WO2017198049A1

WO2017198049A1 - Pyrazolopyrimidine derivative as btk inhibitor and preparation method and pharmaceutical composition thereof

Info

Publication number: WO2017198049A1
Application number: PCT/CN2017/081905
Authority: WO
Inventors: 吴予川
Original assignee: 浙江予川医药科技有限公司
Priority date: 2016-05-16
Filing date: 2017-04-25
Publication date: 2017-11-23
Also published as: CN107383013B; CN107383013A; CN111153906B; CN111153906A

Abstract

Disclosed is a novel pyrazolopyrimidine compound as shown in formula (I), and a stereoisomer, or a pharmaceutically acceptable salt, or a solvate, or a prodrug thereof. In addition, also disclosed are a preparation method for the compound, a pharmaceutical composition and use thereof.

Description

作为BTK抑制剂的吡唑并嘧啶衍生物及其制备方法和药物组合物Pyrazolopyrimidine derivative as BTK inhibitor, preparation method thereof and pharmaceutical composition

技术领域Technical field

本发明实施方案属于药物化学技术领域，具体地说，涉及一种新型高效、选择性好、具有良好的药代动力学性质的、作为BTK抑制剂的吡唑并嘧啶类衍生物及其制备方法和药物组合物。The present invention belongs to the technical field of medicinal chemistry, and in particular to a novel pyrazole pyrimidine derivative as a BTK inhibitor with high efficiency, good selectivity and good pharmacokinetic properties and preparation method thereof And pharmaceutical compositions.

背景background

蛋白激酶是人体内生物酶中的最大家族，包括超500种蛋白质。特别地，对于酪氨酸激酶，其酪氨酸残基上的酚官能团能被磷酸化，从而发挥重要的生物信号传导的作用。酪氨酸激酶家族拥有控制细胞生长、迁移和分化的成员。异常的激酶活性己经被阐明了与许多人体疾病密切相关，这些疾病包括癌症、自身免疫疾病和炎性疾病。Protein kinases are the largest family of biological enzymes in the human body, including over 500 proteins. In particular, for tyrosine kinases, the phenolic function on the tyrosine residue can be phosphorylated to exert important biosignaling effects. The tyrosine kinase family has members that control cell growth, migration, and differentiation. Abnormal kinase activity has been elucidated in close association with many human diseases, including cancer, autoimmune diseases, and inflammatory diseases.

布鲁顿酪氨酸激酶(BTK)是一种细胞质非受体酪氨酸激酶，属于TEC激酶家族(共有5个成员BTK，TEC，ITK，TXK，BMX)一员。BTK基因位于X-染色体的Xq21.33-Xq22，共有19外显子，跨越37.5kb基因组DNA。Bruton's tyrosine kinase (BTK) is a cytoplasmic non-receptor tyrosine kinase belonging to the TEC kinase family (a total of five members BTK, TEC, ITK, TXK, BMX). The BTK gene is located on Xq21.33-Xq22 of the X-chromosome and shares 19 exons spanning 37.5 kb of genomic DNA.

除了T细胞和浆细胞外，BTK表达在几乎所有造血细胞上，尤其在B淋巴细胞发生，分化，信号和生存中发挥必不可少的作用。B细胞是通过B细胞受体(BCR)被活化的，而BTK在BCR信号通路中起到了决定性的作用。B细胞上的BCR被活化后，会引起BTK的激活，然后导致下游的磷脂酶C(PLC)浓度增加，并激活IP3和DAG信号通路。这一信号通路可以促进细胞的增殖、粘附和存活。BTK基因突变会导致一种罕见的遗传性B细胞特异性免疫缺陷疾病，被称为X-连锁无丙种球蛋白血症(X-Iinked agammaglobulinemia，XLA)。在这种疾病中，BTK的功能被抑制，从而导致了B细胞的产生或成熟受阻。患有XLA疾病的男性，体内基本没有B细胞，循环抗体也很少，容易出现严重甚至致命的感染。这有力证明了BTK在B细胞的生长和分化中起着极其重要的作用。 In addition to T cells and plasma cells, BTK expression plays an essential role in almost all hematopoietic cells, especially in the development, differentiation, signaling and survival of B lymphocytes. B cells are activated by the B cell receptor (BCR), and BTK plays a decisive role in the BCR signaling pathway. Activation of BCR on B cells causes activation of BTK, which in turn leads to an increase in downstream phospholipase C (PLC) concentration and activates the IP3 and DAG signaling pathways. This signaling pathway promotes cell proliferation, adhesion and survival. Mutations in the BTK gene result in a rare hereditary B cell-specific immunodeficiency disease known as X-Iinked agammaglobulinemia (XLA). In this disease, the function of BTK is inhibited, resulting in the production or maturation of B cells. Men with XLA disease have almost no B cells in their bodies, and there are few circulating antibodies, which are prone to serious or even fatal infections. This strongly proves that BTK plays an extremely important role in the growth and differentiation of B cells.

小分子BTK抑制剂能与BTK结合，抑制BTK自身磷酸化，阻止BTK的激活。这能阻断BCR通路的信号传导，抑制B淋巴瘤细胞的增殖，破坏瘤细胞的粘附，从而促进瘤细胞的凋亡。并诱导细胞凋亡。这使BTK在B细胞有关的癌症中成为引人注目的药物靶点，尤其是对于B细胞淋巴瘤和白血病，比如非霍奇金淋巴瘤(NHL)、慢性淋巴细胞白血病(CLL)、和抗复发性或难治性套细胞淋巴瘤(MCL)等。Small molecule BTK inhibitors bind to BTK, inhibit BTK autophosphorylation, and prevent BTK activation. This can block the signal transduction of the BCR pathway, inhibit the proliferation of B lymphoma cells, destroy the adhesion of tumor cells, and promote the apoptosis of tumor cells. And induce apoptosis. This makes BTK a compelling drug target in B cell-associated cancers, especially for B-cell lymphomas and leukemias such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), and Recurrent or refractory mantle cell lymphoma (MCL) and the like.

BTK抑制剂除了可以对抗B细胞淋巴瘤和白血病，还可以抑制B细胞自身抗体和细胞因子的产生。在自身免疫性疾病中，B细胞呈递自身抗原，促进T细胞活化分泌致炎症因，既造成组织损伤，同时又激活B细胞产生大量抗体，触发自身免疫反应。T和B细胞相互作用形成反馈调节链，导致自身免疫反应失控，加重组织病理损伤。所以，BTK可以作为自身免疫性疾病，比如类风湿性关节炎、***性红斑狼疮(SLE)、过敏性疾病(例如食道炎等疾病)的药物靶点。In addition to fighting against B-cell lymphoma and leukemia, BTK inhibitors also inhibit B cell autoantibodies and cytokine production. In autoimmune diseases, B cells present autoantigens, promote the activation and secretion of T cells, cause inflammatory factors, cause tissue damage, and activate B cells to produce a large number of antibodies, triggering autoimmune responses. The interaction of T and B cells forms a feedback regulatory chain, leading to uncontrolled autoimmune response and aggravation of histopathological damage. Therefore, BTK can be used as a drug target for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and allergic diseases (such as diseases such as esophagitis).

此外也有报道BTK抑制剂可与化疗药或免疫检查点抑制剂联用，在临床试验中对多种实体瘤表现出较好的治疗效果。In addition, BTK inhibitors have been reported to be useful in combination with chemotherapeutic agents or immunological checkpoint inhibitors, and have shown superior therapeutic effects in a variety of solid tumors in clinical trials.

在目前已上市的药物中，依鲁替尼是由Pharmacyclics和强生公司联合开发的一种不可逆BTK抑制剂，己分别于2013年11月和2014年2月获得FDA批准，用于治疗套细胞淋巴瘤(MCL)和慢性淋巴性白血病(CLL)。不可逆抑制剂，是以比较牢固的共价键(covalent bond)与酶蛋白中的基团结合的一种化学制剂，不可逆抑制剂通常可以使酶失去活性,从而发挥其独特的高生物学活性。依鲁替尼被FDA定为“突破性”新药，它通过与BTK中的半胱氨酸的巯基发生反应，并形成共价键，使BTK酶失活而发挥疗效。然而，依鲁替尼在给药过程中，易被代谢(被代谢酶氧化代谢成双羟化产物或者被其他含巯基的酶、半胱氨酸、谷胱甘肽等进攻而失活)而影响药效。其临床给药剂量达到了560mg每天，而使病人负担加重。此外，依鲁替尼对除BTK外的一些激酶也有一定的抑制作用，尤其是对EGFR的抑制可导致较严重的皮疹、腹泻等不良反应。因此，本领域仍需发展新一类更为高效、选择性好，良好的药代动力学性质的BTK抑制剂用于相关疾病的治疗。 Among the currently marketed drugs, Ibrutinib is an irreversible BTK inhibitor developed by Pharmacyclics and Johnson & Johnson, and was approved by the FDA in November 2013 and February 2014 for the treatment of mantle cell lymphocytes. Tumor (MCL) and chronic lymphocytic leukemia (CLL). An irreversible inhibitor is a chemical that binds a relatively strong covalent bond to a group in an enzyme protein. An irreversible inhibitor can usually inactivate an enzyme to exert its unique high biological activity. Ibrutinib has been designated by the FDA as a "breakthrough" new drug that works by reacting with the thiol group of cysteine in BTK and forming a covalent bond that inactivates the BTK enzyme. However, ibrutinib is easily metabolized during metabolism (digested by metabolic enzymes to be dihydroxylated or inactivated by other thiol-containing enzymes, cysteine, glutathione, etc.) Affect the efficacy. The clinically administered dose reached 560 mg per day, which increased the burden on the patient. In addition, Ibrutinib also has a certain inhibitory effect on some kinases other than BTK, especially the inhibition of EGFR can lead to more serious rash, diarrhea and other adverse reactions. Therefore, there is still a need in the art to develop a new class of BTK inhibitors that are more efficient, selective, and have good pharmacokinetic properties for the treatment of related diseases.

发明概述Summary of invention

以下是对本申请详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。The following is a summary of the subject matter described in detail herein. This Summary is not intended to limit the scope of the claims.

本发明人研发了一种新型吡唑并嘧啶类衍生物，这些新化合物是蛋白激酶BTK的有效、安全、选择性高的抑制剂。The present inventors have developed a novel pyrazolopyrimidine derivative which is an effective, safe and highly selective inhibitor of protein kinase BTK.

本发明实施方案提供一种新型吡唑并嘧啶类衍生物。它是一种新的共价键抑制剂，通过改变其和半胱氨酸反应率，来改善与靶标的亲和性，以提高疗效，选择性和安全性。Embodiments of the invention provide a novel pyrazolopyrimidine derivative. It is a new covalent bond inhibitor that improves its affinity with the target by altering its reactivity with cysteine to improve efficacy, selectivity and safety.

本发明实施方案还提供上述衍生物的制备方法。Embodiments of the invention also provide methods of preparing the above derivatives.

本发明实施方案还提供含有上述衍生物的药物组合物。Embodiments of the invention also provide pharmaceutical compositions containing the above derivatives.

本发明实施方案还提供上述衍生物的用途。Embodiments of the invention also provide the use of the above derivatives.

具体地说，第一方面，在本发明的实施方案中，本发明实施方案提供了一种新型吡唑并嘧啶化合物，如式(I)所示，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：Specifically, in a first aspect, in an embodiment of the present invention, an embodiment of the present invention provides a novel pyrazolopyrimidine compound, as shown in formula (I), a stereoisomer thereof, a tautomer thereof , or a pharmaceutically acceptable salt, or solvate, or prodrug:

其中，among them,

n，m独立地取自于0、1或2；n, m are independently taken from 0, 1 or 2;

L是O，-C(O)-，-C(O)NH-，-CH₂-，S，S(O)，NH或S(O)₂；L is O, -C(O)-, -C(O)NH-, -CH ₂ -, S, S(O), NH or S(O) ₂ ;

A取自于取代或未取代的苯环、或者取代或未取代的杂芳环，并且与母核及L的连接位点可以任选；A is derived from a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and a linking site to the parent nucleus and L may be optional;

B独立地取自于取代或未取代的脂肪环、取代或未取代的杂环、取代或未取代的苯环、或者取代或未取代的杂芳环，并且与L的连接位点可以任选； B is independently taken from a substituted or unsubstituted aliphatic ring, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and the linking site with L may be optionally selected ;

R₁和R₂各自独立地选自氢、未取代的C1-C4烷基、卤素、氰基，或者R₁和R₂与它们相连的碳原子一起形成三元碳环或四元碳环，或者R₁和R₂合并为氧代基；R ₁ and R ₂ are each independently selected from hydrogen, unsubstituted C1-C4 alkyl, halogen, cyano, or R ₁ and R ₂ together with the carbon atom to which they are attached form a ternary carbocyclic or quaternary carbocyclic ring, Or R ₁ and R _{2 are} combined into an oxo group;

Y选自氰基、或

Y is selected from cyano, or

R₃、R₄、R₅和R₆各自独立地选自氢、未取代的C1-C4烷基、羟基取代的C1-C4烷基、C1-C4烷氧基C1-4烷基、卤素、氰基、或-(CH₂)_qN(R^aR^b)，其中，q为1、2、3、或4，R^a和R^b各自独立地选自氢、未取代的C1-C4烷基；R ₃ , R ₄ , R ₅ and R ₆ are each independently selected from hydrogen, unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl, halogen, a cyano group, or -(CH ₂ ) _q N(R ^a R ^b ), wherein q is 1, 2, 3, or 4, and R ^a and R ^b are each independently selected from hydrogen, unsubstituted C1-C4 alkane base;

并规定，当R₁和R₂都是氢时，A是苯基，L是O，B是4-氯苯基、3-氯吡啶-6-基、2,4-二氟苯基；Y是4-羟基丁-2-烯酰基、丁-2-炔酰基、或氰基；And stipulates that when both R ₁ and R ₂ are hydrogen, A is a phenyl group, L is O, and B is 4-chlorophenyl, 3-chloropyridin-6-yl, 2,4-difluorophenyl; Y Is 4-hydroxybut-2-enoyl, but-2-ynyl, or cyano;

或，当R₁和R₂都是氢时，A是苯基，L是C(O)-、或-CH₂-，B是吗啉-4-基；Y为氰基；Or, when both R ₁ and R ₂ are hydrogen, A is phenyl, L is C(O)-, or -CH ₂ -, B is morpholin-4-yl; Y is cyano;

或，当R₁和R₂都是氢时，A是吡唑基，L是S(O)₂、或-CH₂-，B是苯基、或环丙基；Y为氰基；Or, when both R ₁ and R ₂ are hydrogen, A is pyrazolyl, L is S(O) ₂ , or -CH ₂ -, B is phenyl or cyclopropyl; Y is cyano;

或，当R₁和R₂都是氢时，A是吡啶基，L是NH、或O，B是吡喃-4-基、或环己基；Y为氰基。Or, when both R ₁ and R ₂ are hydrogen, A is a pyridyl group, L is NH or O, B is pyran-4-yl or cyclohexyl; and Y is a cyano group.

在本发明的一种实施方案中，本发明实施方案提供的一种如式(I)所示吡唑并嘧啶化合物，其中，n，m独立地取自于0、1或2；L为O，-C(O)-，-C(O)NH-，-CH₂-，NH或S，更优选地为O，-C(O)NH-，NH。In one embodiment of the present invention, an embodiment of the present invention provides a pyrazolopyrimidine compound of the formula (I), wherein n, m are independently taken from 0, 1 or 2; , -C(O)-, -C(O)NH-, -CH ₂ -, NH or S, more preferably O, -C(O)NH-, NH.

在本发明的一种实施方案中，本发明实施方案提供的一种如式(I)所示吡唑并嘧啶化合物，其中，A取自于取代或未取代的苯环或杂芳环，并且与母核及L的连接位点可以任选；B独立地取自于取代或未取代的脂肪环、取代或未取代的杂环、取代或未取代的苯环、或者取代或未取代的杂芳环，并且与L的连接位点可以任选；其中：In one embodiment of the present invention, an embodiment of the present invention provides a pyrazolopyrimidine compound of the formula (I), wherein A is derived from a substituted or unsubstituted benzene or heteroaryl ring, and The attachment site to the parent nucleus and L may be optionally selected; B is independently derived from a substituted or unsubstituted aliphatic ring, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted benzene ring, or a substituted or unsubstituted hetero An aromatic ring, and a linking site with L can be optionally selected; wherein:

所述取代的苯环是指苯环上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基；优选地，所述取代的苯基为氟取代的苯基、或氯取代的苯基，更优选地为2,4- 二氟苯基、或4-氯苯基；The substituted benzene ring means that any position on the benzene ring is substituted with an optional substituent selected from the group consisting of hydrogen, methyl, methoxy, fluoro, chloro, trifluoromethyl, trifluoromethyl. Oxy or cyano; preferably, the substituted phenyl is a fluoro substituted phenyl or a chloro substituted phenyl, more preferably 2,4- Difluorophenyl or 4-chlorophenyl;

所述未取代的杂芳环是指呋喃、吡咯、噻吩、恶唑、异恶唑、吡唑、咪唑、噻唑、异噻唑、恶二唑、三氮唑、噻二唑、四氮唑、吡啶、嘧啶、吡嗪、哒嗪、三嗪；所述取代的杂芳环是指以上基团上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基；更优选地，所述取代的吡啶为氯代吡啶，特别优选地为4-氯-吡啶-2-基；The unsubstituted heteroaryl ring means furan, pyrrole, thiophene, oxazole, isoxazole, pyrazole, imidazole, thiazole, isothiazole, oxadiazole, triazole, thiadiazole, tetrazolium, pyridine , pyrimidine, pyrazine, pyridazine, triazine; said substituted heteroaryl ring means that any position on the above group is substituted by an optional substituent selected from the group consisting of hydrogen, methyl, methoxy a radical, a fluorine, a chlorine, a trifluoromethyl group, a trifluoromethoxy group or a cyano group; more preferably, the substituted pyridine is a chloropyridine, particularly preferably a 4-chloro-pyridin-2-yl group;

所述未取代的脂肪环是指环丙烷、环丁烷、环戊烷、环己烷、环庚烷、环辛烷；所述取代的脂肪环是指以上基团上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基；The unsubstituted aliphatic ring means cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane; the substituted aliphatic ring means any position on the above group is optionally selected Substituted by a substituent selected from hydrogen, methyl, methoxy, fluoro, chloro, trifluoromethyl, trifluoromethoxy or cyano;

所述未取代的杂环是指四氢呋喃、四氢吡喃、四氢吡咯、哌啶、

其中w取自0、1或2；所述取代的杂环是指以上基团上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基。The unsubstituted heterocyclic ring means tetrahydrofuran, tetrahydropyran, tetrahydropyrrole, piperidine,

Wherein w is taken from 0, 1 or 2; the substituted heterocyclic ring means that any position on the above group is substituted by an optional substituent selected from the group consisting of hydrogen, methyl, methoxy, and fluorine. , chlorine, trifluoromethyl, trifluoromethoxy or cyano.

在本发明的一种实施方案中，本发明实施方案提供的一种如式(I)所示吡唑并嘧啶化合物，其中，优选地，R₁和R₂其中一个为氢、而另一个为未取代的C1-C4烷基(甲基、乙基、正丙基、异丙基、正丁基、异丁基、或叔丁基)，或者R₁和R₂与它们相连的碳原子一起形成环丙基；更优选地，R₁和R₂都是氢，或者其中一个是氢而另一个为甲基，或者R₁和R₂与它们相连的碳原子一起形成环丙基。In one embodiment of the present invention, an embodiment of the present invention provides a pyrazolopyrimidine compound of the formula (I), wherein preferably, one of R ₁ and R ₂ is hydrogen and the other is Unsubstituted C1-C4 alkyl (methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl), or R ₁ and R ₂ together with the carbon atom to which they are attached The cyclopropyl group is formed; more preferably, both R ₁ and R ₂ are hydrogen, or one of them is hydrogen and the other is methyl, or R ₁ and R ₂ together with the carbon atom to which they are attached form a cyclopropyl group.

在本发明的一种优选实施方案中，本发明实施方案提供了一种如式(II)所示吡唑并嘧啶化合物，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药： In a preferred embodiment of the invention, an embodiment of the invention provides a pyrazolopyrimidine compound of formula (II), a stereoisomer, tautomer thereof, or pharmaceutically acceptable Salt, or solvate, or prodrug:

其中，L、A、B和Y的定义如上述式(I)；Wherein, L, A, B and Y are as defined above for formula (I);

并规定，A是苯基，L是O，B是4-氯苯基、3-氯吡啶-6-基、2,4-二氟苯基；Y是4-羟基丁-2-烯酰基、丁-2-炔酰基、或氰基；And stipulates that A is a phenyl group, L is O, B is 4-chlorophenyl, 3-chloropyridin-6-yl, 2,4-difluorophenyl; Y is 4-hydroxybut-2-enoyl, But-2-ynyl, or cyano;

或，A是苯基，L是C(O)-、或-CH₂-，B是吗啉-4-基；Y为氰基；Or, A is phenyl, L is C(O)-, or -CH ₂ -, B is morpholin-4-yl; Y is cyano;

或，A是吡唑基，L是S(O)₂、或-CH₂-，B是苯基、或环丙基；Y为氰基；Or, A is pyrazolyl, L is S(O) ₂ or -CH ₂ -, B is phenyl or cyclopropyl; Y is cyano;

或，A是吡啶基，L是NH、或O，B是吡喃-4-基、或环己基；Y为氰基。Or, A is a pyridyl group, L is NH or O, B is pyran-4-yl or cyclohexyl; Y is a cyano group.

在本发明的一种更优选实施方案中，本发明实施方案提供的一种如式(II)所示吡唑并嘧啶化合物为下列化合物中的一种：In a more preferred embodiment of the present invention, a pyrazolopyrimidine compound of the formula (II) according to an embodiment of the present invention is one of the following compounds:

其中，式(II-1)或(II-2)中L、B和Y的定义如上述式(I)；Wherein, L, B and Y in the formula (II-1) or (II-2) are as defined in the above formula (I);

并规定，L是O，B是4-氯苯基、3-氯吡啶-6-基、2,4-二氟苯基；Y是4-羟基丁-2-烯酰基、丁-2-炔酰基、或氰基；And stipulates that L is O, B is 4-chlorophenyl, 3-chloropyridin-6-yl, 2,4-difluorophenyl; Y is 4-hydroxybut-2-enoyl, but-2-yne An acyl group or a cyano group;

或，L是C(O)-、或-CH₂-，B是吗啉-4-基；Y为氰基。Or, L is C(O)-, or -CH ₂ -, B is morpholin-4-yl; Y is cyano.

在本发明的一种优选实施方案中，本发明实施方案提供了一种如式(III)所示的吡唑并嘧啶化合物，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药： In a preferred embodiment of the invention, an embodiment of the invention provides a pyrazolopyrimidine compound of formula (III), a stereoisomer, tautomer thereof, or pharmaceutically acceptable Salt, or solvate, or prodrug:

其中，L、A、B和Y的定义如上述式(I)。Wherein, L, A, B and Y are as defined in the above formula (I).

在本发明的一种更优选实施方案中，本发明实施方案提供的一种如式(III)所示吡唑并嘧啶化合物为下列化合物中的一种：In a more preferred embodiment of the present invention, a pyrazolopyrimidine compound of the formula (III) according to an embodiment of the present invention is one of the following compounds:

其中，式(III-1)、(III-2)、(III-3)和(III-4)中L、B和Y的定义如上述式(I)。Among them, L, B and Y in the formulae (III-1), (III-2), (III-3) and (III-4) are as defined in the above formula (I).

在本发明的一种优选实施方案中，本发明实施方案提供了一种如式(IV)所示的吡唑并嘧啶化合物，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：In a preferred embodiment of the invention, an embodiment of the invention provides a pyrazolopyrimidine compound of formula (IV), a stereoisomer, tautomer thereof, or pharmaceutically acceptable Salt, or solvate, or prodrug:

在本发明的一种更优选实施方案中，本发明实施方案提供的一种如式(IV)所示的吡唑并嘧啶化合物为下列化合物中的一种：In a more preferred embodiment of the present invention, a pyrazolopyrimidine compound of the formula (IV) provided by an embodiment of the present invention is one of the following compounds:

其中，式(IV-1)或(IV-2)中L、B和Y的定义如上述式(I)。Wherein, L, B and Y in the formula (IV-1) or (IV-2) are as defined in the above formula (I).

在本发明的一种更优选实施方案中，本发明实施方案提供的式(I)、(II)、(II-1)、(II-2)、(III)、(III-1)、(III-2)、(III-3)、(III-4)、(IV)、(IV-1)、或(IV-2)化合物，其中，L为O；In a more preferred embodiment of the invention, the formula (I), (II), (II-1), (II-2), (III), (III-1), (provided by the embodiments of the present invention) a compound of III-2), (III-3), (III-4), (IV), (IV-1), or (IV-2), wherein L is O;

B为

B is

Y选自氰基、或

Y is selected from cyano, or

R₃、R₄、R₅和R₆各自独立选自未取代的C1-C4烷基、羟基取代的C1-C4烷基、C1-C4烷氧基C1-4烷基、卤素、氰基、或-(CH₂)_qN(R^aR^b)，其中，q为1、2、3、或4，R^a和R^b各自独立地选自氢、未取代的C1-C4烷基。R ₃ , R ₄ , R ₅ and R ₆ are each independently selected from unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl, halogen, cyano, Or -(CH ₂ ) _q N(R ^a R ^b ), wherein q is 1, 2, 3, or 4, and R ^a and R ^b are each independently selected from hydrogen, unsubstituted C1-C4 alkyl.

在本发明的一种特别优选实施方案中，本发明实施方案提供一种选自下列化合物中的一种的吡唑并嘧啶类化合物，或其药学上可接受的盐，或溶剂化物，或前药： In a particularly preferred embodiment of the present invention, an embodiment of the present invention provides a pyrazolopyrimidine compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof, or one of the following compounds medicine:

在本发明的实施方案中，所述“药学上可接受的盐”是指药学上可接受的酸加成盐和药学上可接受的碱加成盐：In an embodiment of the invention, the "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable acid addition salt and a pharmaceutically acceptable base addition salt:

所述“药学上可接受的酸加成盐”是指能够保留游离碱的生物有效性而无其他副作用的，与无机酸或有机酸所形成的盐。无机酸盐包括但不限于盐酸盐、氢溴酸盐、硫酸盐、磷酸盐等；有机酸盐包括但不限于甲酸盐、乙酸盐、丙酸盐、乙醇酸盐、葡糖酸盐、乳酸盐、草酸盐、马来酸盐、琥珀酸盐、富马酸盐、酒石酸盐、柠檬酸盐、谷氨酸盐、天冬氨酸盐、苯甲酸盐、甲磺酸盐、对甲苯磺酸盐和水杨酸盐等。这些盐可通过本领域已知的方法制备。The "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid capable of retaining the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, phosphates, and the like; organic acid salts include, but are not limited to, formate, acetate, propionate, glycolate, gluconate , lactate, oxalate, maleate, succinate, fumarate, tartrate, citrate, glutamate, aspartate, benzoate, methanesulfonate , p-toluenesulfonate and salicylate. These salts can be prepared by methods known in the art.

所述“药学上可接受的碱加成盐”是指能够保持游离酸的生物有效性而无其它副作用的盐。这些盐是通过将无机碱或有机碱添加至游离酸而制备的。衍生自无机碱的盐包括但不限于钠盐，钾盐，钙盐和镁盐等。衍生自有机碱的盐包括但不限于铵盐，三乙胺盐，赖氨酸盐，精氨酸盐等。这些盐可通过本领域已知的方法制备。The "pharmaceutically acceptable base addition salt" refers to a salt capable of maintaining the bioavailability of the free acid without other side effects. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, calcium and magnesium salts and the like. Salts derived from organic bases include, but are not limited to, ammonium salts, triethylamine salts, lysine salts, arginine salts, and the like. These salts can be prepared by methods known in the art.

在本发明的实施方案中，所述“溶剂化物”是指本发明实施方案的化合物与溶剂形成的配合物。它们或者在溶剂中反应或者从溶剂中沉淀析出或者结晶出来。例如，一个与水形成的配合物称为“水合物”。In an embodiment of the invention, the "solvate" refers to a complex of a compound of an embodiment of the invention with a solvent. They either react in a solvent or precipitate out of a solvent or Crystallize out. For example, a complex formed with water is referred to as a "hydrate."

在本发明的实施方案中，本发明实施方案的化合物可以含有一个或多个手性中心，并以不同的光学活性形式存在。当化合物含有一个手性中心时，化合物包含对映异构体。本发明实施方案包括这两种异构体和异构体的混合物，如外消旋混合物。对映异构体可以通过本专业已知的方法拆分，例如结晶以及手性色谱等方法。当式(I)化合物含有多于一个的手性中心时，可以存在非对映异构体。本发明实施方案包括拆分过的光学纯的特定异构体以及非对映异构体的混合物。非对映异构体可由本领域已知方法拆分，比如结晶以及制备色谱。In an embodiment of the invention, the compounds of the embodiments of the invention may contain one or more chiral centers and exist in different optically active forms. When the compound contains a chiral center, the compound contains the enantiomer. Embodiments of the invention include mixtures of the two isomers and isomers, such as racemic mixtures. Enantiomers can be resolved by methods known in the art, such as crystallization and chiral chromatography. When the compound of formula (I) contains more than one chiral center, diastereomers may be present. Embodiments of the invention include resolved optically pure specific isomers as well as mixtures of diastereomers. Diastereomers can be resolved by methods known in the art, such as crystallization and preparative chromatography.

在本发明的实施方案中，所述的前药是指已知的氨基保护基和羧基保护基，在生理条件下被水解或经由酶反应释放得到母体化合物。具体的前药制备方法可参照(Saulnier,M.G.；Frennesson,D.B.；Deshpande,M.S.；Hansel,S.B and Vysa,D.M.Bioorg.Med.Chem Lett.1994,4,1985-1990.Greenwald,R.B.；Choe,Y.H.；Conover,C.D.；Shum,K.；Wu,D.；Royzen,M.J.Med.Chem.2000,43,475.)。In an embodiment of the invention, the prodrug refers to a known amino protecting group and a carboxy protecting group which are hydrolyzed under physiological conditions or released via an enzymatic reaction to give the parent compound. Specific prodrug preparation methods can be referred to (Saulnier, MG; Frennesson, DB; Deshpande, MS; Hansel, SB and Vysa, DM Bioorg. Med. Chem Lett. 1994, 4, 1985-1990. Greenwald, RB; Choe, YH Conover, CD; Shum, K.; Wu, D.; Royzen, MJ Med. Chem. 2000, 43, 475.).

第二方面，本发明实施方案提供了上述如式(I)所示的吡唑并嘧啶化合物的制备方法，包括如下步骤：In a second aspect, an embodiment of the present invention provides a method for preparing the pyrazolopyrimidine compound represented by the above formula (I), comprising the steps of:

(1)将式(V)化合物与式(VI)化合物反应，得到式(VII)化合物；(1) reacting a compound of the formula (V) with a compound of the formula (VI) to give a compound of the formula (VII);

(2)将式(VII)化合物与式(VIII)化合物反应，得到式(IX)化合物； (2) reacting a compound of the formula (VII) with a compound of the formula (VIII) to give a compound of the formula (IX);

(3)将式(IX)化合物经脱保护基PG得到式(X)化合物；(3) a compound of the formula (IX) is subjected to a deprotection group PG to give a compound of the formula (X);

(4)将式(X)化合物与式(XI)化合物反应，得到式(I)化合物；(4) reacting a compound of the formula (X) with a compound of the formula (XI) to give a compound of the formula (I);

在上述的式(V)、式(VI)、式(VII)、式(VIII)、式(IX)、式(X)和式(XI)中涉及的取代基R₁、R₂、L、A、B、Y和n、m的定义如上面的式(I)，PG为氨基保护基(合适的氨基保护基包括酰基(例如乙酰基)、碳甲酰胺类(carbamate)(例如2',2',2'-三氯乙氧基羰基、Cbz苄氧基羰基或BOC叔丁氧基羰基)和芳基烷基(例如Bn苄基)，其可在适当时通过水解(例如使用酸，例如二噁烷中的盐酸或二氯甲烷中的三氟乙酸)或通过还原方式(例如苄基或苄氧基羰基的氢解、或使用乙酸中的锌还原性除去2',2',2'-三氯乙氧基羰基)而除去。其它合适的氨基保护基包括三氟乙酰基(-COCF3)，其可通过碱催化的水解除去苄氧基、羰基或叔丁氧基羰基，本领域技术人员可参考T.W. Greene‘Protective Groups in Organic Synthesis’(第4版，J.Wiley and Sons，2006))，X为氯、溴或羟基。Substituents R ₁ , R ₂ , L, in the above formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X) and formula (XI), A, B, Y and n, m are as defined above for formula (I), PG is an amino protecting group (suitable amino protecting groups include acyl (eg acetyl), carbamate (eg 2', 2',2'-trichloroethoxycarbonyl, Cbzbenzyloxycarbonyl or BOC-tert-butoxycarbonyl) and arylalkyl (for example Bnbenzyl) which may be hydrolyzed (for example using an acid, if appropriate) For example, hydrochloric acid in dioxane or trifluoroacetic acid in dichloromethane) or by reduction (for example, hydrogenolysis of benzyl or benzyloxycarbonyl, or reduction by zinc in acetic acid 2', 2', 2 '-Trichloroethoxycarbonyl) is removed. Other suitable amino protecting groups include trifluoroacetyl (-COCF3) which can be removed by base catalyzed hydrolysis to remove benzyloxy, carbonyl or tert-butoxycarbonyl. One skilled in the art can refer to TW Greene 'Protective Groups in Organic Synthesis' (4th edition, J. Wiley and Sons, 2006), where X is chlorine, bromine or hydroxyl.

在本发明的实施方案中，本发明实施方案提供的上述如式(I)所示的吡唑并嘧啶化合物的制备方法，其中，式(V)化合物可以参考下列的方法来制备：In an embodiment of the present invention, the present invention provides a method for producing a pyrazolopyrimidine compound represented by the above formula (I), wherein the compound of the formula (V) can be produced by referring to the following method:

在本发明的实施方案中，本发明实施方案还提供了用于上述吡唑并嘧啶化合物合成的中间体化合物，包括但不限于：In an embodiment of the invention, embodiments of the invention also provide intermediate compounds for use in the synthesis of the above pyrazolopyrimidine compounds, including but not limited to:

其中，Bn为苄基，Boc为叔丁氧羰酰基。 Wherein Bn is a benzyl group and Boc is a tert-butoxycarbonyl group.

第三方面，本发明实施方案提供了一种包含有效剂量的一种或多种本发明实施方案上述吡唑并嘧啶化合物或其立体异构体、互变异构体、溶剂化物或其药学上可接受的盐的药物组合物，所述药物组合物还包括药学上可接受的辅料。In a third aspect, the present invention provides an excipient, a stereoisomer, a solvate or a pharmaceutically thereof thereof, comprising an effective amount of one or more of the pyrazolopyrimidine compounds of the present invention or a stereoisomer thereof A pharmaceutical composition of an acceptable salt, the pharmaceutical composition further comprising a pharmaceutically acceptable adjuvant.

本发明实施方案的药物组合物可以被配制为固态、半固态、液态或气态制剂，如片剂、胶囊、粉剂、颗粒剂、膏剂、溶液剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶。The pharmaceutical composition of the embodiment of the present invention may be formulated into a solid, semi-solid, liquid or gaseous preparation such as a tablet, a capsule, a powder, a granule, a plaster, a solution, a suppository, an injection, an inhalant, a gel, and a micro Balls and aerosols.

本发明实施方案的药物组合物可以通过制药领域中公知的方法制备。例如，制备药物组合物的实际方法为本领域技术人员所已知，例如可参见The Science and Practice of Pharmacy(制药科学与实践)，20th Edition (Philadelphia College of Pharmacy and Science,2000)。The pharmaceutical compositions of the embodiments of the present invention can be prepared by methods well known in the pharmaceutical art. For example, practical methods for preparing pharmaceutical compositions are known to those skilled in the art, for example, see The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).

本发明实施方案的药物组合物的给药途径包括但不限于口服、局部、经皮、肌肉、静脉、吸入、肠胃外、舌下、直肠、***及鼻内。例如，适合口服给药的剂型包括胶囊、片剂、颗粒剂以及糖浆等。这些制剂中包含的本发明实施方案的式(I)化合物可以是固体粉末或颗粒；水性或非水性液体中的溶液或是混悬液；油包水或水包油的乳剂等。上述剂型可由活性化合物与一种或多种载体或辅料经由通用的药剂学方法制成。上述的载体需要与活性化合物或其他辅料兼容。对于固体制剂，常用的无毒载体包括但不限于甘露醇、乳糖、淀粉、硬脂酸镁、纤维素、葡萄糖、蔗糖等。用于液体制剂的载体包括但不限于水、生理盐水、葡萄糖水溶液、乙二醇和聚乙二醇等。活性化合物可与上述载体形成溶液或是混悬液。具体的给药方式和剂型取决于化合物本身的理化性质以及所应用疾病的严重程度等。本领域技术人员能够根据上述因素并结合其自身具有的知识来确定具体的给药途径。例如可参见：李俊，《临床药理学》，人民卫生出版社，2008.06；丁玉峰，论临床剂型因素与合理用药，医药导报，26(5)，2007；Howard C.Ansel,Loyd V.Allen,Jr.,Nicholas G.Popovich著，江志强主译，《药物剂型与给药体系》，中国医药科技出版社，2003.05。Routes of administration of the pharmaceutical compositions of the embodiments of the invention include, but are not limited to, oral, topical, transdermal, intramuscular, intravenous, inhalation, parenteral, sublingual, rectal, vaginal, and intranasal. For example, dosage forms suitable for oral administration include capsules, tablets, granules, and syrups and the like. The compound of formula (I) of the embodiments of the invention included in these formulations may be a solid powder or granule; a solution or suspension in an aqueous or non-aqueous liquid; a water-in-oil or oil-in-water emulsion, and the like. The above dosage forms can be prepared from the active compound with one or more carriers or excipients via conventional pharmaceutical methods. The above carriers need to be compatible with the active compound or other excipients. For solid formulations, commonly used non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, and the like. Carriers for liquid preparations include, but are not limited to, water, physiological saline, aqueous dextrose, ethylene glycol, polyethylene glycol, and the like. The active compound can form a solution or suspension with the above carriers. The particular mode of administration and dosage form will depend on the physicochemical properties of the compound itself, as well as the severity of the disease being applied. Those skilled in the art will be able to determine the specific route of administration based on the above factors in combination with their own knowledge. For example, see: Li Jun, "Clinical Pharmacology", People's Health Publishing House, 2008.06; Ding Yufeng, on clinical dosage forms and rational use of drugs, Medical Herald, 26 (5), 2007; Howard C. Ansel, Loyd V. Allen, Jr., Nicholas G. Popovich, Jiang Zhiqiang, “Pharmaceutical Formulation and Drug Delivery System”, China Medical Science and Technology Press, 2003.05.

本发明实施方案的药物组合物可以以每单位剂量含有预定量的活性成分的单位剂型存在。优选的单位剂量组合物为含有日剂量或亚剂量、或其适当分数的活性成分的那些。因此，这种单位剂量可以一天给药多于一次。优选的单位剂量组合物为含有本申请如上所述的日剂量或亚剂量(一天给药多于一次)、或其适当分数的活性成分的那些。The pharmaceutical compositions of the embodiments of the invention may be presented in unit dosage form containing a predetermined amount of active ingredient per unit dose. Preferred unit dosage compositions are those containing a daily or sub-dose, or an appropriate fraction thereof, of the active ingredient. Thus, such unit doses can be administered more than once a day. Preferred unit dosage compositions are those containing a daily or sub-dose (more than one administration per day) as described above, or an appropriate fraction thereof, of the active ingredient.

本发明实施方案的药物组合物以符合医学实践规范的方式配制，定量和给药。本发明实施方案的化合物的“治疗有效量”由要治疗的具体病症、治疗的个体、病症的起因、药物的靶点以及给药方式等因素决定。通常，经胃肠道外给药的剂量可以是1-200mg/kg，口服给药的剂量可以是1-1000mg/kg。The pharmaceutical compositions of the embodiments of the invention are formulated, quantified, and administered in a manner consistent with medical practice. A "therapeutically effective amount" of a compound of an embodiment of the invention is determined by the particular condition to be treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration. Generally, the dose for parenteral administration may be 1-200 mg/kg, and the dose for oral administration may be 1-1000 mg/kg.

本申请中所提供的有效剂量的范围并非意图限制本申请的范围，而是代表优选的剂量范围。但是，最优选的剂量可针对个别个体而进行调整，这是本领域技术人员所了解且可决定的(例如参阅Berkow等人编著，Merck手册，第16版，Merck公司，Rahway,N.J.,1992)。The range of effective dosages provided herein is not intended to limit the scope of the application, but rather to the preferred dosage range. However, the most preferred dose can be adjusted for individual individuals, this is It is known and determinable by those skilled in the art (see, for example, Berkow et al., ed., Merck Handbook, 16th ed., Merck, Rahway, N.J., 1992).

第四方面，本发明实施方案提供了上述吡唑并嘧啶化合物或其立体异构体、互变异构体、溶剂化物或其药学上可接受的盐在制备用于预防或治疗由BTK介导疾病的药物中的用途。In a fourth aspect, the present invention provides the above pyrazolopyrimidine compound or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof for use in the prevention or treatment of BTK-mediated Use in medicines for diseases.

本发明实施方案提供了一种抑制BTK活性的方法，包含给予生物体系本发明实施方案上述吡唑并嘧啶化合物或其立体异构体、互变异构体、溶剂化物或其药学上可接受的盐或包含本发明实施方案上述吡唑并嘧啶化合物或其立体异构体、互变异构体、溶剂化物或其药学上可接受的盐的药物组合物。Embodiments of the present invention provide a method of inhibiting BTK activity, comprising administering to a biological system, the pyrazolopyrimidine compound of the present invention, or a stereoisomer, tautomer, solvate thereof, or a pharmaceutically acceptable thereof thereof. A salt or a pharmaceutical composition comprising the above pyrazolopyrimidine compound of the present invention or a stereoisomer, tautomer, solvate thereof or a pharmaceutically acceptable salt thereof.

在一些实施方式中，所述生物体系是酶、细胞或哺乳动物。In some embodiments, the biological system is an enzyme, a cell, or a mammal.

本发明实施方案还提供了一种预防或治疗由BTK介导的疾病的方法，其包括对有需要的患者联合给予治疗有效剂量的一种或多种本发明实施方案的上述吡唑并嘧啶化合物或其立体异构体、互变异构体、溶剂化物或其药学上可接受的盐和一种或多种选自以下的药物：免疫调节剂、免疫检查点抑制剂、糖皮质激素、非甾体抗炎药、Cox-2特异性抑制剂、TNF-α结合蛋白、干扰素、白细胞介素和化疗药物。Embodiments of the present invention also provide a method of preventing or treating a disease mediated by BTK comprising administering to a patient in need thereof a therapeutically effective amount of one or more of the above pyrazolopyrimidine compounds of the present invention. Or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt thereof and one or more drugs selected from the group consisting of immunomodulators, immunological checkpoint inhibitors, glucocorticoids, non- Steroidal anti-inflammatory drugs, Cox-2 specific inhibitors, TNF-α binding proteins, interferons, interleukins and chemotherapeutic drugs.

在本发明实施方案的实施方式中，所述由BTK介导的疾病包括自身免疫性疾病、炎性疾病、异种免疫性情况或疾病、血栓栓塞疾病和癌症。在一些具体实施方式中，所述癌症包括B细胞性慢性淋巴细胞白血病、急性淋巴细胞性白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、急性髓性白血病、弥漫性大B细胞淋巴瘤、多发性骨髓瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、华氏巨球蛋白血症、实体瘤。在一些具体实施方式中，所述自身免疫性疾病和炎性疾病选自类风湿性关节炎、骨关节炎、青少年关节炎、慢性阻塞性肺疾病、多重硬化、***性红斑狼疮、银屑病、银屑病关节炎、克罗恩病、溃疡性结肠炎和肠道易激综合症。在一些具体实施方式中，所述异种免疫性情况或疾病包括移植物抗宿主病、移植、输血、过敏反应、***反应、I型超敏反应、过敏性结膜炎、过敏性鼻炎或特应性皮炎。In an embodiment of the embodiments of the invention, the BTK mediated diseases include autoimmune diseases, inflammatory diseases, xenogeneic immune conditions or diseases, thromboembolic diseases, and cancer. In some embodiments, the cancer comprises B-cell chronic lymphocytic leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myeloid leukemia, diffuse large B-cell lymphoma , multiple myeloma, mantle cell lymphoma, small lymphocytic lymphoma, Waldenstrom's macroglobulinemia, solid tumor. In some embodiments, the autoimmune disease and inflammatory disease are selected from the group consisting of rheumatoid arthritis, osteoarthritis, juvenile arthritis, chronic obstructive pulmonary disease, multiple sclerosis, systemic lupus erythematosus, psoriasis , psoriatic arthritis, Crohn's disease, ulcerative colitis, and irritable bowel syndrome. In some embodiments, the xenogeneic immune condition or disease comprises graft versus host disease, transplantation, blood transfusion, allergic reaction, allergy, type I hypersensitivity, allergic conjunctivitis, allergic rhinitis or atopy dermatitis.

实验数据证明，本发明实施方案提供的上述吡唑并嘧啶化合物是蛋白激酶BTK的有效、安全的抑制剂。 Experimental data demonstrate that the above pyrazolopyrimidine compounds provided by embodiments of the present invention are potent and safe inhibitors of protein kinase BTK.

详细描述Detailed Description

以下对本发明实施例进行详细说明。应当理解的是，下文所描述的实验、合成方法以及所涉及的中间体等具体实施例是对本申请的阐明，并不限制本申请的范围。需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互任意组合。The embodiments of the present invention are described in detail below. It is to be understood that the specific examples of the experiments, the synthetic methods, and the intermediates involved, which are described below, are illustrative of the present application and are not intended to limit the scope of the application. It should be noted that, in the case of no conflict, the features in the embodiments and the embodiments in the present application may be arbitrarily combined with each other.

本申请中实验所使用的起始原料或购买自试剂供应商或经由本领域公知的方法由已知原料制备。除非另有说明，本申请的实施例应用下述条件：The starting materials used in the experiments in this application were either purchased from a reagent supplier or prepared from known starting materials by methods well known in the art. Unless otherwise stated, embodiments of the present application apply the following conditions:

温度的单位是摄氏度(℃)；室温的定义是18-25℃；The unit of temperature is Celsius (°C); the definition of room temperature is 18-25 ° C;

有机溶剂使用无水硫酸镁或无水硫酸钠干燥；使用旋转蒸发仪在减压升温条件下旋干(例如：15mmHg，30℃)；Drying the organic solvent with anhydrous magnesium sulfate or anhydrous sodium sulfate; using a rotary evaporator to spin dry under reduced pressure (for example: 15 mmHg, 30 ° C);

快速柱色谱分离时使用200-300目硅胶作为载体，薄层色谱表示薄层色谱法；200-300 mesh silica gel is used as a carrier for rapid column chromatography, and thin layer chromatography is used for thin layer chromatography;

通常情况下，反应的进度通过薄层色谱或LC-MS监测；Typically, the progress of the reaction is monitored by thin layer chromatography or LC-MS;

最终产品的鉴定由核磁共振(Bruker AVANCE 300，300MHz)和LC-MS(Bruker esquine 6000，Agilent 1200series)完成。The identification of the final product was performed by nuclear magnetic resonance (Bruker AVANCE 300, 300 MHz) and LC-MS (Bruker esquine 6000, Agilent 1200 series).

实施例1Example 1

(R)-3-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-腈(WS-300)的制备(R)-3-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1- Preparation of phenyl)piperidine-1-carbonitrile (WS-300)

第一步(R)-3-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-甲酸叔丁酯的制备：Preparation of (R)-3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylic acid tert-butyl ester:

(S)-3-羟基哌啶-1-甲酸叔丁酯(1.7g,8.5mmol)和3-碘-1H-吡唑并[3,4-d]嘧啶-4-胺(2g,7.7mmol)溶于四氢呋喃(100mL)。冰浴下一次加入三苯基膦(2.5g,9.5mmol)，缓慢滴加偶氮二甲酸二异丙酯(2.1g,10.40mmol)。撤去冰浴，反应液室温搅拌18小时，减压蒸干。粗产物过硅胶柱纯化(乙酸乙酯:石油醚＝1：1),得产品(R)-3-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-甲酸叔丁酯(3.5g,100％)。(S)-3-Hydroxypiperidine-1-carboxylic acid tert-butyl ester (1.7 g, 8.5 mmol) and 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2 g, 7.7 mmol ) Dissolved in tetrahydrofuran (100 mL). Triphenylphosphine (2.5 g, 9.5 mmol) was added in the ice bath, and diisopropyl azodicarboxylate (2.1 g, 10.40 mmol) was slowly added dropwise. The ice bath was removed, and the reaction mixture was stirred at room temperature for 18 hr and evaporated. The crude product was purified by silica gel column (ethyl acetate: petroleum ether = 1:1) to give product (R)-3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidine- Tert-butyl ester of 1-yl)piperidine-1-carboxylate (3.5 g, 100%).

第二步(R)-3-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-甲酸叔丁酯的制备：Step 2 (R)-3-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine Preparation of -1-yl) piperidine-1-carboxylic acid tert-butyl ester:

(R)-3-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-甲酸叔丁酯(1.3g,2.9mmol)和5-氯-2-(4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)苯氧基)吡啶(1g,3.0mmol)溶于95％乙醇(50mL)。加入碳酸钠(600mg,5.7mmol)和二(三苯基膦)二氯化钯(200mg,0.28mmol)，氩气保护下回流反应18小时，减压蒸干。粗品用乙酸乙酯(50mL)和水(50mL)萃取。有机相用无水硫酸钠干燥，过滤，减压蒸干。残余物制备板纯化(二氯甲烷:甲醇＝50：1)，得产品(R)-3-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-甲酸叔丁酯(960mg,63％)。(R)-3-(4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylic acid tert-butyl ester (1.3 g, 2.9 mmol) 5-Chloro-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)pyridine (1 g, 3.0 mmol) In 95% ethanol (50 mL). Sodium carbonate (600 mg, 5.7 mmol) and bis(triphenylphosphine)palladium dichloride (200 mg, 0.28 mmol) were added, and the mixture was refluxed under argon atmosphere for 18 hr. The crude was extracted with ethyl acetate (50 mL) and water (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified (dichloromethane:methanol = 50:1) to give the product (R)-3-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)benzene tert-butyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (960 mg, 63%).

第三步(R)-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺的制备：The third step (R)-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4 -d]Preparation of pyrimidine-4-amine:

(R)-3-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-甲酸叔丁酯(960mg,1.8mmol)溶于四氢呋喃(10mL)，加入浓盐酸(0.5mL)，反应液60℃搅拌3小时，减压蒸干得粗产物(R)-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐(1.1g,100％)。直接用于下一步反应。(R)-3-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1- Tert-butyl piperidine-1-carboxylate (960 mg, 1.8 mmol) was dissolved in tetrahydrofuran (10 mL), concentrated hydrochloric acid (0.5 mL) was added, and the reaction mixture was stirred at 60 ° C for 3 hr. -3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4 -amine hydrochloride (1.1 g, 100%). Used directly in the next step.

第四步(R)-3-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-腈的制备： Step 4 (R)-3-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine Preparation of -1-yl) piperidin-1-nitrile:

(R)-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐(500mg,1.1mmol)溶于N,N-二甲基甲酰胺(5mL)，加入碳酸铯(600mg,1.9mmol)和溴化氰(400mg,3.8mmol)，反应液室温搅拌5小时，反应液倒入乙酸乙酯(50mL)，水洗(30mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备板纯化(二氯甲烷:甲醇＝50：1)，得到产品(R)-3-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-腈(130mg,27％)。(R)-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d] Pyrimidine-4-amine hydrochloride (500 mg, 1.1 mmol) was dissolved in N,N-dimethylformamide (5 mL), EtOAc (EtOAc (EtOAc) The mixture was stirred at room temperature for 5 hr. EtOAc EtOAc m. The crude product preparation plate was purified (dichloromethane:methanol = 50:1) to give the product (R)-3-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)benzene -1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonitrile (130 mg, 27%).

¹H-NMR(400MHz,DMSO-d6):δppm 8.29-8.25(m,2H),8.02-7.99(m,1H),7.72(d,J＝8.4,2H),7.32(d,J＝8.4,2H),7.18(d,J＝8.8,1H),4.90-4.85(m,1H),3.64-3.62(m,1H),3.56-3.53(m,1H),3.42-3.39(m,1H),3.20-3.15(m,1H),2.17-2.08(m,2H),1.95-1.81(m,2H). ^{1 H-NMR (400MHz, DMSO} -d6): δppm 8.29-8.25 (m, 2H), 8.02-7.99 (m, 1H), 7.72 (d, J = 8.4,2H), 7.32 (d, J = 8.4, 2H), 7.18 (d, J = 8.8, 1H), 4.90-4.85 (m, 1H), 3.64-3.62 (m, 1H), 3.56-3.53 (m, 1H), 3.42-3.39 (m, 1H), 3.20-3.15(m,1H), 2.17-2.08(m,2H),1.95-1.81(m,2H).

MS:m/z 446.8[M+1]MS: m/z 446.8 [M+1]

实施例2Example 2

(R,E)-1-(3-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)-4-羟基丁-2-烯-1-酮(WS-301)的制备(R,E)-1-(3-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d Preparation of pyrimidin-1-yl)piperidin-1-yl)-4-hydroxybut-2-en-1-one (WS-301)

以实施例1第三步反应的产物为起始物，实施例2经过以下的步骤制备：Taking the product of the third step reaction of Example 1 as a starting material, Example 2 was prepared by the following steps:

第一步(R,E)-1-(3-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)-4-羟基丁-2-烯-1-酮的制备：The first step (R,E)-1-(3-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3, Preparation of 4-d]pyrimidin-1-yl)piperidin-1-yl)-4-hydroxybut-2-en-1-one:

(R)-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐(400mg,0.87mmol)和4-羟基-2-丁烯酸(130mg,1.27mmol)溶于四氢呋喃(20mL)，加入HATU(550mg,1.45mmol)和三乙胺(200mg, 1.98mmol)，反应液室温搅拌18小时，减压蒸干。残余物溶于乙酸乙酯(50mL)，水洗(30mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备板纯化(二氯甲烷:甲醇＝10：1)，得到产品化合物(R,E)-1-(3-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)-4-羟基丁-2-烯-1-酮(110mg,22％)。(R)-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d] Pyrimidine-4-amine hydrochloride (400 mg, 0.87 mmol) and 4-hydroxy-2-butenoic acid (130 mg, 1.27 mmol) were dissolved in tetrahydrofuran (20 mL), and HATU (550 mg, 1.45 mmol) and triethylamine ( 200mg, 1.98 mmol), the reaction mixture was stirred at room temperature for 18 hr and evaporated. The residue was dissolved in ethyl acetate (50 mL)EtOAc. The crude product preparation plate was purified (dichloromethane:methanol = 10:1) to give the product compound (R,E)-1-(3-(4-amino-3-(4-((5-chloropyridine-2-) ()oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-hydroxybut-2-en-1-one (110 mg, twenty two%).

H-NMR(400MHz,DMSO-d6):δppm 8.43(s,1H),8.26(d,J＝2.8,1H),8.03-7.99(m,1H),7.72(d,J＝8.0,2H),7.34(d,J＝8.4,2H),7.19(d,J＝8.8,2H),6.80-6.42(m,2H),4.76(brs,1H),4.57-4.54(m,0.5H),4.22-3.98(m,3.3H),3.75(brs,0.3H),3.23-3.21(m,1H),3.05-3.03(m,0.4H),2.32-2.15(m,2H),1.96-1.93(m,1H),1.62-1.58(m,1H).</ RTI> <RTIgt; 7.34 (d, J = 8.4, 2H), 7.19 (d, J = 8.8, 2H), 6.80-6.42 (m, 2H), 4.76 (brs, 1H), 4.57-4.54 (m, 0.5H), 4.22 3.98 (m, 3.3H), 3.75 (brs, 0.3H), 3.23 - 3.21 (m, 1H), 3.05 - 3.03 (m, 0.4H), 2.32 - 2.15 (m, 2H), 1.96-1.93 (m, 1H), 1.62-1.58 (m, 1H).

MS:m/z 506.2[M+1]MS: m/z 506.2 [M+1]

实施例3Example 3

(R)-3-(4-氨基-3-(4-(4-氯苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-腈(WS-302)的制备(R)-3-(4-Amino-3-(4-(4-chlorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1 -Preparation of nitrile (WS-302)

实施例3从对应的起始物开始，采用与实施例1类似的方法制备而得。Example 3 was prepared in a similar manner to Example 1 starting from the corresponding starting material.

1H-NMR(400MHz,DMSO-d6):δppm 8.27(s,1H),7.67-7.69(d,J＝8Hz,2H),7.46-7.48(d,J＝8Hz,2H),7.14-7.20(m,4H),4.84-4.89(m,1H),3.64-3.65(d,J＝4Hz,1H),3.50-3.54(m,1H),3.38-3.41(m,1H),3.14-3.20(m,1H),2.08-2.18(m,2H),1.79-1.93(m,2H).1H-NMR (400MHz, DMSO-d6): δppm 8.27(s, 1H), 7.67-7.69 (d, J=8Hz, 2H), 7.46-7.48 (d, J=8Hz, 2H), 7.14-7.20 (m , 4H), 4.84 - 4.89 (m, 1H), 3.64 - 3.65 (d, J = 4 Hz, 1H), 3.50 - 3.54 (m, 1H), 3.38 - 3.41 (m, 1H), 3.14 - 3.20 (m, 1H), 2.08-2.18 (m, 2H), 1.79-1.93 (m, 2H).

MS:m/z 446.2[M+1]MS: m/z 446.2 [M+1]

实施例4 Example 4

(R,E)-1-(3-(4-氨基-3-(4-(4-氯苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)-4-羟基丁-2-烯-1-酮(WS-303)的制备(R,E)-1-(3-(4-Amino-3-(4-(4-chlorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl Preparation of piperidin-1-yl)-4-hydroxybut-2-en-1-one (WS-303)

实施例4从对应的起始物开始，采用与实施例2类似的方法制备而得。Example 4 was prepared in a similar manner to Example 2 starting from the corresponding starting material.

1H-NMR(400MHz,DMSO-d6):δppm 8.43(s,1H),7.66-7.68(d,J＝8Hz,2H),7.47-7.49(d,J＝8Hz,2H),7.14-7.20(m,4H),6.50-6.75(m,2H),4.54-4.72(m,2H),4.21-4.40(m,6H),4.14(brs,1H),3.18-3.23(brs,1H),2.50(s,1H),2.39-2.42(m,1H),2.21-2.98(m,1H),1.92(brs,1H),1.58-1.60(m,1H).1H-NMR (400MHz, DMSO-d6): δppm 8.43 (s, 1H), 7.66-7.68 (d, J = 8 Hz, 2H), 7.47-7.49 (d, J = 8 Hz, 2H), 7.14-7.20 (m) , 4H), 6.50-6.75 (m, 2H), 4.54-4.72 (m, 2H), 4.21-4.40 (m, 6H), 4.14 (brs, 1H), 3.18-3.23 (brs, 1H), 2.50 (s , 1H), 2.39-2.42 (m, 1H), 2.21-2.98 (m, 1H), 1.92 (brs, 1H), 1.58-1.60 (m, 1H).

MS:m/z 505.2[M+1]MS: m/z 505.2 [M+1]

实施例5Example 5

(R)-3-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-腈(WS-304)的制备(R)-3-(4-Amino-3-(4-(2,4-difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin Preparation of pyridine-1-carbonitrile (WS-304)

实施例5从对应的起始物开始，采用与实施例1类似的方法制备而得。 Example 5 was prepared in a similar manner to Example 1 starting from the corresponding starting material.

¹H-NMR(400MHz,DMSO-d6):δ8.27(s,1H),7.66-7.50(m,5H),7.40-7.36(m,1H),7.35-7.11(m,3H),4.87-4.85(m,1H),3.65-3.60(m,1H),3.54-3.48(m,1H),3.41-3.38(m,1H),3.19-3.16(m,1H),2.15-2.17(m,2H),1.90-1.82(m,2H). ^{1 H-NMR (400MHz, DMSO} -d6): δ8.27 (s, 1H), 7.66-7.50 (m, 5H), 7.40-7.36 (m, 1H), 7.35-7.11 (m, 3H), 4.87- 4.85 (m, 1H), 3.65-3.60 (m, 1H), 3.54-3.48 (m, 1H), 3.41-3.38 (m, 1H), 3.19-3.16 (m, 1H), 2.15-2.17 (m, 2H) ), 1.90 - 1.82 (m, 2H).

MS:m/z 448.2[M+1]MS: m/z 448.2 [M+1]

实施例6Example 6

(R,E)-1-(3-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)-4-羟基丁-2-烯-1-酮(WS-305)的制备(R,E)-1-(3-(4-Amino-3-(4-(2,4-difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine- Preparation of 1-yl)piperidin-1-yl)-4-hydroxybut-2-en-1-one (WS-305)

实施例6从对应的起始物开始，采用与实施例2类似的方法制备而得。Example 6 was prepared in a similar manner to Example 2 starting from the corresponding starting material.

¹H-NMR(400MHz,CDCl₃-d6):δppm 8.46(s,1H),7.62-7.64(m,2H),7.16-7.18(m,1H),7.06-7.18(m,2H),6.93-7.06(m,3H),6.51-6.63(m,1H),5.41-5.46(brs.,1H),4.82-4.89(m,2H),4.28-4.38(m,2H),4.19-4.23(m,1H),3.36-3.77(m,1H),2.32-2.39(m,2H),1.98-2.02(m,2H),2.32-2.39(m,2H). ¹ H-NMR (400 MHz, CDCl ₃ -d6): δ ppm 8.46 (s, 1H), 7.62-7.64 (m, 2H), 7.16-7.18 (m, 1H), 7.06-7.18 (m, 2H), 6.93 7.06 (m, 3H), 6.51-6.63 (m, 1H), 5.41-5.46 (brs., 1H), 4.82-4.89 (m, 2H), 4.28-4.38 (m, 2H), 4.19-4.23 (m, 1H), 3.36-3.77 (m, 1H), 2.32-2.39 (m, 2H), 1.98-2.02 (m, 2H), 2.32-2.39 (m, 2H).

MS:m/z 506.2[M+1]MS: m/z 506.2 [M+1]

实施例7Example 7

5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-306)的制备 5-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2 -Preparation of methylpiperidine-1-carbonitrile (WS-306)

第一步6-甲基哌啶-3-醇的制备Preparation of the first step 6-methylpiperidin-3-ol

将5-羟基-2-甲基-吡啶(1.5g,13.7mmol)溶在20ml乙酸里加入0.4g二氧化铂,混合物在50psi氢气的压强下搅拌16个小时。抽干溶剂得到化合物。5-Hydroxy-2-methyl-pyridine (1.5 g, 13.7 mmol) was dissolved in 20 ml of acetic acid, 0.4 g of platinum dioxide was added, and the mixture was stirred under a pressure of 50 psi of hydrogen for 16 hours. The solvent was evaporated to give the compound.

第二步5-羟基-2-甲基哌啶-1-甲酸叔丁酯的制备Second step Preparation of 5-hydroxy-2-methylpiperidine-1-carboxylic acid tert-butyl ester

将6-甲基哌啶-3-醇(1.10g，7.8mmol)和碳酸钠(1.6g,15.7mmol)加入到二氯甲烷(100mL)溶液中，然后将二碳酸二叔丁酯(1.71g，7.8mmol)滴加到上述反应体系中，反应液在室温条件下反应18小时，薄层色谱(石油醚:乙酸乙酯＝3:1)检测反应完毕，将反应液萃取，滤液旋干，过柱纯化，得到产品5-羟基-2-甲基哌啶-1-甲酸叔丁酯(1.7g)。6-Methylpiperidin-3-ol (1.10 g, 7.8 mmol) and sodium carbonate (1.6 g, 15.7 mmol) were added to a solution of dichloromethane (100 mL), then di-tert-butyl dicarbonate (1.71 g) , 7.8 mmol) was added dropwise to the above reaction system, and the reaction liquid was reacted at room temperature for 18 hours, and the reaction was completed by thin layer chromatography (petroleum ether: ethyl acetate = 3:1), the reaction mixture was extracted, and the filtrate was dried. Purification by column gave the product 5-hydroxy-2-methylpiperidine-1-carboxylic acid tert-butyl ester (1.7 g).

第三步5-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-甲酸叔丁酯的制备：The third step is the preparation of 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidine-1-carboxylic acid tert-butyl ester:

5-羟基-2-甲基哌啶-1-甲酸叔丁酯(7g,32.6mmol)和3-碘-1H-吡唑并[3,4-d]嘧啶-4-胺(8.5g,32.6mmol)溶于四氢呋喃(100mL)。冰浴下一次加入三苯基膦(11g,41.5mmol)，缓慢滴加偶氮二甲酸二异丙酯(9.6g,47.5mmol)。撤去冰浴，反应液室温搅拌18小时，减压蒸干。粗产物过硅胶柱纯化(乙酸乙酯:石油醚＝1：1)，得到产品5-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-甲酸叔丁酯(12.2g,82％)。tert-Butyl 5-hydroxy-2-methylpiperidine-1-carboxylate (7 g, 32.6 mmol) and 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (8.5 g, 32.6 Methyl) was dissolved in tetrahydrofuran (100 mL). Triphenylphosphine (11 g, 41.5 mmol) was added in the next ice bath, and diisopropyl azodicarboxylate (9.6 g, 47.5 mmol) was slowly added dropwise. The ice bath was removed, and the reaction mixture was stirred at room temperature for 18 hr and evaporated. The crude product was purified by silica gel column (ethyl acetate: petroleum ether = 1:1) to give the product 5-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl) tert-Butyl 2-methylpiperidine-1-carboxylate (12.2 g, 82%).

MS:m/z 459.2[M+1]MS: m/z 459.2 [M+1]

第四步5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-甲酸叔丁酯的制备： Step 4 5-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl Preparation of tert-butyl 2-methylpiperidine-1-carboxylate:

5-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-甲酸叔丁酯(1g,2.2mmol)和5-氯-2-(4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)苯氧基)吡啶(800mg,2.4mmol)溶于95％乙醇(50mL)。加入碳酸钠(500mg,4.7mmol)和二(三苯基膦)二氯化钯(150mg,0.21mmol)，氩气保护下回流反应18小时，减压蒸干。粗品在二氯甲烷(50mL)和水(50mL)中进行萃取。有机相用无水硫酸钠干燥，过滤，减压蒸干。残余物制备板纯化(二氯甲烷:甲醇＝50：1)，得到产品5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-甲酸叔丁酯(700mg,60％)。5-(4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidine-1-carboxylic acid tert-butyl ester (1 g, 2.2 mmol) 5-Chloro-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)pyridine (800 mg, 2.4 mmol) In 95% ethanol (50 mL). Sodium carbonate (500 mg, 4.7 mmol) and bis(triphenylphosphine)palladium dichloride (150 mg, 0.21 mmol) were added, and the mixture was refluxed under argon atmosphere for 18 hr. The crude product was extracted in dichloromethane (50 mL) and water (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified (dichloromethane:methanol = 50:1) to give the product 5-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H. -pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidine-1-carboxylic acid tert-butyl ester (700 mg, 60%).

MS:m/z 536.3[M+1]MS: m/z 536.3 [M+1]

第五步3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(6-甲基哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐的制备：The fifth step is 3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(6-methylpiperidin-3-yl)-1H-pyrazolo[3,4 -d]Preparation of pyrimidine-4-amine hydrochloride:

5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-甲酸叔丁酯(700mg,1.3mmol)溶于四氢呋喃(10mL)，加入浓盐酸(1mL)，反应液60℃搅拌3小时，减压蒸干得粗产物3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(6-甲基哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐(550mg,89％)。直接用于下一步反应。5-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2 Methylpiperidine-1-carboxylic acid tert-butyl ester (700 mg, 1.3 mmol) was dissolved in tetrahydrofuran (10 mL), and concentrated hydrochloric acid (1 mL) was evaporated. 4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(6-methylpiperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4 -amine hydrochloride (550 mg, 89%). Used directly in the next step.

MS:m/z 436.4[M+1]MS: m/z 436.4 [M+1]

第六步5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈的制备：Step 6 5-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl Preparation of 2-methylpiperidine-1-carbonitrile:

3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(6-甲基哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐(250mg,0.53mmol)溶于N,N-二甲基甲酰胺(3mL)，加入碳酸铯(250mg,0.77mmol)和溴化氰(250mg,2.36mmol)，反应液室温搅拌6小时，反应液倒入乙酸乙酯(50mL)，水洗(30mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备板纯化(二氯甲烷:甲醇＝50：1)，得产品5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(120mg,49％)。3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(6-methylpiperidin-3-yl)-1H-pyrazolo[3,4-d] Pyrimidine-4-amine hydrochloride (250 mg, 0.53 mmol) was dissolved in N,N-dimethylformamide (3 mL), EtOAc (250 mg, 0.77 mmol) and cyanide bromide (250 mg, 2. The solution was stirred at room temperature for 6 hr. EtOAc EtOAc m. The crude product preparation plate was purified (dichloromethane:methanol = 50:1) to give the product 5-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H. Pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidine-1-carbonitrile (120 mg, 49%).

¹H-NMR(400MHz,DMSO-d6):δppm 11.67(brs,1H),8.20(s,1H),8.06(d,J＝2.4,1H),7.68-7.64(m,3H),7.28(d,J＝8.8,2H),6.95(d,J＝8.4,1H),6.43(brs, 1H),4.94-4.91(m,1H),3.92-3.87(m,1H),3.48-3.44(m,3H),2.49-2.46(m,1H),2.02-1.88(m,3H),1.37(d,J＝6.8,3H). ^{1 H-NMR (400MHz, DMSO} -d6): δppm 11.67 (brs, 1H), 8.20 (s, 1H), 8.06 (d, J = 2.4,1H), 7.68-7.64 (m, 3H), 7.28 (d , J=8.8, 2H), 6.95 (d, J=8.4, 1H), 6.43 (brs, 1H), 4.94-4.91 (m, 1H), 3.92-3.87 (m, 1H), 3.48-3.44 (m, 3H), 2.49-2.46 (m, 1H), 2.02-1.88 (m, 3H), 1.37 (d, J = 6.8, 3H).

MS:m/z 461.4[M+1]MS: m/z 461.4 [M+1]

实施例8Example 8

(E)-1-(5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-4-羟基丁-2-烯-1-酮(WS-307)的制备(E)-1-(5-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine Preparation of -1-yl)-2-methylpiperidin-1-yl)-4-hydroxybut-2-en-1-one (WS-307)

以实施例7第五步反应的产物为起始物，实施例8经过以下的步骤制备：The product of the reaction of the fifth step of Example 7 was used as a starting material, and Example 8 was prepared by the following steps:

第一步(E)-1-(5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-4-羟基丁-2-烯-1-酮的制备：First step (E)-1-(5-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4- Preparation of d]pyrimidin-1-yl)-2-methylpiperidin-1-yl)-4-hydroxybut-2-en-1-one:

3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(6-甲基哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐(250mg,0.53mmol)和4-羟基-2-丁烯酸(100mg,0.98mmol)溶于四氢呋喃(10mL)，加入HATU(300mg,0.79mmol)和三乙胺(100mg,0.99mmol)，反应液室温搅拌18小时，减压蒸干。残余物溶于乙酸乙酯(50mL)，水洗(30mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备板纯化(二氯甲烷:甲醇＝10：1)，得到产品(E)-1-(5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-4-羟基丁-2-烯-1-酮(155mg,50％)。3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(6-methylpiperidin-3-yl)-1H-pyrazolo[3,4-d] Pyrimidine-4-amine hydrochloride (250 mg, 0.53 mmol) and 4-hydroxy-2-butenoic acid (100 mg, 0.98 mmol) were dissolved in tetrahydrofuran (10 mL), and HATU (300 mg, 0.79 mmol) and triethylamine ( 100 mg, 0.99 mmol), the reaction mixture was stirred at room temperature for 18 hr. The residue was dissolved in ethyl acetate (50 mL)EtOAc. The crude product preparation plate was purified (dichloromethane:methanol = 10:1) to give the product (E)-1-(5-(4-amino-3-(4-((5-chloropyridin-2-yl))) Phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidin-1-yl)-4-hydroxybut-2-en-1-one ( 155 mg, 50%).

¹H-NMR(400MHz,DMSO-d6):δppm 8.35(d,J＝7.6,1H),8.14(d,J＝2.8,1H),7.75-7.69(m,3H),7.31(d,J＝7.6,2H),6.99-6.95(m,2H),6.60-6.50(m,1H),5.75(brs,1.7H),5.05(brs,0.4H),4.82-4.70(m,1.6H),4.50-4.35(m,2.5H), 4.10-4.05(m,0.5H),3.90-3.80(m,0.5H),3.50-3.45(m,0.7H),3.10-3.00(m,0.2H),2.60-2.50(m,1.3H),2.07-1.75(m,5.4H),1.43-1.25(m,3.2H). ¹ H-NMR (400 MHz, DMSO-d6): δ ppm 8.35 (d, J = 7.6, 1H), 8.14 (d, J = 2.8, 1H), 7.75 - 7.69 (m, 3H), 7.31 (d, J = 7.6, 2H), 6.99-6.95 (m, 2H), 6.60-6.50 (m, 1H), 5.75 (brs, 1.7H), 5.05 (brs, 0.4H), 4.82-4.70 (m, 1.6H), 4.50 -4.35 (m, 2.5H), 4.10-4.05 (m, 0.5H), 3.90-3.80 (m, 0.5H), 3.50-3.45 (m, 0.7H), 3.10-3.00 (m, 0.2H), 2.60 -2.50 (m, 1.3H), 2.07-1.75 (m, 5.4H), 1.43-1.25 (m, 3.2H).

MS:m/z 520.4[M+1]MS: m/z 520.4 [M+1]

实施例9Example 9

5-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-308)的制备5-(4-Amino-3-(4-(2,4-difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methyl Preparation of piperidine-1-nitrile (WS-308)

实施例9从对应的起始物开始，采用与实施例7类似的方法制备而得。Example 9 was prepared in a similar manner to Example 7 starting from the corresponding starting material.

¹H-NMR(400MHz,DMSO-d6):8.39(s,1H),7.68(d,J＝8.8,1H),7.19-7.08(m,3H),7.03-6.90(m,2H),5.57(brs,2H),4.98-4.94(m,1H),3.97-3.92(m,1H),3.57-3.46(m,2H),2.56-2.51(m,1H),2.06-1.95(m,3H),1.43(d,J＝7.8,3H). ^{1 H-NMR (400MHz, DMSO} -d6): 8.39 (s, 1H), 7.68 (d, J = 8.8,1H), 7.19-7.08 (m, 3H), 7.03-6.90 (m, 2H), 5.57 ( Brs, 2H), 4.98-4.94 (m, 1H), 3.97-3.92 (m, 1H), 3.57-3.46 (m, 2H), 2.56-2.51 (m, 1H), 2.06-1.95 (m, 3H), 1.43 (d, J = 7.8, 3H).

MS:m/z 462.3[M+1]MS: m/z 462.3 [M+1]

实施例10Example 10

(2S,5R)-5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-309)和(2R,5S)-5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-310)的制备 (2S,5R)-5-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine- 1-yl)-2-methylpiperidine-1-carbonitrile (WS-309) and (2R,5S)-5-(4-amino-3-(4-((5-chloropyridin-2-yl))) Preparation of oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidine-1-carbonitrile (WS-310)

实施例10是实施例7的产物经手性拆分后制得。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x 250mm col,27％甲醇,70mL/分钟)。Example 10 was prepared by chiral resolution of the product of Example 7. The resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5μ, 21 x 250 mm col, 27% methanol, 70 mL/min).

WS-309谱图数据：WS-309 spectrum data:

¹H-NMR(400MHz,DMSO-d6):δppm 11.67(brs,1H),8.20(s,1H),8.06(d,J＝2.4,1H),7.68-7.64(m,3H),7.28(d,J＝8.8,2H),6.95(d,J＝8.4,1H),6.43(brs,1H),4.94-4.91(m,1H),3.92-3.87(m,1H),3.48-3.44(m,3H),2.49-2.46(m,1H),2.02-1.88(m,3H),1.37(d,J＝6.8,3H). ^{1 H-NMR (400MHz, DMSO} -d6): δppm 11.67 (brs, 1H), 8.20 (s, 1H), 8.06 (d, J = 2.4,1H), 7.68-7.64 (m, 3H), 7.28 (d , J=8.8, 2H), 6.95 (d, J=8.4, 1H), 6.43 (brs, 1H), 4.94-4.91 (m, 1H), 3.92-3.87 (m, 1H), 3.48-3.44 (m, 3H), 2.49-2.46 (m, 1H), 2.02-1.88 (m, 3H), 1.37 (d, J = 6.8, 3H).

MS:m/z 461.4[M+1]MS: m/z 461.4 [M+1]

WS-310谱图数据：WS-310 spectrum data:

MS:m/z 461.4[M+1]MS: m/z 461.4 [M+1]

实施例11Example 11

(E)-1-((2S,5R)-5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-4-羟基丁-2-烯-1-酮(WS-311)和(E)-1-((2R,5S)-5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-4-羟基丁-2-烯-1-酮(WS-312)的制备 (E)-1-((2S,5R)-5-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3 , 4-d]pyrimidin-1-yl)-2-methylpiperidin-1-yl)-4-hydroxybut-2-en-1-one (WS-311) and (E)-1-(( 2R,5S)-5-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine-1 Of 2-yl-2-methylpiperidin-1-yl)-4-hydroxybut-2-en-1-one (WS-312)

实施例11是实施例8的产品经手性拆分后制得。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x 250mm col,27％甲醇,70mL/分钟)。Example 11 was prepared by chiral resolution of the product of Example 8. The resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5μ, 21 x 250 mm col, 27% methanol, 70 mL/min).

WS-311谱图数据：WS-311 spectrum data:

¹H-NMR(400MHz,DMSO-d6):δppm 8.35(d,J＝7.6,1H),8.14(d,J＝2.8,1H),7.75-7.69(m,3H),7.31(d,J＝7.6,2H),6.99-6.95(m,2H),6.60-6.50(m,1H),5.75(brs,1.7H),5.05(brs,0.4H),4.82-4.70(m,1.6H),4.50-4.35(m,2.5H),4.10-4.05(m,0.5H),3.90-3.80(m,0.5H),3.50-3.45(m,0.7H),3.10-3.00(m,0.2H),2.60-2.50(m,1.3H),2.07-1.75(m,5.4H),1.43-1.25(m,3.2H). ¹ H-NMR (400 MHz, DMSO-d6): δ ppm 8.35 (d, J = 7.6, 1H), 8.14 (d, J = 2.8, 1H), 7.75 - 7.69 (m, 3H), 7.31 (d, J = 7.6, 2H), 6.99-6.95 (m, 2H), 6.60-6.50 (m, 1H), 5.75 (brs, 1.7H), 5.05 (brs, 0.4H), 4.82-4.70 (m, 1.6H), 4.50 -4.35 (m, 2.5H), 4.10-4.05 (m, 0.5H), 3.90-3.80 (m, 0.5H), 3.50-3.45 (m, 0.7H), 3.10-3.00 (m, 0.2H), 2.60 -2.50 (m, 1.3H), 2.07-1.75 (m, 5.4H), 1.43-1.25 (m, 3.2H).

MS:m/z 520.4[M+1]MS: m/z 520.4 [M+1]

WS-312谱图数据：WS-312 spectrum data:

MS:m/z 520.4[M+1]MS: m/z 520.4 [M+1]

实施例12 Example 12

(2S,5R)-5-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-313)和(2R,5S)-5-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-314)的制备(2S,5R)-5-(4-Amino-3-(4-(2,4-difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl )-2-methylpiperidin-1-carbonitrile (WS-313) and (2R,5S)-5-(4-amino-3-(4-(2,4-difluorophenoxy)phenyl) Preparation of -1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidine-1-carbonitrile (WS-314)

实施例12是实施例9的产品经手性拆分后制得。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x 250mm col,27％甲醇,70mL/分钟)。Example 12 was prepared by chiral resolution of the product of Example 9. The resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5μ, 21 x 250 mm col, 27% methanol, 70 mL/min).

WS-313谱图数据：WS-313 spectrum data:

MS:m/z 462.3[M+1]MS: m/z 462.3 [M+1]

WS-314谱图数据：WS-314 spectrum data:

MS:m/z 462.3[M+1]MS: m/z 462.3 [M+1]

实施例13Example 13

6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-腈(WS-315)的制备 6-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4 -Preparation of azaspiro[2.5]octane-4-carbonitrile (WS-315)

第一步4-羟基丁酸甲酯的制备：The first step of the preparation of methyl 4-hydroxybutyrate:

将二氢呋喃-2(3H)-酮(100g，1.163mol)和三乙胺(460g,4.65mol)加入到甲醇(1L)溶液中，反应液在60℃的条件下反应24小时，薄层色谱(石油醚:乙酸乙酯＝2:1)检测反应完毕，将反应液旋干，得到4-羟基丁酸甲酯(120g，87.6％，黄色液体)，直接用于下一步反应。Dihydrofuran-2(3H)-one (100g, 1.163mol) and triethylamine (460g, 4.65mol) were added to a solution of methanol (1L), and the reaction solution was reacted at 60 ° C for 24 hours, thin layer The reaction was completed by chromatography (petroleum ether: ethyl acetate = 2:1), and the reaction mixture was evaporated to dryness to afford methyl 4-hydroxybutyrate (120 g, 87.6%, yellow liquid).

第二步4-氧代丁酸甲酯的制备：The second step is the preparation of methyl 4-oxobutanoate:

将4-羟基丁酸甲酯(120g，1.02mol)加入到二氯甲烷(1.2L)溶液中，然后将氯铬酸吡啶(330g，1.53mol)加入到上述反应液中，室温条件下反应12小时，薄层色谱(石油醚:乙酸乙酯＝3:1)检测反应完毕，将反应液通过硅藻土过滤，旋干，得到4-氧代丁酸甲酯(60g，50％，黄色液体)，直接用于下一步反应。Methyl 4-hydroxybutyrate (120 g, 1.02 mol) was added to a dichloromethane (1.2 L) solution, and then pyridine chlorochromate (330 g, 1.53 mol) was added to the above reaction solution, and the reaction was carried out at room temperature. The reaction was completed by thin layer chromatography (petroleum ether: ethyl acetate = 3:1). The reaction mixture was filtered through celite and dried to give methyl 4-oxobutyrate (60 g, 50%, yellow liquid) ), used directly in the next step.

第三步4-羟基-5-硝基戊酸甲酯的制备：The third step is the preparation of methyl 4-hydroxy-5-nitropentanoate:

冰水浴中，将4-氧代丁酸甲酯(60g，0.46mol)，硝基甲烷(42g，0.69mol),四氢呋喃(300mL)和叔丁醇(300mL)加入到反应瓶中，然后将叔丁醇钾(5g)缓慢加入上述反应体系中，升至室温，反应2小时，薄层色谱(石油醚:乙酸乙酯＝3:1)检测反应完毕，加水(30mL)淬灭反应，旋干溶剂，加入水(300mL)和乙酸乙酯(300mL)分液，有机相用饱和食盐水洗涤，无水硫酸钠干燥，旋干，得到粗品4-羟基-5-硝基戊酸甲酯(45g，淡黄色油状液体)直接用于下一步。In an ice water bath, methyl 4-oxobutanoate (60 g, 0.46 mol), nitromethane (42 g, 0.69 mol), tetrahydrofuran (300 mL) and tert-butanol (300 mL) were added to the reaction flask, then uncle Potassium butoxide (5g) was slowly added to the above reaction system, raised to room temperature, reacted for 2 hours, and the reaction was completed by thin layer chromatography (petroleum ether: ethyl acetate = 3:1). The reaction was quenched with water (30 mL) and dried. The solvent was added with water (300 mL) and ethyl acetate (300 mL). EtOAc. , light yellow oily liquid) used directly in the next step.

第四步5-羟基哌啶-2-酮的制备：The fourth step is the preparation of 5-hydroxypiperidin-2-one:

将4-羟基-5-硝基戊酸甲酯(45g，0.23mol)和钯碳(2.1g)加入到甲醇(500mL)溶液中，反应液在60℃，氢气存在的条件下反应24小时，薄层色谱(石油醚:乙酸乙酯＝1:1)检测反应完毕，将反应液通过硅藻土过滤，滤液旋干，得到5-羟基哌啶-2-酮(10g，黄色固体,38％)，直接用于下一步反应。Methyl 4-hydroxy-5-nitropentanoate (45 g, 0.23 mol) and palladium on carbon (2.1 g) were added to a solution of methanol (500 mL), and the reaction mixture was reacted at 60 ° C in the presence of hydrogen for 24 hours. Thin layer chromatography Ether: ethyl acetate = 1:1) After the reaction was completed, the reaction mixture was filtered through Celite, and the filtrate was evaporated to give 5-hydroxypiperidin-2-one (10 g, yellow solid, 38%). The next step is to react.

第五步1-苄基-5-(苄氧基)哌啶-2-酮的制备：Step 5: Preparation of 1-benzyl-5-(benzyloxy)piperidin-2-one:

室温下，将5-羟基哌啶-2-酮(10g，0.1mol)加入到二甲基亚砜(100mL)中，然后将氢化钠(10g，0.25mol)缓慢加入到上述反应体系中，加入完毕后，将苄溴(43.5g，0.25mol)加入到反应液中，搅拌过夜，薄层色谱(石油醚:乙酸乙酯＝1:1)检测反应完毕，向反应体系中加入饱和氯化铵(100ml)淬灭反应，乙酸乙酯(100mL*3)萃取三次，饱和食盐水洗涤，无水硫酸钠干燥，旋干，过柱纯化，得到1-苄基-5-(苄氧基)哌啶-2-酮(16g，黄色固体，54％)。5-hydroxypiperidin-2-one (10 g, 0.1 mol) was added to dimethyl sulfoxide (100 mL) at room temperature, then sodium hydride (10 g, 0.25 mol) was slowly added to the above reaction system, and added. After completion, benzyl bromide (43.5 g, 0.25 mol) was added to the reaction solution, stirred overnight, and the reaction was completed by thin layer chromatography (petroleum ether: ethyl acetate = 1:1), and saturated ammonium chloride was added to the reaction system. The reaction was quenched (100 ml), EtOAc (EtOAc (EtOAc)EtOAc. Pyridin-2-one (16 g, yellow solid, 54%).

第六步4-苄基-6-(苄氧基)-4-氮杂螺[2.5]辛烷的制备：Step 6 Preparation of 4-benzyl-6-(benzyloxy)-4-azaspiro[2.5]octane:

-78℃氮气保护下，将1-苄基-5-(苄氧基)哌啶-2-酮(15g，50mmol)溶于无水四氢呋喃(150mL)中，然后向反应瓶中缓慢滴加乙基溴化镁(150mL)，滴加完毕后，再将钛酸四丙酯(45g，150mmol)加入到上述反应体系中，滴加完毕，将反应液升至室温，搅拌2小时，薄层色谱(石油醚:乙酸乙酯＝10:1)检测反应完毕，向反应体系中加入饱和氯化铵(100ml)淬灭反应，乙酸乙酯(100mL*3)萃取三次，饱和食盐水洗涤，无水硫酸钠干燥，旋干，过柱纯化，得到4-苄基-6-(苄氧基)-4-氮杂螺[2.5]辛烷(5.1g，黄色固体,31％)。1-benzyl-5-(benzyloxy)piperidin-2-one (15 g, 50 mmol) was dissolved in anhydrous tetrahydrofuran (150 mL) under nitrogen at -78 ° C, then slowly added dropwise to the reaction flask. After the dropwise addition of magnesium bromide (150 mL), tetrapropyl titanate (45 g, 150 mmol) was added to the above reaction system. After the addition was completed, the reaction solution was allowed to warm to room temperature, stirred for 2 hours, and thin layer chromatography (Petroleum ether: ethyl acetate = 10:1) After the reaction was completed, the reaction was quenched by the addition of saturated ammonium chloride (100 ml), and ethyl acetate (100 mL*3) was extracted three times, washed with brine and dried. Drying over sodium sulphate, EtOAc EtOAc (EtOAc)

第七步4-氮杂螺[2.5]辛-6-醇的制备：Step 7: Preparation of 4-azaspiro[2.5]oct-6-ol:

将4-苄基-6-(苄氧基)-4-氮杂螺[2.5]辛烷(5.5g，18mmol)和钯碳(2g,1.8mmol)加入到甲醇(200mL)和氯化氢(2mL)溶液中，反应液在60℃，氢气条件下反应48小时，薄层色谱(石油醚:乙酸乙酯＝10:1)检测反应完毕，将反应液通过硅藻土过滤，滤液旋干，得到4-氮杂螺[2.5]辛-6-醇(2.5g，黄色固体)，直接用于下一步反应。Add 4-benzyl-6-(benzyloxy)-4-azaspiro[2.5]octane (5.5 g, 18 mmol) and palladium on carbon (2 g, 1.8 mmol) to methanol (200 mL) and hydrogen chloride (2 mL) In the solution, the reaction solution was reacted at 60 ° C under hydrogen for 48 hours, and the reaction was completed by thin layer chromatography (petroleum ether: ethyl acetate = 10:1). The reaction solution was filtered through celite, and the filtrate was dried to give 4 - Azaspiro[2.5]oct-6-ol (2.5 g, yellow solid) was used directly in the next step.

第八步6-羟基-4-氮杂螺[2.5]辛烷-4-甲酸叔丁酯的制备：The eighth step of the preparation of 6-hydroxy-4-azaspiro[2.5]octane-4-carboxylic acid tert-butyl ester:

将4-氮杂螺[2.5]辛-6-醇(1g，7.8mmol)和碳酸钠(1.6g,15.7mmol)加入到二氯甲烷(100mL)溶液中，然后将二碳酸二叔丁酯(1.71g，7.8mmol)滴加到上述反应体系中，反应液在室温条件下反应18小时，薄层色谱(石油醚: 乙酸乙酯＝3:1)检测反应完毕，将反应液萃取，滤液旋干，过柱纯化，得到6-羟基-4-氮杂螺[2.5]辛烷-4-甲酸叔丁酯(1.7g)。4-Azaspiro[2.5]oct-6-ol (1 g, 7.8 mmol) and sodium carbonate (1.6 g, 15.7 mmol) were added to a solution of dichloromethane (100 mL) and then di-tert-butyl dicarbonate ( 1.71 g, 7.8 mmol) was added dropwise to the above reaction system, and the reaction solution was reacted at room temperature for 18 hours, and thin layer chromatography (petroleum ether: Ethyl acetate = 3:1) After the completion of the reaction, the reaction mixture was extracted, the filtrate was spun and purified by column to give tert-butyl 6-hydroxy-4-azaspiro[2.5]octane-4-carboxylate (1.7 g) ).

第九步6-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-甲酸叔丁酯的制备：Step 9 6-(4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-azaspiro[2.5]octane-4-carboxylic acid tert-butyl ester Preparation:

6-羟基-4-氮杂螺[2.5]辛烷-4-甲酸叔丁酯(2.5g,11.0mmol)和3-碘-1H-吡唑并[3,4-d]嘧啶-4-胺(3.4g,13.0mmol)溶于四氢呋喃(200mL)。冰浴下一次加入三苯基膦(4.5g,17.2mmol)，缓慢滴加偶氮二甲酸二异丙酯(3.5g,17.3mmol)。撤去冰浴，反应液室温搅拌18小时，减压蒸干。粗产物过硅胶柱纯化(EA:PE＝1：1)，得产品6-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-甲酸叔丁酯(3.6g,70％)。4-Hydroxy-4-azaspiro[2.5]octane-4-carboxylic acid tert-butyl ester (2.5 g, 11.0 mmol) and 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (3.4 g, 13.0 mmol) was dissolved in tetrahydrofuran (200 mL). Triphenylphosphine (4.5 g, 17.2 mmol) was added in the ice bath, and diisopropyl azodicarboxylate (3.5 g, 17.3 mmol) was slowly added dropwise. The ice bath was removed, and the reaction mixture was stirred at room temperature for 18 hr and evaporated. The crude product was purified by silica gel column (EA: PE = 1:1) to give the product 6-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4- Azaspiro[2.5]octane-4-carboxylic acid tert-butyl ester (3.6 g, 70%).

第十步6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-甲酸叔丁酯的制备：Step 10 6-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl Preparation of tert-butyl 4-azaspiro[2.5]octane-4-carboxylate:

6-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-甲酸叔丁酯(500mg,1.1mmol)和5-氯-2-(4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)苯氧基)吡啶(500mg,1.5mmol)溶于95％乙醇(20mL)中。加入碳酸钠(230mg,2.2mmol)和二(三苯基膦)二氯化钯(120mg,0.2mmol)，氩气保护下回流反应18小时，减压蒸干。粗品用二氯甲烷(50mL)和水(50mL)萃取。有机相用无水硫酸钠干燥，过滤，减压蒸干。残余物制备板纯化(二氯甲烷:甲醇＝50：1)，得产品6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-甲酸叔丁酯(420mg,76％)。6-(4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-azaspiro[2.5]octane-4-carboxylic acid tert-butyl ester (500 mg, 1.1 mmol) and 5-chloro-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)pyridine (500 mg, 1.5 mmol) was dissolved in 95% ethanol (20 mL). Sodium carbonate (230 mg, 2.2 mmol) and bis(triphenylphosphine)palladium dichloride (120 mg, 0.2 mmol) were added, and the mixture was refluxed under argon atmosphere for 18 hr. The crude was extracted with dichloromethane (50 mL) and water (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified (dichloromethane:methanol = 50:1) to give the product 6-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H. Pyrazolo[3,4-d]pyrimidin-1-yl)-4-azaspiro[2.5]octane-4-carboxylic acid tert-butyl ester (420 mg, 76%).

第十一步3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐的制备：The eleventh step 3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(4-azaspiro[2.5]oct-6-yl)-1H-pyrazole Preparation of [3,4-d]pyrimidine-4-amine hydrochloride:

6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-甲酸叔丁酯(420mg,0.8mmol)溶于四氢呋喃(10mL)，加入浓盐酸(1mL)，反应液60℃搅拌5小时，减压蒸干得粗产物3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐(450mg)。直接用于下一步反应。 6-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4 - aza-spiro[2.5]octane-4-carboxylic acid tert-butyl ester (420 mg, 0.8 mmol) was dissolved in tetrahydrofuran (10 mL), concentrated hydrochloric acid (1 mL) was added, and the reaction mixture was stirred at 60 ° C for 5 hr. The product 3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(4-azaspiro[2.5]oct-6-yl)-1H-pyrazolo[3, 4-d]pyrimidine-4-amine hydrochloride (450 mg). Used directly in the next step.

第十二步6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-腈的制备：Step 12 6-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1- Preparation of 4-azaspiro[2.5]octane-4-carbonitrile:

3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐(200mg,0.44mmol)溶于N,N-二甲基甲酰胺(2mL)，加入碳酸铯(200mg,0.62mmol)和溴化氰(200mg,1.89mmol)，反应液室温搅拌6小时，反应液倒入乙酸乙酯(50mL)，水洗(30mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备板纯化(二氯甲烷:甲醇＝50：1)，得产品化合物6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-腈(31mg,15.9％)。3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(4-azaspiro[2.5]oct-6-yl)-1H-pyrazolo[3,4 -d]pyrimidin-4-amine hydrochloride (200 mg, 0.44 mmol) dissolved in N,N-dimethylformamide (2 mL), EtOAc (200 mg, 0.62 mmol) The reaction mixture was stirred at room temperature for 6 hr., and the mixture was evaporated to ethyl acetate (50 mL). The crude product preparation plate was purified (dichloromethane:methanol = 50:1) to give the product compound 6-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)- 1H-Pyrazolo[3,4-d]pyrimidin-1-yl)-4-azaspiro[2.5]octane-4-carbonitrile (31 mg, 15.9%).

¹H-NMR(400MHz,DMSO-d6):δppm 8.30-8.26(m,2H),8.02-7.99(m,1H),7.73(d,J＝8.4,2H),7.32(d,J＝8.4,2H),7.18(d,J＝8.8,1H),5.03-5.01(m,1H),3.67-3.57(m,2H),2.39-2.36(m,1H),2.19-2.15(m,2H),1.57-1.53(m,1H),0.98-0.77(m,4H). ^{1 H-NMR (400MHz, DMSO} -d6): δppm 8.30-8.26 (m, 2H), 8.02-7.99 (m, 1H), 7.73 (d, J = 8.4,2H), 7.32 (d, J = 8.4, 2H), 7.18 (d, J = 8.8, 1H), 5.03-5.01 (m, 1H), 3.67-3.57 (m, 2H), 2.39-2.36 (m, 1H), 2.19-2.15 (m, 2H), 1.57-1.53 (m, 1H), 0.98-0.77 (m, 4H).

MS:m/z 473.2[M+1]MS: m/z 473.2 [M+1]

实施例14Example 14

(R)-6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-腈(WS-316)和(S)-6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-腈(WS-317)的制备(R)-6-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1- 4-Azaspiro[2.5]octane-4-carbonitrile (WS-316) and (S)-6-(4-amino-3-(4-((5-chloropyridin-2-yl)) Preparation of oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-azaspiro[2.5]octane-4-carbonitrile (WS-317)

实施例13第十二步反应的产物经手性拆分后可得实施例14化合物。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x 250mm col,27％甲醇,70mL/分钟)。The product of Example 14 was obtained by chiral resolution of the product of the twelfth step of Example 13. The resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5μ, 21 x 250 mm col, 27% methanol, 70 mL/min).

WS-316谱图数据：WS-316 spectrum data:

MS:m/z 473.2[M+1]MS: m/z 473.2 [M+1]

WS-317谱图数据：WS-317 spectrum data:

MS:m/z 473.2[M+1]MS: m/z 473.2 [M+1]

实施例15Example 15

(E)-1-(6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛-4-基)-4-羟基丁-2-烯-1-酮(WS-318)的制备(E)-1-(6-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine Preparation of -1-yl)-4-azaspiro[2.5]octyl-4-yl)-4-hydroxybut-2-en-1-one (WS-318)

以实施例13第十一步反应的产物为起始物，实施例15经过以下的步骤制备： Starting from the product of the eleventh step of Example 13, the preparation of Example 15 was carried out by the following procedure:

第一步(E)-1-(6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛-4-基)-4-羟基丁-2-烯-1-酮的制备：First step (E)-1-(6-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4- Preparation of d]pyrimidin-1-yl)-4-azaspiro[2.5]oct-4-yl)-4-hydroxybut-2-en-1-one:

3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐(220mg,0.48mmol)和4-羟基-2-丁烯酸(80mg,0.78mmol)溶于四氢呋喃(5mL)，加入HATU(230mg,0.61mmol)和三乙胺(100mg,0.99mmol)，反应液室温搅拌18小时，减压蒸干。残余物溶于乙酸乙酯(50mL)，水洗(30mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备板纯化(二氯甲烷:甲醇＝10：1),得产品(E)-1-(6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛-4-基)-4-羟基丁-2-烯-1-酮(30mg,12％)。3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(4-azaspiro[2.5]oct-6-yl)-1H-pyrazolo[3,4 -d]pyrimidine-4-amine hydrochloride (220 mg, 0.48 mmol) and 4-hydroxy-2-butenoic acid (80 mg, 0.78 mmol) were dissolved in tetrahydrofuran (5 mL), and HATU (230 mg, 0.61 mmol) and Ethylamine (100 mg, 0.99 mmol). The residue was dissolved in ethyl acetate (50 mL)EtOAc. The crude product preparation plate was purified (dichloromethane:methanol = 10:1) to give the product (E)-1-(6-(4-amino-3-(4-((5-chloropyridin-2-yl))oxy) Phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-azaspiro[2.5]oct-4-yl)-4-hydroxybut-2-ene-1 - Ketone (30 mg, 12%).

¹H-NMR(400MHz,DMSO-d6):δppm 8.32(s,1H),8.26(d,J＝2.8,1H),8.02-7.99(m,1H),7.73-7.71(m,2H),7.34-7.31(m,2H),7.21-7.17(m,2H),7.08-6.81(m,3H),4.80-4.50(m,3H),4.20-4.00(m,2H),3.55-3.53(m,2H),2.20-2.00(m,2H),1.40-1.10(m,2H),0.90-0.70(m,2H) ^{1 H-NMR (400MHz, DMSO} -d6): δppm 8.32 (s, 1H), 8.26 (d, J = 2.8,1H), 8.02-7.99 (m, 1H), 7.73-7.71 (m, 2H), 7.34 -7.31 (m, 2H), 7.21-7.17 (m, 2H), 7.08-6.81 (m, 3H), 4.80-4.50 (m, 3H), 4.20-4.00 (m, 2H), 3.55-3.53 (m, 2H), 2.20-2.00 (m, 2H), 1.40-1.10 (m, 2H), 0.90-0.70 (m, 2H)

MS:m/z 532.2[M+1]MS: m/z 532.2 [M+1]

实施例16Example 16

(R,E)-1-(6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛-4-基)-4-羟基丁-2-烯-1-酮(WS-319)和(S,E)-1-(6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛-4-基)-4-羟基丁-2-烯-1-酮(WS-320)制备(R,E)-1-(6-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d Pyrimidin-1-yl)-4-azaspiro[2.5]oct-4-yl)-4-hydroxybut-2-en-1-one (WS-319) and (S,E)-1-( 6-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4 -Azaspiro[2.5]oct-4-yl)-4-hydroxybut-2-en-1-one (WS-320) Preparation

实施例15的化合物经手性拆分后可得实施例16的化合物。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x 250mm col,27％甲醇,70mL/分钟)。The compound of Example 15 was obtained after chiral resolution of the compound of Example 15. The resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5μ, 21 x 250 mm col, 27% methanol, 70 mL/min).

WS-319谱图数据：WS-319 spectrum data:

MS:m/z 532.2[M+1]MS: m/z 532.2 [M+1]

WS-320谱图数据：WS-320 spectrum data:

MS:m/z 532.2[M+1]MS: m/z 532.2 [M+1]

实施例17Example 17

6-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-腈(WS-321)的制备6-(4-Amino-3-(4-(2,4-difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-aza Preparation of spiro[2.5]octane-4-carbonitrile (WS-321)

实施例17从对应的起始物开始，采用与实施例13类似的方法制备而得。 Example 17 was prepared in a similar manner to Example 13 starting from the corresponding starting material.

¹H-NMR(400MHz,DMSO-d6):δppm 8.28(s,1H),7.69-7.53(m,6H),7.39-7.37(m,1H),7.18-7.11(m,2H),5.03-4.98(m,1H),3.64-3.56(m,2H),2.19-2.16(m,1H),2.15-2.06(m,2H),1.34-1.32(m,1H),0.97-0.78(m,4H). ^{1 H-NMR (400MHz, DMSO} -d6): δppm 8.28 (s, 1H), 7.69-7.53 (m, 6H), 7.39-7.37 (m, 1H), 7.18-7.11 (m, 2H), 5.03-4.98 (m,1H), 3.64-3.56 (m, 2H), 2.19-2.16 (m, 1H), 2.15-2.06 (m, 2H), 1.34-1.32 (m, 1H), 0.97-0.78 (m, 4H) .

MS:m/z 474.2[M+1]MS: m/z 474.2 [M+1]

实施例18Example 18

(E)-1-(6-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛-4-基)-4-羟基丁-2-烯-1-酮的制备(WS-322)的制备(E)-1-(6-(4-Amino-3-(4-(2,4-difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1- Preparation of -4-Azaspiro[2.5]oct-4-yl)-4-hydroxybut-2-en-1-one (WS-322)

实施例18从对应的起始物开始，采用与实施例15类似的方法制备而得。Example 18 was prepared in a similar manner to Example 15 starting from the corresponding starting material.

¹H-NMR(400MHz,DMSO-d6):δppm 8.40(s,1H),7.70-7.68(m,2H),7.30-7.04(m,7H),4.75-4.70(m,0.73H),4.55-4.50(m,0.17H),4.13-4.10(m,2H),3.61-3.58(m,2H),3.01-2.95(m,0.65H),2.72-2.60(m,0.81H),2.43-2.38(m,1H),1.84-1.78(m,3H),1.31-1.26(m,2H),0.98-0.70(m,2H) ^{1 H-NMR (400MHz, DMSO} -d6): δppm 8.40 (s, 1H), 7.70-7.68 (m, 2H), 7.30-7.04 (m, 7H), 4.75-4.70 (m, 0.73H), 4.55- 4.50 (m, 0.17H), 4.13-4.10 (m, 2H), 3.61-3.58 (m, 2H), 3.01-2.95 (m, 0.65H), 2.72-2.60 (m, 0.81H), 2.43-2.38 ( m, 1H), 1.84-1.78 (m, 3H), 1.31-1.26 (m, 2H), 0.98-0.70 (m, 2H)

MS:m/z 533.2[M+1]MS: m/z 533.2 [M+1]

实施例19Example 19

(2S,5R)-5-(4-氨基-3-(4-(4-氯苯氧)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-323)和(2R,5S)-5-(4-氨基-3-(4-(4-氯苯氧)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-324)的制备 (2S,5R)-5-(4-Amino-3-(4-(4-chlorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2- Methylpiperidine-1-carbonitrile (WS-323) and (2R,5S)-5-(4-amino-3-(4-(4-chlorophenoxy)phenyl)-1H-pyrazolo[3 ,4-d]pyrimidin-1-yl)-2-methylpiperidine-1-carbonitrile (WS-324)

实施例19从对应的起始物开始，采用与实施例7类似的方法先制备得到5-(4-氨基-3-(4-(4-氯苯氧)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈，该化合物经手性拆分后可得化合物WS-323和WS-324。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x 250mm col,27％甲醇,70mL/分钟)。Example 19 Starting from the corresponding starting material, 5-(4-amino-3-(4-(4-chlorophenoxy)phenyl)-1H-pyrazole was prepared in the same manner as in Example 7. [3,4-d]Pyridine-1-yl)-2-methylpiperidine-1-carbonitrile, which was obtained by chiral resolution to give compounds WS-323 and WS-324. The resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5μ, 21 x 250 mm col, 27% methanol, 70 mL/min).

WS-323谱图数据：WS-323 spectrum data:

¹H-NMR(400MHz,CDCl₃):δppm 8.35(d,J＝11.2Hz,1H),7.72-7.67(m,2H),7.36-7.31(m,2H),7.16-7.04(m,2H),7.02-6.99(m,2H),5.54(s,2H),4.96-4.94(m,1H),3.99-3.91(m,1H),3.54-3.45(m,2H),2.54-2.53(m,1H),2.07-1.97(m,3H),1.44-1.40(m,3H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.35 (d, J = 11.2 Hz, 1H), 7.72-7.67 (m, 2H), 7.36-7.31 (m, 2H), 7.16-7.04 (m, 2H) , 7.02-6.99 (m, 2H), 5.54 (s, 2H), 4.96-4.94 (m, 1H), 3.99-3.91 (m, 1H), 3.54-3.45 (m, 2H), 2.54-2.53 (m, 1H), 2.07-1.97 (m, 3H), 1.44-1.40 (m, 3H).

MS:m/z 460.4[M+H]MS: m/z 460.4 [M+H]

WS-324谱图数据：WS-324 spectrum data:

MS:m/z 460.4[M+H]MS: m/z 460.4 [M+H]

实施例20Example 20

(2S,5R)-5-(4-氨基-3-(4-苯氧苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-325)和(2S,5R)-5-(4-氨基-3-(4-苯氧苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-326)的制备 (2S,5R)-5-(4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidine- 1-nitrile (WS-325) and (2S,5R)-5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl Preparation of 2-methylpiperidine-1-carbonitrile (WS-326)

实施例20从对应的起始物开始，采用与实施例9类似的方法先制备得到5-(4-氨基-3-(4-苯氧苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈，该化合物经手性拆分后可得化合物WS-325和WS-326。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x 250mm col,27％甲醇,70mL/分钟)。Example 20 was prepared starting from the corresponding starting material in a similar manner to Example 9 to give 5-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazole[3,4- d]pyrimidin-1-yl)-2-methylpiperidine-1-carbonitrile, which was chirally resolved to give compounds WS-325 and WS-326. The resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5μ, 21 x 250 mm col, 27% methanol, 70 mL/min).

WS-325谱图数据：WS-325 spectrum data:

¹H-NMR(400MHz,CDCl₃):δppm 8.37(s,1H),7.70-7.66(m,2H),7.42-7.38(m,2H),7.20-7.15(m,3H),7.11-7.08(m,2H),5.51(s,2H),4.99-4.94(m,1H),3.99-3.93(m,1H),3.58-3.47(m,2H),2.56-2.52(m,1H),2.06-1.96(m,3H),1.43(d,J＝6.8,3H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.37 (s, 1H), 7.70-7.66 (m, 2H), 7.42-7.38 (m, 2H), 7.20-7.15 (m, 3H), 7.11-7.08 ( m, 2H), 5.51 (s, 2H), 4.99-4.94 (m, 1H), 3.99-3.93 (m, 1H), 3.58-3.47 (m, 2H), 2.56-2.52 (m, 1H), 2.06- 1.96 (m, 3H), 1.43 (d, J = 6.8, 3H).

MS:m/z 426.5[M+H]MS: m/z 426.5 [M+H]

WS-326谱图数据：WS-326 spectrum data:

MS:m/z 426.5[M+1]MS: m/z 426.5 [M+1]

实施例21 Example 21

1-((2S,5R)-5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-2-丙烯基-1-酮(WS-327)和1-((2R,5S)-5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-2-丙烯基-1-酮(WS-328)的制备1-((2S,5R)-5-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d Pyrimidin-1-yl)-2-methylpiperidin-1-yl)-2-propenyl-1-one (WS-327) and 1-((2R,5S)-5-(4-amino- 3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidine-1 Of 2-yl-2-propen-1-one (WS-328)

以实施例7第五步反应的产物为起始物，实施例21经过以下的步骤制备：The product of the reaction of the fifth step of Example 7 was used as a starting material, and Example 21 was prepared by the following procedure:

3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(6-甲基哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐(300mg,0.63mmol)和丙烯酸(70mg,0.98mmol)溶于THF(10mL)，加入HATU(362mg,0.95mmol)和三乙胺(192mg,1.91mmol)，反应液室温搅拌18h，减压蒸干。残余物溶于乙酸乙酯(50mL)，水洗(30mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备板纯化(DCM:MeOH＝10：1),得5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-2-丙烯基-1-酮(58mg,29％)。该化合物经手性拆分后可得实施例21化合物WS-327和WS-328。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x 250mm col,27％甲醇,70mL/分钟)。3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(6-methylpiperidin-3-yl)-1H-pyrazolo[3,4-d] Pyrimidine-4-amine hydrochloride (300 mg, 0.63 mmol) and acrylic acid (70 mg, 0.98 mmol) were dissolved in THF (10 mL), HATU (362 mg, 0.95 mmol) and triethylamine (192 mg, 1.91 mmol). It was stirred at room temperature for 18 h and evaporated to dryness vacuo. The residue was dissolved in ethyl acetate (50 mL)EtOAc. The crude product was purified (DCM: MeOH = 10:1) to give 5-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazole And [3,4-d]pyrimidin-1-yl)-2-methylpiperidin-1-yl)-2-propenyl-1-one (58 mg, 29%). The compound of Example 21, WS-327 and WS-328, was obtained after chiral resolution of the compound. The resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5μ, 21 x 250 mm col, 27% methanol, 70 mL/min).

WS-327谱图数据：WS-327 spectrum data:

¹H-NMR(400MHz,CDCl₃):δppm 8.38(s,1H),8.14(s,1H),7.75-7.73(m,3H),7.31-7.26(m,2H),6.98(d,J＝8.8Hz,1H),6.62-6.59(m,1H),6.30(d,J＝8.4Hz,1H 1H),5.72-5.65(m,1H),5.55(s,2H),4.87-4.80(m,2H),4.14-4.03(m, 1H),3.53-3.47(m,1H),2.67-2.57(m,1H),2.08-1.96(m,2H),1.86-1.83(m,1H),1.34(d,J＝8.8Hz,3H). ^{1 H-NMR (400MHz, CDCl} 3): δppm 8.38 (s, 1H), 8.14 (s, 1H), 7.75-7.73 (m, 3H), 7.31-7.26 (m, 2H), 6.98 (d, J = 8.8 Hz, 1H), 6.62-6.59 (m, 1H), 6.30 (d, J = 8.4 Hz, 1H 1H), 5.72 - 5.65 (m, 1H), 5.55 (s, 2H), 4.87 - 4.80 (m, 2H), 4.14-4.03 (m, 1H), 3.53-3.47 (m, 1H), 2.67-2.57 (m, 1H), 2.08-1.96 (m, 2H), 1.86-1.83 (m, 1H), 1.34 ( d, J = 8.8 Hz, 3H).

MS:m/z 490.2[M+H]MS: m/z 490.2 [M+H]

WS-328谱图数据：WS-328 spectrum data:

¹H-NMR(400MHz,CDCl₃):δppm 8.38(s,1H),8.14(s,1H),7.75-7.73(m,3H),7.31-7.26(m,2H),6.98(d,J＝8.8Hz,1H),6.62-6.59(m,1H),6.30(d,J＝8.4Hz,1H 1H),5.72-5.65(m,1H),5.55(s,2H),4.87-4.80(m,2H),4.14-4.03(m,1H),3.53-3.47(m,1H),2.67-2.57(m,1H),2.08-1.96(m,2H),1.86-1.83(m,1H),1.34(d,J＝8.8Hz,3H). ^{1 H-NMR (400MHz, CDCl} 3): δppm 8.38 (s, 1H), 8.14 (s, 1H), 7.75-7.73 (m, 3H), 7.31-7.26 (m, 2H), 6.98 (d, J = 8.8 Hz, 1H), 6.62-6.59 (m, 1H), 6.30 (d, J = 8.4 Hz, 1H 1H), 5.72 - 5.65 (m, 1H), 5.55 (s, 2H), 4.87 - 4.80 (m, 2H), 4.14-4.03 (m, 1H), 3.53-3.47 (m, 1H), 2.67-2.57 (m, 1H), 2.08-1.96 (m, 2H), 1.86-1.83 (m, 1H), 1.34 ( d, J = 8.8 Hz, 3H).

MS:m/z 490.2[M+H]MS: m/z 490.2 [M+H]

实施例22Example 22

1-((2S,5R)-5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-2-丁炔基-1-酮(WS-329)和1-((2R,5S)-5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-2-丁炔基-1-酮(WS-330)的制备1-((2S,5R)-5-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d Pyrimidin-1-yl)-2-methylpiperidin-1-yl)-2-butynyl-1-one (WS-329) and 1-((2R,5S)-5-(4-amino -3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidine- Preparation of 1-yl)-2-butynyl-1-one (WS-330)

以实施例7第五步反应的产物为起始物，实施例22经过以下的步骤制备：The product of the reaction of the fifth step of Example 7 was used as a starting material, and Example 22 was prepared by the following procedure:

3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(6-甲基哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐(230mg,0.53mmol)和丁-2-炔酸(80mg,0.98mmol)溶于THF(10mL)，加入HATU(300mg,0.79mmol)和三乙胺(100mg,0.99mmol)，反应液室温搅拌18h，减压蒸干。残余物溶于乙酸乙酯(50mL)，水洗(30 mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备板纯化(DCM:MeOH＝10：1),得1-((2S,5R)-5-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-2-丁炔基-1-酮(130mg,50％)。该化合物经手性拆分后可得实施例22化合物WS-329和WS-330。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x 250mm col,27％甲醇,70mL/分钟)。3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(6-methylpiperidin-3-yl)-1H-pyrazolo[3,4-d] Pyrimidine-4-amine hydrochloride (230 mg, 0.53 mmol) and but-2-ynoic acid (80 mg, 0.98 mmol) were dissolved in THF (10 mL), then HATU (300 mg, 0.79 mmol) and triethylamine (100 mg, 0.99) The reaction mixture was stirred at room temperature for 18 h and evaporated to dryness. The residue was dissolved in ethyl acetate (50 mL) and washed (30) mL*3), dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The crude product was purified (DCM: MeOH = 10:1) to give 1-((2S,5R)-5-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy) Phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidin-1-yl)-2-butynyl-1-one (130 mg, 50%) ). The compound of Example 22, WS-329 and WS-330, was obtained after chiral resolution of the compound. The resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5μ, 21 x 250 mm col, 27% methanol, 70 mL/min).

WS-329谱图数据：WS-329 spectrum data:

¹H-NMR(400MHz,CDCl₃):δppm 8.38(s,1H),8.14(s,1H),7.77-7.69(m,3H),7.33-7.29(m,2H),7.00-6.96(m,1H),5.50(s,2H),4.98-4.81(m,3H),3.89-3.87(m,1H),2.57-2.53(m,1H),2.05(s,3H),1.85-1.76(m,3H),1.41(d,J＝6.8Hz,3H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.38 (s, 1H), 8.14 (s, 1H), 7.77-7.69 (m, 3H), 7.33-7.29 (m, 2H), 7.00-6.96 (m, 1H), 5.50 (s, 2H), 4.98-4.81 (m, 3H), 3.89-3.87 (m, 1H), 2.57-2.53 (m, 1H), 2.05 (s, 3H), 1.85-1.76 (m, 3H), 1.41 (d, J = 6.8 Hz, 3H).

MS:m/z 502.3[M+H]MS: m/z 502.3 [M+H]

WS-330谱图数据：WS-330 spectrum data:

MS:m/z 502.3[M+H]MS: m/z 502.3 [M+H]

实施例23Example 23

1-((2S,5R)-5-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-2-丙烯基-1-酮(WS-331)和1-((2R,5S)-5-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-2-丙烯基-1-酮(WS-332)的制备 1-((2S,5R)-5-(4-Amino-3-(4-(2,4-difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine- 1-yl)-2-methylpiperidin-1-yl)-2-propenyl-1-one (WS-331) and 1-((2R,5S)-5-(4-amino-3-( 4-(2,4-Difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidin-1-yl)-2- Preparation of propenyl-1-one (WS-332)

实施例23从对应的起始物开始，采用与实施例21类似的方法制备而得。Example 23 was prepared in a similar manner to Example 21 starting from the corresponding starting material.

WS-331谱图数据：WS-331 spectrum data:

¹H-NMR(400MHz,CDCl₃):δppm 8.37(s,1H),7.64(d,J＝8.8Hz,2H),7.19-7.13(m,1H),7.09(d,J＝8.4Hz,2H),7.04-6.97(m,1H),6.94-6.90(m,1H),6.66-6.54(m,1H),6.31-6.25(m,1H),5.72-5.65(m,1H),5.45(s,2H),4.85-4.78(m,1H),3.85-3.44(m,2H),2.59-2.53(m,1H),2.12-1.89(m,2H),1.85-1.77(m,1H),1.33(d,J＝8.8Hz,3H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.37 (s, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.19-7.13 (m, 1H), 7.09 (d, J = 8.4 Hz, 2H) ), 7.04-6.97 (m, 1H), 6.94-6.90 (m, 1H), 6.66-6.54 (m, 1H), 6.31-6.25 (m, 1H), 5.72-5.65 (m, 1H), 5.45 (s) , 2H), 4.85-4.78 (m, 1H), 3.85-3.44 (m, 2H), 2.59-2.53 (m, 1H), 2.12-1.89 (m, 2H), 1.85-1.77 (m, 1H), 1.33 (d, J = 8.8 Hz, 3H).

MS:m/z 491.4[M+H]MS: m/z 491.4 [M+H]

WS-332谱图数据：WS-332 spectrum data:

MS:m/z 491.4[M+H]MS: m/z 491.4 [M+H]

实施例24Example 24

1-((2S,5R)-5-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-2-丁炔基-1-酮(WS-333)和1-((2R,5S)-5-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-基)-2-丁炔基-1-酮(WS-334)的制备 1-((2S,5R)-5-(4-Amino-3-(4-(2,4-difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine- 1-yl)-2-methylpiperidin-1-yl)-2-butynyl-1-one (WS-333) and 1-((2R,5S)-5-(4-amino-3- (4-(2,4-Difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidin-1-yl)-2 -butynyl-1-one (WS-334) preparation

实施例24从对应的起始物开始，采用与实施例22类似的方法制备而得。Example 24 was prepared in a similar manner to Example 22 starting from the corresponding starting material.

WS-333谱图数据：WS-333 spectrum data:

¹H-NMR(400MHz,CDCl₃):δppm 8.37(s,1H),7.67-7.62(m,2H),7.19-7.13(m,1H),7.11-7.08(m,2H),7.02-6.98(m,1H),6.97-6.90(m,1H),5.49(s,1H),4.81-4.77(m,2H),3.87-3.46(m,1H),2.56-2.53(m,1H),1.98(s,3H),1.32(d,J＝7.2Hz,3H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.37 (s, 1H), 7.67-7.62 (m, 2H), 7.19-7.13 (m, 1H), 7.11-7.08 (m, 2H), 7.02-6.98 ( m, 1H), 6.97-6.90 (m, 1H), 5.49 (s, 1H), 4.81-4.77 (m, 2H), 3.87-3.46 (m, 1H), 2.56-2.53 (m, 1H), 1.98 ( s, 3H), 1.32 (d, J = 7.2 Hz, 3H).

MS:m/z 503.4[M+H]MS: m/z 503.4 [M+H]

WS-334谱图数据：WS-334 spectrum data:

MS:m/z 503.4[M+H]MS: m/z 503.4 [M+H]

实施例25Example 25

(R)-1-(6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-2-丙烯基-1-酮(WS-335)和(S)-1-(6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-2-丙烯基-1-酮(WS-336)的制备 (R)-1-(6-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine -1-yl)-4-azaspiro[2.5]octane-4-yl)-2-propenyl-1-one (WS-335) and (S)-1-(6-(4-amino-) 3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-azaspiro[2.5] Preparation of Octane-4-yl)-2-propenyl-1-one (WS-336)

以实施例13第十一步反应的产物为起始物，实施例25经过以下的步骤制备：Starting from the product of the eleventh step of Example 13, the preparation of Example 25 was carried out by the following procedure:

3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑并[3,4-d]嘧啶-4-胺盐酸盐(50mg,0.11mmol)和丙烯酸(8.03mg,0.11mmol)溶于THF(5mL)，加入HATU(63.67mg,0.17mmol)和三乙胺(33.89mg,0.33mmol)，反应液室温搅拌18h，减压蒸干。残余物溶于乙酸乙酯(50mL)，水洗(30mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备板纯化(DCM:MeOH＝10：1),得1-(6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-2-丙烯基-1-酮(38mg,67％)。该化合物经手性拆分后可得实施例25化合物WS-335和WS-336。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x 250mm col,27％甲醇,70mL/分钟)。3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1-(4-azaspiro[2.5]oct-6-yl)-1H-pyrazolo[3,4 -d]pyrimidin-4-amine hydrochloride (50 mg, 0.11 mmol) and acrylic acid (8.03 mg, 0.11 mmol) were dissolved in THF (5 mL), and HATU (63.67 mg, 0.17 mmol) and triethylamine (33.89 mg, The reaction mixture was stirred at room temperature for 18 h and then evaporated. The residue was dissolved in ethyl acetate (50 mL)EtOAc. The crude product was purified (DCM: MeOH = 10:1) to give 1-(6-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H. Pyrazolo[3,4-d]pyrimidin-1-yl)-4-azaspiro[2.5]octane-4-yl)-2-propenyl-1-one (38 mg, 67%). The compound of Example 25, WS-335 and WS-336, was obtained after chiral resolution of the compound. The resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5μ, 21 x 250 mm col, 27% methanol, 70 mL/min).

WS-335谱图数据：WS-335 spectrum data:

¹H-NMR(400MHz,CDCl₃):δppm 8.40(s,1H),8.17(s,2H),7.78-7.71(m,2H),7.21(d,J＝8.4Hz,2H),6.99(d,J＝8.8Hz,1H),6.91-6.90(m,1H),6.42-6.38(m,1H),5.75-5.73(m,1H),5.61(s,2H),4.98-4.94(m,1H),3.78-3.75(m,1H),2.54-2.49(m,1H),2.30-2.22(m,2H),1.28-1.20(m,3H),1.14-1.10(m,1H),0.90-0.82(m,2H). ^{1 H-NMR (400MHz, CDCl} 3): δppm 8.40 (s, 1H), 8.17 (s, 2H), 7.78-7.71 (m, 2H), 7.21 (d, J = 8.4Hz, 2H), 6.99 (d , J=8.8Hz, 1H), 6.91-6.90 (m, 1H), 6.42-6.38 (m, 1H), 5.75-5.73 (m, 1H), 5.61 (s, 2H), 4.98-4.94 (m, 1H) ), 3.78-3.75 (m, 1H), 2.54-2.49 (m, 1H), 2.30-2.22 (m, 2H), 1.28-1.20 (m, 3H), 1.14-1.10 (m, 1H), 0.90-0.82 (m, 2H).

MS:m/z 502.3[M+H]MS: m/z 502.3 [M+H]

WS-336谱图数据： WS-336 spectrum data:

MS:m/z 502.4[M+H]MS: m/z 502.4 [M+H]

实施例26Example 26

(R)-1-(6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-2-丁炔基-1-酮(WS-337)和(S)-1-(6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-2-丁炔基-1-酮(WS-338)的制备(R)-1-(6-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine -1-yl)-4-azaspiro[2.5]octane-4-yl)-2-butynyl-1-one (WS-337) and (S)-1-(6-(4-amino) -3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-azaspiro[2.5 Of octyl-4-yl)-2-butynyl-1-one (WS-338)

实施例26从对应的起始物开始，采用与实施例22类似的方法制备而得。Example 26 was prepared in a similar manner to Example 22 starting from the corresponding starting material.

WS-337谱图数据：WS-337 spectrum data:

¹H-NMR(400MHz,CDCl₃-d6):δppm 8.40(s,1H),8.15(s,1H),7.79-7.69(m,3H),7.32-7.29(m,2H),6.99-6.96(m,1H),5.54(s,1H),5.04-5.02(m,1H),4.58-4.56(m,1H),4.07-4.05(m,1H),2.53-2.44(m,1H),2.26-2.20(m,2H),2.06(s,3H),2.01-1.98(s,1H),1.43-1.41(m,1H),1.06-1.05(m,1H),1.05-1.04(m,2H). ¹ H-NMR (400 MHz, CDCl ₃ -d6): δ ppm 8.40 (s, 1H), 8.15 (s, 1H), 7.79-7.69 (m, 3H), 7.32-7.29 (m, 2H), 6.99-6. m,1H), 5.54 (s, 1H), 5.04-5.02 (m, 1H), 4.58-4.56 (m, 1H), 4.07-4.05 (m, 1H), 2.53-2.44 (m, 1H), 2.26- 2.20 (m, 2H), 2.06 (s, 3H), 2.01-1.98 (s, 1H), 1.43-1.41 (m, 1H), 1.06-1.05 (m, 1H), 1.05-1.04 (m, 2H).

MS:m/z 514.3[M+H]MS: m/z 514.3 [M+H]

WS-338谱图数据： WS-338 spectrum data:

MS:m/z 514.3[M+H]MS: m/z 514.3 [M+H]

实施例27Example 27

(R)-1-(6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-4-羟基-2-丁炔基-1-酮(WS-339)和(S)-1-(6-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-4-羟基-2-丁炔基-1-酮(WS-340)的制备(R)-1-(6-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidine -1-yl)-4-azaspiro[2.5]octane-4-yl)-4-hydroxy-2-butynyl-1-one (WS-339) and (S)-1-(6- (4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-nitrogen Preparation of Heterospo [2.5] Octane-4-yl)-4-hydroxy-2-butynyl-1-one (WS-340)

实施例27从对应的起始物开始，采用与实施例21类似的方法制备而得。Example 27 was prepared in a similar manner to Example 21 starting from the corresponding starting material.

WS-339谱图数据：WS-339 spectrum data:

¹H-NMR(400MHz,CDCl₃):δppm 8.38(s,1H),8.13(d,J＝2.4Hz,1H),7.76-7.69(m 3H),7.31-7.29(m,2H),6.97(d,J＝8.8Hz,1H),5.63-5.53(m,1H),5.01-4.96(m,1H),4.46-4.40(m,1H),4.26-4.23(m,1H),3.84-3.83(m,1H),2.27-2.21(m,1H),2.12-2.08(m,3H),1.28-1.25(m,1H),1.04-1.02(m,1H),0.93-0.75(m,2H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.38 (s, 1H), 8.13 (d, J = 2.4 Hz, 1H), 7.76-7.69 (m 3H), 7.31-7.29 (m, 2H), 6.97 ( d, J = 8.8 Hz, 1H), 5.63-5.53 (m, 1H), 5.01-4.96 (m, 1H), 4.46-4.40 (m, 1H), 4.26 - 4.23 (m, 1H), 3.84 - 3.83 ( m, 1H), 2.27-2.21 (m, 1H), 2.12-2.08 (m, 3H), 1.28-1.25 (m, 1H), 1.04-1.02 (m, 1H), 0.93-0.75 (m, 2H).

MS:m/z 530.3[M+H]MS: m/z 530.3 [M+H]

WS-340谱图数据： WS-340 spectrum data:

MS:m/z 530.3[M+H]MS: m/z 530.3 [M+H]

实施例28Example 28

4-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-腈(WS-341)的制备4-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine Preparation of -1-nitrile (WS-341)

实施例28从对应的起始物开始，采用与实施例1类似的方法制备而得。Example 28 was prepared in a similar manner to Example 1 starting from the corresponding starting material.

¹H-NMR(400MHz,CDCl₃)δppm 8.37(s，1H),7.64(d,J＝8.4Hz,2H),7.19-7.13(m,1H),7.09(d,J＝8.4Hz,2H),7.02-6.97(m,1H),6.94-6.90(m,1H),5.63(s,2H),4.92-4.86(m,1H),3.66-3.63(m,2H),3.31-3.25(m,2H),2.54-2.44(m,2H),2.10-2.01(m,2H). ¹ H-NMR (400MHz, CDCl ₃ ) δ ppm 8.37 (s, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.19-7.13 (m, 1H), 7.09 (d, J = 8.4 Hz, 2H) , 7.02-6.97 (m, 1H), 6.94-6.90 (m, 1H), 5.63 (s, 2H), 4.92-4.86 (m, 1H), 3.66-3.63 (m, 2H), 3.31-3.25 (m, 2H), 2.54-2.44 (m, 2H), 2.10-2.01 (m, 2H).

MS:m/z 448.3[M+H]MS: m/z 448.3 [M+H]

实施例29Example 29

5-(4-氨基-3-(1-苄基-1H-吡唑-4-基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-342)的制备 5-(4-Amino-3-(1-benzyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidine Preparation of -1-nitrile (WS-342)

实施例29从对应的起始物开始，采用与实施例7类似的方法制备而得。Example 29 was prepared in a similar manner to Example 7 starting from the corresponding starting material.

¹H-NMR(400MHz,CDCl₃):δppm 8.34(s,1H),7.85(d,J＝16.8Hz，2H),7.41-7.26(m,5H),5.54(s,2H),5.39(s,2H),4.92-4.90(m,1H),3.94-3.88(m,1H),3.74-3.72(m,1H),3.57-3.49(m,1H),3.48-3.42(m,1H),2.49(m，1H)，2.00-1.90(m,1H)，1.63-1.54(m,3H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.34 (s, 1H), 7.85 (d, J = 16.8 Hz, 2H), 7.41 - 7.26 (m, 5H), 5.54 (s, 2H), 5.39 (s , 2H), 4.92-4.90 (m, 1H), 3.94-3.88 (m, 1H), 3.74-3.72 (m, 1H), 3.57-3.49 (m, 1H), 3.48-3.42 (m, 1H), 2.49 (m, 1H), 2.00-1.90 (m, 1H), 1.63-1.54 (m, 3H).

MS:m/z 414.3[M+H]MS: m/z 414.3 [M+H]

实施例30Example 30

5-(4-氨基-3-(4-(吗啉甲基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-343)的制备5-(4-Amino-3-(4-(morpholinyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidin-1- Preparation of nitrile (WS-343)

实施例30从对应的起始物开始，采用与实施例7类似的方法制备而得。Example 30 was prepared in a similar manner to Example 7 starting from the corresponding starting material.

¹H-NMR(400MHz,CDCl₃):δppm 8.34(s,1H),7.69(d,J＝7.6Hz，2H),7.53(d,J＝7.6Hz，2H),5.54(s,2H),4.97(m,1H),3.97-3.93(m,1H),3.75-3.74(m,4H),3.60-3.47(m,5H),2.56-2.52(m,4H),2.49(m，1H),2.01-1.96(m,3H),1.44-1.52(d,J＝6.8Hz,3H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.34 (s, 1H), 7.69 (d, J = 7.6 Hz, 2H), 7.53 (d, J = 7.6 Hz, 2H), 5.54 (s, 2H), 4.97 (m, 1H), 3.97-3.93 (m, 1H), 3.75-3.74 (m, 4H), 3.60-3.47 (m, 5H), 2.56-2.52 (m, 4H), 2.49 (m, 1H), 2.01-1.96 (m, 3H), 1.44-1.52 (d, J = 6.8 Hz, 3H).

MS:m/z 433.3[M+H] MS: m/z 433.3 [M+H]

实施例31Example 31

5-(4-氨基-3-(4-苄基苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-344)的制备5-(4-Amino-3-(4-benzylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidine-1-carbonitrile (WS- Preparation of 344)

实施例31从对应的起始物开始，采用与实施例7类似的方法制备而得。Example 31 was prepared in a similar manner to Example 7 starting from the corresponding starting material.

¹H-NMR(400MHz,CDCl₃):δppm 8.36(s,1H),7.65-7.63(m，2H),7.38-7.36(m,2H),7.25-7.23(m,2H),5.55(s,2H),4.97-4.94(m,1H),3.97-3.92(m,1H),3.74-3.72(m,1H),3.58-3.52(m,1H),2.54-2.52(m,1H),2.05-1.96(m,2H)，1.61-1.56(m,3H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.36 (s, 1H), 7.65-7.63 (m, 2H), 7.38-7.36 (m, 2H), 7.25-7.23 (m, 2H), 5.55 (s, 2H), 4.97-4.94 (m, 1H), 3.97-3.92 (m, 1H), 3.74-3.72 (m, 1H), 3.58-3.52 (m, 1H), 2.54-2.52 (m, 1H), 2.05- 1.96 (m, 2H), 1.61-1.56 (m, 3H).

MS:m/z 424.3[M+H]MS: m/z 424.3 [M+H]

实施例32Example 32

5-(4-氨基-3-(4-(苯氨基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-2-甲基哌啶-1-腈(WS-345)的制备5-(4-Amino-3-(4-(phenylamino)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpiperidine-1-carbonitrile Preparation of WS-345)

实施例32从对应的起始物开始，采用与实施例7类似的方法制备而得。 Example 32 was prepared in a similar manner to Example 7 starting from the corresponding starting material.

¹H-NMR(400MHz,CDCl₃):δppm 8.36(s,1H),7.60(d,J＝8.4Hz，2H),7.35-7.29(m,2H),7.20-7.15(m,4H),7.04-7.00(m,1H),5.91(s,2H),5.57(s,1H),4.97-4.93(m,1H),3.98-3.93(m,1H),3.59-3.55(m,1H),3.50-3.46(m,1H),2.58-2.52(m,1H),2.05-2.03(m,2H),2.00-1.97(m,2H),1.44(d,J＝6.8Hz，3H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.36 (s, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.35-7.29 (m, 2H), 7.20-7.15 (m, 4H), 7.04 -7.00 (m, 1H), 5.91 (s, 2H), 5.57 (s, 1H), 4.97-4.93 (m, 1H), 3.98-3.93 (m, 1H), 3.59-3.55 (m, 1H), 3.50 -3.46 (m, 1H), 2.58-2.52 (m, 1H), 2.05-2.03 (m, 2H), 2.00-1.97 (m, 2H), 1.44 (d, J = 6.8 Hz, 3H).

MS:m/z 425.4[M+H]MS: m/z 425.4 [M+H]

实施例33Example 33

(R)-1-(6-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-2-丙烯基-1-酮(WS-346)和(S)-1-(6-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-2-丙烯基-1-酮(WS-347)的制备(R)-1-(6-(4-amino-3-(4-(2,4-difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1- 4-Azaspiro[2.5]octane-4-yl)-2-propenyl-1-one (WS-346) and (S)-1-(6-(4-amino-3-( 4-(2,4-Difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-azaspiro[2.5]octane-4-yl Preparation of 2-propenyl-1-one (WS-347)

实施例33从对应的起始物开始，采用与实施例21类似的方法制备而得。Example 33 was prepared in a similar manner to Example 21 starting from the corresponding starting material.

WS-346谱图数据：WS-346 spectrum data:

¹H-NMR(400MHz,CDCl₃):δppm 8.37(s,1H),7.65(d,J＝8.4Hz,2H),7.19-7.13(m,1H),7.02-6.97(m,1H),6.94-6.90(m,1H),6.39-6.35(m,1H),5.72-5.69(m,1H),5.49-5.46(m,2H),4.95-4.94(m,1H),3.78-3.74(m,1H),2.52-2.47(m,1H),2.30-2.22(m,1H),1.82-1.69(m,2H),1.28-1.20(m,1H),1.14-1.10(m,1H),0.90-0.79(m,2H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.37 (s, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.19-7.13 (m, 1H), 7.02-6.97 (m, 1H), 6.94 -6.90 (m, 1H), 6.39-6.35 (m, 1H), 5.72-5.69 (m, 1H), 5.49-5.46 (m, 2H), 4.95-4.94 (m, 1H), 3.78-3.74 (m, 1H), 2.52-2.47 (m, 1H), 2.30-2.22 (m, 1H), 1.82-1.69 (m, 2H), 1.28-1.20 (m, 1H), 1.14-1.10 (m, 1H), 0.90- 0.79 (m, 2H).

MS:m/z 503.4[M+H]MS: m/z 503.4 [M+H]

WS-347谱图数据： WS-347 spectrum data:

MS:m/z 503.4[M+H]MS: m/z 503.4 [M+H]

实施例34Example 34

(R)-1-(6-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-2-丁炔基-1-酮(WS-348)和(S)-1-(6-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-2-丁炔基-1-酮(WS-349)的制备(R)-1-(6-(4-amino-3-(4-(2,4-difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1- 4-Azaspiro[2.5]octane-4-yl)-2-butynyl-1-one (WS-348) and (S)-1-(6-(4-amino-3- (4-(2,4-Difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-azaspiro[2.5]octane-4- Preparation of 2-butynyl-1-one (WS-349)

实施例34从对应的起始物开始，采用与实施例26类似的方法制备而得。Example 34 was prepared in a similar manner to Example 26 starting from the corresponding starting material.

WS-348谱图数据：WS-348 spectrum data:

¹H-NMR(400MHz,CDCl₃):δppm 8.37(s,1H),7.69-7.65(m,2H),7.16-7.13(m,1H),7.11-7.07(m,2H),7.02-6.97(m,1H),6.94-6.92(m,1H),5.47(s,2H),5.04-4.91(m,1H),4.59-4.30(m,1H),4.06-3.65(m,1H),2.52-2.45(m,1H),2.25-2.17(m,2H),2.05(s,3H),1.09-0.95(m,1H),0.89-0.86(m,2H),0.77-0.72(m,1H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.37 (s, 1H), 7.69-7.65 (m, 2H), 7.16-7.13 (m, 1H), 7.11-7.07 (m, 2H), 7.02-6.97 ( m,1H), 6.94-6.92 (m, 1H), 5.47 (s, 2H), 5.04-4.91 (m, 1H), 4.59-4.30 (m, 1H), 4.06-3.65 (m, 1H), 2.52- 2.45 (m, 1H), 2.25-2.17 (m, 2H), 2.05 (s, 3H), 1.09-0.95 (m, 1H), 0.89-0.86 (m, 2H), 0.77-0.72 (m, 1H).

MS:m/z 515.4[M+H]MS: m/z 515.4 [M+H]

WS-349谱图数据： WS-349 spectrum data:

MS:m/z 515.4[M+H]MS: m/z 515.4 [M+H]

实施例35Example 35

(R)-1-(6-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-4-羟基-2-丁炔基-1-酮(WS-350)和(S)-1-(6-(4-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)-4-氮杂螺[2.5]辛烷-4-基)-4-羟基-2-丁炔基-1-酮(WS-351)的制备(R)-1-(6-(4-amino-3-(4-(2,4-difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1- 4-Azaspiro[2.5]octane-4-yl)-4-hydroxy-2-butynyl-1-one (WS-350) and (S)-1-(6-(4- Amino-3-(4-(2,4-difluorophenoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-azaspiro[2.5] octyl Preparation of alk-4-yl)-4-hydroxy-2-butynyl-1-one (WS-351)

实施例35从对应的起始物开始，采用与实施例21类似的方法制备而得。Example 35 was prepared in a similar manner to Example 21 starting from the corresponding starting material.

WS-350谱图数据：WS-350 spectrum data:

¹H-NMR(400MHz,CDCl₃):δppm 8.37(s,1H),7.66(d,J＝8,2H),7.21-7.15(m,1H),7.11-7.08(m,2H),7.02-6.97(m,1H),6.94-6.86(m,1H),5.44(s,2H),5.03-4.92(m,1H),4.56-4.39(m,2H),4.32-4.13(m,2H),3.85-3.72(m,1H),2.70-2.43(m,1H),2.33-2.24(m,1H),2.09-2.03(m,2H),1.11-1.00(m,2H),0.88-0.68(m,2H). ¹ H-NMR (400 MHz, CDCl ₃ ): δ ppm 8.37 (s, 1H), 7.66 (d, J=8, 2H), 7.21-7.15 (m, 1H), 7.11-7.08 (m, 2H), 7.02- 6.97 (m, 1H), 6.94-6.86 (m, 1H), 5.44 (s, 2H), 5.03-4.92 (m, 1H), 4.56-4.39 (m, 2H), 4.32-4.13 (m, 2H), 3.85-3.72(m,1H), 2.70-2.43(m,1H),2.33-2.24(m,1H),2.09-2.03(m,2H),1.11-1.00(m,2H),0.88-0.68(m , 2H).

MS:m/z 531.4[M+H]MS: m/z 531.4 [M+H]

WS-351谱图数据： WS-351 spectrum data:

MS:m/z 531.4[M+H]MS: m/z 531.4 [M+H]

实施例36Example 36

3-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)吡咯烷-1-腈(WS-352)制备3-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine Preparation of -1-nitrile (WS-352)

实施例36从对应的起始物开始，采用与实施例1类似的方法制备而得。Example 36 was prepared in a similar manner to Example 1 starting from the corresponding starting material.

¹H-NMR(400MHz,CD₃OD)δppm 8.37(s,1H),7.72(d,J＝8.8Hz,2H),7.32-7.26(m,1H),7.21-7.18(m,1H),7.16(d,J＝8.4Hz,2H),7.06-7.01(m,1H),5.62-5.32(s,1H),3.97-3.92(m,1H),3.87-3.80(m,2H),3.71-3.68(m,1H),2.53-2.48(m,2H),2.20-2.00(m,1H). ¹ H-NMR (400 MHz, CD ₃ OD) δ ppm 8.37 (s, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.32-7.26 (m, 1H), 7.21-7.18 (m, 1H), 7.16 (d, J = 8.4 Hz, 2H), 7.06-7.01 (m, 1H), 5.62-5.32 (s, 1H), 3.97-3.92 (m, 1H), 3.87-3.80 (m, 2H), 3.71-3.68 (m, 1H), 2.53-2.48 (m, 2H), 2.20-2.00 (m, 1H).

MS:434.2[M+H]MS: 434.2 [M+H]

实施例37Example 37

(R)-3-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)吡咯烷-1-腈(WS-353)和(S)-3-(4-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑并[3,4-d]嘧啶-1-基)吡咯烷-1-腈(WS-354)的制备 (R)-3-(4-Amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1- Pyrrolidin-1-carbonitrile (WS-353) and (S)-3-(4-amino-3-(4-((5-chloropyridin-2-yl)oxy)phenyl)-1H- Preparation of pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carbonitrile (WS-354)

实施例36的化合物经手性拆分后可得实施例37的化合物WS-353和WS-354。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x 250mm col,27％甲醇,70mL/分钟)。Compounds of Example 36 were resolved by chirality to afford compounds WS-353 and WS-354 of Example 37. The resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5μ, 21 x 250 mm col, 27% methanol, 70 mL/min).

WS-353谱图数据：WS-353 spectrum data:

MS:434.2[M+H]MS: 434.2 [M+H]

WS-354谱图数据：WS-354 spectrum data:

MS:434.2[M+H]MS: 434.2 [M+H]

实施例38～47Examples 38 to 47

采用与本专利实施例1～37类似的方法，可制备实施例38～47所示结构的化合物。The compounds of the structures shown in Examples 38 to 47 can be produced by a method similar to that of Examples 1 to 37 of the present patents.

实施例48Example 48

BTK激酶活性抑制实验BTK kinase activity inhibition assay

在基于时间分辨荧光共振能量转移方法的试验中，测试本申请公开的化合物对BTK激酶活性的抑制作用。重组的Btk与本申请公开的化合物在室温下在含有50mM Tris pH7.4、10mM MgCl₂、2mM MnCl₂、0.1mM EDTA、1mM DTT、20nM SEB、0.1％BSA、0.005％tween-20的试验缓冲液中预先温育1小时。通过加入ATP(在ATP Km浓度下)和肽底物(Biotin-AVLESEEELYSSARQ-NH2)来引发反应。在室温下温育1小时后，加入等体积的含有50mM HEPES pH7.0、800mM KF、20mM EDTA、0.1％BSA、连接Eu穴合物的p-Tyr66抗体和链霉亲和素标记的XL665的终止液以终止反应。盘在室温下再温育1小时，然后在BMG PHERAstar FS仪器上读取TR-FRET信号(ex337nm，em 620nm/665nm)。基于615nm处的荧光与665nm处的荧光的比值，计算化合物浓度增加情况下残余酶活性。各化合物的IC₅₀通过Graphpad Prism软件的四参数逻辑方程拟合数据而得到。 The inhibition of BTK kinase activity by the compounds disclosed herein was tested in a test based on a time resolved fluorescence resonance energy transfer method. Recombinant Btk and the compounds disclosed herein are tested at room temperature in a test buffer containing 50 mM Tris pH 7.4, 10 mM MgCl ₂ , 2 mM MnCl ₂ , 0.1 mM EDTA, 1 mM DTT, 20 nM SEB, 0.1% BSA, 0.005% tween-20 The solution was incubated for 1 hour in advance. The reaction was initiated by the addition of ATP (at ATP Km concentration) and the peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After incubation for 1 hour at room temperature, an equal volume of XL665 containing 50 mM HEPES pH 7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, attached Eu cryptate and p-Tyr66 antibody and streptavidin-labeled XL665 were added. Stop the solution to stop the reaction. The plates were incubated for an additional hour at room temperature and then the TR-FRET signal (ex337 nm, em 620 nm / 665 nm) was read on a BMG PHERAstar FS instrument. Based on the ratio of the fluorescence at 615 nm to the fluorescence at 665 nm, the residual enzyme activity in the case of an increase in compound concentration was calculated. IC ₅₀ of each compound obtained by the four-parameter logistic equation Graphpad Prism software fitting the data.

按照上述的实验方法，本申请中的部分化合物表现出较强的BTK激酶抑制活性(IC₅₀<1000nM)，其中一些优选化合物的激酶抑制活性很强(IC₅₀<100nM)。具体结果见下表。According to the above experimental methods, some of the compounds in the present application exhibited strong BTK kinase inhibitory activity (IC ₅₀ <1000 nM), and some of the preferred compounds have strong kinase inhibitory activity (IC ₅₀ <100 nM). The specific results are shown in the table below.

激酶抑制活性等级分为A、B、C，具体地A(IC₅₀<100nM)，B(100nM<IC₅₀<1000nM)，C(IC₅₀>1000nM)The kinase inhibitory activity levels are classified into A, B, C, specifically A (IC ₅₀ <100 nM), B (100 nM < IC ₅₀ <1000 nM), C (IC ₅₀ >1000 nM)

实施例49 Example 49

体外激酶选择性实验In vitro kinase selectivity assay

采用基于时间分辨荧光共振能量转移方法建立了EGFR，ITK激酶活性检测平台；采用Z’-Lyte方法建立了LCK，SRC，LYN激酶活性检测平台；采用Lance Ultra方法建立了TEC和JAK3激酶活性检测平台，分别测试本申请公开的化合物对不同激酶活性的抑制作用。每个化合物分别在11个浓度下测定酶活性数据，用Graphpad Prism软件计算该化合物的IC₅₀值。The detection platform of EGFR and ITK kinase activity was established by time-resolved fluorescence resonance energy transfer method. The detection platform of LCK, SRC and LYN kinase activity was established by Z'-Lyte method. The detection platform of TEC and JAK3 kinase activity was established by Lance Ultra method. The compounds disclosed herein were tested for inhibition of different kinase activities, respectively. Each compound activity data were determined at 11 concentrations of the compound IC ₅₀ value calculated using Graphpad Prism software.

按照上述的实验方法，本申请中的一些化合物表现出很强的激酶选择性，明显优于对照化合物依鲁替尼。结果见下表。According to the above experimental methods, some of the compounds in the present application showed strong kinase selectivity, which was significantly better than the control compound ibrutinib. See the table below for the results.

化合物编号Compound number	LCKLCK	SRCSRC	LYNLYN	EGFREGFR	ITKITK	TECTEC
WS-311WS-311	CC	CC	CC	BB	CC	AA
WS-349WS-349	BB	BB	BB	CC	CC	AA
依鲁替尼Ibrutinib	AA	AA	AA	AA	AA	AA

实施例50Example 50

B细胞抑制实验B cell inhibition assay

在活体外短暂暴露至BTK抑制剂足以抑制正常人类B细胞中的B细胞激活。此方案模拟活体内细胞至抑制剂的预测暴露，并且显示尽管清洗抑制剂但对B细胞的抑制仍得以保持。Short exposure to BTK inhibitors in vitro is sufficient to inhibit B cell activation in normal human B cells. This protocol mimics the predicted exposure of cells to inhibitors in vivo and shows that inhibition of B cells is maintained despite the washing inhibitor.

CD B细胞是使用若赛特赛普(RosetteSep)人类B细胞富集混合剂通过阴性选择自健康供体血液纯化得到。将细胞铺板于生长培养基(10％RPMI+10％胎牛血清)中并添加指定浓度的抑制剂。于37℃下培育1小时后，将细胞洗涤三次，每次洗涤均利用于生长培养基中进行8倍稀释。随后将细胞用10μg/mL IgM F(ab')2在37℃下剌激18小时。随后用抗CD69-PE抗体对细胞进行染色并通过流式细胞术使用标准条件进行分析。依照以上的方法进行测定，本申请中优选的化合物对B细胞具有较强的抑制活性，其IC₅₀值小于1nM。CD B cells were purified from healthy donor blood by negative selection using the RosetteSep Human B Cell Enrichment Mix. Cells were plated in growth medium (10% RPMI + 10% fetal bovine serum) and inhibitors of the indicated concentrations were added. After incubating for 1 hour at 37 ° C, the cells were washed three times, and each wash was used for 8-fold dilution in growth medium. The cells were then stimulated with 10 μg/mL IgM F(ab') 2 for 18 hours at 37 °C. Cells were subsequently stained with anti-CD69-PE antibody and analyzed by flow cytometry using standard conditions. According to the above method, the preferred compound of the present application has a strong inhibitory activity against B cells and has an IC ₅₀ value of less than 1 nM.

实施例51 Example 51

T细胞抑制实验T cell inhibition experiment

CD T细胞是使用若赛特赛普(RosetteSep)人类T细胞富集混合剂通过阴性选择自健康供体血液纯化得到。将细胞铺板于生长培养基(10％RPMI+10％胎牛血清)中并添加指定浓度的抑制剂。于37℃下培育1小时后，将细胞洗涤三次，每次洗涤均利用于生长培养基中进行10倍稀释。随后将细胞用anti-CD3/CD28包被珠(珠/细胞比例为1:1)在37℃下剌激18小时。随后用抗CD69-PE抗体对细胞进行染色并通过流式细胞术使用标准条件进行分析。CD T cells were purified from healthy donor blood by negative selection using the RosetteSep Human T Cell Enrichment Mix. Cells were plated in growth medium (10% RPMI + 10% fetal bovine serum) and inhibitors of the indicated concentrations were added. After incubating for 1 hour at 37 ° C, the cells were washed three times, and each wash was used for 10-fold dilution in growth medium. The cells were then challenged with anti-CD3/CD28 coated beads (bead/cell ratio of 1:1) for 18 hours at 37 °C. Cells were subsequently stained with anti-CD69-PE antibody and analyzed by flow cytometry using standard conditions.

依照以上的方法进行测定，本申请中优选的化合物对T细胞具有很弱的抑制活性或无抑制，其IC₅₀值大于4000nM。According to the above method, the preferred compound in the present application has a weak inhibitory activity or no inhibition on T cells, and its IC ₅₀ value is greater than 4000 nM.

实施例52Example 52

人全血B细胞抑制实验Human whole blood B cell inhibition experiment

人全血(hWB)获自健康志愿者，通过静脉穿刺将血液收集到用肝素钠抗凝化的Vacutainer管中。测试化合物在PBS中稀释至10倍所需初始药物浓度)，接着在10％的在PBS中的DMSO中三倍系列稀释，以得到9点的剂量响应曲线。将5.5μL的每种化合物稀释液一式两份添加到aiil 96孔V型底的板上；向对照和无刺激孔中添加5.5μL的10％在PBS中的DMSO。向每孔添加人全血(100μL)，在混合后将板在37C，5％CO₂，100％湿度温育30分钟。在搅拌下向每孔(无刺激孔除外)添加羊F(ab')2抗人IgM(Southern Biotech)(10μL的500μg/mL溶液，50μg/mL最终浓度)，并且将板温育另外20小时。在20小时温育结束时，将样品与荧光探针标记的20μL APC小鼠抗人CD69(BD Pharmingen)在37C，5％CO₂，100％湿度温育30分钟。包括用于补偿调节和初始电压设置的诱导对照、未染色的和单染色剂。然后将样品用1ml的IX Pharmingen Lyse Buffer(BD Pharmingen)裂解，并且将板在1500rpm离心5分钟。通过抽吸除去上清液，将残留的团粒用另外1ml的IX Pharmingen Lyse Buffer再次裂解，并且将板如前离心。吸出上清液，将残留的团粒在FACs缓冲液(PBS+1％FBQ中洗涤。离心后并除去上清液后，将团粒重悬浮在150μL的FACs缓冲液中。将样品转移至适于在BD LSR II流式细胞器的HTS 96孔体系上运行的96孔板。采用适合所用荧光团的激发和发射波长，获取数据并且采用Cell Quest Software获得百分比阳性细胞值。结果最初用FACS分析软件(Flow Jo)分析。IC₅₀值使用XLfit v3，公式201计算。Human whole blood (hWB) was obtained from healthy volunteers and blood was collected by venipuncture into a Vacutainer tube that was anticoagulated with sodium heparin. Test compounds were diluted to 10 times the required initial drug concentration in PBS), followed by three-fold serial dilutions in 10% DMSO in PBS to give a 9 point dose response curve. 5.5 μL of each compound dilution was added to the aiil 96-well V-bottom plate in duplicate; 5.5 μL of 10% DMSO in PBS was added to the control and non-stimulated wells. Human whole blood (100 μL) was added to each well, and after mixing, the plates were incubated for 30 minutes at 37 C, 5% CO ₂ , 100% humidity. Add F (ab') 2 anti-human IgM (Southern Biotech) (10 μL of 500 μg/mL solution, 50 μg/mL final concentration) to each well (without stimulation holes) with stirring, and incubate the plate for an additional 20 hours. . At the end of 20 hours incubation, the samples were labeled with fluorescent probes 20μL APC mouse anti-human CD69 (BD Pharmingen) at _{37C, 5% CO 2, 100} % humidity for 30 minutes. Induction controls, unstained, and single stains are included for compensation adjustment and initial voltage settings. The sample was then lysed with 1 ml of IX Pharmingen Lyse Buffer (BD Pharmingen) and the plate was centrifuged at 1500 rpm for 5 minutes. The supernatant was removed by aspiration, and the remaining pellet was again lysed with an additional 1 ml of IX Pharmingen Lyse Buffer, and the plate was centrifuged as before. The supernatant was aspirated and the remaining pellet was washed in FACs buffer (PBS + 1% FBQ. After centrifugation and the supernatant was removed, the pellet was resuspended in 150 μL of FACs buffer. Transfer the sample to a suitable one. 96-well plates run on the HTS 96-well system of the BD LSR II flow cytometer. Data were acquired using excitation and emission wavelengths appropriate for the fluorophore used and percent positive cells were obtained using Cell Quest Software. Results were initially analyzed using FACS analysis (Flow Jo) Analysis. IC ₅₀ values were calculated using XLfit v3, Equation 201.

依照以上的方法进行测定，本申请中优选的化合物对人全血中B细胞具有较强的抑制活性，其IC₅₀值小于200nM。According to the above method, the preferred compound of the present application has a strong inhibitory activity against B cells in human whole blood, and its IC ₅₀ value is less than 200 nM.

实施例53Example 53

化合物在肝微粒体中的稳定性研究Study on the stability of compounds in liver microsomes

1.待测化合物溶解在乙腈中，制成浓度为0.5mM的储备液。1. The test compound was dissolved in acetonitrile to prepare a stock solution having a concentration of 0.5 mM.

2. 2μL储备液加入1.5ml离心管中，然后加入148μL磷酸缓冲液(100mM，pH 7.4)和10μL肝微粒体(蛋白浓度为20mg/ml)悬液(BD Gentest公司)，肝微粒体的种属分别为人，狗，大鼠，小鼠；对照组加入158μL磷酸缓冲液(100mM，pH 7.4)。2. Add 2 μL of the stock solution to a 1.5 ml centrifuge tube, then add 148 μL of phosphate buffer (100 mM, pH 7.4) and 10 μL of liver microsome (protein concentration of 20 mg/ml) suspension (BD Gentest), liver microsomes. The genus was human, dog, rat, and mouse; the control group was added with 158 μL of phosphate buffer (100 mM, pH 7.4).

3.步骤2中制备好的混合体系，于37℃水浴中预孵3分钟，然后加入40μL NADPH发生体系(含有NADP+：6.5mM，葡萄糖6-磷酸：16.5mM,MgCl₂：16.5mM，葡萄糖6-磷酸脱氢酶:2U/ml)启动反应，并于37℃水浴中孵育1小时。3. Prepare the mixed system in step 2, pre-incubated for 3 minutes in a 37 ° C water bath, then add 40 μL of NADPH production system (containing NADP +: 6.5 mM, glucose 6-phosphate: 16.5 mM, MgCl ₂ : 16.5 mM, glucose 6 - Phosphate dehydrogenase: 2 U/ml) The reaction was initiated and incubated for 1 hour in a 37 ° C water bath.

4.反应进行1小时后，将离心管从水浴中取出，并加入400μL乙腈终止反应，然后涡旋震荡3分钟，最后离心(13000rpm，4℃)5分钟，取上清液用HPLC检测剩余药物浓度Cr。4. After the reaction was carried out for 1 hour, the centrifuge tube was taken out from the water bath, and the reaction was terminated by adding 400 μL of acetonitrile, then vortexed for 3 minutes, finally centrifuged (13,000 rpm, 4 ° C) for 5 minutes, and the supernatant was taken for detection of the remaining drug by HPLC. Concentration Cr.

5.平行制备0分钟反应样品的制备方法：步骤2中制备好的混合体系，于37℃水浴中预孵3分钟后取出，加入400μL乙腈，然后加入40μL NADPH发生体系。涡旋震荡3分钟后，离心(13000rpm，4℃)5分钟，取上清液用HPLC检测药物浓度C0。5. Preparation method of parallel preparation 0 minute reaction sample: The prepared mixed system in step 2 was taken out in a 37 ° C water bath for 3 minutes, and then taken out, 400 μL of acetonitrile was added, and then 40 μL of NADPH generation system was added. After vortexing for 3 minutes, centrifugation (13,000 rpm, 4 ° C) for 5 minutes, and the supernatant was taken to detect the drug concentration C0 by HPLC.

6.经60分钟孵育后，药物在孵育体系中的剩余百分比按照下式计算：6. After 60 minutes of incubation, the remaining percentage of drug in the incubation system is calculated as follows:

药物剩余(％)＝Cr÷C0×100％Drug surplus (%) = Cr ÷ C0 × 100%

按照上述的实验方法，本申请中的优选化合物表现出较好的微粒体稳定性，其在各种属的肝微粒体中的剩余百分比>30％。In accordance with the experimental methods described above, the preferred compounds of the present application exhibit better microsomal stability with a residual percentage > 30% in liver microsomes of various genera.

实施例54 Example 54

评价化合物对CYP酶抑制作用Evaluation of compound inhibition of CYP enzyme

CYP酶代谢是药物生物转化的主要途径，其数量和活性大小直接影响药物在体内的活化与代谢。作为外源性化合物的主要代谢酶，细胞色素CYP是重要的药物I相代谢酶，可以催化多种外源性化合物的氧化和还原代谢。CYP酶在药物的消除过程中起着非常重要的作用，同时也是引起联合用药时药物相互作用产生的主要因素。CYP enzyme metabolism is the main pathway for drug biotransformation, and its quantity and activity directly affect the activation and metabolism of drugs in the body. As a major metabolic enzyme of exogenous compounds, cytochrome CYP is an important drug phase I metabolizing enzyme that catalyzes the oxidation and reductive metabolism of various exogenous compounds. The CYP enzyme plays a very important role in the elimination of the drug, and is also the main factor in the drug interaction caused by the combination.

方法：本实验采用cocktail探针药物法同时测定化合物对人源肝微粒体中五种CYP450酶的抑制作用，人源微粒体来自BD Gentest公司。METHODS: This experiment used the cocktail probe drug method to simultaneously determine the inhibitory effect of compounds on five CYP450 enzymes in human liver microsomes. The human microsomes were from BD Gentest.

实验步骤如下：The experimental steps are as follows:

反应在100mM磷酸盐缓冲液中进行，总体积200μL。反应体系中微粒体浓度为0.25mg/mL，待测化合物浓度为20μM、6.67μM、2.22μM、0.74μM、0.25μM，特异性探针底物及浓度分别为非那西汀(CYP1A2)40μM、右美沙芬(CYP2D6)5μM、双氯芬酸(CYP2C9)10μM、S-美芬妥英(CYP2C19)40μM、睾酮(CYP3A4)80μM。孵育体系在37度恒温振荡器中预孵育5分钟，加入NADPH发生体系(含1.3mM NADP+、3.3mM葡萄糖6-磷酸、0.4U/L葡萄糖6-磷酸脱氢酶、3.3mM MgCL2)开始反应。孵育45min后加入等体积的乙腈终止反应，涡旋，13000rpm离心，取上清LC-MS-MS进样测定代谢产物生成量。特异性代谢产物分别为对乙酰氨基酚(CYP1A2)、右啡烷(CYP2D6)、4-羟基双氯芬酸(CYP2C9)、4-羟基美芬妥英(CYP2C19)、6β-羟基睾酮(CYP3A4)。特异性抑制剂分别为呋拉茶碱(CYP1A2)、奎尼丁(CYP2D6)、磺胺苯吡唑(CYP2C9)、反苯环丙胺(CYP2C19)、酮康唑(CYP3A4)。本实验最终结果为计算半数抑制浓度IC₅₀值。IC₅₀＝((50％-低抑制率％)/(高抑制率％-低抑制率％))×(高浓度-低浓度)+低浓度。按照上述的实验方法，本申请的优选化合物对各种CYP酶均只有不强的抑制或无抑制，说明其对其它药物的代谢影响较小。The reaction was carried out in 100 mM phosphate buffer in a total volume of 200 μL. The concentration of the microsomes in the reaction system was 0.25 mg/mL, and the concentration of the test compound was 20 μM, 6.67 μM, 2.22 μM, 0.74 μM, 0.25 μM. The specific probe substrate and concentration were phenacetin (CYP1A2) 40 μM, respectively. Dextromethorphan (CYP2D6) 5 μM, diclofenac (CYP2C9) 10 μM, S-mefenidine (CYP2C19) 40 μM, testosterone (CYP3A4) 80 μM. The incubation system was pre-incubated for 5 minutes in a 37-degree constant temperature shaker, and the reaction was started by adding a NADPH-producing system (containing 1.3 mM NADP+, 3.3 mM glucose 6-phosphate, 0.4 U/L glucose 6-phosphate dehydrogenase, 3.3 mM MgCL2). After incubation for 45 min, the reaction was stopped by adding an equal volume of acetonitrile, vortexed, centrifuged at 13,000 rpm, and the supernatant was subjected to LC-MS-MS injection to determine the amount of metabolite production. The specific metabolites were acetaminophen (CYP1A2), dextrorphan (CYP2D6), 4-hydroxydiclofenac (CYP2C9), 4-hydroxyfenfenin (CYP2C19), and 6β-hydroxytestosterone (CYP3A4). The specific inhibitors were furaphylline (CYP1A2), quinidine (CYP2D6), sulfaphenazole (CYP2C9), tranylcypromine (CYP2C19), ketoconazole (CYP3A4). The end result of this experiment the concentration IC ₅₀ values of inhibition calculated half. IC ₅₀ = ((50% - low inhibition rate %) / (high inhibition rate % - low inhibition rate %)) x (high concentration - low concentration) + low concentration. According to the above experimental method, the preferred compounds of the present application have only strong inhibition or no inhibition on various CYP enzymes, indicating that they have less influence on the metabolism of other drugs.

实施例55Example 55

化合物在大鼠体内的药物代谢动力学研究方法Method for pharmacokinetic study of compounds in rats

1.雄性SD大鼠【华阜康】买入后，在本实验室适应性饲养7天。 1. Male SD rats [Hua Kangkang] were purchased and adapted for 7 days in this laboratory.

2. 9只SD大鼠随机分为3组，每组3只，一组用于灌胃给药，另一组用于尾静脉注射给药。灌胃给药组的大鼠，给药前需过夜禁食。2. Nine SD rats were randomly divided into 3 groups, 3 in each group, one group was administered by intragastric administration, and the other group was administered by tail vein injection. Rats in the gavage-administered group were fasted overnight before administration.

3.大鼠给药后，采用眼眶静脉丛采血的方法在以下时间点采集血样：I.V.:(给药前)，0.08小时，0.25小时，0.5小时，1小时，2小时，4小时，8小时，24小时。P.O.:0.08小时，0.25小时，0.5小时，1小时，2小时，4小时，8小时，24小时。每个采血时间点采血量约为300μl。3. After administration of the rats, blood samples were taken at the following time points by blood collection from the orbital venous plexus: IV: (before administration), 0.08 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours ,24 hours. P.O.: 0.08 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours. The blood collection at each blood collection time point was about 300 μl.

4.采集的血样在4℃以12000rpm的转速离心5分钟，然后采集上层血浆样品，并于-20℃冰箱中保存待测。4. The collected blood samples were centrifuged at 12000 rpm for 5 minutes at 4 ° C, then the upper plasma samples were collected and stored in a refrigerator at -20 ° C for testing.

5.实验操作总结见表4：5. The experimental operation is summarized in Table 4:

表4、化合物在大鼠体内的药物代谢动力学试验设计Table 4. Design of pharmacokinetics of compounds in rats

6.使用LC-MS/MS(UPLC-MS/MS:液相Waters Acquity UPLC(USA)和质谱5500Q Trap(Applied Biosystem/MDS SCIEX)或者HPLC-MS\MS:液相Agilent 1200series(USA)和质谱API 4000(Applied Biosystem/MDS SCIEX))检测血浆中的化合物浓度。典型的检测条件见下表。6. Using LC-MS/MS (UPLC-MS/MS: liquid phase Waters Acquity UPLC (USA) and mass spectrometry 5500Q Trap (Applied Biosystem/MDS SCIEX) or HPLC-MS\MS: liquid phase Agilent 1200 series (USA) and mass spectrometry API 4000 (Applied Biosystem/MDS SCIEX)) detects the concentration of compounds in plasma. Typical test conditions are shown in the table below.

使用药代动力学专业软件WinNonlin【型号：Phoenix^TM

6.1厂家：Pharsight Corporation】计算药代动力学参数【Phoenix 1.1User’s Guide:p251-p300】。Use the pharmacokinetic professional software WinNonlin [Model: Phoenix ^TM

6.1 Manufacturer: Pharsight Corporation] Calculate pharmacokinetic parameters [Phoenix 1.1 User's Guide: p251-p300].

按照上述的实验方法，本申请中已测定的化合物表现出较好的生物利用度(>40％)。According to the above experimental method, the compounds which have been determined in the present application exhibit a good bioavailability (>40%).

实施例56Example 56

hERG结合实验(Dofetillide法)hERG binding assay (Dofetillide method)

依照专利US20050214870A1上描述的方法，可测定化合物对hERG抑制的IC₅₀值。本申请中优选的化合物对hERG只有很弱的抑制作用或无抑制作用，其IC₅₀值大于1000nM。In accordance with the method described in patent US20050214870A1, IC ₅₀ values may be determined for a compound hERG inhibition. Only weak inhibition present in preferred compounds of the application or no hERG inhibition, an IC ₅₀ value greater than 1000nM.

工业实用性Industrial applicability

本发明实施方案所提供的新型吡唑并嘧啶类衍生物，是蛋白激酶BTK的有效、安全、选择性高的抑制剂，可用作治疗BTK介导疾病的药品。 A novel pyrazolopyrimidine derivative provided by an embodiment of the present invention is a protein kinase BTK An effective, safe, and highly selective inhibitor that can be used as a drug for the treatment of BTK-mediated diseases.

Claims

一种新型吡唑并嘧啶化合物，如式(I)所示，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：A novel pyrazolopyrimidine compound, as shown in formula (I), a stereoisomer, tautomer, or pharmaceutically acceptable salt, or solvate thereof, or prodrug:

其中，among them,

n，m独立地取自于0、1或2；n, m are independently taken from 0, 1 or 2;

L是O，-C(O)-，-C(O)NH-，-CH₂-，S，S(O)，NH或S(O)₂；L is O, -C(O)-, -C(O)NH-, -CH ₂ -, S, S(O), NH or S(O) ₂ ;

A取自于取代或未取代的苯环、或者取代或未取代的杂芳环，并且与母核及L的连接位点是任选的；A is derived from a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and the attachment site to the parent nucleus and L is optional;

B独立地取自于取代或未取代的脂肪环、取代或未取代的杂环、取代或未取代的苯环、或者取代或未取代的杂芳环，并且与L的连接位点是任选的；B is independently taken from a substituted or unsubstituted aliphatic ring, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and the attachment site to L is optional of;

R₁和R₂各自独立地选自氢、未取代的C1-C4烷基、卤素、氰基，或者R₁和R₂与它们相连的碳原子一起形成三元碳环或四元碳环，或者R₁和R₂合并为氧代基；R ₁ and R ₂ are each independently selected from hydrogen, unsubstituted C1-C4 alkyl, halogen, cyano, or R ₁ and R ₂ together with the carbon atom to which they are attached form a ternary carbocyclic or quaternary carbocyclic ring, Or R ₁ and R _{2 are} combined into an oxo group;

Y选自氰基、
其中，R₃、R₄、R₅和R₆各自独立地取自于氢、未取代的C1-C4烷基、羟基取代的C1-C4烷基、C1-C4烷氧基C1-4烷基、卤素、氰基、或-(CH₂)_qN(R^aR^b)，其中，q为1、2、3、或4，R^a和R^b各自独立地选自氢、未取代的C1-C4烷基；Y is selected from cyano group,
Wherein R ₃ , R ₄ , R ₅ and R ₆ are each independently derived from hydrogen, unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl , halogen, cyano, or -(CH ₂ ) _q N(R ^a R ^b ), wherein q is 1, 2, 3, or 4, and R ^a and R ^b are each independently selected from hydrogen, unsubstituted C1 -C4 alkyl;

并规定，当R₁和R₂都是氢时，A是苯基，L是O，B是4-氯苯基、3-氯吡啶-6-基、2,4-二氟苯基；Y是4-羟基丁-2-烯酰基、丁-2-炔酰基、或氰基；And stipulates that when both R ₁ and R ₂ are hydrogen, A is a phenyl group, L is O, and B is 4-chlorophenyl, 3-chloropyridin-6-yl, 2,4-difluorophenyl; Y Is 4-hydroxybut-2-enoyl, but-2-ynyl, or cyano;

或，当R₁和R₂都是氢时，A是苯基，L是C(O)-、或-CH₂-，B是吗啉-4-基；Y为氰基； Or, when both R ₁ and R ₂ are hydrogen, A is phenyl, L is C(O)-, or -CH ₂ -, B is morpholin-4-yl; Y is cyano;

或，当R₁和R₂都是氢时，A是吡唑基，L是S(O)₂、或-CH₂-，B是苯基、或环丙基；Y为氰基；Or, when both R ₁ and R ₂ are hydrogen, A is pyrazolyl, L is S(O) ₂ , or -CH ₂ -, B is phenyl or cyclopropyl; Y is cyano;

或，当R₁和R₂都是氢时，A是吡啶基，L是NH、或O，B是吡喃-4-基、或环己基；Y为氰基。Or, when both R ₁ and R ₂ are hydrogen, A is a pyridyl group, L is NH or O, B is pyran-4-yl or cyclohexyl; and Y is a cyano group.
如权利要求1所述的化合物，其中，n，m独立地取自于0、1或2；L为O，-C(O)-，-C(O)NH-，-CH₂-，NH或S，更优选地为O，-C(O)NH-，NH。The compound according to claim 1, wherein n, m are independently taken from 0, 1, or 2; L is O, -C(O)-, -C(O)NH-, -CH ₂ -, NH Or S, more preferably O, -C(O)NH-, NH.
如权利要求1所述的化合物，其中，A取自于取代或未取代的苯环、或者取代或未取代的杂芳环，并且与母核及L的连接位点是任选的；B独立地取自于取代或未取代的脂肪环、取代或未取代的杂环、取代或未取代的苯环、或者取代或未取代的杂芳环，并且与L的连接位点是任选的；The compound according to claim 1, wherein A is derived from a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and the attachment site to the mother nucleus and L is optional; Or taken from a substituted or unsubstituted aliphatic ring, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and a linking site with L is optional;

其中，所述取代的苯环是指苯基上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基；优选地，所述取代的苯环为氟取代的苯基、或氯取代的苯基，更优选地为2,4-二氟苯基、或4-氯苯基；Wherein the substituted benzene ring means that the position on the phenyl group is substituted with an optional substituent selected from the group consisting of hydrogen, methyl, methoxy, fluoro, chloro, trifluoromethyl, Fluoromethoxy or cyano; preferably, the substituted benzene ring is a fluoro substituted phenyl or a chloro substituted phenyl, more preferably 2,4-difluorophenyl, or 4-chlorophenyl ;

所述未取代的杂芳环是指呋喃、吡咯、噻吩、恶唑、异恶唑、吡唑、咪唑、噻唑、异噻唑、恶二唑、三氮唑、噻二唑、四氮唑、吡啶、嘧啶、吡嗪、哒嗪、三嗪；所述取代的杂芳环是指以上基团上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基；更优选地，所述取代的吡啶为氯代吡啶，特别优选地为4-氯-吡啶-2-基；The unsubstituted heteroaryl ring means furan, pyrrole, thiophene, oxazole, isoxazole, pyrazole, imidazole, thiazole, isothiazole, oxadiazole, triazole, thiadiazole, tetrazolium, pyridine , pyrimidine, pyrazine, pyridazine, triazine; said substituted heteroaryl ring means that any position on the above group is substituted by an optional substituent selected from the group consisting of hydrogen, methyl, methoxy a radical, a fluorine, a chlorine, a trifluoromethyl group, a trifluoromethoxy group or a cyano group; more preferably, the substituted pyridine is a chloropyridine, particularly preferably a 4-chloro-pyridin-2-yl group;

所述未取代的脂肪环是指环丙烷、环丁烷、环戊烷、环己烷、环庚烷、环辛烷；所述取代的脂肪环是指以上基团上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基；The unsubstituted aliphatic ring means cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane; the substituted aliphatic ring means any position on the above group is optionally selected Substituted by a substituent selected from hydrogen, methyl, methoxy, fluoro, chloro, trifluoromethyl, trifluoromethoxy or cyano;

所述未取代的杂环是指四氢呋喃、四氢吡喃、四氢吡咯、哌啶、

其中w 取自0、1或2；所述取代的杂环是指以上基团上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基。The unsubstituted heterocyclic ring means tetrahydrofuran, tetrahydropyran, tetrahydropyrrole, piperidine,

Wherein w is taken from 0, 1 or 2; the substituted heterocyclic ring means that any position on the above group is substituted by an optional substituent selected from the group consisting of hydrogen, methyl, methoxy, and fluorine. , chlorine, trifluoromethyl, trifluoromethoxy or cyano.
如权利要求1所述的化合物，其中，R₁和R₂其中一个为氢、而另一个为C1-C4烷基，或者R₁和R₂与它们相连的碳原子一起形成环丙基；更优选地，R₁和R₂都是氢，或者其中一个是氢而另一个为甲基，或者R₁和R₂与它们相连的碳原子一起形成环丙基。The compound according to claim 1, wherein one of R ₁ and R ₂ is hydrogen and the other is C1-C4 alkyl, or R ₁ and R ₂ together with the carbon atom to which they are attached form a cyclopropyl group; Preferably, R ₁ and R ₂ are both hydrogen, or one of them is hydrogen and the other is methyl, or R ₁ and R ₂ together with the carbon atom to which they are attached form a cyclopropyl group.
如权利要求1所述的化合物，其中，优选地，如式(II)所示的吡唑并嘧啶化合物，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：The compound according to claim 1, wherein a pyrazolopyrimidine compound represented by the formula (II), a stereoisomer, a tautomer thereof, or a pharmaceutically acceptable salt, or a solvent is preferred. Compound, or prodrug:

其中，L、A、B和Y的定义如上述式(I)；Wherein, L, A, B and Y are as defined above for formula (I);

并规定，A是苯基，L是O，B是4-氯苯基、3-氯吡啶-6-基、2,4-二氟苯基；Y是4-羟基丁-2-烯酰基、丁-2-炔酰基、或氰基；And stipulates that A is a phenyl group, L is O, B is 4-chlorophenyl, 3-chloropyridin-6-yl, 2,4-difluorophenyl; Y is 4-hydroxybut-2-enoyl, But-2-ynyl, or cyano;

或，A是苯基，L是C(O)-、或-CH₂-，B是吗啉-4-基；Y为氰基；Or, A is phenyl, L is C(O)-, or -CH ₂ -, B is morpholin-4-yl; Y is cyano;

或，A是吡唑基，L是S(O)₂、或-CH₂-，B是苯基、或环丙基；Y为氰基；Or, A is pyrazolyl, L is S(O) ₂ or -CH ₂ -, B is phenyl or cyclopropyl; Y is cyano;

或，A是吡啶基，L是NH、或O，B是吡喃-4-基、或环己基；Y为氰基；Or, A is pyridyl, L is NH, or O, B is pyran-4-yl, or cyclohexyl; Y is cyano;

更优选地，式(II)所示的吡唑并嘧啶化合物为下列化合物中的一种： More preferably, the pyrazolopyrimidine compound represented by the formula (II) is one of the following compounds:

其中，式(II-1)或(II-2)中L、B和Y的定义如上述式(I)；Wherein, L, B and Y in the formula (II-1) or (II-2) are as defined in the above formula (I);

并规定，L是O，B是4-氯苯基、3-氯吡啶-6-基、2,4-二氟苯基；Y是4-羟基丁-2-烯酰基、丁-2-炔酰基、或氰基；And stipulates that L is O, B is 4-chlorophenyl, 3-chloropyridin-6-yl, 2,4-difluorophenyl; Y is 4-hydroxybut-2-enoyl, but-2-yne An acyl group or a cyano group;

或，L是C(O)-、或-CH₂-，B是吗啉-4-基；Y为氰基；Or, L is C(O)-, or -CH ₂ -, B is morpholin-4-yl; Y is cyano;

或：优选地，如式(III)所示的吡唑并嘧啶化合物，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：Or: preferably, a pyrazolopyrimidine compound represented by formula (III), a stereoisomer, tautomer, or pharmaceutically acceptable salt, or solvate thereof, or prodrug:

其中，L、A、B和Y的定义如上述式(I)；Wherein, L, A, B and Y are as defined above for formula (I);

更优选地，式(III)所示的吡唑并嘧啶化合物为下列化合物中的一种： More preferably, the pyrazolopyrimidine compound represented by the formula (III) is one of the following compounds:

其中，式(III-1)、(III-2)、(III-3)和(III-4)中L、B和Y的定义如上述式(I)；或：Wherein, L, B and Y in the formulae (III-1), (III-2), (III-3) and (III-4) are as defined in the above formula (I); or:

优选地，如式(IV)所示的吡唑并嘧啶化合物，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：Preferably, the pyrazolopyrimidine compound of formula (IV), a stereoisomer, tautomer, or pharmaceutically acceptable salt, or solvate thereof, or prodrug:

其中，L、A、B和Y的定义如上述式(I)；Wherein, L, A, B and Y are as defined above for formula (I);

更优选地，式(IV)所示的吡唑并嘧啶化合物为下列化合物中的一种：More preferably, the pyrazolopyrimidine compound represented by the formula (IV) is one of the following compounds:

其中，式(IV-1)或(IV-2)中L、B和Y的定义如上述式(I)。 Wherein, L, B and Y in the formula (IV-1) or (IV-2) are as defined in the above formula (I).
如权利要求5所述的化合物，其中，L为O；The compound of claim 5, wherein L is O;

B为
和B is
with

Y选自氰基、
Y is selected from cyano group,

R₃、R₄、R₅和R₆取自于氢、未取代的C1-C4烷基、羟基取代的C1-C4烷基、C1-C4烷氧基C1-4烷基、卤素、氰基、或-(CH₂)_qN(R^aR^b)，其中，q为1、2、3、或4，R^a和R^b各自独立地选自氢、未取代的C1-C4烷基。R ₃ , R ₄ , R ₅ and R _{6 are} derived from hydrogen, unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl, halogen, cyano Or -(CH ₂ ) _q N(R ^a R ^b ), wherein q is 1, 2, 3, or 4, and R ^a and R ^b are each independently selected from hydrogen, unsubstituted C1-C4 alkyl.
选自下列化合物中的一种，或其药学上可接受的盐，或溶剂化物，或前药：Or one of the following compounds, or a pharmaceutically acceptable salt, or solvate thereof, or a prodrug:
权利要求1-7中任一项所述化合物的制备方法，包括如下步骤：A method of preparing a compound according to any one of claims 1-7, comprising the steps of:

(1)将式(V)化合物与式(VI)化合物反应，得到式(VII)化合物； (1) reacting a compound of the formula (V) with a compound of the formula (VI) to give a compound of the formula (VII);

(2)将式(VII)化合物与式(VIII)化合物反应，得到式(IX)化合物；(2) reacting a compound of the formula (VII) with a compound of the formula (VIII) to give a compound of the formula (IX);

(3)将式(IX)化合物经脱保护基PG得到式(X)化合物；(3) a compound of the formula (IX) is subjected to a deprotection group PG to give a compound of the formula (X);

(4)将式(X)化合物与式(XI)化合物反应，得到式(I)化合物；(4) reacting a compound of the formula (X) with a compound of the formula (XI) to give a compound of the formula (I);

在上述的式(V)、式(VI)、式(VII)、式(VIII)、式(IX)、式(X)和式(XI)中涉及的取代基R₁、R₂、L、B、Y和n、m的定义如上面的式(I)，PG为氨基保护基，X为氯、溴或羟基。Substituents R ₁ , R ₂ , L, in the above formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X) and formula (XI), B, Y and n, m are as defined above for formula (I), PG is an amino protecting group, and X is chlorine, bromine or hydroxyl.
包含权利要求1-7中任一项所述化合物或其药学上可接受的盐的药物组合物。A pharmaceutical composition comprising a compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof.
权利要求1-7中任一项所述化合物或权利要求9所述药物组合物在制备BTK抑制剂药物中的用途。Use of a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 9 for the preparation of a BTK inhibitor medicament.
如权利要求10所述的用途，其中，BTK抑制剂是指预防或治疗由BTK介导的疾病，所述疾病选自自身免疫性疾病、炎性疾病、异种免疫性情况或疾病、血栓栓塞疾病和癌症。 The use according to claim 10, wherein the BTK inhibitor refers to the prevention or treatment of a disease mediated by BTK, which is selected from the group consisting of an autoimmune disease, an inflammatory disease, a heterogeneous immune condition or disease, and a thromboembolic disease. And cancer.