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 to Chinese patent application No. 2022102345275, filed on March 10, 2022. This application cites the above Chinese patent application in full.

[0002] [Technical field] The present invention relates to crystals of isochroman compounds and methods for their preparation, in particular to a compound of formula (I) and its use in the manufacture of a medicament for treating related diseases.

[0003] [Background technology] Malignant tumors are serious diseases that pose serious threats to the life and health of mankind. Current treatment methods mainly include surgical therapy, chemotherapy, targeted therapy, etc. Chemotherapy is a treatment that uses chemical drugs to kill tumor cells and inhibit the proliferation of tumor cells, and is a systemic treatment. Due to the heterogeneity of malignant tumors, chemotherapy remains an important method of treating tumors. However, it is this systemic treatment that brings about serious side effects to chemotherapy. There is a large unmet clinical need for the development of medicines with targeted effects.

[0004] Aldo-keto reductase (AKR1C3) is a member of the aldo-keto reductase family and is mainly 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, such as liver cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, prostate cancer, and acute lymphoblastic leukemia, especially liver cancer, with the proportion of overexpression exceeding 60%.

[0005] At present, AKR1C3 inhibitors are being developed in the clinic, but no good progress has been made. Haoding reported a compound that targets 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 tumor cells that highly express AKR1C3, giving the chemical a significant targeting effect.

[0006] [ka] At present, research on this target is still in its early stages, and only OBI-3424 has entered the Phase 1 / 2 clinical stage, with the main indications being hepatocellular carcinoma (HCC), castration-resistant prostate cancer (CRPC), pancreatic cancer and T-cell acute lymphoblastic leukemia (T-ALL), etc. Its efficacy and safety are still being verified, so this field still needs 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°, and 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 A-type crystal of the compound represented by formula (I) has a powder X-ray diffraction spectrum 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 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 invention, the alcohol is selected from ethanol, isopropanol, and n-butanol.

[0024] In some embodiments of the 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 period is selected from 1 hour to 72 hours.

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

[0028] The present invention further provides use of a type A crystal of the compound represented by formula (I) or a type A crystal 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 anti-proliferative activity against tumor cells that highly express AKR1C3 enzyme, and the compound of the present invention has shown significant anti-tumor effects in multiple in vivo efficacy models. The A-type crystal 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. A particular phrase or term, unless otherwise defined, should be understood as being of ordinary definition, not indefinite or unclear. When a trade name is mentioned 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 the 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. In order 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 the existing embodiments.

[0033] The present invention will be described in detail 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; commercially available compounds are named by manufacturer's catalogue name.

[0036] The present invention uses the following abbreviations: 2 CO 3 stands for potassium carbonate, and [Ru(p-cym)Cl 2 ] 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 dimethylsulfoxide, EtOAc represents ethyl acetate, MeOH represents methanol, n-heptane represents n-heptane, SiO 2 represents 100-200 mesh silica gel powder for column chromatography, and Et3 SiH represents triethylsilane, HOAc represents acetic acid, and NaIO 4 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, K 3 PO 4 stands for potassium phosphate, HP-β-CD stands for 2-hydroxypropyl-β-cyclodextrin, and QW stands for frequency of administration.

[0037] The 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] 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-scattering slit: 1mm Scanning range: 3~40deg Step angle: 0.02 deg Step size: 0.5 seconds

[0039] Differential Scanning Calorimeter (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 sample (2-10 mg) is placed in an aluminum crucible, then placed in a platinum basket to perform the test, and nitrogen gas (N 2 Under the conditions of (a), the sample is heated from 40°C to 500°C 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 by examples, but does not mean to restrict the present invention. The compounds of the present invention can be prepared by various synthesis methods familiar to those skilled in the art, including the specific embodiments listed below, embodiments combined with other chemical synthesis 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. It is clear to those skilled in the art that various changes and modifications can be made in the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0044] Example 1: Preparation of a compound represented by formula (I) [ka]

[0045] Step A: Add DMF (30 L) to the reaction kettle and begin mechanical stirring. Then add Starting Material 1 (4 kg, 35.37 mol) and Starting Material 2 (6.6 kg, 35.71 mol). After addition is complete, control the internal temperature to rise to 30-40 °C. 2 CO 3 (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 completed, 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 under stirring. A large amount of yellow solid was precipitated, which was suction filtered under reduced pressure and the cake was washed with 45 L of water. The mixture was dried in vacuum to obtain intermediate 3. 1 H NMR (400 MHz, DMSO-d 6 ) 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: Add 1,4-dioxane (27 L) and H 2 O (2.7 L) was added 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. Nitrogen gas was continued to be sparged through the kettle for 15 minutes. [Ru(p-cym)Cl 2 ] 2 (355.37 g, 0.580 mol) was added at once, and the temperature was raised to 90° C. and stirred for 17 hours. The reaction solution was cooled to 25° C., and 2.7 L of water was added thereto. The mixture was allowed to stand at about 5° C. for about 23 hours, and a solid precipitated. The cake was filtered and diluted with 10 L of EtOH / H 2 2H 2 O (volume ratio 1:1), stirred at room temperature for 4 hours, and filtered with suction. The cake was added to 8 L of EtOAc / MTBE (volume ratio 2:1), stirred at room temperature for 17 hours, and filtered with suction. The cake was washed with EtOAc / MTBE (volume ratio 2:1) (1 L x 2), and the cake was dried in vacuum to obtain intermediate 5. 1 H NMR (400 MHz, DMSO-d 6 ) 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 the reaction kettle, and intermediate 5 (2200 g, 5.608 mol) was added with stirring, and the reaction temperature was cooled to -60 to -50 °C under the protection of nitrogen gas. DIBAL-H (6700 mL, 6.7 mol) was added dropwise to the kettle. After the addition was completed, the internal temperature was -60 to -50 °C, and stirring was continued for 1.5 h under the protection of nitrogen gas. MeOH (455 mL) was added dropwise to the kettle, and the internal temperature was controlled at -60 to -50 °C. After the addition was completed, 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 aqueous solution) were added in sequence. After the addition was complete, it was stirred at room temperature for 4 hours, allowed to stand for 12 hours, separated and the organic phase was concentrated to give a yellow solid product which was dried in vacuum to give intermediate 6. 1 H NMR (400 MHz, DMSO-d 6 ) 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: Add DCM (9.4 L) to a reaction kettle and add Intermediate 6 (1880.00 g, 4.767 mol), Et 3 SiH (2284 mL, 14.300 mol) was added. Nitrogen flow protection was turned on and the internal temperature was 0-5°C after the addition was complete. Boron trifluoride ethyl ether complex (1765 mL, 614.301 mol) was added dropwise and the internal temperature was controlled at 0-10°C and stirring was continued for 1 hour. The reaction solution was added with K 2 CO 3An aqueous solution (1.05 kg dissolved in 12 L of water) was added in a batch. After the addition was complete, stirring was 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, and a yellow solid precipitated, which was filtered and 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-d 6 ) 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: Add DCM (7.0 L), MeCN (7.0 L), and water (10.5 L) to a reaction kettle, add intermediate 7 (1740.00 g, 4.599 mol), and ruthenium(III) chloride trihydrate (12.03 g, 0.0460 mol) under stirring, and control the internal temperature at 10-20 °C. Add NaIO within 2 h. 4 (2147.72 g, 14.717 mol) was added in batches, and the internal temperature was controlled at 10-20°C. Stirring was continued for 0.5 h after the addition was completed. DCM (14.0 L) was added to the reaction solution and stirred for 5 min. Then, the reaction solution was filtered through diatomaceous earth to remove excess inorganic salts. The filtrate was separated, and aqueous NaOH solution (8 L, 1 mol / L) was added to the organic phase, which was then filtered through diatomaceous earth and separated. The organic phase was washed successively with 8 L of saturated aqueous sodium sulfite solution and 8 L of saturated brine. The organic phase was concentrated to obtain a crude product. The crude product was purified by column chromatography (SiO 2 , n-heptane:EtOAc=100:0-10:1) to obtain intermediate 8. 1 H NMR (400 MHz, CDCl 3) 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 the protection of nitrogen gas, 450mL of anhydrous THF and 147.6mL of (S)-CBS were added to the reaction kettle in sequence, cooled to 0-10°C, 296.2mL of borane dimethyl sulfide complex (10M) was added dropwise to the reaction system, and the reaction was allowed to proceed with magnetic stirring for 1 hour. A THF solution of intermediate 8 (preparation method: 450g of intermediate 7 was weighed and dissolved in 1800mL of THF) was slowly added dropwise to the reaction system, and the temperature was controlled to 0-10°C for 1 hour. After the reaction was completed, 180mL of MeOH was slowly added dropwise to quench the reaction system. 2250mL of 2.0mol / L hydrochloric acid was weighed and slowly added dropwise to the reaction system, and a large amount of solid was precipitated. The cake was obtained by filtration under reduced pressure for later use, the filtrate was separated, the organic phase was separated, the aqueous phase was extracted with 4500 mL of EtOAc, the organic phase was combined, and concentrated under reduced pressure at 35-40°C until it became solid. The concentrate and the cake were combined and added to 1350 mL of ethanol / 2.0 M hydrochloric acid (volume ratio 2:1), heated to 60-70°C, and stirred for 1 hour. The reaction solution was cooled to 20°C-25°C, filtered under reduced pressure, and the cake was rinsed with 900 mL of purified water. The cake (wet product) was added directly to a mixed solution of 1350 mL of ethanol / purified water (volume ratio 1:1) and stirred at 20-25°C for 2 hours. The cake was filtered under reduced pressure, and the cake was 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 the protection of nitrogen gas, 137g of intermediate 9 and 2055mL of anhydrous THF were added to a 5L single-layer glass reaction kettle, and cooled to -40~-30°C with magnetic stirring. The temperature of the reaction system was controlled at -40~-30°C, 56.7mL of phosphorus oxychloride was added at once, and then 106.3mL of 2-tert-butyl-1,1,3,3-tetramethylguanidine was slowly added dropwise, and after the addition was completed, the reaction was carried out for 4~5 hours while maintaining the temperature at -40~-30°C. After the reaction was completed, the reaction solution was cooled to -50~-40°C, and 548g of 2-bromoethylamine hydrobromide was added at once, and then the temperature was controlled at -50~-40°C, and 467.3mL of DIPEA was slowly added dropwise, and after the addition was completed, the reaction was carried out for 12 hours while maintaining the temperature at -50~-40°C. The reaction solution was quenched by slowly adding 2055 mL of purified water dropwise to the reaction solution. The temperature was naturally raised to 0-10°C, and the solution was allowed to stand to separate. The organic layer was separated and concentrated, and the aqueous layer was extracted once with 1781 mL. The concentrated solution and the extracted solution were mixed, and then successively diluted with 1096 mL of 0.1 M hydrochloric acid, 1% K 3 PO 4 The mixture was washed with 1096 mL of aqueous solution and 1096 mL of saturated aqueous solution of sodium chloride. The organic layer was separated and dried by adding 274 g of anhydrous sodium sulfate. It was filtered under vacuum and the cake was rinsed with 274 mL of EtOAc. The filtrate was concentrated under reduced pressure to an oil or semi-solid. The concentrate was transferred to a reaction kettle and 411 mL of isopropyl acetate was added, heated to 75-80°C, stirred to dissolve completely, and then cooled slowly to 10°C, where a large amount of off-white solid precipitated and was allowed to crystallize for 12 hours. It was filtered under vacuum and the cake was rinsed with 50 mL of isopropyl acetate. The cake was dried under vacuum to obtain intermediate 10.

[0052] Step H: Under nitrogen gas protection, add 3 L of acetone and 300 g of intermediate 10 to a reaction kettle, and add 319.4 g of K while stirring. 3 PO 4 The reaction system was heated to 35-45°C and reacted for 12 hours with stirring. 3 PO 4The reaction was continued for 5 hours. The reaction solution was cooled to 20-25°C, suction filtered under vacuum, and the cake was rinsed with 150 mL of EtOAc. The filtrate was concentrated under reduced pressure. 3 PO 4 The cake was transferred to a 5L single layer glass reaction kettle and slurried with 1.5L of EtOAc and washed. Filtered, the filtrate and concentrate were combined, 0.75L of EtOAc was added, washed with 1.5L of ice water, separated, the aqueous layer was extracted once with 1.5L of EtOAc, the organic layers were combined, washed once with 2.25L of 10% brine, and allowed to settle and separate the organic layer. The organic phase was dried over 600g of anhydrous sodium sulfate for 30 minutes. Suction filtered under vacuum, filtered to remove the anhydrous sodium sulfate, and rinsed with 60mL of EtOAc. The filtrate was concentrated under reduced pressure to give an oil. The oil was quickly added to 328mL of isopropyl acetate and allowed to crystallize for 10 hours with magnetic stirring. Suction filtered under vacuum, the cake was rinsed with mother liquor, and suction dried to give the compound of formula (I).

[0053] Example 2: Preparation of A-type crystals of the compound represented by formula (I) In a 2L single-layer glass reaction kettle, 320 g of the compound represented by formula (I) and 1280 mL of isopropyl acetate were added in sequence, and the oil bath was heated to 60-65°C while stirring magnetically. After the solid was completely dissolved, it was filtered while still hot, and the filtrate was cooled to 20-25°C and crystallized for 2 hours. It was suction filtered under vacuum, and the cake was rinsed with 120 mL of isopropyl acetate. The cake was dried in vacuum to obtain a white solid, which was A-type crystals. The XRPD spectrum of A-type 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. The compound represented by formula (I) (0.5 g) was dissolved in a test tube of isopropyl acetate (10 ml), left to stand at 10-15 ° C, opened and slowly evaporated, and cultured for 20 days to obtain a single crystal. 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) Å3, and the Flack value of the absolute configuration parameters was 0.049 (9). The ellipsoid diagram of the single molecule three-dimensional structure of the compound represented by formula (I) is as shown in Figure 4.

[0055] [Table 2] JPEG2025510593000008.jpg17169

[0056] [Table 3] JPEG2025510593000010.jpg247169 JPEG2025510593000011.jpg191169

[0057] [Table 4] JPEG2025510593000013.jpg247169 JPEG2025510593000014.jpg41169

[0058] Experimental Example 1: Antiproliferative activity of compounds of formula (I) against HepG2 cell line Test materials: DMEM medium, penicillin / streptomycin antibiotics were purchased from Vicente, fetal bovine serum was purchased from Biosera. CellTiter-Glo (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] Testing Method: HepG2 cells (liver cancer) were seeded in a white 384-well plate, and each well contained 1000 HepG2 cells in a 25 μM cell suspension. The cell plate was cultured overnight in a carbon dioxide incubator. The test compounds were diluted 3-fold with a pipette to 9 concentrations, i.e., from 200 μM to 30 nM, and two wells were set up under the same conditions. 99 μL of medium was added to the middle plate, and 1 μL / well of the gradient-diluted compound was transferred to the middle plate according to the corresponding position, and 25 μM / well was transferred to the cell plate after uniform mixing. The range of compound concentrations transferred to the cell plate was 1 μM to 0.15 nM. The cell plate was placed in a carbon dioxide incubator and cultured for 5 days. Another cell plate was prepared, and the signal value on the day of drug addition was read as the maximum value (Max value in the formula below) and was included in the 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 by 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 by 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 the compound represented by formula (I) against 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, penicillin / streptomycin antibiotics were purchased from Vicente, fetal bovine serum was purchased from Biosera. CellTiter-Glo (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] Testing Method: Hep3B cells (liver cancer) were seeded in a white 96-well plate, and each well contained 80 μL of cell suspension, with 3000 Hep3B cells. The cell plate was cultured overnight in a carbon dioxide incubator. The test compounds were diluted 5-fold with a pipette into 9 concentrations, i.e., from 2 μM to 5.12 nM, and two wells were set up under the same conditions. 78 μL of medium was added to the middle plate, and 2 μL / well of the gradient-diluted compounds were transferred to the middle plate according to the corresponding positions, and after uniform mixing, 20 μL / well were transferred to the cell plate. The range of compound concentrations transferred to the cell plate was 10 μM to 0.0256 nM. The cell plate was placed in a carbon dioxide incubator and cultured for 3 days. Another cell plate was prepared, and the signal value on the day of drug addition was read as the maximum value (Max value in the formula below), and was included in the data analysis. 25μL 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 by a multi-label analyzer. 25μL 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 by a multi-label analyzer.

[0065] Data Analysis: Convert raw data into percentage inhibition using the equation (Sample-Min) / (Max-Min) × 100% to IC 50 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 represented by formula (I) against 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 showed high selectivity.

[0068] Experimental Example 3: In vivo pharmacodynamic study of compound of formula (I) on human liver cancer Hep G2 orthotopic xenograft tumor model Purpose of the Test: 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, weight: 18-22 g, Fetal bovine serum (PBS), medium EMEM (catalog number: 30-2003), phosphate buffer, double antibody (catalog number: 15240-062), Matrigel, trypsin.

[0070] Experimental Method and Steps 1. Preparation of cell culture: HepG2-luc cells were cultured in vitro in monolayer. The culture conditions were EMEM medium supplemented with 10% heat-inactivated fetal bovine serum, at 37 °C and 5% CO 2 The cells were cultured in an incubator. They were digested with trypsin-EDTA and subcultured twice a week. When the cell saturation reached 80%-90%, the cells were digested with trypsin-EDTA, counted, and subcultured 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 grouping: The animals were anesthetized by intramuscular injection of 60 mg / kg Serta50 + 1.5 mg / kg xylazine, and when the animals were deeply anesthetized, the animals were properly fixed, the abdominal skin was cleaned with 75% alcohol cotton balls, a wound of about 10 mm was opened with surgical scissors, and 0.03 mL (PBS: Matrigel = 1:1) of HepG2-luc cells was inoculated in situ into the left liver lobe of each mouse, and then the muscle layer wound was sutured with absorbent intestine and the skin wound was sutured with a stapler. The operated animals were placed on a heating blanket and kept warm until they woke up. To reduce the pain of the animals, 2 mg / kg meloxicam (administered by subcutaneous injection once a day) was administered for three consecutive days after surgery. 15 animals were randomly selected to detect the increase in the signal, and when the signal began to rise, they were randomly divided into groups according to the bioluminescence signal value and drug treatment was started, and the detailed treatment method is as shown in Table 7.

[0072] [Table 7]

[0073] 3. Experimental Indicators The experimental indicator is whether the tumor growth can be delayed or whether the tumor can be cured. After tumor inoculation, the bioluminescence signal and the animal's body weight 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 a set time). The calculation formula for tumor inhibition rate TGI is: TGI(%)=[1-(T i -T 0 ) / (V i -V 0 )] × 100, where T i is the mean bioluminescence intensity of the treatment group at a set time, and T 0 is the mean bioluminescence intensity at the start of administration. V i is the average bioluminescence intensity of the blank control group at a set time, and V 0 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 the compound of formula (I) in a HepG2 orthotopic xenograft tumor model was evaluated. After 21 days of administration, the compound of formula (I) had a significant inhibitory effect on tumor growth at a dose of 1 mg / kg, both p<0.05 compared to the vehicle control group. Increasing the dose of the 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 body weights of animals in all treatment groups did not vary significantly, with the average body 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 that the compound has a good safety profile. [Brief description of the drawings]

[0082] [Figure 1]1 is an XRPD spectrum of Cu-Kα radiation of the A-type crystal of the compound represented by formula (I). [Diagram 2] 1 is a DSC spectrum of type A crystals of the compound represented by formula (I). [Diagram 3] 1 is a TGA spectrum of type A crystals of the compound represented by formula (I). [Figure 4] FIG. 2 is a crystal X-ray diffraction conformation ellipsoid diagram of the compound represented by formula (I). [Diagram 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 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 represented by 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 represented by 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. Powder X-ray diffraction spectrum shows 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°, 26.78°, 27.76°, 28.78°, 29.78°, 30.74°, 31.76°, 32.78°, 33.76°, 34.78°, 35.78°, 36.78°, 37.78°, 38.78°, 39.78°, 40.78°, 41.78°, 42.78°, 43.78°, 44.78°, 45.78°, 46.78°, 47.78°, 49.78°, 50.78°, 51.78°, 52.78°, 53.78°, 54.78°, 55.78°, 56.78°, 57.78°, 58.78°, 59.78°, 60.78°, 61.78°, 62.78°, 63.78°, 64.78°, 65.78°, 66.78°, 67.78°, 68.78°, 6 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. 5. The A-type crystal of the compound of formula (I) according to claim 4, having a powder X-ray diffraction spectrum essentially as shown in FIG.

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

7. 7. The compound of formula (I) according to claim 6, wherein the DSC spectrum is essentially as shown in FIG.

8. A type A crystal of the compound represented by formula (I) according to any one of claims 1 to 5, which exhibits a weight loss of 1.03% at 100±3°C in a thermogravimetric analysis curve.

9. 9. The A-type crystal of the compound of formula (I) according to claim 8, having a TGA spectrum essentially as shown in FIG.

10. Use of the A-type crystals of the compound of formula (I) according to any one of claims 1 to 9 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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