AZABICYCLOOCTANE DERIVATIVES AS FXR AGONISTS FOR USE IN THE TREATMENT OF LIVER AND GASTROINTESTINAL DISEASES

Abstract

The invention provides methods for modulating the activity of farnesoid X receptors (FXRs) using compounds of Formula (I) or (II). In particular, the invention provides for the use of compounds of Formula (I) or (II), or a stereoisomer, enantionmer or pharmaceutically acceptable salt thereof, for treating or preventing liver and gastrointestinal diseases.

Claims

1. (canceled)

2. (canceled)

3. The method according to claim 19 wherein R.sup.1 is trifluoromethyl or trifluoromethoxy.

4. The method according to claim 19 wherein R.sup.2 is —CO.sub.2R, and R is hydrogen or C.sub.1-6 alkyl.

5. The method according claim 19 wherein R.sup.3 is methyl, methoxy or fluoro.

6. The method according to claim 19 wherein Z is pyridyl.

7. The method according to claim 19 wherein Z is pyrimidinyl.

8. The method according to claim 19 wherein Z is pyrazinyl.

9. The method according to claim 19 wherein said Z is benzothiazolyl.

10. The method according to claim 19 wherein said compound of Formula (I) is selected from methyl 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylate; 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid; methyl 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethyl)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylate; 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethyl)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid; 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-methoxy-1,3-benzothiazole-6-carboxylic acid; methyl 6-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]pyridine-3-carboxylate; 6-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]pyridine-3-carboxylic acid; methyl 5-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]pyrazine-2-carboxylate; 5-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]pyrazine-2-carboxylic acid; and 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethyl)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-6-methylpyrimidine-4-carboxylic acid; or a pharmaceutically acceptable salt thereof.

11. The method according to claim 19 wherein said compound is 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid, or a pharmaceutically acceptable salt thereof.

12. The method according to claim 19 wherein said compound is 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethyl)phenyl]-1,2-oxazol-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid, or a pharmaceutically acceptable salt thereof.

13. The method according to claim 19 wherein said compound is 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-methoxy-1,3-benzothiazole-6-carboxylic acid, or a pharmaceutically acceptable salt thereof.

14. The method according to claim 19 wherein said compound is 6-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]pyridine-3-carboxylic acid, or a pharmaceutically acceptable salt thereof.

15. The method according to claim 19 wherein said compound is 5-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]pyrazine-2-carboxylic acid, or a pharmaceutically acceptable salt thereof.

16. The method according to claim 19 wherein said compound is 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethyl)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-6-methylpyrimidine-4-carboxylic acid, or a pharmaceutically acceptable salt thereof.

17. The method according claim 19 wherein said condition mediated by FXR is bile acid malabsorption.

18. The method according to claim 17, or the compound of Formula (1) for use according to claim 17, wherein said bile acid malabsorption is primary or secondary bile acid diarrhea.

19. A method for treating or preventing a condition mediated by Farnesoid X receptor (FXR) including bile acid malabsorption, bile reflux gastritis, collagenous colitis, lymphocytic colitis, diversion colitis, indeterminate colitis, Alagille syndrome, biliary atresia, ductopenic liver transplant rejection, bone marrow or stem cell transplant associated graft versus host disease, cystic fibrosis liver disease, and parenteral nutrition-associated liver disease, the method comprising administering to a subject a compound of Formula (I) ##STR00036## or a stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof; wherein Z is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl or benzothiazolyl; each of which is optionally substituted with 1-2 R.sup.3 radicals selected from halogen, C.sub.1-6 alkyl or C.sub.1-6 alkoxy; R.sup.1 is haloC.sub.1-6 alkyl or haloC.sub.1-6 alkoxy; R.sup.2 is —CO.sub.2R, —CONR—(CR.sub.2)—CO.sub.2R, —CONR—(CR.sub.2).sub.2—SO.sub.3R or ##STR00037## each R is independently hydrogen or C.sub.1-6 alkyl.

20. The method according to claim 19 further including a second therapeutic agent.

Description

DESCRIPTION OF THE FIGURES

[0116] FIGS. 1A-1D show the effect of a compound of Formula (I) (“Compound A”) on serum markers of cholestasis and liver damage in the chronic treatment rat ANIT model.

[0117] FIG. 1E shows serum FGF15 protein levels following treatment with a compound of Formula (I) (“Compound A”) in the chronic rat ANIT-induced cholestasis model.

MODES OF CARRYING OUT THE INVENTION

[0118] The present invention provides the use of FXR agonists or partial agonists for treating or preventing liver disease and gastrointestinal disease.

[0119] In one aspect, the invention provides the use of a compound of Formula (I)

##STR00004##

[0120] or a stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof;

[0121] wherein Z is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl or benzothiazolyl; each of which is optionally substituted with 1-2 R.sup.3 radicals selected from halogen, C.sub.1-6 alkyl or C.sub.1-6 alkoxy;

[0122] R.sup.1 is haloC.sub.1-6 alkyl or haloC.sub.1-6 alkoxy;

##STR00005##

[0123] R.sup.2 is —CO.sub.2R, —CONR—(CR.sub.2)—CO.sub.2R, —CONR—(CR.sub.2).sub.2—SO.sub.3R or OH and each R is independently hydrogen or C.sub.1-6 alkyl;

[0124] or a compound of Formula (II)

##STR00006##

[0125] or a stereoisomer, enantiomer, a pharmaceutically acceptable salt, an amino acid conjugate or an acyl glucuronide conjugate thereof;

[0126] wherein Z.sup.1 is phenylene, pyridylene, pyrimidinylene, pyrazinylene, pyridazinylene, thiazolylene, benzothiazolyl, benzo[d]isothiazolyl, imidazo[1,2-a]pyridinyl, quinolinyl, 1H-indolyl, pyrrolo[1,2-b]pyridazinyl, benzofuranyl, benzo[b]thiophenyl, 1H-indazolyl, benzo[d]isoxazolyl, quinazolinyl, 1H-pyrrolo[3,2-c]pyridinyl, pyrazolo[1,5-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, pyrazolo[1,5-a]pyridinyl; each of which is optionally substituted with 1-2 R.sup.6 radicals selected from halogen, C.sub.1-6 alkyl, haloC.sub.1-6 alkyl, C.sub.1-6 alkoxy, haloC.sub.1-6 alkoxy, or cyclopropyl;

[0127] R.sup.3 is phenyl, pyridyl, bicyclo[3.1.0]hexanyl, spiro[2.3]hexanyl, bicyclo[3.1.1]heptanyl, spiro[2.5]octanyl, bicyclo[4.1.0]heptanyl, bicyclo[3.1.0]hexan-6-yl, spiro[2.3]hexan-5-yl, bicyclo[3.1.1]heptan-3-yl, spiro[2.5]octan-4-yl, bicyclo[4.1.0]heptan-3-yl, cyclohexyl or cyclopentyl, each of which is optionally substituted with 1-3 R.sup.3a; or R.sup.3 is cyclopropyl optionally substituted with 1-2 R.sup.3a or phenyl;

[0128] R.sup.3a is halogen, C.sub.1-6 alkyl, haloC.sub.1-6 alkyl, C.sub.1-6 alkoxy, haloC.sub.1-6 alkoxy or cyclopropyl;

[0129] R.sup.4 is C.sub.1-3 alkyl, haloC.sub.1-3 alkyl or cyclopropyl optionally substituted with C.sub.1-3 alkyl or haloC.sub.1-3 alkyl;

[0130] R.sup.5 is —X—CO.sub.2R.sup.7, hydroxyC.sub.1-6 alkyl, CONR.sup.7R.sup.8, CONR(CR.sub.2).sub.1-4CO.sub.2R.sup.7, CONR(CR.sub.2).sub.1-4SO.sub.3R.sup.8 or tetrazolyl; wherein X is a bond, C.sub.1-2 alkylene or cyclopropyl; and

[0131] R, R.sup.7 and R.sup.8 are independently hydrogen or C.sub.1-6 alkyl;

[0132] for treating or preventing liver disease or gastrointestinal disease.

[0133] In another aspect, the invention provides the use of a compound of Formula (I) or (II), or a stereoisomer, enantiomer or pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing liver disease or gastrointestinal disease.

[0134] In one embodiment, the invention provides a compound of Formula (I) or (II), or a stereoisomer, enantiomer or pharmaceutically acceptable salt thereof, for use in the treatment or prevention of cholestatic liver disorders, particularly Alagille syndrome, biliary atresia, ductopenic liver transplant rejection, bone marrow or stem cell transplant associated graft versus host disease, cystic fibrosis liver disease and parenteral nutrition-associated liver disease ((PNALD, also known as intestinal failure-associated liver disease).

[0135] Parenteral nutrition-associated liver disease (PNALD) is a serious complication of parenteral nutrition (PN) in infants who do not tolerate enteral feedings, especially those with acquired or congenital intestinal diseases. Recent reports have shown that infusion with lipid emulsions derived from fish oil (FO) rather than soy oil (SO) improves established PNALD, and that reduction of the SO lipid dose in PN solutions attenuates PNALD. One of the components of SO emulsions, phylosterol, has been implicated in PNALD. Mechanistic studies have demonstrated that among the phylosterols present in SO emulsions, stigmasterol was by far the most potent at inhibiting activity of FXR, which regulates transcription of bile acid transporters in cultured hepatocytes. On the basis of in vitro studies, stigmasterol has been suggested as promoting cholestasis through inhibition of the nuclear receptor FXR, which, in turn, would result in reduced hepatocyte expression of a wide variety of FXR-dependent genes, including the principal determinant of bile secretion, the bile salt export pump (BSEP) (Abcb11). (Carter et al., Pediatr. Res. 62: 301-306 (2007); El Kasmi et al., Sci. Transl. Med. 5: 1-10 (2013)).

[0136] In another embodiment, the invention provides a compound of Formula (I) or (II), or a stereoisomer, enantiomer or pharmaceutically acceptable salt thereof, for use in the treatment or prevention of gastrointestinal diseases, particularly bile acid malabsorption or bile acid diarrhea (including primary bile acid diarrhea and secondary bile acid diarrhea), bile reflux gastritis and inflammatory bowel diseases (IBD), particularly collagenous colitis, lymphocytic colitis, diversion colitis, and indeterminate colitis.

[0137] Primary bile acid diarrhea (pBAD) is a common cause of chronic diarrhea, and is characterized by a cycle wherein the feedback regulation of bile acid synthesis is interrupted, resulting in additional bile acid production. Feedback regulation of bile acid synthesis is under the control of an endocrine pathway, wherein activation of the nuclear bile acid receptor FXR induces enteric expression of fibroblast growth factor 15 (FGF15) in rodents and FGF19 in humans. In liver, FGF15 or FGF19 acts together with FXR-mediated expression of small heterodimer partner to repress bile acid synthesis (Jung et al., Journal of Lipid Research 48: 2693-2700 (2007) Walters J R, Nat Rev Gastroenterol Hepatol. 11(7):426-34 (2014)).

[0138] Many patients suffering from pBAD have reduced levels of the ileal hormone fibroblast growth factor 19 (FGF19), an inhibitory regulator of hepatic bile acid synthesis, secreted in response to FXR activation. FGF19 production in the ileum is stimulated by bile acid binding to FXR, and activating transcription. Recent studies show that therapy with an FXR agonist significantly increased FGF19 in the primary and secondary BAD group, which were in turn associated with reduced bile acid synthesis and clinical improvement. (Walters J R et al., Nat Rev Gastroenterol Hepatol. 11(7):426-34 (2014); Walters J R et al., Aliment Pharmacol Ther. 2014 Oct. 20. doi: 10.1111/apt.12999).

[0139] Bile acids from duodenogastric reflux promote inflammation and increase the risk for gastro-esophageal cancers. FXR is a transcription factor regulated by bile acids such as CDCA (chenodeoxycholic acid), and protects the liver and the intestinal tract against bile acid overload. (Lian et al., Biochem J. 438: 315-323 (2011)).

[0140] Collagenous colitis (CC) is an inflammatory bowel disease (IBD) of unknown origin. In a considerable proportion (44%) of patients with collagenous colitis, the patient suffers from the simultaneous occurrence of bile acid malabsorption. (Ung et al., Gut 46: 170-175 (2000)). Bile acid malabsorption is more uncommon in lymphocytic colitis than in collagenous colitis; however, the 75SeHCAT values suggest a role of bile acids in lymphocytic colitis. The conversion of two patients with lymphocytic colitis to collagenous colitis, and disturbed absorption of bile acids in lymphocytic colitis, suggest that lymphocytic colitis and collagenous colitis represent variants of the same disease. (Ung et al., Hepato-Gastroenterology 49: 432-437 (2002)). FXR activation has also been demonstrated to prevent chemically induced intestinal inflammation, with improvement of colitis symptoms. (Gadaleta et al., Gut 60:463-472 (2011)).

[0141] In another aspect, the invention provides the use of a compound of Formula (I) or (II), or a stereoisomer, enantiomer or pharmaceutically acceptable salt thereof, for treating a condition mediated by Farnesoid X receptor (FXR), wherein said condition is bile acid malabsorption (e.g. is primary or secondary bile acid diarrhea), bile reflux gastritis, collagenous colitis, lymphocytic colitis, diversion colitis, indeterminate colitis, Alagille syndrome, biliary atresia, ductopenic liver transplant rejection, bone marrow or stem cell transplant associated graft versus host disease, cystic fibrosis liver disease, or parenteral nutrition-associated liver disease.

[0142] In one embodiment, the compound of Formula (I) or (II) for use in any of the above embodiments has an activity EC.sub.50 value between 0.1 nM and 500 nM, which can be determined using assays known in the art such as for example, the GST-FXR LBD co-activator interaction assay described in PCT/US2011/062724. In another embodiment, the compound of Formula (I) or (II) for use in any of the above embodiments has an EC.sub.50 value between 0.1 nM and 100 nM; between 0.1 nM and 50 nM; or between 0.1 nM and 30 nM. In yet another embodiment, the compound of Formula (I) or (II) for use in any of the above embodiments has an EC.sub.50 value that is <0.1 nM or >500 nM.

[0143] The compounds for use in the methods of the invention may be administered either simultaneously with, or before or after, one or more other therapeutic agent. The compound for use in the methods of the invention may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other agents.

[0144] In combination therapies for use in the methods of the invention, a compound of Formula (I) or (II) and the other therapeutic agent may be manufactured and/or formulated by the same or different manufacturers. Moreover, the compound of Formula (I) or (II) and the other therapeutic may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g. in the case of a kit comprising a compound of Formula (I) or (II) and the other therapeutic agent); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (iii) in the patient themselves, e.g. during sequential administration of a compound of Formula (I) or (II) and the other therapeutic agent.

[0145] Accordingly, the invention provides for the use of a compound of Formula (I) or (II) for treating or preventing a disease or condition mediated by FXR, wherein the medicament is prepared for administration, or administered with, another therapeutic agent. The invention also provides a compound of Formula (I) or (II) for use in a method of treating or preventing a disease or condition mediated by FXR, wherein the compound of Formula (I) or (II) is prepared for administration, or administered with, another therapeutic agent. The invention also provides another therapeutic agent for use in a method of treating or preventing a disease or condition mediated by FXR, wherein the other therapeutic agent is prepared for administration, or administered with, a compound of Formula (I) or (II).

[0146] The invention also provides for the use of a compound of Formula (I) or (II) for treating or preventing a disease or condition mediated by FXR, wherein the patient has previously (e.g. within 24 hrs) been treated with another therapeutic agent. Alternatively, the invention provides for the use of another therapeutic agent for treating or preventing a disease or condition mediated by FXR, wherein the patient has previously (e.g. within 24 hrs) been treated with a compound of Formula (I) or (II).

[0147] The invention further provides pharmaceutical compositions or combinations comprising a compound of Formula (I) or (II) for treating or preventing liver disease and gastrointestinal disease as described herein. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.

[0148] In one embodiment, a compound of Formula (I) or (II) is administered at the daily dosage.

[0149] In another embodiment, a compound of Formula (I) or (II) is administered enterally; and more particularly, orally.

[0150] Unless specified otherwise, a compound for use in the methods of the invention refers to a compound of Formula (I) or (II), pharmaceutically acceptable salt thereof, prodrugs, and inherently formed moieties (e.g., polymorphs, solvates and/or hydrates). The compound for use in the methods of the invention may be stereoisomers (including diastereoisomers and enantiomers), a mixture of stereoisomers or a single stereoisomer, tautomers or isotopically labeled compounds (including deuterium substitutions). Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds.

EXAMPLES

[0151] Examples of a compound of Formula (I) or (II) for use in the methods of the present invention are described in PCT/US2011/062724. The following examples are offered to illustrate, but not to limit, the compounds for use in the methods of the present invention.

Abbreviations

[0152] AcOH acetic acid
ANIT alpha-naphthyl-isothiocyanate
ALP alkaline phosphatase
ALT alanine aminotransferase
AST aspartate aminotransferase
EtOAc ethyl acetate
EtOH ethanol
FGF15/19 Fibroblast Growth Factor (known as FGF19 in humans)
GGT gamma-glutamyl transpeptidase
LLQ lower limit of quantification
MeOH methanol
THF tetrahydrofuran
TBA Total bile acids
TBIL Total bilirubin

Example 1

2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid (1-1B) and 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethyl)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid (1-2B)

[0153] ##STR00007##

Methyl 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylate (1-1A)

[0154] Into a 25-mL round-bottom flask equipped with a stir bar was added sequentially 4-(((1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-yloxy)methyl)-5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazole (1.29 mmol), N,N-dimethylacetamide (3.6 mL), cesium carbonate (3.31 mmol), and methyl 2-bromo-4-fluorobenzo[d]thiazole-6-carboxylate (3.87 mmol). After stirring the resulting slurry at room temperature for 10 minutes, the mixture was then warmed to 60° C. and stirred for 1 h. The reaction slurry was allowed to cool to room temperature, and was diluted with 200 mL of ethyl acetate and washed with water (3×30 mL). The organic extracts were concentrated under vacuum and directly purified using normal phase silica gel chromatography (40 g silica column) with a 15 min gradient of 10% to 60% ethyl acetate/hexanes. Desired fractions were concentrated in vacuo, and the resulting residue crystallized upon standing to give methyl 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylate (I-1A) as a white crystalline solid. MS (m/z): 618.2 (M+1).

2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid (1-1B)

[0155] To a 25-mL round-bottom flask equipped with a stir bar was added the ester (0.89 mmol), THF (4 mL), MeOH (2 mL), and 3 N aqueous KOH solution (1 mL, 3 mmol). The resulting homogenous solution was stirred for 1 hour at 70° C., cooled to room temperature, and then quenched with AcOH (roughly 0.2 mL of glacial acetic, 3 mmol) until pH=6 was achieved (Whatman class pH strip paper). At this time the reaction was diluted with ethyl acetate (40 mL) and washed with water (3×5 mL). The ethyl acetate fraction was concentrated under vacuum to give to an oily residue. To the resulting oil was then added MeOH (6 mL). The oil quickly dissolved, then immediately began to crystallize. Upon standing for 2.5 hrs, the mother liquor was withdrawn and crystals washed (3×2 mL of ice cold MeOH). The crystals were dried via vacuum (10 mm Hg pressure at 45° C. overnight) and then recrystallized from acetonitrile, filtered, and dried under vacuum to give 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid (1-1B). 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethyl)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid (1-2B).

[0156] Examples 1-2A and the corresponding acid 1-2B can be prepared following the same procedures, from the reaction of intermediate 4-((8-azabicyclo[3.2.1]octan-3-yloxy)methyl)-5-cyclopropyl-3-(2-(trifluoromethyl)phenyl)isoxazole.

TABLE-US-00001 Physical Data Ex MS (m/z), .sup.1H NMR 1-1A [00008] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ 8.13 (d, J = 1.6 Hz, 1H), 7.67-7.59 (m, 3H), 7.54-7.50 (m, 2H), 4.41 (s, 2H), 4.31 (bs, 2H), 3.90 (s, 3H), 3.60 (t, J = 4.8 Hz, 1H), 2.31-2.25 (m, 1H), 2.10 (app dt, J = 14.8, 4 Hz, 2H), 2.02-1.91 (m, 4H), 1.83 (app d, J = 14.8 Hz, 2H), 1.19-1.15 (m, 4H). MS (m/z): 618.2 (M + 1). 1-1B [00009] .sup.1H NMR (MeOD, 400 MHz): δ 8.03 (d, J = 1.6 Hz, 1H), 7.57-7.53 (m, 2H), 7.49 (dd, J = 8.1, 1.8 Hz, 2H), 7.41 (app t, J = 7.6, 1H), 4.31 (s, 2H), 4.22 (broad s, 2H), 3.50 (t, J = 4.4 Hz, 1H), 2.22-2.15 (m, 1H), 2.00 (app dt, J = 14.8, 4.0 Hz, 2H), 1.91-1.81 (m, 4H), 1.75 (d, J = 14.4, 2H), 1.10-1.05 (m, 4H). MS (m/z): 604.2 (M + 1). 1-2A [00010] .sup.1H NMR (DMSOd.sub.6, 400 MHz): δ 8.26 (d, J = 1.6 Hz, 1H), 7.92 (d, J = 8 Hz, 1H), 7.84-7.74 (m, 2H), 7.63-7.60 (m, 2H), 4.26 (bs, 4H), 3.84 (s, 3H), 3.52 (t, J = 4 Hz, 1H), 2.39-2.31 (m, 1H), 2.01-1.94 (m, 2H), 1.85-1.74 (m, 6H), 1.18-1.06 (m, 4H). MS (m/z): 602.3 (M + 1). 1-2B [00011] .sup.1H NMR (DMSOd.sub.6, 400 MHz): δ 8.21 (d, J = 1.6 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.84-7.74 (m, 2H), 7.62-7.56 (m, 2H), 4.26 (bs, 4H), 3.52 (t, J = 4 Hz, 1H), 2.39-2.31 (m, 1H), 2.00-1.96 (m, 2H), 1.85-1.73 (m, 6H), 1.19-1.07 (m, 4H). MS (m/z): 588.1 (M + 1).

Example 2

2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-methoxy-1,3-benzothiazole-6-carboxylic acid

[0157] ##STR00012##

[0158] Methyl 2-chloro-4-methoxybenzo[d]thiazole-6-carboxylate (0.48 mmol) and 4-(((1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-yloxy)methyl)-5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazole (0.48 mmol) and diisopropylethylamine (0.1 mL, 0.7 mmol) were sequentially dissolved in dimethylacetamide (1 mL) and heated to 120° C. overnight. The reaction mixture was cooled to room temperature and then diluted with ethyl acetate and aqueous saturated sodium bicarbonate solution. The organics were separated, the aqueous layer was subjected to a further wash with ethyl acetate, and the organics were combined and dried (MgSO.sub.4) then evaporated in vacuo. Methyl 2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-methoxy-1,3-benzothiazole-6-carboxylate (2A) was obtained as a clear oil after purification by silica gel chromatography with a gradient of 0-100% ethyl acetate/hexanes. MS (m/z): 630.1 (M+1).

[0159] The ester (2A) (0.26 mmol) was dissolved in tetrahydrofuran (1 mL) and ethanol (1 mL) and subjected to an aqueous solution of potassium hydroxide (2.5 mmol in 2 mL water). The mixture was heated to 60° C. for 2 hr and then the solvent was removed in vacuo. The mixture was diluted with 5% aqueous citric acid and extracted with ethyl acetate (2×100 mL). The organics were dried (MgSO.sub.4) then evaporated in vacuo. The product was purified by flash silica chromatography with a gradient of 0-100% ethyl acetate/hexanes to give the corresponding acid (2). .sup.1H NMR (MeOD, 400 MHz): δ 8.77 (s, 2H), 7.66-7.58 (m, 2H), 7.51 (app t, J=8.0 Hz, 2H), 4.63 (bs, 2H), 4.40 (s, 2H), 3.55 (t, J=4.4 Hz, 1H), 2.31-2.24 (m, 1H), 1.99-1.88 (m, 4H), 1.86-1.81 (m, 2H), 1.76 (d, J=14.0 Hz, 2H), 1.19-1.15 (m, 4H). MS (m/z): 616.1 (M+1).

Example 3

[0160] The following compounds were prepared from 4-((8-azabicyclo[3.2.1]octan-3-yloxy)methyl)-5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazole and commercially available ethyl 2-chlorobenzo[d]thiazole-6-carboxylate according to the procedures described for the preparation of Example 1 or 2.

TABLE-US-00002 Physical Data Ex MS (m/z), .sup.1H NMR 3A [00013] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ 8.37 (d, J = 1.6 Hz, 1H), 7.85 (dd, J = 8.8, 2 Hz, 1H), 7.71-7.63 (m, 2H), 7.59-7.53 (m, 2H), 7.47 (d, J = 8.4 Hz, 1H), 4.34 (s, 2H), 4.29 (app q J = 7.2 Hz, 2H), 4.22 (s, 2H), 3.56 (t, J = 4.4 Hz, 1H), 2.39-2.32 (m, 1H), 1.98 (dt, J = 14.8, 4 Hz, 2H), 1.85-1.80 (m, 4H), 1.74 (d, J = 14.4 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H), 1.17-1.06 (m, 4H). MS (m/z): 614.2 (M + 1). 3B [00014] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ 8.30 (d, J = 1.6 Hz, 1H), 7.81 (dd, J = 8.4, 1.8 Hz, 1H), 7.71-7.62 (m, 2H), 7.60-7.53 (m, 2H), 7.42 (d, J = 8.4 Hz, 1H), 4.33 (s, 2H), 4.19 (bs, 2H), 3.54 (t, J = 4.4 Hz, 1H), 2.39-2.31 (m, 1H), 1.98 (dt, J = 14.8, 4 Hz, 2H), 1.86- 1.77 (m, 4H), 1.73 (app d, J = 16.4 Hz, 2H), 1.17-1.04 (m, 4H). MS (m/z): 586.2 (M + 1).

Example 4

2-({2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-1,3-benzothiazol-6-yl}formamido)acetic acid (4-1)

[0161] ##STR00015##

[0162] 2-((1R,3r,5S)-3-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methoxy)-8-azabicyclo[3.2.1]octan-8-yl)benzo[d]thiazole-6-carboxylic acid (Example 3) (0.06 mmol) was combined with glycine methyl ester hydrochloride (0.06 mmol), HATU (O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) (0.065 mmol), diisopropylethylacetate (0.05 ml) and dichloromethane (2 mL). The mixture was stirred for 1 hour, then the solvent was removed in vacuo. The residue was suspended in ethyl acetate (15 mL) and washed with sodium bicarbonate solution (5 mL). The organics were combined and dried (MgSO.sub.4) then evaporated in vacuo. The crude product was purified by flash silica chromatography with 0-100% ethyl acetate in hexanes to give the ester (4A).

[0163] The ester (4A) was subjected to a solution of 4N LiOH in water (2 mL) and dioxane (2 mL) and stirred for 2 hours. The solvent was reduced in vacuo and the mixture diluted with 5% citric acid (10 mL) and extracted with ethyl acetate (2×8 mL). The organics were combined and dried (MgSO.sub.4) then evaporated in vacuo. The product was purified with flash silica chromatography with methanol/dichloromethane with a 0-40% gradient to give the title compound as a white solid.

[0164] Examples 4-2, 4-3 and 4-4 can be prepared following the same procedures, using appropriate intermediates.

TABLE-US-00003 Physical Data Ex MS (m/z), 1H NMR 4-1 [00016] .sup.1H NMR (MeOD, 400 MHz): δ 8.07 (d, J = 1.6 Hz, 1H), 7.71 (dd, J = 8.4, 1.6 Hz, 1H), 7.57-7.48 (m, 2H), 7.41 (app t, J = 7.6 Hz, 2H), 7.35 (d, J = 8.4 Hz, 1H), 4.31 (s, 2H), 4.16 (bs, 2H), 3.95 (s, 2H), 3.50 (t, J = 4.4 Hz, 1H), 2.21-2.15 (m, 1H), 2.00 (dt, J = 14.8, 4 Hz, 2H), 1.91-1.81 (m, 4H), 1.72 (d, J = 14.8 Hz, 2H), 1.09-1.05 (m, 4H). MS (m/z): 643.2 (M + 1). 4-2 [00017] MS (m/z): 661.2 (M + 1) 4-3 [00018] .sup.1H NMR (MeOH-d.sub.4, 400 MHz): δ 8.35 (d, J = 2.0 Hz, 1H), 8.29 (dd, J = 9.6, 3.0 Hz, 1H), 7.64 (app dt, J = 7.6, 2.0 Hz, 1H), 7.59 (app dd, J = 8.4, 2.0 Hz, 1H), 7.54- 7.48 (m, 2H), 7.28 (d, J = 9.6 Hz, 1H), 4.56 (br s, 2H), 4.44 (s, 2H), 4.18 (s, 2H), 3.61 (app t, J = 4.4 Hz, 1H), 2.29-2.27 (m, 1H), 2.06-1.89 (m, 8H), 1.19-1.15 (m, 4H). MS (m/z): 587.2 (M + 1). 4-4 [00019] MS (m/z): 645.1 (M + 1)

Example 5

2-({2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-1,3-benzothiazol-6-yl}formamido)ethane-1-sulfonic acid (5-1)

[0165] ##STR00020##

[0166] To a resealable and pressure tolerable vessel was added the following in sequential order: 2-((1R,3r,5S)-3-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl) isoxazol-4-yl)methoxy)-8-azabicyclo[3.2.1]octan-8-yl)benzo[d]thiazole-6-carboxylic acid (Example 3) (0.1 mmol), tetrahydrofuran (1.0 mL), N-methyl morpholine (approximately 0.1 mL, 0.7 mmol). The suspension was stirred at room temperature for a few minutes until complete dissolution of the starting acid. Next was added 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.15 mmol). and the resulting solution was stirred at 50° C. for 20 minutes until a fine white precipitate formed. This precipitate was physically agitated to ensure that all materials were thoroughly mixed. Next the taurine (0.40 mmol) was added as a dimethyl acetamide (4 mL) suspension. The resulting suspension was sealed in the vessel and heated to 80° C. for 2 hours. The mixture was then cooled to room temperature, diluted with ethyl acetate 20 mL and washed with water (2×3 mL). The organics were dried under vacuum, the resulting residue was diluted with 3 mL of MeOH, and the liquid was directly purified using mass-directed reverse phase HPLC using gradient of 20 to 70% acetonitrile/water with ammonium acetate (0.05%) as modifier. The resulting product was cold vacuum concentrated to give the title compound as a white powder.

[0167] Examples 5-2, 5-3 and 5-4 can be prepared following the same procedures, using appropriate intermediates.

TABLE-US-00004 Physical Data Ex MS (m/z), .sup.1H NMR 5-1 [00021] .sup.1H NMR (MeOD, 400 MHz): δ 8.27 (d, J = 1.6 Hz, 1H), 8.00 (dd, J = 8.4, 1.9 Hz, 1H), 7.65-7.53 (m, 2H), 7.42 (app t, J = 7.8 Hz, 2H), 7.35 (d, J = 8.4 Hz, 1H), 4.42 (br s, 4H), 3.81 (t, J = 6.4 Hz, 2H), 3.11 (t, J = 6.4 Hz, 2H), 2.18-1.93 (m, 8H), 1.72 (d, J = 14.8 Hz, 2H), 1.29- 1.15 (m, 4H). MS (m/z): 693.2 (M + 1). 5-2 [00022] MS (m/z): 711.2 (M + 1) 5-3 [00023] MS (m/z): 695.3 (M + 1) 5-4 [00024] .sup.1H NMR (MeOH-d.sub.4, 400 MHz): δ 8.30 (d, J = 2.0 Hz, 1H), 8.26 (d, J = 9.6 Hz, 1H), 7.66 (app dt, J = 7.6, 2.0 Hz, 1H), 7.61 (app dd, J = 8.4, 2.0 Hz, 1H), 7.54-7.49 (m, 2H), 7.25 (dd, J = 9.4, 1.8 Hz, 1H), 4.55 (br s, 2H), 4.49 (s, 2H), 3.78 (t, J = 8.4 Hz, 2H), 3.60 (app t, J = 4.4 Hz, 1H), 3.07 (t, J = 8.4 Hz, 2H), 2.27-2.21 (m, 1H), 2.08-1.93 (m, 8H), 1.21-1.16 (m, 4H). MS (m/z): 637.2 (M + 1).

Example 6

[0168] ##STR00025##

[0169] The following examples can be prepared from the reaction of 4-(((1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-yloxy)methyl)-5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazole (I-1) and the corresponding pyridyl and pyrazinyl derivative following the procedures described in Example 1.

TABLE-US-00005 Physical Data Ex MS (m/z), .sup.1H NMR 6-1A [00026] MS (m/z): 544.2 (M + 1) 6-1B [00027] .sup.1H NMR (MeOD, 400 MHz): δ 8.64 (d, J = 2 Hz, 1H), 7.98 (dd, J = 8.8, 2.4 Hz, 1H), 7.66-7.58 (m, 2H), 7.52-7.48 (m, 2H), 6.64 (d, J = 9.2 Hz, 1H), 4.43 (bs, 2H), 4.38 (s, 2H), 3.52 (t, J = 4.4 Hz, 1H), 2.31- 2.24 (m, 1H), 1.97-1.82 (m, 6H), 1.71 (d, J = 14.4 Hz, 2H), 1.18-1.14 (m, 4H). MS (m/z): 530.2 (M + 1). 6-2A [00028] MS (m/z): 545.2 (M + 1) 6-2B [00029] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ 8.51 (s, 1H), 8.01 (s, 1H), 7.64-7.56 (m, 2H), 7.51-7.46 (m, 2H), 4.41 (bs, 2H), 4.26 (s, 2H), 3.40 (t, J = 4 Hz, 1H), 2.31-2.24 (m, 1H), 1.75-1.67 (m, 6H), 1.57 (d, J = 14.8 Hz, 2H), 1.09-0.97 (m, 4H). MS (m/z): 531.2 (M + 1).

Example 7

[0170] ##STR00030##

[0171] Example 7 was prepared following the procedures in Example 1 from 4-((8-azabicyclo[3.2.1]octan-3-yloxy)methyl)-5-cyclopropyl-3-(2-(trifluoromethyl)phenyl)isoxazole (I-2) and the corresponding pyrimidyl reagent.

TABLE-US-00006 Physical Data Ex MS (m/z), .sup.1H NMR 7A [00031] MS (m/z): 515.3 (M + 1) 7B [00032] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): δ 7.92 (d, J = 7.6 Hz, 1H), 7.83-7.73 (m, 2H), 7.60 (d, J = 7.6 Hz, 1H), 6.94 (s, 1H), 4.50 (bs, 2H), 4.23 (bs, 2H), 3.47-3.40 (m, 1H), 2.38-2.29 (m, 4H), 1.81-1.61 (m, 8H), 1.16-1.04 (m, 4H). MS (m/z): 529.3 (M + 1).

Example 8

[0172] The following examples were prepared according to the procedures described in Kittelmann, M. et al., Adv. Synth. Catal. 2003, 345, 825-829.

TABLE-US-00007 Physical Data Ex MS (m/z), .sup.1H NMR 8-1 [00033] .sup.1H NMR (DMSO-d.sub.6, 600 MHz): δ 12.87 (br s, 1H), 8.31 (d, J =1.2 Hz, 1H), 7.91 (app t, J = 8.0 Hz, 1H), 7.76 (app t, J = 8.0 Hz, 1H), 7.71 (d, J = 9.3 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 5.58 (d, J = 7.3 Hz, 1H), 4.25 (s, 4H), 3.83 (d, J = 8.7 Hz, 1H), 3.52 (app t, J = 4.0 Hz, 1H), 3.43-3.31 (m, 4H), 2.36- 2.32 (m, 1H), 1.96 (dt, J = 14.0, 4.0 Hz, 2H), 1.85-1.73 (m, 6H), 1.18-1.04 (m, 4H). MS (m/z): 764.3 (M + 1). 8-2 [00034] MS (m/z): 780.2 (M + 1) 8-3 [00035] MS (m/z): 706.3 (M + 1)

Example 9

Effect of Test Compound in Chronic Treatment Rat ANIT Model

[0173] A compound of Formula (I) was evaluated in a chronic treatment model of cholestasis over a range of doses from 0.01 to 3 mg/kg. Rats were treated with ANIT (0.1% w/w) in food for 3 days prior to treatment with Compound A at the indicated doses (“Veh”). A non-cholestatic control group was fed standard chow diet without ANIT, and serve as the non-cholestatic control animals (“Control”). After 14 days of oral dosing, the indicated analyte was measured in serum. LLQ, lower limit of quantitation. Mean±SEM; n=5.

[0174] ANIT treatment caused elevation of hepatobiliary injury indicators, such as elevated levels of circulating aspartate aminotransferase (AST) (FIG. 1A), alanine aminotransferase (ALT) (FIG. 1B), bilirubin (FIG. 1C) and bile acids (FIG. 1D) (“Veh” vs “Control”). These data demonstrate that ANIT exposure induced profound cholestasis and hepatocellular damage. In contrast, Compound A improved many of these indicators starting at doses as low as 0.01 mg/kg. Marked reductions of serum bile acid and bilirubin concentrations were observed upon treatment with Compound A. The reduced levels of total bile acids (TBA) levels associated with treatment of Compound A were consistent with the pharmacological action of FXR agonist by reducing accumulation of bile acids in the liver, enhancing bile acid excretion in the biliary tract and inhibiting bile acid synthesis. The improvement in the serum conjugated bilirubin (a direct indicator for hepatic function) by Compound A implies recovery from cholestasis with improved bile excretion.

[0175] Furthermore, Compound A stimulated serum FGF15 expression in the chronic treatment rat ANIT model in a dose dependent manner (FIG. 1E). Serum FGF15 levels were quantified using an FGF15 Meso Scale Discovery (MSD) assay. Mouse FGF15 antibody from R&D Systems (AF6755) was used both as capture and detection antibody in the assay. MSD SULFO-TAG NHS-Ester was used to label the FGF15 antibody. MSD standard 96-well plates were coated with the FGF15 capture antibody and the plates were blocked with MSD Blocker A (R93AA-2). After washing the plate with PBS+0.05% Tween 20, MSD diluent 4 was dispensed into each well and incubated for 30 min. 25 μl of calibrator dilutions or samples (serum or EDTA plasma) were dispensed into each well and incubated with shaking at RT. After washing, detection antibody was added and incubated with shaking for 1 h at RT. After washing and the addition of MSD Read buffer (R92TC-2), the plate was read on an MSD SECTOR Imager 6000. Plots of the standard curve and unknown samples were calculated using MSD data analysis software.

[0176] Activation of FXR in the ileum induces the expression of fibroblast growth factor 15 (FGF15 in rodent; FGF19 in human), a hormone that is secreted in the portal blood and signals to the liver to repress Cyp7a1 expression synergistically with SHP. The direct FXR-dependent induction of FGF15/19 along with FGF15/19's anti-cholestatic properties makes it a convenient serum biomarker for detecting target engagement of FXR agonists. Significant dose-dependent induction of FGF15 observed with treatment of Compound A demonstrate FXR target engagement by Compound A.

[0177] The results demonstrated in FIG. 1 are consistent with the use of a compound of Formula (I) for the treatment of cholestatic liver disorders such as bile acid malabsorption (e.g., primary or secondary bile acid diarrhea), bile reflux gastritis, collagenous colitis, lymphocytic colitis, diversion colitis, indeterminate colitis, Alagille syndrome, biliary atresia, ductopenic liver transplant rejection, bone marrow or stem cell transplant associated graft versus host disease, cystic fibrosis liver disease, and parenteral nutrition-associated liver disease.

[0178] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.