WO2010059853A1 - Modulateurs de tgr5 et leur procédé d'utilisation - Google Patents

Modulateurs de tgr5 et leur procédé d'utilisation Download PDF

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Publication number
WO2010059853A1
WO2010059853A1 PCT/US2009/065188 US2009065188W WO2010059853A1 WO 2010059853 A1 WO2010059853 A1 WO 2010059853A1 US 2009065188 W US2009065188 W US 2009065188W WO 2010059853 A1 WO2010059853 A1 WO 2010059853A1
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Prior art keywords
hydrogen
compound
hydroxy
nhch
unsubstituted
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PCT/US2009/065188
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English (en)
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Roberto Pellicciari
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Intercept Pharmaceuticals, Inc.
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=41668454&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2010059853(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to KR1020117014037A priority Critical patent/KR101667436B1/ko
Priority to PL09760057T priority patent/PL2376519T3/pl
Priority to JP2011537624A priority patent/JP5535233B2/ja
Priority to CN200980154713.0A priority patent/CN102325784B/zh
Priority to AU2009316566A priority patent/AU2009316566B9/en
Priority to ES09760057.1T priority patent/ES2458168T3/es
Priority to KR1020167028159A priority patent/KR101789960B1/ko
Application filed by Intercept Pharmaceuticals, Inc. filed Critical Intercept Pharmaceuticals, Inc.
Priority to MX2011005295A priority patent/MX2011005295A/es
Priority to DK09760057.1T priority patent/DK2376519T3/da
Priority to BRPI0921983A priority patent/BRPI0921983B8/pt
Priority to EP09760057.1A priority patent/EP2376519B1/fr
Priority to CA2744189A priority patent/CA2744189C/fr
Priority to EA201170714A priority patent/EA020140B1/ru
Publication of WO2010059853A1 publication Critical patent/WO2010059853A1/fr
Priority to IL212969A priority patent/IL212969A/en
Priority to ZA2011/04077A priority patent/ZA201104077B/en
Priority to HK12106982.3A priority patent/HK1166327A1/xx
Priority to IL244812A priority patent/IL244812B/en
Priority to IL264796A priority patent/IL264796B/en

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    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
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    • C07J—STEROIDS
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    • C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton

Definitions

  • the invention relates to compounds that modulate TGR5 and compositions useful in methods for the treatment and/or prevention of various diseases.
  • TGR5 receptor is a G-protein-coupled receptor that has been identified as a cell- surface receptor that is responsive to bile acids (BAs).
  • BAs bile acids
  • the primary structure of TGR5 and its responsiveness to bile acids has been found to be highly conserved in TGR5 among human, bovine, rabbit, rat, and mouse, and thus suggests that TGR5 has important physiological functions.
  • TGR5 has been found to be widely distributed in not only lymphoid tissues but also in other tissues. High levels of TGR5 mRNA have been detected in placenta, spleen, and monocytes/macrophages.
  • Bile acids have been shown to induce internalization of the TGR5 fusion protein from the cell membrane to the cytoplasm. Kawamata et al. 2003, J. Bio. Chem., 278, 9435. TGR5 has been found to be identical to hGPCR19 reported by Takeda et al. 2002, FEBS Lett. 520, 97-
  • TGR5 is associated with the intracellular accumulation of cAMP, that is widely expressed in diverse cell types. While the activation of this membrane receptor in macrophages decreases pro-inflammatory cytokine production, (Kawamata, Y., et al. J. Biol. Chem. 2003, 278, 9435-9440) the stimulation of TGR5 by BAs in adipocytes and myocytes enhances energy expenditure (Watanabe, M.et al. Nature. 2006, 439, 484-489). This latter effect involves the cAMP-dependent induction of type 2 iodothyronine deiodinase (D2), which by, locally converting T4 into T3, gives rise to increased thyroid hormone activity.
  • D2 type 2 iodothyronine deiodinase
  • TGR5 knock-out mice show a significant fat accumulation with body weight gain when challenged with a high fat diet, indicating that the lack of TGR5 decreases energy expenditure and elicits obesity (Maruyama, T., et al. J. Endocrinol. 2006, 191, 197-205).
  • bile acid activation of the membrane receptor has also been reported to promote the p ke peptide 1 (GLP-I) in murine enteroendocrine cell lines (Katsuma, S., Biochem. Biophys. Res. Commun. 2005, 329, 386-390).
  • GLP-I p ke peptide 1
  • TGR5 modulators are also useful for the treatment of other diseases e.g., central nervous diseases as well as inflammatory diseases (WO 01/77325 and WO 02/84286).
  • Modulators of TGR5 also provide methods of regulating bile acid and cholesterol homeostasis, fatty acid absorption, and protein and carbohydrate digestion.
  • TGR5 agonists have been described in literature. Recently, 23-alkyl-substituted and 6,23-alkyl-disubstituted derivatives of chenodeoxycholic acid
  • TGR5 methylation at the C 23 -position of natural bile acids (BAs) confers a marked selectivity to TGR5 over FXR (farnesoid X receptor) activation, whereas the 6 ⁇ -alkyl substitution increases the potency at both receptors.
  • TGR5 agonists include 6-Methyl-2-oxo-4-thiophen-2-yl-1,2,3,4-tetrahydro-pyrimidine- 5-carboxylic acid benzyl ester (WO004067008, Takeda Chemical Industries LTD, Japan,
  • TGR5 agonists have also provided for the first time a pharmacological differentiation of genomic versus nongenomic effects of BAs and have also allowed for informative structure-activity relationship studies, for example, the presence of an accessory binding pocket in TGR5 has been found to play a pivotal role in determining ligand selectivity (See, Pellicciari, et al. J. Med. Chem. 2007, 50, 4265-4268).
  • the availability of more potent and selective TGR5 modulators is necessary to further identify additional features affecting receptor activation and characterize the physiological and pharmacological actions of this receptor in order to better understand its relationship to the prevention and treatment of disease.
  • CA cholic acid
  • Cholic acid is a primary bile acid in human and many animal species, also reported as one of the main components together with bilirubin of Calculus Bovis, a highly valued traditional Chinese medicine (Chen, X., Biochem. Pharmacol. 2002, 63, 533-541).
  • Cholic acid (CA) differs from chenodeoxycholic acid (CDCA) and its derivatives described above by the presence at C- 12 of an additional alpha- hydroxyl group oriented on the polar side of the molecule. This "minor" structural difference accounts for the remarkably different physicochemical and biological features of these two bile acids.
  • the present invention relates to TGR5 modulators and their use to treat and/or prevent various diseases.
  • the invention relates to compounds according to formula A:
  • the invention includes a composition comprising a compound of the invention or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, and at least one pharmaceutically acceptable excipient.
  • the invention includes a compound for use in a method of treating or preventing disease in a subject.
  • the invention also includes the use of a composition or compound of the invention or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, in the manufacture of a medicament for a treating or preventing a disease in a subject.
  • the disease is selected from metabolic disease, inflammatory disease, liver disease, autoimmune disease, cardiac disease, kidney disease, cancer, and gastrointestinal disease.
  • Figure 1 is a graph that shows the impact of compound Ih3e on body weight gain in chow and high fat fed mice.
  • Figure 2 is a series of nine graphs (A-I) that show changes in the metabolic profile of high fat fed mice treated with compound Ih3e.
  • A-D relate to liver enzymes.
  • F-I relate to plasma lipids.
  • Figure 3 is a series of graphs (A-B) that show the results of plasma insulin analysis and oral glucose tolerance test in chow and high fat fed mice treated with compound Ih3e.
  • Figure 4 is a graph that shows changes in glucose levels in chow diet mice treated with compound Ih3e.
  • Figure 5 is a series of graphs (A-D) that show insulin release in vivo after a test meal in chow and high fat fed mice treated with compound Ih3e.
  • Figure 6 is a series of graphs (A-D) that shows oxygen consumption and CO2 production as measured by indirect calorimetry in chow and high fat fed mice treated with compound Ih3e
  • Figure 7 are three graphs (A-C) that show the respiratory exchange ratio (RER) value as calculated after indirect calorimetry in chow and high fat fed mice treated with compound Ih3e.
  • Figure 8 is a series of graphs (A-B) that show locomotor activity and food and water intake for chow and high fat fed mice treated with compound Ih3e.
  • Figure 9 is a series of graphs (A-C) that shows changes in organ weight for chow and high fat fed mice treated with compound Ih3e.
  • Figure 10 is a graph that depicts the surface tension plotted against the logarithm of the concentrating of compound Ih3e (mM) in NaCl 0.15M.
  • Figure 11 is a bile flow chart for a duodenal infusion experiment performed using compound Ih3e.
  • Figure 12 is a bile flow chart for a femoral infusion experiment performed using compound Ih3e.
  • Figure 13 is a graph that depicts secretion rates verses time in femoral and duodenal infusion experiments performed using compound Ih3e.
  • Figure 14 is a seriese of graphs (A-D) related to compound Ih3e and its metabolites.
  • Figure 14A that shows compound Ih3e and its main metabolites identified in bile using mass spectrometry in the iv experiment. Data are reported as absolute area values.
  • Figure 14b is a zoom display of Figure 14 A.
  • Figure 14C shows compound Ih3e and its main metabolites identified in bile using mass spectrometry.
  • Figure 14D is a zoom display of Figure 14C.
  • Figure 15 is a graph that shows the stability of compound Ih3e (triangle) and CA (square) in human stool culture.
  • Figure 16 is a bar graph that shows the dose-dependent release of GLP-I ex vivo induced by compound Ih3e.
  • Figure 17B is a bar graph that shows Cox activity in STC-I cells treated for 1 hr with compound Ih3e at the concentration indicated.
  • Figure 17C is a graph showing oxygen consumption in STC-I cells as measured using the XF24 extracellular flux analyzer (Seahorse Bioscience).
  • Figure 17E shows correlation plots for liver mRNA expression of TGR5 and Kir6.2 in the mouse BxD genetic reference population according to a similar strategy as described Figure A.
  • Figure 17F is a bar graph that shows mRNA expression levels of TGR5, CoxIV, and Kir6.2 in STC-I cells transfected for 36 hr with control or mTGR5 shRNA as was measured by real-time quantitative PCR.
  • Figures 18B and 18C are graphs that show intracellular calcium level in NCI-H716 cells transfected with mock vector, hTGR5 expression vector, or hTGR5 siRNA for 36 hr and treated with 1 (B) or 10 ⁇ M (C) of compound Ih3e.
  • Figure 18G is a bar graph that shows GLP-I release in STC-I cells transfected for 36 hr with control, mTGR5 expression vector, or mTGR5 shRNA and then exposed 30 min to compound Ih3e at the indicated concentration.
  • OGTT oral glucose tolerance test
  • Figure 19E is a series of pictures which are representative immunofluorescent insulin- stained pancreatic sections from TGR5-Tg male mice fed with a HF diet for 20 weeks or from male age-matched littermates fed with a CD or a HF diet for the same duration.
  • Figure 19G is a bar graph that shows insulin content in collagenase-isolated pancreatic islets from male TGR5-Tg mice and control littermates fed with a CD or a HF diet as described in ( Figure 19E).
  • Figure 19H is a graph that shows the results of an OGTT in TGR5 ⁇ ⁇ and TGR5 +/+ male mice fed with a HF diet for 8 weeks.
  • the inset represents the average AUC.
  • the data are represented as mean ⁇ SE.
  • Figure 2OA is graph which shows the results of measurement by HPLC of plasma compound Ih3e levels in CD-, HF-, and HF-fed Ih3e-treated male C57BL6/J mice.
  • Figure 2OD is a bar graph that shows organ mass as expressed as percent of the weight of CD-fed control mice.
  • Figure 2OF is a series of bar graphs which show spontaneous horizontal activity and energy expenditure, evaluated by the measurement of oxygen consumption (VO 2 ) and carbon dioxide release (VCO 2 ), that were monitored over a 18 hr period 6 weeks after the initiation of the dietary intervention.
  • the respiratory quotient (RQ) was calculated as the ratio VCO 2 / VO 2 .
  • Bar graphs represent the average AUC.
  • For the RQ, bar graphs represent the average (n 8).
  • the dotted lines illustrate the addition of the uncoupling agent FCCP at successive doses of 250 and 500 nM.
  • Figure 201 is a series of pictures that are representative pictures of oil redO (ORO) staining of cryosections (top panel) and Sirius red staining of paraffin-embedded sections (bottom panel) of the liver at the end of the dietary intervention. Fibrosis is indicated by the arrow.
  • ORO oil redO
  • Figure 2OK and 20 is a series of bar graphs that show plasma levels of liver enzymes
  • Figure 21 A is a graph that shows the result of an OGTT in CD- and HF-fed male C57BL6/J mice supplemented with 30 mg/ kg/d compound Ih3e for 8 weeks following the onset of obesity induced by feeding a HF diet during 10 weeks.
  • the inset represents the average AUC.
  • Figure 2 IB is a graph that shows fasting glycemia and insulinemia (4 hr fasting) in DIO mice after 3 weeks of dietary intervention with compound Ih3e (top panel). Plasma insulin levels during OGTT in DIO mice (bottom panel).
  • Figure 21C is a graph that shows the result of an OGTT in 14-week-old CD-fed db/db male mice treated with 30 mg/kg/d compound Ih3e for 6 weeks.
  • Figure 2 ID is a graph that shows fasting (4 hr) glycemia and insulinemia in db/db mice after 6 weeks of treatment with compound Ih3e (top panel). Plasma insulin levels during OGTT in DIO mice (bottom panel).
  • the evaluation of liver glucose production and its suppression by insulin, as well as the rate of glucose disappearance, was assessed at equilibrium using 3H-glucose (n 5).
  • Figure 22 A is a series of graphs that shows the results of a study where TGR5 +/+ and
  • FIG. 22B is a series of graphs which show plasma insulin levels that were concurrently measured during the OGTT in DIO in TGR5 +/+ (left panel) and TGR5 'f' (right panel) mice before and after 4 weeks' treatment with compound Ih3e.
  • the data are represented as mean ⁇ SE. Student's unpaired t test; *p ⁇ 0.05, vehicle compared to compound Ih3e 7-treated mice.
  • Figure 23 is a graph that shows compounds Ih3e and Ii3e bile flow rates in a femoral infusion experiment at 1 ⁇ mol/min/kg for 1 h and bile flow rate in a femoral experiment as control infusing 3% BSA physiological solution for 1 h.
  • Figure 24 is a graph that shows compound Ih3e and tauro-Ih3e secretion rates vs. time in a femoral experiment at 1 ⁇ mol/min/kg for 1 h.
  • Figure 25 is a graph that shows compound Ii3e and tauro-Ii3e secretion rates vs. time in femoral experiment at 1 ⁇ mol/min/kg for 1 h.
  • Figure 26 is a graph that shows compound Ih3e and its main metabolites identified in bile samples collected during the femoral infusion experiment. Data are reported as absolute area values.
  • Figure 27 is a zoom display of Figure 26.
  • treating means relieving, lessening, reducing, eliminating, modulating, or ameliorating, i.e. causing regression of the disease state or condition.
  • preventing means, to completely or almost completely stop a disease state or condition, from occurring in a patient or subject, especially when the patient or subject is predisposed to such or at risk of contracting a disease state or condition. Preventing can also include inhibiting, i.e. arresting the development, of a disease state or condition, and relieving or ameliorating, i.e. causing regression of the disease state or condition, for example when the disease state or condition may already be present.
  • the term "6-Et,23(S)-MeCA” refers to the compound Ih3e having the chemical
  • compound Ih3e may also be referred to as 6 ⁇ -ethyl-(23S)-methyl-3 ⁇ ,7 ⁇ ,12 ⁇ trihydroxy-5 ⁇ -cholan-24-oic acid.
  • Bile acids are steroid carboxylic acids derived from cholesterol.
  • the primary bile acids are cholic and chenodeoxycholic acids. In the body, these acids are conjugated with glycine or taurine before they are secreted into the bile.
  • Alkyl includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl), branched-chain alkyl groups (e.g., isopropyl, tert-butyl, isobutyl), cycloalkyl (e.g., alicyclic) groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • straight-chain alkyl groups e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octy
  • a straight chain or branched chain alkyl has six or fewer carbon atoms in its backbone, referred to as "lower alkyl" (e.g., C 1 -C 6 for straight chain meaning 1, 2, 3, 4, 5, or 6 carbon atoms, C 3 -C 6 for branched chain meaning 3, 4, 5, or 6 carbon atoms).
  • a straight chain or branched chain alkyl has four or fewer carbon atoms in its backbone.
  • cycloalkyls have 3, 4, 5, 6, 7, or 8 carbon atoms in their ring structure.
  • substituted alkyl refers to an alkyl moieties having a substituent replace one or more hydrogen atoms on at least one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbony
  • Aryl includes groups with aromaticity, including 5- and 6-membered “unconjugated”, or single-ring, aromatic groups that may include from zero to four heteroatoms, as well as “conjugated”, or multicyclic, systems with at least one aromatic ring.
  • aryl groups include benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
  • aryl includes multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine.
  • aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles", “heterocycles,” “heteroaryls” or “heteroaromatics”.
  • the aromatic ring can be substituted at least one ring position with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ure
  • lower alkyl includes an alkyl group, as defined above, but having from one to ten, for example, from one to six, carbon atoms in its backbone structure.
  • alkoxy or "alkoxyl” includes alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom.
  • alkoxy groups or alkoxyl radicals
  • ether includes compounds or moieties which contain an oxygen bonded to two different carbon atoms or heteroatoms.
  • alkoxyalkyl which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to another alkyl group.
  • esters includes compounds and moieties which contain a carbon or a heteroatom bound to an oxygen atom which is bonded to the carbon of a carbonyl group.
  • ester includes alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.
  • alkyl, alkenyl, or alkynyl groups are as defined above.
  • hydroxy or "hydroxyl” includes groups with an -OH or -O ⁇
  • halogen includes fluorine, bromine, chlorine, iodine, etc.
  • perhalogenated generally refers to a moiety wherein all hydrogens are replaced by halogen atoms.
  • An "anionic group,” as used herein, refers to a group that is negatively charged at physiological pH. Anionic groups include carboxylate, sulfate, sulfonate, sulfinate, sulfamate, tetrazolyl, phosphate, phosphonate, phosphinate, or phosphorothioate or functional equivalents thereof.
  • “Functional equivalents” of anionic groups are intended to include bioisosteres, e.g., bioisosteres of a carboxylate group.
  • Bioisosteres encompass both classical bioisosteric equivalents and non-classical bioisosteric equivalents.
  • Classical and non-classical bioisosteres are known in the art (see, e.g., Silverman, R. B. The Organic Chemistry of Drug Design and Drug Action, Academic Press, Inc.: San Diego, Calif, 1992, pp.19-23).
  • Another anionic group is a carboxylate.
  • stable functionality refers to a substitution pattern that contains a labile linkage, e.g., a functionality or bond that is susceptible to hydrolysis or cleavage under physiological conditions (e.g., aqueous solutions in the neutral pH range).
  • a labile linkage e.g., a functionality or bond that is susceptible to hydrolysis or cleavage under physiological conditions (e.g., aqueous solutions in the neutral pH range).
  • unstable functionalities include acetals and ketals.
  • crystal polymorphs or “polymorphs” refer to the existence of more than one crystal form for a compound, salt or solvate thereof. Crystal polymorphs of the bile acid analog compounds are prepared by crystallization under different conditions.
  • R-EMCA refers to the compound 6 ⁇ -ethyl-23(R)-methylcholic acid having the structure:
  • the compounds of the present invention can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules.
  • hydrates include monohydrates, dihydrates, etc.
  • solvates include ethanol solvates, acetone solvates, etc.
  • Solidvates means solvent addition forms that contain either stoichiometric or non stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate, when the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one of the substances in which the water retains its molecular state as H 2 O, such combination being able to form one or more hydrate.
  • the structure of some of the compounds of the invention include asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of the invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. Enantiomers (R- and S-configurations) are named according to the system developed by R.S. Cahn, C. Ingold, and V. Prelog. Further, the structures and other compounds discussed in this application include all atropic isomers thereof.
  • Atropic isomers are a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropic isomers owe their existence to a restricted rotation caused by hindrance of rotation of large groups about a central bond. Such atropic isomers typically exist as a mixture, however as a result of recent advances in chromatography techniques, it has been possible to separate mixtures of two atropic isomers in select cases.
  • “Stable compound” and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
  • the term “analog” refers to a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group).
  • an analog is a compound that is similar to or comparable in function and appearance to the reference compound.
  • derivative e.g., in the term “bile acid derivatives” refers to compounds that have a common core 4-membered ring structure, and are substituted with various groups as described herein.
  • bioisostere refers to a compound resulting from the exchange of an atom or of a group of atoms with another, broadly similar, atom or group of atoms.
  • the bioisosteric replacement may be physicochemically or topologically based.
  • Examples of carboxylic acid bioisosteres include acyl sulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176 (1996).
  • Combination therapy includes the administration of a compound of the invention and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents (i.e., the compound of the invention and at least a second agent).
  • the beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents.
  • Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).
  • “Combination therapy” may, but generally is not, intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention.
  • Combination therapy is intended to embrace administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner.
  • Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents.
  • Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues.
  • the therapeutic agents can be administered by the same route or by different routes.
  • a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally.
  • all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection.
  • the sequence in which the therapeutic agents are administered is not narrowly critical.
  • Combination therapy also embraces the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non- drug therapies (e.g., surgery or mechanical treatments) .
  • the combination therapy further comprises a non-drug treatment
  • the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.
  • parenteral administration and “administered parenterally” as used herein refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • pulmonary refers to any part, tissue or organ whose primary function is gas exchange with the external environment, e.g., O 2 /CO 2 exchange, within a patient. "Pulmonary” typically refers to the tissues of the respiratory tract.
  • pulmonary administration refers to administering the formulations described herein to any part, tissue or organ whose primary function is gas exchange with the external environment (e.g., mouth, nose, pharynx, oropharynx, laryngopharynx, larynx, trachea, carina, bronchi, bronchioles, alveoli).
  • pulmonary also includes a tissue or cavity that is contingent to the respiratory tract, in particular, the sinuses.
  • a "therapeutically effective amount" of a compound of the invention, or a combination of compounds is an amount (quantity or concentration) of compound or compounds.
  • the amount of the compound to be administered to a subject will depend on the particular disorder, the mode of administration, co-administered compounds, if any, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • prophylactically effective amount means an amount (quantity or concentration) of a compound of the present invention, or a combination of compounds, that is administered to prevent or reduce the risk of a disease - in other words, an amount needed to provide a preventative or prophylactic effect.
  • amount of the present compound to be administered to a subject will depend on the particular disorder, the mode of administration, co-administered compounds, if any, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • reducing the risk of, as used herein, means to lower the likelihood or probability of a central nervous system disease, inflammatory disease and/or metabolic disease from occurring in a patient, especially when the patient or subject is predisposed to such occurrence.
  • a "pharmaceutically acceptable salt” or “salt” of a compound of the invention is a product of the compound that contains an ionic bond, and is typically produced by reacting the compound with either an acid or a base, suitable for administering to a subject.
  • pharmaceutically acceptable salts refer to derivatives of the compounds of the invention wherein the parent compound is modified by making acid or base salts thereof.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodide, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric,
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, USA, p. 1445 (1990).
  • compositions, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • phrases "pharmaceutically acceptable carrier” is art-recognized, and includes, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically acceptable carrier is non-pyrogenic.
  • materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alg
  • compositions or “pharmaceutically acceptable composition” is a formulation containing a compound of the invention or salt, solvate, hydrate, or prodrug thereof.
  • the pharmaceutical composition is in bulk or in unit dosage form.
  • the unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial.
  • the quantity of active ingredient (e.g., a formulation of a compound of the invention or salts thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved.
  • active ingredient e.g., a formulation of a compound of the invention or salts thereof
  • the dosage will also depend on the route of administration.
  • routes of administration A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, and the like.
  • Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
  • flash dose refers to compound formulations that are rapidly dispersing dosage forms.
  • immediate release is defined as a release of compound from a dosage form in a relatively brief period of time, generally up to about 60 minutes.
  • modified release is defined to include delayed release, extended release, and pulsed release.
  • pulsed release is defined as a series of releases of drug from a dosage form.
  • a "subject” includes mammals, e.g., humans, companion animals (e.g., dogs, cats, birds, and the like), farm animals (e.g., cows, sheep, pigs, horses, fowl, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, birds, and the like). Typically, the subject is human.
  • companion animals e.g., dogs, cats, birds, and the like
  • farm animals e.g., cows, sheep, pigs, horses, fowl, and the like
  • laboratory animals e.g., rats, mice, guinea pigs, birds, and the like.
  • the subject is human.
  • Compounds of the invention also include prodrugs or physiologically equivalent derivatives.
  • a "prodrug” or “physiologically equivalent derivative” includes a precursor form of the drug which is metabolically converted in vivo to produce the active drug.
  • the invention further contemplates the use of prodrugs which are converted in vivo to the TGR5 modulating compounds used in the methods of the invention (see, e.g., R. B. Silverman, 1992, “The Organic Chemistry of Drug Design and Drug Action", Academic Press, Chp. 8).
  • prodrugs can be used to alter the biodistribution (e.g., to allow compounds which would not typically cross the blood-brain barrier to cross the blood-brain barrier) or the pharmacokinetics of the TGR5 modulating compound.
  • an anionic group e.g., a carboxylate, sulfate or sulfonate
  • an alkyl group e.g., a methyl group
  • a phenyl group e.g., a phenyl group
  • the ester is administered to a subject, the ester is cleaved, enzymatically or non-enzymatically, reductively or hydrolytically, to reveal the anionic group.
  • Such an ester can be cyclic, e.g., a cyclic sulfate or sulfone, or two or more anionic moieties may be esterified through a linking group.
  • An anionic group can be esterified with moieties (e.g., acyloxymethyl esters) which are cleaved to reveal an intermediate TGR5 modulating compound which subsequently decomposes to yield the active TGR5 modulating compound.
  • the prodrug is a reduced form of a carboxylate, sulfate or sulfonate, e.g., an alcohol or thiol, which is oxidized in vivo to the TGR5 modulating compound.
  • an anionic moiety can be esterified to a group which is actively transported in vivo, or which is selectively taken up by target organs.
  • amino acid conjugates refers to conjugates of the compounds of the invention with any suitable amino acid.
  • Taurine NH(CH 2 ) 2 SO 3 H
  • glycine NHCH 2 CO 2 H
  • Suitable amino acid conjugates of the compounds have the added advantage of enhanced integrity in bile or intestinal fluids.
  • Suitable amino acids are not limited to taurine and glycine.
  • the invention encompasses amino acid conjugates of the compounds of the invention. More specifically, the invention includes amino acid conjugates of compound Ih3e. Even more specifically, the invention includes the taurine and glycine conjugates of compound Ih3e.
  • TGR5 modulator means any compound that interacts with the TGR5 receptor. The interaction is not limited to a compound acting as an antagonist, agonist, partial agonist, or inverse agonist of the TGR5 receptor.
  • the compounds of the present invention act as an antagonist of the TGR5 receptor.
  • the compounds of the present invention act as an agonist of the TGR5 receptor.
  • the compounds of the present invention act as a partial agonist of the TGR5 receptor.
  • the compounds of the present invention as an inverse agonist of the TGR5 receptor.
  • the profile of a ligand traditionally, endogenous or synthetic, is characterized by its intrinsic efficacy 'e' originally described by Furchgott in 1966.
  • agonist means a compound that enhances the activity of another molecule or receptor site.
  • An agonist by classical definition, whether a orthosteric, allosteric, inverse or a co-agonist has a property to bind to the receptor, alter its receptor state and result in a biological action. Consequently, agonism is defined as a property of an agonist or a ligand to produce a biological action. In contrast to this, an orthosteric, allosteric, inverse or a co-agonist has a property to bind to the receptor, alter its receptor state and result in a biological action. Consequently, agonism is defined as a property of an agonist or a ligand to produce a biological action. In contrast to this, an
  • antagonism is essentially an agonist with high affinity to the same receptor macromolecule, but with very less or negligible intrinsic efficacy, and thus sterically prevents the biological actions of an agonist.
  • antagonism may be functional or physiological, where an agonist has a direct competition for the receptor site in former and opposing effects via a different receptor-messenger system in the later.
  • a TGR5 agonist is a receptor ligand or compound that binds to TG R5 and increases the concentration of cyclic adenosine monophosphate (cAMP) by at least 20% in cells expressing the receptor.”
  • a TGR5 antagonist would be a compound that antagonizes or blocks the activity of an agonist, thereby effecting a reduction in the concentration of c AMP.
  • the present invention relates to compounds having TGR5 receptor modulating activity and their use to treat and/or prevent various diseases including metabolic disease, inflammatory disease, liver disease, autoimmune disease, cardiac disease, kidney disease, cancer, and gastrolintestinal disease. Further, the present invention relates to compounds of the formulae described herein.
  • the invention relates to a compound of formula A:
  • R 1 is hydrogen, hydroxy, substituted or unsubstituted alkyl, or halogen
  • R 2 is hydrogen or ⁇ -hydroxy
  • R 3 is hydrogen, hydroxy, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H
  • R 4 is hydrogen, substituted or unsubstituted alkyl, or halogen
  • R 5 is unsubstituted or substituted alkyl, or aryl
  • R 6 is hydrogen, unsubstituted or substituted alkyl, or R 5 and R 6 taken together with the carbons to which they are attached form a ring of size 3, 4, 5, or 6 atoms
  • R 7 is hydrogen, substituted or unsubstituted alkyl, or hydroxy
  • Re is hydrogen, substituted or unsubstituted alkyl
  • R9 is hydrogen, substituted or unsubstituted alkyl or taken together Rg and R 9 form
  • R 1 is hydrogen or hydroxy.
  • R 1 is hydroxy.
  • R 1 is hydrogen.
  • R 2 is ⁇ -hydroxy.
  • R 1 is hydroxy and R 2 is ⁇ -hydroxy.
  • R 1 is hydroxy and R 2 is H.
  • R 1 is hydroxy and R 2 is H.
  • At least one Of R 1 or R 2 is hydroxy.
  • At least one OfR 1 or R 2 is hydrogen.
  • R 1 and R 2 are the same.
  • R 1 and R 2 are each ⁇ -hydroxy.
  • R 1 and R 2 are each hydrogen.
  • R 1O is R 3 .
  • R 3 is hydroxyl, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H.
  • R 3 is hydroxyl.
  • R 3 is not hydroxyl.
  • R 3 is NH(CH 2 ) m SO 3 H.
  • R 3 is NH(CH 2 ) m SO 3 H and m is 2.
  • R 3 is NH(CH 2 ) n CO 2 H.
  • R 3 is NH(CH 2 ) n CO 2 H and n is 1.
  • R 4 is hydrogen or unsubstituted alkyl.
  • R 4 is hydrogen.
  • R 4 is unsubstituted alkyl.
  • R 4 is unsubstituted alkyl.
  • R 4 is methyl or ethyl.
  • R 4 is methyl.
  • R 4 is ethyl.
  • R 3 and R 4 are the same.
  • R 3 and R 4 are different.
  • R 3 and R 4 are each hydrogen.
  • R 3 is hydroxyl and R 4 is hydrogen.
  • R 3 is NH(CH 2 ) m SO 3 H and R 4 is hydrogen.
  • R 3 is NH(CH 2 ) m SO 3 H, R 4 is hydrogen, and m is 2.
  • R 3 is NH(CH 2 ) n CO 2 H and R 4 is hydrogen.
  • R 3 is NH(CH 2 ) n CO 2 H, R 4 is hydrogen, and n is 1.
  • R 3 is H and R 4 is unsubstituted alkyl.
  • R 3 is OH and R 4 is methyl.
  • R 3 is OH and R 4 is ethyl.
  • R 3 is OH and R 4 is methyl.
  • R 5 is unsubstituted or substituted alkyl.
  • R 5 is in the S- conf ⁇ guration.
  • R 5 is in the R-conf ⁇ guration.
  • R 5 is methyl or ethyl.
  • R 5 is S-methyl.
  • R 5 is R- methyl.
  • R 5 is S-ethyl.
  • R 5 is R-ethyl.
  • R 5 is substituted alkyl substituted with phenyl.
  • R 5 is benzyl.
  • R 5 is S-benzyl.
  • R 5 is R-benzyl.
  • R 5 is aryl.
  • R 5 is phenyl.
  • R 4 and R 5 are each unsubstituted alkyl.
  • R 4 and R 5 are each unsubstituted alkyl, wherein R 5 is in the S- conf ⁇ guration and R 4 is in the alpha-configuration.
  • R 4 and R 5 are each unsubstituted alkyl and R 1 is hydroxy.
  • R 4 and R 5 are each unsubstituted alkyl and R 2 is hydrogen.
  • R 4 and R 5 are each unsubstituted alkyl, R 1 is hydroxy, and R 2 is hydrogen.
  • R 1 , R 2 , R 3 , and R 4 are hydrogen.
  • R 2 , R 3 , and R 4 are hydrogen.
  • R 2 and R 3 are hydrogen.
  • At least one OfR 1 , R 2 , R 3 , or R 4 is hydrogen.
  • At least two Of R 1 , R 2 , R 3 , or R 4 are hydrogen.
  • At least three OfR 1 , R 2 , R 3 , or R 4 are hydrogen.
  • At least four OfR 1 , R 2 , R 3 , or R 4 are hydrogen.
  • R 1 , R 2 , and R 4 are hydrogen and R 3 is OH.
  • R 2 and R 4 are hydrogen and R 3 is OH.
  • R 2 is hydrogen and R 3 is OH.
  • At least one OfR 1 , R 2 , or R 4 is hydrogen and R 3 is OH. At least two OfR 1 , R 2 , or R 4 are hydrogen and R 3 is OH. R 1 , R 2 and R 4 are hydrogen and R 3 is OH.
  • at least one of R 1 or R 7 is unsubstituted alkyl. At least one OfR 1 or R 7 is methyl. At least one OfR 1 or R 7 is ethyl. At least one OfR 1 or R 7 is propyl. R 1 is methyl. R 1 is ethyl. R 1 is propyl. R 7 is methyl. R 7 is ethyl. R 7 is propyl.
  • Both R 1 and R 7 are unsubstituted alkyl. Both R 1 and R 7 are methyl. Both R 1 and R 7 are ethyl. R 7 is hydrogen. R 7 is hydroxy. R 1 is hydrogen. R 1 is hydroxyl. One OfR 1 or R 7 is unsubstituted alkyl and the other R 1 or R 7 is hydrogen. One Of R 1 or R 7 is unsubstituted alkyl and the other R 1 or R 7 is hydroxy. At least one OfR 1 or R 7 is unsubstituted alkyl and R 5 is unsubstituted or substituted alkyl. At least one OfR 1 or R 7 is methyl and R 5 is methyl.
  • R 7 is hydroxy and both R 1 and R 5 are unsubstituted alkyl.
  • R 1 is hydroxyl and both R 7 and R 5 are unsubstituted alkyl.
  • At least one OfR 1 or R 7 is unsubstituted alkyl and R 5 is unsubstituted or substituted alkyl, further wherein R 5 is in the S-conf ⁇ guration.
  • At least one OfR 1 or R 7 is unsubstituted alkyl and R 5 is unsubstituted or substituted alkyl, further wherein R 5 is in the R- conf ⁇ guration.
  • R 1 is hydroxy and R 7 is methyl.
  • R 1 is methyl and R 7 is hydroxy.
  • R 6 is unsubstituted alkyl.
  • R 6 is methyl.
  • R 6 is ethyl.
  • R 6 is propyl.
  • R 8 is hydrogen.
  • R 8 is unsubstituted alkyl.
  • R 8 is methyl.
  • R 8 is ethyl.
  • R 8 is propyl.
  • R 2 is ⁇ -hydroxy and R 8 is unsubstituted alkyl.
  • R 8 and R9 form a carbonyl.
  • R 10 is R 3 .
  • R 3 is hydroxyl.
  • At least one of R 8 or R9 is hydrogen.
  • R 8 and R 9 are both hydrogen.
  • At least one OfR 8 or R 9 is unsubstituted alkyl.
  • At least one OfR 8 or R 9 is methyl.
  • At least one OfR 8 or R 9 is ethyl.
  • R 10 is SO 3 H.
  • R 2 , R 4 and R 6 are each hydrogen, R 3 is hydroxyl, and one OfR 1 and R 7 is hydrogen or hydroxyl, then the other R 1 or R 7 is not methyl.
  • R 2 is ⁇ -OH; R 3 is hydroxyl; R 4 and R 6 are each hydrogen; and one Of R 1 and R 7 is hydrogen or hydroxyl, then the other R 1 or R 7 is not methyl.
  • the present invention does not include the following compounds: 3 ⁇ ,7 ⁇ - dihydroxy-7 ⁇ -methyl-5 ⁇ -cholanoic acid, 3 ⁇ ,7 ⁇ -dihydroxy-7 ⁇ -methyl-5 ⁇ -cholanoic acid, 3 ⁇ - hydroxy-7 ⁇ -methyl-5 ⁇ -cholanoic acid, 3 ⁇ ,7 ⁇ ,12 ⁇ -trihydroxy-7 ⁇ -methyl-5 ⁇ -cholan-24-oic acid; 3 ⁇ ,7 ⁇ ,12 ⁇ -trihydroxy-7 ⁇ -methyl-5 ⁇ -cholan-24-oic acid; and 3 ⁇ ,12 ⁇ -dihydroxy-7 ⁇ - methyl-5 ⁇ -cholan-24-oic acid.
  • R 3 when R 3 is hydroxyl and one OfR 1 and R 7 is methyl and the other R 1 and R 7 is hydrogen or hydroxyl, then R 2 , R 4 and R 6 are not all hydrogen.
  • R 2 when R 2 is ⁇ -OH, R 3 is hydroxyl, and one OfR 1 and R 7 is methyl and the other R 1 and R 7 is hydrogen or hydroxyl, then R 4 and R 6 are not all hydrogen.
  • the present invention provides a compound of formula I:
  • R 1 is hydrogen, hydroxy, or halogen
  • R 2 is hydrogen or ⁇ -hydroxy
  • R 3 is hydroxy, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H
  • R 4 is hydrogen, unsubstituted or substituted alkyl, or halogen
  • R 5 is unsubstituted or substituted alkyl, or aryl
  • R 6 is hydrogen or R 5 and R 6 taken together with the carbons to which they are attached form a ring of size 3, 4, 5, or 6 atoms
  • m is an integer 0, 1, 2, 3, 4, or 5
  • n is an integer 0, 1, 2, 3, 4, or 5.
  • R 5 when R 5 is methyl, R 1 is hydroxyl, and R 3 is hydroxyl or NHCH 2 CH 2 SO 3 H, then R 4 is not hydrogen.
  • the present invention provides compounds where R 1 is hydrogen or hydroxy.
  • R 1 is hydroxy.
  • R 1 is hydrogen.
  • R 1 is ⁇ -hydroxy.
  • R 1 is ⁇ -hydroxy.
  • the present invention provides compounds where R 1 is halogen.
  • R 1 is fluorine.
  • R 1 is ⁇ -fluorine.
  • R 1 is ⁇ -fluorine.
  • the stereochemistry OfR 1 in the ⁇ - and ⁇ - conf ⁇ gurations is shown below:
  • R 1 alpha ( ⁇ -) configuration R 1 alpha ( ⁇ -) configuratio
  • the present invention provides compounds where R 2 is ⁇ -hydroxy.
  • R 2 is hydrogen.
  • R 1 is ⁇ -hydroxy and R 2 is ⁇ -hydroxy.
  • R 1 is ⁇ -hydroxy and R 2 is H.
  • R 1 is ⁇ - hydroxy and R 2 is H.
  • the present invention provides compounds where at least one OfR 1 or R 2 is hydroxy.
  • at least one OfR 1 or R 2 is hydrogen.
  • R 1 and R 2 are the same.
  • R 1 and R 2 are each ⁇ -hydroxy.
  • R 1 and R 2 are each hydrogen.
  • the present invention provides compounds where R 3 is hydrogen, hydroxyl, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H.
  • R 3 is hydroxy 1.
  • R 3 is not hydroxy 1.
  • R 3 is NH(CH 2 ) m SO 3 H.
  • R 3 is NH(CH 2 ) m SO 3 H and m is 2.
  • R 3 is NH(CH 2 ) n CO 2 H.
  • R 3 is NH(CH 2 ) n CO 2 H and n is 1.
  • R 4 is hydrogen or alkyl.
  • R 4 is hydrogen.
  • R 4 is lower alkyl.
  • R 4 is lower alkyl and the lower alkyl group is in the alpha configuration.
  • R 4 in the alpha configuration means that R 4 has the stereochemistry shown in the structure below.
  • R 4 is halogen.
  • R 4 is fluorine.
  • R 4 is halogen and the halogen is in the alpha configuration.
  • R 4 is ⁇ -fluorine.
  • R 4 is methyl or ethyl.
  • R 4 is methyl.
  • R 4 is ethyl.
  • R 4 is ⁇ -methyl.
  • R 4 is ⁇ -ethyl.
  • R 3 and R 4 are the same.
  • R 3 and R 4 are different.
  • R 3 and R 4 are each hydrogen.
  • R 3 is NH(CH 2 ) m SO 3 H and R 4 is hydrogen.
  • R 3 is hydroxyl and R 4 is hydrogen.
  • R 3 is NH(CH 2 ) m SO 3 H
  • R 4 is hydrogen and m is 2.
  • R 3 is NH(CH 2 ) n CO 2 H and R 4 is hydrogen.
  • R 3 is NH(CH 2 ) n CO 2 H, R 4 is hydrogen and n is 1.
  • R 3 is OH and R 4 is alkyl.
  • R 3 is OH and R 4 is lower alkyl. Lower alkyl is in the alpha configuration.
  • R 3 is OH and R 4 is methyl.
  • R 3 is OH and R 4 is ethyl.
  • R 3 is OH and R 4 is ⁇ -methyl.
  • R 3 is OH and R 4 is ⁇ -ethyl.
  • R 5 is unsubstituted or substituted alkyl.
  • R 5 is unsubstituted or substituted lower alkyl.
  • R 5 is in the S-configuration.
  • R 5 is in the R-configuration.
  • R 5 is methyl or ethyl.
  • R 5 is S-methyl.
  • R 5 is S-ethyl.
  • R 5 is alkyl substituted with phenyl.
  • R 5 is lower alkyl substituted with phenyl.
  • R 5 is benzyl.
  • R 5 is S-benzyl.
  • R 5 is R- benzyl.
  • R 5 is aryl.
  • R 5 is phenyl.
  • R 4 and R 5 are each unsubstituted alkyl.
  • R 4 and R 5 are each lower unsubstituted alkyl.
  • R 4 and R 5 are each lower unsubstituted alkyl and R 5 is in the S- configuration.
  • R 4 and R 5 are each lower unsubstituted alkyl and R 4 is in the alpha configuration.
  • R 4 and R 5 are not hydrogen.
  • R 4 and R 5 are each lower unsubstituted alkyl and R 1 is ⁇ -hydroxy.
  • R 4 and R 5 are each lower unsubstituted alkyl and R 2 is hydrogen.
  • R 4 and R 5 are each lower unsubstituted alkyl, R 1 is ⁇ -hydroxy, and R 2 is hydrogen.
  • R 5 and R 6 taken together with the carbons to which they are attached form a ring size of 3, 4, 5, or 6 atoms.
  • R 5 and R 6 taken together with the carbons to which they are attached form a 3-membered ring.
  • the 3-membered ring has the following ,
  • the 3-membered ring has the following ,
  • R 1 , R 2 , R 3 , and R 4 are hydrogen.
  • R 2 , R 3 , and R 4 are hydrogen.
  • R 2 and R 3 are hydrogen.
  • R 1 , R 2 , and R 4 are hydrogen and R 3 is OH.
  • R 2 and R 4 are hydrogen and R 3 is OH.
  • R 2 is hydrogen and R 3 is OH.
  • At least one OfR 1 , R 2 , R 3 , or R 4 is hydrogen. In another aspect, at least two OfR 1 , R 2 , R 3 , or R 4 are hydrogen. In another aspect, at least three OfR 1 , R 2 , R 3 , or R 4 are hydrogen. In another aspect, R 1 , R 2 , R 3 , and R 4 are hydrogen. In another aspect, at least one OfR 1 , R 2 , or R 4 is hydrogen and R 3 is OH. In another aspect, at least two Of R 1 , R 2 , or R 4 are hydrogen and R 3 is OH. In another aspect, R 1 , R 2 , and R 4 are hydrogen and R 3 is OH. In another aspect, the present invention does not include when R 5 is methyl, R 4 is hydrogen, and R 2 is H or OH.
  • the compound is selected from Compounds Ia, Ib, Ic, Ig, Ih, Ii, Io, Ip, Iq, IaI, IbI, IcI, IgI, IhI, IiI, IU, ImI, InI, Iol, IpI, IqI, Ia2, Ib2, Ic2, Id2, Ie2, If2, Ig2, Ih2, Ii2, 112, Im2, In2, Io2, Ip2, Iq2, Ia3, Ib3, Ic3, Id3, Ie3, If3, Ig3, Ih3, Ii3, 113, Im3, In3, Ia4, Ib4, Ic4, 1(14, Ie4, If4, Ig4, Ih4, Ii4, 114, Im4, In4, Ia5, Ib5, Ic5, Id5, IeS, IfS, IgS, IhS, IiS, 115, ImS, InS, Ib3e, Ic3e, Id3e, Ie3e
  • the compound is not selected from Compounds Id, Ie, If, IdI, H, Im, and In. In another aspect, the compound is not selected from IeI and IfI.
  • composition or medicament comprising a compound of formula I:
  • R 1 is hydrogen, hydroxy, or halogen
  • R 2 is hydrogen or ⁇ -hydroxy
  • R 3 is hydroxy, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H
  • R 4 is hydrogen, unsubstituted or substituted alkyl, or halogen
  • R 5 is unsubstituted or substituted lower alkyl, or aryl
  • R 6 is hydrogen or R 5 and R 6 taken together with the carbons to which they are attached form a ring of size 3, 4, 5, or 6 atoms
  • m is an integer 0, 1, 2, 3, 4, or 5
  • n is an integer 0, 1, 2, 3, 4, or 5.
  • the present invention includes a composition or medicament comprising a compound of formula I with proviso that when R 5 is methyl, R 1 is hydroxyl, and R 3 is hydroxy or NHCH 2 CH 2 SO 3 H, then R 4 is not hydrogen.
  • Another aspect of the invention includes compounds of Formula IA:
  • R 1 is hydrogen, hydroxy, substituted or unsubstituted alkyl, or halogen
  • R 2 is hydrogen or ⁇ -hydroxy
  • R 3 is hydroxy, hydrogen, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H
  • R 4 is hydrogen, substituted or unsubstituted alkyl, or halogen
  • R 5 is unsubstituted or substituted alkyl, or aryl
  • R 6 is hydrogen, unsubstituted or substituted alkyl, or R 5 and R 6 taken together with the carbons to which they are attached form a ring of size 3, 4, 5, or 6 atoms
  • R 7 is hydrogen, substituted or unsubstituted alkyl, or hydroxy
  • m is an integer 0, 1, 2, 3, 4, or 5
  • n is an integer 0, 1, 2, 3, 4, or 5.
  • R 4 is not hydrogen.
  • R 1 is hydrogen or hydroxy.
  • R 1 is hydroxy.
  • R 1 is hydrogen.
  • R 1 is hydroxy and R 2 is ⁇ -hydroxy.
  • R 1 is hydroxy and R 2 is H.
  • R 1 is hydroxy and R 2 is H.
  • At least one OfR 1 or R 2 is hydroxy.
  • At least one OfR 1 or R 2 is hydrogen.
  • R 1 and R 2 are the same.
  • R 1 is hydroxyl and R 2 is ⁇ -hydroxy.
  • R 1 and R 2 are each hydrogen.
  • R 3 is hydrogen, hydroxy, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H.
  • R 3 is hydroxy.
  • R 3 is not hydroxy.
  • R 3 is NH(CH 2 ) m SO 3 H.
  • R 3 is NH(CH 2 ) m SO 3 H and m is 2.
  • R 3 is NH(CH 2 ) n CO 2 H.
  • R 3 is NH(CH 2 ) n CO 2 H and n is 1.
  • R 4 is hydrogen or unsubstituted alkyl.
  • R 4 is hydrogen.
  • R 4 is unsubstituted alkyl.
  • R 4 is unsubstituted alkyl.
  • R 4 is methyl or ethyl.
  • R 4 is methyl.
  • R 4 is ethyl.
  • R 3 and R 4 are the same.
  • R 3 and R 4 are different.
  • R 3 and R 4 are each hydrogen.
  • R 3 is OH and R 4 is hydrogen.
  • R 3 is NH(CH 2 ) m SO 3 H and R 4 is hydrogen.
  • R 3 is NH(CH 2 ) m SO 3 H, R 4 is hydrogen, and m is 2.
  • R 3 is NH(CH 2 ) n CO 2 H and R 4 is hydrogen.
  • R 3 is NH(CH 2 ) n CO 2 H, R 4 is hydrogen, and n is 1.
  • R 3 is OH and R 4 is unsubstituted alkyl.
  • R 3 is OH and R 4 is unsubstituted alkyl.
  • R 3 is OH and R 4 is methyl.
  • R 3 is OH and R 4 is ethyl.
  • R 3 is OH and R 4 is methyl.
  • R 5 is unsubstituted or substituted alkyl.
  • R 5 is in the S-conf ⁇ guration.
  • R 5 is in the R-configuration.
  • R 5 is methyl or ethyl.
  • R 5 is S-methyl.
  • R 5 is R-methyl.
  • R 5 is S- ethyl.
  • R 5 is R-ethyl.
  • R 5 is substituted with phenyl.
  • R 5 is benzyl.
  • R 5 is S-benzyl.
  • R 5 is R- benzyl.
  • R 5 is aryl.
  • R 5 is phenyl.
  • R 4 and R 5 are each unsubstituted alkyl.
  • R 4 and R 5 are each unsubstituted alkyl, further wherein R 5 is in the S-conf ⁇ guration.
  • R 4 and R 5 are each unsubstituted alkyl.
  • R 4 and R 5 are each unsubstituted alkyl and R 1 is hydroxy.
  • R 4 and R 5 are each unsubstituted alkyl and R 2 is hydrogen.
  • R 4 and R 5 are each unsubstituted alkyl, R 1 is hydroxy, and R 2 is hydrogen.
  • R 1 , R 2 , R 3 , and R 4 are hydrogen.
  • R 2 , R 3 , and R 4 are hydrogen.
  • R 2 and R 3 are hydrogen.
  • At least one OfR 1 , R 2 , R 3 , or R 4 is hydrogen.
  • At least two OfR 1 , R 2 , R 3 , or R 4 is hydrogen. At least three OfR 1 , R 2 , R 3 , or R 4 is hydrogen. R 1 , R 2 , R 3 , and R 4 is hydrogen. In one aspect, R 1 , R 2 , and R 4 are hydrogen and R 3 is OH. R 2 and R 4 are hydrogen and R 3 is OH. R 2 is hydrogen and R 3 is OH. At least one OfR 1 , R 2 , or R 4 is hydrogen and R 3 is OH. At least two OfR 1 , R 2 , or R 4 is hydrogen and R 3 is OH. All OfR 1 , R 2 , and R 4 are hydrogen and R 3 is OH. In another aspect, at least one OfR 1 or R 7 is unsubstituted alkyl. At least one OfR 1 or R 7 is unsubstituted alkyl. At least one OfR 1 or R 7 is unsubstituted alkyl. At least one OfR 1 or R 7
  • R 7 is methyl. At least one OfR 1 or R 7 is ethyl. At least one OfR 1 or R 7 is propyl. Both R 1 and R 7 are unsubstituted alkyl. Both R 1 and R 7 are methyl. Both R 1 and R 7 are ethyl. R 1 and R 7 are the same. R 1 and R 7 are different. R 7 is hydrogen. R 7 is hydroxy. One of R 1 or R 7 is unsubstituted alkyl and the remaining R 1 or R 7 is hydrogen. One OfR 1 or R 7 is unsubstituted alkyl and the remaining R 1 or R 7 is hydroxy. At least one OfR 1 or R 7 is unsubstituted alkyl and R 5 is unsubstituted or substituted alkyl. At least one of R 1 or R 7 is methyl and R 5 is methyl.
  • Both R 1 and R 5 are unsubstituted alkyl and R 7 is hydroxy. Both R 7 and R 5 are unsubstituted alkyl and Rl is hydroxy. R 1 or R 7 is unsubstituted alkyl and R 5 is unsubstituted or substituted alkyl further wherein R 5 is in the S-conf ⁇ guration. R 1 or R 7 is unsubstituted alkyl and R 5 is unsubstituted or substituted alkyl, further wherein R 5 is in the R-conf ⁇ guration.
  • R 1 is hydroxy and R 7 is methyl.
  • R 1 is methyl and R 7 is hydroxy.
  • R 6 is unsubstituted alkyl.
  • R 6 is methyl.
  • R 6 is ethyl.
  • R 2 , and R 6 are each hydrogen.
  • R 2 and R 6 are hydrogen and R 5 is unsubstituted alkyl.
  • R 2 and R 6 are hydrogen, R 5 is unsubstituted alkyl, and at least one OfR 1 or R 7 is unsubstituted alkyl.
  • the compound is selected from Compounds Ia6, Ib6, Ic6, Ig6, Ih6, Ii6, Io6, Ip6, Iq6, Ia7, Ib7, Ic7, Ig7, Ih7, Ii7, 117, Im7, In7, Io7, Ip7, Iq7, Ia8, Ib8, Ic8, Id8, Ie8, If8, Ig8, IhS, IiS, 118, ImS, InS, IoS, IpS, IqS, Ia9, Ib9, Ic9, 1(19, Ie9, If9, Ig9, Ih9, 119, 119, Im9, Iii9, Ia10, Ib10, Ic10, Id10, Ie10, If10, Ig10, Ih10, Ii10, IUO, Im10, In10, IaIl, IbIl, IcIl, IdIl, IeIl, mi, IgIl,
  • R 2 , R 4 , and R 6 are each hydrogen, R 3 is hydroxyl, and one OfR 1 and R 7 is hydrogen or hydroxyl, then the other R 1 or R 7 is not methyl.
  • R 2 is ⁇ -OH; R 3 is hydroxyl; R 4 and R 6 are each hydrogen; and one Of R 1 and R 7 is hydrogen or hydroxyl, then the other R 1 or R 7 is not methyl.
  • the present invention does not include the following compounds: 3 ⁇ ,7 ⁇ - dihydroxy-7 ⁇ -methyl-5 ⁇ -cholanoic acid, 3 ⁇ ,7 ⁇ -dihydroxy-7 ⁇ -methyl-5 ⁇ -cholanoic acid, 3 ⁇ - hydroxy-7 ⁇ -methyl-5 ⁇ -cholanoic acid, 3 ⁇ ,7 ⁇ ,12 ⁇ -trihydroxy-7 ⁇ -methyl-5 ⁇ -cholan-24-oic acid; 3 ⁇ ,7 ⁇ ,12 ⁇ -trihydroxy-7 ⁇ -methyl-5 ⁇ -cholan-24-oic acid; and 3 ⁇ ,12 ⁇ -dihydroxy-7 ⁇ - methyl-5 ⁇ -cholan-24-oic acid.
  • R 3 when R 3 is hydroxyl and one OfR 1 and R 7 is methyl and the other R 1 and R 7 is hydrogen or hydroxyl, then R 2 , R 4 and R 6 are not all hydrogen.
  • R 2 when R 2 is ⁇ -OH, R 3 is hydroxyl, and one OfR 1 and R 7 is methyl and the other R 1 and R 7 is hydrogen or hydroxyl, then R 4 and R 6 are not hydrogen.
  • composition or medicament comprising a compound of formula IA:
  • R 1 is hydrogen, hydroxy, substituted or unsubstituted alkyl or halogen
  • R 2 is hydrogen or ⁇ -hydroxy
  • R 3 is hydroxy, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H
  • R 4 is hydrogen, substituted or unsubstituted alkyl, or halogen
  • R 5 is unsubstituted or substituted alkyl, or aryl
  • R 6 is hydrogen, unsubstituted or substituted alkyl, or R 5 and R 6 taken together with the carbons to which they are attached form a ring of size 3, 4, 5, or 6 atoms
  • R 7 is hydrogen, substituted or unsubstituted alkyl, or hydroxy
  • m is an integer O, 1, 2, 3, 4, or 5
  • n is an integer 0, 1, 2, 3, 4, or 5.
  • R 5 when R 5 is methyl, R 1 is hydroxyl, and R 3 is hydroxyl or NHCH 2 CH 2 SO 3 H, then R 4 is not hydrogen.
  • Another aspect of the present invention includes a compound of Formula II:
  • R 1 is hydrogen, hydroxy, substituted or unsubstituted alkyl, or halogen
  • R 2 is hydrogen or ⁇ -hydroxy
  • R 4 is hydrogen, substituted or unsubstituted alkyl, or halogen
  • R 5 is unsubstituted or substituted alkyl, or aryl
  • R 6 is hydrogen, unsubstituted or substituted alkyl, or R 5 and R 6 taken together with the carbons to which they are attached form a ring of size 3, 4, 5, or 6 atoms
  • R 7 is hydrogen, substituted or unsubstituted alkyl, or hydroxy
  • R 8 is hydrogen, substituted or unsubstituted alkyl.
  • R 4 when R 5 is methyl and R 1 is hydroxyl, then R 4 is not hydrogen.
  • R 1 is hydrogen or hydroxy.
  • R 1 is hydroxy.
  • R 1 is hydrogen.
  • R 1 is ⁇ - hydroxy.
  • R 2 is ⁇ -hydroxy.
  • R 1 is hydroxy and R 2 is ⁇ -hydroxy.
  • R 1 is hydroxy and R 2 is H.
  • At least one OfR 1 or R 2 is hydroxy.
  • At least one OfR 1 or R 2 is hydrogen.
  • R 1 and R 2 are the same.
  • R 1 is hydroxyl and R 2 is ⁇ -hydroxy.
  • R 1 and R 2 are each hydrogen.
  • R 4 is hydrogen or unsubstituted alkyl.
  • R 4 is hydrogen.
  • R 4 is unsubstituted alkyl.
  • R 4 is unsubstituted alkyl.
  • R 4 is methyl or ethyl.
  • R 4 is methyl.
  • R 4 is ethyl.
  • R 5 is unsubstituted or substituted alkyl.
  • R 5 is in the S-conf ⁇ guration.
  • R 5 is in the R-configuration.
  • R 5 is methyl or ethyl.
  • R 5 is S-methyl.
  • R 5 is R-methyl.
  • R 5 is S- ethyl.
  • R 5 is R-ethyl.
  • R 5 is substituted with phenyl.
  • R 5 is benzyl.
  • R 5 is S-benzyl.
  • R 5 is R- benzyl.
  • R 5 is aryl.
  • R 5 is phenyl.
  • R 4 and R 5 are each unsubstituted alkyl.
  • R 4 and R 5 are each unsubstituted alkyl, further wherein R 5 is in the S-conf ⁇ guration.
  • R 4 and R 5 are each unsubstituted alkyl and R 1 is hydroxy.
  • R 4 and R 5 are each unsubstituted alkyl and R 2 is hydrogen.
  • R 4 and R 5 are each unsubstituted alkyl, R 1 is hydroxy and R 2 is hydrogen.
  • R 1 , R 2 , and R 4 are hydrogen.
  • R 2 and R 4 are hydrogen.
  • R 2 is hydrogen.
  • At least one OfR 1 , R 2 , or R 4 is hydrogen.
  • At least two Of R 1 , R 2 , or R 4 is hydrogen.
  • All of R 1 , R 2 , or R 4 is hydrogen.
  • R 1 or R 7 is unsubstituted alkyl.
  • R 1 or R 7 is methyl.
  • R 1 or R 7 is ethyl.
  • R 1 or R 7 is propyl. Both R 1 and R 7 are unsubstituted alkyl.
  • R 7 is hydrogen.
  • R 7 is hydroxy.
  • One OfR 1 or R 7 is unsubstituted alkyl and the remaining R 1 or R 7 is hydrogen.
  • R 7 is unsubstituted alkyl and the remaining R 1 or R 7 is hydroxy. At least one OfR 1 or R 7 is unsubstituted alkyl and R 5 is unsubstituted or substituted alkyl. At least one OfR 1 or R 7 is methyl and R 5 is methyl. R 7 is hydroxy and both R 1 and R 5 are unsubstituted alkyl. Rl is hydroxy and both R 7 and R 5 are unsubstituted alkyl. At least one of R 1 or R 7 is unsubstituted alkyl and R 5 is unsubstituted or substituted alkyl, further wherein R 5 is in the S-conf ⁇ guration.
  • At least one OfR 1 or R 7 is unsubstituted alkyl and R 5 is unsubstituted or substituted alkyl, further wherein R 5 is in the R-configuration.
  • R 7 is hydroxy and both R 1 and R 5 are unsubstituted alkyl, further wherein R 5 is in the S-conf ⁇ guration.
  • R 7 is hydroxy and both R 1 and R 5 are unsubstituted alkyl, further wherein R 5 is in the R-configuration.
  • R 1 is hydroxy and both R 7 and R 5 are unsubstituted alkyl, further wherein R 5 is in the S-conf ⁇ guration.
  • R 1 is hydroxy and both R 7 and R 5 are unsubstituted alkyl, further wherein R 5 is in the R- configuration.
  • R 1 is hydroxy and R 7 is methyl.
  • R 1 is methyl and R 7 is hydroxy.
  • R 6 is unsubstituted alkyl.
  • R 6 is methyl.
  • R 6 is ethyl.
  • R 8 is hydrogen.
  • R 8 is unsubstituted alkyl.
  • R 8 is methyl.
  • R 8 is ethyl.
  • R 2 is ⁇ -hydroxy and R 8 is unsubstituted alkyl.
  • the compound is selected from Compounds Ial2, Ibl2, Icl2, Igl2, Ihl2, Iil2, Iol2, Ipl2, Iql2, Ial3, Ibl3, Icl3, Igl3, Ihl3, Iil3, 1113, Iml3, Inl3, Iol3, Ipl3, Iql3, Ial4, Ibl4, Icl4, 1(114, Iel4, Ifl4, Igl4, Ihl4, Iil4, 1114, Iml4, Inl4, Iol4, Ipl4, Iql4, Ial5, Ibl5, Icl5, Id 15, Iel5, Ifl5, Igl5, Ihl5, Iil5, 1115, Iml5, Ial6, Ibl6, Icl6, Id 16, Iel6, Ifl6, Igl6, Ihl6, Iil6, 1116, Iml6, Inl6, Ial7, Ibl7, Icl7, Idl7, Iel7, Ifl7,
  • composition or medicament comprising a compound of formula II: 4 (II), or a salt, solvate, hydrate, or prodrug thereof, and at least one pharmaceutically acceptable excipient
  • R 1 is hydrogen, hydroxy, substituted or unsubstituted alkyl or halogen
  • R 2 is hydrogen or ⁇ -hydroxy
  • R 4 is hydrogen, substituted or unsubstituted alkyl, or halogen
  • R 5 is unsubstituted or substituted alkyl, or aryl
  • R 6 is hydrogen, unsubstituted or substituted alkyl, or R 5 and R 6 taken together with the carbons to which they are attached form a ring of size 3, 4, 5, or 6 atoms
  • R 7 is hydrogen, substituted or unsubstituted alkyl, or hydroxy
  • R 8 is hydrogen or substituted or unsubstituted alkyl.
  • R 5 is methyl
  • R 1 is hydroxyl
  • R 3 is hydroxyl or NH
  • Another aspect of the invention includes a compound according to formula III: R 8
  • R 1 is hydrogen, hydroxy, or halogen
  • R 2 is hydrogen or ⁇ -hydroxy
  • R 3 is hydroxy, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H
  • R 5 is unsubstituted or substituted alkyl, or aryl
  • R 6 is hydrogen or R 5 and R 6 taken together with the carbons to which they are attached form a ring of size 3, 4, 5, or 6 atoms
  • R 7 is hydrogen, unsubstituted or substituted alkyl or hydroxy
  • R 8 is hydrogen, unsubstituted or substituted alkyl
  • R9 is hydrogen, unsubstituted or substituted alkyl or R 8 and R 9 taken together with the carbon to which they are attached form a carbonyl
  • R 10 is R 3 or SO 3 H
  • m is an integer 0, 1, 2, 3, 4, or 5
  • n is an integer 0, 1, 2, 3, 4, or 5.
  • Another aspect of the invention includes a compound according to formula HIA: (IIIA) or a salt, solvate, hydrate, or prodrug thereof, wherein R 1 is hydrogen, hydroxy, or halogen; R 3 is hydroxy, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H; R 5 is unsubstituted or substituted alkyl, or aryl; R 6 is hydrogen or R 5 and R 6 taken together with the carbons to which they are attached form a ring of size 3, 4, 5, or 6 atoms; R 7 is hydrogen, unsubstituted or substituted alkyl or hydroxy; R 8 is hydrogen, unsubstituted or substituted alkyl; R 9 is hydrogen, unsubstituted or substituted alkyl or R 8 and R 9 taken together with the carbon to which they are attached form a carbonyl; R 10 is R 3 or SO 3 H; m is an integer 0, 1, 2, 3, 4, or 5; and n is an integer 0, 1,
  • One aspect of the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 1 is hydroxyl.
  • Another aspect of the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 8 and R 9 taken together with the carbon to which they are attached form a carbonyl and R 10 is R 3 .
  • R 3 is selected from hydroxy, NH(CH 2 ) 2 SO 3 H, and NHCH 2 CO 2 H.
  • R 3 is hydroxy.
  • R 3 is NH(CH 2 ) 2 SO 3 H.
  • R 3 is NHCH 2 CO 2 H.
  • One aspect of the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 6 is hydrogen.
  • One aspect of the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 5 is unsubstituted alkyl. In one aspect, R 5 is methyl.
  • One aspect of the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 5 is in the S-conf ⁇ guration.
  • One aspect of the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 5 is in the S-configuration and R 5 is methyl.
  • One aspect of the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 7 is hydrogen.
  • One aspect of the invention includes a compound selected from Compounds Ig3e, Ih3e, Ii3e, Ig4e, Ih4e, Ii4e, Ig5e, Ih5e, and Ii5e
  • Another aspect of the invention includes a compound according to formula HIB: (HIB) or a salt, solvate, hydrate, or prodrug thereof, wherein R 3 is hydroxy, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H; R 5 is unsubstituted or substituted alkyl, or aryl; R 8 is hydrogen, unsubstituted or substituted alkyl; R9 is hydrogen, unsubstituted or substituted alkyl or R 8 and R 9 taken together with the carbon to which they are attached form a carbonyl; R 10 is R 3 or SO 3 H; m is an integer 0, 1, 2, 3, 4, or 5; and n is an integer 0, 1, 2, 3, 4, or 5.
  • One aspect of the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 5 is unsubstituted alkyl. In one aspect, R 5 is methyl.
  • One aspect of the invention includes a compound or salt, solvate, hydrate, or prodrug thereof, wherein R 5 is in the S-configuration.
  • One aspect of the invention includes a compound or salt, solvate, hydrate, or prodrug thereof, wherein R 5 is in the S-configuration and R 5 is methyl.
  • One aspect of the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 8 and R9 taken together with the carbon to which they are attached form a carbonyl.
  • R 10 is R 3 .
  • R 3 is selected from hydroxy, NH(CH 2 ) 2 SO 3 H, and NHCH 2 CO 2 H. In one aspect, R 3 is hydroxy. In one aspect, R 3 is NH(CH 2 ) 2 SO 3 H. In one aspect, R 3 is NHCH 2 CO 2 H.
  • Another aspect of the invention includes a compound according to formula HIC:
  • R 3 is hydroxy, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H;
  • R 5 is unsubstituted or substituted alkyl, or aryl;
  • m is an integer 0, 1, 2, 3, 4, or 5;
  • n is an integer 0, 1, 2, 3, 4, or 5.
  • R 3 is selected from hydroxy, NH(CH 2 ) 2 SO 3 H, and NHCH 2 CO 2 H. In one aspect, R 3 is hydroxy.
  • R 3 is NH(CH 2 ) 2 SO 3 H. In one aspect, R 3 is NHCH 2 CO 2 H.
  • One aspect of the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 5 is unsubstituted alkyl. In one aspect, R 5 is methyl.
  • One aspect of the invention includes a compound or salt, solvate, hydrate, or prodrug thereof, wherein R 5 is in the S-configuration.
  • One aspect of the invention includes a compound or salt, solvate, hydrate, or prodrug thereof, wherein R 5 is in the S-configuration and R 5 is methyl.
  • Another aspect of the invention includes a compound according to formula IV:
  • R 1 is hydrogen, hydroxy, or halogen
  • R 2 is hydrogen or ⁇ -hydroxy
  • R 3 is hydroxy, NH(CH 2 ) m SO 3 H, or NH(CH 2 ) n CO 2 H
  • R 5 is unsubstituted or substituted alkyl, or aryl
  • R 6 is hydrogen or R 5 and R 6 taken together with the carbons to which they are attached form a ring of size 3, 4, 5, or 6 atoms
  • m is an integer 0, 1, 2, 3, 4, or 5
  • n is an integer 0, 1, 2, 3, 4, or 5.
  • the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 1 is hydroxy. In another aspect, the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 1 is alpha hydroxy. In one aspect, the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 1 is beta hydroxy. In one aspect, the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 1 is methyl.
  • the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 5 is unsubstituted alkyl. In one aspect, the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 5 is methyl. In one aspect, the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 5 is in the R-conf ⁇ guration. In one aspect, the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 5 is in the S-configuration.
  • the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 6 is hydrogen. In one aspect, the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 2 is hydrogen. In one aspect, the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 2 is alpha hydroxy. In one aspect, the invention includes a compound or a salt, solvate, hydrate, or prodrug thereof, wherein R 3 is hydroxyl.
  • One aspect of the invention includes a compound selected from Compounds Ib3e, Ic3e, Id3e, Ie3e, If3e,Ig3e, Ih3e, Ii3e, I13e, Im3e, In3e, Ia4e, Ib4e, Ic4e, Id4e, Ie4e, If4e, Ig4e, Ih4e, Ii4e, II4e, Im4e, In4e, Ia5e, Ib5e, Ic5e, Id5e, Ie5e, If5e, Ig5e, Ih5e, Ii5e, I15e, Im5e, In5e, Ia9e, Ib9e, Ic9e, Id9e, Ie9e, If9e, Ig9e, Ih9e, Ii9e, I19e, Im9e, In9e, Ia10e, Ib10e, Ic10e, Id10e, I
  • the invention includes Compound Ih3e:
  • the invention includes the taurine conjugate of Compound Ih3e:
  • One aspect of the invention includes Compounds Ib3e, Ic3e, Id3e, Ie3e, If3e,Ig3e, Ih3e, Ii3e, II3e.
  • One aspect of the invention includes Compounds Ia9e, Ib9e, Ic9e, Id9e, Ie9e, If9e, Ig9e, Ih9e, Ii9e, I19e, Im9e, In9e, Ia10e, Ib10e, Ic10e, Id10e, Ie10e, If10e. Ig10e, Ih10e, Ii10e, IUOe, Im10e, In10e, IaI Ie. IbI Ie, Ic11e, IdI Ie. IeI Ie. IfI Ie. Ig11e, Ihlle, Iille, 111 Ie, ImI Ie, and InI Ie.
  • One aspect of the invention includes Compounds IaI5e, IbI5e, IcI5e, IdI5e, IeI5e,
  • the invention includes a compound of the invention, wherein the compound is a pharmaceutically acceptable salt.
  • One aspect of the invention includes a composition comprising a compound of the invention or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, and at least one pharmaceutically acceptable excipient.
  • the present invention also includes radiolabeled compounds of the invention
  • Radiolabeled compounds can be prepared using conventional techniques.
  • radiolabeled compounds of the invention can be prepared by reacting the compound of the invention with tritium gas in the presence of an appropriate catalyst to produce radiolabeled compounds having the formulae described herein.
  • the compounds of the invention are tritiated.
  • the invention includes the use of a compound or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, in the manufacture of a medicament for a treating or preventing disease in a subject.
  • the invention also includes a method of treating or preventing disease in a subject by administering a compound of the invention or a pharmaceutically acceptable salt, hydrate, or prodrug thereof.
  • One aspect of the invention includes the use or method, wherein the disease is selected from metabolic disease, inflammatory disease, liver disease, autoimmune disease, cardiac disease, kidney disease, cancer, and gastrointestinal disease.
  • the invention includes a metabolic disease selected from obesity, diabetes, diabesity, metabolic syndrome, insulin resistance, including pre-diabetic insulin resistance, hypertension, and dyslipidemia.
  • the metabolic disease is obesity.
  • the metabolic disease is diabetes.
  • diabetes is selected from pre-diabetes and type II diabetes.
  • the metabolic disease is metabolic syndrome.
  • the metabolic disease is insulin resistance.
  • the metabolic disease is dyslipidemia.
  • the metabolic disease is diabesity. The term "diabesity" refers to a condition wherein the subject has both diabetes and excessive weight.
  • the invention includes an inflammatory disease selected from allergy, osteoarthritis (OA), chronic obstructive pulmonary disease (COPD), appendicitis, bronchial asthma, pancreatitis, allergic rash, and psoriasis.
  • OA osteoarthritis
  • COPD chronic obstructive pulmonary disease
  • the invention includes an autoimmune disease selected from rheumatoid arthritis, multiple sclerosis, and type I diabetes.
  • the invention includes a gastrointestinal disease selected from inflammatory bowel disease (Crohn's disease, ulcerative colitis), short bowel syndrome (post-radiation colitis), microscopic colitis, irritable bowel syndrome (malabsorption), and bacterial overgrowth.
  • the invention includes kidney disease selected from diabetic nephropathy, chronic renal failure, glomerular nephritis, hypertensive nephrosclerosis, chronic glomerulonephritis, chronic transplant glomerulopathy, chronic interstitial nephritis, and polysystic kidney disease.
  • the invention includes cancer selected from colorectal cancer, liver cancer, heptacellular carcinoma, cholangio carcinoma, renal cancer, gastric cancer, pancreatic cancer, prostate cancer, and insulanoma.
  • the invention includes liver disease selected from nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, chronic viral hepatitis, alcoholic liver disease, drug induced hepatitis, hemochromatosis, primary biliary cirrhosis, primary sclerosing cholangitis, portal hypertension, bile desaturation, Gaucher's disease, Wilson's disease, ⁇ l- antitrypsin deficiency, total parenteral nutrition (TPN), cholelithiasis, TPN-associated cholestasis and sepsis.
  • liver disease selected from nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, chronic viral hepatitis, alcoholic liver disease, drug induced hepatitis, hemochromatosis, primary biliary cirrhosis, primary sclerosing cholangitis, portal hypertension, bile desaturation, Gaucher's disease, Wilson's disease, ⁇ l- antitrypsin defic
  • the invention includes the autoimmune disease erythematosus.
  • the invention includes cardiac disease selected from congestive heart failure, myocardial infarction, atherosclerosis, angina pectoris, arteriosclerosis and cerebrovascular disease (hemorrhage, stroke, cerebrovascular infarction).
  • the invention includes a use or method, wherein the compound of the invention is a TGR5 agonist.
  • the selectivity ratio of TGR5 EC 50 to FXR EC 50 is less than 0.05.
  • the invention includes a use or method, wherein the compound or composition is administered to the subject orally, parentally, intravenously, or topically.
  • the subject is human.
  • One aspect of the invention includes a use or method comprising administering to a subject a therapeutically effective amount of the compound of the invention.
  • the invention includes a use or method comprising administering to subject in need thereof.
  • the present invention includes a use or method comprising administering to a subject a prophylatically effective amount of the compound of the invention.
  • the compounds and compositions of the present invention can be administered by various routes, e.g., oral, subcutaneous, intramuscular, intravenous, or intraperitoneal.
  • the referred routes of administering the pharmaceutical compositions are oral, subcutaneous, and intravenous at daily doses of about 0.01-5000 mg, preferably 5-500 mg, of the FXR ligand for a 70 kg adult human per day.
  • the appropriate dose may be administered in a single daily dose or as divided doses presented at appropriate intervals, for example as two, three, four, or more subdoses per day.
  • inert and pharmaceutically acceptable carriers are used.
  • the pharmaceutical carrier can be either solid or liquid.
  • Solid form preparations include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories.
  • a solid carrier can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material.
  • the carrier is generally a finely divided solid that is in a mixture with the finely divided active component, e.g., a compound of the invention.
  • the active ingredient is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • a low- melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into convenient-sized molds and allowed to cool and solidify.
  • Powders and tablets preferably contain between about 5% to about 70% by weight of the active ingredient of the compound of the invention.
  • Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoa butter, and the like.
  • the pharmaceutical compositions can include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the compound of the invention (with or without other carriers) is surrounded by the carrier, such that the carrier is thus in association with the compound.
  • cachets can also be included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.
  • Liquid pharmaceutical compositions include, for example, solutions suitable for oral or parenteral administration, suspensions, and emulsions suitable for oral administration.
  • Sterile water solutions of the active component or sterile solutions of the active component in solvents comprising water, buffered water, saline, PBS, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like.
  • Sterile solutions can be prepared by dissolving the active component (e.g., a compound of the invention) in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • the pH of the preparations typically will be between 3 and 11, more preferably from 5 to 9, and most preferably from 7 and 8.
  • compositions containing compounds of the invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered in an amount sufficient to cure, reverse, or at least partially slow or arrest the symptoms of the disease and its complications.
  • An amount adequate to cure, reverse, or at least partially slow or arrest the symptom of the disease and its complications is defined as a "therapeutically effective dose.”
  • compositions are administered in an amount sufficient to prevent the symptoms of the disease and its complications.
  • An amount adequate to prevent the symptom of the disease and its complications is defined as a "prophylatically effective dose.”
  • Amounts effective for therapeutic use will depend on the severity of the disease or condition and the weight and general state of the patient, but generally range from about 0.1 mg to about 2,000 mg of the compound per day for a 70 kg patient, with dosages of from about 5 mg to about 500 mg of the compound per day for a 70 kg patient being more commonly used.
  • compositions containing compounds of the invention are administered to a patient susceptible to or otherwise at risk of developing disease, in an amount sufficient to delay or prevent the onset of the disease symptoms. Such an amount is defined to be a "prophylactically effective dose.”
  • the precise amounts of the compound again depend on the patient's state of health and weight, but generally range from about 0.1 mg to about 2,000 mg for a 70 kg patient per day, more commonly from about 5 mg to about 500 mg for a 70 kg patient per day.
  • Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical formulations should provide a quantity of a compound of the invention sufficient to effectively treat or prevent disease in the patient.
  • kits for preventing or treating disease according to the use and method of the present invention.
  • the invention includes kit for treating or preventing disease in a subject, wherein the kit comprises a compound of the invention or a salt, solvate, hydrate, or prodrug thereof.
  • the kits typically include a pharmaceutical composition that contains an effective amount of a compound of the invention, as well as informational material containing instructions of how to dispense the pharmaceutical composition, including description of the type of patients who may be treated, the schedule (e.g., dose and frequency) and route of administration, and the like.
  • Methyl chenodeoxycholanoate (1) was protected in 3- and 7-position by treatment with 3,4- dihydro-2H-pyran in dioxane in presence of catalytic amount of /7-toluenesulfonic acid (p- TSA) to give the corresponding 3 ⁇ ,7 ⁇ -tetrahydropyranyloxy analog (2).
  • EXAMPLE 2 Preparation of 23(S)- and 23(R)-methyl-6 ⁇ -methyl-3 ⁇ ,7 ⁇ -dihydroxy-5 ⁇ - cholan-24-oic acid (Ib3, Ic3) The following compounds were prepared by alkylation of 6 ⁇ -methyl-3 ⁇ ,7 ⁇ - dihydroxy-5 ⁇ -cholan-24-oic acid according to the procedure of Example 1.
  • EXAMPLE 4 Preparation of 23(R)- and 23(5)-methyl-6 ⁇ -methyl-3 ⁇ ,7q,12a- trihydroxy-5 ⁇ -cholan-24-oic acid (Ih3, Ii3) The following compounds were prepared by alkylation of 6 ⁇ -methyl-3 ⁇ ,7 ⁇ ,12 ⁇ - trihydroxy-5 ⁇ -cholan-24-oic acid according to the procedure of Example 1.
  • EXAMPLE 5 Preparation of 23(R)- and 23(5)-methyl-3 ⁇ -hydroxy-5 ⁇ -cholan-24-oic acid (Ip, Iq) The following compounds were prepared by alkylation of 3 ⁇ -hydroxy-5 ⁇ -cholan-24- oic acid according to the procedure of Example 1.
  • Reagents and conditions a) pTSA, MeOH, ultrasound, quant, b) CH 2 (OCH 3 ) 2 , P 2 O 5 , CHCl 3 , 97%. c) LDA, MeI, -78°C. d) MeOH, HCl, 45°C. e) MeOH, NaOH, 45°C, 41%. Overall yield: 39.7%.
  • the esters 2 were successively dissolved in EtOH (15 mL) and treated with a solution of 10 N NaOH (10 mL) at reflux for 4 h, cooled, poured onto cold H 2 O (50 mL), acidified with 2 N HCl, and extracted with EtOAc (3 x 15 mL). The organic phase was washed with brine (10 mL), dried (Na 2 SO 4 ), and concentrated under vacuum. The residue was chromatographated on silica gel.
  • Table 1 shows that compounds of the invention are potent and selective TGR5 modulators.
  • the introduction of an alkyl group at the C-23 position of bile acid gives selectivity for the TGR5 receptor with respect to FXR. This is evident by the observation of the biological results obtained for CDCA, 6-MeCDCA and 6,23-diMe-CDCA (23-R,S isomers mixture) on FXR and TGR5 as shown in Table 1.
  • 6,23-diMe-CDCA is 100-fold more potent on TGR5 with respect to the FXR receptor
  • FRET fluorescence resonance energy transfer
  • Tables 2 and 3 show additional compounds evaluated for TGR5 activity. Luciferase activity was determined in CHO cells stably expressing hTGR5 or transiently cotransfected with a hTGR5 expression vector and a cAMP-responsive element (CRE)-driven luciferase reporter gene. Some of the compounds were further submitted to a luciferase reporter assay to score for their capacity to activate the nuclear bile acid receptor FXR. Table 2.
  • CRE cAMP-responsive element
  • *Data represent average values of at least three independent experiments of CRE-driven luciferase reporter assays in TGR5-transfected CHO cells. Units are ⁇ M for EC 50 and % of 10 ⁇ M LCA value for efficacy.
  • a Data represents average values of at least three independent experiments. Value for efficacy are expressed as % of activity vs. 10 ⁇ M LCA (TGR5) or 10 ⁇ M 6ECDCA (FXR). b Plateau activation level not reached; the maximum concentration tested was 125 ⁇ M for Ib and 10O mM for Ii.
  • Tables 2 and 3 can be determined using methods known in the art, for example, as described below.
  • the N1H Mammalian Gene Collection clone MGC:40597 (also named pCMVSPORT6/hTGR5 or pTGR5) and pcDNA3.1(+) were obtained from Invitrogen (Carlsbad, CA).
  • pCRE-Luc and pCMV ⁇ were obtained from Clontech (Palo Alto, CA).
  • pCMX-hFXR and pCMX-mRXR ⁇ were kind gifts from Dr. David J. Mangelsdorf (Howard Hughes Medical Institute, University of Texas Southwestern Medical Center).
  • pEcREx7-Luc was a kind gift from Dr. Richard A. Heyman (X-ceptor Therapeutics, CA).
  • CHO cells Chinese hamster ovary (CHO) cells, NCI-H716 cells, Hep3B cells and COSl cells were obtained from American Type Culture Collection (Manassas, VA). Cell culture medium, serum and supplements were from Invitrogen or Sigma-Aldrich. All CHO cells were maintained in minimum essential medium ⁇ ( ⁇ -MEM) supplemented with 10%(v/v) fetal bovine serum (FBS) and lOO ⁇ M nonessential amino acids (NEAA). NCI-H716 cells were maintained in suspension in RPMI- 1640 supplemented with 10%(v/v) FBS, 1OmM HEPES and ImM sodium pyruvate. Hep3B cells were maintained in Eagle's medium supplemented with 10%(v/v) FBS and lOO ⁇ M NEAA. COSl cells were maintained in
  • DMEM Dulbecco's modified Eagle's medium
  • FBS 10%(v/v) FBS
  • All cell culture medium was supplemented with 100units/ml penicillin and lOO ⁇ g/ml streptomycin sulfate.
  • Cells were grown at 37 °C in an atmosphere of 5% C02, passed every 2-6 days and freshly plated for each experiment.
  • CHO cells were plated in 96-well plates at a density of 3.5xlO4cells/well, cultured for 24h, and then transfected with 150ng of human (h) TGR5 expression plasmid (pCMVSPORT6/hTGRS) and lOOng of cAMP-responsive element (CRE)-driven luciferase reporter plasmid (pCRE-Luc) in each well using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. After 6h incubation, cells were washed once with phosphate-buffered saline (PBS) and medium was exchanged for DMEM containing 0. l%(w/v) bovine serum albumin (BSA).
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • cells were treated for 5h with different concentrations of each compound in fresh DMEM containing 0.1%(w/v) BSA. After treatment, the cells were lysed with 50 ⁇ l of lysis buffer (25mM Tris-Cl (pH7.6), 2mM EDTA, ImM dithiothreitol (DTT), 10%(v/v) glycerol and l%(v/v) triton X-10O) by a freeze- thaw cycle and subjected to luciferase assays as described below.
  • lysis buffer 25mM Tris-Cl (pH7.6), 2mM EDTA, ImM dithiothreitol (DTT), 10%(v/v) glycerol and l%(v/v) triton X-10O
  • COSl cells were plated in 96-well plates at a density of 2.5 xlO4cells/well in DMEM supplemented with 10%(v/v) charcoal-stripped FBS, cultured for 24h, and then transfected with 25ng of hFXR expression plasmid (pCMX-hFXR), 25ng of mouse (m) retinoid X receptor ⁇ (RXR ⁇ ) expression plasmid (pCMX-mRXRa), 50ng of reporter plasmid (pEcREx7-Luc) and 50ng of pCMV ⁇ as internal control in each well, using the Lipofectamine 2000 reagent.
  • luciferase assays 20 ⁇ l of cell lysate was mixed with lOO ⁇ l of luciferase reaction buffer [235 ⁇ M luciferine, 265 ⁇ M ATP and 135 ⁇ M coenzyme A (CoA)] and luminescence was determined with CentroXS3 LB960 (Berthold Technologies, Bad Wildbad, Germany).
  • lO ⁇ l of cell lysate was mixed with lOO ⁇ l of Buffer Z [6OmM Na2HPO4, 1OmM KCl, ImM MgSO4, 5OmM ⁇ -mercaptoethanol and 0.75mg/ml o- nitrophenyl- ⁇ -D-galactopyranoside (ONPG)] and incubated at 37 °C for 0.5-3h. Reactions were stopped by adding 50 ⁇ l of Stop buffer (IM Na2CO3) and the optical density at 420nm was determined.
  • Buffer Z 6OmM Na2HPO4, 1OmM KCl, ImM MgSO4, 5OmM ⁇ -mercaptoethanol and 0.75mg/ml o- nitrophenyl- ⁇ -D-galactopyranoside (ONPG)
  • CHO cells were transfected with 3.8 ⁇ g of hTGR5 expression plasmid (pCMVSPORT6/hTGR5), 3.8 ⁇ g of CRE-driven luciferase reporter plasmid (pCRE-Luc) and 0.4 ⁇ g of neomycin-resistant gene expression plasmid [pcDNA3.1(+)] using Lipofectamine 2000.
  • the transfectants were selected with 400 ⁇ g/ml G418 sulfate and single clones were grown in 96-well plate, independently.
  • TGR5-expressing CHO cell lines were screened by LCA treatments, followed by luciferase assays.
  • NCI-H716 cells were plated in 96-well plates coated with 0.75mg/ml Matrigel (BD Biosciences) according to manufacturer's instructions just prior to use, at a density of 6xlO4cells/well in DMEM supplemented with 10%(v/v) FBS, lOOunits/ml penicillin and lOO ⁇ g/ml streptomycin sulfate, and cultured for 24h, which allowed cell adhesion to the bottom of the plate.
  • Matrigel BD Biosciences
  • CHO-TGR5 cells were plated in 96-well plates at a density of 3.5 x 104cells/well in ⁇ -MEM supplemented with 10%(v/v) FBS, lOO ⁇ M NEAA, 100 units/ml penicillin and lOO ⁇ g of streptomycin sulfate, and cultured for 24h.
  • the cells were washed twice with PBS and medium was exchanged for cAMP assay medium [DMEM containing 0.1%(w/v) BSA and 0.5mM 3-isobutyl-1-methylxanthine (IBMX)]. After incubation for 30 minutes at 37 °C, the cells were treated with each compound in fresh cAMP assay medium for 30 minutes. After treatment, medium was discarded and cAMP amounts were determined using cAMP-Screen kit (Applied Biosystems, Foster City, CA) according to manufacturer's instructions. 50% effective concentrations (EC50) and efficacy determination
  • EC50 values were determined by probit analysis. Efficacy was determined by calculating percentages of 10 ⁇ M LCA value for TGR5 agonist study and 10 ⁇ M 6 ⁇ -Et-CDCA value for FXR agonist study, respectively. After subtracting the average value of the basal (vehicle-treated) condition, values were applied to EC50 and/or efficacy determinations. Calculation of average EC50 and comparison of the EC50 between different compounds were performed after logarithm transformation.
  • the receptor binding assay was performed by measuring the level of cyclic AMP (cAMP) using FRET assay.
  • Human intestinal cell lines (NCI-H716) were plated in 96-well plates coated with 0.75 mg/ml Matrigel (BD Biosciences) according to manufacturer's instructions just prior to use, at a density of 12xl0 3 cells/well in DMEM supplemented with 10 % (v/v) FBS, 100units/ml penicillin and 100 ⁇ g/ml streptomycin sulfate, and cultured for 24 h, which allowed cell adhesion to the bottom of the plate.
  • cAMP cyclic AMP
  • the cells were washed twice with PBS and medium was exchanged for cAMP assay medium [OPTIMEM containing 0.1% (w/v) BSA and 1 mM 3-isobutyl-1-methylxanthine (IBMX)]. After incubation for 60 minutes at 37 °C, the cells were treated with increasing concentrations of compound Ih3 in stimulation buffer (5 mM HEPES, o,l% BSA in HBSS pH 7.4) containing the europium chelate - Streptavidin and the ALEXA Fluor 647-conjugated antibody anti-cAMP (PerkinElmer) for 1 hour at room temperature. The level of intracellular cAMP was determined with Lance kit (PerkinElmer). Litocholic acid was used as control ligand. Z' factor was used to validate assays. Non linear regression curves, without constraints, were performed by using four parameter equation and GraphPad Prism Software (GraphPad Inc.), to obtain the EC50 values.
  • Alphascreen assay Activity on FXR was assayed by using Alphascreen technology in arigiment coactivator assay.
  • AlphaScreen is a bead-based chemistry assay used to study biomolecular interactions. Binding of molecules captured on the beads leads to an energy transfer from one bead to the other, ultimately producing a luminescent signal. When the partners interact, chemical energy is transferred from Donor to Acceptor beads and a signal is produced.
  • the GST-FXR-LBD interacts with the Src-1 peptide.
  • Acceptor beads were used to capture the GST-fusion FXR-LBD whereas the biotinylated- SRC-I peptide was captured by the streptavidin Donor beads.
  • chemical energy is transferred from Donor to Acceptor beads across the complex streptavidin-Donor/Src-1-Biotin/GSTFXR-LBD/Anti-GST-Acceptor and a signal is produced.
  • the assay was performed in white, low- volume, 384-well Optiplates
  • HEPG2 and HEK293T cells were cultured in E-MEM and DMEM respectively, either supplemented with 1% penicillin/streptomycin, 1% L-glutamine and 10% fetal bovine serum. (high glucose) (Invitrogen, Carlsbad, CA). Cells were grown at 37°C in 5% CO2. All the transfections were made using 5:2 Fugene HD Trasfection reagent ( ⁇ l) to DNA ( ⁇ g) respectively (Roche). Twenty-four hours before transfection HEK293T or HepG2 cells were seeded onto a 96-well plate at a density of 10.000 or 15.000 cells/well, respectively.
  • Transient transfections were performed using 100 ng of reporter vector pGL4.29[luc2P/CRE/Hygro] (Promega), 40ng of pGL4.74 (Renilla), as internal control for transfection efficiency, and 10 ng of expression plasmid pCMV-SPORT6-hTGR5
  • the pGEM vector was added to normalize the amounts of DNA transfected in each assay (2 ⁇ g). Twenty-four hours post- transfection the cells were stimulated with increasing concentrations of compound Ih3e for 18 h. Control cultures received vehicle (0.1 % DMSO) alone.
  • the cells were then lysed by adding 75 ⁇ l of Dual-Glo Luciferase Reagent (Promega) to 75 ⁇ l of medium containing cells/well. Renilla luciferase activity was measured by adding a volume of Dual-Glo Stop & GIo reagent and original culture medium. Luciferase activities were expressed as ratio between luciferase unit and renilla luciferase unit. Each data point is the average of triplicate assays. Each experiment was repeated at least three times.
  • Compounds having an alpha-ethyl group at the C-6 position on the bile acid ring are preferred. More specifically, compounds having an alpha-ethyl group at the C-6 position of the 23-methyl cholic acid are the most preferred. As shown in Table 3A above, compounds having an alpha-ethyl group at the C-6 position are surprisingly and unexpectedly more potent than the corresponding C-6 alpha-methyl derivative.
  • EXAMPLE 9 Metabolic acivities of oleanolic acid and 6-Ethyl, 23-Methyl-cholic acid (Ih3e) in a diet-induced obesity mouse model
  • TGR5 agonists oleanolic acid (OA) or 6 ethyl, 23-methyl cholic acid (Ih3e) correct the development of obesity and associated insulin- resistance in vivo.
  • OA/ Ih3e were administered via food administration for 16 weeks to male C57BL6J mice that had been previously subjected for 10 weeks to a high fat diet.
  • OA was observed as a selective TGR5 agonist that did not cause food aversion. Animals treated with a dose of 100 mg/kg/day of OA showed, however, some signs of toxicity, whereas a lower dose was well tolerated. Therefore, OA was administered at the dose of 50 mg/kg/d in this study.
  • Ih3e As a potent and selective TGR5 ligand. No problems with toxicity were expected with Ih3e, which was administered at ⁇ 50 -fold lower concentration.
  • mice 48 male C57BL6J mice (5 weeks of age) were divided in two groups: one group of 24 (group 1, 2&3) animals received chow diet whereas the other 24 received a high fat diet for a period of 10 weeks (group 4,5&6). The animals were then analyzed during a period of 16 weeks. Five groups of 10 animals were assigned as follows: 1 : chow diet
  • Week-2 Body composition was analyzed, for all groups, by dual energy X-ray absorptiometry (dexascan).
  • Week-1 Serum levels of transaminases, glucose, triglycerides, cholesterol, HDL-C, LDL-C and insulin were measured in all groups after a fasting period of 12 h and mice were then placed on the diets as indicated (Day 0).
  • Week 2 Serum levels of transaminases, glucose, triglycerides, cholesterol, HDL-C, LDL-C and insulin was measured in all groups after a fasting period of 12 h (Day 14).
  • Week 4 Glucose tolerance was determined by subjecting all the animals to an intraperitoneal glucose tolerance test (IPGTT). Animals were fasted for 12 h prior to this test. Nocturnal energy expenditure of groups 1, 4, 5 and 6 (chow diet, high fat diet and high fat diet OA / 6Et23MeCDCA (Ih3e) was measured by indirect calorimetry. Week 8: Body weight composition was again analyzed by dexascan for all groups.
  • IPGTT intraperitoneal glucose tolerance test
  • Serum levels of transaminases, glucose, triglycerides, cholesterol, HDL-C, LDL-C and insulin were measured in all groups after a fasting period of 12 h (Day 56).
  • Week 9 Circadian activity of groups 4, 5 and 6 (high fat diet fed mice) was studied during a period of 30 h.
  • Week 10 Measurement of blood pressure and heart rate was performed on groups 4,
  • Circadian activity measurement was performed on groups 1, 2, 3 and 4.
  • Week 12 Glucose tolerance was analyzed by performing an intraperitoneal glucose tolerance test (IPGTT) on groups 4, 5 and 6. During the IPGTT, blood was also collected to analyze insulin levels. Animals were fasted 12 h prior to these tests. Feces were collected in all groups over a 24 h time period and fecal lipids content was measured.
  • Week 16 Cold test was performed on all animals by measuring body temperature of animals exposed to 4°c.
  • TC plasma lipids
  • TG TG
  • HDL-C HDL-C
  • FFAs liver functions
  • LAT alkaline Pase
  • ⁇ -GT alkaline Pase
  • glucose and insulin lipoprotein profiles of selected groups of plasma (size- exclusion chomatography).
  • standard histology HE staining, succinate dehydrogenase staining, oil-red-0 staining and cell morphology
  • tissue lipid content electron microscopy on BAT and muscle to analyze mitochondria
  • RNA isolation for expression studies of selected genes involved in metabolism and energy homeostasis by quantitative RT-PCR
  • Protein extraction for the study of post-translationnal modifications
  • mice Animals housing and handling Mice were group housed (5 animals / cage) in specific pathogen-free conditions with a 12 h: 12 h (on at 7:00) light-dark cycle, in a temperature (20-22°c) and humidity controlled vivarium, according to the European Community specifications. Animals were allowed free access to water and food.
  • Drinking water Chemical composition of the tap water was regularly analyzed to verify the absence of potential toxic substances at the Institut d'Hydrologie, ULP, France. Drinking water was treated with HCl and HClO 4 to maintain pH between 5 and 5,5 and chlorin concentration between 5 and 6 ppm.
  • mice were fed, either with chow diet (16% protein, 3% fat, 5% fiber, 5% ash) or with high fat diet (20% protein, 20% carbohydrate, 60% fat).
  • Oleanolic acid and 6Et23MeCDCA (Ih3e) were mixed with either powdered chow diet or either powdered high fat diet in the following proportions: 0,5g of OA/kg of food for the 50mg/kg/day treatment and 0,08g of 6Et23MeCA (Ih3e) /kg of food for the 10 mg/kg/day treatment.
  • Pellets were then reconstituted. Control groups received food pellets as provided by the company.
  • animals were anesthesized by inhalation of an isoflurane-O 2 mixture.
  • HDL cholesterol content was determined after precipitation of apo B-containing lipoproteins with phosphotungstic acid/Mg (e.g., Roche Diagnostics, Mannheim, Germany). Free fatty acids level was determined with a kit from Wako (e.g., Neuss, Germany) as specified by the provider. Metabolic and endocrine exploration
  • Blood glucose concentration was measured by a Precision Q.I.D analyzer (e.g., Medisense system), using Medisense Precis electrodes (e.g., Abbot Laboratories, Medisense products,
  • IPGTT Intraperitoneal glucose tolerance test
  • IPGTT Intraperitoneal glucose tolerance test
  • mice which were fasted overnight (12 h). Mice were either injected intraperitoneally (IPGTT) with a solution of 20 % glucose in sterile saline (0.9% NaCl) at a dose of 2g glucose/kg body weight. Blood was collected from the tail vein, for glucose and insulin monitoring, prior and 15, 30, 45, 75, 90, 120, 150, 180 min after administration of the glucose solution. The incremental area of the glucose curve was calculated as a measure of insulin sensitivity, whereas the corresponding insulin levels indicate insulin secretory reserves.
  • Energy expenditure was evaluated through indirect calorimetry by measuring oxygen consumption with the Oxymax apparatus (e.g., Columbus Instruments, Columbus, OH) during 12 h.
  • Oxymax apparatus e.g., Columbus Instruments, Columbus, OH
  • This system consists of an open circuit with air coming in and out of plastic cages (one mouse per cage). Animals were allowed free access to food and water.
  • a very precise CO 2 and O 2 sensor measured the difference in O 2 and CO 2 concentrations in both air volumes, which gave the amount of oxygen consumed in a period of time given that the air flow of air coming in the cage was constant.
  • the data coming out of the apparatus was processed in a connected computer, analyzed, and shown in an exportable Excel file. The values were expressed as ml.kg '.h -1 , which is commonly known as the VO 2 .
  • Bone mineral density (BMD in g/cm 2 ) and body composition were determined by using the PIXIMUS software (version 1 Ax, GE Medical Systems).
  • the Visitech BP-2000 Blood Pressure Analysis System is a computer-automated tail 'cuff system that is used for taking multiple measurements on 4 awake mice simultaneously without operator intervention.
  • the mice were contained in individual dark chambers on a heated platform with their tails threaded through a tail cuff.
  • the system measures blood pressure by determining the cuff pressure at which the blood flow to the tail was eliminated.
  • a photoelectric sensor detects the specimen's pulse.
  • the system generates results that have been shown to correspond closely with mean intra-arterial pressure measured simultaneously in the carotid artery. This allows reproducible values of systolic blood pressure and heart beat rate to be obtained. This required training of the animals for one week in the system.
  • E- Circadian activity Spontaneous locomotor activity was measured using individual boxes, each composed with a sliding floor, a detachable cage, and equipped with infra-red captors allowing measurement of ambulatory locomotor activity and rears. Boxes were linked to a computer using an electronic interface (e.g., Imetronic, Pessac, France). Mice were tested for 32 hours in order to measure habituation to the apparatus as well as nocturnal and diurnal activities. The quantity of water consumed was measured during the test period using an automated lickometer.
  • an electronic interface e.g., Imetronic, Pessac, France.
  • High fat fed mice treated with compound Ih3e showed lower blood glucose, liver enzymes, and plasma lipids than high fat fed mice treated with vehicle.
  • the heart rate of high fed mice treated with compound Ih3e also showed a lower heart rate in comparision with high fat fed mice treated with vehicle.
  • Figure 3 shows that compound Ih3e improves glucose tolerance in high fat fed mice. After 10 weeks, plasma insulin levels were increased in both the chow fed and high fat fed mice treated with compound Ih3e in comparison to mice treated with vehicle as shown in Figure 3 A. After 12 weeks, glucose levels were shown to be lower in high fat fed mice treated with compound Ih3e as shown in Figure 3B.
  • Figure 4 shows oral glucose tolerance test (OGTT) results as glucose levels over a period of 200 min in chow diet fed mice treated with compound Ih3e.
  • Figure 5 shows insulin release in vivo after a test meal.
  • Figure 5A shows insulin release over 30 min.
  • Figure 5B shows fold increase in insulin release compared to basal insulin level. Insulin levels peaked to higher levels at -12 minutes in high fat fed mice treated compound Ih3e in comparison with mice treated with vehicle.
  • Figures 5C and 5D show the fold increase in insulin release compared to basal insulin levels. The fold increase in high fat fed mice treated with compound Ih3e was greater at both 15 and 30 min time points as shown in Figure 5D.
  • Figures 6 (graphs A-D) and 7 (graphs A-C) show that compound Ih3e treated mice have an increase in respiratory exchange ratio (RER) upon HFD (high fat diet) which can be explained as linked to their improved insulin sensitivity which maintains their ability to oxidize glucose.
  • Figure 8 (graphs A and B) show locomotor activity and food/water intake of treated high fat fed and treated chow fed mice in comparison to vehicle treated. Food/water intake for mice fed a high fat diet and treated with compound Ih3e showed a slight increase in intake verses mice treated with vehicle.
  • Figure 9 shows changes in organ weight.
  • Figures 9B and 9C show the percentage of change in body weight, liver, kidney, heart, , peri WAT, epi WAT, Sc WAT, and BAT compared to weight in mice fed a chow diet. In all organs, high fat fed mice treated with compound Ih3e showed a reduced percentage change.
  • Solid BA in protonated form for compound Ih3e
  • the saturated solutions after incubation for 1 week, were filtered on a Millipore filter (0.22 ⁇ pm) and the concentration of BA was measured by HPLC-ESI-MS/MS using Cl 8 column (150mm x 2mm i.d., 4 ⁇ m) and mobile phases of water containing 15mM acetic acid pH 5 and acetonitrile. The flow rate was 150 ⁇ l/min.
  • the mass spectrometry acquisition was performed in the multiple reaction monitoring mode using the ESI source in negative ionization. Water solubility was expressed as ⁇ mol/liter.
  • the water solubility of compound Ih3e is 99 pM a value higher than corresponding dihydroxy BA and comparable with that of CA (see Table 4). Table 4.
  • a Ws water solubility refers to BA as protonated species and therefore not evaluated for TCDCA, and TUDCA which are highly soluble (hs).
  • b CMC Critical Micellar Concentration determined in 0,15 M NaCl water solution.
  • c ST CMC Surface Tension at CMC in 0, 15 M NaCl water solution.
  • LogPA 1 -octanol- water partition coefficient of the studied bile acids as ionized species. *: values from literature.
  • the presence of a 23-methyl group in the compound Ih3e does not compromise the water solubility.
  • Compound Ih3e exhibits a solubility value in the range of natural occurring BA and previous studied synthetic analogues. Further, given the relatively good albumin binding of compound Ih3e, circulation of compound Ih3e in the blood may be facilitated, thereby favoring the systemic targeting of TGR5 in peripheral tissues such as muscle and brown adipose tissue. Examples 9, 16 and 17 further support this hypothesis.
  • CMC Critical Micellar Concentration
  • the detergency i.e. the tendency to form micelles was evaluated for all the charged molecules which are soluble in water as Sodium salt (2 unit up the pKa).
  • the critical micellar concentration (CMC) was determined by surface tension (ST) measurements using a maximum bubble-pressure method which give surface tension values slightly affected by potential impurities like static methods are.
  • the tensiometer was a Sensadyne 6000 (Chem- Dyne Research Corp., Milwaukee, WI) equipped with two glass probes of 0.5 and 4.0 mm diameters connected to a source of nitrogen.
  • the surface tension of BA sodium salts solutions in NaCl 0.15 M was measured at various concentrations inside the 0.13-50 mM range.
  • the surface tension values were plotted against the logarithm of the bile salt concentration; the regression lines corresponding to the two parts of the curve (monomelic and micellar phases) were calculated using the method of least squares, and the intersection of the lines was taken as the CMC value. From the ST vs concentration curves the value of the surface tension at the CMC (equilibrium between monomers and multimers species) was also calculated giving information about the detergency power which is related to the size of the micelles with associate surface tension lowering capacity.
  • the CMC was evaluated by surface tension measurements in non equilibrium conditions i.e. in conditions that impurities slightly affect the surface tension results (Fig. 10).
  • Compound Ih3e presents a low CMC but a moderate detergency and surface tension lowering capacity as shown by the surface tension values at the CMC (low detergency means low toxicity to membrane or cells).
  • the aqueous buffer was previously pre-saturated with 1-octanol, 5 ml of 1-octanol pre-saturated with water was then added and the samples were left to equilibrate for 2 weeks under continuous stirring at room temperature After centrifugation the two phases were carefully separated.
  • BA concentration in the water phase was measured with HPLC-ESI ⁇ MS/MS using C18 column (150mm x 2mm i.d., 4 ⁇ m) and, as mobile phases, water containing 15 mM acetic acid pH 5 and acetonitrile. The flow rate was 150 ⁇ al/min and the column was maintained at 45°C.
  • the mass spectrometry acquisition was performed in the multiple reaction monitoring mode using the ESI source in negative ionization.
  • the carboxylated compound Ih3e with three hydroxyl groups in 3 ⁇ ,7 ⁇ and 12 ⁇ position presents a slightly higher lipophilicity in respect to the natural analogue CA, 1.4 vs 1.1 as a result of the presence of an ethyl in 6 position and a methyl in 23 position.
  • the extent of albumin binding was evaluated by equilibrium dialysis at a fixed BA- albumin ratio.
  • BA was dissolved at a concentration of 100 ⁇ M in 5% bovine serum albumin- saline solution (pH 7.2) and left to stand for 24 h at 25°C. Two millilitres of this solution was dialyzed in cellulose sacs having a molecular weight cut-off of 12-14,000 against 25 ml of saline solution. The system was equilibrated by mechanical gently shaking for 72 h at 25°C.
  • BA concentrations of the dialyzed solution (corresponding to the free unbound fraction) and of the starting solution were determined with HPLC-ESI-MS/MS in the same conditions of the previous analysis.
  • the percent of albumin binding was calculate from the initial BA concentration and from the unbound concentration in the dialyzed fraction. Data are reported in Table 4.
  • the percent albumin binding of compound Ih3e is slightly higher than CA and this derives from the presence of the 23 methyl and 6 ethyl groups.
  • Example 11a 7 a-dehydroxylation.
  • TLC Thin-layer chromatography
  • the solvent system used for the separation of conjugated BA was composed of propionic acid/isoamyl acetate/water/N-propanol (3:4: 1 :2, v/v/v/v; solvent I), and that of the unconjugated BA was acetic acid/carbon tetrachloride/isopropyl ether/isoamyl acetate/water/N-propanol/benzene (1 :4:6:8:2:2, v/v/v/v/v/v; solvent II).
  • Separated BA were revealed with 5% phosphomolybdic acid ethanol solution.
  • Example l ib It is known that intestinal bacteria hydro lyze the C24 amide bond of taurine and glycine conjugated BAs and remove the 7 ⁇ -hydroxyl group of CA, leading to the formation of toxic lipophilic secondary BAs such as deoxycholic acid (DCA) (Ridlon, J.M., et al., J. Lipid Res. 2006, 47, 241-259).
  • DCA deoxycholic acid
  • Compound Ih3e appears not to be sensitive to this process and was shown to be highly stable with more than 95% of the compound unmodified after 12 h of incubation. By comparison, more than 50% of CA (cholic acid) was metabolized after 1 h and up to 90% within 8 h ( Figure 15). It is likely that the extended stability of compound Ih3e is related to the alkylation of the C6 position which provides steric hindrance to the bacterial 7 ⁇ -dehydroxylation process.
  • EXAMPLE 12 Biliary secretion and metabolism of compound Ih3e in bile-fistula rat after duodenal (id) and femoral (iv) administration
  • Example 12 A Aim and Rationale Structural modifications of bile acids could affect their hepatic uptake, hepatic transports and secretion and intestinal absorption. Therefore the knowledge of the biliary secretion after both iv and id administration together their metabolism is a key point in compound selection for additional studies.
  • the compound was administered both intravenously (femoral infusion) and orally (duodenal infusion) at the same dose and its biliary secretion rate was evaluated in bile fistula rat model.
  • the differences in the area under the curve of the biliary secretion vs time between iv and id administration account for its intestinal absorption and give information about its biovailability.
  • the hepatic and intestinal metabolism could be also quite different and therefore, the biliary secretion of compound Ih3e and its main hepatic metabolites was determined. Choleretic effect -Duodenal infusion
  • the bile fistula rat model was developed at the University of Bologna Lab facilities. Compound Ih3e was administered at a dose of 1 ⁇ mol/kg/min (1 hour infusion) to a rat group via duodenal infusion (id). Rats had a bile fistula to collect bile samples at different times before, during, and after the infusion. For duodenal infusion experiment 6 rats (250 ⁇ 10 g) were treated. Bile samples were collected every 15 minutes for four hours. In addition, 3 control rats were treated with saline solution under the same conditions for times and sampling (duodenal control rats).
  • Compound Ih3e presented the a potent choleretic effect and this is believed to be related to its structure; a methyl group in the C-23 position partially prevents conjugation and this molecule can undergo a cholehepatic shunt pathway.
  • the duodenal infusion of CDCA slightly increased the bile flow, which did not exceed 80 ⁇ L/min/kg.
  • Bile samples collected during the iv and id experiments were analyzed to determine the biliary secretion of compound Ih3e and its metabolites.
  • Pure crystalline powder of compound Ih3e was obtained from the R. Pellicciari laboratory of Perugia. Stock solutions in methanol at 1 mmol/L were prepared and working solutions were prepared by diluting appropriate volumes of the primary solution. Methanol and acetronitrile were of HPLC-grade purity. Ammonia was 30% and acetic acid was 99.8%. All reagents were obtained from Carlo Erba Reagents. HPLC-grade water was prepared by a Milli-Q system.
  • the final solution was transferred in an autosampler vial, and 10 ⁇ L was injected into the chromatographic column.
  • Bile rat samples were analyzed by liquid chromatography-tandem mass spectrometry (HPLC-M S/M S) using electrospray (ESI) source in negative ionization mode.
  • HPLC-M S/M S liquid chromatography-tandem mass spectrometry
  • ESI electrospray
  • a Waters Alliance 2695 separation module coupled with autosampler was used. Autosampler was maintained at 7°C. Separation was performed on a Synergi Hydro-RP C 18 column (150x2.0mm i.d., 4 ⁇ m particle size), protected by a
  • Nitrogen was used as nebulizer gas at 100 L/h flow rate and as desolvation gas at 930 L/h.
  • Ion source block and desolvation temperatures were set respectively to 80°C and 180°C.
  • Capillary voltage was 3.0 kV.
  • MassLynx software version 4.0 was used for data acquisition and processing. In addition, using mass spectrometry both in single MS or tandem MS/MS configuration experiments were performed to identify metabolites.
  • a 5-point calibration curve was prepared daily and injected in duplicate. Calibration samples were obtained in the 0.1 to 20 ⁇ mol/L concentration range prepared in mobile phase.
  • the data refer to the secretion rate of the compound recovered in bile as such after duodenal and femoral infusion at a dose of lumol/Kg/min.
  • Table 5 shows concentration and secretion values for compound Ih3e obtained from bile rat samples collected during the duodenal infusion (1 h ranging from 75 to 135 min).
  • Table 6 shows concentration and secretion values obtained from bile rat samples collected during the femoral infusion (1 h ranging from 75 to 135 min).
  • Table 6A Tauro-Ih3e concentration and secretion values estimated from rat bile samples collected during the duodenal infusion (1 hour ranging from 75 to 135 minutes)
  • Table 6B Tauro-Ih3e concentration and secretion values estimated from rat bile samples collected during the femoral infusion (1 hour ranging from 75 to 135 minutes)
  • Example 12B The results in the Table shown below reveal that compound Ih3e has a potent choleretic effect, with the maximum bile secretion rate (SVO) being significantly higher than those of CDCA and CA.
  • ⁇ Data represent average values and standard deviations of six independent experiments.
  • the vehicle used for the id administration was saline solution.
  • the vehicle used for the iv administration was 3% BSA saline solution, pH 7.2.
  • SV 0 maximum bile secretion rate (( ⁇ L/ min)/kg bw).
  • S BA maximum BA secretion rate (( ⁇ mol/min)/kg bw).
  • % free percentage of the administered dose recovered in bile of the molecules as such.
  • % conjugate percentage of the administered dose recovered as conjugated BA.
  • Compound Ih3e is mainly secreted as parent compound (unmodified) and was only slightly metabolized by the liver.
  • the main metabolite was the taurine conjugate and the mono glucuronide was present in a low amount.
  • the metabolism is similar for both iv and id administration. Considering the recovery in bile, we expected to identify other metabolites.
  • Figure 14b is a zoom display of Figure 14a.
  • Figure 14c shows compound Ih3e and its main metabolites identified in bile using mass spectrometry in the di experiment. Data are reported as absolute area values.
  • Figure 14d is a zoom display of Figure 14c.
  • compound Ih3e is moderately hydrophilic and has a mild detergency.
  • the intestinal absorption is also efficient, even if it is not complete, and the molecule does not require extensive hepatic metabolism at the administered dose to be secreted into bile.
  • HepG2 cell cytotoxicity was determined by monitoring ATP decrease and HepG2 cell apotosis was determined by monitoring caspase-3 activation. The results are shown in Table 7. Cytotoxicity Cell viability was measured using Perkinelmer ATP-Lite 1 STEP. ATP is a marker for cell viability because it is present in all metabolically active cells and the concentration declines very rapidly when the cells undergo necrosis or apoptosis. HepG2 cells (Ix10 4 ) were seeded in 96 wells plate and stimulated with 10-fold dilutions from 1 nM to 300 ⁇ M of the compound Ih3e for 4h at 37°C.
  • the plates were equilibrate at RT for 10 minutes and 100 ⁇ l of ATP-Lite 1 STEP Reagent was added to 100 ⁇ l of culture medium containing cells. Luminescence was read with Victor Light (PerkinElemr). The experimental signal was subtracted from background. Tamoxifen was used as positive control of cellular cytotoxicity, while negative control was the non treated cells.
  • Caspases participate in the molecular control of apoptosis and TruPoint Caspase-3 Substrate enables sensitive, robust and homogeneous time-resolved fluorescence assay of caspase-3 activity.
  • Human Hepatocytes cells (HepG2) were seeded (1x10 4 ) in 96 well plate with HepG2 medium without sodium pyruvate. The cells were stimulated 4h at 37°C with serial dilutions of test compound Ih3e from InM to 300 ⁇ M in triplicate. Staurosporin was used as positive control of apoptotic cells. Negative controls were: 1. Unstimulated cells; 2. Medium alone without cells; 3. Cells incubated without the caspase substrate. Lyses buffer and Caspase-3 Substrate were added to the cells and 1 hour and 24 hours after fluorescence was measured with EnVision.
  • the cellular necrosis was analyzed by measuring the release of Lactato DeHydroxegenase (LDH) from the necrotic cells using Promega's CytoTox ONE Homogeneous Membrane Integrity Assay.
  • LDH Lactato DeHydroxegenase
  • HepG2 cells IXlO 4
  • Triton 1 % was used as maximum LDH release control.
  • Tamoxifen was used as inducer necrosis.
  • the plated cells were placed back into the incubator for an additional 4 hours.
  • the selectivity of compounds of the invention was evaluated using assay methods known in the art. Specifically, the following assay methods were used: FXR and LXR: Coactivator Recruitment (alphascreen);
  • TGR5 cAMP level on human intestinal cell line (NCI-H716); PXR: Ligands Competition assay (Binding Assay) CAR: Coactivator Recruitment (Lanthascreen) Table 8 shows the results of these assays.
  • Lanthascreen assay (Invitrogen) was used for nulear receptor selectivity assay.
  • the kit uses a terbium-labeled anti-GST antibody, a fluorescein-labeled coactivator peptide, and a NR ligand-binding domain that is tagged with glutathione-S-transferase (GST) in a homogenous mix-and-read assay format.
  • GST glutathione-S-transferase
  • the assays were performed in 384 microwell plate (PerkinElmer).
  • a 20 ⁇ l total assay reaction included 5 nM GST-tagged NRs, 125 nM of coregulator peptide, 5 nM of TB-anti-GST tagged antibody (terbium-anti-glutathione S transferase tagged), 5 mM DTT and varying concentration of compound Ih3e in the assay buffer supplied by Invitrogen.
  • the negative control was devoid of the compound Ih3e but contained everything else contained in the agonist well.
  • TR-FRET measurements were made in the Envision.
  • the emission ratio 520/495 was plotted against varying ligand concentrations. The data was analyzed using GraphPad Prism using the sigmoidal curve equation with variable slope to obtain EC 50 values.
  • the stability of compound Ih3e was determined using methods known in the art. Cellular fraction concentration was lmg/ml over a time course of 0-15-30-60- 120-240-360- 1440 minutes. Positive controls were testosterone (1000 ng/ml); 7-hydroxy coumarine (1296 ng/ml); benzoic acid (2440 ng/ml) over a time course of 0-10-20-40-60-120.
  • the analytical method used was LC/MS separation on Cl 8 column by gradient polarity; acquisition performed in Single Ion Monitoring. The results are shown below in Table 9.
  • Figure 16 shows that compound Ih3e dramatically and dose-dependently induces the release of GLP-I ex vivo.
  • DPP4 inhibitor DPP4i sitagliptin was a kind gift from Dr. C. Ullmer (Hoffmann-La Roche).
  • Compound Ih3e was synthesized as previously described (Macchiarulo et al., 2008; Pellicciari et al., 2007).
  • Cox activity was evaluated by following the oxidation of fully reduced cytochrome c (Sigma) at 550 nm (Feige et al., 2008b, Cell Metab. 8, 347). ATP/ADP ratio and GLP-I release were measured according to the manufacturers' instructions (Biovision and Millipore, respectively). Primary brown adipocytes were prepared as previously described (Watanabe et al., 2006, Nature 439, 484), and ileal explants were prepared according to an established method (Cima et al., 2004, J. Exp. Med. 200, 1635-1646).
  • NCI-H716(40,000 cells) was seeded in 96-well black plates coated with Matrigel (BD Biosciences). Seventy-two hours after transfection, cells were washed twice in assay buffer (HBSSIx, 2OmM HEPES [pH 7.4]) and assayed for intracellular calcium with Fluo-4 AM according to the manufacturer's protocol (Invitrogen).
  • Plasma parameters and hepatic and fecal lipid content were measured as described (Mataki et al., 2007, MoI. Cell. Biol. 27, 8330-8339). Hematoxylin and eosin (H&E), Sirius red, and oil red O staining were performed as described (Mark al., 2007, Curr. Protoc. MoI. Biol. Chapter 29, Unit 29B, 24), and micrographs were taken on wide-field microscopes (Leica) with a CCD camera. For pancreatic sections, islets were sized and counted from four HE-stained alternated sections spaced of 150 ⁇ M using ImageJ software (five animals per group).
  • pancreatic islets were isolated by collagenase digestion of pancreas from HF-fed TGR5-Tg mice according to online-available procedures (for example, see JOVE (Journal of Visualized Experiments website)). Insulin was extracted after O/N incubation at — 20°C in acid ethanol and measured by ELISA on PBS-diluted samples according to the manufacturer's instructions (Mercodia). GLP-I release was measured in vitro, ex vivo, and in vivo according to methods known in the art.
  • mice Animals were housed and bred according to standardized procedures (Argmann and Auwerx, 2006b). Age-matched male mice were used for all experiments. Genetically engineered mouse models (GEMMs), i.e., TGR5-Tg and TGR5 ⁇ ⁇ mice, were generated as described in the Supplemental Data. DIO in the GEMMs or C57BL/6J mice (Charles River) was induced by feeding 8-week-old mice with a HF diet (60% cal/fat, D 12492; Research Diets) for at least 8 weeks, as mentioned in the text and figure legends. In the dietary intervention experiments, Compound Ih3e was mixed with diet (Feige et al., 2008a, Curr. Protoc. MoI. Biol.
  • HF diet 50% cal/fat, D 12492; Research Diets
  • Hyperinsulinemic euglycemic clamp studies were performed as described (Feige et al., 2008b), with minor modifications including a change in the initial insulin bolus (30 mU/kg) and insulin infusion rate (10 mU/min/kg).
  • Plasma GLP-I levels were measured by ELISA (Millipore) on blood collected by retro-orbital puncture.
  • Experiments with db/db mice (Charles River) were performed in 14-week-old animals fed a CD without or with compound Ih3e (30 mg/kg/d) for 6 weeks (Feige et al., 2008a).
  • TGR5 mRNA expression was analyzed via the GeneNetwork liver mRNA database in the BxD genetic reference population as found on the GeneNetwork Univeristy of Tennessee website. A wide range of variation in TGR5 mRNA expression was evident among the different BxD mouse strains.
  • TGR5 mRNA expression was highly significantly correlated with the expression of several genes encoding for subunits of complexes involved in oxidative phosphorylation, such as cytochrome c oxidase (Cox) (e.g., CoxVIla; Figure 17A) and ATP synthase (Atp6vOb, ATPase H + transporting VO subunit B; Atpaf2, ATP synthase mitochondrial Fl complex assembly factor 2; Atpl a3, ATPase Na + /K+ transporting alpha 3 polypeptide; Atp6vl b2, ATPase H + transporting V 1 subunit B isoform 2).
  • Cox cytochrome c oxidase
  • Atpaf2 ATP synthase mitochondrial Fl complex assembly factor 2
  • Atp6vl b2 ATPase H + transporting
  • Example 18 Activation of TGR5 Signaling Pathway Increases Intracellular Calcium Levels and Stimulates GLP-I release in Enteroendocrine L Cells
  • TGR5-mediated GLP-I release triggered by compound Ih3e was further confirmed in the mouse enteroendocrine STC-I cells in which the impact of compound Ih3e on GLP-I release was enhanced by TGR5 overexpression, while being prevented either by RNA interference (Figure 18G) or by MDL-12-330A, further underscoring the cAMP dependence of TGR5 -mediated GLP-I release ( Figure 18H).
  • Figure 18G RNA interference
  • MDL-12-330A MDL-12-330A
  • TGR5 transgenic mice were generated by oocyte injection of the bacterial artificial chromosome (BAC) RP23-278N1.
  • BAC bacterial artificial chromosome
  • TGR5-Tg mice were shown to have integrated six copies of the RP23-278N1 1 BAC clone, leading to a robust TGR5 mRNA expression, restricted to most tissues that express TGR5 normally.
  • Glucose tolerance was markedly improved in TGR5-Tg mice challenged for 10 weeks with a high-fat (HF) diet compared to control HF-fed litter-mates ( Figure 19A), whereas no difference was noticed in mice on chow diet (CD) (data not shown).
  • HF high-fat
  • CD chow diet
  • no differences were observed in body weight gain between wild-type and TGR5-Tg mice on CD or HF diet, demonstrating that improvement of glucose tolerance in TGR5-Tg mice could not be attributed to the confounding effects of weight loss.
  • GLP-I receptor knockout mice display a marked hyperactivity (Hansotia et al., 2007, J. Clin. Invest. 117, 143-152)
  • Ex-4 efficiently and dose-dependently reduced locomotor activity in mice.
  • at 1 nmol/Kg we noticed a significant decrease in locomotor activity while the mice were still eating properly.
  • pancreatic function has been extensively documented during the last decade and ranges from insulin-secretagogue effects to the promotion of pancreatic islet survival and proliferation (Drucker, 2006, Cell Metab. 3, 153-165).
  • immunofluorescent staining of insulin on pancreatic sections revealed that, in contrast to hypertrophic islets with low insulin content, as observed in HF-fed control mice, islets of HF- fed TGR5-Tg mice were not hypertrophic and stained more intensively for insulin (Figure 19E).
  • counting and sizing of pancreatic islets confirmed that TGR5 expression results in the maintenance of a normal islet distribution profile (Figure 19F), likely due to enhanced plasma GLP-I levels.
  • the insulin content of isolated pancreatic islets was significantly higher in HF-fed TGR5-Tg mice than in controls (Figure 19G).
  • TGR5 ⁇ ⁇ germline TGR5 -deficient mice
  • Example 20 The TGR5 Agonist Compound Ih3e Increases Energy Expenditure and Reduces Hepatic Steatosis and Obesity upond High-Fat Feeding
  • liver enzymes were markedly reduced compared to HF-fed controls, correlating with the absence of liver fibrosis in liver sections of Ih3e-treated mice stained with Sirius red ( Figures 201 and 20K).
  • the improvement in liver function was also reflected by the significant drop in plasma triglycerides and nonesterified fatty acids (NEFAs) in HF-fed mice treated with compound Ih3e ( Figure 20L).
  • Example 21 The TGR5 Agonist Compound Ih3e Improves Insulin Sensitivity in Obese Mice
  • This structure modification could in part modify the physicochemical properties, metabolism and pharmacokinetics of the two compounds.
  • the introduction of a C-23 methyl group in the side chain differently oriented produced two isomers where also the carboxy group is differently oriented and its reactivity in the amidation process or in the deconjugation of the amidated form can be different among the two diasteroisomers.
  • the different carboxy group orientation is also responsible for a different hydrophobic/hydrophilic balance of the two molecules which could result in different bilological properties and metabolism.
  • the bile flow during iv infusion of the control 3% BSA saline vehicle maintained a value ranging from 40 to 60 ⁇ L/min/kg for the entire period of the experiments.
  • the iv infusion of compound Ih3e significantly increased the bile flow rate and this phenomenon started 15 minutes after the beginning of the infusion period and continued for at least 2 hours after the end of the infusion period ( Figure 23).
  • the iv infusion of compound Ii3e also increased the bile flow rate but this effect is significantly lower than that observed for the isomer compound Ih3e ( Figure 23).
  • Rat bile samples were analyzed by HPLC-ESI-MS/MS using the ESI source in negative ionization mode.
  • a Waters Alliance 2695 separation module coupled with autosampler was used. The autosampler was maintained at 7°C. Separation was performed on a Synergi Hydro-RP Cl 8 column (150 x 2.0 mm i.d., 4 ⁇ m particle size), protected by a SecurityGuard ODS 4 x 2.0 mm i.d. precolumn, both supplied from Phenomenex.
  • Mobile phase B was increased from 30% to 64% in 10 minutes, then to 100% in 10 minutes, and held constant for 10 minutes.
  • Flow rate was 150 ⁇ L/min and the column was maintained at 45°C.
  • the column effluent was introduced into the ESI source connected to a triple quadruple MS (Quattro-LC, Micromass) operating in multiple reaction monitoring (MRM) acquisition mode.
  • Nitrogen was used as nebulizer gas at 100 L/h flow rate and as desolvation gas at 930 L/h.
  • the ion source block and desolvation temperatures were set respectively to 80°C and 180°C.
  • Capillary voltage was 3.0 kV.
  • MassLynx software version 4.0 was used for data acquisition and processing. In addition, using mass spectrometry both in single MS and tandem MS/MS configurations, experiments were performed to identify metabolites. Quantification
  • the biliary secretion of compound Ii3e after iv infusion is reported in Figure 25.
  • the kinetic profile indicates that the compound is metabolized by the liver more extensively than compound Ih3e.
  • the parent compound is secreted in bile as such and to a less extent as taurine conjugate.:
  • the percentage of conjugation is higher and the maximum secretion rate of the unconjugared form is lower.
  • the conjugation with taurine contributes to improve compound Ii3e recovery in bile which is approx. 70-80% of the administered dose.
  • Other minor metabolites including glucuronides have been identified in bile in trace amount (Fig 28-29).
  • This molecule was mainly secreted as parent compound (unmodified) and was only slightly metabolized by the liver.
  • the main metabolite was the taurine conjugated species and, at very low levels, the mono-glucuronide species was detected (Fig. 26-27).
  • the presence of the methyl group in C-23 position hinders the conjugation process with taurine and glycine which is required for an efficient secretion of almost all naturally occurring carboxylated BAs; this is crucial for dihydroxy BA and to a lesser extent for trihydroxy BA since they are already quite polar. Formation of glucuronides could become relevant if administered at higher doses.
  • This molecule was mainly secreted as parent compound (unmodified) and was also metabolized by the liver to form the taurine conjugated specie and, at very low levels, the mono-glucuronide specie. (Fig. 27-29).
  • Compound Ii3e is secreted in bile in higher percentage than the diasteroisomer compound Ih3e as taurine conjugated form, 20-30% vs 5-10% and this accounts for the different side chain geometry and to a slightly higher lipophilicity of compound Ii3e.
  • Compound Ih3e is moderately hydrophilic and has a mild detergency. Its hepatic uptake seems efficient. The biliary secretion is also efficient considering that the compound is secreted mainly unmodified and, to a limited extent, conjugated with taurine. The intestinal absorption occurs via passive mechanism like naturally occurring unconjugated BA and the kinetics is similar to that of cholic acid slightly lower that dihydroxy bile acids (Aldini R. et al. Steroids 61, 590-7, 1996).
  • Compound Ih3e does not require extensive hepatic metabolism at the administered dose to be secreted into bile.
  • the presence of the methyl group in the C-23 (S) position prevents extensive conjugation with taurine and the molecule can be efficiently secreted unmodified.
  • An increased hepatic residence time of the molecule results from a ductular absorption since this molecule undergoes to a cholehepatic shunt pathway, which is responsible for its potent choleretic effect.
  • Compound Ii3e is the diasteroisomer of compound Ih3e.
  • Compound Ii3e is characterized by a slightly lower hydrophilicity as a result of the different side chain geometry.
  • the C-23 carboxy group is differently oriented and this accounts for the different hydrophilic-hydrophobic balance of the molecule.
  • the molecule requires a more extensive conjugation with taurine in respect to compound Ih3e.
  • the side chain geometry of the last compound probably produces a BA with a lower substrate specificity toward the enzyme responsible for the conjugation mediated by CoA activation process.
  • the final result is that compound Ii3e is secreted in bile in an higher conjugated percentage than compound Ih3e.

Abstract

L'invention concerne des composés de la formule A : (A) ou un sel, un solvate, un hydrate ou un promédicament de ces composés. Les composés de la formule A sont des modulateurs de TGR5 qui s’utilisent dans le traitement de diverses maladies, comprenant les maladies métaboliques, les maladies inflammatoires, les maladies du foie, les maladies auto-immunes, les maladies cardiaques, les maladies du rein, le cancer et les maladies gastro-intestinales.
PCT/US2009/065188 2008-11-19 2009-11-19 Modulateurs de tgr5 et leur procédé d'utilisation WO2010059853A1 (fr)

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MX2011005295A MX2011005295A (es) 2008-11-19 2009-11-19 Moduladores de tgr5 y metodo de uso de los mismos.
EA201170714A EA020140B1 (ru) 2008-11-19 2009-11-19 Модуляторы tgr5 и способы их применения
JP2011537624A JP5535233B2 (ja) 2008-11-19 2009-11-19 Ｔｇｒ５モジュレーターおよびその使用方法
CN200980154713.0A CN102325784B (zh) 2008-11-19 2009-11-19 G蛋白偶联受体5(tgr5)调节剂及其使用方法
AU2009316566A AU2009316566B9 (en) 2008-11-19 2009-11-19 TGR5 modulators and method of use thereof
ES09760057.1T ES2458168T3 (es) 2008-11-19 2009-11-19 Moduladores de TGR5 y método de uso de los mismos
KR1020167028159A KR101789960B1 (ko) 2008-11-19 2009-11-19 Tgr5 조절제 및 그의 사용 방법
BRPI0921983A BRPI0921983B8 (pt) 2008-11-19 2009-11-19 moduladores tgr5, usos dos mesmos e kit
DK09760057.1T DK2376519T3 (da) 2008-11-19 2009-11-19 TGR5-modulatorer og fremgangsmåde til anvendelse deraf
KR1020117014037A KR101667436B1 (ko) 2008-11-19 2009-11-19 Ｔｇｒ５ 조절제 및 그의 사용 방법
PL09760057T PL2376519T3 (pl) 2008-11-19 2009-11-19 Modulatory TGR5 i sposoby ich zastosowania
EP09760057.1A EP2376519B1 (fr) 2008-11-19 2009-11-19 Modulateurs de tgr5 et leur procédé d'utilisation
CA2744189A CA2744189C (fr) 2008-11-19 2009-11-19 Modulateurs de tgr5 et leur procede d'utilisation
IL212969A IL212969A (en) 2008-11-19 2011-05-18 6-Ethyl Acid, 23-Methyl-Calcium, its Conjugates with Glycine and Taurine and Pharmaceutical Preparations Containing Them
ZA2011/04077A ZA201104077B (en) 2008-11-19 2011-06-01 Tgr5 modulators and method of use thereof
HK12106982.3A HK1166327A1 (en) 2008-11-19 2012-07-17 Tgr5 modulators and method of use thereof g 5(tgr5)
IL244812A IL244812B (en) 2008-11-19 2016-03-29 Process for preparing 23(s)-methyl-12α,7α,3α-trihydroxy-6α-ethyl-5β-cholan-24-butyric acid
IL264796A IL264796B (en) 2008-11-19 2019-02-12 History of 6,23-metabolized bile acid, pharmaceutical preparations acceptable in a pharmacy and compounds for use in a method for treating or preventing a disease

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