WO2014111031A1 - Triazine compound, pharmaceutical salt, isomer, or hydrate thereof, and pharmaceutical composition thereof - Google Patents

Triazine compound, pharmaceutical salt, isomer, or hydrate thereof, and pharmaceutical composition thereof Download PDF

Info

Publication number
WO2014111031A1
WO2014111031A1 PCT/CN2014/070733 CN2014070733W WO2014111031A1 WO 2014111031 A1 WO2014111031 A1 WO 2014111031A1 CN 2014070733 W CN2014070733 W CN 2014070733W WO 2014111031 A1 WO2014111031 A1 WO 2014111031A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
group
alkyl
heterocyclyl
hydrate
Prior art date
Application number
PCT/CN2014/070733
Other languages
French (fr)
Chinese (zh)
Inventor
李育飞
阙灵
Original Assignee
四川恒康发展有限责任公司
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 四川恒康发展有限责任公司 filed Critical 四川恒康发展有限责任公司
Priority to CN201480003555.XA priority Critical patent/CN104870437A/en
Publication of WO2014111031A1 publication Critical patent/WO2014111031A1/en

Links

Classifications

    • 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
    • C07D401/12—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 chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D253/00—Heterocyclic compounds containing six-membered rings having three nitrogen atoms as the only ring hetero atoms, not provided for by group C07D251/00
    • C07D253/02—Heterocyclic compounds containing six-membered rings having three nitrogen atoms as the only ring hetero atoms, not provided for by group C07D251/00 not condensed with other rings
    • C07D253/06—1,2,4-Triazines
    • C07D253/065—1,2,4-Triazines having three double bonds between ring members or between ring members and non-ring members
    • C07D253/07—1,2,4-Triazines having three double bonds between ring members or between ring members and non-ring members with hetero atoms, or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D253/00—Heterocyclic compounds containing six-membered rings having three nitrogen atoms as the only ring hetero atoms, not provided for by group C07D251/00
    • C07D253/02—Heterocyclic compounds containing six-membered rings having three nitrogen atoms as the only ring hetero atoms, not provided for by group C07D251/00 not condensed with other rings
    • C07D253/06—1,2,4-Triazines
    • C07D253/065—1,2,4-Triazines having three double bonds between ring members or between ring members and non-ring members
    • C07D253/07—1,2,4-Triazines having three double bonds between ring members or between ring members and non-ring members with hetero atoms, or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D253/075—Two hetero atoms, in positions 3 and 5
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • C07D471/10—Spiro-condensed systems
    • 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
    • C07D487/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
    • C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/224—Phosphorus triamides

Definitions

  • X is selected from N, CH or 0.
  • R 2 , R 3 or R 4 are independently selected from the group consisting of H, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methyl, ethyl, propyl, butyl, vinyl, Propylene, butenyl, trifluoromethyl, trifluoroethyl, chloromethyl, chloroethyl, methoxy, ethyloxy, methylcarbonyl, ethylcarbonyl, piperidinyl, piperazinyl, Pyrrolyl, imidazolyl, pyrazolyl, pyrrolidinyl, pyrrolyl, dihydropyrrolyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, quinolinyl, benzofuranyl, pyrazine Base, pyrazolinyl, thiadiazolyl, hydroxy, cyano,
  • Compounds of the invention include, but are not limited to, the following compounds: Compound 1
  • the compound of the present invention and a pharmaceutically acceptable salt thereof, or an isomer thereof, or a hydrate thereof and/or composition thereof, may be formulated together with a pharmaceutically acceptable excipient or carrier, and the resulting composition may be administered in vivo.
  • Mammals such as men, women, and animals, are used to treat conditions, symptoms, and diseases.
  • the composition may be: a tablet, a pill, a suspension, a solution, an emulsion, a capsule, an aerosol, a sterile injectable solution. Sterile powder, etc.
  • the amount of the compound or pharmaceutical composition of the invention administered to a patient is not fixed and is usually administered in a pharmaceutically effective amount.
  • the amount of the compound actually administered can be determined by the physician on a case-by-case basis, including the condition being treated, the route of administration selected, the actual compound administered, the individual condition of the patient, and the like.
  • the dosage of the compound of the invention depends on the particular use of the treatment, the mode of administration, the condition of the patient, and the judgment of the physician.
  • the proportion or concentration of the compound of the present invention in the pharmaceutical composition depends on various factors including dosage, physicochemical properties, administration route and the like.
  • the intermediate 2 (0.44 mmol) obtained in Step 1 was dissolved in 10 mL of NMP, and 1.5 eq (0.66 mmol) of m-CPBA was added. After the mixture was stirred at room temperature for 30 min, leq (0.44 mmol) of pTSA and 2 eq ( 0.88 mmol) of 4-morpholinylaniline, the resulting mixture was stirred at 110 ° C for 2-4 h, cooled to room temperature, diluted with more than 100 mL of ethyl acetate, then washed with saturated sodium carbonate solution and then washed with water. Concentration in vacuo and reversed phase HPLC gave compound 27.

Abstract

The present invention relates to a triazine compound, pharmaceutical salt, isomer, or hydrate thereof, pharmaceutical composition thereof, the preparation method therefor, a method for preparing a pharmaceutical composition using the compound and at least one pharmaceutically acceptable vector or excipient, and the use of the compound of the present invention in preparing medicine for treating multiple syk kinase- and/or jak kinase- mediated disorders, symptoms, and diseases.

Description

三嗪化合物、 其药用盐、 异构体或水合物及其药物组合物 技术领域  Triazine compound, pharmaceutically acceptable salt, isomer or hydrate thereof and pharmaceutical composition thereof
本发明涉及一种三嗪化合物、其药用盐、异构体或水合物以及包含他们的药物组 合物，尤其涉及一种脾酪氨酸激酶抑制剂。本发明还涉及所述三嗪化合物及其盐的药 物组合物在制备预防或治疗 Syk(Spleen Tyrosine Kinase: 脾酪氨酸激酶)激酶和 /或 JAK (Janus Kinase) 激酶介导的病症或疾病的用途。 背景技术  The present invention relates to a triazine compound, a pharmaceutically acceptable salt, isomer or hydrate thereof, and a pharmaceutical composition comprising the same, and more particularly to a spleen tyrosine kinase inhibitor. The present invention also relates to a pharmaceutical composition of the triazine compound and a salt thereof for the preparation of a disease or disease mediated by the prevention or treatment of Syk (Spleen Tyrosine Kinase) kinase and/or JAK (Janus Kinase) kinase. use. Background technique
随着肿瘤生物学及其相关学科的飞速发展，人们逐渐认识到细胞癌变的本质是细 胞信号转导通路失调导致的细胞无限增殖。诸如光线、温度等的外源性信号和诸如激 素、 神经递质、 细胞因子等的内源性信号， 可通过不同途径产生各种细胞效应。 信号 转导是指各类细胞信号通过细胞膜和信号分子引起的细胞基因表达改变的过程。可以 说，几乎所有重要的生命现象都与细胞内信号转导有关。细胞信号转导过程的异常会 导致细胞生长、 分化、 代谢和生物学行为障碍， 进而引起各种疾病乃至肿瘤的发生。  With the rapid development of tumor biology and related disciplines, it is gradually recognized that the essence of cell carcinogenesis is the infinite proliferation of cells caused by the imbalance of the cell signal transduction pathway. Exogenous signals such as light, temperature, etc., and endogenous signals such as hormones, neurotransmitters, cytokines, etc., can produce various cellular effects through different pathways. Signal transduction is the process by which various cellular signals undergo changes in cellular gene expression caused by cell membranes and signaling molecules. It can be said that almost all important life phenomena are related to intracellular signal transduction. Abnormalities in cellular signal transduction can lead to cell growth, differentiation, metabolism, and biological behavioral disorders, which in turn can cause various diseases and even tumors.
酪氨酸蛋白激酶是细胞信号转导过程中极为重要的物质，具有多种细胞功能，在 正常细胞的调节、通讯和发育生物学方面起着十分重要的作用。酪氨酸激酶的活性过 高， 会导致其下游信号途径激活， 最终导致肿瘤形成。酪氨酸蛋白激酶按照其结构可 分为受体酪氨酸蛋白激酶（RTKs )和非受体酪氨酸蛋白激酶（nrPTKs)。 T淋巴细胞 受体、 B淋巴细胞受体、 免疫球蛋白受体等能募集 nrPTKs而后通过酪氨酸磷酸化形 成信号转导复合物， 再激活下游的信号转导如 JAK/STAT等途径， 促进细胞增殖， 导 致肿瘤形成。  Tyrosine protein kinase is an extremely important substance in the process of cell signal transduction. It has a variety of cellular functions and plays an important role in the regulation, communication and developmental biology of normal cells. Excessive activity of tyrosine kinases leads to activation of downstream signaling pathways that ultimately lead to tumor formation. Tyrosine protein kinases can be classified into receptor tyrosine protein kinases (RTKs) and non-receptor tyrosine protein kinases (nrPTKs) according to their structures. T lymphocyte receptors, B lymphocyte receptors, immunoglobulin receptors, etc. can recruit nrPTKs and then form signal transduction complexes by tyrosine phosphorylation, and then activate downstream signal transduction such as JAK/STAT to promote Cell proliferation leads to tumor formation.
根据现阶段的研究，抑制酪氨酸激酶信号转导的抗肿瘤药物有单克隆抗体和小分 子酪氨酸激酶抑制剂，其中抗体的制备方法和给药方式在一定程度上限制了其临床应 用。现有的小分子酪氨酸激酶抑制剂种类还很少， 针对的作用靶点也少， 常需与常规 化疗、放疗联合以达到更好的疗效。 同时， 现有抑制剂的抗药性和副反应也需要新的 药物和治疗方法。 发明内容 According to the current research, anti-tumor drugs that inhibit tyrosine kinase signal transduction include monoclonal antibodies and small molecule tyrosine kinase inhibitors. The preparation methods and administration methods of antibodies limit their clinical application to some extent. . There are few types of small molecule tyrosine kinase inhibitors available, and there are few targets for targeting. It is often necessary to combine with conventional chemotherapy and radiotherapy to achieve better therapeutic effects. At the same time, the resistance and side effects of existing inhibitors also require new drugs and treatments. Summary of the invention
本发明的目的是提供一种三嗪化合物、其药用盐、异构体或水合物，其制备方法， 以及至少一种本发明化合物和至少一种药学上可接受的载体或赋形剂制备药用组合 物的方法以及一种或多种本发明化合物在制备治疗 SYK激酶和 /或 JAK激酶介导的病 症或疾病的药物中的用途。  The object of the present invention is to provide a triazine compound, a pharmaceutically acceptable salt, isomer or hydrate thereof, a process for the preparation thereof, and at least one compound of the present invention and at least one pharmaceutically acceptable carrier or excipient Methods of pharmaceutical compositions and the use of one or more compounds of the invention in the manufacture of a medicament for the treatment of a SYK kinase and/or JAK kinase mediated condition or disease.
通式 的化合物或其药 物：  a compound of the formula: or a drug thereof:
Figure imgf000003_0001
Figure imgf000003_0001
其中， 选自具有取代基或不具有取代基的芳基、 具有取代基或不具有取代 基的杂芳基、具有取代基或不具有取代基的 d-do烷基和具有取代基或不具有取 代基的 C3-C6杂环基， 其中！^中的所述取代基是独立地选自下述基团的取代基： 、 螺环基、 杂螺环基、 Wherein, it is selected from an aryl group having a substituent or a substituent, a heteroaryl group having a substituent or a substituent, a d-do alkyl group having a substituent or having no substituent, and having or not having a substituent Substituted C 3 -C 6 heterocyclic group, which! The substituent in ^ is a substituent independently selected from the group consisting of: a spiro group, a heterospiro group,
Figure imgf000003_0002
Ci-Cio烷基、 卤代
Figure imgf000003_0002
Ci-Cio alkyl, halogenated
Ci-Cio Ci-Cio Ci-Cio C6— 10芳基、 C6— 10杂芳基、 C3-C6 杂环基羰基烷氧基； 或可选地具有选自卤素原子、 C2— 1Q烯基、 酰胺基、 CN、 酰 基、 d— 1Q烷基酰基、 d— 1Q烷基磺酰基、 d— 1Q烷基、 C3— 6环烷基、 C3-C6杂环基、 烷基氧基和 C3— 6环烷基氧基的取代基的下述基团： 酰胺基、 烷基磺酰基、 羰 基、 c6— 1Q芳基杂环基、 c3-c6杂环基、 c3-c6杂环基酰胺基、 c3-c6杂环基羰基、Ci-Cio Ci-Cio Ci-Cio C 6 — 10 aryl, C 6 —10 heteroaryl, C 3 -C 6 heterocyclylcarbonylalkoxy; or alternatively having a halogen atom, C 2 — 1Q alkenyl groups, amide groups, CN, acyl, D- 1Q alkyl group, an alkylsulfonyl group 1Q D-, D- 1Q alkyl, C 3 - 6 cycloalkyl, C 3 -C 6 heterocyclic group, an alkoxy group and a C 3 - 6 cycloalkyl group substituted by the following groups: an amido group, an alkylsulfonyl group, a carbonyl group, c 6 - 1Q aromatic heterocyclic group, c 3 -c 6 heterocyclyl , c 3 -c 6 heterocyclylamide, c 3 -c 6 heterocyclylcarbonyl,
C3-C6环烷基胺基、 磺酰基、 氨基磺酰基、 磺酰胺基、 C3-C6杂环基磺酰基； R7、 R8、 R9独立地选自 。烷基、 。卤代烷基、 。烷基氧基、 。烷基羰基、 或 C3— 6 环烷基， R8、 R9 可以与 N 形成一个原子数为 3-6 的环； 或 R 10 Y一 C 3 -C 6 cycloalkylamino, sulfonyl, aminosulfonyl, sulfonylamino, C 3 -C 6 heterocyclylsulfonyl; R 7 , R 8 , R 9 are independently selected. Alkyl, . Haloalkyl,. Alkyloxy, . Alkylcarbonyl, or C 3 - 6 cycloalkyl, R 8, R 9 form a 3-6 ring atoms and N; or R 10 Y one
，其中 Y选自 N、 O或 C; Z独立地为 0、 C、 N、
Figure imgf000004_0001
Wherein Y is selected from N, O or C; Z is independently 0, C, N,
Figure imgf000004_0001
R1()不存在或选自 H、 d— 1Q烷基、 C3— 6环烷基、 d— 1Q烷基酰胺基、 d— 1Q烷基磷酰 基、 1Q烷基磺酰基、 d— 1Q烷基羰基、 d— 1Q烷基磺酰胺基、 CN、 d— 1Q烷基胺基、 C3_6杂环基、 d-d。卤代烷基、 C^。芳基、 C^。杂芳基、 酯基、 醛基、 Cw。烷基 氰基、 d— 1() έϊ代烷基磷酰胺基、 d— 1Q烷氧基羰基， R 1 () is absent or selected from H, d- 1Q alkyl, C 3 - 6 cycloalkyl, d- 1Q alkylamido, d- 1Q alkyl phosphoryl group, 1Q alkylsulfonyl, d- 1Q Alkylcarbonyl , d- 1Q alkylsulfonylamino, CN, d- 1Q alkylamino, C 3 -6 heterocyclyl, dd. Haloalkyl, C^. Aryl, C^. Heteroaryl, ester, aldehyde, Cw. Alkyl cyano, d- 1() deuterated alkylphosphonamido, d- 1Q alkoxycarbonyl,
R2、 R3和 R4独立地为 11、 具有取代基或不具有取代基的 C3-C6环烷基、 具有 取代基或不具有取代基的 Crdo烷基、具有取代基或不具有取代基的 d-do烷基 胺基、 d-do烷基氧基、 具有取代基或不具有取代基的 -0>杂环基、 具有取代 基或不具有取代基的 C6— 1Q芳基、 具有取代基或不具有取代基的 C6— 1Q杂芳基； 其 中 R2、 R3和 R4中的所述取代基是独立地选自下述基团的取代基： 卤素、 羟基、 Ci-Cio C2-C10 C2-C10块基、 基幾基、 基、 Ci-Cio l¾ ¾¾¾¾ C1-C10 烷氧基、 d-do烷氧基羰基、 C6-1Q芳基、 C6-1Q杂芳基、 C3-C6杂环基、 C3-C6环烷 基、 磺酰基、 巯基、 氰基、 C3-C6杂环基胺基、 d-do烷基胺基、 C3-C6环烷基胺 基、 C6— 1Q芳基胺基、 C6— 1Q杂芳基胺基、 d-do芳烷基氧基、 C6— 1Q苯并杂环基胺基。 R 2 , R 3 and R 4 are independently 11, a C 3 -C 6 cycloalkyl group having a substituent or not having a substituent, a Crdo alkyl group having a substituent or having no substituent, having a substituent or not having a d-doalkylamino group of a substituent, a d-doalkyloxy group, a ->heterocyclic group having a substituent or a substituent, a C 6 -1Q aryl group having a substituent or having no substituent a C 6 -1Q heteroaryl group having a substituent or having no substituent; wherein the substituent in R 2 , R 3 and R 4 is a substituent independently selected from the group consisting of halogen, hydroxy, Ci-Cio C2-C10 C 2 -C 10 block, benzyl, phenyl, Ci-Cio l3⁄4 3⁄43⁄43⁄4⁄4 C1-C10 alkoxy, d-doalkoxycarbonyl, C 6 - 1Q aryl, C 6 - 1Q heteroaryl, C 3 -C 6 heterocyclic, C 3 -C 6 cycloalkyl, sulfonyl, decyl, cyano, C 3 -C 6 heterocyclylamino, d-doalkylamino, C 3 -C 6 cycloalkylamino group, C 6 -1Q arylamino group, C 6 -1Q heteroarylamino group, d-do aralkyloxy group, C 6 - 1Q benzoheterocyclylamino group .
在本发明中， 优选当 或 为芳基时， 不为烷基。  In the present invention, it is preferably not an alkyl group when it is an aryl group.
Figure imgf000004_0002
Figure imgf000004_0002
其中，  among them,
R5独立地为 11、 卤素、 CN、 羟基、 C3-C6杂环基、 苯基. 螺环基、 杂螺环基、 R 5 is independently 11, halogen, CN, hydroxy, C 3 -C 6 heterocyclic, phenyl. spiro, heterospiro,
Figure imgf000004_0003
C C10烷基、 卤代
Figure imgf000004_0003
CC 10 alkyl, halogenated
C1-C10 ¾¾¾ C1-C10 ¾¾¾$ί¾ Ci-Cio C6- 10方基 c6— 10杂芳基、 C3-C6 杂环基羰基烷氧基； 或可选地具有选自卤素原子、 d— 1Q烷基、 C3— 6环烷基、 C3-C, 杂环基、 d 1Q烷基氧基和 C3— 6环烷基氧基的取代基的下述基团： C6— 1Q芳基杂环基、 酰胺基、 羰基、 c3-c6杂环基、 C3-C6杂环基酰胺基、 C3-C6杂环基羰基、 C3-C6 环烷基胺基、磺酰基、氨基磺酰基、 C3-C6杂环基 ，\^ C1-C10 ¾¾¾ C1-C10 ¾¾¾ $ ί¾ Ci-Cio C 6 - 10 group side c 6 - 10 heteroaryl, C 3 -C 6 heterocyclylcarbonyl alkoxy; or alternatively, selected from a halogen atom, d- 1Q alkyl, C 3 - 6 cycloalkyl, C 3 -C, Heterocyclyl, d 1Q alkyl group and a C 3 - following substituent group 6 cycloalkyl group: C 6 - 1Q aromatic heterocyclic group, an amide group, a carbonyl group, c 3 -c 6 heteroatoms Cyclic group, C 3 -C 6 heterocyclic amide group, C 3 -C 6 heterocyclocarbonyl group, C 3 -C 6 cycloalkylamino group, sulfonyl group, aminosulfonyl group, C 3 -C 6 heterocyclic group ,\^
其中 Y选自 N、 0或 C; Z独立地为 0、 C、 N、
Figure imgf000005_0001
； R1Q不存在或选自 H、 d— 1Q烷基、 C3— 6环烷基、 d— 1Q烷基酰胺基、 d— 1Q烷基磷酰胺基、 d— 1Q烷基磺 酰基、 1Q烷基羰基、 d— 1Q烷基磺酰胺基、 CN、 d— 1Q烷基胺基、 C3— 6杂环基、 Crdo 卤代烷基、 C6— 1Q芳基、 C6— 1Q杂芳基、 酯基、 醛基、 d— 1Q烷基氰基、 C^o 卤代烷基磷酰胺基、 d— 1Q烷氧基羰基， R7、 R8、 R9独立地选自 d— 1Q烷基、 Cwo 卤代烷基、 d— 1Q烷基氧基、 d— 1Q烷基羰基、 d-do卤代烷基或 C3— 6环烷基， R8、 R9可以与 N形成一个原子数为 3-6的环； Wherein Y is selected from N, 0 or C; Z is independently 0, C, N,
Figure imgf000005_0001
; R 1Q absent or selected from H, d- 1Q alkyl, C 3 - 6 cycloalkyl, d- 1Q alkylamido, d- 1Q alkyl phosphoramide group, d- 1Q alkylsulfonyl, 1Q alkylcarbonyl, d- 1Q alkylsulfonylamino group, CN, d- 1Q alkylamino, C 3 - 6 heterocyclyl, CRDO haloalkyl, C 6 - 1Q aryl group, C 6 - 1Q heteroaryl, Ester group, aldehyde group, d- 1Q alkyl cyano group, C^o haloalkylphosphoramido group, d- 1Q alkoxycarbonyl group, R 7 , R 8 , R 9 are independently selected from d- 1Q alkyl group, Cwo haloalkyl, d- 1Q alkyloxy, d- 1Q alkylcarbonyl, d-do-haloalkyl or C 3 - 6 cycloalkyl, R 8, R 9 form a N atoms with 3-6 ring ;
R6独立地为 H或卤素； R 6 is independently H or halogen;
X选自 N、 CH或 0。  X is selected from N, CH or 0.
上述 R2、 R3或 R4独立地选自下述基团： H、 环丙基、 环丁基、 环戊基、 环 己基、 甲基、 乙基、 丙基、 丁基、 乙烯基、 丙烯基、 丁烯基、 三氟甲基、 三氟乙 基、 氯甲基、 氯乙基、 甲氧基、 乙基氧基、 甲基羰基、 乙基羰基、 哌啶基、 哌嗪 基、 吡咯基、 咪唑基、 吡唑基、 吡咯烷基、 吡咯基、 二氢吡咯基、 吡咯啉基、 咪 唑烷基、 咪唑啉基、 吡唑烷基、 喹啉基、 苯并呋喃基、 吡嗪基、 吡唑啉基、 噻二 唑基、 羟基、 氰基、 磺酰基、 The above R 2 , R 3 or R 4 are independently selected from the group consisting of H, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methyl, ethyl, propyl, butyl, vinyl, Propylene, butenyl, trifluoromethyl, trifluoroethyl, chloromethyl, chloroethyl, methoxy, ethyloxy, methylcarbonyl, ethylcarbonyl, piperidinyl, piperazinyl, Pyrrolyl, imidazolyl, pyrazolyl, pyrrolidinyl, pyrrolyl, dihydropyrrolyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, quinolinyl, benzofuranyl, pyrazine Base, pyrazolinyl, thiadiazolyl, hydroxy, cyano, sulfonyl,
Figure imgf000005_0002
Figure imgf000005_0002
步的， 本发明所述化合物为通式 II的化合物: In the step, the compound of the present invention is a compound of the formula II:
Figure imgf000006_0001
Figure imgf000006_0001
II  II
其中， R2、 R3、 R4、 R6、 X、 Y、 Z和 R10如上所述; 更进一步的， 本发明的化合物具有如下结构: Wherein R 2 , R 3 , R 4 , R 6 , X, Y, Z and R 10 are as described above; further, the compound of the present invention has the following structure:
Figure imgf000006_0002
Figure imgf000006_0002
其中， Ru、 R12、 Rn R14独立地选自 H、 d-do垸基、 d-do垸基胺基、 C, -C10卤代烷基、 C3_6环烷基、 C2-C,。烯基、 CrCw卤代垸基、 CrC^烷氧基、 CrC Wherein R u , R 12 , Rn R 14 are independently selected from the group consisting of H, d-dodecyl, d-dodecylamino, C, -C 10 haloalkyl, C 3 -6 cycloalkyl, C 2 - C,. Alkenyl, CrCw halogenated fluorenyl, CrC alkoxy, C r C
Figure imgf000007_0001
Figure imgf000007_0001
本发明化合物包括但不限于下述化合物： 化合物 1 Compounds of the invention include, but are not limited to, the following compounds: Compound 1
™、、\  TM,,\
"^J 剛 -一 M 化合物 2 化合物 3
Figure imgf000007_0002
化合物 4 化合物 5 "^J 刚-一 M compound 2 compound 3
Figure imgf000007_0002
Compound 4 compound 5
Figure imgf000008_0001
Figure imgf000008_0001
化合物 14 化合物 15
Figure imgf000008_0002
Compound 14 compound 15
Figure imgf000008_0002
化合物 16 化合物 17
Figure imgf000009_0001
Compound 16 compound 17
Figure imgf000009_0001
19
Figure imgf000009_0002
19
Figure imgf000009_0002
化合物 20 化合物 21
Figure imgf000009_0003
Compound 20 Compound 21
Figure imgf000009_0003
化合物 22Compound 22
Figure imgf000009_0004
Figure imgf000009_0004
化合物 24 化合物 25
Figure imgf000009_0005
Compound 24 compound 25
Figure imgf000009_0005
化合物 27 化合物 28 Compound 27 Compound 28
Figure imgf000010_0001
化合物 39 化合物 40
Figure imgf000011_0001
Figure imgf000010_0001
Compound 39 Compound 40
Figure imgf000011_0001
化合物 43 Compound 43
F.
Figure imgf000011_0002
F.
Figure imgf000011_0002
化合物 46 Compound 46
顏¾ Yan 3⁄4
國 Ί s National s
、、\  ,,\
剛 化合物 47 化合物 48 化合物 49 化合物 50 化 51 化 just Compound 47 compound 48 compound 49 compound 50
化Chemical
Figure imgf000012_0001
Figure imgf000012_0001
化合物 54 化合物 55 化合物 56 化合物 57
Figure imgf000012_0002
Compound 54 Compound 55 Compound 56 Compound 57
Figure imgf000012_0002
化合物 61 化合物 62 化合物 63 化合物 64 化合物 65 化合物 66 Compound 61 compound 62 compound 63 Compound 64 compound 65 compound 66
化合物 67 化合物 68 化合物 69 化合物 73 化合物 75 Compound 67 compound 68 compound 69 compound 73 compound 75
Figure imgf000013_0001
化合物 76 化合物 77 化合物 78
Figure imgf000013_0001
Compound 76 Compound 77 Compound 78
Q 、  Q,
广 ： F  Wide : F
(
Figure imgf000013_0002
- (
Figure imgf000013_0002
-
化合物 79 化合物 80
Figure imgf000014_0001
化合物 88
Figure imgf000014_0002
化合物 89
Figure imgf000015_0001
化合物 90
Figure imgf000015_0002
化合物 93 Compound 79 Compound 80
Figure imgf000014_0001
Compound 88
Figure imgf000014_0002
Compound 89
Figure imgf000015_0001
Compound 90
Figure imgf000015_0002
Compound 93
Figure imgf000016_0001
Figure imgf000016_0001
 化合物 97 化合物 98
Figure imgf000017_0001
 Compound 97 Compound 98
Figure imgf000017_0001
化合物 101 化合物 102
Figure imgf000017_0002
Compound 101 Compound 102
Figure imgf000017_0002
化合物 105 化合物 106
Figure imgf000017_0003
Compound 105 Compound 106
Figure imgf000017_0003
化合物 107 化合物 108
Figure imgf000017_0004
化合物 110
Figure imgf000018_0001
Compound 107 Compound 108
Figure imgf000017_0004
Compound 110
Figure imgf000018_0001
本发明还提供一种组合物，所述组合物含有本发明所述的化合物或其盐或其异构 体或其水合物和可药用载体或赋形剂。  The present invention also provides a composition comprising the compound of the present invention or a salt thereof or an isomer thereof or a hydrate thereof, and a pharmaceutically acceptable carrier or excipient.
本发明所述的化合物或其盐或其异构体或其水合物或其组合物用于预防或治疗 syk激酶和 /或 jak激酶介导的病症或疾病。  The compound of the present invention or a salt thereof or an isomer thereof or a hydrate thereof or a composition thereof is for use in the prevention or treatment of a syk kinase and/or jak kinase mediated disorder or disease.
一些实施例中，本发明化合物可用作一种或多种 jak激酶抑制剂；一些实施例中， 本发明化合物用作 syk激酶抑制剂； 一些实施例中本发明化合物用作 jak和 syk双重 抑制剂。 一些实施例中， 本发明化合物用作 jakl激酶抑制剂； 一些实施例中， 本发明化 合物用作 jak2激酶抑制剂； 一些实施例中， 本发明化合物用作 jak3激酶抑制剂。 In some embodiments, the compounds of the invention are useful as one or more jak kinase inhibitors; in some embodiments, the compounds of the invention are useful as syk kinase inhibitors; in some embodiments, the compounds of the invention are used as dual inhibition of jak and syk Agent. In some embodiments, the compounds of the invention are useful as jakl kinase inhibitors; in some embodiments, the compounds of the invention are useful as jak2 kinase inhibitors; in some embodiments, the compounds of the invention are useful as jak3 kinase inhibitors.
一些实施例中， 本发明化合物用作与 jak和 /或 syk相关疾病的治疗， 包括任何与 jak和 /或 syk的表达或活性直接或间接相关的疾病或病症，包括过度表达和 /或异常的 活性水平， 也包括通过调节 jak和 /或 syk活性得到预防、 缓解或治愈的疾病。  In some embodiments, the compounds of the invention are used as a treatment for diseases associated with jak and/or syk, including any disease or condition directly or indirectly related to the expression or activity of jak and/or syk, including overexpression and/or abnormality. The level of activity also includes diseases that are prevented, ameliorated or cured by modulating the activity of jak and/or syk.
所述 syk激酶和 /或 jak激酶介导的病症或疾病选自血液病、过敏性哮喘、骨髓纤 维化和类风湿性关节炎。  The syk kinase and/or jak kinase mediated condition or disease is selected from the group consisting of hematological disease, allergic asthma, myelofibrosis, and rheumatoid arthritis.
进一步的， 所述 syk激酶和 /或 jak激酶介导的病症或疾病选自白血病、骨髓增生 性疾病。  Further, the syk kinase and/or jak kinase mediated condition or disease is selected from the group consisting of leukemia and myeloproliferative diseases.
更进一步的， 所述 syk激酶和 /或 jak激酶介导的病症或疾病选自多发性骨髓瘤、 慢性髓原白血病、 原髓细胞白血病、 B细胞淋巴瘤、 单核细胞白血病、 脾大性红细胞 增多、 嗜酸性白细胞增多综合征、 原发性血小板减少症、 ***性巨细胞疾病。  Further, the syk kinase and/or jak kinase mediated condition or disease is selected from the group consisting of multiple myeloma, chronic myeloid leukemia, promyelocytic leukemia, B cell lymphoma, monocytic leukemia, splenomegaly Increased, eosinophilic syndrome, essential thrombocytopenia, systemic giant cell disease.
本发明化合物还可与一种或多种其他药物联合使用以治疗与 jak和 /或 syk相关疾 病， 例如 FAK激酶抑制剂、 类固醇、 免疫抑制剂等。 可以同时或依顺序施用给需要 治疗的患者。 类固醇可以是可的松类固醇， 例如***。  The compounds of the invention may also be used in combination with one or more other drugs to treat diseases associated with jak and/or syk, such as FAK kinase inhibitors, steroids, immunosuppressive agents and the like. It can be administered to patients in need of treatment at the same time or sequentially. The steroid may be a cortisone steroid such as dexamethasone.
可将本发明化合物及其药学上可接受的盐或其异构体或其水合物和 /或组合物与 药学上可接受的赋形剂或载体配制在一起，得到的组合物可在体内给予哺乳动物， 例 如男人、 妇女和动物， 用于治疗病症、 症状和疾病。 组合物可以是： 片剂、 丸剂、 混 悬剂、 溶液剂、 乳剂、 胶囊、 气雾剂、 无菌注射液。 无菌粉末等。  The compound of the present invention, and a pharmaceutically acceptable salt thereof, or an isomer thereof, or a hydrate thereof and/or composition thereof, may be formulated together with a pharmaceutically acceptable excipient or carrier, and the resulting composition may be administered in vivo. Mammals, such as men, women, and animals, are used to treat conditions, symptoms, and diseases. The composition may be: a tablet, a pill, a suspension, a solution, an emulsion, a capsule, an aerosol, a sterile injectable solution. Sterile powder, etc.
一些实施例中， 药学上可接受的赋形剂包括微晶纤维素、 乳糖、柠檬酸钠、碳酸 钙、磷酸氢钙、 甘露醇、羟丙基 - β -环糊精、 β -环糊精（增加）、 甘氨酸、崩解剂（如 淀粉、 交联羧甲基纤维素钠、 复合硅酸盐和高分子聚乙二醇）， 造粒粘合剂 (如聚乙烯 吡咯烷酮、 蔗糖、 明胶和***胶)和润滑剂 (如硬脂酸镁、 甘油和滑石粉)。  In some embodiments, the pharmaceutically acceptable excipients include microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, calcium hydrogen phosphate, mannitol, hydroxypropyl-beta-cyclodextrin, beta-cyclodextrin. (increased), glycine, disintegrants (such as starch, croscarmellose sodium, complex silicates and high molecular weight polyethylene glycols), granulating binders (such as polyvinylpyrrolidone, sucrose, gelatin and Acacia gum) and lubricants (such as magnesium stearate, glycerin and talc).
向患者施用本发明化合物或药物组合物的量不固定，通常按药用有效量给药。同 时， 实际给予的化合物的量可由医师根据实际情况决定， 包括治疗的病症、选择的给 药途径、给予的实际化合物、 患者的个体情况等。本发明化合物的剂量取决于治疗的 具体用途、 给药方式、 患者状态、 医师判断。 本发明化合物在药物组合物中的比例或 浓度取决于多种因素， 包括剂量、 理化性质、 给药途径等。  The amount of the compound or pharmaceutical composition of the invention administered to a patient is not fixed and is usually administered in a pharmaceutically effective amount. At the same time, the amount of the compound actually administered can be determined by the physician on a case-by-case basis, including the condition being treated, the route of administration selected, the actual compound administered, the individual condition of the patient, and the like. The dosage of the compound of the invention depends on the particular use of the treatment, the mode of administration, the condition of the patient, and the judgment of the physician. The proportion or concentration of the compound of the present invention in the pharmaceutical composition depends on various factors including dosage, physicochemical properties, administration route and the like.
本发明还涉及一种用于制备如权利要求所述的通式 I的化合物或其药学上可接 升 体或水合物的 The invention also relates to a compound for the preparation of a compound of the formula I as claimed in the claims or a pharmaceutically acceptable compound thereof Ascending or hydrated
Figure imgf000020_0001
Figure imgf000020_0001
中间体 1 中间体 2 附图说明  Intermediate 1 Intermediate 2 BRIEF DESCRIPTION OF THE DRAWINGS
图 1是化合物 16、 8、 9、 7 、 25在 ΙΟΟηΜ对 JAK2, JAK3 和 SYK的抑制率 ( )； 图 2是化合物 16、 8、 9、 7、 25对 JAK2酶的半数抑制浓度 IC50值； 图 3是化合物 16、 8、 9、 7、 25对 JAK3酶的半数抑制浓度 IC50值； 图 4化合物 16、 8 、 9 、 7、 . 25对 SYK酶的半数抑制浓度 IC50值；  Figure 1 is the inhibition rate of compound 16,8,9,7,25 on JAK2, JAK3 and SYK in ΙΟΟηΜ; Figure 2 is the IC50 value of the half-inhibitory concentration of compound 16,8,9,7,25 on JAK2 enzyme; Figure 3 is a half-inhibitory concentration IC50 value of compound 16,8,9,7,25 for JAK3 enzyme; Figure 4 Compound 1650, 8, 9, 7, 25 of the SYK enzyme half-inhibitory concentration IC50 value;
图 5是化合物 16、 8、 9、 7、 25在 100 nM对 JAK1 的抑制率 ( )；  Figure 5 is the inhibition rate of JAK1 at 100 nM for compounds 16, 8, 9, 7, 25 ( );
图 6是化合物 16、 8、 9、 7、 25对 〗AK1 的半数抑制浓度 IC50值；  Figure 6 is a half-inhibitory concentration IC50 value of compound 16, 8, 9, 7, 25 versus AK1;
图 7是化合物 16、 8、 9、 7、 25对 WSU-DLCL2细胞的半数抑制浓度 IC50值； 图 8是化合物 16、 8、 9、 7、 25对 L1210细胞的半数抑制浓度 IC50值； 图 9是化合物 16、 8、 9、 7、 25对 THP-1细胞的半数抑制浓度 IC50值； 图 10是化合物 16 、 8 、 9、 . 7 、 25对 K562细胞的半数抑制浓度 IC50值； 图 11是化合物 16. 、 8、 . 9、 , 7- 、 25对 HL-60细胞的半数抑制浓度 IC50值； 图 12是化合物 16 、 8 、 9、 . 7 、 25对 RPMI-8226细胞的半数抑制浓度 IC50值 具体实施方式  Figure 7 is a half-inhibitory concentration IC50 value of compounds 16, 8, 9, 7, 25 versus WSU-DLCL2 cells; Figure 8 is a half-inhibitory concentration IC50 value of compounds 16, 8, 9, 7, 25 versus L1210 cells; Is the half-inhibitory concentration IC50 value of compound 16, 8, 9, 7, 25 on THP-1 cells; Figure 10 is the IC50 value of compound 16 , 8, 9, 7, and 25 on K562 cells; Compounds 16., 8, 9, 9, 7-, 25, half-inhibitory concentration IC50 values for HL-60 cells; Figure 12 is the half-inhibitory concentration IC50 of compounds 16 , 8 , 9 , . 7 , 25 for RPMI-8226 cells Value specific implementation
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会 理解， 下列实施例仅用于说明本发明， 而不应视为限定本发明的范围。 实施例中未注 明具体条件者， 按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产 厂商者， 均为可以通过市购获得的常规产品。  The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but the present invention is to be construed as illustrative only. Those which do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the reagents or instruments used are not specified by the manufacturer, they are all commercially available products.
除另有说明外， 本发明使用的縮写为常规术语。 DIPEA指 Ν,Ν-二异丙基乙胺， DCM指二氯甲烷， ΝΜΡ指 1-甲基 -2-吡咯烷酮， m-CPBA指间氯过氧苯甲酸， p-TSA 指对甲苯磺酸， TFA指三氟乙酸， DCE指 1,2-二氯乙烷， DMF指 Ν,Ν-二甲基甲酰胺， HOBt指 1-羟基苯并***， Et表示乙基， eq表示电子当量， OAc表示乙酸根。 实施例 1 Abbreviations used in the present invention are conventional terms unless otherwise stated. DIPEA refers to hydrazine, Ν-diisopropylethylamine, DCM refers to dichloromethane, ΝΜΡ refers to 1-methyl-2-pyrrolidone, m-CPBA refers to m-chloroperoxybenzoic acid, p-TSA Refers to p-toluenesulfonic acid, TFA refers to trifluoroacetic acid, DCE refers to 1,2-dichloroethane, DMF refers to hydrazine, dimethyl-dimethylformamide, HOBt refers to 1-hydroxybenzotriazole, and Et represents ethyl. Eq represents the electron equivalent and OAc represents the acetate. Example 1
以 3- (甲基巯基) -5-氧 -4,5-二氢 -1,2,4-三嗪 -6-羧酸乙酯为起始原料， 其制备方法见 Starting from ethyl 3-(methylindenyl)-5-oxo-4,5-dihydro-1,2,4-triazine-6-carboxylate, the preparation method is as follows.
Journal of Organic Chemistry,Huang,J.J.,1985,vol.50,p2293。 也可以通过购买途径得到， 如 Cgene Tech
Figure imgf000021_0001
Journal of Organic Chemistry, Huang, JJ, 1985, vol. 50, p2293. It can also be obtained through purchase, such as Cgene Tech.
Figure imgf000021_0001
起始原料  Starting materials
中间体 X的制备： 见 Journal of Organic Chemistry,Huang，J.J.，1985，vol.50，p2293。 也可以通过购买途径得到， 如 Cgene Tech。
Figure imgf000021_0002
Preparation of intermediate X: See Journal of Organic Chemistry, Huang, JJ, 1985, vol. 50, p2293. It can also be obtained through purchase, such as Cgene Tech.
Figure imgf000021_0002
中间体 X  Intermediate X
将起始原料在氯化亚砜中回流 4h， 真空浓縮至干， 得到固体， 即中间体 X。 化合物 1的制备：  The starting material was refluxed in thionyl chloride for 4 h and concentrated to dryness in vacuo to give a solid. Preparation of Compound 1:
步骤 1 :  step 1 :
中间体 X(20 g)溶解于 670 mL干燥的乙腈当中， 加入 DIPEA(16.06 mL), 缓慢滴 加 6.52 mL的环丙胺， 滴加完毕后室温搅拌十五分钟。 随后， 加入氨气饱和的甲醇溶 液 670 mL。 室温继续搅拌 2小时， 减压除去溶剂， 加入 190 mL甲醇和 190 mL二氯 甲烷进行打浆处理， 过滤得到滤饼， 将母液浓縮， 再次过滤得到滤饼， 合并两次滤饼 并用正己烷洗涤后真空干燥得到固体中间体 1 (17g)。
Figure imgf000022_0001
Intermediate X (20 g) was dissolved in 670 mL of dry acetonitrile, DIPEA (16.06 mL) was added, and 6.52 mL of cyclopropylamine was slowly added dropwise, and the mixture was stirred at room temperature for fifteen minutes. Subsequently, 670 mL of an ammonia-saturated methanol solution was added. Stirring was continued for 2 hours at room temperature. The solvent was removed under reduced pressure. MeOH (MeOH) and 190 mL of dichloromethane were applied to the mixture, and the mixture was filtered, and filtered to give a cake. The residue was concentrated and filtered to give a filter cake. After vacuum drying, solid intermediate 1 (17 g) was obtained.
Figure imgf000022_0001
中间体 1  Intermediate 1
1H-NMR(400MHZ,DMSO)：  1H-NMR (400 MHZ, DMSO):
0.58-0.62(m, 2H)， 0.79-0.84(m, 2H)， 2.55 (s, 3H)， 2.88-2.91(m,lH), 7.87(s, 1H)， 8.49(s, 1H), 9.13-9.14(s, 1H)  0.58-0.62 (m, 2H), 0.79-0.84 (m, 2H), 2.55 (s, 3H), 2.88-2.91 (m, lH), 7.87 (s, 1H), 8.49 (s, 1H), 9.13- 9.14(s, 1H)
步骤 2:  Step 2:
步骤 1得到的中间体 1 lOOmg ( 0.44mmol)溶解于 lOmL NMP ( 1-甲基 -2-吡咯烷 酮） 中， 加入 1.5eq ( 0.66mmol ) 的 m-CPBA (间氯过氧苯甲酸）， 混合物在室温下搅 拌 30min后加入 leq ( 0.44mmol) 的 p-TSA (对甲苯磺酸） 和 2eq ( 0.88mmol) 的 4- 吗啉基苯胺， 得到的混合液在 11CTC下搅拌 2-4h， 冷却到室温后， 用 lOOmL以上的 乙酸乙酯稀释， 然后用饱和碳酸钠溶液洗涤， 再用水洗， 真空浓縮， 反相 HPLC分离 得到化合物 1。 The intermediate 1 OOmg (0.44 mmol) obtained in the step 1 was dissolved in 10 mL of NMP (1-methyl-2-pyrrolidone), and 1.5 eq (0.66 mmol) of m-CPBA (m-chloroperoxybenzoic acid) was added, and the mixture was After stirring at room temperature for 30 min, leq (0.44 mmol) of p-TSA (p-toluenesulfonic acid) and 2 eq (0.88 mmol) of 4-morpholinylaniline were added, and the resulting mixture was stirred at 11 CTC for 2-4 h, cooled to room temperature. After that, it was diluted with 100 mL or more of ethyl acetate, washed with a saturated sodium carbonate solution, washed with water, concentrated in vacuo, and then purified by reverse-phase HPLC.
Figure imgf000022_0002
Figure imgf000022_0002
化合物 1  Compound 1
化合物 2-17、 97-105、 115-117可以用相应的胺与中间体 1用上述方法制备得到。 化合物 2-17、 97-105、 115-117的结构见表 1， 其中， X为 C, R3、 R6均为 H，Compounds 2-17, 97-105, and 115-117 can be prepared by the above methods using the corresponding amine and intermediate 1. The structures of the compounds 2-17, 97-105, and 115-117 are shown in Table 1, wherein X is C, and R 3 and R 6 are both H.
R4均为环丙基。 R4 is a cyclopropyl group.
Figure imgf000022_0003
Figure imgf000022_0004
Figure imgf000023_0001
ΐεθΐΐΐ/ ΟΖ OAV
Figure imgf000022_0003
Figure imgf000022_0004
Figure imgf000023_0001
Ϊ́εθΐΐΐ/ ΟΖ OAV
Figure imgf000024_0001
Figure imgf000024_0002
Figure imgf000024_0001
Figure imgf000024_0002
?ΐ  ΐ
(r)6r (HHs 化合物 16: (r)6r (HHs Compound 16:
1H-NMR(400MHZ,DMSO)： 0.59-0.68(m,2H), 0.87-0.94(m,2H), 2.85-2.95(m,lH) 3.16(s,3H), 7.69(s,lH), 7.86(d,2H), 8.16(d,2H), 8.32(s,lH), 9.19(s,lH),10.56(s,lH) 化合物 18:  1H-NMR (400MHZ, DMSO): 0.59-0.68 (m, 2H), 0.87-0.94 (m, 2H), 2.85-2.95 (m, lH) 3.16 (s, 3H), 7.69 (s, lH), 7.86 (d, 2H), 8.16 (d, 2H), 8.32 (s, lH), 9.19 (s, lH), 10.56 (s, lH) Compound 18:
中间体 1 (5 g)加入到 150 mL干燥的 DMF当中， 加入间氯过氧苯甲酸 (11.5 g)并 在室温搅拌一个小时， 然后加入对甲苯磺酸水合物 (4.2 g)和 l-BOC-4-(4-氨基苯基)哌 啶 (12.3 g)。 将反应温度升至 120°C搅拌 2小时后， 将其冷却至室温， 加入乙酸乙酯进 行稀释并用饱和碳酸钠水溶液和饱和食盐水进行洗涤，加入无水硫酸镁进行干燥， 减 压浓縮掉 95%乙酸乙酯，大量固体物质析出，过滤并用少量乙酸乙酯洗涤滤饼后得化 合物 18(3 g).  Intermediate 1 (5 g) was added to 150 mL of dry DMF, m-chloroperoxybenzoic acid (11.5 g) was added and stirred at room temperature for one hour, then p-toluenesulfonic acid hydrate (4.2 g) and l-BOC were added. 4-(4-Aminophenyl)piperidine (12.3 g). After the reaction temperature was raised to 120 ° C and stirred for 2 hours, it was cooled to room temperature, diluted with ethyl acetate and washed with saturated aqueous sodium carbonate and brine, dried over anhydrous magnesium sulfate and evaporated. 95% ethyl acetate, a large amount of solid matter was precipitated, filtered and washed with a small amount of ethyl acetate to give compound 18 (3 g).
Figure imgf000025_0001
Figure imgf000025_0001
化合物 18  Compound 18
化合物 19: 将化合物 18与 TFA/DCM ( 1:1 ) (三氟乙酸 /二氯甲烷） 混合后， 室 温下搅拌， 反 相 HPLC柱分离得到化合物 19。
Figure imgf000025_0002
Compound 19: After compound 18 was mixed with TFA/DCM (1:1) (trifluoroacetic acid/dichloromethane), the mixture was stirred at room temperature.
Figure imgf000025_0002
化合物 19  Compound 19
化合物 20: leq化合物 19溶解于 3mL NMP ( 1-甲基 -2-吡咯烷酮） 中， 加入 3eq DIPEA (Ν,Ν-二异丙基乙胺）和 2eq溴乙腈，得到的混合液在室温下搅拌 2h，用 TFA (三氟乙酸） 酸化， 反相 HPLC柱分离得到化合物 20。
Figure imgf000026_0001
Compound 20: leq compound 19 was dissolved in 3 mL of NMP (1-methyl-2-pyrrolidone), 3 eq of DIPEA (Ν, Ν-diisopropylethylamine) and 2 eq of bromoacetonitrile were added, and the resulting mixture was stirred at room temperature. After 2 h, acidification was carried out with TFA (trifluoroacetic acid), and the compound 20 was isolated by reverse phase HPLC column.
Figure imgf000026_0001
化合物 20  Compound 20
化合物 21 : lOOmg 化合物 19溶解于 3mL DMF (Ν,Ν-二甲基甲酰胺） 中， 密封 管中 12CTC下搅拌 2天， 用水稀释， TFA (三氟乙酸） 酸化， 反相 HPLC柱分离得到 化合物 21。 Compound 21: lOOmg Compound 19 was dissolved in 3 mL of DMF (Ν, Ν-dimethylformamide), stirred in a sealed tube at 12 °C for 2 days, diluted with water, acidified with TFA (trifluoroacetic acid), and separated by reversed phase HPLC column. twenty one.
Figure imgf000026_0002
Figure imgf000026_0002
化合物 21  Compound 21
化合物 22: leq化合物 19溶解于 3mL NMP ( 1-甲基 -2-吡咯烷酮） 中， 0°C下向 混合物中加入 3eq DIPEA (Ν,Ν-二异丙基乙胺）和 2eq甲磺酰氯， 得到的混合液搅拌 lh后， 用 TFA (三氟乙酸） 酸化， 反相 HPLC柱分离得到化合物 22。 Compound 22: leq compound 19 was dissolved in 3 mL of NMP (1-methyl-2-pyrrolidone), and 3 eq of DIPEA (Ν, Ν-diisopropylethylamine) and 2 eq of methanesulfonyl chloride were added to the mixture at 0 °C. After the resulting mixture was stirred for 1 h, it was acidified with TFA (trifluoroacetic acid),
Figure imgf000026_0003
Figure imgf000026_0003
化合物 22  Compound 22
化合物 23:  Compound 23:
Figure imgf000026_0004
化合物 23
Figure imgf000026_0004
Compound 23
步骤 1 : 化合物 18(2.5 g)加入 50 mL盐酸饱和的甲醇溶液中， 室温搅拌 3小时后 将溶剂减压旋干得到固体中间体 Y(2.2 g)。  Step 1 : Compound 18 (2.5 g) was added to 50 mL of EtOAc EtOAc.
上述得到的固体中间体 Y (500 mg)加入到 15mL干燥的 DMF溶液当中， 加入溴 代异丙烷 (472 mg， 分两次加入)， 无水碳酸钾 (353 mg)和碘化钾 (212 mg)， 50°C加热搅 拌 20小时后， 减压除掉 DMF, 随后加入 35mL DCM/MeOH (5:1)并过滤， 滤液浓縮 后用  The solid intermediate Y (500 mg) obtained above was added to 15 mL of dry DMF solution, bromoisopropane (472 mg, added in two portions), anhydrous potassium carbonate (353 mg) and potassium iodide (212 mg), After heating and stirring at 50 ° C for 20 hours, DMF was removed under reduced pressure, followed by 35 mL of DCM / MeOH (5:1) and filtered.
Figure imgf000027_0001
Figure imgf000027_0001
1H-NMR(400MHZ,DMSO): 1H-NMR (400 MHZ, DMSO):
0.58-0.65 (m，2H) ,0.82-0.91 (m，2H) ,1.18-1.32 (d,6H) ,1.79-1.93 (m，2H) ,1.95-2.08 (m,2H) ,2.74-2.93 (m,2H) ,3.00-3.15 (m,2H) ,3.39-3.58 (m,3H) ,7.18 (d,2H) ,7.60 ( s,lH) ,7.82 (d,2H) ,8.23 ( s,lH) ,9.08 ( s,lH) ,10.05 ( s,lH)。 化合物 24:  0.58-0.65 (m, 2H), 0.82-0.91 (m, 2H), 1.18-1.32 (d, 6H), 1.79-1.93 (m, 2H), 1.95-2.08 (m, 2H), 2.74-2.93 (m , 2H), 3.00-3.15 (m, 2H), 3.39-3.58 (m, 3H), 7.18 (d, 2H), 7.60 (s, lH), 7.82 (d, 2H), 8.23 (s, lH), 9.08 ( s,lH) , 10.05 ( s,lH). Compound 24:
Figure imgf000027_0002
Figure imgf000027_0002
化合物 24  Compound 24
中间体 Y (720 mg)加入到 72mL 乙腈溶液当中，无水碳酸钾 (640 mg),碘化钾 (370 mg)和溴乙烷 （900 mg， 分批次加入)， 50°C加热搅拌 48小时后， 减压除掉乙腈， 随 后加入 50mL DCM/MeOH(5:l)并过滤， 滤液浓縮用二氯甲烷和甲醇（DCM： MeOH=20:l)过 Intermediate Y (720 mg) was added to 72 mL of acetonitrile, anhydrous potassium carbonate (640 mg), potassium iodide (370) Mg) and ethyl bromide (900 mg, added in portions), heated and stirred at 50 ° C for 48 hours, acetonitrile was removed under reduced pressure, then 50 mL DCM / MeOH (5: 1) was added and filtered. Methyl chloride and methanol (DCM: MeOH = 20: l)
Figure imgf000028_0001
Figure imgf000028_0001
24  twenty four
1H-NMR(400MHZ,DMSO): 1H-NMR (400 MHZ, DMSO):
0.55-0.68 (m，2H) ,0.79-0.81 (m，2H) ,1.22-1.31 (m，3H) ,1.72-1.91 (m，2H) ,1.95-2.12 (m,2H) ,2.73-2.91 (m,2H) ,2.93-3.08 (m,2H) ,3.10-3.22 (m,2H) ,3.48-3.62 (m,2H) ,7.19 (d,2H) ,7.60 ( s,lH) ,7.86 (d,2H) ,8.23 ( s,lH) ,9.09 ( s,lH) ,10.06 ( s,lH) . 化合物 25:  0.55-0.68 (m, 2H), 0.79-0.81 (m, 2H), 1.22-1.31 (m, 3H), 1.72-1.91 (m, 2H), 1.95-2.12 (m, 2H), 2.73-2.91 (m , 2H), 2.93-3.08 (m, 2H), 3.10-3.22 (m, 2H), 3.48-3.62 (m, 2H), 7.19 (d, 2H), 7.60 (s, lH), 7.86 (d, 2H) ), 8.23 ( s, lH) , 9.09 ( s, lH) , 10.06 ( s, lH) . Compound 25:
Figure imgf000028_0002
Figure imgf000028_0002
化合物 25  Compound 25
中间体 Y (500 mg)加入到 15mL干燥的 DMF溶液当中，加入溴代环戊烷 (572 mg, 分两次加入)， 无水碳酸钾 (353 mg)和碘化钾 (212 mg)， 80 °C加热搅拌 20小时后， 减 压除掉 DMF, 随后加入 35mL DCM/MeOH(5:l)并过滤， 滤液浓縮后用二氯甲烷和甲 醇 (DCM: MeOH=20:l)过柱得到化合物 25 (200 mg)。  Intermediate Y (500 mg) was added to 15 mL of dry DMF solution, bromocyclopentane (572 mg, added in two portions), anhydrous potassium carbonate (353 mg) and potassium iodide (212 mg), 80 °C After heating and stirring for 20 hours, DMF was removed under reduced pressure, followed by 35 mL of DCM / MeOH (5: 1) and filtered, and the filtrate was concentrated and then purified to afford compound 25 with dichloromethane and methanol (DCM: MeOH = 20:1) (200 mg).
Figure imgf000028_0003
Figure imgf000028_0003
中间体 25  Intermediate 25
1H-NMR(400MHZ,DMSO)： 0.62-0.63(m,2H),0.85-0.87(m,2H),1.63-1.65(m,4H),1.68-1.71(m,8H) ,1.83-1.91(m,lH),2.1 5-2.16(m,2H),2.86-2.87(m,2H),3.11-3.13(m,2H) ,7.18(d,2H)„7.56(s,lH) ,7.81(d,2H) ,8.21( s,lH) ,9.07(s,lH),9.98(s,lH) 1H-NMR (400 MHZ, DMSO): 0.62-0.63 (m, 2H), 0.85-0.87 (m, 2H), 1.63-1.65 (m, 4H), 1.68-1.71 (m, 8H), 1.83-1.91 (m, lH), 2.1 5-2.16 ( m,2H),2.86-2.87(m,2H),3.11-3.13(m,2H),7.18(d,2H)„7.56(s,lH) ,7.81(d,2H) ,8.21( s,lH) , 9.07 (s, lH), 9.98 (s, lH)
化合物 26:  Compound 26:
Figure imgf000029_0001
Figure imgf000029_0001
化合物 26 中间体 Y(600 mg)加入到 50mL DMF溶液当中，无水碳酸钾 (530 mg)，碘化钾 (256 mg)和溴代环己烷 （lg， 分批次加入)， 100°C加热搅拌 48小时后， 减压除掉 DMF， 随后加入 50mL DCM/MeOH(5:l)并过滤， 滤液浓縮用二氯甲烷和甲醇 (DCM： MeOH=20:l)过柱得化合物 26 (32 mg, yield: 5% )。  Compound 26 Intermediate Y (600 mg) was added to 50 mL of DMF solution, anhydrous potassium carbonate (530 mg), potassium iodide (256 mg) and bromocyclohexane (lg, added in portions), heated and stirred at 100 °C. After 48 hours, DMF was removed under reduced pressure, followed by 50 mL DCM / MeOH (5: 1) and filtered. The filtrate was concentrated and purified with dichloromethane and methanol (DCM: MeOH = 20:1) to afford compound 26 (32 mg , yield: 5%).
1H-NMR(400MHZ,DMSO)：  1H-NMR (400 MHZ, DMSO):
0.61-0.68 0.61-0.68
( m，2H ) ,0.83-0.91(m,2H),1.08-1.48(m,6H),1.56-1.64(m,lH),1.78-1.86(m,4H),1.97-2.04 (m,4H),2.71-2.75(m,lH),2.82-2.87(m,lH),2.90-3.08(m,2H),3.37-3.44(m,2H),7.24(d,2H,8. 8Hz),7.83(d, 2H,8.8Hz)  (m, 2H), 0.83-0.91 (m, 2H), 1.08-1.48 (m, 6H), 1.56-1.64 (m, lH), 1.78-1.86 (m, 4H), 1.97-2.04 (m, 4H) , 2.71-2.75 (m, lH), 2.82-2.87 (m, lH), 2.90-3.08 (m, 2H), 3.37-3.44 (m, 2H), 7.24 (d, 2H, 8. 8 Hz), 7.83 ( d, 2H, 8.8Hz)
实施例 2 Example 2
化合物 27的制备：  Preparation of Compound 27:
步骤 1 :  step 1 :
中间体 X中加入 20mL干燥乙腈溶解后， 加入 450μί (2.6mmol) DIPEA (Ν,Ν- 二异丙基乙胺）和 2.6mmol环丁基胺。混合物在室温下搅拌 5h后，加入 20mL 7N NH3 的甲醇溶液， 得到的混合液搅拌过夜， 真空浓縮， 用 0-7%的 DCM (二氯甲烷） 甲醇 溶液过硅胶柱， 分离得到中间体 2， 收率 75%。 After dissolving 20 mL of dry acetonitrile in Intermediate X, 450 μί (2.6 mmol) of DIPEA (Ν,Ν-diisopropylethylamine) and 2.6 mmol of cyclobutylamine were added. The mixture was stirred 5h at rt, was added 20mL 7N NH 3 in methanol, the resulting mixture was stirred overnight, concentrated in vacuo, 0-7% in DCM (dichloromethane) in methanol through a silica gel column to give intermediate separation 2, the yield is 75%.
Figure imgf000030_0001
Figure imgf000030_0001
中间体 2  Intermediate 2
步骤 2:  Step 2:
步骤 1得到的中间体 2(0.44mmol)溶解于 lOmL NMP中，加入 1.5eq(0.66mmol) 的 m-CPBA， 混合物在室温下搅拌 30min后， 混合物中加入 leq (0.44mmol)的 pTSA 和 2eq (0.88mmol) 的 4-吗啉基苯胺， 得到的混合液在 110°C下搅拌 2-4h， 冷却到室 温后， 用 lOOmL以上的乙酸乙酯稀释， 然后用饱和碳酸钠溶液洗涤， 再用水洗， 真 空浓縮， 反相 HPLC分离得到化合物 27。 The intermediate 2 (0.44 mmol) obtained in Step 1 was dissolved in 10 mL of NMP, and 1.5 eq (0.66 mmol) of m-CPBA was added. After the mixture was stirred at room temperature for 30 min, leq (0.44 mmol) of pTSA and 2 eq ( 0.88 mmol) of 4-morpholinylaniline, the resulting mixture was stirred at 110 ° C for 2-4 h, cooled to room temperature, diluted with more than 100 mL of ethyl acetate, then washed with saturated sodium carbonate solution and then washed with water. Concentration in vacuo and reversed phase HPLC gave compound 27.
Figure imgf000030_0002
Figure imgf000030_0002
化合物 27  Compound 27
化合物 28-45、 106-114、 118可以用中间体 2与相应的胺通过上述方法制备得到。 化合物 28-45、 106-114、 118的结构见表 2， 其中， X为 C， R3、 R6均为 H，Compounds 28-45, 106-114, and 118 can be prepared by the above method using Intermediate 2 and the corresponding amine. The structures of the compounds 28-45, 106-114, and 118 are shown in Table 2, wherein X is C, and R 3 and R 6 are both H.
R4均为环丁基。 R4 is a cyclobutyl group.
Figure imgf000030_0003
Figure imgf000031_0001
Figure imgf000030_0003
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000032_0001
上述表 2 中相应的胺可以市购得到， 不能市购得到的， 可以通过前述方法制备 得到。 化合物 46: 将化合物 43与 TFA/DCM(1:1)混合后， 室温下搅拌， 反应完成后得 到的混合液真空 反相 HPLC分离 化合物 46。 The corresponding amines in Table 2 above are commercially available and are not commercially available and can be prepared by the aforementioned methods. Compound 46: After compound 43 was mixed with TFA/DCM (1:1), the mixture was stirred at room temperature, and the mixture obtained after completion of the reaction was subjected to vacuum reverse phase HPLC to isolate compound 46.
Figure imgf000032_0002
Figure imgf000032_0002
化合物 46  Compound 46
化合物 47: leq化合物 46溶解于 3mL NMP中，加入 3eq DIPEA和 2eq溴乙腈， 得到的混合液在室 得到化合物 47 ; Compound 47: leq compound 46 was dissolved in 3 mL of NMP, 3 eq of DIPEA and 2 eq of bromoacetonitrile were added, and the resulting mixture was obtained in the compound 47 ;
Figure imgf000032_0003
Figure imgf000032_0003
化合物 47  Compound 47
化合物 48: lOOmg 化合物 46溶解于 3mL DMF中，密封管中 120 °C下搅拌 2天， 用水稀释， TFA酸化， 反相 HPLC柱分离得到化合物 48。
Figure imgf000033_0001
Compound 48: 100 mg Compound 46 was dissolved in 3 mL of DMF, stirred in a sealed tube at 120 ° C for 2 days, diluted with water, acidified by TFA, and isolated on a reverse phase HPLC column to afford compound 48.
Figure imgf000033_0001
化合物 48  Compound 48
化合物 49: leq化合物 46溶解于 3mL NMP中， 0°C下向混合物中加入 3eq DIPEA 和 2eq甲磺酰氯， 得到的混合液搅拌 lh后， 用 TFA酸化， 反相 HPLC柱分离得到化 合物 49。 Compound 49: leq compound 46 was dissolved in 3 mL of NMP, and 3 eq of DIPEA and 2 eq of methanesulfonyl chloride were added to the mixture at 0 ° C, and the resulting mixture was stirred for 1 hour, then acidified with TFA, and then purified to afford compound 49.
Figure imgf000033_0002
Figure imgf000033_0002
化合物 49  Compound 49
化合物 50: 100mg( leq) 化合物 46溶解于 5mL DCE、5mL二氧六环和 5eq DIPEA 的混合液中， 搅拌下， 向混合液中加入 20eq丙酮， 得到的反应液搅拌 lh后， 加入 10eq醋酸和 5eq NaBH(OAc)3， 反应液搅拌过夜， 加入 lmL水后， 真空浓縮干燥， 得 到的产物用甲醇 Compound 50: 100 mg (leq) Compound 46 was dissolved in a mixture of 5 mL of DCE, 5 mL of dioxane and 5 eq of DIPEA, and 20 eq of acetone was added to the mixture with stirring. The resulting reaction mixture was stirred for 1 h, then 10 eq of acetic acid and 5 eq NaBH(OAc) 3 , the reaction solution was stirred overnight, added with 1 mL of water, and then concentrated and evaporated in vacuo.
Figure imgf000033_0003
Figure imgf000033_0003
化合物 50  Compound 50
用化合物 46和溴乙烷为原料， 用与化合物 24类似的成方法可以得到化合物 51.
Figure imgf000034_0001
Using compound 46 and ethyl bromide as starting materials, a compound similar to compound 24 can be used to obtain compound 51.
Figure imgf000034_0001
化合物 51  Compound 51
用化合物 46与溴代环戊烷为原料， 用与化合物 25类似的合成方法可以得到化 合物 52. Using compound 46 and bromocyclopentane as starting materials, a compound similar to compound 25 can be used to obtain a compound.
Figure imgf000034_0002
Figure imgf000034_0002
化合物 52  Compound 52
用化合物 46与溴代环己烷为原料， 用与化合物 26类似的合成方法可以得到化 合物 53.
Figure imgf000034_0003
Using compound 46 and bromocyclohexane as starting materials, a compound similar to compound 26 can be used to obtain compound 53.
Figure imgf000034_0003
化合物 53 实施例 3  Compound 53 Example 3
化合物 54-87可以用实施例 1和实施例 2类似的方法制备。  Compound 54-87 can be prepared in a similar manner to Example 1 and Example 2.
表 3列出了化合物 54-87制备所用的区别原料以及得到的目标化合物的结构， 其中， R2、 为 表 3 化合物 54-87所用部分原料及目标化合物结构 Table 3 lists the different starting materials used in the preparation of the compound 54-87 and the structure of the obtained target compound, wherein R 2 , Table 3 Partial raw materials and target compound structures used in compounds 54-87
Figure imgf000035_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000037_0001
实施例 4  Example 4
化合物 88-96的制备:  Preparation of Compound 88-96:
首先制备  First preparation
Figure imgf000037_0002
Figure imgf000037_0002
中间体 3  Intermediate 3
步骤 1 :  step 1 :
在 25毫升的无水乙醇溶液中加入中间体 X( 10毫摩尔）及 R3R4NH(25毫摩尔）， 反应混合物在 35°C下搅拌过夜， 反应结束后减压抽干溶剂， 所得混合物过柱分离得 到产物 3-1。
Figure imgf000038_0001
Intermediate (10 mmol) and R 3 R4NH (25 mmol) were added to a solution of 25 ml of anhydrous ethanol, and the reaction mixture was stirred at 35 ° C overnight. After completion of the reaction, the solvent was evaporated under reduced pressure and the mixture obtained. Column separation gave product 3-1.
Figure imgf000038_0001
3-1  3-1
步骤 2:  Step 2:
在 100 毫升的圆底三口反应瓶中加入步骤 1 得到的产物 3-1 ( 10 毫摩尔），
Figure imgf000038_0002
( 12毫摩尔）， DMF (25 mL), 反应混合物在 75-80°C下搅 拌反应 5小时。 反应结束后过柱分离 （石油醚： 乙酸乙酯 （100: 0-0： 100) 得产物 3-2。
Figure imgf000038_0003
Add the product 3-1 (10 mmol) obtained in Step 1 to a 100 mL round bottom three-neck reaction flask.
Figure imgf000038_0002
(12 mmol), DMF (25 mL), and the reaction mixture was stirred at 75-80 ° C for 5 hours. After the completion of the reaction, the column was separated (petroleum ether: ethyl acetate (100: 0-0: 100) to give product 3-2.
Figure imgf000038_0003
3-2  3-2
步骤 3:  Step 3:
在 100毫升的圆底三口反应瓶中加入步骤 2得到的产物 3-2 ( 10毫摩尔）， LiOH ( IN, 10 mL) ,MeOH(25 mL), 反应混合物在 25 °C下搅拌反应 10小时。 反应结束后 加入 1N盐酸调节 pH至 1。 反应产物用乙酸乙酯萃取， 干燥， 减压除去溶剂， 得产 物 3-3。
Figure imgf000038_0004
The product obtained in Step 2 was added 3-2 (10 mmol), LiOH (IN, 10 mL), MeOH (25 mL), and the reaction mixture was stirred at 25 ° C for 10 hours in a 100 ml round bottom three-neck reaction flask. . After the end of the reaction, 1N hydrochloric acid was added to adjust the pH to 1. The reaction product was extracted with EtOAc.
Figure imgf000038_0004
R2 R 2
3-3 步骤 4: 3-3 Step 4:
在在 100毫升的圆底三口反应瓶中加入 DMF (25毫升），步骤 3得到的产物 3-3 Add DMF (25 ml) to a 100 ml round bottom three-neck reaction vial, product obtained in step 3 3-3
( 10毫摩尔）， 三乙胺 （10毫摩尔）， HOBt ( 10毫摩尔）， 反应混合物在室温搅拌反 应 1小时， 在搅拌下通入无水氨气 （450毫升）， 室温反应 5小时。 反应结束后加入 冰水 100毫升。 乙酸乙酯萃取反应液三次 （100mLx3)， 合并乙酸乙酯萃取液， 以无 水 Na2S04干燥一小时。 减压除去溶剂， 过柱分离得产物 3-4。 (10 mmol), triethylamine (10 mmol), HOBt (10 mmol), and the reaction mixture was stirred at room temperature for 1 hour, and anhydrous ammonia (450 ml) was added under stirring, and reacted at room temperature for 5 hours. After the reaction, 100 ml of ice water was added. The reaction mixture was extracted three times with ethyl acetate (100 mL×3), and ethyl acetate extracts were combined and dried over anhydrous Na 2 SO 4 for one hour. The solvent was removed under reduced pressure and the product was isolated by column.
Figure imgf000039_0001
Figure imgf000039_0001
3-4  3-4
步骤 5:  Step 5:
在 100毫升的圆底三口反应瓶中加入二氯甲烷（25毫升），步骤 4得到的产物 3-4 ( 10毫摩尔） 及三氟乙酸 （5毫升）， 0 °C下搅拌 6小时， 反应完毕后减压除去溶剂 得中间体 3A-1到 3A-10o  Dichloromethane (25 ml) was added to a 100 ml round bottom three-necked reaction flask, and the product obtained in the step 4 (3-4 mmol) and trifluoroacetic acid (5 ml) was stirred at 0 ° C for 6 hours. After completion, the solvent was removed under reduced pressure to give intermediates 3A-1 to 3A-10.
表 4: 部分中间体 3结构  Table 4: Part of the intermediates 3 structure
Figure imgf000039_0002
Figure imgf000040_0001
Figure imgf000039_0002
Figure imgf000040_0001
化合物 88-96制备：  Compound 88-96 Preparation:
在 5毫升的三口瓶中加入无水四氢呋喃（1毫升），分别加入中间体 3A-1到 3A-10  Add anhydrous tetrahydrofuran (1 ml) to a 5 ml three-necked flask and add intermediates 3A-1 to 3A-10, respectively.
）， 0 °C下搅拌 1小时，然后加入 R13S02C1或 R14COCl
Figure imgf000040_0002
0°C下反应 3小时。 反应完毕后过柱分离得化合物), stirring at 0 °C for 1 hour, then adding R 13 S0 2 C1 or R 14 COCl
Figure imgf000040_0002
The reaction was carried out at 0 ° C for 3 hours. After the reaction is completed, the column is separated to obtain a compound.
88-96。 表 5 化合物 88-96结构 88-96. Table 5 Compound 88-96 structure
Figure imgf000040_0003
Figure imgf000040_0003
实施例 5、  Example 5
可通过本领域中已知的各种方法测定本发明化合物在体外和体内抑制 syk激酶和 jak激酶的活性。 IC5Q值越小， 抑制 syk/jak活性的能力越高。 （IC5Q值为使 syk/jak蛋白水 解活性抑制 50%所需化合物的浓度。） The compounds of the invention inhibit the activity of syk kinase and jak kinase in vitro and in vivo by a variety of methods known in the art. The smaller the IC 5Q value, the higher the ability to inhibit syk/jak activity. (IC 5Q is the concentration of the compound required to inhibit syk/jak proteolytic activity by 50%.)
用于检测和测定对 syk的抑制活性的体外测定方法如下：  The in vitro assay for detecting and determining the inhibitory activity against syk is as follows:
抑制 syk酪氨酸磷酸化活性  Inhibition of syk tyrosine phosphorylation activity
通过测定实验化合物抑制 syk介导的 syk特异性的酪氨酸磷酸化的能力，评价候选 分子抑制 syk酪氨酸磷酸化活性的效力。 Candidates were evaluated by measuring the ability of the test compound to inhibit syk-mediated syk-specific tyrosine phosphorylation The potency of the molecule to inhibit syk tyrosine phosphorylation activity.
采用由 Perkin Elmer Life and Analytical Sciences (Boston, MA)开发的 LANCE™ 技术， 测定 SYK酪氨酸磷酸化活性。 LANCE™是指均相时间分辨荧光分析， 该方法 采用例如时间分辨荧光共振能量转移测定技术 （TR-FRET) (通常参见 Perkin Elmer Application Note-如何优化用基于时间分辨荧光分析的 LANCE检测的酪氨酸激酶测定 (How to Optimize a Tyrosine Kinase Assay Using Time Resolved Fluorescence-Based LANCE Detection)中的方法）。该测定主要涉及利用从磷酸特异性铕标记抗体转移到 作为受体的链亲和素 -别藻蓝素的能量转移测定磷酸化底物。  SYK tyrosine phosphorylation activity was determined using the LANCETM technology developed by Perkin Elmer Life and Analytical Sciences (Boston, MA). LANCETM refers to homogeneous time-resolved fluorescence analysis using, for example, time-resolved fluorescence resonance energy transfer assay (TR-FRET) (see generally Perkin Elmer Application Note - How to optimize tyrosine detected by LANCE based on time-resolved fluorescence analysis) Method in How to Optimize a Tyrosine Kinase Assay Using Time Resolved Fluorescence-Based LANCE Detection). This assay is primarily concerned with the determination of phosphorylated substrates by energy transfer from a phospho-specific purine-labeled antibody to a streptavidin-allophycocyanin as a receptor.
为测试候选分子抑制 SYK酪氨酸磷酸化活性的能力，将分子在 30%的 DMSO中复 溶， 按 1:3的比例进行系列稀释， 最终稀释液含 DMSO但不含候选分子。 在测定中， 最终 DMSO浓度为 3%。 通过两部分反应进行激酶实验。 第一部分反应为激酶反应， 反应物含有候选分子、 全长活性重组 SYK酶 （Millipore, CA) 和生物素标记的 SYK 特异性底物即生物素 -DEEDYESP-OH。 第二部分反应涉及激酶反应的终止且同时加 入检测试剂-铕标记的抗磷酸酪氨酸试剂 （Eu-W1024-PY100， Perkin Elmer, Boston, MA)和链亲和素 -别藻蓝素检测试剂（SA-APC,Prozyme， CA)。 在黑色 U形底 96孔微 量板中进行激酶反应。 用含 50mM Tris pH7.5、 5mM Mgcl2 BlmM DTT的缓冲液稀释 至终反应体积为 50μί， 含 ΙηΜ终浓度的活性 SYK酶、 550nM SYK底物和 ΙΟΟμΜ ATP。 使反应在室温下进行 1小时。淬灭缓冲液含有 100 mM Tris pH7.5、 300mM NaCl、 20mM EDTA, 0.02%Brii35和 0.5BSA。 按以下稀释度， 将检测试剂加入反应混合物： 对于 EU-W1024-PY100而言，按 1:500加入，对于 SA-APC而言，按 1:250加入。通过加入 50μΙ^ 含检测试剂的淬灭缓冲液将激酶反应终止， 在室温下检测 1小时。 在抑制剂存在或不 存在的情况下， 在 TR-FRET仪 Analyst HT (Molecular Probes, Sunnyvale, CA) 中检 测磷酸化底物， 用 CriterionHost Release2.0 (Molecular Probes, Sunnycale, CA) 设置 测定条件。 使用的参数设置如下： 激发波长 360nm， 发射波长 665-7.5nm， 分束器 350nm50/50, 闪光 100脉冲， 迟滞 60us， 积分 400us， z-高度 2mm。 与不存在抑制剂的 情况相比， 根据在抑制剂的存在下观察到的最大响应计算 SYK-酪氨酸激酶活性的抑 制。 通过非线性回归分析得出 IC5Q值， 在表 6中以 1。5()范围形式列出。 To test the ability of candidate molecules to inhibit SYK tyrosine phosphorylation activity, the molecules were reconstituted in 30% DMSO and serially diluted 1:3. The final dilution contained DMSO but no candidate molecules. In the assay, the final DMSO concentration was 3%. Kinase experiments were performed by a two-part reaction. The first part of the reaction is a kinase reaction containing a candidate molecule, a full-length active recombinant SYK enzyme (Millipore, CA) and a biotin-labeled SYK-specific substrate, biotin-DEEDYESP-OH. The second part of the reaction involves the termination of the kinase reaction and the simultaneous addition of the detection reagent-铕-labeled anti-phosphotyrosine reagent (Eu-W1024-PY100, Perkin Elmer, Boston, MA) and streptavidin-allophycocyanin detection reagent (SA-APC, Prozyme, CA). The kinase reaction was carried out in a black U-bottom 96-well microplate. It was diluted with a buffer containing 50 mM Tris pH 7.5, 5 mM MgCl 2 BlmM DTT to a final reaction volume of 50 μί, containing ΙηΜ final concentration of active SYK enzyme, 550 nM SYK substrate and ΙΟΟμΜ ATP. The reaction was allowed to proceed at room temperature for 1 hour. The quenching buffer contained 100 mM Tris pH 7.5, 300 mM NaCl, 20 mM EDTA, 0.02% Brii 35 and 0.5 BSA. The test reagent was added to the reaction mixture at the following dilution: For EU-W1024-PY100, add 1:500, for SA-APC, add 1:250. The kinase reaction was terminated by the addition of 50 μM containing quenching buffer containing the detection reagent, and detected at room temperature for 1 hour. Phosphorylated substrates were detected in TR-FRET Analyzer Analyst HT (Molecular Probes, Sunnyvale, CA) in the presence or absence of inhibitors, and assay conditions were set using Criterion Host Release 2.0 (Molecular Probes, Sunnycale, CA). The parameters used are set as follows: excitation wavelength 360 nm, emission wavelength 665-7.5 nm, beam splitter 350 nm 50/50, flash 100 pulses, hysteresis 60 us, integral 400 us, z-height 2 mm. The inhibition of SYK-tyrosine kinase activity was calculated from the maximum response observed in the presence of the inhibitor compared to the absence of the inhibitor. The IC 5Q value was obtained by nonlinear regression analysis and was 1 in Table 6. The 5() range is listed.
用于检测和测定抑制 jak激酶的活性的体外测定方法如下：  In vitro assays for detecting and determining the activity of inhibiting jak kinase are as follows:
可通过本领域中已知的各种方法 （例如测试化合物抑制人血浆 jak激酶活性的能 力的试验）测定本发明化合物对人 jak激酶活性的作用， 通过 IC5Q值测定本发明化合物 显示出的对人 jak激酶抑制的有效亲和力。 得到的 IC5()值在表 6中列出。 The ability to inhibit human plasma jak kinase activity can be determined by various methods known in the art (eg, test compounds) Test of Force) The effect of the compound of the present invention on human jak kinase activity was determined, and the effective affinity of the compound of the present invention for inhibition of human jak kinase was determined by IC 5Q value. The resulting IC 5 () values are listed in Table 6.
表 6提供了本发明化合物在 syk和 jak实验中的活性： +++++=IC5Q < 0.001 μΜ， ++++=0.001μΜ < IC50 < Ο.ΟΙμΜ, +++=0.01μΜ < IC50 < Ο.ΙμΜ, ++=0.1μΜ < IC50 < ΙμΜ,
Figure imgf000042_0001
Table 6 provides the activity of the compounds of the invention in the syk and jak experiments: +++++=IC 5Q < 0.001 μΜ, ++++=0.001 μΜ < IC 50 < Ο.ΟΙμΜ, +++=0.01μΜ < IC 50 < Ο.ΙμΜ, ++=0.1μΜ < IC 50 < ΙμΜ,
Figure imgf000042_0001
表 6 本发明化合物在 syk和 jak实验中的活性  Table 6 Activity of the compounds of the invention in syk and jak experiments
Figure imgf000042_0002
Figure imgf000042_0002
Figure imgf000043_0001
ΐεθΐΐΐ/ ΟΖ OAV +++ ++ 08
Figure imgf000043_0001
Ϊ́εθΐΐΐ/ ΟΖ OAV +++ ++ 08
++ +++ 6L  ++ +++ 6L
++ +++ 8乙  ++ +++ 8 B
++ +++ LL  ++ +++ LL
++ +++ 9L  ++ +++ 9L
++ ++ iL  ++ ++ iL
++ ++ PL  ++ ++ PL
++ ++ £L  ++ ++ £L
++ ++ ZL  ++ ++ ZL
++ +++ \L  ++ +++ \L
++ +++ 0L  ++ +++ 0L
++ ++ 69  ++ ++ 69
++ ++ 89  ++ ++ 89
++ ++ L9  ++ ++ L9
++ ++ 99  ++ ++ 99
++ +++ 99  ++ +++ 99
++ +++ P9  ++ +++ P9
+++ +++ £9  +++ +++ £9
+++ +++ Z9  +++ +++ Z9
+++ ++ 19  +++ ++ 19
+++ ++ 09  +++ ++ 09
+++ ++ 6i  +++ ++ 6i
++ ++ 8S  ++ ++ 8S
++ ++ Li  ++ ++ Li
+++ +++ 9i  +++ +++ 9i
++ +++ ςς  ++ +++ ςς
++ +++ ρς  ++ +++ ρς
++ +++ ζς  ++ +++ ζς
+++ ++ ζς  +++ ++ ζς
ΐεθΐΐΐ/ ΟΖ OAV 81 ++ Ϊ́εθΐΐΐ/ ΟΖ OAV 81 ++
82 ++  82 ++
83  83
84  84
85  85
86  86
87  87
88  88
89  89
90  90
91  91
92  92
93  93
94  94
95  95
96  96
所有本发明化合物均为有效的 Syk和 /或 jak激酶抑制剂， 在某些实施方案中， 本 发明化合物可为 syk/jak双重抑制剂， 也就是说它们在一定程度上既抑制 syk激酶， 又 抑制 jak激酶。 在其他实施方案中， 本发明化合物可选择性抑制 syk激酶， 但不显著抑 制一种或多种 jak激酶。 在其他实施方案中， 本发明化合物可选择性抑制 jak激酶， 但 不显著抑制一种或多种 syk激酶。 实施例 6 部分化合物进行 JAK2, JAK3和 SYK的抑制活性筛选 All of the compounds of the present invention are effective S yk and / or jak kinase inhibitors, in certain embodiments, the compounds of the present invention can be syk / jak dual inhibitors, that is they are to some extent while suppressing syk kinase, Also inhibits the kak kinase. In other embodiments, the compounds of the invention selectively inhibit syk kinase, but do not significantly inhibit one or more jak kinases. In other embodiments, the compounds of the invention selectively inhibit jak kinase, but do not significantly inhibit one or more syk kinases. Example 6 Screening of Inhibitory Activity of JAK3, JAK3 and SYK by Partial Compounds
一. 实验目的  I. Experimental purpose
测定 5个化合物： 16， 8， 9， 7和 25对激酶 JAK2, JAK3和 SYK的抑制活性。 选取 100 nM 的浓度进行初筛， 分别计算相应浓度的化合物对不同酶的活性抑制率。 根据 初筛的结果， 进一步测定上述 5个化合物对 JAK2, JAK3和 SYK酶的半数抑制浓度 IC50 值。  The inhibitory activities of five compounds: 16, 8, 9, 7 and 25 against the kinases JAK2, JAK3 and SYK were determined. The concentration of 100 nM was selected for preliminary screening, and the inhibition rate of the activity of the corresponding enzymes on different enzymes was calculated. Based on the results of the preliminary screening, the IC50 values of the half inhibitory concentrations of the above five compounds against JAK2, JAK3 and SYK enzymes were further determined.
二. 材料和仪器 2104 EnVision® Multilabel Reader (PerkinElmer) Materials and instruments 2104 EnVision® Multilabel Reader (PerkinElmer)
White Opaque 384-well low volume MicroPlate(Cat.3674, Corning)  White Opaque 384-well low volume MicroPlate (Cat. 3674, Corning)
HTRF kinEASE TK (Cat.62TKOPEC, Cisbio)  HTRF kinEASE TK (Cat.62TKOPEC, Cisbio)
JAK2酶 (Cat: 08-045, Carna)  JAK2 enzyme (Cat: 08-045, Carna)
JAK3酶 (Cat: 08-045, Carna)  JAK3 enzyme (Cat: 08-045, Carna)
SYK酶 (Cat: 08-176, Carna)  SYK enzyme (Cat: 08-176, Carna)
ATP 10 mM (Cat.PV3227, Invitrogen)  ATP 10 mM (Cat.PV3227, Invitrogen)
DTT 1 M (Cat.D5545, Sigma)  DTT 1 M (Cat.D5545, Sigma)
MgCl2 1 M (Cat.M8266, Sigma) MgCl 2 1 M (Cat.M8266, Sigma)
MnCl2 1 M (Cat.244589, Sigma) MnCl 2 1 M (Cat.244589, Sigma)
三. 实验步骤  III. Experimental steps
1 试剂配制  1 reagent preparation
JAK2 ， JAK3和 SYK三种激酶试剂配制  JAK2, JAK3 and SYK three kinase reagents
种激酶的反应体系各组分及浓度表  Kinase reaction system components and concentration table
Figure imgf000046_0001
Figure imgf000046_0001
1 X JAK2酶缓冲液 （Enzymatic Buffer) ：  1 X JAK2 Enzyme buffer (Enzymatic Buffer):
ImL I X激酶缓冲液 （Kinase Buffer) 中含有 200 L 5 X酶缓冲液， 5 μ L 1M MgCl2, 1 μ L 1M DTT, 794 μ L ddH20 (去离子水）。 ImL IX kinase buffer (Kinase Buffer) contains 200 L 5 X enzyme buffer, 5 μL 1M MgCl 2 , 1 μL 1M DTT, 794 μL ddH 2 0 (deionized water).
1 X JAK3酶缓冲液 （Enzymatic Buffer) ：  1 X JAK3 Enzyme buffer (Enzymatic Buffer):
ImL 1 X激酶缓冲液中含有 200 μ L 5 X酶缓冲液， 5 μ L 1M MgCl2, 1 L 1M DTT, 794 μ L ddH20。 ImL 1 X kinase buffer contains 200 μL of 5 X enzyme buffer, 5 μL of 1M MgCl 2 , 1 L of 1 M DTT, and 794 μL of ddH 2 0.
I X SYK酶缓冲液：  I X SYK enzyme buffer:
ImL I X激酶缓冲液中含有 200 L 5 X酶缓冲液， 5 μ L 1M MgCl2, 1 L 1M MnCl2, 1 μ L 1M DTT, 793 μ L ddH20。 5 X酪氨酸激酶底物 （Substrate- TK) 和 ATP工作液 ImL IX kinase buffer contains 200 L 5 X enzyme buffer, 5 μL 1M MgCl 2 , 1 L 1M MnCl 2 , 1 μL 1M DTT, 793 μL ddH 2 0. 5 X tyrosine kinase substrate (Substrate-TK) and ATP working solution
酪氨酸激酶底物和 ATP的具体浓度见表 7。  The specific concentrations of tyrosine kinase substrate and ATP are shown in Table 7.
用 I X激酶缓冲液 （Kinase Buffer) 稀释 Substrate-TK (酪氨酸激酶底物） 和 ATP 至反应浓度的 5倍。  Substrate-TK (tyrosine kinase substrate) and ATP were diluted to 5 times the reaction concentration with I X kinase buffer (Kinase Buffer).
5 X酶工作液  5 X enzyme working fluid
三种激酶 （JAK2 ,JAK3和 SYK) 的浓度优化已在之前的工作中完成， 筛选浓度 见表 7。 用 I X激酶缓冲液配制 5 X酶工作液。  The concentration optimization of the three kinases (JAK2, JAK3 and SYK) has been completed in the previous work, and the screening concentration is shown in Table 7. The 5 X enzyme working solution was prepared using I X kinase buffer.
4 X Sa-XL665 工作液  4 X Sa-XL665 working fluid
Sa-XL665在反应中的浓度参见表 1。 用检测缓冲液 （Detection Buffer ) 配制 4 X Sa-XL665 工作液。  See Table 1 for the concentration of Sa-XL665 in the reaction. Prepare 4 X Sa-XL665 working solution with Detection Buffer.
4 X TK-Ab-cryptate 工作液  4 X TK-Ab-cryptate working fluid
用检测缓冲液 将 TK-Ab-Cryptate稀释 100倍作为工作液。  TK-Ab-Cryptate was diluted 100 times as a working solution with assay buffer.
2. 实验流程  2. Experimental process
所有试剂按照上述方法配好后， 除酶外， 平衡到室温以后， 开始进行加样。 生物素标记的酪氨酸激酶 （TK-biotin ) 底物， ATP， 酶以及一定浓度的化合物 在 50 mM Hepes/NaOH H 7.0， 0.02 NaN3 , 0.01% BSA , 0.1 mM原钒酸盐 ( orthovanadate) , 5 mM MgCl2, 1 mM MnCl2, 1 mM DTT溶液中室温反应。 待测 化合物的抑制浓度进行 4倍梯度稀释 ， 使用 2.5%的 DMSO作为共溶剂。 向所有反应孔 中加入 Ιθ μΐ 经 50 mM Hepes/NaOH pH 7.0， 0.1% BSA, 0.8 M KF, 20 mM EDTA稀释 的 Streptavidin-XL665 和 TK antibody europium cryptate(l : 100) (铕荧光纳米颗粒标记 的酪氨酸抗体） 混合检测液， 室温反应 lh后， 用 ENVISION (Perkinelmer) 仪器检测 荧光信号（320 nm刺激, 665 nm， 615 nm发射）。通过全活性孔和背景信号孔计算出 每个孔的抑制率， 复孔取平均值， 同时用专业的画图分析软件 PRISM 5.0 对每个待测 化合物进行半数抑制活性 （IC50 ) 的拟合。 After all the reagents were prepared as described above, the enzyme was added to the room temperature after the enzyme was added to the room temperature. Biotinylated tyrosine kinase (TK-biotin) substrate, ATP, enzyme and a certain concentration of compound in 50 mM Hepes/NaOH H 7.0, 0.02 NaN 3 , 0.01% BSA, 0.1 mM orthovanadate (orthovanadate) , 5 mM MgCl 2 , 1 mM MnCl 2 , 1 mM DTT solution was reacted at room temperature. The inhibitory concentration of the test compound was subjected to a 4-fold gradient dilution using 2.5% DMSO as a cosolvent. Ιθ μΐ was added to all wells. Streptavidin-XL665 and TK antibody europium cryptate (l: 100) diluted with 50 mM Hepes/NaOH pH 7.0, 0.1% BSA, 0.8 M KF, 20 mM EDTA (铕 fluorescent nanoparticle-labeled Tyrosine antibody) The mixture was mixed and assayed for 1 h at room temperature. The fluorescence signal (320 nm stimulation, 665 nm, 615 nm emission) was detected using an ENVISION (Perkinelmer) instrument. The inhibition rate of each well was calculated from the fully active wells and the background signal wells, and the duplicate wells were averaged, and the half-inhibitory activity (IC50) of each test compound was fitted using a professional drawing analysis software PRISM 5.0.
实验加样流程图如下：  The experimental sample flow chart is as follows:
Figure imgf000047_0001
冲液 液
Figure imgf000047_0001
Liquid
τκ 2 L 2 μ  Τκ 2 L 2 μ
Substrate -biotin L 2 L  Substrate -biotin L 2 L
激酶 2 μ L 2 μ L激酶缓冲液 2 μ L  Kinase 2 μL 2 μL Kinase Buffer 2 μL
把板密封， 室温下孵育 10分钟  Seal the plate and incubate for 10 minutes at room temperature
ATP 2 μ L 2 L 2 μ L  ATP 2 μ L 2 L 2 μ L
把板密封， 室温下孵育  Seal the plate and incubate at room temperature
检测步骤 (10 μ L)  Detection step (10 μL)
Sa-XL665 5 L 5 L 5 L  Sa-XL665 5 L 5 L 5 L
TK-Ab-Cry板 5 L 5 L 5 L  TK-Ab-Cry board 5 L 5 L 5 L
把板密封， 室温下孵育 1小时  Seal the plate and incubate for 1 hour at room temperature
320 nm激发， 665 nm， 615 nm发射  320 nm excitation, 665 nm, 615 nm emission
3. 数据分析  3. Data analysis
发射光比率 （ER， Emission Ratio)= 665 nm发射信号 / 615 nm发射信号 抑制率 = (ER 阳性一 ER样品) /(ER 隱一 ER 酣) *100%  Emission Ratio ( ER, Emission Ratio) = 665 nm Emission Signal / 615 nm Emission Signal Repression Rate = (ER Positive - ER Sample) / (ER 隐 ER 酣) *100%
运用软件 Graphpad Prism 5 并采用计算公式 log (抑制剂）对标准化响应 (normalized response) -可变斜率（Variable slope) 进行 IC50曲线拟合并计算出 IC50 值.  Using the software Graphpad Prism 5 and using the calculation formula log (inhibitor) to normalize the response - variable slope (Variable slope) IC50 curve fitting and calculate the IC50 value.
四 结果  Four results
1)待测化合物在 100 nM对 JAK2, JAK3 禾 B SYK抑制活性测定  1) Determination of the inhibitory activity of the test compound at 100 nM against JAK2, JAK3 and B SYK
应用 HTRF kinEASE TK试剂盒检测化合物 16、 8、 9、 7、 25在 100 nM时对 JAK2, JAK3 和 SYK 的抑制活性， 同时选取星孢菌素 作为参考化合物。  The inhibitory activities of compounds 16, 8, 9, 7, 25 on JAK2, JAK3 and SYK at 100 nM were measured using the HTRF kinEASE TK kit, and staurosporin was selected as a reference compound.
图 1显示了所有化合物 16、 8、 9、 7、 25对 JAK2， JAK3 和 SYK的抑制率。 Figure 1 shows the inhibition rates of JAK2, JAK3 and SYK for all compounds 16, 8, 9, 7, and 25.
2)化合物 16、 8、 9、 7、 25对 JAK2, JAK3 和 SYK酶的半数抑制浓度 IC50值测 定 2) Compounds 16, 8, 9, 7, 25 for the half-inhibitory concentration of JAK2, JAK3 and SYK enzymes IC50 value determination
应用 HTRF kinEASE TK试剂盒检测 5个化合物 16、 8、 9、 7、 25对 JAK2， JAK3 和 SYK酶的半数抑制浓度 IC50值。 进行 JAK2禾 B JAK3 测定时， 化合物 16、 8、 9、 7、 25的作用终浓度从 10 μΜ开始， 4倍梯度稀释， 10个浓度； 进行 SYK测定时， 化合物 16、 8、 9、 25从 100 μΜ开始， 4倍梯度稀释， 10个浓度， 化合物 7从 10 μΜ 开始 4倍梯度稀释， 10个浓度， 控制 DMSO的终浓度为 1%， 每个浓度为复孔测试。 并同步检测参考化合物星孢菌素对 JAK2， JAK3 和 SYK酶的半数抑制浓度，从 1 μΜ 开始 4倍梯度稀释， 10个浓度， 控制 DMSO浓度为 1%， 每个浓度为复孔测试。 实 验结果见图 2-4。 实施例 Ί 化合物对 JAK1 的抑制活性筛选 The half-inhibitory concentration IC50 values of the five compounds 16, 8, 9, 7, 25 against JAK2, JAK3 and SYK enzymes were determined using the HTRF kinEASE TK kit. For the determination of JAK2 and B JAK3, the final concentration of the compounds 16, 8, 9, 7, 25 was started from 10 μΜ, 4 times gradient dilution, 10 concentrations; when performing SYK determination, compounds 16, 8, 9, 25 were Starting at 100 μΜ, 4× gradient dilution, 10 concentrations, Compound 7 was diluted 4 fold from 10 μΜ, 10 concentrations, and the final concentration of DMSO was controlled at 1%, and each concentration was a duplicate well test. Simultaneously, the half-inhibitory concentration of the reference compound staurosporin on JAK2, JAK3 and SYK enzymes was detected, and a 4-fold gradient was diluted from 1 μΜ, 10 concentrations, and the DMSO concentration was controlled to 1%, and each concentration was a duplicate well test. The experimental results are shown in Figure 2-4. EXAMPLES 筛选 Screening of inhibitory activity of compounds against JAK1
一. 实验目的  I. Experimental purpose
测定 5个化合物 16、 8、 9、 7、 25 JAK1的抑制活性。 选取 100 nM 的浓度进 行初筛， 分别计算相应浓度的化合物对酶的活性抑制率。根据初筛的结果判断， 进一 步测定上述 5个化合物对 JAK1酶的半数抑制浓度 IC50值。  The inhibitory activities of five compounds 16, 8, 9, 7, 25 JAK1 were determined. The concentration of 100 nM was selected for preliminary screening, and the inhibition rate of the enzyme activity of the corresponding concentration of the compound was calculated. Based on the results of the preliminary screening, the IC50 value of the half inhibitory concentration of the above five compounds against the JAK1 enzyme was further determined.
二. 材料和仪器  Materials and instruments
Spectra Max M5 Plate Reader (Molecular Devices)  Spectra Max M5 Plate Reader (Molecular Devices)
Corning low volume black Opaque 384-well MicroPlate (Cat.3676, Corning) Z' -Lyte™ Kinase Assay试剂盒 - TYR 6 Peptide (Cat: PV4122, Invitrogen) Z' -LYTE™ Tyr 6肽底物 （Cat: PV4123, Invitrogen)  Corning low volume black Opaque 384-well MicroPlate (Cat.3676, Corning) Z'-LyteTM Kinase Assay Kit - TYR 6 Peptide (Cat: PV4122, Invitrogen) Z'-LYTETM Tyr 6 Peptide Substrate (Cat: PV4123 , Invitrogen)
Z' -LYTE™ Tyr 6 磷酸化 -月太 （Cat: PV4124, Invitrogen)  Z'-LYTETM Tyr 6 Phosphorylation - Yuetai (Cat: PV4124, Invitrogen)
5X 激酶缓冲液 (HEPES 250 mM (pH7.5), 50 mM MgC12, 5 mM EGTA , 0.05% Brij-35)(Cat: PV3189, Invitrogen)  5X Kinase Buffer (HEPES 250 mM (pH 7.5), 50 mM MgC12, 5 mM EGTA, 0.05% Brij-35) (Cat: PV3189, Invitrogen)
10 mM ATP (Cat: PV3227, Invitrogen)  10 mM ATP (Cat: PV3227, Invitrogen)
检测试齐 U (Development Reagent) A (Cat: PV3297, Invitrogen)  U.Development Reagent A (Cat: PV3297, Invitrogen)
检测缓冲液 (Development Buffer) (Cat: P3127, Invitrogen)  Development Buffer (Cat: P3127, Invitrogen)
终止试剂 （Stop Reagent) (Cat: P3094, Invitrogen)  Stop Reagent (Cat: P3094, Invitrogen)
JAK1 (Cat: PV4774, Invitrogen)  JAK1 (Cat: PV4774, Invitrogen)
三. 实验步骤  III. Experimental steps
1 试剂配制  1 reagent preparation
I X激酶缓冲液 A  I X Kinase Buffer A
用无菌水稀释 5 X激酶缓冲液到 1 X激酶缓冲液 A， 此时 lx 激酶缓冲液 A含 有 HEPES 50 mM (pH7.5), 10 mM MgCl2, ImM EGTA和 0.01% Brij-35。 整个激酶反 应中， 采用 IX激酶缓冲液 A稀释 ATP溶液， 底物， 酶和化合物。 The 5 X kinase buffer was diluted with sterile water to 1 X Kinase Buffer A, at which point lx Kinase Buffer A contained HEPES 50 mM (pH 7.5), 10 mM MgCl 2 , ImM EGTA and 0.01% Brij-35. ATP solutions, substrates, enzymes and compounds are diluted with IX Kinase Buffer A throughout the kinase reaction.
4 X 化合物工作液  4 X compound working fluid
待测化合物用 DMSO溶解至 10 μΜ作为储存液， 各取 1 加入 24 1 X 激酶 缓冲液 Α中，得到 400 nM 含 4% DMSO的化合物溶液，每个稀释溶液各取 2.5 加 入 384孔板中，这样在最后的 10 激酶反应体系中化合物的终浓度就分别是 100 nM 并含有 1% DMSO, IC50测定时稀释方法与此类似。 2X Z' -LYTE™肽底物 /JAK1 工作液 The test compound was dissolved in DMSO to 10 μL as a stock solution, and each was added to 24 1 X kinase buffer , to obtain 400 nM compound solution containing 4% DMSO, and each diluted solution was added to 2.5 384-well plate. Thus, the final concentration of the compound in the final 10 kinase reaction system was 100 nM and contained 1% DMSO, and the dilution method was similar when IC50 was determined. 2X Z' -LYTETM Peptide Substrate / JAK1 Working Fluid
用 1 X 激酶缓冲液 A配制肽底物和 JAK1酶至反应终浓度的 2倍。例如配制 300 μΐ 2 X Z' -LYTE™肽底物 /JAK1 工作液，用 IX 激酶缓冲液 A将 Z' -LYTE™ Tyr 6 肽底物稀释到 4 μΜ (1.2 μΐ 1 mM Z' -LYTE™ Tyr 6肽底物） ， 同时将 JAK1稀释为 10 ng/μΐ (10 μΐ 310 ng/μΐ JAK1), 对于化合物筛选试验， 加入 5 μΐ 2Χ Z' -LYTE™肽 底物 /JAK1 工作液到酶反应体系中， 最终 Z' -LYTE™肽底物和 JAK1的终浓度都是 分别为 2 μΜ和 50 ng/孔。  The peptide substrate and JAK1 enzyme were formulated with 1 X kinase buffer A to a concentration of 2 times the final concentration. For example, prepare 300 μΐ 2 XZ′ -LYTETM Peptide Substrate/JAK1 Working Solution and dilute Z'-LYTETM Tyr 6 peptide substrate to 4 μΜ (1.2 μΐ 1 mM Z' -LYTETM Tyr with IX Kinase Buffer A. 6 peptide substrate), and simultaneously diluted JAK1 to 10 ng/μΐ (10 μΐ 310 ng/μΐ JAK1). For compound screening test, add 5 μΐ 2Χ Z′ -LYTETM peptide substrate/JAK1 working solution to the enzyme reaction system. The final concentrations of the final Z'-LYTETM peptide substrate and JAK1 were 2 μΜ and 50 ng/well, respectively.
2X磷酸化-肽 (Phospho-peptide) 工作液  2X Phospho-peptide working solution
用 1 X 激酶缓冲液 A溶液配制 2X磷酸化 -肽工作液。 例如吸取 1.2 μΐ of 100 μΜ Z' -LYTE™ Tyr 11磷酸化 -肽至 28.8 W of IX激酶缓冲液 A充分混匀， 此工作液作 为 100% 磷酸化（phosphorylation)对照孔使用，代替 2X Z' -LYTE™肽底物 /JAK1 工 作液， 对于化合物筛选试验， 加入 5 μΐ 2 Χ Z' -LYTETM Phospho-Peptide工作液到酶 反应体系中， 最终 Z' -LYTE™磷酸化-肽的终浓度为 2 μΜ。 A 2X phosphorylated-peptide working solution was prepared using a 1 X Kinase Buffer A solution. For example, pipette 1.2 μΐ of 100 μΜ Z'-LYTETM Tyr 11 Phospho-peptide to 28.8 W of IX Kinase Buffer A and mix thoroughly. This working solution is used as a 100% phosphorylation control well instead of 2X Z'. -LYTETM peptide substrate/JAK1 working solution, for compound screening test, add 5 μΐ 2 Χ Z' -LYTE TM Phospho-Peptide working solution to the enzyme reaction system, and finally the final concentration of Z'-LYTETM phosphorylation-peptide It is 2 μΜ.
4ΧΑΤΡ工作液  4ΧΑΤΡ working fluid
用 1 X激酶缓冲液 Α配制酶工作液。例如取 3 μΐ 10 mM ATP储液 至 lj 72 μΐ IX 激酶缓冲液 Α得到 400 μΜ工作液， 加入 2 .5 μΐ ATP工作液到反应体系中， 最终 ATP的用量为 100 μΜ。  The enzyme working solution was prepared using 1 X kinase buffer. For example, take 3 μΐ 10 mM ATP stock solution to lj 72 μΐ IX kinase buffer Α to get 400 μΜ working solution, add 2.5 μΐ ATP working solution to the reaction system, and finally use ATP 100 μΜ.
3 X检测混合液  3 X detection mixture
根据检测试剂 （Development Reagent ) B 的 COA 说明， 用检测缓冲液 ( Development Buffer) 1： 128倍稀释检测试齐 U (Development Reagent) A配制 3 X 检测混合液， 例如配制 256 μΐ 3Χ检测混合液， 需要 2 μΐ检测试剂 （Development Reagent) B 储液以及 254 μΐ lx检测缓冲液 （ Development Buffer), 充分混匀， 不 要涡旋。 对于化合物筛选试验， 加入 5 W 3 Χ 检测混合液到酶反应体系中。  According to the COA instructions of the Development Reagent B, use the Development Buffer 1:128 dilution test to prepare the 3 X detection mixture, for example, prepare a 256 μΐ 3Χ detection mixture. Require 2 μΐ Development Reagent B stock solution and 254 μl lx Development Buffer, mix well, and do not vortex. For the compound screening test, 5 W 3 Χ was added to detect the mixture into the enzyme reaction system.
2. 实验流程  2. Experimental process
所有试剂按照上述方法配好后， 除酶外， 平衡到室温以后， 开始进行加样。 After all the reagents were prepared as described above, the enzyme was added to the room temperature after the enzyme was added to the room temperature.
Z ' -LYTE™ Tyr 6 肽底物底物， ATP， 酶以及一定浓度的化合物在 50 mM Hepes/NaOH H 7.5, 0.01% NaN3， 5 mM MgC12, 1 mM EGTA和 0.01% Brij-35溶 液中室温反应 60分钟。 向所有反应孔中加入 5 μΐ 经 IX检测缓冲液 （ Development Buffer)稀释的检测试剂（Development Reagent) A混合检测液， 室温反应 lh后， 向 所有反应孔中加入 5 μΐ Stop Reagent来终止反应，用 Spectra Max M5仪器检测荧光信 号 （405 nm刺激, 435 nm和 530 nm发射）。 通过全活性孔和背景信号孔计算出每个 孔的抑制率， 复孔取平均值， 同时用专业的画图分析软件 PRISM 5.0 对每个待测化 合物进行抑制率的图示。 Z ' -LYTETM Tyr 6 peptide substrate, ATP, enzyme and a certain concentration of compound in 50 mM Hepes/NaOH H 7.5, 0.01% NaN3, 5 mM MgC12, 1 mM EGTA and 0.01% Brij-35 solution at room temperature Reaction for 60 minutes. Add 5 μΐ of Development Reagent A mixed test solution diluted with IX Detection Buffer to all wells, and react at room temperature for 1 h. The reaction was stopped by adding 5 μΐ Stop Reagent to all wells and the fluorescence signal (405 nm stimulation, 435 nm and 530 nm emission) was detected using a Spectra Max M5 instrument. The inhibition rate of each well was calculated from the fully active wells and the background signal wells, and the average value of the duplicate wells was plotted, and the inhibition rate of each test compound was graphically analyzed using a professional drawing analysis software PRISM 5.0.
实验加样流程图如下:  The experimental sample flow chart is as follows:
Figure imgf000051_0001
Figure imgf000051_0001
3. 数据分析  3. Data analysis
发射光比率 (ER)= 435 nm发射信号 / 530 nm发射信号  Emission ratio (ER) = 435 nm emission / 530 nm emission
100%抑制对照的平均发射光比率记为： ER100%  The average emission ratio of the 100% inhibition control is recorded as: ER100%
0%抑制对照的平均发射光比率记为： ER0%  The average emission ratio of the 0% inhibition control is recorded as: ER0%
计算抑制率  Calculated inhibition rate
抑制率用以下公式计算：  The inhibition rate is calculated using the following formula:
抑制率 = (ER样品- ER0 )/ (ER100 - ER0 ) X 100  Inhibition rate = (ER sample - ER0) / (ER100 - ER0 ) X 100
运用软件 Graphpad Prism 5 并采用计算公式 log (抑制剂）对标准化响应 ( normalized response) -可变斜率 ( Variable slope)进行 IC50曲线拟合并计算出 IC50 值.  The IC50 curve was fitted to the normalized response - Variable slope using the software Graphpad Prism 5 and the calculation formula log (inhibitor) was used to calculate the IC50 value.
四 结果  Four results
1)待测化合物在 100 nM时对 JAK1 的抑制活性测定  1) Determination of the inhibitory activity of the test compound at 100 nM against JAK1
应用 Z' -Lyte™ Kinase Assay试剂盒 - Tyr 6肽检测 5个化合物在 100 nM时对 JAK1 的抑制活性， 同时选取星孢菌素 作为参考化合物。 图 5显示了所有待测化合 物对 JAK1酶的抑制率。 2)待测化合物对 JAK1 酶的半数抑制浓度测定 The Z'-LyteTM Kinase Assay Kit - Tyr 6 peptide was used to detect the inhibitory activity of 5 compounds on JAK1 at 100 nM, and staurosporin was selected as a reference compound. Figure 5 shows the inhibition rate of the JAK1 enzyme by all the compounds to be tested. 2) Determination of the half-inhibitory concentration of the test compound on JAK1 enzyme
应用 Z' -LyteTM Kinase Assay试剂盒 - Tyr 6肽检测 5个化合物 16、 8、 9、 7、 25 对 JAK1酶的半数抑制浓度 IC50值。 化合物 8、 9、 7、 25从 100 μΜ开始 4倍梯度稀 释， 10个浓度； 化合物 16从 10 μΜ开始 4倍梯度稀释， 10个浓度， 控制 DMSO终 浓度为 1%， 每个浓度为复孔测试。 并同步检测参考化合物星孢菌素对 JAK1酶的半 数抑制浓度， 从 1 μΜ开始 4倍梯度稀释， 10个浓度， 控制 DMSO浓度为 1%， 每个 浓度为复孔测试。 实验结果见图 6。 The half-inhibitory concentration IC50 value of the five compounds 16, 8, 9, 7, 25 against the JAK1 enzyme was determined using the Z'-Lyte TM Kinase Assay Kit - Tyr 6 peptide. Compounds 8, 9, 7, 25 were diluted 4 times from 100 μΜ, 10 concentrations; Compound 16 was diluted 4 fold from 10 μΜ, 10 concentrations, control DMSO final concentration was 1%, each concentration was duplicate test. Simultaneously, the half-inhibitory concentration of the reference compound staurosporin against the JAK1 enzyme was detected, and a 4-fold gradient dilution was performed from 1 μΜ, 10 concentrations, and the DMSO concentration was controlled to 1%, and each concentration was a duplicate well test. The experimental results are shown in Figure 6.
总结：  to sum up:
Figure imgf000052_0001
Figure imgf000052_0001
实施例 8 使用 BaF3/EpoR/JAK2和 BaF3/EpoR/JAK2V617F细胞 （下称 BEJ细胞， 可以通过 QUINTA' S-CARDAMA et al， BLOOD, 15 APRIL 2010， VOLUME 115, NUMBER 15， 3109-3117中第 3110页 METHODS部分第一段所述的方法由商购的细 胞得到。） 对化合物 25、 化合物 26进行体外测定对细胞株生长的影响 Example 8 BaF3/EpoR/JAK2 and BaF3/EpoR/JAK2V617F cells (hereinafter referred to as BEJ cells, which can be passed through QUINTA' S-CARDAMA et al, BLOOD, 15 APRIL 2010, VOLUME 115, NUMBER 15, 3109-3117, 3110 The method described in the first paragraph of the METHODS section is obtained from commercially available cells.) Effect of in vitro determination of compound 25 and compound 26 on cell line growth
药效学研究。  Pharmacodynamic research.
试验方法（参考 QUINTA' S-CARDAMA et al， BLOOD, 15 APRIL 2010， VOLUME 115， NUMBER 15， 3109-3117第 3110页 METHODS部分的 Cell proliferation assay、 immunoblotting部分)：  Test methods (Ref. QUINTA' S-CARDAMA et al, BLOOD, 15 APRIL 2010, VOLUME 115, NUMBER 15, 3109-3117, p. 3110, Cell proliferation assay, immunoblotting part of the METHODS section):
BEJ细胞接种在 2000/孔白色底 96孔板中， 然后加入不同浓度的化合物 （0.2 % DMSO的终浓度）的处理， 并在 37 °C， 5 C02下孵育 48小时。使用 GLO ( Promega 公司） 荧光素酶试剂测定细胞内 ATP。 并根据数据分析 IC50。  BEJ cells were seeded in 2000/well white 96-well plates, then treated with different concentrations of compound (0.2% DMSO final concentration) and incubated for 48 hours at 37 °C, 5 C02. Intracellular ATP was measured using GLO (Promega) luciferase reagent. And based on the data analysis IC50.
同时， 使用 Western Blot测定药物对 JAK2信号传导途径磷酸化的影响。  At the same time, Western Blot was used to determine the effect of drugs on the phosphorylation of JAK2 signaling pathway.
结果： IC50 (化合物 25 ) : 1.248uM  Results: IC50 (Compound 25): 1.248uM
IC50 (化合物 26 ) : 0.7352uM 实施例 9 应用 CCK-8检测试剂盒检测 5个候选化合物 （16、 8、 9、 7、 25)对 4 个肿瘤细胞株 （RPMI-8226, K562, HL-60 & THP-1 ) 的细胞毒性 IC50值； 以及采用 CellTite-Glo检测试剂盒检测 5个候选化合物 （16、 8、 9、 7、 25 ) 对 2个肿瘤细胞株 (WSU-DLCL2 & L1210) 的细胞毒性 IC50值。 IC50 (compound 26) : 0.7352uM Example 9 Cytotoxicity of 5 candidate compounds (16, 8, 9, 7, 25) against 4 tumor cell lines (RPMI-8226, K562, HL-60 & THP-1) was detected using the CCK-8 assay kit. IC50 values; and Cyto IC50 values of 5 candidate compounds (16, 8, 9, 7, 25) against 2 tumor cell lines (WSU-DLCL2 & L1210) were detected using the CellTite-Glo assay kit.
1、 材料和方法  1, materials and methods
细胞株：  Cell line:
RPMI-8226 人多发性骨髓瘤细胞株 （订购于美国 ATCC)  RPMI-8226 Human Multiple Myeloma Cell Line (Order from ATCC, USA)
K562人慢性髓原白血病细胞株 （订购于中科院上海细胞资源中心）  K562 human chronic myeloid leukemia cell line (ordered at the Shanghai Cell Resource Center of the Chinese Academy of Sciences)
HL-60人原髓细胞白血病细胞株 （订购于中科院上海细胞资源中心）  HL-60 human myeloid leukemia cell line (ordered at the Shanghai Cell Resource Center of the Chinese Academy of Sciences)
WSU-DLCL2 人 B细胞淋巴瘤细胞株 （订购于美国 ATCC)  WSU-DLCL2 Human B Cell Lymphoma Cell Line (Order from ATCC, USA)
THP-1人单核细胞白血病细胞株 （订购于中科院上海细胞资源中心）  THP-1 human monocyte leukemia cell line (ordered at the Shanghai Cell Resource Center of the Chinese Academy of Sciences)
L1210 小鼠白血病细胞株 （订购于中科院上海细胞资源中心）  L1210 mouse leukemia cell line (ordered at the Shanghai Cell Resource Center of the Chinese Academy of Sciences)
2、 试剂和耗材：  2. Reagents and consumables:
Cell Counting Kit-8 (Cat# CK04-13, Dojindo)  Cell Counting Kit-8 (Cat# CK04-13, Dojindo)
CellTiter-Glo® Luminescent Cell Viability Assay (Cat# G7572, Promega)  CellTiter-Glo® Luminescent Cell Viability Assay (Cat# G7572, Promega)
96孔培养板 （Cat# 3599， Cat# 3903, Corning Costar)  96-well culture plate (Cat# 3599, Cat# 3903, Corning Costar)
胎牛血清 （Cat#10099-141， GIBCO)  Fetal bovine serum (Cat#10099-141, GIBCO)
培养基 (In vitro gen )  Medium (In vitro gen )
台式酉每标仪 SpectraMax M5 Microplate Reader ( Molecular Devices )  Benchtop 酉 SpectroMax M5 Microplate Reader ( Molecular Devices )
微孔板检测仪 2104 EnVision® Multilabel Reader (PerkinElmer)  Microplate Detector 2104 EnVision® Multilabel Reader (PerkinElmer)
3、 实验步骤  3, the experimental steps
3.1 试剂配制  3.1 reagent preparation
1 ) 培养基的配制  1) Preparation of the medium
Figure imgf000053_0001
L1210 DMEM+10 FBS
Figure imgf000053_0001
L1210 DMEM+10 FBS
2) 化合物的制备：  2) Preparation of the compound:
用 DMSO稀释化合物使终浓度为 10mM。  The compound was diluted with DMSO to a final concentration of 10 mM.
3.2 IC50实验  3.2 IC50 experiment
收集对数生长期细胞， 计数， 用完全培养基重新悬浮细胞， 调整细胞浓度至合适 浓度 （依照细胞密度优化试验结果确定）， 接种 96孔板， 每孔加 100 μ ΐ细胞悬液。 细胞在 37 。C， 100 %相对湿度， 5 % C02培养箱中孵育 24小时。  Count the logarithmic growth phase cells, count, resuspend the cells with complete medium, adjust the cell concentration to the appropriate concentration (determined according to the cell density optimization test results), inoculate 96-well plates, and add 100 μM cell suspension per well. The cells are at 37. C, 100% relative humidity, incubated in a 5 % C02 incubator for 24 hours.
用培养基将待测化合物稀释至 500 μ Μ后按稀释倍数依次梯度稀释 8 次， 按 25 μ ΐ/孔加入细胞。 化合物的作用终浓度从 100 μ Μ至 0 μ Μ， 4倍梯度稀释， 共 10 个浓度点。  The test compound was diluted to 500 μM with medium, and then serially diluted 8 times in dilution order, and added to the cells at 25 μM/well. The final concentration of the compound was from 100 μ Μ to 0 μ Μ, 4 times gradient dilution, for a total of 10 concentration points.
细胞置于 37 。C， 100 %相对湿度， 5 % C02 培养箱中孵育 72小时。  Place the cells in 37 . C, 100% relative humidity, incubate for 72 hours in a 5 % C02 incubator.
对于 RPMI-8226,K562, HL-60 和 THP-1细胞， 直接加入 10 μ ΐ 的 CCK-8 于细 胞培养基中， 置于 37 °C培养箱中孵育 2-4 小时。 轻轻震荡后在 SpectraMax M5 Microplate Reader上测定 450 nm波长处的吸光度， 以 650 nm处吸光度作为参比， 计算抑制率。  For RPMI-8226, K562, HL-60 and THP-1 cells, add 10 μM of CCK-8 directly to the cell culture medium and incubate in a 37 °C incubator for 2-4 hours. The absorbance at 450 nm was measured on a SpectraMax M5 Microplate Reader with gentle shaking, and the absorbance at 650 nm was used as a reference to calculate the inhibition rate.
对于 WSU-DLCL2和 L1210细胞， 直接加入等体积 的 CellTiter-Glo Reagent于 细胞培养基中， 轻轻震荡混匀后置于室温放置 lOmin, 在 2104 EnVision® Multilabel Reader上测定 Luminescence信号值（RLU)， 计算抑制率。  For WSU-DLCL2 and L1210 cells, add an equal volume of CellTiter-Glo Reagent directly to the cell culture medium, gently shake and mix, place at room temperature for 10 min, and measure Luminescence signal value (RLU) on a 2104 EnVision® Multilabel Reader. Calculate the inhibition rate.
3.3 数据处理  3.3 Data Processing
按下式计算药物对肿瘤细胞生长的抑制率： 肿瘤细胞生长抑制率％ = [(Ac-As)/(Ac-Ab)] X 100%  The inhibition rate of the drug on tumor cell growth was calculated as follows: Tumor cell growth inhibition rate % = [(Ac-As) / (Ac-Ab)] X 100%
As: 样品的 OA/RLU (细胞 +CCK-8+待测化合物）  As: OA/RLU of the sample (cell + CCK-8 + test compound)
Ac: 阴性对照的 OA/RLU (细胞 +CCK-8+DMSO)  Ac: negative control OA/RLU (cell + CCK-8 + DMSO)
Ab: 阳性对照的 OA/RLU (培养基 +CCK-8+DMSO)  Ab: positive control OA/RLU (medium + CCK-8 + DMSO)
运用软件 Graphpad Prism 5 并采用计算公式 log(inhibitor)vs. normalized response-Variable slope进行 IC50曲线拟合并计算出 IC50值.  Using the software Graphpad Prism 5 and using the calculation formula log(inhibitor) vs. normalized response-Variable slope, the IC50 curve is fitted and the IC50 value is calculated.
4、 实验结果  4, the experimental results
本实验测试了 5个候选化合物（16、 8、 9、 7、 25)对 6个肿瘤细胞株（RPMI-8226, K562, HL60, WSU-DLCL2, THP-1 和 L1210) 的细胞毒性作用。 实验结果如图 7-12所示，采用作图软件 Graphpad Prism 5.0 拟合出 5个化合物对各个细胞株的曲线 图和 IC50值。 化合物的作用终浓度从 100 μ Μ开始, 4倍梯度稀释, 10个浓度点. 其中 有些化合物在最高作用浓度 100 μ Μ 时对某些细胞株的细胞毒性作用的百分抑制率 未达到 50%或抑制作用不明显,无法拟合出有效可靠的 IC50值， 因此在表格中表示为 IC50>100 μ Μ或无效果 （No effect)。 This experiment tested the cytotoxic effects of five candidate compounds (16, 8, 9, 7, 25) on six tumor cell lines (RPMI-8226, K562, HL60, WSU-DLCL2, THP-1 and L1210). Experimental results As shown in Figures 7-12, the graphs and IC50 values of five compounds against each cell line were fitted using the mapping software Graphpad Prism 5.0. The final concentration of the compound was started from 100 μΜ, 4 times gradient dilution, 10 concentration points. Some of the compounds had a percent inhibition of cytotoxicity to certain cell lines at the highest concentration of 100 μΜ. Or the inhibition is not obvious, and the effective and reliable IC50 value cannot be fitted, so it is expressed in the table as IC50>100 μ Μ or No effect.
Figure imgf000055_0001
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根 据已经公开的所有教导， 可以对那些细节进行各种修改和替换， 这些改变均在本发明 的保护范围之内。 本发明的全部范围由所附权利要求及其任何等同物给出。
Figure imgf000055_0001
Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

权利要求书 claims
1. 通式 I的化合物或 水合物: 1. Compounds or hydrates of general formula I:
Figure imgf000056_0001
Figure imgf000056_0001
R2 I R 2 I
其中， 选自具有取代基或不具有取代基的芳基、 具有取代基或不具有取代 基的杂芳基、具有取代基或不具有取代基的 d-do烷基和具有取代基或不具有取 代基的 C3-C6杂 环基，其中 中的所述取代基是独立地选自下述基团的取代基: 卤素、 CN、 羟基、 C3-C6杂环基、 d— 1Q烷基磺酰基、 苯基、 螺环基、 杂螺环基、 Among them, selected from aryl groups with or without substituents, heteroaryl groups with or without substituents, d-do alkyl groups with or without substituents, and d-do alkyl groups with or without substituents. The substituent is a C 3 -C 6 heterocyclyl group, wherein the substituent in is a substituent independently selected from the following groups: halogen, CN, hydroxyl, C 3 -C 6 heterocyclyl, d— 1Q Alkylsulfonyl, phenyl, spirocyclyl, heterospiryl,
Figure imgf000056_0002
、 C1-C10烷基、 卤代 Ci-Cio Ci-Cio院基氧基、 Ci-Cio院基胺基、 C6— 10芳基、 C6— 10杂芳基、 C3-C6 杂环基羰基烷氧基； 或可选地具有选自卤素原子、 C2— 1Q烯基、 酰胺基、 CN、 酰 基、 d— 1Q烷基酰基、 d— 1Q烷基磺酰基、 d— 1Q烷基、 C3— 6环烷基、 C3-C6杂环基、 烷基氧基和 C3— 6环烷基氧基的取代基的下述基团： 酰胺基、 烷基磺酰基、 羰 基、 C6-10芳基杂环基、 C3-C6杂环基、 C3-C6杂环基酰胺基、 C3-C6杂环基羰 基、 C3-C6环烷基胺基、 磺酰基、 氨基磺酰基、 磺酰胺基、 C3-C6杂环基磺酰基；
Figure imgf000056_0002
, C1-C10 alkyl, halogenated Ci-Cio Ci- Cio acetyloxy, Ci-Cio acetylamine, C 6-10 aryl, C 6-10 heteroaryl , C 3 -C 6 heterocycle base carbonyl alkoxy group; or optionally having a halogen atom, C 2 - 1Q alkenyl group, amide group, CN, acyl group, d- 1Q alkyl acyl group, d - 1Q alkylsulfonyl group, d - 1Q alkyl group , C 3 - 6 cycloalkyl, C 3 -C 6 heterocyclyl, alkyloxy and the following groups of the substituents of C 3 - 6 cycloalkyloxy: amide group, alkylsulfonyl group, carbonyl group , C6-10 arylheterocyclyl, C3-C6heterocyclyl, C3-C6heterocyclylamide, C3-C6heterocyclylcarbonyl, C3 - C6cycloalkylamino , sulfonyl, sulfamate Acyl group, sulfonamide group, C 3 -C 6 heterocyclyl sulfonyl group;
R7、 R8、 R9独立地选自 Cw。烷基、 Cw。卤代烷基、 。烷基氧基、 Cw。烷基羰 基、 或 C3— 6环烷基， R8、 R9 可以与 N 形成一个原子数为 3-6 的环； 或
Figure imgf000056_0003
R 7 , R 8 , R 9 are independently selected from Cw. Alkyl, Cw. Haloalkyl, . Alkyloxy, Cw. Alkylcarbonyl, or C 3-6 cycloalkyl, R 8 and R 9 can form a ring with 3-6 atoms with N ; or
Figure imgf000056_0003
R1()不存在或选自 H、 d— 1Q烷基、 C3— 6环烷基、 d— 1Q烷基酰胺基、 d— 1Q烷基磷酰 胺基、 d— 1Q烷基磺酰基、 d— 1Q烷基羰基、 d— 1Q烷基磺酰胺基、 CN、 d— 1Q烷基胺 基、 C3— 6杂环基、 Cr iQ 卤代烷基、 C6— 1()芳基、 C6— 1()杂芳基、 酯基、 醛基、 d— 1() 烷基氰基、 1() έϊ代烷基磷酰胺基、 d— 1Q烷氧基羰基， R 1( ) does not exist or is selected from H, d- 1Q alkyl group, C 3-6 cycloalkyl group, d- 1Q alkyl amide group, d- 1Q alkyl phosphoryl group Amino group, d- 1Q alkylsulfonyl group, d - 1Q alkylcarbonyl group, d- 1Q alkylsulfonamide group, CN, d- 1Q alkylamino group, C3-6 heterocyclyl group, C r iQ haloalkyl group , C 6 — 1 () aryl group, C 6 — 1 () heteroaryl group, ester group, aldehyde group, d — 1 () alkyl cyano group, 1 () έϊalkyl phosphoramidyl group, d — 1Q alkoxycarbonyl,
R2、 R3和 R4独立地为 11、 具有取代基或不具有取代基的 C3-C6环烷基、 具有 取代基或不具有取代基的 d-do烷基、具有取代基或不具有取代基的 d-do烷基 胺基、 d-do烷基氧基、 具有取代基或不具有取代基的 C3-C6杂环基、 具有取代 基或不具有取代基的 C6— 1Q芳基、 具有取代基或不具有取代基的 C6—1Q杂芳基； 其 中 R2、 和 中的所述取代基是独立地选自下述基团的取代基： 卤素、 羟基、 Ci-Cio C2-Cio C2-C10块基、 基幾基、 基、 Ci-Cio l¾ ¾¾¾¾ C1-C10 烷氧基、 d-do烷氧基羰基、 C6-1Q芳基、 C6-1Q杂芳基、 C3-C6杂环基、 C3-C6环烷 基、 磺酰基、 巯基、 氰基、 C3-C6杂环基胺基、 d-do烷基胺基、 C3-C6环烷基胺 基、 C6— 1Q芳基胺基、 C6— 1Q杂芳基胺基、 d-do芳烷基氧基、 C6— 1Q苯并杂环基胺基。 R 2 , R 3 and R 4 are independently 11, a C 3 -C 6 cycloalkyl group with or without a substituent, a d-do alkyl group with or without a substituent, a substituent or d-do alkylamino group without substituent, d-do alkyloxy group, C 3 -C 6 heterocyclyl group with or without substituent, C 6 with or without substituent - 1Q aryl, C 6 - 1Q heteroaryl with or without substituents; wherein R 2 , the substituents in and are independently selected from the substituents of the following groups: halogen, hydroxyl, Ci-Cio C 2 -Cio C 2 -C 10 alkyl, carbonyl, base, Ci-Cio l¾ ¾¾¾¾ C1-C10 alkoxy, d-do alkoxycarbonyl, C 6 - 1Q aryl, C 6 - 1Q heteroaryl, C 3 -C 6 heterocyclyl, C 3 -C 6 cycloalkyl, sulfonyl, mercapto, cyano, C 3 -C 6 heterocyclyl amino, d-do alkylamino , C 3 -C 6 cycloalkylamino group, C 6 - 1Q arylamine group, C 6 - 1Q heteroarylamino group, d-do aralkyloxy group, C 6 - 1Q benzoheterocyclic amine group base.
2、 如权利要求 1 所述的化合物或其药学上可接受的盐、 异构体或水合物， 2. The compound of claim 1 or its pharmaceutically acceptable salt, isomer or hydrate,
Figure imgf000057_0001
Figure imgf000057_0001
杂环基、 卤代 d-do烷基、 d-do烷基氧基、 d-do烷基胺基、 C6— 1Q芳基、 C6_10 杂芳基、 C3-C6杂环基羰基烷氧基； 或可选地具有选自卤素原子、 d— 1Q烷基、 C3— 6 环烷基、 C3-C6杂环基、 d— 1Q烷基氧基和 C3— 6环烷基氧基的取代基的下述基团： C6— 1Q芳基杂环基、 酰胺基、 羰基、 C3-C6杂环基、 C3-C6杂环基酰胺基、 C3-C6杂 环基羰基、 c3-c6环烷基胺基、 磺酰基、 氨基磺酰基、 c3-c6杂环基磺酰基； 或 ，\^ ，其中 Y选自 N、 O或 C ; Z独立地为 0、 C、 N、
Figure imgf000058_0001
 Heterocyclyl, halogenated d-do alkyl, d-do alkyloxy, d-do alkylamine, C 6 — 1Q aryl, C 6 — 10 heteroaryl, C 3 — C 6 heterocycle base carbonyl alkoxy; or optionally having a halogen atom, d- 1Q alkyl, C 3-6 cycloalkyl, C 3 -C 6 heterocyclyl, d- 1Q alkyloxy and C 3 - The following groups of the substituents of 6 cycloalkyloxy groups: C 6 - 1Q aryl heterocyclyl group, amide group, carbonyl group, C 3 -C 6 heterocyclyl group, C 3 -C 6 heterocyclyl amide group, C3 - C6heterocyclylcarbonyl , c3 - c6cycloalkylamino , sulfonyl, aminosulfonyl, c3 - c6heterocyclylsulfonyl; or ,\^, where Y is selected from N, O or C; Z is independently 0, C, N,
Figure imgf000058_0001
R1()不存在或选自 H、 d— 1Q烷基、 C3— 6环烷基、 d— 1Q烷基酰胺基、 d— 1Q烷基磷酰 胺基、 1Q烷基磺酰基、 d— 1Q烷基羰基、 d— 1Q烷基磺酰胺基、 CN、 d— 1Q烷基胺 基、 C3— 6杂环基、 d-do卤代烷基、 C6— 1Q芳基、 C6— 1Q杂芳基、 酯基、 醛基、 Cwo 烷基氰基、 d— 1Q卤代烷基磷酰胺基、 d— 1Q烷氧基羰基， R7、 R8、 R9独立地选自 Ci— 10院基、 Ci— 10 代院基、 Ci— 10院基氧基、 Ci— 10院基羰基、 Ci— ιο 1¾代院基或 C3— 6 环烷基， R8、 R9可以与 N形成一个原子数为 3-6的环； R 1( ) does not exist or is selected from H, d- 1Q alkyl group, C 3-6 cycloalkyl group, d- 1Q alkyl amide group, d- 1Q alkylphosphoramidyl group, 1Q alkylsulfonyl group, d- 1Q alkylcarbonyl, d- 1Q alkylsulfonamide, CN, d- 1Q alkylamino, C 3-6 heterocyclyl, d-do haloalkyl, C 6-1Q aryl , C 6-1Q hetero Aryl group, ester group, aldehyde group, Cwo alkylcyano group, d- 1Q haloalkylphosphoramidyl group, d- 1Q alkoxycarbonyl group, R 7 , R 8 , R 9 are independently selected from Ci- 10 courtyard group, Ci—10 hydroxyl, Ci—10 oxy, Ci—10 carbonyl, Ci—10 1¾ cycloalkyl or C 3—6 cycloalkyl, R 8 and R 9 can form one atomic number with N It is a ring of 3-6;
R6独立地为 H或卤素； R 6 is independently H or halogen;
X选自 N、 CH或 0。 X is selected from N, CH or 0.
3. 如权利要求 1所述的化合物或其药学上可接受的盐、异构体或水合物， 其 中 R2、 R3或 R4独立地选自下述基团： H、 环丙基、 环丁基、 环戊基、 环己基、 甲基、 乙基、 丙基、 丁基、 乙烯基、 丙烯基、 丁烯基、 三氟甲基、 三氟乙基、 氯 甲基、 氯乙基、 甲氧基、 乙基氧基、 甲基羰基、 乙基羰基、 哌啶基、 哌嗪基、 吡 咯基、 咪唑基、 吡唑基、 吡咯烷基、 吡咯基、 二氢吡咯基、 吡咯啉基、 咪唑烷基、 咪唑啉基、 吡唑烷基、 喹啉基、 苯并呋喃基、 吡嗪基、 吡唑啉基、 噻二唑基、 羟 基、 氰基、 磺酰基、 吡啶基、 嘧啶基、 -CH2CH2OH、 -CH2NHEt。 3. The compound of claim 1 or a pharmaceutically acceptable salt, isomer or hydrate thereof, wherein R 2 , R 3 or R 4 are independently selected from the following groups: H, cyclopropyl, Cyclobutyl, cyclopentyl, cyclohexyl, methyl, ethyl, propyl, butyl, vinyl, propenyl, butenyl, trifluoromethyl, trifluoroethyl, chloromethyl, chloroethyl , methoxy, ethyloxy, methylcarbonyl, ethylcarbonyl, piperidyl, piperazinyl, pyrrolyl, imidazolyl, pyrazolyl, pyrrolidinyl, pyrrolyl, dihydropyrrolyl, pyrroline base, imidazolidinyl, imidazolinyl, pyrazolidinyl, quinolinyl, benzofuranyl, pyrazinyl, pyrazolinyl, thiadiazolyl, hydroxyl, cyano, sulfonyl, pyridyl, pyrimidine base, -CH 2 CH 2 OH, -CH 2 NHEt.
4. 如权利要求 2所述的化合物或其药学上可接受的盐、异构体或水合物， 其 中 4. The compound of claim 2 or a pharmaceutically acceptable salt, isomer or hydrate thereof, wherein
Figure imgf000058_0002
Figure imgf000058_0002
其中， R2、 R3、 R4、 R6、 X Y、 Z和 R1Q如权利要求 1或 2所述。 Among them, R 2 , R 3 , R 4 , R 6 , XY, Z and R 1Q are as described in claim 1 or 2.
5. 如权利要求 4所述的化合物或其药学上可接受的盐、异构体或水合物， 具 有如下结构： 5. The compound of claim 4 or its pharmaceutically acceptable salt, isomer or hydrate, having the following structure:
Figure imgf000059_0001
Figure imgf000059_0001
其中， Ru、 R12、 R13、 R14独立地选自 H、 d-do烷基、 d-d。烷基胺基、 Ci-Cio l¾ ¾¾¾¾ C3_6 i C2-Cio C C10 ¾¾氧基、 Ci-Cio Among them, R u , R 12 , R 13 , and R 14 are independently selected from H, d-do alkyl, and dd. Alkylamino, Ci-Cio l¾ ¾¾¾C 3_6 i C 2 -Cio CC 10 ¾¾oxy, Ci-Cio
羟基、 氰基、 磺酰基、 C6— 1Q芳基、 C3— 6杂环基、 卤素取代的 d-do烷基胺基、 羟 基取代的 - 。烷基胺基。 Hydroxy, cyano, sulfonyl, C 6 — 1Q aryl, C 3 — 6 heterocyclyl, halogen-substituted d-do alkylamino, hydroxyl-substituted -. Alkylamino.
6. 如权利要求 5所述的化合物或其药学上可接受的盐、异构体或水合物， 其 中所述环烷基选自环丙基、 环丁基、 环戊基和环己基。 6. The compound of claim 5 or a pharmaceutically acceptable salt, isomer or hydrate thereof, wherein the cycloalkyl group is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
7. 如权利要求 2所述的化合物或其药学上可接受的盐、异构体或水合物， 其 中 R5选自： 7. The compound of claim 2 or a pharmaceutically acceptable salt, isomer or hydrate thereof, wherein R is selected from:
Figure imgf000059_0002
〇
Figure imgf000059_0002
〇
Figure imgf000060_0001
Figure imgf000060_0001
8. 一种化合物或其药学上可接受的 异构体或水合物， 所述化合物选自
Figure imgf000060_0002
 8. A compound or a pharmaceutically acceptable isomer or hydrate thereof, the compound is selected from
Figure imgf000060_0002
化合物 2 化合物 3 Compound 2 Compound 3
化合物 8 化合物 9
Figure imgf000061_0001
12Compound 8 Compound 9
Figure imgf000061_0001
12
Figure imgf000061_0002
Figure imgf000061_0002
化合物 14 化合物 15
Figure imgf000061_0003
Compound 14 Compound 15
Figure imgf000061_0003
化合物 18 化合物 19
Figure imgf000061_0004
Compound 18 Compound 19
Figure imgf000061_0004
化合物 20 化合物 21
Figure imgf000062_0001
Compound 20 Compound 21
Figure imgf000062_0001
化合物 22 23
Figure imgf000062_0002
Compound 22 23
Figure imgf000062_0002
化合物 24 化合物 25
Figure imgf000062_0003
Figure imgf000062_0004
Compound 24 Compound 25
Figure imgf000062_0003
Figure imgf000062_0004
化合物 31 化合物 32
Figure imgf000063_0001
化合物 43 化合物 44Compound 31 Compound 32
Figure imgf000063_0001
Compound 43 Compound 44
Figure imgf000064_0001
Figure imgf000064_0001
化合物 46Compound 46
Figure imgf000064_0002
Figure imgf000064_0002
化合物 51 化合物 52 、 "s Compound 51 Compound 52 , "s
\、―、■ / \,―,■ /
Figure imgf000065_0001
Figure imgf000065_0001
化合物 54 化合物 55 化合物 56 化合物 57
Figure imgf000065_0002
Compound 54 Compound 55 Compound 56 Compound 57
Figure imgf000065_0002
化合物 61 化合物 62 化合物 63
Figure imgf000065_0003
Compound 61 Compound 62 Compound 63
Figure imgf000065_0003
化合物 67 化合物 68 化合物 69 化 化合物 73
Figure imgf000066_0001
Compound 67 Compound 68 Compound 69 Compound 73
Figure imgf000066_0001
化合物 76 化合物 77 化合物 78
Figure imgf000066_0002
Figure imgf000066_0003
Compound 76 Compound 77 Compound 78
Figure imgf000066_0002
Figure imgf000066_0003
化合物 81 化合物 82Compound 81 Compound 82
Figure imgf000066_0004
Figure imgf000066_0004
化合物 83 化合物 84
Figure imgf000067_0001
Figure imgf000067_0002
Figure imgf000067_0003
Figure imgf000068_0001
化合物 94
Figure imgf000069_0001
Compound 83 Compound 84
Figure imgf000067_0001
Figure imgf000067_0002
Figure imgf000067_0003
Figure imgf000068_0001
Compound 94
Figure imgf000069_0001
化合物 97 化合物 98
Figure imgf000069_0002
化合物 101 化合物 102
Figure imgf000070_0001
Compound 97 Compound 98
Figure imgf000069_0002
Compound 101 Compound 102
Figure imgf000070_0001
化合物 103 化合物 104
Figure imgf000070_0002
Compound 103 Compound 104
Figure imgf000070_0002
化合物 107 化合物 108
Figure imgf000070_0003
Compound 107 Compound 108
Figure imgf000070_0003
化合物 110
Figure imgf000070_0004
化合物 114 Compound 110
Figure imgf000070_0004
Compound 114
Figure imgf000071_0001
Figure imgf000071_0001
9. 一种组合物， 所述组合物含有权利要求 1-8任意一项所述的化合物或其药学 上可接受的盐、 异构体或水合物和可药用载体或赋形剂。 9. A composition containing the compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt, isomer or hydrate thereof and a pharmaceutically acceptable carrier or excipient.
10. 权利要求 1-8任意一项所述的化合物或其药学上可接受的盐、 异构体或水 合物或其组合在制备用于预防或治疗 syk激酶和 /或 jak激酶介导的病症或疾病的药物 中的用途。 10. The compound of any one of claims 1 to 8 or its pharmaceutically acceptable salt, isomer or hydrate or combination thereof in the preparation for the prevention or treatment of syk kinase and/or jak kinase mediated diseases or use in medicines for diseases.
11. 如权利要求 10所述的用途， 其中所述 syk激酶和 /或 jak激酶介导的病症或 疾病选自血液病、 过敏性哮喘、 骨髓纤维化和类风湿性关节炎。 11. The use as claimed in claim 10, wherein the syk kinase and/or jak kinase-mediated disorder or disease is selected from the group consisting of hematological diseases, allergic asthma, myelofibrosis and rheumatoid arthritis.
12. 如权利要求 11所述的用途， 其中所述 syk激酶和 /或 jak激酶介导的病症或 疾病选自白血病、 骨髓增生性疾病。 12. The use as claimed in claim 11, wherein the syk kinase and/or jak kinase-mediated disorder or disease is selected from the group consisting of leukemia and myeloproliferative diseases.
13. 如权利要求 12所述的用途， 其中所述 syk激酶和 /或 jak激酶介导的病症或 疾病选自多发性骨髓瘤、 慢性髓原白血病、 原髓细胞白血病、 B细胞淋巴瘤、 单核细 胞白血病、 脾大性红细胞增多、 嗜酸性白细胞增多综合征、 原发性血小板减少症、 系 统性巨细胞疾病。 13. Use as claimed in claim 12, wherein the syk kinase and/or jak kinase mediated disorder or disease is selected from the group consisting of multiple myeloma, chronic myelogenous leukemia, myelogenous leukemia, B-cell lymphoma, monocytogenes Nuclear cell leukemia, splenomegaly, eosinophilia syndrome, essential thrombocytopenia, systemic giant cell disease.
14. 权利要求 1-8任意一项所述的化合物或其药学上可接受的盐、 异构体或水 合物或其组合用于治疗患有 jak和 /或 syk相关疾病的方法。 14. A method of using the compound of any one of claims 1 to 8 or its pharmaceutically acceptable salt, isomer or hydrate or combination thereof for treating jak and/or syk related diseases.
15. 如权利要求 14所述的方法， 其中所述疾病选自多发性骨髓瘤、 慢性髓原白 血病、 原髓细胞白血病、 B细胞淋巴瘤、 单核细胞白血病、 脾大性红细胞增多、 嗜酸 性白细胞增多综合征、 原发性血小板减少症、 ***性巨细胞疾病。 15. The method of claim 14, wherein the disease is selected from the group consisting of multiple myeloma, chronic myeloid leukemia Blood diseases, myeloid leukemia, B-cell lymphoma, monocytic leukemia, splenomegaly, eosinophilia syndrome, essential thrombocytopenia, systemic giant cell disease.
16. 一种用于制备如权利要求 1所述的通式 I的化合物或其药学上可接受的盐、 异构体或水合物的中间体， 16. An intermediate for preparing the compound of general formula I as claimed in claim 1 or a pharmaceutically acceptable salt, isomer or hydrate thereof,
Figure imgf000072_0001
Figure imgf000072_0001
中间体 1 中间体 2 Intermediate 1 Intermediate 2
PCT/CN2014/070733 2013-01-17 2014-01-16 Triazine compound, pharmaceutical salt, isomer, or hydrate thereof, and pharmaceutical composition thereof WO2014111031A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201480003555.XA CN104870437A (en) 2013-01-17 2014-01-16 Triazine compound, pharmaceutical salt, isomer, or hydrate thereof, and pharmaceutical composition thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201310017606.1 2013-01-17
CN201310017606 2013-01-17

Publications (1)

Publication Number Publication Date
WO2014111031A1 true WO2014111031A1 (en) 2014-07-24

Family

ID=51209029

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/070733 WO2014111031A1 (en) 2013-01-17 2014-01-16 Triazine compound, pharmaceutical salt, isomer, or hydrate thereof, and pharmaceutical composition thereof

Country Status (2)

Country Link
CN (1) CN104870437A (en)
WO (1) WO2014111031A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9382246B2 (en) 2013-12-05 2016-07-05 Pharmacyclics Llc Inhibitors of Bruton's tyrosine kinase
CN109293569A (en) * 2018-11-08 2019-02-01 湘潭大学 A method of the amine reaction that turns that no catalyst participates in prepares carboxamides derivatives

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000076980A1 (en) * 1999-06-10 2000-12-21 Yamanouchi Pharmaceutical Co., Ltd. Novel nitrogen-contaiing heterocyclic derivatives or salts thereof
EP1184376A1 (en) * 1999-06-09 2002-03-06 Yamanouchi Pharmaceutical Co. Ltd. Novel heterocyclic carboxamide derivatives
WO2013047813A1 (en) * 2011-09-30 2013-04-04 大鵬薬品工業株式会社 1,2,4-triazine-6-carboxamide derivative
WO2013171690A1 (en) * 2012-05-16 2013-11-21 Novartis Ag Monocyclic heteroaryl cycloalkyldiamine derivatives
WO2013192049A2 (en) * 2012-06-22 2013-12-27 Portola Pharmaceuticals, Inc. 1,2,4-triazine-6-carboxamide kinase inhibitors

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2308821T3 (en) * 1997-12-15 2008-12-01 Astellas Pharma Inc. NEW DERIVATIVES OF PIRIMIDIN-5-CARBOXAMIDA.

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1184376A1 (en) * 1999-06-09 2002-03-06 Yamanouchi Pharmaceutical Co. Ltd. Novel heterocyclic carboxamide derivatives
WO2000076980A1 (en) * 1999-06-10 2000-12-21 Yamanouchi Pharmaceutical Co., Ltd. Novel nitrogen-contaiing heterocyclic derivatives or salts thereof
WO2013047813A1 (en) * 2011-09-30 2013-04-04 大鵬薬品工業株式会社 1,2,4-triazine-6-carboxamide derivative
WO2013171690A1 (en) * 2012-05-16 2013-11-21 Novartis Ag Monocyclic heteroaryl cycloalkyldiamine derivatives
WO2013192049A2 (en) * 2012-06-22 2013-12-27 Portola Pharmaceuticals, Inc. 1,2,4-triazine-6-carboxamide kinase inhibitors

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9382246B2 (en) 2013-12-05 2016-07-05 Pharmacyclics Llc Inhibitors of Bruton's tyrosine kinase
US9656988B2 (en) 2013-12-05 2017-05-23 Pharmacyclics Llc Inhibitors of Bruton's tyrosine kinase
CN109293569A (en) * 2018-11-08 2019-02-01 湘潭大学 A method of the amine reaction that turns that no catalyst participates in prepares carboxamides derivatives
CN109293569B (en) * 2018-11-08 2021-08-06 湘潭大学 Method for preparing formamide derivative through amine transfer reaction without participation of catalyst

Also Published As

Publication number Publication date
CN104870437A (en) 2015-08-26

Similar Documents

Publication Publication Date Title
TWI542588B (en) Novel heterocyclic compounds
JP2021020957A (en) Methods to induce targeted protein degradation through bifunctional molecules
JP2022017384A (en) Novel ulk1 inhibitors and methods for using the same
TWI433677B (en) Heterocyclic compounds and uses thereof
WO2022061251A1 (en) Compounds and methods for kras modulation and indications therefor
JP2019517559A (en) New compounds
WO2020073945A1 (en) Bicyclic derivative inhibitor, preparation method therefor, and application thereof
KR20190039567A (en) The novel compound
EP3294742B1 (en) New (5,8-dimethyl-9-phenyl-5,8-dihydro-6h-pyrazolo[3,4-h)quinazolin-2-yl)-(1h-pyrazol-5-yl)-amines and theri derivatives as igf-1r/1r inhibitors.
EP2393814A1 (en) Pyrrolopyrimidinyl axl kinase inhibitors
CN114026104B (en) COT modulators and methods of use thereof
CN104936954A (en) Compounds and their methods of use
WO2020035020A1 (en) Imidazopyridine derivative, preparation method therefor, and application thereof in medicine
CN112236429A (en) Heteroaryl compounds as type II IRAK inhibitors and uses thereof
JP6609308B2 (en) Substitutional macrocycles as kinase inhibitors
JP2020514361A (en) 9,10,11,12-Tetrahydro-8H- [1,4] diazepino [5 &#39;, 6&#39;: 4,5] thieno [3,2-F] quinolin-8-one compounds and uses thereof
EP3947375B1 (en) Imidazolonylquinoline compounds and therapeutic uses thereof
CA3172987A1 (en) Small molecule inhibitors of oncogenic chd1l with preclinical activity against colorectal cancer
TW202337432A (en) Annulated 2-amino-3-cyano thiophenes and derivatives for the treatment of cancer
TW202340209A (en) Annulated 2-amino-3-cyano thiophenes and derivatives for the treatment of cancer
WO2014111031A1 (en) Triazine compound, pharmaceutical salt, isomer, or hydrate thereof, and pharmaceutical composition thereof
CN112513027B (en) Indazole amine derivative, preparation method and medical application thereof
WO2020224607A1 (en) Ezh2 inhibitor and use thereof
EP3596064B1 (en) Tosylacetate based compounds and derivatives thereof as phgdh inhibitors
CN110869371B (en) 7-substituted pyrrolotriazine compound or pharmaceutically acceptable salt thereof, and preparation method and application thereof

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: 14740182

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205N DATED 23/09/2015)