Crystal form, preparation method and application of pyridopyrrole compounds

Abstract

A pyridopyrrole compound crystal form, preparation method and application. #imgabs0#

Description

Technical Field

[0001] The present invention relates to a crystal form of a pyridopyrrole compound, and a preparation method and application of the crystal form. Background Art

[0002] Tumor development is often accompanied by excessive cell activation and persistent proliferation. Cyclin-dependent kinases (CDKs) play a crucial role in regulating the cell cycle and transcriptional processes, mediated by intracellular and extracellular signals. In cancer cells, CDK-cyclin activity is often dysregulated. Possible causes include overactivation of signaling pathways, overexpression of cyclins, abnormal CDK amplification, and inactivation or loss of endogenous inhibitors. These findings have inspired the development of tumor treatment technologies through the continuous search for novel CDK inhibitors.

[0003] CDK9, a member of the CDK family, is primarily involved in transcriptional regulation. Heterodimers composed of CDK9 and cyclins (T1, T2a, T2b, and K) form the positive transcription elongation factor (p-TEFb), with approximately 80% of CDK9 binding to cyclin T1. P-TEFb regulates transcriptional elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II, primarily at Ser-2. CDK9 inhibition and transcriptional repression lead to the rapid depletion of short-lived mRNA transcripts and associated proteins (including Myc and Mcl-1), resulting in the death of cancer cells that are highly dependent on these anti-apoptotic proteins. Therefore, targeting CDK9 represents a therapeutic strategy for tumor types that are highly dependent on these anti-apoptotic proteins.

[0004] Currently, CDK9 small molecule inhibitors have entered clinical research for cancer treatment, including Bayer's BAY1251152 and AstraZeneca's AZD4573. These patents include WO2012160034, WO2014076091, WO2009047359, WO2011110612, and US2016376287.

[0005]

[0006] Although much effort has been made to develop CDK9 inhibitors for the treatment of cancer and other diseases, no drugs targeting this target have been marketed to date. Among the drugs undergoing clinical trials, the most common clinically significant grade 3 / 4 and dose-limiting adverse side effect of BAY1251152 is neutropenia, while AZD4573 has poor kinase selectivity and metabolism, limiting its efficacy. Therefore, there is still an urgent need to develop novel, safer and more effective CDK9 inhibitors that can treat a variety of cancers, including leukemia and lymphoma. Summary of the Invention

[0007] The present invention provides a crystalline form A of the compound of formula (I), whose X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2θ angles: 7.22±0.20°, 17.16±0.20° and 22.34±0.20°;

[0008]

[0009] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.22±0.20°, 15.24±0.20°, 15.80±0.20°, 17.16±0.20°, 20.70±0.20°, 22.34±0.20°, 24.46±0.20° and 31.74±0.20°.

[0010] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.22±0.20°, 8.76±0.20°, 15.24±0.20°, 15.80±0.20°, 17.16±0.20°, 19.66±0.20°, 20.70±0.20°, 22.34±0.20°, 24.46±0.20°, 25.84±0.20°, 29.76±0.20° and 31.74±0.20°.

[0011] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.22°, 8.76°, 9.84°, 12.24°, 15.24°, 15.80°, 16.22°, 17.16°, 17.52°, 18.40°, 19.26°, 19.66°, 20.70°, 21.46°, 22.34°, 23.64°, 24.46°, 25.84°, 27.10°, 27.62°, 28.02°, 29.26°, 29.76°, 30.88°, 31.74°, 33.38°, 37.10° and 37.68°.

[0012] In some embodiments of the present invention, the above-mentioned crystal form A has an XRPD pattern as shown in FIG. Figure 1 shown.

[0013] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form A is shown in Table 1.

[0014] Table 1 XRPD analysis data of the crystal form of compound A of formula (I)

[0015]

[0016] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form A has an endothermic peak starting point at 77.71±3°C and 236.85±3°C, respectively.

[0017] In some embodiments of the present invention, the above-mentioned crystal form A has a DSC spectrum as shown below: Figure 2 shown.

[0018] In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned crystal form A shows a weight loss of 3.420% at 200±3°C.

[0019] In some embodiments of the present invention, the TGA spectrum of the above-mentioned A crystal form is as follows Figure 3 shown.

[0020] The present invention also provides a crystal form B of the compound of formula (I), which has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2θ angles: 19.72±0.20°, 21.52±0.20° and 23.20±0.20°;

[0021]

[0022] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 10.74±0.20°, 16.22±0.20°, 19.72±0.20°, 20.58±0.20°, 21.52±0.20°, 22.30±0.20°, 23.20±0.20° and 28.04±0.20°.

[0023] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 7.92±0.20°, 10.74±0.20°, 16.22±0.20°, 17.66±0.20°, 19.72±0.20°, 20.58±0.20°, 21.52±0.20°, 22.30±0.20°, 23.20±0.20°, 23.88±0.20°, 26.54±0.20°, 27.48±0.20° and 28.04±0.20°.

[0024] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 7.92°, 10.74°, 11.42°, 13.52°, 13.76°, 15.86°, 16.22°, 16.52°, 17.66°, 17.90°, 18.22°, 18.92°, 19.72°, 20.58°, 21.52°, 22.30°, 23.20°, 23.88°, 25.32°, 26. 6.12°, 26.54°, 27.14°, 27.48°, 27.72°, 28.04°, 28.52°, 28.96°, 29.20°, 29.74°, 30.24°, 30.58°, 31.56°, 32.54°, 32.82°, 33.38°, 34.36°, 34.75°, 35.44°, 36.00°, 36.56°, 37.08°, 37.96°, 38.74° and 39.50°.

[0025] In some embodiments of the present invention, the above-mentioned B crystal form has an XRPD pattern as shown in FIG. Figure 4 shown.

[0026] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form B is shown in Table 2.

[0027] Table 2 XRPD analysis data of the crystal form B of compound of formula (I)

[0028]

[0029]

[0030] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned B crystal form has an endothermic peak starting point at 257.81±3°C.

[0031] In some embodiments of the present invention, the above-mentioned B crystal form has a DSC spectrum as shown in FIG. Figure 5 shown.

[0032] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned Form B shows a weight loss of 0.326% at 200±3°C.

[0033] In some embodiments of the present invention, the TGA spectrum of the above-mentioned B crystal form is as follows Figure 6 shown.

[0034] The present invention also provides a method for preparing crystal form A, comprising the following steps:

[0035] 1) adding the compound of formula (I) into anhydrous methanol and refluxing;

[0036] 2) the compound of formula (I) is completely dissolved and filtered while hot;

[0037] 3) Distilled water was added dropwise to the filtrate under reflux to precipitate a white solid, and the mixture was cooled naturally to room temperature and stirred at room temperature;

[0038] 4) The mixture is filtered and the filter cake is dried under reduced pressure.

[0039] In some embodiments of the present invention, the reflux temperature is 65°C-80°C, preferably 65°C.

[0040] In some embodiments of the present invention, the stirring time is 10-12 hours, preferably 12 hours.

[0041] The present invention also provides a method for preparing Form B, comprising the following steps:

[0042] The crystal form A of the compound of formula (I) is stirred in ethanol, filtered, and the filter cake is dried under reduced pressure;

[0043] in,

[0044] The stirring temperature is 20°C;

[0045] The stirring time is 20-21 hours.

[0046] The present invention also provides the use of the above-mentioned crystal form A and the above-mentioned crystal form B in the preparation of CDK9 inhibitor drugs.

[0047] Technical Effects

[0048] The compound of formula (I) of the present application has good in vivo efficacy, and its crystal form is stable, is less affected by light, heat and humidity, and has good solubility, and has broad prospects for drug development.

[0049] Definition and Description

[0050] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0051] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0052] The chemical reactions of the present invention are carried out in suitable solvents that are compatible with the chemical transformations of the present invention and the reagents and materials required. To obtain the compounds of the present invention, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0053] The present invention will be described in detail below through examples, which are not intended to limit the present invention in any way.

[0054] All solvents used in the present invention were commercially available and used without further purification.

[0055] The structures of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data on the cultured single crystal using a Bruker D8 venture diffractometer, using CuKα radiation as the light source and scanning mode: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0056] The solvent used in the present invention is commercially available.

[0057] The present invention uses the following abbreviations: DCM stands for dichloromethane; DMF stands for N,N-dimethylformamide; DMSO stands for dimethyl sulfoxide; EtOH stands for ethanol; MeOH stands for methanol; ACN stands for acetonitrile; THF stands for tetrahydrofuran; H2O stands for water; NCS stands for 1-chloropyrrolidine-2,5-dione; NIS stands for N-iodosuccinimide; and DMAC stands for dimethylacetamide.

[0058] Compounds are named according to the conventional nomenclature in the art or using Software naming, commercially available compounds use supplier catalog names. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 : XRPD pattern of Form A;

[0060] Figure 2 : DSC spectrum of crystal form A;

[0061] Figure 3 : TGA spectrum of crystal form A;

[0062] Figure 4 : XRPD pattern of Form B;

[0063] Figure 5 : DSC spectrum of crystal form B;

[0064] Figure 6 : TGA spectrum of crystal form B.

[0065] Instrument parameters

[0066] X-ray powder diffraction (XRPD) instrument information and methods of the present invention

[0067] XRPD was performed using a DX-2700BH X-ray diffractometer from Dandong Haoyuan Company. The test parameters are shown in Table 3.

[0068] Table 3: XRPD test parameters

[0069] Differential Scanning Calorimetry (DSC) Instrument Information and Methods of the Invention

[0070] The DSC spectra were collected on a TA2500 differential scanning calorimeter, and the test parameters are shown in Table 4.

[0071] Table 4: DSC test parameters

[0072] parameter METTLERTOLEDODSC1 Sample tray High-pressure crucible Temperature range 40～350℃ Scan rate (℃ / min) 10 Shielding gas Nitrogen

[0073] Thermogravimetric analysis (TGA) instrument information and method of the present invention

[0074] TGA was collected on a TA5500 thermogravimetric analyzer, and the test parameters are shown in Table 5.

[0075] Table 5: TGA test parameters

[0076] DETAILED DESCRIPTION

[0077] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments, but the specific implementation methods are not intended to limit the content of the present invention.

[0078] Example 1: Preparation of compound of formula (I)

[0079]

[0080] Step 1:

[0081] To a solution of compound 1 (900 g, 5.20 mol, 1 eq) in DMF (3.60 L) at 0°C, NCS (729.37 g, 5.46 mol, 1.05 eq) was added portionwise. The mixture was stirred at 15°C for 12 hours. The reaction of the starting compound 1 was monitored by LCMS and HPLC for complete reaction. While stirring, the reaction solution was slowly poured into a 10% (w%) sodium hydroxide aqueous solution (4.0 L). The mixture was extracted with ethyl acetate (2.0 L x 2). The organic phases were combined, washed with saturated brine (2.0 L x 2), separated, and concentrated to yield a residue. The residue was slurried with dichloromethane (3.0 L) for 2 hours. Filtered, and the filter cake dried to yield compound 2. 1 H NMR (400MHz, CDCl3) δ8.05 (s, 1H), 6.79 (s, 1H), 4.57 (br s, 2H). LCMS(ESI)m / z:208.9(M+1).

[0082] Step 2:

[0083] To a solution of compound 2 (1870 g, 9.01 mol, 1 eq) in acetic acid (5.0 L) was added NIS (2.23 kg, 9.92 mol, 1.1 eq). The mixture was purged with nitrogen three times and then heated to 80°C for 4.0 hours. The reaction was complete as monitored by LCMS and HPLC. The reaction mixture was cooled to 15°C and slowly poured into water (2.5 L) while stirring. A large amount of solid precipitated. The solid was filtered, and the filter cake was washed with water (1 L) and dried to afford the crude product. The crude product was slurried with ethanol (2.0 L). The mixture was filtered, and the filter cake was dried to afford compound 3. LCMS (ESI) m / z: 334.8 (M+1).

[0084] Step 3:

[0085] To a solution of compound 3 (2700 g, 7.48 mol) in toluene (16.2 L) were added compound 4 (1.88 kg, 8.98 mol, 1.20 equiv), dichlorobistriphenylphosphine palladium (262.51 g, 374 mmol, 0.05 equiv), cuprous iodide (142.46 g, 747.99 mmol, 0.1 equiv), and triethylamine (2.27 kg, 22.44 mol, 3.12 L, 3 equiv). The mixture was purged with nitrogen three times and heated to 110°C for 5 hours. The mixture was cooled to 15°C, whereupon a large amount of solid precipitated. The mixture was filtered, and the filter cake was dried under reduced pressure to yield a crude product. The crude product was washed once with water (20 L), filtered, and the filter cake was drained. The filter cake was recrystallized from toluene (12 L) to yield compound 5. LCMS (ESI) m / z: 416.1 (M+1).

[0086] Step 4:

[0087] DMAC (3.15 L) was added to a 5.0-liter three-necked flask. Compound 5 (630 g, 1.50 mol, 1.0 eq) was added with stirring to obtain a suspension. Potassium tert-butoxide (252.98 g, 2.25 mol, 1.50 eq) was added portionwise. After addition, the mixture was stirred at 15°C for 16 hours to obtain a clear solution. While stirring, the reaction solution was slowly added to water (18.9 L), resulting in the precipitation of a large amount of solid. After stirring for half an hour, the mixture was filtered, the filter cake was drained, and the filter cake was slurried with water (12.6 L) for 2 hours. Filtered, and the filter cake was dried to obtain Compound 6.

[0088] Step 5:

[0089] To a 30-liter reactor, dioxane (6.0 L) and water (600 mL) were added, stirring was initiated, and compound 6 (600 g, 1.45 mol, 1 eq), compound 7 (361.20 g, 1.74 mol, 1.20 eq), and cesium carbonate (942.92 g, 2.89 mol, 2.0 eq) were added. The mixture was purged with nitrogen three times, and Pd(dppf)Cl2·CH2Cl2 (29.55 g, 36.17 mmol) was added. The atmosphere was purged with nitrogen three times, and the temperature was raised to 105-110°C for 16 hours. The reaction mixture was cooled to 15°C. The reaction mixture was divided into two batches, each approximately 4.0 L. While stirring, half of the reaction mixture was slowly added to water (15 L). A large amount of solid precipitated, which was filtered and the filter cake was washed with ethanol (3.0 L x 2). The combined filter cakes were dissolved in 15 L of a 5:1 dichloromethane:methanol ratio and filtered. The filtrate was added with mercaptosilica gel (40% w / w, 220 g) and stirred at room temperature for 16 hours. The mixture was then filtered through a pad of celite and the palladium was removed three times using the same method. The filtrate was spin-dried, and the residue was slurried with ethanol (3.0 L), filtered, and the filter cake dried to yield Compound 8. LCMS (ESI) m / z: 416.2 (M+1).

[0090] Step 6: Ethyl acetate (2.35 L) was added to a 5.0 L three-necked flask, stirring was started, and compound 8 (470 g, 1.08 mol) was added. The solid did not completely dissolve to obtain a white suspension. Hydrochloric acid / ethyl acetate (4 mol / L, 2.35 L) was added dropwise using a constant pressure dropping funnel. After the addition was complete, the reaction was stirred at room temperature for 16 hours. The reaction was filtered to obtain the hydrochloride of the compound of formula (I). The hydrochloride of the compound of formula (I) was rinsed with ethyl acetate (2.0 L) and then drained. The filter cake was dissolved in 1.50 L of water, the organic phase was discarded, and the aqueous phase was transferred to a 5.0 L three-necked flask. Under stirring, 1 mol / L sodium hydroxide aqueous solution was added dropwise to a pH of ~11. A large amount of white solid precipitated and was filtered to obtain the compound of formula (I). 1HNMR(400MHz,DMSO-d6)δ11.96(s,1H),9.21(br d,J=8.8Hz,1H),8.97(br d,J=10.2Hz,1H),8.31(s,1H),8.19(s,1H),7.96(s,1H),6.34(d,J=1.4Hz,1H),3.96(s,3H),3.33(br d, J=12.4Hz, 2H), 3.13-2.94 (m, 3H), 2.24 (br d, J=12.2Hz, 2H), 1.98-1.81 (m, 2H); LCMS (ESI) m / z: 316.2 (M+1).

[0091] Example 2: Preparation of Crystalline Form A of Compound (I)

[0092] Anhydrous methanol (9.0 L) was added to a 30-liter low-temperature jacketed reactor, stirring was started, and the compound of formula (I) (300 g, 0.92 mol) was added. The heating was turned on, the external temperature was 80°C, the internal temperature was about 65°C, the methanol began to reflux, and the temperature was maintained at 65°C. The solid was completely dissolved to obtain a clear solution. The reaction solution was filtered while hot, and the filtrate was transferred to the reactor. Distilled water (9.0 L) was added dropwise under reflux. After the addition was completed, a large amount of white solid precipitated. The mixture was naturally cooled to room temperature and stirred at room temperature for 12 hours. The mixture was filtered, the filter cake was transferred to an oven, and dried under reduced pressure to obtain Form A of the compound of formula (I). The XRPD test results are as follows: Figure 1 , TGA and DSC test results are as follows Figure 2 and Figure 3 shown.

[0093] Example 3: Preparation of Crystalline Form B of Compound (I)

[0094] At 20°C, Form A of the compound of formula (I) (50 g, 0.158 mol) was stirred in 250 mL of ethanol for 21 hours. The mixture was filtered, and the filter cake was transferred to an oven and dried under reduced pressure to obtain Form B of the compound of formula (I). The XRPD test results are as follows: Figure 4 , TGA and DSC test results are as follows Figure 5 and Figure 6 shown.

[0095] Example 4: Stability test of the crystal form A of compound of formula (I)

[0096] Purpose of the experiment:

[0097] The stability of Form A of Compound (I) was investigated under influencing factors (high temperature, high humidity and light) and accelerated conditions (40°C / 75% RH and 30°C / 65% RH) to evaluate the solid stability of Form A.

[0098] Experimental methods:

[0099] 1) Approximately 1.5 g of Form A of Compound (I) was weighed and placed into a clean, dry glass bottle. Two portions were weighed and labeled S1-condition-time and S2-condition-time, respectively. Approximately 20 mg was weighed and placed into a clean, dry glass bottle, labeled S3-condition-time. The sample was spread into a thin layer and used as the test sample. The sample was placed under the influencing factor test conditions (40°C, 60°C, 25°C / 75% RH, 25°C / 92.5% RH, light, and light control) and accelerated conditions (40°C / 75% RH and 30°C / 65% RH). The samples were fully exposed. Samples were collected at 5, 10, and 30 days at 40°C, 60°C, 25°C / 75% RH, and 25°C / 92.5% RH; samples were analyzed at 5 and 10 days for the light control; and samples were analyzed at 1, 2, and 3 months under the accelerated conditions. The analytical methods are shown in Table 6.

[0100] Table 6

[0101]

[0102] 2) At the time point of investigation, remove the corresponding test sample and cap it. Remove the Day 0 sample from the refrigerator and analyze it after returning to room temperature. The sample labeled S1-condition-time is used for content and related substance testing; the sample labeled S2-condition-time is used as a backup sample; and the sample labeled S3-condition-time is used for XRPD analysis.

[0103] Experimental results:

[0104] The results of the solid stability test of Form A are shown in Table 7.

[0105] Table 7

[0106]

[0107] * Light (total illumination visible light = 5000 ± 500 lux, UV 90 μw / cm 2 , open); **simultaneously with visible light + UV.

[0108] Experimental conclusion: The crystal form A of compound of formula (I) has good stability.

[0109] Biological testing

[0110] Experimental Example 1: In vitro CDK9 / CyclinT1 enzyme activity test

[0111] Experimental Materials:

[0112] CDK9 / CyclinT1 kinase was purchased from Carna, ADP-Glo detection kit was purchased from Promega, PKDTide substrate and kinase reaction buffer were purchased from Signalchem. Nivo multi-label analyzer was purchased from PerkinElmer.

[0113] Experimental methods:

[0114] Dilute the enzyme, substrate, ATP, and inhibitor using the kinase buffer provided in the kit.

[0115] The test compound was diluted 5-fold to the 8th concentration using a dispenser, that is, from 50 μM to 0.65 nM, with a DMSO concentration of 5%, and a double-well experiment was set up. 1 μL of each inhibitor concentration gradient, 2 μL of CDK9 / CyclinT1 enzyme (4 ng), and a mixture of 2 μL of substrate and ATP (100 μM adenosine triphosphate, 0.2 μg / μL substrate) were added to the microplate. At this time, the final concentration gradient of the compound was 10 μM diluted to 0.13 nM. The reaction system was placed at 25 ° C for 120 minutes. After the reaction was completed, 5 μL of ADP-Glo reagent was added to each well, and the reaction was continued at 25 ° C for 40 minutes. After the reaction was completed, 10 μL of kinase detection reagent was added to each well. After the reaction was completed, chemiluminescence was read using a multi-label analyzer at 25 ° C for 30 minutes, with an integration time of 0.5 seconds. Data analysis:

[0116] The raw data were converted into inhibition rate, IC, using the equation (Sample-Min) / (Max-Min)*100%. 50 The value can be obtained by four-parameter curve fitting (derived by log(inhibitor) vs. response--Variable slope mode in GraphPad Prism). Table 6 provides the inhibitory activity of the compounds of the present invention on CDK9 / CyclinT1 enzyme.

[0117] Experimental conclusion:

[0118] The compound of formula (I) has good activity against CDK9 kinase, and is similar in activity to the reference compounds BAY1251152 and AZD4573.

[0119] Experimental Example 2: In vitro CDK1 / CyclinB1 enzyme activity test

[0120] Experimental Materials:

[0121] The CDK1 / CyclinB1 kinase detection kit was purchased from Promega, and the Nivo multilabel analyzer was purchased from PerkinElmer.

[0122] Experimental methods:

[0123] Dilute the enzyme, substrate, ATP, and inhibitor using the kinase buffer provided in the kit.

[0124] The test compound was diluted five-fold using a pipette to the eighth concentration, from 50 μM to 0.65 nM, using 5% DMSO. A duplicate assay was performed. To the microplate, 1 μL of each inhibitor concentration gradient, 2 μL of CDK1 / Cyclin B1 enzyme (12.5 ng), and 2 μL of a substrate and ATP mixture (25 μM ATP, 0.2 μg / μL substrate) were added, resulting in a final compound concentration gradient of 10 μM to 0.13 nM. The reaction system was incubated at 25°C for 120 minutes. After completion of the reaction, 5 μL of ADP-Glo reagent was added to each well and the reaction continued at 25°C for 40 minutes. After the reaction was complete, 10 μL of kinase detection reagent was added to each well. The reaction was incubated at 25°C for 30 minutes. Chemiluminescence was then read using a multi-label analyzer with an integration time of 0.5 seconds.

[0125] Data Analysis:

[0126] The raw data were converted into inhibition rate, IC, using the equation (Sample-Min) / (Max-Min)*100%. 50 The value can be obtained by four-parameter curve fitting (derived by using the log(inhibitor) vs. response--Variable slope mode in GraphPad Prism). Table 6 provides the inhibitory activity of the compounds of the present invention against CDK1 / CyclinB1 enzymes.

[0127] Experimental conclusion:

[0128] The compound of formula (I) has weak inhibitory activity against CDK1 kinase, so the compound of the present invention exhibits better selectivity against CDK1 than BAY1251152 and AZD4573.

[0129] Experimental Example 3: In vitro CDK2 / CyclinE1 enzyme activity test

[0130] Experimental Materials:

[0131] The CDK2 / Cyclin E1 kinase detection kit was purchased from Promega, and the Nivo multilabel analyzer was purchased from PerkinElmer.

[0132] Experimental methods:

[0133] Dilute the enzyme, substrate, ATP, and inhibitor using the kinase buffer provided in the kit.

[0134] Test compounds were diluted five-fold using a pipette to an eighth concentration, from 50 μM to 0.65 nM, using 5% DMSO. A duplicate assay was performed. To each microplate, 1 μL of each inhibitor concentration gradient, 2 μL of CDK2 / Cyclin E1 enzyme (2 ng), and 2 μL of a substrate and ATP mixture (150 μM ATP, 0.1 μg / μL substrate) were added, resulting in a final compound concentration gradient of 10 μM to 0.13 nM. The reaction system was incubated at 25°C for 60 minutes. Following completion of the reaction, 5 μL of ADP-Glo reagent was added to each well and the reaction continued at 25°C for 40 minutes. After the reaction, 10 μL of kinase detection reagent was added to each well. The reaction was incubated at 25°C for 30 minutes. Luminescence was then measured using a multi-label analyzer with an integration time of 0.5 seconds.

[0135] Data Analysis:

[0136] The raw data were converted into inhibition rate, IC, using the equation (Sample-Min) / (Max-Min)*100%. 50 The value can be obtained by four-parameter curve fitting (derived by using the log(inhibitor) vs. response--Variable slope mode in GraphPad Prism). Table 6 provides the inhibitory activity of the compounds of the present invention against CDK2 / CyclinE1 enzyme.

[0137] Experimental conclusion:

[0138] The compound of formula (I) has weak inhibitory activity against CDK2 kinase, so the compound of the present invention exhibits better selectivity against CDK2 than BAY1251152 and AZD4573.

[0139] Experimental Example 4: In vitro cell activity test

[0140] Experimental Materials:

[0141] IMDM medium, fetal bovine serum, and penicillin / streptomycin antibiotics were purchased from Promega (Madison, WI). MV-4-11 cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences. Nivo multi-label analyzer was used (PerkinElmer).

[0142] Experimental methods:

[0143] MV-4-11 cells were seeded into a white 96-well plate, with 80 μL of cell suspension per well, containing 6,000 MV-4-11 cells. The plate was incubated in a CO2 incubator overnight.

[0144] Use a pipette to dilute the test compound 5-fold to the eighth concentration, from 2 mM to 26 nM, in duplicate. Add 78 μL of culture medium to the middle plate. Transfer 2 μL of the serially diluted compound to each well of the corresponding position. Mix thoroughly, and then transfer 20 μL per well to the cell plate. Final compound concentrations range from 10 μM to 0.13 nM. Incubate the cell plate in a CO2 incubator for 3 days.

[0145] 25 μL of Promega CellTiter-Glo reagent was added to each well of the cell plate and incubated at room temperature for 10 minutes to allow the luminescence signal to stabilize. The cells were read using a PerkinElmer Nivo Multilabel Analyzer.

[0146] Data Analysis:

[0147] The raw data were converted into inhibition rate, IC, using the equation (Sample-Min) / (Max-Min)*100%. 50 The value of can be obtained by four-parameter curve fitting (obtained in the "log (inhibitor) vs. response--Variable slope" mode in GraphPad Prism). Table 6 provides the inhibitory activity of the compounds of the present invention on MV-4-11 cell proliferation.

[0148] Experimental conclusion:

[0149] The compound of formula (I) has good anti-proliferative activity against MV4-11 cells. The experimental results are shown in Table 8:

[0150] Table 8

[0151]

[0152] Experimental Example 5: In vivo efficacy study

[0153] In vivo efficacy studies were performed on BALB / c nude mice subcutaneously implanted with MV4-11 acute myeloid leukemia patient-derived human tumor cell line-based xenografts (CDX).

[0154] Experimental operation:

[0155] BALB / c nude mice, female, 6-8 weeks old, weighing approximately 18-22 g, were kept in a special pathogen-free environment in single ventilated cages (3 mice per cage). All cages, bedding, and water were disinfected before use. All animals had free access to standard certified commercial laboratory diet. A total of 36 mice purchased from Shanghai Lingchang Biological Science and Technology Co., LTD. were used for the study. Each mouse was implanted with tumor cells (10 × 10 6 The tumors were grown in 0.2 ml of phosphate buffered saline (PBS). Dosing was initiated when the mean tumor volume reached approximately 121 cubic millimeters. The test compound was administered weekly by injection at a dose of 10 mg / kg. Tumor volume was measured twice weekly using a two-dimensional caliper. Volume was measured in cubic millimeters and calculated using the following formula: V = 0.5a × b 2 , where a and b are the major and minor diameters of the tumor, respectively. Antitumor efficacy was determined by dividing the mean tumor volume increase in animals treated with the compound by the mean tumor volume increase in untreated animals.

[0156] Experimental conclusion:

[0157] In the MV4-11 acute myeloid leukemia CDX in vivo efficacy model, the compound of formula (I) exhibited good efficacy and safety. The in vivo efficacy results are shown in Table 9:

[0158] Table 9

[0159]

Claims

1. Crystal form A of the compound of formula (I), ， Its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.22°, 8.76°, 9.84°, 12.24°, 15.24°, 15.80°, 16.22°, 17.16°, 17.52°, 18.40°, 19.26°, 19.66°, 20.70°, 21.46°, 22.34°, 23.64°, 24.46°, 25.84°, 27.10°, 27.62°, 28.02°, 29.26°, 29.76°, 30.88°, 31.74°, 33.38°, 37.10° and 37.68°, and its XRPD pattern is shown in Figure 1.

2. The crystal form A according to claim 1, wherein its differential scanning calorimetry curve has an endothermic peak starting point at 77.71±3°C and 236.85±3°C, respectively.

3. The crystal form A according to claim 2, has a DSC spectrum as shown in Figure 2.

4. The crystal form A according to claim 1, wherein the thermogravimetric analysis curve thereof shows a weight loss of 3.420% at 200±3°C.

5. The crystal form A according to claim 4, whose TGA spectrum is shown in Figure 3.

6. The method for preparing the crystalline form A of compound of formula (I) according to claim 1, comprising the following steps: 1) adding the compound of formula (I) into anhydrous methanol and refluxing; 2) The compound of formula (I) is completely dissolved and filtered while hot; 3) Add distilled water dropwise to the filtrate under reflux to precipitate a white solid, cool it naturally to room temperature, and stir at room temperature; 4) The mixture is filtered and the filter cake is dried under reduced pressure.

7. The preparation method according to claim 6, wherein The reflux temperature was 65°C.

8. The preparation method according to claim 6, wherein The stirring time was 12 hours.

9. Use of the crystal form A according to any one of claims 1 to 5 in the preparation of CDK9 inhibitor drugs.

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