Crystalline forms of imidazolinone derivatives

Abstract

The present invention relates to crystalline forms of imidazolinone derivatives, and further to crystalline forms of substituted imidazolinone derivatives, and pharmaceutical compositions, preparation methods, and uses thereof in the manufacture of DNA-PK inhibitors. Specifically, the present invention relates to crystalline forms I to VI of the compound represented by formula (A), and pharmaceutical compositions, preparation methods, and uses thereof in the manufacture of DNA-PK inhibitors. [C1] TIFF2024533674000021.tif39156

Description

[Technical Field]

[0001] The present invention relates to a crystalline form of an imidazolinone derivative or a hydrate or solvate thereof, a method for producing the same or a pharmaceutical composition thereof, and use of the same in the field of producing DNA-PK inhibitors. [Background technology]

[0002] DNA-dependent protein kinase (DNA-PK) is an enzyme complex consisting of the Ku70 / Ku80 heterodimer and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This enzyme complex is activated by DNA and exerts its corresponding functions (George et al., 2019). DNA-PK, a serine / threonine protein kinase, is a member of the PIKK (phosphatidylinositol 3-kinase-related kinase) family and plays an important role in the repair of intracellular DNA double-strand breaks (DSBs), cellular DNA recombination, and antibody DNA rearrangement (V(D)J recombination). It is also involved in physiological processes such as chromosome modification, transcriptional regulation, and telomere maintenance.

[0003] DNA-PK inhibitors can enhance the therapeutic effect of antitumor therapies that cause DNA damage (e.g., IR, chemotherapy, etc.). Although the use of DNA-PK inhibitors interferes to some extent with the DNA repair function of normal cells, normal cells have multiple complementary DNA repair pathways. On the other hand, tumor cells are exposed to strong DNA replication stress and lack effective DNA repair methods, making them more sensitive to DNA-PK inhibitors. Therefore, inhibiting DNA-PK activity in tumor cells can enhance the killing effect of other antitumor therapies.

[0004] The patent (application number: PCT / CN2021 / 087912) describes a novel DNA-PK inhibitor having the structure shown in formula (A), which has good inhibitory effect on DNA-PK activity and has potential for the preparation of antitumor drugs.

[0005] [ka] Summary of the Invention [Means for solving the problem]

[0006] The present invention provides a crystalline form of 4-((7-ethyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)-2-fluoro-5-methylbenzamide (Compound A), which has the following chemical structure: [ka]

[0007] The crystalline form of the present invention exhibits at least one of the following advantages: good solubility, high stability, ease of processing, processing and purification, improved oral drug bioavailability, extended drug shelf life, and ease of manufacturing each dosage form.

[0008] The crystalline forms of the present invention exhibit pharmaceutical advantages over the amorphous form of Compound A. In particular, the crystalline forms have enhanced chemical and physical stability and are more advantageous for the preparation of solid drug dosage forms containing pharmacologically active ingredients.

[0009] The crystalline form of the present invention is present in about 5% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 10% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 15% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 20% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 25% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 30% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 35% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 40% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 45% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 50% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 55% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 60% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 65% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 70% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 75% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 80% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 85% to about 100% by weight of the drug substance. In one embodiment, the crystalline form of the present invention is present in about 90% to about 100% by weight of the drug substance. In some embodiments, the crystalline form of the present invention is present at about 95% to about 100% by weight of the drug substance. In some embodiments, the crystalline form of the present invention is present at about 98% to about 100% by weight of the drug substance. In some embodiments, the crystalline form of the present invention is present at about 99% to about 100% by weight of the drug substance. In some embodiments, substantially all of the drug substance is the crystalline form of the present invention, i.e., the drug substance is substantially phase-pure crystalline.

[0010] Compound A of the present invention is an amorphous form of Compound A unless otherwise specified.

[0011] One embodiment of the crystalline form described in the present invention is anhydrous Compound A (crystalline form I) having characteristic diffraction peaks at the following 2θ positions in a powder X-ray diffraction pattern using Cu-Kα radiation: 9.859°±0.3°, 14.759°±0.3°, 19.679°±0.3°, and 19.961°±0.3°.

[0012] The powder X-ray diffraction pattern of crystalline form I further has characteristic diffraction peaks at 2θ diffraction angles of 4.979°±0.2°, 15.759°±0.2°, 19.339°±0.2°, 22.481°±0.2°, and 24.641°±0.2°.

[0013] Furthermore, the powder X-ray diffraction pattern of crystalline form I has characteristic diffraction peaks at 2θ positions of 12.120°±0.2°, 18.802°±0.2°, and 21.822°±0.2°.

[0014] Furthermore, the powder X-ray diffraction pattern of crystalline form I has characteristic diffraction peaks at the following 2θ positions: 11.483°±0.2°, 20.422°±0.2°, 21.379°±0.2°, 22.142°±0.2°, 25.939°±0.2°, 29.180°±0.2°, and 31.041°±0.2°.

[0015] Furthermore, the powder X-ray diffraction pattern (XRD) of the crystalline form I is essentially as shown in FIG. 1 or FIG.

[0016] One embodiment of the crystalline form according to the present invention is crystalline form II, the powder X-ray diffraction pattern of which is essentially as shown in FIG.

[0017] One embodiment of the crystalline form according to the present invention is crystalline form III, the powder X-ray diffraction pattern of which is essentially as shown in FIG.

[0018] One embodiment of the crystalline form according to the present invention is crystalline form IV, the powder X-ray diffraction pattern of which is essentially as shown in FIG.

[0019] One embodiment of the crystalline form described in the present invention is crystalline form V, the powder X-ray diffraction pattern of which is essentially as shown in FIG.

[0020] One embodiment of the crystalline form of the present invention is crystalline form VI, and the powder X-ray diffraction pattern of crystalline form VI is essentially as shown in FIG.

[0021] The present invention also relates to a method for preparing crystalline Form I, selected from Method 1 or Method 2.

[0022] Method 1: Compound A is dissolved in a solvent, and the temperature is raised to reflux to dissolve the solid. The solution is then allowed to cool and crystallize, followed by filtration and drying to obtain Form I.

[0023] Method 2: Compound A is dissolved in a solvent, and the temperature is raised to 75-85°C to dissolve the solid. The temperature is then lowered to 55-65°C to crystallize the solid, and the temperature is further lowered to 15-25°C to crystallize the solid, followed by filtration and drying to obtain Crystal Form I.

[0024] The solvent is selected from alcohol-based solvents or mixed solvents of alcohol-based solvents and water.

[0025] The present invention also relates to pharmaceutical compositions comprising a therapeutically effective amount of a crystalline compound according to the present invention and one or more pharmaceutically acceptable carriers or excipients.

[0026] The crystalline forms described in the present invention may be used as active pharmaceutical ingredients or pharmaceutical compositions containing them as active ingredients in the manufacture of DNA-PK inhibitor medicaments.

[0027] Here, the DNA-PK inhibitor is used in the manufacture of a drug for treating and preventing cancer.

[0028] A powder X-ray diffraction pattern that is substantially the same as the one disclosed in the present invention also falls within the scope of the present invention.

[0029] Terms used in this specification and claims have the following meanings unless otherwise specified.

[0030] An "effective dose" is an amount of a compound to elicit a physiological or medical response in a tissue, system, or subject, which amount is intended to include an amount of compound sufficient to prevent or alleviate to some extent one or more symptoms of the disease or condition being treated when administered to a subject.

[0031] "I C 50 " means the half-maximal inhibitory concentration, which refers to the concentration at which half of the maximum inhibitory effect is reached.

[0032] The structure of the crystalline forms of the present invention can be analyzed using a variety of analytical techniques known to those skilled in the art, including, but not limited to, powder X-ray diffraction (XRD).

[0033] It will be understood that the numerical values ​​described and claimed in this invention are approximations, and variations in the numerical values ​​can be due to instrument calibration, instrument error, crystal purity, crystal size, sample size, and other factors.

[0034] It will be understood that the crystalline forms of the present invention are not limited to those having characteristic patterns (e.g., XRD) that are exactly the same as those depicted in the drawings disclosed in the present invention, but that any crystalline form having a characteristic pattern that is basically the same as or essentially the same as the pattern depicted in the drawings is included within the scope of the present invention.

[0035] It will be apparent to those skilled in the art from consideration of the specification and operation of the examples that various modifications and variations of the present invention may be made without departing from the scope and spirit of the present invention. [Brief explanation of the drawings]

[0036] [Figure 1] 1 is a powder X-ray diffraction pattern of crystalline form I of Compound A prepared in Example 2 using Cu-Kα radiation. [Figure 2] 1 is a powder X-ray diffraction pattern of crystalline form I of Compound A prepared in Example 3. [Figure 3] 1 is a TGA diagram of crystalline form I of Compound A prepared in Example 3. [Figure 4] 1 is a DSC diagram of crystalline form I of Compound A prepared in Example 3. [Figure 5] FIG. 1 is a DVS diagram of crystalline form I of Compound A prepared in Example 3. [Figure 6] 1 is a powder X-ray diffraction pattern of crystalline form II. [Figure 7] FIG. 1 is a TGA diagram of crystalline form II. [Figure 8] FIG. 1 is a DSC diagram of crystalline form II. [Figure 9] 1 is a powder X-ray diffraction pattern of crystalline form III. [Figure 10] FIG. 1 is a TGA diagram of crystalline form III. [Figure 11] FIG. 1 is a DSC diagram of crystalline form III. [Figure 12] 1 is a powder X-ray diffraction pattern of crystalline form IV. [Figure 13] FIG. 1 is a TGA diagram of crystalline form IV. [Figure 14] FIG. 1 is a DSC diagram of crystalline form IV. [Figure 15] 1 is a powder X-ray diffraction pattern of crystalline form V. [Figure 16] FIG. 1 is a TGA diagram of crystalline form V. [Figure 17] FIG. 1 is a DSC diagram of crystalline form V. [Figure 18] 1 is a powder X-ray diffraction pattern of crystalline form VI. [Figure 19] FIG. 1 is a TGA diagram of crystalline form VI. [Figure 20] FIG. 1 is a DSC diagram of crystalline form VI. DETAILED DESCRIPTION OF THE INVENTION

[0037] The following detailed description of the implementation procedures and beneficial effects of the present invention will be given using specific examples, which are intended to help readers better understand the essence and characteristics of the present invention and are not intended to limit the scope of the present application.

[0038] Unless otherwise specified in the examples, solutions refer to aqueous solutions.

[0039] Unless otherwise specified, the experimental conditions for crystallization were generally room temperature (20°C to 30°C, 30%RH to 70%RH), and the solvent ratios refer to volume ratios.

[0040] Intermediate 1 4-Amino-2-fluoro-5-methylbenzamide (Intermediate 1) 4-amino-2-fluoro-5-methylbenzamide [ka]

[0041] 4-Amino-2-fluoro-5-methylbenzonitrile 1A (300 mg, 2 mmol) and potassium carbonate (41.4 mg, 0.3 mmol) were dissolved in 1 mL of dimethyl sulfoxide, and 300 μL of hydrogen peroxide solution was added in an ice bath. The mixture was then gradually heated to 60 °C and stirred for 2 hours. The reaction was monitored by TLC until completion, and 5 mL of water was added to the reaction solution to precipitate a white solid. The solid was filtered and spin-dried to remove the water, yielding the title compound 4-amino-2-fluoro-5-methylbenzamide intermediate 1 (white solid, 160 mg, 47% yield).

[0042] 1 H NMR (400 MHz DMSO) δ 7.37 (d, 1H), 7.15 (s, 1H), 6.96 (s, 1H), 6.32 (d, 1H), 5.70 (s, 1H), 2.01 (s, 3H). LC-MS m / z (ESI) = 169.10 [M+1]

[0043] Intermediate 2 2-Chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (Intermediate 2) 2-chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one [ka]

[0044] Step 1: Ethyl 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylate (2B) ethyl 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylate Ethyl 2,4-dichloropyrimidine-5-carboxylate 2A (30.00 g, 136.4 mmol) and tetrahydro-2H-pyran-4-amine hydrochloride (18.66 g, 136.4 mmol) were dissolved in acetonitrile (600 mL). After stirring several times, potassium carbonate (46.92 g, 340.9 mmol) was added and the mixture was stirred at room temperature for 4 hours. After monitoring the completion of the reaction by TLC, the mixture was filtered, the residue was washed with ethyl acetate (300 mL), and the filtrate was concentrated to give the crude product. The crude product was purified by column separation (n-hexane:ethyl acetate (v / v) = 1:1) to give the title compound, ethyl 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylate 2B (white solid, 30.0 g, 77.4% yield).

[0045] 1 H NMR (400 MHz DMSO) δ 8.62 (s,1H), 8.32 (d, 1H), 4.30 (q, 2H), 4.21-4.16 (m, 1H), 3.86-3.83 (m, 2H), 3.48-3.42 (m, 2H), 1.88-1.85 (m, 2H), 1.62-1.53 ​​(m, 2H), 1.31 (t, 3H). LC-MS m / z (ESI) = 286.10 [M+1]

[0046] Step 2: 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylic acid (2C) 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylic acid Ethyl 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylate 2B (30 g, 104.99 mmol) was dissolved in tetrahydrofuran / water (200 mL / 200 mL), lithium hydroxide (5.03 g, 209.99 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction was monitored for completion by TLC, concentrated to remove tetrahydrofuran, and the pH was adjusted to 5 with 6N hydrochloric acid to precipitate a white solid. The filter cake was washed twice with petroleum ether, and the solid was collected to give the title compound 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylic acid 2C (white solid, 15.0 g, 55.44% yield).

[0047] 1 H NMR (400 MHz DMSO) δ 8.60 (s, 1H), 8.54 (d, 1H), 4.20-4.15 (m, 1H), 3.86-3.83 (m, 2H), 3.48-3.42 (m, 2H), 1.89-1.86 (m, 2H), 1.60-1.50 (m, 2H). LC-MS m / z(ESI)= 258.10 [M+1].

[0048] Step 3: 2-chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (2D) 2-chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one 2-Chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylic acid 2C (15 g, 58.21 mmol) was dissolved in dimethylacetamide (150 mL), triethylamine (7.38 mL, 58.21 mmol), and diphenyl azidophosphate (12.06 mL, 58.21 mmol) were added, and the mixture was gradually heated to 120 °C and stirred for 1.5 h. After completion of the reaction by TLC, the reaction mixture was poured into ice water, filtered, and the solid was collected. The solid was washed three times with water and concentrated to dryness under vacuum to give the title compound 2-chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one 2D (white solid, 13.0 g, 87.69% yield).

[0049] 1 H NMR (400 MHz DMSO) δ 11.63 (s, 1H), 8.11 (s, 1H), 4.43-4.37 (m, 1H), 3.98-3.94 (m, 2H), 2.59-2.38 (m, 2H), 1.73-1.65 (m, 2H). LC-MS m / z (ESI) = 255.10 [M+1].

[0050] Step 4: 2-Chloro-7-ethyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (Intermediate 2) 2-chloro-7-ethyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one 2-Chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one 2D (400 mg, 1.57 mmol) was dissolved in N,N-dimethylformamide (8 mL), and cesium carbonate (511 mg, 1.57 mmol) and iodoethane (293 mg, 1.88 mmol) were added at 0 °C. The mixture was stirred for 1 h. After monitoring the reaction by TLC until completion, 10 mL of water was added, and the mixture was extracted three times with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated, and the organic solvent was removed by rotary evaporation to give the title compound, 2-chloro-7-ethyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one intermediate 2 (white solid, 290 mg, 65.32% yield).

[0051] 1 H NMR (400 MHz, DMSO-d6) δ 8.44 (s, 1H), 4.50 -4.41 (m, 1H), 3.99 -3.95 (m, 2H), 3.89 (q, 2H), 3.45 (t, 2H), 2.46-2.41 (m, 2H), 1.71-1.67 (m, 2H), 1.25 (t, 3H). LC-MS m / z (ESI) = 283.10 [M+1].

[0052] Example 1 Preparation of Compound A 4-((7-ethyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)-2-fluoro-5-methylbenzamide (Compound A) 4-((7-ethyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)-2-fluoro-5-methylbenzamide [ka]

[0053] 4-Amino-2-fluoro-5-methylbenzamide Intermediate 1 (350 mg, 2.12 mmol), 2-chloro-7-ethyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one Intermediate 2 (150 mg, 0.53 mmol), cesium carbonate (690 mg, 2.12 mmol), and (2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) methanesulfonate (72 mg, 0.08 mmol) were dissolved in 1,4-dioxane (5 mL), vented under nitrogen gas protection, and reacted at 110 °C for 4 h with stirring. The reaction was monitored by TLC until completion, and the concentrated reaction solution was separated and purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 20 / 1). Pre-HPLC was used to obtain the title compound 4-((7-ethyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)-2-fluoro-5-methylbenzamide Compound A (white solid, 38 mg, yield 17.28%).

[0054] 1 H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H), 8.25 (s, 1H), 7.89 (d, 1H), 7.55 (d, 1H), 7.42 (d, 2H), 4.48-4.40 (m, 1H), 3.98 (dd, 2H), 3.85 (q, 2H), 3.43 (t, 2H), 2.58-2.54 (m, 2H), 2.30 (s, 3H) 1.71-1.68 (m, 2H), 1.25 (t, 3H). LC-MS m / z(ESI)= 415.20 [M+1].

[0055] Example 2 Preparation of Crystalline Form I of Compound A 530 g of solid compound A was transferred to a reactor, and 15.9 L of a mixed solution (ethanol:water = 12.7 L:3.2 L) was added. The mixed suspension was heated to reflux (refluxing began at 72 ° C, the solid gradually dissolved, and the temperature was raised to 76 ° C, after which it was maintained stable, the solid dissolved, and a yellow solution was formed). After the solution became clear, it was refluxed with stirring for 1 hour and then left overnight to slowly precipitate crystals. After filtration, the filter cake was air-dried at 60 ° C for 16 hours, then pulverized in a grinder, and the solid was again air-dried at 60 ° C for 16 hours.

[0056] Example 3 Preparation of Crystalline Form I of Compound A A mixed solution of 1.050 kg of compound A, 23.0 kg of ethanol, and 9.3 kg of purified water was added to the reactor in this order, and the reaction solution was heated to 80±5°C and dissolved for 1 hour. The temperature was then lowered to 65°C and maintained at 60±5°C for 1 hour to crystallize, and then lowered to 25°C and maintained at 20±5°C for 2 hours to continue crystallization. After filtration, the product was dried at 60±5°C for 16 hours.

[0057] Example 4 Preparation of Crystalline Form II of Compound A 0.1 kg of compound A, 2.3 kg of ethanol, and 0.9 kg of purified water were added to a reactor in this order to obtain a mixed solution. The mixed solution was heated to 80±5°C and dissolved for 1 hour, then cooled to 60±5°C and maintained for 1 hour, then cooled to 20±5°C and maintained for 2 hours, filtered, and the resulting product was dried at 60±5°C. The product was then added to 1,4-dioxane, stirred at 50°C for 6 days, and vacuum dried at room temperature for 1 day to obtain a solid.

[0058] Example 5 Preparation of Crystalline Form III of Compound A 0.1 kg of compound A, 2.3 kg of ethanol, and 0.9 kg of purified water were added to a reactor in this order to obtain a mixed solution. The mixed solution was heated to 80±5°C and dissolved for 1 hour, then cooled to 60±5°C and maintained at that temperature for 1 hour, then cooled to 20±5°C and maintained at that temperature for 2 hours, filtered, and the resulting product was dried at 60±5°C. The product was then added to 1,4-dioxane, suspended and stirred at 50°C for 4 days, and vacuum dried at room temperature for 2 hours to obtain a solid.

[0059] Example 6 Preparation of Crystalline Form IV of Compound A 0.1 kg of compound A, 2.3 kg of ethanol, and 0.9 kg of purified water were added to a reactor in this order to obtain a mixed solution. The mixed solution was heated to 80±5°C and dissolved for 1 hour, then cooled to 60±5°C and maintained at that temperature for 1 hour, then cooled to 20±5°C and maintained at that temperature for 2 hours, filtered, and the resulting product was dried at 60±5°C. The product was then added to acetone, suspended and stirred at room temperature for 6 days, and left to dry at room temperature to obtain a solid.

[0060] Example 7 Preparation of Crystalline Form V of Compound A 0.1 kg of compound A, 2.3 kg of ethanol, and 0.9 kg of purified water were added to a reactor in this order to obtain a mixed solution. The mixed solution was heated to 80±5°C and dissolved for 1 hour, then cooled to 60±5°C and maintained for 1 hour, then cooled to 20±5°C and maintained for 2 hours, filtered, and the resulting solution was dried at 60±5°C. The product was then added to 1,4-dioxane and suspended and stirred at 50°C for 4 days, and dried under vacuum at room temperature. The resulting solid was then heated to 125°C and cooled to room temperature.

[0061] Example 8 Preparation of Crystalline Form VI of Compound A 0.1 kg of compound A, 2.3 kg of ethanol, and 0.9 kg of purified water were added to a reactor in this order to obtain a mixed solution. The mixed solution was heated to 80±5°C and dissolved for 1 hour, then cooled to 60±5°C and maintained for 1 hour, then cooled to 20±5°C and maintained for 2 hours, filtered, and the resulting product was dried at 60±5°C. The product was then added to acetone and suspended and stirred at room temperature for 6 days. The resulting solid was then left to dry at room temperature and heated to 130°C and cooled to room temperature.

[0062] Test Example 1 The crystalline form I of compound A prepared in Example 2 was subjected to an X-ray diffraction analysis using an X-ray diffractometer from Dandong Haoyuan Instrument Co., Ltd. at room temperature with graphite monochromatic CuKα radiation (λ=1.54) to obtain a powder diffraction pattern, and the data were analyzed.

[0063] The powder X-ray diffraction data of crystalline form I of Compound A prepared in Example 2 is shown in Table 1.

[0064] [Table 1-1] [Table 1-2]

[0065] The powder X-ray diffraction pattern of crystalline form I of Compound A prepared in Example 2 is shown in FIG.

[0066] Test Example 2 XRPD results were collected on an X'Pert3 and Empyrean powder X-ray diffraction analyzer for crystalline Form I of Compound A prepared in Example 3. The scan parameters are shown in Table 2.

[0067] [Table 2]

[0068] The powder X-ray diffraction data of crystalline Form I of Compound A prepared in Example 3 is shown in Table 3.

[0069] [Table 3-1] [Table 3-2]

[0070] The powder X-ray diffraction pattern of crystalline form I of Compound A prepared in Example 3 is shown in FIG.

[0071] Test Example 3 Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were performed on the crystalline form I of Compound A prepared in Example 3. The TGA and DSC data were collected using a TA Q5000 / Discovery 5500 thermogravimetric analyzer and a TA Discovery 2500 differential scanning calorimeter, respectively. Table 4 shows the measurement parameters for TGA and DSC. The test results are shown in Figures 3 and 4, respectively. The TGA spectrum showed a weight loss of 0.95% when heated to 230.0°C, and the DSC spectrum showed an endothermic peak at 238.4°C.

[0072] [Table 4]

[0073] Test Example 4 Dynamic moisture sorption measurements were performed on crystalline Form I of Compound A prepared in Example 3. The dynamic moisture sorption (DVS) curve was collected using a DVS intrinsic system on a Surface Measurement Systems (SMS). The relative humidity at 25°C was corrected for the deliquescence points of LiCl, Mg(NO3)2, and KCl, and the DVS measurement parameters are shown in Table 5. The test results are shown in Figure 5. As can be seen from Figure 5, the weight of a sample of crystalline Form I of Compound A of the present invention changed by less than 0.2% at 25°C from 0% RH to 95% RH to 0% RH, indicating that the sample had little or no wettability. Comparison of the powder X-ray diffraction patterns before and after DVS indicates that there was no change in the crystalline form before and after DVS.

[0074] [Table 5]

[0075] Test Example 5 XRPD results for Form II were collected using the test conditions in Test Example 2. The powder X-ray diffraction data for Form II are shown in Table 6.

[0076] [Table 6]

[0077] The powder X-ray diffraction pattern of crystalline form II is shown in FIG.

[0078] Test Example 6 Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of crystalline Form II were performed under the test conditions of Test Example 3. The test results are shown in Figures 7 and 8, respectively. The TGA spectrum showed a stepwise weight loss of 4.47% when heated to 90°C, and the DSC spectrum showed two endothermic peaks at 97.5°C (peak temperature) and 236.1°C (onset temperature) and one exothermic peak at 155.3°C (peak temperature).

[0079] Test Example 7 XRPD results for Form III were collected using the test conditions in Test Example 2. The powder X-ray diffraction data for Form III are shown in Table 7.

[0080] [Table 7]

[0081] The powder X-ray diffraction pattern of crystalline form III is shown in FIG.

[0082] Test Example 8 Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of crystalline Form III were performed under the test conditions of Test Example 3. The test results are shown in Figures 10 and 11, respectively. The TGA spectrum showed a 2.5% weight loss when heated to 100°C, followed by a stepwise weight loss of 17.7% when heated to 150°C. The DSC spectrum showed one endothermic signal at 112.1°C (onset temperature), presumably due to dehydration or solvent, and two endothermic peaks at 234.7°C and 236.7°C (peak temperatures).

[0083] Test Example 9 XRPD results for Form IV were collected using the test conditions in Example 2. The powder X-ray diffraction data for Form IV are shown in Table 8.

[0084] [Table 8]

[0085] The powder X-ray diffraction pattern of crystalline form IV is shown in FIG.

[0086] Test Example 10 Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of Form IV were performed under the test conditions of Test Example 3. The test results are shown in Figures 13 and 14, respectively. The TGA spectrum shows a stepwise weight loss of 11.3% when heated to 120°C, and the DSC spectrum shows two endothermic peaks at 106.8°C and 235.4°C (onset temperature).

[0087] Test Example 11 XRPD results for Form V were collected using the test conditions in Test Example 2. The powder X-ray diffraction data for Form V are shown in Table 9.

[0088] [Table 9]

[0089] The powder X-ray diffraction pattern of crystalline form V is shown in FIG.

[0090] Test Example 12 Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of crystalline form V were performed under the test conditions of Test Example 3. The test results are shown in Figures 16 and 17, respectively. The TGA spectrum shows that the sample lost 1.8% weight when heated to 230°C, and the DSC spectrum shows two endothermic signals at 234.7°C and 236.7°C (peak temperatures).

[0091] Test Example 13 XRPD results for Form VI were collected using the test conditions in Test Example 2. The powder X-ray diffraction data for Form VI are shown in Table 10.

[0092] [Table 10]

[0093] The powder X-ray diffraction pattern of crystalline Form VI is shown in FIG.

[0094] Test Example 14 Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of Form VI were performed under the conditions of Experimental Example 3. The test results are shown in Figures 19 and 20, respectively. The TGA spectrum shows that the sample lost 1.0% weight when heated to 200°C, and the DSC spectrum shows one endothermic peak at 235.1°C (onset temperature).

[0095] Test Example 15 Crystalline Form I was left under conditions of 80°C / closed and 60°C / closed for one day, and under conditions of 25°C / 60%RH / open and 40°C / 75%RH / open for one week, without any change in crystal form or decrease in purity (physical stability was evaluated by measuring the crystal form by XRPD, and chemical stability was evaluated by measuring the purity by HPLC), indicating that Crystalline Form I has good solid-state stability under the evaluation conditions.

[0096] In vivo test example 1.DNA-PK kinase inhibition test The inhibitory activity of the compounds against DNA-PK kinase was detected using a DNA-PK kinase assay kit (purchased from Promega, Catalog No.: V4107, Lot No.: 0000366495). The results were quantified by chemiluminescence. The specific experimental procedure is as follows:

[0097] i. ADP-fluorescence calibration curve with different concentrations was constructed according to the kit's specifications.

[0098] ii. A 5 μL reaction mixture was prepared in a 384-well white plate, and 1 μL of compound (concentration gradient set to 1 μM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, and 0.013 nM, respectively) was added to each well. 20 units of DNA-PK kinase, 0.2 μg / μL substrate, 10 μg / μL DNA, 50 μM ATP, and 1% DMSO were added.

[0099] iii. The mixture was mixed uniformly, centrifuged (1000 rpm, 30 s), and incubated at 37°C for 60 min.

[0100] iv. 5 μL of ADP-Glo TM The reaction was stopped by adding a reagent, mixed uniformly, centrifuged (1000 rpm, 30 s), and incubated at room temperature for 40 min.

[0101] v. 10 μL of Kinase Detection Reagent was added, the mixture was mixed uniformly by shaking, centrifuged (1000 rpm, 30 s), and incubated at room temperature for 30 min.

[0102] Fluorescence values ​​were measured using a microplate reader (Thermo Fisher, Varoskan LUX). IC values ​​were calculated using GraphPad Prism 8. 50 was calculated and the results are shown in Table 11.

[0103] [Table 11]

[0104] Note: The comparative example is compound 3 in J. Med. Chem (2020), 63(7), 3461-3471, which was prepared according to its preparation method.

[0105] The results show that the compounds of the present invention have more significant inhibitory effects on DNA-PK kinase compared with the comparative examples.

[0106] 2. Pharmacokinetic Measurements 2.1 Test materials ICR mice (purchased from Beijing Weitonghua Laboratory Animal Technology Co., Ltd.)

[0107] 2.2 Experimental Procedure (1) Healthy male ICR mice (18-22 g) were prepared, and 18 mice were used for each compound. They were divided into two groups (iv and po groups) with 9 mice in each group. Three mice were selected at each time point and blood samples were taken.

[0108] (2) After overnight fasting (with free access to water), the compounds of the present invention were dissolved (or suspended) in 5% DMSO and 95% 30% HP-β-CD (v:v) and administered via tail vein (iv, 1 mg / kg) or oral gavage (po, 10 mg / kg), respectively.

[0109] (3) For the iv group, 0.1 mL of blood was collected from the submandibular vein at 5 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after administration, anticoagulated with EDTA-K2, centrifuged at 4°C for 5 min to obtain plasma, and stored at -20°C until measurement.

[0110] (4) In the po group, 0.1 mL of blood was collected from the submandibular vein before administration and 15 min, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration, and the processing method was the same as in the iv group.

[0111] (5) The concentration of the compound of the present invention in plasma was measured by LC / MS / MS.

[0112] (6) Calculation and fitting of the results were performed using Analyst 1.6 from AB.

[0113] The test results show that Compound A and Forms I, II, III, IV, V and VI all have good pharmacokinetic properties.

[0114] Although the present invention has been described in detail in the specification with reference to specific embodiments, it is understood by those skilled in the art that the above embodiments are illustrative and do not limit the present invention. Those skilled in the art may make some improvements and modifications to the present invention without departing from the principles of the present invention, and the technical solutions obtained by these improvements and modifications are also included in the scope of protection of the claims of the present invention. The present specification includes the following aspects. Section 1. A crystal of the compound represented by formula (A). [ka] Section 2. Item 1. The crystal according to Item 1, characterized in that crystalline form I has characteristic diffraction peaks at 2θ positions of 9.859°±0.3°, 14.759°±0.3°, 19.679°±0.3°, and 19.961°±0.3° in a powder X-ray diffraction pattern using Cu-Kα radiation. Section 3. Item 3. The crystal according to Item 2, wherein crystalline form I further has characteristic diffraction peaks at the following 2θ positions in a powder X-ray diffraction pattern using Cu-Kα radiation: 4.979°±0.2°, 15.759°±0.2°, 19.339°±0.2°, 22.481°±0.2°, and 24.641°±0.2°. Section 4. Item 4. The crystal according to Item 3, wherein crystalline form I further has characteristic diffraction peaks at 2θ positions of 12.120°±0.2°, 18.802°±0.2°, and 21.822°±0.2° in a powder X-ray diffraction pattern using Cu-Kα radiation. Section 5. Item 5. The crystal according to Item 4, wherein the powder X-ray diffraction pattern of crystalline form I is essentially as shown in FIG. 1 or FIG. 2. Section 6. Item 6. The crystal according to any one of Items 2 to 5, wherein the TGA curve of crystalline form I is essentially as shown in FIG. Section 7. Item 6. The crystal according to any one of Items 2 to 5, wherein the DSC curve of crystalline form I is essentially as shown in FIG. Section 8. Item 1. The crystal according to item 1, characterized in that the powder X-ray diffraction pattern of crystalline form II is essentially as shown in FIG. 6. Section 9. Item 1. The crystal according to item 1, characterized in that the powder X-ray diffraction pattern of crystalline form III is essentially as shown in FIG. Section 10. Item 1. The crystal according to item 1, wherein the powder X-ray diffraction pattern of crystalline form IV is essentially as shown in FIG. Section 11. Item 1. The crystal according to item 1, wherein the powder X-ray diffraction pattern of crystalline form V is essentially as shown in FIG. 15. Section 12. Item 1. The crystal according to item 1, wherein the powder X-ray diffraction pattern of crystalline form VI is essentially as shown in FIG. 18. Section 13. A method for producing crystalline form I according to any one of items 2 to 7, comprising: The manufacturing method includes: Method 1: dissolving Compound A in a solvent, heating to reflux to dissolve the solid, allowing it to cool and crystallize, and filtering and drying to obtain Crystal Form I; or a method 2 in which compound A is dissolved in a solvent, the temperature is raised to 75°C to 85°C to dissolve the solid, the temperature is lowered to 55°C to 65°C to crystallize, the temperature is further lowered to 15°C to 25°C to crystallize, and the crystal is filtered and dried to obtain crystalline form I; The method for producing the present invention is characterized in that the solvent is selected from an alcohol-based solvent or a mixed solvent of an alcohol-based solvent and water. Section 14. 13. A pharmaceutical composition comprising a therapeutically effective amount of the crystalline form according to any one of items 1 to 12, and a pharmaceutically acceptable carrier or excipient. Section 15. Use of the crystalline form according to any one of Items 1 to 12 or the pharmaceutical composition according to Item 14 in the manufacture of a DNA-PK inhibitor. Section 16. Use of the crystalline form according to any one of Items 1 to 12 or the pharmaceutical composition according to Item 14 in the manufacture of a medicament for treating and preventing cancer.

Claims

1. A crystal of the compound represented by formula (A). 【Chemical 1】

2. 2. The crystal according to claim 1, wherein the crystalline form I has characteristic diffraction peaks at 2θ positions of 9.859°±0.3°, 14.759°±0.3°, 19.679°±0.3°, and 19.961°±0.3° in a powder X-ray diffraction pattern using Cu-Kα radiation.

3. The crystal according to claim 2, characterized in that the crystalline form I further has characteristic diffraction peaks at the following 2θ positions in a powder X-ray diffraction pattern using Cu-Kα radiation: 4.979°±0.2°, 15.759°±0.2°, 19.339°±0.2°, 22.481°±0.2°, and 24.641°±0.2°.

4. The crystal according to claim 3, characterized in that the crystalline form I further has characteristic diffraction peaks at 2θ positions of 12.120°±0.2°, 18.802°±0.2°, and 21.822°±0.2° in a powder X-ray diffraction pattern using Cu-Kα radiation.

5. The crystal according to claim 4, characterized in that the powder X-ray diffraction pattern of crystalline form I is essentially as shown in Table 1 or Table 3 below. 【Table 1-1】 【Table 1-2】 【Table 3-1】 【Table 3-2】

6. 3. The crystal according to claim 2, wherein the TGA curve of crystalline form I shows that it loses weight by 0.95% when heated from 27.3°C to 230.0°C.

7. 3. The crystal according to claim 2, wherein the DSC curve of crystalline form I essentially shows an endothermic peak at 238.4°C.

8. The crystal according to claim 1, characterized in that the powder X-ray diffraction pattern of crystalline form II is essentially as shown in Table 6 below. 【Table 6】

9. 2. The crystal of claim 1, wherein the powder X-ray diffraction pattern of crystalline form III is essentially as shown in Table 7 below. 【Table 7】

10. 2. The crystal of claim 1, wherein the powder X-ray diffraction pattern of crystalline form IV is essentially as shown in Table 8 below. 【Table 8】

11. 2. The crystal of claim 1, wherein the powder X-ray diffraction pattern of crystalline form V is essentially as shown in Table 9 below. 【Table 9】

12. 2. The crystal of claim 1, wherein the powder X-ray diffraction pattern of crystalline form VI is essentially as shown in Table 10 below. 【Table 10】

13. A method for preparing crystalline form I according to claim 2, comprising the steps of: The manufacturing method includes: Method 1, in which Compound A is dissolved in a solvent, the mixture is heated to reflux to dissolve the solid, and then the mixture is allowed to cool to cause crystallization, followed by filtration and drying to obtain Crystal Form I; or a method 2 in which Compound A is dissolved in a solvent, the temperature is raised to 75°C to 85°C to dissolve the solid, the temperature is lowered to 55°C to 65°C to crystallize, the temperature is further lowered to 15°C to 25°C to crystallize, and the crystal is filtered and dried to obtain Crystal Form I; The method for producing the present invention is characterized in that the solvent is selected from an alcohol-based solvent or a mixed solvent of an alcohol-based solvent and water.

14. A pharmaceutical composition comprising a therapeutically effective amount of the crystal of claim 1 and a pharmaceutically acceptable carrier or excipient.

15. Use of the crystal according to claim 1 in the production of a DNA-PK inhibitor.

16. 10. Use of the crystal of claim 1 in the manufacture of a medicament for treating and preventing cancer.

Images