COMPOUNDS FOR MODULATING ACTIVITY OF FXR AND USES THEREOF

Abstract

Provided are a compound for modulating the activity of FXR having a structure of formula (I), a pharmaceutically acceptable salt, an ester or a stereoisomer thereof.

##STR00001##

Claims

1. A compound for modulating the activity of FXR having a structure of formula (I), or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof: ##STR00086## wherein: R.sup.1, R.sup.2 and R.sup.3 are independently selected from H, halogen, unsubstituted or halogen substituted C.sub.1-6alkyl and unsubstituted or halogen substituted C.sub.1-6 alkoxy, provided that at least one of R.sup.2 and R.sup.3 is not hydrogen, R.sup.0 is selected from unsubstituted or halogen substituted C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl and C.sub.4-7 alkylcycloalkyl; X.sup.1 and X.sup.2 are independently selected from H and halogen; moiety —O—Z attaches to the naphthalene ring, wherein Z is a residue selected from 5-10 membered aryl and 5-10 membered heteroaryl optionally having one or more hetero atoms selected from N, O and S, wherein the 5-10 membered aryl or 5-10 membered heteroaryl is substituted by R.sup.4 and is optionally further substituted by R.sup.5; wherein R.sup.4 is selected from —COOH, —CH.sub.2COOH, —NHSO.sub.2CF.sub.3, —SO.sub.2NH—C.sub.1-6 alkyl, —SO.sub.3H, —CONHSO.sub.2—C.sub.1-6 alkyl, —CONHSO.sub.2—C.sub.3-6 cycloalkyl, —CONHSO.sub.2-5-10 membered aryl and —CONHSO.sub.2-5-10 membered aryl substituted by C.sub.1-6 alkyl at the aryl, and wherein R.sup.5 is selected from H, C.sub.1-6 alkyl, halogen, C.sub.1-6 haloalkyl, —O—(C.sub.1-6 alkyl) and —NH—(C.sub.1-6 alkyl).

2. The compound according to claim 1, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein R.sup.1, R.sup.2 and R.sup.3 are independently selected from H, halogen and C.sub.1-3 perfluoroalkoxy, and R.sup.0 is selected from isopropyl and cyclopropyl.

3. The compound according to claim 2, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein R.sup.1, R.sup.2 and R.sup.3 are independently selected from H, Cl, F and —O—CF.sub.3.

4. The compound according to claim 1, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein both of R.sup.1, R.sup.2 and R.sup.3 are Cl, and R.sup.3 is selected from H, F and —O—CH.sub.3; or R.sup.1 is —O—CF.sub.3, and both of R.sup.2 and R.sup.3 are H.

5. The compound according to claim 1, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein Z is a phenyl which is substituted by R.sup.4 and is optionally substituted by R.sup.5; or Z is a 5-10 membered heteroaryl having one or more hetero atoms selected from N, O and S, which is substituted by R.sup.4 and is optionally substituted by R.sup.5.

6. The compound according to claim 5, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein Z is a 5-6 membered heteroaryl having one or more hetero atoms selected from N, O and S, which is substituted by R.sup.4 and is optionally substituted by R.sup.5.

7. The compound according to claim 1, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein Z is a pyridyl, which is substituted by R.sup.4 and is optionally substituted by R.sup.5.

8. The compound according to claim 1, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein R.sup.4 is selected from —COOH, —CH.sub.2COOH, —CONHSO.sub.2—C.sub.1-6 alkyl and —CONHSO.sub.2—C.sub.3-6 cycloalkyl.

9. The compound according to claim 8, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein R.sup.4 is —COOH or —CH.sub.2COOH.

10. The compound according to claim 1, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein R.sup.5 is selected from H, C.sub.1-3 alkyl and halogen.

11. The compound according to claim 1, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein Z is pyridyl; R.sup.4 is —COOH; and R.sup.5 is H or halogen.

12. The compound according to claim 1, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein each halogen present in the compound is fluoro or chloro.

13. The compound according to claim 1, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein the compound is selected from the group consisting of: ##STR00087## ##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098##

14-22. (canceled)

23. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, and a pharmaceutically acceptable auxiliary material.

24. The pharmaceutical composition according to claim 23, wherein the pharmaceutical composition further comprises a second therapeutic agent being useful in the treatment of nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), cholestasis liver tree, chronic liver disease, hepatitis C infection, alcoholic liver disease, hepatic fibrosis, primary sclerosing cholangitis (PSC), gallstones, bile duct atresia, lower urinary tract symptoms and benign prostatic hyperplasia (BPH), ureteral calculi, obesity, type 2 diabetes, atherosclerosis, atherosclerosis, or liver damage caused by hypercholesterolemia or hyperlipidemia.

25. A method for treating a condition or disease mediated by FXR in a subject suffering therefrom, comprising administering to the subject a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof.

26-27. (canceled)

28. The method according to claim 25, further comprising administering a second therapeutic agent.

29. The method according to claim 25, wherein the condition or the disease is selected from the group consisting of nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), cholestasis liver tree, chronic liver disease, hepatitis C infection, alcoholic liver disease, hepatic fibrosis, primary sclerosing cholangitis (PSC), gallstones, bile duct atresia, lower urinary tract symptoms and benign prostatic hyperplasia (BPH), ureteral calculi, obesity, type 2 diabetes, atherosclerosis, atherosclerosis, and liver damage caused by hypercholesterolemia or hyperlipidemia.

30. The compound according to claim 9, or a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, wherein R.sup.4 is —COOH.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, drawings required for the description of the embodiments of the present invention will be briefly described below. Obviously, the drawings in the following description are only some embodiments of the present invention.

[0068] FIG. 1 shows reduction of NAS score after Compound 1 treatment in streptozocin (STZ)+diethylnitrosamine (DEN)+high fat diet (HFD) mice disease model.

[0069] FIG. 2 shows reduction of liver fibrosis after Compound 1 treatment in STZ+DEN+HFD mice disease model.

[0070] FIG. 3 shows reduction of NAS score after Compound 1 treatment in diethylnitrosamine (DEN)+high fat diet (HFD)+cholesterol/cholate (CHOL) treated rats.

[0071] FIG. 4 shows reduction of liver fibrosis after Compound 1 treatment in DEN+HFD+CHOL treated rats.

BEST MODE FOR CARRYING OUT THE INVENTION

[0072] The present invention will be further illustrated with reference to the examples below. It is necessary to state that, the examples below are only for illustration, but not for limitation of the present invention. Various alterations that are made by a person skilled in the art in accordance with teaching from the present invention should be within the scope claimed by the claims of the present invention.

Example 1

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl) methoxy)naphthalen-2-yl)oxy)nicotinic acid (Compound 1)

[0073] ##STR00019##

[0074] (a) Referring to the following reaction equation (Route A), Compound 1A-1 (1.0 g, 2.88 mmol, 1 eq.), Compound 1A-2 (0.46 g, 2.88 mmol, 1 eq.) and cesium carbonate (1.88 g, 5.76 mmol, 2 eq.) were dissolved in DMF (10 ml). The reaction was carried out at 65° C. for 2 h. After cooling, 10 ml water and 10 ml EA (ethyl acetate) were added for extraction, and the organic phase was washed with water and concentrated to dryness to give Compound 1A, 6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-2-ol, 0.8 g, yield: 65.0%. LCMS (ESI): calculated for C.sub.23H.sub.17C.sub.12NO.sub.3; [M+H].sup.+: 426.1, found: 426.1.

##STR00020##

[0075] (b) Referring to the following reaction equation, Compound 1A (0.2 g, 0.47 mmol, 1 eq.), 6-bromonicotinic acid methyl ester (0.1 g, 0.47 mmol, 1 eq.) and cesium carbonate (0.306 g, 0.94 mmol, 2 eq.) were dissolved in DMF (10 ml). The reaction was carried out at 65° C. for 2 h. After cooling, 10 ml water and 10 ml EA were added for extraction, and the organic phase was washed with water and concentrated to dryness to give Compound 1B, methyl 6((6((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalene-2-yl)oxy)nicotinate, 0.21 g, yield: 80.0%. LCMS (ESI): calculated for C.sub.30H.sub.22C.sub.12N.sub.2O.sub.5; [M+H].sup.+: 561.1, found: 561.1.

##STR00021##

[0076] (c) Referring to the following reaction equation, compound 1B (100 mg) was dissolved in methanol (2 ml), then 10% NaOH aqueous solution (1 ml) was added, the temperature was raised to 60° C., and the reaction was carried out for 1 h. The pH of the reaction solution was adjusted to 2 to 4 by adding 1N HCl solution, and 10 ml EA (ethyl acetate) was added for extraction. The organic phase was concentrated and purified on a column (PE/EA/AcOH=1/1/01 elution, wherein PE is petroleum ether) to give the title compound 1 (36 mg, yield: 37.0%).

[0077] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.57 (s, 1H), 8.23 (d, J=7.2 Hz, 1H), 7.74 (dd, J=2.0, 8.8 Hz, 2H), 7.60 (d, J=7.6 Hz, 2H), 7.56 (s, 1H), 7.51 (dd, J=8.8, 7.2 Hz, 1H), 7.33 (s, 1H), 7.26 (d, J=8.8 Hz, 1H), 7.02 (d, J=8.0 Hz, 1H), 6.93 (d, J=6.4 Hz, 1H), 4.98 (s, 2H), 2.57-2.50 (m, 1H), 1.19-1.11 (m, 4H). LCMS (ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1. .sup.13C NMR (400 MHz, DMSO-d.sub.6) δ7.79, 8.87, 8.87, 59.31, 107.74, 110.05, 110.97, 117.64, 119.43, 122.52, 127.55, 128.64, 128.89, 128.89, 129.18, 129.67, 131.73, 131.79, 132.94, 135.10, 135.10, 141.20, 149.11, 150.73, 155.79, 159.68, 163.82, 167.81, 172.61. IR (cm.sup.−1): major stretches at 1591.94 (C═O stretch), 1412.27, 1556.70 (C—C stretch), 1364.37, 1389.89 (C—H deformation), 1218.41, 1250.94 (C═N stretch), 791.88 (C—Cl stretch).

##STR00022##

Example 2

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl) methoxy)naphthalen-2-yl)oxy)pyridazine-3-carboxylic acid (Compound 2)

[0078] ##STR00023##

[0079] Following the procedure of Example 1, the title Compound 2 was obtained by substituting methyl 6-bromopyridazine-3-carboxylate for 6-bromonicotinic acid methyl ester.

[0080] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.52 (s, 1H), 8.25 (d, J=7.2 Hz, 1H), 7.74 (dd, J=2.0, 8.8 Hz, 2H), 7.61 (d, J=7.6 Hz, 2H), 7.52 (dd, J=8.8, 7.2 Hz, 1H), 7.34 (s, 1H), 7.26 (d, J=8.8 Hz, 1H), 7.00 (d, J=8.0 Hz, 1H), 6.95 (d, J=6.4 Hz, 1H), 4.98 (s, 2H), 2.59-2.50 (m, 1H), 1.21-1.11 (m, 4H). LCMS (ESI): calculated for C.sub.28H.sub.19Cl.sub.2N.sub.3O.sub.5; [M+H].sup.+: 548.1, found: 548.1.

Example 3

Preparation of 5-chloro-6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl) isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)nicotinic acid (Compound 3)

[0081] ##STR00024##

[0082] Following the procedure of Example 1, the title Compound 3 was obtained by substituting methyl 5,6-dichloronicotinate for 6-bromonicotinic acid methyl ester.

[0083] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.60 (s, 1H), 7.73 (dd, J=2.0, 8.8 Hz, 2H), 7.59 (d, J=7.6 Hz, 2H), 7.51 (dd, J=8.8, 7.2 Hz, 1H), 7.33 (s, 1H), 7.26 (d, J=8.8 Hz, 1H), 7.01 (d, J=8.0 Hz, 1H), 6.95 (d, J=6.4 Hz, 1H), 5.00 (s, 2H), 1.26-1.12 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.19Cl.sub.3N.sub.2O.sub.5; [M+H].sup.+: 581.0, found: 581.0.

Example 4

Preparation of 2-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl) methoxy)naphthalen-2-yl)oxy)thiazole-5-carboxylic acid (Compound 4)

[0084] ##STR00025##

[0085] Following the procedure of Example 1, the title Compound 4 was obtained by substituting methyl 2-bromothiazole-5-carboxylate for 6-bromonicotinic acid methyl ester.

[0086] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.80 (s, 1H), 7.69 (dd, J=2.0, 8.8 Hz, 2H), 7.59 (d, J=7.6 Hz, 2H), 7.53 (dd, J=8.8, 7.2 Hz, 1H), 7.32 (s, 1H), 7.26 (d, J=8.8 Hz, 1H), 7.01 (d, J=8.0 Hz, 1H), 6.99 (d, J=6.4 Hz, 1H), 5.00 (s, 2H), 1.25-1.12 (m, 5H). LCMS (ESI): calculated for C.sub.27H.sub.18Cl.sub.2N.sub.2O.sub.5S; [M+H].sup.+: 553.0, found: 553.0.

Example 5

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl) methoxy)naphthalen-2-yl)oxy)-5-methylnicotinic acid (Compound 5)

[0087] ##STR00026##

[0088] Following the procedure of Example 1, the title Compound 5 was obtained by substituting methyl 6-bromo-5-methylnicotinate for 6-bromonicotinic acid methyl ester.

[0089] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.78 (s, 1H), 8.35 (d, J=1.5 Hz, 1H), 8.12-7.90 (m, 1H), 7.72-7.61 (m, 2H), 7.54 (s, 3H), 7.28 (m, 2H), 7.15-7.10 (m, 1H), 7.07 (dd, J=7.5, 1.5 Hz, 1H), 6.95 (dd, J=7.6, 1.6 Hz, 1H), 5.41 (s, 2H), 2.99-2.70 (m, 1H), 2.28 (s, 3H), 2.12-1.56 (m, 4H). LCMS (ESI): calculated for C.sub.30H.sub.22Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 561.1, found: 561.1.

Example 6

Preparation of 6((6((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl) methoxy)naphthalen-2-yl)oxy)-N-(cyclopropylsulfonyl)nicotinamide (Compound 6)

[0090] ##STR00027##

[0091] Compound 1 (70 mg) as prepared in Example 1 and cyclopropylsulfonamide (23 mg) were dissolved in 2 ml DCM (dichloromethane), then 40 mg EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 26 mg DMAP (dimethylaminopyridine) were added. After completion of the reaction, 10 ml DCM and 10 ml water was added for extraction. The organic phase was washed with water and concentrated to dryness. The crude product is purified by column (PE/EA/AcOH=2/1/0.01) to give the title Compound 6 (8 mg, yield: 9.6%).

[0092] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.63 (d, J=1.5 Hz, 1H), 8.30 (dd, J=7.5, 1.5 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.73 (d, J=8.0 Hz, 1H), 7.59-7.62 (m, 3H), 7.49-7.53 (m, 1H), 7.35 (s, 1H), 7.26-7.29 (m, 1H), 7.10 (d, J=8.0 Hz, 1H), 6.93-6.96 (m, 1H), 4.98 (s, 2H), 1.02-1.20 (m, 10H). LCMS (ESI): calculated for C.sub.32H.sub.25Cl.sub.2N.sub.3O.sub.6S; [M+H].sup.+: 650.1, found: 650.1.

Example 7

Preparation of 5-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl) methoxy)naphthalen-2-yl)oxy)pyrazine-2-carboxylic acid (Compound 7)

[0093] ##STR00028##

[0094] Following the procedure of Example 1, the title Compound 7 was obtained by substituting methyl 5-chloro-pyridine-2-carboxylate for 6-bromonicotinic acid methyl ester.

[0095] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.63 (s, 1H), 8.30 (s, 1H), 7.79 (d, J=8.8 Hz, 1H), 7.72 (d, J=9.2 Hz, 1H), 7.58-7.63 (m, 4H), 7.49-7.53 (m, 1H), 7.34 (d, J=2.0 Hz, 1H), 6.94 (d, J=9.2 Hz, 1H), 4.98 (s, 2H), 1.11-1.22 (m, 5H). LCMS (ESI): calculated for C.sub.28H.sub.19Cl.sub.2N.sub.3O.sub.5; [M+H].sup.+: 548.1, found: 548.1.

Example 8

Preparation of 2-chloro-6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl) isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)nicotinic acid (Compound 8)

[0096] ##STR00029##

[0097] Following the procedure of Example 1, the title Compound 8 was obtained by substituting methyl 2,6-dichloronicotinate for 6-bromonicotinic acid methyl ester.

[0098] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.98 (br s, 1H), 7.70-7.79 (m, 2H), 7.60 (d, J=8.0 Hz, 2H), 7.47-7.55 (m, 2H), 7.18-7.33 (m, 2H), 6.90-6.95 (m, 2H), 4.98 (s, 2H), 1.11-1.22 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.19Cl.sub.3N.sub.2O.sub.5; [M+H].sup.+: 581.0, found: 581.0.

Example 9

Preparation of 5-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)picolinic acid (Compound 9)

[0099] ##STR00030##

[0100] Following the procedure of Example 1, the title Compound 9 was obtained by substituting methyl 5-bromopicolinate for 6-bromonicotinic acid methyl ester.

[0101] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.46 (d, J=3.1 Hz, 1H), 8.03 (d, J=8.7 Hz, 1H), 7.83 (d, J=8.9 Hz, 1H), 7.72 (d, J=9.1 Hz, 1H), 7.55 (dt, J=28.7, 8.3 Hz, 4H), 7.43 (d, J=8.6 Hz, 1H), 7.39-7.24 (m, 2H), 6.95 (d, J=8.9 Hz, 1H), 4.98 (s, 2H), 1.23-1.02 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

Example 10

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)-2-methylnicotinic acid (Compound 10)

[0102] ##STR00031##

[0103] Following the procedure of Example 1, the title Compound 10 was obtained by substituting methyl 6-chloro-2-methylnicotinate for 6-bromonicotinic acid methyl ester.

[0104] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.21 (d, J=8.6 Hz, 1H), 7.78 (d, J=8.9 Hz, 1H), 7.72 (d, J=9.0 Hz, 1H), 7.62-7.55 (m, 3H), 7.51 (dd, J=9.0, 7.1 Hz, 1H), 7.34 (d, J=2.5 Hz, 1H), 7.27 (dd, J=8.8, 2.4 Hz, 1H), 6.94 (dd, J=8.9, 2.5 Hz, 1H), 6.85 (d, J=8.6 Hz, 1H), 4.98 (s, 2H), 2.52 (s, 3H), 1.24-1.07 (m, 5H). LCMS (ESI): calculated for C.sub.30H.sub.22Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 561.1, found: 561.1.

Example 11

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)picolinic acid (Compound 11)

[0105] ##STR00032##

[0106] Following the procedure of Example 1, the title Compound 11 was obtained by substituting methyl 2,6-dichloronicotinate for 6-bromonicotinic acid methyl ester.

[0107] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.99 (t, J=7.9 Hz, 1H), 7.77 (d, J=8.1 Hz, 2H), 7.71 (d, J=8.9 Hz, 1H), 7.63-7.45 (m, 4H), 7.33 (s, 1H), 7.29 (d, J=9.0 Hz, 1H), 7.22 (d, J=8.2 Hz, 1H), 6.94 (d, J=9.0 Hz, 1H), 4.98 (s, 2H), 1.26-1.01 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

Example 12

Preparation of 2-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)isonicotinic acid (Compound 12)

[0108] ##STR00033##

[0109] Following the procedure of Example 1, the title Compound 12 was obtained by substituting methyl 2-fluoroisonicotinate for 6-bromonicotinic acid methyl ester.

[0110] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.29 (d, J=5.1 Hz, 1H), 7.78 (d, J=8.8 Hz, 1H), 7.59 (t, J=7.7 Hz, 3H), 7.52 (m, 2H), 7.34 (s, 2H), 7.29 (s, 1H), 4.98 (s, 2H), 1.31-1.06 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

Example 13

Preparation of 3-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)picolinic acid (Compound 13)

[0111] ##STR00034##

[0112] Following the procedure of Example 1, the title Compound 13 was obtained by substituting methyl 3-fluoropicolinate for 6-bromonicotinic acid methyl ester.

[0113] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.39 (d, J=4.4 Hz, 1H), 7.77 (d, J=9.0 Hz, 1H), 7.66 (d, J=9.2 Hz, 1H), 7.59 (d, J=8.1 Hz, 2H), 7.54-7.43 (m, 3H), 7.30 (d, J=2.8 Hz, 2H), 7.26-7.16 (m, 1H), 6.95-6.85 (m, 1H), 4.95 (s, 2H), 1.24-1.06 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

Example 14

Preparation of 2-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)benzoic acid (Compound 14)

[0114] ##STR00035##

[0115] Following the procedure of Example 1, the title Compound 14 was obtained by substituting methyl 2-fluorobenzoate for 6-bromonicotinic acid methyl ester.

[0116] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.82 (d, J=7.8 Hz, 1H), 7.73 (d, J=8.9 Hz, 1H), 7.66-7.57 (m, 3H), 7.55-7.45 (m, 2H), 7.26 (d, J=10.6 Hz, 2H), 7.21-7.11 (m, 2H), 6.99 (d, J=8.3 Hz, 1H), 4.94 (s, 2H), 1.27-1.06 (m, 5H). LCMS (ESI): calculated for C.sub.30H.sub.21Cl.sub.2NO.sub.5; [M+H].sup.+: 546.1, found: 546.1

Example 15

Preparation of 2-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)nicotinic acid (Compound 15)

[0117] ##STR00036##

[0118] Following the procedure of Example 1, the title Compound 15 was obtained by substituting methyl 2-chloronicotinate for 6-bromonicotinic acid methyl ester. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.30-8.19 (m, 2H), 7.73 (dd, J=19.2, 9.0 Hz, 2H), 7.60 (d, J=7.9 Hz, 2H), 7.55-7.47 (m, 2H), 7.32 (d, J=2.5 Hz, 1H), 7.26-7.18 (m, 2H), 6.92 (dd, J=8.9, 2.5 Hz, 1H), 4.98 (s, 2H), 1.23-1.10 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

Example 16

Preparation of 3-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)isonicotinic acid (Compound 16)

[0119] ##STR00037##

[0120] Following the procedure of Example 1, the title Compound 16 was obtained by substituting methyl 3-fluoroisonicotinate for 6-bromonicotinic acid methyl ester.

[0121] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.52 (d, J=4.8 Hz, 1H), 8.39 (s, 1H), 7.76 (d, J=8.9 Hz, 1H), 7.73 (d, J=4.9 Hz, 1H), 7.64 (d, J=9.0 Hz, 1H), 7.59 (d, J=7.7 Hz, 2H), 7.50 (dd, J=9.0, 7.0 Hz, 1H), 7.32-7.15 (m, 4H), 6.89 (dd, J=8.9, 2.5 Hz, 1H), 4.95 (s, 2H), 1.27-1.09 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

Example 17

Preparation of 6(6-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl) isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)nicotinic acid (Compound 17)

[0122] ##STR00038##

[0123] Following the procedure of Example 1, the title Compound 17 was obtained by substituting 4-(chloromethyl)-5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl) isoxazole for 1A-1.

[0124] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.62 (s, 1H), 8.27 (d, J=8.1 Hz, 1H), 7.75 (dt, J=31.9, 15.9 Hz, 2H), 7.61 (s, 2H), 7.56-7.43 (m, 2H), 7.36 (s, 1H), 7.29 (d, J=8.6 Hz, 1H), 7.11 (d, J=8.2 Hz, 1H), 7.00 (d, J=8.5 Hz, 1H), 5.03 (s, 2H), 2.44-2.37 (m, 1H), 1.20-1.05 (m, 4H). LCMS (ESI): calculated for C.sub.30H.sub.2, F.sub.3N.sub.2O.sub.6; [M+H].sup.+: 563.1, found: 563.1.

Example 18

Preparation of 6-6((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl) isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)nicotinic acid (Compound 18)

[0125] ##STR00039##

[0126] Following the procedure of Example 1, the title Compound 18 was obtained by substituting 4-(chloromethyl)-5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl) isoxazole for 1A-1.

[0127] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.63 (s, 1H), 8.27 (d, J=7.9 Hz, 1H), 7.87-7.63 (m, 4H), 7.60 (s, 1H), 7.40-7.24 (m, 2H), 7.11 (d, J=8.0 Hz, 1H), 6.96 (d, J=8.6 Hz, 1H), 4.98 (s, 2H), 2.47-2.40 (m, 1H), 1.23-1.08 (m, 4H). LCMS (ESI): calculated for C.sub.29H.sub.19Cl.sub.2FN.sub.2O.sub.5; [M+H].sup.+: 565.1, found: 565.1.

Example 19

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichloro-4-methoxyphenyl) isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)nicotinic acid (Compound 19)

[0128] ##STR00040##

[0129] Following the procedure of Example 1, the title Compound 19 was obtained by substituting 4-(chloromethyl)-5-cyclopropyl-3-(2,6-dichloro-4-methoxyphenyl) isoxazole for 1A-1.

[0130] LCMS (ESI): calculated for C.sub.30H.sub.22C.sub.12N.sub.2O.sub.6; [M+H]+: 577.1, found: 577.1.

Example 20

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-1-fluoronaphthalen-2-yl)oxy)nicotinic acid (Compound 20)

[0131] ##STR00041##

[0132] (a) Referring to the following reaction equation (Route C), Compound 20A-1 (1.0 g, 4.15 mmol, 1 eq.), Compound 20A-2 (0.90 g, 4.15 mmol, 1 eq.) and cesium carbonate (2.70 g, 8.30 mmol, 2 eq.) were dissolved in DMF (10 ml). The reaction was carried out at 65° C. for 2 h. After cooling, 10 ml water and 10 ml EA (ethyl acetate) were added for extraction, and the organic phase was washed with water and concentrated to dryness to give Compound 20A, methyl 6-((6-bromo-1-fluoronaphthalen-2-yl)oxy)nicotinate, 1.2 g, yield: 77.0%. LCMS (ESI): calculated for C.sub.17H.sub.11BrFNO.sub.3; [M+H].sup.+: 376.0, found: 376.0.

##STR00042##

[0133] (b) Referring to the following reaction equation, compound 20A (200 mg, 0.53 mmol, 1 eq) was dissolved in dry THF (2 ml), then KOAc (104 mg, 1.06 mmol, 2 eq), Pd(dppf).sub.2Cl.sub.2 (39 mg, 0.053 mmol, 0.1 eq), and bis(pinacolato)diboron (135 mg, 0.53 mmol, 1 eq) were added under N.sub.2, and the reaction mixture was heated to reflux for 2 h. After cooling, 10 ml water and 10 ml EtOAc were added for extraction, and the organic phase was washed with water and concentrated to dryness. The residue was purified by silica gel column chromatography (petroleum Ether:EtOAc=3:1) to give Compound 20B, methyl 6-((1-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-yl)oxy)nicotinate, 151 mg, yield: 67.1%. LCMS (ESI): calculated for C.sub.23H.sub.23BFNO.sub.5; [M+H].sup.+: 424.2, found: 424.2.

##STR00043##

[0134] (c) Referring to the following reaction equation, compound 20B (100 mg) was dissolved in EtOH (2 ml), then 30% H.sub.2O.sub.2 aqueous solution (1 ml) were added. The reaction mixture was stirred at room temperature for 1 h, quenched with saturated aqueous Na.sub.2SO.sub.3, and extracted with EA. The organic phase was concentrated and purified on a column (PE/EA=3/1) to give the compound 20C (36 mg, yield: 37.0%). LCMS (ESI): calculated for C.sub.17H.sub.12FNO.sub.4; [M+H].sup.+: 314.1, found: 314.1.

##STR00044##

[0135] (d) Referring to the following reaction equation, Compound 20C (0.2 g, 0.47 mmol, 1 eq.), 1A-1 (0.1 g, 0.47 mmol, 1 eq.) and cesium carbonate (0.306 g, 0.94 mmol, 2 eq.) were dissolved in DMF (10 ml) for reacting. The reaction was carried out at 65° C. for 2 h. After cooling, 10 ml water and 10 ml EtOAc were added for extraction, and the organic phase was washed with water and concentrated to dryness to give Compound 20D, 0.21 g, yield: 80.0%.

[0136] LCMS (ESI): calculated for C.sub.30H.sub.21Cl.sub.2FN.sub.2O.sub.5; [M+H].sup.+: 579.1, found: 579.1.

##STR00045##

[0137] (e) Referring to the following reaction equation, compound 20D (100 mg) was dissolved in dry THF (2 ml), then 10% NaOH aqueous solution (1 ml) were added under N.sub.2, and the reaction mixture was heated to reflux for 1 h. The pH of the reaction solution was adjusted to 3 to 4 by adding 1N HCl solution, and 10 ml EA was added for extraction. The organic phase was concentrated and purified on a column (PE/EA/AcOH=1/1/0.01 elution) to give the title compound 20 (36 mg, yield: 37.0%).

##STR00046##

[0138] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.63 (d, J=2.4 Hz, 1H), 8.30 (dd, J=8.7, 2.4 Hz, 1H), 7.92 (d, J=9.0 Hz, 1H), 7.70 (s, 1H), 7.64 (d, J=5.3 Hz, 1H), 7.60 (d, J=6.4 Hz, 1H), 7.57 (d, J=4.3 Hz, 2H), 7.42-7.36 (m, 2H), 7.17 (d, J=8.6 Hz, 1H), 5.09 (s, 2H), 1.22-1.06 (m, 5H). LCMS (ESI): [0139] calculated for C.sub.29H.sub.19Cl.sub.2FN.sub.2O.sub.5; [M+H].sup.+: 565.1, found: 565.1.

Example 21

Preparation of 6-((1-chloro-6-((5-cyclopropyl-3-(2,6-dichlorophenyl) isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)nicotinic acid (Compound 21)

[0140] ##STR00047##

[0141] Following the procedure of Example 20, the title Compound 21 was obtained by substituting 6-bromo-1-chloronaphthalen-2-ol for 20A-1.

[0142] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.63 (s, 1H), 8.27 (d, J=7.9 Hz, 1H), 7.87-7.63 (m, 4H), 7.60 (s, 1H), 7.40-7.24 (m, 2H), 7.11 (d, J=8.0 Hz, 1H), 6.96 (d, J=8.6 Hz, 1H), 4.98 (s, 2H), 2.47-2.40 (m, 1H), 1.23-1.08 (m, 4H). LCMS (ESI): calculated for C.sub.29H.sub.19Cl.sub.3N.sub.2O.sub.5; [M+H].sup.+: 581.0, found: 581.0.

Example 22

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-1-yl)oxy)nicotinic acid (Compound 22)

[0143] ##STR00048##

[0144] (a) Referring to the following reaction equation (Route D), Compound 22A-1 (2.0 g, 12.49 mmol, 1 eq.), Compound 22A-2 (1.71 g, 9.99 mmol, 0.8 eq.) and cesium carbonate (6.09 g, 18.74 mmol, 1.5 eq.) were dissolved in DMF (20 ml) for reacting. The reaction was carried out at 65° C. for 3 h. After cooling, 30 ml water and 30 ml EA (ethyl acetate) were added for extraction, and the organic phase was washed with water and concentrated to dryness. The residue was purified by silica gel column chromatography (petroleum:AcOEt=5:1) to give Compound 22A, methyl 6-((6-hydroxynaphthalen-1-yl)oxy)nicotinate, 1.1 g, yield: 37.3%. LCMS (ESI): calculated for C.sub.17H.sub.13NO.sub.4; [M+H].sup.+: 296.1, found: 296.1.

##STR00049##

[0145] (b) Referring to the following reaction equation, Compound 22A (0.2 g, 0.68 mmol, 1 eq.), 22A-3 (0.2 g, 0.68 mmol, 1 eq.) and cesium carbonate (0.44 g, 1.36 mmol, 2 eq.) were dissolved in DMF (5 ml) for reacting. The reaction was carried out at 40° C. for 2 h. After cooling, 10 ml water and 10 ml EA were added for extraction, and the organic phase was washed with water and concentrated to dryness to give Compound 22B, methyl 6-((6((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-1-yl)oxy) nicotinate, 0.31 g, yield: 81.2%. LCMS (ESI): calculated for C.sub.30H.sub.22Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 561.1, found: 561.1.

##STR00050##

[0146] (c) Referring to the following reaction equation, compound 22B (100 mg) was dissolved in methanol (2 ml), then 10% NaOH aqueous solution (1 ml) was added, the temperature was raised to 60° C., and the reaction was carried out for 0.5 h. The pH of the reaction solution was adjusted to 2 to 4 by adding 1N HCl solution, and 10 ml EA was added for extraction. The organic phase was concentrated on a column (PE/EA/AcOH=1/1/0.01 elution) to give the title compound 22 (42 mg, yield: 43.2%).

[0147] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.11 (br s, 1H), 8.56 (s, 1H), 8.28 (d, J=8.5 Hz, 1H), 7.66 (d. J=8.3 Hz, 1H), 7.56-7.61 (m, 3H), 7.45-7.53 (m, 2H), 7.39 (s, 1H), 7.15 (t, J=9.6 Hz, 2H), 6.9 (d, J=9.2 Hz, 2H), 4.98 (s, 2H), 1.09-1.28 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

##STR00051##

Example 23

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-1-yl)oxy)picolinic acid (Compound 23)

[0148] ##STR00052##

[0149] Following the procedure of Example 22, the title Compound 23 was obtained by substituting methyl 6-fluoropicolinate for 22A-2.

[0150] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ7.99 (t, J=7.8 Hz, 1H), 7.78 (d, J=7.4 Hz, 1H), 7.73 (d, J=9.2 Hz, 1H), 7.63 (d, J=8.3 Hz, 1H), 7.60-7.55 (m, 2H), 7.52-7.44 (m, 2H), 7.40-7.37 (m, 1H), 7.20 (d, J=8.3 Hz, 1H), 7.09 (d, J=7.5 Hz, 1H), 6.94-6.90 (m, 1H), 4.99 (s, 2H), 1.23-1.09 (m, 5H). LCMS(ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

Example 24

Preparation of 2-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-1-yl)oxy)isonicotinic acid (Compound 24)

[0151] ##STR00053##

[0152] Following the procedure of Example 22, the title Compound 24 was obtained by substituting methyl 2-fluoroisonicotinate for 22A-2.

[0153] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.22 (d, J=5.1 Hz, 1H), 7.67-7.62 (m, 2H), 7.58 (d, J=8.0 Hz, 2H), 7.53-7.42 (m, 3H), 7.38 (s, 2H), 7.11 (d, J=7.5 Hz, 1H), 6.89 (dd, J=9.2, 2.4 Hz, 1H), 4.98 (s, 2H), 1.22-1.07 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

Example 25

Preparation of 3-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-1-yl)oxy)picolinic acid (Compound 25)

[0154] ##STR00054##

[0155] Following the procedure of Example 22, the title Compound 25 was obtained by substituting methyl 3-fluoropicolinate for 22A-2.

[0156] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.41-8.37 (m, 1H), 7.90 (d, J=9.2 Hz, 1H), 7.62-7.54 (m, 3H), 7.53-7.47 (m, 2H), 7.43-7.35 (m, 2H), 7.35-7.30 (m, 1H), 6.99-6.94 (m, 1H), 6.77 (d, J=7.6 Hz, 1H), 5.00 (s, 2H), 1.21-1.10 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.20O.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

Example 26

Preparation of 2-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-1-yl)oxy)-4-fluorobenzoic acid (Compound 26)

[0157] ##STR00055##

[0158] Following the procedure of Example 22, the title Compound 26 was obtained by substituting methyl 2,4-difluorobenzoate for 22A-2.

[0159] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.22 (d, J=5.1 Hz, 1H), 7.67-7.61 (m, 2H), 7.57 (s, 2H), 7.52-7.43 (m, 3H), 7.38 (s, 2H), 7.11 (d, J=7.5 Hz, 1H), 6.90 (s, OH), 4.98 (s, 2H), 1.20-1.06 (m, 5H). LCMS (ESI): calculated for C.sub.30H.sub.20Cl.sub.2FNO.sub.5; [M+H].sup.+: 564.1, found: 564.1.

Example 27

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-1-yl)oxy)-2-methylnicotinic acid (Compound 27)

[0160] ##STR00056##

[0161] Following the procedure of Example 22, the title Compound 27 was obtained by substituting methyl 6-chloro-2-methylnicotinate for 22A-2.

[0162] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.20 (d, J=8.5 Hz, 1H), 7.63 (dd, J=8.8, 4.5 Hz, 2H), 7.57 (s, 1H), 7.52-7.43 (m, 1H), 7.38 (d, J=2.7 Hz, 1H), 7.11 (d, J=7.5 Hz, 1H), 6.90 (dd, J=9.2, 2.5 Hz, 1H), 6.81 (d, J=8.6 Hz, 1H), 4.98 (s, 2H), 2.48 (s, 3H), 1.23-1.00 (m, 5H). LCMS (ESI): calculated for C.sub.30H.sub.22Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 561.1, found: 561.1.

Example 28

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-1-yl)oxy)-5-methylnicotinic acid (Compound 28)

[0163] ##STR00057##

[0164] Following the procedure of Example 22, the title Compound 28 was obtained by substituting methyl 6-chloro-5-methylnicotinate for 22A-2.

[0165] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.31 (d, J=2.3 Hz, 1H), 8.19 (d, J=2.3 Hz, 1H), 7.64 (d, J=8.3 Hz, 1H), 7.61-7.55 (m, 3H), 7.53-7.44 (m, 2H), 7.38 (d, J=2.7 Hz, 1H), 7.11 (d, J=7.5 Hz, 1H), 6.88 (dd, J=9.1, 2.5 Hz, 1H), 4.98 (s, 2H), 2.47 (s, 3H), 1.20-1.08 (m, 5H). LCMS (ESI): calculated for C.sub.30H.sub.22Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 561.1, found: 561.1.

Example 29

Preparation of 6-((5-chloro-6-((5-cyclopropyl-3-(2,6-dichlorophenyl)-isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)nicotinic acid (Compound 29)

[0166] ##STR00058##

[0167] Following the procedure of Example 32, the title Compound 29 was obtained by substituting 6-bromo-1-chloronaphthalen-2-ol for 32A-1.

[0168] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.63 (s, 1H), 8.29 (d, J=8.6 Hz, 1H), 7.92 (d, J=8.9 Hz, 1H), 7.70 (s, 1H), 7.65-7.46 (m, 4H), 7.38 (s, 2H), 7.17 (d, J=8.5 Hz, 1H), 5.09 (s, 2H), 1.21-1.02 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.19Cl.sub.3N.sub.2O.sub.5; [M+H].sup.+: 581.0, found: 581.0.

Example 30

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-2-fluoronaphthalen-1-yl)oxy)nicotinic acid (Compound 30)

[0169] ##STR00059##

[0170] Following the procedure of Example 20, the title Compound 30 was obtained by substituting 6-bromo-2-fluoronaphthalen-1-ol for 20A-1.

[0171] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.59 (d, J=2.4 Hz, 1H), 8.31 (dd, J=8.6, 2.4 Hz, 1H), 7.86 (d, J=9.1 Hz, 1H), 7.66 (d, J=6.9 Hz, 1H), 7.59 (s, 1H), 7.56 (dd, J=5.7, 3.3 Hz, 1H), 7.51 (dd, J=9.0, 7.1 Hz, 1H), 7.46-7.39 (m, 2H), 7.25 (d, J=8.6 Hz, 1H), 7.05 (dd, J=9.2, 2.4 Hz, 1H), 5.02 (s, 2H), 1.28-1.08 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.19Cl.sub.2FN.sub.2O.sub.5; [M+H].sup.+: 565.1, found: 565.1.

Example 31

Preparation of 6-((7-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)nicotinic acid (Compound 31)

[0172] ##STR00060##

[0173] Following the procedure of Example 1, the title Compound 31 was obtained by substituting naphthalene-2,7-diol for 1A-2.

[0174] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.64 (s, 1H), 8.28 (d, J=8.6 Hz, 1H), 7.85 (d, J=8.8 Hz, 1H), 7.77 (d, J=9.0 Hz, 1H), 7.58 (d, J=8.1 Hz, 2H), 7.55-7.43 (m, 2H), 7.27 (s, 1H), 7.13 (t, J=9.9 Hz, 2H), 6.89 (d, J=8.9 Hz, 1H), 4.95 (s, 2H), 1.29-1.06 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

Example 32

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-5-fluoronaphthalen-2-yl)oxy)nicotinic acid (Compound 32)

[0175] ##STR00061##

[0176] (a) Referring to the following reaction equation (Route B), Compound 32A-1 (1.0 g, 4.15 mmol, 1 eq.), Compound 1A-1 (1.44 g, 4.15 mmol, 1 eq.) and cesium carbonate (2.70 g, 8.30 mmol, 2 eq.) were dissolved in DMF (10 ml) for reacting. The reaction was carried out at 65° C. for 2 h. After cooling, 10 ml water and 10 ml EA (ethyl acetate) were added for extraction, and the organic phase was washed with water and concentrated to dryness to give Compound 32A, 4-(((6-bromo-1-fluoronaphthalen-2-yl)oxy)methyl)-5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazole, 1.51 g, yield: 71.9%. LCMS (ESI): calculated for C.sub.23H.sub.15BrCl.sub.2FNO.sub.2; [M+H].sup.+: 506.0, found: 506.0.

##STR00062##

[0177] (b) Referring to the following reaction equation, compound 32A (200 mg, 0.39 mmol, 1 eq) was dissolved in dry THF (2 ml), then KOAc (76 mg, 0.78 mmol, 2 eq), Pd(dppf).sub.2Cl.sub.2 (28 mg, 0.039 mmol, 0.1 eq), and bis(pinacolato)diboron (100 mg, 0.39 mmol, 1 eq) were added under N.sub.2, and the reaction mixture was heated to reflux for 2 h. After cooling, 10 ml water and 10 ml EA were added for extraction, and the organic phase was washed with water and concentrated to dryness. The residue was purified by silica gel column chromatography (petroleum:AcOEt=3:1) to give Compound 32B, 5-cyclopropyl-3-(2,6-dichlorophenyl)-4-(((1-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-yl)oxy)methyl)isox azole, 137 mg, yield: 62.8%. LCMS (ESI): calculated for C.sub.29H.sub.27BCl.sub.2FNO.sub.4; [M+H].sup.+: 554.1, found: 554.1.

##STR00063##

[0178] (c) Referring to the following reaction equation, compound 32B (100 mg) was dissolved in EtOH (2 ml), then 30% H.sub.2O.sub.2 aqueous solution (1 ml) were added. The reaction mixture was stirred at room temperature for 1 h, quenched with saturated aqueous Na.sub.2SO.sub.3, and extracted with EA. The organic phase was concentrated and purified on a column (PE/EA=3/1) to give the compound 32C (61 mg, yield: 76.2%). LCMS (ESI): calculated for C.sub.23H.sub.16Cl.sub.2FNO.sub.3; [M+H].sup.+: 444.1, found: 444.1.

##STR00064##

[0179] (d) Referring to the following reaction equation, Compound 32C (50 mg, 0.11 mmol, 1 eq.), 1A-3 (24.3 mg, 0.11 mmol, 1 eq.) and cesium carbonate (71.5 mg, 0.22 mmol, 2 eq.) were dissolved in DMF (1 ml) for reacting. The reaction was carried out at 65° C. for 2 h. After cooling, 5 ml water and 5 ml EA were added for extraction, and the organic phase was washed with water and concentrated to dryness to give Compound 32D, 40 mg, yield: 61.5%. LCMS

[0180] (ESI): calculated for C.sub.30H.sub.21Cl.sub.2FN.sub.2O.sub.5; [M+H].sup.+: 579.1, found: 579.1.

##STR00065##

[0181] (e) Referring to the following reaction equation, compound 32D (30 mg) was dissolved in MeOH (1 ml), then 10% NaOH aqueous solution (0.5 ml) were added under N.sub.2, and the reaction mixture was heated to reflux for 1 h. The pH of the reaction solution was adjusted to 3 to 4 by adding 1N HCl solution, and 5 ml EA was added for extraction. The organic phase was concentrated and purified on a column (PE/EA/AcOH=1/1/0.01 elution) to give the title compound 32 (21 mg, yield: 71.7%).

[0182] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.59 (d, J=2.3 Hz, 1H), 8.31 (dd, J=8.6, 2.4 Hz, 1H), 7.86 (d, J=9.1 Hz, 1H), 7.62 (d, J=5.3 Hz, 1H), 7.59 (s, 1H), 7.56 (dd, J=5.7, 3.3 Hz, 1H), 7.51 (dd, J=9.0, 7.1 Hz, 1H), 7.46-7.40 (m, 2H), 7.25 (d, J=8.6 Hz, 1H), 7.06 (dd, J=5.7, 4.3 Hz, 1H), 5.02 (s, 2H), 1.26-1.09 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.19Cl.sub.2FN.sub.2O.sub.5; [M+H].sup.+: 565.1, found: 565.1.

##STR00066##

Example 33

Preparation of Sodium 6 #6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl) methoxy) naphthalen-2-yl)oxy)nicotinate

[0183] ##STR00067##

[0184] An aq. solution of NaOH (30%, 1.44 g, 1.2 eq) was added to a solution of Compound 1 (4.99 g, 9.12 mmol) in EtOH at r.t. After the reaction mixture was heated at reflux for 6 h, it was cooled to r.t. The solid was collected by filtration, washed with EtOH (10 ml), and dried to give a gray solid (4.07 g, yield: 78.3%).

##STR00068##

Example 34

Preparation of Calcium 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy) naphthalen-2-yl)oxy)nicotinate

[0185] ##STR00069##

[0186] To a solution of Compound 35 (1.00 g, 1.76 mmol) in water (10 ml) was added a solution of CaCl.sub.2 (1.0 g, 20%) in water. White precipitates formed. After the reaction mixture was stirred at r.t. for 4 h, the solid was collected by filtration, washed with water (2.0 ml) to give the product as a white solid (0.80 g, 76.7%).

##STR00070##

Example 35

Preparation of 2-((6((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy) naphthalen-1-yl)oxy)nicotinic acid

[0187] ##STR00071##

[0188] The title compound 35 was prepared according to Route D, following the procedure of Example 22. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.31 (s, 1H), 8.29-8.24 (m, 1H), 8.15-8.10 (m, 1H), 7.67 (d, J=9.1 Hz, 1H), 7.64-7.56 (m, 3H), 7.53-7.47 (m, 1H), 7.47-7.42 (m, 1H), 7.36 (d, J=2.6 Hz, 1H), 7.22-7.17 (m, 1H), 7.06 (d, J=7.4 Hz, 1H), 6.92-6.87 (m, 1H), 4.98 (s, 2H), 1.24-1.08 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

Example 36

Preparation of 6-((5-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy) naphthalen-2-yl)oxy)nicotinic acid

[0189] ##STR00072##

[0190] The title compound 36 was prepared according to Route B, following the procedure of Example 32. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.19 (s, 1H), 8.65 (d, J=2.4 Hz, 1H), 8.29 (dd, J=8.6, 2.4 Hz, 1H), 7.77 (d, J=9.1 Hz, 1H), 7.65-7.54 (m, 3H), 7.51-7.44 (m, 1H), 7.44-7.34 (m, 2H), 7.19 (dd, J=9.2, 2.4 Hz, 1H), 7.14 (d, J=8.7 Hz, 1H), 7.01 (d, J=7.4 Hz, 1H), 5.09 (s, 2H), 1.31-1.07 (m, 6H). LCMS (ESI): calculated for C.sub.29H.sub.20Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 547.1, found: 547.1.

Example 37

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-5-fluoronaphthalen-2-yl)oxy)-2-methylnicotinic acid

[0191] ##STR00073##

[0192] The title compound 37 was prepared according to Route B, following the procedure of Example 32. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.01 (s, 1H), 8.24 (d, J=8.6 Hz, 1H), 7.91 (d, J=9.1 Hz, 1H), 7.68 (s, 1H), 7.62 (d, J=9.1 Hz, 1H), 7.58 (d, J=7.9 Hz, 2H), 7.55-7.51 (m, 1H), 7.43-7.34 (m, 2H), 6.92 (d, J=8.6 Hz, 1H), 5.09 (s, 2H), 2.52 (s, 3H), 1.19-1.08 (m, 4H). LCMS (ESI): calculated for C.sub.30H.sub.21Cl.sub.2FN.sub.2O.sub.5; [M+H].sup.+: 579.1, found: 579.1.

Example 38

Preparation of 6-((7-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-8-fluoronaphthalen-2-yl)oxy)nicotinic acid

[0193] ##STR00074##

[0194] The title compound 38 was prepared according to Route B, following the procedure of Example 32. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.64 (s, 1H), 8.30 (dd, J=8.7, 2.4 Hz, 1H), 7.96 (d, J=8.9 Hz, 1H), 7.70 (d, J=9.0 Hz, 1H), 7.64-7.48 (m, 3H), 7.37 (t, J=8.8 Hz, 1H), 7.27 (d, J=9.2 Hz, 1H), 7.19 (d, J=8.6 Hz, 1H), 6.85 (s, 1H), 5.10 (s, 2H), 2.07-1.89 (m, 1H), 0.94-0.76 (m, 4H). LCMS (ESI): calculated for C.sub.29H.sub.19Cl.sub.2FN.sub.2O.sub.5; [M+H].sup.+: 565.1, found: 565.1.

Example 39

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-5-fluoronaphthalen-1-yl)oxy)-2-methylnicotinic acid

[0195] ##STR00075##

[0196] The title compound 39 was prepared according to Route B, following the procedure of Example 32. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.94 (dd, J=7.9, 5.0 Hz, 1H), 7.83 (d, J=8.1 Hz, 1H), 7.72-7.59 (m, 2H), 7.58-7.49 (m, 2H), 7.49-7.38 (m, 2H), 7.25-6.97 (m, 1H), 6.67 (d, J=7.9 Hz, 1H), 5.52 (d, J=16.9 Hz, 1H), 5.24 (d, J=16.9 Hz, 1H), 2.70-2.96 (M, 1H), 2.61 (s, 3H), 1.05-0.89 (m, 4H). LCMS (ESI): calculated for C.sub.30H.sub.21Cl.sub.2FN.sub.2O.sub.5; [M+H].sup.+: 579.1, found: 579.1.

Example 40

Preparation of 6((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-1-yl)oxy)-5-methylpicolinic acid

[0197] ##STR00076##

[0198] The title compound 40 was prepared according to Route D, following the procedure of Example 22. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.34 (d, J=8.5 Hz, 1H), 7.79 (d, J=8.2 Hz, 1H), 7.65-7.55 (m, 2H), 7.54-7.49 (m, 2H), 7.50-7.40 (m, 2H), 7.22 (t, J=2.3 Hz, 1H), 7.03-6.93 (m, 2H), 5.44 (s, 2H), 2.95-2.58 (m, 1H), 2.22 (s, 3H), 1.01 (m, 4H). LCMS (ESI): calculated for C.sub.30H.sub.22Cl.sub.2N.sub.2O.sub.5; [M+H].sup.+: 561.1, found: 561.1.

Example 41

Preparation of 6-((2,4-dichloro-6-((5-cyclopropyl-3-(2,6-dichlorophenyl) isoxazol-4-yl)methoxy)naphthalen-1-yl)oxy)nicotinic acid

[0199] ##STR00077##

[0200] The title compound 41 was prepared according to Route C, following the procedure of Example 20. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.17 (s, 1H), 8.66 (d, J=2.4 Hz, 1H), 8.34 (dd, J=8.6, 2.4 Hz, 1H), 7.84-7.78 (m, 2H), 7.70-7.65 (m, 2H), 7.64-7.56 (m, 2H), 7.38 (dd, J=9.1, 2.3 Hz, 1H), 7.24 (d, J=8.6 Hz, 1H), 4.92 (s, 2H), 2.45-2.41 (m, 1H), 1.22-1.10 (m, 4H). LCMS (ESI): calculated for C.sub.29H.sub.18Cl.sub.4N.sub.2O.sub.5; [M+H].sup.+: 615.0, found: 615.0.

Example 42

Preparation of 6-((2-chloro-6-((5-cyclopropyl-3-(2,6-dichlorophenyl) isoxazol-4-yl)methoxy)naphthalen-1-yl)oxy)nicotinic acid

[0201] ##STR00078##

[0202] The title compound 42 was prepared according to Route C, following the procedure of Example 20. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.15 (s, 1H), 8.65 (d, J=2.4 Hz, 1H), 8.31 (dd, J=8.6, 2.4 Hz, 1H), 7.76 (d, J=2.4 Hz, 1H), 7.70 (d, J=8.8 Hz, 3H), 7.66-7.59 (m, 1H), 7.51 (d, J=8.8 Hz, 1H), 7.44 (d, J=9.1 Hz, 1H), 7.22 (dd, J=9.1, 2.4 Hz, 1H), 7.19 (d, J=8.6 Hz, 1H), 4.87 (s, 2H), 2.46-2.40 (m, 1H), 1.30-1.09 (m, 4H). LCMS (ESI): calculated for C.sub.29H.sub.19Cl.sub.3N.sub.2O.sub.5; [M+H].sup.+: 581.0, found: 581.0.

Example 43

Preparation of 6-((1-chloro-6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)naphthalen-2-yl)oxy)-2-methylnicotinic acid

[0203] ##STR00079##

[0204] The title compound 43 was prepared according to Route C, following the procedure of Example 20. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.97 (s, 1H), 8.24 (d, J=8.6 Hz, 1H), 8.00 (d, J=9.2 Hz, 1H), 7.80 (d, J=8.9 Hz, 1H), 7.60 (d, J=8.0 Hz, 2H), 7.55-7.49 (m, 1H), 7.47 (s, 1H), 7.43 (d, J=8.8 Hz, 1H), 7.12 (dd, J=9.2, 2.5 Hz, 1H), 6.95 (d, J=8.6 Hz, 1H), 5.02 (s, 2H), 2.47 (s, 3H), 1.23-1.11 (m, 5H). LCMS (ESI): calculated for C.sub.30H.sub.21Cl.sub.3N.sub.2O.sub.5; [M+H].sup.+: 595.1, found: 595.1.

Example 44

Preparation of 2-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-2-fluoronaphthalen-1-yl)oxy)-4-fluorobenzoic acid

[0205] ##STR00080##

[0206] The title compound 44 was prepared according to Route C, following the procedure of Example 20. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.04 (s, 1H), 7.82 (t, J=8.6 Hz, 1H), 7.71 (d, J=9.2 Hz, 1H), 7.57 (d, J=7.8 Hz, 2H), 7.52-7.46 (m, 1H), 7.39 (d, J=2.4 Hz, 1H), 7.37-7.31 (m, 1H), 7.15-7.10 (m, 1H), 7.01 (dd, J=9.3, 2.4 Hz, 1H), 6.88 (dd, J=12.3, 2.4 Hz, 1H), 6.73 (dd, J=8.7, 2.3 Hz, 1H), 5.08 (s, 2H), 1.28-1.05 (m, 4H). LCMS (ESI): calculated for C.sub.30H.sub.19Cl.sub.2F.sub.2NO.sub.5; [M+H].sup.+: 582.1, found: 582.1.

Example 45

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-2-fluoronaphthalen-1-yl)oxy)-2-methylnicotinic acid

[0207] ##STR00081##

[0208] The title compound 45 was prepared according to Route C, following the procedure of Example 20. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.98 (s, 1H), 8.21 (d, J=8.6 Hz, 1H), 7.65 (dd, J=9.3, 1.7 Hz, 1H), 7.57 (d, J=7.7 Hz, 2H), 7.51-7.44 (m, 1H), 7.37 (d, J=2.5 Hz, 1H), 7.35-7.29 (m, 1H), 7.16-7.11 (m, 1H), 6.98 (dd, J=9.2, 2.5 Hz, 1H), 6.87 (d, J=8.5 Hz, 1H), 5.08 (s, 2H), 1.22-1.09 (m, 4H). LCMS (ESI): calculated for C.sub.30H.sub.21Cl.sub.2FN.sub.2O.sub.5; [M+H].sup.+: 579.1, found: 579.1.

Example 46

Preparation of 6((2-chloro-6 ((5-cyclopropyl-3-(2,6-dichlorophenyl) isoxazol-4-yl)methoxy)naphthalen-1-yl)oxy)-2-methylnicotinic acid

[0209] ##STR00082##

[0210] The title compound 46 was prepared according to Route C, following the procedure of Example 20. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.03 (s, 1H), 8.25 (d, J=8.6 Hz, 1H), 7.76 (d, J=9.2 Hz, 1H), 7.69 (d, J=8.2 Hz, 1H), 7.58 (d, J=8.0 Hz, 2H), 7.50 (d, J=8.1 Hz, 1H), 7.49-7.44 (m, 1H), 7.18 (d, J=8.2 Hz, 1H), 7.04 (dd, J=9.2, 2.4 Hz, 1H), 6.95 (d, J=8.6 Hz, 1H), 5.13 (s, 2H), 2.59-2.54 (m, 1H), 2.48 (s, 3H), 1.27-1.15 (m, 4H). LCMS (ESI): calculated for C.sub.30H.sub.21Cl.sub.3N.sub.2O.sub.5; [M+H].sup.+: 595.1, found: 595.1.

Example 47

Preparation of 6-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-2-fluoronaphthalen-1-yl)oxy)-5-methylnicotinic acid

[0211] ##STR00083##

[0212] The title compound 47 was prepared according to Route C, following the procedure of Example 20. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.09 (s, 1H), 8.32 (s, 1H), 8.21 (s, 1H), 7.67-7.55 (m, 3H), 7.55-7.45 (m, 1H), 7.39 (s, 1H), 7.37-7.30 (m, 1H), 7.20-7.09 (m, 1H), 7.03-6.93 (m, 1H), 5.09 (s, 2H), 2.48 (s, 3H), 1.41-1.00 (m, 5H). LCMS (ESI): calculated for C.sub.30H.sub.21Cl.sub.2FN.sub.2O.sub.5; [M+H].sup.+: 579.1, found: 579.1.

Example 48

Preparation of 2-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-2-fluoronaphthalen-1-yl)oxy)nicotinic acid

[0213] ##STR00084##

[0214] The title compound 48 was prepared according to Route C, following the procedure of Example 20. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.29-8.27 (m, 1H), 8.22 (d, J=8.5 Hz, 1H), 8.09-8.06 (m, 1H), 7.53-7.47 (m, 3H), 7.46-7.42 (m, 1H), 7.30-7.25 (m, 2H), 7.08-7.05 (m, 1H), 6.96 (dd, J=8.4, 2.4 Hz, 1H), 5.44 (s, 2H), 2.79 (p, J=6.4 Hz, 1H), 1.20-1.09 (m, 5H). LCMS (ESI): calculated for C.sub.29H.sub.19C.sub.12FN.sub.2O.sub.5; [M+H].sup.+: 565.1, found: 565.1.

Example 49

Preparation of 2-((6-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)-2-fluoronaphthalen-1-yl)oxy)-4-fluorobenzoic acid

[0215] ##STR00085##

[0216] The title compound 49 was prepared according to Route D, following the procedure of Example 22. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.62 (s, 1H), 7.84 (d, J=9.2 Hz, 1H), 7.63-7.55 (m, 3H), 7.54-7.49 (m, 1H), 7.42 (t, J=7.9 Hz, 1H), 7.40-7.32 (m, 2H), 7.06 (t, J=8.8 Hz, 1H), 6.96 (dd, J=9.1, 2.5 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 6.56 (d, J=8.4 Hz, 1H), 4.99 (s, 2H), 1.23-1.09 (m, 4H). LCMS (ESI): calculated for C.sub.30H.sub.20Cl.sub.2FN.sub.2O.sub.5; [M+H].sup.+: 564.1, found: 564.1.

Biology Examples

Example A

[0217] FXR Agonist Binding Ability

[0218] Evaluation of the activation effect of the compound of the present invention with regard to the binding of FXR to its co-stimulatory factor SRC-1 was conducted by using time-resolved analytical techniques. The results are listed in Table 1.

[0219] Experimental Materials

[0220] 1. Protein: glutathione-S-transferase (GST)-labeled human FXR protein (Invitrogen)

[0221] 2. Coactivator: Fluorescein-labeled steroid receptor coactivator (SRC2-2) (Invitrogen)

[0222] 3. Detection reagent: LanthaScreen Time-Resolved Fluorescence Analysis Kit (Invitrogen)

[0223] Experimental Method [0224] 1. The compound was prepared into a 10 mM DMSO stock solution and stored in a −20° C. refrigerator for a long time.

[0225] 2. The compound was diluted to 1 mM before the experiment, and then the compound was diluted 3-fold to 10 concentration points using DMSO. Then use buffer G (Invitrogen, PV4553) to dilute these 10 concentration points by 50 times to become a working fluid. Add 10 μl of each working solution to each well of a 384-well plate.

[0226] 3. Prepare a human FXR protein solution (final concentration 20 nM) in chilled buffer G and add 5 μl of human FXR protein solution to each well of a 384-well plate.

[0227] 4. Prepare a mix of buffer G containing 2 μM fluorescein-labeled steroid receptor coactivator and 20 nM GST antibody.

[0228] 5. Add 5 μl of the mixture prepared in Step 4 to a 384-well plate.

[0229] 6. Centrifuge the 384-well plate at 1000 g for 1 minute.

[0230] 7. Incubate for 1 hour at room temperature in the dark.

[0231] 8. Read the 384-well plate at 520 nm and 495 nm on an Envision 2104 plate reader.

[0232] 9. Calculate the EC.sub.50 value of the activation effect of the compound.

Example B

[0233] FXR Agonist Transactivation Ability

[0234] Evaluation of the ability of the compound of the present invention to promote FXR transactivation was conducted by using luciferase reporter gene expression technology. The results are listed in Table 1.

[0235] Experimental Materials

[0236] 1. Cell line: HEK293T (Invitrogen)

[0237] 2. Expression plasmid: pBIND-hFXR-LBD-GAL4 (Promega), pGL4.35-luciferase (Promega)

[0238] 3. Cell culture medium: 10% serum and penicillin/streptomycin double antibody in DMEM medium

[0239] 4. Detection reagent: Steady-Glo fluorescence detection system (Promega).

[0240] 5. Transfection reagent: TransIT-293 Transfection Reagent (MIRUS BIO)

[0241] Experimental Method

[0242] 1. The compound was prepared into a 10 mM DMSO stock solution and stored in a −20° C. refrigerator for a long time.

[0243] 2. Resuscitation HEK293T cells were seeded in 10 cm culture dishes at a concentration of 5.5×10.sup.6 and incubated for 16 hours at 37° C. in a 5% CO.sub.2 incubator.

[0244] 3. Return the transfection reagent to room temperature before transfection. The Trans-IT solution was added dropwise to Opti-MEM, and mixed by inversion for 5 minutes; the expression plasmid was added, mixed by inversion, and incubated at room temperature for 20 minutes.

[0245] 4. Add the transfection mixture from Step 3 to the prepared 10 cm dish in Step 2 and incubate for 5-6 hours in a 5% CO.sub.2 incubator.

[0246] 5. Dilute the compound 3 folds to 10 concentration points using DMSO; add 25 nl of compound per well in a 384-well plate using an Echo 550 sonic pipette; add HEK293T cells to the 384-well plate at a concentration of 15,000 cells/well; 37° C. incubate for 16-20 hours in a 5% CO.sub.2 incubator.

[0247] 6. Add 25 μl of Steady-Glo Fluorescent Reagent to each well and read the fluorescence on an Envision 2104 plate reader.

[0248] 7. Calculate the EC.sub.50 value of the activation of the compound.

TABLE-US-00001 TABLE 1 FXR agonist binding activity values grouped in the following range: A indicates EC50 < 50 nM; B indicates 50 < EC50 < 500 nM; C indicates EC50 > 500 nM. Compound FXR binding activity Compound 1 B Compound 2 B Compound 3 C Compound 4 B Compound 5 C Compound 6 B Compound 7 C Compound 8 B Compound 9 B Compound 10 B Compound 11 A Compound 12 A Compound 13 B Compound 14 B Compound 15 B Compound 16 A Compound 17 C Compound 18 B Compound 19 C Compound 20 B Compound 21 B Compound 22 A Compound 23 C Compound 24 A Compound 25 B Compound 26 A Compound 27 A Compound 28 A Compound 29 B Compound 30 A Compound 31 A Compound 32 A Compound 33 B Compound 34 B Compound 35 A Compound 36 B Compound 37 B Compound 38 A Compound 39 A Compound 40 C Compound 41 C Compound 42 C Compound 43 B Compound 44 A Compound 45 A Compound 46 B Compound 47 A Compound 48 A Compound 49 B

TABLE-US-00002 TABLE 2 cell activity values grouped in the following range: A indicates EC50 < 200 nM; B indicates 500 < EC50 < 1000 nM; C indicates EC50 > 1000 nM. Compound Cell activity Compound 1 A Compound 2 C Compound 3 C Compound 4 C Compound 5 C Compound 6 C Compound 7 C Compound 8 B Compound 9 C Compound 10 A Compound 11 B Compound 12 C Compound 13 C Compound 14 C Compound 15 C Compound 16 C Compound 17 C Compound 18 C Compound 19 C Compound 20 B Compound 21 A Compound 22 A Compound 23 C Compound 24 A Compound 25 C Compound 26 A Compound 27 A Compound 28 A Compound 29 B Compound 30 A Compound 31 A Compound 32 A Compound 33 A Compound 34 A Compound 35 A Compound 36 C Compound 37 C Compound 38 B Compound 39 A Compound 40 C Compound 41 C Compound 42 C Compound 43 B Compound 44 A Compound 45 A Compound 46 A Compound 47 A Compound 48 A Compound 49 C

Example C

[0249] Mouse Pharmacokinetic Studies

[0250] Typical PK study procedure. For i.v. PK studies, a group of three fasted Male CD-1 mice were dosed with the compound (2.0 mg/kg, 0.50 mg/mL in 5% solutol in saline, clear solution), and 0.02 mL blood was collected at 0.0830, 0.250, 0.500, 1.00, 2.00, 4.00, 8.00, 24.0 h. For PO PK studies, a group of three fasted Male CD-1 mice were dosed with the compound (10 mg/kg, 1 mg/mL in 5% solutol in saline, clear solution), and 0.02 mL blood was collected at 0.250, 0.500, 1.00, 2.00, 4.00, 6.00, 8.00, 24.0 h. The blood was collected into EP tubes (containing EDTA K20.85-1.15 mg) and centrifuged at 3,000 g or 3,200 g at 4° C. for 10 min) and plasma was isolated and divided into two vials. One was used for bioanalysis and one was kept as backup. The samples were kept at −60° C. or below, until being analyzed by LC MS/MS. The samples were analyzed with AB SCIEX INSTRUMENTS LC-MS/MS AU-Triple Quad 6500 Plus and data were processed with Phoenix WinNonlin 6.3 (IV-noncompartmental model and PO-noncompartmental model).

[0251] PK Results

[0252] Pharmacokinetic results in mouse is shown in Table 3.

TABLE-US-00003 TABLE 3 Mouse Pharmacokinetic results IV PO T½ Cl Vd AUC .sub.0-t Cmax Tmax AUC .sub.0-t F Compound (h) (mL/min/kg) (L/kg) (ng•h/mL) (ng/ml) (h) (ng•h/mL) (%) 1 0.85 47.4 1.75 705 641 0.83 1159   33% 10 1.6 9.26 0.66 3553 5503 0.417 9361 52.6% 11 1.44 6.66 0.294 4977 5726 0.5 8329 33.3% 21 1.28 35.8 1.75 969 1480 0.5 2220 46.2% 22 0.837 73.8 1.84 448 894 0.5 718   32% 24 0.94 105 2.94 330 209 0.3 198   12% 26 0.62 35 0.745 969 900 0.5 830   17% 27 1.61 19.7 0.897 1690 2078 0.5 2785 33.2% 28 0.977 28.4 0.663 1177 2130 0.5 1743 29.9% 31 0.614 110 2.77 304 133 0.333 140 9.74% 32 1.03 24 1.36 1395 1395 0.5 3810 54.7% 44 1.23 60.4 2.06 559 384 0.933 646 23.7% 45 1.4 22.1 1.04 1501 627 1.00 1140 15.7% 47 0.638 22.2 0.495 1500 2146 0.5 2140 28.6%

Example D

[0253] Therapeutic efficacy of FXR compounds was evaluated in STZ+DEN+HFD induced NASH model in male C57BL/6 mice. Briefly, newborn male C57BL/6 mice were injected with streptozocin (STZ) at Week 2 to introduce diabetes and diethylnitrosamine (DEN) at Week 4 to promote liver fibrosis. Mice receiving neither STZ nor DEN were used as negative control (Group 1, n=12) and were fed with normal diet. At Week 6, 60 diabetic mice were selected (blood glucose>12 mmol/L after 6 h of fasting) and were fed with high fat diet (HFD, diet that contains 60 kcal % fat). After one week on HFD, animals were randomly assigned into 5 groups based on body weight: Group 2 (n=12), disease model group, no compound treatment; Group 3 (n=10), positive control group, treated with OCA (30 mg/kg); Group 4 (n=12), treated with Compound 1 (3 mg/kg); Group 5 (n=12), treated with Compound 1 (10 mg/kg); Group 6 (n=12), treated with Compound 1 (30 mg/kg). OCA and Compounds 1 were PO QD, for 7 weeks. On the day after last dose, all animals were euthanized and liver tissues were fixed with formalin for pathological evaluation.

[0254] FIG. 1 shows reduction of NAS score after Compound 1 treatment in STZ+DEN+HFD mice disease model. FIG. 2 shows reduction of liver fibrosis after Compound 1 treatment in STZ+DEN+HFD mice disease model. As shown in FIG. 1, treatment of Compound 1 at low dose (3 mg/kg), medium dose (10 mg/kg), and high dose (30 mg/kg), respectively, showed a dose dependent decrease in hepatocyte steatosis (p<0.001). The 30 mg/kg group showed a decreased NAS score by 46.2% as compared with the model group. The positive control group (OCA 30 mg/kg) also showed decrease in NAS score. As shown in FIG. 2, treatment of Compound 1 at low (3 mg/kg) and high doses (30 mg/kg) significantly inhibited the progress of liver fibrosis and the 30 mg/kg doses group lowered cirrhosis percentage by 15.2%. In conclusion, after 7-week treatment at 30 mg/kg per day, Compound 1 significantly decreases in NAS score and liver fibrosis.

Example E

[0255] Therapeutic efficacy of FXR compounds was evaluated in DEN+HFD+CHOL induced NASH model in male SD rats.

[0256] Newborn male rats received DEN injection at Week 2 after birth to generate NASH model. Negative control animals (Group 1, n=10) received no DEN injection. At Week 4, 50 rats that received DEN injections started HFD+CHOL diet (60 kcal % Fat+1.25% Cholesterol+0.5% cholate) for 8 weeks, while negative control animals were still on normal diet. At Week 5, DEN treated rats were randomly assigned into 5 groups (Group 2-6) based on body weight. Disease Group (Group 2, n=10), received no treatment; OCA group (Group 3, n=10) received OCA at 30 mg/kg. Group 4-6 (n=10 each) received Compound 1 at low (3 mg/kg), medium (3 mg/kg), and high doses (3 mg/kg). OCA and Compound 1 were given PO QD, for 7 weeks. On the day after last dose, all animals were euthanized and liver tissues were fixed with formalin for pathological evaluation.

[0257] FIG. 3 shows reduction of NAS score after Compound 1 treatment in DEN+HFD+CHOL treated rats. FIG. 4 shows reduction of liver fibrosis after Compound 1 treatment in DEN+HFD+CHOL treated rats.

[0258] As shown in FIG. 3, treatment of Compound 1 at low (3 mg/kg), medium (10 mg/kg), and high doses (30 mg/kg), respectively, showed a dose dependent decrease in NAS score (all p<0.001). The NAS score of 30 mg/kg group was lowered by 42.9% as compared to the model group (Group 2). The positive control (OCA 30 mg/kg) also showed a decrease in NAS score (p<0.001). As shown in FIG. 4, treatment of Compound 1 at low (3 mg/kg), medium (10 mg/kg), and high doses (30 mg/kg), significantly inhibited the progress of liver fibrosis (both p<0.01). Treatment at 30 mg/kg group lowered cirrhosis percentage by 28.0%.