Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J9/00—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
  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• the invention concerns relates to compounds that modulate TGR5 and compositions containing such compounds useful in methods for the treatment and prevention of disease.
• the compounds of the invention are analogues of chenodeoxycholic acid having a substituent at the C- 16 position.
• 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, 97-101.
• 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.; Fujii, R.; Hosoya, M.; Harada, M.; Yoshida, H.; Miwa, M.; Fukusumi, S.; Habata, Y.; Itoh, T.; Shintani, Y.; Hinuma, S.; Fujisawa, Y.; Fujino, M., A G protein-coupled receptor responsive to bile acids. J. Biol. Chem.
• 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.; Tanaka, K.; Suzuki, J.; Miyoshi, H.; Harada, N.; Nakamura, T.; Miyamoto, Y.; Kanatani, A.; Tamai, Y., Targeted disruption of G protein-coupled bile acid receptor 1 (Gpbarl/MBar) in mice.
• G protein-coupled bile acid receptor 1 G protein-coupled bile acid receptor 1
• TGR5 glucagon-like peptide 1
• GLP-I glucagon-like peptide 1
• TGR5 is an attractive target for the treatment of metabolic diseases e.g., obesity, diabetes (type I and type II), dyslipidemia, non-alcoholic steatohepatitis (NASH), and the metabolic syndrome.
• metabolic diseases e.g., obesity, diabetes (type I and type II), dyslipidemia, non-alcoholic steatohepatitis (NASH), and the metabolic syndrome.
• TGR5 agonists have been so far described in literature. Recently, 23- alkyl-substituted and 6,23-alkyl-disubstituted derivatives of chenodeoxycholic acid, such as the 6 ⁇ -ethyl-23(S)-methyl-chenodeoxycholic acid shown below, have been reported as potent and selective agonists of TGR5 (Pellicciari, R.; Sato, H.; Gioiello, A.; Costantino, G.; Macchiarulo, A.; Sadeghpour, B. M.; Giorgi, G.; Schoonjans, K.; Auwerx, J., Nongenomic actions of bile acids. Synthesis and preliminary characterization of 23-and 6,23-alkyl- substituted bile acid derivatives as selective modulators for the g-protein coupled receptor TGR5. J. Med. Chem. 2007, 50, 4265-4268).
• TGR5 agonists include 6-methyl-2-oxo-4- thiophen-2-yl-l,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid benzyl ester (WO004067008, Takeda Chemical Industries LTD, Japan, 2004) and oleanoic acid (Sato, H.; Genet, C;
• TGR5 agonist isolated from Olea europaea. Biochem. andBiophys. Res. Commun. 2007, 362, 793-798; Ito, F.; Hinuma, K.; Kanzaki, N.; Miki, T.; Kawamata, Y.; Oi, S.; Tawaeaishi, T.; Ishichi, Y.; Hirohashi, M. Preparation of aromatic ring- fused cyclic compounds as TGR5 receptor agonists.
• PN W02004067008, 2004. More recently, the first synthesis of enantiomeric chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) has allowed to assess the specificity of the interaction of natural BAs to TGR5 (Katona, B. W.; Cummins, C. L.; Ferguson, A. D.; Li, T.; Schmidt, D. R.; Mangelsdorf, D. J.; Covey, D. F., Synthesis, Characterization, and Receptor Interaction Profiles of Enantiomeric Bile Acids. J. Med. Chem. 2007, 50, 6048-6058).
• the present invention has identified compounds that are chenodeoxycholic acid analogues which have a substituent at the C- 16 position as well as methods of using these compounds to treat disease.
• the present invention relates to compounds and their use to treat and prevent diseases that involve modulation of the TGR5 receptor, such as metabolic disease, inflammatory disease, liver disease, autoimmune disease, cardiac disease, kidney disease, cancer, and gastrointestinal disease.
• diseases that involve modulation of the TGR5 receptor such as metabolic disease, inflammatory disease, liver disease, autoimmune disease, cardiac disease, kidney disease, cancer, and gastrointestinal disease.
• the compounds of the invention are analogues of chenodeoxycholic acid that are substituted at the C- 16 position of the D ring as shown below.
• the invention includes a compound having the formula A:
• Ri 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 7 is hydrogen, substituted or unsubstituted alkyl, or hydroxy
• Rn is hydroxyl, OSO 3 H, OSO 3 " , OCOCH 3 , OPO 3 H, OPO 3 2" , OC 6 H 8 O 6 " , or hydrogen;
• Ri 2 is hydroxyl, OSO 3 H, OSO 3 " , OCOCH 3 , OPO 3 H, OPO 3 2" , OC 6 H 8 O 6 " , or hydrogen, or taken together Rn and Ri 2 form a carbonyl; and Ri 6 is hydroxyl, alkoxy, and halogen, provided that when R 2 , R 4 , R 7 , and Ri 2 are hydrogen,
• Ri and Rn are OH, then Ri 6 is not alpha OH.
• the invention includes a compound having the formula B: (B) or a salt, solvate, hydrate, or amino acid conjugate thereof, wherein R 1 , R 2 , R 4 , R 7 , Rn, R12, and Ri 6 are as described herein.
• the invention includes a compound having the formula C:
• the invention includes a compound having the formula D:
• the invention includes a compound having the formula E:
• the invention includes a compound having the formula F: (F) or a salt, solvate, hydrate, or amino acid conjugate thereof, wherein R 2 and Ri 6 are as described in claim 1.
• the invention includes a compound or a salt, solvate, hydrate, or amino acid conjugate thereof, wherein Ri is OH.
• the invention includes a compound or a salt, solvate, hydrate, or amino acid conjugate thereof, wherein R 7 is H.
• the invention includes a compound or a salt, solvate, hydrate or amino acid conjugate thereof, wherein R 2 is H.
• the invention includes a compound or a salt, solvate, hydrate or amino acid conjugate thereof, wherein R 2 is alpha-OH.
• the invention includes a compound or a salt, solvate, hydrate or amino acid conjugate thereof, wherein R 2 is beta-OH.
• the invention includes a compound or a salt, solvate, hydrate or amino acid conjugate thereof, wherein R 4 is unsubstituted alkyl.
• the invention includes a compound or a salt, solvate, hydrate or amino acid conjugate thereof, wherein R 4 is ethyl.
• the invention includes a compound or a salt, solvate, hydrate, or amino acid conjugate thereof, wherein Rn is hydroxyl.
• the invention includes a compound or a salt, solvate, hydrate, or amino acid conjugate thereof, wherein Ri 2 is hydrogen.
• the invention includes a compound or a salt, solvate, hydrate, or amino acid conjugate thereof, wherein Ri 6 is alpha hydroxyl.
• the invention includes a compound or salt, solvate, hydrate, or amino acid conjugate thereof, wherein Ri 6 is beta hydroxyl.
• the invention includes a compound, wherein the compound is Compound 10:
• the invention includes a compound, wherein the compound is a pharmaceutically acceptable salt.
• the invention includes a composition comprising a compound of the invention and at least one pharmaceutically acceptable excipient.
• the invention includes a compound or composition for use in a method of treating or preventing a disease, the method comprising administering the compound of the invention.
• the invention includes the use of a compound of the invention in the manufacture of a medicament for a method of treating or preventing disease.
• the invention includes the use of a compound having the formula A:
• R 2 is hydrogen or hydroxy
• R 4 is hydrogen, substituted or unsubstituted alkyl, or halogen
• R 7 is hydrogen, substituted or unsubstituted alkyl, or hydroxy
• Rn is hydroxyl, OSO3H
• OSO 3 " OCOCH 3 , OPO 3 H, OPO 3 2" , OC 6 H 8 O 6 " , or hydrogen; R i2 is hydroxyl, OSO 3 H, OSO 3 " ,
• the invention includes a method of treating disease by administering to a subject a compound having the formula A or a salt, solvate, hydrate, or amino acid conjugate thereof.
• the invention includes the use, 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 the use, wherein the compound or composition is administered to the subject orally, parentally, intravenously, or topically.
• the invention includes the use, wherein the subject is a human.
• Figure 1 is a series of 2 graphs (A-B) that shows the results of FRET assay using compound 10 (Fig. IA) and LCA (Fig. IB) on HEK293T cells transiently transfected with vector alone (V) or TGR5 expressing vector (TGR5).
• Figure 2 is a series of 2 graphs (A-B) that show transactivation assay (CRE-Luc reporter) results for compound 10 and LCA (positive control) using TGR5 expressing
• Figure 3 is a bar graph that shows the results of TGR5 target gene expression assay for compound 10 using intestinal NC1-H716 cells and LCA as a positive control.
• Figure 4 is a series of 3 graphs (A-C) that show the results for compound 10 of in vitro cytotoxicity testing measuring ATP -release after 4 hours of stimulation using human intestinal (NCI-H716).
• LCA is a positive control.
• Figure 5 is a series of 3 graphs (A-C) that shows the results for compound 10 of in vitro cytotoxicity testing measuring ATP -release after 4 hours of stimulation using hepatic (HepG2) cell lines.
• LCA is a positive control.
• Figure 6 is a graph that depicts the surface tension plotted against the logarithm of the concentration of compound 10 (mM) in NaCl 0.15M.
• Figure 7 is a bile flow chart for a duodenal infusion experiment performed using compound 10.
• Figure 8 is a bile flow chart for a femoral infusion experiment performed using compound 10.
• Figure 9 is a graph that depicts secretion rates versus time in femoral and duodenal infusion experiments performed using compound 10.
• Figure 10 is a series of graphs that shows compound 10 and its main metabolite identified in bile using mass spectrometry in an iv experiment. Data are reported as absolute area values.
• the invention includes a compound of formula A:
• Ri is hydrogen, hydroxy, substituted or unsubstituted alkyl, or halogen
• R 2 is hydrogen or hydroxyl
• R 4 is hydrogen, substituted or unsubstituted alkyl, or halogen
• R 7 is hydrogen, substituted or unsubstituted alkyl, or hydroxyl
• Rn is hydroxyl, OSO 3 H, OSO 3 " , OCOCH 3 , OPO 3 H, OPO 3 2" , OC 6 H 8 O 6 " , or hydrogen
• R i2 is hydroxyl, OSO 3 H, OSO 3 " ,
• the invention does not include a compound wherein R 2 , R 4 , R 7 , and Ri 2 are hydrogen, Ri and Rn are OH, and Ri 6 is alpha OH.
• the invention provides a compound of formula A or a salt, solvate, hydrate, or amino acid conjugate, provided that when R 2 , R 4 , R 7 , and Ri 2 are hydrogen and Ri and Rn are OH, then Ri 6 is not alpha OH.
• the invention does not include
• the invention includes a compound having the formula B:
• the invention includes a compound having the formula C:
• the invention includes a compound having the formula D:
• the invention includes a compound having the formula E:
• the invention includes a compound having the formula F:
• the invention includes a compound or a salt, solvate, hydrate, or amino acid conjugate thereof, wherein Ri is OH. In one aspect, the invention includes a compound or a salt, solvate, hydrate, or amino acid conjugate thereof, wherein R 7 is H. In one aspect, the invention includes a compound of a salt, solvate, hydrate, or amino acid conjugate thereof, wherein Ri is OH and R 7 is H.
• the invention includes a compound or a salt, solvate, hydrate or amino acid conjugate thereof, wherein R 2 is H. In one aspect, the invention includes a compound or a salt, solvate, hydrate or amino acid conjugate thereof, wherein R 2 is alpha-OH. In one aspect, the invention includes a compound or a salt, solvate, hydrate or amino acid conjugate thereof, wherein R 2 is beta-OH.
• the invention includes a compound or a salt, solvate, hydrate or amino acid conjugate thereof, wherein R 4 is unsubstituted alkyl. In one aspect, the invention includes a compound or a salt, solvate, hydrate or amino acid conjugate thereof, wherein R 4 is ethyl.
• the invention includes r a compound or a salt, solvate, hydrate, or amino acid conjugate thereof, wherein Rn is hydroxyl. In one aspect, the invention includes a compound or a salt, solvate, hydrate, or amino acid conjugate thereof, wherein Ri 2 is hydrogen.
• the invention includes the compound 10:
• the invention includes a compound, wherein the compound is a pharmaceutically acceptable salt.
• the invention includes a composition comprising a compound or a salt, solvate, hydrate, or amino acid conjugate thereof and at least one excipient. In one aspect, the invention includes a composition comprising a compound or a salt, solvate, hydrate, or amino acid conjugate thereof and at least one pharmaceutically acceptable excipient. In one aspect, the invention includes the use of a compound or a composition of the invention, in the manufacture of a medicament for treating or preventing disease in a subject. In another aspect, the invention includes a method of treating or preventing disease in a subject by administering a compound or a composition of the invention. In one aspect, the invention includes a therapeutically effective amount of a compound or composition of the invention is administered to the subject. In one aspect, the invention includes a prophylactially effective amount of a compound or composition of the invention is administered.
• the invention includes the use of the compound or composition of the invention, in the manufacture of a medicament for a treating or preventing a disease in a subject that involves modulation of the TGR5 receptor.
• the invention includes a method of treating or preventing a disease that involves modulation of the TGR5 receptor in a subject by administering a compound or composition of the invention.
• the invention includes the use, 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 method of treating or preventing a disease selected from metabolic disease, inflammatory disease, liver disease, autoimmune disease, cardiac disease, kidney disease, cancer, and gastrointestinal disease.
• the invention includes the use, wherein the disease is a metabolic disease selected from obesity, diabetes, metabolic syndrome, insulin resistance, hypertension, and dyslipidemia.
• the invention includes a method of treating or preventing a metabolic disease selected from obesity, diabetes, diabesity, metabolic syndrome, insulin resistance, prediabetic insulin resistance, hypertension, and dyslipidemia.
• the invention includes the use, wherein the disease is an inflammatory disease selected from allergy, osteoarthritis, appendicitis, bronchial asthma, pancreatitis, allergic rash, and psoriasis.
• the invention includes a method of treating or preventing an inflammatory disease selected from allergy, osteoarthritis, appendicitis, bronchial asthma, pancreatitis, allergic rash, and psoriasis.
• the invention includes the use, wherein the disease is an autoimmune disease selected from rheumatoid arthritis, multiple sclerosis, and type I diabetes.
• the invention includes a method of treating or preventing an autoimmune disease selected from rheumatoid arthritis, multiple sclerosis, and type I diabetes.
• the invention includes the use, wherein the disease is 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 a method of treating or preventing 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 the use, wherein the disease is kidney disease selected from diabetic nephropathy, chronic renal failure, hypertensive nephrosclerosis, chronic glomerulonephritis, chronic transplant glomerulopathy, chronic interstitial nephritis, and polysystic kidney disease.
• the invention includes a method of treating or preventing kidney disease selected from diabetic nephropathy, chronic renal failure, hypertensive nephrosclerosis, chronic glomerulonephritis, chronic transplant glomerulopathy, chronic interstitial nephritis, and polysystic kidney disease.
• the invention includes the use, wherein the disease is cancer selected from colorectal cancer, liver cancer, heptacellular carcinoma, cholangio carcinoma, renal cancer, gastric cancer, pancreatic cancer, prostate cancer, and insulanoma.
• the invention includes a method of treating or preventing cancer selected from colorectal cancer, liver cancer, heptacellular carcinoma, cholangio carcinoma, renal cancer, gastric cancer, pancreatic cancer, prostate cancer, and insulanoma.
• the invention includes the use, wherein the disease is a 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.
• a 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
• the invention includes a method of treating or preventing a 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.
• a 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
• the invention includes the use, wherein the cardiac disease is selected from congestive heart failure, myocardial infarction, atherosclerosis, angina pectoris, arteriosclerosis and cerebrovascular disease (hemorrhage, stroke, cerebrovascular infarction).
• the invention includes a method of treating or preventing a cardiac disease selected from congestive heart failure, myocardial infarction, atherosclerosis, angina pectoris, arteriosclerosis and cerebrovascular disease (hemorrhage, stroke, cerebrovascular infarction).
• the invention includes a disease involving modulation of the TGR5 receptor.
• the invention includes a compound that is a TGR5 agonist.
• the compound is a selective TGR5 agonist over FXR activator.
• the compound or composition of the invention is administered to the subject orally, parentally, intravenously, or topically.
• the subject is a human.
• 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.
• amino acid conjugates refers to conjugates of the compounds of the formulae of the invention with any suitable amino acid. Such suitable amino acid conjugates of the compounds of the formulae of the invention will have the added advantage of enhanced integrity in bile or intestinal fluids. Suitable amino acids include but are not limited to glycine and taurine. Thus, the present invention encompasses the glycine and taurine conjugates of any of the compounds of the invention.
• 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
• 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.
• a straight chain or branched chain alkyl has six or fewer carbon atoms in its backbone (e.g., Ci-C 6 for straight chain, C3-C6 for branched chain). In some examples, a straight chain or branched chain alkyl has four or fewer carbon atoms in its backbone. Further, cycloalkyls have from three to eight 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
• 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
• Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., tetralin, methylenedioxyphenyl).
• alicyclic or heterocyclic rings which are not aromatic so as to form a multicyclic system (e.g., tetralin, methylenedioxyphenyl).
• 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.
• 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.
• anionic group 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.
• 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).
• unstable functionalities include acetals and ketals.
• the compounds of the present invention for example, the salts of the compounds, can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules.
• Nonlimiting examples of hydrates include monohydrates, dihydrates, etc.
• solvates include ethanol solvates, acetone solvates, etc.
• Solvates 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. It will be noted that the structure of some of the compounds of the invention include asymmetric carbon atoms.
• 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.
• Solid 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.
• 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.
• 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.
• a therapeutically effective amount of a compound when administered to a subject in need of treatment symptoms arising from the disease are ameliorated immediately or after administration of the compound one or more times.
• 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.
• a “salt” of a compound of the invention is a product of the compound that contains an ionic bond and its typically produced by reacting the compound with either an acid or a base.
• a “pharmaceutically acceptable salt” is a salt suitable for administering to a subject.
• a “composition” is a formulation containing a compound of the invention in a form suitable for administration to a subject. In another embodiment, 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.
• One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient.
• the dosage will also depend on the route 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 esterif ⁇ ed through a linking group.
• An anionic group can be esterif ⁇ ed 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 esterif ⁇ ed to a group which is actively transported in vivo, or which is selectively taken up by target organs.
• 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 act 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.
• an "antagonist” 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 TGR5 and increases the concentration of cyclic adenosine monophosphate (cAMP) by at least 20% in cells expressing the receptor.”
• cAMP cyclic adenosine monophosphate
• a TGR5 antagonist would be a compound that antagonizes or blocks the activity of an agonist, thereby effecting a reduction in the concentration of cAMP
• the present invention relates to compounds having TGR5 receptor modulating activity and their use to treat and prevent metabolic diseases such as obesity and insulin sensitivity.
• a compound of the invention is shown below.
• EXAMPLE 1 Synthesis of TGR5 Modulators
• the compounds of the invention, and related derivatives, can be synthesized by methods known to one skilled in the art.
• Reagent and conditions a) MeOH, pTSA, ultrasound, 30 0 C, 2h, 93%. b) Ac 2 O, NaHCO 3 , THF, reflux, 12h, 99%. c) 1. 3-I-benzoic acid, (COCl) 2 , CH 2 Cl 2 , rt, Ih. 2. CaH 2 , BnEt 3 N + Cl " , toluene, reflux, 48h, 80%. d) PhICl 2 , 1 BuOH, CH 2 Cl 2 , hv, O 0 C, Ih, 91%. e) Pyridine, reflux, 12h, 64%. f) 1. BH 3 THF, rt, 2h. 2.
• the 17-chloro derivative 5 (3.3g, 4.46mmol) was dissolved in dry pyridine (130 ml) and refluxed overnight. The solvent was then evaporated under reduced pressure and the residue was purified by flash chromatography eluting with petroleum ether/EtOAc 8:2 to yield 2.24 g (3.18 mmol, 72%) of the desired olefin as white solid.
• the olefin 6 (0.3 g, 0.42 mmol) was dissolved in BH 3 -THF (10.6 ml IM in THF) at 0 0 C and then stirred at room temperature for 2 h. After this time the reaction was cooled at 0 0 C and a mixture of 4M aqueous NaOH (20 ml) and H 2 O 2 (20 ml) was added dropwise and the resulting mixture was stirred at this temperature for 3 h. The reaction was acidified with IN HCl and extracted with CH 2 Cl 2 (3x60 ml). The combined organic layers were dried over anhydrous sodium sulphate and evaporated to dryness under reduced pressure.
• EXAMPLE 2 In vitro TGR5 and FXR Activity
• EXAMPLE 2A TGR5 and FXR Receptor Binding
• Table 1 summarizes the potency and efficacy of a compound of the invention on FXR and TGR5 Receptors Table 1.
• 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 12x10 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-l-methylxanthine (IBMX)]. After incubation for 60 minutes at 37 0 C, the cells were treated with increasing concentrations of compound 10 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
• Activity on FXR was assayed by using Alphascreen technology in a coactivator recruitment 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.
• Anti-GST-coated Acceptor beads were used to capture the GST-fusion FXR-LBD whereas the biotinylated- SRC-I peptide was captured by the streptavidin Donor beads.
• the assay buffer contained 50 mM Tris (pH 7.4), 50 mM KCl, 0.1% BSA, and 1 mM DTT.
• the stimulation times with l ⁇ l of ligands (solubilized in 100% DMSO) were fixed to 30' a room temperature.
• the concentration of DMSO in each well was maintained at a final concentration of 4%.
• the detection mix acceptor and donor beads
• the plates were incubated in the dark for 4 h at room temperature and then were read with an Envision microplate analyzer (PerkinElmer). Dose response curves were performed in triplicate and Z' factor was used to validate the 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.
• 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 10 for 18 h. Control cultures received vehicle (0.1% DMSO) alone.
• the cells were then lysed by adding 75 ⁇ l of Dual-Glo Lucif erase Reagent (Promega) to 75 ⁇ l of medium containing cells/well. Renilla luciferase activity was measured by adding volume: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.
• Efficacy was determined by calculating percentages of 10 ⁇ M LCA value for TGR5 agonist study and 10 ⁇ M 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 done after transformation to logarithms.
• the level of ACC and AMPK gene expression in intestinal NCI-H716 cells was measured using compound 10 and LCA as positive control.
• the mRNA expression level of TGR-5 target genes was measured by Real-Time Polymerase Chain Reaction (Q-RTPCR).
• Total RNA was isolated (Aurum Total RNA Mini Kit BioRad) from NCI-H716 stimulated with 5 ⁇ M compound 10 for 18 hours.
• the RNA was randomly reverse-transcribed with ISCRIPT cDNA SYNTHESIS KIT (BioRad) in 20 ⁇ l reaction volume.
• the amount of gene expression was then calculated as the difference ( ⁇ CT) between the CT value of the sample for the target gene and the mean CT value of that sample for the endogenous control ⁇ 2-Microglobulin.
• Relative expression was calculated as the difference ( ⁇ CT) between the ⁇ CT values of the test sample and of the control sample (WT) for each target gene.
• the relative quantitation value was expressed and shown as 2- ⁇ CT. All PCR primers were intron spanning designed using the software Beacon Designer on published sequence data from the NCBI database. The results are shown in Figure 3.
• 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.
• Human NCI-H716 or HepG2 cells (1x10 4 ) were seeded in 96 wells plate and stimulated with 10-fold dilutions from 1 nM to 300 ⁇ M of the compound 10 for 4h at 37°C. The plates were equilibrated 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 (PerkinElmer). The experimental signal was subtracted from background. Tamoxifen was used as a positive control of cellular cytotoxicity, while untreated cells served as negative control. The results are shown in Figures 4 and 5.
• EXAMPLE 3 Metabolic activities of compounds of the invention in a diet-induced obesity mouse model
• TGR5 agonists ovallic acid (OA) or compounddof the invention (for example, a "test compound”)
• OA leanolic acid
• test compound for example, a "test compound”
• OA/ test compound are administered via food administration for 16 weeks to male C57BL6J mice that were previously subjected for 10 weeks to a high fat diet.
• TGR5 ligands No problems with toxicity are expected with compounds of the invention, which are administered at ⁇ 50 -fold lower concentration.
• mice 48 male C57BL6J mice (5 weeks of age) are divided in two groups: one group of 24 (group 1, 2&3) animals receives chow diet whereas the other group of 24 receives a high fat diet for a period of 10 weeks (group 4,5&6). The animals are then analyzed during a period of 16 weeks. Five groups of 10 animals are assigned as follows: 1 : chow diet
• Week-2 Body composition is 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 are measured in all groups after a fasting period of 12 h and mice are then placed on the diets as indicated (Day 0).
• Week 2 Serum levels of transaminases, glucose, triglycerides, cholesterol, HDL-C, LDL-C and insulin are measured in all groups after a fasting period of 12 h (Day 14).
• Glucose tolerance is determined by subjecting all the animals to an intraperitoneal glucose tolerance test (IPGTT). Animals are 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 / test compound are measured by indirect calorimetry.
• IPGTT intraperitoneal glucose tolerance test
• Week 8 Body weight composition is again analyzed by dexascan for all groups. Serum levels of transaminases, glucose, triglycerides, cholesterol, HDL-C, LDL-C and insulin are 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) is studied during a period of 30 h.
• Week 10 Measurement of blood pressure and heart rate is performed on groups 4, 5 and 6.
• Week 11 Rectal temperature of all animals is measured at room temperature at 10:00 am.
• Circadian activity measurement is performed on groups 1, 2, 3 and 4.
• Week 12 Glucose tolerance is analyzed by performing an intraperitoneal glucose tolerance test (IPGTT) on groups 4, 5 and 6. During the IPGTT, blood is also collected to analyze insulin levels. Animals are fasted 12 h prior to these tests.
• IPGTT intraperitoneal glucose tolerance test
• Feces are collected in all groups over a 24 h time period and fecal lipids content is measured.
• Week 16 Cold test is performed on all animals by measuring body temperature of animals exposed to 4°C.
• Liver, small intestine, adipose tissues (WAT and BAT), pancreas, heart and muscle are collected, weighed and kept for further analyses including: 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 such as acetylation of proteins of interest (e.g. PGC-l ⁇ ).
• mice are 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 are allowed free access to water and food.
• the standard rodent chow diet is obtained from UAR and the high fat diet is obtained from Research Diet. Mice are 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 test compound 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 test compound /kg of food for the 10 mg/kg/day treatment. Pellets are then reconstituted. Control groups receive food pellets without test compound or OA. Due to the consistency of the high fat diet, no water is added in the mix with OA. In the case of the chow diet, which is harder to reconstitute, a minimal amount of water is added to the powder to reconstitute pellets, which are then air-dried. New batches of food are prepared weekly.
• Blood collection Blood is collected either from the retro-orbital sinus under anesthesia or from the tail vein.
• animals are anesthesized with a mixture of ketamine (200 mg/kg) / Xylasine (10 mg/kg) administred by intra-peritoneal injection.
• animals are anesthesized by inhalation of an isoflurane-0 2 mixture.
• Serum triglycerides, total and HDL cholesterol are determined by enzymatic assays. Serum HDL cholesterol content are determined after precipitation of apo B-containing lipoproteins with phosphotungstic acid/Mg (e.g., Roche Diagnostics, Mannheim, Germany). Free fatty acids level are determined with a kit from Wako (e.g., Neuss, Germany) as specified by the provider.
• Wako e.g., Neuss, Germany
• Blood glucose concentration is measured by a Precision Q. I. D analyzer (e.g., Medisense system), using Medisense Precis electrodes (e.g., Abbot Laboratories, Medisense products, Bedford, USA). This method is validated, by comparing Precision Q.I. D analyzer values with classical glucose measurements. The Precision Q. I. D method was chosen since it requires a minimal amount of blood and can hence be employed for multiple measurements such as during an IPGTT. Plasma insulin (e.g., Mercodia, Uppsala, Sweden) is determined by ELISA according to the manufacturer's specifications.
• Plasma insulin e.g., Mercodia, Uppsala, Sweden
• Lipoprotein profiles are obtained by fast protein liquid chromatography, allowing separation of the three major lipoprotein classes VLDL, LDL, and HDL.
• Intraperitoneal glucose tolerance test IPGTT
• IPGTT Intraperitoneal glucose tolerance test
• mice which are fasted overnight (12 h).
• Mice are 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 is 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 is calculated as a measure of insulin sensitivity, whereas the corresponding insulin levels indicate insulin secretory reserves.
• Energy expenditure is 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 are allowed free access to food and water.
• a very precise CO 2 and O 2 sensor measures the difference in O 2 and CO 2 concentrations in both air volumes, which gives the amount of oxygen consumed in a period of time given that the air flow of air coming in the cage is constant.
• the data coming out of the apparatus is processed in a connected computer, analyzed, and shown in an exportable Excel file. The values are expressed as ml.kg ⁇ .h "1 , which is commonly known as the VO 2 . Determination of body fat content by Dexa scanning
• the Dexa analyses are performed by the ultra high resolution PIXIMUS Series Densitometer (0.18 x 0.18 mm pixels, GE Medical Systems, Madison, WI, USA). Bone mineral density (BMD in g/cm 2 ) and body composition are determined by using the PIXIMUS software (version 1.4x, 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 are 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 is 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 requires training of the animals for one week in the system.
• E- Circadian activity Spontaneous locomotor activity is 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 are linked to a computer using an electronic interface (e.g., Imetronic, Pessac, France). Mice are tested for 32 hours in order to measure habituation to the apparatus as well as nocturnal and diurnal activities. The quantity of water consumed is measured during the test period using an automated lickometer.
• an electronic interface e.g., Imetronic, Pessac, France.
• the water solubility was measured for the insoluble protonated species of carboxylated bile acids at a pH 1.
• the water solubility of compound 10 was 120 ⁇ M (see Table 2).
• 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.
• LogP A 1-octanol-water partition coefficient of the studied bile acids as ionized species.
• CMC Critical Micellar Concentration
• 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.6).
• Table 2 shows the results.
• Compound 10 presented a high CMC with a low surface tension lowering capacity which indicated that this compound is a moderate detergent and the micelles have a very low aggregation number.
• the presence of a hydroxyl group in the C- 16 beta position reduces the hydrophobic area responsible for back to back interaction, which is to form micelles and therefore, the micelles are small and poorly detergent. This property confers to the molecule a low toxicity when accumulated in a given biological fluid or organ.
• the octanol/water partition coefficient was measured for molecules in their ionized form and therefore the carboxy analogues were studied at a relatively high pH (8 to 9) to ensure full ionization of the carboxyl group.
• the 1 -Octanol/water partition coefficient (log P) was evaluated using a conventional shake-flask procedure. The experiments were carried out on 0.1 mM bile salt solution buffered at pH 8 with 0.1 M phosphate buffer to ensure complete ionization of the BA; the log P values refer to the BA in the ionized form, not to the protonated species, and the initial concentration of each BA was below its own CMC value.
• 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-
• the extent of albumin binding was evaluated by equilibrium dialysis at a fixed BA- albumin ratio. Each 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 12,000-14,000 Dalton against 25 ml of saline solution. The system was equilibrated by 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 percentage of albumin binding was calculated from the initial BA concentration and from the unbound concentration in the dialyzed fraction. Data are reported in the Table 2.
• the percent binding of compound 10 is higher than CA as a result of the methyl group in the side chain.
• Compound 10 presents an albumin binding compatible with a relatively fast hepatic uptake, similar to natural occurring BA.
• EXAMPLE 5 In vitro metabolic stability in human stools cuture Stability to Intestinal Bacteria. 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 6 Biliary secretion and metabolism of compound 10 in bile-fistula rat after duodenal (id) and femoral (iv) administration
• AUC area under the curve
• the bile fistula rat model was developed at the University of Bologna Lab facilities.
• the compounds were administered at a dose of 1 ⁇ mol/kg/min (1 hour infusion) to a rat group via duodenal infusion (id).
• the rats have a bile fistula to collect bile samples at different times before and during the infusion.
• 6 rats 250 ⁇ 10 g were treated. Bile samples were collected every 15 minutes for four hours.
• 3 control rats were treated with saline solution under the same conditions for times and sampling (duodenal control rats).
• Figure 7 shows bile flow during sample collection (one animal). Duodenal infusion starts after 30 min baseline bile collection and continues for one hour. Compound 10 is not choleretic and the bile flow is similar to control group.
• Final solution was transferred in autosampler vials, and 10 ⁇ L was injected into the chromatographic column.
• Bile rat samples were analyzed by liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) using electrospray (ESI) source in negative ionization mode.
• HPLC-MS/MS liquid chromatography-tandem mass spectrometry
• ESI electrospray
• Flow rate was 150 ⁇ L/min and the column was maintained at 45°C.
• the column effluent was introduced into ESI source connected to a triple quadruple mass spectrometer (Quattro-LC, Micromass) operating in Multiple Reaction Monitoring (MRM) acquisition mode.
• Nitrogen was used as nebulizer gas at 90 L/h flow rate and as desolvation gas at 930 L/h.
• Ion source block and desolvation temperatures were set respectively to 80 0 C and 180 0 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. Quantification
• the data refer to the secretion rate of the analogues recovered in bile as such after duodenal and femoral infusion at a dose of lumol/Kg/min. Major and minor metabolites are reported later.
• Table 3 shows concentration and secretion values for compound 10 obtained from bile rat samples collected during the duodenal infusion (I h ranging from 75 to 135 min). Table 3.
• Table 4 shows concentration and secretion values for compound 10 obtained from bile rat samples collected during the femoral infusion (1 h ranging from 75 to 135 min).
• Table 4 shows concentration and secretion values for compound 10 obtained from bile rat samples collected during the femoral infusion (1 h ranging from 75 to 135 min).
• Table 4. a not calculated n.d.: not detected
• the biliary secretion of compound 10 after iv infusion is not efficient and the maximum secretion rate inlow (Fig. 9).
• the compound is metabolized to form the taurine conjugate and this contributes to slightly improve its recovery.
• the biliary secretion after id administration is much less than the iv experiments suggesting a poor intestinal absorption of the molecule.
• Compound 10 undergoes to an hepatic metabolism like natural occurring BA. After iv administration is secreted in bile as such and mainly conjugated with taurine Minor metabolites such as lactone and a monoglucuronide metabolites has been also found. After id administration the molecule is recovered as such and mainly metabolised to form the taurine conjugates.
• Figure 10a Compound 10 and its main metabolites identified in bile using mass spectrometry in the iv experiment. Data are reported as absolute area values.
• Figure 10b Zoom display of Figure 10a.
• Figure 10c Compound 10 and its main metabolites identified in bile using mass spectrometry in the di experiment. Data are reported as absolute area values.
• Compound 10 is moderately hydrophilic with a low detergency.
• the hepatic uptake is efficient and also the intestinal absorption .
• the compound is secreted in bile as such and mainly as taurine conjugate and the recovery in bile almost complete.
• 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 5. 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.
• Human NCI-H716 or HepG2 cells (1x10 4 ) were seeded in 96 wells plate and stimulated with 10-fold dilutions from 1 nM to 300 ⁇ M of the compound 10 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 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 En Vision.
• the cellular necrosis was analyzed by measuring the release of Lactate DeHydroxegenase (LDH) from the necrotic cells using Promega's CytoTox ONE Homogeneous Membrane Integrity Assay.
• LDH Lactate DeHydroxegenase
• Human hepatocyte 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.
• 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 6 shows the results of these assays. TR-FRET Coactivator Assay
• 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
• 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 10 in the assay buffer supplied by Invitrogen.
• the negative control was devoid of the compound 10 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 EC50 values.
• FXR and LXR Coactivator Recruitment(Alphascreen); TGR-5: cAMP level on human intestinal cell line (NCI-H716); PXR: Ligands Competition assay (Binding Assay); *: data representing inverse agonism

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