USE AND PHARMACEUTICAL COMPOSITION OF PHENYLISOXAZOLYL METHYLENE-NAPHTHALENE-ETHER DERIVATIVES

Abstract

Provided are a method of treating or preventing infection with hepatitis B virus in a human or animal, comprising administering to the human or animal in need thereof a therapeutically effective amount of a phenylisoxazolyl methylene-naphthalene-ether derivative having a structure of formula (I); use of a phenylisoxazolyl methylene-naphthalene-ether derivative having a structure of formula (I) in the preparation of a pharmaceutical composition for anti-hepatitis B vims and a pharmaceutical composition for anti-hepatitis B virus.

##STR00001##

Claims

1. A method of treating or preventing infection with hepatitis B virus in a human or animal, comprising administering to the human or animal in need thereof a therapeutically effective amount of a phenylisoxazolyl methylene-naphthalene-ether derivative having a structure of formula (I), or a pharmaceutically acceptable salt, ester or stereoisomer thereof: ##STR00078## wherein: R.sup.1, R.sup.2 and R.sup.3 are independently selected from H, halogen, unsubstituted or halogen substituted C.sub.1-6 alkyl and unsubstituted or halogen substituted C.sub.1-6 alkoxy, provided that at least one of R.sup.1, 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 or 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; and wherein R4 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 —CONHSO2-5-10 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 method according to claim 1, 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 or cyclopropyl; 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; and wherein the halogen is fluoro or chloro.

3. The method according to claim 2, wherein R.sup.1, R.sup.2 and R.sup.3 are independently selected from H, Cl, F and —O—CF.sub.3; and 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, 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, R.sup.5 is selected from H, C.sub.1-3 alkyl and halogen.

4. The method according to claim 3, wherein Z is pyridyl; R.sup.4 is —COOH; and R.sup.5 is H or halogen.

5. The method according to claim 1, wherein the phenylisoxazolyl methylene-naphthalene-ether derivative is of one of the following structures: ##STR00079## ##STR00080## ##STR00081## ##STR00082##

49. The method according to claim 4, wherein the phenylisoxazolyl methylene-naphthalene-ether derivative is of the following structure: ##STR00083##

7. The method according to claim 1, wherein the method comprises administering the phenylisoxazolylmethylene-naphthalene-ether derivative having the structure of formula (I), or a pharmaceutically acceptable salt, ester or stereoisomer thereof in combination with one or more other anti-HBV agents.

8. The method of claim 7, wherein the other anti-HBV agents are selected from HBV vaccines, HBV DNA polymerase inhibitors, immunomodulators, toll-like receptor (TLR) modulators, interferon alpha receptor ligands, hepatitis b surface antigen (HBsAg) inhibitors, cyclophilin inhibitors, HBV viral entry inhibitors, antisense oligonucleotide targeting viral mRNA, short interfering RNAs (siRNA) and ddRNAi endonuclease modulators, ribonucleotide reductase inhibitors, HBV E antigen inhibitors, covalently closed circular DNA (cccDNA) inhibitors, fatty acid synthase inhibitors, HBV antibodies, CCR2 chemokine antagonists, retinoic acid-inducible gene 1 stimulators, NOD2 stimulators, PD-1 inhibitors, PD-L1 inhibitors, KDM inhibitors, and HBV replication inhibitors.

9. The method of claim 8, wherein the HBV DNA polymerase inhibitor is entecavir or tenofovir, the PD-1 inhibitor is one or more selected from nivolumab, pembrolizumab, pidilizumab, BGB-108, SHR-1210, PDR-001, PF-06801591, IBI-308, cemiplimab, camrelizumab, sintilimab, tislelizumab (BGB-A317), BCD-100, JNJ-63723283, Zimberelimab (GLS-010, WBP-3055), Balstilimab (AGEN2034) and dostarlimab (TSR-042); the PD-L1 inhibitors is one or more selected from atezolizumab (RG-7446), avelumab, BGB-A333, BMS-936559 (MDX-1105), durvalumab, CX-072, GX-P2, KN035 (ASC022), GS-4224 and INCB086550; the antisense oligonucleotide is Ionis-HBVRx or Ionis-HBV-LRx the short interfering RNA is JNJ-3989, or Vir-2218, or DCR-HBVS the fatty acid synthase inhibitor is TVB-2640 and/or TVB-3567; the interferon is Pegasys; and the capsid inhibitor is one or more selected from ABI-H0731, ABI-H2158, ABI-H3733, CB-HBV-001, JNJ-6379, JNJ-0440, QL-007, RG-7907 and RO7049389.

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. A pharmaceutical composition for anti-hepatitis B virus, comprising a therapeutically effective amount of a phenylisoxazolyl methylene-naphthalene-ether derivative having a structure of formula (I), or a pharmaceutically acceptable salt, ester or stereoisomer thereof and one or more other anti-HBV agents, and a pharmaceutically acceptable auxiliary material, ##STR00084## wherein: R.sup.1, R.sup.2 and R.sup.3 are independently selected from H, halogen, unsubstituted or halogen substituted C.sub.1-6 alkyl and unsubstituted or halogen substituted C.sub.1-6 alkoxy, provided that at least one of R.sup.1, 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 X2 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 or 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; and 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.1-4 alkyl).

17. The pharmaceutical composition according to claim 16, 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 or cyclopropyl; 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; and wherein the halogen is fluoro or chloro.

18. The pharmaceutical composition according to claim 17, wherein R.sup.1, R.sup.2 and R.sup.3 are independently selected from H, Cl, F and —O—CF.sub.3; and 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, wherein R is selected from —COOH, —CH.sub.2COOH, —CONHSO.sub.2—C.sub.1-6 alkyl and —CONHSO.sub.2—C.sub.3-6 cycloalkyl, R.sup.5 is selected from H, C.sub.1-3 alkyl and halogen.

19. The pharmaceutical composition according to claim 18, wherein Z is pyridyl; R.sup.4 is —COOH; and R.sup.5 is H or halogen.

20. The pharmaceutical composition according to claim 16, wherein the phenylisoxazolyl methylene-naphthalene-ether derivative is of one of the following structures: ##STR00085## ##STR00086## ##STR00087## ##STR00088##

21. The pharmaceutical composition according to claim 16, wherein the phenylisoxazolyl methylene-naphthalene-ether derivative is of the following structure: ##STR00089##

22. The pharmaceutical composition according to claim 16, wherein the pharmaceutical composition comprises one or more other anti-HBV agents and the phenylisoxazolylmethylene-naphthalene-ether derivative having the structure of formula (I), or a pharmaceutically acceptable salt, ester or stereoisomer thereof.

23. The pharmaceutical composition of claim 22, wherein the other anti-HBV agents are selected from HBV vaccines, HBV DNA polymerase inhibitors, immunomodulators, toll-like receptor (TLR) modulators, interferon alpha receptor ligands, hepatitis b surface antigen (HBsAg) inhibitors, cyclophilin inhibitors, HBV viral entry inhibitors, antisense oligonucleotide targeting viral mRNA, short interfering RNAs (siRNA) and ddRNAi endonuclease modulators, ribonucleotide reductase inhibitors, HBV E antigen inhibitors, covalently closed circular DNA (cccDNA) inhibitors, fatty acid synthesis inhibitors, HBV antibodies, CCR2 chemokine antagonists, retinoic acid-inducible gene 1 stimulators, NOD2 stimulators, PD-1 inhibitors, PD-L1 inhibitors, KDM inhibitors, and HBV replication inhibitors.

24. The pharmaceutical composition of claim 23, wherein the HBV DNA polymerase inhibitor is entecavir or tenofovir, the PD-1 inhibitor is one or more selected from nivolumab, pembrolizumab, pidilizumab, BGB-108, SHR-1210, PDR-001, PF-06801591, IBI-308, cemiplimab, camrelizumab, sintilimab, tislelizumab (BGB-A317), BCD-100, JNJ-63723283, Zimberelimab (GLS-010, WBP-3055), Balstilimab (AGEN2034) and dostarlimab (TSR-042); the PD-L1 inhibitors is one or more selected from atezolizumab (RG-7446), avelumab, BGB-A333, BMS-936559 (MDX-1105), durvalumab, CX-072, GX-P2, KN035 (ASC022), GS-4224 and INCB086550; the antisense oligonucleotide is Ionis-HBVRx or Ionis-HBV-LRx; the short interfering RNA is JNJ-3989, Vir-2218, or DCR-HBVS; the fatty acid synthase inhibitor is TVB-2640 and/or TVB-3567; the interferon is Pegasys; and the capsid inhibitor is one or more selected from ABI-H0731, ABI-H2158, ABI-H3733, CB-HBV-001, JNJ-6379, JNJ-0440, QL-007, RG-7907 and RO7049389.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0096] 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.

[0097] FIG. 1 is EC50 curves of inhibition HBV DNA in PHH Cells;

[0098] FIG. 2 is EC50 curves of HBsAg inhibition in PHH Cells;

[0099] FIG. 3 is EC50 curves of HBV RNA inhibition in PHH Cells.

[0100] FIG. 4 is HBV DNA level in AAV-HBV mouse serum before and after treatment of compound 33.

[0101] FIG. 5 is HBV RNA level in AAV-HBV mouse serum before and after treatment of compound 33.

[0102] FIG. 6 is HBsAg level in AAV-HBV mouse serum before and after treatment of compound 33.

BEST MODE FOR CARRYING OUT THE INVENTION

[0103] 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)

[0104] ##STR00011##

[0105] (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 C23H17Cl2NO3; [M+H]+: 426.1, found: 426.1.

##STR00012##

[0106] (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 C30H22Cl2N2O5; [M+H]+: 561.1, found: 561.1.

##STR00013##

[0107] (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=1/1/0.01 elution, wherein PE is petroleum ether) to give the title compound 1 (36 mg, yield: 37.0%).

[0108] 1H NMR (400 MHz, DMSO-d6) 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 C29H20Cl2N2O5; [M+H]+: 547.1, found: 547.1, 13C NMR (400 MHz, DMSO-d6) δ 7.79, 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-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).

##STR00014##

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)

[0109] ##STR00015##

[0110] 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.

[0111] 1H NMR (400 MHz, DMSO-d6) 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 C28H19Cl2N3O5; [M+H]+: 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)

[0112] ##STR00016##

[0113] Following the procedure of Example 1, the title Compound 3 was obtained by substituting methyl 5,6-dichloronicotinate for 6-bromonicotinic acid methyl ester.

[0114] 1H NMR (400 MHz, DMSO-d6) 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, 511). LCMS (ESI): calculated for C29H19C3N2O5; [M+H]+: 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)

[0115] ##STR00017##

[0116] 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.

[0117] 1H NMR (400 MHz, DMSO-d6) 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 C27H18Cl2N2O5S; [M+H]+: 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)

[0118] ##STR00018##

[0119] 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.

[0120] 1H NMR (400 MHz, DMSO-d6) 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 C30H22Cl2N2O5; [M+H]+: 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)

[0121] ##STR00019##

[0122] 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%).

[0123] 1H NMR (400 MHz, DMSO-d6) 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 C32H25Cl2N3O6S; [M+H]+: 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)

[0124] ##STR00020##

[0125] 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.

[0126] 1H NMR (400 MHz, DMSO-d6) 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 C28H19Cl2N3O5; [M+H]+: 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)

[0127] ##STR00021##

[0128] Following the procedure of Example 1, the title Compound 8 was obtained by substituting methyl 2,6-dichloronicotinate for 6-bromonicotinic acid methyl ester.

[0129] 1H NMR (400 MHz, DMSO-d6) 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, 211), 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 C29H19Cl3N2O5; [M+H]+: 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)

[0130] ##STR00022##

[0131] Following the procedure of Example 1, the title Compound 9 was obtained by substituting methyl 5-bromopicolinate for 6-bromonicotinic acid methyl ester.

[0132] 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J=3.1 Hz, 1H), 8.03 (d, J=8.7 Hz, 11H), 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, 411), 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 C29H20Cl2N2O5; [M+H]+: 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)

[0133] ##STR00023##

[0134] 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.

[0135] 1H NMR (400 MHz, DMSO-d6) 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 C30H22Cl2N2O5; [M+H]+: 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)

[0136] ##STR00024##

[0137] Following the procedure of Example 1, the title Compound 11 was obtained by substituting methyl 2,6-dichloronicotinate for 6-bromonicotinic acid methyl ester.

[0138] 1H NMR (400 MHz, DMSO-d6) 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 C29H20Cl2N2O5; [M+H]+: 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)

[0139] ##STR00025##

[0140] Following the procedure of Example 1, the title Compound 12 was obtained by substituting methyl 2-fluoroisonicotinate for 6-bromonicotinic acid methyl ester.

[0141] 1H NMR (400 MHz, DMSO-d6) 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 C29H20Cl2N2O5; [M+H]+: 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)

[0142] ##STR00026##

[0143] Following the procedure of Example 1, the title Compound 13 was obtained by substituting methyl 3-fluoropicolinate for 6-bromonicotinic acid methyl ester.

[0144] 1H NMR (400 MHz, DMSO-d6) 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 C29H20Cl2N2O5; [M+H]+: 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)

[0145] ##STR00027##

[0146] Following the procedure of Example 1, the title Compound 14 was obtained by substituting methyl 2-fluorobenzoate for 6-bromonicotinic acid methyl ester.

[0147] 1H NMR (400 MHz, DMSO-d6) 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 C30H21Cl2NO5; [M+H]+: 546.1, found: 546.1

Example 15

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

[0148] ##STR00028##

[0149] Following the procedure of Example 1, the title Compound 15 was obtained by substituting methyl 2-chloronicotinate for 6-bromonicotinic acid methyl ester.

[0150] 1H NMR (400 MHz, DMSO-d6) 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 C29H20Cl2N2O5; [M+H]+: 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)

[0151] ##STR00029##

[0152] Following the procedure of Example 1, the title Compound 16 was obtained by substituting methyl 3-fluoroisonicotinate for 6-bromonicotinic acid methyl ester.

[0153] 1H NMR (400 MHz, DMSO-d6) 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 C29H20Cl2N2O5; [M+H]+: 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)

[0154] ##STR00030##

[0155] 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.

[0156] 1H NMR (400 MHz, DMSO-d6) 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, 211), 2.44-2.37 (m, 1H), 1.20-1.05 (m, 4H). LCMS (ESI): calculated for C30H21F3N206; [M+H]+: 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)

[0157] ##STR00031##

[0158] 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.

[0159] 1H NMR (400 MHz, DMSO-d6) 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 C29H19Cl2FN2O5; [M+H]+: 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)

[0160] ##STR00032##

[0161] 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.

[0162] LCMS (ESI): calculated for C30H22Cl2N206; [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)

[0163] ##STR00033##

[0164] (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 C17H11BrFNO3; [M+H]+: 376.0, found: 376.0.

##STR00034##

[0165] (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)2C12 (39 mg, 0.053 mmol, 0.1 eq), and bis(pinacolato)diboron (135 mg, 0.53 mmol, 1 eq) were added under N2, 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 C23H23BFNO5; [M+H]+: 424.2, found: 424.2.

##STR00035##

[0166] (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 Na2SO3, 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 C17H12FNO4; [M+H]+: 314.1, found: 314.1.

##STR00036##

[0167] (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%. LCMS (ESI): calculated for C30H21Cl2FN2O5; [M+H]+: 579.1, found: 579.1.

##STR00037##

[0168] (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 N2, 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%).

##STR00038##

[0169] 1H NMR (400 MHz, DMSO-d6) 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): calculated for C29H19Cl2FN2O5; [M+H]+: 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)

[0170] ##STR00039##

[0171] Following the procedure of Example 20, the title Compound 21 was obtained by substituting 6-bromo-1-chloronaphthalen-2-ol for 20A-1.

[0172] 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.27 (d, J=7.9 Hz, 1H), 7.87-7.63 (m, 411), 7.60 (s, 1H), 7.40-7.24 (m, 211), 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 C29H19Cl3N2O5; [M+H]+: 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)

[0173] ##STR00040##

[0174] (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 C17H13NO4; [M+H]+: 296.1, found: 296.1.

##STR00041##

[0175] (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 C30H22Cl2N2O5; [M+H]+: 561.1, found: 561.1.

##STR00042##

[0176] (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%).

[0177] 1H NMR (400 MHz, DMSO-d6) 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 C29H20Cl2N2O5; [M+H]+: 547.1, found: 547.1.

##STR00043##

Example 23

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

[0178] ##STR00044##

[0179] Following the procedure of Example 22, the title Compound 23 was obtained by substituting methyl 6-fluoropicolinate for 22A-2.

[0180] 1H NMR (400 MHz, DMSO-d6) 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 C29H20Cl2N2O5; [M+H]+: 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)

[0181] ##STR00045##

[0182] Following the procedure of Example 22, the title Compound 24 was obtained by substituting methyl 2-fluoroisonicotinate for 22A-2.

[0183] 1H NMR (400 MHz, DMSO-d6) 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 C29H20Cl2N2O5; [M4+H]+: 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)

[0184] ##STR00046##

[0185] Following the procedure of Example 22, the title Compound 25 was obtained by substituting methyl 3-fluoropicolinate for 22A-2.

[0186] 1H NMR (400 MHz, DMSO-d6) 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 C29H20Cl2N2O5; [M+H]+: 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)

[0187] ##STR00047##

[0188] Following the procedure of Example 22, the title Compound 26 was obtained by substituting methyl 2,4-difluorobenzoate for 22A-2.

[0189] 1H NMR (400 MHz, DMSO-d6) 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 C30H20Cl2FNO5; [M+H]+: 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)

[0190] ##STR00048##

[0191] Following the procedure of Example 22, the title Compound 27 was obtained by substituting methyl 6-chloro-2-methylnicotinate for 22A-2.

[0192] 1H NMR (400 MHz, DMSO-d6) 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 C30H22Cl2N2O5; [M+H]+: 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)

[0193] ##STR00049##

[0194] Following the procedure of Example 22, the title Compound 28 was obtained by substituting methyl 6-chloro-5-methylnicotinate for 22A-2.

[0195] 1H NMR (400 MHz, DMSO-d6) 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 C30H22Cl2N2O5; [M+H]+: 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)

[0196] ##STR00050##

[0197] Following the procedure of Example 32, the title Compound 29 was obtained by substituting 6-bromo-1-chloronaphthalen-2-ol for 32A-1.

[0198] 1H NMR (400 MHz, DMSO-d6) 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 C29H19Cl3N2O5; [M+H]+: 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)

[0199] ##STR00051##

[0200] Following the procedure of Example 20, the title Compound 30 was obtained by substituting 6-bromo-2-fluoronaphthalen-1-ol for 20A-1.

[0201] 1H NMR (400 MHz, DMSO-d6) 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 C29H19Cl2FN2O5; [M+H]+: 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)

[0202] ##STR00052##

[0203] Following the procedure of Example 1, the title Compound 31 was obtained by substituting naphthalene-2,7-diol for 1A-2.

[0204] 1H NMR (400 MHz, DMSO-d6) δ 8.64 (s, 1H1), 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 C29H20Cl2N2O5; [M+H]+: 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)

[0205] ##STR00053##

[0206] (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 C23H15BrCl2FNO2; [M+H]+: 506.0, found: 506.0.

##STR00054##

[0207] (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)2Cl2 (28 mg, 0.039 mmol, 0.1 eq), and bis(pinacolato)diboron (100 mg, 0.39 mmol, 1 eq) were added under N2, 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)isoxazole, 137 mg, yield: 62.8%. LCMS (ESI): calculated for C29H27BCl2FNO4; [M+H]+: 554.1, found: 554.1.

##STR00055##

[0208] (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 Na2SO3, 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 C23H16Cl2FNO3; [M+H]+: 444.1, found: 444.1.

##STR00056##

[0209] (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 (ESI): calculated for C30H21Cl2FN2O5; [M+H]+: 579.1, found: 579.1.

##STR00057##

[0210] (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 N2, 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%).

[0211] 1H NMR (400 MHz, DMSO-d6) 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 C29H19Cl2FN2O5; [M+H]+: 565.1, found: 565.1.

##STR00058##

Example 33

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

[0212] ##STR00059##

[0213] 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. Alter 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%).

##STR00060##

Example 34

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

[0214] ##STR00061##

[0215] To a solution of Compound 35 (1.00 g, 1.76 mmol) in water (10 ml) was added a solution of CaCl2 (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%).

##STR00062##

Example 35

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

[0216] ##STR00063##

[0217] The title compound 35 was prepared according to Route D, following the procedure of Example 22. 1H NMR (400 MHz, DMSO-d6) 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 C29H20Cl2N2O5; [M+H]+: 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

[0218] ##STR00064##

[0219] The title compound 36 was prepared according to Route B, following the procedure of Example 32. 1H NMR (400 MHz, DMSO-d6) 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, 11H), 7.14 (d, J=8.7 Hz, 11H), 7.01 (d, J=7.4 Hz, 1H), 5.09 (s, 2H), 1.31-1.07 (m, 6H). LCMS (ESI): calculated for C29H20Cl2N2O5; [M+H]+: 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

[0220] ##STR00065##

[0221] The title compound 37 was prepared according to Route B, following the procedure of Example 32. 1H NMR (400 MHz, DMSO-d6) 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, 11H), 5.09 (s, 2H), 2.52 (s, 3H), 1.19-1.08 (m, 4H). LCMS (ESI): calculated for C30H21Cl2FN2O5; [M+H]+: 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

[0222] ##STR00066##

[0223] The title compound 38 was prepared according to Route B, following the procedure of Example 32. 1H NMR (400 MHz, DMSO-d6) 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 C29H19Cl2FN2O5; [M+H]+: 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

[0224] ##STR00067##

[0225] The title compound 39 was prepared according to Route B, following the procedure of Example 32. 1H NMR (400 MHz, CDCl3) δ 7.94 (dd, J=7.9, 5.0 Hz, 11H), 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 C30H21Cl2FN2O5; [M+H]+: 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

[0226] ##STR00068##

[0227] The title compound 40 was prepared according to Route D, following the procedure of Example 22. 1H NMR (400 MHz, CDCl3) 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 C30H22Cl2N2O5; [M+H]+: 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

[0228] ##STR00069##

[0229] The title compound 41 was prepared according to Route C, following the procedure of Example 20. 1H NMR (400 MHz, DMSO-d6) 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 C29H18C14N2O5; [M+1]+: 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

[0230] ##STR00070##

[0231] The title compound 42 was prepared according to Route C, following the procedure of Example 20. 1H NMR (400 MHz, DMSO-d6) 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 C29H19Cl3N2O5; [M+H]+: 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

[0232] ##STR00071##

[0233] The title compound 43 was prepared according to Route C, following the procedure of Example 20. 1H NMR (400 MHz, DMSO-d6) 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 C30H21Cl3N2O5; [M+H]+: 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

[0234] ##STR00072##

[0235] The title compound 44 was prepared according to Route C, following the procedure of Example 20. 1H NMR (400 MHz, DMSO-d6) 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, 211), 1.28-1.05 (m, 4H). LCMS (ESI): calculated for C30H19Cl2F2NO5; [M+H]+: 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

[0236] ##STR00073##

[0237] The title compound 45 was prepared according to Route C, following the procedure of Example 20. 1H NMR (400 MHz, DMSO-d6) 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 C30H21Cl2FN2O5; [M+H]+: 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

[0238] ##STR00074##

[0239] The title compound 46 was prepared according to Route C, following the procedure of Example 20. 1H NMR (400 MHz, DMSO-d6) 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, 211), 2.59-2.54 (m, 1H), 2.48 (s, 311), 1.27-1.15 (m, 411). LCMS (ESI): calculated for C30H21Cl3N2O5; [M+H]+: 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

[0240] ##STR00075##

[0241] The title compound 47 was prepared according to Route C, following the procedure of Example 20. 1H NMR (400 MHz, DMSO-d6) 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, 11H), 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 C30H21Cl2FN2O5; [M+H]+: 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

[0242] ##STR00076##

[0243] The title compound 48 was prepared according to Route C, following the procedure of Example 20. 1H NMR (400 MHz, DMSO-d6) 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 C29H19Cl2FN2O5; [M+H]+: 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

[0244] ##STR00077##

[0245] The title compound 49 was prepared according to Route D, following the procedure of Example 22. 1 H NMR (400 MHz, DMSO-d6) 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 C30H20Cl2FN2O5; [M+H]+: 564.1, found: 564.1.

BIOLOGY EXAMPLES

[0246] Compound of Formula (I) in HBV Cell-Based Assay

[0247] PHH (Primary Human Hepatocyte) Assay: Cell Line and Compound Treatment

[0248] The PHH cells were seeded into the 48-well plates at the density of 1.32×105 cells/well. The PHH cells seeding date was defined as day 0. The PHH cells were infected with HBV (D type) at 1600 GE/cell on day 1. The culture medium containing compounds was refreshed on day 2, 4, 6 and day 8. On day 9, the cells were treated with CCK8 and culture supernatant and cells were collected.

[0249] FXR Compounds were tested at 10000.0, 3000.0, 1000.0, 300.0, 100.0, 30.0, and 10.0 nM, and ETV was tested at 0.2000, 0.0667, 0.0222, 0.0074, 0.0025, 0.0008, and 0.0003 nM. 1% DMSO was used as non-treatment control. The supernatants on day 9 were collected, and analyzed for HBV DNA by qPCR, HBV RNA by RT-qPCR, HBsAg and HBeA by ELISA.

[0250] HBV DNA, RNA and Antigen Assay

[0251] DNA in the culture supernatants was isolated with the QIAamp 96 DNA Blood Kit according to the manual and quantified by the qPCR. 80 μl of the culture supernatants sample was used to extract DNA. The elution volume was 120 μl. The PCR mix (8 μl/well) and the samples (2 μl/well) or plasmid standards (2 μl/well) were added to the 384-well optical reaction plate and performed using the following program: 95C for 10 min, then cycling at 95° C. 15 sec, 60° C. 1 min for 40 cycles.

[0252] RNA in the culture supernatants was isolated with the PureLink™ Pro 96 Viral RNA Kit according to the manual. 35 μl of the culture supernatants sample was used to extract RNA. The elution volume was 100 μl. HBV RNA was quantified by RT-qPCR.

[0253] For HepG2-NTCP Assay, HBsAg and HBeAg in the samples harvested on day 9 were measured by the HBsAg ELISA kit (Autobio) and HBeAg ELISA kit (Autobio) according to the manual. For PHH assay, Chemiluminescence Apparatu was used to measure HBsAg according to the Antu HBsAg ELISA kit manual.

[0254] After collecting culture supernatants at the terminal day for each plate, CCK-8 working solution (diluted with fresh culture medium at ratio of 1:9) was added to the cell plates. The plates were incubated at 37° C., 5% CO2 incubator for approximate 30 min. The ODs were measured by microplate reader (OD450 nm/OD630 nm).

[0255] Dose Response Curves

[0256] % HBV DNA inhibition=(1−HBV DNA copy number of test sample/avg. HBV DNA copy number of 1% DMSO control)×100%

[0257] % HBV RNA inhibition=(1−HBV RNA copy number of test sample/avg. HBV RNA copy number of 1% DMSO control)×100%

[0258] % HBsAg inhibition=(1−HBsAg quantity of sample/HBsAg quantity of 1% DMSO control)×100%

[0259] % HBeAg inhibition=(1−HBeAg quantity of sample/HBeAg quantity of 1% DMSO control)×100%

[0260] % Cell viability=value of sample/a value of 1% DMSO×100%.

[0261] The EC50 and CC50 values were determined by dose-response curves fitted by GraphPad Prism using “log (agonist) vs. response-variable slope”.

[0262] Results

[0263] As shown in in FIGS. 1, 2 and 3, in PHH assay, Compound 1, 22, and 27 had dose-dependent inhibition of extracellular HBV DNA HBsAg and HBV RNA. As control, ETV showed significant inhibition of HBV DNA, but no significant inhibition of HBV HBsAg and HBV RNA.

[0264] Compound of Formula (I) in AAV/HBV Animal Model

[0265] AAV/HBV Mouse Model and Compound Treatment

[0266] Male C57BL′6 mice of 5-week old (obtained from Shanghai Jihui Laboratory Animal Care Co., Ltd) were given rAAV8-1.3HBV (1×1011 v.g.) via tail vein to establish infection on Day −28. After 14 days, 21 days, and on Day −4, blood was drawn from submandibular vein to determine the level of HBV DNA, and HBsAg. On Day-4, 40 mice were chosen based on HBV DNA, and HBsAg results, and were randomly assigned to 5 groups. After the mice were infected with HBV virus for 28 days, treatment was given orally once per day, for 4 weeks. Group 1 was given vehicle (0.5% CMC-Na, 10 ml/kg), Group 2 was given positive control ETV (0.1 mg/kg). Groups 3, 4, and 5 were given Compound 33 at 10 mg/kg, 30 mg/kg, and 60 mg/kg, respectively. Blood was drawn from submandibular vein every week to determine the concentration of HBV DNA, HBV RNA, and HBsAg. On Day 28, blood was taken from the heart.

[0267] Blood Sample Analysis

[0268] HBV DNA determination by qPCR. DNA in the mice plasma was isolated with the QIAamp 96 DNA Blood Kit according to the manual and quantified by the qPCR, and performed using the following program: 95C for 10 min, then cycling at 95 V 15 sec, 60° C. 1 min for 40 cycles.

[0269] HBV RNA determination by qPCR. RNA in the mice plasma was isolated with the PureLink™ Pro 96 Viral RNA/DNA Kit according to the manual. 20 μl of the plasma was used to extract RNA

[0270] HBsAg determination by ELISA. HBsAg in the samples was measured by the HBsAg ELISA kit (Autobio) according to the manual. Briefly, after the plasma was diluted 1200× in a coated plate, and incubate with the enzyme conjugated (37V, 60 min), and the plate was washed 5 time. Substrate was added, and it was kept from light at room temperature for 10 min and intensity was measured by a plated reader.

[0271] Data Analysis

[0272] Average±Standard deviation was calculated for each group, and analyzed with Student's t-test.

[0273] Results

[0274] As shown in FIG. 4, after Compound 33 was given to the mice for 28 days, the HBV DNA in the high dose group (60 mg/kg) was significantly lower than that of the vehicle group. On average, the log 10[DNA copy/uL] of the high dose group was 0.77-1.12 lower than the vehicle group (p<0.05).

[0275] As shown in FIG. 5, after Compound 33 was given to the mice for 28 days, the HBV RNA in the high dose group (60 mg/kg) was significantly lower than that of the vehicle group. On average, the log 10[RNA copy/uL] of the high dose group was 0.60-0.66 lower than the vehicle group (p<0.01). ETV treatment showed no significant effect on HBV RNA as compared to the vehicle group.

[0276] As shown in FIG. 6, after Compound 33 was given to the mice for 28 days, the HBsAg in the high dose group (60 mg/kg) was lowered by 0.38 log 10 [IU/ml] as compared to the vehicle group. ETV treatment showed no significant effect on HBsAg as compared to the vehicle group.