CRYSTALLINE FORM OF IMIDAZOLINONE DERIVATIVE

Abstract

The present invention relates to a crystal form of an imidazolinone derivative. The present invention further relates to a crystal form of a substituted imidazolinone derivative and a pharmaceutical composition thereof, and a preparation method therefor and the use thereof in the preparation of a DNA-PK inhibitor. Specifically, the present invention relates to crystal forms I-VI of the compound as represented by formula (A) and a pharmaceutical composition thereof, and a preparation method therefor and the use thereof in the preparation of a DNA-PK inhibitor.

Description

Crystal forms of imidazolidinone derivatives Technical Field

[0001] The present invention relates to an imidazolidinone derivative, or a hydrate or solvate thereof in a crystal form, as well as a preparation method thereof or a pharmaceutical composition thereof and use thereof in the field of preparing DNA-PK inhibitors. Background Art

[0002] DNA-dependent protein kinase (DNA-PK) is a DNA-PK enzyme complex composed of the Ku70 / Ku80 heterodimer and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This enzyme complex requires DNA to be activated and function properly (George et al., 2019). As a serine / threonine protein kinase, DNA-PK belongs to the phosphatidylinositol 3-kinase-related kinase (PIKK) family. It not only plays an important role in repairing double-strand breaks (DSBs) in intracellular DNA and in cellular DNA recombination or antibody DNA rearrangement (V(D)J recombination), but also participates in physiological processes such as chromosome modification, transcriptional regulation, and telomere maintenance.

[0003] Combining DNA-PK inhibitors with anti-tumor therapies that cause DNA damage (such as IR and chemotherapy agents) can enhance therapeutic efficacy. While DNA-PK inhibitors can interfere with the DNA repair function of normal cells to a certain extent, normal cells have multiple DNA repair pathways to supplement this function. Tumor cells, however, face intense DNA replication pressure and lack effective DNA repair pathways, making them more sensitive to DNA-PK inhibitors. Inhibiting DNA-PK activity in tumor cells can enhance the cytotoxicity of other anti-tumor therapies.

[0004] The patent (application number: PCT / CN2021 / 087912) describes a novel DNA-PK inhibitor, the structure of which is shown in Formula (A). It has a good inhibitory effect on DNA-PK activity and has the potential to prepare anti-tumor drugs.

[0005]

[0006] Summary of the Invention

[0007] 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). Compound A has the following chemical structure:

[0008]

[0009] The crystal form of the present invention exhibits at least one of the following advantages: good solubility, high stability, easy handling, processing, and purification, improved oral bioavailability of drugs, extended drug storage life, and ease of manufacture of various dosage forms.

[0010] The crystalline form of the present invention exhibits pharmaceutical advantages over the amorphous form of compound A. In particular, the crystalline form has enhanced chemical and physical stability, which is more conducive to the preparation of solid pharmaceutical dosage forms containing pharmacologically active ingredients.

[0011] The crystalline form of the present invention is present in an amount of about 5% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 10% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 15% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 20% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 25% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 30% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 35% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 40% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 45% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 50% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 55% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 60% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 65% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 70% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 75% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 80% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 85% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 90% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 95% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the present invention is present in an amount of about 98% to about 100% by weight of the drug substance. In certain embodiments, the crystalline form of the invention is present in about 99% to about 100% by weight of the drug substance. In certain embodiments, substantially all of the drug substance is a crystalline form of the invention, i.e., the drug substance is substantially phase-pure crystals.

[0012] Unless otherwise specified, the compound A of the present invention is the amorphous form of the compound A.

[0013] One embodiment of the crystalline form of the present invention is anhydrous compound A (crystalline form I), which has an X-ray powder diffraction pattern using Cu-Kα radiation having characteristic diffraction peaks at the following 2θ positions: 9.859°±0.3°, 14.759°±0.3°, 19.679°±0.3°, and 19.961°±0.3°.

[0014] Among them, in the X-ray powder diffraction of the crystal form I, the 2θ diffraction angle also has characteristic diffraction peaks at 4.979°±0.2°, 15.759°±0.2°, 19.339°±0.2°, 22.481°±0.2°, and 24.641°±0.2°.

[0015] Furthermore, the X-ray powder diffraction pattern of the crystalline form I also has characteristic diffraction peaks at the following 2θ positions: 12.120°±0.2°, 18.802°±0.2°, and 21.822°±0.2°.

[0016] Furthermore, the X-ray powder diffraction pattern of Form I also 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°.

[0017] Furthermore, the X-ray powder diffraction pattern (XRD) of the crystalline form I is substantially as shown in FIG1 or FIG2 .

[0018] One embodiment of the crystal form of the present invention is crystal form II, and the X-ray powder diffraction pattern of crystal form II is substantially as shown in FIG6 .

[0019] One embodiment of the crystal form of the present invention is crystal form III, and the X-ray powder diffraction pattern of crystal form III is substantially as shown in FIG9 .

[0020] One embodiment of the crystal form of the present invention is crystal form IV, and the X-ray powder diffraction pattern of crystal form IV is substantially as shown in FIG12 .

[0021] One embodiment of the crystal form of the present invention is crystal form V, and the X-ray powder diffraction pattern of crystal form V is substantially as shown in FIG15 .

[0022] One embodiment of the crystal form of the present invention is crystal form VI, and the X-ray powder diffraction pattern of crystal form VI is substantially as shown in FIG18 .

[0023] The present invention also relates to a method for preparing Form I, which is selected from Method 1 or Method 2:

[0024] Method 1: Dissolve compound A in a solvent, raise the temperature to reflux, dissolve the solid, then cool naturally to crystallize, filter and dry to obtain Form I;

[0025] Method 2: Dissolve compound A in a solvent, raise the temperature to 75-85°C, dissolve the solid, then cool to 55-65°C for crystallization, then cool to 15-25°C for crystallization, filter and dry to obtain Form I;

[0026] The above-mentioned solvent is selected from alcohol solvents or a mixed solvent of alcohol solvents and water.

[0027] The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of the crystalline compound of the present invention and one or more pharmaceutically acceptable carriers or excipients.

[0028] The crystal form of the present invention can be used as an active pharmaceutical ingredient, or a pharmaceutical composition containing the crystal form as an active ingredient, to prepare DNA-PK inhibitor drugs.

[0029] Among them, DNA-PK inhibitors are used to prepare drugs for treating and preventing cancer.

[0030] The X-ray powder diffraction pattern disclosed in the present invention and those substantially the same also fall within the scope of the present invention.

[0031] Unless stated otherwise, the terms used in the specification and claims have the following meanings.

[0032] An "effective amount" refers to that amount of a compound that will elicit the physiological or medical response of a tissue, system, or subject that is being sought, and includes an amount of the compound that, when administered to a subject, is sufficient to prevent or alleviate to some extent one or more symptoms of the disorder or condition being treated.

[0033] “IC 50 ” refers to the half-maximal inhibitory concentration, which is the concentration at which half of the maximum inhibitory effect is achieved.

[0034] The structure of the crystalline form of the present invention can be analyzed using various analytical techniques known to those skilled in the art, including but not limited to X-ray powder diffraction (XRD).

[0035] It is understood that the numerical values ​​described and protected by the present invention are approximate values. Variations in the numerical values ​​may be due to equipment calibration, equipment errors, crystal purity, crystal size, sample size and other factors.

[0036] It is understood that the crystal form of the present invention is not limited to the characteristic spectra that are exactly the same as the characteristic spectra described in the drawings disclosed in the present invention, such as XRD. Any crystal form having characteristic spectra that are substantially the same or essentially the same as those described in the drawings falls within the scope of the present invention.

[0037] Various modifications and alterations to the present invention will become apparent to those skilled in the art from consideration of the specification and practice of the invention without departing from the scope and spirit of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG1 is an X-ray powder diffraction pattern of Compound A Form I prepared in Example 2 using Cu-Kα radiation.

[0039] FIG2 is an X-ray powder diffraction pattern of Form I of Compound A prepared in Example 3.

[0040] Figure 3 is a TGA chart of Compound A Form I prepared in Example 3.

[0041] FIG4 is a DSC graph of Form I of Compound A prepared in Example 3.

[0042] Figure 5 is a DVS diagram of Form I of Compound A prepared in Example 3.

[0043] FIG6 is an X-ray powder diffraction pattern of Form II.

[0044] FIG7 is a TGA diagram of Form II.

[0045] FIG8 is a DSC graph of Form II.

[0046] FIG9 is an X-ray powder diffraction pattern of Form III.

[0047] FIG10 is a TGA chart of Form III.

[0048] FIG11 is a DSC graph of Form III.

[0049] FIG12 is an X-ray powder diffraction pattern of Form IV.

[0050] FIG13 is a TGA chart of Form IV.

[0051] FIG14 is a DSC chart of Form IV.

[0052] FIG15 is an X-ray powder diffraction pattern of Form V.

[0053] FIG16 is a TGA chart of Form V.

[0054] FIG17 is a DSC chart of Form V.

[0055] FIG18 is an X-ray powder diffraction pattern of Form VI.

[0056] FIG19 is a TGA chart of Form VI.

[0057] FIG20 is a DSC chart of Form VI. DETAILED DESCRIPTION

[0058] The following describes in detail the implementation process of the present invention and the beneficial effects produced by specific embodiments, which is intended to help readers better understand the essence and characteristics of the present invention and is not intended to limit the scope of implementation of this case.

[0059] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0060] Unless otherwise specified, the experimental conditions for crystallization are generally room temperature (20-30° C., 30-70% RH), and the solvent ratio refers to the volume ratio.

[0061] Intermediate 1

[0062] 4-Amino-2-fluoro-5-methylbenzamide (Intermediate 1)

[0063] 4-amino-2-fluoro-5-methylbenzamide

[0064]

[0065] 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. 300 μL of hydrogen peroxide was added under ice-cooling, and the mixture was gradually heated to 60°C and stirred for 2 h. TLC was used to monitor the reaction until completion. 5 mL of water was added to the reaction solution, and a white solid precipitated. The solid was filtered and dried to obtain the title compound, 4-amino-2-fluoro-5-methylbenzamide intermediate 1 (white solid, 160 mg, 47% yield).

[0066] 1 H NMR (400MHz DMSO) δ7.37(d,1H),7.15(s,1H),6.96(s,1H),6.32(d,1H),5.70(s,1H),2.01(s,3H).

[0067] LC-MS m / z (ESI) = 169.10 [M+1]

[0068] Intermediate 2

[0069] 2-Chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (Intermediate 2)

[0070] 2-chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one

[0071]

[0072] first step:

[0073] Ethyl 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylate (2B)

[0074] ethyl 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylate

[0075] 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). Potassium carbonate (46.92 g, 340.9 mmol) was added several times with stirring, and the mixture was stirred at room temperature for 4 h. After completion of the reaction, the mixture was filtered, the residue washed with ethyl acetate (300 mL), and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography (n-hexane:ethyl acetate (v / v) = 1:1) to obtain 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).

[0076] 1 H NMR(400MHz DMSO)δ8.62(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).

[0077] LC-MS m / z (ESI) = 286.10 [M+1]

[0078] Step 2:

[0079] 2-Chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylic acid (2C)

[0080] 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylic acid

[0081] 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 stirred at room temperature for 1 hour. The reaction was monitored for completion by TLC. The mixture was concentrated to remove tetrahydrofuran and the pH was adjusted to 5 with 6N hydrochloric acid. A white solid precipitated and was filtered. The filter cake was washed twice with petroleum ether to obtain 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).

[0082] 1 H NMR (400MHz DMSO) δ8.60(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).

[0083] LC-MS m / z(ESI)=258.10[M+1].

[0084] Step 3:

[0085] 2-Chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (2D)

[0086] 2-chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one

[0087] 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 diphenylphosphoryl azide (12.06 mL, 58.21 mmol) were added, and the temperature was gradually increased to 120°C with stirring for 1.5 hours. After completion of the reaction, monitored by TLC, the reaction solution was poured into ice water, and the solid was collected by filtration, washed three times with water, and concentrated in vacuo 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).

[0088] 1H NMR (400MHz DMSO) δ11.63(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).

[0089] LC-MS m / z(ESI)=255.10[M+1].

[0090] Step 4:

[0091] 2-Chloro-7-ethyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (Intermediate 2)

[0092] 2-chloro-7-ethyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one

[0093] 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). Cesium carbonate (511 mg, 1.57 mmol) and iodoethane (293 mg, 1.88 mmol) were added and stirred at 0°C for 1 h. The reaction was monitored by TLC until completion. Subsequently, 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 and concentrated. The organic solvent was removed by rotary evaporation to obtain 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).

[0094] 1 H NMR(400MHz,DMSO-d6)δ8.44(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).

[0095] LC-MS m / z(ESI)=283.10[M+1].

[0096] Example 1 Preparation of Compound A

[0097] 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)

[0098] 4-((7-ethyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)-2-fluo-5-methylbenzamide

[0099]

[0100] 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 methanesulfonic acid (2-dicyclohexylphosphine-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl) (2'-amino-1,1'-biphenyl-2-yl) palladium (II) (72 mg, 0.08 mmol) were dissolved in 1,4-dioxane (5 mL), vented under nitrogen protection, and stirred at 110 ° C for 4 h. The reaction was monitored by TLC until completion. The concentrated reaction solution was separated and purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 20 / 1), and subjected to Pre-HPLC to give 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, 17.28% yield).

[0101] 1 H NMR(400MHz,DMSO-d6)δ8.53(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).

[0102] LC-MS m / z(ESI)=415.20[M+1].

[0103] Example 2 Preparation of Compound A Crystalline Form I

[0104] Transfer 530g of Compound A solid to a reactor and add 15.9L of a mixed solution (ethanol:water = 12.7L:3.2L). The mixed suspension was heated to reflux (starting at 72°C, the solid gradually dissolved; heating to 76°C, where it remained stable, dissolving the solid, resulting in a yellow solution). Once the solution clarified, reflux with stirring for 1 hour and allow to stand overnight to slowly precipitate crystals. Filter the filter cake, air-dry it at 60°C for 16 hours, then grind it in a mill. The solid was then air-dried again at 60°C for 16 hours.

[0105] Example 3 Preparation of Compound A Crystalline Form I

[0106] A mixed solution of 1.050 kg of Compound A, 23.0 kg of ethanol, and 9.3 kg of purified water was added sequentially to a reactor. The reaction solution was heated to 80±5°C for dissolution for 1 hour, then cooled to 65°C and maintained at 60±5°C for crystallization for 1 hour. The temperature was then further cooled to 25°C and maintained at 20±5°C for crystallization for another 2 hours. Filter the product, and dry it at 60±5°C for 16 hours.

[0107] Example 4 Preparation of Compound A Crystalline Form II

[0108] A mixed solution of 0.1 kg of compound A, 2.3 kg of ethanol, and 0.9 kg of purified water was added sequentially to a reactor. The mixture was heated to 80 ± 5°C and dissolved for 1 hour, then cooled to 60 ± 5°C and held for 1 hour. The temperature was further cooled to 20 ± 5°C and held for 2 hours. The resulting product was filtered and dried at 60 ± 5°C. The product was then added to 1,4-dioxane and stirred at 50°C for 6 days. The solid was then dried under vacuum at room temperature for 1 day to obtain a product.

[0109] Example 5 Preparation of Compound A Crystalline Form III

[0110] A mixed solution of 0.1 kg of compound A, 2.3 kg of ethanol, and 0.9 kg of purified water was added sequentially to a reactor. The mixture was heated to 80 ± 5°C and dissolved for 1 hour, then cooled to 60 ± 5°C and held for 1 hour. The temperature was further cooled to 20 ± 5°C and held for 2 hours. The resulting product was filtered and dried at 60 ± 5°C. The product was then added to 1,4-dioxane and suspended and stirred at 50°C for 4 days. The solid was then dried under vacuum at room temperature for 2 hours.

[0111] Example 6 Preparation of Compound A Crystalline Form IV

[0112] A mixed solution of 0.1 kg of compound A, 2.3 kg of ethanol, and 0.9 kg of purified water was added sequentially to a reaction kettle. The mixture was heated to 80 ± 5°C and dissolved for 1 hour, then cooled to 60 ± 5°C and held for 1 hour. The temperature was further cooled to 20 ± 5°C and held for 2 hours. The resulting solution was filtered and dried at 60 ± 5°C. The product was then added to acetone and suspended and stirred at room temperature for 6 days. The solid was then allowed to dry at room temperature.

[0113] Example 7 Preparation of Compound A Crystalline Form V

[0114] A mixed solution of 0.1 kg of compound A, 2.3 kg of ethanol, and 0.9 kg of purified water was added sequentially to a reactor. The mixture was heated to 80 ± 5°C and dissolved for 1 hour. The temperature was then lowered to 60 ± 5°C and held for 1 hour. The temperature was then further lowered to 20 ± 5°C and held for 2 hours. The resulting solution was filtered and dried at 60 ± 5°C. The product was then added to 1,4-dioxane and suspended and stirred at 50°C for 4 days. After vacuum drying at room temperature, the resulting solid was heated to 125°C and cooled to room temperature.

[0115] Example 8 Preparation of Compound A Crystalline Form VI

[0116] A mixed solution of 0.1 kg of Compound A, 2.3 kg of ethanol, and 0.9 kg of purified water was added sequentially to a reactor. The mixture was heated to 80 ± 5°C and dissolved for 1 hour. The temperature was then lowered to 60 ± 5°C and held for 1 hour. The temperature was further lowered to 20 ± 5°C and held for 2 hours. The resulting solution was filtered and 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 dried at room temperature and heated to 130°C and cooled to room temperature.

[0117] Test Example 1

[0118] The crystal form I of compound A prepared in Example 2 was irradiated with graphite monochromatic Cu Kα radiation (λ=1.54) using an X-ray diffractometer from Dandong Haoyuan Instrument Co., Ltd. at room temperature to obtain a powder diffraction pattern, and the data were analyzed.

[0119] The X-ray powder diffraction data of Compound A Form I prepared in Example 2 are shown in Table 1.

[0120] Table 1 X-ray powder diffraction data of Compound A Form I prepared in Example 2

[0121] Peak number 2θ[°] Interplanar spacing Peak height Half-peak width Relative peak height 14.979 17.73 2889 70.20 223.12 9.85 98.96 45388 30.177 1003 11.48 37.722 10.178 5.74 12.12 7.29 664 120.159 10.65 14.75 95.99 721126 0.162 9615 .7595.61897850.15220.2716.6015.33591300.1533.4816.9985.212730.1871.9917.5235.057880.1192.31018.4044.8171910.2144.91118.8024.71585670.16914.6

[0122] 1219.3394.58618560.192221319.6794.507612500.18232.21419.9614.444511120.16728.61520.4224.34532120.1565.51621.3794.15292340.26561721.8224.06966460.22716.61822.1424.01142240.2325.81922.4 813.95169020.16423.22022.8773.88421760.1884.52124.1983.6751100.1462.82224.6413.60999460.22824.42325.9393.43223330.1428.62426.6813.33841280.1773.32526.9613.3044850.1342.22627.6013.22921 000.2152.62728.0383.1798710.2011.82828.5193.1273810.1462.12929.183.05792410.1556.23029.6243.01311480.223.83130.0772.9688380.28113230.3222.9453610.2751.63330.6192.9174780.29523431.0412 .87873600.2019.33531.7232.81841160.23833632.5242.7508660.2561.73733.3792.6823710.1771.83834.0392.6317840.2842.23935.0012.5616640.1181.64035.5872.52071030.2692.74137.1782.4164420.2061.1

[0123] 4237.7242.3827730.1381.94338.3192.347510.3071.34438.612.33460.1951.24539.1252.30051120.1542.9

[0124] The X-ray powder diffraction pattern of Form I of Compound A prepared in Example 2 is shown in FIG1 .

[0125] Test Example 2

[0126] The XRPD results of Form I of Compound A prepared in Example 3 were collected on X'Pert3 and Empyrean X-ray powder diffraction analyzers, and the scanning parameters are shown in the table.

[0127] Table 2 XRPD test parameters

[0128]

[0129] The X-ray powder diffraction data of Compound A Form I prepared in Example 3 are shown in Table 3.

[0130] Table 3 X-ray powder diffraction data of Compound A Form I prepared in Example 3

[0131]

[0132]

[0133] The X-ray powder diffraction pattern of Form I of Compound A prepared in Example 3 is shown in FIG2 .

[0134] Test Example 3

[0135] Form I of Compound A prepared in Example 3 was subjected to thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). TGA and DSC measurements were collected on a TA Q5000 / Discovery 5500 thermogravimetric analyzer and a TA Discovery 2500 differential scanning calorimeter, respectively. Table 4 lists the TGA and DSC test parameters. The test results are shown in Figures 3 and 4, respectively. The TGA spectrum shows a weight loss of 0.95% upon heating to 230.0°C; the DSC spectrum shows an endothermic peak at 238.4°C.

[0136] Table 4 TGA and DSC test parameters

[0137] Parameters TGADSC method linear heating linear heating sample pan aluminum pan, open aluminum pan, press cover temperature range room temperature ~ 350 ° C 25 ° C ~ 300 ° C heating rate 10 ° C / min 10 ° C / min protective gas nitrogen nitrogen

[0138] Test Example 4

[0139] Form I of Compound A prepared in Example 3 was subjected to dynamic moisture sorption testing. Dynamic moisture sorption (DVS) curves were collected on an SMS (Surface Measurement Systems) DVS Intrinsic. Relative humidity at 25°C was calibrated using the deliquescence points of LiCl, Mg(NO3)2, and KCl. DVS test parameters are listed in Table 5. The test results are shown in Figure 5. As shown in Figure 5, the weight change of Form I of Compound A at 25°C over a range of 0% RH to 95% RH to 0% RH is less than 0.2%, indicating that the sample has little or no hygroscopicity. A comparison of X-ray powder diffraction patterns before and after DVS reveals no change in the crystalline form.

[0140] Table 5 DVS test parameters

[0141]

[0142] Test Example 5

[0143] The test conditions of Test Example 2 were used to collect the XRPD results of Form II. The X-ray powder diffraction data of Form II are shown in Table 6.

[0144] Table 6 X-ray powder diffraction data of Form II

[0145]

[0146] The X-ray powder diffraction pattern of Form II is shown in Figure 6.

[0147] Test Example 6

[0148] Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of Form II were performed using the test conditions of Test Example 3. The test results are shown in Figures 7 and 8, respectively. The TGA spectrum shows a 4.47% step-wise weight loss upon heating to 90°C. The DSC spectrum shows 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).

[0149] Test Example 7

[0150] The test conditions of Test Example 2 were used to collect XRPD results of Form III. The X-ray powder diffraction data of Form III are shown in Table 7.

[0151] Table 7 X-ray powder diffraction data of Form III

[0152]

[0153] The X-ray powder diffraction pattern of Form III is shown in Figure 9.

[0154] Test Example 8

[0155] Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of Form III were performed using the test conditions of Test Example 3. The test results are shown in Figures 10 and 11, respectively. The TGA spectrum shows a 2.5% weight loss upon heating to 100°C, and a 17.7% step-wise weight loss upon further heating to 150°C. The DSC spectrum shows an endothermic signal at 112.1°C (starting temperature), presumably due to dehydration or solvent; and two endothermic peaks at 234.7°C and 236.7°C (peak temperatures).

[0156] Test Example 9

[0157] The test conditions of Test Example 2 were used to collect the XRPD results of Form IV. The X-ray powder diffraction data of Form IV are shown in Table 8.

[0158] Table 8 X-ray powder diffraction data of Form IV

[0159]

[0160]

[0161] The X-ray powder diffraction pattern of Form IV is shown in Figure 12.

[0162] Test Example 10

[0163] Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of Form IV were performed using the test conditions of Test Example 3. The test results are shown in Figures 13 and 14, respectively. The TGA spectrum shows an 11.3% step-wise weight loss when heated to 120°C, and the DSC spectrum shows two endothermic peaks at 106.8°C and 235.4°C (onset temperature).

[0164] Test Example 11

[0165] The test conditions of Test Example 2 were used to collect the XRPD results of Form V. The X-ray powder diffraction data of Form V are shown in Table 9.

[0166]

[0167] Table 9 X-ray powder diffraction data of Form V

[0168] The X-ray powder diffraction pattern of Form V is shown in Figure 15.

[0169] Test Example 12

[0170] Using the test conditions of Test Example 3, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of Form V were performed. The test results are shown in Figures 16 and 17, respectively. The TGA spectrum shows that the sample loses 1.8% of its weight when heated to 230°C, and the DSC spectrum shows two endothermic signals at 234.7°C and 236.7°C (peak temperature).

[0171] Test Example 13

[0172] The test conditions of Test Example 2 were used to collect the XRPD results of Form VI. The X-ray powder diffraction data of Form VI are shown in Table 10.

[0173] Table 10 X-ray powder diffraction data of Form VI

[0174]

[0175] The X-ray powder diffraction pattern of Form VI is shown in Figure 18.

[0176] Test Example 14

[0177] Using the test conditions of Test Example 3, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of Form VI were performed. The test results are shown in Figures 19 and 20, respectively. The TGA spectrum shows that the sample loses 1.0% weight when heated to 200°C, and the DSC spectrum shows an endothermic peak at 235.1°C (onset temperature).

[0178] Test Example 15

[0179] After Form I was placed under 80°C / closed and 60°C / closed conditions for 1 day, and under 25°C / 60% RH / open and 40°C / 75% RH / open conditions for 1 week, no crystal transformation or purity reduction occurred (physical stability was evaluated by XRPD testing of the crystal form, and chemical stability was evaluated by HPLC testing of purity), indicating that Form I has good solid-state stability under the evaluation conditions.

[0180] Biological test cases

[0181] 1. DNA-PK kinase inhibition assay

[0182] The inhibitory activity of the compounds against DNA-PK kinase was detected using a DNA-PK kinase assay kit (purchased from Promega, catalog number: V4107, batch number: 0000366495). The results were quantified using chemiluminescence. The specific experimental protocol is as follows:

[0183] i. Construct a standard curve of ADP-fluorescence at different concentrations according to the kit instructions;

[0184] ii. Prepare a 5 μL reaction system in a 384-well white plate. Add 1 μL of compound (concentration gradient: 1 μM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, 0.013 nM), 20 units of DNA-PK kinase, 0.2 μg / μL substrate, 10 μg / μL DNA, 50 μM ATP, and 1% DMSO to each well.

[0185] iii. Mix well, centrifuge (1000 rpm, 30 s), and incubate at 37°C for 60 min;

[0186] iv. Add 5 μL ADP-Glo TM Reagent to terminate the reaction, mix well, centrifuge (1000 rpm, 30 s), and incubate at room temperature for 40 min;

[0187] v. Add 10 μL Kinase Detection Reagent, shake to mix, centrifuge (1000 rpm, 30 s), and incubate at room temperature for 30 min;

[0188] vi. Fluorescence values ​​were measured using a microplate reader (Thermo Fisher, Varioskan LUX). IC was performed using GraphPad Prism 8. 50 The results are shown in Table 11.

[0189] Table 11 Inhibitory activity against DNA-PK kinase

[0190] Compound number IC 50 (nM) Compound A4.17 + Control Example 100.40

[0191] Note: The reference example is compound 3 of J.Med.Chem (2020), 63(7), 3461-3471, which was prepared according to its preparation method.

[0192] The results showed that compared with the control example, the compound of the present invention had a more significant inhibitory effect on DNA-PK kinase.

[0193] 2. Pharmacokinetic determination

[0194] 2.1 Test materials

[0195] ICR mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.)

[0196] 2.2 Experimental steps

[0197] (1) Prepare healthy male ICR mice (18-22 g). Eighteen mice were used for each compound and divided into two groups (iv and po groups), with nine mice in each group. Three mice were selected for blood collection at each time point.

[0198] (2) After fasting overnight (with free access to water), the compound of the present invention was dissolved (or suspended) in 5% DMSO and 95% 30% HP-β-CD (v:v) as solvents and administered via the tail vein (iv, 1 mg / kg) or oral gavage (po, 10 mg / kg).

[0199] (3) In 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. The blood was anticoagulated with EDTA-K2, centrifuged at 4 °C for 5 min to separate the plasma, and stored at -20 °C for testing.

[0200] (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 treatment method was the same as that of the intravenous group.

[0201] (5) The plasma concentration of the compound of the present invention was determined by LC / MS / MS.

[0202] (6) Analyst 1.6 of AB Company was used to calculate and fit the results.

[0203] The experimental results show that compound A and crystalline form I, crystalline form II, crystalline form III, crystalline form IV, crystalline form V and crystalline form VI all have good pharmacokinetic properties.

[0204] The specification of the present invention describes the specific implementation scheme in detail. Those skilled in the art should recognize that the above implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principles of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A crystal of the compound represented by formula (A): where the crystal is in crystalline form I and has a characteristic diffraction peak at 2θ position 9.859°±0.3°.

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

3. The crystal of claim 2, wherein the crystalline form I further has characteristic diffraction peaks at 2θ positions of 4.979°±0.2°, 15.759°±0.2°, 19.339°±0.2°, 22.481°±0.2° and 24.641°±0.2° in an X-ray powder diffraction pattern obtained under the action of Cu-Kα radiation.

4. The crystal of claim 3, wherein 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 an X-ray powder diffraction pattern obtained under the action of Cu-Kα radiation.

5. The crystal of claim 4, wherein the crystalline form I has an X-ray powder diffraction pattern as shown in Fig. 1 or Fig.

2.

6. The crystal of claim 1, wherein the crystalline form I has characteristic diffraction peaks at 2θ positions 4.979, 9.859, 11.483, 12.12, 14.759, 15.759, 16.601, 16.998, 17.523, 18.404, 18.802, 19.339, 19.679, 19.961, 20.422, 21.379, 21.822, 22.142, 22.481, 22.877, 24.198, 24.641, 25.939, 26.681, 26.961, 27.601, 28.038, 28.519, 29.18, 29.624, 30.077, 30.322, 30.619, 31.041, 31.723, 32.524, 33.379, 34.039, 35.001, 35.587, 37.178, 37.724, 38.319, 38.61 and 39.125 in the X-ray powder diffraction pattern obtained under the action of Cu-Kα radiation.

7. The crystal according to claim 1, wherein the crystalline form I has characteristic diffraction peaks at 2θ positions of 4.93, 9.80, 11.46, 12.08, 14.70, 15.73, 16.63, 18.43, 18.80, 19.30, 19.63, 19.94, 20.45, 21.36, 21.86, 22.47, 22.89, 24.60, 25.90, 26.81, 27.63, 29.14, 29.61, 31.01, 31.73, 33.35, 34.00, 35.03, 35.65, 37.

73. 38.93, 39.20 and 39.81 in the X-ray powder diffraction pattern obtained under the action of Cu-Kα radiation.

8. The crystal according to any one of claims 2 to 7, wherein the crystalline form I has a TGA (thermogravimetric analysis) curve as shown in Fig.

3.

9. The crystal according to any one of claims 2 to 7, wherein the crystalline form I has a DSC (differential scanning calorimetry) curve as shown in Fig.

4.

10. A crystal of the compound represented by formula (A): where the crystal is in crystalline form II and has an X-ray powder diffraction pattern as shown in Fig.

6.

11. The crystal of claim 10, wherein the crystalline form II has characteristic diffraction peaks at 2θ positions of 4.76, 8.61, 9.52, 10.46, 13.78, 14.29, 15.51, 17.18, 18.39, 18.60, 19.08, 19.75, 23.90, 24.12, 24.93, 25.97, 26.29, 27.77, 29.78, 30.87, 34.77, 36.00, 37.69, and 38.38 in an X-ray powder diffraction pattern obtained under the action of Cu-Kα radiation.

12. A crystal of the compound represented by formula (A): where the crystal is in crystalline form III and has an X-ray powder diffraction pattern as shown in Fig.

9.

13. The crystal of claim 12, wherein the crystalline form III has characteristic diffraction peaks at 2θ positions of 8.49, 9.02, 9.27, 13.49, 15.32, 15.78, 17.82, 18.07, 18.28, 18.56, 18.99, 19.97, 20.96, 22.54, 23.04, 23.45, 24.21, 24.65, 25.61, 26.26, 28.16, 29.14, 30.18, and 35.01 in an X-ray powder diffraction pattern obtained under the action of Cu-Kα radiation.

14. A crystal of the compound represented by formula (A): where the crystal is in crystalline form IV and has an X-ray powder diffraction pattern as shown in Fig.

12.

15. The crystal of claim 14, wherein the crystalline form IV has characteristic diffraction peaks at 2θ positions of 6.59, 9.39, 10.55, 11.18, 13.92, 14.41, 14.60, 15.67, 17.36, 18.50, 18.89, 19.73, 20.45, 21.15, 22.28, 24.34, 24.69, 25.69, 26.41, 26.93, 28.88, 29.54, 31.96, 33.93, 34.91 and 37.57 in an X-ray powder diffraction pattern obtained under the action of Cu-Kα radiation.

16. A crystal of the compound represented by formula (A): where the crystal is in crystalline form V and has an X-ray powder diffraction pattern as shown in Fig.

15.

17. The crystal of claim 16, wherein the crystalline form V has characteristic diffraction peaks at 2θ positions of 4.87, 8.91, 9.76, 10.48, 11.35, 12.47, 13.83, 14.96, 15.69, 16.22, 17.84, 18.63, 19.66, 20.80, 21.78, 22.42, 24.93, 25.82, 26.59, 27.42, 28.08, 29.49, 30.84, 32.82 and 36.06 in an X-ray powder diffraction pattern obtained under the action of Cu-Kα radiation.

18. A crystal of the compound represented by formula (A): where the crystal is in crystalline form VI and has an X-ray powder diffraction pattern as shown in Fig.

18.

19. The crystal of claim 18, wherein the crystalline form VI has characteristic diffraction peaks at 2θ positions 10.66, 11.52, 14.44, 14.96, 15.44, 16.39, 18.85, 19.35, 20.78, 21.45, 22.12, 22.41, 23.39, 24.60, 25.27, 30.15 and 33.19 in an X-ray powder diffraction pattern obtained under the action of Cu-Kα radiation.

20. A pharmaceutical composition comprising a therapeutically effective amount of a crystalline form according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier or excipient.

21. Use of a crystalline form according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 20 in the production of a DNA-PK (DNA-dependent protein kinase) inhibitor.

22. The use of a crystalline form according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 20 in the preparation of a medicinal product for the treatment and prevention of cancer.

23. A method for producing crystalline form I according to any one of claims 2 to 9, comprising dissolving Compound A in a solvent, heating to a temperature of 75-85°C until the solid is dissolved and then cooling to 55-65°C for crystallization and further cooling to 15-25°C for crystallization, followed by filtration and drying to obtain crystalline form I; where the solvent is a mixed solvent containing an alcohol base and water.

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