WO2023216956A1 - 喜树碱类化合物及其制备方法和应用 - Google Patents

喜树碱类化合物及其制备方法和应用 Download PDF

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WO2023216956A1
WO2023216956A1 PCT/CN2023/092019 CN2023092019W WO2023216956A1 WO 2023216956 A1 WO2023216956 A1 WO 2023216956A1 CN 2023092019 W CN2023092019 W CN 2023092019W WO 2023216956 A1 WO2023216956 A1 WO 2023216956A1
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cancer
hydrogen
compound
alkyl
ring
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PCT/CN2023/092019
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English (en)
French (fr)
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田强
张毅涛
叶健
李怡乐
宋宏梅
葛均友
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四川科伦博泰生物医药股份有限公司
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Publication of WO2023216956A1 publication Critical patent/WO2023216956A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/12—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 three hetero rings
    • C07D491/14—Ortho-condensed systems
    • C07D491/147—Ortho-condensed systems the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom
    • 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

Definitions

  • the present invention relates to a class of camptothecin compounds with anti-tumor activity and their conjugates, as well as their preparation methods and applications in the medical field.
  • Camptothecin (CPT, Formula 1) is a five-ring quinoline core compound isolated from Camptotheca acuminata, a plant of the Davidia family. It consists of quinoline ring AB, pyrrole ring C, and pyridone ring D. It consists of ⁇ -hydroxylactone ring E, in which the 20-position is in S configuration (see structural formula below). It was introduced into clinical practice in the early 1970s due to its excellent anti-cancer activity. Later, clinical trials were terminated due to severe side effects such as diarrhea and hemorrhagic cystitis.
  • camptothecin can form a ternary complex with cellular DNA topoisomerase I, thereby inhibiting the unwinding of DNA, causing DNA replication to be blocked, and thus causing cell death (Cancer Res. 1989, 49, 6365). Camptothecin and its derivatives have strong anti-tumor activity in animal models of lung cancer, breast cancer, colorectal cancer, ovarian cancer, etc. (Nature Review Cancer. 2006, 6, 789).
  • camptothecin drugs have been approved for tumor treatment (Med.Res.Rev.2015,35,753).
  • Irinotecan is used for the treatment of colorectal cancer; topotecan is used for the treatment of ovarian cancer; belotecan is used for the treatment of ovarian cancer and small cell lung cancer.
  • Camptothecin derivatives include Exatecan, Rubitecan, Karenitecan, Diflomotecan, Lurtotecan, Gimatecan, Namitecan, Simmitecan, Silatecan, Chimmitecan, Elomotecan, etc.
  • Camptothecin drugs or derivatives often have blood toxicity caused by bone marrow suppression, such as neutropenia, leukopenia, thrombocytopenia, anemia, etc., as well as gastrointestinal side effects, such as nausea, vomiting, diarrhea, etc.
  • Clinical studies have found that measures to improve the safety and effectiveness of camptothecin compounds include improving their pharmacokinetic properties, adjusting activity, reducing dosage, or using their conjugates to form antibody conjugates with antibodies. Therefore, the development of camptothecin compounds and their conjugates with novel structures that can enhance effectiveness and improve safety issues still has high clinical demand and application value.
  • the present invention provides novel camptothecin compounds and their conjugates.
  • the camptothecin compounds have good anti-tumor activity, excellent permeability, low efflux rate, and good metabolic stability, and are promising. It is used for the treatment of tumor diseases; its conjugates have broad application prospects for antibody-conjugated drugs.
  • a first aspect of the invention provides compounds or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotopic labels, metabolites and prodrugs thereof, wherein the compound has the following Structure shown:
  • R 1 is selected from hydrogen, fluorine and chlorine
  • R 2 is selected from hydrogen, methyl, fluorine, chlorine and hydroxyl; or R 1 and R 2 are connected to adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring;
  • R 3 is selected from hydrogen, C 1-6 alkyl, deuterated C 1-6 alkyl, C 1-6 haloalkyl and C 1-6 alkoxyalkyl, C 3-6 cycloalkyl and 3-6 yuan Heterocyclyl, or R 3 is connected to the adjacent carbon atom of the benzene ring to form a six-membered carbon ring;
  • A is selected from
  • Ring B is selected from 3-6 membered carbocyclic rings or 3-6 membered heterocyclic groups
  • R 4 is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3 - 6 cycloalkyl or 3-6 membered heterocyclyl;
  • R 5 and R 6 are each independently selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 3-6 cycloalkyl, C 1-6 haloalkyl; or R 5 or R 6 is adjacent to Carbon atoms are connected to form a 3-6 membered ring;
  • R 4 and R 5 are connected to form a 4-6 membered ring
  • n 1-6, such as 1, 2, 3, 4, 5 or 6.
  • R 3 is connected to adjacent carbon atoms of the benzene ring to form a six-membered carbocyclic ring, and the carbon atoms are carbon atoms in the meta position of R 1 and ortho position of R 2 .
  • R 1 is selected from hydrogen, fluorine and chlorine
  • R 2 is selected from hydrogen, methyl, fluorine, chlorine and hydroxyl; or R 1 and R 2 are connected to adjacent carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring;
  • R 3 is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl or connected to adjacent carbon atoms of the benzene ring to form a six-membered carbon ring;
  • A is selected from
  • Ring B is selected from 3-6 membered carbocyclic rings or 3-6 membered heterocyclic groups
  • R 4 is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3 - 6 cycloalkyl or 3-6 membered heterocyclyl;
  • R 5 and R 6 are each independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 haloalkyl; or R 5 or R 6 is connected to adjacent carbon atoms to form 3-6 ring;
  • R 4 and R 5 are connected to form a 4-6 membered ring
  • n 1 or 2.
  • the compound has the structure of Formula (II):
  • R 1 ' is selected from hydrogen, fluorine and chlorine
  • R 2 ' is selected from methyl, fluorine, chlorine and hydroxyl
  • R 3 ' is selected from hydrogen, C 1-6 alkyl, deuterated C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl and 3-6 One-membered heterocyclyl; R 3 ' is preferably hydrogen, C 1-6 alkyl, deuterated C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxyalkyl;
  • R 4 ' is selected from hydrogen, C 1-6 alkyl and C 1-6 haloalkyl; R 4 ' is preferably hydrogen;
  • R 1 ' is fluorine and R 2 ' is methyl, R 3 ' and R 4 ' are not simultaneously hydrogen;
  • R 4 ' is not C 1-6 alkyl.
  • R 1 ' is selected from hydrogen, fluorine and chlorine
  • R 2 ' is selected from methyl, fluorine, chlorine and hydroxyl
  • R 3 ' is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl and 3-6 membered heterocyclyl; R 3 ' is preferably Hydrogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxyalkyl;
  • R 4 ' is selected from hydrogen, C 1-6 alkyl and C 1-6 haloalkyl; R 4 ' is preferably hydrogen;
  • R 1 ' is fluorine and R 2 ' is methyl, R 3 ' and R 4 ' are not simultaneously hydrogen;
  • R 4 ' is not C 1-6 alkyl.
  • R 1 ' is selected from hydrogen, fluorine and chlorine
  • R 2 ' is chlorine or methyl
  • R 3 ' is hydrogen, methyl or deuterated methyl
  • R 4 ' is hydrogen
  • R 1 ' is fluorine
  • R 2 ' is chlorine
  • R 3 ' is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl and 3-6 membered heterocyclyl; R 3 ' is preferably Hydrogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxyalkyl; preferably hydrogen, C 1-6 alkyl or C 1-6 haloalkyl; for example, hydrogen, methyl or deuterium methyl group;
  • R 4 ' is selected from hydrogen, C 1-6 alkyl and C 1-6 haloalkyl; R 4 ' is preferably hydrogen.
  • R 1 ' is chlorine
  • R 2 ' is methyl
  • R 3 ' is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl and 3-6 membered heterocyclyl; R 3 ' is preferably Hydrogen, C 1-6 alkyl, C 1-6 haloalkyl or C 1-6 alkoxyalkyl; preferably hydrogen, C 1-6 alkyl or C 1-6 haloalkyl; for example, hydrogen, methyl or deuterium methyl group;
  • R 4 ' is selected from hydrogen, C 1-6 alkyl and C 1-6 haloalkyl; R 4 ' is preferably hydrogen.
  • the compound has the structure of Formula (III):
  • R 1 is selected from hydrogen, fluorine and chlorine
  • R 2 is selected from hydrogen, methyl, fluorine, chlorine and hydroxyl
  • R 3 is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl and 3-6 membered heterocyclyl, or R 3 and benzene Adjacent carbon atoms in the ring are connected to form a six-membered carbon ring; R 3 is preferably hydrogen;
  • R 4 is selected from hydrogen and C 1-6 alkyl
  • R 5 and R 6 are independently selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 1-6 haloalkyl, C 3 - 6 cycloalkyl and 3-6 membered heterocyclyl, or R 5 And R 6 is connected to adjacent carbon atoms to form a 3-6 membered ring;
  • R 4 and R 5 are connected to form a 4-6 membered ring
  • n 1 or 2;
  • R 1 is fluorine
  • R 2 is methyl
  • R 3 is hydrogen
  • R 4 is hydrogen
  • R 5 and R 6 are not hydrogen at the same time, nor are they connected to adjacent carbon atoms to form a cyclopropyl group
  • R 1 is fluorine
  • R 2 is methyl
  • R 3 is hydrogen
  • R 4 is not alkyl
  • R 3 is connected to adjacent carbon atoms of the benzene ring to form a six-membered carbocyclic ring, and the carbon atoms are carbon atoms in the meta position of R 1 and ortho position of R 2 .
  • the compound has the structure shown in formula (III-1),
  • R 1 is selected from hydrogen, fluorine and chlorine
  • R 2 is selected from hydrogen, methyl, fluorine, chlorine and hydroxyl
  • R 3 is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl and 3-6 membered heterocyclyl; R 3 is preferably hydrogen;
  • R 4 is selected from hydrogen and C 1-6 alkyl
  • R 5 and R 6 are independently selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 3-6 cycloalkyl and 3-6 membered heterocyclyl, or R 5 and R 6 are combined with adjacent carbon Atoms connected to form 3-6 membered rings;
  • R 1 is fluorine
  • R 2 is methyl
  • R 3 is hydrogen
  • R 4 is hydrogen
  • R 5 and R 6 are not hydrogen at the same time, nor are they connected to adjacent carbon atoms to form a cyclopropyl group
  • R 1 is fluorine
  • R 2 is methyl
  • R 3 is hydrogen
  • R 4 is not alkyl
  • R2 is selected from fluorine and chlorine.
  • R is selected from hydrogen, methyl, fluorine, and chlorine.
  • R is selected from methyl and chlorine.
  • R3 is selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxyalkyl, C 3-6 cycloalkyl, and 3-6 membered heterocyclyl .
  • R4 is hydrogen
  • R 5 and R 6 are independently selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 3 - 6 cycloalkyl, and C 2-6 alkenyl, or R 5 and R 6 is connected to adjacent carbon atoms to form a 3-6 membered ring.
  • R 5 and R 6 are independently selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 3 - 6 cycloalkyl, or R 5 and R 6 are connected to adjacent carbon atoms Into a 3-6 membered carbon ring.
  • R 5 and R 6 are independently selected from hydrogen, methyl, vinyl, allyl, cyclopropyl, or R 5 and R 6 are connected to adjacent carbon atoms to form a 3-membered carbocyclic ring.
  • R 5 and R 6 are independently selected from hydrogen, C 1-6 alkyl and C 3 - 6 cycloalkyl, or R 5 and R 6 are connected to adjacent carbon atoms to form a 3-6 membered carbocyclic ring .
  • R 5 and R 6 are independently selected from hydrogen, methyl, and cyclopropyl, or R 5 and R 6 are connected to adjacent carbon atoms to form a 3-membered carbocyclic ring.
  • R4 and R5 are connected to form a 5-membered ring.
  • R 4 and R 5 are connected to form a 5-membered ring and R 6 is hydrogen.
  • R 1 is selected from fluorine and chlorine
  • R 2 is selected from chlorine and methyl
  • R 3 is selected from hydrogen and C 1-6 alkyl, or R 3 is connected to the adjacent carbon atoms of the benzene ring to form a six-membered carbon ring;
  • R 4 is hydrogen
  • R 5 and R 6 are independently selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 3 - 6 cycloalkyl and C 2-6 alkenyl, or R 5 and R 6 are combined with adjacent carbon atoms Connected to form a 3-6 membered ring;
  • R 4 and R 5 are connected to form a 4-6 membered ring.
  • R 3 is connected to adjacent carbon atoms of the benzene ring to form a six-membered carbocyclic ring, and the carbon atoms are carbon atoms in the meta position of R 1 and ortho position of R 2 .
  • R 1 is fluorine
  • R 2 is chlorine
  • R 3 is selected from hydrogen and C 1-2 alkyl, or R 3 is connected to the adjacent carbon atoms of the benzene ring to form a six-membered carbon ring;
  • R 4 is hydrogen
  • R 5 and R 6 are independently selected from hydrogen, C 1-2 alkyl, C 1-2 haloalkyl, C 3 - 6 cycloalkyl and C 2-4 alkenyl, or R 5 and R 6 are combined with adjacent carbon atoms Connected to form a 3-6 membered ring;
  • R 4 and R 5 are connected to form a 4-6 membered ring.
  • R 3 is connected to adjacent carbon atoms of the benzene ring to form a six-membered carbocyclic ring, and the carbon atoms are carbon atoms in the meta position of R 1 and ortho position of R 2 .
  • R 1 is chlorine
  • R 2 is methyl
  • R 3 is selected from hydrogen and C 1-2 alkyl, or R 3 is connected to the adjacent carbon atoms of the benzene ring to form a six-membered carbon ring;
  • R 4 is hydrogen
  • R 5 and R 6 are independently selected from hydrogen, C 1-2 alkyl, C 1-2 haloalkyl, C 3 - 6 cycloalkyl and C 2-4 alkenyl, or R 5 and R 6 are combined with adjacent carbon atoms Connected to form a 3-6 membered ring;
  • R 4 and R 5 are connected to form a 4-6 membered ring.
  • R 3 is connected to adjacent carbon atoms of the benzene ring to form a six-membered carbocyclic ring, and the carbon atoms are carbon atoms in the meta position of R 1 and ortho position of R 2 .
  • R 1 is fluorine
  • R 2 is methyl
  • R 3 is selected from hydrogen and C 1-2 alkyl, or R 3 is connected to the adjacent carbon atoms of the benzene ring to form a six-membered carbon ring;
  • R 4 is hydrogen
  • R 5 and R 6 are independently selected from hydrogen, C 1-2 alkyl, C 1-2 haloalkyl, C 3 - 6 cycloalkyl and C 2-4 alkenyl, or R 5 and R 6 are combined with adjacent carbon atoms Connected to form a 3-6 membered ring; preferably, R 5 and R 6 are not hydrogen at the same time;
  • R 4 and R 5 are connected to form a 4-6 membered ring.
  • R 3 is connected to adjacent carbon atoms of the benzene ring to form a six-membered carbocyclic ring, and the carbon atoms are carbon atoms in the meta position of R 1 and ortho position of R 2 .
  • R 1 is selected from fluorine and chlorine
  • R 2 is selected from methyl and chlorine; preferably chlorine;
  • R 3 is hydrogen, or R 3 is connected to the adjacent carbon atoms of the benzene ring to form a six-membered carbon ring;
  • R 4 is hydrogen
  • R 5 and R 6 are independently selected from hydrogen, C 1-6 alkyl, C 1-6 haloalkyl, C 3 - 6 cycloalkyl and C 2-6 alkenyl, or R 5 and R 6 are combined with adjacent carbon atoms Connected to form a 3-6 membered ring;
  • R 4 and R 5 are connected to form a 4-6 membered ring.
  • R 3 is connected to adjacent carbon atoms of the benzene ring to form a six-membered carbocyclic ring, and the carbon atoms are carbon atoms in the meta position of R 1 and ortho position of R 2 .
  • R 1 is selected from fluorine and chlorine
  • R 2 is selected from chlorine
  • R 3 is hydrogen, or R 3 is connected to the adjacent carbon atoms of the benzene ring to form a six-membered carbon ring;
  • R 4 is hydrogen
  • R 5 and R 6 are independently selected from hydrogen, methyl, trifluoromethyl, cyclopropyl and vinyl, or R 5 and R 6 are connected to adjacent carbon atoms to form a 3-membered ring;
  • R 4 and R 5 are connected to form a 5-membered ring.
  • R 3 is connected to adjacent carbon atoms of the benzene ring to form a six-membered carbocyclic ring, and the carbon atoms are carbon atoms in the meta position of R 1 and ortho position of R 2 .
  • the compound has the structure of Formula (IV):
  • R 1 ' is selected from hydrogen, fluorine and chlorine
  • R 2 ' is selected from hydrogen, methyl, fluorine, chlorine and hydroxyl
  • R 4 ' is selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl and C 1-6 haloalkyl; R 4 ' is preferably from hydrogen;
  • B is selected from C 1-6 alkylene, 3-6 membered carbocyclic group or 3-6 membered heterocyclic group;
  • R 1 ' is fluorine
  • R 2 ' is methyl, and a dotted carbocyclic ring exists
  • R 4 ' is not hydrogen and is not a C 1-6 alkyl group
  • R 2 ' is methyl, R 1 ' is fluorine, and the dotted carbocyclic ring does not exist, the B ring is not a 3-membered carbocyclic ring.
  • R 1 ' is selected from hydrogen, fluorine, and chlorine;
  • R 2 ' is selected from hydrogen, methyl, fluorine, chlorine and hydroxyl
  • R 4 ' is selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl and C 1-6 haloalkyl; R 4 ' is preferably from hydrogen;
  • B is selected from 3-6 membered carbocyclic ring or 3-6 membered heterocyclic group
  • R 1 ' is fluorine
  • R 2 ' is methyl
  • R 4 ' is not hydrogen and is not an alkyl group
  • R 2 ' is methyl, R 1 ' is fluorine, and the dotted carbocyclic ring does not exist, the B ring is not a 3-membered carbocyclic ring.
  • R 1 ' is selected from fluorine and chlorine.
  • R 2 ' is selected from hydrogen, methyl, fluorine, and chlorine.
  • R 2 ' is selected from methyl and chlorine.
  • R 4 ' is hydrogen
  • B is C 1-6 alkylene; preferably, B is methylene, ethylene, propylene, isopropylene; more preferably, B is propylene, isopropylene propyl.
  • Ring B is a 3-6 membered carbocyclic ring; preferably, Ring B is a 3-6 membered saturated carbocyclic ring.
  • the invention provides the following compounds:
  • the present invention also provides compounds of formula (V) or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotopic labels, metabolites and prodrugs thereof:
  • MLED Formula(V) MLED Formula(V)
  • M is a linker connecting the antibody or its antigen-binding fragment
  • L is the connector connecting connectors M and E;
  • E is the structural fragment connecting L and D;
  • D is the structural fragment of a cytotoxic drug.
  • M is selected from the following structures:
  • X is selected from leaving groups, such as chlorine, bromine, -OMs, OTs, and OTf.
  • M is selected from the following structures:
  • L is selected from a structure consisting of one or more of the following: C1-6 alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, D -Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly -Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly -Gly ⁇
  • R' represents hydrogen, C1-6 alkyl or alkyl containing -(CH 2 CH 2 O) r -; r is selected from an integer of 1-10; s is selected from an integer of 1-20.
  • L is selected from the following structures: where s is selected from an integer from 1-20.
  • E is selected from a single bond, -NH- CH2- ,
  • E is -NH- CH2- .
  • the cytotoxic drug is selected from the compounds described in any one of the first aspects of the invention.
  • the cytotoxic drug is selected from compounds 1-1 to 1-2; 2-1 to 2-23; 3-1 to 3-4; or 4-1 to 4- 12.
  • D is selected from the structure of the compound described in the first aspect of the invention after dehydrogenation.
  • D is selected from compounds 1-1 to 1-2; 2-1 to 2-18; 3-1 to 3-18; 4-1 to 4-16; 5-1 to 5-10; 6-1 to 6-10; 7-1 to 7-2; 8-1 to 8-2 or 9-1 to 9-2 after dehydrogenation.
  • D is selected from the following structures:
  • M-L-E-D is selected from the following compounds:
  • the present invention also provides an antibody drug conjugate (ADC) represented by formula (VI): Ab-(MLED) x Formula (VI)
  • Ab is an antibody or its antigen-binding fragment
  • M is a linker site connecting the antibody or its antigen-binding fragment
  • L is the connector connecting connectors M and E;
  • E is the structural fragment connecting L and D;
  • D is the structural fragment of a cytotoxic drug
  • x ranges from 1 to 10.
  • M, L, E, and D in formula (VI) are as described above.
  • the antibody drug conjugate (ADC) is represented by formula (VI-1): Ab-MLED Formula (VI-1)
  • Ab-(MLED) x is selected from the following structures:
  • ADC A-8 ADC A-9:
  • x is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1- 10, 2 ⁇ 3, 2 ⁇ 4, 2 ⁇ 5, 2 ⁇ 6, 2 ⁇ 7, 2 ⁇ 8, 2 ⁇ 9, 2 ⁇ 10, 3 ⁇ 4, 3 ⁇ 5, 3 ⁇ 6, 3 ⁇ 7, 3 ⁇ 8, 3 ⁇ 9, 3 ⁇ 10, 4 ⁇ 5, 4 ⁇ 6, 4 ⁇ 7, 4 ⁇ 8, 4 ⁇ 9, 4 ⁇ 10, 5 ⁇ 6, 5 ⁇ 7, 5 ⁇ 8, 5 ⁇ 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10, preferably 3 ⁇ 9.
  • x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the present invention also provides the following antibody drug conjugates (ADCs) or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, and isotope labels thereof , metabolites and prodrugs:
  • ADCs antibody drug conjugates
  • pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, and isotope labels thereof metabolites and prodrugs:
  • compositions of antibody drug conjugates as described herein.
  • Such compositions may comprise a plurality of ADCs described herein, wherein each ADC comprises a drug-linker described herein, wherein x is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • each antibody molecule in the composition can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 drug-linkers.
  • the compositions are characterized by a "drug-to-antibody” ratio (DAR) in the range of about 1 to about 10.
  • DAR drug-to-antibody ratio
  • the ADC compositions described herein have a DAR of about 1 to about 10, or any subrange therebetween, such as: about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5 , about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7 , about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10 , about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10, or about 9 to 10.
  • the ADC compositions described herein have a DAR of about 3 to 9, such as about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5 to 6, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 8.0, about 4.5 to 5.0, about 4.5 to 5.5, about 4.5 to 6.0, about 4.5 to 6.5, about 4.5 to 7.0, about 4.5 to 7.5 about 5.0 to 8.0, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5
  • the invention also provides a composition comprising one or more antibody-drug conjugates, the composition having a DAR value (drug-to-antibody conjugation ratio) of 7.5 to 8.5, preferably 7.5 to 8.5. 8.0, further preferably 8.0:
  • the invention also provides a composition comprising one or more antibody-drug conjugates, the composition having a DAR value (drug-to-antibody conjugation ratio) of 7.5 to 8.5, preferably 7.5 to 8.5. 8.0, further preferably 7.96:
  • the invention also provides a composition comprising one or more antibody-drug conjugates, the composition having a DAR value (drug-to-antibody conjugation ratio) of 7.5 to 8.5, preferably 7.5 to 8.5. 8.0, further preferably 7.95:
  • the "*" marked in the structural formula of a compound indicates that the marked carbon atom is a chiral carbon atom, and the present invention includes a pair of enantiomers formed by the chiral carbon atom. For example, if a compound contains two different chiral carbon atoms, the present invention includes four optical isomers formed by the chiral carbon atoms.
  • alkyl is defined as a straight or branched saturated aliphatic hydrocarbon radical.
  • an alkyl group has 1 to 12, such as 1 to 6 carbon atoms.
  • C 1-6 alkyl refers to a linear or branched group of 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl (isobutyl, sec-butyl, tert-butyl, n-pentyl and n-hexyl), which is optionally substituted by 1 or more (such as 1, 2 or 3) suitable substituents.
  • alkenyl refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C 2-6 alkenyl", “C 2-4 alkenyl”, etc. Examples include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl Alkenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexenyl Dialkenyl etc.
  • alkynyl refers to a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond. Including, for example, “C 2-6 alkynyl”, “C 4-6 alkynyl” and the like.
  • Examples include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butadiynyl, 1-pentynyl, 2 -Pentynyl, 3-pentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4 -Hexadiynyl, etc.
  • cycloalkyl refers to a saturated cyclic hydrocarbon group, including but not limited to monocycloalkyl and bicycloalkyl (such as spirocycloalkyl, And cycloalkyl and bridged cycloalkyl).
  • C 3-6 cycloalkyl refers to a cycloalkyl group having 3 to 6 ring-forming carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., which may be optionally replaced by 1 or Substitution with multiple (such as 1, 2 or 3) suitable substituents, for example methyl substituted cyclopropyl.
  • carrier refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic structure, a hydrocarbon group connected through ring carbons. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
  • carrier refers to a saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., a monocyclic ring such as a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, Cycloheptane ring, cyclooctane ring, cyclononane ring, or bicyclic ring, including spiro, fused or bridged systems (such as bicyclo[1.1.1]pentane ring, bicyclo[2.2.1]heptane ring, Bicyclo[3.2.1]octane ring or bicyclo[5.2.0]nonane ring, decalin ring, etc.), which may be optionally substituted by 1 or more (such as 1, 2 or 3) suitable substituents Substitution.
  • the term "3-6 membered carbocyclic such as bicycl
  • heterocyclyl or “heterocycle” refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic cyclic structure whose ring atoms are composed of carbon atoms and at least one (e.g. 1, 2 or 3) composed of heteroatoms selected from nitrogen, oxygen and sulfur.
  • a heterocyclyl group can be connected to the rest of the molecule through any ring atom if the valence bond requirements are met.
  • the heterocyclic group in the present invention is preferably a 3-6 membered heterocyclic group.
  • 3-6 membered heterocyclyl refers to a heterocyclic group with 3 to 6 ring atoms, including 3-membered heterocyclyl, 4-membered heterocyclyl, 5-membered heterocyclyl and 6-membered heterocyclyl.
  • Membered heterocyclic groups include nitrogen-containing heterocyclic groups and oxygen-containing heterocyclic groups, such as 4-6-membered heterocyclic groups, such as 4-6-membered nitrogen-containing heterocyclic groups and 4-6-membered oxygen-containing heterocyclic groups.
  • heterocyclyl groups include (but are not limited to) azetidinyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl.
  • the heterocyclyl group in the present invention may be optionally substituted by one or more substituents described in the present invention.
  • the heterocyclyl group in the present invention is optionally condensed with one or more aromatic or non-aromatic rings.
  • oxygen-containing heterocycle refers to the aforementioned heterocycle in which one or more (for example, 1, 2 or 3) ring atoms are oxygen atoms, such as a 5-6 membered oxygen-containing heterocycle. Specific examples include but It is not limited to ethylene oxide ring, tetrahydrofuran ring, furan ring, tetrahydropyran ring, pyran ring, etc.
  • the "nitrogen-containing heterocyclic ring” used in the present invention refers to the heterocyclic ring as described above in which one or more (for example, 1, 2 or 3) ring atoms are nitrogen atoms.
  • haloalkyl refers to an alkyl group substituted by one or more (such as 1, 2 or 3) the same or different halogen atoms, where alkyl is as defined above.
  • C 1-6 haloalkyl refers to a haloalkyl group having 1 to 6 carbon atoms.
  • Common haloalkyl groups include (but are not limited to) -CH 2 F, -CHF 2 , -CF 3 , -CH 2 CF 3 , -CF 2 CF 3 , -CH 2 CH 2 CF 3 , -CH 2 Cl, etc.
  • the haloalkyl groups in the present invention are optionally substituted with one or more substituents described in the present invention.
  • alkoxy refers to a group having the structure "alkyl-O-", where alkyl is as defined above.
  • alkyl is as defined above.
  • Common alkoxy groups include (but are not limited to) methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy Key et al. in the present invention
  • Alkoxy groups are optionally substituted with one or more substituents described herein.
  • alkoxyalkyl refers to an alkyl group substituted by one or more (eg 1, 2, 3 or 4) alkoxy groups, where alkoxy and alkyl are as defined above.
  • C 1-6 alkoxyalkyl refers to an alkoxy group having 1 to 6 carbon atoms substituted by one or more (e.g., 1, 2, 3 or 4) alkoxy groups. of alkyl.
  • Common alkoxyalkyl groups include (but are not limited to) CH 3 O-CH 2 -, C 2 H 5 -O-CH 2 -, C 2 H 5 -O-CH 2 CH 2 -, etc.
  • halo or halogen group is defined to include F, Cl, Br or I.
  • nitrogen oxide refers to an oxide (eg, a mono- or di-oxide) of at least one nitrogen atom in the compound structure of the present application. Nitrogen mono-oxides may exist as a single positional isomer or as a mixture of positional isomers.
  • substituted means that one or more (e.g., one, two, three or four) hydrogens on the designated atom are replaced by a selection from the indicated group, provided that no more than the designated atom is present in the case of normal valence and the substitution forms a stable compound. Combinations of substituents and/or variables are permissible only if such combinations form stable compounds.
  • substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as optionally substituted with one or more substituents in the substituent list, one or more hydrogens on the carbon (to the extent of any hydrogen present) may be present individually and/or together Replaced by independently selected optional substituents. If the nitrogen of a substituent is described as optionally substituted with one or more of the substituents listed, then one or more hydrogens on the nitrogen (to the extent of any hydrogen present) may each be independently selected as optional. substitution of substituents.
  • each substituent is selected independently of the other.
  • each substituent may be the same as or different from another (other) substituent.
  • one or more means 1 or more than 1, such as 2, 3, 4, 5 or 10 under reasonable conditions.
  • the point of attachment of a substituent may be from any suitable position on the substituent.
  • stereoisomer means an isomer formed due to at least one asymmetric center. In compounds with one or more (e.g., one, two, three or four) asymmetric centers, they can give rise to racemic mixtures, single enantiomers, diastereomeric mixtures and individual of diastereomers. Certain individual molecules may also exist as geometric isomers (cis/trans). Similarly, compounds of the present invention may exist as mixtures of two or more structurally distinct forms in rapid equilibrium (often referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, and imine-enamine tautomers. wait.
  • Solid lines may be used in this article solid wedge or virtual wedge Draw the carbon-carbon bonds of the compounds of the invention.
  • the use of solid lines to depict bonds to asymmetric carbon atoms is intended to include all possible stereoisomerisms at that carbon atom. isomers (e.g., specific enantiomers, racemic mixtures, etc.).
  • the use of solid or imaginary wedges to depict bonds to asymmetric carbon atoms is intended to demonstrate that the stereoisomers shown exist. When present in a racemic mixture, solid and imaginary wedges are used to define relative stereochemistry rather than absolute stereochemistry.
  • the compounds of the present invention are intended to exist as stereoisomers (which includes cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, They exist in the form of geometric isomers, rotamers, conformational isomers, atropisomers and mixtures thereof).
  • the compounds of the present invention may exhibit more than one type of isomerism and consist of mixtures thereof (eg, racemic mixtures and pairs of diastereoisomers).
  • the present invention encompasses all possible crystalline forms or polymorphs of the compounds of the invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion.
  • compositions of the present invention may exist in free form for therapeutic use, or, where appropriate, as pharmaceutically acceptable derivatives thereof.
  • pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites or prodrugs that, upon administration to a patient in need thereof, can directly or indirectly Compounds of the invention or metabolites or residues thereof are provided. Therefore, when reference is made herein to "a compound of the invention", it is also intended to encompass the various derivative forms of the compound described above.
  • Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof.
  • Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts, including aspartate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, and the like.
  • Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts, including aluminum salts, arginine salts, choline salts, diethylamine salts, and the like.
  • esters means an ester derived from the compounds of each general formula herein, including physiologically hydrolyzable esters (which can be hydrolyzed under physiological conditions to release the free acid or alcohol form of the compound of the invention).
  • the compounds of the present invention may themselves be esters.
  • the compounds of the invention may exist in the form of solvates, preferably hydrates, wherein the compounds of the invention comprise as structural elements of the crystal lattice of the compounds a polar solvent, in particular such as water, methanol or ethanol.
  • a polar solvent in particular such as water, methanol or ethanol.
  • the amount of polar solvent, especially water, may be present in stoichiometric or non-stoichiometric ratios.
  • metabolites of the compounds of the invention ie substances formed in the body upon administration of the compounds of the invention. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, delipidation, enzymatic hydrolysis, etc. of the administered compound.
  • the invention includes metabolites of the compounds of the invention, including compounds prepared by contacting a compound of the invention with a mammal for a time sufficient to produce metabolites thereof.
  • the invention further includes within its scope prodrugs of the compounds of the invention.
  • prodrugs will be functional group derivatives of the compound that are readily converted in vivo to the desired therapeutically active compound. Therefore, in these instances, the term "administering" as used in the treatment methods of the present invention shall include the treatment of various diseases or conditions with prodrug forms of one or more of the claimed compounds, but in The prodrug form is converted in vivo to the compound described above upon administration to an individual.
  • “Design of Prodrug” ed. H. Bundgaard, Elsevier, 1985, general methods for selecting and preparing suitable prodrug derivatives are described.
  • the invention further includes within its scope isotopic markers of the compounds of the invention which are identical to the compounds of the invention except that one or more atoms are substituted with the same atomic number but an atomic mass or mass number different from that which predominates in nature Atomic substitution for atomic mass or mass number.
  • the invention also encompasses compounds of the invention containing protecting groups.
  • protecting groups In any process for preparing the compounds of the invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any relevant molecules, thereby forming chemically protected forms of the compounds of the invention. This can be achieved by conventional protecting groups, for example, those described in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991 Protecting Groups, these references are incorporated herein by reference.
  • the protecting groups can be removed at an appropriate subsequent stage using methods known in the art.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound described in the first or second aspect of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, or solvent thereof compounds, nitrogen oxides, isotope labels, metabolites or prodrugs, and one or more pharmaceutically acceptable carriers.
  • pharmaceutical composition refers to a composition that can be used as a medicament and contains a pharmaceutically active ingredient (API) (or therapeutic agent) and optionally one or more pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier refers to an excipient that is administered with a therapeutic agent and that is suitable, within the scope of sound medical judgment, for contact with human and/or other animal tissue without undue toxicity, irritation, Allergic reactions or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • the above-mentioned pharmaceutical composition can act systemically and/or locally, which can be achieved through suitable dosage forms.
  • the dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, and aqueous suspensions. , injectable solutions, elixirs, syrups.
  • the above pharmaceutical composition may contain 0.01 mg to 1000 mg of at least one compound of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolism substances or prodrugs.
  • the present invention also provides a method for preparing the above-mentioned pharmaceutical composition or its corresponding preparation form, which includes at least one compound of the present invention or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate substances, nitrogen oxides, isotope labels, metabolites or prodrugs in combination with one or more pharmaceutically acceptable carriers.
  • the present invention provides a pharmaceutical kit product, which includes:
  • the first therapeutic agent at least one compound according to the first or second aspect of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide thereof , isotope markers, metabolites or prodrugs, or the pharmaceutical composition described in the third aspect as the first pharmaceutical composition;
  • kit product may contain 0.01 mg to 1000 mg of at least one compound of the present invention or its pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope markers, metabolism substances or prodrugs.
  • the present invention also provides a preparation method for the above-mentioned pharmaceutical kit, which includes adding at least one compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope thereof
  • a marker, metabolite or prodrug or pharmaceutical composition as described above is combined with the optional presence of at least one other therapeutic agent or a pharmaceutical composition, packaging and/or instructions containing the other therapeutic agent.
  • the compound of the present invention can exhibit a strong effect in inhibiting abnormal cell proliferation.
  • the present application provides compounds of the invention or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotopic labels, metabolites and prodrugs thereof, or pharmaceutical combinations thereof Drugs used to treat diseases involving abnormal cell proliferation.
  • this application also provides the compounds of the present invention or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope markers, metabolites and prodrugs or the above-mentioned drugs Use of the composition in the preparation of a medicament for the treatment of diseases involving abnormal cell proliferation.
  • the disorder involving abnormal cellular proliferation includes, but is not limited to, tumors, such as advanced solid tumors.
  • the application also provides compounds of the invention or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotopic markers, metabolites and prodrugs thereof or pharmaceutical combinations of the invention.
  • the formulations are for in vivo or in vitro administration.
  • the formulation can be administered to a subject to inhibit the proliferation of tumor cells in the subject; alternatively, the formulation can be administered to cells in vitro (eg, cell lines or cells from the subject), To inhibit the proliferation of tumor cells in vitro.
  • Tumors described in the present invention include (but are not limited to): brain tumors, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrium Cancer, colorectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain Cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, prostate tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.
  • the present invention provides a method for treating diseases related to abnormal cell proliferation, which includes the following steps: adding a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, ester, or stereoisomer thereof , polymorphs, solvates, nitrogen oxides, isotopic markers, metabolites and prodrugs or the above pharmaceutical compositions are administered to individuals in need thereof.
  • an effective amount refers to a dose capable of inducing a biological or medical response in a cell, tissue, organ or organism (eg, an individual) and sufficient to achieve the desired preventive and/or therapeutic effect.
  • Dosage regimens can be adjusted to provide the best desired response. For example, it can be administered in a single dose, in divided doses over time, or in a proportionally reduced or increased dose according to the actual situation. It will be understood that, for any particular individual, specific dosage regimens should be adjusted according to need and the professional judgment of the person administering or supervising the administration of the compositions.
  • an effective amount is about 0.001-10000 mg/kg of subject's body weight/day. Where appropriate, the effective amount is about 0.01-1000 mg/kg subject body weight/day.
  • Administration can be from about 0.01 to 1000 mg/kg of subject's body weight every day, every two days, or every three days, and typically from about 0.1 to 500 mg/kg of subject's body weight.
  • Exemplary dosing regimens are once or more daily, or once or more weekly, or once or more monthly. When multiple doses are administered, the intervals between single doses may generally be daily, weekly, monthly, or yearly.
  • administration may be in the form of a sustained-release formulation, in which case less frequent dosing is required.
  • the dosage and frequency of administration may vary based on the half-life of the drug in the subject and may vary based on whether the application is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered over a long period of time at relatively infrequent intervals; in therapeutic applications, it is sometimes necessary to administer relatively high doses at shorter intervals until the progression of the disease is retarded or stopped, preferably until the individual Demonstrates partial or complete improvement in disease symptoms, after which prophylactic application can be adopted.
  • treatment refers to the alleviation or elimination of a targeted disease or condition. If a subject receives a therapeutic amount of a compound of the present invention or a pharmaceutically acceptable form thereof or a pharmaceutical composition of the present invention, at least one indicator and symptom of the subject is observable and/or detectable. Detected remission and/or improvement indicates that the subject has been successfully "treated.” It is understood that treatment includes not only complete treatment, but also less than complete treatment, but achieving some biologically or medically relevant results.
  • administration/administration/administration refers to the administration of a pharmaceutically active ingredient (such as a compound of the present invention) or a pharmaceutical composition containing a pharmaceutically active ingredient (such as a pharmaceutical composition of the present invention)
  • a pharmaceutically active ingredient such as a compound of the present invention
  • a pharmaceutical composition containing a pharmaceutically active ingredient such as a pharmaceutical composition of the present invention
  • Common administration methods include (but are not limited to) oral administration, subcutaneous administration, intramuscular administration, subperitoneal administration, ocular administration, nasal administration, sublingual administration, rectal administration, vaginal administration, etc.
  • needle for refers to a physician's or other caregiver's judgment that an individual needs or would benefit from a preventive and/or therapeutic procedure, based on the physician's or other caregiver's expertise in the individual's area of expertise. factors.
  • the term "individual” refers to a human or non-human animal.
  • Individuals of the present invention include individuals suffering from diseases and/or disorders (patients) and normal individuals.
  • Non-human animals of the present invention include all vertebrates, such as non-mammals, such as birds, amphibians, reptiles, etc., and mammals, such as non-human primates, domestic animals and/or domesticated animals (such as sheep, dogs, cats, cows, pigs, etc.).
  • the fourth aspect of the present invention provides a synthesis method of the compound.
  • R 3 is hydrogen
  • the compound of formula (I) in the present invention can be synthesized and prepared by the following synthetic route.
  • LG is a leaving group selected from methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyl. Oxygen or iodine;
  • this step is performed at a suitable temperature of 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C.
  • this step is performed at a suitable temperature, such as 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 120°C, preferably 120°C;
  • this step is performed in a suitable organic solvent, which may be selected from toluene, xylene, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, Toluene and xylene are preferred;
  • a suitable organic solvent which may be selected from toluene, xylene, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, Toluene and xylene are preferred;
  • this step is performed under acidic conditions
  • Reagents providing acidic conditions include p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, formic acid, sulfuric acid, methanesulfonic acid, preferably p-toluenesulfonic acid.
  • this step is performed at a suitable temperature of 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 85°C, 100°C, preferably 85°C.
  • this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 50°C;
  • this step is performed in a suitable organic solvent, which may be selected from halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane ( 1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylethane Amide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably acetonitrile.
  • halogenated hydrocarbons e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane ( 1,2-DCE), etc.
  • nitriles such as acetonitrile (AN), etc.
  • NMP N,N-
  • this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base.
  • the organic base can be selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py)
  • the inorganic base can be selected from potassium phosphate (K 3 PO4), sodium hydride (NaH), potassium carbonate (K 2 CO 3 ), carbonic acid Sodium (Na 2 CO 3 ), sodium bicarbonate (NaHCO 3 ), cesium carbonate (Cs 2 CO 3 ) and NaOH, preferably Na 2 CO 3 or NaHCO 3 ;
  • this step is performed under a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HBTU and HATU;
  • this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;
  • this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.
  • a suitable organic solvent which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.
  • this step is carried out in a suitable base, including an organic base or an inorganic base
  • the organic base can be selected from DIPEA, TEA, t-BuOK and Py
  • the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably DIPEA.
  • this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 90°C, 100°C, preferably 90°C;
  • this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. and 1,4-dioxane, 1,4-dioxane is preferred.
  • a suitable organic solvent which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. and 1,4-dioxane, 1,4-dioxane is preferred.
  • this step is carried out in a suitable base, including an organic base or an inorganic base
  • the organic base can be selected from DIPEA, TEA, t-BuOK and Py
  • the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably TEA.
  • this step is performed at a suitable temperature, the temperature is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 0 to 25°C;
  • this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.
  • a suitable organic solvent which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.
  • this step is carried out in a suitable base, including an organic base or an inorganic base
  • the organic base can be selected from DIPEA, TEA, t-BuOK and Py
  • the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably TEA.
  • the compound of formula (I) in the present invention can be synthesized and prepared by the following synthesis route.
  • LG is a leaving group selected from methanesulfonyl, trifluoromethanesulfonyloxy and halogen, preferably trifluoromethanesulfonyl. Oxygen or iodine;
  • this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 50°C;
  • this step is performed in a suitable organic solvent, which may be selected from halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane ( 1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylethane Amide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably acetonitrile.
  • halogenated hydrocarbons e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane ( 1,2-DCE), etc.
  • nitriles such as acetonitrile (AN), etc.
  • NMP N,N-
  • this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base.
  • the organic base can be selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py)
  • the inorganic base can be selected from potassium phosphate (K 3 PO4), sodium hydride (NaH), potassium carbonate (K 2 CO 3 ), carbonic acid Sodium (Na 2 CO 3 ), sodium bicarbonate (NaHCO 3 ), cesium carbonate (Cs 2 CO 3 ) and NaOH, preferably Na 2 CO 3 or NaHCO 3 ;
  • this step is performed under a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC and HOBT, preferably HBTU and HATU;
  • this step is performed at a suitable temperature, which is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 25°C;
  • this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.
  • a suitable organic solvent which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.
  • this step is carried out in a suitable base, including an organic base or an inorganic base
  • the organic base can be selected from DIPEA, TEA, t-BuOK and Py
  • the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably DIPEA.
  • this step is performed at a suitable temperature, which is 5°C, 20°C, 25°C, 40°C, 50°C, 60°C, 90°C, 100°C, preferably 90°C;
  • this step is performed in a suitable organic solvent, which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. and 1,4-dioxane, preferably 1,4-dioxane.
  • a suitable organic solvent which may be selected from the group consisting of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. and 1,4-dioxane, preferably 1,4-dioxane.
  • this step is carried out in a suitable base, including an organic base or an inorganic base
  • the organic base can be selected from DIPEA, TEA, t-BuOK and Py
  • the inorganic base can be selected from K 3 PO 4 , NaH, K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 and NaOH, preferably TEA.
  • this step is performed at a suitable temperature, the temperature is 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, preferably 0 to 25°C;
  • this step is performed in a suitable organic solvent, which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.
  • a suitable organic solvent which may be selected from the group consisting of methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, n-heptane, n-hexane, ethyl acetate, preferably N,N-dimethylformamide.
  • this step is performed in a suitable base, including an organic base or an inorganic base
  • the organic base can be selected from DIPEA, TEA, t-BuOK and Py
  • the inorganic base can be selected from K3PO4 ,NaH, K2CO3, Na2CO3, Cs2CO3 and NaOH, preferably TEA.
  • the present invention provides camptothecin compounds represented by formula (I) to formula (IV), pharmaceutical compositions, preparation methods and uses thereof.
  • This type of compound has good anti-tumor activity, has the potential to overcome drug resistance, and can be used to treat abnormal cell proliferation disorders, including but not limited to advanced solid tumors.
  • the measuring instrument of nuclear magnetic resonance used a Bruker 400MHz nuclear magnetic resonance instrument; hexadeuterated dimethyl sulfoxide (DMSO-d 6 ); the internal standard substance was tetramethylsilane (TMS).
  • NMR nuclear magnetic resonance
  • s singlet
  • d doublet
  • t triplet
  • q quartet
  • m multiplet
  • br broad
  • J coupling constant
  • Hz Hertz
  • DMSO-d6 deuterated dimethyl sulfoxide.
  • the delta value is expressed as ppm value.
  • MS mass spectrometry
  • ESI Agilent 6120B
  • Step 1 Preparation of 1-(2-amino-5-chloro-4-fluorophenyl)-2-chloroethyl-1-one
  • reaction solution is extracted with dichloromethane (3x 25mL). The organic phase is washed with saturated brine and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated to obtain the title compound (2g, 7.21mmol), which was directly used in the next reaction.
  • Step 2 (S)-9-chloro-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyran [3',4': Preparation of 6,7]indolazine[1,2-b]quinoline-3,14(4H)-dione
  • Step 3 (S)-11-(aminomethyl)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyran [3',4': Preparation of 6,7]indolazine[1,2-b]quinoline-3,14(4H)-dione
  • the solid was collected by filtration and dried to obtain the hydrochloride of the title compound (180 mg, 386.03 ⁇ mol).
  • the hydrochloride of 1-1 (20 mg, 46.62 ⁇ mol) was added to a mixed solvent of DMF (1 mL) and water (1 mL). After adjusting the pH to 8 with sodium bicarbonate, it was purified by preparative high performance liquid chromatography (purification conditions (S)-9-chloro-11-(chloromethyl)-4-ethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyran [3',4' :6,7]indolezine[1,2-b]quinoline-3,14(4H)-dione (1-1,3mg).
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% formic acid)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% trifluoroacetic acid)
  • Step 1 tert-butyl (N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10, 13,15-Hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolazine[1,2-b]quinolin-1-yl)sulfamoyl) urethane
  • Dissolve tert-butyl alcohol (20.92 mg, 0.282 mmol) in DCM (0.5 mL), cool and stir to 0°C, add dropwise chlorosulfonyl isocyanate (39.94 mg, 0.282 mol, 24.56 ⁇ L), and keep the reaction for 0.5 hours before use.
  • Dissolve ixotecan mesylate 100 mg, 0.188 mol
  • DMF 2 mL
  • drop in TEA 57.11 mg, 0.564 mmol, 78.45 ⁇ L
  • cool and stir to 0°C then drop in the above DCM reaction solution, and recover naturally. React at room temperature for 1 hour.
  • the reaction was monitored by LCMS and the product was obvious.
  • the reaction solution is directly used for the next reaction.
  • Step 2 (1S,9S)-1-sulfamic acid-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H , 13H-benzo[de]pyran[3',4':6,7]indolazine[1,2-b]quinoline-10,13-dione
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% formic acid)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% formic acid)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% trifluoroacetic acid)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% trifluoroacetic acid)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% trifluoroacetic acid)
  • Step 1 (2R,3S,4R,5R,6R)-2-(2-(8-(9-fluorenylmethoxycarbonylamino)-3,6-dioxa-octanoamido)-4- ((((((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7]indolizino[1,2b]quinolin-11-yl)methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl) )-Tetrahydro-2H-pyran-3,4,5-triacetate (A-11-2)
  • Step 2 (2R,3R,4R,5S,6R)-6-(2-(2-(2-(2-(2-aminoethyl)ethoxy)acetamido)-4-(((((( S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4' :6,7]Mesozoindeno[1,2-b]quinolin-11yl)methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro -2Hpyran-2-carboxylic acid (A-11-3)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% formic acid)
  • Step 3 (2R,3R,4R,5S,6R)-6-(4-((((((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,4, 12,14-Tetrahydro-1H-pyrano[3',4':6,7]indolo[1,2-b]quinolin-11-yl)methyl)carbamoyl)oxy) Methyl)-2-(2-(2-(N-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamido)ethylamino)ethoxy)acetamide (Hydroxy)phenoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-carboxylic acid (A-11)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% formic acid)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% formic acid)
  • Step 1 (9H-fluoren-9-yl)methyl ((9S,12S,15S)-1-((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14 -Dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indeno[1,2-b]quinolin-11-yl) -9,12-Dimethyl-3,8,11,14-tetraoxo-5-oxa-2,7,10,13-tetraazahexadecane-15-yl)carbamate ( B-2-1)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% formic acid)
  • Step 2 (S)-2-amino-N-((9S,12S)-1-((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxy Generation-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indeno[1,2-b]quinolin-11-yl)-9- Methyl-3,8,11-trioxo-5-oxa-2,7,10-triazatridecan-12-yl)propionamide (B-2-2)
  • Step 3 N-((9S,12S,15S)-1-((S)-9-chloro-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4, 12,14-tetrahydro-1-pyrano[3',4':6,7]indeno[1,2-b]quinolin-11-yl)-9,12-dimethyl- 3,8,11,14-tetraoxo-5-oxa-2,7,10,13-tetraazahexadecane-15-yl)-6-(2-(methanesulfonyl)pyrimidine-5 -yl)hex-5-ynamide (B-2)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% formic acid)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% formic acid)
  • Example 12 (R)-N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10, 13,15-Hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indeno[1,2-b]quinolin-1-yl)-3 -Hydroxybutanamide (2-26)
  • Mobile phase A acetonitrile
  • mobile phase B water (0.05% formic acid)
  • (1) Cell plating First, use the corresponding culture medium to culture tumor cells HT29, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating.
  • the sources of tumor cells are shown in Table 1.
  • Co-incubation of compounds of the present invention and tumor cells add 90 ⁇ L of cell suspension in a 96-well plate respectively. After the cells adhere, add 10 ⁇ L of bioactive molecules (compounds of the present invention) diluted with culture medium into the wells of the above plate and incubate. 72h.
  • test results show that the compounds of the present invention in Table 2 have a strong inhibitory effect on the proliferation of HT29 colon cancer cells.
  • (1) Cell plating First, use the corresponding culture medium to culture tumor cells HCC1806, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating.
  • the sources of tumor cells are shown in Table 3.
  • Co-incubation of compounds of the present invention and tumor cells add 90 ⁇ L of cell suspension in a 96-well plate respectively. After the cells adhere, add 10 ⁇ L of bioactive molecules (compounds of the present invention) diluted with culture medium into the wells of the above plate and incubate. 72h.
  • test results show that the compounds of the present invention in Table 4 have significant effects on the proliferation of HCC1806 human breast squamous cancer cells.
  • the inhibitory effect shows that the compounds of the present invention in Table 4 have significant effects on the proliferation of HCC1806 human breast squamous cancer cells. The inhibitory effect.
  • (1) Cell plating First, use the corresponding culture medium to culture tumor cells NCI-H358, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating.
  • the sources of tumor cells are shown in Table 5.
  • Co-incubation of compounds of the present invention and tumor cells add 90 ⁇ L of cell suspension in a 96-well plate respectively. After the cells adhere, add 10 ⁇ L of bioactive molecules (compounds of the present invention) diluted with culture medium into the wells of the above plate and incubate. 72h.
  • test results show that the compounds of the present invention in Table 6 have a significant inhibitory effect on the proliferation of NCI-H358 human non-small cell lung cancer cells.
  • (1) Cell plating First, use the corresponding culture medium to culture tumor cells NCI-N87, digest the cells with trypsin, After centrifugation, resuspend the cells for counting and adjust the cells to the appropriate concentration for plating.
  • the sources of tumor cells are shown in Table 7.
  • Co-incubation of compounds of the present invention and tumor cells add 90 ⁇ L of cell suspension in a 96-well plate respectively. After the cells adhere, add 10 ⁇ L of bioactive molecules (compounds of the present invention) diluted with culture medium into the wells of the above plate and incubate. 72h.
  • test results show that the compounds of the present invention in Table 8 have a significant inhibitory effect on the proliferation of NCI-N87 human gastric cancer cells.
  • (1) Cell plating First, use the corresponding culture medium to culture tumor cells MDA-MB-231, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating.
  • the sources of tumor cells are shown in Table 9.
  • Co-incubation of compounds of the present invention and tumor cells add 90 ⁇ L of cell suspension in a 96-well plate respectively. After the cells adhere, add 10 ⁇ L of bioactive molecules (compounds of the present invention) diluted with culture medium into the wells of the above plate and incubate. 72h.
  • test results show that the compounds of the present invention in Table 10 have a significant inhibitory effect on the proliferation of MDA-MB-231 human breast cancer cells.
  • Co-incubation of compounds of the present invention and tumor cells add 90 ⁇ L of cell suspension in a 96-well plate respectively. After the cells adhere, add 10 ⁇ L of bioactive molecules (compounds of the present invention) diluted with culture medium into the wells of the above plate and incubate. 72h.
  • test results show that the compounds of the present invention in Table 12 have a significant inhibitory effect on the proliferation of Jeko-1 human mantle cell lymphoma cells.
  • (1) Cell plating First, use the corresponding culture medium to culture tumor cells MDA-MB-453, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating.
  • the sources of tumor cells are shown in Table 13.
  • Co-incubation of the compounds of the present invention and tumor cells add 90 ⁇ L of cell suspension in a 96-well plate respectively. After the cells adhere, remove the culture medium from the cells and add 10 ⁇ L of bioactive molecules (compounds of the present invention) diluted with the culture medium. into the above plate wells and incubate for 72h.
  • test results show that the compounds of the present invention in Table 14 have a significant inhibitory effect on the proliferation of MDA-MB-453 human breast cancer cells.
  • (1) Cell plating First, use the corresponding culture medium to culture tumor cells HCC827, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating.
  • the sources of tumor cells are shown in Table 15.
  • Co-incubation of the compounds of the present invention and tumor cells After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the above-mentioned plate wells, and incubate for 72 hours.
  • test results show that the compounds of the present invention in Table 16 have an inhibitory effect on the proliferation of HCC827 human non-small cell lung cancer cells.
  • (1) Cell plating First, use the corresponding culture medium to culture tumor cells HCC1954, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating.
  • the sources of tumor cells are shown in Table 17.
  • Co-incubation of the compounds of the present invention and tumor cells After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the above-mentioned plate wells, and incubate for 72 hours.
  • test results show that the compounds of the present invention in Table 18 have an inhibitory effect on the proliferation of HCC1954 human breast cancer cells.
  • (1) Cell plating First, use the corresponding culture medium to culture tumor cells NCI-H1975, digest the cells with trypsin, resuspend the cells after centrifugation, count them, and adjust the cells to an appropriate concentration for plating.
  • the sources of tumor cells are shown in Table 19.
  • Co-incubation of the compounds of the present invention and tumor cells After the cells adhere to the wall, remove the culture medium from the cells, add the diluted bioactive molecules (compounds of the present invention) to the above-mentioned plate wells, and incubate for 72 hours.
  • test results show that the compounds of the present invention in Table 20 have an inhibitory effect on the proliferation of NCI-H1975 human non-small cell lung cancer cells.
  • Complete culture medium Add 10% fetal bovine serum and 1% penicillin/streptomycin to the MEM culture medium.
  • HBSS buffer HBSS containing Ca 2+ and Mg 2+ .
  • Compound stock solution Weigh a certain amount of the compound to be tested and prepare a 10mM stock solution with DMSO.
  • MDCK cells (26th generation) in the logarithmic growth phase were digested and dispersed evenly with 0.05% trypsin-EDTA, and then seeded into the Transwell chamber at 3*10 5 cells/cm 2 , with 200 ⁇ L on the A side and 1000 ⁇ L on the B side. After inoculation, the medium was changed once a day and cultured for 3 to 4 days.
  • TEER cell transmembrane resistance
  • C0 is the initial concentration ( ⁇ M) of the test drug at the donor
  • (dCr/dt) ⁇ Vr is the rate at which the test drug appears at the receiver ( ⁇ M/s)
  • Vr is the solution volume at the receiver.
  • A is the surface area of the polycarbonate membrane (cm 2 ).
  • P app (B ⁇ A) is the apparent permeability coefficient of the test drug from the BL (baso-lateral) end to the AP (apical) end
  • P app (A ⁇ B) is the apparent permeability coefficient of the test drug from the AP end to the BL end. Observational permeability coefficient.
  • ERi and ERa are the efflux rates of the test compound in the presence and absence of P-gp inhibitor, respectively.
  • Recovery rate (%) 100 ⁇ [(Vr ⁇ Cr)+(Vd ⁇ Cd)]/(Vd ⁇ C0)
  • Vd and Vr are the solution volumes on the supply side and receiving side respectively
  • Cd and Cr are the compound concentrations on the supply side and receiving side respectively.
  • control compound 2, control compound 3, control compound 4 and compound 2-19 are all low-permeability compounds, especially the permeability of control compound 2 is relatively low; control compound 1, compound 2-1, compound 2-22 and compound 2-25 are medium-permeability compounds; compound 1-1 It is a highly permeable compound; the efflux ratio data suggests that control compound 1, control compound 2, control compound 3, control compound 4, compound 2-22 and compound 2-25 may be efflux transporter substrates, especially control compound 2, The efflux rate of control compound 3 and control compound 4 is relatively high; compound 1-1, compound 2-1 and compound 2-19 are not substrates of efflux transporters or are weak substrates of efflux transporters.
  • Liver microsomes were used as an in vitro model to evaluate the metabolic stability of compounds in human and monkey liver microsomes.
  • the positive compound testosterone or the substance to be tested (liver microsome solution, 50 ⁇ L) was mixed with PBS (25 ⁇ L). After preincubation (37°C) for 5 min, NADPH (25 ⁇ L) was added to make the final concentration of the positive compound or the substance to be tested be 1 ⁇ M.
  • the final concentration of liver microsomal protein was 0.5 mg/ml, and the final concentration of monkey liver microsomal protein was 1 mg/ml.
  • the test group and the positive compound group were incubated for 0 and 15 min. After the corresponding reaction time, add 300 ⁇ L of ice-cold acetonitrile containing internal standard to terminate the reaction, vortex, and store at -80°C until assay. All incubated samples were duplicates.
  • Prototype remaining rate (%) 100 ⁇ (A incubated sample /A 0h )
  • a incubated sample the peak area ratio of the compound to the internal standard after incubation for the corresponding time;
  • a 0h the peak area ratio of the compound to the internal standard when unreacted.

Abstract

本发明涉及具有抗肿瘤活性的喜树碱类化合物及其制备方法和应用。具体地，本发明涉及下式化合物或其药学上可接受的形式，其药物组合物、制备方法和用途。所述化合物可用作治疗细胞异常增殖方面疾病的药物，式（I）。

Description

喜树碱类化合物及其制备方法和应用
本申请是以CN申请号为202210521215.2，申请日为2022年5月13日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。
技术领域
本发明涉及一类具有抗肿瘤活性的喜树碱类化合物及其缀合物，以及它们的制备方法和在医药领域中的应用。
背景技术
喜树碱(Camptothecin，CPT，式1)是从珙桐科植物喜树(Camptothecaacuminata)中分离得到的一种五环喹啉母核化合物，由喹啉环AB、吡咯环C、吡啶酮环D和α-羟基内酯环E组成，其中20-位为S构型(见下文结构式)。20世纪70年代初由于其优异的抗癌活性而被引入临床，后来由于出现腹泻、出血性膀胱炎等严重副反应，而终止了临床试验。
研究数据表明，喜树碱可与细胞DNA拓扑异构酶Ⅰ形成三元复合物，从而抑制DNA的解旋，导致DNA复制受阻，进而造成细胞死亡(Cancer Res.1989,49,6365)。喜树碱及其衍生物在肺癌、乳腺癌、结直肠癌、卵巢癌等动物体内模型中具有很强的抗肿瘤活性(Nature Review Cancer.2006,6,789)。
目前已有多个喜树碱类药物被批准上市用于肿瘤治疗(Med.Res.Rev.2015,35,753)。伊立替康为结直肠癌治疗药物；拓扑替康用于卵巢癌的治疗；贝洛替康用于卵巢癌和小细胞肺癌的治疗。喜树碱衍生物还有Exatecan,Rubitecan,Karenitecan,Diflomotecan,Lurtotecan,Gimatecan,Namitecan,Simmitecan,Silatecan,Chimmitecan,Elomotecan等。
喜树碱类药物或衍生物多存在骨髓抑制导致的血液毒性，比如中性粒细胞减少、白细胞减少、血小板减少、贫血等，以及胃肠道副作用，比如恶心、呕吐、腹泻等。临床研究发现提升喜树碱类化合物安全性和有效性的措施包括改善其药代性质、调节活性、减少用量或者利用其缀合物与抗体形成抗体偶联药物等。因此，研发结构新颖并能够提升有效性、改善安全性问题的喜树碱类化合物及其缀合物仍有很高的临床需求和应用价值。
发明内容
本发明提供结构新颖的喜树碱类化合物及其缀合物，该喜树碱类化合物具有良好的抗肿瘤活性，优异的渗透性，较低的外排率，以及良好的代谢稳定性，有望用于肿瘤疾病的治疗；其缀合物有广泛的抗体偶联药物应用前景。
本发明第一方面提供化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药，其中所述化合物具有如下所示结构：
其中，
R1选自氢、氟和氯；
R2选自氢、甲基、氟、氯和羟基；或者R1和R2与相邻碳原子连接成5-6元含氧杂环；
R3选自氢、C1-6烷基、氘代C1-6烷基、C1-6卤代烷基和C1-6烷氧烷基，C3-6环烷基和3-6元杂环基，或者R3与苯环相邻碳原子相连成六元碳环；
A选自
B环选自3-6元碳环或3-6元杂环基；
R4选自氢、C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基、C3-6环烷基或3-6元杂环基；
R5和R6各自独立地选自氢、C1-6烷基、C2-6烯基、C3-6环烷基、C1-6卤代烷基；或者R5或R6与相邻碳原子连接成3-6元环；
或者R4和R5连接成4-6元环；
n＝1-6，例如1、2、3、4、5或6。
在一些实施方案中，R3与苯环相邻碳原子相连成六元碳环，所述碳原子为R1间位且R2邻位的碳原子。
在一些实施方案中，式(I)中，
R1选自氢、氟和氯；
R2选自氢、甲基、氟、氯和羟基；或者R1和R2与相邻碳原子连接成5-6元含氧杂环；
R3选自氢、C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基或与苯环相邻碳原子相连成六元碳环；
A选自
B环选自3-6元碳环或3-6元杂环基；
R4选自氢、C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基、C3-6环烷基或3-6元杂环基；
R5和R6各自独立地选自氢、C1-6烷基、C3-6环烷基、C1-6卤代烷基；或者R5或R6与相邻碳原子连接成3-6元环；
或者，R4和R5连接成4-6元环；
n＝1或2。
在一些实施方案中，所述化合物具有式(II)的结构：
在式(II)中，
R1'选自氢、氟和氯；
R2'选自甲基、氟、氯和羟基；
R3'选自氢、C1-6烷基、氘代C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基、C3-6环烷基和3-6元杂环基；R3'优选氢、C1-6烷基、氘代C1-6烷基、C1-6卤代烷基或C1-6烷氧烷基；
R4'选自氢、C1-6烷基和C1-6卤代烷基；R4'优选氢；
当R1'为氟，且R2'为甲基时，R3'和R4'不同时为氢；且
当R1'为氟，且R2’为甲基时，R4’不为C1-6烷基。
在一些实施方案中，在式(II)中，
R1'选自氢、氟和氯；
R2'选自甲基、氟、氯和羟基；
R3'选自氢、C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基、C3-6环烷基和3-6元杂环基；R3'优选氢、C1-6烷基、C1-6卤代烷基或C1-6烷氧烷基；
R4'选自氢、C1-6烷基和C1-6卤代烷基；R4'优选氢；
当R1'为氟，且R2'为甲基时，R3'和R4'不同时为氢；且
当R1’为氟，且R2’为甲基时，R4’不为C1-6烷基。
在一些实施方案中，在式(II)中，
R1'选自氢、氟和氯；
R2'为氯或甲基；
R3'为氢、甲基或氘代甲基；
R4'为氢。
在一些实施方案中，在式(II)中，
R1'为氟；
R2'为氯；
R3'选自氢、C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基、C3-6环烷基和3-6元杂环基；R3'优选氢、C1-6烷基、C1-6卤代烷基或C1-6烷氧烷基；优选氢、C1-6烷基或C1-6卤代烷基；例如，氢、甲基或氘代甲基；
R4'选自氢、C1-6烷基和C1-6卤代烷基；R4'优选氢。
在一些实施方案中，在式(II)中，
R1'为氯；
R2'为甲基；
R3'选自氢、C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基、C3-6环烷基和3-6元杂环基；R3'优选氢、C1-6烷基、C1-6卤代烷基或C1-6烷氧烷基；优选氢、C1-6烷基或C1-6卤代烷基；例如，氢、甲基或氘代甲基；
R4'选自氢、C1-6烷基和C1-6卤代烷基；R4'优选氢。
在一些实施方案中，所述化合物具有式(III)的结构：
在式(III)中，
R1选自氢、氟和氯；
R2选自氢、甲基、氟、氯和羟基；
R3选自氢、C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基、C3-6环烷基和3-6元杂环基，或者R3与苯环相邻碳原子相连成六元碳环；R3优选氢；
R4选自氢和C1-6烷基；
R5和R6独立选自氢、C1-6烷基、C2-6烯基、C1-6卤代烷基、C3-6环烷基和3-6元杂环基，或者R5和R6与相邻碳原子连接成3-6元环；
或者，R4和R5连接成4-6元环；
n＝1或2；
当R1为氟，R2为甲基，R3为氢，且R4为氢时，R5和R6不同时为氢，也不与相邻碳原子连接成环丙基；且
当R1为氟，R2为甲基，且R3为氢时，R4不为烷基。
在一些实施方案中，R3与苯环相邻碳原子相连成六元碳环，所述碳原子为R1间位且R2邻位的碳原子。
在一些实施方案中，所述化合物具有式(III-1)所示结构，

R1选自氢、氟和氯；
R2选自氢、甲基、氟、氯和羟基；
R3选自氢、C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基、C3-6环烷基和3-6元杂环基；R3优选氢；
R4选自氢和C1-6烷基；
R5和R6独立选自氢、C1-6烷基、C1-6卤代烷基、C3-6环烷基和3-6元杂环基，或者R5和R6与相邻碳原子连接成3-6元环；
当R1为氟，R2为甲基，R3为氢，且R4为氢时，R5和R6不同时为氢，也不与相邻碳原子连接成环丙基；且
当R1为氟，R2为甲基，且R3为氢时，R4不为烷基。
在一些实施方案中，R2选自氟和氯。
在一些实施方案中，R2选自氢、甲基、氟和氯。
在一些实施方案中，R2选自甲基和氯。
在一些实施方案中，R3选自氢、C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基、C3-6环烷基和3-6元杂环基。
在一些实施方案中，R4为氢。
在一些实施方案中，R5和R6独立选自氢、C1-6烷基、C1-6卤代烷基、C3-6环烷基和C2-6烯基，或者R5和R6与相邻碳原子连接成3-6元环。
在一些实施方案中，R5和R6独立选自氢、C1-6烷基、C2-6烯基、C3-6环烷基，或者R5和R6与相邻碳原子连接成3-6元碳环。
在一些实施方案中，R5和R6独立选自氢、甲基、乙烯基、烯丙基、环丙基，或者R5和R6与相邻碳原子连接成3元碳环。
在一些实施方案中，R5和R6独立选自氢、C1-6烷基和C3-6环烷基，或者R5和R6与相邻碳原子连接成3-6元碳环。
在一些实施方案中，R5和R6独立选自氢、甲基和环丙基，或者R5和R6与相邻碳原子连接成3元碳环。
在一些实施方案中，当n＝2时，式(III)中为
在一些实施方案中，R4和R5连接成5元环。
在一些实施方案中，R4和R5连接成5元环，R6为氢。
在一些实施方案中，在式(III)或式(III-1)中，
R1选自氟和氯；
R2选自氯和甲基；
R3选自氢和C1-6烷基，或者R3与苯环相邻碳原子相连成六元碳环；
R4为氢；
R5和R6独立选自氢、C1-6烷基、C1-6卤代烷基、C3-6环烷基和C2-6烯基，或者R5和R6与相邻碳原子连接成3-6元环；
或者，R4和R5连接成4-6元环。
在一些实施方案中，R3与苯环相邻碳原子相连成六元碳环，所述碳原子为R1间位且R2邻位的碳原子。
在一些实施方案中，在式(III)或式(III-1)中，
R1为氟；
R2为氯；
R3选自氢和C1-2烷基，或者R3与苯环相邻碳原子相连成六元碳环；
R4为氢；
R5和R6独立选自氢、C1-2烷基、C1-2卤代烷基、C3-6环烷基和C2-4烯基，或者R5和R6与相邻碳原子连接成3-6元环；
或者，R4和R5连接成4-6元环。
在一些实施方案中，R3与苯环相邻碳原子相连成六元碳环，所述碳原子为R1间位且R2邻位的碳原子。
在一些实施方案中，在式(III)或式(III-1)中，
R1为氯；
R2为甲基；
R3选自氢和C1-2烷基，或者R3与苯环相邻碳原子相连成六元碳环；
R4为氢；
R5和R6独立选自氢、C1-2烷基、C1-2卤代烷基、C3-6环烷基和C2-4烯基，或者R5和R6与相邻碳原子连接成3-6元环；
或者，R4和R5连接成4-6元环。
在一些实施方案中，R3与苯环相邻碳原子相连成六元碳环，所述碳原子为R1间位且R2邻位的碳原子。
在一些实施方案中，在式(III)或式(III-1)中，
R1为氟；
R2为甲基；
R3选自氢和C1-2烷基，或者R3与苯环相邻碳原子相连成六元碳环；
R4为氢；
R5和R6独立选自氢、C1-2烷基、C1-2卤代烷基、C3-6环烷基和C2-4烯基，或者R5和R6与相邻碳原子连接成3-6元环；优选地，R5和R6不同时为氢；
或者，R4和R5连接成4-6元环。
在一些实施方案中，R3与苯环相邻碳原子相连成六元碳环，所述碳原子为R1间位且R2邻位的碳原子。
在一些实施方案中，在式(III)或式(III-1)中，
R1选自氟和氯；
R2选自甲基和氯；优选为氯；
R3为氢，或者R3与苯环相邻碳原子相连成六元碳环；
R4为氢；
R5和R6独立选自氢、C1-6烷基、C1-6卤代烷基、C3-6环烷基和C2-6烯基，或者R5和R6与相邻碳原子连接成3-6元环；
或者，R4和R5连接成4-6元环。
在一些实施方案中，R3与苯环相邻碳原子相连成六元碳环，所述碳原子为R1间位且R2邻位的碳原子。
在一些实施方案中，在式(III)或式(III-1)中，
R1选自氟和氯；
R2选自氯；
R3为氢，或者R3与苯环相邻碳原子相连成六元碳环；
R4为氢；
R5和R6独立选自氢、甲基、三氟甲基、环丙基和乙烯基，或者R5和R6与相邻碳原子连接成3元环；
或者，R4和R5连接成5元环。
在一些实施方案中，R3与苯环相邻碳原子相连成六元碳环，所述碳原子为R1间位且R2邻位的碳原子。
在一些实施方案中，所述化合物具有式(IV)的结构：
在式(IV)中，
R1'选自氢、氟和氯；
R2'选自氢、甲基、氟、氯和羟基；
R4'选自氢、C1-6烷基、C3-6环烷基和C1-6卤代烷基；R4'优选自氢；
B选自C1-6亚烷基、3-6元碳环或3-6元杂环基；
当R1’为氟、R2’为甲基、虚线碳环存在时，R4’不为氢且不为C1-6烷基；且
当R2’是甲基，R1’是氟，虚线碳环不存在时，B环不为3元碳环。
在一些实施方案中，在式(IV)中，R1'选自氢、氟和氯；
R2'选自氢、甲基、氟、氯和羟基；
R4'选自氢、C1-6烷基、C3-6环烷基和C1-6卤代烷基；R4'优选自氢；
B选自3-6元碳环或3-6元杂环基；
当R1’为氟、R2’为甲基、虚线碳环存在时，R4’不为氢且不为烷基；且
当R2’是甲基，R1’是氟，虚线碳环不存在时，B环不为3元碳环。
在一些实施方案中，R1'选自氟和氯。
在一些实施方案中，R2'选自氢、甲基、氟和氯。
在一些实施方案中，R2'选自甲基和氯。
在一些实施方案中，R4'为氢。
在一些实施方案中，B为C1-6亚烷基；优选地，B为亚甲基、亚乙基、亚丙基、亚异丙基；更优选地，B为亚丙基、亚异丙基。
在一些实施方案中，B环为3-6元碳环；优选地，B环为3-6元饱和碳环。
在一些实施方案中，本发明提供下述化合物：



另一方面，本发明还提供式(V)化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药：
M-L-E-D
式(V)
其中，
M是连接抗体或其抗原结合片段的接头；
L是连接接头M和E之间的连接子；
E是连接L和D的结构片段；
D是细胞毒性药物的结构片段。
在一些实施方案中，M选自以下结构：
其中，X选自离去基团，比如氯、溴、-OMs、OTs、OTf。
在一些实施方案中，M选自以下结构：
在一些实施方案中，L选自由下述的一个或多个组成的结构：C1-6亚烷基、-N(R’)-、羰基、-O-、Val、Cit、Phe、Lys、D-Val、Leu、Gly、Ala、Asn、Val-Cit、Val-Ala、Val-Lys、Val-Lys(Ac)、Phe-Lys、Phe-Lys(Ac)、D-Val-Leu-Lys、Gly-Gly-Arg、Ala-Ala-Asn、Ala-Ala-Ala、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Gly-Gly-Gly、
其中R’代表氢、C1-6烷基或含-(CH2CH2O)r-的烷基；r选自1-10的整数；s选自1-20的整数。
在一些实施方案中，L选自以下结构：
其中s选自1-20的整数。
在一些实施方案中，E选自单键、-NH-CH2-、
在一些实施方案中，E为-NH-CH2-。
在一些实施方案中，所述细胞毒性药物选自本发明第一方面任一项所述的化合物。
在一些实施方案中，所述细胞毒性药物选自本发明所述的化合物1-1至1-2；2-1至2-23；3-1至3-4；或4-1至4-12。
在一些实施方案中，D选自本发明第一方面所述化合物去氢之后的结构。
在一些实施方案中，D选自本发明所述的化合物1-1至1-2；2-1至2-18；3-1至3-18；4-1至4-16；5-1至5-10；6-1至6-10；7-1至7-2；8-1至8-2或9-1至9-2去氢之后的结构。
在一些实施方案中，D选自以下结构：




在一些实施方案中，M-L-E-D选自以下化合物：
A-1：
A-2：
A-3：
A-4：
A-5：
A-6：
A-7：
A-8：
A-9：
A-10：
A-11：
A-12：
B-1：
B-2：
B-3：
B-4：
B-5:
B-6:
B-7:
B-8:
B-9:
B-10:
B-11:
B-12:
C-1：
C-2：
C-3：
C-4：
C-5：
C-6：
C-7：
C-8：
C-9：
C-10:
C-11:
另一方面，本发明还提供式(VI)所示抗体药物偶联物(ADC)：
Ab-(M-L-E-D)x
式(VI)
其中，
Ab是抗体或其抗原结合片段；
M是连接所述抗体或其抗原结合片段的接头部位；
L是连接接头M和E之间的连接子；
E是连接L和D的结构片段；
D是细胞毒性药物的结构片段；
x为1至10。
在一些实施方案中，式(VI)中的M、L、E、D如前所述。
在一些实施方案中，所述抗体药物偶联物(ADC)如式(VI-1)所示：
Ab-M-L-E-D
式(VI-1)
在一些实施方案中，Ab-(M-L-E-D)x选自以下结构：
ADC A-1：
ADC A-2：
ADC A-3：
ADC A-4：
ADC A-5：
ADC A-6：
ADC A-7：
ADC A-8：ADC A-9：
ADC A-10：
ADC A-11：
ADC A-12：
ADC B-1：
ADC B-2：
ADC B-3：
ADC B-4：
ADC B-5：
ADC B-6：
ADC B-7：
ADC B-8：
ADC B-9：
ADC B-10：
ADC B-11：
ADC B-12：
ADC C-1：
ADC C-2：
ADC C-3：
ADC C-4：
ADC C-5：
ADC C-6：
ADC C-7：
ADC C-8：
ADC C-9：
ADC C-10:
ADC C-11:
在一些实施方案中，所述x为1-10，例如：1～2，1～3，1～4，1～5，1～6，1～7，1～8，1～9，1～10，2～3，2～4，2～5，2～6，2～7，2～8，2～9，2～10，3～4，3～5，3～6，3～7，3～8，3～9，3～10，4～5，4～6，4～7，4～8，4～9，4～10，5～6，5～7，5～8，5～9，5～10，6～7，6～8，6～9，6～10，7～8，7～9，7～10，8～9，8～10，或9～10，优选为3～9。
在一些实施方案中，所述x为1，2，3，4，5，6，7，8，9或10。
在一些实施方案中，本发明还提供下述抗体药物偶联物(ADC)或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药：
Trastuzumab A-11：
Trastuzumab B-1：
Trastuzumab B-2：
在另一方面，本申请提供了如本文所述的抗体药物偶联物(ADC)的组合物。这种组合物可包含多个本文所述的ADC，其中每个ADC包含本文所述的药物-连接体，其中x独立地为1、2、3、4、5、6、7、8、9或10。换言之，所述组合物中的每个抗体分子可以与1、2、3、4、5、6、7、8、9或10个药物-连接体缀合。因此，所述组合物的特征在于“药物-抗体”比(DAR)在约1至约10的范围内。测定DAR的方法是技术人员熟知的，包括使用反相色谱或HPLC-MS的方法。
例如，在任意实施方案中，本文所述的ADC组合物具有约1至约10或其间任何子范围的DAR，例如:约1至2、约1至3、约1至4、约1至5、约1至6、约1至7、约1至8、约1至9、约1至10、约2至3、约2至4、约2至5、约2至6，约2至7，约2至8，约2至9，约2至10，约3至4，约3至5，约3至6，约3至7，约3至8，约3至9，约3至10，约4至5，约4至6，约4至7，约4至8，约4至9，约4至10，约5至6，约5 至7，约5至8，约5至9、约5至10、约6至7、约6至8、约6至9、约6至10、约7至8、约7至9、约7至10、约8至9、约8至10或约9至10。
在某些实施方案中，本文所述ADC组合物的DAR为约3至9，例如约3.0至3.5、约3.0至4.0、约3.0至4.5、约3.0至5.0、约3.0至6.0、约3.5至4.0、约3.5至4.5、约3.5至5.0、约3.5至5.5、约3.5至6.0、约3.5至6.5至6约4.0至4.5，约4.0至5.0，约4.0至5.5，约4.0至6.0，约4.0至6.5，约4.0至7.0，约4.0至8.0，约4.5至5.0，约4.5至5.5，约4.5至6.0，约4.5至6.5，约4.5至7.0，约4.5至7.5约5.0至8.0，约5.5至6.0，约5.5至6.5，约5.5至7.0，约5.5至7.5，约5.5至8.0，约6.0至6.5，约6.0至7.0，约6.0至7.5，约6.0至8.5，约6.5至7.0，约6.5至7.5，约6.5至7.5，约6.5至8.5，约7.0至7.5，约7.5至8.0，约7.5至8.5。
在一些实施方案中，本发明还提供包含一种或多种下述抗体药物偶联物的组合物，所述组合物的DAR值(药物抗体偶联比)为7.5至8.5，优选为7.5至8.0，进一步优选为8.0：
Trastuzumab A-11：
在一些实施方案中，本发明还提供包含一种或多种下述抗体药物偶联物的组合物，所述组合物的DAR值(药物抗体偶联比)为7.5至8.5，优选为7.5至8.0，进一步优选为7.96：
Trastuzumab B-1：
在一些实施方案中，本发明还提供包含一种或多种下述抗体药物偶联物的组合物，所述组合物的DAR值(药物抗体偶联比)为7.5至8.5，优选为7.5至8.0，进一步优选为7.95：
Trastuzumab B-2：
定义
除非在下文中另有定义，本文中所用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。提及本文中使用的技术意图指在本领域中通常所理解的技术，包括那些对本领域技术人员显而易见的技术的变化或等效技术的替换。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本发明。
术语“包括”、“包含”、“具有”、“含有”或“涉及”及其在本文中的其它变体形式为包含性的(inclusive)或开放式的，且不排除其它未列举的元素或方法步骤。
如本文中所使用，化合物结构式中标记的“*”表示被标记的碳原子为手性碳原子，本发明包括由该手性碳原子形成的一对对映异构体。如在一个化合物中含两个不同的手性碳原子，本发明包括由该手性碳原子形成的4个旋光异构体。
如本文中所使用，表示结构片段与分子其他部分相连接的部位。
术语“烷基”定义为直链或支链饱和脂肪族烃基。在一些实施方案中，烷基具有1至12个，例如1至6个碳原子。例如，如本文中所使用，术语“C1-6烷基”指1至6个碳原子的线性或支化的基团(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基和正己基)，其任选地被1或多个(诸如1、2或3个)适合的取代基取代。
术语“烯基”是指含有至少一个碳碳双键的直链或支链的烃基，包括例如“C2-6烯基”、“C2-4烯基”等。其实例包括但不限于：乙烯基、1-丙烯基、2-丙烯基、1-丁烯基、2-丁烯基、1,3-丁二烯基、1-戊烯基、2-戊烯基、3-戊烯基、1,3-戊二烯基、1,4-戊二烯基、1-己烯基、2-己烯基、3-己烯基、1,4-己二烯基等。
术语“炔基”是指含有至少一个碳碳三键的直链或支链烃基。包括例如“C2-6炔基”、“C4-6炔基”等。其实例包括但不限于：乙炔基、1-丙炔基、2-丙炔基、1-丁炔基、2-丁炔基、1,3-丁二炔基、1-戊炔基、2-戊炔基、3-戊炔基、1,3-戊二炔基、1,4-戊二炔基、1-己炔基、2-己炔基、3-己炔基、1,4-己二炔基等。
术语“环烷基”是指饱和环状烃基，包括但不限于单环烷基和双环烷基(诸如螺环烷基、 并环烷基和桥环烷基)。术语“C3-6环烷基”指具有3至6个成环碳原子的环烷基，诸如环丙基、环丁基、环戊基、环己基等，其可任选地被1或多个(诸如1、2或3个)适合的取代基取代，例如甲基取代的环丙基。
术语“碳环”或“碳环基”是指饱和或部分不饱和的非芳族单环或多环结构、通过环碳连接的烃基。其实例包括但不限于环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环庚基和环辛基。
术语“碳环”指饱和或不饱和的非芳族单环或多环(诸如双环)烃环(例如单环，诸如环丙烷环、环丁烷环、环戊烷环、环己烷环、环庚烷环、环辛烷环、环壬烷环，或双环，包括螺环、稠合或桥连系统(诸如双环[1.1.1]戊烷环、双环[2.2.1]庚烷环、双环[3.2.1]辛烷环或双环[5.2.0]壬烷环、十氢化萘环等)，其可任选地被1或多个(诸如1、2或3个)适合的取代基取代。术语“3-6元碳环”是指含3、4、5或6个成环碳原子的碳环。
术语“杂环基”或“杂环”是指饱和或部分饱和的，单环或多环(诸如双环)的非芳香族环状结构，其环原子由碳原子以及至少一个(例如1、2或3个)选自氮、氧和硫的杂原子构成。如果满足价键要求，杂环基可以通过任意一个环原子与分子的其余部分连接。本发明中的杂环基优选为3-6元杂环基。本发明中所使用的术语“3-6元杂环基”是指具有3至6个环原子的杂环基，包括3元杂环基、4元杂环基、5元杂环基和6元杂环基，包括含氮杂环基、含氧杂环基，例如4-6元杂环基，例如4-6元含氮杂环基、4-6元含氧杂环基。常见的杂环基包括(但不限于)氮杂环丁烷基(azetidinyl)、氧杂环丁烷基(oxetanyl)、四氢呋喃基(tetrahydrofuryl)、吡咯烷基(pyrrolidinyl)、吡咯烷酮基(pyrrolidinonyl)、咪唑烷基(imidazolidinyl)、吡唑烷基(pyrazolidinyl)、四氢吡喃基(tetrahydropyranyl)、哌啶基(piperidinyl)、哌嗪基(piperazinyl)、吗啉基(morpholinyl)。本发明中的杂环基可任选地被一或多个本发明所描述的取代基取代。本发明中的杂环基任选地与一或多个芳香环或非芳香环稠合。
术语“含氧杂环”是指一个或多个(例如1、2或3个)环原子为氧原子的如前所述的杂环，例如5-6元含氧杂环，具体实例包括但不限于环氧乙烷环、四氢呋喃环、呋喃环、四氢吡喃环、吡喃环等。本发明所述的“含氮杂环”是指一个或多个(例如1、2或3个)环原子为氮原子的如前所述的杂环。
术语“卤代烷基”是指被一或多个(诸如1、2或3个)相同或不同的卤素原子取代的烷基，其中烷基定义如上所述。例如，本发明中所使用的术语“C1-6卤代烷基”是指具有1至6个碳原子的卤代烷基。常见的卤代烷基包括(但不限于)-CH2F、-CHF2、-CF3、-CH2CF3、-CF2CF3、-CH2CH2CF3、-CH2Cl等。本发明中的卤代烷基任选地被一或多个本发明所描述的取代基取代。
术语“烷氧基”是指具有“烷基-O-”结构的基团，其中烷基定义如上所述。例如C1-6烷氧基、C1-4烷氧基、C1-3烷氧基或C1-2烷氧基等。常见的烷氧基包括(但不限于)甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、戊氧基、己氧基等。本发明中的 烷氧基任选地被一或多个本发明所描述的取代基取代。
术语“烷氧基烷基”是指被一个或多个(例如1、2、3或4个)烷氧基取代的烷基，其中烷氧基及烷基的定义如上所述。例如，本发明中所使用的术语“C1-6烷氧基烷基”是指具有1-6个碳原子、被一个或多个(例如1、2、3或4个)烷氧基取代的烷基。常见的烷氧基烷基包括(但不限于)CH3O-CH2-,C2H5-O-CH2-、C2H5-O-CH2CH2-等。
术语“卤代”或“卤素”基团定义为包括F、Cl、Br或I。
术语“氮氧化物”是指本申请化合物结构中的至少一个氮原子的氧化物(例如单-或二-氧化物)。氮的单-氧化物可以以单一的位置异构体或位置异构体的混合物的形式存在。
术语“取代”指所指定的原子上的一个或多个(例如一个、两个、三个或四个)氢被从所指出的基团的选择代替，条件是未超过所指定的原子在当前情况下的正常原子价并且所述取代形成稳定的化合物。取代基和/或变量的组合仅仅当这种组合形成稳定的化合物时才是允许的。
如果取代基被描述为“任选地被取代”，则取代基可(1)未被取代或(2)被取代。如果取代基的碳被描述为任选地被取代基列表中的一个或多个取代基所取代，则碳上的一个或多个氢(至存在的任何氢的程度)可单独和/或一起被独立地选择的任选的取代基替代。如果取代基的氮被描述为任选地被取代基列表中的一个或多个取代，则氮上的一个或多个氢(至存在的任何氢的程度)可各自被独立地选择的任选的取代基替代。
如果取代基被描述为“独立地选自”一组，则各取代基独立于另一者被选择。因此，各取代基可与另一(其他)取代基相同或不同。
如本文中所使用，术语“一个或多个”意指在合理条件下的1个或超过1个，例如2个、3个、4个、5个或10个。
除非指明，否则如本文中所使用，取代基的连接点可来自取代基的任意适宜位置。
术语“立体异构体”表示由于至少一个不对称中心形成的异构体。在具有一个或多个(例如一个、两个、三个或四个)不对称中心的化合物中，其可产生外消旋混合物、单一对映异构体、非对映异构体混合物和单独的非对映异构体。特定个别分子也可以几何异构体(顺式/反式)存在。类似地，本发明的化合物可以两种或更多种处于快速平衡的结构不同的形式的混合物(通常称作互变异构体)存在。互变异构体的代表性实例包括酮-烯醇互变异构体、苯酚-酮互变异构体、亚硝基-肟互变异构体、亚胺-烯胺互变异构体等。要理解，本申请的范围涵盖所有这样的以任意比例(例如60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％或99％)的异构体或其混合物。
本文中可使用实线实楔形或虚楔形描绘本发明的化合物的碳-碳键。使用实线以描绘键连至不对称碳原子的键欲表明，包括该碳原子处的所有可能的立体异构 体(例如，特定的对映异构体、外消旋混合物等)。使用实或虚楔形以描绘键连至不对称碳原子的键欲表明，存在所示的立体异构体。当存在于外消旋混合物中时，使用实及虚楔形以定义相对立体化学，而非绝对立体化学。除非另外指明，否则本发明的化合物意欲可以立体异构体(其包括顺式及反式异构体、光学异构体(例如R及S对映异构体)、非对映异构体、几何异构体、旋转异构体、构象异构体、阻转异构体及其混合物)的形式存在。本发明的化合物可表现一种以上类型的异构现象，且由其混合物(例如外消旋混合物及非对映异构体对)组成。
本发明涵盖本发明的化合物的所有可能的结晶形式或多晶型物，其可为单一多晶型物或多于一种多晶型物的任意比例的混合物。
还应当理解，本发明的某些化合物可以游离形式存在用于治疗，或适当时，以其药学上可接受的衍生物形式存在。在本发明中，药学上可接受的衍生物包括但不限于，可药用盐、酯、溶剂合物、代谢物或前药，在将它们向需要其的患者给药后，能够直接或间接提供本发明的化合物或其代谢物或残余物。因此，当在本文中提及“本发明的化合物”时，也意在涵盖化合物的上述各种衍生物形式。
本发明的化合物的可药用盐包括其酸加成盐及碱加成盐。
适合的酸加成盐由形成药学可接受盐的酸来形成，包括天冬氨酸盐、延胡索酸盐、葡庚糖酸盐、葡糖酸盐、葡糖醛酸盐、六氟磷酸盐等。
适合的碱加成盐由形成药学可接受盐的碱来形成，包括铝盐、精氨酸盐、胆碱盐、二乙胺盐等。
适合的盐的综述参见Stahl及Wermuth的“Handbook of Pharmaceutical Salts:Properties,Selection,and Use”(Wiley-VCH,2002)。用于制备本发明的化合物的可药用盐的方法为本领域技术人员已知的。
术语“酯”意指衍生自本申请中各个通式化合物的酯，其包括生理上可水解的酯(可在生理条件下水解以释放游离酸或醇形式的本发明的化合物)。本发明的化合物本身也可以是酯。
本发明的化合物可以溶剂合物(优选水合物)的形式存在，其中本发明的化合物包含作为所述化合物晶格的结构要素的极性溶剂，特别是例如水、甲醇或乙醇。极性溶剂特别是水的量可以化学计量比或非化学计量比存在。
在本发明的范围内还包括本发明的化合物的代谢物，即在给药本发明的化合物时体内形成的物质。这样的产物可由例如被给药的化合物的氧化、还原、水解、酰胺化、脱酰胺化、酯化、脱脂化、酶解等产生。因此，本发明包括本发明的化合物的代谢物，包括通过使本发明的化合物与哺乳动物接触足以产生其代谢产物的时间的方法制得的化合物。
本发明在其范围内进一步包括本发明的化合物的前药。通常这样的前药会是所述化合物的官能团衍生物，其易于在体内转化成期望的治疗活性化合物。因此，在这些情况中，用于本发明的治疗方法的术语“给药”应包括用所要求保护的化合物中的一种或多种的前药形式来治疗各种疾病或病症，但是在向个体给药后所述前药形式在体内转化成上述化合物。例如，在“Design of Prodrug”,ed.H.Bundgaard,Elsevier,1985中，描述了选择和制备适合的前药衍生物的常规方法。
本发明在其范围内进一步包括本发明的化合物的同位素标记物，其与本发明的化合物相同，除了一个或多个原子被具有相同原子序数但原子质量或质量数不同于在自然界中占优势的原子质量或质量数的原子替代。
本发明还涵盖含有保护基的本发明的化合物。在制备本发明的化合物的任何过程中，保护在任何有关分子上的敏感基团或反应基团可能是必需的和/或期望的，由此形成本发明的化合物的化学保护的形式。这可以通过常规的保护基实现，例如，在Protective Groups in Organic Chemistry,ed.J.F.W.McOmie,Plenum Press,1973；和T.W.Greene&P.G.M.Wuts,Protective Groups in Organic Synthesis,John Wiley&Sons,1991中所述的那些保护基，这些参考文献通过援引加入本文。使用本领域已知的方法，在适当的后续阶段可以移除保护基。
药物组合物
在第三个方面，本发明提供一种药物组合物，其包含本发明第一方面或第二方面所述的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药，以及一种或多种药学上可接受的载体。
术语“药物组合物”是指可以用作药物的组合物，其包含药物活性成分(API)(或治疗剂)以及可选的一种或多种药学上可接受载体。术语“药学上可接受的载体”是指与治疗剂一同给药的辅料，并且其在合理的医学判断的范围内适于接触人类和/或其它动物的组织而没有过度的毒性、刺激性、过敏反应或与合理的益处/风险比相应的其它问题或并发症。
上述药物组合物可以系统地作用和/或局部地作用，其可以通过适合的剂型来实现。所述剂型包括但不限于片剂、胶囊剂、锭剂、硬糖剂、散剂、喷雾剂、乳膏剂、软膏剂、栓剂、凝胶剂、糊剂、洗剂、软膏剂、水性混悬剂、可注射溶液剂、酏剂、糖浆剂。
上述药物组合物可以包含0.01mg至1000mg的至少一种本发明的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药。
本发明还提供了上述药物组合物或其相应的制剂形式的制备方法，其包括将至少一种本发明的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药与一种或多种药学上可接受的载体组合。
药盒产品
在第四个方面，本发明提供一种药盒产品，其包含：
a)作为第一治疗剂的至少一种本发明第一方面或第二方面所述的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药，或者作为第一药物组合物的第三方面所述的药物组合物；
b)任选存在的作为第二治疗剂的至少一种其他治疗剂，或者作为第二药物组合物的包含其他治疗剂的药物组合物；和
c)任选存在的包装和/或说明书。
上述药盒产品可以包含0.01mg至1000mg的至少一种本发明的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药。
本发明还提供了上述药盒的制备方法，其包括将至少一种本发明的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药或者上述药物组合物与任选存在的至少一种其他治疗剂或者包含其他治疗剂的药物组合物、包装和/或说明书组合。
医药用途
本发明的化合物能够表现出较强的抑制细胞异常增殖方面的作用。
因此，本申请提供了本发明的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药或者上述药物组合物，其用于治疗细胞异常增殖方面的疾病。
另外，本申请还提供了本发明的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药或者上述药物组合物在制备用于治疗细胞异常增殖方面的疾病的药物中的用途。
在一些实施方案中，所述细胞异常增殖方面的疾病包括(但不限于)肿瘤，例如晚期实体瘤。
本申请还提供本发明的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药或者本发明的药物组合物在制备制剂中的用途，所述制剂用于抑制肿瘤细胞的增殖。在某些实施方案中，所述制剂用于体内或者体外施用。例如，所述制剂可被施用至受试者体内，以抑制受试者体内的肿瘤细胞的增殖；或者，所述制剂可被施用至体外细胞(例如细胞系或者来自受试者的细胞)，以抑制体外肿瘤细胞的增殖。
本发明所述肿瘤包括(但不限于)：脑瘤、肺癌、鳞状上皮细胞癌、膀胱癌、胃癌、卵巢癌、腹膜癌、胰腺癌、乳腺癌、头颈癌、子宫颈癌、子宫内膜癌、结直肠癌、肝癌、肾癌、食管腺癌、食管鳞状细胞癌、前列腺癌、雌性生殖道癌、原位癌、淋巴瘤、神经纤维瘤、甲状腺癌、骨癌、皮肤癌、脑癌、结肠癌、睾丸癌、胃肠道间质瘤、前列腺肿瘤、肥大细胞肿瘤、多发性骨髓瘤、黑色素瘤、胶质瘤或肉瘤。
治疗方法
在另一个方面，本发明提供了一种用于治疗细胞异常增殖方面的疾病的方法，其包括下列步骤：将治疗有效量的本发明的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药或者上述药物组合物施用于对其有需求的个体。
术语“有效量”是指能够诱发细胞、组织、器官或生物体(例如个体)产生生物或医学反应，并且足以实现所需预防和/或治疗效果的剂量。
可调整给药方案以提供最佳所需响应。例如，可单次给药，可随时间分剂量给药，或可根据实际情况按比例减少或增加剂量后给药。可以理解的是，对于任何特定个体，具体的给药方案应根据需要以及给药组合物或监督组合物的给药人员的专业判断而调整。
本发明的化合物的给药量将取决于个体情况、疾病或病症的严重性、给药的速率、化合物的处置及处方医师的判断。一般而言，有效量为约0.001-10000mg/kg受试者体重/天。在合适的情况下，有效量为约0.01-1000mg/kg受试者体重/天。可以每天、每两天或每三天给药约0.01-1000mg/kg受试者体重，通常约0.1-500mg/kg受试者体重。示例性的给药方案为每天一次或多次，或者每周一次或多次，或者每月一次或多次。多次给药时，单次剂量之间的间隔通常可以是每天、每周、每月或每年。或者，可以缓释制剂的形式给药，在这种情况下需要较低的给药频率。给药剂量和频率可根据药物在受试者体内的半衰期而不同，也可以根据是预防性应用还是治疗性应用而不同。在预防性应用中，以相对低频率的间隔长期施用相对低的剂量；在治疗性应用中，有时需要以较短的间隔施用相对高的剂量，直至疾病的进展被延缓或停止，优选直至个体表现出疾病症状的部分或完全改善，此后可以采用预防性应用。
术语“治疗”是指减轻或消除所针对的疾病或病症。如果受试者接受了治疗量的本发明的化合物或其药学上可接受的形式或者本发明的药物组合物，该受试者的至少一种指标和症状表现出可观察到的和/或可检测出的缓解和/或改善，则表明该受试者已被成功地“治疗”。可以理解的是，治疗不仅包括完全地治疗，还包括未达到完全地治疗，但实现了一些生物学或医学相关的结果。
术语“施用(administrate/administrating/administration)”(或“给药”)是指将药物活性成分(比如本发明的化合物)或包含药物活性成分的药物组合物(例如本发明的药物组合物) 应用于个体或其细胞、组织、器官、生物流体等部位，以便使药物活性成分或药物组合物与个体或其细胞、组织、器官、生物流体等部位接触的过程。常见的施用方式包括(但不限于)口服施用、皮下施用、肌内施用、腹膜下施用、眼部施用、鼻部施用、舌下施用、直肠施用、阴道施用等。
术语“对其有需求”是指医生或其他护理人员对个体需要或者将要从预防和/或治疗过程中获益的判断，该判断的得出基于医生或其他护理人员在其专长领域中的各种因素。
术语“个体”(或称受试者)是指人类或非人动物。本发明的个体包括患有疾病和/或病症的个体(患者)和正常的个体。本发明的非人动物包括所有脊椎动物，例如非哺乳动物，例如鸟类、两栖类、爬行类等，和哺乳动物，例如非人灵长类、家畜和/或驯化动物(例如绵羊、犬、猫、奶牛、猪等)。
制备方法
本发明第四方面提供了所述化合物的合成方法。
当R3为氢时，本发明中的式(I)化合物可由下述合成路线进行合成制备。
其中，R1、R2、R3、R4和A含义如前文所述，LG为离去基团，选自甲磺酰基、三氟甲磺酰氧基和卤素，优选三氟甲磺酰氧基或碘；
步骤一
化合物式(I)-SM1和化合物式(I)-SM2发生傅克酰基化反应得到式(I)-IM1化合物；
在部分实施方案中，该步骤在适合的温度下进行，所述温度为5℃、20℃、25℃、40℃、50℃、60℃、100℃，优选25℃。
步骤二
化合物式(IV)-IM1和化合物式(I)-SM3在酸性条件下发生闭环反应得到化合物式(I)-IM2；
在部分实施方案中，该步骤在适合的温度下进行，所述温度为20℃、25℃、40℃、 50℃、60℃、100℃、120℃，优选120℃；
在部分实施方案中，该步骤在适合的有机溶剂中进行，所述有机溶剂可选自甲苯、二甲苯、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲基亚砜，优选甲苯和二甲苯；
在部分实施方案中，该步骤在酸性条件下进行；
提供酸性条件的试剂包括对甲苯磺酸、盐酸、三氟乙酸、甲酸、硫酸、甲磺酸，优选为对甲苯磺酸。
步骤三：
化合物式(I)-IM2发生Delépine反应得到化合物式(I)-IM3
在部分实施方案中，该步骤在适合的温度下进行，所述温度为5℃、20℃、25℃、40℃、50℃、60℃、85℃、100℃，优选85℃。
步骤四：
化合物式(I)-IM3与化合物式(I)-SM4发生取代反应得到式(I)-IM4化合物。
在部分实施方案中，该步骤在适合的温度下进行，所述温度为20℃、25℃、40℃、50℃、60℃、100℃、140℃，优选50℃；
在部分实施方案中，该步骤在适合的有机溶剂中进行，所述有机溶剂可选自卤代烃类(例如二氯甲烷(DCM)、氯仿(TCM)、1,2-二氯乙烷(1,2-DCE)等)、腈类(例如乙腈(AN)等)、N-甲基吡咯烷酮(NMP)、N,N-二甲基甲酰胺(DMF)、N,N-二甲基乙酰胺(DMA)、四氢呋喃(THF)、1,4-二氧六环(Dioxane)、二甲基亚砜(DMSO)及其任意组合，优选乙腈。
在部分实施方案中，该步骤在适合的碱存在下进行，所述碱包括有机碱或无机碱，所述有机碱可选自N,N-二异丙基乙胺(DIPEA)、三乙胺(TEA)、叔丁醇钾(t-BuOK)和吡啶(Py)，所述无机碱可选自磷酸钾(K3PO4)、氢化钠(NaH)、碳酸钾(K2CO3)、碳酸钠(Na2CO3)、碳酸氢钠(NaHCO3)、碳酸铯(Cs2CO3)和NaOH，优选Na2CO3或NaHCO3；
步骤五
当A为时，化合物式(I)-IM4与化合物式(I)-SM5-1或化合物式(I)-SM5-2发生缩合反应得到式(I)化合物。R5、R6、n和环B含义如前文所述。
在部分实施方案中，该步骤在合适的缩合试剂下进行，所述缩合试剂可选自HATU、HBTU、EDCI、DCC和HOBT，优选HBTU、HATU；
在部分实施方案中，该步骤在适合的温度下进行，所述温度为20℃、25℃、40℃、50℃、60℃、100℃，优选25℃；
在部分实施方案中，该步骤在适合的有机溶剂中进行，所述有机溶剂可选自甲醇、四氢呋喃、二氯甲烷、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲基亚砜、正庚烷、正己烷、乙酸乙酯，优选N,N-二甲基甲酰胺。
在部分实施方案中，该步骤在适合的碱中进行，所述碱包括有机碱或无机碱，所述有机碱可选自DIPEA、TEA、t-BuOK和Py，所述无机碱可选自K3PO4、NaH、K2CO3、Na2CO3、Cs2CO3和NaOH，优选DIPEA。
当A为R4和R5为氢时，化合物式(I)-IM4与三甲基氰硅烷发生反应得到式(I)化合物。
在部分实施方案中，该步骤在适合的温度下进行，所述温度为5℃、20℃、25℃、40℃、50℃、60℃、90℃、100℃，优选90℃；
在部分实施方案中，该步骤在适合的有机溶剂中进行，所述有机溶剂可选自四氢呋喃、二氯甲烷、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲基亚砜和1,4-二氧六环， 优选1,4-二氧六环。
在部分实施方案中，该步骤在适合的碱中进行，所述碱包括有机碱或无机碱，所述有机碱可选自DIPEA、TEA、t-BuOK和Py，所述无机碱可选自K3PO4、NaH、K2CO3、Na2CO3、Cs2CO3和NaOH，优选TEA。
当A为R5和R6为氢时，化合物式(I)-IM4与氯磺酰异氰酸酯发生反应，再脱除氨基保护基得到式(I)化合物。
在部分实施方案中，该步骤在适合的温度下进行，所述温度为20℃、25℃、40℃、50℃、60℃、100℃，优选0～25℃；
在部分实施方案中，该步骤在适合的有机溶剂中进行，所述有机溶剂可选自甲醇、四氢呋喃、二氯甲烷、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲基亚砜、正庚烷、正己烷、乙酸乙酯，优选N,N-二甲基甲酰胺。
在部分实施方案中，该步骤在适合的碱中进行，所述碱包括有机碱或无机碱，所述有机碱可选自DIPEA、TEA、t-BuOK和Py，所述无机碱可选自K3PO4、NaH、K2CO3、Na2CO3、Cs2CO3和NaOH，优选TEA。
当R3与苯环相邻碳原子相连成六元碳环时，本发明中的式(I)化合物可由下述合成路线进行合成制备。
其中，R1、R2、R3、R4和A含义如前文所述，LG为离去基团，选自甲磺酰基、三氟甲磺酰氧基和卤素，优选三氟甲磺酰氧基或碘；
步骤一：
化合物式(I)-SM6与化合物式(I)-SM4发生取代反应得到式(I)-IM5化合物。
在部分实施方案中，该步骤在适合的温度下进行，所述温度为20℃、25℃、40℃、50℃、60℃、100℃、140℃，优选50℃；
在部分实施方案中，该步骤在适合的有机溶剂中进行，所述有机溶剂可选自卤代烃类(例如二氯甲烷(DCM)、氯仿(TCM)、1,2-二氯乙烷(1,2-DCE)等)、腈类(例如乙腈(AN)等)、N-甲基吡咯烷酮(NMP)、N,N-二甲基甲酰胺(DMF)、N,N-二甲基乙酰胺(DMA)、四氢呋喃(THF)、1,4-二氧六环(Dioxane)、二甲基亚砜(DMSO)及其任意组合，优选乙腈。
在部分实施方案中，该步骤在适合的碱存在下进行，所述碱包括有机碱或无机碱，所述有机碱可选自N,N-二异丙基乙胺(DIPEA)、三乙胺(TEA)、叔丁醇钾(t-BuOK)和吡啶(Py)，所述无机碱可选自磷酸钾(K3PO4)、氢化钠(NaH)、碳酸钾(K2CO3)、碳酸钠(Na2CO3)、碳酸氢钠(NaHCO3)、碳酸铯(Cs2CO3)和NaOH，优选Na2CO3或NaHCO3；
步骤二
当A为时，化合物式(I)-IM5与化合物式(I)-SM5-2发生缩合反应得到式(I)化合物。R5、R6和环B含义如前文所述。
在部分实施方案中，该步骤在合适的缩合试剂下进行，所述缩合试剂可选自HATU、HBTU、EDCI、DCC和HOBT，优选HBTU、HATU；
在部分实施方案中，该步骤在适合的温度下进行，所述温度为20℃、25℃、40℃、50℃、60℃、100℃，优选25℃；
在部分实施方案中，该步骤在适合的有机溶剂中进行，所述有机溶剂可选自甲醇、四氢呋喃、二氯甲烷、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲基亚砜、正庚烷、正己烷、乙酸乙酯，优选N,N-二甲基甲酰胺。
在部分实施方案中，该步骤在适合的碱中进行，所述碱包括有机碱或无机碱，所述有机碱可选自DIPEA、TEA、t-BuOK和Py，所述无机碱可选自K3PO4、NaH、K2CO3、Na2CO3、Cs2CO3和NaOH，优选DIPEA。
当A为R4和R5为氢时，化合物式(I)-IM5与三甲基氰硅烷发生反应得到式(I)化合物。
在部分实施方案中，该步骤在适合的温度下进行，所述温度为5℃、20℃、25℃、40℃、50℃、60℃、90℃、100℃，优选90℃；
在部分实施方案中，该步骤在适合的有机溶剂中进行，所述有机溶剂可选自四氢呋喃、二氯甲烷、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲基亚砜和1,4-二氧六环，优选1,4-二氧六环。
在部分实施方案中，该步骤在适合的碱中进行，所述碱包括有机碱或无机碱，所述有机碱可选自DIPEA、TEA、t-BuOK和Py，所述无机碱可选自K3PO4、NaH、K2CO3、Na2CO3、Cs2CO3和NaOH，优选TEA。
当A为R5和R6为氢时，化合物式(I)-IM5与氯磺酰异氰酸酯发生反应，再脱除氨基保护基得到式(I)化合物。
在部分实施方案中，该步骤在适合的温度下进行，所述温度为20℃、25℃、40℃、50℃、60℃、100℃，优选0～25℃；
在部分实施方案中，该步骤在适合的有机溶剂中进行，所述有机溶剂可选自甲醇、四氢呋喃、二氯甲烷、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲基亚砜、正庚烷、正己烷、乙酸乙酯，优选N,N-二甲基甲酰胺。
在部分实施方案中，该步骤在适合的碱中进行，所述碱包括有机碱或无机碱，所述有机碱可选自DIPEA、TEA、t-BuOK和Py，所述无机碱可选自K3PO4、NaH、 K2CO3、Na2CO3、Cs2CO3和NaOH，优选TEA。
发明的有益效果
本发明提供式(I)-式(IV)所示的喜树碱类化合物，及其药物组合物、制备方法和用途。该类化合物具有良好的抗肿瘤活性，有克服耐药的潜力，可用于治疗细胞异常增殖性病症，所述病症包括但不限于晚期实体瘤。
具体实施方式
以下通过具体实施方式的描述对本申请作进一步说明，但这并非是对本申请的限制。本领域技术人员根据本申请的教导，可以做出各种修改或改进，而不脱离本申请的基本思想和范围。
本发明中的缩写具有以下含义：
以下的实施例中记载的化合物的结构通过核磁共振(1H NMR)或质谱(MS)来确定。
核磁共振(1H NMR)的测定仪器使用Bruker 400MHz核磁共振仪；六氘代二甲基亚砜(DMSO-d6)；内标物质为四甲基硅烷(TMS)。
实施例中使用的核磁共振(NMR)图谱中的缩写示于以下。
s：单峰(singlet)、d：二重峰(doublet)、t：三重峰(triplet)、q：四重峰(quartet)、m：多重峰(multiplet)、br：宽峰(broad)、J：偶合常数、Hz：赫兹、DMSO-d6：氘化二甲基亚砜。δ值用ppm值表示。
质谱(MS)的测定仪器使用Agilent(ESI)质谱仪，型号为Agilent 6120B。
实施例一(S)-11-(氨甲基)-9-氯-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-3,14(4H)-二酮的制备(化合物1-1)
步骤一：1-(2-氨基-5-氯-4-氟苯基)-2-氯乙基-1-酮的制备
向冰水冷却的三氯化硼(1M,36mL)的1,2-二氯乙烷(40mL)溶液中滴加4-氯-3-氟苯胺(4.36g,29.95mmol)的1,2-二氯乙烷(20mL)溶液，后在室温下依次加入2-氯乙腈(2.71g,35.94mmol)和四氯化钛(6.82g,35.94mmol)，升温至回流反应16小时，反应液冷却至室温后加入2.5N盐酸(55.0mL)并再次加热至85℃反应30分钟，反应液冷却至室温后经二氯甲烷萃取(3x 25mL),有机相经饱和食盐水洗涤和无水硫酸钠干燥，滤液过滤浓缩得标题化合物(2g,7.21mmol)，直接用于下一步反应。
ESI-MS(m/z):222[M+H]+.
步骤二：(S)-9-氯-11-(氯甲基)-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-3,14(4H)-二酮的制备
将1-(2-氨基-5-氯-4-氟苯基)-2-氯乙基-1-酮(1-1-2,0.5g,1.80mmol)加入甲苯(10mL)中，然后依次加入(S)-4-乙基-4-羟基-7,8-二氢-1H-吡喃并[3,4-f]吲哚嗪-3,6,10(4H)-三酮(IM-1，500.00mg,1.90mmol)和对甲苯磺酸一水合物(68.53mg,360.29μmol)，反应液加热至120℃反应4小时，反应液浓缩后用乙酸乙酯打浆，过滤收集固体得标题化合物(0.6g,1.34mmol)，直接用于下一步反应。
ESI-MS(m/z):449[M+H]+.
步骤三：(S)-11-(氨甲基)-9-氯-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-3,14(4H)-二酮的制备
将(S)-9-氯-11-(氯甲基)-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-3,14(4H)-二酮(1-1-3,0.1g,222.59μmol)溶于乙醇(5mL)中，加入乌洛托品(94mg,670.54μmol)，反应液加热至85℃反应5小时，反应液冷却至室温后加入 0.2mL浓盐酸，搅拌10分钟后直接浓缩，粗品经乙酸乙酯打浆，过滤收集固体并干燥得标题化合物(180mg,386.03μmol)的盐酸盐。将1-1的盐酸盐(20mg,46.62μmol)加入到DMF(1mL)和水(1mL)的混合溶剂中，用碳酸氢钠调节pH至8后，经制备高效液相色谱纯化(纯化条件如下)可得(S)-9-氯-11-(氯甲基)-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-3,14(4H)-二酮(1-1,3mg)。
色谱柱：SunFire Prep C18OBD 19mm×150mm×5.0μm
流动相A：乙腈；流动相B：水(0.05％甲酸)
结构表征数据如下：
1H NMR(400MHz,DMSO-d6)δ8.62(d,J＝8.1Hz,1H),8.16(d,J＝10.3Hz,1H),7.33(s,1H),6.56(s,1H),5.45(d,J＝8.6Hz,4H),4.35(s,2H),1.97–1.74(m,2H),0.88(t,J＝7.3Hz,3H).
ESI-MS(m/z):430[M+H]+.实施例二(S)-N-((9-氯-4-乙基-8-氟-4-羟基-3,14-二氧-3,4,12,14-四氢-1H-吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-11-基)甲基)-2-羟基乙酰胺的制备(化合物2-1)
将(S)-11-(氨甲基)-9-氯-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-3,14(4H)-二酮(1-1,60mg,128.68μmol,盐酸盐)溶于DMF(1mL)中，依次加入乙醇酸(20mg,262.98μmol)，三乙胺(26.04mg,257.35μmol)和DMTMM(71.21mg,257.35μmol)，反应体系在20℃反应1小时，反应液经制备高效液相色谱纯化得到标题化合物(4mg,8.04μmol)。
色谱柱：SunFire Prep C18OBD 19mm×150mm×5.0μm
流动相A：乙腈；流动相B：水(0.05％三氟乙酸)

结构表征数据如下：
1H NMR(400MHz,DMSO-d6)δ8.87(d,J＝4.1Hz,1H),8.85(s,1H),8.19(d,J＝10.3Hz,1H),7.34(s,1H),6.55(s,1H),5.55(s,2H),5.45(s,2H),4.82(d,J＝6.0Hz,2H),3.82(s,2H),1.87(dd,J＝9.7,7.5Hz,2H),0.87(t,J＝7.3Hz,3H).
ESI-MS(m/z):488[M+H]+.
实施例三(1S,9S)-1-氨磺酰胺基-9-乙基-5-氟-9-羟基-4-甲基-1,2,3,9,12,15-六氢-10H,13H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-10,13-二酮(化合物7-1)
步骤一：叔丁基(N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧基-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-1-基)氨磺酰基)氨基甲酸酯
将叔丁醇(20.92mg,0.282mmol)溶于DCM(0.5mL)，冷却搅拌至0℃滴入氯磺酰异氰酸酯(39.94mg,0.282mol,24.56μL)，保温反应0.5小时备用。将甲磺酸伊喜替康(100mg,0.188mol)溶于DMF(2mL)，滴入TEA(57.11mg,0.564mmol,78.45μL)，冷却搅拌至0℃后滴入上述DCM反应液，自然恢复室温反应1小时。LCMS监控反应，产物明显。反应液直接进行下一步反应。
ESI-MS(m/z):615.1[M+1]+.
步骤二：(1S,9S)-1-氨磺酰胺基-9-乙基-5-氟-9-羟基-4-甲基-1,2,3,9,12,15-六氢-10H，13H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-10,13-二酮
叔丁基(N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧基-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-1-基)氨磺酰基)氨基甲酸酯反应液加入二氯甲烷(1mL)稀释，滴入三氟乙酸(0.5mL),搅拌反应1小时。LCMS监控产物。减压浓缩后制备纯化得标题化合物52.26mg。
ESI-MS(m/z):515.2[M+1]+.
1H NMR(400MHz,DMSO)：δ7.78(d,J＝10.9Hz,1H),7.30(d,J＝9.0Hz,2H),6.98(s,2H),6.53(s,1H),5.55–5.36(m,4H),4.92(d,J＝4.2Hz,1H),3.27(dd,J＝19.1, 6.9Hz,1H),3.14–3.04(m,1H),2.38(s,3H),2.36–2.22(m,2H),1.93–1.79(m,2H),0.87(t,J＝7.3Hz,3H).
色谱柱：SunFire Prep C18OBD 19mm×150mm×5.0μm
流动相A：乙腈；流动相B：水(0.05％甲酸)
实施例四1-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧基-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-1-基)脲(化合物7-2)
将甲磺酸伊喜替康(50mg,0.082mmol)溶于1,4-二氧六环(1mL)，滴入三乙胺(16.65mg,0.165mmol,22.87μL)，搅拌下滴入三甲基硅氰(6.53mg,0.165mmol,8.24μL)，升温90℃反应2小时。LCMS监控反应至原料消失。反应液冷却至室温，滴入MeOH(1mL)，搅拌10分钟，减压浓缩后制备纯化得标题化合物12.92mg。
ESI-MS(m/z):479.1[M+1]+.
1H NMR(400MHz,DMSO)：δ7.78(d,J＝10.9Hz,1H),7.30(s,1H),6.74(d,J＝9.1Hz,1H),6.53(s,1H),5.69(s,2H),5.42(s,2H),5.29(q,J＝19.2Hz,3H),3.15(s,2H),2.39(s,3H),2.20–2.07(m,2H),1.86(dd,J＝10.4,7.4Hz,2H),0.87(t,J＝7.3Hz,3H).
色谱柱：SunFire Prep C18OBD 19mm×150mm×5.0μm
流动相A：乙腈；流动相B：水(0.05％甲酸)
实施例五N-((S)-9-氯-4-乙基-8-氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并 [3',4':6，7]中氮茚并[1,2-b]喹啉-11-基)甲基)-2-环丙基-2-羟基乙酰胺的制备(化合物2-4)
将(S)-11-(氨甲基)-9-氯-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-3,14(4H)-二酮(1-1,30mg,69.8μmol)溶于DMF(1mL)中，依次加入2-环丙基-2-羟基乙酸(16.21mg,139.6μmol)，DIPEA(22.55mg,174.5μmol)和HATU(31.83mg,83.75μmol)，反应体系在20℃反应1小时，反应液直接经制备高效液相色谱纯化得到标题化合物11mg。
色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μm
流动相A：乙腈；流动相B：水(0.05％三氟乙酸)
结构表征数据如下：
1H NMR(400MHz,DMSO-d6)δ8.81(d,J＝8Hz,1H),8.75(m,1H),8.19(d,J＝8Hz,1H),7.34(s,1H),6.55(s,2H),5.53(s,2H),5.45(s,2H),4.82(m,2H),3.57(m,1H),1.86(m,2H),0.97(m,1H),0.87(t,J＝8Hz,3H),0.32(m,2H),0.23(m,2H).
ESI-MS(m/z):529[M+H]+.
实施例六N-(((S)-9-氯-4-乙基-8-氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-11-基)甲基)-2-羟基-3-烯胺的制备(化合物2-22)
将(S)-11-(氨甲基)-9-氯-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-3,14(4H)-二酮(1-1,30mg,73.28μmol)溶于DMF(1mL)中，依次加入2- 羟基-3-烯酸(7.48mg,73.28μmol)，DIPEA(23.68mg,183.19μmol)和HATU(31.42mg,87.93μmol)，反应体系在20℃反应1小时，反应液直接经制备高效液相色谱纯化得到标题化合物5.36mg。
色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μm
流动相A：乙腈；流动相B：水(0.05％三氟乙酸)
结构表征数据如下：
1H NMR(400MHz,DMSO-d6)δ8.58(m,1H),7.97(m,1H),7.63(m,1H),5.59(m,1H),5.51(s,1H),5.40(m,2H),5.18(d,J＝12Hz,1H),4.95(s,2H),4.61(s,2H),4.58(m,1H),1.95(m,2H),0.98(t,J＝8Hz,3H).
ESI-MS(m/z):514[M+H]+.
实施例七N-(((S)-9-氯-4-乙基-8-氟-4-羟基-3,14-二氧-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-11-基)甲基)-3,3,3-三氟-2-羟丙酰胺的制备(化合物2-19)
将(S)-11-(氨甲基)-9-氯-4-乙基-8-氟-4-羟基-1,12-二氢-14H-吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-3,14(4H)-二酮(1-1,22mg,51.18μmol)溶于DMF(1mL)中，依次加入3,3,3-三氟-2-羟基丙酸(8.85mg,61.42μmol)，DIPEA(19.84mg,153.55μmol)和HATU(23.34mg,61.42μmol)，反应体系在20℃反应1小时，反应液直接经制备高效液相色谱纯化得到标题化合物2mg。
色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μm
流动相A：乙腈；流动相B：水(0.05％三氟乙酸)

结构表征数据如下：
1H NMR(400MHz,DMSO-d6)δ8.78(d,J＝8Hz,1H),8.19(d,J＝8Hz 1H),7.34(s,1H),6.55(s,1H),5.91(m,1H),5.85(m,1H),5.54(s,2H),5.45(s,2H),4.86(m,2H),4.58(m,1H),1.86(m,2H),0.87(t,J＝8Hz,3H).
ESI-MS(m/z):556[M+H]+.
实施例八(2R,3R,4R,5S,6R)-6-(4-((((((S)-9-氯-4-乙基-8-氟-4-羟基-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚并[1,2-b]喹啉-11-基)甲基)氨基甲酰基)氧基)甲基)-2-(2-(2-(N-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)乙胺基)乙氧基)乙酰胺基)苯氧基)-3,4,5-三羟基-四氢-2H-吡喃-2-羧酸(A-11)
步骤一:(2R,3S,4R,5R,6R)-2-(2-(8-(9-芴基甲氧基羰基氨基)-3,6-二氧杂-辛酰胺基)-4-((((((S)-9-氯-4-乙基-8-氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲嗪并[1,2b]喹啉-11-基)甲基)氨基甲酰基)氧基)甲基)苯氧基)-6-(甲氧羰基)-四氢-2H-吡喃-3,4,5-三乙酸酯(A-11-2)
将化合物1-1(30mg,69.80μmol)和化合物A-11-1(75.85mg,76.77μmol，参照WO2022253035第81页制备)溶于N,N-二甲基甲酰胺(1mL)中,随后加入HOBT(11.32 mg mg,83.75μmol)和N,N-二异丙基乙胺(22.55mg,174.49μmol),室温搅拌1小时。反应完毕后,旋干反应液得粗品A-11-2，直接进行下一步反应。
步骤二:(2R,3R,4R,5S,6R)-6-(2-(2-(2-(2-氨基乙基)乙氧基)乙酰胺基)-4-((((((S)-9-氯-4-乙基-8-氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-11基)甲基)氨基甲酰基)氧基)甲基)苯氧基)-3,4,5-三羟基四氢-2H吡喃-2-羧酸(A-11-3)
将A-11-2溶于MeOH(1mL)和水(0.5mL)中,加入LiOH·H2O(26.28mg,625.67μmol),室温反应4小时。反应结束后,反应液直接用制备高效液相色谱纯化并冷冻干燥得标题化合物A-11-3(20mg)。
其纯化方法如下:
色谱柱:Waters SunFire Prep C18OBD(5μm*19mm*150mm)
流动相A:乙腈；流动相B:水(0.05％甲酸)
步骤三:(2R,3R,4R,5S,6R)-6-(4-((((((S)-9-氯-4-乙基-8-氟-4-羟基-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚并[1,2-b]喹啉-11-基)甲基)氨基甲酰基)氧基)甲基)-2-(2-(2-(N-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)乙胺基)乙氧基)乙酰胺基)苯氧基)-3,4,5-三羟基-四氢-2H-吡喃-2-羧酸(A-11)
将A-11-3(20mg,21.83μmol)，IM-1(7.98mg,21.83μmol)和N,N-二异丙基乙胺(8.46mg,65.49μmol)溶于DMF(1mL)中,反应液在室温条件下搅拌1小时。反应结束后,反应液直接用制备高效液相色谱纯化并冷冻干燥得标题化合物3.42mg。
其结构表征数据如下:
MS m/z(ESI):1168.2[M+H]+.
其纯化方法如下:
色谱柱:Waters SunFire Prep C18OBD(5μm*19mm*150mm)
流动相A:乙腈；流动相B:水(0.05％甲酸)

实施例九N-((S)-12-苄基-1-((S)-9-氯-4-乙基-8-氟-4-羟基-3,14-二氧代-3,4,12,14-四氢1H-吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-11-基)-3,8,11,14,17-五氧代-5-氧杂-2,7,10,13-四氮杂十五烷-15-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(B-1)
将化合物1-1(30mg,69.80μmol)和化合物IM-2(51.72mg,76.77μmol，参照WO2022253035第92页制备)溶于N,N-二甲基甲酰胺(1mL)中,随后加入HATU(29.17mg,76.77μmol)和N,N-二异丙基乙胺(22.55mg,174.49μmol),室温搅拌1小时。反应完毕后,反应液直接用制备高效液相色谱纯化并冷冻干燥得标题化合物10mg。
其结构表征数据如下:
MS m/z(ESI):1087.3[M+H]+.
其纯化方法如下:
色谱柱:Waters SunFire Prep C18OBD(5μm*19mm*150mm)
流动相A:乙腈；流动相B:水(0.05％甲酸)
实施例十N-((9S,12S,15S)-1-((S)-9-氯-4-乙基-8-氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1-吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-11-基)-9,12-二甲基-3,8,11,14-四氧代-5-氧杂-2,7,10,13-四氮杂十六烷-15-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(B-2)
步骤一:(9H-芴-9-基)甲基((9S,12S,15S)-1-((S)-9-氯-4-乙基-8-氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-11-基)-9,12-二甲基-3,8,11,14-四氧代-5-氧杂-2,7,10,13-四氮杂十六烷-15-基)氨基甲酸酯(B-2-1)
将化合物1-1(50mg,116.33μmol)和化合物IM-3(66.03mg,122.14μmol)溶于N,N-二甲基甲酰胺(1mL)中,随后加入HATU(48.62mg,127.96μmol)和N,N-二异丙基乙胺(37.58mg,290.81μmol),室温搅拌1小时。反应完毕后,反应液浓缩后粗品经高效液相色谱纯化后冷冻干燥得标题化合物100mg。
其纯化方法如下:
色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)
流动相A:乙腈；流动相B:水(0.05％甲酸)
步骤二:(S)-2-氨基-N-((9S,12S)-1-((S)-9-氯-4-乙基-8-氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-11-基)-9-甲基-3,8,11-三氧代-5-氧杂-2,7,10-三氮杂十三烷-12-基)丙酰胺(B-2-2)
将化合物B-2-1(100mg,105.00μmol)溶于N,N-二甲基甲酰胺(1mL)中,加入二乙胺(0.5mL),室温反应1小时。反应结束后,反应液经减压浓缩得粗品,直接用于下一步反应。
步骤三:N-((9S,12S,15S)-1-((S)-9-氯-4-乙基-8-氟-4-羟基-3,14-二氧代-3,4,12,14-四氢-1-吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-11-基)-9,12-二甲基-3,8,11,14-四氧代-5-氧杂-2,7,10,13-四氮杂十六烷-15-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(B-2)
将化合物IM-1(17.66mg,48.39μmol)和N,N-二异丙基乙胺(12.48mg,0.226mmol)溶于含有B-2-2粗品的N,N-二甲基甲酰胺(1mL)中,反应体系在室温条件下搅拌1小时。反应结束后，反应液经减压浓缩得粗品，粗品经高效液相色谱纯化后冷冻干燥得标题化合物5mg。
其结构表征数据如下:
MS m/z(ESI):981.7[M+H]+.
其纯化方法如下:
色谱柱:Waters SunFire Prep C18OBD(5μm*19mm*150mm)
流动相A:乙腈；流动相B:水(0.05％甲酸)
实施例十一:(S)-N-((1S,9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧-2,3,9,10,13,15-六氢-1H,12H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-1-基)-3-羟基丁酰胺(2-25)
将(1S,9S)-1-氨基-4-氯-9-乙基-5-氟-9-羟基-1,2,3,9,12,15-六氢-10H,13H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-10,13-二酮(参照WO2022166762A1制备)(30mg,0.053mmol) 、(S)-3-羟基丁酸(16.5mg,0.158mmol)、HATU(40.0mg,0.105mmol)和DIPEA(20.4mg,0.158mmol)加到DMF(0.5mL)反应体系中,25℃搅拌反应2小时,反应液直接经高效液相色谱制备后冷冻干燥得标题化合物(13.5mg,0.024mmol)。
其分离纯化方法如下:
色谱柱:Waters XBridge Prep C18OBD(5μm*19mm*150mm)
流动相A:乙腈；流动相B:水(0.05％甲酸)
其结构表征数据如下:
ESI-MS(m/z):542.1[M+H]+
1H NMR(400MHz,DMSO-d6)δ8.44(d,J＝8.8Hz,1H),8.09(d,J＝10.4Hz,1H),7.34(s,1H),6.56(m,1H),5.64–5.60(m,1H),5.44(s,2H),5.32–5.25(m,2H),4.63(d,J＝4.8Hz,1H),4.06–4.01(m,1H),3.31–3.26(m,2H),2.28–2.23(m,1H),2.20–2.13(m,3H),1.90–1.82(m,2H),1.08(d,J＝6.4Hz,3H),0.87(t,J＝7.2Hz,3H).
实施例十二:(R)-N-((1S,9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧-2,3,9,10,13,15-六氢-1H,12H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-1-基)-3-羟基丁酰胺(2-26)
将(1S,9S)-1-氨基-4-氯-9-乙基-5-氟-9-羟基-1,2,3,9,12,15-六氢-10H,13H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-10,13-二酮(参照WO2022166762A1制备)(30mg,52.64μmol)、(R)-3-羟基丁酸(10.96mg,105.28μmol)、HATU(30.02mg,78.96μmol)和DIPEA(20.41mg,157.93μmol)加到DMF(2mL)反应体系中,25℃搅拌反应2小时,反应液直接经高效液相色谱制备后冷冻干燥得标题化合物(17mg,31.42μmol)。
其分离纯化方法如下:
色谱柱:Waters XBridge Prep C18OBD(5μm*19mm*150mm)
流动相A:乙腈；流动相B:水(0.05％甲酸)
其结构表征数据如下:
ESI-MS(m/z):542.1[M+H]+
1H NMR(400MHz,DMSO)δ8.43(d,J＝8.8Hz,1H),8.06(d,J＝10.4Hz,1H),7.33(s,1H),6.54(s,1H),5.63-5.55(m,1H),5.43(s,2H),5.27(d,J＝18.8Hz,1H),5.21(d,J＝18.8Hz,1H),4.66(d,J＝4.8Hz,1H),4.09-3.98(m,1H),3.32-3.26(m,2H),2.32-2.25(m,1H),2.24-2.12(m,3H),1.93-1.78(m,2H),1.08(d,J＝6.0Hz,3H),0.87(t,J＝7.2Hz,3H).
抗体药物偶联物的制备例
Trastuzumab-A-11(DAR8)的制备
取1.781mL Trastuzumab抗体(16mg/mL)，用89μL 20mM磷酸盐缓冲液+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，108μL，pH 7.60)溶液混匀，室温放置1.5h。再缓慢加入10倍物质量的溶解在二甲基亚砜的A-11(218μL,10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物Trastuzumab-A-11。质谱法测定DAR值为8.0。
Trastuzumab-B-1(DAR 8)的制备
取1.973mL Trastuzumab抗体(15.2mg/mL)，用98.7μL 20mM磷酸盐缓冲液+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，56.92μL，pH 7.60)溶液混匀，室温放置1.5h。再缓慢加入12倍物质量的溶解在二甲基亚砜的B-1(258.16μL,10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物Trastuzumab-B-1。质谱法测定DAR值为7.96。
Trastuzumab-B-2(DAR 8)的制备
取2.16mL Trastuzumab抗体(16.2mg/mL)，用108μL 20mM磷酸盐缓冲液+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基) 膦，88.42μL，pH 7.60)溶液混匀，室温放置1.5h。再缓慢加入20倍物质量的溶解在二甲基亚砜的B-2(502.38μL,10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物Trastuzumab-B-2。质谱法测定DAR值为7.95。
生物学评价
一、肿瘤细胞增殖抑制试验
1.化合物对HT29细胞增殖的抑制作用
(1)细胞铺板：首先采用相应的培养基培养肿瘤细胞HT29，用胰酶消化细胞，离心后重悬细胞计数，调整细胞至合适的浓度进行铺板。肿瘤细胞来源见表1。
表1.肿瘤细胞来源
本发明化合物和肿瘤细胞共孵育：在96孔板中分别加入90μL细胞悬液，待细胞贴壁后，加入10μL用培养基稀释好的生物活性分子(本发明化合物)到上述板孔中，孵育72h。
体外细胞活性检测：孵育结束后，每孔加入Cell Counting-LiteTM 2.0试剂(Vazyme/诺唯赞)50μL，避光振荡混匀，反应10min后即可进行检测，酶标仪(厂家：BMG，型号：PHERAStar-FS)读数。不含细胞的培养基孔为背景RLU，有细胞但不含化合物的培养孔为细胞对照RLU。细胞抑制率＝1-(样品RLU-背景RLU)/(细胞对照RLU-背景RLU)×100％，按照四参数模型拟合曲线，计算化合物的半数抑制浓度(IC50)，检测结果如表2所示。
(2)数据结果
表2.HT29细胞增殖抑制活性

测试结果表明，表2中本发明的化合物对HT29结肠癌细胞具有较强的增殖抑制作用。
2.化合物对HCC1806细胞增殖的抑制作用
(1)细胞铺板：首先采用相应的培养基培养肿瘤细胞HCC1806，用胰酶消化细胞，离心后重悬细胞计数，调整细胞至合适的浓度进行铺板。肿瘤细胞来源见表3。
表3.肿瘤细胞来源
本发明化合物和肿瘤细胞共孵育：在96孔板中分别加入90μL细胞悬液，待细胞贴壁后，加入10μL用培养基稀释好的生物活性分子(本发明化合物)到上述板孔中，孵育72h。
体外细胞活性检测：孵育结束后，每孔加入Cell Counting-LiteTM 2.0试剂(Vazyme/诺唯赞)50μL，避光振荡混匀，反应10min后即可进行检测，酶标仪(厂家：BMG，型号：PHERA Star-FS)读数。不含细胞的培养基孔为背景RLU，有细胞但不含化合物的培养孔为细胞对照RLU。细胞抑制率＝1-(样品RLU-背景RLU)/(细胞对照RLU-背景RLU)×100％，按照四参数模型拟合曲线，计算化合物的半数抑制浓度(IC50)，检测结果如表4所示。
(2)数据结果
表4.HCC1806细胞增殖抑制活性
测试结果表明，表4中本发明的化合物对HCC1806人乳腺鳞状癌细胞增殖具有显 著的抑制作用。
3.化合物对NCI-H358细胞增殖的抑制作用
(1)细胞铺板：首先采用相应的培养基培养肿瘤细胞NCI-H358，用胰酶消化细胞，离心后重悬细胞计数，调整细胞至合适的浓度进行铺板。肿瘤细胞来源见表5。
表5.肿瘤细胞来源
本发明化合物和肿瘤细胞共孵育：在96孔板中分别加入90μL细胞悬液，待细胞贴壁后，加入10μL用培养基稀释好的生物活性分子(本发明化合物)到上述板孔中，孵育72h。
体外细胞活性检测：孵育结束后，每孔加入Cell Counting-LiteTM 2.0试剂(Vazyme/诺唯赞)50μL，避光振荡混匀，反应10min后即可进行检测，酶标仪(厂家：BMG，型号：PHERA Star-FS)读数。不含细胞的培养基孔为背景RLU，有细胞但不含化合物的培养孔为细胞对照RLU。细胞抑制率＝1-(样品RLU-背景RLU)/(细胞对照RLU-背景RLU)×100％，按照四参数模型拟合曲线，计算化合物的半数抑制浓度(IC50)，检测结果如表6所示。
(2)数据结果
表6.NCI-H358细胞增殖抑制活性
测试结果表明，表6中本发明的化合物对NCI-H358人非小细胞肺癌细胞增殖具有显著的抑制作用。
4.化合物对NCI-N87细胞增殖的抑制作用
(1)细胞铺板：首先采用相应的培养基培养肿瘤细胞NCI-N87，用胰酶消化细胞， 离心后重悬细胞计数，调整细胞至合适的浓度进行铺板。肿瘤细胞来源见表7。
表7.肿瘤细胞来源
本发明化合物和肿瘤细胞共孵育：在96孔板中分别加入90μL细胞悬液，待细胞贴壁后，加入10μL用培养基稀释好的生物活性分子(本发明化合物)到上述板孔中，孵育72h。
体外细胞活性检测：孵育结束后，每孔加入Cell Counting-LiteTM 2.0试剂(Vazyme/诺唯赞)50μL，避光振荡混匀，反应10min后即可进行检测，酶标仪(厂家：BMG，型号：PHERA Star-FS)读数。不含细胞的培养基孔为背景RLU，有细胞但不含化合物的培养孔为细胞对照RLU。细胞抑制率＝1-(样品RLU-背景RLU)/(细胞对照RLU-背景RLU)×100％，按照四参数模型拟合曲线，计算化合物的半数抑制浓度(IC50)，检测结果如表8所示。
(2)数据结果
表8.NCI-N87细胞增殖抑制活性
测试结果表明，表8中本发明的化合物对NCI-N87人胃癌细胞增殖具有显著的抑制作用。
5.化合物对MDA-MB-231细胞增殖的抑制作用
(1)细胞铺板：首先采用相应的培养基培养肿瘤细胞MDA-MB-231，用胰酶消化细胞，离心后重悬细胞计数，调整细胞至合适的浓度进行铺板。肿瘤细胞来源见表9。
表9.肿瘤细胞来源
本发明化合物和肿瘤细胞共孵育：在96孔板中分别加入90μL细胞悬液，待细胞贴壁后，加入10μL用培养基稀释好的生物活性分子(本发明化合物)到上述板孔中，孵育72h。
体外细胞活性检测：孵育结束后，每孔加入Cell Counting-LiteTM 2.0试剂(Vazyme/诺唯赞)50μL，避光振荡混匀，反应10min后即可进行检测，酶标仪(厂家：BMG，型号：PHERA Star-FS)读数。不含细胞的培养基孔为背景RLU，有细胞但不含化合物的培养孔为细胞对照RLU。细胞抑制率＝1-(样品RLU-背景RLU)/(溶媒RLU-背景RLU)×100％，按照四参数模型拟合曲线，计算化合物的半数抑制浓度(IC50)，检测结果如表10所示。
(2)数据结果
表10.MDA-MB-231细胞增殖抑制活性
测试结果表明，表10中本发明的化合物对MDA-MB-231人乳腺癌细胞增殖具有显著的抑制作用。
6.化合物对Jeko-1细胞增殖的抑制作用
(1)细胞铺板：首先采用相应的培养基培养肿瘤细胞Jeko-1，将细胞离心后重悬并细胞计数，最后调整细胞至合适的浓度进行铺板。肿瘤细胞来源见表11。
表11.肿瘤细胞来源
本发明化合物和肿瘤细胞共孵育：在96孔板中分别加入90μL细胞悬液，待细胞贴壁后，加入10μL用培养基稀释好的生物活性分子(本发明化合物)到上述板孔中，孵育72h。
体外细胞活性检测：孵育结束后，每孔加入Cell Counting-LiteTM 2.0试剂(Vazyme/诺唯赞)50μL，避光振荡混匀，反应10min后即可进行检测，酶标仪(厂家：BMG，型号：PHERA Star-FS)读数。不含细胞的培养基孔为背景RLU，有细胞但不含化合物的培养孔为细胞对照RLU。细胞抑制率＝1-(样品RLU-背景RLU)/(细胞对照RLU-背景RLU)×100％，按照四参数模型拟合曲线，计算化合物的半数抑制浓度(IC50)，检测结果如表12所示。
(2)数据结果
表12.Jeko-1细胞增殖抑制活性
测试结果表明，表12中本发明的化合物对Jeko-1人套细胞淋巴瘤细胞增殖具有显著的抑制作用。
7.化合物对MDA-MB-453细胞增殖的抑制作用
(1)细胞铺板：首先采用相应的培养基培养肿瘤细胞MDA-MB-453，用胰酶消化细胞，离心后重悬细胞计数，调整细胞至合适的浓度进行铺板。肿瘤细胞来源见表13。
表13.肿瘤细胞来源
本发明化合物和肿瘤细胞共孵育：在96孔板中分别加入90μL细胞悬液，待细胞贴壁后，移除细胞中培养基，加入10μL用培养基稀释好的生物活性分子(本发明化合物)到上述板孔中，孵育72h。
体外细胞活性检测：孵育结束后，每孔加入Cell Counting-LiteTM 2.0试剂(Vazyme/诺唯赞)50μL，避光振荡混匀，反应10min后即可进行检测，酶标仪(厂家：BMG，型号：PHERA Star-FS)读数。不含细胞的培养基孔为背景RLU，有细胞但不含化合 物的培养孔为细胞对照RLU。细胞抑制率＝1-(样品RLU-背景RLU)/(细胞对照RLU-背景RLU)×100％，按照四参数模型拟合曲线，计算化合物的半数抑制浓度(IC50)，检测结果如表14所示。
(2)数据结果
表14.MDA-MB-453细胞增殖抑制活性
测试结果表明，表14中本发明的化合物对MDA-MB-453人乳腺癌细胞增殖具有显著的抑制作用。
8.化合物对HCC827细胞增殖的抑制作用
(1)细胞铺板：首先采用相应的培养基培养肿瘤细胞HCC827，用胰酶消化细胞，离心后重悬细胞计数，调整细胞至合适的浓度进行铺板。肿瘤细胞来源见表15。
表15.肿瘤细胞来源
本发明化合物和肿瘤细胞共孵育：待细胞贴壁后，移除细胞中培养基，将稀释好的生物活性分子(本发明化合物)加入到上述板孔中，孵育72h。
体外细胞活性检测：孵育结束后，每孔加入Cell Counting-LiteTM 2.0试剂(Vazyme/诺唯赞)50μL，避光振荡混匀，反应10min后即可进行检测，酶标仪(厂家：BMG,型号：PHERAStar-FS)读数。利用不含细胞的培养基的CellCounting-LiteTM获得背景RLU,含有细胞的培养基的Cell Counting-LiteTM获得溶媒RLU。细胞抑制率＝1-(样品RLU-背景RLU)/(溶媒RLU-背景RLU)×100％，按照四参数模型拟合曲线，计算化合物的半数抑制浓度(IC50)。RLU(relative light unit)：相对光单位。检测结果如表16所示。
(2)数据结果
表16.化合物对HCC827细胞的增殖抑制活性
测试结果表明，表16中本发明的化合物对HCC827人非小细胞肺癌细胞增殖具有抑制作用。
9.化合物对HCC1954细胞增殖的抑制作用
(1)细胞铺板：首先采用相应的培养基培养肿瘤细胞HCC1954，用胰酶消化细胞，离心后重悬细胞计数，调整细胞至合适的浓度进行铺板。肿瘤细胞来源见表17。
表17.肿瘤细胞来源
本发明化合物和肿瘤细胞共孵育：待细胞贴壁后，移除细胞中培养基，将稀释好的生物活性分子(本发明化合物)加入到上述板孔中，孵育72h。
体外细胞活性检测：孵育结束后，每孔加入Cell Counting-LiteTM 2.0试剂(Vazyme/诺唯赞)50μL，避光振荡混匀，反应10min后即可进行检测，酶标仪(厂家：BMG,型号：PHERAStar-FS)读数。利用不含细胞的培养基的CellCounting-LiteTM获得背景RLU,含有细胞的培养基的Cell Counting-LiteTM获得溶媒RLU。细胞抑制率＝1-(样品RLU-背景RLU)/(溶媒RLU-背景RLU)×100％，按照四参数模型拟合曲线，计算化合物的半数抑制浓度(IC50)。RLU(relative light unit)：相对光单位。检测结果如表18所示。
(2)数据结果
表18.化合物对HCC1954细胞的增殖抑制活性
测试结果表明，表18中本发明的化合物对HCC1954人乳腺癌细胞的增殖具有抑制作用。
10.化合物对NCI-H1975细胞增殖的抑制作用
(1)细胞铺板：首先采用相应的培养基培养肿瘤细胞NCI-H1975，用胰酶消化细胞，离心后重悬细胞计数，调整细胞至合适的浓度进行铺板。肿瘤细胞来源见表19。
表19.肿瘤细胞来源
本发明化合物和肿瘤细胞共孵育：待细胞贴壁后，移除细胞中培养基，将稀释好的生物活性分子(本发明化合物)加入到上述板孔中，孵育72h。
体外细胞活性检测：孵育结束后，每孔加入Cell Counting-LiteTM 2.0试剂(Vazyme/诺唯赞)50μL，避光振荡混匀，反应10min后即可进行检测，酶标仪(厂家：BMG,型号：PHERAStar-FS)读数。利用不含细胞的培养基的CellCounting-LiteTM获得背景RLU,含有细胞的培养基的Cell Counting-LiteTM获得溶媒RLU。细胞抑制率＝1-(样品RLU-背景RLU)/(溶媒RLU-背景RLU)×100％，按照四参数模型拟合曲线，计算化合物的半数抑制浓度(IC50)。RLU(relative light unit)：相对光单位。检测结果如表20所示。
(2)数据结果
表20.化合物对NCI-H1975细胞的增殖抑制活性
测试结果表明，表20中本发明的化合物对NCI-H1975人非小细胞肺癌细胞的增殖具有抑制作用。
二、渗透性试验
采用MDCK细胞模型评价化合物的渗透性，LC-MS/MS法测定待测物的浓度，计算表观渗透系数(Papp)和外排比[Efflux ratio＝Papp(B→A)/Papp(A→B)]。
1.试剂配制
完全培养基：在MEM培养液中加入10％胎牛血清、1％青/链霉素。
消化液：0.05％胰蛋白酶-EDTA。
HBSS缓冲液：含Ca2+、Mg2+的HBSS。
渗透液：含10mM Hepes、4％BSA的HBSS，pH 7.4。
化合物储备液：称取一定量的待测化合物，用DMSO配制成10mM储备液。
2.细胞培养
将处于对数生长期的MDCK细胞(26代)经0.05％胰蛋白酶-EDTA消化分散均匀后，按照细胞3*105个/cm2接种到Transwell小室中，A侧200μL，B侧1000μL。接种后每天换液一次，培养至3～4天。
3.渗透性实验
将细胞培养基吸除，用渗透液洗3次，A侧添加200μL渗透液，B侧添加1000μL渗透液，采用电压电阻仪测定细胞跨膜电阻值(TEER)。测定细胞TEER值后，将两侧渗透液吸除并加入溶液。120min后，分别吸取A侧和B侧150μL样品，-80℃冻存，待测。随后A侧加入10μg/ml荧光黄，孵育30min后，吸取B侧溶液100μL到96孔白板，通过酶标仪在Ex＝485nm，Em＝530nm波长处测定荧光值，计算透过量(小于1％)
4.样品处理
取80μL待测化合物样品加入320μL含内标的乙腈溶液(30ng/mL Tolbutamide)，涡旋，离心10min(4000rpm，4℃)，取上清液进行LC-MS/MS分析。
5.数据处理
采用Excel 2013计算Papp值以及从B侧→A侧和A侧→B侧的Papp比值，并计算每个对照化合物及待测化合物的回收率。
(1)表观渗透性的计算
根据下式计算化合物的表观渗透系数(Papp)：
Papp＝(dCr/dt)×Vr/(A×C0)
C0是受试药物所在端(donor)的初始浓度(μM)，(dCr/dt)×Vr是在接受端(receiver)受试药物出现的速率(μM/s)，Vr是接收侧的溶液体积(mL)，A是聚碳酯膜的表面积(cm2)。
评价标准：低渗透性:Papp≤0.5(×10-6cm/s)
中渗透性:0.5<Papp<2.5(×10-6cm/s)
高渗透性:Papp≥2.5(×10-6cm/s)
(2)转运体
根据Papp计算外排率(Efflux Ratio，ER)：
ER＝Papp(B→A)/Papp(A→B)
其中，Papp(B→A)为受试药物从BL(baso-lateral)端到AP(apical)端的表观渗透系数，Papp(A→B)为受试药物从AP端到BL端的表观渗透系数。
评价标准：很可能为外排转运体底物(Probably):ERa≥2并且ERa/ERi>2
可能为外排转运体底物(Likely):ERa≥2
外排转运体底物的可能性小或无(Poor or non):ERa<2
其中ERi和ERa分别为P-gp抑制剂存在和不存在时待测化合物的外排率。
回收率计算公式如下：
回收率(％)＝100×[(Vr×Cr)+(Vd×Cd)]/(Vd×C0)
其中Vd和Vr分别为供给侧和接收侧溶液体积，Cd和Cr分别为供给侧和接收侧的化合物浓度。
6.结果
具体数据如下表：
根据Papp(A→B)结果显示对照化合物2、对照化合物3、对照化合物4和化合物 2-19均为低渗透性化合物，特别是对照化合物2的渗透性相对较低；对照化合物1、化合物2-1、化合物2-22和化合物2-25为中等渗透性化合物；化合物1-1为高渗透性化合物；外排比数据提示对照化合物1、对照化合物2、对照化合物3、对照化合物4、化合物2-22和化合物2-25可能是外排转运体底物，特别是对照化合物2、对照化合物3和对照化合物4的外排率相对较高；化合物1-1、化合物2-1和化合物2-19不是外排转运体底物或为外排转运体的弱底物。
三、肝微粒体稳定性试验
采用肝微粒体作为体外模型，来评价化合物在人和猴肝微粒体中的代谢稳定性。
1.材料与方法
1.1主要试验材料
对照化合物及本专利发明的化合物
睾酮Dr.Ehrenstorfer,德国
混合人肝微粒体Xenotech,美国
混合雄性食蟹猴肝微粒体RILD,中国
1.2肝微粒体孵育体系
阳性化合物睾酮或待测物(肝微粒体溶液,50μL)与PBS(25μL)混合,预孵(37℃)5min后,加入NADPH(25μL),使阳性化合物或待测物终浓度为1μM,人肝微粒体蛋白终浓度为0.5mg/ml,猴肝微粒体蛋白终浓度为1mg/ml,待测物组和阳性化合物组孵育0、15min。反应相应时间后加入300μL含内标的冰乙腈终止反应,涡旋,存于-80℃待测。所有孵育样本均为双样本。
1.3样品预处理
将待测化合物涡流1min,离心10min(4℃,4000rpm),取上清液300μL,进行LC-MS/MS分析。
2.数据处理
使用Excel软件，计算孵育体系中原形药物的剩余率(％)：
原形剩余率(％)＝100×(A孵育样品/A0h)
注：A孵育样品：孵育相应时间后化合物与内标峰面积比；A0h：未反应时化合物与内标峰面积比。
3.结果与结论
化合物在人和猴肝微粒体中孵育15min后的原形剩余率如表22所示。
表22化合物在人和猴肝微粒体中孵育15min后的原形剩余率
表22数据显示，相比于对照化合物4，化合物2-25具有明显的肝微粒体稳定性优势。
对照化合物结构：
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解，根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (17)

  1. 化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药，其中所述化合物具有如下所示结构：
    其中，
    R1选自氢、氟和氯；
    R2选自氢、甲基、氟、氯和羟基；或者R1和R2与相邻碳原子连接成5-6元含氧杂环；
    R3选自氢、C1-6烷基、氘代C1-6烷基、C1-6卤代烷基，C1-6烷氧烷基，C3-6环烷基和3-6元杂环基，或者R3与苯环相邻碳原子相连成六元碳环；
    A选自
    B环选自3-6元碳环或3-6元杂环基；
    R4选自氢、C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基、C3-6环烷基或3-6元杂环基；
    R5和R6各自独立地选自氢、C1-6烷基、C2-6烯基、C3-6环烷基、C1-6卤代烷基；或者R5或R6与相邻碳原子连接成3-6元环；
    或者R4和R5连接成4-6元环；
    n＝1-6。
  2. 权利要求1所述的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药，其中所述化合物具有式(II)的结构：

    在式(II)中，
    R1'选自氢、氟和氯；
    R2'选自甲基、氟、氯和羟基；
    R3'选自氢、C1-6烷基、氘代C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基、C3-6环烷基和3-6元杂环基；R3'优选氢、C1-6烷基、氘代C1-6烷基、C1-6卤代烷基或C1-6烷氧烷基；
    R4'选自氢、C1-6烷基和C1-6卤代烷基；R4'优选氢；
    当R1'为氟，且R2'为甲基时，R3'和R4'不同时为氢；且
    当R1'为氟，且R2’为甲基时，R4’不为C1-6烷基。
  3. 权利要求1所述的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药，其中所述化合物具有式(III)的结构：
    在式(III)中，
    R1选自氢、氟和氯；
    R2选自氢、甲基、氟、氯和羟基；
    R3选自氢、C1-6烷基、C1-6卤代烷基、C1-6烷氧烷基、C3-6环烷基和3-6元杂环基，或者R3与苯环相邻碳原子相连成六元碳环；R3优选氢；
    R4选自氢和C1-6烷基；
    R5和R6独立选自氢、C1-6烷基、C1-6卤代烷基、C3-6环烷基、C2-6烯基和3-6元杂环基，或者R5和R6与相邻碳原子连接成3-6元环；
    或者，R4和R5连接成4-6元环；
    n＝1或2；
    当R1为氟，R2为甲基，R3为氢，且R4为氢时，R5和R6不同时为氢，也不与相邻碳原子连接成环丙基；且
    当R1是氟，R2是甲基，且R3是氢时，R4不为C1-6烷基。
  4. 权利要求1所述的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药，其中所述化合物具有式(IV)的结构：
    在式(IV)中，
    R1'选自氢、氟和氯；
    R2'选自氢、甲基、氟、氯和羟基；
    R4'选自氢、C1-6烷基、C3-6环烷基和C1-6卤代烷基；R4'优选自氢；
    B选自C1-6亚烷基、3-6元碳环或3-6元杂环基；
    当R1’为氟、R2’为甲基、虚线碳环存在时，R4’不为氢且不为C1-6烷基；且
    当R2’是甲基，R1’是氟，虚线碳环不存在时，B环不为3元碳环。
  5. 权利要求1所述的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药，所述化合物结构如下所示：




  6. 式(V)所示化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药，其中所述化合物结构如下所示：
    M-L-E-D
    式(V)
    其中，
    M是和抗体或其抗原结合片段的接头部位；
    L是连接接头M和E之间的连接子；
    E是连接L和D的结构片段；
    D是细胞毒性药物的结构片段；
    优选地，M选自以下结构：
    其中，X选自离去基团，比如氯、溴、-OMs、OTs、OTf；
    优选地，M选自以下结构：
    优选地，L选自由下述的一个或多个组成的结构：C1-6亚烷基、-N(R’)-、羰基、-O-、Val、Cit、Phe、Lys、D-Val、Leu、Gly、Ala、Asn、Val-Cit、Val-Ala、Val-Lys、Val-Lys(Ac)、Phe-Lys、Phe-Lys(Ac)、D-Val-Leu-Lys、Gly-Gly-Arg、Ala-Ala-Asn、Ala-Ala-Ala、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Gly-Gly-Gly、 其中R’代表氢、C1-6烷基或含-(CH2CH2O)r-的烷基；r选自1-10的整数；s选自1-20的整数；
    优选地，L选自以下结构：
    其中s选自1-20的整数；
    优选地，E选自单键、-NH-CH2-、
    优选地，E为-NH-CH2-；
    优选地，所述细胞毒性药物选自本权利要求1～5任一项所述的化合物；
    优选地，所述细胞毒性药物选自权利要求5所述的化合物；优选地，D选自权利要求1～5任一项所述-化合物去氢之后的结构；
    优选地，D选自权利要求5任一项所述的化合物去氢之后的结构；
    优选地，D选自以下结构：



    优选地，D选自以下结构：
  7. 根据权利要求6所述的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药，所述化合物结构如下所示：






  8. 式(VI)所示抗体药物偶联物(ADC)：
    Ab-(M-L-E-D)x
    式(VI)
    其中，
    Ab是抗体；
    M是和抗体或其抗原结合片段的接头部位；
    L是连接接头M和E之间的连接子；
    E是连接L和D的结构片段；
    D是细胞毒性药物的结构片段；
    x为1至10；
    M、L、E、D如权利要求1～7中任意一项所述；
    优选地，Ab-(M-L-E-D)x选自以下结构：





  9. 抗体药物偶联物(ADC)，其结构如下所示：

  10. 包含一种或多种下述抗体药物偶联物的组合物，所述组合物的DAR值(药物抗体偶联比)为7.5至8.5，优选为7.5至8.0，进一步优选为8.0：
  11. 包含一种或多种下述抗体药物偶联物的组合物，所述组合物的DAR值(药物抗体偶联比)为7.5至8.5，优选为7.5至8.0，进一步优选为7.96：

  12. 包含一种或多种下述抗体药物偶联物的组合物，所述组合物的DAR值(药物抗体偶联比)为7.5至8.5，优选为7.5至8.0，进一步优选为7.95：
  13. 一种药物组合物，其包含权利要求1-7任一项的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药、或权利要求8-9任意一项的抗体药物偶联物、或权利要求10-12任意一项的组合物，以及一种或多种药学上可接受的载体。
  14. 一种药盒产品，其包含：
    a)作为第一治疗剂的至少一种权利要求1-7任一项的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药、或权利要求8-9任意一项的抗体药物偶联物、或权利要求10-12任意一项的组合物，或者权利要求13的药物组合物；
    b)任选存在的作为第二治疗剂的至少一种其他治疗剂，或者作为第二药物组合物的包含其他治疗剂的药物组合物；和
    c)任选存在的包装和/或说明书。
  15. 权利要求1-9任一项的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药、或权利要求8-9任意一项的抗体药物偶联物、或权利要求10-12任意一项的组合物、权利要求13的药物组合物或者权利要求14的药盒产品在制备用于治疗细胞异常增殖方面的疾病的药物中的用途；
    优选地，所述疾病为肿瘤，例如晚期实体瘤；
    优选地，所述肿瘤选自肺癌、鳞状上皮细胞癌、膀胱癌、胃癌、卵巢癌、腹膜癌、胰腺癌、乳腺癌、头颈癌、子宫颈癌、子宫内膜癌、直肠癌、肝癌、肾癌、食管腺癌、食管 鳞状细胞癌、前列腺癌、雌性生殖道癌、原位癌、淋巴瘤、神经纤维瘤、甲状腺癌、骨癌、皮肤癌、脑癌、结肠癌、睾丸癌、胃肠道间质瘤、肥大细胞肿瘤、多发性骨髓瘤、黑色素瘤、胶质瘤或肉瘤。
  16. 一种治疗细胞异常增殖方面的疾病的方法，其包括以下步骤：将治疗有效量的权利要求1-9任一项的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药、或权利要求8-9任意一项的抗体药物偶联物、或权利要求10-12任意一项的组合物、或者权利要求13的药物组合物或者权利要求14的药盒产品施用于对其有需求的个体；
    优选地，所述疾病为肿瘤，例如晚期实体瘤；
    优选地，所述肿瘤选自肺癌、鳞状上皮细胞癌、膀胱癌、胃癌、卵巢癌、腹膜癌、胰腺癌、乳腺癌、头颈癌、子宫颈癌、子宫内膜癌、直肠癌、肝癌、肾癌、食管腺癌、食管鳞状细胞癌、前列腺癌、雌性生殖道癌、原位癌、淋巴瘤、神经纤维瘤、甲状腺癌、骨癌、皮肤癌、脑癌、结肠癌、睾丸癌、胃肠道间质瘤、肥大细胞肿瘤、多发性骨髓瘤、黑色素瘤、胶质瘤或肉瘤。
  17. 权利要求1-8任一项的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物和前药、或权利要求8-9任意一项的抗体药物偶联物、或权利要求10-12任意一项的组合物、权利要求13的药物组合物或者权利要求14的药盒产品，其用于治疗细胞异常增殖方面的疾病；
    优选地，所述疾病为肿瘤，例如晚期实体瘤；
    优选地，所述肿瘤选自肺癌、鳞状上皮细胞癌、膀胱癌、胃癌、卵巢癌、腹膜癌、胰腺癌、乳腺癌、头颈癌、子宫颈癌、子宫内膜癌、直肠癌、肝癌、肾癌、食管腺癌、食管鳞状细胞癌、前列腺癌、雌性生殖道癌、原位癌、淋巴瘤、神经纤维瘤、甲状腺癌、骨癌、皮肤癌、脑癌、结肠癌、睾丸癌、胃肠道间质瘤、肥大细胞肿瘤、多发性骨髓瘤、黑色素瘤、胶质瘤或肉瘤。
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