CN116270644A

CN116270644A - Pharmaceutical combination of CDK9 inhibitor and BCL-2 inhibitor and application thereof

Info

Publication number: CN116270644A
Application number: CN202310574464.2A
Authority: CN
Inventors: 周福生; 兰炯; 吕强
Original assignee: Genfleet Therapeutics Shanghai Inc
Current assignee: Genfleet Therapeutics Shanghai Inc
Priority date: 2023-05-22
Filing date: 2023-05-22
Publication date: 2023-06-23

Abstract

The invention provides a pharmaceutical composition or kit comprising a therapeutically effective amount of a CDK9 inhibitor and a therapeutically effective amount of a BCL-2 inhibitor, its use 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 BCL-2 inhibitor, the composition being used in combination in cancer cells with a good synergistic effect.

Description

Pharmaceutical combination of CDK9 inhibitor and BCL-2 inhibitor and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a drug product containing a CDK9 inhibitor and a BCL-2 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 invention there is provided a pharmaceutical composition or kit comprising a therapeutically effective amount of a CDK9 inhibitor and a therapeutically effective amount of a BCL-2 inhibitor, said CDK9 inhibitor being a compound of formula (I), a stereoisomer, solvate or a pharmaceutically acceptable salt thereof. The CDK9 inhibitor and the BCL-2 inhibitor have obvious synergistic effects when used in combination.

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 BCL-2 inhibitor specifically inhibits the BCL-2 protein and does not inhibit the BCL-xl or BCL-w protein.

In one embodiment, the BCL-2 inhibitor includes one or more selected from Navitocrax (ABT-263), venetoclax (ABT-199), pelcitaclax (APG-1252), A-1155463, A-1331852, ABT-737, obatocrax, S44563, TW-37, AT101, HA14-1, and Sabutocrax.

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

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

In one embodiment, the CDK9 inhibitor and the BCL-2 inhibitor are present in a mass ratio of 0.001-1000, which may be, for example, 0.001-1000, 0.001-500, 0.004-250, 0.004-242.72, 0.01-100 or 0.1-10.

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 to 1000nM CDK9 inhibitor.

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

In one embodiment, the pharmaceutical composition or kit comprises 4.12-1000 nM BCL-2 inhibitor.

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

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

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

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

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

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

In another aspect of the invention 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 invention 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 manufacture of a medicament or kit or combination of a medicament for the treatment of a CDK9 mediated disease or condition. The CDK9 inhibitor and the BCL-2 inhibitor have obvious synergistic effects when used in combination.

In one embodiment, the BCL-2 inhibitor specifically inhibits a BCL-2 protein.

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

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

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

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

The invention 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, 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 present invention also provides an administration dose of the BCL-2 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, 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 invention 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 BCL-2 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 is a graph showing the growth inhibition matrix of tumor cells HL-60 by the combination of maleate salt of the compound of formula (I) in example 1 and Venetoclax.

FIG. 2 shows the average synergy calculated in the Bliss statistical model of example 1.

Fig. 3 shows the average synergy calculated in the HSA statistical model of example 1.

Fig. 4 shows the average synergy calculated in the Loewe statistical model of example 1.

FIG. 5 shows the average synergy calculated in the ZIP statistical model of example 1.

FIG. 6 is a graph showing the growth inhibition matrix of tumor cells Kaumi-1 by the combined action of maleate salt of the compound of formula (I) in example 2 and Venetoclax.

FIG. 7 shows the average synergy calculated in the Bliss statistical model of example 2.

Fig. 8 shows the average synergy calculated in the HSA statistical model in example 2.

Fig. 9 shows the average synergy calculated in the Loewe statistical model of example 2.

FIG. 10 shows the average synergy calculated in the ZIP statistical model of example 2.

FIG. 11 is a graph showing tumor growth curves (mean.+ -. Standard error) for each group of tumor-bearing mice in example 3.

FIG. 12 is a graph showing tumor growth curves for individual tumor-bearing mice of example 3.

FIG. 13 shows the relative animal weight change (mean.+ -. Standard error) of tumor-bearing mice in example 3.

Detailed Description

I. Definition and description

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 invention prepared from the compounds of the present invention which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present invention 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 invention 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 invention contain basic and acidic functionalities that can be converted to either base or acid addition salts.

Pharmaceutically acceptable salts of the invention 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 invention. In addition, prodrugs can be converted to the compounds 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 agent, the prodrug may be slowly converted to the compound of the invention.

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

The compounds of the 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 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 invention.

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 administration of the compounds of the present application to an organism. The pharmaceutical compositions of the invention 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 invention 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 invention 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 invention is selected from simultaneous, separate and co-administration, or separate and sequential administration.

In the present invention, 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 BCL-2 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 BCL-2 inhibitor are administered separately during different dosing cycles. These modes of administration are all within the scope of the combination administration of the present invention.

The BCL-2 inhibitor comprises a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof. For example, in one embodiment, the pharmaceutical composition of the present invention comprises a compound of formula (I) and Venetoclax @

II), also known as ABT199 or ABT-199, GDC0199, is meant to include the use of Venetoclax or stereoisomers, solvates or pharmaceutically acceptable salts thereof of the compounds of formula (II).

Detailed description of the preferred embodiments

The CDK inhibitor disclosed in the present invention 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.

Cell culture (examples 1-2)

Observing the growth state of cells under an inverted microscope, selecting cells in a logarithmic growth phase, and performing aseptic operation in a biosafety cabinet. Transferring cell suspension into centrifuge tube, centrifuging to precipitate cells, removing supernatant, re-suspending cells with fresh complete culture medium, dispersing into new culture bottle at proper ratio, placing into 37deg.C, 5% CO ₂ Culturing in an incubator.

Combined group cell survival proportion calculation method

The cell survival ratio (% cell survival rate) after the combination of the two drugs is calculated as follows: cell survival ratio = (1- (DMSO control RLU-drug RLU)/(DMSO control RLU-blank control RLU)) ×100%. DMSO control was vehicle control without drug, blank control was medium control without drug and vehicle.

Interpretation of cell survival proportion calculation results

In the graph of the calculation results of cell survival ratio (fig. 1 and 6), the abscissa represents the concentration of maleate salt (effective concentration of drug in the well solution) of the compound of formula (I) in the present application, the ordinate represents the concentration of the drug to be used (effective concentration of drug in the well solution), the abscissa and the ordinate are marked as vertical lines, respectively, the points of intersection of the two vertical lines are marked as the cell survival ratio of the drug to be used in the two concentrations. For example, in fig. 1, the cell viability ratio is 82.8% when the horizontal reading is 37.04 nM and the vertical reading is 0nM, the two concentration scales are perpendicular to each other, and the value of the grid where the two perpendicular lines intersect is 82.8, indicating that the Venetoclax concentration is 0nM, i.e., the cell viability ratio is 82.8% when the maleate salt of the compound of formula (I) is administered alone.

Synergistic effect evaluation method

The effect of the combination of the two drugs on the tumor cells is evaluated by adopting a statistical method. The statistical software used is R systems, the statistical model is Bliss, HSA, loewe, ZIP, when the synergy value is > 5, the synergy value is > 10, the synergy value is obvious, the synergy value is < -5, the antagonism effect, and the synergy value is < -10, the antagonism effect is obvious. And further judging the final efficacy of the combined administration of the two medicaments according to the synergy value.

EXAMPLE 1 Effect of the Compound maleate salt of formula (I) in combination with the BCL-2 inhibitor Venetoclax on HL-60 tumor cells

Human acute promyelocytic leukemia cells HL-60 (ATCC, CCL-240) were inoculated into 96-well plates at a rate of 5000 cells per well, and after cell attachment (24 h), the compound maleate of formula (I) and Venetoclax drug were added to dilute to a gradient concentration of 2 multiple wells per concentration. After 6h, cell viability was measured by the Cell Titer-Glo method, 1/2 volume of Cell Titer Glo lysate (Progema, G7573) was added to each well, shake-plated for 2 min, and left to stand for 10 min, and RLU values were read on instrument Envision.

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

The combined effect is shown in fig. 2-5, in human acute promyelocytic leukemia cells HL-60, the maleate salt of the compound of the formula (I) of 4.12 nM-1000 nM is combined with vennetoclax of 4.12 nM-1000 nM, and the average synergy values calculated in four statistical models of Bliss (fig. 2), HSA (fig. 3), loewe (fig. 4) and ZIP (fig. 5) are 21.302, 21.319, 19.075 and 21.814 respectively, which are all far greater than 5. The compound maleate salt of formula (I) was shown to have a good synergistic effect in combination with Venetoclax in tumor cells.

EXAMPLE 2 Effect of the Compound maleate salt of formula (I) in combination with the BCL-2 inhibitor Venetoclax on Kaumi-1 tumor cells

Human acute lymphoblastic leukemia cells Kasumi-1 (ATCC, CRL-2724) were inoculated into 96-well plates at 4000 cells per well, and after cell attachment (24 h), the compound maleate of formula (I) and Venetoclax drug were added to dilute to a gradient concentration of 2 multiple wells per concentration. After 24h, cell viability was measured by the Cell Titer-Glo method, 1/2 volume of Cell Titer Glo lysate (Progema, G7573) was added to each well, shake-plated for 2 min, and left to stand for 10 min, and RLU values were read on instrument Envision.

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

The combined effect is shown in fig. 7-10, in human acute lymphoblastic leukemia cell Kasumi-1, the maleate salt of the compound of formula (I) of 4.12 nM-1000 nM is combined with vennetoclax of 4.12 nM-1000 nM, and the average synergy values calculated in four statistical models of Bliss (fig. 7), HSA (fig. 8), loewe (fig. 9) and ZIP (fig. 10) are 5.502, 12.611, 9.139 and 5.924 respectively, which are all greater than 5. The compound maleate salt of formula (I) was shown to have a good synergistic effect in combination with Venetoclax in tumor cells.

EXAMPLE 3 in vivo antitumor pharmacodynamic evaluation of the Compound maleate salt of formula (I) in combination with the BCL-2 inhibitor Venetoclax on 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)。

Observing animal health condition every day during experiment, for example, animal weight is reduced by 10%, and administration dosage is halved; weight loss of 15%, stopping administration until weight recovery; or animal tumor volume exceeding 2,000 mm ³ Immediately to euthanize. The health condition is as follows, informing the veterinarian and at the time of euthanasia:

obvious emaciation and body weight reduction of more than 20 percent.

Can not eat and drink water freely.

Animals develop infection, severe tumor destruction, or hematoma.

Animals developed the following clinical symptoms and continued to worsen: hair, bow back, ear, nose and eye or foot blushing, shortness of breath, tics, diarrhea, pain, dehydration, dying.

Experimental animals:

BALB/c nude mice, 6-8 week old, females, purchased from Beijing Veitz Lihua laboratory animal technologies Co., ltd., animal eligibility number 20170011005453, feeding environmental SPF grade.

Experimental drugs:

venetoclax (ABT 199) was purchased from Shanghai and Emotion chemical Co., ltd., lot number 1257044-40-8.

Preparation method comprises injecting maleic acid salt of the compound of formula (I) into tail vein, and dissolving in PBS (0.3402 g KH ₂ PO ₄ Dissolving in 50 mL ultrapure water, adjusting pH=7.0-7.2, and autoclaving); venetoclax vehicle 5% DMSO+50% PEG400+5% Tween80+ddH ₂ O, autoclaving.

The experimental method comprises the following steps:

HL-60 cells were cultured in IMDM medium supplemented with 20% FBS and 1% penicillin-streptomycin at 37deg.C, 5% CO ₂ Is cultured in an incubator of (a). Collecting cells, and adjusting cell density to 2.0X10 ⁸ /mL; the right back of the mice was inoculated subcutaneously at a volume of 0.20. 0.20 mL per animal (50% Matrigel) and at an inoculum size of 2X 10 per animal ⁷ Individual cells. When the tumor grows to 140-230 mm ³ Time%D13 32 tumor-bearing mice were selected and randomly divided into 4 groups of 8 mice each, and the dosing method was performed according to the experimental protocol of table 1. The administration volume of the mice is 10 mL/kg. Animals were weighed daily with an electronic balance and tumor volumes were measured 3 times per week with vernier calipers.

Table 1 dosing regimen for therapeutic agents

Note that: p.o is oral; v is tail vein injection; q.d is once per day; biw is administered 2 times per week for 2 days continuously and stopped for 5 days.

The main evaluation indexes are as follows:

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

Tumor growth inhibition (tumor growth inhibition, TGI):

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

Tumor weight inhibition (tumor weight inhibition, TWI):

inhibition ratio of Tumor Weight (TWI) = (1-TW) _treatment/Dx /TW _control/Dx ) 100%, TW therein _control Average tumor weight (g), TW of control group _treatment Mean tumor weight (g) of the treatment group.

Relative body weight change (Relative change of body weight, RCBW):

RCBW (%) = (BW _i -BW ₀ ) BW 0X 100%; wherein BW is _i Is the weight and BW of animals on a specific day ₀ Is the weight of the animal on the day of initial dosing.

Experimental results and conclusions

Analysis and mapping of all experimental data was done using GraphPad Prism software (GraphPad Software). Tumor volumes of each group were statistically compared to animal body reuse Two-way ANOVA (Dunnett's multiple comparisons test), and tumor weights of each group at endpoint were statistically compared to One-way ANOVA (One-way ANOVA, dunnett's multiple comparisons test). Statistically significant differences were considered when P < 0.05. Tumor volume, weight, and animal body weight are all expressed as Mean ± Standard Error (SEM). The results are shown in Table 2 below.

TABLE 2

Note that: * P < 0.01, p < 0.001, respectively compared to the G1 vehicle control group.

Effect of each treatment group on tumor growth in HL-60 tumor-bearing mice (see fig. 11 and 12); the body weight changes of each group of tumor-bearing mice are shown in FIG. 13.

The results show that on an HL-60 human acute promyelocytic leukemia model, the maleate of the compound of the formula (I) is administrated by tail vein injection twice a week (continuously for 2 days, and the administration is stopped for 5 days) at10 mg/kg, and Venetoclax is orally taken once a day at 100mg/kg, so that the maleate of the compound of the formula (I) and the Venetoclax have no obvious influence on animal weight, and the combined use of the maleate of the compound of the formula (I) and the Venetoclax has obviously better drug effects than the single drug.

The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention 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 invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims

1. MedicineA composition or kit comprising a therapeutically effective amount of a CDK9 inhibitor and a therapeutically effective amount of Venetoclax, said CDK9 inhibitor being a compound of formula (I), a stereoisomer, solvate or a pharmaceutically acceptable salt thereof.

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. A pharmaceutical composition or kit according to claim 1 wherein the mass ratio of CDK9 inhibitor to Venetoclax is from 0.004 to 250.

4. A pharmaceutical composition or kit according to claim 3 wherein the mass ratio of CDK9 inhibitor to Venetoclax is 20 (200-700) or 20 (575-700).

5. A pharmaceutical composition or kit according to claim 1 wherein the CDK9 inhibitor is administered in an amount selected from 0.01-5000 mg/day and the BCL-2 inhibitor is administered in an amount selected from 0.01-1000 mg/day.

6. Use of a pharmaceutical composition or kit according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of a CDK9 mediated disease or condition.

Use of a CDK9 inhibitor, which CDK9 inhibitor is a compound of formula (I), a stereoisomer, solvate or a pharmaceutically acceptable salt thereof, in combination with Venetoclax for the preparation of a medicament or kit or medicament for the treatment of a CDK9 mediated disease or condition.

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 according to claim 7, wherein the mass ratio of CDK9 inhibitor to Venetoclax is from 0.004 to 250.

10. The use according to claim 7, wherein the mass ratio of CDK9 inhibitor to Venetoclax is 20 (200-700) or 20 (575-700).

11. The use according to claim 6 or 7, wherein the CDK9 mediated disease or condition is cancer.

12. The use of claim 11, wherein the cancer is a solid tumor or hematological tumor.

13. The use of claim 11, wherein 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, renal cancer, gastrointestinal stromal tumor, leukemia, lymphoma, and nasopharyngeal carcinoma.