A crystalline form of a triazolopyridine-substituted indazole compound and a method of making the same

Abstract

A crystal form of a triazolopyridine-substituted indazole compound and a preparation method thereof, and specifically discloses a crystal form of a compound shown in formula (I) and a preparation method thereof.

Description

[0001] This invention claims the following priority:

[0002] CN202110722010.6, application date: June 28, 2021. Technical Field

[0003] This invention relates to a crystal form of a triazolopyridine-substituted indazole compound and a method for preparing the same, specifically to the crystal form of the compound shown in formula (I) and a method for preparing the same. Background Technology

[0004] Rheumatoid arthritis (RA) is a chronic inflammatory, systemic autoimmune disease. Early joint manifestations of RA are often difficult to distinguish from other types of inflammatory arthritis. More characteristic signs of RA include joint erosion, rheumatoid nodules, and other extra-articular manifestations. RA affects women more than men (3:1), and the age of onset is between 30 and 55 years.

[0005] The pathogenesis of rheumatoid arthritis is very complex. It mainly involves the presentation of autoantigens by major histocompatibility complex II (MHC-II) positive antigen-presenting cells (APCs) to activated CD4+ T cells, which initiates a specific immune response. At the same time, activated T cells, macrophages and other cells migrate to the synovium, which increases the secretion of various inflammatory cytokines such as TNFα, IL-1 and IL-6, which infiltrate the synovial joint and lead to the corresponding arthritis symptoms.

[0006] Glucocorticoids (GCs) have been widely used to treat inflammatory and immune diseases for decades, including rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), osteoarthritis, rheumatic fever, allergic rhinitis, systemic lupus erythematosus, Crohn's disease, inflammatory bowel disease, and ulcerative colitis.

[0007] Glucocorticoids (GCs) bind to glucocorticoid receptors (GRs), enter the cell nucleus, and influence gene transcription (activation and inhibition), reducing the production of inflammatory factors. Glucocorticoid receptors are members of the conserved nuclear receptor superfamily, belonging to the nuclear transcription factor family. They are widely distributed in various tissues and cells of the body, and almost all cells are their target cells, playing an important regulatory role in the body's development, growth, metabolism, and immune function. GCs often have serious and irreversible side effects, such as osteoporosis, hyperglycemia, diabetes, hypertension, muscle atrophy, and Cushing's syndrome, severely limiting the use of GCs in chronic diseases.

[0008] Examples of GR ligands have been found that can selectively induce transcriptional repression without significant transcriptional activation, reducing the risk of systemic side effects while maintaining anti-inflammatory activity. These are termed selective glucocorticoid receptor modulators (SGRMs). Unlike GC, SGRMs, when binding to GR, induce complete transcriptional repression with only partial transcriptional activation, thus controlling related side effects while maintaining anti-inflammatory activity. Summary of the Invention

[0009] This invention provides the Q crystal form of the compound of formula (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 10.301±0.200°, 17.459±0.200°, 19.141±0.200°.

[0010]

[0011] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned Q crystal form has characteristic diffraction peaks at the following 2θ angles: 10.301±0.200°, 12.779±0.200°, 16.860±0.200°, 17.459±0.200°, 19.141±0.200°, 20.701±0.200°, 21.701±0.200°, 24.400±0.200°.

[0012] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned Q crystal form has characteristic diffraction peaks at the following 2θ angles: 8.060±0.200°, 10.301±0.200°, 12.779±0.200°, 16.860±0.200°, 17.459±0.200°, 17.870±0.200°, 18.639±0.200°, 19.141±0.200°, 20.701±0.200°, 21.701±0.200°, 22.579±0.200°, 24.400±0.200°.

[0013] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned Q-type crystal has characteristic diffraction peaks at the following 2θ angles: 3.861±0.200°, 8.060±0.200°, 9.621±0.200°, 10.301±0.200°, 11.500±0.200°, 12.779±0.200°, 16.860±0. 200°, 17.459±0.200°, 17.870±0.200°, 18.639±0.200°, 19.141±0.200°, 20.701±0.200°, 21.701±0.200°, 22.579±0.200°, 24.400±0.200°, 24.939±0.200°.

[0014] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned Q crystal form has characteristic diffraction peaks at the following 2θ angles: 3.322°, 3.861°, 4.899°, 6.421°, 7.440°, 8.060°, 8.722°, 9.621°, 10.301°, 11.500°, 12.779°, 13.197°, 14.321°, 15.041°, 15.859°, 16.860°, 17.459°, 17.870°, 18.639°, 19.141°, 20.701°, 21.701°, 22.579°, 24.400°, 24.939°, 25.700°, 26.602°, 27.201°.

[0015] This invention provides the Q crystal form of the compound of formula (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 10.301±0.200°, 17.459±0.200°, 19.141±0.200°, and also at 24.400±0.200°, and / or 16.860±0.200°, and / or 12.779±0.200°, and / or 21.701±0.200°, and / or 20.701±0.200°, and / or 8.060±0.200°, and / or 22.579±0.200°, and / or 17.870±0.200°, and / or 18.639±0.200°, and / or 3.861±0.200°. °, and / or 24.939±0.200°, and / or 9.621±0.200°, and / or 11.500±0.200°, and / or 6.421±0.200°, and / or 4.899±0.200°, and / or 26.602±0.200°, and / or 3.322±0.200°, and / or 7.440 ±0.200°, and / or 13.197±0.200°, and / or 25.700±0.200°, and / or 14.321±0.200°, and / or 8.722±0.200°, and / or 15.859±0.200°, and / or 27.201±0.200°, and / or 15.041±0.200°.

[0016] In some embodiments of the present invention, the Q crystal form of the above-mentioned compound has the following XRPD pattern: Figure 1 As shown.

[0017] In some embodiments of the present invention, the XRPD spectrum analysis data of the above-mentioned Q crystal form are shown in Table 1.

[0018] Table 1. XRPD spectrum analysis data of compound Q crystal form (I)

[0019]

[0020]

[0021] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned Q crystal form has an initial value of an endothermic peak at 181.4℃±5℃.

[0022] In some embodiments of the present invention, the DSC spectrum of the above-mentioned Q crystal form is as follows: Figure 2 As shown.

[0023] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned Q crystal form shows a weight loss of 1.47% at 150℃±3℃.

[0024] In some embodiments of the present invention, the TGA pattern of the above-mentioned Q crystal form is as follows: Figure 3 As shown.

[0025] The present invention also provides a method for preparing the Q crystal form of compound (I), comprising:

[0026] (a) Add the compound of formula (I) to a solvent to form a suspension;

[0027] (b) Stir at 50–55°C for 96–120 hours;

[0028] (c) After filtration, vacuum dry for 8–16 hours;

[0029] The solvent is methyl isobutyl ketone and n-heptane in a volume ratio of 1:6.

[0030] The present invention also provides the S-crystal form of the compound of formula (I), whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.019±0.200°, 19.579±0.200°, 20.262±0.200°.

[0031]

[0032] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned S-type crystal has characteristic diffraction peaks at the following 2θ angles: 11.999±0.200°, 13.019±0.200°, 14.961±0.200°, 18.182±0.200°, 19.579±0.200°, 20.262±0.200°, 22.939±0.200°, 24.021±0.200°.

[0033] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned S-type crystal has characteristic diffraction peaks at the following 2θ angles: 3.201±0.200°, 11.463±0.200°, 11.999±0.200°, 13.019±0.200°, 14.523±0.200°, 14.961±0.200°, 18.182±0.200°, 19.579±0.200°, 20.262±0.200°, 22.939±0.200°, 24.021±0.200°, 25.738±0.200°.

[0034] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned S-type crystal has characteristic diffraction peaks at the following 2θ angles: 11.463±0.200°, 11.999±0.200°, 13.019±0.200°, 14.523±0.200°, 14.961±0.200°, 18.182±0.200°, 19.579±0.200°, 20.262±0.200°, 22.939±0.200°, 24.021±0.200°, 25.738±0.200°.

[0035] In some embodiments of the present invention, the XRPD pattern of the above-mentioned S-crystal form is as follows: Figure 5 As shown.

[0036] In some embodiments of the present invention, the XRPD spectra analysis data of the above-mentioned S-crystal form are shown in Table 2:

[0037] Table 2 shows the XRPD spectrum analysis data of compound S crystal form (I).

[0038]

[0039]

[0040] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned S-type crystal has an endothermic peak at 114.9℃±3℃.

[0041] In some embodiments of the present invention, the DSC spectrum of the above-mentioned S-crystal form is as follows: Figure 6 As shown.

[0042] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned S-type crystal shows a weight loss of 0.99% at 90.0℃±3℃ and a weight loss of 2.54% at 120.0℃±3℃.

[0043] In some embodiments of the present invention, the TGA pattern of the above-mentioned S-crystal form is as follows: Figure 7 As shown.

[0044] The present invention also provides a method for preparing the S-crystal form of compound (I), comprising:

[0045] (a) Add the compound of formula (I) to a solvent to form a suspension;

[0046] (b) The suspension gradually becomes clear after stirring at 80-85°C for 2-3 hours;

[0047] (c) Slowly cool to 20-25℃, and a white solid will precipitate;

[0048] (d) After filtration, vacuum dry for 8–16 hours;

[0049] The solvent is selected from ethyl acetate.

[0050] The present invention also provides the use of the above-described Q crystal form and S crystal form, or the Q crystal form and S crystal form prepared according to the above method, in the preparation of drugs for treating rheumatoid arthritis.

[0051] Technical effect

[0052] The compounds of this invention can bind to glucocorticoid receptors, exhibiting both good gene transcription inhibitory activity and general gene transcription activating activity. While enhancing cellular anti-inflammatory activity, they reduce side effects such as hyperglycemia and osteoporosis. Furthermore, they possess good pharmacokinetic properties and oral absorption rates, making them suitable for the treatment of rheumatoid arthritis. The compounds of this invention are crystal stable, have good hygroscopicity, and are minimally affected by light and heat.

[0053] Definitions and Explanations

[0054] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular phrase or term should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0055] The intermediate compounds of the present invention can be prepared by various synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention.

[0056] The chemical reactions in the specific embodiments of this invention are carried out in a suitable solvent, which must be suitable for the chemical changes of this invention and the reagents and materials required therefor. To obtain the compounds of this invention, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0057] The structures of the compounds of this invention can be confirmed by conventional methods well known to those skilled in the art. If this invention relates to the absolute configuration of a compound, that absolute configuration can be confirmed by conventional techniques in the art. For example, single-crystal X-ray diffraction (SXRD) is used, where the cultured single crystal is used to collect diffraction intensity data using a Bruker D8 venture diffractometer with CuKα radiation as the light source. The scanning method is as follows: After scanning and collecting relevant data, the crystal structure can be further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0058] Unless otherwise specified, the exothermic direction of the DSC spectrum is upward.

[0059] The present invention will be described in detail below through embodiments, which are not intended to limit the present invention in any way.

[0060] All solvents used in this invention are commercially available and can be used without further purification.

[0061] The following abbreviations are used in this invention: PhMgBr represents magnesium phenyl bromide; (i-PrO)3Al represents aluminum isopropoxide; T3P represents tri-n-propyl cyclic phosphoric anhydride; Cs2CO3 represents cesium carbonate; K2CO3 represents potassium carbonate; i-PrOH represents isopropanol; MeCN represents acetonitrile; DCM represents dichloromethane; THF represents tetrahydrofuran; EtOAc represents ethyl acetate; MeOH represents methanol; 1,4-dioxane represents 1,4-dioxane; MIBK represents methyl isobutyl ketone; n-Heptane represents n-heptane.

[0062] Compounds are named according to conventional naming principles in this field or Software naming conventions are used; commercially available compounds use supplier catalog names.

[0063] The present invention relates to a powder X-ray diffractometer (XRPD) method.

[0064] Instrument Model: DX-2700BH X-ray Diffractometer

[0065] Test method: Approximately 10–20 mg of sample is used for XRPD detection.

[0066] The detailed XRPD parameters are as follows:

[0067] Optical tube: Cu, kα,

[0068] Phototube voltage: 40kV, Phototube current: 40mA

[0069] Diverging slit: 1mm

[0070] Detector slit: 0.3mm

[0071] Anti-scattering slit: 1mm

[0072] Scan range: 3-40 degrees

[0073] Step size: 0.02 deg

[0074] Scan time: 0.5s

[0075] This invention relates to a differential scanning calorimeter (DSC) method.

[0076] Instrument Model: TA Instruments Discovery DSC 2500 Differential Scanning Calorimeter

[0077] Take a sample (1-5 mg) and place it in a covered aluminum crucible under the protection of dry nitrogen at 50 mL / min for testing. The method is as follows: heat to the set test temperature at 25℃, with a heating rate of 10℃ / min.

[0078] The present invention relates to a thermogravimetric analysis (TGA) method.

[0079] Instrument Model: TA Discovery TGA 5500 Thermogravimetric Analyzer

[0080] Test method: Take a sample (2-5 mg) and place it in a platinum pot under the protection of dry nitrogen at 25 mL / min for testing. The method is: room temperature to 350℃, heating rate is 10℃ / min.

[0081] This invention presents a method for dynamic vapor adsorption analysis (DVS).

[0082] Instrument Model: SMS (Surface Measurement Systems) Dynamic Steam Adsorption Analyzer

[0083] Test conditions: Take a sample (10-15 mg) and place it in the DVS sample tray for testing.

[0084] The detailed DVS parameters are as follows:

[0085] Temperature: 25℃

[0086] Equilibrium: dm / dt = 0.002% / min (shortest: 10min, longest: 180min)

[0087] Drying: Dry at 0% RH for 120 min

[0088] RH (%) test steps:

[0089] 10% (0%RH-90%RH, 90%RH-0%RH)

[0090] 5% (90%RH-95%RH, 95%RH-90%RH)RH(%)

[0091] RH (%) test range: 0%-95%

[0092] The classification of hygroscopicity is shown in Table 3:

[0093] Table 3. Classification of Hygroscopicity Evaluation

[0094] Hygroscopic classification ΔW% deliquescence Absorbs sufficient water to form a liquid Highly hygroscopic ΔW% ≥ 15% Hygroscopic 15% > ΔW% ≥ 2% Slightly hygroscopic 2% > ΔW% ≥ 0.2% None or almost none of the hygroscopic properties ΔW% < 0.2%

[0095] Note: ΔW% represents the moisture gain of the test sample at 25±1℃ and 80±2%RH.

[0096] X-ray single crystal diffraction method of the present invention

[0097] Instrument Model: Single Crystal X-ray Diffractometer (SC-XRD) (D8 VENTURE)

[0098] Test method: The sample was dissolved in 1.5 mL of ethyl acetate at room temperature. The sample solution was placed in a 1 mL semi-sealed centrifuge tube and left to stand in a dark and vibration-proof place. It was allowed to evaporate slowly at room temperature. Colorless needle-like crystals were obtained on the third day. The diffraction experiment temperature was T = 99.99(11) K.

[0099] Instrument parameters:

[0100] Bruker D8 VENTURE CMOS Photon II diffractometer with helios mxmultilayer monochromator.

[0101] Cryogenic system: Oxford Cryostream 800

[0102] Light source: Cu 2.5kW

[0103] - Distance from crystal to CCD detector: d = 45mm

[0104] Pipe pressure: 50kV

[0105] Pipe current: 50mA Attached Figure Description

[0106] Figure 1 The XRPD spectrum of Cu-Kα radiation for the Q crystal form of compound (I) is shown.

[0107] Figure 2 The DSC spectrum of the Q crystal form of compound (I) is shown.

[0108] Figure 3 The TGA spectrum of compound Q of formula (I) is shown.

[0109] Figure 4 The DVS spectrum of the Q crystal form of compound (I) is shown.

[0110] Figure 5The Cu-Kα radiation XRPD spectrum of the S crystal form of compound (I) is shown below.

[0111] Figure 6 The DSC spectrum of the S-crystal form of compound (I) is shown.

[0112] Figure 7 The TGA spectrum of the S-crystal form of compound (I) is shown.

[0113] Figure 8 The DVS spectrum of the S crystal form of compound (I) is shown.

[0114] Figure 9 The ellipsoid diagram of the three-dimensional structure of compound (I);

[0115] Figure 10 The diagram shows the ellipsoidal structure of a single-cell bimolecular compound of formula (I). Detailed Implementation

[0116] The present invention will be described in detail below with reference to embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope thereof.

[0117] Example 1: Preparation of compound (I)

[0118]

[0119]

[0120] Step 1: Synthesis of Compound 5A

[0121] Parallel four-pot reaction: Compound 5B (200 g) was added to a dry three-necked flask, dissolved in dichloromethane (2 L), and p-toluenesulfonic acid (15.59 g) was added with stirring. 2,3-Dihydropyran (206.82 g) was slowly added dropwise. After the addition was complete, the reaction was stirred at 20°C for 16 hours. After the reaction was complete, the four reaction solutions were combined and processed. The reaction solution was added to a saturated sodium bicarbonate solution (8 L), separated, and the aqueous phase was extracted with dichloromethane (2 L × 2). The organic phases were combined, washed with saturated brine (8 L), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 1 / 0 to 5 / 1, v / v) to obtain compound 5A. 1H NMR (400MHz, CDCl3) δppm 8.05-8.12 (m, 1H) 7.95 (d, J = 0.4Hz, 1H) 7.58-7.65 (m, 1H) 7.36-7.44 (m, 1H) 5.66-5.75 (m, 1H )3.96-4.06(m,1H)3.68-3.79(m,1H)2.48-2.60(m,1H)2.02-2.21(m,2H)1.66-1.79(m,3H).

[0122] Step 2: Synthesis of Compound 2

[0123] Dichloromethane (15 L) was added to a dry 50 L reactor. Compound 1 (1.5 kg) was added in batches, and the mixture was stirred until dissolved. N,N'-carbonyldiimidazole (1.4 kg) was then added in batches. After the addition was complete, the mixture was stirred at 20 °C for 4 hours. N-methylmorpholine (1.0 kg) was added to the reaction system, followed by compound 1A (0.84 kg) in batches. After the addition was complete, the mixture was stirred at 20 °C for 12 hours. After the reaction was complete, the reaction mixture was transferred to a separatory funnel, and water (20 L) was added. The mixture was separated, and the organic phase was collected. The organic phase was washed sequentially with saturated citric acid aqueous solution (20 L × 3), saturated sodium bicarbonate aqueous solution (20 L × 2), and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 2.

[0124] Step 3: Synthesis of Compound 3

[0125] Anhydrous tetrahydrofuran (16 L) was added to a 50 L reactor, and stirring was started. Compound 2 (1.6 kg) was then added. The system was cooled to -10 °C, and isopropyl magnesium chloride (574.4 g, 2 mol / L tetrahydrofuran solution) was added dropwise between -10 and -5 °C. After the addition was complete, the system was stirred at -10 °C for 0.5 hours. Phenyl magnesium bromide (1672 g, 3 mol / L diethyl ether solution) was added dropwise between -10 and -5 °C. After the addition was complete, the system was heated to 25 °C and stirred for 12 hours. After the reaction was complete, the reaction solution was slowly quenched in a saturated ammonium chloride solution (15 L) and ice water (3 L). The mixture was separated, and the organic phase was collected. The aqueous phase was extracted with ethyl acetate (10 L × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 3.

[0126] Step 4: Synthesis of Compound 4

[0127] Isopropanol (15 L) was added to a 50 L reactor, followed by a 2 L isopropanol solution of compound 3 (1.7 kg). Aluminum isopropoxide (625.88 g) was then added, resulting in a gray suspension. After the addition was complete, the reaction mixture was heated to 50 °C and stirred for 12 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to remove most of the isopropanol. The residue was dissolved in ethyl acetate (5 L), and the pH was adjusted to 3–4 with a saturated citric acid aqueous solution (20 L). Extraction was then performed with ethyl acetate (10 L × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a red oily compound 4.

[0128] Step 5: Synthesis of the hydrochloride salt of compound 5

[0129] Compound 4 (1.5 kg) was added in portions to hydrochloric acid / ethyl acetate (4 mol / L, 12 L), and the reaction mixture was stirred at 20 °C for 16 hours. After the reaction was complete, methyl tert-butyl ether (10 L) was added to the reaction system, and the mixture was stirred for 2 hours, resulting in the precipitation of a large amount of solid. The mixture was filtered, and the filter cake was washed with methyl tert-butyl ether (5 L) in portions. The filter cake was collected and dried under vacuum to obtain the hydrochloride salt of compound 5 as a white solid. 1 H NMR(400MHz, DMSO-d6)δppm8.08(br s, 3H) 7.41-7.45 (m, 2H) 7.36 (t, J = 7.45Hz, 2H) 7.25-7.31 (m, 1H) 6.09 (d, J = 3.95Hz , 1H) 5.02 (t, J=3.51Hz, 1H) 1.66-1.77 (m, 1H) 0.91-0.95 (m, 3H) 0.85-0.89 (m, 3H).

[0130] Step 6: Synthesis of Compound 6

[0131] Ten parallel batches of reaction: Compound 5 hydrochloride (100 g) was added to acetonitrile (1.5 L), followed by cesium carbonate (302.1 g), then compound 5A (101.4 g) and N,N-dimethylglycine (31.9 g). The mixture was purged with nitrogen three times. Finally, cuprous iodide (11.8 g) was added, and the mixture was purged with nitrogen three times. The mixture was stirred in an oil bath at 110 °C for 72 hours. After the reaction was complete, the reaction solution was cooled to room temperature, and the 10 batches of reaction were combined. The reaction solution was filtered, and the filter cake was washed several times with ethyl acetate (5 L). The filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in methyl tert-butyl ether (10 L), and then saturated citric acid aqueous solution (15 L) was added. After stirring, the mixture was separated. The aqueous phase was extracted with methyl tert-butyl ether (10 L), and the aqueous phase was retained while the organic phase was discarded. The aqueous phase was adjusted to pH 7–8 with sodium hydroxide aqueous solution (1 mol / L), and then further adjusted to pH 8–9 with ammonia. Extraction was then performed with ethyl acetate (10 L × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was purified by column chromatography (eluent: n-heptane / ethyl acetate = 1 / 1–0 / 1) to give a red solid compound 6.

[0132] Step 7: Synthesis of Compound 7

[0133] Parallel two-pot reaction: Ethyl acetate (2 L) was added to a dry 5 L three-necked flask, followed by compound 6 (250 g) and compound 6A (79.75 g). The reaction mixture was cooled to below 10 °C using an ice-water bath, and 628.83 g of 50% ethyl acetate solution of tri-n-propyl cyclophosphine was added dropwise in portions, maintaining a temperature T < 10 °C. Finally, 133.27 g of N-methylmorpholine was added dropwise, maintaining a temperature T < 10 °C. After the addition was complete, the reaction mixture was heated to 20 °C and stirred for 16 hours. After the reaction was complete, the two pots of reaction were combined. The reaction mixture was added to water (4 L), stirred, and separated. The aqueous phase was extracted with ethyl acetate (2 L × 2). The organic phases were combined, washed with saturated sodium bicarbonate aqueous solution (4 L) and saturated brine (4 L), respectively, and concentrated to dryness under reduced pressure to obtain a pale yellow solid compound 7.

[0134] Step 8: Synthesis of the hydrochloride salt of compound 8

[0135] In a dry 3L three-necked flask, a hydrogen chloride / methanol solution (4 mol / L, 2L) was added, followed by the addition of compound 7 (200 g) in portions. After the addition was complete, the mixture was stirred at 25°C for 16 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to remove methanol. The residue was dissolved in ethyl acetate (1 L), and n-heptane (1.5 L) was slowly added dropwise. A solid slowly precipitated from the system, and stirring was continued for 2 hours. The mixture was filtered, and the resulting solid was washed with ethyl acetate:n-heptane = 1:1.5 (500 mL). The solid was collected and dried under vacuum to give the hydrochloride salt of compound 8.

[0136] Step 9: Synthesis of compound (I)

[0137] Add 1,4-dioxane (2.25 L) to a dry 3 L three-necked flask, then add the hydrochloride salt of compound 8 (150 g), start stirring, and then add potassium carbonate (146.72 g) and stir for 10 minutes. Add compound 9 (84.09 g) and (1R,2R)-(-)-N,N-dimethylcyclohexane-1,2-diamine (25.17 g) to the reaction solution, and purge with nitrogen three times. Finally, add cuprous iodide (13.48 g) to the reaction solution, and purge with nitrogen three times. Heat the reaction solution to 110 °C and react under nitrogen protection for 36 hours. After the reaction is complete, cool the reaction solution to room temperature, filter through a diatomaceous earth liner, and wash the filter cake with ethyl acetate (1 L). Wash the filtrate once with a mixture of water (3 L) and ammonia (300 mL), and separate the layers; extract the aqueous phase with ethyl acetate (2 L × 2). The organic phases were combined, washed once with saturated brine (3 L), and concentrated to dryness under reduced pressure to obtain the crude product. The crude product was dissolved in dichloromethane, filtered through a silica gel pad (100-200 mesh silica gel, weight ratio 1:3), and eluted with dichloromethane:methanol = 50:1. The eluent was concentrated to dryness under reduced pressure, and the crude product was recrystallized from ethanol (1.25 L) to obtain crude product (I). Crude product (I) was purified by preparative separation using a Phenomenex luna C18 column (250 mm * 100 mm * 10 μm); mobile phase: [H₂O (0.05% HCl) - ACN]; ACN%: 30%-60%, for 20 min. The prepared solution was concentrated under reduced pressure to remove most of the acetonitrile, and the pH was adjusted to 7-8 with saturated sodium bicarbonate solution. Extraction was performed with ethyl acetate (1 L × 2). The organic phases were combined, concentrated to dryness under reduced pressure to obtain compound (I) (76 g).

[0138] Example 2: Preparation of the Q crystal form of compound (I)

[0139] In a dry 5L three-necked flask, a 1.9L mixture of methyl isobutyl ketone and n-heptane in a 1:6 ratio was added, followed by the addition of compound (I) (76g) in portions. After the addition was complete, the mixture was heated to 50°C and stirred for 96 hours. The reaction mixture was filtered, and the filter cake was washed with 300mL of methyl isobutyl ketone and n-heptane in a 1:6 ratio. The solid was collected and dried under vacuum at 60°C to obtain the Q crystal form of compound (I). 1H NMR (400MHz, DMSO-d6) δppm9.28 (s, 1H) 9.07 (d, J = 0.88Hz, 1H) 8.23-8.32 (m, 2H) 7. 95 (d, J=9.8Hz, 1H)7.76-7.89 (m, 2H)7.50 (d, J=7.16Hz, 2H)7.27-7.34 (m, 2H)7.19 -7.26(m, 2H) 7.17(d, J = 2.14Hz, 1H) 5.33(d, J = 9.54Hz, 1H) 4.24(td, J = 9.69, 4.08Hz, 1H) 2.43(td, J = 6.84, 4.28Hz, 1H) 1.39(t, J = 19.46Hz, 3H) 0.93(t, J = 6.28Hz, 6H). Q-type XRPD, such as... Figure 1 As shown, DSC is as follows Figure 2 As shown, TGA is as follows Figure 3 As shown.

[0140] Example 3: Preparation of the S-crystal form of compound (I)

[0141] 250 mg of compound (I) was weighed and added to a 10 mL single-necked flask. Ethyl acetate (1.75 mL) was added at 20 °C to form a suspension. After adding a magnetic stir bar, the suspension was placed on a magnetically heated stirrer (80 °C) and stirred until completely dissolved. The temperature was then slowly lowered to 20 °C, and a solid precipitated. The solid was filtered under reduced pressure under N2 protection, and then dried under vacuum to obtain the S-crystal form of compound (I).

[0142] At 20°C, 4 g of compound (I) was added to a 100 mL single-necked flask, and 28 mL of ethyl acetate was added to form a suspension. After adding a magnetic stir bar, the suspension sample was placed on a magnetically heated stirrer (80°C) and stirred until completely dissolved, and stirring was continued for 1 hour. Subsequently, the reaction solution was slowly cooled to 20°C, and a solid precipitated. The solid was filtered under reduced pressure under N2 protection, and the filter cake was dried to constant weight in a vacuum drying oven to obtain the S-crystal form of compound (I).

[0143] 1H NMR (400MHz, DMSO-d6) δppm 9.27 (s, 1H) 9.07 (s, 1H) 8.24-8.32 (m, 2H) 7.95 (d, J = 9.88Hz, 1H) 7.78-7. 89 (m, 2H) 7.50 (d, J = 7.46Hz, 2H) 7.26-7.34 (m, 2H) 7.18-7.26 (m, 2H) 7.17 ( d, J=2.20Hz, 1H) 5.33 (d, J=9.66Hz, 1H) 4.23 (td, J=9.66, 4.18Hz, 1H) 2.4 3(td, J=6.63, 4.28Hz, 1H) 1.39 (t, J=19.40Hz, 3H) 0.92 (t, J=6.26Hz, 6H). S-type XRPD, such as Figure 5 As shown, DSC is as follows Figure 6 As shown, TGA is as follows Figure 7 As shown.

[0144] Example 4: Study on the hygroscopicity of the Q crystal form of compound (I)

[0145] Experimental materials:

[0146] SMS DVS Advantage Dynamic Vapor Adsorption Unit

[0147] Experimental methods:

[0148] Take 10-15 mg of compound (I) in crystal form Q and place it in the DVS sample tray for testing.

[0149] Experimental results:

[0150] The DVS spectrum of the Q crystal form of compound (I) is as follows: Figure 4 As shown, ΔW = 0.21%.

[0151] Experimental conclusion:

[0152] The Q crystal form of compound (I) at 25°C showed a 0.21% increase in weight at 80% RH compared to the initial 0% RH, indicating that the sample was slightly hygroscopic.

[0153] Example 5: Study on the hygroscopicity of the S crystal form of compound (I)

[0154] Experimental materials:

[0155] SMS DVS Advantage Dynamic Vapor Adsorption Unit

[0156] Experimental methods:

[0157] Take 10-15 mg of compound S crystal form of formula (I) and place it in the DVS sample tray for testing.

[0158] Experimental results:

[0159] The DVS spectrum of the S crystal form of compound (I) is as follows: Figure 8 As shown, compared with 0% RH, the sample's moisture absorption weight gain ΔW = 0.6% at 80% RH.

[0160] Experimental conclusion:

[0161] The S crystal form of compound (I) at 25°C showed a 0.6% increase in weight at 80% RH compared to the initial 0% RH, indicating that the sample was slightly hygroscopic.

[0162] Example 6: Solid stability test of the Q crystal form of compound (I)

[0163] Based on the "Guidelines for Stability Testing of Active Pharmaceutical Ingredients and Preparations" (Chinese Pharmacopoeia 2015, Part IV, General Chapter 9001), the stability of compound (I) in crystal form Q was investigated under high temperature (60℃, open), high humidity (room temperature / relative humidity 92.5%, open) and strong light (5000 lx, closed) conditions.

[0164] Weigh 15 mg of compound (I) in crystal form Q and place it at the bottom of a glass sample vial, spreading it into a thin layer. For samples placed under high temperature and high humidity conditions, seal the vial opening with aluminum foil, making small holes in the foil to ensure sufficient contact between the sample and ambient air; for samples placed under strong light conditions, seal with a screw cap. Samples placed under different conditions were sampled and tested on day 5, day 10, and month 3 (XRPD). The test results were compared with the initial test results on day 0. The experimental results are shown in Table 4 below:

[0165] Table 4 shows the solid stability test results of compound Q crystal form (I).

[0166]

[0167] Conclusion: Compound Q of formula (I) exhibits good stability under high temperature, high humidity and strong light conditions.

[0168] Example 7: Single-crystal X-ray diffraction analysis of compound (I)

[0169] Single crystal cultivation process: The sample was dissolved in 1.5 mL of ethyl acetate at room temperature. The sample solution was placed in a 1 mL semi-sealed centrifuge tube and allowed to stand in a dark and vibration-free place. It was allowed to evaporate slowly at room temperature. Colorless needle-like crystals were obtained on the third day.

[0170] Single-crystal X-ray diffraction analysis revealed that each unit cell contains two molecules of compound (I) and two molecules of solvent (ethyl acetate). An ellipsoidal diagram of the stereostructure of compound (I) is attached. Figure 9 and10 Crystal structure data and parameters of compounds of formula (I) are shown in Tables 5, 6, 7, 8 and 9.

[0171] Table 5 Crystal data for compound (I)

[0172]

[0173]

[0174] Table 6 shows the atomic coordinates (×10) of the compound crystal of formula (I). 4 and equivalent isotropic shift parameters

[0175]

[0176]

[0177]

[0178] Bond lengths of compounds of formula (I) in Table 7

[0179]

[0180]

[0181] Table 8. Bond angles (°) of compounds of formula (I)

[0182]

[0183]

[0184]

[0185] Table 9. Torsion angle (°) of compounds of formula (I)

[0186]

[0187]

[0188]

[0189] Experimental Example 1: In vitro detection of the inhibitory effect of compounds on hMMP1 transcriptional activity in a luciferase reporter gene screening system

[0190] Experimental objective:

[0191] The human MMP-1 promoter region (containing two AP-1 binding sites and two PEA3 sites, totaling 249 bp, gene bank catalog #AF023338) was cloned upstream of the luciferase reporter gene. The hMMP-1 promoter reporter gene was constructed and transfected into HeLa cells, allowing for easy detection of luciferase production. Stable recombinant hMMP-1 / luciferase cell lines were used for the development and validation of this experiment.

[0192] Culture media and reagents:

[0193] 1. Conventional cell culture medium

[0194] DMEM 90%, FBS 10%, 1mM NEAA, 1mM Sodium Pyruvate, 4mM L-Glutamine, 300μg / mL G418, store at 4°C.

[0195] 2. Refrigerant

[0196] DMEM 75%, FBS 20%, DMSO 5%. Prepare before use.

[0197] 3. Experimental cell culture medium

[0198] DMEM 97%, Charcoal-stripped serum 3%.

[0199] 4. Bright-Glo reagent kit

[0200] Transfer the entire Bright Glo Buffer from one bottle to a brown fluorescent substrate vial, invert and mix until the substrate is completely dissolved, dispense in appropriate amounts, and store at -80°C.

[0201] method

[0202] Preparation of cell suspensions from frozen cells

[0203] 1. Cell thawing

[0204] 1) Thaw the frozen cells quickly and place them in a 37°C water bath with constant stirring until completely dissolved.

[0205] 2) Add the cells to a 10mL centrifuge tube (containing 5mL of preheated cell culture medium), and centrifuge at 1000 rpm for 5 minutes.

[0206] 3) Discard the supernatant and resuspend the cells in 10 mL of preheated cell culture medium.

[0207] 4) Transfer the cell suspension to a 100mM cell culture dish and incubate in a 37℃ 5% CO2 incubator.

[0208] 2. Passage breeding

[0209] 1) When the cells reach 80-90% confluence, perform cell passage culture. pGL6.0-TA-hMMP-1HeLa cells are usually passaged twice a week, with a 1:3 or 1:6 dilution.

[0210] 2) Carefully aspirate all the culture medium, gently rinse the cell layer with an appropriate amount of DPBS, and then aspirate.

[0211] 3) Add an appropriate amount of 0.05% Typsine EDTA and incubate in a CO2 incubator for 3-5 minutes to digest the cells.

[0212] 4) Resuspend the cells in an appropriate amount of preheated cell culture medium and dilute for passage culture.

[0213] 3. Change the culture medium every other day.

[0214] 1) Gently aspirate all the culture medium.

[0215] 2) Add fresh cell culture medium (preheated to 37℃) (10 mL per 100 mm dish).

[0216] 4. Cryopreservation of cells

[0217] 1) Repeat subculture steps 1-4.

[0218] 2) Centrifuge the cells at 1000 rpm for 5 minutes.

[0219] 3) Remove the supernatant, resuspend in cryopreservation solution, count and dilute to 2-3 × 10⁻⁶. 6 Cells / mL. Add 1 mL of suspended cells to each cell cryotube.

[0220] 4) Place the cryovials into the cryovial container, and then transfer the cryovial container to a -80°C freezer overnight.

[0221] 5) Transfer the cryovials to liquid nitrogen (-196°C).

[0222] 5. Inoculate cells

[0223] 1) Carefully aspirate all the culture medium, gently rinse the cell layer with an appropriate amount of DPBS, and then aspirate.

[0224] 2) Add an appropriate amount of 0.05% Typsine EDTA and incubate in a CO2 incubator for 3-5 minutes to digest the cells.

[0225] 3) Resuspend the cells with an appropriate amount of cell culture medium.

[0226] 4) Calculate the required cell quantity. The cell concentration is 5 × 10⁻⁶. 3One hole / hole.

[0227] 5) Dilute the cell suspension with an appropriate cell culture medium.

[0228] 6) The cell suspension is dispensed into disposable sterile sample dispensing containers.

[0229] 7) 30 μL per well was inoculated into a 384-well plate.

[0230] 8) Place the cell plate in a 37℃ 5% CO2 incubator and incubate for 18-24 hours.

[0231] Compound preparation

[0232] 1. PMA:

[0233] PMA was diluted to 1 mM with DMSO, aliquoted and stored at -80°C in the dark for later use.

[0234] 2. Dexamethasone:

[0235] Dilute with DMSO to 30 mM, aliquot and store in a -80°C freezer for later use, protected from light.

[0236] 3. Preparation of a compound at 10 times its concentration:

[0237] The test compound was diluted to 10 mM with DMSO, aliquoted, and stored at -80°C for later use.

[0238] The compounds were diluted with DMSO to 1 mM, 0.25 mM, 0.0625 mM, 0.015625 mM, 0.0039 mM, 0.0009765 mM, 0.000244 mM, 0.000061 mM, 0.00001526 mM, and 0.0000038125 mM, and then diluted 100-fold with serum-free PMA medium containing 100 nM to obtain experimental plates of 10× concentration compounds.

[0239] The final DMSO concentration is 0.1%. PMA should be protected from light during use.

[0240] hMMP1 GR transcriptional repression activity assay

[0241] 1) Cell inoculation: Fresh cells were inoculated at 5 × 10⁻⁶ cells per cell line. 3 30 μL / well was inoculated into a 384 white experimental plate and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0242] 2) Compound preparation: Prepare the compound plate before the experiment, including a 10× concentration of the reference compound (dexamethasone) and the test compound, and finally obtain the 10× concentration compound experimental plate.

[0243] 3) Add the compound: Transfer 3.3 μL of the 10× compound using Bravo and add it to the cell plate. Incubate at 37°C in a 5% CO2 incubator for 18 hours.

[0244] 4) 30 μL of Bright-Glo fluorescence detection reagent was transferred into the cell plate.

[0245] 5) Centrifuge and incubate for 2 minutes

[0246] 6) Measure the fluorescence value using an Envision plate reader.

[0247] Data processing and analysis

[0248] Positive control: 10 nM PMA + 1000 nM dexamethasone (0.1% DMSO)

[0249] Negative control: 10 nM PMA (0.1% DMSO)

[0250] Test compound: highest concentration 1000 nM, 4-fold dilution, 10 wells in total, replicate.

[0251] Dexamethasone: maximum concentration 1000 nM, 4-fold dilution, 10 wells, replicate.

[0252] Concentration curves of the test compounds were generated using the graphing software GraphPad Prism5, and IC50 was calculated. 50 value.

[0253] The experimental results are shown in Table 10.

[0254] Experimental Example 2: In vitro detection of compound activation of MMTV transcriptional activity in a luciferase reporter gene screening system

[0255] Experimental objective:

[0256] The promoter of mouse mammary tumor virus (MMTV) contains a specific binding site that activates GR (GREs). To determine the activity of compounds, a reporter gene (luciferase) was inserted after the MMTV promoter, and this structure is stably expressed in the genome of the HeLa cell line. The MMTV promoter was activated using the test compound to induce luciferase expression, and its activity was detected by luminescence measurement.

[0257] Culture media and reagents:

[0258] 1. Conventional cell culture medium

[0259] DMEM 90%, FBS 10%, Hygromycin 100 μg / mL

[0260] 2. Refrigerant

[0261] DMEM 75%, FBS 20%, DMSO 5%.

[0262] 3. Experimental cell culture medium

[0263] DMEM 97%, Charcoal-stripped serum 3%.

[0264] 4. Bright-Glo reagent kit

[0265] Transfer the entire BrightGloBuffer vial to a brown fluorescent substrate vial, invert and mix until the substrate is completely dissolved, dispense in appropriate amounts, and store at -80°C.

[0266] method

[0267] Preparation of cell suspensions from frozen cells

[0268] 1. Cell thawing

[0269] 1) Place the frozen cells in a 37°C water bath and stir continuously until completely thawed.

[0270] 2) Add the cells to a 15mL centrifuge tube (containing 5mL of preheated cell culture medium), and centrifuge at 1000 rpm for 5 minutes.

[0271] 3) Discard the supernatant and add 5 mL of preheated cell culture medium to resuspend the cells.

[0272] 4) Transfer the cell suspension to a 60mm cell culture dish and incubate in a 37℃ 5% CO2 incubator.

[0273] 2. Passage breeding

[0274] 1) When the cells reach 80-90% confluence, perform cell passage culture. Usually, passage is performed twice a week, with a 1:3 or 1:6 dilution.

[0275] 2) Carefully aspirate all the culture medium, gently rinse the cell layer with an appropriate amount of DPBS, and then aspirate.

[0276] 3) Add an appropriate amount of 0.05% Typsine EDTA and incubate in a CO2 incubator for 3-5 minutes to digest the cells.

[0277] 4) Resuspend the cells in an appropriate amount of preheated cell culture medium and dilute for passage culture.

[0278] 3. Change the culture medium every other day.

[0279] 1) Gently aspirate all the culture medium.

[0280] 2) Add fresh cell culture medium (preheated to 37℃) (10 mL per 100 mm dish).

[0281] 4. Cryopreservation of cells

[0282] 1) Repeat subculture steps 1-4.

[0283] 2) Centrifuge the cells at 1000 rpm for 5 minutes.

[0284] 3) Remove the supernatant, resuspend in cryopreservation solution, count and dilute to (2-3)×10⁻⁶. 6 Cells / mL. Add 1 mL of suspended cells to each cell cryotube.

[0285] 4) Place the cryovials into the cryovial container, and then transfer the cryovial container to a -80°C freezer overnight.

[0286] 5) Transfer the cryovials to liquid nitrogen (-196°C).

[0287] 5. Inoculate cells

[0288] 1) Carefully aspirate all the culture medium, gently rinse the cell layer with an appropriate amount of DPBS, and then aspirate.

[0289] 2) Add an appropriate amount of 0.05% Typsine EDTA and incubate in a CO2 incubator for 3-5 minutes to digest the cells.

[0290] 3) Resuspend the cells with an appropriate amount of cell culture medium.

[0291] 4) Calculate the required cell quantity, with a cell concentration of 4 × 10⁻⁶. 3 One hole / hole.

[0292] 5) Dilute the cell suspension with an appropriate cell culture medium.

[0293] 6) The cell suspension is dispensed into disposable sterile sample dispensing containers.

[0294] 7) 30 μL per well was seeded into a 384-well plate.

[0295] 8) Place the cell plate in a 37℃ 5% CO2 incubator and incubate for 18-24 hours.

[0296] Compound preparation

[0297] 1. Dexamethasone:

[0298] Dilute with DMSO to 30 mM, aliquot and store in a -80°C freezer for later use, protected from light.

[0299] 2. Preparation of a compound at 4 times its concentration:

[0300] The test compound was diluted to 10 mM with DMSO, aliquoted, and stored at -80°C for later use.

[0301] The compound was diluted with DMSO to 1 mM, 0.25 mM, 0.0625 mM, 0.015625 mM, 0.0039 mM, 0.0009765 mM, 0.000244 mM, 0.000061 mM, 0.00001526 mM and 0.0000038125 mM, and then diluted 250 times with serum culture medium containing activated charcoal to obtain 4× concentration experimental plates, which were prepared before use.

[0302] The final experimental concentration of DMSO was 0.1%.

[0303] MMTV_GR transcriptional activation activity assay

[0304] 1) Cell inoculation: Fresh cells were inoculated at a rate of 4 × 10⁻⁶. 3 30 μL / well was seeded into a 384 white transparent plate and incubated at 37°C for 24 hours in a 5% CO2 incubator.

[0305] 2) Compound preparation: Prepare the compound plate before the experiment. Prepare the reference compound (dexamethasone) and the test compound with a 4-fold dilution gradient. Transfer 100 nl to each well using Echo. Then add 25 uL of culture medium to each well. This plate is a 4x compound experimental plate.

[0306] 3) Add the compound: Transfer 10 μL of the 4× compound from the compound experimental plate using Bravo and add it to the cell plate. Incubate at 37°C in a 5% CO2 incubator for 20-24 hours.

[0307] 4) 30 μL of Bright-Glo fluorescence detection reagent was transferred into the cell plate.

[0308] 5) Centrifuge and incubate for 2 minutes

[0309] 6) Measure the fluorescence value using an Envision plate reader.

[0310] Data processing and analysis

[0311] Positive control: 1000 nM dexamethasone (0.1% DMSO)

[0312] Negative control: 0.1% DMSO

[0313] Test compound: highest concentration 1000 nM, 4-fold dilution, 10 wells in total, replicate.

[0314] Dexamethasone: maximum concentration 1000 nM, 4-fold dilution, 10 wells, replicate.

[0315] Concentration curves of the test compounds were generated using the graphing software GraphPad Prism5, and IC50 was calculated. 50 value.

[0316] The experimental results are shown in Table 10.

[0317] Table 10 Results of in vitro screening tests of compounds of the present invention

[0318]

[0319] Conclusion: Compound Q of formula (I) exhibits excellent transcriptional repression activity and considerable transcriptional activation activity.

[0320] Experimental Example 3: In vivo PK study of the Q crystal form of compound (I) in female Lewis rats

[0321] 1. Research Methods and Experimental Design

[0322] 1.1 Test System

[0323] Table 11. Test System

[0324]

[0325] 1.2 Environmental Adaptation / Inspection and Quarantine

[0326] After the animals arrive at WuXi AppTec's animal facility, they will undergo an acclimatization period of at least 3 days. At the end of the acclimatization period, a veterinarian or designated personnel will check the animals' health status to assess their suitability for experimental research.

[0327] 1.3 Animal husbandry

[0328] Animal husbandry in this experimental protocol complies with the U.S. Animal Welfare Act and follows the National Academy of Sciences Research Council's "Guidelines for the Management and Use of Laboratory Animals" (8th Edition) and the Laboratory Animal Management Regulations (2017 Revision), among other regulations and guidelines. Animals will be housed together, but may be housed individually post-surgery, depending on specific experimental requirements, for behavioral or health reasons, or due to the death of a companion. Except in cases requiring fasting, qualified rat growth and reproduction feed will be provided daily, with free access to food. Each batch of feed will have a report on nutritional composition, chemical contaminants, and microbiological analysis, and will be administered to the animals only after veterinary evaluation and approval. Drinking water will be autoclaved reverse osmosis water, supplied in water bottles, with free access to water. Drinking water will be periodically sampled by a qualified third-party testing laboratory for microbiological and environmental contaminant testing, with the test reports reviewed and evaluated by the veterinarian. Animals will use either wood shavings or corncob bedding. If corncob bedding is used, it will be replaced with wood shavings bedding before fasting. Each batch of bedding will have an environmental contaminant analysis report provided by the supplier, and will be used only after veterinary review and approval. Environmental conditions were controlled at room temperature of 20℃–26℃, relative humidity of 40%–70%, and alternating light and dark cycles for 12 hours. The light-dark cycle may be affected by experimental procedures. Temperature and humidity were monitored in real-time using the Vaisala ViewLinc monitoring system.

[0329] 1.4 Administration

[0330] On the day of the experiment, the animals in Group 1 were given a single gavage of compound (I) Q crystal form at a concentration of 0.3 mg / mL. The animals were weighed before administration and the volume of administration was calculated based on their weight.

[0331] 1.5 Selection of Dosage

[0332] Oral administration via gavage (0.3 mg / kg).

[0333] 1.6 Euthanasia

[0334] The animals were euthanized under CO2 anesthesia after the last PK sample was collected.

[0335] 1.7 Animal weight and clinical observation

[0336] On the day of administration, select animals of appropriate weight strictly according to the experimental protocol. Observe the overall health of the animals on the day of administration. Monitor the animals at each sample collection time point and each administration time point. Record any abnormalities accurately throughout the experiment.

[0337] 2 Sample Collection

[0338] 2.1 Drug Solution

[0339] After each formulation was prepared, three portions of the oral administration solution (upper, middle, and lower layers) were collected and analyzed by HPLC-UV to examine the accuracy of the formulation concentration.

[0340] 2.2 Sample Collection and Processing

[0341] Collect approximately 0.2 mL of whole blood at a specified time (or other suitable sampling site) via jugular vein puncture, and record the actual blood collection time in the experimental log. The acceptable error for collection time points is ±1 minute for time points within 1 hour of drug administration, and ±5% for other time points (theoretical time). All blood samples were immediately transferred to labeled commercially available centrifuge tubes containing K2-EDTA. After collection, the blood samples were centrifuged at 3200g for 10 minutes at 4°C, and the supernatant plasma was aspirated, rapidly placed on dry ice, and then stored at -60°C or lower for LC-MS / MS analysis.

[0342] 3 Sample Analysis

[0343] 3.1 Drug Solution

[0344] The concentration of the drug-eluting formulation should be determined using HPLC-UV, and the calibration curve should include at least six concentration levels. The actual measured concentration should be within 80% to 120% of the theoretical concentration; otherwise, pharmacokinetic parameters should be calculated based on the actual dose.

[0345] 3.2 Plasma Samples

[0346] The concentration of the test sample in plasma was determined using LC-MS / MS bioanalytical methods.

[0347] When analyzing samples using LC-MS / MS bioanalytical methods, each run batch must include at least one standard curve and two sets of quality control samples processed simultaneously. The number of quality control samples should be greater than 5% of the number of unknown samples. Calibration standards and quality control samples must be freshly prepared on the day of analysis.

[0348] The acceptance criteria for each analytical batch of standard curves include: each standard curve contains seven non-zero concentration calibration standards. The calculated concentrations of the calibration standards should be within ±20.0% of the labeled values, except for the lowest calibration standard, which should be within ±25.0%. At least 75% of the calibration standards in the standard curve should meet the acceptance criteria. If the lower or upper limit of quantitation of the standard curve does not meet the acceptance criteria, the limit of quantitation will be raised or lowered accordingly.

[0349] The acceptance criteria for quality control samples in each analytical batch include: each set of quality control samples must include at least three concentration levels (low, medium, and high), and the measured concentration of the quality control samples should be within ±20.0% of the labeled value. At least two-thirds of the quality control samples must meet the acceptance criteria.

[0350] Analyte interference: The mass spectrometry response of the analyte in double-blank and single-blank samples should be less than 50% of the limit of quantitation.

[0351] Residue: System residues are confirmed by immediately injecting a blank sample after injecting the highest concentration of calibration standard. The concentration of the analyte in the residue sample is less than or equal to the lower limit of quantitation (LLOQ).

[0352] Experimental results: see Table 12.

[0353] Table 12 shows the in vivo pharmacokinetic results of compound Q crystal form (I).

[0354]

[0355] Experimental conclusion: Compound Q of formula (I) has excellent pharmacokinetic properties.

Claims

1. The Q crystal form of compound (Ⅰ) has X-ray powder diffraction patterns with characteristic diffraction peaks at the following 2θ angles: 10.301±0.200°, 12.779±0.200°, 16.860±0.200°, 17.459±0.200°, 19.141±0.200°, 20.701±0.200°, 21.701±0.200°, 24.400±0.200°. 。 2. The Q crystal form according to claim 1, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.060±0.200°, 10.301±0.200°, 12.779±0.200°, 16.860±0.200°, 17.459±0.200°, 17.870±0.200°, 18.639±0.200°, 19.141±0.200°, 20.701±0.200°, 21.701±0.200°, 22.579±0.200°, 24.400±0.200°.

3. The Q-type crystal according to claim 2, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.861±0.200°, 8.060±0.200°, 9.621±0.200°, 10.301±0.200°, 11.500±0.200°, 12.779±0.200°, 16.860±0. 200°, 17.459±0.200°, 17.870±0.200°, 18.639±0.200°, 19.141±0.200°, 20.701±0.200°, 21.701±0.200°, 22.579±0.200°, 24.400±0.200°, 24.939±0.200°.

4. The Q crystal form according to claim 3, its XRPD pattern is shown in Figure 1.

5. The Q crystal form according to any one of claims 1 to 4, wherein the differential scanning calorimetry curve has an initial value of an endothermic peak at 181.4°C ± 5°C.

6. The Q crystal form according to claim 5, its DSC spectrum is shown in Figure 2.

7. The Q crystal form according to any one of claims 1 to 4, wherein the thermogravimetric analysis curve shows a weight loss of 1.47% at 150°C ± 3°C.

8. The Q crystal form according to claim 7, its TGA spectrum is shown in Figure 3.

9. The S-crystal form of compound (I) has a characteristic diffraction peak at the following 2θ angles in its X-ray powder diffraction pattern: 11.999±0.200°, 13.019±0.200°, 14.961±0.200°, 18.182±0.200°, 19.579±0.200°, 20.262±0.200°, 22.939±0.200°, 24.021±0.200°. 。 10. The S-type according to claim 9, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.201±0.200°, 11.463±0.200°, 11.999±0.200°, 13.019±0.200°, 14.523±0.200°, 14.961±0.200°, 18.182±0.200°, 19.579±0.200°, 20.262±0.200°, 22.939±0.200°, 24.021±0.200°, 25.738±0.200°.

11. The S-crystal form according to claim 10, its XRPD pattern is shown in Figure 5.

12. The S-crystal form according to any one of claims 9 to 11, wherein the differential scanning calorimetry curve has an endothermic peak at 114.9°C ± 3°C.

13. The S-crystal form according to claim 12, its DSC spectrum is shown in Figure 6.

14. The S-type according to any one of claims 9 to 11, wherein the thermogravimetric analysis curve shows a weight loss of 0.99% at 90.0°C ± 3°C and a weight loss of 2.54% at 120.0°C ± 3°C.

15. The S-crystal form according to claim 14, its TGA spectrum is shown in Figure 7.

16. The use of the Q crystal form according to any one of claims 1 to 8 or the S crystal form according to any one of claims 9 to 15 in the preparation of a medicament for treating rheumatoid arthritis.

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