Crystalline form of ATR inhibitor and use thereof

Abstract

Disclosed are a crystalline form of an ATR inhibitor, a preparation method therefor, and the use thereof in the preparation of a drug for treating ATR-related diseases. (I)

Description

Technical FieldProvided are crystal forms of an ATR inhibitor and preparation processes thereof and use for the manufacture of a medicament for treating an ATR-related disease.BackgroundATR (Ataxia Telangiectasia-mutated and Rad3-Related protein kinase) belongs to the PIKKs(phosphatidylinositol-3-kinase-related kinase) family and participates in DNA damage repair to maintaingene stability. ATR protein kinase has a synergistic response on DNA damage, replication stress and cellcycle disturbances. ATR and ATM belong to the PIKK family of serine / threonine protein kinases, and theyare common component of the cell cycle and DNA damage repairing, and other members include Chkl,BRCA1, p53. ATR is mainly responsible for DNA replication stress (duplication fork arrest) and repair ofsingle strand break.When the double-stranded DNA breaks or the replication fork arrests, ATR is activated by thesingle-stranded DNA structure. DNA polymerase stays in the process of DNA replication, and thereplication helicase continues to unwind at the leading end of the DNA replication fork, resulting in theproduction of long single-stranded DNA (ssDNA), which is then bound by the single-stranded DNA andRPA (replication protein A). ATR / ATR acting protein complex is recruited by RPA upon replication stressor DNA damage to the damage site, RPA-single-stranded DNA complex activates the RAD17 / rfc2-5complex to bind to the damage site, DNA-ssDNA junction activates Rad9-HUS-RAD1 (9-1-1) heterotrimer,9-1-1 in turn recruits TopBP1 to activate ATR. Once ATR is activated, ATR promotes DNA repair throughdownstream targets, stabilizing and restarting arrested replication forks and transient cell cycle arrest.These functions are achieved by ATR via mediating the downstream target Chk1. ATR acts as checkpointfor DNA damage in the cell cycle during S phase. It can mediate the degradation of CDC25A through Chk1,thereby delaying the DNA replication process and providing time to repair the replication fork. ATR is alsothe main regulator of G2 / M cell cycle checkpoint, preventing cells from entering mitosis prematurelybefore DNA replication is completed or DNA damage. This ATR-dependent G2 / M cell cycle arrest ismainly mediated by two mechanisms: 1. Degradation of CDC25A; 2. Phosphorylation of Cdc25C by Chk1to bind to 14-3-protein. The binding of Cdc25C to 14-3-3 protein promotes its export from the nucleus andcytoplasmic isolation, thereby inhibiting its ability to dephosphorylate and activate nuclear Cdc2, which inturn prevents entry into mitosis.ATR gene mutations are very rare, and only few patients with Seckel syndrome have ATR genemutations, which are characterized by stunting and microcephaly. Disruption of ATR-related pathways canlead to genome instability, and ATR protein is activated by most cancer chemotherapy. In addition, theduplication of the ATR gene has been described as a risk factor for rhabdomyosarcoma.ATR is essential for cell self-replication and is activated in the S phase to regulate the origin ofreplication and repair damaged replication forks. Damage to the replication forks can increase thesensitivity of cancer cells to platinum and hydroxyurea anticancer agents and reduce the resistance ofcancer cells. Therefore, inhibiting ATR may be an effective method in cancer treatment in the future.SummaryProvided is a Crystal Form A of the compound of formula (I), the Crystal Form A has an X-ray powderdiffraction (XRPD) pattern having characteristic diffraction peaks at the following 2θ angles: 8.10±0.20°,18.33±0.20° and 22.63±0.20°.(I)In some embodiments according to the present disclosure, the Crystal Form A has an X-ray powderdiffraction (XRPD) pattern having characteristic diffraction peaks at the following 2θ angles: 7.46±0.20°,8.10±0.20°, 13.03±0.20°, 15.07±0.20°, 15.58±0.20°, 16.19±0.20°, 18.33±0.20° and 22.63 ±0.20°.In some embodiments according to the present disclosure, the Crystal Form A has an X-ray powderdiffraction (XRPD) pattern having characteristic diffraction peaks at the following 2θ angles: 7.46±0.20°,8.10±0.20°, 13.03±0.20°, 13.46±0.20°, 15.07±0.20°, 15.58±0.20°, 16.19±0.20°, 18.33±0.20°, 21.17±0.20°and 22.63 ±0.20°.In some embodiments according to the present disclosure, the Crystal Form A has an X-ray powderdiffraction (XRPD) pattern having characteristic diffraction peaks at the following 2θ angles: 7.46°, 8.10°,11.24°, 13.03°, 13.46°, 15.07°, 15.58°, 15.98°, 16.19°, 17.70°, 18.33°, 19.60°, 21.17°, 22.63°, 23.84°, 25.56°and 26.57°.In some embodiments according to the present disclosure, the Crystal Form A has an XRPD pattern asshown in Figure 1.In some embodiments according to the present disclosure, the Crystal Form A has an XRPD patternwith Analysis Data shown in Table 1:Table 1: XRPD Pattern Analysis Data of Crystal Form AProvided is a Crystal Form B of a compound of formula (I), wherein the Crystal Form B has an X-raypowder diffraction (XRPD) pattern having characteristic diffraction peaks at the following 2θ angles:8.45±0.20°, 10.87±0.20° and 20.56±0.20°.In some embodiments according to the present disclosure, the Crystal Form B has an X-ray powderdiffraction (XRPD) pattern having characteristic diffraction peaks at the following 2θ angles: 8.45±0.20°,10.87±0.20°, 14.83±0.20°, 15.54±0.20°, 17.33±0.20°, 20.56±0.20°, 22.00±0.20° and 22.63±0.20°.In some embodiments according to the present disclosure, the Crystal Form B has an X-ray powderdiffraction (XRPD) pattern having characteristic diffraction peaks at the following 2θ angles: 8.45±0.20°,10.87±0.20°, 14.83±0.20°, 15.54±0.20°, 17.33±0.20°, 20.08±0.20°, 20.56±0.20°, 22.00±0.20°,22.63±0.20° and 25.26±0.20°.In some embodiments according to the present disclosure, the Crystal Form B has an X-ray powderdiffraction (XRPD) pattern having characteristic diffraction peaks at the following 2θ angles: 8.45°, 9.20°,10.87°, 12.57°, 14.14°, 14.53°, 14.83°, 15.54°, 16.80°, 17.33°, 18.43°, 19.84°, 20.08°, 20.56°, 21.39°,22.00°, 22.44°, 22.63°, 23.26°, 25.26°, 25.85° and 26.98°.In some embodiments according to the present disclosure, the Crystal Form B has an XRPD pattern asshown in Figure 2.In some embodiments according to the present disclosure, the Crystal Form B has an XRPD patternwith Analysis Data shown in Table 2:Table 2: XRPD Pattern Analysis Data of Crystal Form BIn some embodiments according to the present disclosure, the Crystal Form B has a DifferentialScanning Calorimetry curve (DSC) having one onset point of endothermic peak at 174.3±3°C.In some embodiments according to the present disclosure, the Crystal Form B has a DSC pattern asshown in Figure 3.In some embodiments according to the present disclosure, the Crystal Form B has aThermogravimetric Analysis curve (TGA), wherein the weight loss at 150°C±3°C is 1.49%.In some embodiments according to the present disclosure, the Crystal Form B has a TGA pattern asshown in Figure 4.Provided is a process for preparing a Crystal Form A of a compound of formula (I), comprising:1) adding the compound of formula (I) into ethanol solvent;2) adding water;3) stirring for 100-120 h;4) performing recrystallization at room temperature to obtain the Crystal Form A.Provided is a process for preparing a Crystal Form B of a compound of formula (I), comprising:1) adding the compound of formula (I) into a solvent;2) heating to a temperature with stirring for 2.5-120 h;3) performing recrystallization at room temperature to obtain the Crystal Form B.In some embodiments according to the present disclosure, the solvent is: methanol, methyl tertiarybutyl ether, methanol / water (V / V, 1:0.3-1), acetone / water(V / V, 1:1), isopropanol / water (V / V, 1:1), ethylacetate / n-heptane(V / V, 1:1), isopropyl acetate / n-heptane(V / V, 1:1), ethanol / n-heptane(V / V, 1:1),acetonitrile / n-heptane(V / V, 1:1), isopropanol / n-heptane (V / V, 1:1) or dichloromethane / n-heptane (V / V,1:1).In some embodiments according to the present disclosure, the temperature is 25-70°C.Provided is a process for preparing a Crystal Form B of a compound of formula (I), comprising:1) adding the compound of formula (I) into alcohol solvent;2) adding water;3) stirring for 15-20 h;4) performing recrystallization at room temperature to obtain the Crystal Form B.In some embodiments according to the present disclosure, the volume ratio of the alcohol solvent andwater is 1:1-1:4.In some embodiments according to the present disclosure, the alcohol solvent is selected from thegroup consisting of methanol.In some embodiments according to the present disclosure, the concentration range of the compound offormula (I) is selected from the group consisting of 25 mg / mL-50 mg / mL.Provided is also use of the compound of formula (I), the Crystal Form A or the Crystal Form B for themanufacture of a medicament for treating an ATR associated disease.In some embodiments according to the present disclosure, the medicament is for use in treating a solidtumor or a blood tumor.In some embodiments according to the present disclosure, the medicament is for use in treatingcolorectal cancer, gastric cancer, esophageal cancer, primary peritoneal carcinoma, adrenocorticalcarcinoma, renal clear cell carcinoma, prostate cancer, bladder urothelial carcinoma, ovarian cancer, breastcancer, endometrial carcinoma, fallopian tube carcinoma, non-small cell lung cancer or small cell lungcancer.Technical effectThe Crystal Form A and Crystal Form B of the compound of formula (I) according to the presentdisclosure are stable, are less affected by light, heat and humidity, have good drug efficacy in vivo, and arepromising for drugability.Definition and descriptionUnless stated otherwise, the following terms and phrases have the following definitions. A specificterm or phrase should not be considered as indefinite or unclear without specific definition and should beunderstood according to the normal meanings. A tradename used herein shall refer to the correspondingarticle or the active ingredient.The intermediate compounds herein can be prepared by various synthesis processes well-known to aperson skilled in the art, including the specific embodiments listed below, the embodiments by acombination with other chemical synthesis processes, and equivalent alternatives well known to a personskilled in the art. The preferable embodiments include but are not limited to the Examples below.The chemical reaction of the specific embodiments is performed in a suitable solvent, and the solventshould be suitable for the chemical changes of the present disclosure and the required reagents andmaterials. To obtain the compound of the present disclosure, a person skilled in the art can modify or selecta synthesis step or a reaction scheme based on the available embodiments.The present disclosure will be described in a detailed manner and the Examples should be notconsidered as limitation thereto.The solvents used herein are commercially available and can be used without further purification.The solvents used herein can be commercially available. The following abbreviations are used herein:EtOH represents ethanol; MeOH represents methanol; TFA represents trifluoroacetic acid; TsOH representsp-toluenesulfonic acid; mp represents melting point; EtSO3H represents ethanesulfonic acid; MeSO3Hrepresents methanesulfonic acid; THF represents tetrahydrofuran; EtOAc represents ethyl acetate.X-ray powder diffractometer (XRPD)Device:Testing method: about 10-20 mg of sample is used for XRPD detection.Detailed XRPD parameters are as follows:Radiation source: Cu, kα (Kα1=1.540598 Å, Kα2=1.544426 Å, Kα2 / Kα1 intensity ratio: 0.5)Light tube voltage: 45 kV, Light tube current: 40 mADivergence slit: fixed 1 / 8 deg1st soller slit: 0.04 rad2nd soller slit: 0.04 radReceiving slit: noneAnti-scatter slit: 7.5 mmMeasuring time: 5 minScanning angle range: 3-40 degStep width angle: 0.0263 degTime / step: 46.665 secSample plate speed: 15 rpmDifferential Scanning Calorimeter (DSC)Device: TA Q200 / Q2000 / 2500 Differential Scanning CalorimeterTesting method: The sample (about 1-5 mg) is placed in DSC aluminum plate for testing, under 50 mL / minN2, and is heated from 25°C (room temperature) until decomposition at the heating rate of 10 °C / min.Thermal Gravimetric Analyzer (TGA)Device: TA Q5000 / 5500 Thermal Gravimetric AnalyzerTesting method: The sample (about 1-5 mg) is placed in TGA aluminum plate for testing, under 10 mL / minN2, and is heated from room temperature to 350 °C at the heating rate of 10 °C / min.Brief Description of The DrawingsFigure 1 shows the XRPD pattern at Cu-Kα radiation of the Crystal Form A of the compound ofFormula (I);Figure 2 shows the XRPD pattern at Cu-Kα radiation of the Crystal Form B of the compound ofFormula (I);Figure 3 shows the DSC pattern of the Crystal Form B of the compound of Formula (I);Figure 4 shows the TGA pattern of the Crystal Form B of the compound of Formula (I).Detailed DescriptionFor better understanding of the present disclosure, further description will be provided below byreference to the specific examples, which are not any limitation thereto.Example 1: Preparation of the compound of formula (I)Step 1: Preparation of compound 1-SM1-24.0 L of dimethyl sulphoxide was added into a 50 L tank reactor at room temperature, to which were added1-SM1-A (1500.69 g, 8.39 mol), (R)-3-Methylmorpholine (854.97 g, 8.45 mol), potassium carbonate(2891g, 20.92mol) successively, and then 6.0 L of dimethyl sulphoxide was added again for dilution. Afteraddition, the reaction system was stirred at 95°C for 3 h. After detection of complete conversion to1-SM1-1, the temperature was lowered to 45°C and the tank reactor was purged with nitrogen for 5 minand then added with 1,4-dimethylpyrazole-5-boronic acid pinacol ester (1952.44 g, 8.79 mol),Tetrakis(triphenylphosphine)palladium (192.98 g, 0.167 mol). After addition, 2.0 L of dimethyl sulphoxidewas added to rinse the inner wall, and then stirring was performed for 12 h at 104°C under nitrogenatmosphere. After the reaction was completed, the temperature was lowered to 40°C, and the reactionsystem was filtered. The filter cake was rinsed with 20.0 L of ethyl acetate, and the filtrate was poured intothe reactor. 15.0 L of water was added into the reactor, and the reaction system was stirred for 2 min andallowed to stand for liquid separation. The aqueous phase was extracted again with 10.0 L of ethyl acetate,and the organic phases were combined, washed with water (10.0 L), saturated brine (8.0 L*2), respectively.The organic phases were concentrated to give the crude Compound 1-SM1-2 directly for the next reaction.MS m / z: 304.0[M+H]+Step 2: Preparation of compound 1-SM1Hydrogen chloride (1344.0 g, 36.82 mol) was passed into 6.0 L of 1,4-dioxane at -40°C for further use. 5.0L of 1,4-dioxane was added into a 50 L tank reactor. The crude product 1-SM1-2 obtained by concentrationwas dissolved in 5.0 L of 1,4-dioxane and added into the reactor, to which was added 15.0 L of 1,4-dioxanefor dilution under stirring. The temperature was raised to 70°C, and to the reaction liquid was slowly addedthe above-mentioned hydrochloric acid / 1,4-dioxane (1344 g, 6.0 L) and reaction was performed at 98°C for15 h. The temperature was lowered to 40°C and the reaction system was filtered. The filter cake was rinsedwith 15.0 L of ethyl acetate, and the solid was poured into a tank reactor and slurried with 15.0 L of ethylacetate for 30 min. The reaction system was filtered, and the filter cake was rinsed with 5.0 L of ethylacetate. The solid was dried in a vacuum drying cabinet to give Compound 1-SM1.MS m / z: 290.1[M+H]+1H NMR (400 MHz, DMSO-d6) δ ppm 1.34 (br d, J=6.52 Hz, 3 H) 2.08 (s, 3 H) 3.47 - 3.58 (m, 2 H) 3.64 -3.70 (m, 1 H) 3.72 - 3.78 (m, 1 H) 3.87 (s, 3 H) 3.97 (br s, 1 H) 4.07 - 4.32 (m, 1 H) 4.47 (br s, 1 H) 6.61 (s,1 H) 7.44 (s, 1 H)Step 3: Preparation of compound 1-SM2To a solution of compound 1-SM2-1 (2 g, 7.87 mmol), Bis(pinacolato)diboron (4.00 g, 15.74 mmol) and[1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.3 g, 410.00 μmol) in 1,4-dioxane (25.0mL) was added potassium acetate (2.32 g, 23.61 mmol) and the reaction system was purged with nitrogenfor three times. The reaction system was heated at 100°C with stirring for 8 h and filtered. The solution wasconcentrated to give the crude product which was separated with column chromatography to giveCompound 1-SM2.MS-ESI m / z: 302.1 [M+H]+.Step 4: Preparation of compound 1-1At room temperature, 15.0 L of toluene was added into a 50 L tank reactor, and compound 1-SM1 (1500.0g, 4.23 mol), triethylamine (1175.0 ml, 8.45 mol) were added successively, and phosphorus oxychloride(1178.0ml, 12.68mol) was added in portions under stirring. After addition, the reaction solution was stirredat 103-108°C for 1 h and 40 min. After detection of completion of the reaction, the temperature waslowered to 45°C. The reaction solution was transferred to a temporary storage tank and 15.0 L of purifiedwater was added into the tank reactor. Under stirring, the reaction solution was added into the purifiedwater in portions, with the temperature controlled at 20-40 °C. After addition, the pH was adjusted to 6-7with 12.0 L of aqueous sodium hydroxide solution (4M) and the temperature was controlled at 20-40°C.After adjusting the pH, 7.5 L of ethyl acetate was added into the tank reactor with stirring evenly forlayering. The water phase was extracted with 15.0 L of ethyl acetate. The organic phases were combined,washed with 12.0 L of saturated brine, concentrated under reduced pressure to no fraction to give the crudeproduct. The crude product was dissolved with 1.5 L of methyl tertiary butyl ether, to which was added12.0 L of n-heptane in portions with stirring, and the mixture was stirred for 5 min and filtered. The filtercake was rinsed with 5.0 L of n-heptane, and the solid was placed in a tray and allowed to be driedspontaneously to give Compound 1-1.MS m / z: 308.0[M+H]+1H NMR (400 MHz, DMSO-d6) δ ppm 1.29 (br d, J=6.78 Hz, 3 H) 2.17 (s, 3 H) 3.24 - 3.32 (m, 1 H) 3.52(br s, 1 H) 3.63 - 3.69 (m, 1 H) 3.78 (br d, J=11.54 Hz, 1 H) 3.96 (s, 3 H) 4.01 (br s, 1 H) 4.14 (br s, 1 H)4.47 (br s, 1 H) 6.89 (s, 1 H) 7.42 (s, 1 H)Step 5: Preparation of compound 1-2At 20-30°C under nitrogen protection, 2.1 L of dimethyl sulfoxide was added into a 10 L glass kettlewith stirring, to which were added Compound 1-1 (0.21 kg), Compound 1-SM2 (0.306 kg), aqueoussodium carbonate solution (1.3 M, 1.05 L), [1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium (II)(0.00749 kg) successively with an addition funnel. The reaction system was heated to an internaltemperature of 60-70°C, and the temperature was maintained for 4-16 h. The system was cooled to 40-45°C,to which was added dropwise 5.25 L of water within 30 min, and then stirred for 30 min. The system wasfiltered with suction and the filter cake was washed with 2.1 L of water. Drying under vacuum at 45°C gavethe crude product. To the crude product obtained in the previous stage was added 2.625 L of ethyl acetate.After stirred evenly to dissolution, 10.5 L of methyl tertiary butyl ether was added again with furtherstirring for 30 min. After filtration with Buchner funnel covered with celite, the celite layer was washedagain with a mixed solution of 2.1 L of ethyl acetate and methyl tertiary butyl ether (volume ratio 1:4). Thefiltrates were combined, and the organic phase was concentrated to give a concentrate. The upper blackfilter cake of celite was collected, to which was added 1.5 L of ethyl acetate. After stirring at roomtemperature for 1 h, filtration was performed with a Buchner funnel covered with celite, the filter cake waswashed with 0.5 L of ethyl acetate, and the filtrate was concentrated. The concentrates obtained in previoustwo stages were combined.To the previously obtained concentrate was added 1.5 L of ethyl acetate for dissolution, and thenslowly added dropwise to 4.5 L solution of n-heptane under stirring (1.5 h), and then further stirred for 2 h.The system was filtered, and the filter cake was washed with a mixed solution of 0.4 L of ethyl acetate andn-heptane (volume ratio 1: 3). After drying under vacuum, the filter cake was added into a 2 L one-neckflask, to which was added 0.8 L of isopropyl acetate. After reflux for 4 h, the reaction system was slowlycooled to room temperature and stirred overnight. The reaction system was filtered with a Buchner funnel,and the filter cake was washed with 0.3 L of isopropyl acetate. The solid was collected and dried undervacuum to give the product.The product was dissolved in 4.2 L of ethyl acetate, and 42 g of activated carbon was added understirring. The reaction system was stirred overnight under reflux, filtered while hot, and filtered through aBuchner funnel covered with celite. The celite layer was washed again with 2.0 L of ethyl acetate and thefiltrates were combined. The organic phase was concentrated to 3.0 L. 1.2 L of ethyl acetate and 43 g ofactivated carbon were added to the above organic phase, which was stirred under reflux for 8 h and thenfiltered while hot. The reaction system was filtered through a Buchner funnel covered with celite. The celitelayer was washed with 2.0 L of ethyl acetate, and filtrates were combined. The organic phase wasconcentrated and dried under vacuum to give Compound 1-2.MS m / z: 447.0 [M+H]+1H NMR (CHCl3-d, 400MHz): δ = 8.98 (d, J=1.3 Hz, 1H), 8.57 (br s, 1H), 8.27 (s, 1H), 7.58 (d, J=2.0 Hz,1H), 7.49 (t, J=2.8 Hz, 1H), 7.42 (s, 1H), 6.50 (s, 1H), 4.48 (br s, 1H), 4.29 (br d, J=12.5 Hz, 1H), 4.17 (s,3H), 4.13 (dd, J=11.9, 2.9 Hz, 1H), 3.98 (s, 3H), 3.87-3.93 (m, 1H), 3.80-3.87 (m, 1H), 3.69 (td, J=11.9, 3.0Hz, 1H), 3.44 (td, J=12.8, 3.8 Hz, 1H), 2.25 (s, 3H), 1.44 ppm (d, J=7.0 Hz, 3H)Step 6: Preparation of compound of formula (1)At 20°C, lithium aluminum hydride (63.0 mL, 2.5M) was added to Compound 1-2 (35.0 g, 78.39mmol) in tetrahydrofuran (50.0 mL), and the reaction system was stirred at 20°C for 1 h. At 0-5°C, 6.9 mLof water, 6.9 mL of 15% sodium hydroxide and 20.7 mL of water were slowly added into the reactionsolution successively, which was then filtered. The filtrate was concentrated to give the crude product,which was separated by column chromatography (ethyl acetate / petroleum ether: 50-100%) to give theproduct. The above product was dissolved in 20.0 mL of dimethyl sulfoxide at room temperature, slowlyadded dropwise into 400 mL of stirred water , filtered and dried to give the compound of formula (I).MS m / z: 419.1 [M+H]+1H NMR (400 MHz, CHCl3-d) δ ppm 8.39 (br s, 1 H), 8.28 (s, 1 H), 7.58 (s, 1 H), 7.51 (br s, 1 H), 7.42 (s,1 H), 7.35 (t, J=2.76 Hz, 1 H), 6.49 (s, 1 H), 4.89 (s, 2 H), 4.51 (br s, 1 H), 4.30 (br d, J=14.05 Hz, 1 H),4.11 - 4.18 (m, 4 H), 3.81 - 3.94 (m, 2 H), 3.70 (td, J=11.86, 3.14 Hz, 1 H), 3.44 (td, J=12.86, 3.89 Hz, 1 H),2.26 (s, 3 H), 1.45 (d, J=6.78 Hz, 3 H)Example 2: Preparation of Crystal Form A of the compound of formula (1)About 500.0 mg of the compound of formula (I) was weighed and dissolved in 5 mL of ethanol, towhich was added dropwise 15 mL of purified water. After addition, the reaction system was placed on amagnetic stirrer (20°C) and stirred for 120 h. The suspension was filtered to give a solid, which was driedovernight in a vacuum drying cabinet to give Crystal Form A of the compound of formula (I).1H NMR (400MHz, CHCl3-d) δ = 8.39 (br s, 1H), 8.25 (d, J=1.3 Hz, 1H), 7.53 (s, 1H), 7.49 (t, J=2.3 Hz,1H), 7.40 (s, 1H), 7.31 (t, J=2.8 Hz, 1H), 6.46 (s, 1H), 4.86 (s, 2H), 4.48 (br d, J=4.8 Hz, 1H), 4.28 (br d,J=12.5 Hz, 1H), 4.15 - 4.07 (m, 4H), 3.91 - 3.86 (m, 1H), 3.84 - 3.79 (m, 1H), 3.67 (dt, J=3.0, 11.9 Hz, 1H),3.42 (dt, J=3.9, 12.9 Hz, 1H), 2.23 (s, 3H), 1.42 (d, J=7.0 Hz, 3H)Example 3: Preparation of Crystal Form B of the compound of formula (1)About 100 mg of the compound of formula (1) were added into different glass bottles, to which wereadded appropriate amounts of organic solvents or solvent mixtures (Table 3). The above samples werestirred in a constant temperature mixer (40°C) (see Table 3 for stirring time) (protection from light). Then,the solid was filtered and placed in a vacuum drying cabinet (40°C) for drying overnight. All the treatmentsgave Crystal Form B.Table 3. Various appropriate amounts of organic solvents and stirring timeExample 4: Preparation of Crystal Form B of the compound of formula (1)The compound of formula (1) (see Table 4 for mass) was slowly added into methanol solvent at(60-70°C) (see Table 4 for volume), and then water was slowly added (see Table 4 for volume). Afterstirring for 0.5 h at 60°C, the temperature was lowered to 55°C and stirred for 0.5 h. Then, the temperaturewas lowered to 50°C and stirred for 0.5 h. Then, the temperature was lowered to 45°C and stirred for 0.5 h.Then, the temperature was lowered to 40°C and stirred for 0.5 h. Then, the temperature was lowered to35°C and stirred for 0.5 h. Then, the temperature was lowered to 30°C and stirred for 0.5 h. Then, thetemperature was lowered to 20-25°C and stirred for 10 h. The solid was filtered to give Crystal Form B.Table 4. Various appropriate amounts of organic solvents and stirring timeExample 5: Preparation of Crystal Form B of the compound of formula (1)Experimental procedure: About 5.5 g of the compound of formula (1) was slowly added into 50 mL ofmethanol solvent at (60-70°C). After stirring at 60°C for 0.5 h, the temperature was lowered to 25°C andstirred for 2 h, and the solid was filtered to give Crystal Form B.Example 6: Preparation of Crystal Form B of the compound of formula (1)900.0 g of the compound of formula (I) was dissolved in 9.0 L of methanol, to which was addedslowly dropwise 9.0 L of purified water at room temperature (25°C). The reaction system was furtherstirred for 20 h and filtered under reduced pressure. The filter cake was washed with 6.0 L of purified waterand the solid was dried under vacuum to give Crystal Form B of compound of formula (I).MS m / z: 419.0 [M+H]+1H NMR (CHCl3-d, 400MHz): δ = 8.60 (br s, 1H), 8.21 (s, 1H), 7.45 (br s, 1H), 7.42 (br s, 1H), 7.40 (s,1H), 7.25 (br d, J=2.5 Hz, 1H), 6.45 (s, 1H), 4.81 (br s, 2H), 4.47 (br d, J=5.8 Hz, 1H), 4.27 (br d, J=13.8Hz, 1H), 4.07-4.13 (m, 4H), 3.85-3.91 (m, 1H), 3.78-3.84 (m, 1H), 3.66 (td, J=11.9, 3.0 Hz, 1H), 3.41 (td,J=12.8, 3.8 Hz, 1H), 2.22 (s, 3H), 1.41 ppm (d, J=6.8 Hz, 3H)Example 7: Solid stability tests of Crystal Form A under high temperature and high humidityconditionsTwo samples of Crystal Form A were weighed in parallel, each about 100 mg, placed on the bottom ofa glass sample bottle and spread into a thin layer. The samples were sealed with aluminum foil paper, andsome small holes were pierced in the aluminum foil paper to ensure that the sample can fully contact withthe ambient air. The samples were placed at a constant temperature and humidity box under the condition of40°C / 75% humidity. The samples under the above conditions were sampled and tested on day 30, and thetest results were compared with the initial test result on day 0. The test results were shown in Table 5below:Table 5: Solid stability test of Crystal Form AConclusion: Crystal Form A of the compound of formula (I) has good stability and is easy formedicine manufacture.Example 8: Solid physical stability tests of Crystal Form A under different temperature, humidityand light conditionsFour samples of Crystal Form A were weighed in parallel, each about 100 mg, placed on the bottom ofa glass sample bottle and spread into a thin layer. The samples were sealed with aluminum foil paper, andsome small holes were pierced in the aluminum foil paper to ensure that the sample can fully contact withthe ambient air. Four prepared samples were placed in 25°C / 92.5% relative humidity, 60°C, 40°C / 75% andlight conditions, respectively, and the physical stability of the samples on the 10th day was investigated. Atthe same time, a sample of about 100 mg of Crystal Form A was weighed separately, placed on the bottomof the glass sample bottle, sealed with a screw cap, and stored at -20°C for use as control. On the 10th day,all the samples were taken out, returned to room temperature, and the appearance changes of the sampleswere observed. XRPD was used to detect the sample crystal forms. By comparing the accelerated sampleswith the control sample, the solid physical stability of Crystal Form A of the compound of formula (I) wasdetermined. The following Table 6 showed the results of the solid physical stability experiment of CrystalForm A.Table 6 Solid physical stability tests of Crystal Form A under different temperature and humidityconditions and under lightConclusion: Crystal Form A of the compound of formula (I) has good stability and is easy formedicine manufacture.Example 9: Solid physical stability tests of Crystal Form B under high temperature, high humidityand light conditionsTwo samples of Crystal Form B were weighed in parallel for each group, placed on the bottom of aglass sample bottle and spread into a thin layer. The samples were sealed with aluminum foil paper, andsome small holes were pierced in the aluminum foil paper to ensure that the sample can fully contact withthe ambient air. The samples were placed in a constant temperature and humidity or light box underdifferent humidity conditions. The samples placed under the above conditions were sampled and tested onday 5, day 10, day 30, 1 month, 3 month or 6 month. The test results were compared with the initial testresults on day 0. The test results were shown in Table 7-11 below:Table 7. Solid high temperature stability test of Crystal Form B at 60°CConclusion: Crystal Form B of the compound of formula (I) has good high temperature stability and iseasy for medicine manufacture.Table 8. Solid high humidity stability test of Crystal Form B at 25°C / 92.5% RHConclusion: Crystal Form B of the compound of formula (I) has good high humidity stability and iseasy for medicine manufacture.Table 9. Solid light stability test of Crystal Form BConclusion: Crystal Form B of the compound of formula (I) has good light stability.Table 10. Solid stability test of Crystal Form B at 40°C / 75% RHConclusion: Crystal Form B of the compound of formula (I) has good stability and is easy formedicine manufacture.Table 11. Solid stability test of Crystal Form B at 25°C / 65% RHConclusion: Crystal Form B of the compound of formula (I) has good stability and is easy formedicine manufacture.Experimental Example 1: In vitro evaluationIC50 values were determined to evaluate the inhibitory activity of the test compound on human ATRkinase.ATR / ATRIP(h) was incubated in an assay buffer containing 50 nM GST-cMyc-p53 and Mg / ATP(concentration as required). The reaction was initiated by adding a Mg / ATP mixture. After incubation for30 min at room temperature, a stop solution containing EDTA was added to terminate the reaction. Finally,the detecting buffer containing the d2-labeled anti-GST monoclonal antibody and the europium-labeledanti-phospho Ser15 antibody against phosphorylated p53 were added. Then, the plate in time-resolvedfluorescence mode was read and homogeneous time resolution was performed.The fluorescence (HTRF) signal was determined according to the formula: HTRF = HTRF =10000x(Em665 nm / Em620 nm).XLFit version 5.3 (ID Business Solutions) was used to analyze IC50 data. Nonlinear regressionanalysis was used to fit the S-shaped dose response (variable slope) curve. The test result was shown inTable 12:Table 12: In vitro screening test result of the present compoundConclusion: The present compound of formula (I) has a good inhibitory effect on kinase ATR.Experimental Example 2: In vitro cell viability testThe effect of the compound on inhibiting cell proliferation was investigated in this test by detectingthe effect of the compound on cell activity in vitro in tumor cell lines LoVo.CellTiter-Glo cell viability detection by luminescence methodThe following steps were carried out in accordance with the instructions of the Promega CellTiter-GloLuminescence Cell Viability Detection Kit (Promega-G7573).(1). The CellTiter-Glo buffer was melted and allowed to room temperature.(2). The CellTiter-Glo substrate was allowed to room temperature.(3). CellTiter-Glo buffer was added to a bottle of CellTiter-Glo substrate to dissolve the substrate toprepare CellTiter-Glo working solution(4). Vortex was performed slowly for complete dissolution.(5). The cell culture plate was taken out and placed for 30 min to equilibrate to room temperature.(6). 50 μL (half volume of cell culture medium in each well) of CellTiter-Glo working solution wasadded to each well. The cell plate was wrapped with aluminum foil for protection from light.(7). The culture plate was shaken on an orbital shaker for 2 min to induce cell lysis.(8). The culture plate was allowed to stand at room temperature for 10 min to stabilize theluminescence signal.(9). The luminescence signal was detected on the SpectraMax i3x of Molecular Devices plate reader.Data AnalysisThe following formula was used to calculate the inhibition rate (IR) of the test compound: IR (%) = (1- (RLU compound - RLU blank control) / (RLU vehicle control - RLU blank control))*100%.The inhibition rates of different concentrations of compounds were calculated in Excel, and thenGraphPad Prism software was used to draw the inhibition curve and calculate the relevant parameters,including the minimum inhibition rate, the maximum inhibition rate and IC50.The test results were shown in Table 13:Table 13 In vitro LoVo cell proliferation inhibition test resultsConclusion: The present compound of formula (1) TR has good inhibitory effect on LoVo tumor cellswith mutation in the ATM signaling pathway.Experimental Example 3: In vivo pharmacokinetic properties studyTest samples: On the basis of the above tests, some of these compounds with high activity andrepresentative structures were selected for further tests.Experimental procedure: The purpose of this study is to determine the pharmacokinetic parameters ofthe compound and calculate its gavage bioavailability in female Balb / c Nude mice.Six female Balb / c Nude mice were used in this project, three mice were injected intravenously, withthe dosage of 1 mg / kg, where plasma samples at 0 h (before administration) and at 0.0833, 0.25, 0.5, 1, 2, 4,6, 8 and 24 h after administration were collected, and the other three mice were administeredintragastrically with the dose of 10 mg / kg or 25 mg / kg, where plasma samples at 0 h (before administration)and at 0.5, 1, 2, 3, 4, 6, 8, 24 h after administration were collected. LC-MS / MS analysis was performed onthe collected samples and data were collected. The collected analysis data were calculated with PhoenixWinNonlin 6.2.1 software for relevant pharmacokinetic parameters. The test results were shown in Table14.1 and 14.214.1 Results of intravenous injection administrationNote: C0 (nM) is the concentration of the drug in the body at 0 min; Cl (mL / min / kg) is the clearancerate of the drug in the body; Vdss (L / kg) is the volume of distribution of the drug in the body; T1 / 2 (h) is thehalf-life; AUC0-t (nM.h) is the amount of drug exposure in the body; Cmax (nM) is the highest concentrationof the drug in the body; F is the bioavailability.Conclusion: the present compound of formula (1) has good absorption and exposure in intragastricadministration and is suitable for oral administration.Experimental Example 4: Colorectal cancer LoVo CDX in vivo efficacy studyPurpose: LoVo is a colorectal adenocarcinoma tumor cell with MRE11A mutation (MRE11A is a keycomponent of the ATM signaling pathway for DNA double-strand break repair), which is sensitive to ATRinhibitor. This test use the LoVo CDX model of rectal cancer to verify the inhibitory effect of ATR inhibitoron a tumor with defective ATM signaling pathway.Experimental procedure:1. Experimental AnimalsSpecies: MouseStrain: BALB / c nude miceSupplier: Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.Week age and weight: 6-8 weeks old, 18-22 gGender: female2. Cell CultureHuman colon cancer LoVo cells (ECACC, Catalog: 87060101), in vitro monolayer culture, cultureconditions were Ham's F-12 medium with 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mLstreptomycin and 2 mM glutamine, 37°C, 5% CO2 culture.Pancreatin-EDTA was used for routine digestion and passage twice a week. When the cell saturationwas 80%-90%, the cells were collected, counted, and seeded. 0.1 mL (10 x 106) of LoVo cells weresubcutaneously inoculated on the right back of each nude mouse, and group administration was initiatedwhen the average tumor volume reached 173 mm3.3. Preparation of test substance and dosage25.51 mg of compound of formula (1) was weighed and dissolved in 0.500 mL of DMSO. 2.000 mLof propylene glycol and 2.500 mL of deionized water were added, which was vortexed and mixed well andadjusted to pH=6.0 to give a clear solution.Dosage: 25 mg / kg of all test compounds were administered by gavage twice a day, with an interval of8 h within a day.4. Tumor measurement and experimental indicatorsThe diameter of the tumor was measured with a vernier caliper twice a week. The calculation formulafor the tumor volume: V = 0.5a x b2, where a and b represent the long diameter and short diameter of thetumor, respectively.The anti-tumor efficacy of the compound was evaluated by TGI (%) or relative tumor proliferationrate T / C (%).Relative tumor proliferation rate T / C (%) = TRTV / CRTV x 100 % (TRTV: average RTV of thetreatment group; CRTV: average RTV of the negative control group). According to the results of tumormeasurement, the relative tumor volume (RTV) was calculated, and the calculation formula was RTV = Vt / V0, where V0 is the tumor volume measured at the time of group administration (i.e., D0), Vt is the tumorvolume at a certain measurement, and TRTV and CRTV use the data at the same day.TGI (%) represents the tumor growth inhibition rate. TGI (%) = [1-(average tumor volume at the endof a certain treatment group - average tumor volume at the beginning of the treatment group) / (averagetumor volume at the end of treatment in the solvent control group - average tumor volume at the beginningof treatment in the solvent control group)]x100%.After the experiment was completed, the tumor weight was detected and the percentage of T / C weightwas calculated, where T weight and C weight represent the tumor weight of the administration group andthe vehicle control group, respectively.5. Test resultsThe efficacy of the compound in the human colorectal cancer xenograft tumor model was evaluated inthis test, with the solvent control group as the reference. At day 17 of administration, the compound offormula (1) (25 mg / kg) group had T / C and TGI of 27.8% and 90.7%, respectively, as compared to thevehicle control group6. ConclusionIn this experiment, the present compound of formula (1) shows inhibitory effect on the growth ofhuman colorectal cancer LoVo cell subcutaneous xenograft tumor-bearing mice.

Claims

1. A Crystal Form A of the compound of formula (I), wherein, the Crystal Form A has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 8.10±0.20°, 18.33±0.20° and 22.63±0.20°. (I)2. The Crystal Form A according to claim 1, wherein, the Crystal Form A has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 7.46±0.20°, 8.10±0.20°, 13.03±0.20°, 15.07±0.20°, 15.58±0.20°, 16.19±0.20°, 18.33±0.20° and 22.63 ±0.20°.

3. The Crystal Form A according to claim 2, wherein, the Crystal Form A has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 7.46±0.20°, 8.10±0.20°, 13.03±0.20°, 13.46±0.20°, 15.07±0.20°, 15.58±0.20°, 16.19±0.20°, 18.33±0.20°, 21.17±0.20° and 22.63 ±0.20°.

4. The Crystal Form A according to claim 3, wherein, the Crystal Form A has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 7.46°, 8.10°, 11.24°, 13.03°, 13.46°, 15.07°, 15.58°, 15.98°, 16.19°, 17.70°, 18.33°, 19.60°, 21.17°, 22.63°, 23.84°, 25.56° and 26.57°.

5. The Crystal Form A according to claim 4, wherein, the Crystal Form A has an XRPD pattern as shown in Figure 1.

6. A Crystal Form B of the compound of formula (I), wherein, the Crystal Form B has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 8.45±0.20°, 10.87±0.20° and 20.56±0.20°.

7. The Crystal Form B according to claim 6, wherein, the Crystal Form B has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 8.45±0.20°, 10.87±0.20°, 14.83±0.20°, 15.54±0.20°, 17.33±0.20°, 20.56±0.20°, 22.00±0.20° and 22.63±0.20°.

8. The Crystal Form B according to claim 7, wherein, the Crystal Form B has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 8.45±0.20°, 10.87±0.20°, 14.83±0.20°, 15.54±0.20°, 17.33±0.20°, 20.08±0.20°, 20.56±0.20°, 22.00±0.20°, 22.63±0.20° and 25.26±0.20°.

9. The Crystal Form B according to claim 8, wherein, the Crystal Form B has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 8.45°, 9.20°, 10.87°, 12.57°, 14.14°, 14.53°, 14.83°, 15.54°, 16.80°, 17.33°, 18.43°, 19.84°, 20.08°, 20.56°, 21.39°, 22.00°, 22.44°, 22.63°, 23.26°, 25.26°, 25.85° and 26.98°.

10. The Crystal Form B according to claim 9, wherein, the Crystal Form B has an XRPD pattern as shown in Figure 2.

11. The Crystal Form B according to any one of claims 6-10, wherein, the Crystal Form B has a Differential Scanning Calorimetry curve (DSC) having one onset point of endothermic peak at 174.3±3°C.

12. The Crystal Form B according to claim 11, wherein, the Crystal Form B has a DSC pattern as shown in Figure 3.

13. The Crystal Form B according to any one of claims 6-10, wherein, the Crystal Form B has a Thermogravimetric Analysis curve (TGA), wherein the weight loss at 150°C±3°C is 1.49%.

14. The Crystal Form B according to claim 13, wherein, the Crystal Form B has a TGA pattern as shown in Figure 4.

15. A process for preparing a Crystal Form A of a compound of formula (I), comprising: 1) adding the compound of formula (I) into ethanol solvent; 2) adding water; 3) stirring for 100-120 h; 4) performing recrystallization at room temperature to obtain the Crystal Form A.

16. A process for preparing a Crystal Form B of a compound of formula (I), comprising: 1) adding the compound of formula (I) into a solvent; 2) heating to a temperature with stirring for 2.5-120 h; 3) performing recrystallization at room temperature to obtain the Crystal Form B.

17. The preparing process according to claim 16, wherein the solvent is methanol, methyl tertiary butyl ether, methanol / water (V / V, 1:0.3-1), acetone / water (V / V, 1:1), isopropanol / water (V / V, 1:1), ethyl acetate / n-heptane (V / V, 1:1), isopropyl acetate / n-heptane (V / V, 1:1), ethanol / n-heptane (V / V, 1:1), acetonitrile / n-heptane (V / V, 1:1), isopropanol / n-heptane (V / V, 1:1) or dichloromethane / n-heptane(V / V, 1:1).

18. The preparing process according to claim 17, wherein the temperature is 25-70°C.

19. The preparing process according to claim 16, wherein the concentration range of the compound of formula (I) is selected from the group consisting of 25 mg / mL-50 mg / mL.