CN107074839A - Composition and method for treating the metabolism illness related to body weight - Google Patents

Abstract

The present invention relates to compound, pharmaceutical composition and the preparation for being coupled structure A B C with three, the three connections structure A B C have piperidyl, pyrrolinyl or pyridine radicals A rings, thiazole B rings and phenyl C rings, and they further can independently be substituted.Also provide and utilize compound as described herein, pharmaceutical composition and preparation for treating metabolic disorder or the method for cell hyperproliferative diseases.Also provide and lose weight or increase the method for themogenesis during weight loss, methods described utilizes structure A B C compound or the compound of such structural formula, the R in the structural formula as mentioned₁It is H, Et, OMe or n-propyl；Y is CH or (formula)；R₂It is OH, OMe or NH i Pr；R₃It is H, F or Cl；And R₄It is H, Me, Cl, Br, F, OH, OBz, OCH₂COOMe、OCH₂COOH、NH₂、NH‑i‑Pr、NHCOMe、NHSO₂Me, NHBn (formula), OMe, NHBoc, (formula), NHTs, (formula) or (formula)；Its pharmaceutical salts or stereoisomer or the two.

Description

Compositions and methods for treating metabolic and weight-related disorders

Cross References to Related Applications

This international application claims the benefit of priority of pending application U.S. Serial No. 14/270,130 filed on May 5, 2014 under 35 U.S.C. §120, which was filed on October 11, 2013 under 35 U.S.C. §120 The benefit of priority of pending application U.S. Serial No. 14/052,074, which claims the benefit of priority of pending application U.S. Serial No. 14/013,918 filed on August 29, 2013 under 35 U.S.C. § 120, all of which This application is fully incorporated herein by reference.

Federal Funds Instructions

This invention made use of federal funds from National Institutes of Health Grant GM-63115 and Department of Defense Grant No. DAMD 17-03-1-0228. The US Government has certain rights in this invention.

Background of the invention

field of invention

In general, the present invention relates to the fields of medicine and molecular biology of metabolic disorders. In a particular aspect, the field of the invention relates to specific compositions for the treatment of disorders such as obesity due in part to upregulation of thermogenesis. In certain aspects, the composition comprises fatostatin A and analogs or derivatives thereof.

Description of related fields

Metabolic syndrome involves many cardiovascular risk factors, including hypertension, dyslipidemia, obesity, type 2 diabetes, pancreatic β-cell dysfunction ) and atherosclerosis. Diets varying in fat or carbohydrate content favor energy metabolism in animals, including humans. Long-chain fatty acids are a major source of energy and an important component of the lipids that make up cell membranes. They are derived from food and are synthesized de novo from acetyl-CoA. Cholesterol is also derived from food and is synthesized from acetyl-CoA. The conversion of carbohydrates to acylglycerides by the de novo synthesis of fatty acids and cholesterol involves at least 12 and 23 enzymatic reactions, respectively. The expression levels of the genes encoding these enzymes are controlled by three transcription factors, SREBP-1a, -1c and SREBP-2, called sterol regulatory element binding proteins (SREBPs). These membrane-bound proteins are members of one class of the basic helix-loop-helix leuc amino acid zipper family of transcription factors. Unlike other transcription factors that are members of the leuc amino acid zipper, SREBP is synthesized as an ER-membrane-bound precursor that requires proteolysis by two Golgi membrane-bound proteases (site-1 and site-2 proteases) Released to activate transcription of target genes in the nucleus.

Proteolytic activation of SREBP is tightly regulated by sterols through interaction with SREBP cleavage activator protein (SCAP), the ER-membrane bound guard protein of SREBP. When sterols accumulate in the ER membrane, the SCAP/SREBP complex is unable to leave the ER to the Golgi apparatus, and thus the proteolytic processing of SREBP is inhibited. SREBP is a key adipogenesis transcription factor controlling fat metabolism homeostasis.

The prior art lacks novel compositions and methods useful in the treatment of a variety of metabolic disorders. The present invention fulfills this long standing need and desire in the art.

Summary of the invention

The present invention teaches compounds, formulations and pharmaceutical compositions thereof, and uses thereof for the treatment of metabolic disorders such as, but not limited to, diseases involving cellular hyperproliferation (eg, cancer) or body weight-related conditions.

The present invention relates to compounds having the following general structures:

A-B-C

Ring A is pyridine or substituted pyridine, piperidine or substituted piperidine, pyrrolidine or substituted pyrrolidine, thiazole or substituted thiazole, benzene ring or substituted benzene ring. Ring B is thiazole or substituted thiazole, piperazine or substituted piperazine, benzene ring or substituted benzene ring. Ring C is a benzene ring or a substituted benzene ring, pyridine or a substituted pyridine, thiazole or a substituted thiazole.

The present invention also relates to compounds having the following chemical structures:

R ₁ substituent is H, halogen, -OH, -OC _{1 -3} alkoxy, -OC(O)R ₃ ; R ₃ is C ₁ -C ₃ alkyl or aryl, -OCH ₂ -C(O )OR ₄ ; R ₄ is H or C ₁ -C ₃ alkyl, -NHR ₅ ; R ₅ is H, C ₁ -C ₄ alkyl, alkylcyclopropane, benzyl, -NHC(O)C ₁ - C ₃ amide, —NHC(O)OR ₆ carbamate; R ₆ is tert-butyl or benzyl, —NH—SO ₂ —R ₇ sulfonamide, and R ₇ is alkyl or aryl. The R ₂ substituent may be alkyl or R ₈ OC(O)—, and R ₈ is C ₃ -C ₅ alkyl or aryl.

The present invention further relates to compounds having the following chemical structures:

R ₁ substituent is H, halogen, -OH, -OC _{1 -3} alkoxy, -OC(O)R ₃ ; R ₃ is C ₁ -C ₃ alkyl or aryl, -OCH ₂ -C(O )OR ₄ ; R ₄ is H or C ₁ -C ₃ alkyl, -NHR ₅ ; R ₅ is H, C ₁ -C ₄ alkyl, alkylcyclopropane, benzyl, -NHC(O)C ₁ - C ₃ amide, —NHC(O)OR ₆ carbamate; R ₆ is tert-butyl or benzyl, —NH—SO ₂ —R ₇ sulfonamide, and R ₇ is alkyl or aryl. The R ₂ substituent may be alkyl or R ₈ OC(O)—, and R ₈ is C ₃ -C ₅ alkyl or aryl.

The present invention further relates to compounds having the following chemical structures:

R ₉ is H, halogen, -OH, -OC ₁ -C ₃ alkoxy, -OC(O)R ₁₁ ; R ₁₁ is C ₁ -C ₃ alkyl or aryl, -OCH ₂ -C(O) OR ₁₂ ; R ₁₂ is H or C ₁ -C ₃ alkyl, -NHR ₁₃ ; R ₁₃ is H, C ₁ -C ₄ alkyl, alkylcyclopropane, benzyl, -NHC(O)C ₁ - ₃ Amide, -NHC(O)OR ₁₄ carbamate; R ₁₄ is tert-butyl or benzyl, -NH-SO ₂ -R ₁₅ sulfonamide; R ₁₅ is alkyl or aryl or -SO ₂ -NH- R ₁₆ sulfonamide, and R ₁₆ is alkyl or aryl. R ₁₀ is nitrogen or methylene. n is 0 or 1, and when n is 1, Z is -C=O. A can have the following structure:

Wherein R ₁₇ is H or C ₁ -C ₃ alkyl.

The present invention relates to the compounds described herein formulated as pharmaceutical compositions in pharmaceutically acceptable excipients or as formulations comprising food, animal feed substances or medicaments. The present invention also relates to kits comprising a compound described herein or a combination thereof or a pharmaceutical composition or other formulation thereof and a container containing said compound.

The present invention further relates to the following compounds: N-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)methanesulfonamide, 2-(4-(4-bromo Phenyl)thiazol-2-yl)pyrrolidine-1-carboxylic acid tert-butyl ester, 2-(4-(4-bromophenyl)thiazol-2-yl)pyrrolidine-1-carboxylic acid benzyl ester, 4-(4 -Bromophenyl)-2-(pyrrolidin-2-yl)thiazole, 4-(4-bromophenyl)-2-(1-propylpyrrolidin-2-yl)thiazole, 3-(4-( 4-Bromophenyl)thiazol-2-yl)piperidine-1-carboxylic acid tert-butyl ester, 3-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylic acid benzyl ester, 3 -(4-(4-bromophenyl)thiazol-2-yl)-1-propylpiperidine, 4-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylic acid benzyl Ester, (R)-2-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)pyrrolidine-1-carboxylic acid benzyl ester, 3-(4-(4-(methyl Sulfonamido)phenyl)thiazol-2-yl)piperidine-1-carboxylic acid benzyl ester, 4-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)piperidine-1 - benzyl formate, 4-(3-(pyridin-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-N-tosylanilide, (4-(5-chloro-2-methylphenyl)piperazin-1-yl)(4-(tosylamino)phenyl)methanone, 4-(4-((1-methyl-1H -Benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)-N-tosylanilide, 3-chloro-4-methyl-N-(6-(4-(3- (Trifluoromethyl)benzyl)piperazin-1-yl)pyridin-3-yl)benzenesulfonamide, 4-chloro-N-(4-(4-((1-methyl-1H-benzo[ d] imidazol-2-yl)methyl)piperazin-1-yl)phenyl)benzenesulfonamide, (Z)-4-(3-cyano-3-(4-(2,4-dimethyl Phenyl)thiazol-2-yl)allyl)-N-(thiazol-2-yl)benzenesulfonamide, N-(3-(H-imidazo[1,2-a]pyridin-2-yl) Phenyl)-4-methyl-2-phenylthiazole-5-carboxamide, N-(3-(benzo[d]thiazol-2-yl)phenyl)isonicotinamide, 3-(4-chloro Phenyl)-4,5-dihydro-1-phenyl-5-(2-phenylthiazol-4-yl)-1H-pyrazole, N-(4-(6-methylbenzo[d] Thiazol-2-yl)phenyl)-2-(N-m-tolylmethylsulfonamido)acetamide, N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl )-2-(N-p-tolylmethylsulfonamido)acetamide; pharmaceutically acceptable salts thereof; stereoisomers; and any combination thereof.

The present invention further relates to a method for treating a metabolic disorder in an animal, said method comprising administering to a subject 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 stereoisomeric Constructs or their combinations. In a preferred aspect, the metabolic disorder is cancer or a weight-related disorder. The invention relates to related methods further comprising the step of providing a second therapy. The second therapy includes diet therapy, physical therapy, behavioral therapy, surgery, drug therapy and combinations thereof. In some aspects, the metabolic disorder is diabetes, and additional therapy includes diet therapy, physical therapy, and drug therapy.

The present invention further relates to methods for treating cellular hyperproliferative disorders in a patient in need thereof. The method includes the step of administering to the patient a therapeutically effective amount of at least one compound described herein, or a pharmaceutically acceptable salt or stereoisomer thereof, or a combination thereof.

The invention further relates to related methods for reducing the body weight of 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 stereoisomer thereof, or a combination thereof.

The invention further relates to methods for reducing body weight in an animal in need thereof. The method comprises administering to the animal a therapeutically effective amount of a compound having structures A-B-C or as specifically described herein, or a pharmaceutically acceptable salt or stereoisomer thereof, or a combination thereof, in a pharmaceutically acceptable medium. Ring A may be pyridine or substituted pyridine, piperidine or substituted piperidine, pyrrolidine or substituted pyrrolidine, thiazole or substituted thiazole, benzene ring or substituted benzene ring. Ring B can be thiazole or substituted thiazole, piperazine or substituted piperazine, benzene ring or substituted benzene ring. Ring C may be benzene or substituted benzene, pyridine or substituted pyridine, thiazole or substituted thiazole.

The present invention also relates to a method for increasing thermogenesis without reducing lean body mass during weight loss in an animal, said method comprising the step of administering to said animal a therapeutically effective amount of a compound having the following structure: compound of:

or a pharmaceutically acceptable salt or stereoisomer thereof or a combination thereof. R1 substituent can be H, Et, OMe or _n -propyl; Y is CH or _The R2 substituent can be OH, OMe or NH-i-Pr. The _R3 substituent can be H, F or Cl. R ₄ substituents are H, Me, Cl, Br, F, OH, OBz, OCH ₂ COOMe, OCH ₂ COOH, NH ₂ , NH-i-Pr, NHCOMe, NHSO ₂ Me, NHBn, OMe, NHBoc, NHTs, The compound may be a pharmaceutically acceptable salt or stereoisomer thereof or a combination thereof.

The foregoing has described, rather broadly, the features and technical advantages of the present invention so that the following detailed description of the invention may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. Those skilled in the art should also understand that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are characteristic of this invention, both its organization and method of operation, together with other objects and advantages, will be better understood from the following description when considered in conjunction with the accompanying drawings. It should be clearly understood, however, that each drawing is for purposes of illustration and description only and is not to be considered as limiting of the invention.

Brief description of the drawings

For a more complete understanding of the present invention, the following description will now be made in conjunction with the accompanying drawings.

Figures 1A-1B show validation of microarray results by RT-PCR. DU145 cells were treated with DMSO (lane 1) and 5 [mu]M Fatustatin A (lane 2) for 6 hours (Fig. 1A). Total RNA was then extracted and subjected to RT-PCR. (FIG. 1B) Summary of RT-PCR and microarray data. ACL, ATP citrate lyase; HMG CoAR, 3-hydroxy-3-methylglutaryl-CoA reductase; LDLR, low-density lipoprotein receptor, MVD, mevalonate pyrophosphate decarboxylase; SCD , stearoyl-CoA desaturase; INSIG1, insulin-inducible gene 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figures 2A-2C show that phatostatin A inhibits the ability of endogenous SREBP to activate a reporter gene. HEK293 cells were co-transfected with the SRE-1-driven luciferase reporter (pSRE-Luc) (Fig. 2A) and the β-gal reporter under the control of the actin promoter (Fig. 2B). Transfected cells were treated with various concentrations of phatustatin A or DMSO alone in media containing lipid-free serum. After 20 hours of incubation, luciferase activity was determined and data were normalized by β-galactosidase activity. In FIG. 2C , HEK293 cells were transfected with pCMV-SREBP-1c(1-436) and pSRE-Luc, and the transfected cells were treated with or without 20 mM phatustatin A in medium containing lipid-free serum. Each value represents the mean of three independent experiments.

Figures 3A-3H show the effect of Fatuostatin A on SREBP-1 and -2. DU145 cells were treated with DMSO alone or phatuostatin A (1 and 5 μM) for 6 hours. Levels of precursor and mature forms of SREBP-1 (Fig. 3A) or SREBP-2 (Fig. 3B) were examined by Western blot. The lower panel shows a western blot of actin as a loading control. (FIGS. 3C-3H) The localization of SREBP-1 was examined by immunostaining. Cells were treated with DMSO alone (Figure 3C-3E) or 5 μM Fatustatin A (Figure 3F-3H), and then stained with DAPI (Figure 3C and Figure 3F) or with anti-SREBP-1 (Figure 3D and Figure 3G ).

Figures 4A-4G show that knockdown of SREBP-1 by siRNA inhibits insulin-induced adipogenesis. Two stably transfected clones of 3T3-L1 cells whose expression of SREBP-1 was knocked down were established and induced to differentiate into adipocytes. Knockdown cells did not differentiate (Fig. 4D and 4F), whereas 3T3-L1 cells transfected with blank vector (neo) mostly differentiated into adipocytes (Fig. 4B). Figures 4A, 4C and 4E show cells without insulin induction. Figure 4G is a Western blot analysis of clones showing successful knockdown of SREBP-1.

Figures 5A-5B demonstrate that siRNA knockdown of SREBP-1 blocks serum-independent growth of DU145 prostate cancer cells. In Figure 5A, two stably transfected clones of DU145 cells with SREBP-1 expression knocked down were established and incubated in serum-free, 2% fetal bovine serum (FBS), 2% fat-free fetal bovine serum, or 1 μg /mL IGF1 in MEM medium for 3 days. Growth rate was measured by WST-1 assay. Knockdown cells did not grow in MEM medium without serum, with 2% fat-free FB, or 1 μg/mL IGF1, but showed as much growth as control cells in the presence of serum. Experiments were performed in triplicate. Figure 5B is a Western blot showing the extent of SREBP-1 knockdown in clones 1 and 2.

Figures 6A-6G demonstrate the effect of phatuostatin A on mice after fasting/re-introduction to a fat-free diet. Mice were injected intraperitoneally with 30 mg/kg of phatustatin A daily throughout the duration of the experiment (fasted for 48 hours after the start of the day and then fed a fat-free diet for 48 hours). Body weight loss (Figure 6A) and food intake (Figure 6B) were measured at the end of the 48-hour feeding. Figure 6C shows the serum composition of treated and control mice. Figure 6D is a representative Western blot of SREBP-1 in liver extracts from 2 different mice in the control and phatostatin A treated groups. Normalize the amount of protein loaded. Figure 6E is a representative Western blot of a Coomassie-stained gel showing FAS expression of liver extracts (upper panel) and loading control (lower panel). Figures 6F-6G show FAS and ACC activity in liver extracts. Data are mean+SD (n=5); *P<0.05.

Figures 7A-7E show the effect of two weeks of phatostatin A treatment on mice. Mice aged 5-6 months were injected daily for two weeks with 30 mg/kg phatustatin A or 10% DMSO. Figure 7A shows body weight before and after treatment with tustatin A, and Figure 7B shows the amount of body weight lost after treatment. Figure 7C shows serum levels of glucose, cholesterol and triglycerides (TG) in treated and control mice. Figure 7D shows FAS activity in liver extracts. Figure 7E is a representative Western blot analysis of liver extracts from three mice each in the control and treatment groups. The amount of protein loaded was normalized. Data are expressed as mean+SD (n=5); *P<0.05.

Figures 8A-8C show the effect of Fatuostatin A on body weight and food consumption. Two groups of ob/ob male mice (n=5) were injected intraperitoneally with fatostatin A or 10% DMSO in PBS (control group) daily for four weeks. The mice were fed with normal diet, and the weight of the mice and the amount of food consumed were measured on the first day of the experiment and every subsequent day. Figure 8A is a photograph of representative control and phatostatin A treated mice. Figure 8B shows the weight of each mouse within each group measured daily. The mean and variance of the weights are shown. In Figure 8C, food intake was measured daily and expressed as cumulative food intake per mouse over a 28-day period.

Figures 9A-9H demonstrate the serum composition of control and Fatuostatin A treated ob/ob mice. Blood was collected from tail veins of overnight fasted mice, and serum was collected after cell isolation. Composition was determined as described below. Data are expressed as mean ± SD, n = 5 mice per group.

Figures 10A-10D show the effect of Fatuostatin on the liver and adipose tissue of ob/ob mice. Figure 10A shows the livers of fetostatin A-treated mice (left) and controls (right). Figure 10B shows histological analysis of cryosections of livers from control and phatuostatin A ob/ob mice stained with Oil-Red O to detect lipid droplets and counterstained with Mayer's hematoxylin. The livers of three different mice treated with tustatin A exhibited a significant reduction in red-stained lipid droplets (upper panel), compared with treated mice, and controls showed enrichment of red-stained lipid droplets (lower panel). . Figure 10C shows epididymal fat pads isolated from phatuostatin A-treated ob/ob mice (left) and controls (right). Figure 10D shows the average weight of liver and epididymal fat pads isolated from ob/ob control and phatuostatin A treated mice. Data are expressed as mean±SD, n=5 mice per group (*P<0.05).

FIGS. 11A-11B show triglyceride ( FIG. 11A ) and cholesterol ( FIG. 11B ) levels in the liver of control and Fatuostatin A-treated ob/ob mice. Lipids were extracted from the liver, and triglycerides and cholesterol were quantified as described below. Data are expressed as mean ± SD, n = 5 mice per group (*P = 0.0004; ).

Figures 12A-12D demonstrate that Fatuostatin A reduces the expression level and activity of lipogenic enzymes. Figure 12A shows the activity of acetyl-CoA carboxylase (ACC) and Figure 12B shows the activity of fatty acid synthase, assayed in liver extracts of ob/ob mice as described below. Figure 12C is a Western blot analysis of crude liver extracts from three individual ob/ob mice separated by 4-12% NuPAGE MES gels, probed with different antibodies, and detected with ECL. Figure 12D shows the ratio of the intensity of specific bands for different lipogenic enzymes of phatuostatin A relative to control mice after normalization to actin. Data are expressed as mean ± SD, n = 5 mice per group ( *P<0.05).

Figure 13 shows transcript levels of hepatic lipogenic enzymes in controls relative to Fatuostatin A ob/ob mice. The mRNA levels of each gene were normalized to actin. RNA was isolated from control and Fatuostatin A-treated mice (n=5) and measured by real-time quantitative RT-PCR. *P<0.05 vs. control.

Figures 14A-14F show exemplary compounds 1-66 of the invention.

Figure 15 shows a standard luciferase reporter assay utilizing exemplary analogs 2-18.

Figure 16 shows a standard luciferase reporter assay using exemplary analogs 19-34.

Figure 17 provides a standard luciferase reporter assay utilizing exemplary analogs 35-44.

Figures 18A-18D show that Fatuostatin blocks the activation of SREBP. Suppression of the ability of endogenous SREBP to activate the luciferase reporter gene by phatustatin in media containing lipid-free serum. CHO-K1 cells were transfected with the SRE-1-driven luciferase reporter (pSRE-Luc) (Fig. 18A). Transfected cells were treated with different concentrations of fetustatin in medium containing lipid-free serum. Effect of Fatuostatin on CHO-K1 cells co-transfected with pCMV-SREBP-1c(1-436) and pSRE-Luc in medium containing lipid-free serum ( FIG. 18B ). PLAP-BP2 in transfected CHO-K1 cells remained membrane bound unless it was cleaved by S1P in the Golgi apparatus and secreted into the medium (left panel). Treatment with vatustatin (20 μM) or sterols (10 μg/mL cholesterol and 1 μg/mL 25-hydroxycholesterol) affected the cleavage of PLAP-BP2 compared to EtOH control ( FIG. 18C ). Western blot analysis of CHO-K1 cells treated with tustatin. P and N indicate the uncleaved membrane precursor and cleaved nuclear form of SREBP-2, respectively (Fig. 18D).

Figures 19A-19B show that Fattostatin blocks the translocation of SREBP from the ER to the Golgi apparatus. Figure 19A is a Western blot analysis showing the effect of brefeldin A (brefeldin A) on CHO- Effects on K1 cells. Figure 19B is a Western blot analysis using anti-SCAP IgG-9D5 of cells grown in the presence or absence of 20 μM fatustatin or sterols (10 μg/mL cholesterol and 1 μg/mL 25-hydroxycholesterol). The numbers on the right indicate the number of N-linked sugar chains present on the protease-protected SCAP fragment.

20A-20D show the structure of dansyl fatostatin (dansyl fatostatin), fatostatin-polyproline linker-biotin conjugate, and polyproline linker-biotin conjugate and the compounds treated with them. cell. Figure 20A shows how to insert a polyproline linker to better protect the fatustatin molecule (Sato et al., 2007). In FIG. 20B , CHO-K1 cells treated with dansyl-fatuostatin and ER-tracker red show that dansyl-fatuostatin is localized in the ER. Scale bar = 10 μm. Figure 20C shows the interaction of phatostatin with SCAP by binding to neutravidine-agarose beads saturated with biotinylated phatostatin in CHO-K1 membrane extracts Western blot analysis of anti-SCAP, anti-SREBP-1, anti-SREBP-2 and anti-ATF6 antibodies of the protein showed. Figure 20D shows that for competition assays, membrane extracts were pre-incubated with EtOH, cholesterol or phatustatin alone.

Figure 21 shows the effect of phatuostatin on the liver and adipose tissue of ob/ob mice. Liver sections from fatostatin-treated and control mice show red-stained lipid droplets.

Figure 22 shows Western blot analysis of CHO-K1 cells treated with tustatin. P and N indicate the uncleaved membrane precursor and cleaved nuclear form of SREBP-1, respectively.

Figure 23 provides an exemplary synthetic scheme for Fatuostatin, Dansyl Fatuostatin and Fatuostatin-Polyproline Linker-Biotin.

Figure 24 shows the ability of fatostatin analogs to inhibit the ability of endogenous SREBP to activate a luciferase reporter gene in media containing lipid-free serum. CHO-K1 cells were transfected with pSRE-Luc. Transfected cells were treated with different concentrations of fatostatin, dansylfatostatin or isopropylamine derivatives in medium containing lipid-free serum.

Figures 25A-25D show that Fatostatin reduces the expression level and activity of lipogenic enzymes. The activities of acetyl-CoA carboxylase (ACC) (FIG. 25A) and fatty acid synthase (FAS) (FIG. 25B) were measured in ob/ob mouse liver extracts. Western blot analysis of crude liver extracts was performed (Fig. 25C). The ratio of the intensity of the specific bands of the different lipogenic enzymes in the different lipogenic enzymes in control mice after normalization to actin is shown for Fatuostatin ( FIG. 25D ). Data are expressed as mean ± SD, n = 5 mice per group ( *P<0.05).

Figure 26 provides a synthetic scheme for compound 53.

Figure 27 provides a synthetic scheme for Compound 19 and Compound 17.

Figure 28 shows a standard SREBP activation assay using exemplary analogs 45-55 and 19.

Figure 29 shows a standard SREBP activation assay using exemplary analogs 56-61 and 19.

Figure 30 shows a standard SREBP activation assay using exemplary analogs 62-66.

Figure 31 shows inhibitory concentration (sub 50) data for exemplary Compound 53.

Figure 32 shows inhibitory concentration (sub 50) data for exemplary compounds 58 and 61.

Figures 33A-33B show that compound 19 inhibits the growth of breast cancer cell line SUM159. Cells were seeded in 96-well plates at a density of 10000 cells/well in 100 ul of medium containing 2% charcoal stripped serum. After 24 hours, the indicated concentrations of compound 19 were added to the cells for an additional 48 hours. Cell viability was determined using the WST-1 assay. Figure 33A: Shows the effect of different concentrations of Compound 19 on cell growth, evident from the change in absorbance at A450nm. FIG. 33B : 10 μM treatment of HePG2 cells significantly downregulated the expression levels of lipogenic genes (black bars), as determined by RT PCR analysis; values are represented as mean ± SD; *P<0.05.

Figures 34A-34C show that compound 19 inhibits the growth of the human hepatoma cell line HePG2. Cells were seeded in 16-well plates at a density of 100000 cells/well in medium containing 2% charcoal-treated serum. After 24 hours, the indicated concentrations of compound 19 were added to the cells for an additional 48 hours. Figure 34A shows photographs of control and HePG2 cells treated with 25, 50 and 100 [mu]M Compound 19. Figure 34B shows a representative Western blot analysis showing that treated cells (T) had reduced levels of mature and active forms of SREBP-1 and higher levels of precursors compared to untreated controls. Figure 34C shows that 10 μM treatment of HePG2 cells significantly downregulated the expression levels of lipogenic genes and did not affect INSIG2 (which is known not to be regulated by SREBP) as determined by RT PCR analysis. Values are expressed as mean±SD; *P<0.05.

Figures 35A-35F show that Compound 19 inhibits the growth of the human acute lymphoblastic leukemia cell line MOLT-4 and the human multiple myeloma cell line RPMI8226. 10,000 MOLT-4 cells (Figure 35A-35C) and 20,000 RPMI8226 cells (Figure 35D-35F) were seeded in 96-well plates and cultured in RPMI 1640 containing 5% FBS or lipid-free carbon-treated serum (FF-FBS) base at 37C for 24 hours. MOLT-4 and RPMI8226 cells were treated with different concentrations of compound 19 for another 48 hours (None control without DMSO, vehicle with DMSO, 1, 2, 5, 10 and 20 μM in DMSO ; RPMI8226 cells were also treated with 3 μM in DMSO). At the end of 48 hours, cells were subjected to MTT assay to determine viability. Values are expressed as mean±SD; *P<0.05.

Figures 36A-36F show that Compound 17 inhibits the growth of the human acute lymphoblastic leukemia cell line MOLT-4 and the human multiple myeloma cell line RPMI8226. 20,000 MOLT-4 cells (FIGS. 36A-36C) and 20,000 RPMI8226 cells (FIGS. 36D-36F) were seeded in 96-well plates in 96-well plates containing 5% fetal bovine serum (FBS) or 5% lipid-free carbon-treated serum (FF -FBS) in RPMI1640 medium at 37C for 24 hours. Cells were then treated with different concentrations of compound 17 for an additional 48 hours (None control without DMSO, vehicle with DMSO, 1, 2, 3, 5, 10 and 20 μΜ in DMSO). At the end of the 48 hour treatment, cells were subjected to MTT assay to determine viability. Values are expressed as mean±SD (n=3); **P<0.001; ***P<0.0001.

Figures 37A-37D: Body weight and composition (fat and lean) of SD rats after three weeks of feeding RD and HFHC diets. Six- to seven-week-old male SD rats with an initial body weight of approximately 193±7.0 g/rat were fed a normal diet (RD) or a high-fat, high-carbohydrate diet for three weeks. Body weight (Fig. 37B), fat mass (Fig. 37C) and lean weight (leanweight) (Fig. 37D) content were measured using the 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 a HFHC diet for three weeks, after which treatment was initiated by oral gavage with different doses of Compound 19 or cottonseed oil vehicle for an additional eight weeks. Body weight and food intake were measured and cumulative values calculated. (Values are mean+S.E.M; *P<0.05; treated vs control vehicle group).

Figures 39A-39E show body composition fat and lean mass determined by ECHO MRI method. Figure 39A shows the total body weight in grams after compound 19 administration. Figure 39B shows total body fat in grams after compound 19 administration. Figure 39C shows the percentage of fat per animal after compound 19 administration. Figure 39D shows total lean body mass in grams following Compound 19 administration. Figure 39E shows percent lean body mass per gram following Compound 19 administration. Rats in different experimental groups were analyzed for body composition every two weeks. Percentages of fat and lean body mass were calculated from the weight of fat and mass relative to body weight. Labels in Figures 39B-39E correspond to the same groups in Figure 39A. Asterisks indicate significant differences between treatment and control groups (*P<0.05). Data are mean ± S.E.M (n=15).

Figures 40A-40D show the effect of Compound 19 on the liver of HFHC-fed SD rats. Figure 40A: Representative livers of treated (2.5 and 10 mg/kg) and control rats (n=5 per group). Figure 40B: Oil Red O staining of liver cryosections. Oil droplets are shown in red. Bars represent 200 μm. Figure 40C: TG and cholesterol levels in liver tissue from control and treated animals; Figure 40D: Gene expression controlled by SREBP-1 and 2 in treated rats compared to controls, measured by real-time PCR multiple change. ACC: acetyl-CoA carboxylase; ACL: ATP citrate lyase; SCD: stearoyl-CoA desaturase, MVD mevalonate decarboxylase; LDLR; LDL receptor; INSIG-1: insulin-inducible gene 1; mean±SE; *P<0.05.

Figure 41 shows the fold change in gene expression of UCP2 in rats treated with compound 19 compared to controls. Total RNA was extracted from liver tissue of control and rats treated with the indicated doses of Compound 19, and the level of UCP mRNA was determined using real-time PCR.

Detailed description of the invention

General Embodiments of the Invention

The compounds of the invention are used to treat and/or prevent metabolic disorders. For example, dysregulated fatty acid and cholesterol biosynthesis and excess dietary fat intake are associated with a variety of medical complications, including at least obesity, diabetes, hypertension, and cardiovascular disease, and in certain aspects, these conditions use this The compounds of the invention are used for treatment and/or prophylaxis. Epidemiological evidence suggests that metabolic diseases, including obesity, also contribute to the development of invasive forms of cancer, including, but not limited to, prostate cancer.

Upon fat consumption, sterol regulatory element-binding proteins (SREBPs) are hydrolyzed from membrane proteins and translocate into the nucleus, where they activate the transcription of genes involved in cholesterol and fatty acid biosynthesis. The present invention identifies a synthetic small molecule previously known to block adipogenesis and cancer cell growth as a selective inhibitor of SREBP activation, and also provides analogs and derivatives of this molecule. The drug-like molecule tustatin A attenuates the proteolytic activation of SREBP, thereby reducing the intracellular transcription of its responsive genes. In mice, phatustatin A blocks the activation of SREBP-1 in the liver, reduces body weight, lowers blood cholesterol and glucose levels, and downregulates lipogenic enzymes. The activities and expression levels of fatty acid synthase and acetyl-CoA carboxylase were reduced in the liver of treated mice. In certain aspects, phatustatin A is used as a tool for understanding cellular pathways and provides a consensus molecule as a starting point for pharmacological intervention of at least one metabolic disease.

In particular embodiments, small molecules that modulate metabolism-related phenotypes are used as tools for dissecting complex correlations. Fatuostatin A causes two distinct phenotypes in cultured mammalian cells: complete inhibition of insulin-induced adipogenesis in 3T3-L1 mouse fibroblasts; and serum-independent insulin-like in DU145 human prostate cancer cells Selective inhibition of growth factor 1 (IGF1)-dependent growth.

In certain aspects of the invention, Fatuostatin A selectively blocks the activation of SREBP, a key lipogenic transcription factor that activates specific genes involved in cholesterol and fatty acid synthesis. Fatuostatin A was identified as an inhibitor of SREBP consistent with its anti-adipogenic properties and points to a role for SREBP in IGF1 -dependent growth of prostate cancer. The present invention relates to compounds of fatostatin A, preferably analogues and derivatives thereof, which block at least the activation of SREBP-1, for example as shown in experimental mice. Administration of phatustatin A to obese ob/ob mice resulted in weight loss and a significant reduction in visceral fat. Moreover, during weight loss, the expression of uncoupling protein 1, uncoupling protein 2, and uncoupling protein 3 increased, and thermogenesis increased without a decrease in lean body mass.

The present invention relates to the treatment and/or prevention of at least one symptom of a metabolic disorder. The metabolic disorder may be of any type as long as one of its symptoms is improved or prevented by the compound of the present invention. Specifically, metabolic diseases result from one or more inborn errors of metabolism (which may be referred to as genetic disorders), such as caused by defective metabolic enzymes (e.g., enzymes with one or more mutations or disorders, including regulatory proteins and Inherited traits caused by mutations in the transport machinery).

In general, a metabolic disorder can be defined as a disorder that affects cellular energy production. While most metabolic disorders are inherited, some disorders may be acquired as a result of one or more factors, including diet, toxins, infections, and more. Inherited metabolic disorders may result from a genetic defect that results in the absence or improper construction of an enzyme necessary for a certain step in a cellular metabolic process. The largest class of metabolic disorders includes the following: 1) glycogen storage diseases (also known as glycogenosis or dextrinosis), including disorders affecting carbohydrate metabolism; 2) Fat oxidation disorders, which affect fat metabolism and the metabolism of fat components; and 3) mitochondrial disorders, which affect mitochondria. Examples of glycogen storage diseases (GSD) include at least Type I GSD (glucose-6-phosphatase deficiency; von Gierke's disease); Type II GSD (acid maltase deficiency; Pompe's GSD Type III (Glycogen Debranching Enzyme Deficiency; Cori's Disease or Forbe's Disease); GSD Type IV (Glycogen Branching Enzyme Deficiency; Anderson Disease ( Andersen disease); GSD type V (muscle glycogen phosphorylase deficiency; McArdle disease); GSD type VI (hepatic phosphorylase deficiency, Hers' disease); GSD type VII (muscle phosphofructokinase deficiency; Tarui's disease); GSD type IX (phosphorylase kinase deficiency); and GSD type XI (glucose transporter deficiency; Fanconi-Bickel disease) ).

In certain embodiments, a deficiency in fatty acid metabolism can be described as a fat oxidation disorder or a lipid storage disease. For example, they may involve one or more inborn errors of metabolism resulting from deficiencies in enzymes that affect the body's ability to oxidize fatty acids to produce energy in, for example, muscle, liver, and other cell types. Examples of fatty acid metabolism deficiencies include at least: CoA dehydrogenase deficiency; other CoA enzyme deficiencies; carnitine-related disorders; or lipid storage disorders. Examples of CoA dehydrogenase deficiencies include at least: very long-chain acyl-CoA dehydrogenase deficiency (VLCAD); long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD); medium-chain acyl-CoA dehydrogenase deficiency Hydrogenase deficiency (MCAD); short-chain acyl-CoA dehydrogenase deficiency (SCAD); and short-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency (SCHAD). Examples of other CoA 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 palmitoyl Transferase II deficiency (CPT). Examples of lipid storage diseases include acid lipase disease; Wolman disease; cholesterol ester storage disease; Gaucher disease; Pick disease; Fabry disease; Farber's disease; gangliosidoses; Krabbé disease; and metachromatic leukodystrophy leukodystrophy). Other disorders of fatty acid metabolism include at least mitochondrial trifunctional protein deficiency; electron transport flavoprotein (ETF) dehydrogenase deficiency (GAII and MADD); Tangier disease; and acute fatty liver of pregnancy. Examples of mitochondrial disorders include at least progressive external ophthalmoplegia (PEO); diabetes and deafness (DAD); Leber hereditary optic neuropathy (LHON), mitochondrial encephalomyopathy, lactic acid Lactic acidosis and stroke-like syndrome (MELAS); myoclonic epilepsy and ragged-red fibers (Myoclonic epilepsy and ragged-red fibers, MERRF); Leigh syndrome ( Leigh syndrome; subacute sclerosing encephalopathy; neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP); Kearns-Sell syndrome -Sayre syndrome, KSS); Myoneurogenic gastrointestinal encephalopathy (MNGIE). In particular aspects of the invention, the metabolic disorder is or has one or more of the following as one of its complications: obesity, hyperlipemia, diabetes, Fatty liver, hypertension and cardiovascular disease.

The present invention relates to the treatment of diseases associated with cellular hyperproliferation. In specific examples, cellular hyperproliferation can be caused by cancer or other neoplastic diseases or conditions. Without limitation, the hyperproliferative disease may be breast cancer, respiratory tract cancer, brain cancer, reproductive organ cancer, prostate cancer, digestive tract cancer, urinary tract cancer, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer Distant metastases from carcinoma, lymphoma, sarcoma, melanoma, leukemia, multiple myeloma, or solid tumors.

General Compounds Invented

Generally, the present invention provides compounds having the general formula: or pharmaceutically acceptable salts and stereoisomers thereof:

A-B-C

wherein A, B and C may be the same or different, and each may be a 5-, 6- or 7-membered ring or a fused bicyclic ring system, said ring being heterocyclic or non-heterocyclic, substituted or non-substituted The rings, A, B and C are connected directly or through an intervening chain of atoms or linker, and the chain of atoms or linker is a saturated carbon chain or an unsaturated carbon chain with or without additional functional groups.

Preferably, ring A is a 6-membered heterocyclic ring with one heteroatom. The A ring can be substituted. In preferred embodiments, the ring is a pyridine ring; more preferably, the nitrogen atom of the pyridine ring is at position 4 or 2 relative to the position of the B ring. Most preferably, the pyridine ring is substituted with n-propyl on the carbon alpha to the nitrogen heteroatom. In other preferred embodiments, side chains of 1-5 atoms, more preferably 1-5 carbons, are present on the A ring. In other exemplary and non-limiting embodiments, the A ring can be, for example, phenyl, pyrrole, thiophene, furan, pyrimidine, isoquinoline, quinoline, benzofuran, indole, Azole, naphthyl, piperidine, pyrrolidine, imidazole, imidazo[1,2-a]pyridine, benzimidazole, thiazole or benzothiazole.

In one aspect of the invention, ring A is a piperidine ring. Preferably, the nitrogen atom on the piperidine ring is at the 4 position relative to the position of the B ring. In other related aspects, the nitrogen atom on the piperidine ring is at position 3 relative to the position of the B ring. The nitrogen atom of piperidine may be further substituted, and wherein said substitution is selected from the group consisting of alkyl, sulfoxide, sulfone, alkyl or aryl sulfonate, sulfonic acid, and any combination thereof. For example, the substitution can be a propyl group, a tert-butyloxycarbonyl (BOC) or benzyloxycarbonyl group.

In another aspect of the present invention, ring A is a pyrrolidine ring; preferably, the nitrogen atom on the pyrrolidine ring is at the 2-position relative to the position of ring B. The nitrogen atom of the pyrrolidine may be further substituted, and wherein said substitution is selected from the group consisting of alkyl, sulfoxide, sulfone, alkyl or aryl sulfonate, sulfonic acid, and any combination thereof. For example, the substitution can be a propyl group, a tert-butyloxycarbonyl (BOC) or benzyloxycarbonyl group.

Preferably, the B ring is a 5-membered ring with at least two heteroatoms. The B ring can be substituted. In a preferred embodiment, ring B is a thiazole ring. In other exemplary and non-limiting embodiments, the B ring can be, for example Azole, imidazole, iso azole, imidazole, thiophene, furan, pyrimidine, pyrazole, isothiazole, thiazolopyridazine, aryl or pyrazole. The B ring can also be a 6-membered ring with two heteroatoms. For example, ring B is a piperazine ring. Preferably, the C ring is a 6-membered ring, most preferably a benzene ring. The C ring can be substituted. In a preferred embodiment, ring C is methyl substituted. In other exemplary and non-limiting embodiments, the C ring can be, for example, phenyl, pyridine, pyrrole, thiophene, furan, pyrimidine, isoquinoline, quinoline, benzofuran, indole, azole or naphthyl.

Exemplary compounds are provided in Figures 14A-14F and Tables 3 and 4. Referring to compound 1, 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, and the methyl substituted benzene ring corresponds to the C ring of the general formula. It is understood that substitutions are permitted at any position in any of the A, B and C rings, and that any substitution may be the same or different from any other substitution. In addition to those shown in Figures 14A-14F, non-limiting examples of substitutions include the following groups: H (ie, unsubstituted); hydroxyl; C _1-10 alkyl; C _2-10 alkenyl; C _{2 -10} alkynyl; C _3-6 cycloalkyl; Aryl; Heteroaryl; Wherein said alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl are optionally replaced by 1-5 Substituted by a group selected from the group consisting of: hydroxyl, -(C=O) ^Ra ; -(C=O) ^ORa , -(C=O)H, -(C=O)OH, O (CH ₂ ) _n COOR ^a , wherein n=1-10, and wherein R ^a is C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, or C _3-6 cycloalkyl, Aryl, heteroaryl, fluoro, chloro, bromo, iodo, cyano, carboxy, amino, mono- and di-substituted amino, mono- and di-substituted amido, and any combination thereof;- (C=O)R ^a ; -(C=O)OR ^a , -(C=O)H; -(C=O)OH; -O(CH ₂ ) _n COOR ^a , where n=1-10, And wherein R ^a is C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, or C _3-6 cycloalkyl, aryl or heteroaryl, fluorine, chlorine, bromine, iodine; cyano; carboxyl; amino; amido, with substituted monosubstituted and disubstituted amino groups selected from the group consisting of: C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-6 cycloalkyl, aryl, heteroaryl, sulfoxide, sulfone, sulfonate, alkylsulfonate, sulfonic acid and any combination thereof; wherein the alkyl, alkenyl, alkynyl, Cycloalkyl, aryl and heteroaryl are optionally substituted with 1-5 groups selected from the group consisting of: hydroxyl, -(C=O)R ^a , -(C=O)OR ^a , -(C=O)H, -(C=O)OH, -O(CH ₂ ) _n COOR ^a , where n=1-10, and where R ^a is C _1-10 alkyl, C _2-10 Alkenyl, C _2-10 alkynyl, or C _3-6 cycloalkyl, aryl and heteroaryl; fluorine; chlorine; bromine; iodine; cyano; carboxyl; amino; having one or more of the following groups Monosubstituted and disubstituted amino groups: C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl groups, and any combination thereof; and, having a group selected from the group consisting of Substituted mono- and di-substituted amido groups: C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-6 cycloalkyl, aryl, heteroaryl, sulfoxide, Sulfone, sulfonate, alkylsulfonate, sulfonic acid, sulfonate, alkylsulfonate, sulfonic acid and any combination thereof; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aromatic and heteroaryl are optionally substituted with 1-5 groups selected from the group consisting of: hydroxyl; -(C=O)R ^a ; -(C=O)OR ^a , -(C= O)H;-( C=O)OH, -O(CH ₂ ) _n COOR ^a , where n=1-10, and where R ^a is C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, or C _3-6 cycloalkyl, aryl or heteroaryl; fluorine; chlorine; bromine; iodine; cyano; carboxyl; amino; _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl groups, and any combination thereof.

In the present invention, there are compounds having the following general formula or pharmaceutically acceptable salts or stereoisomers thereof: ABC, wherein A, B, and C are the same or different, and wherein each includes a 5-, 6-, or 7-membered ring or a fused bicyclic ring system, the rings being heterocyclic or non-heterocyclic, substituted or non-substituted rings, wherein A, B and C are connected directly or through an intervening chain of atoms or a linker, and wherein the atoms The 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 any one of the substituents may be the same or different from any other substitution, and wherein the substitutions consist of: a) hydroxyl, b) C1-10 alkyl, c) C2-10 alkenyl , d) C2-10 alkynyl, e) C3-6 cycloalkyl, f) aryl, g) heteroaryl, wherein b), c), d), e), f) and/or g) Said substitution of is optionally further substituted with 1-5 groups consisting of: 1) hydroxyl, 2) -(C=O)R ^a , 3) -(C=O)OR ^a , 4) -(C=O)H, 5)-(C=O)OH, 6)-O(CH ₂ ) _n COOR ^a , where n=1-10, 7) halogen, 8) cyano, 9) carboxy, 10) amino group, 11) monosubstituted amino group, 12) disubstituted amino group, 13) amido group, 14) monosubstituted amido group; 15) disubstituted amido group, and any combination thereof, wherein in 2), 3) or 6), R ^a is C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-6 cycloalkyl, aryl or heteroaryl, h)-(C=O) R ^a , i)-(C=O)OR ^a , j)-(C=O)H, k)-(C=O)OH; l)-O(CH ₂ )nCOOR ^a , where n=1- 10, wherein in h), i) or l), R ^a is C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-6 cycloalkyl, aryl or hetero Aryl, m) halogen, n) cyano, o) carboxyl, p) amino, q) monosubstituted amino, r) disubstituted amino, s) amido, t) monosubstituted amido, and u) Disubstituted amido, wherein one or more of the monosubstituted amino, disubstituted amino, monosubstituted amido and disubstituted amido has a substitution selected from the group consisting of: C1-10 Alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-6 cycloalkyl, aryl, heteroaryl, sulfoxide, sulfone, sulfonate, alkylsulfonate, sulfonic acid, and their Any combination, wherein in u), said alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heteroaryl is optionally further substituted with 1-5 groups selected from the group consisting of : i) hydroxyl, ii) -(C=O)R ^a , iii) -(C=O)OR ^a , iv) -(C=O)H, v) -(C=O)OH, vi) -O(CH ₂ ) _n COOR ^a , where n=1-10, wherein in ii), iii) or vi), R ^a is C _1-10 alkyl, C _2-10 alkenyl, C _{2-10 Alkynyl} , C cycloalkyl, aryl or heteroaryl, vii) halogen, viii) cyano, ix) carboxy, x) amino, xi) monosubstituted amino, xii) disubstituted amino, xiii ) amido; xiv) monosubstituted amido, xv) disubstituted amido, and any combination thereof.

Exemplary compounds, compositions, formulations and methods of use

In one embodiment of the invention there is provided a compound having the chemical structure A-B-C, wherein A is pyridine or substituted pyridine, piperidine or substituted piperidine, pyrrolidine or substituted pyrrolidine, thiazole or substituted thiazole, benzene ring or substituted benzene ring; B is thiazole or substituted thiazole, piperazine or substituted piperazine, benzene ring or substituted benzene ring; and C is benzene ring or substituted benzene ring, pyridine or substituted pyridine, thiazole or substituted Thiazoles.

In a preferred aspect, the chemical structure is:

Wherein R ₁ is H, halogen, -OH, -OC _{1 -3} alkoxy, -OC(O)R ₃ ; R ₃ is C ₁ -C ₃ alkyl or aryl, -OCH ₂ -C(O) OR ₄ ; R ₄ is H or C ₁ -C ₃ alkyl, -NHR ₅ ; R ₅ is H, C ₁ -C ₄ alkyl, alkylcyclopropane, benzyl, -NHC(O)C ₁ -C ₃ amide, -NHC(O)OR ₆ carbamate; R ₆ is tert-butyl or fluorenylmethyl or -NH-SO ₂ -R ₇ sulfonamide; R ₇ is alkyl or aryl, R ₂ is Alkyl or R ₈ OC(O)-, and R ₈ is C ₃ -C ₅ alkyl or aryl. In particular, said halogen may be bromine.

In another preferred aspect, the chemical structure is:

Wherein R ₁ is H, halogen, -OH, -OC _{1 -3} alkoxy, -OC(O)R ₃ ; R ₃ is C ₁ -C ₃ alkyl or aryl, -OCH ₂ -C(O) OR ₄ ; R ₄ is H or C ₁ -C ₃ alkyl, -NHR ₅ ; R ₅ is H, C ₁ -C ₄ alkyl, alkylcyclopropane, benzyl, -NHC(O)C ₁ -C ₃ amide, -NHC(O)OR ₆ carbamate; R ₆ is tert-butyl or fluorenylmethyl or -NH-SO ₂ -R ₇ sulfonamide; R ₇ is alkyl or aryl, R ₂ is Alkyl or R ₈ OC(O)-, and R ₈ is C ₃ -C ₅ alkyl or aryl. In particular, said halogen may be bromine.

In another preferred aspect, the chemical structure is:

Wherein R ₉ is H, halogen, -OH, -OC ₁ -C ₃ alkoxyl, -OC(O)R ₁₁ ; R ₁₁ is C ₁ -C ₃ alkyl or aryl, -OCH ₂ -C(O )OR ₁₂ ; R ₁₂ is H or C ₁ -C ₃ alkyl, -NHR ₁₃ ; R ₁₃ is H, C ₁ -C ₄ alkyl, alkylcyclopropane, benzyl, -NHC(O)C ₁ - ₃ amide, -NHC(O)OR ₁₄ carbamate; R ₁₄ is tert-butyl or fluorenylmethyl, -NH-SO ₂ -R ₁₅ sulfonamide; R ₁₅ is alkyl or aryl or -SO ₂ -NH-R sulfonamide _{; R 16 is alkyl or aryl, R 10 is nitrogen} or methylene, n is 0 or 1, and when n is 1, Z is -C=O; and A is

Wherein R ₁₇ is H or C ₁ -C ₃ alkyl.

In another preferred aspect, the chemical structure is:

In a related embodiment, there is provided a pharmaceutical composition comprising the aforementioned compound and a pharmaceutically acceptable excipient. In another related embodiment there is provided the aforementioned compound formulated as a food, animal feed material or medicament. In another related embodiment, there is provided a kit comprising the aforementioned compound and a container containing said compound. The container may be any suitable container suitable for storing medicaments known in the art and commercially available.

In another embodiment of the present invention, the preferred compound is N-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)methanesulfonamide, 2-(4- (4-bromophenyl)thiazol-2-yl)pyrrolidine-1-carboxylic acid tert-butyl ester, 2-(4-(4-bromophenyl)thiazol-2-yl)pyrrolidine-1-carboxylic acid benzyl ester, 4-(4-bromophenyl)-2-(pyrrolidin-2-yl)thiazole, 4-(4-bromophenyl)-2-(1-propylpyrrolidin-2-yl)thiazole, 3- (4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylic acid tert-butyl ester, 3-(4-(4-bromophenyl)thiazol-2-yl)piperidine-1-carboxylic acid Benzyl ester, 3-(4-(4-bromophenyl)thiazol-2-yl)-1-propylpiperidine, 4-(4-(4-bromophenyl)thiazol-2-yl)piperidine- Benzyl 1-carboxylate, (R)-2-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)pyrrolidine-1-carboxylic acid benzyl ester, 3-(4-(4 -(methylsulfonamido)phenyl)thiazol-2-yl)piperidine-1-carboxylic acid benzyl ester, 4-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl) Benzyl piperidine-1-carboxylate, 4-(3-(pyridin-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-N-toluene Sulfonylaniline, (4-(5-chloro-2-methylphenyl)piperazin-1-yl)(4-(tosylamino)phenyl)methanone, 4-(4-((1- Methyl-1H-benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)-N-tosylaniline, 3-chloro-4-methyl-N-(6-(4 -(3-(trifluoromethyl)benzyl)piperazin-1-yl)pyridin-3-yl)benzenesulfonamide, 4-chloro-N-(4-(4-((1-methyl-1H -Benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)phenyl)benzenesulfonamide, (Z)-4-(3-cyano-3-(4-(2,4 -Dimethylphenyl)thiazol-2-yl)allyl)-N-(thiazol-2-yl)benzenesulfonamide, N-(3-(H-imidazo[1,2-a]pyridine- 2-yl)phenyl)-4-methyl-2-phenylthiazole-5-carboxamide, N-(3-(benzo[d]thiazol-2-yl)phenyl)isonicotinamide, 3- (4-chlorophenyl)-4,5-dihydro-1-phenyl-5-(2-phenylthiazol-4-yl)-1H-pyrazole, N-(4-(6-methylbenzene And[d]thiazol-2-yl)phenyl)-2-(N-m-tolylmethylsulfonamido)acetamide, N-(4-(6-methylbenzo[d]thiazole-2- yl)phenyl)-2-(N-p-tolylmethylsulfonamido)acetamide; pharmaceutically acceptable salts thereof; stereoisomers; or any combination thereof.

In another embodiment of the present invention, there is provided a method for treating a metabolic disorder in an animal, said method comprising administering to said animal a therapeutically effective amount of at least one of the aforementioned compounds or a pharmaceutically acceptable salt or stereoisomer thereof or a combination of them.

Further to this embodiment, said method comprises the step of providing said animal with a second therapy. In this further embodiment, said second therapy comprises diet therapy, physical therapy, behavioral therapy, surgery, drug therapy, chemotherapy or a combination thereof. In particular, said second therapy may be lifestyle changes, antihyperglycemic agents, insulin, glucagon-like peptide (GLP), dipeptidyl peptidase-4 inhibitors, thiazolidinediones, lipid-lowering compounds or A combination of two or more of them.

In two embodiments, the metabolic disorder is a weight-related condition, a disease associated with cellular hyperproliferation, hyperlipidemia, diabetes or its complications, fatty liver, hypertension or cardiovascular disease. In particular, the metabolic disorder may be obesity, hypertension, arteriosclerosis, asthma, hyperlipidemia, hyperinsulinemia, non-alcoholic fatty liver liver) and type 2 diabetes caused by insulin resistance. In one aspect of these embodiments, the metabolic disorder is a body weight related disorder, wherein the therapeutically effective amount of the compound increases the expression of uncoupling protein 1, uncoupling protein 2 or uncoupling protein 3. Furthermore, in this aspect, the therapeutically effective amount of the compound increases thermogenesis without reducing lean body mass during weight loss in the animal. In another aspect, the disease associated with cellular hyperproliferation is cancer. In particular, the cancer is breast cancer, respiratory tract cancer, brain cancer, reproductive organ cancer, prostate cancer, digestive tract cancer, urinary tract cancer, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer, lymphoma , sarcomas, melanomas, leukemias, or distant metastases from solid tumors.

In another embodiment, there is provided a method for treating a cell hyperproliferative disease in a patient in need thereof, said method comprising administering to said patient a therapeutically effective amount of at least one of the aforementioned compounds or a pharmaceutically acceptable salt or stereo isomers or their combinations. In particular, the cellular hyperproliferative disease may be a cancer as described above.

In a related embodiment, there is provided a method for treating cancer in a patient in need thereof, said method comprising the step of administering to said patient a therapeutically effective amount of one or more of the foregoing pharmaceutical compositions. The cancer can be as described herein.

In another embodiment, there is provided a method for reducing body weight in an animal in need thereof, said method comprising the step of administering to said animal a therapeutically effective amount of one or more of the foregoing compounds in a pharmaceutically acceptable vehicle.

In another embodiment of the present invention there is provided a method for increasing thermogenesis without reducing lean body mass during weight loss in an animal, said method comprising administering to said animal a therapeutically effective amount of The step of compound or pharmaceutically acceptable salt or stereoisomer thereof or their combination, said compound has following structure:

Wherein the R substituent is H, Et, OMe or _n -propyl; Y is CH or R ₂ is OH, OMe or NH-i-Pr; R ₃ is H, F or Cl; R ₄ is H, Me, Cl, Br, F, OH, OBz, OCH ₂ COOMe, OCH ₂ COOH, NH ₂ , NH-i-Pr, NHCOMe, NHSO ₂ Me, NHBn, OMe, NHBoc, NHTs, Using the methods of the invention, the total body fat of the animals is reduced following administration of the compounds.

In all of these embodiments and aspects thereof, one of ordinary skill in the art can readily determine useful dosages of the compounds of the invention depending on the metabolic disorder to be treated, such as, but not limited to, and the desired outcome. Diseases associated with cellular hyperplasia or weight-related conditions. Typically, the compounds are administered at a dose of about 1 mg/kg to about 100 mg/kg. The compounds can be administered in compositions in the form of food, animal feed material or pharmaceuticals. In a preferred aspect, the animal's body weight is reduced by increasing unlinked thermogenesis with or without a concomitant loss of lean body mass. Typically, the compounds increase the expression of uncoupling proteins. Representative examples of uncoupling proteins include uncoupling protein 1, uncoupling protein 2, and uncoupling protein 3. The methods of the invention are 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, asthma, hyperlipidemia, hyperinsulinemia hyperemia, nonalcoholic fatty liver disease, and type 2 diabetes mellitus caused by insulin resistance. Also, those of ordinary skill in the art will readily recognize the use of providing the animal with a second therapy including, but not limited to, lifestyle changes, antihyperglycemic agents, insulin, glucagon-like Peptides (GLP), dipeptidyl peptidase-4 inhibitors, thiazolidinediones, lipid-lowering compounds and combinations of two or more thereof.

As used in this specification, "a" ("a" or "an") may mean one or more. As used in the claims, the word "a" ("a" or "an") when used in conjunction with the word "comprises" may mean one or more than one. As used herein, "another" may mean at least a second or more. In particular embodiments, for example, aspects of the invention may "consist essentially of" or "consist of" one or more sequences of the invention. 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 practiced in accordance with any other method or composition described herein.

The term "animal" as used herein refers to a mammal, preferably a human, patient, subject or individual, who receives or has been administered one or more compounds, compositions or formulations described herein.

_The term "alkyl" is used herein to refer to a substituted monovalent group (i.e., _-CH3 , _-CH2CH3 , -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 etc.).

The term "alkenyl" is used herein to refer to a substituted monovalent group (i.e., -CH =CH ₂ , -CH=CHCH ₃ , -C=C(CH ₃ ) ₂ , -CH ₂ CH=CH ₂ etc.).

The term "alkynyl" is used herein to refer to a substituted monovalent group (i.e., -C ≡CH, -C≡CCH ₃ , -C≡CCH(CH ₃ ) ₂ , -CH ₂ C≡CH, etc.).

The term "aryloxy" as used herein refers to an aryl group having a bridging oxygen atom, such as phenoxy (-OC ₆ H ₅ ) or benzoyloxy (-OCH ₂ C ₆ H ₅ ). " _Arylamino " means an aryl group having a _bridging amine functionality, eg _-NHCH2C6H5 . " _Arylamido " means an aryl group having a bridging amide group, eg _- (C=O) _NHCH2C6H5 .

The term "alkylene" is used herein to refer to a substituted divalent group (i.e. = _CH2 , =CHCH ₃ , =C(CH ₃ ) ₂ etc.).

The term "cycloalkyl" is used herein to refer to a substituted monovalent group (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cyclo heptyl).

The term "aryl" is used herein to refer to a substituted monovalent group conceptually removed from a monocyclic or bicyclic aromatic hydrocarbon by removing one hydrogen atom. Examples of aryl groups are phenyl, indenyl and naphthyl.

The term "heteroaryl" is used herein to denote a group obtained 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 Substituted monovalent groups. Examples of heteroaryl groups include, but are not limited to: pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, Azolyl, iso Azolyl, thiazolyl, pyridyl, pyrimidinyl, pyrazinyl, benzimidazolyl, indolyl and purinyl. Heteroaryl substituents can be attached to a carbon atom or via a heteroatom. Examples of monocyclic heteroaryl groups include: pyrrolyl, furyl, thienyl, pyrazolyl, Azolyl, iso Azolyl, thiazolyl and pyridyl. Examples of bicyclic heteroaryl groups include: pyrimidinyl, pyrazinyl, benzimidazolyl, indolyl and purinyl. A single ring can have 5 or 6 atoms. Thus, this includes 4-membered monocyclic heteroaryl groups and 5-membered monocyclic heteroaryl groups. Also included are bicyclic heteroaryl groups having one 5-membered ring and one 6-membered ring and bicyclic heteroaryl groups having two 6-membered rings.

The term "halogen" includes iodine, bromine, chlorine and fluorine.

The term "substituted" is understood to include multiple degrees of substitution of substituents. Substitution occurs when the valences on a chemical group or moiety are satisfied by an atom or functional group other than hydrogen. In the case of multiple substitutions, the substituted compounds may be independently substituted by one or more disclosed or claimed substituent moieties, in singular or plural form. Independent substitution means that the (two or more) substituents may be the same or different.

The term "pharmaceutically acceptable salt" 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, etc.; or 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, bitter almond acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid , camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, etc.; Or a salt formed when an aluminum ion is substituted; or a salt formed by coordination with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc.

The term "stereoisomer" means isomeric molecules having the same connectivity of atoms as one or more other molecules, but differing in the arrangement of the atoms in space. This definition includes enantiomers, diastereomers, cis isomers, trans isomers, conformational isomers.

The term "unsubstituted" means that all valences on a chemical group or moiety are saturated with hydrogen.

The term "saturated carbon chain" as used herein refers to straight or branched acyclic saturated hydrocarbons (i.e., -CH ₃ , -CH ₂ -, -CH ₂ CH ₃ , -CH ₂ CH ₂ --CH _{2 CH2CH3} , _{-CH2CH2CH2 , -CH2CH2CH2CH3 , -CH2CH2CH2CH3- , -CH2CH ( CH3 ) 2 , -C ( CH3 ) 3} etc.).

The term "unsaturated carbon chain" as used herein refers to a carbon chain containing at least one carbon-carbon double bond (ie, -CH=CH ₂ , -CH=CH-, -CH=CHCH ₃ , -C=C(CH ₃ ) ₂ , -CH ₂ CH=CH ₂ , -CH ₂ CH=CH-, etc.) or at least one carbon-carbon triple bond (ie, -C≡CH, -C≡C-, -C≡CCH ₃ , -C ≡CCH-, -C≡CCH (CH ₃ ) ₂ , -CH ₂ C≡CH, etc.) straight-chain or branched acyclic unsaturated hydrocarbon.

The invention also includes protected derivatives of the compounds disclosed herein. For example, when the compounds of the present invention contain groups such as hydroxy or carbonyl, these groups can be protected with appropriate protecting groups. For a list of suitable protecting groups see T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, Inc. 1981, the disclosure of which is fully incorporated herein by reference. Protected derivatives of the 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 exist as racemates, racemic mixtures and individual diastereomers and all possible isomers and mixtures thereof, Optical isomers are included and are included within the scope of the present invention. Furthermore, the compounds disclosed herein may exist in tautomeric forms and both tautomeric forms are contemplated within the scope of the invention even though only one tautomeric structure is depicted.

In a specific embodiment of the invention, compositions of the invention target one or more members of the sterol regulatory element binding protein (SREBP) pathway. This pathway involves the proteolytic release of the membrane-bound transcription factor SREBP, which, in particular aspects, facilitates translocation from the cytoplasm to the nucleus. In the nucleus, SREBP binds to elements called sterol regulatory elements (SREs) present in regulatory regions of genes encoding lipid production-related enzymes. After SREBP binds to DNA, it regulates the transcription of target genes, eg, upregulates them.

pharmaceutical preparations

The pharmaceutical compositions of the present invention comprise an effective amount of one or more compositions of the present invention (and additional agents as appropriate) dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable carrier" refers to molecular entities and compositions that do not produce side effects, allergic or other adverse reactions when properly administered to an animal (eg, a human). According to the disclosure of the present invention, the preparation of a pharmaceutical composition containing at least one phatustatin A analog or derivative or other active ingredients is known to those skilled in the art, such as Remington's Pharmaceutical Sciences (Remington Pharmacopoeia), 18th Edition. As illustrated by Mack Printing Company, 1990. Also, for animal (eg, human) administration, it is understood that preparations should meet sterility, pyrogenicity, general safety and purity standards, as required by FDA's Office of Biological Standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, Salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, similar substances, and combinations thereof, as per ordinary skill in the art Known to the person (see eg, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Its use in pharmaceutical compositions is presently contemplated in addition to any conventional carriers incompatible with the active ingredient.

Depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for the route of administration such as injection, the phatustatin A analog or derivative may contain different types of carriers. The present invention may be administered intravenously, intradermally, percutaneously, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, intramucosally, orally, locally, locally, Inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized infusion of directly infiltrated target cells, via catheter, by lavage, in emulsion, lipid composition (e.g., liposomes), or by present Administration by other methods known to those of ordinary skill in the art, or any combination of the foregoing (see, eg, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990).

Fatuostatin A analogs or derivatives can be formulated as compositions in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, for example with free amino groups of proteinaceous compositions, or with inorganic acids, such as, for example, hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid or mandelic acid . Salts formed with the free carboxyl groups can be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or organic bases, such as isopropylamine, trimethylamine, histidine or procaine. After reconstitution, solutions are administered in a manner compatible with the dosage form and in therapeutically effective amounts. The formulations can be readily administered in a variety of dosage forms, for example formulated for parenteral administration, such as injectable solutions, or aerosols for delivery to the lung, or for ingestive administration, such as drug release capsules and the like.

Further, according to the present invention, compositions of the present invention suitable for administration are provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and include liquid, semi-solid (ie paste) or solid carriers. The use of the compositions for administration thereof in the practice of the methods of the present invention is suitable in addition to any conventional medium, agent, diluent or carrier which is deleterious to the recipient or to the therapeutic effect of the composition contained therein, at present. Examples of carriers or diluents include: fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, etc., or combinations thereof. The composition may also contain various antioxidants to retard oxidation of one or more components. In addition, the action of microorganisms can be prevented by preservatives, such as various antibacterial and antifungal agents, which include, but are not limited to, parabens (such as methylparaben, propylparaben ), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof. According to the invention, the composition is combined with the carrier in any conventional and practicable manner, for example by solution, suspension, emulsification, mixing, encapsulation, absorption and the like. These methods are known to those skilled in the art.

In a particular embodiment of the invention, the composition is intimately combined or mixed with a semi-solid or solid carrier. Mixing can be done in any conventional way, such as grinding. Stabilizers may also be added during mixing to protect the composition from loss of therapeutic activity, ie denaturation in the stomach. Examples of stabilizers that can be used in the composition include: buffers, amino acids such as glycine and lysine, sugars such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc. .

In other embodiments, the present invention contemplates the use of pharmaceutical lipid excipient compositions comprising a phatuostatin A analog or derivative, one or more lipids, and an aqueous solvent. As used herein, the term "lipid" is defined to include any of a broad range of substances that are characteristically insoluble in water and extractable with organic solvents. This broad class of compounds is well known to those skilled in the art and when the term "lipid" is used herein it is not limited to any particular structure. Examples include compounds containing long-chain aliphatic hydrocarbons and derivatives thereof. Lipids can be naturally occurring or synthetic (ie, artificially designed or prepared). Lipids, however, are generally a biological substance. Biological lipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids containing ether and ester linkages Lipids and polymeric lipids of fatty acids, and combinations thereof.

Those of ordinary skill in the art are familiar with the range of techniques that can be used to disperse compositions into lipid vehicles. For example, a phatuostatin A analog or derivative can be dispersed into a lipid-containing solution, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, mixed with a lipid, by any means known to those of ordinary skill in the art. Assembled, covalently bound to lipids, contained in lipids as a suspension, contained or complexed with micelles or liposomes, or otherwise associated with lipids or lipid structures. Dispersions may or may not result in the formation of liposomes.

Actual dosages of compositions of the present invention administered to animal patients may be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, past or concurrent therapeutic interventions, patient idiosyncrasies, and route of administration. . Depending on the dose and route of administration, the preferred dosage and/or the frequency of administration of an effective amount may vary according to the response of the subject.

In certain embodiments, pharmaceutical compositions may contain, for example, at least about 0.1% active compound. In other embodiments, the active compound may, for example, constitute from about 2% to about 75% by weight of the unit, or from about 25% to about 60%, and any ranges derivable therein. The amount of active compound in each therapeutically effective composition may, of course, be prepared in such a manner that an appropriate 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, and other pharmacological considerations are within the purview of those skilled in the art of preparing such pharmaceutical formulations, as such, various dosage and treatment regimens will be required.

In other non-limiting examples, the dosage for each administration may also comprise about 1 microgram/kg/body weight, about 5 micrograms/kg/body weight, about 10 micrograms/kg/body weight, about 50 micrograms/kg/body weight, about 100 micrograms/kg/body weight, about 200 micrograms/kg/body weight, about 350 micrograms/kg/body weight, about 500 micrograms/kg/body weight, about 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 50 mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about 350 mg/kg/body weight, about 500 mg/kg/body weight, to about 1000 mg/kg/body weight kg/body weight or higher, and any range that can be derived. In non-limiting examples of ranges derivable from the numbers listed here, based on the above numbers, about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 micrograms/kg/body weight to about 500 mg/kg/body weight may be administered The range of kg/body weight etc.

Digestive Compositions and Preparations

The phatuostatin A analog or derivative is formulated for administration by the digestive route. Digestive routes include all possible routes of administration in which the composition comes into direct contact with the digestive tract. Specifically, the pharmaceutical compositions disclosed herein can be administered orally, buccally, rectally or sublingually. Thus, the 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 capsules, or they may be compressed into tablets, or they may be incorporated directly into the diet.

The active compounds may be combined with excipients and administered in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, sachets, etc. (Mathiowitz et al., 1997; Hwang et al. 1998; US Patent Nos. 5,641,515; 5,580,579; and 5,792,451). Tablets, troches, pills, capsules, etc. may also contain the following: binders, such as, for example, tragacanth, acacia, cornstarch, gelatin, or combinations thereof; excipients, such as, for example, dicalcium phosphate, mannitol, lactose , starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; disintegrants, such as, for example, corn starch, potato starch, alginic acid, or combinations thereof; lubricants, such as, for example, magnesium stearate; sweetening agents such as, for example, sucrose, lactose, saccharin, or combinations thereof; flavoring agents, such as, for example, peppermint, oil of wintergreen, cherry flavoring, orange flavoring, and the like. 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 example, 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. The enteric coating prevents denaturation of the composition in the stomach or upper intestinal tract where the pH is acidic. See, eg, US Patent No. 5,629,001. On reaching the small intestine, the alkaline pH therein dissolves the coating and allows the composition to be released and taken up by specific cells, such as epithelial enterocytes and Peyer's patch M 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. Of course, any material used in the preparation of any unit dosage form should be pharmaceutically pure and substantially nontoxic in the amounts employed. In addition, the active compounds can be incorporated into sustained release formulations and formulations.

For oral administration, the compositions of the present invention may optionally be combined with one or more excipients to form mouthwashes, toothpastes, buccal tablets, oral sprays or sublingual oral formulations. For example, a mouthwash may be prepared by incorporating the active ingredient in the required amount in a suitable solvent such as sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as a solution containing sodium borate, glycerin, and potassium bicarbonate, or dispersed in a toothpaste, or added in a therapeutically effective amount which may include water, binders, abrasives, flavorings, In the combination of foaming agent and wetting agent. Alternatively, the composition may be formulated as a tablet or solution that can be placed under the tongue or otherwise dissolved in the oral cavity.

Other formulations suitable for other ingestive modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, eg, usually inserted into the rectum for administration. After insertion, the suppository softens, melts, or dissolves in the cavity fluid. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing the active ingredient, for example, from about 0.5% to about 10%, and preferably from about 1% to about 2%.

Parenteral Compositions and Preparations

Fatuostatin A analogs or derivatives can be administered parenterally. As used herein, the term "parenteral" includes routes that bypass the digestive tract. Specifically, the route of administration of the pharmaceutical composition disclosed herein may be, for example, but not limited to: intravenous, intradermal, intramuscular, intraarterial, intrathecal, subcutaneous or intraperitoneal. 5,543,158; 5,641,515; and 5,399,363.

Solutions of the active compounds as free base or pharmacologically acceptable salt forms can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can 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 dispersion (US Patent 5,466,468). In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium including, for example, water, ethanol, polyol (ie, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable compounds thereof, and/or vegetable oils. For example, with coatings such as lecithin, for dispersions, by maintaining the desired particle size, and using surfactants, proper fluidity can be maintained. 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. In many cases, it will be preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, such as aluminum monostearate and gelatin.

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. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Sterile aqueous media which can be used in this regard are known to the person skilled in the art from the disclosure of the present invention. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid, or injected at the suggested infusion site (see, e.g., "Remington's Pharmaceutical Sciences "Remington's Pharmacopoeia") 15th ed., pp. 1035-1038 and pp. 1570-1580). Some modification in dosage will be required depending on the status of the subject requiring treatment. In any event, the person responsible for administering will determine the appropriate dosage for an individual subject.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in an appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, 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. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization techniques which yield a powder of the active agent plus any other desired ingredient from a previously sterile-filtered solution thereof. . Powder compositions are combined with a liquid carrier such as water or saline solution, with or without stabilizers.

The active compound phatustatin A analogs or derivatives may be formulated for administration by a variety of routes, such as topical (i.e. transdermal), mucosal (intranasal, vaginal, etc.) and/or inhalation medicine. Pharmaceutical compositions for topical administration may comprise the active compound formulated for application in an application such as an ointment, paste, cream or powder. Ointments include all oils, absorbents, emulsions and water-soluble based compositions for topical application, whereas creams and lotions are those compositions comprising only an emulsion base. Medications for topical administration may contain penetration enhancers to facilitate the absorption of the active ingredient through the skin. Suitable penetration enhancers include glycerol, alcohols, alkylmethylsulfoxides, pyrrolidone and luarocapram. Possible bases for compositions for topical application include: polyethylene glycol, lanolin, cold cream and petrolatum and any other suitable absorbent, emulsion or water-soluble ointment base. Topical formulations may also contain emulsifiers, gelling agents, and antimicrobial preservatives, as desired, to preserve the active ingredients and provide a uniform mixture. Transdermal administration of the present invention may also involve the use of "patches". For example, a patch may provide one or more active substances at a predetermined rate and in a continuous manner over a certain period of time.

The pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalants and/or other aerosol delivery vehicles. Methods of delivering compositions directly to the lung by nasal aerosol have been described, for example, in US Patent Nos. 5,756,353 and 5,804,212. Similarly, drug delivery using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (US Patent No. 5,725,871 ) is also well known in the pharmaceutical arts. Transmucosal delivery of drugs in the form of polytetrafluoroethylene carrier matrices is described in US Patent No. 5,780,045.

The term aerosol refers to a colloidal system of finely divided solids of liquid particles dispersed in a liquefied or pressurized gaseous propellant. Typical aerosols for inhalation according to the invention consist of a suspension of the active ingredient in a liquid propellant or a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers.

combination therapy

To increase the effectiveness of the compositions of the invention, other therapies may be delivered to individuals with metabolic disorders. For example, an obese individual can be administered a composition of the invention in addition to another therapy for obesity. For example, additional obesity treatments include diet therapy, physical therapy (exercise), drug therapy, surgery, and behavioral therapy. Exemplary drug therapies include, for example: (Xenical ), Phentermine, and Sibutramine Exemplary procedures include, for example, liposuction and gastric bypass.

For individuals with diabetes, for example, exemplary additional therapeutic compounds include one or more of the following: Actos (pioglitazone); ACTOSPlus Met; Amaryl (Glycine glimepiride); Avandaryl (Avandia + glimepiride); Avandia (rosiglitazone); Duetact (pioglitazone HCl and glimepiride); Galvus (Vildagliptin); Glipizide (Sulfonlyurea); Glucophage (metformin); Glimepiride; Glucovance (glyburide/metformin); Glyburide (Glyburide); Glyset (miglitol) glycosidase inhibitor; Januvia (sitagliptin phosphate); Glipizide-metformin (Metaglip) (glipizide + metformin); metformin-biguanide (Metformin–biguanide); Prandin (repaglinide); Precose (acarbose ); Rezulin (troglitazone); Starlix (nateglinide). Other diabetes treatments include improving diet and exercise.

Exemplary Measurements of Metabolic Disorder Treatment

In a particular aspect of the invention, an individual is administered one or more compositions of the invention, and the individual is assessed for improvement in at least one symptom of a metabolic disorder. For example, in particular embodiments where the metabolic disorder is obesity, an improvement in obesity during and/or following treatment with one or more compositions of the invention may be determined. Improvement in adiposity can be measured by any standard means, but in particular aspects, amelioration in adiposity is determined, for example, by weight measurement, body mass index (BMI) measurement, and/or body part size measurement (eg, waist circumference measurement). An exemplary method of calculating BMI includes dividing a person's weight in kilograms by the square of his height in meters (weight [kg] height [m] ² ). A BMI above 30 is considered obese, and a BMI between 25 and 29.9 is considered overweight. In other aspects of the invention, improvement in a diabetic individual is detected after administration of the therapy of the invention to said individual. In a specific embodiment, diabetes is monitored by blood testing. For example, a blood test can measure the chemical A1C. The higher the blood sugar, the higher the A1C level will be. In some cases, cholesterol (including HDL and/or LDL cholesterol) and/or triglycerides are measured, such as by standard means in the art. In particular cases, fasting lipoprotein profiles are performed, such as by standard means in the art.

Kit of the present invention

Any of the compositions described herein can be included in a kit. In a non-limiting example, a kit includes a composition suitable for treating and/or preventing one or more metabolic disorders. In other embodiments of the invention, kits include one or more devices for sampling from an individual. For example, the instrument may be one or more of a swab (such as a cotton swab), a toothpick, a scalpel, a spatula, a syringe, and the like. In another embodiment, other compounds are provided in the kit, eg, other compounds useful in the treatment and/or prevention of metabolic disorders. For example, any composition provided in a kit can be packaged in an aqueous medium or in lyophilized form. The containers of the kit will generally include at least one vial, test tube, flask, bottle, syringe or other container form into which the components can be placed, and preferably aliquoted as appropriate. When more than one component is present in the kit, the kit will also typically include a second, third or other additional container into which these additional components can be separately placed.

The following examples are included to illustrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques described in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Fatustatin A reduces the expression of SREBP-responsive genes

Comparison of the gene expression profiles of drug-treated and untreated cells could reveal specific molecular pathways affected by phatustatin A. DU145 cells were treated with tustatin A or DMSO alone, and extracted mRNA samples were analyzed by Affimetrix DNA microarray, mapping 33,000 genes (Table 1).

Table 1. Known or likely SREBP-controlled genes regulated by fetostatin A shown in microarray results

Results showed that 55% of the genes downregulated (<0.7-fold) by phatustatin A were known or likely controlled by sterol regulatory element binding proteins (SREBPs), including LDL receptor, HMG-CoA reductase, and fatty acid Synthase (Horton et al., 2003). Downregulation of representative SREBP-responsive genes was confirmed by RT-PCR experiments (FIGS. 1A-1B). These results suggest that phatuostatin A is a selective inhibitor of the SREBP pathway.

To demonstrate that phatostatin A attenuates the function of SREBP, the ability of endogenous SREBP to activate the transcription of SREBP-responsive reporter genes was assayed in HEK293 cells in the presence or absence of phatostatin A ( FIGS. 2A-2B ). Fatostatin A reduces the activation of a reporter gene in a concentration-dependent manner in which luciferase expression is controlled by three repeats of the sterol regulatory element. In contrast, Fatuostatin A was unable to impair the ability of the exogenously expressed mature form (amino acids 1-500) of SREBP-1 to activate reporter gene activity (Fig. 2C). These results indicated that phatustatin A could selectively block the activation process of SREBP in cells.

Example 2

Fatustatin A blocks the proteolytic activation of SREBP

To examine whether fetustatin A affects the proteolytic activation of SREBP, whole cell lysates of DU145 cells treated with fatustatin A were analyzed by Western blot with an antibody against the NH2 terminus of SREBP-1 ( FIG. 3A ). Fatuostatin A treatment decreased the amount of the 68 KDa mature form of SREBP-1 in a dose-dependent manner, whereas the amount of the 125 KDa precursor form increased. Similar results were obtained for SREBP-2 using an antibody against its COOH terminus (Fig. 3B). These results suggest that phatustatin A directly or indirectly attenuates the proteolytic activation of both SREBP isoforms.

Inhibition of proteolytic activation of SREBP will disrupt nuclear transfer of SREBP. The effect of Fatustatin A on the subcellular localization of SREBP-1 was analyzed by immunofluorescence microscopy using an antibody against the NH2 terminus of SREBP-1. When cells were treated with DMSO alone, SREBP-1 localized almost exclusively in the nucleus in serum-free (fat-free) medium (Fig. 3C-3E). In contrast, when the cells were incubated with Fatustatin A, the immunofluorescence of SREBP-1 decreased in the nucleus with a corresponding increase outside the nucleus (FIGS. 3F-3H), suggesting that Fatustatin A inhibits the nuclear localization of SREBP-1.

Example 3

Confirmation of the Fatostatin Phenotype by Knockdown of SREBP-1

Fatuostatin A causes two phenotypes in cultured cells: (i) inhibition of insulin-induced adipogenesis of 3T3-L1 cells and (ii) inhibition of serum-independent growth of DU145 prostate cancer cells. The first phenotype is fully consistent with the conclusion that because of the known role of SREBP-1 in adipogenesis, phatustatin A is a blocker of SREBP-1 (Tontonoz et al., 1993). In order to confirm that under cell culture conditions, the expression of SREBP-1 in 3T3-L1 cells was silenced by transfecting the expression vector of SREBP-1 specific small interfering RNA (siRNA) (Fig. 4G), and the effect of silencing on insulin-induced role in lipogenesis. As expected, knockdown of SREBP-1 expression completely blocked oil droplet formation in 3T3-L1 cells (Fig. 4D-4F, clones 1 and 2), whereas control cells transfected with an empty vector (neo; Fig. 4A) exhibited As much fat accumulation as parental 3T3-L1 cells (Fig. 4B). These results suggest that the fatostatin A-induced phenotype in 3T3-L1 cells is mediated through inhibition of SREBP-1.

In order to test whether the inhibition of SREBP-1 by Fatustatin A mediates the inhibition of serum-independent growth of DU145 cells, the expression vector of SREBP-1 in DU145 cells was similarly induced by transfection of SREBP-1-specific siRNA expression vector Silencing (Fig. 5B). Control cells transfected with empty vector (neo) were grown in the presence of serum or IGF1, just like parental DU145 cells. In contrast, knockdown cells in which SREBP-1 expression was silenced (clones 1 and 2) exhibited reduced serum-independent IGF1-driven growth with minimal effect on their serum-dependent growth (Fig. 5A).

The requirement for SREBP-1 in serum-independent growth may be due to the absence of external fat sources in serum-free media. In the absence of exogenous fatty acids in serum, cells need to synthesize fatty acids and cholesterol (the building blocks of membranes) to maintain cell growth. To test the importance of fatty acids in cell growth, the growth of SREBP-1 knockdown cells in fat-free serum medium was monitored (Fig. 5A). SREBP-1 silencing impairs cell growth in fat-free medium to the same extent as it does in serum-free IGF1-containing medium. These results suggest that phatustatin A blocks the serum-independent growth of cancer cells by inhibiting SREBP-1.

Example 4

Fatuostatin A reduces body weight, lowers cholesterol and glucose levels and downregulates lipogenic enzymes in mice

The drug-like chemical structure of phatuostatin A prompted the inventors to study its ability to inhibit SREBP-1 in the liver of intact animals. The effect of phatustatin A on hepatic SREBP-1 was examined under adipogenic conditions of prolonged fasting (48 hours) followed by feeding a fat-free high carbohydrate diet for another 48 hours. Mice were injected intraperitoneally with fatustatin A at 30 mg/kg/day for 5 days starting the day before the 48-hour fasting period. After 48 hours of fasting, the treatment group lost more weight than the control group (6.12±0.6 vs. 4.9±0.3 g/mouse; p=0.01) ( FIG. 6A ). No reduction in food intake or overt toxicity was observed during treatment (Fig. 6B). Interestingly, 48 hours after re-feeding a fat-free high-carbohydrate diet, phatostatin A-treated mice showed reduced glucose levels (110±23 vs. 137±14 mg/dl; P=0.06) and gallbladder Trend for sterols (93±20 vs. 120±19 mg/dl; P=0.12) ( FIG. 6C ). Both HDL and LDL were decreased in the treated mice group. However, the reduction in LDL levels appeared to be more pronounced (16±5 vs. 30±6 mg/d1) (Fig. 6C).

The expression level of SREBP-1 in liver extracts was detected by western blotting. Consistent with the results in cell culture, liver extracts from mice treated with Fatustatin A showed reduced amounts of the 68 KDa mature form and increased amounts of the 125 KDa precursor form of SREBP-1 ( FIG. 6D ). Hepatic expression of fatty acid synthase (FAS), a representative SREBP-1 responsive lipogenic enzyme (Boizard et al., 1998), was also determined after treatment. Western blot analysis of liver extracts showed that FAS expression levels were reduced by up to 30% by statustatin A treatment (Fig. 6E). Consistent with the reduced expression, its enzymatic activity in the extracts was similarly reduced. As observed for FAS (Fig. 6G), the activity of acetyl-CoA carboxylase (ACC), also regulated by SREBP-1, was reduced in liver extracts (Fig. 6F). These results suggest that phatuostatin A blocks the activation of SREBP-1 in mouse livers, as was found in cultured cells.

Longer treatment (2 weeks) of another group of mice fed a normal diet resulted in a 10% loss in body weight, while the body weight of the control group did not change (FIGS. 7A-7B). Food intake was similar between the two groups (3.8 and 3.5 g/mouse/day for treated and control mice, respectively). Consistent with the results of mice fed a fasted/re-fed fat-free diet, mice fed a normal diet exhibited significantly lower blood glucose levels and lower triglycerides (TG) and gallbladder Trends in sterol levels (Fig. 7C). FAS activity and its protein levels were also reduced by about 30% (Fig. 7D and 7E).

Example 5

Significance of the invention

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 the SREBP family of transcription factors (Brown and Goldstein, 1997). Small molecules that modulate SREBP function can be used to treat metabolic diseases and can be used as tools to further understand diseases at the molecular level. Cell- and animal-based data suggest that phatustatin A reduces the expression of lipogenic genes by downregulating the amount of the mature SREBP-1 form in the nucleus.

Small molecules that activate SREBP-1 and -2 are reported. These LDL-lowering molecules upregulate LDL receptor expression by stimulating proteolytic activation of SREBP. Although the molecular mechanism of action is not fully understood, data suggest that SCAP is the molecule's primary target. Unlike these molecules, phatustatin A inhibits the activation of SREBP and downregulates the expression of SREBP-responsive genes, including the LDL receptor gene (Table 1).

The animal data for phatustatin A are consistent with the cell culture results. Fetustatin A-treated mice had significantly lower levels of the mature SREBP-1 form and higher levels of the precursor form in liver extracts from mice refed a fat-free diet. On the other hand and as expected, in the liver extracts of the control group, the level of the mature form was higher than that of the precursor form (Horton et al., 1998). Interestingly, the total amount of combined forms appears to be unchanged, the only difference being the distribution between the nuclear (mature) and cytosolic forms (precursors). These data suggest that phatustatin A may not alter the expression level of SREBP-1, but instead enhances the cleavage process of the precursor, resulting in a decrease in its amount and an increase in the mature and active form. The importance of SREBP-1 cleavage in lipogenesis has been demonstrated by experiments using SCAP-deficient mice: under re-feeding conditions, the mouse liver failed to induce the expression of ACC and FAS (Liang et al., 2002).

To assess the physiological significance of reduced nuclear SREBP-1 levels, the levels and activities of ACC and FAS were determined. Their activity in liver extracts was down-regulated in response to phatustatin A treatment. These results are consistent with a role for SREBP-1 as a regulator of the fatty acid synthesis pathway (Shimano, 2000). Shimano et al. revealed that SREBP-1 ^-/- mice fed a high carbohydrate diet failed to induce FAS and ACC levels, confirming the role of SREBP-1 in regulating the expression of lipogenic enzymes.

An interesting observation in phatustatin A-treated mice was a reduction in body weight and blood glucose compared to controls. The decreased body weight may be due to a lower rate of lipogenesis resulting from the downregulation of lipogenic enzymes such as ACC and FAS. Furthermore, as a product of ACC and a potent inhibitor of carnitine palmitoyltransferase, reduction of malonyl-CoA can lead to increased fatty acid oxidation and fat burning. In a specific embodiment, phatustatin A inhibits SREBP-1 cleavage down-regulates lipogenic enzymes, increases fatty acid oxidation, reduces body weight and increases insulin sensitivity, resulting in decreased glucose.

Example 6

Material

Delipidated serum was prepared as described (Goldstein et al., 1983). Fat-free FBS was purchased from Fisher. Rabbit anti-SREBP-1 (sc-8984) and goat anti-actin (sc-1616) polyclonal antibodies 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-isobutyllutein (MIX) were obtained from Sigma.

Preparation of Fatuostatin A

While stirring, a mixture of 2-bromo-4'-methylacetophenone (1.22g, 5.70mmol) and prothionamide (1.03g, 5.70mmol) in ethanol (20ml) was heated at 70°C for 0.5 hours, and then cooled to 0°C. The formed yellow precipitate was filtered, washed with cold ethanol, and dried to obtain phatustatin A hydrobromide (1.78 g, 83%) as yellow needles: ¹ HNMR (DMSO-d ₆ , 600 MHz) d _H 8.88 ( d, J=6.2Hz, 1H), 8.54(s, 1H), 8.46(d, J=1.4Hz, 1H), 8.36(dd, J=1.4, 6.2Hz, ^1H ), 7.99(d, J= 7.6Hz, 2H), 7.31(d, J=7.6Hz, 2H), 3.03(t, J=7.6Hz, 2H), 2.35(s, 3H), 1.80(m, 2H), 0.96(t, J= 7.6, 3H); HRMS (FAB) exact mass calculated for C18H18N2S ₊ H requires m/z _295.1269 , observed m/z _295.1269 .

cell culture

DU145 human androgen-independent prostate cancer cells (ATCC) were maintained at 37 °C in 5% CO in the presence of ₂ mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 1.5 g/L bicarbonate Sodium and Eagle's minimal essential medium with 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin sulfate. 3T3-L1 fibroblasts (ATCC) were maintained at 37°C in the presence of 5.5 mM glucose, 10% fetal bovine serum, 50 μg/mL gentamicin, 0.5 mM glutamine, and 0.5 μg/mL amphotericin B (fungizone ) in Dulbecco's modified Eagle's medium. Human embryonic kidney 293 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin sulfate at 37 °C, 5% _CO2 .

Oligonucleotide Microarray Analysis

In the presence of 1 μg/mL IGF1, DU145 prostate cancer cells were treated with 5 mM phatustatin A or DMSO alone in serum-free medium for 6 h, the total RNA was extracted into TRI reagent (Molecular Research Center), and passed through RNeasy Mini Kit (Qiagen) for further isolation. Purified mRNA was analyzed at the Baylor College of Medicine Microarray Core Facility by the Affymetrix Human Genome U133 Plus 2.0 Array consisting of almost 45,000 probe sets, the The probe sets represent more than 39,000 transcripts from approximately 33,000 validated human genes (Affymetrix, Inc.).

Luciferase reporter assay

On day 0, HEK293 cells were plated in triplicate on 96-well plates at a density of 5 x 10 ³ /well in Dulbecco's modified Dulbecco's modified Eagle's medium. On day 2, cells were transiently co-transfected with the following plasmids by using Lipofectamine reagent (Invitrogen): 0.4 μg/well pSRE-Luc (an SRE-1-driven luciferase reporter construct) and 0.1 μg /well b-gal reporter, where the expression of β-gal is under the control of the actin promoter, in a final volume of 150 mL. After incubating at 37°C for 5 hours, the cells were washed with phosphate-buffered saline, and then treated with 10% lipid-free serum, 100 units/μL penicillin, and 100 μg/mL streptavidin sulfate in the absence or presence of phatustatin A. Cultured in 100 μl of Dulbecco's modified Eagle's medium. After 20 hours of incubation, cells in each well were lysed with 20 μL 1x Reporter Lysis Buffer (Promega), and aliquots were used to assay luciferase (10 μL) and β-galactosidase (10 μL) activities. For luciferase assays, photon production, expressed as counts per second, was detected in a Wallac 1420 ARVOsx multilabel counter (PerkinElmer). For the β-galactosidase assay, the hydrolysis of O-nitrophenyl-β-D-galactosidase was measured by a microplate reader (Tecan) at a wavelength of 405 nm after incubation for 0.5 h at 37°C. Luciferase activity (counts per second) was normalized by [beta]-galactosidase activity (OD units). For overexpression of the N-terminal mature form of SREBP-1c, pCMV-SREBP-1c(1-436) was co-transfected with pSRE-Luc. pSRE-Luc and pCMV-SREBP-1c(1-436) were provided by JL Goldstein (University of Texas Southwestern Medical Center).

RT-PCR experiment

Total RNA was extracted from DU145 cells into TRI reagent (Molecular Research Center) and isolated using RNeasy Mini Kit. RT-PCR was performed on RNA samples using Access RT-PCR System. The RT-PCR reaction solution contained total RNA, 1 μM of each primer, 0.2 mM dNTP, 1 mM MgSO ₄ , AMV reverse transcriptase (2 units) and Tf1 DNA polymerase (2 units), with a final volume of 25 μL. The primer pairs used were as follows: for low density lipoprotein receptor (LDLR) 5'-TCAGAC CGG GAC TGC TTG GAC GGC TCA GTC-3' (SEQ ID NO: 1) and 5'-CCA CTT AGG CAG TGGAAC TCG AAG GCC G-3' (SEQ ID NO:2); for stearoyl-CoA desaturase (SCD) it is 5'-GCC TGCTTG ATA ATA TAT AAA C-3'(SEQ ID NO:3) and 5'- CAC TTG AAT TGA GCT TTA G-3' (SEQ ID NO: 4); 5'-AAG AAA AAG TGT CAG ACA GCT GG-3' (SEQ ID NO: 5) and 5' for ATP citrate lyase (ACL) - TGG ACT GAA GGG GTG TTA GC-3' (SEQ ID NO: 6); for 3-hydroxy-3-methylglutaryl-CoA reductase (HMG CoA R) is 5'-GCC CGA CAG TTC TGA ACT GGA ACA-3' (SEQ ID NO:7) and 5'-GAA CCT GAG ACC TCT CTG AAA GAG-3' (SEQ ID NO:8); 5'-CTG for mevalonate kinase (MVD) CCT GAC TGC CTC AGC-3' (SEQ ID NO:9) and 5'-ACC TCT CCT GAC ACC TGG G-3' (SEQ ID NO:10); for insulin-inducible gene 1 (INSIG1) is 5'-AAG ACT TCA GGG TAA GTC ATC A-3' (SEQ ID NO: 11) and 5'-CGT GTA TAA TGG TGT CTA TCA G-3' (SEQ ID NO: 12). Amplification conditions were as follows: 1 cycle at 94 °C for 4 min, followed by denaturation at 94 °C for 40 s, annealing at 50 °C for 40 s, extension at 68 °C for 2 min, 22 cycles (for SCD and HMG CoAR), Anneal at 58°C for 24 cycles (for LDLR and INSIG1), or at 60°C for 24 cycles (for ATP citrate lyase (ACL)) and at 55°C for 30 cycles (for MVD). Amplified DNA was analyzed by agarose gel and quantified with Scion-image (version 4.02) software.

western blot

DU145 prostate cancer cells were seeded into 6-well plates at a density of ² x 105 cells/well and cultured overnight at 37°C in serum-free MEM. Cells were then treated with DMSO or phatustatin A (1 or 5 mM) in the presence of IGF1 (1 μg/mL). After 6 hours of incubation, cells were harvested into PBS and lysed in SDS buffer. Samples were resolved on 10% SDS-PAGE gels and blotted with rabbit anti-SREBP-1 and anti-SREBP-2 antibodies. Specific bands were visualized with enhanced chemiluminescence (ECL) detection reagent (Amersham).

Immunofluorescence experiment

DU145 prostate cancer cells were seeded onto coverslips overnight in serum-free MEM and then treated with 5 mM phatustatin A or DMSO alone in serum-free MEM containing IGF1 (1 μg/mL). After 6 hours of incubation, cells were fixed in methanol at -20°C for 20 minutes and blocked in PBS containing 5% milk and 0.1% Tween 20 for 1 hour. Samples were incubated with rabbit polyclonal anti-SREBP-1 (Santa Cruz: sc-8984) followed by fluorescein isothiocyanate conjugated anti-rabbit IgG antibody (Chemicon Inc). Coverslips were visualized under a Nikon TE200 fluorescence microscope at ×400 magnification using filters appropriate for fluorescence detection.

siRNA knockdown of SREBP

Complementary oligonucleotides of the sequence (512-531) derived from the SREBP-1 gene were inserted into the pSUPER vector (OligoEngine): 5'-GAT CCC CGC CAC ATT GAG CTC CTC TCT TCA AGA GAG AGA GGA GCT CAA TGTGGC TTT TTG GAAA-3' (SEQ ID NO: 13), and 5'-AGC TTT TCC AAA AAG CCA CAT TGA GCTCCT CTC TCT CTT GAA GGA GGA GCT CAA TGT GGC GGG-3' (SEQ ID NO: 14). The resulting plasmid was transfected into 3T3-L1 or DU145 cells using Fugene 6 (Roche). To establish a stably transfected clone, neomycin derivative G418 (Gibco) was used at a concentration of 500 μg/mL, and a stable transformant was established. The expression level of SREBP-1 was evaluated by Western blot. For adipogenesis experiments, 3T3-L1 cells were seeded into 96-well plates in DMEM containing 10% fetal bovine serum and cultured for an additional 2 days to complete confluency. On day 0, the medium was switched to an induction medium containing 10% fetal bovine serum, 5 μg/mL insulin, 0.5 mM 1-methyl-3-isobutyllutein (MIX) and 1 μM dexamethasone (DEX ) of DMEM. On day 2, the induction medium was removed and replaced with DMEM medium containing 10% fetal bovine serum and 5 μg/mL insulin. On day 10, fatty oil droplets were stained with Oil-Red O. For cell growth experiments, DU145 cells were seeded in 96-well plates at a density of 2,000 cells/well in serum-free or MEM containing 1 μg/mL IGF1, 2% fat-free fetal calf serum, or 2% fetal calf serum. Cell growth was assessed after 3 days by WST-1 assay. The experiment was performed in triplicate.

Animal Studies Using Fatuostatin A

Male mice (129Sv background) were housed under controlled conditions (12 h light/dark cycle; 25°C) at the Animal Care Center at Baylor College of Medicine with ad libitum access to standard laboratory chow (Purina Mills) and water. Fatuostatin A (30 mg/kg; 150 μL) was administered intraperitoneally to 5-6 month old male mice (129Sv background) using two different protocols. The first protocol involved fasting the mice for 48 hours and then re-feeding them with a fat-free diet for another 48 hours. In addition to SREBP, this treatment also induced the activity and levels of lipogenic enzymes such as ACC and FAS. Administration of Fatuostatin A or 10% DMSO in PBS to the control group (n=5) started 24 hours before fasting and continued daily until the end of the experiment.

In the second protocol, two groups of male mice (n=5) were treated daily for 2 weeks with 30 mg/kg phatustatin A or 10% DMSO in PBS. Food intake and body weight were measured daily. At the end of the experiment, the mice were briefly fasted for 4-5 hours, and they were bled to detect serum components. The mice were then sacrificed, and their livers were quickly removed and ground to a powder in liquid nitrogen. Powdered tissue was suspended in 10 ml PBS containing 0.1 mM PMSF, 5 mM benzamidine, and 5 mg/mL protease inhibitor cocktail (Roche), homogenized with a Polytron (3x30 sec, high speed), and briefly sonicated to degrade DNA. Extracts were clarified by centrifugation at 16,000 xg for 20 minutes. Samples were then subjected to western blot analysis with commercially available antibodies against FAS and SREBP-1. FAS and ACC activities were assayed as previously described (Mao et al., 2006).

Example 7

Fatuostatin A prevents fatty liver, attenuates hyperglycemia and induces weight loss in ob/ob mice

Despite efforts to explore the networks that regulate food intake and energy balance, how obesity contributes to these diseases is still not fully understood. To study the effect of phatustatin A on male ob mice, especially its effect on preventing weight gain, diabetic conditions and fatty liver by reducing white fat size. As mentioned before, phatustatin A is an inhibitor of the master control of transcription by inhibiting the action of SREBP-1. Normal mice treated with tustatin A lost weight and had lower glucose and cholesterol levels. Fatuostatin A reduces the active mature form of SREBP-1 in the livers of treated mice compared to controls.

SREBP-1 and -2 play related but distinct roles in fatty acid and cholesterol biosynthesis. SREBP-1 preferentially activates genes required for fatty acid synthesis, while SREBP-2 favors cholesterol production. Since phatustatin A blocks the activation of SREBP-1 and possibly SREBP-2, administration of phatustatin A to obese ob/ob mice transiently modulated fatty acid and cholesterol biosynthesis and revealed interesting patterns in obese mice. type.

Effects of Fatostatin A on Body Weight and Food Intake

The study employed 4-5 week old male ob/ob mice with an average body weight of approximately 23 g/mouse. Fatostatin A (30 mg/kg/day) was delivered intraperitoneally daily, and body weight and food intake were measured. As shown in Figures 8A-8B, the weight gain of treated mice was significantly lower than that of controls. At the end of the first week of treatment, ob control mice injected with DMSO had an increase of an average of 4.82 g/mouse (from 23.58±0.62 to 28.40±1.45), while the fetustatin-treated group had an increase of approximately 3.37 g/mouse (from 23.08 ±1.53 to 26.45±1.2 g/mouse), (p=0.03). After 28 days of treatment, the fatustatin A treated group weighed about 12% less than the control (32.1±1.4 versus 36.2±2.2 g/mouse for fatustatin) (P=0.02). Cumulative food intake was similar in both groups (Fig. 8C). On average, food intake in the treatment group was not significantly different from the control, 5.4±1.5 vs. 5.9±1.4 g/mouse.day, respectively.

Effects of Fatuostatin A on the Characteristics of Glucose and Lipid in Blood

One of the most unique phenotypes in ob/ob mice is the hyperglycemia resulting from the insulin resistance condition. To determine the effect of phatustatin A on blood glucose and lipids, serum levels of glucose, triglycerides and cholesterol were analyzed in ob/ob mice fed a standard diet.

As shown in Figures 9A-9H, after an overnight fast, serum glucose levels in treated animals were approximately 70% lower than controls; 153.2±30.5 and 429.4±87 mg/dl, respectively (P=0.003). Glucose levels in the serum of treated animals became comparable to wild-type mice with a functional ob gene, while control mice given DMSO were hyperglycemic as expected. Interestingly, ketond bodies (β-hydroxybutyrate) increased about 7-fold in treated animals compared to controls; 3.62±1.41 and 0.5±0.37 mg/dl, respectively (P=0.004 ). The high levels of ketone bodies in the Fatuostatin A animals indicated a marked increase in fatty acid oxidation in the liver, the main product of which was ketone bodies secreted into the blood. Also, the increased blood component in the treated mice was unesterified free fatty acid (NEFA) measured in serum, which was about 70% higher than that in the control; 1.93±0.26 and 0.7±0.2 mEq/l, respectively (P= 0.028). This increase in NEFA levels may be due to increased lipolysis from adipose tissue due to an increased demand for fatty acid oxidation. FFA is known to be associated with insulin resistance in animals and humans. However, while FFA levels were elevated in the serum of phatuostatin A-treated ob/ob mice, glucose levels were significantly lower than controls, suggesting improved insulin sensitivity, possibly due to improved insulin signaling. Furthermore, it has recently been found that increased fatty acid oxidation in mouse tissues (liver, fat, and muscle) from mutant acetyl-CoA carboxylase mice (Acc2 ^-/- mutant mice) results in increased lipid accumulation in adipocytes. Higher ketone bodies and increased NEFA in the blood due to hydrolysis. Triglyceride (TG) levels in the serum of treated mice were increased by about 30% compared to controls, 115±11 and 79±12, respectively (P=0.006), which indicated that Fatuostatin A increased TG from the liver secretion and migration. Serum levels of total cholesterol showed a trend towards lower levels in fatostatin-treated animals, 183±16 vs. 219±18 mg/dl (P=0.06). However, LDL was significantly reduced by about 35% (31±3 vs. 48±8; P=0.02), while HDL was reduced by less, about 22% (144±11 and 183±12; P=0.02). Since LDL levels decreased more than HDL in the sera of Fatuostatin A-treated mice, this suggests a desired outcome of Fatuostatin A treatment. Levels of VLDL transporting triglycerides, phospholipids and cholesterol, calculated based on TG levels, increased by approximately 50% (23.1 ± 2.3 compared to 15.8 ± 2.4 mg/dl).

Fatuostatin A reduces epididymal fat size and improves fatty liver

Due to uncontrolled food intake, ob/ob mice become morbidly obese and accumulate excessive fat levels in adipose tissue and in different organs such as the liver, leading to non-alcoholic fatty liver disease and insulin resistance. At approximately 8-9 weeks of age, control untreated mice displayed enlarged liver size and accumulated fat, as indicated by the pale color, compared to those mice treated with Fatustatin A (Fig. 10A). The average weight of the livers of phatostatin A-treated mice was about 32% less than that of controls (1.59±0.2 vs. 2.34±0.15; P=0.06) ( FIG. 10D ). Control mouse liver sections stained with oil red for lipid droplets contained abundant lipid droplets, whereas those sections of phatostatin A-treated mice had no lipid droplets, which were mainly triglycerides (Fig. 10B). It has been shown that transgenic mice overexpressing SREBP 1 develop fatty liver. However, in ob/ob mice lacking SREBP-1 (lep ^ob/ob X Srebp 1 ^-/- ), fatty liver disease was significantly improved, suggesting that SREBP 1 is a major contributor to fatty liver development in ob/ob mice Acting factor.

At the end of 4 weeks of phatustatin A treatment, the body weight of the treated mice was lower than that of the control. Fatostatin A-treated mice were found to have significantly smaller fat pads by measuring the epididymal fat pad as the predominant white adipose tissue (Fig. 10C). The average weight of fat pads was about 20% less than controls (2.7±0.1 vs. 3.6±0.2; P=0.02) ( FIG. 10D ). Smaller fat pads may be due to reduced lipid storage and/or reduced lipogenesis and increased fatty acid oxidation in fat. Previous studies using Acc2 ^-/- mutant mice have shown that lack of ACC2 also leads to less fat in the liver, smaller epididymal fat pads and increased fatty acid oxidation in different tissues including the liver. It is noted here that, due to inhibition of carnitine palmitoyltransferase by malonyl-CoA produced in the absence of ACC2, fatty acid oxidation is increased and mice become hyperinsulin-sensitive and avoid diet-induced obesity and diabetes. In a specific aspect, phatustatin A down-regulates ACC enzymes increases fatty acid oxidation and inhibits fatty acid synthesis in different tissues such as, for example, liver, fat and muscle. TG and cholesterol levels in the livers of fatustatin A-treated ob/ob mice were determined and compared with ob/b controls. As shown in FIG. 11A , TG levels in the livers of treated mice decreased by approximately 65% (14.8±3.7 and 38.7±6.0 mg/gram liver, respectively; P=0.0004). Fatuostatin A also decreased cholesterol levels in the liver by more than 20% (2.8±0.5 and 3.6±0.1; P=0.03) ( FIG. 11B ). These results further confirm the Oil Red O staining and suggest that fatty liver in ob/ob mice, caused in part by increased hepatic lipogenesis, can be completely prevented by phatustatin A treatment. The decrease in these lipids in the liver of treated ob/ob mice was due to the dramatic inhibition of lipogenic enzymes required for the synthesis of TG and cholesterol or its precursors. In addition, lipase hepatic activity was increased due to the increased demand for fatty acid oxidation in different mouse tissues, including the liver, and also increased transfer of these lipids from the liver into the circulation to be oxidized by different fatty acid tissues (such as heart and muscle) use. In a specific aspect, this was associated with higher levels of TG in the blood of ob/ob mice treated with Fatuostatin A.

Fatuostatin A downregulates lipogenic enzymes in ob/b mouse liver

Enzymes in the lipogenesis pathway are regulated by transcription factors such as PPAR and SREBP. The effect of phatustatin A on lipogenic enzyme levels and activities in treated ob/ob mice was examined. The activity of acetyl-CoA carboxylase (ACC), which performs the rate-limiting step in fatty acid synthesis, is determined. ACC catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA (the building block in fatty acid synthesis), which is carried out by another multifunctional enzyme, fatty acid synthase (FAS). In addition to its role in fatty acid synthesis, malonyl-CoA also plays an important role in fatty acid oxidation by inhibiting carnitine palmitoyltransferase 1 (CPT 1). Lipogenic enzymes are significantly induced in ob/ob mice, partially explaining the morbidly obese phenotype of these mice. The activity of ACC in the liver extracts of mice treated with Fatuostatin A decreased by about 40% (3.44±0.44 vs. 5.55±0.57 nmol/min.mg) ( FIG. 12A ). Fatuostatin A-treated ob/ob mice also had significantly downregulated fatty acid synthase activity in liver extracts. FAS activity decreased by more than 70% in treated mice (8.64±1.91 vs. 22.6±1.37 nmol/min.mg) ( FIG. 12B ). Both ACC and FAS activity decreased due to decreased expression levels of both enzymes, as shown by Western blot analysis of both enzymes (Fig. 12C). Its product fatty acids, C14:0 and C16:0, were significantly lower (approximately 50%) in the livers of fetostatin-treated ob/ob mice than in untreated control mice (Table 2).

ACC is acutely regulated by phosphorylation/dephosphorylation mechanisms, leading to inhibition and activation of the enzyme, respectively. As shown in Figure 12C, the level of phospho-ACC was higher in the control group, however, because the expression level of ACC was also higher and to the same extent, this shows that phatuostatin A did not change the specific phosphorylation level (P-ACC/ ACC protein). These results suggest that the downregulation of ACC activity is solely due to decreased enzyme levels, not decreased phosphorylation status (Fig. 12C-12D). It was previously shown that two ACC isoforms exist in the liver; ACC1 (the dominant isoform in liver) and ACC2 (the dominant isoform in muscle), which play distinct roles in regulating lipid synthesis and oxidation, respectively. Decreases in ACC and FAS activity indicated decreased lipogenesis, while fat burning was significantly increased in the liver of FA-treated mice, which was consistent with FA-treated ob/ob mice as shown in Figure 9A-9H Consistent with the 7-fold increase in ketone bodies in the blood. The levels of two key enzymes in fatty acid metabolism also under the transcriptional regulation of SREBP-1, ATP citrate lyase (ACL) and stearate-CoA desaturase 1 (SCD1) were determined. Protein levels of ACL, which converts cytosolic citrate to acetyl-CoA, which is used by ACC to generate malonyl for fatty acid synthesis, were reduced by approximately 70% in liver extracts from mice with phatustatin A Substrate for coenzyme A. This downregulation of ACL levels further amplifies the effect of phatuostatin A on the reduction of the adipogenesis process in adipogenic tissues such as the liver. SCD1 catalyzes the rate-limiting step in monounsaturated fatty acid biosynthesis by introducing a cis double bond at the Δ9 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 constituents of triglycerides and cholesteryl esters, and, in mice including ob/ob mice, SCD1 Deficiency of β leads to increased metabolic rate, reduced obesity, protection from fatty liver and avoidance of diet-induced diabetes. As shown in FIG. 12D , the protein level of SCD1 was decreased by about 50% in the liver extracts of mice treated with Fatuostatin A compared to controls. This is confirmed by the following results: monounsaturated fatty acids C16:1, C18:1 and C20:1 decreased by about 70%, and their elongated products (C18:2)N-6, (C18:3)N-6, ( The desaturation of C20:2)N-6 and (C20-3)N-6 was reduced by about 50% (Table 2). In a particular embodiment of the invention, this reduced effect on SCD1 is an important factor in weight loss and reduces TG levels in the liver and avoids fatty liver. Interestingly, phatustatin A treatment did not alter protein levels of FADS1 or Δ5 desaturase.

Table 2: Gas chromatography-mass spectrometry (GC-MS) analysis of fatty acids in livers of OB/OB-treated mice and their untreated controls

Liver samples (100 mg) were obtained from Fatuostatin A-treated ob/ob mice and untreated control mice and stored at -80°C until fatty acid content analysis. Fatty acids were extracted according to Folch's protocol and quantified by gas chromatography-mass spectrometry (GC-SM). As shown in the table above, fatty acid de novo synthesis products C14:0 and C16:0 decreased by about 50%, monounsaturated fatty acids C16:1, C18:1 and C20:1 and their extended desaturation products (C18:2) N-6 , (C18:3)N-6, (C20:2)N-6, (C20-3)N-6 and (C20:5)N-3 decreased by about 70%. The products of FAS myristate (C14:0) and palmitate (C16:0) decreased by about 50% (P<0.05). C18 levels were unchanged not only from de novo fatty acid synthesis by FAS but also from food and chain elongation systems. Interestingly, there was a strong trend for C20:0 to decrease by about 15% (P+0.059) and C24:0 to increase by 30% (P=0.05). Parallel to the significant reduction in long-chain saturated fatty acids, the results showed a significant reduction of about 70% in the levels of monounsaturated fatty acids C16:1, C18:1 and C20:1 (P<0.004). Also, polyunsaturated long-chain fatty acids (18:2)N-6, (18:3)N-6, (20:2)N-6, (20:3)N-6 and (20:5)N -3 levels drop by 30-60%. These decreases were due to the downregulation of key enzymes in the lipogenesis pathway (FAS, ACC and SCD, ACL) at the transcriptional and translational levels. These results help to explain the effect of phatustatin A in improving fatty liver conditions by reducing the level of triglycerides produced in the liver.

Down-regulation of mRNA levels of lipogenic enzymes

Decreased protein levels may be due to transcriptional or translational regulation. In addition to the adipogenesis transcription factor PPARγ, real-time PCR can also be used to determine the levels of mRNA levels of representative adipogenesis genes ACC1, FAS and SCD1. The mRNA levels of ACC1, FAS and SCD1 decreased by about 80% (Fig. 13). These results are consistent with lower enzyme protein levels and activities, and strongly suggest that phatustatin A reduces adipogenesis by inhibiting the maturation of SREBP-1. In specific embodiments, the downregulation of lipogenic enzymes involves one of its major transcription factors, PPARγ. The mRNA level of this transcription factor was decreased by about 40% in extracts from mice treated with Fatuostatin A (Fig. 13). Since hyperglycemia was reduced and fatty liver was prevented in ob/ob mice treated with Fatustatin A, it was suggested that PPARγ in the liver is, in particular, one of several factors that may influence these pathologies . In conclusion, in treated ob/ob mice, phatustatin A improved fatty liver, attenuated obesity and reduced hyperglycemia through its effect on SREBP-1 by reducing hepatic TG stores. These studies suggest that phatustatin A and its analogs are useful agents against eg obesity, fatty liver and diabetes.

Homozygous male obese (ob/ob) mice (C57BL/6J, The Jackson Laboratory, Bar Harbor, ME) aged 4-5 weeks were housed under controlled conditions (12 hr light/dark cycle; 25°C). Five mice were housed per cage, and upon their arrival, the mice had free access to standard laboratory chow (Purina Mills, Richmond IN) and water for one week. On the first day of the experiment and every subsequent day, the weight of the mice and the amount of food consumed were determined. The weight of the mice and the remaining food were measured between 3-5 p.m. every day, and then ip injection of Fatustatin A (30 mg/kg; 150 μL). Administration of Fatuostatin A or 10% DMSO in PBS to the control group (n=5) continued daily for 4 weeks until the end of the study.

After 28 days of daily injections of Fatuostatin A, the mice were fasted overnight and bled, and whole blood glucose and β-hydroxybutyrate were measured with a Glucometer Precision Xtra (Abbott). For determination of serum composition, glucose, triglyceride and cholesterol measurements were performed by the Comparative Pathology Laboratory (Baylor College of Medicine). Serum non-esterified fatty acids (NEFA) were determined by using the NEFA C kit (Wako Chemicals, Richmond, VA).

Mice were sacrificed and the weights of liver and epididymal fat pad were determined. Cryosections of liver sections from individual animals were stained with Oil Red O to visualize fat droplets (TG) in liver sections as previously described (Abu-Elheiga et al., 2001). The remaining liver tissue was frozen into liquid nitrogen and stored at -80°C for further analysis. Hepatic triglycerides were assayed using the Cholesterol E Kit (Wako) and Infinity Triglyceride Kit (Thermo Electron, Melbourne, Australia) adapted for colorimetric analysis in 96-well plate format as described in reference (Chandler et al., 2003). and cholesterol content.

Enzyme activity and western blot analysis

Frozen liver fractions were ground into powder in liquid nitrogen. Powdered tissue was suspended in 10 ml PBS containing 0.1 mM PMSF, 5 mM benzamidine, and 5 mg/ml protease inhibitor cocktail (Roche) and homogenized with a Polytron (3x30 sec, high speed) and briefly sonicated to reduce Solve DNA. Extracts were clarified by centrifugation at 16,000 xg for 20 minutes. The protein concentration in the supernatant was determined and subjected to western blot analysis with commercially available antibodies against the following enzymes: FAS (BD Biosciences), citrate lyase SCD1, FADS1, ACC and phospho-ACC antibodies. Proteins were visualized with Amersham ECL Plus ^™ Western Blotting Detection Reagent. The intensity of a specific band of the protein of interest is scanned and quantified normalized against β-actin. FAS and ACC activities from liver extracts were assayed as previously described (Mao et al., 2006).

quantitative real-time PCR

Total RNA was prepared from mouse tissues using TRIzol reagent (Invitrogen). Equal amounts of RNA from 5 mice were pooled and treated with DNase I (Turbo DNA-free, Ambion, Inc.). First-strand cDNA was synthesized from 2 μg of DNase I-treated total RNA with Superscript II RNAase H-reverse transcriptase (Invitrogen) using random hexamer primers. Real-time PCR contained 10 ng reverse transcribed total RNA, 0.5 μM forward and reverse primers and 10 μl 2x master mix from DyNAmo HS SYBR Green qPCR kit (Finnzymes) in a final volume of 20 μl. PCR was performed in 96-well plates using DNA Engine OpticonSystem (MJ Research, Inc). All reactions were performed in triplicate and relative amounts of mRNA were calculated using the comparative C(t) method. Cycle threshold C(t) was calculated with Opticon Monitoring software 2.02 (MJ Research). Mouse β-actin mRNA was used as an internal control. Data are expressed as mean ± SD. Differences between two groups were assessed using an unpaired two-tailed Student t-test.

Example 8

Identification of Target Molecules of Fatustatin A and Its Analogs or Derivatives

In certain aspects of the invention, one or more targets of phatustatin A or an analog or derivative thereof are identified. While any suitable method may be used for such identification, in particular embodiments, phatustatin A or an analog or derivative thereof is labeled. Exemplary labels include, for example, biotin.

Example 9

Exemplary compounds and modifications thereof

Figures 14A-14F show exemplary compounds of the invention, their given names are provided in Table 3 and Table 4. Figures 15-17 show exemplary luciferase reporter gene assays for these exemplary compounds at 20 mM by the same method shown in Figure 2A. Adipogenesis assays were performed as described (Choi et al., 2003). Analogs that completely inhibited oil droplet formation in cells were scored as adipogenic inhibiting analogs.

Table 3: Exemplary compounds of the invention

Table 4

Furthermore, the skilled artisan understands that it is appropriate to modify one or more aspects of the exemplary compounds to facilitate the identification of other suitable compounds. For example, when the suitability of a particular compound for the treatment and/or prevention of one or more metabolic disorders is determined, the compound can be modified to identify other related compounds for the same or a different metabolic disorder. In specific embodiments, these changes can be made according to the exemplary chemical groups described herein.

Example 10

Blocking fat synthesis by inhibiting SREBP activation

After fat depletion in cells, sterol regulatory element binding proteins (SREBPs) are released by hydrolysis of membrane proteins and translocate into the nucleus where they activate the transcription of genes involved in cholesterol and fatty acid biosynthesis. In the present invention, it was demonstrated that a small synthetic molecule that blocks adipogenesis is a selective inhibitor of SREBP activation. Diarylthiazole derivatives known as phatustatins impair the proteolytic activation of SREBP, thereby reducing the transcription of lipogenic genes in cells. The molecular target of fatostatin appears to be SREBP cleavage-activating protein (SCAP). Fatuostatin blocks increases in body weight gain, blood glucose, and hepatic lipid accumulation in obese ob/ob mice, even under conditions of uncontrolled food intake.

As described herein, phatustatin inhibits insulin-induced adipogenesis of 3T3-L1 cells and serum-independent growth of DU145 cells (Choi et al., 2003). Gene expression profiles of drug-treated and untreated cells were compared to obtain information on specific molecular pathways affected by fatostatin. DU145 cells were treated with tustatin or DMSO alone, and extracted mRNA samples were analyzed by Affymetrix DNA microarray mapping 33,000 genes. Of these genes (all available from the GenBank database of the National Center for Biotechnology Information on the World Wide Web), 63 genes had at least 35% reduction in transcript levels in response to fatostatin treatment (Table 5). 34 of the affected genes are directly related to fat or sterol synthesis, such as genes encoding biosynthetic enzymes, and 18 of the affected genes have been reported to be controlled by SREBP (Horton et al., 2003). Downregulation of affected SREBP-responsive genes was confirmed by RT-PCR experiments. The high incidence of SREBP-responsive genes and fat/cholesterol biosynthesis genes in the list of down-regulated genes suggested that Fatustatin acts on the SREBP pathway. Table 5 shows the results of the microarray analysis. Eighteen underlined genes have been reported to be controlled by SREBP, and genes in bold are related to fat or sterol synthesis.

table 5

To confirm that fetostatin attenuates the function of SREBP, the ability of endogenous SREBP to activate SREBP-responsive reporter genes in CHO-K1 cells was determined in the presence or absence of fetostatin (Fig. 18A). Fatostatin reduces the activation of a reporter gene whose expression of luciferase is controlled by a sterol regulatory element. Fatostatin had limited effects on the ability of the mature form of exogenously expressed SREBP-1 (amino acids 1-436) to activate the reporter gene (Fig. 18B), suggesting that Fatostatin attenuates the activation process of SREBP-1.

To determine whether fetustatin affects ER-Golgi transfer and proteolytic processing of SREBP, the reporter assay developed by Sakai et al. (1998) was employed. PLAP-BP2 in transfected CHO-K1 cells remains membrane bound unless it is cleaved by S1P in the Golgi apparatus and secreted into the medium. In this assay, a secreted alkaline phosphatase (PLAP-BP2513-1141) fused to a fragment of SREBP-2 lacking the NH2-terminal DNA-binding domain allows monitoring of metastasis and processing (FIG. 18C). When cells were co-transfected with plasmids encoding PLAP-BP2513–1141 and SCAP, PLAP phosphatase was secreted, resulting in a fluorescent signal. Addition of fetustatin or sterols similarly reduced secretion (Fig. 18C). Fatostatin-mediated inhibition of SREBP activation was confirmed by Western blot analysis of SREBP. Treatment of CHO-K1 cells with tustatin decreased the amount of the 68 KDa mature form of SREBP-2 and increased the amount of the 125 KDa precursor form (Fig. 18D). Regarding SREBP-1, similar results were obtained (Fig. 22). These results collectively indicate that phatustatin blocks the activation process of both SREBP isoforms.

The inventors believe that phatustatin attenuates the proteolytic cleavage of SREBP in the Golgi or the ER-Golgi translocation of the SCAP/SREBP complex. Brefeldin A, a natural product that blocks the anterograde transfer of proteins from the ER to the Golgi, is known to render SREBP unresponsive to sterols, and by relocalizing S1P from the Golgi to the ER leads to SREBP is constitutively processed in the ER (DeBose-Boyd et al., 1999). In the presence of brefeldin A, fetostatin had no effect on SREBP processing (Fig. 19A), suggesting that fetostatin does not block proteolysis itself.

To determine whether fetustatin blocks the ER-to-Golgi transfer of the SCAP/SREBP complex, the inventors analyzed the extent of N-linked glycosylation of SCAP in the Golgi. SCAP contains a glycosylated luminal loop that is protected from proteolysis by trypsin and recognizes anti-SCAP IgG-9D5. When SCAP is located in the ER, the two oligosaccharides in the loop are sensitive to endoglycosidase H. When SCAP is transported to the Golgi apparatus, its sugars become resistant to digestion by endoglycosidase H. Translocated SCAPs have higher levels of glycosylation and are more resistant to endoglycosidase H than ER-bound SCAPs. Sterols prevent SCAP from becoming endoglycosidase H resistant by inhibiting ER-Golgi transfer (Nohturfft et al., 1998). Cells were grown in the absence or presence of fatustatin or sterols, and membrane fractions were treated with trypsin and endoglycosidase H sequentially. In cells grown without fatostatin and sterols, tryptic fragments of SCAP were more resistant to endoglycosidase H and had one or two sugar chains (Fig. 19B, lane 1). When cells were grown in the presence of fetustatin or sterol, the SCAP fragment was less resistant to endoglycosidase H and had no or only one sugar chain (Fig. 19B, lanes 2 and 3). Thus, Fatustatin appears to inhibit the transfer of SCAP from the ER to the Golgi apparatus.

The study of the structure-activity relationship of phatustatin showed that when its toluene moiety was modified with various alkyl or aryl sulfonamide groups, the biological activity of the molecule was preserved or even enhanced. A fluorescent derivative, dansyl fatustatin (Fig. 20A) retained its ability to block SREBP activation (Fig. 24) and was used as a microscopic probe. Confocal microscopic analysis revealed the localization of dansyl fatustatin coincident with that of ER-tracker red, a specific marker for ER (Fig. 20B). In contrast, the control dansyl molecule lacking phatustatin failed to localize to any organelles. Selective ER localization suggests that fatostatin binds to proteins in the ER; the most likely candidate is SCAP, a target of cholesterol for the control of SREBP (Radhakrishnan et al., 2004). To test this hypothesis, proteins bound to the phatustatin-polyproline linker-biotin conjugate (Fig. Antibodies to SREBP-2 and ATF6, an unrelated ER-binding transcription factor, were analyzed by Western blot (Ye et al., 2000). The results indicated that Fatuostatin bound to SCAP, but not to other proteins (Fig. 20C). This binding was lost when excess phatustatin was added, but not with excess cholesterol (FIG. 20D), raising the possibility that phatustatin may interact with SCAP at a different site than cholesterol. effect.

Having established the critical role of SREBP in adipogenesis, the pharmacological effects of fetustatin were then examined in ob/ob mice, a mouse model of obesity, under conditions of uncontrolled food intake. Fatostatin was delivered intraperitoneally daily, and food intake and body weight were monitored. The mean daily food intake of treated mice was not significantly different from controls (5.4+1.5 vs. 5.9+1.4 g/mouse/day, respectively, p>0.05), and no overt toxicity was observed during treatment. After 28 days of treatment with fetustatin, treated mice were about 12% lighter than untreated controls (32.1±1.4 and 36.22 g/mouse, respectively, p=0.02). One of the most distinctive phenotypes in ob/ob mice is hyperglycemia due to insulin resistance. Examination of blood components revealed that the average glucose level of treated mice was approximately 70% lower than that of untreated mice (153.2±30.5 vs. 429.4±87 mg/dl, respectively, p=0.003), which was within the range of normal glucose levels . These results are consistent with the reported role of SREBP-1c in the pathogenesis of hepatic insulin resistance (Ide et al., 2004).

Another phenotype in ob/ob mice is excessive accumulation of fat in organs, including nonalcoholic fatty liver disease. In untreated ob/ob mice, enlarged and fatty livers were evident by their pale color, whereas the livers of fatustatin-treated mice appeared normal. Treated mice had ~32% lighter livers and smaller fat pads compared to untreated mice. Oil red staining of liver sections showed that the livers of untreated ob/ob mice contained abundant fat droplets, whereas the livers of treated mice contained lower levels of lipid accumulation ( FIG. 21 ). Triglyceride and cholesterol levels also decreased in the livers of treated mice. Fatostatin prevents fatty liver in ob/ob mice consistent with the reported role of SREBP-1 in fatty liver development: transgenic mice overexpressing SREBP-1 develop fatty liver, whereas ob/ob mice lacking SREBP-1 (lep ^ob/ob X Srebp 1 ^-/- ) have a healthy liver (Yahagi et al., 2002).

Hepatic fat levels in treated mice are thought to be reduced because of reduced hepatic expression of SREBP-responsive lipogenic enzymes. Therefore, the effects of fetustatin on representative SREBP-responsive lipogenic enzymes, including fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase 1 (SCD1), and ATP lemon Effect of liver protein levels and enzyme activity of acid lyase (ACL). Biochemical analysis showed that protein levels and activities of lipogenic enzymes were decreased in liver extracts from phatustatin-treated mice (FIGS. 25A-25C). Thus, fetustatin blocks SREBP-1 processing in the liver, downregulates lipogenic enzymes and reduces hepatic triglyceride storage. Fatuostatin represents the first nonsteroidal synthetic molecule to inhibit SREBP activation.

Luciferase reporter assay. On day 0, CHO-K1 cells were plated onto 96-well plates in medium A (1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium with 5% fetal calf serum, 100 units/mL penicillin and 100 μg/mL streptomycin sulfate). On day 2, using Lipofectamine reagent (Invitrogen), pSRE-Luc (an SRE-1-driven luciferase reporter construct) (Hua et al., 1995) and pAc-β-gal (β-gal reporter , in which the expression of β-gal is controlled by the actin promoter) transiently co-transfected cells. After incubating for 5 hours, the cells were washed with phosphate-buffered saline (PBS), and then cultured in medium B (1:1 ratio of Ham's F-12 medium and Dulbecco's modified Eagle's medium) in the absence or presence of phatustatin. The mixture was incubated with 5% lipid-free serum, 100 units/mL penicillin, 100 μg/mL streptomycin sulfate, 50 mM compactin and 50 mM sodium mevalonate). After 20 hours of incubation, cells in each well were lysed and aliquots were used to assay luciferase and β-galactosidase activities. Luciferase activity was normalized by β-galactosidase activity. For overexpression of the N-terminal mature form of SREBP-1c, pCMV-SREBP-1c(1-436) was co-transfected with pSRE-Luc and pAc-β-gal.

Western blot analysis of SREBP processing. On day 0, CHO-K1 cells were plated onto medium A 100 mm dishes. On day 2, cells were washed with PBS and incubated in medium B in the absence or presence of fetustatin. On day 3, the cells were washed once with cold PBS, and then washed with 10 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1% (w/v) SDS and protease inhibitor cocktail (1 μg/ml pepstatin A, 10 μg/ml leupeptin, 200 μM phenylmethylsulfonyl fluoride) buffer treatment. The protein concentration of each total cell extract was determined (BCA kit; Pierce), and then a 22-33 μg aliquot of the cell extract was mixed with 0.25 volume of buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 25 % glycerol, 0.2% (w/v) bromophenol blue and 5% (v/v) 2-mercaptoethanol) were mixed and heated at 95°C for 7 minutes. Samples were resolved on a 10% SDS-PAGE gel and blotted with a mouse monoclonal antibody (IgG-7D4) against SREBP-2 (Yang et al., 1995). Specific bands were visualized with enhanced chemiluminescence (ECL) detection reagents (Amersham).

Modification of SCAP oligosaccharides. Cell membrane fractions were prepared as described elsewhere herein. The membrane pellet was resuspended in 0.1 mL buffer containing 10 mM Hepes.KOH (pH 7.4), 10 mM KCl, 1.5 mM MgCl ₂ , 1 mM sodium EDTA and 100 mM NaCl. Aliquots of the protein were then incubated for 30 minutes at 30°C in the absence or presence of 1 μg trypsin in a total volume of 58 μL. The reaction was terminated by adding 2 μL (400 units) soybean trypsin inhibitor. For subsequent treatment with Endoglycosidase H, each sample received 10 μl of a solution containing 3.5% (w/v) SDS and 7% (v/v) 2-mercaptoethanol. After heating at 100°C for 10 minutes, each sample received successively the addition of 9 μl of 0.5 M sodium citrate (pH 5.5), 5 μL of a solution containing 17′ protease inhibitor (concentration 1x, corresponding to 10 μg/mL leupeptin, 5 μg/mL mL pepstatin A and 2 μg/mL aprotinin), followed by 1 μL (5 units) endoglycosidase H. The reaction was carried out at 37°C overnight, and was added by adding 20 μL containing 0.25M Tris·HCl (pH 6.8), 2% SDS, 10% (volume/volume) glycerol, 0.05% (weight/volume) bromophenol blue and 4% 2- A buffer of mercaptoethanol was used to stop the reaction. The mixture was then heated at 100°C for 5 minutes and subjected to SDS/PAGE (12% gel).

Confocal microscopy analysis. CHO-K1 cells on -70% confluent glass-bottom 96-well plates (Grainer) were incubated with 0.2 μM ER-tracker Red (Invitrogen) and 5 mM dansyl-fatustatin for 1 hour. Fluorescence images were acquired and analyzed with a Carl Zeiss LSM 510 confocal microscope equipped with a CSU10 spinning disk confocal scanner (Yokogawa Electric Corporation) and an ORCA-CCD camera (Hamamatsu Photonics). Images were analyzed with IPLab software (Solution Systems).

Binding assay. Cell membrane fractions were prepared as described in Supplementary Methods. Membrane fractions were extracted with PBS containing 0.1% FOS-Choline 10 (Hampton Research). Extracts were mixed with Neutravidine-Sepharose beads (10 μί) saturated with biotinylated phatustatin and incubated for 1 hour. Bound proteins were washed four times with PBS containing 0.1% FOS-Choline 10, boiled in 25 [mu]L SDS sample buffer, and subjected to Western blotting. For competition assays, saturating amounts of cholesterol or phatustatin were added to membrane extracts and incubated with the beads.

Animal study procedure. Homozygous male obese (ob/ob) mice (C57BL/6J, The Jackson Laboratory, Bar Harbor, ME) aged 4-5 weeks were housed under controlled conditions (12 hr light/dark cycle; 25°C). Five animals were housed per cage, and upon their arrival, the animals had free access to standard laboratory chow (Purina Mills, Richmond, IN) and water for one week. On the first day of the experiment and every subsequent day, the weight and food intake of each mouse were measured between 3:00 and 5:00 p.m. After weight measurements, treated mice received ip injections of phatustatin (30 mg/kg; 150 μL) and control mice received 10% DMSO in PBS. Daily injections continued for 4 weeks until the end of the study.

blood components. After 28 days of daily injections of Fatustatin, the mice were fasted for 5-6 hours, and whole blood glucose and β-hydroxybutyrate were measured with a Glucometer Precision Xtra (Abbott). Measurements of serum components, glucose, triglycerides and cholesterol were performed by Comparative Pathology Laboratory (Baylor College of Medicine). Serum non-esterified fatty acids (NEFA) were measured using the NEFA C kit (Wako Chemicals, Richmond, VA).

Liver analysis. Mice were sacrificed, and liver and epididymal fat pad weights were determined. Cryosections of liver sections from individual animals were stained with Oil Red O to visualize fat droplets (triglycerides) in liver sections as described (Abu-Elheiga et al., 2001). The remaining liver tissue was frozen in liquid nitrogen and stored at -80°C for further analysis.

Tissue triglyceride and cholesterol content. Hepatic triglyceride and cholesterol levels were determined as described by Chandler et al. (2003) using the Cholesterol E Kit (Wako) and the Infinity Triglyceride Kit (Thermo Electron, Melbourne, Australia).

Synthesis of Fatuostatin 1, Dansyl Fatuostatin, and Fatuostatin-Polyproline Linker-Biotin Conjugate

Figure 23 shows the synthesis of phatustatin 1, dansyl fatuostatin, phatuostatin-polyproline linker-biotin conjugate and the intermediates of the synthesis.

Synthesis of Fatostatin 1

A mixture of prothionamide (1.03g, 5.70mmol) and 2-bromo-4'-methylacetophenone (1.22g, 5.70mmol) in ethanol (20ml) was heated at 70°C with stirring 0.5 hour, then cooled to 0 °C. The formed yellow precipitate was filtered, washed with cold ethanol, and dried to obtain 2-propyl-4-(4-p-tolylthiazol-2-yl)pyridine (fatustatin) 1HBr salt (1.78 g, 83%). mp: 190-193°C; ¹ H NMR (600MHz, DMSO-d ₆ ): δ8.88(d, J=6.2Hz, 1H), 8.54(s, 1H), 8.46(d, J=1.4Hz, 1H ), 8.36(dd, J=1.4, 6.2Hz, 1H), 7.99(d, J=7.6Hz, 2H), 7.31(d, J=7.6Hz, 2H), 3.03(t, J=7.6Hz, 2H ), 2.35 (s, 3H), 1.80 (m, 2H), 0.96 (t, J=7.6Hz, 3H); ¹³ C NMR (150MHz, DMSO-d ₆ ): d161.3, 158.5, 156.9, 146.2, 143.2, 138.4, 130.4, 129.5, 126.3, 122.3, 120.3, 119.5, 35.0, 22.4, 20.9, 13.4; HRMS (m/z): [M+H] ⁺ 295.1269 calculated for C ₁₈ H ₁₉ N ₂ S; found 295.1269.

Synthesis of 4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)aniline 16

2 (1.08g, 3.0mmol), benzophenone imine (0.57g, 3.3mmol), Pd ₂ (dba) ₃ (86mg, 0.15mmol), BINAP (280mg, 0.45 mmol), sodium tert-butoxide (1.44 g, 9.0 mmol) and anhydrous toluene (30 mL) and purged with argon. The pressure tube was sealed and heated in a 100°C bath for 20 hours. After cooling to room temperature, the reaction mixture was chromatographed ( _SiO2 , 4:1 hexane:EtOAc) to afford 1.35 g of 8 (98%) as a yellow oil. Then, to a solution of 8 (1.35 g, 2.9 mmol) in THF (20 mL) was added 2N aqueous HCl (15 mL). After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure, then diluted with EtOAc (100 mL), and washed with saturated _{Na2CO3 (} 50 mL) solution. The aqueous washes were extracted with EtOAc (3 x 40 mL), and the combined EtOAc layers _were dried over _Na2SO4 and concentrated. The crude product was chromatographed ( _SiO2 , 4:1 hexanes:EtOAc) to afford 0.73 g of 4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)aniline 16 as white crystals (82%). ¹ H NMR (600MHz, CDCl ₃ ): δ8.61(d, J=4.8Hz, 1H), 7.80(d, J=8.9Hz, 2H), 7.75(d, J=1.4Hz, 1H), 7.67( dd, J=1.4, 4.8Hz, 1H), 7.36(s, 1H), 6.75(d, J=8.9Hz, 2H), 3.82(brs, 1H), 2.85(t, J=7.6Hz, 2H), 1.83 (m, 2H), 1.01 (t, J=7.6Hz, 3H); ¹³ CNMR (150MHz, CDCl ₃ ): δ164.9, 163.4, 157.3, 150.0, 146.8, 140.8, 127.7, 124.8, 119.2, 117.8, 115.1, 111.3, 40.4, 23.1, 13.9; HRMS (m/z): [ _M +H] ⁺ calcd for _{C17H18N3S 296.1221} ; found 296.1228.

Synthesis of Dansyl Fatuostatin

To a magnetically stirred solution of 16 (50 mg, 0.17 mmol) and pyridine (27 mg, 0.34 mmol) in _CH2Cl2 ( ₅ mL) was added dansyl chloride (50 mg, 0.18 mmol). After stirring for 17 h, the reaction mixture was concentrated under reduced pressure, and the residue was partitioned between EtOAc (50 mL) and saturated NH4Cl solution (20 mL). The aqueous phase was extracted with EtOAc (2 x 20 mL). The combined extracts were washed with saturated NaHCO ₃ solution, dried over Na ₂ SO ₄ and concentrated. The crude product was chromatographed ( _SiO2 , 2:1 hexanes:EtOAc) to afford dansylfatuostatin (65 mg, 73%) as yellow crystals. ¹ H NMR (600MHz, CDCl ₃ ): δ8.60(d, J=4.8Hz, 1H), 8.50(d, J=8.2Hz, 1H), 8.36(d, J=8.3Hz, 1H), 8.21( d, J=8.3Hz, 1H), 7.76(d, J=8.6Hz, 2H), 7.70(d, J=1.5Hz, 1H), 7.62(dd, J=1.5, 4.8Hz, 1H), 7.61( t, J=8.3Hz, 1H) 7.44(s, 1H), 7.43(dd, J=7.5, 8.2Hz, 1H), 7.19(d, J=7.5Hz, 1H), 7.04(d, J=8.6Hz ¹³ C NMR (150MHz, CDCl ₃ ): δ165.5, 163.5, 156.1, 152.2, 150.0, 140.5, 136.7, 134.0, 131.1, 131.0, 130.5, 129.8, 129.7, 128.7, 127.3, 123.1, 128.6, 119.2, 1 , 115.3, 113.8, 45.4, 40.4, 23.1, 13.9; HRMS (m/z): [M ₊ H] ⁺ _calcd for _{C29H29N4O2S2 529.1732} ; found _529.1733 .

Synthesis of tert-butyl 4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenylcarbamate 40

To a magnetically stirred solution of 16 (0.57 g, 1.92 mmol) and 4-(dimethylamino)pyridine (5 mg, 0.4 mmol) in THF (20 mL) was added di-tert-butyl dicarbonate (0.49 g, 2.21 mmol) . After stirring for 17 h, the reaction mixture was concentrated under reduced pressure, and the residue was partitioned between EtOAc (100 mL) and saturated _NH4Cl solution (30 mL). The aqueous phase was extracted with EtOAc (2x50 mL). The combined extracts were washed with saturated NaHCO ₃ solution, dried over Na ₂ SO ₄ and concentrated. The crude product was chromatographed ( _SiO2 , 2:1 hexane:EtOAc) to afford 4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl as a yellow foam tert-Butyl carbamate 40 (0.33 g, 43%). ¹ H NMR (300MHz, CDCl ₃ ): δ8.68(d, J=5.1Hz, 1H), 7.93(d, J=8.4Hz, 2H), 7.79(s, 1H), 7.72(d, J=5.1 Hz, 1H), 7.52(s, 1H), 7.47(d, J=8.4Hz, 2H), 6.58(s, 1H), 2.90(t, J=7.5Hz, 2H,), 1.87(m, 2H) , 1.55(s, 9H), 1.03(t, J=7.5Hz, 3H); ¹³ C NMR (75MHz, CDCl ₃ ): δ163.1, 156.5, 152.3, 149.6, 140.6, 138.5, 128.7, 128.1, 127.0, 118.3, 117.7, 114.3, 112.9, 80.6, 41.0, 28.4, 24.1, 13.7; HRMS (m/z): [ _M +H] ⁺ _calcd for _{C22H26N3O2S 396.1746} ; found 396.1738.

Synthesis of R＝N(Boc)CH ₂ CH ₂ CH ₂ COOH Intermediate

Under _N2 atmosphere, 40 (200 mg, 0.51 mmol) was added to a suspension of NaH (60% dispersion in mineral oil, 24 mg, 0.6 mmol) in DMF (5 mL), and the mixture was stirred at room temperature for 2 Hour. Then, NaI (91 mg, 0.6 mmol) and ethyl 4-bromobutyrate (0.12 g, 0.6 mmol) in DMF (2 mL) were added. After stirring for 18 h, the reaction mixture was poured into water (20 mL) and extracted with EtOAc (2 x 50 mL). The combined extracts _were dried over _Na2SO4 and concentrated. The crude product was chromatographed ( _SiO2 , 2:1 hexanes:AcOEt) to afford 11 (35 mg, 13%) as a yellow oil. To a solution of 11 (30 mg, 2.9 mmol) in THF (1 mL) and MeOH (0.5 mL) was then added 2N aqueous NaOH (0.2 mL). After stirring at room temperature for 18 h, the reaction mixture was concentrated under reduced pressure, then diluted with EtOAc (10 mL), washed with saturated _NH4Cl solution (5 mL). The aqueous washes were extracted with EtOAc (2 x 10 mL), and the combined EtOAc layers _were dried over _Na2SO4 and concentrated. The crude product was chromatographed ( _SiO2 , EtOAc) to yield 14 mg of the R=N(Boc _{) CH2CH2CH2COOH} intermediate (50% ₎ as a white foam. ¹ H NMR (300MHz, CDCl ₃ ): δ8.69(d, J=5.1Hz, 1H), 7.95(d, J=8.4Hz, 2H), 7.80(s, 1H), 7.73(d, J=5.1 Hz, 1H), 7.53(s, 1H), 7.51(d, J=8.4Hz, 2H), 6.58(s, 1H), 2.92(m, 4H), 2.33(m, 2H), 1.90(m, 4H ), 1.50(s, 9H), 1.02(t, J=7.5Hz, 3H); ¹³ C NMR (75MHz, CDCl ₃ ): δ174.2, 162.9, 156.3, 152.9, 149.6, 140.6, 138.5, 129.3, 128.9 , 127.2, 119.0, 117.9, 114.8, 113.3, 80.8, 41.0, 40.3, 32.1, 28.4, 24.1, 21.1, 13.7; HRMS (m/z): [M+H] ⁺ for C ₂₆ H ₃₂ N ₃ O ₄ S The calculated value is 482.2114; the measured value is 482.2120.

Synthesis of 4-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenylamino)-N-isopropylbutanamide

To a solution of R=N(Boc) _{CH2CH2CH2COOH} intermediate ( _{17 mg} , 0.035 mmol), triethylamine (17 μl, 0.14 mmol) and isopropylamine (4 μL, 0.046 mmol) in DMF (0.5 mL) HATU (16 mg, 0.042 mmol) was added to . After stirring for 18 h, the reaction mixture was concentrated under reduced pressure, and the residue was partitioned between EtOAc (20 mL) and saturated _NH4Cl solution (10 mL). The aqueous phase was extracted with EtOAc (2 x 10 mL). The combined extracts were washed with saturated NaHCO ₃ solution, dried over Na ₂ SO ₄ and concentrated. The crude product was chromatographed ( _SiO2 , 2:1 hexanes:EtOAc) to afford the N-Boc protected 4-(4-(2-(2-propylpyridin-4-yl)thiazole as a yellow oil -4-yl)phenylamino)-N-isopropylbutanamide (14 mg, 77%). Then, to a solution of 13 (12 mg, 2.9 mmol) in THF (1 mL) was added TFA (0.2 mL). After stirring at room temperature for 18 h, the reaction mixture was concentrated under reduced pressure, then diluted with EtOAc (20 mL), and washed with saturated _NH4Cl solution (10 mL). The aqueous washes were extracted with EtOAc (2 x 5 mL), and the combined EtOAc layers _were dried over _Na2SO4 and concentrated. The crude product was chromatographed ( _SiO2 , 2:1 hexanes:EtOAc) to afford 6.2 mg of 4-(4-(2-(2-propylpyridin-4-yl)thiazole-4 as a yellow foam -yl)phenylamino)-N-isopropylbutanamide (63%). ¹ H NMR (300MHz, CDCl ₃ ): δ8.69(d, J=5.4Hz, 1H), 8.04(1H, s), 7.96(d, J=8.7Hz, 2H), 7.96(s, 1H), 7.72(d, J=5.4Hz, 1H), 7.51(s, 1H), 6.65(d, J=8.7Hz, 2H), 3.95(m, 1H), 3.03(m, 4H,), 2.35(m, 2H), 1.92(m, 4H), 1.25(d, J=6.3Hz, 6H), 1.03(t, J=7.5Hz, 3H); ¹³ C NMR(75MHz, CDCl ₃ ): δ171.6, 162.9, 156.3, 152.9, 140.6, 138.5, 129.3, 128.9, 127.2, 119.0, 117.9, 114.8, 113.3, 42.2, 41.0, 40.3, 32.1, 24.1, 23.2, 21.1, 13.7; HRMS (m/z): [M+H] ⁺ Calculated for _{C24H31N4OS 423.2219} ; found _423.2216 .

Synthesis of Fatustatin-Polyproline Linker-Biotin Conjugate

Fatuostatin-KPGQFLYELKKPPPPPPPPPPKK (SEQ ID NO: 15)-aminocaproic acid-biotin.

On Rink-Amide MBHA resin by coupling N-α-Fmoc protected amino acid, N-ε-Fmoc-e _- aminocaproic acid, R=N(Boc _{) CH2CH2CH2COOH} intermediate, biotin Conjugates were synthesized and purified by reverse phase HPLC as described (Sato et al., 2007). Conjugate ₃ : _Calcd for _{C155H238N35O29S2 +} needs to be _3119.9 ^. Measured (MALDI-TOF-MS) is 3119.7 [M+H] ⁺ .

plasmid. pSRE-Luc, pCMV-SREBP-1c(1-436), pCMV-PLAP-BP2(513-1141) and pCMV-SCAP were gifts from J.L. Goldstein and M.S. Brown (University of Texas Southwestern Medical Center) (Sakai et al., 1998 ; Hua et al., 1995).

Antibody. Monoclonal anti-SREBP-1 IgG (2A4), anti-SCAP IgG (9D5) and anti-ATF6 IgG (H-280) were purchased from Santa Cruz Biotechnology. Monoclonal anti-SREBP-2 IgG-7D4 was a gift from J.L. Goldstein and M.S. Brown (University of Texas Southwestern Medical Center). Polyclonal anti-FA, anti-ACC, anti-SCD1 and anti-ACL IgG were purchased from BD Biosciences.

cell culture. Chinese hamster ovary K1 (CHO-K1) cells were maintained in Dulbecco's modified Eagle's medium/Ham's F12 medium [1 :1] at 37°C under 5% CO ₂ . Human androgen-independent prostate cancer cells (DU145) were maintained in a solution containing 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM non-essential amino acids and 1.5 g/L sodium bicarbonate and 10% fetal bovine serum, 100 units /mL penicillin and 100 μg/mL streptomycin sulfate in Eagle's minimal essential medium at 37 °C under 5% _CO .

Preparation of cell membrane fractions. Cells were collected, then resuspended in buffer (10 mM Hepes.KOH (pH 7.4), 10 mM KCl, 1.5 mM MgCl ₂ , and 1 mM sodium EDTA), passed through a 22-gauge needle, and centrifuged at 1,000×g for 5 minutes. The post-nuclear supernatant was then centrifuged at 15,000 xg for 30 minutes and the supernatant removed.

Oligonucleotide microarray analysis. In the presence of 1 μg/mL IGF1, DU145 prostate cancer cells were treated with 5 mM phatustatin or DMSO alone in serum-free medium for 6 hours, total RNA was extracted in TRI reagent (Molecular Research Center) and passed through RNeasy Mini Kit (Qiagen) Further separation. Purified mRNA was analyzed at the Baylor College of Medicine Microarray Core Facility by the Affymetrix Human Genome U133 Plus 2.0 Array consisting of nearly 45,000 probe sets representing approximately 33,000 known Over 39,000 transcripts of validated human genes (Affymetrix, Inc.).

RT-PCR experiments. Total RNA was extracted from DU145 cells into TRI reagent (Molecular Research Center) and isolated using RNeasy Mini Kit (Qiagen). RT-PCR was performed on RNA samples using the Access RT-PCR System (Promega). RT-PCR reactions contained total RNA, 1 μM of each primer, 0.2 mM dNTPs, 1 mM MgSO ₄ , AMV reverse transcriptase (2 units) and Tf1 DNA polymerase (2 units) in a final volume of 25 μL. The primer pairs used were as follows: for low density lipoprotein receptor (LDLR) 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 stearoyl-CoA desaturase (SCD) is 5'-GCC TGC TTG ATA ATA TAT AAA C-3' (SEQ ID NO: 18) and 5'-CAC TTG AATTGA GCT TTA G-3' (SEQ ID NO: 19); for ATP citrate lyase (ACL) 5'-AAG AAA AAG TGTCAG ACA GCT GG-3' (SEQ ID NO: 20) and 5'-TGG ACT GAA GGG GTG TTA GC-3' (SEQ ID NO:21); for 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR) is 5'-GCC CGA CAG TTC TGA ACTGGA ACA-3' (SEQ ID NO:22) and 5'-GAA CCT GAG ACC TCT CTG AAA GAG-3' (SEQ ID NO:23); for mevalonate pyrophosphate decarboxylase (MVD) is 5'-CTG CCT GAC TGC CTC AGC-3' (SEQ ID NO:24) and 5'-ACC TCT CCT GAC ACC TGG G-3' (SEQ ID NO:25); for insulin-inducible gene 1 (INSIG1) is 5'-AAG ACT TCA GGG TAA GTC ATC A-3' (SEQ ID NO: 26) and 5'-CGT GTA TAATGG TGT CTA TCA G-3' (SEQ ID NO: 27). Amplification conditions were as follows: 1 cycle of 4 min at 94°C, followed by denaturation at 94°C for 40 s, annealing at 50°C for 40 s, extension at 68°C for 2 min, 22 cycles (for SCD and HMG CoA R), at 58°C °C for 24 cycles (for LDLR and INSIG1), or 60 °C for 24 cycles (for ACL) and 55 °C for 30 cycles (for MVD). Amplified DNA was analyzed by agarose gel and quantified with Scion-image software.

PLAP-BP2 cleavage. On day 0, CHO-K1 cells were plated onto 96-well plates in medium A. On day 2, cells were transiently co-transfected with pCMV-PLAP-BP2(513–1141), pCMV-SCAP, and pAc-β-gal using Lipofectamine reagent (Invitrogen). After 5 hours of incubation, the cells were washed with PBS and then incubated in medium B in the absence or presence of fetustatin (20 μM) or sterols (10 μg/mL cholesterol and 1 μg/mL 25-hydroxycholesterol) . After 20 hours of incubation, aliquots of the medium were assayed for secreted alkaline phosphatase activity. Cells in each well were lysed for determination of β-galactosidase activity. Alkaline phosphatase activity was normalized by the activity of β-galactosidase.

Enzyme activity and Western blot analysis. Frozen liver fractions were ground into powder in liquid nitrogen. Powdered tissue was suspended in 10 ml PBS containing 0.1 mM PMSF, 5 mM benzamidine, and 5 mg/ml protease inhibitor cocktail (Roche) and homogenized with a Polytron (3x30 sec, high speed) and briefly sonicated to degrade DNA. Extracts were clarified by centrifugation at 16,000 xg for 20 minutes. The protein concentration in the supernatant was determined and subjected to western blot analysis with antibodies against FAS, ACC, SCD1 and ACL. The intensity of a specific band of the protein of interest is scanned and normalized against b-actin for quantification. FAS and ACC activities in liver extracts were determined as previously described (Mao et al., 2006).

Example 11

Synthesis of (R)-2-(4-(4-(methylsulfonamido)phenyl)thiazol-2-yl)pyrrolidine-1-carboxylic acid benzyl ester 53

To a solution of L-prolinamide (1.14 g, 10.0 mmol) in acetonitrile (100 mL) was added benzyl chloroformate (1.55 mL, 11.0 mmol) and triethylamine (2.79 mL) at room temperature as described in Figure 26 , 20mmol). The mixture was stirred at room temperature for 12 hours and diluted with AcOEt. The mixture was poured into 0.1M HCl solution and separated between organic and aqueous layers. The organic layer was washed with saturated aqueous NaHCO ₃ and brine, dried (Na ₂ SO ₄ ), filtered, and concentrated. The residue was purified by silica gel column chromatography to yield 67 (2.26 g, 91%) as a colorless oil.

A mixture of 67 (586 mg, 2.36 mmol) and Lawesson's reagent (1.05 g, 2.60 mmol) in toluene (30 mL) was stirred at 90 °C for 1 h. After cooling to room temperature, the solvent was evaporated. The residue was purified by silica gel column chromatography to yield 68 (495 mg, 79%) as a white amorphous solid.

A mixture of 68 (300 mg, 1.13 mmol) and 4-bromobenzoyl bromide (318 mg, 1.13 mmol) in EtOH (10 mL) was stirred at reflux for 2 hours. After evaporation, the residue was purified by silica gel column chromatography to yield 48 (265 mg, 53%) as a white solid.

48 (230mg, 0.519mmol), Pd ₂ (dba) ₃ (9.50mg, 2mol%), BINAP (97%, 20.0mg, 6mol%), sodium tert-butoxide (69.8mg, 0.727mmol) and diphenylmethyl A mixture of ketimine (0.105 mL, 0.623 mmol) in toluene (5.0 mL) was refluxed for 8 hours. After cooling to room temperature, the mixture was filtered through Celite, and the filtrate was evaporated. The residue was purified by silica gel column chromatography to yield 69 (228 mg, 81%) as a yellow amorphous solid.

To a solution of 69 (175 mg, 0.321 mmol) in THF (3 mL) was added aqueous HCl (4M, 0.3 mL), and the mixture was stirred at room temperature for 1 hr. The mixture was neutralized with saturated aqueous NaHCO ₃ and extracted with AcOEt. The organic layer was washed with brine, dried (Na ₂ SO ₄ ), filtered, and concentrated. The residue was purified by silica gel column chromatography to yield 70 (113 mg, 93%) as a colorless oil.

A mixture of 70 (92 mg, 0.24 mmol), methanesulfonyl chloride (0.021 mL, 0.27 mmol) and pyridine (0.043 mL, 0.53 mmol) in _CH2Cl2 ( ₂ mL) was stirred at room temperature for 8 hours. After addition of MeOH, the mixture was diluted with AcOEt and washed with saturated aqueous NaHCO ₃ and brine. The organic layer was dried (Na ₂ SO ₄ ), filtered, and concentrated. The residue was purified by silica gel column chromatography to yield 53 (91 mg, 83%) as a white solid.

¹ H NMR (CDCl ₃ , 600 MHz, two rotamers, ratio=1:0.95) δ 7.80-7.86 (m, 2H), 7.14-7.41 (m, 7H), 6.64 (s, 1H, major rotamer), 6.83 (s, 1H, minor rotamer), 5.07-5.36 (m, 3H), 3.73 (m, 1H), 3.59 (m, 1H), 3.03 (s, 3H, major rotamer), 3.00 (s, 3H, minor rotamer), 2.28-2.39 (m, 2H), 2.00-2.10 (m, 2H); ¹³ C NMR (CDCl ₃ , 150 MHz, Two rotamers showing major peaks) 174.7, 155.4, 154.4, 136.4, 131.9, 128.5, 128.3, 127.9, 127.8, 127.7, 127.7, 120.8, 112.3, 67.1, 59.4, 47.2, 39.4, 32.9, 23.1; HRMS (FAB): Calcd ^{. for C22H24N3O4S2 [M+H]+} _{exact mass 458.1203 ; found} 458.1200.

Example 12

Synthesis of N-(4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)phenyl)methanesulfonamide (19)

As shown in Figure 27, in reaction (a), a mixture of prothionamide (9.01 g, 50.0 mmol) and 4-bromobenzoyl bromide 6a (13.9 g, 50.0 mmol) in EtOH (400 mL) was dissolved in Stir at 70°C for 1 hour. After cooling to room temperature, the precipitate was filtered off with EtOAc and washed with EtOAc and saturated aqueous NaHCO ₃ . The organic layer was washed with brine, dried over _Na2SO4 , and concentrated to yield 4-( ₄ -bromophenyl)-2-(2-propylpyridin-4-yl)thiazole 2 (14.3 g , 79%).

In reaction (b), a pressure tube was filled with 2 (1.08g, 3.0mmol), benzophenone imine (0.57g, 3.3mmol), Pd ₂ dba ₃ (86mg, 0.15mmol), BINAP (280mg, 0.45 mmol), sodium tert-butoxide (1.44 g, 9.0 mmol) and anhydrous toluene (30 mL), and purged with argon. The pressure tube was sealed and heated in a 100°C bath for 20 hours. After cooling to room temperature, the reaction mixture was chromatographed ( _SiO2 , 4:1 hexanes:EtOAc) to afford 1.35 g of 71 (98%) as a yellow oil.

Then, in reaction (c), to a solution of 71 (1.35 g, 2.9 mmol) in THF (20 mL) was added 2M aqueous HCl (15 mL). After stirring at room temperature for 2 h, the reaction mixture was concentrated under reduced pressure, then diluted with EtOAc (100 mL), and washed with saturated _{Na2CO3 (} 50 mL) solution. The aqueous washes were extracted with EtOAc (3 x 40 mL), and the combined EtOAc layers _were dried over _Na2SO4 and concentrated. The crude product was chromatographed ( _SiO2 , 4:1 hexanes:EtOAc) to afford 0.73 g of 4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)aniline 16 as white crystals (82%).

¹ H NMR (600MHz, CDCl ₃ ): δ8.61(d, J=4.8Hz, 1H), 7.80(d, J=8.9Hz, 2H), 7.75(d, J=1.4Hz, 1H), 7.67( dd, J=1.4, 4.8Hz, 1H), 7.36(s, 1H), 6.75(d, J=8.9Hz, 2H), 3.82(brs, 1H), 2.85(t, J=7.6Hz, 2H), 1.83 (m, 2H), 1.01 (t, J=7.6Hz, 3H); ¹³ C NMR (150MHz, CDCl ₃ ): δ164.9, 163.4, 157.3, 150.0, 146.8, 140.8, 127.7, 124.8, 119.2, 117.8 , 115.1, 111.3, 40.4, 23.1, 13.9; HRMS (m/z): [M+H]+ calculated for C17H18N3S 296.1221; found 296.1228.

In reaction (d), to a stirred solution of 16 (800 mg, 2.71 mmol) and pyridine (0.66 mL, 8.1 mmol) in _CH2Cl2 ( ₂₀ mL) was added methanesulfonyl chloride (0.23 mL, 2.97 mmol) at 0 °C ). After stirring for 0.5 h, the mixture was poured into 2M citric acid solution and extracted with EtOAc. The combined extracts were washed with saturated NaHCO ₃ solution and brine, dried over Na ₂ SO ₄ , and concentrated to yield N-(4-(2-(2-propylpyridin-4-yl) as a yellow foam Thiazol-4-yl)phenyl)methanesulfonamide 19 (880 mg, 87%).

¹ H NMR (300MHz, CD ₃ OD): δ8.55(d, J=5.2Hz, 1H), 8.02(d, J=8.8Hz, 2H), 7.95(s, 1H), 7.90(d, J= 1.9Hz, 1H), 7.84(dd, J=1.9, 5.2Hz, 1H), 7.34(d, J=8.8Hz, 2H), 3.00(s, 3H), 2.86(t, J=7.7Hz, 2H) , 1.80(m, 2H), 1.01(t, J=7.3Hz, 3H).m/z=374[M+H] ⁺ .

Example 13

Synthesis of N-isopropyl-4-(2-(2-propylpyridin-4-yl)thiazol-4-yl)aniline (17)

As shown in Figure 27, the method for the synthesis of compound 17 utilizes the same steps ac as described for compound 19 in Example 12. For compound 17, in step e, to a stirred solution of 16 (1.02 g, 3.45 mmol) in _CH2Cl2 ( ₂₀ mL) was added acetone (2.5 mL, 34.5 mmol) and acetic acid (2.0 mL, 34.5 mmol). After the mixture was stirred for ₁ hour, Na(OAc)3BH (1.5 g, 6.9 mmol) was added. The mixture was stirred for 20 hours. The mixture was poured into saturated NaHCO ₃ solution and extracted with EtOAc. The combined extracts _were dried over _Na2SO4 and concentrated. Chromatography ( _SiO2 , 4:1 hexanes/EtOAc) of the crude product yielded 17 (845 mg, 73%) as a white foam. ¹ H NMR (300MHz, CDCl ₃ ): δ8.61(d, J=5.2Hz, 1H), 7.81(d, J=8.5Hz, 2H), 7.76(d, J=1.4Hz, 1H), 7.68( dd, J=1.4, 5.2Hz, 1H), 7.34(s, 1H), 6.65(d, J=8.5Hz, 2H), 3.70(m, 1H), 2.86(t, J=7.6Hz, 2H), 1.83(m, 2H), 1.25(d, J=6.0Hz, 6H), 1.02(t, J=7.4Hz, 3H).m/z=338[M+H] ⁺ .

Example 14

SREBP Activation Assay for Compounds 45-66

Standard SREBP activation assays were performed on the identified exemplary compounds in Table 4 following the method used in Example 1 for Fatuostatin A. In CHOK1 cells, endogenous Ability of SREBP to activate transcription of SREBP-responsive reporter genes. Among them, compound 61 showed about 25% inhibition of SREBP activation, compound 53 showed about 30% inhibition, compound 58 showed about 42% inhibition, and compound 19 showed about 45% inhibition. Inhibitory concentrations were determined for compound 53 (Figure 31 ) and compounds 58 and 61 (Figure 32).

Example 15

Compound 19 inhibits the growth of human breast cancer cell line SUM159 and downregulates the adipogenesis pathway

Treatment of SUM159 cells with different concentrations of Compound 19 for 48 hours resulted in cytostatic and toxic effects (Fig. 33A). Cells were seeded on 96-well plates at a density of 5000 cells/well in 100 μl of medium containing 2% charcoal-treated serum. After 24 hours, various concentrations of compound 19 were added for an additional 48 hours. Cell viability was measured using the WST-1 CellViability and Proliferation Assay (ScienCell Research Laboratory, Carlsbad CA). Changes in mRNA expression levels of lipogenic genes were determined in cells treated with 10 μM. There was a significant downregulation of genes under the control of the transcription factor SREBP. The Insig2 gene, known not to be regulated by these transcription factors, was not affected by this treatment (Fig. 33B).

The effect of compound 19 on the human hepatocellular carcinoma cell line (HePG2) was also demonstrated. As shown in Figures 34A-34B, compound 19 produced high levels of toxicity as shown by marked changes in morphology and growth inhibition of treated cells. When HePG2 cells were treated with 25 μM Compound 19, the levels of mature and active forms were significantly reduced, while the SREBP-1 precursor was increased, and correspondingly, the expression levels of genes controlled by SREBP were increased ( FIGS. 34A-34B ). These results are consistent with and support the conceptual role of compound 19 as an inhibitor of adipogenesis by inhibiting SREBP activation.

Compound 19 inhibits the growth of human acute lymphoblastic leukemia cell line MOLT-4

MOLT-4 cells were grown under two different conditions: under 5% fetal bovine serum (FBS) and under 5% FBS fat-free serum (charcoal-treated serum). 10,000 cells were seeded in 96-well plates for 24 hours before treatment with different concentrations of compound 19 was initiated. Compound 19 dissolved in DMSO was added to the cells for 48 hours before cell growth was determined using the MTT assay. As shown in Figure 35A, cells grew similarly in 5% FBS and fat-free FBS. Compound 19 inhibited about 75-80% of cell growth at 10 and 20 μM under both growth conditions. These results show that compound 19 is equally potent at 10 and 20 μM, even in the presence of exogenous lipids, suggesting that inhibition of de novo lipid synthesis leads to growth inhibition in this cancer cell line. Interestingly, there was a 30-50% growth inhibition at 2 and 5 μM of compound 19 in fat-free FBS (Fig. 35B).

Compound 19 inhibits the growth of human multiple myeloma cell line RPMI8226

RPMI8226 cells were cultured under the same conditions as MOLT-4 cells. 20,000 cells were seeded into 96-well plates for 24 hours and then treated with increasing concentrations of compound 19 as described for MOLT-4 cells. As shown in Figure 35D, cells grew similarly under 5% FBS and fat-free FBS, except that 5 μM Compound 19 in 5% FF FBS inhibited growth by about 1.5 times more. Compound 19 was similarly effective at 10 μM and 20 μM in 5% FBS and 5% FF FBS. A strong trend towards inhibition started at 3 μM ( FIG. 35E ) in 5% FBS condition and 2 μM ( FIG. 35F ) in FBS fat-free serum.

Example 16

Compound 17 inhibits the growth of human acute lymphoblastic leukemia cell line MOLT-4

MOLT-4 cells were grown under two different conditions: under 5% fetal bovine serum (FBS) and under 5% FBS fat-free serum (charcoal-treated serum). 20,000 cells were seeded in 96-well plates for 24 hours before treatment with different concentrations of compound 17 was initiated. Compound 17 dissolved in DMSO was added to the cells for 48 hours before cell growth was determined using the MTT assay. As shown in Figure 36A, 5, 10 and 20 [mu]M strongly inhibited the growth of cells in 5% FBS and 5% fat-free FBS. At 5% FF FBS, 3 [mu]M exhibited approximately 2.5-fold inhibition over 5% FBS. Compound 17 inhibited approximately 85-100% of cell growth at 5, 10 and 20 μΜ under both growth conditions. In both 5% FBS and 5% FBS fat-free serum, there was a strong trend towards inhibition from 1 [mu]M (FIGS. 36B-36C).

Compound 17 inhibits the growth of human multiple myeloma cell line RPMI

RPMI8226 was grown under the same conditions as MOLT-4 cells. 20,000 cells were seeded on 96-well plates for 24 hours and then treated with increasing concentrations of compound 19 as described for MOLT-4 cells. As shown in Figure 36D, the growth initiation of cells under 5% FBS and fat-free FBS was similar, except that in 5% FF FBS, 5, 10 and 20 μM Compound 17 inhibited growth about 1.5-6 times more. Under 5% FBS conditions (FIG. 35E) and 5% FBS fat-free serum (FIG. 35F), there was a strong inhibition starting at 3 [mu]M.

Example 17

Compound 19 upregulates uncoupling protein 2 (UCP2) and reduces body fat

SD male rats were fed a Western diet (high fat high carbohydrate (HFHC)) rich in carbohydrates (35% kcal) and fat (45% kcal) for 3 weeks. Results from MTD studies suggest that the maximum effective dose may be at or below 100 mg/kg. Therefore, 1/40 and 1/10 of 100mg/kg were selected for the effect study. The rats fed with HFHC diet were divided into four groups, 15 animals in each group: (1) control group not receiving any treatment; (2) control group given 1ml cottonseed oil; (3) compound 19 treated with 2.5mg/kg Treated experimental group; (4) Experimental group treated with 10 mg/kg compound 19. Finally, one group (n=5) was fed regular rat chow (RD).

Compound 19 effectively reduces body weight and total body fat

Three weeks of feeding the HFHC diet increased the total body weight of the rats by approximately 12%, compared to rats fed the conventional chow, 332±6.5 versus 375±4.0 g, respectively (FIG. 37A). As expected, rats fed the conventional diet had significantly lower total body fat and % fat than those fed the HFHC diet (44.0±2.5 vs. 62.4±2.4 g/rat and 13.95±0.5 vs. 18.48±0.48 %). Lean percentage was also higher in RD-fed rats compared to HFHC-fed rats (Figures 37B, 37C and 37D).

Effects 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 Sunday through Friday. Doses were calculated based on body weight on the day of administration of drug and control vehicle. As shown in Figure 38, after three weeks of feeding the HFHC diet and before the start of treatment, the animals gained approximately 200 grams (Figure 38A). The control group fed the HFHC diet without cottonseed oil gained similarly to the second control group (control vehicle), suggesting that the vehicle had no effect on body weight change (Figure 38B). Two weeks after the start of treatment, body weight decreased significantly in the 2.5 mg/kg and 10 mg/kg treated groups. At the end of the eight-week treatment, 2.5 mg/kg reduced weight gain by approximately 15% compared to control vehicle (357.7±10.4 g/rat versus 420.9±2.4 g/rat, respectively) ( FIG. 38A ). In 10 mg/kg treated rats, body weight gain decreased more, about 24% lower than the control vehicle group (321.4±10.3 g/rat, respectively). Cumulative food intake was similar between the control and 2.5 mg/kg groups (1370±21 vs. 1320±18 g/rat, respectively), and slightly decreased in the 10 mg/kg group (1245±21, see Figure 2B) . When the ratio of total body weight gain was calculated relative to food intake, approximately 13% and 16% less body weight was gained in the 2.5 and 10 mg/kg groups compared to controls (0.326±0.011, 0.313±0.014 versus 0.373±0. 0.011). In the absence of reduced food intake, the reduced body weight in the 2.5 mg/kg group could be interpreted as meaning that the drug reduced body weight without reducing food intake, as in ob/ob mice observed in.

The reduction in weight gain was primarily due to lower body fat

To determine the effect of Compound 19 on reducing total body fat composition, body composition was determined every two weeks using the ECHOMRI method. After two weeks of treatment, rats treated with 2.5 and 10 mg/kg had lower total body weight than the control and control vehicle groups (Fig. 39A). This reduction was primarily due to lower body fat, with no significant change in lean body mass (Fig. 39B). After eight weeks of treatment, the body weight of 2.5 and 10 mg/kg treated rats was 8% and 15% lower than that of the control group (545.6±9.7, 502.8±7.8 and 592.9±5.4 g/rat, respectively, see FIG. 39A ). The body weight of the 10 mg/kg group reached a value similar to that of rats fed a regular diet. More importantly, the difference in body weight could be mainly attributed to the difference in fat content between the treatment and control groups (Fig. 39B). After eight weeks of treatment, total body fat mass was approximately 25 and 43% lower at 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). Total % fat was also significantly lower, with the 2.5 mg/kg group having similar % fat to the group of rats fed the regular diet, and the 10 mg/kg group having a lower percentage than the regular diet; 26.6 ± 0.53 (control group) ; 21.9±0.82 (2.5 mg/kg group); 18.0±0.6 (10 mg/kg) and 21.2±0.82 (regular diet) ( FIG. 39C ). Although the percent lean body mass was higher at 2.5 and 10 mg/kg compared to the percent lean body mass in the control group, there was no significant difference in lean body mass between all groups (Figures 39D and 39E).

Compound 19 ameliorates fatty liver condition induced by HFHC diet and downregulates gene expression of de novo lipogenesis and potentially through upregulation of uncoupling proteins and thermogenesis

Feeding Sprague-Dawley rats with a high-fat diet not only induces obesity but also causes fatty liver conditions in which high levels of triglycerides accumulate. After eight weeks of treatment, the effect of Compound 19 on the liver of SD rats fed a HFHC diet was examined. As shown in Figure 40A, control rats developed larger and fatter livers compared to treated rats. This was further confirmed by staining frozen liver sections from five animals per group with Oil Red O staining, where fat accumulation was lower in all treatment groups (n=5) compared to controls ( FIG. 40B ). These results were confirmed by determining TG and cholesterol levels in control and treated rats. As shown in Figure 40C, TG and cholesterol levels were 40 and 25% lower, respectively, in the two treated animal groups (Figure 40C). Using real-time PCR, we found that genes controlled by SREBP-1 (ACC, SCD and ACL) and genes controlled by SREBP-2 (MVD, LDLR and INSIG-1) were significantly downregulated by 40-80% (Fig. 40D). These results suggest that compound 19 not only reduces total body fat, but also improves fatty liver condition, and is a promising drug for the treatment of human fatty liver disease.

Upregulation of Uncoupling Protein 2 (UCP2) in Rat Liver Treated with Compound 19

The involvement of UCP in the upregulation of thermogenesis is well documented. They dissipate metabolic energy as heat by lowering the mitochondrial membrane potential. The liver is a very active metabolic organ, especially in lipid synthesis. The present inventors have shown that there is no significant difference in food consumption between control and Compound 19 treated rats. Despite being fed an adipogenic diet, treated animals still accumulated significantly lower body fat, including in the liver. The liver was examined as an example of possible energy uncoupling and the level of UCP2 (the major UCP) in the liver was determined. As shown in Figure 41, using the real-time PCR method, UCP2 levels were significantly higher in the 2.5 and 10 mg/kg treated groups, increasing by approximately 1.5 and 3 fold, respectively. These results strongly suggest that the reduction in lipid content in the liver is due to reduced synthesis and uncoupling, where the energy is partially dissipated through uncoupled thermogenesis.

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Claims (38)

1. compound, it has following chemical constitutions：

A-B-C

Wherein A is pyridine or substituted pyridine, piperidines or substituted piperidines, pyrrolidines or substituted pyrrolidines, thiazole or substitution Thiazole, phenyl ring or substituted phenyl ring；

B is thiazole or substituted thiazole, piperazine or substituted piperazine, phenyl ring or substituted phenyl ring；And

C is phenyl ring or substituted phenyl ring, pyridine or substituted pyridine, thiazole or substituted thiazole.

2. the compound of claim 1, wherein the chemical constitution is：

Wherein R₁It is H, halogen ,-OH ,-O-C_1-3Alkoxy ,-OC (O) R₃；

R₃It is C₁-C₃Alkyl or aryl ,-OCH₂-C(O)OR₄；

R₄It is H or C₁-C₃Alkyl ,-NHR₅；

R₅It is H, C₁-C₄Alkyl, alkylcyclopropane, benzyl ,-NHC (O) C₁-C₃Acid amides ,-NHC (O) O-R₆Carbamate；

R₆It is the tert-butyl group or fluorenyl methyl or-NH-SO₂-R₇Sulfonamide；

R₇It is alkyl or aryl,

R₂It is alkyl or R₈OC (O)-, and

R₈It is C₃-C₅Alkyl or aryl.

3. the compound of claim 2, wherein the halogen is bromine.

4. the compound of claim 1, wherein the chemical constitution is：

Wherein R₁It is H, halogen ,-OH ,-O-C₁-₃Alkoxy ,-OC (O) R₃；

R₃It is C₁-C₃Alkyl or aryl ,-OCH₂-C(O)OR₄；

R₄It is H or C₁-C₃Alkyl ,-NHR₅；

R₅It is H, C₁-C₄Alkyl, alkylcyclopropane, benzyl ,-NHC (O) C₁-C₃Acid amides ,-NHC (O) O-R₆Carbamate；

R₆It is the tert-butyl group or fluorenyl methyl or-NH-SO₂-R₇Sulfonamide；

R₇It is alkyl or aryl,

R₂It is alkyl or R₈OC (O)-, and

R₈It is C₃-C₅Alkyl or aryl.

5. the compound of claim 4, wherein the halogen is bromine.

6. the compound of claim 1, wherein the chemical constitution is：

Wherein R₉It is H, halogen ,-OH ,-O-C₁-C₃Alkoxy ,-OC (O) R₁₁；

R₁₁It is C₁-C₃Alkyl or aryl ,-OCH₂-C(O)OR₁₂；

R₁₂It is H or C₁-C₃Alkyl ,-NHR₁₃；

R₁₃It is H, C₁-C₄Alkyl, alkylcyclopropane, benzyl ,-NHC (O) C₁-₃Acid amides ,-NHC (O) O-R₁₄Carbamate；

R₁₄It is the tert-butyl group or fluorenyl methyl ,-NH-SO₂-R₁₅Sulfonamide；

R₁₅It is alkyl or aryl or-SO₂-NH-R₁₆Sulfonamide；

R₁₆It is alkyl or aryl,

R₁₀It is nitrogen or methylene,

N is 0 or 1, and when n is 1, Z is-C=O；And

A is

Wherein R₁₇It is H or C₁-C₃Alkyl.

7. the compound of claim 1, wherein the chemical constitution is：

8. the compound of claim 1, wherein the chemical constitution is：

9. the compound of claim 1, wherein the chemical constitution is：

10. the compound of claim 1, wherein the chemical constitution is：

11. the compound of claim 1, wherein the chemical constitution is：

12. the compound of claim 1, wherein the chemical constitution is：

13. the compound of claim 1, wherein the chemical constitution is：

14. treating the method for the metabolic disorder in animal, methods described comprises the steps：

The compound or pharmaceutically acceptable salt thereof or stereoisomer of at least one claim 1 of therapeutically effective amount are applied to the animal Or combinations thereof.

15. the method for claim 14, it also comprises the steps：

The second therapy is provided to the animal.

16. the method for claim 15, wherein second therapy include dietetic treatment, physical therapy, behavior therapy, operation, Medicinal treatment or its combination.

17. the method for claim 16, wherein second therapy is lifestyle change, antihyperglycemic agents, insulin, pancreas height Blood glucose element sample peptide (GLP), dipeptidyl peptidase-4 inhibitors, thiazolidinedione, hypolipidemic compounds or group two or more in them Close.

18. the method for claim 14, wherein the metabolic disorder is the disease related to cell hyperplasia, body weight correlation The patient's condition, hyperlipidemia, diabetes or its complication, fatty liver, hypertension or cardiovascular disease.

19. the method for claim 18, wherein the metabolic disorder be obesity, hypertension, artery sclerosis, asthma, hyperlipidemia, Hyperinsulinemia, NASH and the diabetes B caused by insulin resistance.

20. the method for claim 18, wherein the disease related to cell hyperplasia is cancer.

21. the method for claim 20, wherein the cancer is breast cancer, respiratory cancer, the cancer of the brain, genital cancer, prostate It is cancer, digestive system cancer, carcinoma of urethra, cancer eye, liver cancer, cutaneum carcinoma, head and neck cancer, thyroid cancer, accessory thyroid glands cancer, lymthoma, sarcoma, black The DISTANT METASTASES IN of plain knurl, leukaemia or solid tumor.

22. the method for claim 18, wherein the metabolic disorder is the related illness of body weight, the therapeutically effective amount it is described The expression of the albumen 3 of compound increase uncoupling proteins 1, uncoupling protein-3 or uncoupling.

23. the method for claim 18, wherein the metabolic disorder is the related illness of body weight, process is mitigated in the weight of animals In, the compound increase themogenesis of the therapeutically effective amount, without reducing lean body mass.

24. the method for treating the cell hyperproliferative diseases in patient in need, methods described comprises the steps：

To the compound or pharmaceutically acceptable salt thereof or stereoisomer of at least one claim 1 of patient therapeuticallv's effective dose Or combinations thereof.

25. the method for claim 24, wherein the cell hyperproliferative diseases are breast cancer, respiratory cancer, the cancer of the brain, genitals Official's cancer, prostate cancer, digestive system cancer, carcinoma of urethra, cancer eye, liver cancer, cutaneum carcinoma, head and neck cancer, thyroid cancer, accessory thyroid glands cancer, pouring Bar knurl, sarcoma, melanoma, the DISTANT METASTASES IN of leukaemia or solid tumor.

26. the method for the body weight for mitigating the animal for having this to need, methods described comprises the steps：Applied to the animal The compound of one or more claims 1 of therapeutically effective amount in medicinal medium.

27. the method for claim 26, wherein the animal suffers from obesity, hypertension, artery sclerosis, asthma, hyperlipidemia, height Insulinemia, NASH or diabetes B or its combination caused by insulin resistance.

28. preparation, compound of the preparation comprising claim 1 and food, ani-mal feed substance or medicine.

29. for method of the increase themogenesis without reducing lean body mass during the weight of animals mitigation, methods described includes Following step：The preparation of the claim 28 of therapeutically effective amount is applied to the animal.

30. pharmaceutical composition, compound of the described pharmaceutical composition comprising claim 1 and pharmaceutical excipient.

31. the method for treating the cancer in patient in need, methods described comprises the steps：Applied to the patient With the pharmaceutical composition of one or more claims 30 of therapeutically effective amount.

32. kit, it is included：

The compound of claim 1, and

Accommodate the container of the compound.

33. compound, it is：

2- (4- (4- bromophenyls) thiazol-2-yl) pyrrolidines -1- t-butyl formates,

2- (4- (4- bromophenyls) thiazol-2-yl) pyrrolidines -1- benzyl formates,

4- (4- bromophenyls) -2- (pyrrolidin-2-yl) thiazole,

4- (4- bromophenyls) -2- (1- propyl pyrrole alkane -2- bases) thiazole,

3- (4- (4- bromophenyls) thiazol-2-yl) piperidines -1- t-butyl formates,

3- (4- (4- bromophenyls) thiazol-2-yl) piperidines -1- benzyl formates,

3- (4- (4- bromophenyls) thiazol-2-yl) -1- propylpiperdines,

4- (4- (4- bromophenyls) thiazol-2-yl) piperidines -1- benzyl formates,

(R) -2- (4- (4- (sulfonyloxy methyl amido) phenyl) thiazol-2-yl) pyrrolidines -1- benzyl formates

3- (4- (4- (sulfonyloxy methyl amido) phenyl) thiazol-2-yl) piperidines -1- benzyl formates

4- (4- (4- (sulfonyloxy methyl amido) phenyl) thiazol-2-yl) piperidines -1- benzyl formates

4- (3- (pyridine -2- bases)-[1,2,4] triazol [4,3-b] pyridazine -6- bases)-N- tosyl aniline,

(4- (5- chloro-2-methyls phenyl) piperazine -1- bases) (4- tosyls amino) phenyl) ketone,

4- (4- ((1- methyl isophthalic acid H- benzos [d] imidazoles -2- bases) methyl) piperazine -1- bases)-N- tosyl aniline,

The chloro- 4- methyl-N- of 3- (6- (4- (3- (trifluoromethyl) benzyl) piperazine -1- bases) pyridin-3-yl) benzsulfamide

The chloro- N- of 4- (4- (4- ((1- methyl isophthalic acid H- benzos [d] imidazoles -2- bases) methyl) piperazine -1- bases) phenyl) benzsulfamide

(Z) -4- (3- cyano group -3- (4- (2,4- 3,5-dimethylphenyls) thiazol-2-yl) pi-allyl)-N- (thiazol-2-yl) benzene sulfonyl Amine

N- (3- (H- imidazos [1,2-a] pyridine -2- bases) phenyl) -4- methyl -2- phenyl thiazole -5- formamides,

N- (3- (benzo [d] thiazol-2-yl) phenyl) Pyrazinamide,

3- (4- chlorphenyls) -4,5- dihydro -1- phenyl -5- (2- phenyl thiazole -4- bases) -1H- pyrazoles,

N- (4- (6- methyl benzo [d] thiazol-2-yl) phenyl) -2- (tolylmethyl sulfoamido between N-) acetamide；Or

N- (4- (6- methyl benzo [d] thiazol-2-yl) phenyl) -2- (N- p-methylphenyl sulfonyloxy methyls amido) acetamide.

34. for method of the increase themogenesis without reducing lean body mass during the weight of animals mitigation, methods described includes The step of compound for the therapeutically effective amount being applied in the animal in medicinal medium, the compound has following structures：

Wherein R₁It is H, Et, OMe or n-propyl；

Y be CH or

R₂It is OH, OMe or NH-i-Pr；

R₃It is H, F or Cl；And

R₄It is H, Me, Cl, Br, F, OH, OBz, OCH₂COOMe、OCH₂COOH、NH₂、NH-i-Pr、NHCOMe、NHSO₂Me、NHBn、OMe、NHBoc、NHTs、Or its pharmaceutical salts or solid Isomers or combinations thereof.

35. the method for claim 34, wherein the animal is with the related patient's condition of following body weight：Obesity, hypertension, artery are hard Change, asthma, hyperlipidemia, hyperinsulinemia, NASH and the diabetes B caused by insulin resistance.

36. the method for claim 36, it also includes providing the second therapy to the animal, and second therapy includes life side Formula change, antihyperglycemic agents, insulin, glucagon-like peptide (GLP), dipeptidyl peptidase-4 inhibitors, thiazolidinedione, drop Compound or combination two or more in them.

37. the method for claim 1 wherein the compound is configured to food, animal feed material or medicine.

38. the method for claim 38, wherein compound increase uncoupling proteins 1, uncoupling protein-3 and UCPS 3 expression.