WO2015031710A1 - Compositions and methods for the treatment of metabolic and body weight related disorders - Google Patents

Compositions and methods for the treatment of metabolic and body weight related disorders Download PDF

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Publication number
WO2015031710A1
WO2015031710A1 PCT/US2014/053334 US2014053334W WO2015031710A1 WO 2015031710 A1 WO2015031710 A1 WO 2015031710A1 US 2014053334 W US2014053334 W US 2014053334W WO 2015031710 A1 WO2015031710 A1 WO 2015031710A1
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WIPO (PCT)
Prior art keywords
compound
fatostatin
alkyl
thiazol
srebp
Prior art date
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PCT/US2014/053334
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English (en)
French (fr)
Inventor
Motonari Uesugi
Salih J. Wakil
Lutfi Abu-Elheiga
Mizuki WATANABE
Original Assignee
Baylor College Of Medicine
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Publication date
Priority claimed from US14/013,918 external-priority patent/US9085566B2/en
Priority claimed from US14/052,074 external-priority patent/US9187485B2/en
Priority claimed from US14/270,130 external-priority patent/US9233941B2/en
Application filed by Baylor College Of Medicine filed Critical Baylor College Of Medicine
Priority to CA2922703A priority Critical patent/CA2922703A1/en
Priority to CN201480058587.XA priority patent/CN107074839A/zh
Priority to EP14841043.4A priority patent/EP3039022A4/en
Priority to JP2016537878A priority patent/JP2016534124A/ja
Priority to AU2014312227A priority patent/AU2014312227A1/en
Publication of WO2015031710A1 publication Critical patent/WO2015031710A1/en

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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/06—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/76—Nitrogen atoms to which a second hetero atom is attached
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    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/06—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
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    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C07D235/14—Radicals substituted by nitrogen atoms
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    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/50—Nitrogen atoms bound to hetero atoms
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    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
    • C07D277/66—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2 with aromatic rings or ring systems directly attached in position 2
    • C—CHEMISTRY; METALLURGY
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    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
    • C07D295/182—Radicals derived from carboxylic acids
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    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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    • C07D487/04—Ortho-condensed systems

Definitions

  • the present invention utilized federal funding from the National Institutes of Health Grant GM-631 15 and Department of Defense Grant No. DAMD17-03-1 -0228. The United States Government has certain rights in the invention.
  • the present invention generally relates to the fields of medicine and molecular biology of metabolic disorders.
  • the field of the invention relates to particular compositions for the treatment of disorders, such as obesity partly due to upregulation of thermogenesis.
  • the compositions comprise fatostatin A and its analogs or derivatives.
  • Metabolic syndrome covers many cardiovascular risk factors including hypertension, dyslipidaemia, obesity, type 2 diabetes, pancreatic ⁇ — cell dysfunction, and atherosclerosis.
  • a diet varying in fat or carbohydrate contents contributes to energy metabolism of animals including humans.
  • Long chain fatty acids are major source of energy and important components of the lipids that comprise the cellular membranes. They are derived from food and synthesized de novo from acetyl-CoA. Cholesterol is also derived from food and synthesized from acetyl-CoA.
  • the conversion of carbohydrates into acylglycerides through de novo fatty acid and cholesterol synthesis involves at least 12 and 23 enzymatic reactions, respectively.
  • SREBPs sterol regulatory element-binding proteins
  • SREBP-1a SREBP-1a
  • -1 c SREBP-2
  • SREBPs membrane-bound proteins
  • SREBP-1a SREBP-1a
  • -1 c SREBP-2
  • SREBPs SREBP-1a
  • -1 c SREBP-2
  • SREBPs membrane-bound proteins
  • SREBPs are synthesized as an ER-membrane-bound precursor, which needs to be proteolytically released by two proteases bound to the Golgi membrane, Site-1 and Site-2 proteases, in order to activate transcription of target genes in the nucleus.
  • SREBP SREBP cleavage-activating protein
  • SCAP SREBP cleavage-activating protein
  • ER-membrane-bound escort protein of SREBPs an ER-membrane-bound escort protein of SREBPs.
  • the present invention teaches compounds, formulations and pharmaceutical compositions thereof and uses thereof for treating a metabolic disorder, such as, but not limited to, a disease related to cell hyperproliferation, e.g., a cancer, or a weight-related condition.
  • a metabolic disorder such as, but not limited to, a disease related to cell hyperproliferation, e.g., a cancer, or a weight-related condition.
  • the present invention is directed to a compound having the general structure:
  • the A ring is a pyridine or a substituted pyridine, a piperidine or a substituted piperidine, a pyrrolidine or a substituted pyrrolidine, a thiazole or a substituted thiazole, a phenyl ring or a substituted phenyl ring.
  • the B ring is a thioazole or a substituted thioazole, a piperazine or a substituted piperazine, a phenyl ring or a substituted phenyl ring.
  • the C ring is a phenyl ring or a substituted phenyl ring, a pyridine or a substituted pyridine, a thioazole or a substituted thioazole.
  • the present invention also is directed to a compound having the chemical structure
  • the Ri substituents are H , halogen, -OH, -0-Ci- 3 alkoxy, -OC(0)R 3 ;
  • R 3 is C 1 -C 3 alkyl or aryl, -OCH 2 C(0)OR 4 ;
  • R 4 is H or C 1 -C 3 alkyl, -NHR 5 ;
  • R 5 is H , C C 4 alkyl, alkylcyclopropane, benzyl, -NHC(0)d C 3 amide, -NHC(0)0-R 6 carbamate;
  • R 6 is ferf-butyl or benzyl, -NH-SO 2 -R7 sulfonamide and R 7 i: alkyl or aryl.
  • the R 2 substituents may be alkyl or R 8 OC(0)- and R 8 is C3-C5 alkyl or aryl.
  • the present invention is directed further still to a compound having the chemical structure
  • the Ri substituents are H , halogen, -OH, -0-C r3 alkoxy, -OC(0)R 3 ;
  • R 3 is C C 3 alkyl or aryl, -OCH 2 C(0)OR 4 ;
  • R 4 is H or C 1 -C 3 alkyl, -NHR 5 ;
  • R 5 is H , C 1 -C4 alkyl, alkylcyclopropane, benzyl, -NHC(0)Ci C 3 amide, -NHC(0)0-R e carbamate;
  • R 6 is ferf-butyl or benzyl, -NH-SO 2 -R7 sulfonamide and R 7 ii alkyl or aryl.
  • the R 2 substituents may be alkyl or R 8 OC(0)- and R 8 is C 3 -C 5 alkyl or aryl.
  • the present invention is directed further still to a compound having the chemical structure
  • the R 9 is H , halogen, -OH , -O-C1-C3 alkoxy, -OC(0)Rn ;
  • Rn is C1-C3 alkyl or aryl, -OCH 2 -C(0)ORi 2 ;
  • R-12 is H or C1-C3 alkyl, -N H R 13 ;
  • R 13 is H , C1-C4 alkyl, alkylcyclopropane, benzyl, -NHC(0)Cr3amide, -N HC(0)0-R 14 carbamate;
  • R 14 is ferf-butyl or benzyl, -N H-SO2-R15 sulfonamide;
  • R 15 is alkyl or aryl or -SO2-N H-R sulfonamide and
  • R 16 is alkyl or aryl.
  • the R 10 is nitrogen or methylene, n is 0 or 1 and when n is 1 , Z is -C
  • the present invention is directed to the compounds described herein formulated as a pharmaceutical composition in a pharmaceutically acceptable excipient or as a formulation comprising a food, an animal feed material or a medicine.
  • the present invention is also directed to a kit comprising the compounds or combination thereof as described herein or a pharmaceutical composition thereof or other formulation thereof and a container housing the compound.
  • the present invention is directed further still to a compound that is /V-(4-(2-(2-propylpyridin- 4-yl)thiazol-4-yl)phenyl)methanesulfonamide, ferf-butyl-2-(4-(4-bromophenyl)thiazol-2-yl) pyrrolidine- 1 -carboxylate, benzyl 2-(4-(4-bromophenyl)thiazol-2-yl)pyrrolidine-1 -carboxylate, 4-(4-bromophenyl) -2-(pyrrolidin-2-yl)thiazole, 4-(4-bromophenyl)-2-(1 -propylpyrrolidin-2-yl)thiazole, iert-butyl 3-(4-(4- bromophenyl)thiazol-2-yl)piperidine-1 -carboxylate, benzyl 3-(4-(4-bromophenyl)
  • the present invention is directed further still to a method for treating a metabolic disorder in an animal, comprising the step of administering to the individual a therapeutically effective amount of at least one compound having the chemical structure A-B-C or as specifically described herein or a pharmaceutically acceptable salt or a stereoisomer thereof or a combination thereof.
  • the metabolic disorder is a cancer or a weight-related disorder.
  • the present invention is directed to a related method further comprising the step of providing a second therapy.
  • the second therapy comprises dietary therapy, physical therapy, behavior therapy, surgery, drug therapy, and a combination thereof.
  • the metabolic disorder is diabetes and the additional therapy comprises dietary therapy, physical therapy, and drug therapy.
  • the present invention is directed further still to a method for treating a cell hyperproliferative disease in a patient in need thereof.
  • the method comprises the step of administering to the patient a therapeutically effective amount of at least one compound described herein, or a pharmaceutically acceptable salt or a stereoisomer thereof or a combination thereof.
  • the present invention is directed further still to a related method for reducing body weight in an animal in need thereof.
  • the method comprises administering to the patient a therapeutically effective amount of at least one compound described herein, or a pharmaceutically acceptable salt or a stereoisomer thereof or a combination thereof.
  • the present invention is directed further still to a method for reducing body weight in an animal in need thereof.
  • the method comprises administering to the animal, in a pharmaceutically acceptable medium, a therapeutically effective amount of a compound having the structure: A-B-C or as specifically described herein or a pharmaceutically acceptable salt or a stereoisomer thereof or a combination thereof.
  • the A ring may be a pyridine or a substituted pyridine, a piperidine or a substituted piperidine, a pyrrolidine or a substituted pyrrolidine, a thiazole or a substituted thiazole, a phenyl ring or a substituted phenyl ring.
  • the B ring may be a thioazole or a substituted thioazole, a piperazine or a substituted piperazine, a phenyl ring or a substituted phenyl ring.
  • the C ring may be a phenyl ring or a substituted phenyl ring , a pyridine or a substituted pyridine, a thioazole or a substituted thioazole.
  • the present invention also is directed to a method for increasing thermogenesis without reducing lean body mass during weight loss in an animal , comprising the step of administering to the animal, in a pharmaceutically acceptable medium, a therapeutically effective amount of a compound having the structure:
  • the Ri substituents may be H , Et, O e or n-propyl; Y is CH or .
  • the R 2 substituents may be OH , OMe, or NH-i-Pr.
  • the R 3 substituents may be H, F, or CI.
  • the R 4 substituents are H , Me, CI, Br, F, OH, OBz, OCH 2 COOMe, OCH 2 COO NHS0 2 Me, NHBn,
  • the compound may be a pharmaceutically acceptable salt or a stereoisomer thereof or a combination thereof.
  • FIGS. 1A-1 B show confirmation of the microarray results by RT-PCR.
  • DU145 cells were treated with DMSO (lane 1 ) and 5 ⁇ fatostatin A (lane 2) for 6 hrs (FIG. 1A).
  • Total RNA was then extracted and subjected to RT-PCR.
  • FIG. 1 B A summary of the RT-PCR and microarray data.
  • ACL ATP citrate lyase
  • HMG CoAR 3-hydroxy-3-methyl-glutaryl-CoA reductase
  • LDLR low-density lipoprotein receptor
  • MVD mevalonate pyrophosphate decarboxylase
  • SCD stearyl-CoA desaturase
  • INSIG1 insulin-induced gene 1
  • GAPDH glyceraldehyde-3-phosphate dehydrogenase.
  • FIGS. 2A-2C show that fatostatin A suppresses the ability of endogenous SREBPs to activate a reporter gene.
  • HEK293 cells were co-transfected with an SRE-1-driven luciferase reporter (pSRE-Luc) (FIG. 2A) and a ⁇ -gal reporter controlled under an actin promoter (FIG. 2B).
  • the transfected cells were treated by varied concentrations of fatostatin A or DMSO alone in a medium containing lipid-free serum. After 20-hr incubation, luciferase activity was measured, and the data were normalized by ⁇ -galactosidase activity.
  • pSRE-Luc SRE-1-driven luciferase reporter
  • FIG. 2B actin promoter
  • 2C HEK293 cells were transfected with pCMV- SREBP-1 c (1-436) and pSRE-Luc, and the transfected cells were treated with or without 20 mM fatostatin A in a medium containing lipid-free serum. Each value represents the average of three independent experiments.
  • FIGS. 3A-3H show effects of fatostatin A on SREBP-1 and -2.
  • DU145 cells were treated with DMSO alone or fatostatin A (1 and 5 ⁇ ) for 6 hrs.
  • the levels of precursor and mature forms of SREBP-1 (FIG. 3A) or SREBP-2 (FIG. 3B) were examined by western blots. Western blots of actin are shown in lower panel as a loading control.
  • FIGS. 3C-3H Localization of SREBP-1 was examined by immunostaining.
  • Cells were treated with DMSO alone (FIGS. 3C-3E) or 5 ⁇ of fatostatin A (FIGS. 3F-3H) and then stained with DAPI (FIG. 3C and FIG. 3F) or anti-SREBP-1 (FIG. 3D and FIG. 3G).
  • FIGS. 4A-4G show inhibition of the insulin-induced adipogenesis by siRNA knockdown of the SREBP-1.
  • Two stably transfected clones of 3T3-L1 cells in which the expression of SREBP-1 were knocked down were established and induced to differentiate into adipocytes. The knockdown cells were not differentiated (FIG. 4D and FIG. 4F), whereas 3T3-L1 cells transfected with an empty vector (neo) were mostly differentiated into adipocytes (FIG. 4B).
  • FIGS. 4A, 4C and 4E show the cells without the insulin induction.
  • FIG. 4G is a Western blot analysis of the clones indicating the successful knockdown of SREBP-1.
  • FIGS. 5A-5B demonstrate siRNA knockdown of SREBP-1 blocks the serum-independent growth of DU145 prostate cancer cell.
  • FIG. 5A two stably transfected clones of DU145 cells in which the expression of SREBP-1 were knocked down were established and grown in an MEM medium containing no serum, 2% fetal bovine serum (FBS), 2% fat-free fetal bovine serum, or 1 g mL of IGF1 for three days. The growth rates were measured by WST-1 assays. The knockdown cells failed to grow in the MEM medium containing no serum, 2% fat-free FBS, or I g/mL of IGF1 but exhibited as much growth as control cells in the presence of serum. The experiments were performed in triplicate.
  • FIG. 5B are Western blots showing the extents of SREBP-1 knockdown in clones 1 and 2.
  • FIGS. 6A-6G demonstrate effects of fatostatin A on mice after fasting/refeeding fat free diet. Mice were injected with 30 mg/kg of fatostatin A intraperitoneally daily for the entire experiments starting one day before fasting for 48 hrs followed by feeding fat free diet for another 48 hrs. Loss of body weight (FIG. 6A) and food intake (FIG. 6B) were determined at the end of the 48-hr feeding.
  • FIG. 6C shows serum constituents of the treated and control mice.
  • FIG. 6D is a representative western blot for SREBP-1 of the liver extracts from 2 different mice from the control and fatostatin A treated groups. The loaded amounts of proteins were normalized.
  • FIG. 6A-6G demonstrate effects of fatostatin A on mice after fasting/refeeding fat free diet. Mice were injected with 30 mg/kg of fatostatin A intraperitoneally daily for the entire experiments starting one day before fasting for 48 hrs followed by feeding fat free diet for another 48 hrs. Loss
  • FIGS. 6E-6G is a representative western blot showing FAS expression (top panel) and a coomassie stained gel for loading control (bottom) of liver extracts.
  • FIGS. 7A-7E show effects of two-week treatment of fatostatin A on mice. 5-6 month old mice were injected daily for two weeks either with 30 mg/kg fatostatin A or 10% DMSO.
  • FIG. 7A shows body weight before and after the treatment with fatostatin A and
  • FIG. 7B shows the amounts of weight loss after treatment.
  • FIG. 7C show the serum levels of glucose, cholesterol, and triglycerides (TG) in the treated and control mice.
  • FIG. 7D shows FAS activity in liver extracts.
  • FIGS. 8A-8C show effect of fatostatin A on body weight and food consumption.
  • FIG. 8A is a picture of representative control and fatostatin A treated mice.
  • FIG. 8B shows the weight of each mouse within each group was measured daily. The average and variance of the weights are shown.
  • food intake was measured every day and was expressed as cumulative food intake per mouse over the 28 days period.
  • FIGS. 9A-9H demonstrate serum constituents of control and fatostatin A treated ob/ob mice. Blood was collected from tail veins of overnight fasted mice, and serum was collected after separation from cells. Constituents were determined as described below. The data are shown as mean ⁇ SD, n - 5 mice in each group.
  • FIGS. 10A-10D show effect of fatostatin on liver and adipose tissue of ob/ob mice.
  • FIG. 10A shows livers of fatostatin A treated mice (left) and controls (right).
  • FIG. 10B shows histological analyses of frozen sections of livers of the control and fatostatin A ob/ob mice stained with Oil Red- O to detect lipid droplets and counter-stained with Mayer's hematoxylin. Livers of three different mice treated with fatostatin A, showing a dramatic decrease in red-stained droplets (top) and controls showing an abundance of red-stained lipid droplets compared to the treated mice (bottom).
  • FIG. 10A shows livers of fatostatin A treated mice (left) and controls (right).
  • FIG. 10B shows histological analyses of frozen sections of livers of the control and fatostatin A ob/ob mice stained with Oil Red- O to detect lipid droplets and counter-stained with Mayer's he
  • FIG. 10C shows epididymal fat pads isolated from fatostatin A treated ob/ob mice (left) and controls (right).
  • FIGS. 12A-12D demonstrate that fatostatin A reduces the expression levels and activities of lipogenic enzymes.
  • FIG. 12A shows the activity of acetyl-CoA carboxylase (ACC) and
  • FIG.12B shows fatty acid synthase measured in liver extracts of ob/ob mice as described below.
  • FIG. 12C is a Western Blot analysis of liver crude extracts, from three individual ob/ob mice, were separated by 4-12% NuPAGE MES gels and probed with different antibodies and detected with ECL.
  • FIGS. 14A-14F illustrates exemplary compounds 1 -66 of the present invention.
  • FIG. 15 demonstrates a standard luciferase reporter gene assay with exemplary analogues
  • FIG. 16 shows a standard luciferase reporter gene assay with exemplary analogues 19-34.
  • FIG. 17 provides a standard luciferase reporter gene assay with exemplary analogues 35-
  • FIGS. 18A-18D show that fatostatin blocks the activation of SREBP. Suppression by fatostatin of the ability of endogenous SREBPs to activate a luciferase reporter gene in a medium containing lipid-free serum.
  • CHO-K1 cells were transfected with an SRE-1 -driven luciferase reporter (pSRE-Luc) (FIG. 18A). The transfected cells were treated by varied concentrations of fatostatin in a medium containing lipid-free serum. Effect of fatostatin on CHO-K1 cells co-transfected with pCMV- SREBP-1 c(1-436) and pSRE-Luc in a medium containing lipid-free serum (FIG. 18B).
  • PLAP-BP2 in transfected CHO-K1 cells remains membrane-bound unless it is cleaved by S1 P in the Golgi and secreted into the culture medium (left).
  • Treatment with fatostatin (20 ⁇ ) or sterols (10 ⁇ g/mL cholesterol and 1 ⁇ g/mL 25-hydroxycholesterol) affect cleavage of PLAP-BP2 compared to EtOH controls (FIG. 18C).
  • P and N denote the uncleaved membrane precursor and cleaved nuclear forms of SREBP-2, respectively (FIG.
  • FIGS. 19A-19B show that fatostatin blocks the translocation of SREBPs from the ER to the Golgi.
  • FIG. 19A is a Western blot analysis showing effects of brefeldin A on CHO-K1 cells treated with EtOH alone, sterols (10 g/ml cholesterol and 1 ⁇ g ml 25-hydroxycholesterol), or 20 ⁇ fatostatin.
  • FIG. 19B is a Western blot analysis with anti-SCAP lgG-9D5 of cells grown in the absence or presence of 20 ⁇ fatostatin or sterols (10 ⁇ g mL cholesterol and 1 ⁇ g/mL 25- hydroxycholesterol). Numbers on the right denote the number of N-linked sugar chains present on protease-protected SCAP fragments.
  • FIGS. 20A-20D show the structures of dansyl fatostatin, fatostatin-polyproline linker-biotin conjugate and polyproline linker-biotin conjugate and cells treated with the same.
  • FIG. 20A illustrates how the polyproline linker was inserted for better projection of the fatostatin molecule (Sato ei a/., 2007).
  • FIG. 20C illustrates the interaction of fatostatin with SCAP, shown by western blot analysis with anti-SCAP, anti-SREBP-1 , anti-SREBP-2, and anti-ATF6 antibodies of proteins bound to Neutravidine-agarose beads saturated with biotinylated fatostatin in CHO-K1 membrane extract.
  • FIG. 20D shows that for the competition assay, membrane extracts were preincubated with EtOH alone, cholesterol, or fatostatin.
  • FIGS. 21 show effects of fatostatin on liver and adipose tissue of ob/ob mice. Sections of the livers of fatostatin-treated and control mice showing red-stained lipid droplets.
  • FIG. 22 demonstrates a western blot analysis of CHO-K1 cells treated with fatostatin.
  • P and N denote the uncleaved membrane precursor and cleaved nuclear forms of SREBP-1 , respectively.
  • FIG. 23 provides an exemplary synthetic scheme of fatostatin, dansyl fatostatin and fatostatin-polyproline linker-biotin.
  • FIG. 24 shows suppression by fatostatin analogues of the ability of endogenous SREBPs to activate a luciferase reporter gene in a medium containing lipid-free serum.
  • CHO-K1 cells were transfected with pSRE-Luc. The transfected cells were treated by varied concentrations of fatostatin, dansyl fatostatin or isopropylamine derivative in a medium containing lipid-free serum.
  • FIGS. 25A-25D show that fatostatin reduces the expression levels and activities of lipogenic enzymes.
  • Activity of acetyl-CoA carboxylase (ACC) (FIG. 25A) and fatty acid synthase (FAS) (FIG. 25B) were measured in liver extracts of ob/ob mice.
  • FIG. 26 provides a synthetic scheme of compound 53.
  • FIG. 27 provides a synthetic scheme of compound 19 and compound 17.
  • FIG. 28 shows a standard SREBP activation assay with exemplary analogues 45-55 and
  • FIG. 29 shows a standard SREBP activation assay with exemplary analogues 56-61 and 19.
  • FIG. 30 shows a standard SREBP activation assay with exemplary analogues 62-66.
  • FIG. 31 shows an Inhibitory Concentration (sub 50) data of exemplary compound 53.
  • FIG. 32 shows an Inhibitory Concentration (sub 50) data of exemplary compounds 58 and
  • FIGS. 33A-33B show that compound 19 inhibited the growth of breast cancer cell line SUM 159.
  • Cells were seeded onto 96 well plates at a density of 10000 cells/well in 10Oul medium containing 2% charcoal stripped serum. After 24 hours, compound 19 was added to the cells at the indicated concentrations for another 48 hours. Cell viability was determined using WST-1 assay.
  • FIG. 33A shows the effect of different concentrations of compound 19 on cell growth as evident from the changes in the absorbance at A450nm.
  • FIG. 33B Expression levels of lipogenic genes were significantly downregulated by 10 ⁇ treatment of HePG2 cells as determined by RT PCR analysis (black bar); values are depicted as means + SD; *P ⁇ 0.05
  • FIGS. 34A-34C show that compound 19 inhibited the growth of human liver cancer cell line HePG2.
  • Cells were seeded onto 16 well plates at a density of 100000 cells/well in medium containing 2% charcoal stripped serum. After 24 hours, compound 19 was added to the cells at the indicated concentrations for another48 hours.
  • FIG. 34A shows photographs of control and treated HePG2 cells with 25, 50 and 100 ⁇ compound 19.
  • FIG. 34B shows a representative Western blot analysis showing that the treated cells (T) had a decreased level of the mature and active form of SREBP-1 and higher levels of the precursor compared with the untreated controls.
  • 34C shows expression levels of lipogenic genes were significantly downregulated by 10 ⁇ treatment of HePG2 cells and did not affect INSIG2 which is not known to be regulated by SREBP, as determined by RT PCR analysis. Values are depicted as means + SD; *P ⁇ 0.05.
  • FIGS. 35A-35F show that compound 19 inhibited the growth of human acute lymphoblastic leukemia cell line MOLT-4 and human multiple myeloma cell line RP I8226.
  • 10,000 OLT-4 cells (FIGS. 35A-35C) and 20,000 RP I8226 cells (FIGS. 35D-35F) were seeded onto 96 well plate in RPMI 1640 medium containing 5% FBS or fat-free charcoal treated serum (FF-FBS) for 24 hours at 37 C.
  • FF-FBS fat-free charcoal treated serum
  • MOLT-4 and RPMI8226 cells were treated for additional 48 hours with different concentrations of compound 19 (None control without DMSO, Vehicle with DMSO, 1 , 2, 5, 10 and 20 ⁇ in DMSO; RPMI8226 cells also were treated with 3 ⁇ in DMSO). At the end of 48 hours cells were subjected to MTT assay to determine viability. Values are depicted as means + SD; *P ⁇ 0.05.
  • FIGS. 36A-36F show that compound 17 inhibited the growth of human acute lymphoblastic leukemia cell line MOLT-4 and human multiple myeloma cell line RPMI8226.
  • Figures 37A-37D Body weight and composition (fat and lean) in SD rats after three weeks of feeding RD and HFHC Diets. Male SD rats 6-7 weeks old with initial body weight of about 193 + 7.0 gram/rat were fed regular diet (RD) or high fat high carbohydrate diets for three weeks. Body weight ( Figure 37B) fat weight ( Figure 37C) and lean weight (Figure 37D) content was measured using ECHO MRI method 15 . (*P ⁇ 0.05)
  • Figures 38A-38B show cumulative body weight gain (Figure 38A) and food intake (Figure 38B) after eight weeks of treatment with compound 19.
  • SD rats were fed HFHC diet for three weeks before the start of treatment with different doses of compound 19 via oral gavage or cottonseed oil vehicle for an additional eight weeks.
  • Body weight and food intake was measured and cumulative values were calculated. (Values are means+ S.E.M; *P ⁇ 0.05; treated compared to control vehicle group.
  • Figure 39A-39E show body composition fat and lean mass determine by ECHO MRI method.
  • Figure 39A shows total body weight in grams after administration of compound 19.
  • Figure 39B shows total body fat in grams after administration of compound 19.
  • Figure 39C shows the percentage of fat per animal after administration of compound 19.
  • Figure 39D shows total lean weight in gram after administration of compound 19.
  • Figure 39E shows percent lean weight per gram after administration of compound 19. Rats from different experimental groups were subjected to body composition analysis every two weeks. Percent of fat and lean mass was calculated from the weights of fat and mass relative to body weight.
  • the labels in Figure 39B-39E correspond to the same groups as in Figure 39A. Asterisks indicate significant differences between treated and control groups ( * P ⁇ 0.05). Data are means + S.E.M
  • Figures 40A-40D shows the effect of compound 19 on livers of SD rats fed HFHC.
  • Figure 40B Oil Red O staining of frozen sections of livers. Oil droplets are shown in red . Bars are indicating 200 micron.
  • Figure 40C levels of TG and cholesterol in liver tissues from control and treated animals;
  • Figure 40D Fold changes of gene expression controlled by SREBP-1 and 2 in treated rats compared to controls measured by real time PCR.
  • ACC acetyl-CoA carboxylase
  • ACL ATP citrate-lyase
  • SCD steryl-CoA desaturase MVD Mevalonate decarboxylase
  • LDLR LDL receptor
  • INSIG-1 insulin induced gene 1 ; Means are + SE; *P ⁇ 0.05.
  • Figure 41 shows fold changes in gene expression of UCP2 in treated rats with compound 19 compared to controls.
  • Total RNA was extracted from liver tissues of control and compound 19 treated rats with the indicated doses and level of UCP mRNA was determined using real-time PCR.
  • Metabolic disorders are treated and/or prevented with compounds of the present invention.
  • dysregulated biosynthesis of fatty acids and cholesterols and excessive intake of dietary fat are correlated with a number of medical complications including at least obesity, diabetes, hypertension, and cardiovascular diseases, and in certain aspects these conditions are treated and/or prevented with a compound of the invention.
  • Epidemiological evidence indicates that metabolic diseases including obesity also promote the development of an aggressive form of cancers, including but not limited to prostate cancer.
  • sterol regulatory element binding proteins Upon fat depletion, sterol regulatory element binding proteins (SREBPs) are proteolytically released from the membrane and translocated into the nucleus, where they activate the transcription of the genes involved in cholesterol and fatty acid biosynthesis.
  • the present invention identifies a synthetic small molecule previously known to block both adipogenesis and cancer cell growth as a selective inhibitor of the SREBP activation and also provides analogs and derivatives of that molecule.
  • the drug-like molecule fatostatin A impairs the proteolytic activation of SREBPs, thereby reducing the transcription of their responsive genes in cells.
  • fatostatin A blocks the activation of SREBP-1 in the liver, reduces body weight, lowers the levels of blood cholesterol and glucose, and down-regulates lipogenic enzymes. Fatty acid synthase and acetyl-CoA carboxylase activities and their expression levels were decreased in the liver of the treated mice. Fatostatin A serves as a tool to understand cellular pathways and provides a consensus molecule as at least starting point for pharmacological intervention of metabolic diseases, in certain aspects.
  • Fatostatin A causes two distinct phenotypes in cultured mammalian cells: complete inhibition of the insulin-induced adipogenesis of 3T3-L1 mouse fibroblast cells; and selective repression of the serum-independent insulin-like growth factor 1 (IGF1 )-dependent growth of DU145 human prostate cancer cells.
  • IGF1 insulin-like growth factor 1
  • fatostatin A selectively blocks the activation of SREBPs, a key lipogenic transcription factor that activates specific genes involved in cholesterol and fatty acid synthesis.
  • fatostatin A as an inhibitor of SREBPs is consistent with its anti- adipogenic property, and indicates a role of SREBPs in the IGF1 -dependent growth of prostate cancer.
  • the present invention concerns fatostatin A, preferably analog and derivative compounds thereof, that blocks the activation of at least SREBP-1 , for example as shown in experimental mice.
  • Administration of fatostatin A into obese ob/ob mice led to weight loss and marked reduction of visceral fat.
  • expression of uncoupling protein 1 , uncoupling protein 2 and uncoupling protein 3 is increased and thermogenesis was increased without reducing lean body mass during weight loss
  • the present invention concerns treatment and/or prevention of at least one symptom of a metabolic disorder.
  • the metabolic disorder may be of any kind, so long as one of its symptoms is improved or prevented with a compound of the present invention.
  • the metabolic disease is from one or more inborn errors of metabolism (which may be referred to as genetic disorders), such as inherited traits that are due to a defective metabolic enzyme (for example one having one or more mutations or disorders that involve mutations in regulatory proteins and in transport mechanisms).
  • metabolic disorders may be defined as disorders that affect energy production in a cell. Although most metabolic disorders are genetic, some may be acquired as a result of one or more factors, including diet, toxins, infections, and so forth. Genetic metabolic disorders may be caused by genetic defects that result in missing or improperly constructed enzymes necessary for some step in the metabolic process of the cell.
  • the largest categories of metabolic disorders include the following: 1 ) glycogen storage diseases (also referred to as glycogenosis or dextrinosis), which include disorders that affect carbohydrate metabolism; 2) fatty oxidation disorders, which affect fat metabolism and metabolism of fat components; and 3) mitochondrial disorders, which affect mitochondria.
  • GSD glycogen storage diseases
  • GSD type I glucose-6-phosphatase deficiency; von Gierke's disease
  • GSD type II acid maltase deficiency; Pompe's disease
  • GSD type III glycogen debrancher deficiency; Cori's disease or Forbe's disease
  • GSD type IV glycogen branching enzyme deficiency; Andersen disease
  • GSD type V muscle glycogen phosphorylase deficiency; McArdle disease
  • GSD type VI liver phosphorylase deficiency, Hers's disease
  • GSD type VI I muscle phosphofructokinase deficiency; Tarui's disease
  • GSD type IX phosphorylase kinase deficiency
  • GSD type XI glucose transporter deficiency; Fanconi-Bickel disease
  • Fatty acid metabolism deficiencies may be described as fatty oxidation disorders or as lipid storage disorders, in certain embodiments. They may involve one or more inborn errors of metabolism that result from enzyme deficiencies that affect the body's ability to oxidize fatty acids for the production of energy within muscles, liver, and other cell types, for example.
  • fatty acid metabolism deficiencies include at least coenzyme A dehydrogenase deficiencies; other coenzyme A enzyme deficiencies; carnitine-related disorders; or lipid storage disorders.
  • coenzyme A dehydrogenase deficiencies include at least very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCAD); long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency (LCHAD); medium-chain acyl-coenzyme A dehydrogenase deficiency (MCAD); short- chain acyl-coenzyme A dehydrogenase deficiency (SCAD); and short chain L-3-hydroxyacyl-coA dehydrogenase deficiency (SCHAD).
  • VLCAD very long-chain acyl-coenzyme A dehydrogenase deficiency
  • LCHAD long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency
  • MCAD medium-chain acyl-coenzyme A dehydrogenase deficiency
  • SCAD short- chain acyl-coenzyme A dehydrogenase de
  • Examples of other coenzyme A enzyme deficiencies include at least 2,4 Dienoyl-CoA reductase deficiency; 3-hydroxy-3-methylglutaryl-CoA lyase deficiency; and malonyl-CoA decarboxylase deficiency.
  • Examples of carnitine-related deficiencies include at least primary carnitine deficiency; carnitine-acylcarnitine translocase deficiency; carnitine palmitoyltransferase I deficiency (CPT); and carnitine palmitoyltransferase II deficiency (CPT).
  • lipid storage diseases include acid lipase diseases; Wolman disease; cholesteryl ester storage disease; Gaucher disease; Niemann-Pick disease; Fabry disease; Farber's disease; gangliosidoses; Krabbe disease; and metachromatic leukodystrophy.
  • Other fatty acid metabolism disorders include at least mitochondrial trifunctional protein deficiency; electron transfer flavoprotein (ETF) dehydrogenase deficiency (GAM & MADD); Tangier disease; and acute fatty liver of pregnancy.
  • ETF electron transfer flavoprotein
  • mitochondrial diseases include at least progressive external ophthalmoplegia (PEO); Diabetes mellitus and deafness (DAD); Leber hereditary optic neuropathy (LHON) Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like syndrome (MELAS); Myoclonic epilepsy and ragged-red fibers (MERRF); Leigh syndrome; subacute sclerosing encephalopathy; Neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP); Kearns-Sayre syndrome (KSS); Myoneurogenic gastrointestinal encephalopathy (MNGIE).
  • the metabolic disorder is, or has as one of its complications, one or more of the following: obesity, hyperlipemia, diabetes, fatty liver, hypertension, and cardiovascular disease.
  • the present invention concerns treatment of a disease related to cell hyperproliferation.
  • cell hyperproliferation may be caused by a cancer or other neoplastic disease or disorder.
  • the hyperproliferative disease may be cancer of the breast, respiratory tract, brain, reproductive organs, prostate, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid, lymphoma, sarcoma, melanoma, leukemia, multiple myeloma, or a distant metastasis of a solid tumor.
  • the present invention provides compound, or pharmaceutically acceptable salts and stereoisomers thereof, having the general formula:
  • A, B, and C can be the same or different and each may be a 5-, 6-, or 7- membered ring or a fused bicyclic ring system, the ring being a heterocyclic ring or no n -heterocyclic ring, a substituted ring or non-substituted ring, A, B and C are either directly connected or connected through an intervening atom chain or linker and said atom chain or linker is a saturated carbon chain or an unsaturated carbon chain with or without additional functional groups.
  • the A ring is a 6-membered heterocyclic ring with one heteroatom.
  • the A ring may be substituted.
  • the ring is a pyridine ring; more preferably, the nitrogen atom of the pyridine ring is in the 4-position or in the 2-position realtive to the position of the B ring.
  • the pyridine ring is substituted with a n-propyl group on a carbon positioned alpha to the nitrogen heteroatom.
  • the A ring may be phenyl, pyrrole, thiophene, furan, pyrimidine, isoquinoline, quinoline, benzofuran, indole, oxazole, naphthyl, piperidine, pyrrolidine, imidazole, imidazol[1 ,2- a]pyridine, benzoimidazole, thiazole, or benzothiazole, for example.
  • the A ring is a piperidine ring.
  • the nitrogen atom of the piperidine ring is in the 4-position relative to the position of the B ring.
  • nitrogen atom of the piperidine ring is in the 3-position relative to the position of the B ring.
  • the nitrogen atom of the piperidine may be further substituted and wherein the substitutions are selected from the group consisting of alkyi, sulfoxide, sulfone, alkyi or aryl sulfonate, sulfonic acid, and any combination thereof.
  • the substitution may be a propyl group, a ferf-butyloxycarbonyl (BOC) or a benzyloxycarbonyl group.
  • the A ring is a pyrrolidine ring; preferably, the nitrogen atom of the pyrrolidine ring is in the 2-position relative to the position of the B ring.
  • the nitrogen atom of the pyrrolidine may be further substituted and wherein the substitutions are selected from the group consisting of alkyi, sulfoxide, sulfone, alkyi or aryl sulfonate, sulfonic acid, and any combination thereof.
  • the substitution may be a propyl group, a ferf-butyloxycarbonyl (BOC) or a benzyloxycarbonyl group.
  • the B ring is a 5-membered ring with at least two heteroatoms.
  • the B ring may be substituted.
  • the B ring is a thiazole ring.
  • the B ring may be oxazole, imidazol, isooxazole, imidazole, thiphene, furan, pyrimidine, pyrazole, isothiazole, thiazolopyridazine, aryl, or pyrazole, for example.
  • the B ring may further be a 6-membered ring with two heteroatoms.
  • the B ring is a piperazine ring.
  • the C ring is a 6-membered ring, most preferably a phenyl ring.
  • the C ring may be substituted.
  • the C ring is methyl substituted.
  • the C ring may be phenyl, pyridine, pyrrole, thiophene, furan, pyrimidine, isoquinoline, quinoline, benzofuran, indole, oxazole, or naphthyl, for example.
  • Exemplary compounds are provided in FIGS. 14A-14F and Tables 3 and 4.
  • the n-propyl substituted pyridine ring corresponds to the A ring of the general formula
  • the 2,4- substituted thiazole ring corresponds to the B ring of the general formula
  • the methyl substituted phenyl ring corresponds to the C ring of the general formula.
  • substitutions are permissible at any position in any of the A, B, and C rings and any substitutions may be the same or different from any other substitutions.
  • Non-limiting examples of the substitutions include the following groups: H (i.e., unsubstituted); hydroxy; C-
  • a compound, or pharmaceutically acceptable salt or stereoisomer thereof having the general formula: A-B-C, wherein A, B, and C are the same or different and wherein each comprises a 5-, 6-, or 7- membered ring or a fused bicyclic ring system, the ring being a heterocyclic ring or non-heterocyclic ring, a substituted ring, or non-substituted ring, wherein A, B and C are either directly connected or connected through an intervening atom chain or linker and wherein said atom chain or linker is a saturated carbon chain or an unsaturated carbon chain with or without additional functional groups, wherein any one, any two, or all three of A, B, and C are unsubstituted or have one or more substitutions, and wherein any substitution may be the same or different from any other substitution, and wherein the substitutions are consisting of: a) hydroxy, b) C1 -10 alkyl, c) C2-10 al
  • A is a pyridine or a substituted pyridine, a piperidine or a substituted piperidine, a pyrrolidine or a substituted pyrrolidine, a thiazole or a substituted thiazole, a phenyl ring or a substituted phenyl ring;
  • B is a thioazole or a substituted thioazole, a piperazine or a substituted piperazine, a phenyl ring or a substituted phenyl ring;
  • C is a phenyl ring or a substituted phenyl ring, a pyridine or a substituted pyridine, a thioazole or a substituted thioazole.
  • chemical structure is:
  • halogen -OH , -O-C1-3 alkoxy, -OC(0)R 3 ;
  • R 3 is d-C 3 alkyl or aryl, -OCH 2 - C(0)OR 4 ;
  • R 4 is H or Ci-C 3 alkyl, -NHR 5 ;
  • R 5 is H , C1-C4 alkyl, alkylcyclopropane, benzyl, -NHC(0)Ci- C 3 amide, -NHC(0)0-R 6 carbamate;
  • R 6 is iert-butyl or fluorenylmethyl or -NH-SO2-R7 sulfonamide;
  • R 7 is alkyl or aryl,
  • R 2 is alkyl or R 8 OC(0)- and
  • R 8 is C 3 -C 5 alkyl or aryl.
  • the halogen may be bromine.
  • chemical structure is:
  • Ri is H , halogen, -OH , -O-C1-3 alkoxy, -OC(0)R 3 ;
  • R 3 is Ci-C 3 alkyl or aryl, -OCH 2 - C(0)OR 4 ;
  • R 4 is H or C C 3 alkyl, -NHR 5 ;
  • R 5 is H , C C 4 alkyl, alkylcyclopropane, benzyl, -NHC(0)Ci- C 3 amide, -NHC(0)0-R 6 carbamate;
  • R 6 is iert-butyl or fluorenylmethyl or -NH-S0 2 -R 7 sulfonamide;
  • R 7 is alkyl or aryl,
  • R 2 is alkyl or R 8 OC(0)- and
  • R 8 is C 3 -C 5 alkyl or aryl.
  • the halogen may be bromine.
  • the chemical structure is: wherein R 9 is H, halogen, -OH, -O-C1-C3 alkoxy, -OC(0)Rn; Rn is C-1-C3 alkyl or aryl, - OCH 2 -C(0)OR 12 ; R12 is H or C C 3 alkyl, -NHR 13 ; R 13 is H, C C 4 alkyl, alkylcyclopropane, benzyl, - NHC(0)Ci- 3 amide, -NHC(0)0-R 14 carbamate; R 14 is fert-butyl or fluorenylmethyl, -NH-S0 2 -Ri 5 sulfonamide; R 15 is alkyl or aryl or -S0 2 -NH-R 16 sulfonamide; R 16 is alkyl or aryl, R 10 is nitrogen or m A is wherein R 17 is H or C1-C3 alkyl
  • composition comprising the compound as described supra and a pharmaceutically acceptable excipient.
  • the compound as described supra formulated as a food, an animal feed material or a medicine.
  • a kit comprising the compound as described supra and a container housing the compound.
  • the container can be any appropriate container adapted to store a drug known in the art and commercially available.
  • preferred compounds are N-(4-(2-(2- propylpyridin-4-yl)thiazol-4-yl)phenyl)methanesulfonamide, ierf-butyl 2-(4-(4-bromophenyl)thiazol-2- yl)pyrrolidine-1-carboxylate, benzyl 2-(4-(4-bromophenyl)thiazol-2-yl)pyrrolidine-1 -carboxylate, 4-(4- bromophenyl)-2-(pyrrolidin-2-yl)thiazole, 4-(4-bromophenyl)-2-(1 -propylpyrrolidin-2-yl)thiazole, tert- butyl 3-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylate, benzyl 3-(4-(4-bromophenyl)thiazol- 2-yl)
  • a method for treating a metabolic disorder in an animal comprising the step of administering to the animal a therapeutically effective amount of at least one compound as described supra, or a pharmaceutically acceptable salt or a stereoisomer thereof or a combination thereof.
  • the method comprises the step of providing a second therapy to the animal.
  • the second therapy comprises dietary therapy, physical therapy, behavior therapy, surgery, drug therapy, chemotherapy, or a combination thereof.
  • the second therapy may be a lifestyle modification, an antihyperglycemic agent, insulin, glucagon-like peptide (GLP), a dipeptidyl peptidase-4 inhibitor, a thiazolidinedione, a lipid lowering compound or a combination of two or more thereof.
  • the metabolic disorder is a weight related condition, a disease related to cell hyperproliferation, hyperlipemia, diabetes or complications thereof, fatty liver, hypertension, or a cardiovascular disease.
  • the metabolic disorder may be obesity, hypertension, arteriosclerosis, asthma, hyperlipidemia, hyperinsulinemia, non-alcoholic fatty liver and type 2 diabetes caused by insulin resistance.
  • the metabolic disorder is a weight-related disorder where the therapeutically effective amount of the compound increases the expression of uncoupling protein 1 , uncoupling protein 2 or uncoupling protein 3.
  • the therapeutically effective amount of the compound increases thermogenesis without reducing lean body mass during weight loss in the animal.
  • the disease related to cell hyperproliferation is a cancer.
  • the cancer is a cancer of the breast, respiratory tract, brain, reproductive organs, prostate, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid, lymphoma, sarcoma, melanoma, leukemia or a distant metastasis of a solid tumor.
  • a method for treating a cell hyperproliferative disease in a patient in need thereof comprising the step of administering to the patient a therapeutically effective amount of at least one compound as described supra, or a pharmaceutically acceptable salt or a stereoisomer thereof or a combination thereof.
  • the cell hyperproliferative disease may be a cancer as described supra.
  • a method for treating a cancer in a patient in need thereof comprising the step of administering to the patient, a therapeutically effective amount of one or more pharmaceutical compositions as described supra.
  • the cancer may be as described herein.
  • a method for reducing body weight in an animal in need thereof comprising the step of administering to the animal, in a pharmaceutically acceptable medium, a therapeutically effective amount of one or more of the compounds as described supra.
  • a method for increasing thermogenesis without reducing lean body mass during weight loss in an animal comprising the step of administering to the animal, in a pharmaceutically acceptable medium, a therapeutically effective amount of a compound having the structure
  • R-i substituents are H, Et , O e or n-propyl; Y is CH or ; R 2 is OH, OMe, or i-Pr; R 3 is H, F, or CI; R 4 is H, Me, CI, Br, F, OH, OBz, OCH 2 COOMe, OCH 2 COOH, NH 2 , NH-
  • a useful dose of a compound of the present invention depending upon the metabolic disorder, such as, but not limited to, a disease related to cell hyperproliferation or a weight-related disorder, to be treated or the outcome desired.
  • the compound is administered in a dose of from about 1 mg/kg to about 100 mg/kg.
  • the compound may be administered in a composition which is in the form of a food, an animal feed material, or a medicine.
  • body weight is reduced in the animal by an increase in uncoupled thermogenesis with or without a concurrent reduction in lean body mass.
  • the compound increases the expression of uncoupling protein.
  • uncoupling proteins include uncoupling protein 1 , uncoupling protein 2 and uncoupling protein 3.
  • the methods of the present invention would be useful in a variety of situations including, but not limited to, where the animal suffers from a weight-related condition selected from the group consisting of obesity, hypertension, arteriosclerosis, athsma, hyperlipidemia, hyperinsulinemia, non-alcoholic fatty liver and type 2 diabetes caused by insulin resistance.
  • a person having ordinary skill in this art would readily recognize the utility of providing to the animal a second therapy including, but not limited to, a lifestyle modification, an antihyperglycemic agent, insulin, glucagon-like peptide (GLP), a dipeptidyl peptidase-4 inhibitor, a thiazolidinedione, a lipid lowering compound, and a combination of two or more thereof.
  • a lifestyle modification including, but not limited to, an antihyperglycemic agent, insulin, glucagon-like peptide (GLP), a dipeptidyl peptidase-4 inhibitor, a thiazolidinedione, a lipid lowering compound, and a combination of two or more thereof.
  • a or “an” may mean one or more.
  • the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
  • another may mean at least a second or more.
  • aspects of the invention may "consist essentially of or “consist of” one or more sequences of the invention, for example.
  • Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
  • animal refers to a mammal, preferably a human, patient, subject, or individual that receives or has administered to one or more of the compounds, compositions or formulations described herein.
  • alkyl refers to a substituting univalent group derived by conceptual removal of one hydrogen atom from a straight or branched-chain acyclic saturated hydrocarbon (i.e., -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH 2 CH 2 CH 3 , -CH 2 CH(CH 3 ) 2 , - C(CH 3 ) 3 , efc.).
  • alkynyl refers to a substituting univalent group derived by conceptual removal of one hydrogen atom from a straight or branched-chain acyclic unsaturated hydrocarbon containing at least one carbon-carbon triple bond (i.e., -C ⁇ CH, -C ⁇ CCH 3 , - C ⁇ CCH(CH 3 ) 2 , -CH 2 C ⁇ CH, efc.).
  • aryloxy refers to an aryl group with a bridging oxygen atom, such as phenoxy (-OC 6 H 5 ), or benzoxy (-OCH 2 C 6 H 5 ).
  • Arylamino means an aryl group with a bridging amine function such as -NHCH 2 C 6 H 5 .
  • cycloalkyl refers to a substituting univalent group derived by conceptual removal of one hydrogen atom from a saturated monocyclic hydrocarbon (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl).
  • aryl refers to a substituting univalent group derived by conceptual removal of one hydrogen atom from a monocyclic or bicyclic aromatic hydrocarbon. Examples of aryl groups are phenyl, indenyl, and naphthyl.
  • heteroaryl refers to a substituting univalent group derived by the conceptual removal of one hydrogen atom from a monocyclic or bicyclic aromatic ring system containing 1 , 2, 3, or 4 heteroatoms selected from N, O, or S.
  • heteroaryl groups include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl, pyrimidinyl, pyrazinyl, benzimidazolyl, indolyl, and purinyl.
  • Heteraryl substituents can be attached at a carbon atom or through the heteroatom.
  • Examples of moncyclic heteroaryl groups include pyrrolyl, furyl, thienyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and pyridyl.
  • Examples of bicyclic heteroaryl groups include pyrimidinyl, pyrazinyl, benzimidazolyl, indolyl, and purinyl. Individual rings may have 5 or 6 atoms. Thus, this includes a 4-membered moncyclic heteroaryl group and a 5-memebered monocylcic heteroaryl group. It also includes a bicyclic heteroaryl group having one 5-membered ring and one 6-membered ring, and a bicyclic heteroaryl group having two 6-membered rings.
  • halo includes iodo, bromo, chloro and fluoro.
  • substituted shall be deemed to include multiple degrees of substitution by a substitutent.
  • a substitution occurs where a valence on a chemical group or moiety is satisfied by an atome or functional group other than hydrogen.
  • the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally.
  • independently substituted it is meant that the (two or more) substituents can be the same or different.
  • salts refers herein to a salt of a compound that possesses the desired pharmacological activity of the parent compound.
  • Such salts include: (1 ) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4- hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulf
  • an alkali metal ion an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.
  • stereoisomer means an isomeric molecule whose atomic connectivity is the same as one or more other molecules but whose atomic arrangement in space is different. This definition includes enantiomers, diastereomers, c/s-isomers, irans-isomers, conformational isomers.
  • unsubstituted means all that valences on a chemical group or moiety are satisfied by hydrogen.
  • saturated carbon chain refers to a straight or branched-chain acyclic saturated hydrocarbon (i.e., -CH 3 , -CH 2 -, -CH 2 CH 3 , -CH 2 CH 2 - -CH 2 CH 2 CH 3 , -CH 2 CH 2 CH 2 , - CH 2 CH 2 CH 2 CH 3 , -CH 2 CH(CH 3 ) 2 , -C(CH 3 ) 3 , efc.).
  • unsaturated carbon chain refers to a straight or branched-chain acyclic unsaturated hydrocarbon containing at least one carbon-carbon double bond (i.e.
  • the present invention also includes protected derivatives of compounds disclosed herein.
  • compounds of the present invention when compounds of the present invention contain groups such as hydroxyl or carbonyl, these groups can be protected with a suitable protecting group.
  • suitable protective groups can be found in T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, Inc. 1981 , the disclosure of which is incorporated herein by reference in its entirety.
  • the protected derivatives of compounds of the present invention can be prepared by methods well known in the art.
  • the compounds of the present invention may have asymmetric centers, chiral axes, and chiral planes, and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, being included in the present invention.
  • the compounds disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure may be depicted.
  • a composition of the invention targets one or more members of a sterol regulatory element binding protein (SREBP) pathway.
  • SREBP sterol regulatory element binding protein
  • the pathway relates to the proteolytic release of a membrane-bound transcription factor, SREBP, in specific aspects, which facilitates transport from the cytoplasm to the nucleus.
  • SREBP binds elements referred to as the sterol regulatory elements (SREs) present in regulatory regions of the genes that encode enzymes associated with production of lipids.
  • SREs sterol regulatory elements
  • compositions of the present invention comprise an effective amount of one or more compositions of the invention (and additional agent, where appropriate) dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal , such as, for example, a human, as appropriate.
  • the preparation of a pharmaceutical composition that contains at least one fatostatin A analog or derivative or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. , antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
  • the fatostatin A analog or derivative may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation, e.g., aerosol inhalation, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions, e.g., liposomes, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990).
  • the fatostatin A analog or derivative may be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g. , those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.
  • the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent.
  • the carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate.
  • carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof.
  • the composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens, e.g., methylparabens, and propylparabens, chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • various antibacterial and antifungal agents including but not limited to parabens, e.g., methylparabens, and propylparabens, chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
  • the composition is combined or mixed thoroughly with a semi-solid or solid carrier.
  • the mixing can be carried out in any convenient manner such as grinding.
  • Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.
  • stabilizers for use in an composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
  • the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include fatostatin A analog or derivative, one or more lipids, and an aqueous solvent.
  • lipid will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term "lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long- chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance.
  • Bio lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • the fatostatin A analog or derivative may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art.
  • the dispersion may or may not result in the formation of liposomes.
  • the actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject.
  • compositions may comprise, for example, at least about 0.1 % of an active compound.
  • the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, efc, can be administered, based on the numbers described above.
  • the fatostatin A analog or derivative is formulated to be administered via an alimentary route.
  • Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually.
  • these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al. , 1997; Hwang et al. , 1998; U.S. Pat. Nos. 5,641 ,515; 5,580,579; and 5,792,451 ).
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.
  • a binder such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof
  • an excipient such as, for
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U .S. Pat. No. 5,629,001 .
  • the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch cells.
  • a syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
  • suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1 % to about 2%.
  • the fatostatin A analog or derivative may be administered via a parenteral route.
  • parenteral includes routes that bypass the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterial ly, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,537,514, 6,613,308, 5,466,468, 5,543,158; 5,641 ,515; and 5,399,363.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof and/or vegetable oils.
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
  • the active compound fatostatin A analog or derivative may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
  • Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder.
  • Ointments include all oleaginous, adsorption, emulsion and water-solubly based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only.
  • Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin.
  • Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram.
  • Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water- soluble ointment base.
  • Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture.
  • Transdermal administration of the present invention may also comprise the use of a "patch".
  • the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a period of time.
  • compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212.
  • the delivery of drugs using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871 ) are also well-known in the pharmaceutical arts.
  • Transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045.
  • aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant.
  • the typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent.
  • Suitable propellants include hydrocarbons and hydrocarbon ethers.
  • an additional therapy may be delivered to an individual having a metabolic disorder.
  • an individual that is obese may be administered a composition of the invention in addition to another therapy for obesity.
  • Additional obesity therapies include dietary therapy, physical therapy (exercise), drug therapy, surgery, and behavioral therapy, for example.
  • Exemplary drug therapies include, for example, Xenical Orlistat®, Phentermine, and Sibutramine (Meridia®).
  • Exemplary surgeries include liposuction and gastric bypass, for example.
  • exemplary additional compounds for therapy include one or more of the following: Actos (pioglitizone); ACTOSPIus Met; Amaryl (glimepiride); Avandaryl (Avandia + Glimiperide); Avandia (rosiglitazone); Avandamet (rosiglitazone maleate and metformin hydrochloride); Byettap; Duetact (pioglitazone HCI and glimepiride); Galvus (Vildagliptin); Glipizide (Sulfonlyurea); Glucophage (metformin); Glimepiride; Glucovance (glyburide/metformin); Glucotrol XL (glipizide extended release); Glyburide; Glyset (miglitol) glucosidase inhibitor; Januvia (sitagliptin phosphate); Metaglip (glipizide+metformin); Metformin - biguan
  • an individual is given one or more compositions of the present invention and the individual is assessed for an improvement in at least one symptom of the metabolic disorder.
  • an improvement in obesity may be determined during and/or following treatment with one or more compositions of the invention.
  • An improvement in obesity may be measured by any standard means, but in particular aspects the improvement in obesity is measured by weight measurement, body mass index (BMI) measurement, and/or body part size measurement (such as waist measurement), for example.
  • Exemplary methods for calculating BMI includes dividing a person's body weight in kilograms by their height in meters squared (weight [kg] height [m] 2 ). A BMI of 30 or more is considered obese and a BMI between 25 to 29.9 is considered overweight.
  • an individual with diabetes is tested for an improvement following administration to the individual of the therapy of the invention.
  • the monitoring of diabetes occurs by blood test.
  • the blood test may measure the chemical A1 C. The higher the blood sugar, the higher the A1 C level will be.
  • cholesterol including HDL and/or LDL cholesterol
  • triglycerides are measured, such as by standard means in the art.
  • a fasting lipoprotein profile is performed, such as by standard means in the art.
  • the kit comprises a composition suitable for treatment and/or prevention of one or more metabolic disorders.
  • the kit comprises one or more apparatuses to obtain a sample from an individual.
  • Such an apparatus may be one or more of a swab, such as a cotton swab, toothpick, scalpel, spatula, syringe, and so forth, for example.
  • an additional compound is provided in the kit, such as an additional compound for treatment and/or prevention of a metabolic disorder.
  • Any compositions that are provided in the kits may be packaged either in aqueous media or in lyophilized form, for example.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed.
  • Fatostatin A reduces the expression of SREBP-responsive genes
  • fatostatin A impairs the function of SREBPs
  • the ability of endogenous SREBPs to activate transcription of an SREBP-responsive reporter gene was measured in the presence or absence of fatostatin A in HEK293 cells (FIGS. 2A-2B).
  • Fatostatin A decreased in a concentration dependent manner the activation of the reporter gene in which the expression of luciferase is controlled by three repeats of sterol regulatory elements.
  • fatostatin A failed to impair the ability of an exogenously expressed mature form of SREBP-1 (amino acids 1 -500) to activate the reporter gene activity (FIG. 2C).
  • Fatostatin A blocks the proteolytic activation of SREBPS
  • fatostatin A affects the proteolytic activation of SREBPs.
  • whole cell lysates of DU145 cells treated with fatostatin A were analyzed by western blots with an antibody against the NH2 terminus of SREBP-1 (FIG. 3A).
  • the treatment of fatostatin A decreased the amounts of the 68 KDa mature form of SREBP-1 in a dose-dependent manner, while the amounts of the 125 KDa precursor form increased.
  • Similar results were obtained for SREBP-2 with an antibody against its COOH terminus (FIG. 3B).
  • Fatostatin A causes two phenotypes in cultured cells: (i) inhibition of the insulin-induced adipogenesis of 3T3-L1 cells and (ii) repression of the serum-independent growth of DU145 prostate cancer cells.
  • the first phenotype is in complete agreement with the conclusion that fatostatin A is a blocker of SREBP-1 because of the known role of SREBP-1 in lipogenesis (Tontonoz et al., 1993).
  • siRNA small interfering RNA
  • the requirement of SREBP-1 in the serum-independent growth may be due to the lack of external fat sources in the serum-free medium. Without exogenous fatty acids present in the serum, cells need to synthesize fatty acids and cholesterol, the building blocks of membranes, to maintain the cell growth. To test the importance of fatty acids in the cell growth, the growth of the SREBP-1 knockdown cells was monitored in a fat-free serum medium (FIG. 5A). The SREBP-1 silencing impaired the cell growth in a fat-free medium as much as it did in the serum-free IGF1 -containing medium. These results indicate that fatostatin A blocks the serum-independent growth of cancer cells through the inhibition of SREBP-1 .
  • Fatostatin A reduces body weight, lowers cholesterol and glucose levels, and downrequlates lipogenic enzymes in mice
  • fatostatin A The drug-like chemical structure of fatostatin A prompted the inventors to investigate its ability to inhibit SREBP-1 in the liver of whole animals.
  • the effect of fatostatin A on hepatic SREBP- 1 under lipogenic conditions of prolonged fasting (48 hours) followed by feeding fat free high carbohydrate diet for another 48 hours was examined. Mice were intraperitoneally injected with fatostatin A at 30 mg/kg/day for 5 days starting one day prior to the 48-hour fasting period. After 48 hours of fasting, the treated group lost more weight than the control group did (6.12 ⁇ 0.6 compared to 4.9 + 0.3 gram/mouse; p 0.01 .) (FIG. 6A). No reduction of food intake or obvious toxicity were observed during the treatment (FIG.
  • the liver extracts from the mice treated with fatostatin A displayed decreased amounts of the 68 KDa mature form of SREBP-1 and increased amounts of the 125 KDa precursor form (FIG. 6D).
  • Western blot analysis of liver extracts showed that expression levels of FAS was decreased up to 30% by the fatostatin A treatment (FIG. 6E). Consistent with the reduction of the expression, its enzymatic activity in the extracts was similarly decreased.
  • ACC acetyl-CoA carboxylase
  • mice fed with normal diet Longer treatment (two weeks) of another group of mice fed with normal diet resulted in 10% loss of body weight whereas the control group had no change of body weight (FIGS. 7A-7B). Food intake was similar between both groups (3.8 and 3.5 g/mouse/day for treated and control mice, respectively). Consistent with the results of mice fed under fasting/refeeding fat free diet, mice fed with normal diet exhibited significantly lower glucose levels and a trend of lower triglyceride (TG) and cholesterol levels in the blood (FIG. 7C). FAS activity and its protein level were also decreased about 30% (FIGS. 7D and 7E). EXAMPLE 5
  • Bioactive small molecules have proven to be valuable tools for exploring complex cellular processes including metabolic pathways.
  • a key regulator of lipid homeostasis and insulin action is a family of SREBP transcription factors (Brown and Goldstein, 1997).
  • Small molecules that modulate the SREBP functions may find their use in the treatment of metabolic diseases and may serve as tools for further molecular understanding of the diseases.
  • the cell-based and animal data suggest that fatostatin A impairs the expression of lipogenic genes through downregulating the amounts of the mature SREBP-1 form in the nucleus.
  • the animal data of fatostatin A are consistent with the cell culture results.
  • liver extracts of mice treated with fatostatin A under refeeding fat free diet there was a significantly lower level of mature SREBP-1 form and a higher level of the precursor form.
  • the level of the mature form was higher than the precursor form in liver extracts of the control group (Horton et al., 1998).
  • fatostatin A treated mice compared to control is a reduction of body weight and blood glucose.
  • the reduction in body weight may be due to a lower lipogenesis rate, as a result of the downregulation of lipogenic enzymes such as ACC and FAS.
  • the reduction in malonyl-CoA, the product of ACC and a potent inhibitor of carnitine palmitoyl transferase may result in enhanced fatty acid oxidation and fat burning.
  • inhibition of SREBP-1 cleavage by fatostatin A downregulates lipogenic enzymes, enhances fatty acid oxidation, reduces weight, and increases insulin sensitivity resulting in lowering glucose.
  • Lipid-depleted serum was prepared as described (Goldstein ei at., 1983). Fat-free FBS was obtained from Fisher. Rabbit anti-SREBP-1 (sc-8984) and goat anti-actin (sc-1616) polyclonal antibody were purchased from Santa Cruz Biotechnology. Mouse anti-SREBP-2 polyclonal antibody and mouse anti-FAS antibody were obtained from BD Biosciences. Anti-goat IgG HRP and anti- rabbit IgG HRP were obtained from Promega. ProLong Gold antifade reagent with DAPI was obtained from Molecular Probes Invitrogen Detection Technologies. Anti-rabbit IgG FITC was obtained from Chemicon International. Dexamethasone (DEX) and 1 -methyl- 3-isobutylxanthin (MIX) were obtained from Sigma.
  • DEX Dexamethasone
  • MIX 1 -methyl- 3-isobutylxanthin
  • DU145 human androgen-independent prostate cancer cells were maintained in an Eagle's minimum essential medium containing 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 1 .5 g/L sodium biocarbonate with 10% fetal bovine serum, 100 units/mL penicillin, and 100 pg/mL streptomycin sulfate at 37 °C under 5% C0 2 .
  • 3T3-L1 fibroblasts cells were maintained in a Dulbecco's modified Eagle's medium containing 5.5 mM glucose, 10% fetal bovine serum, 50 pg/mL gentamycin, 0.5 mM glutamine, and 0.5 pg/mL fungizone at 37 °C.
  • Human embryonic kidney 293 cells were maintained in a Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/mL penicillin, and 100 pg/mL streptomycin sulfate at 37 °C under 5% C0 2 .
  • DU145 prostate cancer cells were treated with 5 mM of fatostatin A or DMSO alone in the presence of 1 pg /mL of IGF1 for 6 hrs in a serum free medium, total RNA was extracted in a TRI reagent (Molecular Research Center) and further isolated by RNeasy Mini Kit (Qiagen). Purified mRNA was analyzed in Baylor College of Medicine Microarray Core Facility by Affymetrix Human Genome U133 Plus 2.0 Array consisting of almost 45,000 probe sets representing more than 39,000 transcripts derived from approximately 33,000 well-substantiated human genes (Affymetrix, Inc.).
  • HEK293 cells were plated out in triplicate at a density of 5 x 10 3 /well onto a 96- well plate in a Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/mL penicillin, and 100 pg/mL streptomycin sulfate.
  • the cells were transiently co-transfected with the following plasmids by using Lipofectamine reagent (Invitrogen): 0.4 ⁇ g well pSRE-Luc (an SRE-1 -driven luciferase reporter construct), and 0.1 ⁇ g well a b-gal reporter in which the expression of ⁇ -gal is controlled by an actin promoter in a final volume of 150 mL.
  • Lipofectamine reagent Invitrogen
  • pSRE-Luc an SRE-1 -driven luciferase reporter construct
  • 0.1 ⁇ g well a b-gal reporter in which the expression of ⁇ -gal is controlled by an actin promoter in a final volume of 150 mL.
  • the cells were washed with phosphate-buffered saline and then incubated in 100 ⁇ of Dulbecco's modified Eagle's medium with 10% lipid-depleted serum, 100 units ⁇ L penicillin, and 100 pg/mL streptomycin sulfate in the absence or presence of fatostatin A. After 20 hrs of incubation, the cells in each well were lysed with 20 ⁇ _ of 1 x Reporter Lysis Buffer (Promega), and aliquots were used for measurement of luciferase (10 ⁇ ) and ⁇ -galactosidase (10 ⁇ L) activities.
  • luciferase assay photon production was detected as counts per second in a Wallac 1420 ARVOsx multilabel counter (PerkinElmer).
  • ⁇ -galactosidase assays hydrolysis of O-nitrophenyl- ⁇ -D-galactosidase was measured after incubation for 0.5 h at 37 °C by a microplate reader at the wave length of 405 nm (Tecan). The luciferase activity (counts per second) was normalized by the acitivity of ⁇ - galactosidase (OD units).
  • pCMV- SREBP-1 C (1-436) was co-transfected with pSRE-Luc.
  • pSRE-Luc and pCMV-SREBP-1c (1 -436) were provided by J. L. Goldstein (University of Texas Southwestern Medical Center). RT-PCR experiments
  • the primer pairs used are as follows: 5'-TCA GAC CGG GAC TGC TTG GAC GGC TCA GTC -3' (SEQ ID NO: 1 ) and 5'-CCA CTT AGG CAG TGG AAC TCG AAG GCC G -3' (SEQ ID NO: 2) for Low density lipoprotein receptor (LDLR); 5'- GCC TGC TTG ATA ATA TAT AAA C -3' (SEQ ID NO: 3) and 5' - CAC TTG AAT TGA GCT TTA G -3' (SEQ ID NO: 4) for stearoyl-CoA desaturase (SCD); 5' -AAG AAA AAG TGT CAG ACA GCT GG -3' (SEQ ID NO: 5) and 5' - TGG ACT GAA GGG GTG TTA GC -3' (SEQ ID NO: 6) for ATP citrate lyase (ACL); 5'- GCC CGA CAG TTC TGA ACT GGA ACA
  • the amplification conditions are as follows: 1 cycle at 94°C for 4 min, then denatured at 94°C for 40 s, annealed at 50°C for 40 s, and extended at 68°C for 2 min with 22 cycles for SCD and HMG CoA R, annealed at 58°C with 24 cycles for LDLR and INSIG1 , or annealed at 60°C with 24 cycles for ATP citrate lyase (ACL), annealed at 55°C with 30 cycles for MVD.
  • the amplified DNAs were analyzed by an agarose gel and quantified with the Scion-image (version 4.02) software.
  • DU145 prostate cancer cells were seeded on a 6-well plate at a density of 2 x 10 5 cells/well in a serum-free MEM incubated at 37°C for overnight. The cells were then treated with DMSO or fatostatin A (1 or 5 mM) in presence of IGF1 (1 pg /mL). After 6 hrs of incubation, the cells were harvested in PBS and lysated in an SDS buffer. The samples were separated on a 10% SDS-PAGE gel and blotted by using rabbit anti-SREBP-1 and anti-SREBP-2 antibodies. The specific bands were visualized by using enhanced chemiluminescent (ECL) detection reagents (Amersham).
  • ECL enhanced chemiluminescent
  • DU145 prostate cancer cells were seeded on coverslips for overnight in a serum-free MEM, and then treated with 5 mM of fatostatin A or DMSO alone in a serum-free MEM containing IGF1 (1 g/mL). After 6 hrs of incubation, the cells were fixed for 20 min in methanol at -20°C and blocked for 1 hr in a PBS containing 5% milk and 0.1 % Tween 20. The samples were incubated with rabbit polyclonal anti-SREBP-1 (Santa Cruz: sc-8984) and then fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (Chemicon Inc). The coverslips were visualized under a Nikon TE200 fluorescence microscope at x400 magnification with appropriate filters for fluorescence detection. siRNA knockdown of SREBPs
  • Complimentary oligonucleotides derived from the sequence of the SREBP-1 gene (512- 531 ), 5'- GAT CCC CGC CAC ATT GAG CTC CTC TCT TCA AGA GAG AGA GGA GCT CAA TGT GGC TTT TTG GAAA-3' (SEQ ID NO: 1 3), and 5'-AGC TTT TCC AAA AAG CCA CAT TGA GCT CCT CTC TCT CTT GAA GGA GGA GCT CAA TGT GGC GGG-3' (SEQ ID NO: 14), were inserted into a pSUPER vector (OligoEngine). The resulting plasmid was transfected into 3T3-L1 or DU 1 45 cells with Fugene 6 (Roche).
  • neomycin-derivative G41 8 (Gibco) was used at a concentration of 500 Mg/mL, and stable transformants were established. The expression levels of the SREBP-1 were evaluated by western blots.
  • 3T3-L1 cells were seeded onto a 96-well plate in a DMEM with 1 0% fetal bovine serum and incubated for another two days to complete confluence.
  • the medium was switched to the induction medium: DMEM containing 1 0% fetal bovine serum, 5 Mg/mL of insulin , 0.5 mM 1 -methyl- 3-isobutylxanthin (M IX), and 1 ⁇ dexamethazone (DEX).
  • the induction medium was removed and switched to a DMEM medium containing 1 0% fetal bovine serum and 5 Mg/mL of insulin.
  • adipose oil droplets were stained with Oil-Red O.
  • DU 145 cells were seeded onto 96-well plates at density of 2,000 cells/well in an MEM without serum or with 1 g /mL of IGF1 , 2% fat-free fetal bovine serum, or 2% fetal bovine serum. The cell growth was estimated by WST-1 assays after 3 days. The experiments were performed in triplicate. Animal studies with fatostatin A
  • mice Male mice (129Sv background) were housed under controlled conditions (12-hr light/dark cycle; 25°C) in the Animal Care Center at Baylor College of Medicine and had ad libitum access to standard laboratory chow (Purina Mills) and water.
  • Fatostatin A was administered intraperitoneally (30 mg/kg; 1 50 ⁇ ) to 5-6 month old male mice (129Sv background) using two different protocols. First protocol involves fasting the mice for 48 hrs, followed by refeeding fat free diet for another 48 hrs. This treatment induces both activities and levels of lipogenic enzymes such as ACC and FAS in addition to SREBPs.
  • the administration of fatostatin A or 1 0% DMSO in PBS to control groups (n 5) started 24 hrs before the fasting and continued daily until the end of the experiment.
  • PBS containing 0.1 mM PMSF, 5 mM benzamidine, and 5 mg/mL protease inhibitor cocktail (Roche), homogenized using Polytron (3 x 30 Sec, at a high speed), and sonicated briefly to degrade DNA.
  • Fatostatin A prevents fatty liver, reduces hvperqlvcemia and induces weight loss in ob/ob mice
  • fatostatin A is an inhibitor of the master control of transcription by inhibiting the action of SREBP-1.
  • SREBP-1 normal mice treated with fatostatin A lost weight and had lower levels of glucose and cholesterol.
  • Fatostatin A reduced the active mature form of SREBP-1 in the liver of treated mice compared to controls.
  • SREBP-1 and -2 play related but distinct roles in biosynthesis of fatty acids and cholesterol.
  • SREBP-1 preferentially activate the genes required for fatty acid synthesis
  • SREBP-2 favors cholesterogenesis. Since fatostatin A blocks the activation of SREBP-1 and perhaps SREBP-2, administration of fatostatin A into obese ob/ob mice transiently modulates the biosynthesis of both fatty acids and cholesterol and reveals interesting phenotypes in the obese mice.
  • mice were administered intraperitoneally daily, and body weight and food intake were measured. As shown in FIGS. 8A-8B, the increase in body weight of the treated mice was significantly lower than the controls.
  • the accumulative food intake was similar in both groups FIG. 8C).
  • food intake was not significantly different from the controls being 5.4 ⁇ 1 .5 compared to 5.9 ⁇ 1.4 g/mouse. day respectively.
  • ob/ob mice One of the most distinct phenotypes in ob/ob mice is hyperglycemia as a result of insulin resistance conditions.
  • the serum levels of glucose, triglycerides, and cholesterol were analyzed in ob/ob mice fed with standard diet.
  • ketone bodies in fatostatin A animals shows a significant increase in fatty acid oxidation in livers in which the main product is ketone bodies that is secreted in the blood.
  • NEFA free fatty acids
  • TG triglycerides
  • VLDL which transports triglycerides, phospholipids, and cholesterol and is calculated based on TG levels, increased about 50% (23.1 ⁇ 2.3 compared to 15.8 ⁇ 2.4 mg/dl).
  • Fatostatin A reduces the size of epididymal fat and ameliorates fatty liver
  • ob/ob mice become morbidly obese and accumulate excessive levels of fat in fat tissues and in different organs, such as liver-causing non-alcoholic fatty liver conditions and insulin resistance.
  • untreated mice showed enlarged liver size and accumulated fat, as evident from pale color, compared to those treated with fatostatin A (FIG. 1 0A).
  • the treated mice weighed less than the controls.
  • the epididymal fat pads which is the major white fat tissue.
  • the average weight of the fat pads was about 20% less than the controls (2.7 ⁇ 0.1 compared to 3.6 ⁇ 0.2; P - 0.02) (FIG. 10D).
  • the smaller fat pads may be due to decrease in storage of lipids and/or decrease in lipogenesis and enhanced fatty acid oxidation in the adipose.
  • liver of treated ob/ob mice is due to significant inhibition of lipogenic enzymes needed to synthesize TG and cholesterol or their precursors.
  • lipogenic enzymes needed to synthesize TG and cholesterol or their precursors.
  • lipase liver activity due to increased demand for fatty acid oxidation by different mice tissues, including the liver there is an increase in lipase liver activity and also enhanced mobilization of these lipids from liver to the circulation for utilization by different fatty acid oxidizing tissues such as heart and muscles.
  • this is related to the higher level of TG in blood of fatostatin A treated ob/ob mice.
  • Fatostatin A downrequlates lipogenic enzymes in ob/b mice liver
  • Enzymes in lipogenic pathways are regulated by transcription factors, such as PPAR and SREBPs.
  • the effect of fatostatin A on lipogenic enzymes levels and activities in treated ob/ob mice was examined.
  • the activity of acetyl-CoA carboxylase (ACC) which carries out the rate-limiting step in fatty acid synthesis, was determined.
  • ACC catalyzes the carboxylation of acetyl-CoA to yield malonyl-CoA, the building block for fatty acid synthesis, which is carried out by another multifunctional enzyme, fatty acid synthase (FAS).
  • FAS fatty acid synthase
  • This downregulation in ACL level further amplify the effect of fatostatin A on the reduction in the lipogenic process in lipogenic tissues such a liver.
  • SCD1 catalyzes the rate-limiting step in the biosynthesis of monounsaturated fatty acids, by introducing a Cis double bond in ⁇ 9 position of fatty acyl-CoA, such as palmitoyl-CoA and stearoyl- CoA.
  • the products, palmitoleoyl-CoA (16: 1 ) and oleoyl-CoA (18: 1 ) are important components of triglycerides and cholesterol esters and deletion of SCD1 in mice including ob/ob mice resulted in increased metabolic rate, reduced adiposity, preventing fatty liver and protecting against diabetes induced by diet. As shown in FIG. 12D, the protein level of SCD1 was reduced about 50% in liver extracts of fatostatin A treated mice compared to the controls.
  • Liver samples (100 mg) obtained from fatostatin A-treated ob/ob mice and non-treated control mice and stored at - 80°C until analyzed for fatty acid contents.
  • the fatty acids were extracted according to Folch's protocol and quantitatively analyzed using gas chromatography-mass spectrometry (GC-SM).
  • the decrease in protein levels can be attributed to a transcriptional or translational regulation.
  • Real time PCR was used to determined the levels of mRNA levels of representatives of lipogenic genes ACC1 , FAS and SCD1 in addition to the lipogenic transcription factor PPAR ⁇ . There was about 80% reduction in the mRNA levels of ACC1 , FAS and SCD1 (FIG. 13). These results are consistent with lower levels of enzyme proteins and activities, and strongly indicate that fatostatin A lowers lipogenesis by inhibiting the maturation of SREBP-1 .
  • the down regulation of lipogenic enzyme involves one of its main transcription factors, PPAR ⁇ , in specific embodiments. The mRNA level of this transcription factor was reduced by about 40% in extracts of fatostatin A treated mice (FIG. 13).
  • Fatostatin A through its action on SREBP-1 ameliorated fatty liver by reducing hepatic TG storage, reduced adiposity and lowered hyperglycemia in treated ob/ob mice.
  • mice After 28 days of daily injection of fatostatin A mice were fasted overnight and blood was withdrawn and Whole blood glucose and ⁇ -hydroxybutyrate were measured with a Glucometer Precision Xtra (Abbott). For determination of serum constituents. Glucose, triglyceride and cholesterol measurements were done by the Comparative Pathology Laboratory (Baylor College of Medicine). Serum non-esterified fatty acids (NEFA) were measured by using NEFA C kit (Wako Chemicals, Richmond, VA).
  • mice were sacrificed and weights of livers, and Epididymal fat pads were determined.
  • liver slices Frozen sections of Liver slices from individual animals were stained with Oil Red O to visualize the fat droplets (TG) in liver slices as described earlier (Abu-Elheiga ef at. , 2001 ). The remaining liver tissues were frozen in liquid nitrogen and kept at - 80°C for further analysis. Liver triglyceride and cholesterol contents were carried out as described in the reference (Chandler, ef a/. , 2003) using Cholesterol E Kit (Wako) and Infinity Triglyceride Kit (Thermo Electron, Melbourne, Australia), adapted for colorimetric analysis in 96-well plate format.
  • a portion of the frozen liver was ground to powder in liquid nitrogen.
  • the powdered tissues were suspended in 10 ml of PBS containing 0.1 mM PMSF, 5 mM benzamidine, and 5 mg/ml protease inhibitor cocktail (Roche), and homogenized using Polytron (3 x 30 Sec, at high speed) and sonicated briefly to degrade DNA.
  • the extracts were clarified by centrifugation at 16,000 x g for 20 min. Protein concentrations in the supernatant were determined, and subjected to western blot analysis using commercially available antibodies against the following enzymes: FAS (BD Biosciences), citrate lyase SCD1 , FADS1 , ACC and phospho-ACC antibodies.
  • the proteins were visualized using Amersham ECL PlusTM Western Blotting Detection Reagents. The intensity of the specific bands of proteins of interest were scanned and normalized against beta-actin for quantifications. FAS and ACC activities from the liver extracts were determined as described earlier (Mao et al. , 2006).
  • First stranded cDNA was synthesized from 2 g of DNase l-treated total RNA with random hexamer primer using Superscript II RNase H-reverse transcriptase (Invitrogen).
  • the real time PCR contained, in a final volume of 20 ⁇ , 10 ng of reverse transcribed total RNA, 0.5 ⁇ forward and reverse primers, and 10 ⁇ of 2 x master mix from DyNAmo HS SYBR Green qPCR kit (Finnzymes).
  • PCR was carried out in 96-well plate using DNA Engine Opticon System (MJ Research, Inc). All reactions were done in triplicate and the relative amounts of mRNAs were calculated using the comparative C(t) method. The cycle threshold C(t) was calculated using the Opticon Monitor software 2.02 (MJ Research). Mouse ⁇ -actin mRNA was used as the internal control. Data were expressed as the mean + SD. Difference between two groups was assessed using the unpaired two-tailed Student t-test. EXAMPLE 8
  • one or more targets of fatostatin A or its analog or derivative is identified.
  • any suitable method may be employed for such identification, in specific embodiments the fatostatin A or analog or derivative thereof, is labeled.
  • Exemplary labels include biotin, for example.
  • FIGS. 14A-14F illustrate exemplary compounds of the invention, and their given names are provided in Table 3 and Table 4.
  • FIGS. 15-17 demonstrate exemplary luciferase reporter gene assays for these exemplary compounds at 20 mM by the same method shown in FIG. 2A.
  • the adipogenesis assay was performed as decribed (Choi et al., 2003).
  • the analogues that completely inhibited the formation of oil droplets in cells were scored to be adipogenesis-inhibiting analogues.
  • a skilled artisan recognizes that it may be suitable to modify one or more aspects of an exemplary compound to assist in identifying other suitable compounds. For example, upon determination of suitability for a particular compound for treatment and/or prevention of one or more metabolic disorders, the compound may be modified to identify other related compounds for use for the same or a different metabolic disorder. Such alterations may occur in accordance with exemplary chemical groups as described herein, in specific embodiments.
  • sterol regulatory element binding proteins Upon fat depletion in a cell, sterol regulatory element binding proteins (SREBPs) are released proteolytically from the membrane and translocated into the nucleus, where they activate transcription of the genes involved in cholesterol and fatty acid biosynthesis.
  • SREBPs sterol regulatory element binding proteins
  • the diarylthiazole derivative called fatostatin, impairs the proteolytic activation of SREBPs, thereby decreasing the transcription of lipogenic genes in cells.
  • the molecular target of fatostatin appears to be SREBP cleavage-activating protein (SCAP). Fatostatin blocked increases in body weight, blood glucose, and hepatic fat accumulation in obese ob/ob mice, even under uncontrolled food intake.
  • SCAP SREBP cleavage-activating protein
  • fatostatin inhibits the insulin-induced adipogenesis of 3T3-L1 cells and the serum-independent growth of DU145 cells (Choi et al., 2003).
  • Gene expression profiles of drug- treated and untreated cells were compared to gain information about specific molecular pathways affected by fatostatin.
  • DU145 cells were treated with fatostatin or DMSO alone, and the extracted mRNA samples were analyzed by Affymetrix DNA microarrays mapping 33,000 genes. Of those genes (all of which are available at the National Center for Biotechnology Information's GenBank database on the world wide web), transcription levels of 63 genes decreased at least 35% in response to fatostatin treatment (Table 5).
  • Brefeldin A a natural product that blocks anterograde movement of proteins from the ER to the Golgi, is known to render SREBPs unresponsive to sterols, and causes SREBPs to be constitutively processed in the ER by relocating S1 P from the Golgi to the ER (DeBose-Boyd et at., 1999).
  • fatostatin had no impact on the SREBP processing (FIG. 19A), suggesting that fatostatin does not block the proteolysis itself.
  • SCAP contains a glycosylated luminal loop that is protected from proteolysis by trypsin and recognizes anti-SCAP lgG-9D5.
  • the two oligosaccharides in the loop are sensitive to endoglycosidase H when SCAP resides in the ER.
  • SCAP is transported to the Golgi, its sugars become resistant to digestion by endoglycosidase H.
  • the translocated SCAP has higher levels of glycosylation, and is more resistant to endoglycosidase H than ER-bound SCAP.
  • Sterols prevent SCAP from becoming resistant to endoglycosidase H by inhibiting the ER-Golgi translocation (Nohturfft ei a/. , 1998).
  • Cells were grown in the absence or presence of fatostatin or sterols, and membrane fractions were treated successively with trypsin and endoglycosidase H. In cells grown without fatostatin and sterols, a tryptic fragment of SCAP was more resistant to endoglycosidase H and had one or two saccharide chains (FIG. 19B, lane 1 ).
  • the selective ER localization implies that fatostatin binds to a protein in the ER; the most likely candidate is SCAP, the target of cholesterol for the control of SREBP (Radhakrishnan ef a/., 2004).
  • SCAP the target of cholesterol for the control of SREBP
  • proteins bound to a fatostatin- polyproline linker-biotin conjugate (FIG. 20A) (Sato ef a/., 2007) were purified from cell lysates and analyzed by western blots with antibodies against SCAP, SREBP-1 , SREBP-2, and ATF6, an unrelated ER-bound transcription factor (Ye et al. , 2000).
  • Another phenotype of ob/ob mice is excessive accumulation of fat in organs, including nonalcoholic fatty liver. Enlarged and fatty livers were evident from their pale color in the untreated ob/ob mice, while livers of the mice treated with fatostatin appeared normal. Livers of the treated mice averaged -32% less weight, and fat pads were smaller than those of the untreated mice. Oil red staining of the liver sections showed that the livers of the untreated ob/ob mice contained abundant lipid droplets, while livers of the treated mice contain lower levels of lipid accumulation (FIG. 21 ). The triglyceride and cholesterol levels in the livers of the treated mice were also reduced.
  • fatostatin blocks the processing of SREBP-1 in liver, downregulates lipogenic enzymes, and reduces hepatic triglyceride storage.
  • Fatostatin represents the first non-sterol-like synthetic molecule that inhibits the activation of SREBPs.
  • Luciferase reporter assay On day 0, CHO-K1 cells were plated out onto a 96-well plate in medium A (a 1 :1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium, with 5% fetal bovine serum, 100 units/mL penicillin, and 100 g/mL streptomycin sulfate).
  • medium A a 1 :1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium, with 5% fetal bovine serum, 100 units/mL penicillin, and 100 g/mL streptomycin sulfate.
  • the cells were transiently co-transfected with pSRE-Luc (an SRE-1 -driven luciferase reporter construct) (Hua ef a/., 1995) and pAc-p-gal ( ⁇ -gal reporter in which the expression of ⁇ -gal is controlled by an actin promoter), using Lipofectamine reagent (Invitrogen).
  • pSRE-Luc an SRE-1 -driven luciferase reporter construct
  • pAc-p-gal ⁇ -gal reporter in which the expression of ⁇ -gal is controlled by an actin promoter
  • the cells were washed with phosphate-buffered saline (PBS), and then incubated, in the absence or presence of fatostatin, in medium B (a 1 :1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium, with 5% lipid-depleted serum, 100 units/mL penicillin, 100 pg/mL streptomycin sulfate, 50 m compactin, and 50 mM sodium mevalonate). After 20 h of incubation, the cells in each well were lysed, and aliquots were used to measure luciferase and ⁇ -galactosidase activities.
  • PBS phosphate-buffered saline
  • Luciferase activity was normalized by the activity of ⁇ -galactosidase.
  • pCMV-SREBP-1c(1 -436) was co-transfected with pSRE-Luc and ⁇ - ⁇ -gal.
  • CHO-K1 cells were plated out onto a 100 mm dish of medium A.
  • the cells were washed with PBS, and then incubated in medium B in the absence or presence of fatostatin.
  • the cells were washed once with cold PBS, and then treated with buffer containing 10 mM Tris-HCI, pH 7.6, 100 mM NaCI, 1 % (w/v) SDS, and protease inhibitor mixture (1 pg/ml pepstatin A, 10 pg/ml leupeptin, 200 ⁇ phenylmethylsulfonyl fluoride).
  • the protein concentration of each total cell extract was measured (BCA kit; Pierce), after which a 22-33 pg aliquot of cell extract was mixed with 0.25 volume of buffer (250 mM Tris-HCI, pH 6.8, 10% SDS, 25% glycerol, 0.2% (w/v) bromophenol blue, and 5% (v/v) 2- mercaptoethanol), heated for 7 min at 95 °C.
  • the samples were separated on a 10% SDS-PAGE gel and blotted using mouse monoclonal antibody against SREBP-2 (lgG-7D4) (Yang ei a/. , 1995). The specific bands were visualized using enhanced chemiluminescent (ECL) detection reagents (Amersham).
  • ECL enhanced chemiluminescent
  • SCAP oligosaccharides Modification of SCAP oligosaccharides.
  • Cell membrane fractions were prepared as described elsewhere herein. The membrane pellets were resuspended in 0.1 ml_ of buffer containing 10 mM Hepes-KOH (pH 7.4), 10 mM KCI, 1 .5 mM MgCI 2 , 1 mM sodium EDTA, and 100 mM NaCI. Aliquots of protein were then incubated in the absence or presence of 1 pg of trypsin, in a total volume of 58 pl_, for 30 min at 30 °C. Reactions were stopped by addition of 2 ⁇ _ (400 units) of soybean trypsin inhibitor.
  • each sample received 10 ⁇ of solution containing 3.5% (wt vol) SDS and 7% (vol/vol) 2-mercaptoethanol. After heating at 100°C for 10 min, each sample received sequential additions of 9 ⁇ of 0.5 M sodium citrate (pH 5.5), 5 pL of solution containing 17 ' protease inhibitors (a concentration of 1 x, corresponding to 10 pg/mL leupeptin, 5 pg/mL pepstatin A, and 2 pg/mL aprotinin), followed by 1 pL (5 units) of endoglycosidase H.
  • 0.5 M sodium citrate pH 5.5
  • 5 pL of solution containing 17 ' protease inhibitors a concentration of 1 x, corresponding to 10 pg/mL leupeptin, 5 pg/mL pepstatin A, and 2 pg/mL aprotinin
  • the reactions were carried out overnight at 37 °C and stopped by the addition of 20 pL of buffer containing 0.25 M Tris « HCI (pH 6.8), 2% SDS, 10% (vol/vol) glycerol, 0.05% (wt/vol) bromophenol blue, and 4% 2-mercaptoethanol. The mixtures then were heated at 100 °C for 5 min and subjected to SDS/PAGE (12% gels).
  • the membrane fraction was extracted with PBS containing 0.1 % FOS-Choline 10 (Hampton Research).
  • the extract was mixed with Neutravidine-agarose beads (10 pL) saturated with biotinylated fatostatin and incubated for 1 h.
  • the bound proteins were washed four times with PBS containing 0.1 % FOS-Choline 10, boiled in 25 pL of SDS sample buffer, and subjected to western blotting.
  • saturated amounts of cholesterol or fatostatin were added to the membrane extract before incubating with the beads.
  • mice Four to five week-old homozygous male obese (ob/ob) mice (C57BL/6J, The Jackson Laboratory, Bar Harbor, ME) were housed under controlled conditions (12 h light/dark cycle; 25 °C). The animals were housed 5 per cage, and had ad libitum access to standard laboratory chow (Purina Mills, Richmond, IN) and water for one week after their arrival. On first day of the experiment and every day thereafter, the weight of each mouse and the amount of food intake were measured between 3:00 and 5:00 p.m. Following weight measurements, treated mice received an ip injection of fatostatin (30 mg/kg; 150 pL), and control mice received 10% DMSO in PBS. Daily Injections were continued for four weeks, until the end of the study.
  • liver analyses Mice were sacrificed, and weights of livers and epididymal fat pads were determined. Frozen sections of liver slices from individual animals were stained with Oil Red O to visualize the fat droplets (triglycerides) in liver slices, as described (Abu-Elheiga ef al , 2001 ). The remaining liver tissues were frozen in liquid nitrogen and kept at -80 °C for further analysis.
  • Tissue triglyceride and cholesterol contents were determined as described by Chandler et al. (2003), using a Cholesterol E Kit (Wako) and an Infinity Triglyceride Kit (Thermo Electron, Melbourne, Australia).
  • FIG. 23 depicts the synthesis of fatostatin 1 , dansyl fatostatin, fatostatin-polyproline linker- biotin conjugates and the synthetic intermediates.
  • a pressure tube was charged with 2 (1.08 g, 3.0 mmol), benzophenone imine (0.57 g, 3.3 mmol), Pd 2 (dba) 3 (86 mg, 0.15 mmol), BINAP (280 mg, 0.45 mmol), sodium tert-butoxide (1 .44 g, 9.0 mmol), and dry toluene (30 mL) and purged with argon gas.
  • the pressure tube was sealed and heated in a 100 °C bath for 20 h. After being cooled to room temperature, the reaction mixture was chromatographed (Si0 2 , 4:1 hexane:EtOAc) to provide 1.35 g of 8 (98%) as a yellow oil.
  • Fatostatin-KPGQFLYELKKPPPPPPPPPKK (SEQ ID NO: 15)-aminocaproic acid-biotin.
  • Conjugate 3 calcd for
  • Antibodies Monoclonal anti-SREBP-1 IgG (2A4), anti-SCAP IgG (9D5) and anti-ATF6 IgG
  • Polyclonal anti-FAS, anti-ACC, anti-SCD1 and anti-ACL IgG were purchased from BD Biosciences.
  • Human androgen- independent prostate cancer cells (DU145) were maintained in an Eagle's minimum essential medium containing 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 1.5 g/L sodium biocarbonate with 10% fetal bovine serum, 100 units/mL penicillin, and 100
  • Oligonucleotide microarray analysis DU145 prostate cancer cells were treated with 5 mM of fatostatin or DMSO alone in the presence of 1 ⁇ g /mL of IGF1 for 6 hrs in a serum free medium, total RNA was extracted in a TRI reagent (Molecular Research Center) and further isolated by RNeasy Mini Kit (Qiagen). Purified mRNA was analyzed in Baylor College of Medicine Microarray Core Facility by Affymetrix Human Genome U133 Plus 2.0 Array consisting of almost 45,000 probe sets representing more than 39,000 transcripts derived from approximately 33,000 well- substantiated human genes (Affymetrix, Inc.).
  • RNA sample was subjected to RT-PCR by using the Access RT-PCR System (Promega).
  • RT-PCR reactions contained total RNA, 1 ⁇ of each primer, 0.2 mM dNTP, 1 mM MgS0 4 , AMV reverse transcriptase (2 units), and Tfl DNA polymerase (2 units) in a final volume of 25 ⁇ _.
  • the primer pairs used are as follows: 5'- TCA GAC CGG GAC TGC TTG GAC GGC TCA GTC -3' (SEQ ID NO: 16) and 5'-CCA CTT AGG CAG TGG AAC TCG AAG GCC G -3' (SEQ ID NO: 17) for Low density lipoprotein receptor (LDLR); 5'- GCC TGC TTG ATA ATA TAT AAA C -3' (SEQ ID NO: 18) and 5' - CAC TTG AAT TGA GCT TTA G -3' (SEQ ID NO: 19) for stearoyl-CoA desaturase (SCD); 5' -AAG AAA AAG TGT CAG ACA GCT GG -3' (SEQ ID NO: 20) and 5' - TGG ACT GAA GGG GTG TTA GC -3' (SEQ ID NO: 21 ) for ATP citrate lyase (ACL); 5'- GCC CGA CAG TTC TGA ACT GGA
  • the amplification conditions are as follows: 1 cycle at 94°C for 4 min, then denatured at 94°C for 40 s, annealed at 50°C for 40 s, and extended at 68 °C for 2 min with 22 cycles for SCD and HMG CoA R, annealed at 58°C with 24 cycles for LDLR and INSIG1 , or annealed at 60°C with 24 cycles for ACL, annealed at 55°C with 30 cycles for MVD.
  • the amplified DNAs were analyzed by an agarose gel and quantified with the Scion-image software.
  • PLAP-BP2 Cleavage On day 0, CHO-K1 cells were plated out onto a 96-well plate in medium A. On day 2, the cells were transiently co-transfected with pCMV-PLAP-BP2(513-1141 ), pCMV-SCAP and ⁇ - ⁇ -gal, using Lipofectamine reagent (Invitrogen). After incubation for 5 h, the cells were washed with PBS, and then incubated, in the absence or presence of fatostatin (20 ⁇ ) or sterols (10 ⁇ g/mL cholesterol and 1 ⁇ g mL 25-hydroxycholesterol), in medium B.
  • fatostatin (20 ⁇
  • sterols 10 ⁇ g/mL cholesterol and 1 ⁇ g mL 25-hydroxycholesterol
  • Enzymatic activities and western blot analyses A portion of the frozen liver was ground to powder in liquid nitrogen. The powdered tissues were suspended in 10 mL of PBS containing 0.1 mM PMSF, 5 mM benzamidine, and 5 mg/mL protease inhibitor cocktail (Roche), and homogenized using Polytron (3 x 30 Sec, at high speed), and sonicated briefly to degrade DNA. The extracts were clarified by centrifugation at 16,000 x g for 20 min. Protein concentrations in the supernatant were determined, and subjected to western blot analysis using antibodies against FAS, ACC, SCD1 and ACL. The intensity of the specific bands of proteins of interest were scanned and normalized against b-actin for quantifications. FAS and ACC activities from the liver extracts were determined as described earlier (Mao ef a/., 2006).
  • reaction (a) a mixture of prothionamide (9.01 g, 50.0 mmol) and 4- bromophenacyl bromide 6a (13.9 g, 50.0 mmol) in EtOH (400 mL) was stirred at 70 °C for 1 h. After cooled to room temperature, the precipitation was filtered off with EtOAc and washed with EtOAc and saturated aqueous NaHC0 3 . The organic layer was washed with brine, dried over Na 2 S0 4 , and concentrated to give 4-(4-bromophenyl)-2-(2-propylpyridin-4-yl)thiazole 2 (14.3 g, 79%) as a light- yellow solid.
  • reaction (b) a pressure tube was charged with 2 (1.08 g, 3.0 mmol), benzophenone imine (0.57 g, 3.3 mmol), Pd 2 dba 3 (86 mg, 0.15 mmol), BINAP (280 mg, 0.45 mmol), sodium ferf-butoxide (1.44 g, 9.0 mmol), and dry toluene (30 mL) and purged with argon gas.
  • the pressure tube was sealed and heated in a 100°C bath for 20 hrs. After being cooled to room temperature, the reaction mixture was chromatographed (Si0 2 , 4:1 hexane:EtOAc) to provide 1.35 g of 71 (98%) as a yellow oil.
  • reaction (c) to a solution of 71 (1 .35 g, 2.9 mmol) in THF (20 mL) was added 2 M aqueous HCI solution (15 mL). After being stirred at room temperature for 2 hrs, the reaction mixture was concentrated under reduced pressure, then diluted with EtOAc (100 mL) and washed with saturated Na 2 C0 3 (50 mL) solution. The aqueous wash was extracted with EtOAc (3 ⁇ 40 mL), and the combined EtOAc layers were dried over Na 2 S0 4 and concentrated.
  • the procedure to synthesize compound 17 is utilizes the same steps a- c as described for compound 19 in Example 12.
  • acetone 2.5 mL, 34.5 mmol
  • acetic acid 2.0 ml_, 34.5 mmol
  • acetone 2.5 mL, 34.5 mmol
  • acetic acid 2.0 ml_, 34.5 mmol
  • 16 1 .02 g, 3.45 mmol
  • CH 2 CI 2 20 mL
  • Na(OAc) 3 BH 1.5 g, 6.9 mmol
  • Standard SREBP activation assays were performed on the exemplary compounds identified in Table 4 as per the method for Fatostatin A in Example 1.
  • the ability of endogenous SREBPs to activate transcription of an SREBP-responsive reporter gene was measured in the presence or absence of compounds 45-55 and 19 (FIG. 28), compounds 56-61 and 19 (FIG. 29) and compounds 62-66 (FIG. 30) at 5 ⁇ in CHOK1 cells.
  • compound 61 demonstrated inhibition of SREBP activation at about 25%, with compound 53 at about 30%, compound 58 at about 42%, and compound 19 at about 45%.
  • the inhibitory concentrations of compounds 53 (FIG. 31 ) and compounds 58 and 61 (FIG. 32) were determined.
  • Compound 19 inhibited the growth of the human breast cancer cell lines SUM159 and downregulated lipogenic pathways
  • Compound 19 was also demonstrated on human hepatocellular carcinoma cell line (HePG2). As shown in FIGS. 34A-34B, Compound 19 produced a high level of toxicity as evidenced by the dramatic change in the morphology and the inhibition of growth of the treated cells. When HePG2 cells were treated with 25 ⁇ of Compound 19 there was a significant decrease in the level of the mature and active form and an increase in the precursor of SREBP-1 and consequently the expression levels of genes that are controlled by SREBP (FIGS. 34A-34B). These results are consistent and supportive of the conceptual action of compound 19 as an inhibitor of lipogenesis through the inhibition of SREBP activation.
  • Compound 19 inhibits the growth of human acute lymphoblastic leukemia cell line MOLT-4
  • MOLT-4 cells were grown at two different conditions under 5% fetal bovine serum (FBS) and under 5% FBS fat free serum (charcoal treated serum). 10,000 Cells were seeded a 96 well plate for 24 hours before starting the treatment with different concentrations of compound 19. Compound 19 dissolved in DMSO was added to cells for 48 hours before determining cell growth using MTT assay. As shown in FIG. 35A, the growth of the cells under 5% FBS and FBS fat free was similar. Compound 19 inhibited cell growth about 75-80% at 10 and 20 ⁇ under both growth conditions. These results show that at 10 and 20 ⁇ compound 19 was equally effective even in the presence of exogenous lipids, which suggest that inhibition of de novo lipid synthesis result in growth inhibition in this cancer sell line. Interestingly, under FBS fat free serum there was 30-50% growth inhibition at 2 and 5 ⁇ compound 19 (Fig. 35C) and a strong trend of inhibition at 5 ⁇ in 5% FBS condition (FIG. 35B).
  • Compound 19 inhibits the growth of human multiple myeloma cell line RPMI8226
  • RPMI8226 cells were grown under the same conditions as the MOLT-4 cells. 20,000 cells were seeded into 96 well plates for 24 hrs prior to treatment with increasing concentrations of compound 19 as described for MOLT-4 cells. As shown in FIG. 35D, the growth of the cells under 5% FBS and FBS fat free was similar except that 5 ⁇ compound 19 in 5% FF FBS inhibited growth about 1 .5 more than the corresponding amount in 5% FBS. Compound 19 was similarly effective at 10 ⁇ and 20 ⁇ in 5% FBS and 5% FF FBS. There was a strong trend of inhibition starting at 3 ⁇ in 5% FBS condition (FIG. 35E) and FBS fat free serum at 2 ⁇ (FIG. 35F).
  • Compound 17 inhibits the growth of human acute lymphoblastic leukemia cell line MOLT-4
  • MOLT-4 cells were grown at two different conditions under 5% fetal bovine serum (FBS) and under 5% FBS fat free serum (charcoal treated serum). 20,000 cells were seeded into a 96 well plate for 24 hours before starting the treatment with different concentrations of compound 17. Compound 17 dissolved in DMSO was added to cells for 48 hours before determining cell growth using MTT assay. As shown in FIG. 36A, the growth of the cells under 5% FBS and 5% FBS fat free was strongly inhibited at 5, 10 and 20 ⁇ . Under 5% FF FBS, 3 ⁇ exhibited about 2.5 times the inhibition as under 5% FBS conditions. Compound 17 inhibited cell growth about 85-100% at 5, 10 and 20 ⁇ under both growth conditions. Under both 5% FBS and 5% FBS fat free sera there was a strong trend of inhibition starting at 1 ⁇ (FIGS. 36B-36C).
  • FBS fetal bovine serum
  • FBS fat free serum charcoal treated serum
  • Compound 17 inhibits the growth of human multiple myeloma cell line RPMI
  • RPMI8226 cells were grown under the same conditions as the MOLT-4 cells. 20,000 cells were seeded into 96 well plates for 24 hrs prior to treatment with increasing concentrations of compound 19 as described for MOLT-4 cells. As shown in FIG. 36D, the growth of the cells under 5% FBS and FBS fat free was initially similar except that compound 17 in 5% FF FBS inhibited growth about 1 .5 to 6 times more than the corresponding amount in 5% FBS at 5, 10 and 20 ⁇ . There was a strong inhibition starting at 3 ⁇ in 5% FBS condition (FIG. 35E) and 5% FBS fat free serum (FIG. 35F).
  • Compound 19 up regulates uncoupling protein 2 (UCP2) and reduces body fat
  • Compound 19 was effective in reducing body weight and total body fat
  • Feeding the HFHC diet for three weeks increased total body weight of rats about 12% more than the rats that were fed a regular diet, 332 + 6.5 vs. 375 + 4.0 gram respectively (Fig 37A).
  • total body fat and % fat in the rats fed the regular diet was significantly lower than those fed HFHC diet (44.0 + 2.5 vs. 62.4 + 2.4 g/rat and 13.95 + 0.5 vs. 18.48 +0.48 %, respectively).
  • the percent lean was also higher in RD fed rats compared to HFHC fed rats (Figs. 37B, 37C and 37D). Effect of Compound 19 on body weight and composition
  • Body weight was determined daily and food intake was measured every 2-3 days. Rats were given compound 19 in cottonseed oil for six days starting on Sunday until Friday. The dose was calculated based on the body weight at the day of the administration of the drug and the control vehicle. As shown in Figure 38, the animals gained weight about 200 gram after three weeks of feeding the HFHC diet and before the start of the treatment (Fig. 38A). The control group which was fed HFHC diet, but not given cottonseed oil and the second control group (control vehicle) gained similar weight, suggesting the vehicle does not contribute to changes in body weight (Fig. 38B). There was significant reduction in body weight in the 2.5 mg/kg and the 10 mg/kg treated group after two weeks of the start of the treatment.
  • the difference in body weight can be attributed mostly to the difference in fat content between the treated and control groups (Fig. 39B).
  • the total body fat weight was about 25 and 43% lower in 2.5 and 10 mg/kg compared to control-vehicle (114.7 + 5.2, 86.5 + 3.2 and 150.9 + 4.2 gr/rat respectively).
  • the total% fat was also significantly lower, with 2.5 mg/kg group had a similar %fat to the rat group that was fed the regular diet and 10 mg/kg had a lower percentage than the regular diet; 26.6 ⁇ 0.53 (control); 21.9 + 0.82 (2.5 mg/kg); 18.0 + 0.6 (10 mg/kg ) and 21.2 + 0.82 (regular diet) (Fig. 39C).
  • the lean mass weight was not significantly different between all groups, although percent lean mass was higher in 2.5 and 10 mg/kg compared to that of the control groups (Fig. 39D and 39E).
  • Compound 19 ameliorated fatty liver conditions caused by HFHC diet and downreoulated gene expression of de novo lipoqenesis and potentially by upregulation of uncoupling proteins and thermogenesis
  • UCPs are involved in the upregulation of thermogenesis. They dissipate metabolic energy as heat by lowering the mitochondria membrane potential.
  • the liver is a very active metabolic organ, especially in lipid synthesis.
  • the present invention has shown that food consumption was not significantly different between control and rats treated with compound 19. Despite feeding with lipogenic diet the treated animals accumulated significantly lower body fat including the livers.
  • the livers were examined as an example where energy might have been uncoupled and determined the levels of UCP2, the major UCP in liver. As shown in Figure 41 , using real-time PCR method the level of UCP2 was significantly higher and increased about 1 .5 and 3 fold in the 2.5 and 10 mg/kg treated groups respectively.

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