Crystalline forms of 4-(tert-butoxyamino)-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1,3,5-triazin-2-amine
11773079 · 2023-10-03
Assignee
- Chia Tai Tianqing Pharmaceutical Group Co, Ltd. (Jiangsu, CN)
- Lianyungang Runzhong Pharmaceutical Co., Ltd. (Jiangsu, CN)
- Centaurus Biopharma Co., Ltd. (Beijing, CN)
Inventors
- Ticong Huang (Lianyungang, CN)
- Huifeng Xiao (Lianyungang, CN)
- Rui Zhao (Lianyungang, CN)
- Fei Liu (Lianyungang, CN)
- Wei Wei (Lianyungang, CN)
- Hongjiang Xu (Lianyungang, CN)
- Xiaojin Wang (Lianyungang, CN)
- Jingli Wu (Lianyungang, CN)
Cpc classification
International classification
A61K31/53
HUMAN NECESSITIES
C07D251/18
CHEMISTRY; METALLURGY
Abstract
The invention discloses a crystal of 4-(tert-butoxyamino)-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1,3,5-triazin-2-amine compound, a mesylate salt and crystal thereof, a preparation method thereof, a composition containing thereof, and a use thereof for inhibiting activity of mutant IDH2 and treating cancer. ##STR00001##
Claims
1. Crystalline Form A of 4-(tert-butoxyamino)-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1,3,5-triazin-2-amine of Formula I: ##STR00008## wherein the crystalline form is characterized by an X-ray powder diffraction pattern having X-ray powder diffraction peaks (°2θ) expressed by values at 10.1°, 16.1°, 17.5°, 18.9°, and 21.7°±0.2° (2θ).
2. The crystalline Form A according to claim 1, wherein the crystalline Form A is further characterized by an X-ray powder diffraction pattern having additional X-ray powder diffraction peaks (°2θ) expressed by values at 23.5°, 24.4°, and 26.2°±0.2°(2θ).
3. The crystalline Form A according to claim 1, wherein the crystalline Form A is further characterized by an X-ray powder diffraction pattern having additional X-ray powder diffraction peaks (°2θ) expressed by values at 22.4°, 22.8°, 23.5°, 24.0°, 24.4°, 26.2°, and 29.8°±0.2°(2θ).
4. The crystalline Form A according to claim 1, wherein the crystalline Form A is further characterized by an X-ray powder diffraction pattern having additional X-ray powder diffraction peaks (°2θ) expressed by values at 12.3°, 14.3°, 14.6°, 19.7°, 20.1°, 22.4°, 22.8°, 23.5°, 24.0°, 24.4°, 26.2°, and 29.8°±0.2°(2θ).
5. The crystalline Form A according to claim 1, wherein the crystalline Form A is further characterized by an X-ray powder diffraction pattern as shown in
6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the crystalline Form A according to claim 1.
7. A method for treating an isocitrate dehydrogenase 2 mutation-induced cancer in a subject, wherein the 1 method comprises administering to the subject in need thereof the crystalline Form A according to claim 1.
8. The method according to claim 7, wherein the isocitrate dehydrogenase 2 mutation-induced cancer is selected from the group consisting of glioblastoma, myelodysplastic syndrome, myeloproliferative neoplasm, acute myelogenous leukemia, sarcoma, melanoma, non-small cell lung cancer, chondrosarcoma, bile duct cancer, and angioimmunoblastic non-Hodgkin's lymphoma.
9. A process for preparing the crystalline Form A of 4-(tert-butoxyamino)-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1,3,5-triazin-2-amine of Formula I according to claim 1: ##STR00009## wherein the process comprises the following steps: (1) at room temperature, dissolving 4-(tert-butoxyamino)-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1,3,5-triazin-2-amine of the Formula I below in at least one organic solvent selected from the group consisting of acetone, acetonitrile, methanol, ethanol, isopropanol, tert-butanol, tetrahydrofuran, and 1,4-dioxane, or a mixture thereof, to obtain a solution: ##STR00010## (2) concentrating the solution formed in step (1) above under reduced pressure; and (3) drying the crystalline Form A of 4-(tert-butoxyamino)-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1,3,5-triazin-2-amine of the Formula I above.
10. The process according to claim 9, wherein the organic solvent is selected from the group consisting of tetrahydrofuran and 1,4-dioxane, or a mixture thereof.
11. A process for preparing the crystalline Form A of 4-(tert-butoxyamino)-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1,3,5-triazin-2-amine of Formula I according to claim 1: ##STR00011## wherein the process comprises the following steps: (1) slurrying 4-(tert-butoxyamino)-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1,3,5-triazin-2-amine of the Formula I below in at least one organic solvent selected from the group consisting of dichloromethane, diethyl ether, ethylene glycol dimethyl ether, methyl tert-butyl ether, n-hexane, n-heptane, and n-octane, or a mixture thereof: ##STR00012## (2) filtering the slurry formed in step (1) above; and (3) drying the crystalline Form A of 4-(tert-butoxyamino)-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1,3,5-triazin-2-amine of the Formula I above.
12. The process according to claim 11, wherein the organic solvent is selected from the group consisting of dichloromethane, methyl tert-butyl ether, and n-heptane, or a mixture thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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SPECIFIC EMBODIMENTS
(20) The following specific examples are provided to enable those skilled in the art to more clearly understand and practice the invention. They should not be construed as a limitation to the scope of the invention, but as mere illustrations and typical representatives of the invention. Those skilled in the art will understand that there are other synthetic routes involved for preparing the compounds of the present application, and ones provided below are non-limiting examples.
(21) All operations involving raw materials that are susceptible to oxidation or hydrolysis are carried out under a nitrogen protection atmosphere. Unless indicated otherwise, raw materials used in the present invention are commercially available and directly used without further purification. Solvents used in the present invention are commercially available and directly used without any spefical treatments.
(22) Column chromatography was performed using silica gel (200-300 mesh) produced by Qingdao Chemical Co., Ltd. Nuclear magnetic resonance chromatography (NMR) was measured using a Varian VNMRS-400 nuclear magnetic resonance meter; Liquid chromatography-mass spectrometry (LC/MS) was performed using FINNIGAN Thermo LCQ Advantage MAX, Agilent LC 1200 series (column: Waters Symmetry C18, Ø4.6×50 mm, 5 micron, 35° C.) and using ESI (+) ion mode. The melting point tester is BUCHI B-545 melting point apparatus.
Example 1: Preparation of the Compound of Formula I
A: methyl 6-trifluoromethyl-pyridine-2-carboxylate
(23) ##STR00003##
(24) To a solution of 2-bromo-6-trifluoromethylpyridine (1.48 g, 6.55 mmol) in methanol (50.0 mL) were added in sequence palladium acetate (74.0 mg, 0.33 mmol), 1,1′-bis(diphenylphosphino)ferrocene (363.0 mg, 0.655 mmol) and triethylamine (0.92 g, 9.1 mmol) under the protection of nitrogen gas. The reaction solution reacted at 60° C. under a carbon monoxide atmosphere with 2 atmospheric pressures for 18 hours. After the reaction was completed, the reaction solution was cooled to room temperature and filtered. The filtrate was concentrated under vacuum and reduced pressure, and the resulting residue was purified by silica gel column chromatography to afford the title compound (0.9 g, yield: 67.0%).
(25) 2-Bromo-6-trifluoromethylpyridine was commercially available.
B: 6-(6-trifluoromethylpyridin-2-yl)-1,3,5-triazin-2,4(1H,3H)-dione
(26) ##STR00004##
(27) To a solution of sodium ethoxide (11.2 g, 165.0 mmol) in ethanol (200 mL) were added in sequence methyl 6-trifluoromethyl-pyridine-2-carboxylate (10.0 g, 48.7 mmol) and biuret (4.2 g, 40.7 mmol) under the protection of nitrogen gas. The mixture was heated to reflux for 2 hours and then cooled to room temperature. Then the reaction solution was concentrated under vacuum and reduced pressure, and the resulting residue was poured into water and adjusted to pH 7 with 6N hydrochloric acid solution. The resulting solid was filtered, and the filter cake was washed with water and then dried to afford the title compound (5.0 g, yield: 47.5%).
C: 2,4-dichloro-6-(6-trifluoromethylpyridin-2-yl)-1,3,5-triazine
(28) ##STR00005##
(29) A mixture solution of 6-(6-trifluoromethylpyridin-2-yl)-1,3,5-triazin-2,4(1H,3H)-dione (15.0 g, 58.1 mmol) and phosphorus oxychloride (200 mL) reacted at 100° C. for 2 hours under the protection of nitrogen gas and then cooled to room temperature. The reaction solution was concentrated under vacuum and reduced pressure, and the resulting residue was poured into a saturated aqueous solution of sodium bicarbonate. The resulting mixture was extracted with ethyl acetate (2×100 mL), and the organic phase was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated under vacuum to afford the title compound (10.0 g, yield: 58.3%).
D: 4-chloro-6-(6-trifluoromethylpyridin-2-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1,3,5-triazin-2-amine
(30) ##STR00006##
(31) To a solution of 2,4-dichloro-6-(6-trifluoromethylpyridin-2-yl)-1,3,5-triazine (5.0 g, 16.9 mmol) in tetrahydrofuran (100 mL) were added 4-amino-2-trifluoromethylpyridine (3.3 g, 20.3 mmol) and sodium bicarbonate (2.14 g, 25.3 mmol). The mixture reacted at 70° C. for 8 hours and then cooled to room temperature. The reaction solution was then concentrated under vacuum and reduced pressure, and the resulting residue was purified by silica gel column chromatography to afford the title compound (6.5 g, yield: 91.2%).
(32) 4-Amino-2-trifluoromethylpyridine was commercially available.
E: 4-(tert-butoxyamino)-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1,3,5-triazin-2-amine (the Compound of Formula I)
(33) ##STR00007##
(34) To a solution of 4-chloro-6-(6-trifluoromethylpyridin-2-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1,3,5-triazin-2-amine (39.7 g, 94.5 mmol) in tetrahydrofuran (400 mL) were added O-tert-butylhydroxylamine hydrochloride (17.7 g, 140.9 mmol) and sodium bicarbonate (31.7 g, 377 mmol). The mixture reacted at 70° C. for 8 hours and then cooled to room temperature. Then the reaction solution was filtered, the filtrate was concentrated under vacuum and reduced pressure, and the resulting residue was purified by silica gel column chromatography to afford the title compound (30.0 g, yield: 67%).
(35) Mp: 226.1˜228° C.
(36) MS m/z[ESI]: 474.14 [M+H].sup.+.
(37) .sup.1H-NMR (400 MHz, DMSO-d6): δ=11.00 (s, 1H), 10.84 (s, 1H), 8.74 (s, 1H), 8.57 (t, J=6.5 Hz, 2H), 8.31 (t, J=7.9 Hz, 1H), 8.11 (d, J=7.7 Hz, 1H), 7.99 (s, 1H), 1.28 (s, 9H).
Example 2: Preparation of the Crystalline Form A of the Compound of Formula I
(38) At room temperature, to tetrahydrofuran (60 mL) was added the compound of Formula I (5.0 g, 10.6 mmol) obtained in Example 1, stirred and dissolved to obtain a clear solution. Then the solution was concentrated under reduced pressure, and the resulting solid was dried under vacuum at 80° C. for 4 hours, Mp: 224.6° C.˜226° C.
Example 3: Preparation of the Crystalline Form A of the Compound of Formula I
(39) At room temperature, to 1,4-dioxane (100 mL) was added the compound of Formula I (5.0 g, 10.6 mmol) obtained in Example 1, stirred and dissolved to obtain a clear solution. Then the solution was concentrated under reduced pressure, and the resulting solid was dried under vacuum at 80° C. for 4 hours, Mp: 224.6° C.˜226° C.
Example 4: Preparation of the Crystalline Form A of the Compound of Formula I
(40) To the compound of Formula I (5.0 g, 10.6 mmol) obtained in Example 1 was added dichloromethane (50 mL), and slurred under reflux for 2 hours, and then the reaction temperature was lowered to room temperature. The resulting mixture was filtered, and the filter cake was dried under vacuum at 80° C. for 4 hours, Mp: 224.6° C.˜226° C.
Example 5: Preparation of the Crystalline Form A of the Compound of Formula I
(41) To the compound of Formula I (5.0 g, 10.6 mmol) obtained in Example 1 was added n-heptane (50 mL), and slurred at room temperature for 2 hours. The resulting mixture was filtered, and the filter cake was dried under vacuum at 80° C. for 4 hours, Mp: 224.6° C.˜226° C.
Example 6: Preparation of the Crystalline Form B of the Compound of Formula I
(42) At room temperature, to the compound of Formula I (5.0 g, 10.6 mmol) obtained in Example 1 was added ethyl acetate (50 mL), stirred and dissolved to obtain a clear solution. Then the solution was concentrated under reduced pressure, and the resulting solid was dried under vacuum at 80° C. for 4 hours, Mp: 226.1° C.˜228° C.
Example 7: Preparation of the Hydrochloride Salt of the Compound of Formula I
(43) To a solution of the compound of Formula I (1.0 g, 2.1 mmol) obtained in Example 1 in ethyl acetate (10 mL) was added dropwise a prepared solution of 14.6% by mass hydrogen chloride (3.2 mmol) in ethanol (0.8 g) at room temperature. After stirring for 2 hours, a solid was precipitated from the reaction solution, and then filtered. The filter cake was dried under vacuum at 80° C., Mp: 177.2˜179° C.
Example 8: Preparation of the Sulfate of the Compound of Formula I
(44) To a solution of the compound of Formula I (1.0 g, 2.1 mmol) obtained in Example 1 in ethyl acetate (10 mL) was added dropwise a prepared solution (1.9 mL) of 1.67 mol/L dilute sulfuric acid in ethanol at room temperature. After stirring for 2 hours, a solid was precipitated from the reaction solution, and then filtered. The filter cake was dried under vacuum at 80° C., Mp: 181.2˜183° C.
Example 9: Preparation of the p-Toluenesulfonate of the Compound of Formula I
(45) P-toluenesulfonic acid monohydrate (0.6 g, 3.1 mmol) was dissolved in acetone (2 mL) at room temperature. Then, to a solution of the compound of Formula I (1.0 g, 2.1 mmol) obtained in Example 1 in acetone (10 mL) was added dropwise the pre-formulated solution of p-toluenesulfonic acid in acetone. After stirring for 2 hours, a solid was precipitated from the reaction solution, and then filtered. The filter cake was dried under vacuum at 80° C., Mp: 173.9˜176° C.
Example 10: Preparation of the Maleate of the Compound of Formula I
(46) To a solution of the compound of Formula I (2.45 g, 5.2 mmol) obtained in Example 1 in acetone (10 mL) was added maleic acid (0.58 g, 5.0 mmol) at room temperature. After stirring for 2 hours, a solid was precipitated from the reaction solution, and then filtered. The filter cake was dried under vacuum at 80° C., Mp: 171.3˜173° C.
Example 11: Preparation of the Crystalline Form I of the Mesylate of the Compound of Formula I
(47) To a solution of the compound of Formula I (3.0 g, 6.3 mmol) obtained in Example 1 in methyl tert-butyl ether (90 mL) was added dropwise methanesulfonic acid (0.61 g, 6.3 mmol) at room temperature. After stirring for 1 hour, a solid was precipitated from the reaction solution, and then filtered. The filter cake was dried under vacuum at 80° C., Mp: 148.2˜150.2° C.
Example 12: Preparation of the Crystalline Form II of the Mesylate of the Compound of Formula I
(48) To a solution of the compound of Formula I (3.0 g, 6.3 mmol) obtained in Example 1 in methyl tert-butyl ether (90 mL) was added dropwise methanesulfonic acid (0.61 g, 6.3 mmol) at a reflux temperature. After stirring for 1 hour, the reaction temperature was lowered to room temperature, and a solid was precipitated from the reaction solution and then filtered. The filter cake was dried under vacuum at 80° C., Mp: 187.1˜189.1° C.
Example 13: Preparation of the Crystalline Form III of the Mesylate of the Compound of Formula I
(49) To a solution of the compound of Formula I (5.0 g, 10.6 mmol) obtained in Example 1 in ethyl acetate (100 mL) was added dropwise methanesulfonic acid (1.21 g, 12.6 mmol) at room temperature. After stirring for 2 hours, a solid was precipitated from the reaction solution, and then filtered. The filter cake was dried under vacuum at 80° C., Mp: 150.9˜152.9° C.
Example 14: Preparation of the Crystalline Form IV of the Mesylate of the Compound of Formula I
(50) To a solution of the compound of Formula I (5.0 g, 10.6 mmol) obtained in Example 1 in ethyl acetate (100 mL) was added dropwise methanesulfonic acid (3.05 g, 31.5 mmol) at room temperature. After stirring for 2 hours, a solid was precipitated from the reaction solution, and then filtered. The filter cake was dried under vacuum at 80° C. for 4 hours and then further dried under vacuum at 100° C. for 4 hours, Mp: 155.0˜157.0° C.
Example 16: Preparation of the Crystalline Form V of the Mesylate of the Compound of Formula I
(51) To a solution of the compound of Formula I (5.0 g, 10.6 mmol) obtained in Example 1 in acetone (100 mL) was added dropwise methanesulfonic acid (3.05 g, 31.5 mmol) at room temperature. After stirring for 2 hours, a solid was precipitated from the reaction solution, and then filtered. The filter cake was dried under vacuum at 80° C., Mp: 151.8˜153.8° C.
Example 17: Preparation of the Crystalline Form VI of the Mesylate of the Compound of Formula I
(52) To a solution of the compound of Formula I (5.0 g, 10.6 mmol) obtained in Example 1 in acetonitrile (100 mL) was added dropwise methanesulfonic acid (1.21 g, 12.6 mmol) at room temperature. After stirring for 2 hours, a solid was precipitated from the reaction solution, and then filtered. The filter cake was dried under vacuum at 80° C., Mp: 145.5˜147.5° C.
Example 18: Preparation of the Crystalline Form VII of the Mesylate of the Compound of Formula I
(53) To a solution of the compound of Formula I (5.0 g, 10.6 mmol) obtained in Example 1 in acetonitrile (100 mL) was added dropwise methanesulfonic acid (3.05 g, 31.5 mmol) at room temperature. After stirring for 2 hours, a solid was precipitated from the reaction solution, and then filtered. The filter cake was dried under vacuum at 80° C., Mp: 140.1˜142.1° C.
Example 19: Preparation of the Crystalline Form VIII of the Mesylate of the Compound of Formula I
(54) The compound of Formula 1 (3.0 g, 6.3 mmol) was dissolved in methyl tert-butyl ether (90 mL), and the reaction solution was heated to reflux. Then, a solution of methylenesulfonic acid (0.61 g, 6.3 mmol) in methyl tert-butyl ether (60 mL) was slowly added dropwise thereto. The reaction solution was refluxed to react for 1 hour, and then cooled to room temperature followed by filteration. The filter cake was dried under vacuum at 90° C. to afford a white solid, MP: 188.8˜191.2° C.
Example 20: Preparation of the Crystalline Form VIII of the Mesylate of the Compound of Formula I
(55) The compound of Formula 1 (3.0 g, 6.3 mmol) was dissolved in methyl tert-butyl ether (90 mL), and the reaction solution was heated to reflux. Then, a solution of methylenesulfonic acid (0.31 g, 3.1 mmol) in methyl tert-butyl ether (30 mL) was slowly added dropwise thereto. The reaction solution was refluxed to react for 1 hour, and then cooled to room temperature followed by filteration. The filter cake was dried under vacuum at 90° C. to afford a white solid, MP: 188.8˜191.2° C.
Example 21: Hygroscopicity Test of Various Salts of the Compound of Formula I
(56) The various acid addition salts of the compound of Formula I were tested according to “Guiding Principles for Drug Hygroscopicity Test” described in the Chinese Pharmacopoeia, 2010 edition, Part II, Appendix XIX J. The increased weights by hygroscopy of the samples were calculated respectively, and the results are shown in Table 11.
(57) TABLE-US-00011 TABLE 11 Hygroscopicity Test Results Increased Weights by Hygroscopy, Sample Names HR = 80% ± 2(%) The mesylate (Example 12) 1.0 The hydrochloride salt 0.9 (Example 7) The sulfate (Example 8) 1.3 The p-toluenesulfonate 0.7 (Example 9) Maleate (Example 10) 2.2 The crystalline Form VIII of 1.0 the mesylate (Example 19 )
Example 22: Stability Test of Various Salts of the Compound of Formula I
(58) The stability of the various acid addition salts of the compound of Formula I were tested according to the method described in the Chinese Pharmacopoeia, 2010 edition, Part II, Appendix XIX C. The results are shown in Table 12.
(59) TABLE-US-00012 TABLE 12 Stability Test Results Purity Purity after high before temperature and Sample Names test high humidity test The mesylate (Example 12) 98.82% 99.22% The hydrochloride salt (Example 7) 98.85% 99.62% The sulfate (Example 8) 99.30% 93.58% The p-toluenesulfonate (Example 9) 98.79% 97.63% The crystalline Form VIII of the 98.82% 99.22% mesylate (Example 19 )
Example 23: Inhibitory Activity Test Against IDH2
(60) The inhibitory activity of the compound of the present application against IDH2 (R172K, 40-end) was determined by using the following method. The inhibitory activity was expressed as IC.sub.50 values, i.e., the concentration of the compound required to achieve 50% inhibition of IDH2 activity.
(61) Materials and Methods:
(62) The inhibitory activity of a compound against IDH2 (R172K, 40-end) was determined by the decrease of a helper factor NADPH (reduced coenzyme II). The test compound was pre-incubated with an enzyme and NADPH, and then a reaction was initiated by the addition of a-KG, and performed for 120 minutes under a linear condition. Then, the reaction was terminated by the addition of diaphorase (lipoamide dehydrogenase) and the corresponding substrate resazurin. Diaphorase terminated the IDH2m reaction by decreasing the available helper factor NADPH, which oxidized NADPH to NADP (oxidized coenzyme II), and reduced resazurin to highly fluorescent resorufin. The amount of remaining helper factor NADPH after a specific reaction time was quantified via an easily detectable fluorophore.
(63) Specifically, 2.5 μl of a 3-fold gradient diluted test compound was added to a 384-well plate, and then 5 μl of a reaction buffer (20 mM Tris-HCl, PH7.5; 150 mM NaCl; 10 mM MgCl.sub.2; 10 mM MnCl.sub.2; 0.4 mg/ml BSA and 2 mM DTT) containing 80 nM IDH2 (R172K, 40-end) and 40 μM NADPH was added. Then, the resulting test mixture was incubated for 120 minutes at a temperature of 23° C., and then 2.5 μl of the reaction buffer containing 4 mM a-KG was added to initiate the reaction. After incubating for 120 minutes at room temperature, 5 μl of a termination mixture (0.4 U/ml diaphorase and 40 μM resazurin) prepared with the reaction buffer was added to convert resazurin to resorufin to determine the remaining NADPH. After incubating for 10 minutes at a temperature of 23° C., a fluorescence value was determined through Flexstation 3 at Ex535/Em595. The results are shown in Table 13: the compound of Formula I has a better inhibitory activity than AG-221.
(64) TABLE-US-00013 TABLE 13 Inhibitory activity against IDH2 Sample Names IC.sub.50 (nM) The compound of 32 Formula I AG-221 68
Example 24: Pharmacokinetic Test
(65) Healthy male adult rats (7-9 weeks old) were used, each group of animals (3 male rats) was intragastrically administered once at a single dose of 5 mg/kg. The animals in the intragastric administration group were fasted overnight before this study. The fasting time period was from 10 hours before administration to 4 hours after administration.
(66) Blood samples were taken at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h after administration. The animals were anesthetized with isoflurane using a small animal anesthesia machine, and then 0.3 mL whole blood samples were taken from the fundus venous plexus. The blood samples were placed in heparin anticoagulant tubes, and centrifuged for 5 min at 4° C. and 4000 rpm. The resulting plasma samples were transferred to centrifuge tubes, and stored at −80° C. until analysis.
(67) Verified liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to analyze the plasma samples. Plasma concentration-time data of individual animals were analyzed using WinNonlin (Professional Edition, version 6.3; Pharsight Company) software. The non-compartmental model was introduced for concentration analysis. The pharmacokinetic parameters (PK parameters) of the compounds were calculated. The results are shown in Table 14. The compound of Formula I has a good in vivo metabolic level and a long half-life, and its plasma concentration is higher than AG-221 at the same dosage.
(68) TABLE-US-00014 TABLE 14 PK parameters of the compound of Formula I and AG-221 The compound PK parameters of Formula I AG-221 Dosage ( mg/kg) 5 5 T.sub.1/2 (h) 12.0 3.73 T.sub.max (h) 5.0 4.00 C.sub.max (ng/ml) 589 479 AUC(0-∞) (ng*h/mL) 10838 5385
Example 25: Pharmacokinetic Test
(69) 1. Test material Species: SD rats; Level: SPF; Number and gender: 20, males; Body weight: 200˜220 g; Source: Shanghai Xipuer Bikai Experimental Animal Co., Ltd. SCXK (Shanghai) 2013-0016.
(70) 2. Test method
(71) Twenty male SPF SD rats were randomly divided into 4 groups, fasted (free access to water) overnight, and then intragastrically administered with 50 mg/kg of the crystalline Form I of the mesylate of the compound of Formula I (Example 11), 50 mg/kg of the crystalline Form II of the mesylate of the compound of Formula I (Example 12), 50 mg/kg of the crystalline Form VIII of the mesylate of the compound of Formula I (Example 19) and 50 mg/kg of the hydrochloride salt of the compound of Formula I (Example 7), respectively (in basic group). Blood samples (0.2˜0.3 mL) were taken from the orbital of the rats at the blood collection time points 0.25 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 24 h, 30 h and 48 h after administration. The whole blood collected was then placed in EDTA-K2 centrifuge tubes, stored at 4° C., and centrifuged for 10 min at a speed of 4000 rpm at 4° C. within 0.5 h to separate plasma. After the collection of all the plasma, it was stored at −20° C. within 1 h. Pharmacokinetic parameters (PK parameters) were calculated using DAS 3.2.1 software according to the non-compartment statistical moment theory, and the results are shown in Table 15.
(72) TABLE-US-00015 TABLE 15 PK parameters after the intragastric administration of 50 mg/kg (in free base) of the compounds in SD rats The The The crystalline crystalline crystalline Form I Form II Form VIII The of the of the of the hydrochloride mesylate mesylate mesylate salt of the of the of the of the compound PK compound compound compound of parameters of Formula I of Formula I of Formula I Formula I T.sub.max (h) 6.60 8.80 8.80 10.6 C.sub.max (ng/ml) 1728 2102 2102 1642 MRT(0-t) (h) 16.5 15.0 15.0 18.02 T.sub.1/2(h) 10.4 9.53 9.53 13.67 AUC(0-t) 43925 42249 42249 48570.9 (ng*h/mL) AUC(0-∞) 47369 43976 43976 55252.1 (ng*h/mL)