Heterocyclic compounds and their use as cdks inhibitors

Abstract

The present application relates to heterocyclic compounds and their use as CDK inhibitors, belonging to the field of medicinal chemistry, in particular to compounds having the structure of formula (I), stereoisomers, tautomers or mixtures thereof, or pharmaceutically acceptable salts, prodrugs or deuterated compounds thereof. The present application further provides a preparation method of such compounds and their use for modulating cyclin-dependent kinases (CDKs). In addition, the present application also provides the use of such compounds for treating or preventing diseases related to abnormal cell proliferation or CDK-related diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a new heterocyclic compound used as a cyclin-dependent kinase (CDK) inhibitor, a derivative thereof and a stereoisomer thereof, a preparation method thereof, and the use thereof for regulating or inhibiting the activity of a cyclin-dependent kinase, for treating and preventing diseases related to CDK activity, and for preparing a drug for treating the diseases. BACKGROUND

[0002] Cyclin-dependent kinases are a class of serine / threonine protein kinases that regulate various cell cycle processes and gene transcriptional regulation. Currently, 20 different CDKs have been reported, all of which contain a homologous sequence of PSTAIRE and bind to the corresponding regulatory subunit, cyclin, to form an active heterodimer, which is involved in physiological processes such as transcription, metabolism, neural differentiation and development. Among them, CDK1 / 2 / 4 / 6 is involved in regulating cell cycle progression, and CDK7 / 8 / 9 / 12 / 13 is involved in gene transcriptional regulation.

[0003] CDK7 is composed of 346 amino acids and is widely expressed in tissues, and usually binds to cyclin H (Cyclin H) and MAT1 to form a cyclin-activated kinase (CAK) trimer complex. The main physiological functions of CDK7 include indirect regulation of the cell cycle, regulation of transcription initiation, and regulation of super-enhancer-mediated gene expression. When it is a CAK complex, CDK7 activates the activity of T-loop domains of CDK1, CDK2, CDK4 and CDK6, etc. by phosphorylation, thereby indirectly participating in all stages of the cell cycle and promoting the smooth progress of the cell cycle. At the same time, CAK is one of the components of the general transcription complex TFIIH, and CDK7, as the core kinase of TFIIH, mediates the transcription initiation process by phosphorylating the 5th serine of the C-terminal domain (CTD) of RNA polymerase II (POLR2A). The TFIIH complex not only plays a role in RNA transcription, but is also necessary for nucleotide excision repair (NER). In addition, CDK7 also affects the transcription process of a large number of genes (such as transcription factors and signal transduction molecules) through super-enhancers (SE).

[0004] CDK7 is up-regulated in various tumors, such as triple-negative breast cancer (TNBC), high-grade serous ovarian cancer (HGSOC), small cell lung cancer (SCLC), etc., and promotes the expression of genes, including proto-oncogene Myc, through enhancers. CDK7 up-regulation is not only a mechanism of tumorigenesis and progression of various tumors, but also a poor prognosis biomarker of cancer. On the contrary, inhibition of CDK7 by RNAi, small molecule drugs, etc. shows significant anti-tumor activity in various PDX tumor models, achieving complete tumor regression. CDK7 inhibitors can inhibit the proliferation of breast cancer and colorectal cancer in vivo, and play a synergistic role with tamoxifen in the treatment of ER+ breast cancer. Inhibition of CDK7 can hinder cell cycle progression, inhibit cell proliferation, reduce the expression of proto-oncogene c-Myc, etc. and induce apoptosis.

[0005] There is no drug on the market for the CDK7 small molecule inhibitors currently under research. CDK7 inhibitors entering clinical trials include SY-1365 (terminated research) and SY-5609 (I phase clinical trial) of Syros Company, and CT7001 of Carrick Company. SY-5609 is used for the treatment of advanced solid tumors, including breast cancer, ovarian cancer, colorectal cancer, lung cancer, etc. CT7001 is used for the treatment of advanced solid tumors. In addition, there are other CDK7 small molecule inhibitors under preclinical research.

[0006] CDK7 plays a key role in transcriptional regulation and cell cycle regulation. More and more studies have shown that CDK7 inhibitors can effectively inhibit the proliferation of malignant tumors in vitro and in vivo, and can produce synergistic effects with other drugs. The purpose of the present patent research is to provide CDK7 targeted inhibitors for the treatment of tumors and other diseases related to CDK7. SUMMARY

[0007] The present application relates to a heterocyclic compound, a stereoisomer, a tautomer or a mixture thereof, or a pharmaceutically acceptable salt thereof, a solvate, a hydrate, a prodrug or a deuterated compound thereof, the structure of the heterocyclic compound is shown as formula (I):

[0008]

[0009] X 1 , X 2 each independently is CR 3 or N;

[0010] R x is selected from H, halogen, cyano, isocyano, amino, hydroxyl, nitro, C1-C6 alkyl, halo C1-C6 alkyl, C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl; preferably, R x is selected from H, halogen, cyano, isocyano, trifluoromethyl, trichloromethyl, amino, cyclopropyl;

[0011] R 1 , R 2 , R 3 each independently is selected from the group consisting of: H, halogen, cyano, isocyano, amino, hydroxy, mercapto, carboxy, Ci-C6-alkyl, halo-Ci-C6-alkyl, Ci-C6-alkoxy, C3-C6-cycloalkyl, 3- to 6-membered heterocycloalkyl, C3-C6-cycloalkyloxy;

[0012] Ring B is a group selected from the group consisting of:

[0013]

[0014] each R 4 is independently selected from the group consisting of H, halogen, amino, hydroxy, nitro, mercapto, cyano, isocyano, Ci-C6-alkyl, Ci-C6-alkoxy, halo-Ci-C6-alkyl, C3-C6-cycloalkyl, 3- to 6-membered heterocycloalkyl, -S(=0)R a1 , -S-R a1 , -S(=0)2R a1 , -(CH2) n -C(=0)OR a1 , -(CH2) n -C(=0)R a1 , -(CH2) n -R a2 , -(CH2) n -S(=0)2R a1 , -(CH2) n -P(=0)(OR a1 )2, -(CH2) n -P(=0)(R a1 )2, -(CH2) n -P(=0)(OR a1 )(R a1 ), -(CR b )2-R a2 , -NR b -(CH2) n -R a2 , aryl, heteroaryl, -0(CH2) n -R a2 , -0-(C2-C4-alkenylene)-R a1 , wherein said cycloalkyl, heterocycloalkyl, aryl, heteroaryl are substituted or unsubstituted by one or more R b , said heteroatoms being one or more of N, O, or S; preferably said aryl is a C6-Ci0-aryl, said heteroaryl is a 5- to 10-membered heteroaryl; 10 ​

[0015] Each R a1 Independently selected from: H, hydroxyl, amino, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, 3-6 membered heterocyclic alkyl, aryl, heteroaryl; preferably, the aryl group is C6-C6. 14 Aryl, more preferably C6-C 10 The aryl group is more preferably selected from phenyl and naphthyl; preferably, the heteroaryl group is a 5- to 10-membered heteroaryl group, more preferably selected from imidazolyl, furanyl, thiophene, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrroleyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, and pyrazinyl.

[0016] Each R a2 Independently selected from: H, halogen, amino, hydroxyl, cyano, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, 3-6 membered heterocyclic alkyl, aryl, heteroaryl; preferably, the aryl group is C6-C6. 14 Aryl, more preferably C6-C 10 The aryl group is more preferably selected from phenyl and naphthyl; preferably, the heteroaryl group is a 5- to 10-membered heteroaryl group, more preferably selected from imidazolyl, furanyl, thiophene, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrroleyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, and pyrazinyl.

[0017] Each R b Independently selected from: H, amino, hydroxyl, halogen, cyano, C1-C4 alkyl, halogenated C1-C4 alkyl, C3-C4 cycloalkyl;

[0018] n = 0, 1, 2, 3, 4, 5 or 6;

[0019] p = 0, 1, 2, 3 or 4;

[0020] G is a C3-C7 cycloalkyl or a 3-7 heterocyclic alkyl group having one or more substituents or no substituents, wherein the substituents of G are selected from one or more of the following groups: halogen, amino, cyano, isocyano, hydroxy, nitro, carbonyl, aldehyde, acyl, amide, phosphoryl, sulfonyl, ester, carboxyl, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, 3-6 heterocyclic alkyl, C1-C6 alkoxy, C3-C6 cycloalkyloxy, 3-6 heterocyclic alkyloxy, aryl, heteroaryl, C6-C7 cycloalkyloxy, C3-C6 cycloalkyloxy, C3-C6 cycloalkyloxy, aryl, heteroaryl, C6-C7 cycloalkyloxy, C3-C6 cycloalkyloxy, C3-C6 cycloalkyloxy, C3-C6 cycloalkyloxy, aryl, heteroaryl, C3-C7 cycloalkyloxy ... 10 aryloxy group, 5-10 heteroaryloxy group, wherein the heteroatom is one or more of N, O, or S;

[0021] L is selected from: -(CH2) m -,-O(CH2) m -,-(CH2)m O-, -C(=O)-, -NR c -, -O-, -C(=O)O-, -OC(=O)-, -NR c -C(=O)-, -C(=O)-NR c -, -NR c -(CH2) m -, -(CH2) m -NR c -, -S-, -S(=O)-, -S(=O)2-(CH2) m -, -(CH2) m -S(=O)2-, -S(=O)O-,-OS(=O)-, -S(=O)-NR c -, -NR c -S(=O)-, -S(=O)2O-, -OS(=O)2-, -S(=O)2NR c -, -NR c -S(=O)2-, absent; m = 0, 1, 2, 3, 4, 5 or 6;

[0022] each R c is independently selected from the group consisting of H, amino, hydroxyl, halogen, cyano, C1-C4 alkyl, haloC1-C4 alkyl, C3-C4 cycloalkyl;

[0023] D is selected from the group consisting of substituted or unsubstituted C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl, aryl, heteroaryl; or is absent; wherein the substituents of said D are one or more of amino, hydroxyl, halogen, cyano, C1-C4 alkyl, haloC1-C4 alkyl, C3-C4 cycloalkyl; preferably said aryl is C6-C 14 aryl, more preferably C6-C 10 aryl; preferably said heteroaryl is 5-14 membered heteroaryl, more preferably 5-10 membered heteroaryl;

[0024] R y is selected from the group consisting of hydroxyl, halogen, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, haloC1-C6 alkyl, haloC1-C6 alkoxy, -(CH2) i -N(R d )2, -(CH2) i -R e , -NR d -(CH2) i -R e , -(CH2) i -P(=O)(OR d )R e , -(CH2) i-P(=O)(OR d )2, -NR d -S(=O)2R d , -NR d -S(=O)2R e , -S(=O)2R d , -S(=O)2R e , -NR d -S(=O)R d , -NR d -S(=O)R e , R d substituted or unsubstituted R e , is absent;

[0025] Y is N or CR d ; i = 0, 1, 2, 3, or 4;

[0026] each R d , R 5A , R 5B is independently selected from the group consisting of: H, hydroxyl, amino, halogen, cyano, C1-C6alkyl, C1-C6alkoxy, haloC1-C6alkyl; R 5C , R 5D is independently selected from the group consisting of: R d , -N(R d )2, C2-C4alkenyl, C2-C4alkynyl;

[0027] each R e is independently selected from the group consisting of R d substituted C3-C6cycloalkyl, 3-6 membered heterocycloalkyl, aryl, heteroaryl, wherein the heteroatoms are one or more of N, O, or S; preferably, the aryl is C6-C 14 aryl, more preferably C6-C 10 aryl; preferably, the heteroaryl is 5-14 membered heteroaryl, more preferably 5-10 membered heteroaryl.

[0028] In some embodiments of the compounds of the present application, stereoisomers, tautomers, or mixtures thereof, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or deuterated compounds thereof, G is C3-C6cycloalkyl or 3-6 membered heterocycloalkyl;

[0029] L is selected from the group consisting of: -(CH2) m -, -O(CH2) m -, -(CH2) m O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR c-C(=O)-,-C(=O)-NR c -,-NR c -(CH2) m -,-(CH2) m -NR c -,-S(=O)2NR c -, -O-, -S-, -S(=O)-, -S(=O)2-; m=0, 1, 2, 3 or 4;

[0030] D is selected from the following groups, whether substituted or unsubstituted: C5-C6 cycloalkyl, 5-6 membered heterocyclic alkyl, C6-C6 cyclo ... 10 Aryl, 5-10 heteroaryl; wherein the substituent of D is one or more of amino, hydroxyl, halogen, cyano, C1-C4 alkyl, halo-C1-C4 alkyl, C3-C4 cycloalkyl;

[0031] R y Selected from hydroxyl, halogen, amino, cyano, C1-C4 alkyl, C1-C4 alkoxy, halo-C1-C6 alkyl, halo-C1-C6 alkoxy, -(CH2). i -N(R d 2、-(CH2) i -R e -NR d -(CH2) i -R e -NR d -S(=O)2R d , via R d Replaced or unreplaced R e , It does not exist;

[0032] i = 0, 1, 2, 3 or 4;

[0033] Each R d Independently selected from: H, hydroxyl, amino, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl; each R 5A R 5B Each group is independently selected from: H, hydroxyl, amino, halogen, cyano; R 5C Selected from: R d -N(R) d 2. C2-C4 alkenyl, C2-C4 alkynyl;

[0034] Each R e Independently selected from one or more R d The following groups are substituted: C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl, C6-C 10aryl, 5-10 membered heteroaryl, wherein the heteroatoms are one or more of N, O, or S.

[0035] In some embodiments of the compounds of the present application, stereoisomers, tautomers, or mixtures thereof, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or deuterated compounds thereof, G is selected from the following groups:

[0036] is wherein the a end is attached to NH and the b end is attached to L;

[0037] W 1 , W 2 , W 3 each independently N or CR 6 ;

[0038] each R 6 is independently selected from the group consisting of: H, halogen, nitro, amino, cyano, isocyano, hydroxyl, aldehyde, acyl, amido, ester, carboxyl, C1-C6 alkyl, haloC1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C1-C6 alkoxy, C3-C6 cycloalkyloxy, C3-C6 heterocycloalkyloxy, wherein the heteroatoms are one or more of N, O, or S;

[0039] Preferably, each R 6 is independently selected from the group consisting of: H, halogen, amino, hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, CF3, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, C1-C4 ester, amido;

[0040] q = 0, 1, 2, or 3.

[0041] In some embodiments of the compounds of the present application, stereoisomers, tautomers, or mixtures thereof, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or deuterated compounds thereof, L is selected from the group consisting of: -(CH2) m -, -C(=O)-, -C(=O)O-, -OC(=O)-, -NR c -C(=O)-, -C(=O)-NR c -, -NR c -(CH2) m -, -(CH2) m -NR c -, -S(=O)2NR c -, -NR c -S(=O)2-; m = 0, 1, 2, 3, or 4; and each R c is independently selected from the group consisting of: H, amino, hydroxyl, halogen, cyano, C1-C4 alkyl.

[0042] In some embodiments of the compounds of the present application, stereoisomers, tautomers, or mixtures thereof, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or deuterated compounds thereof, D is substituted or unsubstituted C6-C10aryl or 5-10 membered heteroaryl. 10 aryl or 5-10 membered heteroaryl.

[0043] In further embodiments, D is selected from the group consisting of substituted or unsubstituted:

[0044]

[0045] wherein said substituents are one or more of amino, hydroxyl, halogen, cyano, C1-C4alkyl, haloC1-C4alkyl, C3-C4cycloalkyl.

[0046] In still further embodiments, D is selected from the group consisting of substituted or unsubstituted:

[0047]

[0048] wherein a end is connected to L and b end is connected to R y ; or a end is connected to R y and b end is connected to L;

[0049] wherein said substituents are one or more of amino, hydroxyl, halogen, cyano, C1-C4alkyl, haloC1-C4alkyl, C3-C4cycloalkyl.

[0050] In some embodiments of the compounds of the present application, stereoisomers, tautomers, or mixtures thereof, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or deuterated compounds thereof, R y is selected from the group consisting of hydroxyl, halogen, amino, cyano, C1-C4alkyl, C1-C4alkoxy, haloC1-C6alkyl, haloC1-C6alkoxy, null.

[0051] In some embodiments of the compounds of the present application, stereoisomers, tautomers, or mixtures thereof, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or deuterated compounds thereof, R y is selected from the group consisting of -(CH2) i -N(R d )2, -NR d -S(=O)2R d , null;

[0052] i = 0, 1, 2, 3, or 4;

[0053] each R 5A , R 5Beach independently selected from the group consisting of: H, hydroxyl, amino, halogen, cyano; R 5C selected from the group consisting of: R d , -N(R d )2, C2-C4 alkenyl, C2-C4 alkynyl; each R d is independently selected from the group consisting of: H, hydroxyl, amino, halogen, cyano, C1-C4 alkyl, C1-C4 alkoxy, haloC1-C4 alkyl.

[0054] In some embodiments of the compounds of the present application, stereoisomers, tautomers, or mixtures thereof, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or deuterated compounds thereof, R y is selected from the group consisting of: -(CH2) i -R e , -NR d -(CH2) i -R e , R d substituted or unsubstituted R e is absent;

[0055] i = 0, 1, 2, 3, or 4;

[0056] each R d is independently selected from the group consisting of: H, hydroxyl, amino, halogen, cyano, C1-C6 alkyl, C1-C6 alkoxy, haloC1-C6 alkyl.

[0057] each R e is independently selected from the group consisting of R d substituted C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl, C6-C 10 aryl, 5-10 membered heteroaryl, wherein the heteroatoms are N, O, or S.

[0058] In further embodiments of the present application, each R e is independently selected from the group consisting of R d substituted or unsubstituted:

[0059]

[0060] In some specific embodiments of the present application, each R e is independently selected from the group consisting of R d substituted or unsubstituted:

[0061]

[0062] In some embodiments of the compounds of the present application, stereoisomers, tautomers, or mixtures thereof, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or deuterated compounds thereof, each R e is independently selected from the group consisting of substituted or unsubstituted: d substituted or unsubstituted:

[0063]

[0064] In some embodiments of the compounds of the present application, stereoisomers, tautomers, or mixtures thereof, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or deuterated compounds thereof, each R e is independently selected from the group consisting of substituted or unsubstituted: d substituted or unsubstituted:

[0065]

[0066] In some embodiments of the compounds of the present application, stereoisomers, tautomers, or mixtures thereof, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or deuterated compounds thereof, G is C3-C7cycloalkyl or 3-7 membered heterocycloalkyl having 1-3 substituents or no substitution, wherein the substituents of G are one or more selected from the group consisting of halogen, amino, cyano, isocyano, hydroxyl, nitro, carbonyl, aldehyde, acyl, amido, phosphoryl, sulfonyl, ester, carboxyl, C1-C4alkyl, haloC1-C4alkyl, C3-C6cycloalkyl, 3-6 membered heterocycloalkyl, C1-C6alkoxy, C3-C6cycloalkyloxy, 3-6 membered heterocycloalkyloxy, C6-C10aryl, 5-10 membered heteroaryl, C6-C10aryloxy, 5-10 membered heteroaryloxy, wherein the heteroatoms are one or more of N, O, or S; and L, D, and R 10 aryl, 5-10 membered heteroaryl, C6-C 10 aryloxy, C6-C 10 heteroaryloxy, wherein the heteroatoms are one or more of N, O, or S; and L, D, and R y is absent.

[0067] In some specific embodiments of the present application, the compounds have a structure selected from the group consisting of:

[0068]

[0069] The present application also relates to the use of any of the aforementioned compounds, stereoisomers, tautomers, or mixtures thereof, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or deuterated compounds thereof for the treatment or prophylaxis of a disease associated with cyclin-dependent kinase (CDK) activity or expression level, and the use of any of the aforementioned compounds, stereoisomers, tautomers, or mixtures thereof, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, or deuterated compounds thereof for the preparation of a medicament for the treatment or prophylaxis of a disease associated with cyclin-dependent kinase activity or expression level.

[0070] In yet another aspect, there is also provided use of a compound described herein, a stereoisomer, a tautomer, or mixtures thereof, a pharmaceutically acceptable salt, a solvate, a hydrate, a prodrug, or a deuterated compound thereof for treating or preventing a disease responsive to modulation (e.g., promotion or inhibition) of a cyclin-dependent kinase (CDK), and use of the same in the manufacture of a medicament for treating or preventing a disease responsive to modulation (e.g., promotion or inhibition) of a cyclin-dependent kinase (CDK).

[0071] In some preferred embodiments, the CDK is one or more selected from CDK7, CDK12, CDK13. In some embodiments, the CDK is CDK7.

[0072] In yet another aspect, there is also provided use of a compound described herein, a stereoisomer, a tautomer, or mixtures thereof, a pharmaceutically acceptable salt, a solvate, a hydrate, a prodrug, or a deuterated compound thereof in the manufacture of a medicament for treating or preventing a disease associated with abnormal cell proliferation. In some embodiments, the disease associated with abnormal cell proliferation is a tumor. In some further embodiments, the tumor is selected from bladder cancer, breast cancer, lung cancer (including but not limited to small cell lung cancer, or non-small cell lung cancer such as lung adenocarcinoma, lung squamous carcinoma, and lung large cell carcinoma), colorectal cancer, kidney cancer, epidermal cancer, liver cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer (including but not limited to papillary thyroid cancer, follicular thyroid cancer, anaplastic thyroid cancer), nasal cancer, head and neck cancer, prostate cancer, central or peripheral nervous system tumor, skin cancer, angiosarcoma, melanoma, seminoma, leukemia (e.g., acute leukemia such as acute lymphoblastic leukemia, acute non-lymphocytic leukemia; or chronic leukemia such as chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic monocytic leukemia), multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (including but not limited to B-cell lymphoma, T-cell lymphoma).

[0073] In some embodiments, the tumor is selected from breast cancer, ovarian cancer, colorectal cancer, lung cancer. In some embodiments, the tumor is selected from triple negative breast cancer, high-grade serous ovarian cancer, small cell lung cancer.

[0074] The present application further relates to a method of inhibiting CDK function in a cell in vitro or in vivo, comprising contacting said cell with an effective amount of a compound described herein, a stereoisomer, a tautomer, or mixtures thereof, a pharmaceutically acceptable salt, a solvate, a hydrate, a prodrug, or a deuterated compound thereof.

[0075] The present application is also directed to a method of modulating (e.g., inhibiting) cell proliferation, inhibiting cell cycle progression, promoting apoptosis, or a combination of one or more thereof, in vitro or in vivo, comprising contacting a cell with an effective amount of a compound described herein, a stereoisomer, a tautomer, or a mixture thereof, a pharmaceutically acceptable salt, a solvate, a hydrate, a prodrug, or a deuterated compound thereof.

[0076] The present application is also directed to a method of treating or preventing a disease, comprising administering to a subject in need thereof an effective amount of a compound described herein, a stereoisomer, a tautomer, or a mixture thereof, a pharmaceutically acceptable salt, a solvate, a hydrate, a prodrug, or a deuterated compound thereof; wherein the disease is a CDK-related disease, e.g., a disease associated with CDK activity (e.g., a disease caused by abnormal CDK activity), a disease associated with CDK gene mutation, a disease associated with CDK expression (e.g., abnormal expression), a disease associated with upstream pathway activation of CDK, a disease ameliorated by inhibition of CDK, or a disease responsive to inhibition of CDK. Preferably, the disease is a disease associated with CDK activity, a disease associated with CDK expression, or a disease responsive to inhibition of CDK. In some embodiments, the CDK is one or more selected from CDK7, CDK12, CDK13. In some embodiments, the CDK is CDK7.

[0077] The present application is further directed to a method of treating or preventing a disease associated with abnormal cell proliferation, comprising administering to a subject in need thereof an effective amount of a compound described herein, a stereoisomer, a tautomer, or a mixture thereof, a pharmaceutically acceptable salt, a solvate, a hydrate, a prodrug, or a deuterated compound thereof. In some embodiments, the disease associated with abnormal cell proliferation is a tumor. In some preferred embodiments, the tumor is selected from bladder cancer, breast cancer, lung cancer (e.g., small cell lung cancer, or non-small cell lung cancer such as lung adenocarcinoma, lung squamous carcinoma, and lung large cell carcinoma), colorectal cancer, kidney cancer, epidermal cancer, liver cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer (e.g., papillary thyroid carcinoma, follicular thyroid carcinoma, anaplastic thyroid carcinoma), nasal cancer, head and neck cancer, prostate cancer, central or peripheral nervous system tumor, skin cancer, vascular endothelioma, melanoma, seminoma, leukemia (e.g., acute leukemia, such as acute lymphoblastic leukemia, acute non-lymphoblastic leukemia; or chronic leukemia, such as chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic monocytic leukemia), multiple myeloma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma (e.g., B-cell lymphoma, T-cell lymphoma). In some preferred embodiments, the tumor is selected from breast cancer, ovarian cancer, colorectal cancer, lung cancer. In more preferred embodiments, the tumor is selected from triple-negative breast cancer, high-grade serous ovarian cancer, small cell lung cancer.

[0078] The present application also relates to a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a compound of the present application, a stereoisomer, a tautomer, or a mixture thereof, a pharmaceutically acceptable salt, a solvate, a hydrate, a prodrug, or a deuterated compound thereof, and a pharmaceutically acceptable carrier or excipient.

[0079] The present application also relates to a pharmaceutical composition for treating or preventing a disease comprising a compound of the present application, a stereoisomer, a tautomer, or a mixture thereof, a pharmaceutically acceptable salt, a solvate, a hydrate, a prodrug, or a deuterated compound thereof. In some embodiments, the disease is a disease associated with abnormal cell proliferation, a disease responsive to modulation of a cell cycle protein-dependent kinase, or a disease associated with cell cycle protein-dependent kinase activity or expression level. In some embodiments, the cell cycle protein-dependent kinase is selected from CDK7, CDK12, CDK13, preferably CDK7. In some embodiments, the disease associated with abnormal cell proliferation is a tumor, for example, bladder cancer, breast cancer, lung cancer (e.g., small cell lung cancer, or non-small cell lung cancer such as lung adenocarcinoma, lung squamous carcinoma, and lung large cell carcinoma), colorectal cancer, kidney cancer, epidermal cancer, liver cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer (e.g., papillary thyroid carcinoma, follicular thyroid carcinoma, thyroid anaplastic carcinoma), nasal cancer, head and neck cancer, prostate cancer, central or peripheral nervous system tumor, skin cancer, vascular endothelioma, melanoma, seminoma, leukemia (acute leukemia, such as acute lymphoblastic leukemia, acute non-lymphocytic leukemia; or chronic leukemia, such as chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic monocytic leukemia), multiple myeloma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma (e.g., B-cell lymphoma, T-cell lymphoma). In some embodiments, the tumor is selected from breast cancer, ovarian cancer, colorectal cancer, lung cancer. In some embodiments, the tumor is selected from triple-negative breast cancer, high-grade serous ovarian cancer, small cell lung cancer.

[0080] The present application further relates to a compound according to the application, a stereoisomer, a tautomer or a mixture thereof, a pharmaceutically acceptable salt, a solvate, a hydrate, a prodrug or a deuterated compound thereof, for use in the treatment or prevention of a disease associated with abnormal cell proliferation, a disease responsive to modulation of a cell cycle protein dependent kinase or a disease associated with cell cycle protein dependent kinase activity or expression. In some embodiments, the cell cycle protein dependent kinase is selected from CDK7, CDK12, CDK13, preferably CDK7. In some embodiments, the disease associated with abnormal cell proliferation is a tumor, for example, bladder cancer, breast cancer, lung cancer (e.g., small cell lung cancer, or non-small cell lung cancer such as lung adenocarcinoma, lung squamous carcinoma and lung large cell carcinoma), colorectal cancer, kidney cancer, epidermal cancer, liver cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer (e.g., papillary thyroid carcinoma, follicular thyroid carcinoma, anaplastic thyroid carcinoma), nasal cancer, head and neck cancer, prostate cancer, central or peripheral nervous system tumor, skin cancer, vascular endothelioma, melanoma, seminoma, leukemia (acute leukemia, such as acute lymphoblastic leukemia, acute nonlymphocytic leukemia; or chronic leukemia, such as chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic monocytic leukemia), multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (e.g., B-cell lymphoma, T-cell lymphoma). In some embodiments, the tumor is selected from breast cancer, ovarian cancer, colorectal cancer, lung cancer. In some embodiments, the tumor is selected from triple negative breast cancer, high grade serous ovarian cancer, small cell lung cancer.

[0081] Definitions and general terms

[0082] Unless otherwise indicated, the following terms have the meanings set forth below in the specification and claims:

[0083] "Alkyl" refers to an aliphatic hydrocarbon group, referring to a saturated hydrocarbon group. The alkyl moiety can be a straight chain alkyl or a branched chain alkyl. For example, C1-C6 alkyl. C1-C6 alkyl refers to an alkyl group having from 1 to 6 carbon atoms, for example, an alkyl group having 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, neopentyl, n-hexyl, and the like. The alkyl group can be unsubstituted or substituted with one or more substituents including, but not limited to, alkyl, alkoxy, cyano, hydroxyl, carbonyl, carboxyl, aryl, heteroaryl, amine, halogen, sulfonyl, sulfinyl, phosphonyl, and the like.

[0084] C 1-n including C 1-2 , C 1-3 …C1-q q is an integer greater than 1. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, and the like.

[0085] "Alkenyl" refers to an alkyl group in which the starting two atoms form a double bond, i.e., alkenyl is -C(R)=C(R)-R', where R' refers to the remainder of the alkenyl group, and each R can be the same or different. Alkenyl groups can be optionally substituted, including, but not limited to, -CH=CH2, -C(CH3)=CH2, -CH=CHCH3, -C(CH3)=CHCH3, -CH=CHCH2-, and the like. Alkenyl groups can have 2 to 6 carbon atoms, e.g., alkenyl groups having 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms.

[0086] "Akynyl" refers to an alkyl group in which the starting two atoms form a triple bond, i.e., akynyl is -C≡C-R, where R refers to the remainder of the akynyl group. Akynyl groups can be optionally substituted, including, but not limited to, -C≡CH, -C≡CCH3, -C≡CCH2CH3, -C≡C-, and -C≡CCH2-, and the like. Akynyl groups can have 2 to 6 carbon atoms, e.g., akynyl groups having 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms.

[0087] "Ester" refers to a chemical structure having the formula -COOR, where R can be alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and the like. C1-C4 ester refers to a chemical structure having the formula -COOR x1 , where R x1 is C1-C4 alkyl.

[0088] "Amido" refers to a chemical structure having the formula "-CONR x1 R x2 " or "-NR x3 COR x4 ", where R x1 , R x2 , R x3 , and R x4 are independently H or C1-C4 alkyl.

[0089] "Ring" means any covalently closed structure, including, for example, carbocyclic (e.g., aryl or cycloalkyl), heterocyclic (e.g., heteroaryl or heterocycloalkyl), aromatic (e.g., aryl or heteroaryl), non-aromatic (e.g., cycloalkyl or heterocycloalkyl). The ring can be optionally substituted, and can be monocyclic or polycyclic. Typical polycyclic rings generally include bicyclic, tricyclic rings. The rings of the present application generally have 1-20 ring atoms, e.g., 1 ring atom, 2 ring atoms, 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, 11 ring atoms, 12 ring atoms, 13 ring atoms, 14 ring atoms, 15 ring atoms, 16 ring atoms, 17 ring atoms, 18 ring atoms, 19 ring atoms, or 20 ring atoms.

[0090] "Member" means the number of skeletal atoms that make up a ring. Typical 5-membered rings include, for example, cyclopentyl, pyrrole, imidazole, thiazole, furan, and thiophene; typical 6-membered rings include, for example, cyclohexyl, pyridine, pyran, pyrazine, thiopyran, pyridazine, pyrimidine, benzene, and the like. Of these, rings that contain heteroatoms in the skeletal atoms are heterocyclic rings; aromatic groups that contain heteroatoms are heteroaromatic groups; non-aromatic groups that contain heteroatoms are heterocycloalkyl groups, which include heterocycloalkyl groups.

[0091] "Heteroatom" means an atom other than carbon or hydrogen. One or more of the heteroatoms in the heterocyclic rings of the present application can be independently selected from O, S, N, Si, and P, but are not limited thereto.

[0092] The term "alicyclic group" as used herein means a cyclic hydrocarbon substituent that includes one or more rings and that is saturated or partially unsaturated (including one or more double bonds, but no ring has a completely conjugated pi-electron system and is not aromatic). A cycloalkyl group is included as a subgenus of alicyclic groups, i.e., a saturated alicyclic group. Cycloalkyl groups further include monocycloalkyl and polycycloalkyl groups, which include 3-20 carbon atoms that can form rings, preferably 3-10 carbon atoms. Examples of cycloalkyl groups include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecane, cyclododecyl, cyclohexenyl, and the like; polycycloalkyl groups also include cycloalkyl groups that contain spiro, fused, and bridged ring structures. Typical alicyclic groups include, but are not limited to:

[0093]

[0094] Typical alicyclic groups include, but are not limited to, the groups formed from the alicyclics described above.

[0095] The term "aliphatic heterocyclyl" as used herein refers to a cyclic substituent comprising one or more rings that is saturated or partially unsaturated (comprising one or more double bonds, but no ring has a fully conjugated pi-electron system, and is not aromatic) and which comprises one or more (e.g., 1, 2, 3, or 4) heteroatoms in addition to carbon in its ring atoms, wherein the heteroatoms can be N, O, S, or SO2 The aliphatic heterocyclyl group can be monocyclic, fused, bridged, and spiro structures. Aliphatic heterocyclyl groups include the subgenus "heterocycloalkyl", i.e., saturated aliphatic heterocyclyl groups. Heterocycloalkyl groups can be 3- to 10-membered (e.g., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-membered, e.g., comprising 3, 4, 5, 6, 7, 8, 9, or 10 ring atoms) monocyclic or bicyclic or tricyclic. Typical aliphatic heterocycles include, but are not limited to:

[0096]

[0097] Typical aliphatic heterocyclyl groups include, but are not limited to, groups formed from the above aliphatic heterocycles.

[0098] "Aryl" refers to a monocyclic or fused polycyclic (that is, rings that share pairs of adjacent carbon atoms) ring system having a conjugated pi-electron system of 6 to 14 carbon atoms (6- to 14-membered), preferably 6 to 10 atoms, such as phenyl and naphthyl. More preferably phenyl. The aryl ring can be fused to a heteroaryl, heterocycloalkyl, or cycloalkyl ring, wherein the ring that is attached to the parent structure is the aryl ring.

[0099] The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 (e.g., 1, 2, 3, or 4) heteroatoms, 5 to 14 ring atoms (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). The heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl is preferably 5- to 10-membered, comprising 1 to 3 heteroatoms; more preferably 5- or 6-membered, comprising 1 to 2 heteroatoms; preferably, for example, imidazolyl, furanyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, and the like, preferably imidazolyl, thiazolyl, pyrazolyl, or pyrimidinyl, thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring can be fused to an aryl, heterocycloalkyl, or cycloalkyl ring, wherein the ring that is attached to the parent structure is the heteroaryl ring.

[0100] "substituted" means that one or more hydrogen atoms, preferably up to 5 (e.g., 1, 2, 3, 4, 5), more preferably 1 to 3, of a group can be independently replaced with the corresponding number of the substituents as defined herein. It is understood that the substituents are only placed at their possible chemical positions, which can or can not be possible (experimentally or theoretically) by one skilled in the art without undue effort. For example, an amino group with a free hydrogen can not be stable when attached to a carbon atom with an unsaturated (e.g., olefinic) bond. By way of example, a substituted group in the present application can be substituted with a group selected from: C 1-6 alkyl, C 3-6 cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxy, alkoxy, alkylthio, aryloxy, nitro, acyl, halogen, haloalkyl, amino, mercapto, cyano, ester, carboxy, amido, -C=0, haloC 1-6 alkyl, amino-substituted C 1-6 alkyl, hydroxy-substituted C 1-6 alkyl, C 1-6 heteroalkyl, haloC 1-6 heteroalkyl, amino-substituted C 1-6 heteroalkyl, hydroxy-substituted C 1-6 heteroalkyl, alkyl-substituted C 3-6 cycloalkyl, haloC 3-6 cycloalkyl, hydroxy-substituted C 3-6 cycloalkyl, amino-substituted C 3-6 cycloalkyl or C 3-6 heterocycloalkyl, alkyl-substituted C 3-6 heterocycloalkyl, haloC 3-6 heterocycloalkyl, hydroxy-substituted C 3-6 heterocycloalkyl, and amino-substituted C 3-6 heterocycloalkyl, etc.

[0101] "target protein" means a protein molecule or a portion of a protein that is capable of being bound by a selective binding compound. In certain embodiments, the target protein is CDK7.

[0102] "inhibitor" means a compound that causes a decrease in the amount of activity of a target protein or target molecule as compared to the amount of such activity in the absence of the inhibitor. In some embodiments, the inhibitor causes a decrease in the metabolic activity of the target protein. In some embodiments, the inhibitor causes a decrease in the corresponding enzymatic activity.

[0103] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted" means that the group can or can not be substituted; "optionally substituted heterocycloalkyl" means that an alkyl group can or can not be present, including instances where the heterocycloalkyl group is substituted with an alkyl group and instances where the heterocycloalkyl group is not substituted with an alkyl group.

[0104] The term "substituted or unsubstituted" refers herein to the group that can be unsubstituted or substituted with one or more substituents to the extent chemically allowed for such substitution(s), including multiple substitution at the same moiety (to the extent chemically allowed), each substituent can be located at any available position of the group, and can be attached through any available atom of the substituent. "Any available position" refers to any position on the group that is chemically obtainable by methods known in the art or methods taught herein, and which does not result in an excessively unstable molecule. When there are two or more substituents on any group, each substituent is defined independently of any other substituent, and thus can be the same or different.

[0105] The term "halogen" refers to a fluoro, chloro, bromo, or iodo substituent.

[0106] The term "haloalkyl" refers to an alkyl group as defined herein in which one or more hydrogens is replaced by the same or different halogen. Examples of haloalkyl include -CF3, -CH2Cl, -CH2CF3, -CH2CCl3, and the like.

[0107] The term "C1-C6alkoxy" refers to a group R'-O-, where R' is a C1-C6alkyl. "C3-C6cycloalkyloxy" refers to a group R'-O-, where R' is a C3-C6cycloalkyl.

[0108] The term "stereoisomer" refers to compounds having the same chemical structure, but differing in the spatial arrangement of atoms or groups. Stereoisomers of compounds of the context of the present application include, but are not limited to, enantiomers, diastereomers, cis / trans isomers, mesomers, racemates, or mixtures thereof.

[0109] When bonds to chiral carbons are depicted as straight lines in structural formulae herein, it is understood that the structural formulae encompass both the (R) and (S) configurations of the chiral carbon and thus also encompass both enantiomers and mixtures thereof. Similarly, when a compound name is described without specifying the chirality of a chiral carbon, it is understood that the name includes both the (R) and (S) configurations of the chiral carbon and thus also encompasses the individual enantiomers and mixtures thereof. The production of a particular stereoisomer or mixture thereof can be identified in the examples where such stereoisomer or mixture is obtained, but this is by no means a limitation that all stereoisomers and mixtures thereof are excluded from the scope of the present application.

[0110] The present application includes all possible enantiomeric and diastereomeric forms and mixtures thereof in all proportions, for example mixtures of enantiomers and / or diastereomers. Thus, enantiomers are subject matter of the present application in the form of enantiomerically pure forms (left- and right-rotating enantiomers), in the form of racemates and in the form of mixtures of both enantiomers in all proportions. In the case of cis / trans isomers, the present application includes both the cis and trans forms as well as mixtures thereof in all proportions. If desired, the preparation of individual stereoisomers can be achieved by separation of mixtures using conventional methods, for example by chromatography or crystallization, by using stereochemically homogeneous starting materials for synthesis, or by stereoselective synthesis. Optionally, derivatization can be performed prior to separation of the stereoisomers. The separation of stereoisomeric mixtures can be performed at an intermediate stage of the synthesis of the target compound, or using the final racemic product. The absolute stereochemistry can be determined by X-ray crystallography of a crystalline product or a crystalline intermediate that has been derivatized, if necessary, using reagents of known stereochemistry. When the compounds according to the present application are capable of existing in tautomeric forms, all individual tautomers and mixtures thereof are included within the scope of the present application. Unless otherwise specified, the present application includes all such isomers, salts, solvates (including hydrates) or solvated salts of such isomers of the racemates, enantiomers, diastereomers and tautomers, and mixtures thereof.

[0111] The term "fused ring" as used herein refers to a polycyclic structure of 5 to 20 members, in which each ring of the system shares an adjacent pair of ring atoms with other rings of the system, wherein one or more rings can contain one or more double bonds. Preferably, the fused ring is 6 to 14 members, more preferably 7 to 10 members. The fused ring can be bi-, tri-, tetra- or polycyclic, preferably bi- or tri-cyclic, more preferably 5 / 5 or 5 / 6 bi-cyclic, depending on the number of rings comprising the fused ring. The term "fused ring" as defined in the context of the present application includes fused carbocyclic and fused heterocyclic rings. The fused heterocyclic ring is a fused ring containing one or more heteroatoms selected from O, S, N in addition to carbon in the ring-forming atoms, including, for example, fused aliphatic heterocyclic and fused heteroaromatic rings. The fused ring can be aromatic or non-aromatic. Typical fused ring aromatic compounds include, but are not limited to, naphthalene, anthracene, phenanthrene, and the like; fused ring heterocyclic compounds include, but are not limited to, indole, quinoline, purine, and the like.

[0112] The term "bridged ring" refers to a polycyclic structure of 5 to 20 members, in which any two rings share two non-adjacent (i.e., "not directly connected") ring atoms, which can contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system. Preferably, the bridged ring is 6 to 14 members, more preferably 7 to 10 members. The bridged ring can be bi-, tri-, tetra- or polycyclic, preferably bi-, tri- or tetra-cyclic, more preferably bi- or tri-cyclic, depending on the number of rings comprising the bridged ring. The term "bridged ring" as defined in the context of the present application includes bridged carbocyclic and bridged heterocyclic rings. The bridged heterocyclic ring, i.e., containing one or more heteroatoms selected from O, S, N in addition to carbon in the ring-forming atoms.

[0113] The term "spirocyclic" refers to a polycyclic structure of 5 to 20 members, in which a single ring shares a single carbon atom (referred to as a spiro atom) with another ring, which can contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system. Preferably, the spirocyclic ring is 6 to 14 members, more preferably 7 to 10 members. The term "spirocyclic" as defined in the context of the present application includes spirocarbocyclic and spiroheterocyclic rings. The spiroheterocyclic ring, i.e., containing one or more heteroatoms selected from O, S, N in addition to carbon in the ring-forming atoms.

[0114] The term "subject" as used herein includes mammals.

[0115] The mammals as described herein include bovids, equids, ovines, caprines, canids, felids, rodents, primates, wherein the preferred mammal is a human.

[0116] The term "effective amount" as used herein refers to an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, a therapeutically effective amount refers to an amount that is sufficient to cure or at least partially arrest the disease and its complications in an individual already suffering from the disease. A prophylactically effective amount refers to an amount that is effective to prevent, arrest, or delay the onset of a disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight, and gender, the mode of administration of the drug, and other therapies that may be administered at the same time, and the like.

[0117] The amount of the compound described herein, a stereoisomer, a tautomer, or a mixture thereof, a pharmaceutically acceptable salt, a prodrug, or a deuterated compound thereof administered to a subject depends on the type and severity of the disease or condition and the characteristics of the subject, such as general health, age, body weight, and tolerance to drugs, as well as the dosage form and mode of administration of the drug, and the period or interval of administration, and the like. One skilled in the art is capable of determining an appropriate dosage based on these and other factors. Generally, the compound described herein, a stereoisomer, a tautomer, or a mixture thereof, a pharmaceutically acceptable salt, a prodrug, or a deuterated compound thereof is used at a daily dose of about 0.0001-1000 mg / kg body weight per day, which can be administered in one or more doses as appropriate.

[0118] According to certain embodiments of the present application, the pharmaceutically acceptable salt described herein includes inorganic or organic acid salts, and inorganic or organic base salts of the compound. For example, the pharmaceutically acceptable salt includes, but is not limited to, alkali metal salts including sodium salt, potassium salt, lithium salt; alkaline earth metal salts including calcium salt, magnesium salt; metal salts including aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt, and the like; meglumine salt, ammonium salt, t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, monoethanolamine, diethanolamine, triethanolamine, N-methyl-D-glucamine, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-N-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris(hydroxymethyl)aminomethane salt, and the like amine salts; salts of the compound with inorganic or organic acids including hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, hydroxyacetic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, embonic acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthalene formic acid, hydroiodic acid, malic acid, tannic acid, and the like.

[0119] The carriers described in the present application include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, lanolin.

[0120] The term "excipient" used in the present application refers to an additional substance in a pharmaceutical preparation other than the main drug. It is stable in nature, has no compatibility contraindication with the main drug, does not produce side effects, does not affect the therapeutic effect, is not easy to deform, dry, mold, insect-bored at room temperature, is harmless to the human body, has no physiological effect, does not produce chemical or physical action with the main drug, does not affect the content determination of the main drug, etc. For example, the binding agent, filler, disintegrant, lubricant in tablets; preservative, antioxidant, flavoring agent, aromatic agent, cosolvent, emulsifier, solubilizer, osmotic pressure regulator, colorant, etc. in oral liquid preparations can be called excipients, etc.

[0121] In the present application, for the same compound, if the compound name is inconsistent with the structural formula, the compound structural formula is used as the standard. DETAILED DESCRIPTION

[0122] In order to further illustrate the present application, the cell cycle protein-dependent kinase inhibitory activity compound and its preparation method and application of the present application are further described below in conjunction with specific examples, but the protection scope of the present application is not limited thereto.

[0123] Abbreviation term explanation:

[0124] BOC represents tert-butyloxycarbonyl;

[0125] ACN represents acetonitrile;

[0126] (Boc)2O represents di-tert-butyl dicarbonate;

[0127] Cs2CO3 represents cesium carbonate;

[0128] DMF represents N,N-dimethylformamide;

[0129] DMSO represents dimethyl sulfoxide;

[0130] NMP represents N-methyl pyrrolidone;

[0131] NBS represents N-bromosuccinimide;

[0132] DCM represents dichloromethane;

[0133] DIPEA represents N,N-diisopropylethylamine;

[0134] DMAP represents 4-N,N-dimethylaminopyridine;

[0135] THF represents tetrahydrofuran;

[0136] TFA represents trifluoroacetic acid;

[0137] TEA represents triethylamine;

[0138] K2CO3 represents potassium carbonate;

[0139] KOAc represents potassium acetate;

[0140] Pd(dppf)Cl2 represents [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium;

[0141] Pd2(dba)3 represents tris(dibenzylideneacetone)dipalladium;

[0142] Pd(t-Bu3P)2 represents bis(tri-tert-butylphosphine)palladium;

[0143] PCy3 represents tricyclohexylphosphine;

[0144] DIAD represents diisopropyl azodicarboxylate;

[0145] PPh3 represents triphenylphosphine;

[0146] Xantphos represents 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene;

[0147] Pd(OAc)2 represents palladium acetate;

[0148] SEM represents 2-(trimethylsilyl)ethoxymethyl;

[0149] EA represents ethyl acetate;

[0150] Pd(PCy)2Cl2 represents dichlorobis(tricyclohexylphosphine)palladium;

[0151] Xphos Pd G3 represents (2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'- biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) methanesulfonate;

[0152] Brettphos Pd G3 represents (2-dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'- triisopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) methanesulfonate

[0153] Zn(CN)2 represents zinc cyanide;

[0154] Pd(TFA)2represents palladium trifluoroacetate;

[0155] HATU represents 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0156] HOBt represents 1-hydroxybenzotriazole;

[0157] EDCI represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;

[0158] DIEA represents N,N-diisopropylethylamine;

[0159] Py represents pyridine;

[0160] NIS represents N-iodosuccinimide.

[0161] Synthetic methods for the compounds of the present application

[0162] The present application also provides a synthesis method of the compound, and the synthesis method of the present application is mainly prepared from the preparation method reported in the chemical literature or the related synthesis using commercially available chemical reagents as starting materials.

[0163] Example 1 : Preparation of (R)-4-(1 H-indol-3-yl)-6-(pyrrolidin-3-ylamino)-1,7- naphthyridine-3-carbonitrile Specific procedure

[0164] The title compound is synthesized according to the following route

[0165]

[0166] Example 2: Preparation of (S)-4-(1 H-indol-3-yl)-6-(piperidin-3-ylamino)-1,7- naphthyridine-3-carbonitrile

[0167] Step 1: Preparation of tert-butyl 3-(3-cyano-6-fluoro-1,7-naphthyridin-4-yl)-1H-indole-1-carboxylate

[0168] Under nitrogen protection, 4-chloro-6-fluoro-1,7-naphthyridine-3-carbonitrile (400 mg, 1.93 mmol) and (1-(tert-butyl carboxylate)-1H-indol-3-yl)boronic acid (603 mg, 2.31 mmol) were dissolved in dioxane (10 mL) and water (2 mL), then K2CO3 (665 mg, 4.82 mmol) and Pd(dppf)Cl2 (141 mg, 192 μmol) were added, and the system was stirred at 80°C for 4 h. After the reaction was completed, water was added to dilute the reaction solution, and then ethyl acetate was extracted twice, and the organic phase was combined and washed with saturated brine once, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was separated by column chromatography to obtain compound 1A.

[0169]

[0170] MS (ESI) m / z 389.1 (M+H) +

[0171] 1 HNMR (400MHz, CDC13-d) δ = 9.39 (s, 1H), 9.17 (s, 1H), 8.32 (d, J = 8.4 Hz, 1H), 8.04 (s, 1H), 7.52-7.45 (m, 1H), 7.34-7.29 (m, 2H), 7.21 (d, J = 7.8 Hz, 1H), 1.74 (s, 9H)

[0172] Step 2: Preparation of tert-butyl (3R)-3-((3-cyano-4-(lH-indol-3-yl)-l,7- naphthyridin-6-yl)amino)pyrrolidine- 1 -carboxylate

[0173] tert-Butyl 3-(3-cyano-6-fluoro-l,7-naphthyridin-4-yl)-lH-indole-l-carboxylate (300 mg, 772 μmol) was dissolved in DMSO (6 mL), then (R)-tert-butyl 3- aminopyrrolidine-l-carboxylate (215.8 mg, 1.16 mmol) was added, the system was stirred at 120 °C for 3 h. After the reaction was completed, water was added to dilute the reaction solution, then extracted with ethyl acetate twice, the organic phase was combined and washed with saturated brine once, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was separated by column chromatography to obtain compound IB.

[0174]

[0175] MS (ESI) m / z 455.2 (M+H) +

[0176] 1 H NMR (400MHz, CDC13-d) δ = 9.21 (s, 1H), 9.04 (s, 1H), 8.79 (s, 1H), 8.17 (s, 1H), 7.71 (s, 1H), 7.60-7.53 (m, 1H), 7.43-7.38 (m, 1H), 7.37-7.31 (m, 2H), 6.62 (s, 1H), 4.72-4.51 (m, 1H), 4.49-4.28 (m, 1H), 3.25 (s, 1H), 3.04 (d, J = 15.2 Hz, 2H), 2.00-1.96 (m, 1H), 1.88 (d, J = 11.1 Hz, 1H), 1.44 (s, 9H)

[0177] Step 3: Preparation of (R)-4-(lH-indol-3-yl)-6-(pyrrolidin-3-ylamino)-l,7- naphthyridine-3-carbonitrile

[0178] Tert-butyl (3R)-3-((3-cyano-4-(1H-indol-3-yl)-1,7-naphthyridin-6-yl)amino)pyrrolidine-1- carboxylate (100 mg, 220 μmol) was dissolved in DCM (1 mL), then added into 1,4-dioxane (4 M, 1 mL) of hydrogen chloride, the system was reacted at room temperature for 1 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the obtained crude product was directly purified by high pressure preparation separation to obtain the compound of Example 1.

[0179]

[0180] MS (ESI) m / z 355.2 (M+H) +

[0181] 1 H NMR (400 MHz, DMSO-d6) δ = 11.78 (s, 1H), 9.08 (s, 1H), 8.73 (s, 1H), 7.87 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.15-7.08 (m, 1H), 6.84 (s, 1H), 6.59 (s, 1H), 3.98 (s, 1H), 2.91-2.80 (m, 2H), 2.76-2.60 (m, 2H), 1.86 (s, 1H), 1.93-1.54 (m, 1H), 1.54 (d, J = 5.5 Hz, 1H)

[0182] Example 3: Preparation of 6-(((1 R,3S)-3-aminocyclopentyl)amino)-4-(1 H- indol-3-yl)-1,7-naphthyridine-3-carbonitrile

[0183] Referring to the preparation method in Example 1, wherein step 2 is replaced by (S)-3-aminopiperidine-1-carboxylic acid tert-butyl ester instead of (R)-3-aminopyrrolidine-1-carboxylic acid tert-butyl ester, and other steps and raw materials are the same as those in Example 1, to obtain the compound of Example 2.

[0184]

[0185] MS (ESI) m / z 369.2 (M+H) +

[0186] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.12 - 1.24 (m, 1 H), 1.28 - 1.38 (m, 1 H), 1.47 - 1.82 (m, 4 H), 2.02 - 2.11 (m, 1 H), 3.13 - 3.17 (m, 1 H), 3.66 - 3.94 (m, 2 H), 6.55 (d, J=4.89 Hz, 1 H), 7.11 (d, J=5.01 Hz, 2 H), 7.25 (t, J=7.57 Hz, 1 H), 7.34 (d, J=7.87 Hz, 1 H), 7.57 (d, J=8.23 Hz, 1 H), 7.93 (s, 1 H), 8.73 - 8.76 (m, 1 H), 9.05 - 9.09 (m, 1 H), 11.81 - 12.13 (m, 1 H).

[0187] Specific procedure Example 4: Preparation of N-(4-((3R)-3-((3-cyano-4-(1 H-indol-3-yl)-1,7- naphthyridin-6-yl)amino)pyrrolidine-1 -carbonyl)phenyl)acrylamide

[0188] The title compound was prepared according to the procedure described in Example 1, Step 2, substituting (1S,3R)-3-aminocyclopentyl)carbamic acid tert-butyl ester for (R)-3-aminopyrrolidine-1-carboxylic acid tert-butyl ester in Step 2, and using the same procedures and starting materials as in Example 1 for the remaining steps.

[0189]

[0190] MS (ESI) m / z 369.2 (M+H) +

[0191] 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.12 - 1.24 (m, 1 H), 1.28 - 1.38 (m, 1 H), 1.47 - 1.82 (m, 4 H), 2.02 - 2.11 (m, 1 H), 3.13 - 3.17 (m, 1 H), 3.66 - 3.94 (m, 2 H), 6.55 (d, J=4.89 Hz, 1 H), 7.11 (d, J=5.01 Hz, 2 H), 7.25 (t, J=7.57 Hz, 1 H), 7.34 (d, J=7.87 Hz, 1 H), 7.57 (d, J=8.23 Hz, 1 H), 7.93 (s, 1 H), 8.73 - 8.76 (m, 1 H), 9.05 - 9.09 (m, 1 H), 11.81 - 12.13 (m, 1 H).

[0192] Specific procedure Example 5: Preparation of N-(4-((3S)-3-((3-cyano-4-(1 H-indol-3-yl)-1,7- naphthyridin-6-yl)amino)pyrrolidine-1 -carbonyl)thiazol-2-yl)acryloyl amide

[0193] The title compound was prepared according to the procedure described in Example 1, Step 2, substituting (1S,3R)-3-aminocyclopentyl)carbamic acid tert-butyl ester for (R)-3-aminopyrrolidine-1-carboxylic acid tert-butyl ester in Step 2, and using the same procedures and starting materials as in Example 1 for the remaining steps.

[0194]

[0195] Example 6: Preparation of (R)-4-(1 H-indol-3-yl)-6-((1 -(4-(isopropylamino)benzoyl) pyrrolidin-3-yl)amino)-1,7-naphthyridine-3-carbonitrile

[0196] Step 1: Preparation of N-(4-((3R)-3-((3-cyano-4-(lH-indol-3-yl)-l,7- naphthyridin-6-yl)amino)pyrrolidin-l-ylcarbonyl)phenyl)acrylamide

[0197] (R)-4-(lH-indol-3-yl)-6-(pyrrolidin-3-ylamino)-l,7-naphthyridine-3- carbonitrile (50.0 mg, 141 μmol) and 4-acrylamidobenzoic acid (32.4 mg, 169 μmol) were dissolved in DCM (1 mL), then tri-n-propyl phosphine oxide 50% ethyl acetate solution (67.3 mg, 211 μmol) and DIPEA (36.5 mg, 282 μmol) were added, the system was reacted at room temperature for 16 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the obtained crude product was directly purified by high pressure preparative separation to obtain the compound of Example 4.

[0198]

[0199] MS (ESI) m / z 528.2 (M+H) +

[0200] 1 HNMR (400 MHz, DMSO-d6) δ = 11.98 (s, 1H), 10.32 (s, 1H), 9.21-9.02 (m, 1H), 8.77 (d, J = 12.6 Hz, 1H), 7.92 (d, J = 14.5 Hz, 1H), 7.78-7.54 (m, 4H), 7.48 (s, 2H), 7.40-7.06 (m, 3H), 6.97-6.58 (m, 1H), 6.43 (s, 1H), 6.29 (s, 1H), 5.79 (s, 1H), 4.45-3.74 (m, 5H), 2.12 (s, 1H), 1.89 (s, 1H).

[0201] Example 7: Preparation of (R)-N-(4-(3-((3-cyano-4-(1 H-indol-3-yl)-1,7- naphthyridin-6-yl)amino)pyrrolidine-1 -carbonyl phenyl)acetamide

[0202] Referring to the preparation method in Example 4, 2-acrylamidothiazole-4- carboxylic acid was used to replace 4-acrylamidobenzoic acid, and other raw materials were the same as in Example 4 to obtain the compound of Example 5.

[0203]

[0204] MS (ESI) m / z 535.2 (M+H) +

[0205] 1 HNMR (400 MHz, DMSO-d6) δ ppm 12.37 (s, 1 H), 11.97 (s, 1 H), 9.12 (d, J=14.99 Hz, 1 H), 8.78 (d, J=7.28 Hz, 1 H), 7.84 - 7.98 (m, 1 H), 7.73 (s, 1 H), 7.50 - 7.61 (m, 1 H), 7.13 - 7.40 (m, 4 H), 6.69 (d, J=13.45 Hz, 1 H), 6.45 - 6.59 (m, 1 H), 6.32 - 6.45 (m, 1 H), 5.91 (t, J=9.37 Hz, 1 H), 4.30 - 4.54 (m, 1 H), 3.98 - 4.21 (m, 1 H), 3.90 (s, 1 H), 3.56 - 3.77 (m, 2 H), 2.07 (s, 1 H), 1.75 (s, 1 H)

[0206] Example 8: Preparation of (R)-6-((1 -(4-(1 H-imidazol-2-yl)benzoyl)pyrrolidin-3- yl)amino)-4-(1 H-indol-3-yl)-1,7-naphthyridine-3-carbonitrile Example 9: Preparation of (R)-4-(1 H-indol-3-yl)-6-((1 -(4-(4-methyl-1 H-1,2,3- triazol-1 -yl)benzoyl)pyrrolidin-3-yl)amino)-1,7-naphthyridine-3-carbonitrile

[0207] Referring to the preparation method in Example 4, 4-(isopropylamino)benzoic acid is used to replace 4-acrylamidobenzoic acid, and other raw materials are the same as in Example 4 to obtain the compound of Example 6.

[0208]

[0209] MS (ESI) m / z 516.2 (M+H) + .

[0210] 1 HNMR (400 MHz, DMSO-d6) δ ppm 12.37 (s, 1 H), 11.97 (s, 1 H), 9.12 (d, J=14.99 Hz, 1 H), 8.78 (d, J=7.28 Hz, 1 H), 7.84 - 7.98 (m, 1 H), 7.73 (s, 1 H), 7.50 - 7.61 (m, 1 H), 7.13 - 7.40 (m, 4 H), 6.69 (d, J=13.45 Hz, 1 H), 6.45 - 6.59 (m, 1 H), 6.32 - 6.45 (m, 1 H), 5.91 (t, J=9.37 Hz, 1 H), 4.30 - 4.54 (m, 1 H), 3.98 - 4.21 (m, 1 H), 3.90 (s, 1 H), 3.56 - 3.77 (m, 2 H), 2.07 (s, 1 H), 1.75 (s, 1 H)

[0211] Example 10: Preparation of (R)-6-((1 -(4-(1 H-pyrazol-1 -yl)benzoyl)pyrrolidin-3- yl)amino)-4-(1 H-indol-3-yl)-1,7-naphthyridine-3-carbonitrile Example 11 : Preparation of (3R)-N-(4-(3-((3-cyano-4-(1 H-indol-3-yl)-1,7- naphthyridin-6-yl)amino)pyrrolidine-1 -carbonyl ) phenyl)methanesulfonamide

[0212] The preparation method in Example 4 was referred to, 4-acetamidobenzoic acid was used to replace 4-acrylamidobenzoic acid, and other raw materials were the same as in Example 4 to obtain the compound of Example 7.

[0213]

[0214] MS (ESI) m / z 516.2 (M+H) + .

[0215] 1 HNMR (400MHz, DMSO-d6) δ = 11.96 (d, J = 7.9 Hz, 1H), 10.09 (s, 1H), 9.20-8.99 (m, 1H), 8.77 (d, J = 13.0 Hz, 1H), 7.92 (d, J = 16.8 Hz, 1H), 7.59 (t, J = 9.0 Hz, 3H), 7.49-7.40 (m, 2H), 7.38-7.18 (m, 3H), 7.15-6.88 (m, 1H), 6.82-6.55 (m, 1H), 4.43-4.13 (m, 1H), 3.78-3.57 (m, 1H), 3.54-3.39 (m, 3H), 2.22-2.09 (m, 1H), 2.04 (s, 3H), 1.88 (s, 1H)

[0216] Example 12: Preparation of (3R)-4-(1 H-indol-3-yl)-6-((1 -(4-(5-methyl-1,3,4- oxadiazol-2-yl)benzoyl)pyrrolidin-3-yl)amino)-1,7-naphthyridine-3-carbonitrile Specific procedure

[0217] The preparation method in Example 4 was referred to, 4-acetamidobenzoic acid was used to replace 4-acrylamidobenzoic acid, and other raw materials were the same as in Example 4 to obtain the compound of Example 7.

[0218]

[0219] MS (ESI) m / z 516.2 (M+H) + .

[0220] 1H NMR (400 MHz, DMSO-d6) δ ppm 12.60 (s, 1 H), 11.96 (d, J=9.66 Hz, 1 H), 9.00 - 9.20 (m, 1 H), 8.78 (d, J=17.73 Hz, 1 H), 7.86 - 8.00 (m, 3 H), 7.49 - 7.65 (m, 3 H), 7.08 - 7.42 (m, 5 H), 7.04 (s, 1 H), 6.61 - 6.77 (m, 1 H), 4.16 - 4.48 (m, 1 H), 3.74 (s, 1 H), 3.61 (s, 1 H), 3.42 - 3.58 (m, 2 H), 2.03 - 2.23 (m, 1 H), 1.91 (s, 1 H).

[0221] Example 13: Preparation of (R,E)-N-(4-(3-((3-cyano-4-(1 H-indol-3-yl)-1,7- naphthyridin-6-yl)amino)pyrrolidine-1 -carbonyl phenyl)-4-(dimethylamino)but-2-enamide

[0222] The compound of Example 9 was obtained by using 4-(4-methyl-lH-l,2,3-triazol-l- yl)benzoic acid instead of 4-acrylamidobenzoic acid in the preparation method of Example 4, and using the same raw materials as in Example 4.

[0223]

[0224] MS (ESI) m / z 540.2 (M+H) + .

[0225] 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.96 (d, J=9.9 Hz, 1 H), 9.03 - 9.16 (m, 1 H), 8.77 (d, J=16.5 Hz, 1 H), 8.58 (d, J=7.5 Hz, 1 H), 7.85 - 7.98 (m, 3 H), 7.69 (t, J=9.6 Hz, 2 H), 7.57 (d, J=8.4 Hz, 1 H), 7.07 - 7.41 (m, 4 H), 6.60 - 6.77 (m, 1 H), 4.19 - 4.45 (m, 1 H), 3.38 - 3.79 (m, 4 H), 2.32 (d, J=3.1 Hz, 3 H), 1.75 - 2.19 (m, 2 H).

[0226] Specific procedure ​

[0227] The compound of Example 10 was obtained by using 4-(lH-pyrazol-l-yl)benzoic acid instead of 4-acrylamidobenzoic acid in the preparation method of Example 4, and using the same raw materials as in Example 4.

[0228]

[0229] ​ ​

[0230] The title compound was prepared by following the procedure described in Example 4 using 4-(methylsulfonamido)benzoic acid instead of 4-acrylamidobenzoic acid and using the same starting materials as in Example 4.

[0231]

[0232] MS (ESI) m / z 552.2 (M+H) +

[0233] 1 HNMR (400 MHz, DMSO-d6) δ ppm 11.97 (d, J=8.11 Hz, 1 H), 9.94 - 10.12 (m, 1 H), 9.03 - 9.18 (m, 1 H), 8.78 (d, J=12.64 Hz, 1 H), 7.88 - 7.99 (m, 1 H), 7.53 - 7.62 (m, 1 H), 7.43 - 7.52 (m, 2 H), 7.06 - 7.41 (m, 6 H), 6.59 - 6.76 (m, 1 H), 4.17 - 4.44 (m, 1 H), 3.49 - 3.84 (m, 4 H), 3.03 (d, J=5.60 Hz, 3 H), 2.00 - 2.20 (m, 1 H), 1.75 - 1.98 (m, 1 H).

[0234] ​ ​

[0235] The title compound was prepared by following the procedure described in Example 4 using 4-(methylsulfonamido)benzoic acid instead of 4-acrylamidobenzoic acid and using the same starting materials as in Example 4.

[0236]

[0237] ​

[0238] Step 1: Preparation of (3R)-4-(lH-indol-3-yl)-6-((l-(4-(5-methyl-l,3,4-oxadiazol-2- yl)benzoyl)pyrrolidin-3-yl)amino)-l,7-naphthyridine-3-carbonitrile

[0239] To a solution of (R)-4-(lH-indol-3-yl)-6-(pyrrolidin-3-ylamino)-l,7- naphthyridine-3-carbonitrile (100 mg, 282 μmol) in DMF (1 mL) was added DIPEA (255 mg, 1.98 mmol), HOBt (45.7 mg, 338 μmol) and EDCI (64.9 mg, 338 μmol) at room temperature, and stirred for 2 min. Then 4-(5-methyl-l,3,4-oxadiazol-2-yl)benzoic acid (69.1 mg, 338 μmol) was added to the above reaction mixture, and the resulting mixture was stirred at room temperature for 12 h. After the reaction was completed, the reaction mixture was concentrated under reduced pressure. The resulting crude product was directly purified by high pressure preparative separation to give the compound of Example 12.

[0240]

[0241] MS (ESI) m / z 541.2 (M+H) +

[0242] 1 HNMR (400 MHz, DMSO-d6) δ ppm 11.97 (d, J=9.17 Hz, IH), 9.03 - 9.17 (m, IH), 8.78 (d, J=15.65 Hz, IH), 8.00 (t, J=8.68 Hz, 2H), 7.93 (d, J=18.58 Hz, IH), 7.64 - 7.73 (m, 2H), 7.53 - 7.61 (m, IH), 7.08 - 7.41 (m, 4H), 6.60 - 6.77 (m, IH), 4.12 - 4.49 (m, 2H), 3.40 - 3.80 (m, 3H), 2.59 (d, J=4.03 Hz, 3H), 2.04 - 2.22 (m, IH), 1.77 - 2.02 (m, IH).

[0243] ​ ​

[0244] The title compound was synthesized according to the following scheme:

[0245]

[0246] ​

[0247] Step 1: Preparation of (R,E)-N-(4-(3-((3-cyano-4-(lH-indol-3-yl)-l,7-naphthyridin-6- yl)amino)pyrrolidine-l-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide

[0248] (R)-4-(lH-indol-3-yl)-6-(pyrrolidin-3-ylamino)-l,7-naphthyridine-3-carbonitrile (45 mg, 0.13 mmol), (E)-4-(4-(dimethylamino)but-2-enamino)benzoic acid (38.7 mg, 0.156 mmol), DIPEA (50.3 mg, 0.39 mmol) were dissolved in DCM (10 mL), HATU (54.3 mg, 0.143 mmol) was added portionwise, after the addition was completed, the system was reacted at room temperature for 2 h. After the reaction was completed, the reaction liquid was concentrated under reduced pressure, and the obtained crude product was directly purified by high pressure preparative separation to obtain the compound of Example 13.

[0249]

[0250] MS (ESI) m / z 585.3 (M+H) +

[0251] 1 HNMR (400 MHz, DMSO-d6) δ 11.97 (d, J = 9.8 Hz, 1H), 10.22 (s, 1H), 9.11 (d, J = 32.8 Hz, 1H), 8.78 (d, J = 14.4 Hz, 1H), 7.93 (d, J = 17.0 Hz, 1H), 7.69 (d, J = 8.2 Hz, 2H), 7.63 - 7.54 (m, 1H), 7.48 (t, J = 9.7 Hz, 2H), 7.35 (dd, J = 12.6, 6.9 Hz, 2H), 7.24 (q, J = 13.2, 10.5 Hz, 1H), 7.14 (q, J = 10.0, 7.1 Hz, 1H), 6.75 (d, J = 15.0 Hz, 2H), 6.32 - 6.23 (m, 1H), 4.34 (d, J = 27.9 Hz, 2H), 3.73 (s, 1H), 3.61 (d, J = 8.7 Hz, 1H), 3.50 (s, 1H), 3.39 (d, J = 12.4 Hz, 1H), 3.06 (d, J = 5.7 Hz, 2H), 2.18 (s, 6H), 1.87 (s, 1H).

[0252] Example 14: Preparation of (3R)-6-((l-(4-chlorobenzoyl)pyrrolidin-3-yl)amino)-4- (lH-indol-3-yl)-l,7-naphthyridine-3-carbonitrile Preparation of 7-naphthyridine-3-carbonitrile

[0253] The title compound was synthesized according to the following route:

[0254]

[0255] Specific procedure

[0256] Step 1: Preparation of (R,E)-N-(4-(3-((3-cyano-4-(lH-indol-3-yl)-l,7- naphthyridin-6-yl)amino)pyrrolidine-l-carbonyl)phenyl)-4- (dimethylamino)but-2-enamide

[0257] (R)-4-(lH-indol-3-yl)-6-(pyrrolidin-3-ylamino)-l,7-naphthyridine-3- carbonitrile (100 mg, 256 μmol) was dissolved in DCM (10 mL), DIPEA (99.2 mg, 767 μmol) was added to the reaction, the system was cooled to 0 °C, 4-chlorobenzoyl chloride (35.8 mg, 204.7 μmol) was slowly added dropwise to the reaction, after the dropwise addition was completed, the system was reacted at 0 °C for 1 h. After the reaction was completed, saturated aqueous ammonium chloride solution was added to quench the reaction, ethyl acetate was added for extraction, the organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, the obtained crude product was directly purified by high pressure preparation separation to obtain the compound of Example 14.

[0258]

[0259] MS (ESI) m / z 493.1 (M+H) +

[0260] 1 H NMR (400 MHz, DMSO-d6) δ = 11.36 - 12.29 (m, 1H), 9.01 - 9.20 (m, 1H), 8.78 (d, J = 11.25 Hz, 1H), 7.93 (d, J = 15.16 Hz, 1H), 7.43 - 7.63 (m, 5H), 7.18 - 7.40 (m, 3H), 6.85 - 7.15 (m, 1H), 6.57 - 6.74 (m, 1H), 4.19 - 4.44 (m, 1H), 3.58 - 3.78 (m, 1H), 3.45 - 3.57 (m, 1H), 3.40 (d, J = 8.56 Hz, 1H), 3.25 (d, J = 10.15 Hz, 1H), 2.11 (s, 1H), 1.78 - 1.99 (m, 1H).

[0261] Example 15: Preparation of (3R)-4-(lH-indol-3-yl)-6-((l-(isothiazole-5-carbonyl)pyrrolidin- 3-yl)amino)-l,7-naphthyridine-3-carbonitrile Specific procedure

[0262] The compound of Example 15 was obtained according to the preparation method in Example 14, using 4-(methylsulfonamido)benzoic acid instead of 4-chlorobenzoyl chloride, and the other raw materials being the same as in Example 14.

[0263]

[0264] MS (ESI) m / z 450.2 (M+H)+

[0265] 1 H NMR (400 MHz, DMSO-d6) d = 12.01 (s, 1H), 9.13 (d, J = 9.38 Hz, 1H), 8.79 (d, J = 4.38 Hz, 1H), 8.74 (d, J = 1.75 Hz, 1H), 7.91-7.96 (m, 1H), 7.57 (d, J = 8.13 Hz, 1H), 7.30-7.41 (m, 2H), 7.20-7.27 (m, 1H), 7.11 (dd, J = 12.94, 8.32 Hz, 1H), 6.98-7.03 (m, 1H), 6.67-6.73 (m, 1H), 4.40 (s, 2H), 3.98-3.97 (d, J = 13.38 Hz, 1H), 3.81 (s, 1H), 3.61-3.59 (m, 1H), 1.86-2.01 (m, 1H), 1.95 (s, 1H).

[0266] Example 16: Preparation of (3R)-l-acryloylpyrrolidin-3-yl-3-((3-cyano-4-(lH-indol-3-yl)- 1,7-naphthyridin-6-yl)amino)pyrrolidine-1-carboxylate Specific procedure

[0267] The title compound was synthesized according to the following route:

[0268]

[0269] Example 17: Preparation of N-(4-(((3R)-3-((3-cyano-4-(lH-indol-3-yl)-l,7-naphthyridin-6- yl)amino)pyrrolidin-l-yl)methyl)phenyl)-N-methylacrylamide

[0270] Step 1: Preparation of tert-butyl 3-(((4-nitrophenoxy)carbonyl)oxy)pyrrolidine-1- carboxylate

[0271] Tert-butyl 3-hydroxypyrrolidine-1-carboxylate (3.00 g, 16.0 mmol) and DIPEA (3.11 g, 24.0 mmol, 4.19 mL) were dissolved in THF (30.0 mL), and 4-nitrophenyl chloroformate (3.23 g, 16.0 mmol) was added to the above reaction system at 0 °C, and then reacted at room temperature for 2 h. After the reaction was completed, the reaction liquid was directly used for the next step reaction.

[0272]

[0273] Step 2: Preparation of (3R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl-3-((3-cyano-4-(1H- indol-3-yl)-1,7-naphthyridin-6-yl)amino)pyrrolidine-1-carboxylate

[0274] To a solution of 4-(lH-indol-3-yl)-6-((R)-pyrrolidin-3-ylamino)-l,7- naphthyridine-3-carbonitrile (400 mg, 1.13 mmol) and DIPEA (291 mg, 2.26 mmol, 393 μί) in THF (4.00 mL) was added tert-butyl-3-(((4-nitrophenoxy)carbonyl)oxy)pyrrolidine- 1-carboxylate (795 mg, 2.26 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 12 h.

[0275] After the reaction was completed, the reaction mixture was concentrated, and the crude product was separated by column chromatography to obtain compound 16B.

[0276]

[0277] MS (ESI) m / z 568.3 (M+H) +

[0278] 1 HNMR (400 MHz, DMSO-d6) δ ppm 12.02 (s, 1H), 9.13-9.24 (m, 1H), 8.78-8.94 (m, 1H), 7.97 (s, 1H), 7.62 (d, J=8.3 Hz, 1H), 7.27-7.43 (m, 3H), 7.16 (t, J=6.8 Hz, 1H), 6.67-6.77 (m, 1H), 5.09 (s, 1H), 4.31 (s, 1H), 3.11-3.43 (m, 8H), 1.29-1.57 (m, 12H).

[0279] Step 3: Preparation of (3R)-pyrrolidin-3-yl-3-((3-cyano-4-(lH-indol-3-yl)-l,7- naphthyridin-6-yl)amino)pyrrolidine-1-carboxylate

[0280] To a solution of (3R)-l-(tert-butoxycarbonyl)pyrrolidin-3-yl-3-((3-cyano-4-(lH- indol-3-yl)-l,7-naphthyridin-6-yl)amino)pyrrolidine-l-carboxylate (260 mg, 458 μmol) in DCM (2.34 mL) was added TFA (0.26 mL) at room temperature. After the addition was completed, the reaction mixture was stirred at room temperature for 5 h. After the reaction was completed, the reaction mixture was concentrated to obtain compound 16C.

[0281]

[0282] MS (ESI) m / z 468.3 (M+H) +

[0283] 1HNMR (400 MHz, DMSO-d6) δ ppm 12.55-14.35 (m, 3H), 11.95 (s, 1H), 8.90-9.10 (m, 2H), 8.72 (s, 1H), 7.86 (d, J=2.2 Hz, 1H), 7.52 (d, J=8.2 Hz, 1H), 7.01-7.30 (m, 3H), 6.64 (d, J=6.2 Hz, 1H), 5.10 (s, 1H), 4.11-4.41 (m, 1H), 3.29-3.62 (m, 3H), 3.04-3.23 (m, 3H), 1.65-2.18 (m, 4H).

[0284] Step 4: Preparation of (3R)-1-acryloylpyrrolidin-3-yl-3-((3-cyano-4-(1H-indol-3- yl)-1,7-naphthyridin-6-yl)amino)pyrrolidine-1-carboxylate

[0285] (3R)-Pyrrolidin-3-yl-3-((3-cyano-4-(1H-indol-3-yl)-1,7-naphthyridin-6-yl)amino)pyrrolidine- 1-carboxylate (214 mg, 457 μmol) and TEA (185 mg, 1.83 mmol, 254.84 μL) were dissolved in DCM (2.14 mL), acryloyl chloride (41.4 mg, 457 μmol, 37.32 μL) was slowly added into the reaction system at 0 °C, the reaction was carried out at 0 °C for 2 h. After the reaction was completed, the reaction solution was concentrated under vacuum, and the obtained crude product was directly purified by high pressure preparation separation to obtain the compound of Example 16.

[0286]

[0287] MS (ESI) m / z 522.2 (M+H) +

[0288] 1 HNMR (400 MHz, DMSO-d6) δ ppm 11.96 (s, 1H), 9.10 (s, 1H), 8.77 (s, 1H), 7.92 (s, 1H), 7.56 (d, J=8.2 Hz, 1H), 7.19-7.36 (m, 3H), 7.10 (s, 1H), 6.44-6.70 (m, 2H), 6.03-6.16 (m, 1H), 5.55-5.70 (m, 1H), 5.03-5.18 (m, 1H), 4.24 (s, 1H), 3.33-3.81 (m, 7H), 3.16 (d, J=14.8 Hz, 1H), 1.67-2.21 (m, 4H).

[0289] Specific procedure Example 18: Preparation of 4-(lH-indol-3-yl)-6-(((R)-l-((2-methyl-lH-benzo[d]imidazol-5- yl)methyl)pyrrolidin-3-yl)amino)-l,7-naphthyridine-3-carbonitrile

[0290] The title compound was synthesized according to the following route:

[0291]

[0292] Specific procedure

[0293] Step 1: Preparation of N-(4-(((3R)-3-((3-cyano-4-(lH-indol-3-yl)-l,7- naphthyridin-6-yl)amino)pyrrolidin-l-yl)methyl)phenyl)-N-methylacrylamide

[0294] N-(4-(((3R)-3-((3-cyano-4-(lH-indol-3-yl)-l,7-naphthyridin-6-yl)amino)pyrrolidin-l- yl)methyl)phenyl)-N-methylacrylamide (60.0 mg, 120 μmol) was dissolved in DCM (3 mL), then acetic acid (101.6 μg, 1.69 μmol) was added, the system was stirred at room temperature for 1 h, then sodium borohydride (71.7 mg, 339 μmol) was added, and the stirring was continued at room temperature for 16 h. After the reaction was completed, water was added to quench the reaction, and ethyl acetate was added for extraction. The organic phase was washed with saturated brine once, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was directly purified by high pressure preparative separation to obtain the compound of Example 17.

[0295]

[0296] MS (ESI) m / z 528.2 (M+H) +

[0297] 1 HNMR (400 MHz, DMSO-d6) δ = 11.96 (s, 1H), 9.07 (s, 1H), 8.75 (s, 1H), 7.93 (s, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.40-7.28 (m, 3H), 7.27-7.15 (m, 4H), 7.14-6.94 (m, 1H), 6.68-6.55 (m, 1H), 6.18-6.09 (m, 1H), 6.02 (s, 1H), 5.53 (d, J = 11.7 Hz, 1H), 4.19-3.98 (m, 1H), 3.64-3.47 (m, 2H), 3.22 (s, 3H), 2.80-2.55 (m, 2H), 2.46-2.34 (m, 2H), 2.07 (s, 1H), 1.64 (m, 1H).

[0298] Example 19: Preparation of 4-(lH-indol-3-yl)-6-(((R)-l-(4-morpholinobenzyl)pyrrolidin-3- yl)amino)-l,7-naphthyridine-3-carbonitrile Specific procedure

[0299] The compound of Example 18 was obtained by using 2-methyl-lH-benzimidazole-5- carboxaldehyde instead of N-(4-formylphenyl)-N-methylacrylamide in the preparation method of Example 17, and using the same starting materials as in Example 17.

[0300]

[0301] MS (ESI) m / z 499.2 (M+H) +

[0302] 1 HNMR (400 MHz, DMSO-d6) δ ppm 11.98 (d, J=9.76 Hz, IH), 9.06 (s, IH), 8.74 (s, IH), 7.92 (s, IH), 7.57 (dd, J=6.69, 5.32 Hz, IH), 7.07 - 7.40 (m, 6H), 7.01 (d, J=8.13 Hz, 2H), 6.57 - 6.66 (m, IH), 3.96 - 4.18 (m, IH), 3.49 - 3.69 (m, 4H), 2.72 - 2.80 (m, IH), 2.63 - 2.84 (m, IH), 2.45 (s, 3H), 2.00 - 2.12 (m, IH), 1.54 - 1.70 (m, IH).

[0303] Example 20: Preparation of 4-(lH-indol-3-yl)-6-(((R)-l-(4-(4-methyl-lH-l,2,3-triazol-l- yl)benzyl)pyrrolidin-3-yl)amino)-l,7-naphthyridine-3-carbonitrile Specific procedure

[0304] The compound of Example 19 was obtained by using 4-morpholinobenzaldehyde instead of N-(4-formylphenyl)-N-methylacrylamide in the preparation method of Example 17, and using the same starting materials as in Example 17.

[0305]

[0306] MS (ESI) m / z 530.3 (M+H) +

[0307] 1H NMR (400 MHz, DMSO-d6) δ ppm 11.96 (s, 1 H), 9.06 (s, 1 H), 8.75 (s, 1 H), 7.92 (s, 1 H), 7.58 (d, J=8.19 Hz, 1 H), 7.20 - 7.37 (m, 2 H), 6.99 - 7.19 (m, 4 H), 6.86 (d, J=5.75 Hz, 2 H), 6.60 (d, J=15.53 Hz, 1 H), 3.97 - 4.20 (m, 1 H), 3.67 - 3.77 (m, 4 H), 3.37 - 3.50 (m, 3 H), 3.05 (d, J=4.16 Hz, 3 H), 2.54 - 2.77 (m, 2 H), 2.27 - 2.43 (m, 2 H), 1.98 - 2.12 (m, 1 H), 1.54 - 1.71 (m, 1 H).

[0308] Example 21: Preparation of 4-(lH-indol-3-yl)-6-(((R)-l-(4-morpholinobenzyl)pyrrolidin-3- yl)amino)-l,7-naphthyridine-3-carbonitrile Specific procedure

[0309] Referring to the preparation method in Example 17, the Example 20 compound was obtained by replacing N-(4-formylphenyl)-N-methylacrylamide with 4-(4-methyl-lH-l,2,3-triazol-l- yl)benzaldehyde, and using the same starting materials as in Example 17.

[0310]

[0311] MS (ESI) m / z 526.2 (M+H) +

[0312] 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.97 (s, 1 H), 9.08 (s, 1 H), 8.76 (s, 1 H), 8.50 (s, 1 H), 7.94 (s, 1 H), 7.78 (t, J=7.69 Hz, 2 H), 7.55 - 7.61 (m, 1 H), 7.46 (d, J=8.34 Hz, 2 H), 7.18 - 7.37 (m, 3 H), 6.98 - 7.16 (m, 1 H), 6.62 (d, J=19.43 Hz, 1 H), 4.03 - 4.21 (m, 1 H), 3.56 - 3.67 (m, 2 H), 2.68 (s, 1 H), 2.56 - 2.61 (m, 1 H), 2.37 - 2.46 (m, 2 H), 2.33 (s, 3 H), 2.04 - 2.15 (m, 1 H), 1.58 - 1.75 (m, 1 H).

[0313] Example 22: Preparation of 4-(lH-indol-3-yl)-6-(((R)-l-(4-((3-methylazetidin-3-yl)amino)benzyl) pyrrolidin-3-yl)amino)-l,7-naphthyridine-3-carbonitrile Specific procedure

[0314] The procedure described in Reference Example 17 was followed using 4-[(3- methylazetidin-3-yl)amino]benzaldehyde in place of N-(4-formylphenyl)-N- methylacrylamide and the other starting materials were the same as in Example 17 to give the compound of Example 22.

[0315]

[0316] MS (ESI) m / z 528.2 (M+H) +

[0317] 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.97 (s, 1H), 9.06 (s, 1H), 8.75 (s, 1H), 7.92 (dd, J=4.30, 3.20 Hz, 1H), 7.52 - 7.59 (m, 3H), 6.99 - 7.37 (m, 6H), 6.56 - 6.65 (m, 1H), 3.98 - 4.17 (m, 1H), 3.80 (t, J=6.73 Hz, 2H), 3.43 - 3.56 (m, 2H), 2.53 - 2.75 (m, 2H), 2.44 - 2.47 (m, 2H), 2.29 - 2.41 (m, 2H), 1.99 - 2.09 (m, 3H), 1.54 - 1.70 (m, 1H).

[0318] Example 23: Preparation of N-(4-(((3R)-3-((3-cyano-4-(lH-indol-3-yl)-l,7-naphthyridin-6- yl)amino)pyrrolidin-l-yl)methyl)phenyl)-N-methylacrylamide Specific procedure

[0319] The procedure described in Reference Example 17 was followed using 4-[(3- methylazetidin-3-yl)amino]benzaldehyde in place of N-(4-formylphenyl)-N- methylacrylamide and the other starting materials were the same as in Example 17 to give the compound of Example 22.

[0320]

[0321] MS (ESI) m / z 530.3 (M+H) +

[0322] 1H NMR (400 MHz, DMSO-d6) δ 11.96 (s, 1H), 9.07 (s, 1H), 8.75 (s, 1H), 7.93 (s, 1H), 7.58 (d, J = 8.2 Hz, 1H), 7.20 - 7.37 (m, 2H), 7.04 (d, J = 7.0 Hz, 2H), 6.96 (d, J = 8.2 Hz, 2H), 6.55 - 6.64 (m, 1H), 6.29 (d, J = 7.0 Hz, 2H), 5.95 (s, 1H), 4.58 (d, J = 5.62 Hz, 2H), 4.43 (d, J = 5.62 Hz, 2H), 3.95 - 4.16 (m, 2H) 2.32 ~ 2.40 (m, 4H), 2.02 ~ 2.04 (m, 1H), 1.59 (m, 2H), 1.52 (s, 3H)

[0323] Example 24: Preparation of 6-(((S)-l-(4-(lH-imidazol-2-yl)benzoyl)piperidin-3-yl)amino)-4- (lH-indol-3-yl)-l,7-naphthyridine-3-carbonitrile Specific procedure

[0324] The title compound was synthesized according to the following route:

[0325]

[0326] Example 25: Preparation of 4-acryloylamido-N-((lS,3R)-3-((3-cyano-4-(lH-indol-3-yl)- 1,7-naphthyridin-6-yl)amino)cyclopentyl)benzamide

[0327] Step 1: Preparation of 4-(lH-indol-3-yl)-6-(((R)-l-((5-nitropyridin-2-yl)methyl)pyrrolidin-3-yl)amino)-l,7-naphthyridine-3-carbonitrile

[0328] The title compound was synthesized according to the following route:

[0329]

[0330] MS (ESI) m / z 491.2 (M+H) +

[0331] Step 2: Preparation of 6-(((R)-l-((5-amino pyridin-2-yl)methyl)pyrrolidin-3-yl)amino)-4-(lH-indol-3-yl)-l,7-naphthyridine-3-carbonitrile

[0332] Ammonium chloride (218 mg, 4.08 mmol) was slowly added to a solution of 4-(lH- indol-3-yl)-6-(((R)-l-((5-nitropyridin-2-yl)methyl)pyrrolidin-3-yl)amino)-l,7- naphthyridine-3-carbonitrile (200 mg, 407 μmol), zinc (266 mg, 4.08 mmol) in water (0.5 mL) and methanol (0.5 mL) at 0 °C and the reaction was heated to 65 °C for 12 h. After the reaction was complete, the reaction was filtered through celite and the filtrate was concentrated under vacuum. The crude product was purified by high pressure preparative separation to give the compound of Example 23.

[0333]

[0334] MS (ESI) m / z 461.2 (M+H) +

[0335] 1 HNMR (400 MHz, DMSO-d6) δ ppm 11.96 (s, 1H), 9.07 (s, 1H), 8.75 (s, 1H), 7.92 (s, 1H), 7.82 (s, 1H), 7.57 (d, J=7.70 Hz, 1H), 6.94 - 7.42 (m, 6H), 6.87 (s, 1H), 6.51 - 6.67 (m, 1H), 5.16 (s, 2H), 3.93 - 4.25 (m, 1H), 3.48 (s, 1H), 2.67 (s, 2H), 2.27 - 2.46 (m, 2H), 2.04 (s, 1H), 1.62 (s, 1H).

[0336] Specific procedure Example 26: Preparation of N-((lS,3R)-3-((3-cyano-4-(lH-indol-3-yl)-l,7-naphthyridin-6- yl)amino)cyclopentyl)-4-(lH-imidazol-2-yl)benzamide

[0337] The title compound was synthesized according to the route below

[0338]

[0339] ​

[0340] Step 1: Preparation of 6-(((S)-l-(4-(lH-imidazol-2-yl)benzoyl)piperidin-3-yl)amino)- 4-(lH-indol-3-yl)-l,7-naphthyridine-3-carbonitrile

[0341] Step 1 : Preparation of 6-(((S)-l-(4-(lH-imidazol-2-yl)benzoyl)piperidin-3-yl)amino)- 4-(lH-indol-3-yl)-l,7-naphthyridine-3-carbonitrile

[0342]

[0343] MS (ESI) m / z 539.2 (M+H) +

[0344] 1 H NMR (400 MHz, DMSO-d6) δ ppm 12.55 (s, 1H), 11.95 (s, 1H), 8.57 - 9.20 (m, 2H), 8.13 (s, 1H), 7.67 - 8.01 (m, 3H), 7.52 - 7.65 (m, 1H), 6.86 - 7.51 (m, 8H), 6.51 - 6.85 (m, 1H), 2.75 - 3.20 (m, 4H), 1.69 - 2.07 (m, 2H), 1.43 - 1.62 (m, 2H).

[0345] ​ ​

[0346] The title compound was synthesized according to the route below

[0347]

[0348] ​

[0349] Step 1 : Preparation of 6-(((S)-l-(4-(lH-imidazol-2-yl)benzoyl)piperidin-3-yl)amino)- 4-(lH-indol-3-yl)-l,7-naphthyridine-3-carbonitrile

[0350] Dissolve 6-(((1R,3S)-3-aminocyclopentyl)amino)-4-(1H-indol-3-yl)-1,7- naphthyridine-3-carbonitrile (190 mg, 469.26 μmol, HC1) in DCM (2 mL), then add DIPEA (363 mg, 2.82 mmol, 490 μL), 4-acryloylcarboxamidobenzoic acid (107 mg, 563 μmol) and tri-n-propyl phosphoric acid adite 50% ethyl acetate solution (447 mg, 703 μmol, 418 μL) in turn, after the addition, the reaction system is reacted at room temperature for 16 h. After the reaction is completed, the reaction liquid is concentrated, and the crude product is purified by high pressure preparation separation to obtain the compound of Example 25.

[0351]

[0352] MS (ESI) m / z 542.2 (M+H) +

[0353] 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.97 (d, J=1.55 Hz, 1 H), 10.35 (s, 1 H), 9.10 (s, 1 H), 8.76 (s, 1 H), 8.21 - 8.29 (m, 1 H), 7.95 (d, J=2.26 Hz, 1 H), 7.77 - 7.83 (m, 2 H), 7.69 - 7.75 (m, 2 H), 7.58 (d, J=8.23 Hz, 1 H), 7.35 (d, J=7.87 Hz, 1 H), 7.25 (t, J=7.45 Hz, 1 H), 7.17 (t, J=5.54 Hz, 1 H), 7.08 - 7.14 (m, 1 H), 6.59 (d, J=8.46 Hz, 1 H), 6.39 - 6.53 (m, 1 H), 6.23 - 6.33 (m, 1 H), 5.75 - 5.82 (m, 1 H), 4.15 - 4.28 (m, 1 H), 3.93 (d, J=3.81 Hz, 1 H), 2.24 - 2.37 (m, 1 H), 1.77 - 1.97 (m, 2 H), 1.64 (d, J=4.41 Hz, 2 H), 1.42 - 1.55 (m, 1 H).

[0354] ​ ​

[0355] Referring to the preparation method in Example 25, 4-(1H-imidazol-2-yl)benzoic acid is used to replace 4-acryloylcarboxamidobenzoic acid, and other raw materials are the same as those in Example 25 to obtain the compound of Example 26.

[0356]

[0357] MS (ESI) m / z 539.2 (M+H) +

[0358] 1 H NMR (400 MHz, DMSO-d6) δ ppm 11.56 - 12.88 (m, 2 H), 9.10 (s, 1 H), 8.76 (s, 1 H), 8.35 (d, J=7.39 Hz, 1 H), 8.00 (s, 1 H), 7.96 (d, J=12.76 Hz, 2 H), 7.88 (d, J=8.34 Hz, 2 H), 7.59 (d, J=7.87 Hz, 1 H), 7.35 (d, J=7.99 Hz, 1 H), 7.08 - 7.27 (m, 5 H), 6.60 (s, 1 H), 4.15 - 4.34 (m, 1 H), 3.88 - 4.04 (m, 1 H), 2.29 - 2.38 (m, 1 H), 1.78 - 1.97 (m, 2 H), 1.59 - 1.75 (m, 2 H), 1.47 - 1.57 (m, 1 H).

[0359] Example 27: Preparation of 6-(((1R,3S)-3-((4-(1H-pyrazol-1-yl)benzyl)amino)cyclopentyl)amino)-4- (1H-indol-3-yl)-1,7-naphthyridine-3-carbonitrile

[0360] The title compound was synthesized according to the following scheme

[0361]

[0362] Specific procedure

[0363] Step 1: Preparation of 6-(((1R,3S)-3-((4-(1H-pyrazol-1-yl)benzyl)amino)cyclopentyl)amino)-4-(1H-indol-3-yl)-1,7-naphthyridine-3-carbonitrile

[0364] Dissolve 6-(((1R,3S)-3-aminocyclopentyl)amino)-4-(1H-indol-3-yl)-1,7-naphthyridine-3-carbonitrile (100 mg, 271 μmol) and 4-(1H-pyrazol-1-yl)benzaldehyde (56.1 mg, 325 μmol) in DCM (1 mL), then add TEA (13.7 mg, 135 μmol, 18.9 μL) and stir for 1 hour, then add sodium borohydride acetate (115 mg, 542 μmol) and acetic acid (163 μg, 2.71 μmol) to the mixture, and react at room temperature for 48 h. After the reaction is complete, concentrate the reaction solution, and purify the crude product by high-pressure preparative separation to obtain the compound of Example 27.

[0365]

[0366] MS (ESI) m / z 525.2 (M+H) +

[0367] 1 H NMR (400 MHz, DMSO-d6) δ 11.95 (s, 1H), 9.08 (s, 1H), 8.75 (s, 1H), 8.45 (d, J = 2.2 Hz, 1H), 7.94 (s, 1H), 7.72-7.79 (m, 2H), 7.71-7.78 (m, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.2 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.11 (s, 1H), 7.09 (s, 1H), 6.57 (s, 1H), 6.52-6.55 (m, 1H), 3.87 (s, 1H), 3.67 (s, 2H), 2.98 (d, J = 4.6 Hz, 1H), 2.07-2.18 (m, 1H), 1.72 (s, 2H), 1.47-1.63 (m, 2H), 1.31 (s, 2H).

[0368] Example 28: Preparation of 6-[[(3R)-1-[4-(1H-imidazol-2-yl)benzoyl]pyrrolidin-3-yl]amino]-4-(1H- pyrazol-4-yl)-1,7-naphthyridine-3-carbonitrile

[0369] The title compound was synthesized according to the following route

[0370]

[0371] Specific procedure

[0372] Step 1: Preparation of 6-fluoro-4-(lH-pyrazol-4-yl)-l,7-naphthyridine-3- carbonitrile

[0373] Under nitrogen protection, 4-chloro-6-fluoro-l,7-naphthyridine-3-carbonitrile (1.00 g, 4.82 mmol) and 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-lH-pyrazole (1.56 g, 5.30 mmol) were dissolved in dioxane (10 mL) and water (2 mL), then K2CO3 (1.33 g, 9.63 mmol) and Pd(dppf)Cl2 (352 mg, 481 μmol) were added, and the reaction system was reacted at 80 °C for 2 h. After the reaction was completed, water was added to dilute the reaction solution, and then extracted twice with ethyl acetate, and the combined organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by column chromatography to obtain compound 28A.

[0374]

[0375] MS (ESI) m / z 240.1 (M+H) +

[0376] Step 2: Preparation of (R)-tert-butyl 3-((3-cyano-4-(lH-pyrazol-4-yl)-l,7- naphthyridin-6-yl)amino)pyrrolidine- 1 -carboxylate

[0377] Dissolve 6-fluoro-4-(lH-pyrazol-4-yl)-l,7-naphthyridine-3-carbonitrile (200 mg, 836 μmol) and (R)-tert-butyl 3-aminopyrrolidine- 1 -carboxylate (311 mg, 1.67 mmol) in NMP (4 mL), the system is reacted at 130 °C for 16 h. After the reaction is complete, water is added to dilute the reaction, then extracted twice with ethyl acetate, the combined organic phase is washed once with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product is separated by column chromatography to obtain compound 28B.

[0378]

[0379] MS (ESI) m / z 406.2 (M+H) +

[0380] 1 HNMR (400 MHz, DMSO-d6) δ = 13.98 - 13.36 (m, 1H), 9.07 (s, 1H), 8.73 (s, 1H), 8.25 - 8.13 (m, 2H), 7.36 (d, J = 6.4 Hz, 1H), 6.87 (s, 1H), 4.40 (d, J = 13.2 Hz, 1H), 3.63 - 3.51 (m, 1H), 3.39 (d, J = 8.9 Hz, 2H), 3.16 - 3.08 (m, 1H), 2.14 - 2.05 (m, 1H), 1.79 (s, 1H), 1.41 - 1.37 (m, 9H)

[0381] Step 3: Preparation of 4-(lH-pyrazol-4-yl)-6-[[(3R)-pyrrolidin-3-yl]amino]- 1,7-naphthyridine-3-carbonitrile

[0382] Dissolve (R)-tert-butyl 3-((3-cyano-4-(lH-pyrazol-4-yl)-l,7-naphthyridin-6- yl)amino)pyrrolidine- 1 -carboxylate (100 mg, 246 μmol) in DCM (1 mL), add a solution of HC1 in 1,4-dioxane (4 M, 1 mL) to the above solution, and the system is reacted at room temperature for 8 h. After the reaction is complete, the reaction is concentrated under reduced pressure to obtain compound 28C.

[0383]

[0384] MS (ESI) m / z 306.1 (M+H) +

[0385] Step 4: Preparation of 6-[[(3R)-1-[4-(1H-imidazol-2-yl)benzoyl]pyrrolidin-3-yl]amino]-4-(1H-pyrazol-4-yl)-1,7-naphthyridine-3-carbonitrile

[0386] Dissolve 4-(1H-pyrazol-4-yl)-6-[[(3R)-pyrrolidin-3-yl]amino]-1,7-naphthyridine-3-carbonitrile (89.3 mg, 261 μmol) in THF (5 mL), add TEA (79.3 mg, 784 μmol), stir at 0 °C for 10 min, slowly drop 4-(1H-imidazol-2-yl)benzoyl chloride (54.0 mg, 261 μmol) into the mixture, and react at 0 °C for 4 h. After the reaction is completed, concentrate the reaction solution under reduced pressure, and purify the crude product by high-pressure preparative separation to obtain the compound of Example 28.

[0387]

[0388] MS (ESI) m / z 476.2 (M+H) +

[0389] 1 H NMR (400 MHz, DMSO-d6) δ 13.58 (s, 1H), 12.60 (s, 1H), 8.95-9.14 (m, 1H), 8.75 (d, J = 18.58 Hz, 1H), 7.85-8.48 (m, 3H), 7.51-7.66 (m, 2H), 7.42-7.45 (m, 1H), 7.38-7.50 (m, 1H), 7.36-7.51 (m, 1H), 7.20-7.33 (m, 1H), 7.06 (d, J = 6.0 Hz, 1H), 6.89 (d, J = 15.8 Hz, 1H), 4.37-4.57 (m, 1H), 3.84-3.80 (d, J = 13.4 Hz, 1H), 3.42-3.74 (m, 2H), 3.42-3.74 (m, 1H), 1.97 (s, 1H).

[0390] Example 29: Preparation of (S)-5-(1H-indol-3-yl)-3-(piperidin-3-ylamino)isoquinoline-6- carbonitrile

[0391] The synthesis route of the title compound is as follows

[0392]

[0393] Specific procedure

[0394] Step 1: Preparation of methyl 2-chloro-5-(4-methoxy-4-oxobut-1-en-1- yl)isonicotinate

[0395] Methyl 5-bromo-2-chloroisonicotinate (25.0 g, 99.8 mmol) was dissolved in 1,4-dioxane (250 mL) under nitrogen, then bis(triphenylphosphine)palladium (1.02 g, 2.00 mmol), methyl but-3-enoate (14.9 g, 149 mmol) and N,N-dicyclohexylmethanamine (42.8 g, 219 mmol) were added, after the addition was completed, the reaction system was reacted at 110 °C for 16 h. After the reaction was completed, the reaction system was reduced to room temperature, then filtered, the mother liquor was collected and concentrated, and the crude product was separated by column chromatography to obtain compound 29A.

[0396]

[0397] MS (ESI) m / z 270.0 (M+H) +

[0398] 1 H NMR (400 MHz, DMSO-d6) d ppm 8.74 (s, 1H), 7.72-7.84 (m, 1H), 6.93 (d, J=16.10 Hz, 1H), 6.44 (m, 1H), 3.86-3.88 (m, 3H), 3.62-3.65 (m, 3H), 3.35 (s, 2H).

[0399] Step 2: Preparation of methyl 3-chloro-5-hydroxyisoquinoline-6-carboxylate

[0400] Methyl 2-chloro-5-(4-methoxy-4-oxobut-1-en-1-yl)isonicotinate (28.0 g, 103 mmol) was dissolved in THF (260 mL), potassium tert-butoxide (16.3 g, 145 mmol) was added at 0 °C, then the system was reacted at room temperature for 2 h. After the reaction was completed, the reaction liquid was quenched with 20% aqueous citric acid solution, the PH was adjusted to 7.0, then extracted with ethyl acetate three times, the organic phase was combined, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was separated by column chromatography to obtain compound 29B.

[0401]

[0402] MS (ESI) m / z 238.0 (M+H) +

[0403] 1H NMR (400 MHz, DMSO-d6) δ ppm 11.65 (s, 1 H), 9.23 (s, 1 H), 8.09 (s, 1 H), 7.89 (d, J=8.82 Hz, 1 H), 7.66 (d, J=8.82 Hz, 1 H), 3.99 (s, 3 H).

[0404] Step 3: Preparation of 3-chloro-5-hydroxyisoquinoline-6-carboxamide

[0405] In a sealed tube, methyl 3-chloro-5-hydroxyisoquinoline-6-carboxylate (10.0 g, 42.0 mmol) was added to NH3 in MeOH (4 M, 100 mL) and heated to 80 °C for 16 h. After the reaction was complete, the system was cooled to room temperature and then the reaction was concentrated. The crude product was separated by column chromatography to give compound 29C.

[0406]

[0407] MS (ESI) m / z 223.0 (M+H) +

[0408] 1 H NMR (400 MHz, DMSO-d6) δ ppm 14.86-15.18 (m, 1 H), 9.15-9.25 (m, 1 H), 8.80 (s, 1 H), 8.33 (s, 1 H), 8.03-8.07 (m, 2 H), 7.58 (d, J=8.77 Hz, 1 H).

[0409] Step 4: Preparation of 3-chloro-5-hydroxyisoquinoline-6-carbonitrile

[0410] TEA (10.6 g, 105 mmol, 14.6 mL) was added dropwise to a mixture of 3-chloro-5-hydroxy-isoquinoline-6-carboxamide (7.8 g, 35.0 mmol) and tri-n-propyl phosphite 50% in ethyl acetate (55.7 g, 87.6 mmol, 50% purity) in dry THF (100 mL) at 0 °C. The reaction system was stirred at 0 °C for 30 min and then the system was allowed to warm to room temperature for 12 h. After the reaction was complete, the reaction was quenched with water and the aqueous phase was extracted with ethyl acetate three times. The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by column chromatography to give compound 29D.

[0411]

[0412] MS (ESI) m / z 205.0 (M+H) +

[0413] 1HNMR (400 MHz, DMSO-d6) δ ppm 12.15 (s, 1 H) 9.25 (s, 1 H) 8.28 (s, 1 H) 7.69-7.76 (m, 2 H).

[0414] Step 5: Preparation of (S)-tert-butyl 3-((6-cyano-5-hydroxyisoquinolin-3- yl)amino)piperidine-1-carboxylate

[0415] Under nitrogen protection, 3-chloro-5-hydroxyisoquinoline-6-carbonitrile (500 mg, 2.44 mmol) was dissolved in DMF (5 mL), then sodium tert-butoxide (939 mg, 9.77 mmol,), Brettphos Pd G3 (221 mg, 244.36 μmol) and (S)-tert-butyl 3-aminopiperidine-1-carboxylate (978 mg, 4.89 mmol) were added into the reaction solution, the system was reacted at 160 °C for 1 h. After the reaction was completed, the reaction was quenched with water, the aqueous phase was extracted with ethyl acetate three times, the combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was separated by column chromatography to obtain compound 29E.

[0416]

[0417] MS (ESI) m / z 369.2 (M+H) +

[0418] 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.51 (s, 1 H), 7.06 (s, 1 H), 6.77 (d, J=8.38 Hz, 1 H), 6.51 (d, J=7.94 Hz, 1 H), 6.13 (d, J=7.28 Hz, 1 H), 3.77-3.91 (m, 1 H), 3.57-3.67 (m, 2 H), 2.88 (s, 2 H), 1.88-1.97 (m, 2 H), 1.68-1.75 (m, 1 H), 1.30-1.42 (m, 11 H).

[0419] Step 6: Preparation of (S)-tert-butyl 3-((6-cyano-5-(((trifluoromethyl)sulfonyl)oxy)isoquinolin-3-yl)amino)piperidine-1-carboxylate

[0420] (S)-tert-buty\ 3-((6-cyano-5-hydroxyisoquinolin-3-yl)amino)piperidine-l- carboxylate (198 mg, 537 umol) was dissolved in DCM (1 mL), TEA (163 mg, 1.61 mmol) and l,l,l-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (230 mg, 644 μmol) were added to the above solution, the system was reacted at room temperature for 16 h. After the reaction was completed, the reaction was concentrated, and the crude product was separated by column chromatography to obtain compound 29F.

[0421]

[0422] MS (ESI) m / z 501.1 (M+H) +

[0423] 1 H NMR (400 MHz, DMSO-d6) δ ppm 9.19 (s, 1H), 8.16 (d, J=8.38 Hz, 1H), 7.55 (d, J=8.25 Hz, 1H), 7.05 (d, J=7.75 Hz, 1H), 6.68 (s, 1H), 3.64 - 3.83 (m, 2H), 3.17 (s, 1H), 1.99 (s, 2H), 1.75 - 1.86 (m, 1H), 1.52 - 1.61 (m, 1H), 1.40 - 1.46 (m, 2H), 1.23 - 1.38 (m, 9H).

[0424] Step 7: Preparation of (S)-tert-butyl 3-((6-cyano-5-(lH-indol-3-yl)isoquinolin-3- yl)amino)piperidine-l-carboxylate

[0425] (S)-tert-butyl 3-((6-cyano-5-(((trifluoromethyl)sulfonyl)oxy)isoquinolin-3- yl)amino)piperidine-l-carboxylate (84.3 mg, 168 μmol) was dissolved in 1,4- dioxane (1 mL) under nitrogen protection, K2CO3(42.3 mg, 306 μmol), (l-(tert- butoxycarbonyl)-lH-indol-3-yl)boronic acid (40.0 mg, 153 μmol), l,3-bis(2,6- diisopropylphenyl)imidazol-2-ylidene(3-chloropyridine)palladium(II) dichloride (10.4 mg, 15.3 μmol) were added to the above solution, and then the system was raised to 100 °C for 16 h. After the reaction was completed, the reaction was filtered, the filter cake was washed with ethyl acetate 3 times, the filtrate was concentrated, and the crude product was separated by column chromatography to obtain compound 29G.

[0426]

[0427] MS (ESI) m / z 468.2 (M+H)+

[0428] 1 H NMR (400 MHz, DMSO-d6) δ ppm 9.10 (s, 1 H), 8.19 (dd, J=8.29, 2.68 Hz, 1 H), 8.07 (d, J=8.46 Hz, 2 H), 7.79 (s, 2 H), 7.39 (s, 2 H), 7.37 (s, 2 H), 3.74-3.78 (m, 1 H), 3.60-3.69 (m, 2 H), 3.07-3.24 (m, 2 H), 2.52 (s, 2 H), 1.84 (d, J=5.60 Hz, 2 H), 1.68 (s, 1 H), 1.66 (s, 9 H).

[0429] Step 8: Preparation of (S)-5-(1H-indol-3-yl)-3-(piperidin-3-ylamino)isoquinoline-6- carbonitrile

[0430] (S)-3-((6-Cyano-5-(1H-indol-3-yl)isoquinolin-3-yl)amino)piperidine-1-carboxylic acid tert-butyl ester (50.0 mg, 88.0 μmol) was dissolved in DCM (0.9 mL), then TFA (0.1 mL) was added dropwise to the above solution, the system was reacted at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by high pressure preparation to obtain the compound of Example 29.

[0431]

[0432] MS (ESI) m / z 368.2 (M+H) +

[0433] 1H NMR (400 MHz, DMSO-d6) δ ppm 11.65 (s, 1 H), 9.02 (s, 1 H), 7.93 (d, J=8.58 Hz, 1 H), 7.70 (d, J=2.38 Hz, 1 H), 7.54 (d, J=8.11 Hz, 1 H), 7.40 (dd, J=8.52, 1.25 Hz, 1 H), 7.14 - 7.23 (m, 2 H), 7.00 - 7.06 (m, 1 H), 6.60 (dd, J=8.34, 2.86 Hz, 1 H), 6.49 (d, J=6.56 Hz, 1 H), 3.43 - 3.61 (m, 1 H), 2.90 - 2.99 (m, 1 H), 2.71 (dd, J=11.98, 3.76 Hz, 1 H), 2.35 - 2.39 (m, 1 H), 2.16 - 2.30 (m, 2 H), 1.78 - 1.87 (m, 1 H), 1.52 - 1.57 (m, 1 H), 1.28 - 1.34 (m, 1 H), 1.21 - 1.26 (m, 1 H).

[0434] Biological experiment

[0435] Test Example 1: In vitro CDK7 kinase inhibition activity test

[0436] Purpose of experiment

[0437] The method of Mobility-Shift Assay using microfluidic chip technology was used to test the kinase activity, with IC 50 value of compound as index, to evaluate the inhibition of compound on Cyclin-dependent kinase 7 (CDK7).

[0438] Materials of experiment

[0439] CDK7 / CycH / MAT1 (Carna, Cat No: 04-108), Kinase substrate CTD3 (GL, Cat No: 0346885), Caliper substrate C (GL, Cat No: 738989), DMSO (Sigma, Cat No: D8418-1L), 384-well plate (Corning, Cat No: 3573).

[0440] Method of experiment

[0441] The Mobility shift assay was used to detect the IC 50 value of test compound on CDK7 kinase. The concentration of compound tested was 10000 nM starting, 3-fold dilution, 10 concentrations, single hole detection.

[0442] Compound preparation

[0443] The test compound is dissolved in 100% DMSO, prepared into 10 mM stock solution, and stored in a nitrogen cabinet in the dark.

[0444] Kinase reaction process

[0445] (1) Prepare 1 x Kinase buffer.

[0446] (2) Preparation of compound concentration gradient: the initial concentration of the test compound is 10,000 nM, 3-fold dilution, 10 concentrations, single well. Dilute into 100% DMSO solution of 100-fold final concentration in a 384 source plate. Use liquid dispenser Echo 550 to transfer 250 nL of 100-fold final concentration of compound to the target plate 384 well plate.

[0447] (3) Prepare 2.5-fold final concentration of kinase solution with 1 x Kinase buffer.

[0448] (4) Add 10 μL of 2.5-fold final concentration of kinase solution to the compound well and the positive control well, respectively; add 10 μL of 1 x Kinase buffer to the negative control well.

[0449] (5) Centrifuge at 1000 rpm for 30 seconds, shake well after mixing the reaction plate, and incubate at room temperature for 10 minutes.

[0450] (6) Prepare a mixed solution of 5 / 3-fold final concentration of ATP and kinase substrate with 1 x Kinase buffer.

[0451] (7) Add 15 μL of 5 / 3-fold final concentration of ATP and substrate mixed solution to start the reaction.

[0452] (8) Centrifuge the 384 well plate at 1000 rpm for 30 seconds, shake well after mixing, and incubate at room temperature for the corresponding time.

[0453] (9) Add 30 μL of termination detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and shake well.

[0454] (10) Read the conversion rate with Caliper EZ enzyme marker (Caliper EZ Reader).

[0455] Data analysis

[0456] Calculation formula

[0457]

[0458] Where: %Inhibition represents the percentage inhibition rate; Conversion%_sample is the conversion rate reading of the sample; Conversion%_min: the mean value of the negative control wells, representing the conversion rate reading of the wells without enzyme activity; Conversion%_max: the mean value of the positive control wells, representing the conversion rate reading of the wells without compound inhibition.

[0459] Fitting dose-response curve

[0460] Using the concentration log value as the X-axis and the percentage inhibition rate as the Y-axis, the dose-response curve was fitted using the log(inhibitor) vs. response–variable slope function of the analysis software GraphPad Prism 5 to obtain the IC50 value of each compound on enzyme activity.

[0461] The calculation formula is Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)).

[0462] Experimental results

[0463] Table 1

[0464]

[0465]

[0466] Note: IC50 values ​​of 0-10 nM are marked as A; 10-100 nM as B; and 100-50000 nM as C.

[0467] Test Example 3: In vitro proliferation inhibition test

[0468] Experimental Objective

[0469] By detecting the viability of tumor cells after the action of the compound, using IC50... 50 The value was used as an indicator to evaluate the in vitro inhibitory effect of the compound on the proliferation of ovarian cancer cells A2780.

[0470] Experimental materials

[0471] A2780 (Nanjing Kebai, catalog number: CBP60224), CellCounting-Lite™ 2.0 (Novizan, catalog number: 0346885), DMSO (Xiaxia Reagent, catalog number: 20190701), RPMI 1640 (HyClone, catalog number: SH30809.01), Trypsin (HyClone, catalog number: J190002), Multifunctional Microplate Reader (BMG, model: FSX), CO2 incubator (Thermo Scientific, Model: RI-250).

[0472] Experimental method

[0473] (1) Take the logarithmic growth phase cells A2780, trypsin digestion, 1000 rpm centrifugation for 3 minutes, discard the supernatant, count after resuspension of the culture medium, inoculate 5*10 3 Each well in a 96-well plate, and incubate in a 37℃, 5% CO2 incubator for 24h.

[0474] (2) Dissolve the test compound in 100% DMSO to prepare a 10mM stock solution, store at 4℃ in the dark. Take the compound stock solution for 3-fold gradient dilution, take the diluted compound solution and add it to the inoculated 96-well plate, at this time the final drug concentration is 30000nM, 10000nM, 3333nM, 1111nM, 370nM, 123nM, 41nM, 14nM and 5nM, set up control wells and blank wells, and the DMSO content of all wells is 0.5%. Continue to incubate at 37℃, 5% CO2 incubator for 72h.

[0475] (4) Take out the test cell culture plate, equilibrate at room temperature for 30 minutes, add an equal volume of Cell Counting-LiteTM2.0 to the test cell culture, shake well to lyse the cells, and after 10 minutes of standing, use a multifunctional enzyme marker to detect the luminescence signal.

[0476] Data analysis

[0477] Calculation formula

[0478]

[0479] Wherein: Ls represents the luminescence value of the experimental well, Lb represents the blank luminescence value, and Lc represents the luminescence value of the control well.

[0480] Fitting dose-effect curve

[0481] Take the log value of the concentration as the X axis and the percentage inhibition rate as the Y axis, use the analysis software GraphPad Prism 5 to fit the dose-effect curve of log(inhibitor) vs. response-Variable slope, and thus obtain the IC50 value of each compound on tumor cells A2780.

[0482] The calculation formula is Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC50-X)*HillSlope))

[0483] Experimental results

[0484] Table 2

[0485] Example No. 72h IC 50 (nM) Example No. 72h IC 50 (nM) 2 215.3 3 1036 4 63.86 6 639 8 1561 9 326 10 389 11 5615 12 509 13 544.1 14 1010 15 1206 22 448 25 260

[0486] From the above data, it can be seen that the compounds of the present application have a proliferation inhibitory effect on tumor cells.

[0487] The above examples and test examples are illustrative examples for specifically setting forth the present application, and are not intended to limit the scope of protection of the present application. Any improvement, modification, equivalent structure or equivalent process transformation made by using the principles and contents of the present application, or directly or indirectly applied to other related technical fields, are also included in the scope of protection of the present application.

Claims

1. Compounds having the structure of formula (I): Or its pharmaceutically acceptable salt; wherein X 1 is CR 3 or N; X 2 is CR 3 ; R x selected from cyano; R 1 , R 2 , R 3 are each independently selected from the group consisting of: H; Ring B is a group selected from the following group: Each R 4 Independently selected from H; p = 0, 1, 2, 3 or 4; G is a C3-C7 cycloalkyl or a 3-7 membered heterocyclic alkyl having one or more substituents or no substituents; wherein the substituents of G are selected from one or more of the group consisting of amino, C1-C6 alkyl, and wherein the heterocyclic alkyl has one heteroatom selected from N; L is selected from: -(CH2) m -, -C(=O)-, -C(=O)O-, -NR c -C(=O)-, -NR c -(CH2) m -, absent; m = 1 ; each R c is independently selected from: H; D is selected from the group consisting of substituted or unsubstituted 3-6 membered heterocycloalkyl, C6-Ci0aryl, 5-14 membered heteroaryl; wherein the substituents of D are one or more of amino, halo, Ci-C4alkyl; wherein each of the heterocycloalkyl, aryl, and heteroaryl groups has 1-4 heteroatoms selected from N, O, or S; or is absent; 14 D is selected from the group consisting of substituted or unsubstituted 3-6 membered heterocycloalkyl, C6-Ci0aryl, 5-14 membered heteroaryl; wherein the substituents of D are one or more of amino, halo, Ci-C4alkyl; wherein each of the heterocycloalkyl, aryl, and heteroaryl groups has 1-4 heteroatoms selected from N, O, or S; or is absent; R y selected from halogen, amino, Ci-C6alkyl, -(CH2) i -N(R d )2, -(CH2) i -R e , -NR d -(CH2) i -R e , -NR d -S(=O)2R d , R d substituted or unsubstituted R e , is absent; Y is N; i = 0, 1, 2, 3 or 4; each R d , R 5A , R 5B is independently selected from the group consisting of H, amino, C1-C6 alkyl; R 5C , R 5D is independently selected from the group consisting of R d , -N(R d )2; Each R e Independently selected from one or more R d The following groups are substituted: 3-6 membered heterocyclic alkyl groups, 5-14 membered heteroaryl groups, wherein each of the heterocyclic alkyl groups and heteroaryl groups has 1-4 heteroatoms selected from N or O.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof; wherein... G is a C3-C6 cycloalkyl or a 3-6 membered heterocycloalkyl; L is selected from: -(CH2) m - C(=O)-, -C(=O)O-, -NR c - C(=O)-, -NR c -(CH2) m -; m = 1; D is selected from the group consisting of substituted or unsubstituted 5-6 membered heterocycloalkyl, C6-C10 aryl, 5-10 membered heteroaryl; wherein the substituents of said D are one or more of amino, halogen, C1-C4 alkyl; 10 aryl, 5-10 membered heteroaryl; wherein the substituents of said D are one or more of amino, halogen, C1-C4 alkyl; R y selected from halogen, amino, C1-C4alkyl, -(CH2) i -N(R d )2, -(CH2) i -R e , -NR d -(CH2) i -R e , -NR d -S(=O)2R d , R d substituted or unsubstituted R e 、 is absent; i = 0, 1, 2, 3 or 4; Each R d Independently selected from: H, amino, C1-C6 alkyl; each R 5A R 5B Each is independently selected from: H, amino; R 5C Selected from: R d -N(R) d )2; Each R e Independently selected from one or more R d The following groups are substituted: 3-6 membered heterocyclic alkyl groups, 5-10 membered heteroaryl groups.

3. The compound of claim 2 or a pharmaceutically acceptable salt thereof; wherein... G is wherein a is attached to NH and b is attached to L; W 1 , W 2 , W 3 each independently is N or CR 6 ; each R is independently selected from the group consisting of H; q = 0, 1, 2, or 3. 6 each R is independently selected from the group consisting of H; q = 0, 1, 4. The compound of claim 1 or pharmaceutically acceptable salt thereof; wherein D is substituted or unsubstituted C6-Ci0aryl or 5-10 membered heteroaryl. 10 aryl or 5-10 membered heteroaryl.

5. The compound of claim 4 or a pharmaceutically acceptable salt thereof; wherein D is selected from substituted or unsubstituted groups: The substituents thereon are one or more amino, halogen, or C1-C4 alkyl groups.

6. The compound of claim 5 or a pharmaceutically acceptable salt thereof; wherein D is selected from substituted or unsubstituted groups: wherein a is attached to L and b is attached to R y connected; The substituents thereon are one or more amino, halogen, or C1-C4 alkyl groups.

7. The compound of claim 1, or a pharmaceutically acceptable salt thereof; wherein R y is selected from the group consisting of halogen, amino, C1-C4 alkyl, nothing.

8. The compound of claim 1, or a pharmaceutically acceptable salt thereof; wherein R y is selected from -(CH2) i -N(R d )2, -NR d -S(=O)2R d , is absent; i = 0, 1, 2, 3 or 4; each R 5A , R 5B are each independently selected from the group consisting of H, amino; R 5C is selected from the group consisting of R d , -N(R d )2; each R d is independently selected from the group consisting of H, amino, C1-C4 alkyl.

9. The compound of claim 1 or a pharmaceutically acceptable salt thereof; wherein R y Selected from -(CH2) i -R e -NR d -(CH2) i -R e , via R d Replaced or unreplaced R e It does not exist; i = 0, 1, 2, 3 or 4; Each R d Independently selected from: H, amino, C1-C6 alkyl; Each R e Independently selected from one or more R d The following groups are substituted: 3-6 membered heterocyclic alkyl groups, 5-10 membered heteroaryl groups.

10. The compound of claim 9, or a pharmaceutically acceptable salt thereof; wherein each R e is independently selected from the group consisting of substituted or unsubstituted: d substituted or unsubstituted:

11. The compound of claim 10, or a pharmaceutically acceptable salt thereof; wherein each R e is independently selected from substituted or unsubstituted: d is independently selected from substituted or unsubstituted:

12. The compound of claim 9, or a pharmaceutically acceptable salt thereof; wherein each R e is independently selected from substituted or unsubstituted: d is independently selected from substituted or unsubstituted:

13. The compound of claim 12, or a pharmaceutically acceptable salt thereof; wherein each R e is independently selected from substituted or unsubstituted: d substituted or unsubstituted:

14. The compound of claim 1 or a pharmaceutically acceptable salt thereof; wherein... G is C3to C7cycloalkyl or 3 to 7 membered heterocycloalkyl having 1 to 3 substituents or no substitution, wherein the substituents of G are one or more selected from the group consisting of amino, C1to C4alkyl; and L, D and R y is absent.

15. A compound or a pharmaceutically acceptable salt thereof, wherein said compound is selected from the group consisting of:

16. Use of the compound of any one of claims 1-15 in the preparation of a medicament for treating or preventing diseases in response to inhibition of a cyclin-dependent kinase, wherein the cyclin-dependent kinase is selected from CDK7.

17. The use as claimed in claim 16, wherein the disease is a tumor.

18. The use as described in claim 17, wherein the tumor is selected from ovarian cancer.

19. The use as described in claim 17, wherein the tumor is selected from high-grade serous ovarian cancer.

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