WO2021147790A1

WO2021147790A1 - Pyrazolo[1,5-a]pyrazine derivative and preparation method therefor and use thereof

Info

Publication number: WO2021147790A1
Application number: PCT/CN2021/072362
Authority: WO
Inventors: 张盼盼; 颜孙力; 李英; 叶成; 钱文建; 胡泰山; 陈磊
Original assignee: 浙江海正药业股份有限公司
Priority date: 2020-01-22
Filing date: 2021-01-18
Publication date: 2021-07-29
Also published as: CN113150012A; CN113150012B

Abstract

The present invention relates to a pyrazolo[1,5-a]pyrazine derivative and a preparation method therefor and a use thereof in medicines. In particular, the present invention relates to a pyrazolo[1,5-a]pyrazine derivative shown in general formula (I), a preparation method therefor, a pharmaceutically acceptable salt thereof and a use thereof as a therapeutic agent, particularly as a JAKS family kinase inhibitor. The definition of each substituent group in the general formula (I) is the same as that in the description.

Description

吡唑并[1,5-a]吡嗪类衍生物及其制备方法和用途Pyrazolo[1,5-a]pyrazine derivatives and preparation method and use thereof

技术领域Technical field

本发明涉及一种吡唑并[1,5-a]吡嗪类衍生物、其制备方法及含有该衍生物的药物组合物以及其作为治疗剂特别是作为JAKS家族激酶抑制剂的用途。The present invention relates to a pyrazolo[1,5-a]pyrazine derivative, a preparation method thereof, a pharmaceutical composition containing the derivative, and its use as a therapeutic agent, especially as a JAKS family kinase inhibitor.

背景技术Background technique

酪氨酸激酶2(TYK2)是非受体型酪氨酸激酶，隶属于Janus激酶家族(JAKS)，JAKS包括JAK1、JAK2、JAK3和TYK2亚型，这4个家族成员由1100个氨基酸组成，具有高度的同源性，可以分为7个同源结构域(JH)：JH1为高度保守的激酶区，具有催化活性；JH2为激酶样区，是JAK激酶不同于其它酪氨酸激酶的独特之处，该区域不具有催化活性，但是可以调节JH1的活性；JH3-JH4是SH2结构域，可以特异性的识别和结合活化的酪氨酸残基；JH5-JH7是FERM结构域，该区域相对保守，调节JAK与受体的结合。JAK1、JAK2和TYK2在人体中广泛存在，而JAK3只存在于骨髓和淋巴等造血组织中。Tyrosine kinase 2 (TYK2) is a non-receptor tyrosine kinase that belongs to the Janus kinase family (JAKS). JAKS includes JAK1, JAK2, JAK3 and TYK2 subtypes. These four family members are composed of 1100 amino acids and have The high degree of homology can be divided into 7 homology domains (JH): JH1 is a highly conserved kinase domain with catalytic activity; JH2 is a kinase-like domain, which is the unique difference between JAK kinase and other tyrosine kinases At this point, this region does not have catalytic activity, but can regulate the activity of JH1; JH3-JH4 is the SH2 domain, which can specifically recognize and bind to activated tyrosine residues; JH5-JH7 is the FERM domain, which is relatively Conservative, regulates the binding of JAK to the receptor. JAK1, JAK2 and TYK2 are widely present in the human body, while JAK3 only exists in hematopoietic tissues such as bone marrow and lymph.

JAK激酶/信号转导与转录激活子(The Janus kinase/signal transducer and activator of transcription,JAK/STAT)信号通路介导多种细胞因子信号转导，包括白细胞介素、干扰素、***、粒细胞和巨噬细胞集落刺激因子等。细胞因子与其受体结合后引起受体分子的二聚化，使得与受体偶联的JAK激酶相互接近并通过交互的酪氨酸磷酸化而活化，活化的JAK催化受体本身的磷酸化并形成相应的STAT停靠位点，使STAT实现其磷酸化，然后STAT形成同源或异源二聚体并进入细胞核，与相应的靶基因启动子结合而激活基因转录和表达。因此，JAK激酶介导的信号通路在细胞的增殖、分化、凋亡以及炎症反应等多种生命过程中具有重要作用。但是在病理过程中，细胞因子的过多分泌导致JAK/STAT通路的过度激活，引起自身免疫病、肿瘤等疾病的发生，包括银屑病、***性红斑狼疮、类风湿性关节炎、牛皮癣、白癜风、克罗恩综合征、结肠炎等。JAK kinase/signal transducer and activator of transcription (The Janus kinase/signal transducer and activator of transcription, JAK/STAT) signaling pathway mediates a variety of cytokine signal transduction, including interleukin, interferon, erythropoietin , Granulocyte and macrophage colony stimulating factors. The binding of cytokines to their receptors causes the dimerization of receptor molecules, so that JAK kinases coupled to the receptors approach each other and are activated by interactive tyrosine phosphorylation. The activated JAK catalyzes the phosphorylation of the receptor itself. The corresponding STAT docking site is formed to make STAT phosphorylate, and then STAT forms a homodimer or heterodimer and enters the nucleus, and combines with the corresponding target gene promoter to activate gene transcription and expression. Therefore, the signal pathway mediated by JAK kinase plays an important role in cell proliferation, differentiation, apoptosis, and inflammation. However, in the pathological process, excessive secretion of cytokines leads to excessive activation of the JAK/STAT pathway, causing autoimmune diseases, tumors and other diseases, including psoriasis, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, Vitiligo, Crohn's syndrome, colitis, etc.

JAK激酶参与细胞因子的信号传导，调控免疫应答反应，因此，JAK激酶可作为治疗银屑病、类风湿性关节炎等自身免疫性疾病的靶点。目前，辉瑞公司开发的托法替尼已经成功上市，能够有效抑制JAK1、JAK2、JAK3，从而用于治疗类风湿性关节炎；礼来公司开发的JAK1和JAK2激酶抑制剂巴瑞克替尼，作为治疗类风湿性关节炎的药物也已经上市，艾伯维公司开发的JAK1激酶抑制剂upadacitinib于2019年在美国上市，同样是治疗类风湿性关节炎。JAKS激酶家族其它成员开发不断取得的骄人成绩，促使TYK2也成为研究的热点，虽然目前没有该靶点的药物上市，但是数个化合物已经处于临床阶段，TYK2终于也在蛋白激酶的历史进程中初露锋芒。最值得一提的是，BMS公司开发的TYK2抑制剂BMS-986165，用于治疗银屑病，目前正处于临床3期，在治疗中度至重度斑块状银屑病患者的临床2期试验中达到疗效主要终点，副反应小，具有高度的安全性和有效性。相信在不久之后，TYK2抑制剂就会传来捷报，给银屑病患者带来福音。JAK kinase participates in the signal transduction of cytokines and regulates the immune response. Therefore, JAK kinase can be used as a target for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis. At present, tofacitinib developed by Pfizer has been successfully marketed, which can effectively inhibit JAK1, JAK2, and JAK3 for the treatment of rheumatoid arthritis; the JAK1 and JAK2 kinase inhibitor barectinib developed by Eli Lilly, As a drug for the treatment of rheumatoid arthritis, upadacitinib, a JAK1 kinase inhibitor developed by AbbVie, was launched in the United States in 2019. It is also a treatment for rheumatoid arthritis. The outstanding achievements made by other members of the JAKS kinase family have made TYK2 a research hotspot. Although there is currently no drug with this target on the market, several compounds are already in the clinical stage, and TYK2 is finally in the history of protein kinases. Began to show off. The most worth mentioning is that the TYK2 inhibitor BMS-986165 developed by BMS is used for the treatment of psoriasis. It is currently in Phase 3 clinical trials and is in a phase 2 trial for the treatment of patients with moderate to severe plaque psoriasis. It reaches the primary end point of curative effect in medium, with small side effects, and has high safety and effectiveness. It is believed that in the near future, TYK2 inhibitors will have good news and bring good news to patients with psoriasis.

发明内容Summary of the invention

本发明的目的在于提供一种通式(I)所示的吡唑并[1,5-a]吡嗪类衍生物，或其立体异构体、互变异构体或其可药用的盐：The object of the present invention is to provide a pyrazolo[1,5-a]pyrazine derivative represented by the general formula (I), or its stereoisomers, tautomers, or pharmaceutically acceptable Salt:

其中：in:

Z选自键或-(CH ₂) _h-，其中一个或多个亚甲基单元任选进一步被一个或多个选自C ₁-C ₃烷基、C ₁-C ₆烷氧基或卤素的取代基所取代，其中所述的烷基或烷氧基任选进一步被一个或多个卤素取代； Z is selected from bond or -(CH ₂ ) _h -, wherein one or more methylene units are optionally further selected from C ₁ -C ₃ alkyl, C ₁ -C ₆ alkoxy or halogen Wherein the alkyl or alkoxy group is optionally further substituted with one or more halogens;

R ¹选自C ₁-C ₄烷基、C ₃-C ₆环烷基或3～6元杂环基，其中所述的烷基、环烷基或杂环基任选进一步被一个或多个选自C ₁-C ₄烷基、C ₁-C ₆烷氧基、C ₁-C ₄卤代烷基、C ₁-C ₆卤代烷氧基、卤素、羟基、氰基、硝基或氨基的取代基所取代； R ^{1 is} selected from a C ₁ -C ₄ alkyl group, a C ₃ -C ₆ cycloalkyl group or a 3-6 membered heterocyclic group, wherein the alkyl group, cycloalkyl group or heterocyclic group is optionally further substituted by one or more A substitution selected from C ₁ -C ₄ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₄ haloalkyl, C ₁ -C ₆ haloalkoxy, halogen, hydroxyl, cyano, nitro or amino Substitution;

R ²和R ³相同或不同，各自独立地选自氢、C ₁-C ₆烷基或卤素；其中所述的烷基任选进一步被一个或多个卤素所取代； R ² and R ^{3 are the} same or different, and are each independently selected from hydrogen, C ₁ -C ₆ alkyl or halogen; wherein the alkyl group is optionally further substituted with one or more halogens;

R ⁴选自氢、C ₁-C ₆烷基或C ₃-C ₆环烷基，其中所述的烷基或环烷基任选进一步被一个或多个卤素取代； R ^{4 is} selected from hydrogen, C ₁ -C ₆ alkyl or C ₃ -C ₆ cycloalkyl, wherein the alkyl or cycloalkyl is optionally further substituted with one or more halogens;

R ⁵选自氢、氨基或C ₁-C ₆烷基，其中所述的烷基任选进一步被一个或多个卤素取代； R ^{5 is} selected from hydrogen, amino or C ₁ -C ₆ alkyl, wherein the alkyl is optionally further substituted with one or more halogens;

m和n各自独立地选自0、1或2；优选地，m和n为1；m and n are each independently selected from 0, 1 or 2; preferably, m and n are 1;

h选自1、2或3。h is selected from 1, 2 or 3.

本发明优选的技术方案，一种通式(I)所示的化合物、互变异构体或其可药用的盐，其为通式(II)所述的化合物或其立体异构体、互变异构体或其可药用的盐，The preferred technical solution of the present invention is a compound represented by general formula (I), a tautomer or a pharmaceutically acceptable salt thereof, which is a compound represented by general formula (II) or a stereoisomer thereof, Tautomers or pharmaceutically acceptable salts thereof,

其中：in:

R ⁶相同或不同，各自独立地选自C ₁-C ₄烷基或卤素，其中所述的烷基任选进一步被一个或多个卤素所取代； R ^{6 is the} same or different, each independently selected from C ₁ -C ₄ alkyl or halogen, wherein the alkyl is optionally further substituted with one or more halogens;

j选自1、2、3或4；j is selected from 1, 2, 3 or 4;

k选自0，1或2；k is selected from 0, 1 or 2;

R ²～R ⁵、Z、m或n的定义如通式(I)所示。 The definitions of R ² to R ⁵ , Z, m or n are as shown in the general formula (I).

本发明优选的技术方案，一种通式(I)所示的化合物、互变异构体或其可药用的盐，其为通式(III)所述的化合物或其立体异构体、互变异构体或其可药用的盐，The preferred technical solution of the present invention is a compound represented by general formula (I), a tautomer or a pharmaceutically acceptable salt thereof, which is a compound represented by general formula (III) or a stereoisomer thereof, Tautomers or pharmaceutically acceptable salts thereof,

其中：in:

k为2；k is 2;

R ²～R ⁶、Z、j、m或n的定义如通式(II)所示。 The definitions of R ² to R ⁶ , Z, j, m or n are as shown in the general formula (II).

本发明优选的技术方案，一种通式(I)、(II)、(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐，其中R ⁴为甲基或乙基。 The preferred technical solution of the present invention is a compound represented by general formula (I), (II), (III) or its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, wherein R ⁴ is Methyl or ethyl.

本发明优选的技术方案，一种通式(I)、(II)、(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐，其中：The preferred technical solution of the present invention is a compound represented by the general formulas (I), (II), (III) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein:

R ²和R ³相同或不同，各自独立地选自氢原子或甲基； R ² and R ^{3 are the} same or different, and are each independently selected from a hydrogen atom or a methyl group;

R ⁵选自氢原子或甲基。 R ^{5 is} selected from a hydrogen atom or a methyl group.

本发明优选的技术方案，一种通式(II)或(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐，其中R ⁶选自氟或三氟甲基。 The preferred technical solution of the present invention is a compound represented by general formula (II) or (III) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein R ^{6 is} selected from fluorine or tris Fluoromethyl.

本发明优选的技术方案，一种通式(II)或(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐，其中j选自1或2。The preferred technical solution of the present invention is a compound represented by general formula (II) or (III) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein j is selected from 1 or 2.

本发明的典型化合物包括，但不限于：Typical compounds of the present invention include, but are not limited to:

或其立体异构体、互变异构体或其可药用的盐。Or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof.

注：如果在画出的结构和给出的该结构的名称之间有差异，则画出的结构将给予更大的权重。Note: If there is a difference between the drawn structure and the given name of the structure, the drawn structure will be given more weight.

一种通式(I)化合物或其立体异构体、互变异构体或其可药用的盐的制备方法，所述方法包括：A method for preparing a compound of general formula (I) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, the method comprising:

通式(IA)化合物或其盐与通式(IB)化合物在碱性条件下进行缩合反应，得到通式(I)化合物；The compound of general formula (IA) or its salt and the compound of general formula (IB) undergo condensation reaction under basic conditions to obtain the compound of general formula (I);

其中：in:

碱性条件由有机碱提供，所述有机碱选自N,N-二异丙基乙胺、吡啶、三乙胺、哌啶、N-甲基哌嗪、4-二甲氨吡啶或叔丁醇钾，优选为N,N-二异丙基乙胺；The basic conditions are provided by an organic base selected from N,N-diisopropylethylamine, pyridine, triethylamine, piperidine, N-methylpiperazine, 4-dimethylaminopyridine or tert-butyl Potassium alkoxide, preferably N,N-diisopropylethylamine;

R ¹～R ⁵、Z、m或n的定义如通式(I)中所述。 The definitions of R ¹ to R ⁵ , Z, m or n are as described in the general formula (I).

本发明还涉及一种通式(IA)所示的化合物或其立体异构体、互变异构体或其可药用的盐：The present invention also relates to a compound represented by the general formula (IA) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof:

其中：R ²～R ⁵、Z、m或n的定义如通式(I)中所述。 Wherein: R ² to R ⁵ , Z, m or n are defined as described in the general formula (I).

本发明的通式(IA)化合物包括，但不限于：The compounds of general formula (IA) of the present invention include, but are not limited to:

在另一方面，本发明提供一种药物组合物，所述的药物组合物含有有效剂量的通式(I)、(II)或(III)所述的化合物或其立体异构体、互变异构体或其可药用的盐，及可药用的载体、赋形剂或它们的组合。In another aspect, the present invention provides a pharmaceutical composition containing an effective dose of the compound of general formula (I), (II) or (III) or its stereoisomers, tautomers Isomers or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers, excipients, or combinations thereof.

在另一方面，本发明提供一种药物组合物，所述药物组合物还含有抗炎药中的至少一种，所述抗炎药选自非甾体类抗炎药、非特异性环氧合酶-2抑制剂、特异性环氧合酶-2抑制剂、皮质激素类、肿瘤坏死因子受体拮抗剂、水杨酸酯或盐、免疫抑制剂或甲氨蝶呤。In another aspect, the present invention provides a pharmaceutical composition, the pharmaceutical composition also contains at least one of anti-inflammatory drugs, the anti-inflammatory drugs selected from non-steroidal anti-inflammatory drugs, non-specific epoxy Enzyme-2 inhibitors, specific cyclooxygenase-2 inhibitors, corticosteroids, tumor necrosis factor receptor antagonists, salicylates or salts, immunosuppressants, or methotrexate.

在另一方面，本发明提供一种抑制JAKS激酶的方法，其中所述的方法包括，给予病人一种药物组合物，所述的药物组合物含有有效剂量的通式(I)、(II)或(III)所述的化合物或其立体异构体、互变异构体或其可药用的盐，及可药用的载体、赋形剂或它们的组合。In another aspect, the present invention provides a method for inhibiting JAKS kinase, wherein the method includes administering a pharmaceutical composition to the patient, the pharmaceutical composition containing an effective dose of the general formula (I), (II) Or the compound described in (III) or its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers, excipients, or combinations thereof.

在另一方面，本发明提供一种通式(I)、(II)或(III)所述的化合物或其立体异构体、互变异构体或其可药用的盐，或其药物组合物在制备JAK1激酶、JAK2激酶、JAK3激酶和/或TYK2激酶抑制剂中的用途，优选为在制备JAK1激酶、JAK2激酶和/或TYK2激酶抑制剂中的用途。In another aspect, the present invention provides a compound of the general formula (I), (II) or (III) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, or a drug thereof The use of the composition in the preparation of JAK1 kinase, JAK2 kinase, JAK3 kinase and/or TYK2 kinase inhibitors is preferably the use in the preparation of JAK1 kinase, JAK2 kinase and/or TYK2 kinase inhibitors.

在另一方面，本发明提供一种通式(I)、(II)或(III)所述的化合物或其立体异构体、互变异构体或其可药用的盐，或其药物组合物在制备治疗由JAK1激酶、JAK2激酶、JAK3激酶和/或TYK2激酶介导的疾病的药物中的用途，其中优选为在制备治疗由JAK1激酶、JAK2激酶和/或TYK2激酶介导的疾病的药物中的用途，其中所述的由JAK1激酶、JAK2激酶和/或TYK2激酶介导的疾病包括自身免疫性疾病、炎症性疾病和癌症；其中所述的自身免疫性疾病包括哮喘、银屑病、狼疮、多发性硬化症、变应性鼻炎、异位性皮炎、接触性皮炎和迟发过敏反应；其中所述的炎症性疾病包括炎症性肠病、类风湿关节炎，所述炎症性肠病包括克罗恩病和溃疡性结肠炎；其中所述的癌症包括非小细胞肺癌、小细胞肺癌、乳腺癌、胰腺癌、神经胶质瘤、胶质母细胞瘤、卵巢癌、子***、结肠直肠癌、黑色素瘤、子宫内膜癌、***癌、膀胱癌、胃癌、肝癌、胃肠间质瘤、甲状腺癌、白血病、非霍奇金淋巴瘤、鼻咽癌、食道癌、脑瘤、淋巴瘤、多发性骨髓瘤、胆管癌和实体瘤；所述白血病进一步优选选自慢性粒细胞白血病和急性髓细胞性白血病。In another aspect, the present invention provides a compound of the general formula (I), (II) or (III) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, or a drug thereof Use of the composition in the preparation of a medicine for the treatment of diseases mediated by JAK1 kinase, JAK2 kinase, JAK3 kinase and/or TYK2 kinase, wherein preferably in the preparation and treatment of diseases mediated by JAK1 kinase, JAK2 kinase and/or TYK2 kinase Wherein the diseases mediated by JAK1 kinase, JAK2 kinase and/or TYK2 kinase include autoimmune diseases, inflammatory diseases and cancer; wherein the autoimmune diseases include asthma, psoriasis Disease, lupus, multiple sclerosis, allergic rhinitis, atopic dermatitis, contact dermatitis and delayed allergic reaction; wherein the inflammatory diseases include inflammatory bowel disease, rheumatoid arthritis, the inflammatory Bowel diseases include Crohn’s disease and ulcerative colitis; the cancers described therein include non-small cell lung cancer, small cell lung cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, ovarian cancer, and cervical cancer. Cancer, colorectal cancer, melanoma, endometrial cancer, prostate cancer, bladder cancer, stomach cancer, liver cancer, gastrointestinal stromal tumor, thyroid cancer, leukemia, non-Hodgkin's lymphoma, nasopharyngeal cancer, esophageal cancer, brain Tumors, lymphomas, multiple myeloma, cholangiocarcinoma and solid tumors; the leukemia is further preferably selected from chronic myelogenous leukemia and acute myeloid leukemia.

在另一方面，本发明提供一种通式(I)、(II)或(III)所述的化合物或其立体异构体、互变异构体或其可药用的盐，或其药物组合物在制备治疗自身免疫性疾病、炎症性疾病和癌症药物中的用途；其中所述的自身免疫性疾病包括哮喘、银屑病、狼疮、多发性硬化症、变应性鼻炎、异位性皮炎、接触性皮炎和迟发过敏反应；其中所述的炎症性疾病包括炎症性肠病、类风湿关节炎，所述炎症性肠病包括克罗恩病和溃疡性结肠炎；其中所述的癌症包括非小细胞肺癌、小细胞肺癌、乳腺癌、胰腺癌、神经胶质瘤、胶质母细胞瘤、卵巢癌、子***、结肠直肠癌、黑色素瘤、子宫内膜癌、***癌、膀胱癌、胃癌、肝癌、胃肠间质瘤、甲状腺癌、白血病、非霍奇金淋巴瘤、鼻咽癌、食道癌、脑瘤、淋巴瘤、多发性骨髓瘤、胆管癌和实体瘤。In another aspect, the present invention provides a compound of the general formula (I), (II) or (III) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, or a drug thereof Use of the composition in the preparation of drugs for the treatment of autoimmune diseases, inflammatory diseases and cancer; wherein said autoimmune diseases include asthma, psoriasis, lupus, multiple sclerosis, allergic rhinitis, atopic Dermatitis, contact dermatitis and delayed allergic reactions; wherein the inflammatory diseases include inflammatory bowel disease, rheumatoid arthritis, and the inflammatory bowel disease includes Crohn’s disease and ulcerative colitis; wherein the Cancers include non-small cell lung cancer, small cell lung cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, ovarian cancer, cervical cancer, colorectal cancer, melanoma, endometrial cancer, prostate cancer, Bladder cancer, stomach cancer, liver cancer, gastrointestinal stromal tumor, thyroid cancer, leukemia, non-Hodgkin’s lymphoma, nasopharyngeal cancer, esophageal cancer, brain tumor, lymphoma, multiple myeloma, cholangiocarcinoma and solid tumors.

发明的详细说明Detailed description of the invention

除非有相反陈述，否则本发明在说明书和权利要求书中所使用的部分术语定义如下：Unless stated to the contrary, some terms used in the specification and claims of the present invention are defined as follows:

“烷基”当作一基团或一基团的一部分时是指包括C ₁-C ₂₀直链或者带有支链的脂肪烃基团。优选为C ₁-C ₁₀烷基，更优选为C ₁-C ₆烷基。烷基基团的实施例包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代或未取代的。 "Alkyl" when regarded as a group or a part of a group means to include a C ₁ -C ₂₀ linear or branched aliphatic hydrocarbon group. It is preferably a C ₁ -C ₁₀ alkyl group, more preferably a C ₁ -C ₆ alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-di Methylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1 -Ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethyl Butyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl Wait. Alkyl groups can be substituted or unsubstituted.

“环烷基”是指饱和或部分饱和的单环、稠环、桥环和螺环的碳环。优选为C ₃-C ₁₂环烷基，更优选为C ₃-C ₈环烷基，最优选为C ₃-C ₆环烷基。单环环烷基的实施例包括但不限于环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等，优选环丙基、环己烯基。 "Cycloalkyl" refers to saturated or partially saturated monocyclic, fused, bridged, and spirocyclic carbocyclic rings. It is preferably a C ₃ -C ₁₂ cycloalkyl group, more preferably a C ₃ -C ₈ cycloalkyl group, and most preferably a C ₃ -C ₆ cycloalkyl group. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptyl Alkenyl, cyclooctyl, etc., preferably cyclopropyl and cyclohexenyl.

“杂环基”、“杂环”或“杂环的”在本申请中可交换使用，都是指非芳香性杂环基，其中一个或多个成环的原子是杂原子，如氧、氮、硫原子等，包括单环、多环、稠环、桥环和螺环。优选具有5至7元单环或7至10元双-或三环，其可以包含1，2或3个选自氮、氧和/或硫中的原子。“杂环基”的实例包括但不限于吗啉基，氧杂环丁烷基，硫代吗啉基，四氢吡喃基，1，1-二氧代-硫代吗啉基，哌啶基，2-氧代-哌啶基，吡咯烷基，2-氧代-吡咯烷基，哌嗪-2-酮，8-氧杂-3-氮杂-双环[3.2.1]辛基或哌嗪基、。杂环基可以是取代或未取代的。"Heterocyclyl", "heterocyclic" or "heterocyclic" are used interchangeably in this application and all refer to non-aromatic heterocyclic groups, in which one or more ring-forming atoms are heteroatoms, such as oxygen, Nitrogen, sulfur atoms, etc., including monocyclic, polycyclic, fused, bridged, and spirocyclic rings. It preferably has a 5- to 7-membered monocyclic ring or a 7- to 10-membered bi- or tricyclic ring, which may contain 1, 2 or 3 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heterocyclyl" include, but are not limited to, morpholinyl, oxetanyl, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxo-thiomorpholinyl, piperidine Group, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo[3.2.1]octyl or Piperazinyl,. The heterocyclic group may be substituted or unsubstituted.

“烷氧基”是指(烷基-O-)的基团。其中，烷基见本文有关定义。C ₁-C ₆的烷氧基为优先选择。其实例包括，但不限于：甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基等。 "Alkoxy" refers to a (alkyl-O-) group. Among them, the alkyl group is defined in this article. C ₁ -C ₆ alkoxy groups are preferred. Examples thereof include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.

“羟基”指-OH基团。"Hydroxy" refers to the -OH group.

“卤素”是指氟、氯、溴和碘。"Halogen" refers to fluorine, chlorine, bromine and iodine.

“氨基”指-NH ₂。 "Amino" refers to -NH ₂ .

“氰基”指-CN。"Cyano" refers to -CN.

“硝基”指-NO ₂。 "Nitro" refers to -NO ₂ .

“DMSO”指二甲基亚砜。"DMSO" refers to dimethyl sulfoxide.

“BOC”指叔丁氧基羰基。"BOC" means tert-butoxycarbonyl.

“Ts”指对甲苯磺酰基。"Ts" means p-toluenesulfonyl.

“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。"Substituted" refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, independently of each other, substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without too much effort. For example, an amino group or a hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.

本说明书所述的“取代”或“取代的”，如无特别指出，均是指基团可被一个或多个选自以下的基团取代：烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、氨基、卤素、疏基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氨基、卤代烷基、羟烷基、羧基、羧酸酯基。The "substituted" or "substituted" mentioned in this specification, unless otherwise specified, mean that the group can be substituted by one or more groups selected from the following: alkyl, alkenyl, alkynyl, alkoxy , Alkylthio, alkylamino, amino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, Cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl, carboxyl, carboxylate.

“可药用的盐”是指上述化合物能保持原有生物活性并且适合于医药用途的某些盐类。式(I)所表示的化合物的可药用的盐可以为金属盐、与合适的酸形成的胺盐。"Pharmaceutically acceptable salts" refer to certain salts of the above compounds that can maintain the original biological activity and are suitable for medical use. The pharmaceutically acceptable salt of the compound represented by formula (I) may be a metal salt or an amine salt formed with a suitable acid.

“药物组合物”表示含有一种或多种本文所述化合物或其生理学上可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients. Shape agent. The purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.

具体实施方式Detailed ways

以下结合实施例用于进一步描述本发明，但这些实施例并非限制着本发明的范围。The following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention.

实施例Example

实施例给出了式(I)所表示的代表性化合物的制备及相关结构鉴定数据。必须说明，下述实施例是用于说明本发明而不是对本发明的限制。 ¹H NMR图谱是用Bruker仪器(400MHz)测定而得，化学位移用ppm表示。使用四甲基硅烷内标准(0.00ppm)。 ¹H NMR的表示方法：s＝单峰，d＝双重峰，t＝三重峰，m＝多重峰，br＝变宽的，dd＝双重峰的双重峰，dt＝三重峰的双重峰。若提供偶合常数时，其单位为Hz。 The examples show the preparation of representative compounds represented by formula (I) and relevant structural identification data. It must be noted that the following examples are used to illustrate the present invention but not to limit the present invention. ^{The 1} H NMR spectrum was measured with a Bruker instrument (400MHz), and the chemical shift was expressed in ppm. Use tetramethylsilane internal standard (0.00ppm). ¹ H NMR expression method: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of doublet, dt = doublet of triplet. If the coupling constant is provided, its unit is Hz.

质谱是用LC/MS仪测定得到，离子化方式可为ESI或APCI。The mass spectrum is measured by an LC/MS instrument, and the ionization method can be ESI or APCI.

薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm。The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the size of the silica gel plate used for thin layer chromatography (TLC) is 0.15mm～0.2mm, and the size of the thin layer chromatography separation and purification product is 0.4mm. ~0.5mm.

柱层析使用烟台黄海硅胶200～300目硅胶为载体。The column chromatography uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

在下列实施例中，除非另有指明，所有温度为摄氏温度，除非另有指明，各种起始原料和试剂来自市售或者是根据已知的方法合成，市售原料和试剂均不经进一步纯化直接使用，除非另有指明，市售厂家包括但不限于Aldrich Chemical Company，ABCR GmbH&Co.KG，Acros Organics，广赞化工科技有限公司和景颜化工科技有限公司等处购买。In the following examples, unless otherwise specified, all temperatures are degrees Celsius. Unless otherwise specified, various starting materials and reagents are either commercially available or synthesized according to known methods. The commercially available raw materials and reagents are not further processed. Purification is used directly, unless otherwise specified, commercial manufacturers including but not limited to Aldrich Chemical Company, ABCR GmbH & Co. KG, Acros Organics, Guangzan Chemical Technology Co., Ltd. and Jingyan Chemical Technology Co., Ltd. purchase.

CD ₃OD：氘代甲醇。 CD ₃ OD: Deuterated methanol.

CDCl ₃：氘代氯仿。 CDCl ₃ : Deuterated chloroform.

DMSO-d ₆：氘代二甲基亚砜。 DMSO-d ₆ : Deuterated dimethyl sulfoxide.

实施例中无特殊说明，反应中的溶液是指水溶液。There is no special description in the examples, and the solution in the reaction refers to an aqueous solution.

对化合物进行纯化，采用硅胶柱层析洗脱剂体系和薄层色谱法，其中洗脱剂体系选自：A：石油醚和乙酸乙酯体系；B：二氯甲烷和甲醇体系；C：二氯甲烷和乙酸乙酯；其中溶剂的体积比根据化合物的极性不同而不同，也可以加入少量的酸性或碱性试剂进行调节，如醋酸或三乙胺等。The compound was purified using silica gel column chromatography eluent system and thin layer chromatography, wherein the eluent system was selected from: A: petroleum ether and ethyl acetate system; B: dichloromethane and methanol system; C: two Methyl chloride and ethyl acetate; the volume ratio of the solvent varies according to the polarity of the compound, and a small amount of acidic or alkaline reagents can also be added to adjust, such as acetic acid or triethylamine.

实施例1Example 1

((S)-2,2-二氟环丙基)(1R,5S)-3-(6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-4-基)-3,8-二氮杂二环[3.2.1]辛烷-8-基)甲酮((S)-2,2-Difluorocyclopropyl)(1R,5S)-3-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a ]Pyrazin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-yl)methanone

第一步first step

2-溴-1-(1-甲基-1H-吡唑-4-基)乙酮2-bromo-1-(1-methyl-1H-pyrazol-4-yl)ethanone

将1-(1-甲基吡唑)乙酮1a(5.0g,40.28mmol)溶于125mL二氯甲烷和乙醇的混合溶剂(V:V＝4:1)中，冰浴下加入三溴化吡啶(12.9g,40.28mmol)，室温下反应6小时。TLC监测反应至原料消失。在反应液中加入水(200mL)，分液，分出有机相，水相用二氯甲烷(50mL×2)萃取，合并有机相，以无水硫酸钠干燥，过滤，减压浓缩，得到的残留物用硅胶柱层析法(洗脱剂：B体系)纯化，得到2-溴-1-(1-甲基-1H-吡唑-4-基)乙酮1b(6.0g)，产率：73％。Dissolve 1-(1-methylpyrazole)ethanone 1a (5.0g, 40.28mmol) in 125mL of a mixed solvent of dichloromethane and ethanol (V:V=4:1), add tribromide under ice bath Pyridine (12.9 g, 40.28 mmol) was reacted at room temperature for 6 hours. The reaction was monitored by TLC until the starting material disappeared. Water (200mL) was added to the reaction solution, the layers were separated, the organic phase was separated, the aqueous phase was extracted with dichloromethane (50mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain The residue was purified by silica gel column chromatography (eluent: system B) to obtain 2-bromo-1-(1-methyl-1H-pyrazol-4-yl)ethanone 1b (6.0 g), yield : 73%.

MS m/z(ESI):203.0[M+1]MS m/z(ESI):203.0[M+1]

第二步Second step

1-(2-(1-甲基-1H-吡唑-4-基)-2-氧乙基)-1H-吡唑-3,5-二甲酸二乙酯1-(2-(1-methyl-1H-pyrazol-4-yl)-2-oxoethyl)-1H-pyrazole-3,5-dicarboxylic acid diethyl ester

将2-溴-1-(1-甲基-1H-吡唑-4-基)乙酮1b(6.0g,29.6mmol)、3,5-吡唑羧酸二乙酯1c(6.9g,32.5mmol)和碳酸铯(14.4g,44.3mmol)溶于30mLN,N-二甲基甲酰胺中，室温下反应5小时。反应结束后，加入水(100mL)，以乙酸乙酯(200mL)萃取，有机相依次用水(15mL×3)和饱和食盐水(15mL)洗涤，以无水硫酸钠干燥，过滤，减压浓缩，得到1-(2-(1-甲基-1H-吡唑-4-基)-2-氧乙基)-1H-吡唑-3,5-二甲酸二乙酯1d(9.2g)，产率：93％。Combine 2-bromo-1-(1-methyl-1H-pyrazol-4-yl)ethanone 1b (6.0g, 29.6mmol), 3,5-pyrazole diethyl carboxylate 1c (6.9g, 32.5 mmol) and cesium carbonate (14.4g, 44.3mmol) were dissolved in 30mL N,N-dimethylformamide and reacted at room temperature for 5 hours. After the completion of the reaction, water (100mL) was added, extracted with ethyl acetate (200mL), the organic phase was washed with water (15mL×3) and saturated brine (15mL) successively, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Obtain 1-(2-(1-methyl-1H-pyrazol-4-yl)-2-oxoethyl)-1H-pyrazole-3,5-dicarboxylate diethyl 1d (9.2g), yield Rate: 93%.

MS m/z(ESI):335.3[M+1]MS m/z(ESI):335.3[M+1]

第三步third step

4-羟基-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-2-羧酸乙酯4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazine-2-carboxylic acid ethyl ester

将1-(2-(1-甲基-1H-吡唑-4-基)-2-氧乙基)-1H-吡唑-3,5-二甲酸二乙酯1d(9.2g,27.5mmol)和乙酸铵(5.2g,82.6mmol)溶于100ml乙醇中，封管中130℃反应4小时。反应结束后，析出大量黄色固体产物，降至室温，过滤，真空干燥得到4-羟基-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-2-羧酸乙酯1e(6.5g)，产率：82％。The 1-(2-(1-methyl-1H-pyrazol-4-yl)-2-oxyethyl)-1H-pyrazole-3,5-dicarboxylate diethyl 1d (9.2g, 27.5mmol ) And ammonium acetate (5.2g, 82.6mmol) were dissolved in 100ml ethanol, and the tube was sealed and reacted at 130°C for 4 hours. After the reaction, a large amount of yellow solid product was precipitated, which was cooled to room temperature, filtered, and dried under vacuum to obtain 4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a] Ethyl pyrazine-2-carboxylate 1e (6.5 g), yield: 82%.

MS m/z(ESI):288.3[M+1]MS m/z(ESI):288.3[M+1]

第四步the fourth step

4-羟基-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-2-羧酸4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazine-2-carboxylic acid

将4-羟基-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-2-羧酸乙酯1e(4.0g,13.9mmol)和氢氧化钠(1.67g,41.8mmol)溶于90mL甲醇和水的混合溶剂(V:V＝2:1)中，室温下反应2小时。TLC监测反应至原料消失，减压浓缩，加入水(100mL)，加入1.0M稀盐酸调节pH至2-3，搅拌1个小时，过滤，将滤饼50℃烘干，得到4-羟基-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-2-羧酸1f(3.6g)，产率：100％。Combine 4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazine-2-carboxylic acid ethyl ester 1e (4.0g, 13.9mmol) and Sodium hydroxide (1.67 g, 41.8 mmol) was dissolved in 90 mL of a mixed solvent of methanol and water (V:V=2:1), and reacted at room temperature for 2 hours. The reaction was monitored by TLC until the raw materials disappeared, concentrated under reduced pressure, water (100mL) was added, 1.0M diluted hydrochloric acid was added to adjust the pH to 2-3, stirred for 1 hour, filtered, and the filter cake was dried at 50°C to obtain 4-hydroxy-6 -(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazine-2-carboxylic acid 1f (3.6g), yield: 100%.

MS m/z(ESI):260.2[M+1]MS m/z(ESI):260.2[M+1]

第五步the fifth step

6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-4-醇6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazin-4-ol

将4-羟基-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-2-羧酸1f(3.8g,14.7mmol)、1,10-菲啰啉(1.3g,7.3mmol)和乙酸铜(1.3g,7.3mmol)溶于60mLN-甲基吡咯烷酮中，氮气保护下， 165℃下反应7小时。LC-MS监测反应至原料消失，降至室温，加入水(100mL)，以二氯甲烷(300mL)萃取，有机相依次以水(50mL×3)和饱和食盐水(50mL)洗涤，以无水硫酸钠干燥，过滤，减压浓缩，得到的残留物用硅胶柱层析法(洗脱剂：B体系)纯化，得到6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-4-醇1g(2.2g)，产率：70％。The 4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazine-2-carboxylic acid 1f (3.8g, 14.7mmol), 1, 10-Phenanthroline (1.3g, 7.3mmol) and copper acetate (1.3g, 7.3mmol) were dissolved in 60mL of N-methylpyrrolidone, and reacted at 165°C for 7 hours under nitrogen protection. The reaction was monitored by LC-MS until the raw materials disappeared, and the temperature was lowered to room temperature. Water (100 mL) was added, and the mixture was extracted with dichloromethane (300 mL). The organic phase was washed with water (50 mL×3) and saturated brine (50 mL) successively, and then washed with water (50 mL×3) and saturated brine (50 mL). Dry over sodium sulfate, filter, and concentrate under reduced pressure. The residue obtained is purified by silica gel column chromatography (eluent: system B) to obtain 6-(1-methyl-1H-pyrazol-4-yl)pyrazole And [1,5-a]pyrazin-4-ol 1g (2.2g), yield: 70%.

MS m/z(ESI):216.2[M+1]MS m/z(ESI):216.2[M+1]

第六步Sixth step

4-氯-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪4-chloro-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazine

将6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-4-醇1g(2.2g,10.2mmol)溶于50mL乙腈中，室温下缓慢加入三氯氧磷(9.4g,61.3mmol)，加毕，升至80℃回流反应4小时。LC-MS监测反应至原料消失。反应结束后，降至室温，于45℃下浓缩除去溶剂和大部分三氯氧磷，加入水(100mL)，以乙酸乙酯(300mL)萃取，有机相以水(50mL×3)洗涤，分水有机相，水相用乙酸乙酯(100mL)反萃一次，合并有机相，以饱和食盐水(50mL)洗涤，以无水硫酸钠干燥，过滤，减压浓缩，得到的残留物用硅胶柱层析法(洗脱剂：A体系)纯化，得到4-氯-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪1h(600mg)，产率：25％。Dissolve 1g (2.2g, 10.2mmol) of 6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazin-4-ol in 50mL acetonitrile at room temperature Phosphorus oxychloride (9.4g, 61.3mmol) was slowly added, and after the addition, the temperature was raised to 80°C and refluxed for 4 hours. LC-MS monitored the reaction until the starting material disappeared. After the reaction, it was cooled to room temperature, concentrated at 45°C to remove the solvent and most of the phosphorus oxychloride, water (100 mL) was added, and the mixture was extracted with ethyl acetate (300 mL). The organic phase was washed with water (50 mL×3) and separated. The aqueous organic phase, the aqueous phase was back-extracted with ethyl acetate (100 mL) once, the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue obtained was subjected to a silica gel column Purification by chromatography (eluent: system A) to obtain 4-chloro-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazine 1h (600mg ), yield: 25%.

MS m/z(ESI):233.7[M+1]MS m/z(ESI):233.7[M+1]

第七步Seventh step

(1R,5S)-3-(6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-4-基)-3,8-二氮杂二环[3.2.1]辛烷-8-羧酸叔丁酯(1R,5S)-3-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazin-4-yl)-3,8-diazepine Heterobicyclo[3.2.1]octane-8-tert-butyl carboxylate

将4-氯-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪1h(2.0g,8.6mmol)、3,8-二氮杂二环[3.2.1]辛烷-8-羧酸叔丁酯1i(2.7g,12.8mmol)和碳酸钾(3.55g,25.7mmol)溶于25mL二甲基乙酰胺中，120℃下反应3小时。反应结束后，反应液冷却至室温，以乙酸乙酯(150mL)萃取，有机相依次用水(15mL×3)和饱和食盐水(15mL)洗涤，以无水硫酸钠干燥，过滤，减压浓缩，得到(1R,5S)-3-(6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-4-基)-3,8-二氮杂二环[3.2.1]辛烷-8-羧酸叔丁酯1j(3.3g)，产率：94％。Put 4-chloro-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazine 1h (2.0g, 8.6mmol), 3,8-diazepine Dicyclo[3.2.1]octane-8-tert-butyl carboxylate 1i (2.7g, 12.8mmol) and potassium carbonate (3.55g, 25.7mmol) were dissolved in 25mL of dimethylacetamide and reacted at 120℃ for 3 Hour. After the reaction, the reaction solution was cooled to room temperature and extracted with ethyl acetate (150 mL). The organic phase was washed with water (15 mL×3) and saturated brine (15 mL) successively, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. (1R,5S)-3-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazin-4-yl)-3,8-two Azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester 1j (3.3g), yield: 94%.

MS m/z(ESI):410.5[M+1]MS m/z(ESI):410.5[M+1]

第八步Eighth step

4-((1R,5S)-3,8-二氮杂二环[3.2.1]辛烷-3-基)-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪盐酸盐4-((1R,5S)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazole And [1,5-a]pyrazine hydrochloride

将(1R,5S)-3-(6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-4-基)-3,8-二氮杂二环[3.2.1]辛烷-8-羧酸叔丁酯1j(3.3g,8.1mmol)溶于40mL 1,4-二氧六环中，滴加氯化氢的1,4-二氧六环溶液(40mL,2.6mol/L)，室温下反应4小时。反应结束后，过滤，用***洗涤滤饼，真空干燥，得到4-((1R,5S)-3,8-二氮杂二环[3.2.1]辛烷-3-基)-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪盐酸盐1k(2.7g,)，产率：99％。(1R,5S)-3-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazin-4-yl)-3,8-two Azabicyclo[3.2.1]octane-8-tert-butyl carboxylate 1j (3.3g, 8.1mmol) is dissolved in 40mL 1,4-dioxane, and 1,4-dioxane with hydrogen chloride is added dropwise Six ring solution (40mL, 2.6mol/L), react at room temperature for 4 hours. After the reaction is over, filter, wash the filter cake with ether, and dry under vacuum to obtain 4-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-6-( 1-Methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazine hydrochloride 1k (2.7g,), yield: 99%.

MS m/z(ESI):310.4[M+1]MS m/z(ESI):310.4[M+1]

第九步Step 9

((S)-2,2-二氟环丙基)((1R,5S)-3-(6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-4-基)-3,8-二氮杂二环[3.2.1]辛烷-8-基)甲酮((S)-2,2-Difluorocyclopropyl)((1R,5S)-3-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5- a]Pyrazin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-yl)methanone

将4-((1R,5S)-3,8-二氮杂二环[3.2.1]辛烷-3-基)-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪盐酸盐1k(2.7g,7.8mmol)、(S)-2,2-二氟环丙烷-1-羧酸1l(1.4g,11.7mmol)、2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)(4.4g,11.7mmol)、N,N-二异丙基乙胺(3.0g,23.4mmol)溶于60mL二氯甲烷中，25℃下搅拌反应，LC-MS监测到反应原料消失，加入去离子水(100mL)淬灭反应，用乙酸乙酯(250mL)萃取，有机相依次用水(15mL×3)和饱和食盐水(15mL)洗涤，以无水硫酸钠干燥，过滤，减压浓缩，得到的残留物用硅胶柱层析法(洗脱剂：B体系)纯化，得到((S)-2,2-二氟环丙基)((1R,5S)-3-(6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡嗪-4-基)-3,8-二氮杂二环[3.2.1]辛烷-8-基)甲酮1(700mg)，产率：22％。Add 4-((1R,5S)-3,8-diazabicyclo[3.2.1]octan-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyridine Azolo[1,5-a]pyrazine hydrochloride 1k (2.7g, 7.8mmol), (S)-2,2-difluorocyclopropane-1-carboxylic acid 1l (1.4g, 11.7mmol), 2 -(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (4.4g, 11.7mmol), N,N-diisopropylethyl The amine (3.0g, 23.4mmol) was dissolved in 60mL of dichloromethane, and the reaction was stirred at 25°C. LC-MS monitored the disappearance of the reaction materials. The reaction was quenched by adding deionized water (100mL) and extracted with ethyl acetate (250mL) The organic phase was washed with water (15mL×3) and saturated brine (15mL) successively, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was subjected to silica gel column chromatography (eluent: system B) Purification to obtain ((S)-2,2-difluorocyclopropyl)((1R,5S)-3-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1 ,5-a]pyrazin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-yl)methanone 1 (700 mg), yield: 22%.

MS m/z(ESI):414.4[M+1]MS m/z(ESI):414.4[M+1]

¹H NMR(400MHz,d-DMSO)δ8.49(s,1H),8.17(d,J＝6.3Hz,1H),7.96(s,2H),7.01(d,J＝14.6Hz,1H),4.80-4.35(m,4H),3.87(s,3H),3.29(s,1H),3.27-3.09(m,2H),2.13-1.78(m,6H). ¹ H NMR(400MHz,d-DMSO)δ8.49(s,1H), 8.17(d,J=6.3Hz,1H), 7.96(s,2H), 7.01(d,J=14.6Hz,1H), 4.80-4.35 (m, 4H), 3.87 (s, 3H), 3.29 (s, 1H), 3.27-3.09 (m, 2H), 2.13-1.78 (m, 6H).

生物学评价Biological evaluation

测试例1、ADPGlo方法测定本发明化合物对TYK2和JAK1激酶活性Test Example 1. The ADPGlo method determines the activity of the compounds of the present invention on TYK2 and JAK1 kinases

以下方法用于测定本发明化合物在体外条件下对重组人源TYK2和JAK1激酶活性的抑制程度。本方法使用Promega公司的ADP-Glo ^TM Kinase Assay试剂盒(货号V9102)。上述试剂盒是一种发光法激酶检测试剂盒，用于检测激酶反应产生的ADP含量，ADP的含量与激酶活性正相关，通过测定ADP的含量，反映化合物对TYK2和JAK1激酶活性的抑制强弱。详细实验操作可参考试剂盒说明书。重组人源JAK1购于Carna(08-144)，TYK2购自Signalchem(T21-11G)。 The following method is used to determine the degree of inhibition of recombinant human TYK2 and JAK1 kinase activity by the compounds of the present invention under in vitro conditions. This method uses Promega's ADP-Glo ^TM Kinase Assay Kit (Cat. No. V9102). The above kit is a luminescence kinase detection kit for detecting the content of ADP produced by the kinase reaction. The content of ADP is positively correlated with kinase activity. By measuring the content of ADP, it reflects the inhibitory strength of the compound on the kinase activity of TYK2 and JAK1. . For detailed experimental operations, please refer to the kit instructions. Recombinant human JAK1 was purchased from Carna (08-144), and TYK2 was purchased from Signalchem (T21-11G).

将实验流程简述如下：受试化合物首先溶解于DMSO中制备为贮存液，随后按照试剂说明书中提供的缓冲液配方配置缓冲液(20mM MgCl ₂,40mM Tris,50uM DTT,0.1mg/ml BSA,pH 7.4)，使用该缓冲液进行梯度稀释，受试化合物在反应体系中的终浓度范围为16000nM～0.008nM。使用梯度稀释的ATP溶液(来自ADP-Glo ^TM Kinase Assay试剂盒)测定TYK2和JAK1的ATP Km值，依据实验所得的ATP Km值，设定反应体系中的ATP浓度分别设定为7.5uM和25uM。反应在384孔微孔板中进行，首先向孔中加入化合物和一定量的TYK2或JAK1蛋白，并在室温下孵育5分钟，随后向反应液中加入ATP溶液及Axltide(TYK2底物，终浓度为0.1mg/mL)或IRS1(Y608)Peptide(JAK1底物，终浓度为0.025mg/mL)，并在室温下振荡孵育120分钟或60分钟。随后向反应体系中加入5μL ADP-Glo Reagent，并在室温下继续振荡孵育40分钟。之后向反应体系中加入10μL Kinase Detection Reagent，并在室温下继续振荡孵育40分钟。孵育结束后，在酶标仪以Luminescence模式测定各孔的化学发光强度值。通过与对照组(0.1％DMSO)的发光强度比值进行比较计算化合物在各浓度下的百分比抑制率，并通过GraphPad Prism 5软件以化合物浓度对数值-抑制率进行非线性回归分析，获得化合物的IC ₅₀值，见表1。 The experimental procedure is briefly described as follows: the test compound is first dissolved in DMSO to prepare a storage solution, and then the buffer is configured according to the buffer formulation provided in the reagent manual (20mM MgCl ₂ , 40mM Tris, 50uM DTT, 0.1mg/ml BSA, pH 7.4), using the buffer for gradient dilution, the final concentration of the test compound in the reaction system ranges from 16000 nM to 0.008 nM. Measure the ATP Km values of TYK2 and JAK1 using a gradiently diluted ATP solution (from ADP-Glo ^TM Kinase Assay Kit). According to the ATP Km value obtained in the experiment, set the ATP concentration in the reaction system to 7.5uM and 25uM, respectively . The reaction is carried out in a 384-well microtiter plate. First, add the compound and a certain amount of TYK2 or JAK1 protein to the wells, and incubate at room temperature for 5 minutes, then add ATP solution and Axltide (TYK2 substrate, final concentration 0.1mg/mL) or IRS1(Y608) Peptide (JAK1 substrate, final concentration is 0.025mg/mL), and incubate with shaking at room temperature for 120 minutes or 60 minutes. Then add 5μL of ADP-Glo Reagent to the reaction system, and continue to incubate with shaking for 40 minutes at room temperature. Then add 10μL Kinase Detection Reagent to the reaction system, and continue to incubate with shaking for 40 minutes at room temperature. After the incubation, the chemiluminescence intensity value of each well was measured in Luminescence mode on the microplate reader. Calculate the percentage inhibition rate of the compound at each concentration by comparing with the luminous intensity ratio of the control group (0.1% DMSO), and use GraphPad Prism 5 software to perform a nonlinear regression analysis of the compound concentration vs. the inhibition rate to obtain the compound IC ₅₀ value, see Table 1.

表1 本发明化合物对JAK家族酶活性抑制的IC ₅₀数据 _{Table 1 The IC 50} data of the compound of the present invention for inhibiting the activity of JAK family enzymes

结论：从表1可以看出，本发明化合物对于TYK2和JAK1激酶具有较好的抑制作用。Conclusion: It can be seen from Table 1 that the compound of the present invention has a good inhibitory effect on TYK2 and JAK1 kinase.

测试例2、HTRF方法测定本发明化合物对JAK2和JAK3激酶活性Test Example 2. HTRF method to determine the activity of the compounds of the present invention on JAK2 and JAK3 kinases

以下方法用于测定本发明化合物在体外条件下对重组人源JAK2和JAK3激酶活性的抑制程度。本方法使用Cisbio公司的

KinEASE-TK酪氨酸激酶试剂盒(货号62TK0PEB)，该试剂盒原理基于时间分辨荧光能量共振转移(TF-FRET)，通过测定JAK2或JAK3激酶介导的生物素化的多肽底物的磷酸化程度来反映化合物对激酶活性的抑制强弱。详细实验操作可参考试剂盒说明书。重组人源JAK2和JAK3购于Carna bioscience(货号分别为JAK2#08-045，JAK3#08-046)。 The following method is used to determine the degree of inhibition of recombinant human JAK2 and JAK3 kinase activity by the compounds of the present invention under in vitro conditions. This method uses Cisbio's

KinEASE-TK Tyrosine Kinase Kit (Cat. No. 62TK0PEB), the principle of this kit is based on time-resolved fluorescence energy resonance transfer (TF-FRET), which measures the phosphorylation of biotinylated peptide substrates mediated by JAK2 or JAK3 kinase The degree reflects the inhibitory strength of the compound on the kinase activity. For detailed experimental operations, please refer to the kit instructions. Recombinant human JAK2 and JAK3 were purchased from Carna bioscience (product numbers are JAK2#08-045, JAK3#08-046, respectively).

将实验流程简述如下：受试化合物首先溶解于DMSO中制备为贮存液，随后以试剂盒中提供的缓冲液进行梯度稀释，受试化合物在反应体系中的终浓度范围为16000M～0.008nM。使用梯度稀释的ATP溶液(Sigma，A7699-1G)的测定JAK2和JAK3蛋白的ATP Km值浓度，依据获得的Km值，设定反应体系中ATP的浓度分别为2uM和5uM。反应在384孔微孔板中进行，首先向孔中加入化合物和一定量的对应JAK2或JAK3蛋白，并在室温下孵育5-10分钟，随后向反应液中加入ATP溶液和生物素化的多肽底物溶液，并在室温下振荡孵育60分钟。随后向反应中加入偶联有铕系元素化合物的抗磷酸化酪氨酸抗体和偶联有修饰化的别藻蓝蛋白XL665的链酶亲和素，并在室温下继续振荡孵育1小时。孵育结束后，在酶标仪以TF-FRET模式上测定在304nM的激发波长下，各孔发射波长为620nM和665nM的荧光强度，并计算各孔665/620 的荧光强度比值。通过与对照组(0.1％DMSO)的荧光强度比值进行比较，计算化合物在各浓度下的百分比抑制率，并通过GraphPad Prism 5软件以化合物浓度对数值-抑制率进行非线性回归分析，得化合物的IC ₅₀值，见表2。 The experimental procedure is briefly described as follows: the test compound is first dissolved in DMSO to prepare a storage solution, and then the test compound is gradually diluted with the buffer provided in the kit. The final concentration of the test compound in the reaction system ranges from 16000M to 0.008nM. The concentration of ATP Km of JAK2 and JAK3 proteins was determined using a gradiently diluted ATP solution (Sigma, A7699-1G). According to the obtained Km value, the ATP concentration in the reaction system was set to 2uM and 5uM, respectively. The reaction is carried out in a 384-well microtiter plate. First, add the compound and a certain amount of corresponding JAK2 or JAK3 protein to the well, and incubate at room temperature for 5-10 minutes, then add ATP solution and biotinylated peptide to the reaction solution Substrate solution and incubate with shaking at room temperature for 60 minutes. Subsequently, an anti-phosphotyrosine antibody coupled with a europium compound and a streptavidin coupled with a modified allophycocyanin XL665 were added to the reaction, and the reaction was continued to incubate with shaking for 1 hour at room temperature. After the incubation, the fluorescence intensity of each well at 620nM and 665nM emission wavelengths at the excitation wavelength of 304nM was measured on the microplate reader in the TF-FRET mode, and the fluorescence intensity ratio of each well 665/620 was calculated. By comparing with the fluorescence intensity ratio of the control group (0.1% DMSO), the percentage inhibition rate of the compound at each concentration was calculated, and the non-linear regression analysis of the compound concentration vs. the inhibition rate was performed by GraphPad Prism 5 software to obtain the compound's IC ₅₀ value, see Table 2.

表2 本发明化合物对JAK家族酶活性抑制的IC ₅₀数据 _{Table 2 The IC 50} data of the compound of the present invention for inhibiting the activity of JAK family enzymes

结论：从表2可以看出，本发明化合物对于JAK2激酶具有较好的抑制作用，对于JAK3的抑制作用较弱。Conclusion: It can be seen from Table 2 that the compound of the present invention has a good inhibitory effect on JAK2 kinase, but has a weaker inhibitory effect on JAK3.

综上，从表1和表2可以看出，本发明化合物对JAK1、JAK2和TYK2具有选择性抑制作用。In summary, it can be seen from Table 1 and Table 2 that the compound of the present invention has a selective inhibitory effect on JAK1, JAK2 and TYK2.

测试例3、HTRF方法测定本发明化合物对IFN-α2b诱导的Jurkat细胞中p-STAT3的抑制活性Test Example 3. HTRF method to determine the inhibitory activity of the compound of the present invention on p-STAT3 in Jurkat cells induced by IFN-α2b

以下方法用于测定本发明化合物对IFN-α2b诱导的Jurkat细胞中p-STAT3的影响。Jurkat细胞购于中国科学院典型培养物保藏委员会细胞库。细胞培养于含10％胎牛血清、100U青霉素和100μg/mL链霉素的RPMI 1640培养基中；培养在37℃，5％CO ₂培养箱内。p-STAT3通过LANCE Ultra Phosphorylated STAT3(Y705)Cellular Detection Kit试剂盒(PerkinElmer，#TRF4004M)进行测定。 The following method is used to determine the effect of the compound of the present invention on p-STAT3 in Jurkat cells induced by IFN-α2b. Jurkat cells were purchased from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100U penicillin and 100μg/mL streptomycin; cultured in a 37°C, 5% CO ₂ incubator. p-STAT3 was measured by LANCE Ultra Phosphorylated STAT3 (Y705) Cellular Detection Kit (PerkinElmer, #TRF4004M).

实验方法按照试剂盒说明书的步骤操作，简述如下：受试化合物首先溶解于DMSO中制备为贮存液，随后以对应细胞的培养基进行梯度稀释，配制成测试样品，化合物的终浓度范围在10000nM-0.61nM。将一定数量的Jurkat细胞接种至T25或T75培养瓶，置37℃、5％CO ₂培养箱饥饿过夜，之后将已饥饿处理的细胞按200000个细胞(8μl细胞悬液)/孔接板至384孔板中。再将稀释的待测化合物按每孔2μl加入384孔板中，放置于37℃、5％CO ₂培养箱孵育30min，之后加入2ul终浓度为60nM的IFN-α2b激活p-STAT320min。后续用LANCE Ultra Phosphorylated STAT3(Y705)Cellular Detection Kit进行p-STAT3的检测。试剂盒测定方式简述如下：使用LANCE Ultra Lysis Buffer 1裂解细胞40min，之后在384孔板中加入5ul 4X MIX Eu-labeled anti-STAT3(Y705)Antibody(0.5nM final)和ULight labeled anti-STAT3 Antibody(5nM final)。室温孵育4小时后在酶标仪中以TF-FRET模式上测定在304nM的激发波长下，各孔发射波长为620nM和665nM的荧光强度，并计算各孔665/620的荧光强度比值。 The experimental method was operated in accordance with the steps in the kit instructions, briefly as follows: the test compound was first dissolved in DMSO to prepare a stock solution, and then gradually diluted with the medium of the corresponding cell to prepare a test sample. The final concentration of the compound was in the range of 10000nM -0.61nM. Inoculate a certain number of Jurkat cells into T25 or T75 culture flasks, place them in a 37°C, 5% CO ₂ incubator and starve them overnight, and then plate the starved cells at 200,000 cells (8μl cell suspension)/well to 384 Orifice plate. Then add 2μl of diluted test compound per well into a 384-well plate, place it in a 37°C, 5% CO ₂ incubator for 30 min, and then add 2 ul of IFN-α2b at a final concentration of 60 nM to activate p-STAT for 320 min. Follow-up LANCE Ultra Phosphorylated STAT3 (Y705) Cellular Detection Kit for p-STAT3 detection. The test method of the kit is briefly described as follows: Use LANCE Ultra Lysis Buffer 1 to lyse the cells for 40 minutes, and then add 5ul 4X MIX Eu-labeled anti-STAT3 (Y705) Antibody (0.5nM final) and ULight labeled anti-STAT3 Antibody to a 384-well plate. (5nM final). After 4 hours of incubation at room temperature, the fluorescence intensity of each well at 620nM and 665nM emission wavelengths at the excitation wavelength of 304nM was measured in the TF-FRET mode in a microplate reader, and the fluorescence intensity ratio of each well 665/620 was calculated.

将实验数据扣除细胞背景数据，最终以样品浓度对数值为横坐标，Ratio为纵坐标，使用GraphPad Prism 5软件中以化合物浓度对数-抑制率进行非线性回归分析，得到化合物抑制细胞p-STAT3的IC ₅₀值，见表3。 The experimental data was subtracted from the cell background data, and finally the logarithmic value of the sample concentration was used as the abscissa and Ratio was the ordinate. Using GraphPad Prism 5 software, the compound concentration logarithm-inhibition rate was used for nonlinear regression analysis to obtain compound inhibitory cell p-STAT3 The IC ₅₀ value is shown in Table 3.

表3本发明化合物对IFN-α2b诱导的Jurkat细胞中p-STAT3的抑制活性Table 3 Inhibitory activity of the compounds of the present invention on p-STAT3 in Jurkat cells induced by IFN-α2b

实施例编号Example number	IC ₅₀(nM) IC ₅₀ (nM)
11	23.723.7

结论：从表3可以看出，本发明化合物对于IFN-α2b诱导的Jurkat细胞中p-STAT3具有较强的抑制作用。Conclusion: It can be seen from Table 3 that the compound of the present invention has a strong inhibitory effect on p-STAT3 in Jurkat cells induced by IFN-α2b.

测试例3、本发明化合物SD大鼠口服药代动力学研究Test Example 3. Study on oral pharmacokinetics of the compound of the present invention in SD rats

1、实验目的1. The purpose of the experiment

以SD大鼠为受试动物，采用LC/MS/MS法测定大鼠静脉注射或灌胃给予本发明化合物，测定其不同时刻血浆中的药物浓度，研究本发明化合物在大鼠体内的药代动力学特征。Taking SD rats as the test animals, the LC/MS/MS method was used to determine the intravenous injection or intragastric administration of the compound of the present invention in rats, and to determine the drug concentration in plasma at different times to study the pharmacokinetics of the compound of the present invention in rats. Kinetic characteristics.

2、实验方案2. Experimental program

2.1实验药品与动物2.1 Experimental drugs and animals

本发明化合物1Compound 1 of the present invention

健康成年Sprague Dawley(SD)雄性大鼠6只，购自维通利华实验动物技术有限公司，生产许可证号：11400700271077。6 healthy adult Sprague Dawley (SD) male rats, purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd., production license number: 11400700271077.

2.2药物配制与给药2.2 Drug formulation and administration

口服灌胃组：Oral gavage group:

称取适量的样品，加入适量DMSO和PEG200(v/v＝12.1:87.9)，涡旋混匀制成0.5mg/mL溶液。Weigh an appropriate amount of sample, add appropriate amount of DMSO and PEG200 (v/v=12.1:87.9), vortex and mix to make a 0.5 mg/mL solution.

健康成年SD雄性大鼠6只，禁食过夜后分别和灌胃给药(给药剂量10mg/kg)，给药4小时后进食。Six healthy adult male SD rats were fasted overnight and then administered separately and intragastrically (administration dose 10mg/kg), and 4 hours after administration.

2.3样品采集2.3 Sample collection

于给药前和给药后0.083小时、0.25小时、0.5小时、1小时、2小时、4小时、8小时、12小时和24小时经颈静脉采约0.2mL血液，肝素钠抗凝。血液样本采集后置于冰上，离心分离血浆(离心条件：1500g，10分钟)。收集的血浆分析前存放于–40～–20℃。About 0.2 mL of blood was collected from the jugular vein at 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after administration, and heparin sodium was used for anticoagulation. After the blood sample is collected, it is placed on ice, and the plasma is separated by centrifugation (centrifugation conditions: 1500 g, 10 minutes). The collected plasma is stored at -40～-20℃ before analysis.

2.4样品前处理2.4 Sample pretreatment

取10μL血浆样品中加入400μL乙腈(包含内标工作液，其中维拉帕米5ng/mL和格列本脲50ng/mL)，涡旋10分钟，3700转/分钟离心10分钟，取70μL上清液，加入70μL水，涡旋10分钟，取2μL混合液至LC-MS/MS进样分析。Take 10μL of plasma sample and add 400μL of acetonitrile (including internal standard working solution, in which verapamil 5ng/mL and glibenclamide 50ng/mL), vortex for 10 minutes, centrifuge at 3700 rpm for 10 minutes, and take 70μL of supernatant Add 70μL of water, vortex for 10 minutes, take 2μL of the mixed solution to LC-MS/MS for sample injection analysis.

3、药代动力学参数结果3. Results of pharmacokinetic parameters

本发明的化合物和阳性对照的药代动力学参数如下表所示The pharmacokinetic parameters of the compound of the present invention and the positive control are shown in the following table

表4Table 4

结论：从表4可以看出，本发明化合物具有较好的药代动力学性质。Conclusion: It can be seen from Table 4 that the compound of the present invention has good pharmacokinetic properties.

Claims

一种通式(I)所示的化合物或其立体异构体、互变异构体或其可药用的盐：A compound represented by general formula (I) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof:

其中：in:

Z选自键或-(CH ₂) _h-，其中一个或多个亚甲基单元任选进一步被一个或多个选自C ₁-C ₃烷基、C ₁-C ₆烷氧基或卤素的取代基所取代，其中所述的烷基或烷氧基任选进一步被一个或多个卤素取代； Z is selected from bond or -(CH ₂ ) _h -, wherein one or more methylene units are optionally further selected from C ₁ -C ₃ alkyl, C ₁ -C ₆ alkoxy or halogen Wherein the alkyl or alkoxy group is optionally further substituted with one or more halogens;

R ¹选自C ₁-C ₄烷基、C ₃-C ₆环烷基或3～6元杂环基，其中所述的烷基、环烷基或杂环基任选进一步被一个或多个选自C ₁-C ₄烷基、C ₁-C ₆烷氧基、C ₁-C ₄卤代烷基、C ₁-C ₆卤代烷氧基、卤素、羟基、氰基、硝基或氨基的取代基所取代； R ^{1 is} selected from a C ₁ -C ₄ alkyl group, a C ₃ -C ₆ cycloalkyl group or a 3-6 membered heterocyclic group, wherein the alkyl group, cycloalkyl group or heterocyclic group is optionally further substituted by one or more A substitution selected from C ₁ -C ₄ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₄ haloalkyl, C ₁ -C ₆ haloalkoxy, halogen, hydroxyl, cyano, nitro or amino Substitution;

R ²和R ³相同或不同，各自独立地选自氢、C ₁-C ₆烷基或卤素；其中所述的烷基任选进一步被一个或多个卤素所取代； R ² and R ^{3 are the} same or different, and are each independently selected from hydrogen, C ₁ -C ₆ alkyl or halogen; wherein the alkyl group is optionally further substituted with one or more halogens;

R ⁴选自氢、C ₁-C ₆烷基或C ₃-C ₆环烷基，其中所述的烷基或环烷基任选进一步被一个或多个卤素取代； R ^{4 is} selected from hydrogen, C ₁ -C ₆ alkyl or C ₃ -C ₆ cycloalkyl, wherein the alkyl or cycloalkyl is optionally further substituted with one or more halogens;

R ⁵选自氢、氨基或C ₁-C ₆烷基，其中所述的烷基任选进一步被一个或多个卤素取代； R ^{5 is} selected from hydrogen, amino or C ₁ -C ₆ alkyl, wherein the alkyl is optionally further substituted with one or more halogens;

m和n各自独立地选自0、1或2；优选地，m和n为1；m and n are each independently selected from 0, 1 or 2; preferably, m and n are 1;

h选自1、2或3。h is selected from 1, 2 or 3.
根据权利要求1所述的化合物或其立体异构体、互变异构体或其可药用的盐，其为通式(II)所述的化合物或其立体异构体、互变异构体或其可药用的盐，The compound or its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof according to claim 1, which is the compound of general formula (II) or its stereoisomers or tautomers Body or its pharmaceutically acceptable salt,

其中：in:

R ⁶相同或不同，各自独立地选自C ₁-C ₄烷基或卤素，其中所述的烷基任选进一步被一个或多个卤素所取代； R ^{6 is the} same or different, each independently selected from C ₁ -C ₄ alkyl or halogen, wherein the alkyl is optionally further substituted with one or more halogens;

j选自1、2、3或4；j is selected from 1, 2, 3 or 4;

k选自0，1或2；k is selected from 0, 1 or 2;

R ²～R ⁵、Z、m或n的定义如权利要求1中所述。 The definitions of R ² to R ⁵ , Z, m or n are as described in claim 1.
根据权利要求2所述的化合物或其立体异构体、互变异构体或其可药用的盐，其为通式(III)所述的化合物或其立体异构体、互变异构体或其可药用的盐，The compound according to claim 2 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, which is the compound of general formula (III) or its stereoisomer or tautomer Body or its pharmaceutically acceptable salt,

其中：in:

k为2；k is 2;

R ²～R ⁶、Z、j、m或n的定义如权利要求2中所述。 The definitions of R ² to R ⁶ , Z, j, m or n are as described in claim 2.
根据权利要求1～3中任一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，其中R ⁴为甲基或乙基。 The compound according to any one of claims 1 to 3, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein R ⁴ is a methyl group or an ethyl group.
根据权利要求1～3中任一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，其中：The compound or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof according to any one of claims 1 to 3, wherein:

R ²和R ³相同或不同，各自独立地选自氢原子或甲基； R ² and R ^{3 are the} same or different, and are each independently selected from a hydrogen atom or a methyl group;

R ⁵选自氢原子或甲基。 R ^{5 is} selected from a hydrogen atom or a methyl group.
根据权利要求2～3中任一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，其中R ⁶选自氟或三氟甲基。 The compound according to any one of claims 2 to 3, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein R ^{6 is} selected from fluorine or trifluoromethyl.
根据权利要求2～3中任一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，其中j选自1或2。The compound according to any one of claims 2 to 3, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein j is selected from 1 or 2.
根据权利要求1所述的化合物或其立体异构体、互变异构体或其可药用的盐，其中所述的化合物为：The compound or its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof according to claim 1, wherein the compound is:
一种根据权利要求1所述的通式(I)化合物或其立体异构体、互变异构体或其可药用的盐的制备方法，所述方法包括：A method for preparing a compound of general formula (I) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof according to claim 1, said method comprising:

通式(IA)化合物或其盐与通式(IB)化合物在碱性条件下进行缩合反应，得到通式(I)化合物；The compound of general formula (IA) or its salt and the compound of general formula (IB) undergo condensation reaction under basic conditions to obtain the compound of general formula (I);

其中：in:

碱性条件由有机碱提供，所述有机碱选自N,N-二异丙基乙胺、吡啶、三乙胺、哌啶、N-甲基哌嗪、4-二甲氨吡啶或叔丁醇钾，优选为N,N-二异丙基乙胺；The basic conditions are provided by an organic base selected from N,N-diisopropylethylamine, pyridine, triethylamine, piperidine, N-methylpiperazine, 4-dimethylaminopyridine or tert-butyl Potassium alkoxide, preferably N,N-diisopropylethylamine;

R ¹～R ⁵、Z、m或n的定义如权利要求1中所述。 The definitions of R ¹ to R ⁵ , Z, m or n are as described in claim 1.
一种通式(IA)所示的化合物或其立体异构体、互变异构体或其可药用的盐：A compound represented by general formula (IA) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof:

其中：R ²～R ⁵、Z、m或n的定义如权利要求1中所述。 Wherein: R ² ˜R ⁵ , Z, m or n are defined as described in claim 1.
根据权利要求10所述的通式(IA)所示的化合物或其立体异构体、互变异构体或其可药用的盐，其中所述的化合物为：The compound represented by general formula (IA) or its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof according to claim 10, wherein the compound is:
一种药物组合物，所述的药物组合物含有有效剂量的根据权利要求1～8中任何一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，及可药用的载体、赋形剂或它们的组合。A pharmaceutical composition containing an effective dose of the compound or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof according to any one of claims 1-8, And pharmaceutically acceptable carriers, excipients or their combinations.
根据权利要求12所述的药物组合物，其特征在于，所述药物组合物还含有抗炎药中的至少一种，所述抗炎药选自非甾体类抗炎药、非特异性环氧合酶-2抑制剂、特异性环氧合酶-2抑制剂、皮质激素类、肿瘤坏死因子受体拮抗剂、水杨酸酯或盐、免疫抑制剂或甲氨蝶呤。The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition further contains at least one of anti-inflammatory drugs selected from the group consisting of non-steroidal anti-inflammatory drugs, non-specific epoxy Synthase-2 inhibitors, specific cyclooxygenase-2 inhibitors, corticosteroids, tumor necrosis factor receptor antagonists, salicylates or salts, immunosuppressants, or methotrexate.
根据权利要求1～8中任何一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，或根据权利要求12所述的药物组合物在制备JAK1激酶、JAK2激酶、JAK3激酶和/或TYK2激酶抑制剂中的用途，优选为在制备JAK1激酶、JAK2激酶和/或TYK2激酶抑制剂中的用途。The compound according to any one of claims 1 to 8 or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition according to claim 12 in the preparation of JAK1 kinase, The use of JAK2 kinase, JAK3 kinase and/or TYK2 kinase inhibitors is preferably the use in the preparation of JAK1 kinase, JAK2 kinase and/or TYK2 kinase inhibitors.
根据权利要求1～8中任何一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，或根据权利要求12所述的药物组合物在制备治疗由JAK1激酶、JAK2激酶、JAK3激酶和/或TYK2激酶介导的疾病的药物中的用途，其中优选为在制备治疗由JAK1激酶、JAK2激酶和/或TYK2激酶介导的疾病的药物中的用途。The compound according to any one of claims 1 to 8 or its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, or the pharmaceutical composition according to claim 12 in preparation and treatment by JAK1 The use of kinases, JAK2 kinase, JAK3 kinase, and/or TYK2 kinase in medicaments for diseases mediated by JAK1 kinase, JAK2 kinase and/or TYK2 kinase is preferably used in the preparation of drugs for treating diseases mediated by JAK1 kinase, JAK2 kinase and/or TYK2 kinase.
根据权利要求15所述的在制备治疗由JAK1激酶、JAK2激酶、JAK3激酶和/或TYK2激酶介导的疾病的药物中的用途，其中所述的由JAK1激酶、JAK2激酶和/或TYK2激酶介导的疾病包括自身免疫性疾病、炎症性疾病和癌症；其中所述的自身免疫性疾病包括哮喘、银屑病、狼疮、多发性硬化症、变应性鼻炎、异位性皮炎、接触性皮炎和迟发过敏反应；其中所述的炎症性疾病包括炎症性肠病、类风湿关节炎，所述炎症性肠病包括克罗恩病和溃疡性结肠炎；其中所述的癌症包括非小细胞肺癌、小细胞肺癌、乳腺癌、胰腺癌、神经胶质瘤、胶质母细胞瘤、卵巢癌、子***、结肠直肠癌、黑色素瘤、子宫内膜癌、***癌、膀胱癌、胃癌、肝癌、胃肠间质瘤、甲状腺癌、白血病、非霍奇金淋巴瘤、鼻咽癌、食道癌、脑瘤、淋巴瘤、多发性骨髓瘤、胆管癌和实体瘤，其中所述的白血病选自慢性粒细胞白血病和急性髓细胞性白血病。The use of claim 15 in the preparation of a medicament for the treatment of diseases mediated by JAK1 kinase, JAK2 kinase, JAK3 kinase and/or TYK2 kinase, wherein said JAK1 kinase, JAK2 kinase and/or TYK2 kinase are mediated by JAK1 kinase, JAK2 kinase and/or TYK2 kinase. The leading diseases include autoimmune diseases, inflammatory diseases and cancer; the autoimmune diseases described therein include asthma, psoriasis, lupus, multiple sclerosis, allergic rhinitis, atopic dermatitis, contact dermatitis And delayed allergic reactions; wherein the inflammatory diseases include inflammatory bowel disease, rheumatoid arthritis, the inflammatory bowel diseases include Crohn’s disease and ulcerative colitis; wherein the cancer includes non-small cell Lung cancer, small cell lung cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, ovarian cancer, cervical cancer, colorectal cancer, melanoma, endometrial cancer, prostate cancer, bladder cancer, stomach cancer, Liver cancer, gastrointestinal stromal tumor, thyroid cancer, leukemia, non-Hodgkin’s lymphoma, nasopharyngeal cancer, esophageal cancer, brain tumor, lymphoma, multiple myeloma, cholangiocarcinoma and solid tumors, among which leukemias are selected Since chronic myelogenous leukemia and acute myeloid leukemia.
根据权利要求1～8中任何一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，或根据权利要求12所述的药物组合物在制备治疗自身免疫性疾病、炎症性疾病和癌症药物中的用途；其中所述的自身免疫性疾病包括哮喘、银屑病、狼疮、多发性硬化症、变应性鼻炎、异位性皮炎、接触性皮炎和迟发过敏反应；其中所述的炎症性疾病包括炎症性肠病、类风湿关节炎，所述炎症性肠病包括克罗恩病和溃疡性结肠炎；其中所述的癌症包括非小细胞肺癌、小细胞肺癌、乳腺癌、胰腺癌、神经胶质瘤、胶质母细胞瘤、卵巢癌、子***、结肠直肠癌、黑色素瘤、子宫内膜癌、***癌、膀胱癌、胃癌、肝癌、胃肠间质瘤、甲状腺癌、白血病、非霍奇金淋巴瘤、鼻咽癌、食道癌、脑瘤、淋巴瘤、多发性骨髓瘤、胆管癌和实体瘤。The compound according to any one of claims 1 to 8 or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition according to claim 12 is used in the preparation of the treatment of autoimmunity The use of sexual diseases, inflammatory diseases and cancer drugs; wherein the autoimmune diseases include asthma, psoriasis, lupus, multiple sclerosis, allergic rhinitis, atopic dermatitis, contact dermatitis and delayed Allergic reactions; wherein the inflammatory diseases include inflammatory bowel disease, rheumatoid arthritis, the inflammatory bowel diseases include Crohn’s disease and ulcerative colitis; wherein the cancer includes non-small cell lung cancer, Small cell lung cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, ovarian cancer, cervical cancer, colorectal cancer, melanoma, endometrial cancer, prostate cancer, bladder cancer, stomach cancer, liver cancer, Gastrointestinal stromal tumor, thyroid cancer, leukemia, non-Hodgkin’s lymphoma, nasopharyngeal cancer, esophageal cancer, brain tumor, lymphoma, multiple myeloma, cholangiocarcinoma, and solid tumors.