WO2021147952A1

WO2021147952A1 - Pyrimidopyrrole compound

Info

Publication number: WO2021147952A1
Application number: PCT/CN2021/073081
Authority: WO
Inventors: 张国宝; 陈家隽; 周峰; 蒋蕾; 唐锋; 唐任宏; 任晋生
Original assignee: 江苏先声药业有限公司
Priority date: 2020-01-21
Filing date: 2021-01-21
Publication date: 2021-07-29
Also published as: CN115023428A

Abstract

Provided in the present application is a pyrimido five-membered ring compound represented by formula (Ib) or a pharmaceutically acceptable salt thereof, a pharmaceutical composition and a preparation method therefor, and a use thereof as a JAK3 and/or BTK inhibitor.

Description

Pyrimidopyrrole compounds

This application requires a patent application filed with the State Intellectual Property Office of China on January 21, 2020, the patent application number is 202010071941.X, the title of the invention is "Pyrimidopyrrole compounds" and the filed with the State Intellectual Property Office of China on September 22, 2020, The patent application number is 202011003941.2, and the invention title is "pyrimidopyrrole compounds" two prior applications. The full texts of the two applications are incorporated into this application by reference.

Technical field

The present invention relates to a novel pyrimidopyrrole compound or pharmaceutically acceptable salt, a pharmaceutical composition containing them, and its use in the prevention or treatment of kinase-related diseases such as Janus kinase (JAK, especially JAK3) and/or Bruce The use of tyrosine kinase (BTK) related diseases.

Background technique

Autoimmune disease is a type of disease that is caused by abnormal immune function to attack one's own cells or tissues, leading to inflammation and tissue damage, including rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and systemic lupus erythematosus (SLE) Wait. BTK and JAK3 are two important targets for autoimmune diseases.

BTK is a member of the TEC family of non-receptor tyrosine kinases, and its structure includes PH domain, TH domain, SH3 domain, SH2 domain and SH1 domain. BTK plays a key role in the activation of the B cell antigen receptor (BCR) signaling pathway, regulates the development and activation of B cells, and plays an important role in the proliferation of B cells, the expression of pro-inflammatory cytokines, and the secretion of antibodies. (Targeting Bruton's tyrosine kind in B cell malignancies. Nat Rev Cancer. 2014 Apr; 14(4): 219-32), so BTK has become one of the important targets for the treatment of diseases related to abnormal B cell activation, including autoimmune diseases And B-cell lymphoma. Ibrutinib, Acalabrutinib and Zanubrutinib are three approved BTK inhibitors, which mainly treat B-cell lymphoma. They have obvious effects in some patients. However, serious side effects and drug-resistant mutations have also been observed clinically. In 2017, ibrutinib was approved by the US FDA for the treatment of graft-versus-host disease (GVHD), while other BTK inhibitors are currently being actively explored clinically to treat 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 transducer and activator of transcription (STAT) are the downstream substrates of JAK3. JAK3 activates STAT to make it a dimer into the nucleus and regulate the transcription and expression of specific genes. The JAK-STAT signaling pathway plays an important role in the proliferation and differentiation of lymphocytes, as well as the expression of pro-inflammatory cytokines (JAK inhibition as a therapeutic strategy for immune and inflammatory diseases. Nat Rev Drug Discov. 2017 December 28; 17(1): 78; The JAK-STAT Pathway: Impact on Human Disease and Therapeutic Intervention. Annual Review of Medicine. Vol. 66:311-328), therefore JAK3 has become one of the targets of autoimmune diseases and malignant tumors. Tofacitinib is a JAK3 inhibitor approved by the FDA, which has shown good clinical efficacy in RA and IBD. However, there are also certain adverse reactions, including serious infections, liver damage, etc., which are considered to be related to Tofacitinib's insufficient selectivity for JAK1/2 (JAK inhibition as a therapeutic strategy for immune and inflammatory diseases. Nat Rev Drug Discov. 2017 December 28 ;17(1):78;JAK-inhibitors.New players in the field of immune-mediated diseases, beyond rheumatoid arthritis.Rheumatology(Oxford).2019 February 1;58(Suppl 1):i43-i54).

In addition to the separate clinical effects of BTK and JAK3 inhibitors, while inhibiting the BTK/JAK3 signaling pathway will show a synergistic effect. Several studies have shown that in the collagen-induced rat arthritis model (CIA), both BTK and JAK are inhibited at the same time. It is observed that joint swelling is significantly relieved, the number of osteoclasts is reduced, and the pathological score is also significantly improved. The curative effect is better than that of single drug. Function (2016 ACR/ARHP Annual Meeting. Abstract 484; 2013 ACR/ARHP Annual Meeting. Abstract 2353). Abbvie Company launched ABBV599 (a combination of BTK inhibitor and JAK inhibitor) for RA and SLE in Phase II clinical trials in September 2018 and June 2019, respectively. Another dual-target inhibitor of BTK/JAK3, DWP212525, also demonstrated disease relief and joint protection in the mouse CIA model (2019 ACR/ARHP Annual Meeting. Abstract 965).

In view of the huge autoimmune disease market and the unmet market demand, based on the functions of BTK and JAK3 in autoimmune diseases and the existing clinical effects, it is necessary to develop good activity against BTK and JAK3, with good selectivity and toxicity. A dual-target small molecule inhibitor with low side effects.

Summary of the invention

The present invention provides a compound represented by formula (Ib) or a pharmaceutically acceptable salt thereof:

in:

X is selected from O or NH;

R ¹ is selected H, or R ^a is optionally substituted by the following groups: C ₁ -C ₁₀ alkyl, C ₃ -C ₁₄ cycloalkyl, 3-14 membered heterocyclyl, C ₆ -C ₁₀ aryl group or 5-10 membered heteroaryl;

R ^{a is} selected from F, Cl, Br, I, OH, CN, =O, NO ₂ or the following groups optionally substituted by R ^b _{: 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, C ₃ -C ₁₄ cycloalkyl, 3-14 membered heterocyclyl, C ₁ -C ₁₀ alkoxy, C ₃ -C ₁₄ cycloalkyloxy, 3-14 membered heterocyclyloxy, C ₂ -C ₁₀ alkenyl , 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 is} selected from F, Cl, Br, I, OH, CN, =O, NO ₂ , NH ₂ , SH, C ₁ -C ₁₀ alkyl, C ₃ -C ₁₄ cycloalkyl, 3-14 membered heterocycle Group, C ₁ -C ₁₀ alkoxy, C ₃ -C ₁₄ cycloalkyloxy, 3-14 membered heterocyclyloxy, C ₂ -C ₁₀ alkenyl, 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 is} selected from H, F, Cl, Br, I, C ₃ -C ₁₄ cycloalkyl or phenyl, the C ₃ -C ₁₄ cycloalkyl or phenyl is optionally substituted ^{by R d;}

R ^{d is} selected from F, Cl, Br, I, OH, CN or a C ₁ -C ₄ alkyl group optionally substituted with a group selected from F, Cl, Br, I, OH;

R ^{3 is} _{selected from H, F, Cl, Br, I or a C 1} -C ₁₀ alkyl group optionally substituted with a group selected from F, Cl, Br, I, OH;

R ^{4 is} selected from H, F, Cl, Br, I, OH, CN or the following groups ^{optionally substituted by R c} _{: C 1} -C ₁₀ alkyl, C ₁ -C ₁₀ alkoxy;

R ⁵ , R ⁶ , and R ^{7 are} independently selected from H, F, Cl, Br, I, CN, or the following groups ^{optionally substituted by R e} _{: C 1} -C ₁₀ alkyl, C ₃ -C ₁₀ cycloalkane Group or 3-10 membered heterocyclic group;

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

n is selected from 0 or 1;

m is selected from 1 or 2;

The conditions are: when R ² =Cl, X=O, n=1 and R ^{1 is} not methyl or cyclopropyl; when R ² =H, X=O and R ^{1 is} not ethyl, CH ₂ CHF _2. CH ₂ CH ₂ OH, cyclopropyl,

When R ² =F, R ^{1 is} not 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, which C ₃ -C ₁₄ cycloalkyl or phenyl is optionally substituted ^{by R d.}

In some embodiments, R ^{2 is} selected from C ₃ -C ₁₀ cycloalkyl or phenyl, which C ₃ -C ₁₀ cycloalkyl or phenyl is optionally substituted ^{by R d.}

In some embodiments, R ^{2 is} selected from C ₃ -C ₆ cycloalkyl or phenyl, which C ₃ -C ₆ cycloalkyl or phenyl is optionally substituted ^{by R d.}

In some embodiments, R ^{2 is} selected from cyclopropyl or phenyl, which cyclopropyl or phenyl is optionally substituted with ^Rd.

In some embodiments, R ^{2 is} selected from C ₃ -C ₆ cycloalkyl or phenyl.

In some embodiments, R ^{2 is} selected from cyclopropyl or phenyl.

In some embodiments, R ^{2 is} selected from H, F, Cl, Br, or I.

In some embodiments, R ^{2 is} selected from H, F, or Cl.

In some embodiments, R ^{3 is} _{selected from H, F, Cl, Br, I, or a C 1} -C ₆ alkyl group optionally substituted with a group selected from F, Cl, Br, I, OH.

In some embodiments, R ^{3 is} _{selected from H, F, Cl, Br, I, or a C 1} -C ₄ alkyl group optionally substituted with a group selected from F, Cl, Br, I, OH.

In some embodiments, R ^{3 is} selected from H, F, Cl, Br, or I.

In some embodiments, R ^{3 is} selected from H or F.

In some embodiments, R ^{1 is} selected from H or the following groups ^{optionally substituted with Ra} _{: C 1} -C ₁₀ alkyl, C ₃ -C ₁₀ cycloalkyl, 3-10 membered heterocyclyl, C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl.

In some embodiments, R ¹ is selected from C ₁ -C ₁₀ alkyl group or a 3-10 membered heterocyclic group, the C ₁ -C ₁₀ alkyl, or 3-10 membered heterocyclyl optionally substituted with R ^a.

In some embodiments, R ¹ is selected from C ₁ -C ₆ alkyl, or 4-6 membered heterocyclyl group, a C ₁ -C ₆ alkyl, or 4-6 membered heterocyclyl optionally substituted with R ^a.

In some embodiments, R ^{1 is} selected from a C ₁ -C ₆ alkyl group or a 4-6 membered heterocyclic group, the 4-6 membered heterocyclic group contains an O atom and/or a N atom, and the C ₁ -C ₆ alkyl, or 4-6 membered heterocyclyl optionally substituted with R ^a.

In some embodiments, R ^{1 is} selected from a C ₁ -C ₆ alkyl group or a 4-6 membered heterocyclic group, the 4-6 membered heterocyclic group contains O and/or N as ring atoms, and the C _1- C ₆ alkyl, or 4-6 membered heterocyclyl optionally substituted with R ^a.

In some embodiments, R ^{1 is} selected from a C ₁ -C ₆ alkyl group or a 4-6 membered heterocyclic group, the 4-6 membered heterocyclic group contains one O atom or one N atom, and the C ₁ -C ₆ alkyl, or 4-6 membered heterocyclyl optionally substituted with R ^a.

In some embodiments, R ^{1 is} selected from a C ₁ -C ₆ alkyl group or a 4-6 membered heterocyclic group, the 4-6 membered heterocyclic group contains one O or one N as a ring atom, and the C _1- C ₆ alkyl, or 4-6 membered heterocyclyl optionally substituted with R ^a.

In some embodiments, R ^a is selected from F, Cl, Br, I, OH, CN, = O, NO 2 , or optionally substituted R ^b group consisting _{_{of: NH 2, SH, C 1}} -C 10 alkyl Group, C ₃ -C ₁₀ cycloalkyl, 3-10 membered heterocyclyl, C ₁ -C ₁₀ alkoxy, C ₃ -C ₁₀ cycloalkyloxy, 3-10 membered heterocyclyloxy, C ₂ -C ₁₀ alkenyl, 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 is} selected from F, Cl, Br, I, OH, CN, =0, NO ₂ , NH ₂ , SH, C ₁ -C ₁₀ alkyl, C ₃ -C ₁₀ cycloalkyl, 3-10 membered heterocyclyl, C ₁ -C ₁₀ alkoxy, C ₃ -C ₁₀ cycloalkyloxy, 3-10 membered heterocyclyloxy, C ₂ -C ₁₀ alkenyl, 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 F, Cl, Br, I, OH, CN, C 1 -C 10 alkyl or C ₃ -C ₁₀ cycloalkyl.

In some embodiments, R ^a is selected from F, Cl, Br, I or C ₁ -C ₆ alkyl.

In some embodiments, R ^a is selected from F or C ₁ -C ₆ alkyl.

In some embodiments, R ^a is selected from C ₁ -C ₆ alkyl.

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

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

In some embodiments, R ^a is selected from methyl.

In some embodiments, R ^{1 is} selected from methyl, trifluoromethyl, ethyl, oxetanyl, tetrahydrofuranyl, N-methylpyrrolidinyl, tetrahydropyranyl, or N-methylpiperidine base.

In some embodiments, R ^{1 is} selected from methyl, ethyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, or N-methylpiperidinyl.

In some embodiments, R ^{1 is} selected from ethyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, or N-methylpiperidinyl.

In some embodiments, R ^{1 is} selected from methyl, trifluoromethyl, ethyl,

In some embodiments, R ^{1 is} selected from methyl, ethyl,

In some embodiments, R ^{1 is} selected from ethyl,

In some embodiments, R ^{4 is} selected from H, F, Cl, Br, I, OH, CN, or the following groups ^{optionally substituted with R c} _{: C 1} -C ₁₀ alkyl.

In some embodiments, R ^{4 is} selected from H, F, Cl, Br, I, OH, CN, or C ₁ -C ₆ alkyl.

In some embodiments, R ^{4 is} selected from H or C ₁ -C ₆ alkyl.

In some embodiments, R ^{4 is} selected from H or methyl.

In some embodiments, R ^{5 is} selected from H, F, Cl, Br, I, CN, or _{C 1} -C ₁₀ alkyl optionally substituted with ^{R e.}

In some embodiments, R ^{5 is} selected from H, F, Cl, Br, I, CN.

In some embodiments, R ^{5 is} selected from H or CN.

In some embodiments, R ⁶ and R ^{7 are} independently selected from H, F, Cl, Br, I, CN, or C ₁ -C ₁₀ alkyl ^{optionally substituted by R e.}

In some embodiments, R ⁶ and R ^{7 are} independently selected from H, CN, or _{C 1} -C ₁₀ alkyl optionally substituted with ^{R e.}

In some embodiments, R ⁶ and R ^{7 are} independently selected from H, CN, or C ₁ -C ₆ alkyl.

In some embodiments, R ⁶ and R ^{7 are} independently selected from H or C ₁ -C ₆ alkyl.

In some embodiments, R ⁶ and R ^{7 are} independently selected from H or tert-butyl.

In some embodiments, the n is selected from 1.

In some embodiments, the n is selected from zero.

In some embodiments, the m is selected from 1.

In some embodiments, the 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:

in:

X is selected from O or NH;

R ^{2 is} selected from C ₃ -C ₁₄ cycloalkyl or phenyl, the C ₃ -C ₁₄ cycloalkyl or phenyl is optionally substituted ^{by R d;}

R ^{c is} 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 is} selected from C ₃ -C ₆ cycloalkyl or phenyl.

In some embodiments, R ^{2 is} selected from cyclopropyl or phenyl.

In some embodiments, R ^{3 is} selected from H, F, Cl, Br, or I.

In some embodiments, R ^{3 is} selected from H or F.

In some embodiments, R ^a is selected from C ₁ -C ₆ alkyl.

In some embodiments, R ^a is selected from methyl. In some embodiments, R ^{1 is} selected from methyl, ethyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, or N-methylpiperidinyl.

In some embodiments, R ^{1 is} selected from methyl, ethyl,

In some embodiments, R ^{1 is} selected from ethyl,

In some embodiments, R ^{4 is} selected from H or C ₁ -C ₆ alkyl.

In some embodiments, R ^{4 is} selected from H or methyl.

In some embodiments, the n is selected from 1.

In some embodiments, the n is selected from zero. 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 , and R ³ 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 (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 are 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 ¹ , R ² and R ³ are as defined above.

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

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

The present invention also provides a pharmaceutical composition, which comprises a compound represented by formula (Ib) or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable excipients.

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

Further, the present invention relates to a compound represented by formula (Ib) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for preparing prevention or treatment of Janus kinase (JAK, especially JAK3) and/or Bruton's tyrosine The use of kinases (BTK) related diseases in medicine.

Further, the present invention relates to a compound represented by formula (Ib) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the prevention or treatment of Janus kinase (JAK, especially JAK3) and/or Bruton's tyrosine kinase (BTK) Use in related diseases.

Further, the present invention relates to a compound of formula (Ib) or a pharmaceutically acceptable salt thereof for preventing or treating diseases related to Janus kinase (JAK, especially JAK3) and/or Bruton's tyrosine kinase (BTK), or Pharmaceutical composition.

The present invention also relates to a method for treating diseases related to Janus kinase (JAK, particularly JAK3) and/or Bruton's tyrosine kinase (BTK), which method comprises administering to a patient a therapeutically effective dose containing the compound of the present invention A pharmaceutical preparation of a compound of formula (Ib) or a pharmaceutically acceptable salt thereof.

In the preferred embodiment of the present invention, the diseases related to Janus kinase (JAK, especially JAK3) and/or Bruton's tyrosine kinase (BTK) include but are not limited to tumors (such as B-cell lymphoma) and autoimmunity Diseases (such as rheumatoid arthritis, inflammatory bowel disease and systemic lupus erythematosus) and so on.

Definition and description of terms

Unless otherwise stated, the definitions of groups and terms described in the specification and claims of the present invention include definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples. Etc., can be combined and combined with each other arbitrarily. Such combination and the group definition and compound structure after the combination should fall within the scope of the description of the present invention.

In the present invention

The place indicates the 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, and organic acids and bases.

The term "stereoisomer" refers to the isomers produced by the different arrangements of atoms in the molecule in space, including cis and trans isomers, enantiomers, diastereomers and conformational isomers.

The compound of the present invention may have an asymmetric carbon atom (optical center) or a double bond. Racemates, enantiomers, diastereomers, geometric isomers and individual isomers are all included in the scope of the present invention.

The schematic diagram of the racemate or enantiomerically pure compound herein is from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise specified, use wedge keys and virtual wedge keys

Represents the absolute configuration of a three-dimensional center, with black real and virtual keys

Represents the cis-trans configuration of an alicyclic compound. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, they include E and Z geometric isomers. Likewise, all tautomeric forms are included within the scope of the present 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 Conformers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to Within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present invention. The asymmetric atom-containing compound of the present application can be isolated in an optically pure form or a racemic form. The optically active pure form can be resolved from the racemic mixture or synthesized by using chiral raw materials or chiral reagents.

The term "tautomer" refers to an isomer of a functional group resulting from the rapid movement of an atom in a molecule at two positions. The compounds of the present invention may exhibit tautomerism. Tautomeric compounds can exist in two or more mutually convertible species. Proton shift tautomers result from the migration of covalently bonded hydrogen atoms between two atoms. Tautomers generally exist in an equilibrium form, and an attempt to separate a single tautomer usually produces a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties of the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the ketone type is dominant; in phenol, the enol type is dominant. The present invention encompasses all tautomeric forms of the compound.

The term "pharmaceutical composition" means a mixture of one or more of the compounds described in the text or their physiologically/pharmaceutically acceptable salts or prodrugs and 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 the organism.

The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence of the specific atom is normal and the substituted compound is stable. When the substituent is oxo (ie =O), it means that two hydrogen atoms are replaced, and the oxo will not occur on the aromatic group.

The term "optional" or "optionally" means that the event or situation described later can occur or not occur, and the description includes occurrence of said event or situation and non-occurrence of said event or situation. For example, the ethyl group is "optionally" substituted by halogen, meaning that the ethyl group can be unsubstituted (CH ₂ CH ₃ ), monosubstituted (such as CH ₂ CH ₂ F), or polysubstituted (such as CHFCH ₂ F, CH ₂ CHF ₂ etc.) or completely substituted (CF ₂ CF ₃ ). Those skilled in the art can understand that for any group containing one or more substituents, any substitution or substitution pattern that is impossible to exist in space and/or cannot be synthesized will not be introduced.

When any variable (such as R) occurs more than once 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" should be understood to mean a linear or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl Group, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.; "C ₁ -C ₆ alkyl" It should be understood to mean a linear or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, and 6 carbon atoms. "C ₁ -C ₄ alkyl" should be understood to mean a linear or branched saturated monovalent hydrocarbon group having 1, 2, 3, or 4 carbon atoms. The term "alkoxy" can be understood as "alkyloxy" or "alkyl-O", preferably, "C ₁ -C ₁₀ alkoxy" may include "C ₁ -C ₆ alkoxy" and " C ₁ -C ₄ alkoxy".

The term "C ₂ -C ₁₀ alkenyl" should be understood to preferably mean a linear or branched monovalent hydrocarbon group, 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", more preferably "C ₂ -C ₄ alkenyl", still more preferably C ₂ or C ₃ alkenyl. It should be understood that where the alkenyl group contains more than one double bond, the double bonds may be separated from each other or conjugated. The alkenyl groups such as vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-butanyl -2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2 -Alkenyl, 2-methylprop-1-enyl, (E)-1-methylprop-1-enyl, (Z)-1-methylprop-1-enyl.

The term "C ₂ -C ₁₀ alkynyl" should be understood to mean a linear or branched monovalent hydrocarbon group containing one or more triple bonds and having 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms, "C ₂ -C ₆ alkynyl" is preferred, "C ₂ -C ₄ alkynyl" is more preferred, and C ₂ or C ₃ alkynyl is still more preferred. The alkynyl group is, for example, ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3-ynyl, 1-methylprop- 2-alkynyl.

The term "C ₃ -C ₁₄ cycloalkyl" should be understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring, which has 3 to 14 carbon atoms. The term "C ₃ -C ₁₀ cycloalkyl" should be understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring, which has 3 to 10 carbon atoms. The term "C ₃ -C ₆ cycloalkyl" should be 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 ring. According to the present invention, the bicyclic hydrocarbon ring includes a bridged ring, a spiro ring or a parallel ring structure.

The term "cycloalkyloxy" can be understood as "cycloalkyl-O", preferably, "C ₃ -C ₁₄ cycloalkyloxy" may include "C ₃ -C ₁₀ cycloalkyloxy" and " C ₃ -C ₆ cycloalkyloxy".

The term "3-14 membered heterocyclic group" shall be understood as a saturated or partially saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3-14 ring atoms, which contains 1-5, preferably 1-3 selected from Heteroatoms of N, O and S. The term "3-10 membered heterocyclic group" means a saturated or partially saturated monovalent monocyclic or bicyclic hydrocarbon ring containing 1-5, preferably 1-3 heteroatoms selected from N, O and S. The term "4-6 membered heterocyclyl" should be understood as a saturated or partially saturated monovalent or bicyclic hydrocarbon ring with 4, 5, 6 ring atoms, which contains 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 ring, such as azetidinyl, oxetanyl; 5-membered ring, such as tetrahydrofuranyl, dioxolyl, pyrrole Alkyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or 6-membered ring, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazinyl Or trithiaalkyl; or partially saturated 6-membered ring such as tetrahydropyridyl; or 7-membered ring such as diazacycloheptanyl. Optionally, the heterocyclic group may be benzo-fused. The heterocyclic group may be bicyclic, such as but not limited to a 5, 5-membered ring, such as hexahydrocyclopenta[c]pyrrole-2(1H)-yl ring, or a 5, 6-membered bicyclic ring, such as hexahydropyrrole And [1,2-a]pyrazine-2(1H)-yl ring. The ring containing the nitrogen atom may be partially unsaturated, that is, it may contain one or more double bonds, such as but not limited to 2,5-dihydro-1H-pyrrolyl, 4H-[1,3,4]thiadi Azinyl, 4,5-dihydrooxazolyl or 4H-[1,4]thiazinyl, or it may be benzo-fused, such as but not limited to dihydroisoquinolinyl. According to the present invention, the heterocyclic group is non-aromatic. The bicyclic hydrocarbon ring includes a bridged ring, a spiro ring or a parallel ring structure.

The term "3-14 membered heterocyclyloxy" can be understood as "3-14 membered heterocyclyl-O", preferably, "3-14 membered heterocyclyloxy" may include "3-10 membered heterocyclic基oxy".

The term "C 6 -C 10 aryl" should be understood to preferably mean a monovalent or partially aromatic monocyclic or bicyclic hydrocarbon ring 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"), such as indanyl or indenyl, or having 10 A ring of three carbon atoms ("C ₁₀ aryl"), such as tetrahydronaphthyl, dihydronaphthyl, or naphthyl.

The term "C ₆ -C ₁₀ aryloxy" can be understood as "C ₆ -C ₁₀ aryl-O".

"5-10 membered heteroaryl" should be understood to include monovalent monocyclic, bicyclic or tricyclic aromatic ring systems having 5, 6, 7, 8, 9, 10 ring atoms, especially 5 or 6 or 9 or 10 ring atoms, and it contains 1 to 5, preferably 1 to 3 heteroatoms independently selected from N, O and S and, in addition, may be benzo-fused in each case. In particular, the heteroaryl group is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiol Diazolyl, etc. and their benzo derivatives, such as benzofuranyl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazole Group, indazolyl, indolyl, isoindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc., and their benzo derivatives, such as quinolinyl, quinoline Azolinyl, isoquinolinyl, etc.; or azepine, indazinyl, purinyl, etc. and their benzo derivatives; or cinolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, Naphthyridinyl, pterridinyl, etc.

The term "5-10 membered heteroaryloxy" can be understood as "5-10 membered heteroaryl-O".

The present application also includes compounds of the present application that are the same as those described herein, but have one or more atoms replaced by an isotope-labeled atom having an atomic weight or mass number different from the atomic weight or mass number commonly found in nature. Examples of isotopes that can be bound to the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 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, etc.

Certain isotope-labeled compounds of the application (such as ^{those labeled with 3} H and ¹⁴ C) can be used in compound and/or substrate tissue distribution analysis. Tritiated (ie ³ H) and carbon-14 (ie ¹⁴ C) isotopes are especially preferred due to their ease of preparation and detectability. Positron emission 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. Generally, the isotopically-labeled compounds of the present application can be prepared by the following procedures similar to those disclosed in the schemes and/or examples below, by replacing non-isotopically-labeled reagents with isotope-labeled reagents.

In addition, substitution with heavier isotopes (such as deuterium (ie ² H)) can provide certain therapeutic advantages resulting from higher metabolic stability (for example, increased in vivo half-life or reduced dosage requirements), and therefore in certain situations The following may be preferred, where the deuterium substitution can be partial or complete, and partial deuterium substitution refers to the substitution of at least one hydrogen by at least one deuterium.

All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.

The term "treatment" means administering the compound or formulation described in this application 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 disease state in mammals, especially when such mammals are susceptible to the disease state, but have not been diagnosed as having the disease state;

(ii) Suppress the disease or disease state, that is, curb its development;

(iii) Alleviation of the disease or disease state, even if the disease or disease state subsides.

The term "therapeutically effective amount" means (i) treatment or prevention of a particular disease, condition or disorder, (ii) reduction, amelioration or elimination of one or more symptoms of a particular disease, condition or disorder, or (iii) prevention or delay The amount of the compound of the present invention for 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 it can be routinely determined by those skilled in the art. Determined by its own knowledge and this disclosure.

The term "excipients" refers to pharmaceutically acceptable inert ingredients. Examples of types of the term "excipient" include, without limitation, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients can enhance the handling characteristics of the pharmaceutical preparation, that is, make the preparation more suitable for direct compression by increasing fluidity and/or adhesion. Examples of typical "pharmaceutically acceptable carriers" suitable for the above formulations are: sugars, starches, cellulose and its derivatives and other auxiliary materials commonly used in pharmaceutical formulations.

The term "pharmaceutically acceptable excipients" refers to those excipients that have no obvious stimulating effect on the organism and will not damage the biological activity and performance of the active compound. Suitable auxiliary materials 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", "comprise" or "comprise" and their English variants such as comprises or comprising should be understood in an open, non-exclusive meaning, that is, "including but not limited to".

The pharmaceutical composition of the present application can be prepared by combining the compound of the present application with suitable pharmaceutically acceptable excipients, for example, can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, and powders. , Granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.

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

The pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing method, dissolution method, granulation method, sugar-coated pill method, grinding method, emulsification method, freeze-drying method, etc.

In some embodiments, the pharmaceutical composition is in an oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These auxiliary materials enable the compound of the present application to be formulated into tablets, pills, lozenges, sugar-coated agents, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.

The solid oral composition can be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: mixing the active compound with solid excipients, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain tablets Or the core of the dragee. Suitable excipients include but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like.

The pharmaceutical composition may also be suitable for parenteral administration, such as a sterile solution, suspension or lyophilized product in a suitable unit dosage form.

In all the methods of administration of the compound of general formula (Ib) described herein, the daily dose is 0.01 to 100 mg/kg body weight, preferably 0.05 to 50 mg/kg body weight, more preferably 0.1 to 30 mg/kg body weight, alone or The form of divided doses. The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.

The chemical reaction in the specific embodiment of the present invention is completed in a suitable solvent, and the solvent must be suitable for the chemical change of the present invention and the required reagents and materials. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction schemes on the basis of the existing embodiments.

Detailed ways

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

The solvent used in the present invention is commercially available. Commercially available compounds use supplier catalog names.

The structure of the compound is determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). The unit of NMR shift is 10 ^-6 (ppm). The solvents measured by NMR are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS); "IC ₅₀ "refers to the half inhibitory concentration, which refers to the half of the maximum inhibitory effect concentration. The following eluent can be formed by two or more solvents to form a mixed eluent, and the percentage is the volume ratio of each solvent. For example, "methanol/dichloromethane: 0-8%" means mixed elution during gradient elution. The volume dosage of methanol:dichloromethane in the reagent is 0:100-8:100.

The present invention uses the following acronyms:

Example 1 Preparation of Compound 001

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

Synthetic route and specific synthetic steps:

Step 1: Synthesis of 2,4-Dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (1b)

At room temperature 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) was dissolved in dichloromethane (100mL), and the reaction solution was stirred at 110°C for 16 hours until the reaction was complete. The mixture was poured into water and extracted with dichloromethane. The organic phases were combined and then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The crude product obtained was purified by silica gel column purification (EA:PE=0-20%) to obtain the title product 1b( 13.0g).

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

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

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

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

The third step: 2,4-Dichloro-5-cyclopropyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d] Synthesis of 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] palladium dichloride (176mg, 0.25mmol), silver oxide (286mg, 1.25mmol) and potassium phosphate (1.6g, 7.5mmol) were sequentially added to the dioxane (12mL) solution, and then stirred at 90°C for 2 hours under a nitrogen atmosphere until the reaction was complete. The mixture was filtered, the liquid phase was poured into water and extracted with ethyl acetate, the organic phases were combined and then dried and filtered with anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure, and the crude product obtained was purified by silica gel column (EA:PE=0-20%). The title product 1e (500 mg).

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

The fourth step: tert-butyl (R)-3-((2-chloro-5-cyclopropyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7hydro- Synthesis of 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 at room temperature 1e (480mg, 1.3mmol), (R)-3-aminopiperidine-1-carboxylic acid tert-butyl ester 1f (528mg, 2.6mmol) and N,N-diisopropylethylamine (521mg, 3.9mmol) dissolved In isopropanol (5 mL), the reaction solution was placed in a sealed tube and stirred at 110°C for 16 hours until the reaction was complete. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined and then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The crude product obtained was purified by silica gel column (EA:PE=0-20%) to obtain the title product 1g. (580mg).

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

The fifth step: tert-butyl (R)-3-((2-((1-ethyl-1H-pyrazol-4-yl)amino)-5-cyclopropyl-7-((2-(三Synthesis of (methylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidine-1 carboxylate (1i)

Add tert-butyl(R)-3-((2-chloro-5-cyclopropyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo [2,3-d]pyrimidin-4-yl)amino)piperidine-1 carboxylate 1g (560mg, 1.1mmol), 1-ethyl-1H-pyrazole-4-amine 1h (238mg, 2.2mmol) And trifluoroacetic acid (123mg, 1.1mmol) were dissolved in isopropanol (6mL), and the reaction solution was stirred at 110°C for 16 hours in a sealed tube until the reaction was complete. The reaction solution was _{adjusted to pH=9 with saturated NaHCO 3} solution, poured into water and extracted with ethyl acetate, the organic phases were combined and then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the crude product obtained was purified with a silica gel column (EA: PE=0-10%) to obtain the title product 1i (100mg).

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

The 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]pyrimidine-2,4-diamine (1j)

Add tert-butyl(R)-3-((2-((1-ethyl-1H-pyrazol-4-yl)amino)-5-cyclopropyl-7-((2-(trimethyl) at room temperature (Methylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidine-1 carboxylate 1i (100mg, 0.2mmol) dissolved in dichloromethane In a solution of methane (1 mL) and dioxane hydrochloride (1 mL, 4M), the reaction solution was stirred at room temperature for 2 hours until the reaction was complete. The reaction solution was _{adjusted to pH=9 with saturated NaHCO 3} solution, poured into water and extracted with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the title product 1j (80 mg, crude product).

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

The seventh step: (R)-1-(3-((5-cyclopropyl-2-((1-ethyl-1H-pyrazol-4-yl)amino)-7-((2-(三(Methylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)prop-2-en-1-one ( 1l) Synthesis

(R)-5-cyclopropyl-N2-(1-ethyl-1H-pyrazol-4-yl)-N4-(piperidin-3-yl)-7-((2-(三(Methylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine 1j (80mg, 0.2mmol) was dissolved in dichloromethane (2mL), Add triethylamine (64mg, 0.6mmol) and acryloyl chloride 1k (38mg, 0.4mmol) at -10°C. The reaction solution continued to react at -10°C for 30 minutes until the reaction was complete. The reaction solution was diluted with water (20 mL), extracted with dichloromethane (20 mL*2), and the organic phases were combined and spin-dried to obtain 11 (100 mg) of the title product.

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

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

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

The ninth step: (R)-1-(3-((5-cyclopropyl-2-((1-ethyl-1H-pyrazol-4-yl)amino)-7H-pyrrolo[2,3 -d) Synthesis of 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) and added Sodium hydroxide (2mL) solution, the reaction solution was stirred and reacted at room temperature for 1 hour until the reaction was complete. The reaction solution was diluted with water (20 mL), extracted with ethyl acetate (30 mL*2), the combined organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered and spin-dried to obtain the crude product. Purified by HPLC to obtain the title product 001 (15.82mg).

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

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

¹ H NMR (400MHz, DMSO-d ₆ ) δ 10.59 (d, J = 8.3 Hz, 1H), 8.43 (s, 1H), 7.85 (d, J = 13.2 Hz, 1H), 7.43 (d, J = 22.1Hz, 1H), 6.89 (d, J = 5.7 Hz, 1H), 6.45 (s, 1H), 6.07 (dd, J = 38.5, 16.3 Hz, 1H), 5.70 (ddd, J = 91.0, 51.4, 9.1 Hz, 2H), 4.30 (s, 1H), 4.03 (q, J = 7.2 Hz, 3H), 3.80 (d, J = 10.9 Hz, 1H), 3.68 (d, J = 14.3 Hz, 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 = 9.4 Hz, 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]pyrimidine- 4-yl)amino)piperidin-1-yl)prop-2-en-1-one

Synthetic route and specific synthetic steps:

The first step: 2,4-Dichloro-5-phenyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine (2c) Synthesis

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] palladium dichloride (112mg, 0.15mmol) and sodium carbonate (1.2 g, 11.4mmol) was added to the dioxane (20mL) and water (4mL) solution successively, and then stirred under nitrogen at 85°C for 2 hours until the reaction was complete. The mixture was filtered, the liquid phase was poured into water and extracted with ethyl acetate, the organic phases were combined and then dried and filtered with anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure, and the crude product obtained was purified by silica gel column (EA:PE=0-20%). The title product 2c (900 mg).

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

The second step: tert-butyl (R)-3-((2-chloro-5-phenyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7hydro-pyrrole Synthesis of [2,3-d]pyrimidin-4-yl)amino)piperidine-1 carboxylate (2e)

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

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

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

Add tert-butyl(R)-3-((2-chloro-5-phenyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[ 2,3-d]pyrimidin-4-yl)amino)piperidine-1 carboxylate 2e (230mg, 0.4mmol), 1-ethyl-1H-pyrazole-4-amine 2f (89mg, 0.8mmol) and Trifluoroacetic acid (46mg, 0.4mmol) was dissolved in isopropanol (3mL), and the reaction solution was stirred at 110°C for 16 hours until the reaction was complete. The reaction solution was _{adjusted to pH=9 with saturated NaHCO 3} solution, poured into water and extracted with ethyl acetate, the organic phases were combined and then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the crude product obtained was purified with a silica gel column (EA: PE = 0-10%) purification to obtain the title product 2g (130mg).

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

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

Add tert-butyl(R)-3-((2-((1-ethyl-1H-pyrazol-4-yl)amino)-5-phenyl-7-((2-(trimethyl Silyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidine-1 carboxylate 2g (130mg, 0.2mmol), dissolved in dichloromethane In a solution of methane (1 mL) and dioxane hydrochloride (1 mL), the reaction solution was stirred at room temperature for 16 hours until the reaction was complete. The reaction solution was _{adjusted to pH=9 with saturated NaHCO 3} solution, poured into water and extracted with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the title product 2h (100 mg, crude product).

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

The fifth step: (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) synthesis

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

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

The sixth step: (R)-1-(3-((2-((1-ethyl-1H-pyrazol-4-yl)amino)-5-phenyl-7H-pyrrolo[2,3- d) Synthesis of 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) and NaOH was added (2mL) solution, the reaction solution was stirred at room temperature for 30 minutes to complete the reaction. The reaction solution was diluted with water (20 mL), extracted with ethyl acetate (30 mL*2), the combined organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered and spin-dried to obtain the crude product. Purified by HPLC to obtain the title compound 002 (29.31 mg).

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

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

1H NMR (400MHz, DMSO) δ 11.19 (s, 1H), 8.57 (s, 1H), 7.88 (d, J = 9.7 Hz, 1H), 7.44 (dd, J = 20.4, 13.3 Hz, 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)-7Hpyrrolo[2,3- d)pyrimidin-4-yl)amino)-4-fluoropiperidine

Synthetic route and specific synthetic steps:

The preparation method is the same as in Example 2, except that cyclopropylboronic acid (3b) replaces phenylboronic acid (2b) in Example 2, and (3S,4R)-3-amino-4-fluoropiperidine-1-carboxylate Tert-butyl ester (3d) instead of (R)-3-aminopiperidine-1-carboxylic acid tert-butyl ester (2d) in Example 2, and purified by HPLC to obtain the title compound 003 (8.84 mg) .

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

¹ H NMR (400MHz, DMSO) δ 10.68 (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 = 7.6 Hz, 3H), 0.80 (d, J = 7.2 Hz, 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 steps:

Step 1: Synthesis of 4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (4i)

At room temperature, 4-nitro-1H-pyrazole 4g (500mg, 4.4mmol), tetrahydro-2H-pyran-4-ol 4h (451mg, 4.4mmol), triphenylphosphine (1.4g, 5.3mmol) Dissolve in anhydrous tetrahydrofuran (20 mL) and add di-tert-butyl azodicarboxylate (1.32 g, 5.7 mmol). The system was stirred at room temperature for 16 hours under nitrogen. It was quenched by adding water (30 mL), and extracted with ethyl acetate (30 mL*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 obtain the title product 4i (600 mg).

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

Step 2: Synthesis of 1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole-4-amine (4b)

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

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

The third step: 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- Synthesis of formate (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-pyrazole-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 the reaction flask, and the mixture was reacted under nitrogen at 120°C for 16h. After completion, the solvent was spin-dried, and the residue was purified by thin-layer column chromatography (methanol/dichloromethane: 0-8%) to obtain the target product 4c (260 mg).

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

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

Add (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, 0.31 mmol) was dissolved in dichloromethane (3mL), 4M hydrochloric acid·dioxane (6mL) was added and stirred for 1 hour, then spin-dried, the residue was dissolved in dichloromethane (3mL) at room temperature, and three Fluoroacetic acid (3 mL) and stir for 1 hour. The mixture was spin-dried to obtain the crude product 4d (140 mg).

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

The fifth step: (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) synthesis

(R)-(5-cyclopropyl-4-(piperidin-3-ylamino)-2-((1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole -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 ( 10 mL), the mixture was cooled to 0°C and acrylic acid chloride 4e (35 mg, 0.6031 mmol) dissolved in dichloromethane (1 mL) 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 obtain the crude product 4f (270 mg).

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

The sixth step: (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)

Add (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, 0.31 mmol) was dissolved in tetrahydrofuran (2mL) and 2M sodium hydroxide (2mL) was added. The mixture was stirred for 2 hours, spin-dried tetrahydrofuran, and then extracted with dichloromethane. The organic layer was spin-dried, dissolved in acetonitrile and purified by HPLC to obtain the title compound 004 (58.0 mg).

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

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

¹ H NMR (400MHz, DMSO-d ₆ ) δ 10.60 (br s, 1H), 8.44 (s, 1H), 7.92 (br s, 1H), 7.46 (d, J = 16.0 Hz, 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 steps:

The preparation method is the same as that in Example 4, except that tetrahydrofuran-3-ol (5h) replaces tetrahydro-2H-pyran-4-ol (4h), and purified by HPLC to obtain the title compound 005 (57.98mg) ).

High performance liquid chromatography: Waters waters (waters) 2767 preparative chromatograph, mobile phase A (Mobile phase A): 0.1% FA in water, mobile phase B (Mobile phase B): acetonitrile, column: Sunfire( Prep C18 OBD 19*250mm 10μm), Gradient: 10% ACN runs for 3 minutes, 30%-35% ACN runs for 14 minutes, 95% ACN runs for 17 minutes, and 10% ACN runs for 21 minutes to complete purification and obtain the target compound.

LCMS: Rt: 1.09 min; 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

Synthetic route and specific synthetic steps:

The preparation method is the same as that in Example 4, the difference is that 3-oxetanol (6b) replaces the tetrahydro-2H-pyran-4-ol (4h) in Example 4, and HPLC method is used. Purification gave the title compound 006 (24.48 mg).

High performance liquid chromatography: Waters waters (waters) 2767 preparative chromatograph, mobile phase A (Mobile phase A): 10mmol% TFA in water, mobile phase B (Mobile phase B): acetonitrile, column: Sunfire( Prep C18 OBD 19*250mm 10μm), Gradient: 10% ACN runs for 4 minutes, 40%-45% ACN runs for 14 minutes, 95% ACN runs for 14 minutes, and 10% ACN runs for 17 minutes to complete purification and obtain the target compound.

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

¹ H NMR (400MHz, DMSO) δ 11.52-11.16 (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

Synthetic route and specific synthetic steps:

The preparation method is the same as that in Example 4, the difference is that 1-methylpiperidin-4-ol (7h) replaces the tetrahydro-2H-pyran-4-ol (4h) in Example 4, and it is prepared by HPLC Purification by chromatography gave the title compound 007 (13.03 mg).

High performance liquid chromatography: Waters waters (waters) 2767 preparative chromatograph, mobile phase A (Mobile phase A): 0.1% FA in water, mobile phase B (Mobile phase B): acetonitrile, column: Sunfire( Prep C18 OBD 19*250mm 10μm), Gradient: 10% ACN runs for 3 minutes, 35%-40% ACN runs for 15 minutes, 95% ACN runs for 19 minutes, and 10% ACN runs for 22 minutes to complete purification and obtain the target compound.

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

¹ H NMR (400MHz, DMSO-d6) δ 10.61 (s, 1H), 8.43 (s, 1H), 7.91 (s, 1H), 7.45 (d, J = 14.2 Hz, 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 = 10.8 Hz, 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

Synthetic route and specific synthetic steps:

Step 1: Synthesis of 1-methyl-4-(4-nitro-1H-pyrazol-1-yl)piperidine (8c)

Dissolve triphenylphosphine (34.8g, 132.8mmol) in anhydrous tetrahydrofuran (300mL) solution and cool to 0°C, then slowly inject DIAD (26.8g, 132.8mmol) and stir at 0°C for 10 minutes. A large amount of white precipitate formed in the solution.

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

Post-treatment: the reaction solution was concentrated under reduced pressure, and the obtained crude product was dissolved in ethyl acetate (150 mL), and then extracted with 3 mol/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 (200 mL×2). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title product 8c (16.5 g).

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

Step 2: 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, and then 10% Pd/ C (3.3g), the reaction solution was stirred overnight under a hydrogen atmosphere. After the reaction, the reaction solution was filtered, and the filtrate was spin-dried to obtain the title product 8d (12.8g)

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

The third step: tert-butyl(3S,4R)-3-((2-chloro-5-cyclopropyl-7-((2-(trimethylsilyl)ethoxy)methyl)- Synthesis of 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] Pyrimidine 8e (357.0mg, 1.0mmol), compound tert-butyl (3S, 4R)-3-amino-4-fluoropiperidine-1-carboxylate 8f (545.0mg, 2.5mmol) and DIPEA (387.0mg) All were dissolved in ethanol (8 mL), and then the mixture was added to a stuffy tank at 120°C and stirred overnight. After the reaction, the reaction solution was concentrated and spin-dried. The obtained crude product was separated and purified by normal phase column to obtain 8 g (256 mg) of the title product. LCMS: Rt: 1.941 min; MS m/z (ESI): 540.2 [M+H] ⁺ .

The fourth step: tert-butyl(3S,4R)-3-((5-cyclopropyl-2-((1-(1-methylpiperidin-4-yl)-1H-pyrazole-4- Yl)amino)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-4-fluoro Synthesis of piperidine-1-carboxylate (8h)

The intermediate 1-(1-methylpiperidin-4-yl)-1H-pyrazole-4-amine 8d (254mg, 1.41mmol), the 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) were added to dioxane In the ring (8 mL), under the protection of nitrogen, heat to 110°C and stir overnight. After the reaction, the reaction solution was cooled to room temperature and filtered. The filtrate was directly purified by reverse phase column (Prep-HPLC (Boston Prime C18 150*30mm*5μm; A%: water (containing 0.225% FA); B%: ACN 10%-40%, 8-20min) to obtain the title product 8h (153.0mg).

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

The fifth step: (5-cyclopropyl-4-(((3S,4R)-4-fluoropiperidin-3-yl)amino)-2-((1-(1-methylpiperidine-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-fluoropiper Pyridine-1-carboxylate was dissolved in TFA (3 mL) for 8 h (153 mg, 0.22 mmol), and then stirred at room temperature for 1 h. After the completion of the reaction, it was spin-dried to obtain the title product 8i (185mg, purity: 66.8%) directly into the next reaction.

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

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

Add (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 a mixture of acetone (3mL) and H ₂ O (3mL) Add solid potassium carbonate to adjust the pH to 9-10, and then heat the reaction solution to 40°C and stir overnight. LCMS detected that the intermediate 8i was completely converted to the title product 8j, and the reaction solution was directly subjected to the next reaction.

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

The seventh step: 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)

Cool the reaction solution from the previous step to 0°C, continue adding potassium carbonate solid to adjust the pH to 9-10, and then dissolve acryloyl chloride 8k (91mg, 0.66mmol) in acetone (1mL) and add dropwise to the reaction solution. After the addition was completed, the reaction solution was stirred at 0°C for 1 h, and LCMS detected the disappearance of the raw materials. After the reaction, the reaction solution is filtered and sent directly to preparation (Prep-HPLC(Boston Prime C18 150*30mm*5μm; A%: water (including 0.225% FA)); B%: ACN 13%-43%, 8-15min ) Was purified to obtain the target product 008 (9.58 mg).

LCMS: Rt: 5.287 min; 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).

Example 9 Preparation of Compound 009

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

Synthetic route and specific synthetic steps:

The first step: (2S,5R)-5-((2-chloro-5-cyclopropyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[ Synthesis of 2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidine-1-carboxylic acid benzyl ester (9c)

Respectively mix 2,4-dichloro-5-cyclopropyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine at room temperature (9a, 500mg, 1.39mmol), (2S, 5R)-5-amino-2-methylpiperidine-1-carboxylic acid benzyl ester (9b, 346mg, 1.39mmol) and DIPEA (448mg, 3.48mmol) were added to In isopropanol (10.0 mL) solution, the reaction system was heated to 110° C., and the reaction was stirred at this temperature for 16 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (ethyl acetate/petroleum ether = 1/10) to obtain 9c (385 mg, 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: (2S,5R)-5-((5-cyclopropyl-2-((1-methyl-1H-pyrazol-4-yl)amino)-7-((2-(trimethyl (Silyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidine-1-carboxylic acid benzyl ester (9e)

Under nitrogen protection at room temperature, add Pd ₂ (dba) ₃ (32.0mg, 0.035mmol) and BINAP (21.8mg, 0.035mmol) to 9c (200mg, 0.351mmol), 1-methyl-1H-pyrazole-4- In the 1,4-dioxane (10.0 mL) solution of amino (9d, 40.8 mg, 0.421 mmol) and Cs ₂ CO ₃ (285.9 mg, 0.878 mmol), the reaction system was heated to 100°C and stirred for 16 hours, then cooled To room temperature. After filtration, the filter cake was washed with ethyl acetate (30.0 mL). After the solution was concentrated under reduced pressure, it was purified by Chem-flash (acetonitrile/0.1% trifluoroacetic acid) to obtain 9e (195 mg, 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) Synthesis of -7-((2-(Trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (9f)

Under the protection of nitrogen at room temperature, Pd/C (5 mg) was added to 9e (195 mg, 0.309 mmol) of methanol (5.0 mL), and the reaction system was stirred at room temperature under hydrogen for 16 hours. The reaction mixture was filtered under reduced pressure, the filter cake was washed with methanol (20.0 mL), the filtrates were combined, and evaporated to dryness under reduced pressure. The residue was purified by Chem-flash (acetonitrile/0.1% trifluoroacetic acid) to obtain 9f (136 mg, 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) Synthesis of -7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (9g)

At 0°C, trifluoroacetic acid (1mL) was added to the dichloromethane solution (5.0mL) of 9f (136mg, 0.273mmol), the reaction solution was stirred at room temperature for 2 hours, and the reaction solution was concentrated under reduced pressure to give a yellow The oil was added to a methanol solution (5.0 mL) of DIPEA (1.0 mL), and the reaction solution was stirred at 50°C 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 9 g (95 mg, yield: 94.8%).

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

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

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

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

¹ H NMR (400M Hz, DMSO-d ₆ ) δ 10.59 (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]pyrimidine -4-yl)amino)pyrrolidin-1-yl)prop-2-en-1-one

Synthetic route and specific synthetic steps:

The first step: (R)-3-((2-chloro-5-cyclopropyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2 Synthesis of ,3-d)pyrimidin-4-yl)amino)pyrrolidine-1-carboxylic acid tert-butyl ester (10c)

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

The second step: ((R)-3-((5-cyclopropyl-2-((1-methyl-1H-pyrazol-4-yl)amino)-7-((2-(trimethyl Synthesis of (silyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidine-1-carboxylic acid tert-butyl ester (10e)

Under nitrogen protection at room temperature, add Pd ₂ (dba) ₃ (72.3mg, 0.079mmol), BINAP (49.1mg, 0.079mmol) to 10c (400mg, 0.79mmol), 1-methyl-1H-pyrazole-4 -In a 1,4-dioxane (30 mL) solution of amine (10d, 92 mg, 0.94 mmol) and cesium carbonate (772 mg, 2.37 mmol), the reaction system was heated to 100° C., stirred for 16 hours, and cooled to room temperature. It was 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 obtain 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[ Synthesis of 2,3-d]pyrimidine-2,4-diamine (10f)

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

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

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

10f (190mg, 0.56mmol) was dissolved in _{a mixture of tetrahydrofuran (4.0mL) and H 2} 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, stirred at 0°C for 2 hours, filtered, and purified (Prep-HPLC (Boston Prime C18 150*30mm*5μm; A%: water (containing 0.225% FA)); B%: ACN 13%-43% , 8-10min) to obtain 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 ) δ 10.83 (brs, 1H), 8.88 (brs, 1H), 7.84 (d, J = 2.4 Hz, 1H), 7.49 (d, J = 3.6 Hz, 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

Synthetic route and specific synthetic steps:

Step 1: Synthesis of 2-cyano-4,4-dimethyl-2-pentenoic acid (11a)

Dissolve cyanoacetic acid (5.00g, 58.0mmol) in anhydrous methanol (50.0mL), add trimethylacetaldehyde (5.00g, 58.0mmol) and sodium hydroxide (4.64g, 116mmol), stir for 1 hour at 40°C . The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (methanol/dichloromethane=1/10) to obtain Intermediate 11a (4.2 g, 65.0%).

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

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

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

Synthetic route and specific synthetic steps:

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

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

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

The second step: (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 Synthesis of (12e)

Intermediate 12c (700mg, 1.2mmol) and 1-(trifluoromethyl)-1H-pyrazole-4-amine 12d (253mg, 1.4mmol) were dissolved in anhydrous dioxane (30.0mL) and added Tris(dibenzylideneacetone)dipalladium (112mg, 123μmol), binaphthylphosphine (76mg, 123μmol) and cesium carbonate (1.0g, 3.1mmol) were stirred at 100°C for 16 hours under a nitrogen atmosphere. After cooling, water (10.0 mL) was added to dilute, and ethyl acetate (20.0 mL*2) was used for extraction. The organic phase was washed with saturated sodium chloride aqueous solution (20.0 mL), dried, filtered and concentrated. The residue was purified by column chromatography (ethyl acetate/petroleum ether = 1/2) to obtain Intermediate 12e (720 mg, 85.6%).

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

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

Intermediate 12e (370 mg, 540 μmol) was dissolved in anhydrous methanol (15.0 mL), and wet palladium on carbon (40 mg, 10% Pd) was added. Stir at 25°C for 16 hours under a hydrogen atmosphere (15 psi). The reaction solution was filtered and concentrated to obtain crude product 12f (270 mg), which was used directly in the next step without purification.

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

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

The crude product 12f (270 mg, 490 μmol) was dissolved in anhydrous dichloromethane (10.0 mL). Trifluoroacetic acid (2.0mL) was added and stirred at 25°C for 6 hours. The crude product obtained by concentration of the reaction solution under reduced pressure was dissolved in methanol (6.0mL) and water (3.0mL). Lithium hydroxide (21mg, 490μmol) was added. The temperature was raised to 50°C and stirred for 2 hours. The reaction solution was cooled and concentrated by filtration. After adding water to the residue, a solid precipitated out, and 12 g (260 mg) of a crude green solid product was obtained by filtration, which was directly used in the next step without purification.

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

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

At 0°C, 12 g (260 mg, 495 μmol) of the crude product was dissolved in tetrahydrofuran (4.0 mL) and water (2.0 mL). Potassium phosphate (228mg, 990μmol) and acryloyl chloride (45mg, 495μmol) were added, and the temperature was raised to 25°C and stirred for 2 hours. Dilute with water (10.0mL), extract with ethyl acetate (15.0mL*2), wash the organic phase with saturated aqueous sodium chloride (15.0mL), dry, filter and concentrate. The residue is purified by preparation (Prep-HPLC (Boston Prime C18) 150*30mm*5μm; A%: water (containing 0.225% FA); B%: ACN 22%-52%)) to obtain the product 012 (25.4 mg, 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)-7Hpyrrolo[2,3-d]pyrimidine-4- (Yl)oxy)nonyl-1-yl)prop-2-en-1-one

Synthetic route and specific synthetic steps:

The first step: 4-((2,5-Dichloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H pyrrolo[2,3-d]pyrimidin-4-yl) Synthesis of tert-butyl oxy)azepane-1 carboxylate (13c)

At room temperature, NaH (60mg, 1.5mmol) was added in multiple batches to 4-hydroxyazepane-1-carboxylic acid tert-butyl ester 13b (323mg, 1.5mmol) in anhydrous DMSO (10.0mL) solution, After adding and stirring for 15 minutes, add 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 heated to 55°C, reacted for 15 minutes, cooled to room temperature, saturated ammonium chloride solution (50.0mL) was added to the reaction solution, extracted three times with dichloromethane (50.0mL*3), combined the organic phases, and water ( 50.0mL) and saturated brine (5.0mL) each washed once, the organic phase was dried and filtered with anhydrous sodium sulfate, the filtrate was concentrated under reduced pressure and purified by silica gel column (ethyl acetate: petroleum ether = 1:10), 13c (620.4 mg, yield: 78%) was obtained.

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

The second step: 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-carboxylic acid tert-butyl ester (13e)

Under nitrogen protection at room temperature, 13c (620.4mg, 1.17mmol), 1-methyl-1H-pyrazole-4-amine 13d (170mg, 1.76mmol) and Cs ₂ CO ₃ (763mg, 2.34mmol) in 1,4 -In the dioxane (20.0 mL) solution, the reaction system was heated to 100° C., stirred for 16 hours, and cooled to room temperature. After filtration, the filter cake was washed with tetrahydrofuran (50.0 mL), concentrated under reduced pressure and purified to obtain the product 13e (595.8 mg, yield: 86.2%).

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

¹ H NMR (400MHz, CDCl ₃ ) δ 7.84 (s, 1H), 7.60 (s, 1H), 6.87 (s, 1H), 6.64 (d, J = 7.6 Hz, 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: ((4-(Azepan-4-oxy)-5-chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)-7Hpyrrolo[ Synthesis of 2,3-d]pyrimidin-7-yl)methanol (13f)

At room temperature, TFA (12.0 mL) was added to the intermediate 13e (6.0 mL) solution, and after stirring for 2 hours at room temperature, the reaction solution was concentrated to dryness under reduced pressure, and the resulting crude product 13f was directly used in the next reaction.

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

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

Dissolve the crude product 13f (1mmol) in a mixed solvent of acetone (12.0ml) and water (8.0mL), adjust the pH to 10 with potassium carbonate, heat to 35°C, stir for 16 hours, and cool the reaction solution to 0°C. Then, acryloyl chloride (126 mg, 1.4 mmol) was dissolved in acetone (1.0 mL) and added dropwise to the reaction solution. After the dripping was completed, the reaction solution was stirred at 0°C for 1 hour. The reaction solution was filtered and spin dry purified (Prep-HPLC (Boston Prime C18 150*30mm*5μm; A%: water (containing 0.225% FA); B%: ACN 22%-52%)) to obtain 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, 2.4 Hz, 1H), 5.47 (d, J = 4.8 Hz, 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, 2.4 Hz, 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)-7Hpyrrolo [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

Synthetic route and specific synthetic steps:

The first step: 1-((3S,4R)4-fluoro-3-((2-((1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)amine) -7((2-(Trimethylsilyl)ethoxy)methyl)-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-piperidine-1-carboxylic acid tert-butyl ester Synthesis of (14c)

Under the protection of nitrogen at room temperature, 1-((3S,4R)4-fluoro-3-((2-chloro)-7((2-(trimethylsilyl)ethoxy)methyl)-7H Pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-tetrahydropyrrole-1-carboxylic acid tert-butyl ester 14a (125mg, 0.25mmol) and 1-(1-methylpiperidin-4-yl) )-1H-pyrazole-4-ammonia 14b (67.5mg, 0.38mmol) was dissolved in 1,4-dioxane (10.0mL), and then under stirring, Pd ₂ (dba) ₃ (22.8mg, 0.025 mmol), BINAP (31.1mg, 0.2mmol) and cesium carbonate (162.8mg, 0.5mmol), the reaction system was heated to the 120 series, stirred for 12 hours, cooled to room temperature and filtered, the filter cake was washed with ethyl acetate (60.0mL) The filtrate was concentrated under reduced pressure and purified (dichloromethane: methanol = 15:1) to obtain 14c (120 mg, 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 = 46.3 Hz, 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: 1-((3S,4R)4-fluoro-3-((2-((1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)amine) Synthesis of -7H pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-piperidine (14d)

At 0°C, trifluoroacetic acid (5.0 mL) was added to a dichloromethane solution (5.0 mL) of 14c (125 mg, 0.19 mmol), and stirred at room temperature for 16 hours. After concentration, the crude product was dissolved in _{a mixture of THF/H 2} O (6.0 mL, 2:1), LiOH (125.0 mg, 5.2 mmol) was added, and the mixture was stirred at room temperature for 1 h. The crude product was purified by filtration to obtain 14d (87 mg, yield). Rate: 99%).

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

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

At 0°C, 14d (87 mg, 0.21 mmol) was added to _{the mixture of tetrahydrofuran (2.0 mL) and H 2} O (1.0 mL), and potassium phosphate (233.5 mg, 1.1 mmol) was added. After stirring for 10 minutes, a solution of acryloyl chloride (29.0 mg, 0.32 mmol) dissolved in tetrahydrofuran (1.0 mL) was slowly added dropwise. After stirring for 2 hours, it was filtered and the filtrate was prepared and purified (Prep-HPLC (Boston Prime C18 150*30mm*5μm; A%: water (containing 0.225% FA); B%: ACN 20%-50%)) to obtain product 014 (35.11 mg, yield: 35.7%).

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

¹ H NMR (400MHz, DMSO-d ₆ ) δ 11.32 (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 = 32.5 Hz, 1H), 3.41 (s, 1H), 2.97 (d, J = 8.2 Hz, 1H), 2.29 (d, J = 24.4 Hz, 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-pyrrole And [2,3-d]pyrimidin-4-yl)oxy)piperidin-1-yl)prop-2-en-1-one

Synthetic route and specific synthetic steps:

The first step: (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-carboxylic acid tert-butyl ester ( 15g) Synthesis

Intermediate 15e (100mg, 0.20mmol), (R)-1-(tetrahydrofuran-3-yl)-1H-pyrazole-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 solution was stirred at 95°C for 16 hours under argon protection. After cooling, the reaction solution was filtered with 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 15 g (150 mg, yield: 90%).

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

¹ H NMR (400MHz, CDCl ₃ ) δ 8.04 (s, 1H), 7.65 (s, 1H), 6.96 (d, J = 3.6 Hz, 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: 4-(((3S,4R)-4-fluoropiperidin-3-yl)oxy)-N-(1-((R)-tetrahydrofuran-3-yl)-1H-pyrazole- Synthesis of 4-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (15h)

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

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

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

Dissolve 15h (100mg, 0.26mmol) in a mixture of tetrahydrofuran (0.5mL) and water (0.3mL), add potassium phosphate solid (110mg, 0.52mmol) and stir at 25℃, add acryloyl chloride (23mg, 0.26 A solution of mmol) in tetrahydrofuran (0.5 mL) was added dropwise and stirred for 1 hour. The reaction solution was concentrated under reduced pressure and preparatively purified (Prep-HPLC (Boston Prime C18 150*30mm*5μm; A%: water (containing 0.225% FA); B%: ACN 15%-40%)) to obtain 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 ) δ 11.31 (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

Synthetic route and specific synthetic steps:

The first step: (3S,4R)-3-((2-chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H pyrrolo[2,3-d]pyrimidine -4-yl)oxy)-4-fluoropyridine-1-carboxylic acid tert-butyl ester (16c) synthesis

At room temperature, NaH (91mg, 2.27mmol) was added in batches of (3S, 4R)-4-fluoro-3-hydroxypiperidine-1-carboxylic acid tert-butyl ester 16b (500mg, 2.27mmol) in DMSO (15.0 mL) in the solution, add and stir for 15 minutes, then add 2,4-dichloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H pyrrolo[2,3-d]pyrimidine 16a (720mg, 2.27mmol) was added, the reaction system was heated to 55°C, reacted for 15 minutes, 50.0mL saturated NH ₄ Cl solution was added to the reaction solution, extracted three times with dichloromethane (50.0mL*3), the organic phases were combined, Washed with water (50.0mL) and saturated brine (50mL) each time, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and purified (ethyl acetate: petroleum ether = 1:8) to obtain 16c (748mg, yield : 58%).

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

¹ H NMR (400MHz, CDCl ₃ ) δ 7.20 (d, J = 3.6 Hz, 1H), 6.63 (d, J = 3.6 Hz, 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: (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-carboxylic acid tert-butyl ester Synthesis of (16e)

Under nitrogen protection at room temperature, add Pd ₂ (dba) ₃ (14.6mg, 0.016mmol) and BINAP (9.7mg, 0.016mmol) to 16c (81mg, 0.162mmol), (R)-1-(1-methylpyrrole) Alkyl-3-yl)-1H-pyrazole-4-amine 16d (53.5mg, 0.324mmol) and Cs ₂ CO ₃ (105mg, 0.324mmol) in 1,4-dioxane (5.0mL) solution, The reaction system was heated to 100°C, stirred for 16 hours, and cooled to room temperature. After filtering, the filter cake was washed with 50.0 mL of tetrahydrofuran, and the solution was concentrated under reduced pressure and the crude product obtained was purified to obtain 16e (67.6 mg, yield: 66.2%).

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

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

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

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

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

The crude product 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°C, and stirred for 16 hours. The reaction solution was cooled to 0°C, and then acryloyl chloride (45 mg, 0.48 mmol) was dissolved in acetone (1.0 mL) and added dropwise to the reaction solution. After the addition is complete, the reaction solution is filtered and spin-dried. The obtained crude product was purified (Prep-HPLC (Boston Prime C18150*30mm*5μm; A%: water (containing 0.225% FA); B%: ACN 15%-50%)) to obtain the target product 016 (15.18 mg, yield: 31%).

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

¹ H NMR (400M Hz, DMSO-d ₆ ) δ 11.32 (s, 1H), 9.02 (s, 1H), 8.01 (s, 1H), 7.65 (d, J = 10.8 Hz, 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-(oxetan-3-yl)-1H-pyrazol-4-yl)amino)-7H-pyrrole And [2,3-d]pyrimidin-4-yl)oxy)piperidin-1-yl)prop-2-en-1-one

Synthetic route and specific synthetic steps:

Step 1: Synthesis of 2-chloro-4-(((3S,4R)-4-fluoropiperidin-3-yl)oxy)-7H-pyrrolo[2,3-d]pyrimidine (17b)

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

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

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

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

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

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

Under stirring at 0°C, 17d (45.5 mg, 0.122 mmol) was dissolved in a mixture of tetrahydrofuran (5.0 mL) and water (2.0 mL), potassium phosphate (64.7 mg, 0.305 mmol) was added and stirred. Acrylic chloride (13.3 mg, 0.146 mmol) was added dropwise to the reaction solution, and stirred at room temperature for 2 hours. The reaction solution was filtered, and the filtrate was prepared and purified (Prep-HPLC (Boston Prime C18 150*30mm*5μm; A%: water (containing 0.225% FA); B%: ACN 22%-52%)) to obtain 017 (2.15 mg, yield Rate: 4.2%).

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

¹ H NMR (400MHz, DMSO-d ₆ ) δ 11.34 (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]pyrimidine -4-yl)amino)-2-methylpyrrolidin-1-yl)prop-2-en-1-one

Synthetic route and specific synthetic steps:

The first step: (2S, 4R)-4-((2-chloro-5-fluoro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2 Synthesis of ,3-d)pyrimidin-4-yl)amino)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (18c)

2,4-Dichloro-5-fluoro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine 18a (300mg, 0.98mmol), (2S, 4R)-4-amino-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester 18b (197mg, 0.98mmol) was added to DIPEA (287mg, 2.25mmol) in isopropanol (10.0 mL) in the solution, stirred at 110 ℃ for 16 hours. Add water (100.0mL) to terminate the reaction, extract with ethyl acetate (100.0mL*2), combine the organic phases, wash with saturated brine (100.0mL), concentrate the organic phase under reduced pressure, and purify (ethyl acetate: petroleum ether = 1:10 ) To obtain 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 = 8.0 Hz, 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: (2S, 4R)-4-((5-fluoro-2-((1-methyl-1H-pyrazol-4-yl)amino)-7-((2-(trimethylform Synthesis of (silyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (18e)

Under nitrogen protection at room temperature, add Pd ₂ (dba) ₃ (84.2mg, 0.092mmol), BINAP (57.2mg, 0.092mmol) to 18c (480mg, 0.92mmol), 1-methyl-1H-pyrazole-4 -In a 1,4-dioxane (20.0 mL) solution of amine 18d (116 mg, 1.19 mmol) and cesium carbonate (749 mg, 2.3 mmol), the reaction was heated to 120° C. and stirred overnight, cooled to room temperature and filtered. The reaction solution was concentrated under reduced pressure and purified (ethyl acetate: petroleum ether=1:1) to obtain 18e (480 mg, 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 -Synthesis of pyrrolo[2,3-d]pyrimidine-2,4-diamine (18f)

18e (480 mg, 0.85 mmol) was added to a dichloromethane solution (6.0 mL) of trifluoroacetic acid (3.0 mL) at room temperature, and the reaction solution was stirred at 0°C for 6 hours. The reaction solution was quenched with saturated sodium bicarbonate (50.0ml), extracted with ethyl acetate (20.0mL*2), and the organic phase was concentrated under reduced pressure. The crude product was added to LiOH (522mg, 12.7mmol) in tetrahydrofuran/aqueous solution ( 4.0 mL/1.0 mL), the reaction solution was stirred at 25°C for 2 hours. The reaction solution was concentrated under reduced pressure and reversed phase to obtain Intermediate 18f (80 mg, yield: 28%).

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

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

Dissolve 18f (80mg, 0.24mmol) in a mixture of tetrahydrofuran (6.0mL) and water (2.0mL), add potassium phosphate (127.0mg, 0.60mmol) and stir, add acryloyl chloride (30.5mg, 0.34mmol) in tetrahydrofuran (0.5mL) The solution was added dropwise and stirred at 25°C for 1 hour. The reaction solution was concentrated under reduced pressure and purified by Prep-HPLC (acetonitrile/water/1‰TFA) to obtain 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 ) δ 10.69 (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).

Test examples of biological activity and related properties

Test Example 1: BTK kinase activity inhibition test

Experimental principle: After BTK kinase is incubated with the compound, it reacts with the substrate under the action of ATP. The ADP produced by the reaction was quantified using Promega's ADP-GLO detection kit to reflect the enzyme activity.

Experimental instruments: Labcyte Echo650 pipetting system; Perkin Elmer Envision microplate reader; Eppendorf 5810 centrifuge.

Experimental Materials:

试剂Reagent	品牌brand	货号Item No.
Tris hydrochloride溶液Tris hydrochloride solution	SigmaSigma	T2663T2663
BRIJ 35 detergent(10％)BRIJ 35 detergent (10%)	MerckMerck	203728203728
MgCl ₂溶液 MgCl ₂ solution	SigmaSigma	M1028M1028
ADP-Glo激酶检测试剂盒ADP-Glo Kinase Detection Kit	PromegaPromega	V9102V9102
BTKBTK	Carna bioscienceCarna bioscience	08-18008-180
Poly(4:1Glu,Tyr)Poly(4:1Glu, Tyr)	SigmaSigma	P0275P0275
384孔板384-well plate	Perkin ElmerPerkin Elmer	60072906007290

Experimental method: Use the Echo pipetting system to transfer the test compound to a 384-well plate, add 2 μL/well of BTK, and incubate for 30 minutes. Then add 3μL/well of the mixed solution of substrate Poly(4:1Glu, Tyr) and ATP to start the enzyme reaction. The final concentration of the compound starts from 3 μM or 300 nM or 100 nM, and is diluted 3 times. The final concentration of enzyme in the reaction is 1.7ng/well, the final concentration of ATP is 36μM, and the final concentration of substrate is 0.1mg/mL. After reacting for 1 hour, add 5μL/well of ADP-GLO reagent and incubate for 40 minutes. Then add 10μL/well kinase reaction detection reagent and incubate for 30 minutes. Read the fluorescence signal with Envision microplate reader, and calculate the inhibition rate and the half inhibition concentration (IC ₅₀ ).

The biological activity of the compound of the present invention was determined by the above test, and the measured IC ₅₀ value is shown in Table 1 below.

_{Table 1 The IC 50 of} Example Compounds Inhibiting BTK Kinase Activity

实施例化合物编号Example compound number	IC ₅₀(nM) IC ₅₀ (nM)	实施例化合物编号Example compound number	IC ₅₀(nM) IC ₅₀ (nM)
001001	6.536.53	010010	3.923.92
002002	7.417.41	011011	5.355.35
003003	3.263.26	012012	9.079.07
004004	3.833.83	013013	4.004.00
005005	4.834.83	014014	0.970.97
006006	6.606.60	015015	2.142.14
007007	1.771.77	016016	1.901.90
008008	2.482.48	017017	3.633.63
009009	3.433.43	018018	1.391.39

Test Example 2: JAK3 kinase activity inhibition test

Experimental principle: After the JAK3 kinase is incubated with the compound, it reacts with the substrate under the action of ATP. The ADP produced by the reaction was quantified using Promega's ADP-GLO detection kit to reflect the enzyme activity.

laboratory apparatus:

Labcyte Echo650 Pipetting System

Perkin Elmer Envision Microplate Reader

Eppendorf 5810 centrifuge.

Experimental Materials:

试剂Reagent	品牌brand	货号Item No.
Tris hydrochloride溶液Tris hydrochloride solution	SigmaSigma	T2663T2663
BRIJ 35 detergent(10％)BRIJ 35 detergent (10%)	MerckMerck	203728203728
MgCl ₂溶液 MgCl ₂ solution	SigmaSigma	M1028M1028
ADP-Glo激酶检测试剂盒ADP-Glo Kinase Detection Kit	PromegaPromega	V9102V9102
JAK3JAK3	Carna bioscienceCarna bioscience	08-04608-046
Poly(4:1Glu,Tyr)Poly(4:1Glu, Tyr)	SigmaSigma	P0275P0275
384孔板384-well plate	Perkin ElmerPerkin Elmer	60072906007290

experimental method:

Use the Echo pipetting system to transfer the test compound to a 384-well plate, and add 2 μL/well of JAK3, and incubate for 30 minutes. Then add 3μL/well of the mixed solution of substrate Poly(4:1Glu, Tyr) and ATP to start the enzyme reaction. The final concentration of the compound starts from 3 μM or 300 nM or 100 nM, and is diluted 3 times. In the reaction, the final concentration of enzyme was 1.9ng/well, the final concentration of ATP was 36μM, and the final concentration of substrate was 0.1mg/mL. After reacting for 1 hour, add 5μL/well of ADP-GLO reagent and incubate for 40 minutes. Then add 10μL/well kinase reaction detection reagent and incubate for 30 minutes. Read the fluorescence signal with Envision microplate reader, and calculate the inhibition rate and the half inhibition concentration (IC ₅₀ ).

The biological activity of the compound of the present invention was determined by the above test, and the measured IC ₅₀ value is shown in Table 2 below.

_{Table 2 The IC 50 of the} compound of the present invention inhibiting JAK3 kinase activity

实施例化合物编号Example compound number	IC ₅₀(nM) IC ₅₀ (nM)	实施例化合物编号Example compound number	IC ₅₀(nM) IC ₅₀ (nM)
001001	1.661.66	010010	1.271.27
002002	0.610.61	011011	7.237.23
003003	0.550.55	012012	1.741.74

004004	1.361.36	013013	0.580.58
005005	1.411.41	014014	0.510.51
006006	4.744.74	015015	0.250.25
007007	1.481.48	016016	0.890.89
008008	3.043.04	017017	0.840.84
009009	1.811.81	018018	0.460.46

Test Example 3: Inhibition of BTK phosphorylation in Ramos cells

Experimental principle: After incubating Ramos cells with compounds and stimulants, use Cisbio's BTK phosphorylation detection kit to detect the transfer of fluorescence energy by homogeneous time-resolved fluorescence (HTRF) method, thereby reflecting the inhibitory effect on phosphorylation .

laboratory apparatus:

仪器instrument	品牌brand	型号model
生物安全柜Biological safety cabinet	ESCOESCO	CLASSⅡ BSCCLASSⅡ BSC
离心机Centrifuge	EppendorfEppendorf	58105810
CO ₂培养箱 CO ₂ incubator	ESCOESCO	CCL-170B-8CCL-170B-8
细胞计数仪Cell counter	CountStarCountStar	IC1000IC1000
EnvisionEnvision	Perkin ElmerPerkin Elmer	//

Experimental Materials:

试剂Reagent	提供商Provider	货号Item No.
RPMI-1640培养基RPMI-1640 medium	GIBCOGIBCO	A10491-01A10491-01
灭活胎牛血清(HI-FBS)Inactivated Fetal Bovine Serum (HI-FBS)	GIBCOGIBCO	1010014710100147
HBSS溶液HBSS solution	GIBCOGIBCO	14025-09214025-092
anti-human IgM抗体anti-human IgM antibody	Jackson ImmunoJackson Immuno	109-006-129109-006-129
BTK phospho-Y223 HTRF检测试剂盒BTK phospho-Y223 HTRF detection kit	CisbioCisbio	63ADK017PEH63ADK017PEH
384孔板384-well plate	Perkin ElmerPerkin Elmer	60076806007680
RamosRamos	ATCCATCC	CRL-1596CRL-1596

experimental method:

Echo pipetting system with the test compound were transferred to 384-well plates, Ramos cells were adjusted to a density of 1X10 ⁷ cells / mL, was added 10μL / well of the cell suspension, incubated in an incubator at 37 ℃, 5% CO ₂ in Hour. Then add 5 μL/well of the stimulator anti-human IgM antibody, the final concentration of the stimulant is 10 μg/mL, and incubate for 10 minutes. The final concentration of the compound is 1 μM starting with a 4-fold dilution. Add 5μL/well of cell lysate and incubate at room temperature for 30 minutes. Use Cisbio's BTK phospho-Y223 kit to detect the phosphorylation degree of BTK, and finally read the fluorescence signal at 665nm and 615nm of the emission light on the Envision microplate reader, and calculate the inhibition rate and the half inhibition concentration (IC ₅₀ ).

The biological activity of the compound of the present invention was determined by the above test, and the measured IC ₅₀ value is shown in Table 3 below.

Table 3 The inhibitory activity of the compounds of the present invention on BTK phosphorylation in Ramos cells

实施例化合物编号Example compound number	IC ₅₀(nM) IC ₅₀ (nM)	实施例化合物编号Example compound number	IC ₅₀(nM) IC ₅₀ (nM)
001001	23.7623.76	010010	53.4953.49

002002	62.2462.24	011011	5.045.04
003003	2.632.63	012012	6.156.15
004004	5.075.07	013013	10.8510.85
005005	10.9010.90	014014	2.022.02
006006	11.3411.34	015015	17.8917.89
007007	2.392.39	016016	4.234.23
008008	5.785.78	017017	9.669.66
009009	5.735.73	018018	4.014.01

Test Example 4: Inhibition of STAT5 phosphorylation in CTLL-2 cells

Experimental principle: This experiment is to evaluate the effect of compounds on the phosphorylation of STAT5, a downstream substrate of JAK3. After incubating CTLL-2 cells with the compound and stimulant, use Perkin Elmer's p-STAT5 (Tyr694/699) detection kit to detect the fluorescence between the donor beads and the acceptor beads through the time-resolved fluorescence method Energy transfer, which reflects the inhibition of phosphorylation.

laboratory apparatus:

仪器instrument	品牌brand	型号model
生物安全柜Biological safety cabinet	Thermo ScientificThermo Scientific	1300 Series A21300 Series A2
离心机Centrifuge	EppendorfEppendorf	57025702
CO ₂培养箱 CO ₂ incubator	Thermo ScientificThermo Scientific	371371
细胞计数仪Cell counter	InvitrogenInvitrogen	C10281C10281
EnvisionEnvision	Perkin ElmerPerkin Elmer	//
EchoEcho	LabcyteLabcyte	655655

Experimental Materials:

试剂Reagent	品牌brand	货号Item No.
胎牛血清(FBS)Fetal Bovine Serum (FBS)	GIBCOGIBCO	10099-14110099-141
二甲基亚砜(DMSO)Dimethyl sulfoxide (DMSO)	SigmaSigma	D8418-1LD8418-1L
IL-2IL-2	R&DR&D	402-ML-020402-ML-020
CTLL-2CTLL-2	ATCCATCC	TIB-214TIB-214
AlphaLISA SureFire Ultra p-STAT5(Tyr694/699)检测试剂盒AlphaLISA SureFire Ultra p-STAT5 (Tyr694/699) Detection Kit	Perkin ElmerPerkin Elmer	ALSU-PST5-B500ALSU-PST5-B500

experimental method:

CTLL-2 cells were seeded in a 384-well plate, 1.5×10 ⁴ cells/15 μl/well, the compound was transferred to the 384-well plate with Echo _{, and incubated in a 37°C, 5% CO 2} incubator for 30 minutes. Then add 5μL/well of stimulant IL-2, the final concentration is 1ng/mL, and incubate for 30 minutes. The final concentration of the compound starts from 3 μM and is diluted 3 times. Add 5μL/well of cell lysate and incubate at room temperature for 10 minutes. Perkin Elmer's AlphaLISA p-STAT5 (Tyr694/699) detection kit was used to detect the phosphorylation degree of STAT5, and finally the AlphaLISA signal was read on the Envision microplate reader, and the inhibition rate and the half inhibitory concentration (IC ₅₀ ) were calculated.

The biological activity of the compound of the present invention was determined by the above test, and the measured IC ₅₀ value is shown in Table 4 below.

Table 4 The inhibitory activity of the compounds of the present invention on STAT5 phosphorylation in CTLL-2 cells

Example compound number

IC ₅₀ (nM)

Example compound number

IC ₅₀ (nM)

001001	11.3111.31	009009	46.8546.85
003003	19.8519.85	010010	143.82143.82
004004	44.5744.57	012012	13.2913.29
005005	18.0318.03	013013	17.2017.20
006006	13.9213.92	015015	47.0847.08
007007	185.27185.27	017017	76.9876.98
008008	200.28200.28	018018	27.2027.20

Test Example 5: Occupation of BTK target in mouse spleen

Experimental principle: This experiment is to evaluate the occupancy of the BTK target by the compound in the mouse spleen. Homogenize the frozen spleen sample, and then incubate it with a biotin-labeled probe compound. The BTK protein that is not occupied by the compound binds to the probe, and the BTK protein that is occupied by the compound cannot bind to the probe. It is detected by the ELISA method. Reflects the occupation of the BTK target by the compound.

laboratory apparatus:

仪器instrument	品牌brand	型号model
离心机Centrifuge	EppendorfEppendorf	58105810
离心机Centrifuge	EppendorfEppendorf	5430R5430R
组织研磨仪Tissue grinder	美壁Beautiful wall	LD48LD48
EnvisionEnvision	Perkin ElmerPerkin Elmer	//

Experimental Materials:

试剂Reagent	品牌brand	货号Item No.
Anti-BTK抗体Anti-BTK antibody	Cell signalCell signal	8547S8547S
链霉亲和素包被板Streptavidin coated plate	R&D SystemsR&D Systems	CP004CP004
CNX-500CNX-500	MCEMCE	HY-100338HY-100338
ELISA显色液ELISA color developing solution	R&D SystemsR&D Systems	DY999DY999
ELISA终止液ELISA stop solution	R&D SystemsR&D Systems	DY994DY994
RIPA裂解液RIPA Lysis Solution	SigmaSigma	R0278R0278
Anti-rabbit IgG抗体Anti-rabbit IgG antibody	Cell signalCell signal	7074S7074S
BCA Protein Assay试剂盒BCA Protein Assay Kit	PiercePierce	2322523225

experimental method:

Using C57BL/6N female mice, the test compound was prepared in 2% Tween80/0.5% methycellulose solution for intragastric administration at a dose of 10 mg/kg, and the spleen was taken 0.5 h or 24 h after administration and stored in dry ice. The frozen spleen samples were homogenized and the protein concentration was detected with the BCA kit. The spleen homogenate after the protein concentration was adjusted to be the same was incubated with the probe compound CNX-500 for 1 hour, and the final concentration of CNX-500 was 1 μM. Then transfer 100 μL/well to the streptavidin-coated plate and incubate overnight. Discard the supernatant and wash, add anti-BTK antibody and incubate for 2 hours. Discard the supernatant and wash, add HRP-labeled Anti-rabbit IgG antibody, and incubate for 1 hour. The supernatant is discarded and washed, and the color is developed for 10-15 minutes with the color developing solution. After the reaction is terminated, the absorbance value is read at a wavelength of 450 nm with Envision. To calculate the occupancy ratio, the calculation formula for the occupancy ratio is:

in:

Signal _max means: the signal generated by the control sample after adding the probe compound;

The signal _min means: the signal generated after the control sample is not added with the probe compound;

The signal _compound to be tested means: the signal generated by adding the probe compound to the sample of the compound to be tested.

The experimental results are shown in Table 5.

Table 5 Occupation of BTK target in mouse spleen by compounds of the present invention

Test Example 6: Inhibition of IL-2 induced phosphorylation of STAT5 in mouse whole blood

Experimental principle: This experiment is to evaluate the effect of compounds on the phosphorylation of STAT5, a downstream substrate of JAK3. After oral administration of mice, whole blood was taken, and the stimulant IL-2 was added to incubate for 15 minutes, and the level of STAT5 phosphorylation in lymphocytes was detected by flow cytometry, thereby reflecting the inhibitory effect of the compound on the JAK3 target.

laboratory apparatus:

仪器instrument	品牌brand	型号model
生物安全柜Biological safety cabinet	ESCOESCO	CLASSⅡ BSCCLASSⅡ BSC
离心机Centrifuge	EppendorfEppendorf	58105810
CO ₂培养箱 CO ₂ incubator	ESCOESCO	CCL-170B-8CCL-170B-8
Flow cytometerFlow cytometer	BD BiosciencesBD Biosciences	Canto IICanto II
-4℃冷藏箱-4℃ freezer	海尔Haier	HYC-650HYC-650

Experimental Materials:

试剂Reagent	品牌brand	货号Item No.
AF647 Mouse Anti-pStat5抗体AF647 Mouse Anti-pStat5 antibody	BD BiosciencesBD Biosciences	562076562076
BV 421 anti-mouse CD8抗体BV 421 anti-mouse CD8 antibody	BiolegendBiolegend	100738100738
BV421 Anti-Mouse CD3抗体BV421 Anti-Mouse CD3 antibody	BD BiosciencesBD Biosciences	740014740014
FITC Anti-Mouse CD4抗体FITC Anti-Mouse CD4 antibody	BD BiosciencesBD Biosciences	553047553047
IL-2IL-2	R&DR&D	402-ML-020402-ML-020
小鼠Fc封闭抗体Mouse Fc blocking antibody	BiolegendBiolegend	156603156603
裂解固定液Lysis Fixative	BD BiosciencesBD Biosciences	558049558049
细胞染色液Cell staining solution	BiolegendBiolegend	420201420201
96孔板96-well plates	CorningCorning	37993799
Perm Buffer IIIPerm Buffer III	BD BiosciencesBD Biosciences	558050558050

96-well deep well plate

Axygen

P-96-450V-C

experimental method:

Using C57BL/6N female mice, the compound to be tested was prepared in a 2% Tween80/0.5% methycellulose solution and administered by gavage at a dose of 10 mg/kg. After 0.5 hours of administration, whole blood was taken and placed in a sodium heparin anticoagulation tube middle. Seed 80μL/well of whole blood in a 96-well plate, add mouse Fc blocking antibody, and then add 5μL/well of detection antibody. In different batches of experiments, the detection antibody is CD8 antibody or CD3 antibody/CD4 antibody mixture. Add 10μL/well of stimulator IL-2, incubate for 15 minutes, the final concentration of stimulant is 200ng/mL. Transfer 60μL/well of whole blood to a 96-well deep well plate, add 350μL/well of lysis fixative, and incubate for 10 minutes. After centrifugation and discarding the supernatant, the cells were resuspended in 100 μL/well Perm Buffer III and incubated for 30 minutes. Discard the supernatant by centrifugation, add 50μL/well of pSTAT5 antibody, and incubate for 30 minutes. After centrifugation and discarding the supernatant, the cells were resuspended in the staining solution, the signal of pSTAT5 was detected by flow cytometry, and the inhibition rate was calculated. The calculation formula of the inhibition rate is:

in:

Signal _max means: the signal generated by the control sample after adding the stimulant IL-2;

Signal _min means: the signal produced by the control sample without the stimulant IL-2;

Signal _{test compound} means: the signal generated by adding the stimulant IL-2 to the sample of the test compound.

The experimental results are shown in Table 6.

Table 6 The inhibitory effect of the compound of the present invention on IL-2 induced STAT5 phosphorylation in mouse whole blood

In the foregoing, the embodiments of the present invention have been described. However, the present invention is not limited to the above-mentioned embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

The compound represented by formula (Ib) or a pharmaceutically acceptable salt thereof:

X is selected from O or NH;

R 1 is selected H, or R a is optionally substituted by the following groups: C 1 -C 10 alkyl, C 3 -C 14 cycloalkyl, 3-14 membered heterocyclyl, C 6 -C 10 aryl group or 5-10 membered heteroaryl;

R a is selected from F, Cl, Br, I, OH, CN, =O, NO 2 or the following groups optionally substituted by R b : 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, C 3 -C 14 cycloalkyl, 3-14 membered heterocyclyl, C 1 -C 10 alkoxy, C 3 -C 14 cycloalkyloxy, 3-14 membered heterocyclyloxy, C 2 -C 10 alkenyl , 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 is selected from F, Cl, Br, I, OH, CN, =O, NO 2 , NH 2 , SH, C 1 -C 10 alkyl, C 3 -C 14 cycloalkyl, 3-14 membered heterocycle Group, C 1 -C 10 alkoxy, C 3 -C 14 cycloalkyloxy, 3-14 membered heterocyclyloxy, C 2 -C 10 alkenyl, 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 is selected from H, F, Cl, Br, I, C 3 -C 14 cycloalkyl or phenyl, the C 3 -C 14 cycloalkyl or phenyl is optionally substituted by R d;

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

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

R 4 is selected from H, F, Cl, Br, I, OH, CN or the following groups optionally substituted by R c : C 1 -C 10 alkyl, C 1 -C 10 alkoxy;

R 5 , R 6 , and R 7 are independently selected from H, F, Cl, Br, I, CN, or the following groups optionally substituted by R e : C 1 -C 10 alkyl, C 3 -C 10 cycloalkane Group or 3-10 membered heterocyclic group;

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

n is selected from 0 or 1;

m is selected from 1 or 2;

The conditions are: when R 2 =Cl, X=O, n=1 and R 1 is not methyl or cyclopropyl; when R 2 =H, X=O and R 1 is not ethyl, CH 2 CHF 2. CH 2 CH 2 OH, cyclopropyl,
When R 2 =F, R 1 is not ethyl or CH 2 CHF 2 .
The compound represented by formula (Ib) or a pharmaceutically acceptable salt thereof according to claim 1, wherein X is selected from NH.
The formula (Ib) compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein, R & lt selected from H or an optionally substituted following groups R a: C 1 -C 10 alkyl Group, C 3 -C 10 cycloalkyl group, 3-10 membered heterocyclic group, C 6 -C 10 aryl group or 5-10 membered heteroaryl group.
The compound represented by formula (Ib) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 is selected from a C 1 -C 10 alkyl group or a 3-10 membered heterocyclic group, and the C 1 -C 10 alkyl or 3-10 membered heterocyclyl optionally substituted with R a.
The compound of formula (Ib) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 2 is selected from C 3 -C 14 cycloalkyl or phenyl, and the C 3 -C 14 Cycloalkyl or phenyl is optionally substituted by Rd.
The compound of formula (Ib) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 2 is selected from cyclopropyl or phenyl, and the cyclopropyl or phenyl is optionally substituted by R d replace.
The compound represented by formula (Ib) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 2 is 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 Its pharmaceutically acceptable salt:

Wherein: X, R 1 , R 2 , R 3 , R 4 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 (I) or Its pharmaceutically acceptable salt:

Wherein, R 1 , R 2 , and R 3 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 (IIb) or Its pharmaceutically acceptable salt:

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 Its pharmaceutically acceptable salt:

Wherein, X, R 1 , R 2 , R 3 , R 4 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 (II) or Its pharmaceutically acceptable salt:

Wherein, R 1 , R 2 , and R 3 are as defined in claim 1.
The compound represented by 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 the compound of any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.
Use of the compound of any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating diseases related to Janus kinase (JAK) and/or Bruton's tyrosine kinase (BTK).
The use according to claim 15, said Janus kinase (JAK) and/or Bruton's tyrosine kinase (BTK) related diseases are tumors or autoimmune diseases.