CRYSTAL OF PDE3/PDE4 DUAL INHIBITOR AND USE THEREOF
20230115817 · 2023-04-13
Inventors
Cpc classification
C07C309/30
CHEMISTRY; METALLURGY
International classification
Abstract
Provided are a crystal of a tricyclic compound as shown in formula (I) or a pharmaceutically acceptable salt thereof and a preparation method therefor, and the use thereof in preparing a drug for treating PDE3- and/or PDE4-related diseases.
##STR00001##
Claims
1. A crystalline form of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of a compound of formula (I), wherein the pharmaceutically acceptable salt is sulfate, p-toluenesulfonate, methanesulfonate or maleate: ##STR00036##
2. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 1, wherein the crystalline form is a crystalline form of the compound of formula (I) comprising 4, 5, 6, 7 or 8 diffraction peaks in an X-ray powder diffraction (XRPD) pattern using Cu Kα radiation selected from the following 2θ angles: 4.14±0.2°, 6.56±0.2°, 6.98±0.2°, 8.20±0.2°, 11.50±0.2°, 12.66±0.2°, 13.94±0.2° and 16.35±0.2°; or having diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation at the following 2θ angles: 4.14±0.2°, 6.98±0.2°, 8.20±0.2° and 11.50±0.2°; or having diffraction peaks at the following 2θ angles: 4.14±0.2°, 6.56±0.2°, 6.98±0.2°, 8.20±0.2°, 11.50±0.2°, 12.66±0.2°, 13.94±0.2° and 16.35±0.2°; or having diffraction peaks at the following 2θ angles: 4.14±0.2°, 6.56±0.2°, 6.98±0.2°, 8.20±0.2°, 9.35±0.2°, 11.50±0.2°, 12.66±0.2°, 13.94±0.2°, 14.52±0.2°, 16.35±0.2°, 21.52±0.2° and 24.57±0.2°; or the crystalline form is a crystalline form of the compound of formula (I) having an XRPD pattern using Cu Kα radiation as shown in
3. (canceled)
4. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 2, wherein the crystalline form is a crystalline form of the compound of formula (I) having endothermic peaks in a differential scanning calorimetry curve at 146.23±2° C. and/or 162.19±2° C.; or having exothermic peaks in a differential scanning calorimetry curve at 172.65±2° C. and/or 241.73±2° C.
5. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 1, wherein the crystalline form is a crystalline form of the compound of formula (I) comprising 5, 6, 7, 8, 9, 10 or 11 diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation selected from the following 2θ angles: 5.81±0.2°, 8.38±0.2°, 11.16±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 16.76±0.2°, 17.95±0.2°, 20.83±0.2°, 24.73±0.2° and 26.13±0.2°; or having diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation at the following 2θ angles: 5.81±0.2°, 13.96±0.2°, 15.01±0.2°, 17.95±0.2° and 24.73±0.2°; or having diffraction peaks at the following 2θ angles: 5.81±0.2°, 8.38±0.2°, 11.16±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 16.76±0.2°, 17.95±0.2°, 20.83±0.2°, 24.73±0.2° and 26.13±0.2°; or having diffraction peaks at the following 2θ angles: 5.81±0.2°, 8.38±0.2°, 9.13±0.2°, 11.16±0.2°, 11.60±0.2°, 12.82±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 16.76±0.2°, 17.95±0.2°, 18.91±0.2°, 20.83±0.2°, 24.36±0.2°, 24.73±0.2°, 25.78±0.2° and 26.13±0.2°; or the crystalline form is a crystalline form of the compound of formula (I) having an XRPD pattern using Cu Kα radiation as shown in
6. (canceled)
7. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 5, wherein the crystalline form is a crystalline form of the compound of formula (I) having an exothermic peak in a differential scanning calorimetry curve at 247.70±2° C.
8. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 1, wherein the crystalline form is a crystalline form of the compound of formula (I) comprising 4, 5, 6, 7, 8 or 9 diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation selected from the following 2θ angles: 4.57±0.2°, 6.41±0.2°, 7.18±0.2°, 11.58±0.2°, 12.84±0.2°, 13.21±0.2°, 14.34±0.2°, 16.05±0.2° and 23.41±0.2°; or having diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation at the following 2θ angles: 4.57±0.2°, 6.41±0.2°, 7.18±0.2° and 14.34±0.2°; or having diffraction peaks at the following 2θ angles: 4.57±0.2°, 6.41±0.2°, 7.18±0.2°, 11.58±0.2°, 12.84±0.2°, 13.21±0.2°, 14.34±0.2°, 16.05±0.2° and 23.41±0.2°; or having diffraction peaks at the following 2θ angles: 4.57±0.2°, 6.41±0.2°, 7.18±0.2°, 9.07±0.2°, 11.58±0.2°, 12.84±0.2°, 13.21±0.2°, 14.34±0.2°, 16.05±0.2°, 18.15±0.2°, 19.26±0.2°, 20.85±0.2° and 23.41±0.2°; or the crystalline form is a crystalline form of the compound of formula (I) having an XRPD pattern using Cu Kα radiation as shown in
9. (canceled)
10. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 8, wherein the crystalline form is a crystalline form of the compound of formula (I) having exothermic peaks in a differential scanning calorimetry curve at 152.26±2° C. and/or 247.92±2° C.
11. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 1, wherein the crystalline form is a crystalline form of the compound of formula (II) ##STR00037## comprising 4, 5, 6, 7 or 8 diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation selected from the following 2θ angles: 4.84±0.2°, 9.58±0.2°, 10.93±0.2°, 11.97±0.2°, 14.31±0.2°, 14.75±0.2°, 16.49±0.2° and 24.42±0.2°; or having diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation at the following 2θ angles: 4.84±0.2°, 9.58±0.2°, 11.97±0.2° and 14.75±0.2°; or having diffraction peaks at the following 2θ angles: 4.84±0.2°, 9.58±0.2°, 10.93±0.2°, 11.97±0.2°, 14.31±0.2°, 14.75±0.2°, 16.49±0.2° and 24.42±0.2°; or having diffraction peaks at the following 2θ angles: 4.84±0.2°, 9.58±0.2°, 10.93±0.2°, 11.97±0.2°, 12.72±0.2°, 13.93±0.2°, 14.31±0.2°, 14.75±0.2°, 16.49±0.2°, 17.91±0.2°, 19.25±0.2°, 19.90±0.2°, 20.57±0.2°, 24.42±0.2° and 25.70±0.2°; or the crystalline form is a crystalline form of the compound of formula (II) having an XRPD pattern using Cu Kα radiation as shown in
12. (canceled)
13. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 1, wherein the crystalline form is a crystalline form of the compound of formula (III) ##STR00038## comprising 3, 4, 5 or 6 diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation selected from the following 20 angles: 6.53±0.2°, 10.87±0.2°, 12.48±0.2°, 13.11±0.2°, 16.58±0.2° and 25.03±0.2°; or having diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation at the following 2θ angles: 6.53±0.2°, 12.48±0.2° and 13.11±0.2°; or having diffraction peaks at the following 20 angles: 6.53±0.2°, 10.87±0.2°, 12.48±0.2°, 13.11±0.2°, 16.58±0.2° and 25.03±0.2°; or having diffraction peaks at the following 2θ angles: 6.53±0.2°, 10.87±0.2°, 12.48±0.2°, 13.11±0.2°, 14.04±0.2°, 16.58±0.2°, 25.03±0.2°, 25.56±0.2° and 26.66±0.2°; or the crystalline form is a crystalline form of the compound of formula (III) having an XRPD pattern using Cu Kα radiation as shown in
14. (canceled)
15. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 1, wherein the crystalline form is a crystalline form of the compound of formula (IV) ##STR00039## comprising 4, 5, 6, 7 or 8 diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation selected from the following 2θ angles: 11.22±0.2°, 12.58±0.2°, 16.43±0.2°, 17.90±0.2°, 18.85±0.2°, 22.62±0.2°, 24.45±0.2° and 25.87±0.2°; or having diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation at the following 2θ angles: 11.22±0.2°, 18.85±0.2°, 22.62±0.2° and 24.45±0.2°; or having diffraction peaks at the following 2θ angles: 11.22±0.2°, 12.58±0.2°, 16.43±0.2°, 17.90±0.2°, 18.85±0.2°, 22.62±0.2°, 24.45±0.2° and 25.87±0.2°; or having diffraction peaks at the following 2θ angles: 11.22±0.2°, 12.58±0.2°, 16.43±0.2°, 17.08±0.2°, 17.90±0.2°, 18.85±0.2°, 19.23±0.2°, 19.72±0.2°, 22.62±0.2°, 23.27±0.2°, 24.45±0.2° and 25.87±0.2°; or having diffraction peaks at the following 2θ angles: 11.22±0.2°, 12.58±0.2°, 13.88±0.2°, 15.49±0.2°, 16.04±0.2°, 16.43±0.2°, 17.08±0.2°, 17.90±0.2°, 18.54±0.2°, 18.85±0.2°, 19.23±0.2°, 19.72±0.2°, 20.02±0.2°, 20.51±0.2°, 22.62±0.2°, 23.27±0.2°, 24.45±0.2°, 24.83±0.2°, 25.42±0.2°, 25.87±0.2°, 26.09±0.2° and 29.53±0.2°; or the crystalline form is a crystalline form of the compound of formula (IV) having an XRPD pattern using Cu Kα radiation as shown in
16. (canceled)
17. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 15, wherein the crystalline form is a crystalline form of the compound of formula (IV) having an at endothermic peak at 191.35±2° C. and/or an exothermic peak at 222.21±2° C. in a differential scanning calorimetry curve.
18. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 1, wherein the crystalline form is a crystalline form of the compound of formula (V) ##STR00040## comprising 4, 5, 6, 7 or 8 diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation selected from the following 2θ angles: 5.83±0.2°, 6.62±0.2°, 9.50±0.2°, 10.98±0.2°, 17.16±0.2°, 19.05±0.2°, 24.71±0.2° and 25.16±0.2°; or having diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation at the following 2θ angles: 5.83±0.2°, 6.62±0.2°, 9.50±0.2° and 10.98±0.2°; or having diffraction peaks at the following 2θ angles: 5.83±0.2°, 6.62±0.2°, 9.50±0.2°, 10.98±0.2°, 17.16±0.2°, 19.05±0.2°, 24.71±0.2° and 25.16±0.2°; or having diffraction peaks at the following 2θ angles: 5.83±0.2°, 6.62±0.2°, 9.50±0.2°, 10.98±0.2°, 11.59±0.2°, 13.23±0.2°, 16.27±0.2°, 17.16±0.2°, 19.05±0.2°, 21.63±0.2°, 24.71±0.2° and 25.16±0.2°; or the crystalline form is a crystalline form of the compound of formula (V) having an XRPD pattern using Cu Kα radiation as shown in
19. (canceled)
20. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 1, wherein the pharmaceutically acceptable salt is sulfate, p-toluenesulfonate, methanesulfonate or maleate; the sulfate of the compound of formula (I) is selected from a compound of formula (II), ##STR00041## the p-toluenesulfonate of the compound of formula (I) is selected from a compound of formula (III), ##STR00042## the methanesulfonate of the compound of formula (I) is selected from a compound of formula (IV), ##STR00043## or the maleate of the compound of formula (I) is selected from a compound of formula (V), ##STR00044##
21. The crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 1, wherein the crystalline form is in the form of a crystalline composition, comprising the crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, wherein the crystalline form accounts for 50% or more of the weight of the crystalline composition.
22. A pharmaceutical composition, comprising a therapeutically effective amount of the crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 1.
23. A method for preparing a compound of formula (I), comprising: preparing the compound of formula (I) by method 1 or method 2; wherein the method 1 comprises: (1) reacting compound 1-2a to give compound BB-4; and (2) reacting compound BB-4 with 5-hydroxy-3-methyl-1,2,3-triazole-4-carboxylic acid to give the compound of formula (I), ##STR00045## the method 2 comprises: reacting compound 1-4b to give the compound of formula (I) ##STR00046##
24. The method according to claim 23, wherein the compound of formula (I) is prepared by the method 1, and wherein compound 1-2a is prepared by reacting compound BB-1 with compound 1-1a, ##STR00047## or the compound of formula (I) is prepared by the method 2, and wherein compound 1-4b is prepared by reacting compound BB-4 with compound 1-3b, ##STR00048##
25. (canceled)
26. The method according to claim 23, wherein compound BB-4 is prepared by reacting compound 1-1b, ##STR00049##
27. The method according to claim 26, wherein compound 1-1b is prepared by reacting compound BB-1 with compound a ##STR00050## wherein X is selected from the group consisting of halogens; X is selected from the group consisting of Cl and Br; or X is selected from Br.
28-30. (canceled)
31. A method for preventing or treating a condition associated with PDE3 and/or PDE4 in a mammal, comprising administering to a mammal in need thereof a therapeutically effective amount of the crystalline form of the compound of formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutically acceptable salt of a compound of formula (I) according to claim 1, or the crystalline composition or the pharmaceutical composition thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0257] In order to better understand the content of the present application, further description is given with reference to specific examples, but the specific embodiments are not intended to limit the content of the present application.
Synthesis of Intermediate BB-1
[0258] ##STR00027##
Step 1: Synthesis of Compound BB-1-2
[0259] A mixture of compound BB-1-1 (21.10 g) and ethyl cyanoacetate (11.00 g, 10.38 mL) was stirred at 100° C. for 16 h in nitrogen atmosphere. After completion of the reaction, the mixture was cooled to 70° C., ethanol (30 mL) was slowly and dropwise added, and a large amount of solid was precipitated. The resulting mixture was filtered, and the filter cake was dried under reduced pressure to give product BB-1-2.
[0260] .sup.1H NMR (400 MHz, DMSO-d6) δ=8.26 (t, J=5.2 Hz, 1H), 6.86 (d, J=8.0 Hz, 1H), 6.79 (br s, 1H), 6.71 (d, 8.0 Hz, 1H), 4.00 (q, J=6.8 Hz, 2H), 3.72 (s, 3H), 3.59 (s, 2H), 3.31-3.23 (m, 2H), 2.64 (t, J=7.2 Hz, 2H), 1.32 (t, J=6.8 Hz, 3H). MS-ESI m/z: 263.1[M+H].sup.+.
Step 2: Synthesis of Compound BB-1-3
[0261] In nitrogen atmosphere, phosphorus oxychloride (379.50 g, 230.00 mL) was heated to 85° C., and compound BB-1-2 (26.00 g) was added in portions. The reaction mixture was stirred at 85° C. for 2 h for reaction. After the reaction was completed, most of the phosphorus oxychloride was removed by reduced pressure distillation. To the residue was added dichloromethane (200 mL) and the mixture was washed with water (100 mL×2). The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure. The resulting crude product was purified by slurrying with ethyl acetate (20 mL) to give compound BB-1-3.
[0262] .sup.1H NMR (400 MHz, CD.sub.3OD) δ=7.16 (s, 1H), 6.83 (s, 1H), 4.62 (s, 1H), 4.12 (q, J=6.8 Hz, 2H), 3.86 (s, 3H), 3.35 (d, J=6.4 Hz, 2H), 2.84 (t, J=6.4 Hz, 2H), 1.44 (t, J=6.8 Hz, 3H). MS-ESI m/z: 245.1[M+H].sup.+.
Step 3: Synthesis of Compound BB-1-4
[0263] Compound BB-1-3 (1.00 g) was added to 98% concentrated sulfuric acid (12.88 g, 128.69 mmol, 7.00 mL) in portions at 0° C. The reaction mixture was stirred at 27° C. for 3 h. After the reaction was completed, the mixture was added to cold water (15 mL), and then aqueous sodium hydroxide solution (4 mol/L, 32 mL) was added dropwise to adjust to a neutral pH, followed by extraction with ethyl acetate (100 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to give compound BB-1-4.
[0264] MS-ESI m/z: 263.1[M+H].sup.+.
Step 4: Synthesis of Compound BB-1-5
[0265] Sodium (2.42 g) was added in portions to ethanol (80 mL) at 0° C. After the mixture was stirred at 28° C. for 0.5 h, compound BB-1-4 (6.90 g) was added to the solution in portions, and the mixture was stirred at 80° C. for 0.5 h. Then, diethyl carbonate (9.32 g, 9.51 mL) was added in one portion, and the mixture was stirred for 5 h at 80° C. After the reaction was completed, the mixture was cooled to room temperature, ice water (30 mL) was slowly added, and then diluted hydrochloric acid (2 mol/L, 53 mL) was added to adjust the mixture to a neutral pH. A large amount of solid was precipitated. The mixture was filtered, and the resulting filter cake was purified by slurrying with ethanol (10 mL) to give compound BB-1-5.
[0266] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=11.22 (br s, 1H), 7.35 (s, 1H), 6.95 (s, 1H), 6.22 (s, 1H), 4.09 (q, J=6.8 Hz, 2H), 3.90 (br s, 2H), 3.83 (s, 3H), 2.89 (br s, 2H), 1.35 (t, J=6.8 Hz, 3H). MS-ESI m/z: 289.1[M+H].sup.+.
Step 5: Synthesis of Compound BB-1-6
[0267] Compound BB-1-5 (5.00 g) was dissolved in phosphorus oxychloride (30 mL) at room temperature. The reaction mixture was stirred at 100° C. for 16 h in nitrogen atmosphere. After the reaction was completed, most of the solvent was removed by reduced pressure distillation. Water (100 mL) was added and the resulting mixture was extracted with dichloromethane (150 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to give compound BB-1-6. MS-ESI m/z: 306.9[M+H].sup.+.
Step 6: Synthesis of Compound BB-1
[0268] Compound BB-1-6 (925.67 mg) was dissolved in isopropanol (8 mL) at room temperature, and 2,4,6-trimethylaniline (2.10 g) was added. The reaction mixture was stirred at 90° C. for 15 h in nitrogen atmosphere. After the reaction was completed, the mixture was cooled to room temperature and concentrated under reduced pressure, and the resulting residue was purified by slurrying with ethanol (6 mL) to give compound BB-1.
[0269] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=8.85 (br s, 1H), 7.27 (s, 1H), 6.97 (s, 1H), 6.90 (s, 2H), 6.45 (s, 1H), 4.10 (q, J=6.8 Hz, 2H), 3.90 (t, J=6.0 Hz, 2H), 3.86 (s, 3H), 2.87 (t, J=6.0 Hz, 2H), 2.45 (s, 3H), 2.11 (s, 6H), 1.37 (t, J=6.8 Hz, 3H). MS-ESI m/z: 406.2[M+H].sup.+.
Synthesis of compound BB-4
[0270] ##STR00028##
Step 1: Synthesis of Compound BB-4-1
[0271] Compound BB-1 (1.00 g) was dissolved in 2-butanone (35 mL) at room temperature, and 2-(2-bromoethyl)isoindoline-1,3-dione (3.76 g), potassium carbonate (3.07 g) and sodium iodide (2.22 g) were added successively. The reaction mixture was stirred at 85° C. for 72 h in nitrogen atmosphere. After the reaction was completed, the mixture was concentrated to remove most of the organic solvent before water (30 mL) and ethyl acetate (25 mL×3) were added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the desiccant and concentrated under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (eluent: petroleum ether:ethyl acetate=15:1-3:1) to give compound BB-4-1.
[0272] MS-ESI m/z: 579.3[M+H].sup.+.
Step 2: Synthesis of Compound BB-4
[0273] Compound BB-4-1 (500.00 mg) was dissolved in trichloromethane (3 mL) and ethanol (3 mL) at room temperature, and hydrazine hydrate (152.67 mg, 85% purity) was added. The mixture was stirred at 28° C. for 16 h in nitrogen atmosphere. After the reaction was completed, the mixture was concentrated to remove most of the organic solvent before water (15 mL) and dichloromethane (15 mL×3) were added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to give compound BB-4.
[0274] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=6.95 (s, 1H), 6.85 (br s, 2H), 6.66 (s, 1H), 5.31 (s, 1H), 4.14 (t, J=6.8 Hz, 2H), 4.05 (q, J=6.8 Hz, 2H), 3.91 (t, J=6.4 Hz, 2H), 3.62 (s, 3H), 2.90-2.86 (m, 4H), 2.22 (s, 3H), 1.95 (br s, 6H), 1.33 (t, J=6.8 Hz, 3H). MS-ESI m/z: 449.2[M+H].sup.+.
Example 1: Preparation of Compound of Formula (I)
[0275] Method 1
##STR00029##
[0276] 5-Hydroxy-3-methyl-1,2,3-triazole-4-carboxylic acid (18.50 mg) was dissolved in DCM (1 mL) at 20° C. HATU (8.80 mg) and triethylamine (57.40 μL) were added and the mixture was stirred for 2 h. Compound BB-4 (50 mg) was added and the system was stirred at the temperature for 16 h. The mixture was diluted to 10 mL with DCM, washed with water (30 mL×3), dried over anhydrous sodium sulfate, and filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure to remove the solvent to give a crude product. The crude product was separated and purified by prep-HPLC to give the target compound of formula (I) in the form of a yellow solid.
[0277] .sup.1H NMR (400 MHz, CD.sub.3OD) δ=6.94 (s, 2H), 6.87 (s, 1H), 6.77 (s, 1H), 5.52 (s, 1H), 4.48 (t, J=6.0 Hz, 2H), 4.15 (s, 3H), 4.12-4.08 (m, 2H), 4.01 (t, J=6.0 Hz, 2H), 3.87 (t, J=6.0 Hz, 2H), 3.69 (s, 3H), 2.94 (t, J=6.0 Hz, 2H), 2.29 (s, 3H), 2.06 (s, 6H), 1.41 (t, J=6.8 Hz, 3H). MS m/z [M+H].sup.+ 574.1.
Method 2
[0278] ##STR00030##
Step One: Preparation of Compound 1-1a
[0279] N-Boc-ethanolamine (50 g, 48.08 mL) and p-toluenesulfonyl chloride (70.96 g) were dissolved in methyl tert-butyl ether (500 mL), potassium hydroxide (52.21 g) was added, and the mixture was heated to 80° C. and refluxed for 4 h. The mixture was cooled to room temperature and poured into a mixture of ice and water (1000 mL) to separate the organic phases. The aqueous phase was extracted with methyl tert-butyl ether (100 mL×2). The organic phases were combined, washed with cold water (1000 mL), dried over anhydrous sodium sulfate, and filtered to remove the desiccant. The filtrate was concentrated under reduced pressure to remove the solvent to give crude product 1-1a (28.5 g) in the form of a yellow oily liquid, which was directly used in the next step without purification.
[0280] .sup.1H NMR (400 MHz, CHLOROFORM-d) δ (ppm) 2.15 (s, 4H), 1.47 (s, 9H).
Step Two: Preparation of Compound 1-2a
[0281] Compound BB-1 (2 g) and compound 1-1a (4.24 g) were dissolved in acetonitrile (17 mL), and lithium carbonate (2.19 g) and water (3 mL) were added. The system was heated to 100° C. and stirred for 70 h. The mixture was cooled, let stand, and filtered to remove the precipitate. The filtrate was concentrated under reduced pressure to remove the solvent and mixed with 50 mL of water. The mixture was extracted with ethyl acetate (20 mL×3). The organic phases were combined, washed with 0.5 M sodium hydroxide solution (100 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to remove the solvent to give a crude product. The crude product was separated and purified by column chromatography to give the target compound 1-2a (1.08 g) in the form of a pale yellow solid.
[0282] MS-ESI (m/z): 549.2 [M+1].sup.+
[0283] .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ (ppm) 6.91 (s, 2H), 6.85 (s, 1H), 6.75 (s, 1H), 5.49 (s, 1H), 4.36 (t, J=5.9 Hz, 2H), 4.09 (q, J=7.0 Hz, 2H), 4.00 (t, J=6.1 Hz, 2H), 3.68 (s, 3H), 3.56-3.48 (m, 2H), 2.91 (t, J=6.3 Hz, 2H), 2.27 (s, 3H), 2.06 (s, 6H), 1.39 (s, 12H).
Step Three: Preparation of Compound BB-4
[0284] Compound 1-2a (1 g) and 4 M hydrogen chloride in methanol (20 mL) were mixed uniformly. The system was heated to 60° C. and stirred for 2 h. The mixture was concentrated under reduced pressure to remove the solvent to give a crude product in the form of a pale yellow oily liquid. 20 mL of petroleum ether and 5 mL of ethyl acetate were added to the crude product. The mixture was stirred for 30 min and then filtered. The solid was collected and dried to give the target compound BB-4 (810 mg, 91.63% yield) in the form of a pale yellow solid.
[0285] .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ (ppm) 7.16 (s, 2H), 6.98 (s, 1H), 6.80 (s, 1H), 5.68 (s, 1H), 4.72 (br s, 2H), 4.22 (br s, 2H), 4.16 (q, J=7.0 Hz, 2H), 3.69 (s, 3H), 3.48 (br s, 2H), 3.08 (br s, 2H), 2.37 (s, 3H), 2.30 (s, 6H), 1.43 (t, J=7.0 Hz, 3H).
Step Four: Preparation of Compound (I)
[0286] 5-Hydroxy-3-methyl-1,2,3-triazole-4-carboxylic acid (18.50 mg) was dissolved in DCM (1 mL) at 20° C. HATU (8.80 mg) and triethylamine (57.40 pt) were added and the mixture was stirred for 2 h. Compound BB-4 (50 mg) was added and the system was stirred at the temperature for 16 h. The mixture was diluted to 10 mL with DCM, then washed with water (30 mL×3), dried over anhydrous sodium sulfate, and filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure to remove the solvent to give a crude product. The crude product was separated and purified by prep-HPLC to give the target compound of formula (I) (22 mg, 37.2% yield) in the form of a yellow solid.
[0287] MS-ESI (m/z):574.5 [M+1].sup.+
[0288] .sup.1H NMR (400 MHz, METHANOL-d.sub.4) δ (ppm) 6.94 (s, 2H), 6.87 (s, 1H), 6.75 (s, 1H), 5.51 (s, 1H), 4.47 (br t, J=6.0 Hz, 2H), 4.14 (s, 3H), 4.10 (q, J=7.0 Hz, 2H), 4.01 (br t, J=5.9 Hz, 2H), 3.85 (br t, J=5.9 Hz, 2H), 3.68 (s, 3H), 2.92 (br t, J=6.0 Hz, 2H), 2.29 (s, 3H), 2.05 (s, 6H), 1.41 (t, J=6.9 Hz, 3H).
Method 3
[0289] ##STR00031##
Step One: Preparation of Compound 1-1b
[0290] 2-Butanone (12 L) was added to a 50-L jacketed kettle at 20° C. Compound BB-1 (615 g) and 2-bromoacetamide (612.4 g) were added with stirring. Anhydrous potassium phosphate (1.57 kg) and sodium iodide (665.4 g) were added to the system. The reaction system was heated to 90° C. and stirred for 18 h in nitrogen atmosphere. The reaction mixture was cooled to room temperature before water (12 L) was added. The mixture was stirred for 1 h, and filtered to give a solid. The resulting solid was dried in vacuum to give the target compound 1-1b (575 g, 81.96% yield) in the form of a yellow solid.
[0291] MS-ESI (m/z):463.23 [M+1].sup.+
[0292] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=7.49 (br s, 1H), 7.06 (br s, 1H), 6.95 (s, 1H), 6.82 (s, 2H), 6.69 (s, 1H), 5.33 (s, 1H), 4.66 (s, 2H), 4.07 (q, J=6.9 Hz, 2H), 3.91 (br t, J=6.0 Hz, 2H), 3.63 (s, 3H), 2.90 (br t, J=5.9 Hz, 2H), 2.20 (s, 3H), 1.92 (s, 6H), 1.33 (t, J=7.0 Hz, 3H).
Step Two: Preparation of Compound BB-4
[0293] Tetrahydrofuran (8.6 L) was added to a 50-L dry jacketed kettle at 20° C., and a borane-dimethyl sulfide solution (10 M, 950 mL) was added dropwise to the kettle with stirring. After addition, the system was cooled to 0° C. before compound 1-1b (440 g) was added. The cooling unit was turned off. The mixture was naturally warmed to 20-25° C., and stirred for another 18 h. The reaction mixture was cooled to 0° C., methanol (˜1.5 L) was added dropwise to quench the reaction until no bubbles were generated, and a 3 M hydrochloric acid solution (˜800 mL) was added dropwise to the reaction mixture to adjust to pH 2-3. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was dissolved in dichloromethane, and a saturated sodium bicarbonate solution was added to adjust the mixture to pH 7-8. The phases were separated, and the aqueous phase was extracted with dichloromethane (3000 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure. The resulting product was separated and purified by flash column chromatography to give the target compound BB-4 (125 g).
[0294] MS-ESI (m/z):449.25 [M+1].sup.+
[0295] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=7.91 (br s, 2H), 6.96 (s, 1H), 6.87 (s, 2H), 6.69 (s, 1H), 5.35 (s, 1H), 4.41 (br t, J=5.8 Hz, 2H), 4.07 (q, J=6.8 Hz, 2H), 3.92 (br t, J=5.8 Hz, 2H), 3.63 (s, 3H), 3.21 (br s, 2H), 2.90 (br t, J=5.8 Hz, 2H), 2.22 (s, 3H), 1.98 (s, 6H), 1.33 (t, J=7.0 Hz, 3H).
Step Three: Preparation of Compound 1-4b
[0296] Compound 1-3b (68.68 g) was dissolved in dichloromethane (700 mL) at 20° C., N,N-carbonyldiimidazole (61.56 g) was added, and the system was stirred at 25° C. for 5 h. The mixture was washed with saturated brine (700 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dissolved in DMF (675 mL), compound BB-4 (95 g) was added in portions, and the system was stirred at 20-25° C. for 18 h. Methanol (675 mL) was added to the reaction mixture, and the mixture was stirred for 1 h and filtered to give the target compound 1-4b (120 g, 81.49% yield).
[0297] MS-ESI (m/z):694.33 [M+1].sup.+
[0298] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=8.36 (t, J=6.1 Hz, 1H), 7.26 (d, J=8.6 Hz, 2H), 6.96-6.87 (m, 3H), 6.82 (s, 2H), 6.66 (s, 1H), 5.14 (s, 2H), 4.31 (br t, J=6.3 Hz, 2H), 4.16 (s, 3H), 4.06 (q, J=6.8 Hz, 2H), 3.86 (br t, J=5.8 Hz, 2H), 3.75-3.66 (m, 5H), 3.62 (s, 3H), 2.86 (br t, J=5.9 Hz, 2H), 2.21 (s, 3H), 1.91 (s, 6H), 1.33 (t, J=6.9 Hz, 3H).
Step Four: Preparation of Compound (I)
[0299] Compound 1-4b (120 g) was dissolved in a mixed solvent of trifluoroacetic acid (1000 mL) and glacial acetic acid (200 mL) at 20° C., and the system was heated to 90-100° C. and stirred for 48 h. The reaction mixture was concentrated under reduced pressure to remove most of the solvent. The residue was dissolved in DCM (1000 mL), and a saturated sodium bicarbonate solution was added to adjust to pH 7-8. The organic phase and the aqueous phase were separated. The aqueous phase was extracted with DCM (1000 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered to remove the desiccant. The filtrate was concentrated under reduced pressure to remove the solvent to give the target compound of formula (I) (80 g, 80.60% yield). MS-ESI (m/z): 574.27 [M+1].sup.+
[0300] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=7.73-7.65 (m, 1H), 6.94 (s, 1H), 6.84 (s, 2H), 6.66 (s, 1H), 5.32 (s, 1H), 4.35 (t, J=6.1 Hz, 2H), 4.12-4.01 (m, 5H), 3.88 (t, J=6.1 Hz, 2H), 3.70 (q, J=6.1 Hz, 2H), 3.62 (s, 3H), 2.87 (br t, J=6.0 Hz, 2H), 2.21 (s, 3H), 1.93 (s, 6H), 1.33 (t, J=6.9 Hz, 3H), 0.90-0.90 (m, 1H).
Example 2: Preparation of Crystalline Form a of Compound of Formula (I)
[0301] 50 mg of the compound of formula (I) was added to a 4-mL glass bottle, 1 mL of anhydrous methanol was added, and the mixture was heated to 40° C. and stirred for 48 h. The mixture was naturally cooled to room temperature, centrifuged to separate the solid, and dried in vacuum to give 34 mg of solid, namely the crystalline form A. The XRPD pattern is shown in
Example 3: Preparation of Crystalline Form B of Compound of Formula (I)
[0302] 50 mg of the compound of formula (I) was added to a 4-mL glass bottle, 1 mL of anhydrous ethanol and 0.2 mL of water were added, and the mixture was heated to 40° C. and stirred for 48 h. The mixture was naturally cooled to room temperature, centrifuged to separate the solid, and dried in vacuum to give 46 mg of solid, namely the crystalline form B. The XRPD pattern is shown in
Example 4: Preparation of Crystalline Form C of Compound of Formula (I)
[0303] 50 mg of the compound of formula (I) was added to a 4-mL glass bottle, 1 mL of acetonitrile was added, and the mixture was heated to 40° C. and stirred for 48 h. The mixture was naturally cooled to room temperature, centrifuged to separate the solid, and dried in vacuum to give 37 mg of solid, i.e., the crystalline form C. The XRPD pattern is shown in
Example 5: Preparation of Salt of Compound of Formula (I)
[0304] 100 mg of the compound of formula (I) was added to a 4 mL glass bottle, and 2 mL of anhydrous tetrahydrofuran was added. The mixture was heated to 70° C. and stirred for 1 h to fully dissolve the compound, and then cooled to 40° C. A solution prepared from the corresponding acid or base (the specific chemicals and amounts are shown in Table 8) and 0.1 mL of water was added to the mixture, and the mixture was stirred at the temperature for 12 h. The mixture was centrifuged to separate a solid precipitate, and the precipitate was dried in vacuum to give a solid, i.e., the corresponding salt of the crystalline form of the compound of formula (I).
TABLE-US-00008 TABLE 8 Preparation of salt forms XRPD/ TGA/DSC characterization Salt form Acid/base Amount .sup.1HNMR data
Example 6: Stability Study of Solid Crystalline Form B of the Compound of Formula (I) High Performance Liquid Chromatography (HPLC)
[0305] The chromatographic conditions of the HPLC method are seen in table below: Chromatographic column: Zorbax SB C-18, 4.6 mm×150 mm, 5 μm (PDS-HPLC-007)
[0306] Mobile phase A: 0.1% TFA in water
[0307] Mobile phase B: 100% ACN
[0308] Preparation of sample: the sample was dissolved in a mixed solvent of acetonitrile and water (acetonitrile:water=50:50 (v/v))
Solid Stability Testing
[0309] The stability of the compound in the following conditions was examined, and samples were taken at different time points to detect the content. About 5 mg of the crystalline form B of the compound of formula (I) was accurately weighted in duplicate, transferred to a dry and clean glass bottle, spread into a thin layer as test samples, and placed in experimental conditions of influential factors ((60° C.), (relative humidity 92.5%), illumination (total illumination of 1.2×106 Lux.Math.hr/near UV energy of 200 w.Math.hr/m2), (40° C., relative humidity 75%), or (60° C., relative humidity 75%)). The samples were covered with aluminum foils having holes, and thus completely exposed to the conditions. Sampling analysis was performed at 5 days, 10 days, 1 month, 2 months and 3 months. The samples were completely exposed to illumination (visible light of 1200000 Lux, UV of 200 W) at room temperature. The results are shown in Table 9.
TABLE-US-00009 TABLE 9 Results of solid stability sample content assay (5 d, 10 d, 1 M, 2 M and 3 M) RRT/Norm % Total impurities % Day 0 0.11 60° C.-5 days 0.10 60° C.-10 days 0.11 92.5% RH-5 days 0.11 92.5% RH-10 days 0.10 In the dark 0.11 Illumination 0.10 40° C.-75% RH-10 days 0.10 60° C.-75% RH-10 days 0.10 40° C.-75% RH-1 month 0.10 60° C.-75% RH-1 month 0.10 40° C.-75% RH-2 months 0.08 40° C.-75% RH-3 months 0.09
[0310] As can be seen, the crystalline form of the compound of formula (I) of the present application has good stability in the conditions of high temperature, high humidity or illumination without the increase of impurities during the test.
Example 7: Hygroscopicity Study of Crystalline Form B of Compound of Formula (I)
[0311] Instrument model: SMS DVS Advantage
[0312] Test conditions: the sample (10-20 mg, the crystalline form B prepared in Example 3) was placed in DVS sample tray for testing.
[0313] The detailed DVS parameters were as follows:
[0314] Temperature: 25° C.
[0315] Balancing: dm/dt=0.01%/min (shortest: 10 min, longest: 180 min)
[0316] Drying: drying at 0% RH for 120 min
[0317] RH (%) test gradient: 10%
[0318] Range of RH (%) test gradient: 0%-90%-0% The resulting dynamic vapor sorption (DVS) plot is shown in
[0319] As can be seen from
Experimental Example 1: In Vitro Detection of the Inhibitory Activity of the Compound Against PDE 3A Enzyme
[0320] Objective: to determine the AMP/GMP expression based on fluorescence polarization, i.e., to trace binding of AMP/GMP to antibody so as to indicate enzyme activity.
[0321] Reagents:
[0322] Buffer solution: 10 mM Tris-HCl (pH 7.5), 5 mM MgCl.sub.2, 0.01% Brij 35, 1 mM dithiothreitol (DTT), and 1% DMSO.
[0323] Enzyme: recombinant human PDE3A (Gene accession number: NM_000921; amino acid 669-end) was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag, with the molecular weight being 84 kDa.
[0324] Enzyme substrate: 1 μM cAMP
[0325] Detection: Transcreener®AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer.
[0326] Procedures:
[0327] 1. The recombinant human PDE3A enzyme and enzyme substrate (1 μM cAMP) were each dissolved in newly prepared experimental buffer solution;
[0328] 2. The PDE3A enzyme buffer solution was transferred into reaction wells;
[0329] 3. The compound which was dissolved in 100% DMSO was added to the reaction wells containing PDE3A enzyme buffer solution by acoustic technique (echo 550; millilambda range) and the mixture was incubated for 10 min at room temperature;
[0330] 4. The enzyme substrate buffer solution was added to the above reaction wells to initiate the reaction;
[0331] 5. The resulting mixture was incubated at room temperature for 1 h;
[0332] 6. The detection mixture (Transcreener®AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer) was added to stop the reaction, and the resulting mixture was incubated for 90 min while slowly mixing. The measurement range of fluorescence polarization was Ex/Em=620/688.
[0333] Data analysis: the fluorescence polarization signal was converted to nM based on AMP/GMP standard curve and the percentage enzyme activity relative to DMSO control calculated by Excel. GraphPad Prism was used for curve fitting (drawing medical icon). The results are shown in Table 10.
Experimental Example 2: In Vitro Detection of the Inhibitory Activity of the Compound Against PDE4B Enzyme
[0334] Objective: to determine the AMP/GMP expression based on fluorescence polarization, i.e., to trace binding of AMP/GMP to antibody so as to indicate enzyme activity.
[0335] Reagents:
[0336] Buffer solution: 10 mM Tris-HCl (pH 7.5), 5 mM MgCl.sub.2, 0.01% Brij 35, 1 mM DTT, and 1% DMSO.
[0337] Enzyme: recombinant human PDE4B (Gene accession number: NM_002600; amino acid 305-end) was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag, with the molecular weight being 78 kDa.
[0338] Enzyme substrate: 1 μM cAMP
[0339] Detection: Transcreener®AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer.
[0340] Procedures:
[0341] 1. The recombinant human PDE4B enzyme and enzyme substrate (1 μM cAMP) were each dissolved in newly prepared buffer solution;
[0342] 2. The PDE4B enzyme buffer solution was transferred into reaction wells;
[0343] 3. The compound which was dissolved in 100% DMSO by acoustic technique (echo 550; millilambda range) was added to the reaction wells containing PDE4B enzyme buffer solution and the mixture was incubated for 10 minutes at room temperature;
[0344] 4. The enzyme substrate buffer solution was added to the above reaction wells to initiate the reaction;
[0345] 5. The resulting mixture was incubated at room temperature for 1 h;
[0346] 6. The detection mixture (Transcreener®AMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer) was added to stop the reaction, and the resulting mixture was incubated for 90 min while slowly mixing. The measurement range of fluorescence polarization was Ex/Em=620/688.
[0347] Data analysis: the fluorescence polarization signal was converted to nM based on AMP/GMP standard curve and the percentage enzyme activity relative to DMSO control calculated by Excel. GraphPad Prism was used for curve fitting (drawing medical icon). The results are shown in Table 10:
TABLE-US-00010 TABLE 10 Results of in vitro screening test for the compound PDE3A PDE4B Compound IC.sub.50(nM) IC.sub.50(nM) Compound of 0.03 0.41 formula (I)
[0348] The compound of the present application has significant dual inhibitory effect on PDE3 and PDE4.
Experimental Example 3: Pharmacokinetic Study in Beagle Dogs
[0349] In this study, male beagle dogs were selected as test animals, and LC-MS/MS was used for quantitatively measuring the drug concentration in plasma of beagle dogs at different time points after intravenous injection or intragastric administration of the compound of formula (I) so as to evaluate the pharmacokinetics of the compound of formula (I) in beagle dogs.
[0350] The clear solution of the compound of formula (I) was injected into two beagle dogs of 10-12 kg via the cephalic vein or saphenous vein, and the clear solution of the compound of formula (I) was administered intragastrically to two beagle dogs of 10-12 kg (fasted overnight). The animals were all subjected to a blood collection of approximately 500 μL each time from peripheral veins at 0.0333, 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h post-dose, and the blood was transferred into commercial centrifuge tubes containing 0.85-1.15 mg of K.sub.2 EDTA.2H.sub.2O anticoagulant, and plasma was separated by centrifugation at 3000 g for 10 min at 4° C. The plasma concentration was measured by LC-MS/MS, and the relevant pharmacokinetic parameters were calculated using pharmacokinetic software WinNonlin™ Version 6.3 (Pharsight, Mountain View, Calif.) using non-compartmental model linear-log trapezoidal method.
TABLE-US-00011 TABLE 11 Pharmacokinetic parameters of the compound in beagle dogs Pharma- Intravenous injection (0.5 mg/kg) Intragastric administration (3 mg/kg) cokinetics Plasma Area under plasma Peak Time Area under plasma Bio- in clearance Half- concentration- concen- to concentration- avail- beagle (mL/ life time curve tration peak time curve ability dogs min/kg) (h) (0-inf, nM .Math. h) (nM) (h) (0-inf, nM .Math. h) (%) Compound of 70.3 0.3 210 59.4 0.6 123 7.5 formula (I)
[0351] The compound of the present application has high in vivo plasma clearance, low systemic exposure in plasma by oral administration and low oral bioavailability.
Experimental Example 4: Inhibitory Effect on Activity of Isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of Human Liver Microsomal Cytochrome P450
[0352] A total of 5 specific probe substrates of 5 isoenzymes of CYP, i.e., phenacetin (CYP1A2), diclofenac (CYP2C9), (S)-mephenytoin (CYP2C19), dextromethorphan (CYP2D6) and midazolam (CYP3A4) were each co-incubated with human liver microsomes and the compound of formula (I), and then reduced nicotinamide adenine dinucleotide phosphate (NADPH) was added to initiate the reaction. After the reaction was completed, the samples were treated, and the concentrations of 5 metabolites (acetaminophen, 4′-hydroxydiclofenac, 4′-hydroxymephenytoin, dextrorphan and 1′-hydroxymidazolam) generated from the specific substrates were quantitatively detected by LC-MS/MS to calculate the corresponding half maximal inhibitory concentrations (IC.sub.50).
TABLE-US-00012 TABLE 12 Inhibitory effect of compound of formula (I) on five CYP enzymes IC.sub.50 (μM) Compound No. CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4 Compound of 50 31 50 50 50 formula (I)
[0353] The compound of the present application has low inhibitory effect on the 5 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomal cytochrome P450.
Experimental Example 5: Pharmacodynamic Study in Cigarette Smoke-Induced Rat Acute Lung Injury Model
[0354] Animals
[0355] Male Sprague-Dawley rats (supplied by Shanghai SLAC Laboratory Animal Co., Ltd.), SPF grade, approximately 200 g.
[0356] Procedures
[0357] 1. Animals were randomly divided into 6 groups according to body weight after arrival and a one-week acclimation;
[0358] 2. On days 1-3 of the experiment, the corresponding compound of each group was atomized for 30 min. The animals in the model group and the treatment groups were exposed to cigarette smoke for 1 h, and after a 4-h interval, the animals were exposed to cigarette smoke again for 1 h. Cigarette smoke was given twice daily for 3 consecutive days. The control group animals were exposed to room air;
[0359] 3. On day 4 of the experiment, the corresponding compound of each group was atomized for 30 min, and then the animals in the model group and the treatment groups were given the atomized 150 ng/mL LPS for 15 min by inhalation. After 3 h (from the time starting atomization), the animals were exposed to cigarette smoke for 1 h, and then the lung function (Penh and F) of the animals was examined; bronchoalveolar lavage fluid was collected for cell counting after the animals were euthanized with CO.sub.2.
[0360] 4. Administration
[0361] Administration mode: the test compound and reference compound were given by atomization at the maximum atomization rate (approximately 12 mL) with the whole-body exposure atomization device for 30 min.
[0362] Administration frequency: the drug or solvent were given by atomization for 30 min in every morning before exposure to cigarette smoke, and were given before the inhalation of the atomized LPS on day 4.
[0363] 5. Measurements of pharmacodynamic endpoints
[0364] (1) Total white blood cells in BALF (bronchoalveolar lavage fluid);
[0365] (2) Mch challenge pulmonary function test (airway resistance index Penh);
TABLE-US-00013 TABLE 13 Grouping Compound concentration Number of in solution for Time of Group animals atomization administration Model group 10 — 30 min before the first cigarette smoke exposure every day Low dose group 10 0.05 mg/ml 30 min before the first of the compound cigarette smoke exposure of formula (I) every day High dose group 10 0.15 mg/ml 30 min before the first of the compound cigarette smoke exposure of formula (I) every day
[0366] The results are shown in
[0367] The compound of the present application can reduce the total number of white blood cells in BALF and the airway resistance index Penh in a cigarette smoke-induced rat acute lung injury model.
Experimental Example 6: In Vitro Detection of the Inhibitory Activity of the Compound Against TNF-α in Human Peripheral Blood Mononuclear Cells
[0368] Objective: to measure the anti-inflammatory activity at cellular level of the test compound based on the level of TNF-α in human peripheral blood mononuclear cells (hPBMCs).
[0369] Procedures:
[0370] 1. Normal human whole blood was collected into an EDTA anticoagulation tube;
[0371] 2. The PBMCs were separated by Ficoll density gradient centrifugation, and then counted, and the cell concentration was adjusted to 2×10.sup.6/mL;
[0372] 3. To each well of a U-bottom 96-well plate were added 2×10.sup.5 cells, 1 ng/mL LPS, and solutions of the compound of formula (I) in DMSO at concentrations of 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 100 pM and 10 pM, with a system volume of 200 μL per well;
[0373] 4. The mixture was incubated for 24 h, and then the supernatant was collected;
[0374] 5. The level of TNF-α in the supernatant was detected by ELISA, an inhibition curve was fitted using Graphpad Prism software, and the IC.sub.50 was calculated.
[0375] The results are shown in Table 14:
TABLE-US-00014 TABLE 14 Results of in vitro test for the compound hPBMC Compound IC.sub.50(nM) Compound of 29.18 formula (I)
[0376] The compound of the present application has significant inhibitory effect on TNF-α in human peripheral blood mononuclear cells (hPBMCs).