Anti-tumor combined pharmaceutical composition and application thereof

Abstract

The invention provides a combined pharmaceutical composition of a CDK8 / 19 inhibitor and azacitidine and application of the combined pharmaceutical composition in preparation of antitumor drugs. Particularly, the compound shown in the formula (B) and azacitidine are combined for use, so that a relatively good synergistic effect is shown in a cell proliferation inhibition experiment and an in-vivo tumor model. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, specifically to a combined pharmaceutical composition of a CDK8 / 19 inhibitor and azacitidine and its use in the preparation of anti-tumor drugs. Background Art

[0002] CDK8 and CDK19 are tumor regulatory transcriptional kinases with high homology in both structure and function. Different from other kinases in the CDK family, such as CDK1, CDK2, and CDK4 / 6, CDK8 does not play a role in cell cycle regulation. Therefore, blocking CDK8 does not inhibit the growth of normal cells. However, since CDK8 plays a crucial role in the formation of the phenotype of pluripotent stem cells, knocking out CDK8 in embryonic stem cells will lead to embryonic development arrest. CDK8 plays an important role in regulating transcription by binding to the mediator complex or phosphorylating transcription factors. Numerous genetic and biochemical studies have identified CDK8 as a key oncogenic driver in many cancers. Specifically, CDK8-mediated activation of the oncogenic Wnt-β-catenin signal, transcription of estrogen-induced genes, and inhibition of super-enhancer-related genes contribute to tumorigenesis in colorectal, breast, and hematological malignancies, respectively. Currently, overexpression of CDK8 has been observed in approximately 50% of colorectal cancers, melanomas, and breast cancers, and is associated with poor prognosis. In addition, studies have also shown that CDK8 inhibitors can provide an important method for anti-cancer treatment as a single drug or in combination with various anti-tumor therapies or agents that activate the immune system. For example, it can be used to treat blood diseases (such as the treatment of AML and myelodysplastic syndrome (MDS)).

[0003] Azacitidine (Aza) is a cytosine nucleoside analogue that exerts anti-tumor effects by causing DNA demethylation and direct cytotoxic effects on abnormal hematopoietic cells in the bone marrow. The concentration at which azacitidine has the greatest inhibitory effect on DNA methylation in vitro does not significantly inhibit DNA synthesis. The incidence of high-risk MDS and AML is high, and the prognosis is poor (the five-year survival rate is only about 30%). In particular, for elderly or frail patients who cannot tolerate high-dose chemotherapy and patients with accompanying high-risk diseases, the treatment options are very limited: Azacitidine is the standard first-line therapy for high-risk MDS; The combination therapy of the Bcl-2 inhibitor Venetoclax and azacitidine has begun to be used in the treatment of AML that cannot tolerate traditional intensive chemotherapy; However, azacitidine has relatively significant blood, liver, and kidney toxicity, and Venetoclax also has significant blood toxicity and is extremely prone to drug resistance and recurrence. Therefore, for MDS and AML patients who are elderly or frail and cannot tolerate intensive chemotherapy, there is still an unmet medical need, and there is an urgent need for a combination of azacitidine and new drugs to better improve its safety and effectiveness.

[0004] CDK8 / 19 inhibitors disclosed in the prior art:

[0005]

[0006] Patent CN202211453864.X discloses a small molecule CDK8 / 19 inhibitor, the structure of which is shown in formula (B). This small molecule inhibitor has good CDK8 / 19 kinase inhibitory activity and various cell anti-proliferative activities. At the same time, this molecule has good pharmacokinetic properties and in vivo efficacy, and is expected to be developed into a clinical drug.

[0007]

[0008] There is no prior art report on the combined use of CDK8 / 19 inhibitors and azacitidine, and it is also unpredictable whether there are clinical benefits in this combination. The present invention aims to provide a combined pharmaceutical composition of a CDK8 / 19 inhibitor and azacitidine. Summary of the Invention

[0009] The purpose of the present invention is to provide a combined pharmaceutical composition of a CDK8 / 19 inhibitor and azacitidine and its use in the preparation of anti-tumor drugs.

[0010] Specifically,

[0011] The present invention provides a combined pharmaceutical composition, comprising a CDK8 / 19 inhibitor and azacitidine.

[0012] In some embodiments of the present invention, the above-mentioned CDK8 / 19 inhibitor is selected from SNX631, D-SNX631, Senexin B, Senexin C, BI-1347, RVU120, the compound shown in formula (B), and any combination thereof:

[0013]

[0014] In some embodiments of the present invention, the above-mentioned CDK8 / 19 inhibitor is selected from the compound shown in formula (B):

[0015]

[0016] The present invention also provides the use of the above-mentioned combined pharmaceutical composition in the preparation of drugs for treating hematological tumors.

[0017] In some embodiments of the present invention, the hematological tumors in the above-mentioned use are selected from acute myeloid leukemia, myelodysplastic syndrome, and myeloproliferative hematological tumors.

[0018] In some embodiments of the present invention, the hematological tumors in the above-mentioned use are selected from hematological tumors insensitive to azacitidine.

[0019] In some embodiments of the present invention, in the above-mentioned use, the CDK8 / 19 inhibitor and azacitidine can be administered simultaneously, sequentially, or at intervals.

[0020] The present invention also provides a method for treating a tumor, which comprises administering to the subject an effective therapeutically amount of a CDK8 / 19 inhibitor and azacitidine.

[0021] In some embodiments of the present invention, the CDK8 / 19 inhibitor in the above-mentioned treatment method is selected from SNX631, D-SNX631, Senexin B, Senexin C, BI-1347, RVU120, the compound shown in formula (B), and any combination thereof:

[0022]

[0023] In some embodiments of the present invention, the CDK8 / 19 inhibitor in the above-mentioned treatment method is selected from the compound shown in formula (B):

[0024]

[0025] In some embodiments of the present invention, the tumor in the above-mentioned treatment method is selected from hematological tumors.

[0026] In some embodiments of the present invention, the hematological tumors in the above-mentioned treatment method are selected from acute myeloid leukemia, myelodysplastic syndrome, and myeloproliferative hematological tumors.

[0027] In some embodiments of the present invention, the hematological tumors in the above-mentioned treatment method are selected from hematological tumor cells insensitive to azacitidine.

[0028] In some embodiments of the present invention, the CDK8 / 19 inhibitor and azacitidine in the above-mentioned treatment method can be administered simultaneously, sequentially, or at intervals.

[0029] The present invention provides the use of a CDK8 / 19 inhibitor and azacitidine in combination in the preparation of a medicament for treating a tumor.

[0030] In some embodiments of the present invention, in the above-mentioned use, the CDK8 / 19 inhibitor is selected from the compound shown in formula (B):

[0031]

[0032] In some embodiments of the present invention, in the above-mentioned use, the tumor is selected from hematological tumors.

[0033] In some embodiments of the present invention, in the above-mentioned use, the hematological tumors are selected from acute myeloid leukemia, myelodysplastic syndrome, and myeloproliferative hematological tumors.

[0034] In some embodiments of the present invention, in the above use, the hematological tumor is selected from hematological tumors insensitive to azacitidine.

[0035] In some embodiments of the present invention, in the above use, the CDK8 / 19 inhibitor and azacitidine can be administered simultaneously, sequentially, or at intervals.

[0036] In some embodiments of the present invention, in the above use, the dosage of the CDK8 / 19 inhibitor is 60 mg / kg, administered once a day.

[0037] In some embodiments of the present invention, in the above use, the dosage of azacitidine is 2 mg / kg, administered once a day.

[0038] In some embodiments of the present invention, in the above use, the CDK8 / 19 inhibitor is administered orally.

[0039] In some embodiments of the present invention, in the above use, the daily dosage of CDK8 / 19 is 1 - 1000 mg, administered once a day or twice a day.

[0040] In some embodiments of the present invention, in the above use, azacitidine is subcutaneously or intravenously injected at 75 mg / m2 per day for 7 days, and in subsequent treatment cycles, it is administered once every 4 weeks. If no beneficial effect is found after 2 treatment cycles and no toxicity occurs except nausea and vomiting, the dose can be increased to 100 mg / m2.

[0041] In some embodiments of the present invention, in the above use, azacitidine is subcutaneously or intravenously injected at 5 - 75 mg / m2 per day for 7 days, and in subsequent treatment cycles, it is administered once every 4 weeks. If no beneficial effect or poor effect is found after 2 treatment cycles and no toxicity occurs except nausea and vomiting, the dose can be increased to 100 mg / m2.

[0042] Technical effects

[0043] Research data show that the combined use of CDK8 / 19 and azacitidine of the present invention, especially the combined use of the compound of formula (B) and azacitidine, exhibits good synergistic effects in both cell proliferation inhibition experiments and in vivo tumor models. The CDK8 / 19 inhibitor and azacitidine show potential for clinical combination use, which can significantly reduce the dosage of azacitidine and improve the effectiveness of azacitidine, suggesting that it may expand the population of patients who can use azacitidine.

[0044] Definitions and explanations

[0045] Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered indefinite or unclear if it is not specifically defined, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commodity or its active ingredient.

[0046] The term "combination pharmaceutical composition" means that two drugs are separately contained in different dosage unit formulations and can be administered simultaneously, sequentially, or at intervals.

[0047] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0048] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, prepared from a compound having specific substituents found in the present invention and a relatively non-toxic acid or base. When a compound of the present invention contains a relatively acidic functional group, the base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of base in a pure solution or a suitable inert solvent. When a compound of the present invention contains a relatively basic functional group, the acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compounds containing acid radicals or basic groups by conventional chemical methods. Generally, the preparation method of such salts is to react these compounds in the form of free acids or bases with a stoichiometric amount of an appropriate base or acid in water, an organic solvent, or a mixture of both.

[0049] The term "effective therapeutic amount" means an amount of a compound of the present invention or a pharmaceutically acceptable salt that is sufficient to treat a disorder with a reasonable effect / risk ratio applicable to any medical treatment and / or prevention. However, it should be recognized that the total daily dosage of the pharmaceutically acceptable salts and compositions of the compounds of formula I of the present invention must be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dose level will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the specific compound employed; the duration of the treatment; drugs used in combination with or simultaneously with the specific compound employed; and similar factors well known in the medical art. For example, it is common practice in the art to start the dosage of a compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is obtained.

[0050] The term "administer" means physically introducing a composition comprising a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those skilled in the art, including but not limited to oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, buccal, intranasal, by inhalation, vaginal, intraocular, by topical administration, subcutaneous, intra-adipose, intra-articular, intraperitoneal, and intrathecal. In certain specific embodiments, administration is by oral administration.

[0051] The term "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In certain embodiments, the subject is a human. The terms "subject" and "patient" may be used interchangeably in certain contexts herein.

[0052] "Synergy" or "synergistic effect" means that when two drugs are used in combination, their overall effect is greater than the sum of the effects of each drug used alone, their directions of action are the same, and they achieve an effect of mutual enhancement. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a graph showing the in vitro synergistic effect of the compound of formula (B) and azacitidine on cell proliferation inhibition.

[0054] Figure 2 It is a graph showing the in vivo pharmacodynamic synergistic effect of the compound of formula (B) and azacitidine. DETAILED DESCRIPTION OF THE INVENTION

[0055] The present invention will be described in detail below by way of examples, but these examples are only for illustration and do not limit the scope of the present invention in any way. Similarly, the present invention is not limited to any specific preferred embodiments described herein. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the technical features of the present invention still fall within the protection scope of the present invention. Example 1: Cell Proliferation Inhibition Test of the Compound of Formula (B) in Combination with Azacitidine

[0056] 1. Experimental Purpose

[0057] To study the effect of the compound of formula (B) in combination with azacitidine on the proliferation inhibition of hematological tumor cell lines by the CTG (Cell Titer-Glo Luminescent viability assay) detection method, so as to evaluate the effect of their combination.

[0058] 2. Experimental Materials

[0059] 2.1 Cell Information

[0060] Table 1. Cell Information

[0061] Serial number Cell name Source tissue Cell source 1 MV-4-11 Acute myeloid leukemia ATCC(CRL-9591)

[0062] 2.2 Sample Information

[0063] Table 2. Sample Information

[0064] Name Batch number Purity Storage conditions Source Compound of formula (B) ZYJ-F2077P70 99.38％ 2-8℃ Qilu Pharmaceutical Co., Ltd. Azacitidine T2001L41KA 100％ 2-8℃ Shandong Anhong Pharmaceutical Co., Ltd.

[0065] 2.3 Other Reagents and Consumables

[0066] Table 3. Information of Other Reagents and Consumables

[0067] Name Manufacturer Article number FBS Gibco 10099-141C RPMI1640 Gibco 11875-093 Cell Titer-Glo Luminescent viability assay kit Promega G7570 384-well white clear-bottom cell culture plate Greiner 781098

[0068] 3. Experimental Methods

[0069] 3.1 Experimental Design

[0070] Analyze the inhibitory effects of compound (B) and azacitidine on the proliferation of several hematological tumor cells, with two replicates set. The concentration range of compound (B) is 0.15 - 1000 nM, and the concentration range of azacitidine is 1.52 - 10000 nM.

[0071] 3.2 Experimental Procedures

[0072] (1) Preparation of the working solution concentration of the compound

[0073] Prepare the compound to be tested and add it to the specified wells of the T8 cassette according to the software prompt during dosing.

[0074] (2) Cell seeding and drug treatment

[0075] One day before compound treatment, centrifuge the cells, count them with a cell counter, dilute the cells to the specified concentration with the corresponding culture medium according to the counting results, and use a multi-channel pipetting system to add the cells to the corresponding 384-well microplate as required, and culture them overnight in a 37°C incubator.

[0076] Take out the cells cultured overnight, add the compound using the D300E according to the microplate layout diagram, and culture them in the incubator for 5 - 7 days. After the incubation is completed, take out the culture plate, equilibrate it to room temperature for 30 minutes, take out the pre-prepared Cell reagent, equilibrate it to room temperature for 30 minutes and mix well, use a multi-channel pipetting system, add 20 μL of Cell reagent to each well, shake it at 500 revolutions per minute on a microplate thermostatic shaker for 10 minutes, and let it stand at room temperature for 10 minutes. The above operations need to be carried out under light-proof conditions. Use an Envision microplate reader (PerkinElmer) to detect the luminescence signal.

[0077] 3.3 Data Analysis

[0078] The SynergyFinder online analysis software (https: / / synergyfinder.fimm.fi / ) was used to evaluate the drug synergy effect, and the ZIP (Zero-inflated Poisson) regression mixture model was used for calculation.

[0079] 4. Experimental results

[0080] Table 4. Results of inhibitory effect on cell proliferation of the compound of formula (B) in combination with azacitidine

[0081]

[0082] 5 Experimental conclusions

[0083] As can be seen from Table 4 and Figure 1 it can be seen that the compound of formula (B) and azacitidine have good synergistic effects on inhibiting cell proliferation (the darker the color in the red area, the stronger the synergistic effect. A synergistic effect score greater than 3 indicates a synergistic effect, and greater than 10 indicates a strong synergistic effect).

[0084] Example 2: In vivo pharmacodynamic study of the compound of formula (B) in combination with azacitidine on the MV4-11 xenograft tumor model

[0085] 1. Experimental purpose

[0086] To evaluate the in vivo efficacy of the compound of formula (B) in combination with azacitidine on the subcutaneous xenograft tumor BALB / c Nude mouse model of human myelomonocytic leukemia MV4-11 cells.

[0087] 2. Experimental materials

[0088] 2.1 Experimental animals and breeding environment

[0089] Species: Mouse

[0090] Strain: BALB / c Nude mouse

[0091] Arrival age: 6-8 weeks old

[0092] Gender: Female

[0093] Body weight: 18-22 g

[0094] Supplier: Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0095] Animal certificate: 20230216Abzz0619000783

[0096] 2.2 Compound information

[0097] Table 5. Compound information

[0098] Name Batch number Purity Storage conditions Source Compound of formula (B) ZYJ-F2077P70 99.38％ 2-8℃ Qilu Pharmaceutical Co., Ltd. Azacitidine T2001L41KA 100％ 2-8℃ Shandong Anhong Pharmaceutical Co., Ltd.

[0099] 3. Experimental methods and procedures

[0100] 3.1 Cell culture

[0101] Human myelomonocytic leukemia MV4-11 cells (ATCC, CRL-9591) were cultured in vitro as a monolayer. The culture conditions were RPMI1640 medium supplemented with 10% fetal bovine serum and 1% antibiotics, and cultured in a 37°C, 5% CO2 cell culture incubator. Routine subculture was performed twice a week. When the cell confluence reached 80%-90% and the cell number reached the required amount, the cells were harvested, counted, and inoculated.

[0102] 3.2 Tumor cell inoculation and grouping

[0103] 100 μL of PBS containing 10×10 6 MV4-11 cells was mixed with 100 μL of Matrigel (1:1) in equal volume and then subcutaneously inoculated into the back of the right forelimb of each mouse. When the average tumor volume reached 142 mm 3 , random grouping was performed and drug administration was started. The experimental grouping and drug administration plan are shown in the following table.

[0104] Table 6. Experimental animal grouping and drug administration plan

[0105]

[0106]

[0107] Note:

[0108] 1. N: Number of mice in each group

[0109] 2. Drug administration volume parameter: 10 μL / g according to the mouse body weight

[0110] 3. Vehicle: 5% DMSO + 5% Solutol + 90% Saline

[0111] 3.4 Preparation of test substances

[0112] Table 7. Preparation method of test substances

[0113]

[0114] Note: The drug needs to be gently and fully mixed before drug administration. The compound preparation cycle is daily preparation.

[0115] 3.5 Daily observation of experimental animals

[0116] The formulation and any modifications of this experimental protocol have been evaluated and approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec (Nantong) Co., Ltd. The use and welfare of experimental animals are carried out in accordance with the regulations of the American Association for the Accreditation of Laboratory Animal Care International (AAALAC). The health status and mortality of animals are monitored daily. Routine examinations include observing tumor growth and the effects of drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake (only visually), body weight changes (body weight is measured twice a week), external signs, or other abnormal conditions. The number of animal deaths and side effects within each group are recorded based on the number of animals in each group.

[0117] 3.5 Tumor measurement and experimental indicators

[0118] The tumor diameter is measured twice a week using vernier calipers. The formula for calculating the tumor volume is: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively.

[0119] The antitumor efficacy of the compound is evaluated using the tumor growth inhibition rate TGI (%). Calculation of TGI (%): TGI (%) = [1 - (average tumor volume at the end of drug administration in a certain treatment group - average tumor volume at the start of drug administration in that treatment group) / (average tumor volume at the end of treatment in the vehicle control group - average tumor volume at the start of treatment in the vehicle control group)] × 100%.

[0120] 3.6 Statistical analysis

[0121] Statistical analysis is performed using SPSS software based on the mean and standard error of the mean (SEM) data of tumor volumes at the end of the experiment. Statistical analysis is carried out using the T-Test. A p < 0.05 is considered to indicate a significant difference.

[0122] 4. Experimental results

[0123] Table 8. Tumor growth inhibition rate

[0124]

[0125] Note:

[0126] a. The p-value is analyzed using the independent samples T-Test (when the variances are homogeneous) or

[0127] the Mann-Whitney test (when the variances are heterogeneous) with the Vehicle group as the control based on the tumor volumes of different groups; a p < 0.05 is considered to indicate a significant difference.

[0128] b. The p-values were analyzed for the tumor volumes in different groups. For the group of compound (B) + Azacitidine vs. each single-drug group, an independent samples T-Test (when the variances were homogeneous) or Mann-Whitney test (when the variances were heterogeneous) was used. A significant difference was considered when p < 0.05.

[0129] 5. Experimental conclusions

[0130] As can be seen from Table 8 and Figure 2 it can be seen that compound (B) has a significant tumor inhibitory effect on the subcutaneous xenograft tumor model of human myelomonocytic leukemia MV4-11 cells, and the combination of compound (B) and Azacitidine has a synergistic effect on the proliferation of subcutaneous xenograft tumors of MV4-11 cells.

[0131] Preparation of compound (B):

[0132]

[0133] Reaction route:

[0134]

[0135] Operation steps:

[0136] Step A: Dissolve 5-bromoisoindoline hydrochloride (500 mg, 2.13 mmol) in dichloromethane (10 mL), add triethylamine (647.2 mg, 6.40 mmol), and dropwise add acetyl chloride (251 mg, 3.20 mmol). React overnight at room temperature.

[0137] After TLC monitoring showed the disappearance of the starting material, add dilute hydrochloric acid (1 M, 10 mL) and dichloromethane (15 mL), stir, and separate the layers. The organic phase was first washed with saturated sodium chloride solution (10 mL), then dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 1-(5-bromoisoindolin-2-yl)ethan-1-one (370 mg).

[0138] MS(ESI) M / Z: 240.3 [M+H] + .

[0139] Step B: 1-(5-Bromodihydroisoindol-2-yl)ethan-1-one (370 mg, 1.54 mmol), tert-butyl 1,4-diazepane-1-carboxylate (370.4 mg, 1.85 mmol), sodium tert-butoxide (207.3 mg, 2.16 mmol), 1,1'-binaphthalene-2,2'-bis(diphenylphosphine) (143.9 mg, 0.23 mmol), and tris(dibenzylideneacetone)dipalladium(0) (70.6 mg, 0.077 mmol) were dissolved in toluene (5 mL). The mixture was purged with nitrogen three times and then refluxed at 120 °C for 3 h.

[0140] After monitoring by TLC showed the disappearance of the starting materials, the reaction mixture was cooled. Water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 2). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain tert-butyl 4-(2-acetyl-dihydroisoindol-5-yl)-1,4-diazepane-1-carboxylate (400 mg).

[0141] MS(ESI) M / Z: 360.6 [M+H] + .

[0142] Step C: tert-Butyl 4-(2-acetyl-dihydroisoindol-5-yl)-1,4-diazepane-1-carboxylate (400 mg, 1.11 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1.5 mL) was added. The mixture was stirred at room temperature for 2 h.

[0143] After monitoring by TLC showed the disappearance of the starting materials, the reaction mixture was concentrated under reduced pressure and dried under high vacuum to obtain the crude product 1-(5-(1,4-diazepan-1-yl)dihydroisoindol-2-yl)ethan-1-one trifluoroacetate, which was directly used in the next step.

[0144] Step D: The crude product 1-(5-(1,4-diazepan-1-yl)dihydroisoindol-2-yl)ethan-1-one trifluoroacetate, 2,4-dichloronicotinonitrile (192.5 mg, 1.11 mmol), and N,N-diisopropylethylamine (717.3 mg, 5.55 mmol) were added to acetonitrile (5 mL). The mixture was refluxed at 80 °C overnight.

[0145] After monitoring by LCMS showed the disappearance of the starting materials, the reaction mixture was cooled. Water (10 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 2). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 4-(4-(2-acetyl-dihydroisoindol-5-yl)-1,4-diazepan-1-yl)-2-chloronicotinonitrile (330 mg).

[0146] MS(ESI) M / Z: 396.5 [M+H] + .

[0147] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.02 - 7.96 (m, 1H), 7.13 – 7.07 (m, 1H), 6.96 (d, J = 6.5 Hz, 1H), 6.79 – 6.70 (m, 2H), 4.69 (d, J = 24 Hz, 2H), 4.48 (d, J = 20.4 Hz, 2H), 3.97 – 3.89 (m, 2H), 3.78 – 3.68 (m, 4H), 3.53 (t, J = 5.9 Hz, 2H), 2.06 – 1.88 (m, 5H).

[0148] Step E: 4-(4-(2-Acetyl-2,3-dihydro-1H-isoindol-5-yl)-1,4-diazepan-1-yl)-2-chloronicotinonitrile (120 mg, 0.30 mmol), mercaptoacetamide (82.9 mg, 0.91 mmol), and sodium methoxide (24.6 mg, 0.46 mmol) were added to methanol (2.5 mL), and the mixture was reacted at 65 °C for 4 h. Then sodium methoxide (24.6 mg, 0.46 mmol) was added, and the reaction was carried out overnight in a microwave tube at 90 °C.

[0149] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by preparative method to obtain 38.55 mg of the compound of formula (B).

[0150] MS(ESI) M / Z: 451.6 [M+H] + .

[0151] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.40 (dd, J = 5.4, 0.8 Hz, 1H), 7.18 – 7.06 (m, 4H), 7.04 – 6.97 (m, 2H), 6.79 – 6.69 (m, 2H), 4.72 (d, J = 18.4 Hz, 2H), 4.51 (d, J = 15.1 Hz, 2H), 3.81 – 3.72 (m, 2H), 3.60 – 3.52 (m, 2H), 3.31 – 3.26 (m, 2H), 3.23 – 3.14 (m, 2H), 2.20 – 2.10 (m, 2H), 2.04 (s, 3H).

[0152] The reference compound 15U was prepared according to the preparation method in WO2005100365:

[0153]

[0154] Example 4: Biological Activity Experiment

[0155] (1) In vitro CDK Enzymology Experiment

[0156] The inhibitory effects on the kinase activities of CDK8 / CycC / Med12 and CDK19 / CycC were tested in vitro using the ADP-Glo Luminescent method, and the half-maximal inhibitory concentration IC50 of the compound against the CDK8 kinase activity was obtained. 50

[0157] The inhibitory effects on the kinase activities of CDK2 / CycE1, CDK7 / cyclinH / MAT1, and CDK9 / cyclinT1 were tested in vitro using the time-resolved fluorescence resonance energy transfer detection (LanceUltra) method, and the half-maximal inhibitory concentration IC50 of the compound against the CDK2 / CDK7 / CDK9 kinase activities was obtained.

[0158] 1. Experimental Materials

[0159] CDK8 was purified from Vivid Biosciences, CDK19 was purchased from BPS Bioscience, the substrate MBP was purchased from signalChem, the ADP-Glo kit was purchased from Promega, DMSO was purchased from Sigma, and the 384-well plate was purchased from Corning.

[0160] CDK2 / CDK7 / CDK9 was purchased from Carna, and the substrate 18 / substrate 8 was purchased from Gil Biochemical.

[0161] 2. Experimental Methods

[0162] (1) Prepare 1×Kinase buffer; add FAM-labeled peptide and ATP to 1×Kinase buffer to make 2.5×Peptide solution.

[0163] (2) Preparation of compound concentration gradient: The test concentration of the test compound starts from 10 μM, is diluted 3-fold, with 10 concentrations, and duplicate wells are detected. Dilute it into a 100% DMSO solution with a final concentration of 100-fold in a 384-source plate. Use the dispenser Echo650 to transfer 50 nL of the compound with a final concentration of 100-fold to the destination 384-well plate. Add 200 nL of DMSO to the positive and negative control wells.

[0164] (3) Add the corresponding kinase to 1×Kinase buffer to prepare the kinase solution.

[0165] ​(4) Add 2.5 μL of kinase solution to the compound well and the positive control well respectively; add 2.5 μL of 1× Kinase buffer to the negative control well.

[0166] (5) Centrifuge at 1000 rpm for 30 seconds, mix the reaction plate by oscillation, and incubate at room temperature for 10 minutes.

[0167] (6) Prepare a mixed solution of 2-fold final concentration of ATP and substrate by adding the MBP-labeled polypeptide and ATP to 1× Kinase buffer.

[0168] (7) Add 2.5 μL or 10 μL of the mixed solution of 2-fold final concentration of ATP and substrate to initiate the reaction.

[0169] (8) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, mix by oscillation, and incubate at 37 °C for 2 hours or 1 hour.

[0170] (9) Add 5 μL of ADP-Glo reagent 1 to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, mix by oscillation, and incubate at 37 °C for 1 hour. Transfer 10 μL of ADP-Glo reagent 2 to the 384-well plate; incubate and react at 37 °C for 1 hour.

[0171] Or

[0172] Add 20 μL of the termination detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, mix by oscillation, and let it stand at room temperature for 1 hour.

[0173] (10) Read the RLU value using an Envision 2104 Multilabel Reader.

[0174] Calculation formula:

[0175] Copy the RLU reading value, and convert the above data into the inhibition percentage through the formula.

[0176] % inhibition = (max - Sample RLU) / (max - min) * 100.

[0177] Or

[0178] Copy the numerical ratio of the fluorescence reading (Lance signal ratio (665 nm / 615 nm)); convert the above data into the inhibition percentage through the formula.

[0179] % inhibition = (max - Sample Lance signal ratio) / (max - min) * 100.

[0180] Wherein: "min" is the reading of the control sample well without adding enzyme for reaction; "max" is the reading of the control well with DMSO added.

[0181] Fitting the dose-effect curve

[0182] Import the data into MS Excel and perform curve fitting using the XLFit excel add-in version 5.4.0.8; fitting formula: Y = Bottom + (Top - Bottom) / (1 + (IC50 / X)^HillSlope).

[0183] 3. Experimental results

[0184] Table 9 Inhibitory activity of compound of formula (B) against CDK kinase

[0185]

[0186] (2) Cell proliferation inhibition experiment

[0187] In this experiment, the fluorescence method for measuring the intracellular ATP content (CellTiter - Glo) was used to detect the inhibitory proliferation effect of the compound on MV4 - 11 and KG - 1 cell lines, and the half - inhibitory concentration IC of the compound on the above cell lines was obtained. 50 .

[0188] 1. Experimental materials

[0189] IMDM medium, fetal bovine serum (FBS), 100X Pen / Strep, GlutaMAX - I Supplement were purchased from GIBCO; MV4 - 11 and KG - 1 cell lines were purchased from the American Type Culture Collection (ATCC); Cell Titer - Glo luminescent cell viability detection reagent was purchased from Promega.

[0190] 2. Experimental methods

[0191] 1) Inoculate cells in a 384 - well culture plate at a density of 300 MV4 - 11 or KG - 1 cells per well, 50 μL per well. Place it in an incubator (37 °C, 5% CO 2 ) and incubate overnight.

[0192] 2) Day 0: Use D300e (TECAN) to add 50 nL of gradient - diluted test compound (starting concentration is 10 μM, 9 concentrations, diluted at a 1:3 ratio) to the cells in the culture plate, and the final concentration of DMSO is 0.4%. Place the culture plate in a cell culture incubator and incubate for 168 hours (37 °C, 5% CO 2 ). Add 20 nL of DMSO to each well as the blank control.

[0193] 3) Day 7: Add 30 μL of Cell Titer-Glo reagent to each well, shake at 500 rpm for 10 minutes, centrifuge at 1000 rpm for 1 minute, and incubate in the dark at room temperature for 20 minutes to stabilize the luminescence signal.

[0194] 4) Detect the luminescence signal using an Envision microplate reader (PerkinElmer).

[0195] 5) Use GraphPad Prism 6 software for data analysis and calculate the IC of the compound. 50 。

[0196] 3. Experimental Results

[0197] Table 10 Inhibitory Activity of Compounds of Formula (B) on Cell Proliferation

[0198]

[0199] (III) Direct Inhibition Test of Human Liver Microsomal CYP450 Enzymes

[0200] In this experiment, specific substrates of each CYP450 enzyme subtype were used as probes to co-incubate with human liver microsomes, and the method of detecting the formation of metabolites was used to detect the direct inhibitory effect of the compound on each CYP450 subtype of human liver microsomes, and the half-maximal inhibitory concentration IC of the compound on the above CYP subtypes was obtained. 50 。

[0201] 1. Test Materials

[0202] Human liver microsomes were purchased from BD Gentest at a concentration of 20 mg / mL; potassium phosphate buffer, 100 mM; magnesium chloride, 300 mM; NADP (nicotinamide adenine dinucleotide phosphate), 10 mM; termination solution, acetonitrile (containing 200 nM alprazolam, 200 nM labetalol and 200 nM tolbutamide as internal standards).

[0203] Positive controls, test compounds and substrates are shown in Table 11.

[0204] Table 11 Positive Controls, Test Compounds and Substrates

[0205]

[0206] 2. Experimental Method

[0207] Incubation was carried out in 96-well deep plates. The following volumes were dispensed into each well of the incubation plate: 169 μL of the main solution and 1 μL of working solutions of test compounds or positive control compounds at multiple concentrations. The incubation plate was placed in a water bath and preheated at 37 °C for 5 minutes. The experiment was performed in duplicate.

[0208] 10 μL of substrate was added to the incubation plate, and then 20 μL of 10 mM NADPH solution was added to initiate the reaction at a final concentration of 1 mM. Incubation was carried out in a 37 °C water bath for 20 minutes.

[0209] After 20 minutes of incubation, the reaction was quenched by adding 300 μL of quenching solution (cold acetonitrile containing 3% formic acid, 200 nM alprazolam, 200 nM labetalol, and 200 nM tolbutamide). The plate was centrifuged at 3,220 g at 4 °C for 50 minutes. 150 μL of the supernatant was transferred to a new plate. The supernatant could be diluted with 150 μL of pure water. The sample was thoroughly mixed and analyzed using UPLC-MS / MS.

[0210] Inhibition of CYP450 in human liver microsomes was measured by the percentage decrease in activity of labeled metabolite formation compared to the uninhibited solvent control (= 100% activity). The mean enzyme activity (as a percentage of the uninhibited control) for each concentration was plotted against the inhibitor concentration and fitted to an IC 50 curve.

[0211] 3. Experimental Results

[0212] Table 12 Half-maximal inhibitory concentration of CYP2C19 enzyme

[0213]

[0214] Results: As can be seen from the above table, the IC 50 of the compound of formula (B) of the present invention for the CYP2C19 enzyme was significantly higher than 15 U, indicating that the compound of the present invention has a lower risk of direct inhibition of the CYP450 enzyme.

[0215] (IV) In Vivo Pharmacokinetic Studies in Animals

[0216] Using rats and dogs as test animals, after intravenous bolus injection and oral injection of the compound of the present invention, plasma samples were collected at specific time points, the concentration of the compound in plasma was detected by LC-MS / MS, and PK parameters were calculated to reflect the pharmacokinetic behavior of the compound of the present invention in the plasma of rats and dogs.

[0217] 1. Test Protocol

[0218] 1.1 Test Drugs:

[0219] 15 U and the compound of formula (B).

[0220] 1.2 Test animals

[0221] Rats, Sprague-Dawley, male, supplied by Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0222] Dogs, Beagle Dog, male, supplied by Jiangsu Marshall Biotechnology Co., LTD

[0223] 1.3 Drug administration

[0224] Drug administration information of compound (B) and 15U compound in rats: IV administration dose is 1 mg / kg, administration volume is 5 mL / kg; PO administration dose is 5 mg / kg, administration volume is 10 mL / kg, 5 vol% DMSO / 30 vol% PEG400 (solubilizer) / 65% Saline (normal saline).

[0225] Drug administration information of compound (B) and 15U compound in dogs: For compound (B), IV administration dose is 0.5 mg / kg, administration volume is 2 mL / kg; PO administration dose is 1 mg / kg, administration volume is 2 mL / kg, 5 vol% DMSO / 30 vol% PEG400 (solubilizer) / 65% Saline (normal saline). For 15U compound, IV administration dose is 0.5 mg / kg, administration volume is 1 mL / kg; PO administration dose is 3 mg / kg, administration volume is 2 mL / kg, 5 vol% DMSO / 30 vol% PEG400 (solubilizer) / 65% Saline (normal saline).

[0226] 1.4 Experimental equipment

[0227] The centrifuge was purchased from Eppendorf, and the pipette was purchased from Eppendorf.

[0228] 1.5 Sample collection

[0229] After drug administration in rats and dogs, at 0.0833 (IV), 0.25, 0.5, 1, 2, 4, 8 and 24 hours, 0.2 mL and 0.5 mL of venous blood were collected respectively, placed in EDTA-K2 tubes, centrifuged at 4°C and 2000 g for 10 min to separate plasma, and stored at -80°C.

[0230] 1.6 Sample treatment

[0231] Treatment of rat plasma samples:

[0232] 1) Add 200 μL of acetonitrile to 50 μL of plasma sample, vortex mix and then centrifuge for 15 minutes.

[0233] 2) Dilute the treated supernatant with water and analyze the concentration of the compound to be measured by LC / MS / MS.

[0234] Treatment of canine plasma samples:

[0235] 1) Add 200 μL of acetonitrile to 30 μL of plasma sample for precipitation, vortex mix, and then centrifuge for 15 minutes.

[0236] 2) Dilute the treated supernatant with water and analyze the concentration of the compound to be measured by LC / MS / MS.

[0237] 2. Experimental results

[0238] Pharmacokinetic parameters were calculated using WinNonlin 6.1. The pharmacokinetic parameters are shown in Tables 13 and 14. Among them, Cmax represents the maximum plasma drug concentration, CL represents the clearance rate, Vss represents the steady-state volume of distribution, T1 / 2 represents the terminal elimination half-life, MRTInf represents the mean residence time, AUC represents the area under the plasma concentration-time curve, and F represents the bioavailability.

[0239] Table 13 Pharmacokinetic parameters of intravenous injection and oral administration of some compounds of the present invention in rats

[0240]

[0241] Table 14 Pharmacokinetic parameters of intravenous injection and oral administration of some compounds of the present invention in dogs

[0242]

[0243]

[0244] Note: " / " indicates not determined

[0245] Results: As can be seen from Tables 13 and 14, the compounds of the present invention have good pharmacokinetic properties in rats and dogs.

Claims

1. Use of a CDK8 / 19 inhibitor in combination with azacitidine in the preparation of a drug for treating tumors.

2. The use according to claim 1, characterized in that The CDK8 / 19 inhibitor is selected from SNX631, D-SNX631, Senexin B, Senexin C, BI-1347, RVU120, a compound represented by formula (B) and any combination thereof:

3. The use according to any one of claims 1 to 2, characterized in that The tumor is a blood tumor.

4. The use according to claim 3, characterized in that The blood tumor is selected from acute myeloid leukemia, myelodysplastic syndrome and myeloproliferative blood tumor.

5. The use according to claim 4, characterized in that The blood tumor is selected from azacitidine-insensitive blood tumors.

6. The use according to any one of claims 1 to 5, characterized in that CDK8 / 19 inhibitors and azacitidine can be administered simultaneously, sequentially or intermittently.

7. The use according to any one of claims 1 to 6, characterized in that CDK8 / 19 inhibitors are administered orally at a daily dose of 1-1000 mg, once a day or twice a day.

8. The use according to any one of claims 1 to 7, characterized in that Azacitidine is administered subcutaneously or intravenously at a dose of 5-75 mg / m2 daily for 7 days, followed by a treatment cycle every 4 weeks. If no beneficial effect or inadequate effect is observed after 2 treatment cycles and no toxicity occurs except nausea and vomiting, the dose can be increased to 100 mg / m2.

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