Crystal form of fused tricyclic derivative and preparation method thereof

Abstract

The invention relates to a crystal form of a fused tricyclic derivative and a preparation method thereof. Specifically, the invention relates to a crystal form of a compound (3S, 4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropane-2-yl)-3-methyl-3, 4-dihydro-5-oxa-1, 2a-diazabenaphthen-6-yl) pyrimidine-2-yl) amino) tetrahydro-2H-pyran-3-ol, a pharmaceutically acceptable salt thereof, and a corresponding preparation method thereof.

Description

A crystal form of a fused tricyclic derivative and a preparation method thereof

[0001] This application claims priority to Chinese patent application No. 202210915848.1, filed August 1, 2022. This application incorporates the entire text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to a crystal form and a preparation method of a fused tricyclic derivative, and belongs to the field of pharmaceuticals. Background Art

[0003] Cyclin-dependent kinases (CDKs) are important cellular enzymes that play an important role in regulating eukaryotic cell division and proliferation. The cyclin-dependent kinase catalytic unit is activated by the regulatory subunits called cyclins. At least 16 mammalian 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).

[0004] PCT / CN2022 / 074509 provides a new class of cyclin-dependent kinase inhibitors, and finding development forms of the compounds disclosed in this application has important clinical significance.

[0005] Summary of the Invention

[0006] The present disclosure provides a crystalline form of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropane-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol (a compound represented by formula (I)), a pharmaceutically acceptable salt thereof, and a corresponding preparation method.

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

[0008] In an optional embodiment, the crystal form A of the compound represented by formula (I) provided by the present disclosure has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, with 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 crystal form A of the compound represented by formula (I) provided by the present disclosure has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, and 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 an optional embodiment, the present disclosure provides a crystal form A of the compound represented by formula (I), with an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, as shown in FIG2 .

[0011] The present disclosure provides a method for preparing crystal form A of a compound represented by formula (I), comprising the following steps:

[0012] Method 1

[0013] a) dissolving the compound represented by formula (I) in solvent 1;

[0014] b) adding solvent 2 to precipitate crystals;

[0015] The solvent 1 is selected from a ketone solvent or an ester solvent, and the solvent 2 is an ether solvent.

[0016] In an optional 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.

[0017] Method 2

[0018] The compound represented by formula (I) is dissolved in solvent 3, stirred and crystallized, wherein the solvent 3 is selected from ester solvents and ketone solvents; preferably, the ester solvent is selected from ethyl acetate and isopropyl acetate, and the ketone solvent is methyl isobutyl ketone.

[0019] The present disclosure provides a crystal form B of a compound represented by formula (I), which has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with characteristic peaks at 11.3, 15.1, 20.9, 22.8, and 23.7.

[0020] In an optional embodiment, the present disclosure provides a crystalline form B of the compound represented by formula (I) having an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, with 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.

[0021] In an optional embodiment, the present disclosure provides a crystalline form B of the compound represented by formula (I), with an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, having 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.

[0022] In an optional embodiment, the present disclosure provides a crystal form B of the compound represented by formula (I), with an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, as shown in FIG3 .

[0023] The present disclosure provides a method for preparing crystal form B of a compound represented by formula (I), comprising the following steps: dissolving the compound represented by formula (I) in a substituted lower alkane, and stirring to crystallize.

[0024] In an optional embodiment, the substituted lower alkane is nitromethane.

[0025] The present disclosure provides a crystal form C of a compound represented by formula (I), which has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with characteristic peaks at 4.8, 10.1, 12.0, and 15.0.

[0026] In an optional embodiment, the present disclosure provides a crystal form C of the compound represented by formula (I), with an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, having characteristic peaks at 4.8, 7.6, 10.1, 12.0, 15.0, 19.7, 21.2, and 23.5.

[0027] In an optional embodiment, the present disclosure provides a crystal form C of the compound represented by formula (I), and the X-ray powder diffraction pattern represented by the diffraction angle 2θ is shown in FIG4 .

[0028] The present disclosure provides a method for preparing the crystal form C of the compound represented by formula (I), which is selected from the following methods:

[0029] Method 1

[0030] The compound represented by formula (I) is dissolved in solvent 4, and evaporated and crystallized, wherein the solvent 4 is selected from a substituted lower alkane or a ketone solvent; optionally, the substituted lower alkane is selected from dichloromethane or 1,2-dichloroethane, and the ketone solvent is methyl isobutyl ketone;

[0031] Method 2

[0032] The compound represented by formula (I) is dissolved in solvent 5, stirred and crystallized, wherein the solvent 5 is selected from an aromatic hydrocarbon solvent; optionally, the aromatic hydrocarbon solvent is selected from p-xylene or toluene;

[0033] Method 3

[0034] a) dissolving the compound represented by formula (I) in solvent 6;

[0035] b) adding solvent 7 to precipitate crystals;

[0036] The solvent 6 is selected from alcohol solvents, ketone solvents, nitrile solvents, and ether solvents, 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.

[0037] The present disclosure provides a D-type crystal of a compound represented by formula (I), which has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, and has characteristic peaks at 10.0, 10.5, 17.0, 18.7, and 23.9.

[0038] In an optional embodiment, the D crystal form of the compound represented by formula (I) provided by the present disclosure has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, and 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.

[0039] In an optional embodiment, the D form of the compound represented by formula (I) provided by the present disclosure has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, and 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.

[0040] In an optional embodiment, the present disclosure provides a D-type crystal of the compound represented by formula (I), with an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, as shown in FIG5 .

[0041] The present disclosure provides a method for preparing the D crystal form of the compound represented by formula (I), comprising the following steps: mixing the compound represented by formula (I) with a solvent 8, stirring and crystallizing, wherein 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.

[0042] The present disclosure provides a crystalline form E of a compound represented by formula (I), which has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with characteristic peaks at 4.8, 11.4, 14.4, 15.1, 16.4, 19.3, and 22.9.

[0043] In an optional embodiment, the E form of the compound represented by formula (I) provided by the present disclosure has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, with 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.

[0044] In an optional embodiment, the E form of the compound represented by formula (I) provided by the present disclosure has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, and 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.

[0045] In an optional embodiment, the present disclosure provides an X-ray powder diffraction pattern of Form E of the compound represented by formula (I) expressed in terms of a diffraction angle 2θ, as shown in FIG6 .

[0046] The present disclosure provides a method for preparing the E crystal form of the compound represented by formula (I), which is selected from the following methods:

[0047] Method 1

[0048] The compound represented by formula (I) is dissolved in solvent 9, stirred and crystallized, wherein the solvent 9 is a nitrile solvent, and optionally, the nitrile solvent is acetonitrile;

[0049] Method 2

[0050] a) dissolving the compound represented by formula (I) in solvent 10,

[0051] b) adding solvent 11 to precipitate crystals;

[0052] The solvent 10 is selected from ketone solvents, and the solvent 11 is an ether solvent; optionally, the ketone solvent is acetone, and the ether solvent is isopropyl ether.

[0053] The present disclosure provides a crystalline form F of a compound represented by formula (I), which has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with characteristic peaks at 11.1, 11.4, 14.3, 15.1, 15.7, 19.2, and 22.0.

[0054] In an optional embodiment, the present disclosure provides a crystalline form F of the compound represented by formula (I), having an X-ray powder diffraction pattern expressed in terms of a diffraction angle 2θ, with 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.

[0055] In an optional embodiment, the F form of the compound represented by formula (I) provided by the present disclosure has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, and 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.

[0056] In an optional embodiment, the present disclosure provides a crystal form F of the compound represented by formula (I), with an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, as shown in FIG7 .

[0057] The present disclosure provides a method for preparing the F crystal form of the compound represented by formula (I), which is selected from the following methods:

[0058] Method 1

[0059] The compound represented by formula (I) is dissolved in solvent 12, stirred and crystallized, wherein the solvent 12 is a ketone solvent; optionally, the ketone solvent is methyl isobutyl ketone;

[0060] Method 2

[0061] The compound represented by formula (I) is dissolved in tetrahydrofuran, and after clarification, isopropyl ether and seed crystals of the F crystal form are added to precipitate crystals.

[0062] The present disclosure provides a G crystal form of a compound represented by formula (I), which has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with characteristic peaks at 11.3, 15.3, 16.1, 17.9, 18.6, and 20.8.

[0063] In an optional embodiment, the G form of the compound represented by formula (I) provided by the present disclosure has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, with characteristic peaks at 11.3, 15.3, 16.1, 17.9, 18.6, 20.8, 22.8, 23.1, 23.8, and 25.8.

[0064] In an optional embodiment, the G form of the compound represented by formula (I) provided by the present disclosure has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, and 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.

[0065] In an optional embodiment, the present disclosure provides a G-type crystal of the compound represented by formula (I), with an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, as shown in FIG8 .

[0066] The present disclosure provides a method for preparing the G crystal form of the compound represented by formula (I), comprising the following steps: dissolving the compound represented by formula (I) in a mixed solvent of an alcohol solvent and an ether solvent, stirring and crystallizing; optionally, the alcohol solvent is selected from methanol, and the ether solvent is isopropyl ether.

[0067] The present disclosure provides a H crystal form of a compound represented by formula (I), which has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with characteristic peaks at 11.7, 12.3, 15.5, 19.4, and 22.3.

[0068] In an optional embodiment, the H crystal form of the compound represented by formula (I) provided by the present disclosure has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, with characteristic peaks at 11.7, 12.3, 15.5, 18.2, 19.4, 21.3, 22.3, 24.9, 27.1, and 28.1.

[0069] In an optional embodiment, the H crystal form of the compound represented by formula (I) provided by the present disclosure has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, and 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.

[0070] In an optional embodiment, the present disclosure provides an H-type compound represented by formula (I), and an X-ray powder diffraction pattern represented by a diffraction angle 2θ, as shown in FIG9 .

[0071] The present disclosure provides a method for preparing the crystalline form H of the compound represented by formula (I), which is selected from the following methods:

[0072] Method 1

[0073] The compound represented by formula (I) is dissolved in solvent 13, stirred and crystallized, wherein the solvent 13 is selected from an alcohol solvent, a mixed solvent of an alcohol solvent and water, a ketone solvent or an ester solvent; optionally, the alcohol 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;

[0074] Method 2

[0075] The compound represented by formula (I) is mixed with a solvent 14, and the mixture is beaten and crystallized, wherein the solvent 14 is selected from water or an ether solvent; optionally, the ether solvent is selected from methyl tert-butyl ether or isopropyl ether;

[0076] Method 3

[0077] a) dissolving the compound represented by formula (I) in solvent 15;

[0078] b) adding solvent 16 to precipitate crystals;

[0079] 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. Another aspect of the present disclosure provides a pharmaceutically acceptable salt of the compound represented by formula (I), wherein the pharmaceutically acceptable salt is selected from a hydrochloride, a sulfate, a methanesulfonate, a p-toluenesulfonate, a phosphate, a citrate, or a malate.

[0080] In an optional embodiment, the ratio of the compound represented by formula (I) to the acid molecule is selected from about 3:1-1:3, specifically about 1:1, 1:2 or 1:3.

[0081] The present disclosure provides a method for preparing a pharmaceutically acceptable salt of a compound represented by formula (I), comprising the step of reacting a 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.

[0082] In an optional embodiment, the present disclosure provides a method for preparing a pharmaceutically acceptable salt of a compound represented by formula (I), wherein the reaction occurs under the action of a solvent.

[0083] In an optional embodiment, 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.

[0084] In an optional embodiment, the nitrile solvent is acetonitrile.

[0085] In an optional embodiment, the alcohol solvent is ethanol.

[0086] The pharmaceutically acceptable salt of the compound represented by formula (I) provided in the present disclosure may be in any crystalline form or amorphous form.

[0087] The substituted lower alkanes described in the present disclosure can be selected from nitromethane, dichloromethane, and chloroform; the nitrile solvent is selected from acetonitrile or propionitrile; the alcohol solvent refers to a C1-C6 alcohol, which can be selected from methanol, ethanol, n-propanol, isopropanol, and n-butanol; the ketone solvent is selected from acetone, 2-butanone, and methyl isobutyl ketone; the ether solvent is selected from isopropyl ether, tetrahydrofuran, dioxane, and propylene glycol methyl ether; the ester solvent is selected from ethyl acetate, methyl acetate, and isopropyl acetate; and the lower alkane is selected from n-hexane or n-heptane.

[0088] In the preparation method provided by the present disclosure, the ratio of the solvent can be 0.1-100 times (w / v) 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.

[0089] The preparation method of the crystal form provided in the present disclosure includes the step of separating the solid and the liquid, which can be specifically performed by filtration, centrifugation or complete evaporation of the solvent.

[0090] The preparation method of the crystalline form provided in the present disclosure may optionally include a drying step.

[0091] The present disclosure also provides a pharmaceutical composition comprising any of the aforementioned crystal forms or pharmaceutically acceptable salts, or a mixture thereof, and a pharmaceutical excipient selected from pharmaceutically acceptable excipients.

[0092] The present disclosure also provides a method for preparing a pharmaceutical composition, comprising the step of mixing any of the aforementioned crystal forms or pharmaceutically acceptable salts with a pharmaceutically acceptable excipient.

[0093] The present disclosure also provides use of any of the aforementioned crystal forms or pharmaceutically acceptable salts, or the aforementioned composition, in the preparation of a medicament for treating or preventing diseases associated with cyclin-dependent kinases.

[0094] The present disclosure also provides use of any of the aforementioned crystal forms or pharmaceutically acceptable salts, or the aforementioned composition, in the preparation of a medicament for treating or preventing cancer.

[0095] In an optional embodiment, 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.

[0096] The "2θ or 2θ angle" mentioned in the present disclosure refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degree; the error range of each characteristic peak 2θ is ±0.2 (including the case where the number exceeding 1 decimal place is rounded off), which can be -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -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, 0.20.

[0097] The drying temperature in the present disclosure is generally 25°C to 100°C, preferably 40°C to 70°C. Drying can be performed at normal pressure or reduced pressure, with a pressure of less than -0.08 MPa.

[0098] The "excipients" described in this disclosure include, but are not limited to, any adjuvant, carrier, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent or emulsifier that has been approved by the U.S. Food and Drug Administration for use by humans or livestock animals.

[0099] The "beating" mentioned in the present disclosure refers to a purification method that utilizes the property that a substance has poor solubility in a solvent but impurities have good solubility in a solvent. Beating purification can remove color, change the crystal form or remove a small amount of impurities.

[0100] The starting material used in the crystal form preparation method of the present invention can be any form of compound, including but not limited to: amorphous, any crystal form, hydrate, solvate, etc.

[0101] 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%.

[0102] The contents disclosed in PCT / CN2022 / 074509 are incorporated into this disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1. XRPD spectrum of the amorphous form of the compound represented by formula (I).

[0104] Figure 2. XRPD spectrum of Form A of the compound represented by formula (I).

[0105] Figure 3. XRPD spectrum of Form B of the compound represented by formula (I).

[0106] Figure 4. XRPD spectrum of Form C of the compound represented by formula (I).

[0107] Figure 5. XRPD spectrum of Form D of the compound represented by formula (I).

[0108] Figure 6. XRPD spectrum of Form E of the compound represented by formula (I).

[0109] Figure 7. XRPD spectrum of Form F of the compound represented by formula (I).

[0110] Figure 8. XRPD spectrum of Form G of the compound represented by formula (I).

[0111] Figure 9. XRPD spectrum of Form H of the compound represented by formula (I). DETAILED DESCRIPTION

[0112] The present disclosure is further described below with reference to the following embodiments, but these embodiments are not intended to limit the scope of the present disclosure.

[0113] Example

[0114] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). -6 The unit of ppm is given. NMR measurements were performed using a Bruker AVANCE NEO 500M NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), and tetramethylsilane (TMS) was used as the internal standard.

[0115] MS was measured using an Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS liquid spectrometer-mass spectrometer (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS).

[0116] waters ACQuity UPLC-QD / SQD (Manufacturer: waters, MS model: waters ACQuity Qda Detector / waters SQ Detector)

[0117] THERMO Ultimate 3000-Q Exactive (Manufacturer: THERMO, MS model: THERMO Q Exactive)

[0118] High performance liquid chromatography (HPLC) analysis was performed using Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLC e2695-2489 high pressure liquid chromatographs.

[0119] Chiral HPLC analysis was performed using an Agilent 1260 DAD high performance liquid chromatograph.

[0120] High performance liquid chromatography (HPLC) was performed using Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson GX-281 preparative chromatographs.

[0121] Chiral preparations were performed using a Shimadzu LC-20AP preparative chromatograph.

[0122] The CombiFlash rapid preparation instrument used was Combiflash Rf200 (TELEDYNE ISCO).

[0123] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The specification of the silica gel plate used in thin layer chromatography (TLC) is 0.15mm~0.2mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm~0.5mm.

[0124] Silica gel column chromatography generally uses Yantai Huanghai Silica Gel 200-300 mesh silica gel as the carrier.

[0125] Average kinase inhibition rate and IC 50 The values ​​were determined using a NovoStar microplate reader (BMG, Germany).

[0126] The known starting materials disclosed herein can be synthesized by methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui Chemicals, and other companies.

[0127] Unless otherwise specified in the examples, all reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.

[0128] Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1 L.

[0129] Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

[0130] The pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0131] The hydrogenation reaction is usually carried out by evacuating the chamber and filling it with hydrogen, and the operation is repeated three times.

[0132] A CEM Discover-S 908860 microwave reactor was used for the microwave reaction.

[0133] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0134] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0135] The reaction progress in the examples was monitored by thin layer chromatography (TLC). The developing solvent used in the reaction, the column chromatography eluent system used for purifying the compound, and the developing solvent system for thin layer chromatography included: A: dichloromethane / methanol system. The volume ratio of the solvent was adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0136] Test conditions of the instruments used in the experiments in this disclosure:

[0137] 1. Differential Scanning Calorimeter (DSC)

[0138] Instrument model: Mettler Toledo DSC 3+STARe System

[0139] Purge gas: nitrogen; nitrogen purge rate: 50mL / min

[0140] Heating rate: 10.0℃ / min

[0141] Temperature range: 25-350℃ or 25-300℃

[0142] 2. X-ray Powder Diffraction (XRPD)

[0143] Instrument model: BRUKER D8Discover X-ray powder diffractometer

[0144] Radiation: Monochromatic Cu-Kα radiation (λ=1.5406)

[0145] Scanning mode: θ / 2θ, scanning range (2θ range): 3 to 45°

[0146] Voltage: 40kV, Current: 40mA

[0147] 3. Thermogravimetric Analysis (TGA)

[0148] Instrument model: Mettler Toledo TGA2

[0149] Purge gas: nitrogen; nitrogen purge rate: 50mL / min

[0150] Heating rate: 10.0℃ / min

[0151] Temperature range: 30-350℃ or 30-300℃

[0152] 4. DVS stands for dynamic moisture adsorption

[0153] The test was performed using a Surface Measurement Systems advantage 2 at 25°C with humidity ranging from 50%-95%-0%-95%-50% RH in 10% steps. The judgment criterion was that the mass change dM / dT for each gradient was less than 0.002%, TMAX was 360 min, and the cycle was repeated twice.

[0154] Example 1. Preparation of free amorphous

[0155] 10 mg of the compound represented by formula (I) was added to 0.5 mL of water, stirred at room temperature, centrifuged, and then vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was amorphous, and the XRPD spectrum is shown in Figure 1.

[0156] Example 2. Preparation of Free Form A

[0157] 10 mg of the compound represented by formula (I) was dissolved in 0.05 mL of isopropyl acetate, cooled to 5°C, stirred to crystallize, filtered, and the filter cake collected and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified the product as Form A. The XRPD spectrum is shown in Figure 2, and the positions of its characteristic peaks are shown in Table 1. The DSC spectrum showed endothermic peaks at 87.79°C and 254.08°C. The TGA spectrum showed a weight loss of 10.07% from 30°C to 150°C.

[0158] Table 1

[0159] Example 3. Preparation of Free Form A

[0160] 10 mg of the compound represented by formula (I) was added to 0.05 mL of ethyl acetate and dissolved. The mixture was cooled to 5°C, stirred for crystallization, filtered, and the filter cake was collected and dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was Form A.

[0161] Example 4. Preparation of Free Form A

[0162] 10 mg of the compound represented by formula (I) was added to 0.05 mL of methyl isobutyl ketone to dissolve the mixture, cooled to 5°C, stirred for crystallization, filtered, and the filter cake was collected and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form A.

[0163] Example 5. Preparation of Free Form A

[0164] 10 mg of the compound represented by formula (I) was dissolved in 0.05 mL of acetone, 0.6 mL of isopropyl ether was added, and the mixture was stirred for crystallization. The mixture was centrifuged and then vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form A.

[0165] Example 6. Preparation of Free Form A

[0166] 10 mg of the compound represented by 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 for crystallization. The mixture was centrifuged and then vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form A.

[0167] Example 7. Preparation of Free Form A

[0168] 10 mg of the compound represented by formula (I) was dissolved in 0.05 mL of ethyl acetate, and 0.8 mL of methyl tert-butyl ether was added and evaporated for crystallization. X-ray powder diffraction analysis showed that the product was Form A.

[0169] Example 8. Preparation of Free Form B

[0170] 10 mg of the compound represented by formula (I) was dissolved in 0.05 mL of nitromethane, stirred at room temperature to crystallize, filtered, and the filter cake collected and vacuum dried to obtain a solid. X-ray powder diffraction analysis identified the product as Form B. The XRPD spectrum is shown in Figure 3, and the positions of its characteristic peaks are shown in Table 2. The DSC spectrum showed endothermic peaks at 154.21°C and 254.73°C. The TGA spectrum showed a weight loss of 4.42% from 30°C to 150°C.

[0171] Table 2

[0172] Example 9. Preparation of Free Form C

[0173] 10 mg of the compound represented by formula (I) was dissolved in 0.05 mL of dichloromethane, evaporated, and crystallized to obtain a solid. X-ray powder diffraction analysis identified the product as Form C. The XRPD spectrum is shown in Figure 4 , and the positions of its characteristic peaks are shown in Table 3 . The DSC spectrum showed endothermic peaks at 125.96°C and 253.40°C. The TGA spectrum showed a weight loss of 4.37% between 30°C and 150°C.

[0174] Table 3

[0175] Example 10. Preparation of Free Form C

[0176] 10 mg of the compound represented by formula (I) was added to 0.05 mL of 1,2-dichloroethane, dissolved, evaporated, and crystallized to obtain a solid. X-ray powder diffraction analysis showed that the product was Form C.

[0177] Example 11. Preparation of Free Form C

[0178] 10 mg of the compound represented by formula (I) was added to 0.05 mL of methyl isobutyl ketone, dissolved, evaporated, and crystallized to obtain a solid. X-ray powder diffraction analysis showed that the product was Form C.

[0179] Example 12. Preparation of Free Form C

[0180] 10 mg of the compound represented by formula (I) was added to 1 mL of p-xylene, stirred to separate crystals, centrifuged, and then vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form C.

[0181] Example 13. Preparation of Free Form C

[0182] 10 mg of the compound represented by formula (I) was added to 1 mL of toluene, stirred to separate crystals, centrifuged, and then vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form C.

[0183] Example 14. Preparation of Free Form C

[0184] 10 mg of the compound represented by formula (I) was added to 0.1 mL of isopropyl alcohol, and 1 mL of isopropyl ether was added and stirred to precipitate a solid. The solid was centrifuged and then dried in vacuo to obtain a solid. X-ray powder diffraction analysis showed that the product was Form C.

[0185] Example 15. Preparation of Free Form C

[0186] 10 mg of the compound represented by formula (I) was added to 0.1 mL of acetone, and 1 mL of isopropyl ether was added and stirred to precipitate a solid. The solid was centrifuged and then dried in vacuo to obtain a solid. X-ray powder diffraction analysis showed that the product was Form C.

[0187] Example 16. Preparation of Free Form C

[0188] 10 mg of the compound represented by formula (I) was added to 0.1 mL of ACN (acetonitrile), and 1 mL of isopropyl ether was added and stirred to precipitate a solid. The solid was centrifuged and then dried in vacuo to obtain a solid. X-ray powder diffraction analysis showed that the product was Form C.

[0189] Example 17. Preparation of Free Form C

[0190] 10 mg of the compound represented by formula (I) was added to 0.1 mL of THF (tetrahydrofuran), and 1 mL of isopropyl ether was added and stirred to precipitate a solid. The solid was centrifuged and then dried in vacuo to obtain a solid. X-ray powder diffraction analysis showed that the product was Form C.

[0191] Example 18. Preparation of Free Form C

[0192] 10 mg of the compound represented by formula (I) was added to 0.1 mL of EtOH, and 1 mL of isopropyl ether was added and stirred to precipitate a solid, which was centrifuged and then dried in vacuo to obtain a solid. X-ray powder diffraction analysis showed that the product was Form C.

[0193] Example 19. Preparation of Free Form D

[0194] 10 mg of the compound represented by formula (I) was added to 1 mL of isopropyl ether, stirred at 60°C for 2 hours, centrifuged, and vacuum dried to obtain a solid. X-ray powder diffraction analysis identified the product as Form D. The XRPD spectrum is shown in Figure 5, and the positions of its characteristic peaks are shown in Table 4. The DSC spectrum revealed an endothermic peak at 196.66°C. The TGA spectrum showed a weight loss of 0.63% between 30°C and 150°C.

[0195] Table 4

[0196] Example 20. Preparation of Free Form D

[0197] 100 mg of the compound represented by formula (I) was added to 10 mL of isopropyl ether, and about 1-2 mg of seed crystals were added. The mixture was slurried at 60°C for 2 h, centrifuged, and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form D.

[0198] Example 21. Preparation of Free Form D

[0199] 50 mg of the compound represented by formula (I) was added to 0.1 mL of ethanol and stirred at 60°C to dissolve. 0.4 mL of isopropyl ether was added, followed by 1-2 mg of Form D seed crystals. The mixture was stirred at 60°C for 2 h until a solid precipitated. The solid was centrifuged and then dried under vacuum to obtain a solid. X-ray powder diffraction analysis confirmed that the product was Form D.

[0200] Example 22. Preparation of Free Form D

[0201] 50 mg of the compound represented by formula (I) was added to 0.1 mL of methanol and stirred at 60°C to dissolve. 0.4 mL of isopropyl ether was added, followed by 1-2 mg of Form D seed crystals. The mixture was stirred at 60°C for 2 h until a solid precipitated. The solid was centrifuged and then dried under vacuum to obtain a solid. X-ray powder diffraction analysis confirmed that the product was Form D.

[0202] Example 23. Preparation of Free Form D

[0203] 700 mg of the compound represented by formula (I) was added to 1.4 mL of ethanol and stirred at 60°C to dissolve. 4.2 mL of isopropyl ether was added, followed by 1-2 mg of Form D seed crystals. The mixture was stirred at 60°C for 2 h until a solid precipitated. The solid was centrifuged and then dried under vacuum to obtain a solid. X-ray powder diffraction analysis confirmed that the product was Form D.

[0204] Example 24. Preparation of Free Form D

[0205] 10 mg of the compound represented by formula (I) was added to 0.4 mL of ethanol / isopropyl ether (1 / 20), stirred at 60°C, and dissolved. Stirring was continued for 1 hour to precipitate a solid, which was centrifuged and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form D.

[0206] Example 25. Preparation of Free Form D

[0207] 10 mg of the compound represented by formula (I) was added to 0.4 mL of methanol / isopropyl ether (1 / 20), stirred at 60°C, and dissolved. Stirring was continued for 1 hour to precipitate a solid, which was centrifuged and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form D.

[0208] Example 26. Preparation of Free Form E

[0209] 10 mg of the compound represented by formula (I) was added to 0.05 mL of acetonitrile to dissolve the mixture. The mixture was cooled to 5°C and stirred at low temperature to crystallize. The mixture was filtered, the filter cake collected, and vacuum dried to obtain a solid. X-ray powder diffraction analysis identified the product as Form E. The XRPD spectrum is shown in Figure 6, and the positions of its characteristic peaks are shown in Table 5. The DSC spectrum showed endothermic peaks at 35.46°C and 137.39°C. The TGA spectrum showed a weight loss of 6.04% from 30°C to 160°C.

[0210] Table 5

[0211] Example 27. Preparation of Free Form E

[0212] 100 mg of the compound represented by formula (I) was added to 0.5 mL of acetone to dissolve, 3 mL of isopropyl ether was added, the temperature was lowered to 5°C, stirred for crystallization, filtered, the filter cake was collected, and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form E.

[0213] Example 28. Preparation of Free Form F

[0214] 100 mg of the compound represented by formula (I) was added to 0.5 mL of methyl isobutyl ketone to dissolve the mixture. The mixture was stirred at room temperature to crystallize, filtered, and the filter cake was collected and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified the product as Form F. The XRPD spectrum is shown in Figure 7, and the positions of its characteristic peaks are shown in Table 6. The DSC spectrum showed endothermic peaks at 32.47°C and 137.02°C. The TGA spectrum showed a weight loss of 6.99% from 30°C to 160°C.

[0215] Table 6

[0216] Example 29. Preparation of Free Form F

[0217] 100 mg of the compound represented by formula (I) was added to 2 mL of tetrahydrofuran, stirred at 60° C. to dissolve, 10 mL of isopropyl ether and Form F seed crystals were added, and stirred at 60° C. for 2 h to precipitate a solid. The solid was filtered, the filter cake was collected, and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form F.

[0218] Example 30. Preparation of Free Form G

[0219] 50 mg of the compound represented by formula (I) was dissolved in 0.1 mL of methanol and 0.4 mL of isopropyl ether. The mixture was stirred at 60°C for 2 h to precipitate a solid. The filter cake was collected by filtration and vacuum dried to obtain a solid. X-ray powder diffraction analysis identified the product as Form G. The XRPD spectrum is shown in Figure 8, and the positions of its characteristic peaks are shown in Table 7. The DSC spectrum showed an endothermic peak at 173.41°C. The TGA spectrum showed a weight loss of 1.18% from 30°C to 190°C.

[0220] Table 7

[0221] Example 31. Preparation of Free Form H

[0222] 10 mg of the compound represented by formula (I) was added to 0.05 mL of ethanol and heated at 60°C with stirring to dissolve. The mixture was then suspended at 50°C-5°C with stirring and ramping to precipitate. The filter cake was collected by filtration and vacuum dried to obtain a solid. X-ray powder diffraction analysis identified the product as Form H. The XRPD spectrum is shown in Figure 9, and the positions of its characteristic peaks are shown in Table 8. The DSC spectrum showed an endothermic peak at 206.30°C. The TGA spectrum showed a weight loss of 0.29% between 33°C and 218°C.

[0223] Table 8

[0224] Example 32. Preparation of Free Form H

[0225] 10 mg of the compound represented by formula (I) was added to 0.05 mL of isopropanol and heated at 60°C with stirring to dissolve. The mixture was then suspended at 50°C-5°C with stirring and ramping to precipitate. The filter cake was collected by filtration and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline Form H.

[0226] Example 33. Preparation of Free Form H

[0227] 10 mg of the compound represented by formula (I) was added to 0.05 mL of n-propanol and heated at 60°C with stirring to dissolve. The mixture was then suspended at 50°C to 5°C with stirring and ramping to precipitate. The filter cake was collected by filtration and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form H.

[0228] Example 34. Preparation of Free Form H

[0229] 10 mg of the compound represented by formula (I) was added to 0.05 mL of 2-butanone, heated and stirred at 60°C to dissolve, then suspended at 50°C-5°C with stirring and ramping to precipitate. The filter cake was collected by filtration and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form H.

[0230] Example 35. Preparation of Free Form H

[0231] 10 mg of the compound represented by formula (I) was added to 0.05 mL of a 1 / 1 methanol / water solution and heated at 60°C with stirring to dissolve. The mixture was then suspended at 50°C to 5°C with stirring and ramping to precipitate. The filter cake was collected by filtration and vacuum dried to obtain a solid. X-ray powder diffraction analysis confirmed the product to be Form H.

[0232] Example 36. Preparation of Free Form H

[0233] 10 mg of the compound represented by formula (I) was added to 0.25 mL of ethyl acetate and heated at 60°C with stirring to dissolve. The mixture was then suspended at 50°C to 5°C with stirring and ramping to precipitate. The filter cake was collected by filtration and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form H.

[0234] Example 37. Preparation of Free Form H

[0235] 10 mg of the compound represented by formula (I) was added to 0.25 mL of methyl isobutyl ketone (MIK) and heated at 60°C with stirring to dissolve. The mixture was then suspended at 50°C to 5°C with stirring and ramping to precipitate. The filter cake was collected by filtration and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form H.

[0236] Example 38. Preparation of Free Form H

[0237] 10 mg of the compound represented by formula (I) was added to 1 mL of water and heated with stirring at 60°C until insoluble. The mixture was suspended at 50°C-5°C with stirring and ramping, and then filtered. The filter cake was collected and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form H.

[0238] Example 39. Preparation of Free Form H

[0239] 10 mg of the compound represented by formula (I) was added to 1 mL of methyl tert-butyl ether and heated with stirring at 60°C until insoluble. The mixture was then suspended and stirred at 50°C to 5°C, and the mixture was stirred and slurried. The mixture was filtered, the filter cake collected, and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline Form H.

[0240] Example 40. Preparation of Free Form H

[0241] 10 mg of the compound represented by formula (I) was added to 1 mL of isopropyl ether and heated at 60°C with stirring until insoluble. The mixture was suspended at 50°C-5°C with stirring and ramping, then filtered, the filter cake collected, and vacuum dried to obtain a solid. X-ray powder diffraction analysis confirmed that the product was Form H.

[0242] Example 41. Preparation of Free Form H

[0243] 10 mg of the compound represented by formula (I) was added to 0.15 mL of isopropanol at 60°C and stirred to dissolve. 1 mL of water was added and stirred to precipitate. The solid was centrifuged and then dried in vacuo to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0244] Example 42. Preparation of Free Form H

[0245] 10 mg of the compound represented by formula (I) was added to 0.15 mL of isopropanol at 60°C and stirred for dissolution. 1 mL of n-heptane was added and stirred to precipitate. The solid was centrifuged and then dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0246] Example 43. Preparation of Free Form H

[0247] 10 mg of the compound represented by formula (I) was added to 0.15 mL of isopropyl alcohol at 60°C and stirred to dissolve. 1 mL of isopropyl ether was added and stirred to precipitate. The solid was centrifuged and then dried in vacuo to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0248] Example 44. Preparation of Free Form H

[0249] 10 mg of the compound represented by formula (I) was added to 0.1 mL of acetone at 60°C and stirred for dissolution. 1 mL of water was added to precipitate the solution, which was centrifuged and then dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0250] Example 45. Preparation of Free Form H

[0251] 10 mg of the compound represented by formula (I) was added to 0.1 mL of acetone at 60°C and stirred for dissolution. 1 mL of n-heptane was added to precipitate the solution, which was centrifuged and then vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0252] Example 46. Preparation of Free Form H

[0253] 10 mg of the compound represented by formula (I) was added to 0.1 mL of acetone at 60°C and stirred for dissolution. 1 mL of isopropyl ether was added to precipitate the solution, which was centrifuged and then vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form H.

[0254] Example 47. Preparation of Free Form H

[0255] 10 mg of the compound represented by formula (I) was added to 0.05 mL of tetrahydrofuran and stirred at 60°C for dissolution. 1 mL of methyl tert-butyl ether was added to precipitate the solution. The solution was centrifuged and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was Form H.

[0256] Example 48. Preparation of Free Form H

[0257] 10 mg of the compound represented by formula (I) was added to 0.05 mL of tetrahydrofuran and stirred at 60°C for dissolution. 1 mL of isopropyl ether was added to precipitate the solution, which was centrifuged and then vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0258] Example 49. Study on the hygroscopicity of the free crystal form

[0259] Surface Measurement Systems intrinsic DVS was used at 25°C, with a humidity range of 0%-95% in 10% increments. The judgment criteria were a mass change dM / dT of less than 0.002% per gradient, TMAX 360 min, and two cycles.

[0260] Table 9

[0261] Example 50. Study on factors affecting the stability of crystal form

[0262] The free state samples were laid out in the open and their stability was investigated under conditions of light (4500 Lux), high temperature (40°C, 60°C), and high humidity (RH 75%, RH 92.5%). The sampling period was 30 days.

[0263] Table 10

[0264] Table 11

[0265] Table 12

[0266] Table 13

[0267] Table 14

[0268] Table 15

[0269] The results of the influencing factor experiments showed that the crystal forms D / E / F / G / H and the free amorphous form had good chemical stability under high temperature and high humidity conditions. The crystal forms D / E / F / G / H and the free amorphous form had good physical stability.

[0270] Example 51. Long-term / accelerated stability of crystal forms

[0271] The free state samples were sealed in aluminum foil bags and placed under 25°C / 60% RH and 40°C / 75% RH conditions to investigate their stability. The results are shown below.

[0272] Table 16

[0273] Experimental results show that Form H exhibits good physical and chemical stability after 3 months of storage under long-term accelerated conditions. Forms D, E, F, and G also exhibit good physical and chemical stability after 6 months of storage under long-term accelerated conditions. The free amorphous form exhibits accelerated deliquescence but exhibits good physical and chemical stability under other conditions.

[0274] Example 52. Preparation of hydrochloride

[0275] 100 mg of the compound represented by formula (I) was added to 2 mL of acetonitrile, and 115.3 μl of 2M hydrochloric acid ethanol solution was added and stirred for 5 h. The sample dissolved clearly, and 5 mL of isopropyl ether was added and stirred to precipitate. The solid was centrifuged and dried in vacuo to obtain a solid. Ion chromatography results showed a chloride ion content of 6.7%.

[0276] Example 53. Preparation of sulfate

[0277] 100 mg of the compound represented by formula (I) was added to 2 mL of acetonitrile, and 115.3 μl of 2M sulfuric acid ethanol solution was added and stirred for 5 h. The sample dissolved clearly, and 5 mL of isopropyl ether was added and stirred to precipitate. The solid was centrifuged and dried in vacuo to obtain a solid. Ion chromatography results showed that the sulfate ion content was 14.9%.

[0278] Example 54. Preparation of Methanesulfonate

[0279] 100 mg of the compound represented by formula (I) was added to 2 mL of acetonitrile, and 115.3 μl of 2M methanesulfonic acid ethanol solution was added and stirred for 5 h. The sample dissolved clearly, and 5 mL of isopropyl ether was added and stirred to precipitate. The solid was centrifuged and dried in vacuo to obtain a solid. Ion chromatography results showed that the content of methanesulfonate ion was 17.8%.

[0280] Example 55. Preparation of p-toluenesulfonate

[0281] 100 mg of the compound represented by formula (I) was added to 2 mL of acetonitrile, and 115.3 μl of 2M p-toluenesulfonic acid ethanol solution was added and stirred for 5 h. The sample dissolved clearly, and 5 mL of isopropyl ether was added and stirred to precipitate. The mixture was centrifuged and vacuum dried to obtain a solid. Ion chromatography results showed that the p-toluenesulfonate ion content was 34.3%.

[0282] Example 56. Preparation of phosphate

[0283] 10 mg of the compound represented by formula (I) was added to 0.2 mL of acetonitrile, and 11.5 μl of 2 M ethanolic phosphoric acid solution was added, stirred for 5 h, centrifuged, and vacuum dried to obtain a solid.

[0284] Example 57. Preparation of citrate

[0285] 10 mg of the compound represented by formula (I) was added to 0.2 mL of acetonitrile, and 11.5 μl of 2 M citric acid ethanol solution was added, stirred for 5 h, centrifuged, and vacuum dried to obtain a solid.

[0286] Example 58. Preparation of malate

[0287] 10 mg of the compound represented by formula (I) was added to 0.2 mL of acetonitrile, and 11.5 μl of 2 M citric acid ethanol solution was added, stirred for 5 h, centrifuged, and vacuum dried to obtain a solid.

[0288] Example 59.

[0289] (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 Isomer 1

[0290] first step

[0291] 6-Bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-2-carbaldehyde 4a

[0292] 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 sequentially to 30 mL of 1,4-dioxane. The reaction was allowed to proceed at 95°C for 8 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was collected. The residue was concentrated under reduced pressure and purified by C-18 reverse-phase chromatography to afford the title compound 4a (930 mg, 44% yield).

[0293] MS (ESI) m / z 299.1, 301.1 [M+H] +

[0294] Step 2

[0295] 1-(6-Bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-2-yl)ethan-1-ol 4b

[0296] Under a nitrogen 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 mixture was cooled to -20°C, and a solution of methylmagnesium bromide in tetrahydrofuran (3 mol / L, 1.5 mL, 4.5 mmol) was added dropwise. The mixture was allowed to react at -20°C for 4 hours. The reaction was quenched by the addition of 5 mL of water. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C-18 reverse-phase chromatography to afford the title compound 4b (830 mg, 85% yield).

[0297] MS (ESI) m / z 315.2, 317.2 [M+H] +

[0298] Step 3

[0299] 1-(6-Bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-2-yl)ethan-1-one 4c

[0300] At room temperature, 1-(6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-2-yl)ethan-1-one (600 mg, 1.9 mmol) was dissolved in 20 mL of tetrahydrofuran, and Dess-Martin periodinane (2.0 g, 4.8 mmol) was added. The mixture was heated to 80°C and reacted for 2 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was collected. The residue was concentrated under reduced pressure and purified by C-18 reverse phase chromatography to obtain the title compound 4c (350 mg, yield: 59%).

[0301] MS (ESI) m / z 313.1, 315.1 [M+H] +

[0302] Step 4

[0303] 2-(6-Bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-2-yl)propan-2-ol 4d

[0304] Under a nitrogen atmosphere, 1-(6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-2-yl)ethan-1-one (350 mg, 1.1 mmol) was dissolved in 20 mL of tetrahydrofuran. The mixture was cooled to -20°C, and a solution of methylmagnesium bromide in tetrahydrofuran (3 mol / L, 0.7 mL, 2.1 mmol) was added dropwise. The mixture was allowed to react at -20°C for 4 hours. The reaction was quenched by the addition of 5 mL of water. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C-18 reverse-phase chromatography to afford the title compound 4d (260 mg, 71% yield).

[0305] MS (ESI) m / z 329.2, 331.2 [M+H] +

[0306] Step 5

[0307] 2-(6-(2,5-dichloropyrimidin-4-yl)-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-2-yl)propan-2-ol 4e

[0308] Under a nitrogen atmosphere, 2-(6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthyl-2-yl)propan-2-ol (260 mg, 0.8 mmol), pinacol 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 reacted 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-phosphoryladamantane (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. The mixture was reacted at 80°C for 1 hour. The reaction mixture was cooled to room temperature, filtered, and the filtrate was collected. The residue was concentrated under reduced pressure and purified by C-18 reverse-phase chromatography to give the title compound 4e (129 mg, 41% yield).

[0309] MS (ESI) m / z 397.3 [M+H] +

[0310] Step 6

[0311] (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 Isomer 1

[0312] (3S,4R)-4-((5-chloro-4-((R)-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 Isomer 2

[0313] Under a nitrogen atmosphere, 2-(6-(2,5-dichloropyrimidin-4-yl)-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthen-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 reaction was carried out at 85°C for 3 hours. The reaction solution 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 obtain a crude mixture 4f. The crude product was separated by chiral separation [Column:

[0314] DAICEL CHIRALPAK AD (250mm*30mm, 10μm), Condition: 0.1% NH3·H2O ETOH, Begin B: 40%; End B: 40%; Flow Rate (ml / min): 60)] to give the title compound isomer 1 (25.2mg, yield: 17%) and the title compound isomer 2 (22mg, yield: 15%).

[0315] Analytical methods

[0316] Column: DAICEL CHIRALCEL AD-3 (100mm*4.6mm, 3μm);

[0317] Mobile phase: A: CO2B: ethanol (0.05% DEA), Gradient: from 5% to 40% of B in 2min and hold 40% for 1.2min, then 5% of B for 0.8min;

[0318] FlowRate: 4 mL / min;

[0319] ABPR: 1500psi;

[0320] Temperature: 35℃.

[0321] The compound with a retention time of 1.887 min was defined as isomer 2;

[0322] MS (ESI) m / z 478.1 [M+H] +

[0323] 1H NMR (400MHz, DMSO-d6) δ = 8.37 (s, 1H), 7.45 (br s, 1H), 6.92 (d, J = 11.3Hz, 1H), 5.82 (br s, 1H), 5.23 (q, J = 6.4Hz, 1H), 4.47 (d, J = 11.0Hz, 1H), 4.22 (br d, J=11.3Hz, 1H), 3.89-3.67(m, 3H), 3.39-3.25(m, 4H), 3.03(br t, J=10.2Hz, 1H), 1.95(br d, J=10.5Hz, 1H), 1.68 (s, 3H), 1.62 (s, 3H), 1.45 (d, J=6.5Hz, 3H)

[0324] The compound with a retention time of 2.078 min was defined as isomer 1;

[0325] MS (ESI) m / z 478.1 [M+H] +

[0326] 1 H NMR (400MHz, DMSO-d6) δ = 8.37 (s, 1H), 7.45 (br s, 1H), 6.92 (d, J = 11.5Hz, 1H), 5.82 (s, 1H), 5.29-5.19 (m, 1H), 4.93 (d, J = 5.5Hz, 1H), 4.47 (d, J = 11.3Hz, 1H), 4.21 (br d, J=10.3Hz, 1H), 3.85-3.75(m, 3H), 3.53-3.40(m, 2H), 3.03(br t, J=10.4Hz, 1H), 1.94(br s, 1H), 1.67 (s, 3H), 1.62 (s, 3H), 1.45 (d, J = 6.5Hz, 3H), 1.06 (t, J = 7.0Hz, 1H)

[0327] Biological evaluation

[0328] The present invention is further described and explained below in conjunction with test examples, but these examples are not intended to limit the scope of the present invention.

[0329] Test Example 1: Detection of Cyclin-Dependent Kinase Activity of Disclosed Compounds

[0330] 1. Experimental Materials

[0331] Compound A is example A94 of WO 2019 / 207463A1, which was synthesized according to the method disclosed in the patent.

[0332] 2. Kinase activity test (CDK4 / CyclinD1, CDK6 / CyclinD3) Mobility shift assay to measure kinase activity

[0333] In vitro CDK kinase activity was assessed using the Mobility Shift Assay. The starting concentration of the test compound for CDK inhibition was 300 nM, with 10 concentrations tested in duplicate. Staurosporine was used as a standard control.

[0334] Prepare 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). Add the appropriate amount of kinase to the 1x kinase buffer to prepare a 2.5x enzyme solution; prepare a 5x compound dilution solution (1x kinase buffer, 10% DMSO) corresponding to the test compound concentration; and add the appropriate amount of FAM-labeled peptide and ATP to the 1x kinase buffer to prepare a 2.5x substrate solution. 5 μl of 5x compound dilution and 10 μl of 2.5x enzyme solution were added to the reaction wells of a 384-well reaction plate, mixed, and incubated at room temperature for 10 minutes. 10 μl of 2.5x substrate solution was then added to the 384-well plate, and the plate was centrifuged at 1000 rpm for 1 minute. The plate was incubated at 28°C for 60 minutes (biochemical incubator model: SPX-100B-Z). 30 μl of stop solution was added to the 384-well plate to terminate the reaction, and the plate was centrifuged at 1000 rpm for 1 minute. Conversion rate data were read on a Caliper EZ Reader II (excitation wavelength: 400 nm, emission wavelengths: 445 nm and 520 nm).

[0335] IC of the compound 50 The values ​​were fitted using XLFit Excel add-in version 5.4.0.8. The fitting formula is:

[0336] Y=Bottom+(Top-Bottom) / (1+(IC50 / X)^HillSlope).

[0337] 3. Kinase activity test (CDK1 / CyclinB, CDK 9 / CyclinT1)

[0338] In vitro CDK (CDK2, CDK9) kinase activity assays were performed with a starting concentration of 1 uM, 3-fold dilutions, a total of 10 concentrations, and replicates. PHA-793887 was used as a control compound.

[0339] Prepare 1x kinase reaction buffer (40mM Tris-HCl, pH 7.4, 20mM MgCl2, 0.1mg / ml BSA, 50µM DTT), 1 volume of 5x kinase reaction buffer, and 4 volumes of water. Add DTT (final concentration 50µM). Use an Echo655 to transfer 50µL of the diluted compound working solution (final DMSO concentration 1%) to each well of a reaction plate (784075, Greiner). Seal the plate with film and centrifuge at 1000g for 1 minute. Prepare 2x enzyme (0.3ng / µL CDK2 / CyclinE1 or CDK9 / CyclinT1) in 1x kinase reaction buffer. Add 2.5µL of the above kinase solution to each well. Seal the plate with film and centrifuge at 1000g for 1 minute. Let stand at room temperature for 10 minutes. Prepare a 2× kinase substrate and ATP mixture using 1× kinase reaction buffer. The 2× CDK2 / CylinE1 kinase substrate consists of 0.4 mg / ml Histone H1 and 30 μM ATP. Add 2.5 μL of the 2× Histone H1 and ATP mixture to the reaction plate and centrifuge at 1000 g for 30 seconds to start the reaction. After the kinase assay is allowed to react at room temperature for 120 minutes, add 4 μL of ADP-Glo ​​reagent and react at room temperature for 40 minutes. Then, add 8 μL of kinase detection reagent and react at room temperature for 40 minutes. The luminescent signal is read using Envision 2104. Data analysis is as follows.

[0340] a) Percent inhibition: %inhibition = 100 - (Signalcmpd - SignalAve_PC) / (SignalAve_VC - SignalAve_PC) × 100.

[0341] SignalAve_PC: The average value of all positive control wells in the whole plate.

[0342] SignalAve_VC: The average value of all negative control wells in the whole plate.

[0343] b) Compound IC50: calculated using GraphPad 8.0 using the following nonlinear fitting formula.

[0344] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope))

[0345] X: log value of compound concentration; Y: percentage of compound inhibition

[0346] The CDK (CDK1, CDK4, CDK6, CDK9) kinase biochemical inhibitory activity of the disclosed compounds was determined by the above assay, and the measured IC 50 See Table 17 and Table 18 for values.

[0347] Table 17.

[0348] Table 18.

[0349] Test Example 2: CYP inhibition experiment

[0350] The metabolic reactions of representative substrates of the five major human CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) were evaluated using pooled human liver microsomes from 150 donors (Corning, Cat. No. 452117). The effects of various concentrations of the test compounds on the metabolic reactions of phenacetin (CYP1A2), diclofenac sodium (CYP2C9), S-mephenytoin (CYP2C19), bufuralol hydrochloride (CYP2D6), and midazolam (CYP3A4 / 5) were determined by liquid chromatography-tandem mass spectrometry (LC / MS / MS).

[0351] A 200 μL reaction mixture (100 mmol / L phosphate buffer, pH 7.4, containing 0.3% DMSO, 0.6% acetonitrile, and 0.1% methanol, by volume) containing 30 μM phenacetin, 10 μM diclofenac sodium, 35 μM S-mephenytoin, 5 μM bufuralol hydrochloride, 3 μM midazolam, 1 mM NADPH, test compounds (at concentrations of 0.1, 0.3, 1, 3, 10, and 30 μmol / L), positive compounds, or a blank control, was incubated with human liver microsomes (0.2 mg / mL) at 37°C for 5 minutes. Then, 200 μL of acetonitrile containing 3% formic acid and 40 nM internal standard verapamil was added, and the mixture was centrifuged at 4000 rpm for 50 minutes. The mixture was cooled on ice for 20 minutes and then centrifuged at 4000 rpm for 20 minutes to precipitate the protein. 200 μL of the supernatant was used for LC / MS / MS analysis.

[0352] The peak area was calculated based on the chromatogram. The residual activity ratio (%) was calculated using the following formula:

[0353] Peak area ratio = metabolite peak area / internal standard peak area

[0354] Residual activity ratio (%) = peak area ratio of the test compound group / peak area ratio of the blank group

[0355] CYP half-maximal inhibitory concentration (IC 50 ) was calculated using Excel XLfit 5.3.1.3.

[0356] The measured CYP half-maximal inhibitory concentration (IC 50 ) values ​​are shown in Table 19 below.

[0357] Table 19. The half-maximal inhibitory concentration (IC) of the compounds of the present invention on CYP 50 )

[0358] Test Example 3: Solubility Test

[0359] The thermodynamic solubility of the compounds was determined in phosphate buffer at pH 7.4. Sample supernatants and standards of known concentrations were analyzed by LC / MS / MS.

[0360] 1. Materials and Reagents

[0361] Compound A (Compound A is example A94 of WO 2019 / 207463A1, synthesized according to the method disclosed in the patent).

[0362] NaH2PO4·2H2O (analytical grade), NaH2PO4 (analytical grade), NaOH (analytical grade).

[0363] 1.5 mL flat-bottom glass tubes (BioTech Solutions); molded Teflon caps (BioTech Solutions), Teflon-coated stirring bars (BioTech Solutions), Eppendorf Comfort Thermomixer, and 96-well deep-well plates.

[0364] 2. Preparation of 0.01 M sodium phosphate buffer at pH 7.4

[0365] Weigh 15.6 g of NaH₂PO₄·2H₂O into a 1 L glass bottle and dissolve in 1 L of deionized water. The pH of the solution should be approximately 4.7. Adjust the pH to 7.4 with 10 M NaOH.

[0366] 3. Solubility determination process

[0367] Accurately weigh 1 mg of powder of each compound and place it into a glass tube. Add 1 mL of pH 7.4 phosphate buffer per mg to the tube. Add a stir bar to each tube, close the lid, and place the sample plate containing the tubes in an Eppendorf Comfort Thermomixer. Incubate at 25°C, 1100 rpm for 24 hours. After the incubation period, open the lid, remove the stir bar using a magnet, and record the contents of each tube. Centrifuge the plate at 25°C, 4000 rpm for 30 minutes. Aspirate 750 μL of the supernatant. Rinse the pipette tip with acetonitrile for 5 seconds, then with pure water for 5 seconds. The first 50 μL of the solution was then discarded, and the remaining 700 μL was added to another 96-well sample plate containing glass tubes. The sample was then centrifuged again for 30 minutes (25°C, 4000 rpm). 10 μL of the sample from the second centrifugation was 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 then added to 990 μL of a 1:1 mixture of acetonitrile and water containing the internal standard (10,000x sample). The sample dilution factor may vary depending on the solubility value and LC / MS signal response.

[0368] Table 20. Phenomenon records and dilution factors

[0369] 4. Preparation of Standards

[0370] Accurately weigh 1 mg of compound powder and add it to a glass tube. Add 1 mL of DMSO per mg to each tube. Add a stir bar to each tube and cap. Place the tray containing the standard tubes in an Eppendorf Comfort Thermomixer and incubate at 1,100 rpm at 25°C for 2 hours to allow the powder to fully dissolve. Observe for complete dissolution of the solids and note any compounds that do not dissolve completely in the DMSO solution. Pipette 10 μL of the 1 mg / mL standard into 990 μL of a 1:1 mixture of acetonitrile and water containing an internal standard to obtain a 10 μg / mL standard. Pipette 10 μL of the 10 μg / mL standard into 990 μL of a 1:1 mixture of acetonitrile and water containing an internal standard to obtain a 0.1 μg / mL standard. Sample dilution may vary depending on LC / MS signal response. Samples were analyzed by LC / MS / MS. All compounds were tested individually.

[0371] 5. Data calculation

[0372] All calculations were performed using Microsoft Excel. Samples were analyzed by LC / MS / MS and quantified based on standards of known concentrations. The solubility of the test compound was calculated using the following formula: [Sample] = Area Ratio sample × DF sample × [STD] / Area Ratio STD

[0373] DF: dilution factor.

[0374] Table 21. Solubility of some compounds of the present invention

[0375] Test Example 5: PXR induction experiment

[0376] 1. Evaluate the potential of test compounds to induce drug-metabolizing enzyme activity via in vitro PXR activation. EC50 values ​​were determined at various concentrations of test compound (30, 10, 3.33, 1.11, 0.370, and 0.123 μM) upon in vitro PXR activation. The positive control, rifampicin, was used at concentrations of 20, 5, 1.25, 0.312, 0.0781, and 0.195 μM.

[0377] 2. Materials and Reagents

[0378] 1) DPX2 (human PXR gene and fluorescent reporter gene stably transfected into HepG2 cells) cells were purchased from Puracyp (Carlsbad, CA).

[0379] 2) The client provided the test compound, and the control drug (rifampicin) was purchased from Sigma (St. Louis, MO).

[0380] 3) CellTiter-Fluor TM The cell viability assay kit and One-Glo fluorescence assay kit were purchased from Promega (Madison, WI), fetal bovine serum (FBS) was purchased from Corning (Manassas, VA), the MTS3 shaker was purchased from IKA Labortechnik (Staufen, Germany), DMEM, penicillin, and streptomycin were purchased from local suppliers, Hygromycin B and G418 were purchased from Merck (Darmstadt, Germany), and cell culture medium and DPX2 cells were purchased from Puracyp Inc.

[0381] 3. Experimental steps

[0382] 3.1 Seed Plate Preparation

[0383] 1) Add 50 mL of FBS to 450 mL of cell culture medium.

[0384] 2) DPX2 cells were cultured in a T-75 culture flask in an incubator at 37° C., 5% CO 2 , and 95% relative humidity, and the cells were digested when the cells covered 80-90% of the bottom of the culture flask.

[0385] 3) Wash the surface of the T-75 cultured cells with 8 mL of PBS, discard the PBS, add 3 mL of trypsin, and digest at 37°C for about 5 minutes, or until the cells are digested and suspended in trypsin. Add 10 mL of excess serum-containing culture medium to neutralize the trypsin.

[0386] 4) Transfer the cell suspension to a conical-bottom centrifuge tube and centrifuge at 120 g for 10 minutes. Resuspend the cells in seed culture medium and adjust the concentration to 4 x 10 5 cells / mL. Add 100 μL of diluted cells to each well of a 96-well cell culture plate. Place the plate in an incubator at 37°C for 24 hours and prepare for the PXR activation assay.

[0387] 3.2 Dosing treatment

[0388] 1) Prepare the test compound and positive compound (rifampicin) in DMSO and dilute the compound in serum-free medium at 37°C. Final concentrations of rifampicin as the positive control are 20, 5, 1.25, 0.312, 0.0781, and 0.195 μM, and final concentrations of the test compound are 30, 10, 3.33, 1.11, 0.370, and 0.123 μM. The final DMSO concentration is 0.1%. Add 1 μL of DMSO to 1 mL of preincubated medium as a solvent control.

[0389] 2) Take the cell culture plate out of the incubator, discard the culture medium, add 100 μL of the test compound and the positive compound to the appropriate wells, two parallel wells per group, and place the cell plate in the incubator for 24 hours.

[0390] 3.3 Quantitative determination of PXR activation

[0391] 1) Two days after drug treatment, cultures can be used for quantitative detection of PXR activation.

[0392] 2) CellTiter-Fluor TM Equilibrate the Cell Viability Assay Kit and One-Glo Luciferase Reagent to room temperature. Add GF-AFC substrate (10 μL) to Assay Buffer (10 mL) to create a 2X reagent, then dilute to 1X with 10 mL of PBS. Add ONE-Glo substrate to ONE-Glo Luciferase Assay Buffer.

[0393] 3) Remove the cell culture plate from the incubator, discard the culture medium in each well, wash twice with PBS, and add 1X CellTiter-Fluor TM The reagent was added to the sterilized sample reservoir, 50 μL was drawn into each well using a dispenser, and incubated at 37°C for 30 minutes.

[0394] 4) Remove the 96-well cell plate from the incubator and measure the fluorescence value of each well using a microplate reader in fluorescence mode with an excitation wavelength of 400 nm and an emission wavelength of 505 nm.

[0395] 5) Pour ONE-Glo reagent into the sample reservoir, then use a dispenser to dispense 50 μL into each well. Gently mix the reagent and incubate at room temperature for 5 minutes. After incubation, read the luminescence value of each well using a luminometer.

[0396] 4. Calculation of cell induction value

[0397] 4.1 Cell viability

[0398] Cell viability calculation formula:

[0399] Percent cell viability(%)=I(sample) / (I(vehicle)x 100

[0400] I(sample) is the fluorescence intensity of the sample, and I(vehicle) refers to the fluorescence intensity of cells after 0.1% DMSO.

[0401] 4.2 Calculation of cell induction value

[0402] All data were calculated using Microsoft Excel.

[0403] The activity of luciferase is expressed as RFU / RLU, where RLU is the average luminescence intensity of two parallel assays at each concentration of each compound, and RFU is the average fluorescence intensity of two parallel assays at each concentration of each compound.

[0404] The formula for calculating the induction multiple is:

[0405] Table 22. Some of the measured PXR induction values

Claims

1. The X-ray powder diffraction pattern of Form A of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropane-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, expressed in terms of diffraction angle 2θ, is 9.6, 10.0, 11.7, 15.0, Preferably, there are characteristic peaks at 7.8, 9.6, 10.0, 11.7, 15.0, 18.9, 19.4, 20.1, 21.1, and 21.7; most preferably, there are 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.

2. The crystalline form B of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropane-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol has an X-ray powder diffraction pattern expressed in terms of a diffraction angle 2θ, with characteristic peaks at 11.3, 15.1, 20.9, 22.8, and 23.7; preferably, at 7.4, 11.3, 14.3, 15.1, 16.3, 18. most preferably, there are 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.

3. Form C of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropane-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthen-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, having an X-ray powder diffraction pattern expressed in terms of a diffraction angle 2θ, and having characteristic peaks at 4.8, 10.1, 12.0, and 15.0; preferably, having characteristic peaks at 4.8, 7.6, 10.1, 12.0, 15.0, 19.7, 21.2, and 23.

5.

4. The D crystal form of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropane-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ degrees, has characteristic peaks at 10.0, 10.5, 17.0, 18.7, and 23.9; preferably, at 10.0, 1 most preferably, there are 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.

5. The X-ray powder diffraction pattern of the E 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-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, expressed in terms of diffraction angle 2θ, is 4.8 , 11.4, 14.4, 15.1, 16.4, 17.9, 19.3, 19.9, 20.9, 22.9, 23.5, there are characteristic peaks; preferably, there are 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, 23.5; more preferably, there are 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, 30.

5.

6. The F crystalline form of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropane-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol has an X-ray powder diffraction pattern expressed in terms of a diffraction angle 2θ, with characteristic peaks at 11.1, 11.4, 14.3, 15.1, 15.7, 19.2, and 22.0; preferably, at 9.1, 11.1, 11.4, 13.3, 14.3, , 23.6, 24.2, 24.6, 25.2, 25.6, 26.1, 28.7, 30.3, 35.0, have characteristic peaks.

7. Form G 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, having an X-ray powder diffraction pattern expressed in terms of a diffraction angle 2θ, and having characteristic peaks at 11.3, 15.3, 16.1, 17.9, 18.6, and 20.8; preferably, at 11.3, 15.3, 16.1, 17.9, 18.6, and 20.

8. most preferably, there are 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.

8. The H crystalline form of the compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropane-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthylene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, having an X-ray powder diffraction pattern expressed in terms of a diffraction angle 2θ, and having characteristic peaks at 11.7, 12.3, 15.5, 19.4, and 22.3; preferably, at 11.7, 12.3, 15.5, 18.2 , 19.4, 21.3, 22.3, 24.9, 27.1, and 28.1, and most preferably, there are 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.

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 Form A 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-diazaacenaphthyl-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 1 or 9, comprising the following steps: Method 1 a) dissolving the compound represented by formula (I) in solvent 1; b) adding 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; 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 solvent 3, stirred and crystallized, wherein the solvent 3 is selected from ester solvents and ketone solvents; preferably, the ester solvent is selected from ethyl acetate and isopropyl acetate, and the ketone solvent is methyl isobutyl ketone.

11. A method for preparing Form B 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-diazaacenaphthyl-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 2 or 9, comprising the following steps: The compound represented by formula (I) is dissolved in a substituted lower alkane, stirred and crystallized; preferably, the substituted lower alkane is nitromethane.

12. A method for preparing Crystal Form C 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-diazaacenaphthyl-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 3 or 9, selected from the following methods: Method 1 The compound represented by formula (I) is dissolved in solvent 4, and evaporated to crystallize, wherein the solvent 4 is selected from substituted lower alkanes or ketone solvents; preferably, the substituted lower alkanes are 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 solvent 5, stirred and crystallized, wherein the solvent 5 is selected from aromatic hydrocarbon solvents; preferably, the aromatic hydrocarbon solvent is selected from p-xylene or toluene; Method 3 a) dissolving the compound represented by formula (I) in solvent 6; b) adding solvent 7 to precipitate crystals; The solvent 6 is selected from alcohol solvents, ketone solvents, nitrile solvents, and ether solvents, 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.

13. A method for preparing the D-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-diazaacenaphthyl-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 4 or 9, comprising the following steps: The compound represented by formula (I) is dissolved in solvent 8, stirred and crystallized, wherein the solvent 8 is an ether solvent or a mixed solvent of an alcohol solvent and an ether solvent; preferably, the alcohol solvent is methanol, ethanol or isopropanol, and the ether solvent is isopropyl ether.

14. A method for preparing the E 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-diazaacenaphthyl-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 5 or 9, selected from the following methods: Method 1 Dissolve the compound represented by formula (I) in solvent 9, stir and crystallize, wherein the solvent 9 is a nitrile solvent, preferably, the nitrile solvent is acetonitrile; Method 2 a) dissolving the compound represented by formula (I) in solvent 10; b) adding solvent 11 to precipitate crystals; The solvent 10 is selected from ketone solvents, and the solvent 11 is an ether solvent; preferably, the ketone solvent is acetone, and the ether solvent is isopropyl ether.

15. A method for preparing the crystalline form F 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-diazaacenaphthyl-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 6 or 9, selected from the following methods: Method 1 The compound represented by formula (I) is dissolved in solvent 12, stirred and crystallized, wherein the solvent 12 is a ketone solvent; preferably, the ketone solvent is methyl isobutyl ketone; Method 2 The compound represented by formula (I) is dissolved in tetrahydrofuran, and after clarification, isopropyl ether and seed crystals of the F crystal form are added to precipitate crystals.

16. A method for preparing the crystalline form G 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-diazaacenaphthyl-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 7 or 9, comprising the following steps: The compound represented by formula (I) is dissolved in a mixed solvent of an alcohol solvent and an ether solvent, and stirred for crystallization; preferably, the alcohol solvent is selected from methanol, and the ether solvent is isopropyl ether.

17. A method for preparing the H 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-diazaacenaphthyl-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 8 or 9, selected from the following methods: Method 1 The compound represented by formula (I) is dissolved in solvent 13, stirred and crystallized, wherein the solvent 13 is selected from an alcohol solvent, a mixed solvent of an alcohol solvent and water, a ketone solvent, and an ester solvent; preferably, the alcohol 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, and the mixture is beaten and crystallized, wherein the solvent 14 is selected from water and an ether solvent; preferably, the ether solvent is selected from methyl tert-butyl ether or isopropyl ether; Method 3 a) dissolving the compound represented by formula (I) in solvent 15; b) adding solvent 16 to precipitate crystals; The solvent 15 is selected from alcohol solvents, ketone solvents, and ether solvents, and the solvent 16 is selected from lower alkanes, ether solvents 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.

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-diazaacenaphthyl-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, sulfate, methanesulfonate, p-toluenesulfonate, phosphate, citrate, or malate.

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-diazaacenaphthyl-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to claim 18, comprising the step of reacting a free base with an acid molecule selected from hydrochloric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, phosphoric acid, citric acid or malic acid.

20. A pharmaceutical composition comprising the crystalline form or compound according to any one of claims 1 to 9 or 18, and optionally selected from pharmaceutically acceptable excipients.

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

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

23. Use of the crystalline form or compound according to any one of claims 1 to 9 or 18, or the composition according to claim 20, in the preparation of a medicament for treating or preventing cancer; preferably, 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.

Images