Compound for treating metabolic diseases and preparation method and use thereof

Abstract

Provided are a compound for treating metabolic diseases having the structure as shown in formula (I) or formula (II), or a racemate, stereoisomer, geometric isomer, tautomer, solvate, hydrate, metabolite, pharmaceutically acceptable salt or prodrug thereof. The compound is an activator of FXR and/or a TGR5 receptor, and thus has the activity of activating FXR and/or a TGR5 receptor, and can be used in the preparation of drugs for treating chronic liver diseases, metabolic diseases or portal hypertension.

Claims

1. A compound as shown in Formula (I), or a racemate, a stereoisomer, a geometric isomer, a tautomer, or a pharmaceutically acceptable salt thereof: ##STR00061## wherein, R.sub.1 is hydrogen, substituted or unsubstituted alkyl or halogen; each of the R.sub.2 is independently selected from the group consisting of substituted or unsubstituted alkyl, halogen, hydroxyl, nitro, cyano, sulphonic acid group and carboxyl; m is 0, 1, 2, 3 or 4; each of the R.sub.3 is independently selected from the group consisting of substituted or unsubstituted alkyl, halogen, hydroxyl and aryl; and n is 0, 1, 2, 3 or 4.

2. The compound according to claim 1, wherein R.sub.1 is hydrogen, substituted or unsubstituted C.sub.1-5 alkyl or halogen.

3. The compound according to claim 1, wherein R.sub.1 is hydrogen, fluorine, chlorine, bromine, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted propyl, or substituted or unsubstituted isopropyl.

4. The compound according to claim 1, wherein each of the R.sub.2 is independently selected from the group consisting of fluorine, chlorine, bromine, nitro, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, and substituted or unsubstituted isopropyl; m is 0 or 1; each of the R.sub.3 is independently selected from the group consisting of fluorine, chlorine, bromine, substituted or unsubstituted methyl, and substituted or unsubstituted ethyl; and n is 0 or 1.

5. The compound according to claim 1, which is selected from the group consisting of: ##STR00062## ##STR00063## or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof.

6. A method of producing the compound according to claim 1, comprising: reacting Compound (10) with Compound (16) to obtain a compound as shown in Formula (I), ##STR00064## wherein, R.sub.1 is hydrogen, substituted or unsubstituted alkyl, and halogen; each of the R.sub.2 is independently selected from the group consisting of substituted or unsubstituted alkyl, halogen, hydroxyl, nitro, sulfonyl hydroxide and carboxyl; m is 0, 1, 2, 3 or 4; each of the R.sub.3 is independently selected from the group consisting of substituted or unsubstituted alkyl, halogen, hydroxyl and aryl; and n is 0, 1, 2, 3 or 4.

7. The method according to claim 6, wherein the reaction is performed under the catalysis of tert-butylmagnesium chloride and with 1,4-dioxane as the solvent.

8. A method for activating receptor FXR and/or TGR5 comprising administering to a subject or sample in need thereof a therapeutically effective amount of the compound according to claim 1.

9. A method for treating or ameliorating receptor FXR and/or TGR5 mediated disease, comprising administering to a subject in need thereof a therapeutically effective amount of the compound according to claim 1.

10. The method according to claim 9, wherein the receptor FXR and/or TGR5 mediated disease is selected from chronic liver disease, metabolic disease and portal hypertension.

11. The method according to claim 10, wherein the chronic liver disease is selected from primary biliary cholestatic cirrhosis, primary sclerosing cholangitis, liver fibrosis-related disease, drug-induced cholestasis, progressive familial intrahepatic cholestasis, cholestasis of pregnancy, alcoholic liver disease and non-alcoholic fatty liver disease; the portal hypertension is a portal hypertension with increased portal pressure caused by liver fibrosis, cirrhosis, splenomegaly or another reason; and the metabolic disease is selected from hypercholesterolemia, dyslipidemia, cholesterol gallstones and hypertriglyceridemia.

12. A compound as shown in Formula (II), or a racemate, a stereoisomer, a geometric isomer, a tautomer, or a pharmaceutically acceptable salt thereof: ##STR00065## wherein, R.sub.4 is hydrogen, substituted or unsubstituted alkyl or halogen; each of the R.sub.5 is independently selected from the group consisting of substituted or unsubstituted alkyl, halogen, hydroxyl, cyano, nitro, sulphonic acid group, and carboxyl; R.sub.6 is selected from the group consisting of substituted or unsubstituted C.sub.1-5 alkyl, aryl, heteroaryl, and cyclohexyl; x is 0, 1, 2, 3 or 4; and y is 0 or 1.

13. The compound according to claim 12, wherein R.sub.4 is hydrogen, substituted or unsubstituted C.sub.1-6 alkyl or halogen.

14. The compound according to claim 12, wherein R.sub.4 is hydrogen, fluorine, chlorine, bromine, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted propyl or substituted or unsubstituted isopropyl; each of the R.sub.5 is independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, hydroxyl, cyano, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, and substituted or unsubstituted isopropyl; and R.sub.6 is selected from the group consisting of substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted propyl, substituted or unsubstituted isopropyl, phenyl, pyridyl and cyclohexyl.

15. The compound according to claim 12, which is selected from the group consisting of: ##STR00066## ##STR00067## or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof.

16. The compound according to claim 1, wherein each of the R.sub.2 is independently cyano; m is 0 or 1; each of the R.sub.3 is independently selected from the group consisting of fluorine, chlorine, bromine, substituted or unsubstituted methyl, and substituted or unsubstituted ethyl; and n is 0 or 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a mass spectrum of Compound 14 in Example 1;

(2) FIG. 2 is a mass spectrum of Compound 16 in Example 1;

(3) FIG. 3 is a proton nuclear magnetic resonance spectrum of Compound 16 in Example 1;

(4) FIG. 4 is a mass spectrum of Compound 1 in Example 1;

(5) FIG. 5 is a proton nuclear magnetic resonance spectrum of Compound 1 in Example 1;

(6) FIG. 6 is the bile discharge amount at different time points in different groups of rats;

(7) FIG. 7 is the effect on portal vein pressure in rats of each dose group of tested Compound 1-H;

(8) FIG. 8 is the average percentage change of portal vein pressure in rats of each dose group of tested Compound 1-H;

(9) FIG. 9 is the appearance of liver and spleen of rats in each dose group of tested Compound 1-H;

(10) FIG. 10 is serologic indicators of rats in each dose group of test compound 1-H; and

(11) FIG. 11 is Masson's trichrome stain and HE stain of liver pathological sections of rats in each dose group of tested Compound 1-H.

DETAILED DESCRIPTION

(12) In order to further illustrate the present disclosure, the compounds for treating metabolic diseases and preparation method and use thereof provided by the present disclosure are described in detail below with reference to the examples. The proportions of the solvents in the following examples are volume ratios unless otherwise stated. For example, ethyl acetate:petroleum ether=1:5 represents that the volume ratio of ethyl acetate to petroleum ether is 1:5.

Example 1 Synthesis of Compound 1 (i.e. Compound 1-H)

(13) 1. Synthesis of Compound 2

(14) ##STR00014##

(15) 300 mL of tetrahydrofuran was added into a 1000 mL round-bottom flask, then 30 g of chenodeoxycholic acid (as shown in Formula a) (76.42 mmol) was added, and 3 mL of perchloric acid was added, and 100 mL of formic acid was added with stirring at room temperature. The temperature was controlled to be 50° C. and the reaction was carried out under stirring for 15 hours. The solvent was removed by concentration under reduced pressure. The residue was diluted with 4 L of water, the solid was collected by filtration, and 2 L of dichloromethane was added to dissolve the solid. The solution was dried with anhydrous sodium sulfate, filtered and then concentrated under reduced pressure. The crude product was dispersed and stirred with 4 L of petroleum ether, and a white solid was collected by filtration to obtain 32.8 g of Compound 2, with a yield of 98%.

(16) 2. Synthesis of Compound 3

(17) ##STR00015##

(18) Under protection of argon, 100 g of Compound 2 (222.92 mmol), 3 L of carbon tetrachloride, 199.1 g of lead acetate (446.41 mmol), and 113.64 g of iodine (447.40 mmol) were sequentially added into a 5 L round-bottom flask. The obtained reaction solution was stirred at 20° C. for 24 hours under irradiation of a tungsten lamp (250 watts). 3 L of saturated sodium thiosulfate was added to quench the reaction. The solid was removed by filtration. The filtrate was extracted twice with dichloromethane, 3 L each time. The organic phases were combined, 2 L of saturated sodium thiosulfate were added respectively, washed twice, and then washed once with 2 L of water and once with 2 L of saturated aqueous solution of sodium chloride. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. 110 g of light yellow solid Compound 3 was obtained, with a yield of 93%.

(19) 3. Synthesis of Compound 4

(20) ##STR00016##

(21) Compound 3 (250 g, 471.27 mmol), 150.9 g of potassium benzoate (941.88 mmol), 2 L of N,N-dimethylformamide were added sequentially into a 3 L round-bottom flask at room temperature. The reaction solution was stirred at 80° C. for 15 hours and concentrated under reduced pressure. The residue was dissolved in 3 L of dichloromethane. The solution was washed twice with 1.6 L of water, and then once with 1.6 L of saturated sodium thiosulfate and once with 1.6 L of saturated sodium chloride sequentially. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. 310 g of brown Compound 4 was obtained and used for the next reaction without purification.

(22) 4. Synthesis of Compound 5

(23) ##STR00017##

(24) 1 L of methanol was added into a 2 L round-bottom flask, and 35 g of crude Compound 4 was dissolved in the methanol (1000 mL), then 20 mL of concentrated hydrochloric acid was added. The reaction solution was stirred at 20° C. for 24 hours, and concentrated under reduced pressure. The residue was dissolved in 3 L of dichloromethane. The solution was washed twice with 800 mL of a saturated aqueous solution of sodium bicarbonate and once with 800 mL of a saturated aqueous solution of sodium chloride. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (dichloromethane:methanol=80:1). 22.8 g of Compound 5 was obtained (a yield of 60%, a yellow solid).

(25) 5. Synthesis of Compound 6

(26) ##STR00018##

(27) 1 L of chloroform was added into a 2 L round-bottom flask, and 35 g (74.68 mmol) of Compound 5 was dissolved in chloroform solvent, then a mixture of pyridinium chlorochromate (17.687 g, 82.30 mmol) and silica gel (140 g) were added, and then 35 mL of acetic acid was added dropwise. The reaction solution was stirred at 20° C. for 24 hours, and the reaction solution was diluted with a mixed solution of 4 L of dichloromethane and 40 mL of methanol. A solid was obtained by filtration. The filtrate was respectively washed twice with 1.2 L of water and once with 1.2 L of a saturated sodium chloride solution. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (dichloromethane:methanol=100:1) to obtain 23 g of Compound 6 (a yield of 60%, a yellow solid).

(28) 6. Synthesis of Compound 7

(29) ##STR00019##

(30) 1.2 L of tetrahydrofuran was added into a 5 L three-neck flask, diisopropylamine (182 g, 1.80 mol) was dissolved in tetrahydrofuran, 2.5 M n-butyllithium in tetrahydrofuran solution (721 mL) was added dropwise at −78° C., stirred at −78° C. for half an hour after the completion of the addition. Then trimethylsilyl chloride (160.3 g, 1.50 mol) was added dropwise at −78° C., and stirred at −78° C. for half an hour. Then, a mixed solution of Compound 6 (70 g, 0.15 mol) and tetrahydrofuran (900 mL) was added dropwise to the above-mentioned solution at −78° C., and stirred at −78° C. for half an hour. Triethylamine (272.3 g, 2.70 mol) was added dropwise at −78° C. Then the temperature was raised to 20° C., and stirring was carried out for 2 hours. 2.1 L of a saturated aqueous solution of sodium bicarbonate was added to quench the reaction. The reaction solution was extracted with 6 L of ethyl acetate. The organic phases were combined, respectively washed twice with 1.2 L of water, five times with a saturated aqueous solution of ammonium chloride, and once with 1.2 L of a saturated aqueous solution of sodium chloride. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated. 145 g of crude Compound 7 was obtained and used directly for the next step of reaction.

(31) 7. Synthesis of Compound 8

(32) ##STR00020##

(33) Under protection of argon, a mixed solution of Compound 7 (40 g, 69.07 mmol) and dichloromethane (600 mL) was added into a three-neck flask. Acetaldehyde (6.506 g, 147.86 mmol) was added to the solution, and boron trifluoride-diethyl etherate (69.99 g, 492.89 mmol) was added dropwise at −60° C. The reaction solution was stirred at 20° C. for 24 hours. The pH of the reaction solution was adjusted to 7 to 8 with a saturated aqueous solution of sodium hydrogen carbonate. The reaction solution was diluted with 100 mL of water, and then extracted three times with 700 mL of dichloromethane, and the organic phases were combined. The organic phase was respectively washed twice with 1 L of water, once with 1 L of a saturated aqueous solution of sodium chloride, and then dried with anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (dichloromethane:methanol=100:1) to obtain 10 g of Compound 8 (a yield of 37%, a white solid).

(34) 8. Synthesis of Compound 9

(35) ##STR00021##

(36) Under protection of nitrogen, 100 mL of tetrahydrofuran, Compound 8 (5 g, 12.87 mmol), methanol 25 mL, cerium(III) chloride (19.17 g, 51.46 mmol), and sodium borohydride (1.85 g, 51.39 mmol) were added into a 250 mL three-neck flask. The solution was reacted at 20° C. for 24 hours. Then 3 L of a saturated aqueous solution of ammonium chloride was added to quench the reaction. The reaction solution was concentrated under reduced pressure. The residue was diluted with 300 mL of water and extracted three times with 600 mL of dichloromethane, and the organic phases were combined. The organic phase was respectively washed once with 300 mL of a saturated aqueous solution of sodium bicarbonate, once with 300 mL of a saturated aqueous solution of sodium chloride, and then dried with anhydrous sodium sulfate, filtered and concentrated. 4.8 g of Compound 9 was obtained (a yield of 96%, a yellow solid).

(37) 9. Synthesis of Compound 10

(38) ##STR00022##

(39) 80 mL of methanol, Compound 9 (8 g, 20.48 mmol) and 10% Pd/C (4 g, 3.77 mmol) were added into a 250 mL round-bottom flask, and hydrogen was introduced and reacted at 40° C. for 24 hours. The reaction solution was filtered and concentrated. The residue was purified by silica gel column chromatography (dichloromethane:methanol=100:1) to obtain 5.6 g of Compound 10 (a yield of 70%, a white solid).

(40) 10. Synthesis of Compound 11

(41) ##STR00023##

(42) Under protection of argon, a mixed solution of (2S)-2-amino-3-phenylpropionic acid (250 g, 1.51 mol) and 3.5 L of water were added into a 5 L three neck flask, then formic acid (491.75 g, 6.04 mol) was added. 4% Pd/C (250 g, 94.34 mmol) was added at 20° C. Hydrogen was introduced and the reaction was carried out at 20° C. for 48 hours. The resultant was filtered and concentrated. The crude product was recrystallized from methanol, and the solid was collected by filtration to obtain 190 g of Compound 11 (a yield of 65%, a white solid).

(43) .sup.1H-NMR: (CD3OD, 400 MHz, ppm): 7.25-7.40 (m, 5H), 3.86 (t, J=6.9 Hz, 1H), 3.37-3.31 (m, 1H), 3.24 (dd, J=14.6, 6.8 Hz, 1H), 2.85 (s, 6H).

(44) 11. Synthesis of Compound 12

(45) ##STR00024##

(46) Under protection of nitrogen, 700 mL of a mixed solution of tetrahydrofuran and diisopropylamine (188.6 g) was added into a 3 L three neck flask. n-Butyllithium (2.5 M tetrahydrofuran, 746 mL) was added dropwise within 30 minutes at −78° C. The temperature of the reaction was raised to 20° C., and the reaction was carried out under stirring for 20 minutes. Then, a mixed solution of ethyl acetate (164.4 g, 1.87 mol) and tetrahydrofuran (200 mL) was added dropwise at −78° C. within 30 minutes, and the solution was stirred at −78° C. for 1 hour. A mixed solution of 4-pyridinecarboxaldehyde (100 g, 933.62 mmol) and 250 mL of tetrahydrofuran were added dropwise at −78° C. within 25 minutes. The temperature of the reaction was raised to 0° C., and the reaction was carried out under stirring for 1.5 hours. 3.6 L of 1N hydrochloric acid was added to quench the reaction. The solution was extracted three times with 1 L of ethyl acetate. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether=1:1) to obtain yellow oily Compound 12 (a yield of 80%, 146 g).

(47) 12. Synthesis of Compound 13

(48) ##STR00025##

(49) Under protection of nitrogen, a mixed solution of Compound 12 (29.7 g, 153.69 mmol) and dichloromethane (300 mL) was added to a 1 L three-neck flask, and then N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (36.9 g, 192.49 mmol), 4,4-dimethylaminopyridine (1.56 g, 12.77 mmol), Compound 11 (25 g, 128.06 mmol) were added. The reaction solution was stirred at 20° C. for 5 hours, and after the completion of the reaction, the solution was diluted with 300 mL of dichloromethane. The reaction mixture was washed twice with 500 mL of a saturated aqueous solution of sodium carbonate, once with 500 mL of water, and once with 500 mL of a saturated aqueous solution of sodium chloride. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether=1:5). The obtained crude product was purified again by Flash-Prep-HPLC, petroleum ether (containing 0.1% triethylamine)/ethyl acetate (containing 0.1% triethylamine)=35:65, to increase the polarity to ethyl acetate (containing 0.1% triethylamine), to obtain yellow solid Compound 13 (a yield of 32%, 15 g).

(50) 13. Synthesis of Compound 14

(51) ##STR00026##

(52) Under protection of nitrogen, 900 mL of diethyl ether was added into a 2 L three-neck flask, the temperature was decreased to −20° C., and lithium aluminum hydride (18.5 g, 487.48 mmol) was added. Then, a mixed solution of Compound 13 (30 g, 80.98 mmol) and diethyl ether (300 mL) was added dropwise at −20° C. The reaction solution was stirred at −20° C. for 3 hours. 400 mL of ethyl acetate was added to quench the reaction. Then, 40 mL of a saturated aqueous solution of sodium sulfate and 150 mL of methanol were added, stirred at 20° C. for 1 hour, filtered and concentrated. The residue was purified by silica gel column chromatography (dichloromethane:methanol=20:1). The crude product was recrystallized from ethyl acetate to obtain white solid Compound 14 (PH-MKA-MT2002-8, 7.74 g, a yield of 62%).

(53) LCMS: (ES, m/z): 154[M+H].sup.+.

(54) The mass spectrum is as shown in FIG. 1.

(55) The liquid phase data is as shown in Table 1 below:

(56) TABLE-US-00001 TABLE 1 Compound 14 liquid phase data Peak position Peak area 0.64 min 810059
14. Synthesis of Compound 15

(57) ##STR00027##

(58) Under protection of argon, 400 mL of tetrahydrofuran and 250 mL of diethyl ether were added to a 3 L three-neck flask. Phosphoryl chloride (77.2 g, 503.49 mmol, 1.00 eq.) was added to the three-neck flask. A mixed solution of sodium 4-nitrophenolate (70 g, 503.20 mmol), triethylamine (50.96 g, 503.61 mmol), tetrahydrofuran (400 mL) and diethyl ether (250 mL) was slowly added to the reaction system at −78° C. The temperature of the reaction was raised to 20° C. and the reaction was carried out for 24 hours. The solid was filtered and the filtrate was concentrated. The crude product was purified by vacuum distillation (5 mmHg), and the fraction at 180° C. was collected to obtain bright yellow oily Compound 15 (38 g, a yield of 29%).

(59) P-NMR: (CDCl.sub.3, 121.5 MHz, ppm): 3.35

(60) 15. Synthesis of Compound 16

(61) ##STR00028##

(62) Under protection of nitrogen, a mixed solution of Compound 14 (27 g, 176.27 mmol), pyridine (210 mL) and triethylamine (3.024 g, 29.88 mmol) was added into a 1000 mL three-neck flask. A mixed solution of Compound 15 (45 g, 175.79 mmol) and pyridine (360 mL) was added dropwise at 0° C. to 5° C. The reaction solution was stirred at 20° C. for 5 hours. After the disappearance of the raw material, sodium 4-nitrophenolate (113.4 g, 704.35 mmol, 4.00 equiv) was added. After the completion of the addition, the solution was stirred at 40° C. for 4 hours, and then stirred at 20° C. for 16 hours. The reaction solution was concentrated. The residue was dissolved in 1.5 L of hydrochloric acid (2 mol/L), and washed twice with 1.5 L of ethyl acetate. The separated aqueous phase was adjusted to a pH of 7 to 8 with sodium bicarbonate, and then extracted three times with 1.2 L of dichloromethane. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated. The residue was purified by Flash-Prep-HPLC to obtain off-white solid Compound 16 (35 g, a yield of 59%).

(63) LC-MS: (ES, m/z): 337[M+H].sup.+.

(64) .sup.1H-NMR: (DMSO, 400 MHz, ppm): 8.62-8.62 (m, 2H), 8.35-8.31 (m, 2H), 7.59-7.57 (m, 2H), 7.44-7.42 (m, 2H), 5.91 (dd, J=10.1, 3.7 Hz, 1H), 4.71-4.56 (m, 2H), 2.32-2.17 (m, 2H).

(65) P-NMR: (DMSO, 162 MHz, ppm): −14.08.

(66) The mass spectrum is as shown in FIG. 2.

(67) The liquid phase data is as shown in Table 2 below:

(68) TABLE-US-00002 TABLE 2 Compound 16 liquid phase data Peak position Peak area 1.088 5970276 1.278 14725

(69) The hydrogen spectrum is as shown in FIG. 3.

(70) 16. Synthesis of Compound 1 (i.e. Compound 1-H)

(71) ##STR00029##

(72) Under protection of argon, a mixed solution of Compound 10 (500 mg, 1.27 mmol) and 1,4-dioxane (6 mL) was added to a 25 mL three-neck flask, tert-butylmagnesium chloride (6.37 mL, 1 mol/L) was added dropwise at 20° C., stirred at 20° C. for 4 hours, Then, a mixed solution of Compound 16 (855.8 mg, 2.55 mmol) and 1,4-dioxane (15 mL) was added dropwise at 65° C., and stirred at 65° C. for 2 hours. 100 mL of saturated ammonium chloride solution was added to quench the reaction. The reaction solution was extracted twice with 100 mL dichloromethane. The organic phases were combined. The organic phase was washed once with 150 mL of saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (dichloromethane:methanol=20:1). The crude product was purified by Flash-Prep-HPLC to obtain off-white solid Compound 1 (i.e. Compound 1-H, 30 mg), and the purity was 96.8%.

(73) LC-MS: (ES, m/z): 590.2[M+H].sup.+.

(74) .sup.1H-NMR: (CD3OD, 400 MHz, ppm): 8.60-8.58 (m, 2H), 7.51-7.49 (m, 2H), 5.76 (m, 1H), 4.68 (ddt, J=15.0, 7.2, 4.4 Hz, 1H), 4.58-4.44 (m, 1H), 4.30-4.19 (m, 2H), 3.45 (tt, J=10.6, 4.5 Hz, 1H), 3.12-3.02 (m, 1H), 2.34-2.23 (m, 2H), 2.08-1.77 (m, 6H), 1.74-1.41 (m, 9H), 1.41-1.02 (m, 10H), 1.01 (d, J=6.4 Hz, 3H), 0.95 (s, 3H), 0.86 (t, J=7.4 Hz, 3H), 0.70 (s, 3H).

(75) P-NMR: (CD3OD, 162 MHz, ppm): −4.998.

(76) The mass spectrum is as shown in FIG. 4.

(77) The liquid phase data is as shown in Table 3 below:

(78) TABLE-US-00003 TABLE 3 Compound 1 liquid phase data Peak position Peak area 3.722 285 3.819 249727

(79) The hydrogen spectrum is as shown in FIG. 5.

Example 2 Synthesis of Compound 2-0

(80) 1. Synthesis of Compound 2-1

(81) ##STR00030##

(82) A volume of anhydrous diethyl ether was added into a three-neck flask, then phenol (1000 g, 1.0 eq) and triethylamine (1070 g, 1.0 eq) were added. Phosphoryl chloride (1600 g, 1.0 eq) was added dropwise with stirring at 0° C., and the reaction was carried out under protection of nitrogen for 3 hours. The reaction system was allowed to stand and warm back to room temperature and stay overnight. After the completion of the reaction, the triethylamine salt was quickly filtered off. The filtrate was collected and concentrated, and dried to obtain an oily product Compound 2-1, which was used directly in the next step of reaction without further purification.

(83) 2. Synthesis of Compound PH-MIK-001-21

(84) ##STR00031##

(85) A volume of anhydrous dichloromethane (20 L) was added into a three-neck flask, then triethylamine (770 g, 2.0 eq.) was added. The reaction system was placed at −78° C., a solution of Compound 2-1 (800 g, 1.0 eq) dissolved in anhydrous dichloromethane, and (S)-3-(Chloroamino)-1-isopropoxybutan-2-one (685 g, 1.0 eq) were added dropwise, and the reaction was carried out for 2 h after the addition. The reaction system was allowed to stand and warm back to room temperature for overnight. The solvent was removed. The residue was washed with ethyl acetate and then filtered. The filtrate was concentrated and dried to obtain Compound PH-MIK-001-21 (800 g), which was used directly in the next step of reaction without further purification.

(86) 3. Synthesis of Compound 2-3

(87) ##STR00032##

(88) Compound 2-2 (700 g, 1.73 mol, 1.00 eq.) was added into tetrahydrofuran solvent, placed at −25° C. under protection of nitrogen and TMSCl (840 g, 7.79 mol, 4.5 eq) was added dropwise. After 1 hour of reaction, LDA (5.2 L, 2.0 M in THF, 1.00 eq.) was added dropwise for approximately 1 hour. After the completion of the addition, the reaction was carried out at the same temperature for 2 hours. After the completion of the reaction, a pre-cooled aqueous solution of citric acid was added to quench the reaction. The water phase was discarded. The organic phase was dried with anhydrous sodium sulfate and concentrated to obtain 820 g of a yellow oily crude product of Compound 2-3, which was used directly in the next step of reaction without further purification.

(89) 4. Synthesis of Compound 2-4

(90) ##STR00033##

(91) Crude compound 2-3 (820 g, 1.49 mol, 1.00 eq.) was added to 4.5 L of anhydrous dichloromethane and stirred at −60° C. Acetaldehyde (120 g, 2.72 mol, 1.8 eq.) was added dropwise for about 1 hour, and then BF3.Et2O (695 mL, 3.6 eq.) was added dropwise for about 1.5 hour. After the completion of the addition, the reaction system was allowed to react at this temperature for 2 hours, then continued to react at room temperature for 3 hours. The reaction system was cooled to lower than 0° C., a 50% aqueous solution of sodium hydroxide (320 mL) was added to quench the reaction, and stirred well for 10 minutes. Finally, the water phase was discarded. The organic phase was collected, washed with an aqueous solution of sodium chloride, dried with anhydrous sodium sulfate, and concentrated to obtain 750 g of yellow oily crude product of Compound 2-4, which was used directly in the next step of reaction without further purification.

(92) 5. Synthesis of Compound 2-5

(93) ##STR00034##

(94) The crude product of Compound 2-4 was dissolved in a mixed solution of methanol/water (2.5/0.5 L), and added to a sodium hydroxide solution (a 50% aqueous solution of sodium hydroxide, 180 mL) with stirring at room temperature. The reaction system was allowed to react at 50° C. for 4 hours. After the completion of the reaction, the methanol solution was concentrated, and acidified with citric acid and extracted twice with ethyl acetate. The organic phase was collected and washed with a saturated aqueous solution of sodium chloride, dried with anhydrous sodium sulfate and concentrated. Finally, recrystallization was performed in ethyl acetate to obtain 440 g of yellow solid Compound 2-5. The total yield of three steps was 60%.

(95) LC-MS: (ES, m/z): 415 [M−H].sup.−.

(96) 6. Synthesis of Compound 2-6

(97) ##STR00035##

(98) Compound 2-5 (100 g, 0.24 mol, 1.00 eq.) was dissolved in water (1000 mL), and sodium hydroxide (18 g, 0.45 mol, 1.88 eq.) was added with stirring. After it was dissolve completely, Pd/C (10%, 10 g, 0.1 eq.) was added under protection of nitrogen. After the completion of the addition, it was displaced with hydrogen and maintained at a hydrogen pressure of 5 atm, and reacted at 100° C. for 4 hours. After the completion of the reaction, the solid was filtered off, and the filtrate was acidified with hydrochloric acid, and extracted twice with ethyl acetate, washed with a saturated aqueous solution of sodium chloride, dried with anhydrous sodium sulfate and concentrated. Finally, recrystallization was performed in methyl tert-butyl ether:n-hexane=1:2 to obtain 75 g of white solid Compound 2-6 (a yield of 70%).

(99) LC-MS: (ES, m/z): 417 [M−H].sup.−.

(100) 7. Synthesis of Compound 2-7

(101) ##STR00036##

(102) Compound 2-6 (215 g, 0.514 mol, 1.0 eq.) was dissolved in formic acid (2 L), and 15 mL of perchloric acid was added dropwise with stirring. The reaction was carried out at 45° C. for 16 hours. After the completion of the reaction, the reaction system was allowed to cool to room temperature. 1.5 L of acetic anhydride was added dropwise during 1.0 hour. After the completion of the addition, the solution was stirred well for 30 minutes. The mixture was poured into an ice bath, extracted twice with diethyl ether. The collected organic phase was washed with water, dried with anhydrous sodium sulfate, and concentrated to obtain yellow solid Compound 2-7, which was used directly in the next step of reaction without further purification.

(103) LC-MS: (ES, m/z): 445 [M−H].sup.−.

(104) 8. Synthesis of Compound 2-8

(105) ##STR00037##

(106) The crude product Compound 2-7 (220 g, 0.49 mol, 1.0 eq.) was dissolved in 1.5 L of trifluorineacetic acid, trifluorineacetic anhydride (425 mL, 3.67 mol, 7.5 eq.) was added with stirring. The reaction was carried out at 0 to 5° C. for 1.5 hours and then sodium nitrite (103 g, 1.48 mmol, 3.0 eq.) was added portionwise. After the completion of the addition, the reaction mixture was reacted at 0 to 5° C. for 1.5 hours, and then at 40° C. overnight. After the completion of the reaction, the reaction mixture was neutralized with a 2M sodium hydroxide aqueous solution, extracted twice with ethyl acetate. The collected organic phase was washed twice with a saturated aqueous solution of sodium chloride, dried with anhydrous sodium sulfate and concentrated to obtain 160 g of Compound 2-8, a yellow solid.

(107) 9. Synthesis of Compound 2-9

(108) ##STR00038##

(109) The crude product Compound 2-8 (160 g, 0.39 mol, 1.0 eq.) was dissolved in a solution of methanol:water=1:1, 40% aqueous solution of potassium hydroxide was added dropwise. After the completion of the addition, the reaction system was allowed to react at 90° C. for 72 hours. After the completion of the reaction, the reaction system was neutralized with a 6 N aqueous solution of hydrochloric acid. Methanol was removed by concentration under reduced pressure. The residue was extracted three times with ethyl acetate. The collected organic phase was washed with a saturated aqueous solution of sodium chloride, dried with anhydrous sodium sulfate and concentrated to obtain 150 g of off-white crude product of solid Compound 2-9, which was used directly in the next step of reaction without further purification.

(110) LC-MS: (ES, m/z): 403 [M−H].sup.−.

(111) 10. Synthesis of Compound 2-10

(112) ##STR00039##

(113) The crude product Compound 2-9 (150 g, 0.38 mol, 1.0 eq.) was dissolved in tetrahydrofuran/water (2.5 L, v/v=4/1). Sodium borohydride (72 g, 1.90 mol, 5.0 eq.) was added portionwise at 0° C. After the completion of the addition, the reaction system was allowed to react at room temperature for 2 hours. After the completion of the reaction, water and methanol was added at 0° C. to quench the reaction system. The solvent was concentrated, washed with water, acidified with 1N hydrochloric acid and extracted three times with ethyl acetate. The organic phase was collected, washed three times with a saturated aqueous solution of sodium chloride, dried with anhydrous sodium sulfate and concentrated to obtain 135 g of yellow solid crude product of Compound 2-10, which was used directly in the next step of reaction without further purification.

(114) LC-MS: (ES, m/z): 405 [M−H].sup.−.

(115) 11. Synthesis of Compound 2-11

(116) ##STR00040##

(117) The crude product Compound 2-10 (135 g, 0.33 mol, 1.0 eq.) was added to dried methanol (2.5 L), and then p-toluenesulfonic acid (227 g, 1.32 mol, 4.0 eq.) was added. The reaction mixture was stirred well overnight. After the completion of the reaction, the reaction was neutralized with an aqueous solution of sodium bicarbonate. The solvent was concentrated. The residue was extracted twice with ethyl acetate. The organic phase was collected, washed with an aqueous solution of sodium bicarbonate and a saturated aqueous solution of sodium chloride, dried with anhydrous sodium sulfate and concentrated to obtain 120 g of yellow crude sticky product Compound 2-11, which was used directly in the next step of reaction without further purification.

(118) LC-MS: (ES, m/z): 421 [M+H].sup.+.

(119) 12. Synthesis of Compound 2-12

(120) ##STR00041##

(121) The crude product Compound 2-11 (120 g, 0.286 mol, 1.0 eq.) was dissolved in dichloromethane (2 L). 2,6-Lutidine (160 mL, 1.43 mol, 5.0 eq.) and

(122) trifluorinemethanesulfonic acid tert-butyldimethylsilyl ester (190 mL, 0.86 mol, 3.0 eq.) was added respectively at 0° C. The reaction system was stirred at room temperature for 24 hours. After the completion of the reaction, it was quenched with an aqueous solution of sodium bisulfate until neutral. The aqueous phase was separated and extracted twice with dichloromethane. The organic phase was washed with an aqueous solution of sodium bisulfate, a saturated aqueous solution of sodium carbonate and a saturated aqueous solution of sodium chloride, dried with anhydrous sodium sulfate and concentrated to obtain 135 g of yellow crude sticky product Compound 2-12.

(123) 13. Synthesis of Compound 2-13

(124) ##STR00042##

(125) The crude product Compound 2-12 (135 g, 0.2 mol, 1.0 eq.) was dissolved in dried tetrahydrofuran (1.2 L), and dried methanol (24 mL) was added under protection of nitrogen at 0° C. Then lithium borohydride (290 mL, 2 M in THF, 0.6 mol, 3 eq.) was added portionwise. The mixed reagent was allowed to react at room temperature overnight. After the completion of the reaction, the reaction system was quenched with 1 M aqueous solution of sodium hydroxide and extracted three times with ethyl acetate. The organic phase was collected, washed with a saturated aqueous solution of sodium chloride, dried with anhydrous sodium sulfate and concentrated to obtain 120 g of yellow oily crude product Compound 2-13, which was used directly in the next step of reaction without further purification.

(126) 14. Synthesis of Compound 2-14

(127) ##STR00043##

(128) The crude product Compound 2-13 (120 g, 193 mmol, 1.0 eq.) and N-methylimidazole (126 g, 8.0 eq) were added to anhydrous tetrahydrofuran (1400 mL). The compound PH-MIK-001-21 (382.5 g, 6.5 eq) dissolved in anhydrous tetrahydrofuran was added dropwise with thorough stirring. After the completion of the addition, the reaction system was stirred at room temperature overnight. After the completion of the reaction, the solvent was removed by concentration under reduced pressure, purified with by silica gel column to obtain 80 g of yellow oily Compound 2-14.

(129) 16. Synthesis of Compound 2-0

(130) ##STR00044##

(131) Compound 2-14 (80 g, 88 mmol, 1.0 eq.) was dissolved in dried methanol (1200 mL), and concentrated hydrochloric acid (242 mL, 2.9 mol, 20 eq.) was added dropwise to the above-mentioned reaction system. After the completion of the addition, the mixture was allowed to react at room temperature for 4 days. After the completion of the reaction, methanol was removed by concentration under reduced pressure. The remaining system was neutralized with water and sodium bicarbonate, extracted three times with dichloromethane. The organic phase was collected, washed with a saturated aqueous solution of sodium chloride, dried with anhydrous sodium sulfate and concentrated. The residue was purified by Flash-Prep-HPLC to obtain Compound 2-0, which is an off-white solid (a yield of 97.3%).

Example 3

(132) 1. Synthesis of Compound PH-MIK-001-22

(133) ##STR00045##

(134) 20 L anhydrous dichloromethane was added into a three-neck flask, and then triethylamine (770 g, 2.0 eq.) was added. The reaction system was placed at −78° C., and a solution of Compound 2-1 (prepared according to the preparation method of Example 2, 800 g, 1.0 eq.) dissolved in anhydrous dichloromethane and (S)-3-(Chloroamino)-1-isopropoxybutan-2-one (631 g, 1.0 eq.) were added dropwise. After the completion of the reaction, the reaction was carried out for 2 hours. Then the reaction system was allowed to stand and warm back to room temperature for overnight. The solvent was removed. The residue was washed with ethyl acetate and then filtered. The filtrate was concentrated and dried to obtain Compound PH-MIK-001-22 (760 g), which was used directly in the next step of reaction.

(135) 2. Synthesis of Compound 3-1

(136) ##STR00046##

(137) The crude product Compound 2-13 (120 g, 1.0 eq.) and N-methylimidazole (126 g, 8.0 eq.) were added to anhydrous tetrahydrofuran. A solution of compound PH-MIK-001-22 (401 g, 6.5 eq.) dissolved in anhydrous tetrahydrofuran was added dropwise with thorough stirring. After the completion of the addition, the reaction system was stirred at room temperature overnight. After the completion of the reaction, the solvent was removed by concentration under reduced pressure, purified with silica gel column to obtain 75 g of yellow oily Compound 3-1.

(138) 3. Synthesis of Compound 3-0

(139) ##STR00047##

(140) Compound 3-1 (75 g, 88 mmol, 1.0 eq.) was dissolved in dried methanol (1200 mL), concentrated hydrochloric acid (242 mL, 2.9 mol, 20 eq.) was added dropwise to the above-mentioned reaction system. After the completion of the dropwise addition, the solution was allowed to react at room temperature for 4 days. After the completion of the reaction, methanol was removed by concentration under reduced pressure. The remaining system was neutralized with water and sodium bicarbonate, and extracted three times with dichloromethane. The organic phase was collected, and washed with a saturated aqueous solution of sodium chloride, dried with anhydrous sodium sulfate and concentrated. The residue was purified by Flash-Prep-HPLC to obtain Compound 3-0, which was a 35 g off-white solid (a yield of 96.9%).

(141) LC-MS: (ES, m/z): 662.21 [M+H].sup.+.

(142) In addition to the compounds of which the preparation methods were specifically given, the compounds in Table 4 below were also prepared according to the preparation methods of Examples 1 to 3. Specific experimental steps were omitted, and the raw materials and reagents were all commercially available.

(143) TABLE-US-00004 TABLE 4 LC-MS Compound (m/z) numbers Compound structures [M + H].sup.+ Compound 1-IP 604.32 Compound 1-Cl 624.10 Compound 1-Me1 0 604.45 Compound 1-F 608.76 Compound 1-Cl2 624.81 Compound 1-Me2 575.72 Compound 1-Me3 610.96 Compound 4-0 680.10 Compound 5-0 720.94 Compound 6-0 711.12 Compound 7-0 707.96 Compound 8-0 697.45 Compound 9-0 0 762.20

Example 4 Application Example

(144) (A) the Effect of the Compounds of the Present Disclosure on 17α-Ethinylestradiol (E2-17α) Induced Cholestasis in Rats

(145) 1. Preparation of Test Solutions and Reagents

(146) Preparation of 1.0% CMC-Na solution: 1 p CMC-Na powder was weighed and added to 100 mL (50° C.) of distilled water, the mixture was rapidly stirred with a stirrer for 1 hour to fully swell, thereby to obtain a 1% CMC-Na solution.

(147) Preparation of E2-17α solution in propylene glycol: 54/0.98=56 mp of E2-17α was weighed, dissolved in 9 mL of propane-1,2-diol, shaked by a vortexer for 1 hour, thereby to obtain a 7 mp/mL suspension of E2-17α.

(148) 2. Grouping and Administration Method

(149) Sprague Dawley rats, weighing 250 to 350 g, were randomly grouped into: blank control group, E2-17α group (model group) and test compound groups, including E2-17α+Compound 1-H (5 mg/Kg) group, E2-17α+Compound 1-IP (5.1 mg/Kg) group, E2-17α+Compound 1-Cl (5.3 mg/Kg), E2-17α+Compound 1-Me 1 (5.1 mg/Kg) group, E2-17α+Compound 2-0 (5.1 mg/Kg) group, E2-17α+Compound 6-0 (5.9 mg/Kg) group, and positive control E2-17α+UDCA (15 mg/kg) group and obeticholic acid (OCA, 6 mg/kg+10 mg/kg) group. UDCA, i.e. ursodeoxycholic acid, is a bile acid derivative drug that has been marketed for the treatment of gallstones, cholestatic liver disease, fatty liver, and various types of hepatitis.

(150) 17α-Ethinylestradiol (E2-17α) was administered by subcutaneous injection into the neck for model establishment for 7 days. Meantime, the test compounds were suspended in a 1% CMC-Na solution and administered by intragastric administration for 7 days during the E2-17α modeling. The grouping is as shown in Table 5 below.

(151) TABLE-US-00005 TABLE 5 Grouping and Administration Method Numbers Admini- Volumes of stration of Grouping Animals Drugs Methods Administration Control 14 Equal volume CMC-Na solution s.c. + i.g. 0.1 mL/100 g E2-17α 14 E2-17α (in propane-1,2-diol) + s.c. 0.1 mL/100 g (7 mg/kg) equal volume CMC-Na solution E2-17α + 14 E2-17α (in propane-1,2-diol) + s.c. + i.g. 0.5 mL/100 g Compound 1-H Compound 1-H (5 mg/kg) CMC-Na solution E2-17α + 14 E2-17α (in propane-1,2-diol) + s.c. + i.g. 0.5 mL/100 g Compound 1-IP Compound 1-IP-CMC-Na (5.1 mg/kg) solution E2-17α + 14 E2-17α (in propane-1,2-dio1) + s.c. + i.g. 0.5 mL/100 g Compound 1-Cl Compound 1-Cl-CMC-Na (5.3 mg/kg) solution E2-17α + 14 E2-17α (in propane-1,2-diol) + s.c. + i.g. 0.5 mL/100 g Compound 1-Me1 Compound 1-Me1-CMC-Na (5.1 mg/kg) solution E2-17α + 14 E2-17α (in propane-1,2-diol) + s.c. + i.g. 0.5 mL/100 g Compound 2-0 Compound 2-0-CMC-Na (5.6 mg/kg) solution E2-17α + 14 E2-17α (in propane-1,2-diol) + s.c. + i.g. 0.5 mL/100 g Compound 6-0 Compound 6-0-CMC-Na (5.9 mg/Kg ) solution E2-17α + 14 E2-17α (in propane-1,2-diol) + s.c. + i.g. Calculated based UDCA UDCA pure aqueous solution on the actual drug (15 mg/kg) dosage E2-17α + OCA 14 E2-17α (in propane-1,2-diol) + s.c. + o.p. Calculated based (10 mg/kg) OCA-CMC-Na solution on the actual drug dosage (Note: s.c, subcutaneous injection; i.g, intragastric) Note: each administration group was given 17α-ethinylestradiol (E2-17α) at the same time.
3. Experimental Steps:

(152) E2-17α in propylene glycol was administered to rats for 7 days to establish the model, and different test samples in CMC-Na solution were administered by intragastric administration. On the 8.sup.th day, 1 mL of rat fasting blood sample was collected for detection of serum biochemical indicators, and E2-17α in propylene glycol was continuously administered to establish the model, and different test samples CMC-Na solution were administered by intragastric administration. After 0.5 hours of administration on the 9.sup.th day, the rats were weighed and anesthetized with 10% urethane (10 mL/kg) via intraperitoneal injection. After anesthesia, the rat was fixed on a board. A midline abdominal incision was made from the upper acetabular area, and the incision was about 2 to 3 cm. The indoor temperature should be maintained during the experiment, the body temperature of the rats was maintained and bile outflow was promoted. Handle of ophthalmic forceps was used to turn the liver up and find the duodenum and stomach junction. A suture (size 0) was placed at the junction to reserve. On the duodenum, about 2 cm away from the pylorus, a thin tube with a yellow transparent tube perpendicular to the duodenum could be found passing through the pancreas, that is, the common bile duct. At the junction of the common bile duct and the duodenum, the common bile duct was separated by an ophthalmic curved forceps, but was careful not to break the small blood vessels around the bile duct and avoided stimulating the pancreatic tissue by hand. After the separation, two sutures were place, and the suture close to the intestine end was ligated, as a traction suture, ophthalmic scissors were used to obliquely cut a small opening in the wall of the tube, a prepared pancreatic juice collection tube was inserted into the small opening. A mixture of yellow bile and pancreatic juice flowed out immediately after the insertion. The tube was ligated and fixed, and this tube was used to collect bile. After completion of the intubation, the skin was sutured. The other end of the bile duct intubation was taken out, flowing out to a fixed 0.5 mL centrifuge tube. Bile was continuously collected 8 times once every 15 minutes, until 120 minutes.

(153) 4. Measurement of Indicators:

(154) 1) Collection of Bile

(155) After completion of the intubation, the skin was sutured to prevent water evaporation of the abdominal cavity. The other end of the bile duct intubation was taken out, and the bile flowed out to a fixed 0.5 mL collection tube. Bile was continuously collected 8 times once every 15 minutes for a total of 120 minutes. Before the experiment, the collection tube was accurately weighed using an analytical balance. After the bile was collected in the experiment, the total weight of the bile and the collection tube was weighed, and the weight of the bile in the tube was calculated by subtraction and converted to a volume based on 1 g/mL. After collection of the bile, blood was taken from the inferior vena cava, and serum biochemical indicators were measured.

(156) 2) Detection of Indicators in Serum and Liver Samples

(157) The blood sample was allowed to stand in a 37° C. water bath for 10 minutes, centrifuged at 3000 rpm (1368 g)×10 min, the serum was separated conventionally, dispensed and frozen at −20° C. for storage. The indicators to be detected in serum included ALT, AST, and ALP. The detection methods of the above indicators were all carried out in accordance with the relevant instructions of the kit.

(158) 5. Statistical Analysis

(159) The data were analyzed using SPSS18.0 statistics software. Measurement data were compared between the two groups using the t test. Multiple groups of means were compared using one-way analysis of variance (ANOVA) and multiple comparisons between groups. Data mean±standard error (Mean±SEM.) or mean±standard deviation (Mean±SD.).

(160) 6. Test Results

(161) The bile excretion rates at different time points in each rat group are shown in Table 6.

(162) TABLE-US-00006 TABLE 6 Bile discharge amount at different time points in different rat groups (x ± s, mg/kg/min) Com- Com- Com- Com- Com- Com- pound pound pound pound pound pound Control E2-17α UDCA 1-H 1-IP 1-C1 1-Mel 2-0 6-0 OCA Time (n = 12) (n = 11) (n = 12) (n = 12) (n = 12) (n = 11) (n = 11) (n = 11) (n = 11) (n = 11)  15 63.39 ± 49.85 ± 52.36 ± 62.24 ± 53.48 ± 57.80 ± 54.43 ± 52.89 ± 51.32 ± 51.23 ± min 16.75 15.13 13.30 14.36 13.30 11.47 12.13 10.87  9.43 11.45  30 60.51 ± 47.08 ± 47.70 ± 58.68 ± 51.73 ± 54.80 ± 48.85 ± 53.29 ± 52.04 ± 49.73 ± min 11.21 10.53 11.01 12.90 11.01 12.37 10.54  8.76  7.38 10.22  45 58.82 ± 44.81 ± 45.14 ± 55.89 ± 50.34 ± 48.17 ± 46.34 ± 47.13 ± 48.31 ± 47.97 ± min 10.62 11.72 10.63 11.65 10.63 14.50 14.76 12.34 11.45  9.98  60 55.56 ± 42.74 ± 44.58 ± 52.06 ± 47.63 ± 46.64 ± 45.74 ± 46.64 ± 45.96 ± 44.84 ± min  7.98 10.55 10.99 10.46 10.99 11.80 11.67 11.78 10.87 6.6  75 53.23 ± 42.02 ± 43.09 ± 51.47 ± 44.27 ± 45.83 ± 42.73 ± 45.06 ± 44.06 ± 43.77 ± min  9.84  9.93 10.63 10.06 10.63 14.55 13.42 11.63 12.75  7.39  90 50.76 ± 41.33 ± 40.08 ± 48.30 ± 43.56 ± 45.65 ± 42.12 ± 43.01 ± 43.11 ± 41.42 ± min  9.73 12.68 12.08 11.81 12.08 15.01 14.83 15.25 12.52  8.05 105 46.39 ± 39.85 ± 43.72 ± 45.36 ± 43.72 ± 43.37 ± 41.53 ± 43.26 ± 42.34 ± 38.56 ± min 10.82 12.08 11.41 11.46 11.41 14.34 12.28 10.76 11.63  8.68 120 45.70 ± 38.02 ± 41.11 ± 44.73 ± 42.36 ± 44.21 ± 40.25 ± 41.38 ± 40.21 ± 37.33 ± min  9.37 10.54 11.24 13.20 13.30 16.91 15.74 10.58 13.17  9.62 Note: 17α-ethinylestradiol (E2-17α) was also administered to each of the above administration groups.

(163) Bile discharge amounts at different time points in rats in different dose groups of test Compound 1-H are compared in FIG. 6. As can be seen from Table 6 and FIG. 6, compared with the control group, the bile flow rate in the model group decreased; compared with the E2-17α group, the bile flow rate of the Compound 1-H, Compound 1-IP, Compound 1-Cl, Compound 1-Mel, Compound 2-0 and Compound 6-0 groups significantly increased (compared with E2-17α, *P<0.05).

(164) According to the data in Table 6, comparing the effect of promoting bile excretion of the compounds in the present disclosure with the currently marketed ursodesoxycholic acid (UDCA): the bile discharge amounts of the test compounds of the present disclosure at a dose of 5 mg/kg to 5.6 mg/kg were equivalent to that of ursodesoxycholic acid at a dose of 15 mg/kg; 105 minutes after administration, the bile discharge amounts of the test Compound 1-H group and Compound 1-Cl group were significantly higher than that of the ursodeoxycholic acid group. Comparing the effect of promoting bile excretion of the compounds in the present disclosure with the currently marketed obeticholic acid (OCA): the bile discharge amounts of the test compounds of the present disclosure at a dose of 5 mg/kg to 5.6 mg/kg were higher than the bile discharge amounts of obeticholic acid at a dose of 10 mg/kg, and with the prolongation of the time after administration, the effect of promoting bile excretion of obeticholic acid gradually reduced, while the effect of promoting bile excretion of the compounds of the present disclosure was still higher than that of the model group (the E2-17α group) 120 minutes after administration. The intensity of promoting bile excretion, i.e. amount of bile discharge, of the compounds of the present disclosure was better than those of obeticholic acid and ursodesoxycholic acid; the action time promoting bile excretion of the compounds of the present disclosure was also longer than those of obeticholic acid and ursodesoxycholic acid.

(165) The serum ALT, AST, and ALP levels of each group of rats after intragastric administration are shown in Table 7.

(166) TABLE-US-00007 TABLE 7 Serum biochemical properties of rats in each group after intragastric administration (n = 12, x ± s) ALT AST ALP Grouping n (U/L) (U/L) (U/L) Control 12 64.17 ± 13.42 196.67 ± 11.14 191.75 ± 18.59 E2-17α 12 95.67 ± 10.03 280.58 ± 20.95 257.42 ± 18.22 UDCA 11 85.55 ± 13.80 263.36 ± 15.35 242.18 ± 12.10 E2-17α + 11 72.36 ± 14.74 228.82 ± 17.74 218.64 ± 18.94 Compound 1-H E2-17α + 12 87.42 ± 11.72 258.83 ± 10.82 230.92 ± 14.00 Compound 1-IP E2-17α + 11 77.36 ± 11.42 230.89 ± 12.38 226.65 ± 12.38 Compound 1-Cl E2-17α + 12 80.64 ± 13.22 252.18 ± 16.59 235.64 ± 16.26 Compound 1-Me1 E2-17α + 12 78.64 ± 10.65 236.59 ± 13.06 220.35 ± 12.92 Compound 2-0

(167) As can be seen from Table 7, Compound 1-H, Compound 1-IP, Compound 1-Cl, Compound 1-Mel, and Compound 2-0 could respectively lower the values of ALT, AST, and ALP.

(168) 7. Conclusion: Compound 1-H, Compound 1-IP, Compound 1-Cl, Compound 1-Mel, Compound 2-0 and Compound 6-0 in the examples of the present disclosure have a significant effect on ameliorating intrahepatic cholestasis, promoting bile excretion, and thus has a therapeutic effect on diseases associated with bile excretion disorders. The compounds in the examples of the present disclosure can also correspondingly reduce the levels of ALT, AST and ALP, indicating that the compounds in the examples of the present disclosure have certain effects on liver damage repair.

(169) (B) The Effect of the Compound of the Present Disclosure on Cirrhotic Portal Hypertension

1. Preparation of Test Samples And Solutions

(170) 1) Preparation of saline solution of TAA: 10 g of thioacetamide (TAA) was weighed and dissolved in 100 mL of saline solution, and then filtered through a 0.22 μm sterile membrane filter into a sterile bottle.

(171) 2) Preparation of 0.5% CMC-Na suspension of test compound: 400 mg of Compound 1-H, Compound 1-IP, Compound 1-Cl, Compound 1-Mel or Compound 2-0 were weighed and dissolved in 100 mL of 0.5% CMC-Na solution, and shaken by a vortexer for 30 minutes, thereby to obtain 20 mg/Kg (high dose) groups. That is, CMC-Na suspension of 4 mg/mL of Compounds 1-H, 1-IP, 1-Cl, 1-Mel or 2-0. For the middle and low dose groups, drug preparation methods were the same.

2. Grouping and Administration Method

(172) 1) Modeling Method

(173) A rat model of cirrhotic portal hypertension was established by intraperitoneal injection of TAA. Except that the blank control group was intraperitoneally injected with saline, remaining animals were intraperitoneally injected (i.p.) with 10% thioacetamide (TAA) in saline solution to establish the model. The dose was 200 mg/Kg, and the administration volume was 2 mL/Kg. All experimental animals were intraperitoneally injected with TAA or an equal volume of saline once every three days. Before each dose, the rat shaving beddings should be replaced by clean ones in advance. The body weight was measured once every two modeling administration. The body weight of the rats increased or decreased by 10% compared with the previous one, the dose of TAA increased or decreased by 50% correspondingly. The administration was individualized to strictly control body weight change of each rat, which could effectively improve the success rate and uniformity of modeling.

(174) 2) Animal Grouping

(175) The successfully modeled animals were randomly grouped, 12 rats per group, and 20 rats in the blank control group. The groupings are shown in the table below. Portal hypertension was induced by TAA in rats, and a portal hypertension model was established after 13 days. The test compound was administered once a day for 10 days. The specific groupings are shown as follows:

(176) TABLE-US-00008 TABLE 8 Experimental animal groups and administration doses Administration Administration Volumes of Grouping n Methods Doses (mg/Kg) Administration Control 20 i.v. + i.g. N.S. — TAA 12 i.v. + i.g. N.S. — TAA + 1-H 12 i.g.  5 0.5 mL/100 g 5 mg/Kg TAA + 1-H 12 i.g. 10 0.5 mL/100 g 10 mg/Kg TAA + 1-H 12 i.g. 20 0.5 mL/100 g 20 mg/Kg TAA + 1-IP 12 i.g.  5 0.5 mL/100 g 5 mg/Kg TAA + 1-IP 12 i.g. 10 0.5 mL/100 g 10 mg/Kg TAA + 1-IP 12 i.g. 20 0.5 mL/100 g 20 mg/Kg TAA + 1-Cl 12 i.g.  5 0.5 mL/100 g 5 mg/Kg TAA + 1-Cl 12 i.g. 10 0.5 mL/100 g 10 mg/Kg TAA + 1-Cl 12 i.g. 20 0.5 mL/100 g 20 mg/Kg TAA + 1-Me1 12 i.g.  5 0.5 mL/100 g 5 mg/Kg TAA + 1-Me1 12 i.g. 10 0.5 mL/100 g 10 mg/Kg TAA + 1-Me1 12 i.g. 20 0.5 mL/100 g 20 mg/Kg TAA + 2-0 12 i.g.  5 0.5 mL/100 g 5 mg/Kg TAA + 2-0 12 i.g. 10 0.5 mL/100 g 10 mg/Kg TAA + 2-0 12 i.g. 20 0.5 mL/100 g 20 mg/Kg (Note: i.v., intravenous; i.g., intragastric; N.S., saline)
3) Administration Method

(177) Intragastric administration was adopted. Specific operation was the same as that of the intragastric operation in the examples of cholestasis.

3. Experimental Steps

(178) Animal vital signs and behavioral activities, gastrointestinal reactions, etc. were observed daily during the test to observe the general state of the animal for abnormalities. The injection site was observed every day for signs of erythema, edema, hemorrhage or mass development. All animals were weighed once before administration, and the overall changes in rat constitution during modeling and administration were observed.

(179) At the end of the administration, orbital blood was collected from the rats. The blood sample was placed in a 37° C. water bath for 10 minutes, centrifuged for 10 minutes at 3000 r/min (1370 g). Upper layer of the serum was taken and detected directly, or temporarily stored at −80° C., for serologic indicator measurement. After the blood samples were collected, the rats were fed with foods and water for 3 days. The blood volume was gradually restored for subsequent pressure measurement.

(180) 1) Measurement of Portal Vein Pressure

(181) Rats were fasted for 12 hours before the experiment, and were anesthetized with 10% chloral hydrate via intraperitoneal injection. The dose of anesthetic was 2.5 mL/Kg. After the animal was anesthetized, it was immobilized on an operating table. The abdomen was opened about 3 cm along the abdominal white line. After laparotomy, the duodenum was flipped, the portal vein was found. No. 4 intravenous needle was used to connect to the pressure transformer. All the valves of the transformer were opened. The needle was inserted into the portal vein, after blood flowing back, continued to insert about lcm. After the pressure was stable, all valves were closed. Stable portal vein pressure was measured. The pressure transformer was connected to a biological function test system (BL-420F).

(182) 2) Mean Arterial Pressure Measurement.

(183) The skin along the midline was incised using scissors. One side of the common carotid artery was separated. The common carotid artery was cannulated to measure the mean arterial pressure. The measurement method was the same as that of the portal vein.

4. Indicator Detection

(184) 1) 1.5 mL of orbital blood was collected before the experiment, of which the steps were the same as the aforesaid steps, to measure serum ALT, AST, ALP, γ-GT and TBA contents.

(185) 2) A small piece at same part of the liver of the rat was cut and fixed in a 10% formaldehyde solution for tissue section pathology observation. An appropriate size of the liver was cut off, and the blood on the surface was washed off quickly with saline, put into a sterile EP tube, cryopreserved in liquid nitrogen for subsequent experiments.

5. Test Results

(186) 1) Portal vein pressure: Compared with the blank control group, the portal vein pressure in the TAA group (model group) increased (∇P<0.01), indicating that cirrhotic portal hypertension had occurred; compared with the TAA group, the portal vein pressure in the 5 mg/Kg group, the 10 mg/Kg group, and the 20 mg/Kg group of each test compound significantly decreased (#P<0.05, ΔP<0.01).

(187) FIG. 7 shows the effect of each dose group of test compound 1-H on portal vein pressure.

(188) Percentage difference from the mean value of portal vein pressure of the TAA group, the mean percentage change (%) of each component of the administration group was obtained. Wherein, the percentage of portal vein pressure reduction was >10%, which can be considered clinically meaningful and related to the risk of portal hypertension. As can been seen from the figure, treated by administering Compound 1-H, the mean percentage changes of portal vein pressure of TAA+Compound 1-H 5 mg/Kg group, TAA+Compound 1-H 10 mg/Kg group, TAA+Compound 1-H 20 mg/Kg group were all greater than 10%. Therefore, it can be considered that using the Compound 1-H to reduce portal hypertension is clinically meaningful (P<0.01, P<0.05). FIG. 8 shows the mean percentage changes of portal vein pressure in rats in each dose group of test compound 1-H.

(189) 2) Appearance of the liver: The appearance of the liver and spleen of rats in each dose group of test Compound 1-H is shown in FIG. 9. FIG. 9 (A) shows the control (blank control) group. The liver of the rats in the blank control group was bright red, the surface was smooth and shiny, the boundary was clear, and the size and liver wet weight were normal. FIG. 9 (B) shows the TAA (model) group. The liver of the rats in the TAA group was reddish-brown, the boundary was unclear, and the liver surface was coarse and heterogeneous. Nodular hyperplasia formed more spherical protrusions on some rat liver surface. Hepatic lobe size of some rats with cirrhosis was abnormal, and the main manifestations were: left lateral segment, central hepatic segments and two discoid papillary segments increased in volume, while left middle segment and caudate lobe showed different degree of atrophy, but the overall volume increased and the wet weight increased; the size of the spleen of most rats was normal or slightly atrophic. FIG. 9 (E) shows the TAA+5 mg/Kg Compound 1-H group. After administration of compound 1-H, compared with the TAA group, the liver cirrhosis nodules of rats in the TAA+5 mg/Kg Compound 1-H group was milder, the bulge significantly reduced, the atrophy or hyperplasia changes of the liver lobe was lighter, but the surface was still heterogeneous compared with the blank control group. FIG. 9 (F) shows the TAA+10 mg/Kg Compound 1-H, and FIG. 9 (G) shows the TAA+20 mg/Kg Compound 1-H group, the liver and spleen appearance was similar to that of the TAA+5 mg/Kg Compound 1-H group.

(190) 3) Blood biochemical indicators: Serum indicators of rats in each dose group of test compound 1-H are shown in Figure. 10. FIG. 10 (A) shows the serum ALT levels of rats in each group (Mean±SEM, n=12). Compared with the blank control group, ALT level in the TAA group increased (*P<0.05); ALT levels of the Compound 1-H 5 mg/Kg group, Compound 1-H 10 mg/Kg group, and Compound 1-20 mg/Kg group decreased (#P<0.05). FIG. 10 (B) shows serum AST levels (Mean±SEM, n=12) of rats in each group. Compared with the blank control group, AST level of the TAA group increased (*P<0.05); AST levels of the Compound 1-H 5 mg/Kg group, Compound 1-H 10 mg/Kg group, and Compound 1-H 20 mg/Kg group decreased (#P<0.05). FIG. 10 (C) shows serum ALP levels (Mean±SEM, n=12) of rats in each group. Compared with the blank control group, ALP level of the TAA group increased (∇P<0.01); ALT levels in all administration groups decreased (#P<0.05, ΔP<0.01). FIG. 10 (D) is serum ALB contents (Mean±SEM, n=12) of rats in each group. FIG. 10 (E) shows serum γ-GT levels (Mean±SEM, n=12) of rats in each group. Compared with the blank control group, γ-GT level of the TAA group increased (∇P<0.01), γ-GT levels in all administration groups decreased (#P<0.05, ΔP<0.01). FIG. 10 (F) shows serum TBA contents (Mean±SEM, n=12) of rats in each group. Compared with the blank control group, TBA level in the TAA group increased (∇P<0.01), TBA levels in all administration groups decreased (ΔP<0.01).

(191) 4) Liver tissue section: The Masson staining and HE staining images of liver pathological sections of rats in each dose group of test compound 1-H are shown in FIG. 11. FIGS. (1) to (3) are respectively Masson stained (100× field of view), HE stained (100× field of view) and HE stained (200× field of view) of the same sample. Wherein FIG. 11(A) is microscopic photographs of the control (blank control) group. In the blank control group, there was no obvious hyperplasia of liver fibrous tissue, the morphology of the portal tube area was normal, and there was no hyperplasia of the bile duct. The morphological structure of hepatocytes was normal, and no cell necrosis or inflammatory cell infiltration was observed. FIG. 11 (B) is microscopic photographs of the TAA (model) group. Compared with the blank control group, the fibrous tissue of the TAA group was hyperplastic and formed pseudolobules, there was hyperplasia of the bile ducts in the portal tube area accompanied by round lymphocytic infiltration, and symptoms of liver fibrosis were developed. FIG. 11 (C) is microscopic photographs of the TAA+5 mg/Kg Compound 1-H group. The group mainly showed that the interstitial space of the fibrous tissue was thinner, the cell morphologic structure was basically normal, the inflammatory cell infiltration was milder, and there was no lots of hyperplasia of the bile duct in the portal tube area, which proved that the symptoms of liver fibrosis were ameliorated. FIG. 11 (D) is microscopic photographs of the TAA+10 mg/Kg Compound 1-H group. The morphopathologic structure of the group was similar to that of the TAA+5 mg/Kg Compound 1-H group. FIG. 11 (E) is microscopic photographs of the TAA+20 mg/Kg Compound 1-H group. The morphopathologic structure of the group was similar to that of the TAA+5 mg/Kg Compound 1-H group. Pathological indicators shows that compound 1-H had certain improvement effects on the hyperplasia of liver fibrous tissue and pseudolobule formation, which was mainly characterized by thinning of fibrous septa.

6. Conclusion

(192) Rat cirrhotic portal hypertension was successfully induced by intraperitoneal injection of TAA, and the test compounds prepared in the examples of the present disclosure were administered after the model was established. The experimental results show that using 5 mg/Kg, 10 mg/Kg and 20 mg/Kg three doses of the test compounds can (1) reduce the portal vein pressure in rats, the percentage of portal vein pressure reduction was >10%; (2) improve liver cirrhosis nodular hyperplasia, lobar atrophy or hyperplasia of the liver; (3) improve the levels of various blood indicators in rats, ALT, AST, ALP, ALB, γ-GT and TBA all decreased; (4) improve tissue morphology, alleviate liver fibrosis lesions.

(193) The above-mentioned results can be explained by that the test compounds of the present disclosure improves the intrahepatic high blood flow resistance in the function of relieving hepatic vasoconstriction by activating the FXR and TGR5 receptors, and at the same time has certain effects on ameliorating organic lesions such as liver fibrosis, and promoting the decrease of blood flow resistance in the liver. In addition, due to its own effect of increasing bile secretion and discharge, serum TBA, ALP and γ-GT levels decrease, reducing further damage to liver cells by toxic substances after cholestatic, and reducing levels of ALT and AST.

(194) In summary, the compounds of the present disclosure can better reduce rat cirrhotic portal hypertension, and also has certain effects on ameliorating organic lesions such as liver fibrosis.

(195) The above description of the examples is merely to assist in understanding the method and its core idea of the present invention. It should be pointed out that a person having ordinary skill in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention, and these modifications and changes also fall in the protection scope of the present invention.