Crystalline forms of purine derivatives and pharmaceutical compositions thereof

Abstract

This invention relates to crystalline forms of substituted purine derivatives and pharmaceutical compositions thereof, methods of preparation thereof, and use in the preparation of DNA-PK inhibitors. Specifically, it relates to crystalline forms of compounds of formula (A) and pharmaceutical compositions thereof, methods of preparation thereof, and use in the preparation of DNA-PK inhibitors.

Description

Technical Field

[0001] This invention relates to a purine derivative, or the crystal form of its hydrate or solvate, as well as its preparation method or pharmaceutical composition and its use in the preparation of DNA-PK inhibitors. Background Technology

[0002] DNA-dependent protein kinases (DNA-PKs) are DNA-PK enzyme complexes composed of Ku70 / Ku80 heterodimers and DNA-dependent protein kinase catalytic subunits (DNA-PKcs). These enzyme complexes require DNA involvement to activate and function (George et al., 2019). As a serine / threonine protein kinase, DNA-PK belongs to the PIKK (phosphatidylinositol 3-kinase-related kinase) family. It plays a crucial role not only in repairing intracellular DNA double-strand breaks (DSBs) and in cellular DNA recombination or antibody-mediated DNA rearrangement (V(D)J recombination), but also in physiological processes such as chromosome modification, transcriptional regulation, and telomere maintenance.

[0003] In normal physiological processes, various factors can lead to DNA splits (DSBs): DSBs often occur as intermediate products during somatic cell DNA recombination, a process crucial for the formation of the functional immune system in all vertebrates; single-strand or double-strand breaks can also occur when the replication fork encounters damaged bases during DNA replication; DNA can also produce DSBs due to attack by reactive oxygen species (ROS) during normal metabolism (Cannan & Pederson, 2016). Furthermore, various exogenous factors can also cause DSBs, such as ionizing radiation (IR) and chemotherapeutic agents (e.g., topoisomerase II inhibitors) (George et al., 2019). If DSBs are not repaired or are incorrectly repaired, mutations and / or chromosomal aberrations will occur, ultimately leading to cell death. To cope with the harm caused by DSBs, eukaryotic cells have evolved various mechanisms to repair damaged DNA to maintain cell viability and genome stability. In eukaryotic cells, the most important DNA repair mechanism is non-homologous end-joining (NHEJ). This method of directly joining broken DNA does not require the participation of homologous DNA fragments and can occur at any stage of the cell cycle. NHEJ is a dynamic process mediated by DNA-PK that requires the participation of multiple proteins and signaling pathways. The basic process is as follows: (1) Ku70 / Ku80 heterodimers recognize and bind to the ends of double-stranded DNA breaks; (2) recruit proteins such as DNA-PKcs and XRCC4-DNA ligase IV complex to both sides of the DNA double-strand breaks; (3) DNA-PKcs autophosphorylate and activate their own kinase activity; (4) DNA-PKcs act as an adhesive to join the ends of the broken DNA and prevent exonucleases from degrading the DNA; (5) process the DNA to remove unjoinable ends or other forms of damage at the breaks; (6) XRCC4-DNA ligase IV complex repairs the DNA ends (in some cases, DNA polymerase may be needed to synthesize new ends before ligation). Phosphorylation of DNA-PKcs can induce conformational changes in proteins and regulate the activity of various proteins during NHEJ (such as Artemis, Ku70, Ku80, and DNA ligase), which is crucial for DNA repair. Therefore, phosphorylated DNA-PKcs (pDNA-PKcs) are often used as markers of cellular DSBs.

[0004] Previous studies have shown that DNA-PK activity is associated with the development and progression of various tumors: for example, DNA-PKcs in melanoma can promote angiogenesis and tumor metastasis; DNA-PKcs expression is significantly upregulated in multiple myeloma; and the content of Ku protein is significantly increased in radiotherapy-resistant thyroid tumors (Ihara, Ashizawa, Shichijo, & Kudo, 2019). Therefore, combining DNA-PK inhibitors with anti-tumor therapies that cause DNA damage (such as immunotherapy and chemotherapy agents) could be considered to improve efficacy. The use of DNA-PK inhibitors can interfere with the DNA repair function of normal cells to some extent. However, normal cells have multiple DNA repair pathways as a supplement, while tumor cells face strong DNA replication pressure and lack effective DNA repair mechanisms. Inhibiting the activity of DNA-PK in tumor cells can enhance the killing effect of other anti-tumor drugs on tumor cells. Summary of the Invention

[0005] This invention provides crystal forms I and II of 3-(7-methyl-2-[(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-8oxo-8,9-dihydro-7-hydro-purine-9-yl)adamantane-1-carboxynitrile (compound A), which has the following chemical structure:

[0006]

[0007] The crystal form of the present invention exhibits at least the following advantages: good solubility, high stability, easy handling, processing and purification, improved oral bioavailability of drugs, extended drug shelf life, and easy manufacturing of various dosage forms.

[0008] The crystal form of this invention exhibits pharmaceutical advantages over the amorphous form of compound A. In particular, the crystal form enhances chemical and physical stability, which is more advantageous for preparing solid drug dosage forms containing pharmacologically active ingredients.

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

[0010] Unless otherwise specified, compound A in this invention refers to its amorphous form.

[0011] One embodiment of the crystal form described in this invention is an anhydrous compound A (crystal form I), which, when subjected to Cu-Kα radiation, exhibits characteristic diffraction peaks at the following 2θ positions in its X-ray powder diffraction pattern: 9.4472°±0.3°, 18.836°±0.3°, and 23.79°±0.3°.

[0012] In the X-ray powder diffraction of crystal form I, the 2θ diffraction angles also show characteristic diffraction peaks at 7.414°±0.2°, 13.514°±0.2°, 15.119°±0.2°, 15.43°±0.2°, 16.601°±0.2°, and 23.496°±0.2°.

[0013] Furthermore, the X-ray powder diffraction pattern of crystal form I also shows characteristic diffraction peaks at the following 2θ positions: 13.077°±0.2°, 17.027°±0.2°, 17.527°±0.2°, 24.55°±0.2°, and 27.407°±0.2°.

[0014] Furthermore, the X-ray powder diffraction pattern of crystal form I also shows characteristic diffraction peaks at the following 2θ positions: 4.7909°±0.2°, 10.404°±0.2°, 13.790°±0.2°, 18.01°±0.2°, 21.389°±0.2°, 22.064°±0.2°, 25.154°±0.2°, and 25.912°±0.2°.

[0015] Furthermore, the X-ray powder diffraction (XRD) pattern of crystal form I is basically as shown in Figure 1.

[0016] One embodiment of the crystal form described in this invention is an anhydrous compound A (crystal form II), which, when subjected to Cu-Kα radiation, exhibits characteristic diffraction peaks at the following 2θ positions in its X-ray powder diffraction pattern: 7.8763°±0.3°, 16.633°±0.3°, 18.059°±0.3°, and 27.085°±0.3°.

[0017] Among them, in the X-ray powder diffraction of crystal form II, the 2θ diffraction angles also have characteristic diffraction peaks at 11.619°±0.2°, 17.075°±0.2°, 22.088°±0.2°, and 27.915°±0.2°.

[0018] Furthermore, the X-ray powder diffraction pattern of crystal form II also shows characteristic diffraction peaks at the following 2θ positions: 9.243°±0.2°, 10.912°±0.2°, 13.019°±0.2°, 14.591°±0.2°, 22.504°±0.2°, and 23.475°±0.2°.

[0019] Furthermore, the X-ray powder diffraction pattern of crystal form II also shows characteristic diffraction peaks at the following 2θ positions: 9.624°±0.2°, 10.156°±0.2°, 19.26°±0.2°, 23.807°±0.2°, and 25.080°±0.2°.

[0020] Furthermore, the X-ray powder diffraction (XRD) pattern of crystal form II is basically as shown in Figure 2.

[0021] The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of the crystalline compound described herein, and one or more pharmaceutically acceptable carriers or excipients.

[0022] The crystal form described in this invention, or the pharmaceutical composition thereof, which is an active pharmaceutical ingredient, can be used to prepare DNA-PK inhibitor drugs.

[0023] DNA-PK inhibitors are used to prepare drugs for the treatment and prevention of cancer.

[0024] The X-ray powder diffraction pattern disclosed in this invention, and those substantially the same, also fall within the scope of this invention.

[0025] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0026] "Effective dose" refers to the amount of a compound that causes physiological or medical translation in an tissue, system, or subject. This amount is sought, including the amount of a compound, when administered to a subject, sufficient to prevent the occurrence of one or more symptoms of the treated disease or condition or to alleviate them to some extent.

[0027] IC 50 "Half-inhibition concentration" refers to the concentration at which half of the maximum inhibitory effect is achieved.

[0028] The crystal structure 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).

[0029] It is understood that the numerical values ​​described and protected in this invention are approximate. Variations within these values ​​may be attributed to equipment calibration, equipment errors, crystal purity, crystal size, sample size, and other factors.

[0030] It is understood that the crystal forms of the present invention are not limited to those that are exactly the same as the characteristic spectra described in the accompanying drawings, such as XRD. Any crystal form having a characteristic spectra that are substantially the same or essentially the same as those described in the accompanying drawings falls within the scope of the present invention.

[0031] Various modifications and changes to this invention will be apparent to those skilled in the art upon consideration of the description and embodiments thereof without departing from the scope and spirit of the invention. Attached Figure Description

[0032] Figure 1 shows the X-ray powder diffraction pattern of compound A, crystal form I, using Cu-Kα radiation.

[0033] Figure 2 shows the X-ray powder diffraction pattern of compound A crystal form II using Cu-Kα radiation. Detailed Implementation

[0034] The following detailed embodiments illustrate the implementation process and beneficial effects of the present invention, aiming to help readers better understand the essence and characteristics of the present invention, and are not intended to limit the scope of implementation of this case.

[0035] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0036] Unless otherwise specified, the experimental conditions for crystallization are generally room temperature (20-30℃, 30-70% RH), and the solvent ratio refers to the volume ratio.

[0037] Example 1: Preparation of Compound A

[0038] 3-(7-methyl-2-[(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-8oxo-8,9-dihydro-7-hydro-purine-9-yl)adamantane-1-carboxynitrile (Compound A)

[0039] 3-(7-methyl-2-[(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-8-oxo-8,9-dihydro-7H-purin-9-yl)adamantane-1-carbonitrile

[0040]

[0041]

[0042] first step:

[0043] 3-Aminoadamantane-1-carboxylic acid tert-butyl ester (1b)

[0044] tert-butyl 3-aminoadamantane-1-carboxylate

[0045] Compound 1a (10 g, 51.21 mmol) was dissolved in thionyl chloride (70 mL) and refluxed at 90 °C for 1 h. The reaction solution was directly concentrated, and after redissolving in toluene (50 mL), excess thionyl chloride was removed by concentration. Tert-butanol (60 mL) was added in an ice bath, and the reaction was carried out at room temperature for 1 h. The reaction was monitored by TLC until it was complete. The reaction solution was directly concentrated, and the solid was collected to give the target compound 1b (white solid, 12 g, yield 93.22%).

[0046] LC-MS m / z(ESI)=252.20[M+1].

[0047] Step Two:

[0048] 4-((3-(tert-butoxycarbonyl)adamantane-1-yl)amino)-2-chloropyrimidine-5-carboxylic acid ethyl ester (1c)

[0049] ethyl4-(((1s,3r,5R,7S)-3-(tert-butoxycarbonyl)adamantan-1-yl)amino)-2-chloropyrimidine-5-carboxylate

[0050] Compound 2,4-dichloropyrimidine-5-carboxylic acid ethyl ester (12 g, 54.29 mmol), compound 1b (13.65 g, 54.29 mmol), and potassium carbonate (15.01 g, 108.58 mmol) were dissolved in acetonitrile (150 mL). The reaction mixture was reacted at room temperature for 16 h. After the reaction was monitored by TLC, the mixture was filtered, and the solid was washed with a small amount of acetonitrile. The filtrates were combined and concentrated. The crude product was separated by column chromatography (petroleum ether / ethyl acetate (v / v) = 1 / 1) to give the target compound 1c (white solid, 15 g, yield 63.38%).

[0051] 1 H NMR (400MHz, DMSO-d6) δ8.63(s,1H),8.36(s,1H),4.30(q,2H),2.00–2.18(m,8H),1.61-1.73(m,6H),1.38(s,9H),1.31(t,3H).

[0052] Step 3:

[0053] 4-((3-(tert-butoxycarbonyl)adamantane-1-yl)amino)-2-chloropyrimidine-5-carboxylic acid (1d)

[0054] 4-((3-(tert-butoxycarbonyl)adamantan-1-yl)amino)-2-chloropyrimidine-5-carboxylicacid

[0055] Compound 1c (15 g, 34.41 mmol) was dissolved in 200 mL of tetrahydrofuran and 200 mL of water. Lithium hydroxide (1.65 g, 68.82 mmol) was added, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC until complete. The tetrahydrofuran was removed by concentration, and the pH was adjusted to 5 with 6N hydrochloric acid. A white solid precipitated out. The mixture was filtered, and the filter cake was washed twice with petroleum ether. The solid was collected to give the title compound 1d (white solid, 14 g, yield 99.75%).

[0056] 1 H NMR (400MHz, DMSO-d6) δ8.65(s,1H),8.58(s,1H),2.01–2.17(m,8H),1.57–1.77(m,6H),1.38(s,9H).

[0057] LC-MS m / z(ESI)=408.10[M+1].

[0058] Step 4:

[0059] 3-(2-chloro-8-oxo-8,9-dihydro-7-hydro-purine-9-yl)adamantane-1-carboxylic acid tert-butyl ester (1e)

[0060] tert-butyl-3-(2-chloro-8-oxo-8,9-dihydro-7H-purin-9-yl)adamantane-1-carboxylate

[0061] Compound 1d (15 g, 36.77 mmol) was dissolved in N,N-dimethylacetamide (150 mL). Diphenyl azidophosphate (7.91 mL, 36.77 mmol) and triethylamine (5.11 mL, 36.77 mmol) were added in an ice bath. The reaction mixture was stirred at room temperature for 1 h, then heated to 120 °C and reacted for another 3 h. The reaction was monitored by TLC until complete (dichloromethane / methanol (v / v) = 4 / 1). The reaction mixture was allowed to cool naturally to room temperature and slowly poured into 600 mL of ice water. A large amount of solid appeared. The mixture was filtered, the solid was collected, and slurried with ethyl acetate (150 mL). The solid was then dried under vacuum to give the target compound 1e (white solid, 7.0 g, yield 47.02%).

[0062] 1H NMR (400MHz, DMSO-d6) δ11.56(s,1H),8.07(s,1H),2.44–2.57(m,6H),2.23(s,2H),1.58–1.80(m,6H),1.39(s,9H).

[0063] Step 5:

[0064] tert-Butyl 3-(2-chloro-8-oxo-8,9-dihydro-7-hydro-purine-9-yl)adamantane-1-carboxylic acid tert-butyl ester (1f)

[0065] tert-butyl3-(2-chloro-7-methyl-8-oxo-8,9-dihydro-7H-purin-9-yl)adamantane-1-carboxylate

[0066] Compound 1e (5 g, 12.35 mmol) was dissolved in dimethylformamide (40 mL), and cesium carbonate (6.04 g, 18.52 mL) and dimethyl sulfate (1.4 mL, 14.82 mmol) were added at 0 °C. The reaction was carried out at room temperature for 2 h. The reaction was monitored by TLC until it was complete. 100 mL of water was added, and a solid precipitated. The solid was filtered and dried to give the target compound 1f (white solid, 5.0 g, yield 96.64%).

[0067] 1 H NMR (400MHz, DMSO-d6) δ8.31(s,1H),3.29(s,3H),2.43–2.56(m,6H),2.24(s,2H),1.54–1.80(m,6H),1.38(s,9H).

[0068] Step 6:

[0069] tert-Butyl 3-(7-methyl-2-[(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-8-oxo-8,9-dihydro-7-hydro-purine-9-yl)adamantane-1-carboxylic acid tert-butyl ester (1g)

[0070] tert-butyl3-(7-methyl-2-[(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-8-oxo-8,9-dihydro-7H-purin-9-yl)adamantane-1-carboxylate

[0071] 7-Methyl-[1,2,4]triazolo[1,5-a]pyridine-6-amine (500 mg, 3.37 mmol), compound 1f (1.41 g, 3.37 mmol), cesium carbonate (2.31 g, 7.08 mmol), and [(2-di-cyclohexylphosphino-3,6-dimethoxy-2′,4′,6′-triisopropyl-1,1′-biphenyl)-2-(2′-amino-1,1′-biphenyl)]palladium(II)methanesulfonate (310 mg, 0.34 mmol) were dissolved in dioxane (10 mL), under nitrogen protection and purged, and stirred at 100 °C for 4 h. After the reaction was monitored by TLC, the reaction solution was poured into ice water, the solid was collected, and the solid was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 100 / 1) to obtain 1 g of the target compound (white solid, 1.4 g, yield 78.29%).

[0072] 1 H NMR(400MHz,DMSO-d6)δ9.08(s,1H),8.58(s,1H),8.36(s,1H),8.10(s,1H),7.68(s ,1H),3.24(s,3H),2.33-2.61(m,6H),2.14(s,2H),1.51-1.67(m,6H),1.32(s,9H).

[0073] LC-MS m / z(ESI)=531.3[M+1].

[0074] Step 7:

[0075] 3-(7-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl]amino]amino)-8-oxo-7,8-dihydro-9H-purine-9-yl)adamantane-1-carboxylic acid (1h)

[0076] 3-(7-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-8-oxo-7,8-dihydro-9H-purin-9-yl)adamantane-1-carboxylic acid

[0077] 1 g (1.4 g, 2.64 mmol) of compound was dissolved in 100 mL of 4N dioxane hydrochloride solution. The mixture was reacted at room temperature for 16 h, concentrated, and prepared under medium pressure to obtain the target compound 1 h (light yellow solid, 1.4 g, yield 99%).

[0078] 1H NMR(400MHz,DMSO-d6)δ12.15(s,1H),9.07(s,1H),8.57(s,1H),8.36(s,1H),8.09(s,1H), 7.68(s,1H),3.24(s,3H),2.41-2.58(m,6H),2.38(s,3H),2.14(s,2H),1.56–1.71(m,6H).

[0079] LC-MS m / z(ESI)=475.20[M+1].

[0080] Step 8:

[0081] 3-(7-methyl-2-[(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-8-oxo-8,9-dihydro-7-hydro-purine-9-yl)adamantane-1-carboxamide (1i)

[0082] 3-(7-methyl-2-[(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-8-oxo-8,9-dihydro-7H-purin-9-yl)adamantane-1-carboxamide

[0083] Compound 1h (0.5 g, 1.05 mmol), ammonium chloride (0.56 g, 10.50 mmol), and triethylamine (0.73 mL, 5.25 mmol) were dissolved in N,N-dimethylformamide (15 mL). HATU (0.6 g, 1.58 mmol) was added in an ice bath. The mixture was reacted at room temperature for 1 h. The reaction was quenched with water (30 mL). The mixture was extracted with ethyl acetate (30 mL × 3). The organic phase was dried and concentrated to give the target compound 1i (white solid, 0.14 g, yield 28.16%).

[0084] 1 H NMR(400MHz,DMSO-d6)δ9.08(s,1H),8.56(s,1H),8.36(s,1H),8.08(s,1H),7.68(s,1H), 6.97(s,1H),6.74(s,1H),3.24(s,3H),2.33-2.62(m,9H),2.15(s,2H),1.51-1.73(m,4H).

[0085] LC-MS m / z(ESI)=474.3[M+1].

[0086] Step 9:

[0087] 3-(7-methyl-2-[(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-8oxo-8,9-dihydro-7-hydro-purine-9-yl)adamantane-1-carboxynitrile (Compound A)

[0088] 3-(7-methyl-2-[(7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-8-oxo-8,9-dihydr o-7H-purin-9-yl)adamantane-1-carbonitrile

[0089] Compound 1i (130 mg, 0.27 mmol) was dissolved in dichloromethane (20 mL), and pyridine (90 mg, 1.08 mmol) and trifluoroacetic anhydride (170 mg, 0.81 mmol) were added in an ice bath. The reaction was continued for 1 h while maintaining the temperature. Methanol (20 mL) was added, and the crude product was concentrated. The crude product was redissolved in ethyl acetate (50 mL), dried over 15% NaHCO3 (50 mL), and saturated brine (50 mL), respectively. The product was then concentrated to give compound A (pale yellow solid, 60 mg, yield 48.78%).

[0090] 1H NMR(400MHz,DMSO-d6)δ9.07(s,1H),8.65(s,1H),8.37(s,1H),8.11(s,1H),7.70(s,1H),3.25(s,3 H),2.75(s,2H),2.44-2.51(m,4H),2.38(s,3H),2.15(s,2H),1.91-1.94(m,4H),1.53-1.62(m,2H).

[0091] LC-MS m / z(ESI)=456.2[M+1].

[0092] Example 2 Preparation of crystal form I of compound A

[0093] Compound A was recrystallized in dichloromethane to obtain crystal form I of compound A.

[0094] Example 3 Preparation of Compound A (Crystal Form II)

[0095] Compound A was recrystallized in a dichloromethane / methanol (volume ratio 9 / 1) mixture to obtain crystal form II of compound A.

[0096] Test Example 1

[0097] Powder diffraction patterns of compound A in crystal forms I and II were obtained using a Rigbu xtalab co-electrode diffractometer (Japan) at room temperature under monochromatic Cu-Kα radiation of graphite (λ = 1.54), voltage 45 kV, current 40 mA, and a scanning range (2θ angle) of 3°–60°. Using Olex2, the structure was analyzed using the SHELXT structure analysis program, and the data package was refined using the SHELXL-97 direct method with minimum variance.

[0098] The X-ray powder diffraction data of compound A crystal form I are shown in Table 1, and the X-ray powder diffraction pattern of compound A crystal form I is shown in Figure 1.

[0099] Table 1. X-ray powder diffraction data of compound A, crystal form I.

[0100] Peak 2θ angle (degrees) Interplanar spacing Peak height Half peak width relative peak height 1 4.7909 18.430 1715 0.2631 5.00 2 7.414 11.914 8298 0.289 26.21 3 9.4472 9.3540 35184 0.2743 100.00

[0101] 4 10.404 8.496 1611 0.280 5.28 5 10.78 8.199 212 0.59 1.29 6 11.964 7.392 633 0.286 2.28 7 13.077 6.7646 6619 0.285 18.76 8 13.514 6.5466 6458 0.311 20.02 9 13.790 6.416 1732 0.32 5.44 10 14.096 6.278 1420 0.27 3.86 11 15.119 5.855 5685 0.34 20.31 12 15.43 5.738 5114 0.41 21.84 13 16.601 5.3357 5661 0.372 21.97 14 17.027 5.2031 4245 0.274 12.15 15 17.527 5.056 2258 0.49 11.48 16 18.01 4.921 1656 0.34 5.86 17 18.23 4.862 1212 0.28 3.48 18 18.836 4.7075 7500 0.462 36.15 19 19.702 4.5024 1669 0.261 4.54 20 20.471 4.335 566 0.20 1.20 21 21.389 4.1509 1832 0.48 9.24 22 22.064 4.0254 1835 0.300 5.75 23 23.496 3.7832 8375 0.289 24.08 24 23.79 3.737 6576 0.60 39.02 25 24.55 3.623 1337 0.9 12.22 26 25.154 3.5375 3029 0.293 8.83 27 25.912 3.4357 2677 0.367 9.78 28 26.676 3.3390 1176 0.23 2.66 29 27.407 3.2516 4893 0.337 16.41 30 28.39 3.141 668 0.69 4.62 31 29.454 3.0301 662 0.41 2.71 32 31.46 2.842 486 0.48 2.33

[0102] 33 31.92 2.801 262 0.46 1.19 34 32.480 2.7544 1100 0.303 3.32 35 32.997 2.7124 776 0.29 2.26 36 33.402 2.6805 338 0.29 0.96 37 34.26 2.6152 336 0.33 1.09 38 35.28 2.5418 195 0.30 0.60 39 36.47 2.462 301 0.62 1.92 40 37.87 2.3739 366 0.41 1.55 41 41.113 2.1938 179 0.21 0.38 42 41.74 2.1620 80 0.22 0.17 43 42.50 2.1254 135 0.27 0.36 44 43.14 2.095 164 0.66 1.09 45 44.11 2.0513 163 0.24 0.40 46 45.49 1.992 224 0.41 0.97 47 45.97 1.9724 93 0.26 0.26 48 48.02 1.893 114 0.75 0.87 49 48.83 1.8635 265 0.36 0.97 50 50.71 1.7988 124 0.29 0.36 51 52.13 1.7530 159 0.59 0.94 52 53.27 1.7181 78 0.56 0.43 53 57.93 1.5905 55 0.8 0.86

[0103] The X-ray powder diffraction data of compound A crystal form II are shown in Table 2, and the X-ray powder diffraction pattern of compound A crystal form II is shown in Figure 2.

[0104] Table 2. X-ray powder diffraction data of compound A, crystal form II.

[0105] Peak 2θ angle (degrees) Interplanar spacing Peak height Half peak width relative peak height 1 4.01 22.0 37 0.94 1.69 2 4.860 18.17 291 0.203 2.86

[0106] 3 5.50 16.06 85 0.40 1.64 4 7.8763 11.216 8195 0.2114 71.72 5 8.419 10.494 121 0.14 0.72 6 9.243 9.561 4450 0.198 34.85 7 9.624 9.183 1820 0.297 21.40 8 10.156 8.703 2964 0.205 24.06 9 10.912 8.102 3916 0.245 41.70 10 11.619 7.610 3444 0.335 50.28 11 13.019 6.794 3259 0.369 43.09 12 13.601 6.505 920 0.39 12.97 13 13.924 6.355 494 0.18 3.21 14 14.591 6.066 3074 0.293 32.32 15 14.828 5.9696 2286 0.170 13.95 16 15.075 5.872 1791 0.218 13.98 17 15.725 5.631 830 0.30 9.81 18 16.057 5.515 1519 0.21 12.49 19 16.633 5.325 4907 0.45 88.15 20 17.075 5.189 4877 0.298 57.80 21 18.059 4.908 5877 0.427 100.00 22 19.26 4.605 2245 0.26 23.17 23 19.52 4.543 842 0.18 6.02 24 20.38 4.354 843 0.22 6.52 25 20.6 4.32 992 0.29 10.34 26 21.553 4.120 1191 0.29 12.19 27 22.088 4.0211 3984 0.327 46.35 28 22.504 3.948 2719 0.33 31.68 29 22.88 3.883 1255 0.40 17.87 30 23.475 3.7866 4161 0.287 42.46 31 23.807 3.734 2391 0.33 27.78

[0107] 32 25.080 3.548 2026 0.317 22.87 33 25.754 3.4564 1305 0.27 12.57 34 27.085 3.2895 6518 0.289 83.52 35 27.915 3.1935 2958 0.383 50.10 36 29.325 3.0431 223 0.29 2.28 37 31.639 2.8256 2103 0.142 18.64 38 32.57 2.747 119 0.22 1.47 39 33.960 2.6376 431 0.41 6.34 40 34.55 2.5940 483 0.48 8.29 41 35.74 2.510 118 0.23 1.38 42 36.40 2.466 243 0.72 8.94 43 39.05 2.305 139 0.28 2.12 44 42.82 2.110 280 1.28 14.97 45 45.353 1.9980 803 0.271 9.10 46 46.44 1.9536 164 0.42 2.88 47 48.34 1.881 88 0.33 1.04 48 50.10 1.819 91 0.37 1.43 49 50.68 1.800 99 0.44 1.81 50 52.62 1.738 64 0.38 0.87 51 56.450 1.62877 1675 0.073 6.17

[0108] Test Example 2

[0109] DNA-PK kinase inhibition assay

[0110] The inhibitory activity of the compound against DNA-PK kinase was detected using a DNA-PK kinase assay kit (purchased from Promega, catalog number: V4107, lot number: 0000366495). The results were quantified using chemiluminescence. The specific experimental protocol is as follows:

[0111] i. Construct standard curves for different concentrations of ADP-fluorescence according to the kit instructions;

[0112] ii. Prepare a 5 μL reaction system in a 384-well white plate. Add 1 μL of compound A (with concentration gradients of 1 μM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, and 0.013 nM) to each well, along with 20 units of DNA-PK kinase, 0.2 μg / μL of substrate, 10 μg / μL of DNA, 50 μM ATP, and 1% DMSO.

[0113] iii. Mix well, centrifuge (1000 rpm, 30 s), and incubate at 37°C for 60 min;

[0114] iv. Add 5 μL of ADP-Glo TM The reaction was terminated with Reagent, mixed well, centrifuged (1000 rpm, 30 s), and incubated at room temperature for 40 min.

[0115] v. Add 10 μL Kinase Detection Reagent, vortex to mix, centrifuge (1000 rpm, 30 s), and incubate at room temperature for 30 min;

[0116] vi. Measure the fluorescence value using a microplate reader (Thermo Fisher, Varioskan LUX). Perform IC50 analysis using a GraphPad Prism8. 50 The calculation results are shown in Table 3.

[0117] Table 3. Inhibitory activity of compound A against DNA-PK kinase

[0118] Compound numbering <![CDATA[IC 50 (nM)]]> Compound A 0.08 Comparison Example 100.20

[0119] Note: The control example is compound 3 of J.Med.Chem (2020), 63(7), 3461-3471, which was prepared according to its preparation method.

[0120] The results showed that, compared with the control, the compound of the present invention had a more significant inhibitory effect on DNA-PK kinase.

[0121] This invention specification provides a detailed description of specific embodiments. Those skilled in the art should recognize that the above embodiments are exemplary and should not be construed as limiting the invention. For those skilled in the art, various improvements and modifications can be made to the invention without departing from its principles, and the resulting technical solutions also fall within the protection scope of the claims of this invention.

Claims

1. Crystal of the compound shown in formula (A): The compound shown in formula (A) is crystal form I. Crystal form I is subjected to Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ positions: 9.4472°±0.3°, 18.836°±0.3°, and 23.79°±0.3°.

2. The crystal according to claim 1, characterized in that, Crystal form I, when subjected to Cu-Kα radiation, exhibits characteristic diffraction peaks at the following 2θ positions in its X-ray powder diffraction pattern: 7.414°±0.2°, 13.514°±0.2°, 15.119°±0.2°, 15.43°±0.2°, 16.601°±0.2°, and 23.496°±0.2°.

3. The crystal according to claim 2, characterized in that, Crystal form I, when subjected to Cu-Kα radiation, exhibits characteristic diffraction peaks at the following 2θ positions in its X-ray powder diffraction pattern: 13.077°±0.2°, 17.027°±0.2°, 17.527°±0.2°, 24.55°±0.2°, and 27.407°±0.2°.

4. The crystal according to claim 3, characterized in that, The X-ray powder diffraction pattern of crystal form I is shown in Figure 1.

5. Crystal of the compound shown in formula (A): Its features are, The compound shown in formula (A) is crystal form II. Crystal form II is subjected to Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ positions: 7.8763°±0.3°, 16.633°±0.3°, 18.059°±0.3°, and 27.085°±0.3°.

6. The crystal according to claim 5, characterized in that, Crystal form II, when subjected to Cu-Kα radiation, exhibits characteristic diffraction peaks at the following 2θ positions in its X-ray powder diffraction pattern: 11.619°±0.2°, 17.075°±0.2°, 22.088°±0.2°, and 27.915°±0.2°.

7. The crystal according to claim 6, characterized in that, Crystal form II, when subjected to Cu-Kα radiation, exhibits characteristic diffraction peaks at the following 2θ positions in its X-ray powder diffraction pattern: 9.243°±0.2°, 10.912°±0.2°, 13.019°±0.2°, 14.591°±0.2°, 22.504°±0.2°, and 23.475°±0.2°.

8. The crystal according to claim 7, characterized in that, The X-ray powder diffraction pattern of crystal form II is shown in Figure 2.

9. A pharmaceutical composition comprising a therapeutically effective amount of the crystals according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier or excipient.

10. Use of the crystal according to any one of claims 1 to 8, or the pharmaceutical composition according to claim 9, in the preparation of a DNA-PK inhibitor.

11. Use of the crystal of any one of claims 1 to 8, or the pharmaceutical composition of claim 9, in the preparation of a medicament for treating and preventing cancer.

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