Crystal form of fluoropyridopyrrole compound and preparation method thereof

Abstract

The present invention discloses a crystalline form of a fluoropyrido-pyrrole compound of formula (1), a preparation method thereof and a use thereof. In the present invention, the compound of formula (1) has a strong inhibitory activity against the ATR enzyme and has a certain anti-cancer activity. The present invention prepares the compound of formula (1) into various crystalline forms to obtain a product form that is more stable than an amorphous compound and is less affected by light, heat and humidity, and has a broader prospect for drug development.

Description

Technical Field

[0001] The present invention relates to a crystal form of a fluoropyridopyrrole compound represented by formula (1) and a preparation method thereof. Background Art

[0002] The DNA damage response (DDR) ensures the integrity of the genome of living cells through a variety of signaling pathways. Intracellular proteins directly recognize abnormal DNA structures and activate kinases associated with the DDR pathway to respond to extensive DNA damage and increased replication stress in cancer cells. The DDR pathway enables cells to survive in the face of genomic instability and replication stress, or mediate senescence or programmed cell death of irreparably damaged cells. Defects in DDR genes promote driver gene mutations, tumor heterogeneity, and escape from apoptosis through multiple pathways, thereby promoting tumor growth.

[0003] ATR (ataxia telangiectasia mutated and RAD-3-related protein kinase) belongs to the PIKKs (phosphatidylinositol-3-kinase-related kinase) family and is involved in DNA damage repair to maintain gene stability. ATR protein kinase produces a synergistic response to DNA damage, replication stress and cell cycle disturbance. ATR and ATM belong to the PIKK family of serine / threonine protein kinases. They are common components of the cell cycle and DNA damage repair, and others include Chk1, BRCA1, and p53. ATR is mainly responsible for the repair of DNA replication stress (replication fork stalling) and single-strand breaks.

[0004] ATR is activated by single-stranded DNA structures when double-strand breaks occur or replication forks stall. DNA polymerases are arrested in the DNA replication process, and replication helicases continue to unwind at the front of the DNA replication fork, resulting in the generation of long single-stranded DNA (ssDNA), which is then bound by single-stranded DNA and RPA (replication protein A). During replication stress or DNA damage, the ATR / ATR-acting protein complex recruited by RPA goes to the damage site, and the RPA-ssDNA complex activates the RAD17 / rfc2-5 complex to bind to the damage site, activating the Rad9-HUS1-RAD1 (9-1-1) heterotrimer at the DNA-ssDNA junction, and 9-1-1 in turn recruits TopBP1 to activate ATR. Once ATR is activated, ATR promotes DNA repair, stabilizes and restarts stalled replication forks and transient cell cycle arrest through downstream targets. These functions are achieved by ATR through the mediation of the downstream target Chk1. ATR acts as a DNA damage cell cycle checkpoint in the S phase. It can mediate the degradation of CDC25A through Chk1, thereby delaying the DNA replication process and providing time for repairing the replication fork. ATR is also the main regulator of the G2 / M cell cycle checkpoint, preventing cells from entering mitosis prematurely before DNA replication is completed or DNA damage occurs. This ATR-dependent G2 / M cell cycle arrest is mainly mediated by two mechanisms: 1. Degradation of CDC25A. 2. Phosphorylation of Cdc25C by Chk1 to bind to 14-3-protein. The binding of Cdc25C to 14-3-3 protein promotes its export from the nucleus and cytoplasmic sequestration, thereby inhibiting its ability to dephosphorylate and activate nuclear Cdc2, which in turn prevents entry into mitosis.

[0005] The ATM gene often mutates in tumor cells, indicating that loss of ATM activity is beneficial to the survival of cancer cells. Inactivation of ATM kinase makes cells more dependent on ATR-mediated signaling pathways, and the combined inactivation of ATR and ATM can induce synthetic lethality in cancer cells. Therefore, inhibiting ATR may be an effective method for future cancer treatment. Summary of the invention

[0006] The present invention has found that the compound of formula (1) has a strong inhibitory activity against the ATR enzyme; at the same time, it has a good inhibitory effect on LoVo tumor cells with a missing ATM signaling pathway; at the same time, the PK parameters such as exposure and bioavailability of the compound of the present invention are good and suitable for medication; in addition, the compound of the present invention can significantly inhibit the growth of human gastric cancer SNU-601 xenograft tumors and is relatively tolerant to mice.

[0007]

[0008] The compound of formula (1) of the present invention is prepared by Suzuki coupling reaction of compound 1-C and intermediate I.

[0009]

[0010] The present invention provides a crystalline form I of a compound of formula (1), wherein the X-ray powder diffraction (XRPD) spectrum has characteristic diffraction peaks at at least one or more of the following 2θ angles: 7.90±0.20°, 9.35±0.20° and 18.81±0.20°:

[0011]

[0012] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned Form I has characteristic diffraction peaks at at least one or more of the following 2θ angles: 7.90±0.20°, 9.35±0.20°, 12.05±0.20°, 12.40±0.20°, 15.78±0.20°, 18.41±0.20°, 18.81±0.20°, and 20.14±0.20°.

[0013] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned Form I has characteristic diffraction peaks at at least the following 2θ angles: 7.90±0.20°, 9.35±0.20°, 12.05±0.20°, 12.40±0.20°, 15.78±0.20°, 16.13±0.20°, 16.48±0.20°, 17.73±0.20°, 18.41±0.20°, 18.81±0.20°, 20.14±0.20°, 20.63±0.20°, and 22.61±0.20°.

[0014] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned Form I has characteristic diffraction peaks at at least one or more of the following 2θ angles: 7.90±0.20°, 9.35±0.20°, 12.05±0.20°, 12.40±0.20°, 14.60±0.20°, 15.78±0.20°, 16.13±0.20°, 16.48±0.20°, 17.73±0.20°, 18.41±0.20°, 18.81±0.20°, 20.14±0.20°, 20.63±0.20°, 21.32±0.20°, 22.04±0.20°, and 22.61±0.20°.

[0015] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned Form I has characteristic diffraction peaks at least at the following 2θ angles: 7.90±0.20°, 9.35±0.20°, and / or 18.81±0.20°, and / or 12.05±0.20°, and / or 12.40±0.20°, and / or 15.78±0.20°, and / or 18.41±0.20°. °, and / or 20.14±0.20°, and / or 16.13±0.20°, and / or 16.48±0.20°, and / or 17.73±0.20°, and / or 20.63±0.20°, and / or 22.61±0.20°, and / or 14.60±0.20°, and / or 21.32±0.20°, and / or 22.04±0.20°.

[0016] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned Form I has characteristic diffraction peaks at at least one or more of the following 2θ angles: 7.90°, 9.35°, 12.05°, 12.40°, 14.60°, 15.78°, 16.13°, 16.48°, 17.73°, 18.41°, 18.81°, 20.14°, 20.63°, 21.32°, 22.04°, and 22.61°.

[0017] In some embodiments of the present invention, the above-mentioned Form I has an XRPD pattern substantially as shown in the attached Figure 1 shown.

[0018] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form I is shown in Table 1:

[0019] Table 1 XRPD analysis data of the crystal form of compound I of formula (1)

[0020]

[0021]

[0022] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned Form I has endothermic peaks at 64.8±3°C, 153.8±3°C and 252.4±3°C.

[0023] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned Form I shows a weight loss of approximately 6.71% at 180.0±3°C.

[0024] In some embodiments of the present invention, the DSC curve and TGA curve of the above-mentioned Form I are as shown in the attached Figure 2 shown.

[0025] The present invention provides a crystal form II of the compound of formula (1), whose X-ray powder diffraction (XRPD) spectrum has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.76±0.20°, 15.93±0.20° and 17.94±0.20°:

[0026]

[0027] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned II crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 7.97±0.20°, 8.76±0.20°, 15.93±0.20°, 17.94±0.20°, 18.61±0.20°, 24.53±0.20°, 25.10±0.20° and 25.81±0.20°.

[0028] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned II crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 7.97±0.20°, 8.76±0.20°, 10.99±0.20°, 12.98±0.20°, 14.70±0.20°, 15.93±0.20°, 16.47±0.20°, 17.94±0.20°, 18.61±0.20°, 20.45±0.20°, 21.98±0.20°, 22.40±0.20°, 2 2.90±0.20°, 23.87±0.20°, 24.53±0.20°, 25.10±0.20°, 25.81±0.20°, 26.06±0.20°, 27.20±0.20°, 27.98±0.20°, 28.71±0.20°, 29.37±0.20°, 29.86±0.20°, 30.29±0.20°, 31.56±0.20°, 32.14±0.20°, 35.31±0.20° and 38.12±0.20°.

[0029] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned II crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.76±0.20°, 15.93±0.20°, and / or 17.94±0.20°, and / or 7.97±0.20°, and / or 18.61±0.20°, and / or 24.53±0.20°, and / or 25.10±0.20°, and / or 25.81±0.20°, and / or 10.99±0.20°, and / or 12.98±0.20°, and / or 14.70±0.20°, and / or 16.47±0.20°, and / or 20.45±0.20°. .20°, and / or 21.98±0.20°, and / or 22.40±0.20°, and / or 22.90±0.20°, and / or 23.87±0.20°, and / or 26.06±0.20°, and / or 27.20±0.20°, and / or 27.98±0.20°, and / or 28.71±0.20°, and / or 29.37±0.20°, and / or 29.86±0.20°, and / or 30.29±0.20°, and / or 31.56±0.20°, and / or 32.14±0.20°, and / or 35.31±0.20°, and / or 38.12±0.20°.

[0030] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned II crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 7.97°, 8.76°, 10.99°, 12.98°, 14.70°, 15.93°, 16.47°, 17.94°, 18.61°, 20.45°, 21.98°, 22.40°, 22.90°, 23.87°, 24.53°, 25.10°, 25.81°, 26.06°, 27.20°, 27.98°, 28.71°, 29.37°, 29.86°, 30.29°, 31.56°, 32.14°, 35.31° and 38.12°.

[0031] In some embodiments of the present invention, the above-mentioned Form II has an XRPD pattern substantially as shown in the attached Figure 3 shown.

[0032] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form II is shown in Table 2:

[0033] Table 2 XRPD analysis data of the crystal form of compound II of formula (1)

[0034]

[0035]

[0036] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned Form II has an endothermic peak at 222.1±3°C and 256.5±3°C respectively.

[0037] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned Form II shows a weight loss of about 1.02% at 250±3°C.

[0038] In some embodiments of the present invention, the DSC curve and TGA curve of the above-mentioned II crystal form are as follows: Figure 4 shown.

[0039] The present invention provides a crystal form III of a compound of formula (1), wherein the X-ray powder diffraction (XRPD) spectrum has characteristic diffraction peaks at at least one or more of the following 2θ angles: 14.848±0.20°, 17.653±0.20° and 19.135±0.20°:

[0040]

[0041] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned III crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 10.518±0.20°, 12.262±0.20°, 14.848±0.20°, 17.146±0.20°, 17.653±0.20°, 19.135±0.20°, 20.365±0.20° and 25.798±0.20°.

[0042] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned III crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 10.518±0.20°, 12.262±0.20°, 14.848±0.20°, 15.854±0.20°, 17.146±0.20°, 17.653±0.20°, 17.840±0.20°, 19.135±0.20°, 20.110±0.20°, 20.365±0.20°, 24.140±0.20° and 25.798±0.20°.

[0043] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned III crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 6.125±0.20°, 7.266±0.20°, 7.911±0.20°, 8.682±0.20°, 9.699±0.20°, 10.518±0.20°, 10.894±0.20° °, 11.520±0.20°, 12.262±0.20°, 12.821±0.20°, 14.848±0.20°, 15.162±0.20°, 15.854±0.20°, 16.280±0.20°, 17.146±0.20°, 17.653±0.20°, 17.840±0.20°. 18.227±0.20°, 18.536±0.20°, 19.135±0.20°, 19.679±0.20°, 20.110±0.20°, 20.365±0.20°, 21.035±0.20°, 21.274±0.20°, 21.696±0.20°, 21.388±0.20°, 22.742±0.20°, 23.113±0.20°, 24.140±0.20°, 24.652±0.20°, 25.132±0.20°, 25.798 ±0.20°, 26.660±0.20°, 26.981±0.20°, 27.621±0.20°, 28.027±0.20°, 28.710±0.20°, 29.335±0.20°, 29.921±0.20°, 30.613±0.20°, 31.184±0.20°, 31.916±0.20°, 32.705±0.20°, 33.293±0.20°, 34.064±0.20°, 36.932±0.20° and 38.188±0.20°.

[0044] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned III crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 14.848±0.20°, 17.653±0.20°, and / or 19.135±0.20°, and / or 10.518±0.20°, and / or 12.262±0.20°, and / or 17.146±0.20°, and / or 20.365±0.20°, and / or 25.798±0.20°, and / or 6.130±0.20°, and / or 7.270±0.20°, and / or 7.911±0.20°, and / or 8.682±0.20°, and / or 9.700±0.20°, and / or 10.890±0.20°, and / or 11.520±0.20°, and / or 12.821±0.20°, and / or 15.162±0.20°, and / or 15.854±0.20°, and / or 16.280±0.20°, and / or 17.840±0.20°, and / or 18.227±0.20°, and / or 18.536±0.20°, and / or 19.679±0.2 0°, and / or 20.110±0.20°, and / or 21.035±0.20°, and / or 21.274±0.20°, and / or 21.696±0.20°, and / or 21.388±0.20°, and / or 22.742±0.20°, and / or 23.113±0.20°, and / or 24.14±0.20°, and / or 24.652±0.20°, and / or 25.132±0.20°, and / or 26.660±0.20°, and / or 26.981±0.20°, and / or 27.6 21±0.20°, and / or 28.027±0.20°, and / or 28.710±0.20°, and / or 29.335±0.20°, and / or 29.921±0.20°, and / or 30.613±0.20°, and / or 31.184±0.20°, and / or 31.916±0.20°, and / or 32.710±0.20°, and / or 33.290±0.20°, and / or 34.064±0.20°, and / or 36.932±0.20°, and / or 38.190±0.20°.

[0045] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned III crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 6.125°, 7.266°, 7.911°, 8.682°, 9.699°, 10.518°, 10.894°, 11.520°, 12.262°, 12.821°, 14.848°, 15.162°, 15.854°, 16.280°, 17.146°, 17.653°, 17.840°, 18.227°, 18.536°, 19.135°, 19.679°, 20. .110°, 20.365°, 21.035°, 21.274°, 21.696°, 21.388°, 22.742°, 23.113°, 24.140°, 24.652°, 25.132°, 25.798°, 26.660°, 26.981°, 27.621°, 28.027°, 28.710°, 29.335°, 29.921°, 30.613°, 31.184°, 31.916°, 32.705°, 33.293°, 34.064°, 36.932°, and 38.188°.

[0046] In some embodiments of the present invention, the above-mentioned III crystal form has an XRPD pattern substantially as shown in the attached Figure 5 shown.

[0047] In some embodiments of the present invention, the XRPD spectrum analysis data of the above-mentioned III crystal form is shown in Table 3:

[0048] Table 3 XRPD analysis data of the crystal form of compound III of formula (1)

[0049]

[0050]

[0051] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned Form III shows a weight loss of about 5.34% at 150±3°C.

[0052] In some embodiments of the present invention, the TGA curve of the above-mentioned III crystal form is as follows: Figure 6 shown.

[0053] The present invention provides a crystal form IV of a compound of formula (1), wherein the X-ray powder diffraction (XRPD) spectrum has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.2794±0.20°, 15.7523±0.20° and 17.5827±0.20°:

[0054]

[0055] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned IV crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.2794±0.20°, 8.4208±0.20°, 15.7523±0.20°, 16.3613±0.20°, 17.5827±0.20°, 18.7041±0.20°, 24.7696±0.20° and 24.9328±0.20°.

[0056] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned IV crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.2794±0.10°, 8.4208±0.10°, 15.7523±0.10°, 16.3613±0.10°, 17.5827±0.10°, 18.7041±0.10°, 24.7696±0.10° and 24.9328±0.10°.

[0057] In some embodiments of the present invention, the X-ray powder diffraction pattern of the IV crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.2794±0.20°, 8.4208±0.20°, 10.6855±0.20°, 12.3408±0.20°, 13.0615±0.20°, 14.6650±0.20°, 15.7523±0.20°, 16.3613±0.20°, 17.5827±0.20°, 18.7041±0.20°, 20.04 70±0.20°, 20.8774±0.20°, 21.7465±0.20°, 22.1529±0.20°, 23.9930±0.20°, 24.7696±0.20°, 24.9238±0.20°, 26.2785±0.20°, 27.1454±0.20°, 28.2680±0.20°, 29.7925±0.20°, 30.8621±0.20°, 32.4019±0.20° and 33.3887±0.20°.

[0058] In some embodiments of the present invention, the X-ray powder diffraction pattern of the IV crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.2794±0.10°, 8.4208±0.10°, 10.6855±0.10°, 12.3408±0.10°, 13.0615±0.10°, 14.6650±0.10°, 15.7523±0.10°, 16.3613±0.10°, 17.5827±0.10°, 18.7041±0.10°, 20.04 70±0.10°, 20.8774±0.10°, 21.7465±0.10°, 22.1529±0.10°, 23.9930±0.10°, 24.7696±0.10°, 24.9238±0.10°, 26.2785±0.10°, 27.1454±0.10°, 28.2680±0.10°, 29.7925±0.10°, 30.8621±0.10°, 32.4019±0.10° and 33.3887±0.10°.

[0059] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned IV crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.2794±0.20°, 15.7523±0.20°, and / or 17.5827±0.20°, and / or 8.4208±0.20°, and / or 16.3613±0.20°, and / or 18.7041±0.20°, and / or 24.7696±0.20°, and / or 24.9328±0.20°, and / or 10.6855±0.20°, and / or 12.3408±0.20°, and / or 13.0615±0.2 0°, and / or 14.6650±0.20°, and / or 20.0470±0.20°, and / or 20.8774±0.20°, and / or 21.7465±0.20°, and / or 22.1529±0.20°, and / or 23.9930±0.20°, and / or 26.2785±0.20°, and / or 27.1454±0.20°, and / or 28.2680±0.20°, and / or 29.7925±0.20°, and / or 30.8621±0.20°, and / or 32.4019±0.20°, and / or 33.3887±0.20°.

[0060] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned IV crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.2794±0.10°, 15.7523±0.10°, and / or 17.5827±0.10°, and / or 8.4208±0.10°, and / or 16.3613±0.10°, and / or 18.7041±0.10°, and / or 24.7696±0.10°, and / or 24.9328±0.10°, and / or 10.6855±0.10°, and / or 12.3408±0.10°, and / or 13.0615±0.10°. 0°, and / or 14.6650±0.10°, and / or 20.0470±0.10°, and / or 20.8774±0.10°, and / or 21.7465±0.10°, and / or 22.1529±0.10°, and / or 23.9930±0.10°, and / or 26.2785±0.10°, and / or 27.1454±0.10°, and / or 28.2680±0.10°, and / or 29.7925±0.10°, and / or 30.8621±0.10°, and / or 32.4019±0.10°, and / or 33.3887±0.10°.

[0061] In some embodiments of the present invention, the X-ray powder diffraction pattern of the IV crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.2794°, 8.4208°, 10.6855°, 12.3408°, 13.0615°, 14.6650°, 15.7523°, 16.3613°, 17.5827°, 18.7 041°, 20.0470°, 20.8774°, 21.7465°, 22.1529°, 23.9930°, 24.7696°, 24.9238°, 26.2785°, 27.1454°, 28.2680°, 29.7925°, 30.8621°, 32.4019° and 33.3887°.

[0062] In some embodiments of the present invention, the above-mentioned IV crystal form has an XRPD pattern substantially as shown in the attached Figure 7 shown.

[0063] In some embodiments of the present invention, the XRPD pattern analysis data of the above IV crystal form are shown in Table 4:

[0064] Table 4 XRPD analysis data of the crystal form of compound IV of formula (1)

[0065]

[0066] The present invention provides a V-form of the compound of formula (1), whose X-ray powder diffraction (XRPD) spectrum has characteristic diffraction peaks at least at the following 2θ angles: 8.4539±0.20°, 17.3745±0.20° and 25.1766±0.20°:

[0067]

[0068] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned V crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.4539±0.20°, 16.1904±0.20°, 17.3745±0.20°, 18.2963±0.20°, 23.3932±0.20°, 25.1766±0.20°, 26.1776±0.20° and 27.0062±0.20°.

[0069] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned V crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.4539±0.20°, 9.4597±0.20°, 10.9365±0.20°, 11.9742±0.20°, 15.7873±0.20°, 16.1904±0.20, 16.8510±0.20°, 17.3745±0.20°, 18.2963±0.20°, 18.6558±0.20°, 18.9325±0.20° , 19.9943±0.20°, 21.2656±0.20°, 22.1742±0.20°, 23.3932±0.20°, 24.0827±0.20°, 25.1766±0.20°, 26.1776±0.20°, 26.6250±0.20°, 27.0062±0.20°, 27.6261±0.20°, 28.5886±0.20°, 29.4495±0.20°, 31.7776±0.20° and 32.9126±0.20°.

[0070] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned V crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.4539±0.20°, 17.3745±0.20°, and / or 25.1766±0.20°, and / or 16.1904±0.20°, and / or 18.2963±0.20°, and / or 23.3932±0.20°, and / or 26.1776±0.20°, and / or 27.0062±0.20°, and / or 9.4597±0.20°, and / or 10.9365±0.20°, and / or 11.9742±0.20°, and / or 15. 7873±0.20°, and / or 16.8510±0.20°, and / or 18.6558±0.20°, and / or 18.9325±0.20°, and / or 19.9943±0.20°, and / or 21.2656±0.20°, and / or 22.1742±0.20°, and / or 24.0827±0.20°, and / or 26.6250±0.20°, and / or 27.6261±0.20°, and / or 28.5886±0.20°, and / or 29.4495±0.20°, and / or 31.7776±0.20°, and / or 32.9126±0.20°.

[0071] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned V crystal form has characteristic diffraction peaks at at least one or more of the following 2θ angles: 8.4539°, 9.4597°, 10.9365°, 11.9742°, 15.7873°, 16.1904°, 16.8510°, 17.3745°, 18.2963°, 18.6558°, 18.9325°, 19.9943°, 21.2656°, 22.1742°, 23.3932°, 24.0827°, 25.1766°, 26.1776°, 26.6250°, 27.0062°, 27.6261°, 28.5886°, 29.4495°, 31.7776° and 32.9126°.

[0072] In some embodiments of the present invention, the XRPD pattern of the above-mentioned Form V is substantially as shown in the attached Figure 8 shown.

[0073] In some embodiments of the present invention, the XRPD spectrum analysis data of the above-mentioned Form V is shown in Table 5:

[0074] Table 5 XRPD analysis data of the crystal form V of the compound of formula (1)

[0075]

[0076] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned V crystal form has an endothermic peak at 236.3±1°C and 256.3±3°C (starting temperature), and an exothermic peak at 237.9±1°C. The endothermic peak at 256.3±3°C (starting temperature) has an enthalpy value of 99.61 J / g, and the endothermic peak near 236.3°C partially overlaps with the exothermic peak near 237.9°C, and the enthalpy value cannot be calculated.

[0077] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned V crystal form shows a weight loss of about 2.0% at 150±3°C.

[0078] In some embodiments of the present invention, the DSC curve and TGA curve of the above-mentioned V crystal form are as follows: Fig. 9 shown.

[0079] The present invention also provides use of the above crystal form in preparing ATR enzyme inhibitors.

[0080] The present invention also provides the use of the crystal form in preparing products for treating diseases related to ATR enzyme activation.

[0081] ATR kinase is currently regarded as a potential target for cancer treatment. Unlike normal cells, a basic characteristic of tumor cells is genomic instability and easy mutation, which are usually accompanied by a large number of functional deficiencies in stabilizing and repairing genomic DNA. Therefore, cancer cells are more dependent on ATR kinase for self-repair. ATR and the signaling pathways it participates in are essential for genome stability and the occurrence, development and treatment of tumors. Previously, a large amount of functional and preclinical experimental data showed that ATR kinase inhibitors can directly and efficiently kill tumor cells.

[0082] Based on the inhibitory activity of the crystal form of the present invention on the ATR enzyme, the disease related to the ATR enzyme activation is selected from cancer.

[0083] The present invention also provides use of the above crystal form and an ATM enzyme inhibitor in preparing a combined drug for treating cancer.

[0084] Wherein, the cancer is selected from breast cancer, cervical cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, ovarian cancer, pancreatic cancer, testicular cancer, bladder cancer, myeloma, non-small cell lung cancer, leukemia, lymphoma, melanoma, esophageal cancer, connective tissue cancer, mesothelioma, prostate cancer, bone cancer, and kidney cancer.

[0085] The present invention also provides a pharmaceutical composition, which comprises the crystal form as described above.

[0086] The present invention also provides a method for preparing the crystalline form of the compound of formula (1), which comprises the following contents:

[0087] The compound of formula (1) was loaded onto a silica gel column and purified by gradient elution using 10-60% tetrahydrofuran / petroleum ether and then by gradient elution using 0-10% methanol / dichloromethane to obtain Form I of the compound of formula (1).

[0088] The present invention also provides a method for preparing the crystal form of the compound II of the above formula (1), which comprises the following contents:

[0089] Take the compound of formula (1), add ethanol, heat to 40-65°C to dissolve, then reduce the temperature to 25°C±2°C, crystallize, and the obtained solid is the II crystal form.

[0090] In the preparation of Form II, the concentration and amount of ethanol used are sufficient to dissolve the compound and allow solid to precipitate when cooled.

[0091] The present invention also provides a method for preparing the crystal form of the compound III of the above formula (1), which comprises the following contents:

[0092] The compound of formula (1) was purified by silica gel column, with the eluent being petroleum ether:ethyl acetate = 1:1, petroleum ether:tetrahydrofuran = 2:1, and dichloromethane:methanol = 25:1. The purified product was added with methanol, refluxed, cooled to 28°C ± 2°C, and crystallized to obtain Form III.

[0093] The present invention also provides a method for preparing the crystal form IV of the compound of formula (1), which comprises the following steps: taking the crystal form II, heating it to 240°C±5°C, and cooling it to obtain the crystal form IV.

[0094] The present invention also provides a method for preparing the crystal form V of the compound of formula (1), which comprises the following contents:

[0095] Method 1: Take the compound of formula (1), stir in 40-60% v / v ethanol at 60°C±5°C to obtain Form V;

[0096] Or method 2: add the compound of formula (1) to ethanol under stirring, then add ethanol, reflux at 80°C±5°C, add V crystal seed crystals under reflux, continue stirring, slowly cool to 24-28°C, crystallize to obtain V crystal.

[0097] The temperature change in the present invention can be controlled by conventional equipment in experiments or production.

[0098] The various crystal forms of the present invention can be prepared by using different methods such as solution dissolution and crystallization, solvent beating and crystallization, and incomplete dissolution and adding another crystal seed to induce crystallization.

[0099] In the present invention, crystal form II and crystal form V correspond to the kinetically stable crystal form and the relatively thermodynamically stable crystal form, respectively. According to the basic principle of crystallization theory, when crystal form II is dissolved and precipitated, crystal form II is obtained by rapid crystallization, and crystal form V can be obtained by slow crystallization by adding seed crystal V for induction. In addition, crystal form II is slurried in a solvent with a certain solubility. Since the saturated solubility of crystal form V is lower than that of crystal form II, when solid-liquid equilibrium occurs, solution precipitation occurs to obtain crystal form V, thereby realizing the process of slurrying and crystallization.

[0100] "Crystallization" refers to the precipitation of another phase when a substance is in a non-equilibrium state, and this phase is precipitated in the form of crystals.

[0101] "Gradient elution" means that in the same elution cycle, the concentration ratio of the mobile phase is continuously changed to a certain extent for elution.

[0102] The product, pharmaceutical composition or combination drug of the present invention may be added with an appropriate amount of pharmaceutically acceptable excipients.

[0103] The term "pharmaceutically acceptable" as used herein refers to any substance that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the host.

[0104] The pharmaceutically acceptable excipients described in the present invention are a general term for all additional materials in the drug except the main drug. The excipients should have the following properties: (1) no toxic effects on the human body and almost no side effects; (2) stable chemical properties and not easily affected by temperature, pH, storage time, etc.; (3) no incompatibility with the main drug and no impact on the efficacy and quality inspection of the main drug; (4) no interaction with the packaging material.

[0105] The excipients in the present invention include but are not limited to fillers (diluents), lubricants (glidants or anti-adhesives), dispersants, wetting agents, adhesives, regulators, solubilizers, antioxidants, antibacterial agents, emulsifiers, disintegrants, etc. Adhesives include syrup, gum arabic, gelatin, sorbitol, tragacanth, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose or hydroxypropyl methyl cellulose, etc.), gelatin slurry, syrup, starch slurry or polyvinyl pyrrolidone, etc.; fillers include lactose, powdered sugar, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salts (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; lubricants include micropowder silica gel, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc.; disintegrants include starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate , pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinyl pyrrolidone or microcrystalline cellulose, etc.; wetting agents include sodium dodecyl sulfate, water or alcohol, etc.; antioxidants include sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutyl benzoic acid, etc.; antibacterial agents include 0.5% phenol, 0.3% cresol, 0.5% trichlorobutanol, etc.; regulators include hydrochloric acid, citric acid, potassium hydroxide (sodium), sodium citrate and buffers (including sodium dihydrogen phosphate and disodium hydrogen phosphate), etc.; emulsifiers include polysorbate-80, sorbitan monophosphate, Pluronic F-68, lecithin, soybean lecithin, etc.; solubilizers include Tween-80, bile, glycerol, etc.

[0106] The administration method of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0107] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0108] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0109] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.

[0110] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0111] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0112] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0113] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0114] The compounds of the present invention can also be used in injection preparations, wherein the injection is selected from liquid injection (water injection), sterile powder for injection (powder injection) or injection tablets (referring to molded tablets or machine-pressed tablets made by aseptic operation of drugs, dissolved in injection water before use, for subcutaneous or intramuscular injection).

[0115] The powder for injection contains at least an excipient in addition to the above-mentioned compound. The excipient in the present invention is a component intentionally added to the drug, which should not have pharmacological properties in the amount used, but the excipient can help the processing, dissolution or dissolution of the drug, delivery of the drug through a targeted administration route or stability.

[0116] The excipient of the present invention can be selected from one or a combination of two or more of carbohydrates, inorganic salts, and polymers, wherein the carbohydrates include monosaccharides, oligosaccharides, or polysaccharides.

[0117] Monosaccharides are sugars that cannot be hydrolyzed any further. They are the basic units of molecules that make up various disaccharides and polysaccharides. They can be divided into trioses, tetroses, pentoses, hexoses, etc. Monosaccharides in nature are mainly pentoses and hexoses. For example, glucose is aldohexose and fructose is ketohexose.

[0118] Oligosaccharides, also known as oligosaccharides, are polymers formed by the condensation of a few monosaccharides (2-10).

[0119] Polysaccharides are high-molecular carbohydrates composed of sugar chains linked by glycosidic bonds, consisting of at least 10 monosaccharides.

[0120] The sterile powder for injection of the present invention can be obtained by conventional aseptic packaging or freeze drying processes.

[0121] In the present invention, the compound of formula (1) has strong inhibitory activity against ATR enzyme and has certain anti-cancer activity. The present invention prepares the compound of formula (1) into various crystal forms to obtain a product form that is more stable than the amorphous compound and less affected by light, heat and humidity, and has a broader prospect for drug development.

[0122] Unless otherwise specified, the terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0123] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed herein, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

[0124] The chemical reactions of the specific embodiments of the present invention are carried out in a suitable solvent, which must be suitable for the chemical changes of the present invention and the reagents and materials required. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction processes based on the existing embodiments.

[0125] The structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data of the cultured single crystal using a Bruker D8venture diffractometer, the light source is CuKα radiation, and the scanning mode is: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0126] The present invention will be described in detail below by way of examples, which are not intended to limit the present invention in any way.

[0127] All solvents used in the present invention were commercially available and used without further purification.

[0128] The solvent used in the present invention is commercially available.

[0129] Instruments and analytical methods

[0130] 1. X-ray powder diffractometer (XRPD)

[0131] Instrument model: Bruker D8advance X-ray diffractometer

[0132] Test method: About 10-20 mg of sample was used for XRPD detection.

[0133] The detailed XRPD parameters are as follows:

[0134] Light pipe: Cu, kα,

[0135] Light tube voltage: 40kV, light tube current: 40mA

[0136] Divergence slit: 0.60mm

[0137] Detector slit: 10.50mm

[0138] Anti-scatter slit: 7.10mm

[0139] Scanning range: 4-40 degrees

[0140] Step diameter: 0.02deg

[0141] Step size: 0.12 seconds

[0142] Sample plate speed: 15rpm

[0143] 2. Differential Scanning Calorimeter (DSC)

[0144] Instrument model: TAQ2000 differential scanning calorimeter

[0145] Test method: Take a sample (~1 mg) and place it in a DSC aluminum pan for testing. Under 50 mL / min N2 conditions, heat the sample from 30°C to 300°C at a heating rate of 10°C / min.

[0146] 3. Thermogravimetric analysis (TGA)

[0147] Instrument model: TAQ5000 thermogravimetric analyzer

[0148] Test method: Take a sample (2-5 mg) and place it in a TGA platinum pan for testing. Under 25 mL / min N2 conditions, heat the sample from 30°C (room temperature) to 300°C or 20% weight loss at a heating rate of 10°C / min. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figure 1 : XRPD pattern of Form I of the compound of formula (1);

[0150] Figure 2 : DSC curve and TGA curve of Form I of the compound of formula (1);

[0151] Figure 3 : XRPD pattern of Form II of the compound of formula (1);

[0152] Figure 4 : DSC curve and TGA curve of crystal form II of the compound of formula (1);

[0153] Figure 5 : XRPD pattern of Form III of the compound of formula (1);

[0154] Figure 6 : TGA spectrum of Form III of the compound of formula (1);

[0155] Figure 7 : XRPD pattern of Form IV of the compound of formula (1);

[0156] Figure 8: XRPD pattern of Form V of the compound of formula (1);

[0157] Fig. 9 : DSC curve and TGA curve of Form V of the compound of formula (1);

[0158] Fig.10 : Chromatogram of related substances of compound II of formula (I) crystalline form. DETAILED DESCRIPTION

[0159] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments, but the specific implementation methods are not intended to limit the content of the present invention.

[0160] Intermediate I

[0161]

[0162] Step 1: Synthesis of compound I-2

[0163] Add sodium hydrogen (220.36 mg, 5.51 mmol, 60%) to a solution of compound I-1 (500 mg, 3.67 mmol) in tetrahydrofuran (30 mL), and react at room temperature 20°C for 1 hour. Add triisopropylsilane (849.80 mg, 4.41 mmol) and react at room temperature 20°C for 2.5 hours. After the reaction is completed, add 10 mL of saturated aqueous ammonium chloride solution to the reaction solution to quench the reaction. Add 60 mL of water and extract with ethyl acetate (70 mL*3). The organic phase is washed once with saturated brine, dried over anhydrous sodium sulfate, filtered to obtain the filtrate and dried under reduced pressure. The crude product is separated by column chromatography (ethyl acetate / petroleum ether: 0-20%) to obtain compound I-2. MSm / z:293.0[M+H] +

[0164] Step 2: Synthesis of Compound I

[0165] Compound I-2 (200 mg, 683.84 μmol) was placed in a three-necked flask, anhydrous tetrahydrofuran (12 mL) was added, and nitrogen was replaced. The reaction solution was cooled to -78 °C, lithium diisopropylamide (2M, 683.84 μL, 2 eq) was added, and stirred for 30 minutes, trimethyl borate (99.48 mg, 957.38 μmol, 108.13 μL, 1.4 eq) was added, and the mixture was heated to room temperature of 18 °C and stirred for 1 hour. After the reaction was completed, 10 mL of saturated aqueous ammonium chloride solution was added to the reaction solution and stirred for 15 minutes. The mixture was extracted with ethyl acetate (30 mL*3), the organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered to obtain the filtrate, and dried under reduced pressure. Intermediate I was obtained.

[0166] MSm / z:337.1[M+H]+ .

[0167] Example 1

[0168]

[0169] Step 1: Synthesis of compound 1-B

[0170] To a solution of compound 1-A (3.22 g, 11.76 mmol, 1 eq) in N,N-dimethylformamide (20.00 mL) at room temperature, (R)-3-methylmorpholine (2.38 g, 23.51 mmol, 2 eq) and potassium carbonate (3.25 g, 23.51 mmol, 63.60 μL, 2 eq) were added, and then stirred at 130 ° C in a nitrogen atmosphere for 18 hours. The reaction system was diluted with water (60 mL), washed with ethyl acetate (50 mL × 3), washed with saturated brine (50 mL), and dried over anhydrous sodium sulfate. After filtering off the desiccant, the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate: 0% to 20%) to obtain compound 1-B. MS-ESIm / z: 339.0 [M+H] +

[0171] Step 2: Synthesis of Compound 1-C

[0172] Palladium acetate (34.81 mg, 155.06 μmol, 0.07 eq) and tricyclohexylphosphine (93.18 mg, 332.28 μmol, 107.72 μL, 0.15 eq) were added to a solution of compound 1-B (0.75 g, 2.22 mmol, 1 eq), 1,4-dimethyl-1H-1,2,3-triazole (258.16 mg, 2.66 mmol, 1.2 eq) and potassium carbonate (918.48 mg, 6.65 mmol, 3 eq) in N,N-dimethylacetamide (2 mL), and nitrogen was bubbled. The reaction was stirred in a microwave at 110 ° C for 1 hour. After the reaction solution was cooled, it was filtered and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (petroleum ether / ethyl acetate: 8% to 50%) to obtain compound 1-C. MS-ESIm / z: 308.1 [M+H] +

[0173] Step 3: Synthesis of the compound of formula (1)

[0174] 1-C (160 mg, 519.86 μmol) was placed in a microwave reactor, ethylene glycol dimethyl ether (5 mL) was added, intermediate I (174.82 mg, 519.86 μmol), an aqueous solution of sodium carbonate (2M, 1.82 mL) and [1,1-bis(diphenylphosphino)ferrocene] dichloropalladium dichloromethane complex (42.45 mg, 51.99 μmol) were added, nitrogen was bubbled for 2 minutes, microwave heated to 110 ° C and stirred for 0.5 hours. After the reaction was completed, 60 mL of water was added to the reaction solution, and the reaction solution was extracted with 210 mL (70*3) of ethyl acetate. The organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered to obtain the filtrate, and dried under reduced pressure. The crude product was separated by column chromatography (tetrahydrofuran / petroleum ether: 20-80%) to obtain the compound of formula (1).

[0175] MSm / z:408.2[M+H] +

[0176] 1 HNMR(400MHz, CDCl3)δppm1.37(brd,J=6.27Hz,3H)2.42(s,3H)3.27-3.40(m,1H)3.67(brt,J=10.92Hz,1H)3.84(brs,2H)4.0 0-4.13(m,5H)4.38(brd,J=4.02Hz,1H)6.46(brs,1H)7.08(brs,1H)7.14(brs,1H)7.54(brs,1H)8.40(brs,1H)8.85(brs,1H)

[0177] Example 2: Preparation of Form I

[0178] Take the compound of formula (1) and pass it through a silica gel column ( 40g Silica Flash Column, Eluent of 10-60% tetrahydrofuran / petroleum ether @ 40 mL / min, gradient elution) was used for purification. 25g Silica Flash Column, Eluent of 0-10% methanol / dichloromethane @35 mL / min, gradient elution) was used for further purification and concentration to obtain Form I of the compound of formula (1).

[0179] The above silica gel column is a commercially available preparative column, which performs gradient elution by itself.

[0180] Example 3: Preparation of Form II

[0181] The compound of formula (1) (2 g) was placed in a single-mouth bottle, and ethanol (20 mL) was added. The mixture was heated to 50°C-60°C and stirred. If the mixture was not dissolved, the heating was stopped and the temperature was gradually lowered to 25°C and stirred for 14 hours. The suspension was filtered under reduced pressure and the filter cake was dried in a vacuum drying oven at 45°C for 4 hours to obtain the II crystal form of the compound of formula (1).

[0182] Example 4: Preparation of Form III

[0183] The compound of formula (1) (5 g) was placed in a single-mouth bottle, methanol (25 mL) was added, and the mixture was heated until reflux began. After reflux for 0.5 hours, the heating was stopped and the temperature was lowered to about 28°C. The mixture was stirred for 2 hours, and the suspension was filtered to obtain the III crystal form of the compound of formula (1).

[0184] Example 5: Preparation of Form IV

[0185] Crystal form II (5-10 mg) of the compound of formula (1) is heated to 240° C. and cooled to obtain crystal form IV.

[0186] Example 6: Preparation of Form V

[0187] Method 1:

[0188] The compound of formula (1) was suspended and stirred in EtOH / H2O (1:2, v / v) at 60°C for 2 days to obtain Form V.

[0189] Method 2:

[0190] Under stirring, add the compound of formula (1) (2 g) to ethanol (15 mL), then add ethanol (9 mL), raise the temperature to 80°C (reflux), and stir for 2 hours. Add V crystal seed (40 mg) under reflux, continue stirring for 1 hour, slowly cool to 24-28°C, and stir for about 100 hours. Filter, wash the filter cake with ethanol (5 mL), collect the solid, and vacuum dry the solid at 50°C for 16 hours to obtain V crystal of the compound of formula (1).

[0191] Experimental Example 1: In vitro cell activity experiment

[0192] By measuring IC 50 The inhibitory activity of the test compound on human ATR kinase was evaluated by using the kinase inhibitory value.

[0193] ATR / ATRIP(h) was incubated in assay buffer containing 50 nM GST-cMyc-p53 and Mg / ATP (10 uM). 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 5% EDTA solution was added to the reaction mixture. 2The plate was then read in time-resolved fluorescence mode and homogeneous time-resolved fluorescence was performed.

[0194] The fluorescence (HTRF) signal was determined according to the formula HTRF = 10000 x (Em665nm / Em620nm).

[0195] Table 6 In vitro ATR enzyme activity test results

[0196] Compound No. <![CDATA[IC 50 (nM)]]> Compound of formula (1) 33

[0197] The experimental results show that the compound of the present invention has strong inhibitory activity against ATR enzyme. At the same time, the present invention uses BAY1895344 as a reference control, and it is found that the enzymatic activity of the compound of formula (1) of the present invention is about 20 times higher than that of the reference control.

[0198] Experimental Example 2: In vitro cell activity experiment

[0199] This experiment studies the inhibitory effect of compounds on cell proliferation by detecting the effects of compounds on in vitro cell activity in the tumor cell line LoVo.

[0200] CellTiter-Glo Luminescent Cell Viability Assay The following steps were performed according to the instructions of the Promega CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega-G7573).

[0201] (1) Thaw CellTiter-Glo buffer and allow to cool to room temperature.

[0202] (2) Allow CellTiter-Glo Substrate to cool to room temperature.

[0203] (3) Prepare CellTiter-Glo working solution by adding CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to dissolve the substrate.

[0204] (4) Slowly vortex to fully dissolve.

[0205] (5) Take out the cell culture plate and let it stand for 30 minutes to equilibrate to room temperature.

[0206] (6) Add 50 μL (equal to half the volume of the cell culture medium in each well) of CellTiter-Glo working solution to each well. Wrap the cell plate with aluminum foil to avoid light.

[0207] (7) Shake the culture plate on an orbital shaker for 2 minutes to induce cell lysis.

[0208] (8) Place the culture plate at room temperature for 10 minutes to stabilize the luminescent signal.

[0209] (9). Detect luminescent signals on a SpectraMax i3x of Molecular Devices plate reader.

[0210] Data analysis

[0211] The inhibition rate (IR) of the test compound was calculated using the following formula: IR (%) = (1-(RLU compound-RLU blank control) / (RLU solvent control-RLU blank control))*100%. The inhibition rates of different concentrations of the compound were calculated in Excel, and then the inhibition curves were plotted and related parameters were calculated using GraphPadPrism software, including the minimum inhibition rate, maximum inhibition rate, and IC 50 .

[0212] The experimental results are shown in Table 7:

[0213] Table 7 In vitro LoVo cell proliferation inhibition test results

[0214] Compound No. <![CDATA[IC of LoVo cell proliferation 50 (μM)]]> Compound of formula (1) 0.102

[0215] The experimental results show that the compound of the present invention has a good inhibitory effect on LoVo tumor cells with missing ATM signaling pathway.

[0216] Experimental Example 3: In vivo pharmacokinetic properties study

[0217] Test sample: Based on the above experiments, the compound of formula (1) was selected for further experiments.

[0218] Experimental method: The purpose of this study is to determine the pharmacokinetic parameters of the compound of formula (1) and calculate its oral bioavailability in female Balb / c Nude mice. This project uses 4 female Balb / c Nude mice. Two mice were intravenously injected with a dose of 1 mg / kg, and plasma samples were collected at 0h (before administration) and 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, and 24h after administration. The other two mice were orally gavaged with a dose of 10 mg / kg or 25 mg / kg, and plasma samples were collected at 0h (before administration) and 0.25, 0.5, 1, 2, 4, 6, 8, and 24h after administration. The collected samples were then analyzed by LC / MS / MS and data were collected. The collected analysis data were used to calculate relevant pharmacokinetic parameters using PhoenixWinNonlin6.2.1 software.

[0219] The experimental results are shown in Tables 8 and 9:

[0220] 8 In vivo pharmacokinetics - intravenous administration results

[0221] <![CDATA[C0(nM)]]> Cl(mL / min / kg) <![CDATA[V dss (L / kg)]]> <![CDATA[T 1 / 2 (h)]]> <![CDATA[AUC 0-t (nM.h)]]> 2804 15.8 1.65 1.32 2549

[0222] 9 In vivo pharmacokinetics - oral administration results

[0223] Dosage (mg / kg) <![CDATA[C max (nM)]]> <![CDATA[T 1 / 2 (h)]]> <![CDATA[AUC 0-t (nM.h)]]> F(％) 10 9565 1.4 22992 91.6 25 17396 3.8 82019 -

[0224] Note: C0 (nM) is the drug concentration in the body at 0 minutes; Cl (mL / min / kg) is the drug clearance rate in the body; V dss (L / kg) is the volume of distribution of the drug in the body; T 1 / 2 (h) is half-life; AUC 0-t (nM.h) is the drug exposure in vivo; Cmax (nM) is the maximum drug concentration in vivo.

[0225] Experimental conclusion: The compounds of the present invention have good in vivo pharmacokinetic properties such as exposure and bioavailability.

[0226] Experimental Example 4: In vivo efficacy study of human gastric cancer cell SNU-601CDX

[0227] Purpose:

[0228] The main purpose of this study is to investigate the antitumor efficacy of the test substance in the human gastric cancer cell SNU-601 xenograft tumor model.

[0229] Experimental methods:

[0230] 1. Experimental Animals

[0231] Species: Mouse strain: CB17SCID mouse

[0232] Supplier: Huafukang Laboratory Animal Technology Co., Ltd.

[0233] Age: 6-8 weeks

[0234] Gender: Female

[0235] 2. Cell Culture

[0236] Human gastric cancer SNU-601 cells, sourced from KCLB (Cat. No. 00601), were maintained and subcultured by Huiyuan Biotech (Shanghai) Co., Ltd. The in vitro culture conditions were RPMI1640 medium (containing 300 mg / L L-glutamine) with 10% fetal bovine serum, 25 mM HEPES and 25 mM sodium bicarbonate added, cultured in a 37°C 5% CO2 incubator, and subcultured two to three times a week. When the cell number reached the requirement, the cells were harvested and counted. 0.2 mL (5×106) of SNU-601 cells (resuspended in DPBS:Matrigel=1:1) were subcutaneously inoculated on the right back of each mouse, and group dosing began when the average tumor volume reached 147.61 mm3.

[0237] 3. Dosage of the test substance

[0238] Dosage: The compound of formula (1) was orally administered at three doses of 15 mg / kg (on for 3 days and rest for 4 days), 10 mg / kg (on for 3 days and rest for 4 days) and 5 mg / kg (continuous administration).

[0239] 4. Tumor Measurement and Experimental Indicators

[0240] Tumor diameter was measured with a vernier caliper three times a week. The formula for calculating tumor volume was: V = 0.5 × a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The relative tumor volume (RTV) is calculated as: RTV (%) = (Vt / V1) × 100; the animal body weight change (BWC) is calculated as: BWC (%) = (BWt–BW1) / BW1 × 100, where V1 and BW1 refer to the tumor volume and body weight of an animal on the day of group administration, and Vt and BWt refer to the tumor volume and body weight of an animal measured at a certain time.

[0241] The anti-tumor efficacy of the compound is evaluated by TGI (%) or relative tumor proliferation rate T / C (%). Relative tumor proliferation rate T / C (%) = TRTV / CRTV × 100 (TRTV: average RTV of the treatment group; CRTV: average RTV of the negative control group). The relative tumor volume (RTV) is calculated based on the results of tumor measurement. The calculation formula is RTV = Vt / V1, where V1 is the tumor volume measured at the time of group administration (i.e., D1), Vt is the tumor volume at a certain measurement, and TRTV and CRTV are taken from the same day.

[0242] TGI (%) reflects the tumor growth inhibition rate. TGI (%) = [(1-(average tumor volume at the end of drug administration in a treatment group - average tumor volume at the beginning of drug administration in the treatment group)) / (average tumor volume at the end of treatment in the solvent control group - average tumor volume at the beginning of treatment in the solvent control group)] × 100.

[0243] After the experiment, the tumor weight was measured and the percentage of Tweight / Cweight was calculated. Tweight and Cweight represent the tumor weights of the drug-treated group and the vehicle control group, respectively.

[0244] 5. Experimental Results

[0245] In this experiment, the human gastric cancer cell SNU601 xenograft tumor model was used to evaluate the in vivo efficacy of the test compound. During the entire dosing period, no animal stopped taking the drug due to a body weight loss of more than 10%, and the efficacy test was terminated on the 21st day after dosing.

[0246] On day 21 after administration, the tumor volume of the vehicle group reached 890.01±184.62mm 3 Compared with the vehicle control group, the compound of formula (1) showed certain anti-tumor effects at the dosages of 15 mg / kg, 10 mg / kg and 5 mg / kg. The corresponding tumor volumes were 108.74±9.67 mm 3 , 136.74±14.46mm3 and 229.99±24.42mm 3 The tumor inhibition rates TGI were 104.81% (p < 0.01), 101.08% (p < 0.01)) and 88.61% (p < 0.01)) respectively.

[0247] During the entire experimental period, the body weight of the animals in the group treated with the compound of formula (1) did not decrease significantly, and the animals were in normal condition.

[0248] In summary, the compounds of the present invention can significantly inhibit the growth of human gastric cancer SNU-601 xenograft tumors and are relatively tolerable to mice.

[0249] Experimental Example 5: Solid Stability Test of Compound II of Formula (I) under High Temperature, High Humidity and Light Conditions

[0250] According to the influencing factors and accelerated test conditions, accurately weigh about 20 mg of the crystal form II of the compound of formula (I) and place it in a dry and clean glass bottle. Weigh 2 portions, mark them as S1 condition-time and S2 condition-time respectively, and then weigh 15 mg and place it in a dry and clean glass bottle, mark it as S3-condition-time, spread it into a thin layer, and use it as the formal test sample, and place it under the influencing factor test conditions (60°C, 25°C / 92.5%RH (relative humidity), light, light control) and accelerated conditions (40°C / 75%RH and 60°C / 75%RH), and the samples are completely exposed. Samples are taken and analyzed at 60°C, 25°C / 92.5%RH, light, and light control at 5 days and 10 days. Samples are taken and analyzed at 40°C / 75%RH and 60°C / 75%RH at 1 month, 2 months, and 3 months. The HPLC analysis method of the relevant substances is shown in Table 10, and the chromatogram is shown in Table 10. Fig.10 The analysis results of the sample content and related substances of the solid form stability of compound II of formula (1) are shown in Table 11.

[0251] Table 10 High performance liquid chromatography analysis method for related substances

[0252]

[0253]

[0254] Table 11: Content of solid form stability samples of compound II of formula (1) and related substance analysis results (5 days, 10 days, 1 month)

[0255] Stability conditions time Crystal form after experiment Total impurities (%) content(%) 60℃ Day 0 Form II 0.35 100.0 60℃ 5 days Form II 0.35 100.4 60℃ 10 days Form II 0.34 101.1 25℃, 92.5% humidity 5 days Form II 0.30 101.0 25℃, 92.5% humidity 10 days Form II 0.35 101.3 illumination 10 days Form II 0.39 101.2 Protect from light 10 days Form II 0.35 103.5 40℃-75% humidity 1 month Form II 0.51 100.3 60℃-75% humidity 1 month Form II 0.50 100.0 40℃-75% humidity 2 months Form II 0.53 100.2 60℃-75% humidity 2 months Form II 0.54 100.4 40℃-75% humidity 3 months Form II 0.47 99.9 60℃-75% humidity 3 months Form II 0.48 100.1

[0256] Conclusion: The crystal form of the compound II of formula (1) did not change in the solid influence factor and accelerated test for 3 months, and had good physical stability. In the analysis of related substances, the total amount of impurities did not increase significantly, and the content was stable.

[0257] Experimental conclusion: The crystal form of the present invention has good stability and is easy to store, transport and manufacture as a medicine.

Claims

1. Form II of the compound of formula (1), by Cu-kα radiation, has an X-ray powder diffraction pattern having characteristic diffraction peaks at least at the following 2θ angles: 8.76±0.20°, 15.93±0.20° and 17.94±0.20°:

2. The II crystal form according to claim 1, characterized in that: Its X-ray powder diffraction spectrum also includes characteristic diffraction peaks at the following 2θ angles: 7.97±0.20°, 18.61±0.20°, and 24.53±0.20°.

3. The II crystal form according to claim 1 or 2, characterized in that: Its X-ray powder diffraction pattern also includes characteristic diffraction peaks at the following 2θ angles: 10.99±0.20°, 12.98±0.20°, 14.70±0.20°, 16.47±0.20°, 20.45±0.20°, 25.10±0.20°, and 25.81±0.20°.

4. The II crystal form according to claim 3, characterized in that: Its X-ray powder diffraction pattern has characteristic diffraction peaks at least at the following 2θ angles: 7.97°, 8.76°, 10.99°, 12.98°, 14.70°, 15.93°, 16.47°, 17.94°, 18.61°, 20.45°, 21.98°, 22.40°, 22.90°, 23.87°, 24.53°, 25.10°, 25.81°, 26.06°, 27.20°, 27.98°, 28.71°, 29.37°, 29.86°, 30.29°, 31.56°, 32.14°, 35.31° and 38.12°.

5. The II crystal form according to claim 4, characterized in that: Its XRPD pattern is basically as shown in Figure 3.

6. The II crystal form according to claim 1, characterized in that: The differential scanning calorimetry curve of Form II has endothermic peaks at 222.1±3°C and 256.5±3°C; the thermogravimetric analysis curve shows weight loss at 250±3°C.

7. Form V of the compound of formula (1), by Cu-kα radiation, has an X-ray powder diffraction pattern having characteristic diffraction peaks at least at the following 2θ angles: 8.4539±0.20°, 9.4597±0.20°, 17.3745±0.20°, 18.2963±0.20° and 25.1766±0.20°:

8. The V crystal form according to claim 7, characterized in that: Its X-ray powder diffraction pattern also includes characteristic diffraction peaks at the following 2θ angles: 15.7873±0.20°, 16.1904±0.20°, 16.8510±0.20°, 17.3745±0.20°, 21.2656±0.20°, 23.3932±0.20°, and 26.6250±0.20°.

9. The V crystal form according to claim 7 or 8, characterized in that: Its X-ray powder diffraction pattern also includes characteristic diffraction peaks at the following 2θ angles: 10.9365±0.20°, 11.9742±0.20°, 18.6558±0.20°, 18.9325±0.20°, 19.9943±0.20°, 22.1742±0.20°, 24.0827±0.20°, 26.1776±0.20°, and 27.0062±0.20°.

10. The V crystal form according to claim 9, characterized in that: Its X-ray powder diffraction pattern has characteristic diffraction peaks at least at the following 2θ angles: 8.4539°, 9.4597°, 10.9365°, 11.9742°, 15.7873°, 16.1904°, 16.8510°, 17.3745°, 18.2963°, 18.6558°, 18.9325°, 19.9943°, 21.2656°, 22.1742°, 23.3932°, 24.0827°, 25.1766°, 26.1776°, 26.6250°, 27.0062°, 27.6261°, 28.5886°, 29.4495°, 31.7776° and 32.9126°.

11. The V crystal form according to claim 10, characterized in that: Its XRPD spectrum is basically shown in Figure 8.

12. The V crystal form according to claim 7, characterized in that: The differential scanning calorimetry curve has endothermic peaks at 236.3±1℃ and 256.3±3℃, and an exothermic peak at 237.9±1℃; the thermogravimetric analysis curve has weight loss at 150±3℃.

13. Use of the crystal form according to any one of claims 1 to 12 in the preparation of ATR enzyme inhibitors.

14. Use of the crystal form according to any one of claims 1 to 12 in the preparation of products for treating diseases related to ATR enzyme activation.

15. The use according to claim 14, characterized in that: The disease related to ATR enzyme activation is selected from cancer.

16. Use of the crystal form according to any one of claims 1 to 12 and an ATM enzyme inhibitor in the preparation of a combined drug for treating cancer.

17. The use according to claim 15 or 16, characterized in that: The cancer is selected from breast cancer, cervical cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, ovarian cancer, pancreatic cancer, testicular cancer, bladder cancer, myeloma, non-small cell lung cancer, leukemia, lymphoma, melanoma, esophageal cancer, connective tissue cancer, mesothelioma, prostate cancer, bone cancer, and kidney cancer.

18. A pharmaceutical composition, characterized in that: It includes the crystal form described in any one of claims 1-12.

19. A method for preparing a crystalline form of compound II of formula (1) according to any one of claims 1 to 6, characterized in that: It includes the following: Take the compound of formula (1), add ethanol, heat to 40-65°C to dissolve, then reduce the temperature to 25°C±2°C, crystallize, and the obtained solid is the II crystal form.

20. A method for preparing the crystal form V of the compound of formula (1) according to any one of claims 7 to 12, characterized in that: It includes the following: Method 1: Compound (1) is stirred in 40-60% v / v ethanol at 60°C ± 5°C to obtain Form V; Or method 2: add compound (1) to ethanol under stirring, then add ethanol, reflux at 80°C±5°C, add V crystal seed crystals under reflux, continue stirring, slowly cool to 24-28°C, crystallize, and obtain V crystal.

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