CN116178366A

CN116178366A - Crystal forms of fluoropyridine pyrrole compound and preparation method thereof

Info

Publication number: CN116178366A
Application number: CN202111420177.3A
Authority: CN
Inventors: 周建光; 葛广存; 李林; 段吴平; 李不鱼
Original assignee: Shenzhen Lingfang Biomedical Technology Co ltd
Current assignee: Shenzhen Lingfang Biomedical Technology Co ltd
Priority date: 2021-11-26
Filing date: 2021-11-26
Publication date: 2023-05-30
Also published as: WO2023093447A1

Abstract

The invention discloses a crystal form of a fluoropyridine pyrrole compound shown in a formula (1), a preparation method and application thereof. In the invention, the compound shown in the formula (1) has stronger inhibition activity against ATR enzyme and certain anticancer activity. The compound of the formula (1) is prepared into various crystal forms, so that the product forms which are more stable than amorphous compounds and less influenced by light, heat and humidity are obtained, and the preparation method has wider medicament prospect.

Description

Crystal forms of fluoropyridine pyrrole compound and preparation method thereof

Technical Field

The invention relates to a crystal form of a fluoropyridine pyrrole compound shown in a formula (1) and a preparation method thereof.

Background

DNA Damage Response (DDR) ensures the integrity of the genome of living cells by a wide variety of signaling pathways. Intracellular proteins will directly recognize aberrant DNA structures and activate related kinases of the DDR pathway to cope with extensive DNA damage and increased replication pressure within cancer cells. The DDR pathway can either survive in the face of genomic instability and replication stress, or mediate aging or programmed death of irreparable damaged cells. Defects of DDR genes promote mutation of driving genes, tumor heterogeneity and apoptosis escape through various ways, so that the effect of promoting tumor growth is achieved.

ATR (telangiectasia ataxia mutation and RAD-3 related protein kinase) belongs to PIKKs (phosphatidylinositol-3-kinase-related kinase) family, and is involved in damage repair of DNA to maintain gene stability. ATR protein kinase damage to DNA, replicative stress and cell cycle disturbances produce a synergistic response. ATR and ATM belong to the PIKK family of serine/threonine protein kinases, which are integral components of cell cycle and DNA damage repair, among others, chkl, BRCAl, p53.ATR is mainly responsible for DNA replication stress (replication fork arrest), repair work of single strand breaks.

ATR is activated by DNA single strand structure when DNA double strand breaks undergo excision or replication fork arrest. The DNA polymerase remains in the process of DNA replication, and the replication helicase continues to unwind at the front of the DNA replication fork, resulting in the production of long single stranded DNA (ssDNA) which is then bound by the single stranded DNA and RPA (replication protein a). Replication stress or DNA damage by RPA recruited ATR/ATR action protein complexes to the injury site, RPA-single stranded DNA complexes activated RAD17/rfc2-5 complexes binding to the injury site, DNA-ssDNA junctions activated Rad9-HUS1-RAD1 (9-1-1) heterotrimers, 9-1-1 in turn recruited TopBP1 to activate ATR. Once ATR is activated, ATR promotes DNA repair, stabilizes and restarts the arrested replication fork and transient cell cycle arrest by downstream targets. These functions are achieved by mediating the downstream target Chk1 by ATR. ATR acts as a DNA-damaging cell cycle checkpoint in S phase. It can mediate the degradation of CDC25A through Chk1, thus delay the replication process of DNA, offer time for repairing replication fork. ATR is also the primary regulator of the G2/M cell cycle checkpoint, preventing cells from prematurely entering mitosis before DNA replication is complete or DNA damage. This ATR-dependent G2/M cell cycle arrest is mediated primarily by two mechanisms: 1. degradation of CDC 25A. 2. Cdc25C was phosphorylated by Chk1 to bind 14-3-protein. 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.

ATM genes are frequently mutated in tumor cells, indicating that loss of ATM activity favors cancer cell survival. ATM kinase inactivation makes cells more dependent on ATR-mediated signaling pathways, and combined inactivation of ATR and ATM can induce synthetic lethality in cancer cells. Thus, inhibition of ATR may be an effective approach in future cancer treatments.

Disclosure of Invention

The research of the invention discovers that the compound of the formula (1) has stronger inhibition activity against ATR enzyme; meanwhile, the anti-tumor cell has a good inhibition effect on LoVo tumor cells with the loss of ATM signal channels; meanwhile, the compound has good PK parameters such as exposure, bioavailability and the like, and is suitable for medication; in addition, the compound of the invention can obviously inhibit the growth of human gastric cancer SNU-601 xenograft tumor and is relatively tolerant to mice.

The compound shown in the formula (1) is prepared by carrying out Suzuki coupling reaction on a compound 1-C and an intermediate I.

The present invention provides a form I of a compound of formula (1) having an X-ray powder diffraction (XRPD) pattern having characteristic diffraction peaks at least at one or more of the following 2Θ angles: 7.90±0.20°, 9.35±0.20° and 18.81±0.20°:

in some aspects of the invention, the X-ray powder diffraction pattern of form I above has characteristic diffraction peaks at least at 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 °, 20.14.+ -. 0.20 °.

In some aspects of the invention, the X-ray powder diffraction pattern of form I above has characteristic diffraction peaks at least at 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 °, 22.61.+ -. 0.20 °.

In some aspects of the invention, the X-ray powder diffraction pattern of form I above has characteristic diffraction peaks at least at 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 °, 22.61.+ -. 0.20 °.

In some aspects of the invention, the X-ray powder diffraction pattern of form I above 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°.

In some aspects of the invention, the X-ray powder diffraction pattern of form I above has characteristic diffraction peaks at least at 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 °, 22.61 °.

In some embodiments of the invention, form I above has an XRPD pattern substantially as shown in figure 1.

In some aspects of the invention, XRPD pattern analytical data for form I above is shown in table 1:

TABLE 1 XRPD resolution data for form I of Compound (1)

In some aspects of the invention, the differential scanning calorimetry curve of form I has peaks with endothermic peaks at 64.8±3 ℃, 153.8±3 ℃ and 252.4±3 ℃.

In some aspects of the invention, the thermogravimetric analysis of form I above loses about 6.71% weight at 180.0±3 ℃.

In some embodiments of the invention, the DSC curve and TGA curve of the form I are shown in FIG. 2.

The present invention provides a form II of a compound of formula (1) having an X-ray powder diffraction (XRPD) pattern having characteristic diffraction peaks at least at one or more of the following 2Θ angles: 8.76 ± 0.20 °, 15.93 ± 0.20 ° and 17.94 ± 0.20 °:

In some aspects of the invention, the X-ray powder diffraction pattern of form II has characteristic diffraction peaks at least at 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 °.

In some aspects of the invention, the X-ray powder diffraction pattern of form II has characteristic diffraction peaks at least at 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 °, 22.90.+ -. 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 °, 30.29.+ -. 0.20 °, 31.56.+ -. 0.20 °, 32.14.+ -. 0.20 °, 35.31.+ -. 0.20 ° and 38.12.+ -. 0.20 °.

In some aspects of the invention, the X-ray powder diffraction pattern of form II has characteristic diffraction peaks at least at 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 °, 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°.

In some aspects of the invention, the X-ray powder diffraction pattern of form II has characteristic diffraction peaks at least at 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 °.

In some embodiments of the invention, form II above has an XRPD pattern substantially as shown in figure 3.

In some aspects of the invention, XRPD pattern analytical data for the form II are shown in table 2:

TABLE 2 XRPD resolution data for form II of Compound (1)

In some embodiments of the invention, the differential scanning calorimetry curve of form II has an endothermic peak at 222.1±3 ℃ and 256.5±3 ℃.

In some aspects of the invention, the thermogravimetric analysis of form II above loses about 1.02% weight at 250±3 ℃.

In some embodiments of the invention, the DSC curve and TGA curve of form II are shown in FIG. 4.

The present invention provides a form III of a compound of formula (1) having an X-ray powder diffraction (XRPD) pattern having characteristic diffraction peaks at least at one or more of the following 2Θ angles: 14.848±0.20°, 17.653±0.20° and 19.135 ±0.20°:

In some aspects of the invention, the X-ray powder diffraction pattern of form III has characteristic diffraction peaks at least at 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 °.

In some aspects of the invention, the X-ray powder diffraction pattern of form III has characteristic diffraction peaks at least at 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°.

In some aspects of the invention, the X-ray powder diffraction pattern of form III has characteristic diffraction peaks at least at 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 °; 21.035 + -0.20 °, and 21.035 + -0.20 °, and 21.035 + -0.20 °, 21.035 + -0.20 ° and 21.035 + -0.20 °.

In some aspects of the invention, the X-ray powder diffraction pattern of form III has characteristic diffraction peaks at least at one or more of the following 2θ angles: 14.848.+ -. 0.20 °, 17.653.+ -. 0.20 °, and/or.+ -. 0.20 °, and/or + -0.20 DEG, and/or + -0.20 deg., and/or + -0.20 deg., and/or + -0.20 DEG, and/or 24.14.+ -. 0.20 °, and/or.+ -. 0.20 °, and/or 24.14±0.20°, and/or ±0.20°; and/or + -0.20 deg..

In some aspects of the invention, the X-ray powder diffraction pattern of form III has characteristic diffraction peaks at least at 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 °, 15.162 °, 17.653 °, 15.162 °, 15.162 °; 15.162 °, 15.162 °; 15.162 °, 15.162 °.

In some embodiments of the invention, form III above has an XRPD pattern substantially as shown in figure 5.

In some aspects of the invention, XRPD pattern analytical data for the above form III are shown in table 3:

TABLE 3 XRPD resolution data for form III of Compound (1)

In some aspects of the invention, the thermogravimetric analysis of the form III described above loses about 5.34% weight at 150±3 ℃.

In some embodiments of the invention, the TGA profile of form III is shown in fig. 6.

The present invention provides crystalline form IV of a compound of formula (1) having an X-ray powder diffraction (XRPD) pattern having characteristic diffraction peaks at least at one or more of the following 2Θ angles: 8.2794 ±0.20°, 15.7523 ±0.20° and 17.5827 ±0.20°:

in some aspects of the invention, the X-ray powder diffraction pattern of form IV above has characteristic diffraction peaks at least at 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°.

In some aspects of the invention, the X-ray powder diffraction pattern of form IV above has characteristic diffraction peaks at least at 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 °.

In some aspects of the invention, the X-ray powder diffraction pattern of form IV above has characteristic diffraction peaks at least at 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°, 20.0470 ±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 °.

In some aspects of the invention, the X-ray powder diffraction pattern of form IV above has characteristic diffraction peaks at least at 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°, 20.0470 ±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°.

In some aspects of the invention, the X-ray powder diffraction pattern of form IV above has characteristic diffraction peaks at least at 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 ±0.20°, 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°, and/or 52±0.20°, and/or 20.

In some aspects of the invention, the X-ray powder diffraction pattern of form IV above has characteristic diffraction peaks at least at one or more of the following 2θ angles: 8.2794 + -0.10 deg., 15.7523 + -0.10 deg., and/or 17.5827 + -0.10 deg., and/or 8.4208 + -0.10 deg., and/or 16.3613 + -0.10 deg., and/or 18.7041 + -0.10 deg., and/or 24.7696 + -0.10 deg., and/or 24.9328 + -0.10 deg., and/or 10.6855 + -0.10 deg., and/or 12.3408 + -0.10 deg., and/or 13.0615 + -0.10 deg., and/or 14.6650 + -0.10 deg., and/or 20.0470 + -0.10 deg., and/or 20.8774 + -0.10 deg., and/or 21.7465 + -0.10 deg., and/or 22.1529 + -0.10 deg., and/or 23.9930 + -0.10 deg., and/or 26.2785 + -0.10 deg., and/or 27.1454 + -0.10 deg., and/or 28.2680 + -0.10 deg., and/or 29.7925 + -0.10 deg., and/or 30.8621 + -0.10 deg., and/or 32.4019 + -0.10 deg., and/or 3723 + -0.10 deg., and/or 33.3887 + -0.10 deg., and/or 5210 deg.

In some aspects of the invention, the X-ray powder diffraction pattern of form IV above has characteristic diffraction peaks at least at 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 °, 18.7041 °, 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 °.

In some aspects of the invention, the XRPD pattern of form IV is substantially as shown in figure 7.

In some aspects of the invention, XRPD pattern analytical data for the IV crystalline form described above is shown in table 4:

TABLE 4 XRPD resolution data for form IV of Compound (1)

The present invention provides a crystalline form V of a compound of formula (1) having an X-ray powder diffraction (XRPD) pattern having characteristic diffraction peaks at least at the following 2Θ angles: 8.4539 ±0.20°, 17.3745 ±0.20° and 25.1766 ±0.20°:

in some aspects of the invention, the X-ray powder diffraction pattern of the V-form has characteristic diffraction peaks at least at 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°.

In some aspects of the invention, the X-ray powder diffraction pattern of the V-form has characteristic diffraction peaks at least at 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 °.

In some aspects of the invention, the X-ray powder diffraction pattern of the V-form has characteristic diffraction peaks at least at 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°.

In some aspects of the invention, the X-ray powder diffraction pattern of the V-form has characteristic diffraction peaks at least at 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 °.

In some aspects of the invention, form V has an XRPD pattern substantially as shown in figure 8.

In some aspects of the invention, XRPD pattern analytical data for the V crystalline form described above are shown in table 5:

TABLE 5 XRPD resolution data for form V of Compound (1)

In some embodiments of the present invention, the V-form differential scanning calorimetry curve has an endothermic peak at 236.3±1 ℃ and 256.3±3 ℃ (initial temperature), 1 exothermic peak at 237.9±1 ℃, the endothermic peak at 256.3±3 ℃ (initial temperature) corresponds to an enthalpy of 99.61J/g, and the endothermic peak near 236.3 ℃ overlaps with the exothermic peak near 237.9 ℃, and the enthalpy cannot be calculated.

In some aspects of the invention, the thermogravimetric analysis of the form V described above loses about 2.0% weight at 150±3 ℃.

In some embodiments of the invention, the DSC curve and TGA curve of the above-described form V are shown in FIG. 9.

The invention also provides application of the crystal form in preparation of ATR enzyme inhibitors.

The invention also provides application of the crystal form in preparing a product for treating related diseases caused by ATR enzyme activation.

ATR kinase is currently considered a potential cancer therapeutic target. Because, in contrast to normal cells, a fundamental feature of tumor cells is genomic instability and susceptibility to mutation, which is often accompanied by a large number of functional deletions in stabilizing and repairing genomic DNA, cancer cells rely more on ATR kinase for self-repair, ATR and its involved signaling pathways are critical for genomic stabilization as well as tumor development, development and treatment. Previously, a large number of functional and preclinical experimental data indicate that ATR kinase inhibitors are directly highly effective in killing tumor cells.

Based on the inhibitory activity of the crystal form of the invention on ATR enzyme, the related diseases caused by ATR enzyme activation are selected from cancers.

The invention also provides application of the crystal form and an ATM enzyme inhibitor in preparing a combined medicament for treating cancers.

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, mesothelial cancer, prostate cancer, bone cancer, and renal cancer.

The invention also provides a pharmaceutical composition comprising the crystalline form as described above.

The invention also provides a preparation method of the compound I crystal form of the formula (1), which comprises the following steps:

purifying the compound of the formula (1) by gradient elution with 10-60% tetrahydrofuran/petroleum ether, and then gradient elution with 0-10% methanol/dichloromethane to obtain the compound of the formula (1) in the form I.

The invention also provides a preparation method of the compound II crystal form of the formula (1), which comprises the following steps:

and (3) adding ethanol into the compound shown in the formula (1), heating to 40-65 ℃ for dissolution, cooling to 25+/-2 ℃, and crystallizing to obtain a solid substance, namely the crystal form II.

In the preparation of the II crystal form, the concentration and the dosage of the ethanol only need to meet the requirement that the compound is dissolved and dissolved, and solid precipitation exists when the temperature is reduced.

The invention also provides a preparation method of the compound III crystal form of the formula (1), which comprises the following steps:

purifying the compound of formula (1) by a silica gel column, wherein the eluent is petroleum ether, ethyl acetate=1:1, petroleum ether, tetrahydrofuran=2:1, and dichloromethane, methanol=25:1 in sequence; purifying the product, adding methanol, refluxing, cooling to 28+/-2 ℃, and crystallizing to obtain the III crystal form.

The invention also provides a preparation method of the compound IV crystal form of the formula (1), which comprises the following steps: and heating the crystal form II to 240+/-5 ℃ and cooling to obtain the crystal form IV.

The invention also provides a preparation method of the compound V crystal form of the formula (1), which comprises the following steps:

the method comprises the following steps: taking a compound of the formula (1), and stirring in 40-60% V/V ethanol at 60+/-5 ℃ to obtain a V crystal form;

or a second method: adding a compound of the formula (1) into ethanol under stirring, adding ethanol, refluxing at 80+/-5 ℃, adding a crystal seed of the V crystal form under the refluxing state, continuously stirring, slowly cooling to 24-28 ℃, and crystallizing to obtain the V crystal form.

The temperature change in the invention can be controlled by adopting conventional equipment in experiments or production.

The preparation of various crystal forms can use different methods such as solution crystallization, solvent beating crystallization, incomplete crystallization, and crystallization induced by another seed crystal.

In the invention, the II crystal form and the V crystal form respectively correspond to a kinetic stable crystal form and a thermodynamic relatively stable crystal form, and according to the basic principle of crystallization theory, when the II crystal form is separated out, the II crystal form is obtained by rapid crystallization, and the V crystal form can be obtained by adding seed crystal V for induction and slow crystallization. In addition, the crystal form II is pulped in a solvent with certain solubility, and as the saturated solubility of the crystal form V is lower than that of the crystal form II, solution precipitation can occur to obtain the crystal form V when solid and liquid are balanced, so that the pulping and crystal transformation process is realized.

By "devitrification" is meant that when the material is in an unbalanced state, additional phases are precipitated, which are precipitated in the form of crystals.

The term "gradient elution" means that the concentration ratio of the mobile phase is continuously changed to a certain extent in the same elution period.

The product, the pharmaceutical composition or the combination drug can be added with a proper amount of pharmaceutically acceptable auxiliary materials.

The term "pharmaceutically acceptable" as used herein is meant to include any substance which does not interfere with the effectiveness of the biological activity of the active ingredient and which is not toxic to the host.

The pharmaceutically acceptable auxiliary materials are the general names of all additional materials except the main drugs in the medicine, and the auxiliary materials have the following properties: (1) no toxic or side effect to human body; (2) The chemical property is stable, and is not easily influenced by temperature, pH, preservation time and the like; (3) No incompatibility with the main medicine, and no influence on the curative effect and quality inspection of the main medicine; (4) does not interact with the packaging material.

Adjuvants in the present invention include, but are not limited to, fillers (diluents), lubricants (glidants or anti-adherents), dispersants, wetting agents, binders, conditioning agents, solubilizing agents, antioxidants, bacteriostats, emulsifiers, disintegrants, and the like. The binder comprises syrup, acacia, gelatin, sorbitol, tragacanth, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethylcellulose, ethylcellulose or hydroxypropyl methylcellulose), gelatin slurry, syrup, starch slurry or polyvinylpyrrolidone; the filler comprises lactose, sugar powder, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salt (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; the lubricant comprises aerosil, magnesium stearate, talcum powder, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol and the like; disintegrants include starch and its derivatives (e.g., sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinylpyrrolidone, microcrystalline cellulose, etc.; the wetting agent comprises sodium dodecyl sulfate, water or alcohol, etc.; the antioxidant comprises sodium sulfite, sodium bisulphite, sodium metabisulfite, dibutyl benzoic acid and the like; the bacteriostat comprises 0.5% phenol, 0.3% cresol, 0.5% chlorobutanol and the like; the regulator comprises hydrochloric acid, citric acid, potassium hydroxide (sodium), sodium citrate, buffer (including sodium dihydrogen phosphate and disodium hydrogen phosphate), etc.; the emulsifier comprises polysorbate-80, sorbitan without acid, pluronic F-68, lecithin, soybean lecithin, etc.; the solubilizer comprises Tween-80, bile, glycerol, etc.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

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

Compositions for parenteral injection may comprise 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 nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms of the compounds of the present invention for topical administration 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 which may be required if necessary.

The compounds of the invention can likewise be used in injectable formulations. Wherein the injection is selected from liquid injection (water injection), sterile powder for injection (powder injection) or tablet for injection (refers to a stamped tablet or a machine pressed tablet prepared by a sterile operation method for medicines), and is dissolved by water for injection when in use for subcutaneous or intramuscular injection.

Wherein the powder for injection contains at least an excipient in addition to the above-mentioned compounds. The excipients described in the present invention, which are components intentionally added to a drug, should not have pharmacological properties in the amounts used, however, the excipients may aid in processing, dissolution or dissolution of the drug, delivery by targeted route of administration, or stability.

The excipient of the present invention may be selected from one or a combination of two or more of carbohydrate, inorganic salt and polymer. Wherein the carbohydrate comprises monosaccharide, oligosaccharide or polysaccharide.

Monosaccharides are sugars which cannot be hydrolyzed any more, and are the basic units of molecules constituting various disaccharides and polysaccharides, and can be classified into triose, tetrose, pentose, hexose, etc., and monosaccharides in nature are mainly pentose and hexose, for example, glucose is aldohexose and fructose is ketohexose.

Oligosaccharides, also called oligosaccharides, are polymers of condensation of a few monosaccharides (2-10).

Polysaccharides are polymeric sugar high molecular carbohydrates composed of glycosidically bonded sugar chains, at least more than 10 monosaccharides.

The sterile powder for injection in the invention can be obtained by conventional sterile packaging or freeze drying processes.

In the invention, the compound shown in the formula (1) has stronger inhibition activity against ATR enzyme and certain anticancer activity. The compound of the formula (1) is prepared into various crystal forms, so that the product forms which are more stable than amorphous compounds and less influenced by light, heat and humidity are obtained, and the preparation method has wider medicament prospect.

Unless otherwise indicated, the terms and phrases used herein are intended to have the following meanings. A particular phrase or terminology, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.

Intermediate compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth herein, embodiments formed by combining them with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention.

The chemical reactions of the embodiments of the present invention are accomplished in a suitable solvent that is compatible with the chemical changes of the present invention and the reagents and materials required therefor. In order to obtain the compounds of the present invention, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes on the basis of the embodiments already present.

The compounds of the present invention may be structured by conventional methods well known to those skilled in the art, and if the present invention relates to the absolute configuration of a compound, the absolute configuration may be confirmed by conventional means in the art. For example, single crystal X-ray diffraction (SXRD), the grown single crystal is collected from diffraction intensity data using a bruker d8 v-ture diffractometer, and the light source is cuka radiation, scanning:

after scanning and collecting the relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by a direct method (Shellxs 97).

The present invention will be specifically described by the following examples, which are not meant to limit the present invention in any way.

All solvents used in the present invention are commercially available and can be used without further purification.

The solvent used in the present invention is commercially available.

Instrument and analysis method

1. Powder X-ray diffraction (XRPD)

Instrument model: brookfield D8 advanced X-ray diffractometer

The testing method comprises the following steps: about 10-20 mg of the sample was used for XRPD detection.

The detailed XRPD parameters are as follows:

light pipe: cu, kα,

light pipe voltage: 40kV, light pipe current: 40mA

Divergence slit: 0.60mm

Detector slit: 10.50mm

Anti-scatter slit: 7.10mm

Scanning range: 4-40deg

Step diameter: 0.02deg

Step size: 0.12 second

Sample disk rotational speed: 15rpm

2. Differential thermal analysis (DifferentialScanningCalorimeter, DSC)

Instrument model: TAQ2000 differential scanning calorimeter

The testing method comprises the following steps: samples (1 mg) were taken and tested in DSC aluminum pans and heated from 30℃to 300℃at a rate of 10℃per minute at 50mL/min N2.

3. Thermogravimetric analysis (ThermalGravimetricAnalyzer, TGA)

Instrument model: TAQ5000 thermogravimetric analyzer

The testing method comprises the following steps: samples (2-5 mg) were taken and tested in a TGA platinum pan and heated from 30 ℃ to 300 ℃ or 20% weight loss at a temperature ramp rate of 10 ℃/min under 25mL/min N2 conditions.

Drawings

Fig. 1: XRPD pattern of form I of compound of formula (1);

fig. 2: DSC profile and TGA profile of form I of the compound of formula (1);

fig. 3: XRPD pattern of form II of compound of formula (1);

fig. 4: DSC profile and TGA profile of form II of the compound of formula (1);

fig. 5: XRPD pattern of form III of compound of formula (1);

fig. 6: TGA profile of form III of the compound of formula (1);

fig. 7: XRPD pattern of form IV of compound of formula (1);

fig. 8: XRPD pattern of form V of compound of formula (1);

Fig. 9: DSC profile and TGA profile of form V of the compound of formula (1);

fig. 10: chromatograms of substances related to the crystal form II of the compound of the formula (I).

Detailed Description

For a better understanding of the present invention, reference will now be made to the following examples, which are not intended to limit the scope of the present invention.

Intermediate I

Step 1: synthesis of Compound I-2

To a solution of compound I-1 (500 mg,3.67 mmol) in tetrahydrofuran (30 mL) was added sodium hydrogen (220.36 mg,5.51mmol, 60%) and the mixture was reacted at room temperature of 20℃for 1 hour. Triisopropylsilane (849.80 mg,4.41 mmol) was added thereto, and the reaction was carried out at room temperature of 20℃for 2.5 hours. After the completion of the reaction, 10mL of a saturated aqueous ammonium chloride solution was added to the reaction mixture to quench the reaction. 60mL of water was added and extracted with ethyl acetate (70 mL. Times.3). The organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered to obtain a 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 ]] ⁺

Step 2: synthesis of Compound I

Compound I-2 (200 mg, 683.84. Mu. Mol) was placed in a three-necked flask, and anhydrous tetrahydrofuran (12 mL) was added thereto, followed by nitrogen substitution. The reaction solution was cooled to-78 ℃, lithium diisopropylamide (2 m,683.84 μl,2 eq) was added, and stirred for 30 minutes, trimethyl borate (99.48 mg,957.38 μl,108.13 μl,1.4 eq) was added, and the mixture was stirred for 1 hour at 18 ℃ at room temperature. After the completion of the reaction, 10mL of a saturated aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was stirred for 15 minutes. Extraction was performed with ethyl acetate (30 ml x 3), the organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered to obtain a filtrate and dried under reduced pressure. Intermediate I is obtained.

MSm/z:337.1[M+H] ⁺ 。

Example 1

/>

Step 1: synthesis of Compound 1-B

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

Step 2: synthesis of Compound 1-C

To a solution of compound 1-B (0.75 g,2.22mmol,1 eq), 1, 4-dimethyl-1H-1, 2, 3-triazole (258.16 mg,2.66mmol,1.2 eq) and potassium carbonate (918.48 mg,6.65mmol,3 eq) in N, N-dimethylacetamide (2 mL) were added palladium acetate (34.81 mg, 155.06. Mu. Mol,0.07 eq) and tricyclohexylphosphine (93.18 mg, 332.28. Mu. Mol, 107.72. Mu.l, 0.15 eq) and the reaction was stirred with nitrogen for 1 hour at 110 ℃. After the reaction liquid is cooled, filtering, decompressing and concentrating to obtain a crude product, and purifying the crude product by column chromatography (petroleum ether/ethyl acetate: 8% -50%) to obtain the compound 1-C. MS-ESim/z 308.1[ M+H ] ] ⁺

Step 3: synthesis of Compound of formula (1)

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

MSm/z:408.2[M+H] ⁺

¹ HNMR(400MHz,CDCl ₃ )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)4.00-4.13(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)

Example 2: preparation of form I

Taking the compound of formula (1), passing through a silica gel column

40g/>

SilicaFlashColumn, eluentof 10-60% tetrahydrofuran/petroleum ether @40mL/min, gradient elution) and then purified again (& lt->

25g

Silica FlashColumn, eluentof 0-10% methanol/dichloromethane @35mL/min, gradient elution) and concentrating to obtain the compound of formula (1) in form I.

The silica gel column is a commercially available preparation column which performs gradient elution by itself.

Example 3: preparation of form II

The compound (2 g) of formula (1)) was placed in a single-necked flask, and ethanol (20 mL) was added. Heating to 50-60 ℃, stirring, stopping heating, gradually reducing to 25 ℃, stirring for 14 hours, decompressing and filtering the suspension, and drying the filter cake in a vacuum drying oven at 45 ℃ for 4 hours to obtain the II crystal form of the compound shown in the formula (1).

Example 4: preparation of form III

The compound (5 g) of the above formula (1) was placed in a single-necked flask, methanol (25 mL) was added thereto, and the mixture was heated until reflux was started, and after 0.5 hour of reflux, the heating was stopped and the temperature was lowered. Cooling to about 28 ℃, stirring for 2 hours, and filtering the suspension to obtain a crystal form III of the compound of the formula (1).

Example 5: preparation of form IV

And (3) heating the crystal form II (5-10 mg) of the compound of the formula (1) to 240 ℃ and cooling to obtain the crystal form IV.

Example 6: preparation of form V

The method comprises the following steps:

compounds of formula (1) in EtOH/H ₂ And (3) suspending and stirring at 60 ℃ in O (1:2, V/V) for 2 days to obtain the V crystal form.

The second method is as follows:

to ethanol (15 mL) was added, while stirring, the compound of formula (1) (2 g), ethanol (9 mL) was added, and the temperature was raised to 80℃and stirred for 2h. The crystal form V seed (40 mg) was added under reflux, stirring was continued for 1 hour, cooling was slowly performed to 24-28℃and stirring was continued for about 100 hours. The mixture was filtered, the filter cake was washed with ethanol (5 mL), and the solid was collected and dried in vacuo at 50℃for 16 hours to give the V crystalline form of the compound of formula (1).

Experimental example 1: in vitro cell Activity assay

By measuring IC ₅₀ The inhibitory activity of the test compounds on ATR kinase in humans was evaluated.

ATR/ATRIP (h) was incubated in assay buffer containing 50 nGST-cMyc-p 53 and Mg/ATP (10 uM). The reaction is initiated by the addition of Mg/ATP mixtures. After incubation for 30 minutes at room temperature, the reaction was stopped by adding a stop solution containing EDTA. Finally, add the mixture containing d ² Detection buffer for labeled anti-GST monoclonal antibody and europium-labeled anti-phosphoSer 15 antibody for anti-phospho p 53. The plates were then read in time resolved fluorescence mode and homogeneously time resolved.

Fluorescence (HTRF) signal was determined according to the formula htrf=10000× (Em 665nm/Em620 nm).

TABLE 6 in vitro ATR enzyme Activity assay results

Numbering of compounds	IC ₅₀ (nM)
		A compound of formula (1)	33

Experimental results show that the compound has stronger inhibition activity against ATR enzyme. Meanwhile, the invention uses BAY1895344 as a reference control, and the enzymatic activity of the compound shown in the formula (1) is found to be about 20 times higher than that of the reference control.

Experimental example 2: in vitro cell Activity assay

The present experiment investigated the effect of compounds in inhibiting cell proliferation by detecting their effect on in vitro cell activity in the tumor cell line LoVo.

Cell Titer-Glo luminescence assay the following steps were performed according to the instructions of Promega cell Titer-Glo luminescence assay cell Activity assay kit (Promega-G7573).

(1) CellTiter-Glo buffer was thawed and left to stand to room temperature.

(2) Place CellTiter-Glo substrate to room temperature.

(3) CellTiter-Glo working solution was prepared by adding CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to dissolve the substrate.

(4) Slowly vortex to dissolve thoroughly.

(5) The cell culture plates were removed and allowed to stand for 30 minutes to equilibrate to room temperature.

(6) Add 50. Mu.L of CellTiter-Glo working fluid per well (equal to half the volume of cell culture fluid per well). The cell plates were wrapped with aluminum foil paper to protect from light.

(7) The plates were shaken on an orbital shaker for 2 minutes to induce cell lysis.

(8) The plates were left at room temperature for 10 minutes to stabilize the luminescence signal.

(9) The luminescence signal was detected on a SpectraMaxi3xof molecular devices reader.

Data analysis

The Inhibition Rate (IR) of the test compound was calculated using the following formula: IR (%) = (1- (RLU compound-RLU placebo)/(RLU vehicle control-RLU placebo)) × 100%. The inhibition rates of compounds at different concentrations were calculated in Excel, and then the inhibition graphs were made using GraphPadPrism software and related parameters were calculated, including minimum inhibition rate, maximum inhibition rate and IC ₅₀ 。

The experimental results are shown in table 7:

TABLE 7 in vitro LoVo cell proliferation inhibition assay results

Numbering of compounds	LoVo cell proliferation IC ₅₀ (μM)
		A compound of formula (1)	0.102

Experimental results show that the compound has a good inhibition effect on LoVo tumor cells with the loss of ATM signal channels.

Experimental example 3: in vivo pharmacokinetic property study

Sample supply: based on the above experiments, further experiments were performed by selecting the compounds of formula (1).

The experimental method comprises the following steps: the purpose of this study was to determine the pharmacokinetic parameters of the compound of formula (1) and calculate its oral bioavailability in female Balb/c Nude mice. The project used 4 female Balb/cNude mice, two mice were given intravenous doses of 1mg/kg, plasma samples of 0h (pre-dose) and 0.0833,0.25,0.5,1,2,4,6,8, 24h post-dose were collected, the other two mice were given oral gavage, the dose was 10mg/kg or 25mg/kg, plasma samples of 0h (pre-dose) and 0.25,0.5,1,2,4,6,8, 24h post-dose were collected, LC/MS analysis was performed on the collected samples and data were collected, and the collected analysis data was used to calculate the relevant pharmacokinetic parameters using phoenix winnonlin6.2.1 software.

The experimental results are shown in tables 8 and 9:

8 in vivo pharmacokinetics-intravenous administration results

C ₀ (nM)	Cl(mL/min/kg)	V _dss (L/kg)	T _1/2 (h)	AUC _0-t (nM.h)
					2804	15.8	1.65	1.32	2549

9 in vivo pharmacokinetics-oral administration results

Dosage (mg/kg)	C _max (nM)	T _1/2 (h)	AUC _0-t (nM.h)	F(％)
					10	9565	1.4	22992	91.6
25	17396	3.8	82019	-

Note that: c (C) ₀ (nM) in vivo drug concentration at 0 min; cl (mL/min/kg) is the clearance rate of the medicine in vivo; v (V) _dss (L/kg) is the in-vivo distribution volume of the drug; t (T) _1/2 (h) Is half-life; AUC (AUC) _0-t (nM.h) is in vivo drug exposure; cmax (nM) is the highest concentration of drug in vivo.

Conclusion of experiment: the compound has good in vivo pharmacokinetic properties such as exposure, bioavailability and the like.

Experimental example 4: in vivo drug efficacy study of human gastric cancer cell SNU-601CDX

The purpose of the experiment is as follows:

the main purpose of the research is to research the anti-tumor efficacy of the test object on a human gastric cancer cell SNU-601 xenograft tumor model.

The experimental method comprises the following steps:

1. experimental animal

Species: mouse strain: CB17SCID mice

The suppliers: animal technology Co., ltd

Week-old: 6-8 weeks of age

Gender: female

2. Cell culture

Human gastric cancer SNU-601 cells, derived from KCLB (cat# 00601), were maintained for passage by Shanghai Biotechnology Inc. In vitro culture conditions were RPMI1640 medium (containing 300 mg/LL-glutamine) supplemented with 10% foetal calf serum, 25mM HEPES and 25mM sodium bicarbonate, 5% CO2 incubator culture at 37℃and two to three passages a week. When the cell number reaches the requirement, the cells are harvested and counted. 0.2mL (5X 106) of SNU-601 cells (resuspended in DPBS: matrigel=1:1) were subcutaneously inoculated in the right back of each mouse and group dosing was started when the average tumor volume reached 147.61mm 3.

3. Dosage of test substance

Dosage of administration: the compound of formula (1) was orally administered in three doses of 15mg/kg (4 days of 3 days of rest), 10mg/kg (4 days of 3 days of rest) and 5mg/kg (continuous administration), respectively.

4. Tumor measurement and experimental index

Tumor diameters were measured with a vernier caliper three times a week. The calculation formula of the tumor volume is: v=0.5×a×b ² A and b represent the major and minor diameters of the tumor, respectively. The Relative Tumor Volume (RTV) was calculated as: RTV (%) = (Vt/V1) ×100; the calculation formula of the animal weight change (BWC) is: BWC (%) = (BWt-BW 1)/bw1×100, where V1 and BW1 refer to tumor volume and body weight of a given animal on the day of group administration, and Vt and BWt refer to tumor volume and body weight of a given animal measured at a time.

The tumor-inhibiting effect of the compound was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%) =trtv/crtv×100 (TRTV: treatment group mean RTV; CRTV: negative control group mean RTV). The Relative Tumor Volume (RTV) is calculated from the result 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 measured at a certain time, and TRTV and CRTV take the same day data.

TGI (%) reflects the tumor growth inhibition rate. TGI (%) = [ (1- (mean tumor volume at the end of the treatment group-mean tumor volume at the beginning of the treatment group))/(mean tumor volume at the end of the treatment with solvent control group-mean tumor volume at the beginning of the treatment with solvent control group) ]x100.

Tumor weights will be measured after the end of the experiment and the percentage of Twaight/Cweight calculated, twaight and Cweight representing tumor weights in the dosing group and vehicle control group, respectively.

5. Experimental results

In this experiment, the in vivo efficacy of the test compound was evaluated using a human gastric cancer cell SNU601 xenograft tumor model. Throughout the administration period, no animals stopped taking the drug due to weight loss exceeding 10%, and the drug effect test was ended on day 21 after administration.

On day 21 post-dose, tumor volume of vehicle group reached 890.01 ± 184.62mm ³ . The compound of formula (1) showed a certain antitumor effect at the doses of 15mg/kg, 10mg/kg and 5mg/kg compared with the vehicle control group. Their corresponding tumor volumes are 108.74 + -9.67 mm respectively ³ 136.74 + -14.46 mm3 and 229.99 + -24.42 mm ³ The tumor inhibition rates TGI were 104.81% (p < 0.01), 101.08% (p < 0.01)) and 88.61% (p < 0.01), respectively.

Throughout the experiment, the animals of the group to which the compound of formula (1) was administered had no significant decrease in body weight and no abnormality in animal status.

In conclusion, the compound disclosed by the invention can obviously inhibit the growth of human gastric cancer SNU-601 xenograft tumors and is relatively tolerant to mice.

Experimental example 5: solid stability test of Compound II Crystal form of formula (I) under high temperature, high humidity and illumination conditions

According to the influence factors and the accelerated test conditions, accurately weighing about 20mg of the compound II crystal form of the formula (I) and placing the compound II crystal form in a dry and clean glass bottle, weighing 2 parts, respectively marking as S1 condition-time and S2 condition-time, weighing 15mg, placing the compound II crystal form in the dry and clean glass bottle, marking as S3 condition-time, spreading the compound II crystal form into a thin layer to serve as a formal test sample, and placing the test sample under the influence factor test conditions (60 ℃,25 ℃/92.5%RH (relative humidity), illumination contrast) and the accelerated conditions (40 ℃/75%RH and 60 ℃/75%RH), wherein the test sample is a complete exposure lofting. 60 ℃,25 ℃/92.5% RH, light control, 5 days, 10 days sampling analysis. 40 ℃/75% RH and 60 ℃/75% RH were sampled and analyzed at 1 month, 2 months, and 3 months. The high performance liquid chromatography method of the related substances is shown in table 10, and the chromatogram is shown in fig. 10. The content of the solid stability sample of the compound II crystal form of the formula (1) and the analysis results of related substances are shown in Table 11.

High performance liquid chromatography method for substances related to Table 10

TABLE 11 solid stability sample content of Compound II form of formula (1) and analysis results of related substances (5 days, 10 days, 1 month)

Stability conditions	Time	Post-experimental crystalline forms	Total impurity (%)	Content (%)
					60℃	Day	0	Crystal form II	0.35	100.0
60℃	For 5 days	Crystal form II	0.35	100.4
					60℃	For 10 days	Crystal form II	0.34	101.1
25 ℃,92.5% humidity	For 5 days	Crystal form II	0.30	101.0
					25 ℃,92.5% humidity	For 10 days	Crystal form II	0.35	101.3
Illumination of	For 10 days	Crystal form II	0.39	101.2
					Light-shielding	For 10 days	Crystal form II	0.35	103.5
Humidity of 40-75%	1 month	Crystal form II	0.51	100.3
					60-75% humidity	1 month	Crystal form II	0.50	100.0
Humidity of 40-75%	2 months of	Crystal form II	0.53	100.2
					60-75% humidity	2 months of	Crystal form II	0.54	100.4
Humidity of 40-75%	For 3 months	Crystal form II	0.47	99.9
					60-75% humidity	For 3 months	Crystal form II	0.48	100.1

Conclusion: the crystal form of the compound II in the formula (1) has good physical stability in the solid influencing factors and acceleration test for 3 months, and the crystal form of the raw material compound is unchanged. In the analysis of related substances, the total amount of impurities is not obviously increased, and the content is stable.

Conclusion of experiment: the invention has good crystal form stability, and is easy for storage, transportation and patent medicine of products.

Claims

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

2. Form II according to claim 1, characterized in that: the X-ray powder diffraction pattern also includes characteristic diffraction peaks at the following 2θ angles: 7.97 + -0.20 deg., 18.61 + -0.20 deg., 24.53 + -0.20 deg..

3. Form II according to claim 1 or 2, characterized in that: the 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 °, 25.81+ -0.20 °.

4. A form II of claim 3, wherein: the X-ray powder diffraction pattern has characteristic diffraction peaks at least at the following 2 theta 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 form II of claim 4, wherein: the XRPD pattern is substantially as shown in figure 3.

6. Form II according to claim 1, characterized in that: the differential scanning calorimetric curve of the II crystal form has an endothermic peak at 222.1+/-3 ℃ and 256.5+/-3 ℃; thermogravimetric analysis curves lost weight at 250±3 ℃.

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

8. form V according to claim 7, characterized in that: the 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 °, 26.6250 + -0.20 °.

9. Form V according to claim 7 or 8, characterized in that: the 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 °, 27.0062 + -0.20 °.

10. Form V according to claim 9, characterized in that: the X-ray powder diffraction pattern has characteristic diffraction peaks at least at the following 2 theta 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. Form V according to claim 10, characterized in that: the XRPD pattern is substantially as shown in figure 8.

12. Form V according to claim 7, characterized in that: the differential scanning calorimetric curve has an endothermic peak at 236.3+ -1deg.C and 256.3+ -3deg.C and an exothermic peak at 237.9+ -1deg.C; the thermogravimetric analysis curve has a weight loss at 150.+ -. 3 ℃.

13. Use of the crystalline form of any one of claims 1-12 in the preparation of an ATR enzyme inhibitor.

14. Use of a crystalline form according to any one of claims 1-12 for the preparation of a product for the treatment of a disease associated with ATR enzyme activation.

15. Use according to claim 14, characterized in that: the related diseases caused by ATR enzyme activation are selected from cancers.

16. Use of a crystalline form according to any one of claims 1 to 12 in combination with an ATM enzyme inhibitor for the manufacture of a medicament for the treatment of cancer.

17. 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, gastric cancer, ovarian cancer, pancreatic cancer, testicular cancer, bladder cancer, myeloma, non-small cell lung cancer, leukemia, lymphoma, melanoma, esophageal cancer, connective tissue cancer, mesothelial cancer, prostate cancer, bone cancer, and renal cancer.

18. A pharmaceutical composition characterized by: comprising the crystalline form of any one of claims 1-12.

19. A process for the preparation of crystalline form II of compound of formula (1) according to any one of claims 1 to 6, characterized in that: it comprises the following contents:

20. A process for the preparation of crystalline form V of compound of formula (1) according to any one of claims 7 to 12, characterized in that: it comprises the following contents:

the method comprises the following steps: taking the compound (1), and stirring in 40-60% V/V ethanol at 60+/-5 ℃ to obtain a V crystal form;

or a second method: adding the compound (1) into ethanol under stirring, adding ethanol, refluxing at 80+/-5 ℃, adding the crystal seed of the V crystal form under the refluxing state, continuously stirring, slowly cooling to 24-28 ℃, and crystallizing to obtain the V crystal form.