技术领域Technical Field
本发明一般性涉及新的具有SHP2抑制活性的取代的炔基杂环化合物、其制备方法、其药物组合物，还涉及这类化合物及其药物组合物治疗受益于SHP2酶抑制的疾病的用途，例如治疗癌症。The present invention generally relates to novel substituted alkynyl heterocyclic compounds having SHP2 inhibitory activity, methods for preparing the same, pharmaceutical compositions thereof, and uses of the compounds and pharmaceutical compositions thereof for treating diseases that benefit from SHP2 enzyme inhibition, such as cancer.
背景技术Background Art
癌症是严重威胁人类健康及生命一类疾重大疾病，尤其是癌症近几年的发病率及死亡率呈快速上升趋势，己超越心血管疾病成为人类健康的头号杀手。肿瘤的增殖、凋亡、转移等与细胞内外的一系列信号传导通路中某个环节的异常密切相关。在这些信号传导途径中，蛋白的磷酸化和去磷酸化至关重要，这个可逆的过程受到激酶和磷酸酶的共同调控。蛋白质酪氨酸激酶(PTK)的磷酸化和蛋白质酪氨酸磷酸酶(PTP)的去磷酸化即是这样一对可逆过程，它们之间保持动态平衡以维持细胞的正常生理功能。反之异常的磷酸化能导致癌症、炎症、糖尿病和其它疾病的产生。Cancer is a serious disease that threatens human health and life. In particular, the incidence and mortality of cancer have been rising rapidly in recent years, surpassing cardiovascular disease to become the number one killer of human health. Tumor proliferation, apoptosis, metastasis, etc. are closely related to the abnormality of a certain link in a series of signal transduction pathways inside and outside the cell. In these signal transduction pathways, protein phosphorylation and dephosphorylation are crucial. This reversible process is jointly regulated by kinases and phosphatases. Phosphorylation of protein tyrosine kinase (PTK) and dephosphorylation of protein tyrosine phosphatase (PTP) are such a pair of reversible processes, and they maintain a dynamic balance to maintain the normal physiological function of cells. On the contrary, abnormal phosphorylation can lead to cancer, inflammation, diabetes and other diseases.
SHP2蛋白是由ptpnll基因编码的一种非受体型蛋白质酪氨酸磷酸酶，广泛表达于各组织，参与胚胎发育、新陈代谢、免疫反应以及肿瘤发生等重要的生理病理过程。SHP2 protein is a non-receptor protein tyrosine phosphatase encoded by the ptpnll gene. It is widely expressed in various tissues and participates in important physiological and pathological processes such as embryonic development, metabolism, immune response and tumorigenesis.
SHP2蛋白由N末端的两个串联SH2结构域(N-SH2和C-SH2)，PTP催化结构域和具有调节作用的C末端尾部组成。SH2结构域是一个构象开关，可以介导SHP2蛋白与含磷酸酪氨酸的激活剂(例如胰岛素受体底物1-IRS1和GRB2相关结合蛋白1-GAB1)的相互作用以及SH2结构域与PTP催化结构域的分子内相互作用。在未受刺激的情况下，SHP2结构域与PTP结构域结合，封闭催化活性位点，使SHP2磷酸酶活性处于自抑制状态。当SH2结构域结合激活剂，抑制性分子内相互作用解除，SHP2磷酸酶处于开放构象，允许SHP2底物定位至催化活性位点并发挥磷酸酶功能。SHP2的这种活性转换的特性，使得各种关于SHP2的突变都有可能破坏SHP2自抑制状态，导致SHP2蛋白磷酸酶活性过度激活，进而引发癌变。实验和临床数据均证实，SHP2在大多数癌症中起到了促进作用，作为第一个被发现的促进癌症发展的酪氨酸磷酸酶，其在癌症领域得到了极大地关注，它的磷酸酶活性在细胞内信号调控中起到了重要作用。The SHP2 protein consists of two tandem SH2 domains (N-SH2 and C-SH2) at the N-terminus, a PTP catalytic domain, and a C-terminal tail with a regulatory function. The SH2 domain is a conformational switch that mediates the interaction between the SHP2 protein and phosphotyrosine-containing activators (such as insulin receptor substrate 1-IRS1 and GRB2-associated binding protein 1-GAB1) and the intramolecular interaction between the SH2 domain and the PTP catalytic domain. In the absence of stimulation, the SHP2 domain binds to the PTP domain, blocking the catalytic active site and placing the SHP2 phosphatase activity in an autoinhibitory state. When the SH2 domain binds to the activator, the inhibitory intramolecular interaction is released, and the SHP2 phosphatase is in an open conformation, allowing the SHP2 substrate to locate to the catalytic active site and perform the phosphatase function. This activity conversion characteristic of SHP2 makes it possible for various mutations in SHP2 to destroy the SHP2 autoinhibitory state, leading to overactivation of the SHP2 protein phosphatase activity, and then inducing carcinogenesis. Both experimental and clinical data have confirmed that SHP2 plays a promoting role in most cancers. As the first tyrosine phosphatase discovered to promote cancer development, it has received great attention in the field of cancer. Its phosphatase activity plays an important role in intracellular signal regulation.
SHP2参与调控由细胞因子、生长因子和激素激活的细胞信号转导途径，包括RAS/ERK，JAK/STAT，PI3K/AKT与NF-κB信号通路，进而调控细胞增殖、分化、细胞周期维持和迁移等生理功能。同时，SHP2还介导了MEK等激酶被抑制之后的代偿性激活途径，从而促进肿瘤耐药的发生。作为PD-1受体的下游分子，SHP2还参与T细胞抑制性信号的传导。已有研究表明，SHP2是PD-1信号传导的下游分子，它不仅抑制T细胞活化而且促进T细胞的失能。因此，靶向SHP2可恢复或增强T细胞介导的抗肿瘤免疫功能。另外SHP2可以通过失活信号转导及转录激活因子STAT1抑制IFN-γ介导的免疫反应。SHP2 is involved in regulating cell signal transduction pathways activated by cytokines, growth factors and hormones, including RAS/ERK, JAK/STAT, PI3K/AKT and NF-κB signaling pathways, thereby regulating physiological functions such as cell proliferation, differentiation, cell cycle maintenance and migration. At the same time, SHP2 also mediates compensatory activation pathways after kinases such as MEK are inhibited, thereby promoting the occurrence of tumor resistance. As a downstream molecule of the PD-1 receptor, SHP2 is also involved in the conduction of T cell inhibitory signals. Studies have shown that SHP2 is a downstream molecule of PD-1 signaling, which not only inhibits T cell activation but also promotes T cell dysfunction. Therefore, targeting SHP2 can restore or enhance T cell-mediated anti-tumor immune function. In addition, SHP2 can inhibit IFN-γ-mediated immune responses by inactivating the signal transducer and transcription activator STAT1.
近年来，SHP2激活突变和高表达在白血病、实体瘤、黑色素瘤、恶性胶质瘤、肺癌、乳腺癌和努男综合症中陆续被发现，与肿瘤的发生、发展和预后密切相关。目前，SHP2已被研究用作临床肿瘤的靶分子。传统SHP2抑制剂(例如II-B08、PHPS1)作用机制均为与SHP2的PTP催化结构域结合，阻止酪氨酸磷酸化的底物进入催化位点，从而抑制SHP2的磷酸酶活性。然而，由于各种磷酸酶PTP催化结构域高度保守、极性和带电环境，使得SHP2传统抑制剂在特异性与生物利用度方面具有较大的缺陷，限制了其临床应用。因此，开发具有高特异性、高安全性、细胞膜渗透性强的SHP2抑制剂是决定SHP2是否可以成为新型肿瘤干预靶点的关键，SHP2蛋白变构抑制剂成为目前研究的主要方向。In recent years, SHP2 activation mutations and high expression have been found in leukemia, solid tumors, melanoma, malignant glioma, lung cancer, breast cancer and Nunan syndrome, and are closely related to the occurrence, development and prognosis of tumors. At present, SHP2 has been studied as a target molecule for clinical tumors. The mechanism of action of traditional SHP2 inhibitors (such as II-B08 and PHPS1) is to bind to the PTP catalytic domain of SHP2, prevent tyrosine phosphorylated substrates from entering the catalytic site, and thus inhibit the phosphatase activity of SHP2. However, due to the highly conserved, polar and charged environment of the PTP catalytic domains of various phosphatases, traditional SHP2 inhibitors have great defects in specificity and bioavailability, which limits their clinical application. Therefore, the development of SHP2 inhibitors with high specificity, high safety and strong cell membrane permeability is the key to determining whether SHP2 can become a new tumor intervention target, and SHP2 protein allosteric inhibitors have become the main direction of current research.
目前尚且没有SHP2抑制剂被批准上市，处于临床研究阶段的化合物有三个，其中加科思公司的JAB-3068进展最快，已被批准II期临床研究，诺华的TNO155和新锐制药公司的RMC-4630都处于临床I期研究阶段。Currently, no SHP2 inhibitors have been approved for marketing. There are three compounds in the clinical research stage. Among them, JAB-3068 of Jacobios has made the fastest progress and has been approved for Phase II clinical trials. TNO155 of Novartis and RMC-4630 of Newray Pharmaceuticals are both in Phase I clinical research.
发明内容Summary of the invention
本发明提供一种式(I)表示的化合物或其药学上可接受的盐、溶剂化物、多晶型物、互变异构体、代谢物或前药，The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof,
其中，in,
A选自如下两并杂环或三并杂环；A is selected from the following dicyclic heterocyclic or tricyclic heterocyclic rings;
R1和R2各自独立地选自氢、卤素和C1-6烷基；R1 andR2 are each independently selected from hydrogen, halogen andC1-6 alkyl;
X选自氢、C1-6烷基、C3-8环烷基、C6-10芳基和C5-10杂芳基，所述C1-6烷基和C3-8环烷基可任选地被一个或多个卤素、-OH、-O-C1-6烷基、-NH2、或C1-6烷基取代，所述C6-10芳基和C5-10杂芳基可与不饱和的脂环、杂脂环、螺环稠合，并且可任选地被一个或多个卤素、-CF3、-OH、-CN、-O-C1-6烷基、-NR3R4、-O-C(O)NR3R4、-NH-(CO)-C1-6烷基、-S-CH2-CONH2、C1-6烷基、C3-6环烷基、C6-10芳基、或C6-10杂芳基取代；X is selected from hydrogen, C1-6 alkyl, C3-8 cycloalkyl, C6-10 aryl and C5-10 heteroaryl, the C1-6 alkyl and C3-8 cycloalkyl may be optionally substituted by one or more halogen, -OH, -OC1-6 alkyl, -NH2 , or C1-6 alkyl, the C6-10 aryl and C5-10 heteroaryl may be condensed with an unsaturated alicyclic, heteroalicyclic, spirocyclic ring, and may be optionally substituted by one or more halogen, -CF3 , -OH, -CN, -OC1-6 alkyl, -NR3 R4 , -OC(O)NR3 R4 , -NH—(CO)—C1-6 alkyl, -S—CH2 —CONH2 , C1-6 alkyl, C3-6 cycloalkyl, C6-10 aryl, or C6-10 heteroaryl;
R3和R4各自独立地选自氢和C1-6烷基；R3 andR4 are each independently selected from hydrogen andC1-6 alkyl;
Y选自C6-10芳基、C5-10杂芳基和C3-12杂脂环基，所述芳基、杂芳基和杂脂环基可任选地被一个或多个卤素、-OH、-O-C1-6烷基、-NH2、C1-6烷基、或C3-6环烷基取代，所述烷基或环烷基可任选地被卤素、-OH、-O-C1-6烷基、或-NH2取代；Y is selected from C6-10 aryl, C5-10 heteroaryl and C3-12 heteroalicyclic, wherein the aryl, heteroaryl and heteroalicyclic may be optionally substituted by one or more halogen, -OH, -OC1-6 alkyl, -NH2 , C1-6 alkyl, or C3-6 cycloalkyl, wherein the alkyl or cycloalkyl may be optionally substituted by halogen, -OH, -OC1-6 alkyl, or -NH2 ;
Z选自单键、-S-和-C≡C-。Z is selected from a single bond, -S- and -C≡C-.
根据本发明的一些实施方式，本发明的式(I)化合物中，具有下式II～X：According to some embodiments of the present invention, the compound of formula (I) of the present invention has the following formula II to X:
其中，R1和R2各自独立地选自氢、卤素和C1-6烷基；wherein R1 and R2 are each independently selected from hydrogen, halogen and C1-6 alkyl;
X选自氢、C1-6烷基、C3-8环烷基、C6-10芳基和C5-10杂芳基，所述C1-6烷基和C3-8环烷基可任选地被一个或多个卤素、-OH、-O-C1-6烷基、-NH2、或C1-6烷基取代，所述C6-10芳基和C5-10杂芳基可与不饱和的脂环、杂脂环、螺环稠合，并且可任选地被一个或多个卤素、-CF3、-OH、-CN、-O-C1-6烷基、-NR3R4、-O-C(O)NR3R4、-NH-(CO)-C1-6烷基、-S-CH2-CONH2、C1-6烷基、C3-6环烷基、C6-10芳基、或C6-10杂芳基取代；X is selected from hydrogen, C1-6 alkyl, C3-8 cycloalkyl, C6-10 aryl and C5-10 heteroaryl, the C1-6 alkyl and C3-8 cycloalkyl may be optionally substituted by one or more halogen, -OH, -OC1-6 alkyl, -NH2 , or C1-6 alkyl, the C6-10 aryl and C5-10 heteroaryl may be condensed with an unsaturated alicyclic, heteroalicyclic, spirocyclic ring, and may be optionally substituted by one or more halogen, -CF3 , -OH, -CN, -OC1-6 alkyl, -NR3 R4 , -OC(O)NR3 R4 , -NH—(CO)—C1-6 alkyl, -S—CH2 —CONH2 , C1-6 alkyl, C3-6 cycloalkyl, C6-10 aryl, or C6-10 heteroaryl;
R3和R4各自独立地选自氢和C1-6烷基；R3 andR4 are each independently selected from hydrogen andC1-6 alkyl;
Y选自C6-10芳基、C5-10杂芳基和C3-12杂脂环基，所述芳基、杂芳基和杂脂环基可任选地被一个或多个卤素、-OH、-O-C1-6烷基、-NH2、C1-6烷基、或C3-6环烷基取代，所述烷基或环烷基可任选地被卤素、-OH、-O-C1-6烷基、或-NH2取代。Y is selected from C6-10 aryl, C5-10 heteroaryl and C3-12 heteroalicyclic, wherein the aryl, heteroaryl and heteroalicyclic may be optionally substituted by one or more halogen, -OH, -OC1-6 alkyl, -NH2 , C1-6 alkyl, or C3-6 cycloalkyl, and the alkyl or cycloalkyl may be optionally substituted by halogen, -OH, -OC1-6 alkyl, or -NH2 .
在一些实施方式中，R1和R2各自独立地选自氢和C1-6烷基；In some embodiments, R1 and R2 are each independently selected from hydrogen and C1-6 alkyl;
在一些实施方式中，X选自氢、C1-6烷基、C3-8环烷基、C6-10芳基和C5-10杂芳基，所述C1-6烷基和C3-8环烷基可任选地被一个或多个卤素、-OH、-O-C1-6烷基、-NH2、或C1-6烷基取代，所述C6-10芳基和C5-10杂芳基可任选地被一个或多个卤素、-CF3、-OH、-CN、-O-C1-6烷基、-NR3R4、-O-C(O)NR3R4、-NH-(CO)-C1-6烷基、-S-CH2-CONH2、C1-6烷基、C3-6环烷基、C6-10芳基、或C5-10杂芳基取代，In some embodiments, X is selected from hydrogen, C1-6 alkyl, C3-8 cycloalkyl, C6-10 aryl and C5-10 heteroaryl, the C1-6 alkyl and C3-8 cycloalkyl may be optionally substituted with one or more halogen, -OH, -OC1-6 alkyl, -NH2 , or C1-6 alkyl, the C6-10 aryl and C5-10 heteroaryl may be optionally substituted with one or more halogen, -CF3 , -OH, -CN, -OC1-6 alkyl, -NR3 R4 , -OC(O)NR3 R4 , -NH-(CO)-C1-6 alkyl, -S-CH2 -CONH2 , C1-6 alkyl, C3-6 cycloalkyl, C6-10 aryl, or C5-10 heteroaryl,
R3和R4各自独立地选自氢和C1-6烷基；R3 andR4 are each independently selected from hydrogen andC1-6 alkyl;
在一些实施方式中，Y选自C6-10芳基、C5-10杂芳基和C3-12杂脂环基，所述芳基、杂芳基和杂脂环基可任选地被一个或多个-OH、-NH2、或C1-6烷基取代；In some embodiments, Y is selected from C6-10 aryl, C5-10 heteroaryl and C3-12 heteroalicyclic, wherein the aryl, heteroaryl and heteroalicyclic may be optionally substituted with one or more -OH, -NH2 , or C1-6 alkyl;
在一些实施方式中，Y选自C3-12杂脂环基，所述杂脂环基可任选地被一个或多个-OH、-NH2、或C1-6烷基取代；In some embodiments, Y is selected from C3-12 heteroalicyclic groups, which may be optionally substituted with one or more -OH, -NH2 , or C1-6 alkyl groups;
在一些实施方式中，Y选自C3-12杂脂环基，所述杂脂环基可任选地被一个或多个-NH2或C1-6烷基取代；In some embodiments, Y is selected from C3-12 heteroalicyclic groups, which may be optionally substituted with one or more -NH2 or C1-6 alkyl groups;
根据本发明的一些实施方式，本发明提供以下化合物：According to some embodiments of the present invention, the present invention provides the following compounds:
另一方面，本发明提供一种药物组合物，其包含本发明的化合物或其药学上可接受的盐、溶剂化物、多晶型物、或互变异构体。在一些实施方案中，本发明的药物组合物还包括药学上可接受的辅料。In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof. In some embodiments, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable excipient.
另一方面，本发明提供治疗与SHP2相关的疾病的方法，其包括对需要该治疗的哺乳动物，优选人类，给予治疗有效量的本发明化合物或其药学上可接受的盐、溶剂化物、多晶型物、或互变异构体、或其药物组合物。In another aspect, the present invention provides a method for treating a disease associated with SHP2, comprising administering a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, or a pharmaceutical composition thereof to a mammal, preferably a human, in need of such treatment.
另一方面，本发明提供本发明化合物或其药学上可接受的盐、溶剂化物、多晶型物、或互变异构体、或其药物组合物在制备治疗与SHP2相关的疾病的药物中的用途。In another aspect, the present invention provides use of a compound of the present invention or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for treating a disease associated with SHP2.
在本发明的部分实施方式中，所述与SHP2相关的疾病为白血病、黑色素瘤、恶性胶质瘤、肺癌、乳腺癌或努男综合症。In some embodiments of the present invention, the disease associated with SHP2 is leukemia, melanoma, malignant glioma, lung cancer, breast cancer or Noonan syndrome.
某些化学术语Some chemical terms
本发明所述的“化合物”包括所有立体异构体、几何异构体、互变异构体和同位素。The “compounds” described in the present invention include all stereoisomers, geometric isomers, tautomers and isotopes.
本发明化合物可以是不对称的，例如，具有一个或多个立体异构体。除非另有说明，所有立体异构体都包括，如对映异构体和非对映异构体。本发明的含有不对称碳原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来。光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。The compounds of the invention may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the invention containing asymmetric carbon atoms can be isolated in optically pure forms or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.
本发明化合物还包括互变异构体形式。互变异构体形式来源于一个单键与相邻的双键交换并一起伴随一个质子的迁移。The compounds of the present invention also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond accompanied by the migration of a proton.
本发明还包括所有同位素的原子，无论是在中间体或最后的化合物。同位素的原子包括具有相同的原子数，但不同质量数。例如，氢的同位素包括氚和氘。The present invention also includes all isotopes of atoms, whether in the intermediates or the final compounds. Isotopes of atoms include atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.
在通式I-V化合物的上述定义中，本文中所用的术语具有如下含义：In the above definition of compounds of general formula I-V, the terms used herein have the following meanings:
术语“卤素”是指氟、氯、溴或碘，优选氟、氯或溴。The term "halogen" refers to fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
术语“烷基”是指由碳原子和氢原子组成的直链或支链的饱和烃基团，如C1-20烷基，优选为C1-6烷基，例如甲基、乙基、丙基(例如正丙基和异丙基)、丁基(例如正丁基、异丁基、仲丁基或叔丁基)、戊基(例如正戊基、异戊基、新戊基)、正己基、2-甲基己基等。所述烷基可以是非取代的或是被取代的，所述的取代基包括但不限于烷基、烷基氧基、氰基、羧基、芳基、杂芳基、氨基、卤素、磺酰基、亚磺酰基、磷酰基和羟基。The term "alkyl" refers to a straight or branched saturated hydrocarbon group consisting of carbon atoms and hydrogen atoms, such as aC1-20 alkyl group, preferably aC1-6 alkyl group, such as a methyl group, an ethyl group, a propyl group (such as n-propyl and isopropyl), a butyl group (such as n-butyl, isobutyl, sec-butyl or tert-butyl), a pentyl group (such as n-pentyl, isopentyl, neopentyl), a n-hexyl group, a 2-methylhexyl group, etc. The alkyl group may be unsubstituted or substituted, and the substituents include, but are not limited to, alkyl, alkyloxy, cyano, carboxyl, aryl, heteroaryl, amino, halogen, sulfonyl, sulfinyl, phosphoryl and hydroxyl.
术语“C1-6烷基”是指由碳原子和氢原子组成的直链或支链的饱和的脂肪烃基团，其通过单键与分子的其余部分连接，其具有1-6个碳原子。所述烷基可以是非取代的或是被一个或多个选自烷基、烷氧基、氨基、卤素和羟基的取代基所取代。非取代的烷基的非限制性实例包括但不限于诸如甲基、乙基、丙基、2-丙基、正丁基、异丁基、叔-丁基、正-戊基、2-甲基丁基、新戊基、正己基、2-甲基己基等。The term "C1-6 alkyl" refers to a straight or branched saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond and has 1 to 6 carbon atoms. The alkyl group may be unsubstituted or substituted with one or more substituents selected from alkyl, alkoxy, amino, halogen and hydroxy. Non-limiting examples of unsubstituted alkyl groups include, but are not limited to, groups such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-methylhexyl, etc.
术语“环烷基”是指由碳原子和氢原子组成的全碳单环的饱和烃基团，如C3-20环烷基，优选为C3-6环烷基，例如环丙基、环丁基、环戊基、环己基等。所述环烷基可以是非取代的或是被取代的，所述的取代基包括但不限于烷基、烷基氧基、氰基、羧基、芳基、杂芳基、氨基、卤素、磺酰基、亚磺酰基、磷酰基和羟基。The term "cycloalkyl" refers to a monocyclic saturated hydrocarbon group consisting of carbon atoms and hydrogen atoms, such as aC3-20 cycloalkyl, preferably aC3-6 cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The cycloalkyl may be unsubstituted or substituted, and the substituents include, but are not limited to, alkyl, alkyloxy, cyano, carboxyl, aryl, heteroaryl, amino, halogen, sulfonyl, sulfinyl, phosphoryl and hydroxyl.
术语“芳基”是指具有完全共轭的π电子体系的全碳单环或稠合环，其具有6-14个碳原子，优选具有6-12个碳原子，最优选具有6个碳原子。芳基可以是非取代的或被一个或多个取代基所取代，所述取代基的实例包括但不限于烷基、烷基氧基、芳基、芳烷基、氨基、卤素、羟基、磺酰基、亚磺酰基、磷酰基和杂脂环基。非取代的芳基的非限制性实例包括但不限于苯基、萘基和蒽基。The term "aryl" refers to a fully conjugated π electron system of all-carbon monocyclic or fused rings having 6-14 carbon atoms, preferably 6-12 carbon atoms, and most preferably 6 carbon atoms. Aryl can be unsubstituted or substituted with one or more substituents, examples of which include but are not limited to alkyl, alkyloxy, aryl, aralkyl, amino, halogen, hydroxyl, sulfonyl, sulfinyl, phosphoryl and heteroalicyclic. Non-limiting examples of unsubstituted aryl include but are not limited to phenyl, naphthyl and anthracenyl.
术语“杂芳基”是指5-12个环原子的单环或稠合环，具有5、6、7、8、9、10、11或12个环原子，其中含有1、2、3或4个选自N、O、S的环原子，其余环原子为C，且具有完全共轭的π-电子体系。杂芳基可以是非取代的或取代的，所述的取代基包括但不限于烷基、烷基氧基、芳基、芳烷基、氨基、卤素、羟基、氰基、硝基、羰基和杂脂环基。非取代的杂芳基的非限制性实例包括但不限于吡咯基、呋喃基、噻吩基、咪唑基、噁唑基、吡唑基、吡啶基、嘧啶基、吡嗪基、喹啉基、异喹啉基、四唑基、三嗪基。The term "heteroaryl" refers to a monocyclic or fused ring of 5-12 ring atoms, having 5, 6, 7, 8, 9, 10, 11 or 12 ring atoms, wherein 1, 2, 3 or 4 ring atoms are selected from N, O, S, and the remaining ring atoms are C, and have a completely conjugated π-electron system. Heteroaryl can be unsubstituted or substituted, and the substituents include, but are not limited to, alkyl, alkyloxy, aryl, aralkyl, amino, halogen, hydroxyl, cyano, nitro, carbonyl and heteroalicyclic. Non-limiting examples of unsubstituted heteroaryl include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, triazinyl.
术语“杂脂环”是指具有3-12个环原子的单环或稠合环，具有3、4、5、6、7、8、9、10、11或12个环原子，其中1或2个环原子是选自N、O、S(O)n(其中n为0、1或2)的杂原子，其余环原子为C。这样的环可以是饱和的或不饱和的(例如具有一个或多个双键)，但是不具有完全共轭的π-电子体系。3元饱和杂脂环的实例包括但不限于4元饱和杂脂环的实例包括但不限于5元饱和杂脂环的实例包括但不限于6元饱和杂脂环的实例包括但不限于7元饱和杂脂环的实例包括但不限于5元不饱和杂脂环的实例包括但不限于6元不饱和杂脂环的实例包括但不限于The term "heteroalicyclic ring" refers to a monocyclic or fused ring having 3-12 ring atoms, having 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 ring atoms, of which 1 or 2 ring atoms are heteroatoms selected from N, O, S(O)n (where n is 0, 1 or 2), and the remaining ring atoms are C. Such rings may be saturated or unsaturated (e.g., have one or more double bonds), but do not have a completely conjugated π-electron system. Examples of 3-membered saturated heteroalicyclic rings include, but are not limited to Examples of 4-membered saturated heteroalicyclic rings include, but are not limited to Examples of 5-membered saturated heteroalicyclic rings include, but are not limited to Examples of 6-membered saturated heteroalicyclic rings include, but are not limited to Examples of 7-membered saturated heteroalicyclic rings include, but are not limited to Examples of 5-membered unsaturated heteroalicyclic rings include, but are not limited to Examples of 6-membered unsaturated heteroalicyclic rings include, but are not limited to
术语“杂脂环基”是指“杂脂环”分子去掉1个氢原子后余下的基团。杂脂环基可以是非取代的或者其中的氢原子任选地被取代基取代，所述的取代基包括但不限于烷基、烷氧基、＝O、芳基、芳烷基、-COOH、-CN、氨基、卤素和羟基。The term "heteroalicyclic group" refers to the group remaining after removing one hydrogen atom from a "heteroalicyclic" molecule. The heteroalicyclic group may be unsubstituted or the hydrogen atoms therein may be optionally substituted with substituents, including but not limited to alkyl, alkoxy, =O, aryl, aralkyl, -COOH, -CN, amino, halogen and hydroxyl.
本发明还提供了制备上述式化合物的方法，包括以下合成方案：The present invention also provides a method for preparing the compound of the above formula, comprising the following synthesis scheme:
合成方案1：Synthesis Scheme 1:
化合物式1-6可以使用合成方案1合成。4，6-二氯-1H-吡唑并[3，4-d]嘧啶与适当的保护剂反应得到中间体1-1。中间体1-1与氢氧化钠溶液反应，得到中间体1-2。中间体1-2与碘甲烷和碱反应得到中间体1-3。中间体1-3与含NH或硼酸酯的化合物反应得到中间体1-4。中间体1-4脱去保护基得到中间体1-5。中间体1-5与N-碘代丁二酰亚胺反应得到化合物1-6。Compound 1-6 can be synthesized using Synthesis Scheme 1. 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine is reacted with an appropriate protecting agent to obtain intermediate 1-1. Intermediate 1-1 is reacted with sodium hydroxide solution to obtain intermediate 1-2. Intermediate 1-2 is reacted with iodomethane and a base to obtain intermediate 1-3. Intermediate 1-3 is reacted with a compound containing NH or borate to obtain intermediate 1-4. Intermediate 1-4 is deprotected to obtain intermediate 1-5. Intermediate 1-5 is reacted with N-iodosuccinimide to obtain compound 1-6.
合成方案2：Synthesis Scheme 2:
化合物式2-5可以使用合成方案2合成。4，6-二氯-1H-吡唑并[3，4-d]嘧啶与N-碘代丁二酰亚胺反应得碘代中间体2-1。中间体2-1和氨水反应到中间体2-2。中间体2-2和适当的保护剂反应得到中间体2-3。中间体2-3用氯乙醛闭环得中间体2-4。中间体2-4与含NH或硼酸酯的化合物反应得到化合物2-5。Compound 2-5 can be synthesized using Synthesis Scheme 2. 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine reacts with N-iodosuccinimide to obtain iodinated intermediate 2-1. Intermediate 2-1 reacts with aqueous ammonia to obtain intermediate 2-2. Intermediate 2-2 reacts with an appropriate protective agent to obtain intermediate 2-3. Intermediate 2-3 is ring-closed with chloroacetaldehyde to obtain intermediate 2-4. Intermediate 2-4 reacts with a compound containing NH or borate to obtain compound 2-5.
合成方案3：Synthesis Scheme 3:
化合物式3-3可以通过合成方案3合成。6-氯-1H-吡唑并[3，4-b]吡嗪与N-碘代丁二酰亚胺反应得到碘代中间体3-1。中间体3-1和适当的保护剂反应得到中间体3-2。中间体3-2与含NH或硼酸酯的化合物反应得到化合物3-3。Compound 3-3 can be synthesized by Synthesis Scheme 3. 6-Chloro-1H-pyrazolo[3,4-b]pyrazine reacts with N-iodosuccinimide to obtain iodinated intermediate 3-1. Intermediate 3-1 reacts with a suitable protecting agent to obtain intermediate 3-2. Intermediate 3-2 reacts with a compound containing NH or borate to obtain compound 3-3.
合成方案4：Synthesis Scheme 4:
最终化合物4-1可以通过合成方案4合成。R1代表端基乙炔、硼酸或硼酸酯、巯基，R2代表碘或溴，两个原料在钯催化剂和碱存在下反应得到产物4-1。若4-1上的基团带有保护基则采用相应的方法脱除保护基。The final compound 4-1 can be synthesized by Synthesis Scheme 4.R1 represents terminal acetylene, boric acid or boric acid ester, thiol,R2 represents iodine or bromine, and the two raw materials react in the presence of a palladium catalyst and a base to obtain the product 4-1. If the group on 4-1 has a protecting group, the protecting group is removed by a corresponding method.
上述合成方案只是列举了本发明中部分化合物的制备方法，按照本领域的公知技术，技术人员在上述合成方案的基础上，采用类似的方法也可合成本发明中的化合物。The above synthesis scheme only lists the preparation methods of some compounds in the present invention. According to the known technology in the art, technicians can also synthesize the compounds in the present invention by using similar methods based on the above synthesis scheme.
本发明的化合物或其盐可以作为活性物质单独给药，优选以其药物组合物的形式给药。The compound of the present invention or a salt thereof can be administered alone as an active substance, or preferably in the form of a pharmaceutical composition thereof.
本发明另一方面提供一种药物组合物，其含有通式I、II、III、IV或V的化合物或其药学上可接受的盐、溶剂化物、多晶型、代谢物作为活性成份，以及一种或多种药学上可接受的载体。Another aspect of the present invention provides a pharmaceutical composition comprising a compound of Formula I, II, III, IV or V or a pharmaceutically acceptable salt, solvate, polymorph, metabolite thereof as an active ingredient, and one or more pharmaceutically acceptable carriers.
“药物组合物”是指一种或多种本发明的化合物或其盐与在本领域中通常接受的用于将生物活性化合物输送至有机体(例如人)的载体的制剂。药物组合物的目的是有利于对有机体给予本发明的化合物。"Pharmaceutical composition" refers to a preparation of one or more compounds of the present invention or their salts with carriers generally accepted in the art for delivering biologically active compounds to organisms (eg, humans). The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present invention to an organism.
术语“药学上可接受的载体”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些载体。“药学上可接受的载体”是指与活性成份一同给药的、有利于活性成份给药的惰性物质，包括但不限于被美国食品药品管理局许可为可接受的用于人或动物(例如家畜)的任何助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味增强剂、表面活性剂、润湿剂、分散剂、崩解剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。所述载体的非限制性实例包括碳酸钙、磷酸钙、各种糖和各类淀粉、纤维素衍生物、明胶、植物油和聚乙二醇。The term "pharmaceutically acceptable carrier" refers to those carriers that have no significant irritation to the organism and do not impair the biological activity and performance of the active compound. "Pharmaceutically acceptable carrier" refers to an inert substance that is administered together with the active ingredient and is conducive to the administration of the active ingredient, including but not limited to any glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, disintegrant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or animals (e.g., livestock). Non-limiting examples of the carrier include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycol.
以纯的形式或以适宜的药物组合物形式的本发明化合物或其药物可接受的盐的给药可通过提供类似用途的药剂的任何可接受的给药模式来进行。本发明的药物组合物可通过将本发明的化合物与适宜的药物可接受的载剂、稀释剂或赋形剂组合而制备。本发明的药物组合物可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、溶液剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶等等。The administration of the compounds of the present invention or their pharmaceutically acceptable salts in pure form or in the form of suitable pharmaceutical compositions can be carried out by any acceptable mode of administration of medicaments of similar use. The pharmaceutical composition of the present invention can be prepared by combining the compounds of the present invention with suitable pharmaceutically acceptable carriers, diluents or excipients. The pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols, etc.
给予本发明化合物或其药学上可接受的盐或其药物组合物的典型途径包括但不限于口服、直肠、透黏膜、经肠给药，或者局部、经皮、吸入、肠胃外、舌下、阴道内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。优选的给药途径是口服给药。Typical routes of administration of the compounds of the invention or their pharmaceutically acceptable salts or their pharmaceutical compositions include, but are not limited to, oral, rectal, transmucosal, enteral administration, or topical, transdermal, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration. The preferred route of administration is oral administration.
本发明的药物组合物可以采用本领域周知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。The pharmaceutical composition of the present invention can be manufactured by methods well known in the art, such as conventional mixing methods, dissolving methods, granulating methods, making sugar-coated pills methods, grinding methods, emulsifying methods, freeze-drying methods, and the like.
在优选的实施方案中，药物组合物是口服形式。对于口服给药，可以通过将活性化合物与本领域熟知的药学上可接受的载体混合，来配制该药物组合物。这些载体能使本发明的化合物被配制成片剂、丸剂、锭剂、糖衣剂、胶囊剂、液体、凝胶剂、浆剂、悬浮剂等，用于对患者的口服给药。In a preferred embodiment, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with a pharmaceutically acceptable carrier well known in the art. These carriers enable the compounds of the present invention to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.
可以通过常规的混合、填充或压片方法来制备固体口服药物组合物。例如，可通过下述方法获得：将所述的活性化合物与固体赋形剂混合，任选地碾磨所得的混合物，如果需要则加入其它合适的辅剂，然后将该混合物加工成颗粒，得到了片剂或糖衣剂的核心。适合的辅料包括但不限于：粘合剂、稀释剂、崩解剂、润滑剂、助流剂、甜味剂或矫味剂等。如微晶纤维素、葡萄糖溶液、阿拉伯胶浆、明胶溶液、蔗糖和淀粉糊；滑石、淀粉、硬脂酸镁、硬脂酸钙或硬脂酸；乳糖、蔗糖、淀粉、甘露糖醇、山梨糖醇或磷酸二钙；二氧化硅；交联羧甲基纤维素钠、预交化淀粉、淀粉羟乙酸钠、藻酸、玉米淀粉、马铃薯淀粉、甲基纤维素、琼脂、羧甲基纤维素、交联聚乙烯吡咯烷酮等。可以根据通常药物实践中公知的方法任选地对糖衣剂的核心进行包衣，尤其使用肠溶包衣。Solid oral pharmaceutical compositions can be prepared by conventional mixing, filling or tableting methods. For example, they can be obtained by the following method: the active compound is mixed with a solid excipient, the mixture is optionally ground, other suitable adjuvants are added if necessary, and then the mixture is processed into particles to obtain the core of a tablet or a sugar-coated agent. Suitable adjuvants include, but are not limited to, adhesives, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, etc. Such as microcrystalline cellulose, glucose solution, acacia gum, gelatin solution, sucrose and starch paste; talc, starch, magnesium stearate, calcium stearate or stearic acid; lactose, sucrose, starch, mannitol, sorbitol or dicalcium phosphate; silicon dioxide; cross-linked sodium carboxymethylcellulose, pre-cross-linked starch, sodium starch glycolate, alginic acid, corn starch, potato starch, methylcellulose, agar, carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, etc. The core of the sugar-coated agent can be optionally coated according to methods known in common pharmaceutical practice, especially using enteric coatings.
药物组合物还可适用于肠胃外给药，如合适的单位剂型的无菌溶液剂、混悬剂或冻干产品。能够使用适当的赋形剂，例如填充剂、缓冲剂或表面活性剂。The pharmaceutical composition may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms. Appropriate excipients such as fillers, buffers or surfactants can be used.
本发明再一方面涉及通式I至通式VI的化合物或其药学上可接受的盐、溶剂化物、多晶型、代谢物等在治疗受益于SHP2抑制的疾病的药物中的用途。所述的受益于SHP2抑制的疾病选自癌症。Another aspect of the present invention relates to the use of compounds of Formula I to Formula VI or pharmaceutically acceptable salts, solvates, polymorphs, metabolites, etc. thereof in drugs for treating diseases that benefit from SHP2 inhibition. The diseases that benefit from SHP2 inhibition are selected from cancer.
本发明提供的取代的炔基杂环类化合物具有非常好的SHP2抑制活性，有望成为高效的SHP2抑制剂类药物。The substituted alkynyl heterocyclic compounds provided by the present invention have very good SHP2 inhibitory activity and are expected to become highly effective SHP2 inhibitor drugs.
具体实施方式DETAILED DESCRIPTION
下面的具体实施例，其目的是使本领域的技术人员能更清楚地理解和实施本发明。它们不应该被认为是对本发明范围的限制，而只是本发明的示例性说明和典型代表。本领域技术人员应该理解：还有形成本发明化合物的其它合成途径，下面提供的是非限制性的实施例。The following specific examples are intended to enable those skilled in the art to more clearly understand and implement the present invention. They should not be considered as limiting the scope of the present invention, but are merely exemplary and typical representations of the present invention. It will be appreciated by those skilled in the art that there are other synthetic pathways for forming the compounds of the present invention, and non-limiting examples are provided below.
凡涉及易氧化或易水解的原料的所有操作都在氮气保护下进行。除非另有说明，本发明使用的原料都是市场上直接买到未经进一步纯化直接使用的。All operations involving raw materials that are easily oxidized or hydrolyzed are carried out under nitrogen protection. Unless otherwise specified, the raw materials used in the present invention are directly purchased from the market and used directly without further purification.
柱层析色谱采用青岛化工有限公司生产的硅胶(200-300目)。薄层色谱采用E.Merck公司生产的预制板(硅胶60 PF254，0.25毫米)。手性化合物分离和对映体过量值(ee)测定使用Agilent LC 1200 series(柱子：CHIRALPAK AD-H，毫米，5微米，30℃)。核磁共振色谱(NMR)使用Varian VNMRS-400核磁共振仪测定；液质联用(LC/MS)使用FINNIGAN Thermo LCQ Advantage MAX，Agilent LC 1200 series(柱子：Waters SymmetryC18，毫米，5微米，35℃)，采用ESI(+)离子模式。Column chromatography used silica gel (200-300 mesh) produced by Qingdao Chemical Co., Ltd. Thin layer chromatography used prefabricated plates (silica gel 60 PF254 , 0.25 mm) produced by E. Merck. Chiral compound separation and enantiomeric excess (ee) determination were performed using Agilent LC 1200 series (column: CHIRALPAK AD-H, mm, 5 μm, 30°C). Nuclear magnetic resonance chromatography (NMR) was measured using a Varian VNMRS-400 nuclear magnetic resonance instrument; liquid chromatography-mass spectrometry (LC/MS) was performed using a FINNIGAN Thermo LCQ Advantage MAX, Agilent LC 1200 series (column: Waters Symmetry C18, mm, 5 μm, 35 °C), using ESI (+) ion mode.
实验部分Experimental Section
中间体1：(3S，4S)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺二盐酸盐Intermediate 1:(3S, 4S)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine dihydrochloride
(3S，4S)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺二盐酸盐按照专利WO2017216706中中间体14的方法合成。(3S, 4S)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine dihydrochloride was synthesized according to the method of intermediate 14 in patent WO2017216706.
中间体2：6-氯-5-甲基-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-d]嘧啶-4(5H)-酮Intermediate 2:6-Chloro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
步骤1：4，6-二氯-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-d]嘧啶Step 1: 4,6-Dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine
将4，6-二氯-1H-吡唑并[3，4-d]嘧啶(2.7g)、3，4-二氢-2H-吡喃(2.4g)、对甲苯磺酸(0.25g)加入四氢呋喃(25mL)中，回流8小时，降至室温后，减压浓缩并用硅胶柱色谱分离(石油醚∶乙酸乙酯，10∶1)得4，6-二氯-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-d]嘧啶(3.0g)。MS m/z[LC-MS]：273.03[M+1]。4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (2.7 g), 3,4-dihydro-2H-pyran (2.4 g), p-toluenesulfonic acid (0.25 g) were added to tetrahydrofuran (25 mL), refluxed for 8 hours, cooled to room temperature, concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 10: 1) to obtain 4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine (3.0 g). MS m/z [LC-MS]: 273.03 [M+1].
步骤2：6-氯-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-d]嘧啶-4(5H)-酮Step 2: 6-Chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
将4，6-二氯-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-d]嘧啶(3.0g)和20％氢氧化钠溶液(4mL)加入乙腈(40mL)中，室温搅拌过夜，减压浓缩后用硅胶柱色谱分离(石油醚∶乙酸乙酯，6∶1)得6-氯-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-d]嘧啶-4(5H)-酮(2.4g)。MS m/z[LC-MS]：255.07[M+1]。1HNMR(400MHz，DMSO-d6)：δ＝13.300(1H，brs)，8.113(1H，s)，5.693(1H，m)，3.908(1H，m)，3.645(1H，m)，2.286(1H，m)，1.967(1H，m)，1.820(1H，m)，1.708(1H，m)，1.523(2H，m)。4,6-Dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine (3.0 g) and 20% sodium hydroxide solution (4 mL) were added to acetonitrile (40 mL), stirred at room temperature overnight, concentrated under reduced pressure, and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 6: 1) to obtain 6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (2.4 g). MS m/z[LC-MS]: 255.07[M+1].1 HNMR (400MHz, DMSO-d6 ): δ = 13.300 (1H, brs), 8.113 (1H, s), 5.693 (1H, m), 3.908 (1H, m), 3.645 (1H, m), 2.286 (1H, m), 1.967 (1H, m), 1.820 (1H, m), 1. 708(1H,m), 1.523(2H,m).
步骤3：6-氯-5-甲基-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-d]嘧啶-4(5H)-酮Step 3: 6-Chloro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
将6-氯-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-d]嘧啶-4(5H)-酮(2.4g)、碳酸钾(3.8g)加入N，N-二甲基甲酰胺(40mL)中，然后滴加碘甲烷(1.0mL)，室温搅拌4小时。反应液倒入200mL水中，用二氯甲烷萃取，萃取液用饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩后用硅胶柱色谱分离(石油醚∶乙酸乙酯，8∶1)得6-氯-5-甲基-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-d]嘧啶-4(5H)-酮(1.8g)。MS m/z[LC-MS]：269.08[M+1]。1HNMR(400MHz，DMSO-d6)：δ＝8.150(1H，s)，5.710(1H，m)，3.911(1H，m)，3.659(1H，m)，3.567(3H，s)，2.310(1H，m)，1.974(1H，m)，1.822(1H，m)，1.708(1H，m)，1.530(2H，m)。6-Chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (2.4 g) and potassium carbonate (3.8 g) were added to N,N-dimethylformamide (40 mL), and then iodomethane (1.0 mL) was added dropwise and stirred at room temperature for 4 hours. The reaction solution was poured into 200 mL of water and extracted with dichloromethane. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 8: 1) to obtain 6-chloro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (1.8 g). MS m/z[LC-MS]: 269.08[M+1].1 HNMR (400MHz, DMSO-d6 ): δ = 8.150 (1H, s), 5.710 (1H, m), 3.911 (1H, m), 3.659 (1H, m), 3.567 (3H, s), 2.310 (1H, m), 1.974 (1H, m), 1.822 (1H, m), 1.708 (1H, m), 1.530 (2H, m).
中间体3：9-碘-5-氯-7-(4-甲氧基苄基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶Intermediate 3:9-iodo-5-chloro-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidine
步骤1：3-碘-4，6-二氯-1H-吡唑并[3，4-d]嘧啶Step 1: 3-iodo-4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine
将4，6-二氯-1H-吡唑并[3，4-d]嘧啶(4.0g)、N-碘代丁二酰亚胺(5.72g)加入乙腈(25mL)中，加热至100℃微波反应25分钟，降至室温后，减压浓缩并用硅胶柱色谱分离(石油醚∶乙酸乙酯，8∶1)得3-碘-4，6-二氯-1H-吡唑并[3，4-d]嘧啶(5.4g)。MS m/z[LC-MS]：314.87[M+1]。4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (4.0 g) and N-iodosuccinimide (5.72 g) were added to acetonitrile (25 mL), heated to 100°C for microwave reaction for 25 minutes, cooled to room temperature, concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 8: 1) to obtain 3-iodo-4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (5.4 g). MS m/z [LC-MS]: 314.87 [M+1].
步骤2：3-碘-6-氯-1H-吡唑并[3，4-d]嘧啶-4-胺Step 2: 3-iodo-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine
将3-碘-4，6-二氯-1H-吡唑并[3，4-d]嘧啶(1.2g)和浓氨水(25％-28％，2mL)加入乙腈(20mL)中，室温搅拌过夜，减压浓缩后用硅胶柱色谱分离(石油醚∶乙酸乙酯，3∶1)得3-碘-6-氯-1H-吡唑并[3，4-d]嘧啶-4-胺(1.1g)。MS m/z[LC-MS]：295.92[M+1]。3-iodo-4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (1.2 g) and concentrated aqueous ammonia (25%-28%, 2 mL) were added to acetonitrile (20 mL), stirred at room temperature overnight, concentrated under reduced pressure, and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 3:1) to obtain 3-iodo-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1.1 g). MS m/z [LC-MS]: 295.92 [M+1].
步骤3：3-碘-6-氯-1-(4-甲氧基苄基)-1H-吡唑并[3，4-d]嘧啶-4-胺Step 3: 3-iodo-6-chloro-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
将3-碘-6-氯-1H-吡唑并[3，4-d]嘧啶-4-胺(1.1g)、碳酸钾(1.3g)加入N，N-二甲基甲酰胺(20mL)中，然后滴加4-甲氧基苄氯(0.5mL)，室温搅拌2小时。反应液倒入150mL水中，用二氯甲烷萃取，萃取液用饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩后用硅胶柱色谱分离(石油醚∶乙酸乙酯，4∶1)得3-碘-6-氯-1-(4-甲氧基苄基)-1H-吡唑并[3，4-d]嘧啶-4-胺(0.90g)。MS m/z[LC-MS]：415.98[M+1]。3-iodo-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1.1 g) and potassium carbonate (1.3 g) were added to N,N-dimethylformamide (20 mL), and then 4-methoxybenzyl chloride (0.5 mL) was added dropwise and stirred at room temperature for 2 hours. The reaction solution was poured into 150 mL of water and extracted with dichloromethane. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1) to obtain 3-iodo-6-chloro-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.90 g). MS m/z[LC-MS]: 415.98[M+1].
步骤4：9-碘-5-氯-7-(4-甲氧基苄基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶Step 4: 9-iodo-5-chloro-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidine
将3-碘-6-氯-1-(4-甲氧基苄基)-1H-吡唑并[3，4-d]嘧啶-4-胺(700mg)和氯乙醛(2mL)加入乙腈(20mL)中，加热至100℃封管反应5小时，倒入水中，用饱和碳酸钠水溶液调pH值到9～10，用二氯甲烷萃取，萃取液用饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩后用硅胶柱色谱分离(石油醚∶乙酸乙酯，8∶1)得9-碘-5-氯-7-(4-甲氧基苄基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶(460mg)。MS m/z[LC-MS]：439.98[M+1]。1HNMR(400MHz，DMSO-d6)：δ＝8.097(1H，d，J＝1.6Hz)，7.629(1H，d，J＝1.6Hz)，7.261(2H，d，J＝8.4Hz)，6.898(2H，d，J＝8.4Hz)，5.381(2H，s)，3.72(3H，s)。3-iodo-6-chloro-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (700 mg) and chloroacetaldehyde (2 mL) were added to acetonitrile (20 mL), heated to 100°C and sealed for reaction for 5 hours, poured into water, adjusted the pH value to 9-10 with saturated sodium carbonate aqueous solution, extracted with dichloromethane, washed with saturated saline, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 8: 1) to obtain 9-iodo-5-chloro-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidine (460 mg). MS m/z[LC-MS]: 439.98[M+1].1 HNMR (400MHz, DMSO-d6 ): δ = 8.097 (1H, d, J = 1.6Hz), 7.629 (1H, d, J = 1.6Hz), 7.261 (2H, d, J = 8.4Hz), 6.898 (2H, d, J = 8.4Hz), 5.381 (2H, s), 3.72 (3H, s).
中间体4：6-氯-3-碘-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[4，3-b]吡嗪Intermediate 4:6-Chloro-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[4,3-b]pyrazine
6-氯-3-碘-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[4，3-b]吡嗪按照专利WO2018057884中第83页实施例29中相同中间体的方法合成。6-Chloro-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[4,3-b]pyrazine was synthesized according to the method of the same intermediate in Example 29 on page 83 of patent WO2018057884.
中间体5：1-(3-碘-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-4-甲基哌啶-4-基氨基甲酸叔丁酯Intermediate 5: tert-butyl1-(3-iodo-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-4-methylpiperidin-4-ylcarbamate
步骤1：4-甲基-1-(5-甲基-4-氧代-1-(四氢-2H-吡喃-2-基)-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)哌啶-4-基氨基甲酸叔丁酯Step 1: tert-Butyl 4-methyl-1-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamate
将中间体2(538mg)、4-甲基哌啶-4-基氨基甲酸叔丁酯(514mg)、二异丙基乙基胺(1mL)加入四氢呋喃(20mL)中，加热至120℃搅拌2小时，减压旋蒸除去溶剂，用硅胶柱色谱分离(石油醚∶乙酸乙酯，5∶1)得4-甲基-1-(5-甲基-4-氧代-1-(四氢-2H-吡喃-2-基)-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)哌啶-4-基氨基甲酸叔丁酯(800mg)。MS m/z[LC-MS]：447.27[M+1]。Intermediate 2 (538 mg), tert-butyl 4-methylpiperidin-4-ylcarbamate (514 mg), and diisopropylethylamine (1 mL) were added to tetrahydrofuran (20 mL), heated to 120° C. and stirred for 2 hours, and the solvent was removed by rotary evaporation under reduced pressure. The product was separated by silica gel column chromatography (petroleum ether: ethyl acetate, 5:1) to obtain tert-butyl 4-methyl-1-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamate (800 mg). MS m/z [LC-MS]: 447.27 [M+1].
步骤2：4-甲基-1-(5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)哌啶-4-基氨基甲酸叔丁酯Step 2: tert-Butyl 4-methyl-1-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamate
将4-甲基-1-(5-甲基-4-氧代-1-(四氢-2H-吡喃-2-基)-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)哌啶-4-基氨基甲酸叔丁酯(445mg)加入甲醇(10mL)中，再加入1M盐酸溶液(2mL)，室温搅拌过夜，用饱和碳酸氢钠水溶液调pH值到8～9，旋蒸除去有机溶剂，再用乙酸乙酯萃取，萃取液干燥浓缩后用硅胶柱色谱分离(石油醚∶乙酸乙酯，4∶1)得4-甲基-1-(5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)哌啶-4-基氨基甲酸叔丁酯(305mg)。MS m/z[LC-MS]：363.22[M+1]。4-Methyl-1-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamic acid tert-butyl ester (445 mg) was added to methanol (10 mL), and then 1M hydrochloric acid solution (2 mL) was added, and stirred at room temperature overnight, and the pH value was adjusted to 8-9 with saturated sodium bicarbonate aqueous solution, and the organic solvent was removed by rotary evaporation, and then extracted with ethyl acetate. The extract was dried and concentrated, and then separated by silica gel column chromatography (petroleum ether: ethyl acetate, 4: 1) to obtain 4-methyl-1-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamic acid tert-butyl ester (305 mg). MS m/z [LC-MS]: 363.22 [M+1].
步骤3：1-(3-碘-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-4-甲基哌啶-4-基氨基甲酸叔丁酯Step 3: tert-Butyl 1-(3-iodo-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-4-methylpiperidin-4-ylcarbamate
将4-甲基-1-(5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)哌啶-4-基氨基甲酸叔丁酯(300mg)、N-碘代丁二酰亚胺(177mg)加入乙腈(5mL)中，加热至100℃微波反应25分钟，降至室温，减压浓缩后用硅胶柱色谱分离(石油醚∶乙酸乙酯，4∶1)得1-(3-碘-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-4-甲基哌啶-4-基氨基甲酸叔丁酯(320mg)。MS m/z[LC-MS]：489.11[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.545(1H，s)，6.583(1H，s)，3.317(3H，s)，3.168(2H，m)，2.994(2H，m)，2.102(1H，m)，1.529(2H，m)，1.356(9H，s)，1.230(3H，s)。4-Methyl-1-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamic acid tert-butyl ester (300 mg) and N-iodosuccinimide (177 mg) were added to acetonitrile (5 mL), heated to 100° C. for microwave reaction for 25 minutes, cooled to room temperature, concentrated under reduced pressure, and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1) to obtain 1-(3-iodo-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-4-methylpiperidin-4-ylcarbamic acid tert-butyl ester (320 mg). MS m/z[LC-MS]: 489.11[M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 13.545 (1H, s), 6.583 (1H, s), 3.317 (3H, s), 3.168 (2H, m), 2.994 (2H, m), 2.102 (1H, m), 1.529 (2H, m), 1.356 (9H, s), 1.230 (3H,s).
中间体6：((3S，4S)-8-(3-碘-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯Intermediate 6: tert-butyl ((3S,4S)-8-(3-iodo-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate
步骤1：6-(((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-1-(四氢-2H-吡喃-2-基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Step 1: 6-(((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照中间体5中步骤1的方法，用中间体1替代4-甲基哌啶-4-基氨基甲酸叔丁酯，得6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-1-(四氢-2H-吡喃-2-基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮。MS m/z[LC-MS]：403.25[M+1]。Referring to the method of step 1 in intermediate 5, intermediate 1 was used to replace tert-butyl 4-methylpiperidin-4-ylcarbamate to obtain 6-((3S, 4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one. MS m/z[LC-MS]: 403.25[M+1].
步骤2：((3S，4S)-3-甲基-8-(5-甲基-4-氧代-1-(四氢-2H-吡喃-2-基)-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯Step 2: tert-Butyl ((3S,4S)-3-methyl-8-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4.5]decan-4-yl)carbamate
将6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-1-(四氢-2H-吡喃-2-基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮(400mg)、三乙胺(500mg)加入二氯甲烷(10mL)中，冰水浴冷却下滴加二碳酸二叔丁酯(260mg)，滴加完后升至室温搅拌4小时，依次用水和饱和食盐水洗，有机相干燥浓缩后用硅胶柱色谱(石油醚∶乙酸乙酯，4∶1)分离得((3S，4S)-3-甲基-8-(5-甲基-4-氧代-1-(四氢-2H-吡喃-2-基)-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯(420mg)。MSm/z[LC-MS]：503.3[M+1]。6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (400 mg) and triethylamine (500 mg) were added to dichloromethane (10 mL), and di-tert-butyl dicarbonate (260 mg) was added dropwise under ice-water cooling. After the addition was complete, the mixture was heated to room temperature and stirred. The mixture was stirred for 4 hours, washed with water and saturated brine in sequence, and the organic phase was dried and concentrated, and then separated by silica gel column chromatography (petroleum ether: ethyl acetate, 4: 1) to obtain tert-butyl ((3S, 4S)-3-methyl-8-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate (420 mg). MSm/z[LC-MS]: 503.3[M+1].
步骤3：((3S，4S)-3-甲基-8-(5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯Step 3: tert-Butyl ((3S,4S)-3-methyl-8-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4.5]decan-4-yl)carbamate
参照中间体5中步骤2的方法，用((3S，4S)-3-甲基-8-(5-甲基-4-氧代-1-(四氢-2H-吡喃-2-基)-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯替代4-甲基-1-(5-甲基-4-氧代-1-(四氢-2H-吡喃-2-基)-4，5-二氢-1H-吡唑[3，4-d]嘧啶-6-基)哌啶-4-基氨基甲酸叔丁酯，得((3S，4S)-3-甲基-8-(5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯。MS m/z[LC-MS]：419.24[M+1]。Referring to the method of step 2 in intermediate 5, 4-methyl-1-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamic acid tert-butyl ester was used instead of 4-methyl-1-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate. =((3S,4S)-3-methyl-8-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamic acid tert-butyl ester. MS m/z[LC-MS]: 419.24[M+1].
步骤4：((3S，4S)-8-(3-碘-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯Step 4: tert-Butyl ((3S,4S)-8-(3-iodo-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)carbamate
参照中间体5中步骤3的方法，用((3S，4S)-3-甲基-8-(5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯替代4-甲基-1-(5-甲基-4-氧代-4，5-二氢-1H-吡唑[3，4-d]嘧啶-6-基)哌啶-4-基氨基甲酸叔丁酯，得((3S，4S)-8-(3-碘-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯。MS m/z[LC-MS]：545.14[M+1]。1HNMR(400MHz，DMSO-d6)：δ＝13.562(1H，s)，6.988(1H，s)，4.134(1H，m)，3.848(1H，m)，3.638(1H，d，J＝8.4Hz)，3.487(1H，d，J＝8.4Hz)，3.328(3H，s)，3.184(1H，m)，3.092(2H，m)，1.62-1.76(3H，m)，1.50-1.60(1H，m)，1.37(9H，s)，0.994(3H，d，J＝6.0Hz)。Referring to the method of step 3 in intermediate 5, tert-butyl ((3S,4S)-3-methyl-8-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate was used to replace tert-butyl 4-methyl-1-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamate to obtain tert-butyl ((3S,4S)-8-(3-iodo-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate. MS m/z[LC-MS]: 545.14[M+1].1 HNMR (400MHz, DMSO-d6 ): δ = 13.562 (1H, s), 6.988 (1H, s), 4.134 (1H, m), 3.848 (1H, m), 3.638 (1H, d, J = 8.4Hz), 3.487 (1H, d, J = 8.4Hz), 3.328 (3H, s), 3 .184 (1H, m), 3.092 (2H, m), 1.62-1.76 (3H, m), 1.50-1.60 (1H, m), 1.37 (9H, s), 0.994 (3H, d, J = 6.0Hz).
中间体7：((3S，4S)-8-(9-碘-7-(4-甲氧基苄基)-7H-咪唑并[1，2-c]吡唑并[4，3-4]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯Intermediate 7:tert-Butyl ((3S,4S)-8-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-4]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate
步骤1：(3S，4S)-8-(9-碘-7-(4-甲氧基苄基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Step 1: (3S,4S)-8-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照中间体5中步骤1的方法，用中间体3替代中间体2，用中间体1替代4-甲基哌啶-4基氨基甲酸叔丁酯，得(3S，4S)-8-(9-碘-7-(4-甲氧基苄基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺。MSm/z[LC-MS]：574.14[M+1]。Referring to the method of step 1 in intermediate 5, intermediate 3 was used to replace intermediate 2, and intermediate 1 was used to replace tert-butyl 4-methylpiperidin-4-ylcarbamate to obtain (3S, 4S)-8-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine. MSm/z[LC-MS]: 574.14[M+1].
步骤2：((3S，4S)-8-(9-碘-7-(4-甲氧基苄基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯Step 2: tert-Butyl ((3S,4S)-8-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)carbamate
参照中间体6中步骤2的方法，用(3S，4S)-8-(9-碘-7-(4-甲氧基苄基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺替代6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-1-(四氢-2H-吡喃-2-基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮，得((3S，4S)-8-(9-碘-7-(4-甲氧基苄基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯。MS m/z[LC-MS]：674.2[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.707(1H，d，J＝1.2Hz)，7.437(1H，d，J＝1.2Hz)，7.251(2H，d，J＝8.4Hz)，6.850(2H，d，J＝8.4Hz)，5.391(2H，s)，4.163(1H，m)，3.913(1H，m)，3.673(3H，s)，3.35-3.55(6H，m)，1.72-1.90(3H，m)，1.60-1.72(1H，m)，1.352(9H，s)，1.007(3H，d，J＝6.4Hz)。Referring to the procedure of step 2 of intermediate 6, substituting (3S,4S)-8-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine for 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-thiazolyl-2-ol -5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one to give ((3S,4S)-8-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamic acid tert-butyl ester. MS m/z[LC-MS]: 674.2[M+1].1 H NMR (400MHz, DMSO-d6 ): δ=7.707 (1H, d, J=1.2Hz), 7.437 (1H, d, J=1.2Hz), 7.251 (2H, d, J=8.4Hz), 6.850 (2H, d, J=8.4Hz), 5.391 (2H, s), 4.163 (1H, m), 3 .913 (1H, m), 3.673 (3H, s), 3.35-3.55 (6H, m), 1.72-1.90 (3H, m), 1.60-1.72 (1H, m), 1.352 (9H, s), 1.007 (3H, d, J = 6.4Hz).
中间体8：((3S，4S)-8-(3-碘-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-b]吡嗪-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯Intermediate 8: tert-butyl((3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)carbamate
步骤1：(3S，4S)-8-(3-碘-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-b]吡嗪-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Step 1: (3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照中间体5中步骤1的方法，用中间体4替代中间体2，用中间体1替代4-甲基哌啶-4-基氨基甲酸叔丁酯，得(3S，4S)-8-(3-碘-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-b]吡嗪-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺。MS m/z[LC-MS]：499.13[M+1]。Referring to the method of step 1 in intermediate 5, intermediate 4 was used to replace intermediate 2, and intermediate 1 was used to replace tert-butyl 4-methylpiperidin-4-ylcarbamate to obtain (3S, 4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine. MS m/z[LC-MS]: 499.13[M+1].
步骤2：((3S，4S)-8-(3-碘-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-b]吡嗪-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯Step 2: tert-Butyl ((3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)carbamate
参照中间体6中步骤2的方法，用(3S，4S)-8-(3-碘-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-b]吡嗪-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺替代6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-1-(四氢-2H-吡喃-2-基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮，得((3S，4S)-8-(3-碘-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-b]吡嗪-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯。MSm/z[LC-MS]：599.19[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝8.410(1H，s)，6.988(1H，s)，5.71(1H，m)，4.14(1H，m)，3.84-3.92(2H，m)，3.65(2H，m)，3.472(1H，d，J＝8.4Hz)，3.18(1H，m)，3.09(2H，m)，2.31(1H，m)，1.98(1H，m)，1.62-1.83(5H，m)，1.49-1.60(3H，m)，1.38(9H，s)，1.004(3H，d，J＝6.0Hz)。Referring to the procedure of step 2 of intermediate 6, substituting (3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine for 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine -5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one to give ((3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamic acid tert-butyl ester. MS m/z [LC-MS]: 599.19 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 8.410 (1H, s), 6.988 (1H, s), 5.71 (1H, m), 4.14 (1H, m), 3.84-3.92 (2H, m), 3.65 (2H, m), 3.472 (1H, d, J = 8.4Hz), 3.18 (1H, m), 3.09 (2H, m), 2.31 (1H, m), 1.98 (1H, m), 1.62-1.83 (5H, m), 1.49-1.60 (3H, m), 1.38 (9H, s), 1.004 (3H, d, J=6.0Hz).
中间体9：((3S，4S)-8-(3-碘-1-(四氢-2H-吡喃-2-基)-1H-吡唑并[3，4-b]吡嗪-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯Intermediate 9: tert-butyl((3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)carbamate
步骤1：8-溴-7-氯-5-(甲硫基)咪唑并[1，2-c]嘧啶Step 1: 8-Bromo-7-chloro-5-(methylthio)imidazo[1,2-c]pyrimidine
5-溴-6-氯-2-(甲硫基)嘧啶-4-胺(1.0g)和40％氯乙醛水溶液(1.2mL)溶于二氧六环(6mL)中，氮气保护下回流14小时，冷却至室温后过滤，得8-溴-7-氯-5-(甲硫基)咪唑并[1，2-c]嘧啶(900mg)。MS m/z[LC-MS]：277.92[M+1]。5-Bromo-6-chloro-2-(methylthio)pyrimidin-4-amine (1.0 g) and 40% chloroacetaldehyde aqueous solution (1.2 mL) were dissolved in dioxane (6 mL), refluxed for 14 hours under nitrogen protection, cooled to room temperature and filtered to obtain 8-bromo-7-chloro-5-(methylthio)imidazo[1,2-c]pyrimidine (900 mg). MS m/z [LC-MS]: 277.92 [M+1].
步骤2：8-溴-7-氯-5-(甲基亚磺酰基)咪唑并[1，2-c]嘧啶Step 2: 8-Bromo-7-chloro-5-(methylsulfinyl)imidazo[1,2-c]pyrimidine
8-溴-7-氯-5-(甲硫基)咪唑并[1，2-c]嘧啶(557mg)溶于二氯甲烷(20mL)中，加入间氯过氧化苯甲酸(85％，487mg)，室温搅拌6小时，反应液依次用饱和碳酸氢钠水溶液、水、饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液旋蒸浓缩，硅胶柱色谱分离纯化(石油醚∶乙酸乙酯，5∶1)得8-溴-7-氯-5-(甲基亚磺酰基)咪唑并[1，2-c]嘧啶(420mg)。MS m/z[LC-MS]：293.91[M+1]。8-Bromo-7-chloro-5-(methylthio)imidazo[1,2-c]pyrimidine (557 mg) was dissolved in dichloromethane (20 mL), and m-chloroperoxybenzoic acid (85%, 487 mg) was added. The mixture was stirred at room temperature for 6 hours. The reaction solution was washed with saturated sodium bicarbonate aqueous solution, water, and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by rotary evaporation. The mixture was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate, 5: 1) to obtain 8-bromo-7-chloro-5-(methylsulfinyl)imidazo[1,2-c]pyrimidine (420 mg). MS m/z[LC-MS]: 293.91[M+1].
步骤3：(3S，4S)-8-(8-溴-7-氯咪唑并[1，2-c]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Step 3: (3S,4S)-8-(8-bromo-7-chloroimidazo[1,2-c]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照中间体5中步骤1的方法，用8-溴-7-氯-5-(甲基亚磺酰基)咪唑并[1，2-c]嘧啶替代中间体2，用中间体1替代4-甲基哌啶-4-基氨基甲酸叔丁酯，得(3S，4S)-8-(8-溴-7-氯咪唑并[1，2-c]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺。MS m/z[LC-MS]：400.06[M+1]。Referring to the method of step 1 in intermediate 5, 8-bromo-7-chloro-5-(methylsulfinyl)imidazo[1,2-c]pyrimidine was used to replace intermediate 2, and intermediate 1 was used to replace tert-butyl 4-methylpiperidin-4-ylcarbamate to obtain (3S,4S)-8-(8-bromo-7-chloroimidazo[1,2-c]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine. MS m/z[LC-MS]: 400.06[M+1].
步骤4：((3S，4S)-8-(8-溴-7-氯咪唑并[1，2-c]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯Step 4: tert-Butyl ((3S,4S)-8-(8-bromo-7-chloroimidazo[1,2-c]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate
参照中间体6中步骤2的方法，用(3S，4S)-8-(8-溴-7-氯咪唑并[1，2-c]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺替代6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-1-(四氢-2H-吡喃-2-基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮，得((3S，4S)-8-(8-溴-7-氯咪唑并[1，2-c]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯。MS m/z[LC-MS]：500.11[M+1]。1HNMR(400MHz，DMSO-d6)：δ＝7.79(1H，d，J＝1.6Hz)，7.62(1H，d，J＝1.6Hz)，Referring to the method of step 2 in intermediate 6, (3S,4S)-8-(8-bromo-7-chloroimidazo[1,2-c]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine was used to replace 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one to obtain tert-butyl ((3S,4S)-8-(8-bromo-7-chloroimidazo[1,2-c]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate. MS m/z[LC-MS]: 500.11[M+1].1 HNMR (400MHz, DMSO-d6 ): δ=7.79 (1H, d, J=1.6Hz), 7.62 (1H, d, J=1.6Hz),
中间体10：2-乙炔基-1，1-二氟环丙烷Intermediate 10:2-ethynyl-1,1-difluorocyclopropane
2-乙炔基-1，1-二氟环丙烷按照专利WO2015066413中中间体3.1.35d的方法合成。2-Ethynyl-1,1-difluorocyclopropane was synthesized according to the method of intermediate 3.1.35d in patent WO2015066413.
中间体11：1-乙炔基-1-甲基环丙烷Intermediate 11:1-ethynyl-1-methylcyclopropane
1-乙炔基-1-甲基环丙烷按照专利WO2011059784中实施例4的方法合成。1-Ethynyl-1-methylcyclopropane was synthesized according to the method of Example 4 in patent WO2011059784.
中间体12：(2-(2-氯-3-(4，4，5，5-四甲基-1，3，2-二氧杂环戊硼烷-2-基)苯基)乙炔基)三甲基硅烷Intermediate 12:(2-(2-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethynyl )trimethylsilane
步骤1：(2-(3-溴-2-氯苯基)乙炔基)三甲基硅烷Step 1: (2-(3-bromo-2-chlorophenyl)ethynyl)trimethylsilane
将1-溴-2-氯-3-碘苯(560mg)、乙炔基三甲基硅烷(196mg)、碘化亚铜(34mg)、二(三苯基膦)二氯化钯(121mg)和三乙胺(533mg)加入四氢呋喃(10mL)中，用高纯氮气置换反应体系中的空气三次，回流12小时，降至室温后，减压浓缩并用硅胶柱色谱分离(以正己烷为洗脱液)得(2-(3-溴-2-氯苯基)乙炔基)三甲基硅烷(424mg)。1HNMR(400MHz，CDCl3)：δ＝7.55(1H，m)，7.43(1H，m)，7.03(1H，m)，0.25(9H，m)。1-Bromo-2-chloro-3-iodobenzene (560 mg), ethynyltrimethylsilane (196 mg), cuprous iodide (34 mg), bis(triphenylphosphine)palladium dichloride (121 mg) and triethylamine (533 mg) were added to tetrahydrofuran (10 mL), and the air in the reaction system was replaced with high-purity nitrogen three times. The mixture was refluxed for 12 hours, cooled to room temperature, concentrated under reduced pressure and separated by silica gel column chromatography (using n-hexane as eluent) to obtain (2-(3-bromo-2-chlorophenyl)ethynyl)trimethylsilane (424 mg).1 HNMR (400 MHz, CDCl3 ): δ=7.55 (1H, m), 7.43 (1H, m), 7.03 (1H, m), 0.25 (9H, m).
步骤2：(2-(2-氯-3-(4，4，5，5-四甲基-1，3，2-二氧杂环戊硼烷-2-基)苯基)乙炔基)三甲基硅烷Step 2: (2-(2-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)ethynyl)trimethylsilane
将(2-(3-溴-2-氯苯基)乙炔基)三甲基硅烷(232mg)、联硼酸频哪醇酯(244mg)、(1，1′-双(二苯膦基)二茂铁)二氯化钯(56mg)和无水乙酸钾(157mg)加入二氧六环(2mL)中，用高纯氮气置换反应体系中的空气三次，加热至90℃反应12小时，反应混合物不经处理可直接进行下步偶联反应。MS m/z[LC-MS]：335.14[M+1]。(2-(3-bromo-2-chlorophenyl)ethynyl)trimethylsilane (232 mg), pinacol diboron (244 mg), (1,1′-bis(diphenylphosphino)ferrocene)palladium dichloride (56 mg) and anhydrous potassium acetate (157 mg) were added to dioxane (2 mL), and the air in the reaction system was replaced with high-purity nitrogen three times, heated to 90° C. for 12 hours, and the reaction mixture was directly used for the next coupling reaction without treatment. MS m/z [LC-MS]: 335.14 [M+1].
中间体13：5-(4，4，5，5-四甲基-1，3，2-二氧杂环戊硼烷-2-基)-3H-螺[苯并呋喃-2，1′-环丙烷]-3-酮Intermediate 13:5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-spiro[benzofuran-2,1′-cyclopropane]-3-one
原料5-溴-3H-螺[苯并呋喃-2，1′-环丙烷]-3-酮参照文献Chemical andPharmaceutical Bulletin；vol.32；nb.9；(1984)；p.3532-3550合成。The raw material 5-bromo-3H-spiro[benzofuran-2,1′-cyclopropane]-3-one was synthesized according to the literature Chemical and Pharmaceutical Bulletin; vol. 32; nb. 9; (1984); p. 3532-3550.
将5-溴-3H-螺[苯并呋喃-2，1′-环丙烷]-3-酮按照中间体12步骤2的方法进行反应得到相应硼酸酯，反应混合物不经处理可直接进行下步偶联反应。MS m/z[LC-MS]：287.15[M+1]。5-Bromo-3H-spiro[benzofuran-2,1′-cyclopropane]-3-one was reacted according to the method of step 2 of intermediate 12 to obtain the corresponding boronate ester, and the reaction mixture was directly used for the next coupling reaction without treatment. MS m/z [LC-MS]: 287.15 [M+1].
中间体14：2-(3-氯-[1，1′-联苯]-4-基)-4，4，5，5-四甲基-1，3，2-二氧杂环戊硼烷Intermediate 14:2-(3-chloro-[1,1′-biphenyl]-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
原料4-溴-3-氯-1，1′-联苯按照文献Journal of the Chemical Society；(1964)；p.3786-3790合成。The raw material 4-bromo-3-chloro-1,1′-biphenyl was synthesized according to the document Journal of the Chemical Society; (1964); p. 3786-3790.
将4-溴-3-氯-1，1′-联苯按照中间体12步骤2的方法进行反应得到相应硼酸酯，反应混合物不经处理可直接进行下步偶联反应。MS m/z[LC-MS]：315.13[M+1]。4-Bromo-3-chloro-1,1′-biphenyl was reacted according to the method of step 2 of intermediate 12 to obtain the corresponding borate ester, and the reaction mixture was directly used for the next coupling reaction without treatment. MS m/z [LC-MS]: 315.13 [M+1].
实施例1：6-(4-氨基-4-甲基哌啶-1-基)-3-(2-(2-氯苯基)乙炔基)-5-甲基-1H-吡唑并[3，4-d]嘧啶-4(5H)-酮Example 1:6-(4-amino-4-methylpiperidin-1-yl)-3-(2-(2-chlorophenyl)ethynyl)-5-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
步骤1：1-(3-(2-((2-氯苯基)乙炔基)-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-4-甲基哌啶-4-基)氨基甲酸叔丁酯Step 1: tert-Butyl 1-(3-(2-((2-chlorophenyl)ethynyl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-4-methylpiperidin-4-yl)carbamate
将中间体5(150mg)、1-氯-2-乙炔基苯(50mg)、碘化亚铜(12mg)、三乙胺(100mg)、二(三苯基膦)二氯化钯(21mg)和四氢呋喃(10mL)加入封管中，氮气置换，加热至80℃搅拌过夜。冷却到室温，倒入水中，用二氯甲烷萃取，萃取液用饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱分离(石油醚∶乙酸乙酯，6∶1)得1-(3-((2-(2-氯苯基)乙炔基)-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-4-甲基哌啶-4-基)氨基甲酸叔丁酯(75mg)。MS m/z[LC-MS]：498.0[M+1]。Intermediate 5 (150 mg), 1-chloro-2-ethynylbenzene (50 mg), cuprous iodide (12 mg), triethylamine (100 mg), bis(triphenylphosphine)palladium dichloride (21 mg) and tetrahydrofuran (10 mL) were added to a sealed tube, replaced with nitrogen, heated to 80°C and stirred overnight. The mixture was cooled to room temperature, poured into water, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 6: 1) to obtain tert-butyl 1-(3-((2-(2-chlorophenyl)ethynyl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-4-methylpiperidin-4-yl)carbamate (75 mg). MS m/z [LC-MS]: 498.0 [M+1].
步骤2：6-(4-氨基-4-甲基哌啶-1-基)-3-(2-(2-氯苯基)乙炔基)-5-甲基-1H-吡唑并[3，4-d]嘧啶-4(5H)-酮Step 2: 6-(4-amino-4-methylpiperidin-1-yl)-3-(2-(2-chlorophenyl)ethynyl)-5-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
将1-(3-(2-((2-氯苯基)乙炔基)-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-4-甲基哌啶-4-基)氨基甲酸叔丁酯(70mg)加入4M氯化氢的二氧六环溶液(3mL)中，室温搅拌1小时，旋干，加入10％碳酸钠水溶液(10mL)，用二氯甲烷萃取，萃取液用无水硫酸钠干燥，过滤，滤液减压浓缩，用薄层硅胶色谱分离(二氯甲烷∶甲醇，10∶1)得6-(4-氨基-4-甲基哌啶-1-基)-3-(2-(2-氯苯基)乙炔基)-5-甲基-1H-吡唑并[3，4-d]嘧啶-4(5H)-酮(35mg)。MS m/z[LC-MS]：397.16[M+1]。1HNMR(400MHz，DMSO-d6)：δ＝7.70-8.40(3H，brs)，7.66(1H，dd，J＝8.0Hz，1.6Hz)，7.59(1H，d，J＝8.0Hz)，7.46(1H，td，J＝8.0Hz，1.6Hz)，7.41(1H，t，J＝8.0Hz)，3.36-3.46(5H，m)，3.08-3.16(2H，m)，1.83-1.91(2H，m)，1.72-1.80(2H，m)，1.34(3H，s)。Tert-butyl 1-(3-(2-((2-chlorophenyl)ethynyl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-4-methylpiperidin-4-yl)carbamate (70 mg) was added to a 4M solution of hydrogen chloride in dioxane (3 mL), stirred at room temperature for 1 hour, spin-dried, added with 10% aqueous sodium carbonate solution (10 mL), extracted with dichloromethane, and the extract was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by thin layer silica gel chromatography (dichloromethane: methanol, 10: 1) to give 6-(4-amino-4-methylpiperidin-1-yl)-3-(2-(2-chlorophenyl)ethynyl)-5-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (35 mg). MS m/z[LC-MS]: 397.16[M+1].1 HNMR (400MHz, DMSO-d6 ): δ=7.70-8.40 (3H, brs), 7.66 (1H, dd, J=8.0Hz, 1.6Hz), 7.59 (1H, d, J=8.0Hz), 7.46 (1H, td, J=8.0Hz, 1.6Hz), 7.41 (1H, t, J=8.0Hz), 3.36-3.46(5H,m), 3.08-3.16(2H,m), 1.83-1.91(2H,m), 1.72-1.80(2H,m), 1.34(3H,s).
实施例2：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((2，4-二氟苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 2:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((2,4-difluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：455.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.2-13.9(1H，brs)，7.69(1H，td，J＝8.4Hz，6.4Hz)，7.45(1H，td，J＝9.6Hz，2.8Hz)，7.19(1H，td，J＝8.4Hz，2.8Hz)，6.71-6.84(2H，brs)，4.13-4.17(1H，m)，3.79(1H，d，J＝8.8Hz)，3.59(1H，d，J＝8.8Hz)，3.34-3.48(6H，m)，2.85-2.96(2H，m)，1.82-1.91(2H，m)，1.66-1.73(1H，m)，1.56-1.62(1H，m)，1.17(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 455.20 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=13.2-13.9 (1H, brs), 7.69 (1H, td, J=8.4 Hz, 6.4 Hz), 7.45 (1H, td, J=9.6 Hz, 2.8 Hz), 7.19 (1H, td, J=8.4 Hz, 2.8 Hz), 6.71-6.84 (2H, brs), 4.13-4.17 (1H, m), 3 .79(1H,d,J=8.8Hz), 3.59(1H,d,J=8.8Hz), 3.34-3.48(6H,m), 2.85-2.96(2H,m), 1.82-1.91(2H,m), 1.66-1.73(1H,m), 1.56-1.62(1H,m), 1.17(3 H, d, J=6.4Hz).
实施例3：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((2-氯苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 3:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((2-chlorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：453.18[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.66(1H，dd，J＝7.6Hz，2.0Hz)，7.59(1H，dd，J＝8.0Hz，1.6Hz)，7.46(1H，td，J＝7.6Hz，1.6Hz)，7.41(1H，td，J＝7.6Hz，1.6Hz)，6.71-6.84(3H，brs)，4.01-4.08(1H，m)，3.64(1H，d，J＝8.8Hz)，3.47(1H，d，J＝8.8Hz)，3.39(3H，s)，3.22-3.34(2H，m)，2.90-3.07(3H，m)，1.79-1.87(1H，m)，1.69-1.78(1H，m)，1.50-1.63(2H，m)，1.06(3H，d，J＝6.0Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 453.18 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.66 (1H, dd, J=7.6 Hz, 2.0 Hz), 7.59 (1H, dd, J=8.0 Hz, 1.6 Hz), 7.46 (1H, td, J=7.6 Hz, 1.6 Hz), 7.41 (1H, td, J=7.6 Hz, 1.6 Hz), 6.71-6.84 (3H, brs), 4.01-4.08 (1H, m), 3.6 4(1H,d,J=8.8Hz), 3.47(1H,d,J=8.8Hz), 3.39(3H,s), 3.22-3.34(2H,m), 2.90-3.07(3H,m), 1.79-1.87(1H,m), 1.69-1.78(1H,m), 1.50-1.63(2H, m), 1.06 (3H, d, J=6.0Hz).
实施例4：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((3-氮苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 4:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((3-nitrophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：453.18[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.60(1H，t，J＝2.0Hz)，7.45-7.54(3H，m)，4.01-4.06(1H，m)，3.63(1H，d，J＝8.4Hz)，3.46(1H，d，J＝8.4Hz)，3.39(3H，s)，3.26-3.34(2H，m)，2.89-3.07(3H，m)，1.80-1.86(1H，m)，1.69-1.77(1H，m)，1.51-1.61(2H，m)，1.06(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 453.18 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ=7.60 (1H, t, J=2.0Hz), 7.45-7.54 (3H, m), 4.01-4.06 (1H, m), 3.63 (1H, d, J=8.4Hz), 3.46 (1H, d, J=8.4Hz), 3.39 (3H, s), 3.2 6-3.34(2H,m), 2.89-3.07(3H,m), 1.80-1.86(1H,m), 1.69-1.77(1H,m), 1.51-1.61(2H,m), 1.06(3H,d,J=6.4Hz).
实施例5：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((2-甲氧基苯基)乙炔基)-5-甲基-1，5-二氧-4H-吡唑并[3，4-d]嘧啶-4-酮Example 5:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((2-methoxyphenyl)ethynyl)-5-methyl-1,5-dioxo-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：449.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝12.80-13.80(1H，brs)，7.46(1H，dd，J＝7.6Hz，1.6Hz)，7.41(1H，td，J＝7.6Hz，1.6Hz)，7.09(1H，d，J＝8.4Hz)，6.98(1H，t，J＝7.6Hz)，4.01-4.08(1H，m)，3.84(3H，s)，3.64(1H，d，J＝8.8Hz)，3.47(1H，d，J＝8.8Hz)，3.38(3H，s)，3.26-3.34(2H，m)，2.88-3.05(3H，m)，1.79-1.88(1H，m)，1.69-1.76(1H，m)，1.50-1.62(2H，m)，1.07(3H，d，J＝6.0Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 449.23 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=12.80-13.80 (1H, brs), 7.46 (1H, dd, J=7.6 Hz, 1.6 Hz), 7.41 (1H, td, J=7.6 Hz, 1.6 Hz), 7.09 (1H, d, J=8.4 Hz), 6.98 (1H, t, J=7.6 Hz), 4.01-4.08 (1H, m), 3.84 (3H, s), 3.6 4(1H,d,J=8.8Hz), 3.47(1H,d,J=8.8Hz), 3.38(3H,s), 3.26-3.34(2H,m), 2.88-3.05(3H,m), 1.79-1.88(1H,m), 1.69-1.76(1H,m), 1.50-1.62(2H, m), 1.07 (3H, d, J=6.0Hz).
实施例6：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-3-(苯基乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 6:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl -3-(phenylethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：419.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.54-7.57(2H，m)，7.42-7.47(3H，m)，4.03-4.09(1H，m)，3.66(1H，d，J＝8.4Hz)，3.49(1H，d，J＝8.4Hz)，3.39(3H，s)，3.26-3.35(2H，m)，2.90-3.04(3H，m)，1.82-1.88(1H，m)，1.72-1.78(1H，m)，1.52-1.64(2H，m)，1.08(3H，d，J＝6.8Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 419.22 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.54-7.57 (2H, m), 7.42-7.47 (3H, m), 4.03-4.09 (1H, m), 3.66 (1H, d, J = 8.4Hz), 3.49 (1H, d, J = 8.4Hz), 3.39 (3H, s), 3.26- 3.35 (2H, m), 2.90-3.04 (3H, m), 1.82-1.88 (1H, m), 1.72-1.78 (1H, m), 1.52-1.64 (2H, m), 1.08 (3H, d, J=6.8Hz).
实施例7：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-(((2-氟苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 7:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-(((2-fluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：437.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.62(1H，td，J＝7.6Hz，1.6Hz)，7.47-7.53(1H，m)，7.35(1H，t，J＝8.8Hz)，7.28(1H，t，J＝8.0Hz)，4.01-4.07(1H，m)，3.63(1H，d，J＝8.4Hz)，3.46(1H，d，J＝8.4Hz)，3.39(3H，s)，3.25-3.34(2H，m)，2.88-3.07(3H，m)，1.80-1.87(1H，m)，1.68-1.76(1H，m)，1.50-1.62(2H，m)，1.06(3H，d，J＝6.0Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 437.21 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.62 (1H, td, J=7.6Hz, 1.6Hz), 7.47-7.53 (1H, m), 7.35 (1H, t, J=8.8Hz), 7.28 (1H, t, J=8.0Hz), 4.01-4.07 (1H, m), 3.63 (1H, d, J=8.4Hz), 3.46 (1H, d, J=8.4Hz), 3.39 (3H, s), 3.25-3.34 (2H, m), 2.88-3.07 (3H, m), 1.80-1.87 (1H, m), 1.68-1.76 (1H, m), 1.50-1.62 (2H, m), 1.06 (3H, d, J=6.0Hz).
实施例8：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((3-氨基苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 8:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((3-aminophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：434.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.3-13.8(1H，brs)，7.05(1H，t，J＝8.0Hz)，6.73(1H，s)，6.65(1H，d，J＝7.6Hz)，6.61(1H，d，J＝7.6Hz)，5.30(2H，s)，4.08-4.16(1H，m)，3.75(1H，d，J＝8.8Hz)，3.54(1H，d，J＝8.8Hz)，3.39(3H，s)，3.26-3.37(3H，m)，2.88-2.98(2H，m)，1.80-1.90(2H，m)，1.56-1.70(2H，m)，1.14(3H，5.6Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 434.23 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=13.3-13.8 (1H, brs), 7.05 (1H, t, J=8.0Hz), 6.73 (1H, s), 6.65 (1H, d, J=7.6Hz), 6.61 (1H, d, J=7.6Hz), 5.30 (2H, s), 4.08-4.16 (1H, m), 3.75 (1H, d , J=8.8Hz), 3.54 (1H, d, J=8.8Hz), 3.39 (3H, s), 3.26-3.37 (3H, m), 2.88-2.98 (2H, m), 1.80-1.90 (2H, m), 1.56-1.70 (2H, m), 1.14 (3H, 5.6Hz).
实施例9：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-3-(吡啶-3-基乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 9:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl -3-(pyridin-3-ylethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：420.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.71(1H，s)，8.74(1H，s)，8.62(1H，d，J＝4.8Hz)，7.96-8.04(3H，m)，7.49(1H，dd，J＝8.0hz，4.8Hz)，4.16-4.22(1H，m)，3.83(1H，d，J＝9.2Hz)，3.64(1H，d，J＝9.2Hz)，3.40-3.50(3H，m)，3.39(3H，s)，2.84-2.94(2H，m)，1.82-1.92(2H，m)，1.70-1.76(1H，m)，1.58-1.64(1H，m)，1.20(3H，d，J＝6.0Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 420.22 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ = 13.71 (1H, s), 8.74 (1H, s), 8.62 (1H, d, J = 4.8Hz), 7.96-8.04 (3H, m), 7.49 (1H, dd, J = 8.0hz, 4.8Hz), 4.16-4.22 (1H, m), 3.83 (1H, d, J = 9.2Hz), 3.6 4(1H,d,J=9.2Hz), 3.40-3.50(3H,m), 3.39(3H,s), 2.84-2.94(2H,m), 1.82-1.92(2H,m), 1.70-1.76(1H,m), 1.58-1.64(1H,m), 1.20(3H,d,J=6.0Hz ).
实施例10：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-3-(吡啶-4-基乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 10:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl -3-(pyridin-4-ylethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：420.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝8.65(2H，d，J＝5.6Hz)，7.51(2H，d，J＝5.6Hz)，4.10-4.16(1H，m)，3.77(1H，d，J＝8.8Hz)，3.56(1H，d，J＝8.8Hz)，3.40(3H，s)，3.22-3.32(3H，m)，2.87-2.99(2H，m)，1.80-1.90(2H，m)，1.65-1.70(1H，m)，1.55-1.61(1H，m)，1.15(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 420.22 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 8.65 (2H, d, J = 5.6Hz), 7.51 (2H, d, J = 5.6Hz), 4.10-4.16 (1H, m), 3.77 (1H, d, J = 8.8Hz), 3.56 (1H, d, J = 8.8Hz), 3.40 (3H, s), 3 .22-3.32(3H,m), 2.87-2.99(2H,m), 1.80-1.90(2H,m), 1.65-1.70(1H,m), 1.55-1.61(1H,m), 1.15(3H,d,J=6.4Hz).
实施例11：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((3，5-二甲氧基苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 11:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((3,5-dimethoxyphenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：479.24[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.48-13.80(1H，brs)，6.68(2H，s)，6.58(1H，s)，4.08-4.16(1H，m)，3.76(6H，s)，3.74(1H，d，J＝8.8Hz)，3.56(1H，d，J＝8.8Hz)，3.35-3.43(5H，m)，3.16-3.22(1H，m)，2.86-2.99(2H，m)，1.78-1.88(2H，m)，1.64-1.69(1H，m)，1.54-1.61(1H，m)，1.14(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 479.24 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 13.48-13.80 (1H, brs), 6.68 (2H, s), 6.58 (1H, s), 4.08-4.16 (1H, m), 3.76 (6H, s), 3.74 (1H, d, J = 8.8Hz), 3.56 (1H, d, J = 8.8Hz ), 3.35-3.43(5H,m), 3.16-3.22(1H,m), 2.86-2.99(2H,m), 1.78-1.88(2H,m), 1.64-1.69(1H,m), 1.54-1.61(1H,m), 1.14(3H,d,J=6.4Hz).
实施例12：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-(3-羟基-3-甲基丁-1-炔-1-基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 12:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-(3-hydroxy-3-methylbut-1-yn-1-yl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：401.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.36-13.48(1H，brs)，5.52(1H，s)，4.08-4.14(1H，m)，3.73(1H，d，J＝8.8Hz)，3.55(1H，d，J＝8.8Hz)，3.37(3H，s)，3.11-3.21(2H，m)，2.84-2.96(3H，m)，1.77-1.86(2H，m)，1.63-1.67(1H，m)，1.54-1.58(1H，m)，1.45(6H，s)，1.13(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 401.23 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 13.36-13.48 (1H, brs), 5.52 (1H, s), 4.08-4.14 (1H, m), 3.73 (1H, d, J = 8.8Hz), 3.55 (1H, d, J = 8.8Hz), 3.37 (3H, s), 3.11-3. 21 (2H, m), 2.84-2.96 (3H, m), 1.77-1.86 (2H, m), 1.63-1.67 (1H, m), 1.54-1.58 (1H, m), 1.45 (6H, s), 1.13 (3H, d, J=6.4Hz).
实施例13：4-((6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-3-基)乙炔基)苯甲腈Example 13:4-((6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)benzonitrile
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：444.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.91(2H，d，J＝8.0Hz)，7.73(2H，d，J＝8.0Hz)，4.06-4.13(1H，m)，3.72(1H，d，J＝8.8Hz)，3.53(1H，d，J＝8.8Hz)，3.40(3H，s)，3.34-3.38(2H，m)，3.09-3.13(1H，m)，2.89-3.01(2H，m)，1.77-1.88(2H，m)，1.62-1.66(1H，m)，1.54-1.58(1H，m)，1.12(3H，d，J＝6.8Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 444.22 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ=7.91 (2H, d, J=8.0Hz), 7.73 (2H, d, J=8.0Hz), 4.06-4.13 (1H, m), 3.72 (1H, d, J=8.8Hz), 3.53 (1H, d, J=8.8Hz), 3.40 (3H, s), 3.3 4-3.38(2H,m), 3.09-3.13(1H,m), 2.89-3.01(2H,m), 1.77-1.88(2H,m), 1.62-1.66(1H,m), 1.54-1.58(1H,m), 1.12(3H,d,J=6.8Hz).
实施例14：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((4-氯苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 14:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((4-chlorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：453.18[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.57(2H，d，J＝8.0Hz)，7.51(2H，d，J＝8.0Hz)，4.08-4.14(1H，m)，3.74(1H，d，J＝8.8Hz)，3.54(1H，d，J＝8.8Hz)，3.39(3H，s)，3.34-3.38(2H，m)，3.15-3.18(1H，m)，2.87-2.99(2H，m)，1.79-1.89(2H，m)，1.63-1.67(1H，m)，1.55-1.59(1H，m)，1.14(3H，d，J＝6.0Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 453.18 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ=7.57 (2H, d, J=8.0Hz), 7.51 (2H, d, J=8.0Hz), 4.08-4.14 (1H, m), 3.74 (1H, d, J=8.8Hz), 3.54 (1H, d, J=8.8Hz), 3.39 (3H, s) 4-3.38(2H,m), 3.15-3.18(1H,m), 2.87-2.99(2H,m), 1.79-1.89(2H,m), 1.63-1.67(1H,m), 1.55-1.59(1H,m), 1.14(3H,d,J=6.0Hz).
实施例15：(3S，4S)-8-(3-((2-氯苯基)乙炔基)-1H-吡唑并[4，3-b]吡嗪-6基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 15:(3S,4S)-8-(3-((2-chlorophenyl)ethynyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：423.17[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝8.30(1H，s)，7.68(1H，dd，J＝7.2Hz，2.0Hz)，7.43(1H，d，J＝8.0Hz)，7.23-7.32(2H，m)，4.17-4.22(1H，m)，3.96-4.06(2H，m)，3.82(1H，d，J＝8.4Hz)，3.70(1H，d，J＝8.4Hz)，3.52-3.59(1H，m)，3.42-3.49(1H，m)，3.01(1H，d，J＝4.0Hz)，1.88-2.03(2H，m)，1.67-1.79(2H，m)，1.24(3H，d，J＝6.8Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 423.17 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=8.30 (1H, s), 7.68 (1H, dd, J=7.2Hz, 2.0Hz), 7.43 (1H, d, J=8.0Hz), 7.23-7.32 (2H, m), 4.17-4.22 (1H, m), 3.96-4.06 (2H, m), 3.82 (1H, d, J=8.4Hz), 3.70 (1H, d, J = 8.4Hz), 3.52-3.59 (1H, m), 3.42-3.49 (1H, m), 3.01 (1H, d, J = 4.0Hz), 1.88-2.03 (2H, m), 1.67-1.79 (2H, m), 1.24 (3H, d, J = 6.8Hz).
实施例16：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-(3-氨基-3-甲基丁-1-炔-1-基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 16:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-(3-amino-3-methylbut-1-yn-1-yl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：400.25[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.33-13.46(1H，brs)，4.08-4.14(1H，m)，3.73(1H，d，J＝8.8Hz)，3.55(1H，d，J＝8.8Hz)，3.37(3H，s)，3.11-3.21(2H，m)，2.85-2.96(3H，m)，1.76-1.86(2H，m)，1.63-1.67(1H，m)，1.54-1.59(1H，m)，1.43(6H，s)，1.14(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 400.25 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 13.33-13.46 (1H, brs), 4.08-4.14 (1H, m), 3.73 (1H, d, J = 8.8Hz), 3.55 (1H, d, J = 8.8Hz), 3.37 (3H, s), 3.11-3.21 (2H, m), 2. 85-2.96 (3H, m), 1.76-1.86 (2H, m), 1.63-1.67 (1H, m), 1.54-1.59 (1H, m), 1.43 (6H, s), 1.14 (3H, d, J=6.4Hz).
实施例17：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-3-((噻吩-2-基)乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 17:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-methyl -3-((thiophen-2-yl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：425.18[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.71(1H，dd，J＝5.2Hz，0.8Hz)，7.45(1H，dd，J＝3.6Hz，0.8Hz)，7.14(1H，dd，J＝5.2Hz，3.6Hz)，4.07-4.13(1H，m)，3.72(1H，d，J＝8.8Hz)，3.53(1H，d，J＝8.8Hz)，3.39(3H，s)，3.32-3.38(2H，m)，3.11(1H，d，J＝5.2Hz)，2.88-2.99(2H，m)，1.77-1.88(2H，m)，1.61-1.66(1H，m)，1.53-1.58(1H，m)，1.12(3H，d，J＝6.8Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 425.18 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.71 (1H, dd, J=5.2 Hz, 0.8 Hz), 7.45 (1H, dd, J=3.6 Hz, 0.8 Hz), 7.14 (1H, dd, J=5.2 Hz, 3.6 Hz), 4.07-4.13 (1H, m), 3.72 (1H, d, J=8.8 Hz), 3.53 (1H, d, J=8.8 Hz), 3.39 (3H, s), 3.32-3.38 (2H, m), 3.11 (1H, d, J = 5.2Hz), 2.88-2.99 (2H, m), 1.77-1.88 (2H, m), 1.61-1.66 (1H, m), 1.53-1.58 (1H, m), 1.12 (3H, d , J=6.8Hz).
实施例18：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((2-氨基苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 18:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((2-aminophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：434.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.21(1H，d，J＝8.0Hz)，7.09(1H，t，J＝8.0Hz)，6.71(1H，d，J＝8.4Hz)，6.51(1H，t，J＝7.2Hz)，6.01(2H，s)，4.41-4.46(1H，m)，4.12-4.20(1H，m)，3.82(1H，d，J＝8.4Hz)，3.58(1H，d，J＝8.4Hz)，3.45-3.49(1H，m)，3.42(3H，s)，3.13-3.15(1H，m)，2.85-2.95(2H，m)，1.84-1.98(2H，m)，1.66-1.72(2H，m)，1.58-1.63(2H，m)，1.19(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 434.23 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.21 (1H, d, J=8.0 Hz), 7.09 (1H, t, J=8.0 Hz), 6.71 (1H, d, J=8.4 Hz), 6.51 (1H, t, J=7.2 Hz), 6.01 (2H, s), 4.41-4.46 (1H, m), 4.12-4.20 (1H, m), 3.82 (1H, d, J=8.4 Hz), 3.70 (1H, d, J=8. .58 (1H, d, J=8.4Hz), 3.45-3.49 (1H, m), 3.42 (3H, s), 3.13-3.15 (1H, m), 2.85-2.95 (2H, m), 1.84-1.98 (2H, m), 1.66-1.72 (2H, m), 1.58-1.63 (2H, m) ), 1.19 (3H, d, J=6.4Hz).
实施例19：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-(环丙基乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 19:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-(cyclopropylethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：383.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.20-13.58(1H，brs)，4.02-4.08(1H，m)，3.90-4.00(1H，m)，3.65(1H，d，J＝8.4Hz)，3.47(1H，d，J＝8.4Hz)，3.35(3H，s)，3.24-3.30(1H，m)，3.11-3.15(1H，m)，2.86-2.99(3H，m)，1.70-1.85(2H，m)，1.50-1.62(2H，m)，1.08(3H，d，J＝6.4Hz)，0.91-0.94(2H，m)，0.75-0.83(2H，m)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 383.22 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ = 13.20-13.58 (1H, brs), 4.02-4.08 (1H, m), 3.90-4.00 (1H, m), 3.65 (1H, d, J = 8.4Hz), 3.47 (1H, d, J = 8.4Hz), 3.35 (3H, s), 3.24-3.30 (1H, m), 3.11- 3.15 (1H, m), 2.86-2.99 (3H, m), 1.70-1.85 (2H, m), 1.50-1.62 (2H, m), 1.08 (3H, d, J = 6.4Hz), 0.91-0.94 (2H, m), 0.75-0.83 (2H, m).
实施例20：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((3-氨基-4-氟苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 20:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-3-((3-amino-4-fluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：452.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.07(1H，d，J＝8.0Hz)，6.94-6.99(1H，m)，6.87-6.91(1H，m)，4.26-4.32(1H，m)，3.93(1H，d，J＝8.0Hz)，3.82(1H，d，J＝8.4Hz)，3.47-3.58(5H，m)，3.39-3.42(1H，m)，2.96-3.08(2H，m)，1.86-2.02(3H，m)，1.71-1.75(1H，m)，1.30(3H，d，J＝7.2Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 452.22 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ=7.07 (1H, d, J=8.0Hz), 6.94-6.99 (1H, m), 6.87-6.91 (1H, m), 4.26-4.32 (1H, m), 3.93 (1H, d, J=8.0Hz), 3.82 (1H, d, J=8.4Hz ), 3.47-3.58 (5H, m), 3.39-3.42 (1H, m), 2.96-3.08 (2H, m), 1.86-2.02 (3H, m), 1.71-1.75 (1H, m), 1.30 (3H, d, J = 7.2Hz).
实施例21：2-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((2-氰基苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 21:2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((2-cyanophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：444.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.75(1H，d，J＝8.0Hz)，7.63-7.68(1H，m)，7.51-7.58(1H，m)，7.45(1H，m)，4.14-4.20(1H，m)，3.81(1H，d，J＝8.8Hz)，3.68(1H，d，J＝8.8Hz)，3.53(3H，s)，3.30-3.43(2H，m)，2.94-3.16(3H，m)，1.52-1.86(4H，m)，0.86(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 444.22 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.75 (1H, d, J = 8.0Hz), 7.63-7.68 (1H, m), 7.51-7.58 (1H, m), 7.45 (1H, m), 4.14-4.20 (1H, m), 3.81 (1H, d, J = 8.8Hz), 3.68 (1 H, d, J = 8.8Hz), 3.53 (3H, s), 3.30-3.43 (2H, m), 2.94-3.16 (3H, m), 1.52-1.86 (4H, m), 0.86 (3H, d, J = 6.4Hz).
实施例22：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-3-((2-(三氟甲基)苯基)乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 22:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-methyl -3-((2-(trifluoromethyl)phenyl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：487.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.82(1H，d，J＝7.6Hz)，7.68(1H，d，J＝8.4Hz)，7.52(1H，m)，7.44(1H，m)，4.13-4.19(1H，m)，3.79(1H，d，J＝8.8Hz)，3.67(1H，d，J＝8.8Hz)，3.53(3H，s)，3.31-3.41(2H，m)，2.98-3.16(3H，m)，1.46-1.90(4H，m)，0.95(3H，d，J＝6.8Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 487.21 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.82 (1H, d, J = 7.6Hz), 7.68 (1H, d, J = 8.4Hz), 7.52 (1H, m), 7.44 (1H, m), 4.13-4.19 (1H, m), 3.79 (1H, d, J = 8.8Hz), 3.67 (1H, d, J=8.8Hz), 3.53 (3H, s), 3.31-3.41 (2H, m), 2.98-3.16 (3H, m), 1.46-1.90 (4H, m), 0.95 (3H, d, J=6.8Hz).
实施例23：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((2，6-二氟苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 23:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((2,6-difluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：455.2[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.51-7.59(1H，m)，7.22-7.27(2H，m)，4.04-4.11(1H，m)，3.69(1H，d，J＝8.4Hz)，3.51(1H，d，J＝8.8Hz)，3.38(3H，s)，3.32-3.6(1H，m)，3.11-3.15(1H，m)，3.05-3.08(1H，m)，2.88-3.01(2H，m)，1.73-1.86(2H，m)，1.60-1.66(1H，m)，1.52-1.58(1H，m)，1.10(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 455.2 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.51-7.59 (1H, m), 7.22-7.27 (2H, m), 4.04-4.11 (1H, m), 3.69 (1H, d, J = 8.4Hz), 3.51 (1H, d, J = 8.8Hz), 3.38 (3H, s), 3.32-3. 6(1H,m), 3.11-3.15(1H,m), 3.05-3.08(1H,m), 2.88-3.01(2H,m), 1.73-1.86(2H,m), 1.60-1.66(1H,m), 1.52-1.58(1H,m), 1.10(3H,d,J=6.4Hz).
实施例24：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-3-(丙-1-炔-1-基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 24:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl -3-(prop-1-yn-1-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：357.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.12-13.58(1H，brs)，4.02-4.08(1H，m)，3.65(1H，d，J＝8.4Hz)，3.47(1H，d，J＝8.4Hz)，3.35(3H，s)，3.24-3.30(1H，m)，3.11-3.15(1H，m)，2.86-2.99(3H，m)，2.05(3H，s)，1.70-1.85(2H，m)，1.50-1.62(2H，m)，1.07(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 357.21 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 13.12-13.58 (1H, brs), 4.02-4.08 (1H, m), 3.65 (1H, d, J = 8.4Hz), 3.47 (1H, d, J = 8.4Hz), 3.35 (3H, s), 3.24-3.30 (1H, m), 3. 11-3.15 (1H, m), 2.86-2.99 (3H, m), 2.05 (3H, s), 1.70-1.85 (2H, m), 1.50-1.62 (2H, m), 1.07 (3H, d, J=6.4Hz).
实施例25：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-(咪唑并[1，2-b]哒嗪-3-基乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 25:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-(imidazo[1,2-b]pyridazin-3-ylethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：460.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.66-1.84(1H，brs)，8.67(1H，dd，J＝4.8Hz，2.4Hz)，8.24(1H，dd，J＝8.8Hz，1.6Hz)，8.19(1H，s)，7.37(1H，dd，J＝8.8Hz，4.8Hz)，4.40-4.43(1H，m)，4.16-4.20(1H，m)，3.83(1H，d，J＝8.8Hz)，3.62(1H，d，J＝8.8Hz)，3.43-3.50(1H，m)，3.39(3H，s)，3.34-3.36(1H，m)，2.85-2.94(2H，m)，1.85-1.94(2H，m)，1.70-1.73(1H，m)，1.58-1.62(1H，m)，1.19(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 460.22 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=13.66-1.84 (1H, brs), 8.67 (1H, dd, J=4.8 Hz, 2.4 Hz), 8.24 (1H, dd, J=8.8 Hz, 1.6 Hz), 8.19 (1H, s), 7.37 (1H, dd, J=8.8 Hz, 4.8 Hz), 4.40-4.43 (1H, m), 4.16-4.20 (1H, m), 3.83 (1H, d, J=8.8Hz), 3.62 (1H, d, J=8.8Hz), 3.43-3.50 (1H, m), 3.39 (3H, s), 3.34-3.36 (1H, m), 2.85-2.94 (2H, m), 1.85-1.94 (2H, m), 1.70-1.73 (1H, m), 1.58- 1.62 (1H, m), 1.19 (3H, d, J=6.4Hz).
实施例26：(3S，4S)-8-(9-((2-氟苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 26:(3S,4S)-8-(9-((2-fluorophenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：446.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.71(1H，m)，7.68(1H，t，J＝8.0Hz)，7.50-7.55(2H，m)，7.37(1H，t，J＝8.0Hz)，7.31(1H，t，J＝8.0Hz)，4.11-4.16(1H，m)，3.76(1H，d，J＝8.0Hz)，3.58-3.64(2H，m)，3.14-3.32(4H，m)，1.89-1.99(2H，m)，1.72-1.77(1H，m)，1.62-1.68(1H，m)，1.14(3H，d，J＝6.8Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 446.21 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ=7.71 (1H, m), 7.68 (1H, t, J=8.0Hz), 7.50-7.55 (2H, m), 7.37 (1H, t, J=8.0Hz), 7.31 (1H, t, J=8.0Hz), 4.11-4.16 (1H, m), 3.76 ( 1H, d, J = 8.0Hz), 3.58-3.64 (2H, m), 3.14-3.32 (4H, m), 1.89-1.99 (2H, m), 1.72-1.77 (1H, m), 1.62-1.68 (1H, m), 1.14 (3H, d, J = 6.8Hz).
实施例27：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-乙炔基-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 27:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-3-ethynyl -5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
步骤1：((3S，4S)-3-甲基-8-(5-甲基-4-氧代-3-((三甲基硅基)乙炔基)-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯Step 1: tert-Butyl ((3S,4S)-3-methyl-8-(5-methyl-4-oxo-3-((trimethylsilyl)ethynyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4.5]decan-4-yl)carbamate
参照实施例1中步骤1的方法，用中间体6代替中间体5，三甲基硅基乙炔代替1-氯-2-乙炔基苯，得到目标化合物。MS m/z[LC-MS]：515.28[M+1]。Referring to the method of step 1 in Example 1, intermediate 6 was used instead of intermediate 5, and trimethylsilyl acetylene was used instead of 1-chloro-2-ethynylbenzene to obtain the target compound. MS m/z [LC-MS]: 515.28 [M+1].
步骤2：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-乙炔基-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Step 2: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-3-ethynyl-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
将((3S，4S)-3-甲基-8-(5-甲基-4-氧代-3-((三甲基硅基)乙炔基)-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯(40mg)溶于二氯甲烷(1mL)，加入1M四丁基氟化铵的四氢呋喃溶液(0.5mL)，室温搅拌0.5小时。再加入4M氯化氢的二氧六环溶液(3mL)，室温搅拌1小时，旋干，加入10％碳酸钠水溶液(10mL)，用二氯甲烷萃取，萃取液用无水硫酸钠干燥，过滤，滤液减压浓缩，用薄层硅胶色谱分离得，得到目标化合物(15mg)。MS m/z[LC-MS]：343.19[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.29-13.74(1H，brs)，4.35(1H，s)，4.05-4.12(1H，m)，3.70(1H，d，J＝8.8Hz)，3.52(1H，d，J＝8.8Hz)，3.36(3H，s)，3.22-3.35(2H，m)，3.08-3.12(1H，m)，2.86-2.98(2H，m)，1.74-1.88(2H，m)，1.60-1.64(1H，m)，1.52-1.56(1H，m)，1.11(3H，d，J＝6.0Hz)。Dissolve tert-butyl ((3S,4S)-3-methyl-8-(5-methyl-4-oxo-3-((trimethylsilyl)ethynyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate (40 mg) in dichloromethane (1 mL), add 1M tetrabutylammonium fluoride tetrahydrofuran solution (0.5 mL), and stir at room temperature for 0.5 hours. Then add 4M hydrogen chloride dioxane solution (3 mL), stir at room temperature for 1 hour, spin dry, add 10% sodium carbonate aqueous solution (10 mL), extract with dichloromethane, dry the extract with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and separate by thin layer silica gel chromatography to obtain the target compound (15 mg). MS m/z[LC-MS]: 343.19[M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 13.29-13.74 (1H, brs), 4.35 (1H, s), 4.05-4.12 (1H, m), 3.70 (1H, d, J = 8.8Hz), 3.52 (1H, d, J = 8.8Hz), 3.36 (3H, s), 3.22-3. 35(2H,m), 3.08-3.12(1H,m), 2.86-2.98(2H,m), 1.74-1.88(2H,m), 1.60-1.64(1H,m), 1.52-1.56(1H,m), 1.11(3H,d,J=6.0Hz).
实施例28：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-3-(3，3，3-三氟丙-1-炔-1-基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 28:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl -3-(3,3,3-trifluoroprop-1-yn-1-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：452.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝4.06-4.12(1H，m)，3.71(1H，d，J＝8.8Hz)，3.52(1H，d，J＝8.8Hz)，3.38(3H，s)，3.24-3.36(2H，m)，3.10-3.14(1H，m)，2.89-3.01(2H，m)，1.76-1.86(2H，m)，1.61-1.65(1H，m)，1.53-1.57(1H，m)，1.11(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 452.22 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=4.06-4.12 (1H, m), 3.71 (1H, d, J=8.8 Hz), 3.52 (1H, d, J=8.8 Hz), 3.38 (3H, s), 3.24-3.36 (2H, m), 3.10-3.14 (1H, m), 2.89-3.01 (2H, m), 1.76-1.86 (2H, m), 1.61-1.65 (1H, m), 1.53-1.57 (1H, m), 1.11 (3H, d, J=6.4 Hz).
实施例29：(3S，4S)-8-(8-((3-氨基苯基)乙炔基)-7-氯咪唑并[1，2-c]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 29:(3S,4S)-8-(8-((3-aminophenyl)ethynyl)-7-chloroimidazo[1,2-c]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：437.19[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.75(1H，d，J＝1.6Hz)，7.59(1H，d，J＝1.6Hz)，7.10(1H，t，J＝8.0Hz)，6.98(1H，t，J＝1.6Hz)，6.95(1H，d，J＝7.6Hz)，6.73(1H，dd，J＝8.0Hz，1.6Hz)，4.56(1H，s)，4.23-4.29(1H，m)，3.84-3.93(3H，m)，3.79(1H，d，J＝9.2Hz)，3.35-3.39(1H，m)，3.23(1H，d，J＝4.8Hz)，1.94-2.05(2H，m)，1.83-1.87(1H，m)，1.74-1.78(1H，m)，1.26(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 437.19 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.75 (1H, d, J=1.6 Hz), 7.59 (1H, d, J=1.6 Hz), 7.10 (1H, t, J=8.0 Hz), 6.98 (1H, t, J=1.6 Hz), 6.95 (1H, d, J=7.6 Hz), 6.73 (1H, dd, J=8.0 Hz, 1.6 Hz), 4.56 (1H, s), 4.23-4.2 9(1H,m), 3.84-3.93(3H,m), 3.79(1H,d,J=9.2Hz), 3.35-3.39(1H,m), 3.23(1H,d,J=4.8Hz), 1.94-2.05(2H,m), 1.83-1.87(1H,m), 1.74-1.78(1H, m), 1.26 (3H, d, J=6.4Hz).
实施例30：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((2-氨基吡啶-3-基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 30:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((2-aminopyridin-3-yl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：435.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.97(1H，d，J＝5.2Hz)，7.60(1H，dd，J＝7.6Hz，2.0Hz)，6.73(2H，s)，6.55(1H，dd，J＝7.6Hz，5.2Hz)，4.09-4.16(1H，m)，3.77(1H，d，J＝8.8Hz)，3.55(1H，d，J＝8.8Hz)，3.41(3H，s)，3.22-3.39(2H，m)，2.86-2.97(3H，m)，1.82-1.92(2H，m)，1.65-1.68(1H，m)，1.56-1.61(1H，m)，1.15(3H，d，J＝6.8Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 435.23 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.97 (1H, d, J=5.2Hz), 7.60 (1H, dd, J=7.6Hz, 2.0Hz), 6.73 (2H, s), 6.55 (1H, dd, J=7.6Hz, 5.2Hz), 4.09-4.16 (1H, m), 3.77 (1H, d, J=8.8Hz), 3.55 (1H, d, J=8.8Hz), 3.41 (3H, s), 3.22-3.39 (2H, m), 2.86-2.97 (3H, m), 1.82-1.92 (2H, m), 1.65-1.68 (1H, m), 1.56-1.61 (1H, m), 1.15 (3H, d, J=6.8Hz).
实施例31：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((1-羟基环丙基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 31:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((1-hydroxycyclopropyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：399.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.38-13.54(1H，brs)，6.35(1H，s)，4.05-4.11(1H，m)，3.68(1H，d，J＝9.2Hz)，3.51(1H，d，J＝8.4Hz)，3.35(3H，s)，3.17-3.32(2H，m)，3.02-3.08(1H，m)，2.84-2.98(2H，m)，1.72-1.84(2H，m)，1.59-1.64(1H，m)，1.51-1.56(1H，m)，1.09(3H，d，J＝6.0Hz)，0.97-0.99(2H，m)，0.85-0.93(2H，m)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 399.22 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ = 13.38-13.54 (1H, brs), 6.35 (1H, s), 4.05-4.11 (1H, m), 3.68 (1H, d, J = 9.2Hz), 3.51 (1H, d, J = 8.4Hz), 3.35 (3H, s), 3.17-3.32 (2H, m), 3.02-3.08 (1H,m), 2.84-2.98(2H,m), 1.72-1.84(2H,m), 1.59-1.64(1H,m), 1.51-1.56(1H,m), 1.09(3H,d,J=6.0Hz), 0.97-0.99(2H,m), 0.85-0.93(2H,m).
实施例32：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-3-((1-甲基-1H-咪唑-4-基)乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 32:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl -3-((1-methyl-1H-imidazol-4-yl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：423.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.54(1H，s)，7.69(1H，s)，7.58(1H，s)，4.15-4.20(1H，m)，3.82(1H，d，J＝8.8Hz)，3.66(3H，s)，3.63(1H，d，J＝8.8Hz)，3.36-3.47(5H，m)，2.82-2.93(3H，m)，1.82-1.92(2H，m)，1.70-1.74(1H，m)，1.57-1.60(1H，m)，1.19(3H，d，J＝6.8Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 423.23 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 13.54 (1H, s), 7.69 (1H, s), 7.58 (1H, s), 4.15-4.20 (1H, m), 3.82 (1H, d, J = 8.8Hz), 3.66 (3H, s), 3.63 (1H, d, J = 8.8Hz), 3.36 -3.47(5H,m), 2.82-2.93(3H,m), 1.82-1.92(2H,m), 1.70-1.74(1H,m), 1.57-1.60(1H,m), 1.19(3H,d,J=6.8Hz).
实施例33：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-3-(吡啶-2-基乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 33:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-methyl -3-(pyridin-2-ylethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物.MS m/z[LC-MS]：420.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝12.92-13.88(1H，brs)，8.61(1H，d，J＝5.2Hz)，7.86(1H，td，J＝8.0Hz，1.6Hz)，7.62(1H，d，J＝7.6Hz)，7.43(1H，dd，J＝7.6Hz，5.2Hz)，4.04-4.10(1H，m)，3.68(1H，d，J＝8.8Hz)，3.51(1H，d，J＝8.8Hz)，3.36-3.42(5H，m)，2.89-3.06(3H，m)，1.72-1.84(2H，m)，1.60-1.64(1H，m)，1.52-1.56(1H，m)，1.09(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates.MS m/z[LC-MS]: 420.22[M+1].1 H NMR (400MHz, DMSO-d6 ): δ＝12.92-13.88(1H, brs), 8.61(1H, d, J＝5.2Hz), 7.86(1H, td, J＝8.0Hz, 1.6Hz), 7.62(1H, d, J＝7.6Hz), 7.43(1H, dd, J＝7.6Hz, 5.2Hz), 4.04-4.10(1H, m), 3.6 8(1H,d,J=8.8Hz), 3.51(1H,d,J=8.8Hz), 3.36-3.42(5H,m), 2.89-3.06(3H,m), 1.72-1.84(2H,m), 1.60-1.64(1H,m), 1.52-1.56(1H,m), 1.09(3H, d, J=6.4Hz).
实施例34：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-3-((1-甲基-1H-吡唑-4-基)乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 34:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl -3-((1-methyl-1H-pyrazol-4-yl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：423.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.22-1.82(1H，brs)，7.85(1H，s)，7.70(1H，s)，4.04-4.10(1H，m)，3.85(3H，s)，3.68(1H，d，J＝8.8Hz)，3.51(1H，d，J＝8.8Hz)，3.32-3.42(5H，m)，2.88-3.04(3H，m)，1.72-1.86(2H，m)，1.60-1.64(1H，m)，1.52-1.56(1H，m)，1.09(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 423.23 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 13.22-1.82 (1H, brs), 7.85 (1H, s), 7.70 (1H, s), 4.04-4.10 (1H, m), 3.85 (3H, s), 3.68 (1H, d, J = 8.8Hz), 3.51 (1H, d, J = 8.8 Hz), 3.32-3.42 (5H, m), 2.88-3.04 (3H, m), 1.72-1.86 (2H, m), 1.60-1.64 (1H, m), 1.52-1.56 (1H, m), 1.09 (3H, d, J = 6.4Hz).
实施例35：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-甲基-3-((1-甲基-1H-吡唑-3-基)乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 35:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl -3-((1-methyl-1H-pyrazol-3-yl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：423.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.22-1.82(1H，brs)，7.78(1H，d，J＝2.4Hz)，6.52(1H，d，J＝2.4Hz)，4.04-4.10(1H，m)，3.85(3H，s)，3.68(1H，d，J＝8.8Hz)，3.51(1H，d，J＝8.8Hz)，3.32-3.42(5H，m)，2.88-3.04(3H，m)，1.72-1.86(2H，m)，1.60-1.64(1H，m)，1.52-1.56(1H，m)，1.09(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 423.23 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 13.22-1.82 (1H, brs), 7.78 (1H, d, J = 2.4Hz), 6.52 (1H, d, J = 2.4Hz), 4.04-4.10 (1H, m), 3.85 (3H, s), 3.68 (1H, d, J = 8.8Hz), 3.5 1(1H,d,J=8.8Hz), 3.32-3.42(5H,m), 2.88-3.04(3H,m), 1.72-1.86(2H,m), 1.60-1.64(1H,m), 1.52-1.56(1H,m), 1.09(3H,d,J=6.4Hz).
实施例36：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-(2-氯-3-乙炔基苯基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 36:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-(2-chloro-3-ethynylphenyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
步骤1：((3S，4S)-8-(3-(2-氯-3-((三甲基硅基)乙炔基)苯基)-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯Step 1: tert-Butyl ((3S,4S)-8-(3-(2-chloro-3-((trimethylsilyl)ethynyl)phenyl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)carbamate
将中间体6(160mg)、含中间体12的反应液(0.8mL)、四(三苯基膦)钯(18mg)、磷酸钾(185mg)加入二氧六环和水的混合溶剂(10∶1，5mL)中，用氮气置换反应体系中的空气三次，加热至80℃搅拌过夜。冷却到室温，倒入水中，用二氯甲烷萃取，萃取液用饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱分离(石油醚∶乙酸乙酯，3∶1)得((3S，4S)-8-(3-(2-氯-3-((三甲基硅基)乙炔基)苯基)-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯(80mg)。MS m/z[LC-MS]：625.27[M+1]。Intermediate 6 (160 mg), reaction solution containing intermediate 12 (0.8 mL), tetrakis(triphenylphosphine)palladium (18 mg), potassium phosphate (185 mg) were added to a mixed solvent of dioxane and water (10:1, 5 mL), the air in the reaction system was replaced with nitrogen three times, heated to 80°C and stirred overnight. The mixture was cooled to room temperature, poured into water, extracted with dichloromethane, the extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 3:1) to obtain tert-butyl ((3S, 4S)-8-(3-(2-chloro-3-((trimethylsilyl)ethynyl)phenyl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate (80 mg). MS m/z[LC-MS]: 625.27[M+1].
步骤2：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-(2-氯-3-乙炔基苯基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Step 2: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-3-(2-chloro-3-ethynylphenyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
将((3S，4S)-8-(3-(2-氯-3-((三甲基硅基)乙炔基)苯基)-5-甲基-4-氧代-4，5-二氢-1H-吡唑并[3，4-d]嘧啶-6-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)氨基甲酸叔丁酯(80mg)加入4M氯化氢的二氧六环溶液(3mL)中，室温搅拌1小时，旋干，加入10％碳酸钠水溶液(10mL)，用二氯甲烷萃取，萃取液用无水硫酸钠干燥，过滤，滤液减压浓缩，用薄层硅胶色谱分离(二氯甲烷∶甲醇，10∶1)得6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-(2-氯-3-乙炔基苯基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮(38mg)。MS m/z[LC-MS]：453.18[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.70(1H，d，J＝8.0Hz)，7.49(1H，d，J＝7.6Hz)，7.34-7.40(1H，m)，4.25-4.32(1H，m)，3.94(1H，d，J＝10.4Hz)，3.83(1H，d，J＝10.4Hz)，3.71-3.73(1H，m)，3.46-3.68(6H，m)，2.94-3.09(2H，m)，1.87-2.05(2H，m)，1.70-1.77(1H，m)，1.56-1.62(1H，m)，1.29(3H，d，J＝7.6Hz)。Tert-butyl ((3S,4S)-8-(3-(2-chloro-3-((trimethylsilyl)ethynyl)phenyl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate (80 mg) was added to a 4M solution of hydrogen chloride in dioxane (3 mL), stirred at room temperature for 1 hour, dried by rotation, and a 10% aqueous sodium carbonate solution was added. (10 mL), extracted with dichloromethane, the extract was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and separated by thin layer silica gel chromatography (dichloromethane: methanol, 10: 1) to give 6-((3S, 4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-(2-chloro-3-ethynylphenyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (38 mg). MS m/z [LC-MS]: 453.18 [M+1].1 H NMR (400MHz, CD3 OD): δ=7.70 (1H, d, J=8.0Hz), 7.49 (1H, d, J=7.6Hz), 7.34-7.40 (1H, m), 4.25-4.32 (1H, m), 3.94 (1H, d, J=10.4Hz), 3.83 (1H, d, J=10.4 Hz), 3.71-3.73 (1H, m), 3.46-3.68 (6H, m), 2.94-3.09 (2H, m), 1.87-2.05 (2H, m), 1.70-1.77 (1H, m), 1.56-1.62 (1H, m), 1.29 (3H, d, J=7.6Hz).
实施例37：6-(4-氨基-4-甲基哌啶-1-基)-3-((2-氟-3，5-二甲氧基苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 37:6-(4-amino-4-methylpiperidin-1-yl)-3-((2-fluoro-3,5-dimethoxyphenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：441.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.21-13.90(1H，brs)，8.22(2H，s)，6.81(1H，dd，J＝7.2Hz，2.8Hz)，6.60(1H，dd，J＝4.4Hz，2.8Hz)，3.84(3H，s)，3.76(3H，s)，3.38-3.46(2H，m)，3.37(3H，s)，3.07-3.14(2H，m)，1.86-1.95(2H，m)，1.73-1.82(2H，m)，1.35(3H，s)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 441.21 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 13.21-13.90 (1H, brs), 8.22 (2H, s), 6.81 (1H, dd, J = 7.2Hz, 2.8Hz), 6.60 (1H, dd, J = 4.4Hz, 2.8Hz), 3.84 (3H, s), 3.76 (3H, s), 3. 38-3.46 (2H, m), 3.37 (3H, s), 3.07-3.14 (2H, m), 1.86-1.95 (2H, m), 1.73-1.82 (2H, m), 1.35 (3H, s).
实施例38：6-(4-氨基-4-甲基哌啶-1-基)-3-((2，5-二氟苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 38:6-(4-amino-4-methylpiperidin-1-yl)-3-((2,5-difluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：399.18[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.63-13.90(1H，brs)，8.13(2H，s)，7.47-7.51(1H，m)，7.34-7.44(2H，m)，3.41-3.44(2H，m)，3.37(3H，s)，3.08-3.14(2H，m)，1.85-1.92(2H，m)，1.73-1.79(2H，m)，1.35(3H，s)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 399.18 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=13.63-13.90 (1H, brs), 8.13 (2H, s), 7.47-7.51 (1H, m), 7.34-7.44 (2H, m), 3.41-3.44 (2H, m), 3.37 (3H, s), 3.08-3.14 (2H, m), 1.85-1.92 (2H, m), 1.73-1.79 (2H, m), 1.35 (3H, s).
实施例39：(S)-1-(4-(9-(2，3-二氯苯基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-1H-吡唑-1-基)丙烷-2-胺Example 39:(S)-1-(4-(9-(2,3-dichlorophenyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin -5-yl)-1H-pyrazol-1-yl)propan-2-amine
参照实施例36中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：427.10[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝8.78-8.92(1H，brs)，8.28-8.42(1H，brs)，8.24(1H，s)，7.77(1H，dd，J＝8.0Hz，1.6Hz)，7.69(1H，dd，J＝7.6Hz，1.6Hz)，7.48-7.51(2H，m)，4.22-4.54(2H，m)，3.50-3.85(1H，m)，1.02-1.32(3H，m)。The target compound can be synthesized by referring to the method in Example 36 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 427.10 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=8.78-8.92 (1H, brs), 8.28-8.42 (1H, brs), 8.24 (1H, s), 7.77 (1H, dd, J=8.0 Hz, 1.6 Hz), 7.69 (1H, dd, J=7.6 Hz, 1.6 Hz), 7.48-7.51 (2H, m), 4.22-4.54 (2H, m), 3.50-3.85 (1H, m), 1.02-1.32 (3H, m).
实施例40：3-((1H-吡唑-4-基)乙炔基)-6-(4-氨基-4-甲基哌啶-1-基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 40:3-((1H-pyrazol-4-yl)ethynyl)-6-(4-amino-4-methylpiperidin-1-yl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d ]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：353.19[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.19-13.86(1H，brs)，8.23(2H，s)，7.94(2H，s)，3.37-3.44(2H，m)，3.35(3H，s)，3.05-3.13(2H，m)，1.86-1.94(2H，m)，1.72-7.8(2H，m)，1.34(3H，s)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 353.19 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=13.19-13.86 (1H, brs), 8.23 (2H, s), 7.94 (2H, s), 3.37-3.44 (2H, m), 3.35 (3H, s), 3.05-3.13 (2H, m), 1.86-1.94 (2H, m), 1.72-7.8 (2H, m), 1.34 (3H, s).
实施例41：6-(4-氨基-4-甲基哌啶-1-基)-5-甲基-3-(嘧啶-5-基乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 41:6-(4-amino-4-methylpiperidin-1-yl)-5-methyl-3-(pyrimidin-5-ylethynyl)-1,5-dihydro -4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：365.18[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.61-13.94(1H，brs)，9.22(1H，s)，8.99(2H，s)，7.78-8.22(2H，brs)，3.39-3.46(2H，m)，3.38(3H，s)，3.08-3.15(2H，m)，1.83-1.91(2H，m)，1.72-1.79(2H，m)，1.34(3H，s)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 365.18 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=13.61-13.94 (1H, brs), 9.22 (1H, s), 8.99 (2H, s), 7.78-8.22 (2H, brs), 3.39-3.46 (2H, m), 3.38 (3H, s), 3.08-3.15 (2H, m), 1.83-1.91 (2H, m), 1.72-1.79 (2H, m), 1.34 (3H, s).
实施例42：6-(4-氨基-4-甲基哌啶-1-基)-3-((3，5-二(三氟甲基)苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 42:6-(4-amino-4-methylpiperidin-1-yl)-3-((3,5-bis(trifluoromethyl)phenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：499.17[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝8.17-8.21(3H，m)，3.37(3H，s)，3.12-3.28(4H，m)，1.54-1.61(2H，m)，1.46-1.52(2H，m)，1.10(3H，s)。Referring to the method in Example 1, the target compound can be synthesized using appropriate starting materials and intermediates. MS m/z [LC-MS]: 499.17 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=8.17-8.21 (3H, m), 3.37 (3H, s), 3.12-3.28 (4H, m), 1.54-1.61 (2H, m), 1.46-1.52 (2H, m), 1.10 (3H, s).
实施例43：6-(4-氨基-4-甲基哌啶-1-基)-5-甲基-3-((3-(三氟甲基)苯基)乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 43:6-(4-amino-4-methylpiperidin-1-yl)-5-methyl-3-((3-(trifluoromethyl)phenyl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：431.18[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.58-13.86(1H，brs)，8.08-8.18(2H，brs)，7.85-7.87(2H，m)，7.81(1H，d，J＝7.6Hz)，7.69(1H，t，J＝7.6Hz)，3.40-3.46(2H，m)，3.38(3H，s)，3.08-3.14(2H，m)，1.86-1.93(2H，m)，1.73-1.80(2H，m)，1.35(3H，s)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 431.18 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=13.58-13.86 (1H, brs), 8.08-8.18 (2H, brs), 7.85-7.87 (2H, m), 7.81 (1H, d, J=7.6 Hz), 7.69 (1H, t, J=7.6 Hz), 3.40-3.46 (2H, m), 3.38 (3H, s), 3.08-3.14 (2H, m), 1.86-1.93 (2H, m), 1.73-1.80 (2H, m), 1.35 (3H, s).
实施例44：6-(4-氨基-4-甲基哌啶-1-基)-5-甲基-3-(噻吩-3-基乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 44:6-(4-amino-4-methylpiperidin-1-yl)-5-methyl-3-(thien-3-ylethynyl)-1,5-dihydro -4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：369.14[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝8.21(2H，s)，7.95(1H，d，J＝1.6Hz)，7.69(1H，dd，J＝4.8Hz，2.8Hz)，7.27(1H，d，J＝4.8Hz)，3.65-3.73(2H，m)，3.39(3H，s)，3.01-3.06(2H，m)，1.89-1.97(2H，m)，1.76-1.84(2H，m)，1.38(3H，s)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 369.14 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=8.21 (2H, s), 7.95 (1H, d, J=1.6 Hz), 7.69 (1H, dd, J=4.8 Hz, 2.8 Hz), 7.27 (1H, d, J=4.8 Hz), 3.65-3.73 (2H, m), 3.39 (3H, s), 3.01-3.06 (2H, m), 1.89-1.97 (2H, m), 1.76-1.84 (2H, m), 1.38 (3H, s).
实施例45：6-(4-氨基-4-甲基哌啶-1-基)-3-((3-氟苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 45:6-(4-amino-4-methylpiperidin-1-yl)-3-((3-fluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：381.19[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝12.74-13.90(1H，brs)，7.70-8.90(2H，brs)，7.46-7.51(1H，m)，7.36-7.40(2H，m)，7.28-7.33(1H，m)，3.38-3.46(2H，m)，3.37(3H，s)，3.07-3.14(2H，m)，1.91-1.98(2H，m)，1.75-1.80(2H，m)，1.35(3H，s)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 381.19 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=12.74-13.90 (1H, brs), 7.70-8.90 (2H, brs), 7.46-7.51 (1H, m), 7.36-7.40 (2H, m), 7.28-7.33 (1H, m), 3.38-3.46 (2H, m), 3.37 (3H, s), 3.07-3.14 (2H, m), 1.91-1.98 (2H, m), 1.75-1.80 (2H, m), 1.35 (3H, s).
实施例46：6-(4-氨基-4-甲基哌啶-1-基)-3-((2，3-二氟苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 46:6-(4-amino-4-methylpiperidin-1-yl)-3-((2,3-difluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：399.18[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.43-7.47(1H，m)，7.30-7.37(1H，m)，7.17-7.23(1H，m)，3.51-3.59(5H，m)，3.21-3.29(2H，m)，1.99-2.06(2H，m)，1.88-1.94(2H，m)，1.48(3H，s)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 399.18 [M+1].1 H NMR (400 MHz, CD3 OD): δ = 7.43-7.47 (1H, m), 7.30-7.37 (1H, m), 7.17-7.23 (1H, m), 3.51-3.59 (5H, m), 3.21-3.29 (2H, m), 1.99-2.06 (2H, m), 1.88-1.94 (2H, m), 1.48 (3H, s).
实施例47：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((2，3-二氟苯基)乙炔基)-5-甲基-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 47:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-((2,3-difluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：455.20[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.43-7.47(1H，m)，7.30-7.37(1H，m)，7.17-7.22(1H，m)，4.22-4.28(1H，m)，3.89(1H，d，J＝8.8Hz)，3.76(1H，d，J＝8.8Hz)，3.46-3.54(5H，m)，3.24-3.29(1H，m)，3.97-3.12(2H，m)，1.91-2.02(2H，m)，1.80-1.86(1H，m)，1.68-1.74(1H，m)，1.40(3H，s)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 455.20 [M+1].1 H NMR (400MHz, CD3 OD): δ=7.43-7.47(1H,m), 7.30-7.37(1H,m), 7.17-7.22(1H,m), 4.22-4.28(1H,m), 3.89(1H,d,J=8.8Hz), 3.76(1H,d,J=8.8Hz), 3 .46-3.54(5H,m), 3.24-3.29(1H,m), 3.97-3.12(2H,m), 1.91-2.02(2H,m), 1.80-1.86(1H,m), 1.68-1.74(1H,m), 1.40(3H,s).
实施例48：6-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-((2-氟苯基)乙炔基)-1，5-二氢-4H-吡唑并[3，4-d]嘧啶-4-酮Example 48:6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-3-((2-fluorophenyl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：423.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.19-13.37(1H，brs)，7.61(1H，td，J＝7.2Hz，1.6Hz)，7.46-7.52(1H，m)，7.34(1H，t，J＝8.4Hz)，7.27(1H，td，J＝8.4Hz，0.8Hz)，3.96-4.19(3H，m)，3.77(1H，d，J＝8.8Hz)，3.54(1H，d，J＝8.8Hz)，3.13-3.25(m，3H)，1.64-1.75(2H，m)，1.53-1.62(1H，m)，1.43-1.50(1H，m)，1.14(3H，d，J＝6.0Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 423.20 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=13.19-13.37 (1H, brs), 7.61 (1H, td, J=7.2Hz, 1.6Hz), 7.46-7.52 (1H, m), 7.34 (1H, t, J=8.4Hz), 7.27 (1H, td, J=8.4Hz, 0.8Hz), 3.96-4.19 (3H, m), 3. 77 (1H, d, J = 8.8Hz), 3.54 (1H, d, J = 8.8Hz), 3.13-3.25 (m, 3H), 1.64-1.75 (2H, m), 1.53-1.62 (1H, m), 1.43-1.50 (1H, m), 1.14 (3H, d, J = 6.0Hz).
实施例49：(3S，4S)-8-(9-((2，3-二氟苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 49:(3S,4S)-8-(9-((2,3-difluorophenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：464.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.70(1H，m)，7.48-7.59(3H，m)，7.28-7.33(1H，m)，4.13-4.21(1H，m)，3.81(1H，d，J＝8.4Hz)，3.60-3.72(3H，m)，3.13-3.25(3H，m)，1.90-2.01(2H，m)，1.62-1.80(2H，m)，1.17(3H，d，J＝5.6Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 464.20 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.70 (1H, m), 7.48-7.59 (3H, m), 7.28-7.33 (1H, m), 4.13-4.21 (1H, m), 3.81 (1H, d, J=8.4 Hz), 3.60-3.72 (3H, m), 3.13-3.25 (3H, m), 1.90-2.01 (2H, m), 1.62-1.80 (2H, m), 1.17 (3H, d, J=5.6 Hz).
实施例50：(3S，4S)-8-(9-((3-氯-2-氟苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 50:(3S,4S)-8-(9-((3-chloro-2-fluorophenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：480.17[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.62-7.72(3H，m)，7.52(1H，d，J＝1.6Hz)，7.33(1H，t，J＝7.6Hz)，4.14-4.20(1H，m)，3.82(1H，d，J＝8.8Hz)，3.62-3.72(3H，m)，3.12-3.24(3H，m)，1.91-1.99(2H，m)，1.74-1.81(1H，m)，1.64-1.71(1H，m)，1.17(3H，d，J＝6.0Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 480.17 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.62-7.72 (3H, m), 7.52 (1H, d, J = 1.6Hz), 7.33 (1H, t, J = 7.6Hz), 4.14-4.20 (1H, m), 3.82 (1H, d, J = 8.8Hz), 3.62-3.72 (3H, m ), 3.12-3.24(3H,m), 1.91-1.99(2H,m), 1.74-1.81(1H,m), 1.64-1.71(1H,m), 1.17(3H,d,J=6.0Hz).
实施例51：(3S，4S)-8-(9-((2-氟-3，5-二甲氧基苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 51:(3S,4S)-8-(9-((2-fluoro-3,5-dimethoxyphenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：506.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.69(1H，s)，7.50(1H，s)，6.84(1H，dd，J＝6.8Hz，2.8Hz)，6.70-6.69(1H，m)，4.14-4.19(1H，m)，3.85(3H，s)，3.77-3.81(4H，m)，3.60-3.69(3H，m)，3.12-3.22(4H，m)，1.89-1.99(2H，m)，1.63-1.68(1H，m)，1.64-1.68(1H，m)，1.16(3H，d，J＝6.8Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 506.23 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.69 (1H, s), 7.50 (1H, s), 6.84 (1H, dd, J = 6.8Hz, 2.8Hz), 6.70-6.69 (1H, m), 4.14-4.19 (1H, m), 3.85 (3H, s), 3.77-3.81 (4H) , m), 3.60-3.69 (3H, m), 3.12-3.22 (4H, m), 1.89-1.99 (2H, m), 1.63-1.68 (1H, m), 1.64-1.68 (1H, m), 1.16 (3H, d, J = 6.8Hz).
实施例52：(3S，4S)-8-(9-((2-氟-5-(三氟甲基)苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 52:(3S,4S)-8-(9-((2-fluoro-5-(trifluoromethyl)phenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：514.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝8.04-8.07(1H，m)，7.89-7.94(1H，m)，7.70(1H，s)，7.64(1H，t，J＝8.8Hz)，7.52(1H，s)，4.15-4.21(1H，m)，3.81(1H，d，J＝8.0Hz)，3.61-3.70(3H，m)，3.13-3.23(3H，m)，1.90-2.01(2H，m)，1.73-1.80(1H，m)，1.62-1.70(1H，m)，1.17(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 514.20 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 8.04-8.07 (1H, m), 7.89-7.94 (1H, m), 7.70 (1H, s), 7.64 (1H, t, J = 8.8Hz), 7.52 (1H, s), 4.15-4.21 (1H, m), 3.81 (1H, d, J = 8.0Hz), 3.61-3.70(3H,m), 3.13-3.23(3H,m), 1.90-2.01(2H,m), 1.73-1.80(1H,m), 1.62-1.70(1H,m), 1.17(3H,d,J=6.4Hz).
实施例53：(3S，4S)-3-甲基-8-(9-((3-(三氟甲基)苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 53:(3S,4S)-3-methyl-8-(9-((3-(trifluoromethyl)phenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：496.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.90-7.93(2H，m)，7.83(1H，d，J＝7.6Hz)，7.69-7.74(2H，m)，7.53(1H，s)，4.15-4.21(1H，m)，3.83(1H，d，J＝9.2Hz)，3.63-3.71(3H，m)，3.34(1H，d，J＝5.2Hz)，3.10-3.19(2H，m)，1.92-2.01(2H，m)，1.75-1.79(1H，m)，1.65-1.70(1H，m)，1.18(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 496.21 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.90-7.93 (2H, m), 7.83 (1H, d, J = 7.6Hz), 7.69-7.74 (2H, m), 7.53 (1H, s), 4.15-4.21 (1H, m), 3.83 (1H, d, J = 9.2Hz), 3.63-3. 71 (3H, m), 3.34 (1H, d, J = 5.2Hz), 3.10-3.19 (2H, m), 1.92-2.01 (2H, m), 1.75-1.79 (1H, m), 1.65-1.70 (1H, m), 1.18 (3H, d, J = 6.4Hz).
实施例54：(3S，4S)-8-(9-((2，5-二氟苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 54:(3S,4S)-8-(9-((2,5-difluorophenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：464.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.70(1H，m)，7.48-7.59(2H，m)，7.35-7.44(2H，m)，4.13-4.21(1H，m)，3.82(1H，d，J＝8.4Hz)，3.61-3.72(3H，m)，3.13-3.25(3H，m)，1.91-2.01(2H，m)，1.62-1.81(2H，m)，1.17(3H，d，J＝5.6Hz)。。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 464.20 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.70 (1H, m), 7.48-7.59 (2H, m), 7.35-7.44 (2H, m), 4.13-4.21 (1H, m), 3.82 (1H, d, J=8.4 Hz), 3.61-3.72 (3H, m), 3.13-3.25 (3H, m), 1.91-2.01 (2H, m), 1.62-1.81 (2H, m), 1.17 (3H, d, J=5.6 Hz).
实施例55：(3S，4S)-8-(9-((2-氟-3-(三氟甲基)苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 55:(3S,4S)-8-(9-((2-fluoro-3-(trifluoromethyl)phenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：514.20[M+1]。1H NMR(400MHz，CD3OD)：δ＝8.11(1H，t，J＝6.8Hz)，7.72-7.77(2H，m)，7.52(1H，d，J＝1.2Hz)，7.42(1H，t，J＝8.0Hz)，4.27-4.32(1H，m)，3.96(1H，d，J＝8.8Hz)，3.79-3.89(3H，m)，3.41(1H，d，J＝4.0Hz)，3.17-3.28(2H，m)，1.95-2.09(3H，m)，1.78-1.82(1H，m)，1.30(3H，d，J＝6.8Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 514.20 [M+1].1 H NMR (400MHz, CD3 OD): δ=8.11 (1H, t, J=6.8Hz), 7.72-7.77 (2H, m), 7.52 (1H, d, J=1.2Hz), 7.42 (1H, t, J=8.0Hz), 4.27-4.32 (1H, m), 3.96 (1H, d, J=8.8 Hz), 3.79-3.89 (3H, m), 3.41 (1H, d, J = 4.0Hz), 3.17-3.28 (2H, m), 1.95-2.09 (3H, m), 1.78-1.82 (1H, m), 1.30 (3H, d, J = 6.8Hz).
实施例56：3-(5-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-9-基)-2-氯苯基二甲基氨基甲酸酯Example 56:3-(5-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-9-yl)-2-chlorophenyl dimethylcarbamate
参照实施例36中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：525.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.85(1H，s)，7.64(1H，d，J＝1.6Hz)，7.60(1H，dd，J＝7.2Hz，1.6Hz)，7.45(1H，t，J＝7.6Hz)，7.36-7.39(2H，m)，4.15-4.21(1H，m)，3.84(1H，d，J＝9.2Hz)，3.60-3.72(3H，m)，3.06-3.20(6H，m)，2.91(3H，s)，1.90-1.99(2H，m)，1.74-1.81(1H，m)，1.64-1.70(1H，m)，1.19(3H，d，J＝6.4Hz)。Referring to the method in Example 36, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 525.22 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=13.85 (1H, s), 7.64 (1H, d, J=1.6Hz), 7.60 (1H, dd, J=7.2Hz, 1.6Hz), 7.45 (1H, t, J=7.6Hz), 7.36-7.39 (2H, m), 4.15-4.21 (1H, m), 3.84 (1H, d, J=9.2 Hz), 3.60-3.72 (3H, m), 3.06-3.20 (6H, m), 2.91 (3H, s), 1.90-1.99 (2H, m), 1.74-1.81 (1H, m), 1.64-1.70 (1H, m), 1.19 (3H, d, J = 6.4Hz).
实施例57：3-((5-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-9-基)乙炔基)-2-氟苯腈Example 57:3-((5-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-9-yl)ethynyl)-2-fluorobenzonitrile
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：471.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.99-8.06(2H，m)，7.72(1H，s)，7.48-7.52(2H，m)，4.09-4.15(1H，m)，3.46(1H，d，J＝8.4Hz)，3.55-3.66(3H，m)，3.12-3.25(3H，m)，1.87-1.99(2H，m)，1.62-1.74(2H，m)，1.13(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 471.21 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.99-8.06 (2H, m), 7.72 (1H, s), 7.48-7.52 (2H, m), 4.09-4.15 (1H, m), 3.46 (1H, d, J=8.4 Hz), 3.55-3.66 (3H, m), 3.12-3.25 (3H, m), 1.87-1.99 (2H, m), 1.62-1.74 (2H, m), 1.13 (3H, d, J=6.4 Hz).
实施例58：(3S，4S)-8-(9-(环丙基乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 58:(3S,4S)-8-(9-(cyclopropylethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin -5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：392.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.56-13.84(1H，brs)，7.65(1H，d，J＝0.8Hz)，7.45(1H，d，J＝0.8Hz)，4.05-4.13(1H，m)，3.94-4.02(1H，m)，3.71(1H，d，J＝8.4Hz)，3.46-3.60(3H，m)，3.02-3.23(3H，m)，1.80-1.99(3H，m)，1.64-1.74(1H，m)，1.10(3H，d，J＝6.4Hz)，0.90-0.94(2H，m)，0.75-0.82(2H，m)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 392.22 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 13.56-13.84 (1H, brs), 7.65 (1H, d, J = 0.8Hz), 7.45 (1H, d, J = 0.8Hz), 4.05-4.13 (1H, m), 3.94-4.02 (1H, m), 3.71 (1H, d, J = 8.4 Hz), 3.46-3.60 (3H, m), 3.02-3.23 (3H, m), 1.80-1.99 (3H, m), 1.64-1.74 (1H, m), 1.10 (3H, d, J = 6.4Hz), 0.90-0.94 (2H, m), 0.75-0.82 (2H, m).
实施例59：N-(3-((5-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-9-基)乙炔基)-2-氟苯基)乙酰胺Example 59:N-(3-((5-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-9-yl)ethynyl)-2-fluorophenyl)acetamide
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：503.23[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.98(1H，t，J＝7.6Hz)，7.75(1H，d，J＝1.6Hz)，7.59(1H，t，J＝7.2Hz)，7.50(1H，d，J＝1.6Hz)，7.19(1H，t，J＝8.0Hz)，4.26-4.32(1H，m)，3.95(1H，d，J＝9.2Hz)，3.77-3.85(3H，m)，3.33-3.36(1H，m)，3.14-3.24(2H，m)，2.18(3H，s)，1.99-2.10(2H，m)，1.89-1.95(1H，m)，1.76-1.83(1H，m)，1.29(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 503.23 [M+1].1 H NMR (400 MHz, CD3 OD): δ = 7.98 (1H, t, J = 7.6 Hz), 7.75 (1H, d, J = 1.6 Hz), 7.59 (1H, t, J = 7.2 Hz), 7.50 (1H, d, J = 1.6 Hz), 7.19 (1H, t, J = 8.0 Hz), 4.26-4.32 (1H, m), 3.95 (1H, d, J = 9. 2Hz), 3.77-3.85 (3H, m), 3.33-3.36 (1H, m), 3.14-3.24 (2H, m), 2.18 (3H, s), 1.99-2.10 (2H, m), 1.89-1.95 (1H, m), 1.76-1.83 (1H, m), 1.29 (3H, d, J=6.4Hz).
实施例60：(3S，4S)-8-(9-((3-氨基-2-氟苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 60:(3S,4S)-8-(9-((3-amino-2-fluorophenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：461.22[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.73(1H，d，J＝2.0Hz)，7.49(1H，d，J＝2.0Hz)，7.03-7.07(1H，m)，6.93(1H，t，J＝7.6Hz)，6.88(1H，td，J＝8.0Hz，2.0Hz)，4.26-4.32(1H，m)，3.95(1H，d，J＝9.2Hz)，3.76-3.85(3H，m)，3.35(1H，d，J＝4.4Hz)，3.17-3.25(2H，m)，1.99-2.10(2H，m)，1.88-1.96(1H，m)，1.76-1.83(1H，m)，1.29(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 461.22 [M+1].1 H NMR (400 MHz, CD3 OD): δ=7.73 (1H, d, J=2.0 Hz), 7.49 (1H, d, J=2.0 Hz), 7.03-7.07 (1H, m), 6.93 (1H, t, J=7.6 Hz), 6.88 (1H, td, J=8.0 Hz, 2.0 Hz), 4.26-4.32 (1H, m), 3.95 (1H, m). d, J=9.2Hz), 3.76-3.85 (3H, m), 3.35 (1H, d, J=4.4Hz), 3.17-3.25 (2H, m), 1.99-2.10 (2H, m), 1.88-1.96 (1H, m), 1.76-1.83 (1H, m), 1.29 (3H, d, J=6 .4Hz).
实施例61：5-(5-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-9-基)-3H-螺[苯并呋喃-2，1′-环丙烷]-3-酮Example 61:5-(5-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-9-yl)-3H-spiro[benzofuran-2,1′-cyclopropane]-3-one
参照实施例36中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：486.23[M+1]。1H NMR(400MHz，CD3OD)：δ＝8.93(1H，s)，7.87(1H，d，J＝8.8Hz)，7.73(1H，s)，7.49(1H，s)，7.38(1H，d，J＝8.4Hz)，4.25-4.32(1H，m)，3.98(1H，d，J＝9.2Hz)，3.76-3.88(3H，m)，3.44(1H，d，J＝4.4Hz)，3.11-3.29(2H，m)，2.04-2.11(2H，m)，1.93-1.98(1H，m)，1.74-1.84(3H，m)，1.55-1.58(2H，m)，1.31(3H，d，J＝6.4Hz)。Referring to the method in Example 36, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 486.23 [M+1].1 H NMR (400 MHz, CD3 OD): δ=8.93 (1H, s), 7.87 (1H, d, J=8.8Hz), 7.73 (1H, s), 7.49 (1H, s), 7.38 (1H, d, J=8.4Hz), 4.25-4.32 (1H, m), 3.98 (1H, d, J=9.2Hz), 3.76-3.88 (3H, m ), 3.44 (1H, d, J = 4.4Hz), 3.11-3.29 (2H, m), 2.04-2.11 (2H, m), 1.93-1.98 (1H, m), 1.74-1.84 (3H, m), 1.55-1.58 (2H, m), 1.31 (3H, d, J = 6.4Hz).
实施例62：(3S，4S)-8-(9-((2，3-二氯苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 62:(3S,4S)-8-(9-((2,3-dichlorophenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：496.14[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.61-7.74(3H，m)，7.51(1H，d，J＝1.6Hz)，7.31(1H，t，J＝7.6Hz)，4.11-4.20(1H，m)，3.78(1H，d，J＝8.4Hz)，3.61-3.74(3H，m)，3.10-3.24(3H，m)，1.89-1.99(2H，m)，1.73-1.85(1H，m)，1.64-1.72(1H，m)，1.16(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 496.14 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.61-7.74 (3H, m), 7.51 (1H, d, J = 1.6Hz), 7.31 (1H, t, J = 7.6Hz), 4.11-4.20 (1H, m), 3.78 (1H, d, J = 8.4Hz), 3.61-3.74 (3H, m ), 3.10-3.24(3H,m), 1.89-1.99(2H,m), 1.73-1.85(1H,m), 1.64-1.72(1H,m), 1.16(3H,d,J=6.4Hz).
实施例63：(3S，4S)-8-(9-((2-氟-3-甲氧基苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 63:(3S,4S)-8-(9-((2-fluoro-3-methoxyphenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：476.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.70(1H，m)，7.50(1H，m)，7.16-7.32(3H，m)，4.10-4.22(1H，m)，3.87(3H，s)，3.78(1H，d，J＝8.4Hz)，3.57-3.69(3H，m)，3.12-3.26(3H，m)，1.87-2.01(2H，m)，1.71-1.79(1H，m)，1.62-1.69(1H，m)，1.15(3H，d，J＝5.6Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 476.22 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.70 (1H, m), 7.50 (1H, m), 7.16-7.32 (3H, m), 4.10-4.22 (1H, m), 3.87 (3H, s), 3.78 (1H, d, J=8.4 Hz), 3.57-3.69 (3H, m), 3.12-3.26 (3H, m), 1.87-2.01 (2H, m), 1.71-1.79 (1H, m), 1.62-1.69 (1H, m), 1.15 (3H, d, J=5.6 Hz).
实施例64：(3S，4S)-8-(9-(3-氯-[1，1′-联苯基]-4-基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 64:(3S,4S)-8-(9-(3-chloro-[1,1′-biphenyl]-4-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例36中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：514.21[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.84(1H，s)，7.70-7.72(5H，m)，7.49(2H，t，J＝7.6Hz)，7.38-7.42(1H，m)，7.35(1H，d，J＝1.6Hz)，4.26-4.32(1H，m)，3.97(1H，d，J＝8.8Hz)，3.78-3.87(3H，m)，3.39(1H，d，J＝3.6Hz)，3.17-3.27(2H，m)，1.99-2.12(2H，m)，1.91-1.98(1H，m)，1.78-1.84(1H，m)，1.30(3H，d，J＝6.8Hz)。The target compound can be synthesized by referring to the method in Example 36 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 514.21 [M+1].1 H NMR (400 MHz, CD3 OD): δ=7.84 (1H, s), 7.70-7.72 (5H, m), 7.49 (2H, t, J=7.6Hz), 7.38-7.42 (1H, m), 7.35 (1H, d, J=1.6Hz), 4.26-4.32 (1H, m), 3.97 (1H, d, J=8.8Hz), 3.78 -3.87(3H,m), 3.39(1H,d,J=3.6Hz), 3.17-3.27(2H,m), 1.99-2.12(2H,m), 1.91-1.98(1H,m), 1.78-1.84(1H,m), 1.30(3H,d,J=6.8Hz).
实施例65：2-((3-(5-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-7H-咪唑并[1，2-c]吡唑并[43-e]嘧啶-9-基)-2-氯苯基)硫基)乙酰胺Example 65:2-((3-(5-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-7H-imidazo[1,2-c]pyrazolo[4.3-e]pyrimidin-9-yl)-2-chlorophenyl)thio)acetamide
参照实施例36中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：527.18[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.71-7.77(1H，m)，7.53(1H，d，J＝6.8Hz)，7.36-7.46(3H，m)，4.29-4.35(1H，m)，4.01(1H，d，J＝10.0Hz)，3.81-3.91(3H，m)，3.76(2H，s)，3.52-3.54(1H，m)，3.17-3.24(2H，m)，2.03-2.22(2H，m)，1.95-2.04(1H，m)，1.80-1.86(1H，m)，1.33(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 36 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 527.18 [M+1].1 H NMR (400MHz, CD3 OD): δ = 7.71-7.77 (1H, m), 7.53 (1H, d, J = 6.8Hz), 7.36-7.46 (3H, m), 4.29-4.35 (1H, m), 4.01 (1H, d, J = 10.0Hz), 3.81-3.91 (3H, m), 3.76 (2H, s), 3.52-3.54 (1H, m), 3.17-3.24 (2H, m), 2.03-2.22 (2H, m), 1.95-2.04 (1H, m), 1.80-1.86 (1H, m), 1.33 (3H, d, J = 6.4Hz).
实施例66：3-((5-((3S，4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-9-基)乙炔基)-2-氟苯酚Example 66:3-((5-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-9-yl)ethynyl)-2-fluorophenol
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：462.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝10.14-10.29(1H，brs)，7.68(1H，d，J＝1.2Hz)，7.50(1H，d，J＝1.2Hz)，7.01-7.08(3H，m)，4.16-4.23(1H，m)，3.84(1H，d，J＝9.2Hz)，3.63-3.73(3H，m)，3.40(1H，d，J＝4.4Hz)，3.08-3.19(2H，m)，1.89-2.01(2H，m)，1.76-1.82(1H，m)，1.64-1.72(1H，m)，1.19(3H，d，J＝6.8Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 462.21 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 10.14-10.29 (1H, brs), 7.68 (1H, d, J = 1.2Hz), 7.50 (1H, d, J = 1.2Hz), 7.01-7.08 (3H, m), 4.16-4.23 (1H, m), 3.84 (1H, d, J = 9.2 Hz), 3.63-3.73 (3H, m), 3.40 (1H, d, J = 4.4Hz), 3.08-3.19 (2H, m), 1.89-2.01 (2H, m), 1.76-1.82 (1H, m), 1.64-1.72 (1H, m), 1.19 (3H, d, J = 6.8Hz).
实施例67：(3S，4S)-8-(9-((2，2-二氟苯并[d][1，3]二氧杂环戊烯-4-基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 67:(3S,4S)-8-(9-((2,2-difluorobenzo[d][1,3]dioxol-4-yl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：508.19[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.74(1H，d，J＝1.6Hz)，7.55(1H，d，J＝8.0Hz)，7.51(1H，d，J＝1.6Hz)，7.26(1H，d，J＝8.0Hz)，7.20(1H，t，J＝8.0Hz)，4.26-4.32(1H，m)，3.95(1H，d，J＝8.8Hz)，3.77-3.88(3H，m)，3.39(1H，d，J＝4.4Hz)，3.16-3.25(2H，m)，2.01-2.02(2H，m)，1.89-1.96(1H，m)，1.76-1.83(1H，m)，1.29(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 508.19 [M+1].1 H NMR (400 MHz, CD3 OD): δ=7.74 (1H, d, J=1.6Hz), 7.55 (1H, d, J=8.0Hz), 7.51 (1H, d, J=1.6Hz), 7.26 (1H, d, J=8.0Hz), 7.20 (1H, t, J=8.0Hz), 4.26-4.32 (1H, m), 3.95 (1H, d, J=8.8Hz), 3.77-3.88 (3H, m), 3.39 (1H, d, J=4.4Hz), 3.16-3.25 (2H, m), 2.01-2.02 (2H, m), 1.89-1.96 (1H, m), 1.76-1.83 (1H, m), 1.29 (3H, d, J=6.4Hz ).
实施例68：(3S，4S)-8-(9-((2-氟-3-甲基苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 68:(3S,4S)-8-(9-((2-fluoro-3-methylphenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：460.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.80(1H，m)，7.62-7.70(2H，m)，7.52(1H，d，J＝1.6Hz)，7.41-7.20(1H，m)，4.13-4.20(1H，m)，3.81(1H，d，J＝8.8Hz)，3.60-3.72(3H，m)，3.27-3.29(1H，m)，3.12-3.21(2H，m)，2.35(3H，s)，1.89-1.97(2H，m)，1.61-1.79(2H，m)，1.18(3H，d，J＝6.4Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 460.23 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.80 (1H, m), 7.62-7.70 (2H, m), 7.52 (1H, d, J = 1.6Hz), 7.41-7.20 (1H, m), 4.13-4.20 (1H, m), 3.81 (1H, d, J = 8.8Hz), 3.60-3 .72(3H,m), 3.27-3.29(1H,m), 3.12-3.21(2H,m), 2.35(3H,s), 1.89-1.97(2H,m), 1.61-1.79(2H,m), 1.18(3H,d,J=6.4Hz).
实施例69：(3S，4S)-8-(9-((2-氟-3-异丙基苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 69:(3S,4S)-8-(9-((2-fluoro-3-isopropylphenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：504.25[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.70(1H，d，J＝1.4Hz)，7.51(1H，d，J＝1.4Hz)，7.18-7.32(3H，m)，4.55(1H，m)，4.10-4.21(1H，m)，3.77(1H，d，J＝8.4Hz)，3.57-3.68(3H，m)，3.10-3.25(3H，m)，1.87-2.02(2H，m)，1.70-1.79(1H，m)，1.60-1.70(1H，m)，1.32(6H，d，J＝6.8Hz)，1.16(3H，d，J＝5.6Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 504.25 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.70 (1H, d, J = 1.4Hz), 7.51 (1H, d, J = 1.4Hz), 7.18-7.32 (3H, m), 4.10-4.21 (1H, m), 3.77 (1H, d, J = 8.4Hz), 3.57- 3.68(3H,m), 3.10-3.25(3H,m), 1.87-2.02(2H,m), 1.70-1.79(1H,m), 1.60-1.70(1H,m), 1.32(6H,d,J=6.8Hz), 1.16(3H,d,J=5.6Hz).
实施例70：(3S，4S)-8-(9-((3-(二氟甲氧基)-2-氟苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 70:(3S,4S)-8-(9-((3-(difluoromethoxy)-2-fluorophenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：512.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.76(1H，d，J＝1.6Hz)，7.51(1H，d，J＝1.6Hz)，7.38(1H，t，J＝56.0Hz)，7.21-7.35(3H，m)，4.12-4.22(1H，m)，3.79(1H，d，J＝8.4Hz)，3.58-3.68(3H，m)，3.12-3.26(3H，m)，1.88-2.01(2H，m)，1.70-1.80(1H，m)，1.61-1.70(1H，m)，1.18(3H，d，J＝5.6Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 512.20 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ=7.76 (1H, d, J=1.6Hz), 7.51 (1H, d, J=1.6Hz), 7.38 (1H, t, J=56.0Hz), 7.21-7.35 (3H, m), 4.12-4.22 (1H, m), 3.79 (1H, d, J=8 .4Hz), 3.58-3.68(3H,m), 3.12-3.26(3H,m), 1.88-2.01(2H,m), 1.70-1.80(1H,m), 1.61-1.70(1H,m), 1.18(3H,d,J=5.6Hz).
实施例71：(3S，4S)-8-(9-((3-(二氟甲基)-2-氟苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 71:(3S,4S)-8-(9-((3-(difluoromethyl)-2-fluorophenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：496.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.84-7.88(1H，m)，7.70-7.74(2H，m)，7.51(1H，d，J＝1.6Hz)，7.41-7.18(1H，m)，7.28(1H，t，J＝54.0Hz)，4.13-4.19(1H，m)，3.80(1H，d，J＝8.8Hz)，3.60-3.71(3H，m)，3.27-3.28(1H，m)，3.12-3.21(2H，m)，1.90-1.99(2H，m)，1.61-1.78(2H，m)，1.17(3H，d，J＝6.8Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 496.21 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.84-7.88 (1H, m), 7.70-7.74 (2H, m), 7.51 (1H, d, J = 1.6Hz), 7.41-7.18 (1H, m), 7.28 (1H, t, J = 54.0Hz), 4.13-4.19 (1H, m), 3 .80 (1H, d, J = 8.8Hz), 3.60-3.71 (3H, m), 3.27-3.28 (1H, m), 3.12-3.21 (2H, m), 1.90-1.99 (2H, m), 1.61-1.78 (2H, m), 1.17 (3H, d, J = 6.8Hz).
实施例72：(3S，4S)-3-甲基-8-(9-((2，3，4-三氟苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 72:(3S,4S)-3-methyl-8-(9-((2,3,4-trifluorophenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：482.19[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.70(1H，d，J＝1.2Hz)，7.52-7.59(1H，m)，7.51(1H，d，J＝1.2Hz)，7.40-7.47(1H，m)，4.13-4.19(1H，m)，3.80(1H，d，J＝8.8Hz)，3.60-3.71(3H，m)，3.28-3.29(1H，m)，3.12-3.21(2H，m)，1.90-1.99(2H，m)，1.72-1.79(1H，m)，1.64-1.70(1H，m)，1.16(3H，d，J＝6.8Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 482.19 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.70 (1H, d, J = 1.2Hz), 7.52-7.59 (1H, m), 7.51 (1H, d, J = 1.2Hz), 7.40-7.47 (1H, m), 4.13-4.19 (1H, m), 3.80 (1H, d, J = 8.8Hz), 3.60-3.71(3H,m), 3.28-3.29(1H,m), 3.12-3.21(2H,m), 1.90-1.99(2H,m), 1.72-1.79(1H,m), 1.64-1.70(1H,m), 1.16(3H,d,J=6.8Hz).
实施例73：(3S，4S)-8-(9-((3-氯-2，5-二氟苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 73:(3S,4S)-8-(9-((3-chloro-2,5-difluorophenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：498.16[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.85-8.11(1H，brs)，7.77-7.83(1H，m)，7.74(0.5H，d，J＝1.6Hz)，7.70(0.5H，d，J＝1.6Hz)，7.59-7.63(1H，m)，7.52(0.5H，d，J＝1.6Hz)，7.49(0.5H，d，J＝1.6Hz)，4.18-4.28(1H，m)，3.66-3.88(4H，m)，3.43(1H，d，J＝4.4Hz)，3.11-3.21(2H，m)，1.91-2.02(2H，m)，1.66-1.86(2H，m)，1.14-1.28(3H，m)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 498.16 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.85-8.11 (1H, brs), 7.77-7.83 (1H, m), 7.74 (0.5H, d, J=1.6 Hz), 7.70 (0.5H, d, J=1.6 Hz), 7.59-7.63 (1H, m), 7.52 (0.5H, d, J=1.6 Hz), 7.49 (0.5H, d, J=1.6 Hz). , d, J = 1.6Hz), 4.18-4.28 (1H, m), 3.66-3.88 (4H, m), 3.43 (1H, d, J = 4.4Hz), 3.11-3.21 (2H, m), 1.91-2.02 (2H, m), 1.66-1.86 (2H, m), 1.14-1.28 (3H ,m).
实施例74：(3S，4S)-8-(9-((2-氟-3-(2，2，2-三氟乙氧基)苯基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 74:(3S,4S)-8-(9-((2-fluoro-3-(2,2,2-trifluoroethoxy)phenyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：544.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.73(1H，d，J＝1.6Hz)，7.50(1H，d，J＝1.6Hz)，7.20-7.34(3H，m)，4.46(2H，m)，4.10-4.22(1H，m)，3.78(1H，d，J＝8.4Hz)，3.57-3.68(3H，m)，3.12-3.25(3H，m)，1.87-2.01(2H，m)，1.70-1.79(1H，m)，1.62-1.70(1H，m)，1.17(3H，d，J＝5.6Hz)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 544.21 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 7.73 (1H, d, J = 1.6Hz), 7.50 (1H, d, J = 1.6Hz), 7.20-7.34 (3H, m), 4.46 (2H, m), 4.10-4.22 (1H, m), 3.78 (1H, d, J = 8.4Hz), 3.5 7-3.68 (3H, m), 3.12-3.25 (3H, m), 1.87-2.01 (2H, m), 1.70-1.79 (1H, m), 1.62-1.70 (1H, m), 1.17 (3H, d, J=5.6Hz).
实施例75：(3S，4S)-8-(9-(33-二甲基丁-1-炔-1-基)-7H-咪唑并[1，2-c]吡唑并[4.3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 75:(3S,4S)-8-(9-(3,3-dimethylbut-1-yn-1-yl)-7H-imidazo[1,2-c]pyrazolo[4.3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：408.25[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.65(1H，d，J＝1.2Hz)，7.45(1H，d，J＝1.2Hz)，4.04-4.16(1H，m)，3.70(1H，d，J＝8.6Hz)，3.40-3.60(3H，m)，3.00-3.24(3H，m)，1.81-1.99(3H，m)，1.64-1.75(1H，d，J＝6.8Hz)，1.27(9H，s)，1.08(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 408.25 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.65 (1H, d, J=1.2 Hz), 7.45 (1H, d, J=1.2 Hz), 4.04-4.16 (1H, m), 3.70 (1H, d, J=8.6 Hz), 3.40-3.60 (3H, m), 3.00-3.24 (3H, m), 1.81-1.99 (3H, m), 1.64-1.75 (1H, d, J=6.8 Hz), 1.27 (9H, s), 1.08 (3H, d, J=6.4 Hz).
实施例76：(3S，4S)-8-(9-(3-氯-3-甲基丁-1-炔-1-基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 76:(3S,4S)-8-(9-(3-chloro-3-methylbut-1-yn-1-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：428.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.46-13.81(1H，brs)，7.68(1H，d，J＝1.2Hz)，7.50(1H，d，J＝1.2Hz)，4.02-4.17(1H，m)，3.71(1H，m)，3.42-3.61(3H，m)，2.99-3.25(3H，m)，1.80-2.00(9H，m)，1.64-1.79(1H，d，J＝6.8Hz)，1.10(3H，d，J＝6.8Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 428.20 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=13.46-13.81 (1H, brs), 7.68 (1H, d, J=1.2 Hz), 7.50 (1H, d, J=1.2 Hz), 4.02-4.17 (1H, m), 3.71 (1H, m), 3.42-3.61 (3H, m), 2.99-3.25 (3H, m), 1.80-2.00 (9H, m), 1.64-1.79 (1H, d, J=6.8 Hz), 1.10 (3H, d, J=6.8 Hz).
实施例77：(3S，4S)-3-甲基-8-(9-(3-甲基丁-1-炔-1-基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 77:(3S,4S)-3-methyl-8-(9-(3-methylbut-1-yn-1-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：394.24[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.65(1H，d，J＝1.2Hz)，7.45(1H，d，J＝1.2Hz)，4.04-4.16(1H，m)，3.70(1H，d，J＝8.6Hz)，3.40-3.60(3H，m)，2.98-3.24(4H，m)，1.81-1.99(3H，m)，1.64-1.75(1H，m)，1.28(6H，d，J＝6.8Hz)，1.07(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 394.24 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.65 (1H, d, J=1.2 Hz), 7.45 (1H, d, J=1.2 Hz), 4.04-4.16 (1H, m), 3.70 (1H, d, J=8.6 Hz), 3.40-3.60 (3H, m), 2.98-3.24 (4H, m), 1.81-1.99 (3H, m), 1.64-1.75 (1H, m), 1.28 (6H, d, J=6.8 Hz), 1.07 (3H, d, J=6.4 Hz).
实施例78：(3S，4S)-3-甲基-8-(9-((1-甲基环丙基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 78:(3S,4S)-3-methyl-8-(9-((1-methylcyclopropyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：406.24[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.58-13.84(1H，brs)，7.63(1H，d，J＝0.8Hz)，7.43(1H，d，J＝0.8Hz)，4.05-4.14(1H，m)，3.72(1H，d，J＝8.6Hz)，3.46-3.60(3H，m)，3.02-3.23(3H，m)，1.80-1.99(3H，m)，1.64-1.74(1H，m)，1.26(3H，s)，1.10(3H，d，J＝6.4Hz)，0.92-0.95(2H，m)，0.72-0.80(2H，m)。The target compound can be synthesized by referring to the method in Example 1 using appropriate starting materials and intermediates. MS m/z [LC-MS]: 406.24 [M+1].1 H NMR (400MHz, DMSO-d6 ): δ = 13.58-13.84 (1H, brs), 7.63 (1H, d, J = 0.8Hz), 7.43 (1H, d, J = 0.8Hz), 4.05-4.14 (1H, m), 3.72 (1H, d, J = 8.6Hz), 3.46-3.60 (3H, m), 3.02-3.23(3H,m), 1.80-1.99(3H,m), 1.64-1.74(1H,m), 1.26(3H,s), 1.10(3H,d,J=6.4Hz), 0.92-0.95(2H,m), 0.72-0.80(2H,m).
实施例79：(3S，4S)-8-(9-((2，2-二氟环丙基)乙炔基)-7H-咪唑并[1，2-c]吡唑并[4，3-e]嘧啶-5-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺Example 79:(3S,4S)-8-(9-((2,2-difluorocyclopropyl)ethynyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine
参照实施例1中的方法，采用合适的起始原料和中间体可以合成得到目标化合物。MS m/z[LC-MS]：428.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.68(1H，d，J＝1.2Hz)，7.48(1H，d，J＝1.2Hz)，4.05-4.17(2H，m)，3.71(1H，d，J＝8.6Hz)，3.46-3.60(3H，m)，3.02-3.23(3H，m)，1.80-1.99(3H，m)，1.64-1.78(2H，m)，1.52(1H，m)，1.12(3H，d，J＝6.4Hz)。Referring to the method in Example 1, the target compound can be synthesized by using appropriate starting materials and intermediates. MS m/z [LC-MS]: 428.20 [M+1].1 H NMR (400 MHz, DMSO-d6 ): δ=7.68 (1H, d, J=1.2 Hz), 7.48 (1H, d, J=1.2 Hz), 4.05-4.17 (2H, m), 3.71 (1H, d, J=8.6 Hz), 3.46-3.60 (3H, m), 3.02-3.23 (3H, m), 1.80-1.99 (3H, m), 1.64-1.78 (2H, m), 1.52 (1H, m), 1.12 (3H, d, J=6.4 Hz).
化合物对SHP2的体外酶学活性抑制作用的测定Determination of the inhibitory effect of compounds on SHP2 enzymatic activity in vitro
本专利中SHP2的酶学活性检测采用快速荧光法进行，使用DiFMUP作为替代底物进行反应并且优化建立了高通量的筛选平台。化合物对SHP2的抑制活性的检测在此平台进行操作。具体方法如下：将终浓度1nM的SHP2与2.5μM的二磷酸化的IRS1肽段(序列：H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide)混合物在23℃预孵育30分钟。将化合物从0.2mM开始用100％DMSO进行5倍的梯度稀释(共7个浓度)，每个浓度取2μL的化合物加入48μL的反应缓冲液(60mM HEPES，pH 7.2，75mM NaCl，75mM KCl，1mM EDTA，0.05％Tween 20，5mM DTT)中进行稀释混匀。取5μL稀释后的化合物加入黑色384孔板中(OptiPlate-384，货号6007270，购买于PerkinElmer)，然后加入10μL的预孵育的SHP2和IRS1肽段混合液，离心混匀，23℃孵育30分钟。加入5μL替代底物DiFMUP(终浓度50μM，货号D6567，购买于Invitrogen)加入反应中并在23℃下孵育60分钟。然后通过加入5μL 160μMbpV(Phen)溶液(SC-22137，购买于Santa)终止反应。反应终止后立即使用酶标仪(Perkin-Elmer)分别在340nm和450nm的激发和发射波长检测测荧光信号，数据使用GraphPad Prism软件计算得到该化合物的IC50值。In this patent, the enzymatic activity detection of SHP2 is carried out by rapid fluorescence method, DiFMUP is used as an alternative substrate for reaction and a high-throughput screening platform is optimized and established. The detection of the inhibitory activity of the compound on SHP2 is operated on this platform. The specific method is as follows: a mixture of SHP2 with a final concentration of 1nM and a diphosphorylated IRS1 peptide (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) is pre-incubated at 23°C for 30 minutes. The compound is diluted 5 times with 100% DMSO starting from 0.2mM (7 concentrations in total), and 2μL of the compound is added to 48μL of reaction buffer (60mM HEPES, pH 7.2, 75mM NaCl, 75mM KCl, 1mM EDTA, 0.05% Tween 20, 5mM DTT) for dilution and mixing. 5 μL of the diluted compound was added to a black 384-well plate (OptiPlate-384, Catalog No. 6007270, purchased from PerkinElmer), and then 10 μL of the pre-incubated SHP2 and IRS1 peptide mixture was added, centrifuged and mixed, and incubated at 23°C for 30 minutes. 5 μL of the alternative substrate DiFMUP (final concentration 50 μM, Catalog No. D6567, purchased from Invitrogen) was added to the reaction and incubated at 23°C for 60 minutes. The reaction was then terminated by adding 5 μL of 160 μM bpV (Phen) solution (SC-22137, purchased from Santa). Immediately after the reaction was terminated, the fluorescence signal was detected using a microplate reader (Perkin-Elmer) at excitation and emission wavelengths of 340 nm and 450 nm, respectively, and the data were calculated using GraphPad Prism software to obtain the IC50 value of the compound.
化合物对SHP2阳性细胞增殖抑制作用的测定Determination of the inhibitory effect of compounds on the proliferation of SHP2 positive cells
人非小细胞肺癌细胞系NCI-H358细胞使用RPMI-1640培养基(货号C11875500BT，购买于Biological Industries)加10％的胎牛血清(FBS，货号04-001-1ACS，购买于Biological Industries，BI)和1％青霉素/链霉素双抗(P/S，货号15070-063，购买于Cibco)进行培养，培养条件为37℃，5％CO2。进行化合物检测的前一天，将NCI-H358细胞以2000个细胞/195μL/孔的浓度铺于196孔板(货号3917，购买于Corning)中。24小时后将化合物从10mM开始用100％DMSO进行3倍的梯度稀释(共10个浓度)，然后每个浓度取2μL的化合物加入48μL的不合血清和双抗的培养基中进行稀释。稀释后的化合物每个浓度取5μL加入铺好的细胞悬液中，将化合物与细胞在细胞培养箱中共孵育72小时(3天)，吸尽培养基后加入25μL的Cell-Titer Glo(G7570，购买于Promega)试剂再次孵育5-10分钟。之后在Envision上读取荧光值，数据使用GraphPad Prism软件计算得到该化合物对细胞增殖的抑制的IC50值。Human non-small cell lung cancer cell line NCI-H358 cells were cultured using RPMI-1640 medium (Cat. No. C11875500BT, purchased from Biological Industries) plus 10% fetal bovine serum (FBS, Cat. No. 04-001-1ACS, purchased from Biological Industries, BI) and 1% penicillin/streptomycin dual antibody (P/S, Cat. No. 15070-063, purchased from Cibco), and the culture conditions were 37°C, 5% CO2. One day before the compound detection, NCI-H358 cells were plated in a 196-well plate (Cat. No. 3917, purchased from Corning) at a concentration of 2000 cells/195 μL/well. After 24 hours, the compound was diluted 3 times from 10 mM with 100% DMSO (a total of 10 concentrations), and then 2 μL of the compound was taken at each concentration and added to 48 μL of serum-free and dual antibody-free culture medium for dilution. 5 μL of each diluted compound concentration was added to the cell suspension, and the compound and cells were co-incubated in a cell culture incubator for 72 hours (3 days). After the culture medium was completely aspirated, 25 μL of Cell-Titer Glo (G7570, purchased from Promega) reagent was added and incubated again for 5-10 minutes. The fluorescence value was then read on Envision, and the data were calculated using GraphPad Prism software to obtain the IC50 value of the compound's inhibition of cell proliferation.
人急性成髓细胞白血病细胞系Kasumi-1细胞使用RPMI-1640培养基(货号C11875500BT，购买于Biological Industries)加20％的胎牛血清(FBS，货号04-001-1ACS，购买于Biological Industries，BI)和1％青霉素/链霉素双抗(P/S，货号15070-063，购买于Gibco)进行培养，培养条件为37℃，5％CO2。进行化合物检测的前一天，将Kasumi-1细胞细胞以3000个细胞/195μL/孔的浓度铺于196孔板(货号3599，购买于Corning)中。24小时后将化合物从10mM开始用100％DMSO进行3倍的梯度稀释(共10个浓度)，然后每个浓度取2μL的化合物加入48μL的不含血清和双抗的培养基中进行稀释。稀释后的化合物每个浓度取5μL加入铺好的细胞悬液中，将化合物与细胞在细胞培养箱中共孵育72小时(3天)，加入35μL的Cell-Titer Blue(G8082，购买于Promega)试剂再次孵育4小时。之后在Flexstation III上读取荧光值(560nm激发，590nm检测)，数据使用GraphPad Prism软件计算得到该化合物对细胞增殖的抑制的IC50值。Kasumi-1 cells, a human acute myeloblastic leukemia cell line, were cultured using RPMI-1640 medium (Cat. No. C11875500BT, purchased from Biological Industries) plus 20% fetal bovine serum (FBS, Cat. No. 04-001-1ACS, purchased from Biological Industries, BI) and 1% penicillin/streptomycin double antibody (P/S, Cat. No. 15070-063, purchased from Gibco) at 37°C and 5% CO2. One day before the compound detection, Kasumi-1 cells were plated in a 196-well plate (Cat. No. 3599, purchased from Corning) at a concentration of 3000 cells/195 μL/well. After 24 hours, the compound was diluted 3-fold from 10 mM with 100% DMSO (a total of 10 concentrations), and then 2 μL of the compound was added to 48 μL of medium without serum and double antibody for dilution at each concentration. 5 μL of each diluted compound concentration was added to the cell suspension, the compound and cells were co-incubated in a cell culture incubator for 72 hours (3 days), 35 μL of Cell-Titer Blue (G8082, purchased from Promega) reagent was added and incubated again for 4 hours. After that, the fluorescence value was read on Flexstation III (560 nm excitation, 590 nm detection), and the data were calculated using GraphPad Prism software to obtain the IC50 value of the compound's inhibition of cell proliferation.
表1实施例化合物对SHP2酶及细胞增殖抑制活性Table 1 Inhibitory activity of the compounds in the examples on SHP2 enzyme and cell proliferation
表1的试验数据表明，本发明提供的化合物具有很好的SHP2激酶抑制活性，同时对SHP2阳性表达细胞的增殖也有很好的抑制活性。The test data in Table 1 show that the compounds provided by the present invention have good SHP2 kinase inhibitory activity and also have good inhibitory activity on the proliferation of SHP2 positive expression cells.
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