Crystals of isochroman compounds

Abstract

The present invention relates to a crystal of an isochroman compound and a method for preparing the same, specifically to a compound of formula (I) and its use in the manufacture of a medicament for treating related diseases. [Formula 1] JPEG2025510593000023.jpg28169

Description

Detailed Description of the Invention

[0001] This application claims priority from Chinese Patent Application No. 2022102345275, filed on March 10, 2022. This application cites the above Chinese patent application in its entirety.

[0002] [Technical Field] The present invention relates to crystals of isochroman compounds and methods for preparing the same, in particular to compounds of formula (I) and their use in the manufacture of medicaments for treating related diseases.

[0003] [Background technology] Malignant tumors are serious diseases that pose a serious threat to human life and health. Current treatments mainly include surgery, chemotherapy, and targeted therapy. Chemotherapy is a systemic treatment that uses chemical drugs to kill tumor cells and inhibit their proliferation. Due to the heterogeneity of malignant tumors, chemotherapy remains an important method for treating tumors. However, this systemic treatment causes significant side effects. There is a significant unmet clinical need for the development of drugs with targeted effects.

[0004] Aldo-keto reductase (AKR1C3) is a member of the aldo-keto reductase family and is primarily involved in hormone synthesis and toxin clearance. AKR1C3 can be overexpressed by factors such as smoking, alcohol, and hepatitis B or C infection. AKR1C3 is overexpressed in various intractable cancers, including liver cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, prostate cancer, and acute lymphoblastic leukemia, particularly liver cancer, with overexpression rates exceeding 60%.

[0005] Currently, AKR1C3 inhibitors are being developed in clinical settings, but little progress has been made. Haoding has reported a compound targeting the AKR1C3 enzyme, OBI-3424. OBI-3424 is a selective prodrug that releases a potent DNA alkylating agent in tumor cells that highly express the AKR1C3 enzyme, selectively killing these cells and providing the chemical with a significant targeting effect.

[0006] [ka] Currently, research into this target is still in its early stages, with only OBI-3424 having entered Phase 1 / 2 clinical trials, with its main indications being hepatocellular carcinoma (HCC), castration-resistant prostate cancer (CRPC), pancreatic cancer, and T-cell acute lymphoblastic leukemia (T-ALL), and its efficacy and safety are still being verified. Therefore, this field still requires further exploration and research.

[0007] Summary of the Invention A type crystal of the compound represented by formula (I). [ka] Its powder X-ray diffraction spectrum using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ angles: 8.25±0.20°, 19.21±0.20°, and 21.76±0.20°.

[0008] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (I) has characteristic diffraction peaks in its powder X-ray diffraction spectrum at the following 2θ angles: 8.25±0.20°, 13.27±0.20°, 15.19±0.20°, 16.43±0.20°, 17.94±0.20°, and 19.21±0.20°.

[0009] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (I) has characteristic diffraction peaks in its powder X-ray diffraction spectrum at the following 2θ angles: 8.25±0.20°, 13.27±0.20°, 15.19±0.20°, 16.43±0.20°, 17.94±0.20°, 19.21±0.20°, and 21.76±0.20°.

[0010] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (I) has characteristic diffraction peaks in its powder X-ray diffraction spectrum at the following 2θ angles: 8.25±0.20°, 15.19±0.20°, 16.43±0.20°, 17.94±0.20°, 19.21±0.20°, 21.76±0.20°, 23.56±0.20°, and 26.66±0.20°.

[0011] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (I) has a powder X-ray diffraction spectrum exhibiting the following 2θ angles: 8.25±0.20°, 12.56±0.20°, 12.89±0.20°, 13.27±0.20°, 15.19±0.20°, 16.01±0.20°, 16.43±0.20°, 17.60±0.20°, 17.94±0.20°, 19.21±0.20°, 19.91±0.20°, 20.21±0.20°, 20.52±0.20°, 21.25±0.20°, 21.76±0.20°, 23.02±0.20°, 23. It has characteristic diffraction peaks at 56±0.20°, 24.71±0.20°, 25.12±0.20°, 25.88±0.20°, 26.66±0.20°, 27.10±0.20°, 27.43±0.20°, 27.84±0.20°, 28.38±0.20°, 29.13±0.20°, 30.01±0.20°, 30.83±0.20°, 31.69±0.20°, 32.26±0.20°, 33.35±0.20°, 35.01±0.20°, 35.52±0.20°, 36.51±0.20°, 37.70±0.20°, and 39.24±0.20°.

[0012] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (I) has a powder X-ray diffraction spectrum exhibiting the following 2θ angles: 8.25°, 12.56°, 12.89°, 13.27°, 15.19°, 16.01°, 16.43°, 17.60°, 17.94°, 19.21°, 19.91°, 20.21°, 20.52°, 21.25°, 21.76°, It has characteristic diffraction peaks at 23.02°, 23.56°, 24.71°, 25.12°, 25.88°, 26.66°, 27.10°, 27.43°, 27.84°, 28.38°, 29.13°, 30.01°, 30.83°, 31.69°, 32.26°, 33.35°, 35.01°, 35.52°, 36.51°, 37.70°, and 39.24°.

[0013] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (I) has a powder X-ray diffraction spectrum having characteristic diffraction peaks at the following 2θ angles: 8.25±0.20°, 19.21±0.20°, 21.76±0.20°, and / or 12.56±0.20°, and / or 12.89±0.20°, and / or 13.27±0.20°, and / or 15.19±0.20°. ±0.20°, and / or 16.01±0.20°, and / or 16.43±0.20°, and / or 17.60±0.20°, and / or 17.94±0.20°, and / or 19.91±0.20°, and / or 20.21±0.20°, and / or 20.52±0.20°, and / or 21.25±0.20°, and / or 23.02±0.20°, and / or 23.56±0. 20°, and / or 24.71±0.20°, and / or 25.12±0.20°, and / or 25.88±0.20°, and / or 26.66±0.20°, and / or 27.10±0.20°, and / or 27.43±0.20°, and / or 27.84±0.20°, and / or 28.38±0.20°, and / or 29.13±0.20°, and / or 30.01±0.20° , and / or 30.83±0.20°, and / or 31.69±0.20°, and / or 32.26±0.20°, and / or 33.35±0.20°, and / or 35.01±0.20°, and / or 35.52±0.20°, and / or 36.51±0.20°, and / or 37.70±0.20°, and / or 39.24±0.20°.

[0014] In some embodiments of the present invention, the analytical data of the powder X-ray diffraction spectrum of the A-type crystal of the compound represented by formula (I) is as shown in Table 1. [Table 1]

[0015] In some embodiments of the present invention, the powder X-ray diffraction spectrum of the A-type crystal of the compound represented by formula (I) is essentially as shown in FIG.

[0016] In some embodiments of the present invention, the A-type crystals of the compound represented by formula (I) have an endothermic peak value at 89.2±3°C in the differential scanning calorimetry curve.

[0017] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (I) has a DSC spectrum essentially as shown in FIG.

[0018] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (I) exhibits a weight loss of 1.03% at 100±3°C in its thermogravimetric analysis curve.

[0019] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (I) has a TGA spectrum essentially as shown in FIG.

[0020] A method for producing type A crystals of the compound of formula (I), comprising the steps of adding a compound of formula (I) in any form (crystalline or amorphous) to a solvent, stirring at a predetermined temperature for a predetermined time, filtering, and drying the cake to obtain type A crystals. [ka]

[0021] In some embodiments of the present invention, the solvent is selected from alcohols, acetone, methyl isobutyl ketone, ethyl acetate, isopropyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran, toluene, dichloromethane, 1,4-dioxane, acetonitrile, and alkanes.

[0022] In some embodiments of the invention, the solvent is a mixture of any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of alcohols, acetone, methyl isobutyl ketone, ethyl acetate, isopropyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran, toluene, dichloromethane, 1,4-dioxane, acetonitrile, and alkanes.

[0023] In some embodiments of the present invention, the alcohol is selected from ethanol, isopropanol, and n-butanol.

[0024] In some embodiments of the present invention, the alkane is selected from n-hexane, n-heptane, and cyclohexane.

[0025] In some embodiments of the present invention, the predetermined temperature is selected from the range of 0°C to 65°C.

[0026] In some embodiments of the present invention, the predetermined time is selected from the range of 1 hour to 72 hours.

[0027] In some embodiments of the present invention, the weight ratio of the compound represented by the predetermined formula (I) to the solvent is selected from the range of 1:1-30.

[0028] The present invention further provides use of type A crystals of the compound represented by formula (I) or type A crystals produced by the method described above in the manufacture of a medicament for treating liver cancer, prostate cancer, pancreatic cancer and / or T-cell acute lymphoblastic leukemia.

[0029] [Technical effect] The compound of the present invention has significant antiproliferative activity against tumor cells highly expressing AKR1C3 enzyme, and has shown significant antitumor effects in multiple in vivo efficacy models. The crystal form A of the compound of the present invention is easy to prepare, has good physical and chemical stability, and has high industrial applicability and economic value.

[0030] [Definitions and Explanations] Unless otherwise stated, the following terms and phrases used herein have the following meanings. Unless otherwise defined, a particular phrase or term should be understood to have its ordinary definition, rather than being indefinite or unclear. When a trade name appears in this specification, it refers to the corresponding product or its active ingredient.

[0031] The intermediate compounds of the present invention can be prepared by various synthetic methods familiar to those skilled in the art, including the specific embodiments listed below, embodiments in combination with other chemical synthetic methods, and equivalent alternative methods familiar to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

[0032] The chemical reactions of specific embodiments of the present invention are completed in suitable solvents, which should be suitable for the chemical reactions of the present invention and the reagents and materials required therefor. To obtain the compounds of the present invention, those skilled in the art may need to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0033] The present invention will be specifically described below with reference to examples, but these examples do not limit the present invention in any way.

[0034] All solvents used in this invention are commercially available and may be used as is without further purification.

[0035] Compounds are named manually or by ChemDraw® software, and commercially available compounds are named by manufacturer's catalogue name.

[0036] The present invention uses the following abbreviations: K2CO3 represents potassium carbonate, [Ru(p-cym)Cl2]2 represents (p-cymene)ruthenium(II) chloride dimer, EtOH represents ethanol, MTBE represents tert-butyl methyl ether, DCM represents dichloromethane, PE represents petroleum ether, DIBAL-H represents diisobutylaluminum hydride, DMF represents N,N-dimethylformamide, DMSO represents dimethyl sulfoxide, EtOAc represents ethyl acetate, MeOH represents methanol, and n-heptane represents n-heptane. SiO2 represents 100-200 mesh silica gel powder for column chromatography, Et3SiH represents triethylsilane, HOAc represents acetic acid, NaIO4 represents sodium periodate, THF represents tetrahydrofuran, DIPEA represents N,N-diisopropylethylamine, (S)-CBS represents (S)-3,3-diphenyl-1-methylpyrrolidino[1,2-c]-1,3,2-oxaborole, K3PO4 represents potassium phosphate, HP-β-CD represents 2-hydroxypropyl-β-cyclodextrin, and QW represents the frequency of administration.

[0037] X-ray powder diffractometer (XRPD) method of the present invention Instrument model: Dandong Haoyuan DX-2700BH X-ray diffractometer Test method: Approximately 10-20 mg of sample is used for XRPD detection.

[0038] The detailed XRPD parameters are as shown below. X-ray tube: Cu, kα, (λ=1.54184Å). Tube voltage: 40 kV, tube current: 30 mA Divergence slit: 1mm Detector slit: 0.3 mm Anti-scatter slit: 1mm Scanning range: 3~40deg Step angle: 0.02 deg Step width: 0.5 seconds

[0039] Differential Scanning Calorimetry (DSC) Method of the Present Invention Instrument model: METTLER TOLEDO DSC1 Differential Scanning Calorimeter Test method: The test is performed by placing a sample (2-6 mg) in a 30 μL DSC gold-plated high-pressure crucible and heating the sample from 40°C to 350°C at a heating rate of 10°C / min.

[0040] The Thermogravimetric Analysis (TGA) Method of the Present Invention Instrument model: TA TGA550 Thermogravimetric Analyzer Test method: The test is performed by placing a sample (2-10 mg) in an aluminum crucible, then placing it in a platinum basket. The sample is heated to 40-500°C under nitrogen gas (N2) conditions at a gas flow rate of 40 mL / min and a heating rate of 10°C / min.

[0041] Single crystal X-ray diffraction detector parameters of the present invention Instrument model: Bruker D8 VENTURE CMOS Photon II diffractometer. Cryogenic system: Oxford Cryostream 800 Light source: Cu:λ=1.54184Å, 2.5kW Distance from crystal to detector: d = 45 mm Tube voltage: 50kV Tube current: 50mA.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an XRPD spectrum of Cu-Kα radiation of the A-type crystal of the compound represented by formula (I). FIG. 2 is a DSC spectrum of type A crystals of the compound represented by formula (I). FIG. 3 is a TGA spectrum of type A crystals of the compound represented by formula (I). FIG. 4 is a crystal X-ray diffraction conformation ellipsoid diagram of the compound of formula (I). FIG. 5 is a tumor growth signal-time curve. FIG. 6 is a schematic representation of tumor weights at the end of the experiment. FIG. 7 shows the animal weight-time curve.

[0043] [Mode for Carrying Out the Invention] The present invention will be specifically described below using examples, but this does not mean to limit the present invention in any way. The compounds of the present invention can be prepared by various synthetic methods familiar to those skilled in the art, including the specific embodiments listed below, embodiments combined with other chemical synthetic methods, and equivalent alternative methods familiar to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention. 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 of the present invention.

[0044] Example 1: Preparation of compounds of formula (I) [ka]

[0045] Step A: DMF (30 L) was added to a reaction kettle and mechanical stirring was initiated. Next, starting material 1 (4 kg, 35.37 mol) and starting material 2 (6.6 kg, 35.71 mol) were added. After the addition was complete, the internal temperature was controlled to rise to 30-40°C, and K2CO3 (12.2 kg, 88.27 mol) was added in batches. Gas was generated, and the internal temperature was controlled at 40-50°C. After the addition was complete, the mixture was stirred at 45-55°C for 16 hours. 42 L of ice water was added to the reaction solution, and 42 L of 4 mol / L hydrochloric acid was slowly added with stirring. A large amount of yellow solid precipitated, which was suction filtered under reduced pressure and washed with 45 L of water. The cake was dried in vacuo to obtain intermediate 3. 1H NMR (400 MHz, DMSO-d6) δ 13.81 - 13.38 (m, 1H), 8.55 (d, J = 2.1 Hz, 1H), 8.24 - 8.12 (m, 2H), 7.97 (ddd, J = 1.6, 8.1, 10.0 Hz, 1H), 7.50 (ddd, J = 0.7, 4.8, 7.9 Hz, 1H), 7.29 (d, J = 8.7 Hz, 1H).

[0046] Step B: 1,4-Dioxane (27 L) and HO (2.7 L) were added to a reaction kettle, and with stirring, Intermediate 3 (3230.60 g, 11.613 mol), Intermediate 4 (3230.60 g, 11.613 mol), guanidine carbonate (1047.08 g, 5.812 mol), and HOAc (662 mL, 11.575 mol) were added sequentially. Nitrogen gas was continuously bubbled through the kettle for 15 minutes. [Ru(p-cym)Cl] (355.37 g, 0.580 mol) was added in one portion, and the mixture was heated to 90 °C and stirred for 17 hours. The reaction solution was cooled to 25 °C, and 2.7 L of water was added. The mixture was allowed to stand at approximately 5 °C for approximately 23 hours, at which point a solid precipitated. The cake was filtered, and the cake was added to 10 L of EtOH / HO (volume ratio 1:1), stirred at room temperature for 4 hours, and filtered under suction. The cake was added to 8 L of EtOAc / MTBE (volume ratio 2:1), stirred at room temperature for 17 hours, and filtered under suction. The cake was washed with EtOAc / MTBE (volume ratio 2:1) (1 L x 2), and dried under vacuum to obtain intermediate 5. 1 H NMR (400 MHz, DMSO-d6) δ 8.64 (s, 1H), 8.18 - 8.11 (m, 1H), 7.92 (ddd, J = 1.4, 8.2, 10.0 Hz, 1H), 7.80 (s, 1H), 7.60 (s, 1H), 7.51 - 7.40 (m, 4H), 7.35 (d, J = 6.9 Hz, 2H), 5.42 (d, J = 0.9 Hz, 2H).

[0047] Step C: Anhydrous DCM (22 L) was added to a reaction kettle, and Intermediate 5 (2200 g, 5.608 mol) was added with stirring. The reaction temperature was cooled to -60 to -50 °C under nitrogen gas protection. DIBAL-H (6700 mL, 6.7 mol) was added dropwise to the kettle. After the addition was complete, the internal temperature was -60 to -50 °C, and stirring was continued for 1.5 h under nitrogen gas protection. MeOH (455 mL) was added dropwise to the kettle, and the internal temperature was controlled at -60 to -50 °C. After the addition was complete, the internal temperature was slowly raised to 0 °C, and isopropanol (7.3 L), dichloromethane (15 L), and L(+) potassium sodium tartrate tetrahydrate aqueous solution (3.5 kg, 22 L of aqueous solution) were added sequentially. After the addition was complete, the mixture was stirred at room temperature for 4 hours, allowed to stand for 12 hours, allowed to separate, and the organic phase was concentrated to give a yellow solid product which was dried in vacuo to give intermediate 6. 1 H NMR (400 MHz, DMSO-d6) δ 8.10 (s, 1H), 8.03 - 8.00 (m, 1H), 7.98 (s, 1H), 7.61 (ddd, J = 1.4, 8.2, 10.1 Hz, 1H), 7.52 (s, 1H), 7.48 - 7.41 (m, 2H), 7.40 - 7.33 (m, 4H), 7.29 (d, J = 5.8 Hz, 1H), 5.94 (d, J = 5.6 Hz, 1H), 4.94 (dd, J = 1.3, 14.4 Hz, 1H), 4.69 (dd, J = 0.9, 14.4 Hz, 1H).

[0048] Step D: DCM (9.4 L) was added to a reaction kettle, and while stirring, Intermediate 6 (1880.00 g, 4.767 mol) and EtSiH (2284 mL, 14.300 mol) were added. Nitrogen flow protection was turned on, and after the addition was complete, the internal temperature was 0-5°C. Boron trifluoride ethyl etherate (1765 mL, 614.301 mol) was added dropwise, and the internal temperature was controlled at 0-10°C, with stirring continued for 1 hour. Aqueous KCO solution (1.05 kg dissolved in 12 L of water) was added in batches to the reaction solution. After the addition was complete, stirring continued for 20 minutes. The reaction mixture was suction filtered under reduced pressure to obtain a cake. The filtrate was separated, and the resulting organic phase was concentrated to 5 L. A yellow solid precipitated, which was filtered. The resulting cake was combined with the previous cake. The cake was dried under vacuum to obtain Intermediate 7. 1 H NMR (400 MHz, DMSO-d6) δ 8.08 (s, 1H), 8.04 (s, 1H), 7.99 (td, J = 1.5, 4.8 Hz, 1H), 7.58 (ddd, J = 1.6, 8.2, 10.2 Hz, 1H), 7.52 (s, 1H), 7.48 - 7.40 (m, 2H), 7.38 - 7.30 (m, 4H), 4.84 (s, 2H), 4.76 (d, J = 1.3 Hz, 2H).

[0049] Step E: To a reaction kettle was added DCM (7.0 L), MeCN (7.0 L), and water (10.5 L). While stirring, Intermediate 7 (1740.00 g, 4.599 mol) and ruthenium(III) chloride trihydrate (12.03 g, 0.0460 mol) were added, controlling the internal temperature at 10-20 °C. NaIO (2147.72 g, 14.717 mol) was added in batches over a 2-hour period, controlling the internal temperature at 10-20 °C. Stirring was continued for 0.5 hours after the addition was complete. DCM (14.0 L) was added to the reaction solution, which was stirred for 5 minutes. The reaction solution was then filtered through diatomaceous earth to remove excess inorganic salts. The filtrate was separated, and the organic phase was added with aqueous NaOH (8 L, 1 mol / L), filtered through diatomaceous earth, and separated. The organic phase was washed sequentially with 8 L of saturated aqueous sodium sulfite and 8 L of saturated brine. The organic phase was concentrated to give the crude product. The crude product was purified by column chromatography (SiO, n-heptane: EtOAc = 100:0 to 10:1) to give intermediate 8. 1 H NMR (400 MHz, CDCl3) δ 8.05 (td, J = 1.6, 4.8 Hz, 1H), 7.76 (s, 1H), 7.52 (ddd, J = 1.6, 7.8, 9.5 Hz, 1H), 7.45 (s, 1H), 7.22 (ddd, J = 0.7, 4.8, 7.8 Hz, 1H), 4.86 (s, 2H), 4.32 (s, 2H).

[0050] Step F: Under nitrogen gas protection, 450 mL of anhydrous THF and 147.6 mL of (S)-CBS were added to the reaction kettle and cooled to 0-10°C. 296.2 mL of borane dimethyl sulfide complex (10 M) was added dropwise to the reaction system and allowed to react with magnetic stirring for 1 hour. A THF solution of intermediate 8 (preparation method: 450 g of intermediate 7 was weighed and dissolved in 1800 mL of THF) was slowly added dropwise to the reaction system, and the temperature was controlled at 0-10°C for 1 hour. After the reaction was complete, 180 mL of MeOH was slowly added dropwise to quench the reaction. 2250 mL of 2.0 mol / L hydrochloric acid was weighed and slowly added dropwise to the reaction system, resulting in the precipitation of a large amount of solid. The cake was filtered under reduced pressure for further use. The filtrate was separated, the organic phase separated, and the aqueous phase extracted with 4500 mL of EtOAc. The combined organic phases were concentrated under reduced pressure at 35-40°C until a solid was obtained. The concentrate and cake were combined and added to 1350 mL of a 2:1 ethanol / 2.0 M hydrochloric acid solution, heated to 60-70°C, and stirred for 1 hour. The reaction solution was cooled to 20-25°C, filtered under reduced pressure, and the cake was rinsed with 900 mL of purified water. The wet cake was added directly to 1350 mL of a 1:1 ethanol / purified water solution and stirred at 20-25°C for 2 hours. The cake was filtered under reduced pressure and rinsed with 900 mL of purified water. The cake was placed on a tray in a vacuum drying oven and dried under vacuum at 40°C to obtain Intermediate 9.

[0051] Step G: Under nitrogen gas protection, 137 g of Intermediate 9 and 2055 mL of anhydrous THF were placed in a 5 L single-layer glass reaction kettle and cooled to -40 to -30 °C with magnetic stirring. The reaction temperature was controlled at -40 to -30 °C, and 56.7 mL of phosphorus oxychloride was added in one portion. 106.3 mL of 2-tert-butyl-1,1,3,3-tetramethylguanidine was then slowly added dropwise. After the addition was complete, the reaction mixture was cooled to -50 to -40 °C, and 548 g of 2-bromoethylamine hydrobromide was added in one portion. 467.3 mL of DIPEA was then slowly added dropwise. After the addition was complete, the reaction mixture was maintained at -50 to -40 °C and allowed to react for 12 hours while maintaining the temperature at -50 to -40 °C. The reaction was quenched by slowly adding 2055 mL of purified water dropwise. The mixture was allowed to warm to 0-10°C and allowed to stand for phase separation. The organic layer was separated and concentrated, and the aqueous layer was extracted once with 1781 mL of water. The concentrated solution and the extracted solution were combined and washed sequentially with 1096 mL of 0.1 M hydrochloric acid, 1096 mL of 1% aqueous K3PO4, and 1096 mL of saturated brine. The organic layer was separated and dried over 274 g of anhydrous sodium sulfate. The mixture was suction filtered under vacuum, and the cake was rinsed with 274 mL of EtOAc. The filtrate was concentrated under reduced pressure to an oil or semisolid. The concentrate was transferred to a reaction kettle, and 411 mL of isopropyl acetate was added. The mixture was heated to 75-80°C and stirred to dissolve completely. The mixture was then slowly cooled to 10°C. A large amount of off-white solid precipitated and crystallized for 12 hours. The mixture was suction filtered under vacuum, and the cake was rinsed with 50 mL of isopropyl acetate. The cake was dried in vacuo to give intermediate 10.

[0052] Step H: Under nitrogen gas protection, 3 L of acetone and 300 g of Intermediate 10 were sequentially added to a reaction kettle, followed by the addition of 319.4 g of K3PO4 with stirring. The reaction mixture was heated to 35-45°C and stirred for 12 hours. After that, 212.9 g of K3PO4 was added and the reaction was continued for 5 hours. The reaction mixture was cooled to 20-25°C, suction filtered under vacuum, and the cake was rinsed with an additional 150 mL of EtOAc. The filtrate was concentrated under reduced pressure. The K3PO4 cake was transferred to a 5 L single-layer glass reaction kettle and slurried with 1.5 L of EtOAc. After filtration, the filtrate and concentrate were combined, 0.75 L of EtOAc was added, and the mixture was washed with 1.5 L of ice water. The aqueous layer was extracted once with 1.5 L of EtOAc. The combined organic layers were washed once with 2.25 L of 10% brine, and the organic layer was allowed to settle. The organic phase was dried over 600 g of anhydrous sodium sulfate for 30 minutes. The mixture was suction filtered under vacuum, filtered to remove the anhydrous sodium sulfate, and rinsed with 60 mL of EtOAc. The filtrate was concentrated under reduced pressure to give an oil. The oil was quickly added to 328 mL of isopropyl acetate and allowed to crystallize for 10 hours with magnetic stirring. The mixture was suction filtered under vacuum, and the cake was rinsed with mother liquor and dried under vacuum to give the compound of Formula (I).

[0053] Example 2: Preparation of Type A Crystals of the Compound of Formula (I) 320 g of the compound represented by Formula (I) and 1280 mL of isopropyl acetate were added sequentially to a 2 L single-layer glass reaction kettle. The oil bath was heated to 60-65°C with magnetic stirring. After the solid was completely dissolved, the mixture was filtered while still hot. The filtrate was cooled to 20-25°C and allowed to crystallize for 2 hours. The mixture was suction filtered under vacuum, and the cake was rinsed with 120 mL of isopropyl acetate. The cake was dried under vacuum to obtain a white solid, which was Type A crystals. The XRPD spectrum of Type A crystals is shown in Figure 1, the DSC spectrum is shown in Figure 2, and the TGA spectrum is shown in Figure 3.

[0054] Example 3: Single crystal X-ray diffraction analysis of the compound of formula (I) The method for producing the single crystal is as follows. Compound (I) (0.5 g) was dissolved in a test tube containing 10 ml of isopropyl acetate, allowed to stand at 10-15°C, opened, and allowed to slowly evaporate. Single crystals were obtained after 20 days of incubation. The crystal system was monoclinic, the space group was P2(1), the unit cell parameters were a = 10.0634(3) Å, b = 8.8860(2) Å, c = 10.8632(3) Å, α = γ = 90, β = 95.3960(10), the volume was V = 967.12(4) Å, and the absolute configuration parameter Flack was 0.049(9). An ellipsoidal diagram of the single molecule three-dimensional structure of compound (I) is shown in Figure 4.

[0055] [Table 2] JPEG0007803596000007.jpg17169

[0056] [Table 3] JPEG0007803596000009.jpg247169 JPEG0007803596000010.jpg191169

[0057] [Table 4] JPEG0007803596000012.jpg247169 JPEG0007803596000013.jpg41169

[0058] Experimental Example 1: Antiproliferative activity of compounds of formula (I) against HepG2 cell line Test materials: DMEM medium and penicillin / streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera. CellTiter-Glo (a chemiluminescent detection reagent for cell viability) reagent was purchased from Promega. HepG2 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Nivo multimode microplate reader (PerkinElmer).

[0059] Experimental Method: HepG2 cells (liver cancer) were seeded into a white 384-well plate, with each well containing 1,000 HepG2 cells at a 25 μM cell suspension. The cell plate was incubated overnight in a carbon dioxide incubator. Test compounds were diluted 3-fold using a pipette to nine concentrations, from 200 μM to 30 nM, and duplicate wells were placed under the same conditions. 99 μL of medium was added to the middle plate, and 1 μL per well of the gradient-diluted compound was transferred to the corresponding position in the middle plate. After uniform mixing, 25 μM per well was transferred to the cell plate. The compound concentrations transferred to the cell plate ranged from 1 μM to 0.15 nM. The cell plate was placed in a carbon dioxide incubator and incubated for 5 days. A separate cell plate was prepared, and the signal value on the day of drug addition was recorded as the maximum value (Max value in the formula below) and used for data analysis. 20 μL of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. The data was read using a multi-label analyzer. 20 μL of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. The data was read using a multi-label analyzer.

[0060] Data Analysis: Convert raw data into percentage inhibition using the equation (Sample-Min) / (Max-Min) × 100% to IC 50The values ​​were obtained by curve fitting using four parameters (obtained by log(inhibitor) vs. response--Variable slope motor in GraphPad Prism). Table 5 provides the inhibitory activity of compounds represented by formula (I) on HepG2 cell proliferation.

[0061] [Table 5]

[0062] Conclusion: The compound of formula (I) has excellent antiproliferative activity against HepG2, which highly expresses AKR1C3.

[0063] Experimental Example 2: Antiproliferative activity of compounds of formula (I) against Hep3B cell line Test materials: EMEM medium and penicillin / streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera. CellTiter-Glo (a chemiluminescent detection reagent for cell viability) reagent was purchased from Promega. Hep3B cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Nivo multimode microplate reader (PerkinElmer).

[0064] Experimental Method: Hep3B cells (liver cancer) were seeded into a white 96-well plate, with 80 μL of cell suspension per well, containing 3,000 Hep3B cells. The cell plate was incubated overnight in a carbon dioxide incubator. Test compounds were diluted 5-fold using a pipette to nine concentrations, from 2 μM to 5.12 nM, and duplicate wells were placed under the same conditions. 78 μL of medium was added to the middle plate, and 2 μL / well of the gradient-diluted compound was transferred to the corresponding positions in the middle plate. After uniform mixing, 20 μL / well was transferred to the cell plate. The compound concentrations transferred to the cell plate ranged from 10 μM to 0.0256 nM. The cell plate was placed in a carbon dioxide incubator and incubated for 3 days. A separate cell plate was prepared, and the signal value on the day of drug addition was recorded as the maximum value (Max value in the formula below) and used for data analysis. 25 μL of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. The data was read using a multi-label analyzer. 25 μL of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. The data was read using a multi-label analyzer.

[0065] Data Analysis: Convert raw data into percentage inhibition using the equation (Sample-Min) / (Max-Min) × 100% to IC 50 The values ​​were obtained by curve fitting using four parameters (obtained by log(inhibitor) vs. response--Variable slope motor in GraphPad Prism). Table 6 provides the inhibitory activity of compounds of formula (I) on Hep3B cell proliferation.

[0066] [Table 6]

[0067] Conclusion: The compound of formula (I) had no antiproliferative activity against Hep3B cells with low expression of AKR1C3 and exhibited high selectivity.

[0068] Experimental Example 3: In vivo pharmacodynamic study of the compound of formula (I) on human liver cancer Hep G2 orthotopic xenograft tumor model Objective of the experiment: This study evaluated the antitumor efficacy of the compounds using a HepG2 orthotopic xenograft tumor nude mouse model.

[0069] Test materials: Female Balb / C nude mice, 6-8 weeks old, weighing 18-22 g, were incubated in PBS, EMEM medium (catalog number: 30-2003), phosphate buffered saline, double antibody (catalog number: 15240-062), Matrigel, and trypsin.

[0070] Experimental Methods and Steps 1. Cell culture preparation: HepG2-luc cells were cultured in vitro as a monolayer in EMEM medium supplemented with 10% heat-inactivated fetal bovine serum in a 37°C, 5% CO2 incubator. They were digested with trypsin-EDTA twice a week and subcultured. When cell saturation reached 80%-90%, the cells were digested with trypsin-EDTA, counted, and then transferred to 166.67 x 10 cells in PBS and Matrigel (PBS:Matrigel = 1:1). 6 The cells were resuspended at a density of 1000 cells / mL.

[0071] 2. Tumor cell inoculation and group assignment: Animals were anesthetized with an intramuscular injection of 60 mg / kg Serta50 plus 1.5 mg / kg xylazine. Once the animals were deeply anesthetized, they were properly immobilized, the abdominal skin was cleaned with a 75% alcohol swab, and a 10 mm wound was created with surgical scissors. 0.03 mL of HepG2-luc cells (PBS:Matrigel = 1:1) were inoculated in situ into the left liver lobe of each mouse. The muscle wound was then sutured with absorbent intestinal tissue, and the skin wound was sutured with staples. The operated animals were placed on a warming blanket and kept warm until they woke up. To alleviate pain, 2 mg / kg meloxicam (administered subcutaneously once daily) was administered for three consecutive days after surgery. Fifteen animals were randomly selected to detect the signal increase. When the signal began to rise, they were randomly divided into groups according to the bioluminescence signal value and drug treatment was initiated. The detailed treatment method is shown in Table 7.

[0072] [Table 7]

[0073] 3. Experimental Indicators The experimental endpoint is whether tumor growth can be delayed or whether tumors can be cured. After tumor inoculation, bioluminescence signals and animal weights were detected once a week until the end of the observation period. The bioluminescence signal value can be used to calculate T / C (where T is the treatment group, and C is the average bioluminescence intensity value of the blank control group at the set time). The formula for calculating tumor inhibition rate (TGI) is: TGI (%) = [1-(T i -T0) / (V i -V0)] × 100, where T i is the mean bioluminescence intensity of the treatment group at a set time, and T0 is the mean bioluminescence intensity at the start of administration. V i is the average bioluminescence intensity of the blank control group at the set time, and V0 is the average bioluminescence intensity at the start of administration.

[0074] 4. Inhibitory effect of the compound on the growth of subcutaneously transplanted HepG2 liver cancer tumors in nude mice In this experiment, the efficacy of compound of formula (I) was evaluated in a HepG2 orthotopic xenograft tumor model. After 21 days of administration, compound of formula (I) had a significant inhibitory effect on tumor growth at a dose of 1 mg / kg, with p<0.05 in all cases compared to the vehicle control group. Increasing the dose of compound of formula (I) to 3 mg / kg enhanced the tumor inhibitory effect.

[0075] Experimental results: As shown in Figures 5 and 6 and Tables 8, 9 and 10.

[0076] [Table 8]

[0077] [Table 9]

[0078] [Table 10]

[0079] [Table 11]

[0080] 5. Changes in weight In this model, the weights of animals in all treatment groups did not vary significantly, with the average weight loss across animals not exceeding 5%, as detailed in FIG.

[0081] 6. Conclusion: The compound represented by formula (I) has a significant effect of inhibiting tumor growth, and the body weight of the animals in the administration group did not decrease significantly, indicating good safety. [Brief explanation of the drawings]

[0082] [Figure 1] 1 is an XRPD spectrum of Cu-Kα radiation of type A crystal of the compound represented by formula (I). [Figure 2] 1 is a DSC spectrum of type A crystals of the compound represented by formula (I). [Figure 3] 1 is a TGA spectrum of type A crystals of the compound represented by formula (I). [Figure 4] FIG. 1 is a crystal X-ray diffraction conformation ellipsoid diagram of the compound represented by formula (I). [Figure 5] Tumor growth signal-time curve. [Figure 6] FIG. 1 is a schematic representation of tumor weights at the end of the experiment. [Figure 7] Animal weight-time curve.

Claims

1. A type A crystal of the compound represented by formula (I), which has characteristic diffraction peaks at the following 2θ angles: 8.25±0.20°, 19.21±0.20°, and 21.76±0.20° in a powder X-ray diffraction spectrum using Cu-Kα radiation. 【Chemistry 1】

2. The A-type crystal of the compound of formula (I) according to claim 1, wherein the powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 8.25±0.20°, 13.27±0.20°, 15.19±0.20°, 16.43±0.20°, 17.94±0.20°, 19.21±0.20°, and 21.76±0.20°.

3. The A-type crystal of the compound of formula (I) according to claim 1, wherein the powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 8.25±0.20°, 15.19±0.20°, 16.43±0.20°, 17.94±0.20°, 19.21±0.20°, 21.76±0.20°, 23.56±0.20°, and 26.66±0.20°.

4. The powder X-ray diffraction spectrum exhibited the following 2θ angles: 8.25°, 12.56°, 12.89°, 13.27°, 15.19°, 16.01°, 16.43°, 17.60°, 17.94°, 19.21°, 19.91°, 20.21°, 20.52°, 21.25°, 21.76°, 23.02°, 23.56°, 24.71°, 25.12°, 25.88° 4. A type A crystal of the compound of formula (I) according to claim 3, having characteristic diffraction peaks at 26.66°, 27.10°, 27.43°, 27.84°, 28.38°, 29.13°, 30.01°, 30.83°, 31.69°, 32.26°, 33.35°, 35.01°, 35.52°, 36.51°, 37.70°, and 39.24°.

5. A type A crystal of the compound represented by formula (I) according to any one of claims 1 to 4, wherein a differential scanning calorimetry curve has an endothermic peak value at 89.2±3°C.

6. A type A crystal of the compound represented by formula (I) according to any one of claims 1 to 4, which exhibits a weight loss of 1.03% at 100±3°C in a thermogravimetric analysis curve.

7. Use of type A crystals of the compound of formula (I) according to any one of claims 1 to 4 in the manufacture of a medicament for treating liver cancer, prostate cancer, pancreatic cancer and / or T-cell acute lymphoblastic leukemia.

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