WO2024239512A1 - Pharmaceutical combination and use thereof in treatment of cancer - Google Patents

Abstract

Provided in the present invention are a pharmaceutical composition or kit comprising a therapeutically effective amount of a CDK9 inhibitor and a therapeutically effective amount of at least one drug for treating cancers; the use thereof in the preparation of a drug or pharmaceutical combination or kit for treating cancers, and the combined use of a CDK9 inhibitor and one or more of a BTK inhibitor, a BCL-2 inhibitor, a DNA methyltransferase inhibitor. The composition has a better synergistic effect in cancer cells when used in combination.

Description

Drug combinations and their use in treating cancer Technical Field

The present invention relates to the field of medical technology, and in particular to a drug combination or drug product comprising a CDK9 inhibitor and use thereof in treating cancer, or a method for combining a CDK9 inhibitor with other drugs for treating cancer.

Background Art

The proliferation and division of eukaryotic cells is a precise and complex regulatory process. The proliferation process is completed through the cell cycle, and the orderly progress of the cell cycle is through its strict molecular regulatory mechanism. At present, it has been found that there are three main types of molecules involved in cell cycle regulation: cyclin-dependent kinases (CDK), cyclins, and cyclin-dependent kinase inhibitors (CKI), among which CDK is at the center. 13 members of the CDK family have been found (CDK1-CDK13). Studies have found that abnormal expression levels or (and) kinase activity of CDK9 can cause abnormal expression of multiple proteins in cells or (and) their mRNA levels. It has been reported that CDK9 pathway dysregulation exists in a variety of hematological malignancies. It can be said that CDK9 is one of the most critical molecules in the occurrence and development of tumors (Shapiro GI. J Clin Oncol, 2006, 24: 1770-83; Boffo S, Damato A, Alfano L, et al., Journal of Experimental & Clinical Cancer Research, 2018, 37(1): 36).

Cyclin-dependent kinase (CDK) inhibitors have been shown to be useful in the treatment of cancer. Although CDK inhibitors as monotherapy have efficacy in certain cancers, there is still a need to develop effective doses and dosing regimens for combining CDK inhibitors with other cancer therapeutics to treat or prevent diseases, disorders or conditions involving cyclin-dependent kinase (CDK) activity.

Summary of the invention

The present invention provides a pharmaceutical composition, a medicine kit or a medicine combination for cancer, and uses thereof. The medicine combination has a good synergistic effect when used in combination in cancer cells.

Pharmaceutical composition or kit or drug combination

In one aspect of the present invention, a pharmaceutical composition or a drug kit is provided, which comprises a therapeutically effective amount of a CDK9 inhibitor and a therapeutically effective amount of at least one drug for treating cancer, wherein the CDK9 inhibitor is a compound of formula (I), a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof; and the drug for treating cancer is one or more of a BCL-2 inhibitor, a BTK inhibitor and a DNA methyltransferase inhibitor.

In one embodiment, the pharmaceutically acceptable salts of the compound of formula (I) include maleate and/or fumarate salts.

In one embodiment, the BCL-2 inhibitor is selected from one or more of Navitoclax, Venetoclax (ABT-199), Pelcitoclax (APG-1252), A-1155463, A-1331852, ABT-737, Obatoclax, S44563, TW-37, AT101, HA14-1 and Sabutoclax.

In one embodiment, the BCL-2 inhibitor is Venetoclax (ABT-199).

In one embodiment, the BTK inhibitor is selected from one or more of ibrutinib, zanubrutinib, spebrutinib (AVL-292), olmutinib (HM-71224), acalabrutinib, CNX-774, CGI1746, LFM-A13, CNX-774, ONO-4059 and RN486.

In one embodiment, the BTK inhibitor is selected from one or both of ibrutinib and zanubrutinib.

In one embodiment, the DNA methyltransferase inhibitor is Azacitidine.

In one embodiment, the pharmaceutical composition or kit,

Comprising a compound of formula (I), a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof and Venetoclax; or,

Comprising a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, Venetoclax and Azacitidine; or,

Comprising a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof and ibrutinib; or,

It includes a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof and Zanubrutinib.

In one embodiment, the mass ratio of the CDK9 inhibitor to the drug for treating cancer is 0.001-1000.

In one embodiment, the mass ratio of the CDK9 inhibitor to the BCL-2 inhibitor is 0.001-1000, for example, 0.001-1000, 0.001-500, 0.004-250, 0.004-242.72, 0.01-100, 0.01-10, 0.01-1, 0.01-0.05 or 0.1-10.

In one embodiment, the pharmaceutical composition or kit comprises 1-1000 nM CDK9 inhibitor and 1-7000 nM BCL-2 inhibitor. The content of the CDK9 inhibitor can further be 4.115-1000 nM, 12.346-1000 nM or 37.037-1000 nM.

In one embodiment, the pharmaceutical composition or kit comprises 1-1000nM CDK9 inhibitor and 1-1000nM BCL-2 inhibitor.

In one embodiment, the pharmaceutical composition or kit comprises 4.12-1000 nM CDK9 inhibitor, and may further comprise 10-100 nM, 10-1000 nM, nM or 100-1000 nM, for example, it can be 4.12 nM, 12.35 nM, 37.04 nM, 111.11 nM, 333.33 nM or 1000 nM.

In one embodiment, the pharmaceutical composition or kit comprises 10 mg/kg of CDK9 inhibitor or a drug dose equivalent to 10 mg/kg of CDK9 inhibitor.

In one embodiment, the pharmaceutical composition or drug kit contains 4.12-1000nM BCL-2 inhibitor, and may further be 10-100nM, 10-1000nM or 100-1000nM, for example, 4.12nM, 12.35nM, 37.04nM, 111.11nM, 333.33nM or 1000nM.

In one embodiment, the pharmaceutical composition or kit comprises 100 mg/kg BCL-2 inhibitor or a drug dose equivalent to 100 mg/kg BCL-2 inhibitor.

In one embodiment, the pharmaceutical composition or kit comprises 1-1000nM maleate salt of the compound of formula (I) and 1-10000nM Venetoclax (ABT-199).

In one embodiment, the pharmaceutical composition or kit comprises 1-1000 nM maleate salt of the compound of formula (I) and 1-1000 nM Venetoclax (ABT-199).

In one embodiment, the pharmaceutical composition or kit comprises 4.12-1000 nM maleate salt of the compound of formula (I) and 4.12-7000 nM Venetoclax (ABT-199).

In one embodiment, the pharmaceutical composition or kit comprises 4.12-1000 nM maleate salt of the compound of formula (I) and 4.12-1000 nM Venetoclax (ABT-199).

In one embodiment, the pharmaceutical composition or kit comprises a drug dosage equivalent to 10 mg/kg maleate salt of the compound of formula (I) and 100 mg/kg Venetoclax (ABT-199).

In one embodiment, the pharmaceutical composition or kit comprises a maleate salt of the compound of formula (I) and Venetoclax (ABT-199) in a mass ratio of 0.004-250, for example, 0.004-242.72, 0.004-1, 0.01-1, 0.01-0.05, 0.01-0.03, 0.02-0.03, 0.02-0.035, 0.025-0.03 or 0.1.

In one embodiment, the pharmaceutical composition or kit comprises a maleate salt of the compound of formula (I) and Venetoclax (ABT-199) in a mass ratio of (4.12-1000):(4.12-1000), for example, 20:(200-1400), 20:(900-1400), 20:(200-700) or 20:(575-700).

In one embodiment, the pharmaceutical composition or kit comprises 1-1000nM CDK9 inhibitor and 1-3000nM BTK inhibitor.

In one embodiment, the pharmaceutical composition or kit comprises 1-1000nM CDK9 inhibitor and 1-1000nM BTK inhibitor.

In one embodiment, the pharmaceutical composition or drug kit contains 25-100nM CDK9 inhibitor, and may further be 25-50nM or 50-100nM, for example, 25nM, 50nM or 100nM.

In one embodiment, the pharmaceutical composition or drug kit contains 1-10nM BTK inhibitor, and may further be 2.5-10nM or 1-2.5nM, and may specifically be 1nM, 2.5nM or 10nM.

In one embodiment, the pharmaceutical composition or kit comprises 1-1000 nM maleate salt of the compound of formula (I) and 1-3000 nM Ibrutinib.

In one embodiment, the pharmaceutical composition or kit comprises 1-1000 nM maleate salt of the compound of formula (I) and 1-1000 nM Ibrutinib.

In one embodiment, the pharmaceutical composition or kit comprises 1-1000 nM maleate salt of the compound of formula (I) and 1-1000 nM Zanubrutinib.

In one embodiment, the pharmaceutical composition or kit comprises 25-100 nM maleate salt of the compound of formula (I) and 1-10 nM Ibrutinib.

In one embodiment, the pharmaceutical composition or kit comprises 50-100 nM maleate salt of the compound of formula (I) and 1-10 nM Ibrutinib.

In one embodiment, the pharmaceutical composition or kit comprises 25-100 nM maleate salt of the compound of formula (I) and 2.5-10 nM Ibrutinib.

In one embodiment, the pharmaceutical composition or kit comprises 50-100 nM maleate salt of the compound of formula (I) and 2.5-10 nM Ibrutinib.

In one embodiment, the pharmaceutical composition or kit comprises 100 nM maleate salt of the compound of formula (I) and 1-10 nM Ibrutinib.

In one embodiment, the pharmaceutical composition or kit comprises 25-100 nM maleate salt of the compound of formula (I) and 1-10 nM Zanubrutinib.

In one embodiment, the pharmaceutical composition or kit comprises 50-100 nM maleate salt of the compound of formula (I) and 1-10 nM Zanubrutinib.

In one embodiment, the pharmaceutical composition or kit comprises 25-100 nM maleate salt of the compound of formula (I) and 2.5-10 nM Zanubrutinib.

In one embodiment, the pharmaceutical composition or kit comprises 50-100 nM maleate salt of the compound of formula (I) and 2.5-10 nM Zanubrutinib.

In one embodiment, the pharmaceutical composition or kit comprises 100 nM maleate salt of the compound of formula (I) and 1-10 nM Zanubrutinib.

In one embodiment, the mass ratio of the CDK9 inhibitor to the BTK inhibitor is 0.001-1000, for example, it can be 0.001-1000, 0.001-500, 0.004-250, 0.01-100, 0.005-1, 0.01-10, 0.01-1, 0.1-10, 1-100, 2.5-100 or 0.1-2, and can further be 1, 2.5, 5, 10, 20, 25, 40, 50 or 100.

In one embodiment, the pharmaceutical composition or kit comprises a maleate salt of the compound of formula (I) and ibrutinib in a weight ratio of 1-100, further 2.5-100, for example, 1, 2.5, 5, 10, 20, 25, 40, 50 or 100.

In one embodiment, the pharmaceutical composition or kit comprises a maleate salt of the compound of formula (I) and Zanubrutinib in a mass ratio of 1-100, and further can be 2.5-100, for example, 1, 2.5, 5, 10, 20, 25, 40, 50 or 100.

In one embodiment, the pharmaceutical composition or kit comprises a maleate salt of the compound of formula (I) and ibrutinib in a mass ratio of 0.001- 1, and can further be 0.005-1, for example, can be 0.01-1, 0.01-0.08, 0.01-0.06 or 0.01-0.05.

In one embodiment, the pharmaceutical composition or kit comprises a maleate salt of the compound of formula (I) and Zanubrutinib in a mass ratio of 0.001-1, further 0.005-1, for example 0.01-1, 0.01-0.08, 0.01-0.06 or 0.01-0.05.

In one embodiment, the pharmaceutical composition or kit comprises 1-1000nM CDK9 inhibitor and 1-10000nM BCL-2 inhibitor and 0.1-50μM azacitidine.

In one embodiment, the pharmaceutical composition or kit comprises 1-1000nM CDK9 inhibitor and 1-1000nM BCL-2 inhibitor and 0.1-50μM azacitidine.

In one embodiment, the pharmaceutical composition or kit comprises 10-100 nM CDK9 inhibitor and 3-3000 nM BCL-2 inhibitor and 1.25-5 μM azacitidine.

In one embodiment, the pharmaceutical composition or kit comprises 10-100nM CDK9 inhibitor and 10-10000nM BCL-2 inhibitor and 2.5-10μM azacitidine.

In one embodiment, the pharmaceutical composition or kit comprises 10-100 nM CDK9 inhibitor and 10-10000 nM BCL-2 inhibitor and 12.5-10 μM azacitidine.

In one embodiment, the pharmaceutical composition or kit comprises 3-100 nM CDK9 inhibitor and 10-1000 nM BCL-2 inhibitor and 3.125-12.5 μM azacitidine.

In one embodiment, the pharmaceutical composition or kit comprises 3-10 nM CDK9 inhibitor and 10-1000 nM BCL-2 inhibitor and 3.125-12.5 μM azacitidine.

In one embodiment, the pharmaceutical composition or kit comprises the maleate salt of the compound of formula (I), Venetoclax (ABT-199) and azacitidine, and the mass ratio thereof is (0.004-250):1:(0.004-250), for example, it can be (0.004-242.72):1:(1-100), (0.004-1):1:(1-50), (0.01-1):1:(1-30), (0.01-0.05):1:(1-30), (0.01-0.03):1:(1-30), (0.02-0.03):1:(1-30), (0.02-0.035):1:(1-30), (0.025-0.03):1:(1-30) or (0.1):1:(1-30).

In one embodiment, the pharmaceutical composition or kit comprises the maleate salt of the compound of formula (I), Venetoclax (ABT-199) and azacitidine in a mass ratio of (1-100):(0.3-3000):(313-5000), (1-100):(0.3-3000):(313-5000), (3-1000):(1-10000):(625-10000) or (1-300):(1-10000):(3125-50000).

In one embodiment, the dosage of the CDK9 inhibitor is selected from 0.01-5000 mg/day, and the dosage of the drug for treating cancer is selected from 0.01-1000 mg/day.

In one embodiment, the pharmaceutical composition or kit is used to treat a CDK9-mediated disease or condition or cancer.

In another aspect of the present invention, there is provided use of the pharmaceutical composition or the pharmaceutical kit in the preparation of a drug for treating a disease or disease state mediated by CDK9.

Another aspect of the present invention provides a kind of:

A pharmaceutical combination of a compound of formula (I), a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof and Venetoclax; or,

A combination of a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, Venetoclax and Azacitidine; or,

A pharmaceutical combination of a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof and ibrutinib; or,

A pharmaceutical combination of a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof and zanubrutinib;

It is used to treat a disease or condition mediated by CDK9.

Purpose or method

In another aspect of the present invention, there is provided a use of a CDK9 inhibitor and a drug for treating cancer in the preparation of a drug or a drug kit or a drug combination for treating a disease or disease state mediated by CDK9, wherein the CDK9 inhibitor is a compound of formula (I), a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof; and the drug for treating cancer is one or more of a BCL-2 inhibitor, a BTK inhibitor and a DNA methyltransferase inhibitor.

The dosage of each inhibitor and the mass ratio of one or more of the CDK9 inhibitor and the BCL-2 inhibitor, the BTK inhibitor and the DNA methyltransferase inhibitor are partially consistent with the aforementioned drug combination.

In one embodiment, the pharmaceutically acceptable salts of the compound of formula (I) include maleate and/or fumarate salts.

In one embodiment, the BCL-2 inhibitor includes one or more selected from Navitoclax (ABT-263), Venetoclax (ABT-199), Pelcitoclax (APG-1252), A-1155463, A-1331852, ABT-737, Obatoclax, S44563, TW-37, AT101, HA14-1 and Sabutoclax.

In one embodiment, the BCL-2 inhibitor is Venetoclax (ABT-199).

In one embodiment, the BTK inhibitor includes one or more selected from ibrutinib, zanubrutinib, spebrutinib (AVL-292), olmutinib (HM-71224), acalabrutinib, CNX-774, CGI1746, LFM-A13, CNX-774, ONO-4059 and RN486.

In one embodiment, the BTK inhibitor is selected from Ibrutinib and Zanubrutinib.

In one embodiment, the DNA methyltransferase inhibitor is Azacitidine.

In one embodiment, the mass ratio of the CDK9 inhibitor to the BCL-2 inhibitor in the application is 0.001-1000, for example, 0.001-1000, 0.001-500, 0.004-250, 0.01-100 or 0.1-10.

In one embodiment, the pharmaceutical composition or drug kit contains 4.12-1000nM CDK9 inhibitor, and may further be 10-100nM, 10-1000nM or 100-1000nM, for example, 4.12nM, 12.35nM, 37.04nM, 111.11nM, 333.33nM or 1000nM.

In one embodiment, the CDK9 inhibitor in the application is a maleate salt of the compound of formula (I), and the BCL-2 inhibitor is Venetoclax (ABT-199), and their mass ratio is 0.004-250, for example, 0.004-242.72, 0.004-1, 0.01-1, 0.01-0.05, 0.01-0.03, 0.02-0.03, 0.02-0.035, 0.025-0.03 or 0.1.

In one embodiment, the CDK9 inhibitor in the application is a maleate salt of the compound of formula (I), and the BCL-2 inhibitor is Venetoclax (ABT-199), and the mass ratio thereof is (4.12-1000):(4.12-1000), for example, it can be 20:(200-1400), 20:(900-1400), 20:(200-700) or 20:(575-700).

In one embodiment, the mass ratio of the CDK9 inhibitor and the BTK inhibitor in the application is 0.001-1000, for example, it can be 0.001-1000, 0.001-500, 0.004-250, 0.01-100, 0.005-1, 0.01-10, 0.01-1, 0.1-10, 2.5-100 or 0.1-2, and can further be 1, 2.5, 5, 10, 20, 25, 40, 50 or 100.

In one embodiment, in the application, the CDK9 inhibitor is a maleate of the compound of formula (I), and the BTK inhibitor is ibrutinib, and the mass ratio thereof is 1-100, and can further be 2.5-100, for example, can be 1, 2.5, 5, 10, 20, 25, 40, 50 or 100.

In one embodiment, the application, the CDK9 inhibitor is a maleate of the compound of formula (I), and the BTK inhibitor is ibrutinib, and the mass ratio thereof is 0.001-1, and can further be 0.005-1, for example, can be 0.01-1, 0.01-0.08, 0.01-0.06, or 0.01-0.05.

In one embodiment, in the application, the CDK9 inhibitor is a maleate of the compound of formula (I), and the BTK inhibitor is Ibrutinib, and the mass ratio thereof is 1-100, and can further be 2.5-100, for example, can be 1, 2.5, 5, 10, 20, 25, 40, 50 or 100.

In one embodiment, in the application, the CDK9 inhibitor is a maleate of the compound of formula (I), and the BTK inhibitor is zanubrutinib, and the mass ratio thereof is 0.001-1, and may further be 0.005-1, for example, 0.01-1, 0.01-0.08, 0.01-0.06 or 0.01-0.05.

In one embodiment, a combination of a CDK9 inhibitor (e.g., a compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof), a BCL-2 inhibitor (e.g., Venetoclax), and a DNA methyltransferase inhibitor (e.g., azacitidine) is provided, wherein the mass ratio of the CDK9 inhibitor, the BCL-2 inhibitor, and the DNA methyltransferase inhibitor is (0.004-250):1:(0.004-250), for example, it can be (0.004-242.7 2):1:(1-100), (0.004-1):1:(1-50), (0.01-1):1:(1-30), (0.01-0.05):1:(1-30), (0.01-0.03):1:(1-30), (0.02-0.03):1:(1-30), (0.02-0.035):1:(1-30), (0.025-0.03):1:(1-30) or (0.1):1:(1-30).

In one embodiment, the mass ratio of CDK9 inhibitor, BCL-2 inhibitor and DNA methyltransferase inhibitor is (1-100):(0.3-3000):(313-5000), (1-100):(0.3-3000):(313-5000), (3-1000):(1-10000):(625-10000) or (1-300):(1-10000):(3125-50000).

The present invention also provides a dosage of a CDK9 inhibitor, which is selected from 0.01-5000 mg/day, preferably 1-1500 mg/day or 1-500 mg/day or 1-100 mg/day, and can be selected from 10 mg/day, 15 mg/day, 20 mg/day, 25 mg/day, 30 mg/day, 35 mg/day, 40 mg/day, 45 mg/day, 50 mg/day, 75 mg/day, 100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 300mg/day, 350mg/day, 400mg/day, 450mg/day, 500mg/day, 550mg/day, 600mg/day, 650mg/day, 700mg/day, 750mg/day, 800mg/day, 850mg/day, 900mg/day, 950mg/day, 1000mg/day, 1200mg/day, 1250mg/day, 1300mg/day, 1400mg/day, 1500mg/day. Or selected from 0.01-5000 mg/week, preferably 0.01-500 mg/week or 1-100 mg/week, optionally 10 mg/week, 15 mg/week, 20 mg/week, 25 mg/week, 30 mg/week, 35 mg/week, 40 mg/week, 45 mg/week, 50 mg/week, 75 mg/week, 100 mg/week, 150 mg/week, 200 mg/week, 250 mg/week, 300 mg/week, 350 mg/week 50mg/week, 400mg/week, 450mg/week, 500mg/week, 550mg/week, 600mg/week, 650mg/week, 700mg/week, 750mg/week, 800mg/week, 850mg/week, 900mg/week, 950mg/week, 1000mg/week, 1200mg/week, 1250mg/week, 1300mg/week, 1400mg/week, 1500mg/week.

The present invention also provides a dosage of the drug for treating cancer (one or more of the BCL-2 inhibitor, BTK inhibitor and DNA methyltransferase inhibitor), which is selected from 0.01-1000 mg/day, preferably 1-500 mg/day or 50-500 mg/day, and can be selected from 10 mg/day, 25 mg/day, 50 mg/day, 75 mg/day, 100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 300 mg/day, 320 mg/day, 350 mg/day, 400 mg/day, 420 mg/day, 450 mg/day, 500 mg/day, 550 mg/day, 600 mg/day, 650 mg/day, 700 mg/day, 750 mg/day, 800 mg/day, 850 mg/day, 900 mg/day, 950 mg/day, 1000 mg/day.

In one embodiment, the CDK9-mediated disease or condition is cancer.

Another aspect of the present invention provides a pharmaceutical composition or a kit, comprising a therapeutically effective amount of a compound of formula (I), a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of at least one drug for treating cancer, wherein the drug for treating cancer is selected from one or more of Venetoclax, Ibrutinib, Zanubrutinib and Azacitidine.

In one embodiment, the aforementioned CDK9-mediated disease, disease state or cancer is selected from one or both of solid tumors and hematological tumors.

In one embodiment, the CDK9-mediated disease or disease state or cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, prostate cancer, bladder cancer, liver cancer, skin cancer, glioma, breast cancer, melanoma, malignant glioma, rhabdomyosarcoma, ovarian cancer, astroglioma, Ewing's sarcoma, retinoblastoma, epithelial cell carcinoma, colon cancer, renal cancer, gastrointestinal stromal tumor, leukemia, lymphoma and nasopharyngeal carcinoma.

In one embodiment, the cancer is selected from a leukemia, such as acute promyelocytic leukemia, acute lymphoid leukemia, myelomonocytic leukemia, or acute myeloid leukemia.

In one embodiment, the cancer is selected from leukemia, for example, chronic lymphocytic leukemia, acute B-cell leukemia, acute megakaryocytic leukemia, acute promyelocytic leukemia, acute lymphocytic leukemia, myelomonocytic leukemia or acute myeloid leukemia.

In one embodiment, the cancer is selected from lymphoma. For example, histiocytic lymphoma, small lymphocytic lymphoma; peripheral T-cell lymphoma, including peripheral T-cell lymphoma-not otherwise specified, angioimmunoblastic T-cell lymphoma, extranodal NK/T-cell lymphoma, anaplastic large cell lymphoma, intranodal peripheral T-cell lymphoma with follicular helper T cell phenotype and T-prolymphocytic leukemia; B-cell lymphoma, including diffuse large B-cell lymphoma, mucosa-associated lymphoid tissue lymphoma, marginal zone lymphoma, mantle cell lymphoma and follicular lymphoma; and Hodgkin's lymphoma.

In one embodiment, the disease or condition is selected from MDS-RAEB (myelodysplastic syndrome-excessive blasts), histiocytic lymphoma, acute B-cell leukemia, acute megakaryocytic leukemia, acute myeloid leukemia, and acute promyelocytic leukemia.

Another aspect of the present invention provides a method for treating cancer, comprising administering a therapeutically effective amount of the above-mentioned CDK9 inhibitor and a therapeutically effective amount of the above-mentioned drug for treating cancer to a subject in need thereof, wherein the therapeutically effective amount of the CDK9 inhibitor and the therapeutically effective amount of the drug for treating cancer can be administered simultaneously, independently formulated and co-administered, or independently formulated and administered sequentially.

In one embodiment, the method for treating cancer further comprises administering to the subject other therapies selected from one or more of radiotherapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, and phototherapy. The other therapies may be in the form of adjuvant therapy or neoadjuvant therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a growth inhibition matrix diagram of the combined action of the maleate salt of the compound of formula (I) in Example 1 and Venetoclax on tumor cells HL-60.

FIG. 2 shows the average synergy value calculated in the Bliss statistical model in Example 1.

FIG. 3 shows the average synergy value calculated in the HSA statistical model in Example 1.

FIG. 4 shows the average synergy value calculated in the Loewe statistical model in Example 1.

FIG. 5 shows the average synergy value calculated in the ZIP statistical model in Example 1.

FIG6 is a growth inhibition matrix diagram of the combined action of the maleate salt of the compound of formula (I) and Venetoclax on tumor cells Kasumi-1 in Example 2.

FIG. 7 shows the average synergy value calculated in the Bliss statistical model in Example 2.

FIG. 8 shows the average synergy value calculated in the HSA statistical model in Example 2.

FIG. 9 shows the average synergy value calculated in the Loewe statistical model in Example 2.

FIG. 10 shows the average synergy value calculated in the ZIP statistical model in Example 2.

FIG. 11 is a growth inhibition matrix diagram of the combined action of the maleate salt of the compound of formula (I) and Venetoclax on tumor cells MV-4-11 in Example 3.

FIG. 12 shows the average synergy values calculated in the Bliss statistical model in Example 3.

FIG. 13 shows the average synergy values calculated in the HSA statistical model in Example 3.

FIG. 14 shows the average synergy values calculated in the Loewe statistical model in Example 3.

FIG. 15 shows the average synergy value calculated in the ZIP statistical model in Example 3.

Figure 16 is a growth inhibition matrix diagram of tumor cell MOLM-13 by the combination of maleate of the compound of formula (I) in Example 4 and Venetoclax.

FIG. 17 shows the average synergy values calculated in the Bliss statistical model in Example 4.

FIG. 18 shows the average synergy values calculated in the HSA statistical model in Example 4.

FIG. 19 shows the average synergy values calculated in the Loewe statistical model in Example 4.

FIG. 20 shows the average synergy value calculated in the ZIP statistical model in Example 4.

FIG. 21 shows the average synergy value calculated in the ZIP statistical model in Example 5.

FIG. 22 shows the average synergy values calculated in the HSA statistical model in Example 5.

FIG. 23 shows the average synergy values calculated in the Bliss statistical model in Example 5.

FIG. 24 shows the average synergy values calculated in the Loewe statistical model in Example 5.

FIG. 25 shows the average synergy value calculated in the ZIP statistical model in Example 6.

FIG. 26 shows the average synergy values calculated in the HSA statistical model in Example 6.

FIG. 27 shows the average synergy values calculated in the Bliss statistical model in Example 6.

FIG. 28 shows the average synergy values calculated in the Loewe statistical model in Example 6.

FIG. 29 shows the average synergy value calculated in the ZIP statistical model in Example 7.

FIG. 30 shows the average synergy values calculated in the HSA statistical model in Example 7.

FIG. 31 shows the average synergy values calculated in the Bliss statistical model in Example 7.

FIG. 32 shows the average synergy values calculated in the Loewe statistical model in Example 7.

FIG. 33 is the tumor growth curve of each group of tumor-bearing mice in Example 8 (mean ± standard error).

FIG. 34 is a tumor growth curve of a single tumor-bearing mouse in each group in Example 8.

FIG. 35 shows the relative animal body weight changes of tumor-bearing mice in Example 8 (mean ± standard error).

FIG. 36 shows the immunoblotting results of OCI-LY-10 cell viability-related proteins using the maleate salt of the compound of formula (I) in Example 9 in combination with Ibrutinib or Zanubrutinib.

Figure 37 shows the effects of the maleate salt of the compound of formula (I) in Example 9 in combination with Ibrutinib or Zanubrutinib on the expression of c-MYC, MCL-1, BFL-1 and BIM proteins in OCI-LY10 cells.

FIG38 shows the effect of the maleate salt of the compound of formula (I) in Example 9 in combination with Ibrutinib or Zanubrutinib on the expression of Cl-Caspase3 protein in OCI-LY10 cells.

FIG. 39 shows the results of the inhibition of OCI-LY-10 cell viability by the maleate salt of the compound of formula (I) in Example 10 in combination with Ibrutinib.

FIG40 shows the results of the inhibition of OCI-LY-10 cell viability by the maleate salt of the compound of formula (I) in Example 10 in combination with Zanubrutinib.

DETAILED DESCRIPTION

I. Definition and Explanation

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

It should be understood that in the present invention, when two or three drugs are used in combination, or in the pharmaceutical composition or kit, the mass ratio between the drugs includes the ratio calculated based on the drugs included therein. For example, in one embodiment, the pharmaceutical composition or kit includes 100nM maleate of the compound of formula (I) and 1-200nM Zanubrutinib. It can be understood that it also includes a pharmaceutical composition or kit in which the mass ratio of maleate of the compound of formula (I) to Zanubrutinib is 100:(1-200). Similarly, it also includes the use of a drug combination or combined medication in which the mass ratio of maleate of the compound of formula (I) to Zanubrutinib is 100:(1-200).

As used herein, the term "cancer treatment drug" refers to other cancer therapeutic agents other than the compound of formula (I) of the present invention, its stereoisomers, solvates or pharmaceutically acceptable salts thereof, preferably a drug for treating blood tumors, more preferably a drug for treating lymphoma or leukemia.

As used herein, the term "inhibit" is used relative to a control. One skilled in the art will readily determine the appropriate control for each experiment. For example, a reduced response in a subject or cell treated with a compound is compared to a response in a subject or cell not treated with the compound. The disclosure of all ranges in the present invention should be considered as disclosure of all subranges and all point values within the range. For example: the disclosure of 1-1000 should be considered as also disclosing ranges of 1-200, 200-300, etc., while also disclosing point values of 200, 300, 400, 500, 600, 700, 800, 900 and 1000.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The compounds may be present in the pharmaceutical composition as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" refers to salts of the compounds of the invention, prepared from compounds having specific substituents discovered by the invention with relatively nontoxic acids or bases. When the compounds of the invention contain relatively acidic functional groups, base addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the invention contain relatively basic functional groups, acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, fumaric acid and methanesulfonic acid, and also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups, and thus can be converted into any base or acid addition salt.

Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare.

In addition to the form of salt, the compound provided by the present invention also exists in the form of prodrugs. The prodrug of the compound described herein easily undergoes chemical changes under physiological conditions to be converted into the compound of the present invention. In addition, the prodrug can be converted to the compound of the present invention by chemical or biochemical methods in an in vivo environment. For example, when the prodrug is placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent, the prodrug can be slowly converted into a compound of the present invention.

Certain compounds of the invention may exist in unsolvated or solvated forms, including hydrated forms. Solvated forms are equivalent and are included within the scope of the present invention. Solvated forms are generally equivalent to unsolvated forms and are included within the scope of the present invention. Certain compounds of the present invention may exist in polymorphic or amorphous forms. Generally, for applications contemplated by the present invention, all physical forms are equivalent and are included within the scope of the present invention.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, atropisomers (or also referred to as rotational isomers), etc., and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention.

The compounds disclosed herein may exist as atropisomers, which are conformational stereoisomers that occur when rotation around a single bond in a molecule is prevented or greatly slowed due to steric interactions with other parts of the molecule. The compounds disclosed herein include all atropisomers as pure individual atropisomer preparations, enriched preparations of each, or non-specific mixtures of each. If the rotation barrier around a single bond is high enough and the interconversion between conformations is slow enough, separation and separation of isomeric species can be allowed. Separation and separation of isomeric species are appropriately represented by the well-known and widely accepted symbols "M" or "P". The term "cancer" refers to a disease characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread to other parts of the body locally or through the bloodstream and lymphatic system. Examples of various cancers are described herein, including but not limited to non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, prostate cancer, bladder cancer, liver cancer, skin cancer, glioma, breast cancer, melanoma, malignant glioma, rhabdomyosarcoma, ovarian cancer, astroglioma, Ewing's sarcoma, retinoblastoma, epithelial cell cancer, colon cancer, renal cancer, gastrointestinal stromal tumor, leukemia, lymphoma, and nasopharyngeal carcinoma, etc. The terms "tumor" and "cancer" are used interchangeably herein, for example, the two terms include solid and liquid, such as diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include precancerous lesions as well as malignant cancers and tumors.

As used herein, an "effective amount" or "therapeutically effective amount" includes an amount sufficient to improve or prevent the symptoms or symptoms of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

"Subject", "individual" or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, mice, apes, humans, farm animals, sport animals, and pets.

The amount of compound administered may depend on the subject being treated, the subject's age, health, sex, and weight, the type of concurrent treatment (if any), the severity of the condition, the nature of the desired effect, the manner and frequency of treatment, and the judgment of the prescribing physician. The frequency of administration may also depend on the pharmacodynamic effect on arterial oxygen tension. However, the most preferred dosage may be adjusted for individual subjects, as will be understood by those skilled in the art and can be determined without undue experimentation. This generally includes adjusting the standard dosage (e.g., if the patient is of low weight, then reducing the dosage).

The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or their salts and a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to facilitate the administration of the drug or drug combination of the present application to an organism. The pharmaceutical composition of the invention may further include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and/or adjuvants such as preservatives, wetting agents, emulsifiers and dispersants.

The "pharmaceutical composition" of the present invention can also be administered to patients or subjects in need of such treatment in any suitable manner, such as oral, parenteral, rectal, pulmonary or topical administration. When used for oral administration, the pharmaceutical composition can be prepared into oral preparations, such as oral solid preparations, such as tablets, capsules, pills, granules, etc.; or, oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When prepared into oral preparations, the pharmaceutical preparations may also contain suitable fillers, binders, disintegrants, lubricants, etc.

The term "pharmaceutically acceptable carrier" refers to those excipients that have no significant irritation to the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, liposomes, polymer micelles or inorganic nanocarriers.

The pharmaceutical composition of the present invention can be prepared into any pharmaceutically acceptable dosage form for oral, nasal, topical (including oral and sublingual), rectal, vaginal and/or parenteral administration, for example, it can be formulated into tablets, lozenges, capsules, pills, solutions, suspensions, syrups, injections, suppositories, inhalants or sprays.

The term "synergism" refers to a phenomenon in which two or more (eg, three) drugs used in combination act more effectively than their individual actions, as opposed to antagonism.

The term "treatment" includes prevention and treatment, for example, treatment of a CDK9-mediated disease includes prevention and/or treatment of a CDK9-mediated disease.

The "combined" administration method of the present invention is selected from simultaneous administration, independent formulation and co-administration or independent formulation and sequential administration.

In the present invention, the so-called "combination" or "combination" is a mode of administration, which includes various situations in which two or more drugs are administered successively or simultaneously. The so-called "simultaneous" here means that the CDK9 inhibitor and the cancer treatment drug (BCL-2 inhibitor, BTK inhibitor or DNA methyltransferase inhibitor) are administered in the same administration cycle, for example, the two drugs are administered within 2 days or 1 day. The so-called "sequential or sequential" administration includes the situation in which the CDK9 inhibitor and one or more of the cancer treatment drugs are administered separately in different administration cycles. These modes of administration all belong to the combined administration described in the present invention.

The BCL-2 inhibitor of the present invention includes stereoisomers, solvates or pharmaceutically acceptable salts thereof. For example, in one embodiment, the present The pharmaceutical composition of the invention comprises a compound of formula (I) and Venetoclax Also known as ABT199 or ABT-199, GDC0199, which means including the use of Venetoclax or a stereoisomer, solvate or a pharmaceutically acceptable salt thereof of the compound represented by formula (II).

The BTK inhibitor of the present invention includes itself, its stereoisomers, solvates or pharmaceutically acceptable salts thereof. For example, in one embodiment, the pharmaceutical composition of the present invention includes a compound of formula (I) and ibrutinib Zanubrutinib This means that it includes a stereoisomer, solvate or a pharmaceutically acceptable salt of the compound of formula (I) and ibrutinib or the compound of formula (IV), or a stereoisomer, solvate or a pharmaceutically acceptable salt of the compound of formula (I) and zabutinib or the compound of formula (III).

The DNA methyltransferase inhibitors of the present invention include the compounds thereof, their stereoisomers, solvates or pharmaceutically acceptable salts thereof. For example, Azacitidine includes compounds of formula (V): and its solvates or pharmaceutically acceptable salts.

II. Specific implementation methods

The CDK inhibitor disclosed in the present invention is an effective and selective CDK9 inhibitor, and its structural formula is shown in the following formula (I), and its name is 4-(((4-(5-chloro-2-(((1R,4r)-4-(((R)-1-methoxypropyl-2-yl)amino)cyclohexyl)amino)pyridin-4-yl)thiazol-2-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile. The preparation method of the compound of formula (I) and its pharmaceutically acceptable salt is published in international patent applications PCT/CN 2018/070108 and PCT/CN2020/094527.

One stereoisomer of the compound of formula (I) is 4-(((4-(5-chloro-2-(((1S,4r)-4-(((S)-1-methoxypropan-2-yl)amino)cyclohexyl)amino)pyridin-4-yl)thiazol-2-yl)amino)methyl)-tetrahydro-2H-pyran-4-carbonitrile.

Another stereoisomer of the compound of formula (I) is 4-(((4-(5-chloro-2-(((1R,4s)-4-(((S)-1-methoxypropan-2-yl)amino)cyclohexyl)amino)pyridin-4-yl)thiazol-2-yl)amino)methyl)-tetrahydro-2H-pyran-4-carbonitrile.

Another stereoisomer of the compound of formula (I) is 4-(((4-(5-chloro-2-(((1S,4s)-4-(((R)-1-methoxypropan-2-yl)amino)cyclohexyl)amino)pyridin-4-yl)thiazol-2-yl)amino)methyl)-tetrahydro-2H-pyran-4-carbonitrile.

Cell culture

Observe the cell growth status under an inverted microscope, select cells in the logarithmic growth phase, and perform aseptic operations in a biosafety cabinet. Transfer the cell suspension to a centrifuge tube, centrifuge the cells, discard the supernatant, resuspend the cells with fresh complete medium, and then disperse them into new culture bottles in appropriate proportions and culture them in a 37°C, 5% _CO2 incubator.

Calculation method of cell survival ratio in combination group

The calculation formula of cell survival rate (% cell survival rate) after the combination of the two drugs is: cell survival rate = (1-(DMSO control RLU-drug RLU)/(DMSO control RLU-blank control RLU)) × 100%. DMSO control is a solvent control without drug, and blank control is a culture medium control without drug and solvent.

Interpretation of cell survival ratio calculation results

In the cell survival ratio calculation result diagram (Figure 1, Figure 6), the abscissa is the concentration of the maleate salt of the compound of formula (I) of the present application (effective concentration of the drug in the well solution), and the ordinate is the concentration of the combined drug (effective concentration of the drug in the well solution). The abscissa and ordinate concentration scales are used as vertical points to draw vertical lines, and the value in the grid where the intersection of the two vertical lines is located is the cell survival ratio of the drug combination group using the two concentrations. For example, in Figure 1, the abscissa reading is 37.04nM, the ordinate reading is 0nM, and the two concentration scales are used as vertical points to draw vertical lines. The value in the grid where the intersection of the two vertical lines is 82.8, then the cell survival ratio is 82.8%, indicating that the concentration of Venetoclax is 0nM, that is, when the maleate salt of the compound of formula (I) is used alone, the cell survival ratio is 82.8%.

Synergy effect evaluation method

Statistical methods are used to evaluate the combined effect of two drugs on tumor cells. For example, the statistical software used is R systems, and the statistical models are Bliss, HSA, Loewe, and ZIP. When the synergy value > 5, there is a synergistic effect, the synergy value > 10 means there is a significant synergistic effect, the synergy value <-5 means there is an antagonistic effect, and the synergy value <-10 means there is a significant antagonistic effect. The synergy value is used to further determine the final efficacy of the combination of two drugs. For example, the statistical software used is SynergyFinder, and the statistical models are Bliss, HSA, Loewe, and ZIP. When the synergy value > 10 means there is a significant synergistic effect, and the synergy value <-10 means there is a significant antagonistic effect. The synergy value is used to further determine the final efficacy of the combination of multiple drugs.

Example 1 Effect of the maleate salt of the compound of formula (I) combined with the BCL-2 inhibitor Venetoclax on HL-60 tumor cells

Human acute promyelocytic leukemia HL-60 cells (ATCC, CCL-240) were inoculated into 96-well plates at a number of 5000 cells per well. After the cells adhered to the wall (24 hours), the maleate salt of the compound of formula (I) and the Venetoclax drug were added to dilute to a certain gradient concentration, and 2 replicate wells were added for each concentration. After 6 hours, the cell viability was detected using the Cell Titer-Glo method. 1/2 volume of Cell Titer Glo lysis buffer (Progema, G7573) was added to each well, the plate was shaken for 2 minutes, and then allowed to stand for 10 minutes. The RLU value was read on the instrument Envision.

The calculation results of cell survival ratio are shown in Figure 1. In human acute promyelocytic leukemia HL-60 cells, the cell survival rate of the maleate salt of the compound of formula (I) at 37.04 nM is 82.8%, the cell survival rate of Venetoclax at 1000 nM is 64.5%, and the cell survival rate of 37.04 nM maleate salt of the compound of formula (I) combined with 1000 nM Venetoclax is 26.4%.

The combined effect is shown in Figures 2-5. In human acute promyelocytic leukemia cells HL-60, the maleate salt of the compound of formula (I) is combined with Venetoclax. The average synergistic values calculated by four statistical models, Bliss (Figure 2), HSA (Figure 3), Loewe (Figure 4), and ZIP (Figure 5), are 21.302, 21.319, 19.075, and 21.814, respectively, which are all much greater than 5. This indicates that the maleate salt of the compound of formula (I) and Venetoclax have a good synergistic effect in tumor cells.

Example 2 Effect of the maleate salt of the compound of formula (I) in combination with the BCL-2 inhibitor Venetoclax on Kasumi-1 tumor cells

Human acute lymphoblastic leukemia cells Kasumi-1 (ATCC, CRL-2724) were inoculated into 96-well plates at a number of 4000 cells per well. After the cells adhered to the wall (24 hours), the maleate salt of the compound of formula (I) and the Venetoclax drug were added to dilute to a certain gradient concentration, and the drug was added to 2 replicate wells for each concentration. After 24 hours, the cell viability was detected using the Cell Titer-Glo method. 1/2 volume of Cell Titer Glo lysis buffer (Progema, G7573) was added to each well, the plate was shaken for 2 minutes, and then allowed to stand for 10 minutes. The RLU value was read on the instrument Envision.

The calculation results of the cell survival ratio are shown in Figure 6. In human acute lymphoblastic leukemia Kasumi-1 cells, the cell survival rate of the maleate salt of the compound of formula (I) at 37.04 nM is 54.4%, the cell survival rate of Venetoclax at 1000 nM is 40.9%, and the cell survival rate of 37.04 nM maleate salt of the compound of formula (I) combined with 1000 nM Venetoclax is 2.7%.

The combined effect is shown in Figures 7-10. In human acute lymphoblastic leukemia cells Kasumi-1, the maleate salt of the compound of formula (I) is combined with Venetoclax. The average synergistic values calculated by four statistical models, Bliss (Figure 7), HSA (Figure 8), Loewe (Figure 9), and ZIP (Figure 10), are 5.502, 12.611, 9.139, and 5.924, respectively, all greater than 5. This indicates that the maleate salt of the compound of formula (I) and Venetoclax have a good synergistic effect in tumor cells.

Example 3 Effect of the maleate salt of the compound of formula (I) in combination with the BCL-2 inhibitor Venetoclax on MV-4-11 tumor cells

Human myelomonocytic leukemia cells MV-4-11 (ATCC, CRL-9591) were inoculated into 96-well plates at a number of 5000 cells per well. After the cells adhered to the wall (24 hours), the maleate salt of the compound of formula (I) and the drug Venetoclax were added to dilute to a certain gradient concentration, and the drugs were added to 2 replicate wells for each concentration. After 6 hours, the cells were plated with Cell Titer-Glo method was used to detect cell viability. 1/2 volume of CellTiter Glo lysis buffer (Progema, G7573) was added to each well, the plate was shaken for 2 minutes, allowed to stand for 10 minutes, and the RLU value was read on the Envision instrument.

The calculation results of cell survival ratio are shown in Figure 11. In human myelomonocytic leukemia cells MV-4-11, the cell survival rate of the maleate salt of the compound of formula (I) at 37.04 nM was 66.8%, the cell survival rate of Venetoclax at 1000 nM was 32.9%, and the cell survival rate of 37.04 nM maleate salt of the compound of formula (I) combined with 1000 nM Venetoclax was 3.8%.

The combined effect is shown in Figures 12-15. In human myelomonocytic leukemia cells MV-4-11, the maleate salt of the compound of formula (I) is combined with Venetoclax. The average synergistic values calculated by four statistical models, Bliss (Figure 12), HSA (Figure 13), Loewe (Figure 14), and ZIP (Figure 15), are 7.914, 15.803, 13.773, and 9.201, respectively, which are all much greater than 5. This indicates that the maleate salt of the compound of formula (I) and Venetoclax have a good synergistic effect when used in combination in tumor cells.

Example 4 Effect of the maleate salt of the compound of formula (I) in combination with the BCL-2 inhibitor Venetoclax on MOLM-13 tumor cells

Human acute myeloid leukemia MOLM-13 cells (ADDEXBIO, C0003003) were inoculated into 96-well plates at a number of 2000 cells per well. After the cells adhered to the wall (24 hours), the maleate salt of the compound of formula (I) and the Venetoclax drug were added to dilute to a certain gradient concentration, and the drug was added to 2 replicate wells for each concentration. After 6 hours, the cell viability was detected using the Cell Titer-Glo method. 1/2 volume of Cell Titer Glo lysis buffer (Progema, G7573) was added to each well, the plate was shaken for 2 minutes, and then allowed to stand for 10 minutes. The RLU value was read on the instrument Envision.

The calculation results of cell survival ratio are shown in Figure 16. In human acute myeloid leukemia MOLM-13 cells, the cell survival rate of the maleate salt of the compound of formula (I) at 37.04 nM was 19.0%, the cell survival rate of Venetoclax at 1000 nM was 38.1%, and the cell survival rate of 37.04 nM maleate salt of the compound of formula (I) combined with 1000 nM Venetoclax was 5.4%.

The combined effect is shown in Figures 17-20. In human acute myeloid leukemia cells MOLM-13, the maleate salt of the compound of formula (I) is combined with Venetoclax. The average synergistic values calculated by four statistical models, Bliss (Figure 17), HSA (Figure 18), Loewe (Figure 19), and ZIP (Figure 20), are 14.368, 18.692, 16.632, and 14.961, respectively, which are much greater than 5. This indicates that the maleate salt of the compound of formula (I) and Venetoclax have a good synergistic effect in tumor cells.

Example 5 Effect of the maleate salt of the compound of formula (I) in combination with the BCL-2 inhibitor Venetoclax and Azacitidine on MV-4-11 tumor cells

Cell culture: Observe the cell growth status under an inverted microscope, select cells in the logarithmic growth phase, and perform aseptic operations in a biosafety cabinet. Transfer the cell suspension to a centrifuge tube, centrifuge the cells, discard the supernatant, resuspend the cells with fresh complete culture medium, and then disperse them into new culture bottles in appropriate proportions and culture them in a 37°C, 5% _CO2 incubator.

Human myelomonocytic leukemia cells MV-4-11 (ATCC, CRL-9591) were seeded into 384-well plates at a density of 2000 cells/35 μL per well and cultured in a 37° C., 5% CO ₂ incubator overnight. After the cells adhered (24 h), 5 μL of maleate of the compound of formula (I) diluted into a certain gradient concentration (final concentration of 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 3 nM, 1 nM) and 5 μL of venetoclax (final concentration of 3000 nM, 949 nM, 300 nM, 95 nM, 30 nM, 9 nM, 3 nM, 1 nM, 0.3 nM) and 5 μL of azacitidine (final concentration of 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.313 μM) were added, and 2 replicate wells were added for each concentration. At the same time, a DMSO control group (cells were treated with culture medium containing 0.3% DMSO) and a blank control group (culture medium without cells and drugs) were set up. After 6 hours of drug action, the cell viability was detected using the Cell Titer-Glo method. 25 μL of Cell Titer Glo lysis buffer (Progema, G7573) was added to each well, the plate was shaken for 2 minutes, allowed to stand for 10 minutes, and the RLU value was read on the Envision instrument.

Data analysis and processing: Calculate the cell proliferation inhibition rate according to the following formula: Inhibition rate (Inhibition rate %) = (1-(RLU _Compound - RLU _{Blank control} )/(RLU _{DMSO control -} RLU _{Blank control} )) × 100%. RLU _Compound is the reading value of the cell compound treatment well, RLU _{Blank control} is the reading value of the blank control well, and RLU _{DMSO control} is the reading value of the cell DMSO control well.

The combined effect is shown in Figures 21-24. In human myelomonocytic leukemia cells MV-4-11, 10nM-100nM maleate of the compound of formula (I) was combined with 3nM-3000nM venetoclax and 1.25μM-5μM azacitidine. The average synergy calculated by four statistical models: ZIP (Figure 21), HSA (Figure 22), Bliss (Figure 23), and Loewe (Figure 24) was 1.25μM-1.5μM. The values were 29.5 (p=1.26E-83), 36.35 (p=1.38E-93), 30.02 (p=7.69E-84), and 18.18 (p=7.01E-93), respectively, all much greater than 10, and the synergistic value of the three-drug combination was greater than the synergistic value of the two-drug combination, indicating that the maleate salt of the compound of formula (I) combined with Venetoclax and azacitidine has a strong synergistic effect in tumor cells.

Example 6 Effect of the maleate salt of the compound of formula (I) in combination with the BCL-2 inhibitor Venetoclax and Azacitidine on Kasumi-1 tumor cells

Cell culture: Observe the cell growth status under an inverted microscope, select cells in the logarithmic growth phase, and perform aseptic operations in a biosafety cabinet. Transfer the cell suspension to a centrifuge tube, centrifuge the cells, discard the supernatant, resuspend the cells with fresh complete culture medium, and then disperse them into new culture bottles in appropriate proportions and culture them in a 37°C, 5% _CO2 incubator.

Human acute myeloid leukemia Kasumi-1 cells (ATCC, CRL-2724) were seeded into 384-well plates at a density of 3000 cells/35 μL per well and cultured in a 37° C., 5% CO ₂ incubator overnight. After the cells adhered (24 h), 5 μL of maleate of the compound of formula (I) diluted into a certain gradient concentration (final concentration of 1000nM, 300nM, 100nM, 80nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 3nM) and 5 μL of venetoclax (final concentration of 10000nM, 3163nM, 1000nM, 316nM, 100nM, 32nM, 10nM, 3nM, 1nM) and 5 μL of azacitidine (final concentration of 10μM, 5μM, 2.5μM, 1.25μM, 0.625μM) were added, and 2 replicate wells were added for each concentration. At the same time, a DMSO control group (cells were treated with culture medium containing 0.3% DMSO) and a blank control group (culture medium without cells and drugs) were set up. After 6 hours of drug action, the cell viability was detected using the Cell Titer-Glo method. 25 μL of Cell Titer Glo lysis buffer (Progema, G7573) was added to each well, the plate was shaken for 2 minutes, allowed to stand for 10 minutes, and the RLU value was read on the Envision instrument.

Data analysis and processing: Calculate the cell proliferation inhibition rate according to the following formula: Inhibition rate (Inhibition rate %) = (1-(RLU _Compound - RLU _{Blank control} )/(RLU _{DMSO control -} RLU _{Blank control} )) × 100%. RLU _Compound is the reading value of the cell compound treatment well, RLU _{Blank control} is the reading value of the blank control well, and RLU _{DMSO control} is the reading value of the cell DMSO control well.

The combined effect is shown in Figures 25-28. In human acute myeloid leukemia cells Kasumi-1, 10nM-1000nM maleate of the compound of formula (I) was combined with 10nM-10000nM venetoclax and 2.5μM-10μM azacitidine. The average calculated by four statistical models: ZIP (Figure 25), HSA (Figure 26), Bliss (Figure 27), and Loewe (Figure 28) The synergistic values were 21.85 (p=2.96E-94), 31.78 (p=3.24E-98), 22.47 (p=1.49E-91), and 23.46 (p=6.74E-76), respectively, all much greater than 10, and the synergistic value of the three-drug combination was greater than the synergistic value of the two-drug combination, indicating that the maleate salt of the compound of formula (I) combined with Venetoclax and azacitidine has a strong synergistic effect in tumor cells.

Example 7 Effect of the maleate salt of the compound of formula (I) in combination with the BCL-2 inhibitor Venetoclax and Azacitidine on HL-60 tumor cells

Cell culture: Observe the cell growth status under an inverted microscope, select cells in the logarithmic growth phase, and perform aseptic operations in a biosafety cabinet. Transfer the cell suspension to a centrifuge tube, centrifuge the cells, discard the supernatant, resuspend the cells with fresh complete culture medium, and then disperse them into new culture bottles in appropriate proportions and culture them in a 37°C, 5% _CO2 incubator.

Human acute promyelocytic leukemia HL-60 cells (ATCC, CCL-240) were seeded into 384-well plates at a density of 2000 cells/35 μL per well and cultured in a 37° C., 5% CO ₂ incubator overnight. After the cells adhered (24 h), 5 μL of maleate of the compound of formula (I) diluted into a certain gradient concentration (final concentration of 300 nM, 100 nM, 80 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 3 nM, 1 nM) and 5 μL of venetoclax (final concentration of 10000 nM, 3163 nM, 1000 nM, 316 nM, 100 nM, 32 nM, 10 nM, 3 nM, 1 nM) and 5 μL of azacitidine (final concentration of 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM) were added, and 2 replicate wells were added for each concentration. At the same time, a DMSO control group (cells were treated with culture medium containing 0.3% DMSO) and a blank control group (culture medium without cells and drugs) were set up. After 24 hours of drug action, the cell viability was detected using the Cell Titer-Glo method. 25 μL of CellTiter Glo lysis buffer (Progema, G7573) was added to each well, the plate was shaken for 2 minutes, allowed to stand for 10 minutes, and the RLU value was read on the Envision instrument.

Data analysis and processing: Calculate the cell proliferation inhibition rate according to the following formula: Inhibition rate (Inhibition rate %) = (1-(RLU _Compound - RLU _{Blank control} )/(RLU _{DMSO control -} RLU _{Blank control} )) × 100%. RLU _Compound is the reading value of the cell compound treatment well, RLU _{Blank control} is the reading value of the blank control well, and RLU _{DMSO control} is the reading value of the cell DMSO control well.

The combined effect is shown in Figures 29-32. In human acute promyelocytic leukemia cells HL-60, 3nM-100nM maleate of the compound of formula (I) was combined with 10nM-1000nM venetoclax and 3.125μM-12.5μM azacitidine. The averages calculated by four statistical models, ZIP (Figure 29), HSA (Figure 30), Bliss (Figure 31), and Loewe (Figure 32) were The average synergistic values were 11.07 (p=2.75E-26), 26.48 (p=2.53E-63), 11.17 (p=1.04E-26), and 17.6 (p=3.38E-65), respectively, all greater than 10, and the synergistic value of the three-drug combination was greater than the synergistic value of the two-drug combination, indicating that the maleate salt of the compound of formula (I) combined with Venetoclax and azacitidine has a good synergistic effect in tumor cells.

Example 8 In vivo antitumor pharmacodynamic evaluation of the maleate salt of the compound of formula (I) in combination with the BCL-2 inhibitor Venetoclax in the HL-60 human acute promyelocytic leukemia model

Materials and Reagents:

HL-60 (ATCC, CCL-240), penicillin-streptomycin (Gibco, 15140-122), IMDM (Gibco, 12440053), FBS (Gibco, 10099-141C), KH ₂ PO ₄ (General reagent, G82821B), DMSO (AMRESCO, 231).

During the experiment, the health status of the animals was observed every day. If the weight of the animals decreased by 10%, the dosage was halved; if the weight of the animals decreased by 15%, the dosage was stopped until the weight decreased. If the animal recovers or the tumor volume exceeds 2,000 mm ³ , euthanize immediately. If the health condition shows any of the following, notify the veterinarian and euthanize:

Obvious weight loss, with body weight loss greater than 20%.

No free access to food and water.

Animals develop infections, severe rupture of tumors, or hematomas.

Animals show the following clinical symptoms and continue to worsen: piloerection, arched back, pale ears, nose, eyes or feet, rapid breathing, convulsions, diarrhea, pain, dehydration, and moribundity.

Experimental Animals:

BALB/c nude mice, 6-8 weeks old, female, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., animal certificate number 20170011005453, and kept in an SPF environment.

Experimental drugs:

Venetoclax (ABT199) was purchased from Shanghai Yu'ang Chemical Co., Ltd., batch number 1257044-40-8.

Preparation method: maleate salt of the compound of formula (I) was injected via tail vein, the solvent was 50 mM PBS (0.3402 g KH ₂ PO ₄ was dissolved in 50 mL ultrapure water, pH was adjusted to 7.0-7.2, and sterilized by autoclave); Venetoclax solvent was 5% DMSO + 50% PEG400 + 5% Tween80 + ddH ₂ O, sterilized by autoclave.

Experimental methods and steps:

HL-60 cells were cultured in IMDM medium supplemented with 20% FBS and 1% penicillin-streptomycin at 37°C in an incubator with 5% CO _2. Cells were collected and the cell density was adjusted to 2.0×10 ⁸ /mL; the cells were subcutaneously inoculated on the right back of the mice, with an inoculation volume of 0.20mL (containing 50% Matrigel) per animal and an inoculation amount of 2×10 ⁷ cells per animal. When the tumor grew to 140-230mm ³ (D13), 32 tumor-bearing mice were selected and randomly divided into 4 groups, with 8 mice in each group. The dosing method was carried out according to the experimental scheme in Table 1. The dosing volume for mice was 10mL/kg. The animals were weighed daily with an electronic balance, and the tumor volume was measured with a vernier caliper 3 times a week.

Table 1 Dosage regimen of therapeutic drugs

Note: po means oral administration; iv means tail vein injection; qd means once a day; biw means twice a week, with continuous administration for 2 days and rest for 5 days.

The main evaluation indicators are:

Tumor volume: Tumor volume (TV) = (L×W ² )/2, where L is the long diameter of the tumor and W is the wide diameter of the tumor.

Tumor growth inhibition (TGI):

TGI (%) = [1-(avT _i-0 /avC _i-0 )] × 100%; wherein avTi-0 is the average tumor volume of the dosing group on a specific day, minus the average tumor volume of the dosing group on the day the dosing started; wherein avCi-0 is the average tumor volume of the vehicle control group on a specific day, minus the average tumor volume of the vehicle control group on the day the dosing started.

Tumor weight inhibition (TWI):

Tumor weight inhibition rate (TWI) = (1-TW _treatment/Dx /TW _control/Dx ) × 100%; where TW _control : average tumor weight of the control group (g), TW _treatment : average tumor weight of the treatment group (g).

Relative change of body weight (RCBW):

RCBW (%) = (BWi _{- BW0} ) / BW0 x 100%; wherein _BWi is the body weight of the animal on a specific day, and _BW0 is the body weight of the animal on the day when dosing begins.

Experimental results and conclusions

All experimental data were analyzed and plotted using GraphPad Prism software (GraphPad Software). The tumor volume and animal weight of each group were statistically compared using two-way ANOVA (Dunnett's multiple comparisons test), and the tumor weight of each group at the end point was statistically compared using one-way ANOVA (Dunnett's multiple comparisons test). When P < 0.05, it was considered to be statistically significant. Tumor volume, weight and animal weight are expressed as mean ± standard error (SEM). The results are shown in Table 2 below.

Table 2

Note: **p<0.01, ***p<0.001, compared with the G1 solvent control group, respectively.

The effects of each treatment group on the tumor growth of HL-60 tumor-bearing mice are shown in Figure 33 and Figure 34 ; the changes in body weight of tumor-bearing mice in each group are shown in Figure 35 .

The results showed that in the HL-60 human acute promyelocytic leukemia model, the maleate salt of the compound of formula (I) was injected into the tail vein at 10 mg/kg twice a week (for 2 consecutive days and 5 days of drug withdrawal), and Venetoclax was orally administered once a day at 100 mg/kg, which had no significant effect on the body weight of the animals. Both of them could significantly inhibit the growth of HL-60 tumors, and the combination of the maleate salt of the compound of formula (I) and Venetoclax was significantly superior to the efficacy of each drug alone.

Example 9: The maleate salt of the compound of formula (I) was combined with the BTK inhibitors Ibrutinib and Zanubrutinib to evaluate the expression of the target protein in the lymphoma cell line OCI-LY-10 model

Experimental methods

1) Cell culture

Human lymphoma cell line OCI-LY10 cells (Shangcheng Beina Chuanglian Biotechnology Co., Ltd., BNCC 337742) were cultured in IMDM medium containing 10% fetal bovine serum at 37°C and 5% CO ₂ incubator. The cell growth state was observed under an inverted microscope, and cells in the logarithmic growth phase were selected and inoculated at 5×10e ⁶ /well. After drug treatment, the target protein was detected.

2) Cell drug treatment

a. Test drug configuration

The maleate salt of the compound of formula (I) (molecular weight: 751.25), Ibrutinib (molecular weight 440.51, MedChemExpress, HY-10997-61547), and Zanubrutinib (molecular weight 471.55, MedChemExpress, HY-101474A-79640) were weighed separately, dissolved in DMSO to a concentration of 10 mM, and stored in a -20°C refrigerator.

b. Dosing treatment

On the first day, 10 mM Ibrutinib or Zanubrutinib stock solution was diluted with DMSO and added to the cells at certain concentrations (10 nM, 1 nM, 0 nM).

On the second day (24 hours later), a 10 mM stock solution of the maleate salt of the compound of formula (I) was diluted with DMSO and added to the cells containing Ibrutinib or Zanubrutinib at 100 nM. After 6 hours, the cells were collected and the subsequent target protein detection experiments were performed.

3) Experiments related to target protein detection

a. Total cell protein extraction and quantification:

Take out the cell culture well plate, transfer the cell suspension to a centrifuge tube, collect the cells by centrifugation, wash once with 4°C pre-cooled PBS solution, and then resuspend the cell pellet with an appropriate amount of pre-cooled protein lysis buffer (RIPA Buffer (10×), purchased from CST, Catalog No.: 9806, containing 1/100 volume of protease inhibitors (Halt Protease Inhibitor Cocktail 1 (100×), purchased from Thermo Fisher, Catalog No. 78439) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2 (100×), purchased from sigma, Catalog No.: P5726; Phosphatase Inhibitor Cocktail 3 (100×), purchased from sigma, Catalog No. P0044)), let stand on ice for 30 minutes; centrifuge at 13300 rpm, 4°C for 20 minutes, transfer the supernatant to a new centrifuge tube, and temporarily store it on wet ice. Take an appropriate amount of protein lysate and determine the protein concentration using the BCA method. According to the protein concentration, add an appropriate amount of LDS-loading buffer (NuPAGE LDS Sample Buffer (4×), purchased from Thermo Fisher, Catalog No. NP0007) and reducing agent (NuPAGE Sample Reducing Agent (10×), purchased from Thermo Fisher, Catalog No. NP0009) to the protein lysate, mix well, adjust the protein concentration, then place in a 98°C metal bath for 10 minutes, cool to room temperature, and then transfer to a -80°C refrigerator for storage.

b.SDS-PAGE gel electrophoresis and protein transfer:

Install the electrophoresis tank and precast gel, then load the sample according to the plan, load 20μg protein (cell protein extracted in step a) in each well, and perform electrophoresis. When the bromophenol blue indicator front approaches the bottom of the gel, stop the electrophoresis. Remove the gel card slot, carefully separate the protein gel, and transfer it to the nitrocellulose transfer membrane for protein transfer. After the protein transfer is completed, cut the transfer membrane near the molecular weight of the target protein according to the molecular weight of the protein standard and mark it.

c. Protein immunoblotting staining: The transfer membrane was first rinsed with deionized water, then protein blocking solution was added, and incubated at room temperature for 1 hour with slow shaking, and then antibody buffer containing primary antibody (protein blocking solution containing 0.05% Tween-20) was added (primary antibody includes: MCL-1 (D2W9E) Rabbit mAb, purchased from CST, No. 94296; BFL1, purchased from CST, Catalog No. 14093; BIM, purchased from CST, Catalog No. 2933; c-MYC (D84C12), purchased from abcam, Catalog No.: ab32072; Cleaved Caspase-3 (Asp175) (5A1E), purchased from CST, Catalog No. 9664; GAPDH (D16H11, purchased from CST, Catalog No. 5174) was used as a control, and incubated at 4°C with slow shaking overnight. Rinse three times with PBST solution, 5-10 minutes each time. Add antibody buffer containing secondary antibody (HRP conjugated anti-rabbit IgG antibody), incubate at room temperature with slow shaking for 1 hour, and then rinse three times with PBST solution, 5-10 minutes each time. Add HRP developer and detect on the machine.

d. Data analysis and processing: Use Image Studio Ver 5.0 software for scanning and analysis, and record the expression level of target protein.

Experimental Results

The effects of the maleate salt of the compound of formula (I) in combination with Ibrutinib and Zanubrutinib on the protein immunoblotting results of the target protein (Figure 36), c-MYC, MCL-1, BFL-1 and BIM protein expression (Figure 37), and Cl-Caspase3 protein expression (Figure 38) are shown in Figures 36-38. The maleate salt of the compound of formula (I) alone, combined with Ibrutinib and Zanubrutinib, respectively, reduced the expression of pro-cell survival proteins c-MYC, MCL-1, and BFL-1, and the combination of the other two drugs increased the expression of pro-apoptotic protein BIM and a significant increase in Cl-Caspase3 (an indicator protein of cell apoptosis).

Example 10: Evaluation of the efficacy of the maleate salt of the compound of formula (I) in combination with the BTK inhibitors Ibrutinib and Zanubrutinib in the lymphoma cell line OCI-LY-10 model

Experimental methods

1) Cell culture

Human lymphoma cell line OCI-LY10 cells (Shangcheng Beina Chuanglian Biotechnology Co., Ltd., BNCC 337742) were cultured in IMDM medium containing 10% fetal bovine serum at 37°C and 5% CO ₂ incubator. The cell growth state was observed under an inverted microscope, and cells in the logarithmic growth phase were selected and inoculated at 4000/well. Two secondary wells were set for each drug-addition group. After drug-addition treatment, cell viability was detected.

2) Cell drug treatment

a. Test drug configuration

The maleate salt (molecular weight: 751.25) of the compound of formula (I), Ibrutinib (molecular weight: 440.51), and Zanubrutinib (molecular weight: 47.56) were weighed separately, dissolved in DMSO to a concentration of 10 mM, and stored in a -20°C refrigerator.

b. Dosing treatment

On the first day, 10 mM Ibrutinib or Zanubrutinib stock solution was added to the cells according to a certain concentration gradient (10 nM, 2.5 nM, 1 nM, 0 nM).

On the second day (24 hours later), 10 mM maleate stock solution of the compound of formula (I) was added to the cells containing Ibrutinib according to a certain concentration gradient (100 nM, 50 nM, 0 nM), or added to the cells containing Zanubrutinib according to a certain concentration gradient (100 nM, 50 nM, 25 nM, 0 nM).

On the third day (24 h later), follow-up cell viability assays were performed.

3) Cell viability determination and data analysis

a. Cell viability assay: Prepare CellTiter Glo lysis buffer according to the instructions of Cell Titer Glo reagent (Progema, G7573). Remove cells from the incubator and observe under a microscope; add 1/2 volume of Cell Titer Glo lysis buffer to each well, shake in the dark for 2 minutes to fully lyse the cells, then place in the dark at room temperature for 10 minutes, and read the chemiluminescent signal (RLU) with an enzyme reader.

b. Data analysis and processing

The RLU reading of each well minus the RLU reading of the blank well (culture medium containing only 0.1% DMSO and no cells) is used as the cell viability value of each well. Cell viability is calculated according to the following formula: Relative ratio of cell viability = ((RLU compound-RLU blank)/(RLU DMSO–RLU blank))×100%. RLU compound is the reading of the cell compound-treated well, RLU blank is the reading of the blank well, and RLU DMSO is the reading of the cell DMSO-treated well. Prism GraphPad 6 for windows v6.01 software was used for plotting.

Experimental Results

The results of the inhibition of OCI-LY-10 cell viability by the maleate salt of the compound of formula (I) in combination with Ibrutinib and Zanubrutinib are shown in Figure 39 and Table 3, Figure 40 and Table 4. Figure 39: **P<0.01, ***P<0.001; Figure 40: *P<0.05, **P<0.01, ***P<0.001. The P values were calculated using GraphPad Prism based on the results of Tables 3 and 4.

Table 3

Table 4

The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims (14)

A pharmaceutical composition or a drug kit, comprising a therapeutically effective amount of a CDK9 inhibitor and a therapeutically effective amount of at least one drug for treating cancer, wherein the CDK9 inhibitor is a compound of formula (I), a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof; and the drug for treating cancer is selected from one or more of a BCL-2 inhibitor, a BTK inhibitor and a DNA methyltransferase inhibitor.
The pharmaceutical composition or kit according to claim 1, wherein the pharmaceutically acceptable salt of the compound of formula (I) comprises maleate and/or fumarate. The pharmaceutical composition or kit according to claim 1, wherein the BCL-2 inhibitor is selected from one or more of Navitoclax, Venetoclax, Pelcitoclax, A-1155463, A-1331852, ABT-737, Obatoclax, S44563, TW-37, AT101, HA14-1 and Sabutoclax; The BTK inhibitor is selected from one or more of ibrutinib, zanubrutinib, sepretinib, omotinib, acalabrutinib, CNX-774, CGI1746, LFM-A13, CNX-774, ONO-4059 and RN486; The DNA methyltransferase inhibitor is Azacitidine. The pharmaceutical composition or kit according to claim 1, wherein the pharmaceutical composition comprises a compound of formula (I), a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof and Venetoclax; or Comprising a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, Venetoclax and Azacitidine; or, Comprising a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof and ibrutinib; or, It includes a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof and Zanubrutinib. The pharmaceutical composition or kit according to claim 1, wherein the dosage of the CDK9 inhibitor is selected from 0.01-500 mg/day or 0.01-500 mg/week, and the dosage of the drug for treating cancer is selected from 0.01-1000 mg/day. The pharmaceutical composition or kit according to any one of claims 1 to 5, which is used to treat a disease or disease state mediated by CDK9. Use of the pharmaceutical composition or kit according to any one of claims 1 to 5 in the preparation of a medicament for treating a disease or disease state mediated by CDK9. A pharmaceutical combination of a compound of formula (I), a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof and Venetoclax; or, A pharmaceutical combination of a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, Venetoclax and Azacitidine; or, A pharmaceutical combination of a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof and ibrutinib; or, A pharmaceutical combination of a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof and Zanubrutinib; which is used for treating a disease or disease state mediated by CDK9. Use of a CDK9 inhibitor and a drug for treating cancer in the preparation of a drug or a drug kit or a drug combination for treating a CDK9-mediated disease or disease state, wherein the CDK9 inhibitor is a compound of formula (I), a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof; and the drug for treating cancer is one or more of a BCL-2 inhibitor, a BTK inhibitor and a DNA methyltransferase inhibitor.
The use according to claim 9, wherein the pharmaceutically acceptable salt of the compound of formula (I) comprises maleate and/or fumarate. The use according to claim 9, wherein the BCL-2 inhibitor is selected from Navitoclax, Venetoclax, Pelcitoclax, A-1155463, One or more of A-1331852, ABT-737, Obatoclax, S44563, TW-37, AT101, HA14-1, and Sabutoclax; The BTK inhibitor is selected from one or more of ibrutinib, zanubrutinib, sepretinib, omotinib, acalabrutinib, CNX-774, CGI1746, LFM-A13, CNX-774, ONO-4059 and RN486; The DNA methyltransferase inhibitor is Azacitidine. The pharmaceutical composition or kit according to claim 7, the pharmaceutical combination according to claim 8, or the use according to claim 9, wherein the CDK9-mediated disease or disease state is cancer. The pharmaceutical composition or kit according to claim 7, the pharmaceutical combination according to claim 8, or the use according to claim 9, wherein the CDK9-mediated disease or disease state is selected from one or both of solid tumors and hematological tumors. The pharmaceutical composition or kit according to claim 7, the pharmaceutical combination according to claim 8, or the use according to claim 9, wherein the CDK9-mediated disease or disease state is selected from one or more of non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, prostate cancer, bladder cancer, liver cancer, skin cancer, glioma, breast cancer, melanoma, malignant glioma, rhabdomyosarcoma, ovarian cancer, astroglioma, Ewing's sarcoma, retinoblastoma, epithelial cell carcinoma, colon cancer, renal cancer, gastrointestinal stromal tumor, leukemia, lymphoma and nasopharyngeal carcinoma.