Crystalline form of a fused tricyclic derivative and method of preparation

Abstract

This disclosure relates to the crystal form and preparation method of a fused tricyclic derivative. Specifically, it relates to the 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, its pharmaceutically acceptable salt, and the corresponding preparation method.

Description

[0001] This application claims priority to Chinese patent application 202210915848.1, filed on August 1, 2022. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the crystal form and preparation method of a condensed tricyclic derivative, belonging to the pharmaceutical field. Background Technology

[0003] Cyclin-dependent kinases (CDKs) are important cellular enzymes that play a crucial role in regulating eukaryotic cell division and proliferation. The catalytic unit of a CDK is known as the activation of the regulatory subunits of 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 discloses a new class of cyclin-dependent kinase inhibitors. Finding developmental forms of the compounds disclosed in this application has significant clinical implications. Summary of the Invention

[0005] This disclosure provides the crystal form of compound (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropane-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazanephrine-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol (the compound shown in formula (I)), its pharmaceutically acceptable salts, and the corresponding preparation methods.

[0006]

[0007] This disclosure provides a crystal form A of the compound shown in formula (I), and an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, which has characteristic peaks at 9.6, 10.0, 11.7, 15.0, 21.1, and 21.7.

[0008] In an optional embodiment, the X-ray powder diffraction pattern of the compound of formula (I) provided in this disclosure, expressed in terms of diffraction angle 2θ, 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 X-ray powder diffraction pattern of the A crystal form of the compound shown in formula (I) provided in this disclosure, expressed as a diffraction angle 2θ, 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 X-ray powder diffraction pattern of the compound of formula (I) provided in this disclosure, expressed in terms of the diffraction angle 2θ, is shown in the attached figure. Figure 2 As shown.

[0011] This disclosure provides a method for preparing crystal form A of the compound shown in formula (I), comprising the following steps:

[0012] Method 1

[0013] a) Dissolve the compound shown in formula (I) in solvent 1;

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

[0015] Solvent 1 is selected from ketone solvents or ester solvents, and 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 shown in formula (I) is dissolved in solvent 3 and stirred to crystallize. 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] This disclosure provides a B-type crystal form of the compound shown in formula (I), whose X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 11.3, 15.1, 20.9, 22.8, and 23.7.

[0020] In an optional embodiment, the X-ray powder diffraction pattern of the B crystal form of the compound of formula (I) provided in this disclosure, expressed as a diffraction angle 2θ, 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.

[0021] In an optional embodiment, the X-ray powder diffraction pattern of the B crystal form of the compound shown in formula (I) provided in this disclosure, expressed as a diffraction angle 2θ, 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.

[0022] In an optional embodiment, the X-ray powder diffraction pattern of the B crystal form of the compound of formula (I) provided in this disclosure, expressed in terms of the diffraction angle 2θ, is shown in the attached figure. Figure 3 As shown.

[0023] This disclosure provides a method for preparing the B crystal form of the compound shown in formula (I), comprising the following steps: dissolving the compound shown in formula (I) in a substituted lower alkane and stirring to induce crystallization.

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

[0025] This disclosure provides a C-crystal form of the compound shown in formula (I), and its X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 4.8, 10.1, 12.0, and 15.0.

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

[0027] In an optional embodiment, the X-ray powder diffraction pattern of the C-crystal form of the compound of formula (I) provided in this disclosure, expressed in terms of the diffraction angle 2θ, is shown in the attached figure. Figure 4 As shown.

[0028] This disclosure provides a method for preparing the C-crystal form of the compound shown in formula (I), selected from the following methods:

[0029] Method 1

[0030] The compound shown in formula (I) is dissolved in solvent 4 and volatilized to crystallize. Solvent 4 is selected from substituted lower alkanes or ketone solvents. Optionally, the substituted lower alkanes are selected from dichloromethane or 1,2-dichloroethane, and the ketone solvent is methyl isobutyl ketone.

[0031] Method 2

[0032] The compound shown in formula (I) is dissolved in solvent 5 and stirred to crystallize. Solvent 5 is selected from aromatic hydrocarbon solvents; optionally, the aromatic hydrocarbon solvent is selected from p-xylene or toluene.

[0033] Method 3

[0034] a) Dissolve the compound shown in formula (I) in solvent 6;

[0035] b) Add 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] This disclosure provides a D-crystal form of the compound shown in formula (I), and an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, which 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 of formula (I) provided in this disclosure 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 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0039] In an optional embodiment, the D crystal form of the compound shown in formula (I) provided in this disclosure 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 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0040] In an optional embodiment, the X-ray powder diffraction pattern of the D crystal form of the compound of formula (I) provided in this disclosure, expressed in terms of the diffraction angle 2θ, is shown in the attached figure. Figure 5 As shown.

[0041] This disclosure provides a method for preparing the D crystal form of the compound shown in formula (I), comprising the following steps: mixing the compound shown in formula (I) with solvent 8, stirring to induce crystallization, wherein the solvent 8 is an ether solvent, or a mixture 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] This disclosure provides an E crystal form of the compound shown in formula (I), and an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, which has 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 crystal form of the compound shown in formula (I) provided in this 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 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0044] In an optional embodiment, the X-ray powder diffraction pattern of the E crystal form of the compound shown in formula (I) provided in this disclosure, expressed as a diffraction angle 2θ, 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 X-ray powder diffraction pattern of the E crystal form of the compound represented by formula (I) provided in this disclosure, expressed in terms of the diffraction angle 2θ, is shown in the attached figure. Figure 6 As shown.

[0046] This disclosure provides a method for preparing the E crystal form of the compound shown in Chinese formula (I), selected from the following methods:

[0047] Method 1

[0048] The compound shown in formula (I) is dissolved in solvent 9 and stirred to crystallize. Solvent 9 is a nitrile solvent, and optionally, the nitrile solvent is acetonitrile.

[0049] Method 2

[0050] a) Dissolve the compound shown in formula (I) in solvent 10.

[0051] b) Add 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] This disclosure provides an F-crystal form of the compound shown in formula (I), whose X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 11.1, 11.4, 14.3, 15.1, 15.7, 19.2, and 22.0.

[0054] In an optional embodiment, the F crystal form of the compound shown in formula (I) provided in this disclosure 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 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0055] In an optional embodiment, the F crystal form of the compound shown in formula (I) provided in this disclosure 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 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0056] In an optional embodiment, the X-ray powder diffraction pattern of the F crystal form of the compound of formula (I) provided in this disclosure, expressed in terms of the diffraction angle 2θ, is shown in the attached figure. Figure 7 As shown.

[0057] This disclosure provides a method for preparing the F-crystal form of the compound shown in formula (I), selected from the following methods:

[0058] Method 1

[0059] The compound shown in formula (I) is dissolved in solvent 12 and stirred to crystallize. Solvent 12 is a ketone solvent. Optionally, the ketone solvent is methyl isobutyl ketone.

[0060] Method 2

[0061] The compound shown in formula (I) was dissolved in tetrahydrofuran, and after the solution was cleared, isopropyl ether and F-type seed crystals were added to precipitate crystals.

[0062] This disclosure provides a G-crystal form of the compound shown in formula (I), and its X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 11.3, 15.3, 16.1, 17.9, 18.6, and 20.8.

[0063] In an optional embodiment, the G crystal form of the compound shown in formula (I) provided in this 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 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0064] In an optional embodiment, the G crystal form of the compound shown in formula (I) provided in this disclosure 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 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0065] In an optional embodiment, the X-ray powder diffraction pattern of the G crystal form of the compound represented by formula (I) provided in this disclosure, expressed in terms of the diffraction angle 2θ, is shown in the attached figure. Figure 8 As shown.

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

[0067] This disclosure provides an H-crystal form of the compound shown in formula (I), whose X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has 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 shown in formula (I) provided in this 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 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0069] In an optional embodiment, the H crystal form of the compound shown in formula (I) provided in this disclosure 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 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0070] In an optional embodiment, the X-ray powder diffraction pattern of the H crystal form of the compound of formula (I) provided in this disclosure, expressed in terms of the diffraction angle 2θ, is shown in the attached figure. Figure 9 As shown.

[0071] This disclosure provides a method for preparing the H crystal form of the compound shown in formula (I), selected from the following methods:

[0072] Method 1

[0073] The compound shown in formula (I) is dissolved in solvent 13 and stirred to crystallize. Solvent 13 is selected from alcohol solvents, mixed solvents of alcohol solvent and water, ketone solvents or ester solvents. 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 shown in formula (I) is mixed with solvent 14 and slurryed to crystallize. Solvent 14 is selected from water or ether solvents. Optionally, the ether solvent is selected from methyl tert-butyl ether or isopropyl ether.

[0076] Method 3

[0077] a) Dissolve the compound shown in formula (I) in solvent 15;

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

[0079] 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; 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 this disclosure provides a pharmaceutically acceptable salt of the compound shown in formula (I), wherein the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, methanesulfonate, p-toluenesulfonate, phosphate, citrate, or malate.

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

[0081] This disclosure provides a method for preparing a pharmaceutically acceptable salt of the compound shown in formula (I), 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.

[0082] In an optional embodiment, the method for preparing the pharmaceutically acceptable salt of the compound shown in formula (I) provided in this disclosure involves a reaction in the presence 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 salts of the compounds represented by formula (I) provided in this disclosure may be of any crystal form or amorphous.

[0087] The substituted lower alkanes described in this disclosure may be selected from nitromethane, dichloromethane, and chloroform; the nitrile solvents may be selected from acetonitrile or propionitrile; the alcohol solvents may be C1-C6 alcohols, such as methanol, ethanol, n-propanol, isopropanol, and n-butanol; the ketone solvents may be selected from acetone, 2-butanone, and methyl isobutyl ketone; the ether solvents may be selected from isopropyl ether, tetrahydrofuran, dioxane, and propylene glycol methyl ether; the ester solvents may be selected from ethyl acetate, methyl acetate, and isopropyl acetate; and the lower alkanes may be selected from n-hexane or n-heptane.

[0088] In the preparation method provided in this disclosure, the ratio of solvent can be 0.1-100 times (w / v) of the compound shown in 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 any value between any two numbers.

[0089] The method for preparing the crystal form disclosed herein includes a solid-liquid separation step, which may specifically be filtration, centrifugation, or complete evaporation of the solvent.

[0090] The method for preparing the crystal form provided in this disclosure may optionally include a drying step.

[0091] This disclosure also provides a pharmaceutical composition comprising any of the aforementioned crystal forms or pharmaceutically acceptable salts, or mixtures thereof, and pharmaceutical excipients optionally selected from pharmaceutically acceptable excipients.

[0092] This disclosure also provides a method for preparing a pharmaceutical composition, comprising the step of mixing the aforementioned arbitrary crystal form or pharmaceutically acceptable salt with a pharmaceutically acceptable excipient.

[0093] This disclosure also provides the use of any of the aforementioned crystal forms or pharmaceutically acceptable salts, or the aforementioned compositions, in the preparation of medicaments for the treatment or prevention of diseases related to cyclin-dependent kinases.

[0094] This disclosure also provides the use of any of the aforementioned crystal forms or pharmaceutically acceptable salts, or the aforementioned compositions, in the preparation of medicaments for the treatment or prevention of cancer.

[0095] In an optional implementation, 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, stomach cancer, and thyroid cancer.

[0096] The "2θ or 2θ angle" mentioned in this disclosure refers to the diffraction angle, where θ is the Bragg angle, and the unit is ° or degree; the error range of 2θ for each characteristic peak is ±0.2 (including the case where the number has more than one decimal place after rounding), and 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 described in this disclosure is generally 25℃~100℃, preferably 40℃~70℃. It can be dried under normal pressure or reduced pressure, with a pressure <-0.08MPa.

[0098] The “excipients” described in this disclosure include, but are not limited to, any adjuvants, carriers, flow aids, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, or emulsifiers that have been approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0099] The "pulping" described in this disclosure refers to a purification method that utilizes the characteristic that substances have poor solubility in solvents, but impurities have good solubility in solvents. Pulping purification can remove color, change crystal form, or remove a small amount of impurities.

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

[0101] In this disclosure, the ratio of the compound shown in formula (I) to the acid is within a reasonable error range of ±10%. It can be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0102] The contents disclosed in PCT / CN2022 / 074509 are also incorporated in this disclosure. Attached Figure Description

[0103] Figure 1 The amorphous XRPD spectrum of the compound shown in formula (I).

[0104] Figure 2 XRPD spectrum of the A crystal form of the compound shown in formula (I).

[0105] Figure 3 XRPD spectrum of the B crystal form of the compound shown in formula (I).

[0106] Figure 4 XRPD spectrum of the C crystal form of the compound shown in formula (I).

[0107] Figure 5 XRPD spectrum of the D crystal form of the compound shown in formula (I).

[0108] Figure 6 XRPD spectrum of the E crystal form of the compound shown in formula (I).

[0109] Figure 7 XRPD spectrum of the F crystal form of the compound shown in formula (I).

[0110] Figure 8 XRPD spectrum of the G crystal form of the compound shown in formula (I).

[0111] Figure 9 XRPD spectrum of the H crystal form of the compound shown in formula (I). Detailed Implementation

[0112] The following embodiments are used to further describe this disclosure, but these embodiments are not intended to limit the scope of this disclosure.

[0113] Example

[0114] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰ ohms. -6 The unit (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), with tetramethylsilane (TMS) as the internal standard.

[0115] MS measurements were performed using an Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS liquid chromatography-mass spectrometry system (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS).

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

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

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

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

[0122] The CombiFlash rapid preparation system uses a CombiFlash Rf200 (TELEDYNE ISCO).

[0123] Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15 mm to 0.2 mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4 mm to 0.5 mm.

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

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

[0126] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

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

[0128] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0129] A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon with a volume of approximately 1L.

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

[0131] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0132] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

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

[0134] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20℃~30℃.

[0135] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system for column chromatography used to purify the compounds, and the developing solvent system for TLC included: A: dichloromethane / methanol system. The volume ratio of the solvent was adjusted according to the polarity of the compounds. Small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0136] The testing conditions of the instruments used in the experiments in this disclosure are as follows:

[0137] 1. Differential Scanning Calorimeter (DSC)

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

[0139] Purging gas: nitrogen; Nitrogen purging rate: 50 mL / 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] Rays: Monochromatic Cu-Kα rays (λ = 1.5406)

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

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

[0147] 3. Thermogravimetric Analysis (TGA)

[0148] Instrument model: Mettler Toledo TGA2

[0149] Purging gas: nitrogen; Nitrogen purging rate: 50 mL / min

[0150] Heating rate: 10.0℃ / min

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

[0152] 4. DVS is a dynamic moisture adsorption method.

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

[0154] Example 1. Preparation of free amorphous material

[0155] 10 mg of the compound shown in 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 below. Figure 1 As shown.

[0156] Example 2. Preparation of free state A crystal form

[0157] 10 mg of the compound shown in formula (I) was dissolved in 0.05 mL of isopropyl acetate, cooled to 5 °C, stirred to induce crystallization, filtered, and the filter cake was collected and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified the product as crystal form A, and the XRPD spectrum is shown below. Figure 2 As shown in Table 1, the positions of its characteristic peaks are as follows. The DSC spectrum shows that the endothermic peaks have peak values ​​of 87.79℃ and 254.08℃. The TGA spectrum shows a weight loss of 10.07% between 30 and 150℃.

[0158] Table 1

[0159]

[0160] Example 3. Preparation of free state A crystal form

[0161] 10 mg of the compound shown in formula (I) was dissolved in 0.05 mL of ethyl acetate, cooled to 5 °C, stirred to induce 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 crystal form A.

[0162] Example 4. Preparation of free state A crystal form

[0163] 10 mg of the compound shown in formula (I) was dissolved in 0.05 mL of methyl isobutyl ketone, cooled to 5 °C, stirred to induce 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 crystal form A.

[0164] Example 5. Preparation of free state A crystal form

[0165] 10 mg of the compound shown in formula (I) was dissolved in 0.05 mL of acetone, followed by the addition of 0.6 mL of isopropyl ether. The mixture was stirred to induce crystallization, centrifuged, and then dried under vacuum to obtain a solid. X-ray powder diffraction analysis revealed that the product was crystal form A.

[0166] Example 6. Preparation of free state A crystal form

[0167] 10 mg of the compound shown in formula (I) was dissolved in 0.05 mL of methyl isobutyl ketone, followed by the addition of 0.6 mL of isopropyl ether. The mixture was stirred to induce crystallization, centrifuged, and then dried under vacuum to obtain a solid. X-ray powder diffraction analysis revealed that the product was crystal form A.

[0168] Example 7. Preparation of free state A crystal form

[0169] 10 mg of the compound shown in formula (I) was dissolved in 0.05 mL of ethyl acetate, and then 0.8 mL of methyl tert-butyl ether was added, followed by evaporation and crystallization. X-ray powder diffraction analysis showed that the product was crystal form A.

[0170] Example 8. Preparation of free B crystal form

[0171] 10 mg of the compound shown in formula (I) was dissolved in 0.05 mL of nitromethane, stirred at room temperature to induce crystallization, filtered, and the filter cake was collected and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified the product as crystal form B, and the XRPD spectrum is shown below. Figure 3 As shown in Table 2, the positions of its characteristic peaks are as follows. The DSC spectrum shows endothermic peaks at 154.21℃ and 254.73℃. The TGA spectrum shows a weight loss of 4.42% between 30 and 150℃.

[0172] Table 2

[0173]

[0174]

[0175] Example 9. Preparation of free carbon crystal form

[0176] 10 mg of the compound shown in formula (I) was dissolved in 0.05 mL of dichloromethane, and the solution was evaporated and crystallized to obtain a solid. X-ray powder diffraction analysis identified the product as crystal form C, and the XRPD spectrum is shown below. Figure 4 As shown in Table 3, the positions of its characteristic peaks are as follows. The DSC spectrum shows endothermic peaks at 125.96℃ and 253.40℃. The TGA spectrum shows a weight loss of 4.37% between 30-150℃.

[0177] Table 3

[0178]

[0179] Example 10. Preparation of free carbon crystal form

[0180] 10 mg of the compound shown in formula (I) was dissolved in 0.05 mL of 1,2-dichloroethane, and the solution was evaporated and crystallized to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form C.

[0181] Example 11. Preparation of free carbon crystal form

[0182] 10 mg of the compound shown in formula (I) was dissolved in 0.05 mL of methyl isobutyl ketone, and the solution was evaporated to crystallize, yielding a solid. X-ray powder diffraction analysis showed that the product was crystalline form C.

[0183] Example 12. Preparation of free carbon crystal form

[0184] 10 mg of the compound shown in formula (I) was added to 1 mL of p-xylene, stirred to induce crystallization, centrifuged, and then dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form C.

[0185] Example 13. Preparation of free carbon crystal form

[0186] 10 mg of the compound shown in formula (I) was added to 1 mL of toluene, stirred to induce crystallization, centrifuged, and then dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form C.

[0187] Example 14. Preparation of free carbon crystal form

[0188] 10 mg of the compound shown in formula (I) was added to 0.1 mL of isopropanol, followed by the addition of 1 mL of isopropyl ether. After stirring, a solid precipitated, which was then centrifuged and dried under vacuum to obtain the solid. X-ray powder diffraction analysis showed that the product was crystalline form C.

[0189] Example 15. Preparation of free carbon crystal form

[0190] 10 mg of the compound shown in formula (I) was added to 0.1 mL of acetone, followed by the addition of 1 mL of isopropyl ether. After stirring, a solid precipitated, which was then centrifuged and dried under vacuum to obtain the solid. X-ray powder diffraction analysis showed that the product was crystalline form C.

[0191] Example 16. Preparation of free carbon crystal form

[0192] 10 mg of the compound shown in formula (I) was added to 0.1 mL of ACN (acetonitrile), followed by the addition of 1 mL of isopropyl ether. After stirring, a solid precipitated, which was then centrifuged and dried under vacuum to obtain the solid. X-ray powder diffraction analysis showed that the product was crystalline form C.

[0193] Example 17. Preparation of free carbon crystal form

[0194] 10 mg of the compound shown in formula (I) was added to 0.1 mL of THF (tetrahydrofuran), followed by the addition of 1 mL of isopropyl ether. After stirring, a solid precipitated, which was then centrifuged and dried under vacuum to obtain the solid. X-ray powder diffraction analysis showed that the product was crystalline form C.

[0195] Example 18. Preparation of free carbon crystal form

[0196] 10 mg of the compound shown in formula (I) was added to 0.1 mL of EtOH, followed by the addition of 1 mL of isopropyl ether. After stirring, a solid precipitated, which was then centrifuged and dried under vacuum to obtain the solid. X-ray powder diffraction analysis showed that the product was crystalline form C.

[0197] Example 19. Preparation of free D-type crystals

[0198] 10 mg of the compound shown in formula (I) was added to 1 mL of isopropyl ether, stirred at 60 °C for 2 h, centrifuged, and then vacuum dried to obtain a solid. X-ray powder diffraction analysis identified the product as crystal form D, and the XRPD spectrum is shown below. Figure 5As shown in Table 4, the positions of its characteristic peaks are as follows. The DSC spectrum shows that the endothermic peak has a peak value of 196.66℃. The TGA spectrum shows that the weight loss is 0.63% from 30 to 150℃.

[0199] Table 4

[0200]

[0201]

[0202] Example 20. Preparation of free D-type crystals

[0203] 100 mg of the compound shown in formula (I) was added to 10 mL of isopropyl ether, along with approximately 1–2 mg of seed crystals. The mixture was stirred at 60 °C for 2 h, centrifuged, and then vacuum dried to obtain a solid. X-ray powder diffraction analysis revealed that the product was crystal form D.

[0204] Example 21. Preparation of free D-type crystals

[0205] 50 mg of the compound shown in formula (I) was added to 0.1 mL of ethanol and stirred at 60 °C until dissolved. 0.4 mL of isopropyl ether was added, followed by 1–2 mg of crystal form D seed crystals. The mixture was stirred at 60 °C for 2 h to precipitate a solid. After centrifugation, the solid was dried under vacuum. X-ray powder diffraction analysis confirmed that the product was crystal form D.

[0206] Example 22. Preparation of free D-crystal form

[0207] 50 mg of the compound shown in formula (I) was added to 0.1 mL of methanol and stirred at 60 °C until dissolved. 0.4 mL of isopropyl ether was added, followed by 1–2 mg of crystal form D seed crystals. The mixture was stirred at 60 °C for 2 h to precipitate a solid. After centrifugation, the solid was dried under vacuum. X-ray powder diffraction analysis confirmed that the product was crystal form D.

[0208] Example 23. Preparation of free D-type crystals

[0209] 700 mg of the compound shown in formula (I) was added to 1.4 mL of ethanol and stirred at 60 °C until dissolved. 4.2 mL of isopropyl ether was then added, followed by 1–2 mg of crystal form D seed crystals. The mixture was stirred at 60 °C for 2 h to precipitate a solid. After centrifugation, the solid was dried under vacuum. X-ray powder diffraction analysis confirmed that the product was crystal form D.

[0210] Example 24. Preparation of free D-type crystals

[0211] 10 mg of the compound shown in formula (I) was added to 0.4 mL of ethanol / isopropyl ether (1 / 20), stirred at 60 °C until dissolved, and stirred for another 1 h to precipitate a solid. After centrifugation, the solid was dried under vacuum. X-ray powder diffraction analysis showed that the product was crystal form D.

[0212] Example 25. Preparation of free D-type crystals

[0213] 10 mg of the compound shown in formula (I) was added to 0.4 mL of methanol / isopropyl ether (1 / 20), stirred at 60 °C until dissolved, and stirred for another 1 h to precipitate a solid. After centrifugation, the solid was dried under vacuum. X-ray powder diffraction analysis showed that the product was crystal form D.

[0214] Example 26. Preparation of free E-crystal form

[0215] 10 mg of the compound shown in formula (I) was added to 0.05 mL of acetonitrile for dissolution. The solution was cooled to 5 °C, stirred at low temperature to induce crystallization, filtered, and the filter cake was collected and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified the product as crystal form E. The XRPD spectrum is shown below. Figure 6 As shown in Table 5, the positions of its characteristic peaks are as follows. The DSC spectrum shows that the endothermic peaks have peak values ​​of 35.46℃ and 137.39℃. The TGA spectrum shows a weight loss of 6.04% between 30 and 160℃.

[0216] Table 5

[0217]

[0218]

[0219] Example 27. Preparation of free E-crystal form

[0220] 100 mg of the compound shown in formula (I) was dissolved in 0.5 mL of acetone, 3 mL of isopropyl ether was added, the temperature was lowered to 5 °C, the mixture was stirred to crystallize, filtered, the filter cake was collected, and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was crystal form E.

[0221] Example 28. Preparation of free F-type crystals

[0222] 100 mg of the compound shown in formula (I) was dissolved in 0.5 mL of methyl isobutyl ketone, stirred at room temperature to induce crystallization, filtered, and the filter cake was collected and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified the product as crystal form F, and the XRPD spectrum is shown below. Figure 7 As shown in Table 6, the positions of its characteristic peaks are as follows. The DSC spectrum shows endothermic peaks at 32.47℃ and 137.02℃. The TGA spectrum shows a weight loss of 6.99% between 30-160℃.

[0223] Table 6

[0224]

[0225]

[0226] Example 29. Preparation of free F-type crystals

[0227] 100 mg of the compound shown in formula (I) was added to 2 mL of tetrahydrofuran and stirred at 60 °C until dissolved. 10 mL of isopropyl ether and F crystal seed were added and stirred at 60 °C for 2 h to precipitate the solid. The solid was filtered, the filter cake was collected, and vacuum dried to obtain the solid. X-ray powder diffraction analysis showed that the product was crystal form F.

[0228] Example 30. Preparation of free G crystal form

[0229] 50 mg of the compound shown in 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 solid was filtered, the filter cake was collected, and dried under vacuum. X-ray powder diffraction analysis identified the product as crystal form G. The XRPD spectrum is shown below. Figure 8 As shown in Table 7, the positions of its characteristic peaks are as follows. The DSC spectrum shows that the endothermic peak has a peak value of 173.41℃. The TGA spectrum shows that the weight loss is 1.18% from 30 to 190℃.

[0230] Table 7

[0231]

[0232]

[0233]

[0234] Example 31. Preparation of free H crystal form

[0235] 10 mg of the compound shown in formula (I) was added to 0.05 mL of ethanol, heated and stirred at 60 °C until dissolved, and then suspended and stirred at 50 °C-5 °C to precipitate. The precipitate was filtered, the filter cake was collected, and vacuum dried to obtain a solid. X-ray powder diffraction analysis identified the product as crystal form H, and the XRPD spectrum is shown below. Figure 9 As shown in Table 8, the positions of its characteristic peaks are as follows. The DSC spectrum shows that the endothermic peak has a peak value of 206.30℃. The TGA spectrum shows that the weight loss is 0.29% from 33 to 218℃.

[0236] Table 8

[0237]

[0238]

[0239] Example 32. Preparation of free H crystal form

[0240] 10 mg of the compound shown in formula (I) was added to 0.05 mL of isopropanol, heated and stirred at 60 °C until dissolved, and then suspended and stirred at 50 °C-5 °C to precipitate. The precipitate was filtered, the filter cake was collected, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0241] Example 33. Preparation of free H crystal form

[0242] 10 mg of the compound shown in formula (I) was added to 0.05 mL of n-propanol, heated and stirred at 60 °C until dissolved, and then suspended and stirred at 50 °C-5 °C to precipitate. The precipitate was filtered, the filter cake was collected, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0243] Example 34. Preparation of free H crystal form

[0244] 10 mg of the compound shown in formula (I) was added to 0.05 mL of 2-butanone, heated and stirred at 60 °C until dissolved, and then suspended and stirred at 50 °C-5 °C to precipitate. The precipitate was filtered, the filter cake was collected, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0245] Example 35. Preparation of free H crystal form

[0246] 10 mg of the compound shown in formula (I) was added to 0.05 mL of methanol / water = 1 / 1 solvent, heated and stirred at 60 °C until dissolved, and then suspended and stirred at 50 °C-5 °C to precipitate. The precipitate was filtered, the filter cake was collected, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0247] Example 36. Preparation of free H crystal form

[0248] 10 mg of the compound shown in formula (I) was added to 0.25 mL of ethyl acetate solvent and heated and stirred at 60 °C until dissolved. The solution was then suspended at 50 °C-5 °C with stirring to precipitate the product. The precipitate was filtered, collected, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0249] Example 37. Preparation of free H crystal form

[0250] 10 mg of the compound shown in formula (I) was added to 0.25 mL of methyl isobutyl ketone solvent and heated and stirred at 60 °C until dissolved. The solution was then suspended at 50 °C-5 °C with stirring to precipitate the product. The precipitate was filtered, collected, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0251] Example 38. Preparation of free H crystal form

[0252] 10 mg of the compound shown in formula (I) was added to 1 mL of water, heated and stirred at 60 °C until it did not dissolve. The mixture was then suspended at 50 °C-5 °C, stirred and slurried, 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 crystalline form H.

[0253] Example 39. Preparation of free H crystal form

[0254] 10 mg of the compound shown in formula (I) was added to 1 mL of methyl tert-butyl ether. The mixture was heated to 60 °C and stirred until insoluble. The solution was then suspended at 50 °C - 5 °C, stirred, and slurried. The mixture was filtered, the filter cake was collected, and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0255] Example 40. Preparation of free H crystal form

[0256] 10 mg of the compound shown in formula (I) was added to 1 mL of isopropyl ether. The mixture was heated to 60 °C with stirring until it did not dissolve. The solution was then suspended at 50 °C - 5 °C with stirring and slurrying. The mixture was filtered, the filter cake was collected, and vacuum dried to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0257] Example 41. Preparation of free H crystal form

[0258] 10 mg of the compound shown in formula (I) was added to 0.15 mL of isopropanol and stirred at 60 °C to dissolve. After adding 1 mL of water, the product was stirred to precipitate, centrifuged, and then dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0259] Example 42. Preparation of free H crystal form

[0260] 10 mg of the compound shown in formula (I) was added to 0.15 mL of isopropanol and stirred at 60 °C to dissolve. After adding 1 mL of n-heptane, the mixture was stirred to precipitate, centrifuged, and then dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0261] Example 43. Preparation of free H crystal form

[0262] 10 mg of the compound shown in formula (I) was added to 0.15 mL of isopropanol and stirred at 60 °C to dissolve. After adding 1 mL of isopropyl ether, the mixture was stirred to precipitate. The precipitate was then centrifuged and dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0263] Example 44. Preparation of free H crystal form

[0264] 10 mg of the compound shown in formula (I) was added to 0.1 mL of acetone and stirred at 60 °C to dissolve. After adding 1 mL of water, precipitation occurred. The solid was obtained by centrifugation and vacuum drying. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0265] Example 45. Preparation of free H crystal form

[0266] 10 mg of the compound shown in formula (I) was added to 0.1 mL of acetone and stirred at 60 °C to dissolve. After adding 1 mL of n-heptane, precipitation occurred. The solid was obtained by centrifugation and vacuum drying. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0267] Example 46. Preparation of free H crystal form

[0268] 10 mg of the compound shown in formula (I) was added to 0.1 mL of acetone and stirred at 60 °C to dissolve. After adding 1 mL of isopropyl ether, the product precipitated, centrifuged, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0269] Example 47. Preparation of free H crystal form

[0270] 10 mg of the compound shown in formula (I) was added to 0.05 mL of tetrahydrofuran and stirred at 60 °C to dissolve. After adding 1 mL of methyl tert-butyl ether, the product precipitated, centrifuged, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0271] Example 48. Preparation of free H crystal form

[0272] 10 mg of the compound shown in formula (I) was added to 0.05 mL of tetrahydrofuran and stirred at 60 °C to dissolve. After adding 1 mL of isopropyl ether, the product precipitated, centrifuged, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was crystalline form H.

[0273] Example 49. Study on the hygroscopicity of the free crystal form

[0274] Using Surface Measurement Systems intrinsic DVS, at 25℃, the humidity range was 0%-95%, with a step size of 10%. The judgment criterion was that the mass change dM / dT for each gradient was less than 0.002%, TMAX 360 min, and two cycles were performed.

[0275] Table 9

[0276]

[0277]

[0278] Example 50. Stability Study of Factors Affecting Crystal Form

[0279] The free-state samples were laid flat in the open and the stability of the samples was investigated under light (4500 Lux), high temperature (40℃, 60℃), and high humidity (RH 75%, RH 92.5%) conditions. The sampling period was 30 days.

[0280] Table 10

[0281]

[0282] Table 11

[0283]

[0284]

[0285] Table 12

[0286]

[0287] Table 13

[0288]

[0289]

[0290] Table 14

[0291]

[0292] Table 15

[0293]

[0294] Experimental results on influencing factors show that both crystalline D / E / F / G / H and free amorphous states exhibit good chemical stability under high temperature and high humidity. Both crystalline D / E / F / G / H and free amorphous states also demonstrate good physical stability.

[0295] Example 51. Long-term / accelerated stability of crystal form

[0296] The free-state sample was sealed in an aluminum foil bag and its stability was investigated under conditions of 25℃ / 60%RH and 40℃ / 75%RH, respectively. The results are shown below.

[0297] Table 16

[0298]

[0299]

[0300] Experimental results show that crystal form H exhibits good physical and chemical stability after 3 months of long-term accelerated storage. Crystal forms D, E, F, and G also exhibit good physical and chemical stability after 6 months of long-term accelerated storage. The free amorphous form undergoes accelerated deliquescence, but maintains good physical and chemical stability under other conditions.

[0301] Example 52. Preparation of hydrochloride

[0302] 100 mg of the compound shown in formula (I) was added to 2 mL of acetonitrile, followed by 115.3 μl of 2M hydrochloric acid-ethanol solution. The mixture was stirred for 5 h until the sample dissolved completely. 5 mL of isopropyl ether was added and stirred to precipitate the solid. The precipitate was then centrifuged and dried under vacuum to obtain a solid. Ion chromatography results showed a chloride ion content of 6.7%.

[0303] Example 53. Preparation of sulfate

[0304] 100 mg of the compound shown in formula (I) was added to 2 mL of acetonitrile, followed by 115.3 μl of 2M sulfuric acid ethanol solution. The mixture was stirred for 5 h until the sample dissolved completely. 5 mL of isopropyl ether was added and stirred to precipitate the solid. The precipitate was then centrifuged and dried under vacuum to obtain a solid. Ion chromatography results showed that the sulfate ion content was 14.9%.

[0305] Example 54. Preparation of methanesulfonate

[0306] 100 mg of the compound shown in formula (I) was added to 2 mL of acetonitrile, followed by 115.3 μl of 2M methanesulfonic acid ethanol solution. The mixture was stirred for 5 h until the sample dissolved completely. 5 mL of isopropyl ether was added and stirred to precipitate the solid. The precipitate was then centrifuged and dried under vacuum to obtain a solid. Ion chromatography results showed that the methanesulfonate ion content was 17.8%.

[0307] Example 55. Preparation of p-Toluenesulfonate

[0308] 100 mg of the compound shown in formula (I) was added to 2 mL of acetonitrile, followed by 115.3 μl of 2 M p-toluenesulfonic acid ethanol solution. The mixture was stirred for 5 h until the sample dissolved completely. 5 mL of isopropyl ether was added and stirred to precipitate the solid. The solid was then centrifuged and dried under vacuum. Ion chromatography results showed that the p-toluenesulfonate ion content was 34.3%.

[0309] Example 56. Preparation of Phosphate

[0310] 10 mg of the compound shown in formula (I) was added to 0.2 mL of acetonitrile, 11.5 μl of 2 M phosphate ethanol solution was added, and the mixture was stirred for 5 h. After centrifugation, the solid was dried under vacuum.

[0311] Example 57. Preparation of citrate

[0312] 10 mg of the compound shown in formula (I) was added to 0.2 mL of acetonitrile, 11.5 μl of 2 M citric acid ethanol solution was added, and the mixture was stirred for 5 h. After centrifugation, the solid was dried under vacuum.

[0313] Example 58. Preparation of malate

[0314] 10 mg of the compound shown in formula (I) was added to 0.2 mL of acetonitrile, 11.5 μl of 2 M citric acid ethanol solution was added, and the mixture was stirred for 5 h. After centrifugation, the solid was dried under vacuum.

[0315] Example 59.

[0316] (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropane-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol isomer 1

[0317]

[0318]

[0319] first step

[0320] 6-Bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazanephrine-2-carboxaldehyde 4a

[0321] Under a nitrogen atmosphere, 6-bromo-8-fluoro-2,3-dimethyl-3,4-dihydro-5-oxa-1,2a-diazanephrine (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 carried out 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 reversed-phase chromatography to give the title compound 4a (930 mg, yield: 44%).

[0322] MS(ESI) m / z 299.1, 301.1 [M+H] +

[0323] Step 2

[0324] 1-(6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazanephrine-2-yl)ethane-1-ol 4b

[0325] Under a nitrogen atmosphere, 930 mg (3.1 mmol) of 6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazanephrine-2-carboxaldehyde was dissolved in 20 mL of tetrahydrofuran. The mixture was cooled to -20 °C, and a solution of methyl magnesium bromide in tetrahydrofuran (3 mol / L, 1.5 mL, 4.5 mmol) was added dropwise. The reaction was continued at -20 °C for 4 hours. The reaction was quenched with 5 mL of water. The reaction solution was concentrated under reduced pressure, and the residue was purified by C-18 reversed-phase chromatography to give the title compound 4b (830 mg, yield: 85%).

[0326] MS(ESI) m / z 315.2, 317.2 [M+H] +

[0327] Step 3

[0328] 1-(6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazanephrine-2-yl)ethane-1-one 4c

[0329] At room temperature, 1-(6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazanephrine-2-yl)ethane-1-one (600 mg, 1.9 mmol) was dissolved in 20 mL of tetrahydrofuran, and Desmartin oxidant (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 reversed-phase chromatography to give the title compound 4c (350 mg, yield: 59%).

[0330] MS(ESI) m / z 313.1, 315.1 [M+H] +

[0331] Step 4

[0332] 2-(6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazanephrine-2-yl)propane-2-ol 4d

[0333] Under a nitrogen atmosphere, 1-(6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazanephrine-2-yl)ethane-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 methyl magnesium bromide in tetrahydrofuran (3 mol / L, 0.7 mL, 2.1 mmol) was added dropwise. The reaction was continued at -20 °C for 4 hours. The reaction was quenched with 5 mL of water. The reaction solution was concentrated under reduced pressure, and the residue was purified by C-18 reversed-phase chromatography to give the title compound 4d (260 mg, yield: 71%).

[0334] MS(ESI) m / z 329.2, 331.2 [M+H] +

[0335] Step 5

[0336] 2-(6-(2,5-dichloropyrimidin-4-yl)-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazanephrine-2-yl)propane-2-ol 4e

[0337] Under a nitrogen atmosphere, 2-(6-bromo-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazanephrine-2-yl)propane-2-ol (260 mg, 0.8 mmol), pinacol diborate (305 mg, 1.2 mmol), potassium acetate (157 mg, 1.6 mmol), and [1,1′-bis(diphenylphosphine)ferrocene]palladium dichloride (117 mg, 0.2 mmol) were dissolved sequentially in 5 mL of 1,4-dioxane. The reaction was carried out at 100 °C for 2 hours. The reaction solution was cooled to room temperature, and 1,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6-phosphoadamantane (58 mg, 0.2 mmol), potassium carbonate (221 mg, 1.6 mmol), 2,4-dichloro-5-fluoropyrimidine (220 mg, 1.2 mmol), tris(dibenzylacetone)palladium (183 mg, 0.2 mmol), and 1 mL of water were added. The reaction mixture was reacted at 80 °C for 1 hour. 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 reversed-phase chromatography to give the title compound 4e (129 mg, yield: 41%).

[0338] MS(ESI) m / z 397.3 [M+H] +

[0339] Step 6

[0340] (3S,4R)-4-((5-chloro-4-((S)-8-fluoro-2-(2-hydroxypropane-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol isomer 1

[0341] (3S,4R)-4-((5-chloro-4-((R)-8-fluoro-2-(2-hydroxypropane-2-yl)-3-methyl-3,4-dihydro-5-oxa-1,2a-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol isomer 2

[0342] Under a nitrogen atmosphere, 2-(6-(2,5-dichloropyrimidin-4-yl)-8-fluoro-3-methyl-3,4-dihydro-5-oxa-1,2a-diazanephrine-2-yl)propane-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 sequentially 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. After concentration under reduced pressure, the residue was purified by C-18 reversed-phase chromatography to obtain crude product mixture 4f. The crude product was separated by chiral resolution. [Column:]

[0343] DAICEL CHIRALPAK AD (250mm*30mm, 10μm), Condition: 0.1% NH3·H2O ETOH, Begin B: 40%; End B: 40%; Flow Rate (ml / min): 60)] yielded title compound isomer 1 (25.2 mg, yield: 17%) and title compound isomer 2 (22 mg, yield: 15%).

[0344] Analytical methods

[0345] Column: DAICEL CHIRALCEL AD-3 (100mm*4.6mm, 3μm);

[0346] Mobile phase: A: CO2B: ethanol (0.05% DEA), Gradient: from 5% to 40% of B in2min and hold 40% for 1.2min, then 5% of B for 0.8min;

[0347] FlowRate: 4 mL / min;

[0348] ABPR: 1500psi;

[0349] Temperature: 35℃.

[0350] The compound with a retention time of 1.887 min was defined as isomer 2;

[0351] MS(ESI)m / z 478.1[M+H]+

[0352] 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)

[0353] The compound with a retention time of 2.078 min was defined as isomer 1;

[0354] MS(ESI) m / z 478.1 [M+H] +

[0355] 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)

[0356] Biological evaluation

[0357] The present invention will be further described and explained below with reference to test examples, but these embodiments are not intended to limit the scope of the present invention.

[0358] Test Example 1: Detection of the activity of the disclosed compound on cyclin-dependent kinases.

[0359] 1. Experimental Materials

[0360]

[0361]

[0362] Compound A is example A94 of WO 2019 / 207463A1, synthesized according to the method disclosed in that patent.

[0363] 2. Kinase activity assay (CDK4 / Cyclin D1, CDK6 / Cyclin D3): Mobility shift assay to measure kinase activity.

[0364] In vitro CDK kinase activity was tested using the Mobility Shift Assay. In the experiment, the initial concentration for testing the inhibitory effect of the test compound on CDK activity was 300 nM, diluted 3-fold, for a total of 10 concentrations, and tested in replicates. Staurosporine was used as a standard control.

[0365] Prepare 1-fold kinase buffer (CDK2) (50 mM HEPES, pH 7.5, 0.0015% Brij-35), 1-fold 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 an appropriate amount of kinase to the 1-fold kinase buffer to prepare a 2.5-fold enzyme solution; prepare a 5-fold dilution of the compound at the corresponding test concentration (1-fold kinase buffer, 10% DMSO); add an appropriate amount of FAM-labeled peptide and ATP to the 1-fold kinase buffer to prepare a 2.5-fold substrate solution. Add 5 μl of 5-fold compound dilution and 10 μl of 2.5-fold enzyme solution to the wells of a 384-well plate, mix well, and incubate at room temperature for 10 minutes. Then add 10 μl of 2.5-fold substrate solution to the 384-well plate and centrifuge at 1000 rpm for 1 minute. Incubate the plate at 28°C for 60 minutes (biochemical incubator model: SPX-100B-Z). Add 30 μl of stop solution to the 384-well plate to terminate the reaction and centrifuge at 1000 rpm for 1 minute. Finally, read the conversion data (excitation wavelength: 400 nm, emission wavelengths: 445 nm and 520 nm) on a Caliper EZ Reader II.

[0366] IC of the compound 50 The values ​​were fitted using XLFit Excel add-in version 5.4.0.8. Fitting formula:

[0367] Y=Bottom+(Top-Bottom) / (1+(IC50 / X)^HillSlope).

[0368] 3. Kinase activity assay (CDK1 / CyclinB, CDK9 / CyclinT1)

[0369] The in vitro CDK (CDK2, CDK9) kinase activity assay started at a concentration of 1 μM, was diluted 3-fold, and a total of 10 concentrations were obtained, with replicates performed. Compound PHA-793887 was used as a control compound.

[0370] Prepare 1x kinase reaction buffer (40mM Tris-HCl, pH 7.4, 20mM Mg2Cl2, 0.1mg / ml BSA, 50uM DTT), 1 volume of 5x kinase reaction buffer, and 4 volumes of water, adding DTT (final concentration 50uM). Using an Echo 655, transfer 50nL of the diluted compound working solution (DMSO final concentration 1%) to each well of a Greiner 784075 reaction plate. Seal the plate with sealing film and centrifuge at 1000g for 1 minute. Prepare 2x enzyme (0.3ng / uLCDK2 / CyclinE1 or CDK9 / CyclinT1) using 1x kinase reaction buffer. Add 2.5uL of the above kinase solution to each well, seal the plate with sealing film, centrifuge at 1000g for 1 minute, and incubate at room temperature for 10 minutes. A 2× kinase substrate and ATP mixture was prepared using 1× kinase reaction buffer. The 2× CDK2 / CylinE1 kinase substrate consisted of 0.4 mg / ml Histone H1 and 30 μM ATP. 2.5 μL of the 2× Histone H1 and ATP mixture was added to the reaction plate, and the plate was centrifuged at 1000g for 30 seconds to begin the reaction. After the kinase assay was performed at room temperature for 120 minutes, 4 μL of ADP-Glo ​​reagent was added, and the reaction was continued at room temperature for 40 minutes. Then, 8 μL of the kinase detection reagent was added, and the reaction was continued at room temperature for 40 minutes. The luminescence signal was read using an Envision 2104. Data analysis is as follows.

[0371] a) Inhibition percentage: %inhibition = 100 - (Signalcmpd - SignalAve_PC) / (SignalAve_VC - SignalAve_PC) × 100.

[0372] SignalAve_PC: The average value of all positive control wells on the entire board.

[0373] SignalAve_VC: The average value of all negative control wells on the entire board.

[0374] b) Compound IC50: Calculated using GraphPad 8.0 with the following nonlinear fitting formula.

[0375] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope))

[0376] X: Log value of compound concentration; Y: Percentage of compound inhibition

[0377] The biochemical inhibitory activity of the CDK (CDK1, CDK4, CDK6, CDK9) kinases of the disclosed compounds was determined by the above experiments, and the measured IC50 values ​​were... 50 The values ​​are shown in Tables 17 and 18.

[0378] Table 17.

[0379]

[0380] Table 18.

[0381]

[0382]

[0383] Test Example 2: CYP Inhibition Experiment

[0384] The representative substrate metabolism responses of the five major human CYP subtypes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) were evaluated using a mixture of 150 donor human liver microsomes (purchased from Corning, catalog number 452117). The effects of different concentrations of the analytes on the metabolic responses of phenacetin (CYP1A2), diclofenac sodium (CYP2C9), S-metphenytoin (CYP2C19), bufrolol hydrochloride (CYP2D6), and midazolam (CYP3A4 / 5) were determined by liquid chromatography-tandem mass spectrometry (LC / MS / MS).

[0385] 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) of 30 μM phenacetin, 10 μM diclofenac sodium, 35 μM S-mphenanthridine, 5 μM bromide hydrochloride, 3 μM midazolam, 1 mM NADPH, the test compound (concentrations of 0.1, 0.3, 1, 3, 10, and 30 μmol / L, respectively), a positive compound, or a blank control, and mixed human liver microsomes (0.2 mg / mL) was incubated at 37 °C for 5 min. Then, 200 μL of acetonitrile solution containing 3% formic acid and 40 nM internal standard verapamil was added, and the mixture was centrifuged at 4000 rpm for 50 min. The mixture was cooled on ice for 20 min, and then centrifuged at 4000 rpm for 20 min to precipitate the protein. 200 μL of the supernatant was analyzed by LC / MS / MS.

[0386] Peak area is calculated from the chromatogram. Residual activity percentage (%) is calculated using the following formula:

[0387] Peak area ratio = Metabolite peak area / Internal standard peak area

[0388] Residual activity percentage (%) = Peak area percentage of the test compound group / Peak area percentage of the blank group

[0389] CYP half-maximal inhibitory concentration (IC50) 50 This was calculated using Excel XLfit 5.3.1.3.

[0390] The measured CYP half-maximal inhibitory concentration (IC50) 50 The values ​​are shown in Table 19 below.

[0391] Table 19. Half-maximal inhibitory concentrations (IC50) of the compounds of the present invention against CYP 50 )

[0392]

[0393]

[0394] Test Example 3: Solubility Test Experiment

[0395] The thermokinetic solubility of the compound in phosphate buffer at pH 7.4 was determined. Sample supernatants and standards of known concentrations were analyzed by LC / MS / MS.

[0396] 1. Materials and reagents

[0397] Compound A (Compound A is example A94 of WO 2019 / 207463A1, synthesized according to the method disclosed in that patent).

[0398] NaH2PO4·2H2O (analytical grade), NaH2PO4 (analytical grade), NaOH (analytical grade).

[0399] 1.5mL flat-bottomed glass tube (BioTech Solutions); molded PTFE cap (BioTech Solutions); PTFE-coated stirring rod (BioTech Solutions); Eppendorf Comfort thermostatic mixer and 96-well deep-hole plate.

[0400] 2. Preparation of 0.01M sodium phosphate buffer at pH 7.4

[0401] Weigh 15.6g of NaH2PO4·2H2O and place it in a 1L glass bottle. Add 1L of deionized water to dissolve it. The pH of the solution is approximately 4.7. Then, adjust the pH to 7.4 using 10M NaOH.

[0402] 3. Solubility Measurement Process

[0403] Accurately weigh 1 mg of powder for each compound and place it in a glass tube. Add phosphate buffer (pH 7.4) to each glass tube at a rate of 1 mL per milligram. Add a stir bar to each glass tube, then cap the tube. Place the sample tray containing the glass tubes in an Eppendorf Comfort Mixer and incubate at 25°C and 1100 rpm for 24 hours. After incubation, open the cap, remove the stir bar using a magnet, and record the observations in each glass tube. Centrifuge the plate at 25°C and 4000 rpm for 30 minutes. Take 750 μL of the supernatant. Wash the pipette tip with acetonitrile for 5 seconds, then with pure water for 5 seconds. The first 50 μL of waste liquid was then discharged, and the remaining 700 μL was added to another 96-well sample dish containing glass tubes. The sample was then centrifuged for 30 minutes (25°C, 4000 rpm). 10 μL of the second centrifuged sample was added to 990 μL of a 1:1 mixture of acetonitrile and water containing the internal standard (100-fold sample). Another 10 μL of the diluted solution was added to 990 μL of the same 1:1 mixture (10,000-fold sample). The sample dilution factor may vary depending on solubility values ​​and LC / MS signal response.

[0404] Table 20. Records of Phenomena and Dilution Factors

[0405]

[0406]

[0407] 4. Preparation of Standards

[0408] Accurately weigh 1 mg of compound powder and add it to a glass tube. Add DMSO to each glass tube at a rate of 1 mL per milligram. Add a stir bar to each glass tube, then cap the tube. Place the dish containing the standard glass tubes in an Eppendorf Comfort thermostatic mixer and incubate at 25°C and 1,100 rpm for 2 hours to allow the powder to dissolve completely. Observe whether the solid is completely dissolved and record the amount of compound that is not completely dissolved in the DMSO solution. Add 10 μL of the 1 mg / mL standard to 990 μL of a mixture of acetonitrile and water (1:1) containing the internal standard to obtain a 10 μg / mL standard. Add 10 μL of the 10 μg / mL standard to 990 μL of a mixture of acetonitrile and water (1:1) containing the internal standard to obtain a 0.1 μg / mL standard. The sample dilution factor may vary depending on the LC / MS signal response. The samples were analyzed by LC / MS / MS. All compounds were tested individually.

[0409] 5. Data Calculation

[0410] All calculations were performed using Microsoft Excel. Samples were analyzed by LC / MS / MS, and quantification was performed using standards of known concentrations. The solubility of the analyte was calculated using the following formula: [Sample] = Area Ratio sample × DF sample × [STD] / Area Ratio STD

[0411] DF: Dilution factor.

[0412] Table 21. Solubility of some compounds in this invention

[0413]

[0414] Test Example 5: PXR Induction Experiment

[0415] 1. To evaluate the potential of the test compounds to induce the activity of drug-metabolizing enzymes through in vitro activation of PXR. EC50 values ​​of the test compounds at different concentrations (30, 10, 3.33, 1.11, 0.370, and 0.123 μM) were obtained by in vitro activation of PXR. Positive control rifampin concentrations were 20, 5, 1.25, 0.312, 0.0781, and 0.195 μM.

[0416] 2. Materials and Reagents

[0417] 1) DPX2 cells (human PXR gene and fluorescent reporter gene stably transfected into HepG2 cells) were purchased from Puracyp (Carlsbad, CA).

[0418] 2) The client provided the compound to be tested, and the control drug (rifampin) was purchased from Sigma (St. Louis, MO).

[0419] 3) CellTiter-Fluor TM Cell viability assay kits and One-Glo fluorescence assay kits were purchased from Promega (Madison, WI), fetal bovine serum (FBS) was purchased from Corning (Manassas, VA), MTS3 shakers were purchased from IKA Labortechnik (Staufen, Germany), DMEM, penicillin, and streptomycin were purchased from local supplier Hygromycin B, and G418 was purchased from Merck (Darmstadt, Germany). Cell culture medium and DPX2 cells were purchased from Puracyp Inc.

[0420] 3. Experimental Procedure

[0421] 3.1 Seed Board Preparation

[0422] 1) Add 50 mL of FBS to 450 mL of cell culture medium.

[0423] 2) DPX2 cells were cultured in T-75 culture flasks at 37°C, 5% CO2, and 95% relative humidity. The cells were digested when they reached 80-90% confluence with the bottom of the culture flask.

[0424] 3) Wash the surface of T-75 cultured cells with 8 mL PBS, discard the PBS, add 3 mL trypsin, digest at 37°C for about 5 minutes, or until the cells are digested and suspended in the trypsin, then add 10 mL of excess serum-containing culture medium to neutralize the trypsin.

[0425] 4) Transfer the cell suspension to a conical-bottom centrifuge tube and centrifuge at 120g for 10 minutes. Resuspend the cells in seeding medium and adjust the concentration to 4 x 10⁻⁶. 5 cells / mL. Add 100 μL of diluted cells to each well of a 96-well cell culture plate. Place the culture plate in an incubator and incubate at 37°C for 24 hours, then prepare for the PXR activation experiment.

[0426] 3.2 Chemical Dosing Treatment

[0427] 1) Prepare the test compound and positive compound (rifampin) using DMSO, and dilute the compound with serum-free medium at 37°C. The final concentrations of the positive control rifampin were 20, 5, 1.25, 0.312, 0.0781, and 0.195 μM, and the final concentrations of the test compound were 30, 10, 3.33, 1.11, 0.370, and 0.123 μM. The final concentration of DMSO was 0.1%. 1 μL of DMSO was added to 1 mL of pre-incubated medium as a solvent control.

[0428] 2) Remove the cell culture plate from the incubator, discard the culture medium, add 100 μL of the test compound and positive compound to the appropriate wells, two in a row for each group, and incubate the cell plate in the incubator for 24 hours.

[0429] 3.3 Quantitative determination of PXR activation

[0430] 1) Two days after drug treatment, the culture can be used for quantitative detection of PXR activation.

[0431] 2) CellTiter-Fluor TM The cell viability assay kit and One-Glo Luciferase reagent were equilibrated to room temperature. 10 μL of GF-AFC substrate was added to Assay Buffer (10 mL) to form a 2X reagent, and then diluted to 1X with 10 mL of PBS; ONE-Glo substrate was added to ONE-Glo Luciferase Assay Buffer.

[0432] 3) Remove the cell culture plate from the incubator, discard the culture medium from each well, wash twice with PBS, and add 1X CellTiter-Fluor... TM Add the reagent to the sterile sample container, and use a multi-pipette to add 50 μL to each well. Incubate at 37°C for 30 minutes.

[0433] 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.

[0434] 5) Pour the ONE-Glo reagent into the sample container, then use a multi-channel pipette to add 50 μL to each well. Gently mix the reagent and incubate at room temperature for 5 minutes. After incubation, use a photometer to read the luminescence value of each well.

[0435] 4. Calculation of cell induction value

[0436] 4.1 Cell viability

[0437] Formula for calculating cell viability:

[0438] Percent cell viability(%)=I(sample) / (I(vehicle)x100

[0439] I(sample) is the fluorescence intensity of the sample, and I(vehicle) refers to the fluorescence intensity of 0.1% DMSO on the cells.

[0440] 4.2 Calculation of Cell Induction Value

[0441] All data was calculated using Microsoft Excel.

[0442] Luciferase activity is represented by RFU / RLU, where RLU is the average luminescence intensity of two parallel samples for each concentration of each compound, and RFU is the average fluorescence intensity of two parallel samples for each concentration of each compound.

[0443] Formula for calculating induction fold:

[0444]

[0445] Table 22. Partially measured PXR induction values

[0446]

Claims

1. The X-ray powder diffraction pattern 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, as represented by the diffraction angle 2θ, shows characteristic peaks at 9.6, 10.0, 11.7, 15.0, 21.1, and 21.7, wherein the error range of the 2θ values ​​is ±0.

2. 。 2. The crystal form as described in claim 1, wherein the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ 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.

3. The crystal form as described in claim 1, wherein the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ 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.

4. The X-ray powder diffraction pattern of the crystal form as described in claim 1, expressed in terms of the diffraction angle 2θ, is shown in Figure 2.

5. The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, of the B 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, at 7.4, 9.5, and 11. Characteristic peaks are found at positions 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. The error range of the 2θ values ​​is ±0.

2. 。 6. The X-ray powder diffraction pattern of the crystal form as described in claim 5, expressed in terms of the diffraction angle 2θ, is shown in Figure 3.

7. The C-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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol), expressed as a diffraction angle 2θ, has characteristic peaks at 4.8, 7.6, 10.1, 12.0, 15.0, 19.7, 21.2, and 23.5, wherein the error range of the 2θ values ​​is ±0.

2. 。 8. The X-ray powder diffraction pattern of the crystal form as described in claim 7, expressed in terms of the diffraction angle 2θ, is shown in Figure 4.

9. 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol), expressed as a diffraction angle 2θ, has characteristic peaks at 10.0, 10.5, 17.0, 18.7, and 23.9, wherein the error range of the 2θ values ​​is ±0.

2. 。 10. The crystal form as described in claim 9, wherein the X-ray powder diffraction pattern 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.

11. The crystal form as described in claim 9, wherein the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ 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.

12. The crystal form as described in claim 9, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is shown in Figure 5.

13. The E 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol), expressed as an X-ray powder diffraction pattern with a diffraction angle of 2θ, has characteristic peaks at 4.8, 11.4, 14.4, 15.1, 16.4, 19.3, and 22.9, wherein the error range of the 2θ values ​​is ±0.

2. 。 14. The crystal form as described in claim 13, wherein the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ 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.

15. The crystal form as described in claim 13, wherein the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ 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.

16. The X-ray powder diffraction pattern of the crystal form as described in claim 13, expressed in terms of the diffraction angle 2θ, is shown in Figure 6.

17. The F-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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol), expressed as a diffraction angle 2θ, has characteristic peaks at 11.1, 11.4, 14.3, 15.1, 15.7, 19.2, and 22.0, wherein the error range of the 2θ values ​​is ±0.

2. 。 18. The crystal form as described in claim 17, wherein the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ 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.

19. The crystal form as described in claim 17, wherein the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ 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.

20. The X-ray powder diffraction pattern of the crystal form as described in claim 17, expressed in terms of the diffraction angle 2θ, is shown in Figure 7.

21. The G 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol), expressed as an X-ray powder diffraction pattern in terms of diffraction angle 2θ, has characteristic peaks at 11.3, 15.3, 16.1, 17.9, 18.6, and 20.8, wherein the error range of the 2θ values ​​is ±0.

2. 。 22. The crystal form as described in claim 21, wherein the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ 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.

23. The crystal form as described in claim 21, wherein the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ 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.

24. The crystal form as described in claim 21, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is shown in Figure 8.

25. The H-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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol), expressed as a diffraction angle 2θ, has characteristic peaks at 11.7, 12.3, 15.5, 19.4, and 22.3, wherein the error range of the 2θ values ​​is ±0.

2. 。 26. The crystal form as described in claim 25, wherein the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ 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.

27. The crystal form as described in claim 25, wherein the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ 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.

28. The crystal form as described in claim 25, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is shown in Figure 9.

29. A method for preparing the A 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to any one of claims 1-4, comprising the following steps: Method 1 a) Dissolve the compound shown in formula (I) in solvent 1; b) Add solvent 2 to precipitate crystals; Solvent 1 is selected from ketone solvents or ester solvents, solvent 2 is an ether solvent, 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 shown in formula (I) is dissolved in solvent 3 and stirred to crystallize. Solvent 3 is selected from ester solvents and ketone solvents. The ester solvent is selected from ethyl acetate and isopropyl acetate. The ketone solvent is methyl isobutyl ketone.

30. A method for preparing the B-phase 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol) according to any one of claims 5-6, comprising the following steps: The compound shown in formula (I) is dissolved in a substituted lower alkane and stirred to crystallize; the substituted lower alkane is nitromethane.

31. A method for preparing the C-phase 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, of formula (I) according to any one of claims 7-8, selected from the following methods: Method 1 The compound shown in formula (I) is dissolved in solvent 4 and volatilized to crystallize. Solvent 4 is selected from substituted lower alkanes or ketone solvents. The substituted lower alkanes are selected from dichloromethane or 1,2-dichloroethane. The ketone solvent is methyl isobutyl ketone. Method 2 The compound shown in formula (I) is dissolved in solvent 5 and stirred to crystallize. Solvent 5 is selected from aromatic hydrocarbon solvents; the aromatic hydrocarbon solvent is selected from p-xylene or toluene. Method 3 a) Dissolve the compound shown in formula (I) in solvent 6; b) Add solvent 7 to precipitate crystals; Solvent 6 is selected from alcohol solvents, ketone solvents, nitrile solvents, and ether solvents; solvent 7 is an ether solvent; alcohol solvent is selected from isopropanol or ethanol; ketone solvent is acetone; nitrile solvent is acetonitrile; and ether solvent is selected from tetrahydrofuran or isopropyl ether.

32. A method for preparing the D-phase 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol) according to any one of claims 9-12, comprising the following steps: The compound shown in formula (I) is dissolved in solvent 8 and stirred to crystallize. Solvent 8 is an ether solvent or a mixture of an alcohol solvent and an ether solvent. The alcohol solvent is methanol, ethanol or isopropanol, and the ether solvent is isopropyl ether.

33. A method for preparing the E 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, of formula (I) according to any one of claims 13-16, selected from the following methods: Method 1 The compound shown in formula (I) is dissolved in solvent 9 and stirred to crystallize. Solvent 9 is a nitrile solvent, and the nitrile solvent is acetonitrile. Method 2 a) Dissolve the compound shown in formula (I) in 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; the ketone solvent is acetone, and the ether solvent is isopropyl ether.

34. A method for preparing the F-phase 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, of formula (I) according to any one of claims 17-20, selected from the following methods: Method 1 The compound shown in formula (I) is dissolved in solvent 12 and stirred to crystallize. Solvent 12 is a ketone solvent; the ketone solvent is methyl isobutyl ketone. Method 2 The compound shown in formula (I) was dissolved in tetrahydrofuran, and after the solution was cleared, isopropyl ether and F-type seed crystals were added to precipitate crystals.

35. A method for preparing the G-phase 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol according to any one of claims 21-24, comprising the following steps: The compound shown in formula (I) is dissolved in a mixed solvent of an alcohol solvent and an ether solvent, and stirred to crystallize; the alcohol solvent is selected from methanol, and the ether solvent is isopropyl ether.

36. A method for preparing the H-phase 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-diazanephrine-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, of formula (I) according to any one of claims 25-28, selected from the following methods: Method 1 The compound shown in formula (I) is dissolved in solvent 13, and stirred to induce crystallization. Solvent 13 is selected from alcohol solvents. The solvents include mixed solvents of alcohols and water, ketone solvents, and ester solvents; wherein the alcohol solvents are selected from methanol, ethanol, isopropanol, and n-propanol, the ketone solvents are selected from acetone, 2-butanone, and methyl isobutyl ketone, and the ester solvent is ethyl acetate; Method 2 The compound shown in formula (I) is mixed with solvent 14, and the mixture is slurried and crystallized. Solvent 14 is selected from water and ether solvents. The ether solvent is selected from methyl tert-butyl ether or isopropyl ether; Method 3 a) Dissolve the compound shown in formula (I) in solvent 15; b) Add solvent 16 to precipitate crystals; Solvent 15 is selected from alcohol solvents, ketone solvents, and ether solvents, and solvent 16 is selected from lower solvents. The solvents are alkanes, ethers, or water; 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.

37. A pharmaceutically acceptable salt 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-diazanephrine-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, wherein the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, methanesulfonate, p-toluenesulfonate, phosphate, citrate, or malate. 。 38. A method for preparing a pharmaceutically acceptable salt 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-diazaacenaphthene-6-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol, 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 benzoic acid. Fruit acids.

39. A pharmaceutical composition comprising the crystal form or compound of any one of claims 1-28 or 37, and optionally a pharmaceutically acceptable excipient.

40. A method for preparing a pharmaceutical composition, comprising the step of mixing the crystal form or compound of any one of claims 1-28 or 37 with a pharmaceutically acceptable excipient.

41. Use of the crystal form or compound of any one of claims 1-28 or 37, or the composition of claim 39, in the preparation of a medicament for the treatment or prevention of diseases related to cyclin-dependent kinases.

42. Use of the crystal form or compound of any one of claims 1-28 or 37, or the composition of claim 39, in the preparation of a medicament for treating or preventing cancer.

43. The use according to claim 42, wherein 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, stomach cancer, and thyroid cancer.

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