Bifunctional compounds and uses thereof

Abstract

The application discloses a bifunctional compound and use thereof, the bifunctional compound is a bifunctional compound with K-L-S structure, wherein: K is a targeting group of cyclin 7CDK7;S is a covalent group targeting cysteine or lysine on CDK7 protein;L is a divalent linking group chemically connecting the targeting group K and the covalent group S;The bifunctional compound or does not contain divalent linking group L, and the targeting group K is directly connected with the covalent group S.The bifunctional compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or stereoisomer and the pharmaceutical composition containing the same show pharmacological activity related to degradation, inhibition of target protein CDK7, and can be used for preventing and treating CDK7 related diseases or disorders.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicinal chemistry, in particular to a bifunctional compound and uses thereof. BACKGROUND

[0002] CDKs are serine / threonine protein kinases that regulate either transcription (CDK 7-13 and 19-20) or cell cycle progression (CDK 1-6 and 14-18). Almost all CDK activation is through: (1) binding to a Cyclin; (2) phosphorylation of the T-Loop by CDK Activating Kinase (CAK). CAK is a ternary complex consisting of CDK7, Cyclin H, and RING-finger protein MAT1. CDK7 is unique in that it is involved in both transcriptional and cell cycle regulation.

[0003] CDK7 is activated upon autophosphorylation of Thr170 in the T-loop and binding to Cyclin H. However, this autophosphorylation is not required for CAK activity, as it is matched by the interaction of CDK7 with MAT1. MAT1 is required for the ternary complex, as the assembly of CDK7 with Cyclin H is not stable. Unlike other CDKs, which have only one phosphorylation site in the T-loop, CDK7 has a second phosphorylation site, Ser164, whose phosphorylation enhances its binding affinity to Cyclin H. Both of these residues of CDK7 are phosphorylated by CDK1 and CDK2, which in turn regulate cell cycle regulators to affect gene expression.

[0004] Aberrant CDK7 expression is detected in multiple cancer types and is associated with aggressive clinicopathological features and poor prognosis. CDK7 is amplified in hepatocellular carcinoma, gastric cancer, and colorectal cancer (CRC) and prostate cancer. Immunohistochemical analysis of 173 gastric cancer specimens showed elevated CDK 7 levels, which were associated with tumor grade. Similarly, CDK7 protein was overexpressed in most oral squamous cell carcinoma specimens and was associated with increased T-stage and decreased disease-free survival, suggesting that it can serve as a prognostic biomarker. CDK7 protein and mRNA levels were upregulated in cancerous breast tissue compared to adjacent normal breast tissue. Interestingly, elevated expression of CDK7 was associated with poor clinical outcome in triple-negative breast cancer (TNBC) patients, but better prognosis in ER+ breast cancer patients.

[0005] Currently, there are some reports on inhibitors of CDK7, such as WO2020093006, WO2019057825, WO2022130304. However, as an example of anti-cancer drugs, traditional small molecules inhibit the activity of target proteins by targeting binding, induce apoptosis of cancer cells, but the target proteins in tumor cells often recover their activity and acquire drug resistance through overexpression of target proteins or new mutations of target proteins. The defects of traditional small molecule inhibitors make small molecule drugs gradually decline, and revolutionary new technologies need to be introduced into the research and development of small molecule drugs.

[0006] In the face of this phenomenon, researchers have found a new method of using covalent small molecules to knock out functional target proteins instead of simply inhibiting the activity of target proteins. As can be seen from patents WO2021009568A1, WO2021099842A1, pentafluorobenzenesulfonamide derivatives can covalently bind to cysteine on the target protein, induce protein conformational changes, expose protein hydrophobic groups, and be recognized by the endogenous ubiquitination system to promote its degradation. In addition, reference J. Am. Chem. Soc. 2021, 143, 4766-4774, by adding an N-acyl-N-alkyl sulfonamide group to the traditional small molecule inhibitor, it can covalently bind to a specific lysine on the target protein and disrupt the function of the target protein.

[0007] For diseases or disorders related to abnormal amplification of CDK7, small molecule covalent preparations capable of targeted degradation therapy are of great significance. Therefore, the present application is proposed. SUMMARY

[0008] The purpose of the present application is to overcome the shortcomings of the prior art and provide a bifunctional compound with K-L-S structure and its use in the preparation of a medicament for treating, inhibiting or preventing CDK7 related diseases.

[0009] The above-mentioned purpose of the present application is achieved by the following technical solutions:

[0010] A bifunctional compound with K-L-S structure, or a pharmaceutically acceptable salt, ester, hydrate, solvate or stereoisomer thereof, wherein:

[0011] K is a targeting group of cyclin 7 CDK7;

[0012] S is a covalent group targeting cysteine or lysine on CDK7 protein;

[0013] L is a bivalent linking group chemically linking the targeting group K and the covalent group S;

[0014] The bifunctional compound does not contain a bivalent linking group L, and the targeting group K is directly connected to the covalent group S.

[0015] Preferably, the targeting group K has any of the following structures:

[0016]

[0017]

[0018] Preferably, the covalent group S is:

[0019]

[0020] wherein: R1, R2, R3 are the same or different; R1, R2, R3 are independently selected from the group consisting of: H, F, Cl, Br, OCH3, CHF2, OCH2F, OCHF2, CHF2, CH2F, CF3, CN, CH3, OH, NH2,

[0021] Preferably, the divalent linking group L is selected from the group consisting of:

[0022] one or a combination of any of the following: substituted or unsubstituted hydrocarbyl, hydrocarbyloxy, oxycarbyl, cyclohydrocarbyl, heterocyclohydrocarbyl, acylhydrocarbyl, hydrocarbylacyl, carbonylhdrocarbyl, hydrocarbonylcarbonyl, amido-hydrocarbyl, hydrocarbylamido, aryl, oligopeptidyl groups having two attachment sites;

[0023] The divalent linking group L links K and S with a chemical covalent bond to form a complete bifunctional compound.

[0024] More preferably, the hydrocarbyl group includes saturated hydrocarbyl, unsaturated hydrocarbyl, aromatic hydrocarbyl, oxygen-hetero-hydrocarbyl, nitrogen-hetero-hydrocarbyl, sulfur-hetero-hydrocarbyl, phosphorus-hetero-hydrocarbyl, and mixed-hetero-hydrocarbyl groups having a chain length of 1 to 20 atoms; wherein the hetero-hydrocarbyl group contains 1 to 5 heteroatoms and the valence of the heteroatoms is satisfied by hydrogen, oxygen, nitrogen, etc. in the corresponding bonding mode as needed.

[0025] More preferably, the divalent linking group L is selected from the group consisting of the following structures:

[0026]

[0027]

[0028]

[0029]

[0030] Preferably, the bifunctional compound having the structure K-L-S is selected from the group consisting of the following structures:

[0031]

[0032]

[0033]

[0034] A pharmaceutical composition comprising the bifunctional compound or a pharmaceutically acceptable salt, ester, hydrate, solvate or stereoisomer thereof as described above.

[0035] Preferably, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient or carrier or diluent.

[0036] More preferably, the pharmaceutically acceptable excipient comprises one or more of a binder, a filler, a disintegrant, a lubricant and a glidant.

[0037] More preferably, the pharmaceutically acceptable carrier comprises one or more of a cream, a lotion, a gel, a liposome and a nanoparticle.

[0038] Preferably, the composition is suitable for oral administration or for injection.

[0039] Use of any of the bifunctional compounds described above or any of the compositions described above for the manufacture of a medicament for the prevention or treatment of a hyperproliferative disorder.

[0040] Use of any of the bifunctional compounds described above or any of the compositions described above for the manufacture of a medicament for the treatment of a CDK7-related disease.

[0041] Preferably, the CDK7-related disease is a CDK7-related malignancy or cancer.

[0042] More preferably, the malignant tumor or cancer is selected from the group consisting of: lung: bronchus (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchus) carcinoma, bronchus adenoma, sarcoma, lymphoma, chondroma, mesothelioma; gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyoma, lymphoma), stomach (carcinoma, lymphoma, leiomyoma), pancreas (ductal adenocarcinoma, insulinoma, glucomonas, gastrinoma, carcinoid tumor, vasoactive intestinal peptide tumor), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hematoma, leiomyoma); genitourinary tract: kidney (adenocarcinoma, Wilms' tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumor, lipoma); liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; biliary tract: gallbladder carcinoma, ampullary carcinoma, cholangiocarcinoma; nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningeal sarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, neuroglioma, epididymal tumor, germ cell tumor (pinealoma), glioblastoma multiforme, oligodendroglioma, neuroglioma, retinoblastoma, congenital tumor), spinal cord neurofibroma, meningioma, glioma, sarcoma); gynecological: uterus (endometrial carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa cell tumor, clear cell carcinoma, dysplasia, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, rhabdomyosarcoma (embryonal), fallopian tubes (carcinoma), breast (carcinoma); hematology: blood (myeloid leukemia (acute and chronic), acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma); skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; adrenal gland: neuroblastoma.

[0043] Preferably, the malignant tumor is one or more of non-small cell lung cancer, prostate cancer, pancreatic cancer, colorectal cancer, cervical cancer, bladder cancer, liver cancer, breast cancer.

[0044] Beneficial effects:

[0045] The present application provides a kind of bifunctional compound with K-L-S structure or its pharmaceutically acceptable salt, ester, hydrate, solvate or stereoisomer and the pharmaceutical composition containing it, they can be used as CDK7 cyclin 7 regulator, show pharmacological activity related to degradation, inhibition of target protein CDK7, can be used for preventing and treating CDK7 related diseases or conditions. DETAILED DESCRIPTION

[0046] The following will be specifically introduced with examples the substantial content of the present application, but not limited to the protection scope of the present application.

[0047] The bifunctional compound disclosed in the present application, or its pharmaceutically acceptable salt, ester or stereoisomer, comprises a targeting group K of cyclin CDK7, a covalent ligand group S of cysteine or lysine and a bivalent linking group (L) chemically connecting the targeting group (K) and the ligand group (S), so that the target protein (cyclin CDK7) is disordered, and then the activity of the protein is affected or inhibited.

[0048] The K and S described in the present application are only shown as examples in terms of quantity and position, and are not used to limit the compound, and in actual cases, those skilled in the art can adjust or change according to needs.

[0049] In some embodiments, the bifunctional compound includes a targeting group K and a covalent ligand group S, and W and T are covalently connected to the corresponding sites of the bivalent linking group L respectively, forming a protein degradation targeting chimera, which is represented by the following general formula:

[0050] K-L-S.

[0051] In some embodiments, the bifunctional compound only includes a targeting group K and a covalent ligand group S, i.e. in the form of K-S.

[0052] In some embodiments, the targeting group K of cyclin CDK7 has the structure as shown below:

[0053]

[0054] Wherein S is selected from:

[0055]

[0056] Wherein R1, R2, R3 can be the same or different; R1, R2, R3 are selected from: H, F, Cl, Br, OCH3, CHF2, OCH2F, OCHF2, CHF2, CH2F, CF3, CN, CH3, OH, NH2,

[0057] In some embodiments, the structure of the bivalent linker L is selected from one or a combination of several of the following: substituted or unsubstituted hydrocarbyl, hydrocarbyloxy, oxycarbyl, cyclohydrocarbyl, heterocyclohydrocarbyl, acylhydrocarbyl, hydrocarbylacyl, carbonylhdrocarbyl, hydrocarbylcarbonyl, amido hydrocarbyl, hydrocarbylamido, aryl, and oligopeptidyl groups having two attachment sites, the bivalent linker (L) covalently linking W and T to form the complete target molecule; wherein hydrocarbyl includes, but is not limited to, saturated hydrocarbyl, unsaturated hydrocarbyl, aromatic hydrocarbyl, oxygen heterocarbyl, nitrogen heterocarbyl, sulfur heterocarbyl, phosphorus heterocarbyl, and mixed heterocarbyl groups of different heteroatoms, and the chain length of the hydrocarbyl or heterocarbyl group is from 1 to 20 atoms, and when it is a heterocarbyl group, the heterocarbyl group contains from 1 to 5 heteroatoms and the valence of the heteroatoms is satisfied by hydrogen, oxygen, nitrogen, and the like as needed in the corresponding bonding.

[0058] Further, the chemical linker L is selected from the following structures:

[0059]

[0060]

[0061]

[0062] In some embodiments, the bifunctional compounds include the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, hydrates, solvates.

[0063]

[0064]

[0065]

[0066]

[0067] The above compounds have good biological activity and can be used for treating CDK7 related diseases.

[0068] The present application also provides a pharmaceutical composition comprising any of the above compounds or pharmaceutically acceptable salts, esters, hydrates, solvates, or stereoisomers thereof.

[0069] Further, at least one pharmaceutically acceptable excipient or carrier or diluent is also included.

[0070] Further, the pharmaceutically acceptable excipient includes one or more of a binder, a filler, a disintegrant, a lubricant, and a glidant.

[0071] Further, the pharmaceutically acceptable carrier includes one or more of a cream, a lotion, a gel, a liposome, and a nanoparticle.

[0072] Further, the composition is suitable for oral administration or injection administration.

[0073] The present application also provides a use of any of the above-mentioned compounds or pharmaceutically acceptable salts or esters or hydrates or solvates or isomers thereof or a pharmaceutical composition in the manufacture of a medicament for treating, inhibiting or preventing a hyperproliferative disorder. The present application also provides a method of treating, inhibiting or preventing a hyperproliferative disorder, comprising administering to a subject an effective amount of the above-mentioned compound and / or pharmaceutical composition, thereby treating the relevant disease.

[0074] In some embodiments, the hyperproliferative disorder is a malignancy or cancer associated with CDK7.

[0075] Further, the malignant tumor or cancer is selected from the group consisting of: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and hamartoma; lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondroma, mesothelioma; gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vasoactive intestinal peptide tumor), small bowel (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hematoma, leiomyoma); genito-urinary tract: kidney (adenocarcinoma, Wilms' tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid odema, lipoma); liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; biliary tract: gall bladder carcinoma, ampullary carcinoma, cholangiocarcinoma; bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteocartilaginous exostosis), benign chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, oligodendroglioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, neurona! tumor), spinal cord neurofibroma, meningioma, glioma, sarcoma); gynecological: uterus (endometrial carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumors, arrhenoblastoma, dysplasia, malignant mole), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, rhabdomyosarcoma (embryonal), fallopian tubes (carcinoma); hematologic: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disease, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma); skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloid, psoriasis; adrenal gland: neuroblastoma.

[0076] In some embodiments, the malignancy is one or more of non-small cell lung cancer, small cell lung cancer, pancreatic cancer, colorectal cancer, cholangiocarcinoma, cervical cancer, bladder cancer, liver cancer, or breast cancer.

[0077] The present application is described in detail below by way of Examples, but it is not meant to present any unfavorable limitations on the present application. The present application has been described in detail herein, and specific embodiments thereof have been disclosed with particularity, but it will be apparent that various changes and modifications can be made therein without departing from the spirit and scope of the present application.

[0078] Example 1

[0079]

[0080] Step A: 3-isopropyl-5-methyl-N-(piperidin-4-yl)pyrazolo[l,5-a]pyrimidin-7-amine (100 mg, 0.37 mmol, 1 eq) and tert-butyl 2-oxo-7-azaspiro[3.5]nonane-7-carboxylate (89 mg, 0.37 mmol, 1 eq) were added to 5 mL dry dichloromethane solution with one drop of acetic acid at room temperature, stirred for 1 min, sodium triacetoxyborohydride (0.24 g, 1.11 mmol, 3 eq) was added to the solution, and stirred at room temperature for 5 h. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 140 mg of white solid with a yield of 60% and a purity of 90%.

[0081] 1 H NMR (400 MHz, CDCl3) δ 7.74 (s, 1H), 6.45 (s, 1H), 5.71 (s, 1H), 3.58 (s, 1H), 3.33-3.16 (m, 5H), 3.08 (d, J = 11.6 Hz, 2H), 2.97 (t, J = 8.1 Hz, 1H), 2.43 (s, 3H), 2.36 (d, J = 11.3 Hz, 2H), 2.15-2.08 (m, 2H), 2.05-1.99 (m, 3H), 1.94-1.84 (m, 3H), 1.53-1.39 (m, 4H), 1.37 (s, 9H), 1.24 (d, J = 6.9 Hz, 6H).

[0082] Step B: To a solution of tert-butyl 2-(4-[(5-methyl-3-(propan-2-yl)pyrazolo[l,5- a]pyrimidin-7-yl)amino]piperidin-l-yl)-7-azaspiro[3.5]nonane-7-carboxylate (150 mg, 0.27 mmol, 1 eq) and 4 M hydrogen chloride solution in dioxane (0.5 mL) was added in 3 mL of dichloromethane and stirred at room temperature for 5 h. After completion of the reaction, the solvent was removed under reduced pressure to get white solid 130 mg, 90% yield, which was used directly for the next step without purification.

[0083] Step C: To a solution of l-(7-azaspiro[3.5]non-2-yl)-N-(5-methyl-3-(propan-2- yl)pyrazolo[l,5-a]pyrimidin-7-yl)piperidin-4-amine (100 mg, 0.25 mmol, 1 eq) and N,N- diisopropylethylamine (48 mg, 0.38 mmol, 1.5 eq) in 3 mL of dry dichloromethane, a solution of pentafluorobenzenesulfonyl chloride (0.032 g, 0.25 mmol, 1 eq) in 1 mL of dichloromethane was added slowly and stirred at this temperature for 1 h. After completion of the reaction, the solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 10: 1) to get white solid 91.8 mg, 63% yield, 95% purity.

[0084] 1 H NMR (400 MHz, CDC13) δ 7.81 (s, 1H), 6.12 (d, J = 8.1 Hz, 1H), 5.74 (s, 1H), 3.50 (d, J = 12.2 Hz, 1H), 3.41 - 3.22 (m, 3H), 3.18 (t, J = 5.5 Hz, 2H), 2.87 - 2.60 (m, 3H), 2.50 (s, 3H), 2.10 (d, J = 12.7 Hz, 2H), 2.05 - 1.93 (m, 4H), 1.76 - 1.70 (m, 8H), 1.33 (d, J = 6.9 Hz, 6H). 19 F NMR (376 MHz, CDC13) δ -134.31 - -134.47 (m, 2F), -143.96 - -147.69 (m, IF), -156.89 - -162.71 (m, IF). LC-MS: 627.06 [M+H] + .

[0085] Example 2

[0086]

[0087] Step A: To a solution of 3-isopropyl-5-methyl-N-(piperidin-4-yl)pyrazolo[l,5- a]pyrimidin-7-amine (100 mg, 0.37 mmol, 1 eq) and l-[(tert-butoxy)carbonyl]piperidine- 4-carboxylic acid (85 mg, 0.37 mmol, 1 eq) and N,N-diisopropylethylamine (240 mg, 1.85 mmol, 5 eq) in 2 mL of N,N-dimethylformamide, 2-(7-azobenzotriazole)-N,N,N',N'- tetramethyluronium hexafluorophosphate (210 mg, 0.55 mmol, 1.5 eq) was added slowly and stirred for 10 min. After completion of the reaction, the solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to get 130 mg of white solid with 70% yield and 90% purity.

[0088] 1 H NMR (400 MHz, CDC13) δ 7.81 (d, J = 1.9 Hz, 1H), 6.14 (d, J = 8.0 Hz, 1H), 5.79 (d, J = 1.9 Hz, 1H), 4.54 (d, J = 13.7 Hz, 1H), 4.15 (s, 3H), 3.96 (d, J = 14.0 Hz, 1H), 3.74 (d, J = 9.9 Hz, 1H), 3.29 (tt, J = 11.5, 5.9 Hz, 2H), 2.94 (q, J = 10.6, 9.2 Hz, 1H), 2.83 - 2.60 (m, 4H), 2.52 (s, 3H), 2.25 - 2.12 (m, 2H), 1.73 - 1.53 (m, 4H), 1.46 (d, J = 1.9 Hz, 9H), 1.33 (d, J = 6.9 Hz, 6H).

[0089] Step B: To a solution of tert-butyl 4-(4-[(5-methyl-3-(propan-2-yl)pyrazolo[l,5- a]pyrimidin-7-yl)amino]piperidine-l-carbonyl)piperidine-l-carboxylate (150 mg, 0.27 mmol, 1 eq) and 4 M hydrogen chloride solution in dioxane (0.5 mL) was added to 3 mL of dichloromethane and stirred at room temperature for 5 h. After completion of the reaction, the solvent was removed under reduced pressure to get 95 mg of white solid with 95% yield which was used directly for the next reaction without purification.

[0090] Step C: N-(5-methyl-3-(propan-2-yl)pyrazolo[l,5-a]pyrimidin-7-yl)-l-(piperidine-4- carbonyl)piperidin-4-amine (95 mg, 0.25 mmol, 1 eq) and N,N-diisopropylethylamine (97 mg, 0.75 mmol, 3 eq) were dissolved in 3 mL of dry dichloromethane in an ice bath, a solution of pentafluorobenzenesulfonyl chloride (0.067 g, 0.25 mmol, 1 eq) in dichloromethane (1 mL) was added slowly and stirred at this temperature for 1 h. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography (dichloromethane:methanol = 10: 1) to obtain 109 mg of a white solid, yield 80%, purity 95%, MS: 623.2 [M+H].

[0091] 1 H NMR (400 MHz, CDC13) δ 7.81 (s, 1H), 6.15 (d, J = 8.1 Hz, 1H), 5.78 (s, 1H), 4.49 (d, J = 13.6 Hz, 1H), 3.88 (d, J = 12.4 Hz, 3H), 3.80 - 3.68 (m, 1H), 3.29 (h, J = 7.2 Hz, 2H), 2.95 (t, J = 11.6 Hz, 3H), 2.67 (tt, J = 9.2, 4.3 Hz, 1H), 2.52 (s, 3H), 2.17 (s, 2H), 2.00 - 1.80 (m, 4H), 1.59 (d, J = 11.4 Hz, 2H), 1.33 (d, J = 6.9 Hz, 6H). 19 F NMR (376 MHz, CDC13) δ -134.35 (dd, J = 21.5, 5.8 Hz, 2F), -145.27 (s, IF), -156.89 - -161.29 (m, 2F). LC-MS: 614.96 [M+H] + .

[0092] Example 3

[0093]

[0094] The synthesis process was referred to that of Example 1 compound, to obtain 126.7 mg of a white solid, yield 72%, purity 95%, MS: 623.2 [M+H]. 1H NMR (400 MHz, CDC13) δ 7.81 (s, 1H), 6.11 (d, J = 8.1 Hz, 1H), 5.75 (s, 1H), 3.58 (ddd, J = 14.2, 9.2, 5.8 Hz, 2H), 3.53 - 3.47 (m, 1H), 3.43 (q, J = 8.3 Hz, 1H), 3.30 (hept, J = 6.9 Hz, 1H), 3.20 (dd, J = 9.9, 7.1 Hz, 1H), 2.87 (d, J = 11.5 Hz, 1H), 2.78 (d, J = 11.7 Hz, 1H), 2.51 (s, 3H), 2.49 - 2.44 (m, 1H), 2.40 - 2.29 (m, 2H), 2.28 - 2.14 (m, 2H), 2.12 - 2.00 (m, 3H), 1.74 - 1.60 (m, 3H), 1.33 (d, J = 6.9 Hz, 6H). 19 F NMR (376 MHz, CDC13) δ -134.89 - -135.36 (m, 2F), -145.80 - -146.49 (m, 1F), -158.17 - -158.67 (m, 2F). LC-MS: 587.02 [M+H] + .

[0095] Example 4

[0096]

[0097] Step A: tert-Butyl (3R)-3-hydroxypyrrolidine-1-carboxylate (90 mg, 0.48 mmol, 1.3 eq) was added to triethylamine (220 μL, 1.67 mmol, 4.5 eq) in 2 mL dry dichloromethane, then 4-nitrophenyl chloroformate (100 mg, 0.52 mmol, 1.4 eq) was added and stirred at room temperature for 3 h, then the reaction was cooled to 0 °C, 3-isopropyl 5-methyl-N-(piperidin-4-yl)pyrazolo[l,5-a]pyrimidin-7-amine (100 mg, 0.37 mmol, 1 eq) was added and the reaction was stirred at 30-45 °C for 5 h. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 20: 1) to obtain a yellow solid 143 mg, yield 69%, purity 90%.

[0098] 1H NMR (400 MHz, CDC13) δ 7.85 (s, 1H), 6.28 (d, J = 8.1 Hz, 1H), 5.81 (s, 1H), 5.25 (s, 1H), 4.12 (q, J = 7.2 Hz, 1H), 3.71 (dd, J = 9.0, 5.0 Hz, 1H), 3.65 - 3.48 (m, 3H), 3.44 (d, J = 13.6 Hz, 1H), 3.28 (p, J = 6.9 Hz, 1H), 3.08 (t, J = 12.5 Hz, 2H), 2.52 (s, 3H), 2.07 (d, J = 19.1 Hz, 4H), 1.79 - 1.55 (m, 3H), 1.48 (s, 11H), 1.29 (d, J = 6.9 Hz, 6H).

[0099] Step B: To a solution of (3R)-1-[(tert-butoxy)carbonyl]pyrrolidin-3-yl 4-[(5-methyl-3- (prop-2-yl)pyrazolo[1,5-a]pyrimidin-7-yl)amino]piperidine-1 -carboxylate (102 mg, 0.21 mmol, 1 eq) and 4M hydrochloric acid in ethyl acetate (0.5 mL) was added at room temperature and stirred for 5 h at room temperature. After the reaction was completed, the solvent was removed under reduced pressure to obtain 70 mg of white solid, 80% yield, which was directly used in the next step without purification.

[0100] Step C: (3R)-Pyrrolidin-3-yl 4-[(5-methyl-3-(prop-2-yl)pyrazolo[1,5-a]pyrimidin-7- yl)amino]piperidine-1 -carboxylate (80 mg, 0.21 mmol, 1 eq) and N,N-diisopropylethylamine (41 mg, 0.32 mmol, 1.5 eq) were dissolved in 3 mL of dry dichloromethane in an ice bath, and a solution of pentafluorobenzenesulfonyl chloride (0.056 g, 0.21 mmol, 1 eq) in dichloromethane (1 mL) was slowly added and stirred at this temperature for 1 h. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain 90 mg of white solid, 60% yield, 95% purity.

[0101] 1H NMR (400 MHz, CDC13) δ 7.82 (s, 1H), 6.14 (s, 1H), 5.78 (s, 1H), 5.28 (s, 1H), 4.16 (s, 1H), 3.94 (s, 1H), 3.74 - 3.52 (m, 5H), 3.30 (hept, J = 6.9 Hz, 1H), 3.04 (ddt, J = 14.3, 11.3, 3.5 Hz, 2H), 2.53 (s, 3H), 2.20 (tt, J = 6.6, 3.5 Hz, 2H), 2.16 - 2.07 (m, 2H), 1.66 - 1.50 (m, 2H), 1.34 (d, J = 6.9 Hz, 6H). 19 F NMR (376 MHz, CDC13) δ -135.17 (dd, J = 86.2, 22.2 Hz, 2F), -145.73 (s, 1F), -158.25 (t, J = 21.5 Hz, 2F). LC-MS: 617.05 [M+H] + .

[0102] Example 5

[0103]

[0104] The synthesis procedure was referenced to the synthesis procedure of Example 1 compound to give 75 mg of white solid in 47% yield, 95% purity.

[0105] 1 H NMR (400 MHz, CDC13) δ 7.82 (s, 1H), 6.15 (s, 1H), 5.79 (s, 1H), 5.32 - 5.21 (m, 1H), 4.14 (s, 1H), 3.94 (s, 1H), 3.75 - 3.52 (m, 5H), 3.31 (hept, J = 6.9 Hz, 1H), 3.04 (ddt, J = 14.2, 11.2, 3.4 Hz, 2H), 2.53 (s, 3H), 2.20 (ddd, J = 9.5, 6.4, 3.6 Hz, 2H), 2.12 (s, 3H), 1.63 - 1.51 (m, 2H), 1.34 (d, J = 6.9 Hz, 6H). 19 F NMR (376 MHz, CDC13) δ -135.17 (dd, J = 85.9, 21.9 Hz, 2F), -145.54 - -146.00 (m, 1F), -158.23 (d, J = 25.5 Hz, 2F). LC-MS: 616.98 [M+H] + .

[0106] Example 6

[0107]

[0108] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 25 mg of white solid in 17% yield with 90% purity.

[0109] 1 H NMR (400 MHz, CD3OD) δ 8.17 (s, 1H), 6.54 (s, 1H), 4.21 (s, 3H), 3.88 (s, 2H), 3.63 (s, 2H), 3.47 (s, 2H), 3.20 (s, 4H), 2.66 (s, 3H), 2.30 (d, J = 14.5 Hz, 2H), 2.17 (d, J = 13.0 Hz, 2H), 1.35 (t, J = 5.2 Hz, 6H). LC-MS: 573.02 [M+H] + .

[0110] Example 7

[0111]

[0112] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 5 mg of white solid in 3.15% yield with 95% purity.

[0113] 1 H NMR (400 MHz, CD3OD) δ 8.17 (s, 1H), 6.54 (s, 1H), 4.21 (s, 3H), 3.88 (s, 2H), 3.63 (s, 2H), 3.47 (s, 2H), 3.20 (s, 4H), 2.66 (s, 3H), 2.30 (d, J = 14.5 Hz, 2H), 2.17 (d, J = 13.0 Hz, 2H), 1.35 (t, J = 5.2 Hz, 6H). LC-MS: 573.02 [M+H] + .

[0114] Example 8

[0115]

[0116] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 24 mg of white solid in 19% yield with 90% purity.

[0117] 1H NMR (400 MHz, CD3OD) δ 8.17 (s, 1H), 6.57 (s, 1H), 4.35 - 4.16 (m, 1H), 3.91 (d, J = 12.0 Hz, 2H), 3.76 (d, J = 12.5 Hz, 2H), 3.22 (td, J = 12.9, 12.0, 6.0 Hz, 2H), 3.12 (d, J = 7.0 Hz, 2H), 2.83 - 2.73 (m, 2H), 2.66 (s, 3H), 2.38 - 2.16 (m, 4H), 2.10 - 1.87 (m, 2H), 1.46 (dd, J = 12.1, 4.1 Hz, 1H), 1.41 (d, J = 3.9 Hz, 1H), 1.35 (d, J = 6.9 Hz, 6H), 0.01 (s, 1H). LC-MS: 600.96 [M+H] + .

[0118] Example 9

[0119]

[0120] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 7.7 mg of white solid in 3.83% yield with 95% purity.

[0121] 1 H NMR (400 MHz, CD3OD) δ 8.17 (s, 1H), 6.57 (s, 1H), 4.35 - 4.16 (m, 1H), 3.91 (d, J = 12.0 Hz, 2H), 3.76 (d, J = 12.5 Hz, 2H), 3.22 (td, J = 12.9, 12.0, 6.0 Hz, 2H), 3.12 (d, J = 7.0 Hz, 2H), 2.83 - 2.73 (m, 2H), 2.66 (s, 3H), 2.38 - 2.16 (m, 4H), 2.10 - 1.87 (m, 2H), 1.46 (dd, J = 12.1, 4.1 Hz, 1H), 1.41 (d, J = 3.9 Hz, 1H), 1.35 (d, J = 6.9 Hz, 6H), 0.01 (s, 1H). LC-MS: 600.96 [M+H] + .

[0122] Example 10

[0123]

[0124] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 40 mg of white solid in 41.71% yield with 95% purity.

[0125] 1H NMR (400 MHz, CDC13) δ 7.80 (s, 1H), 6.09 (d, J = 8.2 Hz, 1H), 5.75 (s, 1H), 3.90 (s, 1H), 3.49 (d, J = 10.5 Hz, 1H), 3.29 (hept, J = 7.0 Hz, 1H), 2.86 (d, J = 11.4 Hz, 2H), 2.62 (t, J = 11.7 Hz, 2H), 2.49 (s, 3H), 2.37 (t, J = 7.2 Hz, 2H), 2.17 (d, J = 10.9 Hz, 2H), 2.08 (d, J = 12.7 Hz, 2H), 1.80 (d, J = 12.4 Hz, 2H), 1.68 (q, J = 10.4, 9.8 Hz, 2H), 1.46 (q, J = 7.1, 6.6 Hz, 2H), 1.33 (d, J = 6.9 Hz, 7H). LC-MS: 614.96 [M+H] + .

[0126] Example 11

[0127]

[0128] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 30 mg of white solid in 23.13% yield with 90% purity.

[0129] 1 H NMR (400 MHz, CDC13) δ 7.79 (s, 1H), 6.11 (d, J = 8.2 Hz, 1H), 5.75 (s, 1H), 3.83 (dd, J = 12.0, 3.7 Hz, 1H), 3.72 (dd, J = 10.4, 5.7 Hz, 1H), 3.49 (dd, J = 8.2, 4.1 Hz, 1H), 3.28 (p, J = 6.9 Hz, 1H), 2.83 (t, J = 10.7 Hz, 0H), 2.79 - 2.72 (m, 2H), 2.49 (s, 4H), 2.21 (d, J = 6.9 Hz, 3H), 2.13 (s, 0H), 2.08 - 2.01 (m, 1H), 1.87 - 1.74 (m, 1H), 1.73 - 1.61 (m, 2H), 1.32 (d, J = 6.9 Hz, 6H). LC-MS: 600.92 [M+H] + .

[0130] Example 12

[0131]

[0132] Step A: To a solution of 1-(methoxycarbonyl)cyclopropane-1 -carboxylic acid (600 mg, 4.16 mmol, 1 eq) and tert-butyl 3-amino pyrrolidine-1 -carboxylate (774.80 mg, 4.16 mmol, 1 eq) and 2-(7-azobenzotriazol-1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1581.76 mg, 4.16 mmol, 1 eq) and N,N-diisopropyl ethyl amine (1075.28 mg, 8.32 mmol, 3 eq) in 15 mL dichloromethane was added at room temperature and stirred for 3 h at room temperature. After completion of the reaction, the solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to get 1.6 g of clear oil in 86% yield and 70% purity. LC-MS: 213.14 [M+H] + .

[0133] Step B: To a solution of tert-butyl 3-(1-(methoxycarbonyl)cyclopropanecarboxamido)pyrrolidine-1-carboxylate (300 mg, 0.071 mmol, 1 eq) and lithium hydroxide (91.97 mg, 3.84 mmol, 4 eq) in 5 mL methanol and 3 mL water was added at room temperature and stirred for 2 h at room temperature. After completion of the reaction, it was adjusted to pH = 3 with 1 M aqueous hydrogen chloride solution and extracted with ethyl acetate and the solvent was removed under reduced pressure to get 230 mg of white solid in 80% yield which was used as such for the next reaction without purification. LC-MS: 225.09 [M+H] + .

[0134] Step C: To a solution of 1-[(1-[(tert-butoxy)carbonyl]pyrrolidin-3-yl)carbamoyl]cyclopropane-1-carboxylic acid (50 mg, 0.17 mmol, 1 eq) and N-(5-methyl-3-(propan-2-yl)pyrazolo[1,5-a]pyrimidin-7-yl)piperidin-4-amine (46.47 mg, 0.17 mmol, 1 eq) and 2-(7-azobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (64.64 mg, 0.17 mmol, 1 eq) and N,N-diisopropyl ethyl amine (43.94 mg, 0.34 mmol, 2 eq) in 5 mL dry dichloromethane was added at room temperature and stirred for 1 h at room temperature. After completion of the reaction, the solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1 : 1) to get 131 mg of yellow oily material in 99% yield and 70% purity. LC-MS: 554.13 [M+H] + .

[0135] Step D: tert-Butyl 3-(l-(4-[(5-methyl-3-(propan-2-yl)pyrazolo[l,5- a]pyrimidin-7-yl)amino]piperidine-l-carbonyl)cyclopropanecarboxamido)pyrrolidine- 1-carboxylate (130 mg, 0.034 mmol, 1 eq) and 4 M hydrogen chloride in dioxane (1 mL) were dissolved in 1 mL of dry 1,4-dioxane at room temperature and stirred for 3 h at room temperature. After the reaction was completed, the solvent was removed under reduced pressure to obtain a white solid 100 mg, yield 94%, purity 10%. LC-MS: 454.22 [M+H] + .

[0136] Step E: l-(4-[(5-methyl-3-(propan-2-yl)pyrazolo[l,5-a]pyrimidin-7-yl)amino]piperidine- 1-carbonyl)-N-(pyrrolidin-3-yl)cyclopropane-l-carboxamide (100 mg, 0.22 mmol, 1 eq) and triethylamine (100 mg, 0.22 mmol, 1 eq) were dissolved in 4 mL of dry dichloromethane at room temperature and a solution of pentafluorobenzenesulfonyl chloride (87.97 mg, 0.66 mmol, 1.5 eq) in dichloromethane (1 mL) was slowly added and the reaction was stirred at this temperature for 1 h. After the reaction was completed, the solvent was removed under reduced pressure and the residue was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 0: 1) to obtain a white solid 27.8 mg, yield 18%, purity 95%.

[0137] 1H NMR (400 MHz, CDC13) δ 7.79 (s, 1H), 6.24 (d, J = 6.5 Hz, 1H), 6.18 (d, J = 8.1 Hz, 1H), 5.80 (s, 1H), 4.47 (q, J = 5.4 Hz, 1H), 4.30 (s, 2H), 3.77 (d, J = 8.7 Hz, 1H), 3.68 (dt, J = 10.1, 7.5 Hz, 1H), 3.57 (dd, J = 10.6, 5.5 Hz, 1H), 3.47 (ddd, J = 21.0, 10.1, 4.4 Hz, 2H), 3.30 (p, J = 6.9 Hz, 1H), 3.25 - 3.14 (m, 2H), 2.52 (s, 3H), 2.27 (dt, J = 14.1, 7.2 Hz, 1H), 2.19 (dd, J = 12.7, 5.5 Hz, 2H), 1.99 (dd, J = 12.9, 7.3 Hz, 1H), 1.66 (s, 4H), 1.56 - 1.36 (m, 2H), 1.34 (d, J = 6.9 Hz, 6H). LC-MS: 684.00 [M+H] + .

[0138] Example 13

[0139]

[0140] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 16 mg of white solid in 21.17% yield with 95% purity.

[0141] 1 H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 6.14 (s, 1H), 5.78 (s, 1H), 4.24 (s, 2H), 4.15 (t, J = 8.3 Hz, 3H), 4.00 (d, J = 8.2 Hz, 2H), 3.70 (s, 1H), 3.31 (s, 1H), 3.10 (s, 2H), 2.93 (s, 1H), 2.53 (s, 3H), 2.16 (s, 2H), 1.62 (d, J = 13.2 Hz, 3H), 1.34 (d, J = 7.0 Hz, 6H). LC-MS: 616.94 [M+H] + .

[0142] Example 14

[0143]

[0144] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 32 mg of white solid in 25.36% yield with 95% purity.

[0145] 1 H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 6.12 (d, J = 8.0 Hz, 1H), 5.78 (s, 1H), 4.50 (d, J = 14.1 Hz, 1H), 3.94 (s, 3H), 3.77 - 3.69 (m, 1H), 3.27 (dt, J = 25.8, 10.0 Hz, 2H), 2.94 (t, J = 12.2 Hz, 1H), 2.80 (s, 1H), 2.68 (s, 2H), 2.52 (s, 3H), 2.29 (d, J = 6.6 Hz, 2H), 2.14 (d, J = 11.6 Hz, 2H), 1.90 (d, J = 13.2 Hz, 2H), 1.80 (s, 2H), 1.57 (t, J = 10.6 Hz, 2H), 1.34 (d, J = 7.0 Hz, 6H). LC-MS: 628.97 [M+H] + .

[0146] Example 15

[0147]

[0148] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 20 mg of white solid in 30.67% yield with 90% purity.

[0149] 1 H NMR (400 MHz, CDC13) δ 7.76 (s, 1H), 7.64 (d, J = 4.9 Hz, 2H), 7.49 (d, J = 7.3 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 6.73 (s, 1H), 6.18 (d, J = 7.6 Hz, 1H), 4.94 (s, 1H), 4.73 (s, 2H), 4.14 - 3.94 (m, 6H), 3.59 - 3.47 (m, 2H), 3.09 - 3.00 (m, 1H), 2.86 (t, J = 11.9 Hz, 2H), 2.16 (d, J = 11.1 Hz, 2H), 2.02 (s, 2H), 1.70 (s, 1H), 1.53 (q, J = 10.8 Hz, 3H), 1.29 (d, J = 6.8 Hz, 6H). LC-MS: 722.91 [M+H] + .

[0150] Example 16

[0151]

[0152] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to afford 58 mg of white solid, yield 32.35%, purity 90%. 1 H NMR (400 MHz, CDC13) δ 7.82 (s, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.63 (s, 1H), 7.51 (d, J = 7.2 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 6.74 (s, 1H), 6.60 (d, J = 7.0 Hz, 1H), 4.90 (s, 1H), 4.77 (d, J = 6.2 Hz, 2H), 4.34 (s, 1H), 4.05 (s, 1H), 3.97 (d, J = 11.4 Hz, 2H), 3.69 (d, J = 13.9 Hz, 1H), 3.62 - 3.49 (m, 3H), 3.34 (d, J = 12.1 Hz, 1H), 3.12 (t, J = 9.7 Hz, 1H), 3.02 (p, J = 7.1 Hz, 1H), 2.07 - 1.95 (m, 3H), 1.78 (s, 2H), 1.52 (s, 2H), 1.28 (d, J = 6.8 Hz, 6H). LC-MS: 722.98 [M+H] + .

[0153] Example 17

[0154]

[0155] The synthetic procedure was referred to the synthetic procedure of Example 1 compound to afford 33.6 mg of white solid in 44% yield with 95% purity. 1 H NMR (400 MHz, CDC13) δ 7.63 (s, 1H), 7.48 - 7.35 (m, 3H), 6.67 (s, 1H), 4.83 (s, 1H), 4.74 (d, J = 6.1 Hz, 2H), 4.07 - 3.94 (m, 3H), 3.74 (s, 4H), 3.53 (t, J = 11.4 Hz, 2H), 3.30 (s, 5H), 3.03 (p, J = 6.9 Hz, 1H), 2.04 (s, 2H), 1.53 (tt, J = 15.1, 7.7 Hz, 2H), 1.29 (d, J = 6.9 Hz, 6H). LC-MS: 709.05 [M+H] + .

[0156] Example 18

[0157]

[0158] The synthetic procedure was referred to the synthetic procedure of Example 1 compound to afford 33.6 mg of white solid in 44% yield with 95% purity.

[0159] 1 H NMR (400 MHz, CDC13) δ 7.63 (s, 1H), 7.48 - 7.35 (m, 3H), 6.67 (s, 1H), 4.83 (s, 1H), 4.74 (d, J = 6.1 Hz, 2H), 4.07 - 3.94 (m, 3H), 3.74 (s, 4H), 3.53 (t, J = 11.4 Hz, 2H), 3.30 (s, 5H), 3.03 (p, J = 6.9 Hz, 1H), 2.04 (s, 2H), 1.53 (tt, J = 15.1, 7.7 Hz, 2H), 1.29 (d, J = 6.9 Hz, 6H). LC-MS: 709.05 [M+H] + .

[0160] Example 19

[0161]

[0162] The synthetic process was referred to the synthetic process of Example 1 compound to afford 34 mg of white solid in 28.36% yield, 90% purity.

[0163] 1 H NMR (400 MHz, CDC13) δ 7.81 (s, 1H), 7.37 (d, J = 8.2 Hz, 2H), 6.88 (d, J = 8.2 Hz, 2H), 6.15 (d, J = 8.1 Hz, 1H), 5.79 (s, 1H), 4.26 (s, 2H), 3.86 - 3.65 (m, 1H), 3.47 - 3.36 (m, 8H), 3.33 - 3.25 (m, 0H), 3.21 (d, J = 10.8 Hz, 1H), 2.51 (s, 3H), 2.16 (d, J = 13.0 Hz, 2H), 1.65 (d, J = 11.8 Hz, 2H), 1.34 (d, J = 6.9 Hz, 7H). LC-MS: 691.92 [M+H] + .

[0164] Example 20

[0165]

[0166] The synthetic process was referred to the synthetic process of Example 1 compound to afford 34 mg of white solid in 28.36% yield, 90% purity.

[0167] 1 H NMR (400 MHz, CDC13) δ 7.81 (s, 1H), 7.37 (d, J = 8.2 Hz, 2H), 6.88 (d, J = 8.2 Hz, 2H), 6.15 (d, J = 8.1 Hz, 1H), 5.79 (s, 1H), 4.26 (s, 2H), 3.86 - 3.65 (m, 1H), 3.47 - 3.36 (m, 8H), 3.33 - 3.25 (m, 0H), 3.21 (d, J = 10.8 Hz, 1H), 2.51 (s, 3H), 2.16 (d, J = 13.0 Hz, 2H), 1.65 (d, J = 11.8 Hz, 2H), 1.34 (d, J = 6.9 Hz, 7H). LC-MS: 691.92 [M+H] + .

[0168] Example 21

[0169]

[0170] The synthetic process was referred to the synthetic process of Example 1 compound to afford 34 mg of white solid in 28.36% yield, 90% purity.

[0171] 1H NMR (400 MHz, CDC13) δ 7.81 (s, 1H), 6.13 (d, J = 8.1 Hz, 1H), 5.78 (s, 1H), 4.52 (d, J = 13.6 Hz, 1H), 3.94 (d, J = 12.2 Hz, 3H), 3.73 (d, J = 9.4 Hz, 1H), 3.30 (dt, J = 13.9, 7.0 Hz, 2H), 2.94 (d, J = 10.8 Hz, 3H), 2.72 (t, J = 11.8 Hz, 2H), 2.52 (s, 3H), 2.21 (d, J = 36.0 Hz, 2H), 1.90 (d, J = 11.6 Hz, 2H), 1.82 (s, 3H) 1.59 (s, 2H), 1.34 (d, J = 7.0 Hz, 6H). LC-MS: 698.08 [M+H] + .

[0172] Example 22

[0173]

[0174] The synthetic procedure was referred to the synthetic procedure of Example 1 compound to afford 32.4 mg of white solid in 31% yield with 95% purity. 1 H NMR (400 MHz, CDC13) δ 7.81 (s, 1H), 6.13 (d, J = 8.1 Hz, 1H), 5.78 (s, 1H), 4.52 (d, J = 13.6 Hz, 1H), 3.94 (d, J = 12.2 Hz, 3H), 3.73 (d, J = 9.4 Hz, 1H), 3.30 (dt, J = 13.9, 7.0 Hz, 2H), 2.94 (d, J = 10.8 Hz, 3H), 2.72 (t, J = 11.8 Hz, 2H), 2.52 (s, 3H), 2.21 (d, J = 36.0 Hz, 2H), 1.90 (d, J = 11.6 Hz, 2H), 1.82 (s, 3H) 1.59 (s, 2H), 1.34 (d, J = 7.0 Hz, 6H). LC-MS: 698.08 [M+H] + .

[0175] Example 23

[0176]

[0177] The synthetic procedure was referred to the synthetic procedure of Example 1 compound to afford 32.4 mg of white solid in 31% yield with 95% purity.

[0178] 1H NMR (400 MHz, CD3OD) δ 7.87 (s, 1H), 6.11 (s, 1H), 5.08 (s, 1H), 4.39 - 4.30 (m, 2H), 4.11 (d, J = 13.6 Hz, 2H), 4.07 (s, 2H), 3.86 (d, J = 21.6 Hz, 1H), 3.27 (t, J = 6.8 Hz, 1H), 3.11 (d, J = 31.5 Hz, 2H), 2.52 (s, 3H), 2.09 (d, J = 10.1 Hz, 2H), 1.71 - 1.56 (m, 2H), 1.31 (d, J = 7.0 Hz, 6H). LC-MS: 602.97 [M+H] + .

[0179] Example 24

[0180]

[0181] The synthetic procedure was referred to the synthetic procedure of Example 12 compound to afford 13 mg of white solid in 10% yield, 90% purity.

[0182] 1 H NMR (400 MHz, CD3OD) δ 7.90 (s, 1H), 6.14 (s, 1H), 4.50 - 4.34 (m, 1H), 4.34 - 4.20 (m, 1H), 4.19 - 4.05 (m, 1H), 3.95 (s, 1H), 3.76 - 3.52 (m, 3H), 3.51 - 3.35 (m, 2H), 3.32 - 3.25 (m, 2H), 3.01 (s, 1H), 2.51 (s, 3H), 2.23 (s, 1H), 2.14 (d, J = 14.1 Hz, 2H), 2.11 - 2.01 (m, 1H), 2.00 - 1.86 (m, 1H), 1.62 (s, 2H), 1.34 (d, J = 6.8 Hz, 6H), 1.31 (s, 2H). LC-MS: 684.00 [M+H] + .

[0183] Example 25

[0184]

[0185] The synthetic procedure was referred to the synthetic procedure of Example 1 compound to afford 59.4 mg of white solid in 38% yield, 90% purity. 1H NMR (400 MHz, CDC13) δ 7.81 (s, 1H), 6.13 (s, 1H), 5.78 (s, 1H), 4.52 (s, 1H), 3.92 (d, J = 12.6 Hz, 3H), 3.73 (s, 1H), 3.27 (d, J = 22.7 Hz, 3H), 2.64 (t, J = 12.0 Hz, 2H), 2.53 (s, 3H), 2.46 - 2.30 (m, 2H), 2.17 (s, 2H), 1.84 (s, 2H), 1.66 - 1.53 (m, 8H), 1.34 (s, 6H). LC-MS: 643.42 [M+H] + .

[0186] Example 26

[0187]

[0188] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 31 mg of white solid in 42% yield, 90% purity.

[0189] 1 H NMR (400 MHz, CDC13) δ 7.81 (s, 1H), 6.13 (s, 1H), 5.78 (s, 1H), 4.52 (s, 1H), 3.92 (d, J = 12.6 Hz, 3H), 3.73 (s, 1H), 3.27 (d, J = 22.7 Hz, 3H), 2.64 (t, J = 12.0 Hz, 2H), 2.53 (s, 3H), 2.46 - 2.30 (m, 2H), 2.17 (s, 2H), 1.84 (s, 2H), 1.66 - 1.53 (m, 8H), 1.34 (s, 6H). LC-MS: 643.42 [M+H] + .

[0190] Example 27

[0191]

[0192] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 20.5 mg of white solid in 22% yield, 90% purity.

[0193] 1H NMR (400 MHz, MeOD) δ 7.86 (s, 1H), 6.04 (s, 1H), 3.31 - 3.17 (m, 4H), 3.04 - 2.95 (m, 2H), 2.50 - 2.41 (m, 5H), 2.29 (t, J = 11.7 Hz, 2H), 2.10 (d, J = 13.1 Hz, 2H), 1.85 (d, J = 12.3 Hz, 2H), 1.77 (t, J = 10.9 Hz, 4H), 1.43 (dt, J = 11.4, 6.7 Hz, 2H). LC-MS: 628.23 [M+H] + .

[0194] Example 28

[0195]

[0196] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 31.1 mg of white solid in 61% yield with 95% purity.

[0197] 1 H NMR (400 MHz, MeOD) δ 7.64 (d, J = 10.1 Hz, 1H), 7.53 - 7.38 (m, 3H), 7.30 (t, J = 6.7 Hz, 1H), 5.48 (s, 1H), 4.76 (d, J = 3.0 Hz, 2H), 3.92 (d, J = 12.2 Hz, 3H), 3.82 (dt, J = 23.2, 5.5 Hz, 2H), 3.62 (d, J = 5.6 Hz, 1H), 3.48 (ddd, J = 28.6, 16.6, 6.2 Hz, 7H), 2.97 (h, J = 7.9, 7.1 Hz, 1H), 2.04 - 1.97 (m, 1H), 1.91 (s, 2H), 1.63 (s, 1H), 1.51 (d, J = 11.9 Hz, 2H), 1.25 (dd, J = 7.0, 3.6 Hz, 6H). LC-MS: 724.24 [M+H] + .

[0198] Example 29

[0199]

[0200] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 17 mg of white solid in 16% yield with 95% purity.

[0201] 1H NMR (400 MHz, MeOD) δ 7.70 (s, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.50 - 7.42 (m, 2H), 7.32 (s, 1H), 4.79 (s, 2H), 4.61 (s, 1H), 3.95 (d, J = 11.2 Hz, 3H), 3.68 (s, 1H), 3.51 (t, J = 11.1 Hz, 2H), 3.27 (s, 4H), 3.09 - 2.97 (m, 2H), 2.84 (s, 1H), 2.68 (s, 4H), 2.60 (d, J = 11.4 Hz, 1H), 1.93 (s, 3H), 1.66 - 1.47 (m, 4H), 1.39 (s, 2H), 1.30 (d, J = 7.0 Hz, 6H). LC-MS: 793 [M+H] + .

[0202] Example 30

[0203]

[0204] Step A: To a solution of N-(5-methyl-3-(propyl-2-yl)pyrazolo[l,5-a]pyrimidin-7- yl)piperidin-4-amine (60 mg, 0.22 mmol, 1 eq) in 2 mL of acetonitrile was added tert-butyl (2S)-2-[(4-methylbenzenesulfonyl)methyl]morpholine-4-carboxylate (78 mg, 0.22 mmol, 1 eq) and potassium carbonate (91 mg, 0.66 mmol, 3 eq). The reaction mixture was stirred at room temperature for 4 h. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 62 mg of white solid in 59% yield with 99% purity.

[0205] Step B: (R)-tert-butyl 2-((4-((3-isopropyl-5-methylpyrazolo[l,5-a]pyrimidin-7-yl)amino)piperidin-l-yl)methyl)morpholine-4-carboxylate (62 mg, 0.13 mmol, 1 eq) and 4 M hydrogen chloride dioxane solution (0.5 mL) were added to 3 mL of dichloromethane at room temperature and stirred for 5 h. After the reaction was completed, the solvent was removed under reduced pressure to give 50 mg of white solid in 90% yield, which was used directly in the next step without purification.

[0206] Step C: To a solution of N-(5-methyl-3-(propan-2-yl)pyrazolo[l,5-a]pyrimidin-7- yl)-l-{[(2S)-morpholin-2-yl]methyl}piperidin-4-amine (60 mg, 0.16 mmol, 1 eq) in 4 mL of dichloromethane was added N,N-diisopropylethylamine (93 mg, 0.72 mmol, 4.5 eq) and pentafluorobenzenesulfonyl chloride (51 mg, 0.19 mmol, 1.2 eq) in an ice bath. The reaction mixture was stirred at room temperature for 5 min. Thin layer chromatography showed that the reaction had completed. The solvent was removed under reduced pressure and the residue was separated by silica gel column chromatography (dichloromethane:methanol = 10: 1) to give 20 mg of white solid, yield 22%, purity 90%.

[0207] 1 H NMR (400 MHz, MeOD) δ 7.84 (s, 1H), 6.00 (s, 1H), 3.96 (d, J = 15.0 Hz, 1H), 3.77 (d, J = 13.4 Hz, 2H), 3.70 - 3.60 (m, 3H), 3.25 (q, J = 6.9 Hz, 1H), 3.00 - 2.82 (m, 3H), 2.61 (t, J = 11.1 Hz, 1H), 2.54 (dd, J = 13.4, 6.5 Hz, 1H), 2.46 (s, 3H), 2.42 - 2.29 (m, 2H), 2.06 (d, J = 12.6 Hz, 2H), 1.82 - 1.69 (m, 2H), 1.31 (d, J = 6.8 Hz, 6H). LC-MS: 603.93 [M+H] + .

[0208] Example 31

[0209]

[0210] The synthetic procedure was referred to the synthetic procedure of Example 1 compound to give 20 mg of white solid, yield 28%, purity 90%.

[0211] 1H NMR (400 MHz, MeOD) δ 7.70 (s, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.41 (s, 1H), 7.30 (d, J = 7.6 Hz, 1H), 4.78 (s, 2H), 3.93 (d, J = 11.0 Hz, 3H), 3.68 (s, 2H), 3.55 - 3.45 (m, 2H), 3.29 (s, 5H), 3.01 (p, J = 7.0 Hz, 1H), 1.90 (s, 2H), 1.74 - 1.46 (m, 9H), 1.29 (d, J = 7.0 Hz, 6H). LC-MS: 778.39 [M+H] + .

[0212] Example 32

[0213]

[0214] The synthetic procedure was referred to the synthetic procedure of Example 1 compound to afford 15 mg of white solid in 17% yield, 90% purity.

[0215] 1 H NMR (400 MHz, MeOD) δ 7.68 (s, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.43 (dd, J = 14.4, 6.7 Hz, 2H), 7.30 (d, J = 7.7 Hz, 1H), 4.77 (s, 2H), 4.03 - 3.89 (m, 3H), 3.66 (d, J = 42.9 Hz, 3H), 3.49 (t, J = 7.2 Hz, 5H), 3.28 (s, 2H), 2.99 (q, J = 7.0 Hz, 1H), 1.95 - 1.74 (m, 4H), 1.64 - 1.49 (m, 4H), 1.40 (s, 2H), 1.27 (d, J = 7.0 Hz, 6H). LC-MS: 763.06 [M+H] + .

[0216] Example 33

[0217]

[0218] The synthetic procedure was referred to the synthetic procedure of Example 1 compound to afford 28.7 mg of white solid in 40% yield, 90% purity. 1H NMR (400 MHz, MeOD) δ 7.64 (d, J = 10.1 Hz, 1H), 7.53 - 7.38 (m, 3H), 7.30 (t, J = 6.7 Hz, 1H), 5.48 (s, 1H), 4.76 (d, J = 3.0 Hz, 2H), 3.92 (d, J = 12.2 Hz, 3H), 3.82 (dt, J = 23.2, 5.5 Hz, 2H), 3.62 (d, J = 5.6 Hz, 1H), 3.48 (ddd, J = 28.6, 16.6, 6.2 Hz, 7H), 2.97 (h, J = 7.9, 7.1 Hz, 1H), 2.04 - 1.97 (m, 1H), 1.91 (s, 2H), 1.63 (s, 1H), 1.51 (d, J = 11.9 Hz, 2H), 1.25 (dd, J = 7.0, 3.6 Hz, 6H). LC-MS: 750.24 [M+H] + .

[0219] Example 34

[0220]

[0221] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 10.2 mg of white solid in 23% yield, 90% purity.

[0222] 1 H NMR (400 MHz, MeOD) δ 7.64 (d, J = 10.1 Hz, 1H), 7.53 - 7.38 (m, 3H), 7.30 (t, J = 6.7 Hz, 1H), 5.48 (s, 1H), 4.76 (d, J = 3.0 Hz, 2H), 3.92 (d, J = 12.2 Hz, 3H), 3.82 (dt, J = 23.2, 5.5 Hz, 2H), 3.62 (d, J = 5.6 Hz, 1H), 3.48 (ddd, J = 28.6, 16.6, 6.2 Hz, 7H), 2.97 (h, J = 7.9, 7.1 Hz, 1H), 2.04 - 1.97 (m, 1H), 1.91 (s, 2H), 1.63 (s, 1H), 1.51 (d, J = 11.9 Hz, 2H), 1.25 (dd, J = 7.0, 3.6 Hz, 6H). LC-MS: 750.24 [M+H] + .

[0223] Example 35

[0224]

[0225] Step A: To a solution of tert-butyl 5-cyclopropyl-3-(3-methyl-2-(3-(tetramethyl-1,3,2- dioxaborolan-2-yl)phenyl)butanamide)-1H-pyrazole-1-carboxylate (200 mg, 0.39 mmol, 1 eq) in 3 mL of tetrahydrofuran and 1 mL of water was added tert-butyl 4-bromo-5,6-dihydropyridine-1(2H)- carboxylate (100 mg, 0.39 mmol, 1 eq), [n-butyldi(1-adamantyl)phosphine]methanesulfonic acid palladium(II) (2-amino-1,1'-biphenyl-2-yl) (43 mg, 0.058 mmol, 0.15 eq) and potassium phosphate (249 mg, 1.17 mmol, 3 eq) at room temperature. The reaction mixture was stirred at 60 °C under nitrogen protection for 4 h. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography to obtain 120 mg of white solid with a yield of 54% and a purity of 90%.

[0226] Step B: tert-Butyl 4-(3-(1-(1-(tert-butoxycarbonyl)-5-cyclopropyl-1H-pyrazol-3-ylamino)-3- methyl-1-oxobutan-2-yl)phenyl)-3,6-dihydropyridine-1(2H)-carboxylate (120 mg, 0.21 mmol, 1 eq) and 4 M hydrogen chloride dioxane solution (0.5 mL) were added to a 3 mL dichloromethane solution at room temperature and stirred for 5 h at room temperature. After the reaction was completed, the solvent was removed under reduced pressure to obtain 60 mg of white solid with a yield of 70%, which was directly used in the next step without purification.

[0227] Step C: N-(5-cyclopropyl-1H-pyrazol-3-yl)-3-methyl-2-(3-(1,2,3,6-tetrahydropyridin-4-yl)phenyl)butanamide (60 mg, 0.16 mmol, 1 eq) and N,N-diisopropylethylamine (93 mg, 0.72 mmol, 4.5 eq) were dissolved in 4 mL of dry dichloromethane, and pentafluorobenzenesulfonyl chloride (51 mg, 0.19 mmol, 1.2 eq) was slowly added and stirred at this temperature for 1 h. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain 10 mg of white solid with a yield of 10.22% and a purity of 90%.

[0228] 1H NMR (400 MHz, MeOD) δ 7.47 (s, 1H), 7.31 (d, J = 5.3 Hz, 3H), 6.13 (s, 2H), 4.03 (s, 2H), 3.62 (t, J = 5.7 Hz, 2H), 3.22 (d, J = 10.8 Hz, 1H), 2.68 (s, 2H), 2.48 - 2.37 (m, 1H), 1.09 (d, J = 6.5 Hz, 3H), 1.02 - 0.92 (m, 3H), 0.73 (d, J = 6.7 Hz, 3H), 0.71 (dd, J = 5.1, 2.4 Hz, 2H). LC-MS: 595.01 [M+H] + .

[0229] Example 36

[0230]

[0231] The synthetic procedure was referenced to the synthetic procedure of Example 35 compound to yield 4 mg of white solid, 5.36% yield, 90% purity.

[0232] 1 H NMR (400 MHz, MeOD) δ 7.30 (s, 1H), 7.27 (d, J = 5.1 Hz, 2H), 7.15 (t, J = 5.4 Hz, 1H), 6.13 (s, 1H), 4.02 (d, J = 12.1 Hz, 2H), 3.20 (d, J = 10.6 Hz, 1H), 2.84 (t, J = 12.2 Hz, 2H), 2.67 (d, J = 12.2 Hz, 1H), 2.46 - 2.35 (m, 1H), 2.06 (d, J = 9.9 Hz, 2H), 1.95 (d, J = 11.9 Hz, 2H), 1.09 (d, J = 6.5 Hz, 3H), 1.04 - 0.84 (m, 5H), 0.73 (d, J = 6.7 Hz, 3H). LC-MS: 619.2 [M+H] + .

[0233] Example 37

[0234]

[0235] The synthetic procedure was referenced to the synthetic procedure of Example 1 to yield 7.2 mg of white solid, 11% yield, 90% purity.

[0236] 1H NMR (400 MHz, MeOD) δ 7.87 (s, 1H), 6.06 (s, 1H), 3.70-3.65 (m, 1H), 3.62-3.50 (m, 7H), 3.37-3.32 (m, 2H), 3.30-3.23 (m, 2H), 2.95 (d, J = 11.6 Hz, 2H), 2.48 (s, 3H), 2.41-2.31 (m, 2H), 2.09 (d, J = 12.5 Hz, 2H), 1.91 (t, J = 7.2 Hz, 2H), 1.78 (q, J = 11.7 Hz, 2H), 1.64 (s, 2H), 1.54 (s, 2H), 1.32 (d, J = 6.8 Hz, 6H). LC-MS: 684.45 [M+H] + .

[0237] Example 38

[0238]

[0239] The synthetic procedure was referenced to the synthetic procedure of Example 1 to yield 4.6 mg of white solid in 12% yield, 90% purity.

[0240] 1 H NMR (400 MHz, MeOD) δ 7.88 (s, 1H), 4.03 (s, 1H), 3.68 (d, J = 13.5 Hz, 2H), 3.39 (d, J = 12.3 Hz, 1H), 3.29 - 3.20 (m, 1H), 3.12 (d, J = 12.2 Hz, 2H), 2.93 (d, J = 10.0 Hz, 2H), 2.49 (s, 5H), 2.11 (s, 2H), 1.95 (s, 1H), 1.92 - 1.67 (m, 7H), 1.37 (d, J = 7.9 Hz, 1H), 1.32 (d, J = 6.9 Hz, 6H). LC-MS: 644.3 [M+H] + .

[0241] Example 39

[0242]

[0243] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 4 mg of white solid in 5.36% yield, 90% purity.

[0244] 1H NMR (400 MHz, MeOD) δ 7.91 (s, 1H), 7.71 (s, 1H), 7.67 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.2 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 4.79 (s, 2H), 4.49 (d, J = 9.9 Hz, 1H), 4.00 (s, 1H), 3.80 - 3.67 (m, 5H), 3.56 (d, J = 6.1 Hz, 2H), 3.07 - 2.85 (m, 3H), 2.72 (s, 3H), 2.38 - 2.27 (m, 1H), 2.22 - 2.07 (m, 3H), 1.76 (s, 2H), 1.29 (d, J = 6.8 Hz, 6H). LC-MS: 722.3 [M+H] + .

[0245] Example 40

[0246]

[0247] The synthetic procedure was referred to the synthetic procedure of Example 1 compound to afford 9 mg of white solid, yield 5.24%, purity 90%.

[0248] 1 H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.64 (d, J = 7.0 Hz, 2H), 7.51 (d, J = 8.2 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 6.65 (d, J = 9.1 Hz, 1H), 6.10 (d, J = 7.6 Hz, 1H), 4.75 (d, J = 6.5 Hz, 2H), 4.16 - 4.07 (m, 1H), 4.01 (d, J = 12.1 Hz, 2H), 3.89 (s, 1H), 3.03 (dt, J = 13.7, 6.9 Hz, 2H), 2.87 (t, J = 12.7 Hz, 4H), 2.41 (s, 3H), 2.33 (s, 2H), 2.17 (d, J = 9.9 Hz, 2H), 2.10 (s, 2H), 1.49 (d, J = 24.2 Hz, 4H), 1.29 (d, J = 7.0 Hz, 6H). LC-MS: 736.3 [M+H] + .

[0249] Example 41

[0250]

[0251] The synthetic procedure was referred to the synthetic procedure of Example 1 compound to afford 11.5 mg of white solid, yield 21%, purity 99.28%. 1H NMR (400 MHz, MeOD) d 7.92 (s, 1H), 7.70 (d, J = 7.0 Hz, 2H), 7.58 (d, J = 7.7 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 4.76 (s, 2H), 4.03 (d, J = 38.8 Hz, 2H), 3.85 (d, J = 12.3 Hz, 1H), 3.64 (d, J = 12.6 Hz, 1H), 3.06 - 2.89 (m, 5H), 2.74 (s, 3H), 2.14 (s, 2H), 1.95 (d, J = 13.3 Hz, 3H), 1.73 (d, J = 11.9 Hz, 2H), 1.62 (d, J = 10.9 Hz, 1H), 1.27 (d, J = 6.9 Hz, 6H). LC-MS: 736.3 [M+H] + .

[0252] Example 42

[0253]

[0254] Step A: 4-(bromomethyl)benzene-1-sulfonyl chloride (50 mg, 0.19 mmol, 1 eq) in 1 mL acetonitrile solution was added potassium fluorohydride (34.13 mg, 0.44 mmol, 2.3 eq) in water (2 mL) solution, then stirred at 25 °C for 1 h under nitrogen protection. TLC showed complete reaction. The mixture was extracted with ethyl acetate, then concentrated under reduced pressure, purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to give the product. 23 mg, yield 48.9%, white solid.

[0255] Step B-C: The synthesis process was referred to the synthesis process of Example 1 compound, 46 mg white solid was obtained, 99% yield.

[0256] Step D: N-(5-methyl-3-(propan-2-yl)pyrazolo[1,5-a]pyrimidin-7-yl)-1-(piperidine-4- carbonyl)piperidin-4-amine (23 mg, 0.060 mmol, 1 eq), 4-(bromomethyl)benzene-1- sulfonyl fluoride (0.015 g, 0.060 mmol, 1 eq) and potassium carbonate (0.025 g, 0.18 mmol, 3 eq) in 0.5 mL dichloromethane solution was stirred at 25 °C for 2 h under nitrogen protection. TLC showed complete reaction. The mixture was adjusted to pH = 9-10, then concentrated under reduced pressure, purified by preparation to give the product 14.2 mg, yield 42%, purity 84%, white solid.

[0257] 1H NMR (400 MHz, CDC13) δ 7.99 (d, J = 8.0 Hz, 2H), 7.85 (s, 1H), 7.65 (d, J = 7.9 Hz, 2H), 6.17 (d, J = 8.2 Hz, 1H), 5.82 (s, 1H), 4.57 (d, J = 13.2 Hz, 1H), 3.97 (s, 1H), 3.77 (s, 1H), 3.65 (s, 2H), 3.34 (dt, J = 14.0, 7.0 Hz, 2H), 2.94 (d, J = 11.3 Hz, 3H), 2.56 (s, 3H), 2.17 (d, J = 30.8 Hz, 4H), 1.96 (s, 3H), 1.72 (d, J = 12.6 Hz, 4H), 1.38 (d, J = 6.9 Hz, 6H). LC-MS: 557.30 [M+H] + .

[0258] Example 43

[0259]

[0260] The synthetic procedure was referenced to the synthetic procedure of Example 42 compound to yield 14 mg of white solid in 12% yield, 95% purity.

[0261] 1 H NMR (400 MHz, CDC13) δ 7.99 (d, J = 8.0 Hz, 2H), 7.85 (s, 1H), 7.65 (d, J = 7.9 Hz, 2H), 6.17 (d, J = 8.2 Hz, 1H), 5.82 (s, 1H), 4.57 (d, J = 13.2 Hz, 1H), 3.97 (s, 1H), 3.77 (s, 1H), 3.65 (s, 2H), 3.34 (dt, J = 14.0, 7.0 Hz, 2H), 2.94 (d, J = 11.3 Hz, 3H), 2.56 (s, 3H), 2.17 (d, J = 30.8 Hz, 4H), 1.96 (s, 3H), 1.72 (d, J = 12.6 Hz, 4H), 1.38 (d, J = 6.9 Hz, 6H). LC-MS: 557.30 [M+H] + .

[0262] Example 44

[0263]

[0264] The synthetic procedure was referenced to the synthetic procedure of Example 51 compound to yield 25.3 mg of white solid in 43% yield, 95% purity.

[0265] 1H NMR (400 MHz, MeOD) δ 7.66 (s, 1H), 7.43 (d, J = 7.4 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.33 - 7.27 (m, 1H), 7.27 - 7.21 (m, 1H), 4.71 (s, 2H), 4.04 - 3.84 (m, 5H), 3.51 (t, J = 11.9 Hz, 2H), 2.99 (dq, J = 14.7, 7.7, 7.3 Hz, 1H), 2.88 - 2.78 (m, 2H), 2.49 (s, 1H), 1.92 (s, 2H), 1.91 - 1.83 (m, 2H), 1.83 - 1.74 (m, 2H), 1.54 (q, J = 10.2, 9.4 Hz, 2H), 1.30 (d, J = 3.2 Hz, 1H), 1.27 (d, J = 7.0 Hz, 6H). LC-MS: 723.2 [M+H] + .

[0266] Example 45

[0267]

[0268] The synthetic procedure was referred to the synthetic procedure of Example 51 compound to afford 5.8 mg of white solid in 7.9% yield with 98% purity.

[0269] 1 H NMR (400 MHz, MeOD) δ 7.66 (s, 1H), 7.43 (d, J = 7.4 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.33 - 7.27 (m, 1H), 7.27 - 7.21 (m, 1H), 4.71 (s, 2H), 4.04 - 3.84 (m, 5H), 3.51 (t, J = 11.9 Hz, 2H), 2.99 (dq, J = 14.7, 7.7, 7.3 Hz, 1H), 2.88 - 2.78 (m, 2H), 2.49 (s, 1H), 1.92 (s, 2H), 1.91 - 1.83 (m, 2H), 1.83 - 1.74 (m, 2H), 1.54 (q, J = 10.2, 9.4 Hz, 2H), 1.30 (d, J = 3.2 Hz, 1H), 1.27 (d, J = 7.0 Hz, 6H). LC-MS: 723.2 [M+H]

[0270] LC-MS: 735.3 [M+H] + .

[0271] Example 46

[0272]

[0273] The synthetic procedure was referred to the synthetic procedure of Example 51 compound to afford 5.8 mg of white solid in 7.9% yield with 98% purity.

[0274] 1H NMR(400MHz,MeOD)δ8.22(d,J=8.0Hz,2H),7.98–7.84(m,4H),7.76(d,J=8.0Hz,1H),7 .64(d,J=7.8Hz,1H),7.50(t,J=8.0Hz,1H),4.85(s,2H),4.52(s,2H),4.32(s,1H),4.1 2(s,1H),3.56(s,2H),3.51(s,1H),3.24(s,2H),3.21(s,2H),3.08(d,J=20.4Hz,2H),2 .92(s,3H),2.21(s,2H),2.11(s,2H),1.87(s,2H),1.76(s,2H),1.31(d,J=6.9Hz,6H).

[0275] LC-MS: 678.30 [M+H] + .

[0276] Example 47

[0277]

[0278] The synthesis process was the same as that of the compound in Example 51, yielding 13 mg of a white solid, 22% yield, and 80% purity.

[0279] 1 H NMR(400MHz,MeOD)δ8.22(d,J=8.1Hz,2H),8.03–7.86(m,4H),7.58(d,J=9.1Hz,1H),7.5 1(t,J=7.5Hz,1H),7.45(d,J=7.3Hz,1H),4.61(s,2H),4.58–4.53(m,1H),4.06(d,J=37.4 Hz,2H),3.84(s,1H),3.62(d,J=6.6Hz,2H),3.51(d,J=30.9Hz,6H),3.16(dt,J=47.8,13. 9Hz, 4H), 2.94 (s, 3H), 2.25 (d, J = 41.1Hz, 6H), 1.31 (d, J = 6.8Hz, 6H). LC-MS: 678.30 [M+H] + .

[0280] Example 48

[0281]

[0282] Step A: To a solution of (3S)-3-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester (50 mg, 0.27 mmol, 1 eq) and triethylamine (0.17 mL, 1.22 mmol. 4.5 eq) in 0.5 mL of acetonitrile, a solution of 4-nitrophenyl chloroformate (54.42 mg, 0.27 mmol, 1 eq) in acetonitrile (0.5 mL) was added at 0 °C and the mixture was stirred at this temperature. The reaction was monitored by spotting on a plate and when complete, a solution of N-(5-methyl-3-(propan-2-yl)pyrazolo[1,5-a]pyrimidin-7-yl)piperidin-4-amine (73.81 mg, 0.27 mmol, 1 eq) in acetonitrile (0.5 mL) was added to the mixture, which was then stirred at 25 °C for 1 h. TLC showed that the reaction was complete. The mixture was diluted with 1 M sodium hydroxide solution, extracted with ethyl acetate and then concentrated under reduced pressure. The product was purified by preparative plate (dichloromethane / methanol = 10 / 1) to give the product. The product 40 mg, 30.7% yield, as a yellow solid.

[0283] Step B: A solution of (3S)-1-[(tert-butoxy)carbonyl]pyrrolidin-3-yl 4-[(5-methyl-3- (propan-2-yl)pyrazolo[1,5-a]pyrimidin-7-yl)amino]piperidine-1-carboxylate (130 mg, 0.27 mmol, 1 eq) and trifluoroacetic acid (0.5 mL) in 1 mL of dichloromethane was stirred at 25 °C for 0.5 h. TLC showed that the starting material was completely reacted. The mixture was adjusted to pH = 9-10 and then concentrated under reduced pressure to give the product 93 mg, 90% yield, as a yellow solid.

[0284] Step C: A solution of (3S)-pyrrolidin-3-yl 4-[(5-methyl-3-(propan-2-yl)pyrazolo[1,5-a]pyrimidin-7- yl)amino]piperidine-1-carboxylate (30 mg, 0.078 mmol, 1 eq), 4-(bromomethyl)benzene-1-sulfonyl fluoride (21.72 mg, 0.086 mmol, 1.1 eq) and potassium carbonate (32.34 mg, 0.23 mmol, 3 eq) in 1 mL of dichloromethane was stirred at 25 °C for 2 h under nitrogen protection. After the reaction was completed, it was extracted with ethyl acetate and then concentrated under reduced pressure. The product was obtained by preparative purification. The product 9.9 mg, 22.83% yield, 90% purity, as a white solid.

[0285] 1H NMR (400 MHz, MeOD) δ 8.22 (d, J = 8.2 Hz, 2H), 8.17 (s, 1H), 7.95 (d, J = 8.2 Hz, 2H), 6.59 (s, 1H), 5.39 (s, 1H), 4.64 (d, J = 2.5 Hz, 2H), 4.56 (q, J = 7.0 Hz, 1H), 4.25 (d, J = 13.5 Hz, 2H), 3.69 (s, 3H), 3.51 (s, 1H), 3.25 (p, J = 6.9 Hz, 1H), 3.07 (s, 2H), 2.68 (s, 3H), 2.58 (s, 1H), 2.31 (s, 1H), 2.07 (s, 2H), 1.87 - 1.73 (m, 2H), 1.38 (d, J = 6.9 Hz, 6H). LC-MS: 559.20 [M+H] + .

[0286] Example 49

[0287]

[0288] The synthetic procedure was referred to the synthetic procedure of Example 51 compound to afford 21.9 mg of white solid in 24% yield with 97% purity. 1 H NMR (400 MHz, CDCl3) δ 7.63 (s, 1H), 7.59 (d, J = 7.3 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.31 (s, 1H), 7.20 (t, J = 7.6 Hz, 1H), 4.66 (s, 2H), 4.00 (d, J = 11.7 Hz, 3H), 3.87 (d, J = 13.0 Hz, 2H), 3.55 (t, J = 10.5 Hz, 2H), 3.01 (d, J = 6.7 Hz, 1H), 2.93 (q, J = 5.9 Hz, 2H), 2.48 - 2.36 (m, 1H), 2.03 (d, J = 12.2 Hz, 2H), 1.90 (s, 4H), 1.57 (d, J = 9.9 Hz, 2H), 1.27 (d, J = 7.8 Hz, 6H). LC-MS: 665.40 [M+H] + .

[0289] Example 50

[0290]

[0291] The synthetic procedure was referred to the synthetic procedure of Example 48 compound to afford 10.8 mg of white solid in 10% yield with 81% purity.

[0292] 1H NMR (400 MHz, MeOD) δ 8.26 - 8.09 (m, 3H), 7.94 (d, J = 8.1 Hz, 2H), 6.60 (s, 1H), 4.69 - 4.36 (m, 4H), 4.31 (d, J = 13.6 Hz, 1H), 4.20 (dd, J = 13.0, 3.6 Hz, 1H), 4.00 (t, J = 11.9 Hz, 1H), 3.88 - 3.71 (m, 2H), 3.58 - 3.36 (m, 5H), 3.24 (dt, J = 13.4, 7.5 Hz, 2H), 3.01 (t, J = 11.4 Hz, 1H), 2.69 (s, 3H), 2.40 - 2.17 (m, 4H), 1.37 (d, J = 6.9 Hz, 6H). LC-MS: 545.30 [M+H] + .

[0293] Example 51

[0294]

[0295] Step A: 4-chloro-2-(methylsulfanyl)-8-(propan-2-yl)pyrazolo[l,5-a][l,3,5]triazine (400 mg, 1.65 mmol, 1 eq), (3-nitrophenyl)methanamine (351.47 mg, 2.31 mmol, 1.4 eq) and N,N-diisopropylethylamine (639.74 mg, 4.95 mmol, 3 eq) in 4 mL of acetonitrile was stirred at 25 °C for 2 h. Thin layer chromatography showed that the starting material was completely reacted. The mixture was extracted with ethyl acetate, then concentrated under reduced pressure, purified by preparative thin layer chromatography (dichloromethane / methanol = 10 / 1) to give the product. The product 200 mg, 33% yield, as a solid.

[0296] Step B: 2-(methylsulfanyl)-N-[(3-nitrophenyl)methyl]-8-(propan-2-yl)pyrazolo[l,5-a][l,3,5]triazin-4-amine (300 mg, 0.84 mmol, 1 eq) in 3 mL of dichloromethane was added m-chloroperoxybenzoic acid (579 mg, 3.36 mmol, 4 eq) at 0 °C, then stirred at 25 °C for 15 min. After the reaction, sodium sulfite saturated solution was added to quench the reaction, then extracted with ethyl acetate, the organic phase was collected, washed with citric acid solution and saturated sodium chloride aqueous solution respectively, concentrated under reduced pressure to give the product 253 mg, yield 78%, as a white solid.

[0297] Step C: 2-methylsulfonyl-N-[(3-nitrophenyl)methyl]-8-(propan-2-yl)pyrazolo[l,5- a] [l,3,5]triazin-4-amine (248 mg, 0.64 mmol, 1 eq) and 4-aminotetrahydropyran (323.68 mg, 3.2 mmol, 5 eq) were stirred in 0.5 mL N-methylpyrrolidin-2-one at 120 °C for 2 h. The mixture was washed with a micro-amount of water and filtered, then the filter cake was collected to give the product 114 mg, 43% yield, as a yellow solid.

[0298] Step D: N4-[(3-nitrophenyl)methyl]-N2-(oxetan-4-yl)-8-(propan-2-yl)pyrazolo[l,5- a] [l,3,5]triazin-2,4-diamine (115 mg, 0.28 mmol, 1 eq) was dissolved in 2 mL tetrahydrofuran, 10% wet palladium on carbon (10 mg) and palladium hydroxide (10 mg) were added, then stirred at room temperature under hydrogen for 2 h. Thin layer chromatography showed the reaction was complete. The mixture was filtered and concentrated under reduced pressure to give the product 90 mg, 84% yield, as a solid.

[0299] Step E: N4-[(3-aminophenyl)methyl]-N2-(oxetan-4-yl)-8-(propan-2-yl)pyrazolo[l,5- a] [l,3,5]triazin-2,4-diamine (34 mg, 0.089 mmol, 1 eq), l-[(tert-butoxy)carbonyl]piperidine- 3-carboxylic acid (20.41 mg, 20.41 mg, 1 eq), 2-(7-azobenzotriazol)-N,N,N',N'- tetramethyluronium hexafluorophosphate (67.68 mg, 0.18 mmol, 2 eq) and N,N- diisopropylethylamine (34.51 mg, 0.27 mmol, 3 eq) were stirred in 1 mL dichloromethane at 25 °C for 2 h. After the reaction was complete, the product was obtained by preparative purification. The product 30 mg, 56% yield.

[0300] Step F: To tert-butyl 3-[(3-{[(2-[(oxan-4-yl)amino]-8-(propan-2-yl)pyrazolo[l,5- a] [l,3,5]triazin-4-yl)amino]methyl}phenyl)carbamoyl]piperidine-l-carboxylate (20 mg, 0.034 mmol, 1 eq) in 1 mL dioxane was added 4 M hydrochloric acid in dioxane (0.5 mL), then stirred at 25 °C for 2 h. After the reaction was complete, the product was concentrated under reduced pressure to give 16 mg, 96% yield.

[0301] Step G: N-(3-{[(2-[(oxan-4-yl)amino]-8-(propan-2-yl)pyrazolo[l,5- a] [l,3,5]triazin-4-yl)amino]methyl}phenyl)piperidine-3-carboxamide (16 mg, 0.032 mmol, leq), 4-(bromomethyl)benzene- 1 -sulfonyl fluoride (8.1 mg, 0.032 mol, leq) and potassium carbonate (13 mg, 0.096 mmol, 3 eq) in 1 mL of acetonitrile were stirred at 25 °C for 2 h. After the reaction was complete, the solid product was obtained by prep purification 6.1 mg, yield 28%, purity 87%.

[0302] 1 H NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 9.45 (s, 1H), 8.70 (s, 1H), 7.81 - 7.65 (m, 3H), 7.56 (d, J = 7.7 Hz, 1H), 7.48 (d, J = 8.1 Hz, 3H), 7.26 (t, J = 7.8 Hz, 1H), 7.08 (t, J = 8.9 Hz, 1H), 4.65 - 4.56 (m, 2H), 4.38 (s, 2H), 3.84 (s, 4H), 3.19 (s, 1H), 3.16 - 2.86 (m, 5H), 2.80 (s, 1H), 2.16 - 1.80 (m, 4H), 1.70 (d, J = 14.7 Hz, 4H), 1.25 (d, J = 7.1 Hz, 6H). LC-MS: 663.30 [M+H] + .

[0303] Example 52

[0304]

[0305] The synthetic procedure was referred to the synthetic procedure of Example 51 compound to give white solid 21.4 mg, yield 63%, purity 96%.

[0306] 1H NMR (400 MHz, DMSO-d6) δ 10.07 (s, 1H), 8.69 (s, 1H), 8.30 (dd, J = 8.1, 5.7 Hz, 2H), 7.93 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 97.6 Hz, 3H), 7.26 (t, J = 7.9 Hz, 1H), 7.07 (s, 1H), 6.00 (s, 1H), 4.57 (d, J = 32.2 Hz, 5H), 3.84 (s, 5H), 3.12 (s, 1H), 2.94 (d, J = 6.9 Hz, 1H), 2.69 (s, 4H), 1.76 (d, J = 71.6 Hz, 2H), 1.41 (d, J = 33.0 Hz, 2H), 1.25 (d, J = 7.6 Hz, 6H). LC-MS: 677.30 [M+H] + .

[0307] Example 53

[0308]

[0309] The synthetic procedure was referred to the synthetic procedure of Example 51 compound to afford 2.5 mg of white solid in 3.88% yield with 78% purity.

[0310] 1 H NMR (400 MHz, MeOD) δ 10.22 (s, 1H), 9.01 (s, -1H), 8.62 (s, 1H), 8.26 (d, J = 8.2 Hz, 2H), 8.03 (s, 1H), 7.83 (d, J = 8.1 Hz, 2H), 7.73 (s, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 4.57 (s, 2H), 4.51 (s, 2H), 4.13 (d, J = 32.5 Hz, 4H), 3.80 (s, 4H), 2.92 (s, 1H), 2.43 - 2.34 (m, 2H), 1.22 (d, J = 8.3 Hz, 6H). LC-MS: 677.30 [M+H] + .

[0311] Example 54

[0312]

[0313] The synthetic procedure was referred to the synthetic procedure of Example 1 compound to afford 34.9 mg of white solid in 60% yield with 95% purity.

[0314] 1H NMR (400 MHz, MeOD) δ 8.22 - 8.11 (m, 3H), 7.93 (d, J = 8.1 Hz, 2H), 6.61 (s, 1H), 4.52 (s, 2H), 4.31 (s, 1H), 3.88 - 3.74 (m, 1H), 3.67 (d, J = 12.4 Hz, 2H), 3.44 (s, 2H), 3.27 (p, J = 6.9 Hz, 2H), 3.09 (d, J = 13.1 Hz, 3H), 2.69 (s, 3H), 2.51 - 2.14 (m, 8H), 1.96 (t, J = 5.7 Hz, 4H), 1.37 (d, J = 6.8 Hz, 6H). LC-MS: 569.30 [M+H] + .

[0315] Example 55

[0316]

[0317] The synthetic procedure was referenced to the synthetic procedure of Example 1 compound to yield 55.9 mg of white solid in 96% yield, 97% purity.

[0318] 1 H NMR (400 MHz, MeOD) δ 8.22 - 8.11 (m, 3H), 7.93 (d, J = 8.1 Hz, 2H), 6.61 (s, 1H), 4.52 (s, 2H), 4.31 (s, 1H), 3.88 - 3.74 (m, 1H), 3.67 (d, J = 12.4 Hz, 2H), 3.44 (s, 2H), 3.27 (p, J = 6.9 Hz, 2H), 3.09 (d, J = 13.1 Hz, 3H), 2.69 (s, 3H), 2.51 - 2.14 (m, 8H), 1.96 (t, J = 5.7 Hz, 4H), 1.37 (d, J = 6.8 Hz, 6H). LC-MS: 569.30 [M+H] + .

[0319] Example 56

[0320]

[0321] Step A: 4-chloro-2-(methylsulfanyl)-8-(propan-2-yl)pyrazolo[l,5-a][l,3,5]triazine (500 mg, 2.06 mmol, 1 eq), tert-butyl 4-aminopiperidine-l-carboxylate (580 mg, 2.88 mmol, 1.4 eq) and N,N-diisopropylethylamine (800 mg, 6.18 mmol, 3 eq) were taken in 20 mL of acetonitrile and stirred at 25 °C for 2 h under nitrogen atmosphere. After completion of the reaction, it was extracted with ethyl acetate and concentrated under reduced pressure to get the crude product 980 mg as white solid.

[0322] Step B: tert-butyl 4-[(2-(methylsulfanyl)-8-(propan-2-yl)pyrazolo[l,5-a][l,3,5]triazin-4- yl)amino]piperidine-l-carboxylate (300 mg, 0.74 mmol, 1 eq) was taken in 8 mL of dry dichloromethane and m-chloroperoxybenzoic acid (510.81 mg, 2.96 mmol, 4 eq) was added slowly and stirred at this temperature for 5 h. After completion of the reaction, 10 mL of dichloromethane and 4 mL of saturated sodium sulfite solution was added to the reaction and extracted in a separatory funnel, the organic phase was collected and washed with citric acid solution and saturated aqueous sodium chloride solution separately and concentrated under reduced pressure to get the white solid. White solid 324 mg, yield 100%.

[0323] Step C: tert-butyl 4-[(2-methylsulfonyl-8-(propan-2-yl)pyrazolo[l,5-a][l,3,5]triazin-4- yl)amino]piperidine-l-carboxylate (108 mg, 0.25 mmol, 1 eq) and 4-aminotetrahydropyran (126.44 mg, 1.25 mmol, 5 eq) were taken in 0.5 mL of N-methylpyrrolidone and stirred at 120 °C for 2 h. The mixture was washed with a trace amount of water and filtered to collect the filter cake to get the product 73 mg, yield 64.5% as a yellow solid.

[0324] Step D: tert-butyl 4-[(2-[(oxan-4-yl)amino]-8-(propan-2-yl)pyrazolo[l,5-a][l,3,5]triazin-4- yl)amino]piperidine-l-carboxylate (1 eq, 0.16 mmol, 73 mg) and trifluoroacetic acid (0.5 mL) were taken in 2 mL of dichloromethane and stirred at 25 °C for 2 h. After completion of the reaction, the pH of the mixture was adjusted to 9-10 and then concentrated under reduced pressure to get the product 57 mg, yield 99.83% as a yellow oil.

[0325] Step E: N2-(oxan-4-yl)-N4-(piperidin-4-yl)-8-(propan-2-yl)pyrazolo[1,5- a][1,3,5]triazine-2,4-diamine (57 mg, 0.16 mmol, 1 eq), N-[(1s,4s)-4- formylcyclohexyl]tert-butylcarbamate (36.37 mg, 0.16 mol, 1 eq) and sodium cyanoborohydride (20 mg, 0.32 mmol, 2 eq) were dissolved in 2 mL of methanol, after adding one drop of acetic acid, stirred at 25 °C for 4 h under nitrogen protection. After the reaction was completed, extracted with ethyl acetate, then concentrated under reduced pressure, purified by scraping the board to obtain 80 mg of product, yield 88%, as a yellow solid.

[0326] Step F: N-[(1s,4s)-4-[(4-[(2-[(oxan-4-yl)amino]-8-(propan-2-yl)pyrazolo[1,5- a][1,3,5]triazin-4-yl)amino]piperidin-1-yl)methyl]cyclohexyl]tert-butylcarbamate (1 eq, 0.18 mmol, 0.18 mol) and 4 M hydrogen chloride dioxane solution (0.5 mL) were stirred in 1 mL of dioxane solution at 25 °C for 2 h. Thin layer chromatography showed that the reaction was complete. The pH of the mixture was adjusted to 9-10, then concentrated under reduced pressure to obtain 80 mg of product, yield 97%, as a white solid.

[0327] Step G: N2-(oxan-4-yl)-8-(propan-2-yl)-N4-(1-{[(1s,4s)-4-aminocyclohexyl]methyl}piperidin- 4-yl)pyrazolo[1,5-a][1,3,5]triazine-2,4-diamine (1 eq, 0.085 mmol, 40 mg) and N,N- diisopropylethylamine (3 eq, 0.26 mmol, 0.042 mL) were added dropwise to five fluorobenzene sulfonyl chloride (1 eq, 0.085 mmol, 22.66 mg) in 1 mL of dichloromethane solution and stirred at 0 °C for 1 h. After the reaction was completed, the product was obtained by preparative purification, 12.6 mg, yield 20.52%, purity 97%, as a white solid.

[0328] 1H NMR (400 MHz, MeOD) δ 7.91 (s, 1H), 4.43 (s, 1H), 4.24 (s, 1H), 4.03 (d, J = 11.8 Hz, 2H), 3.74 (d, J = 12.4 Hz, 2H), 3.60 (t, J = 11.5 Hz, 2H), 3.31 - 2.98 (m, 5H), 2.37 (d, J = 14.0 Hz, 2H), 2.12 (d, J = 12.5 Hz, 2H), 2.01 (t, J = 14.3 Hz, 4H), 1.90 (d, J = 13.6 Hz, 3H), 1.75 (s, 2H), 1.45 (q, J = 11.6 Hz, 3H), 1.32 (d, J = 7.0 Hz, 6H), 1.19 (q, J = 12.1, 11.6 Hz, 2H). LC-MS: 701.30 [M+H] + .

[0329] Example 57

[0330]

[0331] The synthetic procedure was referred to the synthetic procedure of Example 56 compound to afford 5 mg of white solid in 6.45% yield, 97% purity.

[0332] 1 H NMR (400 MHz, MeOD) δ 7.89 (s, 1H), 4.69 (s, 1H), 4.44 (s, 1H), 4.07 - 3.97 (m, 2H), 3.93 - 3.85 (m, 1H), 3.82 (d, J = 9.3 Hz, 1H), 3.73 (d, J = 12.7 Hz, 2H), 3.52 (s, 1H), 3.18 (s, 2H), 3.07 (dt, J = 18.6, 6.5 Hz, 3H), 2.36 (d, J = 8.3 Hz, 3H), 2.08 (s, 3H), 1.98 (d, J = 12.6 Hz, 2H), 1.89 (d, J = 13.5 Hz, 2H), 1.44 (q, J = 11.8 Hz, 3H), 1.32 (d, J = 6.9 Hz, 6H), 1.20 (t, J = 12.6 Hz, 2H) LC-MS: 687.20 [M+H] + .

[0333] Example 58

[0334]

[0335] The synthetic procedure was referred to the synthetic procedure of Example 56 compound to afford 26 mg of white solid in 99% yield, 99% purity.

[0336] 1H NMR (400 MHz, MeOD) δ 7.90 (s, 1H), 4.41 (s, 1H), 4.22 (s, 1H), 4.13 - 4.06 (m, 1H), 4.02 (d, J = 11.9 Hz, 2H), 3.78 (s, 3H), 3.59 (t, J = 11.6 Hz, 2H), 3.44 (d, J = 11.0 Hz, 2H), 3.25 (t, J = 9.3 Hz, 5H), 3.09 (t, J = 6.9 Hz, 1H), 2.35 (d, J = 14.8 Hz, 2H), 2.03 (d, J = 12.8 Hz, 4H), 1.84 - 1.74 (m, 2H), 1.60 (t, J = 14.0 Hz, 2H), 1.45 (d, J = 12.5 Hz, 2H), 1.32 (d, J = 6.8 Hz, 6H). LC-MS: 703.20 [M+H] + .

[0337] Example 59

[0338]

[0339] The synthetic procedure was referred to the synthetic procedure of Example 1 compound to give 5.5 mg of white solid in 97% yield, 98% purity.

[0340] 1 H NMR (400 MHz, MeOD) δ 8.18 (s, 1H), 6.55 (s, 1H), 4.24 (s, 1H), 4.12 - 4.00 (m, 1H), 3.79 (s, 3H), 3.43 (d, J = 11.3 Hz, 2H), 3.29 (s, 1H), 3.25 (t, J = 7.1 Hz, 4H), 2.67 (s, 3H), 2.33 (s, 2H), 2.21 (d, J = 13.6 Hz, 3H), 2.02 (d, J = 11.3 Hz, 1H), 1.79 (d, J = 13.4 Hz, 1H), 1.67 - 1.56 (m, 1H), 1.45 (t, J = 12.4 Hz, 1H), 1.37 (d, J = 6.9 Hz, 6H). LC-MS: 617.20 [M+H] + .

[0341] Example 60

[0342]

[0343] The synthetic procedure was referred to the synthetic procedure of Example 56 compound to give 6.6 mg of white solid in 10% yield, 97% purity.

[0344] 1H NMR (400 MHz, MeOD) δ 7.82 (s, 1H), 4.31 (d, J = 71.4 Hz, 2H), 3.68 (dd, J = 37.3, 12.5 Hz, 4H), 3.25 (s, 2H), 3.06 (t, J = 9.7 Hz, 3H), 2.94 (s, 3H), 2.42 - 2.26 (m, 4H), 1.99 (d, J = 12.5 Hz, 4H), 1.89 (d, J = 13.4 Hz, 4H), 1.56 - 1.38 (m, 3H), 1.31 (d, J = 6.9 Hz, 6H), 1.19 (d, J = 13.1 Hz, 2H). LC-MS: 714.30 [M+H] + .

[0345] Example 61

[0346]

[0347] The synthesis procedure was referenced to the synthesis procedure of Example 56 compound. White solid 10.9 mg was obtained in 10.46% yield, 97% purity.

[0348] 1 H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H), 9.05 (s, 2H), 8.29 (d, J = 8.3 Hz, 2H), 7.93 (d, J = 8.2 Hz, 2H), 7.77 (s, 1H), 4.41 (t, J = 6.2 Hz, 2H), 4.17 (s, 3H), 3.64 (d, J = 12.0 Hz, 3H), 3.42 (t, J = 11.7 Hz, 3H), 3.06 (s, 2H), 2.96 (d, J = 6.7 Hz, 3H), 2.26 - 1.99 (m, 6H), 1.96 - 1.84 (m, 4H), 1.80 (s, 1H), 1.54 (d, J = 11.7 Hz, 2H), 1.41 (t, J = 12.0 Hz, 2H), 1.24 (d, J = 6.9 Hz, 6H), 1.07 (d, J = 12.9 Hz, 2H). LC-MS: 643.30 [M+H] + .

[0349] Example 62

[0350]

[0351] Step A-G: The synthesis procedure was referenced to the synthesis procedure of Example 56 compound. Yellow solid 25 mg was obtained in 91% yield.

[0352] Step F: To a solution of 4-{[(8-(propan-2-yl)-4-[(1-{[(1s,4s)-4- (pentafluorophenylsulfonamido)cyclohexyl]methyl}piperidin-4-yl)amino]pyrazolo[1,5- a][1,3,5]triazin-2-yl)amino]methyl}piperidine-1-carboxylate benzyl ester (1 eq, 0.018 mmol, 15 mg) in 33% hydrogen bromide in acetic acid (0.5 mL) was added was added under nitrogen protection and stirred for 0.5 h in an ice bath. After the reaction was completed, more methyl tert-butyl ether was added to slurry and filtered to get residue. The residue was purified by prep to get the product. This product 3.3 mg, 26% yield, 99% purity, as a white solid.

[0353] 1 H NMR (400 MHz, MeOD) δ 7.83 (s, 1H), 3.73 (d, J = 12.2 Hz, 1H), 3.46 (d, J = 12.8 Hz, 5H), 3.05 (td, J = 20.3, 17.6, 9.8 Hz, 6H), 2.37 (d, J = 14.5 Hz, 3H), 2.02 (dd, J = 29.1, 12.9 Hz, 6H), 1.90 (d, J = 13.5 Hz, 4H), 1.55 (d, J = 13.2 Hz, 2H), 1.44 (q, J = 12.2 Hz, 3H), 1.31 (d, J = 7.2 Hz, 6H), 1.24 - 1.15 (m, 2H). LC-MS: 714.30 [M+H] + .

[0354] Example 63

[0355]

[0356] The synthesis procedure was referenced to the synthesis procedure of Example 56 compound to get 6.5 mg, 8.39% yield, 98% purity as a white solid.

[0357] 1H NMR (400 MHz, MeOD) δ 7.91 - 7.76 (m, 1H), 7.42 - 7.28 (m, 5H), 5.12 (s, 2H), 4.63 (d, J = 13.6 Hz, 2H), 4.40 (s, 1H), 3.85 - 3.65 (m, 3H), 3.56 (s, 1H), 3.18 (ddd, J = 44.3, 18.7, 9.1 Hz, 6H), 3.02 (d, J = 6.4 Hz, 1H), 2.37 (d, J = 14.0 Hz, 2H), 2.02 (q, J = 16.7, 14.9 Hz, 6H), 1.89 (d, J = 13.5 Hz, 2H), 1.55 (d, J = 11.8 Hz, 2H), 1.49 (s, 1H), 1.46 - 1.37 (m, 2H), 1.18 (q, J = 12.3 Hz, 2H). LC-MS: 834.30 [M+H] + .

[0358] Example 64

[0359]

[0360] The synthetic procedure was referred to the synthetic procedure of Example 56 compound to afford 84 mg of white solid, 99% purity, 23% yield.

[0361] 1H NMR (400 MHz, MeOD) δ 7.89 (s, 1H), 4.40 (d, J = 61.8 Hz, 2H), 4.08 (d, J = 11.7 Hz, 1H), 3.89 - 3.70 (m, 3H), 3.64 (d, J = 11.8 Hz, 2H), 3.55 - 3.39 (m, 3H), 3.30 (s, 1H), 3.25 (q, J = 9.9, 7.6 Hz, 4H), 3.14 - 3.05 (m, 1H), 2.96 (d, J = 5.1 Hz, 3H), 2.39 - 2.01 (m, 8H), 1.85 (dd, J = 41.2, 12.8 Hz, 2H), 1.70 - 1.55 (m, 1H), 1.45 (d, J = 12.7 Hz, 1H), 1.31 (d, J = 6.9 Hz, 6H). LC-MS: 716.30 [M+H]+.

[0362] Example 65

[0363]

[0364] The synthetic procedure was referred to the synthetic procedure of Example 56 compound to afford 84 mg of white solid, 99% purity, 23% yield. 1H NMR (400 MHz, MeOD) δ 7.86 (s, 1H), 4.62 - 4.28 (m, 2H), 3.73 (d, J = 12.5 Hz, 2H), 3.51 (d, J = 12.3 Hz, 3H), 3.23 (s, 2H), 3.10 (t, J = 6.8 Hz, 1H), 3.04 (d, J = 6.5 Hz, 1H), 2.33 (t, J = 18.7 Hz, 5H), 2.10 (d, J = 13.8 Hz, 2H), 1.98 (d, J = 12.6 Hz, 2H), 1.90 (d, J = 13.2 Hz, 4H), 1.44 (q, J = 12.3 Hz, 2H), 1.31 (d, J = 6.9 Hz, 6H), 1.19 (q, J = 12.0 Hz, 2H). LC-MS: 700.30 [M+H] + .

[0365] Example 66 Enzyme activity experiment

[0366] The following compounds and comparative examples in the above examples section were tested for biological activity.

[0367] The biological activity test experiment was carried out as follows:

[0368] The CDK7 kinase IC50 value of the test compound was detected (in Wuxi Bai'ao company)

[0369] 1. Compound preparation

[0370] The compound powder was dissolved in 100% DMSO to prepare a 10 mM stock solution, which was further diluted to 0.5 mM as the starting concentration, and then diluted by 3 times successively to obtain 10 different concentrations of compound solution. The compound and enzyme were pre-incubated for 0 minutes and 60 minutes, respectively, using a duplicate hole detection. Staurosporine compound was used as a positive control and tested together with SY5609. The activity of the compound on CDK2, CDK7 and CDK9 CDK kinases was detected using the Mobility shift assay method.

[0371]

[0372] Positive control compound

[0373] 2. Kinase reaction process

[0374] (1) The compound solution and the positive control were diluted 8.3 times with double distilled water and added to the 384-well plate, 2 μL / well for each concentration;

[0375] (2) 6 nM CDK7 / Cyclin H / MAT1 kinase solution was added to the compound hole and the positive control hole, respectively;

[0376] (3) Incubate at room temperature for 0 and 60 minutes;

[0377] (4) Add 2 mM ATP and 2 pM 5-FAM-CDK7 peptide substrate (5-FAM- YSPTSPSYSPTSPSYSPTSPSKKKK) solution (8 nM CDK9 / Cyclin T1 polymer and 2 pM 5-FAM GSRTPMY-NH2 peptide substrate for CDK9 inhibition reaction; 0.5 nM CDK2 / Cyclin E1 polymer and 2 pM 5-FAM- YSPTSPSYSPTSPSYSPTSPSKKKK peptide substrate for CDK2 inhibition reaction);

[0378] (5) Incubate the 384-well plate at 27°C for 50 minutes (incubate for 20 minutes for CDK2 inhibition reaction; incubate for 30 minutes for CDK9 inhibition reaction);

[0379] (6) Add 4 pL of 150 mM EDTA to stop the kinase reaction;

[0380] (7) Read the conversion rate using Caliper / LabChip EZ Reader (Perkin Elmer);

[0381] (8) Curve fitting to obtain IC50(nM) value by Graphpad Prism 8 software 50 Numerical value.

[0382] Through the above detection, the inhibition activity IC50(nM) value of the test sample on CDK7 kinase is shown in Table 1.

[0383] Table 1

[0384]

[0385]

[0386] Example 67 Cell Anti-proliferation Experiment

[0387] I. Experimental materials and equipment

[0388] Human breast cancer cell HCC70 and ovarian cancer cell OVCAR-3 were purchased from Suzhou Tawo Biotechnology Co., Ltd. RPMI1640 medium (Basal Media), DMSO (dimethyl sulfoxide), CTG Cell Viability Detection Reagent (MCE), 0.25% EDTA-Tripsin, 1X PBS (phosphate buffer, pH 7.2), 96-well plate (Beyotime), fetal bovine serum (FBS), penicillin-streptomycin solution (100X) (Beyotime), high-speed refrigerated centrifuge (Thermo Fisher X1R), cell counter (Countstar IC1000), multifunctional microplate reader (Molecular Devices SpectraMax iD5).

[0389] II. Experimental preparation

[0390] 1. Cell plating

[0391] A) The tumor cells were cultured in RPMI 1640 (containing 10% FBS and 1x penicillin-streptomycin) at 37°C, 5% CO2 and saturated humidity to 80-90% density.

[0392] B) Remove the culture medium in the T75 culture flask;

[0393] C) Rinse the cells with 10 mL of 1X PBS twice;

[0394] D) Add 2 mL of 0.25% EDTA-Tripsin, put it in a 37°C, 5% CO2 incubator for 5 minutes, transfer to a 15 mL centrifuge tube, centrifuge at 400g for 3 minutes, discard the supernatant to obtain the cell pellet;

[0395] E) Resuspend the cell pellet with 3 mL of RPMI 1640 medium and count.

[0396] F) Adjust the cell suspension to the appropriate density so that the 96-well plate is 4000 cells / well, with a volume of 90 μL per well, and incubate in a 37°C, 5% CO2 incubator overnight.

[0397] 2. Compound treatment

[0398] Compound dilution

[0399] A) Prepare the gradient dilution solution of the test compound: Prepare a 10 mM stock solution of the test compound. Dissolve 10 μL of the 10 mM stock solution in 90 μL of RPMI 1640 medium to obtain a dilution of the compound at a concentration of 1 mM, and then perform 3-fold serial dilution with 1640 medium, for a total of 9 concentrations. The 9 concentrations of the compound after dilution are as follows: 1000 μM, 333.33 μM, 111.11 μM, 37.04 μM, 12.35 μM, 4.12 μM, 1.37 μM, 0.46 μM, 0.15 μM

[0400] B) After thorough mixing, add 10 μL of the compound solution to the corresponding wells of the 96-well plate, so that the final concentrations of the compound are 100 μM, 33.33 μM, 11.11 μM, 3.70 μM, 1.24 μM, 0.41 μM, 0.14 μM, 0.046 μM, and 0.015 μM, respectively. Set up one well with 10 μL of 0.1% DMSO as a control.

[0401] C) Place the 96-well plate in a 37°C, 5% CO2 incubator for 72 h.

[0402] 3. CTG detection

[0403] A) Take an appropriate amount of detection reagent, thawed and equilibrated to room temperature, according to the amount of CTG Cell Viability Detection Reagent per well of the 96-well plate, i.e., Vcell:VCTG Reagent = 1:1).

[0404] B) Take out the 96-well cell culture plate and equilibrate it at room temperature for 10 min.

[0405] C) Add 100 μL of CTG Cell Viability Detection Reagent to each well of the 96-well plate.

[0406] D) Shake well at room temperature for 2 min to promote complete lysis of the cells.

[0407] E) Incubate at room temperature for 10 min to allow the luminescent signal to stabilize.

[0408] F) Perform chemiluminescence detection on a multifunctional enzyme labeler (Molecular Devices SpectraMax iD5).

[0409] 4. Data analysis

[0410] Calculate the cell viability (%) using the following formula:

[0411] % Cell Viability = 100% x Lum_Sample / Lum_HC

[0412] Lum_Sample: cell reading of compound addition

[0413] Lum_HC: cell reading of 0.1% DMSO control

[0414] The IC50 values were obtained by curve fitting through GraphPad Prism 8 software. As shown in Table 2,

[0415] Table 2

[0416]

[0417]

[0418] The experimental results show that the compound of the present application has lower or equivalent IC50 values compared with SY5609, showing stronger or equivalent binding force to CDK7. The difference in binding force may lead to different target selectivity of the compound, thereby treating different diseases.

[0419] The above examples serve to specifically introduce the essential content of the present application, but those skilled in the art should know that the protection scope of the present application should not be limited to the specific examples.

Claims

1. A bifunctional compound or a pharmaceutically acceptable salt, stereoisomer thereof, characterized in that, The bifunctional compound has any one of the following structures:

2. A pharmaceutical composition, characterized by: The pharmaceutical composition comprises the bifunctional compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer thereof.

3. The pharmaceutical composition of claim 2, wherein: The pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient, carrier or diluent.

4. Use of the bifunctional compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer thereof for the manufacture of an antitumor drug, wherein the tumor is breast cancer or ovarian cancer.

5. Use of the pharmaceutical composition of claim 2 or 3 for the manufacture of an antitumor drug, wherein the tumor is breast cancer or ovarian cancer.

Images