Amide compound, preparation method and uses thereof

Abstract

Disclosed are amide compounds, preparation method and uses thereof, specifically, the compounds represented by formula I or pharmaceutically acceptable salts, wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, Q, X and n are defined as in the description. Also disclosed are a method for preparing the compounds of formula I, a composition containing the compounds, and the uses of the same in the preparation of medicaments for regulating blood lipid and/or preventing gallstone. The compounds of formula I disclosed in the present invention have stability in vitro, good solubility in the pharmaceutical organic solvents and favorable bioavailability in animals.

Claims

1. A compound and pharmaceutically acceptable salts thereof, wherein the compound is selected from: ##STR00011## ##STR00012## ##STR00013## ##STR00014## ##STR00015##

2. A pharmaceutical composition, comprising any one compound of claim 1, and a pharmaceutically acceptable additive.

3. The pharmaceutical composition according to the claim 2, in the form of tablet, pill, powder, liquid, suspension, emulsion, granule, capsule, suppository or ampoule.

Description

DETAILED DESCRIPTION OF THE INVENTION

Experiment 1

Preparation of Compound 01

(1) 1 g of 2-(4-(2-(4-chlorobenzamido)ethyl)phenoxy)-2-methylpropanoic acid, 0.57 g of dicyclohexylcarbodiimide, 0.5 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into a 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (820 mg). MS (ESI): 495 (M+H.sup.+). .sup.1H-NMR: 7.720-7.698 (2H, d), 7.629 (1H, s), 7.605-7.584 (2H, d), 7.281 (1H, s), 7.232-7.210 (2H, d), 7.158 (1H, s), 7.011-6.99 (2H, d), 6.810-6.751 (4H, m), 4.559-4.547 (2H, s), 3.484-3.473 (2H, m), 2.764-2.729 (2H, m), 1.381 (6H, s).

Experiment 2

Preparation of Compound 02

(2) 2 g of 2-(4-(2-(4-chlorobenzamido)ethyl)phenoxy)-2-methylpropanoic acid, 1.2 g of dicyclohexylcarbodiimide, 0.5 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (1.3 g). MS (ESI): 838(M+H.sup.+).

Experiment 3

Preparation of Compound 03

(3) 1 g of 2-methyl-2-(4-(4-chlorobenzoyl)phenoxy)propanoic acid, 0.57 g of dicyclohexylcarbodiimide, 0.5 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (820 mg). MS (ESI): 452 (M+H.sup.+). .sup.1H-NMR: 7.8-7.85 (2H, d), 7.7-7.8 (4H, m), 7.6(1H, m), 7.42-7.53 (2H, d), 7.04-7.13 (2H, d), 6.9-7, 1 (214, d), 6.84 (1H, s), 4.7 (2H, d), 1.65 (6H, s).

Experiment 4

Preparation of Compound 04

(4) 2 g of 2-methyl-2-(4-(4-chlorobenzoyl)phenoxy)propanoic acid, 1.2 g of dicyclohexylcarbodiimide, 0.5 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (1.32 g). MS (ESI): 752(M+H.sup.+).

Experiment 5

Preparation of Compound 05

(5) 1 g of 2,2-dimethyl-5-(2,5-dimethylphenoxy)pentanoic acid, 0.57 g of dicyclohexylcarbodiimide, 0.5 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (720 mg). MS (ESI): 384 (M+H.sup.+). .sup.1H-NMR: 8.017 (1H, s), 7.815-7.794 (1H, d), 6.992-6.914 (4H, m), 6.651-6.595 (3H, m), 4.625-4.614 (2H, d), 3.939-3.910 (2H, m), 2.954 (3H, s), 2.887 (3H, s), 1.778-1.732 (4H, m), 1.272 (6H, s).

Experiment 6

Preparation of Compound 06

(6) 2 g of 2,2-dimethyl-5-(2,5-dimethylphenoxy)pentanoic acid, 1.2 g of dicyclohexylcarbodiimide, 0.5 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (1.2 g). MS (ESI): 616 (M+H.sup.+).

Experiment 7

Preparation of Compound 07

(7) 1 g of 2-(4-(2,2-dichlorocyclopropyl)phenoxy)-2-methylpropanoic acid, 0.57 g of dicyclohexylcarbodiimide, 0.5 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (760 mg). MS (ESI): 422 (M+H.sup.+). .sup.1H-NMR: 7.899-7.877 (1H, d), 7.720 (1H, s), 7.167-7.147 (4H, m), 6.992-6.892 (4H, m), 4.732-4.711 (2H, d), 1.953 (1H, m), 1.554 (6H, s), 1.225 (2H, m).

Experiment 8

Preparation of Compound 08

(8) 2 g of 2-(4-(2,2-dichlorocyclopropyl)phenoxy)-2-methylpropanoic acid, 1.2 g of dicyclohexylcarbodiimide, 0.6 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (1.15 g). MS (ESI): 694 (M+H.sup.+). .sup.1H-NMR: 8.085-8.064 (1H, d), 7.987-7.967 (2H, d), 7.169-7.026 (6H, m), 6.982-6.916 (4H, m), 4.764-4.753 (2H, d), 2.866-2.820 (2H, m), 1.755 (12H, m), 1.599-1.437 (4H, m).

Experiment 9

Preparation of Compound 09

(9) 2 g of 2-(4-chlorophenoxy)-2-methylpropanoic acid, 2.11 g of dicyclohexylcarbodiimide, 1.4 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (2.89 g). MS (ESI): 334 (M+H.sup.+).

Experiment 10

Preparation of Compound 10

(10) 4 g of 2-(4-chlorophenoxy)-2-methylpropanoic acid, 4.45 g of dicyclohexylcarbodiimide, 1.4 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (5.11 g). MS (ESI): 530 (M+H.sup.+).

Experiment 11

Preparation of Compound 11

(11) 2 g of 2,2-(cyclohexylidene-bis(p-phenyleneoxy))bis(2-methylbutyric acid), 1.2 g of dicyclohexylcarbodiimide, 0.6 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (2.16 g). MS (ESI): 588 (M+H.sup.+).

Experiment 12

Preparation of Compound 12

(12) 4 g of 2,2-(cyclohexylidene-bis(p-phenyleneoxy))bis(2-methylbutyric acid), 2.48 g of dicyclohexylcarbodiimide, 0.6 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (3.56 g). MS (ESI): 707 (M+H.sup.+).

Experiment 13

Preparation of Compound 13

(13) 2 g of 2,2-diethyl-5-(3,5-dimethylphenoxy)pentanoic acid, 1.9 g of dicyclohexylcarbodiimide, 1.1 g of 2-amino-1-(3-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through recrystallization in ethyl alcohol, to give the title compound (2.88 g). MS (ESI): 412 (M+H.sup.+).

Experiment 14

Preparation of Compound 14

(14) 2 g of 2,2-dimethyl-5-(3-methyl-4-methoxyphenoxy)pentanoic acid, 1.8 g of dicyclohexylcarbodiimide, 1.2 g of 2-amino-1-(3-methyl-4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through recrystallization in ethyl alcohol, to give the title compound (2.78 g). MS (EST): 414 (M+H.sup.+).

Experiment 15

Preparation of Compound 15

(15) 2 g of 2-methyl-2-isopropyl-5-(3-chloro,6-methylphenoxy)pentanoic acid, 1.7 g of dicyclohexylcarbodiimide, 1.1 g of 3-amino-1-(4-hydroxyphenyl)propyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the title compound (2.03 g). MS (ESI): 461 (M+H.sup.+).

Experiment 16

Preparation of Compound 16

(16) 2 g of 2-ethyl-4-(2,5-dimethylphenoxy)butyric acid, 1.7 g of dicyclohexylcarbodiimide, 1.1 g of 2-methyl-2-amino-1-(3-hydroxy-5-methoxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the title compound (2.03 g). MS (EST): 412 (M+H.sup.+).

Experiment 17

Preparation of Compound 17

(17) 2 g of 2,2-dimethyl-5-(2,5-dimethylphenoxy)pentanoic acid, 1.7 g of dicyclohexylcarbodiimide, 1.1 g of 2-methyl-2-amino-1-(3-chloro-4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through column chromatography; the product was refluxed for 30 minutes in 5 g butyric acid and ammonia gas, washed with water and 1 mold, sodium hydroxide in water, and the crude product obtained was purified through chromatography on silica gel column, to give the title compound (2.03 g). MS (EST): 433 (M+H.sup.+).

Experiment 18

Preparation of Compound 18

(18) 2 g of 2,2-dimethyl-5-(2,5-dimethylphenoxy)pentanoic acid, 1.5 g of oxalyl chloride and 10 ml of dichloromethane were placed into the 50 ml single-neck flask for about 1 hour until no gas evolved, and were evaporated to dryness, to give the light yellow oil for use. 1.1 g of (4-aminocyclopropyl)(4-hydroxyphenyl)methylthioketone and 60 ml dichloromethane were placed into the 100 ml single-neck flask, and the oil was dropped slowly into it, reacted for 1 hour at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the title compound (1.1 g). MS (ESI): 468 (M+H.sup.+).

Experiment 19

Preparation of Compound 19

(19) 1 g of 2,2-dimethyl-5-(2,5-dimethylphenoxy)pentanoic acid, 0.57 g of dicyclohexylcarbodiimide, 0.5 g of 2-amino-1-(3,5-dihydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (725 mg). MS (ESI): 400 (M+H.sup.+).

Experiment 20

Preparation of Compound 20

(20) 1 g of 2-(4-chlorophenyl)-2-cyclopropylpropanoic acid, 0.57 g of dicyclohexylcarbodiimide, 0.5 g of 2-amino-1-(4-hydroxyphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (500 mg). MS (ESI): 374 (M+H.sup.+).

Experiment 21

Preparation of Compound 21

(21) 1 g of 2,2-dimethyl-5-(2,5-dimethylphenoxy)pentanoic acid, 0.57 g of dicyclohexylcarbodiimide, 0.5 g of 2-amino-1-(4-hydroxymethylphenyl)ethyl ketone and 60 ml of dichloromethane were placed into the 100 ml single-neck flask, reacted for 4 hours at room temperature, evaporated to dryness, then purified through chromatography on silica gel column, to give the target compound (723 mg). MS (ESI): 398 (M+H.sup.+).

Experiment 22

Materials and Methods for Screening with the Compounds Prepared in Experiment 1 to 21 in the Hyperlipemia Model of SD Rat

(22) 1. Reagents

(23) Simvastatin tablet (20 mg*7, Hangzhou MSD Pharmaceutical Co., Ltd. Lot: 20090115);

(24) Ursodesoxycholic acid (Bio Basic Inc., Lot: YY0201B207Y);

(25) Lard oil: commercially available;

(26) Cholesterol (Shanghai Lanji Sci-tech Development Co., Ltd. Lot: 090720);

(27) Propylthiouracil (Shanghai Lanji Sci-tech Development Co., Ltd. Lot: 090505);

(28) Deoxycholic acid (Shanghai Lanji Sci-tech Development Co., Ltd. Lot: 090615);

(29) Tween 80 (Sinopharm Chemical Reagent Co., Ltd. CP, Lot: F20090507);

(30) 1,2-propylene glycol (Sinopharm Chemical Reagent Co., Ltd. AR, Lot: T20070125);

(31) 2. Animals

(32) SD rats, male, 150-180 g, supplied by Shanghai Slaccas Laboratory Animal Company Limited.

(33) 3. Instruments

(34) YP2001N electronic balance, from Shanghai Precision Scientific Instrument Co., Ltd.

(35) Hitachi Automatic Biochemical Analyzer 7080.

(36) 4. Methods

(37) Preparation of fat emulsion: 500 g lard oil was weighed and placed into a vessel, heated for melting, and upon heating up to the temperature of 100 C., 200 g cholesterol was added and fully dissolved, then 20 g propylthiouracil was added. After full stirring and dissolving, 500 ml Tween 80 was added to give an oil phase. Meanwhile, 600 ml distilled water and 400 ml 1,2-propylene glycol were heated to 60 C. in water bath, and then 40 g sodium deoxycholate was added with stirring, until full dissolution, to give an aqueous phase. The aqueous phase was added into the oil phase and fully and uniformly mixed, to prepare the fat emulsion.

(38) Formulation of the solution for the compounds: an appropriate amount of compounds was triturated with appropriate amount of Tween 80 for homogeneous dispersion, and then sufficient CMC-Na solution was added and fully triturated, suspended to give the solution for the compounds.

(39) The animals were fed for acclimation for 3 days, with 8 animals as blank control (Control) according to weight, and the rest was subject to intragastric administration of fat emulsion at 1 ml/100 g weight at A.M. 9:00-11:00 daily for continuous 2 weeks; the animals were fasted for 12 hours, and had 1 ml blood sampled at orbital cavity, which was measured with the Hitachi Automatic Biochemical Analyzer 7080 for serum cholesterol (CHO), triglyceride (TO), low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C), with the animals having CHO of 4-7 mmol/L for experiment.

(40) The animals dosed with fat emulsion for 2 weeks were divided into model group (Model), simvastatin group (Sim, 10 mg/kg) and compound group (40 mg/kg) according to weight, and continued to receive intragastric administration with fat emulsion; at the same time, the dosed group was administered with respective amount of drug, and the model group was administered with equal volume of solvent. Fat emulsion was dosed by gavage at A.M., and drug was dosed at P.M. The animals were weighed at Monday, and were observed. The animals were dosed for continuous 21 days, and fasted for 12 hours, with 1 ml blood sampling at orbital cavity. The liver was exposed and observed for pathologic condition; after slicing and weighing, it was fixed into the 4% formaldehyde solution for pathologic examination.

(41) The Hitachi Automatic Biochemical Analyzer 7080 was used to measure serum cholesterol (CHO), triglyceride (TG), low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C).

(42) 5. Data Statistics

(43) Laboratory data were expressed as XSD (Standard Deviation), and comparison between groups was carried out by t-testing.

(44) 6. Results: Effect of the Compounds on Blood Lipid in Animal

(45) TABLE-US-00001 TABLE 1 blood lipid level in the dosed rats (X SD, mmol/L) Dose CHO1 TG HDL-C LDL-C Grouping (mg/kg) Concentration Concentration Concentration Concentration Blank (healthy) 1.82 0.34 0.44 0.13 1.22 0.25 0.36 0.08 Model 19.17 .315 1.95 0.21 1.82 0.33 18.01 2.76 Simvastatin 10 12.52 1.34 1.41 0.16 1.52 0.32 11.92 1.44 Compound 01 40 14.84 1.06 0.66 0.13 1.56 0.25 13.90 1.23 Compound 02 40 17.29 3.23 0.68 0.18 1.68 0.38 16.01 2.99 Compound 03 40 12.63 2.77 0.99 0.18 1.54 0.20 11.11 2.45 Compound 04 40 10.02 1.80 1.15 0.12 1.82 0.19 9.84 1.37 Compound 05 40 15.30 2.45 1.31 0.15 1.54 0.24 14.38 2.12 Compound 06 40 16.37 2.26 1.53 0.16 1.49 0.29 15.78 2.00 Compound 07 40 14.99 1.45 1.39 0.16 1.74 0.46 13.90 1.21 Compound 08 40 12.92 2.10 0.83 0.13 2.12 0.27 11.41 1.90 Compound 09 40 13.64 2.14 0.95 0.14 2.18 0.22 11.64 2.00 Compound 10 40 9.73 1.79 0.94 0.15 2.66 0.24 8.27 1.46 Compound 11 40 12.18 3.12 1.13 0.14 1.79 0.34 11.37 3.66 Compound 12 40 20.17 1.27 0.75 0.25 1.895 0.39 19.31 1.52 Compound 13 40 20.17 1.27 0.75 0.25 1.895 0.39 19.31 1.52 Compound 14 40 20.66 3.23 0.99 0.28 2.25 0.22 20.78 3.46 Compound 15 40 15.00 2.75 1.34 0.48 1.89 0.26 14.04 3.12 Compound 16 40 9.48 1.27 0.78 0.25 1.32 0.39 7.13 1.52 Compound 17 40 10.54 3.23 0.88 0.28 1.82 0.22 8.13 3.46 Compound 18 40 15.04 2.73 1.25 0.33 1.56 0.51 13.47 3.68 Compound 19 40 11.12 1.01 0.94 0.06 1.36 0.66 10.76 1.25 Compound 20 40 19.88 0.84 1.72 0.26 2.63 0.57 18.48 0.43 Compound 21 40 14.42 2.75 2.00 0.52 1.69 0.26 12.15 3.12

(46) From the results, the compound 01 to compound 21 substantially have a certain blood lipid-lowering action, while enable reduction of cholesterol, triglyceride and low-density lipoprotein.

Experiment 23

Screening with the Compounds Prepared in Experiment 1 to 21 in the Cholelithiasis Model of Golden Hamster

(47) Materials and Methods

(48) 1. Reagents:

(49) Simvastatin tablet (20 mg*7, Hangzhou MSD Pharmaceutical Co., Ltd. Lot: 20090115)

(50) Ursodesoxycholic acid (Bio Basic Inc., Lot: YY0201B207Y)

(51) 2. Animals:

(52) 68 male golden hamsters, 50-60 g, supplied by Shanghai Slaccas Laboratory Animal Company Limited.

(53) 3. Instruments:

(54) YP2001N electronic balance, from Shanghai Precision Scientific Instrument Co., Ltd.

(55) Hitachi Automatic Biochemical Analyzer 7080.

(56) 4. Methods:

(57) The animals were fed for acclimation for 3 days, with 8 animals as blank control (Control) according to weight. The control was dosed with normal rat feedstuff, and the rest was dosed with feedstuff leading to cholelithiasis (sucrose 32.1%, cheese 64.2%, cholesterol 0.2%, salt 3%, vitamin B1 20.1%, concentrated fish liver oil 0.4%). The animals were fed for acclimation for another 7 days, and divided into model group (Model), simvastatin group (Sim, 10 mg/kg), Ursodesoxycholic acid group (UDCA, 40 mg/kg), and compound group, n=6/group.

(58) The Sim group was administered with simvastatin 10 mg/kg, the UDCA group was administered with ursodesoxycholic acid 40 mg/kg, the dosing group was administered with 40 mg/kg drug, with administration all starting at the day of grouping, and at P.M. 2:00-3:00 daily. The animals were weighed at Monday, and observed for hair color, stool, and variation of activation level.

(59) The animals were dosed for continuous 45 days and then fasted for 12 hours; were anesthetized with 30 mg/kg sodium secobarbital via intraperitoneal injection; had 1 ml blood sampled via abdominal aorta; had gallbladder exposed, with an orifice on the gallbladder being clamped by the ophthalmological pliers and bile in the gallbladder being sucked with 1 ml syringe.

(60) 5. Biochemical Measurement of Bile:

(61) The Hitachi Automatic Biochemical Analyzer 7080 was used to measure cholesterol (CHO), total total bilirubin (TBIL), total bile acid (TBA) and total protein (TP) in bile.

(62) 6. Data Statistics:

(63) Laboratory data was expressed as XSD, and comparison of measurement data between groups was carried out by t-testing, with non-parametric testing for enumeration data.

(64) TABLE-US-00002 7. Laboratory results: Table 2. level of components in golden hamster bile Total total Number Cholesterol bilirubin Total bile acid of (CHO) (TBIL) (TBA) Total protein Group animals (mmol/L) (mol/L) (mmol/L) (TP) (g/L) Blank (healthy) 8 0.90 0.21 68.1 24.2 17.44 10.12 25.83 11.24 Model 6 1.16 0.72 73.8 34.6 10.12 5.66 37.89 17.23 Simvastatin (10 mg/kg) 6 1.07 0.63 79.2 34.2 7.68 3.31 35.52 18.04 Ursodesoxycholic 6 0.98 0.47 89.4 45.3 12.23 3.04 28.63 10.37 acid (40 mg/kg) Compound 11 6 0.82 0.58** 48.9 22.10 17.46 7.77** 28.66 8.48 (80 mg/kg) Compound 16 6 0.97 0.62** 55.0 29.38 19.74 12.14** 40.70 26.17 (40 mg/kg) Compound 20 6 0.92 0.66** 75.92 .072 16.80 9.62** 32.94 19.18 (20 mg/kg) **refers to a very significant statistic difference.

(65) From results of bile analysis, the contents of bile acid in each of the compound groups were significantly higher than that in the model group, and were better than that of simvastatin and ursodesoxycholic acid in effect of increased bile acid.

Experiment 24

Testing for Acute Toxicity in Mice

(66) The compounds was dissolved into an appropriate amount of Tween-80, and suspended and dispersed uniformly into a given amount of CMC-Na solution, which was administered orally and by gavage at a dose of 5 g/kg, with no toxicity associated with administration being observed, and no death of animals occurred during observation of continuous 14 days. From the results, the sample Compound 01, Compound 02, Compound 03, Compound 04, Compound 05, Compound 06, Compound 07, Compound 08, Compound 09, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19, Compound 20 and Compound 21 were safe.

Experiment 25

Preparation of Tablets

(67) TABLE-US-00003 Formulation Dosage Compound 11 200 mg Microcrystalline cellulose 200 mg Crosslinked Polyvinylpyrrolidone 20 mg Pregelatinized starch 50 mg Magnesium stearate 5 mg

(68) Preparation method: according to the above formulation, the comminuted and sieved compound 11, microcrystalline cellulose, pre-gelatinized starch and cross-linked polyvinylpyrrolidone were mixed uniformly, and then mixed with 5% ethyl alcohol solution, granulated, dried, and then the granules obtained was mixed with the lubricant and pressed into tablets; wherein the compound 11 was comminuted and sieved through the 60-mesh sieve, the microcrystalline cellulose, pregelatinized starch and crosslinked polyvinylpyrrolidone were comminuted and sieved through the 80-mesh sieve, the granulation gave the particles with particle size of 20 mesh, and the drying was carried out at the temperature of preferable 90 C., and moisture was controlled within 3% by mass.

Experiment 26

Preparation of Capsules

(69) TABLE-US-00004 Formulation Dosage Compound 16 200 mg Filler Lactose 250 mg Lubricant Magnesium stearate 2 mg

(70) Preparation method: according to the above formulation, the medicament was mixed uniformly with each of the raw materials as adjuvant, and filled into the capsule shells.

Experiment 27

Preparation of Injection

(71) TABLE-US-00005 Raw materials mg/mL Compound 02 100 Suspending agent Gum tragacanth 1.0 Wetting agent Isopropanol 1.0 Water for injection Ad. 1 mL

(72) Preparation method: according to the above formulation, a mortar was used to grind the compound 02 or salt thereof and the wetting agent for uniform mixing, and then mixed uniformly with the suspending agent, preservative and water for injection, and then regrinded, wherein the grinding provided the particle size of 0.5 m.