pharmaceutically acceptable salts of pyrazoloheteroaryl derivatives and their crystalline forms

Abstract

The present disclosure relates to pharma- ceutically acceptable salts of pyrazoloheteroaryl derivatives and their crystalline forms.Specifically, the present disclosure relates to pharma- ceutically acceptable salts of the compound represented by formula (I) and their crystalline forms.The novel crystalline forms of the present disclosure have good physicochemical properties. [Formula 1] TIFF2024521129000020.tif49168

Description

Technical Field

[0001] This application claims the priority of Chinese Patent Application No. 2021105586705 with a filing date of May 21, 2021. The full text of the above Chinese patent application is incorporated herein by reference.

[0002] The present disclosure relates to pharmaceutically acceptable salts of pyrazoloheteroaryl derivatives and crystal forms thereof, specifically, pharmaceutically acceptable salts of the compounds represented by formula (I) and crystal forms thereof.

Background Art

[0003] In both normal cells and tumor cells, thousands to tens of thousands of DNA damages occur every day. Thus, DNA damage repair plays a very important role in maintaining genomic stability and cell survival. Compared with normal cells, tumor cells can withstand greater replication stress, have more endogenous DNA damages, and often have a deletion of one or more DNA damage repair pathways. As a result, the survival of tumor cells becomes more dependent on proper DNA damage repair.

[0004] [[ID=!9]] Homologous recombination repair is the primary method of repairing DNA double-strand breaks. It uses the homologous sequence of an undamaged sister chromatid as a repair template to replicate the DNA sequence at the damaged site, resulting in highly accurate DNA repair. This repair method primarily occurs during the G2 and S phases of cells. ATR is a key enzyme in the homologous recombination repair pathway and belongs to the PIKK family. When the ATR / ATRIP complex binds to damaged DNA coated with replication protein A (RPA), ATR is activated and phosphorylates downstream proteins such as Chk1 and SMARCAL, regulating various checkpoints in the cell cycle, causing cell cycle arrest, ensuring the stability of damaged DNA, increasing dNTP concentration, and promoting DNA damage repair. The fact that DNA damage repair occurring during the S phase of the cell cycle is primarily achieved through the ATR pathway suggests that ATR is crucial for ensuring cell proliferation. Analysis of clinical tumor samples clearly shows that elevated ATR expression levels have been observed in many tumor tissues, such as those of gastric cancer, liver cancer, colorectal cancer, ovarian cancer, and pancreatic cancer. Furthermore, high levels of ATR are often associated with low survival rates in patients with ovarian and pancreatic cancer. This indicates that ATR is an important target for tumor therapy.

[0005] WO2021098811A relates to a series of novel ATR inhibitors, of which the compound shown in formula (I) exhibits excellent ATR inhibitory activity, and its structure is as follows: [ka] The crystalline structure of pharmaceutical active ingredients and their intermediates tends to affect the chemical stability of the drug. Depending on crystallization and storage conditions, the crystalline structure of the compound may change, sometimes leading to the formation of other crystalline forms. Generally, amorphous products lack a regular crystalline structure and tend to have other defects, such as poor product stability, fine precipitated crystals, difficulty in filtration, tendency to solidify, and poor fluidity. Therefore, it is necessary to improve the various properties of the above-mentioned products, and to thoroughly investigate the discovery of novel crystalline forms that have high purity and good chemical stability. [Overview of the Initiative]

[0006] This disclosure provides novel salt forms, crystalline forms, and methods for producing the same of a compound represented by formula (I). [ka]

[0007] This disclosure provides pharmaceutically acceptable salts of compounds represented by formula (I), which are selected from hydrochloride, sulfate, hydrobromide, mesylate, p-toluenesulfonate, maleate, phosphate, formate, acetate, succinate, fumarate, citrate, malate, hippurate, or oxalate.

[0008] In some embodiments, the pharmaceutically acceptable salt is a mesylate, maleate, or oxalate.

[0009] In some embodiments, the molar ratio of the compound represented by formula (I) to sulfuric acid in the above sulfate is 3:1 to 1:3, preferably 1:0.5 or 1:1.

[0010] In some embodiments, the molar ratio of the compound represented by formula (I) to maleic acid in the above maleate is 3:1 to 1:3, preferably 1:0.5 or 1:1.

[0011] In some embodiments, the molar ratio of the compound represented by formula (I) to p-toluenesulfonic acid in the above-mentioned p-toluenesulfonate is 3:1 to 1:3, preferably 1:1 or 1:2.

[0012] In some embodiments, the molar ratio of the compound represented by formula (I) to mesylic acid in the above mesylate is 3:1 to 1:3, preferably 1:1 or 1:2.

[0013] In some embodiments, the molar ratio of the compound represented by formula (I) to oxalic acid in the above oxalate is 3:1 to 1:3, preferably 1:0.5 or 1:1.

[0014] This disclosure further provides crystalline forms of hydrochloride salts of compounds represented by formula (I), the crystalline forms of which are as follows: The powder X-ray diffraction pattern shows characteristic peaks at 2θ angles of 6.0, 8.3, 12.1, 14.3, 14.9, 16.7, and 26.7 for the crystalline form a of the hydrochloride salt. Crystalline form b of the hydrochloride salt, whose powder X-ray diffraction pattern has characteristic peaks at 2θ angles of 6.0, 12.1, 18.2, 23.6, and 24.4.

[0015] In some embodiments, the powder X-ray diffraction pattern of crystalline form a of the hydrochloride salt has characteristic peaks at 2θ angles of 6.0, 8.3, 9.1, 12.1, 14.3, 14.9, 16.7, 18.3, 19.4, 23.5, 24.3, 26.3, and 26.7.

[0016] In some embodiments, the powder X-ray diffraction pattern of crystalline form a of the hydrochloride salt is as shown in Figure 1.

[0017] In some embodiments, the powder X-ray diffraction pattern of crystalline form b of the hydrochloride salt has characteristic peaks at 2θ angles of 6.0, 8.4, 9.0, 12.1, 16.5, 18.2, 23.6, 24.4, 26.2, 29.5, 33.9, and 35.5.

[0018] In some embodiments, the powder X-ray diffraction pattern of the crystalline form b of the above hydrochloride is as shown in FIG. 2.

[0019] The present disclosure further provides a crystalline form α of a sulfate of a compound represented by formula (I), the powder X-ray diffraction pattern of which has peaks characteristic of 2θ angles of 5.8, 7.6, 13.7, 15.4 and 20.4.

[0020] In some embodiments, the powder X-ray diffraction pattern of the crystalline form α of the above sulfate has peaks characteristic of 2θ angles of 5.8, 7.6, 13.7, 15.4, 16.4, 16.9, 18.0, 18.5, 19.2, 20.4, 23.0, 23.9 and 25.9.

[0021] In some embodiments, the powder X-ray diffraction pattern of the crystalline form α of the above sulfate is as shown in FIG. 3.

[0022] The present disclosure further provides a crystalline form of a hydrobromide of the compound represented by formula (I), and the above crystalline form is as follows: Crystalline form I of the hydrobromide, the powder X-ray diffraction pattern of which has peaks characteristic of 2θ angles of 6.0, 8.1, 14.7, 25.9 and 27.0, Crystalline form II of the hydrobromide, the powder X-ray diffraction pattern of which has peaks characteristic of 2θ angles of 9.3, 11.6, 13.0, 16.8, 18.7 and 24.6.

[0023] In some embodiments, the powder X-ray diffraction pattern of the crystalline form I of the above hydrobromide has peaks characteristic of 2θ angles of 6.0, 8.1, 1,4.7, 17.3, 18.8, 22.0, 25.9, 27.0 and 27.8.

[0024] In some embodiments, the powder X-ray diffraction pattern of the crystalline form I of the above hydrobromide is as shown in FIG. 4.

[0025] In some embodiments, the powder X-ray diffraction pattern of the hydrobromide salt in crystalline form II has characteristic peaks at 2θ angles of 8.2, 9.3, 11.6, 13.0, 15.5, 16.8, 17.6, 18.7, 19.3, 19.8, 21.3, 22.4, 23.3, 24.6, 25.4, 26.1, 26.4, 27.9, 28.7, 31.0, 31.7, 32.2, 34.1, 34.9, 35.5, 36.3, 37.0, 37.7, 38.3, 40.1, and 42.1.

[0026] In some embodiments, the powder X-ray diffraction pattern of crystal form II of the hydrobromide salt is as shown in Figure 5.

[0027] This disclosure further provides crystalline forms of mesylate salts of compounds represented by formula (I), the crystalline forms of which are as follows: The powder X-ray diffraction pattern has characteristic peaks at 2θ angles of 10.0, 16.8, 17.8, 18.4, and 20.6, indicating the crystalline form α of the mesylate, or Crystalline form β of the mesylate has a powder X-ray diffraction pattern characterized by peaks at 2θ angles of 5.9, 8.4, 14.5, 16.8, 19.8 and 26.0. In some embodiments, the powder X-ray diffraction pattern of crystalline form α of the mesylate has characteristic peaks at 2θ angles of 7.7, 10.0, 12.9, 13.8, 14.3, 15.1, 16.8, 17.8, 18.4, 20.3, 20.6, 21.9, 23.1, 24.2, 25.3, 26.1, 26.7, 28.3, 29.0, 30.7, 35.0 and 43.1.

[0028] In some embodiments, the powder X-ray diffraction pattern of the crystalline form α of the mesylate is as shown in Figure 6.

[0029] In some embodiments, the powder X-ray diffraction pattern of the crystalline form β of the mesylate has characteristic peaks at 2θ angles of 5.9, 8.4, 13.6, 14.5, 16.8, 18.5, 19.8, 20.9, 21.6, 23.3, 26.0, 26.7, and 27.4.

[0030] In some embodiments, the powder X-ray diffraction pattern of the crystalline form β of the mesylate is as shown in Figure 7.

[0031] This disclosure further provides crystalline form I of the maleate of the compound represented by formula (I), wherein the powder X-ray diffraction pattern has characteristic peaks at 2θ angles of 10.1, 17.1, 18.0, 19.0 and 24.3.

[0032] In some embodiments, the powder X-ray diffraction pattern of crystalline form I of the maleate has characteristic peaks at 2θ angles of 7.2, 9.4, 10.1, 12.8, 13.2, 14.2, 14.8, 15.7, 17.1, 18.0, 19.0, 22.0, 23.4, 24.3, 25.2, 27.5, and 29.1.

[0033] In some embodiments, the powder X-ray diffraction pattern of crystalline form I of maleic acid is as shown in Figure 8.

[0034] This disclosure further provides a crystalline form a of the p-toluenesulfonate of the compound represented by formula (I), the powder X-ray diffraction pattern of which has characteristic peaks at 2θ angles of 6.5, 8.6, 12.0, 14.5, 21.2, and 22.2.

[0035] In some embodiments, the powder X-ray diffraction pattern of crystalline form a of the p-toluenesulfonate has characteristic peaks at 2θ angles of 6.5, 8.6, 9.9, 12.0, 13.1, 14.5, 16.7, 18.9, 19.7, 21.2, 22.2, 24.2, 26.3, and 27.6.

[0036] In some embodiments, the powder X-ray diffraction pattern of crystalline form a of the above-mentioned p-toluenesulfonate is as shown in Figure 9.

[0037] This disclosure further provides a crystalline form a of the oxalate of the compound represented by formula (I), the powder X-ray diffraction pattern of which has characteristic peaks at 2θ angles of 5.5, 9.1, 11.0, 13.0, 15.5, 16.5 and 20.2.

[0038] In some embodiments, the powder X-ray diffraction pattern of crystalline form a of the oxalate has characteristic peaks at 2θ angles of 5.5, 9.1, 11.0, 13.0, 15.5, 16.5, 20.2, 22.0, 22.5, 23.1, 24.9, 26.2, 27.8 and 30.8.

[0039] In some embodiments, the powder X-ray diffraction pattern of crystalline form a of the oxalate is as shown in Figure 10.

[0040] This disclosure further provides a method for producing crystalline form a of the hydrochloride salt of a compound represented by formula (I), the method comprising mixing a methyl tert-butyl ether (MTBE) solution containing the compound represented by formula (I) with hydrochloric acid, and slurring and crystallizing the mixture.

[0041] This disclosure further provides a method for producing crystalline form b of the hydrochloride salt of a compound represented by formula (I), the method comprising heating the hydrochloride salt a of the compound represented by formula (I) to 90°C and collecting the crystals.

[0042] This disclosure further provides a method for producing crystalline form α of a sulfate of a compound represented by formula (I), the method comprising mixing a solution containing the compound represented by formula (I) and a solvent selected from methyl tert-butyl ether or ethyl acetate (EA) / (n-heptane)heptane with sulfuric acid, and slurring and crystallizing the solution.

[0043] This disclosure further provides a method for producing crystalline form I of a hydrobromide salt of a compound represented by formula (I), the method comprising mixing a solution containing the compound represented by formula (I) and a solvent selected from methyl tert-butyl ether or ethyl acetate / n-heptane with hydrobromic acid, and slurring and crystallizing the mixture.

[0044] This disclosure further provides a method for producing crystalline form II of the hydrobromide salt of the compound represented by formula (I), the method comprising mixing a solution containing the compound represented by formula (I) and a solvent selected from ethyl acetate / n-heptane with hydrobromic acid, and slurring and crystallizing the solution.

[0045] This disclosure further provides a method for producing crystalline form α of a mesylate of a compound represented by formula (I), the method comprising mixing a solution containing the compound represented by formula (I) and methyl tert-butyl ether with methanesulfonic acid, and slurring and crystallizing the mixture.

[0046] This disclosure further provides a method for producing crystalline form β of a mesylate of a compound represented by formula (I), the method comprising mixing a solution containing the compound represented by formula (I) and a solvent selected from methyl tert-butyl ether or ethyl acetate / n-heptane with methanesulfonic acid, and slurring and crystallizing the solution.

[0047] This disclosure further provides a method for producing crystalline form I of a maleate of a compound represented by formula (I), the method comprising mixing a solution containing the compound represented by formula (I) and a solvent selected from methyl tert-butyl ether or ethyl acetate / n-heptane with maleic acid, and slurring and crystallizing the solution.

[0048] This disclosure further provides a method for producing crystalline form a of p-toluenesulfonate of the compound represented by formula (I), the method comprising mixing a solution containing the compound represented by formula (I) and methyl tert-butyl ether with p-toluenesulfonic acid, and slurring and crystallizing the mixture.

[0049] This disclosure further provides a method for producing crystalline form a of the oxalate of a compound represented by formula (I), the method comprising mixing a solution containing the compound represented by formula (I) and a solvent selected from methyl tert-butyl ether or ethyl acetate / n-heptane with oxalic acid, and slurring and crystallizing the mixture.

[0050] The structure and crystal form are determined and studied based on the crystal form obtained as described in this disclosure using powder X-ray diffraction (XRPD) and differential scanning calorimetry (DSC).

[0051] The crystallization method for the crystalline form described herein is a conventional method, such as crystallization by volatilization, crystallization by cooling, or crystallization at room temperature.

[0052] The starting material used in the method for producing the crystalline form according to this disclosure may be any form of the compound represented by formula (I), and the specific form includes, but is not limited to, amorphous, any crystalline form, hydrate, solvate, etc.

[0053] This disclosure further provides pharmaceutical compositions comprising a pharmaceutically acceptable salt of a compound represented by formula (I), and one or more pharmaceutically acceptable vectors or excipients.

[0054] This disclosure further provides pharmaceutical compositions comprising a pharmaceutically acceptable salt crystalline form of the compound represented by formula (I), and one or more pharmaceutically acceptable vectors or excipients.

[0055] This disclosure further provides a method for producing a pharmaceutical composition, comprising the step of mixing a pharmaceutically acceptable salt of a compound represented by formula (I) with one or more pharmaceutically acceptable vectors or excipients.

[0056] This disclosure further provides a method for producing a pharmaceutical composition, comprising the step of mixing a pharmaceutically acceptable salt crystalline form of a compound represented by formula (I) with one or more pharmaceutically acceptable vectors or excipients.

[0057] This disclosure further provides the use of a pharmaceutically acceptable salt or a pharmaceutically acceptable salt crystalline form or pharmaceutical composition of the compound represented by formula (I) described herein in the manufacture of agents for inhibiting ATR kinase.

[0058] This disclosure further provides the use of a pharmaceutically acceptable salt or a pharmaceutically acceptable crystalline form or pharmaceutical composition of the compound represented by formula (I) described herein in the manufacture of a drug for treating hyperproliferative diseases.

[0059] This disclosure further provides the use of a pharmaceutically acceptable salt or a pharmaceutically acceptable crystalline form or pharmaceutical composition of the compound represented by formula (I) described herein in the manufacture of a drug for the treatment of neoplastic diseases.

[0060] The tumors described herein are selected from melanoma, brain tumors, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, lung cancer, kidney cancer, breast cancer, cervical cancer, ovarian cancer, prostate cancer, skin cancer, neuroblastoma, glioma, sarcoma, bone cancer, uterine cancer, endometrial cancer, head and neck tumors, multiple myeloma, B-cell lymphoma, polycythemia vera, leukemia, thyroid tumors, bladder cancer, and gallbladder cancer.

[0061] Unless otherwise stated in the specification and claims of this application, the scientific and technical terms used herein have the meanings generally understood by those skilled in the art. However, to better understand this disclosure, some definitions and interpretations of relevant terms are provided below. In the event of any conflict between the definitions and interpretations provided herein and the meanings generally understood by those skilled in the art, the definitions and interpretations provided herein shall prevail.

[0062] The term "slurry formation" as used in this disclosure refers to a purification method that utilizes the property that a substance has poor solubility in a solvent, but impurities in the solvent have good solubility. Purification by slurry formation can decolorize, change the crystal form, or remove small amounts of impurities.

[0063] The term "powder X-ray diffraction pattern or XRPD" as used in this disclosure means that, based on the Bragg equation 2d sin θ = nλ (where λ is the wavelength of the X-rays and n is an arbitrary positive integer for the order of diffraction, usually taking the first-order diffraction peak, n=1), the Bragg equation can be satisfied when X-rays are incident on a crystal at a sweep angle θ (complementary angle of incidence, also called the Bragg angle), or incident on a specific atomic plane having a d-lattice interplane distance in some crystal samples, thereby measuring the powder X-ray diffraction pattern of this set.

[0064] The "powder X-ray diffraction pattern or XRPD" described herein is a pattern obtained by using Cu-Kα radiation in a powder X-ray diffractometer.

[0065] As described in this disclosure, “differential scanning calorimetry or DSC” refers to measuring the temperature difference and heat flow difference between a sample and a reference object in order to characterize all physical and chemical changes related to thermal effects during heating or constant temperature of a sample and to obtain phase transition information of the sample.

[0066] In this disclosure, “2θ or 2θ angle” refers to the diffraction angle, where θ is the Bragg angle, the unit is degrees or degrees, and the tolerance range for 2θ is ±0.3, ±0.2, or ±0.1.

[0067] The "lattice plane spacing or lattice plane spacing (d value)" as described in this disclosure refers to the selection of three unit vectors a, b, and c that connect two adjacent lattice points that are not parallel to each other in a spatial lattice, and how these three unit vectors divide the lattice into juxtaposed parallelepiped units; this is called the lattice plane spacing. The spatial lattice is divided by connecting the determined parallelepiped units to obtain a set of linear lattices, which are called the spatial lattice or crystal lattice. The lattice and crystal lattice reflect the periodicity of the crystal structure in geometric points and lines, respectively, and different crystal planes have different interplanar spacings (i.e., the distance between two adjacent parallel crystal planes), with units of Å or angstroms.

[0068] "Optionally" or "optionally" means that the event or situation described thereafter may or may not occur, and this description includes both cases in which the event or situation occurs and cases in which it does not occur. For example, "a heterocyclyl group optionally substituted with an alkyl group" means that an alkyl group may or may not be present, and this description includes both cases in which the heterocyclyl group is substituted with an alkyl group and cases in which the heterocyclyl group is not substituted with an alkyl group.

[0069] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, along with other components such as physiologically / pharmaceutically acceptable vectors and excipients. Pharmaceutical compositions are intended to facilitate administration to a living organism and contribute to the absorption of the active ingredient, thereby exerting further biological activity.

[0070] The terms “solvate” or “solvent compound” mean that the agents of this disclosure form pharmaceutically acceptable solvates with one or more solvent molecules, including, but not limited to, water, ethanol, methyl tert-butyl ether, acetone, n-heptane, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0071] The term "vector," as used with the drugs of this disclosure, refers to a system that can alter how a drug enters and is distributed within the human body, control the rate of drug release, and deliver the drug to a target organ. The release and targeting systems of drug vectors can reduce drug degradation and loss, decrease side effects, and improve bioavailability. For example, polymeric surfactants as vectors can self-assemble to form various forms of aggregates due to their unique amphiphilic structure. Preferred examples include micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, making them good drug vectors. [Brief explanation of the drawing]

[0072] [Figure 1] This is the XRPD pattern of crystalline form a of the hydrochloride salt of the compound shown in formula (I). [Figure 2] This is the XRPD pattern of crystalline form b of the hydrochloride salt of the compound shown in formula (I). [Figure 3] This is the XRPD pattern of the crystalline form α of the sulfate of the compound shown in formula (I). [Figure 4] This is the XRPD pattern of crystalline form I of the hydrobromide salt of the compound shown in formula (I). [Figure 5] This is the XRPD pattern of crystalline form II of the hydrobromide salt of the compound shown in formula (I). [Figure 6] This is the XRPD pattern of crystalline form α of the mesylate salt of the compound shown in formula (I). [Figure 7] This is the XRPD pattern of the crystalline form β of the mesylate salt of the compound shown in formula (I). [Figure 8] This is the XRPD pattern of crystalline form I of the maleate of the compound shown in formula (I). [Figure 9] This is the XRPD pattern of crystalline form a of the p-toluenesulfonate of the compound shown in formula (I). [Figure 10] This is the XRPD pattern of crystalline form a of the oxalate of the compound shown in formula (I). [Figure 11] This is the DSC pattern of crystalline form a of the hydrochloride salt of the compound shown in formula (I). [Figure 12] This is the DSC pattern of the crystalline form α of the sulfate of the compound shown in formula (I). [Figure 13] This is the DSC pattern of crystalline form I of the hydrobromide salt of the compound shown in formula (I). [Figure 14] This is the DSC pattern of crystal form II of the hydrobromide salt of the compound shown in formula (I). [Figure 15] This is the DSC pattern of crystal form α of the mesylate salt of the compound shown in formula (I). [Figure 16]This is the DSC pattern of the crystalline form β of the mesylate salt of the compound shown in formula (I). [Figure 17] This is the DSC pattern of crystal form I of the maleate salt of the compound shown in formula (I). [Figure 18] This is the DSC pattern of crystalline form a of the p-toluenesulfonate of the compound shown in formula (I). [Figure 19] This is the DSC pattern of crystalline form a of the oxalate of the compound shown in formula (I). [Modes for carrying out the invention]

[0073] The present disclosure will be described in more detail below in combination with examples, but the examples of the present disclosure are merely for illustrating the technical concepts of the present disclosure and do not limit the substance or scope of the present disclosure.

[0074] The structure of the compound is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR shift (δ) is expressed in units of 10⁻⁶ (ppm). NMR measurements are performed using a Bruker AVANCE-400 nuclear magnetic resonance spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD) as the measurement solvents, and tetramethylsilane (TMS) as the internal standard.

[0075] For the MS measurements, an Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS liquid chromatograph was used (manufacturer: Agilent, MS model number: 6110 / 6120 Quadrupole MS).

[0076] The following detectors were used: Waters ACQuity UPLC-QD / SQD (Manufacturer: Waters, MS Model Number: Waters ACQuity Qda Detector / Waters SQ Detector) and THERMO Ultimate 3000-Q Exactive (Manufacturer: THERMO, MS Model Number: THERMO Q Exactive).

[0077] High-performance liquid chromatography (HPLC) analysis was performed using Agilent HPLC 1260DAD, Agilent HPLC 1260VWD, and Waters HPLC e2695-2489.

[0078] For chiral HPLC analysis, an Agilent 1260 DAD high-performance liquid chromatograph was used.

[0079] For preparative high-performance liquid chromatography, the following instruments were used: Waters 2545-2767 preparative chromatograph, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson GX-281.

[0080] For chiral preparative chromatography, a Shimadzu LC-20AP preparative chromatograph was used.

[0081] For the CombiFlash high-speed preparative chromatograph, the CombiFlash Rf200 (TELEDYNE ISCO) was used.

[0082] For thin-layer chromatography, Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates are used. The specifications of the silica gel plates used for thin-layer chromatography (TLC) are 0.15 mm to 0.2 mm, and the specifications for separation and purification of products by thin-layer chromatography are 0.4 mm to 0.5 mm.

[0083] In silica gel column chromatography, silica gel of 200-300 mesh size, manufactured by Yantai Huanghai Silica Gel, was commonly used as a vector.

[0084] The average kinase inhibition rate and IC50 value are measured using the NovoStar plate reader (BMG, Germany).

[0085] The known starting materials relating to this disclosure may be synthesized by or in accordance with methods known in the art, or may be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., and Dalui Chemicals.

[0086] In the examples, unless otherwise specified, all reactions can be carried out in an argon or nitrogen atmosphere.

[0087] An argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen balloon with a volume of approximately 1 liter.

[0088] A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon with a volume of approximately 1 liter.

[0089] For the pressurized hydrogenation reaction, a Parr 3916EKX type hydrogenator and either a QL-500 type hydrogen generator or an HC2-SS type hydrogenator were used.

[0090] The hydrogenation reaction typically involved repeating the process of evacuating and filling with hydrogen three times.

[0091] A CEM Discover-S 908860 microwave reactor was used for the microwave reaction.

[0092] In the examples, unless otherwise specified, "solution" refers to an aqueous solution.

[0093] In the examples, unless otherwise specified, the reaction temperature is room temperature, which is 20°C to 30°C.

[0094] Thin-layer chromatography (TLC) was used to monitor the progress of the reaction in the examples. The developing solvent used in the reaction, the eluent system for column chromatography used for purifying the compound, and the developing solvent system for thin-layer chromatography included A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system, and C: petroleum ether / ethyl acetate system. The volume ratio of the solvents was adjusted according to the polarity of the compound and may be adjusted by adding small amounts of triethylamine and basic or acidic reagents such as acetic acid.

[0095] THP stands for tetrahydropyranyl group.

[0096] Test conditions for the equipment used in the test:

[0097] 1. Differential Scanning Calorimeter (DSC) Device model number: Mettler Toledo DSC 3+ Purge gas: Nitrogen Heating rate: 10.0℃ / min Temperature range: 25℃~300℃

[0098] 2. X-ray diffraction pattern (X-ray Powder Diffraction, XRPD) Equipment model number: BRUKER D8 Discover Powder X-ray Diffractometer Radiation: Monochromatic Cu-Kα radiation (λ=1.5418Å) Scanning method: θ / 2θ, Scanning range (2θ range): 3°~50° Voltage: 40kV, Current: 40mA

[0099] 3. Ion chromatography Equipment model number: DIONEX INTEGRION HPIC ion chromatograph (USA) Measurement method: Conductance, Separation column: DionexIonPac™-AS11-HC Eluent:EGC-500-KOH Flow rate: 1.4mL / min [Examples]

[0100] Example 1 (R)-2-methyl-2-(1-methyl-5-(3-methylmorpholine)-3-(1H-pyrazole-3-yl)-1H-pyrazolo[4,3-b]pyridine-7-yl)propionitrile I [ka]

[0101] Step 1 (R,E)-1-methyl-4-((1-(3-methylmorpholine)ethylidene)amino)-1H-pyrazole-5-carboxylate methyl 1c Compound (R)-1-(3-methylmorpholine)ethane-1-one 1b (2.5 g, 17.7 mmol, prepared by the method disclosed in "Intermediate-1 of Examples on page 86 of the specification in patent application WO2016020320A1") was dissolved in 1,2-dichloroethane, cooled in ice water under argon gas protection, and phosphorus oxychloride (7.4 g, 48.3 mmol) was gradually added dropwise. After the addition was complete, the mixture was stirred at room temperature for 30 minutes, compound 4-amino-1-methyl-1H-pyrazole-5-methyl formate 1a (2.5 g, 16.1 mmol, Jiangsu Aikang Bio) was added, and the mixture was heated to 80°C and reacted with stirring for 2 hours. The mixture was cooled to room temperature, concentrated under reduced pressure, diluted with dichloromethane (200 mL), cooled in ice water, neutralized to pH 8-9 by adding saturated sodium bicarbonate solution dropwise, washed the organic phase with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, mixed the filtrate with silica gel, and purified using eluent system C by silica gel column chromatography to obtain the title compound 1c (4.8 g) with a yield of 94%. MS m / z (ESI): 281.2 [M+1]

[0102] Step 2 (R)-1-methyl-5-(3-methylmorpholine)-1H-pyrazolo[4,3-b]pyridine-7-phenol 1d Compound 1c (2.6 g, 9.3 mmol) was dissolved in tetrahydrofuran (20 mL), cooled in ice water, and lithium bis(trimethylsilyl)amide (27.8 mL, 1 M tetrahydrofuran solution, 27.8 mmol) was gradually added. The reaction was carried out at 0°C for 1 hour. Methanol (10 mL) was added to quench the reaction, and the mixture was mixed with silica gel. Purification was performed using eluent system A by silica gel column chromatography to obtain the title compound 1d (400 mg) in a yield of 55.8%. MS m / z (ESI): 249.0 [M+1]

[0103] Step 3 (R)-4-(7-chloro-1-methyl-1H-pyrazolo[4,3-b]pyridin-5-yl)-3-methylmorpholine 1e Compound 1d (400 mg, 1.6 mmol) was dissolved in 3.0 mL of phosphorus oxychloride and heated to 90°C, stirring for 2.0 hours. The reaction mixture was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was diluted with dichloromethane (50 mL). The mixture was cooled in ice water, and saturated sodium bicarbonate solution was added to neutralize the pH to 8-9. The reaction was carried out with stirring for 0.5 hours, then allowed to stand and separated. The organic phase was collected, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was mixed with silica gel. The filtrate was purified using eluent system C by silica gel column chromatography to obtain the title compound 1e (240 mg) in a yield of 56%. MS m / z (ESI): 267.0 [M+1]

[0104] Step 4 (R)-2-methyl-2-(1-methyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propionitrile 1g Compound 1e (240 mg, 0.91 mmol) and compound isobutyronitrile 1f (620 mg, 8.9 mmol, Shanghai Bi De) were dissolved in 30 mL of tetrahydrofuran, cooled in a dry ice acetone bath under argon gas protection, lithium bis(trimethylsilyl)amide (8.9 mL, 1 M tetrahydrofuran solution, 8.9 mmol) was added dropwise, the mixture was stirred at low temperature for 0.5 hours, then allowed to rise naturally to room temperature and stirred for 1 hour, water was added to quench the reaction, the organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified using eluent system C by silica gel column chromatography to obtain 1 g (200 mg) of the title compound, with a yield of 74%. MS m / z (ESI): 300.1 [M+1]

[0105] Step 5 (R)-2-(3-bromo-1-methyl-5-(3-methylmorpholinyl)-1H-pyrazolo[4,3-b]pyridin-7-yl)-2-methylpropionitrile 1h Dissolve 1 g (200 mg, 0.67 mmol) in 5 mL of 1,4-dioxane, add sodium hydroxide solution (0.66 mL, 2 M solution, 1.32 mmol), cool with ice water, add liquid bromine (427 mg, 2.67 mmol), stir at low temperature for 10 minutes, allow to rise naturally to room temperature, and react with stirring for 1 hour. Dilute with ethyl acetate, wash the organic phase with saturated sodium thiosulfate solution, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify with eluent system C by silica gel column chromatography to obtain 1 h (140 mg) of the title compound, with a yield of 55%. MS m / z (ESI): 377.9 [M+1]

[0106] Step 6 2-Methyl-2-(1-methyl-5-((R)-3-methylmorpholinyl)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-yl)-1H-pyrazolo[4,3-b]pyridine-7-yl)propionitrile 1i 1H (20 mg, 0.05 mmol), tetrakis(triphenylphosphine)palladium (18 mg, 0.015 mmol), sodium carbonate (11 mg, 0.10 mmol), and 1-(tetrahydro-2H-pyran-2-yl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (29 mg, 0.10 mmol, Shanghai Bi De) were dissolved in 4 mL of ethylene glycol dimethyl ether, 1 mL of water was added, and the mixture was heated to 120°C in a microwave under argon gas protection and reacted for 1 hour. The reaction mixture was cooled to room temperature, 20 mL of water was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, concentrated under reduced pressure, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The mixture was purified using eluent system C by silica gel column chromatography to obtain the title compound 1i (20 mg) in a yield of 84%. MS m / z (ESI): 450.1 [M+1]

[0107] Step 7 (R)-2-methyl-2-(1-methyl-5-(3-methylmorpholine)-3-(1H-pyrazole-3-yl)-1H-pyrazolo[4,3-b]pyridine-7-yl)propionitrile I Compound 1i (20 mg, 0.04 mmol) was dissolved in 5 mL of dichloromethane, and 5 mL of trifluoroacetic acid was added dropwise. After the addition was complete, the mixture was reacted with stirring for 4 hours. The reaction mixture was concentrated under reduced pressure, and a 7 M ammonia-methanol solution was added dropwise to adjust the pH to 8-9. The mixture was concentrated under reduced pressure again, and purified using eluent system A by silica gel column chromatography to obtain the title compound I (7.0 mg) in a yield of 43%. MS m / z (ESI): 366.0 [M+1] 1H NMR (400 MHz, CD3OD):δ 7.58 (s, 1H), 7.03 (s, 1H), 6.86 (s, 1H), 4.39 (s, 4H), 4.04 - 3.82 (m, 2H), 3.74 (s, 2H), 3.58 (td, 1H), 3.26 (dd, 1H), 1.88 (d, 6H), 1.19 (d, 3H).

[0108] Example 2

[0109] 0.25 mL of MTBE solution containing approximately 10 mg of the compound shown in formula (I) obtained in Example 1 was mixed with 22.5 μL of 1.2 mol / L hydrochloric acid ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. The product was defined as crystalline form a of the hydrochloride salt by powder X-ray diffraction detection, and the XRPD pattern is shown in Figure 1, with its characteristic peak positions shown in Table 1.

[0110] [Table 1]

[0111] Example 3

[0112] Crystal form a of the hydrochloride salt of the compound shown in formula (I) was heated to 90°C, and the morphological transition of the crystal form was detected. The resulting product was defined as crystal form b of the hydrochloride salt, and its XRPD pattern is shown in Figure 2. Its characteristic peak positions are shown in Table 2.

[0113] [Table 2]

[0114] Example 4

[0115] 0.25 mL of MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 14.7 μL of 1.8 mol / L sulfuric acid ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. The product was defined as the crystalline form α of the hydrochloride salt by powder X-ray diffraction detection, and a sulfate ion content of 17.9% was detected by ion chromatography. The XRPD spectrum is shown in Figure 3, and the characteristic peak positions are shown in Table 3.

[0116] [Table 3]

[0117] Example 5

[0118] 0.25 mL of MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 36 μL of 0.75 mol / L hydrobromide ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. The product was defined as crystalline form I of hydrobromide by powder X-ray diffraction detection, and the XRPD pattern is shown in Figure 4, with its characteristic peak positions shown in Table 4.

[0119] [Table 4]

[0120] Example 6

[0121] 0.25 mL of an EA / heptane (1:1) solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 72 μL of 0.75 mol / L hydrobromic acid ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. The product was defined as hydrobromide crystal form II by powder X-ray diffraction detection, and the XRPD pattern is shown in Figure 5, with its characteristic peak positions shown in Table 5.

[0122] [Table 5]

[0123] Example 7

[0124] 0.25 mL of MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 17.7 μL of 1.5 mol / L methanesulfonic acid ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. The product was defined as the crystalline form α of the mesylate by powder X-ray diffraction detection, and a 20.0% mesylate ion content was detected by ion chromatography. The XRPD pattern is shown in Figure 6, and its characteristic peak positions are shown in Table 6.

[0125] [Table 6]

[0126] Example 8

[0127] 0.25 mL of MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 35.4 μL of 1.5 mol / L methanesulfonic acid ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. The product was defined as the crystalline form β of the mesylate by powder X-ray diffraction detection, and the XRPD spectrum is shown in Figure 7, with its characteristic peak positions shown in Table 7.

[0128] [Table 7]

[0129] Example 9

[0130] 0.4 mL of MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 30 μL of 1 mol / L maleic acid ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. The product was defined as crystalline form I of maleate by powder X-ray diffraction detection, and a maleate ion content of 23.7% was detected by ion chromatography. The XRPD pattern is shown in Figure 8, and its characteristic peak positions are shown in Table 8.

[0131] [Table 8]

[0132] Example 10

[0133] 0.4 mL of MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 30 μL of 1 mol / L p-toluenesulfonic acid ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. The product was defined as crystalline form a of p-toluenesulfonate by powder X-ray diffraction detection, and the p-toluenesulfonic acid ion content of 34.6% was detected by ion chromatography. The XRPD pattern is shown in Figure 9, and its characteristic peak positions are shown in Table 9.

[0134] [Table 9]

[0135] Example 11

[0136] 0.4 mL of MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 30 μL of 1 mol / L oxalic acid ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. The product was defined as crystalline form a of oxalate by powder X-ray diffraction detection, and a 10.9% oxalate ion content was detected by ion chromatography. The XRPD spectrum is shown in Figure 10, and the characteristic peak positions are shown in Table 10.

[0137] [Table 10]

[0138] Example 12

[0139] Crystal form α of mesylate, crystal form α of sulfate, crystal form I of maleate, crystal form a of p-toluenesulfonate, and crystal form a of oxalate were sealed in aluminum foil bags and their stability was examined under the conditions of -20°C, 4°C, 25°C / 60%RH, and 40°C / 75%RH. The results are as follows.

[0140] [Table 11]

[0141] In long-term accelerated stabilization experiments, all salt forms exhibited excellent physical stability, and crystalline form a of the oxalate was relatively stable chemically. Under conditions of 40°C and 75% RH, crystalline form α of the mesylate and crystalline form I of the maleate slightly decomposed, while crystalline form α of the sulfate and crystalline form a of the p-toluenesulfonate were found to be somewhat less stable.

[0142] Example 13

[0143] 0.2 mL of an ethanol / water (V / V, 9:1) solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 18.5 μL of 1.5 mol / L ethanol-phosphate solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. Powder X-ray diffraction detection revealed that the product was an amorphous phosphate.

[0144] Example 14

[0145] A 0.25 mL MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 10.2 μL of 2.7 mol / L formic acid ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. Powder X-ray diffraction detection revealed that the product was amorphous formate.

[0146] Example 15

[0147] 0.25 mL of ethyl acetate / n-heptane (V / V, 9:1) solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 15.4 μL of 1.8 mol / L ethanol acetate solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. Powder X-ray diffraction detection revealed that the product was amorphous acetate.

[0148] Example 16

[0149] 0.25 mL of ethyl acetate / n-heptane (V / V, 9:1) solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 33 μL of 0.9 mol / L succinic acid ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. Powder X-ray diffraction detection revealed that the product was amorphous succinate.

[0150] Example 17

[0151] A 0.4 mL ethanol / water (V / V, 9:1) solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 60 μL of 0.5 mol / L fumarate ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. Powder X-ray diffraction detection revealed that the product was amorphous fumarate.

[0152] Example 18

[0153] A 0.4 mL solution of MTBE containing approximately 10 mg of the compound shown in formula (I) was mixed with 30 μL of 1 mol / L citrate ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. Powder X-ray diffraction detection revealed that the product was amorphous citrate.

[0154] Example 19

[0155] 0.25 mL of ethyl acetate / n-heptane (V / V, 9:1) solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 30 μL of 1 mol / L malic acid ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. Powder X-ray diffraction detection revealed that the product was amorphous malate.

[0156] Example 20

[0157] 0.25 mL of ethyl acetate / n-heptane (V / V, 9:1) solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 60 μL of 0.5 mol / L hippuric acid ethanol solution to form a slurry. The solid was centrifuged and vacuum-dried to obtain the product. Powder X-ray diffraction detection revealed that the product was amorphous hippurate.

[0158] Biological evaluation

[0159] Test example

[0160] Test Example 1: Inhibitory effect of the compound relating to this disclosure on ATR enzyme.

[0161] The following method is for measuring the inhibitory effect of the compounds related to this disclosure on ATR enzymes. The experimental method is briefly described below.

[0162] 1. Materials and equipment for the experiment 1. ATR enzyme (Eurofins Pharma Discovery Services, 14-953-M) 2. GST-tagged P53 protein (Eurofins Pharma Discovery Services, 14-952-M) 3. 384-well plate (Thermo Scientific, 267462) 4. U-shaped bottom 96-well plate (Corning, 3795) 5. Europium cryptotate-labeled anti-phosphorylated P53 protein antibody (cisbio, 61P08KAE) 6. Anti-GST antibody that binds to d2 (cisbio, 61GSTDLF) 7. ATP solution (Promega, V916B) 8. EDTA (Thermo Scientific, AM9260G) 9. HEPES (Gibco, 15630-080) 10. Plate readers (BMG, PHERAsta)

[0163] 2. Experimental Procedure A 1 nm ATR enzyme, a 50 nm P53 protein, 7.435 μM ATP, and small molecule compounds at various concentrations (starting at 1 μM and diluted to 11 concentrations with a 3-fold gradient) were mixed and incubated at room temperature for 2 hours. A stop solution (12.5 mm HEPES, 250 mm EDTA) was added and mixed uniformly. Further addition included 0.42 ng / well of europium cryptotate-labeled anti-phosphorylated P53 protein antibody and 25 ng / well of d2-binding anti-GST antibody. After incubation overnight at room temperature, fluorescence signals at 620 nm and 665 nm were detected using PHERAstar. Data were processed using GraphPad software.

[0164] 3. Experimental Data The inhibitory activity of the compounds relating to this disclosure against ATR enzyme can be measured by the above tests, and the measured IC 50 The values ​​are shown in Table 12.

[0165] [Table 12]

[0166] Conclusion: The compounds disclosed herein exhibit good inhibitory activity against ATR enzymes.

[0167] Experiment Example 2: Cell Proliferation Experiment

[0168] The following method detects the ATP content inside cells, thereby determining IC 50 The inhibitory effect of the compounds relating to this disclosure on LoVo cell proliferation is evaluated based on their size. The experimental method is briefly described below.

[0169] 1. Materials and equipment for the experiment 1. LoVo, human colon cancer tumor cells (Nanjing Kebai, CBP60032) 2. Fetal bovine serum (GIBCO, 10091-148) 3. F-12K medium (Gibco, 21127030) 4. CellTite-Glo reagent (Promega, G7573) 5. 96-well cell culture plate (corning, 3903) 6. Pancreatin (Invitrogen, 25200-072) 7. Plate readers (BMG, PHERAsta) 8. Cell counter (Shanghai Rui▲Gyoku▼Biological Technology Co., Ltd., IC1000)

[0170] 2. Experimental Procedure LoVo cells were cultured in F-12K medium containing 10% FBS and passaged 2-3 times per week with a passage ratio of 1:3 or 1:5. During passage, the cells were digested with pancreatin, transferred to a centrifuge tube, centrifuged at 1200 rpm for 3 minutes, the supernatant residue was discarded, and the cells were resuspended in fresh medium. 90 μL of the cell suspension was added to a 96-well cell culture plate, resulting in a density of 3.88 × 10⁶. 4 The culture medium was at a concentration of cells / mL, and only 100 μL of complete medium was added to the outer perimeter of a 96-well plate. The culture plates were incubated in an incubator for 24 hours (37°C, 5% CO2).

[0171] The test sample was diluted to 2 ml with DMSO, and then diluted 3-fold sequentially to 10 different concentrations, creating blank and control wells. 5 μL of the test compound solution prepared to a gradient concentration was taken and added to 95 μL of fresh culture medium. 10 μL of the above-mentioned drug-containing medium solution was then added to the culture plate. The culture plate was incubated in an incubator for 3 days (37°C, 5% CO2). In a 96-well cell culture plate, 50 μL of CellTiter-Glo reagent was added to each well, and the plates were left at room temperature in the dark for 5 to 10 minutes. The chemiluminescence signal values ​​were read using PHERAstar, and the data was processed using GraphPad software.

[0172] 3. Experimental Data The inhibitory activity of the compounds relating to this disclosure against LoVo cell proliferation can be measured by the above tests, and the measured IC 50 The values ​​are shown in Table 13.

[0173] [Table 13]

[0174] Conclusion: The compounds disclosed herein exhibit good inhibitory activity against ATR enzymes.

[0175] Pharmacokinetic evaluation

[0176] Test Example 3: Pharmacokinetic study of the compound relating to this disclosure 1. Summary Using rats as test animals, the plasma drug concentrations of the compounds described herein were measured at various time points after intragastric administration by LC / MS / MS. The pharmacokinetic behavior of the compounds described herein in rats was studied, and their pharmacokinetic characteristics were evaluated.

[0177] 2. Test Plan 2.1 Test reagent The compound of Example 1. 2.2 Test animals Twelve healthy adult SD rats, roughly half male and half female, purchased from Weitonglihua Laboratory Animals Co., Ltd., were divided into three groups, each consisting of four rats. 2.3 Manufacturing of pharmaceuticals A certain amount of pharmaceutical product was weighed out, and a colorless, transparent solution was prepared by adding 5% DMSO, 5% Tween 80, and 90% physiological saline. 2.4 Administration SD rats were fasted overnight before being administered the substance intragastricly. The dose was 2 mg / kg in all cases, and the volume of administration was 10.0 mL / kg in all cases.

[0178] 3.Operation Rats were administered the compound of Example 1 intragastricly. 0.2 mL of blood was collected from the orbit before administration and at 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, and 24.0 hours after administration. The blood was placed in an EDTA-K2kk anticoagulation test tube, and the plasma was separated by centrifugation at 4°C and 11000 rpm for 5 minutes. The plasma was stored at -20°C, and the rats were fed 2 hours after administration. The content of the test compound in rat plasma after intragastric administration of different concentrations of the drug was measured: 25 μL of rat plasma was collected at each time point after administration, 50 μL of internal standard solution and 175 μL of acetonitrile were added, the mixture was vortexed for 5 minutes, and the mixture was centrifuged for 10 minutes (4000 rpm). 1 μL of the supernatant was taken from the plasma sample and analyzed by LC / MS / MS.

[0179] 4. Results of pharmacokinetic parameters

[0180] [Table 14]

[0181] Conclusion: The compounds described herein exhibit good pharmacokinetic absorption and offer significant advantages in pharmacokinetics.

Claims

1. A crystal of the hydrochloride salt of a compound represented by formula (I), 【Chemistry 1】 A type a crystal having a powder X-ray diffraction pattern with characteristic peaks at 2θ angles of 6.0, 8.3, 12.1, 14.3, 14.9, 16.7, and 26.7, with an error range of ±0.2 for the 2θ angles.

2. A crystal of the hydrochloride salt of a compound represented by formula (I), 【Chemistry 2】 A b-type crystal having a powder X-ray diffraction pattern with characteristic peaks at 2θ angles of 6.0, 12.1, 18.2, 23.6, 24.4, and 29.5, with an error range of ±0.2 for the 2θ angles.

3. A crystalline sulfate of the compound represented by formula (I), 【Transformation 3】 An α-type crystal having a powder X-ray diffraction pattern with characteristic peaks at 2θ angles of 5.8, 7.6, 13.7, 15.4, and 20.4, with an error range of ±0.2 for the 2θ angles.

4. A crystal of hydrobromide salt of a compound represented by formula (I), 【Chemistry 4】 A type I crystal having a powder X-ray diffraction pattern with characteristic peaks at 2θ angles of 6.0, 8.1, 14.7, 25.9, and 27.0, with an error range of ±0.2 for the 2θ angles.

5. A crystal of hydrobromide salt of a compound represented by formula (I), 【Transformation 5】 A type II crystal having a powder X-ray diffraction pattern with characteristic peaks at 2θ angles of 9.3, 11.6, 13.0, 16.8, 18.7, and 24.6, with an error range of ±0.2 for the 2θ angles.

6. A crystal of a mesylate salt of a compound represented by formula (I), 【Transformation 6】 (i) Powder X-ray diffraction pattern having characteristic peaks at 2θ angles of 10.0, 16.8, 17.8, 18.4 and 20.6, or (ii) Powder X-ray diffraction pattern having characteristic peaks at 2θ angles of 7.7, 10.0, 12.9, 13.8, 14.3, 15.1, 16.8, 17.8, 18.4, 20.3, 20.6, 21.9, 23.1, 24.2, 25.3, 26.1, 26.7, 28.3, 29.0, 30.7, 35.0 and 43.

1. An α-type crystal having the error range of the 2θ angle being ±0.

2.

7. A crystal of a mesylate salt of a compound represented by formula (I), 【Transformation 7】 A β-type crystal having a powder X-ray diffraction pattern with characteristic peaks at 2θ angles of 5.9, 8.4, 14.5, 16.8, 19.8, and 26.0, with an error range of ±0.2 for the 2θ angles.

8. A crystal of the maleate of the compound represented by formula (I), 【Transformation 8】 (i) Powder X-ray diffraction pattern having characteristic peaks at 2θ angles of 10.1, 17.1, 18.0, 19.0 and 24.3, or (ii) Powder X-ray diffraction pattern having characteristic peaks at 2θ angles of 7.2, 9.4, 10.1, 12.8, 13.2, 14.2, 14.8, 15.7, 17.1, 18.0, 19.0, 22.0, 23.4, 24.3, 25.2, 27.5 and 29.

1. An I-shaped crystal having the error range of the 2θ angle being ±0.

2.

9. A crystal of p-toluenesulfonate of a compound represented by formula (I), 【Chemistry 9】 A type A crystal having a powder X-ray diffraction pattern with characteristic peaks at 2θ angles of 6.5, 8.6, 12.0, 14.5, 21.2, and 22.2, with an error range of ±0.2 for the 2θ angles.

10. A crystal of the oxalate of the compound represented by formula (I), 【Chemistry 10】 (i) Powder X-ray diffraction pattern having characteristic peaks at 2θ angles of 5.5, 9.1, 11.0, 13.0, 16.5 and 20.2, or (ii) Powder X-ray diffraction pattern having characteristic peaks at 2θ angles of 5.5, 9.1, 11.0, 13.0, 15.5, 16.5, 20.2, 22.0, 22.5, 23.1, 24.9, 26.2, 27.8 and 30.

8. an a-type crystal having the error range of the 2θ angle being ±0.

2.

11. A method for producing crystals of a mesylate salt of a compound represented by formula (I) as described in claim 6, comprising mixing a solution containing the compound represented by formula (I) and methyl tert-butyl ether with methanesulfonic acid, forming a slurry, and crystallizing, wherein the mesylate salt is crystalline, and the crystalline mesylate salt is in the α form.

12. A method for producing crystals of a maleate salt of a compound represented by formula (I) according to claim 8, comprising mixing a solution containing the compound represented by formula (I) and a solvent selected from methyl tert-butyl ether or ethyl acetate / n-heptane with maleic acid, forming a slurry, and crystallizing it.

13. A method for producing oxalate crystals of a compound represented by formula (I) according to claim 10, comprising mixing a solution containing the compound represented by formula (I) and a solvent selected from methyl tert-butyl ether or ethyl acetate / n-heptane with oxalic acid, forming a slurry, and crystallizing the crystals.

14. A pharmaceutical composition comprising a crystalline salt of a compound represented by formula (I) as described in any one of claims 1 to 10, and one or more pharmaceutically acceptable vectors or excipients.

15. A method for producing a pharmaceutical composition, comprising the step of mixing a crystal of a salt of a compound represented by formula (I) as described in any one of claims 1 to 10 with one or more pharmaceutically acceptable vectors or excipients.

16. A pharmaceutical composition according to claim 14 for inhibiting ATR kinase.

17. The pharmaceutical composition according to claim 14 for treating hyperproliferative diseases.

18. A pharmaceutical composition according to claim 14 for treating neoplastic diseases.

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