WO2020224607A1 - Ezh2抑制剂及其用途 - Google Patents

Ezh2抑制剂及其用途 Download PDF

Info

Publication number
WO2020224607A1
WO2020224607A1 PCT/CN2020/088861 CN2020088861W WO2020224607A1 WO 2020224607 A1 WO2020224607 A1 WO 2020224607A1 CN 2020088861 W CN2020088861 W CN 2020088861W WO 2020224607 A1 WO2020224607 A1 WO 2020224607A1
Authority
WO
WIPO (PCT)
Prior art keywords
methyl
methoxy
oxo
diazoheptino
chloro
Prior art date
Application number
PCT/CN2020/088861
Other languages
English (en)
French (fr)
Inventor
李进
陈弘道
刘浏
杨民民
Original Assignee
南京药石科技股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京药石科技股份有限公司 filed Critical 南京药石科技股份有限公司
Priority to US17/608,950 priority Critical patent/US20220281872A1/en
Priority to CN202080010425.4A priority patent/CN113365989A/zh
Publication of WO2020224607A1 publication Critical patent/WO2020224607A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/06—Peri-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • This application belongs to the field of chemistry and medicine, and specifically relates to an EZH2 inhibitor and its use.
  • Drosophila Pc genes are a group of epigenetic effectors.
  • EZH2 is the catalytic component of Drosophila Pc gene repressor complex 2 (PRC2).
  • Drosophila Pc gene repressor complex 2 is a kind of protein by making 27 on histone H3. Lysine (H3K27) is a conserved multi-subunit complex that is methylated to inhibit gene transcription.
  • EZH2 plays a key role in regulating gene expression patterns that determine cell fate, such as differentiation and self-renewal. The overexpression of EZH2 in some cancer cells is associated with cell proliferation, cell invasion, chemoresistance, and metastasis.
  • EZH2 expression has been observed in many solid tumors (including prostate cancer, breast cancer, skin cancer, bladder cancer, liver cancer, pancreatic cancer, head and neck cancer), and is related to cancer invasion and metastasis.
  • Recurrent somatic mutations of EZH2 have been identified in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL).
  • DLBCL diffuse large B-cell lymphoma
  • FL follicular lymphoma
  • EZH2 inhibitors currently being developed are still in the clinical stage, such as EPZ6438 (Tazemetostat, clinical phase II), CPI-1205 (clinical phase I/II), PF-6821497 (clinical phase I), SHR-2554 (clinical phase I/ Phase II), etc., there is no EZH2 inhibitor successfully marketed, so EZH2 lead compounds with novel compound structures and good activity are still in urgent need of research and development.
  • the present application discloses a class of compounds that can be used as EZH2 inhibitors and their use in the preparation of drugs for the treatment of EZH2-mediated diseases.
  • the application provides a compound represented by formula (I), or a pharmaceutically acceptable salt, ester, prodrug, complex, solvate, hydrate or isomer thereof:
  • R 1 is selected from hydrogen, halogen, C 1 -C 6 alkyl, hydroxyl, cyano or amino;
  • R 2 is selected from -CR 4 R 5 , -NR 4 R 5 , 4-15 membered heterocyclic group, C 6 -C 10 aryl group, 5-14 membered heteroaryl group, wherein the heterocyclic group, aryl group or The heteroaryl group is optionally substituted by one or more R 6 , and R 6 is selected from halogen, hydroxy, cyano, amino or C 1 -C 6 alkyl optionally substituted by hydroxy or cyano;
  • R 3 is selected from hydrogen, halogen, C 1 -C 6 alkyl, hydroxyl, cyano or amino;
  • R 4 and R 5 are each independently selected from C 1 -C 6 alkyl, C 1 -C 6 alkoxy or 4-15 membered heterocyclic group;
  • U, V and L are each independently selected from C or N, and at least one is N;
  • X and Y are each independently selected from C 1 -C 6 alkyl, C 1 -C 6 fluoroalkyl, C 1 -C 6 alkoxy or C 1 -C 6 fluoroalkoxy;
  • R 7 is selected from hydrogen, halogen, C 1 -C 6 alkyl, hydroxyl, cyano or amino;
  • n 0, 1 or 2;
  • formula (I) is formula (Ia):
  • R 1 is selected from hydrogen, halogen, C 1 -C 6 alkyl, hydroxyl, cyano or amino;
  • R 2 is selected from -CR 4 R 5 , -NR 4 R 5 , 4-15 membered heterocyclic group, C 6 -C 10 aryl group, 5-14 membered heteroaryl group, wherein the heterocyclic group, aryl group or The heteroaryl group is optionally substituted by one or more R 6 , and R 6 is selected from halogen, hydroxy, cyano, amino or C 1 -C 6 alkyl optionally substituted by hydroxy or cyano;
  • R 3 is selected from hydrogen, halogen, C 1 -C 6 alkyl, hydroxyl, cyano or amino;
  • R 4 and R 5 are each independently selected from C 1 -C 6 alkyl, C 1 -C 6 alkoxy or 3-12 membered heterocyclic group;
  • U, V and L are each independently selected from C or N, and at least one is N;
  • R 7 is selected from hydrogen, halogen, C 1 -C 6 alkyl, hydroxyl, cyano or amino;
  • n 0, 1 or 2;
  • the heterocyclic group is monocyclic or polycyclic (including 2 or more fused rings, including spiro, fused or bridged systems, such as bicyclic ring systems), saturated or unsaturated non-aromatic 4 A to 15-membered ring system, including 1 to 14 ring-forming carbon atoms and 1 to 10 ring-forming heteroatoms, the ring-forming heteroatoms are independently selected from O, S, N, P, and B.
  • a 4- to 12-membered heterocycloalkyl is a monocyclic or polycyclic, saturated or unsaturated, non-aromatic 4- to 12-membered ring system that contains one or more ring-forming heteroatoms.
  • the aryl group is an all-carbon monocyclic or fused-ring polycyclic aromatic group with a conjugated ⁇ -electron system.
  • the aryl group may have 6 to 10 carbon atoms in one or more rings. Most commonly, aryl groups have 6 carbon atoms in the ring.
  • a C6-10 aryl group is an aromatic group containing 6 to 10 carbon atoms, such as phenyl or naphthyl.
  • the heteroaryl group is a monocyclic or fused-ring polycyclic aromatic heterocyclic group having one or more heteroatom ring members (ring atoms) in at least one ring, and the heteroatom ring members are independently of each other Selected from O, S and N.
  • Heteroaryl groups have 5 to 14 ring-forming atoms, including 1 to 13 carbon atoms and 1 to 8 heteroatoms selected from O, S, and N.
  • a heteroaryl group has 5 to 10 ring-forming atoms, including one to four heteroatoms.
  • a heteroaryl group has 5 to 8 ring-forming atoms, including one, two, or three heteroatoms.
  • a 5-membered heteroaryl group is a monocyclic heteroaryl group as defined above, which has 5 ring-forming atoms in the monocyclic heteroaryl ring;
  • a 6-membered heteroaryl group is a monocyclic heteroaryl group as defined above Group, having 6 ring-forming atoms in a monocyclic heteroaryl ring;
  • 5- to 10-membered heteroaryl is a monocyclic or bicyclic heteroaryl group as defined above, which has a monocyclic or bicyclic heteroaryl ring 5, 6, 7, 8, 9 or 10 ring-forming atoms.
  • R 1 is selected from halogen or C 1 -C 6 alkyl
  • R 2 is selected from -CR 4 R 5 , -NR 4 R 5 , 5-6 membered heteroaryl, wherein the heteroaryl is optionally substituted by one or more R 6 , and R 6 is optionally C 1 -C 6 alkyl substituted with hydroxy or cyano;
  • R 3 is selected from hydrogen or halogen
  • R 4 and R 5 are each independently selected from C 1 -C 6 alkyl, C 1 -C 6 alkoxy or 4-6 membered heterocyclic group;
  • U, V and L are each independently selected from C or N, and at least one is N;
  • R 7 is selected from hydrogen or C 1 -C 6 alkyl
  • n 0 or 1;
  • R 1 is selected from chloro or methyl
  • R 2 is selected from the following substituents:
  • R 3 is selected from hydrogen or chlorine
  • U, V and L are each independently selected from C or N, and at least one is N;
  • R 7 is selected from hydrogen or methyl
  • n 0 or 1;
  • the compound is any of the following compounds:
  • this application provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned compound, or a pharmaceutically acceptable salt, ester, prodrug, complex, solvate, hydrate or isomer thereof; And pharmaceutically acceptable carriers or excipients.
  • this application provides the aforementioned compounds, or pharmaceutically acceptable salts, esters, prodrugs, complexes, solvates, hydrates or isomers thereof, for the preparation of drugs for the treatment of EZH2-mediated diseases the use of.
  • the EZH2-mediated disease may be cancer, and the cancer is lung cancer, gastric cancer, liver cancer, breast cancer, nasopharyngeal cancer, pancreatic cancer, ovarian cancer, cervical cancer, colorectal cancer, glioma, melanoma, Prostate cancer, kidney cancer, esophageal cancer, mesothelioma, head and neck cancer, bladder cancer, salivary gland cancer, leukemia or lymphoma, especially anaplastic large cell lymphoma or non-Hodgkin lymphoma.
  • R 8 is halogen
  • R 9 is a hydroxyl protecting group.
  • the hydroxy protecting group can be a silicon protecting group, such as trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl; benzyl, such as benzyl Group, p-methoxybenzyl; alkoxymethyl or propenyl.
  • R 8 is bromo and R 9 is benzyl.
  • the compound of formula (II) or (III) can be used to prepare the compound of formula (I).
  • Compound 5 uses the similar synthetic procedure of compound 1 in Example 1 to 1,4-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane- 2-yl)-1H-pyrazole was prepared as starting material.
  • Compound 6 uses the similar synthetic procedure of compound 5 in Example 5 to 9-bromo-7,10-dichloro-2-((4-methoxy-6-methyl-2-oxo-1,2- Dihydropyridin-3-yl)methyl)-3,4-dihydro-[1,4]diazoheptino[6,7,1-HI]indole-1(2H)-one Manufactured from starting materials.
  • Example 7 10-Chloro-9-(1-(2-hydroxyethyl)-3,5-dimethyl-1H-pyrazol-4-yl)-2-((4-methoxy-6- Methyl-2-oxo-1,2-)dihydropyridin-3-yl)methyl)-3,4-dihydro-[1,4]diazoheptino[6,7,1- HI]Indole-1(2H)-one(7)
  • Compound 7 uses the similar synthetic procedure of compound 1 in Example 1, to 2-(3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxylboron) Pentan-2-yl)-1H-pyrazol-1-yl)ethan-1-ol was prepared as starting material.
  • Compound 8 uses the similar synthetic procedure of compound 1 in Example 1, to 2-(3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxylboron) Pentan-2-yl)-1H-pyrazol-1-yl)acetonitrile was prepared as starting material.
  • Example 9 10-Chloro-9-(methoxy(oxetan-3-yl)methyl)-2-((4-methoxy-6-methyl-2-oxo-1, 2-Dihydropyridin-3-yl)methyl)-3,4dihydro-[1,4]diazoheptino[6,7,1-HI]indole-1(2H)-one( 9)
  • intermediate 12.1 (20.00 g, 75.61 mmol, 1.0 eq.) was dissolved in AcOH (100 mL), NIS (20.41 g, 90.73 mmol, 1.2 eq.) was added, and the mixture was stirred at ambient temperature for 16 hours. The reaction mixture was poured into cold water, and the precipitate was collected by filtration. The obtained solid was dissolved in DCM, dried over MgSO 4 and concentrated to dryness to obtain crude intermediate 12.2 (20.00 g) as a brown solid, which was directly used in the next step without further purification.
  • intermediate 13.2 (800 mg, 2.90 mmol, 1.0 eq) was dissolved in TFA (10 mL), KNO 3 (1.47 g, 14.5 mmol, 5.0 eq.) was added, and the resulting mixture was stirred for 4 hours.
  • the reaction mixture was poured into cold water and extracted with DCM. The organic phase was washed with brine, dried over MgSO 4 and concentrated to dryness to give crude intermediate 13.3 (1.00 g) as a brown solid.
  • Compound 16 uses a similar synthesis method to compound 1 in Example 1, starting from intermediate 1.9 and tert-butyl 5-methyl-1,2,3-oxthiazolidine-3-carboxylate 2,2-dioxide Manufactured from starting materials.
  • 1 H NMR 400MHz, DMSO-d 6 ) 7.59 (s, 1H), 7.40 (s, 1H), 6.58 (d, 1H), 6.34 (d, 1H), 5.47-5.51 (m, 1H), 4.63 ( brs, 1H), 4.36-4.38 (m, 1H), 3.91 (s, 3H), 3.79 (s, 2H), 2.03-2.35 (m, 9H), 1.59 (s, 3H).
  • Compound 17 uses a similar synthesis method to compound 1 in Example 1, starting from intermediate 1.9 and tert-butyl 4-methyl-1,2,3-oxthiazolidine-3-carboxylate 2,2-dioxide Manufactured from starting materials.
  • 1 H NMR 400MHz, DMSO-d 6 ) 11.56 (s, 1H), 7.62 (s, 1H), 7.42 (s, 1H), 6.56 (s, 1H), 6.14 (s, 1H), 4.91-4.94 ( m, 1H), 4.34 (brs, 3H), 4.02 (brs, 1H), 3.77 (s, 3H), 2.02-2.28 (m, 9H), 0.66 (s, 3H).
  • the human recombinant PRC2 complex is composed of human recombinant EZH2, AEBP2, EED, RbAp48 and SUZ12, co-expressed in an insect cell/baculovirus expression system, and purchased from Reaction Biology (Malvern, PA).
  • Biotin-labeled histone H3 peptide (21-44) substrate was purchased from Anaspec (Fremont, CA).
  • the HTRF detection kit including the monoclonal antibody histone H3K27Me1-Eu(K) fluorescent donor and streptavidin-labeled XL665 fluorescent acceptor was from Cisbio (Shanghai, China).
  • S-adenosylmethionine (SAM), Tween 20, dimethyl sulfoxide (DMSO) and Tris buffer were obtained from Sigma at the highest level of purity.
  • the experiment was performed in a buffer composed of 50 mM Tris, pH 9.0, 0.1% BSA and 0.01% Tween20, and 1 mM DTT was added before the experiment.
  • 2.5 ⁇ L of 4% DMSO compound solution and 5 ⁇ L of PRC2/SAM mixed solution pre-incubated for 30 minutes were added to the buffer, and added to a white low-volume 384-well microtiter plate.
  • the mixture solution was incubated at room temperature for 15 minutes with gentle shaking.
  • the methyl transfer reaction was initiated by adding 2.5 ⁇ L of biotin-H3(21-44) peptide substrate solution in the buffer.
  • the final concentrations of PRC2, SAM, biotin-H3 peptide substrate and DMSO were 2ng/ ⁇ L, 30 ⁇ M, 10 ⁇ M and 1%, respectively.
  • the reaction is protected from light and shaken gently at room temperature for 120 minutes, and then 10 ⁇ L of the monoclonal antibody histone H3K27Me1-Eu(K) fluorescent donor and streptavidin-labeled XL665 fluorescent acceptor in the detection buffer from the manufacturer are added to the reaction mixture and incubated 60 minutes.
  • the final concentrations of these two reagents were 50ng/mL and 62.5nM, respectively.
  • the plate was read on the Envision multi-template reader of PerkinElmer (Waltham MA, USA) with an excitation wavelength of 320nm and emission wavelengths of 665 and 615nm.
  • the IC 50 value of the inhibitor was obtained by fitting a sigmoidal dose-response curve (variable slope, four parameters) of the fluorescence intensity at 665 and 615 nm to the inhibitor concentration using Prism 7 (La Jolla, CA).
  • Karpass422 and WSUDLCL-2 cells were purchased from ECACC (European Standard Cell Collection) and DSMZ (German Collection of Microorganisms).
  • RMPI1640 medium, penicillin-streptomycin, heat-inactivated fetal bovine serum (FBS) and horse serum were purchased from ThermoFisher (Waltham MA, USA).
  • Corning 96-well cell culture plates were purchased from ThermoFisher (Waltham MA, USA).
  • the assay plate After removing 100 ⁇ L of the old medium from the cell plate, 100 ⁇ L of the medium with the dissolved test compound is added to the assay plate, which is prepared by adding 1 ⁇ L of the compound DMSO series solution to 199 ⁇ L of cell culture medium, and is aligned with the original preparation. the same. Cell viability was measured using Cell Counting Kit-8 (CCK8) obtained from Beyotime Biotehnology (Shanghai, China). Add 20 ⁇ L of CCK8 reagent to each well of the assay plate, and incubate it at 37°C for 2 hours. The OD at 450 nm was read with a FlexStation 3 microplate reader (Molecular Devices, Sunnyvale, CA 94089 USA). A sigmoid dose response model (variable slope, four parameters) was used in Prism 7 (La Jolla, CA) to determine the concentration (IC 50 value) of the compound that inhibited cell survival by 50%.
  • CCK8 Cell Counting Kit-8
  • Table 2 Enzyme inhibitory activity against wild-type PRC2/EZH2 and cell growth inhibitory activity against Karpas422 and WSUDLCL-2 B cell non-Hodgkin's lymphoma cell lines

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

本申请提供一种由式(I)表示的化合物，或其药学上可接受的盐、酯、前药、络合物、溶剂化物、水合物或异构体；以及其用于制备用于治疗EZH2介导的疾病的药物的用途。

Description

EZH2抑制剂及其用途 技术领域
本申请属于化学医药领域，具体涉及一种EZH2抑制剂及其用途。
背景技术
表观遗传改变在细胞演化过程(包括细胞增生、细胞分化与细胞存活)中扮演着重要角色。通过变更CpG岛甲基化模式、组蛋白修饰与DNA结合蛋白的异常调控，可诱导发生肿瘤抑制基因的表观遗传沉默与致癌基因的活化。果蝇Pc基因是一组表观遗传效应器。EZH2(zeste同系物2的增强子)是果蝇Pc基因阻抑物复合物2(PRC2)的催化组分，果蝇Pc基因阻抑物复合物2是一种通过使组蛋白H3上的27位赖氨酸(H3K27)甲基化来抑制基因转录的保守多亚基复合物。EZH2在调节基因表达模式(其调节决定细胞命运，例如分化与自我更新)中扮演着关键角色。某些癌细胞中过度表达EZH2，和细胞增生、细胞侵袭、化学抗性与转移有关联。
EZH2表达的上调已在很多实体瘤(包括前列腺癌、乳腺癌、皮肤癌、膀胱癌、肝癌、胰腺癌、头颈癌)中观察到，并和癌症侵袭、转移等相关。在弥散性大B细胞淋巴瘤(DLBCL)与滤泡性淋巴瘤(FL)中已鉴定出EZH2的再发性体细胞突变。
目前在开发的EZH2抑制剂均仍处于临床阶段，如EPZ6438(Tazemetostat，临床Ⅱ期)、CPI-1205(临床Ⅰ/Ⅱ期)、PF-6821497(临床Ⅰ期)、SHR-2554(临床Ⅰ/Ⅱ期)等，暂无成功上市的EZH2抑制剂，因此具有新颖化合物结构和良好活性的EZH2先导化合物仍亟需进行研究开发。
发明内容
本申请公开了一类可作为EZH2抑制剂的化合物以及其在制备用于治疗EZH2介导的疾病的药物的用途。
一方面，本申请提供了如式(I)表示的化合物，或其药学上可接受的盐、酯、前药、络合物、溶剂化物、水合物或异构体：
Figure PCTCN2020088861-appb-000001
其中，R 1选自氢、卤素、C 1-C 6烷基、羟基、氰基或氨基；
R 2选自-CR 4R 5、-NR 4R 5、4-15元杂环基、C 6-C 10芳基、5-14元杂芳基，其中所述杂环基、芳基或杂芳基任选地被一个或多个R 6所取代，R 6选自卤素、羟基、氰基、氨基或可选地被羟基或氰基取代的C 1-C 6烷基；
R 3选自氢、卤素、C 1-C 6烷基、羟基、氰基或氨基；
R 4和R 5各自独立地选自C 1-C 6烷基、C 1-C 6烷氧基或4-15元杂环基；
U、V和L分别独立地选自C或N，且至少一个为N；
X和Y各自独立地选自C 1-C 6烷基、C 1-C 6氟代烷基、C 1-C 6烷氧基或C 1-C 6氟代烷氧基；
R 7选自氢、卤素、C 1-C 6烷基、羟基、氰基或氨基；
n＝0、1或2；并且
Figure PCTCN2020088861-appb-000002
代表单键或双键。
在一些实施方案中，式(I)是式(Ia)：
Figure PCTCN2020088861-appb-000003
其中R 1选自氢、卤素、C 1-C 6烷基、羟基、氰基或氨基；
R 2选自-CR 4R 5、-NR 4R 5、4-15元杂环基、C 6-C 10芳基、5-14元杂芳基，其中所述杂环基、芳基或杂芳基任选地被一个或多个R 6所取代，R 6选自卤素、羟基、氰基、氨基或可选地被羟基或氰基取代的C 1-C 6烷基；
R 3选自氢、卤素、C 1-C 6烷基、羟基、氰基或氨基；
R 4和R 5各自独立地选自C 1-C 6烷基、C 1-C 6烷氧基或3-12元杂环基；
U、V和L分别独立地选自C或N，且至少一个为N；
R 7选自氢、卤素、C 1-C 6烷基、羟基、氰基或氨基；
n＝0、1或2；并且
Figure PCTCN2020088861-appb-000004
代表单键或双键。
所述杂环基即单环或多环(包括2个或更多个稠合在一起的环，包括螺、稠合或桥连系统，例如双环系统)、饱和或不饱和的非芳族4至15元环系统，包含1至14个成环碳原子和1至10个成环杂原子，所述成环杂原子彼此独立地选自O、S、N、P和B。杂环烷基基团还可任选地含有一个或多个氧代(即，＝O)或硫羰(即，＝S)基团。例如，4至12元杂环烷基是单环或多环、饱和或不饱和的非芳族4至12元环系统，其包含一个或多个成环杂原子。
所述芳基即具有共轭π电子系统的全碳单环或稠环多环芳族基团。芳基基团在一个或多 个环中可具有6至10个碳原子。最常见的是，芳基基团在环中具有6个碳原子。例如，C6-10芳基是含有6至10个碳原子的芳族基团，例如苯基或萘基。
所述杂芳基，即在至少一个环中具有一个或多个杂原子环成员(成环原子)的单环或稠环多环芳族杂环基团，所述杂原子环成员彼此独立地选自O、S和N。杂芳基基团具有5至14个成环原子，包括1至13个碳原子和1至8个选自O、S和N的杂原子。在一些实施方案中，杂芳基基团具有5至10个成环原子，包括一至四个杂原子。杂芳基基团还可含有一至三个氧代或硫羰(即，＝S)基团。在一些实施方案中，杂芳基基团具有5至8个成环原子，包括一个、两个或三个杂原子。例如，5元杂芳基基团是如上定义的单环杂芳基基团，在单环杂芳基环中具有5个成环原子；6元杂芳基是如上定义的单环杂芳基基团，在单环杂芳基环中具有6个成环原子；5～10元杂芳基是如上定义的单环或双环杂芳基基团，在单环或双环杂芳基环中具有5、6、7、8、9或10个成环原子。
在另一些实施方案中，R 1选自卤素或C 1-C 6烷基；
R 2选自-CR 4R 5、-NR 4R 5、5-6元杂芳基，其中所述杂芳基任选地被一个或多个R 6所取代，R 6为可选地被羟基或氰基取代的C 1-C 6烷基；
R 3选自氢或卤素；
R 4和R 5各自独立地选自C 1-C 6烷基、C 1-C 6烷氧基或4-6元杂环基；
U、V和L分别独立地选自C或N，且至少一个为N；
R 7选自氢或C 1-C 6烷基；
n＝0或1；并且
Figure PCTCN2020088861-appb-000005
代表单键或双键。
在另一些实施方案中，R 1选自氯或甲基；
R 2选自下列取代基：
Figure PCTCN2020088861-appb-000006
R 3选自氢或氯；
U、V和L分别独立地选自C或N，且至少一个为N；
R 7选自氢或甲基；
n＝0或1；并且
Figure PCTCN2020088861-appb-000007
代表单键或双键。
在另一些实施方案中，所述化合物是以下化合物中的任意一种：
10-氯-9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(1)
Figure PCTCN2020088861-appb-000008
10-氯-9-(乙基(氧杂环丁烷-3-基)氨基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(2)
Figure PCTCN2020088861-appb-000009
10-氯-9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4,6,7-四氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(3)
Figure PCTCN2020088861-appb-000010
10-氯-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-9-(1,3,5-三甲基-1H-吡唑-4-基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(4)
Figure PCTCN2020088861-appb-000011
10-氯-9-(1,4-二甲基-1H-吡唑-5-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(5)
Figure PCTCN2020088861-appb-000012
7,10-二氯-9-(1,4-二甲基-1H-吡唑-5-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(6)
Figure PCTCN2020088861-appb-000013
10-氯-9-(1-(2-羟乙基)-3,5-二甲基-1H-吡唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-)二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(7)
Figure PCTCN2020088861-appb-000014
2-(4-(10-氯-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-1-氧代-1,2,3,4-四氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-9-基)-3,5-二甲基-1H-吡唑-1-基)乙腈(8)
Figure PCTCN2020088861-appb-000015
10-氯-9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(9)
Figure PCTCN2020088861-appb-000016
(R)-10-氯-9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(10)
Figure PCTCN2020088861-appb-000017
(S)-10-氯-9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(11)
Figure PCTCN2020088861-appb-000018
5-氯-4-(3,5-二甲基异噁唑-4-基)-7-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-8,9二氢-[1,4]重氮基庚英并[6,7,1-HI]吲唑-6(7H)-酮(12)
Figure PCTCN2020088861-appb-000019
5-氯-4-(3,5-二甲基异噁唑-4-基)-7-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-8,9二氢-2,7,9a-三氮杂苯并[cd]薁-6(7H)-酮(13)
Figure PCTCN2020088861-appb-000020
9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-10-甲基-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(14)
Figure PCTCN2020088861-appb-000021
9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-10-甲基-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(15)
Figure PCTCN2020088861-appb-000022
10-氯-9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-4-甲基-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(16)
Figure PCTCN2020088861-appb-000023
10-氯-9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3-甲基-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(17)
Figure PCTCN2020088861-appb-000024
另一方面，本申请提供一种药物组合物，其包含治疗有效量的前述化合物，或其药学上可接受的盐、酯、前药、络合物、溶剂化物、水合物或异构体；以及药学上可接受的载体或赋形剂。
另一方面，本申请提供前述化合物，或其药学上可接受的盐、酯、前药、络合物、溶剂化物、水合物或异构体用于制备用于治疗EZH2介导的疾病的药物的用途。
所述的EZH2介导的疾病可以为癌症，所述的癌症为肺癌、胃癌、肝癌、乳腺癌、鼻咽癌、胰腺癌、卵巢癌、宫颈癌、结肠直肠癌、胶质瘤、黑色素瘤、前列腺癌、肾癌、食道癌、间皮瘤、头颈癌、膀胱癌、唾腺癌、白血病或淋巴瘤，特别是间变性大细胞淋巴瘤或非霍奇金淋巴瘤。
另一方面，本申请提供一种由式(Ⅱ)或式(Ⅲ)表示的化合物：
Figure PCTCN2020088861-appb-000025
其中，R 8为卤素，R 9为羟基保护基。所述羟基保护基可以为硅类保护基，如三甲基硅基、叔丁基二甲基硅基、叔丁基二苯基硅基、三异丙基硅基；苄基类，如苄基、对甲氧基苄基；烷氧基甲基类或丙烯基。
在一些实施方案中，R 8为溴，R 9为苄基。
所述式(Ⅱ)或式(Ⅲ)化合物可用于制备式(Ⅰ)化合物。
具体实施方式
实施例1 10-氯-9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3- 基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(1)
Figure PCTCN2020088861-appb-000026
搅拌下，将4-溴-3-氯-2-甲基苯胺(70.00g,318mmol,1.0eq.)溶于AcOH(500mL)，加入AcONa(36.70g,445mmol,1.4eq.)，然后在冰水浴温度下分批加入NIS(78.75g,349mmol,1.1eq.)，反应混合物在环境温度下搅拌16小时。将反应混合物浓缩至干，并用EtOAc/水稀释。有机相用NaOH的水溶液洗涤，MgSO 4干燥，并浓缩至干。将得到的残余物用PE/EtOAc＝10/1洗涤，得到中间体1.1(60.50g，55％产率)，为灰白色固体。 1H NMR(400MHz,DMSO-d 6)δ(ppm)7.79(s,1H),5.34(s,2H),2.31(s,3H)。
搅拌下，将中间体1.1(60.00g,173mmol,1.0eq.)，CuI(4.94g,26.1mmol,0.15eq.)和Pd(PPh 3) 2Cl 2(18.21g,26.1mmol,0.15eq.)溶于TEA(500mL)，在氮气气氛下加入三甲基乙炔基硅(38.70g,208mmol,1.2eq.)，反应混合物在环境温度下搅拌4小时。将反应物通过硅藻土垫过滤，并用EtOAc洗涤。合并有机相，浓缩至干。通过柱色谱(硅胶，PE/EtOAc＝100/1)纯化残余物，得到中间体1.2(55.00g，100％产率)，为黄色油状物。
搅拌下，将中间体1.2(47.00g,149mmol,1.0eq.)溶于DMF(500mL)，分批加入t-BuOK(35.90g,297mmol,2.0eq.)，反应混合物加热至80℃反应16个小时。浓缩反应混合物，残余物用EtOAc/水稀释。有机相用盐水洗涤，MgSO 4干燥，并浓缩至干。通过柱色谱(硅胶，PE/EtOAc＝20/1)纯化残余物，得到中间体1.4(28.30g，78％产率)，为浅黄色油状物。
搅拌下，将中间体1.4(22.30g,90.2mmol,1.0eq.)溶于THF(200mL)，冰水浴温度下加入Boc 2O(29.50g,135.2mmol,1.5eq.)，然后分批加入DMAP(2.18g,18.1mmol,0.2eq.)，反应混合物在环境温度下搅拌5小时。将反应混合物用EtOAc/水稀释。有机相用盐水洗涤， MgSO 4干燥，并浓缩至干。通过柱色谱(硅胶，PE/EtOAc＝30/1)纯化残余物，得到中间体1.5(32.05g，95％产率)，为白色固体。
搅拌下，将中间体1.5(6.00g,17.4mmol,1.0eq.)溶于CCl 4(100mL)，加入(PhCO) 2O(0.42g,1.74mmol,0.1eq.)，然后加入NBS(3.25g,18.3mmol,1.05eq.)，反应混合物加热至85℃反应3小时。通过柱色谱(硅胶，PE/EtOAc＝100/1)纯化残余物，得到中间体1.6(6.00g，82％产率)，为白色固体。
搅拌下，将中间体1.6(6.00g,14.2mmol,1.0eq.)溶于MeCN(60mL)，加入N-甲基-N-氧化吗啉(3.34g,28.4mmol,2.0eq.)的水溶液，反应混合物在环境温度下搅拌16个小时。将反应混合物用EtOAc/水稀释。有机相用盐水洗涤，MgSO 4干燥，并浓缩至干。通过柱色谱(硅胶，PE/EtOAc＝50/1)纯化残余物，得到中间体1.7(3.50g，69％产率)，为白色固体。 1H NMR(400MHz,DMSO-d 6)δ(ppm)10.39(s,1H),8.27(s,1H),7.80(d,J＝3.7Hz,1H),6.80(d,J＝3.7Hz,1H),1.57(s,9H)。
搅拌下，将中间体1.7(3.00g,11.6mmol,1.0eq.)溶于水(3mL)和1,4-二氧六环(30mL)，加入NaH 2PO 4(2.19g,13.9mmol,1.2eq.)，2-甲基-2-丁烯(1.65g,23.2mmol,2.0eq.)，然后在冰水浴温度下分批加入NaClO 2(1.16g,11.6mmol,1.0eq.)，反应混合物在环境温度下搅拌3个小时。将反应混合物用EtOAc/水稀释。有机相用盐水洗涤，MgSO 4干燥，并浓缩至干，得到中间体1.8(3.00g，96％产率)，为白色固体。
搅拌下，将中间体1.8(3.00g,8.02mmol,1.0eq.)溶于甲醇(30mL)，滴加浓硫酸(5mL)，反应混合物加热至60℃反应16小时。反应混合物用EtOAc/水稀释。有机相用盐水洗涤，MgSO 4干燥，并浓缩至干。通过柱色谱(硅胶，PE/EtOAc＝10/1)纯化残余物，得到中间体1.9(0.30g，13％产率)，为白色固体。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.49(brs,1H),8.17(s,1H),7.52-7.53(m,1H),6.55-6.56(m,1H),3.97(s,3H)。
搅拌下，将中间体1.9(300mg,1.46mmol,1.0eq.)溶于DMF(5mL)，在冰水浴温度下加入NaH(60％分散于油中,487mg,2.19mmol,1.5eq.)，再加入叔丁基-2,2-二氧代1,2λ 6,3-氧杂噻唑-3-羧酸甲酯(2.0eq.)反应混合物在环境温度下搅拌1小时。将反应混合物用EtOAc/水稀释。有机相用盐水洗涤，经MgSO 4干燥，并浓缩至干，得到粗制中间体1.10(>100％产率)，其不经进一步纯化直接用于下一步骤。
将中间体1.10(粗品)溶于HCl/1,4-二氧六环溶液(5mL)中，在环境温度下搅拌30分钟。将反应混合物浓缩至干，得到粗制中间体1.11(540mg，>100％产率)，其不经进一步纯化直接用于下一步骤。
将中间体1.11(540mg，粗品)和K 2CO 3(450mg，3.26mmol，2.0eq.)溶于甲醇(5mL)中加热至回流5小时。反应混合物浓缩至干，并通过柱色谱(硅胶，PE/EtOAc＝10/1)纯化残余物，得到中间体1.12(300mg，61％产率)，为白色固体。 1H NMR(400MHz,DMSO-d 6)δ(ppm)8.54(t,J＝6.8Hz,1H),8.10(s,1H),7.49(d,J＝3.2Hz,1H),6.54(d,J＝3.2Hz,1H),4.29-4.31(m,2H),3.52-3.56(m,2H)。
搅拌下，将中间体1.12(300mg,1.0mmol,1.0eq.)溶于DMF(5mL)，加入t-BuOK(170mg,1.5mmol,1.5eq.)，所得混合物再搅拌10分钟。加入2-(苄氧基)-3-(氯甲基)-4-甲氧基-6-甲基吡啶(305mg,1.1mmol,1.1eq.)，所得混合物再搅拌3小时。将反应混合物用EtOAc/水稀释。有机相用盐水洗涤，MgSO 4干燥，并浓缩至干。通过柱色谱(硅胶，PE/EtOAc＝10/1)纯化残余物，得到中间体1.13(220mg，41％产率)，为黄色固体。
搅拌下，将中间体1.13(200mg,0.37mmol,1.0eq.)和3,5-二甲基异噁唑-4-硼酸频哪醇酯(126mg,0.56mmol,1.5eq.)溶于1,4-二氧六环(3mL)，加入K 3PO 4(236mg,1.11mmol,3.0eq.)和催化量的Pd(dppf)Cl 2，反应混合物加热至90℃反应1小时。将反应混合物浓缩至干，并通过柱色谱(硅胶，PE/EtOAc＝5/1)纯化残余物，得到中间体1.14(103mg，50％产率)，为白色固体。
搅拌下，将中间体1.14(103mg,0.18mmol,1.0eq.)溶于DCM(5mL)，加入TFA(1mL)，反应混合物在环境温度下反应3小时。将反应混合物浓缩至干，并用DCM/饱和NaHCO 3稀释。将有机相用盐水洗涤，MgSO 4干燥，并浓缩至干。通过柱色谱(硅胶，DCM/MeOH＝30/1)纯化残余物，得到化合物1(29mg，34％产率)，为白色固体。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.58(br,1H),7.64(s,1H),7.46(d,J＝3.2Hz,1H),6.55(d,J＝3.2Hz,1H),6.16(s,1H),4.62(br,2H),4.19(br,2H),3.81(s,3H),3.65(m,2H),2.23-2.24(m,6H),2.06(s,3H)。
实施例2 10-氯-9-(乙基(氧杂环丁烷-3-基)氨基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(2)
Figure PCTCN2020088861-appb-000027
搅拌下，将中间体1.13(300mg,0.55mmol,1.0eq.)和氧杂环丁烷-3-胺(83mg,1.11mmol,2.0eq.)溶于1,4-二氧六环(5mL)，加入催化量的Pd(dba) 2和XPhos，然后再加入固态的Cs 2CO 3(542mg,1.67mmol,3.0eq.)，混合物在氮气气氛下搅拌回流2小时。将反应混合物用水稀释，并用EtOAc萃取。将有机相用盐水洗涤，MgSO 4干燥，并浓缩至干。通过柱色谱(硅胶，PE/EtOAc＝3/1)纯化残余物，得到中间体2.1(200mg，68％产率)，为黄色固体。
搅拌下，将中间体2.1(200mg,0.38mmol,1.0eq.)溶于DMF(5mL)，在冰水浴温度下分批加入NaH(60％,18mg,0.75mmol,2.0eq.)，搅拌20分钟。加入EtI(117mg,0.75mmol,2.0eq.)，反应混合物在环境温度下搅拌16小时。将反应混合物用水稀释，并用EtOAc萃取。将有机相用盐水洗涤，MgSO 4干燥，并浓缩至干，得到粗制中间体2.2(250mg，100％产率)，为黄色油状物，其不经进一步纯化直接用于下一步骤。
化合物2使用实施例1中化合物1相似的合成步骤，以中间体2.2作为起始原料制得。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.56(br,1H),7.38(s,1H),7.35(d,J＝3.2Hz,1H),6.43(d, J＝3.1Hz,1H),6.16(s,1H),4.56(brs,5H),4.32(brs,2H),4.12(brs,2H),3.82(s,3H),3.59(m,2H),3.01(q,J＝6.6Hz,2H),2.22(s,3H),0.88(t,J＝6.6Hz,3H)。
实施例3 10-氯-9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4,6,7-四氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(3)
Figure PCTCN2020088861-appb-000028
搅拌下，将中间体1.12(500mg,1.67mmol,1.0eq.)溶于AcOH(5mL)，分批加入固态NaBH 3CN(316mg,5.02mmol,3.0eq.)，反应混合物在环境温度下反应16小时。将反应混合物用饱和Na 2CO 3处理直至达到pH＝8。将混合物用EtOAc萃取，经MgSO 4干燥，并浓缩至干，得到粗制中间体3.1(350mg，70％产率)，为黄色固体，其不经进一步纯化直接用于下一步骤。
化合物3使用实施例1中化合物1相似的合成步骤，以中间体3.1作为起始原料制得。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.51(s,1H),6.99(s,1H),6.13(s,1H),4.49(s,1H),3.79(s,3H),3.51(t,J＝8.7Hz,2H),3.39-3.40(m,2H),3.24-3.25(m,2H),2.91-2.99(m,2H),2.22(s,3H),2.21(s,3H),2.05(s,3H)。
实施例4 10-氯-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-9-(1,3,5-三甲基-1H-吡唑-4-基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(4)
Figure PCTCN2020088861-appb-000029
化合物4使用实施例1中化合物1相似的合成步骤，以1,3,5-三甲基-4-(4,4,5,5-四甲基-1,3,2-二氧硼戊烷-2-基)-1H-吡唑作为起始原料制得。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.57(s,1H),7.48(s,1H),7.40(d,J＝3.2Hz,1H),6.50(d,J＝3.2Hz,1H),6.16(s,1H),4.61(brs,2H),4.18(brs,2H),3.81(s,3H),3.72(s,3H),3.64(s,2H),2.23(s,3H),2.04(s,3H),1.96(s,3H)。
实施例5 10-氯-9-(1,4-二甲基-1H-吡唑-5-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(5)
Figure PCTCN2020088861-appb-000030
化合物5使用实施例1中化合物1相似的合成步骤，以1,4-二甲基-5-(4,4,5,5-四甲基-1,3,2-二氧硼戊烷-2-基)-1H-吡唑作为起始原料制得。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.59(s,1H),7.63(s,1H),7.48(d,J＝3.1Hz,1H),7.33(s,1H),6.58(d,J＝3.1Hz,1H),6.16(s,1H),4.63(brs,2H),4.21(brs,2H),3.81(s,3H),3.67(s,2H),3.53(s,3H),2.23(s,3H),1.84(s,3H)。
实施例6 7,10-二氯-9-(1,4-二甲基-1H-吡唑-5-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(6)
Figure PCTCN2020088861-appb-000031
化合物6使用实施例5中化合物5相似的合成步骤，以9-溴-7,10-二氯-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮作为起始原料制得。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.58(s,1H),7.74(s,1H),7.53(s,1H),7.35(s,1H),6.16(s,1H),4.62(brs,2H),4.19(brs,2H),3.82(s,3H),3.68(s,2H),3.53(s,3H),2.23(s,3H),1.84(s,3H)。
实施例7 10-氯-9-(1-(2-羟乙基)-3,5-二甲基-1H-吡唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-)二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(7)
Figure PCTCN2020088861-appb-000032
化合物7使用实施例1中化合物1相似的合成步骤，以2-(3,5-二甲基-4-(4,4,5,5-四甲基-1,3,2-二氧硼戊烷-2-基)-1H-吡唑-1-基)乙-1-醇作为起始原料制得。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.58(s,1H),7.48(s,1H),7.41(s,1H),6.51(s,1H),6.16(s,1H),4.88(m,1H),4.62(m,2H),4.17(m,2H),4.05-4.10(m,2H),3.81(s,3H),3.74-3.75(m,2H),3.64(s,2H),2.23(s,3H),2.07(s,3H),1.98(s,3H)。
实施例8 2-(4-(10-氯-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-1-氧代-1,2,3,4-四氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-9-基)-3,5-二甲基-1H-吡唑-1-基)乙腈(8)
Figure PCTCN2020088861-appb-000033
化合物8使用实施例1中化合物1相似的合成步骤，以2-(3,5-二甲基-4-(4,4,5,5-四甲基-1,3,2-二氧硼戊烷-2-基)-1H-吡唑-1-基)乙腈作为起始原料制得。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.57(s,1H),7.54(s,1H),7.43(d,J＝3.2Hz,1H),6.52(d,J＝3.2Hz,1H),6.16(s,1H),5.42(s,2H),4.62(brs,2H),4.18(brs,2H),3.82(s,3H),3.65(s,2H),2.23(s,3H),2.12(s,3H),1.99(s,3H)。
实施例9 10-氯-9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(9)
Figure PCTCN2020088861-appb-000034
搅拌下，将中间体1.13(500mg,0.92mmol,1.0eq.)和B 2(pin) 2(351mg,1.38mmol,1.5eq.)溶于1,4-二氧六环(10mL)，加入AcOK(272mg,2.77mmol,3.0eq.)，再加入催化量的Pd(dppf)Cl 2，反应混合物在氮气气氛下加热回流12小时。将反应混合物用水稀释，并用EtOAc萃取。将有机相用盐水洗涤，MgSO 4干燥，并浓缩至干。通过柱色谱(硅胶，PE/EtOAc＝1/1)纯化获得的残余物，得到中间体9.1(500mg，92％产率)，为粗制白色固体。
搅拌下，将中间体9.1(500mg,0.85mmol,1.0eq.)和氧杂环丁烷-3-甲醛(110mg,1.27mmol,1.5eq.)溶于THF(10mL)，加入固体K 2CO 3(352mg,2.55mmol,3.0eq.)，再加入催化量的PdCl 2和三(1-萘基)膦。反应混合物在氮气气氛下加热回流12小时。将反应混合物用水稀释，并用EtOAc萃取。将有机相用盐水洗涤，MgSO 4干燥，并浓缩至干。通过柱色谱(硅胶，DCM/MeOH＝50/1)纯化残余物，得到中间体9.2(150mg，32％产率)，为黄色固体。
搅拌下，将中间体9.2(150mg,0.27mmol,1.0eq.)和MeI(59mg,0.41mmol,1.5eq.)溶于THF(5mL)，在冰水浴温度下加入固态t-BuOK(46mg,0.41mmol,1.5eq.)，将反应混 合物再搅拌1小时。将反应混合物用水稀释，并用EtOAc萃取。将有机相用盐水洗涤，MgSO 4干燥，并浓缩至干，得到粗制中间体9.3(150mg，98％产率)，为黄色油状物，其不经进一步纯化直接用于下一步骤。
搅拌下，将中间体9.3(20mg,0.04mmol,1.0eq.)溶于THF(5mL)，加入催化量的Pd(OH) 2，反应混合物在氢气气氛和环境温度下搅拌12小时。过滤反应混合物以除去催化剂，并将滤液浓缩至干。通过柱色谱(硅胶，DCM/MeOH＝10/1)纯化残余物，得到化合物9(10mg，62％产率)，为白色固体。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.56(s,1H),7.68(s,1H),7.40(d,J＝3.1Hz,1H),6.54(d,J＝3.2Hz,1H),6.16(s,1H),5.08(d,J＝6.6Hz,1H),4.43-4.64(m,6H),4.14(brs,2H),3.81(s,3H),3.60(s,2H),3.32-3.38(m,1H),3.21(s,3H),2.22(s,3H)。
实施例10 (R)-10-氯-9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(10)和(S)-10-氯-9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(11)
Figure PCTCN2020088861-appb-000035
通过化合物9的手性分离制备化合物10和化合物11。
色谱柱：CHIRALCEL OZ-H(OZH0CD-VC005)
柱规格：0.46cmI.D.*15cmL
进样量：10.0uL
流动相：MeOH＝100％
流速：1.0mL/min
波长：UV 214nm
HPLC设备：ShimadzuLC-20AD
1H NMR(400MHz,DMSO-d 6)δ(ppm)11.56(s,1H),7.68(s,1H),7.40(d,J＝3.1Hz,1H),6.54(d,J＝3.2Hz,1H),6.16(s,1H),5.08(d,J＝6.6Hz,1H),4.43-4.64(m,6H),4.14(brs,2H),3.81(s,3H),3.60(s,2H),3.32-3.38(m,1H),3.21(s,3H),2.22(s,3H)；e.e.>98％；化合物10：保留时间11.56分钟，[α]＝76.13°(MeOH,c＝0.790g/100mL)；化合物11：保留时间8.86分钟，[α]＝-86.31°(MeOH,c＝0.723g/100mL)。
实施例11 5-氯-4-(3,5-二甲基异噁唑-4-基)-7-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-8,9二氢-[1,4]重氮基庚英并[6,7,1-HI]吲唑-6(7H)-酮(12)
Figure PCTCN2020088861-appb-000036
搅拌下，将2-氨基-6-氯苯甲酸甲酯(20.00g,110mmol,1.0eq.)溶于DMF(150mL)，在冰水浴温度下分批加入NBS(23.22g,130mmol,1.2eq.)，反应混合物在环境温度下搅拌3小时。将反应混合物倒入水中，并用EtOAc萃取。将有机相用盐水洗涤，MgSO 4干燥，并浓缩至干。通过柱色谱(硅胶，PE/EtOAc＝9/1)纯化残余物，得到中间体12.1(25.00g)，为棕色油状物。
搅拌下，将中间体12.1(20.00g,75.61mmol,1.0eq.)溶于AcOH(100mL)，加入NIS(20.41g,90.73mmol,1.2eq.)，并在环境温度下搅拌16小时。将反应混合物倒入冷水中，过滤收集沉淀物。将获得的固体溶解在DCM中，经MgSO 4干燥，并浓缩至干，得到粗制中间体12.2(20.00g)，为棕色固体，其不经进一步纯化直接用于下一步骤。
搅拌下，将中间体12.2(100mg,0.34mmol,1.0eq.)，MeB(OH) 2(103mg,0.41mmol,1.2eq.)和K 3PO 4(219mg,1.03mmol,3.0eq.)溶于1,4-二氧六环(5mL)和水(1mL)的混合溶剂，加入催化量的Pd(dppf)Cl 2，反应混合物在氮气气氛下加热回流4小时。将反应混合物用水稀释，并用EtOAc萃取。将有机相用盐水洗涤，MgSO 4干燥，并浓缩至干。通过柱色谱(硅胶，PE/EtOAc＝10/1)纯化残余物，得到中间体12.3(100mg)。
搅拌下，将中间体12.3(100mg,0.36mmol,1.0eq.)溶于氯仿(5mL)，加入AcOK(11mg,0.11mmol,0.3eq.)和Ac 2O(84mg,0.83mmol,2.3eq.)，反应混合物加热至回流。加入亚硝酸异戊酯(92mg,0.79mmol,2.2eq.)，搅拌反应混合物直至反应完成。将反应混合物倒入冷水中，并用DCM萃取。将有机相用盐水洗涤，经MgSO 4干燥，并浓缩至干，得到粗制中间体12.4(100mg)，其不经进一步纯化直接用于下一步骤。
化合物12使用实施例1中化合物1相似的合成方法制得。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.58(brs,1H),8.22(s,1H),7.93(s,1H),6.17(s,1H),4.64(s,2H),4.39(s,2H),3.73-3.81(m,5H),2.23-2.26(m,6H),2.06(s,3H)。
实施例12 5-氯-4-(3,5-二甲基异噁唑-4-基)-7-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-8,9二氢-2,7,9a-三氮杂苯并[cd]薁-6(7H)-酮(13)
Figure PCTCN2020088861-appb-000037
搅拌下，将中间体12.1(5.00g,18.9mmol,1.0eq.)和(2-氧代乙基)氨基甲酸叔丁酯(4.65g,28.36mmol,1.5eq.)溶于DCE(25mL)，加入NaBH(OAc) 3(12.01g,56.71mmol,3.0eq.)，反应混合物在环境温度下搅拌16小时。将反应混合物倒入冷水中，并用DCM萃取。将有机相用盐水洗涤，MgSO 4干燥，并浓缩至干。通过柱色谱(硅胶，PE/EtOAc＝6/1)纯化残余物，得到中间体13.1(6.60g)，为黄色油状物。
搅拌下，将中间体13.1溶于DCM(10mL)，加入TFA(10mL)，反应混合物在环境温度下搅拌2小时。将反应混合物浓缩至干。将残余物溶于MeOH(100mL)中，并加入固体K 2CO 3。反应混合物加热至回流36小时。将反应混合物浓缩至干，并将所得残余物用水处理。通过过滤收集沉淀物，得到粗制中间体13.2(100％产率)，将其直接用于下一步骤。
搅拌下，将中间体13.2(800mg,2.90mmol,1.0eq)溶于TFA(10mL)，加入KNO 3(1.47g,14.5mmol,5.0eq.)，所得混合物搅拌4小时。将反应混合物倒入冷水中，并用DCM萃取。将有机相用盐水洗涤，经MgSO 4干燥，并浓缩至干，得到粗制中间体13.3(1.00g)，为棕色固体。
搅拌下，将中间体13.3(粗品1g)溶于DMF(10mL)和水(5mL)的混合溶剂，加入连二亚硫酸钠(5.43g,43.4mmol)，反应混合物加热至70℃反应3小时。将反应混合物倒入冷水中，并用EtOAc萃取。将有机相用盐水洗涤，经MgSO 4干燥，并浓缩至干，得到粗制中间体13.4(1.80g)，为棕色固体。
搅拌下，将中间体13.4(粗品1.80g)加入原甲酸三甲酯中，加热至回流过夜。将反应混合物浓缩至干，得到粗制中间体13.5(产率>100％)，将其不经进一步纯化直接用于下一步骤。
化合物13使用实施例1中化合物1相似的合成方法制得。 1H NMR(400MHz,DMSO-d 6)δ(ppm)12.11(brs,1H),7.93(s,1H),7.71(s,1H),5.99(s,1H),4.87-5.00(m,2H),4.23-4.33(m,2H),4.06-4.10(m,1H),3.92(s,3H),3.74-3.79(m,1H),2.14-2.38(m,9H)。
实施例13 9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-10-甲基-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(14)
Figure PCTCN2020088861-appb-000038
化合物14使用实施例1中化合物1相似的合成方法，以2-氨基-6-甲基-苯甲酸甲酯为起始原料制得。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.57(brs,1H),7.56(s,1H),7.26(d,J＝3.0Hz,1H),6.44(d,J＝3.0Hz,1H),6.16(s,1H),4.32-4.87(m,7H),4.13(brs,2H),3.81(s,3H),3.56(brs,2H),3.38-3.43(m,1H),3.15(s,3H),2.52(s,3H),2.25(s,3H)。
实施例14 9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-10-甲基-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(15)
Figure PCTCN2020088861-appb-000039
化合物15使用实施例14中化合物13相似的合成方法制得。 1H NMR(400MHz,DMSO-d 6)δ(ppm)11.57(s,1H),7.43(s,1H),7.33(d,J＝3.2Hz,1H),6.46(d,J＝3.2Hz,1H),6.16(s,1H),4.64(brs,2H),4.18(brs,2H),3.81(s,3H),3.62(brs,2H),2.27(s,3H),2.22(s,3H),2.20(s,3H),2.00(s,3H)。
实施例15 10-氯-9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-4-甲基-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(16)
Figure PCTCN2020088861-appb-000040
化合物16使用实施例1中化合物1相似的合成方法，由中间体1.9和5-甲基-1,2,3-噁噻 唑烷-3-羧酸叔丁酯2,2-二氧化物作为起始原料制得。 1H NMR(400MHz,DMSO-d 6)7.59(s,1H),7.40(s,1H),6.58(d,1H),6.34(d,1H),5.47-5.51(m,1H),4.63(brs,1H),4.36-4.38(m,1H),3.91(s,3H),3.79(s,2H),2.03-2.35(m,9H),1.59(s,3H)。
实施例16 10-氯-9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3-甲基-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(17)
Figure PCTCN2020088861-appb-000041
化合物17使用实施例1中化合物1相似的合成方法，由中间体1.9和4-甲基-1,2,3-噁噻唑烷-3-羧酸叔丁酯2,2-二氧化物作为起始原料制得。 1H NMR(400MHz,DMSO-d 6)11.56(s,1H),7.62(s,1H),7.42(s,1H),6.56(s,1H),6.14(s,1H),4.91-4.94(m,1H),4.34(brs,3H),4.02(brs,1H),3.77(s,3H),2.02-2.28(m,9H),0.66(s,3H)。
本文描述的代表性化合物的结构及表征显示在表1中。
表1：代表化合物的结构、 1HNMR和分子量数据
Figure PCTCN2020088861-appb-000042
Figure PCTCN2020088861-appb-000043
Figure PCTCN2020088861-appb-000044
实施例17
PRC2/EZH2生化抑制试验
材料
人重组PRC2复合物由人重组EZH2，AEBP2，EED，RbAp48和SUZ12构成，在昆虫细胞/杆状病毒表达系统中共表达，购自Reaction Biology(Malvern，PA)。生物素标记的组蛋白H3肽(21-44)底物购自Anaspec(Fremont，CA)。包括单抗组蛋白H3K27Me1-Eu(K)荧光供体和链霉亲和素标记的XL665荧光受体的HTRF检测试剂盒来自Cisbio(中国上海)。S-腺苷甲硫氨酸(SAM)，吐温20，二甲基亚砜(DMSO)和Tris缓冲液以最高纯度水平从Sigma获得。
PRC2/EZH2 HTRF酶抑制试验的通用程序
实验在由50mM Tris，pH 9.0，0.1％BSA和0.01％Tween20组成的缓冲液中进行，实验开始前加入1mM DTT。将2.5μL的4％DMSO的化合物溶液和5μL预培养30分钟的PRC2/SAM混合溶液加入缓冲液，将其加入白色低体积384孔微量滴定板中。将该混合物溶液在室温下轻摇培育15分钟。通过在缓冲液中添加2.5μL生物素-H3(21-44)肽底物溶液来引发甲基转移反应。PRC2，SAM，生物素-H3肽底物和DMSO的最终浓度分别为2ng/μL、30μM、10μM和1％。反应避光室温轻摇120分钟，然后将10μL来自制造商的检测缓冲液中的单抗组蛋白H3K27Me1-Eu(K)荧光供体和链霉亲和素标记的XL665荧光受体加入反应混合物培育60分钟。这两种试剂的最终浓度分别为50ng/mL和62.5nM。在PerkinElmer(Waltham MA，USA)的Envision多模板读数器上读取平板，激发波长为320nm，发射波长为665和615nm。抑制剂的IC 50值通过使用Prism 7(La Jolla，CA)拟合665和615nm处的荧光强度比抑制剂浓度的S形剂量-响应曲线(可变斜率，四个参数)获得。
细胞增殖抑制试验
Karpass422和WSUDLCL-2细胞购自ECACC(欧洲标准细胞收藏中心)和DSMZ(德国微生物菌种保藏中心)。RMPI1640培养基，青霉素-链霉素，热灭活的胎牛血清(FBS)和马血清购自ThermoFisher(Waltham MA,USA)。康宁96孔细胞培养板购自ThermoFisher(Waltham MA,USA)。
为了评价合成的化合物对B细胞非霍奇金淋巴瘤Karpass422和WSUDLCL-2细胞增殖的抑制能力，将指数生长的细胞分别接种于含有20％和10％马血清的RMPI1640培养基和1％青霉素-链霉素，浓度分别为2000和3000细胞/ml，96孔板，每孔100μL，并孵育在37℃，5％CO 2培养箱中过夜。将化合物在DMSO中制备为10点、3倍系列稀释液，以6mM开始。将来自化合物储备板的1μL DMSO溶液添加到99μL细胞培养基(测定中化合物的最终最高浓度为30μM，并且DMSO的最终浓度为0.5％)。将培养基中的100μL化合物溶液添加到Karpass422细胞板的每个孔。在添加化合物溶液之后，将测定板在37℃、5％CO 2下孵育12 天。在该孵育期间，每4天更换具有新溶解的测试化合物的细胞培养基。从细胞板中取出100μL旧培养基后，100μL具有溶解的测试化合物的培养基被加入到测定板中，其通过将1μL化合物DMSO系列溶液加入到199μL细胞培养基中制备，且与最初的制备布局相同。使用从Beyotime Biotehnology(上海，中国)获得的细胞计数试剂盒-8(CCK8)测量细胞活力。向测定板的每个孔中加入20μL CCK8试剂，将其在37℃下孵育2小时。用FlexStation 3微板读取器(Molecular Devices,Sunnyvale,CA 94089 USA)读取450nm处的O.D.。在Prism 7(La Jolla,CA)中使用S形剂量反应模型(可变斜率，四个参数)确定化合物使细胞成活率抑制50％的浓度(IC 50值)。
本文描述的代表性化合物的结果显示在表2中。
表2：针对野生型PRC2/EZH2的酶抑制活性和针对Karpas422和WSUDLCL-2 B细胞非霍奇金淋巴瘤细胞系的细胞生长抑制活性
Figure PCTCN2020088861-appb-000045

Claims (14)

  1. 一种由式(I)表示的化合物，或其药学上可接受的盐、酯、前药、络合物、溶剂化物、水合物或异构体：
    Figure PCTCN2020088861-appb-100001
    其中，R 1选自氢、卤素、C 1-C 6烷基、羟基、氰基或氨基；
    R 2选自-CR 4R 5、-NR 4R 5、4-15元杂环基、C 6-C 10芳基、5-14元杂芳基，其中所述杂环基、芳基或杂芳基任选地被一个或多个R 6所取代，R 6选自卤素、羟基、氰基、氨基或可选地被羟基或氰基取代的C 1-C 6烷基；
    R 3选自氢、卤素、C 1-C 6烷基、羟基、氰基或氨基；
    R 4和R 5各自独立地选自C 1-C 6烷基、C 1-C 6烷氧基或4-15元杂环基；
    U、V和L分别独立地选自C或N，且至少一个为N；
    X和Y各自独立地选自C 1-C 6烷基、C 1-C 6氟代烷基、C 1-C 6烷氧基或C 1-C 6氟代烷氧基；
    R 7选自氢、卤素、C 1-C 6烷基、羟基、氰基或氨基；
    n＝0、1或2；并且
    Figure PCTCN2020088861-appb-100002
    代表单键或双键。
  2. 根据权利要求1所述的化合物，其中式(I)是式(Ia)：
    Figure PCTCN2020088861-appb-100003
    其中R 1选自氢、卤素、C 1-C 6烷基、羟基、氰基或氨基；
    R 2选自-CR 4R 5、-NR 4R 5、4-15元杂环基、C 6-C 10芳基、5-14元杂芳基，其中所述杂环基、芳基或杂芳基任选地被一个或多个R 6所取代，R 6选自卤素、羟基、氰基、氨基或可选地被羟基或氰基取代的C 1-C 6烷基；
    R 3选自氢、卤素、C 1-C 6烷基、羟基、氰基或氨基；
    R 4和R 5各自独立地选自C 1-C 6烷基、C 1-C 6烷氧基或4-15元杂环基；
    U、V和L分别独立地选自C或N，且至少一个为N；
    R 7选自氢、卤素、C 1-C 6烷基、羟基、氰基或氨基；
    n＝0、1或2；并且
    Figure PCTCN2020088861-appb-100004
    代表单键或双键。
  3. 根据权利要求1或2所述的化合物，其中：
    R 1选自卤素或C 1-C 6烷基；
    R 2选自-CR 4R 5、-NR 4R 5、5-6元杂芳基，其中所述杂芳基任选地被一个或多个R 6所取代，R 6为可选地被羟基或氰基取代的C 1-C 6烷基；
    R 3选自氢或卤素；
    R 4和R 5各自独立地选自C 1-C 6烷基、C 1-C 6烷氧基或4-6元杂环基；
    U、V和L分别独立地选自C或N，且至少一个为N；
    R 7选自氢或C 1-C 6烷基；
    n＝0或1；并且
    Figure PCTCN2020088861-appb-100005
    代表单键或双键。
  4. 根据权利要求1或2所述的化合物，其中：
    R 1选自氯或甲基；
    R 2选自下列取代基：
    Figure PCTCN2020088861-appb-100006
    R 3选自氢或氯；
    U、V和L分别独立地选自C或N，且至少一个为N；
    R 7选自氢或甲基；
    n＝0或1；并且
    Figure PCTCN2020088861-appb-100007
    代表单键或双键。
  5. 根据权利要求1或2所述的化合物，其是以下化合物中的任意一种：
    10-氯-9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(1)
    Figure PCTCN2020088861-appb-100008
    10-氯-9-(乙基(氧杂环丁烷-3-基)氨基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(2)
    Figure PCTCN2020088861-appb-100009
    10-氯-9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4,6,7-四氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(3)
    Figure PCTCN2020088861-appb-100010
    10-氯-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-9-(1,3,5-三甲基-1H-吡唑-4-基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(4)
    Figure PCTCN2020088861-appb-100011
    10-氯-9-(1,4-二甲基-1H-吡唑-5-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(5)
    Figure PCTCN2020088861-appb-100012
    7,10-二氯-9-(1,4-二甲基-1H-吡唑-5-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(6)
    Figure PCTCN2020088861-appb-100013
    10-氯-9-(1-(2-羟乙基)-3,5-二甲基-1H-吡唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-)二氢吡啶-3-基)甲基)-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(7)
    Figure PCTCN2020088861-appb-100014
    2-(4-(10-氯-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-1-氧代-1,2,3,4-四氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-9-基)-3,5-二甲基-1H-吡唑-1-基)乙腈(8)
    Figure PCTCN2020088861-appb-100015
    10-氯-9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(9)
    Figure PCTCN2020088861-appb-100016
    (R)-10-氯-9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(10)
    Figure PCTCN2020088861-appb-100017
    (S)-10-氯-9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(11)
    Figure PCTCN2020088861-appb-100018
    5-氯-4-(3,5-二甲基异噁唑-4-基)-7-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-8,9二氢-[1,4]重氮基庚英并[6,7,1-HI]吲唑-6(7H)-酮(12)
    Figure PCTCN2020088861-appb-100019
    5-氯-4-(3,5-二甲基异噁唑-4-基)-7-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基) -8,9二氢-2,7,9a-三氮杂苯并[cd]薁-6(7H)-酮(13)
    Figure PCTCN2020088861-appb-100020
    9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-10-甲基-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(14)
    Figure PCTCN2020088861-appb-100021
    9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-10-甲基-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(15)
    Figure PCTCN2020088861-appb-100022
    10-氯-9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-4-甲基-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(16)
    Figure PCTCN2020088861-appb-100023
    10-氯-9-(3,5-二甲基异噁唑-4-基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3-甲基-3,4-二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(17)
    Figure PCTCN2020088861-appb-100024
  6. 根据权利要求1或2所述的化合物，其是以下化合物中的任意一种：
    10-氯-9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(9)
    Figure PCTCN2020088861-appb-100025
    (R)-10-氯-9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(10)
    Figure PCTCN2020088861-appb-100026
    (S)-10-氯-9-(甲氧基(氧杂环丁烷-3-基)甲基)-2-((4-甲氧基-6-甲基-2-氧代-1,2-二氢吡啶-3-基)甲基)-3,4二氢-[1,4]重氮基庚英并[6,7,1-HI]吲哚-1(2H)-酮(11)
    Figure PCTCN2020088861-appb-100027
  7. 一种药物组合物，其包含治疗有效量的根据权利要求1或2所述的化合物，或其药学上可接受的盐、酯、前药、络合物、溶剂化物、水合物或异构体；以及药学上可接受的载体或赋形剂。
  8. 权利要求1或2所述的化合物，或其药学上可接受的盐、酯、前药、络合物、溶剂化物、水合物或异构体用于制备用于治疗EZH2介导的疾病的药物的用途。
  9. 根据权利要求8所述的用途，其中所述的疾病为癌症。
  10. 根据权利要求9所述的用途，其中所述的癌症为肺癌、胃癌、肝癌、乳腺癌、鼻咽癌、胰腺癌、卵巢癌、宫颈癌、结肠直肠癌、胶质瘤、黑色素瘤、前列腺癌、肾癌、食道癌、间皮瘤、头颈癌、膀胱癌、唾腺癌、白血病或淋巴瘤。
  11. 根据权利要求9所述的用途，其中所述的癌症为间变性大细胞淋巴瘤或非霍奇金淋巴瘤。
  12. 一种由式(Ⅱ)或式(Ⅲ)表示的化合物：
    Figure PCTCN2020088861-appb-100028
    Figure PCTCN2020088861-appb-100029
    其中，R 8为卤素，R 9为羟基保护基。
  13. 根据权利要求12所述的化合物，其中R 8为溴，R 9为苄基。
  14. 权利要求12所述的化合物用于制备权利要求1或2所述化合物的用途。
PCT/CN2020/088861 2019-05-07 2020-05-07 Ezh2抑制剂及其用途 WO2020224607A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/608,950 US20220281872A1 (en) 2019-05-07 2020-05-07 Ezh2 inhibitor and use thereof
CN202080010425.4A CN113365989A (zh) 2019-05-07 2020-05-07 Ezh2抑制剂及其用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910376057.4A CN111909157B (zh) 2019-05-07 2019-05-07 Ezh2抑制剂及其用途
CN201910376057.4 2019-05-07

Publications (1)

Publication Number Publication Date
WO2020224607A1 true WO2020224607A1 (zh) 2020-11-12

Family

ID=73051433

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/088861 WO2020224607A1 (zh) 2019-05-07 2020-05-07 Ezh2抑制剂及其用途

Country Status (3)

Country Link
US (1) US20220281872A1 (zh)
CN (2) CN111909157B (zh)
WO (1) WO2020224607A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022179584A1 (zh) * 2021-02-26 2022-09-01 南京药石科技股份有限公司 新型ezh2抑制剂及其用途

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112574218A (zh) * 2020-12-30 2021-03-30 广州安岩仁医药科技有限公司 吲哚或氮杂苯并吡咯类化合物的合成方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012118812A2 (en) * 2011-02-28 2012-09-07 Epizyme, Inc. Substituted 6,5-fused bicyclic heteroaryl compounds
WO2015077194A1 (en) * 2013-11-22 2015-05-28 Bristol-Myers Squibb Company Inhibitors of lysine methyl transferase
WO2015193768A1 (en) * 2014-06-17 2015-12-23 Pfizer Inc. Aryl fused lactams as ezh2 modulators
CN105452246A (zh) * 2013-06-06 2016-03-30 葛兰素史密斯克莱知识产权（第2号）有限公司 Zeste增强子同源物2的抑制剂
CN107922434A (zh) * 2015-08-27 2018-04-17 伊莱利利公司 Ezh2抑制剂
WO2019222272A1 (en) * 2018-05-14 2019-11-21 Nuvation Bio Inc. Anti-cancer nuclear hormone receptor-targeting compounds

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ597833A (en) * 2009-09-28 2014-01-31 Hoffmann La Roche Benzoxazepin pi3k inhibitor compounds and methods of use
US20130310379A1 (en) * 2010-11-19 2013-11-21 Constellation Pharmaceuticals Modulators of methyl modifying enzymes, compositions and uses thereof
WO2012151512A2 (en) * 2011-05-04 2012-11-08 Constellation Pharmaceuticals, Inc. Bromodomain inhibitors and uses thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012118812A2 (en) * 2011-02-28 2012-09-07 Epizyme, Inc. Substituted 6,5-fused bicyclic heteroaryl compounds
CN105452246A (zh) * 2013-06-06 2016-03-30 葛兰素史密斯克莱知识产权（第2号）有限公司 Zeste增强子同源物2的抑制剂
WO2015077194A1 (en) * 2013-11-22 2015-05-28 Bristol-Myers Squibb Company Inhibitors of lysine methyl transferase
WO2015193768A1 (en) * 2014-06-17 2015-12-23 Pfizer Inc. Aryl fused lactams as ezh2 modulators
CN107922434A (zh) * 2015-08-27 2018-04-17 伊莱利利公司 Ezh2抑制剂
WO2019222272A1 (en) * 2018-05-14 2019-11-21 Nuvation Bio Inc. Anti-cancer nuclear hormone receptor-targeting compounds

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AMANTULLAH ANSARI ET AL.: "Novel 3-methylindoline inhibitors of EZH2: Design, synthesis and SAR", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 27, no. 2, 25 November 2016 (2016-11-25), pages 217 - 222, XP029862595 *
QIN, YU ET AL.: "EZH2 inhibitors and antitumor therapy", CHEMISTRY OF LIFE, vol. 37, no. 3, 15 June 2017 (2017-06-15), pages 401 - 407, XP055752078 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022179584A1 (zh) * 2021-02-26 2022-09-01 南京药石科技股份有限公司 新型ezh2抑制剂及其用途

Also Published As

Publication number Publication date
CN111909157B (zh) 2023-02-03
US20220281872A1 (en) 2022-09-08
CN113365989A (zh) 2021-09-07
CN111909157A (zh) 2020-11-10

Similar Documents

Publication Publication Date Title
JP2021020957A (ja) 二官能性分子によって標的化タンパク質分解を誘導する方法
EP2850082B1 (en) 1-(3,3-dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido(2,3-d)pyrimidin-6-yl)phenyl)urea as Raf kinase inhibitor for the treatment of cancer
WO2018045957A1 (zh) 一种cdk4/6抑制剂及其制备方法和应用
WO2020073945A1 (zh) 双环类衍生物抑制剂、其制备方法和应用
KR20040062591A (ko) 인돌리논 화합물에 의한 급성 골수성 백혈병의 치료방법
WO2016054987A1 (zh) Egfr抑制剂及其制备和应用
EP3600287B1 (en) Inhibitors of kinase networks and uses thereof
WO2021143701A1 (zh) 嘧啶-4(3h)-酮类杂环化合物、其制备方法及其在医药学上的应用
EA004989B1 (ru) Трициклические ингибиторы поли(adp-рибозо)полимераз
US20220227729A1 (en) Identification and use of kras inhibitors
WO2014040549A1 (zh) 炔杂芳环化合物及其应用
WO2016011979A1 (zh) 2，4-二取代7H-吡咯并[2，3-d]嘧啶衍生物、其制法与医药上的用途
US10689364B2 (en) Isoindoline or isoquinoline compounds, a process for their preparation and pharmaceutical compositions containing them
JP2020514361A (ja) ９，１０，１１，１２−テトラヒドロ−８ｈ−［１，４］ジアゼピノ［５’，６’：４，５］チエノ［３，２−ｆ］キノリン−８−オン化合物およびその使用
JP2023513854A (ja) 大環状化合物およびその使用
WO2020125759A1 (zh) 作为wnt信号通路抑制剂的化合物及其医学应用
TW201934546A (zh) 嘧啶類化合物、其製備方法及其醫藥用途
CN112771046A (zh) 其他被取代的三唑并喹喔啉衍生物
CN116768861A (zh) Sos1蛋白降解靶向嵌合体及其组合物、制剂和用途
WO2020224607A1 (zh) Ezh2抑制剂及其用途
WO2019080723A1 (zh) 多取代吡啶酮类衍生物、其制备方法及其医药用途
CN111542522A (zh) 可用作激酶抑制剂的被取代的吡唑并嘧啶
CA3194868A1 (en) Heterocyclic cullin ring ubiquitin ligase compounds and uses thereof
WO2021185298A1 (zh) Egfr酪氨酸激酶抑制剂及其用途
CN112574207B (zh) Erk1/2蛋白激酶抑制剂及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20802824

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20802824

Country of ref document: EP

Kind code of ref document: A1