A pyrazole compound and its preparation method and application

Abstract

The present invention belongs to the field of pharmaceutical synthesis, and specifically relates to a pyrazole compound, a preparation method thereof and an application thereof. The structure of the pyrazole compound is shown in Formula I, R1 is Cl or F; R2 is H, Br, Cl or methyl. The present invention proposes a CDK9-HDAC dual inhibitor, which can significantly induce LSCs differentiation and inhibit AML cell proliferation, and exhibits effective and selective inhibition of CDK9, HDAC1, HDAC2 and HDAC3 at low nanomolar concentrations. In addition, it also has good oral bioavailability, providing a new strategy for targeting LSCs to treat AML.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical synthesis, and specifically relates to a pyrazole compound and a preparation method and application thereof. Background Art

[0002] Acute myeloid leukemia (AML) is a heterogeneous clonal disease characterized by the proliferation and accumulation of myeloblasts in the bone marrow, which ultimately leads to hematopoietic dysfunction. Unfortunately, the relapse rate of AML patients receiving existing therapies is high. Leukemic cancer stem cells (LSCs) are key players in the occurrence and development of leukemia and are considered to be the main cause of leukemia relapse and drug resistance. Recent studies have shown that a large number of LSCs detected at diagnosis may be associated with a high risk of relapse and poor survival in leukemia patients after treatment, and accurate detection of LSCs at diagnosis is very useful for the treatment of leukemia patients. Therefore, it is necessary to develop new and effective anti-AML therapies, namely therapeutic strategies targeting LSCs. DNA and chromatin modifications are crucial to the pathogenesis of AML, and they are closely related to abnormal epigenetic events, which will enhance oncogene expression, block differentiation, and accumulate LSCs. More and more research results have shown that histone deacetylases (HDACs) are an important family of epigenetic proteins that are essential for the maintenance of LSCs. Targeting HDACs is considered a promising and effective chemotherapeutic solution for the treatment of AML.

[0003] In addition, cyclin-dependent kinases (CDKs) are a family of serine / threonine protein kinases whose catalytic ability depends on binding to specific cyclins. Some CDKs (CDK1, CDK2, CDK4, and CDK6) play a key role in regulating cell cycle progression, and another family of CDKs that regulate transcription has been identified, including CDK7 and CD K9. CDK9, as the catalytically active part of the elongation factor P-TEFb, can phosphorylate the serine 2 (ser2) residue of RNA pol II and is required for efficient transcription elongation. Recent studies have shown that CDK9 is considered a promising target for cancer treatment. Summary of the invention

[0004] Based on previous literature research, the present invention proposes that CDK9 and HDACs dual inhibitors will provide synergistic effects on the treatment of AML; both may not only be closely related to regulating the epigenetics of cancer cells, but may also induce the differentiation of cancer stem cells. Based on the above research, the present invention prepares a series of CDK9-HDAC dual inhibitors, which are found to significantly induce LSCs differentiation and inhibit AML cell proliferation, and exhibit effective and selective inhibition of CDK9, HDAC1, HDAC2 and HDAC3 at low nanomolar concentrations. In addition, it also has good oral bioavailability, providing a new strategy for targeting LSCs to treat AML. When prepared as a drug or a pharmaceutical composition (the dosage form may be an injection or oral solution, etc.), it may also include a pharmaceutical carrier (disintegrant (sodium starch glycolate or sodium carboxymethyl cellulose, etc.) or a binder (microcrystalline cellulose or hydroxymethyl cellulose, etc.) or an excipient, etc.

[0005] The details are as follows:

[0006] A pyrazole compound, the structure of which is shown in Formula I:

[0007]

[0008] R1 is Cl or F; R2 is H, Br, Cl or methyl.

[0009] Further preferably, the structure of the pyrazole compound is as shown in one of 1-8:

[0010]

[0011] The present invention also provides a method for preparing the above pyrazole compounds.

[0012]

[0013] The beneficial effects of the present invention are as follows: the present invention proposes a CDK9-HDAC dual inhibitor, which can significantly induce LSCs differentiation and inhibit AML cell proliferation, and exhibits effective and selective inhibition of CDK9, HDAC1, HDAC2 and HDAC3 at low nanomolar concentrations. In addition, it has good oral bioavailability, providing a new strategy for targeting LSCs to treat AML. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure shows the experimental results of AML cell apoptosis induced by Chidamide and compound 1;

[0015] Figure 2 Shown is the effect of Chidamide and Compound 1 on the stemness of AML cells;

[0016] Figure 3 Shown is the effect of chidamide and compound 1 on CD11b of AML cells + The results of the subgroup impact plot;

[0017] Figure 4 Shown is the effect of chidamide and compound 1 on the LSC biomarker CD34 in AML cells + CD38 - Expression impact result diagram;

[0018] Figure 5 Shown is the PK study results of compound 1. DETAILED DESCRIPTION

[0019] The following will provide a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments and drawings, so as to fully understand the purpose, scheme and effects of the present invention.

[0020] Embodiment 1:

[0021] The preparation of compound 1, the structure of which is shown below:

[0022]

[0023] The specific preparation method is:

[0024] (1) Preparation of compound (1-1), the structure of which is shown below:

[0025]

[0026] The specific steps are as follows: Under an ice bath and argon protection, 4-nitropyrazole-3-carboxylic acid (1.50 g, 9.55 mmol) was dissolved in dry dichloromethane (80 mL), N, N-dimethylformamide (1 mL) was added, and then oxalyl chloride (1.0 mL, 11.46 mmol) was added. After 5 minutes, the ice bath was removed, and the reaction was continued at room temperature for 2 hours. The reaction solution was concentrated, and the obtained crude acid chloride was directly used for the next step. The crude acid chloride obtained in the previous step was redissolved in dry dichloromethane (80 mL), and methyl 4-aminobenzoate (1.44 g, 9.55 mmol) was added under ice bath conditions. After 5 minutes, the ice bath was removed, and the reaction was continued at room temperature for 3 hours. The liquid in the bottle was concentrated using a rotary evaporator. Saturated NH4Cl solution and hydrochloric acid solution (1 mol L -1 ) The reaction solution was washed and extracted with ethyl acetate (3×250 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane: methanol = 60:1 to 40:1) to obtain compound 1-1 (white solid, 2.25 g, yield: 81%).

[0027] The NMR test results of compound 1-1 are: 1 H NMR (600MHz, DMSO-d6) δ11.03(s,1H),8.90(s,1H),7.99(d,J=8.6Hz,2H),7.86(d,J=8.5Hz,2H),3.85(s,3H). 13 C NMR (100MH z, DMSO-d6) δ166.2, 143.2, 133.0, 130.9, 125.3, 119.6, 52.4.

[0028] (2) Preparation of compound (1-2), the structure of which is shown below:

[0029]

[0030] The specific steps are as follows: dissolve compound 1-1 (3.32 g, 11.43 mmol) in ethanol (140 mL), add water (70 mL), then add iron powder (2.55 g, 45.80 mmol), stir evenly at room temperature, and then add glacial acetic acid (3 mL). Slowly heat the reaction system to 90 ° C for 5 h, filter the reaction solution with diatomaceous earth while hot, concentrate the filtrate, wash the reaction solution with saturated NaHCO3 solution, extract with n-butanol (3×250 mL), dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the crude product with silica gel column chromatography (dichloromethane: methanol = 20:1 to 15:1) to obtain compound 1-2 (white solid, 1.86 g, yield: 60%).

[0031] The NMR test results of compound 1-2 are: 1 H NMR (600MHz, DMSO-d6) δ14.36(s,1H),11.04(s,1H),8.94(s,1H),8.05-7.94(m,2H),7.91-7.80(m,2H),3.86(s,3H). 13 C NMR (150MHz, DMSO-d6) δ166.4,163.4,144.0,134.6,131.8,130.5,124.1,119.7,115.8,52.3.

[0032] (3) Preparation of compound (1-3), the structure of which is shown below:

[0033]

[0034] The specific steps are as follows: In an ice bath and argon protection environment, compound 1-2 (2.00g, 7.70mmol) was dissolved in dry dichloromethane (80mL), 2,6-dichlorobenzoyl chloride (2.15mL, 15.38mmol), N,N-dimethylformamide (2.0mL) and triethylamine (3.2mL, 23.07mmol) were added, and the reaction system was slowly heated to room temperature for 4h. The reaction solution was concentrated, washed with saturated NaHCO3 solution, extracted with ethyl acetate (3×250mL), the organic phase was dried with anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane: methanol = 60:1 to 55:1) to obtain compound 1-3 (white solid, 1.93g, yield: 58%).

[0035] The NMR test results of compound 1-3 are: 1 H NMR(600MHz,DMSO-d6)δ13.67(s,1H),10.66(s,1H),10.13(s,1H),8.46(d,J=1.4Hz,1H), 8.01-7.95(m,2H),7.95-7.86(m,2H),7.59-7.55(m,2H),7.52-7.47(m,1H),3.82(s,3H). 13 C NMR (150MHz, DMSO-d6) δ166.3,162.6,161.3,143.3,136.0,133.8,132.2,131.8,130.4,128.8,124.9,122.3,122.2,120.3,52.4,49.1.

[0036] (4) Preparation of compound (1-4), the structure of which is shown below:

[0037]

[0038] The specific steps are as follows: Under ice bath, compound 1-3 (1.55 g, 3.60 mmol) was dissolved in tetrahydrofuran (12 mL), solid lithium hydroxide (0.90 g, 21.50 mmol) was added, water (12 mL) was slowly added, and the ice bath was removed after 5 min. After stirring at room temperature for 3 h, the liquid in the bottle was concentrated by rotary evaporator. Hydrochloric acid solution (1 mol L -1 ) The reaction solution was washed with ethyl acetate (4×200 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane: methanol = 20:1 to 10:1) to obtain compound 1-4 (white solid, 0.99 g, yield: 66%).

[0039] The NMR test results of compound 1-4 are: 1 H NMR(600MHz,DMSO-d6)δ13.74(s,1H),12.85(s,1H),10.63(s,1H),10.16(s,1H),8.45(s,1H ),7.94(d,J=8.9Hz,2H),7.92-7.86(m,2H),7.63-7.58(m,2H),7.53(dd,J=9.1,7.1Hz,1H). 13 C NMR (150MHz, DMSO-d6) δ167.6,161.3,142.6,136.0,132.2,131.8,130.5,128.8,122.3,120.2.

[0040] (5) Preparation of compound 1: Compound 1-4 (178 mg, 0.43 mmol) was dissolved in N,N-dimethylformamide (3 mL), and o-phenylenediamine (69 mg, 0.64 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (98 mg, 0.51 mmol), and 4-dimethylaminopyridine (52 mg, 0.43 mmol) were added. After stirring at room temperature for 8 h, ethyl acetate (30 mL) was added to the reaction system for extraction. The reaction solution was washed twice with saturated NaCl solution, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane: methanol = 47:1 to 43:1) to obtain compound 1 (yellow solid, 50 mg, yield: 23%).

[0041] The chemical test results of compound 1 are: 1 H NMR(400MHz,DMSO-d6)δ13.65(s,1H),10.57(s,1H),10.16(s,1H),9.58(s,1H),8.48(s,1H),7.96(s ,4H),7.61-7.49(m,3H),7.17(s,1H),6.99(s,1H),6.80(s,1H),6.60(s,1H),4.88(d,J=16.3Hz,2H). 13 C NMR(100MH z,DMSO-d6)δ165.2,162.5,162.4,162.4,161.4,143.6,141.6,136.0,133.8,132.2,131.8,1 30.0,128.9,128.8,127.2,126.9,123.9,122.2,120.1,120.0,120.0,116.8,116.7,116.6.HR MS(ESI):m / z calculated for C24 H 18 Cl2N6NaO3 + :531.0710, found 531.0712.

[0042] Embodiment 2:

[0043] (1) Preparation of compound 2, the structure of which is shown below:

[0044]

[0045] The specific preparation method is similar to the preparation method of compound 1, except that one of the raw materials used is 4,5-dimethyl-o-phenylenediamine, which replaces the o-phenylenediamine in the preparation method of compound 1 in an equimolar manner.

[0046] The NMR test results of compound 2 are: 1 H NMR(400MHz,DMSO-d6)δ13.65(s,1H),10.58(s,1H),10.16(s,1H),9.53(s,1H),8.48(s,1H),7 .94(s,4H),7.56(d,J=28.3Hz,3H),6.92(s,1H),6.58(s,1H),4.60(s,2H),2.12-2.05(m,6H). 13 C NMR(100MHz,DMSO-d6)δ166.3,165.0,162.5,161.3,141.5,141.2,136.0,135.9,134.4,133.8,132.2,13 1.8,131.5,130.0,128.8,128.8,128.0,124.1,122.2,121.6,120.2,120.0,118.4,118.1,19.6,18.9.HR MS(ESI):m / z calculated for C 26 H 22 Cl2N6NaO3 + :559.1023, found 559.1028.

[0047] (2) Preparation of compound 3, the structure of which is shown below:

[0048]

[0049] The specific preparation method is similar to the preparation method of compound 1, except that one of the raw materials used is 4,5-dibromo-o-phenylenediamine, which replaces the o-phenylenediamine in the preparation method of compound 1 in an equimolar manner.

[0050] The NMR test results of compound 3 are:1 H NMR(400MHz,DMSO-d6)δ13.66(s,1H),10.58(s,1H),10.15(s,1H),9.60(s,1H),8.47(s,1H),7 .95(d,J=18.8Hz,4H),7.58(d,J=7.8Hz,2H),7.53(d,J=9.8Hz,2H),7.14(s,1H),5.41(s,2H). 13 C NMR(100MHz,DMSO-d6)δ165.5,162.5,161.3,144.6,144.6,141.9,136.0,133.8,132.2,131.8,131.8,13 1.0,129.5,129.1,128.8,128.8,124.5,122.3,122.2,120.8,120.0,120.0,119.7,107.7.HRMS(ESI):m / z calculated for C 24 H 16 Br2Cl2N6NaO3 + :686.8920, found 686.8922.

[0051] (3) Preparation of compound 4, the structure of which is shown below:

[0052]

[0053] The specific preparation method is similar to the preparation method of compound 1, except that one of the raw materials used is 4,5-dichloro-o-phenylenediamine, which replaces the o-phenylenediamine in the preparation method of compound 1 in an equimolar manner.

[0054] The NMR test results of compound 4 are: 1 H NMR(400MHz,DMSO-d6)δ13.66(s,1H),10.58(s,1H),10.15(s,1H),9.61(s,1H),8.47(s ,1H),7.95(d,J=0.9Hz,4H),7.63-7.48(m,4H),7.44(s,1H),6.97(s,1H),5.40(s,2H). 13C NMR(100MHz,DMSO-d6)δ165.6,162.5,161.3,144.0,141.9,136.0,133.8,132.2,131.8,131.8,129.5,129.1,1 29.1,128.8,128.8,128.3,128.1,123.8,122.3,122.2,120.0,120.0,116.5,116.5.HRMS(ESI):m / zcalculated for C 24 H 16 Cl4N6NaO3 + :598.9930, found 598.9935.

[0055] (4) Preparation of compound 5, the structure of which is shown below:

[0056]

[0057] The specific preparation method is similar to the preparation method of compound 1, except that one of the raw materials used is 2,6-difluorobenzoyl chloride, which replaces the 2,6-dichlorobenzoyl chloride in the preparation method of compound (1-3) in an equimolar amount.

[0058] The NMR test results of compound 5 are: 1 H NMR(400MHz,DMSO-d6)δ13.64(s,1H),10.60(s,1H),10.32(s,1H),9.61(s,1H),8.45(s,1H),8.08-7.87(m,4H), 7.65(s,1H),7.29(d,J=18.6Hz,2H),7.16(s,1H),6.98(s,1H),6.78(s,1H),6.66(s,1H),4.89(d,J=11.0Hz,2H). 13 C NMR(100MHz,DMSO-d6)δ165.2,162.5,161.4,143.6,143.6,141.6,136.0,132.2,131.8,131.8,130.0,12 8.9,128.9,128.8,128.8,127.2,126.9,123.9,122.2,120.0,120.0,116.7,116.7,116.6.HRMS(ESI):m / z calculated for C 24 H 18 F2N6NaO3 + :499.1301,found499.1303.

[0059] (5) Preparation of compound 6, the structure of which is shown below:

[0060]

[0061] The specific preparation method is similar to the preparation method of compound 1, except that one of the raw materials used is 2,6-difluorobenzoyl chloride, which is an equimolar replacement for 2,6-dichlorobenzoyl chloride in the preparation method of compound (1-3); the second raw material used is 4,5-dimethyl-o-phenylenediamine, which is an equimolar replacement for o-phenylenediamine in the preparation method of compound 1.

[0062] The NMR test results of compound 6 are: 1 H NMR(400MHz,DMSO-d6)δ13.63(s,1H),10.57(s,1H),10.32(s,1H),9.52(s,1H),8.46(s,1H),7.97(s,4H),7 .66(s,1H),7.27(d,J=3.2Hz,2H),6.94(s,1H),6.59(s,1H),4.59(d,J=17.0Hz,2H),2.11(d,J=16.2Hz,6H). 13 C NMR(100MHz,DMSO-d6)δ165.0,162.6,160.6,158.9,157.1,141.5,141.1,134.4,133.7,133.3,130.1,128.8,1 28.0,124.1,122.4,122.1,121.7,120.1,118.1,114.3,112.9,112.9,112.8,112.8,19.6,18.9.HRMS(ESI):m / z calculated for C 26 H 22 F2N6NaO3 + :527.1614, found 527.1618.

[0063] (6) Preparation of compound 7, the structure of which is shown below:

[0064]

[0065] The specific preparation method is similar to the preparation method of compound 1, except that one of the raw materials used is 2,6-difluorobenzoyl chloride, which is an equimolar replacement for 2,6-dichlorobenzoyl chloride in the preparation method of compound (1-3); the second raw material used is 4,5-dibromo-o-phenylenediamine, which is an equimolar replacement for o-phenylenediamine in the preparation method of compound 1.

[0066] The NMR test results of compound 7 are: 1 H NMR(400MHz,DMSO-d6)δ13.66(s,1H),10.61(s,1H),10.30(s,1H),9.62(s,1H),8.46(s,1H),8.11- 7.92(m,4H),7.66(s,1H),7.56(s,1H),7.28(d,J=18.9Hz,2H),7.15(s,1H),5.42(d,J=17.6Hz,1H). 13 C NMR (100MHz, MeOD) δ166.9,162.3,162.1,143.1,141.5,135.2,132.1,131.4,129.8,129.6,129.0,128. 5,128.5,128.1,128.1,127.6,127.1,124.8,123.1,122.1,119.5,119.5,118.8,117.1.HRMS(ESI):m / z calculated for C 24 H 16 Br2F2N6NaO3 + :654.9511, found 654.9512.

[0067] (7) Preparation of compound 8, the structure of which is shown below:

[0068]

[0069] The specific preparation method is similar to the preparation method of compound 1, except that one of the raw materials used is 2,6-difluorobenzoyl chloride, which is an equimolar replacement for 2,6-dichlorobenzoyl chloride in the preparation method of compound (1-3); the second raw material used is 4,5-dichloro-o-phenylenediamine, which is an equimolar replacement for o-phenylenediamine in the preparation method of compound 1.

[0070] The NMR test results of compound 8 are: 1 H NMR(400MHz,DMSO-d6)δ13.66(s,1H),10.59(s,1H),10.31(s,1H),9.63(s,1H),8.46(s,1H ),7.98(s,4H),7.66(s,1H),7.48(s,1H),7.28(d,J=21.4Hz,2H),6.98(s,1H),5.43(s,2H). 13C NMR(100MHz,DMSO-d6)δ165.6,162.6,161.0,158.6,157.1,144.0,144.0,141.9,133.7,129.5,129.1,12 9.1,128.3,128.1,128.1,123.8,122.4,122.1,120.1,120.1,116.5,116.5,113.0,112.7.HRMS(ESI):m / z calculated for C 24 H 16 Cl2F2N6NaO3 + :567.0521, found 567.0523.

[0071] Embodiment 3:

[0072] (1) Enzyme research

[0073] The in vitro enzyme inhibitory activities of CDK9 and HDAC were preliminarily evaluated using the test compounds at concentrations of 100 and 1000 nM, respectively (Table 1). The results showed that all the synthetic derivatives exhibited significant inhibitory effects on CDK9, and the IC 50 The values ​​were all less than 100nM. At the same time, we used Hela cell nuclear extracts to conduct preliminary tests on the HDA C inhibitory activity of the compounds, and found that all phenylenediamine derivatives showed relatively high inhibitory effects on HDAC. Among them, compound 1 was found to be the most effective type I HDACs inhibitor. The inhibition rate of type I HDACs reached 100% at a concentration of 1μM.

[0074] Table 1 Enzyme inhibition study of compounds

[0075]

[0076] (2) CCK-8 assay

[0077] In order to elucidate the growth inhibitory effect of small molecule compounds on four AML cell lines, AT7519 and chidamide were used as positive drugs. The structure of AT7519 is shown below:

[0078]

[0079] The study found that compound 1 and compound 2 showed strong growth inhibition effects on all four tested cells. More interestingly, compared with the positive compound cedabenbine, U937 and Kasumi-1 were more sensitive to compound 1, while HL60 and SKNO-1 were less sensitive to compound 1. The CCK-8 test results showed that these results indicate that compound 1 may be an effective active compound for the treatment of AML.

[0080] Table 2 Antiproliferative activity of compounds on four AML cell lines

[0081]

[0082] (3) Enzyme selectivity of compound 1 against HDACs and CDKs isoforms

[0083] Following the surprising results of compound 1 on four AML cell lines, the effects of compound 1 on the enzymatic activity of other HDAC and CDK subtypes were explored. The results showed that compound 1 showed stronger inhibitory effects on HDAC1 and HDAC3 than the positive drug cedabendine (IC 50 The two compounds showed similar inhibitory effects on HDAC2 and HDAC8, but had little effect on HDAC4, HDAC5, HDAC7 and HDAC9 (IC 50 >1000nM). The study found that compound 1 is a moderate inhibitor of HDAC6 with an IC 50 The results showed that the activity of compound 1 on AML cells was different from that of the positive drug cedabenbine. In addition, compound 1 was identified as a potent CDK9 inhibitor (IC 50 =22 nM), because compound 1 can more selectively inhibit CDK9 compared with other CDK isoforms, namely CDK1, CDK2, CDK4, and CDK6.

[0084] Table 3 Enzyme activity selectivity of compound 1 against HDACs and CDKs isoforms

[0085]

[0086]

[0087] (4) Analysis of the apoptosis-inducing effect of compound 1 on two different AML cell lines

[0088] The study found that when AML cells were treated with compound 1, apoptosis could be clearly observed under a microscope, so both HL60 and U937 cells were treated with different concentrations of compound 1 and cedabenbine for 48 hours using a single vehicle as a control. They were then stained with FITC-Annexin V and propidium iodide (PI). The percentage of apoptotic AML cells was measured by flow cytometry. ( Figure 1 , wherein, (A) flow cytometry analysis of apoptosis of HL60 cells treated with chidamide and different concentrations of compound 1 for 48 hours; (B) flow cytometry analysis of apoptosis of U937 cells treated with chidamide and different concentrations of compound 1 for 48 hours; (C) quantitative analysis of apoptotic tumor cells in A. Data are expressed as mean ± SD, student T test, *P < 0.05, **P < 0.01, ***P < 0.001; (D) quantitative analysis of apoptotic tumor cells in B. Data are expressed as mean ± SD, student T test, *P < 0.05, **P < 0.01, ***P < 0.001). The results showed that compound 1 significantly induced apoptosis of HL60 and U937 cells in a dose-dependent manner.

[0089] (5) Effect of compound 1 on the stemness of AML cells

[0090] To further evaluate whether compound 1 can inhibit leukemia stem cells (LSC), soft agar colony formation assay was performed on AML cells using cedabenbine as a control. The results showed that after treatment with compound 1, the number of colonies of HL60 and U937 cells was significantly reduced in a dose-dependent manner ( Figure 2 , Inhibitory effects of cedabenbine and compound 1 on AML cell colony formation; Colony formation assay was performed to measure the ability of U937 and HL60 cells to form colonies. (A) (B) Representative images of long-term growth assays of U937 and HL60 cells after continuous treatment with cedabenbine and compound 1 for 15 days. On the right is the quantification of colony numbers. Data are expressed as mean ± SD, student T test, *P < 0.05, **P < 0.01). The results show that compound 1 can significantly reduce the stemness of AML cells.

[0091] (6) Effect of compound 1 on the expression of myeloid differentiation marker CD11b in AML cell lines

[0092] The effect of compound 1 on the expression of the myeloid differentiation marker CD11b in AML cell lines was tested, using chidamide as a control to determine the effect of compound 1 on LSCs ( Figure 3 , Chidamide and compound 1 affect AML cell CD11b +(A) HL60 cells were treated with chidamide and compound 1 for 24 h, and CD11b was analyzed by flow cytometry. + Cells; (B) U937 cells were treated with chidamide and compound 1 for 24 hours, and CD11b was analyzed by flow cytometry + cells; (C) HL60 cells CD11b in A + Quantitative analysis of subpopulations. Data are expressed as mean ± SD, student T test, *P < 0.05, **P < 0.01, ***P < 0.001; (D) U937 cell CD11b in B + Quantitative analysis of subpopulations. Data are expressed as mean ± SD, student T test, *P < 0.05, **P < 0.01, ***P < 0.001). The results showed that treatment with cedabendine and compound 1 significantly increased the expression of myeloid differentiation marker CD11b in HL60 and U937 cells in a dose-dependent manner.

[0093] (7) Effect of Compound 1 on LSC biomarker CD34 in AML cells + CD38 - The impact of expression

[0094] The direct effect of compound 1 on LSCs was then evaluated, with chidamide used as a control. + CD38 - ) to obtain the proportion of LSC in the AML cell population. The results are as follows Figure 4 As shown (Chidamide and Compound 1 changed CD34 + CD38 - AML cell subpopulations. (A) HL60 cells were treated with chidamide and compound 1 for 24 hours, and CD34 + CD38 - (B) U937 cells were treated with cedabendine and compound 1 for 24 hours, and CD34 was analyzed by flow cytometry. + CD38 - Expression of subpopulations; (C) CD34 shown in A + CD38 + Quantification of AML cell subsets. Data are expressed as mean ± SD, student T test, *P < 0.05, **P < 0.01, ***P < 0.001; (D) CD34 + CD38 +Quantification of AML cell subsets. Data are expressed as mean ± SD, student T test, *P < 0.05, **P < 0.01, ***P < 0.001). Flow cytometric analysis showed that CD38 in U937 and HL60 cells treated with compound 1 - The proportion of cells was significantly reduced in a dose-dependent manner. More importantly, after treatment with compound 1, CD34 + CD38 + The proportion of cells increased significantly, which is a cell population predicting a good prognosis. In general, incubation with different concentrations of compound 1 can significantly differentiate AML cells and reduce the number of LSCs in AML cells.

[0095] (8) Study on the pharmacokinetic properties of compound 1

[0096] The SD rat model was used for preliminary pharmacokinetic (PK) studies. The plasma concentration-time curve of compound 1 was statistically analyzed after a single dose of compound 1 (eg Figure 5 PK parameters are determined by The calculations are listed in Table 4. The test results show that the lead compound exhibits sufficient oral bioavailability (56.7%), but the plasma clearance rate is relatively fast. This indicates that the lead compound 1 is suitable for oral administration.

[0097] Table 4 PK parameters

[0098]

[0099] The above is only a preferred embodiment of the present invention. The present invention is not limited to the above implementation. As long as the technical effect of the present invention is achieved by the same means, it should belong to the protection scope of the present invention. Within the protection scope of the present invention, its technical scheme and / or implementation method can have various modifications and changes.

Claims

1. A pyrazole compound, the structure of which is shown in Formula I: Formula I; R1 is Cl or F; R2 is H, Br, Cl or methyl.

2. The pyrazole compound according to claim 1, characterized in that Its structure is shown in one of 1-8: 。 3. A method for preparing a pyrazole compound according to claim 1 or 2, characterized in that: 。 4. Use of the pyrazole compound according to claim 1 or 2 in the preparation of a dual inhibitor of CDK9 and HDACs, characterized in that: The HDACs are HDAC1, HDAC2, HDAC3, HDAC6 and HDAC8.

5. Use of the pyrazole compound according to claim 1 or 2 in the preparation of a drug for treating acute myeloid leukemia, characterized in that: The structure of the pyrazole compound is as follows: 。 6. A pharmaceutical composition, characterized in that The invention comprises the pyrazole compound according to claim 1 or 2, and also comprises a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that The dosage form of the pharmaceutical composition is injection or oral solution.

8. The pharmaceutical composition according to claim 6, characterized in that The carrier is a disintegrant or a binder.

9. The pharmaceutical composition according to claim 8, characterized in that The disintegrant is sodium starch glycolate or sodium carboxymethyl cellulose; the binder is microcrystalline cellulose or hydroxymethyl cellulose.

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