkyl;
  • the antibody-drug conjugate or the pharmaceutically acceptable salt or solvent compound thereof is provided, wherein the peptide residue of L 3 is selected from one, two or more Amino acid (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E) or aspartic acid (D) Polypeptide residues formed by amino acids.
  • they are amino acid residues formed by one, two or more amino acids selected from phenylalanine and glycine; more preferably are tetrapeptide residues; most preferably are GGFG (glycine-glycine-phenylalanine- Glycine) composed of tetrapeptide residues.
  • L 4 is selected from -NR 7 (CR 8 R 9 ) q -, -C(O)NR 7 , -C(O)NR 7 (CH 2 ) q -or a chemical bond, wherein q is selected from an integer of 0-6, Preferably it is -NR 7 (CR 8 R 9 ) q -;
  • R 5 , R 6 and R 7 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a halogenated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, and an alkoxyalkyl group.
  • R 8 and R 9 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a halogenated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a hetero Cyclic, aryl, substituted aryl or heteroaryl.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided, wherein L 4 is selected from -NR 7 (CR 8 R 9 ) q -, and R 7 is selected from A hydrogen atom, a deuterium atom or an alkyl group, R 8 and R 9 are the same or different, and each independently has a hydrogen atom, a deuterium atom or an alkyl group, and q is 1 or 2; L 4 is preferably -NR 7 CR 8 R 9 -; L 4 is more preferably -NHCH 2 -.
  • a preferred solution of one aspect of the present invention provides a drug-linker compound represented by the general formula (LXD 2 ) or its tautomer, meso, racemate, enantiomer, non- The enantiomer or its mixture form, or its pharmaceutically acceptable salt or solvate, which is a compound represented by the general formula (La-XD 2 ) or its pharmaceutically acceptable salt or solvate:
  • Z is selected from, but not limited to -YC(O)-, -CH 2 -C(O)-NR 5 -YC(O)- or -C(O)-YC(O)-, wherein Y is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl, or linear or linear-cyclic heteroalkyl of 1-8 atoms, the heteroalkyl includes a group selected from N, O or S 1-3 atoms, the C 1-8 alkyl, cycloalkyl, linear or linear cyclic heteroalkyl are each independently selected from deuterated atoms, halogen, hydroxyl, carboxyl, cyano, nitro Substituted by one or more substituents of a group, an amino group, an alkyl group, a heteroalkyl group, a substituted alkyl group, a carboxy group, an alkoxy group or a cycloalkyl group;
  • L 2 is selected from -NR 6 (CH 2 CH 2 O) p CH 2 CH 2 C(O)-, -NR 6 (CH 2 CH 2 O) p CH 2 C(O)-, -S(CH 2 ) p C(O)- or chemical bond, where p is preferably an integer of 0-20, preferably a chemical bond;
  • L 3 is selected from a peptide residue consisting of 2-7 amino acids, wherein the optional amino acid is further selected from deuterium atom, halogen, hydroxyl, cyano, amino, nitro, alkyl, substituted alkyl, alkoxy and cyclic Alkyl group or substituted cycloalkyl group is substituted by one or more substituents;
  • R is selected from deuterium atom, C 1-6 alkyl or substituted alkyl, aryl, substituted aryl or heteroaryl;
  • R a is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, an aryl group, a substituted aryl group or Heteroaryl
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl
  • R a , R b together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • R 5 , R 6 and R 7 are the same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a halogenated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, and an alkoxy group Alkyl, heterocyclyl, aryl, substituted aryl or heteroaryl;
  • R 8 and R 9 are the same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a halogenated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, Heterocyclic group, aryl group, substituted aryl group or heteroaryl group.
  • n is selected from an integer of 0 to 4, preferably 0 or 1, more preferably 0.
  • a preferred solution of one aspect of the present invention provides a drug-linker compound represented by the general formula (LXD 2 ) or its tautomer, meso, racemate, enantiomer, non- Enantiomers or their mixtures, or their pharmaceutically acceptable salts or solvates, which are compounds represented by the general formula (Lb-XD 2 ) or their pharmaceutically acceptable salts or solvates:
  • Z is selected from, but not limited to -YC(O)-, -CH 2 -C(O)-NR 5 -YC(O)- or -C(O)-YC(O)-, wherein Y is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl, or linear or linear-cyclic heteroalkyl of 1-8 atoms, the heteroalkyl includes a group selected from N, O or S 1-3 atoms, the C 1-8 alkyl, cycloalkyl, linear or linear cyclic heteroalkyl are each independently selected from deuterated atoms, halogen, hydroxyl, carboxyl, cyano, nitro Substituted by one or more substituents of a group, an amino group, an alkyl group, a heteroalkyl group, a substituted alkyl group, a carboxy group, an alkoxy group or a cycloalkyl group;
  • R is selected from deuterium atom, C 1-6 alkyl or substituted alkyl, aryl, substituted aryl or heteroaryl;
  • R a is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, an aryl group, a substituted aryl group or Heteroaryl
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl
  • R a , R b together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • R 7 is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a haloalkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, a substituted aryl group or Heteroaryl
  • R 8 and R 9 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a halogenated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic ring Group, aryl, substituted aryl, or heteroaryl.
  • n is selected from an integer of 0 to 4, preferably 0 or 1, more preferably 0.
  • the antibody-drug conjugates or tautomers, mesoisomers, racemates, enantiomers, diastereomers or mixtures thereof are provided , Or a pharmaceutically acceptable salt or solvate thereof, which is an antibody-drug conjugate represented by the general formula (LX-Dr) or a pharmaceutically acceptable salt or solvate thereof, which is selected from, but not limited to in:
  • the antibody-drug conjugates or tautomers, mesoisomers, racemates, enantiomers, diastereomers or mixtures thereof are provided , Or its pharmaceutically acceptable salt or solvate, which is the antibody-drug conjugate represented by the general formula (Ab-LXD r ) or its pharmaceutically acceptable salt or solvate:
  • R is selected from deuterium atom, C 1-6 alkyl or substituted alkyl, aryl, substituted aryl or heteroaryl;
  • X is selected from -C(O)-CR a R b -O-(CR 3 R 4 ) n -, -C(O)-CR a R b -NH-(CR 3 R 4 ) n -or -C( O)-CR a R b -(CR 3 R 4 ) n -S-;
  • R a is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, an aryl group, a substituted aryl group or Heteroaryl
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl
  • R a , R b together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • R 3 and R 4 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group , Cycloalkyl or heterocyclic group;
  • R 3 and R 4 together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • n is selected from an integer from 0 to 4.
  • n is selected from an integer or decimal of 1-10;
  • Ab is an antibody, antibody fragment or protein
  • L is the connecting unit.
  • the provided antibody-drug conjugate or its pharmaceutically acceptable salt or solvate is the antibody-drug conjugate represented by the general formula (Ab-LXD 2) or its A pharmaceutically acceptable salt or solvate;
  • R is selected from deuterium atom, C 1-6 alkyl or substituted alkyl, aryl, substituted aryl or heteroaryl;
  • R a is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, an aryl group, a substituted aryl group or Heteroaryl; preferably C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl;
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl; preferably C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl;
  • R a , R b together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • n 0 or 1
  • n is selected from an integer or a decimal number from 1 to 10; preferably n is 2 to 8, which can be an integer or a decimal; more preferably n is 3 to 8, which can be an integer or a decimal;
  • the antibody-drug conjugate represented by the general formula (Ab-LX-Dr) or a pharmaceutically acceptable salt or solvate thereof is provided, which is of the general formula (Ab-L a -The antibody-drug conjugate shown in XD 2 ) or a pharmaceutically acceptable salt or solvate thereof;
  • Z is selected from but not limited to -YC(O)-, -CH 2 -C(O)-NR 5 -YC(O)- or -C(O)-YC(O)-, where Y, Selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl or linear or linear-cyclic heteroalkyl of 1-8 atoms, the heteroalkyl contains selected from N, O or 1-3 atoms of S, the C 1-8 alkyl, cycloalkyl, linear or linear cyclic heteroalkyl are each independently selected from deuterated atoms, halogen, hydroxyl, carboxyl, cyano , Nitro, amino, alkyl, heteroalkyl, substituted alkyl, carboxy, alkoxy or cycloalkyl substituted by one or more substituents;
  • L 2 is selected from without limitation -NR 6 (CH 2 CH 2 O) p CH 2 CH 2 C(O)-, -NR 6 (CH 2 CH 2 O) p CH 2 C(O)-, -S (CH 2 ) p C(O)- or a chemical bond, where p is preferably an integer from 0 to 20, preferably a chemical bond;
  • L 3 is non-limitingly selected from peptide residues composed of 2-7 amino acids, wherein the optional amino acid is further selected from deuterium atom, halogen, hydroxyl, cyano, amino, nitro, alkyl, substituted alkyl, alkane One or more substituents in oxy and cycloalkyl or substituted cycloalkyl;
  • R is selected from deuterium atom, C 1-6 alkyl or substituted alkyl, aryl, substituted aryl or heteroaryl;
  • R a is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, an aryl group, a substituted aryl group or Heteroaryl
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl
  • R a , R b together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclic group;
  • R 3 and R 4 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group , Cycloalkyl or heterocyclic group;
  • R 3 and R 4 together with the carbon atoms to which they are connected form a C 3-6 cycloalkyl group or a heterocyclic group;
  • R 5 , R 6 and R 7 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a halogenated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, and an alkoxyalkyl group , Heterocyclic group, aryl group, substituted aryl group or heteroaryl group;
  • R 8 and R 9 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a halogenated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic ring Group, aryl, substituted aryl, or heteroaryl.
  • n is selected from an integer of 0 to 4, preferably 0 or 1, more preferably 0;
  • n is selected from 1-10, and can be an integer or a decimal
  • Ab is an antibody, antibody fragment or protein.
  • the antibody-drug conjugate represented by the general formula (Ab-LXD r ) or a pharmaceutically acceptable salt or solvate thereof is provided, which is of the general formula (Ab-L b- XD 2 ) the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof;
  • the antibody-drug conjugate represented by the general formula (Ab-L-X-Dr) of the present invention is selected from the following structures without limitation:
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof wherein the Ab is an antibody, an antibody fragment or an antigen-binding fragment thereof, the antibody It is selected from a chimeric antibody, a humanized antibody or a fully human antibody; preferably a monoclonal antibody.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof wherein the antibody, antibody fragment or antigen-binding fragment thereof is non-limitingly selected from: Anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV -1 antibody, anti-CanAg/AFP antibody, anti-cladin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT/CD117 antibody, anti-STEAP1 antibody, Anti-SLAMF7/CS1 antibody, anti-NaPi2B/SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody
  • a method for preparing the antibody-drug conjugate represented by the general formula (Ab-L a -XD 2 ) or a pharmaceutically acceptable salt or solvate thereof which includes the following step:
  • Ab is an antibody, an antibody fragment or an antigen-binding fragment thereof
  • R, R a , R b , R 7 , R 8 , R 9 , L 2 , L 3 , Z and n are as defined in the above general formula (Ab-La-XD 2 ).
  • composition which contains the compound represented by the general formula (D) according to the present invention or its tautomers, mesosomes, racemates, Enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • Another aspect of the present invention further relates to an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, which comprises a ligand and a drug linked to the ligand, wherein the drug is selected from the group of the present invention
  • the compound represented by the general formula (D), the compound represented by the general formula (LX-Dr), or its tautomers, mesosomes, racemates, enantiomers, and non-pairs Enantiomers, or mixtures thereof, or pharmaceutically acceptable salts thereof preferably the drug is linked to the ligand via a linker, and the ligand is preferably a monoclonal antibody.
  • an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof which comprises combining the compound represented by the general formula (D) of the present invention, the general formula The compound represented by the formula (LX-Dr), or its tautomer, meso, racemate, enantiomer, diastereomer, or a mixture thereof, or a mixture thereof.
  • the step of connecting the pharmaceutically acceptable salt with the ligand is preferably connected via a linker, and the ligand is preferably a monoclonal antibody.
  • Another aspect of the present invention further relates to the antibody-drug conjugate or compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof, for use as a medicine.
  • Another aspect of the present invention further relates to the antibody-drug conjugate or compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof in the preparation of a medicament for the treatment or prevention of tumors.
  • the antibody-drug conjugate or compound of the present invention or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof in the preparation of a medicament for the treatment or prevention of tumors.
  • Another aspect of the present invention further relates to the antibody-drug conjugates or compounds of the present invention, or pharmaceutically acceptable salts or solvates thereof, or pharmaceutical compositions used in the preparation of drugs for the treatment and/or prevention of cancer.
  • the cancer is selected from without limitation: breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer , Lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma And leukemia and other solid tumors or blood tumors.
  • Another aspect of the present invention further relates to a method for treating and/or preventing tumors, the method comprising administering to a patient in need thereof a therapeutically effective dose of the antibody-drug conjugate or compound of the present invention, or A pharmaceutically acceptable salt or solvate thereof or a pharmaceutical composition containing the same.
  • Another aspect of the present invention further relates to a method for treating or preventing cancer, the method comprising administering to a patient in need thereof a therapeutically effective dose of the antibody-drug conjugate or compound of the present invention, or a pharmacological agent thereof.
  • the cancer is selected from, without limitation, breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer , Liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma Solid tumors or hematological tumors such as cell tumors, glioma multiforme, sarcomas, lymphomas and leukemias.
  • the active compound can be formulated into a form suitable for administration by any appropriate route, and the active compound is preferably in a unit dose form, or in a form in which the patient can self-administer in a single dose.
  • the expression of the unit dose of the compound or composition of the present invention can be tablet, capsule, cachet, bottled syrup, powder, granule, lozenge, suppository, regenerating powder or liquid preparation.
  • the dosage of the compound or composition used in the treatment method of the present invention will generally vary with the severity of the disease, the weight of the patient, and the relative efficacy of the compound.
  • a suitable unit dose may be 0.1-1000 mg.
  • the pharmaceutical composition of the present invention may contain one or more excipients, which are non-limitingly selected from the following ingredients: fillers (diluents), binders, wetting agents, disintegrants Or excipients and so on.
  • the composition may contain 0.1 to 99% by weight of the active compound.
  • the pharmaceutical composition containing the active ingredient may be in a form suitable for oral administration, such as tablets, dragees, lozenges, water or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or Elixirs.
  • the oral composition can be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions can have binders, fillers, lubricants, disintegrants or pharmaceutically acceptable wetting agents, etc.
  • the similar composition may also contain one or more ingredients selected from the group consisting of sweeteners, flavoring agents, coloring agents and preservatives to provide pleasing and palatable pharmaceutical preparations.
  • Aqueous suspensions contain the active substance and excipients suitable for the preparation of aqueous suspensions for mixing.
  • the aqueous suspension may also contain one or more preservatives, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents.
  • Oil suspensions can be formulated by suspending the active ingredients in vegetable oils.
  • the oil suspension may contain thickeners.
  • the above-mentioned sweeteners and flavoring agents can be added to provide a palatable preparation.
  • the pharmaceutical composition can also be dispersible powders and granules used to prepare aqueous suspensions to provide active ingredients by adding one or more of a water-mixed dispersant, wetting agent, suspending agent or preservative. Other excipients such as sweeteners, flavoring agents and coloring agents may also be added. These compositions are preserved by adding antioxidants such as ascorbic acid.
  • the pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous solution.
  • Acceptable solvents or solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution.
  • the sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase.
  • the active ingredient is dissolved in a mixture of soybean oil and lecithin.
  • the oil solution is added to the mixture of water and glycerin to form a microemulsion.
  • the injection or microemulsion can be injected into the patient's bloodstream by local large-scale injection.
  • a continuous intravenous delivery device can be used.
  • An example of such a device is the Deltec CADD-PLUS.TM. 5400 intravenous pump.
  • the pharmaceutical composition may be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration.
  • the suspension can be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents mentioned above.
  • the sterile injection preparation may also be a sterile injection solution or suspension prepared in a parenterally acceptable non-toxic diluent or solvent.
  • sterile fixed oil can be conveniently used as a solvent or suspending medium.
  • the compounds of the present invention can be administered in the form of suppositories for rectal administration.
  • These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid in the rectum and thus will melt in the rectum to release the drug.
  • suitable non-irritating excipient include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights and mixtures of fatty acid esters of polyethylene glycol.
  • the dosage of the drug depends on many factors, including but not limited to the following factors: the activity of the specific compound used, the age of the patient, the weight of the patient, the health of the patient, and the behavior of the patient. , The patient’s diet, time of administration, mode of administration, rate of excretion, combination of drugs, etc.; in addition, the best treatment mode, such as the mode of treatment, the daily dosage of the compound of the general formula, or the type of pharmaceutically acceptable salt can be based on Traditional treatment plan to verify.
  • Figure 1 shows the in vivo efficacy of ADC on A431 tumor-bearing mouse model
  • Figure 2 is a graph showing the in vitro activity data of compounds 6, 12, 14 and 16 of the present invention on Fadu cells;
  • Figure 3 is a graph showing the in vitro activity data of compounds 6, 12, 14 and 16 of the present invention on A431 cells;
  • Figure 4 is a graph showing the in vitro activity data of compounds 6, 12, 14 and 16 of the present invention on Bxpc-3 cells;
  • Figure 5 is a graph showing the in vitro activity data of compounds 6, 12, 14 and 16 of the present invention on SW620 cells.
  • the applicant intends to include the formulation of the trade name product, the generic drug and the active drug part of the trade name product.
  • antibody is a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell.
  • the role of antibodies is to present drugs to target cell populations that bind to antibodies. These antibodies include but are not limited to protein hormones, lectins, growth factors, antibodies or other molecules that can bind to cells.
  • the antibody is referred to as Ab.
  • the antibody can form a link with the linking unit through its heteroatoms, preferably an antibody or an antigen-binding fragment thereof, and the antibody is selected from a chimeric antibody, a humanized antibody, and a fully human Antibody or murine antibody; preferably a monoclonal antibody.
  • drug refers to a cytotoxic drug.
  • the drug is denoted as D, which can have chemical molecules in tumor cells that strongly disrupt their normal growth.
  • cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to lack of specificity, while killing tumor cells, it will also cause normal cell apoptosis, leading to serious side effects.
  • the term includes toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g.
  • linker unit or “linker fragment” or “linker unit” refers to a chemical structure fragment or bond with one end connected to a ligand and the other end to a drug, and other linkers can also be connected before being connected to the drug.
  • the preferred scheme of the present invention is expressed as L and L 1 -L 4 , where the L 1 end is connected to the ligand, and the L 4 end is connected to the structural unit X and then connected to the drug (D).
  • the linker including extensions, spacers and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the linker may be a "breakable linker" that facilitates the release of the drug in the cell.
  • breakable linker For example, acid-labile linkers (such as hydrazone), protease-sensitive (such as peptidase-sensitive) linkers, light-labile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al. Cancer Research 52:127-131, 1992); U.S. Patent No. 5,208,020).
  • antibody-drug conjugate refers to that the antibody is connected to a biologically active drug through a stable linking unit.
  • ADC antibody-drug conjugate
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the antibodies of the present invention are preferably specific antibodies against cell surface antigens on target cells.
  • Non-limiting examples are the following antibodies: anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, and anti-MUC16 antibody , Anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg/AFP antibody, anti-cladin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT/CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7/CS1 antibody, anti-NaPi2B/SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) Antibody, anti-MUC1/CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PT
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FRI, CDRI, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the antibodies of the present invention include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.
  • murine-derived antibody in the present disclosure refers to the preparation of antibodies with mice based on knowledge and skills in the art. During preparation, the test object is injected with a specific antigen, and then the hybridization of the antibody with the desired sequence or functional characteristics is separated and expressed
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma secreting murine-derived specific monoclonal antibodies, then clone the variable region genes from the murine hybridoma cells, and then clone the constant region genes of the human antibody as needed, and replace the murine variable region genes. It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.
  • humanized antibody is also called CDR-grafted antibody (CDR-grafted antibody), refers to the mouse CDR sequence grafted into the human antibody variable region framework, that is, different types of human germline antibody framework sequences It can overcome the heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components.
  • CDR-grafted antibody refers to the mouse CDR sequence grafted into the human antibody variable region framework, that is, different types of human germline antibody framework sequences It can overcome the heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components.
  • Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or public references. Such as humans.
  • the germline DNA sequences of the heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com/ac.uk/vbase ), as well as in Kabat, EA, etc., Found in 1991 Sequences of Proteins of Immunological Interest, 5th edition.
  • the human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations. Maintain activity.
  • the humanized antibodies of the present invention also include humanized antibodies that are further subjected to affinity maturation of the CDRs by phage display.
  • the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the present invention is a fully human monoclonal antibody.
  • the related technologies of fully human antibody preparation mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of antibodies.
  • binding fragments contained in "antigen-binding fragments” include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, including through the hinge region A bivalent fragment of two Fab fragments connected by a disulfide bridge, (iii) Fd fragment composed of VH and CH1 domains; (iv) Fv fragment composed of VH and VL domains of one arm of an antibody; (v ) A single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) can optionally be passed A combination
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, recombination methods can be used to connect them through a synthetic linker so that it can be produced as a single protein in which the VL and VH regions are paired to form a monovalent molecule.
  • Chain referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426 and Huston et al. (1 988) Proc. Nat L. Acad. Sci. USA 85: 5879-5883).
  • Such single chain antibodies are also intended to be included in the "antigen-binding fragment" of the term antibody.
  • the antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or lgM antibodies.
  • Fab is an antibody fragment that has a molecular weight of about 50,000 and has antigen-binding activity among fragments obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), wherein the H chain is N-terminal About half of the side and the entire L chain are joined together by disulfide bonds.
  • F(ab')2 is an antibody with a molecular weight of about 100,000 obtained by digesting the lower part of the two disulfide bonds in the hinge region of IgG with the enzyme pepsin and has antigen-binding activity and contains two Fab regions connected at the hinge position. Fragment.
  • Fab' is an antibody fragment with a molecular weight of about 50,000 and antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of F(ab')2 described above.
  • the Fab' can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into a prokaryotic organism or eukaryotic organism to express Fab'.
  • single-chain antibody means an antibody heavy chain variable domain (or region; (H) and antibody light chain variable domain (or region; VL) connected by a linker.
  • Such scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variations thereof Body composition, for example, using 1-4 repeated variants (Holliger et al. (1993), proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • linkers that can be used in the present invention are described by Alfthan et al. (1995) , Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immuno 1.31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999) , J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • CDR refers to one of the six hypervariable regions in the variable domain of an antibody that mainly contribute to antigen binding.
  • One of the most commonly used definitions of the 6 CDRs is provided by Kabat E.A. et al. (1991) Sequences of Proteins of Immunological Interest. NIH Publication 91-3242).
  • the Kabat definition of CDR only applies to the CDR1, CDR2, and CDR3 of the light chain variable domain (CDR L1, CDRL2, CDRL3 or L1, L2, L3), and the CDR2 and CDR3 of the heavy chain variable domain.
  • CDR3 CDR H2, CDR H3 or H2, H3
  • antibody framework refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable structure without CDR.
  • epitopes refers to the site on an antigen where an immunoglobulin or antibody specifically binds.
  • Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, et al. (1996).
  • antibodies bind with an affinity (KD) of approximately less than 10 -7 M, such as approximately less than 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors invented herein can replicate autonomously in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of the host cell after being introduced into the host cell, so as to follow The host genome replicates together (e.g., a non-episomal mammalian vector).
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human FR regions to the non-human CDR region.
  • the human FR germline sequence can be obtained from the 1mMunoGeneTics (MGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351 obtain.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacteria, microorganisms, plant or animal cells.
  • Bacteria that are easily transformed include members of the enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.
  • the engineered antibody or antigen-binding fragment of the present invention can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by conventional techniques. For example, use A or G Sepharose FF column with adjusted buffer for purification.
  • the bound antibody was eluted by the PH gradient method, and the antibody fragments were detected by SDS-PAGE and collected.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • peptide refers to a fragment of a compound between amino acids and proteins. It is composed of two or more amino acid molecules connected to each other by peptide bonds. They are structural and functional fragments of proteins, such as hormones, enzymes, etc. in essence. All are peptides.
  • sucrose refers to a biological macromolecule composed of three elements: C, H, and O, which can be divided into monosaccharides, disaccharides and polybran.
  • fluorescent probe refers to the characteristic fluorescence in the ultraviolet-visible-near-infrared region, and its fluorescent properties (excitation and emission wavelength, intensity, lifetime, polarization, etc.) can vary with the properties of the environment, such as polarity, refractive index A type of fluorescent molecules that change sensitively due to changes in viscosity, viscosity, etc., which non-covalently interact with nucleic acids (DNA or RNA), proteins or other macromolecular structures to change one or several fluorescent properties, which can be used for research The nature and behavior of macromolecular substances.
  • toxic drug refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction. Including toxins and other compounds that can be used in tumor treatment.
  • toxin refers to any substance that can have a harmful effect on the growth or proliferation of cells, and can be small molecule toxins and their derivatives from bacteria, fungi, plants or animals, including camptothecin derivatives such as ixati Health, maytansinoid and its derivatives (CN101573384) such as DM1, DM3, DM4, auristatin F (AF) and its derivatives, such as MMAF, MMAE, 3024 (WO 2016/127790 A1), diphtheria toxin, external Toxin, ricin A chain, abrin A chain, modeccin, ⁇ -toxin (sarcin), Aleutitesfordii toxic protein, dianthin toxic protein , Phytolaca americana toxic protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, jatropha curcin, croton, sapaonaria officinalis Inhibitors, gelonin, mitogellin, restrictoc
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably containing 1 to 10 carbons The most preferred is an alkyl group containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-Dimethylpropyl, 2,2-Dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -Methylhexyl, 3-methylhexyl, 4-methylhe
  • lower alkyl groups containing 1 to 6 carbon atoms More preferred are lower alkyl groups containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and sec-butyl.
  • Alkyl groups may be substituted or unsubstituted.
  • substituents When substituted, substituents may be substituted at any available attachment point.
  • the substituents are preferably one or more of the following groups, which are independently selected from alkanes Group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane Oxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein the alkyl group is as defined above.
  • alkylene refers to a saturated linear or branched aliphatic hydrocarbon group, which has two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, which is A straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, more preferably an alkylene group containing 1 to 6 carbon atoms.
  • Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene [-CH(CH 3 )-], 1,2-ethylene (-CH 2 -) CH 2 )-, 1,1-propylene [-CH(CH 2 CH 3 )-], 1,2-propylene [-CH 2 CH(CH 3 )-], 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and 1,5-butylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -) Wait.
  • the alkylene group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is preferably independently optionally selected from alkyl, alkenyl, alkynyl , Alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy , Cycloalkylthio, heterocycloalkylthio and oxo groups are substituted by one or more substituents.
  • alkoxy refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where the definition of alkyl or cycloalkyl is as described above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • the alkoxy group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent.
  • the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 Carbon atoms, most preferably 3 to 8 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Groups, cyclooctyl, etc.; polycyclic cycloalkyls include spiro, fused, and bridged cycloalkyls.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) u (where u is an integer of 0 to 2) heteroatoms, but does not include the ring part of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocyclic groups include spirocyclic, condensed, and bridged heterocyclic groups.
  • the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, non-limiting examples thereof include:
  • the heterocyclic group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio, oxo.
  • aryl refers to a 6 to 14-membered all-carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) group with a conjugated electron system, preferably 6 to 10 members, such as benzene And naphthyl, preferably phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, and non-limiting examples thereof include:
  • the aryl group may be substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio base.
  • heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen.
  • Heteroaryl groups are preferably 5 to 10 members, more preferably 5 or 6 members, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrakis Azole and so on.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:
  • Heteroaryl groups may be optionally substituted or unsubstituted.
  • the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.
  • amino protecting group is to keep the amino group unchanged when other parts of the molecule react, and to protect the amino group with a group that is easy to remove.
  • Non-limiting examples include 9-fluorenylmethyloxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with 1-3 substituents selected from halogen, alkoxy or nitro.
  • the amino protecting group is preferably 9-fluorenylmethyloxycarbonyl.
  • cycloalkylalkyl refers to an alkyl group substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, where alkyl is as defined above and where cycloalkyl is as defined above.
  • haloalkyl refers to an alkyl group substituted with one or more halogens, where the alkyl group is as defined above.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms, where the alkyl group is as defined above.
  • hydroxy refers to the -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH 2 .
  • nitro refers to -NO 2 .
  • amido refers to -C(O)N-(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.
  • carboxylate group refers to -C(O)O-(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.
  • the present invention also includes compounds of formula (D) in various deuterated forms.
  • Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom.
  • Those skilled in the art can synthesize the compound of formula (D) in the deuterated form with reference to relevant literature.
  • Commercially available deuterated starting materials can be used when preparing the deuterated form of the compound of formula (D), or they can be synthesized using conventional techniques using deuterated reagents.
  • Deuterated reagents include, but are not limited to, deuterated borane and tri-deuterated. Borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide, etc.
  • heterocyclic group optionally substituted by an alkyl group means that an alkyl group may but need not be present, and the description includes the case where the heterocyclic group is substituted by an alkyl group and the case where the heterocyclic group is not substituted by an alkyl group.
  • Substituted refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1-3 hydrogen atoms independently of each other, substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (through experiment or theory) possible or impossible substitutions without too much effort. For example, an amino group or a hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
  • pharmaceutical composition means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • pharmaceutically acceptable salt refers to the salt of the antibody-drug conjugate of the present invention, or the salt of the compound described in the present invention. Such salts have It is safe and effective, and has due biological activity.
  • the antibody-drug conjugate compound of the present invention contains at least one amino group, so it can form a salt with an acid.
  • Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride , Hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, diphosphate Hydrogen salt, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, P-toluenesulfonate.
  • solvate or “solvent compound” means that the antibody-drug conjugate compound of the present invention forms a pharmaceutically acceptable solvate with one or more solvent molecules.
  • solvent molecules include water, ethanol, acetonitrile, Isopropanol, DMSO, ethyl acetate.
  • drug loading refers to the average number of cytotoxic drugs loaded on each antibody in the molecule. It can also be expressed as the ratio of the amount of drug to the amount of antibody. The range of drug loading can be 0-12 per antibody (Ab). One, preferably 1-10 cytotoxic drugs (D). In the embodiment of the present invention, the drug loading is expressed as m, which may be an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 as an example. Conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test and HPLC characteristics can be used to identify the average number of drug products per ADC molecule after the coupling reaction.
  • the cytotoxic drug is coupled to the N-terminal amino group of the ligand and/or the epsilon-amino group of the lysine residue through the linking unit.
  • the cytotoxic drug can be coupled to the antibody in the coupling reaction.
  • the number of drug molecules will be less than the theoretical maximum.
  • ligand cytotoxic drug conjugates including:
  • carrier used in the drug of the present invention refers to a system that can change the way the drug enters the body and its distribution in the body, control the release rate of the drug, and deliver the drug to the targeted organ.
  • the drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure.
  • Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to contain drug molecules, and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.
  • excipient is an addition to the main drug in a pharmaceutical preparation, and can also be referred to as an adjuvant.
  • adjuvant such as binders, fillers, disintegrants, lubricants in tablets; base parts in semi-solid preparations ointments and creams; preservatives, antioxidants, flavors, fragrances, and auxiliary agents in liquid preparations
  • Solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.
  • diluent is also called a filler, and its main purpose is to increase the weight and volume of the tablet.
  • the addition of diluent not only guarantees a certain volume, but also reduces the deviation of the dosage of the main components, and improves the compression molding of the drug.
  • an absorbent should be added to absorb the oily substance to keep it in a “dry” state, so as to facilitate the manufacture of tablets.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous solution.
  • the acceptable solvents and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution.
  • the sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase.
  • the active ingredient is dissolved in a mixture of soybean oil and lecithin.
  • the oil solution is added to the mixture of water and glycerin to form a microemulsion.
  • the injection or microemulsion can be injected into the patient's bloodstream by local large-scale injection.
  • a continuous intravenous delivery device can be used.
  • An example of such a device is the Deltec CADD-PLUS.TM. 5400 intravenous pump.
  • the pharmaceutical composition may be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration.
  • the suspension can be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents mentioned above.
  • the sterile injection preparation may also be a sterile injection solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent, for example, a solution prepared in 1,3-butanediol.
  • sterile fixed oil can be conveniently used as a solvent or suspending medium. For this purpose, any blended fixed oil including synthetic mono- or diglycerides can be used.
  • fatty acids such as oleic acid can also be used to prepare injections.
  • the present invention relates to a cleavable linker with a specific structure and a drug with a specific structure, and an antibody drug conjugate (ADC) composed of a linker, a drug and an antibody.
  • ADC antibody drug conjugate
  • This type of ADC is a complex formed by linking a toxic substance to an antibody via a spacer.
  • the antibody-conjugated drug (ADC) is degraded in the body to release active molecules, which play a corresponding role.
  • the following terms and phrases as used herein are intended to have the following meanings.
  • the brand name includes the product formula, generic drugs, and active pharmaceutical ingredients of the brand name product.
  • alkyl refers to a saturated hydrocarbon group having 1-20 carbon atoms.
  • alkylene groups include, but are not limited to, methyl (-CH 3 ), ethyl (-CH 2 -CH 3 ), n-propyl, n-butyl, n-pentyl, and n-hexyl.
  • R', R" and R"' each independently refers to hydrogen, unsubstituted C 1-8 alkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C 1-8 alkyl, C 1- 8 alkoxy or C 1-8 thioalkoxy, or unsubstituted aryl -C 1-4 alkyl .
  • -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.
  • Acid amino acid refers to an amino acid whose isoelectric point is less than 7. Acidic amino acid molecules often contain one or more acidic groups such as carboxyl groups, which can be effectively ionized into negative ions in the structure to increase hydrophilicity. Acidic amino acids can be natural or unnatural amino acids.
  • Natural amino acids refer to amino acids synthesized by biology. Natural amino acids are generally L-type, but there are a few exceptions, such as glycine, including natural and biosynthetic.
  • Unnatural amino acid refers to an amino acid obtained by synthetic means.
  • the ligand unit is a targeting agent that specifically binds to the target part.
  • the ligand can specifically bind to cellular components or to cellular components or to other target molecules of interest.
  • the target moiety or target is usually on the surface of the cell.
  • the role of the ligand unit is to deliver the drug unit to a specific target cell population with which the ligand unit interacts.
  • Ligands include but are not limited to proteins, polypeptides and peptides, as well as non-proteins such as sugars. Suitable ligand units include, for example, antibodies, such as full-length (complete) antibodies and antigen-binding fragments thereof.
  • the ligand unit is a non-antibody targeting agent
  • it can be a peptide or polypeptide, or a non-protein molecule.
  • targeting agents include interferons, lymphokines, hormones, growth factors and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell binding molecules or substances.
  • the linker is covalently attached to the sulfur atom of the ligand.
  • the sulfur atom is the sulfur atom of a cysteine residue, which forms an interchain disulfide bond of the antibody.
  • the sulfur atom is a sulfur atom of a cysteine residue that has been introduced into the ligand unit, which forms an interchain disulfide bond of the antibody.
  • the sulfur atom is the sulfur atom of the cysteine residue that has been introduced into the ligand unit (for example, by site-directed mutagenesis or chemical reaction).
  • the sulfur atom to which the linker binds is selected from the cysteine residues that form the interchain disulfide bond of the antibody or the frontal cysteine residues that have been introduced into the ligand unit (for example, by site-directed mutagenesis or chemical reaction).
  • the EU index in Kabat Kabat (Kabat EA et al., (1991)) "Sequences of proteins of Immunological Interest" (Sequences of proteins of Immunological Interest), fifth edition, NIH publication 91-3242) Numbering system.
  • antibody or “antibody unit” includes any part of the structure of an antibody within the scope to which it belongs. This unit can bind, reactively associate, or complex a receptor, antigen, or target other receptor unit possessed by the cell population.
  • the antibody can be any protein or protein molecule that can bind, complex, or react with a part of the cell population to be treated or biologically modified.
  • the antibody constituting the antibody-drug conjugate of the present invention preferably maintains its original antigen binding ability in the wild state. Therefore, the antibody of the present invention can, preferably, specifically bind to the antigen.
  • the antigens involved include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, and molecules related to tissue growth and differentiation (as known or predicted Functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (as known or predicted to be functional).
  • TAA tumor-associated antigens
  • the tumor-related factor may be a cluster differentiation factor (such as CD protein).
  • Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies directed against cell surface receptors and tumor-associated antigens.
  • tumor-associated antigens are well-known in the industry and can be prepared by antibody preparation methods and information well-known in the industry.
  • researchers are trying to find transmembrane or other tumor-related peptides. These targets can be specifically expressed on the surface of one or more cancer cells, but little or no expression on the surface of one or more non-cancer cells.
  • tumor-associated polypeptides are more overexpressed on the surface of cancer cells than on the surface of non-cancer cells. Confirming that such tumor-related factors can greatly improve the specific targeting properties of antibody-based treatment of cancer.
  • Tumor-associated antigens include, but are not limited to the following tumor-associated antigens (1)-(36).
  • the antigen-related information well-known in the industry is marked as follows, including name, other names, and gene bank accession number.
  • Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases, such as Genbank.
  • Antibodies targeting corresponding tumor-associated antigens include all amino acid sequence variants and homologs, which have at least 70%, 80%, 85%, 90%, or 95% homology with the sequence confirmed in the reference, or have the same
  • the tumor-associated antigen sequences in the cited literature have completely identical biological properties and characteristics.
  • inhibitor refers to reducing the detectable amount, or preventing it completely.
  • cancer refers to a physiological condition or disease characterized by unregulated cell growth.
  • Tumor includes cancer cells.
  • autoimmune disease is a disease or disorder that originates from the tissues or proteins of an individual.
  • 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 compound may contain at least one amino or carboxyl group, and therefore may form an addition salt with the corresponding acid or base.
  • 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, tannate, pantothenate, bitartrate, ascorbate, salicylate, Formate, this format, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, potassium salt, sodium salt, etc.
  • pharmaceutically acceptable salts have more than one point atom in the structure. Examples in which multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counter-examples.
  • 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 digested in the lysosome, and the small molecule drug is released.
  • An active molecule composed of amino acid residues of an antibody. The resulting changes in the structure of the drug molecule do not reduce its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate neighboring cells. Therefore, such active drugs cannot kill adjacent tumor cells that do not express the targeted antigen (antigen-negative cells) (bystander effect) (Ducry et al., 2010, Bioconjugate Chem. 21:5-13).
  • reaction solution was directly concentrated under reduced pressure and purified by preparative high performance liquid chromatography (acetonitrile/pure water system).
  • the target peak was collected and reduced After the acetonitrile was removed by pressure, it was lyophilized to obtain about 2 mg of compound 16 as a yellow solid with a yield of about 60%.
  • reaction solution is directly concentrated under reduced pressure and purified by preparative high performance liquid chromatography (acetonitrile/pure water system).
  • the target peak is collected and the acetonitrile is removed under reduced pressure.
  • compound 4 (4mg, 8.9umol) was added to a 10mL bottle, 1mL absolute ethanol, 0.2mL DMF and 0.15mL NMM were added to dissolve, cooled to 0°C in an ice water bath, and compound 21 (4.1mg, 4eq is prepared by the method disclosed in the patent application "WO2013106717”), 1-hydroxybenzotriazole HOBt (3.7mg, 4eq) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt Salt EDCI (5.1mg, 4eq), naturally warmed to room temperature and reacted for 2 hours, TLC monitored the end of the reaction, the reaction solution was directly concentrated under reduced pressure, purified by preparative high performance liquid chromatography (acetonitrile/pure water system), and collected After removing the acetonitrile under reduced pressure, the target peak was lyophilized to obtain about 1.5 mg of compound 22A, yellow solid, about 1.5 mg of compound 22B, yellow solid
  • Patent No: WO 2020063673 It is prepared by referring to the synthetic method of the known method "Patent No: WO 2020063673".
  • Patent No: WO 2020063673 It is prepared by referring to the synthetic method of the known method "Patent No: WO 2020063673".
  • compound 82 was fed (28 mg, 100 umol) to obtain 19 mg of target compound, yield 25%, MS m/z: [M+H] + 762.4.
  • compound 88 was fed (34 mg, 70 umol) to obtain 45 mg of target compound, yield 86%, MS m/z: [M+H] + 750.9.
  • compound 110 was fed (measured according to compound 109) to obtain 1.1 mg of target compound, with a two-step yield of about 13%, MS m/z: [M+H] + 1259.8.
  • the antibody molecules whose monomer ratio is greater than 95% are exchanged into a phosphate buffer containing EDTA with 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 exchange the liquid into PBS, and remove the uncoupled payload.
  • the sample was centrifuged at 14000 rpm for 5 minutes, and the supernatant was taken for analysis.
  • Mobile phase A: 50mM PB, 300mM NaCl, 200mM Arg, 5% IPA, pH6.5;
  • the mobile phase A was eluted isocratically for 30 min, flow rate: 0.714 mL/min, column temperature 25°C, detection wavelength: 280 nm.
  • the sample was centrifuged at 14000 rpm for 5 minutes, and the supernatant was taken for analysis.
  • Mobile phase A: 1.5M ammonium sulfate, 0.025M anhydrous sodium phosphate, pH 7.0;
  • the mobile phase A equilibrates the chromatographic column, the mobile phase A and B are gradient eluted, and the flow rate is 0.8mL/min;
  • Example 25 Prepare according to the general coupling method, and use Trastuzumab as the antibody to obtain ADC: Tras-6
  • Example 26 Prepare according to the general coupling method, and use Tr000 as the antibody to obtain ADC: Tr000-6
  • Example 27 Prepare according to the general coupling method, and use Trastuzumab as the antibody to obtain ADC:Tras-9
  • Example 28 Prepared according to the general coupling method, using Trastuzumab as the antibody to obtain ADC:Tras-12
  • Example 29 Prepare according to the general coupling method, and use Tr000 as the antibody to obtain ADC: Tr000-12
  • Example 30 Prepare according to the general coupling method, and use Trastuzumab as the antibody to obtain ADC: Tras-14
  • Example 31 Prepare according to the general coupling method, and use Trastuzumab as the antibody to obtain ADC: Tras-16
  • Example 32 Prepare according to the general coupling method, and use Tr000 as the antibody to obtain ADC: Tr000-16
  • Example 33 Prepare according to the general coupling method, and use Trastuzumab as the antibody to obtain ADC: Tras-18
  • Example 34 Prepared according to the general coupling method, using Trastuzumab as the antibody to obtain ADC: Tras-20A
  • Example 35 Prepared according to the general coupling method, using Trastuzumab as the antibody to obtain ADC: Tras-22B
  • Example 36 Prepared according to the general coupling method, using Trastuzumab as the antibody to obtain ADC: Tras-PEG-6
  • Example 37 Prepare according to the general coupling method, and use Trastuzumab as the antibody to obtain ADC: Tras-PEG-9
  • Example 38 Prepared according to the general coupling method, using Trastuzumab as the antibody to obtain ADC: Tras-PEG-12
  • Example 39 Prepared according to the general coupling method, using Trastuzumab as the antibody to obtain ADC: Tras-PEG-14
  • Example 40 Prepare according to the general coupling method, and use Trastuzumab as the antibody to obtain ADC: Tras-PEG-16
  • Example 41 Prepared according to the general coupling method, using Trastuzumab as the antibody to obtain ADC: Tras-PEG-18
  • Example 42 Prepared according to the general coupling method, using Trastuzumab as the antibody to obtain ADC: Tras-PEG-20A
  • Example 43 Prepared according to the general coupling method, using Trastuzumab as the antibody to obtain ADC: Tras-PEG-22B
  • UV lamp in the biological safety cabinet 30 minutes in advance, and then turn on the ventilation for 3 minutes.
  • the dilution of antibody solution prepare the test solution with the initial concentration of 5uM and 300uL in the first column of the V-type 96-well plate with the detection medium, and then add 240uL of the detection medium in the second column to the 10th column. Take 60uL from the first row of uniformity and add it to the second row, mix up and down 10 times with a row gun, discard the pipette tip, and operate the next 7 concentrations in sequence.
  • MTS reagent After 4 days, take out the MTS reagent. After thawing at room temperature and avoiding light, vortex and mix thoroughly. In a biological safety cabinet, add 20 ⁇ L CellTiter One Solution Reagen MTS reagent to each 100 ⁇ L cell culture volume along the side wall of the well, and gently tap the plate surface. , After mixing the MTS solution evenly, place it in a cell culture incubator and incubate for 2 hours in the dark. After the reaction, the 96-well plate was taken out, the OD490nm absorbance value was detected in the microplate reader, and the data was recorded, sorted, analyzed and stored.
  • camptothecin derivatives of the present invention have obvious in vitro anti-tumor activity (nanomolar level), which is significantly less toxic than the toxin used by Daiichi Sankyo (control compound 16).
  • the designed and synthesized camptothecin derivatives are expected to become safer anti-tumor ADC drugs.
  • an A431 tumor-bearing mouse model was established to evaluate the in vivo efficacy of the toxin ADC coupled drug. That is, 1 ⁇ 10 6 A431 cells were injected subcutaneously into the right side of BALB/c nude mice aged 4-6 weeks. After the average tumor size of the mice grew to 180-200mm3, the mice were randomly divided into groups, 5 in each group, at 0 , 7, 14, and 21 days were given blank control (buffer solution blank), antibody drug conjugate Tr000-6, both were administered intravenously at a dose of 10 mg/kg. The tumor volume measurement data is displayed as the mean tumor volume ⁇ SE at the time of measurement.

Abstract

一种抗肿瘤药用的喜树碱类衍生物及其抗体-药物偶联物。通过一系列分子结构改造，获得更优的喜树碱类抗肿瘤药物，以更适合作为抗体偶联用药物。

Description

一种喜树碱衍生物及其偶联物 技术领域
本发明涉及作为抗肿瘤药用的喜树碱类衍生物及其抗体-药物偶联物。具体的说，本发明涉及通过一系列分子结构改造，获得更优的喜树碱类抗肿瘤药物，以更适合作为抗体偶联用药物。
背景技术
抗体-药物偶联物(antibody drug conjugate,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，小分子药物基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，达到治疗的目的。与传统的化疗药物相比，抗体-药物偶联物能精准地结合肿瘤细胞并降低对正常细胞的影响[Mullard A,(2013)Nature Reviews Drug Discovery,12:329-332；DiJoseph JF,Armellino DC,(2004)Blood,103:1807-1814]。
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ADC新药Adcetris TM上市，用于治疗霍奇金淋巴瘤以及复发性间变性大细胞淋巴瘤[Nat.Biotechnol(2003)21(7):778-784；WO2004010957；WO2005001038；US7090843A；US7659241；WO2008025020]。，临床应用已经证明了此类药物的安全性和有效性。
2013年2月，作为第一个被美国FDA批准用于治疗实体肿瘤的ADC药物，Kadcyla(ado-trastezumab emtansine,T-DM1)，用于治疗HER2阳性同时对曲妥珠单抗(Tratuzumab,商品名：Herceptin)和紫杉醇有抗药性的晚期或者转移性乳腺癌患者(WO2005037992；US8088387)。
ADC常用毒素包括MMAE，T-DM1，PBD等剧毒类毒素，此类毒素毒性强，即便在形成ADC药物以后，治疗窗也较窄。
通过抑制拓扑异构酶Ⅰ而具有抗肿瘤作用的喜树碱类衍生物，是一类用于抗体-药物偶联物的小分子。依喜替康作为其中代表化合物，由第一三共公司开发，该药前期作为单独化疗药物使用推进至三期临床，主要适应症为骨癌、前列腺癌、乳腺癌、胰腺癌等，因为副作用大，治疗窗窄，最终直接给药的依喜替康未能成功上市。
为克服依喜替康直接给药的副作用，第一三共公司将依喜替康开发作为ADC毒素使用，并已成功上市。第一个上市的以依喜替康为毒素的ADC，代号DS-8201，针对HER2靶点，用了单个抗体连接8个毒素的形式。但是，第一三共在后续临床中针对其他靶点如 Trop2设计的以依喜替康为毒素的ADC药物，因为安全性的问题，不得已降低至抗体药物比：DAR＝4。偶联药物数目的减少，会降低ADC药物的治疗指数。
本发明所需要解决的技术问题，就是探索发现更优的抗肿瘤喜树碱类衍生物，降低化合物毒性，提高其在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ADC药物。在对ADC类药物及喜树碱衍生物构效关系综合理解的基础上，本发明设计合成出一系列具有显著抗肿瘤活性的喜树碱类衍生物及其抗体偶联物，所得的喜树碱类衍生物的毒性显著降低，最终通过体内抗肿瘤活性测定，证明其良好的抗肿瘤活性。
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类衍生物及其偶联物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或者其混合物形式，或其药学上可接受的盐或溶剂化物，其中所述抗体-药物偶联物包含式D所示的抗肿瘤喜树碱类化合物。
Figure PCTCN2020115807-appb-000001
其中：
R选自氘原子、C 1-6烷基、氘代烷基、取代烷基、芳基、取代的芳基或杂芳基，优选C 1-6烷基或取代烷基；
R 1选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、羧基、羧基、杂环基、芳基、取代芳基或杂芳基；
R 2选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、羧基、羧基、杂环基、芳基、取代芳基或杂芳基；
X选自-C(O)-CR aR b-O-(CR 3R 4) n-、-C(O)-CR aR b-NH-(CR 3R 4) n-或-C(O)-CR aR b-(CR 3R 4) n-S-；
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R b选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或者杂环基；
或者，R 3、R 4连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
n选自0至4的整数。
本发明的一些实施方案中，提供的喜树碱类衍生物或其互变异构体、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，或药学上可接受的盐或溶剂化物，包含具有通式D 1所示的化合物或其药学上可接受的盐或溶剂化物；
Figure PCTCN2020115807-appb-000002
其中：
R选自氘原子、C 1-6烷基或取代烷基、芳基、取代芳基或杂芳基；
X选自-C(O)-CR aR b-(CR 3R 4) n-O-、-C(O)-CR aR b-(CR 3R 4) n-NH-或-C(O)-CR aR b-(CR 3R 4) n-S-；
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R b选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、羟基、氨基、氰基、硝基、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟烷基、环烷基或者杂环基；
或者，R 3、R 4连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或者杂环基；
n选自0至4的整数；
其中，式D 1中的波浪线或表示氢原子，或与接头单元或与结合肿瘤靶细胞所表达抗原的抗体共价连接。
本发明的一些实施方案中，提供的喜树碱类衍生物或其药学上可接受的盐或溶剂化物，结构单元-X-为-C(O)-CR aR b-(CR 3R 4) n-O-或者结构单元-X-为-C(O)-CR aR b-(CH 2) n-O-；
其中：
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R b选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或者杂环基；
或者，R 3、R 4连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或者杂环基；
n为0或1。
本发明的一些实施方案中，提供的喜树碱类衍生物或者其药学上可接受的盐或溶剂化物，其中结构单元-X-的O端与连接单元L相连，结构单元-X-选自，但不局限于：
Figure PCTCN2020115807-appb-000003
本发明的一方面，提供了一种通式(D 2)所示的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，或其药学上可接受的盐或溶剂化物，
Figure PCTCN2020115807-appb-000004
其中：
R选自氘原子、C 1-6烷基或取代烷基、芳基、取代的芳基或杂芳基，优选C 1-6烷基或取代烷基；
R a选自环烷基或者环烷基烷基，优选C 3-6环烷基烷基或者C 3-6环烷基；
R b选自氢原子、氘原子、烷基、卤代烷基、环烷基或者环烷基烷基，优选氢原子或者氘原子；
或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
n为0或者1；
其中，式D 2中的波浪线或表示氢原子，或与接头单元或与结合靶细胞所表达抗原的抗体共价连接。
本发明一方面的一个优选方案，提供的通式(D)所示的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、或其药学上接受的盐或溶剂化物，包含具有通式(D 3)所示的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、或其药学上接受的盐或溶剂化物：
Figure PCTCN2020115807-appb-000005
其中：
R选自氘原子、C 1-6烷基或取代烷基、芳基、取代的芳基或杂芳基；
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；优选C 3-6环烷基烷基或者C 3-6环烷基；
R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；优选C 3-6环烷基烷基或者C 3-6环烷基；
或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
n为0或者1；
本发明所示的通式(D)所示的化合物包括但不局限于:
Figure PCTCN2020115807-appb-000006
Figure PCTCN2020115807-appb-000007
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、其混合物形式或其药学上可接受的盐或溶剂化物；包括上述任一化合物的喜树碱衍生物类抗肿瘤药，非限制性地用于制备肺癌、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药物。
在本发明的另一个优选的实施例中，根据本发明所述的抗体-药物偶联物或者其药学上可接受的盐或者溶剂化合物，其中接头单元-L-为-L 1-L 2-L 3-L 4-，其L 1端与抗体相连，L 4端与X相连；
其中，L 1选自，但不局限于：-Y-C(O)-、-CH 2-C(O)-NR 5-Y-C(O)-或者-C(O)-Y-C(O)-，其中Y非限制性地选自C 1-8烷基、C 1-8烷基-环烷基或1-8个原子的直链或直链-环状杂烷基，所述杂烷基包含选自N、O或者S的1-3个原子，所述的C 1-8烷基、环烷基、直链或直链环状杂烷基各自独立的被选自氘代原子、卤素、羟基、氰基、硝基、氨基、烷基、杂烷基、取代烷基、羧基、烷氧基或环烷基的一个或者多个取代基所取代；
本发明的另一些实施方案中，抗体-药物偶联物或者其药学上可接受的盐或溶剂化物，其中连接单元L 1选自，但不局限于：-(琥珀酰亚胺-3-基-N-)-(CH 2) o-C(O)-、-(琥珀酰亚胺-3-基-N)-CH 2-环己基-C(O)-、-(琥珀酰亚胺-3-基-N-)-(CH 2CH 2O) o-C(O)-、-CH 2-C(O)-NR 5-(CH 2) o-C(O)-或者-C(O)-(CH 2) o-C(O)-，其中o选自2-8的整数，优选为5；
L 2选自-NR 6(CH 2CH 2O) pCH 2CH 2C(O)-、-NR 6(CH 2CH 2O) pCH 2C(O)-、-S(CH 2) pC(O)- 或者化学键，其中p选自0-20的整数；
本发明的一些实施方案中，提供的抗体-药物偶联物或者其药学上可接受的盐或溶剂化物，其中连接单元L 2选自-NR 6(CH 2CH 2O) pCH 2C(O)-或者化学键，其中p选自0-12的整数。
L 3非限制性地选自由2-7个氨基酸构成的肽残基，其中任选氨基酸进一步被选自氘原子、卤素、羟基、氰基、氨基、硝基、烷基、取代烷基、烷氧基和环烷基或取代环烷基中的一个或多个取代基所取代；
本发明的一些实施方案中，提供的抗体-药物偶联物或者其药学上可接受的盐或溶剂化合物，其中所述的L 3的肽残基为由一个、两个或者多个选择苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨酸(E)或者天冬氨酸(D)中的氨基酸形成的多肽残基。优选为由一个、两个或者多个选自苯丙氨酸和甘氨酸的氨基酸形成的氨基酸残基；更优选的为四肽残基；最优选的为GGFG(甘氨酸-甘氨酸-苯丙氨酸-甘氨酸)组成的四肽残基。
L 4选自-NR 7(CR 8R 9) q-、-C(O)NR 7、-C(O)NR 7(CH 2) q-或者化学键，其中q选自0-6的整数，优选为-NR 7(CR 8R 9) q-；
R 5、R 6和R 7相同或者不同，且各自独立地为氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R 8和R 9相同或者不同，且各自独立地为氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
本发明的另一些实施方案中，提供的抗体-药物偶联物或者其药学上可接受的盐或溶剂化物，其中L 4选自-NR 7(CR 8R 9) q-，R 7选自氢原子、氘原子或者烷基，R 8和R 9相同或者不同，且各自独立地位氢原子、氘原子或者烷基，q为1或者2；L 4优选为-NR 7CR 8R 9-；L 4更优选为-NHCH 2-。
本发明一方面的一个优选方案，提供的通式(L-X-D 2)所示的药物-连接子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、或其药学上接受的盐或溶剂化物，其为通式(La-X-D 2)所示的化合物或其药学上接受的盐或溶剂化物：
Figure PCTCN2020115807-appb-000008
其中：
Z选自，但不局限于-Y-C(O)-、-CH 2-C(O)-NR 5-Y-C(O)-或-C(O)-Y-C(O)-，其中Y，选自C 1-8烷基、C 1-8烷基-环烷基或1-8个原子的直链或直链-环状杂烷基，所述杂烷基包含选自N、O或者S的1-3个原子，所述的C 1-8烷基、环烷基、直链或直链环状杂烷基各自独立的被选自氘代原子、卤素、羟基、羧基、氰基、硝基、氨基、烷基、杂烷基、取代烷基、羧基、烷氧基或环烷基的一个或者多个取代基所取代；
L 2选自-NR 6(CH 2CH 2O) pCH 2CH 2C(O)-、-NR 6(CH 2CH 2O) pCH 2C(O)-、-S(CH 2) pC(O)-或化学键，其中p优选为0-20的整数，优选为化学键；
L 3选自由2-7个氨基酸构成的肽残基，其中任选氨基酸进一步被选自氘原子、卤素、羟基、氰基、氨基、硝基、烷基、取代烷基、烷氧基和环烷基或者取代环烷基中的一个或多个取代基所取代；
R选自氘原子、C 1-6烷基或取代烷基、芳基、取代芳基或杂芳基；
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R b选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
R 5、R 6和R 7相同或者不同，且各自独立地选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R 8和R 9相同或者不同，且各自独立地选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
n选自0至4的整数，优选为0或1，更优选为0。
本发明一方面的一个优选方案，提供的通式(L-X-D 2)所示的药物-连接子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、或其药学上接受的盐或溶剂化物，其为通式(Lb-X-D 2)所示的化合物或其药学上接受的盐或溶剂化物：
Figure PCTCN2020115807-appb-000009
其中：
Z选自，但不局限于-Y-C(O)-、-CH 2-C(O)-NR 5-Y-C(O)-或-C(O)-Y-C(O)-，其中Y，选自C 1-8烷基、C 1-8烷基-环烷基或1-8个原子的直链或直链-环状杂烷基，所述杂烷基包含选自N、O或者S的1-3个原子，所述的C 1-8烷基、环烷基、直链或直链环状杂烷基各自独立的被选自氘代原子、卤素、羟基、羧基、氰基、硝基、氨基、烷基、杂烷基、取代烷基、羧基、烷氧基或环烷基的一个或者多个取代基所取代；
R选自氘原子、C 1-6烷基或取代烷基、芳基、取代芳基或杂芳基；
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R b选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
R 7选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R 8和R 9相同或者不同，且各自独立地氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
n选自0至4的整数，优选为0或1，更优选为0。
本发明的一些实施方案中，提供的抗体-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、非对映异构体或者其混合物形式，或其药学上可接受的盐或者溶剂化物，其为通式(L-X-Dr)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，其选 自，但不局限于：
Figure PCTCN2020115807-appb-000010
Figure PCTCN2020115807-appb-000011
Figure PCTCN2020115807-appb-000012
Figure PCTCN2020115807-appb-000013
本发明的一些实施方案中，提供的抗体-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、非对映异构体或者其混合物形式，或者其药学上可接受的盐或溶剂化物，其为通式(Ab-L-X-D r)所示的抗体-药物偶联物或者其药学上可接受的盐或溶剂化物：
Figure PCTCN2020115807-appb-000014
其中：
R选自氘原子、C 1-6烷基或取代烷基、芳基、取代的芳基或杂芳基；
X选自-C(O)-CR aR b-O-(CR 3R 4) n-、-C(O)-CR aR b-NH-(CR 3R 4) n-或-C(O)-CR aR b-(CR 3R 4) n-S-；
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R b选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
或者，R 3、R 4连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或者杂环基；
n选自0至4的整数；
m选自1-10的整数或小数；
Ab为抗体、抗体片段或蛋白；
L为连接单元。
本发明的另一些实施方案中，提供的抗体-药物偶联物或者其药学上可接受的盐或溶剂化物，其为通式(Ab-L-X-D 2)所示的抗体-药物偶联物或者其药学上可接受的盐或溶剂化物；
Figure PCTCN2020115807-appb-000015
其中：
R选自氘原子、C 1-6烷基或取代烷基、芳基、取代的芳基或杂芳基；
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；优选C 3-6环烷基烷基或者C 3-6环烷基；
R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；优选C 3-6环烷基烷基或者C 3-6环烷基；
或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
n为0或者1；
m选自1至10的整数或小数；优选n为2至8，可以为整数，也可以为小数；更优选n为3至8，可以为整数，也可以为小数；
Ab为抗体；L为接头单元。
本发明的另一些实施方案中，提供的通式(Ab-L-X-Dr)所示的抗体-药物偶联物或者其药学上可接受的盐或溶剂化物，其为通式(Ab-L a-X-D 2)所示的抗体-药物偶联物或者其药学上可接受的盐或溶剂化物；
Figure PCTCN2020115807-appb-000016
其中：Z选自，但不局限于-Y-C(O)-、-CH 2-C(O)-NR 5-Y-C(O)-或-C(O)-Y-C(O)-，其中 Y，选自C 1-8烷基、C 1-8烷基-环烷基或者1-8个原子的直链或者直链-环状杂烷基，所述杂烷基包含选自N、O或者S的1-3个原子，所述的C 1-8烷基、环烷基、直链或者直链环状杂烷基各自独立的被选自氘代原子、卤素、羟基、羧基、氰基、硝基、氨基、烷基、杂烷基、取代烷基、羧基、烷氧基或者环烷基的一个或者多个取代基所取代；
L 2非限制性地选自-NR 6(CH 2CH 2O) pCH 2CH 2C(O)-、-NR 6(CH 2CH 2O) pCH 2C(O)-、-S(CH 2) pC(O)-或化学键，其中p优选为0-20的整数，优选为化学键；
L 3非限制性地选自由2-7个氨基酸构成的肽残基，其中任选氨基酸进一步被选自氘原子、卤素、羟基、氰基、氨基、硝基、烷基、取代烷基、烷氧基和环烷基或者取代环烷基中的一个或多个取代基所取代；
R选自氘原子、C 1-6烷基或取代烷基、芳基、取代的芳基或杂芳基；
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R b选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或者杂环基；
或者，R 3、R 4连同其所连接碳原子构成C 3-6环烷基或者杂环基；
R 5、R 6和R 7相同或者不同，且各自独立地氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R 8和R 9相同或者不同，且各自独立地氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
n选自0至4的整数，优选为0或1，更优选为0；
m选自1-10，可以为整数也可以为小数；
Ab为抗体、抗体片段或蛋白。
本发明的另一些实施方案中，提供的通式(Ab-L-X-D r)所示的抗体-药物偶联物或其药学上可接受的盐或者溶剂化物，其为通式(Ab-L b-X-D 2)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化物；
Figure PCTCN2020115807-appb-000017
其中：
Ab、R a、R b、R 7-R 9、Z、m和n如通式(Ab-La-X-Dr)所述。
本发明通式(Ab-L-X-Dr)所示的抗体-药物偶联物非限制性地选自以下结构：
Figure PCTCN2020115807-appb-000018
Figure PCTCN2020115807-appb-000019
Figure PCTCN2020115807-appb-000020
本发明的另一些实施方案中，所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述的Ab为抗体、抗体片段或者其抗原结合片段，所述的抗体选自嵌合抗体、人源化抗体或全人源抗体；优选为单克隆抗体。
本发明的另一些实施方案中，所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述的抗体、抗体片段或者其抗原结合片段非限制性地选自：抗EGFRvIII抗体、抗DLL-3抗体、抗PSMA抗体、抗CD70抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM-1抗体、抗LRRC15抗体、抗LIV-1抗体、抗CanAg/AFP抗体、抗cladin 18.2抗体、抗Mesothelin抗体、抗HER2(ErbB2)抗体、抗EGFR抗体、抗c-MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7-H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗TROP-2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P-cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体或抗CD123抗体。
本发明的另一方面，提供了一种制备如通式(Ab-L a-X-D 2)所示的抗体-药物偶联物或者其药学上可接受的盐或溶剂化物的方法，其包括如下步骤：
Figure PCTCN2020115807-appb-000021
通过抗体、抗体片段或者其抗原结合片段与通式(L a-X-D 2)偶联反应，得到通式(Ab-La-X-D 2)；
其中：Ab为抗体、抗体片段或者其抗原结合片段；
R、R a、R b、R 7、R 8、R 9、L 2、L 3、Z及n如上述通式(Ab-La-X-D 2)所定义。
本发明的另一方面，进一步涉及一种药物组合物，其含有根据本发明所述的通式(D)所示的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，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明的另一方面，进一步涉及一种抗体-药物偶联物或其药学上可接受的盐或溶剂化物，其包含配体和连接至配体的药物，其中所述药物选自本发明所述的通式(D)所示的化合物、通式(L-X-Dr)所示的化合物、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，优选药物通过接头连接至配体，优选配体为单克隆抗体。
本发明的另一方面，进一步涉及一种抗体-药物偶联物或其药学上可接受的盐或溶剂化物的制备方法，包含将本发明所述的通式(D)所示的化合物、通式(L-X-Dr)所示的化合物、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，或其可药用的盐与配体连接的步骤，优选通过接头连接，优选配体为单克隆抗体。
本发明的另一方面，进一步涉及本发明所述的抗体-药物偶联物或化合物、或其药学上可接受的盐或溶剂化物，或其药物组合物，其用作药物。
本发明的另一方面，进一步涉及本发明所述的抗体-药物偶联物或化合物、或其药学上可接受的盐或溶剂化物，或其药物组合物在制备用于治疗或预防肿瘤的药物中的用途。
本发明的另一方面，进一步涉及本发明所述的抗体-药物偶联物或化合物、或其药学上可接受的盐或溶剂化物，或药物组合物在制备治疗和/或预防癌症的药物的用途，所述癌症非限制性地选自：乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、肺癌、结肠癌、直肠癌、结直肠癌、骨癌、皮肤癌、甲状腺癌、胰腺癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、多形性胶质细胞瘤、肉瘤、淋巴瘤和白血病等实体瘤或血液肿瘤。
本发明的另一方面，进一步涉及一种用于治疗和/或预防肿瘤的方法，该方法包括向需要其的患者施用治疗有效剂量的本发明所述的抗体-药物偶联物或化合物、或其药学上可接受的盐或溶剂化物或包含其的药物组合物。
本发明的另一方面，进一步涉及一种用于治疗或预防癌症的方法，该方法包括向需要其的患者施用治疗有效剂量的本发明所述的抗体-药物偶联物或化合物、或其药学上可接受的盐或溶剂合物或包含其的药物组合物；所述癌症非限制性地选自：乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、肺癌、结肠癌、直肠癌、结直肠癌、骨癌、皮肤癌、甲状腺癌、胰腺癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、多形性胶质细胞瘤、肉瘤、淋巴瘤和白血病等实体瘤或血液肿瘤。
可将活性化合物制成适合于通过任何适当途径给药的形式，活性化合物优选是以单位剂量的方式，或者是以患者可以以单剂自我给药的方式。本发明化合物或组合物的单位剂量的表达方式可以是片剂、胶囊、扁囊剂、瓶装药水、药粉、颗粒剂、锭剂、栓剂、再生药粉或液体制剂。
本发明治疗方法中所用化合物或组合物的剂量通常将随疾病的严重性、患者的体重和化合物的相对功效而改变。不过，作为一般性指导，合适的单位剂量可以是0.1-1000mg。
本发明的药物组合物除活性化合物外，可含有一种或多种辅料，所述辅料非限制性地选自以下成分：填充剂(稀释剂)、粘合剂、润湿剂、崩解剂或赋形剂等。根据给药方法的不同，组合物可含有0.1至99重量％的活性化合物。
含活性成分的药物组合物可以是适用于口服的形式，例如片剂、糖锭剂、锭剂、水或油混悬液、可分散粉末或颗粒、乳液、硬或软胶囊，或糖浆剂或酏剂。可按照本领域任何己知制备药用组合物的方法制备口服组合物，此类组合物可有粘合剂、填充剂、润滑剂、崩解剂或药学上可接受的润湿剂等，此类组合物还可以含有一种或多种选自以下的成分：甜味剂、矫味剂、着色剂和防腐剂，以提供悦目和可口的药用制剂。
水悬浮液含有活性物质和用于混合的适宜制备水悬浮液的赋形剂。水混悬液也可以含有一种或多种防腐剂例、一种或多种着色剂、一种或多种矫味剂和一种或多种甜味剂。
油混悬液可通过使活性成分悬浮于植物油中配制而成。油悬浮液可含有增稠剂。可加入上述的甜味剂和矫味剂，以提供可口的制剂。
药物组合物还可以是用于制备水混悬液的可分散粉末和颗粒提供活性成分，通过加入水混合分散剂、湿润剂、悬浮剂或防腐剂中的一种或多种。也可加入其他赋形剂例如甜味剂、矫味剂和着色剂。通过加入抗氧化剂例如抗坏血酸保存这些组合物。
本发明的药物组合物也可以是水包油乳剂的形式。
药物组合物可以是无菌注射水溶液形式。可以使用的可接受的溶媒或溶剂有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。例如将活性成分溶于大豆油和卵磷脂的混合物中。然后将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入患者的血流中。或者，最好按可保持本发明化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的实例是Deltec CADD-PLUS.TM.5400型静脉注射泵。
药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按己知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在肠胃外可接受的无毒稀释剂或溶剂中制备的无菌注射溶液或混悬液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。
可按用于直肠给药的栓剂形式给予本发明化合物。可通过将药物与在普通温度下为固体但在直肠中为液体，因而在直肠中会溶化而释放药物的适宜的无刺激性赋形剂混合来制备这些药物组合物。此类物质包括可可脂、甘油明胶、氢化植物油、各种分子量的聚乙二醇和聚乙二醇的脂肪酸酯的混合物。
如本领域技术人员所熟知的，药物的给药剂量依赖于多种因素，包括但并非限定于以下因素：所用具体化合物的活性、患者的年龄、患者的体重、患者的健康状况、患者的行 为、患者的饮食、给药时间、给药方式、排泄的速率、药物的组合等；另外，最佳的治疗方式如治疗的模式、通式化合物的日用量或可药用的盐的种类可以根据传统的治疗方案来验证。
附图说明
图1为ADC对A431荷瘤小鼠模型的体内药效；
图2为本发明化合物6、12、14和16对Fadu细胞的体外活性数据图；
图3为本发明化合物6、12、14和16对A431细胞的体外活性数据图；
图4为本发明化合物6、12、14和16对Bxpc-3细胞的体外活性数据图；
图5为本发明化合物6、12、14和16对SW620细胞的体外活性数据图。
具体实施方式
除非另有限定，本文所用的所有技术和科学术语均与本发明所属领域普通技术人员的通常理解一致。虽然也可采用与本文所述相似或等同的任何方法和材料实施或测试本发明，但本文描述了优选的方法和材料。描述和要求保护本发明时，依据以下定义使用下列术语。
当本发明中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有相同的下述含义。
术语“抗体”是能识别和结合目标细胞相关的抗原或受体的大分子化合物。抗体的作用是将药物呈递给与抗体结合的目标细胞群，这些抗体包括但不限于蛋白类激素、凝集素、生长因子、抗体或其他能与细胞结合的分子。在本发明实施方式中，抗体表示为Ab，抗体可通过其杂原子与连接单元形成连接键，优选为抗体或其抗原结合片段，所述抗体选自嵌合抗体、人源化抗体、全人抗体或鼠源抗体；优选为单克隆抗体。
术语“药物”是指细胞毒性药物，药物表示为D，能在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。该术语包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32和Lu的放射性同位素)，毒性药物，化疗药物，抗生素和核溶酶，优选为毒性药物。
术语“接头单元”或“连接片段”或“连接单元”是指一端与配体连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。本发明的优选方案表示为L 和L l-L 4，其中L l端与配体相连，L 4端与结构单元X相连后与药物(D)相连。
连接头，包括延伸物、间隔物和氨基酸单元，可以通过本领域己知方法合成，诸如US2005-0238649A1中所记载的。连接头可以是便于在细胞中释放药物的“可断裂接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等Cancer Research 52:127-131,1992)；美国专利No.5,208,020)。
术语“抗体-药物偶联物"，指抗体通过稳定的连接单元与具有生物活性的药物相连。在本发明中优选为抗体-药物偶联物(antibody drug conjugate,ADC)，指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的毒性药物相连。
本发明所用氨基酸三字母代码和单字母代码如J.biol.Chem,1968,243,3558.中所述。
术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。本发明所述的抗体优选为针对靶细胞上细胞表面抗原的特异性抗体，非限制性实施例为以下抗体：抗EGFRvIII抗体、抗DLL-3抗体、抗PSMA抗体、抗CD70抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM-1抗体、抗LRRC15抗体、抗LIV-1抗体、抗CanAg/AFP抗体、抗cladin 18.2抗体、抗Mesothelin抗体、抗HER2(ErbB2)抗体、抗EGFR抗体、抗c-MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7-H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗TROP-2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P-cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD33抗体、抗 CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体或抗CD123抗体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FRI，CDRI，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本发明的抗体包括鼠源抗体、嵌合抗体、人源化抗体和全人源抗体，优选人源化抗体和全人源抗体。
术语“鼠源抗体"在本公开中为根据本领域知识和技能用鼠制备抗体。制备时用特定抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交
术语“嵌合抗体(chimeric antibody)"，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。
术语“人源化抗体(humanized antibody)也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网 www.mrccpe.com/ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本发明的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。进一步描述参与人源化可使用小鼠抗体的方法的文献包括，例如Queen等，Proc.,Natl.Acad.Sci.USA,88,2869,1991和Winter及其同事的方法[Jones.,Nature,321,522,(1986)],Riechmann,等[Nature,332,323-327,1988)，Verhoeyen,等，Science,239,1534(1988)]。
术语“全人源抗体”、“全人抗体”或“完全人源抗体”，也称“全人源单克隆抗体”，其抗体 的可变区和恒定区都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。本发明为全人源单克隆抗体。全人抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。
术语“抗原结合片段”是指抗体的保持特异性结合抗原的能力的一个或多个片段。己显示可利用全长抗体的片段来进行抗体的抗原结合功能。“抗原结合片段"中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341:544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426和Huston等人(1 988)Proc.NatL.Acad.Sci.USA85：5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段"中。使用本领域技术人员己知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1,IgG2，IgG3或IgG4亚型)，IgA1,IgA2，IgD，IgE或lgM抗体。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50，000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
F(ab’)2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
Fab’是通过切割上述F(ab’)2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。
此外，可以通过将编码抗体的Fab’片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab’来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域 (或区域；(H)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8：725-731,Choi等人(2001)，Eur.J.Immuno 1.31:94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001),Cancer Immunol.描述。
术语"CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR Ll、CDRL2、CDRL3或Ll、L2、L3)，以及重链可变结构域的CDR2和CDR3(CDR H2、CDR H3或H2、H3)。
术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通常以独特的空间构象包括至少3,4,5,6,7,8,9,10,11,12,13,14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology,第66卷，G.E.Morris,等.(1996)。
术语“特异性结合”“选择性结合”“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如大约小于10 -8M、10 -9M或10 -10M或更小的亲和力(KD)结合。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输己与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中发明的载体能够在己引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载 体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从1mMunoGeneTics(MGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指己经向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
术语“肽”是指介于氨基酸和蛋白质之间的化合物片段，由2个或2个以上氨基酸分子通过肽键相互连接而成，是蛋白质的结构与功能片段，如激素、酶类等本质上都是肽。
术语“糖”是指由C、H、O三种元素组成的生物大分子，可分为单糖、二糖和多糠等。
术语“荧光探针”是指在紫外-可见-近红外区有特征荧光，并且其荧光性质(激发和发射波长、强度、寿命和偏振等)可随所处环境的性质，如极性、折射率、粘度等改变而灵敏地改变的一类荧光性分子，其与核酸(DNA或RNA)、蛋白质或其他大分子结构非共价相互作用而使一种或几种荧光性质发生改变，可用于研宄大分子物质的性质和行为。
术语“毒性药物”是指抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质。包括毒 素和其他能用于肿瘤治疗的化合物。
术语“毒素”是指能够对细胞的生长或增殖产生有害效果的任何物质，可以是来自细菌、真菌、植物或动物的小分子毒素及其衍生物，包括喜树碱类衍生物如伊沙替康，美登木素生物碱及其衍生物(CN101573384)如DM1、DM3、DM4,auristatin F(AF)及其衍生物，如MMAF、MMAE、3024(WO 2016/127790 A1)，白喉毒素、外毒素、蓖麻毒蛋白(ricin)A链、相思豆毒蛋白(abrin)A链、modeccin、α-帚曲霉素(sarcin)、油桐(Aleutites fordii)毒蛋白、香石竹(dianthin)毒蛋白、美洲商陆(Phytolaca americana)毒蛋白(PAPI、PAPII和PAP-S)、苦瓜(Momordica charantia)抑制物、麻疯树毒蛋白(curcin)、巴豆毒蛋白(crotin)、肥皂草(sapaonaria officinalis)抑制物、白树毒蛋白(gelonin)、丝林霉素(mitogellin)局限曲霉素(restrictocin)、酚霉素(phenomycin)、依诺霉素(enomycin)和单端孢菌素(trichothecenes)。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2，3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2，3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、 卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷基如上所定义。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子，更优选含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH 2-)、1,1-亚乙基[-CH(CH 3)-]、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基[-CH(CH 2CH 3)-]、1,2-亚丙基[-CH 2CH(CH 3)-]、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)和1,5-亚丁基(-CH 2CH 2CH 2CH 2CH 2-)等。亚烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个取代基所取代。
术语“烷氧基"指-O-(烷基)和-O-(非取代的环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O) u(其中u是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选环烷基环包含3至10个环原子。单环杂环基的非限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和 桥环的杂环基。
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，其非限制性实例包括：
Figure PCTCN2020115807-appb-000022
等。
杂环基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“芳基”指具有共轭的兀电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元，例如苯基和萘基，优选苯基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
Figure PCTCN2020115807-appb-000023
等。
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
Figure PCTCN2020115807-appb-000024
Figure PCTCN2020115807-appb-000025
等。
杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“氨基保护基”是为了使分子其它部位进行反应时氨基保持不变，用易于脱去的基团对氨基进行保护。非限制性实施例包含9-芴甲氧羰基、叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基等。这些基团可任选地被选自卤素、烷氧基或硝基中的1-3个取代基所取代。所述氨基保护基优选为9-芴甲氧羰基。
术语“环烷基烷基”指烷基被一个或多个环烷基取代，优选被一个环烷基取代，其中烷基如上所定义，其中环烷基如上所定义。
术语“卤代烷基”指烷基被一个或多个卤素取代，其中烷基如上所定义。
术语“氘代烷基”指烷基被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH 2。术语“硝基”指-NO 2。
术语“酰胺基"指-C(O)N-(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
术语“羧酸酯基"指-C(O)O-(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
本发明还包括各种氘化形式的式(D)化合物。与碳原子连接的各个可用的氢原子可独立地被氘原子替换。本领域技术人员能够参考相关文献合成氘化形式的式(D)化合物。在制备氘代形式的式(D)化合物时可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂包括但不限于氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团"意味着烷基可以但不必须存在，该说明包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
“取代”或“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1-3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或 不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的盐”或“可药用盐”是指本发明抗体-药物偶联物的盐，或本发明中所述的化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性，本发明抗体-药物偶联化合物至少含有一个氨基，因此可以与酸形成盐，药学上可接受的盐的非限制性实例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
术语“溶剂化物”或“溶剂化合物”指本发明的抗体-药物偶联化合物与一种或多种溶剂分子形成可药用的溶剂化物，溶剂分子的非限制性实例包括水、乙醇、乙腈、异丙醇、DMSO、乙酸乙酯。
术语“载药量”是分子中每个抗体上加载的细胞毒性药物平均数量，也可以表示为药物量和抗体量的比值，药物载量的范围可以是每个抗体(Ab)连接0-12个，优选1-10个细胞毒性药物(D)。在本发明的实施方式中，载药量表示为m,示例性的可以为1,2，3，4，5，6，7，8，9，10的均值。可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的药物品均数量。
本发明的一个实施方式中，细胞毒性药物通过连接单元偶联在配体的N端氨基和/或赖氨酸残基的ε-氨基上，一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。
可以用以下非限制性方法控制配体细胞毒性药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
常规的药物组合物的制备见中国药典。
术语“载体"用于本发明的药物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药 物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
术语“赋形剂”是在药物制剂中除主药以外的附加物，也可称为辅料。如片剂中的黏合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。如淀粉、乳糖、钙的无机盐、微晶纤维素等。
药物组合物可以是无菌注射水溶液形式。可在使用的可接受的溶媒和溶剂中有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。例如将活性成分溶于大豆油和卵磷脂的混合物中。然后将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入患者的血流中。或者，最好按可保持本发明化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的实例是Deltec CADD-PLUS.TM.5400型静脉注射泵。
药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按己知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液，例如1,3-丁二醇中制备的溶液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。为此目的，可使用包括合成甘油单或二酯在内的任何调和固定油。此外，脂肪酸例如油酸也可以制备注射剂。
本发明涉及一类可裂解的特定结构的连接子和特定结构的药物，及由连接子、药物与抗体组成的抗体药物偶联物(ADC)。此类ADC是经由间隔物将一种毒性物质连于抗体而形成的复合物。该抗体偶联药物(ADC)在体内经降解而释放出活性分子，从而起到相应的作用。
本发明的合成方法
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“烷基”是指具有1-20个碳原子的饱和烃基团。亚烷基基团的实施例包括但不限于甲基(-CH 3),乙基(-CH 2-CH 3),正丙基，正丁基，正戊基和正己基。
术语“取代烷基”指烷基中的氢被取代基团取代，除非文中另有说明，烷基的取代基可以是选自下组的多种基团：-卤素、-OR’、-NR’R”、-SR’、-SiR’R”R”’、-OC(O)R’、-C(O)R’、-CO 2R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R”’、-NR”C(O) 2R’、-NH-C(NH 2)＝NH、-NR’C(NH 2)＝NH、-NH-C(NH 2)＝NR’、-S(O)R’、-S(O) 2R’、-S(O) 2NR’R”、-NR’S(O) 2R”、-CN和-NO 2，取代基数量为0至(2m’+1)，其中m’为该基团中碳原子的总数。R’、R”和R”’各自独立的指代氢、未取代的C 1-8烷基、未取代的芳基、由1-3个卤素取代的芳基、未取代的C 1-8烷基、C 1-8烷氧基或C 1-8硫代烷氧基、或未取代的芳基-C 1-4烷基。R’和R”连接于同一个氮原子时，它们可与该氮原子一起形成3-，4-，5-，6-或7-元环。例如，-NR’R”包括1-吡咯烷基和4-吗啉基。
“酸性氨基酸”指氨基酸的等电点小于7，酸性氨基酸分子中往往带有1个或多个羧基等酸性基团，在结构中可有效电离为负离子形式而增加亲水性。酸性氨基酸可以为天然的，也可为非天然的氨基酸。
“天然氨基酸”指由生物合成的氨基酸。天然氨基酸一般情况下是L-型的，但也有少数例外，比如甘氨酸，包括天然的和生物体合成的。
“非天然氨基酸”指通过合成手段所获得的氨基酸。
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)的药学上可接受的有机或无机盐。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)。
下面结合具体实施例，进一步阐述本发明。这些实施例仅用于说明本发明而不是用于限制本发明的范围。除非另行定义，文中所使用的所有专业与科学用语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：
Figure PCTCN2020115807-appb-000026
化合物2的合成：
向伊喜替康甲磺酸盐1(150.0mg,282.4umol，采用专利申请“EP0737683A1”所公开的方法制备)中加入8mL DMF，冰水浴冷却至0℃，滴加5滴三乙胺，调节pH至7-8，冰水浴下滴加2滴溴化苄，升温至室温(25℃)下反应1小时，TLC监测反应完全，反应液减压浓缩，所得粗产品经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物2约110mg，黄色固体，收率约74％，MS m/z:[M+H] +526.3。
化合物3的合成：
室温下，于25mL单口瓶中依次加入化合物2(110mg,209.4umol),4mL甲酸溶解，向所得亮黄色溶液中加入1mL甲醛(40％水溶液)，升温至50℃下反应1小时后，TLC监测反应完全，冷却至室温，反应液经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物3约45mg，黄色粉末状固体，收率约40％，MS m/z:[M+H] +540.6。
化合物4的合成：
室温下，于25mL单口瓶中加入化合物3(23mg,42.7umol),加入2mL DMF溶解，向所得亮黄色溶液中加入23mg 5％Pd/C，氢气气球置换体系内气氛，维持室温下反应1.5小时后，HPLC监测反应完全，过滤除去Pd/C，所得反应液浓缩后，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物4约10mg，黄色粉末状固体，收率约52％，MS m/z:[M+H] +450.5。
化合物5的合成：
室温下，于5mL瓶中加入化合物4(10mg,22.3umol),加入2mL DMF溶解，向所得溶液中依次加入DIPEA(7.8uL,2eq)，苄氧基乙酰氯(8.2mg,2eq)，维持室温下反应 0.5小时，TLC监测反应完全，所得反应液浓缩后，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物5约11mg，黄色粉末状固体，收率约83％，MS m/z:[M+H] +598.3。
化合物6的合成：
室温下，于25mL单口瓶中加入化合物5(11mg,18.4umol),加入1mL DMF溶解，向所得溶液中加入11mg 10％Pd/C，氢气气球置换体系内气氛，维持室温下反应1小时，TLC监测反应完全，过滤除去Pd/C，所得反应液浓缩后，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物6约2.6mg，黄色粉末状固体，收率约28％，MS m/z:[M+H] +508.2。
实施例2：
Figure PCTCN2020115807-appb-000027
化合物7的合成：
向伊喜替康甲磺酸盐1(100.0mg,188.3umol，采用专利申请“EP0737683A1”所公开的方法制备)中加入4mL DMF，冰水浴冷却至0℃，滴加三乙胺(105uL，4eq)，冰水浴下滴加碘乙烷(34uL，1.5eq)，升温至室温(25℃)下反应4小时后，反应液减压浓缩，所得粗产品经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物7约11mg，黄色固体，收率约13％，MS m/z:[M+H] +464.2。
化合物8的合成：
室温下，于25mL瓶中加入化合物7(10mg,22umol),加入2mL DMF溶解，向所得溶液中依次加入DIPEA(8uL,2eq)，苄氧基乙酰氯(8mg,2eq)，维持室温下反应0.5小时，TLC监测反应完全，所得反应液浓缩后，经制备级高效液相色谱法纯化(乙腈/纯 水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物8约8mg，黄色固体，收率约60％，MS m/z:[M+H] +612.7。
化合物9的合成：
室温下，于25mL单口瓶中加入化合物8(8mg,13.1umol),加入2mL DMF溶解，向所得溶液中加入8mg 10％Pd/C，氢气气球置换体系内气氛，维持室温下反应3小时后，过滤除去Pd/C，TLC监测反应完全，所得反应液浓缩后，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物9约3.5mg，黄色固体，收率约51％，MS m/z:[M+H] +522.2。
实施例3：
Figure PCTCN2020115807-appb-000028
化合物10的合成：
向伊喜替康甲磺酸盐1(100.0mg,188.3umol，采用专利申请“EP0737683A1”所公开的方法制备)中加入4mL DMF，冰水浴冷却至0℃，滴加三乙胺(105uL，4eq)，冰水浴下滴加烯丙基溴(24.4uL，1.5eq)，升温至室温(25℃)下反应4小时，反应液减压浓缩，所得粗产品经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物10约13mg，黄色固体，收率约15％，MS m/z:[M+H] +476.2。
化合物11的合成：
室温下，于25mL瓶中加入化合物10(10mg,21umol),加入2mL DMF溶解，向所得溶液中依次加入DIPEA(8uL,2eq)，苄氧基乙酰氯(8mg,2eq)，维持室温下反应0.5小时，HPLC监测反应完全，所得反应液浓缩后，经制备级高效液相色谱法纯化(乙腈/纯 水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物11约7mg，黄色固体，收率约53％，MS m/z:[M+H] +624.6。
化合物12的合成：
室温下，于25mL单口瓶中加入化合物11(6mg,10umol),加入2mL DMF溶解，向所得溶液中加入6mg 10％Pd/C，氢气气球置换体系内气氛，维持室温下反应3小时后，过滤除去Pd/C，TLC监测反应完全，所得反应液浓缩后，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物12约2.7mg，黄色固体，收率约50％，MS m/z:[M+H] +536.2。
实施例4：
Figure PCTCN2020115807-appb-000029
化合物14的合成：
室温下，于10mL瓶中加入化合物4(3mg,6.7umol),加入2mL DMF溶解，冰水浴冷却至0℃，向所得溶液中依次加入DIPEA(2.2uL,2eq)，1-羟基环丙基甲酸13(1.4mg,2eq)以及4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(DMTMM,4mg,2eq)，维持0℃下反应2小时，TLC监测反应完全，0℃下向所得反应液中加入5mL饱和NaCl水溶液淬灭反应，水相经乙酸乙酯(10mL×3)萃取，合并有机相，无水硫酸钠干燥，过滤，滤液经减压浓缩，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物14约1.6mg，黄色固体，收率约45％，MS m/z:[M+H] +534.2。
实施例5：
Figure PCTCN2020115807-appb-000030
化合物16的合成:
室温下，于10mL瓶中加入化合物4(3mg,6.7umol),加入1mL无水乙醇、0.2mL DMF以及0.15mL NMM溶解，冰水浴冷却至0℃，向所得溶液中依次加入L-(+)-乳酸(2.4mg,4eq)，1-羟基苯并三唑HOBt(3.7mg,4eq)以及1-(3-二甲氨基丙基)-3-乙基碳二亚胺基盐酸盐EDCI(5.1mg,4eq)，自然升温至室温后反应2小时，HPLC监测反应结束，直接将反应液减压浓缩，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物16约2mg,黄色固体，收率约60％，MS m/z:[M+H] +522.2。
实施例6：
Figure PCTCN2020115807-appb-000031
化合物18的合成:
室温下，于10mL瓶中加入化合物4(3mg,6.7umol),加入1mL无水乙醇、0.2mL DMF以及0.15mLNMM溶解，冰水浴冷却至0℃，向所得溶液中依次加入L-扁桃酸(4.1mg,4eq)，1-羟基苯并三唑HOBt(3.7mg,4eq)以及1-(3-二甲氨基丙基)-3-乙基碳二亚胺基盐酸盐EDCI(5.1mg,4eq)，自然升温至室温后反应4小时，TLC检测反应结束，直接将反应液减压浓缩，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物18约2.4mg,黄色粉末状固体，收率约61％，MS m/z:[M+H] +584.1。
实施例7：
Figure PCTCN2020115807-appb-000032
化合物20的合成：
室温下，于10mL瓶中加入化合物4(3mg,6.7umol),加入1mL无水乙醇、0.2mL DMF以及0.15mLNMM溶解，冰水浴冷却至0℃，向所得溶液中依次加入三氟乳酸(3.9mg,4eq)，1-羟基苯并三唑HOBt(3.7mg,4eq)以及1-(3-二甲氨基丙基)-3-乙基碳二亚胺基盐酸盐EDCI(5.1mg,4eq)，自然升温至室温后反应1小时，直接将反应液减压浓缩，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物20A约1.2mg，黄色粉末状固体，化合物20B约1.2mg,黄色粉末状固体，收率约61％，MS m/z:[M+H] +576.4。
实施例8：
Figure PCTCN2020115807-appb-000033
化合物22的合成：
室温下，于10mL瓶中加入化合物4(4mg,8.9umol),加入1mL无水乙醇、0.2mL DMF以及0.15mLNMM溶解，冰水浴冷却至0℃，向所得溶液中依次加入化合物21(4.1mg,4eq采用专利申请“WO2013106717”公开的方法制备)，1-羟基苯并三唑HOBt(3.7mg, 4eq)以及1-(3-二甲氨基丙基)-3-乙基碳二亚胺基盐酸盐EDCI(5.1mg,4eq)，自然升温至室温后反应2小时，TLC监测反应结束，直接将反应液减压浓缩，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物22A约1.5mg，黄色固体，化合物22B约1.5mg,黄色固体，收率约59％，MS m/z:[M+H] +576.1。
实施例9：
Figure PCTCN2020115807-appb-000034
化合物25的合成：
0℃下，于50mL单口瓶中加入化合物23(800mg,4.8mmol),加入50mL DMF向所得溶液中加入DIPEA(0.8mL,1eq)，以及化合物24(1767.0mg,1eq)，自然升温至室温后反应2小时，直接将反应液减压浓缩，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物25约727mg，黄色固体，收率约32％，MS m/z:[M+H] +475.5。
化合物26的合成：
室温下，于25mL单口瓶中加入化合物25(727mg,1.5mmol),加入15mL DMF溶解，向所得溶液中加入750mg 5％Pd/C，氢气气球置换体系内气氛，维持室温下反应3小时，TLC监测反应完成后，过滤除去Pd/C，所得反应液浓缩后，直接用于下一步反应。
化合物27的合成：
室温下，于25mL瓶中加入化合物26,加入15mL DMF溶解，冰水浴冷却至0℃，向所得溶液中依次加入DIPEA(47uL,2eq)，化合物4(1343.3mg,2eq)以及4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(DMTMM,884mg,2eq)，维持0℃下反应2小时，HPLC监测反应结束，向所得反应液中加入5mL饱和NaCl水溶液淬灭反应，水相经乙酸乙酯(10mL X 3)萃取，合并有机相，无水硫酸钠干燥，过滤，滤液经减压浓缩，经制备 级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物27约311mg，黄色固体，两步收率约25％，MS m/z:[M+H] +816.9。
化合物28的合成：
0℃下，于25mL单口瓶中加入化合物27(310mg,0.38mmol),加入5mL DMF溶解，向所得溶液中加入Diethylamine(80uL,0.76mmol)，自然升温至室温，维持室温下反应3小时后，所得反应液浓缩后，直接用于下一步反应。
化合物30的合成：
室温下，于10mL单口瓶中加入化合物28,加入5mL DMF溶解，冰水浴冷却至0℃，向所得溶液中依次加入DIPEA(126uL,2eq)，化合物29(178mg,1eq)以及4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(DMTMM,224mg,2eq)，维持0℃下反应2小时，TLC监测反应完成，向所得反应液中加入10mL饱和NaCl水溶液淬灭反应，水相经乙酸乙酯(20mL X 3)萃取，合并有机相，无水硫酸钠干燥，过滤，滤液经减压浓缩，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物30约15mg，黄色固体，两步收率约1.4％，MS m/z:[M+H] +1048.1。
实施例10：
Figure PCTCN2020115807-appb-000035
化合物31的合成：
室温下，于25mL瓶中加入化合物26(50mg,0.13mmol),加入5mL DMF溶解，冰水浴冷却至0℃，向所得溶液中依次加入DIPEA(43uL,2eq)，化合物7(60mg,1eq)以及4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(DMTMM,77mg,2eq)，维持0℃下反应4小时，TLC监测反应结束，向所得反应液中加入15mL水淬灭反应，水相经乙酸乙酯(20mL X 3)萃取，合并有机相，无水硫酸钠干燥，过滤，滤液经减压浓缩，经制备 级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物31约36mg，黄色固体，收率约33％，MS m/z:[M+NH 4] +847.4。
化合物32的合成：
0℃下，于25mL单口瓶中加入化合物31(36mg,43umol),加入5mL DMF溶解，向所得溶液中加入Diethylamine(9uL,87umol)，自然升温至室温，维持室温下反应2小时后，所得反应液浓缩后，直接用于下一步反应。
化合物33的合成：
室温下，于10mL单口瓶中加入化合物32,加入2mL DMF溶解，冰水浴冷却至0℃，向所得溶液中依次加入DIPEA(7uL,2eq)，化合物29(30mg,1.5eq)以及4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(DMTMM,25mg,2eq)，维持0℃下反应3小时，TLC监测反应结束，向所得反应液中加入5mL饱和NaCl水溶液淬灭反应，水相经乙酸乙酯(10mL X 3)萃取，合并有机相，无水硫酸钠干燥，过滤，滤液经减压浓缩，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物33约6mg，黄色固体，两步收率约8.4％，MS m/z:[M+H] +1662.4。
实施例11：
Figure PCTCN2020115807-appb-000036
化合物34的合成：
参照化合物27的合成，化合物26投料48mg，得到黄色固体化合物34，23mg，收率22％，MS m/z:[M+H] +828.4。
化合物35的合成：
室温下，于25mL瓶中加入化合物34(23mg,28umol),加入5mL DMF溶解，向所得溶液中加入10％Pd/C(23mg)，H 2气球置换反应瓶中气氛三次，HPLC监测反应完全后， 过滤除去Pd/C，所得滤液经油泵减压浓缩后，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物35约11mg，黄色固体，收率约47％，MS m/z:[M+H] +830.6。
化合物36的合成：
参照化合物28的合成，化合物35投料11mg，得到黄色固体化合物36，黄色固体约6mg，收率74％，MS m/z:[M+H] +608.5。
化合物37的合成：
参照化合物30的合成，投料量6mg，得到2.6mg黄色固体化合物37，收率约25％，MS m/z:[M+H] +1062.6。
实施例12：
Figure PCTCN2020115807-appb-000037
化合物39的合成：
参照化合物25的合成，投料化合物38(31mg,161umol)，得到化合物39，52mg，收率65％，MS m/z:[M+H] +501.4。
化合物40的合成：
参照化合物26的合成，投料化合物39(51mg,100umol)，得到化合物40，未经纯化直接用于下一步反应。
化合物41的合成：
参照化合物27的合成，化合物40(按化合物39计量)，得到化合物41，21mg，两步收率25％，MS m/z:[M+H] +842.4。
化合物42的合成：
参照化合物28的合成，投料化合物41(21mg,25umol)，得到黄色固体化合物42，约9mg，收率58％，MS m/z:[M+H] +620.2。
化合物43的合成：
参照化合物30的合成，投料量5mg，得到1.4mg黄色固体化合物43，收率约16％，MS m/z:[M+H] +1074.5。
实施例13：
Figure PCTCN2020115807-appb-000038
化合物45的合成：
参照化合物25的合成，投料化合物44(25mg,139umol)，得到黄色固体化合物45，50mg，收率74％，MS m/z:[M+H] +488.3。
化合物46的合成：
参照化合物26的合成，投料化合物45(50mg,100umol)，得到黄色固体化合物46，未经纯化直接用于下一步反应。
化合物47的合成：
参照化合物27的合成，化合物46投料按化合物45计量，得到黄色固体化合物47，28mg，两步收率33％，MS m/z:[M+H] +830.4。
化合物48的合成：
参照化合物28的合成，化合物47投料27mg，得到黄色固体化合物48，约11mg，收率56％，MS m/z:[M+H] +608.5。
化合物49的合成：
参照化合物30的合成，化合物48投料量10mg，得到2.6mg黄色固体化合物49，收率约15％，MS m/z:[M+H] +1062.4。
实施例14：
Figure PCTCN2020115807-appb-000039
化合物50的合成：
参照已知文献“Organic Letters,2012,14(18),4910-4913.”的合成方法制备。
化合物51的合成：
参照化合物25的合成，投料化合物50(30mg,124umol)，得到黄色油状化合物51，47mg，收率82％，MS m/z:[M+H] +461.1。
化合物52的合成：
参照化合物26的合成，投料化合物51(46mg,100umol)，未经纯化直接用于下一步反应。
化合物53的合成：
参照化合物27的合成，化合物52投料按化合物51计量，得到黄色固体化合物53，35mg，两步收率39％，MS m/z:[M+H] +892.1。
化合物54的合成：
参照化合物28的合成，化合物53投料10mg，得到黄色固体化合物54，约4mg，收率54％，MS m/z:[M+H] +670.0。
化合物55的合成：
参照化合物30的合成，化合物54投料量4mg，得到1.8mg黄色固体化合物55，收率约27％，MS m/z:[M+H] +1124.4。
实施例15：
Figure PCTCN2020115807-appb-000040
化合物56的合成：
参照已知方法“Patent No:WO 2020063673”的合成方法制备。
化合物57的合成：
参照化合物25的合成，投料化合物56(50mg,214umol)，得到黄色油状化合物24mg，收率21％，MS m/z:[M+H] +543.1。
化合物58的合成：
参照化合物26的合成，投料化合物57(24mg,44umol)，未经纯化直接用于下一步反应。
化合物59的合成：
参照化合物27的合成，化合物58投料按化合物57计量，得到15mg化合物59，两步收率39％，MS m/z:[M+H] +884.3。
化合物60的合成：
参照化合物28的合成，化合物59投料15mg，得到化合物60，约4mg，收率36％，MS m/z:[M+H] +662.7。
化合物61的合成：
参照化合物30的合成，化合物60投料量4mg，得到2.2mg黄色固体化合物61，收率约33％，MS m/z:[M+H] +1116.7。
实施例16：
Figure PCTCN2020115807-appb-000041
化合物65的合成：
参照已知方法“Patent No:WO 2020063673”的合成方法制备。
化合物66的合成：
参照化合物25的合成，投料化合物65(30mg,146umol)，得到黄色油状化合物27mg，收率36％，MS m/z:[M+H] +515.2。
化合物67的合成：
参照化合物26的合成，投料化合物66(27mg,53umol)，未经纯化直接用于下一步反应。
化合物68的合成：
参照化合物27的合成，化合物67投料按化合物66计量，得到9mg化合物68，两步收率20％，MS m/z:[M+H] +856.7。
化合物69的合成：
参照化合物28的合成，化合物68投料9mg，得到化合物69，约2.5mg，收率38％， MS m/z:[M+H] +634.2。
化合物70的合成：
参照化合物30的合成，化合物69投料量2.5mg，得到1.2mg黄色固体化合物61，收率约28％，MS m/z:[M+H] +1088.5。
实施例17：
Figure PCTCN2020115807-appb-000042
化合物71的合成：
0℃下，于25mL单口瓶中加入化合物25(40mg,84umol),加入5mL DMF，滴加Diethylamine(13uL,1.5eq)，自然升温至室温后反应2小时，直接将反应液减压浓缩，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物71约17mg，收率约80％，MS m/z:[M+H] +253.1。
化合物73的合成：
室温下，于25mL瓶中加入化合物71(17mg,67umol),加入5mL DMF溶解，冰水浴冷却至0℃，向所得溶液中依次加入DIPEA(22uL,2eq)，化合物72(67mg,2eq)以及4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(DMTMM,40mg,2eq)，维持0℃下反应4小时，向所得反应液中加入5mL饱和NaCl水溶液淬灭反应，水相经乙酸乙酯(10mL X 3)萃取，合并有机相，无水硫酸钠干燥，过滤，滤液经减压浓缩，经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物73约21mg，收率约43％，MS m/z:[M+H] +736.4。
化合物74的合成：
0℃下，于25mL单口瓶中加入化合物73(21mg,29umol),加入5mL DMF溶解，向所得溶液中加入Diethylamine(6uL,58umol)，自然升温至室温，维持室温下反应5小 时后，所得反应液浓缩后，直接用于下一步反应。
化合物76的合成：
室温下，于5mL单口瓶中加入化合物74,按化合物73计量，加入1.5mL DMF溶解，冰水浴冷却至0℃，向所得溶液中依次加入DIPEA(10uL,2eq)，化合物75(17mg,1.5eq)以及4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(DMTMM,17mg,2eq)，维持0℃下反应1小时，升温至室温反应3小时后，直接经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物76约11mg，黄色固体，两步收率约43％，MS m/z:[M+H] +878.4。
化合物77的合成：
室温下，于5mL单口瓶中加入化合物76(11mg,13umol),加入1mL DMF溶解，向所得溶液中加入11mg 10％Pd/C，氢气气球置换体系内气氛，维持室温下反应1小时后，过滤除去Pd/C，所得反应液浓缩后，直接用于下一步反应。
化合物78的合成：
室温下，于5mL单口瓶中加入化合物77(按化合物76计量)，加入1mL DMF溶解，冰水浴冷却至0℃，向所得溶液中依次加入DIPEA(5uL,2eq)，化合物4(6mg,1eq)以及4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(DMTMM,8mg,2eq)，维持0℃下反应1小时，反应液直接经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物78约1.5mg，两步收率约9％，MS m/z:[M+H] +1219.6。
实施例18：
Figure PCTCN2020115807-appb-000043
化合物79的合成：
室温下，于5mL单口瓶中加入化合物77(4mg,5umol)，加入1mL DMF溶解，冰水浴冷却至0℃，向所得溶液中依次加入DIPEA(1.7uL,2eq)，化合物7(2.3mg,1eq)以及4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(DMTMM,2.9mg,2eq)，维持0℃下反应2小时，反应液直接经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物79约1.3mg，收率约16％，MS m/z:[M+H] +1233.5。
实施例19：
Figure PCTCN2020115807-appb-000044
化合物80的合成：
室温下，于5mL单口瓶中加入化合物10(12mg,25umol),加入2mL DMF溶解，向所得溶液中加入12mg 10％Pd/C，氢气气球置换体系内气氛，维持室温下反应2小时后，过滤除去Pd/C，所得反应液浓缩后，直接用于下一步反应。
化合物81的合成：
室温下，于5mL单口瓶中加入化合物80(按化合物10计量)，加入2mL DMF溶解，冰水浴冷却至0℃，向所得溶液中依次加入DIPEA(9uL,2eq)，化合物77(20mg,1eq)以及4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(DMTMM,15mg,2eq)，维持0℃下反应2小时，反应液直接经制备级高效液相色谱法纯化(乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干，得到化合物81约3.3mg，两步收率约11％，MS m/z:[M+H] +1247.6。
实施例20：
Figure PCTCN2020115807-appb-000045
化合物82的合成：
参照化合物71的合成，化合物39投料(200mg,400umol)，得到目标化合物28mg，收率25％，MS m/z:[M+H] +279.0。
化合物83的合成：
参照化合物73的合成，化合物82投料(28mg,100umol)，得到目标化合物19mg，收率25％，MS m/z:[M+H] +762.4。
化合物84的合成：
参照化合物74的合成，化合物83投料(19mg,25umol)，得到目标化合物8mg，收率59％，MS m/z:[M+H] +540.2。
化合物85的合成：
参照化合物76的合成，化合物84投料(8mg,15umol)，得到目标化合物6mg，收率45％，MS m/z:[M+H] +904.4。
化合物86的合成：
参照化合物77的合成，化合物85投料(6mg,6.7umol)，所得反应液经浓缩后直接用于下一步反应。
化合物87的合成：
参照化合物78的合成，化合物86投料(按化合物85计量)，得到目标化合物0.9mg，两步收率约11％，MS m/z:[M+H] +1245.5。
实施例21：
Figure PCTCN2020115807-appb-000046
化合物88的合成：
参照化合物71的合成，化合物45投料(250mg,512umol)，得到目标化合物34mg，收率14％，MS m/z:[M+H] +489.4。
化合物89的合成：
参照化合物73的合成，化合物88投料(34mg,70umol)，得到目标化合物45mg，收率86％，MS m/z:[M+H] +750.9。
化合物90的合成：
参照化合物74的合成，化合物89投料(45mg,60umol)，得到目标化合物18mg，收率57％，MS m/z:[M+H] +528.1。
化合物91的合成：
参照化合物76的合成，化合物90投料(18mg,34umol)，得到目标化合物11mg，收率36％，MS m/z:[M+H] +892.8。
化合物92的合成：
参照化合物77的合成，化合物91投料(11mg,12umol)，所得反应液经浓缩后直接用于下一步反应。
化合物93的合成：
参照化合物78的合成，化合物92投料(按化合物91计量)，得到目标化合物2.8mg，两步收率约19％，MS m/z:[M+H] +1233.5。
实施例22：
Figure PCTCN2020115807-appb-000047
化合物94的合成：
参照化合物71的合成，化合物51投料(210mg,382umol)，得到目标化合物30mg，收率24％，MS m/z:[M+H] +329.0。
化合物95的合成：
参照化合物73的合成，化合物94投料(30mg,91umol)，得到目标化合物27mg，收率36％，MS m/z:[M+H] +812.3。
化合物96的合成：
参照化合物74的合成，化合物95投料(30mg,33umol)，得到目标化合物10mg， 收率51％，MS m/z:[M+H] +590.3。
化合物97的合成：
参照化合物76的合成，化合物96投料(10mg,17umol)，得到目标化合物6mg，收率37％，MS m/z:[M+H] +954.0。
化合物98的合成：
参照化合物77的合成，化合物97投料(6mg,6.3umol)，所得反应液经浓缩后直接用于下一步反应。
化合物99的合成：
参照化合物78的合成，化合物98投料(按化合物97计量)，得到目标化合物1.8mg，两步收率约22％，MS m/z:[M+H] +1295.4。
实施例23：
Figure PCTCN2020115807-appb-000048
化合物100的合成：
参照化合物71的合成，化合物57投料(200mg,369umol)，得到目标化合物36mg，收率30％，MS m/z:[M+H] +321.1。
化合物101的合成：
参照化合物73的合成，化合物100投料(36mg,112umol)，得到目标化合物25mg，收率28％，MS m/z:[M+H] +804.8。
化合物102的合成：
参照化合物74的合成，化合物101投料(25mg,31umol)，得到目标化合物12mg，收率67％，MS m/z:[M+H] +582.5。
化合物103的合成：
参照化合物76的合成，化合物102投料(12mg,21umol)，得到目标化合物9mg，收率45％，MS m/z:[M+H] +946.9。
化合物104的合成：
参照化合物77的合成，化合物103投料(9mg,10umol)，所得反应液经浓缩后直接用于下一步反应。
化合物105的合成：
参照化合物78的合成，化合物86投料(按化合物85计量)，得到目标化合物3.2mg，两步收率约25％，MS m/z:[M+H] +1287.3。
实施例24：
Figure PCTCN2020115807-appb-000049
化合物106的合成：
参照化合物71的合成，化合物66投料(300mg,583umol)，得到目标化合物41mg，收率24％，MS m/z:[M+H] +293.1。
化合物107的合成：
参照化合物73的合成，化合物106投料(41mg,140umol)，得到目标化合物29mg，收率27％，MS m/z:[M+H] +776.1。
化合物108的合成：
参照化合物74的合成，化合物107投料(29mg,37umol)，得到目标化合物7mg，收率34％，MS m/z:[M+H] +554.4。
化合物109的合成：
参照化合物76的合成，化合物108投料(7mg,13umol)，得到目标化合物6mg，收率50％，MS m/z:[M+H] +918.2。
化合物110的合成：
参照化合物77的合成，化合物109投料(6mg,6.5umol)，所得反应液经浓缩后直接用于下一步反应。
化合物111的合成：
参照化合物78的合成，化合物110投料(按化合物109计量)，得到目标化合物1.1mg，两步收率约13％，MS m/z:[M+H] +1259.8。
1)通用偶联方法：
将通过初步的纯化后单体率大于95％的抗体分子，使用超滤离心管换液至含有EDTA的磷酸盐缓冲液中，浓度10mg/mL。加入10倍于抗体摩尔分子数的TCEP，室温下反应2h。使用超滤离心管换液至pH 6.5的磷酸缓冲液中，再加入10倍于抗体摩尔分子数的DHAA，室温下反应2h。然后加入15倍于抗体摩尔分子数的payload，室温下反应4h。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。
2)抗体-药物偶联DAR的检测：
单体率检测条件：
样品14000rpm离心5分钟，取上清液进样分析。
仪器：Waters e2695(2489UV/Vis)；
色谱柱：TSKgel G3000SWXL(7.8×300mm，5μm)；
流动相：A：50mM PB，300mM NaCl，200mM Arg，5％IPA，pH6.5；
流动相A等度洗脱30min，流速：0.714mL/min，柱温25℃，检测波长：280nm。
DAR检测条件：
样品14000rpm离心5分钟，取上清液进样分析。
仪器：Waters H-class(TUV)；
色谱柱：Proteomix HIC Butyl-NP5(4.6×35mm，5μm)；
流动相：A：1.5M硫酸铵，0.025M无水磷酸钠，pH 7.0；
B：0.025M无水磷酸钠，25％IPA，pH7.0；
流动相A平衡色谱柱，流动相A和B梯度洗脱，流速0.8mL/min；柱温25℃，检测
波长：214nm。
实施例25：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-6
Figure PCTCN2020115807-appb-000050
实施例26：按通用偶联方法制备，以Tr000为抗体，得ADC:Tr000-6
Figure PCTCN2020115807-appb-000051
实施例27：按通用偶联方法制备，以Trastuzumab为抗体，得ADC:Tras-9
Figure PCTCN2020115807-appb-000052
实施例28：按通用偶联方法制备，以Trastuzumab为抗体，得ADC:Tras-12
Figure PCTCN2020115807-appb-000053
实施例29：按通用偶联方法制备，以Tr000为抗体，得ADC:Tr000-12
Figure PCTCN2020115807-appb-000054
实施例30：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-14
Figure PCTCN2020115807-appb-000055
实施例31：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-16
Figure PCTCN2020115807-appb-000056
实施例32：按通用偶联方法制备，以Tr000为抗体，得ADC：Tr000-16
Figure PCTCN2020115807-appb-000057
实施例33：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-18
Figure PCTCN2020115807-appb-000058
实施例34：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-20A
Figure PCTCN2020115807-appb-000059
实施例35：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-22B
Figure PCTCN2020115807-appb-000060
实施例36：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-PEG-6
Figure PCTCN2020115807-appb-000061
实施例37：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-PEG-9
Figure PCTCN2020115807-appb-000062
实施例38：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-PEG-12
Figure PCTCN2020115807-appb-000063
实施例39：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-PEG-14
Figure PCTCN2020115807-appb-000064
实施例40：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-PEG-16
Figure PCTCN2020115807-appb-000065
实施例41：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-PEG-18
Figure PCTCN2020115807-appb-000066
实施例42：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-PEG-20A
Figure PCTCN2020115807-appb-000067
实施例43：按通用偶联方法制备，以Trastuzumab为抗体，得ADC：Tras-PEG-22B
Figure PCTCN2020115807-appb-000068
体外活性测试
1)实验材料
细胞：A431,SW620,BXPC3,Fadu来源于中国科学院细胞库
细胞培养基DMEM：Gibco
FBS：BIOWEST
2)培养基的配制
生长培养基(with 10％FBS,Penicillin/streptomycin(100U/mL)
检测培养基(with 1％FBS,Penicillin/streptomycin(100U/mL)
3)操作
提前30min开启生物安全柜紫外灯照射，然后后开通风3min。将生长培养基、检测培养基、D-PBS和胰酶放入37℃恒温水浴锅里预热，之后用酒精对表面进行消毒，放入生物安全柜中。将汇合率在80％左右的细胞放于生物安全柜中，吸掉旧培养基，用D-PBS润洗，吸弃，用胰酶消化2～3min，后加入生长培养基中和，离心1200rpm，3min。吸去离心上清，用4mL检测培养基混匀，取100uL计数(其中取出50uL细胞液，加入50uL Trypan Blue Stain并混匀，混匀后计数)。按照预先优化好的细胞铺板密度铺板，80ul/孔铺于96孔板中，孔E11、F11只加80uL检测培养基，边缘孔加入150uL的DPBS。铺板24h后加入稀释抗体，每孔20uL和设置对照，第11列只加入20uL的检测培养基，每个浓度设置2个复孔，加入后于细胞涡旋震荡器上混匀，550rpm，3min。
抗体溶液的稀释：用检测培养基在V型96孔板第一列中配制起始浓度为5uM，300uL的供试品品溶液，后面第2到10列分别加入240uL的检测培养基，从混匀的 第一列中取60uL加入到第二列，用排枪上下混匀10次，弃枪头，后面7个浓度依次操作。
4)检测
4天后取出MTS试剂，常温避光解冻后，充分涡旋混匀后，在生物安全柜中，沿孔侧壁按每100μL细胞培养体积加入20μL CellTiter One Solution Reagen MTS试剂，轻轻拍动板面，使MTS溶液混合均匀后，放于细胞培养箱中避光静置孵育2h。反应结束后，取出96孔板，于酶标仪中检测OD490nm吸光值，并进行数据记录、整理、分析和存储。
5)结果
表1 喜树碱类衍生物对靶标细胞的体外增殖抑制的IC 50值：
Figure PCTCN2020115807-appb-000069
细胞实验结果表明，本发明所述的喜树碱衍生物具有明显的体外抗肿瘤活性(纳摩尔水平)，相比于第一三共所用毒素(对照化合物16)毒性明显降低，因此，本发明设计合成的喜树碱类衍生物有望成为更安全的抗肿瘤ADC药物。
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1×10 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180-200mm3，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Tr000-6，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。

Claims (22)

  1. 一种喜树碱类衍生物，提供了一种通式D所示的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药学上可接受的盐或溶剂化物：
    Figure PCTCN2020115807-appb-100001
    其中：
    R选自氘原子、C 1-6烷基、氘代烷基、取代烷基、芳基、取代芳基或杂芳基；
    R 1选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
    R 2选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
    X选自-C(O)-CR aR b-(CR 3R 4) n-O-、-C(O)-CR aR b-(CR 3R 4) n-NH-或-C(O)-CR aR b-(CR 3R 4) n-S-；
    R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
    或者，R 3、R 4连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    n选自0至4的整数。
  2. 根据权利要求1所述的喜树碱类衍生物或其互变异构体、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药学上可接受的盐或溶剂化物，其特征在于：通式D 1所示的化合物或其药学上可接受的盐或溶剂化物：
    Figure PCTCN2020115807-appb-100002
    其中：
    R选自氘原子、C 1-6烷基、氘代烷基、取代烷基、芳基、取代芳基或杂芳基；
    X选自-C(O)-CR aR b-(CR 3R 4) n-O-、-C(O)-CR aR b-(CR 3R 4) n-NH-或-C(O)-CR aR b-(CR 3R 4) n-S-；
    R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    R b选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、羟基、氨基、氰基、硝基、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟烷基、环烷基或杂环基；
    或者，R 3、R 4连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    n选自0至4的整数；
    其中，式D 1中的波浪线或表示氢原子，或与接头单元或与结合靶细胞所表达抗原的抗体共价连接。
  3. 根据权利要求1或2中所述的喜树碱类衍生物或其互变异构体、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，或其药学上可接受的盐或溶剂化物，其特征在于：
    X为-C(O)-CR aR b-(CR 3R 4) n-O-；
    R选自氘原子、C 1-6烷基、氘代烷基、取代烷基、芳基、取代芳基或杂芳基；
    R 3和R 4相同或不同，各自独立地选自氢原子、氘原子、卤素或烷基；
    R a为C 3-6环烷基烷基或C 3-6环烷基；
    R b选自氢原子、卤代烷基或C 3-6环烷基；优选氢原子；
    或者，R a和R b与其相连接的碳原子一起形成C 3-6环烷基；
    n为0或者1。
  4. 根据权利要求1或2中所述的喜树碱类衍生物或其互变异构体、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，或其药学上可接受的盐或溶剂化物，其特征在于：所述喜树碱类衍生物包含式D 2所示的结构：
    Figure PCTCN2020115807-appb-100003
    其中：
    R选自氘原子、C 1-6烷基、氚代烷基、取代烷基、芳基、取代的芳基或杂芳基；
    R a选自环烷基或者环烷基烷基；
    R b选自氢原子、氘原子、烷基、卤代烷基、环烷基或者环烷基烷基；
    或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    n为0或者1；
    其中，式D 2中的波浪线或表示氢原子，或与接头单元或与结合靶细胞所表达抗原的抗体共价连接。
  5. 根据权利要求1或2所述的喜树碱类衍生物或其互变异构体、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，或其药学上可接受的盐或溶剂化物，其特征在于：X非限制性地选自：
    Figure PCTCN2020115807-appb-100004
    Figure PCTCN2020115807-appb-100005
  6. 根据权利要求1或2中所述的喜树碱类衍生物或其药学上可接受的盐或溶剂化物，其特征在于：其为通式D 3所示的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、或其药学上可接受的盐或溶剂化物：
    Figure PCTCN2020115807-appb-100006
    其中：
    R选自氘原子、C 1-6烷基、氚代烷基、取代烷基、芳基、取代芳基或杂芳基；
    R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；优选C 3-6环烷基或者环烷基烷基；
    R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    n为0或者1；
  7. 根据权利要求1、2或6中所述的喜树碱类衍生物或其药学上可接受的盐或溶剂化物，其特征在于：其非限制性地选自以下所示的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、或其药学上接受的盐或溶剂化物：
    Figure PCTCN2020115807-appb-100007
  8. 一种通式如(-L-X-D 2)所示的药物-连接子化合物或者其药学上可接受的盐或溶剂化物；
    Figure PCTCN2020115807-appb-100008
    其中：
    R选自氘原子、C 1-6烷基、氘代烷基、取代烷基、芳基、取代的芳基或杂芳基；
    R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；优选C 3-6环烷基或者环烷基烷基；
    R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    n为0或者1；
    L为连接单元；
    其中，式-L-X-D 2中的波浪线或表示氢原子，或与接头单元或与结合靶细胞所表达抗原的抗体共价连接。
  9. 根据权利要求8中所述的药物-连接子化合物或者其药学上可接受的盐或溶剂化物， 其特征在于：其中X的O端与连接单元L相连。
  10. 根据权利要求8或9中所述的药物-连接子化合物或其药学上可接受的盐或者溶剂化物，其特征在于：其中连接单元-L-为-L 1-L 2-L 3-L 4-,，其L 1端与抗体相连，L 4端与X相连；
    其中:
    L 1选自：-(琥珀酰亚胺-3-基-N)-Y-C(O)-、-CH 2-C(O)-NR 5-Y-C(O)-或-C(O)-Y-C(O)-，
    其中，Y选自C 1-8烷基、C 1-8烷基-环烷基或者1-8个原子的直链或者直链-环状杂烷基；所述杂烷基包含选自N、O或者S的1-3个原子，所述的C 1-8烷基、环烷基、直链或者直链环状杂烷基各自独立地被选自氘代原子、卤素、羟基、氰基、硝基、氨基、烷基、羧基、杂烷基、取代烷基、烷氧基或者环烷基的一个或者多个取代基所取代；
    L 2选自：-NR 6(CH 2CH 2O) pCH 2CH 2C(O)-、-NR 6(CH 2CH 2O) pCH 2C(O)-、-S(CH 2) pC(O)-或者化学键，其中p选自0-20的整数；
    L 3选自：由2-7个氨基酸构成的肽残基，其中任选氨基酸进一步被选自氘原子、卤素、羟基、氰基、氨基、硝基、烷基、取代烷基、烷氧基和环烷基或者取代环烷基中的一个或多个取代基所取代；
    L 4选自：-NR 7(CR 8R 9) q-、-C(O)NR 7、-C(O)NR 7(CH 2) q-或者化学键，其中q选自0-6的整数；
    R 5、R 6和R 7相同或者不同，且各自独立地选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    R 8和R 9相同或者不同，且各自独立地选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
  11. 根据权利要求8-10中任一所述的药物-连接子化合物或其药学上可接受的盐或溶剂化合物，其特征在于：
    L 1选自：-(琥珀酰亚胺-3-基-N)-Y-C(O)-、-CH 2-C(O)-NR 5-Y-C(O)-或者-C(O)-Y-C(O)-，
    其中Y选自C 1-8烷基、C 1-8烷基-环烷基或者1-8个原子的直链或者直链-环状杂烷基；所述杂烷基包含选自N、O或者S的1-3个原子，所述的C 1-8烷基、环烷基、直链或者直链环状杂烷基各自独立地选自氘代原子、卤素、羟基、氰基、硝基、氨基、烷基、羧基、杂烷基、取代烷基、烷氧基或者环烷基的一个或者多个取代基所取代；
    L 2选自：-NR 6(CH 2CH 2O) pCH 2CH 2C(O)-、-NR 6(CH 2CH 2O) pCH 2C(O)-、-S(CH 2) pC(O)-或者化学键，其中p选自0-20的整数。
    L 3选自：由2-7个氨基酸构成的肽残基，其中任选氨基酸进一步被选自氘原子、卤素、羟基、氰基、氨基、硝基、烷基、取代烷基、羧基、烷氧基和环烷基或者取代环烷基中的一个或多个取代基所取代；优选为苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨酸(E)或者天冬氨酸(D)中的氨基酸形成的多肽残基；优选为由一个、两个或者多个选自苯丙氨酸和甘氨酸的氨基酸形成的氨基酸残基；最优选的为GGFG(甘氨酸-甘氨酸-苯丙氨酸-甘氨酸)组成的四肽残基；
    L 4选自：-NR 7(CR 8R 9) q-、-C(O)NR 7、-C(O)NR 7(CH 2) q-或者化学键，其中q选自0-6的整数，L 4优选为-NR 7CR 8R 9-；L 4更优选为-NHCH 2-。
    R 5、R 6和R 7相同或者不同，且各自独立地选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    R 8和R 9相同或者不同，且各自独立地氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
  12. 一种通式(L-X-D 2)所示的药物-连接子化合物或者其药学上可接受的盐或溶剂化物；
    Figure PCTCN2020115807-appb-100009
    其中：
    Z选自：-Y-C(O)-、-CH 2-C(O)-NR 5-Y-C(O)-或者-C(O)-Y-C(O)-，其中Y非限制性地选自C 1-8烷基、C 1-8烷基-环烷基或1-8个原子的直链或直链-环状杂烷基，所述杂烷基包含选自N、O或者S的1-3个原子，所述的C 1-8烷基、环烷基、直链或直链环状杂烷基各自独立地被选自氘代原子、卤素、羟基、氰基、硝基、氨基、烷基、杂烷基、取代烷基、烷氧基、羧基或环烷基的一个或者多个取代基所取代；
    L 2选自：-NR 6(CH 2CH 2O) pCH 2CH 2C(O)-、-NR 6(CH 2CH 2O) pCH 2C(O)-、-S(CH 2) pC(O)-或化学键，其中p选自0-20的整数；
    L 3选自：由2-7个氨基酸构成的肽残基，其中任选氨基酸进一步被选自氘原子、卤素、羟基、氰基、氨基、硝基、烷基、取代烷基、烷氧基和环烷基或者取代环烷基中的一个或多个取代基所取代；
    R选自氘原子、C 1-6烷基或取代烷基、芳基、取代芳基或杂芳基；
    R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、杂环基、芳基、取代芳基或杂芳基；
    R b选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    R 5、R 6和R 7相同或者不同，且各自独立地氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    R 8和R 9相同或者不同，且各自独立地氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
    n选自0至4的整数。
  13. 根据权利要求12所述的通式(L-X-D 2)所示的化合物或者其药学上可接受的盐或溶剂化物，其特征在于：其为通式(L b-X-D 2)所示的化合物，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；
    Figure PCTCN2020115807-appb-100010
    其中：
    Z选自：-Y-C(O)-、-CH 2-C(O)-NR 5-Y-C(O)-或者-C(O)-Y-C(O)-，其中Y非限制性地选自C 1-8烷基、C 1-8烷基-环烷基或1-8个原子的直链或直链-环状杂烷基，所述杂烷基包含选自N、O或者S的1-3个原子，所述的C 1-8烷基、环烷基、直链或直链环状杂烷基各自独立地被选自氘代原子、卤素、羟基、氰基、硝基、氨基、烷基、杂烷基、取代烷基、烷氧基、羧基或环烷基的一个或者多个取代基所取代；
    R选自氘原子、C 1-6烷基、氘代烷基、取代烷基、芳基、取代芳基或杂芳基；
    R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、杂环基、芳基、取代芳基或杂芳基；
    R b选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    R 5和R 7相同或者不同，且各自独立地选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    R 8和R 9相同或者不同，且各自独立地选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    n选自0至4的整数。
  14. 根据权利要求1或13中所述的通式(L-X-D 2)所示的药物-连接子化合物或者其药学上可接受的盐或溶剂化物，其特征在于：选自以下结构：
    Figure PCTCN2020115807-appb-100011
    Figure PCTCN2020115807-appb-100012
    Figure PCTCN2020115807-appb-100013
    Figure PCTCN2020115807-appb-100014
    Figure PCTCN2020115807-appb-100015
  15. 一种包括权利要求1-14任一所述的喜树碱衍生物或喜树碱-连接子化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或混合物形式，或其药学上可接受的盐或溶剂化物，与抗体连接形成如通式(Ab-L-X-Dr)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化物：
    Figure PCTCN2020115807-appb-100016
    其中：
    X选自-C(O)-CR aR b-(CR 3R 4) n-O-、-C(O)-CR aR b-(CR 3R 4) n-NH-或-C(O)-CR aR b-(CR 3R 4) n-S-；
    R选自氘原子、C 1-6烷基、氘代烷基、取代烷基、芳基、取代芳基或杂芳基；
    R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    R b选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R a、R b连同其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    R 3、R 4相同或者不同，且分别独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
    或者，R 3、R 4连同其所连接碳原子构成C 3-6环烷基或杂环基；
    n选自0至4的整数；
    m选自1-10的整数或小数；
    Ab为抗体、抗体片段或蛋白；
    L为连接单元。
  16. 根据权利要求15中所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：Ab为抗体，可通过其杂原子与连接单元形成连接键，所述抗体选自嵌合抗体、人源化抗体、全人源抗体或鼠源抗体。
  17. 根据权利要求15或16所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：所述抗体为单克隆抗体，其选自：抗EGFRvIII抗体、抗DLL-3抗体、抗PSMA抗体、抗CD70抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM-1抗体、抗LRRC15抗体、抗LIV-1抗体、抗CanAg/AFP抗体、抗cladin 18.2抗体、抗Mesothelin抗体、抗HER2(ErbB2)抗体、抗EGFR抗体、抗c-MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7-H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗TROP-2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P-cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体或抗CD123抗体。
  18. 根据权利要求15或16中所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于，非限制性地选自以下结构：
    Figure PCTCN2020115807-appb-100017
    Figure PCTCN2020115807-appb-100018
    Figure PCTCN2020115807-appb-100019
    Figure PCTCN2020115807-appb-100020
    其中：
    m选自1-10的整数或小数；
    Ab为抗体、抗体片段或者与抗原连接的片段。
  19. 一种制备如权利要求15-18所述通式(Ab-L a-X-Dr)所示抗体-药物偶联物或其药学上可接受的盐或溶剂化物的方法，其特征在于，包括如下步骤：
    Figure PCTCN2020115807-appb-100021
    通过抗体、抗体片段或者其抗原结合片段与通式(La-X-D 2)偶联反应，得到通式(Ab-L a-X-D 2)。
  20. 一种药物组合物，其含有治疗有效量的根据权利要求1-19中任一项所述的抗体-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，或其药学上可接受的盐或溶剂化物，以及药学上可接受的载体、稀释剂或赋形剂。
  21. 一种包括权利要求1-19中任一项所述的喜树碱类衍生物或其抗体-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 其药学上可接受的盐或溶剂化合物，以及药学上可接受的载体、稀释剂或者赋形剂，在制备用于治疗或预防肿瘤的药物中的用途。
  22. 根据权利要求21中所述的用途，其特征在于：所述的癌症优非限制性地选自：乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、肺癌、结肠癌、直肠癌、结直肠癌、骨癌、皮肤癌、甲状腺癌、胰腺癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、多形性胶质细胞瘤、肉瘤、淋巴瘤和白血病等实体瘤或血液肿瘤。
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