A salt form, a crystal form and a preparation method thereof of an IDH1 mutant inhibitor

Abstract

The present invention discloses a salt form, a crystal form and a preparation method of an IDH1 mutant inhibitor. #imgabs0#

Description

[0001] This application claims the priority of a Chinese patent application filed with the China Patent Office on December 23, 2019, with application number 201911335601.7 and invention name “A salt form, a crystal form and a preparation method of an IDH1 mutant inhibitor”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to a salt form and a crystal form of an IDH1 mutant inhibitor and a preparation method thereof. Background Art

[0003] Isocitrate dehydrogenase is an important enzyme in the citric acid cycle, catalyzing the oxidative decarboxylation of isocitrate to 2-oxoglutarate (i.e., 2-α-ketoglutarate, α-KG). The protein encoded by the gene IDH 1 is a NADP(+)-dependent isocitrate dehydrogenase found in the cytoplasm and peroxisomes, which contains a PTS-1 peroxidase targeting signal sequence. The presence of this enzyme in the oxisome suggests a role in the regeneration of internal NADPH.

[0004] Non-mutant, such as wild-type IDH, catalyzes the oxidative decarboxylation of isocitrate while reducing NAD + (NADP + ) to NADP(NADPH):

[0005] Isocitrate + NAD + (NADP + )→α-KG+CO2+NADP(NADPH)+H +

[0006] IDH 1 / 2 mutant proteins (IDH 1 / 2m) have been found in a variety of tumors, including glioma, acute myeloid leukemia (AML), chondrosarcoma, intrahepatic cholangiocarcinoma, melanoma, prostate cancer, and angioimmunoblastic T-cell lymphoma. Among gliomas, more than 70% of non-primary glioblastomas have IDH1 mutations, and 92.7% of IDH1 mutant tumors have arginine replaced by histidine (i.e., IDH1 R132H) (Hartmann C, Acta Neuropathol. 2009 Oct; 118(4): 469-74)

[0007] IDH mutant protein has a new protein function, namely catalyzing the reduction of α-KG to produce the oncometabolite 2-hydroxyglutarate (2-HG). The production of 2-HG is believed to contribute to the formation and development of cancer (Dang L, Nature, 2009 Dec 10; 462(7274): 739-44). The level of 2-HG produced in normal cells is very low, but cells with IDH mutations can produce high levels of 2-HG. High levels of 2-HG can also be found in tumors with IDH mutations.

[0008] Therefore, inhibition of mutant IDH and its new activity is a potential approach for cancer treatment, and there is a need to obtain inhibitors of IDH mutants to inhibit their new role in producing 2-HG.

[0009] Acta Neuropathol (2017, Vol(133), Issue 4, 629-644) disclosed the specific structure of compound BAY1436032.

[0010] Summary of the invention

[0011] The present invention provides a crystal form A of a compound of formula (I), characterized in that its X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 9.78±0.20°, 12.06±0.20°, and 20.37±0.20°.

[0012]

[0013] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.66±0.20°, 9.78±0.20°, 12.06±0.20°, 17.43±0.20°, 18.02±0.20°, 18.81±0.20°, 20.37±0.20°, 23.10±0.20°.

[0014] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 7.66±0.20°, 9.78±0.20°, 12.06±0.20°, 17.43±0.20°, 18.02±0.20°, 18.81±0.20°, 20.37±0.20°, 20.91±0.20°, 22.46±0.20°, 23.10±0.20°.

[0015] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 4.88°, 6.03°, 7.66°, 9.78°, 10.57°, 11.65°, 12.06°, 13.13°, 13.55°, 14.70°, 14.94°, 15.34°, 15.73°, 16.02°, 16.64°, 17.43°, 18.02° , 18.34°, 18.81°, 19.25°, 19.60°, 20.37°, 20.91°, 21.17°, 22.03°, 22.46°, 23.10°, 23.68°, 24.25°, 25.16°, 25.51°, 26.44°, 27.14°, 28.77°, 29.42°, 30.97°, 31.76°, 32.29°, 33.16°.

[0016] In some embodiments of the present invention, the above-mentioned Form A has an XRPD pattern as shown in Figure 1 shown.

[0017] In some embodiments of the present invention, the XRPD spectrum analysis data of the above-mentioned Form A is shown in Table 1:

[0018] Table 1. XRPD spectrum analysis data of Form A

[0019]

[0020] In some embodiments of the present invention, the above-mentioned crystal form A has a differential scanning calorimetry curve with an endothermic peak at 248.7±3.0°C.

[0021] In some embodiments of the present invention, the DSC spectrum of the above-mentioned A crystal form is as follows Figure 2 shown.

[0022] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned crystal form A shows a weight loss of 0.97% at 230.℃±3.0℃.

[0023] In some embodiments of the present invention, the TGA spectrum of the above-mentioned A crystal form is as follows Figure 3 shown.

[0024] The present invention also provides a crystal form B of the compound of formula (I), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 11.66±0.20°, 16.69±0.20°, 17.69±0.20°.

[0025] 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: 7.48±0.20°, 11.66±0.20°, 15.83±0.20°, 16.69±0.20°, 17.69±0.20°, 19.68±0.20°, 21.79±0.20°, 22.90±0.20°.

[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: 7.48±0.20°, 11.66±0.20°, 12.47±0.20°, 15.83±0.20°, 16.69±0.20°, 17.69±0.20°, 19.68±0.20°, 21.79±0.20°, 22.90±0.20°, 23.84±0.20°.

[0027] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction at the following 2θ angles: 7.48°, 11.66°, 12.47°, 14.56°, 15.83°, 16.06°, 16.69°, 17.69°, 18.13°, 19.68°, 21.19°, 21.79°, 22.12°, 22.90°, 23.84°, 25.50°, 29.72°.

[0028] In some embodiments of the present invention, the above-mentioned B crystal form has an XRPD pattern as shown in Figure 4 shown.

[0029] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form B is shown in Table 2:

[0030] Table 2. XRPD pattern analysis data of Form B

[0031]

[0032] In some embodiments of the present invention, the above-mentioned B crystal form has a differential scanning calorimetry curve with an endothermic peak at 246.8±3.0°C.

[0033] In some embodiments of the present invention, the DSC spectrum of the above-mentioned B crystal form is as follows Figure 5 shown.

[0034] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned B crystal form shows a weight loss of 0.54% at 230.0°C±3.0°C.

[0035] In some embodiments of the present invention, the TGA spectrum of the above-mentioned B crystal form is as follows Figure 6 shown.

[0036] The present invention also provides a crystal form C of the compound of formula (I), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.59±0.20°, 18.19±0.20°, 19.74±0.20°.

[0037] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned C crystal form has characteristic diffraction peaks at the following 2θ angles: 9.59±0.20°, 12.17±0.20°, 12.65±0.20°, 18.19±0.20°, 18.87±0.20°, 19.74±0.20°, 21.27±0.20°, 23.05±0.20°.

[0038] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 9.59±0.20°, 11.60±0.20°, 12.17±0.20°, 12.65±0.20°, 18.19±0.20°, 18.87±0.20°, 19.74±0.20°, 21.27±0.20°, 22.20±0.20°, 23.05±0.20°.

[0039] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned C crystal form has characteristic diffraction at the following 2θ angles: 7.70°, 9.59°, 10.78°, 11.60°, 12.17°, 12.65°, 17.70°, 18.19°, 18.87°, 19.74°, 20.12°, 21.27°, 22.20°, 23.05°, 25.25°, 25.61°, 26.43°, 28.53°.

[0040] In some embodiments of the present invention, the above-mentioned C crystal form has an XRPD pattern as shown in Figure 7 shown.

[0041] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form C are shown in Table 3:

[0042] Table 3. XRPD pattern analysis data of Form C

[0043]

[0044] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned C crystal form has an endothermic peak at 248.6±3.0°C.

[0045] In some embodiments of the present invention, the DSC spectrum of the above-mentioned C crystal form is as follows Figure 8 shown.

[0046] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned C crystal form shows a weight loss of 3.01% at 230.0°C±3.0°C.

[0047] In some embodiments of the present invention, the TGA spectrum of the above-mentioned C crystal form is as follows Fig. 9 shown.

[0048] The present invention also provides compounds of formula (II).

[0049]

[0050] The present invention also provides a D crystal form of the compound of formula (II), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 16.85°±0.20°, 19.93°±0.20°, 21.89°±0.20°.

[0051] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned D crystal form has characteristic diffraction peaks at the following 2θ angles: 7.31±0.20°, 8.48±0.20°, 13.08±0.20°, 16.85±0.20°, 17.44±0.20°, 19.93±0.20°, 21.89±0.20°, 22.49±0.20°.

[0052] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned D crystal form has characteristic diffraction at the following 2θ angles: 7.31°, 8.48°, 13.08°, 14.64°, 16.85°, 17.44°, 19.93°, 21.89°, 22.49°, and 25.87°.

[0053] In some embodiments of the present invention, the above-mentioned D crystal form has an XRPD pattern as shown in Fig.10 shown.

[0054] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned D crystal form are shown in Table 4:

[0055] Table 4. XRPD pattern analysis data of Form D

[0056]

[0057] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned D crystal form has an endothermic peak at 211.6±3.0°C.

[0058] In some embodiments of the present invention, the DSC spectrum of the above-mentioned D crystal form is as follows Fig.11 shown.

[0059] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned D crystal form shows a weight loss of 2.50% at 210.0°C±3.0°C.

[0060] In some embodiments of the present invention, the TGA spectrum of the above-mentioned D crystal form is as follows Fig.12 shown.

[0061] The present invention also provides compounds of formula (III).

[0062]

[0063] The present invention also provides a crystalline form E of the compound of formula (III), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 15.07°±0.20°, 20.07°±0.20°, 25.50°±0.20°.

[0064] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned E crystal form has characteristic diffraction peaks at the following 2θ angles: 11.80±0.20°, 15.07±0.20°, 17.71±0.20°, 20.07±0.20°, 22.54±0.20°, 24.42±0.20°, 25.50±0.20°, 26.20±0.20°.

[0065] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned E crystal form has characteristic diffraction at the following 2θ angles: 11.10°, 11.80°, 15.07°, 16.70°, 17.71°, 20.07°, 20.48°, 22.54°, 24.42°, 25.50°, 26.20°, 28.99°, 30.63°, 35.34°, 38.41°, and 39.31°.

[0066] In some embodiments of the present invention, the above-mentioned E crystal form has an XRPD pattern as shown in Fig.13 shown.

[0067] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned E crystal form is shown in Table 5:

[0068] Table 5. XRPD pattern analysis data of Form E

[0069]

[0070] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned E crystal form has an endothermic peak at 163.5±3.0°C and 240.4±3.0°C, respectively.

[0071] In some embodiments of the present invention, the DSC spectrum of the above-mentioned E crystal form is as follows Fig.14 shown.

[0072] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned E crystal form shows a weight loss of 0.29% at 140.0°C±3.0°C.

[0073] In some embodiments of the present invention, the TGA spectrum of the above-mentioned E crystal form is as follows Fig.15 shown.

[0074] The present invention also provides compounds of formula (IV).

[0075]

[0076] The present invention also provides a crystalline form F of the compound of formula (IV), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.19°±0.20°, 11.11°±0.20°, 22.37°±0.20°.

[0077] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned F crystal form has characteristic diffraction peaks at the following 2θ angles: 8.19±0.20°, 11.11±0.20°, 15.74±0.20°, 17.12±0.20°, 18.16±0.20°, 21.78±0.20°, 22.37±0.20°, 23.86±0.20°.

[0078] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned F crystal form has characteristic diffraction at the following 2θ angles: 8.19°, 8.53°, 11.11°, 15.74°, 17.12°, 18.16°, 18.95°, 19.50°, 21.12°, 21.78°, 22.37°, 22.77°, 23.86°, 25.66°.

[0079] In some embodiments of the present invention, the above-mentioned F crystal form has an XRPD pattern as shown in Fig.16 shown.

[0080] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned F crystal form are shown in Table 6:

[0081] Table 6. XRPD pattern analysis data of Form F

[0082]

[0083] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned F crystal form has an endothermic peak at 141.5±3.0°C.

[0084] In some embodiments of the present invention, the DSC spectrum of the above-mentioned F crystal form is as follows Fig.17 shown.

[0085] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned F crystal form shows a weight loss of 1.08% at 130.0°C±3.0°C.

[0086] In some embodiments of the present invention, the TGA spectrum of the above-mentioned F crystal form is as follows Fig.18 shown.

[0087] The present invention also provides use of the above compound or crystal form A or crystal form B or crystal form C or crystal form D or crystal form E or crystal form F in the preparation of drugs for treating intrahepatic cholangioma and the like.

[0088] Technical Effects

[0089] Each crystal form of the compound of the present invention has the advantages of being stable, having good hygroscopicity, and being less affected by light and heat, and has broad prospects for drug development. The compound of formula (I) has a good inhibitory effect on mutant IDH1R132H and IDH1R132C at the enzymatic level, and has no inhibitory effect on wild-type IDH protein. At the cellular level, the compound of formula (I) has a good 2-HG inhibitory effect on U87MG brain glioma cells with IDH1R132H mutation. The compound of formula (I) has good pharmacokinetic properties in mice.

[0090] Definition and Description

[0091] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0092] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

[0093] The chemical reactions of the specific embodiments of the present invention are carried out in a suitable solvent, which must be suitable for the chemical changes of the present invention and the reagents and materials required. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction processes based on the existing embodiments.

[0094] The present invention will be described in detail below by way of examples, which are not intended to limit the present invention in any way.

[0095] All solvents used in the present invention were commercially available and used without further purification.

[0096] The solvent used in the present invention can be obtained commercially. The present invention uses the following abbreviations: EtOH represents ethanol; MeOH represents methanol; EtOAc represents ethyl acetate; TFA represents trifluoroacetic acid; TsOH represents p-toluenesulfonic acid; mp represents melting point; THF represents tetrahydrofuran; K2CO3 represents potassium carbonate; NaOH represents sodium hydroxide; DMSO represents dimethyl sulfoxide; MeSO3H represents methanesulfonic acid; EDCI represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0097] Compounds are named according to the conventional nomenclature in the art or using The software names were used, and commercially available compounds were named using the supplier's catalog names.

[0098] The X-ray powder diffractometer (XRPD) method of the present invention

[0099] Instrument model: X'Pert3 X-ray diffractometer from PANalytical

[0100] Test method: Approximately 10 mg of sample was used for XRPD detection.

[0101] The detailed XRPD parameters are as follows:

[0102] Radiation source: Cu, kα ( Kα2 / Kα1 intensity ratio: 0.5)

[0103] Light tube voltage: 45kV, light tube current: 40mA

[0104] Divergence slit: fixed 1 / 8deg

[0105] First Soller slit: 0.04 rad, Second Soller slit: 0.04 rad

[0106] Receiving slit: None, Anti-scattering slit: 7.5mm

[0107] Measurement time: 5min

[0108] Scanning angle range: 3-40 degrees

[0109] Step width angle: 0.0263deg

[0110] Step length: 46.665 seconds

[0111] Sample plate speed: 15rpm

[0112] Differential Scanning Calorimeter (DSC) method of the present invention

[0113] Instrument model: TA Discovery DSC2500 differential scanning calorimeter.

[0114] Test method: Take a sample (1-10 mg) and place it in a covered aluminum crucible. Heat the sample from room temperature to 300°C (or 350°C) at a rate of 10°C / min under the protection of 50mL / min dry N2. At the same time, the TA software records the heat change of the sample during the heating process.

[0115] Thermogravimetric analysis (TGA) method of the present invention

[0116] Instrument model: TA Discovery TGA 5500 Thermogravimetric Analyzer

[0117] Test method: Take a sample (2-15 mg) and place it in a platinum crucible. Use segmented high-resolution detection to heat the sample from room temperature to 350°C at a heating rate of 10°C / min under the protection of 50mL / min dry N2. At the same time, the TA software records the weight change of the sample during the heating process.

[0118] Dynamic Vapor Sorption (DVS) method of the present invention

[0119] Instrument model: SMS DVS intrinsic dynamic moisture adsorption instrument

[0120] Test conditions: Weigh 10 mg of sample and place it in the DVS sample pan for testing.

[0121] The detailed DVS parameters are as follows:

[0122] Temperature: 25℃

[0123] Balance: dm / dt=0.01% / min

[0124] Drying: Drying at 0% RH for 120 minutes

[0125] RH(%) test level: 5%

[0126] RH(%) test step range: 0%-95%-0%

[0127] The moisture absorption evaluation is classified as follows:

[0128] Hygroscopicity classification Water adsorption standard (ΔW%) Deliquescent Absorb enough water to become liquid Very hygroscopic ΔW%≥15% Hygroscopic 15%>ΔW%≥2% Slightly hygroscopic 2%>ΔW%≥0.2% Non-hygroscopic ΔW%<0.2%

[0129] Note: ΔW% indicates the weight gain of the test product at 25±1℃ and 80±2%RH (European Pharmacopoeia 6.0). BRIEF DESCRIPTION OF THE DRAWINGS

[0130] Figure 1 is the XRPD spectrum of the crystal form A of the compound of formula (I) using Cu-Kα radiation;

[0131] Figure 2 is the DSC spectrum of the crystalline form A of the compound of formula (I);

[0132] Figure 3 is the TGA spectrum of the crystalline form A of the compound of formula (I);

[0133] Figure 4 is the XRPD spectrum of the crystal form B of the compound of formula (I) using Cu-Kα radiation;

[0134] Figure 5 is the DSC spectrum of the crystalline form B of the compound of formula (I);

[0135] Figure 6 is the TGA spectrum of the crystalline form B of the compound of formula (I);

[0136] Figure 7 is the XRPD spectrum of the crystal form C of the compound of formula (I) using Cu-Kα radiation;

[0137] Figure 8 is the DSC spectrum of the crystalline form C of the compound of formula (I);

[0138] Fig. 9 is the TGA spectrum of the crystalline form C of the compound of formula (I);

[0139] Fig.10 is the XRPD spectrum of the crystal form D of the compound of formula (II) using Cu-Kα radiation;

[0140] Fig.11 is the DSC spectrum of the crystal form D of the compound of formula (II);

[0141] Fig.12 is the TGA spectrum of the crystal form D of the compound of formula (II);

[0142] Fig.13 is the XRPD spectrum of the crystal form E of the compound of formula (III) using Cu-Kα radiation;

[0143] Fig.14 is the DSC spectrum of the crystalline form E of the compound of formula (III);

[0144] Fig.15 is the TGA spectrum of the crystalline form E of the compound of formula (III);

[0145] Fig.16 is the XRPD spectrum of the crystal form F of the compound of formula (IV) under Cu-Kα radiation;

[0146] Fig.17 is the DSC spectrum of the crystalline form F of the compound of formula (IV);

[0147] Fig.18 is the TGA spectrum of the crystalline form F of the compound of formula (IV);

[0148] Fig.19 This is the DVS spectrum of the crystal form B of compound of formula (I). DETAILED DESCRIPTION

[0149] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments, but the specific implementation methods are not intended to limit the content of the present invention.

[0150] Example 1: Preparation of the compound of formula (I)

[0151]

[0152] Step 1:

[0153] Add THF (4.05 L) to a 50 L high and low temperature reactor at once, control the internal temperature at 20-30 ° C, add compound 1 (1345.47 g, 7.91 mol), and then add triethyl phosphoacetate (1912.00 g, 8.36 mol). Control the internal temperature and drop the prepared K2CO3 aqueous solution (3.97 L, 4 M). After the addition, stir the system at 20-30 ° C for 17 hours to stop the reaction. Add water (12 L) to the reactor, stir for 2 hours, filter the system, rinse the filter cake with water (5 L), and vacuum dry (-0.1 Mpa, 50 ° C) to obtain compound 2.

[0154] Step 2:

[0155] Add ethyl acetate (4.55L) and n-hexane (4.55L) to a 50L high and low temperature reactor, control the internal temperature at 30-40°C, then add compound 2 (1710.00g, 7.15mol), K2CO3 (1001.00g, 7.17mol) and (R)-1,2,3,4-tetrahydro-1-naphthylamine (1111.50g, 7.49mol) in sequence, control the internal temperature at 70-80°C, stir for 16 hours, and stop the reaction. Control the internal temperature at 70-75°C, discharge the system into three 5L barrels under stirring, cool naturally to 30-40°C, precipitate a large amount of solid, and crush it to obtain a crude product. Add n-hexane (17L) and water (5.1L) to the reactor, control the internal temperature at 30-40°C, add the above crude product, and stir for 4 hours. After filtration, the filter cake was rinsed with a mixed solvent of n-hexane (5 L) and water (5 L), the filter cake was drained, and vacuum dried (-0.1 MPa, 50° C.) to obtain compound 3.

[0156] Step 3:

[0157] Add compound 3 (500.00 g, 1.36 mol), wet palladium carbon (50 g, 10% content) and THF (5 L) to a 10 L autoclave, replace with argon three times, replace with hydrogen three times, introduce hydrogen (2.8 Mpa), and stir at 25-35 ° C for 1 hour. Add hydrogen to 2.8 Mpa, continue stirring for 1 hour, add hydrogen to 2.8 Mpa, stir for 16.5 hours, and stop the reaction. The reaction solution is filtered through a diatomaceous earth layer, the filtrate is collected, the filter cake is rinsed with THF twice (500 mL*2), and the filtrate is combined for about 6 L. The filtrate containing compound 4 is directly used in the next step, and the yield is calculated according to the theoretical 100%.

[0158] Step 4:

[0159] Add compound 4 (1837.36 g, 5.43 mol, 18.5 L THF solution) to a 50 L high and low temperature reactor at one time, control the internal temperature at 20-30 ° C, add 4-trifluoromethoxyphenylisothiocyanate (1194.00 g, 5.45 mol), heat to 40-45 ° C and stir for 1 hour, then add EDCI (1150.00 g, 6.00 mol), heat to 65-70 ° C and stir for 17 hours. Cool the reaction system to 30-40 ° C, filter, and collect the filtrate. The filtrate is concentrated (-0.1 MPa, 50 ° C) to obtain compound 5. Directly put it into the next step, and the yield is calculated according to the theory of 100%.

[0160] Step 5:

[0161] Compound 5 (2832.31 g, 5.41 mol) and EtOH (11.37 L) were added to a 50 L high and low temperature reactor in sequence. NaOH (437.13 g, 10.93 mol) was dissolved in water (5.65 L) and slowly added to the reactor. The dropping speed was controlled according to the temperature change, and the internal temperature was controlled at 25-35 ° C. After the addition, the mixture was stirred at 25-35 ° C for 16 hours and the reaction was stopped. Dilute hydrochloric acid (1 M) was slowly added to the system to adjust the pH to 5-6, and about 11 L of dilute hydrochloric acid was consumed. After the adjustment was completed, the mixture was stirred at 25-35 ° C for 2 hours, and then filtered and dried. The filter cake was washed with a mixed solvent of EtOH and water (EtOH: water = 1: 1, 5.6 L) and dried. The obtained filter cake was dried under reduced pressure (-0.1 MPa, 50 ° C) to obtain the compound of formula (I). 1 H NMR (400MHz, CD3OD) δ7.60 (d, J=8.6Hz, 2H), 7.33-7.15 (m, 5H), 7.04 (br t, J=7.0Hz, 1H), 6.83 (br d, J=7.6Hz, 1H), 6.74 (br d, J=7.9Hz, 1H), 6.41 (br d, J=8.1Hz, 1H), 5.88 (br t, J=8.1Hz, IH), 3.15-3.01 (m, 1H), 3.00-2.85 (m, 3H), 2.57 (br t, J=7.4Hz, 2H), 2.30 (br s, 2H), 2.13 (br s, 1H), 2.01 (br d, J=16.1Hz, 1H).

[0162] Example 2: Preparation of Crystalline Form A of Compound (I)

[0163] The compound of formula (I) (360 g) was dissolved in DMSO (400 mL) and heated to 80°C. Under stirring, a mixed solution of EtOH (2480 mL) and water (1080 mL) was added dropwise to the solution. After about 2 hours, the addition was completed and the heating was stopped. The solution was slowly cooled to 20°C, stirred for 58 hours, and then filtered. The obtained filter cake was soaked in EtOH (500 mL) for 10 minutes, filtered and drained. Repeat three times, collect the filter cake, and vacuum dry to constant weight to obtain the A crystal form of the compound of formula (I).

[0164] Example 3: Preparation of Crystalline Form B of Compound (I)

[0165] Weigh the crystal form A (20 mg) of the compound of formula (I) into a reaction bottle, add acetone (0.3 mL) to form a suspension. The suspension is magnetically stirred at room temperature for 6 days, centrifuged, the supernatant is removed, and the solid is vacuum dried at 50° C. for two hours to obtain the crystal form B of the compound of formula (I).

[0166] Weigh the crystal form A (20 mg) of the compound of formula (I) into a reaction bottle, add ethyl acetate (0.3 mL) to form a suspension. The suspension is magnetically stirred at room temperature for 6 days, centrifuged, the supernatant is removed, and the solid is vacuum dried at 50° C. for 2 hours to obtain the crystal form B of the compound of formula (I).

[0167] Example 4: Preparation of Crystalline Form B of Compound (I)

[0168] EtOH (7344 mL), DMSO (1836 mL) and compound (I) (2295.00 g, 4.63 mol) were added to a 50 L high and low temperature reactor in sequence. After the addition, the internal temperature was controlled at 80-85 ° C to completely dissolve the compound. Then, the mixture was filtered while hot to remove insoluble impurities, and the filtrate was poured into the reactor again, and the temperature in the reactor was controlled at 82 ° C. After the temperature stabilized, a mixed solution of EtOH: water = 2: 1 was slowly added to the system, and the situation in the reactor was observed while adding the mixture, until slight turbidity appeared during the addition, and then the mixture quickly dissolved and cleared (the temperature was 78 ° C at this time, and 1200 mL of solvent was consumed). 6 g of the B form of compound (I) was added to the system in batches as seed crystals. After the seed crystals were added, the system became turbid, and the heating was stopped, and the temperature was naturally lowered to 25-35 ° C, and stirred for 16.5 hours. The temperature was controlled at 25-35°C, and 17160 mL of a mixed solution of EtOH: water = 2: 1 was slowly added dropwise to the system. After the addition was completed, stirring was continued at 25-35°C. After a total of 24 hours of stirring, stirring was stopped, and the mixture was filtered and dried. The filter cake was washed with a solvent of EtOH: water = 2: 1 (5 L * 2) and dried. The obtained solid was added to a reaction kettle, and EtOH (5.5 L) was added. The mixture was stirred at 25-35°C for 1.5 hours, stirring was stopped, and the mixture was filtered and dried. The filter cake was vacuum dried at 50°C to constant weight to obtain Form B of the compound of formula (I).

[0169] Example 5: Preparation of Crystalline Form C of Compound (I)

[0170] Weigh the crystal form A (20 mg) of the compound of formula (I) into a reaction bottle, add THF (0.3 mL) to form a suspension. The suspension is magnetically stirred at room temperature for 6 days, n-hexane is added dropwise until insoluble matter precipitates, centrifuged, the supernatant is removed, and the solid is vacuum dried at 50° C. for two hours to obtain the crystal form C of the compound of formula (I).

[0171] Example 6: Preparation of Crystalline Form D of Compound (II)

[0172] Weigh the Form A (20 mg) of the compound of formula (I) into a reaction bottle, add acetone (0.3 mL) containing MeSO3H (3 μL), stir magnetically at room temperature for 5 days, centrifuge, remove the supernatant, and vacuum dry the solid at 50°C for two hours to obtain Form D of the compound of formula (II).

[0173] Example 7: Preparation of Crystalline Form E of Compound (III)

[0174] Weigh the crystal form A (20 mg) of the compound of formula (I) and D-glucuronic acid (8.5 mg) into a reaction bottle, add acetone (0.3 mL), stir magnetically at room temperature for 5 days, centrifuge, remove the supernatant, and vacuum dry the solid at 50° C. for two hours to obtain the crystal form E of the compound of formula (III).

[0175] Example 8: Preparation of Crystalline Form F of Compound (IV)

[0176] Weigh the crystal form A (20 mg) of the compound of formula (I) and maleic acid (5.1 mg) into a reaction bottle, add ethyl acetate (0.3 mL), stir magnetically at room temperature for 5 days, add n-heptane until solid precipitates, separate, collect the filter cake, and vacuum dry at 50° C. for two hours to obtain the crystal form F of the compound of formula (IV).

[0177] Example 9: Hygroscopicity study of the crystalline form B of compound of formula (I)

[0178] Experimental Materials:

[0179] SMS DVS Intrinsic Dynamic Vapor Sorption Instrument

[0180] Experimental methods:

[0181] 10.03 mg of the crystalline form B of the compound of formula (I) was weighed and placed in a DVS sample pan for testing.

[0182] Experimental results:

[0183] The DVS spectrum of the crystal form B of the compound of formula (I) is as follows Fig.19 As shown, ΔW = 0.09%.

[0184] Experimental conclusion:

[0185] The weight gain of the crystal form B of the compound of formula (I) at 25° C. and 80% RH is 0.09%, which means it is non-hygroscopic.

[0186] Example 10: Solid Stability Test of Crystalline Form B of Compound (I)

[0187] According to the "Guidelines for Stability Testing of APIs and Preparations" (Chinese Pharmacopoeia 2015 Edition Part IV General Rules 9001), the stability of the crystalline form E of compound of formula (I) under high temperature (60°C, open), high humidity (room temperature / relative humidity 92.5%, open) and strong light (5000lx, sealed) conditions was investigated.

[0188] Accurately weigh about 10 mg of the compound of formula (I) and place it in a dry and clean glass bottle, spread it into a thin layer, cover it with aluminum foil, poke a small hole, and place it under the influencing factor test conditions (60°C, 92.5% RH) and accelerated conditions (40°C / 75% RH and 60°C / 75% RH). The samples placed under light (visible light 1200000Lux, ultraviolet 200W) conditions use transparent glass bottles, one group is fully exposed, and one group is fully wrapped with aluminum foil. Place 2 copies for each condition at each time point, and the samples for XRPD detection are placed separately. The samples placed under different conditions are sampled and tested for XRPD at the planned test endpoint, and the test results are compared with the initial test results on day 0. The test results are shown in Table 7 below:

[0189] Table 7 Solid stability test results of compound B of formula (I)

[0190]

[0191]

[0192] Conclusion: The crystal form B of compound of formula (I) has good stability under high temperature, high humidity, strong light and accelerated conditions.

[0193] Experimental Example 1: IDH1 in vitro enzyme activity test

[0194] IDH1 mutants catalyze the NADPH-dependent reduction of α-KG (α-ketoglutarate) to 2-HG (2-hydroxyglutarate), and the consumed NADPH can be read out using fluorescence.

[0195] Reagents:

[0196] Basic reaction buffer: 50 mM KH2PO4, pH 7.5, 10 mM MgCl2, 10% glycerol, 150 mM NaCl, 0.05% BSA (bovine serum albumin), 2 mM b-ME (2-mercaptoethanol), 0.003% Brij35 (oxyethylene lauryl ether)

[0197] Substrates and cofactors:

[0198] IDH1wt (wild type): 65 μM isocitrate + 50 μM NADP

[0199] IDH1(R132H): 1500μMα-KG+15μM NADPH

[0200] IDH1(R132C): 500μMα-KG+15μM NADPH

[0201] Reaction process:

[0202] 1.33X enzyme (no enzyme in control wells), buffer and NADP or NADPH (control wells) were added to the wells of the reaction plate. The test compound was dissolved in 100% DMSO and added to the enzyme mixture (Echo550, nanoliter level). After simple centrifugation and incubation for 60 minutes, a mixture of 4X substrate and cofactor was added to start the reaction. After simple centrifugation and shaking, incubation was carried out at room temperature for 45 minutes. A mixture of 3X lipoamide dehydrogenase and ninazuridine was prepared and added to the reaction solution to test the amount of NADPH generated or remaining. After simple centrifugation and incubation at room temperature for 10 minutes, the multifunctional microplate reader Envision was used to measure (Ex / Em=535 / 590nm).

[0203] The experimental results are shown in Tables 8 and 9:

[0204] Table 8 IDH1 in vitro enzyme (IDH1 R132H) activity IC 50 Test Results

[0205]

[0206] Table 9 IDH1 in vitro enzyme (IDH1 R132C, WT) activity IC 50 Test Results

[0207]

[0208] Conclusion: The compound of formula (I) has a good inhibitory effect on mutant IDH1R132H and IDH1R132C at the enzymatic level, but has no inhibitory effect on wild-type IDH protein.

[0209] Experimental Example 2: IDH1 cytological activity test

[0210] In this study, the compound was co-incubated with the IDH1 mutant cell line, and the 2HG content in the cell culture supernatant was detected by LC-MS to determine the inhibitory activity of the compound against the IDH1 mutant. IDH1 catalyzes the reduction of isocitrate to α-ketoglutarate (α-KG) in the body, while the IDH1 mutant further catalyzes the reduction of α-KG to 2-hydroxyglutarate (2HG).

[0211] The U87MG-IDH1-R132H cell line is a stable cell line that can stably express the IDH1-R132H mutant after transfecting U87MG cells with IDH1-R132H, while the HT1080 cell line contains the endogenous IDH1-R132C mutant. The experimental process is as follows:

[0212] 1) The compound was diluted 3-fold with DMSO and added to the cell culture plate, with a total of 10 concentrations, and each concentration was duplicated. The negative control well contained only DMSO, and the positive control well contained BAY1436032 at a final concentration of 5 μM. The final DMSO concentration in all wells was 0.5%.

[0213] 2) The IDH1 mutant cell line was seeded into a cell culture plate containing the compound at a density of 40,000 cells / well, and the cells and the compound were co-incubated in a 37° C., 5% CO 2 incubator for 3 days.

[0214] 3) After 3 days, take 10 μl of cell culture supernatant, dilute it 21 times with 200 μl ddH2O water to 210 μl and mix well. Take out 50 μl of the dilution and add 200 μl of precipitation agent (containing 0.4 μg / ml D-2-hydroxyglutaric acid 13 C5 acetonitrile). After centrifugation at 4000 rpm for 10 minutes, 100 μl of the supernatant was taken and the 2-HG content was detected by LC-MS.

[0215] 4) In parallel, the ATPlite 1Step kit was used to detect the effect of the compound on the cell viability of the IDH1 mutant cell line according to the instructions.

[0216] 5) Using the 2HG content data and the inhibition rate percentage (inhibition rate %) of the compound at each concentration on the IDH1 mutant, the calculation formula is:

[0217] Inhibition rate % = (CPD-ZPE) / (HPE-ZPE)×100%

[0218] The cell viability data was used to calculate the cytotoxicity percentage (cytotoxicity%) of the compound against IDH1 mutant cell lines using the following formula:

[0219] Cytotoxicity % = (1-CPD / ZPE) × 100%

[0220] CPD: signal value of compound well

[0221] ZPE: Average signal of negative control wells, with 0.5% DMSO replacing compound

[0222] HPE: Average signal of positive control wells

[0223] 6) The inhibition rate % and cytotoxicity % were fitted to the dose-effect curve using GraphPad Prism software, and the IC of the test compound was obtained. 50 value.

[0224] The experimental results are shown in Table 10:

[0225] Table 10 IDH1 in vitro cell (U87MG) activity IC50 Test Results

[0226]

[0227] Conclusion: At the cellular level, the compound of formula (I) has a good 2-HG inhibitory effect on U87MG glioma cells with IDH1R132H mutation.

[0228] Experimental Example 3: Pharmacokinetic evaluation in mice

[0229] Purpose:

[0230] The pharmacokinetic parameters of the compound of formula (I) in mice were detected.

[0231] Experimental protocol:

[0232] 1) Experimental drug: compound of formula (I);

[0233] 2) Experimental animals: 8 male CD-1 mice aged 7-10 weeks were divided into 2 groups, 4 mice in each group;

[0234] 3) Drug preparation: For the tail vein injection group, weigh an appropriate amount of drug and dissolve it in DMSO: 20% hydroxypropyl beta-cyclodextrin (HPbCD)

[0235] =10:90 mixed solvent, prepared into a 0.5 mg / mL solution; for the oral administration group, weigh an appropriate amount of drug and dissolve it in DMSO:

[0236] Cremophor EL: 5% sulfobutyl cyclodextrin (Captisol) = 5:10:85, prepared into a suspension.

[0237] Experimental operation:

[0238] The first group of animals were given a single injection of the drug at a dose of 1 mg / kg and a concentration of 0.5 mg / mL by tail vein, and the second group of animals were given a dose of 20 mg / kg and a concentration of 2 mg / mL by oral gavage. Plasma samples were collected from the animals at 0.0833 (tail vein injection group only), 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. The drug concentration in the plasma samples was determined by LC-MS / MS method, and the kinetic parameters of the test drugs were shown in Table 11:

[0239] Table 11 Pharmacokinetic evaluation parameters in mice

[0240]

[0241] Conclusion: The compound of formula (I) has good pharmacokinetic properties in mice.

Claims

1. A crystalline form of the compound of formula (I), characterized in that Its X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 7.66±0.20°, 9.78±0.20°, 12.06±0.20°, 17.43±0.20°, 18.02±0.20°, 18.81±0.20°, 20.37±0.20°, 23.10±0.20°; 2. According to the crystal form A according to claim 1, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.66±0.20°, 9.78±0.20°, 12.06±0.20°, 17.43±0.20°, 18.02±0.20°, 18.81±0.20°, 20.37±0.20°, 20.91±0.20°, 22.46±0.20°, 23.10±0.20°.

3. According to the A crystal form described in claim 2, its XRPD spectrum is shown in Figure 1.

4. Form B of the compound of formula (I), characterized in that Its X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 7.48±0.20°, 11.66±0.20°, 15.83±0.20°, 16.69±0.20°, 17.69±0.20°, 19.68±0.20°, 21.79±0.20°, 22.90±0.20°; 5. The crystal form B according to claim 4, has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 7.48±0.20°, 11.66±0.20°, 12.47±0.20°, 15.83±0.20°, 16.69±0.20°, 17.69±0.20°, 19.68±0.20°, 21.79±0.20°, 22.90±0.20°, 23.84±0.20°.

6. The B crystal form according to claim 5, has an XRPD pattern as shown in Figure 4.

7. The crystal form B according to any one of claims 4 to 6, wherein the differential scanning calorimetry curve thereof has an endothermic peak at 246.8±3.0°C.

8. The B crystal form according to claim 7, whose DSC spectrum is shown in Figure 5.

9. The crystal form B according to any one of claims 4 to 6, wherein the weight loss of the crystal form B at 230.0°C±3.0°C is 0.54%.

10. The B crystal form according to claim 9, whose TGA spectrum is shown in Figure 6.

11. Crystal form C of the compound of formula (I), characterized in that Its X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 9.59±0.20°，12.17±0.20°，12.65±0.20°，18.19±0.20°，18.87±0.20°，19.74±0.20°，21.27±0.20°，23.05±0.20°； 12. The crystal form C according to claim 11, has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 9.59±0.20°, 11.60±0.20°, 12.17±0.20°, 12.65±0.20°, 18.19±0.20°, 18.87±0.20°, 19.74±0.20°, 21.27±0.20°, 22.20±0.20°, 23.05±0.20°.

13. The crystal form C according to claim 12, whose XRPD pattern is shown in Figure 7.

14. Compound represented by formula (II):

15. Crystal form D of the compound of formula (II), characterized in that Its X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 7.31±0.20°，8.48±0.20°，13.08±0.20°，16.85±0.20°，17.44±0.20°，19.93±0.20°，21.89±0.20°，22.49±0.20°； 16. The D crystal form according to claim 15, whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.31°, 8.48°, 13.08°, 14.64°, 16.85°, 17.44°, 19.93°, 21.89°, 22.49°, 25.87°.

17. The D crystal form according to claim 16, whose XRPD pattern is shown in Figure 10.

18. Use of the compound according to claim 14, or the crystal form A according to any one of claims 1 to 3, or the crystal form B according to any one of claims 4 to 10, or the crystal form C according to any one of claims 11 to 13, or the crystal form D according to any one of claims 15 to 17 in the preparation of a drug for treating intrahepatic cholangioma.

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