CN116270658B

CN116270658B - Pharmaceutical combination of CDK9 inhibitor and BTK inhibitor and application thereof

Info

Publication number: CN116270658B
Application number: CN202310588624.9A
Authority: CN
Inventors: 黄晓静; 刘�东; 周福生; 兰炯; 吕强
Original assignee: Genfleet Therapeutics Shanghai Inc
Current assignee: Genfleet Therapeutics Shanghai Inc
Priority date: 2023-05-24
Filing date: 2023-05-24
Publication date: 2023-10-27
Anticipated expiration: 2043-05-24
Also published as: CN116270658A

Abstract

The application provides a pharmaceutical composition or kit comprising a therapeutically effective amount of a CDK9 inhibitor and a therapeutically effective amount of a BTK inhibitor, the use thereof in the manufacture of a medicament or pharmaceutical combination or kit for the treatment of cancer and the use of a combination of a CDK9 inhibitor and a BTK inhibitor, the composition being used in combination in cancer cells with a good synergistic effect.

Description

Pharmaceutical combination of CDK9 inhibitor and BTK inhibitor and application thereof

Technical Field

The application relates to the technical field of medicines, in particular to a drug product containing a CDK9 inhibitor and a BTK inhibitor and application thereof in treating cancers.

Background

Proliferation and division of eukaryotic cells is a precise and complex regulatory process. Proliferation is accomplished by the cell cycle, which proceeds orderly through its strict molecular regulatory mechanisms. Three major classes of molecules have been found to be involved in cell cycle regulation: cyclin-dependent kinase (CDK), cyclin-dependent kinase (cyclin-dependent kinases, CDK), cyclin-dependent kinase inhibitor (cyclin-dependent kinaseinhibitors, CKI), wherein CDK is centrally located. The CDK family has found 13 members (CDK 1-CDK 13) and studies have found that abnormalities in CDK9 expression levels or (and) kinase activity can lead to abnormalities in intracellular expression of various proteins or (and) mRNA levels thereof. It has been reported that CDK9 pathway disorders exist in a variety of hematological malignancies, and CDK9 is one of the most critical molecules in the course of tumorigenesis and progression (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 proven useful in the treatment of cancer. Although CDK inhibitors as monotherapy have efficacy in certain cancers, there remains a need to develop effective doses and dosing regimens for the administration of CDK inhibitors in combination with other cancer therapeutic agents to treat, prevent diseases, disorders or conditions involving Cyclin Dependent Kinase (CDK) activity.

Disclosure of Invention

In one aspect of the application there is provided a pharmaceutical composition or kit comprising a therapeutically effective amount of a CDK9 inhibitor and a therapeutically effective amount of a BTK inhibitor, said CDK9 inhibitor being a compound of formula (I), a stereoisomer, solvate or a pharmaceutically acceptable salt thereof. The CDK9 inhibitor and the BTK inhibitor have obvious synergistic effect when used in combination.

（I）

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

In one embodiment, the BTK inhibitor comprises one or more selected from Ibrutinib (Ibrutinib), zebutinib (Zanubrutinib), capetinib (Spibrutinib, AVL-292), omatinib (Olmeutinib, HM-71224), acartinib (Acalabrutinib), CNX-774, CGI1746, LFM-A13, CNX-774, ONO-4059, and RN 486.

In one embodiment, the BTK inhibitor is selected from Ibrutinib (Ibrutinib) and zebutinib (zambutinib).

In one embodiment, the pharmaceutical composition or kit comprises a maleate salt of a compound of formula (I) and Ibrutinib (ibutinib) or a maleate salt of a compound of formula (I) and zebutinib (zambutinib).

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

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 kit comprises 25-100 nM CDK9 inhibitor, further may be 25-50 nM or 50-100 nM, e.g., may be 25-nM, 50nM or 100 nM.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In another aspect of the application there is provided the use of a pharmaceutical composition or kit as described above in the manufacture of a medicament for the treatment of a CDK9 mediated disease or condition.

In another aspect of the application there is provided the use of a CDK9 inhibitor, which CDK9 inhibitor is a compound of formula (I), a stereoisomer, solvate or a pharmaceutically acceptable salt thereof, in the preparation of a medicament or kit or combination of medicaments for treating a CDK9 mediated disease or condition. The CDK9 inhibitor and the BTK inhibitor have obvious synergistic effect when used in combination.

（I）

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

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

The application also provides an administration dose of a CDK9 inhibitor selected from 0.01-5000 mg/day, preferably 1-1500 mg/day, optionally 10 mg/day, 50 mg/day, 100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 300 mg/day, 350 mg/day, 400 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, 8500 mg/day, 900 mg/day, 950 mg/day, 1000 mg/day, 1200 mg/day, 1250 mg/day, 1300 mg/day, 1400 mg/day, 1500 mg/day.

The application also provides a dose of BTK inhibitor selected from 0.01-1000 mg/day, preferably 1-500 mg/day, optionally 10 mg/day, 50 mg/day, 100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 300 mg/day, 350 mg/day, 400 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, 8500 mg/day, 900 mg/day, 950 mg/day, 1000 mg/day.

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

In one embodiment, the cancer is a solid tumor or hematological tumor.

In one embodiment, the cancer is non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, pancreatic cancer, prostate cancer, bladder cancer, liver cancer, skin cancer, glioma, breast cancer, melanoma, glioblastoma, rhabdomyosarcoma, ovarian cancer, astroglioma, ewing's sarcoma, retinoblastoma, epithelial cell carcinoma, colon cancer, kidney cancer, gastrointestinal stromal tumor, leukemia, lymphoma, and nasopharyngeal carcinoma.

In one embodiment, the disease or condition is selected from the group consisting of MDS-RAEB (myelodysplastic syndrome-primitive cytopenia), histiocytic lymphoma, acute B-cell leukemia, acute megakaryoblastic leukemia, acute myeloid leukemia, and acute promyelocytic leukemia.

In another aspect of the application there is provided a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a CDK9 inhibitor as described above and a therapeutically effective amount of a BTK inhibitor as described above, wherein the therapeutically effective amount of the CDK9 inhibitor and the therapeutically effective amount of the other cancer therapeutic agent may be administered simultaneously, separately formulated and co-administered or separately formulated and administered sequentially.

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

Drawings

FIG. 1 shows the immunoblotting results of the maleate salt of the compound of formula (I) in example 1 in combination with Ibrutinib or Zanubrutinib, respectively, on OCI-LY-10 cell viability-related proteins.

FIG. 2 shows the effect of the maleate salt of the compound of formula (I) in example 1 in combination with Ibrutinib or Zanubrutinib, respectively, on the expression of OCI-LY10 cell c-MYC, MCL-1, BFL-1 and BIM proteins.

FIG. 3 shows the effect of the maleate salt of the compound of formula (I) in example 1 in combination with Ibrutinib or Zanubrutinib, respectively, on Cl-Caspase3 protein expression in OCI-LY10 cells.

FIG. 4 shows the results of inhibition of OCI-LY-10 cell viability by the compound of formula (I) maleate salt in combination with Ibrutinib in example 2.

FIG. 5 shows the results of inhibition of OCI-LY-10 cell viability by the compound of formula (I) maleate salt in combination with Zanubrutinib in example 2.

Description of the embodiments

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.

As used herein and unless otherwise indicated, the terms "comprising," "including," "having," "containing," and their grammatical equivalents are generally understood to be open-ended and not to be limiting, e.g., not to exclude other, unrecited elements or steps.

As used herein, the term "inhibit" is used relative to a control. One skilled in the art will readily determine the appropriate controls 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 this disclosure should be considered to be a 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 from 1-200, 200-300, etc., as well as 200, 300, 400, 500, 600, 700, 800, 900, and 1000, etc.

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

The compounds may be present in the pharmaceutical composition as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present application prepared from the compounds of the present application which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present application contain relatively acidic functional groups, base addition salts may be obtained by contacting neutral forms of such compounds with a sufficient amount of a base in pure solution or in 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 present application contain relatively basic functional groups, the acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of an acid in pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts including acids 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-toluic acid, citric acid, tartaric acid, fumaric acid, and methanesulfonic acid; also included are salts of amino acids (e.g., arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the application contain basic and acidic functionalities that can be converted to either base or acid addition salts.

Pharmaceutically acceptable salts of the application can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.

In addition to salt forms, the compounds provided herein exist in prodrug forms. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to convert to the compounds of the application. In addition, prodrugs can be converted to the compounds of the present application 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 agent, the prodrug may be slowly converted to the compound of the application.

Certain compounds of the application may exist in unsolvated forms or solvated forms, including hydrated forms. In general, solvated forms, which are equivalent to unsolvated forms, are intended to be encompassed within the scope of the present application. Solvated forms are generally equivalent to unsolvated forms and are intended to be encompassed within the scope of the present application. Certain compounds of the application may exist in polymorphic or amorphous forms. In general, all physical forms are equivalent for the applications contemplated by the present application and are intended to be within the scope of the present application.

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

The compounds disclosed herein may exist as atropisomers, which are conformational stereoisomers that occur when rotation about a single bond in a molecule is prevented or greatly slowed down 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 unspecified mixtures of each. Separation and isolation of the isomeric species may be tolerated if the rotational barrier around the single bond is sufficiently high and interconversion between conformations is sufficiently slow. The separation and isolation of the isomeric species is suitably indicated by the well known and widely accepted symbols "M" or "P". The term "cancer" refers to a disease characterized by uncontrolled growth of abnormal cells. Cancer cells may spread to other parts of the body locally or through the blood stream and lymphatic system. Examples of various cancers are described herein, including but not limited to non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, pancreatic cancer, prostate cancer, bladder cancer, liver cancer, skin cancer, glioma, breast cancer, melanoma, glioblastoma, rhabdomyosarcoma, ovarian cancer, astroglioma, ewing's sarcoma, retinoblastoma, epithelial cell carcinoma, colon cancer, renal cancer, gastrointestinal stromal tumor, leukemia, lymphoma, and nasopharyngeal carcinoma, among others. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms include solid and liquid, such as diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes premalignant lesions, malignant cancers and tumors.

An "effective amount" or "therapeutically effective amount" as used herein includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the route of administration and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

"subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, apes, humans, farm animals, athletic animals, and pets.

The amount of compound administered may depend on the subject being treated, the age, health, sex and weight of the subject, the type of concurrent therapy (if any), the severity of the condition, the nature of the effect desired, the manner and frequency of treatment, and the discretion of the prescribing physician. The frequency of administration may also depend on the pharmacodynamic effect on the arterial oxygen partial pressure. However, the most preferred dosage may be adjusted according to the individual subject, as understood by those skilled in the art and can be determined without undue experimentation. This typically involves adjusting a standard dose (e.g., reducing the dose if the patient is low in weight).

The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or salts thereof and a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to facilitate the administration of the compounds of the application to an organism. The pharmaceutical compositions of the application may include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and/or adjuvants, such as preserving, wetting, emulsifying and dispersing agents.

The "pharmaceutical composition" of the application may also be administered to a patient or subject in need of such treatment in any suitable manner of administration, such as oral, parenteral, rectal, pulmonary or topical administration, and the like. When used for oral administration, the pharmaceutical composition may be formulated into an oral preparation, for example, an oral solid preparation such as a tablet, capsule, pill, granule, etc.; or oral liquid preparations such as oral solutions, oral suspensions, syrups, etc. When formulated into an oral formulation, the pharmaceutical formulation may further comprise suitable fillers, binders, disintegrants, lubricants, etc.

The term "pharmaceutically acceptable carrier" refers to those excipients which do not significantly stimulate the organism and which do not impair the biological activity and properties of the active compound. Suitable excipients are well known to the person skilled in the art, for example carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, liposomes, polymeric micelles or inorganic nanocarriers and the like.

The pharmaceutical compositions of the application may be formulated in any pharmaceutically acceptable dosage form for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration, e.g., as tablets, troches, capsules, pills, solutions, suspensions, syrups, injections, suppositories, inhalants or sprays.

The term "synergistic effect" refers to the phenomenon that the effect of two drugs applied in combination is more effective than their effect alone, as opposed to antagonism.

The term "treating" includes prophylaxis and treatment, e.g., treatment of a CDK9 mediated disorder includes prophylaxis and/or treatment of a CDK9 mediated disorder.

The "combined" mode of administration of the present application is selected from simultaneous, separate and co-administration, or separate and sequential administration.

In the present application, the term "combination" or "combination" is intended to include each case where two drugs are administered sequentially or simultaneously, and "simultaneously" as used herein means that the CDK9 inhibitor and the BTK inhibitor are administered within the same administration period, for example, within 2 days, or within 1 day. By "sequential or sequential" administration is meant to include the case where the CDK9 inhibitor and the BTK inhibitor are administered separately in different dosing cycles. These modes of administration are all within the scope of the combination administration of the present application.

The BTK inhibitor of the application includes a stereoisomer, solvate or pharmaceutically acceptable salt thereof. For example, in one embodiment, the pharmaceutical composition of the present application comprises a compound of formula (I) and ibrutinib @II) or zebutinib (>III), i.e. comprising stereoisomers, solvates or pharmaceutically acceptable salts thereof of the compound of formula (I) and ibrutinib or of the compound of formula (II), or comprising stereoisomers, solvates or pharmaceutically acceptable salts thereof of the compound of formula (I) and zebutide or of the compound of formula (III).

The CDK inhibitor disclosed in the present application is a potent and selective CDK9 inhibitor, the structural formula of which is shown in the following formula (I), and the preparation methods of the compound of the formula (I) and pharmaceutically acceptable salts thereof are disclosed in International application PCT/CN 2018/070108 and PCT/CN2020/094527, wherein the name of the CDK inhibitor 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.

(I)

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

Experimental method

1) Cell culture

Human lymphoma cell line OCI-LY10 cells (BNCC 337742, north Nata-invasive Union Biotechnology Co., ltd.) were cultured using IMDM medium containing 10% fetal bovine serum, 37℃and 5% CO ₂ Culturing in an incubator. Observing the growth state of cells under an inverted microscope, selecting cells in logarithmic growth phase, inoculating fine cells according to 5×10e6/wellAnd (3) detecting target proteins after the cells are treated by adding medicines.

2) Cell drug addition treatment

a. Test drug configuration

Separately weighing maleate (molecular weight: 751.25), ibrutinib (molecular weight: 440.51,MedChemExpress, HY-10997-61547), zanubrutinib (molecular weight: 471.55,MedChemExpress, HY-101474A-79640), dissolving in DMSO to give a concentration of 10 mM, and storing in a refrigerator at-20deg.C.

b. Dosing treatment

On the first day, 10 mM of the Ibrutinib or Zanubrutinib stock was diluted with DMSO at a concentration of: (10 nM, 1 nM,0 nM) were added to the cells, respectively.

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

3) Target protein detection related experiments

a. Cell total protein extraction and quantification:

taking out the cell culture well plate, transferring the cell suspension into a centrifuge tube, centrifuging to collect cells, washing once with a PBS solution precooled at 4 ℃, then resuspending the cell pellet with an appropriate amount of precooled protein lysate (RIPA Buffer (10×), available from CST under the trade designation 9806, containing 1/100 volume of protease inhibitor (Halt Protease Inhibitor Cocktail 1 (100×), available from Thermo Fisher under the trade designation 78439) and phosphatase inhibitor (Phosphatase Inhibitor Cocktail 2 (100×), available from sigma under the trade designation P5726; phosphatase Inhibitor Cocktail 3 (100×), available from sigma under the trade designation P0044)), standing on ice for 30 min;13300 The mixture was centrifuged at 4℃for 20 min at rpm, and the supernatant was transferred to a new centrifuge tube and stored on wet ice for a while. And taking a proper amount of protein lysate, and measuring the protein concentration by using a BCA method. According to the protein concentration, an appropriate amount of LDS-loading buffer (NuPAGE LDS Sample Buffer (4×), available from Thermo Fisher, cat# NP 0007) and reducing agent (NuPAGE Sample Reducing Agent (10×), available from Thermo Fisher, cat# NP 0009) were added to the protein lysate, mixed well, the protein concentration was adjusted, then cooled to room temperature in a 98℃metal bath for 10 min, and then transferred to a-80℃refrigerator for storage.

b. SDS-PAGE gel electrophoresis and protein transfer:

the electrophoresis tank and the pre-gel were mounted, and then 20. Mu.g of protein (cellular protein extracted in step a) was loaded per well according to the plan for electrophoresis. Electrophoresis was stopped when bromophenol blue indicated that the front was near the bottom of the gel. The gel clamping groove is removed, the protein gel is carefully separated and transferred onto a nitrocellulose transfer membrane, and protein transfer is performed. After finishing the protein transfer, cutting a transfer film near the molecular weight of the target protein according to the molecular weight of the protein standard substance, and marking.

c. Western blot staining: transfer membranes were rinsed once with deionized water, then added with protein blocking solution, incubated at room temperature with slow shaking for 1 hr, then added with antibody buffer containing primary antibody (protein blocking solution containing 0.05% Tween-20) (primary antibody comprising MCL-1 (D2W 9E) Rabbit mAb, purchased from CST, stock number 94296; BFL1, purchased from CST, stock number 14093; BIM, purchased from CST, stock number 2933; C-MYC (D84C 12), purchased from abcam, stock number ab32072; clear Caspase-3 (Asp 175) (5A 1E), purchased from CST, stock number 9664), incubated at 4℃with slow shaking for three times with PBST solution, each time 5-10 min, added with antibody buffer containing secondary antibody (HRP conjugated anti-rabb antibody), incubated at room temperature with slow shaking for 1 hr, then rinsed three times with PBST solution, each time with 6H11, each time with shaking for 5-10 min, and developed on a machine.

d. And (3) data analysis processing: scanning and analysis were performed using Image Studio Ver 5.0 software, and the target protein expression was recorded.

Experimental results

The effect of the combination of the compounds of formula (I) maleate and Ibrutinib, zanubrutinib on the Western blot results of target proteins (FIG. 1), c-MYC, MCL-1, BFL-1 and BIM protein expression (FIG. 2), and Cl-Caspase3 protein expression (FIG. 3), respectively, are shown in FIGS. 1-3. The maleate salt of the formula (I) alone and Ibrutinib, zanubrutinib in combination respectively reduce the expression of the pro-cell survivin c-MYC, MCL-1 and BFL-1, and the combination of the other two drugs increases the expression of the pro-apoptotic protein BIM and the significant increase of Cl-Caspase3 (index protein of apoptosis).

Experimental method

1) Cell culture

Human lymphoma cell line OCI-LY10 cells (BNCC 337742, north Nata-invasive Union Biotechnology Co., ltd.) were cultured using IMDM medium containing 10% fetal bovine serum, 37℃and 5% CO ₂ Culturing in an incubator. Observing the growth state of cells under an inverted microscope, selecting cells in the logarithmic growth phase, inoculating the cells according to 4000/hole, arranging two auxiliary holes in each dosing group, and detecting the activity of the cells after dosing treatment.

2) Cell drug addition treatment

a. Test drug configuration

Separately, the maleate salt (molecular weight: 751.25), ibutinib (molecular weight: 440.51), zanubrutinib (molecular weight: 47.56) of the compound of formula (I) was weighed, dissolved in DMSO to a concentration of 10 mM, and stored in a refrigerator at-20 ℃.

b. Dosing treatment

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

The following day (24 hours later), 10 mM of the maleate salt of the compound of formula (I) was added to cells containing Ibrutinib at a concentration gradient (100 nM,50nM,0 nM) or Zanubrutinib at a concentration gradient (100 nM,50nM,25nM,0 nM).

On the third day (after 24 hours), a subsequent cell viability assay was performed.

3) Cell viability assay and data analysis

a. Cell viability assay: cellTiter Glo lysate was prepared according to the instructions of CellTiter Glo reagent (Progema, G7573). From the cells in the incubator, microscopic observation; 1/2 volume of Cell Titer Glo lysate was added to each well, the cells were allowed to lyse well by shaking in the dark for 2 minutes, then left in the dark at room temperature for 10 minutes, and chemiluminescent signals (RLU) were read with an microplate reader.

b. Data analysis processing

RLU readings from each well were subtracted from RLU readings from blank wells (medium with 0.1% DMSO alone and without cells) as cell viability values from each well. Cell viability was calculated according to the following formula: cell viability relative proportion= ("RLU compound-RLU blanc)/(RLU DMSO-RLU blanc)) ×100%. RLU compound is read from cell compound treated wells, RLU blank is read from blank wells, and RLU DMSO is read from cell DMSO treated wells. Mapping was performed using Prism GraphPad 6 for windows v6.01 software.

Experimental results

The results of inhibition of OCI-LY-10 cell viability by the combination of the compounds of formula (I) maleate and Ibrutinib, zanubrutinib, respectively, are shown in fig. 4 and table 1, and fig. 5 and table 2. Fig. 4: * *P<0.01, ***P<0.001; fig. 5: *P<0.05, **P<0.01, ***P<0.001。PValues were calculated using GraphPad Prism based on the results of tables 1 and 2.

TABLE 1

TABLE 2

The foregoing descriptions of specific exemplary embodiments of the present application are presented for purposes of illustration and description. It is not intended to limit the application to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the application and its practical application to thereby enable one skilled in the art to make and utilize the application in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims and their equivalents.

Claims

1. A pharmaceutical composition or kit comprising a therapeutically effective amount of a CDK9 inhibitor and a therapeutically effective amount of a BTK inhibitor, said CDK9 inhibitor being a compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; the BTK inhibitor is ibrutinib or zebutinib;

（I）；

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

2. The pharmaceutical composition or kit according to claim 1, wherein the pharmaceutically acceptable salt of the compound of formula (I) comprises a maleate salt and/or a fumarate salt.

3. The pharmaceutical composition or kit of claim 1, wherein the pharmaceutical composition or kit comprises 100nM of the compound maleate salt of formula (I) and 1-10 nM ibrutinib; or (b)

The pharmaceutical composition or kit comprises 100nM of the compound maleate salt of formula (I) and 1-10 nM zebutinib.

4. Pharmaceutical composition or kit according to claim 1, wherein the mass ratio of CDK9 inhibitor to BTK inhibitor is from 0.001 to 1000.

5. The pharmaceutical composition or kit according to claim 1, wherein the pharmaceutical composition or kit comprises the compound of formula (I) maleate salt and ibrutinib in a mass ratio of 5, 10, 40 or 100; or alternatively, the first and second heat exchangers may be,

the pharmaceutical composition or the kit comprises the compound of formula (I) maleate and zebutinib in a mass ratio of 5, 10, 20, 40, 50 or 100.

6. Use of a pharmaceutical composition or kit according to any one of claims 1-5 for the manufacture of a medicament for the treatment of lymphoma.

7. Use of a CDK9 inhibitor and a BTK inhibitor in the preparation of a medicament or kit for the treatment of lymphoma, said CDK9 inhibitor being a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof; the BTK inhibitor is ibrutinib or zebutinib;

（I）

8. The use according to claim 7, wherein the pharmaceutically acceptable salts of the compounds of formula (I) comprise maleate and/or fumarate salts.

9. The use of claim 7, wherein the pharmaceutical composition or kit comprises 100nM of the compound maleate salt of formula (I) and 1-10 nM ibrutinib; or (b)

10. The use according to claim 7, wherein the mass ratio of CDK9 inhibitor to BTK inhibitor is from 0.001 to 1000.

11. The use according to claim 7, wherein the pharmaceutical composition or kit comprises the compound maleate salt of formula (I) and ibrutinib in a mass ratio of 5, 10, 40 or 100; or alternatively, the first and second heat exchangers may be,