WO2024140653A1

WO2024140653A1 - Selective cdk12/13 inhibitor and application thereof

Info

Publication number: WO2024140653A1
Application number: PCT/CN2023/141851
Authority: WO
Inventors: 孙韡; 全旭; 李晴晴; 王明逍; 郭靖; 白磊; 张剑; 张小猛; 蒋美玲
Original assignee: 南京圣和药业股份有限公司
Priority date: 2022-12-27
Filing date: 2023-12-26
Publication date: 2024-07-04
Also published as: CN118255746A

Abstract

The present invention belongs to the field of pharmaceutical chemistry, relates to a compound serving as a CDK12/13 inhibitor and to application of the compound, and specifically provides a compound shown in formula (I), or an isomer, pharmaceutically acceptable salt, solvate, crystal, or prodrug thereof, methods for their preparation, pharmaceutical compositions containing said compounds, and the use of said compounds or compositions in the treatment of diseases associated with CDK12/13.

Description

Selective CDK12/13 inhibitors and their applications

Technical Field

The present invention belongs to the field of medicinal chemistry, and specifically relates to compounds or their isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs as selective CDK12/13 inhibitors, their preparation methods, and pharmaceutical compositions containing these compounds and the use of these compounds or compositions for treating diseases related to CDK12/13.

Background technique

Members of the cyclin-dependent kinase (CDK) family play key regulatory roles in cell proliferation. There are more than 20 known mammalian CDKs. CDK12 and CDK13 were identified in a cDNA screen for cell cycle regulators.

CDK12 (also known as CrkRS, CRK7) is a protein composed of 1490 amino acids with a molecular weight of 164kDa. Its protein structure can be divided into three main domains: N-terminal domain, kinase domain and C-terminal domain. In CDK12, the RS domain at the N-terminus is very conserved and plays an important role in mRNA splicing. CDK12 has multiple functions, such as phosphorylating the CTD region of RNA polymerase Pol II, which can promote the elongation of transcription; can interact with RNA processing factors to regulate the RNA shearing process; mediate the phosphorylation of transcribed RNA polymerase II, regulate mRNA 3' end processing; and regulate the polyadenylation and transcription of introns (Cancer Res., 2021, 81, 18-26).

The CDK that is most closely related to CDK12 is CDK13 (also known as CDC2L, CDC2L5, CHED). CDK13 is the largest CDK, consisting of 1512 amino acids. Its kinase domain is 92% similar to the kinase domain of CDK12, but there are significant differences in their structures outside this region. Similar to CDK12, CDK13 can also form heterodimers with CyclinK to play a kinase role. It can also catalyze the phosphorylation of Ser2 and Ser5 in the CTD region of RNA polymerase Pol II like CDK12 to play subsequent functions. Studies have shown (Cell Reports, 2016, 14, 320-331) that in addition to this common biochemical function, CDK12 and CDK13 regulate different gene clusters. In addition, CDK13 can also regulate the expression of snRNA and snoRNA.

Studies have found that CDK12 and CDK13 are closely related to a variety of cancers and pathways, and mutations, amplifications, deletions and fusions of the CDK12/13 genome have been found in cancer. Studies have shown that CDK12/13 gene mutations and overexpression are widely present in colorectal cancer, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, endometrial cancer, bladder cancer, pancreatic cancer and other cancers, and patients with high expression of CDK12/13 in a variety of tumors have a poor prognosis.

Most of the existing small molecule CDK inhibitors are pan-CDK inhibitors. This type of inhibitor usually has defects such as insufficient efficacy, high toxicity, and a narrow safety window. Studies have shown that CDK1 and CDK7 play an important role in cell cycle regulation and transcription in normal cells. Therefore, the development of a new generation of selective CDK inhibitors has certain clinical application significance for improving efficacy and reducing toxic side effects. Given that CDK12/13 plays an important role in the occurrence and development of multiple tumors such as gastric cancer, colorectal cancer, and breast cancer, the development of selective CDK12/13 inhibitors is of great significance. Selective inhibitors should have good clinical application prospects.

Summary of the invention

One object of the present invention is to provide a class of compounds having CDK12/13 inhibitory activity represented by general formula (I), general formula (II) or general formula (III) or their isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs.

Another object of the present invention is to provide a method for preparing the compound of general formula (I), general formula (II) or general formula (III) of the present invention or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug.

Another object of the present invention is to provide a composition comprising a compound of the general formula (I), general formula (II) or general formula (III) of the present invention or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug and a pharmaceutically acceptable carrier, as well as a composition comprising a compound of the general formula (I), general formula (II) or general formula (III) of the present invention or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug and another one or more drugs.

Another object of the present invention is to provide a method for treating diseases associated with CDK12/13 using the compounds of the general formula (I), general formula (II) or general formula (III) of the present invention or their isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs, as well as the use of the compounds of the general formula (I), general formula (II) or general formula (III) of the present invention or their isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs in the preparation of drugs for treating diseases associated with CDK12/13.

In view of the above-mentioned invention object, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a compound represented by general formula (I) or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof,

in,

_X1 and _X2 are each independently selected from _CH2 , CH, NH, N and S;

_R is selected from halogen, hydroxy, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxyl, cyano, amino, oxygen, sulfur, mercapto, alkylthio, monoalkylamino, alkylacylamino, alkylacyl, aminoacyl, alkylaminoacyl, dialkylamino, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkylthio, heterocyclylalkylthio, arylalkylthio and heteroarylalkylthio, which is optionally substituted with one or more alkyl, alkoxy, alkylamino, halogen, hydroxy, amino, nitro, cyano, haloalkyl, alkylacyl, aminoacyl or alkylaminoacyl;

_R2 , _R3 and _R4 are each independently selected from hydrogen, halogen, hydroxy, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy alkyl, alkylamino, alkylthio, alkylamino, alkylacylamino, alkylacyl, aminoacyl, alkylaminoacyl, dialkylamino, and oxo groups;

_R is selected from halogen, hydroxy, carbonyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxyl, cyano, amino, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, optionally substituted with one or more alkyl, alkenyl, alkynyl, alkoxy, alkylamino, halogen, hydroxy, amino, haloalkyl, alkylacyl, aminoacyl or alkylaminoacyl;

p and q are each independently selected from 0, 1, 2, 3 and 4; and

is a single bond or a double bond, two of which All are single keys, or one is a single bond and the other is a double bond.

In a second aspect, the present invention provides a compound represented by general formula (II) or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof,

Wherein, _X1 , _X2 , _R1 , _R2 , _R3 , _R4 , _R5 , p, q and As defined in general formula (I).

In a third aspect, the present invention provides a compound represented by general formula (III) or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof,

Wherein, _X1 , _X2 , _R1 , _R2 , _R3 , _R4 , _R5 , p, q and As defined in formula (I) and formula (II).

In some preferred embodiments, the compound of the present invention is a compound of general formula (I), general formula (II) or general formula (III) or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof, wherein the group Selected from

In some specific embodiments, the compound of the present invention is a compound of formula (I), formula (II) or formula (III) or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof, wherein the group for Said Can be

In some embodiments, the compound of the present invention is a compound of formula (I), formula (II) or formula (III) or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof, wherein:

_R1 is selected from halogen, hydroxy, _C1-6 alkyl, halo-substituted _C1-6 alkyl, hydroxy _C1-6 alkyl, _C1-6 alkoxy, halo-substituted _C1-6 alkoxy, hydroxy _C1-6 alkoxy, nitro, carboxyl, cyano, amino, oxygen, sulfur, mercapto, _C1-6 alkylthio, mono- _C1-6 alkylamino, _C1-6 alkylacylamino, _C1-6 alkylacyl, aminoacyl, _C1-6 alkylaminoacyl, di- _C1-6 alkylamino, _C3-8 cycloalkyl, _C3-8 heterocyclyl, _C6-12 aryl, _C5-12 heteroaryl _, _C3-8 _{cycloalkylC1-6} alkylthio, _C3-8 heterocyclylC1-6 alkylthio, _C6-12 _arylC1-6 alkylthio and _C5-12 heteroarylC C _1-6 alkylthio, which is optionally substituted with one or more C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylamino, halogen, hydroxy, amino, nitro, cyano, halogenated C _1-3 alkyl, C _1-3 alkylacyl, aminoacyl or C _1-3 alkylaminoacyl;

Further preferably, _R1 is selected from halogen, hydroxyl, _C1-3 alkyl _, halogenated C1-3 alkyl, hydroxy _C1-3 alkyl, _C1-3 alkoxy, halogenated _C1-3 alkoxy, hydroxy _C1-3 alkoxy, nitro, carboxyl, cyano, amino, oxygen, sulfur, thiol, _C1-3 alkylthio, mono _C1-3 alkylamino, C1-3 alkylacylamino, _C1-3 alkylacyl, aminoacyl, _C1-3 alkylaminoacyl, di- _C1-3 alkylamino, _C3-8 cycloalkyl _{, C3-8 heterocyclyl, C6-12 aryl, C5-12 heteroaryl, C3-8 cycloalkyl C1-3 alkylthio, C3-8 heterocyclyl C1-3} _alkylthio _, _C6-12 _aryl _C1-3 _alkylthio _and _C5-12 _heteroaryl _C C _1-3 alkylthio, which is optionally substituted with one or more C _1-3 alkyl, C _1-3 alkoxy, C _1-3 alkylamino, halogen, hydroxy, amino, nitro, cyano, halogenated C _1-3 alkyl, C _1-3 alkylacyl, aminoacyl or C _1-3 alkylaminoacyl;

More preferably, _R1 is selected from halogen, hydroxy, methyl, ethyl, propyl, isopropyl, isobutyl, trifluoromethyl, trifluoroethyl, hydroxy _C1-3 alkyl, _C1-3 alkoxy, halogenated _C1-3 alkoxy, hydroxy _C1-3 alkoxy, nitro, carboxyl, cyano, amino, oxygen, sulfur, thiol, _C1-3 alkylthio, mono _C1-3 alkylamino, C1-3 alkylacylamino, _C1-3 alkylacyl, aminoacyl, _C1-3 alkylaminoacyl, di- _C1-3 alkylamino, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxa _- _C3-6 cyclic group, aza _-C3-6 cyclic group, _C6-12 aryl, aza- _C5-12 heteroaryl, oxa _-C5-12 heteroaryl, aza _-C5-12 heteroaryl, _C3-6 cycloalkyl C C _1-3 alkylthio, oxa-C _3-6 heterocyclyl C _1-3 alkylthio, aza-C _3-6 heterocyclyl C _1-3 alkylthio, aza-C _3-6 heterocyclyl C _1-3 alkylthio, C _6-12 aryl C _1-3 alkylthio, aza-C _5-12 heteroaryl C _1-3 alkylthio, oxa-C _5-12 heteroaryl C _1-3 alkylthio, aza-C _5-12 heteroaryl C _1-3 alkylthio, which is optionally substituted with one or more hydroxyl, methyl, ethyl, propyl, isopropyl, isobutyl, trifluoromethyl, trifluoroethyl, C _1-3 alkoxy, C _1-3 alkylamino, halogen, hydroxyl, amino, nitro, cyano, halogenated C _1-3 alkyl, C _1-3 alkylacyl, aminoacyl or C _1-3 alkylaminoacyl.

In some specific embodiments, according to the compound of formula (I), formula (II) or formula (III) or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug of the present invention, _R1 is selected from Trifluoromethyl,

R ₂ , R ₃ and R ₄ are each independently selected from hydrogen, halogen, hydroxy, alkyl, halogenated C _1-6 alkyl, hydroxy C _1-6 alkyl, C _1-6 alkoxy, halogenated C _1-6 alkoxy, hydroxy C _1-6 alkoxy, nitro, carboxyl, cyano, amino, C _1-6 alkylthio, mono C _1-6 alkylamino, C _1-6 alkylacylamino, C _1-6 alkylacyl, aminoacyl, C _1-6 alkylaminoacyl, di-C _1-6 alkylamino and oxo;

Further preferably, R ₂ , R ₃ and R ₄ are each independently selected from hydrogen, halogen, hydroxy, C _1-3 alkyl, halogenated C _1-3 alkyl, hydroxy C _1-3 alkyl, C _1-3 alkoxy, halogenated C _1-3 alkoxy, hydroxy C _1-3 alkoxy, nitro, carboxyl, cyano, amino, C _1-3 alkylthio, mono C _1-3 alkylamino, C _1-3 alkylacylamino, C _1-3 alkylacyl, aminoacyl, C _1-3 alkylaminoacyl, di-C _1-3 alkylamino and oxo groups;

Still further preferably, R ₂ , R ₃ and R ₄ are each independently selected from hydrogen, halogen, hydroxy, methyl, ethyl, propyl, isopropyl, isobutyl, trifluoromethyl, trifluoroethyl, hydroxy C _1-3 alkyl, C _1-3 alkoxy, halogenated C _1-3 alkoxy, hydroxy C _1-3 alkoxy, nitro, carboxyl, cyano, amino, oxygen, sulfur, mercapto, C _1-3 alkylthio, mono C _1-3 alkylamino, C _1-3 alkylacylamino, C _1-3 alkylacyl, aminoacyl, C _1-3 alkylaminoacyl, di-C _1-3 alkylamino and oxo.

In some specific embodiments, according to the compounds of formula (I), (II) or (III) of the present invention or their isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs, _R2 is selected from hydrogen, methyl, ethyl, propyl, fluorine, chlorine, bromine, _R3 is selected from hydrogen, methyl, ethyl, propyl, fluorine, chlorine, bromine, and _R4 is selected from hydrogen, methyl, ethyl, propyl, fluorine, chlorine, bromine, trifluoromethyl and oxo.

_R5 is selected from halogen, hydroxy, carbonyl, _C1-6 alkyl, halogenated C1-6 alkyl, hydroxy _C1-6 alkyl, _C1-6 alkoxy, halogenated _C1-6 alkoxy, hydroxy _C1-6 alkoxy, nitro _{, carboxyl, cyano, amino, C1-6 alkylcarbonyl, C2-6} _{alkenylcarbonyl} _, _C2-6 alkynylcarbonyl, which is optionally substituted with one or more C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkoxy, C _1-6 alkylamino, halogen, hydroxy, amino, halogenated C _1-6 alkyl, C _1-6 alkylacyl, aminoacyl or C _1-6 alkylaminoacyl;

Further preferably, R ₅ is selected from halogen, hydroxy, carbonyl, C _1-3 alkyl, halogenated C _1-3 alkyl, hydroxy C _1-3 alkyl, C _1-3 alkoxy, halogenated C _1-3 alkoxy, hydroxy C _1-3 alkoxy, nitro, carboxyl, cyano, amino, C _1-3 alkylcarbonyl, C _2-4 alkenylcarbonyl, C _2-4 alkynylcarbonyl, which is optionally substituted with one or more C _1-3 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, C _1-3 alkoxy, C _1-3 alkylamino, halogen, hydroxy, amino, halogenated C _1-3 alkyl, C _1-3 alkylacyl, aminoacyl or C _1-3 alkylaminoacyl;

Still further preferably, R ₅ is selected from halogen, hydroxy, carbonyl, methyl, ethyl, propyl, isopropyl, isobutyl, trifluoromethyl, trifluoroethyl, hydroxy C _1-3 alkyl, C _1-3 alkoxy, halogenated C _{1-3 alkoxy, hydroxy C 1-3} _alkoxy , nitro, carboxyl, cyano, amino, C _1-3 alkylcarbonyl, C 2-4 alkenylcarbonyl, C _2-4 alkynylcarbonyl, which is optionally substituted _by one or more C _1-3 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, C _1-3 alkoxy, C _1-3 alkylamino, halogen, hydroxy, amino, halogenated C _1-3 alkyl, C _1-3 alkylacyl, aminoacyl or C _1-3 alkylaminoacyl.

In some specific embodiments, according to the compound of formula (I), formula (II) or formula (III) of the present invention or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug, _R5 is selected from

In some embodiments, the compound of the present invention is a compound of formula (I), formula (II) or formula (III) or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof, wherein p and q are each independently selected from 0, 1, 2 and 3.

In some embodiments, according to the compounds of general formula (I), general formula (II) or general formula (III) of the present invention, or their isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs, p is 0, and q is 1, 2 or 3. In other specific embodiments, according to the compounds of general formula (I), general formula (II) or general formula (III) of the present invention, or their isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs, p is 1, and q is 0, 1, 2 or 3. In other specific embodiments, according to the compounds of general formula (I), general formula (II) or general formula (III) of the present invention, or their isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs, p is 1, and q is 0, 1, 2 or 3.

In some specific embodiments, the compound of general formula (I), general formula (II) or general formula (III) according to the present invention or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug, for In other specific embodiments, the compound of general formula (I), general formula (II) or general formula (III) or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug according to the present invention, for In other specific embodiments, the compound of general formula (I), general formula (II) or general formula (III) or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug according to the present invention, for

The present invention provides the following specific compounds or their isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs:

In a fourth aspect, the present invention provides a pharmaceutical composition comprising a compound represented by Formula I, Formula (II) or Formula (III) of the present invention or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof.

In some embodiments, the present invention provides compounds of Formula I, Formula (II) or Formula (III) of the present invention or isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs thereof, and pharmaceutical compositions comprising compounds of Formula I, Formula (II) or Formula (III) of the present invention or isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs thereof, wherein the compounds or pharmaceutical compositions are used to treat diseases associated with CDK12/13.

In some embodiments, the present invention provides a pharmaceutical composition comprising a compound represented by Formula I, Formula (II) or Formula (III) of the present invention or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug and a pharmaceutically acceptable carrier.

The compound shown in the general formula I, general formula (II) or general formula (III) of the present invention or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation, so as to be suitable for oral or parenteral administration. The method of administration includes, but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal and oral routes. The preparation can be administered by any route, such as by infusion or push injection, by the route of absorption through the epithelium or skin mucosa (such as oral mucosa or rectum, etc.). Administration can be systemic or local. Examples of oral administration preparations include solid or liquid dosage forms, specifically, tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions, etc. The preparation can be prepared by methods known in the art and include carriers, diluents or excipients conventionally used in the field of pharmaceutical preparations.

In a fifth aspect, the present invention provides a method for treating diseases associated with CDK12/13 using a compound represented by Formula I, Formula (II) or Formula (III) of the present invention or its isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs, or a pharmaceutical composition comprising the same, as well as use of the same in the preparation of drugs for treating diseases associated with CDK12/13.

In some embodiments, the present invention provides compounds represented by Formula I, Formula (II) or Formula (III) of the present invention or their isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs, or pharmaceutical compositions comprising the same for treating diseases associated with abnormal activity of CDK12/13.

In some preferred embodiments, the present invention provides a compound represented by general formula I, general formula (II) or general formula (III) of the present invention or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof, or a pharmaceutical composition comprising the same for use in treating CDK12/13-related diseases. The invention relates to a method for treating a disease associated with CDK12/13 and a use thereof in the preparation of a medicament for treating a disease associated with CDK12/13, wherein the disease associated with CDK12/13 includes but is not limited to proliferative diseases, metabolic diseases, inflammatory disorders, autoimmune diseases and infectious diseases. In some embodiments, the proliferative disease associated with CDK12/13 of the present invention is cancer.

In some embodiments, the present invention provides a method for treating a disease mediated by CDK12/13, comprising administering a therapeutically effective amount of a compound of formula I, formula (II) or formula (III) of the present invention, or an isomer thereof, a pharmaceutically acceptable salt, solvate, crystal or prodrug or pharmaceutical composition thereof, comprising administering a therapeutically effective amount of a compound of formula I, formula (II) or formula (III) of the present invention, or an isomer thereof, a pharmaceutically acceptable salt, solvate, crystal or prodrug or pharmaceutical composition thereof. In some embodiments, the present invention provides a method for treating a proliferative disease, comprising administering a therapeutically effective amount of a compound of formula I, formula (II) or formula (III) of the present invention, or an isomer thereof, a pharmaceutically acceptable salt, solvate, crystal or prodrug thereof. In some embodiments, the present invention provides a method for inhibiting tumor cell growth and/or metastasis, comprising administering a therapeutically effective amount of a compound of formula I, formula (II) or formula (III) of the present invention, or an isomer thereof, a pharmaceutically acceptable salt, solvate, crystal or prodrug thereof.

In some embodiments, the diseases associated with CDK12/13 described in the present invention include but are not limited to: acoustic neuroma, adenocarcinoma, adrenal cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendothelial sarcoma, angiosarcoma), adnexal cancer, benign monoclonal gammopathy, bile cancer (e.g., bile duct cancer), bladder cancer, breast cancer (e.g., breast adenocarcinoma, breast papillary carcinoma, breast cancer, breast medullary carcinoma, triple-negative breast cancer), brain cancer (e.g., meningioma; glioma, such as astrocytoma, oligodendroglioma; medulloblastoma), bronchial carcinoma, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial cancer, ependymoma, endothelial sarcoma (e.g., Kaposi's sarcoma (Kaposi's sarcoma), multiple idiopathic hemorrhagic sarcomas), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarinoma), Ewing sarcoma (e.g., sarcoma), eye cancer (e.g., intraocular melanoma, retinoblastoma), familial eosinophilia, gallbladder cancer, stomach cancer (e.g., gastric adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma), oral cancer (e.g., oral squamous cell carcinoma (OSCC), pharyngeal cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal carcinoma, oropharyngeal cancer)), hematopoietic cancer (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myeloid leukemia (CML) (e.g., B-cell CML, T-cell CML) and chronic lymphocytic leukemia (CLL) (e.g., B-cell CL L, T-cell CLL); lymphomas such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., "Waldenstrom's macroglobulinemia ( macroglobulinemia)”), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and primary central nervous system (CNS) lymphomas; and T-cell NHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (eg, mycosis fungiodes, Sezary syndrome syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia/lymphomas as described above; and multiple myeloma (MM), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumor, immune cell amyloidosis, kidney cancer (e.g., Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular carcinoma (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disease myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelofibrosis, chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic leukemia syndrome (HES)), neuroblastoma, neurofibroma (e.g., multiple neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma, ovarian clear cell carcinoma, ovarian serous cystadenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), islet cell tumor), penile cancer (e.g., Paget's disease of the penis and scrotum), disease), pinealoma, primary neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary duct cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small intestine cancer (e.g., adnexal cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary thyroid carcinoma, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer, and vulvar cancer (e.g., Paget's disease of the vulva), medulloblastoma, adenoid cystic carcinoma, melanoma, glioblastoma.

In some preferred embodiments, the present invention provides a compound represented by general formula I, general formula (II) or general formula (III) of the present invention or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug, or a pharmaceutical composition comprising the same for use in a method for treating a disease associated with CDK12/13 and in the preparation of a medicament for treating a disease associated with CDK12/13, wherein the disease associated with CDK12/13 includes but is not limited to: breast cancer, esophageal cancer, bladder cancer, lung cancer, hematopoietic system cancer, lymphoma, medulloblastoma, medulloblastoma, rectal adenocarcinoma, colon cancer, gastric cancer, pancreatic cancer, liver cancer, adenoid cystic carcinoma, prostate cancer, lung cancer, head and neck squamous cell carcinoma, brain cancer, hepatocellular carcinoma, melanoma, oligodendroglioma, glioblastoma, testicular cancer, ovarian clear cell carcinoma, ovarian serous cystadenocarcinoma, thyroid cancer, multiple myeloma (AML), renal cell carcinoma, mantle cell lymphoma, triple-negative breast cancer, and non-small cell lung cancer.

In some specific embodiments, the CDK12/13-mediated cancer is breast cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, colon cancer, kidney cancer, bladder cancer, head and neck cancer, thyroid cancer, esophageal cancer, gastric cancer, pancreatic cancer, ovarian cancer, gallbladder cancer, cervical cancer, prostate, skin cancer, blood system tumors, nervous system tumors, melanoma, osteosarcoma or Kaposi's sarcoma.

Definition of Terms

Unless stated otherwise, the terms used in the specification and claims have the following meanings.

"Hydrogen", "carbon" and "oxygen" in the compounds of the present invention include all isotopes thereof. Isotopes should be understood to include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include protium, tritium and deuterium, isotopes of carbon include ¹² C, ¹³ C and ¹⁴ C, isotopes of oxygen include ¹⁶ O and ¹⁸ O, etc.

The "isomer" of the present invention refers to a molecule with the same atomic composition and connection mode, but different three-dimensional spatial arrangement, including but not limited to diastereomers, enantiomers, cis-trans isomers, and mixtures thereof, such as racemic mixtures. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane polarized light. When describing optically active compounds, the prefix D, L or R, S is used to indicate the absolute configuration of the chiral center of the molecule. The prefix D, L or (+), (-) is used to name the sign of rotation of plane polarized light of the compound, (-) or L means that the compound is left-handed, and the prefix (+) or D means that the compound is right-handed. The chemical structure of these stereoisomers is the same, but their stereostructures are different. Specific stereoisomers can be enantiomers, and a mixture of isomers is usually called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may result in no stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers, devoid of optical activity.

Depending on the choice of starting materials and process, the compounds of the invention may exist in the form of one of the possible isomers or a mixture thereof, such as a racemate and a diastereomeric mixture (depending on the number of asymmetric carbon atoms). Optically active (R)- or (S)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.

Any resulting mixture of stereoisomers can be separated into the pure or substantially pure geometric isomers, enantiomers, diastereomers on the basis of the differences in the constituent physicochemical properties, for example, by chromatography and/or fractional crystallization.

The "halogen" of the present invention refers to fluorine, chlorine, bromine, and iodine. The "halogenated" of the present invention refers to substitution with fluorine, chlorine, bromine, or iodine.

The "alkyl" of the present invention refers to a straight or branched saturated aliphatic hydrocarbon group, preferably a straight or branched group containing 1 to 6 carbon atoms, and further preferably a straight or branched group containing 1 to 3 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, etc. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be at any available point of attachment.

The "alkenyl" of the present invention refers to a straight or branched hydrocarbon chain radical consisting only of carbon atoms and hydrogen atoms, containing at least one carbon-carbon double bond, and having 2 to 12 carbon atoms. In some embodiments, the alkenyl group comprises 2 to 8 carbon atoms. In other embodiments, the alkenyl group comprises 2 to 4 carbon atoms. The alkenyl group is connected to the rest of the molecule by a single bond, such as vinyl, allyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, etc. The alkenyl group is optionally substituted with one or more other substituents, such as alkyl, halogen, cyano, nitro, oxygen, sulfur, imino, haloalkyl, alkoxy, cycloalkyl, heterocyclic radical, heteroaryl, heteroaryl, etc.

The term "alkynyl" in the present invention refers to a straight or branched hydrocarbon chain radical consisting only of carbon atoms and hydrogen atoms, containing at least one carbon- A alkynyl group is a 1-carbon triple bond and has 2 to 12 carbon atoms. In some embodiments, the alkynyl group contains 2 to 8 carbon atoms. In other embodiments, the alkynyl group contains 2 to 4 carbon atoms. The alkynyl group is connected to the rest of the molecule by a single bond, such as ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. The alkynyl group is optionally substituted with one or more other substituents, such as alkyl, halogen, cyano, nitro, oxygen, sulfur, imino, haloalkyl, alkoxy, cycloalkyl, heterocyclyl, heteroaryl, heteroaryl, etc.

The "carbonyl group" and "acyl group" of the present invention both refer to -C(O)-.

The "sulfonyl group" of the present invention refers to -S(O) ₂ -.

The "sulfonamide group" of the present invention refers to -S(O) ₂ NH-.

The "haloalkyl group" of the present invention refers to an alkyl group substituted with at least one halogen.

The "hydroxyalkyl group" of the present invention refers to an alkyl group substituted with at least one hydroxy group.

"Alkoxy" of the present invention refers to -O-alkyl. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, n-propoxy, isopropoxy, isobutoxy, sec-butoxy, etc. Alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituent can be at any available point of attachment.

The "cycloalkyl" of the present invention refers to a cyclic saturated hydrocarbon group. Suitable cycloalkyl groups may be substituted or unsubstituted monocyclic, bicyclic or tricyclic saturated hydrocarbon groups having 3 to 12 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The "heterocyclyl" of the present invention refers to a group of a 3- to 12-membered non-aromatic ring system having 1 to 4 ring heteroatoms, each of which is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon ("3-12 membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as long as the valence permits. The heterocyclyl group can be either monocyclic ("monocyclic heterocyclyl") or a fused, bridged or spiral ring system (e.g., a bicyclic system (also known as a "bicyclic heterocyclyl")) and can be saturated or partially unsaturated. Suitable heterocyclyls include, but are not limited to, piperidinyl, azetidinyl, aziridine, tetrahydropyrrolyl, piperazinyl, dihydroquinazolinyl, oxacyclopropyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, etc. Each example of a heterocyclyl group can be optionally substituted or unsubstituted, and when substituted, the substituent can be at any usable point of attachment.

The "aryl" of the present invention refers to an aromatic system that may contain a monocyclic or fused polycyclic ring, preferably a monocyclic or fused bicyclic ring, containing 6 to 12 carbon atoms, preferably containing about 6 to about 10 carbon atoms. Suitable aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, fluorenyl, indanyl. Aryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents may be at any usable point of attachment.

The "heteroaryl" of the present invention refers to an aromatic group in which at least one carbon atom is replaced by a heteroatom, preferably composed of 5-12 atoms (5-12-membered heteroaryl), and more preferably composed of 5-10 atoms (5-10-membered heteroaryl), wherein the heteroatom is O, S, or N. The heteroaryl groups include, but are not limited to, imidazolyl, pyrrolyl, furanyl, thienyl, pyrazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, tetrazolyl, indolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, isoindolyl, benzopyrazolyl, benzimidazolyl, benzofuranyl, benzopyranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzisoxazolyl, benzisothiazolyl, quinolyl, isoquinolyl, quinazolinyl, cinnolinyl, quinoxalinyl, benzoxazinyl, benzothiazinyl, imidazopyridinyl, pyrimidopyrazolyl, pyrimidoimidazolyl, etc. The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituents may be at any available point of attachment.

The "pharmaceutically acceptable salt" of the present invention refers to the salt of the compound of the present invention, which is safe and effective when used in mammals and has the desired biological activity.

The "solvate" of the present invention refers to a complex formed by the combination of a solute (such as an active compound, a salt of an active compound) and a solvent (such as water) in a conventional sense. The solvent refers to a solvent known to or easily determined by a person skilled in the art. If it is water, the solvate is usually referred to as a hydrate, such as a hemihydrate, a monohydrate, a dihydrate, a trihydrate or an alternative thereof.

The in vivo effects of the compounds of formula (I) may be partially exerted by one or more metabolites formed in the human or animal body after the compound of formula (I) is administered. As described above, the in vivo effects of the compounds of formula (I) may also be exerted via the metabolism of precursor compounds ("prodrugs"). "Prodrugs" of the present invention refer to compounds that are converted into compounds of the present invention under physiological conditions in an organism due to reactions with enzymes, gastric acid, etc., i.e., compounds that are converted into compounds of the present invention by oxidation, reduction, hydrolysis, etc. of enzymes and/or compounds that are converted into compounds of the present invention by hydrolysis reactions of gastric acid, etc.

The term "crystalline" in the present invention refers to a solid whose internal structure is formed by regularly repeating constituent atoms (or groups thereof) in three dimensions, and is distinguished from an amorphous solid that does not have such a regular internal structure.

The "pharmaceutical composition" of the present invention refers to a mixture comprising any one of the compounds of the present invention, including corresponding isomers, prodrugs, solvates, pharmaceutically acceptable salts or chemically protected forms thereof, and one or more pharmaceutically acceptable carriers and/or one or more other drugs. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to an organism. The composition is generally used to prepare a drug for the treatment and/or prevention of a disease mediated by one or more kinases.

The "pharmaceutically acceptable carrier" of the present invention refers to a carrier that does not cause significant irritation to an organism and does not interfere with the biological activity and properties of the administered compound, including all solvents, diluents or other excipients, dispersants, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. Unless any conventional carrier medium is incompatible with the compound of the present invention. Some examples of pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, as well as cellulose and cellulose acetate; malt, gelatin, etc.

The "excipient" of the present invention refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Excipients may include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols.

Detailed ways

The present invention is further described in detail below in conjunction with the examples, but the present invention is not limited to these examples. The materials used in the following examples are all commercially available unless otherwise specified.

Intermediate 1: Preparation of tert-butyl 3-amino-5-cyclopropyl-1H-pyrazole-1-carboxylate

Sodium hydroxide (36.43 g, 910.75 mmol, 11.2 eq) was dissolved in a mixed solution of dichloromethane (133 mL) and water (162 mL), and 5-cyclopropyl-1H-pyrazole-3-amine (10.0 g, 81.19 mmol, 1.0 eq) was added. The mixed solution was placed in an ice bath and cooled to 0°C. Under this condition, di-tert-butyl dicarbonate (21.26 g, 97.43 mmol, 1.2 eq) was added. After stirring at 0°C for 5 min, the mixture was moved to room temperature and stirred overnight. Diluted with water, extracted with dichloromethane 3 times. The organic phases were combined, dried, filtered, and concentrated. Purification by column chromatography gave 12.8 g of the title compound. ESI-MS m/z: 124.1 [M+H-Boc] ⁺ .

Intermediate 2: 5-((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-amine

Step 1: Preparation of 1-((5-(tert-butyl)oxazol-2-yl)methyl)thiourea

Dissolve 5-tert-butyl-2-(chloromethyl)oxazole (2.49 g, 14.34 mmol, 1.0 equiv) and thiourea (1.09 g, 14.34 mmol, 1.0 equiv) in 25 mL of anhydrous ethanol, protect with argon, stir at 80°C for 3 h, and monitor the reaction. After the reaction is complete, concentrate to obtain 3.05 g of the title compound. ESI-MS m/z: 214.09 [M+H] ⁺ .

Step 2: Preparation of 5-((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-amine

1-((5-(tert-butyl)oxazol-2-yl)methyl)thiourea (3.05 g, 14.33 mmol, 1.0 eq), 2-amino-5-bromothiazole (2.56 g, 14.33 mmol, 1.0 eq) and tetra-n-butylammonium sulfate (142 mg, 0.24 mmol, 0.017 eq) were added to a mixed solution of sodium hydroxide (8.59 g, 214.41 mmol, 15.0 eq) in 25 mL of water and 25 mL of toluene, and stirred at room temperature for 3 h. The reaction was monitored by LC-MS. After completion, dilute with water, extract with ethyl acetate three times, combine the organic phases, dry, filter, concentrate, and chromatograph on a C18 column to obtain the title compound. ESI-MS m/z: 270.08 [M+H] ⁺ .

Example 1: 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide

Step 1: Preparation of methyl 2-(4-bromo-1H-pyrazol-1-yl)propanoate

4-Bromo-1H-pyrazole (5.0 g, 34.02 mmol, 1.0 eq), methyl 2-bromopropionate (6.82 g, 40.82 mmol, 1.2 eq) and potassium carbonate (9.4 g, 68.04 mmol, 2.0 eq) were added to anhydrous N,N-dimethylformamide and reacted at room temperature for 2.0 h. The reaction was monitored by LC-MS. After the reaction of the raw materials was completed, water was added to quench the reaction, and ethyl acetate was used for extraction. The organic phases were combined, washed with water 3 times, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 8.0 g of the title compound. ESI-MS m/z: 233.1 [M+H] ⁺ .

Step 2: Preparation of 2-(4-bromo-1H-pyrazol-1-yl)propanoic acid

Dissolve methyl 2-(4-bromo-1H-pyrazol-1-yl)propanoate (8.0 g, 34.3 mmol, 1.0 eq) in 50 mL of a mixed solvent (methanol: water = 10:1), add lithium hydroxide (3.29 g, 137.3 mmol, 4.0 eq), and react at room temperature for 2.0 h. LC-MS tracking and monitoring the reaction, after the reaction is completed, add water to quench, adjust the pH to 3-4, extract with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 2.62 g of the title compound. ESI-MS m/z: 219.0 [M + H] ⁺ .

Step 3: Preparation of tert-butyl 3-(2-(4-bromo-1H-pyrazol-1-yl)propionamido)-5-cyclopropyl-1H-pyrazole-1-carboxylate

Dissolve 2-(4-bromo-1H-pyrazol-1-yl)propionic acid (500 mg, 2.28 mmol, 1.0 eq) in 10 mL of anhydrous dichloromethane, add a drop of N,N-dimethylformamide, cool to 0°C in an ice bath, add oxalyl chloride (589 mg, 4.56 mmol, 2.0 eq), stir at 0°C for 20 min, move to room temperature and react for 1.0 h. LC-MS tracking and monitoring the reaction, the reaction is complete. After the solvent is dried, dissolve in 10 mL of anhydrous dichloromethane, add N,N-diisopropylethylamine (1.18 g, 2.51 mmol, 3.0 eq), cool to 0°C in an ice bath, add 3-amino-5-cyclopropyl-1H-pyrazole -1-carboxylic acid tert-butyl ester (585 mg, 2.51 mmol, 1.1 eq) was moved to room temperature for reaction for 1.0 h. The reaction was monitored by LC-MS and the reaction was complete. Concentration and sand making, column chromatography purification, to obtain 758 mg of the title compound. ESI-MS m/z: 324.1 [M + H-Boc] ⁺ .

Step 4: Preparation of tert-butyl 5-(1-((1-(tert-butoxycarbonyl)-5-cyclopropyl-1H-pyrazol-3-yl)amino)-1-oxopropan-2-yl)-1H-pyrazol-4-yl)-3,6-dihydropyridine-1(2H)-carboxylate

tert-Butyl 3-(2-(4-bromo-1H-pyrazol-1-yl)propionamido)-5-cyclopropyl-1H-pyrazole-1-carboxylate (758 mg, 1.79 mmol, 1.0 eq), tert-butyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-1(2H)-pyridinecarboxylate (775 mg, 2.51 mmol, 1.4 eq), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (131 mg, 0.18 mmol, 0.1 eq) and cesium carbonate (1.75 g, 5.37 mmol, 3.0 eq) were dissolved in 11 mL of a mixed solvent (1,4-dioxane: water = 10:1), replaced with nitrogen three times, and heated to reflux at 100°C for 2.0 h. The reaction was monitored by LC-MS. After the reaction was completed, the mixture was washed with saturated brine, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and sanded, and purified by column chromatography to obtain 860 mg of the title compound. ESI-MS m/z: 427.2 [M+H-Boc] ⁺ .

Step 5: Preparation of N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)propanamide

Dissolve tert-butyl 5-(1-((1-(tert-butoxycarbonyl)-5-cyclopropyl-1H-pyrazol-3-yl)amino)-1-oxopropan-2-yl)-1H-pyrazol-4-yl)-3,6-dihydropyridine-1(2H)-carboxylate (860 mg, 1.63 mmol, 1.0 eq) in 10 mL of anhydrous dichloromethane, cool to 0°C in an ice bath, add 1 mL of trifluoroacetic acid, and move to room temperature for 24 h. LC-MS tracking and monitoring of the reaction, the reaction is complete. The reaction solution is concentrated under reduced pressure to remove excess trifluoroacetic acid. Add 10 mL of dichloromethane, cool to 0°C in an ice bath, slowly drop N,N-diisopropylethylamine to adjust the pH to 9-10, and remove the solvent to obtain 1.47 g of the title compound. ESI-MS m/z: 327.2[M+H] ⁺ .

Step 6: Preparation of 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide

N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)propanamide (1.47 g, 2.66 mmol, 1.0eq) was dissolved in 15mL of anhydrous dichloromethane, cooled to 0°C in an ice bath, and N,N-diisopropylethylamine (1.03g, 7.98mmol, 3.0eq) and acryloyl chloride (120mg, 1.33mmol, 0.5eq) were added. The reaction was continued for 0.5h in an ice bath. The reaction was monitored by LC-MS and the reaction was completed. The solvent was dried and purified by C18 column to obtain 240mg of the title compound. ESI-MS m/z: 381.3[M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ12.12(s,1H),10.57(s,1H),8.08-7.93(m,1H),7.65(d,J＝16.0Hz,1H),7.05–6.83(m,1H),6.25-6.05(m,3H),5.75-5.66(m,1H),5.17(q,J＝8.0Hz,1H),4.35-4.21(m,2H),3.74-3.58(m,2H),2.30-2.16(m,2H),1.88-1.79(m,1H),1.65(d,J＝8.0Hz,3H),0.96-0.82(m,2H),0.70-0.54(m,2H).

The racemate of 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide (240 mg) was separated by chiral preparative column. (Method: Chiral column: ChiralCel OD, 250×30 mm I.D., 10 μm, elution: isocratic: B 35%, A = carbon dioxide, B = ethanol (0.1% ammonia water)) to obtain isomer 1 (retention time 1.730, 116 mg) and isomer 2 (retention time 2.912, 116 mg) of the title compound.

Isomer 1 (Example 2): ESI-MS m/z: 381.3 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.12(s,1H),10.57(s,1H),8.08-7.93(m,1H),7.65(d,J＝16.0Hz,1H),7.05–6.83(m,1H),6.25-6.05(m,3H),5.75-5.66(m,1H),5.17(q,J＝8.0Hz,1H),4.35-4.21(m,2H),3.74-3.58(m,2H),2.30-2.16(m,2H),1.88-1.79(m,1H),1.65(d,J＝8.0Hz,3H),0.96-0.82(m,2H),0.70-0.54(m,2H).

Isomer 2 (Example 3): ESI-MS m/z: 381.3 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.12(s,1H),10.57(s,1H),8.08-7.93(m,1H),7.65(d,J＝16.0Hz,1H),7.05–6.83(m,1H),6.25-6.05(m,3H),5.75-5.66(m,1H),5.17(q,J＝8.0Hz,1H),4.35-4.21(m,2H),3.74-3.58(m,2H),2.30-2.16(m,2H),1.88-1.79(m,1H),1.65(d,J＝8.0Hz,3H),0.96-0.82(m,2H),0.70-0.54(m,2H).

Example 4: 2-(4-(1-acryloyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide

The preparation method is the same as that of Example 1, except that the raw material 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-1(2H)-pyridinecarboxylic acid tert-butyl ester is replaced with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-1(2H)-pyridinecarboxylic acid tert-butyl ester to obtain the title compound. ESI-MS m/z: 381.1 [M+H] ⁺ _. ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.11(s,1H),10.55(s,1H),7.91(s,1H),7.61(s,1H),6.92–6.73(m,1H),6.12(t,J＝8.0Hz,2H),6.02-5.95(m,1H),5.72-5.66(m,1H),5.16(q,J＝8.0Hz,1H),4.23-4.06(m,2H),3.77-3.66(m,2H),2.42–2.33(m,2H),1.88–1.79(m,1H),1.63(d,J＝8.0Hz,3H),0.94–0.83(m,2H),0.67–0.58(m,2H).

Example 5: 2-(4-(1-acryloyl-2,5-dihydro-1H-pyrrol-3-yl)-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide

The preparation method is the same as that of Example 1, except that the raw material 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-3,6-dihydro-1(2H)-pyridinecarboxylic acid tert-butyl ester is replaced with 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-2,5-dihydropyrrole-1-carboxylic acid tert-butyl ester to obtain the title compound. ESI-MS m/z: 367.2 [M+H] ⁺ _. ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.13(s,1H),10.63(d,J＝12.0Hz,1H),8.05(d,J＝20.0Hz,1H),7.70(d,J＝8.0Hz,1H),6.72-6.55(m,1H),6.20(d,J＝16.0Hz,1H),6.11(s,1H),6.03(s,1H),5.80-5.66(m,1H),5.26-5.13(m,1H),4.68-4.43(m,2H),4.42-4.19(m,2H),1.89-1.78(m,1H),1.65(d,J＝8.0Hz,3H),0.97-0.79(m,2H),0.70-0.50(m,2H).

Example 6: 2-(4-(1-(1-propynyl)-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide

The preparation method is the same as that of Example 1, except that the raw material acryloyl chloride is replaced with but-2-ynoic acid, N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(1,2,5,6-tetrahydropyridine-3-yl)-1H-pyrazol-1-yl) propionamide (90 mg, 0.28 mmol), but-2-ynoic acid (47 mg, 0.56 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (107 mg, 0.56 mmol) and N,N-dimethylformamide (108 mg, 0.84 mmol) are added to N,N-dimethylformamide solution (3 mL), stirred at room temperature for 2 h, LC-MS tracking and monitoring the reaction, and the reaction is completed. The reaction solution is diluted with water, extracted with ethyl acetate three times, the organic phases are combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain 20 mg of the title compound. ESI-MS m/z: 393.2 [M+H] ⁺ _. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.11 (s, 1H), 10.55 (s, 1H), 7.97 (s, 1H), 7.63 (s, 1H), 6.12 (s, 2H), 5.24–5.11 (m, 1H), 4.50–4.15 (m, 2H), 3.90–3.56 (m, 2H), 2.33–2.15 (m, 2H), 2.05 (s, 3H), 1.89–1.79 (m, 1H), 1.74–1.55 (m, 3H), 0.98–0.81 (m, 2H), 0.72–0.55 (m, 2H).

Example 7: N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(1-(2-fluoroacryloyl)-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl) Propionamide

The preparation method is the same as that of Example 1, except that the raw material acryloyl chloride is replaced with 2-fluoroacrylic acid, N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl) propionamide (80 mg, 0.245 mmol, 1.0 eq) is dissolved in 5 mL of anhydrous dichloromethane, 2-fluoroacrylic acid (44 mg, 0.49 mmol, 2.0 eq), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (186 mg, 0.49 mmol, 2.0 eq) and N,N-diisopropylethylamine (158 mg, 1.227 mmol, 5.0 eq) are added and reacted at room temperature for 1 hour. After LC-MS tracking monitoring, the reaction is complete. The mixture was diluted with water, extracted with ethyl acetate, concentrated, and purified by column chromatography to obtain 16 mg of the title compound. ESI-MS m/z: 399.2 [M+H] ⁺ _. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.12 (s, 1H), 10.56 (s, 1H), 7.98 (s, 1H), 7.64 (s, 1H), 6.19-6.08 (m, 2H), 5.37-5.25 (m, 2H), 5.17 (q, J=8.0 Hz, 1H), 4.24 (s, 2H), 3.63 (t, J=4.0 Hz, 2H), 2.36-2.21 (m, 2H), 1.88-1.79 (m, 1H), 1.64 (d, J=8.0 Hz, 3H), 0.96-0.84 (m, 2H), 0.69-0.56 (m, 2H).

Example 8: 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)-N-(5-cyclopropylthiazol-2-yl)propanamide

The preparation method is the same as that of Example 1, except that the raw material 3-amino-5-cyclopropyl-1H-pyrazole-1-carboxylic acid tert-butyl ester is replaced by 5-cyclopropylthiazole-2-amine to obtain 500 mg of the title compound. ESI-MS m/z: 398.2 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.30(s,1H),8.15-7.97(m,1H),7.68(d,J＝16.0Hz,1H),7.18(s,1H),7.06-6.80(m,1H),6.25-6.08(m,2H),5.78-5.65(m,1H),5.27(q,J＝8.0Hz,1H),4.29(d,J＝16.0Hz,2H),3.76-3.58(m,2H),2.31-2.13(m,2H),2.07-1.93(m,1H),1.70(d,J＝8.0Hz,3H),1.02–0.82(m,2H),0.72-0.50(m,2H).

The racemate of 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)-N-(5-cyclopropylthiazol-2-yl)propanamide (500 mg) was separated by chiral preparative column. (Method: Chiral column: ChiralCel OD, 250×30 mm ID, 10 μm, elution: isocratic: B 40%, A=carbon dioxide, B=ethanol) to obtain Isomer 1 (retention time 1.722, 219 mg) and Isomer 2 (retention time 2.824, 210 mg) of the title compound.

Isomer 1 (Example 9): ESI-MS m/z: 398.2 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.32(s,1H),8.15-7.97(m,1H),7.68(d,J＝16.0Hz,1H),7.19(s,1H),7.06-6.80(m,1H),6.25-6.08(m,2H),5.78-5.65(m,1H),5.27(q,J＝8.0Hz,1H),4.29(d,J＝16.0Hz,2H),3.76-3.58(m,2H),2.31-2.13(m,2H),2.07-1.93(m,1H),1.70(d,J＝8.0Hz,3H),1.02–0.82(m,2H),0.72-0.50(m,2H).

Isomer 2 (Example 10): ESI-MS m/z: 398.2 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.28(s,1H),8.15-7.97(m,1H),7.68(d,J＝16.0Hz,1H),7.19(s,1H),7.06-6.80(m,1H),6.25-6.08(m,2H),5.78-5.65(m,1H),5.27(q,J＝8.0Hz,1H),4.29(d,J＝16.0Hz,2H),3.76-3.58(m,2H),2.31-2.13(m,2H),2.07-1.93(m,1H),1.70(d,J＝8.0Hz,3H),1.02–0.82(m,2H),0.72-0.50(m,2H).

Example 11: 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)-N-(5-(trifluoromethyl)-1H-pyrazol-3-yl)propanamide

The preparation method is the same as that of Example 1, except that the raw material 3-amino-5-cyclopropyl-1H-pyrazole-1-carboxylic acid tert-butyl ester is replaced by 5-(trifluoromethyl)-1H-pyrazole-3-amine to obtain the title compound. ESI-MS m/z: 409.12 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 13.41 (s, 1H), 11.14 (s, 1H), 8.16–7.97 (m, 1H), 7.70 (d, J=16.0 Hz, 1H), 7.05–6.80 (m, 1H), 6.40 (s, 1H), 6.24–6.07 (m, 2H), 5.71 (t, J=8.0 Hz, 1H), 5.21 (q, J=8.0 Hz, 1H), 4.38–4.20 (m, 2H), 3.75–3.59 (m, 2H), 2.30–2.15 (m, 2H), 1.71 (d, J=8.0 Hz, 3H).

Example 12: 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)-N-(5-(trifluoromethyl)thiazol-2-yl)propanamide

The preparation method is the same as that of Example 1, except that the raw material 3-amino-5-cyclopropyl-1H-pyrazole-1-carboxylic acid tert-butyl ester is replaced by 5-(trifluoromethyl)thiazol-2-amine to obtain the title compound. ESI-MS m/z: 425.9 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 13.16 (s, 1H), 8.26-8.00 (m, 2H), 7.70 (d, J＝16.0 Hz, 1H), 7.14-6.76 (m, 1H), 6.28-6.04 (m, 2H), 5.71 (t, J＝8.0 Hz, 1H), 5.44-5.29 (m, 1H), 4.42-4.19 (m, 2H), 3.75-3.57 (m, 2H), 2.33-2.13 (m, 2H), 1.75 (d, J＝ 8.0Hz,3H).

Example 13: 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)-N-(5-((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)propanamide

The preparation method is the same as that of Example 1, except that the raw material 3-amino-5-cyclopropyl-1H-pyrazole-1-carboxylic acid tert-butyl ester is replaced by 5-((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazole-2-amine, and the raw material is used to prepare 720 mg of the title compound. ESI-MS m/z: 527.15 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.67(s,1H),8.18-7.98(m,1H),7.69(d,J＝16.0Hz,1H),7.43(s,1H),7.05-6.81(m,1H),6.70(s,1H),6.26-6.06(m,2H),5.78-5.66(m,1H),5.28(q,J＝8.0Hz,1H),4.39-4.21(m,2H),4.06(s,2H),3.74-3.56(m,2H),2.30-2.15(m,2H),1.71(d,J＝8.0Hz,3H),1.15(s,9H).

2-(4-(1-Acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)-N-(5-((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)propanamide (750 mg) was separated by chiral preparative column. (Method: Chiral column: ChiralPak AD, 250×30 mm I.D., 10 μm, elution: isocratic: B 30%, A = carbon dioxide, B = isopropanol (0.1% ammonia water)) to obtain isomer 1 (retention time 3.974, 354 mg) and isomer 2 (retention time 5.494, 334 mg) of the title compound.

Isomer 1 (Example 14): ESI-MS m/z: 527.15 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.67(s,1H),8.18-7.98(m,1H),7.69(d,J＝16.0Hz,1H),7.43(s,1H),7.05-6.81(m,1H),6.70(s,1H),6.26-6.06(m,2H),5.78-5.66(m,1H),5.28(q,J＝8.0Hz,1H),4.39-4.21(m,2H),4.06(s,2H),3.74-3.56(m,2H),2.30-2.15(m,2H),1.71(d,J＝8.0Hz,3H),1.15(s,9H).

Isomer 2 (Example 15): ESI-MS m/z: 527.15 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.67(s,1H),8.18-7.98(m,1H),7.69(d,J＝16.0Hz,1H),7.43(s,1H),7.05-6.81(m,1H),6.70(s,1H),6.26-6.06(m,2H),5.78-5.66(m,1H),5.28(q,J＝8.0Hz,1H),4.39-4.21(m,2H),4.06(s,2H),3.74-3.56(m,2H),2.30-2.15(m,2H),1.71(d,J＝8.0Hz,3H),1.15(s,9H).

Example 16: 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)acetamide

Step 1: Preparation of ethyl 2-(4-bromo-1H-pyrazol-1-yl)acetate

Dissolve 4-bromopyrazole (7.0 g, 47.6 mmol, 1.0 eq), ethyl 2-bromoacetate (9.53 g, 57.1 mmol, 1.2 eq), and potassium carbonate (13.16 g, 95.2 mmol, 2.0 eq) in 100 mL of anhydrous N,N-dimethylformamide, stir at room temperature for 3.5 h, and monitor the reaction by LC-MS. After the reaction is completed, add water to quench the reaction, extract the aqueous phase with ethyl acetate twice, wash with water three times, wash with saturated brine, dry with anhydrous sodium sulfate, and concentrate to obtain 9.7 g of the title compound. ESI-MS m/z: 232.9 [M+H] ⁺ .

Step 2: Preparation of 2-(4-bromo-1H-pyrazol-1-yl)acetic acid

Dissolve ethyl 2-(4-bromo-1H-pyrazol-1-yl)acetate (4.5 g, 19.3 mmol, 1.0 eq) in 50 mL of methanol, add 50 mL of water, then add lithium hydroxide (1.85 g, 77.2 mmol, 4.0 eq), and stir at room temperature for 2 h. LC-MS tracking and monitoring of the reaction, the reaction is complete. Remove methanol by rotary evaporation, adjust the pH to 3 with 2M dilute hydrochloric acid solution, extract with ethyl acetate 3 times, dry over anhydrous sodium sulfate, and spin dry to obtain 3.0 g of the title compound. ESI-MS m/z: 204.9 [M + H] ⁺ .

Step 3: Preparation of tert-butyl 3-(2-(4-bromo-1H-pyrazol-1-yl)acetamide)-5-cyclopropyl-1H-pyrazole-2-carboxylate

Dissolve 2-(4-bromo-1H-pyrazol-1-yl)acetic acid (1.0 g, 4.88 mmol, 1.0 eq) in 20 mL of anhydrous dichloromethane, add oxalyl chloride (1.26 g, 9.76 mmol, 2.0 eq) at 0°C, stir at room temperature for 1 h, monitor the reaction by LC-MS until complete, spin dry, dissolve in 20 mL of anhydrous dichloromethane solution, and directly use for the next step. Dissolve tert-butyl 3-amino-5-cyclopropyl-1H-pyrazole-1-carboxylate (1.2 g, 5.37 mmol, 1.1 eq) in dichloromethane, add the dichloromethane solution of the above-prepared acyl chloride dropwise at 0°C, and stir at room temperature for 1 h. Monitor the reaction by LC-MS until complete. Add water to quench the reaction, separate the liquids, extract the aqueous phase with dichloromethane twice, combine the organic phases, dry over anhydrous sodium sulfate, and concentrate to obtain Sand, column chromatography purification, to obtain the title compound 1.3g. ESI-MS m/z: 309.9 [M + H-Boc] ⁺ .

Step 4: Preparation of tert-butyl 5-(1-(2-((1-(tert-butoxycarbonyl)-5-cyclopropyl-1H-pyrazol-3-yl)amino)-2-oxoethyl)-1H-pyrazol-4-yl)-3,6-dihydropyridine-1(2H)-carboxylate

3-(2-(4-bromo-1H-pyrazol-1-yl)acetamide)-5-cyclopropyl-1H-pyrazole-2-carboxylic acid tert-butyl ester (400 mg, 0.975 mmol, 1.0 eq), 5-(4,4,5,5-tetramethyl-1,3,2-dioxybenzoic acid-2-yl)-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (422 mg, 1.365 mmol, 1.4 eq), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (71 mg, 0.098 mmol, 0.1 eq), and cesium carbonate (953 mg, 2.925 mmol, 3.0 eq) were added to 10 mL of 1,4-dioxane, and 1 mL of water was added. The mixture was protected by argon and heated to 100°C with stirring for 4 h. The reaction was monitored by LC-MS. After the reaction was completed, the product was filtered, concentrated and sanded, and purified by column chromatography to obtain 287 mg of the title compound. ESI-MS m/z: 413.2 [M+H-Boc] ⁺ .

Step 5: Preparation of N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)acetamide

Dissolve tert-butyl 5-(1-(2-((1-(tert-butyloxycarbonyl)-5-cyclopropyl-1H-pyrazol-3-yl)amino)-2-oxoethyl)-1H-pyrazol-4-yl)-3,6-dihydropyridine-1(2H)-carboxylate (270 mg) in 3 mL of anhydrous dichloromethane, add trifluoroacetic acid (342 mg, 3 mmol), and stir at room temperature for 6 h. LC-MS tracking and monitoring of the reaction, the reaction was completed. Adjust the pH to 7 with N,N-diisopropylethylamine, spin dry, and directly use for the next step reaction. ESI-MS m/z: 313.1[M+H] ⁺ .

Step 6: Preparation of 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)acetamide

Dissolve N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(1,2,5,6-tetrahydropyridin-3-yl)-1H-pyrazol-1-yl)acetamide in 10 mL of anhydrous dichloromethane, add N,N-diisopropylethylamine (136 mg, 1.054 mmol), cool to 0°C, add acryloyl chloride (48 mg, 0.527 mmol), stirred at room temperature for 2 h. LC-MS followed the reaction and the reaction was complete. Water was added to quench the reaction, and ethyl acetate was extracted three times. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and spin-dried. Column chromatography was used for purification to obtain 37 mg of the title compound. ESI-MS m/z: 367.2 [M + H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.10(s,1H),10.56(s,1H),7.88(d,J＝24.0Hz,1H),7.66(d,J＝16.0Hz,1H),7.00-6.81(m,1H),6.22-6.05(m,3H),5.75-5.66(m,1H),4.92(s,2H),4.27(d,J＝16.0Hz,2H),3.74-3.60(m,2H),2.29-2.16(m,2H),1.90-1.79(m,1H),0.97-0.82(m,2H),0.70-0.55(m,2H).

Example 17: 2-(4-(1-acryloyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)acetamide

The preparation method is the same as the preparation method of Example 16, except that 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-1(2H)-picolinic acid tert-butyl ester is replaced by 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-1(2H)-picolinic acid tert-butyl ester, and the raw material is used to obtain the title compound. ESI-MS m/z: 367.2 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.09 (s, 1H), 10.54 (s, 1H), 7.79 (s, 1H), 7.62 (s, 1H), 6.95-6.68 (m, 1H), 6.18-6.05 (m, 2H), 6.01-5.93 (m, 1H), 5.72-5.65 (m, 1H), 4.90 (s, 2H), 4.23-4.06 (m, 2H), 3.78-3.66 (m, 2H), 2.42-2.30 (m, 2H), 1.89-1.79 (m, 1H), 0.98-0.82 (m, 2H), 0.68-0.57 (m, 2H).

Example 18: 2-(4-(1-acryloyl-2,5-dihydro-1H-pyrrol-3-yl)-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)acetamide

The preparation method is the same as the preparation method of Example 16, except that 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-1(2H)-picolinic acid tert-butyl ester is replaced by 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydropyrrole-1-carboxylic acid tert-butyl ester, and the raw material is used to obtain the title compound. ESI-MS m/z: 353.2 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.10 (s, 1H), 10.60 (s, 1H), 7.98-7.85 (m, 1H), 7.71 (d, J=12.0 Hz, 1H), 6.73-6.53 (m, 1H), 6.28–6.07 (m, 2H), 6.02 (s, 1H), 5.80-5.67 (m, 1H), 4.95 (d, J=4.0 Hz, 2H), 4.66-4.21 (m, 4H), 1.92-1.78 (m, 1H), 0.95-0.81 (m, 2H), 0.70-0.54 (m, 2H).

Example 19: 2-(2-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)thiazol-4-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide

Step 1: Preparation of methyl 2-(2-aminothiazol-4-yl)acetate

Dissolve methyl 4-chloro-3-oxobutanoate (10.0 g, 66.4 mmol, 1.0 eq) and thiourea (5.05 g, 66.4 mmol, 1.0 eq) in 200 mL of anhydrous ethanol, heat to 80°C and stir for 12 h. Follow the reaction with LC-MS. After the reaction is complete, concentrate to 100 mL of ethanol, filter, wash the filter cake with methyl tert-butyl ether three times, and dry to obtain 9.9 g of the title compound. ESI-MS m/z: 173.1 [M+H] ⁺ .

Step 2: Preparation of methyl 2-(2-bromothiazol-4-yl)acetate

Add 2-(2-aminothiazole-4-yl)acetic acid methyl ester (9.65g, 56.1mmol) and cuprous bromide (17.36g, 168.3mmol) into a three-necked flask, protect with argon, add 200mL acetonitrile, cool to 0℃, slowly drop tert-butyl nitrite (24.14g, 168.3mmol), stir for 30min, heat to 70℃ and stir for 3h, monitor the reaction by LC-MS, after the reaction is complete, add water to quench the reaction, extract the aqueous phase with ethyl acetate 3 times, dry with anhydrous sodium sulfate, concentrate and make sand, purify by column chromatography, and obtain 7.3g of the title compound. ESI-MS m/z: 236.0[M+H] ⁺ .

Step 3: Preparation of methyl 2-(2-bromothiazol-4-yl)propanoate

Dissolve methyl 2-(2-bromothiazol-4-yl)acetate (1.5 g, 6.36 mmol) in 20 mL of anhydrous tetrahydrofuran, protect with argon, cool to -78°C, slowly add 2 M lithium diisopropylamide (4.77 mL, 9.54 mmol), add iodomethane (1.083 g, 7.63 mmol), stir and react at room temperature for 2 h, monitor the reaction by LC-MS, after the reaction is complete, add saturated ammonium chloride solution to quench the reaction, separate the liquids, extract with ethyl acetate three times, dry over anhydrous sodium sulfate, concentrate and make sand, purify by column chromatography to obtain 800 mg of the title compound. ESI-MS m/z: 249.98 [M+H] ⁺ .

Step 4: Preparation of 2-(2-bromothiazol-4-yl)propanoic acid

Dissolve methyl 2-(2-bromothiazol-4-yl)propionate (800 mg, 3.2 mmol) in 10 mL of methanol, add 10 mL of tetrahydrofuran, then add 10 mL of water, then add lithium hydroxide (307 mg, 12.8 mmol), and stir at room temperature for 2 h. LC-MS tracking and monitoring of the reaction, the reaction is complete. Remove methanol and tetrahydrofuran by rotary evaporation, adjust the pH to 3 with 2M dilute hydrochloric acid solution, extract 3 times with ethyl acetate, dry over anhydrous sodium sulfate, and spin dry to obtain 742 mg of the title compound. ESI-MS m/z: 235.9 [M + H] ⁺ .

Step 5: Preparation of tert-butyl 3-(2-(2-bromothiazol-4-yl)propylamino)-5-cyclopropyl-1H-pyrazole-1-carboxylate

Dissolve 2-(2-bromothiazol-4-yl)propionic acid (722mg, 3.06mmol) in 20mL of anhydrous dichloromethane, add 10 drops of N,N-dimethylformamide, add oxalyl chloride (777mg, 6.12mmol) at 0℃, stir at room temperature for 1h, monitor the reaction by LC-MS, spin dry, dissolve in 20mL of anhydrous dichloromethane solution, and directly react in the next step. Dissolve tert-butyl 3-amino-5-cyclopropyl-1H-pyrazole-1-carboxylate (752mg, 3.366mmol) in dichloromethane, add the dichloromethane solution of the above-prepared acyl chloride dropwise at 0℃, stir at room temperature for 1h. Monitor the reaction by LC-MS, and the reaction is complete. Add water to quench the reaction, separate the liquids, extract the aqueous phase with dichloromethane twice, combine the organic phases, dry over anhydrous sodium sulfate, concentrate and make sand, and purify by column chromatography to obtain 480mg of the title compound. ESI-MS m/z:341.0[M+H-Boc] ⁺ .

Step 6: Preparation of tert-butyl 5-(4-(1-((1-(tert-butoxycarbonyl)-5-cyclopropyl-1H-pyrazol-3-yl)amino)-1-oxopropan-2-yl)-2-thiazolidinyl)-3,6-dihydropyridine-1(2H)-carboxylate

3-(2-(2-bromothiazol-4-yl)propylamino)-5-cyclopropyl-1H-pyrazole-1-carboxylic acid tert-butyl ester (450mg, 1.02mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxybenzoic acid-2-yl)-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (442mg, 1.428mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (75mg, 0.102mmol), cesium carbonate (997mg, 3.06mmol) were added to 10mL of anhydrous 1,4-dioxane, and then 1mL of water was added, and the temperature was raised to 100℃ and stirred for 4h. The reaction was monitored by LC-MS tracking. After the reaction was completed, the mixture was filtered, dried, concentrated and sanded, and purified by column chromatography to obtain 470mg of the title compound. ESI-MS m/z:444.1[M+H-Boc] ⁺ .

Step 7: Preparation of N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(2-(1,2,5,6-tetrahydropyridin-3-ylthiazol-4-yl)propanamide

Dissolve tert-butyl 5-(4-(1-((1-(tert-butyloxycarbonyl)-5-cyclopropyl-1H-pyrazol-3-yl)amino)-1-oxopropane-2-yl)-2-thiazolidinyl)-3,6-dihydropyridine-1(2H)-carboxylate (400 mg) in 10 mL of anhydrous dichloromethane, add trifluoroacetic acid (1.7 g, 15 mmol), and stir at room temperature for 1 h. LC-MS tracking and monitoring of the reaction, the reaction was complete. Adjust the pH to 7 with N,N-diisopropylethylamine, spin dry, and directly use for the next step reaction. ESI-MS m/z[M+H] ⁺ :344.15.

Step 8: Preparation of 2-(2-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)thiazol-4-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide

The compound N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(2-(1,2,5,6-tetrahydropyridin-3-ylthiazole-4-yl)propionamide was dissolved in 10 mL of anhydrous dichloromethane, and N,N-diisopropylethylamine (476 mg, 3.68 mmol) was added. The mixture was cooled to 0°C, and acryloyl chloride (67 mg, 0.736 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction was monitored by LC-MS and the reaction was completed. Water was added to quench the reaction, and the mixture was extracted with dichloromethane three times. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by C18 column chromatography to obtain 77 mg of the title compound. ESI-MS m/z: 398.08 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.04(s,1H),10.39(s,1H),7.34(s,1H),6.94–6.62(m,2H),6.24–6.04(m,2H),5.71(d,J＝12.0Hz,1H),4.47(s,2H),4.11–4.00(m,1H),3.77-3.60(m,2H),2.40–2.25(m,2H),1.90–1.78(m,1H),1.43(d,J＝8.0Hz,3H),0.98–0.79(m,2H),0.70–0.55(m,2H).

Example 20: 2-(2-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)thiazol-4-yl)-N-(5-cyclopropylthiazol-2-yl)propanamide

The preparation method is the same as that of Example 19, except that the raw material 3-amino-5-cyclopropyl-1H-pyrazole-1-carboxylic acid tert-butyl ester is replaced by 5-cyclopropylthiazole-2-amine to obtain the title compound. ESI-MS m/z: 414.94 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.13 (s, 1H), 7.40 (s, 1H), 7.16 (s, 1H), 6.97–6.59 (m, 2H), 6.24–6.04 (m, 1H), 5.71 (s, 1H), 4.46 (s, 2H), 4.22–4.10 (m, 1H), 3.76–3.61 (m, 2H), 2.40–2.25 (m, 2H), 2.06–1.95 (m, 1H), 1.48 (d, J=8.0 Hz, 3H), 1.00–0.87 (m, 2H), 0.70–0.56 (m, 2H).

Example 21: 2-(2-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)thiazol-5-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide

The preparation method is the same as that of Example 19, except that the raw material 2-(2-aminothiazol-4-yl)acetic acid methyl ester is replaced by 2-(2-aminothiazol-5-yl)acetic acid methyl ester to obtain the title compound. ESI-MS m/z: 398.08 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.09(s,1H),10.58(s,1H),7.61(s,1H),6.95–6.73(m,1H),6.70–6.57(m,1H),6.24–6.04(m,2H),5.71(d,J＝12.0Hz,1H),4.54–4.38(m,2H),4.21(q,J＝8.0Hz,1H),3.77–3.59(m,2H),2.41–2.23(m,2H),1.89–1.76(m,1H),1.43(d,J＝4.0Hz,3H),0.95–0.84(m,2H),0.70–0.55(m,2H).

Example 22: 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-3-methyl-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide

The preparation method is the same as that of Example 1, except that the raw material 4-bromopyrazole is replaced by 4-bromo-3-methyl-1H-pyrazole to obtain the title compound. ESI-MS m/z: 395.0 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.13(s,1H),10.56(s,1H),8.00-7.78(m,1H),7.04–6.81(m,1H),6.24-6.05(m,2H),5.91(d,J＝12.0Hz,1H),5.71(d,J＝12.0Hz,1H),5.08(q,J＝8.0Hz,1H),4.25(d,J＝12.0Hz,2H),3.73-3.59(m,2H),2.36-2.15(m,5H),1.89-1.79(m,1H),1.61(d,J＝8.0Hz,3H),0.94-0.82(m,2H),0.69-0.55(m,2H).

2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-3-methyl-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide (200 mg) was separated by chiral preparative column. (Method: Chiral column: ChiralCel OD, 250×30 mm I.D., 10 μm, elution: isocratic: B 40%, A = carbon dioxide, B = ethanol (0.1% ammonia water)) to obtain isomer 1 (retention time 1.555, 94 mg) and isomer 2 (retention time 2.607, 92 mg) of the title compound.

Isomer 1 (Example 23): ESI-MS m/z: 395.0 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H), 10.56 (s, 1H), 8.00-7.78 (m, 1H), 7.04–6.81 (m, 1H), 6.24-6.05 (m, 2H), 5.91 (d, J = 12.0 Hz, 1H), 5.71 (d, J = 12.0 Hz, 1H), 5.08 (q, J = 8.0 Hz, 1H), 4.25 (d, J = 12.0 Hz, 2H), 3.73-3.59 (m, 2H), 2.36-2.15 (m, 5H), 1.89-1.79 (m, 1H), 1.61 (d, J = 8.0 Hz, 3H), 0.94-0.82 (m, 2H), 0.69-0.55 (m, 2H).

Isomer 2 (Example 24): ESI-MS m/z: 395.0 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H), 10.56 (s, 1H), 8.00-7.78 (m, 1H), 7.04-6.81 (m, 1H), 6.24-6.05 (m, 2H), 5.91 (d, J = 12.0 Hz, 1H), 5.71 (d, J = 12.0 Hz, 1H), 5.08 (q, J = 8.0 Hz, 1H), 4.25 (d, J = 12.0 Hz, 2H), 3.73-3.59 (m, 2H), 2.36-2.15 (m, 5H), 1.89-1.79 (m, 1H), 1.61 (d, J = 8.0 Hz, 3H), 0.94-0.82 (m, 2H), 0.69-0.55 (m, 2H).

Example 25: 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-3-methyl-1H-pyrazol-1-yl)-N-(5-cyclopropylthiazol-2-yl)propanamide

The preparation method is the same as that of Example 22, except that the raw material 3-amino-5-cyclopropyl-1H-pyrazole-1-carboxylic acid tert-butyl ester is replaced with 5-cyclopropylthiazole-2-amine to obtain the title compound. ESI-MS m/z: 412.0 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.32(s,1H),8.09–7.82(m,1H),7.19(s,1H),7.06–6.78(m,1H),6.16(t,J＝8.0Hz,1H),6.01–5.85(m,1H),5.80–5.64(m,1H),5.35–5.05(m,1H),4.40–4.15(m,2H),3.82–3.54(m,2H),2.35–2.13(m,4H),2.13–1.94(m,2H),1.67(s,3H),1.05–0.80(m,2H),0.75–0.50(m,2H).

Example 26: 2-(4-(1-acryloyl-1,2,5,6-tetrahydropyridin-3-yl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)-N-(5-cyclopropyl-1H-pyrazin-3-yl)propanamide

The preparation method is the same as that of Example 22, except that the raw material 4-bromopyrazole is replaced by 4-bromo-3-(trifluoromethyl)-1H-pyrazole to obtain the title compound. ESI-MS m/z: 449.0 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.17 (s, 1H), 10.80 (s, 1H), 8.40–8.15 (m, 1H), 7.00-6.79 (m, 1H), 6.26–6.07 (m, 2H), 6.06-5.92 (m, 1H), 5.71 (d, J=8.0 Hz, 1H), 5.38–5.19 (m, 1H), 4.38–4.12 (m, 2H), 3.66 (s, 2H), 2.37–2.15 (m, 2H), 1.91–1.79 (m, 1H), 1.72 (d, J＝4.0 Hz, 3H), 1.00-1.76 (m, 2H), 0.72–0.52 (m, 2H).

Example 27: 2-(1-acryloyl-2'-oxo-1,2,5,6-tetrahydro-[3,4'-bipyridyl]-1'(2'H)-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide

Step 1: Preparation of methyl 2-(4-bromo-2-oxopyridin-1(2H)-yl)propanoate

Dissolve 4-bromopyridin-2(1H)-one (3.00 g, 17.24 mmol, 1.0 eq) in anhydrous N,N-dimethylformamide (30 mL), add methyl 2-bromopropionate (3.45 g, 20.69 mmol, 1.2 eq) and potassium carbonate (4.77 g, 34.48 mmol, 2.0 eq), and stir at room temperature for 3.5 h. LC-MS tracking and monitoring the reaction, after the reaction is completed, add water to quench the reaction, extract with ethyl acetate twice, wash with water three times, wash with saturated brine, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify with column chromatography to obtain 3.09 g of the title compound. ESI-MS m/z: 260.0 [M+H] ⁺ .

Step 2: Preparation of 2-(1-(tert-butyloxycarbonyl)-2'-oxo-1,2,5,6-tetrahydro-[3,4'-bipyridyl]-1'(2'H)-yl)propanoic acid

Add methyl 2-(4-bromo-2-oxopyridin-1(2H)-yl)propanoate (3.038 g, 11.68 mmol, 1.0 eq) to a reaction flask, then add 1-BOC-3,6-dihydro-2H-pyridine-5-boronic acid pinacol ester (5.056 g, 16.35 mmol, 1.4 eq), 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (855 mg, 1.168 mmol, 0.1 eq) and cesium carbonate (11.417 g, 35.04 mmol, 3.0 eq), then add 1,4-dioxane (25 mL) and water (2.5 mL). Heat and stir at 100°C for 3 h under argon protection. The reaction was monitored by LC-MS. After the reaction was completed, the reaction solution was filtered, the filtrate was diluted with water, and extracted with ethyl acetate three times. The pH of the aqueous phase was adjusted to 5-6 with dilute hydrochloric acid. Solids precipitated, filtered, and dried to obtain 0.98 g of the title compound. ESI-MS m/z: 349.2 [M+H] ⁺ .

Step 3: Preparation of tert-butyl 1'-(1-((1-(tert-butoxycarbonyl)-5-cyclopropyl-1H-pyrazol-3-yl)amino)-1-oxopropan-2-yl)-2'-oxo-1',2',5,6-tetrahydro-[3,4'-bipyridine]-1(2H)-carboxylate

Dissolve 2-(1-(tert-Butyloxycarbonyl)-2'-oxo-1,2,5,6-tetrahydro-[3,4'-bipyridyl]-1'(2'H)-yl)propanoic acid (1.00 g, 2.87 mmol, 1.0 eq) in N,N-dimethylformamide (15 mL), add tert-butyl 3-amino-5-cyclopropyl-1H-pyrazole-1-carboxylate (961 mg, 4.31 mmol, 1.5 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (825 mg, 4.31 mmol, 1.5 eq) and 1-hydroxybenzotriazole (776 mg, 5.74 mmol, 2.0 eq), and heat and stir at 80°C to react overnight. The reaction was monitored by LC-MS. After the reaction was completed, the mixture was diluted with water, extracted with ethyl acetate three times, washed with water twice, washed with saturated sodium bicarbonate once, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain 209 mg of the title compound. ESI-MS m/z: 454.3 [M+H-Boc] ⁺ .

Step 4: Preparation of N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(2'-oxo-1,2,5,6-tetrahydro-[3,4'-bipyridyl]-1'(2'H)-yl)propanamide

Dissolve 1'-(1-((1-(tert-butoxycarbonyl)-5-cyclopropyl-1H-pyrazol-3-yl)amino)-1-oxopropane-2-yl)-2'-oxo-1',2',5,6-tetrahydro-[3,4'-bipyridine]-1(2H)-carboxylic acid tert-butyl ester (155 mg, 0.28 mmol, 1.0 eq) in anhydrous dichloromethane (3 mL), add trifluoroacetic acid (0.2 mL), and stir at room temperature for 2 h. LC-MS tracking and monitoring the reaction, after the reaction is completed, concentrate under reduced pressure. Add 3 mL of dichloromethane, adjust the pH to about 9 with N,N-diisopropylethylamine, and remove the solvent to obtain 99 mg of the crude product of the title compound. ESI-MS m/z: 354.2[M+H] ⁺ .

Step 5: Preparation of 2-(1-acryloyl-2'-oxo-1,2,5,6-tetrahydro-[3,4'-bipyridyl]-1'(2'H)-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)propanamide

N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(2'-oxo-1,2,5,6-tetrahydro-[3,4'-bipyridine]-1'(2'H)-yl)propanamide (99 mg, 0.28 mmol, 1.0 eq) was dissolved in anhydrous N,N-dimethylformamide (3 mL), acryloyl chloride (23 mg, 0.25 mmol, 0.9 eq) and N,N-diisopropylethylamine (0.3 mL) were added, and the mixture was stirred at room temperature for 2 h. The reaction was monitored by LC-MS. After completion of the reaction, the mixture was concentrated under reduced pressure. Purification by column chromatography gave 20 mg of the title compound. ESI-MS m/z: 408.2[M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.09(s,1H),10.64(s,1H),7.74-7.58(m,1H),7.04–6.82(m,1H),6.70–6.55(m,1H),6.52–6.24(m,2H),6.21–6.02(m,2H),5.79–5.64(m,1H),5.53(q,J＝8.0Hz,1H),4.45–4.24(m,2H),3.76-3.58(m,2H),2.41–2.25(m,2H),1.89–1.79(m,1H),1.56(d,J＝8.0Hz,3H),0.95-0.83(m,2H),0.67-0.57(m,2H).

Example 28: 2-(1'-acryloyl-1',2',5',6'-tetrahydro-[2,3'-bipyridyl]-5-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2,2-difluoroacetamide

Step 1: Preparation of methyl 2-(6-chloropyridin-3-yl)-2,2-difluoroacetate

Dissolve methyl 2-(6-chloropyridin-3-yl)acetate (3g, 16.2mmol, 1.0eq) in anhydrous tetrahydrofuran (30mL). After nitrogen protection, slowly add lithium hexamethyldisilazide (65mL, 64.7mmol, 4.0eq) dropwise after ice bath at -78℃, and stir for 2h at -78℃. Add N-fluorobisbenzenesulfonamide (40g, 129.3mmol, 8.0eq) dropwise and stir for 2.5h. Monitor the reaction by LC-MS. After the reaction is complete, add saturated aqueous ammonium chloride solution to quench, add ethyl acetate (3*30mL) to extract, combine the organic phases, dry and concentrate, and purify by column chromatography to obtain 0.95g of the title compound. ESI-MS m/z: 222.1[M+H] ⁺ .

Step 2: Preparation of 2-(6-chloropyridin-3-yl)-2,2-difluoroacetic acid

Dissolve methyl 2-(6-chloropyridin-3-yl)-2,2-difluoroacetate (950 mg, 4.3 mmol, 1.0 eq) in methanol (10 mL), cool to 0°C in an ice bath, add a solution of lithium hydroxide (413 mg, 17.2 mmol, 4.0 eq) in water (10 mL), move to room temperature, and react for 1 h. Monitor the reaction by LC-MS. After the reaction is complete, concentrate under reduced pressure, dilute with water (10 mL), extract with ethyl acetate (3*10 mL), adjust the pH of the aqueous phase to 4 with 1N hydrochloric acid aqueous solution, concentrate under reduced pressure, and purify by column chromatography to obtain 600 mg of the title compound. ESI-MS m/z: 208.2[M+1] ⁺ .

Step 3: Preparation of (2-(6-chloropyridin-3-yl)-N-(5-cyclopropyl-1-(2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)-2,2-difluoroacetamide

Dissolve 2-(6-chloropyridin-3-yl)-2,2-difluoroacetic acid (500 mg, 2.41 mmol) in 10 mL of anhydrous dichloromethane, add oxalyl chloride (612 mg, 4.82 mmol) at 0°C, stir at room temperature for 1 h, monitor the reaction by LC-MS, and after the reaction is complete, spin dry, dissolve in 10 mL of anhydrous dichloromethane solution, and directly use for the next step. Amine (613 mg, 2.41 mmol) was dissolved in dichloromethane, and the dichloromethane solution of the above-prepared acyl chloride was added dropwise at 0°C, and the reaction was stirred at room temperature for 1 hour. The reaction was monitored by LC-MS tracking, and the reaction was completed. Water was added to quench the reaction, and the liquid was separated and extracted with dichloromethane twice. The organic phases were combined, dried over anhydrous sodium sulfate, spin-dried, and purified by column chromatography to obtain 600 mg of the title compound. ESI-MS m/z: 443.1 [M + H] ⁺ .

Step 4: Preparation of tert-butyl 5-(2-((5-cyclopropyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)amino)-1,1-difluoro-2-oxoethyl)-5',6'-dihydro-[2,3'-bipyridine]-1'(2'H)-carboxylate

(2-(6-chloropyridin-3-yl)-N-(5-cyclopropyl-1-(2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)-2,2-difluoroacetamide (200 mg, 0.45 mmol, 1.0 eq) was weighed into a 25 mL reaction bottle, dioxane (5 mL) was added to dissolve, and tert-butyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxybenzoic acid-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (195 mg, 0.63 mmol, 1.4 eq), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (33 mg, 0.045 mmol, 0.1 eq), cesium carbonate (441 mg, 1.35 mmol, 3.0 eq), and water (0.5 mL) were added in sequence. Stir overnight at 100°C under argon protection. The reaction was monitored by LC-MS and the reaction was complete. Concentration and column chromatography purification were performed to obtain 166 mg of the title compound. ESI-MS m/z: 590.7 [M+H] ⁺ .

Step 5: Preparation of N-(5-cyclopropyl-1H-pyrazol-3-yl)-2,2-difluoro-2-(1',2',5',6'-tetrahydro-[2,3'-bipyridyl]-5-yl)acetamide

Dissolve 5-(2-((5-cyclopropyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)amino)-1,1-difluoro-2-oxoethyl)-5',6'-dihydro-[2,3'-bipyridine]-1'(2'H)-carboxylic acid tert-butyl ester (156 mg, 0.26 mmol, 1.0 eq) in dichloromethane (3 mL), add trifluoroacetic acid (342 mg, 3 mmol, 15.0 eq) in an ice bath and react at room temperature for 4 h. LC-MS monitors the reaction. After the reaction is completed, spin dry the reaction solution, add N,N-diisopropylethylamine to adjust the pH to about 9, spin remove the solvent, and directly use it for the next step reaction. ESI-MS m/z: 360.2[M+H] ⁺ .

Step 6: Preparation of 2-(1'-acryloyl-1',2',5',6'-tetrahydro-[2,3'-bipyridyl]-5-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2,2-difluoroacetamide

Dissolve N-(5-cyclopropyl-1H-pyrazol-3-yl)-2,2-difluoro-2-(1',2',5',6'-tetrahydro-[2,3'-bipyridyl]-5-yl)acetamide in anhydrous dichloromethane (3 mL), cool to 0°C in an ice bath, add N,N-diisopropylethylamine (168 mg, 1.3 mmol, 5.0 eq) and acryloyl chloride (24 mg, 0.26 mmol, 1.0 eq), move to room temperature, and react for 1 h. Monitor the reaction by LC-MS. After the reaction is complete, add dichloromethane (8 mL) was used for dilution, and water (3×8 mL) was added for extraction. The organic phase was dried, concentrated, and purified by column chromatography to obtain 43 mg of the title compound. ESI-MS m/z: 414.2 [M+1] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.33 (s, 1H), 11.30 (s, 1H), 8.80 (s, 1H), 8.03 (d, J=8.0 Hz, 1H), 7.85 (t, J=8.0 Hz, 1H), 7.03–6.79 (m, 2H), 6.20–6.07 (m, 2H), 5.71 (d, J=12.0 Hz, 1H), 4.62–4.49 (m, 2H), 3.77–3.61 (m, 2H), 2.45–2.30 (m, 2H), 1.91–1.81 (m, 1H), 0.97–0.82 (m, 2H), 0.71–0.58 (m, 2H).

Example 29: 2-(1'-acryloyl-1',2',3',6'-tetrahydro-[2,4'-bipyridyl]-5-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2,2-difluoroacetamide

The preparation method is the same as that of Example 28, except that 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-1(2H)-picolinic acid tert-butyl ester is replaced by 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-1(2H)-picolinic acid tert-butyl ester, and the raw material is used to prepare the title compound. ESI-MS m/z: 414.2 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.33(s,1H),11.29(s,1H),8.80(s,1H),8.03(d,J＝8.0Hz,1H),7.75(d,J＝8.0Hz,1H),6.96–6.72(m,2H),6.14(t,J＝8.0Hz,2H),5.71(dd,J＝12.0,4.0Hz,1H),4.43–4.17(m,2H),3.84-3.70(m,2H),2.72–2.54(m,2H),1.91–1.81(m,1H),0.97–0.82(m,2H),0.71–0.55(m,2H).

Example 30: 2-(6-(1-acryloyl-2,5-dihydro-1H-pyrrol-3-yl)pyridin-3-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2,2-difluoroacetamide

The preparation method is the same as that of Example 28, except that the raw material 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-1(2H)-picolinic acid tert-butyl ester is replaced with 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydropyrrole-1-carboxylic acid tert-butyl ester to obtain the title compound. ESI-MS m/z: 400.14 [M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ12.35(s,1H),11.31(s,1H),8.83(d,J＝4.0Hz,1H),8.11–8.05(m,1H),7.93–7.87(m,1H),6.93-6.86(m,1H),6.76–6.58(m,1H),6.26-6.11(m,2H),5.74(d,J＝8.0Hz,1H),4.88-4.62(m,2H),4.62–4.39(m,2H),1.90–1.82(m,1H),0.95-0.84(m,2H),0.69-0.60(m,2H).

Example 31: 2-(1'-Acryloyl-1',2',5',6'-tetrahydro-[2,3'-bipyridyl]-5-yl)-N-(5-((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)propanamide

Step 1: Preparation of tert-butyl 5-(1-methoxy-1-oxo-2-propyl)-5',6'-dihydro-[2,3'-bipyridine]-1'(2'H)-carboxylate

Dissolve methyl 2-(6-chloropyridin-3-yl)propanoate (0.5 g, 2.50 mmol, 1.0 eq), tert-butyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-1(2H)-picolinate (1.08 g, 3.50 mmol, 1.4 eq), cesium carbonate (2.44 g, 7.50 mmol, 3.0 eq), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (183 mg, 0.25 mmol, 0.1 eq) in 10 mL of 1,4-dioxane and 1 mL of water, protect with Ar, stir at 100 ° C for 2.5 h, and monitor the reaction. LC-MS tracking and monitoring the reaction, after the reaction is completed, dilute with water, extract with ethyl acetate, dry, concentrate, and purify by column chromatography to obtain 0.86 g of the title compound. ESI-MS m/z:347.2[M+H]+.

Step 2: Preparation of 2-(1'-(tert-butoxycarbonyl)-1',2',5',6'-tetrahydro-[2,3'-bipyridyl]-5-yl)propanoic acid

Dissolve 5-(1-methoxy-1-oxo-2-propyl)-5',6'-dihydro-[2,3'-bipyridine]-1'(2'H)-carboxylic acid tert-butyl ester (0.86 g, 2.50 mmol) and lithium hydroxide (437 mg, 18.0 mmol) in 15 mL tetrahydrofuran and 3 mL water, stir at room temperature for 2.5 h, monitor the reaction by LC-MS, and after the reaction is completed, adjust the pH to about 5 with 1 M HCl solution, extract with ethyl acetate, dry, and concentrate to obtain 816 mg of the title compound. ESI-MS m/z: 333.2 [M+H] ⁺ .

Step 3: Preparation of tert-butyl 5-(1-((5-(5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)amino)-1-oxo-2-propyl)-5',6'-dihydro-[2,3'-bipyridine]-1'(2'H)-carboxylate

2-(1'-(tert-Butyloxycarbonyl)-1',2',5',6'-tetrahydro-[2,3'-bipyridyl]-5-yl)propanoic acid (794 mg, 2.40 mmol, 1.2 eq) was dissolved in 20 mL of dichloromethane, and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (989 mg, 2.60 mmol, 1.3 eq) and N,N-dimethylformamide (780 mg, 6.00 mmol, 3.0 eq) were added. The mixture was stirred at room temperature for 10 min, and then 5-((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazole-2-amine (539 mg, 2.00 mmol, 1.0 eq) was added. The mixture was stirred at room temperature for 2 h. The reaction was monitored by LC-MS. After the reaction was completed. Dilute with water, extract with dichloromethane, combine the organic layers, dry, concentrate, and purify by column chromatography to obtain 700 mg of the title compound. ESI-MS m/z: 583.9 [M+H] ⁺ .

Step 4: Preparation of N-(5-(((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)-2-(1',2',5',6'-tetrahydro-[2,3'-bipyridyl]-5-yl)propanamide

Dissolve 5-(1-((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)amino)-1-oxo-2-propyl)-5',6'-dihydro-[2,3'-bipyridine]-1'(2'H)-carboxylic acid tert-butyl ester (700 mg, 1.20 mmol) in 15 mL of anhydrous dichloromethane, add 3.42 g of trifluoroacetic acid, stir at room temperature for 2 h, and monitor the reaction. After the reaction is completed, adjust the pH to about 8 with saturated sodium bicarbonate solution, extract with ethyl acetate, combine the organic layers, dry, and concentrate to obtain 714 mg of the title compound. ESI-MS m/z: 484.12 [M+H] ⁺ .

Step 5: Preparation of 2-(1'-acryloyl-1',2',5',6'-tetrahydro-[2,3'-bipyridyl]-5-yl)-N-(5-((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)propanamide

N-(5-(((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)-2-(1',2',5',6'-tetrahydro-[2,3'-bipyridyl]-5-yl)propanamide (714mg, 1.48mmol, 1.0eq) was dissolved in anhydrous dichloromethane, cooled to 0°C, N,N-dimethylformamide (574mg, 4.44mmol, 3.0eq) was added, and then acryloyl chloride (72mg, 1.18mmol, 0.8eq) was slowly added dropwise, and then stirred at 0°C for 10min, stirred at room temperature for 15min, and the reaction was monitored by LC-MS. After the reaction was completed. Dilute with water, extract with dichloromethane, combine the organic phases, dry, concentrate, and purify by column chromatography to obtain 315mg of the title compound. ESI-MS m/z: 538.10[M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ12.53(s,1H),8.51(s,1H),7.79–7.69(m,1H),7.64(t,J＝8.0Hz,1H),7.39(s,1H),6.94-6.72(m,2H),6.69(s,1H),6.20-6.04(m,1H),5.69(d,J＝8.0Hz,1H),4.58–4.42(m,2H),4.10–3.94(m,3H),3.73–3.61(m,2H),2.40–2.25(m,2H),1.46(d,J＝8.0Hz,3H),1.09(s,9H).

2-(1'-Acryloyl-1',2',5',6'-tetrahydro-[2,3'-bipyridine]-5-yl)-N-(5-((5-(tert-butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)propanamide (200 mg) was separated by chiral preparative column. (Method: Chiral column: ChiralPak AD, 250×30 mm I.D., 10 μm, elution: isocratic: B 40%, A = carbon dioxide, B = isopropanol (0.1% ammonia water)) to obtain isomer 1 (retention time 1.484, 91 mg) and isomer 2 (retention time 2.146, 92 mg) of the title compound.

Isomer 1 (Example 32): ESI-MS m/z: 538.10 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.53(s,1H),8.51(s,1H),7.79–7.69(m,1H),7.64(t,J＝8.0Hz,1H),7.39(s,1H),6.94-6.72(m,2H),6.69(s,1H),6.20-6.04(m,1H),5.69(d,J＝8.0Hz,1H),4.58–4.42(m,2H),4.10–3.94(m,3H),3.73–3.61(m,2H),2.40–2.25(m,2H),1.46(d,J＝8.0Hz,3H),1.09(s,9H).

Isomer 2 (Example 33): ESI-MS m/z: 538.10 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.53(s,1H),8.51(s,1H),7.79–7.69(m,1H),7.64(t,J＝8.0Hz,1H),7.39(s,1H),6.94-6.72(m,2H),6.69(s,1H),6.20-6.04(m,1H),5.69(d,J＝8.0Hz,1H),4.58–4.42(m,2H),4.10–3.94(m,3H),3.73–3.61(m,2H),2.40–2.25(m,2H),1.46(d,J＝8.0Hz,3H),1.09(s,9H).

Experimental Example 1: Evaluation of in vitro kinase activity of compounds

1. Experimental Materials

Compound preparation: The compounds of the present invention prepared in the above examples were all prepared into 10 mM stock solutions with DMSO. Subsequently, starting from the highest starting concentration of 10 μM, 10 concentration gradients were diluted 3 times with DMSO and centrifuged at 1000 g for 1 min for subsequent experiments. 1% DMSO was used as a solvent control. The positive internal reference for CDK12 and CDK13 was THZ531 (purchased from MCE), and the positive internal reference for CDK7 and CDK9 was PHA-793887 (purchased from Selleck).

Reagents: ADP-Glo Kinase Assay (Promega, V9102), CDK7/CycH/MATA (Biortus, BP487-492-479), CDK9/CyclinT1 (Biortus, BP480), CDK12/CyclinK (Biortus, BP1642), CDK13/CyclinK (Biortus, BP1646/1648/691), ATP (Promega, V910B), DTT (Sigma, 48316), DMSO (Sigma, D8418).

Instrument: LabChip EZ Reader, purchased from Caliper, USA.

2. Experimental Methods

2.1.1X Kinase Buffer Preparation

Prepare 1X kinase buffer by mixing 1 volume of 5X enzyme buffer with 4 volumes of distilled water. Prepare a 1.5 mM DTT solution.

2.2. Compound screening

Add DMSO or test compound solution to the 384-well assay plate via Echo, seal the assay plate, and centrifuge the compound plate at 1000g for 1 min. Prepare 2X enzyme solution (CDK7/CycH/MATA, CDK9/CyclinT1, CDK12/CyclinK, CDK13/CyclinK) in 1x kinase buffer, add 2.5uL 2X enzyme solution to the 384-well assay plate, centrifuge the plate at 1000g for 1 min, and leave at room temperature for 10 min.

Prepare a 2X substrate (0.4 mg/mL MBP in CDK7/CycH/MAT1, 0.4 mg/mL PDK tide in CDK9/CyclinT1, 160 μM pS7-CTD in CDK12/CyclinK and CDK13/CyclinK) and ATP (2000 μM ATP in CDK7/CycH/MAT1, 2000 μM ATP in CDK9/CyclinT1, 500 μM ATP in CDK12/CyclinK, 120 μM ATP in CDK13/CyclinK) mixture in 1X kinase buffer and add 2.5 μL of the 2X substrate and ATP mixture to the well plate. This step is the start step of the reaction. Centrifuge the plate at 1000 g for 1 min, seal the assay plate, and leave at room temperature for 120 min. Add 4 μL of ADP-Glo reagent to the plate and centrifuge at 1000 g for 1 min. Incubate at room temperature for 60 min. Add 8 μL of kinase assay reagent to the plate and incubate at room temperature for 60 min. Centrifuge at 1000 g for 1 min. Measure luminescence signal on Envision 2104 plate reader.

3. Data Analysis

3.1 Calculation of inhibition rate (%)

GraphPad Prism 6.0 software was used to calculate IC ₅₀ and draw the dose-response curve of the compound. The inhibition rate was calculated as follows: %inhibition = 100-(Signalcmpd-SignalAve_PC)/(SignalAve_VC-SignalAve_PC)×100. (Signalcmpd: luminescent signal value of the test compound group; SignalAve_PC: luminescent signal value of the positive internal reference group; SignalAve_VC: luminescent signal value of the solvent control group)

3.2 Calculate _IC50 and draw the effect-dose curve of the compound

The inhibition rate of the corresponding compound concentration and the logarithmic value of the concentration were fitted into a nonlinear regression (dose response-variable slope) using GraphPad 6.0 to calculate the IC50.

Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC ₅₀ -X) × HillSlope)) (Bottom: lowest inhibition rate; Top: highest inhibition rate; HillSlope: steepness of the proliferation inhibition curve)

X: log of inhibitor concentration; Y: %Inhibition.

Table 1

“-” means not detected.

It can be seen from the above experimental results that the compounds of the present invention have good inhibitory activity against CDK12/13, and have low inhibitory effects on CDK7 and CDK9 and high selectivity.

Experimental Example 2: Evaluation of in vitro cell activity of compounds

1. Experimental Materials

Test compound: The compounds of the present invention prepared in the above examples, each compound was prepared into a 20mM stock solution with DMSO, the stock solution was diluted with DMSO, the highest concentration was diluted 10 times to 2mM, and then diluted 4 times with DMSO in sequence to 500.00nM, 125.00nM, 31.25nM, 7.81nM, 1.95nM, 0.49nM, 0.12nM, 0.03nM. After the compound was diluted with DMSO, it was diluted 100 times with cell culture medium before administration, and the negative control well was culture medium plus DMSO of the same volume as the administration well.

Human breast cancer cell lines MDA-MB-231, HCC1937, and human gastric cancer cell lines SNU-1 and AGS were purchased from the American Type Culture Collection (ATCC).

Reagents: RPMI-1640, purchased from Invitrogen, USA; L-15, purchased from Invitrogen, USA; FBS, purchased from Invitrogen, USA; pancreatin, purchased from Invitrogen, USA; P/S, purchased from Invitrogen, USA; DMSO, purchased from Sigma, USA; Luminescent Cell Viability Assay Kit was purchased from Progema, USA; Trypan blue staining solution was purchased from Progema, USA.

2. Experimental Methods

2.1 Cell culture:

Cell recovery: Dissolve the cells in a 37-degree water bath, then transfer to 15 mL of preheated culture medium, centrifuge at 1000 rpm for 5 min, discard the culture medium, resuspend the cells in 15 mL of fresh culture medium, transfer to a T75 culture flask, and culture in an incubator at 37°C, 5% _CO2 . After 24 hours, replace the cells with fresh culture medium.

Cell passaging: Transfer the revived cells to a 15 mL sterile centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the culture medium, Mix with 1 mL of serum-containing culture medium, add to a 10 cm culture dish according to a certain ratio, and culture in an incubator at 37 degrees and 5% CO ₂ .

2.2 Experimental steps:

Day 0: Plate. After the cells grow to a confluence of 80%-90% in the culture flask, discard the culture medium in the dish, wash the dead cells with 1 mL of PBS, discard the PBS, add 1 mL of trypsin for digestion, and when the cells become brighter and rounder under the microscope, terminate the digestion with an equal volume of culture medium, gently blow off the cells with a pipette, collect all the suspension in the dish into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard all the supernatant in the tube after centrifugation, mix the cells in the tube with 1 mL of culture medium, and count them. The cells were plated into 96-well plates at a density of 5000 cells/well for HCC1937, 3000 cells/well for MDA-MB-231, 3000 cells/well for SNU-1, and 3000 cells/well for AGS. The volume of culture medium + cells in each well was 100 μL. The four outer edges of the plate were filled with 200 μL PBS to prevent the culture medium at the edge from evaporating quickly, resulting in large differences in the culture conditions of the inner plate wells. After plating, the plates were placed in culture medium for overnight culture.

Day 1: Dosing. The drugs were prepared as described in the above “Test Compounds”, and the drugs diluted with culture medium were added to the 96-well plate prepared on the first day, 100 μL per well. The final drug concentrations in the plate were 10 μM, 2.5 μM, 0.625 μM, 0.15625 μM, 0.0390625 μM, 0.009765625 μM, 0.002441406 μM, 0.000610352 μM, 0.000152588 μM, 0 μM from left to right.

Day 3: Detection. After 3 days of drug treatment, discard the culture medium in the wells, take out the CellTiter-Glo Luminescent Cell Viabillity Assay 30 minutes in advance, and equilibrate to room temperature. Use 10% FBS culture medium to dilute Celltiter-Glo reagent 1:1 (equal volume), add 150μL diluted Celltiter-Glo reagent to each well, and shake at room temperature for 2 minutes. After incubating at room temperature for 10 minutes, take 100μL to detect the chemiluminescent signal, shake, and read the injection detection conditions for 500ms.

Data processing and analysis

(1) Calculate the inhibition rate of each well relative to the solvent control well based on the A.U. value derived from the microplate reader:

Inhibition (%) = 100 - (A.U. experimental well - A.U. blank well) / (A.U. solvent control well - A.U. blank well) × 100

(2) Based on different drug concentrations and their corresponding inhibition rates, the _IC50 curve was drawn using GraphicPad 5.0 software, and the data was analyzed to obtain the final _IC50 value.

The experimental results are shown in Table 2.

Table 2

“-” means not detected.

It can be seen from the above experiments that the compounds of the present invention exhibit good inhibitory activity against both human breast cancer cells and human gastric cancer cells.

Although the present invention has been described in detail above, it is understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the spirit and scope of the present invention. The scope of the present invention is not limited to the detailed description above, but should be attributed to the claims.

Claims

A compound represented by general formula (I) or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug,

in,

X1 and X2 are each independently selected from CH2 , CH, NH, N and S;

R is selected from halogen, hydroxy, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxyl, cyano, amino, oxygen, sulfur, mercapto, alkylthio, monoalkylamino, alkylacylamino, alkylacyl, aminoacyl, alkylaminoacyl, dialkylamino, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkylthio, heterocyclylalkylthio, arylalkylthio and heteroarylalkylthio, which is optionally substituted with one or more alkyl, alkoxy, alkylamino, halogen, hydroxy, amino, nitro, cyano, haloalkyl, alkylacyl, aminoacyl or alkylaminoacyl;

R 2 , R 3 and R 4 are each independently selected from hydrogen, halogen, hydroxy, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxyl, cyano, amino, alkylthio, monoalkylamino, alkylacylamino, alkylacyl, aminoacyl, alkylaminoacyl, dialkylamino and oxo;

R is selected from halogen, hydroxy, carbonyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxyl, cyano, amino, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, optionally substituted with one or more alkyl, alkenyl, alkynyl, alkoxy, alkylamino, halogen, hydroxy, amino, haloalkyl, alkylacyl, aminoacyl or alkylaminoacyl;

p and q are each independently selected from 0, 1, 2, 3 and 4; and

is a single bond or a double bond, two of which All are single keys, or one is a single bond and the other is a double bond.
The compound according to claim 1 or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug,

Among them Selected from
The compound according to claim 1 or 2, or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug,

wherein R1 is selected from halogen, hydroxy, C1-6 alkyl, halogenated C1-6 alkyl, hydroxy C1-6 alkyl, C1-6 alkoxy, halogenated C1-6 alkoxy C 1-6 alkylthio, halogen, hydroxy, amino, nitro, cyano, halogenated C 1-6 alkyl, C 3-8 cycloalkyl, C 3-8 heterocyclyl, C 6-12 aryl, C 5-12 heteroaryl, C 3-8 cycloalkylC 1-6 alkylthio, C 3-8 heterocyclylC 1-6 alkylthio, C 6-12 arylC 1-6 alkylthio and C 5-12 heteroarylC 1-6 alkylthio, which are optionally substituted by one or more C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylamino , halogen , hydroxy , amino, nitro, cyano, halogenated C 1-3 alkyl , C 3-8 The aminoacyl group may be substituted with a C 1-3 alkylacyl group, an aminoacyl group or a C 1-3 alkylaminoacyl group.
The compound according to any one of claims 1 to 3 or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug, wherein R 2 , R 3 and R 4 are each independently selected from hydrogen, halogen, hydroxy, alkyl, halogenated C 1-6 alkyl, hydroxy C 1-6 alkyl, C 1-6 alkoxy, halogenated C 1-6 alkoxy, hydroxy C 1-6 alkoxy, nitro, carboxyl, cyano, amino, C 1-6 alkylthio, mono C 1-6 alkylamino, C 1-6 alkylacylamino, C 1-6 alkylacyl, aminoacyl, C 1-6 alkylaminoacyl, di-C 1-6 alkylamino and oxo.
The compound according to any one of claims 1 to 4 or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug, wherein R 5 is selected from halogen, hydroxy, carbonyl, C 1-6 alkyl, halogenated C 1-6 alkyl, hydroxy C 1-6 alkyl, C 1-6 alkoxy , halogenated C 1-6 alkoxy, hydroxy C 1-6 alkoxy, nitro, carboxyl, cyano, amino, C 1-6 alkylcarbonyl, C 2-6 alkenylcarbonyl, C 2-6 alkynylcarbonyl, which is optionally substituted with one or more C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 alkylamino, halogen, hydroxy, amino, halogenated C 1-6 alkyl, C 1-6 alkylacyl, aminoacyl or C 1-6 alkylaminoacyl.
The compound according to any one of claims 1 to 5, or an isomer, a pharmaceutically acceptable salt, a solvate, a crystal or a prodrug thereof, wherein p and q are each independently selected from 0, 1, 2 and 3.
A compound represented by general formula (II) or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug,

Wherein, X1 , X2 , R1 , R2 , R3 , R4 , R5 , p, q and As defined in the general formula (I) in claims 1-6.
The compound according to any one of claims 1 to 7 or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug, wherein the compound is selected from the following compounds:
A pharmaceutical composition comprising the compound according to any one of claims 1 to 8 or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug and a pharmaceutically acceptable carrier.
Use of the compound according to any one of claims 1 to 8 or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug, or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating a disease associated with CDK12/13.