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

A crystal form of an ATR inhibitor and its application

[0001] This application claims the following priority:

[0002] CN201910722102.7, application date: 2019.08.06. Technical Field

[0003] This invention relates to a crystal form of an ATR inhibitor and a method for preparing the same, as well as its application in the preparation of medicaments for treating ATR-related diseases. Background Technology

[0004] ATR (capillary dilatation ataxia mutation and RAD-3-related protein kinase) belongs to the PIKK (phosphatidylinositol-3-kinase-associated kinase) family and participates in DNA damage repair to maintain gene stability. ATR protein kinases produce synergistic responses to DNA damage, replication stress, and cell cycle disruption. ATR and ATM both belong to the PIKK family of serine / threonine protein kinases; they are common components of cell cycle and DNA damage repair. Other PIKK families include Chkl, BRC1A, and p53. ATR is primarily responsible for repairing DNA replication stress (replication fork arrest) and single-strand breaks.

[0005] When DNA double-strand breaks occur, resulting in excision or replication fork arrest, the ATR is activated by single-stranded DNA structures. DNA polymerase remains in the DNA replication process, while replication helicase continues to unwind at the tip of the replication fork, leading to the production of long single-stranded DNA (ssDNA), which then binds to RPA (replication protein A). During replication stress or DNA damage, the ATR / ATR-acting protein complex recruited by RPA to the damage site, where the RPA-ssDNA complex activates the RAD17 / rfc2-5 complex, which binds to the damage site. The Rad9-HUS1-RAD1 (9-1-1) heterotrimer is activated at the DNA-ssDNA junction, and 9-1-1, in turn, recruits TopBP1 to activate the ATR. Once activated, the ATR promotes DNA repair, stabilizes and restarts stalled replication forks, and induces transient cell cycle arrest through downstream targets. These functions are achieved by the ATR mediating its downstream target, Chk1. The ATR acts as a cell cycle checkpoint for DNA damage in S phase. It can delay DNA replication by mediating the degradation of CDC25A through Chk1, thus providing time for the repair of replication forks. ATR is also a major regulator of the G2 / M cell cycle checkpoint, preventing premature mitosis before DNA replication is complete or DNA damage occurs. This ATR-dependent G2 / M cell cycle arrest is mainly mediated through two mechanisms: 1. Degradation of CDC25A. 2. Phosphorylation of Cdc25C by Chk1, causing it to bind to the 14-3-protein. The binding of Cdc25C to the 14-3-3 protein promotes its export from the nucleus and cytoplasmic isolation, thereby inhibiting its ability to dephosphorylate and activate nuclear Cdc2, which in turn prevents entry into mitosis.

[0006] Mutations in the ATR gene are extremely rare, occurring only in a small minority of patients with Seckel syndrome, characterized by developmental delay and microcephaly. Disruptions in ATR-related pathways lead to genomic instability, and the ATR protein is activated by most cancer chemotherapy therapies. Furthermore, duplication of the ATR gene has been described as a risk factor for rhabdomyosarcoma.

[0007] ATR is essential for cell self-replication and is activated in the S phase to regulate the origin of replication and repair damaged replication forks. Damage to replication forks can increase the sensitivity of cancer cells to platinum-based and hydroxyurea-based anticancer drugs, and reduce their drug resistance. Therefore, inhibiting ATR may be an effective approach in future cancer treatment.

[0008] Summary of the Invention

[0009] The present invention provides an X-ray powder diffraction pattern of the A crystal form of the compound of formula (Ⅰ) with characteristic diffraction peaks at the following 2θ angles: 8.10±0.20°, 18.33±0.20° and 22.63±0.20°.

[0010]

[0011] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A-type crystal has 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°.

[0012] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A-type crystal has 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°.

[0013] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A-type crystal has 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°.

[0014] In some embodiments of the present invention, the XRPD pattern of the above-mentioned A crystal form is shown in Figure 1.

[0015] In some embodiments of the present invention, the XRPD spectra analysis data of the above-mentioned A-type crystal form are shown in Table 1:

[0016] Table 1. XRPD pattern analysis data for crystal form A

[0017]

[0018]

[0019] The present invention provides an X-ray powder diffraction pattern of the B crystal form of the compound of formula (I) with characteristic diffraction peaks at the following 2θ angles: 8.45±0.20°, 10.87±0.20° and 20.56±0.20°.

[0020] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has 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°.

[0021] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has 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°.

[0022] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has 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°.

[0023] In some embodiments of the present invention, the XRPD pattern of the B crystal form is shown in Figure 2.

[0024] In some embodiments of the present invention, the XRPD spectra analysis data of the above-mentioned B crystal form are shown in Table 2:

[0025] Table 2. XRPD pattern analysis data of crystal form B

[0026]

[0027]

[0028] In some embodiments of the present invention, the differential scanning calorimetry (DSC) curve of the B crystal form has an endothermic peak starting point at 174.3±3℃.

[0029] In some embodiments of the present invention, the DSC spectrum of the B crystal form is shown in Figure 3.

[0030] In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned B crystal form shows a weight loss of 1.49% at 150℃±3℃.

[0031] In some embodiments of the present invention, the TGA spectrum of the B crystal form is shown in Figure 4.

[0032] This invention provides a method for preparing the crystal form of compound A of formula (Ⅰ), comprising:

[0033] 1) Add the compound of formula (Ⅰ) to an ethanol solvent;

[0034] 2) Add water again;

[0035] 3) Stir for 100–120 hours;

[0036] 4) Prepared by recrystallization at room temperature.

[0037] This invention provides a method for preparing the crystal form of compound B of formula (I), comprising:

[0038] 1) Add the compound of formula (Ⅰ) to the solvent;

[0039] 2) Heat to a certain temperature and stir for 2.5 to 120 hours;

[0040] 3) Recrystallization at room temperature yielded crystal form B.

[0041] In some embodiments of the present invention, the solvent is: methanol, methyl tert-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).

[0042] In some embodiments of the present invention, the temperature is 25–70°C.

[0043] This invention provides a method for preparing the crystal form of compound B of formula (I), comprising:

[0044] 1) Add the compound of formula (Ⅰ) to an alcohol solvent;

[0045] 2) Add water again;

[0046] 3) Stir for 15–20 hours;

[0047] 4) Prepared by recrystallization at room temperature.

[0048] In some embodiments of the present invention, the volume ratio of the above-mentioned alcohol solvent to water is 1:1 to 1:4.

[0049] In some embodiments of the present invention, the alcohol solvent is selected from methanol.

[0050] In some embodiments of the present invention, the concentration range of the compound of formula (I) is selected from 25 mg / mL to 50 mg / mL.

[0051] The present invention also provides the use of the compound of formula (I), the crystal form A, or the crystal form B above in the preparation of a medicament for treating ATR-related diseases.

[0052] In some embodiments of the present invention, the above-described application is characterized in that the drug is a drug for treating solid tumors or hematologic malignancies.

[0053] In some embodiments of the present invention, the above-described application is characterized in that the drug is used to treat colorectal cancer, gastric cancer, esophageal cancer, primary peritoneal cancer, adrenocortical carcinoma, clear cell renal cell carcinoma, prostate cancer, urothelial carcinoma of the bladder, ovarian cancer, breast cancer, endometrial cancer, fallopian tube cancer, non-small cell lung cancer, or small cell lung cancer.

[0054] Technical effect

[0055] The compound of formula (Ⅰ) of this invention has stable crystal forms A and B, is less affected by light, heat and humidity, and has good efficacy for in vivo administration, and has broad prospects for drug development.

[0056] Definitions and Explanations

[0057] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular phrase or term should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0058] The intermediate compounds of the present invention can be prepared by various synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention.

[0059] The chemical reactions in the specific embodiments of this invention are carried out in a suitable solvent, which must be suitable for the chemical changes of this invention and the reagents and materials required therefor. To obtain the compounds of this invention, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0060] The present invention will be described in detail below through embodiments, which are not intended to limit the present invention in any way.

[0061] All solvents used in this invention are commercially available and can be used without further purification.

[0062] The solvents used in this invention are commercially available. The following abbreviations are used in this invention: EtOH represents ethanol; MeOH represents methanol; TFA represents trifluoroacetic acid; TsOH represents p-toluenesulfonic acid; mp represents melting point; EtSO3H represents ethanesulfonic acid; MeSO3H represents methanesulfonic acid; THF represents tetrahydrofuran; and EtOAc represents ethyl acetate.

[0063] The present invention relates to an X-ray powder diffractometer (XRPD) method.

[0064] Instrument Model:

[0065] Test method: Approximately 10-20 mg of sample is used for XRPD detection.

[0066] The detailed XRPD parameters are as follows:

[0067] X-ray source: Cu,kα ( Kα2 / Kα1 intensity ratio: 0.5

[0068] Phototube voltage: 45kV, Phototube current: 40mA

[0069] Diverging slit: Fixed 1 / 8deg

[0070] First Sola Slit: 0.04 rad

[0071] Second Sola Slit: 0.04 rad

[0072] Receiving slit: None

[0073] Anti-scattering slit: 7.5mm

[0074] Measurement time: 5 min

[0075] Scanning angle range: 3-40 degrees

[0076] Step width angle: 0.0263deg

[0077] Step length: 46.665 seconds

[0078] Sample tray rotation speed: 15 rpm

[0079] This invention relates to a differential scanning calorimeter (DSC) method.

[0080] Instrument Model: TA Q200 / Q2000 / 2500 Differential Scanning Calorimeter

[0081] Test method: Take a sample (about 1-5 mg) and place it in a DSC aluminum dish for testing. Under 50 mL / min N2 conditions, heat the sample from 25 °C (room temperature) to before sample decomposition at a heating rate of 10 °C / min.

[0082] The present invention relates to a thermogravimetric analysis (TGA) method.

[0083] Instrument Model: TA Q5000 / 5500 Thermogravimetric Analyzer

[0084] Test method: Take a sample (about 1-5 mg) and place it in a TGA aluminum dish for testing. Under N2 conditions of 10 mL / min, heat the sample from room temperature to 350℃ at a heating rate of 10℃ / min. Attached Figure Description

[0085] Figure 1 shows the Cu-Kα radiation XRPD spectrum of compound A of formula (I);

[0086] Figure 2 shows the Cu-Kα radiation XRPD spectrum of compound B of formula (I);

[0087] Figure 3 shows the DSC spectrum of compound B of formula (I);

[0088] Figure 4 shows the TGA spectrum of compound B of formula (I). Detailed Implementation

[0089] To better understand the content of this invention, further explanation will be provided below with reference to specific embodiments. However, the specific implementation methods are not intended to limit the content of this invention.

[0090] Example 1: Preparation of compound (I)

[0091]

[0092] Step 1: Preparation of compound 1-SM1-2

[0093] At room temperature, 4.0 L of dimethyl sulfoxide (DMSO) was first added to a 50 L reactor. While stirring, 1-SM1-A (1500.69 g, 8.39 mol), (R)-3-methylmorpholine (854.97 g, 8.45 mol), and potassium carbonate (2891 g, 20.92 mol) were added sequentially. Then, 6.0 L of DMSO was added again for dilution. After the addition was complete, the reactor was stirred at 95 °C for 3 hours. Once the reaction was completely converted to 1-SM1-1, the temperature was lowered to 45 °C, and nitrogen gas was purged into the reactor for 5 minutes. Then, 1,4-dimethylpyrazole-5-phenanthrol borate (1952.44 g, 8.79 mol) and tetrakis(triphenylphosphine)palladium (192.98 g, 0.167 mol) were added. After the addition was complete, 2.0 L of DMSO was added to rinse the inner wall of the reactor, and the reactor was stirred at 104 °C under a nitrogen atmosphere for 12 hours. After the reaction was completed, the temperature was lowered to 40°C, filtered, and the filter cake was washed with 20.0 L of ethyl acetate. The filtrate was poured into a reactor, and 15.0 L of water was added. The mixture was stirred for 2 minutes and allowed to stand for separation. The aqueous phase was extracted again with 10.0 L of ethyl acetate. The organic phases were then combined and washed with water (10.0 L) and saturated brine (8.0 L * 2), respectively. The organic phase was concentrated to obtain crude compound 1-SM1-2, which was directly used in the next reaction.

[0094] MS m / z: 304.0 [M+H] +

[0095] Step 2: Preparation of compound 1-SM1

[0096] Hydrogen chloride (1344.0 g, 36.82 mol) was bubbled into 6.0 L of 1,4-dioxane at -40°C for later use. First, 5.0 L of 1,4-dioxane was added to a 50 L reactor. The concentrated crude product 1-SM1-2 was dissolved in 5.0 L of 1,4-dioxane and added to the reactor. While stirring, 15.0 L of 1,4-dioxane was added for dilution. The temperature was raised to 70°C, and the self-prepared hydrochloric acid / 1,4-dioxane (1344 g, 6.0 L) was slowly added to the reaction solution. The reaction was carried out at 98°C for 15 hours. The temperature was lowered to 40°C, and the mixture was filtered. The filter cake was washed with 15.0 L of ethyl acetate. The solid was poured into a reactor and slurried with 15.0 L of ethyl acetate for 30 minutes. The mixture was filtered, and the filter cake was washed with 5.0 L of ethyl acetate. The solid was dried in a vacuum drying oven to obtain compound 1-SM1.

[0097] MS m / z: 290.1 ​​[M+H] +

[0098] 1H NMR (400MHz, DMSO-d6) δppm 1.34 (br d, J=6.52Hz, 3H) 2.08 (s, 3H) 3.47-3.58 (m, 2H) 3.64-3.70 (m, 1H) 3.72-3.78 (m, 1H) 3.87 (s, 3H) 3.97 (br s,1H)4.07-4.32(m,1H)4.47(br s,1H)6.61(s,1H)7.44(s,1H)

[0099] Step 3: Preparation of compound 1-SM2

[0100]

[0101] Potassium acetate (2.32 g, 23.61 mmol) was added to a solution of compound 1-SM2-1 (2 g, 7.87 mmol), bis(diphenylphosphine)ferrocene palladium chloride (0.3 g, 410.00 μmol), and 1,4-dioxane (25.0 mL). The mixture was purged three times with nitrogen, and the reaction was heated and stirred at 100 °C for 8 hours. The solution was filtered, concentrated, and the crude product was obtained. The crude product was then separated by column chromatography to obtain compound 1-SM2.

[0102] MS-ESI m / z: 302.1 [M+H] + .

[0103] Step 4: Preparation of Compound 1-1

[0104]

[0105] At room temperature, 15.0 L of toluene was first added to a 50 L reactor. While stirring, compound 1-SM1 (1500.0 g, 4.23 mol), triethylamine (1175.0 ml, 8.45 mol), and phosphorus oxychloride (1178.0 ml, 12.68 mol) were added sequentially. After the additions were complete, the reaction mixture was stirred at 103–108 °C for 1 hour and 40 minutes. After the reaction was complete, the temperature was lowered to 45 °C, and the reaction mixture was transferred to a temporary storage container. 15.0 L of purified water was added to the reactor, and the reaction mixture was added in batches while stirring, maintaining the temperature at 20–40 °C. After the additions were complete, the pH was adjusted to 6–7 with 12.0 L of 4 M sodium hydroxide aqueous solution, maintaining the temperature at 20–40 °C. After pH adjustment, 7.5 L of ethyl acetate was added to the reactor and stirred until homogeneous. The phases separated, and the aqueous phase was extracted with 15.0 L of ethyl acetate. The combined organic phases were washed with 12.0 L of saturated brine. The organic phase was concentrated under reduced pressure until no fraction was obtained to give a crude product. The crude product was dissolved in 1.5 L of methyl tert-butyl ether, and 12.0 L of n-heptane was added in batches with stirring. After stirring for 5 minutes, the mixture was filtered. The filter cake was washed with 5.0 L of n-heptane and the filter cake was filtered again. The solid was placed in a tray and allowed to air dry to give compound 1-1.

[0106] MS m / z: 308.0 [M+H] +

[0107] 1 H NMR (400MHz, DMSO-d6) δppm 1.29 (br d, J=6.78Hz, 3H) 2.17 (s, 3H) 3.24-3.32 (m, 1H) 3.52 (br s, 1H) 3.63-3.69 (m, 1H) 3.78 (br d,J=11.54Hz,1H)3.96(s,3H)4.01(br s,1H)4.14(br s,1H)4.47(br s,1H)6.89(s,1H)7.42(s,1H)

[0108]

[0109] Step 5: Preparation of compounds 1-2

[0110] Under nitrogen protection at 20–30°C, 2.1 L of dimethyl sulfoxide was added to a 10 L glass reactor. While stirring, compound 1-1 (0.21 kg), compound 1-SM2 (0.306 kg), sodium carbonate aqueous solution (1.3 M, 1.05 L), and 1,1'-bis(diphenylphosphine)ferrocene palladium chloride (0.00749 kg) were added sequentially via a feeding funnel. The mixture was heated to an internal temperature of 60–70°C and maintained at this temperature for 4–16 hours. The system was then cooled to 40–45°C, and 5.25 L of water was added dropwise over 30 minutes. Stirring continued for another 30 minutes. The mixture was then filtered, and the filter cake was washed with 2.1 L of water. The crude product was obtained by vacuum drying at 45°C. 2.625 L of ethyl acetate was added to the crude product obtained in the first stage, and after stirring until dissolved, 10.5 L of methyl tert-butyl ether was added again, and stirring was continued for 30 minutes. The mixture was then filtered through a Buchner funnel lined with diatomaceous earth. The diatomaceous earth layer was washed again with a mixed solution of 2.1 L of ethyl acetate and methyl tert-butyl ether (volume ratio 1:4). The filtrates were combined, and the organic phase was concentrated to obtain a concentrate. The black filter cake on top of the diatomaceous earth was collected, and 1.5 L of ethyl acetate was added. After stirring at room temperature for 1 hour, the mixture was filtered through a Buchner funnel lined with diatomaceous earth. The filter cake was washed with 0.5 L of ethyl acetate, and the filtrate was concentrated. The concentrates obtained from the two separate reactions were combined.

[0111] The concentrate obtained in the first stage was dissolved in 1.5 L of ethyl acetate, and then slowly added dropwise to 4.5 L of n-heptane solution under stirring (1.5 h). Stirring was continued for 2 h, and the mixture was filtered. The filter cake was washed with a mixture of 0.4 L of ethyl acetate and n-heptane (volume ratio 1:3). After vacuum drying, the filter cake was added to a 2 L single-necked flask along with 0.8 L of isopropyl acetate. The mixture was refluxed for 4 h, then slowly cooled to room temperature and stirred overnight. The mixture was filtered using a Buchner funnel, and the filter cake was washed with 0.3 L of isopropyl acetate. The solid was collected and vacuum dried to obtain the product.

[0112] The product was dissolved in 4.2 L of ethyl acetate. 42 g of activated carbon was added with stirring, and the mixture was stirred overnight under reflux. The solution was hot-filtered and filtered through a Buchner funnel lined with diatomaceous earth. The diatomaceous earth layer was washed again with 2.0 L of ethyl acetate. The filtrates were combined, and the organic phase was concentrated to 3.0 L. 1.2 L of ethyl acetate and 43 g of activated carbon were added to the organic phase. The mixture was stirred under reflux for 8 h, hot-filtered, and filtered through a Buchner funnel lined with diatomaceous earth. The diatomaceous earth layer was washed with 2.0 L of ethyl acetate. The filtrates were combined, and the organic phase was concentrated and dried under vacuum to give compounds 1-2.

[0113] MS m / z: 447.0 [M+H] +

[0114] 1H NMR (CHCl3-d, 400MHz): δ = 8.98 (d, J = 1.3Hz, 1H), 8.57 (br s,1H),8.27(s,1H),7.58(d,J=2.0Hz,1H),7.49(t,J=2.8Hz,1H),7.42(s,1H),6.50(s,1H),4.48(br s,1H),4.29(br d,J=12.5Hz,1H),4.17(s,3H),4.13(dd,J=11.9,2.9Hz,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=12.8,3.8Hz,1H),2.25(s,3H),1.44ppm(d,J=7.0Hz,3H)

[0115]

[0116] Step 6: Preparation of Compound 1 of Formula (1)

[0117] At 20°C, 63.0 mL of lithium aluminum hydride (2.5 M) was added to 50.0 mL of tetrahydrofuran containing 35.0 g (78.39 mmol) of compounds 1-2, and the reaction was stirred at 20°C for 1 hour. At 0-5°C, 6.9 mL of water, 6.9 mL of 15% sodium hydroxide, and 20.7 mL of water were slowly added sequentially to the reaction mixture, followed by filtration. The filtrate was concentrated to obtain a crude product, which was then separated by column chromatography (ethyl acetate / petroleum ether: 50-100%) to obtain the product. The product was dissolved in 20.0 mL of dimethyl sulfoxide at room temperature and slowly added dropwise to 400 mL of stirred water. The mixture was filtered and dried to obtain compound (I).

[0118] MS m / z: 419.1 [M+H] +

[0119] 1H NMR(400MHz,CHCl3-d)δppm 8.39(br s,1H),8.28(s,1H),7.58(s,1H),7.51(br s,1H),7.42(s,1H),7.35(t,J=2.76Hz,1H),6.49(s,1H),4.89(s,2H),4.51(br s,1H),4.30(br d,J=14.05Hz,1H),4.11-4.18(m,4H),3.81-3.94(m,2H),3.70(td,J=11.86,3. 14Hz,1H),3.44(td,J=12.86,3.89Hz,1H),2.26(s,3H),1.45(d,J=6.78Hz,3H)

[0120] Example 2: Preparation of the crystal form of compound A of formula (1)

[0121] Weigh approximately 500.0 mg of compound (I) and dissolve it in 5 mL of ethanol. Add 15 mL of purified water dropwise. After the addition is complete, stir the mixture on a magnetic stirrer (20 °C) for 120 hours. Filter the suspension to obtain a solid, and dry it overnight in a vacuum drying oven to obtain crystal form A of compound (I).

[0122] 1 H NMR(400MHz,CHCl3-d)δ=8.39(br s,1H),8.25(d,J=1.3Hz,1H),7.53(s,1H),7.49(t,J=2.3Hz,1H),7.40(s,1H),7.31(t,J=2.8Hz,1H),6.46(s,1H),4.86(s,2H),4.48(br d,J=4.8Hz,1H),4.28(br d,J=12.5Hz,1H),4.15-4.07(m,4H),3.91-3.86(m,1H),3.84-3.79(m,1H),3.67(dt, J=3.0,11.9Hz,1H),3.42(dt,J=3.9,12.9Hz,1H),2.23(s,3H),1.42(d,J=7.0Hz,3H)

[0123] Example 3: Preparation of the crystal form of compound B of formula (1)

[0124] Approximately 100 mg of compound (1) was added to different glass bottles, and appropriate amounts of organic solvent or solvent mixtures were added to each (Table 3). The samples were then placed in a constant temperature mixer (40°C) and stirred (stirring time is shown in Table 3) (protected from light). The solids were then filtered and dried overnight in a vacuum drying oven (40°C) to obtain crystal form B.

[0125] Table 3. Appropriate amounts of organic solvents and stirring times

[0126] Solvent (mL) Stirring Time (h) Acetone 0.7 + Water 0.7 120 Isopropanol 1 + Water 172 Methanol 1 + Water 1120 Methyl tert-butyl ether 172 Ethyl acetate 1 + n-heptane 1120 Isopropyl acetate 1 + n-heptane 1120 Ethanol 1 + n-heptane 1120 Acetonitrile 1 + n-heptane 1120 Isopropanol 1 + n-heptane 1120 Dichloromethane 2 + n-heptane 2 120

[0127] Example 4: Preparation of the crystal form of compound B of formula (1)

[0128] Compound of formula (1) (mass shown in Table 4) was slowly added to methanol solvent at a temperature of (60-70℃) (volume shown in Table 4), followed by slow dropwise addition of water (volume shown in Table 4). The mixture was stirred at 60℃ for 0.5 h, then cooled to 55℃ and stirred for 0.5 h, then cooled to 50℃ and stirred for 0.5 h, then cooled to 45℃ and stirred for 0.5 h, then cooled to 40℃ and stirred for 0.5 h, then cooled to 35℃ and stirred for 0.5 h, then cooled to 30℃ and stirred for 0.5 h, and finally cooled to 20-25℃ and stirred for 10 h. The solid was then filtered to obtain crystal form B.

[0129] Table 4. Appropriate amounts of organic solvents and stirring times

[0130] Formula (1) (g) Solvent (mL) Stirring time (h) 3 Methanol 30 13.5 2 Methanol 40 + Water 40 13.5 2 Methanol 40 + Water 20 13.5 2 Methanol 40 + Water 13.3 13.5

[0131] Example 5: Preparation of the crystal form of compound B of formula (1)

[0132] Experimental procedure: Take about 5.5g of compound (1) and slowly add it to 50mL of methanol solvent at a temperature of (60-70℃). Stir at 60℃ for 0.5h, then cool down to 25℃ and stir for 2h. Filter the solid to obtain crystal form B.

[0133] Example 6: Preparation of the crystal form of compound B of formula (1)

[0134] 900.0 g of compound (I) was dissolved in 9.0 L of methanol, and 9.0 L of purified water was slowly added dropwise at room temperature (25 °C). The mixture was then stirred for 20 hours. The mixture was filtered under reduced pressure, the filter cake was washed with 6.0 L of purified water, and the solid was dried under vacuum to obtain the B crystal form of compound (I).

[0135] MS m / z: 419.0 [M+H] +

[0136] 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 = 2.5Hz, 1H), 6.45 (s, 1H), 4.81 (br s,2H),4.47(br d,J=5.8Hz,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 =11.9,3.0Hz,1H),3.41(td,J=12.8,3.8Hz,1H),2.22(s,3H),1.41ppm(d,J=6.8Hz,3H)

[0137] Example 7: Solid stability test of crystal form A under high temperature and high humidity conditions

[0138] Two parallel portions of crystal form A, approximately 100 mg each, were weighed and placed at the bottom of a glass sample vial, spreading them into a thin layer. The vial opening was sealed with aluminum foil, with small holes punched in the foil to ensure sufficient contact between the sample and ambient air. The vial was then placed in a constant temperature and humidity chamber at 40℃ / 75% humidity. Samples placed under these conditions were collected and tested on day 30. The test results were compared with the initial test results from day 0. The experimental results are shown in Table 5 below.

[0139] Table 5 Solid stability test of crystal form A

[0140] Time Point (Day) Appearance Crystal Form Content (%) Total Impurities (%) 0 White Powder Crystal Form A 99.6 10.3 930 White Powder Crystal Form A 99.5 90.45

[0141] Experimental conclusion: Compound A of formula (I) has good crystal stability and is easy to prepare into a drug.

[0142] Example 8: Solid physical stability test of crystal form A under different temperature, humidity and light conditions

[0143] Four parallel samples of crystal form A, each approximately 100 mg, were weighed and placed in a thin layer at the bottom of a glass sample vial. The vials were sealed with aluminum foil, with small holes punched in the foil to ensure sufficient contact between the samples and ambient air. The four samples were placed under conditions of 25℃ / 92.5% relative humidity, 60℃, 40℃ / 75% relative humidity, and light exposure, respectively, to examine their physical stability on day 10. Simultaneously, a separate sample of approximately 100 mg of crystal form A was weighed and placed at the bottom of a glass sample vial, sealed with a screw cap, and stored at -20℃ as a control. On day 10, all samples were removed, allowed to return to room temperature, and their appearance was observed. The crystal form was then determined using XRPD. The solid-state physical stability of compound A (I) was determined by comparing the accelerated sample with the control sample. Table 6 below shows the experimental results of the solid-state physical stability of crystal form A.

[0144] Table 6. Solid physical stability tests of crystal form A under different temperature, humidity, and light conditions.

[0145]

[0146] Experimental conclusion: Compound A of formula (I) has good crystal stability and is easy to prepare into a drug.

[0147] Example 9: Solid physical stability test of B crystal form under high temperature, high humidity and light conditions

[0148] Two B-type samples were weighed in parallel for each group and placed at the bottom of a glass sample vial, spreading them into a thin layer. The vial opening was sealed with aluminum foil, and small holes were punched in the foil to ensure full contact between the sample and the ambient air. The vials were then placed in constant temperature and humidity chambers or light chambers under different humidity conditions. Samples placed under the above conditions were taken for testing on the 5th, 10th, 30th, 1 month, 3 months, or 6 months. The test results were compared with the initial test results on day 0. The test results are shown in Tables 7-11 below:

[0149] Table 7. Stability test of solid B-type crystals at 60°C

[0150] Time Point (Day) Crystal Form Content (%) Total Impurities (%) 0B Crystal Form 99.0 0.42 5B Crystal Form 101.5 0.43 10B Crystal Form 101.6 0.39 30B Crystal Form 99.4 0.41

[0151] Experimental conclusion: Compound B of formula (I) has good high-temperature stability and is easy to prepare into a drug.

[0152] Table 8. Stability test of solid B crystal form at high humidity 25℃ / 92.5% RH

[0153] Time Point (Day) Crystal Form Content (%) Total Impurities (%) 0B Crystal Form 99.0 0.42 5B Crystal Form 98.9 0.44 10B Crystal Form 100.1 0.41 30B Crystal Form 99.7 0.41

[0154] Experimental conclusion: Compound B of formula (I) has good high humidity stability and is easy to prepare into a drug.

[0155] Table 9. Solid-state light stability test for B-type crystals

[0156] Time point (day) Crystal form content (%) Total impurities (%) 0B crystal form 99.0 0.42 5B crystal form 101.0 0.44

[0157] 10B crystal form 100.70.42

[0158] Experimental conclusion: Compound B of formula (I) has good light stability.

[0159] Table 10. Stability test of solid B crystal form at 40℃ / 75%RH

[0160] Time Point (Day) Crystal Form Content (%) Total Impurities (%) 0B Crystal Form 99.0 0.42 1 Month B Crystal Form 100.6 0.45 2 Month B Crystal Form 100.4 0.42 3 Month B Crystal Form 98.8 0.44 6 Month B Crystal Form 99.8 0.43

[0161] Experimental conclusion: Compound B of formula (I) has good crystal stability and is easy to prepare into a drug.

[0162] Table 11. Stability test of B crystal form at 25℃ / 65%RH

[0163] Time Point (Day) Crystal Form Content (%) Total Impurities (%) 0B Crystal Form 99.0 0.42 3 Months B Crystal Form 98.4 0.45 6 Months B Crystal Form 99.8 0.43

[0164] Experimental conclusion: Compound B of formula (I) has good crystal stability and is easy to prepare into a drug.

[0165] Experimental Example 1: In vitro evaluation

[0166] By measuring IC 50 The value is used to evaluate the inhibitory activity of the test compound on human ATR kinase.

[0167] ATR / ATRIP(h) was incubated in assay buffer containing 50 nM GST-cMyc-p53 and Mg / ATP (at the required concentration). The reaction was initiated by adding the Mg / ATP mixture. After incubation at room temperature for 30 minutes, the reaction was terminated by adding a stop solution containing EDTA. Finally, a solution containing d... 2 Detection buffer and antiphosphorylated p-labeled anti-GST monoclonal antibody53 Europium-labeled anti-Ser15 phosphate antibody was then used. The plates were then read in time-resolved fluorescence mode and homogeneous time-resolved chromatography was performed.

[0168] The fluorescence (HTRF) signal is determined using the formula HTRF = 10000 × (Em665nm / Em620nm).

[0169] Analyzing ICs using XLFit version 5.3 (ID Business Solutions) 50 Data. Nonlinear regression analysis was used to fit the S-shaped dose-response (variable slope) curve. The experimental results are shown in Table 12:

[0170] Table 12 Results of in vitro screening tests of the compounds of the present invention

[0171] Compound number ATR average IC 50 (nM) Formula (1) Compound 29

[0172] Conclusion: The compound of formula (I) of this invention has a good inhibitory effect on the kinase ATR.

[0173] Experimental Example 2: In vitro cell viability assay

[0174] This study investigated the inhibitory effect of the compound on cell proliferation by examining its influence on in vitro cell activity in the LoVo tumor cell line.

[0175] CellTiter-Glo luminescence assay for cell viability

[0176] The following steps were performed in accordance with the instructions for the PromegaCellTiter-Glo chemiluminescence immunoassay kit (Promega-G7573).

[0177] (1). Melt the CellTiter-Glo buffer and let it come to room temperature.

[0178] (2). Place the CellTiter-Glo substrate at room temperature.

[0179] (3) Add CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to dissolve the substrate, thereby preparing CellTiter-Glo working solution.

[0180] (4) Slow vortexing to ensure complete dissolution.

[0181] (5) Remove the cell culture plate and let it stand for 30 minutes to allow it to equilibrate to room temperature.

[0182] (6) Add 50 μL of CellTiter-Glo working solution (equivalent to half the volume of cell culture medium in each well) to each well. Wrap the cell plate with aluminum foil to protect it from light.

[0183] (7) Shake the culture plate on a track shaker for 2 minutes to induce cell lysis.

[0184] (8) The culture plate was placed at room temperature for 10 minutes to stabilize the luminescence signal.

[0185] (9) Detect the light emission signal on the SpectraMax i3x of Molecular Devices reader.

[0186] Data Analysis

[0187] The inhibition rate (IR) of the detected compound is calculated using the following formula: IR (%) = (1 – (RLU compound – RLU blank control) / (RLU solvent control – RLU blank control)) * 100%. The inhibition rates of different compound concentrations are calculated in Excel, and then inhibition curves are plotted and relevant parameters, including minimum inhibition rate, maximum inhibition rate, and IC50, are calculated using GraphPad Prism software. 50 .

[0188] The experimental results are shown in Table 13:

[0189] Table 13 Results of in vitro LoVo cell proliferation inhibition assay

[0190] Formula (1) compound IC 50 (μM) 0.51

[0191] Experimental conclusion: The compound TR of formula (1) of this invention has a good inhibitory effect on LoVo tumor cells with mutations in the ATM signaling pathway.

[0192] Experimental Example 3: In vivo pharmacokinetic properties study

[0193] Test samples: Based on the above tests, select some highly active and structurally representative compounds for further testing.

[0194] Methods: The purpose of this study was to determine the pharmacokinetic parameters of this compound and calculate its bioavailability by gavage administration in female Balb / c Nude mice. Six female Balb / c Nude mice were used. Three mice were administered the compound intravenously at a dose of 1 mg / kg, and plasma samples were collected at 0 h (before administration) and 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post-administration. The other three mice were administered the compound by gavage at doses of 10 mg / kg or 25 mg / kg, and plasma samples were collected at 0 h (before administration) and 0.5, 1, 2, 3, 4, 6, 8, and 24 h post-administration. The collected samples were then analyzed by LC-MS / MS, and the relevant pharmacokinetic parameters were calculated using Phoenix WinNonlin 6.2.1 software. The results are shown in Tables 14.1 and 14.2.

[0195] 14.1 Results of intravenous administration

[0196] Formula (1) Compound (1 mg / kg IV) C0 (nM) 1955 Cl (mL / min / kg) 34.3 Vd ss (L / kg) 2.21T 1 / 2 (h)2.57AUC 0-t (nM.h)1087

[0197] 14.2 Results of gavage administration

[0198] Formula (1) compound (10 mg / kg) C max (nM)6500T 1 / 2 (h)2.02AUC 0-t (nM.h)14983F(%)129.0

[0199] Note: C0 (nM) is the drug concentration in vivo at 0 minutes; Cl (mL / min / kg) is the drug clearance rate in vivo; Vd ss (L / kg) is the volume of distribution of the drug in the body; T 1 / 2 (h) represents the half-life; AUC 0-t (nM.h) represents the in vivo drug exposure; C max (nM) represents the highest concentration of the drug in vivo; F represents bioavailability.

[0200] Experimental conclusion: The compound of formula (1) of the present invention has good absorption and exposure when administered by gavage, and is suitable for oral administration.

[0201] Case Study 4: In vivo efficacy study of LoVo CDX in colorectal cancer

[0202] Experimental objective:

[0203] LoVo is a colorectal adenocarcinoma cell line with MRE11A mutations (MRE11A is a key component of the ATM signaling pathway involved in DNA double-strand break repair) that is sensitive to ATR inhibitors. This study will use a LoVo rectal cancer CDX model to verify the inhibitory effect of ATR inhibitor monotherapy on tumors with ATM signaling pathway defects.

[0204] Experimental methods:

[0205] 1. Laboratory animals

[0206] Species: Mouse

[0207] Strain: BALB / c nude mice

[0208] Supplier: Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0209] Age and weight: 6-8 weeks old, weight 18-22 grams

[0210] Sex: Female

[0211] 2. Cell Culture

[0212] Human colon cancer LoVo cells (ECACC, catalog number: 87060101) were cultured in vitro in a monolayer under Ham's F-12 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM glutamine at 37°C and 5% CO2. Cells were passaged twice weekly using trypsin-EDTA digestion. When cell saturation reached 80%-90%, cells were harvested, counted, and seeded. 0.1 mL (10 x 10⁶ cells) of LoVo cells were subcutaneously seeded into the right posterior dorsal region of each nude mouse, resulting in an average tumor volume of 173 mm². 3 Dosing will begin in groups at that time.

[0213] 3. Preparation and dosage of the test substance

[0214] Weigh 25.51 mg of compound (1) and dissolve it in 0.500 mL of DMSO. Add 2.000 mL of propylene glycol and 2.500 mL of deionized water, vortex to mix, and adjust the pH to 6.0 to obtain a clear solution.

[0215] Dosage: All test compounds were administered at 25 mg / kg twice daily by gavage, with an 8-hour interval between doses per day.

[0216] 4. Tumor Measurement and Laboratory Indicators

[0217] The tumor diameter was measured twice a week using calipers. The formula for calculating tumor volume is: V = 0.5a × b 2, where a and b represent the long and short diameters of the tumor, respectively.

[0218] The antitumor efficacy of the compounds was evaluated using TGI (%) or relative tumor proliferation rate (T / C) (%). Relative tumor proliferation rate (T / C) (%) = TRTV / CRTV × 100% (TRTV: mean RTV in the treatment group; CRTV: mean RTV in the negative control group). Relative tumor volume (RTV) was calculated based on tumor measurements using the formula RTV = Vt / V0, where V0 is the tumor volume measured at the time of administration (D0), and Vt is the tumor volume at a specific measurement. TRTV and CRTV data were taken from the same day.

[0219] TGI (%) reflects the tumor growth inhibition rate. TGI (%) = [1 - (mean tumor volume at the end of treatment - mean tumor volume at the beginning of treatment) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the beginning of treatment in the solvent control group)] × 100%.

[0220] After the experiment, tumor weight will be measured and the T / Cweight percentage will be calculated. Tweight and Cweight represent the tumor weight of the drug administration group and the solvent control group, respectively.

[0221] 5. Test Results

[0222] This study evaluated the efficacy of the compound in a human colorectal cancer xenograft model, with the solvent control group as a reference. After 17 days of administration, the T / C and TGI of the compound (1) group (25 mg / kg) were 27.8% and 90.7% respectively compared with the solvent control group.

[0223] 6. Conclusion

[0224] In this experiment, the compound of formula (1) of the present invention had a certain inhibitory effect on the growth of tumor-bearing mice in the subcutaneous xenograft model of human colorectal cancer LoVo cells.

Claims

1. The A crystal form of the compound of formula (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.10 ± 0.20°, 18.33 ± 0.20°, and 22.63 ± 0.20°.

2. The A crystal form according to claim 1, whose X-ray powder diffraction pattern has 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 A crystal form according to claim 2, whose X-ray powder diffraction pattern has 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 A crystal form according to claim 3, whose X-ray powder diffraction pattern has 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 A crystal form according to claim 4, whose XRPD pattern is shown in Figure 1.

6. The B crystal form of the compound of formula (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.45 ± 0.20°, 10.87 ± 0.20°, and 20.56 ± 0.20°.

7. The B crystal form according to claim 6, whose X-ray powder diffraction pattern has 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 B crystal form according to claim 7, whose X-ray powder diffraction pattern has 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 B crystal form according to claim 8 has characteristic diffraction peaks in the X-ray powder diffraction pattern 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 XRPD pattern of the B crystal form according to claim 9 is shown in Figure 2.

11. The B crystal form according to claims 6-10 has a starting point of an endothermic peak in the differential scanning calorimetry curve (DSC) at 174.3 ± 3 °C.

12. The DSC pattern of the B crystal form according to claim 11 is shown in Figure 3.

13. The B crystal form according to claims 6-10 has a weight loss of 1.49% at 150 °C ± 3 °C in the thermogravimetric analysis curve (TGA).

14. The TGA pattern of the B crystal form according to claim 13 is shown in Figure 4.

15. A method for preparing the A crystal form of the compound of formula (I), comprising: 1) adding the compound of formula (I) to an ethanol solvent; 2) adding water; 3) stirring for 100-120 hours; 4) recrystallizing at room temperature to obtain.

16. A method for preparing the B crystal form of the compound of formula (I), comprising: 1) adding the compound of formula (I) to a solvent; 2) heating to a certain temperature and stirring for 2.5-120 hours; 3) recrystallizing at room temperature to obtain the B crystal form.

17. The preparation method according to claim 16, wherein, the solvent is: methanol, methyl tert-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 preparation method according to claim 17, wherein, the temperature is 25-70 °C.

19. The preparation method according to claim 16, wherein, the concentration range of the compound of formula (I) is selected from 25 mg / mL to 50 mg / mL.

20. Use of the A crystal form according to any one of claims 1-5 or the B crystal form according to any one of claims 6-14 in the preparation of a drug for treating ATR-related diseases.

21. The use according to claim 20, characterized in that, the drug is a drug for treating solid tumors or hematological tumors.

22. The use according to claim 20, characterized in that, The drug is a drug for treating colorectal cancer, gastric cancer, esophageal cancer, primary peritoneal cancer, adrenocortical carcinoma, renal clear cell carcinoma, prostate cancer, bladder urothelial carcinoma, ovarian cancer, breast cancer, endometrial cancer, fallopian tube cancer, non-small cell lung cancer or small cell lung cancer.

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