CN115023428A

CN115023428A - Pyrimidopyrroles

Info

Publication number: CN115023428A
Application number: CN202180010090.0A
Authority: CN
Inventors: 张国宝; 陈家隽; 周峰; 蒋蕾; 唐锋; 唐任宏; 任晋生
Original assignee: Jiangsu Simcere Pharmaceutical Co Ltd
Current assignee: Jiangsu Simcere Pharmaceutical Co Ltd
Priority date: 2020-01-21
Filing date: 2021-01-21
Publication date: 2022-09-06
Also published as: WO2021147952A1

Abstract

The application provides a pyrimido five-membered ring compound shown in formula (Ib) or pharmaceutically acceptable salt thereof, a pharmaceutical composition and a preparation method thereof, and application of the pyrimido five-membered ring compound as JAK3 and/or BTK inhibitor.

Description

Pyrimidopyrroles

The application requires the priority of two prior applications, namely a patent application number 202010071941.X entitled "pyrimidopyrrole compound" submitted to the intellectual property office of China on 21/1/2020 and a patent application number 202011003941.2 entitled "pyrimidopyrrole compound" submitted to the intellectual property office of China on 22/9/2020. The entire disclosures of both applications are incorporated by reference into this application.

Technical Field

The invention relates to a novel pyrimidopyrrole compound or a pharmaceutically acceptable salt, a pharmaceutical composition containing the same and application thereof in preventing or treating kinase related diseases such as Janus kinase (JAK, particularly JAK3) and/or Bruton Tyrosine Kinase (BTK) related diseases.

Background

Autoimmune diseases are diseases in which an attack on self-cells or tissues is caused by an immune dysfunction, resulting in inflammation and tissue damage, and include Rheumatoid Arthritis (RA), Inflammatory Bowel Disease (IBD), and Systemic Lupus Erythematosus (SLE). BTK and JAK3 are two important targets for autoimmune diseases.

BTK is a member of the TEC family of non-receptor tyrosine kinases and structurally includes the PH domain, TH domain, SH3 domain, SH2 domain and SH1 domain. BTK plays a key role in the activation process of a B cell antigen receptor (BCR) signal channel, regulates the development and activation of B cells, and plays an important role in the proliferation of the B cells, the expression of proinflammatory cytokines and the secretion of antibodies (Targeting Bruton's tyrosine kinases in B cell receptors. Nat Rev cancer.2014 Apr; 14(4):219-32), so BTK becomes one of important targets for treating diseases related to abnormal activation of the B cells, including autoimmune diseases and B cell lymphoma. Ibrutinib, Acarabutinib and Zanbutinib are three approved BTK inhibitors, mainly used for treating B cell lymphoma, and have obvious curative effect in partial patients, but serious side effect and drug resistance mutation are also observed clinically. Ibrutinib was approved by the U.S. FDA for the treatment of Graft Versus Host Disease (GVHD) in 2017, while other BTK inhibitors are currently actively being clinically explored for the treatment of autoimmune diseases, including RA, SLE, and Multiple Sclerosis (MS).

JAK3 is a member of the JAK family of non-receptor tyrosine kinases. The JAK kinase family has 4 members, JAK-1, JAK-2, JAK-3 and TYK-2. Signal Transducers and Activators of Transcription (STATs) are downstream substrates of JAK3, and JAK3 activates STATs to dimerize them into nuclei, regulating transcriptional expression of specific genes. The JAK-STAT signaling Pathway plays an important role in lymphocyte proliferation and differentiation and expression of proinflammatory cytokines (JAK inhibition as a Therapeutic Pathway for immune and inflammatory diseases Nat Rev Drug Discov.2017 Decumber 28; 17(1) 78; The JAK-STAT Pathway: Impact on Human Disease and Therapeutic interaction.Annual Review of medicine. Vol.66: 311) 328), so JAK3 becomes one of The targets of autoimmune diseases and malignant tumors. Tofacitinib is an FDA-approved JAK3 inhibitor, which exhibits good clinical efficacy in RA and IBD. But also have certain adverse effects, including severe infections, liver damage, etc., which are believed to be associated with insufficient selectivity of Tofacitinib for JAK1/2 (JAK inhibition as a thermal protocol for immune and inflammatory diseases. Nat Rev Drug disorders.2017 Decumber 28; 17(1): 78; JAK-inhibition. New plasma in the field of immune-mediated diseases, and bed and rhematoid arthritis. Rheumatology (Oxford) 2019 Feb 1; 58 (supply 1): i43-i 54).

In addition to the separate clinical effects of the BTK and JAK3 inhibitors, inhibition of the BTK/JAK3 signaling pathway at the same time would show synergistic efficacy. Several studies indicate that BTK and JAK are simultaneously inhibited in a collagen-induced arthritis model (CIA) in rats, joint swelling is obviously relieved, the number of osteoclasts is reduced, pathological scores are also obviously improved, and the curative effect is superior to that of a single drug (2016 ACR/ARHP annular meeting. abstract 484; 2013 ACR/ARHP annular meeting. abstract 2353). Abbvie initiated a second clinical trial of ABBV599(BTK inhibitor and JAK inhibitor in combination) against RA and SLE in 2018, 9 and 2019, 6, respectively. Another dual-target inhibitor to BTK/JAK3 DWP212525 also exhibited remission of disease and protection of joints in the mouse CIA model (2019 ACR/ARHP annular meeting. abstract 965).

In view of the huge autoimmune disease market and the unmet market demand, based on the functions of BTK and JAK3 on autoimmune diseases and the existing clinical effects, there is a need to develop a dual-target small molecule inhibitor with good activity, good selectivity and low toxic and side effects on BTK and JAK 3.

Disclosure of Invention

The invention provides a compound shown as a formula (Ib) or a pharmaceutically acceptable salt thereof:

wherein:

x is selected from O or NH;

R ¹ selected from H or optionally substituted by R ^a Substituted of the following groups: c ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₄ Cycloalkyl, 3-14 membered heterocyclyl, C ₆ -C ₁₀ Aryl or 5-10 membered heteroaryl;

R ^a selected from F, Cl, Br, I, OH, CN, O, NO ₂ Or optionally substituted with R ^b Substituted of the following groups: NH (NH) ₂ 、SH、S(O)NH ₂ 、S(O)(C ₁ -C ₁₀ Alkyl), S (O) ₂ (C ₁ -C ₁₀ Alkyl), P (O) (C) ₁ -C ₁₀ Alkyl group), C ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₄ Cycloalkyl, 3-14 membered heterocyclyl, C ₁ -C ₁₀ Alkoxy radical, C ₃ -C ₁₄ Cycloalkyloxy, 3-14 membered heterocyclyloxy, C ₂ -C ₁₀ Alkenyl radical, C ₂ -C ₁₀ Alkynyl, C ₆ -C ₁₀ Aryl, 5-10 membered heteroaryl, C ₆ -C ₁₀ Aryloxy or 5-10 membered heteroaryloxy;

R ^b selected from F, Cl, Br, I, OH, CN, ═ O, NO ₂ 、NH ₂ 、SH、C ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₄ Cycloalkyl, 3-14 membered heterocyclyl, C ₁ -C ₁₀ Alkoxy radical, C ₃ -C ₁₄ Cycloalkyloxy, 3-14 membered heterocyclyloxy, C ₂ -C ₁₀ Alkenyl radical, C ₂ -C ₁₀ Alkynyl, C ₆ -C ₁₀ Aryl, 5-10 membered heteroaryl, C ₆ -C ₁₀ Aryloxy or 5-10 membered heteroaryloxy;

R ² selected from H, F, Cl, Br, I, C ₃ -C ₁₄ Cycloalkyl or phenyl, said C ₃ -C ₁₄ Cycloalkyl or phenyl optionally substituted by R ^d Substitution;

R ^d selected from F, Cl, Br, I, OH, CN or C optionally substituted by a group selected from F, Cl, Br, I, OH ₁ -C ₄ An alkyl group;

R ³ selected from H, F, Cl, Br, I or C optionally substituted by a group selected from F, Cl, Br, I, OH ₁ -C ₁₀ An alkyl group;

R ⁴ selected from H, F, Cl, Br, I, OH, CN or optionally by R ^c Substituted of the following groups: c ₁ -C ₁₀ Alkyl radical, C ₁ -C ₁₀ An alkoxy group;

R ⁵ 、R ⁶ 、R ⁷ independently selected from H, F, Cl, Br, I, CN or optionally substituted by R ^e Substituted of the following groups: c ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₀ Cycloalkyl or 3-10 membered heterocyclyl;

R ^c 、R ^e independently selected from F, Cl, Br, I, OH;

n is selected from 0 or 1;

m is selected from 1 or 2;

with the following conditions: when R is ² When Cl, X is O, n is 1 and R is ¹ Is not methyl or cyclopropyl; when R is ² When H, X is O and R ¹ Is not ethyl, CH ₂ CHF ₂ 、CH ₂ CH ₂ OH, cyclopropyl,

when R is ² When F is equal to R ¹ Not being ethyl or CH ₂ CHF ₂ 。

In some embodiments, X is selected from NH.

In some embodiments, X is selected from O.

In some embodiments, R ² Is selected from C ₃ -C ₁₄ Cycloalkyl or phenyl, said C ₃ -C ₁₄ Cycloalkyl or phenyl optionally substituted by R ^d And (4) substitution.

In some embodiments, R ² Is selected from C ₃ -C ₁₀ Cycloalkyl or phenyl, said C ₃ -C ₁₀ Cycloalkyl or phenyl optionally substituted by R ^d And (4) substitution.

In some embodiments, R ² Is selected from C ₃ -C ₆ Cycloalkyl or phenyl, said C ₃ -C ₆ Cycloalkyl or phenyl optionally substituted by R ^d And (4) substitution.

In some embodiments, R ² Selected from cyclopropyl or phenyl, optionally substituted by R ^d And (4) substitution.

In some embodiments, R ² Is selected from C ₃ -C ₆ Cycloalkyl or phenyl.

In some embodiments, R ² Selected from cyclopropyl or phenyl.

In some embodiments, R ² Selected from H, F, Cl, Br or I.

In some embodiments, R ² Selected from H, F or Cl.

In some embodiments, R ³ Selected from H, F, Cl, Br, I or C optionally substituted by a group selected from F, Cl, Br, I, OH ₁ -C ₆ An alkyl group.

In some embodiments, R ³ Selected from H, F, Cl, Br, I or C optionally substituted by a group selected from F, Cl, Br, I, OH ₁ -C ₄ An alkyl group.

In some embodiments, R ³ Selected from H, F, Cl, Br or I.

In some embodiments, R ³ Selected from H or F.

In some embodiments, R ¹ Selected from H or optionally substituted by R ^a Substituted of the following groups: c ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₀ Cycloalkyl, 3-to 10-membered heterocyclic group, C ₆ -C ₁₀ Aryl or 5-10 membered heteroaryl.

In some embodiments, R ¹ Is selected from C ₁ -C ₁₀ Alkyl or 3-10 membered heterocyclyl, said C ₁ -C ₁₀ Alkyl or 3-10 membered heterocyclyl is optionally substituted by R ^a And (4) substitution.

In some embodiments, R ¹ Is selected from C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl, C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl is optionally substituted by R ^a And (4) substitution.

In some embodiments, R ¹ Is selected from C ₁ -C ₆ An alkyl group or a 4-6 membered heterocyclic group, said 4-6 membered heterocyclic group containing an O atom and/or an N atom, said C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl is optionally substituted by R ^a And (4) substitution.

In some embodiments, R ¹ Is selected from C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl, said 4-6 membered heterocyclyl containing O and/or N as ring atoms, said C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl is optionally substituted by R ^a And (4) substitution.

In some embodiments, R ¹ Is selected from C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl, said 4-6 membered heterocyclyl containing one O atom or one N atom, said C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl is optionally substituted by R ^a And (4) substitution.

In some embodiments, R ¹ Is selected from C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl, said 4-6 membered heterocyclyl containing one O or one N as a ring atom, said C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl is optionally substituted by R ^a And (4) substitution.

In some embodiments, R ^a Selected from F, Cl, Br, I, OH, CN, ═ O, NO ₂ Or optionally substituted with R ^b Substituted of the following groups: NH ₂ 、SH、C ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₀ Cycloalkyl, 3-10 membered heterocyclyl, C ₁ -C ₁₀ Alkoxy radical, C ₃ -C ₁₀ Cycloalkyloxy, 3-10 membered heterocyclyloxy, C ₂ -C ₁₀ Alkenyl radical, C ₂ -C ₁₀ Alkynyl, C ₆ -C ₁₀ Aryl, 5-10 membered heteroaryl, C ₆ -C ₁₀ Aryloxy or 5-10 membered heteroaryloxy.

In some embodiments, R ^b Selected from F, Cl, Br, I, OH, CN, ═ O, NO ₂ 、NH ₂ 、SH、C ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₀ Cycloalkyl, 3-10 membered heterocyclyl, C ₁ -C ₁₀ Alkoxy radical, C ₃ -C ₁₀ Cycloalkyloxy, 3-10 membered heterocyclyloxy, C ₂ -C ₁₀ Alkenyl radical, C ₂ -C ₁₀ Alkynyl, C ₆ -C ₁₀ Aryl, 5-10 membered heteroaryl, C ₆ -C ₁₀ Aryloxy or 5-10 membered heteroaryloxy.

In some embodiments, R ^a Selected from F, Cl, Br, I, OH, CN, C ₁ -C ₁₀ Alkyl or C ₃ -C ₁₀ A cycloalkyl group.

In some embodiments, R ^a Selected from F, Cl, Br, I or C ₁ -C ₆ An alkyl group.

In some embodiments, R ^a Selected from F or C ₁ -C ₆ An alkyl group.

In some embodiments, R ^a Is selected from C ₁ -C ₆ An alkyl group.

In some embodiments, R ^a Selected from F or methyl.

In some embodiments, R ^a Is selected from F.

In some embodiments, R ^a Selected from methyl.

In some embodiments, R ¹ Selected from methyl, trifluoromethyl, ethyl, oxetanyl, tetrahydrofuryl, N-methylpyrrolidinyl, tetrahydropyranyl or N-methylpiperidinyl.

In some embodiments, R ¹ Selected from methyl, ethyl, oxetanyl, tetrahydrofuryl, tetrahydropyranyl or N-methylpiperidinyl.

In some embodiments, R ¹ Selected from ethyl, oxetanyl, tetrahydrofuryl, tetrahydropyranyl or N-methylpiperidinyl.

In some embodiments, R ¹ Selected from methyl, trifluoromethyl, ethyl,

In some embodiments, R ¹ Selected from methyl, ethyl,

In some embodiments, R ¹ Selected from ethyl,

In some embodiments, R ⁴ Selected from H, F, Cl, Br, I, OH, CN or optionally substituted by R ^c Substituted of the following groups: c ₁ -C ₁₀ An alkyl group.

In some embodiments, R ⁴ Selected from H, F, Cl, Br, I, OH, CN or C ₁ -C ₆ An alkyl group.

In some embodiments, R ⁴ Is selected from H or C ₁ -C ₆ An alkyl group.

In some embodiments, R ⁴ Selected from H or AAnd (4) a base.

In some embodiments, R ⁵ Selected from H, F, Cl, Br, I, CN or optionally substituted by R ^e Substituted C ₁ -C ₁₀ An alkyl group.

In some embodiments, R ⁵ Selected from H, F, Cl, Br, I, CN.

In some embodiments, R ⁵ Selected from H or CN.

In some embodiments, R ⁶ 、R ⁷ Independently selected from H, F, Cl, Br, I, CN or optionally substituted by R ^e Substituted C ₁ -C ₁₀ An alkyl group.

In some embodiments, R ⁶ 、R ⁷ Independently selected from H, CN or optionally substituted by R ^e Substituted C ₁ -C ₁₀ An alkyl group.

In some embodiments, R ⁶ 、R ⁷ Independently selected from H, CN or C ₁ -C ₆ An alkyl group.

In some embodiments, R ⁶ 、R ⁷ Independently selected from H or C ₁ -C ₆ An alkyl group.

In some embodiments, R ⁶ 、R ⁷ Independently selected from H or tert-butyl.

In some embodiments, the n is selected from 1.

In some embodiments, the n is selected from 0.

In some embodiments, m is selected from 1.

In some embodiments, m is selected from 2.

In some embodiments, the compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is selected from a compound of formula (Ia), or a pharmaceutically acceptable salt thereof:

wherein:

x is selected from O or NH;

R ^a selected from F, Cl, Br, I, OH, CN, O, NO ₂ Or optionally substituted with R ^b Substituted of the following groups: NH (NH) ₂ 、SH、S(O)NH ₂ 、S(O)(C ₁ -C ₁₀ Alkyl), S (O) ₂ (C ₁ -C ₁₀ Alkyl), P (O) (C) ₁ -C ₁₀ Alkyl), C ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₄ Cycloalkyl, 3-14 membered heterocyclyl, C ₁ -C ₁₀ Alkoxy radical, C ₃ -C ₁₄ Cycloalkyloxy, 3-14 membered heterocyclyloxy, C ₂ -C ₁₀ Alkenyl radical, C ₂ -C ₁₀ Alkynyl, C ₆ -C ₁₀ Aryl, 5-10 membered heteroaryl, C ₆ -C ₁₀ Aryloxy or 5-10 membered heteroaryloxy;

R ^b selected from F, Cl, Br, I, OH, CN, ═ O, NO ₂ 、NH ₂ 、SH、C ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₄ Cycloalkyl, 3-14 membered heterocyclyl, C ₁ -C ₁₀ Alkoxy radical, C ₃ -C ₁₄ Cycloalkyloxy, 3-14 membered heterocyclyloxy, C ₂ -C ₁₀ Alkenyl radical, C ₂ -C ₁₀ Alkynyl, C ₆ -C ₁₀ Aryl, 5-to 10-membered heteroaryl, C ₆ -C ₁₀ Aryloxy or 5-10 membered heteroaryloxy;

R ² is selected from C ₃ -C ₁₄ Cycloalkyl or phenyl, said C ₃ -C ₁₄ Cycloalkyl or phenyl optionally substituted by R ^d Substitution;

R ⁴ selected from H, F, Cl, Br, I, OH, CN or optionally substituted by R ^c Substituted of the following groups: c ₁ -C ₁₀ Alkyl radical, C ₁ -C ₁₀ An alkoxy group;

R ^c selected from F, Cl, Br, I, OH;

n is selected from 0 or 1.

In some embodiments, X is selected from NH.

In some embodiments, R ² Selected from cyclopropyl or phenyl, said cyclopropyl or phenyl being optionally substituted by R ^d And (4) substitution.

In some embodiments, R ² Is selected from C ₃ -C ₆ Cycloalkyl or phenyl.

In some embodiments, R ² Selected from cyclopropyl or phenyl.

In some embodiments of the present invention, the substrate is,R ³ selected from H, F, Cl, Br, I or C optionally substituted by a group selected from F, Cl, Br, I, OH ₁ -C ₆ An alkyl group.

In some embodiments, R ³ Selected from H, F, Cl, Br or I.

In some embodiments, R ³ Selected from H or F.

In some embodiments, R ¹ Is selected from C ₁ -C ₁₀ Alkyl or 3-10 membered heterocyclic group, said C ₁ -C ₁₀ Alkyl or 3-10 membered heterocyclyl is optionally substituted by R ^a And (4) substitution.

In some embodiments, R ¹ Is selected from C ₁ -C ₆ Alkyl or 4-6 membered heterocyclic group, said C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl is optionally substituted by R ^a And (4) substitution.

In some embodiments, R ¹ Is selected from C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl, said 4-6 membered heterocyclyl containing O and/or N as ring atoms, said C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl is optionally substitutedR ^a And (4) substitution.

In some embodiments, R ¹ Is selected from C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl, said 4-6 membered heterocyclyl containing one O or one N as ring atoms, said C ₁ -C ₆ Alkyl or 4-6 membered heterocyclyl is optionally substituted by R ^a And (4) substitution.

In some embodiments, R ^a Selected from F, Cl, Br, I, OH, CN, ═ O, NO ₂ Or optionally substituted with R ^b Substituted of the following groups: NH (NH) ₂ 、SH、C ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₀ Cycloalkyl, 3-10 membered heterocyclyl, C ₁ -C ₁₀ Alkoxy radical, C ₃ -C ₁₀ Cycloalkyloxy, 3-10 membered heterocyclyloxy, C ₂ -C ₁₀ Alkenyl radical, C ₂ -C ₁₀ Alkynyl, C ₆ -C ₁₀ Aryl, 5-10 membered heteroaryl, C ₆ -C ₁₀ Aryloxy or 5-10 membered heteroaryloxy.

In some embodiments, R ^b Selected from F, Cl, Br, I, OH, CN, ═ O, NO ₂ 、NH ₂ 、SH、C ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₀ Cycloalkyl, 3-to 10-membered heterocyclic group, C ₁ -C ₁₀ Alkoxy radical, C ₃ -C ₁₀ Cycloalkyloxy, 3-10 membered heterocyclyloxy, C ₂ -C ₁₀ Alkenyl radical, C ₂ -C ₁₀ Alkynyl, C ₆ -C ₁₀ Aryl, 5-10 membered heteroaryl, C ₆ -C ₁₀ Aryloxy or 5-10 membered heteroaryloxy.

In some embodiments, R ^a Is selected from C ₁ -C ₆ An alkyl group.

In some embodiments, R ^a Selected from methyl. In some embodiments, R ¹ Selected from methyl, ethyl, oxetanyl, tetrahydrofuryl, tetrahydropyranyl or N-methylpiperidinyl.

In some embodiments, R ¹ Selected from methyl, ethyl,

In some embodiments, R ¹ Selected from ethyl,

In some embodiments, R ⁴ Is selected from H or C ₁ -C ₆ An alkyl group.

In some embodiments, R ⁴ Selected from H or methyl.

In some embodiments, the n is selected from 1.

In some embodiments, the n is selected from 0. In some embodiments, the compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is selected from a compound of formula (I), or a pharmaceutically acceptable salt thereof:

wherein: r ¹ 、R ² 、R ³ As defined in formula (Ib).

In some embodiments, the compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is selected from a compound of formula (IIb), or a pharmaceutically acceptable salt thereof:

wherein: r ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ 、R ⁷ X, m and n are as defined in formula (Ib).

In some embodiments, the compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is selected from a compound of formula (IIa), or a pharmaceutically acceptable salt thereof:

wherein, X, R ¹ 、R ² 、R ³ 、R ⁴ And n is as defined above.

In some embodiments, the compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is selected from a compound of formula (II), or a pharmaceutically acceptable salt thereof:

wherein R is ¹ 、R ² 、R ³ As defined above.

In some embodiments, the compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is selected from the following compounds, or pharmaceutically acceptable salts thereof:

in some embodiments, the compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is selected from the following compounds or pharmaceutically acceptable salts:

the invention also provides a pharmaceutical composition which comprises the compound shown in the formula (Ib) or pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials.

The invention also provides a pharmaceutical composition which comprises the compound shown in the formula (Ib) or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or excipient.

Further, the invention relates to an application of the compound shown in the formula (Ib) or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in preparing a medicament for preventing or treating Janus kinase (JAK, particularly JAK3) and/or Bruton Tyrosine Kinase (BTK) related diseases.

Further, the invention relates to an application of the compound shown in the formula (Ib) or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in preventing or treating Janus kinase (JAK, particularly JAK3) and/or Bruton Tyrosine Kinase (BTK) related diseases.

Further, the present invention relates to a compound of formula (Ib) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the prevention or treatment of a disease associated with Janus kinases (JAK, particularly JAK3) and/or Bruton's Tyrosine Kinase (BTK).

The invention also relates to a method of treating a disease associated with Janus kinases (JAKs, particularly JAK3) and/or Bruton's Tyrosine Kinase (BTK), the method comprising administering to a patient a therapeutically effective amount of a pharmaceutical formulation comprising a compound of formula (Ib) as described herein or a pharmaceutically acceptable salt thereof.

Preferred embodiments of the present invention include diseases related to Janus kinases (JAK, particularly JAK3) and/or Bruton's Tyrosine Kinase (BTK) including, but not limited to, tumors (e.g., B cell lymphoma), autoimmune diseases (e.g., rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus), and the like.

Definition and description of terms

Unless otherwise indicated, the definitions of groups and terms described in the specification and claims of the present invention, including definitions thereof as examples, exemplary definitions, preferred definitions, definitions described in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. The definitions of the groups and the structures of the compounds in such combinations and after the combination are within the scope of the present invention as defined in the specification.

In the invention

And (b) represents a connection site.

The term "pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of non-toxic acids or bases, including salts of inorganic acids and bases, organic acids and bases.

The term "stereoisomer" refers to isomers resulting from the different arrangement of atoms in a molecule, including cis-trans isomers, enantiomers, diastereomers, and conformers.

The compounds of the present invention may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, enantiomers, diastereomers, geometric isomers and individual isomers are included within the scope of the present invention.

The illustration of the racemic or enantiomerically pure compounds herein is from Maehr, J.chem.Ed.1985, 62: 114-120. Using wedge and virtual wedge keys, unless otherwise specified

Representing an absolute configuration of a solid center, with black solid and dotted bonds

Represents a cis-trans configuration of an alicyclic compound. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, they include the E, Z geometric isomer unless otherwise specified. Likewise, all tautomeric forms are included within the scope of the invention.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-) -and (+) -enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, as well as racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention. The compounds of the present application containing asymmetric atoms can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from a racemic mixture or synthesized by using chiral starting materials or chiral reagents.

The term "tautomer" refers to an isomer of a functional group resulting from the rapid movement of an atom in two positions in a molecule. The compounds of the invention may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer often result in a mixture whose physicochemical properties are consistent with the mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; whereas in phenol the enol type predominates. The present invention encompasses all tautomeric forms of the compounds.

The term "pharmaceutical composition" denotes a mixture of one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof with other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to an organism.

The term "substituted" means that any one or more hydrogen atoms on a particular atom is replaced with a substituent, so long as the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., ═ O), meaning that two hydrogen atoms are substituted, oxo does not occur on the aryl.

The terms "optionally" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, ethyl is "optionally" substituted with halo, meaning that ethyl may be unsubstituted (CH) ₂ CH ₃ ) Monosubstituted (e.g. CH) ₂ CH ₂ F) Polysubstituted (e.g. CHFCH) ₂ F、CH ₂ CHF ₂ Etc.) or completely substituted (CF) ₂ CF ₃ ). It will be appreciated by those skilled in the art that any group containing one or more substituents will not incorporate any substitution or substitution pattern which is not sterically impossible and/or cannot be synthesized.

When any variable (e.g., R) occurs more than one time in the composition or structure of a compound, its definition in each case is independent.

The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

The term "C ₁ -C ₁₀ Alkyl "is understood to mean a straight-chain or branched, saturated monovalent hydrocarbon radical having 1,2, 3,4, 5,6, 7, 8, 9 or 10 carbon atoms. The alkyl group is, for example, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an isopentyl group, a 2-methylbutyl group, a 1-ethylpropyl group, a 1, 2-dimethylpropyl group, a neopentyl group, a 1, 1-dimethylpropyl group, a 4-methylpentyl group, a 3-methylpentyl group, a 2-methylpentyl group, a 1-methylpentyl group, a 2-ethylbutyl group, a 1-ethylbutyl group, a 3, 3-dimethylbutyl group, a 2, 2-dimethylbutyl group, a 1, 1-dimethylbutyl group, a 2, 3-dimethylbutyl group, a 1, 3-dimethylbutyl group, or a 1, 2-dimethylbutyl group; "C ₁ -C ₆ Alkyl "is understood to mean a straight-chain or branched, saturated monovalent hydrocarbon radical having 1,2, 3,4, 5,6 carbon atoms. "C ₁ -C ₄ Alkyl "is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having 1,2, 3,4 carbon atoms. The term "alkoxy" is understood to mean "alkyloxy" or "alkyl-O", preferably "C ₁ -C ₁₀ Alkoxy groups "may contain" C ₁ -C ₆ Alkoxy "and" C ₁ -C ₄ Alkoxy ".

The term "C ₂ -C ₁₀ Alkenyl "is understood to preferably mean a straight-chain or branched, monovalent hydrocarbon radical which contains one or more double bonds and has 2,3, 4,5, 6, 7, 8, 9, 10 carbon atoms, preferably" C ₂ -C ₆ Alkenyl group ", more preferably" C ₂ -C ₄ Alkenyl ", even more preferably C ₂ Or C ₃ An alkenyl group. It is understood that in the case where the alkenyl group contains more than one double bond, the double bonds may be separated or conjugated to each other. Such as vinyl, allyl, (E) -2-methylvinyl, (Z) -2-methylvinyl, (E) -but-2-enyl, (Z) -but-2-enyl, (E) -but-1-alkenyl, (Z) -but-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, (E) -1-methylprop-1-enyl, (Z) -1-methylprop-1-enyl.

The term "C ₂ -C ₁₀ Alkynyl "is understood to mean a straight-chain or branched monovalent hydrocarbon radical comprising one or more triple bonds and having 2,3, 4,5, 6, 7, 8, 9, 10 carbon atoms, preferably" C ₂ -C ₆ Alkynyl ", more preferably" C ₂ -C ₄ Alkynyl ", even more preferably C ₂ Or C ₃ Alkynyl. Said alkynyl is for example ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3-ynyl, 1-methylprop-2-ynyl.

The term "C ₃ -C ₁₄ Cycloalkyl "is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 14 carbon atoms. The term "C ₃ -C ₁₀ Cycloalkyl "is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 10 carbon atoms. The term "C ₃ -C ₆ Cycloalkyl "is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3,4, 5,6 carbon atoms. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or bicyclic hydrocarbon groups such as decalin rings. According to the present invention, the bicyclic hydrocarbon ring includes a bridged ring, a spiro ring or a fused ring structure.

The term "cycloalkyloxy" is understood to mean "cycloalkyl-O", preferably "C ₃ -C ₁₄ Cycloalkyloxy "may contain" C ₃ -C ₁₀ Cycloalkyloxy "and" C ₃ -C ₆ Cycloalkyloxy ".

The term "3-14 membered heterocyclyl" is to be understood as a saturated or partially saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 14 ring atoms, comprising 1 to 5, preferably 1 to 3 heteroatoms selected from N, O and S. The term "3-10 membered heterocyclyl" means a saturated or partially saturated monovalent monocyclic or bicyclic hydrocarbon ring comprising 1-5, preferably 1-3 heteroatoms selected from N, O and S. The term "4-6 membered heterocyclyl" is to be understood as a saturated or partially saturated monovalent monocyclic or bicyclic hydrocarbon ring having 4,5, 6 ring atoms, comprising 1-5, preferably 1-3 heteroatoms selected from N, O and S. In particular, the heterocyclic group may include, but is not limited to: 4-membered rings such as azetidinyl, oxetanyl; 5-membered rings such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6-membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, or trithianyl; or a partially saturated 6-membered ring such as tetrahydropyridinyl; or a 7-membered ring such as diazepanyl. Optionally, the heterocyclic group may be benzo-fused. The heterocyclyl group may be bicyclic, for example but not limited to a 5,5 membered ring, such as a hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl ring, or a 5,6 membered bicyclic ring, such as a hexahydropyrrolo [1,2-a ] pyrazin-2 (1H) -yl ring. The nitrogen atom containing ring may be partially unsaturated, i.e., it may contain one or more double bonds, such as, but not limited to, 2, 5-dihydro-1H-pyrrolyl, 4H- [1,3,4] thiadiazinyl, 4, 5-dihydrooxazolyl, or 4H- [1,4] thiazinyl, or it may be benzo-fused, such as, but not limited to, dihydroisoquinolinyl. According to the invention, the heterocyclic radical is non-aromatic. The bicyclic hydrocarbon ring includes a bridged ring, a spiro ring or a fused ring structure.

The term "3-14 membered heterocyclyloxy" is understood to mean "3-14 membered heterocyclyl-O", preferably "3-14 membered heterocyclyloxy" may comprise "3-10 membered heterocyclyloxy".

The term "C ₆ -C ₁₀ Aryl "is to be understood as preferably meaning a mono-or bicyclic hydrocarbon ring of monovalent or partially aromatic character having 6, 7, 8, 9, 10 carbon atoms. In particular a ring having 6 carbon atoms ("C) ₆ Aryl "), such as phenyl; or a ring having 9 carbon atoms ("C) ₉ Aryl group), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C) ₁₀ Aryl), such as tetralinyl, dihydronaphthyl, or naphthyl.

The term "C ₆ -C ₁₀ Aryloxy "is to be understood as meaning" C ₆ -C ₁₀ aryl-O'.

"5-to 10-membered heteroaryl" is understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring systems: which has 5,6, 7, 8, 9, 10 ring atoms, in particular 5 or 6 or 9 or 10 ring atoms, and which contains 1 to 5, preferably 1 to 3 heteroatoms independently selected from N, O and S and, in addition, can be benzo-fused in each case. In particular, heteroaryl is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl and the like and their benzo derivatives, such as benzofuryl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl, and the like; or azocinyl, indolizinyl, purinyl and the like and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl and the like.

The term "5-10 membered heteroaryloxy" is understood to mean "5-10 membered heteroaryl-O".

The present application also includes isotopically-labeled compounds of the present application, which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine, such as respectively ² H、 ³ H、 ¹¹ C、 ¹³ C、 ¹⁴ C、 ¹³ N、 ¹⁵ N、 ¹⁵ O、 ¹⁷ O、 ¹⁸ O、 ³¹ P、 ³² P、 ³⁵ S、 ¹⁸ F、 ¹²³ I、 ¹²⁵ I and ³⁶ cl, and the like.

Certain isotopically-labeled compounds of the present application (e.g., with ³ H and ¹⁴ c-labeled ones) can be used in compound and/or substrate tissue distribution assays. Tritiated (i.e. by tritiation) ³ H) And carbon-14 (i.e. ¹⁴ C) Isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes, such as ¹⁵ O、 ¹³ N、 ¹¹ C and ¹⁸ f can be used in Positron Emission Tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the present application can generally be prepared by following procedures analogous to those disclosed in the schemes and/or in the examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

In addition, heavier isotopes are used (such as deuterium (i.e., deuterium) ² H) Substitution, where deuterium substitution may be partial or complete, partial deuterium substitution refers to substitution of at least one hydrogen with at least one deuterium, may provide some therapeutic advantage resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and thus may be preferred in some circumstances.

All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

The term "treating" means administering a compound or formulation described herein to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) preventing the occurrence of a disease or condition in a mammal, particularly when such mammal is predisposed to the disease condition but has not yet been diagnosed as having it;

(ii) inhibiting the disease or disease state, i.e., arresting its development;

(iii) alleviating the disease or condition, i.e., causing regression of the disease or condition.

The term "therapeutically effective amount" means an amount of a compound of the invention that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the compound of the present invention that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art, in view of their own knowledge and this disclosure.

The term "adjuvant" refers to a pharmaceutically acceptable inert ingredient. Examples of classes of the term "excipient" include, without limitation, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients enhance the handling characteristics of the pharmaceutical formulation, i.e., make the formulation more amenable to direct compression by increasing flowability and/or cohesiveness. Examples of typical "pharmaceutically acceptable carriers" suitable for use in the above formulations are: saccharides, starches, cellulose and its derivatives and the like are commonly used as excipients in pharmaceutical preparations.

The term "pharmaceutically acceptable adjuvants" refers to those adjuvants which do not have a significant irritating effect on the organism and do not impair the biological activity and properties of the active compound. Suitable adjuvants are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

The words "comprise", "comprises", "comprising" or "includes" and variations thereof such as "comprises" or "comprising" are to be understood in an open, non-exclusive sense, that is, to mean "including but not limited to".

The pharmaceutical compositions of the present application can be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable excipients, and can be formulated, for example, into solid, semisolid, liquid, or gaseous formulations such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, aerosols, and the like.

Typical routes of administration of a compound of the present application or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

The pharmaceutical compositions of the present application can be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

In some embodiments, the pharmaceutical composition is in an oral form. For oral administration, the pharmaceutical compositions may be formulated by mixing the active compounds with pharmaceutically acceptable excipients well known in the art. These adjuvants enable the compounds of the present application to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions and the like, for oral administration to a patient.

Solid oral compositions may be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: the active compounds are mixed with solid adjuvants, optionally the mixture obtained is milled, if desired with further suitable adjuvants, and the mixture is then processed to granules, to give tablets or dragee cores. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like.

The pharmaceutical compositions may also be adapted for parenteral administration, as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.

In all methods of administration of the compounds of general formula (Ib) described herein, the daily dose is from 0.01 to 100mg/kg body weight, preferably from 0.05 to 50mg/kg body weight, more preferably from 0.1 to 30mg/kg body weight, in single or divided doses. The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, examples of the present invention.

The chemical reactions of the embodiments of the present invention are carried out in a suitable solvent that is compatible with the chemical changes of the present invention and the reagents and materials required therefor. In order to obtain the compounds of the present invention, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes based on the existing embodiments.

Detailed Description

The following examples illustrate the technical solutions of the present invention in detail, but the scope of the present invention includes but is not limited thereto.

The solvent used in the present invention can be commercially available. Commercially available compounds are under the supplier catalogue name.

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) and/or Mass Spectrometry (MS). NMR shift in units of 10 ^-6 (ppm). Solvents for NMR determination are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol and the like, and an internal standard is Tetramethylsilane (TMS); ' IC ₅₀ "half inhibitory concentration" means the concentration at which half of the maximum inhibitory effect is achieved. The following eluents may be mixed eluents formed from two or more solvents in the percentage of each solvent by volume, such as "methanol/dichloromethane: 0-8% "indicates that methanol in the mixed eluent during gradient elution: the volume consumption of the dichloromethane is 0: 100-8: 100.

The invention employs the following abbreviations:

EXAMPLE 1 preparation of Compound 001

(R) -1- (3- ((5-cyclopropyl-2- ((1-ethyl-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

the first step is as follows: synthesis of 2, 4-dichloro-5-iodo-7H-pyrrolo [2,3-d ] pyrimidine (1b)

2, 4-dichloro-7H-pyrrolo [2,3-d ] pyrimidine 1a (10g,53.2mmol), N-iodosuccinimide (14.4g,79.8mmol) and 2,4, 6-trimethylaniline (718mg,0.53mmol) were dissolved in dichloromethane (100mL) at room temperature, and the reaction was stirred at 110 ℃ for 16 hours until completion. The mixture was poured into water and extracted with dichloromethane, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column (EA: PE ═ 0 to 20%) to give the title product 1b (13.0 g).

LCMS:Rt:1.708min；MS m/z(ESI):313.8[M+H] ⁺ 。

The second step: synthesis of 2, 4-dichloro-5-iodo-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine (1c)

2, 4-dichloro-5-iodo-7H-pyrrolo [2,3-d ] pyrimidine 1b (7g,22.4mmol) was dissolved in tetrahydrofuran (70mL) at room temperature, sodium hydride (3.58g,89.6mmol, 60%) and 2- (trimethylsilyl) ethoxymethyl chloride (5.6g,33.6mmol) were added sequentially at 0 deg.C, and the reaction was stirred at 0 deg.C for 0.5 hours until completion. The reaction was quenched by slowly adding 50mL of water to the reaction solution, the mixture was poured into water and extracted with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column (EA: PE ═ 0 to 20%) to obtain the title product 1c (3.5 g).

LCMS:Rt:1.230min；MS m/z(ESI):444.0[M+H] ⁺ 。

The third step: synthesis of 2, 4-dichloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine (1e)

2, 4-dichloro-5-iodo-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine 1c (1.1g,2.5mmol), cyclopropylboronic acid 1d (341mg,2.8mmol), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (176mg,0.25mmol), silver oxide (286mg,1.25mmol) and potassium phosphate (1.6g,7.5mmol) were added in succession to a solution of dioxane (12mL) at room temperature and then stirred at 90 ℃ under nitrogen for 2 hours to completion of the reaction. The mixture was filtered, the liquid phase was poured into water and extracted with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column (EA: PE ═ 0 to 20%) to give the title product 1e (500 mg).

LCMS:Rt:1.983min；MS m/z(ESI):358.0[M+H] ⁺ 。

The fourth step: synthesis of tert-butyl (R) -3- ((2-chloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7 h-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidine-1-carboxylate (1g)

2, 4-dichloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine 1e (480mg,1.3mmol), (R) -3-aminopiperidine-1-carboxylic acid tert-butyl ester 1f (528mg,2.6mmol) and N, N-diisopropylethylamine (521mg,3.9mmol) were dissolved in isopropanol (5mL) at room temperature, and the reaction solution was stirred in a stoppered tube at 110 ℃ for 16H until the reaction was complete. The reaction mixture was poured into water, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column (EA: PE 0-20%) to obtain the title product 1g (580 mg).

LCMS:Rt:2.251min；MS m/z(ESI):522.2[M+H] ⁺ 。

The fifth step: synthesis of tert-butyl (R) -3- ((2- ((1-ethyl-1H-pyrazol-4-yl) amino) -5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidine-1-carboxylate (1i)

Reacting tert-butyl (R) -3- ((2-chloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d) at room temperature]Pyrimidin-4-yl) amino) piperidine-1-carboxylic acid ester 1g (560mg,1.1mmol), 1-ethyl-1H-pyrazol-4-amine 1H (238mg,2.2mmol) and trisFluoroacetic acid (123mg,1.1mmol) was dissolved in isopropanol (6mL), and the reaction solution was stirred at 110 ℃ for 16 hours in a sealed tube until the reaction was complete. NaHCO for reaction liquid ₃ The saturated solution was adjusted to pH 9 with base, poured into water and extracted with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column (EA: PE ═ 0 to 10%) to give the title product 1i (100 mg).

LCMS:Rt:1.440min；MS m/z(ESI):597.3[M+H] ⁺ 。

And a sixth step: (R) -5-cyclopropyl-N ² - (1-ethyl-1H-pyrazol-4-yl) -N ⁴ - (piperidin-3-yl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d]Synthesis of pyrimidine-2, 4-diamine (1j)

Reacting tert-butyl (R) -3- ((2- ((1-ethyl-1H-pyrazol-4-yl) amino) -5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] at room temperature]Pyrimidin-4-yl) amino) piperidine-1-carboxylic acid ester 1i (100mg,0.2mmol) was dissolved in a solution of dichloromethane (1mL) and dioxane hydrochloride (1mL,4M), and the reaction was stirred at room temperature for 2 hours until completion. NaHCO for reaction liquid ₃ The saturated solution was adjusted to pH 9, poured into water and extracted with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate filtered, and the filtrate was concentrated under reduced pressure to give the title product 1j (80mg, crude).

LCMS:Rt:1.201min；MS m/z(ESI):497.3[M+H] ⁺ 。

The seventh step: synthesis of (R) -1- (3- ((5-cyclopropyl-2- ((1-ethyl-1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one (1l)

(R) -5-cyclopropyl-N2- (1-ethyl-1H-pyrazol-4-yl) -N4- (piperidin-3-yl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine-2, 4-diamine 1j (80mg,0.2mmol) was dissolved in dichloromethane (2mL) at room temperature and triethylamine (64mg,0.6mmol) and acryloyl chloride 1k (38mg,0.4mmol) were added at-10 ℃. The reaction solution was allowed to continue to react at-10 ℃ for 30 minutes until the reaction was complete. The reaction was diluted with clear water (20mL), extracted with dichloromethane (20mL x2) and the combined organic phases were spin dried to give the title product 1l (100 mg).

LCMS:Rt:1.796min；MS m/z(ESI):551.3[M+H] ⁺ 。

Eighth step: synthesis of (R) -1- (3- ((5-cyclopropyl-2- ((1-ethyl-1H-pyrazol-4-yl) amino) -7- (hydroxymethyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one (1m)

(R) -1- (3- ((5-cyclopropyl-2- ((1-ethyl-1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d) at room temperature]Pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one 1l (100mg,0.2mmol) and trifluoroacetic acid (103mg,0.9mmol) were dissolved in a dichloromethane (5mL) solution, and the reaction solution was stirred at room temperature for 0.5 hour until the reaction was complete. NaHCO for reaction liquid ₃ The saturated solution was adjusted to pH 9, poured into water and extracted with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to give the title product 1m (80mg, crude).

LCMS:Rt:1.003min；MS m/z(ESI):451.2[M+H] ⁺ 。

The ninth step: synthesis of (R) -1- (3- ((5-cyclopropyl-2- ((1-ethyl-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one (001)

(R) -1- (3- ((5-cyclopropyl-2- ((1-ethyl-1H-pyrazol-4-yl) amino) -7- (hydroxymethyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one 1m (80mg,0.2mmol) was dissolved in tetrahydrofuran (3mL) at room temperature, a sodium hydroxide (2mL) solution was added, and the reaction solution was stirred at room temperature for 1 hour until the reaction was complete. The reaction was diluted with water (20mL), extracted with ethyl acetate (30mL _ 2), and the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, and the crude product obtained was filtered off. Purification by high performance liquid preparative chromatography gave the title product 001(15.82 mg).

High performance liquid preparative chromatography, Watts (waters)2767 preparative chromatograph, mobile phase A (Mobile phase A) 10 mmol% FA in water, mobile phase B (Mobile phase B) acetonitrile, chromatographic column Sunfire (Prep C18 OBD 19 250mm 10 μm), Gradient (Gradient) 10% ACN running for 4min, 45% -55% ACN running for 14min, 95% ACN running for 14min, 10% ACN running for 17min to finish purification, and obtaining the target compound.

LCMS:Rt:0.933min；MS m/z(ESI):421.2[M+H] ⁺ 。

¹ H NMR(400MHz,DMSO-d ₆ )δ10.59(d,J＝8.3Hz,1H),8.43(s,1H),7.85(d,J＝13.2Hz,1H),7.43(d,J＝22.1Hz,1H),6.89(d,J＝5.7Hz,1H),6.45(s,1H),6.07(dd,J＝38.5,16.3Hz,1H),5.70(ddd,J＝91.0,51.4,9.1Hz,2H),4.30(s,1H),4.03(q,J＝7.2Hz,3H),3.80(d,J＝10.9Hz,1H),3.68(d,J＝14.3Hz,2H),1.97(s,1H),1.85(s,2H),1.73(s,1H),1.57(s,1H),1.39–1.28(m,3H),0.76(d,J＝9.4Hz,2H),0.59–0.41(m,2H).

EXAMPLE 2 preparation of Compound 002

(R) -1- (3- ((2- ((1-ethyl-1H-pyrazol-4-yl) amino) -5-phenyl-7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

the first step is as follows: synthesis of 2, 4-dichloro-5-phenyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine (2c)

2, 4-dichloro-5-iodo-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine 2a (1.7g,3.8mmol), phenylboronic acid 2b (466mg,3.8mmol), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (112mg,0.15mmol) and sodium carbonate (1.2g,11.4mmol) were added sequentially to a solution of dioxane (20mL) and water (4mL) at room temperature and then stirred at 85 ℃ under nitrogen for 2 hours to completion. The mixture was filtered, the liquid phase was poured into water and extracted with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column (EA: PE ═ 0 to 20%) to give the title product 2c (900 mg).

LCMS:Rt:1.994min；MS m/z(ESI):394.0[M+H] ⁺ 。

The second step is that: synthesis of tert-butyl (R) -3- ((2-chloro-5-phenyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7 h-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidine-1-carboxylic acid ester (2e)

2, 4-dichloro-5-phenyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine 2c (900mg,2.3mmol), (R) -3-aminopiperidine-1-carboxylic acid tert-butyl ester 2d (930mg,4.6mmol) and N, N-diisopropylethylamine (900mg,6.9mmol) were dissolved in isopropanol (10mL) at room temperature and the reaction solution was stirred at 110 ℃ for 16H until the reaction was complete. The reaction mixture was poured into water, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column (EA: PE 0-20%) to obtain the title product 2e (230 mg).

LCMS:Rt:2.467min；MS m/z(ESI):558.2[M+H] ⁺ 。

The third step: synthesis of tert-butyl (R) -3- ((2- ((1-ethyl-1H-pyrazol-4-yl) amino) -5-phenyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidine-1-carboxylate (2g)

Reacting tert-butyl (R) -3- ((2-chloro-5-phenyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d) at room temperature]Pyrimidin-4-yl) amino) piperidine-1-carboxylic acid ester 2e (230mg,0.4mmol), 1-ethyl-1H-pyrazol-4-amine 2f (89mg,0.8mmol) and trifluoroacetic acid (46mg,0.4mmol) were dissolved in isopropanol (3mL), and the reaction solution was stirred at 110 ℃ for 16 hours until the reaction was complete. NaHCO for reaction liquid ₃ The saturated solution was adjusted to pH 9 with base, poured into water and extracted with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column (EA: PE ═ 0 to 10%) to obtain 2g (130mg) of the title product.

LCMS:Rt:1.749min；MS m/z(ESI):633.3[M+H] ⁺ 。

The fourth step: synthesis of (R) - (2- ((1-ethyl-1H-pyrazol-4-yl) amino) -5-phenyl-4- (piperidin-3-ylamino) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methanol (2H)

Reacting tert-butyl (R) -3- ((2- ((1-ethyl-1H-pyrazol-4-yl) amino) -5-phenyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] at room temperature]Pyrimidin-4-yl) amino) piperidine-1-carboxylate 2g (130mg,0.2mmol) was dissolved in dichloromethane (1mL) and dioxane hydrochloride (1mL) and the reaction was stirred at room temperature for 16h to completion. NaHCO for reaction liquid ₃ The saturated solution was alkalified to pH 9, poured into water and extracted with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate filtered and the filtrate was concentrated under reduced pressure to give the title product 2h (100mg, crude).

LCMS:Rt:0.682min；MS m/z(ESI):433.3[M+H] ⁺ 。

The fifth step: synthesis of (R) -1- (3- ((2- ((1-ethyl-1H-pyrazol-4-yl) amino) -7- (hydroxymethyl) -5-phenyl-7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one (2j)

(R) - (2- ((1-Ethyl-1H-pyrazol-4-yl) amino) -5-phenyl-4- (piperidin-3-ylamino) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methanol 2H (90mg,0.2mmol) was dissolved in dichloromethane (2mL) at room temperature, and triethylamine (64mg,0.6mmol) and acryloyl chloride 2i (38mg,0.4mmol) were added at-10 ℃. The reaction solution was allowed to continue to react at-10 ℃ for 30 minutes until the reaction was complete. The reaction was diluted with clear water (20mL), extracted with dichloromethane (20mL x2) and the combined organic phases were spin dried to give the title product 2j (90 mg).

LCMS:Rt:1.253min；MS m/z(ESI):487.2[M+H] ⁺ 。

And a sixth step: synthesis of (R) -1- (3- ((2- ((1-ethyl-1H-pyrazol-4-yl) amino) -5-phenyl-7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one (002)

(R) -1- (3- ((2- ((1-ethyl-1H-pyrazol-4-yl) amino) -7- (hydroxymethyl) -5-phenyl-7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one 2j (90mg,0.2mmol) was dissolved in tetrahydrofuran (3mL) at room temperature, a NaOH (2mL) solution was added, and the reaction solution was stirred at room temperature for 30 minutes until the reaction was complete. The reaction was diluted with clear water (20mL), extracted with ethyl acetate (30mL x2), and the combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, and filtered to dry the resulting crude product. Purification by high performance liquid preparative chromatography gave the title compound 002(29.31 mg).

High performance liquid preparative chromatography, Waters 2767 preparative chromatograph, mobile phase A (mobile phase A) 10 mmol% FA in water, mobile phase B (mobile phase B) acetonitrile, chromatographic column Sunfire (Prep C18 OBD 19 250mm 10 μm), Gradient (Gradient) 10% ACN for 3min, 40% -45% ACN for 13min, 95% ACN for 15min, 10% ACN for 19min to finish purification, and obtaining the target compound.

LCMS:Rt:1.043min；MS m/z(ESI):457.2[M+H] ⁺ 。

1H NMR(400MHz,DMSO)δ11.19(s,1H),8.57(s,1H),7.88(d,J＝9.7Hz,1H),7.44(dd,J＝20.4,13.3Hz,5H),7.30(dd,J＝9.5,4.4Hz,1H),6.92–6.40(m,2H),6.03(dd,J＝38.0,16.7Hz,1H),5.58(dd,J＝99.3,10.1Hz,1H),5.06(d,J＝7.7Hz,1H),4.26(s,1H),4.05(q,J＝6.9Hz,2H),3.69(dd,J＝35.0,14.2Hz,2H),3.52(s,2H),1.92(s,1H),1.49(s,2H),1.34(d,J＝6.9Hz,4H).

EXAMPLE 3 preparation of Compound 003

1-acryloyl- (3S,4R) -3- ((5-cyclopropyl-2- ((1-ethyl-1H-pyrazol-4-yl) amino) -7H pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoropiperidine

Synthetic route and specific synthetic procedure:

the preparation was identical to example 2, except that cyclopropylboronic acid (3b) was used instead of phenylboronic acid (2b) in example 2 and tert-butyl (3S,4R) -3-amino-4-fluoropiperidine-1-carboxylate (3d) was used instead of tert-butyl (R) -3-aminopiperidine-1-carboxylate (2d) in example 2, and purification was carried out by high performance liquid preparative chromatography to give the title compound 003(8.84 mg).

High performance liquid preparative chromatography, Waters 2767 preparative chromatograph, mobile phase A (Mobile phase A) 10 mmol% FA in water, mobile phase B (Mobile phase B) acetonitrile, chromatographic column Sunfire (Prep C18 OBD 19 250mm 10 μm), Gradient (Gradient) 10% ACN running for 4min, 45% -55% ACN running for 14min, 95% ACN running for 14min, 10% ACN running for 17min to finish purification, and obtaining the target compound.

LCMS:Rt:0.869min；MS m/z(ESI):439.1[M+H] ⁺ 。

¹ H NMR(400MHz,DMSO)δ10.68(s,1H),8.48(s,1H),7.94-7.78(m,1H),7.48-7.34(m,1H),6.95-6.84(m,0.5H),6.72-6.61(m,0.5H),6.51(s,1H),6.21-5.85(m,2H),5.78-5.52(m,1H),5.25-5.00(m,1H),4.58-4.42(m,1H),4.06-3.88(m,3H),3.64-3.54(m,1H),3.42-3.28(s,1.5H),3.14-3.06(m,0.5H),2.15-1.75(m,3H),1.32(d,J＝7.6Hz,3H),0.80(d,J＝7.2Hz,2H),0.64-0.48(m,2H).

EXAMPLE 4 preparation of Compound 004

(R) -1- (3- ((5-cyclopropyl-2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one

Synthetic route and specific synthetic procedure:

the first step is as follows: synthesis of 4-nitro-1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazole (4i)

4g (500mg, 4.4mmol) of 4-nitro-1H-pyrazole, 4H (451mg, 4.4mmol) of tetrahydro-2H-pyran-4-ol and 1.4g (5.3 mmol) of triphenylphosphine were dissolved in anhydrous tetrahydrofuran (20mL) at room temperature, and di-tert-butyl azodicarboxylate (1.32g, 5.7mmol) was added. The system was stirred at room temperature under nitrogen for 16 hours. Water (30mL) was added to quench and extracted with ethyl acetate (30mL x 3). The organic layer was collected, dried, filtered and spin dried. The residue was purified by thin layer column chromatography (ethyl acetate/petroleum ether: 10-50%) to give the title product 4i (600 mg).

LCMS:Rt:1.26min；MS m/z(ESI):198.1[M+H] ⁺ 。

The second step is that: synthesis of 1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-amine (4b)

4-Nitro-1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazole 4i (600mg, 3.2mmol) was dissolved in methanol (10mL) at room temperature, and wet palladium on carbon (30mg) was added. The mixture was reacted at room temperature for 3 hours under a hydrogen atmosphere. It was then filtered and the filtrate was spin-dried to give crude 4b (480 mg).

LCMS:Rt:0.376min；MS m/z(ESI):168.4[M+H] ⁺ 。

The third step: synthesis of tert-butyl (R) -3- ((5-cyclopropyl-2- (((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidine-1-carboxylate (4c)

(R) -tert-butyl 3- ((2-chloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) aminopiperidine-1-carboxylate 4a (i.e. 1g in example 1 above) (180mg, 0.34mmol), 1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-amine 4b (170mg, 1.02mmol), 1,1 '-binaphthyl-2, 2' -diphenylphosphine (21mg,0.034mmol), cesium carbonate (326mg,1mmol), tris (dibenzylideneacetone) dipalladium (31mg,0.034mmol) and 1, 4-dioxane (10mL) were added to a reaction flask at room temperature, the mixture was reacted under nitrogen at 120 ℃ for 16 h. After the completion of the reaction, the solvent was spin-dried, and the residue was purified by thin layer column chromatography (methanol/dichloromethane: 0-8%) to obtain the objective product 4c (260 mg).

LCMS:Rt:1.306min；MS m/z(ESI):653.1[M+H] ⁺ 。

The fourth step: synthesis of (R) - (5-cyclopropyl-4- (piperidin-3-ylamino) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methanol (4d)

(3R) -tert-butyl 3- ((5-cyclopropyl-2- ((1- (tetrahydrofuran-3-yl) -1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidine-1-carboxylate 4c (200mg, 0.31mmol) was dissolved in dichloromethane (3mL) at room temperature, 4M hydrochloric acid dioxane (6mL) was added and stirred for 1 hour, followed by spin-drying, the residue was dissolved in dichloromethane (3mL) at room temperature, trifluoroacetic acid (3mL) was added and stirred for 1 hour. The mixture was spin dried to give crude 4d (140 mg).

LCMS:Rt:0.875min；MS m/z(ESI):453.1[M+H] ⁺ 。

The fifth step: synthesis of (R) -1- (3- ((5-cyclopropyl-7- (hydroxymethyl) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one (4f)

(R) - (5-cyclopropyl-4- (piperidin-3-ylamino) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methanol 4d (140mg, 0.31mmol) and triethylamine (94mg, 0.93mmol) were dissolved in dichloromethane (10mL) at room temperature, the mixture was cooled to 0 ℃ and propenyl acid chloride 4e (35mg,0.6031mmol) dissolved in dichloromethane (1mL) was added and stirred for 2 hours. After the reaction was quenched with water, the solvent was spin-dried and the residue was extracted with dichloromethane. The organic layer was collected, dried and spin dried to give crude 4f (270 mg).

LCMS:Rt:1.127min；MS m/z(ESI):507.1[M+H] ⁺ 。

And a sixth step: synthesis of (R) -1- (3- ((5-cyclopropyl-2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one (004)

(R) -1- (3- ((5-cyclopropyl-7- (hydroxymethyl) -2- ((1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one 4f (157mg, 0.31mmol) was dissolved in tetrahydrofuran (2mL) with 2M sodium hydroxide (2mL) added. The mixture was stirred for 2 hours, tetrahydrofuran was spin-dried, and then extracted with dichloromethane. The organic layer was spun dry, dissolved in acetonitrile and purified by high performance liquid preparative chromatography to give the title compound 004(58.0 mg).

High performance liquid preparative chromatography, Waters 2767 preparative chromatograph, mobile phase A (mobile phase A) 0.1% FA in water, mobile phase B (mobile phase B) acetonitrile, chromatographic column Sunfire (Prep C18 OBD 19 250mm 10 μm), Gradient (Gradient) 10% ACN running for 3min, 30% -35% ACN running for 14min, 95% ACN running for 17min, 10% ACN running for 21min to finish purification, and obtaining the target compound.

LCMS:Rt:0.830min；MS m/z(ESI):477.2[M+H] ⁺ 。

¹ H NMR(400MHz,DMSO-d ₆ )δ10.60(br s,1H),8.44(s,1H),7.92(br s,1H),7.46(d,J＝16.0Hz,1H),6.95-6.54(m,1H),6.45(s,1H),6.20-5.94(m,1H),5.86-5.74(m,1H),5.74-5.45(m,1H),4.36-4.19(m,2H),3.96,3.93(two s,3H),3.85–3.51(m,5H),2.01–1.80(m,7H),1.75-1.47(m,2H),0.88-0.67(m,2H),0.58-0.36(m,2H).

EXAMPLE 5 preparation of Compound 005

1- ((3R) -3- ((5-cyclopropyl-2- ((1- (tetrahydrofuran-3-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one

Synthetic route and specific synthetic procedure:

the preparation was carried out as in example 4, except that tetrahydrofuran-3-ol (5H) was used instead of tetrahydro-2H-pyran-4-ol (4H), and purification by high performance liquid preparative chromatography gave the title compound 005(57.98 mg).

LCMS:Rt:1.09min；MS m/z(ESI):463.1[M+H] ⁺ 。

1H NMR(400MHz,DMSO-d6)δ10.62(d,J＝7.2Hz,1H),8.48(s,1H),7.92(d,J＝8.8Hz,1H),7.47(d,J＝15.2Hz,1H),6.92-6.58(m,1H),6.46(s,1H),6.13-5.99(m,1H),5.84-5.49(m,2H),4.94-4.88(m,1H),4.32(s,1H),3.96–3.51(m,8H),2.34–2.18(m,2H),1.96–1.59(m,5H),0.76(d,J＝8.8Hz,2H),0.52–0.48(m,2H).

EXAMPLE 6 preparation of Compound 006

(R) -1-acryloyl-3- ((5-cyclopropyl-2- ((1-oxetan-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidine

The synthesis route and the specific synthesis steps are as follows:

the preparation was carried out according to the same manner as in example 4 except that 3-oxetanyl alcohol (6b) was used instead of tetrahydro-2H-pyran-4-ol (4H) in example 4, and purified by high performance liquid preparative chromatography to give the title compound 006(24.48 mg).

High performance liquid preparative chromatography, Watts (waters)2767 preparative chromatograph, mobile phase A (Mobile phase A) 10 mmol% TFA in water, mobile phase B (Mobile phase B) acetonitrile, chromatographic column Sunfire (Prep C18 OBD 19 250mm 10 μm), Gradient (Gradient) 10% ACN running for 4min, 40% -45% ACN running for 14min, 95% ACN running for 14min, 10% ACN running for 17min to finish purification, and obtaining the target compound.

LCMS:Rt:0.809min；MS m/z(ESI):449.1[M+H] ⁺ 。

¹ H NMR(400MHz,DMSO)δ11.52-11.16(m,1H),9.89-9.51(m,1H),8.09-8.04(m,1H),7.72-7.65(m,1H),6.97-6.89(m,1H),6.79-6.51(m,2H),6.20-5.70(m,2H),5.54-5.51(m,1H),4.99-4.83(m,3H),4.39-4.01(m,3H),4.04-3.40(m,4H),2.10-1.88(m,2H),1.88-1.45(m,2H),1.00-0.79(m,2H),0.62-0.43(m,2H).

EXAMPLE 7 preparation of Compound 007

(R) -1- (3- ((5-cyclopropyl-2- ((1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

the preparation was identical to example 4, except that 1-methylpiperidin-4-ol (7H) was substituted for tetrahydro-2H-pyran-4-ol (4H) in example 4, and purified by high performance liquid preparative chromatography to give the title compound 007(13.03 mg).

High performance liquid preparative chromatography, Waters 2767 preparative chromatograph, mobile phase A (mobile phase A) 0.1% FA in water, mobile phase B (mobile phase B) acetonitrile, chromatographic column Sunfire (Prep C18 OBD 19 250mm 10 μm), Gradient (Gradient) 10% ACN running for 3min, 35% -40% ACN running for 15min, 95% ACN running for 19min, 10% ACN running for 22min to finish purification, and obtaining the target compound.

LCMS:Rt:0.728min；MS m/z(ESI):490.2[M+H] ⁺ 。

¹ H NMR(400MHz,DMSO-d6)δ10.61(s,1H),8.43(s,1H),7.91(s,1H),7.45(d,J＝14.2Hz,1H),6.88(s,1H),6.45(s,1H),6.06(m,1H),5.84–5.35(m,2H),4.43–3.34(m,8H),2.87(d,J＝10.8Hz,2H),2.29–1.41(m,12H),0.76(d,J＝7.8Hz,2H),0.58–0.37(m,2H).

EXAMPLE 8 preparation of Compound 008

1- ((3S,4R) -3- ((5-cyclopropyl-2- ((1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoropiperidin-1-yl) prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

the first step is as follows: synthesis of 1-methyl-4- (4-nitro-1H-pyrazol-1-yl) piperidine (8c)

Triphenylphosphine (34.8g,132.8mmol) was dissolved in anhydrous tetrahydrofuran (300mL) and cooled to 0 deg.C, followed by slow injection of DIAD (26.8g,132.8mmol) and stirring at 0 deg.C for 10min, at which time a large amount of white precipitate formed.

Compound 4-nitro-1H-pyrazole 8a (10.0g,88.5mmol) and compound 1-methylpiperidin-4-ol 8b (11.2g,97.4mmol) were both dissolved in anhydrous tetrahydrofuran (100mL) and then slowly added dropwise to the above solution. After the addition was complete, the ice bath was removed and the reaction solution was stirred at room temperature overnight.

And (3) post-treatment: the reaction solution was concentrated under reduced pressure, and the resulting crude product was dissolved in ethyl acetate (150mL), followed by extraction with 3mol/L hydrochloric acid solution (100 mL). The collected aqueous phase was adjusted to pH 9-10 with saturated potassium carbonate solution and then extracted with ethyl acetate (200mLx 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give the title product 8c (16.5 g).

LCMS:Rt:0.405min；MS m/z(ESI):211.1[M+H] ⁺ 。

The second step is that: synthesis of 1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-amine (8d)

The compound 1-methyl-4- (4-nitro-1H-pyrazol-1-yl) piperidine 8C (16.5g, 78.6mmol) was dissolved in ethanol (200mL) at room temperature, 10% Pd/C (3.3g) was then added, and the reaction solution was stirred under a hydrogen atmosphere overnight. After completion of the reaction, the reaction solution was filtered, and the filtrate was spin-dried to obtain the title product 8d (12.8g)

LCMS:Rt:1.47min；MS m/z(ESI):181.1[M+H] ⁺ 。

The third step: synthesis of tert-butyl (3S,4R) -3- ((2-chloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoropiperidine-1-carboxylate (8g)

The compound 2, 4-dichloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] is reacted at room temperature]Pyrimidine 8e (357.0mg,1.0mmol), the compounds tert-butyl (3S,4R) -3-amino-4-fluoropiperidine-1-carboxylate 8f (545.0mg,2.5mmol) and DIPEA (387.0mg) were dissolved in ethanol (8mL), and the mixture was added to a closed pot and stirred at 120 ℃ overnight. After the reaction is finished, the reaction solution is concentrated and dried. The resulting crude product was purified by normal phase column separation to give 8g (256mg) of the title product. LCMS: Rt:1.941 min; MS M/z (ESI) 540.2[ M + H] ⁺ 。

The fourth step: synthesis of tert-butyl (3S,4R) -3- ((5-cyclopropyl-2- ((1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoropiperidine-1-carboxylate (8H)

Intermediate 1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-amine 8d (254mg, 1.41mmol), intermediate tert-butyl (3S,4R) -3- ((2-chloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) amino) -4-fluoropiperidine-1-carboxylate 8g (256mg, 0.47mmol), Pd ₂ (dba) ₃ (0.1eq), BINAP (0.1eq) and Cs ₂ CO ₃ (3eq) to dioxane (8mL)Under the protection of nitrogen, the mixture is heated to 110 ℃ and stirred overnight. After the reaction was completed, the reaction solution was cooled to room temperature and filtered. The filtrate was directly purified by reverse phase column (Prep-HPLC (Boston Prime C18150 x 30mm x 5 μm; A%: water (containing 0.225% FA); B%: ACN 10% -40%, 8-20min) to give the title product 8h (153.0 mg).

LCMS:Rt:1.279min；MS m/z(ESI):684.4[M+H] ⁺ 。

The fifth step: synthesis of (5-cyclopropyl-4- (((3S,4R) -4-fluoropiperidin-3-yl) amino) -2- ((1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methanol (8i)

The intermediate tert-butyl (3S,4R) -3- ((5-cyclopropyl-2- ((1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoropiperidine-1-carboxylate 8H (153mg,0.22mmol) was dissolved in TFA (3mL) and then stirred at room temperature for 1H. After the completion of the reaction, it was spin-dried to obtain the title product 8i (185mg, purity: 66.8%) which was directly charged into the next reaction.

LCMS:Rt:1.171min；MS m/z(ESI):484.1[M+H] ⁺ 。

And a sixth step: 5-cyclopropyl-N ⁴ - ((3S,4R) -4-Fluoropiperidin-3-yl) -N ² - (1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl) -7H-pyrrolo [2,3-d]Synthesis of pyrimidine-2, 4-diamine (8j)

Mixing (5-cyclopropyl-4- (((3S,4R) -4-fluoropiperidin-3-yl) amino) -2- ((1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2, 3-d)]Pyrimidin-7-yl) methanol 8i (185mg) dissolved in acetone (3mL) and H ₂ To the mixture of O (3mL), potassium carbonate solid was added to adjust the pH to 9-10, and then the reaction was heated to 40 ℃ and stirred overnight. LCMS detected complete conversion of intermediate 8i to the title product 8j, which was carried forward directly to the next reaction.

LCMS:Rt:1.45min；MS m/z(ESI):454.1[M+H] ⁺ 。

The seventh step: synthesis of 1- ((3S,4R) -3- ((5-cyclopropyl-2- ((1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoropiperidin-1-yl) prop-2-en-1-one (008)

The reaction solution from the previous step was cooled to 0 ℃, potassium carbonate solid was further added to adjust the PH to 9-10, and then acryloyl chloride 8k (91mg,0.66mmol) was dissolved in acetone (1mL) and added dropwise to the reaction solution. After the addition was complete, the reaction was stirred at 0 ℃ for 1h and the disappearance of starting material was detected by LCMS. After the reaction was completed, the reaction mixture was filtered and directly sent to preparative chromatography (Prep-HPLC (Boston Prime C18150 x 30mm x 5 μm; A%: water (containing 0.225% of FA); B%: ACN 13% -43%, 8-15min) for purification to obtain the objective 008(9.58 mg).

LCMS:Rt:5.287min；MS m/z(ESI):508.7[M+H] ⁺ 。

¹ H NMR(400MHz,DMSO-d6)δ10.66(s,1H),8.50(s,1H),7.91(s,1H),7.43(d,J＝6.0Hz,1H),6.95-6.76(m,1H),6.47(s,1H),6.16-6.10(m,1H),5.88-5.61(m,2H),5.13-4.94(m,1H),4.89(s,1H),3.99-3.46(m,5H), 2.84(d,J＝10.8Hz,2H),2.19(s,3H),2.04-1.72(m,9H),0.87-0.69(m,2H),0.60-0.51(m,1H),0.46-0.40(m,1H).

Preparation of compound 009 of example 9

1- ((2S,5R) -5- ((5-cyclopropyl-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpiperidin-1-yl) prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

the first step is as follows: synthesis of benzyl (2S,5R) -5- ((2-chloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpiperidine-1-carboxylate (9c)

2, 4-dichloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine (9a,500mg,1.39mmol), (benzyl 2S,5R) -5-amino-2-methylpiperidine-1-carboxylate (9b,346mg,1.39mmol), and DIPEA (448mg,3.48mmol), respectively, were added to a solution of isopropanol (10.0mL) at room temperature, the reaction was heated to 110 ℃ and stirred at this temperature for 16 hours. The reaction solution was concentrated under reduced pressure to give a crude product, which was then purified by column chromatography (ethyl acetate/petroleum ether: 1/10) to give 9c (385mg, yield: 48.6%).

LCMS:Rt:2.157min；MS m/z(ESI):570.2[M+H]；

¹ H NMR(400M Hz,DMSO-d ₆ )δ7.45-7.39(m,4H),7.35-7.32(m,1H),6.77(s,1H),5.72(d,J＝8.0Hz,1H),5.46(s,2H),5.20(dd,J＝17.6,12.4Hz,2H),4.60-4.55(m,2H),4.26-4.20(m,1H),3.54(t,J＝8.0Hz,2H),2.73(t,J＝11.6Hz,1H),2.21(s,1H),2.12-2.08(m,1H),1.93-1.88(m,1H),1.70-1.65(m,2H),1.31-1.28(m,1H),1.25(d,J＝6.8Hz,3H),0.93(t,J＝8.0Hz,3H),0.72-0.71(m,2H),0.03(s,9H).

The second step is that: synthesis of benzyl (2S,5R) -5- ((5-cyclopropyl-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpiperidine-1-carboxylate (9e)

Pd is added under the protection of nitrogen at room temperature ₂ (dba) ₃ (32.0mg,0.035mmol) and BINAP (21.8mg,0.035mmol) were added to 9c (200mg, 0.351mmol), 1-methyl-1H-pyrazol-4-amino (9d,40.8mg, 0.421mmol) and Cs ₂ CO ₃ (285.9mg,0.878mmol) in 1, 4-dioxane (10.0mL), the reaction was warmed to 100 ℃ and stirred for 16 hours, and cooled to room temperature. Filtration and cake washing with ethyl acetate (30.0mL), the solution was concentrated under reduced pressure and purified by Chem-flash (acetonitrile/0.1% trifluoroacetic acid) to give 9e (195mg, yield: 88.0%).

LCMS:Rt:2.148min；MS m/z(ESI):631.3[M+H]。

The third step: 5-cyclopropyl-N ² - (1-methyl-1H-pyrazol-4-yl) -N ⁴ - ((3R,6S) -6-methylpiperidin-3-yl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Synthesis of pyrimidine-2, 4-diamine (9f)

Pd/C (5mg) was added to 9e (195mg,0.309mmol) in methanol (5.0mL) at room temperature under a nitrogen blanket, and the reaction was stirred under hydrogen at room temperature for 16 hours. The reaction mixture was filtered under reduced pressure, the filter cake was washed with methanol (20.0mL), the filtrates were combined, evaporated to dryness under reduced pressure, and the residue was purified by Chem-flash (acetonitrile/0.1% trifluoroacetic acid) to give 9f (136mg, yield: 88.3%).

LCMS:Rt:1.212min；MS m/z(ESI):497.3[M+H]。

The fourth step: 5-cyclopropyl-N ² - (1-methyl-1H-pyrazol-4-yl) -N ⁴ - ((3R,6S) -6-methylpiperidin-3-yl) -7H-pyrrolo [2, 3-d)]Synthesis of pyrimidine-2, 4-diamine (9g)

Trifluoroacetic acid (1mL) was added to a 9f (136mg,0.273mmol) solution in methylene chloride (5.0mL) at 0 ℃ and the reaction solution was stirred at room temperature for 2 hours, and the reaction solution was concentrated under reduced pressure to give a yellow oil which was added to a methanol solution (5.0mL) of DIPEA (1.0mL) and the reaction solution was stirred at 50 ℃ for 6 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by Chem-flash (acetonitrile/0.1% trifluoroacetic acid) to obtain 9g (95mg, yield: 94.8%).

LCMS:Rt:1.124min；MS m/z(ESI):367.2[M+H]。

The fifth step: synthesis of 1- ((2S,5R) -5- ((5-cyclopropyl-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpiperidin-1-yl) prop-2-en-1-one (009)

9g (95mg,0.259mmol) were dissolved in tetrahydrofuran (2.0mL) and H ₂ To the mixture of O (3.0mL) was added potassium phosphate solid (137.5mg,0.648mmol), and acryloyl chloride (9h,28.2mg,0.311mmol) was added dropwise to the reaction mixture with stirring at 0 ℃ and stirred at room temperature for 2 hours. The reaction mixture was filtered, and the filtrate was purified by Prep-HPLC (formic acid system) (A%: water (containing 0.225% of FA); B%: ACN 13% -43%, 8-10min) to give 009(18.35mg, yield: 16.8%).

LCMS:Rt:4.416min；MS m/z(ESI):421.5[M+H]；

¹ H NMR(400M Hz,DMSO-d ₆ )δ10.59(s,1H),8.40(s,1H),7.81(s,1H),7.38(s,1H),6.92(brs,1H),6.43(s,1H),6.11-6.07(m,1H),5.87(d,J＝8.4Hz,1H),5.64(brs,1H),4.79-4.28(m,2H),4.13(brs,2H),3.71(s,3H),2.76-2.70(m,1H),2.03-1.99(m,1H),1.87-1.85(m,2H),1.72-1.67(m,2H),1.23(s,3H),0.83(d,J＝8.0Hz,1H),0.61-0.57(m,1H),0.47-0.43(m,1H).

EXAMPLE 10 preparation of Compound 010

(R) -1- (3- ((5-cyclopropyl-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) pyrrolidin-1-yl) prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

the first step is as follows: synthesis of tert-butyl (R) -3- ((2-chloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) pyrrolidine-1-carboxylate (10c)

10a (500mg,1.4mmol), (R) -3-aminopyrrolidine-1-carboxylic acid tert-butyl ester (10b,313mg,1.68mmol) and DIPEA (543mL,4.2mmol) were added to a solution of isopropanol (10.0mL) at room temperature, respectively. The reaction was sealed and heated to 110 ℃ and stirred at this temperature for 16 hours. The reaction solution was concentrated under reduced pressure to give a crude product, which was then purified by column (petroleum ether/ethyl acetate: 4/1) to give 10c (450mg, yield: 49.3%). LCMS: Rt 1.320 min; MS M/z (ESI) 508.3[ M + H ].

The second step is that: synthesis of tert-butyl ((R) -3- ((5-cyclopropyl-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) pyrrolidine-1-carboxylate (10e)

Pd is added at room temperature under the protection of nitrogen ₂ (dba) ₃ (72.3mg,0.079mmol), BINAP (49.1mg,0.079mmol) were added to 10c (400mg,0.79mmol), 1-methyl-1H-pyrazol-4-amine (10d,92mg,0.94mmol) and caesium carbonate (772mg,2.37mmol)1, 4-dioxane (30mL), the reaction system was warmed to 100 deg.C, stirred for 16 hours, and cooled to room temperature. Quenched with water (30ml), extracted with ethyl acetate (50.0ml × 2), the organic phase was concentrated and purified by column (petroleum ether/ethyl acetate 2/1) to give 10e (361mg, yield: 80.4%).

LCMS:Rt:1.474min；MS m/z(ESI):569.3[M+H]；

¹ H NMR(400MHz,DMSO-d ₆ )δ8.82(s,1H),8.00(s,1H),7.60(s,2H),6.74(d,J＝0.8Hz,1H),6.07(s,1H),5.45(s,2H),4.82-4.75(m,1H),3.90(s,3H),3.79-3.76(m,1H),3.59(t,J＝8.0Hz,3H),3.48(s,1H),3.43(s,1H),2.34(s,1H),2.14(s,1H),1.51(d,J＝8.8Hz,9H),0.94-0.91(m,4H),0.62(s,2H),0.00(s,9H).

The third step: (R) -5-cyclopropyl-N ² - (1-methyl-1H-pyrazol-4-yl) -N ⁴ - (pyrrolidin-3-yl) -7H-pyrrolo [2,3-d]Synthesis of pyrimidine-2, 4-diamine (10f)

10e (361mg,0.63mmol) was added to a solution of trifluoroacetic acid (1.0mL) in dichloromethane (3.0mL) at room temperature and the reaction was stirred at room temperature for 2h to afford a single product. The reaction mixture was concentrated under reduced pressure, added to a tetrahydrofuran/water (4.0mL/2mL) lithium hydroxide solution (100mg,2.3mmol), and the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure to give 10f (190mg, yield: 88.8%).

LCMS:Rt:1.245min；MS m/z(ESI):339.5[M+H]。

The fourth step: synthesis of (R) -1- (3- ((5-cyclopropyl-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) pyrrolidin-1-yl) prop-2-en-1-one (010)

10f (190mg,0.56mmol) was dissolved in tetrahydrofuran (4.0mL) and H ₂ To the mixture of O (2.0mL), potassium phosphate solid (297mg,1.4mmol) was added and stirred at room temperature, 10g (71mg,0.78mmol) of acryloyl chloride was added dropwise and stirred at 0 ℃ for 2 hours, followed by filtration to afford purification (Prep-HPLC (Boston Prime C18150: 30 mm: 5 μm; A%: water (containing 0.225% FA); B%: ACN 13% -43%, 8-10min) to afford 010(25.59mg, yield: 11.2%).

LCMS:Rt:6.005min；MS m/z(ESI):393.2[M+H]。

¹ H NMR(400M Hz,DMSO-d ₆ )δ10.83(brs,1H),8.88(brs,1H),7.84(d,J＝2.4Hz,1H),7.49(d,J＝3.6Hz,1H),6.67-6.54(m,1H),6.51(s,1H),6.18-6.12(m,1H),5.68(t,J＝12.8Hz,1H),4.81-4.69(m,1H),4.03-3.99(m,1H),3.79(s,3H),3.74-3.59(m,2H),3.54-3.44(m,2H),2.34-2.17(m,1H),2.07-1.99(m,2H),0.79(t,J＝2.8Hz,2H),0.50(d,J＝2.4Hz,2H).

EXAMPLE 11 preparation of Compound 011

(E) -2- ((2S,5R) -5- ((5-cyclopropyl-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpiperidine-1-carbonyl) -4, 4-dimethylpent-2-enenitrile

The synthesis route and the specific synthesis steps are as follows:

the first step is as follows: synthesis of 2-cyano-4, 4-dimethyl-2-pentenoic acid (11a)

Cyanoacetic acid (5.00g,58.0mmol) was dissolved in anhydrous methanol (50.0mL), trimethylacetaldehyde (5.00g,58.0mmol) and sodium hydroxide (4.64g,116mmol) were added, and the mixture was stirred at 40 ℃ for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (methanol/dichloromethane ═ 1/10) to give intermediate 11a (4.2g, 65.0%).

¹ H NMR(400MHz,CDCl3)δ＝7.74(d,J＝0.8Hz,1H),1.40-1.30(s,9H).

The second step is that: (E) synthesis of (E) -2- ((2S,5R) -5- ((5-cyclopropyl-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpiperidine-1-carbonyl) -4, 4-dimethylpent-2-enenitrile (011)

Intermediate 009(250mg, 682. mu. mol) and 2-cyano-4, 4-dimethyl-2-pentenoic acid 11a (115mg, 750. mu. mol) were dissolved in N, N-dimethylformamide (4.0mL), triethylamine (207mg,2.1mmol) and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (259mg, 682. mu. mol) were added, and stirred at 25 ℃ for 16 hours. The reaction mixture was filtered and separated and purified by Prep-HPLC (Boston Prime C18150: 30 mm: 5 μm; mobile phase: A%: water (containing 0.225% of FA); B%: ACN 22% -52%, 9min) to give 011(5.9mg, yield: 1.7%).

LCMS:Rt:3.573min；MS m/z(ESI):502.5[M+H].

¹ H NMR(400MHz,METHANOL-d ₄ )δ＝7.89(s,1H),7.49(s,1H),6.79(br s,1H),6.47(d,J＝1.2Hz,1H),4.22(br s,1H),3.85(s,3H),3.23-2.87(m,1H),2.11-1.78(m,5H),1.45-1.10(m,12H),0.96-0.82(m,2H),0.71-0.49(m,1H),0.59(br d,J＝5.3Hz,1H)

EXAMPLE 12 preparation of Compound 012

1- ((2S,5R) -5- ((5-cyclopropyl-2- ((1- (trifluoromethyl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpiperidin-1-yl) prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

synthesis of (2S,5R) -benzyl 5- ((2-chloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpiperidine-1-carboxylate (12c) as first step

2, 4-dichloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine 12a (2.0g,5.6mmol) was dissolved in anhydrous isopropanol (30.0mL), diisopropylethylamine (1.4g,11.2mmol) and benzyl (2S,5R) -5-amino-2-methylpiperidine-1-carboxylate 12b (1.52g,6.1mmol) were added and stirred at 80 ℃ for 16 hours. After cooling, the reaction was concentrated under reduced pressure and the residue was purified by column chromatography (ethyl acetate/petroleum ether: 1/4) to give intermediate 12c (1.3g, 40.9%).

LCMS:Rt:1.116min；MS m/z(ESI):570.2[M+H].

Second step Synthesis of (2S,5R) -benzyl-5- ((5-cyclopropyl-2- ((1- (trifluoromethyl) -1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpiperidine-1-carboxylate (12e)

Intermediate 12c (700mg,1.2mmol) and 1- (trifluoromethyl) -1H-pyrazol-4-amine 12d (253mg,1.4mmol) were dissolved in anhydrous dioxane (30.0mL), tris (dibenzylideneacetone) dipalladium (112mg, 123. mu. mol), binaphthyl-diphenylphosphine (76mg, 123. mu. mol) and cesium carbonate (1.0g,3.1mmol) were added, and stirring was performed at 100 ℃ for 16 hours under a nitrogen atmosphere. After cooling, water (10.0mL) was added to dilute the solution, extracted with ethyl acetate (20.0mL _ 2), and the organic phase was washed with saturated aqueous sodium chloride (20.0mL), dried, filtered and concentrated. The residue was purified by column chromatography (ethyl acetate/petroleum ether-1/2) to give intermediate 12e (720mg, 85.6%).

LCMS:Rt:1.240min；MS m/z(ESI):685.3[M+H].

The third step is 5-cyclopropyl-N ⁴ - ((3R,6S) -6-methylpiperidin-3-yl) -N ² - (1- (trifluoromethyl) -1H-pyrazol-4-yl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d]Synthesis of pyrimidine-2, 4-diamine (12f)

Intermediate 12e (370mg, 540. mu. mol) was dissolved in dry methanol (15.0mL) and wet palladium on carbon (40mg, 10% Pd) was added. Stirred under hydrogen (15psi) at 25 ℃ for 16 h. The reaction was concentrated by filtration to give crude 12f (270mg) which was used in the next step without purification.

LCMS:Rt:1.239min；MS m/z(ESI):551.3[M+H].

The fourth step is 5-cyclopropyl-N ⁴ - ((3R,6S) -6-methylpiperidin-3-yl) -N ² - (1- (trifluoromethyl) -1H-pyrazol-4-yl) -7H-pyrrolo [2,3-d]Synthesis of pyrimidine-2, 4-diamine (12g)

Crude 12f (270mg, 490. mu. mol) was dissolved in dry dichloromethane (10.0 mL). Trifluoroacetic acid (2.0mL) was added thereto, and after stirring at 25 ℃ for 6 hours, the reaction mixture was concentrated under reduced pressure to obtain a crude product, which was dissolved in methanol (6.0mL) and water (3.0mL), and lithium hydroxide (21mg, 490. mu. mol) was added thereto, and the mixture was heated to 50 ℃ and stirred for 2 hours. The reaction solution was cooled, filtered and concentrated, and after adding water to the residue, a solid precipitated and filtered to give 12g (260mg) of a crude green solid, which was used in the next step without purification.

LCMS:Rt:2.543min；MS m/z(ESI):421.1[M+H].

Fifth step Synthesis of 1- ((2S,5R) -5- ((5-cyclopropyl-2- ((1- (trifluoromethyl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpiperidin-1-yl) prop-2-en-1-one (012)

At 0 deg.C, 12g (260mg, 495. mu. mol) of crude was dissolved in tetrahydrofuran (4.0mL) and water (2.0 mL). Potassium phosphate (228mg, 990. mu. mol) and acryloyl chloride (45mg, 495. mu. mol) were added thereto, and the mixture was stirred at 25 ℃ for 2 hours. Diluted with water (10.0mL), extracted with ethyl acetate (15.0mL x2), the organic phase washed with saturated aqueous sodium chloride (15.0mL), dried, filtered and concentrated, and the residue purified by preparative purification (Prep-HPLC (Boston Prime C18150 x 30mm x 5 μm; a%: water (containing 0.225% FA); B%: ACN 22% -52%)) to give the product 012(25.4mg, 10.8%).

LC-MS:Rt:2.892min；MS m/z(ESI):475.2[M+H]；

¹ H NMR(400MHz,DMSO-d ₆ )δ＝10.77(br s,1H),8.95(s,1H),8.39(br s,1H),7.89(br s,1H),6.78(br s,1H),6.52(s,1H),6.18-5.94(m,2H),5.63(br s,1H),4.92-3.93(m,3H),3.17-2.72(m,1H),2.04(br s,1H),1.88(br s,2H),1.70(br s,2H),1.22(br s,3H),0.85(br d,J＝7.8Hz,2H),0.66-0.37(m,1H),0.66-0.37(m,2H).

EXAMPLE 13 preparation of Compound 013

(R) -1- (4- ((5-chloro-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7H pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) nonyl-1-yl) prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

the first step is as follows: synthesis of tert-butyl 4- ((2, 5-dichloro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) azepane-1 carboxylate (13c)

NaH (60mg,1.5mmol) was added in portions to a solution of tert-butyl 4-hydroxyazepan-1-carboxylate 13b (323mg,1.5mmol) in anhydrous DMSO (10.0mL) at room temperature, stirring was completed for 15 minutes, and then 2,4, 5-trichloro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine 13a (527mg,1.5mmol) was added. The reaction system was warmed to 55 ℃, reacted for 15 minutes, cooled to room temperature, and a saturated ammonium chloride solution (50.0mL) was added to the reaction solution, extracted three times with dichloromethane (50.0mL × 3), the organic phases were combined, washed once with water (50.0mL) and saturated brine (5.0mL), the organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure and purified by a silica gel column (ethyl acetate: petroleum ether ═ 1:10) to give 13c (620.4mg, yield: 78%).

LCMS:Rt:1.775min；MS m/z(ESI):533.5[M+H].

The second step is that: synthesis of tert-butyl 4- ((5-chloro-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) azepane-1-carboxylate (13e)

13c (620.4mg,1.17mmol), 1-methyl-1H-pyrazol-4-amine 13d (170mg,1.76mmol) and Cs are added under the protection of nitrogen at room temperature ₂ CO ₃ (763mg,2.34mmol) in 1, 4-dioxane (20.0mL), the reaction was warmed to 100 deg.C, stirred for 16 hours, and cooled to room temperature. Filtration and washing of the filter cake with tetrahydrofuran (50.0mL) were carried out, followed by concentration under reduced pressure and purification to give the product 13e (595.8mg, yield: 86.2%).

LCMS:Rt:1.480min；MS m/z(ESI):592.2[M+H].

¹ H NMR(400MHz,CDCl ₃ )δ7.84(s,1H),7.60(s,1H),6.87(s,1H),6.64(d,J＝7.6Hz,1H),5.59-5.54(m,1H),5.49(s,2H),3.97(s,3H),3.83-3.66(m,2H),3.60(t,J＝8.0Hz,2H),3.49-3.39(m,2H),2.23-2.08(m,4H),1.93-1.82(m,2H),1.54(s,9H),0.98(t,J＝8.4Hz,2H),0.00(s,9H).

The third step: synthesis of ((4- (azepan-4-oxy) -5-chloro-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methanol (13f)

After TFA (12.0mL) was added to a solution of intermediate 13e (6.0mL) at room temperature and stirred at room temperature for 2 hours, the reaction was concentrated to dryness under reduced pressure and the crude 13f was used directly in the next reaction.

LCMS:Rt:0.763min；MS m/z(ESI):392.0[M+H].

The fourth step: synthesis of 1- (4- ((5-chloro-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) azepan-1-yl) prop-2-en-1-one (013 and 013-1)

Crude 13f (1mmol) was dissolved in a mixed solvent of acetone (12.0mL) and water (8.0mL), the pH was adjusted to 10 with potassium carbonate, the mixture was heated to 35 ℃ and stirred for 16 hours, the reaction solution was cooled to 0 ℃ and then acryloyl chloride (126mg, 1.4mmol) was dissolved in acetone (1.0mL) and added dropwise to the reaction solution. After the completion of the dropwise addition, the reaction mixture was stirred at 0 ℃ for 1 hour. The reaction was filtered and purified by spin-drying (Prep-HPLC (Boston Prime C18150 x 30mm x 5 μm; A%: water (containing 0.225% FA); B%: ACN 22% -52%)) to give the product.

013(14.28mg, yield: 6.9%):

LCMS:Rt:6.567min；MS m/z(ESI):416.1[M+H]；

¹ H NMR(400MHz,DMSO-d ₆ )δ11.47(s,1H),9.01(s,1H),7.83(s,1H),7.49(s,1H),7.03(d,J＝1.6Hz,1H),6.84-6.74(m,1H),6.18-6.11(m,1H),5.66(dd,J＝10.4,2.4Hz,1H),5.47(d,J＝4.8Hz,1H),3.81(s,3H),3.79-3.76(m,2H),3.63-3.57(m,1H),3.47-3.40(m,1H),2.04(t,J＝4.8Hz,3H),1.98-1.91(m,1H),1.84-1.76(m,2H).

013-1(13.82mg, yield: 6.7%):

LCMS:Rt:6.567min；MS m/z(ESI):416.1[M+H]；

¹ H NMR(400MHz,DMSO-d ₆ )δ11.47(s,1H),9.01(s,1H),7.83(s,1H),7.49(s,1H),7.03(d,J＝2.0Hz,1H),6.84-6.74(m,1H),6.18-6.11(m,1H),5.66(dd,J＝8.0,2.4Hz,1H),5.50-5.46(m,1H),3.80(s,3H),3.76-3.71(m,2H),3.64-3.57(m,1H),3.46-3.39(m,1H),2.04(t,J＝5.2Hz,3H),1.98-1.91(m,1H),1.84-1.78(m,2H).

EXAMPLE 14 preparation of Compound 014

1- ((3S,4R) 4-fluoro-3- ((2- ((1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl) amino) -7H pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) -piperidin-1-yl) prop-2-en-1-one

1-((3S,4R)-4-fluoro-3-((2-((1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)piperidin-1-yl)prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

the first step is as follows: synthesis of 1- ((3S,4R) 4-fluoro-3- ((2- ((1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl) amino) -7((2- (trimethylsilyl) ethoxy) methyl) -pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) -piperidine-1-carboxylic acid tert-butyl ester (14c)

1- ((3S,4R) 4-fluoro-3- ((2-chloro) -7((2- (trimethylsilyl) ethoxy) methyl) -7H pyrrolo [2, 3-d) was reacted at room temperature under a nitrogen blanket]Pyrimidin-4-yl) oxy) -tetrahydropyrrole-1-carboxylic acid tert-butyl ester 14a (125mg,0.25mmol) and 1- (1-methylpiperidin-4-yl) -1H-pyrazole-4-amine 14b (67.5mg,0.38mmol) were dissolved in 1, 4-dioxane (10.0mL) and Pd was added with stirring ₂ (dba) ₃ (22.8mg,0.025mmol), BINAP (31.1mg,0.2mmol) and cesium carbonate (162.8mg,0.5mmol), the reaction system was warmed to 120 systems, stirred for 12 hours, cooled to room temperature, filtered, the filter cake was washed with ethyl acetate (60.0mL), and the filtrate was concentrated under reduced pressure and purified (dichloromethane: methanol ═ 15:1) to give 14c (120mg, yield: 74).5％)。

LCMS:Rt:1.307min；MS m/z(ESI):645.3[M+H].

¹ H NMR(400MHz,DMSO-d ₆ )δ9.24(s,1H),8.12(brs,1H),7.68(s,1H),7.21(d,J＝3.6Hz,1H),6.42(d,J＝3.4Hz,1H),5.60(s,3H),5.18(d,J＝46.3Hz,1H),4.48(s,1H),4.24(s,1H),4.04(s,1H),3.63(t,J＝8.0Hz,2H),3.04(d,J＝28.0Hz,3H),2.41(s,3H),2.20(s,3H),2.11(s,6H),1.47(s,2H),1.35(s,1H),1.08(s,6H),0.99–0.92(m,2H),0.00(s,9H).

The second step is that: synthesis of 1- ((3S,4R) 4-fluoro-3- ((2- ((1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) -piperidine (14d)

Trifluoroacetic acid (5.0mL) was added to a solution of 14c (125mg,0.19mmol) in dichloromethane (5.0mL) at 0 ℃ and stirred at room temperature for 16 h. After concentration, the crude product is dissolved in THF/H ₂ To a mixture of O (6.0mL,2:1) was added LiOH (125.0mg,5.2mmol), and the mixture was stirred at room temperature for 1 hour to obtain a crude product, which was purified by filtration to obtain 14d (87mg, yield: 99%).

LCMS:Rt:0.427min；MS m/z(ESI):415.2[M+H].

The third step: synthesis of 1- ((3S,4R) 4-fluoro-3- ((2- ((1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) -piperidin-1-yl) prop-2-en-1-one (014)

14d (87mg,0.21mmol) was added to tetrahydrofuran (2.0mL) and H at 0 deg.C ₂ To the mixture of O (1.0mL), potassium phosphate (233.5mg,1.1mmol) was added. After stirring for 10 minutes, a solution of acryloyl chloride (29.0mg,0.32mmol) in tetrahydrofuran (1.0mL) was added slowly dropwise. After stirring for 2 hours, filtration and preparative purification of the filtrate (Prep-HPLC (Boston Prime C18150 x 30mm x 5 μm; A%: water (containing 0.225% FA); B%: ACN 20% -50%)) gave product 014(35.11mg, yield: 35.7%).

LCMS:Rt:5.937min；MS m/z(ESI):469.3[M+H].

¹ H NMR(400MHz,DMSO-d ₆ )δ11.32(s,1H),8.92(s,1H),8.23(s,1H),7.95(s,1H),7.53(s,1H),7.02-6.77(m,1H),6.66-6.41(m,1H),6.27-5.90(m,1H),5.78-5.37(m,1H),5.19(d,J＝48.3Hz,1H),3.96(m,8H),3.72(d,J＝32.5Hz,1H),3.41(s,1H),2.97(d,J＝8.2Hz,1H),2.29(d,J＝24.4Hz,4H),2.00(s,4H).

EXAMPLE 15 preparation of Compound 015

1- ((3S,4R) -4-fluoro-3- ((2- ((1- ((R) -tetrahydrofuran-3-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) piperidin-1-yl) prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

the first step is as follows: synthesis of tert-butyl (3S,4R) 4-fluoro-3- ((2- (((1- ((R) -tetrahydrofuran-3-yl) -1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) piperidine-1-carboxylate (15g)

Intermediate 15e (100mg,0.20mmol), (R) -1- (tetrahydrofuran-3-yl) -1H-pyrazol-4-amine 15f (61mg,0.40mmol), Pd (dppf) Cl ₂ (15mg,0.02mmol), XantPhos (12mg,0.02mmol) and cesium carbonate (130mg,0.40mmol) were added to 1, 4-dioxane (2.0mL) and the reaction was stirred under argon at 95 ℃ for 16 h. After cooling, the reaction solution was filtered through celite, and the filter cake was washed with ethyl acetate (10.0 mL). The filtrate was concentrated and purified under reduced pressure (petroleum ether/ethyl acetate ═ 2:1) to obtain 15g (150mg, yield: 90%).

LCMS:Rt:0.73min；MS m/z(ESI):618.4[M+H].

¹ H NMR(400MHz,CDCl ₃ )δ8.04(s,1H),7.65(s,1H),6.96(d,J＝3.6Hz,1H),6.67(brs,1H),6.52(d,J＝3.6Hz,1H),5.57(s,2H),5.46-5.37(m,1H),5.19-5.03(m,2H),4.25-4.20(m,1H),4.17-4.11(m,1H),4.05-4.01(m,3H),3.90-3.69(m,2H),3.63-3.59(m,3H),2.56-2.50(m,1H),2.43-2.38(m,1H),2.31-2.21(m,1H),2.03-1.89(m,1H),1.59-1.35(m,9H),0.98(t,J＝8.8Hz,2H),0.00(s,9H).

The second step is that: synthesis of 4- (((3S,4R) -4-fluoropiperidin-3-yl) oxy) -N- (1- ((R) -tetrahydrofuran-3-yl) -1H-pyrazol-4-yl) -7H-pyrrolo [2,3-d ] pyrimidin-2-amine (15H)

15g (150mg,0.24mmol) was added to trifluoroacetic acid (3.0mL) at 25 ℃ and the reaction was stirred for 30 minutes at 25 ℃. The reaction mixture was concentrated under reduced pressure, added to a solution of lithium hydroxide monohydrate in tetrahydrofuran (3.0 mL)/water (0.5mL) (pH 12), and stirred at 25 ℃ for 30 minutes. Concentrated under reduced pressure, diluted with 5.0mL of water and extracted with ethyl acetate (5.0mL × 3). The organic phase was concentrated to give 15h (120mg, yield: > 99%).

LCMS:Rt:1.15min；MS m/z(ESI):388.2[M+H].

The third step: synthesis of 1- ((3S,4R) -4-fluoro-3- ((2- ((1- ((R) -tetrahydrofuran-3-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) piperidin-1-yl) prop-2-en-1-one (015)

15h (100mg,0.26mmol) was dissolved in a mixture of tetrahydrofuran (0.5mL) and water (0.3mL), potassium phosphate solid (110mg,0.52mmol) was added and stirred at 25 deg.C, a solution of acryloyl chloride (23mg,0.26mmol) in tetrahydrofuran (0.5mL) was added dropwise and stirred for 1 h. The reaction solution was concentrated under reduced pressure and preparative purification (Prep-HPLC (Boston Prime C18150: 30 mm: 5 μm; A%: water (containing 0.225% FA); B%: ACN 15% -40%)) gave 015(8.5mg, yield: 7%).

LCMS:Rt:5.07min；MS m/z(ESI):442.3[M+H].

¹ H NMR(400M Hz,DMSO-d ₆ )δ11.31(s,1H),8.94(s,1H),7.94(s,1H),7.54-7.50(m,1H),6.94(s,1H),6.90-6.49(m,1H),6.21-6.16(m,1H),6.11-5.93(m,1H),5.73-5.41(m,2H),5.24-4.94(m,2H),4.06-3.96(m,3H),3.88-3.60(m,5H),2.39-2.32(m,1H),2.26-2.06(m,1H),2.08-1.76(m,2H).

EXAMPLE 16 preparation of Compound 016

1- ((3S,4R) -4-fluoro-3- ((2- ((1- ((R) -1-methylpyrrolidin-3-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) piperidin-1-yl) prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

the first step is as follows: synthesis of tert-butyl (3S,4R) -3- ((2-chloro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) -4-fluoropyridine-1-carboxylate (16c)

NaH (91mg,2.27mmol) was added in portions to a solution of tert-butyl (3S,4R) -4-fluoro-3-hydroxypiperidine-1-carboxylate 16b (500mg,2.27mmol) in DMSO (15.0mL) at room temperature, stirring was completed for 15 minutes, and then 2, 4-dichloro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] was added]Pyrimidine 16a (720mg,2.27mmol) was added, the reaction was warmed to 55 ℃ for 15 minutes, 50.0mL of saturated NH was added ₄ The reaction mixture was added to a Cl solution, extracted three times with dichloromethane (50.0mL × 3), the organic phases were combined, washed once with water (50.0mL) and saturated brine (50mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified (ethyl acetate: petroleum ether ═ 1:8) to give 16c (748mg, yield: 58%).

LCMS:Rt:1.893min；MS m/z(ESI):501.2[M+H].

¹ H NMR(400MHz,CDCl ₃ )δ7.20(d,J＝3.6Hz,1H),6.63(d,J＝3.6Hz,1H),5.61(s,2H),5.50-5.46(m,1H),5.12-5.00(m,1H),3.95-3.62(m,4H),3.56(t,J＝8.4Hz,2H),2.31-2.21(m,1H),2.03-1.95(m,1H),1.32(s,9H),0.95(t,J＝5.2Hz,2H),0.00(s,9H).

The second step is that: synthesis of tert-butyl (3S,4R) 4-fluoro-3- ((2- ((1- ((R) -1-methylpyrrolidin-3-yl) -1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) piperidine-1-carboxylate (16e)

Pd is added under the protection of nitrogen at room temperature ₂ (dba) ₃ (14.6mg,0.016mmol) and BINAP (9.7mg,0.016mmol) were added to 16c (81mg,0.162mmol), (R) -1- (1-methylpyrrolidin-3-yl) -1H-pyrazol-4-amine 16d (53.5mg,0.324mmol) and Cs ₂ CO ₃ (105mg,0.324mmol) in 1, 4-dioxane (5.0mL), the reaction was warmed to 100 deg.C, stirred for 16h, and cooled to room temperature. Filtration and washing of the filter cake with 50.0mL of tetrahydrofuran, the crude product obtained after concentration of the solution under reduced pressure was purified to give 16e (67.6mg, yield: 66.2%).

LCMS:Rt:1.420min；MS m/z(ESI):631.8[M+H].

The third step: synthesis of (4- ((3S,4R) -4-fluoropiperidin-3-yl) oxy) -2- ((1- ((R) -1-methylpyrrolidin-3-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-7-yl) methanol (16f)

TFA (8.0mL) was added to a solution of 16e (595.8mg,1mmol) in dichloromethane (4.0mL) at room temperature, stirred at room temperature for 2 hours, concentrated under reduced pressure, dichloromethane (12.0mL) was added, and the excess trifluoroacetic acid was removed by concentration under reduced pressure. Concentrated under reduced pressure and the crude 16f obtained was used directly in the next reaction.

LCMS:Rt:0.750min；MS m/z(ESI):431.0[M+H]。

The fourth step: synthesis of 1- ((3S,4R) -4-fluoro-3- ((2- ((1- ((R) -1-methylpyrrolidin-3-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) piperidin-1-yl) prop-2-en-1-one (016)

Crude 16f (0.11mmol) was dissolved in a mixed solvent of acetone (6.0mL) and water (4.0mL), adjusted to pH 10 with potassium carbonate, heated to 35 ℃ and stirred for 16 hours. The reaction solution was cooled to 0 ℃, and then acryloyl chloride (45mg, 0.48mmol) was dissolved in acetone (1.0mL) and added dropwise to the reaction solution. After the dropwise addition, the reaction solution was filtered and spin-dried. The resulting crude product was purified (Prep-HPLC (Boston Prime C18150: 30 mm: 5 μm; A%: water (containing 0.225% FA); B%: ACN 15% -50%)) to give the title product 016(15.18mg, yield: 31%).

LCMS:Rt:3.617min；MS m/z(ESI):455.2[M+H].

¹ H NMR(400M Hz,DMSO-d ₆ )δ11.32(s,1H),9.02(s,1H),8.01(s,1H),7.65(d,J＝10.8Hz,1H),6.95(s,1H),6.92-6.49(m,1H),6.18(d,J＝14.4Hz,1H),6.10-5.93(m,1H),5.73-5.41(m,2H),5.25-5.12(m,2H),4.09-3.95(m,2H),3.86-3.76(m,3H),3.68-3.51(m,2H),3.44-3.31(m,2H),2.97-2.90(m,3H),2.33-2.25(m,1H),2.15-2.04(m,1H),1.98-1.93(m,1H).

EXAMPLE 17 preparation of Compound 017

1- ((3S,4R) -4-fluoro-3- ((2- ((1- (oxetanyl-3-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) piperidin-1-yl) prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

the first step is as follows: synthesis of 2-chloro-4- (((3S,4R) -4-fluoropiperidin-3-yl) oxy) -7H-pyrrolo [2,3-d ] pyrimidine (17b)

Trifluoroacetic acid (0.5mL) was added to a dichloromethane solution (5.0mL) of tert-butyl (3S,4R) -3- ((2-chloro-7- ((2-trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) -4-fluoropiperidine-1-carboxylate 17a (180mg,0.359mmol) at 0 ℃, the reaction solution was stirred at room temperature for 16 hours, concentrated under reduced pressure, the resulting crude product was added to a methanol solution (5.0mL) of DIPEA (1.0mL), and the reaction solution was stirred at 30 ℃ for 2 hours. The solution was concentrated under reduced pressure and purified (acetonitrile/0.1% trifluoroacetic acid) to obtain 17b (97.2mg, yield: 99%).

LCMS:Rt:0.757min；MS m/z(ESI):271.1[M+H].

The second step: synthesis of 4- (((3S,4R) -4-fluoropiperidin-3-yl) oxy) -N- (1- (oxetan-3-yl) -1H-pyrazol-4-yl) -7H-pyrrolo [2,3-d ] pyrimidin-2-amine (17d)

Under nitrogen protection, 17b (97.2mg,0.359mmol) and 1- (oxetan-3-yl) -1H-pyrazol-4-amine 17c,60.0mg,0.431mmol were dissolved in 1, 4-bisOxohexacyclo (10.0mL) and then Pd was added with stirring ₂ (dba) ₃ (32.9mg,0.036mmol), BINAP (22.4mg,0.036mmol) and cesium carbonate (292.4mg,0.897mmol), the reaction was warmed to 100 deg.C, stirred for 16 hours, cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate (60.0 mL). The filtrate was concentrated under reduced pressure to obtain crude product, which was purified to obtain 17d (45.5mg, yield: 33.9%).

LCMS:Rt:0.490min；MS m/z(ESI):374.4[M+H].

The third step: synthesis of 1- ((3S,4R) -4-fluoro-3- ((2- ((1- (oxetan-3-yl) -1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) piperidin-1-yl) prop-2-en-1-one (017)

17d (45.5mg,0.122mmol) was dissolved in a mixture of tetrahydrofuran (5.0mL) and water (2.0mL) with stirring at 0 ℃ and potassium phosphate (64.7mg,0.305mmol) was added and stirred. Acryloyl chloride (13.3mg,0.146mmol) was added dropwise to the reaction solution, and the mixture was stirred at room temperature for 2 hours. The reaction was filtered and the filtrate was purified (Prep-HPLC (Boston Prime C18150 x 30mm x 5 μm; A%: water (containing 0.225% FA); B%: ACN 22% -52%)) to give 017(2.15mg, yield: 4.2%).

LCMS:Rt:5.527min；MS m/z(ESI):428.1[M+H].

¹ H NMR(400MHz,DMSO-d ₆ )δ11.34(s,1H),9.00(s,1H),8.04(s,1H),7.65-7.62(m,1H),6.94(s,1H),6.56-6.17(m,1H),6.12-5.92(m,1H),5.74-5.59(m,1H),5.50-5.43(m,2H),5.25-5.12(m,1H),4.92-4.85(m,4H),4.04-4.00(m,1H),3.90-3.75(m,2H),3.70-3.62(m,1H),3.47-3.39(m,1H),2.14-1.90(m,2H).

EXAMPLE 18 preparation of Compound 018

1- ((2S, 4R) -4- ((5-fluoro-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidin-1-yl) prop-2-en-1-one

The synthesis route and the specific synthesis steps are as follows:

the first step is as follows: synthesis of tert-butyl (2S, 4R) -4- ((2-chloro-5-fluoro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidine-1-carboxylate (18c)

2, 4-dichloro-5-fluoro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine 18a (300mg,0.98mmol), (tert-butyl 2S, 4R) -4-amino-2-methylpyrrolidine-1-carboxylate 18b (197mg,0.98mmol) was added to a solution of DIPEA (287mg,2.25mmol) in isopropanol (10.0mL) and stirred at 110 ℃ for 16H. The reaction was terminated by adding water (100.0mL), ethyl acetate (100.0mL × 2) was extracted, the organic phases were combined, washed with saturated brine (100.0mL), the organic phase was concentrated under reduced pressure, and purified (ethyl acetate: petroleum ether: 1:10) to give 18c (380mg, yield: 85%).

LCMS:Rt:1.997min；MS m/z(ESI):500.1[M+H].

¹ H NMR(400MHz,DMSO-d ₆ )δ7.64(s,1H),7.40(s,1H),5.47(d,J＝9.6Hz,2H),4.59(s,1H),3.87(s,2H),3.55(t,J＝8.0Hz,2H),3.25(s,1H),2.48-2.45(m,1H),1.87(s,1H),1.48(s,9H),1.32(d,J＝6.4Hz,3H),0.89(d,J＝8.4Hz,2H),0.00(s,9H).

The second step: synthesis of tert-butyl (2S, 4R) -4- ((5-fluoro-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidine-1-carboxylate (18e)

Pd is added at room temperature under the protection of nitrogen ₂ (dba) ₃ (84.2mg,0.092mmol), BINAP (57.2mg,0.092mmol) were added to a solution of 18c (480mg,0.92mmol), 1-methyl-1H-pyrazol-4-amine 18d (116mg,1.19mmol) and cesium carbonate (749mg,2.3mmol) in 1, 4-dioxan (20.0mL), the reaction was warmed to 120 ℃ and stirred overnight, cooled to room temperature and filtered. The reaction mixture was concentrated under reduced pressure and purified (ethyl acetate: petroleum ether: 1) to give 18e (480mg, yield: 93%).

LCMS:Rt:1.650min；MS m/z(ESI):561.2[M+H].

The third step: 5-fluoro-N ² - (1-methyl-1H-pyrazol-4-yl) -N ⁴ - ((3R, 5S) -5-methylpyrrolidin-3-yl) -7H-pyrrolo [2,3-d]Synthesis of pyrimidine-2, 4-diamine (18f)

18e (480mg,0.85mmol) was added to a solution of trifluoroacetic acid (3.0mL) in dichloromethane (6.0mL) at room temperature, and the reaction was stirred at 0 ℃ for 6 hours. The reaction was quenched with saturated sodium bicarbonate (50.0mL), extracted with ethyl acetate (20.0mL x2), the organic phase was concentrated under reduced pressure, the crude product was added without purification to a tetrahydrofuran/water solution of LiOH (522mg,12.7mmol) (4.0mL/1.0mL) and the reaction was stirred for 2 hours at 25 ℃. The reaction mixture was concentrated under reduced pressure, and the resulting solution was subjected to reverse phase reaction to give intermediate 18f (80mg, yield: 28%).

LCMS:Rt:1.07min；MS m/z(ESI):331.2[M+H].

The fourth step: synthesis of 1- ((2S, 4R) -4- ((5-fluoro-2- ((1-methyl-1H-pyrazol-4-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidin-1-yl) prop-2-en-1-one (018)

18f (80mg,0.24mmol) was dissolved in a mixture of tetrahydrofuran (6.0mL) and water (2.0mL), potassium phosphate (127.0mg,0.60mmol) was added and stirred, a solution of acryloyl chloride (30.5mg,0.34mmol) in tetrahydrofuran (0.5mL) was added dropwise and stirred at 25 ℃ for 1 hour. The reaction mixture was concentrated under reduced pressure and purified by Prep-HPLC (acetonitrile/water/1% TFA) to give 018(15.7mg, yield: 17.0%).

LCMS:Rt:6.327min；MS m/z(ESI):385.1[M+H].

¹ H NMR(400MHz,DMSO-d ₆ )δ10.69(s,1H),8.62(s,1H),7.80(s,1H),7.45(s,1H),6.68-6.53(m,3H),6.15(t,J＝12.0Hz,1H),5.68-5.63(m,1H),4.65-4.58(m,1H),4.22-4.05(m,2H),3.74(s,3H),3.48-3.25(m,1H),2.55-2.42(m,1H),1.94-1.83(m,1H),1.29(d,J＝6.0Hz,3H).

Examples of biological Activity and related Properties

Test example 1: BTK kinase Activity inhibition assay

The experimental principle is as follows: after incubation of the BTK kinase with the compound, it is reacted with the substrate under the action of ATP. ADP produced by the reaction was quantified using ADP-GLO detection kit from Promega to reflect the enzyme activity.

An experimental instrument: labcyte Echo650 pipetting system; envision microplate reader from Perkin Elmer; eppendorf 5810 centrifuge.

Experimental materials:

reagent	Brand	Goods number
Tris hydrochloride solution	Sigma	T2663
BRIJ 35 detergent(10％)	Merck	203728
MgCl 2 solution	Sigma	M1028
ADP-Glo kinase detection kit	Promega	V9102
BTK	Carna bioscience	08-180
Poly(4:1Glu,Tyr)	Sigma	P0275
384 orifice plate	Perkin Elmer	6007290

The experimental method comprises the following steps: test compounds were transferred to 384-well plates using an Echo pipetting system, and 2. mu.L/well of BTK was added and incubated for 30 minutes. Then, 3. mu.L/well of a mixed solution of substrate Poly (4:1Glu, Tyr) and ATP was added to initiate an enzyme reaction. The final concentration of compound was 3-fold diluted starting from 3 μ M or 300nM or 100nM, respectively. The final concentration of enzyme in the reaction was 1.7 ng/well, the final concentration of ATP was 36. mu.M, and the final concentration of substrate was 0.1 mg/mL. After 1 hour of reaction, 5. mu.L/well of ADP-GLO reagent was added and incubated for 40 minutes. Then 10. mu.L/well of kinase reaction detection reagent was added and incubated for 30 minutes. The fluorescence signal was read with an Envision microplate reader and the inhibition rate, median Inhibitory Concentration (IC) was calculated ₅₀ )。

The biological activity of the compounds of the invention is determined by the above assay, the IC determined ₅₀ The values are given in table 1 below.

TABLE 1 IC inhibition of BTK kinase activity by compounds of the examples ₅₀

Example Compound numbering	IC ₅₀ (nM)	Example Compound numbering	IC ₅₀ (nM)
001	6.53	010	3.92
002	7.41	011	5.35
003	3.26	012	9.07
004	3.83	013	4.00
005	4.83	014	0.97
006	6.60	015	2.14
007	1.77	016	1.90
008	2.48	017	3.63
009	3.43	018	1.39

Test example 2: JAK3 kinase Activity inhibition assay

The experimental principle is as follows: after incubation with compound, JAK3 kinase reacts with the substrate under the action of ATP. ADP produced by the reaction was quantified using ADP-GLO detection kit from Promega to reflect the enzyme activity.

An experimental instrument:

echo650 pipetting system of Labcyte company

Envision microplate reader manufactured by Perkin Elmer

Eppendorf corporation 5810 centrifuge.

Experimental materials:

reagent	Brand	Goods number
Tris hydrochloride solution	Sigma	T2663
BRIJ 35 detergent(10％)	Merck	203728
MgCl 2 solution	Sigma	M1028
ADP-Glo kinase detection kit	Promega	V9102
JAK3	Carna bioscience	08-046
Poly(4:1Glu,Tyr)	Sigma	P0275
384 orifice plate	Perkin Elmer	6007290

The experimental method comprises the following steps:

test compounds were transferred to 384-well plates using an Echo pipetting system, and 2 μ L/well of JAK3 was added and incubated for 30 minutes. Then, 3. mu.L/well of a mixed solution of substrate Poly (4:1Glu, Tyr) and ATP was added to initiate an enzyme reaction. The final concentration of compound was 3-fold diluted starting from 3 μ M or 300nM or 100nM, respectively. The final concentration of enzyme in the reaction was 1.9 ng/well, the final concentration of ATP was 36. mu.M, and the final concentration of substrate was 0.1 mg/mL. After 1 hour of reaction, 5. mu.L/well of ADP-GLO reagent was added and incubated for 40 minutes. Then 10. mu.L/well of kinase reaction detection reagent was added and incubated for 30 minutes. The fluorescence signal was read with an Envision microplate reader and the inhibition rate, median Inhibitory Concentration (IC) was calculated ₅₀ )。

The biological activity of the compounds of the invention is determined by the above assay, the IC determined ₅₀ The values are given in Table 2 below.

Table 2 IC inhibition of JAK3 kinase activity by compounds of the invention ₅₀

Example Compound numbering	IC ₅₀ (nM)	Number of example Compounds	IC ₅₀ (nM)
001	1.66	010	1.27
002	0.61	011	7.23
003	0.55	012	1.74

004	1.36	013	0.58
005	1.41	014	0.51
006	4.74	015	0.25
007	1.48	016	0.89
008	3.04	017	0.84
009	1.81	018	0.46

Test example 3: inhibition of BTK phosphorylation in Ramos cells

The experimental principle is as follows: after Ramos cells are incubated with the compound and the stimulating agent, the transfer of fluorescence energy is detected by a homogeneous phase time-resolved fluorescence (HTRF) method using a BTK phosphorylation detection kit of Cisbio, so as to reflect the inhibition effect on phosphorylation.

An experimental instrument:

instrument for measuring the position of a moving object	Brand	Model number
Biological safety cabinet	ESCO	CLASSⅡ BSC
Centrifugal machine	Eppendorf	5810
CO 2 incubator	ESCO	CCL-170B-8
Cell counter	CountStar	IC1000
Envision	Perkin Elmer	/

Experimental materials:

reagent	Providers of	Goods number
RPMI-1640 medium	GIBCO	A10491-01
Inactivated fetal bovine serum (HI-FBS)	GIBCO	10100147
HBSS solution	GIBCO	14025-092
anti-human IgM antibody	Jackson Immuno	109-006-129
BTK phospho-Y223 HTRF detection kit	Cisbio	63ADK017PEH
384 orifice plate	Perkin Elmer	6007680
Ramos	ATCC	CRL-1596

The experimental method comprises the following steps:

test compounds were transferred to 384-well plates using an Echo pipetting system, and the Ramos cell density was adjusted to 1X10 ⁷ cells/mL, 10. mu.L/well of cell suspension was added, 5% CO at 37 ℃ ₂ Was incubated for 1 hour. Then 5. mu.L/well of a stimulator anti-human IgM antibody was added to the cells at a final concentration of 10. mu.g/mL, and the cells were incubated for 10 minutes. The final concentration of compound was 1 μ M starting, 4-fold diluted. 5 μ L/well of cell lysate was added and incubated for 30 minutes at room temperature. The phosphorylation degree of BTK is detected by using a BTK phospho-Y223 kit of Cisbio company, and finally, fluorescent signals under the conditions of emitting light of 665nm and 615nm are read on an Envision microplate reader, and the inhibition rate and the median Inhibitory Concentration (IC) are calculated ₅₀ )。

The biological activity of the compounds of the invention is determined by the above assay, the IC determined ₅₀ The values are given in Table 3 below.

TABLE 3 inhibitory Activity of the Compounds of the present invention on BTK phosphorylation in Ramos cells

Example Compound numbering	IC ₅₀ (nM)	Example Compound numbering	IC ₅₀ (nM)
001	23.76	010	53.49

002	62.24	011	5.04
003	2.63	012	6.15
004	5.07	013	10.85
005	10.90	014	2.02
006	11.34	015	17.89
007	2.39	016	4.23
008	5.78	017	9.66
009	5.73	018	4.01

Test example 4: inhibition of STAT5 phosphorylation in CTLL-2 cells

The experimental principle is as follows: this experiment was performed to evaluate the effect of compounds on phosphorylation of STAT5, a downstream substrate of JAK 3. After CTLL-2 cells were incubated with compounds and stimulators, the fluorescence energy transfer between donor beads and acceptor beads was detected by time-resolved fluorescence using the p-STAT5(Tyr694/699) detection kit from Perkin Elmer to reflect the inhibition of phosphorylation.

An experimental instrument:

instrument for measuring the position of a moving object	Brand	Model number
Biological safety cabinet	Thermo Scientific	1300 Series A2
Centrifugal machine	Eppendorf	5702
CO 2 incubator	Thermo Scientific	371
Cell counter	Invitrogen	C10281
Envision	Perkin Elmer	/
Echo	Labcyte	655

Experimental materials:

reagent	Brand	Goods number
Fetal Bovine Serum (FBS)	GIBCO	10099-141
Dimethyl sulfoxide (DMSO)	Sigma	D8418-1L
IL-2	R&D	402-ML-020
CTLL-2	ATCC	TIB-214
AlphaLISA SureFire Ultra p-STAT5(Tyr694/699) detection kit	Perkin Elmer	ALSU-PST5-B500

The experimental method comprises the following steps:

CTLL-2 cells were plated in 384-well plates, 1.5X10 ⁴ Cells/15. mu.l/well, compounds were transferred to 384-well plates with Echo at 37 ℃ with 5% CO ₂ Was incubated for 30 minutes in the incubator (1). Then 5. mu.L/well of stimulator IL-2 was added to a final concentration of 1ng/mL and incubated for 30 minutes. Compound final concentrations were started at 3 μ M, 3-fold dilutions. Add 5. mu.L/well of cell lysate and incubate for 10min at room temperature. The phosphorylation degree of STAT5 is detected by an AlphaLISA p-STAT5(Tyr694/699) detection kit of Perkin Elmer company, and finally, an AlphaLISA signal is read on an Envision microplate reader to calculate the inhibition rate and the median Inhibitory Concentration (IC) ₅₀ )。

The biological activity of the compounds of the invention is determined by the above assay, the IC determined ₅₀ The values are given in Table 4 below.

TABLE 4 inhibitory Activity of the Compounds of the invention on STAT5 phosphorylation in CTLL-2 cells

Example Compound numbering

IC ₅₀ (nM)

Number of example Compounds

IC ₅₀ (nM)

001	11.31	009	46.85
003	19.85	010	143.82
004	44.57	012	13.29
005	18.03	013	17.20
006	13.92	015	47.08
007	185.27	017	76.98
008	200.28	018	27.20

Test example 5: occupancy of BTK targets in mouse spleen

The experimental principle is as follows: this experiment was to evaluate the occupancy of BTK targets by compounds in mouse spleen. Frozen spleen samples were homogenized and then incubated with biotinylated probe compounds, and the BTK protein not occupied by the compound bound to the probe, whereas the BTK protein already occupied by the compound did not bind to the probe and was detected by ELISA, reflecting the occupancy of the BTK target by the compound.

An experimental instrument:

instrument for measuring the position of a moving object	Brand	Model number
Centrifugal machine	Eppendorf	5810
Centrifugal machine	Eppendorf	5430R
Tissue grinder	Beauty wall	LD48
Envision	Perkin Elmer	/

Experimental materials:

reagent	Brand	Goods number
Anti-BTK antibodies	Cell signal	8547S
Streptavidin-coated plate	R&D Systems	CP004
CNX-500	MCE	HY-100338
ELISA developing solution	R&D Systems	DY999
ELISA stop solution	R&D Systems	DY994
RIPA lysate	Sigma	R0278
Anti-rabbitIgG antibody	Cell signal	7074S
BCA Protein Assay kit	Pierce	23225

The experimental method comprises the following steps:

using C57BL/6N female mice, test compounds were formulated in 2% Tween 80/0.5% methocellulose solution for administration by intragastric administration at a dose of 10mg/kg, and spleens were removed and stored in dry ice at 0.5h or 24 h after administration. Frozen spleen samples were homogenized and assayed for protein concentration using the BCA kit. Spleen homogenates with the adjusted protein concentration were incubated with the probe compound CNX-500 for 1 hour at a final CNX-500 concentration of 1. mu.M. Then transfer 100. mu.L/well to streptavidin coated plate and incubate overnight. The supernatant was discarded and washed, and incubated for 2 hours with anti-BTK antibody. The supernatant was discarded and washed, and an Anti-rabbitIgG antibody labeled with HRP was added and incubated for 1 hour. The supernatant was discarded and washed, and then developed with a developing solution for 10 to 15 minutes, after the reaction was terminated, the absorbance value was read at a wavelength of 450nm using Envision. And calculating the occupation ratio, wherein the occupation ratio is calculated by the formula:

wherein:

signal _max Represents: the signal generated by the control sample after addition of the probe compound;

signal _min Represents: signal generated in the control sample without the probe compound;

signal _{Test compound} Represents: the signal generated upon addition of the probe compound to a sample of the test compound.

The results of the experiment are shown in Table 5.

TABLE 5 occupancy of BTK targets in mouse spleen by Compounds of the invention

Test example 6: inhibition of IL-2-induced STAT5 phosphorylation in mouse whole blood

The experimental principle is as follows: this experiment was performed to evaluate the effect of compounds on phosphorylation of STAT5, a downstream substrate of JAK 3. After oral administration, the mice are taken as whole blood, added with a stimulant IL-2 and incubated for 15 minutes, and the STAT5 phosphorylation level in lymphocytes is detected by a flow cytometry technology, so that the inhibition effect of the compound on JAK3 targets is reflected.

An experimental instrument:

instrument	Brand	Model number
Biological safety cabinet	ESCO	CLASSⅡ BSC
Centrifugal machine	Eppendorf	5810
CO 2 incubator	ESCO	CCL-170B-8
Flow cytometer	BD Biosciences	Canto II
Refrigerator at-4 DEG C	Haier	HYC-650

Experimental materials:

reagent	Brand	Goods number
AF647 Mouse Anti-pStat5 antibody	BD Biosciences	562076
BV421 anti-mouse CD8 antibody	Biolegend	100738
BV421 Anti-Mouse CD3 antibody	BD Biosciences	740014
FITC Anti-Mouse CD4 antibody	BD Biosciences	553047
IL-2	R&D	402-ML-020
Mouse Fc blocking antibodies	Biolegend	156603
Lysis stationary liquid	BD Biosciences	558049
Cell staining solution	Biolegend	420201
96-well plate	Corning	3799
Perm Buffer III	BD Biosciences	558050

96-hole deep hole plate

Axygen

P-96-450V-C

The experimental method comprises the following steps:

using C57BL/6N female mice, test compounds were formulated in 2% Tween 80/0.5% methylcellulose solution and administered by gavage at a dose of 10mg/kg, and whole blood was placed in heparin sodium anticoagulation tube after 0.5 hour of administration. The whole blood is seeded into a 96-well plate at 80 mu L/well, the mouse Fc blocking antibody is added, then 5 mu L/well of the detection antibody is added, and the detection antibody is CD8 antibody or CD3 antibody/CD 4 antibody mixed liquor in different batches of experiments. Add 10. mu.L/well stimulator IL-2 and incubate for 15min at a final stimulator concentration of 200 ng/mL. Whole blood was transferred to a 96-well deep-well plate at 60. mu.L/well and incubated for 10 minutes with addition of 350. mu.L/well of lysis fixative. After centrifugation to remove supernatant, cells were resuspended in 100. mu.L/well Perm Buffer III and incubated for 30 minutes. The supernatant was discarded by centrifugation, 50. mu.L/well of pSTAT5 antibody was added, and the mixture was incubated for 30 minutes. After centrifugation and removal of the supernatant, the cells were resuspended in a staining solution, the signal of pSTAT5 was detected by flow cytometry, and the inhibition rate was calculated. The inhibition rate calculation formula is as follows:

wherein:

signal _max Represents: the signal generated by the control group sample after adding the stimulant IL-2;

signal _min Represents: the control group samples generate signals after adding no stimulant IL-2;

signal _{Test compound} Represents: the signal generated upon addition of the stimulator IL-2 to a sample of the test compound.

The results of the experiment are shown in Table 6.

TABLE 6 inhibition of IL-2 induced STAT5 phosphorylation in mouse whole blood by compounds of the invention

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

A compound of formula (Ib):

x is selected from O or NH;

R ¹ selected from H or optionally substituted by R ^a Substituted of the following groups: c ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₄ Cycloalkyl, 3-14 membered heterocyclyl, C ₆ -C ₁₀ Aryl or 5-10 membered heteroaryl;

R ^a selected from F, Cl, Br, I, OH, CN, ═ O, NO ₂ Or optionally substituted with R ^b Substituted of the following groups: NH (NH) ₂ 、SH、S(O)NH ₂ 、S(O)(C ₁ -C ₁₀ Alkyl), S (O) ₂ (C ₁ -C ₁₀ Alkyl), P (O) (C) ₁ -C ₁₀ Alkyl group), C ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₄ Cycloalkyl, 3-14 membered heterocyclyl, C ₁ -C ₁₀ Alkoxy radical, C ₃ -C ₁₄ Cycloalkyloxy, 3-14 membered heterocyclyloxy, C ₂ -C ₁₀ Alkenyl radical, C ₂ -C ₁₀ Alkynyl, C ₆ -C ₁₀ Aryl, 5-10 membered heteroaryl, C ₆ -C ₁₀ Aryloxy or 5-10 membered heteroaryloxy;

R ^b selected from F, Cl, Br, I, OH, CN, O, NO ₂ 、NH ₂ 、SH、C ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₄ Cycloalkyl, 3-14 membered heterocyclyl, C ₁ -C ₁₀ Alkoxy radical, C ₃ -C ₁₄ Cycloalkyloxy, 3-14 membered heterocyclyloxy, C ₂ -C ₁₀ Alkenyl radical, C ₂ -C ₁₀ Alkynyl, C ₆ -C ₁₀ Aryl, 5-10 membered heteroaryl, C ₆ -C ₁₀ Aryloxy or 5-10 membered heteroaryloxy;

R ² selected from H, F, Cl, Br, I, C ₃ -C ₁₄ Cycloalkyl or phenyl, said C ₃ -C ₁₄ Cycloalkyl or phenyl optionally substituted by R ^d Substitution;

R ^d selected from F, Cl, Br, I, OH, CN or C optionally substituted by a group selected from F, Cl, Br, I, OH ₁ -C ₄ An alkyl group;

R ³ selected from H, F, Cl, Br, I or C optionally substituted by a group selected from F, Cl, Br, I, OH ₁ -C ₁₀ An alkyl group;

R ⁴ selected from H, F, Cl, Br,I. OH, CN or optionally substituted R ^c Substituted of the following groups: c ₁ -C ₁₀ Alkyl radical, C ₁ -C ₁₀ An alkoxy group;

R ⁵ 、R ⁶ 、R ⁷ independently selected from H, F, Cl, Br, I, CN or optionally substituted by R ^e Substituted of the following groups: c ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₀ Cycloalkyl or 3-10 membered heterocyclyl;

R ^c 、R ^e independently selected from F, Cl, Br, I, OH;

n is selected from 0 or 1;

m is selected from 1 or 2;

with the following conditions: when R is ² When Cl, X is O, n is 1 and R is ¹ Is not methyl or cyclopropyl; when R is ² When H, X is O and R ¹ Is not ethyl, CH ₂ CHF ₂ 、CH ₂ CH ₂ OH, cyclopropyl,
when R is ² When F is equal to R ¹ Not being ethyl or CH ₂ CHF ₂ 。
The compound of formula (Ib), or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein X is selected from NH.
The compound of formula (Ib), or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein R is ¹ Selected from H or optionally substituted by R ^a Substituted of the following groups: c ₁ -C ₁₀ Alkyl radical, C ₃ -C ₁₀ Cycloalkyl, 3-10 membered heterocyclyl, C ₆ -C ₁₀ Aryl or 5-10 membered heteroaryl.
The compound of formula (Ib), or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein R is ¹ Is selected from C ₁ -C ₁₀ Alkyl or 3-10 membered heterocyclic group, said C ₁ -C ₁₀ Alkyl or 3-10 membered heterocyclyl is optionally substituted by R ^a And (4) substitution.
The compound of formula (Ib), or a pharmaceutically acceptable salt thereof, according to claim 1, wherein R is ² Is selected from C ₃ -C ₁₄ Cycloalkyl or phenyl, said C ₃ -C ₁₄ Cycloalkyl or phenyl optionally substituted by R ^d And (4) substitution.
The compound of formula (Ib), or a pharmaceutically acceptable salt thereof, according to claim 1, wherein R is ² Selected from cyclopropyl or phenyl, said cyclopropyl or phenyl being optionally substituted by R ^d And (4) substitution.
The compound of formula (Ib), or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein R is ² Selected from H, F, Cl, Br or I.
The compound of formula (Ib), or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is selected from a compound of formula (Ia), or a pharmaceutically acceptable salt thereof:

wherein: x, R ¹ 、R ² 、R ³ 、R ⁴ And n is as defined in claim 1.
The compound of formula (Ib), or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is selected from a compound of formula (I):

wherein R is ¹ 、R ² 、R ³ As defined in claim 1.
The compound of formula (Ib), or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein the compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is selected from a compound of formula (IIb), or a pharmaceutically acceptable salt thereof:

wherein: r ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ 、R ⁷ X, m and n are as defined in claim 1.
The compound of formula (Ib), or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is selected from a compound of formula (IIa), or a pharmaceutically acceptable salt thereof:

wherein, X, R ¹ 、R ² 、R ³ 、R ⁴ And n is as defined in claim 1.
The compound of formula (Ib), or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is selected from a compound of formula (II):

wherein R is ¹ 、R ² 、R ³ As defined in claim 1.
The compound of formula (Ib), or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the compound is selected from one of the following structures:
a pharmaceutical composition comprising a compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.
Use of a compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention or treatment of a disease associated with Janus kinase (JAK) and/or Bruton's Tyrosine Kinase (BTK).
The use according to claim 15, wherein the Janus kinase (JAK) and/or Bruton's Tyrosine Kinase (BTK) -associated disease is a tumor or an autoimmune disease.