A crystal form of a cyclopropyl-substituted benzofuran compound and a preparation method thereof

Abstract

The application relates to a crystal form of a cyclopropyl-substituted benzofuran compound and a preparation method thereof, and particularly discloses a crystal form of a compound of formula (I) and a preparation method thereof.

Description

[0001] This invention claims the following priority:

[0002] CN202110214161.0, application date: February 25, 2021. Technical Field

[0003] This invention relates to the crystal form of a cyclopropyl-substituted benzofuran compound and its preparation method, specifically to the crystal form of the compound of formula (I) and its preparation method. Background Technology

[0004] Pancreatic adenocholinergic acicular cells have acetyl-CoA receptors (AChR) and cholecystokinin receptors (CCKR) on their membranes. Both receptors are dependent on Ca2+. 2+ The former, under the action of acetylcholine, activates phospholipase (PLC) to generate inositol 1,4,5-triphosphate (IP3). The latter, under the action of cholecystokinin, has its receptor bind to adenosine diphosphate ribose cyclase via an unknown pathway to generate nicotinic adenosine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate ribose (CADPR). IP3 and ryanodine receptors on the endoplasmic reticulum are activated by IP3 and NAADP / CADPR, respectively, releasing stored Ca2+. 2+ Released from the endoplasmic reticulum into the cytoplasm. With intracellular Ca... 2+ Empty. Ca 2+ Depletion of the reservoir causes Ca2+ located on the endoplasmic reticulum 2+ The receptor STIM1 protein oligomerizes and moves toward the nearest endoplasmic reticulum-cell membrane junction, opening the Orai channel on the plasma membrane and allowing Ca2+ to enter. 2+ Influx, causing intracellular Ca2+ 2+ Excessive levels can prematurely activate proenzymes, inducing the production of inflammatory factors within cells.

[0005] Factors such as alcohol and kidney stones can induce calcium deficiency. 2+ Released from the endoplasmic reticulum, while endoplasmic reticulum Ca 2+ The reduction in existing levels stimulates the hyperactivation of cellular CRAC channels (specifically, Orai channels), leading to a surge in calcium levels. 2+ Influx of calcium into pancreatic acinar cells significantly increases intracellular calcium concentration, leading to premature activation of zymogen granules into trypsin. Trypsin then activates other pancreatic digestive enzymes, ultimately resulting in pancreatic autodigestion and necrosis. CRAC inhibitors can inhibit calcium influx. 2+ The influx of CRAC inhibitors prevents pancreatic necrosis. CRAC inhibitors can inhibit the influx of calcium... 2+ The release of these substances prevents pancreatic necrosis.

[0006] In developed countries, gallstones and alcohol use obstructing the common bile duct are the most common causes of acute pancreatitis, accounting for 70-80%. Gallstone-induced pancreatitis is caused by ductal obstruction and the effect of bile acids on pancreatic acinar cells. Gallstones allow bile to reflux into the pancreatic duct system, and once inside the acinar cells, bile acids activate calcium entry into these cells via CRAC channels. This leads to acute pancreatitis and exocrine cell necrosis of the pancreas through the activation of unregulated digestive enzymes, cytokine production, and the infiltration of inflammatory cells into the pancreas. Alcohol use is the second most common cause of acute pancreatitis, but the correlation between alcohol and pancreatitis is not fully understood. While alcohol use is commonly associated with both acute and chronic pancreatitis, alcohol itself does not cause pancreatitis. Instead, it appears that metabolic byproducts of alcohol may be the cause of the disease in some patients. Researchers have demonstrated that specific ethanol metabolites called fatty acid ethyl esters (FAEEs) can induce a sustained release of intracellular calcium ions, thereby activating CRAC channels. The resulting high intracellular calcium levels are similar to those caused by gallstones and can trigger the disease. Summary of the Invention

[0007] This invention provides crystal form A of the compound of formula (I).

[0008]

[0009] Its characteristic feature is that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.892±0.200°, 17.583±0.200°, 18.959±0.200°, 22.113±0.200°, 25.029±0.200°.

[0010] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A-type crystal has characteristic diffraction peaks at the following 2θ angles: 8.892±0.200°, 12.617±0.200°, 16.793±0.200°, 17.583±0.200°, 18.959±0.200°, 22.113±0.200°, 25.029±0.200°, 26.512±0.200°.

[0011] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A-type crystal has characteristic diffraction peaks at the following 2θ angles: 8.892±0.200°, 11.883±0.200°, 12.617±0.200°, 13.206±0.200°, 16.793±0.200°, 17.583±0.200°, 18.018±0.200°, 18.959±0.200°, 22.113±0.200°, 25.029±0.200°, 25.858±0.200°, 26.512±0.200°.

[0012] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A-type crystal has characteristic diffraction peaks at the following 2θ angles: 4.474°, 8.892°, 9.571°, 10.288°, 10.533°, 11.076°, 11.883°, 12.617°, 13.206°, 13.653°, 14.886°, 15.788°, 16.301°, 16.793°, 17.583°, 1 8.018°, 18.959°, 19.574°, 19.981°, 20.661°, 20.964°, 21.651°, 22.113°, 23.031°, 23.747°, 24.107°, 25.029°, 25.414°, 25.858°, 26.512°, 28.797°, 30.039°, 31.672°, 32.596°, 35.275°.

[0013] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A-type crystal has characteristic diffraction peaks at the following 2θ angles: 3.616°, 4.474°, 8.892°, 9.571°, 10.288°, 10.533°, 11.076°, 11.883°, 12.617°, 13.206°, 13.653°, 14.886°, 15.788°, 16.301°, 16.793°, 17.58°. 3°, 18.018°, 18.959°, 19.574°, 19.981°, 20.661°, 20.964°, 21.651°, 22.113°, 23.031°, 23.747°, 24.107°, 25.029°, 25.414°, 25.858°, 26.512°, 28.797°, 30.039°, 31.672°, 32.596°, 35.275°.

[0014] In some embodiments of the present invention, the XRPD pattern of the above-mentioned crystal form A is essentially as follows: Figure 1 As shown.

[0015] In some embodiments of the present invention, the XRPD spectra analysis data of the above-mentioned A-type crystal form are shown in Table 1:

[0016] Table 1. XRPD pattern analysis data for crystal form A

[0017]

[0018]

[0019] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned A crystal form has peak values ​​of endothermic peaks at 138.4℃±3℃ and 163.4℃±3℃.

[0020] In some embodiments of the present invention, the DSC spectrum of the above-mentioned A-type crystal is essentially as follows: Figure 2 As shown.

[0021] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned A crystal form shows a weight loss of 3.33% at 140.0℃±3℃.

[0022] In some embodiments of the present invention, the TGA pattern of the above-mentioned A-type crystal is essentially as follows: Figure 3 As shown.

[0023] This invention also provides crystal form B of the compound of formula (I),

[0024]

[0025] Its characteristic feature is that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.797±0.200°, 17.578±0.200°, 18.811±0.200°, 21.997±0.200°.

[0026] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 4.389±0.200°, 8.797±0.200°, 11.786±0.200°, 17.578±0.200°, and 21.997±0.200°.

[0027] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 4.389±0.200°, 8.797±0.200°, 9.473±0.200°, 11.786±0.200°, 15.710±0.200°, 16.739±0.200°, and 21.997±0.200°.

[0028] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 4.389±0.200°, 8.797±0.200°, 13.168±0.200°, 16.739±0.200°, 17.578±0.200°, 18.811±0.200°, 21.997±0.200°, 26.443±0.200°.

[0029] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 4.389±0.200°, 8.797±0.200°, 9.473±0.200°, 11.786±0.200°, 13.168±0.200°, 15.710±0.200°, 16.739±0.200°, 17.578±0.200°, 18.811±0.200°, and 21.997±0.200°.

[0030] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B-type crystal has characteristic diffraction peaks at the following 2θ angles: 4.389±0.200°, 8.797±0.200°, 11.786±0.200°, 13.168±0.200°, 16.739±0.200°, 17.578±0.200°, 18.811±0.200°, 20.617±0.200°, 21.997±0.200°, 24.970±0.200°, 25.804±0.200°, and 26.443±0.200°.

[0031] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B-type crystal has characteristic diffraction peaks at the following 2θ angles: 8.797±0.200°, 17.578±0.200°, 21.997±0.200°, and / or 4.389±0.200°, and / or 9.473±0.200°, and / or 10.172±0.200°, and / or 10.356±0.200°, and / or 10.953±0.200°, and / or 11.786±0.200°, and / or 12.569±0.200°, and / or 13.1 68±0.200°, and / or 13.454±0.200°, and / or 13.991±0.200°, and / or 14.828±0.200°, and / or 15.710±0.200°, and / or 16.231±0.200°, and / or 16.739±0.200°, and / or 17.949±0.200°, and / or 18.811±0.200°, and / or 19.577±0.200°, and / or 19.875±0.200°, and / or 20.617±0.200°, and / or 20.849± 0.200°, and / or 22.875±0.200°, and / or 23.704±0.200°, and / or 24.165±0.200°, and / or 24.970±0.200°, and / or 25.449±0.200°, and / or 25.804±0.200°, and / or 26.443±0.200°, and / or 26.942±0.200°, and / or 27.841±0.200°, and / or 28.731±0.200°, and / or 29.022±0.200°, and / or 29.525±0.2 00°, and / or 30.033±0.200°, and / or 30.962±0.200°, and / or 31.747±0.200°, and / or 32.609±0.200°, and / or 33.12±0.200°, and / or 33.979±0.200°, and / or 34.568±0.200°, and / or 35.104±0.200°, and / or 35.421±0.200°, and / or 36.374±0.200°, and / or 37.075±0.200°, and / or 38.979±0.200°.

[0032] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B-type crystal has characteristic diffraction peaks at the following 2θ angles: 4.389°, 8.797°, 9.473°, 10.172°, 10.356°, 11.786°, 12.569°, 13.168°, 13.991°, 15.710°, 16.739°, 17.578°, 17.949°, 18.811°, 19.577°, 19.875°, 20.617°, 20.849°, 21.997°, 24.970°, 25.804°, 26.443°, 26.942°, 27.841°, 28.731°, 30.033°, and 30.962°.

[0033] In some embodiments of the present invention, the XRPD pattern of the above-mentioned B crystal form is essentially as follows: Figure 4 As shown.

[0034] In some embodiments of the present invention, the XRPD spectra analysis data of the above-mentioned B crystal form are shown in Table 2:

[0035] Table 2. XRPD pattern analysis data of crystal form B

[0036]

[0037]

[0038] In some embodiments of the present invention, the differential scanning calorimetry curve of the B crystal form has an initial value of an endothermic peak at 165.7℃±5℃.

[0039] In some embodiments of the present invention, the DSC pattern of the above-mentioned B crystal form is essentially as follows: Figure 5 As shown.

[0040] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned B crystal form shows a weight loss of 1.14% at 150℃±3℃.

[0041] In some embodiments of the present invention, the TGA pattern of the B crystal form described above is essentially as follows: Figure 6 As shown.

[0042] The present invention also provides the use of crystal form A or crystal form B in the preparation of drugs for treating acute pancreatitis.

[0043] Technical effect

[0044] The compound of this invention has good PK properties and therapeutic effect on acute pancreatitis. It has stable crystal form, good hygroscopicity, and is less affected by light and heat.

[0045] Definitions and Explanations

[0046] 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.

[0047] 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.

[0048] In the DSC spectrum of this invention, peaks with heat flux less than 0 are endothermic peaks, and peaks with heat flux greater than 0 are exothermic peaks.

[0049] 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.

[0050] 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 is further analyzed using the direct method (Shelx 97) to confirm the absolute configuration. The invention will be described in detail below through examples, which are not intended to limit the invention in any way.

[0051] All solvents used in this invention are commercially available and can be used without further purification.

[0052] Compounds are named according to conventional naming principles in the field or using Software naming conventions are used; commercially available compounds use supplier catalog names.

[0053] The present invention relates to a powder X-ray diffractometer (XRPD) method.

[0054] Instrument Model: Bruker D2PHASER X-ray Diffractometer

[0055] Test method: Approximately 10–20 mg of sample is used for XRPD detection.

[0056] The detailed XRPD parameters are as follows:

[0057] Optical tube: Cu,kα,

[0058] Phototube voltage: 30kV, Phototube current: 40mA

[0059] Diverging slit: 0.60mm

[0060] Detector slit: 10.50mm

[0061] Anti-scattering slit: 7.10mm

[0062] Scan range: 3-40 degrees

[0063] Step size: 0.02deg

[0064] Step length: 0.5 seconds

[0065] Sample tray rotation speed: 15 rpm

[0066] This invention relates to a differential scanning calorimeter (DSC) method.

[0067] Instrument Model: TA Q2000 Differential Scanning Calorimeter

[0068] Test method: Take a sample (~1mg) and place it in a DSC aluminum pot for testing. Under N2 conditions of 50mL / min, heat the sample from 30℃ (room temperature) to 300℃ (or 350℃) at a heating rate of 10℃ / min.

[0069] The present invention relates to a thermogravimetric analysis (TGA) method.

[0070] Instrument Model: TA Q5000IR Thermogravimetric Analyzer

[0071] Test method: Take a sample (2-5 mg) and place it in a TGA platinum pot for testing. Under N2 conditions of 25 mL / min, heat the sample from room temperature to 350℃ or lose 20% of its weight at a heating rate of 10℃ / min.

[0072] This invention presents a method for dynamic vapor adsorption analysis (DVS).

[0073] Instrument Model: SMS DVS Advantage Dynamic Vapor Adsorption Analyzer

[0074] Test conditions: Take a sample (10-15 mg) and place it in the DVS sample tray for testing.

[0075] The detailed DVS parameters are as follows:

[0076] Temperature: 25℃

[0077] Equilibrium: dm / dt = 0.01% / min (shortest: 10min, longest: 180min)

[0078] Drying: Dry at 0% RH for 120 min

[0079] RH (%) test step: 10%

[0080] RH (%) test range: 0%-90%-0%

[0081] The classification of hygroscopicity is shown in Table 3:

[0082] Table 3. Classification of Hygroscopicity Evaluation

[0083] 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%

[0084] Note: ΔW% represents the moisture gain of the test sample at 25±1℃ and 80±2%RH. Attached Figure Description

[0085] Figure 1 The Cu-Kα radiation XRPD spectrum of compound A of formula (I) is shown.

[0086] Figure 2 The DSC spectrum of compound A of formula (I) is shown.

[0087] Figure 3 The TGA spectrum of compound A of formula (I) is shown.

[0088] Figure 4 The Cu-Kα radiation XRPD spectrum of compound B of formula (I) is shown.

[0089] Figure 5 The DSC spectrum of compound B of formula (I) is shown.

[0090] Figure 6 The TGA spectrum of compound B of formula (I) is shown.

[0091] Figure 7 The DVS spectrum of compound A of formula (I) is shown.

[0092] Figure 8 The results of serum amylase (AMY) level testing for compounds of formula (I);

[0093] Figure 9 The results are the serum lipase (LPS) levels of the compound of formula (I). Detailed Implementation

[0094] 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.

[0095] Example 1: Preparation of compound CC-6

[0096]

[0097] Step 1: Synthesis of compound BB-3-2

[0098] BB-3-1 (2 g, 11.49 mmol) and anhydrous dichloromethane (50 mL) were added to a pre-dried single-necked flask, followed by triethylamine (3.49 g, 34.48 mmol, 4.80 mL), N,N-dimethylaminopyridine (140.43 mg, 1.15 mmol), and 2,6-difluorobenzoyl chloride (4.46 g, 25.29 mmol, 3.19 mL). The mixture was reacted at 40 °C for 3 hours. The crude product was directly concentrated under reduced pressure and purified by rapid column chromatography (petroleum ether:ethyl acetate = 10:1-5:1) to obtain BB-3-2. MS (ESI) m / z: 453.9 [M+H] + .

[0099] Step 2: Synthesis of compound BB-3

[0100] Add BB-3-2 (5 g, 11.01 mmol) and solvents tetrahydrofuran (60 mL) and methanol (60 mL) to a pre-dried flask, followed by 2M sodium hydroxide aqueous solution (24.56 mL). Stir at 25 °C for 1 hour, add 50 mL of water, extract with ethyl acetate (150 mL × 3), combine the organic phases, wash with 20 mL of saturated brine, dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product, and purify by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1-2:1) to obtain BB-3. MS (ESI) m / z: 314 [M+H] + . 1H NMR (400MHz, CDCl3) δppm 9.50 (s, 1H), 8.53 (br s, 1H), 8.34 (d, J = 1.60Hz, 1H), 7.52 (tt, J = 8.40, 6.00Hz, 1H), 7.07 (t, J = 8.00Hz, 2H).

[0101] Step 3: Synthesis of Compound 6-1

[0102] Add raw material 3-2 (0.1 g, 422.84 μmol) and solvent acetonitrile (2 mL) to a pre-dried flask, followed by reagents p-toluenesulfonic acid (218.44 mg, 1.27 mmol), sodium nitrite (58.35 mg, 845.69 μmol), and potassium iodide (175.48 mg, 1.06 mmol). Stir at 25 °C for 0.5 hours. Add 10 mL of saturated sodium bicarbonate aqueous solution to the system, extract with ethyl acetate (30 mL * 3), combine the organic phases, wash with 5 mL of saturated brine, dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product, and purify by silica gel column chromatography (petroleum ether) to obtain 6-1. 1 HNMR (400MHz, CDCl3) δppm 7.75 (s, 1H), 6.94 (s, 1H), 3.66-3.85 (m, 3H).

[0103] Step 4: Synthesis of Compound 6-2

[0104] In a pre-dried flask, raw material 6-1 (0.07 g, 201.51 μmol) and solvent diisopropylamine (2 mL) were added, followed by reagents dichloro(bis(triphenylphosphine)palladium (7.07 mg, 10.08 μmol), cuprous iodide (3.84 mg, 20.15 μmol), and triphenylphosphine (5.29 mg, 20.15 μmol). The mixture was stirred at 25 °C for 0.5 h, followed by the addition of cyclopropylacetylene (13.32 mg, 201.51 μmol, 16.71 μL). The mixture was stirred at 70 °C for 12 h, and 5 mL of water was added to the system. The mixture was extracted with ethyl acetate (20 mL * 3), and the organic phases were combined. The mixture was washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was then purified by silica gel column chromatography (petroleum ether) to obtain 6-2. 1 H NMR (400MHz, CDCl3) δppm 7.32 (s, 1H), 6.99 (s, 1H), 3.78 (s, 3H), 1.35-1.51 (m, 1H), 0.80-0.86 (m, 2H), 0.73-0.79 (m, 2H).

[0105] Step 5: Synthesis of Compound 6-3

[0106] Add raw material 6-2 (0.05 g, 175.09 μmol) and anhydrous ethanol (3 mL) to a pre-dried microwave tube, followed by the addition of reagent p-toluenesulfonic acid monohydrate (33.31 mg, 175.09 μmol). Microwave reaction at 125 °C for 1 hour, then concentrate under reduced pressure and purify by silica gel column chromatography (petroleum ether) to obtain 6-3. 1 H NMR (400MHz, CDCl3) δppm 7.54(s,1H),7.43(s,1H),6.20(s,1H),1.84-2.01(m,1H),0.92-0.98(m,2H),0.85-0.92(m,2H).

[0107] Step 6: Synthesis of Compound 6-4

[0108] In a pre-dried flask, feedstock 6-3 (0.1 g, 368.27 μmol), bis-pinacolborate (140.28 mg, 552.41 μmol), and anhydrous dioxane (2 mL) were added, followed by potassium acetate (108.43 mg, 1.10 mmol), [1,1-bis(diphenylphosphine)ferrocene]palladium dichloromethane (30.07 mg, 36.83 μmol). The mixture was stirred at 100 °C for 12 hours. 5 mL of water was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 6-4. MS (ESI) m / z: 319 [M+H] + .

[0109] Step 7: Synthesis of compound CC-6

[0110] Add raw material 6-4 (0.1 g, 313.87 μmol), BB-3 (65.72 mg, 209.25 μmol), and solvent dioxane (2 mL) / acetonitrile (1 mL) / water (0.5 mL) to a pre-dried flask. Then add reagents potassium carbonate (57.84 mg, 418.49 μmol) and [1,1-bis(diphenylphosphine)ferrocene]palladium dichloromethane (17.09 mg, 20.92 μmol). Stir at 100 °C for 2 hours. Add 5 mL of water to the system, extract with ethyl acetate (30 mL x 3), combine the organic phases, wash with 5 mL of saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the crude product. Perform preparative HPLC analysis (column: Phenomenex Gemini-NX C18 75 x 30 mm x 3 μm; mobile phase: [water (10 mM... [NH4HCO3)-ACN]; ACN%: 50%-80%, 10.5 min) to obtain CC-6 after purification. 1H NMR (400MHz, CDCl3) δppm 9.79 (s, 1H), 8.71 (d, J = 1.60Hz, 1H), 8.44 (br s,1H),7.67(s,1H),7.58(s,1H),7.44-7.56(m,1H),7.08(t,J=8.00Hz,2H),6.38(s,1H),2.01-2.14(m,1H),0.95-1.13(m,4H). FNMR (400MHz, CDCl3) δppm-110.929.

[0111] Example 2: Preparation of compound (I)

[0112]

[0113] Synthesis route:

[0114]

[0115] Step 1:

[0116] The starting compound CC-6 (24.7 g, 58.01 mmol) and N,N-dimethylacetamide (250 mL) were added to the reaction solution, followed by di-tert-butylchloromethyl phosphate (37.51 g, 145.02 mmol), cesium carbonate (47.25 g, 145.02 mmol), and potassium iodide (962.91 mg, 5.80 mmol). The reaction solution was stirred at 40 °C for 16 hours. 2000 mL of water and 300 mL of ethyl acetate were added to the reaction solution, and the mixture was stirred for 3 hours. The mixture was then extracted with ethyl acetate (300 mL × 3), and the organic phases were combined and dried over anhydrous sodium sulfate. The crude product was then concentrated under reduced pressure. The crude product was recrystallized from dichloromethane and n-heptane (dichloromethane:n-heptane = 1:6, 600 mL), concentrated under reduced pressure to obtain the crude product, which was then slurried with ethyl acetate and n-heptane (ethyl acetate:n-heptane = 1:5, 100 mL), filtered, and the filter cake was washed with ethyl acetate and n-heptane (ethyl acetate:n-heptane = 1:5). The filter cake was collected and the residual solvent was removed under vacuum to obtain compound 18-2. MS (ESI) m / z: 438 [M-209] + .

[0117] Step 2:

[0118] Compound 18-2 (2.0 g, 3.09 mmol) and acetonitrile (10 mL) were added to a reaction flask, followed by a buffer solution of disodium hydrogen phosphate and citric acid (pH = 3, 10 mL). The reaction mixture was stirred at 50 °C for 16 hours. After cooling, the mixture was filtered, and then 400 mL of ethyl acetate and 400 mL of deionized water were added. The mixture was allowed to stand for separation, and the organic phase was washed with deionized water (100 mL × 3) until the pH was around 7. A saturated sodium bicarbonate aqueous solution (200 mL) was added to the organic phase, and after standing for separation, the aqueous phase was extracted with ethyl acetate (100 mL × 3), and the organic phase was discarded. 200 mL of ethyl acetate was added to the sodium bicarbonate aqueous phase, and then 1 M potassium bisulfate was slowly added to neutralize to pH = 4. After standing and separation, the aqueous phase was extracted with ethyl acetate (200 mL × 3), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 22-1. MS (ESI) m / z: 438 [M-97] + . 1 H NMR (400MHz, CDCl3) δ8.64-8.91(m,2H),7.51-7.61(m,1H),7.45(s,1H),7.28-7.3(m,1H),6.82(br s,3H),6.29(s,1H),5.84(br s,2H),1.96-2.04(m,1H),0.94-1.06(m,4H).

[0119] Step 3:

[0120] The starting compound 22-1 (0.6 g, 1.03 mmol), acetone (10 mL), and deionized water (1 mL) were added to a reaction flask, followed by the addition of tris(hydroxymethyl)aminomethane (249.58 mg, 2.06 mmol). The reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was filtered, and the filter cake was transferred to a flask and concentrated under vacuum to remove residual solvent, yielding compound (I). 1 H NMR(400MHz,D2O)δ8.75(br s,1H),8.44(br s,1H),7.19-7.36(m,2H),7.13(br s,1H),6.82(br s,2H),6.07(s,1H),5.65(br s, 2H), 3.65 (s, 12H), 1.87 (br d, J = 4.4Hz, 1H), 0.88 ( br d, J = 7.2Hz, 2H), 0.74 ( br d, J = 3.2Hz, 2H). MS(ESI)m / z:438[M+H-340] + ,536[M M+H-242] + .

[0121] Example 3: Preparation of compound (I)

[0122]

[0123] Step 1:

[0124] At 20°C, trifluoroacetic acid (17.5 L) and dichloromethane (8.75 L) were added to the reactor, followed by raw material 1 (3.5 kg, 15.80 mol, 1 equivalent). The temperature was lowered to 0-5°C, and N-iodosuccinimide (4.09 kg, 18.17 mol, 1.15 equivalent) was added in batches. After the addition was complete, the system was slowly heated to 20°C, and the reaction was allowed to proceed for 16 hours. After the reaction was completed, samples were taken for HPLC monitoring. The mixture was allowed to stand, and the supernatant was extracted. 7 L of ethanol was added and the mixture was stirred for 0.5 hours. The resulting suspension was filtered to obtain the crude product. The crude product was then slurried with 14 L of ethanol and stirred for 16 hours. The resulting suspension was filtered to obtain compound 2. 1 HNMR (400MHz, DMSO-d6)7.98(s,1H),7.34(s,1H),3.85(s,3H).

[0125] Step 2:

[0126] 2-Methyltetrahydrofuran (25 L) was added to the reactor at 20 °C, followed by raw material 2 (2.5 kg, 7.2 mol, 1 equivalent), compound 3 (618.43 g, 9.36 mol, 1.3 equivalent), triethylamine (2.18 kg, 21.59 mol, 3 equivalent), and cuprous iodide (68.53 g, 359.84 mmol, 0.05 equivalent). The nitrogen flow was adjusted, and dichlorobis(triphenylphosphine)palladium(II) (101.03 g, 143.94 mmol, 0.02 equivalent) was added. The reaction was carried out at 25 °C for 16 hours. After the reaction was completed, the reaction solution was filtered through diatomaceous earth. The filter cake was washed with 6.5 L of ethyl acetate, and the resulting organic phase was washed with 13 L of 1N potassium hydrogen sulfate solution, 13 L of saturated sodium bicarbonate solution, and 13 L of saturated brine. The solution was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was slurried in 15 L of a mixed solvent of n-heptane and ethyl acetate in a ratio of 20:1. After stirring for 1 hour, the mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 4. 1 H NMR (400MHz, DMSO-d6) 7.50 (s, 1H), 7.38 (s, 1H), 3.82 (s, 3H), 1.49–1.60 (m, 1H), 0.86–0.94 (m, 2H), 0.69–0.76 (m, 2H). This step, the same experimental procedure, was performed on 6 batches.

[0127] Step 3:

[0128] At 20°C, ethanol (25 L) was added to the reactor, followed by raw material 4 (2.5 kg, 8.75 mol, 1 equivalent). Concentrated sulfuric acid (858.65 g, 8.75 mol, 1 equivalent) was then added to the reactor. After the addition was complete, the system was slowly heated to an internal temperature of 80°C, and the reaction was allowed to proceed for 16 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in 20 L of ethyl acetate. The organic phase was washed with saturated sodium bicarbonate aqueous solution (10 L * 2) and saturated brine (6.5 L * 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in 60 L of n-heptane, passed through a silica gel adsorption column, and washed with n-heptane (20 L * 2). The filtrate was concentrated under reduced pressure to obtain compound 5. 1 HNMR(400MHz, DMSO-d6)7.96(s,1H),7.76(s,1H),6.59(s,1H),2.08-2.18(m,1H),1.00-1.07(m,2H),0.87-0.94(m,2H).

[0129] Step 4:

[0130] At 20°C, ethanol (30L) and water (3L) were added to the reactor. Then, raw material 5 (1.5kg, 5.52mol, 1 equivalent), compound 6 (2.04kg, 8.29mol, 1.5 equivalent), and potassium phosphate (2.93kg, 13.81mol, 2.5 equivalent) were added. The nitrogen flow was adjusted, and [1,1-bis(di-tert-butylphosphine)ferrocene]palladium(II) dichloride (180.02g, 276.21mmol, 0.05 equivalent) was added. After the addition was complete, the system was slowly heated to an internal temperature of 80°C, and the reaction was allowed to proceed for 16 hours. After the reaction was completed, 6L of water was added to the reaction solution, and the mixture was concentrated to dryness under reduced pressure. Add 45 L of a 1:1 mixture of n-heptane and ethyl acetate to form a suspension. After stirring for 1 hour, pass the suspension through an adsorption chromatography column. The filter cake is stirred for 0.5 hours with 12 L of the 1:1 mixture of n-heptane and ethyl acetate, and filtered again. This process is repeated twice, and the filtrates are collected. The combined filtrates are washed with 5 x 10 L of water, then with 2 x 10 L of saturated brine. The organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product is slurried with 7.5 L of a 10:1 mixture of n-heptane and ethyl acetate for 16 hours, filtered, and the filter cake is concentrated under reduced pressure to obtain compound 7. 1¹H NMR (400MHz, DMSO-d⁶) 8.17(s, ¹H), 7.96(s, ¹H), 7.64(s, ¹H), 7.59(s, ¹H), 6.60(d, J = 4.4Hz, ³H), 2.10–2.20(m, ¹H), 1.00–1.07(m, ²H), 0.88–0.95(m, ²H). This step, the same experimental procedure, was performed on 5 batches.

[0131] Step 5:

[0132] At 20°C, 8 L of dichloromethane was added to the reactor, followed by raw material 7 (1.6 kg, 5.60 mol, 1 equivalent), and pyridine (1.11 kg, 14.00 mol, 2.5 equivalent). The temperature was lowered to 0-5°C, and a solution of compound 8 (1.05 kg, 5.94 mol, 1.06 equivalent) in 1.6 L of dichloromethane was slowly added dropwise. After the addition was complete, the temperature was slowly raised to 25°C, and the reaction was allowed to proceed for 16 hours. After the reaction was completed, 3.2 L of water was added to the reaction solution and the mixture was stirred for 2 hours. The suspension was filtered, and the filter cake was washed with water (6.4 L * 2) and ethanol (1.6 L) to obtain the crude product. The crude product was then mixed with 6 L of methyl tert-butyl ether and 2 L of ethanol and stirred at room temperature for 16 hours. The mixture was filtered, and the filter cake was concentrated under reduced pressure to obtain compound 9. 1 HNMR(400MHz,DMSO-d6)11.81(s,1H),9.50(s,1H),8.74(s,1H),7.76(s,1H),7.75(s,1H),7.58- 7.67(m,1H),7.27(m,2H),6.67(s,1H),2.13-2.23(m,1H),1.03-1.10(m,2H),0.93-0.98(m,2H).

[0133] Step 6:

[0134] N,N-dimethylacetamide (13 L) was added to a 50 L reactor (15 °C). Then, compound 9 (1.3 kg, 3.05 mol, 1 equivalent) was added to the N,N-dimethylacetamide solution, followed by compound 10 (1.97 kg, 7.63 mol, 2.5 equivalent), cesium carbonate (2.49 kg, 7.63 mol, 2.5 equivalent), and potassium iodide (50.68 g, 305.3 mmol, 0.1 equivalent). The reaction solution was a yellow suspension. After the addition was complete, the system was slowly heated to 50 °C and stirred for 40 hours. HPLC monitoring showed that the reactant content was <5%. The mechanical stirring was turned off, and the reaction solution was transferred to a separatory container containing 50 L of water and 16 L of ethyl acetate and stirred for 3 hours. After stirring, the mixture was allowed to stand and separate into layers. The aqueous phase was extracted with ethyl acetate (16 L), and the organic phase was washed successively with 10 L of water and 10 L of brine. The aqueous and brine phases were then extracted with 5 L of ethyl acetate. After separation, this organic phase was combined with the previously obtained organic phase. The combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a reddish-brown viscous crude product. The crude product was completely dissolved in dichloromethane (6 L), and n-heptane (48 L) was slowly added with stirring. The mixture was stirred for 3 hours, forming a yellow solution and a tar-like reddish-brown solid adhering to the walls. The yellow solution was poured off, and n-heptane (12 L) was added to the solution, with stirring continuing for 16 hours to form a yellow suspension. The yellow suspension was filtered through a benchtop filter, the solid was collected, and residual solvent was removed under vacuum to obtain a yellow powdery crude product. The filtrate obtained after filtration was concentrated under reduced pressure to obtain a yellow, viscous crude product. This crude product was then dissolved in dichloromethane (1.2 L), and n-heptane (18 L) was slowly added under stirring for 16 hours. A large reddish-brown solid was formed. The solvent was poured off, and the reddish-brown solid was slurried with a mixed solution of ethyl acetate and n-heptane (ethyl acetate: n-heptane = 1:1, 400 mL). After stirring for 1 hour, the mixture was filtered. The filter cake was washed with a mixed solution of ethyl acetate and n-heptane (1:4, 200 mL * 3), and the solid was collected. The residual solvent was removed under vacuum to obtain a yellow powdery crude product. The two yellow powdery products were combined, and then a mixed solution of ethyl acetate and n-heptane (ethyl acetate: n-heptane = 1:4, 2 L) was added and slurried for 16 hours to obtain a yellow suspension. The yellow suspension was filtered, the solid was collected, and the residual solvent was removed under vacuum to obtain compound 11. 1 HNMR(400MHz, CDCl3)δ8.33-8.90(m,2H),7.46-7.65(m,2H),7.33(br s,1H),6.87(br s,2H),6.34(s,1H),5.89(br d,J=4.8Hz,2H),1.96-2.10(m,1H),1.29-1.61(m,18H),0.97-1.07(m,4H).LCMS(ESI):m / z:438[M-209]+ .

[0135] Step 7:

[0136] Acetonitrile (8.5 L) was added to a 50 L reactor (15 °C), followed by compound 11 (850 g, 1.314 mol) to the acetonitrile solution. A buffer solution with pH 3 prepared from disodium hydrogen phosphate dihydrate (62.3 g, 0.35 mol, 1.75 L deionized water) and citric acid (141.85 g, 0.675 mol, 6.75 L deionized water) was then added. Heating was initiated, and the system was slowly heated to 46 °C and stirred for 18 hours. 1 M citric acid (800 mL) was added dropwise to the reaction mixture, and heating and stirring continued for another 24 hours after the addition was complete. The reaction was then analyzed by HPLC to confirm completion. After cooling, the reaction mixture was separated into separate solutions. Add (40L) ethyl acetate and (30L) water to the separatory process. After standing and separating the layers, separate the aqueous phase and wash the organic phase with water (10L*3). Combine the aqueous phases and wash with 10L ethyl acetate. Combine the organic phases and add 10L of saturated sodium bicarbonate solution prepared with deionized water to the organic phase. Stir for 1 hour and let stand to separate the layers. If no separation occurs after standing, add 10L of deionized water and separate the layers. Wash the organic phase with 30L water. Combine the aqueous phases with the sodium bicarbonate aqueous phase. Extract the sodium bicarbonate aqueous phase with ethyl acetate (10L) and retain the sodium bicarbonate aqueous phase. 20 L of ethyl acetate was added to the sodium bicarbonate aqueous phase, and then 1 M potassium bisulfate (14 L) prepared with deionized water was slowly added under stirring to neutralize to pH 4. No temperature rise was observed during this process. After stirring for 0.5 hours, the mixture was allowed to stand and separate into layers. The aqueous phase was extracted with ethyl acetate (20 L x 3). The organic phases were combined and dried over anhydrous sodium sulfate. The filtrate was then concentrated under reduced pressure to obtain the crude product compound 12. 1 HNMR(400MHz,CDCl3)δ1H NMR(400MHz,DMSO-d6)δ8.51-9.03(m,2H),7.62-7.78(m,2H),7.51(brd,J=18.8Hz,1H),7.12(br s,2H),6.65(s,1H),5.73(br d,J=4.8Hz,2H),2.11-2.23(m,1H),1.01-1.10(m,2H),0.89-0.96(m,2H).LCMS(ESI):m / z:438[M-97]+.

[0137] Step 8:

[0138] The weighed compound 12 (440 g, 0.858 mol, 1 equivalent) was dissolved in 6.6 L of acetone and added to a 50 L reactor (15 °C). Then, the weighed tris(hydroxymethyl)aminomethane (207.99 g, 1.72 mol, 2 equivalents) was dissolved in 0.66 L of deionized water and added to the reactor all at once. After the addition was complete, the heating was started, and the system was slowly heated to 25 °C and stirred for 16 hours. 1 HNMR showed that the reaction was complete. The reaction solution was filtered, and the filter cake was washed with acetone (1L*2). The filter cake was collected, and the residual solvent was removed under vacuum to obtain compound (I). 1 HNMR(400MHz,DMSO-d6)δ8.87(br s,1H),8.59(br s,1H),7.70(s,2H),7.40-7.56(m,1H),7.09(br s,2H),6.64(s,1H),5.56(br d,J=4.4Hz,2H),5.3(brs,12H),3.31(s,12H),2.12-2.22(m,1H),1.02-1.09(m,2H),0.91-0.98(m,2H).LCMS(ESI):m / z:438[M-339] + .

[0139] Example 4: Preparation of crystal form A of compound (I)

[0140] Compound (I) (35.6 g, 45.01 mmol, 98.37% purity, 1 equivalent) and ethyl acetate and methanol (ethyl acetate:methanol = 3:1, 712 mL) were added to a reaction flask at 25 °C. The reaction mixture was stirred at 60 °C for 16 hours. XRPD analysis showed that the reaction was complete. The reaction mixture was filtered, and the filter cake was collected, concentrated under reduced pressure, and dried. Crystal form A was obtained, and its XRPD spectrum is shown below. Figure 1 As shown, the DSC spectrum is as follows: Figure 2 As shown, the TGA spectrum is as follows: Figure 3 As shown. 1 H NMR(400MHz,DMSO-d6)δ8.87(br s,1H),8.59(br s,1H),7.70(s,2H),7.47-7.50(m,1H),7.10(d,J=7.2Hz,2H),6.64(s,1H),5.51(d,J=4.8Hz,2H),3.85(br s,12H),3.32(s,12H),2.13-2.19(m,1H),1.04-1.07(m,2H),0.93-0.95(m,2H).LCMS(ESI):m / z:438[M-339] + .

[0141] Example 5: Preparation of crystal form B of compound (I)

[0142] A mixed solution (2-methyltetrahydrofuran:methanol = 3:1, 15.6L) consisting of 11.7L of 2-methyltetrahydrofuran and 3.9L of methanol was added to a dry and clean 50L reactor (15℃). Stirring was started, and 520g (0.668mol, 1 equivalent) of compound (I) was added to the reactor. After the addition was complete, heating was started, and the system was slowly heated to 60℃ and stirred for 16 hours. XRPD analysis showed that the reaction was complete. The reaction solution was filtered, and the filter cake was washed successively with 2-methyltetrahydrofuran (2L*2) and methyl tert-butyl ether (2L*2). The filter cake was collected, and residual solvent was removed under vacuum to obtain crystal form B, whose XRPD spectrum is shown below. Figure 4 As shown, the DSC spectrum is as follows: Figure 5 As shown, the TGA spectrum is as follows: Figure 6 As shown. 1 H NMR(400MHz,DMSO-d6)δ8.87(br s,1H),8.59(br s,1H),7.70(d,J=3.2Hz,2H),7.47-7.50(m,1H),7.09(s,2H),6.64(s,1H),5.57(s,2H),5.46(brs,12 H)3.34(s,12H),2.13-2.18(m,1H),1.03-1.06(m,2H),0.93-0.94(m,2H).LCMS(ESI):m / z:438[M-339] + .

[0143] Example 6: Study on the hygroscopicity of crystal form A of formula (I)

[0144] Experimental materials:

[0145] SMS DVS Advantage Dynamic Vapor Adsorption Unit

[0146] Experimental methods:

[0147] Take 10-15 mg of compound A (I) and place it in the DVS sample tray for testing.

[0148] Experimental results:

[0149] The DVS spectrum of compound A of formula (I) is as follows: Figure 7 As shown, △W = 1.308%.

[0150] Experimental conclusion:

[0151] The hygroscopic weight gain of compound A in formula (I) at 25°C and 80% RH is 1.308%, indicating slight hygroscopicity.

[0152] Example 7: Thermodynamic solubility test of compound (I)

[0153] Weigh approximately 2 mg of the test compound into a Whatman vial and add 450 μL of phosphate buffer (50 mM, pH 7.4). Press the Whatman vial stopper close to the liquid surface to ensure uniform contact between the filter membrane in the stopper and the liquid surface. Shake well and vortex for two minutes, recording the dissolution of the test compound in the Whatman vial. Place the Whatman vial on a shaker at room temperature and shake at 600 rpm for 24 hours. Slowly press the stopper of the Whatman vial to the bottom to obtain the supernatant. Check all compounds to ensure there is no precipitate in the supernatant to prevent the filter membrane in the stopper from breaking. Prepare linear solutions (3 standard solutions, 1, 20, 200 μM, n = 1) using diluent. Take out 10 μL of the supernatant and dilute it 100 times. Simultaneously inject the obtained diluent and the original solution with the linear solution into a high-performance liquid chromatograph for analysis. Calculate the results using the external standard method based on the peak area and dilution factor.

[0154] The experimental results are shown in Table 4:

[0155] Table 4: Solubility of the compounds of this invention

[0156] Test compound Thermodynamic solubility (pH: 7.4) Compound of formula (I) >200mg / mL

[0157] Conclusion: Compound (I) has excellent solubility in water.

[0158] Example 8: Solid-state accelerated stability study of compound B of formula (I)

[0159] According to the "Guidelines for Stability Testing of Active Pharmaceutical Ingredients and Preparations" (Chinese Pharmacopoeia 2015 Edition, Part IV, General Chapter 9001), the stability of compound (I) B crystal form under accelerated testing conditions was investigated. Approximately 10 mg of compound (I) B crystal form was weighed and placed at the bottom of a glass sample vial, spreading it into a thin layer. The vial was sealed with aluminum foil and small holes were punched. The vials were placed at (40℃ / 75%RH) and (60℃ / 75%RH) for one month. The samples after storage were characterized by XRPD, and the results were compared with the initial test results at day 0. The results are shown in Table 5. Under all stability conditions, the crystal form of compound (I) B crystal form remained unchanged.

[0160] Table 5: Experimental results of solid stability of compound B of formula (I)

[0161] Test conditions Conditions for taking points Crystallization changes Initial crystallization B / B crystal form 40℃ / 75%RH January B crystal form 60℃ / 75%RH January B crystal form

[0162] Experimental conclusion: Compound B of formula (I) has good stability.

[0163] Biological test data:

[0164] Experimental Example 1: CRAC in vitro cell activity test of the compounds of this invention

[0165] 1. Experimental materials:

[0166] 1.1 Reagents and consumables, see Table 6:

[0167] Table 6. Reagents and Consumables

[0168] Names of reagents and consumables brand Product Number 1 384-hole transparent black microporous plate Corning 3712 2 384-well flat-bottomed transparent microporous plate Greiner 781201 3 384-well pointed-bottom transparent microplate Corning 3656 4 10cm cell culture dish Corning 430167 5 15mL centrifuge tube Corning 430791 6 1.5mL transparent tube Axegen MCT-150-C 7 Fluo-8 Calcium Flow Detection Reagent Abcam Ab112129 8 HEPES Gibco 15630-080 9 Probenecid Thermo P36400 10 Sodium chloride Sinopharm Group 10019318 11 Potassium chloride Sinopharm Group 10016318 12 Sodium bicarbonate Sinopharm Group 10018390 13 Magnesium chloride hexahydrate Sinopharm Group 1001218 14 Calcium chloride Sinopharm Group 10005861 15 Sodium hydroxide Sinopharm Group 10019718 16 glucose Sigma 101185414 17 EGTA Amresco 732 18 MEME cell culture medium Gibco 61100

[0169] 19 FBS serum Biosera FB-1058 / 500 20 DPBS Invitrogen 14190 21 0.25% Trypsin-EDTA Invitrogen 25200 22 DMSO Sigma D5879 23 Penicillin / Streptomycin Biosera 70013

[0170] 1.2 Instruments, see Table 7:

[0171] Table 7. Instruments

[0172] instrument brand 1 Bravo pipetting workstation Agilent 2 Echo 550 Liquid Workstation Labcyte 3 FLIPR Testing Platform MD 4 Cell incubator Thermo 5 benchtop high-speed centrifuge Eppendorf

[0173] 1.3 Cell line: RBL-2H3, derived from the HDB cell bank.

[0174] 2. Experimental steps and methods:

[0175] 2.1 Cell Plating

[0176] 1) Prepare the biosafety cabinet and preheat the relevant reagents. Observe the cells daily, and when 85% of the area of ​​the 10cm culture dish is confluent with cells, perform cell passage.

[0177] 2) Remove the cell culture dish and the culture medium. Wash the cell surface with DPBS and remove the DPBS. Digest the cells with 1 mL of 0.25% Trypsin-EDTA for 1-3 minutes, then add 2 mL of culture medium to stop the digestion. Gently pipette the cells until they detach from the culture dish.

[0178] 3) Adjust the cell density to 15,000 cells per well using growth medium, with a volume of 25 μL of culture medium per well.

[0179] 4) The cell culture plates were placed in an incubator at 37°C and 5% CO2 and cultured until the density reached 80%.

[0180] 2.2 Detection

[0181] 1) Remove the cell culture plate from the incubator, invert and centrifuge at 300 rpm for 30 seconds to remove the culture medium, add 20 μL of buffer (ultrapure water, 40 mM sodium chloride, 100 mM potassium chloride, 17 mM sodium bicarbonate, 0.1 mM ethylene glycol diaminoethyl ether tetraacetic acid (EGTA), 12 mM glucose, 1 mM magnesium chloride, 5 mM hydroxyethylpiperazine ethanethioic acid (Hepes), 2.5 mM probenecid, 2 μM FLOO8) to each well, and incubate for 30 minutes.

[0182] 2) Preparation of the compound plate. The compound was dissolved in DMSO. According to the required concentration, the compound was prepared in a compound plate (Greiner784201) using an Echo550 and dissolved in a calcium-free buffer (ultrapure water, 40mM NaCl, 100mM KCl, 17mM NaHCO3, 12mM glucose, 1mM MgCl2, 5mM cell culture medium, 4μM thapsigargin). 10μL of the compound was added to the cell culture plate using FLIPR and incubated at room temperature for 20 minutes. Conclusion: The compound of this invention has a significant inhibitory effect on KDM5A.

[0183] 3) Prepare an induction buffer containing calcium ions (4mM CaCl2, 40mM NaCl, 100mM KCl, 17mM NaHCO3, 12mM glucose, 1mM MgCl2, 5mM Hepes), add 10μL of the induction buffer to the cell culture plate using FLIPR, and collect calcium flow signals for 260 seconds.

[0184] Data processing: ScreenWorks, Excel, XLfit, and GraphPad were used to analyze and plot the acquired signal results. The experimental results are shown in Table 8.

[0185] Table 8: FLIPR assay results for RBL-3H cell inhibition of Ca 2+ IC 50 Test Results

[0186] Test compound <![CDATA[IC 50 (nM)]]> Compound CC-6 136

[0187] Conclusion: Compound CC-6 has a significant inhibitory effect on CRAC channels.

[0188] Experimental Example 2: Pharmacokinetic Evaluation in Mice

[0189] Experimental Objective: Using male C57BL / 6 mice as test animals, this study employed LC / MS / MS to determine the plasma drug concentrations of the test compound at different time points after intravenous or intraperitoneal administration. The aim was to investigate the pharmacokinetic behavior of the test compound in mice and evaluate its pharmacokinetic characteristics.

[0190] Drug preparation: Weigh an appropriate amount of sample and prepare a clear solution of 0.3 mg / mL or 0.5 mg / mL using 40% PEG400 + 20% Solutol + 40% H2O (volume ratio).

[0191] Dosage regimen: Two healthy male C57BL / 6 mice, purchased from Beijing Vital River Laboratory Animal Co., Ltd., were used and fed a normal diet. The intravenous administration group received a 0.5 mg / mL solution, with an administration volume of 2 mL / kg; the dosage was 1 mg / kg. The intraperitoneal administration group received a 0.3 mg / mL solution, with an administration volume of 10 mL / kg; the dosage was 3 mg / kg.

[0192] Procedure: After animal administration, 25 μL of blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours and placed in commercially available anticoagulant tubes pre-filled with EDTA-K2. The plasma was separated by centrifugation for 10 minutes and stored at -60°C. The concentration of the target compound in the plasma sample was determined by LC / MS / MS.

[0193] The experimental results are shown in Table 9.

[0194] Table 9: Pharmacokinetic parameters of compound CC-6 in mice

[0195]

[0196] Conclusion: In the pharmacokinetic evaluation experiment in mice, compound CC-6 administered via intravenous and intraperitoneal routes exhibited high plasma exposure and ideal pharmacokinetic properties.

[0197] Experimental Example 3: Pharmacokinetic Evaluation in Mice

[0198] Experimental Objective: Using male C57BL / 6 mice as test animals, plasma drug concentrations of a compound at different time points after intravenous administration were determined by LC / MS / MS. The pharmacokinetic behavior of the compound in mice was investigated, and its pharmacokinetic characteristics were evaluated.

[0199] Drug preparation: Weigh an appropriate amount of sample and prepare a clear solution of 5 mg / mL with sterile physiological saline.

[0200] Dosage regimen: Two healthy male C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Co., Ltd. They were fed a normal diet, and the administration volume was 10 mL / kg; the dosage was 50 mg / kg.

[0201] Procedure: After animal administration, 25 μL of blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours and placed in commercially available anticoagulant tubes pre-filled with EDTA-K2. The plasma was separated by centrifugation for 10 minutes and stored at -60°C. The concentrations of the corresponding compounds in the plasma samples were determined by LC / MS / MS.

[0202] Experimental results: see Table 10.

[0203] Table 10: Pharmacokinetic parameters of the compounds in mice

[0204]

[0205] Note: NA indicates that the data does not exist, and DNAUC represents the exposure normalized to molar dose.

[0206] Conclusion: In the pharmacokinetic evaluation experiment in mice, compound (I) was rapidly eliminated from plasma after administration. Simultaneously, a large amount of compound CC-6 was detected as early as 5 minutes after administration, and the in vivo exposure of compound CC-6 was comparable after administration of compound (I) at the same molar dose.

[0207] Experiment Example 4: Efficacy Experiment of Frog Skin Extract-Induced Acute Pancreatitis in Mice

[0208] Experimental Objective: To investigate the efficacy of compound (I) in treating acute pancreatitis by intraperitoneal injection of basal saponin in male C57BL / 6 mice.

[0209] Drug preparation: Weigh an appropriate amount of sample and prepare a clear solution of compound (I) with sterile physiological saline at a concentration of 5 mg / mL.

[0210] Experimental protocol: Healthy male C57BL / 6 mice were used to induce a pancreatitis model via intraperitoneal injection of 50 μg / kg of phytospermia extract, administered seven times at 1-hour intervals. One hour after the seventh injection, serum samples were collected to measure amylase and lipase levels. Compound (I) was administered intravenously in four groups (G1-G4). G1 was the healthy control group, G2 was the model group, and G3-4 were the treatment groups. For G1-3, the first dose of phytospermia extract or solvent was administered 0.5 hours before the first injection, and the second dose was administered 0.5 hours after the fourth injection. For G4, the first dose was administered 0.5 hours before the first injection, without a second dose. G3: 25 mg / kg, twice daily (bid); G4: 50 mg / kg, once daily (qd).

[0211] Experimental results: see Figure 8Plasma amylase levels, analyzed by one-way ANOVA, showed that *** represented a significant difference from G1 (p < 0.001); ### represented a significant difference from G2 (p < 0.001); and ¥ represented a significant difference from G3 (p < 0.05). Figure 9 Plasma lipase levels, according to one-way ANOVA, *** represents a significant difference from G1 (P<0.001); ### represents a significant difference from G2 (P<0.001); ￥￥￥ represents a significant difference from G3 (p<0.001).

[0212] Conclusion: In a mouse model of acute pancreatitis induced by tazocin, compound (I) significantly reduced serum amylase (AMY) and lipase (LPS) levels, indicating that it can significantly improve the typical symptoms of acute pancreatitis and shows excellent potential for treating acute pancreatitis.

Claims

1. The A-crystal form of the compound of formula (I), ， characterized in that Its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.892±0.200°, 12.617±0.200°, 16.793±0.200°, 17.583±0.200°, 18.959±0.200°, 22.113±0.200°, 25.029±0.200°, 26.512±0.200°.

2. The A-type crystal according to claim 1, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.892±0.200°, 11.883±0.200°, 12.617±0.200°, 13.206±0.200°, 16.793±0.200°, 17.583±0.200°, 18.018±0.200°, 18.959±0.200°, 22.113±0.200°, 25.029±0.200°, 25.858±0.200°, 26.512±0.200°.

3. The A-type crystal according to claim 2, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.616°, 4.474°, 8.892°, 9.571°, 10.288°, 10.533°, 11.076°, 11.883°, 12.617°, 13.206°, 13.653°, 14.886°, 15.788°, 16.301°, 16.793°, 17.58°. 3°, 18.018°, 18.959°, 19.574°, 19.981°, 20.661°, 20.964°, 21.651°, 22.113°, 23.031°, 23.747°, 24.107°, 25.029°, 25.414°, 25.858°, 26.512°, 28.797°, 30.039°, 31.672°, 32.596°, 35.275°.

4. The XRPD pattern of the A crystal form according to any one of claims 1 to 3 is shown in Figure 1.

5. The A-type crystal according to any one of claims 1 to 3, wherein the differential scanning calorimetry curve has peak values ​​of endothermic peaks at 138.4°C±3℃ and 163.4°C±3℃.

6. The A-type crystal according to claim 5, its DSC spectrum is shown in Figure 2.

7. The A crystal form according to any one of claims 1 to 3, wherein the thermogravimetric analysis curve shows a weight loss of 3.33% at 140.0°C ± 3°C.

8. The TGA spectrum of the A crystal form according to claim 7 is shown in Figure 3.

9. The B-crystal form of compound (I), ， characterized in that Its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.389±0.200°, 8.797±0.200°, 13.168±0.200°, 13.991°±0.200°, 16.739±0.200°, 17.578±0.200°, 18.811±0.200°, 21.997±0.200°, 26.443±0.200°, and 26.942°±0.200°.

10. The B-type crystal according to claim 9, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.389±0.200°, 8.797±0.200°, 11.786±0.200°, 13.168±0.200°, 13.991°±0.200°, 16.739±0.200°, 17.578±0.200°, 18.811±0.200°, 20.617±0.200°, 21.997±0.200°, 24.970±0.200°, 25.804±0.200°, 26.443±0.200°, 26.942°±0.200°.

11. The B-type crystal according to claim 10, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.389°, 8.797°, 9.473°, 10.172°, 10.356°, 11.786°, 12.569°, 13.168°, 13.991°, 15.710°, 16.739°, 17.578°, 17.949°, 18.811°, 19.577°, 19.875°, 20.617°, 20.849°, 21.997°, 24.970°, 25.804°, 26.443°, 26.942°, 27.841°, 28.731°, 30.033°, 30.962°.

12. The B crystal form according to any one of claims 9 to 11, its XRPD pattern is shown in Figure 4.

13. The B crystal form according to any one of claims 9 to 11, wherein the differential scanning calorimetry curve has an initial value of an endothermic peak at 165.7°C ± 5°C.

14. The B crystal form according to claim 13, its DSC spectrum is shown in Figure 5.

15. The B crystal form according to any one of claims 9 to 11, wherein the thermogravimetric analysis curve shows a weight loss of 1.14% at 150°C ± 3°C.

16. The B crystal form according to claim 15, its TGA spectrum is shown in Figure 6.

17. The use of crystal form A according to any one of claims 1 to 8 or crystal form B according to any one of claims 9 to 16 in the preparation of a drug for treating acute pancreatitis.

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