SALT FORM AND CRYSTAL FORM OF MUTANT IDH1 INHIBITOR AND PREPARATION METHOD THEREFOR
20230045991 · 2023-02-16
Assignee
Inventors
- Yaxian CAI (Shanghai, CN)
- Bao YUE (Shanghai, CN)
- Peng YU (Shanghai, CN)
- Changqing WEI (Shanghai, CN)
- Wenyuan QIAN (Shanghai, CN)
Cpc classification
C07D235/30
CHEMISTRY; METALLURGY
International classification
Abstract
A salt form and crystal form of a mutant IDHI inhibitor and a preparation method therefor.
##STR00001##
Claims
1. A crystalline form A of compound represented by formula (I), wherein its X-ray powder diffraction spectrum shows characteristic diffraction peaks at 2 θ angles of 9.78±0.20°, 12.06±0.20° and 20.37±0.20°; ##STR00011##
2. The crystalline form A according to claim 1, wherein its X-ray powder diffraction spectrum shows characteristic diffraction peaks at 2 θ angles of 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° and 23.10±0.20°; or wherein its X-ray powder diffraction spectrum shows characteristic diffraction peaks at 2 θ angles of 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° and 23.10±0.20°; or wherein its XRPD spectrum is shown in
3-4. (canceled)
5. A crystalline form B of compound represented by formula (I), wherein its X-ray powder diffraction spectrum shows characteristic diffraction peaks at 2 θ angles of 11.66±0.20°, 16.69±0.20° and 17.69±0.20°.
6. The crystalline form B according to claim 5, wherein its X-ray powder diffraction spectrum shows characteristic diffraction peaks at 2 θ angles of 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° and 22.90±0.20°; or wherein its X-ray powder diffraction spectrum shows characteristic diffraction peaks at 2 θ angles of 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° and 23.84±0.20°, or wherein its XRPD spectrum is shown in
7-8. (canceled)
9. The crystalline form B according to claim 5, wherein its differential scanning calorimetry graph shows an endothermic peak at 246.8±3.0° C.
10. The crystalline form B according to claim 9, wherein its DSC graph is shown in
11. The crystalline form B according to claim 5, wherein its thermogravimetric analysis graph shows a weight loss of 0.54% at 230.0° C.±3.0° C.
12. The crystalline form B according to claim 11, wherein its TGA graph is shown in
13. A crystalline form C of compound represented by formula (I), wherein its X-ray powder diffraction spectrum shows characteristic diffraction peaks at 2 θ angles of 9.59±0.20°, 18.19±0.20° and 19.74±0.20°.
14. The crystalline form C according to claim 13, wherein its X-ray powder diffraction spectrum shows characteristic diffraction peaks at 2 θ angles of 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° and 23.05±0.20°; or wherein its X-ray powder diffraction spectrum shows characteristic diffraction peaks at 2 θ angles of 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° and 23.05±0.20°, or wherein its XRPD spectrum is shown in
15-16. (canceled)
17. A compound represented by formula (II), ##STR00012##
18. A crystalline form D of the compound represented by formula (II) according to claim 17, wherein its X-ray powder diffraction spectrum shows characteristic diffraction peaks at 2 θ angles of 16.85°±0.20°, 19.93°±0.20° and 21.89°±0.20°.
19. The crystalline form D according to claim 18, wherein its X-ray powder diffraction spectrum shows characteristic diffraction peaks at 2 θ angles of 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° and 22.49±0.20°.
20. The crystalline form D according to claim 19, wherein its X-ray powder diffraction spectrum shows characteristic diffraction peaks at 2 θ angles of 7.31°, 8.48°, 13.08°, 14.64°, 16.85°, 17.44°, 19.93°, 21.89°, 22.49° and 25.87°.
21. The crystalline form D according to claim 20, wherein its XRPD spectrum is shown in
22. A method for treating tumor in intrahepatic bile duct, comprising administering a therapeutically effective amount of the compound according to claim 17 to a subject in need thereof.
23. A method for treating tumor in intrahepatic bile duct, comprising administering a therapeutically effective amount of the crystalline form A according to claim 1 to a subject in need thereof.
24. A method for treating tumor in intrahepatic bile duct, comprising administering a therapeutically effective amount of the crystalline form B according to claim 5 to a subject in need thereof.
25. A method for treating tumor in intrahepatic bile duct, comprising administering a therapeutically effective amount of the crystalline form C according to claim 13 to a subject in need thereof.
26. A method for treating tumor in intrahepatic bile duct, comprising administering a therapeutically effective amount of the crystalline form D according to claim 18 to a subject in need thereof.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION
[0125] In order to better understand the content of the present disclosure, it will be further illustrated below in conjunction with specific examples, but the specific examples do not limit the content of the present disclosure.
Example 1
Preparation of Compound Represented by Formula (I)
[0126] ##STR00007##
Step 1:
[0127] THF (4.05 L) was added at one time to a 50 L high-low temperature reaction kettle and the internal temperature was controlled at 20˜30° C. Compound 1 (1345.47 g, 7.91 mol) was added, followed by triethyl phosphoryl acetate (1912.00 g, 8.36 mol). The internal temperature was controlled, and the prepared aqueous K.sub.2CO.sub.3 solution (3.97 L, 4 M) was added dropwise. After the addition, the system was stirred at 20-30° C. for 17 h and then the reaction was terminated. Water (12 L) was added to the reaction kettle and stirred for 2 h. The system was filtered, and the filter cake was rinsed with water (5 L) and dried under vacuum (−0.1 Mpa, 50° C.) to obtain compound 2.
Step 2:
[0128] Ethyl acetate (4.55 L) and n-hexane (4.55 L) were added to a 50 L high-low temperature reaction kettle and the internal temperature was controlled at 30˜40° C. Then compound 2 (1710.00 g, 7.15 mol) was added, followed by K.sub.2CO.sub.3 (1001.00 g, 7.17 mol) and (R)-1,2,3,4-tetrahydro-1-naphthylamine (1111.50 g, 7.49 mol), and the internal temperature was controlled to 70˜80° C. The system was stirred for 16 h and the reaction was terminated. The internal temperature was controlled at 70˜75° C., and the system was released into three 5L barrels under stirring and cooled to 30˜40° C. naturally to precipitate a large amount of solid, which was triturated to obtain a crude product. N-hexane (17 L) and water (5.1 L) were added to the reaction kettle and the internal temperature was controlled to 30˜40° C. The above crude product was added and stirred for 4 h. After filtration, the filter cake was rinsed with a mixed solvent of n-hexane (5 L) and water (5 L), sucked and dried under vacuum −0.1 Mpa, 50° C.) to obtain compound 3.
Step 3:
[0129] Compound 3 (500.00 g, 1.36 mol), wet palladium carbon (50 g, 10% content) and THF (5L) were added to a 10L autoclave. The system was replaced with argon three times followed by hydrogen three times, introduced with hydrogen (2.8 Mpa) and stirred at 25˜35° C. for 1 h. The system was supplemented with hydrogen to 2.8 Mpa and continued to be stirred for 1 h. Then the system was supplemented with hydrogen to 2.8 Mpa again and stirred for 16.5 h, and the reaction was terminated. The reaction solution was filtered through a celite layer. The filtrate was collected. The filter cake was rinsed twice with THF (500 mL*2), and the filtrate was combined for about 6 L. The filtrate containing compound 4 was directly used in the next step, and the yield was calculated as theoretical 100%.
Step 4:
[0130] Compound 4 (1837.36 g, 5.43 mol, 18.5 L of the THF solution) was added at one time to a 50 L high-low temperature reaction kettle and the internal temperature was controlled at 20˜30° C. Then 4-trifluoromethoxy phenyl isothiocyanate (1194.00 g, 5.45 mol) was added, heated to 40˜45° C. and stirred for 1 h. Then EDCI (1150.00 g, 6.00 mol) was added, heated to 65˜70° C. and stirred for 17 h. The reaction system was cooled to 30˜40° C. and filtered, and the filtrate was collected. The filtrate was concentrated (−0.1 MPa, 50° C.) to obtain compound 5, which was directly used in the next step, and the yield was calculated as theoretical 100%.
Step 5:
[0131] Compound 5 (2832.31 g, 5.41 mol) and EtOH (11.37 L) were added to a 50 L high-low temperature reaction kettle in sequence. NaOH (437.13 g, 10.93 mol) was dissolved in water (5.65 L) and the mixture was slowly added dropwise to the kettle with the dripping speed controlled according to the temperature change, and the internal temperature was controlled to 25˜35° C. After the addition, the system was stirred at 25˜35° C. for 16 h and then the reaction was terminated. Dilute hydrochloric acid (1M) was lowly added dropwise to the system to adjust pH=5-6, with about 11 L of dilute hydrochloric acid consumed. After the adjustment, the system was continued to be stirred at 25˜35° C. for 2 h, then filtered and sucked. The filter cake was washed with a mixed solvent of EtOH and water (EtOH: water =1:1, 5.6 L) and sucked. The obtained filter cake was dried under reduced pressure (−0.1 MPa, 50° C.) to obtain compound represented by formula (I). .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.60 (d, J=8.6 Hz, 2H), 7.33-7.15 (m, 5H), 7.04 (br t, J=7.0 Hz, 1H), 6.83 (br d, J=7.6 Hz, 1H), 6.74 (br d, J=7.9 Hz, 1H), 6.41 (br d, J=8.1 Hz, 1H), 5.88 (br t, J=8.1 Hz, 1H), 3.15-3.01 (m, 1H), 3.00-2.85 (m, 3H), 2.57 (br t, J=7.4 Hz, 2H), 2.30 (br s, 2H), 2.13 (br s, 1H), 2.01 (br d, J=16.1 Hz, 1H).
Example 2
Preparation of Crystalline Form A of Compound Represented by Formula (I)
[0132] The compound represented by 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. The dropwise addition was completed after about 2 h, and then the heating was stopped. The solution was cooled slowly to 20° C., continued to be stirred for 58 h and then filtered. The obtained filter cake was soaked in EtOH (500 mL) for 10 min, filtered and sucked. The procedure was repeated three times. The filter cake was collected and dried to constant weight under vacuum to obtain crystalline form A of the compound represented by formula (I).
Example 3
Preparation of Crystalline Form B of Compound Represented by Formula (I)
[0133] The crystalline form A of the compound represented by formula (I) (20 mg) was added to a reaction flask, and acetone (0.3 mL) was added to form a suspension. The suspension was magnetically stirred at room temperature for 6 days, centrifuged and removed of the supernatant. Solid was dried under vacuum at 50° C. for two hours to obtain crystalline form B of the compound represented by formula (I).
[0134] The crystalline form A of the compound represented by formula (I) (20 mg) was added to a reaction flask, and ethyl acetate (0.3 mL) was added to form a suspension. The suspension was magnetically stirred at room temperature for 6 days, centrifuged and removed of the supernatant. Solid was dried under vacuum at 50° C. for two hours to obtain crystalline form B of the compound represented by formula (I).
Example 4
Preparation of Crystalline Form B of Compound Represented by Formula (I)
[0135] EtOH (7344 mL), DMSO (1836 mL) and the compound represented by formula (I) (2295.00 g, 4.63 mol) were added to a 50 L high-low temperature reaction kettle in sequence. After the addition, the internal temperature was controlled at 80-85° C. to achieve complete dissolution. Then the system was filtered while hot to remove insoluble impurities. The filtrate was poured into the kettle again, and the temperature in the kettle was controlled at 82° C. After the temperature was stable, a mixed solution of EtOH: water=2:1 was slowly added dropwise to the system, and the situation in the kettle was observed during the dropwise addition. When the phenomenon of slight turbidity and then rapid dissolving during the dropwise addition appeared (the temperature at this time was 78° C., and 1200 mL of solvent was consumed), 6 g of crystalline form B of the compound represented by formula (I) was added to the system in batches as a seed crystal. After the seed crystal was added, the system became turbid, and then heating was stopped. The system was naturally cooled to 25-35° C. and stirred for 16.5 h. The temperature was controlled at 25-35° C., and 17160 mL of a mixed solution of EtOH: water=2:1 was slowly added dropwise into the system. After the dropwise addition, the system was continued to be stirred at 25-35° C. and stopped after a total of 24 h of stirring, then filtered and sucked. The filter cake was washed with a solvent of EtOH: water=2:1 (5L*2), and sucked. The obtained solid was added to the reaction kettle, added with EtOH (5.5 L). The mixture was stirred at 25-35° C. for 1.5 h, which was then stopped, and then filtered and sucked. The filter cake was dried under vacuum at 50° C. to constant weight to obtain crystalline form B of the compound represented by formula (I).
Example 5
Preparation of Crystalline Form C of Compound Represented by Formula (I)
[0136] The crystalline form A of the compound represented by formula (I) (20 mg) was added to a reaction flask, and THF (0.3 mL) was added to form a suspension. The suspension was magnetically stirred at room temperature for 6 days, and n-hexane was added dropwise until insoluble matter was precipitated. The system was centrifuged and removed of the supernatant. Solid was dried under vacuum at 50° C. for two hours to obtain crystalline form C of the compound represented by formula (I).
Example 6
Preparation of Crystalline Form D of Compound Represented by Formula (II)
[0137] The crystalline form A of the compound represented by formula (I) (20 mg) was added to a reaction flask, and acetone (0.3 mL) containing MeS0˜3H (3 μL) was added. The system was magnetically stirred at room temperature for 5 days, centrifuged and removed of the supernatant. Solid was dried under vacuum at 50° C. for two hours to obtain crystalline form D of the compound represented by formula (II).
Example 7
Preparation of Crystalline Form E of Compound Represented by Formula (III)
[0138] The crystalline form A of the compound represented by formula (I) (20 mg) and D-glucuronic acid (8.5 mg) were added to a reaction flask, and acetone (0.3 mL) was added. The system was magnetically stirred at room temperature for 5 days, centrifuged and removed of the supernatant. Solid was dried under vacuum at 50° C. for two hours to obtain crystalline form E of the compound represented by formula (III).
Example 8
Preparation of Crystalline Form F of compound represented by formula (IV)
[0139] The crystalline form A of the compound represented by formula (I) (20 mg) and maleic acid (5.1 mg) were added to a reaction flask, and ethyl acetate (0.3 mL) was added. The system was magnetically stirred at room temperature for 5 days, and n-heptane was added until solid was precipitated. After centrifugation, the filter cake was collected and dried under vacuum at 50° C. for two hours to obtain crystalline form F of compound represented by formula (IV).
Example 9
Study on Hygroscopicity of Crystalline Form B of Compound Represented by Formula (I)
Experimental Materials:
[0140] SMS DVS intrinsic Dynamic Vapor Sorption Instrument
Experimental Method:
[0141] 10.03 mg of crystalline form B of the compound represented by formula (I) was weighed out and placed in a DVS sample tray for testing.
Experimental Results:
[0142] The DVS graph of the crystalline form B of the compound represented by formula (I) is shown in
Experimental Conclusions:
[0143] The hygroscopic weight gain of the crystalline form B of the compound represented by formula (I) at 25° C. and 80% RH was 0.09%, indicating it was non-hygroscopic.
Example 10
Solid Stability Test of Crystalline Form B of Compound Represented by Formula (I)
[0144] According to the “Guidelines for Stability Test of Raw Materials and Preparations” (General Principles 9001 of Part Four of Chinese Pharmacopoeia 2015 Edition), crystalline form E of the compound represented by formula (I) was investigated for stability at high temperature (60° C., open), high humidity (room temperature/relative humidity 92.5%, open) and strong light (5000 lx, closed).
[0145] About 10 mg of compound represented by formula (I) was accurately weighed out, placed in a dry and clean glass bottle, spread out into a thin layer and covered with aluminum foil with small holes under the influence factor test conditions (60° C., 92.5% RH) and accelerated conditions (40° C./75% RH and 60° C./75% RH). The samples placed under the condition of light (visible light 1200000 Lux, ultraviolet 200 W) were in transparent glass bottles, with one group fully exposed and the other fully packaged with aluminum foil. Samples in duplicate were placed under each condition at each time point, and the samples for XRPD detection were placed separately. Samples placed under different conditions were sampled for XRPD detection at the planned test end point, and the test results were compared with the initial test results at Day 0. The test results are shown in Table 7 below:
TABLE-US-00008 TABLE 7 Solid stability test results of crystalline form B of compound represented by formula (I) Test condition Time point Crystalline form — 0 days Crystalline form B 60° C., open 5 days Crystalline form B 10 days Crystalline form B 25° C./92.5% RH, open 5 days Crystalline form B 10 days Crystalline form B Light (total illuminance = 5 days Crystalline form B 1.2 × 10.sup.6 Lux2 × 10, 10 days Crystalline form B Near UV = 200 w .Math. hr/m.sup.2, open) 40° C./75% RH, open 1 month Crystalline form B 2 months Crystalline form B 3 months Crystalline form B 60° C./75% RH, open 1 month Crystalline form B 2 months Crystalline form B Conclusion: The crystalline form B of compound represented by formula (I) had good stability under conditions of high temperature, high humidity and strong light and accelerated conditions. Experimental example 1: IDH1 enzyme activity test in vitro
[0146] IDH1 mutant catalyzes the NADPH-dependent reduction of α-KG (α-ketoglutaric acid) to 2-HG (2-hydroxyglutaric acid), and the consumed NADPH can be read out by fluorescence.
Reagents:
[0147] Basic reaction buffer: 50 mM KH.sub.2PO.sub.4, pH 7.5, 10 mM MgCl.sub.12, 10% glycerol, 150 mM NaCl, 0.05% BSA (bovine serum albumin), 2 mM b-ME (2-mercaptoethanol), 0.003% Brij35 (oxyethylene lauryl ether)
Substrates and cCofactors:
[0148] IDH1 wt (wild type): 65 μM isocitrate +50 μM NADP
[0149] IDH1 (R132H): 1500 μM α-KG +15 μM NADPH
[0150] IDH1 (R132C): 500 μM α-KG +15 μM NADPH
Reaction Process:
[0151] 1.33× enzyme (no control wells), buffer and NADP or NADPH (control wells) were added to the wells of a reaction plate. The compounds to be tested were dissolved in 100% DMSO, then added to the enzyme mixture (Echo550, nanoliter level) and incubated for 60 min after brief centrifugation. A mixture of 4× substrate and cofactor was added to start the reaction, briefly centrifuged, shaken, and incubated at room temperature for 45 min. A mixture of 3× lipoamide dehydrogenase and resazurin was prepared, added to the reaction solution to test the amount of the generated or remaining NADPH, incubated at room temperature for 10 min after simple centrifugation, and measured using a multifunctional microplate reader Envision (Ex/Em=535/590 nm).
[0152] Experimental results: as shown in Table 8 and Table 9:
TABLE-US-00009 TABLE 8 IC.sub.50 results of in vitro enzymatic activity test on IDH1 (IDH1 R132H) Compound No. Structure IDH1 R132H (nM) Compound represented by formula (I)
TABLE-US-00010 TABLE 9 IC.sub.50 results of in vitro enzymatic activity test on IDH1 (IDH1 R132C, WT) IDH1 R132C IDH1 Compound No. Structure (nM) WT (nM) Compound represented by formula (I)
Experimental Example 2
IDH1 Cytological Activity Test
[0153] In this study, after co-incubating the compound with IDH1 mutant cell lines, the 2HG content in the cell culture supernatant was detected by LC-MS to determine the inhibitory activity of the compound on IDH1 mutants. IDH1 catalyzes the reduction of isocitrate to α-ketoglutaric acid (α-KG) in vivo, while IDH1 mutants further catalyze the reduction of α-KG to 2-hydroxyglutaric acid (2HG).
[0154] U87MG-IDH1-R132H cell line is a stable transfected cell line that can stably express IDH1-R132H mutant by transfecting U87MG cells with IDH1-R132H, and HT1080 cell line contains endogenous IDH1-R132C mutant.
[0155] The experimental process is as follows:
[0156] 1) The compound was 3-fold serially diluted with DMSO and added to a cell culture plate, with a total of 10 concentrations, each concentration in duplicate. Negative control wells contained DMSO only, and positive control wells contained BAY1436032 at a final concentration of 5 μM. The final concentration of DMSO in all wells was 0.5%.
[0157] 2) The IDH1 mutant cell line was seeded into the compound-containing cell culture plate at a density of 40,000 cells/well. Cells were co-incubated with compounds for 3 days in a 37° C., 5% CO.sub.2 incubator.
[0158] 3) After 3 days, 10 μl of cell culture supernatant was taken, diluted 21 times with 200 μl of ddH.sub.2O to 210 μl and mixed well. 50 μl of the diluted solution was taken and added with 200 μl of a precipitant (acetonitrile containing 0.4 μg/ml D-2-hydroxyglutaric acid .sup.13C5). After centrifugation at 4000 rpm for 10 min, 100 μl of the supernatant was taken to detect the content of 2-HG by LC-MS.
[0159] 4) At the same time, the effects of the compound on the cell viability of IDH1 mutant cell lines was detected in parallel using the ATPlite 1Step kit according to the instructions.
[0160] 5) The percentage inhibition (% inhibition rate) of IDH1 mutants by each concentration of the compound was calculated using the 2HG content data according to a formula of:
% Inhibition rate=(CPD−ZPE)/(HPE−ZPE)×100%;
and the percentage cytotoxicity (% cytotoxicity) of the compound against the IDH1 mutant cell line was calculated using the cell viability data according to a formula of:
% Cytotoxicity=(1−CPD/ZPE)×100%.
CPD: Signal value of compound wells
ZPE: Average signal of negative control wells, with 0.5% DMSO instead of compound
HPE: Average signal of positive control wells
[0161] 6) The % inhibition rate and % cytotoxicity were fitted into a dose-response curve by the GraphPad Prism software to obtain the IC.sub.50 value of the test compound.
[0162] The experimental results are shown in Table 10:
TABLE-US-00011 TABLE 10 IC.sub.50 results of in vitro cell activity test of IDH1 (U87MG) U87MG Compound No. Structure IDH1-R132H (nM) Compound represented by formula (I)
Experimental Example 3
Pharmacokinetic Evaluation in Mice
Experimental Purpose:
[0163] The pharmacokinetic parameters of the compound represented by formula (I) were detected in mice.
Experimental Program:
[0164] 1) Experimental drug: compound represented by formula (I);
[0165] 2) Experimental animals: 8 male CD-1 mice aged 7-10 weeks, divided into 2 groups, 4 mice in each group;
[0166] 3) Drug preparation: for the tail vein injection group, an appropriate amount of the drug was weighed out and dissolved in a mixed solvent of DMSO: 20% hydroxypropyl beta-cyclodextrin (HPbCD)=10:90 to prepare a solution of 0.5 mg/mL; for the gavage administration group, an appropriate amount of the drug was weighed out and dissolved in a mixed solvent of DMSO: polyoxyethylene castor oil EL (Cremophor EL): 5% sulfobutylcyclodextrin (Captisol)=5:10:85 to prepare into a suspension.
Experimental Procedure:
[0167] Animals in group 1 were given a single dose of 1 mg/kg drug at a concentration of 0.5 mg/mL via tail vein injection, and animals in group 2 were given the compound at a dose of 20 mg/kg at a concentration of 2 mg/mL by gavage. Animals were cross-collected for plasma samples at 0.0833 (the tail vein injection group only), 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h after administration. The drug concentration in the plasma samples was determined by LC-MS/MS method, and the kinetic parameters of the tested drugs were obtained in Table 11:
TABLE-US-00012 TABLE 11 Pharmacokinetic evaluation parameters in mice Tail vein injection group Area under Initial volume of the curve Clearance rate concentration distribution Half-life AUC.sub.0-last Cl (mL/min/kg) C.sub.0 (nM) Vd (L/Kg) T.sub.1/2 (h) (nM .Math. h) 2.17 3541 0.690 2.90 15463 Gavage group administration Area under Highest Time at highest the curve concentration concentration AUC.sub.0-last Bioavailability C.sub.max (nM) T.sub.1/2 (h) (nM .Math. h) F (%) 33350 1.00 306609 99.1 Conclusion: The compound represented by formula (I) had good pharmacokinetic properties in mice.