Crystalline forms and preparation methods of fused tricyclic derivatives

Abstract

The present disclosure relates to crystalline forms and preparation methods of fused tricyclic derivatives, specifically to the crystalline form of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, its pharmaceutically acceptable salts, and the corresponding preparation methods.

Description

[Technical Field]

[0001] This application claims priority from Chinese Patent Application No. 202210915848.1, filed on August 1, 2022. The entire text of the above Chinese patent application is incorporated herein by reference.

[0002] The present disclosure relates to crystalline forms and preparation methods of fused tricyclic derivatives, which belong to the pharmaceutical field. [Background technology]

[0003] Cyclin-dependent kinases (CDKs) are important cellular enzymes that play a key role in regulating eukaryotic cell division and proliferation. The catalytic unit of cyclin-dependent kinases is activated by regulatory subunits called cyclins. At least 16 mammalian cellular cyclins have been identified (Annu. Rev. Pharmacol. Toxicol. (1999) 39:295-312). Cyclin B / CDK1, cyclin A / CDK2, cyclin E / CDK2, cyclin D / CDK4, cyclin D / CDK6, and possibly other heterodynes are important regulators of cell cycle progression. Other functions of cyclin / CDK heterodynes include transcriptional regulation, DNA repair, differentiation, and apoptosis (Annu. Rev. Cell. Dev. Biol. (1997) 13:261-291). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Annu.Rev.Pharmacol.Toxicol.(1999)39:295-312 [Non-patent document 2] Annu.Rev.Cell.Dev.Biol. (1997)13:261-291 Summary of the Invention [Problem to be solved by the invention]

[0005] PCT / CN2022 / 074509 provides novel cyclin-dependent kinase inhibitors, and the discovery of development forms of the compounds disclosed in that application has important clinical implications. [Means for solving the problem]

[0006] (Summary of the Invention) The present disclosure provides crystalline forms of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol (compound of formula (I)), pharmaceutically acceptable salts thereof, and corresponding methods of preparation. [ka]

[0007] The present disclosure provides a type A crystal of the compound represented by formula (I), which has a powder X-ray diffraction pattern expressed in 2θ angles with characteristic peaks at 9.6, 10.0, 11.7, 15.0, 21.1, and 21.7.

[0008] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ angles, of the A-type crystal of the compound of Formula (I) provided by the present disclosure has characteristic peaks at 7.8, 9.6, 10.0, 11.7, 15.0, 18.9, 19.4, 20.1, 21.1, and 21.7.

[0009] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angles 2θ, of the crystalline form A of the compound of Formula (I) provided by the present disclosure has characteristic peaks at 7.8, 9.6, 10.0, 11.7, 15.0, 18.9, 19.4, 20.1, 21.1, 21.7, 23.5, 26.7, and 29.4.

[0010] In a selected embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ angles, of the crystalline form A of the compound of formula (I) provided by the present disclosure is shown in FIG.

[0011] The present disclosure provides a method for preparing crystalline Form A of the compound of formula (I), comprising the steps of: Method one a) dissolving a compound of formula (I) in solvent 1; b) Add solvent 2 to precipitate crystals, The solvent 1 is selected from ketone solvents or ester solvents, and the solvent 2 is an ether solvent.

[0012] In an alternative embodiment, the ketone solvent is selected from acetone or methyl isobutyl ketone, the ester solvent is ethyl acetate, and the ether solvent is selected from isopropyl ether or methyl tert-butyl ether.

[0013] Method 2 The compound represented by formula (I) is dissolved in a solvent 3 and stirred to cause crystallization, and the solvent 3 is selected from an ester solvent and a ketone solvent, preferably, the ester solvent is selected from ethyl acetate and isopropyl acetate, and the ketone solvent is methyl isobutyl ketone.

[0014] The present disclosure provides type B crystals of the compound represented by formula (I), which have characteristic peaks at 11.3, 15.1, 20.9, 22.8, and 23.7 in their powder X-ray diffraction pattern expressed in terms of diffraction angle 2θ.

[0015] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ angles, of the B-type crystal of the compound of Formula (I) provided by the present disclosure has characteristic peaks at 7.4, 11.3, 14.3, 15.1, 16.3, 18.0, 20.0, 20.9, 22.8, 23.2, 23.7, 25.1, and 26.0.

[0016] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angles 2θ, of the B-type crystals of the compound of Formula (I) provided by the present disclosure has characteristic peaks at 7.4, 9.5, 11.3, 12.4, 12.8, 13.7, 14.3, 15.1, 16.3, 18.0, 19.2, 20.0, 20.9, 22.8, 23.2, 23.7, 25.1, 26.0, 26.8, 28.2, 30.4, 32.7, 33.6, 34.3, 35.5, 38.4, 39.0, and 40.5.

[0017] In a selected embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ angles, of the B-type crystal of the compound of formula (I) provided by the present disclosure is shown in FIG.

[0018] The present disclosure provides a method for preparing type B crystals of the compound of formula (I), comprising the steps of: dissolving the compound of formula (I) in a substituted lower alkane and stirring to allow crystallization.

[0019] In an alternative embodiment, the substituted lower alkane is nitromethane.

[0020] The present disclosure provides a C-type crystal of the compound represented by formula (I), which has a powder X-ray diffraction pattern expressed in 2θ angles with characteristic peaks at 4.8, 10.1, 12.0, and 15.0.

[0021] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ angles, of the C-type crystal of the compound of formula (I) provided by the present disclosure has characteristic peaks at 4.8, 7.6, 10.1, 12.0, 15.0, 19.7, 21.2, and 23.5.

[0022] In a selected embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ angles, of the C-type crystal of the compound of formula (I) provided by the present disclosure is shown in FIG.

[0023] The present disclosure provides a method for preparing crystalline Form C of the compound of formula (I), the method being selected from the following methods: Method one The compound of formula (I) is dissolved in a solvent 4, and then evaporated to crystallize, the solvent 4 being selected from a substituted lower alkane or a ketone solvent, optionally the substituted lower alkane being selected from dichloromethane or 1,2-dichloroethane, and the ketone solvent being methyl isobutyl ketone; Method 2 The compound represented by formula (I) is dissolved in a solvent 5, and the solution is stirred to crystallize the compound, and the solvent 5 is selected from aromatic hydrocarbon solvents, and optionally, the aromatic hydrocarbon solvent is selected from p-xylene or toluene; Method three a) dissolving the compound of formula (I) in a solvent 6; b) Solvent 7 is added to precipitate crystals, The solvent 6 is selected from an alcohol solvent, a ketone solvent, a nitrile solvent, and an ether solvent, and the solvent 7 is an ether solvent. Optionally, the alcohol solvent is selected from isopropanol or ethanol, the ketone solvent is acetone, the nitrile solvent is acetonitrile, and the ether solvent is selected from tetrahydrofuran or isopropyl ether.

[0024] The present disclosure provides a D-type crystal of the compound represented by formula (I), which has a powder X-ray diffraction pattern expressed in 2θ angles with characteristic peaks at 10.0, 10.5, 17.0, 18.7, and 23.9.

[0025] In an optional embodiment, the powder X-ray diffraction pattern of the D-type crystal of the compound of Formula (I) provided by the present disclosure, expressed in terms of diffraction angle 2θ, has characteristic peaks at 10.0, 10.5, 11.0, 13.4, 15.9, 17.0, 18.3, 18.7, 20.8, 23.9, and 28.0.

[0026] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angles 2θ, of the D-type crystal of the compound of Formula (I) provided herein has characteristic peaks at 10.0, 10.5, 11.0, 12.5, 13.4, 15.9, 17.0, 18.3, 18.7, 20.1, 20.8, 22.3, 23.9, 26.7, 28.0, 30.5, 31.0, 32.1, 33.1, 33.8, and 34.6.

[0027] In a selected embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ angles, of the D-form crystal of the compound of formula (I) provided by the present disclosure is shown in FIG.

[0028] The present disclosure provides a method for preparing crystalline Form D of the compound of formula (I), comprising the steps of: The compound represented by formula (I) is mixed with a solvent 8 and stirred to crystallize, and the solvent 8 is an ether solvent or a mixed solvent of an alcohol solvent and an ether solvent, optionally, the alcohol solvent is methanol, ethanol, or isopropanol, and the ether solvent is isopropyl ether.

[0029] The present disclosure provides Form E crystals of the compound represented by formula (I), which have a powder X-ray diffraction pattern expressed in 2θ angles with characteristic peaks at 4.8, 11.4, 14.4, 15.1, 16.4, 19.3, and 22.9.

[0030] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ, of the E-form crystal of the compound of Formula (I) provided by the present disclosure has characteristic peaks at 4.8, 7.4, 11.4, 14.4, 15.1, 16.4, 17.9, 19.3, 19.9, 20.9, 22.9, and 23.5.

[0031] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angles 2θ, of the E-form crystal of the compound of Formula (I) provided herein has characteristic peaks at 4.8, 7.4, 9.6, 11.4, 14.4, 15.1, 16.4, 17.9, 19.3, 19.9, 20.9, 22.9, 23.5, 24.2, 25.2, 25.9, 26.8, and 30.5.

[0032] In an alternative embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ angles, of the E-form crystal of the compound of formula (I) provided by the present disclosure is shown in FIG.

[0033] The present disclosure provides a method for preparing Form E crystals of the compound of formula (I), the method being selected from the following methods: Method one The compound represented by formula (I) is dissolved in a solvent 9 and stirred to crystallize, and the solvent 9 is a nitrile solvent, and optionally, the nitrile solvent is acetonitrile.

[0034] Method 2 a) dissolving a compound of formula (I) in a solvent 10; b) Add solvent 11 to precipitate crystals; The solvent 10 is selected from ketone solvents, and the solvent 11 is an ether solvent, and optionally, the ketone solvent is acetone, and the ether solvent is isopropyl ether.

[0035] The present disclosure provides crystalline form F of the compound represented by formula (I), which has a powder X-ray diffraction pattern expressed in 2θ angles with characteristic peaks at 11.1, 11.4, 14.3, 15.1, 15.7, 19.2, and 22.0.

[0036] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angles 2θ, of the F-type crystals of the compound of Formula (I) provided herein has characteristic peaks at 9.1, 11.1, 11.4, 13.3, 14.3, 15.1, 15.7, 16.6, 17.9, 19.2, 20.0, 21.1, 22.0, 23.6, 24.6, and 26.1.

[0037] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angles 2θ, of the F-type crystal of the compound of Formula (I) provided herein has characteristic peaks at 4.8, 9.1, 9.8, 11.1, 11.4, 12.0, 13.3, 14.3, 15.1, 15.7, 16.6, 17.9, 19.2, 20.0, 21.1, 22.0, 22.5, 23.6, 24.2, 24.6, 25.2, 25.6, 26.1, 28.7, 30.3, and 35.0.

[0038] In an alternative embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ angles, of the F-type crystal of the compound of formula (I) provided by the present disclosure is shown in FIG.

[0039] The present disclosure provides a method for preparing crystalline Form F of the compound of Formula (I), the method being selected from the following methods: Method one The compound of formula (I) is dissolved in a solvent 12 and stirred to crystallize, and the solvent 12 is a ketone solvent, optionally, the ketone solvent is methyl isobutyl ketone; Method 2 The compound represented by formula (I) is dissolved in tetrahydrofuran, clarified, and isopropyl ether and seed crystals of F-type crystals are added to precipitate crystals.

[0040] The present disclosure provides a G-type crystal of the compound represented by formula (I), which has a powder X-ray diffraction pattern expressed in 2θ angles with characteristic peaks at 11.3, 15.3, 16.1, 17.9, 18.6, and 20.8.

[0041] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ angles, of the G-form crystal of the compound of Formula (I) provided by the present disclosure has characteristic peaks at 11.3, 15.3, 16.1, 17.9, 18.6, 20.8, 22.8, 23.1, 23.8, and 25.8.

[0042] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ, of the G-form crystal of the compound of Formula (I) provided herein has characteristic peaks at 9.1, 11.3, 12.6, 13.8, 14.7, 15.3, 16.1, 17.0, 17.5, 17.9, 18.6, 19.6, 20.8, 21.6, 22.8, 23.1, 23.8, 24.3, 25.3, 25.8, 27.3, 27.8, 29.3, 30.8, 32.3, 33.6, 34.5, and 35.0.

[0043] In a selected embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ angles, of the G-form crystal of the compound of formula (I) provided by the present disclosure is shown in FIG.

[0044] The present disclosure provides a method for preparing type G crystals of the compound of formula (I), comprising the steps of: dissolving the compound of formula (I) in a mixed solvent of an alcoholic solvent and an etheric solvent, and stirring to crystallize; optionally, the alcoholic solvent is selected from methanol, and the etheric solvent is isopropyl ether.

[0045] The present disclosure provides H-type crystals of the compound represented by formula (I), which have characteristic peaks at 11.7, 12.3, 15.5, 19.4, and 22.3 in the powder X-ray diffraction pattern expressed in 2θ angles.

[0046] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ, of the H-form crystal of the compound of Formula (I) provided by the present disclosure has characteristic peaks at 11.7, 12.3, 15.5, 18.2, 19.4, 21.3, 22.3, 24.9, 27.1, and 28.1.

[0047] In an optional embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angles 2θ, of the H-form crystalline form of the compound of Formula (I) provided herein has characteristic peaks at 10.1, 11.7, 12.3, 12.8, 13.4, 15.5, 17.1, 18.2, 19.4, 20.5, 21.3, 22.3, 23.2, 24.5, 24.9, 25.6, 26.4, 27.1, 28.1, 28.4, 30.7, 31.2, 32.1, 33.3, 36.0, 37.0, and 39.2.

[0048] In a selected embodiment, the powder X-ray diffraction pattern, expressed in terms of diffraction angle 2θ angles, of the H-form crystal of the compound of formula (I) provided by the present disclosure is shown in FIG.

[0049] The present disclosure provides a method for preparing crystalline Form H of the compound of formula (I), the method being selected from the following methods: Method one The compound represented by formula (I) is dissolved in a solvent 13, and the solution is stirred to cause crystallization. The solvent 13 is an alcoholic solvent, a mixed solvent of an alcoholic solvent and water, a ketone solvent, or an ester solvent, wherein the alcoholic solvent is selected from methanol, ethanol, isopropanol, and n-propanol, the ketone solvent is selected from acetone, 2-butanone, and methyl isobutyl ketone, and the ester solvent is ethyl acetate; Method 2 The compound represented by formula (I) is mixed with a solvent 14, slurried, and crystallized, the solvent 14 being selected from water or an ether-based solvent; Optionally, the ether solvent is selected from methyl tert-butyl ether or isopropyl ether; Method three a) dissolving the compound of formula (I) in a solvent 15; b) Add solvent 16 to precipitate crystals; The solvent 15 is selected from an alcohol solvent, a ketone solvent, and an ether solvent, and the solvent 16 is selected from a lower alkane, an ether solvent, or water. Optionally, the alcohol solvent is selected from isopropanol, the ketone solvent is acetone, the lower alkane is n-heptane, and the ether solvent is tetrahydrofuran, isopropyl ether, or methyl tert-butyl ether.

[0050] Another aspect of the present disclosure provides a pharmaceutically acceptable salt of a compound of formula (I), wherein the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, mesylate, p-toluenesulfonate, phosphate, citrate, or malate.

[0051] In an alternative embodiment, the ratio of the compound of formula (I) to the acid molecule may be selected from about 3:1 to 1:3, specifically about 1:1, 1:2, or 1:3.

[0052] The present disclosure provides a method for preparing a pharmaceutically acceptable salt of a compound of formula (I), comprising reacting the free base with an acid molecule selected from hydrochloric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, phosphoric acid, citric acid, or malic acid.

[0053] In an alternative embodiment, the present disclosure provides a method for preparing a pharmaceutically acceptable salt of a compound of Formula (I) by reacting in the presence of a solvent.

[0054] In alternative embodiments, the solvent is a nitrile solvent, an alcohol solvent, an ester solvent, an aromatic hydrocarbon solvent, an ether solvent, a ketone solvent, or a substituted lower alkane solvent.

[0055] In an alternative embodiment, the nitrile solvent is acetonitrile.

[0056] In an alternative embodiment, the alcoholic solvent is ethanol.

[0057] The pharmaceutically acceptable salts of the compounds of formula (I) provided by this disclosure may be in any crystalline or amorphous form.

[0058] The substituted lower alkane according to the present disclosure may be selected from nitromethane, dichloromethane, and chloroform, the nitrile solvent is selected from acetonitrile or propionitrile, and the alcohol solvent is C 1- The C6 alcohol may be selected from methanol, ethanol, n-propanol, isopropanol, and n-butanol; the ketone solvent may be selected from acetone, 2-butanone, and methyl isobutyl ketone; the ether solvent may be selected from isopropyl ether, tetrahydrofuran, dioxane, and propylene glycol methyl ether; the ester solvent may be selected from ethyl acetate, methyl acetate, and isopropyl acetate; and the lower alkane may be selected from n-hexane or n-heptane.

[0059] In the preparation method provided by the present disclosure, the ratio of the solvent may be 0.1 to 100 times (w / v) the ratio of the compound represented by formula (I), specifically, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or a value between any two numbers.

[0060] The present disclosure provides methods for preparing crystalline forms that include separating the solid from the liquid, which may be by filtration, centrifugation, or complete evaporation of the solvent.

[0061] The methods for preparing the crystalline forms provided by this disclosure may include an optional step that includes drying.

[0062] The present disclosure also provides a pharmaceutical composition comprising any of the above crystalline forms or pharmaceutically acceptable salts, or a mixture thereof, and optionally a pharmaceutical additive selected from a pharmaceutically acceptable excipient.

[0063] The present disclosure also provides a method for preparing a pharmaceutical composition, comprising mixing any of the crystalline forms or pharmaceutically acceptable salts described above with a pharmaceutically acceptable excipient.

[0064] The present disclosure also provides the use of any of the crystalline forms or pharmaceutically acceptable salts thereof, or the composition, in the preparation of a medicament for treating or preventing a disease associated with a cyclin-dependent kinase.

[0065] The present disclosure also provides the use of any of the crystalline forms or pharmaceutically acceptable salts thereof, or the composition, in the preparation of a medicament for treating or preventing cancer.

[0066] In alternative embodiments, the cancer is selected from breast cancer, ovarian cancer, bladder cancer, uterine cancer, prostate cancer, lung cancer, esophageal cancer, head and neck cancer, intestinal cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, and thyroid cancer.

[0067] The term "2θ or 2θ angle" as used herein refers to the diffraction angle, where θ is the Bragg angle and is expressed in ° or degrees, and the error range of 2θ for each characteristic peak is ±0.2 (including rounding to the nearest decimal place), and may be any of the following: -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, - It may be 0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20.

[0068] The drying temperature described in the present disclosure is generally 25°C to 100°C, preferably 40°C to 70°C, and may be drying at normal pressure or reduced pressure, with the pressure being <-0.08 MPa.

[0069] "Excipients" as referred to in this disclosure include, but are not limited to, any auxiliary agent, carrier, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, or emulsifier that has already been approved by the U.S. Food and Drug Administration and is acceptable for use in humans or domestic animals.

[0070] The term "slurrying" used in this disclosure refers to a purification method that utilizes the properties of a substance that has poor solubility in a solvent but good solubility of impurities in the solvent, and purification by slurrying can decolorize, change the crystal form, or remove small amounts of impurities.

[0071] In the method for preparing the crystalline form of the present disclosure, the starting material used may be a compound in any form, and specific forms include, but are not limited to, amorphous, any crystalline form, hydrate, solvate, etc.

[0072] The ratio of the compound represented by formula (I) to the acid in the present disclosure is within a reasonable error range of ±10%, and may be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0073] The contents disclosed in PCT / CN2022 / 074509 are incorporated by reference into the present disclosure. [Brief explanation of the drawings]

[0074] [Figure 1] 1 is an amorphous XRPD pattern of the compound of formula (I). [Figure 2] 1 is an XRPD pattern of type A crystals of the compound of formula (I). [Figure 3] 1 is an XRPD pattern of type B crystals of the compound of formula (I). [Figure 4] 1 is an XRPD pattern of the C-type crystal of the compound represented by formula (I). [Figure 5] 1 is an XRPD pattern of type D crystal of the compound represented by formula (I). [Figure 6]1 is an XRPD pattern of type E crystals of the compound of formula (I). [Figure 7] 1 is an XRPD pattern of the F-type crystal of the compound represented by formula (I). [Figure 8] 1 is an XRPD pattern of the G-type crystal of the compound of formula (I). [Figure 9] 1 is an XRPD pattern of H-type crystals of the compound of formula (I). DETAILED DESCRIPTION OF THE INVENTION

[0075] The present disclosure will be further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure.

[0076] Example The structure of the compound is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR shifts (δ) are 10 -6 The NMR data are shown in ppm. A Bruker AVANCE NEO 500M nuclear magnetic resonance spectrometer was used for the NMR measurements, and the solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS).

[0077] For MS measurements, a liquid chromatograph mass spectrometer Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS (manufacturer: Agilent, MS model number: 6110 / 6120 Quadrupole MS) was used.

[0078] Waters ACQuity UPLC-QD / SQD (Manufacturer: Waters, MS Model Number: Waters ACQuity Qda Detector / Waters SQ Detector) THERMO Ultimate 3000-Q Exactive (Manufacturer: THERMO, MS Model Number: THERMO Q Exactive) High-performance liquid chromatography (HPLC) analysis was performed using high-pressure liquid chromatographs Agilent HPLC 1200DAD, Agilent HPLC 1200VWD, and Waters HPLC e2695-2489.

[0079] For chiral HPLC analysis, a high performance liquid chromatograph, Agilent 1260 DAD, was used.

[0080] For high-performance liquid preparative chromatography, preparative chromatographs Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 were used.

[0081] For chiral separation, a preparative chromatograph Shimadzu LC-20AP was used.

[0082] Combiflash Rf200 (TELEDYNE ISCO) was used as the CombiFlash high-speed preparative chromatograph.

[0083] Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plates are used for thin layer chromatography. The silica gel plate specifications used for thin layer chromatography (TLC) are 0.15 mm to 0.2 mm, and the specifications for separating and purifying products by thin layer chromatography are 0.4 mm to 0.5 mm.

[0084] For silica gel column chromatography, 200-300 mesh silica gel manufactured by Yantai Huanghai Silica Gel was generally used as the carrier.

[0085] Kinase mean inhibition rate and IC 50 The values ​​were measured using a plate reader NovoStar (BMG, Germany).

[0086] Known starting materials of the present disclosure may be synthesized by or according to methods known in the art, or may be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc.), and Darui Chemical.

[0087] In the examples, unless otherwise specified, all reactions can be carried out under an argon or nitrogen gas atmosphere.

[0088] An argon or nitrogen gas atmosphere refers to an argon or nitrogen gas balloon with a volume of approximately 1 L connected to the reaction flask.

[0089] A hydrogen atmosphere refers to a hydrogen balloon with a volume of approximately 1 L connected to the reaction flask.

[0090] For the pressurized hydrogenation reaction, a Parr 3916EKX hydrogenator and a Seiran QL-500 hydrogen generator or an HC2-SS hydrogenator were used.

[0091] The hydrogenation reaction was usually carried out by repeating the procedure of evacuating and refilling with hydrogen three times.

[0092] A CEM Discover-S 908860 microwave reactor was used for the microwave reactions.

[0093] In the examples, unless otherwise specified, the solution refers to an aqueous solution.

[0094] In the examples, unless otherwise specified, the reaction temperature is room temperature, 20°C to 30°C.

[0095] In the examples, thin layer chromatography (TLC) was used to monitor the reaction progress. The developing solvent used in the reaction, the eluent system of column chromatography for purifying the compound, and the developing solvent system of thin layer chromatography comprised A: dichloromethane / methanol system, and the volume ratio of the solvents was adjusted according to the polarity of the compound, and may be adjusted by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.

[0096] In this disclosure, the test conditions of the equipment used in the experiments: 1. Differential Scanning Calorimeter (DSC) Equipment model number: Mettler Toledo DSC 3+STARe System Purge gas: Nitrogen gas, Nitrogen gas purge rate: 50 mL / min Heating rate: 10.0℃ / min Temperature range: 25 to 350°C or 25 to 300°C 2. X-ray Powder Diffraction (XRPD) Model number: BRUKER D8 DISCOVER Powder X-ray Diffractometer Radiation: Monochromatic Cu-Kα radiation (λ=1.5406) Scanning method: θ / 2θ, Scanning range (2θ range): 3 to 45° Voltage: 40kV, Current: 40mA 3. Thermogravimetric Analysis (TGA) Model number: Mettler Toledo TGA2 Purge gas: Nitrogen gas, Nitrogen gas purge rate: 50 mL / min Heating rate: 10.0℃ / min Temperature range: 30 to 350°C or 30 to 300°C 4. DVS is dynamic moisture absorption The detection was performed using Surface Measurement Systems advantage2 at 25°C, with the humidity starting from 50%-95%-0%-95%-50% RH and changing in 10% steps. The criteria were that the mass change dM / dT at each gradient was less than 0.002%, TMAX was 360 min, and two cycles were performed.

[0097] Example 1. Preparation of free-state amorphous 10 mg of the compound of formula (I) was added to 0.5 mL of water, stirred at room temperature, centrifuged, and dried under vacuum to obtain a solid. The product was found to be amorphous by powder X-ray diffraction, and the XRPD pattern is shown in Figure 1.

[0098] Example 2. Preparation of free-form A crystals 10 mg of the compound of formula (I) was added to 0.05 mL of isopropyl acetate, clarified, cooled to 5°C, and stirred to crystallize. The crystallized product was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was identified as type A crystal by powder X-ray diffraction. The XRPD pattern is shown in Figure 2, and its characteristic peak positions are listed in Table 1. The DSC pattern showed endothermic peaks at 87.79°C and 254.08°C. The TGA pattern showed a weight loss of 10.07% between 30 and 150°C.

[0099] [Table 1]

[0100] Example 3. Preparation of free-form A crystals 10 mg of the compound of formula (I) was added to 0.05 mL of ethyl acetate, clarified, cooled to 5°C, stirred to cause crystallization, filtered, and the filter cake was collected and dried under vacuum to obtain a solid, which was found to be type A crystal by powder X-ray diffraction.

[0101] Example 4. Preparation of free-form A crystals 10 mg of the compound of formula (I) was added to 0.05 mL of methyl isobutyl ketone, clarified, cooled to 5°C, stirred to crystallize, filtered, and the filter cake was collected and dried under vacuum to obtain a solid, which was found to be type A crystal by powder X-ray diffraction.

[0102] Example 5. Preparation of free-form crystals of type A 10 mg of the compound of formula (I) was dissolved in 0.05 mL of acetone, 0.6 mL of isopropyl ether was added, and the mixture was stirred to crystallize. After centrifugation, the mixture was dried under vacuum to obtain a solid. The product was found to be type A crystals by powder X-ray diffraction.

[0103] Example 6. Preparation of free-form A crystals 10 mg of the compound of formula (I) was dissolved in 0.05 mL of methyl isobutyl ketone, 0.6 mL of isopropyl ether was added, and the mixture was stirred to crystallize. After centrifugation, the mixture was dried under vacuum to obtain a solid. The product was found to be type A crystals by powder X-ray diffraction.

[0104] Example 7. Preparation of free-form A crystals 10 mg of the compound of formula (I) was dissolved in 0.05 mL of ethyl acetate, and 0.8 mL of methyl tert-butyl ether was added, followed by evaporation to crystallize. The product was found to be type A crystals by powder X-ray diffraction.

[0105] Example 8. Preparation of free-form B crystals 10 mg of the compound of formula (I) was added to 0.05 mL of nitromethane, clarified, and stirred at room temperature to allow crystallization. The crystal was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was identified as type B crystals by powder X-ray diffraction. The XRPD pattern is shown in Figure 3, and its characteristic peak positions are listed in Table 2. The DSC pattern showed endothermic peaks at 154.21 °C and 254.73 °C. The TGA pattern showed a weight loss of 4.42% between 30 and 150 °C.

[0106] [Table 2]

[0107] Example 9. Preparation of free-form C crystals 10 mg of the compound of formula (I) was added to 0.05 mL of dichloromethane, clarified, and evaporated to crystallize, yielding a solid. The product was identified as type C crystals by powder X-ray diffraction. The XRPD pattern is shown in Figure 4, and its characteristic peak positions are listed in Table 3. The DSC pattern showed endothermic peaks at 125.96 °C and 253.40 °C. The TGA pattern showed a weight loss of 4.37% between 30 and 150 °C.

[0108] [Table 3]

[0109] Example 10. Preparation of free-form C crystals 10 mg of the compound of formula (I) was added to 0.05 mL of 1,2-dichloroethane, clarified, evaporated, and crystallized to obtain a solid, which was found to be type C crystals by powder X-ray diffraction.

[0110] Example 11. Preparation of free-form C crystals 10 mg of the compound of formula (I) was added to 0.05 mL of methyl isobutyl ketone, and the mixture was clarified and evaporated to crystallize, obtaining a solid. The product was found to be type C crystals by powder X-ray diffraction.

[0111] Example 12. Preparation of free-form C crystals 10 mg of the compound of formula (I) was added to 1 mL of p-xylene, stirred to crystallize, centrifuged, and dried under vacuum to obtain a solid. The product was found to be type C crystals by powder X-ray diffraction.

[0112] Example 13. Preparation of free-form C crystals 10 mg of the compound of formula (I) was added to 1 mL of toluene, stirred to cause crystallization, centrifuged, and then dried under vacuum to obtain a solid. The product was found to be type C crystals by powder X-ray diffraction.

[0113] Example 14. Preparation of free-form C crystals 10 mg of the compound of formula (I) was added to 0.1 mL of isopropanol, and 1 mL of isopropyl ether was added thereto, followed by stirring to precipitate a solid. The solid was centrifuged and then dried under vacuum to obtain a solid. The product was found to be type C crystals by powder X-ray diffraction.

[0114] Example 15. Preparation of free-form C crystals 10 mg of the compound of formula (I) was added to 0.1 mL of acetone, and 1 mL of isopropyl ether was added thereto, followed by stirring to precipitate a solid. The solid was centrifuged and then dried under vacuum to obtain a solid. The product was found to be type C crystals by powder X-ray diffraction.

[0115] Example 16. Preparation of free-form C crystals 10 mg of the compound of formula (I) was added to 0.1 mL of ACN (acetonitrile), and 1 mL of isopropyl ether was added, followed by stirring to precipitate a solid. The solid was centrifuged and then dried under vacuum to obtain a solid. The product was found to be type C crystals by powder X-ray diffraction.

[0116] Example 17. Preparation of free-form C crystals 10 mg of the compound of formula (I) was added to 0.1 mL of THF (tetrahydrofuran), and 1 mL of isopropyl ether was added, followed by stirring to precipitate a solid. The solid was centrifuged and then dried under vacuum to obtain a solid. The product was found to be type C crystals by powder X-ray diffraction.

[0117] Example 18. Preparation of free-form C crystals 10 mg of the compound of formula (I) was added to 0.1 mL of EtOH, and 1 mL of isopropyl ether was added and stirred to precipitate a solid. The solid was centrifuged and then dried under vacuum to obtain a solid. The product was found to be type C crystals by powder X-ray diffraction.

[0118] Example 19. Preparation of free-form D crystals 10 mg of the compound of formula (I) was added to 1 mL of isopropyl ether, stirred at 60°C for 2 hours, centrifuged, and dried under vacuum to obtain a solid. The product was identified as type D crystal by powder X-ray diffraction. The XRPD pattern is shown in Figure 5, and its characteristic peak positions are listed in Table 4. The DSC pattern showed an endothermic peak at 196.66°C. The TGA pattern showed a weight loss of 0.63% between 30 and 150°C.

[0119] [Table 4]

[0120] Example 20. Preparation of free-form D crystals 100 mg of the compound of formula (I) was added to 10 mL of isopropyl ether, and approximately 1 to 2 mg of seed crystals was added. The mixture was slurried at 60°C for 2 hours, centrifuged, and then dried under vacuum to obtain a solid. The product was found to be type D crystals by powder X-ray diffraction.

[0121] Example 21. Preparation of free-form D crystals 50 mg of the compound of formula (I) was added to 0.1 mL of ethanol, clarified by stirring at 60°C, 0.4 mL of isopropyl ether was added, and 1 to 2 mg of seed crystals of D-type crystals were added. The mixture was stirred at 60°C for 2 hours to precipitate a solid, which was centrifuged and dried under vacuum to obtain a solid. The product was identified as D-type crystals by powder X-ray diffraction.

[0122] Example 22. Preparation of free-form D crystals 50 mg of the compound of formula (I) was added to 0.1 mL of methanol, clarified by stirring at 60°C, 0.4 mL of isopropyl ether was added, and 1 to 2 mg of seed crystals of D-type crystals were added. The mixture was stirred at 60°C for 2 hours to precipitate a solid, which was centrifuged and dried under vacuum to obtain a solid. The product was identified as D-type crystals by powder X-ray diffraction.

[0123] Example 23. Preparation of free-form D crystals 700 mg of the compound of formula (I) was added to 1.4 mL of ethanol, clarified by stirring at 60°C, 4.2 mL of isopropyl ether was added, and 1 to 2 mg of seed crystals of D-type crystals were added. The mixture was stirred at 60°C for 2 hours to precipitate a solid, which was centrifuged and then dried under vacuum to obtain a solid. The product was identified as D-type crystals by powder X-ray diffraction.

[0124] Example 24. Preparation of free-form D crystals 10 mg of the compound of formula (I) was added to 0.4 mL of ethanol / isopropyl ether (1 / 20), stirred at 60°C to clarify, and stirred for 1 hour to precipitate a solid. After centrifugation, the solid was dried under vacuum to obtain a D-type crystal, which was detected by powder X-ray diffraction.

[0125] Example 25. Preparation of free-form D crystals 10 mg of the compound of formula (I) was added to 0.4 mL of methanol / isopropyl ether (1 / 20), stirred at 60°C to clarify, and stirred for 1 hour to precipitate a solid. After centrifugation, the solid was dried under vacuum to obtain a D-type crystal, which was detected by powder X-ray diffraction.

[0126] Example 26. Preparation of free-form E crystals 10 mg of the compound of formula (I) was added to 0.05 mL of acetonitrile for clarification, cooled to 5°C, and stirred at low temperature to crystallize. The crystal was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was identified as E-type crystals by powder X-ray diffraction. The XRPD pattern is shown in Figure 6, and its characteristic peak positions are listed in Table 5. The DSC pattern showed endothermic peaks at 35.46°C and 137.39°C. The TGA pattern showed a weight loss of 6.04% between 30 and 160°C.

[0127] [Table 5]

[0128] Example 27. Preparation of free-form E crystals 100 mg of the compound represented by formula (I) was added to 0.5 mL of acetone to clarify the mixture, and 3 mL of isopropyl ether was added thereto. The mixture was cooled to 5°C and stirred to cause crystallization. The mixture was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was found to be E-type crystals by powder X-ray diffraction.

[0129] Example 28. Preparation of free-form F crystals 100 mg of the compound of formula (I) was added to 0.5 mL of methyl isobutyl ketone and clarified. The crystallization was carried out by stirring at room temperature, followed by filtration. The filter cake was collected and dried under vacuum to obtain a solid. The product was identified as type F crystals by powder X-ray diffraction. The XRPD pattern is shown in Figure 7, and its characteristic peak positions are listed in Table 6. The DSC pattern showed endothermic peaks at 32.47 °C and 137.02 °C. The TGA pattern showed a weight loss of 6.99% between 30 and 160 °C.

[0130] [Table 6]

[0131] Example 29. Preparation of free-form F crystals 100 mg of the compound of formula (I) was added to 2 mL of tetrahydrofuran, and the mixture was stirred at 60°C to clarify the reaction. 10 mL of isopropyl ether and seed crystals of F-type crystals were added, and the mixture was stirred at 60°C for 2 hours to precipitate a solid. The solid was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was identified as F-type crystals by powder X-ray diffraction.

[0132] Example 30. Preparation of free-form G crystals 50 mg of the compound of formula (I) was added to 0.1 mL of methanol and 0.4 mL of isopropyl ether solvent, and the mixture was clarified and stirred at 60 °C for 2 h. The solid precipitated was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was identified as G-type crystals by powder X-ray diffraction. The XRPD pattern is shown in Figure 8, and its characteristic peak positions are listed in Table 7. The DSC pattern showed an endothermic peak at 173.41 °C. The TGA pattern showed a weight loss of 1.18% between 30 and 190 °C.

[0133] [Table 7]

[0134] Example 31. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.05 mL of ethanol, heated to 60°C with stirring to clarify, suspended at 50°C to 5°C, and stirred while increasing or decreasing the temperature to precipitate. The suspension was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was identified as H-type crystals by powder X-ray diffraction. The XRPD pattern is shown in Figure 9, and its characteristic peak positions are listed in Table 8. The DSC pattern showed an endothermic peak at 206.30°C. The TGA pattern showed a weight loss of 0.29% between 33 and 218°C.

[0135] [Table 8]

[0136] Example 32. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.05 mL of isopropanol, clarified by heating and stirring at 60°C, suspended at 50°C to 5°C, and precipitated by stirring while increasing and decreasing the temperature. The precipitate was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0137] Example 33. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.05 mL of n-propanol, clarified by heating and stirring at 60°C, suspended at 50°C to 5°C, and precipitated by stirring while increasing and decreasing the temperature. The precipitate was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0138] Example 34. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.05 mL of 2-butanone, clarified by heating and stirring at 60°C, suspended at 50°C to 5°C, and precipitated by stirring while increasing and decreasing the temperature. The precipitate was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0139] Example 35. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.05 mL of a 1 / 1 methanol / water solvent, clarified by stirring at 60°C, suspended at 50°C to 5°C, and precipitated by stirring while heating and cooling. The precipitate was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0140] Example 36. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.25 mL of ethyl acetate solvent, clarified by heating and stirring at 60°C, suspended at 50°C to 5°C, and precipitated by stirring while heating and cooling. The precipitate was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0141] Example 37. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.25 mL of methyl isobutyl ketone solvent, clarified by heating and stirring at 60°C, suspended at 50°C to 5°C, and precipitated by stirring while increasing and decreasing the temperature. The precipitate was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0142] Example 38. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 1 mL of water, heated and stirred at 60°C to insolubilize, suspended at 50°C to 5°C, stirred while increasing and decreasing the temperature to form a slurry, filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0143] Example 39. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 1 mL of methyl tert-butyl ether, insolubilized by heating and stirring at 60°C, suspended at 50°C to 5°C, stirred while heating and cooling to form a slurry, filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0144] Example 40. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 1 mL of isopropyl ether, heated and stirred at 60°C to insolubilize, suspended at 50°C to 5°C, stirred while increasing and decreasing the temperature to form a slurry, filtered, and the filter cake was collected and dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0145] Example 41. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.15 mL of isopropanol and dissolved by stirring at 60°C. 1 mL of water was added and the mixture was stirred to precipitate. After centrifugation, the mixture was dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0146] Example 42. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.15 mL of isopropanol and dissolved by stirring at 60°C. 1 mL of n-heptane was added and stirred to precipitate. After centrifugation, the mixture was dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0147] Example 43. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.15 mL of isopropanol and dissolved by stirring at 60°C. 1 mL of isopropyl ether was added, and the mixture was stirred to precipitate. After centrifugation, the mixture was dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0148] Example 44. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.1 mL of acetone and dissolved by stirring at 60°C. 1 mL of water was added to precipitate the product, which was centrifuged and dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0149] Example 45. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.1 mL of acetone and dissolved by stirring at 60°C. 1 mL of n-heptane was added to precipitate the product, which was centrifuged and dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0150] Example 46. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.1 mL of acetone and dissolved by stirring at 60°C. 1 mL of isopropyl ether was added to precipitate the solid, which was centrifuged and dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0151] Example 47. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.05 mL of tetrahydrofuran and dissolved by stirring at 60°C. 1 mL of methyl tert-butyl ether was added to precipitate the solid. After centrifugation, the solid was dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0152] Example 48. Preparation of free H-type crystals 10 mg of the compound of formula (I) was added to 0.05 mL of tetrahydrofuran and dissolved by stirring at 60°C. 1 mL of isopropyl ether was added to precipitate the solid. After centrifugation, the solid was dried under vacuum to obtain a solid. The product was found to be H-type crystals by powder X-ray diffraction.

[0153] Example 49. Hygroscopicity study of the free crystalline form Surface Measurement Systems intrinsic DVS was used to measure humidity in the range of 0% to 95% at 25°C with a 10% step size. The criteria were that the mass change dM / dT for each gradient was less than 0.002%, TMAX was 360 min, and two cycles were performed.

[0154] [Table 9]

[0155] Example 50. Stability study of factors influencing crystal form The free samples were opened and spread out, and the stability of the samples was examined under the conditions of light irradiation (4500 Lux), high temperature (40°C, 60°C), and high humidity (RH 75%, RH 92.5%). The sampling examination period was 30 days.

[0156] [Table 10]

[0157] [Table 11] [Table 12]

[0158] [Table 13]

[0159] [Table 14]

[0160] [Table 15]

[0161] The experimental results of the influencing factors showed that the D / E / F / G / H crystals and free amorphous forms have good chemical stability at high temperature and humidity. The D / E / F / G / H crystals and free amorphous forms have good physical stability.

[0162] Example 51. Long-term / accelerated stability of crystalline forms The free samples were sealed in aluminum foil bags and left at 25°C / 60% RH and 40°C / 75% RH, respectively, to examine their stability. The results are shown below.

[0163] [Table 16]

[0164] As the experimental results show, H-type crystals exhibit good physical and chemical stability after 3 months under long-term accelerated conditions. D-type, E-type, F-type, and G-type crystals exhibit good physical and chemical stability after 6 months under long-term accelerated conditions. Free amorphous materials accelerate deliquescence and exhibit good physical and chemical stability under other conditions.

[0165] Example 52. Preparation of hydrochloride salt 100 mg of the compound of formula (I) was added to 2 mL of acetonitrile, 115.3 μL of 2 M hydrochloric acid ethanol solution was added, and the mixture was stirred for 5 hours. The sample was then clarified, and 5 mL of isopropyl ether was added and stirred to precipitate. The mixture was centrifuged and dried under vacuum to obtain a solid. Ion chromatography showed that the chloride ion content was 6.7%.

[0166] Example 53. Preparation of sulfate salt 100 mg of compound of formula (I) was added to 2 mL of acetonitrile, 115.3 μL of 2 M sulfuric acid ethanol solution was added, and the mixture was stirred for 5 hours. The sample was then clarified, and 5 mL of isopropyl ether was added and stirred to precipitate. The mixture was centrifuged and dried under vacuum to obtain a solid. Ion chromatography showed that the sulfate ion content was 14.9%.

[0167] Example 54. Preparation of mesylate salt 100 mg of compound of formula (I) was added to 2 mL of acetonitrile, 115.3 μL of 2 M methanesulfonic acid ethanol solution was added, and the mixture was stirred for 5 hours. The sample was then clarified, and 5 mL of isopropyl ether was added and stirred to precipitate. The mixture was centrifuged and dried under vacuum to obtain a solid. Ion chromatography showed that the methanesulfonate ion content was 17.8%.

[0168] Example 55. Preparation of p-toluenesulfonic acid salt 100 mg of compound of formula (I) was added to 2 mL of acetonitrile, 115.3 μL of 2 M p-toluenesulfonic acid ethanol solution was added, and the mixture was stirred for 5 hours. The sample was then clarified, and 5 mL of isopropyl ether was added and the mixture was stirred to precipitate. The mixture was centrifuged and dried under vacuum to obtain a solid. Ion chromatography showed that the p-toluenesulfonic acid ion content was 34.3%.

[0169] Example 56. Preparation of phosphate salts 10 mg of the compound represented by formula (I) was added to 0.2 mL of acetonitrile, and 11.5 μL of a 2 M ethanol solution of phosphoric acid was added thereto, followed by stirring for 5 hours, centrifuging, and drying under vacuum to obtain a solid.

[0170] Example 57. Preparation of citrate salt 10 mg of the compound represented by formula (I) was added to 0.2 mL of acetonitrile, and 11.5 μL of a 2 M citric acid ethanol solution was added thereto, followed by stirring for 5 hours, centrifuging, and drying under vacuum to obtain a solid.

[0171] Example 58. Preparation of Malate 10 mg of the compound represented by formula (I) was added to 0.2 mL of acetonitrile, and 11.5 μL of a 2 M citric acid ethanol solution was added thereto, followed by stirring for 5 hours, centrifuging, and drying under vacuum to obtain a solid.

[0172] Example 59. (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol Isomer 1 [ka] [ka]

[0173] Step 1 6-Bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-2-formaldehyde 4a Under a nitrogen atmosphere, 6-bromo-8-fluoro-2,3-dimethyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene (2.0 g, 7 mmol) and selenium dioxide (3.1 g, 28 mmol) were added to 30 mL of 1,4-dioxane. The reaction was allowed to proceed at 95°C for 8 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was collected and concentrated under reduced pressure. The residue was purified by C-18 reverse-phase chromatography to give the title compound 4a (930 mg, yield: 44%). MS(ESI) m / z 299.1,301.1 [M+H] +

[0174] Step 2 1-(6-Bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-2-yl)ethan-1-ol 4b Under a nitrogen gas atmosphere, 6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-2-carbaldehyde (930 mg, 3.1 mmol) was dissolved in 20 mL of tetrahydrofuran. The temperature was lowered to -20 °C, and a tetrahydrofuran solution of methylmagnesium bromide (3 mol / L, 1.5 mL, 4.5 mmol) was added dropwise. The reaction was allowed to proceed at -20 °C for 4 hours. 5 mL of water was added to quench the reaction. The reaction solution was concentrated under reduced pressure, and the residue was purified by C-18 reverse-phase chromatography to obtain the title compound 4b (830 mg, yield: 85%). MS(ESI) m / z 315.2, 317.2 [M+H] +

[0175] Step 3 1-(6-Bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-2-yl)ethan-1-one 4c 1-(6-Bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-2-yl)ethan-1-one (600 mg, 1.9 mmol) was dissolved in 20 mL of tetrahydrofuran at room temperature, and Dess-Martin oxidant (2.0 g, 4.8 mmol) was added. The mixture was heated to 80 °C and reacted for 2 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was collected and concentrated under reduced pressure. The residue was purified by C-18 reverse-phase chromatography to give the title compound 4c (350 mg, yield: 59%). MS(ESI) m / z 313.1, 315.1 [M+H] +

[0176] Step 4 2-(6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-2-yl)propan-2-ol 4d Under a nitrogen atmosphere, 1-(6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-2-yl)ethan-1-one (350 mg, 1.1 mmol) was dissolved in 20 mL of tetrahydrofuran. The temperature was lowered to -20 °C, and a tetrahydrofuran solution of methylmagnesium bromide (3 mol / L, 0.7 mL, 2.1 mmol) was added dropwise. The reaction was allowed to proceed at -20 °C for 4 hours. 5 mL of water was added to quench the reaction. The reaction solution was concentrated under reduced pressure, and the residue was purified by C-18 reverse-phase chromatography to obtain the title compound 4d (260 mg, yield: 71%). MS(ESI) m / z 329.2,331.2 [M+H] +

[0177] Step 5 2-(6-(2,5-dichloropyrimidin-4-yl)-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-2-yl)propan-2-ol 4e In a nitrogen atmosphere, 2-(6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-2-yl)propan-2-ol (260 mg, 0.8 mmol), bis(pinacolato)diboron (305 mg, 1.2 mmol), potassium acetate (157 mg, 1.6 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (117 mg, 0.2 mmol) were dissolved in 5 mL of 1,4-dioxane. The mixture was allowed to react at 100°C for 2 hours. The reaction mixture was cooled to room temperature, and 1,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6-phosphaadamantane (58 mg, 0.2 mmol), potassium carbonate (221 mg, 1.6 mmol), 2,4-dichloro-5-fluoropyrimidine (220 mg, 1.2 mmol), tris(dibenzylideneacetone)dipalladium (183 mg, 0.2 mmol), and 1 mL of water were added, followed by reaction at 80° C. for 1 hour. The reaction mixture was cooled to room temperature, filtered, and the filtrate was collected and concentrated under reduced pressure. The residue was purified by C-18 reverse-phase chromatography to give the title compound 4e (129 mg, 41% yield). MS(ESI) m / z 397.3 [M+H] +

[0178] Step 6 (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol isomer 1 (3S,4R)-4-((5-chloro-4-((R)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol isomer 2 Under a nitrogen atmosphere, 2-(6-(2,5-dichloropyrimidin-4-yl)-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-2-yl)propan-2-ol (124 mg, 0.31 mmol), (3S,4R)-4-aminotetrahydro-2H-pyran-3-ol (19 mg, 0.16 mmol), (S)-(-)-2,2''-bis(diphenylphosphino)-1,1''-binaphthyl (118 mg, 0.5 mmol), and palladium acetate (14 mg, 0.06 mmol) were dissolved in 5 mL of tetrahydrofuran. Cesium carbonate (202 mg, 0.62 mmol) was added, and the mixture was allowed to react at 85°C for 3 hours. The reaction mixture was cooled to room temperature, filtered, the filtrate was collected and concentrated under reduced pressure, and the residue was purified by C-18 reverse phase chromatography to give crude product mixture 4f.

[0179] The crude product was subjected to chiral separation [Column: DAICEL CHIRALPAK AD (250 mm × 30 mm, 10 μm), Condition: 0.1% NH . HO ETOH, Begin B: 40%, End B: 40%, Flow Rate (mL / min): 60)] to obtain the title compound isomer 1 (25.2 mg, yield: 17%) and the title compound isomer 2 (22 mg, yield: 15%). Analysis method Column:DAICEL CHIRALCEL AD-3(100mm×4.6mm, 3μm), Mobile phase: A:CO2, B:ethanol (0.05% DEA), gradient: B increased from 5% to 40% in 2 min, held at 40% for 1.2 min, then held at 5% B for 0.8 min. Flow Rate: 4 mL / min ABPR: 1500 psi, Temperature: 35℃.

[0180] The compound with a retention time of 1.887 min was defined as isomer 2. MS(ESI) m / z 478.1[M+H]+ 1H NMR (400MHz, DMSO-d6) δ = 8.37 (s, 1H), 7.45 (br s, 1H), 6.92 (d, J=11.3 Hz, 1H), 5.82 (br s, 1H), 5.23 (q, J=6.4 Hz, 1H), 4.47 (d, J=11.0 Hz, 1H), 4.22 (br d, J=11.3 Hz, 1H), 3.89 - 3.67 (m, 3H), 3.39 - 3.25 (m, 4H), 3.03 (br t, J=10.2 Hz, 1H), 1.95 (br d, J=10.5 Hz, 1H), 1.68 (s, 3H), 1.62 (s, 3H), 1.45 (d, J=6.5 Hz, 3H) The compound with a retention time of 2.078 min was defined as Isomer 1. MS(ESI) m / z 478.1[M+H] + 1 H NMR (400MHz, DMSO-d6) δ = 8.37 (s, 1H), 7.45 (br s, 1H), 6.92 (d, J=11.5 Hz, 1H), 5.82 (s, 1H), 5.29 - 5.19 (m, 1H), 4.93 (d, J=5.5 Hz, 1H), 4.47 (d, J=11.3 Hz, 1H), 4.21 (br d, J=10.3 Hz, 1H), 3.85 - 3.75 (m, 3H), 3.53 - 3.40 (m, 2H), 3.03 (br t, J=10.4 Hz, 1H), 1.94 (br s, 1H), 1.67 (s, 3H), 1.62 (s, 3H), 1.45 (d, J=6.5 Hz, 3H), 1.06 (t, J=7.0 Hz, 1H)

[0181] Biological evaluation The present invention will be further explained below in conjunction with test examples, but these examples are not intended to limit the scope of the present invention.

[0182] Test Example 1: Detection of activity of the disclosed compounds on cyclin-dependent kinases 1. Experimental materials

[0183] [Table 17]

[0184] Compound A is Example A94 of WO 2019 / 207463A1 and was synthesized by referring to the method disclosed in the patent application.

[0185] 2. Kinase activity test (CDK4 / CyclinD1, CDK6 / CyclinD3) Measurement of kinase activity by mobility shift assay In vitro CDK kinase activity was tested using a mobility shift assay. The starting concentration of the test compounds for CDK activity inhibition was 300 nM, and the compounds were diluted three-fold to a total of 10 concentrations in parallel wells. Staurosporine was used as a standard control.

[0186] 1x kinase buffer (CDK2) (50 mM HEPES, pH 7.5, 0.0015% Brij-35), 1x kinase buffer (CDK4) (20 mM HEPES, pH 7.5, 0.01% Triton X-100), and stop solution (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA) were prepared. A 2.5x enzyme solution was prepared by adding the appropriate amount of kinase to the 1x kinase buffer. Compound dilutions (1x kinase buffer, 10% DMSO) corresponding to 5x the compound test concentration were prepared. A 2.5x substrate solution was prepared by adding the appropriate amount of FAM-labeled polypeptide and ATP to the 1x kinase buffer. 5 μL of 5x compound dilution solution and 10 μL of 2.5x enzyme solution were added to the reaction wells of a 384-well reaction plate, mixed evenly, and then incubated at room temperature for 10 minutes. Then, 10 μL of 2.5x substrate solution was added to the 384-well plate and centrifuged at 1000 rpm for 1 minute. The reaction plate was incubated at 28°C for 60 minutes (biochemical incubator model number: SPX-100B-Z). 30 μL of stop solution was added to the 384-well reaction plate to stop the reaction, and centrifuged at 1000 rpm for 1 minute. Finally, the conversion rate data (excitation wavelength: 400 nm, emission wavelength: 445 nm and 520 nm) were read using Caliper EZ Reader II.

[0187] Compound IC 50 The values ​​were fitted using the XLFit excel add-in version 5.4.0.8. The fitting formula is: Y=Bottom+(Top-Bottom) / (1+(IC50 / X)^HillSlope).

[0188] 3. Kinase activity test (CDK1 / CyclinB, CDK9 / CyclinT1) The starting concentration for in vitro CDK (CDK2, CDK9) kinase activity testing was 1 μM, and the starting concentration was diluted 3-fold to a total of 10 concentrations, which were tested in parallel wells. Compound PHA-793887 was used as a control compound.

[0189] 1x kinase reaction buffer (40 mM Tris-HCl, pH 7.4, 20 mM Mg2Cl2, 0.1 mg / mL BSA, 50 μM DTT), 1x volume of 5x kinase reaction buffer, and 4x volume of water were prepared, and DTT (final concentration 50 μM) was added. 50 nL of diluted compound working solution (final concentration of 1% DMSO) was transferred to each well of a reaction plate (Product Code: 784075, Greiner) using an Echo 655. The reaction plate was sealed with plate sealing film and centrifuged at 1000 x g for 1 minute. 2x enzyme (0.3 ng / μL CDK2 / Cyclin E1 or CDK9 / Cyclin T1) was prepared in 1x kinase reaction buffer, and 2.5 μL of the above kinase solution was added to each well. The reaction plate was sealed with plate sealing film, centrifuged at 1000 x g for 1 minute, and left at room temperature for 10 minutes. A 2x kinase substrate and ATP mixture was prepared in 1x kinase reaction buffer. The 2x CDK2 / CylinE1 kinase substrate consisted of 0.4 mg / mL histone H1 and 30 μM ATP. 2.5 μL of the 2x histone H1 and ATP mixture was added to the reaction plate and centrifuged at 1000 g for 30 seconds to initiate the reaction. The kinase assay was performed at room temperature for 120 minutes, after which 4 μL of ADP-Glo ​​reagent was added and incubated at room temperature for 40 minutes. 8 μL of kinase detection reagent was then added and incubated at room temperature for 40 minutes. The luminescence signal was read using an Envision 2104.

[0190] The data analysis is as follows: a) Percent inhibition: inhibition%=100−(Signalcmpd−SignalAve_PC) / (SignalAve_VC−SignalAve_PC)×100. SignalAve_PC: The average value of all positive control wells across the plate. SignalAve_VC: average value of all negative control wells across the plate.

[0191] b) Compound IC50: Obtained by calculation using the following nonlinear fitting equation in GraphPad 8.0.

[0192] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope)) X: logarithmic value of compound concentration, Y: percentage of compound inhibition The biochemical inhibitory activity of the compounds of the present disclosure against CDK (CDK1, CDK4, CDK6, CDK9) kinases was measured by the above test, and the measured IC 50 The values ​​are shown in Tables 17 and 18.

[0193] [Table 18]

[0194] [Table 19]

[0195] Test Example 2: CYP inhibition experiment One hundred fifty donors were mixed with human liver microsomes (purchased from Corning, product number 452117) to evaluate the metabolic reactions of representative substrates of the five major human CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Liquid chromatography tandem mass spectrometry (LC / MS / MS) was used to measure the effects of different concentrations of the target compound on the metabolic reactions of phenacetin (CYP1A2), diclofenac sodium (CYP2C9), S-mephenytoin (CYP2C19), bufuralol hydrochloride (CYP2D6), and midazolam (CYP3A4 / 5).

[0196] 30 μM phenacetin, 10 μM diclofenac sodium, 35 μM S-mephenytoin, 5 μM bufuralol hydrochloride, 3 μM midazolam, 1 mM NADPH, the compound to be tested (at concentrations of 0.1, 0.3, 1, 3, 10, and 30 μmol / L), the positive compound, or the blank control, and human liver microsomes (0.2 mg / mL) were mixed in 200 μL of 100 mmol / L phosphate buffer, pH 7.4, containing 0.3% DMSO, 0.6% acetonitrile, and 0.1% methanol by volume. The mixture was then incubated at 37°C for 5 minutes. 200 μL of acetonitrile solution containing 3% formic acid and 40 nM internal standard verapamil was then added, followed by centrifugation at 4000 rpm for 50 minutes. The mixture was then cooled on ice for 20 minutes and centrifuged at 4000 rpm for another 20 minutes to precipitate proteins. 200 μL of the supernatant was taken and subjected to LC / MS / MS analysis.

[0197] The peak area was calculated from the chromatogram. The residual activity ratio (%) was calculated using the following formula: Peak area ratio = metabolite peak area / internal standard peak area Residual activity ratio (%) = Peak area ratio of compounds waiting to be measured / Peak area ratio of blank group CYP half-inhibitory concentration (IC 50 ) was calculated using Excel XLfit 5.3.1.3.

[0198] Measured CYP half-inhibitory concentrations (IC 50 ) values ​​are shown below in Table 19.

[0199] [Table 20]

[0200] Test Example 3: Solubility test experiment The thermodynamic solubility of the compounds in phosphate buffer at pH 7.4 was measured. The supernatants of the samples and standards of known concentrations were all detected by LC / MS / MS.

[0201] 1. Materials and reagents Compound A (Compound A is example A94 of WO 2019 / 207463A1, and (obtained with reference to the disclosed method).

[0202] NaH2PO42H2O (analytical reagent), NaH2PO4 (analytical reagent), NaOH (analytical reagent).

[0203] 1.5 mL flat-bottom glass tubes (BioTech Solutions), molded polytetrafluoroethylene lids (BioTech Solutions), polytetrafluoroethylene-coated stir bars (BioTech Solutions), an Eppendorf comfort-type thermostatic mixer, and a 96-well deep-well plate.

[0204] 2. Preparation of 0.01 M Sodium Phosphate Buffer, pH 7.4 15.6 g of NaH2PO42H2O was weighed into a 1 L glass flask and dissolved in 1 L of deionized water. The pH of the solution was about 4.7, and then the pH was adjusted to 7.4 with 10 M NaOH.

[0205] 3. Solubility measurement process 1 mg of each compound was accurately weighed and placed in a glass tube. A phosphate buffer solution of 1 mL per 1 mg was added. A stir bar was placed in each tube, then the lid was replaced. The sample tray containing the tubes was placed in an Eppendorf thermostatic mixer and incubated at 25°C and 1100 rpm for 24 hours. After incubation, the lid was opened, the stir bar was removed by magnetic attraction, and the activity in each tube was recorded. The plate was then centrifuged at 25°C and 4000 rpm for 30 minutes. 750 μL of the supernatant was aspirated. The pipette head was washed with acetonitrile for 5 seconds and then with pure water for 5 seconds. The previous 50 μL of waste solution was then discarded, and the remaining 700 μL was placed in a separate 96-well sample plate containing glass tubes and centrifuged for another 30 minutes (25°C, 4000 rpm). 10 μL of the secondary centrifuged sample was aspirated and added to 990 μL of a 1:1 mixture of acetonitrile and water containing the internal standard (100x sample). 10 μL of the diluted solution was aspirated and added to 990 μL of a 1:1 mixture of acetonitrile and water containing the internal standard (10,000x sample). The dilution factor of the sample may vary depending on the solubility value and LC / MS signal response.

[0206] [Table 21]

[0207] 4. Preparation of standards Accurately weigh 1 mg of compound powder and place it in each glass tube. Add DMSO to each tube. The loading volume was 1 mL per 1 mg. Each tube was fitted with a stir bar, then capped. The tubes containing the standards were placed in an Eppendorf thermostatic mixer and incubated at 25°C and 1,100 rpm for 2 hours to fully dissolve the powder. The complete dissolution of the solid was observed, and any compounds that were not completely dissolved in the DMSO solution were recorded. Ten μL of the 1 mg / mL standard was aspirated and added to 990 μL of a 1:1 mixture of acetonitrile and water containing the internal standard to obtain a 10 μg / mL standard. Ten μL of the 10 μg / mL standard was aspirated and added to 990 μL of a 1:1 mixture of acetonitrile and water containing the internal standard to obtain a 0.1 μg / mL standard. The dilution factor of the sample may vary depending on the LC / MS signal response. The samples were analyzed by LC / MS / MS. All compounds were tested singly.

[0208] 5. Data calculation All calculations were also performed using Microsoft Excel. Samples were analyzed by LC / MS / MS and quantified using standards of known concentration. The solubility of the compound to be measured was calculated using the following formula: [Sample] = Area Ratio sample × DF sample × [STD] / Area Ratio STD DF: dilution factor.

[0209] [Table 22]

[0210] Test Example 5: PXR induction experiment 1. The potential of the target compounds to induce drug-metabolizing enzyme activity by activating PXR in vitro was evaluated. EC50 values ​​were obtained by measuring the activity of the target compounds at different concentrations (30, 10, 3.33, 1.11, 0.370, and 0.123 μM). The concentrations of the positive control, rifampin, were 20, 5, 1.25, 0.312, 0.0781, and 0.195 μM.

[0211] 2. Materials and reagents 1) DPX2 cells (HepG2 cells stably transfected with the human PXR gene and a fluorescent reporter gene) were purchased from Puracyp (Carlsbad, CA).

[0212] 2) Compounds awaiting measurement were provided by the client, and the control drug (rifampin) was purchased from Sigma (St. Louis, MO).

[0213] 3) CellTiter-Fluor TM The Cell Activity Detection Reagent Kit and One-Glo Fluorescence Detection Reagent Kit were purchased from Promega (Madison, WI), fetal bovine serum (FBS) was purchased from Corning (Manassas, VA), an MTS3 shaker was purchased from IKA Labortechnik (Staufen, Germany), DMEM, penicillin, and streptomycin were purchased from a local supplier, Hygromycin B and G418 were purchased from Merck (Darmstadt, Germany), and cell culture medium and DPX2 cells were purchased from Puracyp Inc.

[0214] 3. Experimental Procedure 3.1 Seed plate preparation 1) 50 mL of FBS was added to 450 mL of cell culture medium.

[0215] 2) DPX2 cells were cultured in a T-75 culture flask in an incubator at 37°C, 5% CO2, and 95% relative humidity. When the cells had grown to cover 80-90% of the bottom of the culture flask, they were digested.

[0216] 3) The cell surface of the T-75 culture flask was washed with 8 mL of PBS, the PBS was removed by suction, 3 mL of pancreatin was added, and the cells were digested at 37°C for approximately 5 minutes. Alternatively, the cells were suspended in pancreatin until digested, and 10 mL of excess serum-containing medium was added to neutralize the pancreatin.

[0217] 4) The cell suspension was transferred to a conical-bottom centrifuge tube and centrifuged at 120 g for 10 minutes. The cells were then suspended in seed plate medium to a concentration of 4 x 10 5 The diluted cells were added to each well of a 96-well cell culture plate (100 μL each). The culture plate was placed in an incubator and incubated at 37°C for 24 hours before the PXR activation experiment.

[0218] 3.2 Medication Processing 1) Test compounds and a positive compound (rifampin) were prepared in DMSO and diluted in serum-free medium at 37°C. The final concentrations of rifampin, the positive control, were 20, 5, 1.25, 0.312, 0.0781, and 0.195 μM, and the final concentrations of compounds to be measured were 30, 10, 3.33, 1.11, 0.370, and 0.123 μM. The final DMSO concentration was 0.1%. 1 μL of DMSO was added to 1 mL of pre-incubated medium to serve as a solvent control.

[0219] 2) Remove the cell culture plate from the incubator, discard the medium, and add 100 μL of the target compound and the positive compound to the appropriate wells, with two parallel wells for each group. Place the cell plate in the incubator and incubate for 24 hours.

[0220] 3.3 Quantitative measurement of PXR activation 1) After 2 days of drug treatment, cultures may be subjected to quantitative detection of PXR activation.

[0221] 2) CellTiter-Fluor TM The Cell Activity Detection Reagent Kit and One-Glo Luciferase Reagent were equilibrated to room temperature. GF-AFC (10 μL) substrate was added to Assay Buffer (10 mL) to form a 2X reagent, which was then diluted to 1X with 10 mL of PBS. ONE-Glo substrate was added to the ONE-Glo Luciferase Assay Buffer.

[0222] 3) Remove the cell culture plate from the incubator, discard the medium in each well, wash the plate twice with PBS, and then add 1X CellTiter-Fluor. TM The reagent was added to a sterile injection channel, and 50 μL was drawn up with a multichannel pipette and added to each well, followed by incubation at 37°C for 30 minutes.

[0223] 4) The 96-well cell plate was removed from the incubator, and the fluorescence value of each well was measured using a plate reader in fluorescence mode with an excitation wavelength of 400 nm and an emission wavelength of 505 nm.

[0224] 5) After the ONE-Glo reagent was poured into the injection channel, 50 μL was taken with a multichannel pipette and added to each well. The reagent was gently mixed evenly and left to incubate at room temperature for 5 minutes to ensure uniform mixing. After incubation was complete, the luminescence value of each well was read using a photometer.

[0225] 4. Calculation of cell induction value 4.1 Cell activity Formula for calculating cell viability: Percent cell viability(%)=I(sample) / (I(vehicle)×100 I(sample) is the fluorescence intensity of the sample, and I(vehicle) refers to the fluorescence intensity for cells with 0.1% DMSO.

[0226] 4.2 Calculation of cell induction value All data were calculated using Microsoft Excel.

[0227] Luciferase activity is expressed as RFU / RLU, where RLU is the average luminescence intensity value of the duplicate parallels for each concentration of each compound, and RFU is the average fluorescence intensity value of the duplicate parallels for each concentration of each compound.

[0228] Formula for calculating induction factor:

number

[0229] Table 23

Claims

1. The compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol is an A-type crystal, and the powder X-ray diffraction pattern, expressed as diffraction angles 2θ angles, is 9.6, 10.0, 1 having characteristic peaks at 1.7, 15.0, 21.1, and 21.7, preferably having characteristic peaks at 7.8, 9.6, 10.0, 11.7, 15.0, 18.9, 19.4, 20.1, 21.1, and 21.7, and most preferably having characteristic peaks at 7.8, 9.6, 10.0, 11.7, 15.0, 18.9, 19.4, 20.1, 21.1, 21.7, 23.5, 26.7, and 29.4; A type crystal.

2. The B-type crystal of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol has characteristic peaks at 11.3, 15.1, 20.9, 22.8, and 23.7, preferably 7.4, 11.3, 14. and most preferably, characteristic peaks at 7.4, 9.5, 11.3, 12.4, 12.8, 13.7, 14.3, 15.1, 16.3, 18.0, 19.2, 20.0, 20.9, 22.8, 23.2, 23.7, 25.1, 26.0, 26.8, 28.2, 30.4, 32.7, 33.6, 34.3, 35.5, 38.4, 39.0, 40.

5. B type crystal.

3. a C-type crystal of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, in which the powder X-ray diffraction pattern expressed in terms of diffraction angles 2θ has characteristic peaks at 4.8, 10.1, 12.0, and 15.0, preferably 4.8, 7.6, 10.1, 12.0, 15.0, 19.7, 21.2, and 23.5; C type crystal.

4. The compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol is a D-type crystal, and the powder X-ray diffraction pattern expressed as a diffraction angle 2θ angle has characteristic peaks at 10.0, 10.5, 17.0, 18.7, and 23.

9. and preferably has characteristic peaks at 10.0, 10.5, 11.0, 13.4, 15.9, 17.0, 18.3, 18.7, 20.8, 23.9, and 28.0, and most preferably has characteristic peaks at 10.0, 10.5, 11.0, 12.5, 13.4, 15.9, 17.0, 18.3, 18.7, 20.1, 20.8, 22.3, 23.9, 26.7, 28.0, 30.5, 31.0, 32.1, 33.1, 33.8, and 34.

6. D-type crystal.

5. The compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol is an E-form crystal, and the powder X-ray diffraction pattern, expressed as diffraction angles 2θ angles, is 4.8, 11.4, 14.4, 15.1, 16.4, 19.3, 20. and preferably has characteristic peaks at 4.8, 7.4, 11.4, 14.4, 15.1, 16.4, 17.9, 19.3, 19.9, 20.9, 22.9, and 23.5, and more preferably has characteristic peaks at 4.8, 7.4, 9.6, 11.4, 14.4, 15.1, 16.4, 17.9, 19.3, 19.9, 20.9, 22.9, 23.5, 24.2, 25.2, 25.9, 26.8, and 30.

5. E type crystal.

6. The F-type crystal of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, wherein the powder X-ray diffraction pattern expressed by diffraction angle 2θ angles has characteristic peaks at 11.1, 11.4, 14.3, 15.1, 15.7, 19.2, and 22.0, preferably 9.1, 11.1, and most preferably, characteristic peaks at 4.8, 9.1, 9.8, 11.1, 11.4, 12.0, 13.3, 14.3, 15.1, 15.7, 16.6, 17.9, 19.2, 20.0, 21.1, 22.0, 23.6, 24.6, 26.1, 28.7, 30.3, 35.

0. F type crystal.

7. The compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol has a G-type crystal, and the powder X-ray diffraction pattern expressed in 2θ angles has characteristic peaks at 11.3, 15.3, 16.1, 17.9, 18.6, and 20.8, and preferably has a G-type crystal at 11. and most preferably, characteristic peaks at 9.1, 11.3, 12.6, 13.8, 14.7, 15.3, 16.1, 17.0, 17.5, 17.9, 18.6, 19.6, 20.8, 21.6, 22.8, 23.1, 23.8, 24.3, 25.3, 25.8, 27.3, 27.8, 29.3, 30.8, 32.3, 33.6, 34.5, 35.

0. G-type crystal.

8. The H-type crystal of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol has characteristic peaks at 11.7, 12.3, 15.5, 19.4, and 22.3 in a powder X-ray diffraction pattern expressed in 2θ angles, and preferably has characteristic peaks at 11. and most preferably, characteristic peaks at 10.1, 11.7, 12.3, 12.8, 13.4, 15.5, 17.1, 18.2, 19.4, 20.5, 21.3, 22.3, 23.2, 24.5, 24.9, 25.6, 26.4, 27.1, 28.1, 28.4, 30.7, 31.2, 32.1, 33.3, 36.0, 37.0, 39.

2. H type crystal.

9. The crystalline form according to any one of claims 1 to 8, characterized in that the error range of the 2θ value is ±0.

2.

10. A method for preparing type A crystals of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 1 or 9, comprising the following steps: Method one a) dissolving the compound of formula (I) in solvent 1; b) Add solvent 2 to precipitate crystals; The solvent 1 is selected from a ketone solvent or an ester solvent, and the solvent 2 is an ether solvent, and preferably the ketone solvent is selected from acetone or methyl isobutyl ketone, the ester solvent is ethyl acetate, and the ether solvent is selected from isopropyl ether or methyl tert-butyl ether; Method 2 The compound represented by formula (I) is dissolved in a solvent 3, and the resulting solution is stirred to crystallize the compound, and the solvent 3 is selected from an ester solvent and a ketone solvent, and preferably, the ester solvent is selected from ethyl acetate and isopropyl acetate, and the ketone solvent is methyl isobutyl ketone. method.

11. A method for preparing type B crystals of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 2 or 9, comprising the following steps: dissolving the compound represented by formula (I) in a substituted lower alkane and stirring to cause crystallization, preferably the substituted lower alkane is nitromethane; method.

12. A method for preparing C-type crystals of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 3 or 9, the method being selected from the following methods: Method one The compound represented by formula (I) is dissolved in a solvent 4, and the solvent 4 is evaporated and crystallized, and the solvent 4 is selected from a substituted lower alkane or a ketone solvent, and preferably, the substituted lower alkane is selected from dichloromethane or 1,2-dichloroethane, and the ketone solvent is methyl isobutyl ketone; Method 2 the compound represented by formula (I) is dissolved in a solvent 5, and the resulting solution is stirred to cause crystallization, the solvent 5 being selected from aromatic hydrocarbon solvents, and preferably, the aromatic hydrocarbon solvent being selected from p-xylene or toluene; Method three a) dissolving the compound of formula (I) in solvent 6; b) Solvent 7 is added to precipitate crystals; The solvent 6 is selected from an alcohol solvent, a ketone solvent, a nitrile solvent, and an ether solvent, and the solvent 7 is an ether solvent, preferably, the alcohol solvent is selected from isopropanol or ethanol, the ketone solvent is acetone, the nitrile solvent is acetonitrile, and the ether solvent is selected from tetrahydrofuran or isopropyl ether; method.

13. 10. A method for preparing D-type crystals of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 4 or 9, comprising the following steps: dissolving the compound represented by formula (I) in a solvent 8 and stirring to cause crystallization; the solvent 8 is an ether-based solvent or a mixed solvent of an alcohol-based solvent and an ether-based solvent; preferably, the alcohol-based solvent is methanol, ethanol, or isopropanol, and the ether-based solvent is isopropyl ether; method.

14. A method for preparing E-form crystals of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 5 or 9, the method being selected from the following methods: Method one The compound represented by formula (I) is dissolved in a solvent 9, and the solution is stirred to crystallize the compound, the solvent 9 being a nitrile solvent, and preferably, the nitrile solvent is acetonitrile; Method 2 a) dissolving a compound of formula (I) in a solvent 10; b) Adding solvent 11 to precipitate crystals; The solvent 10 is selected from ketone-based solvents, and the solvent 11 is an ether-based solvent. Preferably, the ketone-based solvent is acetone, and the ether-based solvent is isopropyl ether. method.

15. A method for preparing F-type crystals of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 6 or 9, the method being selected from the following methods: Method one The compound represented by formula (I) is dissolved in a solvent 12 and stirred to crystallize, the solvent 12 being a ketone solvent, preferably methyl isobutyl ketone; Method 2 The compound of formula (I) is dissolved in tetrahydrofuran, the solution is clarified, and isopropyl ether and seed crystals of F-type crystals are added to precipitate crystals. method.

16. A method for preparing the G-type crystal of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 7 or 9, comprising the steps of: dissolving the compound of formula (I) in a mixed solvent of an alcoholic solvent and an etheric solvent, and stirring to cause crystallization; preferably, the alcoholic solvent is selected from methanol and the etheric solvent is isopropyl ether; method.

17. A method for preparing H-form crystals of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 8 or 9, the method being selected from the following methods: Method one The compound represented by formula (I) is dissolved in a solvent 13, and the solution is stirred to cause crystallization, and the solvent 13 is an alcohol-based solvent, a solvent selected from a mixed solvent of an alcoholic solvent and water, a ketone solvent, and an ester solvent, preferably, the alcoholic solvent is selected from methanol, ethanol, isopropanol, and n-propanol, the ketone solvent is selected from acetone, 2-butanone, and methyl isobutyl ketone, and the ester solvent is ethyl acetate; Method 2 The compound represented by formula (I) is mixed with a solvent 14, slurried, and crystallized, the solvent 14 being selected from water and an ether-based solvent; Preferably, the ether solvent is selected from methyl tert-butyl ether or isopropyl ether; Method three a) dissolving the compound of formula (I) in a solvent 15; b) Add solvent 16 to precipitate crystals; The solvent 15 is selected from an alcohol solvent, a ketone solvent, and an ether solvent, and the solvent 16 is selected from a lower alkane, an ether solvent, or water. Preferably, the alcohol solvent is selected from isopropanol, the ketone solvent is acetone, the lower alkane is n-heptane, and the ether solvent is tetrahydrofuran, isopropyl ether, or methyl tert-butyl ether. method.

18. A pharmaceutically acceptable salt of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol selected from the group consisting of hydrochloride, sulfate, mesylate, p-toluenesulfonate, phosphate, citrate, and malate.

19. 19. A method for preparing a pharmaceutically acceptable salt of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropan-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 18, comprising reacting the free base with an acid molecule, wherein the acid molecule is selected from hydrochloric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, phosphoric acid, citric acid, or malic acid. method.

20. A pharmaceutical composition comprising a crystalline form or compound according to any one of claims 1 to 9 or 18, and optionally a pharmaceutically acceptable excipient. Pharmaceutical compositions.

21. A method for preparing a pharmaceutical composition, comprising mixing a crystalline form or a compound according to any one of claims 1 to 9 or 18 with a pharmaceutically acceptable excipient. Preparation method.

22. The crystalline form or compound of any one of claims 1 to 9 or 18, or the composition of claim 20, in the preparation of a medicament for treating or preventing a disease associated with cyclin-dependent kinases, use.

23. 20. Use of a crystalline form or compound according to any one of claims 1 to 9 or 18, or a composition according to claim 20, in the preparation of a medicament for treating or preventing cancer, preferably said cancer being selected from breast cancer, ovarian cancer, bladder cancer, uterine cancer, prostate cancer, lung cancer, esophageal cancer, head and neck cancer, intestinal cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer and thyroid cancer. use.

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