PHARMACEUTICAL COMPOSITION OF TRICYCLIC PDE3/PDE4 DUAL INHIBITOR COMPOUND

Abstract

A pharmaceutical composition of a tricyclic PDE3/PDE4 dual inhibitor compound and a preparation method therefor, specifically relating to a pharmaceutical composition of a compound of formula (I) or a pharmaceutically acceptable salt thereof, a preparation method thereof, and use thereof

Claims

1. A pharmaceutical composition, comprising: a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a surfactant, ##STR00006##

2. The pharmaceutical composition according to claim 1, further comprising a metal chelating agent.

3. The pharmaceutical composition according to claim 1, further comprising a buffering agent.

4. The pharmaceutical composition according to claim, further comprising an osmotic pressure regulator.

5. The pharmaceutical composition according to claim 1, further comprising a diluent.

6. The pharmaceutical composition according to claim 1, comprising the compound of formula (I) or the pharmaceutically acceptable salt thereof, the surfactant, and at least one of the buffering agent, the osmotic pressure regulator, the metal chelating agent and the diluent.

7. The pharmaceutical composition according to claim 1, wherein the surfactant is a non-ionic surfactant; or, the surfactant is one or more selected from the group consisting of polyoxyethylene glycol, polypropylene glycol alkyl ether, alkyl polyglucoside, octylphenol polyoxyethylene ether, alkylphenol polyoxyethylene ether, glycerin alkyl ester, polyoxyethylene sorbitan fatty acid ester, sorbitan alkyl ester, sorbitan fatty acid ester, cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, a block copolymer of polyethylene glycol and polypropylene glycol, and polyethoxylated tallow amine; or, the surfactant is one or more selected from the group consisting of a Tween and a Span; or, the surfactant is one or more selected from the group consisting of Tween 20, Tween 80 and Span 20; or or, the surfactant has a concentration of about 0.01 mg/mL to about 8 mg/mL.

8. The pharmaceutical composition according to claim 3, wherein the buffering agent is one or more selected from the group consisting of sulfuric acid, hydrochloric acid, sodium hydroxide, citric acid, sodium citrate, lactic acid, sodium lactate, acetic acid, sodium acetate, trisodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, tartaric acid, sodium tartrate, glycine, boric acid and phthalic acid; or, the buffering agent is selected from the group consisting of citric acid, a citrate, tartaric acid, a tartrate, phosphoric acid and a phosphate; or, the buffering agent is selected from the group consisting of a citrate, a tartrate and a phosphate; or, the buffering agent has a concentration of about 0.01 mg/mL to about 50 mg/mL; or, the buffering agent is used to control a pH of the pharmaceutical composition between about 3.0 and about 8.5.

9. The pharmaceutical composition according to claim 4, wherein the osmotic pressure regulator is one or more selected from the group consisting of sodium chloride, potassium chloride, glucose, mannitol and xylitol.

10. The pharmaceutical composition according to claim 2, wherein the metal chelating agent is one or more selected from the group consisting of edetic acid, disodium edetate and calcium disodium edetate.

11. The pharmaceutical composition according to claim 5, wherein the diluent is one or more selected from the group consisting of water, ethanol and glycerol.

12. The pharmaceutical composition according to claim 1, comprising the compound of formula (I), the surfactant, the buffering agent, the osmotic pressure regulator, the metal chelating agent and the diluent, wherein the compound of formula (I) has a concentration of 0.002 mg/mL to 50 mg/mL, the surfactant has a concentration of 0.02 mg/mL to 3 mg/mL, the buffering agent has a concentration of 0.1 mg/mL to about 25 mg/mL, the osmotic pressure regulator has a concentration of 5 mg/mL to 9 mg/mL, and the metal chelating agent has a concentration of 0.01 mg/mL to about 5 mg/mL.

13. The pharmaceutical composition according to claim 1, comprising the compound of formula (I), Tween 80, sodium dihydrogen phosphate or monohydrate thereof, disodium hydrogen phosphate, sodium chloride, disodium edetate and water; wherein optionally, the compound of formula (I) has a concentration of 0.002 mg/mL to 50 mg/mL, Tween 80 has a concentration of 0.02 mg/mL to 3 mg/mL, sodium dihydrogen phosphate and disodium hydrogen phosphate have a concentration of 0.1 mg/mL to 25 mg/mL, sodium chloride has a concentration of 5 mg/mL to 9 mg/mL, and disodium edetate has a concentration of 0.01 mg/mL to about 5 mg/mL.

14. The pharmaceutical composition according to claim 1, wherein the compound of formula (I) is a crystalline form of the compound of formula (I) which comprises 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 20 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 20: 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 20: 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°.

15. The pharmaceutical composition according to claim 1, wherein the compound of formula (I) or the pharmaceutically acceptable salt thereof has a particle size of X50<10 μm; or the compound of formula (I) or the pharmaceutically acceptable salt thereof has the particle size of X50<5 μm and X90<10 μm.

16. The pharmaceutical composition according to claim 1, wherein the mass ratio of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the surfactant is about 1:200 to 100:1, and even more preferably about 1:1 to 15:1, and the mass of the compound of formula (I) or the pharmaceutically acceptable salt thereof is based on the compound of formula (I).

17. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is in the form of a suspension.

18. A method for preparing the pharmaceutical composition according to claim 6, comprising: mixing the surfactant, the compound of formula (I) or the pharmaceutically acceptable salt thereof, and at least one selected from the group consisting of: the metal chelating agent, the buffering agent, the diluent, and the osmotic pressure regulator.

19. 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 pharmaceutical composition according to claim 1.

20. (canceled)

21. The method according to claim 19, wherein the condition associated with PDE3 and/or PDE4 is selected from the group consisting of asthma or chronic obstructive pulmonary disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0085] FIG. 1 is an XRPD pattern of the crystalline form of the compound of formula (I);

[0086] FIG. 2 is a DSC pattern of the crystalline form of the compound of formula (I);

[0087] FIG. 3 is a TGA pattern of the crystalline form of the compound of formula (I);

[0088] FIG. 4 is a DVS pattern of the crystalline form of the compound of formula (I);

[0089] FIG. 5 shows the total number of white blood cells in BALF;

[0090] FIG. 6 shows the methacholine (Mch) challenge pulmonary function test (airway resistance index Penh).

DETAILED DESCRIPTION

[0091] The following specific examples are intended to allow those skilled in the art to clearly understand and implement the present application. These specific examples should not be considered as limit to the scope of the present application, but merely as exemplary description and representative of the present application.

EXAMPLES

[0092] Synthesis of intermediate BB-1

##STR00003## ##STR00004##

[0093] Step 1: synthesis of compound BB-1-2 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.

[0094] .sup.1H NMR (400 MHz, DMSO-d6) 6=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]+.

[0095] Step 2: synthesis of compound BB-1-3 Phosphorus oxychloride (379.50 g, 230.00 mL) was heated to 85° C. in nitrogen atmosphere and compound BB-1-2 (26.00 g) was added in portions. The reaction mixture was stirred at 85° C. for 2 h. 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.

[0096] .sup.1H NMR (400 MHz, CD3OD) 6=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]+.

[0097] Step 3: synthesis of compound BB-1-4 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 the pH to neutral, followed by extraction with ethyl acetate (100 mL×3). Then 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.

[0098] MS-ESI m/z: 263.1[M+H]+.

[0099] Step 4: synthesis of compound BB-1-5 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.

[0100] .sup.1H NMR (400 MHz, DMSO-d6) 6=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

[0101] Step 5: synthesis of compound BB-1-6 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]+. Step 6: synthesis of compound BB-1 Compound BB-1-6 (925.67 mg) was dissolved in isopropanol (8 mL) at room temperature, and then 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.

[0102] .sup.1H NMR (400 MHz, DMSO-d6) 6=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]+.

[0103] Synthesis of compound BB-4

##STR00005##

[0104] Step 1: synthesis of compound BB-4-1 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.

[0105] MS—ESI m/z: 579.3[M+H]+.

[0106] Step 2: synthesis of compound BB-4 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 x 3) were added for extraction. Then 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.

[0107] .sup.1H NMR (400 MHz, DMSO-d6) 6=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]+.

Example 1: Preparation of compound of formula (I)

[0108] 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, followed by addition of compound BB-4 (50 mg) and stirring 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.

[0109] .sup.1H NMR (400 MHz, CD3OD) 6=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+1-1]+574.1.

Example 2: Preparation of crystalline form of compound of formula (I)

[0110] 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 crystalline form. The XRPD pattern is shown in FIG. 1, the DSC pattern is shown in FIG. 2, and the TGA pattern is shown in FIG. 3.

Examples 3-36

[0111] Procedures: [0112] 1) various excipients were added to a preparation tank, and the mixture was stirred for dissolving to give an excipient solution; [0113] 2) the compound of formula (I) was added to the prepared excipient solution, and the mixture was stirred to form a uniform suspension; [0114] 3) the suspension was further subjected to a high-pressure homogenizer, a microjet, or a sand mill, and the like, and the particle size of the compound of formula (I) in the preparation was controlled at X50<5 μm and X90<10 μm; and [0115] 4) a product was obtained.

[0116] The amounts of the specific excipients and the products are shown in the following Table 2.

TABLE-US-00002 TABLE 2 Concentration Composition of excipient solution of compound Disodium Sodium Calcium of formula Tween Tween Span hydrogen dihydrogen Tartaric Citric Sodium Disodium disodium (I) in the Example 20 80 20 phosphate phosphate acid acid chloride edetate edetate Water product No. (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) (mL) (mg/mL)  3 0.5 / / / / / / / / / 1000 1  4 / 0.5 / / / / / / / / 1000 1  5 / / 0.5 / / / / / / / 1000 1  6 0.5 / 0.05 / / / / / / / 1000 1  7 / 0.5 0.05 / / / / / / / 1000 1  8 0.5 0.05 2 4 / / / / / 1000 1  9 / 0.5 / 2 4 / / / / / 1000 1 10 / 0.5 / 2 4 / / / 2 / 1000 1 11 0.5 / / 2 4 / / / 2 / 1000 1 12 0.5 / 0.05 2 4 / / / 2 / 1000 1 13 / 0.5 0.05 2 4 / / / 2 / 1000 1 14 0.5 0.5 0.05 2 4 / / / 2 / 1000 1 15 0.5 0.5 0.05 2 4 / / 6.5 2 / 1000 1 16 / 0.5 / 2 4 / / 6.5 2 / 1000 1 17  0.05 / 0.01 2 4 / / 6.5 / / 1000 0.02 18 0.5 / 0.05 2 4 / / 6.5 / / 1000 6 19 2   / 1 12.8 / / / / / / 1000 50 20 2   / 1 2 4 / / / / / 1000 50 21 / 0.05 0.01 2 4 / / 6.5 / / 1000 0.02 22 / 0.5 0.05 2 4 / / 6.5 / / 1000 6 23 / 2 1 12.8 / / / / / / 1000 50 24 / 2 1 1.4 3.6 / / 8.5 / / 1000 50 25 / 0.1 / 2 4 / / 6.5 0.01 / 1000 0.002 26 / 0.3 / 2 4 / / 6.5 0.5 / 1000 0.002 27 / 1 / 1.4 3.6 / / 8.5 1.5 / 1000 1 28 / 0.1 / 2 4 / / 8.5 0.1 / 1000 1 29 / 2 / 0.7 1.7 / / 9 2 / 1000 50 30 / 0.02 / 2 4 / / 6.5 0.01 / 1000 1 31 / 0.02 / 2 4 / / 6.5 0.03 / 1000 1 32 0.2 0.2 / 2 4 / / 6.5 0.02 / 1000 1 33 / 2 / 0.7 1.7 / / 9 / 2 1000 50 34 / 2 / 0.7 1.7 / / / 1 1 1000 50 35* / 2 / / / 3 8 1 / 1000 50 36* / 2 / / / / 3 8 1 / 1000 50 *In Example 35 and Example 36, after mixing and dissolving the various excipients in the step 1), an appropriate amount of sodium hydroxide was added to adjust to pH 5.0 to 7.0.

[0117] Experimental Example 1: Stability study of solid crystalline form of the compound of formula (I)

[0118] High performance liquid chromatography (HPLC) [0119] The chromatographic conditions of the HPLC method are seen in table below: [0120] Chromatography column: Zorbax SB C-18, 4.6 mm×150 mm, 5 μm (PDS-HPLC-007) [0121] Mobile phase A: 0.1% TFA in water [0122] Mobile phase B: 100% ACN

[0123] Preparation of sample: the sample was dissolved with a mixed solvent of acetonitrile and water (acetonitrile: water=50:50 (v/v))

[0124] Solid stability stakeout method

[0125] 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 of the compound of formula (I) prepared in Example 2 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 1.2×10.sup.6 Luxhr/near UV energy of 200 whr/m.sup.2), (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 experimental results are shown in Table 3.

TABLE-US-00003 TABLE 3 Results of solid stability sample content assay (5 days, 10 days, 1 month, 2 months, 3 months) Total RRT/Norm % 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

[0126] 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.

[0127] Experimental Example 2: Hygroscopicity study of crystalline form of the compound of formula (I)

[0128] Instrument model: SMS DVS Advantage

[0129] Test conditions: the sample (10-20 mg, the crystalline form prepared in Example 3) was placed in DVS sample tray for testing.

[0130] The detailed DVS parameters are as follows:

[0131] Temperature: 25° C.

[0132] Balancing: dm/dt=0.01%/min (shortest: 10 min, longest: 180 min)

[0133] Drying: drying at 0% RH for 120 min

[0134] RH (%) test gradient: 10%

[0135] Range of RH (%) test gradient: 0%-90%-0%. The resulting dynamic vapor sorption (DVS) plot is shown in FIG. 4.

[0136] As can be seen from FIG. 4, the crystalline form of the compound of formula (I) of the present application has a small hygroscopicity.

[0137] Experimental Example 3: In vitro detection of the inhibitory activity of the compound against PDE 3A enzyme

[0138] 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.

[0139] Reagents:

[0140] 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.

[0141] 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.

[0142] Enzyme substrate: 1 μM cAMP Detection: TranscreenerOAMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer.

[0143] Procedures: [0144] 1. The recombinant human PDE3A enzyme and enzyme substrate (μM cAMP) were each dissolved in newly prepared experimental buffer solution; [0145] 2. The PDE3A enzyme buffer solution was transferred into reaction wells; [0146] 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; [0147] 4. The enzyme substrate buffer solution was added to the above reaction wells to initiate the reaction; [0148] 5. The resulting mixture was incubated at room temperature for 1 h; [0149] 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/μm=620/688. [0150] 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 4. [0151] Experimental Example 4: In vitro detection of the inhibitory activity of the compound against PDE 4B enzyme [0152] 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. [0153] Reagents: [0154] Buffer solution: 10 mM Tris—HCl (pH 7.5), 5 mM MgCl.sub.2, 0.01% Brij 35, 1 mM DTT, and 1% DMSO. [0155] 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 molecular weight being 78 kDa. [0156] Enzyme substrate: 1 μM cAMP Detection: TranscreenerOAMP2/GMP2 antibody and AMP2/GMP2 AlexaFluor633 tracer. [0157] Procedures: [0158] 1. The recombinant human PDE4B enzyme and enzyme substrate (1 μM cAMP) were each dissolved in newly prepared buffer solution. [0159] 2. The PDE4B enzyme buffer solution was transferred into reaction wells. [0160] 3. The compound dissolved in 100% DMSO was added to the reaction wells containing PDE4B enzyme buffer solution by acoustic technique (echo 550; millilambda range) and the mixture was incubated for 10 min at room temperature. [0161] 4. The enzyme substrate buffer solution was added to the above reaction wells to initiate reaction. [0162] 5. The resulting mixture was incubated at room temperature for 1 h. [0163] 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. [0164] 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).

[0165] The results are shown in Table 4:

TABLE-US-00004 TABLE 4 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)

[0166] The active ingredient in the pharmaceutical composition of the present application has significant dual inhibitory effect on PDE3 and PDE4.

[0167] Experimental Example 5: Pharmacokinetic study in beagle dogs 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.

[0168] 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 (overnight fasted). The animals were all subjected to approximately 500-pt blood collection 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 K2 EDTA.2H20 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 WinNonlinTM Version 6.3 (Pharsight, Mountain View, Calif.), a pharmacokinetic software, with a non-compartmental model linear-log trapezoidal method.

TABLE-US-00005 TABLE 5 Pharmacokinetic parameters of the compound in beagle dogs Intravenous injection (0.5 mg/kg) Intragastric administration (3 mg/kg) Area under Area under plasma plasma Plasma Half- concentration- Peak Time concentration- Pharmacokinetics clearance life time curve concentration to peak time curve Bioavailability in beagle dogs (mL/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)

[0169] The active ingredient in the pharmaceutical composition of the present application has high in vivo plasma clearance, low systemic exposure in plasma by oral administration and low oral bioavailability.

[0170] Experimental Example 6: Inhibitory effect on activity of isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomal cytochrome P450 A total of 5 specific probe substrates of 5 isoenzymes of CYP, namely 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 l′-hydroxymidazolam) generated from the specific substrates were quantitatively detected by lC-MS/MS to calculate the correcnondino- half maximal inhibitory concentrationc (IC.sub.50)

TABLE-US-00006 TABLE 6 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)

[0171] The active ingredient in the pharmaceutical composition of the present application has low inhibitory effect on the 5 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomal cytochrome P450.

[0172] Experimental Example 7: Pharmacodynamic study in cigarette smoke-induced rat acute lung injury model

[0173] Animals

[0174] Male Sprague-Dawley rats (supplied by Shanghai SLAC Laboratory Animal Co., Ltd.), SPF grade, approximately 200 g.

[0175] Procedures [0176] 1. Animals were randomly divided into 6 groups according to body weight after arrival and a one-week acclimation; [0177] 2. On days 1-3 of the experiment, the corresponding compound of each group was atomized for 30 min. Then 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; [0178] 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 μg/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 (BALF) was collected for cell counting after the animals were euthanized with CO2. [0179] 4. Administration 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.

[0180] 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. [0181] 5. Measurements of pharmacodynamic endpoints [0182] (1) Total white blood cells in BALF (bronchoalveolar lavage fluid); [0183] (2) Mch challenge pulmonary function test (airway resistance index Penh);

TABLE-US-00007 TABLE 7 Grouping Compound Number concentration of in solution for Group animals atomization Time of 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 of formula (I) exposure every day High dose group 10 0.15 mg/mL 30 min before the first of the compound cigarette smoke of formula (I) exposure every day

[0184] The experimental results are shown in FIG. 5 and FIG. 6.

[0185] As can be seen from FIG. 5 and FIG. 6, the active ingredient in the pharmaceutical composition of the present application can effectively reduce the number of white blood cells in the bronchoalveolar lavage fluid and the airway resistance index Penh.

[0186] Experimental Example 8: In vitro detection of the inhibitory activity of the compound against TNF-α in human peripheral blood mononuclear cells 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).

[0187] Procedures: [0188] 1. Normal human whole blood was collected into an EDTA anticoagulation tube; [0189] 2. The PBMC was separated by Ficoll density gradient centrifugation and counted, and the cell concentration was adjusted to 2×10.sup.6 cells/mL; [0190] 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 04, 10 04, 1 μM, 100 nM, 10 nM, 1 nM, 100 pM and 10 pM, with a system volume of 200 μL per well; [0191] 4. The mixture was incubated for 24 h, and then the supernatant was collected; [0192] 5. The level of TNF-α in the supernatant was detected by ELISA, an inhibition curve was fitted using Graphpad Prism software, and the IC50 was calculated.

[0193] The results are shown in Table 8:

TABLE-US-00008 TABLE 8 Results of in vitro test for the compound hPBMC Compound IC.sub.50 (nM) Compound of 29.18 formula (I)

[0194] Therefore, the active ingredient in the pharmaceutical composition of the present application shows potent anti-inflammatory activity, and has significant inhibitory effect on TNF-α in human peripheral blood mononuclear cells (hPBMCs).

[0195] Experimental Example 9: Stability Test

[0196] The product obtained in Example 28 was placed for 6 months in an accelerating condition (40° C. 2° C./RH 25%±5%) and a long-term condition (30° C.±2° C./RH 65%±5%), and the results are shown in Table 10.

TABLE-US-00009 TABLE 9 Example 28 Accelerated Long - term for 6 for 6 Items Month 0 months months Related Total 0.71 0.94 0.98 substance (%) impurities Particle size X.sub.10 0.7 0.7 0.7 (μm) X.sub.50 1.4 1.5 1.5 X.sub.90 2.5 2.9 2.6

[0197] As can be seen, the pharmaceutical composition of the present application shows good stability in the accelerating condition and the long-term condition without significant increase of impurities and particle size.