Crystalline forms of imidazolinone derivatives

Abstract

To provide a crystal form of an imidazolinone derivative, further a crystal form of a substituted imidazolinone derivative, a method for producing the same, a pharmaceutical composition containing the crystal form, and use of the crystal form in production of a DNA-PK inhibitor.SOLUTION: Provided are crystal forms I-VI of a compound represented by formula (A), and a pharmaceutical composition, a preparation method and use thereof in the preparation of a DNA-PK inhibitor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[0002] DNA-dependent protein kinase (DNA-PK) is a ubiquitous kinase that binds to the Ku70 / Ku 8 DNA consisting of a heterodimer of 0 and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) The A-PK enzyme complex is activated only by the interaction of DNA and performs the corresponding function. (George et al., 2019). DNA-PK, a serine / threonine protein kinase, exerts belongs to the PIKK (phosphatidylinositol 3-kinase-related kinase) family. , repair of intracellular DNA double-strand breaks (DSBs) and recombination of cellular DNA Not only does it play an important role in the antibody DNA rearrangement process (V(D)J recombination), but it also It is also involved in physiological processes such as chromophore modification, transcriptional regulation, and telomere maintenance.

[0003] DNA-PK inhibitors are useful in combination with anti-tumor therapies that cause DNA damage (e.g., IR, chemotherapeutic agents, etc.). The use of DNA-PK inhibitors can enhance the therapeutic effect. Although this interferes with DNA repair to some extent, multiple complementary DNA repair pathways exist in normal cells. On the other hand, tumor cells are exposed to strong DNA replication stress and lack effective DNA repair methods. Therefore, tumor cells are more sensitive to DNA-PK inhibitors and inhibit DNA-PK activity. This can improve the killing effect of other antitumor therapies on tumor cells.

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

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

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

[0007] The crystalline form of the present invention has good solubility, high stability, ease of processing, processing and purification, and is an oral drug. Improved bioavailability, extended drug shelf life, and ease of manufacturing each dosage form. Demonstrate at least one advantage.

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

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

[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 has a powder X-ray diffraction pattern of 2 θ position: 9.859°±0.3°, 14.759°±0.3°, 19.679°±0.3°, 19.961°±0.3° The anhydrous compound A (crystal form I) has typical diffraction peaks.

[0012] Here, the powder X-ray diffraction pattern of crystalline form I further shows a 2θ diffraction angle of 4.979°±0. Characteristic angles are 2°, 15.759°±0.2°, 19.339°±0.2°, 22.481°±0.2°, and 24.641°±0.2°. It has a diffraction peak.

[0013] Furthermore, in the powder X-ray diffraction pattern of the crystalline form I, the 2θ positions are 12.120°±0.2°, 18.8 It has characteristic diffraction peaks at 21.822°±0.2° and 21.822°±0.2°.

[0014] Furthermore, in the powder X-ray diffraction pattern of the crystalline form I, the 2θ positions are 11.483°±0.2°, 20.42° 2°±0.2°, 21.379°±0.2°, 22.142°±0.2°, 25.939°±0.2°, 29.180°±0.2°, It has a characteristic diffraction peak at 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. 2. is.

[0016] One embodiment of the crystalline form described in the present invention is crystalline form II, and the powder X-ray diffraction pattern of crystalline form II is The pattern is basically as shown in Figure 6.

[0017] One embodiment of the crystalline form according to the present invention is crystalline form III, and the powder X-ray diffraction pattern of said crystalline form III is The turn is basically as shown in Figure 9.

[0018] One embodiment of the crystalline form according to the present invention is crystalline form IV, and the powder X-ray diffraction pattern of crystalline form IV is The pattern is basically as shown in Figure 12.

[0019] One embodiment of the crystalline form according to the present invention is crystalline form V, and the powder X-ray diffraction pattern of crystalline form V is The design is basically as shown in Figure 15.

[0020] One embodiment of the crystalline form of the present invention is crystalline form VI. The powder X-ray diffraction pattern of crystalline form VI is The design is basically as shown in Figure 18.

[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, heated to reflux to dissolve the solid, and then allowed to cool to crystallize. Form I is obtained by precipitation, filtration and drying.

[0023] Method 2: Compound A is dissolved in a solvent, and the temperature is raised to 75-85°C to dissolve the solid, and then the temperature is raised to 55-6 The temperature is lowered to 5°C to allow crystallization, and then the temperature is further lowered to 15-25°C to allow crystallization, and the crystals are filtered and dried. Obtain form I.

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

[0025] The present invention also provides a method for treating a crystalline compound of the present invention in a therapeutically effective amount, comprising administering to a subject a therapeutically effective amount of a crystalline compound of the present invention and one or more pharmaceutical agents. The present invention relates to pharmaceutical compositions comprising a physiologically acceptable carrier or excipient.

[0026] The crystalline form described in the present invention can be used as an active pharmaceutical ingredient or in a pharmaceutical composition containing it as an active ingredient. is used in the production of DNA-PK inhibitor drugs.

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

[0028] Those having substantially the same powder X-ray diffraction pattern as disclosed in the present invention are also within the scope of the present invention. Belongs to.

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

[0030] An "effective dose" is a dose sufficient to elicit a physiological or medical response in a tissue, system, or subject. is the amount of a compound of formula (I) that, when administered to a subject, will cause a decrease in the severity of the disease or condition being treated. sufficient to prevent or to alleviate to some extent the occurrence of one or more of the symptoms of The amount of compound that is required to be contained in a sufficient amount.

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

[0032] The crystalline structures 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. The variation in the crystals is due to instrument calibration, instrument error, crystal purity, crystal size, sample size, and This may be due to the size of the tube and other factors.

[0034] The crystalline form of the present invention has the same characteristic pattern as that shown in the drawings disclosed in the present invention. The patterns are not limited to the optical patterns (e.g., XRD) that are essentially the same as or essentially identical to the patterns shown in the drawings. Any crystalline form having the same characteristic pattern as that of the compound of formula (I) is included within the scope of the present invention. It will be understood that.

[0035] The operation contents of the specification and examples of the present invention may be modified without departing from the scope and spirit of the present invention. Upon consideration, various modifications and variations to the present invention will be apparent to those skilled in the art. [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 specific examples will explain in detail the implementation procedures of the present invention and the beneficial effects that result. However, these are intended to help the reader better understand the nature and features of the present invention. However, it does not 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). , the solvent ratio refers to the volume ratio.

[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), potassium carbonate (41. Dissolve 4 mg of 4-aminobenzoate (0.3 mmol) in 1 mL of dimethyl sulfoxide, add 300 μL of hydrogen peroxide solution in an ice bath, and The temperature was then gradually raised to 60°C and stirred for 2 hours. The reaction was monitored by TLC until completion. 5 mL of water was added to the reaction mixture to precipitate a white solid, which was then filtered and spin-dried to remove the water. The title compound 4-amino-2-fluoro-5-methylbenzamide Intermediate 1 (white solid, 160 mg, yield The success rate was 47%.

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

[0044] Step 1: 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylic acid ethyl ester Lu (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), tetrahydro 2H-pyran-4-amine hydrochloride (18.66 g, 136.4 mmol) was dissolved in acetonitrile (600 mL) and mixed with 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 reaction for completion, the mixture was filtered and the residue was washed with ethyl acetate (300 mL). The filtrate was concentrated to obtain a crude product, which was purified by column separation (n-hexane:acetic acid Ethyl (v / v) = 1:1), the title compound 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin Ethyl limidine-5-carboxylate 2B (white solid, 30.0 g, yield 77.4%) was obtained.

[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 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylic acid ethyl ester Dissolve 2B (30 g, 104.99 mmol) in tetrahydrofuran / water (200 mL / 200 mL) and add lithium hydroxide. The reaction was confirmed to be complete by TLC. The mixture was filtered and concentrated to remove tetrahydrofuran, and the pH was adjusted to 5 with 6N hydrochloric acid to give a white solid. The solid was precipitated, filtered, the filter cake was washed twice with petroleum ether, and the solid was collected and Compound 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylic acid 2 C (white solid, 15.0 g, yield 55.44%) was obtained.

[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( Dissolve 15 g (58.21 mmol) in dimethylacetamide (150 mL) and add triethylamine (7.38 mL, 5 Add diphenyl azidophosphate (12.06 mL, 58.21 mmol) and then heat to 120°C. The temperature was gradually raised to 40°C and the reaction was carried out for 1.5 hours with stirring. The completion of the reaction was monitored by TLC. The reaction mixture was poured into ice water, filtered to collect the solid, washed three times with water, and concentrated to dryness in vacuo to give the standard The title compound is 2-chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one 2D (white solid, 13.0 g, yield 87.69%) was obtained.

[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-purine-8- On (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(4 00 mg, 1.57 mmol) in N,N-dimethylformamide (8 mL), and cesium carbonate (511 mg , 1.57 mmol) and iodoethane (293 mg, 1.88 mmol) were added and the reaction was carried out with stirring for 1 hour. After monitoring by TLC until the reaction was complete, 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. The title compound 2-chloro-7-ethyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydroxide was removed. l-8H-purin-8-one Intermediate 2 (white solid, 290 mg, yield 65.32%) was obtained.

[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-purine -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 methanesulfonic acid (2-dicyclo Hexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl) (2'-amino-1,1'-biphenyl-2-yl)palladium(II) (72 mg, 0.08 mmol) in 1,4-dioxadiphenyl The mixture was dissolved in ethanol (5 mL), ventilated under nitrogen gas protection, and reacted at 110°C for 4 h with stirring. The reaction mixture was monitored by TLC until completion, and the concentrated reaction mixture was purified by silica gel column chromatography. The product was separated and purified by pre-HPLC (dichloromethane / methanol (v / v) = 20 / 1). The compound 4-((7-ethyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-pyran-4-yl)- Compound A (white solid, 38 mg, yield) The result was a 17.28% success rate.

[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 reaction vessel, 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, and the solid gradually dissolved). After the temperature was raised to 76°C and kept stable, the solid dissolved and a yellow solution was formed. After the mixture became clear, it was refluxed for 1 hour with stirring and then allowed to stand overnight to allow crystals to slowly precipitate. After filtering, the filter cake was dried by air at 60°C for 16 hours, and then crushed in a crusher. The coated film was then dried in air 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. The reaction mixture was heated to 80±5°C and dissolved for 1 hour, then the temperature was lowered to 65°C and maintained at 60±5°C. The temperature was then lowered to 25°C, and the temperature was maintained at 20±5°C for 2 hours. After filtration, the product was dried at 60±5°C for 16 hours.

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

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

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

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

[0061] Example 8 Preparation of Crystalline Form VI of Compound A Add 0.1 kg of compound A, 2.3 kg of ethanol, and 0.9 kg of purified water to the reactor in this order to form a mixed solution. The mixture was heated to 80±5°C and dissolved for 1 hour, then cooled to 60±5°C and maintained at this temperature for 1 hour. The temperature was then lowered to 20±5°C and maintained for 2 hours, and the filtered product was dried at 60±5°C. After that, the product was added to acetone and stirred at room temperature for 6 days, and then left to dry at room temperature. The resulting solid was 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 analyzed using an X-ray diffraction apparatus from Dandong Haoyuan Instrument Co., Ltd. Powder diffraction patterns were obtained using graphite monochromated CuKα radiation (λ = 1.54) at room temperature. , analyzed the data.

[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 Crystalline Form I of Compound A prepared in Example 3 was analyzed by X'Pert3 and Empyrean powder X-ray diffraction analysis. XRPD results were collected by the spectrometer, and 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) of crystalline form I of Compound A prepared in Example 3, respectively. Thermal analysis (DSC) was performed using a TA Q5000 / Discovery 5500 thermogravimetric analyzer and a DSC. The data were collected using a TA Discovery 2500 differential scanning calorimeter and a TA Discovery 2500 differential scanning calorimeter. Table 4 shows the results of the TGA and DSC measurements. The test results are shown in Figures 3 and 4, respectively, and the TGA spectrum The weight loss was 0.95% when heated to 230.0°C, and the DSC spectrum showed The peak value of the endothermic peak is shown to be 238.4°C.

[0072] [Table 4]

[0073] Test Example 4 The dynamic water sorption measurement was carried out on the crystalline form I of Compound A prepared in Example 3. DVS curves were collected by DVS Intrinsic from SMS (Surface Measurement Systems). The relative humidity in °C was corrected by the deliquescence points of LiCl, Mg(NO3)2, and KCl, and the DVS measurement parameters were expressed. The test results are shown in Figure 5. As can be seen from Figure 5, the compound A of the present invention The weight of the sample of crystalline form I was 0.01% when heated from 0%RH to 95%RH to 0%RH at 25°C. The wettability of the sample was less than 2%, indicating that the sample had little or no wettability. Comparison of the powder X-ray diffraction patterns of the above shows that there is no change in the crystalline form before and after DVS.

[0074] [Table 5]

[0075] Test Example 5 XRPD results for Crystalline Form II were collected using the test conditions in Test Example 2. Powder X-ray Diffraction of Crystalline Form II The data 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 using the test conditions of Test Example 3. The test results are shown in Figures 7 and 8, respectively. It showed a stepwise weight loss of 4.47% when heated to 97.5°C, and the DSC spectrum There are two endothermic peaks at 155.3°C (peak temperature) and 236.1°C (onset temperature). It is shown that there is one exothermic peak at .

[0079] Test Example 7 XRPD results for crystalline Form III were collected using the test conditions in Test Example 2. Powder X-ray diffraction of crystalline Form III The folding data is 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 using the test conditions of Test Example 3. The test results are shown in Figures 10 and 11, respectively. When heated to 00°C, the weight decreased by 2.5%, and when heated further to 150°C, the weight decreased by 17.7%. The DSC spectrum showed that there was a dehydration or solvent-induced There is one endothermic signal that is presumed to be the peak temperature of the α-glucan derivative, and two endothermic signals at 234.7°C and 236.7°C (peak temperatures). It is shown that there is a thermal peak.

[0083] Test Example 9 XRPD results for crystalline Form IV were collected using the test conditions in Test Example 2. Powder X-ray Diffraction of Crystalline Form IV The data 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 crystalline form IV using the test conditions of Test Example 3 The test results are shown in Figures 13 and 14, respectively. When heated to 106.8°C, there was a stepwise weight loss of 11.3%, and the DSC spectrum showed temperatures of 106.8°C and 235.4°C. Two endothermic peaks are shown at (onset temperature).

[0087] Test Example 11 XRPD results for crystalline form V were collected using the test conditions in Test Example 2. The data is 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 using the test conditions of Test Example 3. The test results are shown in Figures 16 and 17, respectively. In the TGA spectrum, When the sample was heated to 230°C, the weight decreased by 1.8% and the DSC spectrum showed It is shown that there are two endothermic signals at 236.7°C (peak temperature) and 236.7°C (peak temperature).

[0091] Test Example 13 XRPD results for Form VI were collected using the test conditions in Test Example 2. Powder X-ray Diffraction of Form VI The data 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 crystalline form VI were performed using the test conditions of Test Example 3. The test results are shown in Figures 19 and 20, respectively. In the TGA spectrum, When the sample was heated to 200°C, it lost 1.0% of its weight and the DSC spectrum showed a temperature of 235.1°C (opening point). It is shown that there is one endothermic peak at the initial temperature.

[0095] Test Example 15 Crystalline Form I was left under 80°C / closed and 60°C / closed conditions for 1 day, but the temperature remained the same at 25°C / 60%RH / open and 40°C / open conditions. Even after leaving it under ℃ / 75%RH / open conditions for one week, there was no change in the crystal form or a decrease in purity (X Crystalline form was measured by RPD to assess physical stability, and purity was measured by HPLC to assess chemical stability. The qualitative evaluation was carried out.) It is shown 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 evaluated using a DNA-PK kinase assay kit (Promega). The results were detected 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. Prepare 5 μL of reaction mixture in a 384-well white plate, and add 1 μL of each compound (concentration The gradients were set to 1 μM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, and 0.013 nM, respectively. units 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 the reagent, mixed evenly, and centrifuged (1000 rpm, 30 s), and incubated at room temperature for 40 min.

[0101] v. Add 10 μL of Kinase Detection Reagent and shake to mix evenly. The mixture was combined, centrifuged (1000 rpm, 30 s), and incubated at room temperature for 30 min.

[0102] vi. Fluorescence values ​​were measured using a microplate reader (Thermo fisher, Varoskan LUX). GraphPad Prism 8 was used to calculate IC 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, and its preparation method was prepared accordingly.

[0105] The results show that the compounds of the present invention exhibit more significant inhibition of DNA-PK kinase than the comparative compounds. It has been shown to have harmful effects.

[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 divided into two groups (iv and po) using 18 mice per compound. The animals were divided into 2 groups (9 animals per group), and 3 animals were selected at each time point for blood sampling.

[0108] (2) After overnight fasting (water was allowed ad libitum), the rats were incubated in a solvent of 5% DMSO and 95% 30% HP-β-CD (v:v) as described in the present study. The compounds were dissolved (or formed a suspension) in the tail vein (iv, 1 mg / kg) and administered orally by gavage. The animals were administered po (10 mg / kg).

[0109] (3) In the iv group, the injection was administered via the submandibular vein at 5 minutes, 15 minutes, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after administration. 0.1 mL of blood was collected from the peritoneum, anticoagulated with EDTA-K2, and centrifuged at 4°C for 5 minutes to obtain plasma. The cells were then stored at -20°C.

[0110] (4) The po group was administered 15 min, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration. 0.1 mL of blood was collected from the submandibular vein and processed in the same manner as in the venous 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] From the test results, Compound A and crystalline forms I, II, III, IV, V, and Both Forms VI are shown to have good pharmacokinetic properties.

[0114] Although the present invention has been described in detail in the specification with reference to specific embodiments, the above embodiments It will be understood by those skilled in the art that the embodiments are illustrative and not limiting of the present invention. Those skilled in the art may make minor improvements and modifications to the present invention without departing from the principles of the present invention. The technical solutions obtained by these improvements and modifications are also included in the claims of the present invention. Included in the scope of protection.

Claims

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

2. Crystalline Form I has a powder X-ray diffraction pattern with Cu-Kα radiation with a 2θ position of 9.859°±0.3°; It has characteristic diffraction peaks at 14.759°±0.3°, 19.679°±0.3°, and 19.961°±0.3°. The crystal according to claim 1, characterized by:

3. Crystalline Form I has an additional powder X-ray diffraction pattern with Cu-Kα radiation at the 2θ position: 4.979°±0 .2°, 15.759°±0.2°, 19.339°±0.2°, 22.481°±0.2°, 24.641°±0.2° The crystal according to claim 2, characterized in that it has a diffraction peak.

4. Crystalline Form I has an additional 2θ position of 12.120°±1 in the powder X-ray diffraction pattern using Cu-Kα radiation. It is characterized by having characteristic diffraction peaks at 0.2°, 18.802°±0.2°, and 21.822°±0.2°. The crystal according to claim 3.

5. The powder X-ray diffraction pattern of crystalline form I is essentially as shown in Figure 1 or Figure 2. The crystal according to claim 4, wherein

6. The TGA curve of crystalline form I is essentially as shown in Figure 3. The crystal described in any one of claims 1 to 4.

7. The DSC curve of crystalline form I is essentially as shown in Figure 4. The crystal described in any one of claims 1 to 4.

8. The powder X-ray diffraction pattern of crystalline Form II is essentially as shown in Figure 6. The crystal described in claim 1.

9. The powder X-ray diffraction pattern of crystalline Form III is characterized by being essentially as shown in Figure 9. The crystal according to claim 1.

10. The powder X-ray diffraction pattern of crystalline Form IV is characterized by being essentially as shown in Figure 12. The crystal according to claim 1.

11. The powder X-ray diffraction pattern of crystalline form V is essentially as shown in Figure 15. The crystal described in claim 1.

12. The powder X-ray diffraction pattern of crystalline Form VI is characterized by being essentially as shown in Figure 18. The crystal according to claim 1.

13. A method for preparing crystalline form I according to any one of claims 2 to 7, comprising the steps of: The manufacturing method includes: Compound A is dissolved in a solvent, heated to reflux to dissolve the solid, and then allowed to cool and crystallize. , filtering and drying to obtain crystalline form I; or Compound A is dissolved in a solvent, and the temperature is raised to 75-85°C to dissolve the solid, and then the temperature is lowered to 55-65°C. The temperature is then lowered to 15-25°C to allow crystallization, and the mixture is filtered and dried to obtain crystalline form I. Method 2 is selected, The solvent is selected from alcohol-based solvents and mixed solvents of alcohol-based solvents and water. A manufacturing method characterized by:

14. A therapeutically effective amount of the crystalline form of any one of claims 1 to 12 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a compound or excipient.

15. DNA-P of the crystalline form according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 14 Use in the manufacture of K inhibitors.

16. The crystalline form according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 14 is used to treat cancer. Use in the manufacture of drugs for treatment and prevention.

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