WO2019170063A1 - 吲嗪类化合物、其制备方法及用途 - Google Patents

吲嗪类化合物、其制备方法及用途 Download PDF

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WO2019170063A1
WO2019170063A1 PCT/CN2019/076904 CN2019076904W WO2019170063A1 WO 2019170063 A1 WO2019170063 A1 WO 2019170063A1 CN 2019076904 W CN2019076904 W CN 2019076904W WO 2019170063 A1 WO2019170063 A1 WO 2019170063A1
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group
alkyl
optionally substituted
substituted
compound
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PCT/CN2019/076904
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English (en)
French (fr)
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陈旭星
陈奕
黄颖
耿美玉
张琼
丁健
姚虞财
沈倩倩
沈雁雁
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上海海和药物研究开发有限公司
中国科学院上海药物研究所
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Priority to JP2020568587A priority Critical patent/JP7010443B2/ja
Priority to ES19763371T priority patent/ES2902203T3/es
Priority to EP19763371.2A priority patent/EP3763712B1/en
Priority to US16/978,501 priority patent/US11661421B2/en
Priority to CA3092149A priority patent/CA3092149C/en
Publication of WO2019170063A1 publication Critical patent/WO2019170063A1/zh

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    • 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
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • 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
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present invention belongs to the field of medicinal chemistry, and in particular, the present invention relates to a class of azine compounds or pharmaceutically acceptable salts thereof, and a process for the preparation thereof.
  • the pyridazine compound of the present invention can be used for the preparation of a medicament for treating a disease associated with Drosophila zeste gene enhancer homolog 1/2 (EZH1/2) such as a malignant tumor.
  • EZH1/2 Drosophila zeste gene enhancer homolog 1/2
  • Epigenetics means that the nucleotide sequence of a gene does not change, but gene expression undergoes heritable changes. This phenomenon plays an important role in regulating processes including cell proliferation, differentiation, survival, and apoptosis.
  • An important mechanism of epigenetic regulation is histone covalent modification.
  • DNA surrounds the basic structural nucleosomes that form chromatin around histones.
  • Two molecules of H2A, H2B, H3 and H4 in each nucleosome form a histone octamer.
  • Enzymes that catalyze the methylation of histones are called histone methyltransferases (HMTs).
  • Polycomb repressive complex 2 a polycomb repressive complex 2 is a multiprotein complex that functions to catalyze the methylation of histidine H3 at position 27 (H3K27), resulting in silencing of related genes. .
  • the catalytic subunit of PRC2 is EZH1 or EZH2.
  • EZH1 or EZH2 alone has no catalytic function and must be combined with EED (embryonic ectoderm development) and SUZ12 (suppressor of zeste 12 homolog) to exert transmethylation.
  • EZH2 is highly expressed in cells of various tumors (such as breast cancer, colorectal cancer, endometrioma, gastric cancer, liver cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer and bladder cancer). Tumor cell proliferation, invasion, drug resistance and migration are closely related.
  • EZH2 has been found to be mutated in 8-24% of non-Hodgkin's lymphomas, such as Y641F, Y641N, Y641S, Y641H, A677G and A687V. These mutants have enhanced dimethylation and trimethylation catalytic functions of histone H3 at position 27 of lysine compared to wild-type EZH2.
  • Overexpression or mutation of EZH2 causes an increase in the level of 27 lysine trimethylation products (H3K27me3) in H3.
  • High levels of H3K27me3 play an important role in tumor cell proliferation and survival. Abnormal EZH2 activity leads to the development of tumors.
  • the multiple target genes regulated by EZH2 are tumor suppressor genes, and the silencing of tumor suppressor genes may be one of the important mechanisms. Indirect inhibition of EZH2 by siRNA or shRNA or indirect inhibition of EZH2 with the SAH hydrolase inhibitor 3-deazaneplanocin A (3-DZNep) can significantly reduce the proliferation and invasion of tumor cells in vitro and the growth of tumors in vivo.
  • EZH2 also plays an important role in the differentiation of T cells. EZH2 reduces the expression of Th1/Th2 cytokines, such as IFN- ⁇ , IL-4, IL-5, etc., inhibits Th1/Th2-dependent T cell migration, and activates regulatory T cells. In the tumor microenvironment, inhibition of the secretion of chemokines such as CXCL9 and CXCL10 in Th1 cells by EZH2 is an important mechanism of tumor immune escape.
  • Th1/Th2 cytokines such as IFN- ⁇ , IL-4, IL-5, etc.
  • An object of the present invention is to provide a pyridazine compound or a pharmaceutically acceptable salt thereof.
  • Another object of the present invention is to provide a process for preparing a pyridazine compound or a pharmaceutically acceptable salt thereof.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the azine compound or a pharmaceutically acceptable salt thereof.
  • Another object of the present invention is to provide a pyridazine compound or a pharmaceutically acceptable salt thereof or a composition containing the pyridazine compound or a pharmaceutically acceptable salt thereof for the preparation of an antitumor, prevention and/or preparation
  • the use of drugs for the treatment of autoimmune diseases is to provide a pyridazine compound or a pharmaceutically acceptable salt thereof or a composition containing the pyridazine compound or a pharmaceutically acceptable salt thereof for the preparation of an antitumor, prevention and/or preparation.
  • a pyridazine compound of the formula I a pharmaceutically acceptable salt thereof, an enantiomer, a diastereomer, a tautomer, a solvate, and more Crystal form or prodrug,
  • R 1 is selected from
  • R 10 is selected from the group consisting of H, halogen, -NH 2 , -NO 2 , optionally substituted C1-C6 alkyl, and optionally substituted C1-C4 alkoxy;
  • R 2 is selected from the group consisting of H, halogen, cyano and optionally substituted C1-C6 alkyl;
  • R 6 and R 6 ' are each independently selected from the group consisting of: H, methyl, ethyl, propyl and cyclopropyl, and at least one of R 6 and R 6 ' is H;
  • X is an optionally substituted saturated or unsaturated 4-7 membered cyclic hydrocarbon group or an optionally substituted 4-7 membered saturated or unsaturated heterocyclic group, said heterocyclic group comprising 1-2 selected from O, Heteroatoms of N, S, P;
  • R 4 and R 5 are each independently selected from H, halogen, -COOH, -CN, optionally substituted C1-C6 alkyl, optionally substituted 6-16 membered aryl, optionally substituted 5-16 membered hetero Aryl, optionally substituted 4-8 membered saturated or unsaturated cycloalkyl, optionally substituted 4-8 membered saturated or unsaturated heterocyclic group, optionally substituted C1-C6 alkylcarbonyl, optionally substituted -C(O)O-(C1-C6 alkyl), -C(O)(NR a R b ), boronic acid group, optionally substituted C2-C8 alkenyl group, optionally substituted C2-C8 alkynyl group, An optionally substituted C1-C6 alkylsulfone group, an optionally substituted C1-C6 alkylsulfoxide group, and an optionally substituted C1-C6 alkylfluorenyl group; wherein said
  • R 7 is selected from H and optionally substituted C 1 -C 6 alkyl
  • R 8 and R 9 are each independently selected from the group consisting of hydrogen, deuterium, and optionally substituted C1-C6 alkyl;
  • the optionally substituted substituent in R 2 , R 7 , R 8 , R 9 , R 10 , R a and R b means having one or more selected from the group consisting of (e.g. 1, 2, 3 or 4) Substituents: H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, nitro, -OH, amino, methoxy, dimethylamino.
  • R 10 is selected from the group consisting of H, halogen, -NH 2 , -NO 2 , optionally substituted C1-C6 alkyl, and optionally substituted C1-C4 alkoxy;
  • R 2 is selected from the group consisting of H, halogen, cyano and optionally substituted C1-C6 alkyl;
  • R 6 and R 6 ' are each independently selected from the group consisting of: H, methyl, ethyl, propyl and cyclopropyl, and at least one of R 6 and R 6 ' is H;
  • X is an optionally substituted saturated or unsaturated 4-7 membered cyclic hydrocarbon group, or an optionally substituted 4-7 membered saturated or unsaturated heterocyclic group; wherein said heterocyclic group contains 1-2 selected from O a hetero atom of N, S, P; the substituent in X is selected from the group consisting of: -OH, halogen, tert-butyloxycarbonyl (Boc), -NR s R t , C1-substituted by 1-3 R x C4 alkyl, substituted with 1-3 R x is C1-C4 alkoxy, substituted with 1-3 R x is C1-C4 alkyl carbonyl group, substituted with 1-3 R x is C1-C4 alkoxy a carbonyl group and a C1-C4 alkylsulfonyl group substituted by 1 to 3 R x ; each R x is independently selected from the group consisting of: H, halogen, methyl
  • R s and R t are each independently selected from the group consisting of: H, C1-C4 alkyl, C1-C4 haloalkyl,
  • R 4 and R 5 are each independently selected from H, halogen, -COOH, -CN, optionally substituted C1-C6 alkyl, optionally substituted 6-16 membered aryl, optionally substituted 5-16 membered hetero Aryl, optionally substituted 4-8 membered saturated or unsaturated cycloalkyl, optionally substituted 4-8 membered saturated or unsaturated heterocyclic group, optionally substituted C1-C6 alkylcarbonyl, optionally substituted -C(O)O-(C1-C6 alkyl), -C(O)(NR a R b ), boronic acid group, optionally substituted C2-C8 alkenyl group, optionally substituted C2-C8 alkynyl group, An optionally substituted C1-C6 alkylsulfone group, an optionally substituted C1-C6 alkylsulfoxide group, and an optionally substituted C1-C6 alkylfluorenyl group; said heteroary
  • Each R 45 is independently selected from the group consisting of: H, -OH, halogen, -Boc, halogenated C1-C4 alkyl, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 acyl, dimethylamino,
  • a Amino group, diethylamino group, methylethylamino group, ethylamino group, R 451 is selected from H, C 1 -C 4 alkyl; wherein R a , R b are each independently selected from H, optionally substituted C 1 -C 6 alkyl, optionally substituted 3-8 membered cycloalkyl, and optionally a substituted 4-8 membered heterocyclic group, or R a and R b are bonded to N to form an optionally substituted 4-8 membered heterocyclic ring; wherein said heterocyclic ring contains 1-3 selected from N, O, S and a hetero atom of P;
  • R c and R d are each independently selected from the group consisting of: H, C1-C4 alkyl, C1-C4 haloalkyl,
  • R 7 is selected from H and optionally substituted C 1 -C 6 alkyl
  • R 8 and R 9 are each independently selected from the group consisting of hydrogen, deuterium, and optionally substituted C1-C6 alkyl;
  • the substituted substituent in R 2 , R 7 , R 8 , R 9 , R 10 , R a and R b has one or more (e.g. 1, 2, 3 or 4) substituents selected from the group consisting of the lower group : H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, nitro, -OH, amino, methoxy and dimethylamino.
  • R 2 is an optionally substituted C1-C4 alkyl group; preferably a methyl group.
  • R 3 is selected from the group consisting of
  • R h is selected from H, substituted with 1-3 C1-C4 alkyl R x, R x is substituted with 1-3 C1-C4 alkyl carbonyl group, substituted with 1-3 R x C1-C4 Alkoxycarbonyl, C1-C4 alkylsulfonyl substituted by 1-3 R x and Boc;
  • R j is selected from: -OH, halo, substituted with 1-3 R x C1-C4 alkyl, substituted with 1-3 R x is C1-C4 alkoxy and -NR s R t;
  • R x is selected from the group consisting of: H, halogen and -OH;
  • R s and R t are each independently selected from the group consisting of: H, C1-C4 alkyl, C1-C4 haloalkyl,
  • R 6 and R 6 ' are each independently selected from the group consisting of: H, methyl and ethyl, and at least one of R 6 and R 6 ' is H;
  • R 3 is selected from the group consisting of:
  • R h is selected from the group consisting of H and R x (C1-C3 alkyl);
  • R j is selected from the group consisting of: -OH, halogen, R x (C1-C3 alkyl), R x (C1-C3 alkoxy), and -N(C1-C3 alkyl) 2 ;
  • R x is selected from the group consisting of: H, halogen, trifluoromethyl and difluoromethyl;
  • R 6 and R 6 ' are each independently selected from the group consisting of: H, methyl and ethyl, and at least one of R 6 and R 6 ' is H;
  • R 3 is selected from the group consisting of:
  • R h is selected from a fluorine-substituted C1-C3 alkyl group
  • R 6 and R 6 ' are each independently selected from the group consisting of: H and methyl, and at least one of R 6 and R 6 ' is H.
  • R 4 or R 5 are each independently selected from: H, R 55 C1-C4 alkyl, -CN, halogen, R 55 C1-C4 alkylcarbonyl, R 55 (C1-C4 alkoxylate) Carbonyl, -COOH, -C(O)(NR a R b ), And R 55 C1-C3 alkynyl;
  • n is the number of R 55 substituents selected from: 1, 2 and 3;
  • R 55 is selected from the group consisting of: H, R 551 C1-C4 alkyl, R 551 C3-C8 cycloalkyl, halogen, -CN, -NR c R d , (R 551 C1-C4 alkyl) O-, R 551 C1 -C4 alkyl sulfone group, R 551 (C1-C6)alkyl OC(O)-, -COOH and -C(O)(NR a R b ); wherein R 551 is H, -OH, halogen, halogenated C1-C4 alkyl, C1 -C4 alkyl, C1-C4 alkoxy, amino, dimethylamino, methylamino, diethylamino, methylethylamino, ethylamino,
  • R a and R b are each independently selected from the group consisting of: H, C1-C4 alkyl, R b" (C1-C4)alkyl, phenyl, halophenyl, and R b ' substituted heterocyclic; or R a together with R b and the N attached form an R b ' substituted 4-8 membered heterocyclic group containing 1-2 heteroatoms selected from N and O;
  • R b" is selected from the group consisting of: H, -OH, C1-C3 alkoxy, dimethylamino,
  • R b ' is selected from the group consisting of H, C1-C4 alkyl, Boc and C1-C4 acyl;
  • R 4 or R 5 are each independently selected from: H,
  • n is the number of R 55 substituents selected from: 1 and 2;
  • R 55 is selected from the group consisting of H, C1-C4 alkyl, halogen, -NH 2 , (C1-C 2 alkyl)NH- and dimethylamino.
  • R 3 is
  • R 6 and R 6 ' are each independently selected from the group consisting of: H, methyl, ethyl and propyl, and at least one of R 6 and R 6 ' is H;
  • X is selected from the group consisting of an optionally substituted pyranyl group, an optionally substituted piperidinyl group, an optionally substituted piperazinyl group, and an optionally substituted morpholinyl group, and the substituent in X is selected from the group consisting of: C1-C4 alkane And a halogenated C1-C4 alkyl group; preferably, the substituent in X is a trifluoroethyl group;
  • R 2 is a methyl group
  • R 10 is selected from the group consisting of unsubstituted or halogenated C1-C4 alkyl groups and unsubstituted or halogenated C1-C4 alkoxy groups;
  • R 4 and R 5 are each independently selected from H, an optionally substituted pyridyl group, an optionally substituted pyrimidinyl group, an optionally substituted pyridazinyl group, an optionally substituted pyrazolyl group, an optionally substituted triazolyl group, and An optionally substituted pyrrolopyridinyl group;
  • R 4 and R 5 are selected from the group consisting of: H, C1-C4 alkyl, halogen and -NR c R d ;
  • R c and R d are each independently selected from the group consisting of: H, C1-C4 alkyl and halogenated C1-C4 alkyl.
  • R 2 is a methyl group
  • R 3 is R 6 and R 6 ' are each independently selected from H or methyl, and at least one of R 6 and R 6 ' is H;
  • X is selected from the group consisting of morpholinyl, trifluoroethylpiperazinyl and trifluoroethylpiperidinyl; further preferably, morpholinyl, 2,2,2-trifluoroethylpiperazinyl or 2,2,2 -trifluoroethylpiperidinyl;
  • R 4 and R 5 are independently selected from the group consisting of H, dimethylaminopyridine and methylaminopyridine; further preferably, H, 2-dimethylaminopyridine or 2-methylaminopyridine;
  • R 7 , R 8 and R 9 are hydrogen
  • R 10 is a methoxy group.
  • the pyridazine compound of formula I is selected from the group consisting of:
  • the pyridazine compound of formula I is selected from the group consisting of:
  • a process for the preparation of a pyridazine compound of the above formula I or a pharmaceutically acceptable salt thereof which comprises one of the following methods:
  • Method one includes the following steps:
  • the base is selected from the group consisting of sodium hydride, potassium t-butoxide, sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate and sodium carbonate;
  • the Lewis acid is selected from the group consisting of titanium tetrachloride, tetraethyl oxytitanium, titanium tetraisopropoxide, boron trifluoride, copper chloride and aluminum trichloride;
  • R 1 , R 2 , R 4 , R 5 , R 6 , R 6 ' , R 7 , R 8 and R 9 are as defined above, and R k is a C1-C4 straight or branched alkyl group;
  • Ring X is an optionally substituted 4-7 membered saturated or unsaturated heterocyclic group containing at least one N atom, the optional substituent of which is defined as a substituent of X as described above.
  • Method two includes the following steps:
  • the reducing agent is selected from one or a combination of lithium, sodium, magnesium, zinc, lithium aluminum hydride and zinc copper couple;
  • the inert solvent is selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, 1,4- a combination of one or more of dioxane, dichloromethane, chloroform, carbon tetrachloride, and ethyl acetate;
  • R 1 , R 2 , R 4 , R 5 , R 6 , R 6 ' , R 7 , R 8 and R 9 are as defined above, and R k is a C1-C4 straight or branched alkyl group;
  • Ring Y is an optionally substituted saturated or unsaturated 4-7 membered cyclic hydrocarbon group or an optionally substituted 4-7 membered saturated or unsaturated heterocyclic group; wherein said heterocyclic group contains 1-2 selected from O a hetero atom of N, S and P, the substituent of which is optionally substituted as defined above for X.
  • a pharmaceutical composition comprising
  • a pyridazine compound of the above formula I a pharmaceutically acceptable salt, an enantiomer, a diastereomer, a tautomer, a solvate, a polymorph thereof or Prodrug;
  • the pharmaceutical composition further comprises other pharmaceutically acceptable therapeutic agents, particularly other anti-tumor drugs.
  • the therapeutic agent includes, but is not limited to, a drug antitumor drug acting on the chemical structure of DNA such as cisplatin, an antitumor drug affecting nucleic acid synthesis such as methotrexate (MTX), 5-fluorouracil (5FU), etc., affecting nucleic acid transcription
  • Anti-tumor drugs such as doxorubicin, epirubicin, aclarithromycin, phosfomycin, etc., antitumor drugs acting on tubulin synthesis such as paclitaxel, vinorelbine, etc., aromatase inhibitors such as ammonia Mitt, lantron, letrozole, ruined, etc., cell signaling pathway inhibitors such as epidermal growth factor receptor inhibitors Imatinib, Gefitinib, Erlotinib ), Lapatinib, etc.
  • a pyridazine compound of the formula I as described above a pharmaceutically acceptable salt thereof, an enantiomer thereof, a diastereomer thereof, a tautomer thereof
  • a solvate, polymorph or prodrug selected from the group consisting of:
  • the disease associated with EZH1/2 mutation, activity or expression is selected from the group consisting of a tumor and an autoimmune disease.
  • the disease associated with EZH1/2 mutation, activity or expression is selected from the group consisting of B cell lymphoma, malignant rhabdomyomas, synovial sarcoma, breast cancer, colorectal cancer, endometrium Tumor, stomach cancer, liver cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, nasopharyngeal cancer and bladder cancer.
  • the term “about” means that the value can vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms "containing” or “including” may be open, semi-closed, and closed. In other words, the terms also include “consisting essentially of,” or “consisting of.”
  • reaction can be carried out and purified using the manufacturer's instructions for use of the kit, or in a manner well known in the art or as described in the present invention.
  • the above techniques and methods can generally be carried out according to conventional methods well known in the art, as described in the various summaries and more specific references cited and discussed in this specification.
  • group and its substituents can be selected by those skilled in the art to provide stable structural moieties and compounds.
  • substituent When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes the chemically equivalent substituent obtained when the structural formula is written from right to left.
  • substituent -CH 2 O- is equivalent to -OCH 2 -.
  • C1-C6 alkyl refers to an alkyl group as defined below having a total of from 1 to 6 carbon atoms.
  • the total number of carbon atoms in the simplified symbol does not include carbon that may be present in the substituents of the group.
  • halogen means fluoro, chloro, bromo or iodo.
  • Haldroxy means an -OH group.
  • Hydroalkyl means an alkyl group as defined below which is substituted by a hydroxy group (-OH).
  • Niro means -NO 2 .
  • Amino means -NH 2 .
  • Substituted amino means an amino group substituted with one or two alkyl, alkylcarbonyl, aralkyl, heteroaralkyl groups as defined below, for example, monoalkylamino, dialkylamino, alkyl Amido, aralkylamino, heteroarylalkylamino.
  • Carboxyl means -COOH.
  • alkyl means a fully saturated straight or branched hydrocarbon chain group, It consists solely of carbon atoms and hydrogen atoms, has, for example, 1 to 12 (preferably 1 to 8, more preferably 1 to 6) carbon atoms, and is bonded to the rest of the molecule by a single bond, for example including but not limited to Methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl , n-hexyl, heptyl, 2-methylhexyl, 3-methylhexyl, octyl, decyl and decyl.
  • alkyl means a fully saturated straight or branched hydrocarbon chain group, It consists solely of carbon atoms and hydrogen atoms, has, for example, 1 to 12 (preferably 1 to 8, more
  • alkenyl as a group or part of another group means consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, having, for example, 2 to 14 (preferably 2 to 10) And more preferably 2 to 6) carbon atoms and a straight or branched hydrocarbon chain group attached to the remainder of the molecule by a single bond, such as, but not limited to, vinyl, propenyl, allyl, butyl- 1-Alkenyl, but-2-enyl, pent-1-enyl, pentane-1,4-dienyl and the like.
  • cycloalkyl as a group or part of another group means a stable non-aromatic monocyclic or polycyclic hydrocarbon group consisting solely of carbon atoms and hydrogen atoms, which may include a fused ring. a system, a bridged ring system or a spiro ring system having from 3 to 15 carbon atoms, preferably from 3 to 10 carbon atoms, more preferably from 3 to 8 carbon atoms, and which is saturated or unsaturated and may be via any suitable
  • the carbon atom is attached to the rest of the molecule by a single bond. Unless otherwise specifically indicated in the specification, a carbon atom in a cyclic hydrocarbon group may be optionally oxidized.
  • cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclooctyl, 1H- Indenyl, 2,3-indanyl, 1,2,3,4-tetrahydro-naphthyl, 5,6,7,8-tetrahydro-naphthyl, 8,9-dihydro-7H-benzene And cyclohepten-6-yl, 6,7,8,9-tetrahydro-5H-benzocycloheptenyl, 5,6,7,8,9,10-hexahydro-benzocyclooctenyl , fluorenyl, bicyclo [2.2.1] heptyl, 7,7-dimethyl-bicyclo[2.2.1]hept
  • heterocyclyl as a group or part of another group means consisting of 2 to 14 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, phosphorus, oxygen and sulfur.
  • a heterocyclic group may be a monocyclic, bicyclic, tricyclic or more cyclic ring system, which may include a fused ring system, a bridged ring system or a spiro ring system;
  • the nitrogen, carbon or sulfur atom may optionally be oxidized; the nitrogen atom may optionally be quaternized; and the heterocyclic group may be partially or fully saturated.
  • the heterocyclic group may be attached to the remainder of the molecule via a carbon atom or a hetero atom and through a single bond.
  • one or more of the rings may be an aryl or heteroaryl group as defined hereinafter, provided that the point of attachment to the rest of the molecule is a non-aromatic ring atom.
  • the heterocyclic group is preferably a stable 4 to 11 membered non-aromatic monocyclic, bicyclic, bridged or spiro group containing from 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur.
  • heterocyclic groups include, but are not limited to, pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, 2,7-diaza-spiro[3.5]fluorene.
  • Alkan-7-yl 2-oxa-6-aza-spiro[3.3]heptane-6-yl, 2,5-diaza-bicyclo[2.2.1]heptan-2-yl, aza Cyclobutane, pyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrofuranyl, oxazinyl, dioxocyclopentyl, tetrahydroisoquinolinyl, decahydroisoquinolinyl, imidazolinyl, Imidazolidinyl, quinazolinyl, thiazolidinyl, isothiazolidinyl, isoxazolidinyl, indanyl, octahydroindenyl, octahydroisodecyl, pyrrolidinyl, pyrazolidinyl , phthalimido and the like.
  • aryl as a group or part of another group means a conjugated hydrocarbon ring system group having 6 to 18 carbon atoms, preferably having 6 to 10 carbon atoms.
  • an aryl group may be a monocyclic, bicyclic, tricyclic or more cyclic ring system, and may also be fused to a cycloalkyl or heterocyclic group as defined above, provided that the aryl group is via The atoms on the aromatic ring are connected to the rest of the molecule by a single bond.
  • aryl groups include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, anthracenyl, 2,3-dihydro-1H-isoindolyl, 2-benzoxazolinone, 2H-1, 4-benzoxazine-3(4H)-one-7-yl and the like.
  • arylalkyl refers to an alkyl group as defined above substituted with an aryl group as defined above.
  • heteroaryl as a group or part of another group means having from 1 to 15 carbon atoms (preferably having from 1 to 10 carbon atoms) and from 1 to 6 selected from nitrogen in the ring. a 5- to 16-membered conjugated ring system of a hetero atom of oxygen and sulfur. Unless otherwise specifically indicated in the specification, a heteroaryl group may be a monocyclic, bicyclic, tricyclic or more cyclic ring system, and may also be fused to a cycloalkyl or heterocyclic group as defined above, provided that The aryl group is attached to the remainder of the molecule via a single bond through an atom on the aromatic ring.
  • the nitrogen, carbon or sulfur atom in the heteroaryl group can be optionally oxidized; the nitrogen atom can optionally be quaternized.
  • the heteroaryl group is preferably a stable 5- to 12-membered aromatic group containing from 1 to 5 heteroatoms selected from nitrogen, oxygen and sulfur, more preferably from 1 to 4 selected
  • heteroaryl groups include, but are not limited to, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, Benzimidazolyl, benzopyrazolyl, fluorenyl, furyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, pyridazinyl, isodecyl, oxazolyl, isoxazolyl , fluorenyl, quinolyl, isoquinolyl, diaza naphthyl, naphthyridinyl, quinoxalinyl, pteridinyl, oxazolyl, porphyrin, phenanthryl, phenanthroline, acridine Base, phenazinyl
  • heteroarylalkyl refers to an alkyl group as defined above which is substituted by a heteroaryl group as defined above.
  • “optionally” means that the subsequently described event or condition may or may not occur, and that the description includes both the occurrence and non-occurrence of the event or condition.
  • “optionally substituted aryl” means that the aryl group is substituted or unsubstituted, and the description includes both the substituted aryl group and the unsubstituted aryl group.
  • substituted or “substituted by” as used herein, unless the substituent is specifically recited, means that one or more hydrogen atoms on a given atom or group are independently or more, e.g., three or four substituents, the substituents are independently selected from: deuterium (D), halogen, -OH, a mercapto group, a cyano group, -CD 3, -C 1 -C 6 alkyl (preferably -C 1-3 alkyl), C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, cycloalkyl (preferably 3-8 membered cycloalkyl), aryl, heterocyclic (preferably a 3-8 membered heterocyclic group), a heteroaryl group, an aryl-C 1 -C 6 alkyl group, a heteroaryl-C 1 -C 6 alkyl group, a C 1 -C 6 haloalkyl group
  • substituents When one atom or group is substituted with a plurality of substituents, the substituents may be the same or different.
  • a chemical moiety is generally considered to be a chemical entity that is embedded or attached to a molecule.
  • Stepoisomer refers to a compound composed of the same atoms bonded by the same bond but having a different three-dimensional structure.
  • the invention will cover various stereoisomers and mixtures thereof.
  • the compounds of the present invention are intended to include E- and Z-geometric isomers unless otherwise stated.
  • Tautomer refers to an isomer formed by the transfer of a proton from one atom of a molecule to another atom of the same molecule. All tautomeric forms of the compounds of the invention will also be embraced within the scope of the invention.
  • the compounds of the invention may contain one or more chiral carbon atoms, and thus may give rise to enantiomers, diastereomers, and other stereoisomeric forms.
  • Each chiral carbon atom can be defined as (R)- or (S)- based on stereochemistry.
  • the invention is intended to include all possible isomers, as well as racemic and optically pure forms thereof.
  • the preparation of the compounds of the invention may employ racemates, diastereomers or enantiomers as starting materials or intermediates.
  • Optically active isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as by crystallization and chiral chromatography.
  • pharmaceutically acceptable salt includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salt” means a salt formed with an inorganic or organic acid which retains the bioavailability of the free base without any other side effects.
  • Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate , trifluoroacetate, propionate, hexanoate, octoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, hexane Acid salt, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate , cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, me
  • the "pharmaceutically acceptable base addition salt” means a salt formed with an inorganic base or an organic base capable of maintaining the bioavailability of the free acid without other side effects.
  • Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like.
  • Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, the following salts: primary amines, secondary amines and tertiary amines, substituted amines, including naturally substituted amines, cyclic amines, and basic ion exchange resins.
  • ammonia isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, bicyclo Hexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, hydrazine, piperazine, piperazine Pyridine, N-ethylpiperidine, polyamine resin, and the like.
  • Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.
  • pharmaceutical composition refers to a formulation of a compound of the invention and a medium generally accepted in the art for delivery of a biologically active compound to a mammal, such as a human.
  • the medium includes a pharmaceutically acceptable carrier.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, thereby facilitating the absorption of the active ingredient and thereby exerting biological activity.
  • pharmaceutically acceptable refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compound of the invention, and is relatively non-toxic, ie, the substance can be administered to an individual without causing undesirable organisms. The reaction or in an undesirable manner interacts with any of the components contained in the composition.
  • pharmaceutically acceptable excipients include, but are not limited to, any adjuvants, carriers, excipients, glidants, supplements approved by the relevant government authorities for acceptable use by humans or domestic animals.
  • the "tumor” of the present invention includes, but is not limited to, glioma, sarcoma, melanoma, articular chondroma, cholangiocarcinoma, leukemia, gastrointestinal stromal tumor, histiocytic lymphoma, non-small cell lung cancer, small cell lung cancer. , pancreatic cancer, lung squamous cell carcinoma, lung adenocarcinoma, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell carcinoma, cervical cancer, ovarian cancer, intestinal cancer, nasopharyngeal cancer, brain cancer, bone cancer, esophageal cancer, melanin Tumor, kidney cancer, oral cancer and other diseases.
  • preventing include the possibility of reducing the occurrence or progression of a disease or condition by a patient.
  • treatment and other similar synonyms as used herein includes the following meanings:
  • an "effective amount,” “therapeutically effective amount,” or “pharmaceutically effective amount,” as used herein, refers to at least one agent or compound that, after administration, is sufficient to alleviate one or more symptoms of the disease or condition being treated to some extent. The amount. The result can be a reduction and/or alleviation of signs, symptoms or causes, or any other desired change in the biological system.
  • an "effective amount” for treatment is an amount of a composition comprising a compound disclosed herein that is required to provide a significant conditional relief effect in the clinic.
  • An effective amount suitable for any individual case can be determined using techniques such as dose escalation testing.
  • administering refers to a method of delivering a compound or composition to a desired site for biological action. These methods include, but are not limited to, oral routes, duodenal routes, parenteral injections (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration.
  • parenteral injections including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion
  • topical administration and rectal administration.
  • the techniques of administration of the compounds and methods described herein are well known to those skilled in the art, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, those discussed in Pa.
  • the compounds and compositions discussed herein are administered orally.
  • pharmaceutical combination means a pharmaceutical treatment obtained by mixing or combining more than one active ingredient, It includes both fixed and unfixed combinations of active ingredients.
  • fixed combination refers to the simultaneous administration of at least one compound described herein and at least one synergistic agent to a patient in the form of a single entity or a single dosage form.
  • unfixed combination refers to the simultaneous administration, combination or sequential administration of at least one of the compounds described herein and at least one synergistic formulation to the patient in the form of separate entities. These are also applied to cocktail therapy, for example the administration of three or more active ingredients.
  • the intermediate compound functional groups may need to be protected by a suitable protecting group.
  • suitable protecting groups include trialkylsilyl or diarylalkylsilyl groups (e.g., tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl) , tetrahydropyranyl, benzyl, and the like.
  • Suitable protecting groups for amino, mercapto and fluorenyl include t-butoxycarbonyl, benzyloxycarbonyl and the like.
  • Suitable mercapto protecting groups include -C(O)-R" (wherein R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl and the like.
  • Suitable carboxy protecting groups include alkyl, aryl or aralkyl esters.
  • Protecting groups can be introduced and removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Greene, T. W. and P. G. M. Wuts, Protective Groups in Organi Synthesis, (1999), 4th Ed., Wiley.
  • the protecting group can also be a polymeric resin.
  • a oxazine compound of the formula I or a pharmaceutically acceptable salt thereof is provided.
  • a novel composition for the prevention and treatment of a disease associated with an EZH1/2 mutation is provided.
  • iPrOH isopropanol
  • EtOH ethanol
  • DCM dichloromethane
  • TFA trifluoroacetic acid
  • MeOH methanol
  • NaOH sodium hydroxide
  • HCl hydrogen chloride
  • TEA triethylamine
  • 4-dioxane NaH: sodium hydride
  • H 2 O water
  • HATU 2-(7-oxobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate Ester
  • DMF N,N-dimethylformamide
  • THF tetrahydrofuran
  • NBS N-bromosuccinimide
  • MeCN acetonitrile
  • DIPEA N,N-diisopropylethylamine
  • pyridine pyridine
  • ethyl acetate ethyl acetate
  • K 2 CO 3 potassium carbonate
  • Example 1 1-(2-(Dimethylamino)pyridin-4-yl)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridine-3 Preparation of -yl)methyl)-6-methyl-5-(1-morphinolinylpropyl-1-en-1-yl)pyridazin-7-carboxamide:
  • Step 1 Preparation of 3-bromo-1-(2-oxobutyl)-1H-pyrrole-2-carbaldehyde: 3-bromo-1H-pyrrole-2-carbaldehyde (200 mg)
  • Preparation of references In a solution of DMF (5 ml) of WO2012029942, 1-bromobutan-2-one (192 mg) and potassium carbonate (222 mg) were sequentially added and stirred at room temperature overnight. The reaction mixture was extracted with EtOAc EtOAc (EtOAc)EtOAc. Yellow solid (215 mg, yield 76%).
  • MS (ESI) m/z 216 [M+H-CHO] + .
  • Step 2 Preparation of ethyl 1-bromo-6-methyl-5-propionylpyridazine-7-carboxylate: 3-bromo-1-(2-oxopropyl)-1H-pyrrole-2- Ethyl acetate (345 mg) and potassium carbonate (182 mg) were added to a solution of formaldehyde (215 mg) in isopropyl alcohol (5 ml), and the mixture was reacted at 100 ° C for 5 hours. The reaction mixture was cooled, filtered, EtOAcjjjjjjjjjjjjjjjjjjjjjjjjj 28%). MS (ESI) m/z 338 [M+H] + .
  • Step 3 Preparation of ethyl 1-(2-(dimethylamino)pyridin-4-yl)-6-methyl-5-propionylpyridazine-7-carboxylate: 100 ml single port in dry nitrogen-protected The compound was added to the vial: 1-bromo-6-methyl-5-propionylpyridazine-7-carboxylic acid ethyl ester (159 mg), N,N-dimethyl-5-(4,4,5,5 -tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (352 mg), Pd(dppf)Cl 2 (18 mg), cesium carbonate (308 mg) to 6 ml of a mixed solution of 1,4-dioxane and water (5:1, v/v).
  • Step 4 1-(2-(Dimethylamino)pyridin-4-yl)-6-methyl-5-(1-morphinolinylpropyl-1-en-1-yl)pyridazin-7-carboxylate
  • isopropyl acid in a reaction flask of ethyl 1-(2-(dimethylamino)pyridin-4-yl)-6-methyl-5-propionylpyridazin-7-carboxylate (97 mg) 2 ml of morpholine and 1 ml of titanium tetraisopropoxide were added, and the mixture was heated to 100 ° C and stirred overnight. After the reaction mixture was cooled, EtOAc ⁇ Solid (110 mg, yield: 93%). MS (ESI) m / z 463 [M+H] + .
  • Step 5 1-(2-(Dimethylamino)pyridin-4-yl)-6-methyl-5-(1-morphinolinylpropyl-1-en-1-yl)pyridazin-7-carboxylic acid
  • 1-(2-(dimethylamino)pyridin-4-yl)-6-methyl-5-(1-morphinolinylpropyl-1-en-1-yl)pyridazine-7- A solution of isopropyl carboxylic acid (110 mg) in methanol (2.5 ml) was slowly added to 2M aqueous sodium hydroxide (2.5 ml), and the mixture was stirred at 60 ° C overnight, and the mixture was neutralized to pH 5 with dilute hydrochloric acid (1M).
  • Step 6 1-(2-(Dimethylamino)pyridin-4-yl)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridine-3- Preparation of methyl)-6-methyl-5-(1-morphinolinylpropyl-1-en-1-yl)pyridazin-7-carboxamide: 1-(2-(dimethylamino) N,N-dimethyl group of pyridin-4-yl)-6-methyl-5-(1-morphinolinylpropyl-1-en-1-yl)pyridazine-7-carboxylic acid (70 mg) 3-(Aminomethyl)-4-methoxy-6-methylpyridine-2(1H)-one hydrochloride (41 mg) was added to the formamide solution (2 mL).
  • Example 2 1-(2-(Dimethylamino)pyridin-4-yl)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridine-3 Preparation of -yl)methyl)-6-methyl-5-(1-morphinolinylvinyl)pyridazine-7-carboxamide:
  • Step 1 Preparation of 1-(2-oxopropyl)-1H-pyrrole-2-carbaldehyde: The procedure was the same as in Step 1 of Example 1: Yield: 30%.
  • 1 H NMR (CDCl 3 , 400 MHz) ⁇ ppm 9.49 (s, 1H), 7.00 (d, J 8.0 Hz, 1H), 6.87 (brs, 1H), 6.32-6.31 (m, 1H), 5.09 (s, 2H) ), 2.23 (s, 3H).
  • Step 2 Preparation of 5-acetyl-6-methylpyridazine-7-carboxylic acid ethyl ester: The procedure was the same as in Step 2 of Example 1, and the yield was 48%.
  • Step 3 Preparation of ethyl 5-acetyl-1-bromo-6-methylpyridazine-7-carboxylate: 5-acetyl-6-methylpyridazine-7- in a 100 ml dry single-mouth bottle Ethyl carboxylate (500 mg, 2 mmol) was dissolved in 20 mL of THF. bromo succinimide (320 mg, 1.8 mmol) was added portionwise at 0 ° C, and the mixture was stirred at 0 ° C for 20 min.
  • Step 4 Preparation of ethyl 5-acetyl-1-(2-(dimethylamino)pyridin-4-yl)-6-methylpyridazine-7-carboxylate: The procedure is the same as the procedure in Example 1. 3.
  • Step 5 Preparation of 1-(2-(dimethylamino)pyridin-4-yl)-6-methyl-5-(1-morphinolinylvinyl)pyridazine-7-carboxylic acid isopropyl ester: operation The procedure is the same as step 4 in Example 1.
  • Step 6 Preparation of 1-(2-(dimethylamino)pyridin-4-yl)-6-methyl-5-(1-morphinolinylvinyl)pyridazine-7-carboxylic acid: procedure as in the same example Step 5 in 1.
  • Step 7 1-(2-(Dimethylamino)pyridin-4-yl)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridine-3- Preparation of methyl)-6-methyl-5-(1-morphinolinylvinyl)pyridazine-7-carboxamide: The procedure was the same as in Step 6 of Example 1.
  • Example 3 N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-methyl-1-(6- Preparation of methylpyrazin-3-yl)-5-(1-(4-(2,2,2-trifluoroethyl)piperazin-1-yl)vinyl)pyridazine-7-carboxamide:
  • Step 1 5-acetyl-6-methyl-1-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridazine-7 -
  • ethyl carboxylate In a dry 50 mL three-necked flask, ethyl 5-acetyl-1-bromo-6-methylpyridazine-7-carboxylate (2.0 g), Pd(dppf)Cl 2 was added in sequence. (454 mg), pinacol borate (3.15 g) and potassium acetate (1.22 g) were dissolved in 1,4-dioxane (50 ml) and stirred at 110 ° C overnight.
  • Step 2 Preparation of ethyl 5-acetyl-6-methyl-1-(6-methylpyridazin-3-yl)pyridazine-7-carboxylate: The procedure was the same as step 3 in Example 1. The yield was 48%. MS (ESI) m/z 338 [M+H] + .
  • Step 3 6-Methyl-1-(6-methylpyridazin-3-yl)-5-(1-(piperazin-1-yl)vinyl)pyridazine-7-carboxylic acid isopropyl ester
  • Step 4 6-Methyl-1-(6-methylpyridazin-3-yl)-5-(1-(4-(2,2,2-trifluoroethyl)piperazin-1-yl)
  • vinyl)pyridazine-7-carboxylic acid isopropyl ester The crude product (600 mg) obtained in the previous step was dissolved in 5 ml of anhydrous tetrahydrofuran, followed by triethylamine (272 mg), 2, 2, 2-Trifluoroethyl trifluoromethanesulfonate (625 mg), EtOAc (EtOAc m. ) m/z 502 [M+H] + .
  • Step 5 6-Methyl-1-(6-methylpyridazin-3-yl)-5-(1-(4-(2,2,2-trifluoroethyl)piperazin-1-yl) Operation of vinyl)pyridazine-7-formic acid: The same procedure as in step 5 of Example 1 was carried out.
  • Step 6 N-((4-Methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-methyl-1-(6-A
  • Preparation of carbazin-3-yl)-5-(1-(4-(2,2,2-trifluoroethyl)piperazin-1-yl)vinyl)pyridazine-7-carboxamide Operation In the same manner as in Step 6 of Example 1, the yield in two steps was 70%.
  • Step 1 5-(1-(1-(tert-Butyloxycarbonyl)piperidin-4-alkenyl)ethyl)-1-(2-(dimethylamino)pyridin-4-yl)-6-
  • methyl oxazine-7-carboxylate To a 100 ml round bottom flask containing 3.5 g of zinc powder, add 40 ml of dilute hydrochloric acid hydrochloric acid (content 1%), stir at room temperature for 30 minutes, and filter. The solid was washed successively with water, diethyl ether and ethanol, and then the activated zinc powder was drained with an oil pump to leave a residual solvent.
  • Activated zinc powder (2.98 g) was added to a 250 ml three-necked flask, nitrogen was distilled off, transferred to an ice bath, 25 ml of anhydrous tetrahydrofuran was added, and titanium tetrachloride (21 ml) was slowly added dropwise to the reaction solution. After completion, the mixture was transferred to room temperature and stirred for 15 minutes, and transferred to 70 ° C to reflux. After 2 hours, the three-necked flask was transferred to room temperature, and after 15 minutes, it was transferred to an ice bath.
  • Step 2 1-(2-(Dimethylamino)pyridin-4-yl)-6-methyl-5-(1-(piperidin-4-alkenyl)ethyl)pyridazine-7-carboxylic acid
  • ethyl ester Add compound 5-(1-(1-(tert-butyloxycarbonyl)piperidin-4-alkenyl)ethyl)-1-(2-(2) in a dry 50 ml single-mouth bottle Methylamino)pyridin-4-yl)-6-methylpyridazine-7-carboxylate (92 mg), 2 ml of dichloromethane and 0.5 ml of trifluoroacetic acid, stirred at room temperature for 1 hour, and the reaction mixture was saturated. The mixture was neutralized with aq. EtOAc (EtOAc) (EtOAc) MS (ESI) m/z 436 [M+H] + .
  • Step 3 1-(2-(Dimethylamino)pyridin-4-yl)-6-methyl-5-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-
  • alkenylene)ethyl)pyridazine-7-carboxylate The procedure is the same as step 4 in Example 3. A yellow solid (33 mg, yield 96%) was obtained.
  • Step 4 1-(2-(Dimethylamino)pyridin-4-yl)-6-methyl-5-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-
  • alkenylene)ethyl)pyridazine-7-carboxylic acid The procedure is the same as step 5 in Example 1. A yellow solid (21 mg, 70% yield) was obtained. MS (ESI) m / z 487 [M+H] + .
  • Step 5 1-(2-(Dimethylamino)pyridin-4-yl)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridine-3- Methyl)-6-methyl-5-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-alkenyl)ethyl)pyridazine-7-carboxamide
  • Step 5 The procedure is the same as step 6 in Example 1.
  • Step 1 Preparation of ethyl 5-acetyl-6-methyl-1-(2-(methylamino)pyridin-4-yl)pyridazine-7-carboxylate: the procedure is the same as step 3 in Example 1, The yield was 42%. MS (ESI) m/z 352 [M+H] + .
  • Step 2 6-Methyl-1-(2-(methylamino)pyridin-4-yl)-5-(1-(piperidin-4-alkenyl)ethyl)pyridazine-7-carboxylic acid
  • the procedure is the same as in step 1 of Example 4. The yield was 78%.
  • Step 3 6-Methyl-1-(2-(methylamino)pyridin-4-yl)-5-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-ar
  • alkenyl)ethyl)pyridazine-7-carboxylate The procedure is the same as step 4 in Example 3. The yield was 64%.
  • Step 4 6-Methyl-1-(2-(methylamino)pyridin-4-yl)-5-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-ar
  • alkenyl)ethyl)pyridazine-7-carboxylic acid The procedure was the same as step 5 in Example 1.
  • Step 5 N-((4-Methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-6-methyl-1-(2-( Methylamino)pyridin-4-yl)-5-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-alkenyl)ethyl)pyridazine-7-carboxamide
  • the procedure is the same as step 6 in Example 1.
  • HTRF Homogeneous Time-Resolved Fluorescence
  • the PRC2 complex (EZH2Y641F/EED/SUZ12/RbAp48/AEBP2) histone methyltransferase was purchased from Cisbio; the substrate H3(1-50)K27me1 was a product of GL Biochem; methyl donor S -(5'-Adenosyl)-L-methionine chloride dihydrochloride (SAM) was purchased from Sigma-aldrich; Eu-labeled H3K27me3, Streptavidin-XL665 and the buffer required for the reaction were purchased from Cisbio.
  • SAM methyl donor S -(5'-Adenosyl)-L-methionine chloride dihydrochloride
  • ELISA Enzyme-linked immunosorbent assay
  • the PRC2 complex (EZH2/EED/SUZ12/RbAp48/AEBP2) histone methyltransferase used in the experiment was purchased from BPS; the substrate Biotin H3 (21-44) me0 was produced by AnaSpec; SAM was purchased from Sigma. For the GL Biochem product; methyl donor SAM was purchased from Sigma-aldrich; H3K27me3 antibody was purchased from BPS.
  • TBS-T [20 mM Tris-HCl (pH 7.2-7.4, room temperature), 150 mM NaCl, 0.1% (v/v) Tween-20] washed plate 3 times, 3% BSA blocked for 10 min, added anti-H3K27me3 antibody room temperature shaker wet Incubate for 1 h. The plate was washed 3 times with TBS-T, and 3% BSA per well was blocked for 10 min. Subsequently, a horseradish peroxidase-labeled secondary antibody was added and reacted in a wet box at room temperature for 1 h.
  • Table 1 shows the IC 50 values of some compounds.

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Abstract

本发明提供了一类式（I）所示的吲嗪类化合物或其药学上可接受的盐、其制备方法及用途。所述的吲嗪类化合物对于野生型和/或突变型的EZH2或EZH1有抑制作用，有望成为新型抗肿瘤或治疗自身免疫性疾病的药物。

Description

吲嗪类化合物、其制备方法及用途 技术领域
本发明属于药物化学领域，具体地，本发明涉及一类吲嗪类化合物或其药学上可接受的盐、制备方法。本发明的吲嗪类化合物可用于制备治疗与果蝇zeste基因增强子同源物1/2(EZH1/2)相关的疾病如恶性肿瘤等的药物。
背景技术
表观遗传是指基因的核苷酸序列不发生改变，但基因表达却发生了可遗传的改变。这种现象在调节包括细胞增殖，分化，存活以及凋亡等过程中扮演着重要角色。表观遗传调控的一个重要机制是组蛋白共价修饰。在真核细胞中，DNA包绕在组蛋白周围形成染色质的基本结构核小体。每一个核小体中各有两分子的H2A，H2B，H3和H4形成组蛋白八聚体。每个组蛋白的N端氨基酸末端会发生多种共价修饰，例如甲基化、乙酰化、磷酸化、泛素化等，从而控制基因的表达。催化组蛋白甲基化的酶被称为组蛋白甲基转移酶(HMTs)。
多梳抑制复合物2，即PRC2(polycomb repressive complex 2)，是一种多蛋白复合体，具有催化组蛋白H3的27位赖氨酸(H3K27)甲基化的功能，从而造成相关基因的沉默。PRC2的催化亚基为EZH1或EZH2，单独存在的EZH1或EZH2没有催化功能，必须与EED(embryonic ectoderm development)和SUZ12(suppressor of zeste 12 homolog)结合之后才能发挥转甲基作用。EZH2在多种肿瘤(例如乳腺癌，结直肠癌，子宫内膜瘤，胃癌，肝癌，肾癌，肺癌，黑色素瘤，卵巢癌，胰腺癌，前列腺癌和膀胱癌等)细胞中高度表达，与肿瘤细胞的增殖，入侵，耐药和迁移等过程密切相关。
近年来，发现8-24％的非霍奇金淋巴瘤中EZH2发生了突变，如Y641F，Y641N，Y641S，Y641H，A677G和A687V等。与野生型的EZH2相比，这些突变体对组蛋白H3的27位赖氨酸的二甲基化和三甲基化催化功能增强。EZH2过度表达或者发生突变，均会引起H3的27位赖氨酸三甲基化产物(H3K27me3)水平的升高，高水平的H3K27me3对肿瘤细胞的增殖和存活起着重要作用。异常的EZH2活性导致肿瘤的发生发展，EZH2调控的多个目标基因是抑瘤基因，抑瘤基因的沉默可能是其中一个重要机制。通过siRNA或者shRNA下调EZH2或者用SAH水解酶抑制剂3-deazaneplanocin A(3-DZNep)间接抑制EZH2可以明显降低肿瘤细胞的体外增殖和入侵以及体内肿瘤的生长。
EZH2在T细胞分化过程中也起到重要作用。EZH2降低Th1/Th2细胞因子的表达，如IFN-γ，IL-4，IL-5等，抑制Th1/Th2依赖的T细胞迁移，活化调节性T细胞。在肿瘤微环境中，通过EZH2抑制Th1细胞中趋化因子如CXCL9和CXCL10的分泌，是肿瘤免疫逃逸的一个重要机制。
综上所述，目前本领域尚迫切需要研发出能够抑制野生型和/或突变型的EZH1/2的有效药物。
发明内容
本发明的目的在于提供一种吲嗪类化合物或其药学上可接受的盐。
本发明的另一目的是提供一种制备吲嗪类化合物或其药学上可接受的盐的方法。
本发明的另一目的是提供一种包含所述的吲嗪类化合物或其药学上可接受的盐的药物组合物。
本发明的另一目的是提供所述吲嗪类化合物或其药学上可接受的盐或者含有所述吲嗪类化合物或其药学上可接受的盐的组合物在制备抗肿瘤、预防和/或治疗自身免疫性疾病的药物中的应用。
本发明第一方面，提供一种式I所示的吲嗪类化合物、其药学上可接受的盐、对映异构体、非对映异构体、互变异构体、溶剂化物、多晶型物或前药，
Figure PCTCN2019076904-appb-000001
其中，
R 1选自
Figure PCTCN2019076904-appb-000002
R 10选自H、卤素、-NH 2、-NO 2、任选取代的C1-C6烷基和任选取代的C1-C4烷氧基；
R 2选自H、卤素、氰基和任选取代的C1-C6烷基；
R 3为
Figure PCTCN2019076904-appb-000003
R 6和R 6’各自独立地选自：H、甲基、乙基、丙基和环丙基，且R 6和R 6’至少 有一个为H；
X为任选取代的饱和或不饱和的4-7元环烃基或任选取代的4-7元饱和或不饱和的杂环基，所述的杂环基包含1-2个选自O、N、S、P的杂原子；
R 4和R 5分别独立地选自H、卤素、-COOH、-CN、任选取代的C1-C6烷基、任选取代的6-16元芳基、任选取代的5-16元杂芳基、任选取代的4-8元饱和或不饱和环烷基、任选取代的4-8元饱和或不饱和杂环基、任选取代的C1-C6烷基羰基、任选取代的-C(O)O-(C1-C6烷基)、-C(O)(NR aR b)、硼酸基、任选取代的C2-C8烯基、任选取代的C2-C8炔基、任选取代的C1-C6烷基砜基、任选取代的C1-C6烷基亚砜基和任选取代的C1-C6烷基巯基；其中，所述的杂芳基或杂环基包含1-3个选自N、O、S、P的杂原子；其中，R a、R b各自独立地选自H、任选取代的C1-C6烷基、任选取代的3-8元环烷基和任选取代的4-8元杂环基，或R a与R b与N相连形成任选取代的4-8元杂环；其中所述的杂环包含1-3个选自N、O、S、P的杂原子；
R 7选自H和任选取代的C1-C6烷基；
R 8和R 9各自独立地选自氢、氘和任选取代的C1-C6烷基；
R 2、R 7、R 8、R 9、R 10、R a和R b中所述任选取代的取代基指具有选自以下的一个或多个(如1、2、3或4个)取代基：H、卤素、C1-C4烷基、C1-C4卤代烷基、硝基、-OH、氨基、甲氧基、二甲氨基。
在另一优选例中，
R 1为
Figure PCTCN2019076904-appb-000004
R 10选自H、卤素、-NH 2、-NO 2、任选取代的C1-C6烷基和任选取代的C1-C4烷氧基；
R 2选自H、卤素、氰基和任选取代的C1-C6烷基；
R 3为
Figure PCTCN2019076904-appb-000005
R 6和R 6’各自独立地选自：H、甲基、乙基、丙基和环丙基，且R 6和R 6’至少有一个为H；
X为任选取代的饱和或不饱和4-7元环烃基、或任选取代的4-7元饱和或不饱和杂环基；其中，所述的杂环基包含1-2个选自O、N、S、P的杂原子；X中所述取代基选自：-OH、卤素、叔丁基氧羰基(Boc)、-NR sR t、被1-3个R x取代的C1-C4烷基、被1-3个R x取代的C1-C4烷氧基、被1-3个R x取代的C1-C4烷基羰基、被1-3个 R x取代的C1-C4烷氧基羰基和被1-3个R x取代的C1-C4烷基磺酰基；各R x独立的选自：H、卤素、甲氨基、二甲氨基、氨基、-OH、甲氧基和乙氧基；
R s和R t各自独立地选自：H、C1-C4烷基、C1-C4卤代烷基、
Figure PCTCN2019076904-appb-000006
Figure PCTCN2019076904-appb-000007
R 4和R 5分别独立地选自H、卤素、-COOH、-CN、任选取代的C1-C6烷基、任选取代的6-16元芳基、任选取代的5-16元杂芳基、任选取代的4-8元饱和或不饱和环烷基、任选取代的4-8元饱和或不饱和杂环基、任选取代的C1-C6烷基羰基、任选取代的-C(O)O-(C1-C6烷基)、-C(O)(NR aR b)、硼酸基、任选取代的C2-C8烯基、任选取代的C2-C8炔基、任选取代的C1-C6烷基砜基、任选取代的C1-C6烷基亚砜基和任选取代的C1-C6烷基巯基；所述的杂芳基或杂环基包含1-3个选自N、O、S、P的杂原子；且R 4和R 5中所述取代基选自：卤素、-CN、R 45(C1-C4烷基)、R 45(C1-C4烷氧基)、R 45(C1-C4烷基)酰基、R 45(C1-C4烷基)砜基、R 45(C3-C8环烷基)、R 45(4-8元杂环基)和-NR cR d；所述的杂环基为包含1-2个选自N和O的杂原子的杂环基；
各R 45独立的选自：H、-OH、卤素、-Boc、卤代C1-C4烷基、C1-C4烷基、C1-C4烷氧基、C1-C4酰基、二甲胺基、甲氨基、二乙胺基、甲乙氨基、乙胺基、
Figure PCTCN2019076904-appb-000008
Figure PCTCN2019076904-appb-000009
R 451选自H、C1-C4烷基；其中，R a、R b各自独立地选自H、任选取代的C1-C6烷基、任选取代的3-8元环烷基和任选取代的4-8元杂环基，或R a与R b与N相连形成任选取代的4-8元杂环；其中所述的杂环包含1-3个选自N、O、S和P的杂原子；
R c和R d各自独立地选自：H、C1-C4烷基、C1-C4卤代烷基、
Figure PCTCN2019076904-appb-000010
Figure PCTCN2019076904-appb-000011
Figure PCTCN2019076904-appb-000012
R 7选自H和任选取代的C1-C6烷基；
R 8和R 9各自独立地选自氢、氘和任选取代的C1-C6烷基；
R 2、R 7、R 8、R 9、R 10、R a和R b中所述取代的取代基具有选自下组的一个或多个(如1、2、3或4个)取代基：H、卤素、C1-C4烷基、C1-C4卤代烷基、硝基、-OH、氨基、甲氧基和二甲氨基。
在另一优选例中，R 2为任选取代的C1-C4烷基；较佳地为甲基。
在另一优选例中，R 3选自下组：
Figure PCTCN2019076904-appb-000013
Figure PCTCN2019076904-appb-000014
其中R h选自H、被1-3个R x取代的C1-C4烷基、被1-3个R x取代的C1-C4烷基羰基、被1-3个R x取代的C1-C4烷氧基羰基、被1-3个R x取代的C1-C4烷基磺酰基和Boc；
R j选自：-OH、卤素、被1-3个R x取代的C1-C4烷基、被1-3个R x取代的C1-C4烷氧基和-NR sR t；
R x选自：H、卤素和-OH；
R s和R t各自独立地选自：H、C1-C4烷基、C1-C4卤代烷基、
Figure PCTCN2019076904-appb-000015
Figure PCTCN2019076904-appb-000016
R 6和R 6’各自独立地选自：H、甲基和乙基，且R 6和R 6’至少有一个为H；
较佳地，R 3选自下组：
Figure PCTCN2019076904-appb-000017
Figure PCTCN2019076904-appb-000018
其中R h选自H和R x(C1-C3烷基)；
R j选自：-OH、卤素、R x(C1-C3烷基)、R x(C1-C3烷氧基)和-N(C1-C3烷基) 2；
R x选自：H、卤素、三氟甲基和二氟甲基；
R 6和R 6’各自独立地选自：H、甲基和乙基，且R 6和R 6’至少有一个为H；
最佳地，
R 3选自下组：
Figure PCTCN2019076904-appb-000019
R h选自氟取代的C1-C3烷基；
R 6和R 6’各自独立地选自：H和甲基，且R 6和R 6’至少有一个为H。
在另一优选例中，R 4或R 5各自独立地选自：H、R 55C1-C4烷基、-CN、卤素、R 55C1-C4烷基羰基、R 55(C1-C4烷氧基)羰基、-COOH、-C(O)(NR aR b)、
Figure PCTCN2019076904-appb-000020
Figure PCTCN2019076904-appb-000021
Figure PCTCN2019076904-appb-000022
和R 55C1-C3炔基；
n为R 55取代基的数目，选自：1、2和3；
R 55选自：H、R 551C1-C4烷基、R 551C3-C8环烷基、卤素、-CN、-NR cR d、(R 551C1-C4烷基)O-、R 551C1-C4烷基砜基、
Figure PCTCN2019076904-appb-000023
R 551(C1-C6)烷基OC(O)-、-COOH和-C(O)(NR aR b)；其中R 551为H、-OH、卤素、卤代C1-C4烷基、C1-C4烷基、C1-C4烷氧基、氨基、二甲胺基、甲氨基、二乙胺基、甲乙氨基、乙胺基、
Figure PCTCN2019076904-appb-000024
Figure PCTCN2019076904-appb-000025
R a、R b各自独立地选自：H、C1-C4烷基、R b”(C1-C4)烷基、苯基、卤代苯基，和R b’取代的杂环基；或R a与R b与相连的N一起形成R b’取代的4-8元杂环基，所述杂环基包含1-2个选自N和O的杂原子；
R b”选自：H、-OH、C1-C3烷氧基、二甲胺基、
Figure PCTCN2019076904-appb-000026
Figure PCTCN2019076904-appb-000027
R b’选自H、C1-C4烷基、Boc和C1-C4酰基；
较佳地，
R 4或R 5各自独立地选自：H、
Figure PCTCN2019076904-appb-000028
Figure PCTCN2019076904-appb-000029
n为R 55取代基的数目，选自：1和2；
R 55选自：H、C1-C4烷基、卤素、-NH 2、(C1-C2烷基)NH-和二甲胺基。
在另一优选例中，R 3为
Figure PCTCN2019076904-appb-000030
R 6和R 6’各自独立地选自：H、甲基、乙基和丙基，且R 6和R 6’至少有一个为H；
X选自：任选取代的吡喃基、任选取代的哌啶基、任选取代的哌嗪基和任选取代的吗啉基，且X中所述取代基选自：C1-C4烷基和卤代C1-C4烷基；较佳地，X中所述取代基为三氟乙基；
R 2为甲基；
R 10选自：未取代或卤代的C1-C4烷基和未取代或卤代的C1-C4烷氧基；
R 4和R 5分别独立地选自H、任选取代的吡啶基、任选取代的嘧啶基、任选取代的哒嗪基、任选取代的吡唑基、任选取代的三唑基和任选取代的吡咯并吡啶基；
R 4和R 5中所述取代基选自：H、C1-C4烷基、卤素和-NR cR d；
R c和R d各自独立地选自：H、C1-C4烷基和卤代C1-C4烷基。
在另一优选例中，
R 2为甲基；
R 3为
Figure PCTCN2019076904-appb-000031
R 6和R 6’各自独立地选自H或甲基，且R 6和R 6’至少有一个为H；
X选自吗啉基、三氟乙基哌嗪基和三氟乙基哌啶基；进一步优选为，吗啉基、2,2,2-三氟乙基哌嗪基或2,2,2-三氟乙基哌啶基；
R 4和R 5独立地选自H、二甲氨基吡啶和甲基氨基吡啶；进一步优选为，H、2-二甲氨基吡啶或2-甲基氨基吡啶；
R 7、R 8和R 9为氢；
R 10为甲氧基。
在另一优选例中，所述的式I所示的吲嗪类化合物选自下列化合物中：
Figure PCTCN2019076904-appb-000032
Figure PCTCN2019076904-appb-000033
在另一优选例中，所述的式I所示的吲嗪类化合物选自下列化合物中：
Figure PCTCN2019076904-appb-000034
本发明第二方面，提供一种制备上述式I所示的吲嗪类化合物或其药学上可接受的盐的方法，所述方法包括以下方法之一：
方法一：
Figure PCTCN2019076904-appb-000035
方法一包括如下步骤：
(1)在碱存在下，化合物a与化合物b发生缩合反应生成化合物c；
所述的碱选自氢化钠、叔丁醇钾、氢氧化钠、氢氧化钾、碳酸钾、碳酸铯和碳酸钠中的一种或几种的组合；
(2)在路易斯酸的存在下，化合物c和仲胺发生脱水反应生成烯胺化合物d；
所述的路易斯酸选自四氯化钛、四乙基氧钛、四异丙氧钛、三氟化硼、氯化铜和三氯化铝中的一种或几种的组合；
(3)化合物d水解生成化合物e；
(4)化合物e与胺f发生缩合反应，生成化合物I-1，
其中，R 1、R 2、R 4、R 5、R 6、R 6’、R 7、R 8和R 9的定义如上所述，且R k为C1-C4直链或支链烷基；圈X为任选取代的4-7元饱和或不饱和的杂环基，所述的杂环基包含至少一个N原子，其任选取代基的定义如上所述的X的取代基。
方法二：
Figure PCTCN2019076904-appb-000036
方法二包括如下步骤：
(5)惰性溶剂中，在三氯化钛或四氯化钛及还原性试剂的存在下，化合物c与相应的酮发生麦克默里反应生成化合物g；
所述还原剂选自锂、钠、镁、锌、氢化铝锂和锌铜偶中的一种或几种的组合；所述的惰性溶剂选自四氢呋喃、2-甲基四氢呋喃、1,4-二氧六环、二氯甲烷、氯仿、四氯化碳和乙酸乙酯的一种或几种的组合；
(6)化合物g发生水解反应，生成化合物h；
(7)化合物g与胺f发生缩合反应，生成化合物I-2；
其中，R 1、R 2、R 4、R 5、R 6、R 6’、R 7、R 8和R 9的定义如上所述，且R k为C1-C4直链或支链烷基；圈Y为任选取代的饱和或不饱和4-7元环烃基或任选取代的4-7元饱和或不饱和杂环基；其中，所述的杂环基包含1-2个选自O、N、S和P的杂原子，其任选取代基的定义如上所述的X的取代基。
本发明的第三方面，提供了一种药物组合物，所述的药物组合物包括
(1)上述的式I所示的吲嗪类化合物、其药学上可接受的盐、对映异构体、非对映异构体、互变异构体、溶剂化物、多晶型物或前药；和
(2)药学上可接受的载体。
优选地，该药物组合物还进一步包括其他药学上可接受的治疗剂，特别是其他抗肿瘤药物。所述治疗剂包括但不限于：作用于DNA化学结构的药物抗肿瘤药如顺铂，影响核酸合成的抗肿瘤药物如甲氨蝶呤(MTX)、5-氟尿嘧啶(5FU)等，影响核酸转录的抗肿瘤药物如阿霉素、表阿霉素、阿克拉霉素、光辉霉素等，作用于微管蛋白合成的抗肿瘤药物如紫杉醇、长春瑞滨等，芳香化酶抑制剂如氨鲁米特、兰特隆、来曲唑、瑞宁德等，细胞信号通路抑制剂如表皮生长因子受体抑制剂伊马替尼(Imatinib)、吉非替尼(Gefitinib)、埃罗替尼(Erlotinib)、拉帕替尼(Lapatinib)等。
本发明第四方面，提供一种如上所述的式I所示的吲嗪类化合物、其药学上可接受的盐、其对映异构体、非对映异构体、互变异构体、溶剂化物、多晶型物或前药的用途，所述用途选自下组：
(a)制备预防或治疗与EZH1/2突变、活性或表达量相关的疾病的药物；
(b)体外非治疗性地抑制EZH1/2及其突变体的活性；和/或
(c)体外非治疗性地抑制肿瘤细胞的增殖。
在一优选例中，所述与EZH1/2突变、活性或表达量相关的疾病选自下组：肿 瘤和自身免疫性疾病。
在另一优选例中，所述与EZH1/2突变、活性或表达量相关的疾病选自下组：B细胞淋巴瘤、恶性横纹肌瘤、滑膜肉瘤、乳腺癌，结直肠癌、子宫内膜瘤、胃癌、肝癌、肾癌、肺癌、黑色素瘤、卵巢癌、胰腺癌、前列腺癌、鼻咽癌和膀胱癌。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
术语说明
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
基团定义
可在参考文献(包括Carey and Sundberg"ADVANCED ORGANIC CHEMISTRY 4TH ED."Vols.A(2000)and B(2001),Plenum Press,New York)中找到对标准化学术语的定义。除非另有说明，否则采用本领域技术范围内的常规方法，如质谱、NMR、IR和UV/VIS光谱法和药理学方法。除非提出具体定义，否则本文在分析化学、有机合成化学以及药物和药物化学的有关描述中采用的术语是本领域已知的。可在化学合成、化学分析、药物制备、制剂和递送，以及对患者的治疗中使用标准技术。例如，可利用厂商对试剂盒的使用说明，或者按照本领域公知的方式或本发明的说明来实施反应和进行纯化。通常可根据本说明书中引用和讨论的多个概要性和较具体的文献中的描述，按照本领域熟知的常规方法实施上述技术和方法。在本说明书中，可由本领域技术人员选择基团及其取代基以提供稳定的结构部分和化合物。
当通过从左向右书写的常规化学式描述取代基时，该取代基也同样包括从右向左书写结构式时所得到的在化学上等同的取代基。举例而言，-CH 2O-等同于-OCH 2-。
本文所用的章节标题仅用于组织文章的目的，而不应被解释为对所述主题的限制。本申请中引用的所有文献或文献部分包括但不限于专利、专利申请、 文章、书籍、操作手册和论文，均通过引用方式整体并入本文。
在本文中定义的某些化学基团前面通过简化符号来表示该基团中存在的碳原子总数。例如，C1-C6烷基是指具有总共1至6个碳原子的如下文所定义的烷基。简化符号中的碳原子总数不包括可能存在于所述基团的取代基中的碳。
除前述以外，当用于本申请的说明书及权利要求书中时，除非另外特别指明，否则以下术语具有如下所示的含义。
在本申请中，术语“卤素”是指氟、氯、溴或碘。
“羟基”是指-OH基团。
“羟基烷基”是指被羟基(-OH)取代的如下文所定义的烷基。
“羰基”是指-C(＝O)-基团。
“硝基”是指-NO 2。
“氰基”是指-CN。
“氨基”是指-NH 2。
“取代的氨基”是指被一个或两个如下文所定义的烷基、烷基羰基、芳烷基、杂芳烷基取代的氨基，例如，单烷基氨基、二烷基氨基、烷基酰氨基、芳烷基氨基、杂芳烷基氨基。
“羧基”是指-COOH。
在本申请中，作为基团或是其它基团的一部分(例如用在卤素取代的烷基等基团中)，术语“烷基”是指完全饱和的直链或支链的烃链基，仅由碳原子和氢原子组成、具有例如1至12个(优选为1至8个，更优选为1至6个)碳原子，且通过单键与分子的其余部分连接，例如包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、2-甲基丁基、2,2-二甲基丙基、正己基、庚基、2-甲基己基、3-甲基己基、辛基、壬基和癸基等。就本发明而言，术语“烷基”指含有1至6个碳原子的烷基。
在本申请中，作为基团或是其它基团的一部分，术语“烯基”意指仅由碳原子和氢原子组成、含有至少一个双键、具有例如2至14个(优选为2至10个，更优选为2至6个)碳原子且通过单键与分子的其余部分连接的直链或支链的烃链基团，例如但不限于乙烯基、丙烯基、烯丙基、丁-1-烯基、丁-2-烯基、戊-1-烯基、戊-1,4-二烯基等。
在本申请中，作为基团或是其它基团的一部分，术语“环烃基”意指仅由碳 原子和氢原子组成的稳定的非芳香族单环或多环烃基，其可包括稠合环体系、桥环体系或螺环体系，具有3至15个碳原子，优选具有3至10个碳原子，更优选具有3至8个碳原子，且其为饱和或不饱和并可经由任何适宜的碳原子通过单键与分子的其余部分连接。除非本说明书中另外特别指明，环烃基中的碳原子可以任选地被氧化。环烷基的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、环己二烯基、环庚基、环辛基、1H-茚基、2,3-二氢化茚基、1,2,3,4-四氢-萘基、5,6,7,8-四氢-萘基、8,9-二氢-7H-苯并环庚烯-6-基、6,7,8,9-四氢-5H-苯并环庚烯基、5,6,7,8,9,10-六氢-苯并环辛烯基、芴基、二环[2.2.1]庚基、7,7-二甲基-二环[2.2.1]庚基、二环[2.2.1]庚烯基、二环[2.2.2]辛基、二环[3.1.1]庚基、二环[3.2.1]辛基、二环[2.2.2]辛烯基、二环[3.2.1]辛烯基、金刚烷基、八氢-4,7-亚甲基-1H-茚基和八氢-2,5-亚甲基-并环戊二烯基等。
在本申请中，作为基团或是其它基团的一部分，术语“杂环基”意指由2至14个碳原子以及1至6个选自氮、磷、氧和硫的杂原子组成的稳定的3元至20元非芳香族环状基团。除非本说明书中另外特别指明，否则杂环基可以为单环、双环、三环或更多环的环体系，其可包括稠合环体系、桥环体系或螺环体系；其杂环基中的氮、碳或硫原子可任选地被氧化；氮原子可任选地被季铵化；且杂环基可为部分或完全饱和。杂环基可以经由碳原子或者杂原子并通过单键与分子其余部分连接。在包含稠环的杂环基中，一个或多个环可以是下文所定义的芳基或杂芳基，条件是与分子其余部分的连接点为非芳香族环原子。就本发明的目的而言，杂环基优选为包含1至3个选自氮、氧和硫的杂原子的稳定的4元至11元非芳香性单环、双环、桥环或螺环基团，更优选为包含1至3个选自氮、氧和硫的杂原子的稳定的4元至8元非芳香性单环、双环、桥环或螺环基团。杂环基的实例包括但不限于：吡咯烷基、吗啉基、哌嗪基、高哌嗪基、哌啶基、硫代吗啉基、2,7-二氮杂-螺[3.5]壬烷-7-基、2-氧杂-6-氮杂-螺[3.3]庚烷-6-基、2,5-二氮杂-双环[2.2.1]庚烷-2-基、氮杂环丁烷基、吡喃基、四氢吡喃基、噻喃基、四氢呋喃基、噁嗪基、二氧环戊基、四氢异喹啉基、十氢异喹啉基、咪唑啉基、咪唑烷基、喹嗪基、噻唑烷基、异噻唑烷基、异噁唑烷基、二氢吲哚基、八氢吲哚基、八氢异吲哚基、吡咯烷基、吡唑烷基、邻苯二甲酰亚氨基等。
在本申请中，作为基团或是其它基团的一部分，术语“芳基”意指具有6至18个碳原子(优选具有6至10个碳原子)的共轭烃环体系基团。就本发明的目 的而言，芳基可以为单环、双环、三环或更多环的环体系，还可以与上文所定义的环烷基或杂环基稠合，条件是芳基经由芳香环上的原子通过单键与分子的其余部分连接。芳基的实例包括但不限于苯基、萘基、蒽基、菲基、芴基、2,3-二氢-1H-异吲哚基、2-苯并噁唑啉酮、2H-1,4-苯并噁嗪-3(4H)-酮-7-基等。
在本申请中，术语“芳基烷基”是指被上文所定义的芳基所取代的上文所定义的烷基。
在本申请中，作为基团或是其它基团的一部分，术语“杂芳基”意指环内具有1至15个碳原子(优选具有1至10个碳原子)和1至6个选自氮、氧和硫的杂原子的5元至16元共轭环系基团。除非本说明书中另外特别指明，否则杂芳基可为单环、双环、三环或更多环的环体系，还可以与上文所定义的环烷基或杂环基稠合，条件是杂芳基经由芳香环上的原子通过单键与分子的其余部分连接。杂芳基中的氮、碳或硫原子可任选地被氧化；氮原子可任选地被季铵化。就本发明的目的而言，杂芳基优选为包含1至5个选自氮、氧和硫的杂原子的稳定的5元至12元芳香性基团，更优选为包含1至4个选自氮、氧和硫的杂原子的稳定的5元至10元芳香性基团或者包含1至3个选自氮、氧和硫的杂原子的5元至6元芳香性基团。杂芳基的实例包括但不限于噻吩基、咪唑基、吡唑基、噻唑基、噁唑基、噁二唑基、异噁唑基、吡啶基、嘧啶基、吡嗪基、哒嗪基、苯并咪唑基、苯并吡唑基、吲哚基、呋喃基、吡咯基、三唑基、四唑基、三嗪基、吲嗪基、异吲哚基、吲唑基、异吲唑基、嘌呤基、喹啉基、异喹啉基、二氮萘基、萘啶基、喹噁啉基、蝶啶基、咔唑基、咔啉基、菲啶基、菲咯啉基、吖啶基、吩嗪基、异噻唑基、苯并噻唑基、苯并噻吩基、噁三唑基、噌啉基、喹唑啉基、苯硫基、中氮茚基、邻二氮杂菲基、异噁唑基、吩噁嗪基、吩噻嗪基、4,5,6,7-四氢苯并[b]噻吩基、萘并吡啶基、[1,2,4]三唑并[4,3-b]哒嗪、[1,2,4]三唑并[4,3-a]吡嗪、[1,2,4]三唑并[4,3-c]嘧啶、[1,2,4]三唑并[4,3-a]吡啶、咪唑并[1,2-a]吡啶、咪唑并[1,2-b]哒嗪、咪唑并[1,2-a]吡嗪等。
在本申请中，术语“杂芳基烷基”是指被上文所定义的杂芳基所取代的上文所定义的烷基。
在本申请中，“任选地”表示随后描述的事件或状况可能发生也可能不发生，且该描述同时包括该事件或状况发生和不发生的情况。例如，“任选地被取代的芳基”表示芳基被取代或未被取代，且该描述同时包括被取代的芳基与未被取代的芳基。例如，在没有明确列出取代基的情况下，本文所用的术语“被取 代的”或“被……取代”意指给定的原子或基团上的一个或多个氢原子独立地被一个或多个、例如1、2、3或4个取代基取代，所述取代基独立地选自：氘(D)、卤素、-OH、巯基、氰基、-CD 3、-C 1-C 6烷基(优选-C 1-3烷基)、C 2-C 6烯基、C 2-C 6炔基、环烷基(优选3-8元环烷基)、芳基、杂环基(优选3-8元杂环基)、杂芳基、芳基-C 1-C 6烷基-、杂芳基-C 1-C 6烷基-、C 1-C 6卤代烷基-、-OC 1-C 6烷基(优选-OC 1-C 3烷基)、-OC 2-C 6烯基、-OC 1-C 6烷基苯基、-C 1-C 6烷基-OH(优选-C 1-C 4烷基-OH)、-C 1-C 6烷基-SH、-C 1-C 6烷基-O-C 1-C 6烷基、-OC 1-C 6卤代烷基、-NH 2、-C 1-C 6烷基-NH 2(优选-C 1-C 3烷基-NH 2)、-N(C 1-C 6烷基) 2(优选-N(C 1-C 3烷基) 2)、-NH(C 1-C 6烷基)(优选-NH(C 1-C 3烷基))、-N(C 1-C 6烷基)(C 1-C 6烷基苯基)、-NH(C 1-C 6烷基苯基)、硝基、-C(O)-OH、-C(O)OC 1-C 6烷基(优选-C(O)OC 1-C 3烷基)、-CONRiRii(其中Ri和Rii是H、D和C 1-6烷基，优选C 1-3烷基)、-NHC(O)(C 1-C 6烷基)、-NHC(O)(苯基)、-N(C 1-C 6烷基)C(O)(C 1-C 6烷基)、-N(C 1-C 6烷基)C(O)(苯基)、-C(O)C 1-C 6烷基、-C(O)杂芳基(优选-C(O)-5-7元杂芳基)、-C(O)C 1-C 6烷基苯基、-C(O)C 1-C 6卤代烷基、-OC(O)C 1-C 6烷基(优选-OC(O)C 1-C 3烷基)、-S(O) 2-C 1-C 6烷基、-S(O)-C 1-C 6烷基、-S(O) 2-苯基、-S(O) 2-C 1-C 6卤代烷基、-S(O) 2NH 2、-S(O) 2NH(C 1-C 6烷基)、-S(O) 2NH(苯基)、-NHS(O) 2(C 1-C 6烷基)、-NHS(O) 2(苯基)和-NHS(O) 2(C 1-C 6卤代烷基)，其中所述的烷基、环烷基、苯基、芳基、杂环基和杂芳基中的每一个任选被一个或多个选自以下的取代基进一步取代：卤素、-OH、-NH 2、环烷基、3-8元杂环基、C 1-C 4烷基、C 1-C 4卤代烷基-、-OC 1-C 4烷基、-C 1-C 4烷基-OH、-C 1-C 4烷基-O-C 1-C 4烷基、-OC 1-C 4卤代烷基、氰基、硝基、-C(O)-OH、-C(O)OC 1-C 6烷基、-CON(C 1-C 6烷基) 2、-CONH(C 1-C 6烷基)、-CONH 2、-NHC(O)(C 1-C 6烷基)、-NH(C 1-C 6烷基)C(O)(C 1-C 6烷基)、-SO 2(C 1-C 6烷基)、-SO 2(苯基)、-SO 2(C 1-C 6卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 6烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 6烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 6卤代烷基)。当一个原子或基团被多个取代基取代时，所述取代基可以相同或不同。本文所用术语“部分”、“结构部分”、“化学部分”、“基团”、“化学基团”是指分子中的特定片段或官能团。化学部分通常被认为是嵌入或附加到分子上的化学实体。
“立体异构体”是指由相同原子组成，通过相同的键键合，但具有不同三维结构的化合物。本发明将涵盖各种立体异构体及其混合物。
当本发明的化合物中含有烯双键时，除非另有说明，否则本发明的化合物旨在包含E-和Z-几何异构体。
“互变异构体”是指质子从分子的一个原子转移至相同分子的另一个原子而形成的异构体。本发明的化合物的所有互变异构形式也将包含在本发明的范围内。
本发明的化合物或其药学上可接受的盐可能含有一个或多个手性碳原子，且因此可产生对映异构体、非对映异构体及其它立体异构形式。每个手性碳原子可以基于立体化学而被定义为(R)-或(S)-。本发明旨在包括所有可能的异构体，以及其外消旋体和光学纯形式。本发明的化合物的制备可以选择外消旋体、非对映异构体或对映异构体作为原料或中间体。光学活性的异构体可以使用手性合成子或手性试剂来制备，或者使用常规技术进行拆分，例如采用结晶以及手性色谱等方法。
制备/分离个别异构体的常规技术包括由合适的光学纯前体的手性合成，或者使用例如手性高效液相色谱法拆分外消旋体(或盐或衍生物的外消旋体)，例如可参见Gerald Gübitz and Martin G.Schmid(Eds.),Chiral Separations,Methods and Protocols,Methods in Molecular Biology,Vol.243,2004；A.M.Stalcup,Chiral Separations,Annu.Rev.Anal.Chem.3:341-63,2010；Fumiss et al.(eds.),VOGEL’S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5.sup.TH ED.,Longman Scientific and Technical Ltd.,Essex,1991,809-816；Heller,Acc.Chem.Res.1990,23,128。
在本申请中，术语“药学上可接受的盐”包括药学上可接受的酸加成盐和药学上可接受的碱加成盐。
“药学上可接受的酸加成盐”是指能够保留游离碱的生物有效性而无其它副作用的，与无机酸或有机酸所形成的盐。无机酸盐包括但不限于盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐等；有机酸盐包括但不限于甲酸盐、乙酸盐、2,2-二氯乙酸盐、三氟乙酸盐、丙酸盐、己酸盐、辛酸盐、癸酸盐、十一碳烯酸盐、乙醇酸盐、葡糖酸盐、乳酸盐、癸二酸盐、己二酸盐、戊二酸盐、丙二酸盐、草酸盐、马来酸盐、琥珀酸盐、富马酸盐、酒石酸盐、柠檬酸盐、棕榈酸盐、硬脂酸盐、油酸盐、肉桂酸盐、月桂酸盐、苹果酸盐、谷氨酸盐、焦谷氨酸盐、天冬氨酸盐、苯甲酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、海藻酸盐、抗坏血酸盐、水杨酸盐、4-氨基水杨酸盐、萘二磺酸盐等。这些盐可通过本专业已知的方法制备。
“药学上可接受的碱加成盐”是指能够保持游离酸的生物有效性而无其它 副作用的、与无机碱或有机碱所形成的盐。衍生自无机碱的盐包括但不限于钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐、铝盐等。优选的无机盐为铵盐、钠盐、钾盐、钙盐及镁盐。衍生自有机碱的盐包括但不限于以下的盐：伯胺类、仲胺类及叔胺类，被取代的胺类，包括天然的被取代胺类、环状胺类及碱性离子交换树脂，例如氨、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、乙醇胺、二乙醇胺、三乙醇胺、二甲基乙醇胺、2-二甲氨基乙醇、2-二乙氨基乙醇、二环己胺、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、胆碱、甜菜碱、乙二胺、葡萄糖胺、甲基葡萄糖胺、可可碱、嘌呤、哌嗪、哌啶、N-乙基哌啶、聚胺树脂等。优选的有机碱包括异丙胺、二乙胺、乙醇胺、三甲胺、二环己基胺、胆碱及咖啡因。这些盐可通过本专业已知的方法制备。
在本申请中，“药物组合物”是指本发明化合物与本领域通常接受的用于将生物活性化合物输送至哺乳动物(例如人)的介质的制剂。该介质包括药学上可接受的载体。药物组合物的目的是促进生物体的给药，利于活性成分的吸收进而发挥生物活性。
本文所用术语“药学上可接受的”是指不影响本发明化合物的生物活性或性质的物质(如载体或稀释剂)，并且相对无毒，即该物质可施用于个体而不造成不良的生物反应或以不良方式与组合物中包含的任意组分相互作用。
在本申请中，“药学上可接受的赋形剂”包括但不限于任何被相关的政府管理部门许可为可接受供人类或家畜使用的佐剂、载体、赋形剂、助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。
本发明所述“肿瘤”包括但不限于神经胶质瘤、肉瘤、黑色素瘤、关节软骨瘤、胆管瘤、白血病、胃肠间质瘤、组织细胞性淋巴瘤、非小细胞肺癌、小细胞肺癌、胰腺癌、肺鳞癌、肺腺癌、乳腺癌、前列腺癌、肝癌、皮肤癌、上皮细胞癌、宫颈癌、卵巢癌、肠癌、鼻咽癌、脑癌、骨癌、食道癌、黑色素瘤、肾癌、口腔癌等疾病。
本文所用术语“预防的”、“预防”和“防止”包括使病患减少疾病或病症的发生或恶化的可能性。
本文所用的术语“治疗”和其它类似的同义词包括以下含义：
(i)预防疾病或病症在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病或病症，但尚未被诊断为已患有该疾病或病症时；
(ii)抑制疾病或病症，即遏制其发展；
(iii)缓解疾病或病症，即，使该疾病或病症的状态消退；或者
(iv)减轻该疾病或病症所造成的症状。
本文所使用术语“有效量”、“治疗有效量”或“药学有效量”是指服用后足以在某种程度上缓解所治疗的疾病或病症的一个或多个症状的至少一种药剂或化合物的量。其结果可以为迹象、症状或病因的消减和/或缓解，或生物系统的任何其它所需变化。例如，用于治疗的“有效量”是在临床上提供显著的病症缓解效果所需的包含本文公开化合物的组合物的量。可使用诸如剂量递增试验的技术测定适合于任意个体病例中的有效量。
本文所用术语“服用”、“施用”、“给药”等是指能够将化合物或组合物递送到进行生物作用的所需位点的方法。这些方法包括但不限于口服途径、经十二指肠途径、胃肠外注射(包括静脉内、皮下、腹膜内、肌内、动脉内注射或输注)、局部给药和经直肠给药。本领域技术人员熟知可用于本文所述化合物和方法的施用技术，例如在Goodman and Gilman,The Pharmacological Basis of Therapeutics,current ed.；Pergamon；and Remington’s,Pharmaceutical Sciences(current edition),Mack Publishing Co.,Easton,Pa中讨论的那些。在优选的实施方案中，本文讨论的化合物和组合物通过口服施用。
本文所使用术语“药物组合”、“药物联用”、“联合用药”、“施用其它治疗”、“施用其它治疗剂”等是指通过混合或组合不止一种活性成分而获得的药物治疗，其包括活性成分的固定和不固定组合。术语“固定组合”是指以单个实体或单个剂型的形式向患者同时施用至少一种本文所述的化合物和至少一种协同药剂。术语“不固定组合”是指以单独实体的形式向患者同时施用、合用或以可变的间隔时间顺次施用至少一种本文所述的化合物和至少一种协同制剂。这些也应用到鸡尾酒疗法中，例如施用三种或更多种活性成分。
本领域技术人员还应当理解，在下文所述的方法中，中间体化合物官能团可能需要由适当的保护基保护。这样的官能团包括羟基、氨基、巯基及羧酸。合适的羟基保护基包括三烷基甲硅烷基或二芳基烷基甲硅烷基(例如叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基或三甲基甲硅烷基)、四氢吡喃基、苄基等。合适的氨基、脒基及胍基的保护基包括叔丁氧羰基、苄氧羰基等。合适的巯基保护基包括-C(O)-R”(其中R”为烷基、芳基或芳烷基)、对甲氧基苄基、三苯甲基等。合适的羧基保护基包括烷基、芳基或芳烷基酯类。
保护基可根据本领域技术人员已知的和如本文所述的标准技术来引入和除去。保护基的使用详述于Greene,T.W.与P.G.M.Wuts,Protective Groups in Organi Synthesis,(1999),4th Ed.,Wiley中。保护基还可为聚合物树脂。
有益效果
1.提供了一种如式I所示的吲嗪类化合物或其药学上可接受的盐。
2.提供了一种结构新颖的用于预防和治疗与EZH1/2突变相关的疾病的药物组合物。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
各实施例中， 1H NMR由Varian Mercury-400型核磁共振仪记录，化学位移以δ(ppm)表示；质谱由Finnigan/MAT-95(EI)与Finnigan LCQ/DECA and Micromass Ultra Q-TOF(ESI)型质谱仪记录；反相制备HPLC分离用硅胶为200-300目。
其中，化学式或英文字母缩写代表的试剂中文名称表如下：
iPrOH：异丙醇；EtOH：乙醇；DCM：二氯甲烷；TFA：三氟乙酸；MeOH：甲醇；NaOH：氢氧化钠；HCl：氯化氢；TEA：三乙胺；1,4-dioxane：1,4-二氧六环；NaH：氢化钠；H 2O：水；HATU：2-(7-氧化苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯；DMF：N,N-二甲基甲酰胺；THF：四氢呋喃；NBS：N-溴代丁二酰亚胺；MeCN：乙腈；DIPEA：N,N-二异丙基乙胺；DMSO：二甲基亚砜；pyridine：吡啶；ethyl acetate：乙酸乙酯；K 2CO 3：碳酸钾；Cs 2CO 3：碳酸铯；Pd(dppf)Cl 2：[1,1’-双(二苯基膦基)二茂铁]二氯化钯；KOAc：醋酸钾；LiOH：氢氧化锂。
实施例1：1-(2-(二甲氨基)吡啶-4-基)-N-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-6-甲基-5-(1-吗啡啉基丙基-1-烯-1-基)吲嗪-7-甲酰胺的制备：
Figure PCTCN2019076904-appb-000037
步骤1：3-溴-1-(2-氧代丁基)-1H-吡咯-2-甲醛的制备：往3-溴-1H-吡咯-2-甲醛(200毫克)(其制备参考文献：WO2012029942)的DMF(5毫升)溶液中，依次加入1-溴代丁烷-2-酮(192毫克)、碳酸钾(222毫克)，室温搅拌过夜。反应液用乙酸乙酯萃取三次，依次用水和饱和食盐水洗涤，合并有机相，减压蒸馏，粗产品用快速分离柱分离(石油醚：乙酸乙酯＝10:1，v/v)，得到黄色固体(215毫克，产率为76％)。MS(ESI)m/z 216[M+H-CHO] +。
步骤2：1-溴-6-甲基-5-丙酰基吲嗪-7-羧酸乙酯的制备：往3-溴-1-(2-氧代丙基)-1H-吡咯-2-甲醛(215毫克)的异丙醇溶液(5毫升)中，分别加入乙酸乙酯(345毫克)、碳酸钾(182毫克)，100℃反应5小时。反应液冷却后过滤，乙酸乙酯洗涤，滤液减压浓缩，粗产品经快速分离柱分离(石油醚：乙酸乙酯＝10:1，v/v)，得到黄色固体(83毫克，产率为28％)。MS(ESI)m/z 338[M+H] +。
步骤3：1-(2-(二甲氨基)吡啶-4-基)-6-甲基-5-丙酰基吲嗪-7-羧酸乙酯的制备：在干燥的氮气保护的100毫升单口瓶中加入化合物：1-溴-6-甲基-5-丙酰基吲嗪-7-羧酸乙酯(159毫克)、N,N-二甲基-5-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)吡啶-2-胺(352毫克)、Pd(dppf)Cl 2(18毫克)、碳酸铯(308毫克)到6毫升1,4-二氧六环和水的混合溶液中(5：1，v/v)中。多次抽换氮气，与充满氮气的气球相通，在110℃油浴中搅拌过夜。反应液用二氯甲烷萃取(100毫升)，用水(50毫升×2)洗涤，饱和食盐水(50毫升)洗涤。有机相用无水硫酸钠干燥，过滤浓缩得到粗产品。经柱层析(石油醚：乙酸乙酯＝3:1)纯化，得到黄绿色固体(97mg，收率：54％)。MS(ESI)m/z 380[M+H] +。
步骤4：1-(2-(二甲氨基)吡啶-4-基)-6-甲基-5-(1-吗啡啉基丙基-1-烯-1-基)吲嗪 -7-羧酸异丙酯的制备：往1-(2-(二甲氨基)吡啶-4-基)-6-甲基-5-丙酰基吲嗪-7-羧酸乙酯(97毫克)反应瓶中加入2毫升吗啉和1毫升四异丙氧基钛，升温至100℃搅拌过夜。反应液冷却后，用二氯甲烷(15毫升)稀释，并加入0.5毫升水，减压浓缩，残留物中再加入30毫升二氯甲烷，过滤，滤液用无水硫酸钠干燥，过滤浓缩得到黄色固体(110毫克，收率：93％)。MS(ESI)m/z 463[M+H] +。
步骤5：1-(2-(二甲氨基)吡啶-4-基)-6-甲基-5-(1-吗啡啉基丙基-1-烯-1-基)吲嗪-7-甲酸的制备：往1-(2-(二甲氨基)吡啶-4-基)-6-甲基-5-(1-吗啡啉基丙基-1-烯-1-基)吲嗪-7-羧酸异丙酯(110毫克)的甲醇(2.5毫升)溶液中缓慢加入2M氢氧化钠水溶液(2.5毫升)，升至60℃搅拌过夜，反应液用稀盐酸(1M)中和至pH为5，经反相快速分离柱分离(乙腈：水＝1:4，v/v)，得到淡黄绿色固体(70毫克，产率为70％)。MS(ESI)m/z 421[M+H] +。
步骤6：1-(2-(二甲氨基)吡啶-4-基)-N-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-6-甲基-5-(1-吗啡啉基丙基-1-烯-1-基)吲嗪-7-甲酰胺的制备：往1-(2-(二甲氨基)吡啶-4-基)-6-甲基-5-(1-吗啡啉基丙基-1-烯-1-基)吲嗪-7-甲酸(70毫克)的N,N-二甲基甲酰胺溶液中(2毫升)，依次加入3-(氨基甲基)-4-甲氧基-6-甲基吡啶-2(1H)-酮盐酸盐(41毫克)，(其合成参照文献：WO2015023915)，HATU(95毫克)，二异丙基乙胺(51毫克)，室温搅拌反应过夜。经制备分离纯化，得到黄色固体(5毫克，收率：9％)。 1H NMR(400MHz,CDCl 3)δ8.06(d,J＝5.3Hz,1H),7.89(s,1H),7.86(d,J＝5.0Hz,1H),7.38(d,J＝2.8Hz,1H),6.97(d,J＝2.9Hz,1H),6.74(d,J＝5.3Hz,1H),6.63(s,1H),5.86(s,1H),5.01(q,J＝6.8Hz,1H),4.66–4.51(m,2H),3.89(s,3H),3.71–3.58(m,4H),3.05(s,6H),2.84(dd,J＝14.6,10.1Hz,4H),2.35(s,3H),2.15(s,3H),1.40(d,J＝6.8Hz,3H)；MS(ESI)m/z 571[M+H] +。
实施例2：1-(2-(二甲氨基)吡啶-4-基)-N-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-6-甲基-5-(1-吗啡啉基乙烯基)吲嗪-7-甲酰胺的制备：
Figure PCTCN2019076904-appb-000038
步骤1：1-(2-氧代丙基)-1H-吡咯-2-甲醛的制备：操作步骤同实施例1中步骤1：收率为30％。 1H NMR(CDCl 3,400MHz)δppm 9.49(s,1H),7.00(d,J＝8.0Hz,1H),6.87(brs,1H),6.32-6.31(m,1H),5.09(s,2H),2.23(s,3H)。
步骤2：5-乙酰基-6-甲基吲嗪-7-羧酸乙酯的制备：操作步骤同实施例1中步骤2，收率为48％。 1H NMR(CDCl 3,400MHz)δppm 9.20(s,1H),7.21(brs,1H),6.87(t,J＝4.0Hz,1H)，6.74(d，J＝4.0Hz,1H),4.34(q,J＝7.2Hz,2H),2.64(s,3H),2.44(s,3H),1.39(t,J＝7.2Hz,3H)。
步骤3：5-乙酰基-1-溴-6-甲基吲嗪-7-羧酸乙酯的制备：在100毫升干燥单口瓶中，将5-乙酰-6-甲基吲嗪-7-羧酸乙酯(500mg，2mmol)溶于20mL四氢呋喃中，在0℃下分批加入溴代丁二酰亚胺(320mg，1.8mmol),混合物在0℃搅拌20min，减压蒸馏除溶剂得到粗品，粗品经柱层析(石油醚：乙酸乙酯＝20:1，v/v)分离得黄色油状物141mg，产率为26％。 1H NMR(CDCl 3,400MHz)δppm 8.15(s,1H),7.17(d,J＝2.8Hz,1H),6.89(d,J＝2.8Hz,1H),4.37(q,J＝14Hz,2H),2.63(s,3H),2.44(s,3H),1.42(t,J＝14Hz,3H)。
步骤4：5-乙酰基-1-(2-(二甲氨基)吡啶-4-基)-6-甲基吲嗪-7-羧酸乙酯的制备：操作步骤同实施例1中的步骤3。
步骤5：1-(2-(二甲氨基)吡啶-4-基)-6-甲基-5-(1-吗啡啉基乙烯基)吲嗪-7-羧酸异丙酯的制备：操作步骤同实施例1中的步骤4。
步骤6：1-(2-(二甲氨基)吡啶-4-基)-6-甲基-5-(1-吗啡啉基乙烯基)吲嗪-7-甲酸的制备：操作步骤同实施例1中的步骤5。
步骤7：1-(2-(二甲氨基)吡啶-4-基)-N-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-6-甲基-5-(1-吗啡啉基乙烯基)吲嗪-7-甲酰胺的制备：操作步骤同实施例1中的步骤6。
实施例3：N-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-6-甲基-1-(6-甲基哒嗪-3-基)-5-(1-(4-(2,2,2-三氟乙基)哌嗪-1-基)乙烯基)吲嗪-7-甲酰胺的制备：
Figure PCTCN2019076904-appb-000039
步骤1：5-乙酰基-6-甲基-1-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)吲嗪-7-羧酸乙酯的制备：在干燥的50mL三口瓶中依次加入5-乙酰基-1-溴-6-甲基吲嗪-7-羧酸乙酯(2.0克)，Pd(dppf)Cl 2(454毫克)，联硼酸频那醇酯(3.15克)以及醋酸钾(1.22克)溶于1,4-二氧六环(50毫升)，110℃搅拌过夜。反应完毕后，反应液用乙酸乙酯萃取(100毫升×3)，用水(30毫升×2)洗涤，饱和食盐水(30毫升)洗涤，有机相用无水硫酸钠干燥，过滤浓缩得到粗产品。经柱层析(石油醚：乙酸乙酯＝10：1，v/v)纯化，得到黄色固体(910毫克，产率：41％)。MS(ESI)m/z372[M+H] +。
步骤2：5-乙酰基-6-甲基-1-(6-甲基哒嗪-3-基)吲嗪-7-羧酸乙酯的制备：操作步骤同实施例1中的步骤3。产率48％。MS(ESI)m/z 338[M+H] +。
步骤3：6-甲基-1-(6-甲基哒嗪-3-基)-5-(1-(哌嗪-1-基)乙烯基)吲嗪-7-羧酸异丙酯的制备：操作步骤同实施例1中的步骤4。MS(ESI)m/z 420[M+H] +。
步骤4：6-甲基-1-(6-甲基哒嗪-3-基)-5-(1-(4-(2,2,2-三氟乙基)哌嗪-1-基)乙烯基)吲嗪-7-羧酸异丙酯的制备：将上一步制得的粗品(600毫克)溶于5毫升无水四氢呋喃中，依次加入三乙胺(272毫克)、2,2,2-三氟乙基三氟甲磺酸酯(625毫克)，室温搅拌过夜，减压浓缩，粗产品经柱层析分离得到黄色固体(335毫克，两步产率为57％)MS(ESI)m/z 502[M+H] +。
步骤5：6-甲基-1-(6-甲基哒嗪-3-基)-5-(1-(4-(2,2,2-三氟乙基)哌嗪-1-基)乙烯基)吲嗪-7-甲酸的操作：操作同实施例1中的步骤5。
步骤6：N-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-6-甲基-1-(6-甲基哒嗪-3-基)-5-(1-(4-(2,2,2-三氟乙基)哌嗪-1-基)乙烯基)吲嗪-7-甲酰胺的制备：操作同实施例1中的步骤6，两步产率为70％。 1H NMR(400MHz,DMSO-d 6)δ11.44(s, 1H),8.48(s,1H),8.09(t,J＝4.6Hz,1H),7.93(d,J＝8.9Hz,1H),7.49(d,J＝5.7Hz,1H),7.48(s,1H),7.39(d,J＝3.0Hz,1H),6.11(s,1H),4.54(s,1H),4.26(d,J＝4.6Hz,2H),4.15(s,1H),3.85(s,3H),3.18(q,J＝10.0Hz,2H),2.98–2.78(m,4H),2.67–2.60(m,4H),2.59(s,3H),2.22(s,3H),2.18(s,3H)；MS(ESI)m/z 610[M+H] +。
实施例4：1-(2-(二甲氨基)吡啶-4-基)-N-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-6-甲基-5-(1-(1-(2,2,2-三氟乙基)哌啶-4-亚烯基)乙基)吲嗪-7-甲酰胺的制备：
Figure PCTCN2019076904-appb-000040
步骤1：5-(1-(1-(叔丁基氧羰基)哌啶-4-亚烯基)乙基)-1-(2-(二甲氨基)吡啶-4-基)-6-甲基吲嗪-7-羧酸乙酯的制备：往盛有3.5克锌粉的100毫升圆底烧瓶中，加入40毫升的稀盐酸盐酸(含量1％)，室温下搅拌30分钟，抽滤，依次用水，乙醚和乙醇洗涤固体，然后活化后的锌粉用油泵抽干残留溶剂，备用。往250毫升三口烧瓶中加入活化的锌粉(2.98克)，抽换氮气，转移至冰浴，加入25毫升无水四氢呋喃，并向反应液中缓慢滴加四氯化钛(21毫升)，加完后转移至室温下继续搅拌15分钟，移至70℃下回流。2小时后，将三口烧瓶转移至室温，15分钟后转移至冰浴。然后将1-叔丁氧羰基哌啶酮(2.05克)，5-乙酰基-1-(2-(二甲氨基)吡啶-4-基)-6-甲基吲嗪-7-羧酸乙酯(1.18克)和吡啶(2.68克)的四氢呋喃溶液(50毫升)慢慢滴加至三口烧瓶中，加完后转移至室温下继续搅拌15分钟，然后在70℃油浴下回流12小时。冷却至室温，用饱和氯化铵水溶液淬灭，加入水和乙酸乙酯，静置5分钟，保留上层有机相，减压浓缩，经柱层析分离(乙酸乙酯：石油醚＝1:10至1:3，v/v)，得到黄色固体(185毫克，收率：11％)。 1H NMR(400MHz,CDCl 3)δ8.47(s,1H),8.21(d,J＝5.3Hz,1H),7.19(d,J＝2.7Hz,1H),7.03(d,J＝2.8Hz,1H),6.81(dd,J＝ 5.3,1.3Hz,1H),6.73(s,1H),4.35(q,J＝7.1Hz,2H),3.16(s,6H),2.53(s,2H),2.38(s,3H),2.00(s,2H),1.96(s,3H),1.85(d,J＝13.1Hz,4H),1.45(s,9H),1.39(t,J＝7.1Hz,3H)；MS(ESI)m/z 533[M+H] +。
步骤2：1-(2-(二甲氨基)吡啶-4-基)-6-甲基-5-(1-(哌啶-4-亚烯基)乙基)吲嗪-7-羧酸乙酯的制备：在干燥的50毫升单口瓶中加入化合物5-(1-(1-(叔丁基氧羰基)哌啶-4-亚烯基)乙基)-1-(2-(二甲氨基)吡啶-4-基)-6-甲基吲嗪-7-羧酸乙酯(92毫克)，2毫升二氯甲烷和0.5毫升三氟乙酸，室温搅拌反应1小时，反应液用饱和碳酸氢钠溶液中和，二氯甲烷萃取(50毫升)，用水(50毫升×2)洗涤，饱和食盐水(50毫升)洗涤，有机相浓缩得到黄色液体(29mg，收率：39％)。MS(ESI)m/z 433[M+H] +。
步骤3：1-(2-(二甲氨基)吡啶-4-基)-6-甲基-5-(1-(1-(2,2,2-三氟乙基)哌啶-4-亚烯基)乙基)吲嗪-7-羧酸乙酯的制备：操作步骤同实施例3中的步骤4。得到黄色固体(33毫克，产率为96％)。MS(ESI)m/z 515[M+H] +。
步骤4：1-(2-(二甲氨基)吡啶-4-基)-6-甲基-5-(1-(1-(2,2,2-三氟乙基)哌啶-4-亚烯基)乙基)吲嗪-7-甲酸的制备：操作步骤同实施例1中的步骤5。得到黄色固体(21毫克，产率为70％)。MS(ESI)m/z 487[M+H] +。
步骤5：1-(2-(二甲氨基)吡啶-4-基)-N-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-6-甲基-5-(1-(1-(2,2,2-三氟乙基)哌啶-4-亚烯基)乙基)吲嗪-7-甲酰胺的制备：操作步骤同实施例1中的步骤6。得到黄色固体(11毫克，产率为40％) 1H NMR(400MHz,CDCl 3)δ11.91(s,1H),8.07(d,J＝5.3Hz,1H),7.80(s,1H),7.73(s,1H),7.12(d,J＝2.7Hz,1H),6.96(d,J＝2.7Hz,1H),6.75(d,J＝5.4Hz,1H),6.62(s,1H),5.85(s,1H),4.57(qd,J＝14.7,5.3Hz,2H),3.87(s,3H),3.07(d,J＝7.8Hz,6H),2.98(dd,J＝19.1,9.6Hz,2H),2.85–2.77(m,2H),2.56(d,J＝3.9Hz,2H),2.25(s,3H),2.16(s,3H),2.01(d,J＝6.3Hz,4H),1.92(s,3H)；MS(ESI)m/z 637[M+H] +。
实施例5：N-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-6-甲基-1-(2-(甲氨基)吡啶-4-基)-5-(1-(1-(2,2,2-三氟乙基)哌啶-4-亚烯基)乙基)吲嗪-7-甲酰胺的制备：
Figure PCTCN2019076904-appb-000041
步骤1：5-乙酰基-6-甲基-1-(2-(甲氨基)吡啶-4-基)吲嗪-7-羧酸乙酯的制备：操作步骤同实施例1中步骤3，产率为42％。MS(ESI)m/z 352[M+H] +。
步骤2：6-甲基-1-(2-(甲氨基)吡啶-4-基)-5-(1-(哌啶-4-亚烯基)乙基)吲嗪-7-羧酸乙酯的制备：操作步骤同实施例4中步骤1。产率为78％。MS(ESI)m/z 419[M+H] +。
步骤3：6-甲基-1-(2-(甲氨基)吡啶-4-基)-5-(1-(1-(2,2,2-三氟乙基)哌啶-4-亚烯基)乙基)吲嗪-7-羧酸乙酯的制备：操作步骤同实施例3中的步骤4。产率为64％。MS(ESI)m/z 501[M+H] +。
步骤4：6-甲基-1-(2-(甲氨基)吡啶-4-基)-5-(1-(1-(2,2,2-三氟乙基)哌啶-4-亚烯基)乙基)吲嗪-7-甲酸的制备：操作步骤同实施例1中的步骤5。MS(ESI)m/z 473[M+H] +。
步骤5：N-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-6-甲基-1-(2-(甲氨基)吡啶-4-基)-5-(1-(1-(2,2,2-三氟乙基)哌啶-4-亚烯基)乙基)吲嗪-7-甲酰胺的制备：操作步骤同实施例1中的步骤6。 1H NMR(400MHz,CDCl 3)δ12.26(s,1H),7.94(d,J＝5.4Hz,1H),7.82(s,1H),7.12(d,J＝2.8Hz,1H),6.96(d,J＝2.8Hz,1H),6.80–6.74(m,1H),6.57(s,1H),5.86(s,1H),4.57(qd,J＝14.7,5.4Hz,2H),3.87(s,3H),2.97(dd,J＝19.1,9.6Hz,2H),2.86(s,3H),2.84–2.76(m,2H),2.56(s,2H),2.26(s,3H),2.18(s,3H),2.01(s,2H),1.91(s,3H),1.86(s,2H)；MS(ESI)m/z 623[M+H] +。
实施例6：生物活性测定
1、化合物对PRC2复合物(EZH2Y641F)活性测定：
检测方法：均相时间分辨荧光法(Homogeneous Time-Resolved Fluorescence，HTRF)
材料：实验中所用PRC2复合物(EZH2Y641F/EED/SUZ12/RbAp48/AEBP2)组蛋白甲基转移酶购自Cisbio公司；底物H3(1-50)K27me1为GL Biochem公司产品；甲基供体S-(5’-Adenosyl)-L-methionine chloride dihydrochloride(SAM)购自Sigma-aldrich；Eu标记的H3K27me3、Streptavidin-XL665及反应所需缓冲液均购自Cisbio。
实验方法：每孔加入PRC2复合物(EZH2Y641F/EED/SUZ12/RbAp48/AEBP2)、H3(1-50)me1底物、甲基供体SAM及化合物，总反应体系为10μL，室温避光反应4h。每孔加入5μL Eu标记的H3K27Me3抗体和5μL Streptavidin–XL665，混匀，室温孵育1h后，用多标记微孔板分析系统(PerkinElmer Envision)测量620nm和665nm处的荧光值，并计算每孔的HTRF信号比(665nm/620nm)。用SoftMax Pro 5.4.1软件计算化合物的IC 50值。
2、化合物对PRC2复合物(EZH2wild type)活性测定：
检测方法：酶联免疫吸附测定(Enzyme-linked immunosorbent assay,ELISA)
材料：实验中所用PRC2复合物(EZH2/EED/SUZ12/RbAp48/AEBP2)组蛋白甲基转移酶购自BPS公司；底物Biotin H3(21-44)me0为AnaSpec公司产品；SAM购自Sigma，为GL Biochem公司产品；甲基供体SAM购自Sigma-aldrich；H3K27me3抗体购自BPS。
实验方法：采用终浓度为100nM中性亲和素100μL/孔包被96孔板，置于湿盒中摇床过夜，随后每孔加入100μL 3％BSA室温封闭1h。封闭好的96孔板中，每孔加入PRC2复合物(EZH2/EED/SUZ12/RbAp48/AEBP2)、H3(21-44)me0底物、甲基供体SAM及化合物，总反应体系为100μL，放置入湿盒内，室温摇床上反应1h。TBS-T[20mM Tris-HCl(pH 7.2-7.4，室温)，150mM NaCl，0.1％(v/v)Tween-20]洗板3次，3％BSA封闭10min，加入抗H3K27me3抗体室温摇床湿盒孵育1h。再用TBS-T洗板3次，每孔3％BSA封闭10min。随后加入辣根过氧化物酶标记的二抗，室温摇床湿盒内反应1h。最后继续TBS-T洗3次，加入2mg/ml OPD显色液(100μL/孔)显色，并用2M H 2SO 4(50μL/孔)终止反应，酶标仪490nm读值，用SoftMax Pro 5.4.1软件计算化合物的IC 50。
其结果如表1。
表1显示了部分化合物的IC 50值
Figure PCTCN2019076904-appb-000042
Figure PCTCN2019076904-appb-000043
注：“/”表示未测定
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种式I所示的吲嗪类化合物、其药学上可接受的盐、对映异构体、非对映异构体、互变异构体、溶剂化物、多晶型物或前药，
    Figure PCTCN2019076904-appb-100001
    其中，
    R 1选自
    Figure PCTCN2019076904-appb-100002
    R 10选自H、卤素、-NH 2、-NO 2、任选取代的C1-C6烷基和任选取代的C1-C4烷氧基；
    R 2选自H、卤素、氰基和任选取代的C1-C6烷基；
    R 3为
    Figure PCTCN2019076904-appb-100003
    R 6和R 6’各自独立地选自：H、甲基、乙基、丙基和环丙基，且R 6和R 6’至少有一个为H；
    X为任选取代的饱和或不饱和的4-7元环烃基或任选取代的4-7元饱和或不饱和的杂环基，所述的杂环基包含1-2个选自O、N、S和P的杂原子；
    R 4和R 5分别独立地选自H、卤素、-COOH、-CN、任选取代的C1-C6烷基、任选取代的6-16元芳基、任选取代的5-16元杂芳基、任选取代的4-8元饱和或不饱和环烷基、任选取代的4-8元饱和或不饱和杂环基、任选取代的C1-C6烷基羰基、任选取代的-C(O)O-(C1-C6烷基)、-C(O)(NR aR b)、硼酸基、任选取代的C2-C8烯基、任选取代的C2-C8炔基、任选取代的C1-C6烷基砜基、任选取代的C1-C6烷基亚砜基和任选取代的C1-C6烷基巯基；其中，所述的杂芳基或杂环基包含1-3个选自N、O、S、P的杂原子；其中，R a、R b各自独立地选自H、任选取代的C1-C6烷基、任选取代的3-8元环烷基和任选取代的4-8元杂环基，或R a与R b与N相连形成任选取代的4-8元杂环；其中所述的杂环包含1-3个选自N、O、S和P的杂原子；
    R 7选自H和任选取代的C1-C6烷基；
    R 8和R 9各自独立地选自氢、氘和任选取代的C1-C6烷基；
    R 2、R 7、R 8、R 9、R 10、R a和R b中所述任选取代的取代基指具有选自以下的一个或多个取代基：H、卤素、C1-C4烷基、C1-C4卤代烷基、硝基、-OH、氨基、甲氧基和二甲氨基。
  2. 如权利要求1所述的式I所示的吲嗪类化合物、其药学上可接受的盐、对映异构体、非对映异构体、互变异构体、溶剂化物、多晶型物或前药，其特征在于，
    R 1为
    Figure PCTCN2019076904-appb-100004
    R 10选自H、卤素、-NH 2、-NO 2、任选取代的C1-C6烷基和任选取代的C1-C4烷氧基；
    R 2选自H、卤素、氰基和任选取代的C1-C6烷基；优选地，R 2为任选取代的C1-C4烷基；进一步优选地为甲基；
    R 3为
    Figure PCTCN2019076904-appb-100005
    R 6和R 6’各自独立地选自：H、甲基、乙基、丙基和环丙基，且R 6和R 6’至少有一个为H；
    X为任选取代的饱和或不饱和4-7元环烃基、或任选取代的4-7元饱和或不饱和杂环基；其中，所述的杂环基包含1-2个选自O、N、S、P的杂原子；X中所述取代基选自：-OH、卤素、叔丁基氧羰基、-NR sR t、被1-3个R x取代的C1-C4烷基、被1-3个R x取代的C1-C4烷氧基、被1-3个R x取代的C1-C4烷基羰基、被1-3个R x取代的C1-C4烷氧基羰基和被1-3个R x取代的C1-C4烷基磺酰基；各R x独立的选自：H、卤素、甲氨基、二甲氨基、氨基、-OH、甲氧基和乙氧基；
    R s和R t各自独立地选自：H、C1-C4烷基、C1-C4卤代烷基、
    Figure PCTCN2019076904-appb-100006
    Figure PCTCN2019076904-appb-100007
    R 4和R 5分别独立地选自H、卤素、-COOH、-CN、任选取代的C1-C6烷基、任选取代的6-16元芳基、任选取代的5-16元杂芳基、任选取代的4-8元饱和或不饱和环烷基、任选取代的4-8元饱和或不饱和杂环基、任选取代的C1-C6烷基羰基、任 选取代的-C(O)O-(C1-C6烷基)、-C(O)(NR aR b)、硼酸基、任选取代的C2-C8烯基、任选取代的C2-C8炔基、任选取代的C1-C6烷基砜基、任选取代的C1-C6烷基亚砜基和任选取代的C1-C6烷基巯基；所述的杂芳基或杂环基包含1-3个选自N、O、S、P的杂原子；且R 4和R 5中所述取代基选自：卤素、-CN、R 45(C1-C4烷基)、R 45(C1-C4烷氧基)、R 45(C1-C4烷基)酰基、R 45(C1-C4烷基)砜基、R 45(C3-C8环烷基)、R 45(4-8元杂环基)和-NR cR d；所述的杂环基为包含1-2个选自N和O的杂原子的杂环基；
    各R 45独立的选自：H、-OH、卤素、叔丁基氧羰基、卤代C1-C4烷基、C1-C4烷基、C1-C4烷氧基、C1-C4酰基、二甲胺基、甲氨基、二乙胺基、甲乙氨基、乙胺基、
    Figure PCTCN2019076904-appb-100008
    Figure PCTCN2019076904-appb-100009
    R 451选自H、C1-C4烷基；其中，R a、R b各自独立地选自H、任选取代的C1-C6烷基、任选取代的3-8元环烷基和任选取代的4-8元杂环基，或R a与R b与N相连形成任选取代的4-8元杂环；其中所述的杂环包含1-3个选自N、O、S和P的杂原子；
    R c和R d各自独立地选自：H、C1-C4烷基、C1-C4卤代烷基、
    Figure PCTCN2019076904-appb-100010
    Figure PCTCN2019076904-appb-100011
    R 7选自H和任选取代的C1-C6烷基；
    R 8和R 9各自独立地选自氢、氘和任选取代的C1-C6烷基；
    R 2、R 7、R 8、R 9、R 10、R a和R b中所述取代的取代基具有选自下组的一个或多个取代基：H、卤素、C1-C4烷基、C1-C4卤代烷基、硝基、-OH、氨基、甲氧基和二甲氨基。
  3. 如权利要求1所述的式I所示的吲嗪类化合物、其药学上可接受的盐、对映异构体、非对映异构体、互变异构体、溶剂化物、多晶型物或前药，其特征在于，
    R 3选自下组：
    Figure PCTCN2019076904-appb-100012
    Figure PCTCN2019076904-appb-100013
    其中R h选自H、被1-3个R x取代的C1-C4烷基、被1-3个R x取代的C1-C4烷基羰基、被1-3个R x取代的C1-C4烷氧基羰基、被1-3个R x取代的C1-C4烷基磺酰基和叔丁基氧羰基；
    R j选自：-OH、卤素、被1-3个R x取代的C1-C4烷基、被1-3个R x取代的C1-C4烷氧基和-NR sR t；
    R x选自：H、卤素和-OH；
    R s和R t各自独立地选自：H、C1-C4烷基、C1-C4卤代烷基、
    Figure PCTCN2019076904-appb-100014
    Figure PCTCN2019076904-appb-100015
    R 6和R 6’各自独立地选自：H、甲基和乙基，且R 6和R 6’至少有一个为H；
    较佳地，R 3选自下组：
    Figure PCTCN2019076904-appb-100016
    Figure PCTCN2019076904-appb-100017
    Figure PCTCN2019076904-appb-100018
    其中R h选自H和R x(C1-C3烷基)；
    R j选自：-OH、卤素、R x(C1-C3烷基)、R x(C1-C3烷氧基)和-N(C1-C3烷基) 2；
    R x选自：H、卤素、三氟甲基和二氟甲基；
    R 6和R 6’各自独立地选自：H、甲基和乙基，且R 6和R 6’至少有一个为H；
    最佳地，
    R 3选自下组：
    Figure PCTCN2019076904-appb-100019
    R h选自氟取代的C1-C3烷基；
    R 6和R 6’各自独立地选自：H和甲基，且R 6和R 6’至少有一个为H。
  4. 如权利要求1所述的式I所示的吲嗪类化合物、其药学上可接受的盐、对映异构体、非对映异构体、互变异构体、溶剂化物、多晶型物或前药，其特征在于，R 4或R 5各自独立地选自：H、R 55C1-C4烷基、-CN、卤素、R 55C1-C4烷基羰基、R 55(C1-C4烷氧基)羰基、-COOH、-C(O)(NR aR b)、
    Figure PCTCN2019076904-appb-100020
    Figure PCTCN2019076904-appb-100021
    Figure PCTCN2019076904-appb-100022
    和R 55C1-C3炔基；
    n为R 55取代基的数目，选自：1、2和3；
    R 55选自：H、R 551C1-C4烷基、R 551C3-C8环烷基、卤素、-CN、-NR cR d、(R 551C1-C4烷基)O-、R 551C1-C4烷基砜基、
    Figure PCTCN2019076904-appb-100023
    R 551(C1-C6)烷基OC(O)-、-COOH和-C(O)(NR aR b)；其中R 551为H、-OH、卤素、卤代C1-C4烷基、C1-C4烷基、C1-C4烷氧基、氨基、二甲胺基、甲氨基、二乙胺基、甲乙氨基、乙胺基、
    Figure PCTCN2019076904-appb-100024
    Figure PCTCN2019076904-appb-100025
    R a、R b各自独立地选自：H、C1-C4烷基、R b”(C1-C4)烷基、苯基、卤代苯基，和R b’取代的杂环基；或R a与R b与相连的N一起形成R b’取代的4-8元杂环基，所述杂环基包含1-2个选自N和O的杂原子；
    R b”选自：H、-OH、C1-C3烷氧基、二甲胺基、
    Figure PCTCN2019076904-appb-100026
    Figure PCTCN2019076904-appb-100027
    R b’选自H、C1-C4烷基、叔丁基氧羰基和C1-C4酰基；
    较佳地，
    R 4或R 5各自独立地选自：H、
    Figure PCTCN2019076904-appb-100028
    Figure PCTCN2019076904-appb-100029
    n为R 55取代基的数目，选自：1和2；
    R 55选自：H、C1-C4烷基、卤素、-NH 2、(C1-C2烷基)NH-和二甲胺基。
  5. 如权利要求1所述的式I所示的吲嗪类化合物、其药学上可接受的盐、对映异构体、非对映异构体、互变异构体、溶剂化物、多晶型物或前药，其特征在于，
    R 3为
    Figure PCTCN2019076904-appb-100030
    R 6和R 6’各自独立地选自：H、甲基、乙基和丙基，且R 6和R 6’至少有一个为H；
    X选自：任选取代的吡喃基、任选取代的哌啶基、任选取代的哌嗪基和任选取代的吗啉基，且X中所述取代基选自：C1-C4烷基和卤代C1-C4烷基；较佳地，X中所述取代基为三氟乙基；
    R 2为甲基；
    R 10选自：未取代或卤代的C1-C4烷基和未取代或卤代的C1-C4烷氧基；
    R 4、R 5分别独立地选自H、任选取代的吡啶基、任选取代的嘧啶基、任选取代的哒嗪基、任选取代的吡唑基、任选取代的三唑基和任选取代的吡咯并吡啶基；
    R 4和R 5中所述取代基选自：H、C1-C4烷基、卤素和-NR cR d；
    R c和R d各自独立地选自：H、C1-C4烷基和卤代C1-C4烷基。
  6. 如权利要求1所述的式I所示的吲嗪类化合物、其药学上可接受的盐、对映异构体、非对映异构体、互变异构体、溶剂化物、多晶型物或前药，其特征在于，
    R 2为甲基；
    R 3为
    Figure PCTCN2019076904-appb-100031
    R 6和R 6’各自独立地选自H或甲基，且R 6和R 6’至少有一个为H；
    X选自吗啉基、三氟乙基哌嗪基和三氟乙基哌啶基；进一步优选为，吗啉基、2,2,2-三氟乙基哌嗪基或2,2,2-三氟乙基哌啶基；
    R 4和R 5独立地选自H、二甲氨基吡啶和甲基氨基吡啶；进一步优选为，H、2-二甲氨基吡啶或2-甲基氨基吡啶；
    R 7、R 8和R 9为氢；
    R 10为甲氧基。
  7. 如权利要求1所述的式I所示的吲嗪类化合物、其药学上可接受的盐、对映异构体、非对映异构体、互变异构体、溶剂化物、多晶型物或前药，其特征在于，其中，所述式I所示的吲嗪类化合物选自下列化合物：
    Figure PCTCN2019076904-appb-100032
  8. 一种制备权利要求1所述的式I所示的吲嗪类化合物或其药学上可接受的盐的方法，所述方法包括以下方法之一：
    方法一：
    Figure PCTCN2019076904-appb-100033
    方法一包括如下步骤：
    (1)在碱存在下，化合物a与化合物b发生缩合反应生成化合物c；
    (2)在路易斯酸的存在下，化合物c和仲胺发生脱水反应生成烯胺化合物d；
    (3)化合物d水解生成化合物e；
    (4)化合物e与胺f发生缩合反应，生成化合物I-1，
    其中，R 1、R 2、R 4、R 5、R 6、R 6’、R 7、R 8和R 9的定义如权利要求1中所述，且R k为C1-C4直链或支链烷基；圈X为任选取代的4-7元饱和或不饱和的杂环基，所述的杂环基包含至少一个N原子，其任选取代基的定义如权利要求1中所述的X的取代基；
    方法二：
    Figure PCTCN2019076904-appb-100034
    方法二包括如下步骤：
    (5)惰性溶剂中，在三氯化钛或四氯化钛及还原性试剂的存在下，化合物c与 相应的酮发生麦克默里反应生成化合物g；
    (6)化合物g发生水解反应，生成化合物h；
    (7)化合物g与胺f发生缩合反应，生成化合物I-2；
    其中，R 1、R 2、R 4、R 5、R 6、R 6’、R 7、R 8和R 9的定义如权利要求1中所述，且R k为C1-C4直链或支链烷基；圈Y为任选取代的饱和或不饱和4-7元环烃基或任选取代的4-7元饱和或不饱和杂环基；其中，所述的杂环基包含1-2个选自O、N、S和P的杂原子，其任选取代基的定义如权利要求1中所述的X的取代基。
  9. 一种药物组合物，其特征在于，所述药物组合物包括：
    (1)治疗有效量的选自权利要求1～7中任意一项所述的吲嗪类化合物、其药学上可接受的盐、其对映异构体、非对映异构体、互变异构体、溶剂化物、多晶型物或前药作为活性成分；和
    (2)药学上可接受的载体。
  10. 一种如权利要求1～7中任意一项所述的吲嗪类化合物、其药学上可接受的盐、其对映异构体、非对映异构体、互变异构体、溶剂化物、多晶型物或前药或权利要求9所述的药物组合物的用途，所述用途选自下组：
    (a)制备用于预防或治疗与EZH1/2突变、活性或表达量相关的疾病的药物；
    (b)体外非治疗性地抑制EZH2及其突变体的活性；和/或
    (c)体外非治疗性地抑制肿瘤细胞的增殖。
  11. 如权利要求11所述的用途，其特征在于，所述与EZH1/2突变、活性或表达量相关的疾病选自下组：肿瘤和自身免疫性疾病。
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