Amorphous pyrrolidine derivative as PPAR agonist and preparation method thereof

Abstract

The present invention relates to an amorphous pyrrolidine derivative as a PPAR agonist and a preparation method thereof.

Claims

1. An amorphous form of a compound represented by formula (I), ##STR00004## wherein an X-ray powder diffraction pattern of the amorphous form is shown in FIG. 1.

2. The amorphous form according to claim 1, wherein a differential scanning calorimetry curve of the amorphous form has starting points of two endothermic peaks at 69.28±3° C. and 239.33±3° C.

3. The amorphous form according to claim 2, wherein a DSC pattern of the amorphous form is shown in FIG. 2.

4. The amorphous form according to claim 1, wherein a thermogravimetric analysis curve of the amorphous form has a weight loss reached 0.9958% at 120.00±3° C.

5. The amorphous form according to claim 4, wherein a TGA pattern of the amorphous form is shown in FIG. 3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a Cu-Kα radiated XRPD spectrogram of the amorphous form of the compound of formula (I).

(2) FIG. 2 is a DSC spectrogram of the amorphous form of the compound of formula (I).

(3) FIG. 3 is a TGA spectrogram of the amorphous form of the compound of formula (I).

DETAILED DESCRIPTION

(4) Hereinafter the present invention is described in details by ways of examples, but it is not intended to limit the present invention in any adverse manner. The present invention has been described in details herein, and further disclosed specific embodiments. It will be apparent to those skilled in the art that a variety of modifications and improvements can be made to the embodiments of the present invention without departing from the principle and scope of the present invention.

EXAMPLE 1

Preparation of the Compound of Formula (I)

(5) ##STR00003##

(6) Step 1: Preparation of Compound B

(7) At 25° C., acetonitrile (30 L) was added into a 50 L reactor, start stirring, and then Compound A (2.00 kg, 13.32 mol, 1.0 eq), ethyl bromoisobutyrate (7.79 kg, 39.95 mol, 3.0 eq) and potassium carbonate (5.52 kg, 39.95 mol, 3.0 eq) were added. The reaction solution was stirred at 80° C. for 16 h. The reaction temperature was cooled to 25° C., and then filtered. The filtrate was concentrated under reduced pressure. The resultant residue was dissolved in ethyl acetate (5 L). The filter cake was washed with ethyl acetate (5 L×2), and the ethyl acetate solutions were combined. The combined organic phases were washed with aq. NaOH (1 mol/L, 5 L/time) until showed that no spot of raw material A appeared in the organic phase by TLC (petroleum ether: ethyl acetate=5:1). The organic phase was washed with aqueous saturated solution of sodium chloride (5 L×2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, to give 1.51 kg of compound B, yield: 42.9%.

(8) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 9.85 (s, 1H), 7.50 (s, 2H), 4.31-4.23 (m, 2H), 2.25 (s, 6H), 1.45 (s, 6H), 1.33 (t, J=7.2 Hz, 1H).

(9) Step 2: Preparation of Compound C

(10) In a dry ice-ethanol bath (−60° C.), gaseous HCl (3.67 kg, 100.54 mol, 5.3 eq) was introduced into ethanol (12 L), and the system temperature was controlled below 0° C. Ethanol (13 L) and the freshly prepared ethanol solution of HCl were added to a 50 L reactor. The mixture was stirred, and naturally warmed to 25° C. Then, compound B (5.01 kg, 18.97 mol, 1.0 eq) was added. After complete dissolution of the materials, p-methylthioacetophenone (2.83 kg, 17.07 mol, 0.9 eq) was added in portions. The mixture was stirred at 25° C. for 16 h. The reaction system was suction-filtered. The filter cake was dissolved in ethyl acetate (30 L), washed with water (10 L×2), aq. NaOH (1 N, 8 L×2), and aqueous saturated solution of sodium chloride (8 L×2), dried over anhydrous sodium sulfate (1.5 kg), filtered, and concentrated under reduced pressure to give 6.10 kg of compound C, yield: 76.8%.

(11) MS m/z (ESI): 413.1 [M+1].

(12) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.95 (d, J=8.28 Hz, 2H), 7.71 (d, J=15.56 Hz, 1H), 7.42 (d, J=15.56 Hz, 1H), 7.31-7.28 (m, 4H), 4.30 (q, J=7.28 Hz, 2H), 2.54 (s, 3H), 2.25 (s, 6H), 1.50 (s, 6H), 1.36 (t, J=7.15 Hz, 3H).

(13) Step 3: Preparation of Compound D

(14) N,N-dimethylformamide (15 L) was added to a 50 L reactor, start stirring, and trimethylsulfoxonium iodide (3.78 kg, 16.01 mol 1.2 eq) was added, then cooled to 0° C., and potassium tert-butoxide (1.79 kg, 16.01 mol, 1.2 eq) was added in portions. After the mixture was stirred at 0° C. for 30 min, a solution of compound C (5.5 kg, 13.34 mol, 1.0 eq) in N,N-dimethylformamide (15 L) was slowly added. The mixture was stirred at 0° C. for 2 h. The reaction liquor was slowly poured into ice water (0-5° C., 30 L), and then extracted with petroleum ether/ethyl acetate (1:1, 10 L×3). The combined organic phase was washed with water (10 L×2) and aqueous saturated solution of sodium chloride (10 L×2), dried over anhydrous sodium sulfate (2 kg), filtered, and concentrated under reduced pressure to give 5.48 kg of compound D, yield: 96.3%.

(15) MS m/z (ESI): 427.2 [M+1].

(16) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.91 (d, J=8.5 Hz, 2H), 7.27 (d, J=8.5 Hz, 2H), 6.81-6.70 (m, 1H), 6.75 (s, 1H), 4.29 (q, J=7.0 Hz, 2H), 2.83-2.74 (m, 1H), 2.56 (m, 1H), 2.51 (s, 3H), 2.18 (s, 6H), 1.84 (m, 1H), 1.46 (s, 6H), 1.35 (t, J=7.2 Hz, 3H).

(17) Step 4: Preparation of Compound E

(18) Ethanol (35.0 L) was added to a dry 50 L reactor, start stirring, and then compound D (5.45 kg, 12.79 mol, 1.0 eq) and glacial acetic acid (2.30 kg, 38.37 mol, 3.0 eq) were added. After heating the reaction mixture to 80° C., zinc powder (2.45 kg, 38.37 mol, 3.0 eq) was added in portions. The resultant suspension was continuously stirred at 80° C. for 16 h. The reaction liquor was filtered, and the filter cake was washed with ethyl acetate (3 L×2). The combined organic phase was concentrated under reduced pressure. The concentrated solution was dissolved in ethyl acetate (10 L), pump into a 50 L separatory funnel. Ethyl acetate (15 L) and water (10 L) were pumped into the 50 L separatory funnel and stirred for 5 minutes., then stood for phase separation. The organic phase was washed sequentially with 10% of aq. sodium carbonate (10 L×2) and aqueous saturated solution of sodium chloride (10 L×1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 5.15 kg of compound E, yield: 92.9%.

(19) MS m/z (ESI): 429.2 [M+1].

(20) .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.74 (d, J=8.5 Hz, 2H), 7.17 (d, J=8.5 Hz, 2H), 6.71 (s, 2H), 4.21 (q, J=7.1 Hz, 2H), 2.82 (t, J=7.3 Hz, 2H), 2.51 (t, J=7.7 Hz, 2H), 2.45 (s, 3H), 2.09 (s, 6H), 1.94 (quin, J=7.5 Hz, 2H), 1.39 (s, 6H), 1.28 (t, J=7.0 Hz, 3H)

(21) Step 5: Preparation of Compound F

(22) Anhydrous dichloromethane (20 L) was added to a 50 L reactor, start stirring, then compound E (5.21 kg, 12.02 mol, 1.0 eq) and 2,6-lutidine (4.50 kg, 42.07 mol, 3.5 eq) were added. The mixture was cooled to 0° C. Trimethylsilyl trifluoromethanesulfonate (8.01 kg, 36.06 mol, 3.0 eq) was added to the reaction solution, and the mixture was continuously stirred at 0° C. for around 30 min. The reaction liquor was detected by TLC (petroleum ether:ethyl acetate=5:1). Cold water (5-10° C., 10 L) was pumped into a 50 L separatory funnel, and then pump into the reaction liquor under stirring. After stirring for 5 min, the phases were separated. The organic phase was washed with aqueous saturated solution of sodium chloride (10 L), and concentrated under reduced pressure. The concentrated solution was added to a mixed solution of methanol/water (2:1, 30 L) and stirred for about 20 min. Yellow solids were precipitated. The mixture was filtered, dried under reduced pressure to give a crude product as yellow solid.

(23) Anhydrous toluene (35 L) was added to a 50 L reactor, start stirring, and the crude product which was dried under reduced pressure was added into the reactor. The mixture was cooled to 0° C., and Dichloro-dicyano-benzoquinone (2.99 kg, 13.22 mol, 1.1 eq) was added to the reactor in portions. The mixture was continuously stirred at 0° C. for lh. The reaction liquor was detected by TLC (petroleum ether:ethyl acetate=5:1). Water (50 L) and sodium sulfite (3.00 kg) were added to a 120 L barrel. After being stirred to clear, the reaction liquor was slowly poured into the sodium sulfite solution, and ethyl acetate (15 L) was added. The mixture was quickly stirred for 10 min to precipitate a large amount of yellow solid, and then filtered by suction. The filter cake was washed with petroleum ether/ethyl acetate (3:1, 10 L×2). The filtrates were combined, and then the organic phase was separated. The organic phase was washed with 5% sodium sulfite (10 L×2) and aqueous saturated solution of sodium chloride (10 L×2), dried over anhydrous sodium sulfate (2.00 kg), filtered, and concentrated under reduced pressure (40-50° C.) to give 4.29 kg of crude product. Then, the crude product was added to 12 L of anhydrous ethanol, stirring at 25° C. for 0.5 h, then filtered. The filter cake was collected, and dried under reduced pressure to give 3.50 kg of compound F, yield: 69.9%.

(24) MS m/z (ESI): 427.2 [M+1].

(25) .sup.1H NMR (400 MHz, CHLOROFORM-d) □δ ppm 7.85 (d, J=8.5 Hz, 2H), 7.28-7.27 (m, 2H), 7.22-7.14 (m, 1H), 7.02 (s, 1H), 6.83 (s, 2H), 4.30 (s, 2H), 3.53 (d, J=6.8 Hz, 2H), 2.55 (s, 3H), 2.21 (s, 6H), 1.49 (s, 6H), 1.38 (s, 3H).

(26) Step 6: Preparation of Compound G

(27) 2- Methyltetrahydrofuran (30 L) was added to a dry 50 L reactor, start stirring, and compound F (3.0 kg, 6.50 mol, 1.0 eq) and trifluoroacetic acid (37.05 g, 0.33 mol, 0.05 eq) were added. Then, N-(methoxymethyl)-N-(trimethylsilylmethyl)benzylamine (1.85 kg, 7.80 mol, 1.2 eq) was added slowly. The internal temperature was controlled below 30° C. After the dropwise addition, the mixture was continuously stirred at 25° C. for 12 h. The reaction liquor was bump into a 50 L separatory funnel, washed sequentially with 5% of aq. sodium carbonate (10 L×2) and aqueous saturated solution of sodium chloride(10 L×2), dried over anhydrous sodium sulfate (2 kg), filtered, and concentrated under reduced pressure to give 3.92 kg of compound G.

(28) MS m/z (ESI): 560.0 [M+1].

(29) .sup.1H NMR (400 MHz, CHLOROFORM-d) □δ ppm 7.71 (d, J=8.5 Hz, 1H), 7.31-7.20 (m, 9H), 6.71 (s, 1H), 4.30-4.23 (m, 2H), 3.76-3.36 (m, 6H), 3.07-2.96 (m, 2H), 2.68 (t, J=7.3 Hz, 2H), 2.51 (s, 3H), 2.10-2.03 (m, 6H), 1.36-1.22 (m, 9H).

(30) Step 7: Preparation of Compound H

(31) Compound G (3.92 kg, 5.04 mol, 1.0 eq) was dissolved in anhydrous ethanol (20 L), and the solution was added into a 50 L reactor, start stirring. NaOH (604.8 g, 15.12 mol, 3.0 eq) was dissolved in water (6 L), and then the solution was slowly added to the reaction solution. After addition, the mixture was continuously stirred at 25° C. for 16 h. The reaction solution was concentrated under reduced pressure to remove most of the solvent (ethanol). The concentrated solution was bumped into a 50 L separatory funnel, stirred, and then ethyl acetate (20 L) was added. The mixture was washed with 10% of aq. KHSO.sub.4 (10 L×2) and aqueous saturated solution of sodium chloride(10 L×2), dried over anhydrous sodium sulfate (1.5 kg), and concentrated under reduced pressure until about 8 L of solvent was remained and a large amount of solid was precipitated. The concentration was stopped, and the mixture was cooled to 25° C. The concentrated suspension was filtered, and the filter cake was washed with ethyl acetate (2 L×3), dried by suction, and dried under reduced pressure in a vacuum drying chamber to give 2.44 kg of compound H, yield: 89.85%.

(32) MS m/z (ESI): 532.1[M+1].

(33) .sup.1H NMR (400 MHz, CHLOROFORM-d) □δ ppm 7.66 (dd, J=4.0, 8.3 Hz, 2H), 7.41-7.33 (m, 2H), 7.23-7.08 (m, 5H), 6.79 (d, J=2.0 Hz, 2H), 4.12 (q, J=7.0 Hz, 2H), 3.93-3.60 (m, 4H), 3.48-3.32 (m, 1H), 2.97-2.84 (m, 1H), 2.67 (d, J=7.8 Hz, 2H), 2.57-2.49 (m, 3H), 2.25-2.13 (m, 6H), 1.46 (s, 6H), 1.36 (t, J=7.2 Hz, 3H).

(34) Step 8: Preparation of Compound I

(35) Acetonitrile (24 L) and isopropanol (6 L) were added to a dry 50 L reactor, start stirring, then compound H (3.04 kg, 5.65 mol, 1.0 eq) was added. At 80° C., (S)-(−)-(1-naphthyl)ethamine (724.79 g) was slowly added. After addition, the mixture was continuously stirred at 80° C. for 1 h. The heating was stopped. The mixture was naturally cooled, and continuously stirred at 30° C. for 16 h. The stirring was stopped. The mixture was filtered by suction, and the filter cake was washed with isopropanol (2 L×2). After suction to dry, the solid was transferred into a rotatory evaporator to dry under reduced pressure to give 1.63 kg of compound I.

(36) Chiral resolution conditions: Chiral column: Chiralpak AD-3 100×4.6 mm I.D., 3 μm; mobile phase: 40% methanol (0.05% DEA)-CO.sub.2; flow rate: 4 mL/min; column temperature: 40° C.

(37) The retention time of compound I: 1.604 min.

(38) Step 9: Preparation of Compound J

(39) Anhydrous ethanol (30 L) and anhydrous methanol (4.5 L) were added to a dry 50 L reactor, start stirring, and compound I (2.93 kg, 4.17 mol, 1.0 eq) was added. The mixture was heated to 80° C. and stirred for 1 h. The heating was stopped, and the mixture was naturally cooled. The mixture was continuously stirred at 30° C. for 16 h. The suspension was filtered by suction, and the filter cake was washed with ethanol (2 L×2), and dried under reduced pressure. methanol (1.5 L) and ethyl acetate (15 L) were added to the residue, and the mixture was stirred and then pump into a 50 L liquid separator. The organic phase was washed with 10% of aq. KHSO.sub.4 (10 L×5), aqueous saturated solution of sodium chloride (5 L×2), dried over anhydrous sodium sulfate (1 kg), filtered, and concentrated under reduced pressure to give 0.97 kg of compound J.

(40) Chiral resolution conditions: Chiral column: Chiralpak AD-3 100×4.6 mm I.D., 3 μm; mobile phase: 40% methanol (0.05% DEA)-CO.sub.2; flow rate: 4 mL/min; column temperature: 40° C.

(41) The retention of compound J: 1.576 min.

(42) Step 10: Preparation of the Compound of Formula (I)

(43) Anhydrous dichloromethane (7 L) was added to a dry 50 L reactor, start stirring, and compound J (700 g, 1.31 mol, 1.0 eq) and triethylamine (1.33 kg, 13.1 mol, 10.0 eq) were added. At 0° C., phenyl chloroformate (2.24 kg, 13.1 mol, 10.0 eq) was added dropwise to the reaction liquor. After completion of addition, the mixture was continuously stirred at 0° C. for 1 h. A solution of potassium carbonate (543.37 g, 3.94 mol, 3.0 eq) in pure water (3 L) was added to the reaction solution. The mixture was heated to 40° C. and continuously stirred for 20 min. Then, LiOH (165.60 g, 3.94 mol, 3.0 eq) was added, and the mixture was continuously stirred at 25° C. for 20 min. The reaction mixture was subjected to phase separation. The organic phase was washed with aqueous saturated solution of sodium chloride (2 L), dried over anhydrous sodium sulfate (500 g), filtered, and concentrated under reduced pressure. The concentrated solution was dissolved in ethyl acetate (1.5 L), then heptane (5.6 L) was slowly added under high-speed stirring. After addition, the mixture was continuously stirred for 30 min, and filtered. The filter cake was added to heptane/ethyl acetate (4:1, 3.5 L×3), and stirred at a high speed to beat for 30 min, and filtered. The resultant filter cake was dissolved in t-butyl methyl ether (5 L), and washed sequentially with 5% of aq. KHSO.sub.4 (1.5 L×2), deionized water (1 L×2), dried over anhydrous sodium sulfate (300 g), filtered, and concentrated under reduced pressure. The resultant solid was dried in a vacuum oven (40-45° C.) to give the compound of formula (I).

(44) MS m/z (ESI): 584.1 [M+23].

(45) .sup.1H NMR (400 MHz, MeOD-d.sub.4) □δ ppm 7.65 (d, J=6.8 Hz, 2H), 7.43-7.37 (m, 2H), 7.29-7.22 (m, 3H), 7.16 (t, J=7.2 Hz, 2H), 6.87 (s, 2H), 4.13-3.92 (m, 2H), 3.89-3.80 (m, 1H), 3.69 (dd, J=5.4, 10.7 Hz, 1H), 3.58-3.49 (m, 1H), 2.81 (d, J=14.1 Hz, 2H), 2.71-2.63 (m, 1H), 2.55 (s, 3H), 2.23 (s, 6H), 1.43 (s, 6H).

(46) Chiral resolution conditions: Chiral column: Chiralpak AD-3 100×4.6 mm I.D., 3 μm; mobile phase: 40% of methanol (0.05% DEA) in CO.sub.2; flow rate: 2.8 mL/min; column temperature: 40° C.

(47) The retention time of the compound of formula (I): 2.018 min.

EXAMPLE 2

Preparation of Amorphous Form of the Compound of Formula (I)

(48) The temperature was controlled at 25° C., and the crude compound of formula (I) (310.5 g) as pale yellow solid was added to a 3 L reaction flask. Then, heptane (1500 mL) was added. After completion of addition, the reaction was stirred at 25° C. for 2 h, then filtered. The filter cake was washed with n-heptane (500 mL), then filtered to give a crude product. The crude product was dried in a vacuum drying chamber, and subjected to XRPD detection for its form. The obtained final product was in an amorphous form.

(49) The Cu-Kα radiated XRPD pattern of the obtained final product is shown in FIG. 1; the DSC pattern is shown in FIG. 2; and the TGA pattern is shown in FIG. 3.

EXAMPLE 3

Preparation of Amorphous Form of the Compound of Formula (I)

(50) The compound of formula (I) (40.0 mg) was weighed and added to a 4.0 mL glass vial, and 150 μL of ethyl acetate was added to form a suspension. The suspension was stirred on a magnetic stirrer at 40° C. for 2 days, then the sample was centrifuged. The supernate was placed in a fuming cupboard until the solvent was dried off. Then, the resultant solid was dried in a vacuum drying chamber at 40° C. overnight. The resultant final product was amorphous form which was the same as that of Example 1.

EXAMPLE 4

Preparation of Amorphous Form of the Compound of Formula (I)

(51) The compound of formula (I) (39.9 mg) was weighed and added to a 4.0 mL glass vial, and 150 μL of tetrafuran was added to form a suspension. After the suspension was stirred at 40° C. on a magnetic stirrer for 2 days, the sample was centrifuged, and the supernate was placed in a fuming cupboard until the solvent was dried off. Then, the obtained solid was dried in a vacuum drying chamber at 40° C. overnight. The resultant final product was amorphous form which was the same as that of Example 1.

EXAMPLE 5

Solid Stability Test of Amorphous Form of the Compound of Formula (I) Under High Temperature and High Humidity Conditions

(52) Samples (each about 100 mg) of the amorphous form of the compound of formula (I) were weighed in duplicates, and placed on the bottom of a glass sample bottle to spread out into a thin layer. The mouth of the glass sample bottle was sealed with aluminum foil which was pierced to form some holes thereon to ensure that the samples could sufficiently contact with the ambient air. The samples were placed in a constant temperature and humidity chamber at 40° C./75% humidity. The samples stored under the above conditions were sampled and tested on Days 10, 30, 60, and 90. The test results were compared with the initial test results of Day 0. The HPLC analysis method is shown in Table 1. The test results are shown in Table 2 below:

(53) TABLE-US-00001 TABLE 1 HPLC Analysis Method Column: Waters Xbridge C18 (3.5 μm, 150 mm*4.6 mm) Flow Rate: 1.0 mL/min Detection 220 nm Wavelength: Column 35° C. Temperature: Injection Volume: 10 μL Runtime: 50 min Mobile Phase: Mobile phase A: 0.1% TFA-water Mobile phase B: acetonitrile Time (min) Mobile phase A (%) Mobile phase B (%) Gradient: 0.00 90 10 5.00 65 35 35.00 5 95 38.00 5 95 40.00 90 10 45.00 90 10 Diluent Water:acetonitrile = 50:50 (v/v) ProbeWash Water:acetonitrile = 50:50 (v/v)

(54) TABLE-US-00002 TABLE 2 Solid Stability Test of Amorphous form of the Compound of Formula (I) Time Point (Day) Appearance Purity (%) Total Impurity (%) 0 Off-white powder 97.37 2.63 10 Off-white powder 97.49 2.51 30 Off-white powder 97.31 2.69 60 Off-white powder 97.59 2.41 90 Off-white powder 97.28 2.72

(55) The above experimental data shows that the amorphous form of the compound of formula (I) provided in the present invention does not exhibit a significant change in content and impurities at high temperature and humidity, and has relatively high stability at high temperature and humidity.

EXAMPLE 6

Solid Physical Stability Test of Amorphous Form of the Compound of Formula (I) at High Humidity

(56) Samples (each about 100 mg) of the amorphous form of the compound of formula (I) were weighed in duplicates, and placed on the bottom of a glass sample bottle to spread out into a thin layer. The mouth of the glass sample bottle was sealed with aluminum foil which was pierced to form some holes thereon to ensure that the samples could sufficiently contact with the ambient air. The prepared samples were placed under the relative conditions of 25° C./92.5%, and detected for their physical stability on Day 10. Meanwhile, a sample (about 100 mg) of the amorphous form of the compound of formula (I) was separately weighed, placed on the bottom of the glass sample bottle which was sealed with a screw cap, and stored at −20° C. as control. On Day 10, all the samples were taken out and returned to room temperature. The samples were visually observed for their appearance changes, and detected by XRPD for their forms. By comparing the accelerated sample with the control sample, the solid physical stability of the compound of formula (I) was determined. Table 3 as below shows results of the solid physical stability experiments of the amorphous form of the compound of formula (I).

(57) TABLE-US-00003 TABLE 3 Solid Physical Stability Tests of the Amorphous Form of the Compound of Formula (I) at High Humidity Item Time Point 25° C./92.5% Relative Humidity (Open) Form Day 10 Amorphous Appearance Day 10 Off-white powder

(58) The above experimental data shows that the amorphous form of the compound of formula (I) provided in the present invention does not exhibit form and appearance changes at high humidity, and has relatively high stability at high humidity.

EXAMPLE 7

Stability Tests of the Amorphous Form of the Compound of Formula (I) in Various Solvents

(59) A plurality of samples of the amorphous form of the compound of formula (I) (each about 20 mg) were respectively added to 0.3-0.4 mL of a single or mixed solvent as listed in the table below, and stirred at 40° C. After being stirred for 2 days, if the sample was still in a state of a solution or close to a solution, it was filtered, followed by natural evaporation of solvent; and if the sample was still a suspension, the sample was centrifuged for collecting the precipitates, and the supernate was placed in a fuming cupboard until the solvent was dried off. Then, the precipitates and the solids obtained after solvent evaporation were placed in a vacuum drying oven at 40° C. overnight. The solids in all the samples were collected and detected by XRPD for their states. The results are shown in Table 4.

(60) TABLE-US-00004 TABLE 4 Stability tests of the amorphous form of the compound of formula (I) in various solvents No. Solvent Appearance Results 1 Methanol Solid precipitated after Amorphous evaporation of solvent 2 Ethanol Suspension (2 days)/Solid Both precipitated after amorphous evaporation of solvent. 3 Acetonitrile Suspension (2 days)/Solid Both precipitated after amorphous evaporation of solvent. 4 Acetone Suspension (2 days)/Solid Both precipitated after amorphous evaporation of solvent. 5 Ethyl acetate Suspension (2 days)/Solid Both precipitated after amorphous evaporation of solvent. 6 Tetrafuran Suspension (2 days)/Solid Both precipitated after amorphous evaporation of solvent. 7 1,4-dioxane Suspension (2 days)/Solid Both precipitated after amorphous evaporation of solvent. 8 Methanol:water = 3:1 Solid precipitated after Amorphous evaporation of solvent. 9 Ethanol:water = 3:1 Suspension (2 days)/Solid Both precipitated after amorphous evaporation of solvent. 10 Acetonitrile:water = 3:1 Suspension (2 days)/Solid Both precipitated after amorphous evaporation of solvent. 11 Acetone:water = 3:1 Suspension (2 days)/Solid Both precipitated after amorphous evaporation of solvent.

(61) The above experimental data shows that the amorphous form of the compound of formula (I) provided in the present invention does not exhibit any change of form, and has relatively high stability.

Bioactivity Test

EXPERIMENTAL EXAMPLE 8

In Vitro Evaluation

(62) Principles of in Vitro Test of PPAR Agonist Activity

(63) Nuclear Hormone Receptor (NHR) Test

(64) PathHunter's NHR protein interaction and nuclear transfer test are used to detect the activation ability of nuclear hormone receptor in a uniform, non-imaging experiment. This technology is called as enzyme fragment complementation (ETC), and is developed by DiscoverX.

(65) NHR protein test is based on the detection of the protein-protein interaction between a standard length of NHR protein in an activated state and a nuclear fusion protein containing a steroid receptor co-activating peptide (SRCP) region and one or more standard LXXLL acting sequences.

(66) NHR is labeled on the ProLink™ component of an EFC test system, and the SRCP region and the enzyme receptor component (EA) are fused and expressed in the nucleus. When bound to the ligand, the NHR will transfer to the nucleus and obtain the SRCP region in which a complementary effect is produced, thereby generating an equivalent amount of activated-galactosidase (-Gal), accompanied by the generation of chemical light signals. The benefits associated with this pathway include reduced incubation time of the compound, direct test of an NHR target, use of human NHR sequences with a standard length, and selection of some new classes of compounds based on the disruption of protein-protein interactions.

(67) The NHR NT test detected the transfer of an NHR between the cytoplasmic and nuclear compartments. The receptor was labeled on the ProLink™ component of the EFC test system, while the EA and nuclear sequence were fused, thereby limiting the expression of EA on the nucleus. Nucleus transfer leaded to the complementary effect with EA, thereby generating one equivalent of activated galactosidase (-Gal), accompanied by the generation of chemical light signals.

(68) Cell Processing:

(69) 1. PathHunter NHR cell strains were spread from a frozen stock in accordance with standard operations.

(70) 2. Cells were seeded onto a 384-well white cell plate of 20 uL/well, and incubated at 37° C. for an appropriate time prior to test. The culture medium contained activated carbon dextran with filtered serum to reduce the level of hormone expression.

(71) Experiments of Agonist Mode:

(72) 1. As for the measurement of an agonist activity, it is required to incubate the cells with the compound to induce a response.

(73) 2. The compound was formulated with a buffer solution to a stock solution, which was 5×diluted.

(74) 3. 5 uL of 5×diluted solution of the compound was added to the cells, and incubated at 37° C. (or room temperature) for 3-16 h, ensuring that the final concentration of the media was 1%).

(75) Experiments of Inhibitor Mode:

(76) 1. As for the measurement of inhibitory activity, it is required to incubate the cells with the anti-agonist, and then challenged with an agonist at the EC80 concentration.

(77) 2. The compound was formulated with a buffer solution to a stock solution, which was 5×diluted.

(78) 3. 5 uL of 5×diluted solution of the compound was added to the cells, and incubated at 37° C. (or room temperature) for 60 min, ensuring that the final concentration of the media was 1%).

(79) 4. 5 uL of EC80 agonist which was 6×diluted with a buffer solution was added to the cells, and incubated at 37° C. (or room temperature) for 3-16 h.

(80) Signal Detection:

(81) 1. The experimental signals were generated by 12.5 uL or 15 uL (50% v/v) of the PathHunter test reagent mixture which was once added, then needed to be incubated at room temperature for 1 h.

(82) 2. The chemiluminescence signals generated by the microplate were to be detected by a PerkinElmer Envision instrument.

(83) Data Analysis:

(84) 1. The activity of the compound was analyzed by CIBS data analysis software (ChemInnovation, CA).

(85) 2. For experiments of agonist mode, the percentage activity is calculated by the following formula:
% activity=100%×(the mean RLU of the compound to be tested−the mean background RLU of the medium)/(the mean of maximum control of the ligand−the mean background RLU of the medium)

(86) 3. For experiments of antagonist mode, the percentage activity is calculated by the following formula:
% Inhibition=100%×(1−(the mean RLU of the compound to be tested−the mean background RLU of the medium)/(the mean RLU of the EC80 control compound−the mean background RLU of the medium))

(87) 4. It should be noted that the response of the ligand causes a decrease in the activity of the receptor (have inverse agonist with a continuous active target). The activity of these inverse agonist is calculated by the following formula:
% Inverse agonist activity=100%×((the mean background RLU of the medium−the mean RLU of the compound to be tested)/(the mean background RLU of the medium−the mean RLU of the maximum control of ligand))

(88) The experimental results are shown in Table 5:

(89) TABLE-US-00005 TABLE 5 In Vitro Screening Experimental Results of the Compound of the Present Invention PPAR Alpha PPAR Delta PPAR Gamma Maximum Maximum Maximum EC.sub.50 stimulus-response EC.sub.50 stimulus-response EC.sub.50 stimulus-response Compound nM value % nM value % nM value % GW7647 A 100% / / / / L-165,041 / / A 100% / / Troglitazone / / / / E 100% GFT-505 E I C II E II (Elafibranor) Compound A I A II E I of formula (I) Note 1: Based on 100% of the maximum stimulus-response value of in vitro platforms of the known PPAR α agonist GW7647, PPAR δ agonist L-165,041, and PPAR γ agonist Troglitazone, the maximum response values of other compounds are compared with the maximum stimulus-response value to give the corresponding maximum value of stimulus-response. It is generally considered that a compound with the maximum stimulus-response value greater than 80% is a full agonist, a compound with the maximum stimulus-response value greater than 50% and less than 80% is a partial agonist, and a compound with the maximum stimulus-response value less than 50% has an incomplete agonistic effect. Note 2: A   100 nM; 100 nM < B   150 nM; 150 nM < C   200 nM; 200 nM < D   250 nM; E > 250 nM. Note 3: 100%   I    80%; 80% > II   50%; III < 50%.

(90) The above experimental data shows that the compound of formula (I) has a significant activation effect on PPAR Alpha and Delta receptors, and a selective activation effect on a PPAR Gamma receptor.

(91) Based on the above evaluation experiments on the stability and activity of the amorphous form of the compound of formula (I), it can be seen that: unlike the conventional knowledge of amorphous drugs in the field, the amorphous form of the compound of formula (I) provided in the present invention form has a high stability in terms of chemical properties and physical forms. Also, the amorphous form of the compound of formula (I) produces an obvious inhibition effect on cytokines associated with PPAR-related pathways, and has significant effects in improving liver injury, NAS Score and liver fibrosis. It can be seen that the amorphous form of the compound of formula (I) has good medicinal prospects.