Pharmaceutically acceptable salts of pyrazolo-heteroaryl derivatives and crystalline forms thereof

Abstract

The present disclosure relates to a pharmaceutically acceptable salt of a pyrazolo-heteroaryl derivative and a crystalline form thereof. In particular, the present disclosure relates to a pharmaceutically acceptable salt of a compound represented by Formula (I) and a crystalline form thereof. The novel crystal form of the present disclosure has good physicochemical properties.

Description

[0001] This application claims priority to Chinese patent application 2021105586705, filed on May 21, 2021. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This disclosure relates to a pharmaceutically acceptable salt of a pyrazolone aryl derivative and its crystalline form, and more particularly to a pharmaceutically acceptable salt of the compound represented by formula (I) and its crystalline form. Background Technology

[0003] Both normal and tumor cells experience thousands of DNA damage events daily. This makes DNA damage repair crucial for maintaining genome stability and cell survival. Compared to normal cells, tumor cells endure greater replication stress, carry more endogenous DNA damage, and frequently exhibit the absence of one or more DNA damage repair pathways. This makes tumor cell survival even more dependent on the successful completion of DNA damage repair.

[0004] Homologous recombination repair is the primary mechanism for repairing DNA double-strand breaks. It uses the homologous sequence of an undamaged sister chromatid as a template to replicate the damaged DNA sequence, precisely repairing the DNA. This repair primarily occurs during the G2 and S phases of the cell cycle. ATR, a key enzyme in the homologous recombination repair pathway and belonging to the PIKK family, is activated when the ATR / ATRIP complex binds to damaged DNA covered by replication protein A (RPA). ATR then 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, and increasing dNTP concentration, thus promoting DNA damage repair. The repair of DNA damage occurring in the S phase of the cell cycle is mainly accomplished by the ATR pathway, indicating that ATR is crucial for ensuring cell proliferation. Analysis of clinical tumor samples shows elevated ATR expression levels in various tumor tissues, including gastric cancer, liver cancer, colorectal cancer, ovarian cancer, and pancreatic cancer. Furthermore, high ATR levels are often associated with lower survival rates in patients with ovarian and pancreatic cancer. Therefore, ATR is an important target for cancer therapy.

[0005] WO2021098811A relates to a series of novel ATR inhibitors, among which the compound shown in formula (I) exhibits good ATR inhibitory activity, and its structure is shown below:

[0006]

[0007] The crystal structure of pharmaceutical active ingredients and their intermediates often affects their chemical stability. Different crystallization and storage conditions can lead to changes in the crystal structure of compounds, sometimes even resulting in other crystal forms. Generally, amorphous products lack regular crystal structures and often have other defects, such as poor product stability, fine crystals, difficulty in filtration, easy agglomeration, and poor flowability. Therefore, it is essential to improve the various properties of these products, and we need to conduct in-depth research to find new crystal forms with high purity and good chemical stability. Summary of the Invention

[0008] This disclosure provides a new salt form of the compound shown in formula (I), its crystal form, and a method for its preparation.

[0009]

[0010] This disclosure provides a pharmaceutically acceptable salt of the compound represented by formula (I), said pharmaceutically acceptable salt being selected from hydrochloride, sulfate, hydrobromide, methanesulfonate, p-toluenesulfonate, maleate, phosphate, formate, acetate, succinate, fumarate, citrate, malate, hippurate, or oxalate.

[0011] In some embodiments, the pharmaceutically acceptable salt is a mesylate, maleate, or oxalate.

[0012] In some embodiments, the molar ratio of the sulfate compound of formula (I) to sulfuric acid is 3:1 to 1:3, preferably 1:0.5 or 1:1.

[0013] In some embodiments, the molar ratio of the maleate salt of formula (I) to maleic acid is 3:1 to 1:3, preferably 1:0.5 or 1:1.

[0014] In some embodiments, the molar ratio of the compound of formula (I) to p-toluenesulfonate is 3:1 to 1:3, preferably 1:1 or 1:2.

[0015] In some embodiments, the molar ratio of the compound of formula (I) to methanesulfonic acid in the methanesulfonate is 3:1 to 1:3, preferably 1:1 or 1:2.

[0016] In some embodiments, the molar ratio of the oxalate compound of formula (I) to oxalic acid is 3:1 to 1:3, preferably 1:0.5 or 1:1.

[0017] This disclosure also provides a crystal form of the hydrochloride salt of the compound shown in formula (I), wherein the crystal form is:

[0018] The hydrochloride crystal form a has characteristic peaks in its X-ray powder diffraction pattern at 2θ angles of 6.0, 8.3, 12.1, 14.3, 14.9, 16.7 and 26.7.

[0019] The hydrochloride crystal form b has characteristic peaks in its X-ray powder diffraction pattern at 2θ angles of 6.0, 12.1, 18.2, 23.6, and 24.4.

[0020] In some embodiments, the X-ray powder diffraction pattern of the hydrochloride crystal form a 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.

[0021] In some embodiments, the X-ray powder diffraction pattern of the hydrochloride crystal form a is as follows: Figure 1 As shown.

[0022] In some embodiments, the X-ray powder diffraction pattern of the hydrochloride crystal form b 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.

[0023] In some embodiments, the X-ray powder diffraction pattern of the hydrochloride crystal form b is as follows: Figure 2 As shown.

[0024] This disclosure also provides a sulfate crystal form α of the compound shown in formula (I), whose X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 5.8, 7.6, 13.7, 15.4 and 20.4.

[0025] In some embodiments, the X-ray powder diffraction pattern of the sulfate crystal form α has characteristic peaks at 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.

[0026] In some embodiments, the X-ray powder diffraction pattern of the sulfate crystal form α is as follows: Figure 3 As shown.

[0027] This disclosure also provides a crystal form of the hydrobromide salt of the compound shown in formula (I), wherein the crystal form is:

[0028] Hydrobromide crystal form I has characteristic peaks in its X-ray powder diffraction pattern at 2θ angles of 6.0, 8.1, 14.7, 25.9 and 27.0.

[0029] Hydrobromide crystal form II has characteristic peaks in its X-ray powder diffraction pattern at 2θ angles of 9.3, 11.6, 13.0, 16.8, 18.7 and 24.6.

[0030] In some embodiments, the X-ray powder diffraction pattern of the hydrobromide crystal form I has characteristic peaks at 2θ angles of 6.0, 8.1, 14.7, 17.3, 18.8, 22.0, 25.9, 27.0 and 27.8.

[0031] In some embodiments, the X-ray powder diffraction pattern of the hydrobromide crystal form I is as follows: Figure 4 As shown.

[0032] In some embodiments, the X-ray powder diffraction pattern of the hydrobromide crystal 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.

[0033] In some embodiments, the X-ray powder diffraction pattern of the hydrobromide crystal form II is as follows: Figure 5 As shown.

[0034] This disclosure also provides a crystal form of the methanesulfonate salt of the compound shown in formula (I), wherein the crystal form is:

[0035] The α-methanesulfonate crystal form exhibits characteristic peaks in its X-ray powder diffraction pattern at 2θ angles of 10.0, 16.8, 17.8, 18.4, and 20.6; or

[0036] The methanesulfonate crystal form β has characteristic peaks in its X-ray powder diffraction pattern at 2θ angles of 5.9, 8.4, 14.5, 16.8, 19.8, and 26.0. In some embodiments, the methanesulfonate crystal form α has characteristic peaks in its X-ray powder diffraction pattern 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.

[0037] In some embodiments, the X-ray powder diffraction pattern of the methanesulfonate crystal form α is as follows: Figure 6 As shown.

[0038] In some embodiments, the X-ray powder diffraction pattern of the methanesulfonate crystal form β 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.

[0039] In some embodiments, the X-ray powder diffraction pattern of the methanesulfonate crystal form β is as follows: Figure 7 As shown.

[0040] This disclosure also provides a maleate crystal form I of the compound shown in formula (I), whose X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 10.1, 17.1, 18.0, 19.0 and 24.3.

[0041] In some embodiments, the X-ray powder diffraction pattern of maleate crystal form I 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.

[0042] In some embodiments, the X-ray powder diffraction pattern of maleate crystal form I is as follows: Figure 8 As shown.

[0043] This disclosure also provides a p-toluenesulfonate crystal form a of the compound shown in formula (I), whose X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 6.5, 8.6, 12.0, 14.5, 21.2 and 22.2.

[0044] In some embodiments, the X-ray powder diffraction pattern of the p-toluenesulfonate crystal form a 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.

[0045] In some embodiments, the X-ray powder diffraction pattern of the p-toluenesulfonate crystal form a is as follows: Figure 9 As shown.

[0046] This disclosure also provides an oxalate a crystal form of the compound shown in formula (I), whose X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 5.5, 9.1, 11.0, 13.0, 15.5, 16.5 and 20.2.

[0047] In some embodiments, the X-ray powder diffraction pattern of the oxalate crystal form a 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.

[0048] In some embodiments, the X-ray powder diffraction pattern of the oxalate crystal form a is as follows: Figure 10 As shown.

[0049] This disclosure further provides a method for preparing the hydrochloride crystal form a of the compound shown in formula (I), the method comprising: mixing a solution of methyl tert-butyl ether (MTBE) containing the compound shown in formula (I) with hydrochloric acid, and then slurrying and crystallizing.

[0050] This disclosure further provides a method for preparing the hydrochloride crystal form b of the compound shown in formula (I), the method comprising: heating the hydrochloride a of the compound shown in formula (I) to 90°C and collecting the crystals.

[0051] This disclosure further provides a method for preparing the sulfate crystal form α of the compound shown in formula (I), the method comprising: mixing a solution containing the compound shown in formula (I) and a solvent with sulfuric acid, and slurrying to precipitate crystals, wherein the solvent is selected from methyl tert-butyl ether or ethyl acetate (EA) / (n-heptane)heptane.

[0052] This disclosure further provides a method for preparing hydrobromide crystal form I of the compound shown in formula (I), the method comprising: mixing a solution containing the compound shown in formula (I) and a solvent with hydrobromic acid, and slurrying to precipitate crystals, wherein the solvent is selected from methyl tert-butyl ether or ethyl acetate / n-heptane.

[0053] This disclosure further provides a method for preparing hydrobromide crystal form II of the compound shown in formula (I), the method comprising: mixing a solution containing the compound shown in formula (I) and a solvent with hydrobromic acid, and slurrying to precipitate crystals, wherein the solvent is selected from ethyl acetate / n-heptane.

[0054] This disclosure further provides a method for preparing the methanesulfonate crystal form α of the compound shown in formula (I), the method comprising: mixing a solution containing the compound shown in formula (I) and methyl tert-butyl ether with methanesulfonic acid, and then slurrying and crystallizing.

[0055] This disclosure further provides a method for preparing the methanesulfonate crystal form β of the compound shown in formula (I), the method comprising: mixing a solution containing the compound shown in formula (I) and a solvent with methanesulfonic acid, and slurrying to precipitate crystals, wherein the solvent is selected from methyl tert-butyl ether or ethyl acetate / n-heptane.

[0056] This disclosure further provides a method for preparing maleate crystal form I of the compound shown in formula (I), the method comprising: mixing a solution containing the compound shown in formula (I) and a solvent with maleic acid, and slurrying to precipitate crystals, wherein the solvent is selected from methyl tert-butyl ether or ethyl acetate / n-heptane.

[0057] This disclosure further provides a method for preparing the p-toluenesulfonate crystal form a of the compound shown in formula (I), the method comprising: mixing a solution containing the compound shown in formula (I) and methyl tert-butyl ether with p-toluenesulfonic acid, and then slurrying and crystallizing.

[0058] This disclosure further provides a method for preparing the oxalate crystal form a of the compound shown in formula (I), the method comprising: mixing a solution containing the compound shown in formula (I) and a solvent with oxalic acid, and slurrying to precipitate crystals, wherein the solvent is selected from methyl tert-butyl ether or ethyl acetate / n-heptane.

[0059] The crystal forms obtained in this disclosure were subjected to structural determination and crystal form study by X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC).

[0060] The crystallization method of the crystal form disclosed herein is conventional, such as volatilization, cooling crystallization, or crystallization at room temperature.

[0061] The starting material used in the crystal form preparation method disclosed herein can be any form of the compound shown in formula (I), including but not limited to: amorphous, arbitrary crystal form, hydrate, solvate, etc.

[0062] This disclosure further provides a pharmaceutical composition comprising a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable carriers or excipients.

[0063] This disclosure further provides a pharmaceutical composition comprising a pharmaceutically acceptable salt crystal form of a compound of formula (I), and one or more pharmaceutically acceptable carriers or excipients.

[0064] This disclosure further provides a method for preparing a pharmaceutical composition, comprising the step of mixing a pharmaceutically acceptable salt of a compound of formula (I) with one or more pharmaceutically acceptable carriers or excipients.

[0065] This disclosure further provides a method for preparing a pharmaceutical composition, comprising the step of mixing a pharmaceutically acceptable salt of a compound of formula (I) with one or more pharmaceutically acceptable carriers or excipients.

[0066] This disclosure further provides the use of a pharmaceutically acceptable salt or crystal form of a pharmaceutically acceptable salt or a pharmaceutical composition thereof of the compound of formula (I) described herein in the preparation of a medicament for inhibiting ATR kinase.

[0067] This disclosure further provides the use of a pharmaceutically acceptable salt or crystal form of a pharmaceutically acceptable salt or a pharmaceutical composition thereof of the compound of formula (I) described herein in the preparation of a medicament for treating a proliferative disease.

[0068] This disclosure further provides the use of a pharmaceutically acceptable salt or crystal form of a pharmaceutically acceptable salt or a pharmaceutical composition thereof of the compound represented by formula (I) in the preparation of a medicament for treating tumor diseases.

[0069] The tumors described in this disclosure 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.

[0070] In the specification and claims of this application, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. However, for a better understanding of this disclosure, definitions and explanations of some related terms are provided below. Furthermore, in the event of any discrepancy between the definitions and explanations of terms provided herein and their commonly understood meanings by those skilled in the art, the definitions and explanations provided herein shall prevail.

[0071] The "pulping" described in this disclosure refers to a purification method that utilizes the characteristic that substances have poor solubility in solvents, but impurities have good solubility in solvents. Pulping purification can remove color, change crystal form, or remove a small amount of impurities.

[0072] The “X-ray powder diffraction pattern or XRPD” described in this disclosure refers to the X-ray powder diffraction pattern obtained when X-rays are incident on an atomic surface of a crystal or part of a crystal sample with a lattice spacing of d at a grazing angle θ (the complementary angle of the incident angle, also known as the Bragg angle) with a grazing angle θ.

[0073] The “X-ray powder diffraction pattern or XRPD” described in this disclosure is a pattern obtained by using Cu-Kα radiation in an X-ray powder diffractometer.

[0074] The “differential scanning calorimetry or DSC” described in this disclosure refers to measuring the temperature difference and heat flow difference between the sample and the reference material during the sample heating or isothermal process, in order to characterize all physical and chemical changes related to thermal effects and obtain phase transition information of the sample.

[0075] The “2θ or 2θ angle” mentioned in this disclosure refers to the diffraction angle, where θ is the Bragg angle, and the unit is ° or degree. The error range of 2θ is ±0.3 or ±0.2 or ±0.1.

[0076] The "interplanar spacing or interplanar spacing (d-value)" described in this disclosure refers to the use of three non-parallel unit vectors a, b, and c to connect adjacent lattice points in a space lattice. These vectors divide the lattice into juxtaposed parallelepiped units, known as the interplanar spacing. The space lattice is divided according to these defined parallelepiped unit lines, resulting in a linear grid called a space lattice or crystal lattice. Lattices and crystal lattices respectively use geometric points and lines to reflect the periodicity of a crystal structure. Different crystal planes have different interplanar spacings (i.e., the distance between two adjacent parallel crystal planes); the unit is d / d. Or E.

[0077] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of the event or environment. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0078] The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0079] The term "solvent" or "solvent compound" refers to a pharmaceutically usable solvate formed by the present disclosure of a drug with one or more solvent molecules, non-limiting examples of which include water, ethanol, methyl tert-butyl ether, acetone, n-heptane, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0080] The term "carrier" is used in the context of the drugs disclosed herein, referring to a system that can alter the way a drug enters the body and its distribution within the body, control the rate of drug release, and deliver the drug to the target organ. Drug carrier release and targeting systems can reduce drug degradation and loss, decrease side effects, and improve bioavailability. For example, high-molecular-weight surfactants, due to their unique amphiphilic structure, can self-assemble to form various forms of aggregates, preferably such as micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules while also exhibiting good membrane permeability, making them excellent drug carriers. Attached Figure Description

[0081] Figure 1 The XRPD pattern of the hydrochloride crystal form a of the compound shown in formula (I) is shown.

[0082] Figure 2 The XRPD pattern of the hydrochloride crystal form b of the compound shown in formula (I) is shown.

[0083] Figure 3 The image shows the XRPD pattern of the sulfate crystal form α of the compound shown in formula (I).

[0084] Figure 4 The image shows the XRPD pattern of the hydrobromide crystal form I of the compound shown in formula (I).

[0085] Figure 5 The XRPD spectrum of the hydrobromide crystal form II of the compound shown in formula (I) is shown.

[0086] Figure 6 The image shows the XRPD pattern of the methanesulfonate crystal form α of the compound shown in formula (I).

[0087] Figure 7 The image shows the XRPD pattern of the methanesulfonate crystal form β of the compound shown in formula (I).

[0088] Figure 8 The XRPD pattern of maleate crystal form I of the compound shown in formula (I) is shown.

[0089] Figure 9 The image shows the XRPD pattern of p-toluenesulfonic acid crystal form a of the compound shown in formula (I).

[0090] Figure 10 The XRPD pattern of the oxalate crystal form a of the compound shown in formula (I) is shown.

[0091] Figure 11 The DSC spectrum is shown for the hydrochloride crystal form a of the compound shown in formula (I).

[0092] Figure 12 The DSC spectrum is shown for the sulfate crystal form α of the compound shown in formula (I).

[0093] Figure 13 The image shows the DSC spectrum of the hydrobromide crystal form I of the compound shown in formula (I).

[0094] Figure 14 The DSC spectrum is shown for the hydrobromide crystal form II of the compound shown in formula (I).

[0095] Figure 15 The DSC spectrum is shown for the methanesulfonate crystal form α of the compound shown in formula (I).

[0096] Figure 16 The DSC spectrum is shown for the methanesulfonate crystal form β of the compound shown in formula (I).

[0097] Figure 17 The DSC spectrum is shown for maleate crystal form I of the compound shown in formula (I).

[0098] Figure 18 The image shows the DSC spectrum of p-toluenesulfonate crystal form a of the compound shown in formula (I).

[0099] Figure 19 The image shows the DSC spectrum of the oxalate crystal form a of the compound shown in formula (I). Detailed Implementation

[0100] The present disclosure will be explained in more detail below with reference to the embodiments. The embodiments of the present disclosure are only used to illustrate the technical solutions of the present disclosure and are not intended to limit the substance and scope of the present disclosure.

[0101] The structure of the compounds was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) are given in units of 10⁻⁶ (ppm). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer with deuterated dimethyl sulfoxide (DMSO-d₆), deuterated chloroform (CDCl₃), and deuterated methanol (CD₃OD) as solvents, and tetramethylsilane (TMS) as the internal standard.

[0102] MS measurements were performed using an Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS liquid chromatography-mass spectrometry system (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS).

[0103] Waters ACQuity UPLC-QD / SQD (Manufacturer: Waters, MS Model: Waters ACQuity QdaDetector / Waters SQ Detector) THERMO Ultimate 3000-Q Exactive (Manufacturer: THERMO, MS Model: THERMO Q Exactive)

[0104] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent HPLC 1260DAD, Agilent HPLC 1260VWD, and Waters HPLC e2695-2489 high-performance liquid chromatograph.

[0105] Chiral HPLC analysis was performed using an Agilent 1260 DAD high-performance liquid chromatograph.

[0106] High performance liquid chromatography (HPLC) was performed using Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson GX-281 preparative chromatographs.

[0107] Chiral preparation was performed using a Shimadzu LC-20AP preparative chromatograph.

[0108] The CombiFlash rapid preparation system uses a CombiFlash Rf200 (TELEDYNE ISCO).

[0109] Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15 mm to 0.2 mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4 mm to 0.5 mm.

[0110] Silica gel column chromatography generally uses Yantai Huanghai silica gel with a mesh size of 200-300 as the carrier.

[0111] The average inhibition rate and IC50 value of the kinase were determined using a NovoStar microplate reader (BMG GmbH, Germany).

[0112] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0113] Unless otherwise specified in the examples, the reactions can be carried out under an argon or nitrogen atmosphere.

[0114] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0115] A hydrogen atmosphere refers to a reaction vessel connected to a hydrogen balloon with a volume of approximately 1L.

[0116] The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0117] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0118] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

[0119] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0120] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20℃~30℃.

[0121] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system used for column chromatography to purify the compounds, and the developing solvent system for TLC included: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system, and C: petroleum ether / ethyl acetate system. The volume ratio of the solvent was adjusted according to the polarity of the compounds, and small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0122] THP stands for tetrahydropyranyl.

[0123] Test conditions for the instruments used in the experiment:

[0124] Differential Scanning Calorimeter (DSC)

[0125] Instrument model: Mettler Toledo DSC 3+

[0126] Purge gas: Nitrogen

[0127] Heating rate: 10.0℃ / min

[0128] Temperature range: 25-300℃

[0129] 2. X-ray diffraction (XRPD)

[0130] Instrument Model: BRUKER D8 Discover X-ray Powder Diffractometer

[0131] Rays: Monochromatic Cu-Kα rays

[0132] Scanning mode: θ / 2θ, scanning range (2θ range): 3~50°

[0133] Voltage: 40kV, Current: 40mA

[0134] 3. Ion chromatography

[0135] Instrument Model: DIONEX INTEGRION HPIC Ion Chromatograph (USA)

[0136] Detection method: conductivity; Separation column: Dionex IonPacTM-AS11-HC

[0137] Rinse solution: EGC-500-KOH

[0138] Flow rate: 1.4 ml / min

[0139] Example 1

[0140] (R)-2-methyl-2-(1-methyl-5-(3-methylmorphorline)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propionitrile I

[0141]

[0142]

[0143] first step

[0144] (R,E)-1-methyl-4-((1-(3-methylmorphorline)ethoxy)amino)-1H-pyrazole-5-carboxylic acid methyl ester 1c

[0145] Compound (R)-1-(3-methylmorpholine) ethyl-1-one 1b (2.5 g, 17.7 mmol, prepared by the method disclosed in the example of intermediate-1 on page 86 of patent application "WO2016020320A1") was dissolved in 1,2-dichloroethane under argon protection and cooled in ice water. Phosphorus oxychloride (7.4 g, 48.3 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 30 minutes. Then, compound methyl 4-amino-1-methyl-1H-pyrazole-5-carboxylate 1a (2.5 g, 16.1 mmol, Jiangsu Aikon Biotechnology) was added, and the mixture was heated to 80°C and stirred for 2 hours. The mixture was cooled to room temperature, concentrated under reduced pressure, and the residue was diluted with 200 mL of dichloromethane. It was then cooled in ice water and neutralized to pH 8–9 by adding saturated sodium bicarbonate solution. The organic phase was washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was mixed with silica gel. The filtrate was purified by silica gel column chromatography using eluent system C to obtain the title compound 1c (4.8 g), yield: 94%.

[0146] MS m / z(ESI): 281.2 [M+1]

[0147] Step 2

[0148] (R)-1-Methyl-5-(3-methylmorphorline)-1H-pyrazolo[4,3-b]pyridine-7-phenol 1d

[0149] Compound 1c (2.6 g, 9.3 mmol) was dissolved in tetrahydrofuran (20 mL), cooled in ice water, and then bis(trimethylsilylaminolithium) (27.8 mL, 1 M tetrahydrofuran solution, 27.8 mmol) was slowly added. The reaction was carried out at 0 °C for 1 hour. The reaction was quenched with methanol (10 mL), mixed with silica gel, and purified by silica gel column chromatography with eluent system A to give the title compound 1d (400 mg), yield: 55.8%.

[0150] MS m / z(ESI): 249.0 [M+1]

[0151] Step 3

[0152] (R)-4-(7-chloro-1-methyl-1H-pyrazolo[4,3-b]pyridin-5-yl)-3-methylmorpholine 1e

[0153] Compound 1d (400 mg, 1.6 mmol) was dissolved in 3.0 mL of phosphorus oxychloride and heated to 90 °C with stirring for 2.0 h. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the residue was diluted with 50 mL of dichloromethane. The solution was then cooled in ice water, neutralized to pH 8–9 with saturated sodium bicarbonate solution, and stirred for 0.5 h. The mixture was allowed to stand and separated, and the organic phase was collected, washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was mixed with silica gel. The filtrate was purified by silica gel column chromatography with eluent system C to give the title compound 1e (240 mg), yield: 56%.

[0154] MS m / z(ESI): 267.0 [M+1]

[0155] Step 4

[0156] (R)-2-methyl-2-(1-methyl-5-(3-methylmorphorline)-1H-pyrazolo[4,3-b]pyridin-7-yl)propionitrile 1g

[0157] Compound 1e (240 mg, 0.91 mmol) and compound isobutyronitrile 1f (620 mg, 8.9 mmol, Shanghai Bide) were dissolved in 30 mL of tetrahydrofuran under argon protection and cooled in a dry ice-acetone bath. Bistrimethylsilylaminolithium (8.9 mL, 1 M tetrahydrofuran solution, 8.9 mmol) was added dropwise, and the mixture was stirred at low temperature for 0.5 h. After naturally warming to room temperature, the mixture was stirred for 1 h. The reaction was quenched with water, and the organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was purified by silica gel column chromatography with eluent system C to give 1 g (200 mg) of the title compound, yield: 74%.

[0158] MS m / z(ESI): 300.1 [M+1]

[0159] Step 5

[0160] (R)-2-(3-bromo-1-methyl-5-(3-methylmorphorline)-1H-pyrazolo[4,3-b]pyridin-7-yl)-2-methylpropionitrile 1h

[0161] 1 g (200 mg, 0.67 mmol) was dissolved in 5 mL of 1,4-dioxane, and sodium hydroxide solution (0.66 mL, 2 M, 1.32 mmol) was added. The mixture was cooled with ice water, and liquid bromine (427 mg, 2.67 mmol) was added. The mixture was stirred at low temperature for 10 minutes, and then allowed to rise naturally to room temperature with stirring for 1 hour. The mixture was diluted with ethyl acetate, and the organic phase was washed with saturated sodium thiosulfate solution, then washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was purified by silica gel column chromatography with eluent system C to give the title compound 1 h (140 mg), yield: 55%.

[0162] MS m / z(ESI): 377.9 [M+1]

[0163] Step 6

[0164] 2-Methyl-2-(1-Methyl-5-((R)-3-methylmorphorline)-3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propionitrile 1i

[0165] 1 h (20 mg, 0.05 mmol), tetraphenylphosphine 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-dioxoborhecyclopentan-2-yl)-1H-pyrazole (29 mg, 0.10 mmol, Shanghai Bide) were dissolved in 4 mL of ethylene glycol dimethyl ether. 1 mL of water was added, and the mixture was heated to 120 °C for 1 hour under argon protection using a microwave oven. The reaction mixture was cooled to room temperature, and 20 mL of water was added. Extraction was performed with ethyl acetate (20 mL × 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. Purification was performed by silica gel column chromatography with eluent system C to give the title compound 1i (20 mg), yield: 84%.

[0166] MS m / z (ESI): 450.1 [M+1]

[0167] Step 7

[0168] (R)-2-methyl-2-(1-methyl-5-(3-methylmorphorline)-3-(1H-pyrazol-3-yl)-1H-pyrazolo[4,3-b]pyridin-7-yl)propionitrile I

[0169] 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 stirred for 4 hours. The reaction solution was concentrated under reduced pressure, and the pH was adjusted to 8-9 by adding 7 M ammonia-methanol solution. The solution was then concentrated under reduced pressure, and purified by silica gel column chromatography using eluent system A to give title compound I (7.0 mg), yield: 43%.

[0170] MS m / z(ESI): 366.0 [M+1]

[0171] 1H NMR (400MHz, CD3OD): δ7.58(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).

[0172] Example 2

[0173] 0.25 mL of an MTBE solution containing approximately 10 mg of the compound of formula (I) obtained in Example 1 was mixed with 22.5 μL of a 1.2 mol / L hydrochloric acid-ethanol solution and slurried. The solid was separated by centrifugation and vacuum dried to obtain the product. X-ray powder diffraction analysis identified the product as hydrochloride crystal form a, and the XRPD spectrum is shown below. Figure 1 As shown in Table 1, the positions of its characteristic peaks are as follows.

[0174] Table 1

[0175]

[0176]

[0177] Example 3

[0178] After heating the hydrochloride crystal form a of the compound shown in formula (I) to 90°C, the crystal form transformation was detected, and the product was defined as hydrochloride crystal form b. The XRPD spectrum is shown below. Figure 2 As shown in Table 2, the positions of its characteristic peaks are as follows.

[0179] Table 2

[0180]

[0181] Example 4

[0182] 0.25 mL of an MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 14.7 μL of a 1.8 mol / L sulfuric acid ethanol solution and slurried. The solid was separated by centrifugation and vacuum dried to obtain the product. X-ray powder diffraction analysis identified the product as sulfate α-form, and ion chromatography showed a sulfate ion content of 17.9%. The XRPD spectrum is shown below. Figure 3 As shown in Table 3, the positions of its characteristic peaks are as follows.

[0183] Table 3

[0184]

[0185]

[0186] Example 5

[0187] A 0.25 mL solution of MTBE containing approximately 10 mg of the compound shown in formula (I) was mixed with 36 μL of a 0.75 mol / L hydrobromic acid ethanol solution and slurried. The solid was separated by centrifugation and vacuum dried to obtain the product. X-ray powder diffraction analysis identified the product as hydrobromide crystal form I. The XRPD spectrum is shown below. Figure 4 As shown in Table 4, the positions of its characteristic peaks are as follows.

[0188] Table 4

[0189]

[0190] Example 6

[0191] A 0.25 mL solution of EA / heptane (1:1) containing approximately 10 mg of the compound shown in formula (I) was mixed with 72 μL of a 0.75 mol / L hydrobromic acid ethanol solution and slurried. The solid was separated by centrifugation and vacuum dried to obtain the product. X-ray powder diffraction analysis identified the product as hydrobromide crystal form II. The XRPD spectrum is shown below. Figure 5 As shown in Table 5, the positions of its characteristic peaks are as follows.

[0192] Table 5

[0193]

[0194]

[0195] Example 7

[0196] 0.25 mL of an MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 17.7 μL of a 1.5 mol / L methanesulfonic acid ethanol solution and slurried. The solid was separated by centrifugation and vacuum dried to obtain the product. X-ray powder diffraction analysis identified the product as methanesulfonate crystal form α, and ion chromatography showed a methanesulfonate ion content of 20.0%. The XRPD spectrum is shown below. Figure 6 As shown in Table 6, the positions of its characteristic peaks are as follows.

[0197] Table 6

[0198]

[0199]

[0200] Example 8

[0201] 0.25 mL of an MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 35.4 μL of a 1.5 mol / L methanesulfonic acid ethanol solution and slurried. The solid was separated by centrifugation and vacuum dried to obtain the product. X-ray powder diffraction analysis identified the product as methanesulfonate crystal form β, and the XRPD spectrum is shown below. Figure 7 As shown in Table 7, the positions of its characteristic peaks are as follows.

[0202] Table 7

[0203]

[0204] Example 9

[0205] 0.4 mL of an MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 30 μL of a 1 mol / L maleic acid ethanol solution and slurried. The solid was separated by centrifugation and vacuum dried to obtain the product. X-ray powder diffraction analysis identified the product as maleate crystal form I. Ion chromatography analysis showed that its maleate ion content was 23.7%. The XRPD spectrum is shown below. Figure 8 As shown in Table 8, the positions of its characteristic peaks are as follows.

[0206] Table 8

[0207]

[0208]

[0209] Example 10

[0210] 0.4 mL of an MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 30 μL of a 1 mol / L p-toluenesulfonic acid ethanol solution and slurried. The solid was separated by centrifugation and vacuum dried to obtain the product. X-ray powder diffraction analysis identified the product as p-toluenesulfonate crystal form a, and ion chromatography analysis showed that its p-toluenesulfonate ion content was 34.6%. The XRPD spectrum is shown below. Figure 9 As shown in Table 9, the positions of its characteristic peaks are as follows.

[0211] Table 9

[0212]

[0213] Example 11

[0214] 0.4 ml of an MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 30 μL of a 1 mol / L oxalate ethanol solution and slurried. The solid was separated by centrifugation and vacuum dried to obtain the product. X-ray powder diffraction analysis identified the product as oxalate crystal form a, and ion chromatography showed that its oxalate ion content was 10.9%. The XRPD spectrum is shown below. Figure 10 As shown in Table 10, the positions of its characteristic peaks are as follows.

[0215] Table 10

[0216]

[0217] Example 12

[0218] The stability of methanesulfonate crystal form α, sulfate crystal form α, maleate crystal form I, p-toluenesulfonate crystal form a, and oxalate crystal form a was investigated by sealing them in aluminum foil bags and placing them under conditions of -20℃, 4℃, 25℃ / 60%RH, and 40℃ / 75%RH, respectively. The results are as follows.

[0219] Table 11

[0220]

[0221]

[0222] Long-term / accelerated stability tests showed that all salt forms had good physical stability. In terms of chemical stability, oxalate crystal form a was relatively stable, while methanesulfonate crystal form α and maleate crystal form I showed slight degradation at 40℃ and 75% RH. Sulfate crystal form α and p-toluenesulfonate crystal form a had slightly poor stability.

[0223] Example 13

[0224] 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 a 1.5 mol / L ethanolic phosphoric acid solution and slurried. The solid was separated by centrifugation and dried under vacuum to obtain the product. X-ray powder diffraction analysis showed that the product was an amorphous phosphate.

[0225] Example 14

[0226] 0.25 mL of an MTBE solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 10.2 μL of a 2.7 mol / L formic acid ethanol solution and slurried. The solid was separated by centrifugation and dried under vacuum to obtain the product. X-ray powder diffraction analysis showed that the product was an amorphous formate salt.

[0227] Example 15

[0228] A 0.25 mL solution of ethyl acetate / n-heptane (V / V, 9:1) containing approximately 10 mg of the compound shown in formula (I) was mixed with 15.4 μL of a 1.8 mol / L ethanolic acetic acid solution and slurried. The solid was separated by centrifugation and dried under vacuum to obtain the product. X-ray powder diffraction analysis showed that the product was an amorphous acetate.

[0229] Example 16

[0230] A 0.25 mL solution of ethyl acetate / n-heptane (V / V, 9:1) containing approximately 10 mg of the compound shown in formula (I) was mixed with 33 μL of a 0.9 mol / L ethanolic succinate solution and slurried. The solid was separated by centrifugation and dried under vacuum to obtain the product. X-ray powder diffraction analysis showed that the product was an amorphous succinate.

[0231] Example 17

[0232] 0.4 ml of an ethanol / water (V / V, 9:1) solution containing approximately 10 mg of the compound shown in formula (I) was mixed with 60 μL of a 0.5 mol / L fumarate ethanol solution and slurried. The solid was separated by centrifugation and vacuum dried to obtain the product. X-ray powder diffraction analysis showed that the product was an amorphous fumarate.

[0233] Example 18

[0234] A 0.4 mL solution of MTBE containing approximately 10 mg of the compound shown in formula (I) was mixed with 30 μL of a 1 mol / L citric acid ethanol solution and slurried. The solid was separated by centrifugation and dried under vacuum to obtain the product. X-ray powder diffraction analysis showed that the product was an amorphous citrate salt.

[0235] Example 19

[0236] A 0.25 mL solution of ethyl acetate / n-heptane (V / V, 9:1) containing approximately 10 mg of the compound shown in formula (I) was mixed with 30 μL of a 1 mol / L ethanolic solution of malic acid and slurried. The solid was separated by centrifugation and dried under vacuum to obtain the product. X-ray powder diffraction analysis showed that the product was an amorphous malate.

[0237] Example 20

[0238] A 0.25 mL solution of ethyl acetate / n-heptane (V / V, 9:1) containing approximately 10 mg of the compound shown in formula (I) was mixed with 60 μL of a 0.5 mol / L hippuric acid ethanol solution and slurried. The solid was separated by centrifugation and dried under vacuum to obtain the product. X-ray powder diffraction analysis showed that the product was an amorphous hippurate.

[0239] Test example:

[0240] Biological evaluation

[0241] Test Example 1: The inhibitory effect of the disclosed compound on ATR enzyme.

[0242] The following method was used to determine the inhibitory effect of the disclosed compound on ATR enzymes. The experimental method is briefly described below:

[0243] I. Experimental Materials and Instruments

[0244] 1.ATR enzyme (Eurofins Pharma Discovery Services, 14-953-M)

[0245] 2. GST-tagged P53 protein (Eurofins Pharma Discovery Services, 14-952-M)

[0246] 3.384-well plate (Thermo Scientific, 267462)

[0247] 4. U-shaped bottom 96-hole plate (Corning, 3795)

[0248] 5. Antibody against phosphorylated P53 protein labeled with europium cavitation compounds (cisbio, 61P08KAE)

[0249] 6. Anti-GST antibody linked to d2 (cisbio, 61GSTDLF)

[0250] 7. ATP solution (Promega, V916B)

[0251] 8.EDTA (Thermo Scientific, AM9260G)

[0252] 9. Hepes (Gibco, 15630-080)

[0253] 10. Microplate reader (BMG, Pherasta)

[0254] II. Experimental Procedure

[0255] A mixture of 1 nM ATR enzyme, 50 nM P53 protein, 7.435 μM ATP, and small molecule compounds at different concentrations (1 μM initial concentration, 11 concentrations serially diluted 3-fold) was incubated at room temperature for 2 hours. Then, stop solution (12.5 mM HEPES, 250 mM EDTA) was added and mixed thoroughly. Next, 0.42 ng / well of labeled europium cavitation compound anti-phosphorylated P53 protein antibody and 25 ng / well of linked d2 anti-GST antibody were added. After incubation overnight at room temperature, fluorescence signals at 620 nm and 665 nm were detected using Pherastar. Data were processed using GraphPad software.

[0256] III. Experimental Data

[0257] The inhibitory activity of the disclosed compound against ATR enzymes can be determined by the above experiments, and the measured IC50 value is... 50 The values ​​are shown in Table 12.

[0258] Table 12 IC50 of the disclosed compounds against ATR enzyme inhibition 50 .

[0259] Example number <![CDATA[IC 50 / nM]]> Max Inhibition (%) 1 3 100

[0260] Conclusion: The compound disclosed herein exhibits good inhibitory activity against ATR enzymes.

[0261] Test Example 2: Cell Proliferation Experiment

[0262] The following method detects intracellular ATP levels and, based on IC50... 50 The inhibitory effect of the disclosed compound on LoVo cell proliferation was evaluated. The experimental methods are briefly described below:

[0263] I. Experimental Materials and Instruments

[0264] 1. LoVo, human colon cancer tumor cells (Nanjing Kebai, CBP60032)

[0265] 2. Fetal bovine serum (GIBCO, 10091-148)

[0266] 3. F-12K medium (Gibco, 21127030)

[0267] 4. CellTite-Glo reagent (Promega, G7573)

[0268] 5.96-well cell culture plate (corning, 3903)

[0269] 6. Pancreatic enzyme (Invitrogen, 25200-072)

[0270] 7. Microplate reader (BMG, Pherasta)

[0271] 8. Cell counter (Shanghai Ruiyu Biotechnology Co., Ltd., IC1000)

[0272] II. Experimental Procedure

[0273] LoVo cells were cultured in F-12K medium containing 10% FBS, passaged 2-3 times per week at a passage ratio of 1:3 or 1:5. During passage, cells were digested with trypsin and transferred to centrifuge tubes, centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in fresh medium. 90 μL of the cell suspension was added to each 96-well cell culture plate at a density of 3.88 × 10⁻⁶ cells / well. 4 Cells / ml, add only 100 μL of complete culture medium to the periphery of the 96-well plate. Incubate the plate in an incubator for 24 hours (37°C, 5% CO2).

[0274] Dilute the test sample to 2 mM with DMSO, and then serially dilute it 3-fold to 10 concentrations, setting up blank and control wells. Add 5 μL of the prepared gradient concentration test compound solution to 95 μL of fresh culture medium. Then add 10 μL of the above drug-containing culture medium solution to the culture plate. Incubate the culture plate in an incubator for 3 days (37℃, 5% CO2). Add 50 μL of CellTiter-Glo reagent to each well of a 96-well cell culture plate, incubate at room temperature in the dark for 5-10 min, and read the chemiluminescence signal values ​​in a Pherastar instrument. Data are processed using GraphPad software.

[0275] III. Experimental Data

[0276] The inhibitory activity of this disclosed compound on LoVo cell proliferation can be determined by the above experiments, and the measured IC50 value is... 50 The values ​​are shown in Table 13.

[0277] Table 13 IC50 of the disclosed compounds on the inhibition of LoVo cell proliferation 50 .

[0278] Example number <![CDATA[IC 50 / nM]]> Max Inhibition (%) 1 43 93

[0279] Conclusion: The compound disclosed herein exhibits good inhibitory activity against ATR enzymes.

[0280] Pharmacokinetic evaluation

[0281] Test Example 3: Pharmacokinetic Test of the Compounds Disclosed

[0282] 1. Abstract

[0283] Using rats as test animals, the plasma drug concentration at different time points after gavage administration of the compound of Example 1 was determined by LC / MS / MS. The pharmacokinetic behavior of this compound in rats was investigated to evaluate its pharmacokinetic characteristics.

[0284] 2. Test Plan

[0285] 2.1 Test Drugs

[0286] Compound of Example 1.

[0287] 2.2 Experimental Animals

[0288] Twelve healthy adult SD rats, half male and half female, were randomly divided into three groups of four rats each and purchased from Vital River Laboratory Animal Co., Ltd.

[0289] 2.3 Drug Preparation

[0290] Weigh a certain amount of the drug and add 5% DMSO, 5% Tween 80 and 90% physiological saline to prepare a colorless and clear solution.

[0291] 2.4 Administration

[0292] SD rats were fasted overnight and then administered the drug by gavage at a dose of 2 mg / kg and a volume of 10.0 mL / kg.

[0293] 3. Operation

[0294] In Example 1, compound was administered to rats via gavage. Blood samples of 0.2 mL were collected from the orbital cavity 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 samples were placed in EDTA-K2kk anticoagulant tubes, centrifuged at 11,000 rpm for 5 minutes at 4°C to separate the plasma, and stored at -20°C. The rats were fed 2 hours after administration.

[0295] To determine the content of the target compound in rat plasma after gavage administration of different concentrations of the drug: 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 centrifuged for 10 minutes (4000 rpm), and 1 μL of the supernatant of the plasma sample was taken for LC / MS / MS analysis.

[0296] 4. Pharmacokinetic Parameter Results

[0297] Table 14 shows the pharmacokinetic parameters of the compounds disclosed in this paper:

[0298]

[0299] Conclusion: The compound disclosed herein exhibits good pharmacokinetic absorption and significant pharmacokinetic advantages.

Claims

1. A pharmaceutically acceptable salt of the compound shown in formula (I), said pharmaceutically acceptable salt being selected from hydrochloride, sulfate, hydrobromide, methanesulfonate, p-toluenesulfonate, maleate, phosphate, formate, acetate, succinate, fumarate, citrate, malate, hippurate, or oxalate. 。 2. A pharmaceutically acceptable salt of the compound of formula (I) as claimed in claim 1, wherein the pharmaceutically acceptable salt is selected from methanesulfonate, maleate or oxalate.

3. The crystal form of the hydrochloride salt of the compound according to formula (I) of claim 1, wherein the crystal form is: hydrochloride crystal form a, the X-ray powder diffraction pattern of which has characteristic peaks at 2θ angles of 6.0, 8.3, 12.1, 14.3, 14.9, 16.7 and 26.

7.

4. The crystal form a of the hydrochloride salt of the compound of formula (I) according to claim 3, and its X-ray powder diffraction pattern is shown in Figure 1.

5. The crystal form of the hydrochloride salt of the compound according to formula (I) of claim 1, wherein the crystal form is: hydrochloride crystal form b, the X-ray powder diffraction pattern of which has characteristic peaks at 2θ angles of 6.0, 12.1, 18.2, 23.6 and 24.

4.

6. The crystal form b of the hydrochloride salt of the compound of formula (I) according to claim 5, and its X-ray powder diffraction pattern is shown in Figure 2.

7. A sulfate crystal form α of the compound shown in formula (I), whose X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 5.8, 7.6, 13.7, 15.4 and 20.

4.

8. The sulfate crystal form α of the compound of formula (I) according to claim 7 has the X-ray powder diffraction pattern shown in Figure 3.

9. A crystal form of the hydrobromide of the compound shown in formula (I), said crystal form being: hydrobromide crystal form I, the X-ray powder diffraction pattern of which has characteristic peaks at 2θ angles of 6.0, 8.1, 14.7, 25.9 and 27.

0.

10. The crystal form I of the hydrobromide of the compound of formula (I) according to claim 9, the X-ray powder diffraction pattern of which is shown in Figure 4.

11. A crystal form of the hydrobromide of the compound shown in formula (I), said crystal form being: hydrobromide crystal form II, the X-ray powder diffraction pattern of which has characteristic peaks at 2θ angles of 9.3, 11.6, 13.0, 16.8, 18.7 and 24.

6.

12. The crystal form II of the hydrobromide according to claim 11 has the X-ray powder diffraction pattern shown in Figure 5.

13. A crystal form of a methanesulfonate salt of the compound shown in formula (I), said crystal form being: methanesulfonate crystal form Its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 10.0, 16.8, 17.8, 18.4 and 20.

6.

14. The crystal form of the methanesulfonate of the compound of formula (I) according to claim 13. Its X-ray powder diffraction pattern shows 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.

15. The crystal form of the methanesulfonate of the compound of formula (I) according to claim 13. Its X-ray powder diffraction pattern is shown in Figure 6.

16. A crystal form of a methanesulfonate salt of the compound shown in formula (I), said crystal form being a methanesulfonate crystal form. Its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 5.9, 8.4, 14.5, 16.8, 19.8 and 26.

0.

17. A maleate crystal form I of the compound shown in formula (I), the X-ray powder diffraction pattern of which has characteristic peaks at 2θ angles of 10.1, 17.1, 18.0, 19.0 and 24.

3.

18. The maleate crystal form I of the compound of formula (I) according to claim 17, wherein the X-ray powder diffraction pattern 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.

19. The maleate crystal form I of the compound of formula (I) according to claim 17, the X-ray powder diffraction pattern of which is shown in Figure 8.

20. A p-toluenesulfonate crystal form a of the compound shown in formula (I), the X-ray powder diffraction pattern of which has characteristic peaks at 2θ angles of 6.5, 8.6, 12.0, 14.5, 21.2 and 22.

2.

21. The p-toluenesulfonate crystal form a of the compound of formula (I) according to claim 20, the X-ray powder diffraction pattern of which is shown in Figure 9.

22. An oxalate a crystal form of the compound shown in formula (I), the X-ray powder diffraction pattern of which has characteristic peaks at 2θ angles of 5.5, 9.1, 11.0, 13.0, 16.5 and 20.

2.

23. The oxalate a crystal form of the compound of formula (I) according to claim 22, wherein the X-ray powder diffraction pattern 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.

24. The oxalate a crystal form of the compound of formula (I) according to claim 22, the X-ray powder diffraction pattern of which is shown in Figure 10.

25. The crystal form of the salt of the compound of formula (I) according to any one of claims 3-24, wherein, The error range of the 2θ angle is ±0.

2.

26. A method for preparing the methanesulfonate crystal form of the compound of formula (I) as described in any one of claims 13-15 or 25. The method, the method comprising: A solution containing the compound shown in formula (I) and methyl tert-butyl ether is mixed with methanesulfonic acid and then slurried to crystallize.

27. A method for preparing maleate crystal form I of the compound of formula (I) as described in any one of claims 17-19 or 25, the method comprising: A solution containing the compound of formula (I) and a solvent is mixed with maleic acid, and then slurryed to crystallize. The solvent is selected from methyl tert-butyl ether or ethyl acetate / n-heptane.

28. A method for preparing the oxalate crystal form a of the compound of formula (I) as described in any one of claims 22-25, the method comprising: A solution containing the compound of formula (I) and a solvent is mixed with oxalic acid, and then slurryed to crystallize. The solvent is selected from methyl tert-butyl ether or ethyl acetate / n-heptane.

29. A pharmaceutical composition comprising a pharmaceutically acceptable salt of the compound of formula (I) according to claim 1 or a crystal form of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 3-25, and one or more pharmaceutically acceptable carriers or excipients.

30. A method for preparing a pharmaceutical composition, comprising the step of mixing a pharmaceutically acceptable salt of the compound of formula (I) according to claim 1 or a crystal form of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 3-25 with one or more pharmaceutically acceptable carriers or excipients.

31. Use of a salt of the compound of formula (I) according to claim 1, or a crystal form of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 3-25, or the pharmaceutical composition according to claim 29 in the preparation of a medicament for inhibiting ATR kinase.

32. The use according to claim 31, wherein the inhibition of ATR kinase is used to treat hyperproliferative disorders.

33. The use according to claim 31, wherein the inhibition of ATR kinase is used to treat tumor diseases.

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