Design, synthesis, and structure-activity relationship of carbamate-tethered aryl propanoic acids as novel PPARalpha/gamma dual agonists

We have developed a new class of PPARalpha/gamma dual agonists, which show excellent agonistic activity in PPARalpha/gamma transactivation assay. In particular, (R)-9d was identified as a potent PPARalpha/gamma dual agonist with EC(50)s of 0.377 microM in PPARalpha and 0.136 microM in PPARgamma, respectively. Interestingly, the structure-activity relationship revealed that the stereochemistry of the identified PPARalpha/gamma dual agonists significantly affects their agonistic activities in PPARalpha than in PPARgamma.     

A peroxisome proliferator-activated receptor alpha/gamma dual agonist with a unique in vitro profile and potent glucose and lipid effects in rodent models of type 2 diabetes and dyslipidemia

LSN862 is a novel peroxisome proliferator-activated receptor (PPAR)alpha/gamma dual agonist with a unique in vitro profile that shows improvements on glucose and lipid levels in rodent models of type 2 diabetes and dyslipidemia. Data from in vitro binding, cotransfection, and cofactor recruitment assays characterize LSN862 as a high-affinity PPARgamma partial agonist with relatively less but significant PPARalpha agonist activity. Using these same assays, rosiglitazone was characterized as a high-affinity PPARgamma full agonist with no PPARalpha activity. When administered to Zucker diabetic fatty rats, LSN862 displayed significant glucose and triglyceride lowering and a significantly greater increase in adiponectin levels compared with rosiglitazone. Expression of genes involved in metabolic pathways in the liver and in two fat depots from compound-treated Zucker diabetic fatty rats was evaluated. Only LSN862 significantly elevated mRNA levels of pyruvate dehydrogenase kinase isozyme 4 and bifunctional enzyme in the liver and lipoprotein lipase in both fat depots. In contrast, both LSN862 and rosiglitazone decreased phosphoenol pyruvate carboxykinase in the liver and increased malic enzyme mRNA levels in the fat. In addition, LSN862 was examined in a second rodent model of type 2 diabetes, db/db mice. In this study, LSN862 demonstrated statistically better antidiabetic efficacy compared with rosiglitazone with an equivalent side effect profile. LSN862, rosiglitazone, and fenofibrate were each evaluated in the humanized apoA1 transgenic mouse. At the highest dose administered, LSN862 and fenofibrate reduced very low-density lipoprotein cholesterol, whereas, rosiglitazone increased very low-density lipoprotein cholesterol. LSN862, fenofibrate, and rosiglitazone produced maximal increases in high-density lipoprotein cholesterol of 65, 54, and 30%, respectively. These findings show that PPARgamma full agonist activity is not necessary to achieve potent and efficacious insulin-sensitizing benefits and demonstrate the therapeutic advantages of a PPARalpha/gamma dual agonist.     

Design, synthesis, and evaluation of 2-alkoxydihydrocinnamates as PPAR agonists

A series of 2-alkoxydihydrocinnamates were synthesized as PPARgamma and PPARalpha dual agonists. In vitro studies in cell model showed that these compounds were efficacious. Compound 1g was found to be a potent PPARalpha/gamma dual agonist and will be further evaluated for the treatment of type II diabetes.     

Biochemical mechanism of insulin sensitization, lipid modulation and anti-atherogenic potential of PPAR alpha/gamma dual agonist: Ragaglitazar

The current goal in the treatment of diabetes is not only to enhance the glycemic control but also to improve the associated cardiovascular risk factors. Among many of the strategies available, a co-ligand of PPARalpha and gamma in a single molecule which combines the insulin sensitizing potential of PPARgamma and the beneficial lipid modulating properties of PPARalpha agonism, has gained attention in the recent past. Here we report the biochemical mechanism by which a dual PPAR alpha/gamma agonist Ragaglitazar (Raga) achieves this goal. The PPARalpha component of Raga appears to contribute to a significant increase in beta oxidation, ApoA1 secretion and inhibition of TG biosynthesis in HepG2 cells. These effects of Raga at 60 microM were similar to that shown by Fenofibrate (Feno) at 250 microM. The PPARgamma component of Raga showed significant G3PDH activity and TG accumulation with a corresponding increase in aP2 expression in 3T3L1 cells. Significantly reduced levels of IL-6 and TNFalpha were observed in the culture supernatants of Raga treated 3T3L1 cells. Raga resulted in significant insulin dependent glucose uptake in 3T3L1 with a corresponding increase in GLUT4 expression. Further, Raga showed a significant cholesterol efflux with a corresponding increase in ABCA1 protein expression in THP-1 macrophages. In conclusion, Raga activates both PPARalpha and gamma regulated pathway in adipocytes as well as in hepatocytes which together contributes for its insulin sensitizing and lipid lowering activity. In addition the dual activation of PPAR alpha/gamma also shows an athero-protective potential by inducing reverse cholesterol efflux and inhibiting the pro-inflammatory cytokines.     

Dual PPARalpha /gamma activation provides enhanced improvement of insulin sensitivity and glycemic control in ZDF rats

Improvement of insulin sensitivity and lipid and glucose metabolism by coactivation of both nuclear peroxisome proliferator-activated receptor (PPAR)gamma and PPARalpha potentially provides beneficial effects over existing PPARgamma and alpha preferential drugs, respectively, in treatment of type 2 diabetes. We examined the effects of the dual PPARalpha/gamma agonist ragaglitazar on hyperglycemia and whole body insulin sensitivity in early and late diabetes stages in Zucker diabetic fatty (ZDF) rats and compared them with treatment with the PPARgamma preferential agonist rosiglitazone. Despite normalization of hyperglycemia and Hb A(1c) and reduction of plasma triglycerides by both compounds in both prevention and early intervention studies, ragaglitazar treatment resulted in overall reduced circulating insulin and improved insulin sensitivity to a greater extent than after treatment with rosiglitazone. In late-intervention therapy, ragaglitazar reduced Hb A(1c) by 2.3% compared with 1.1% by rosiglitazone. Improvement of insulin sensitivity caused by the dual PPARalpha/gamma agonist ragaglitazar seemed to have beneficial impact over that of the PPARgamma-preferential activator rosiglitazone on glycemic control in frankly diabetic ZDF rats.     

A rapid, homogeneous, fluorescence polarization binding assay for peroxisome proliferator-activated receptors alpha and gamma using a fluorescein-tagged dual PPARalpha/gamma activator

Peroxisome proliferator-activated receptors (PPARs) and other members of the nuclear hormone receptor family are important drug targets for the treatment of metabolic diseases. PPARalpha and PPARgamma play crucial roles in lipid and glucose metabolism, respectively. Therefore, screening methods that help to rapidly identify activators of these receptors should be of considerable value. A homogeneous fluorescence polarization (FP) ligand binding assay capable of rapidly identifying ligands that bind to both PPARalpha and PPARgamma has been developed using purified PPARalpha or PPARgamma ligand binding domains and a fluorescein-labeled analog (FLA) of a potent dual PPARalpha/gamma activator. FLA activator showed good binding affinity toward both PPARalpha (K(i)=0.7microM) and PPARgamma (K(i)=0.4microM). The binding of FLA activator was rapid and reached a plateau within 10 min. The resulting FP signal was stable for at least 18h. The FP binding assay performed robustly in a 384-well format, and the average Z' value was 0.77. There was a good correlation between the binding potency (IC(50) values) and rank order of binding potency for a panel of standard PPAR ligands obtained in FP binding assay and scintillation proximity assay or gel filtration binding assays using (3)H-labeled PPARalpha (r(2)=0.99) and PPARgamma (r(2)=0.99) ligands. There was also a good correlation of IC(50) values obtained by FP binding assay and scintillation proximity assay for the clinically used PPAR activators. Thus, the FP binding assay with a single fluorescein-labeled PPARalpha/gamma dual activator offers a homogeneous nonradioactive, sensitive, robust, and less expensive high-throughput assay for detecting compounds that bind to both PPARgamma and PPARalpha. Using this FP binding assay, we have identified a large number of PPARalpha/gamma dual activators. A similar assay platform may be easily adapted to other members of the nuclear hormone receptor family.     

Comparison of the effects of pioglitazone and rosiglitazone on macrophage foam cell formation

In order to elucidate the antiatherogenic effects of pioglitazone (a peroxisome proliferator-activated receptor [PPAR]gamma agonist with PPARalpha agonistic activity) and rosiglitazone (a more selective PPARgamma agonist), we examined gene expression and cholesteryl ester accumulation in THP-1-derived macrophages. Pioglitazone enhanced the mRNA expression of the proatherogenic factors CD36 and adipophilin, but was approximately 10 times less potent than rosiglitazone. The potencies of the two agents appeared to correspond to their PPARgamma agonistic activities in this respect. However, both agents were similarly potent in enhancing the mRNA expression of the antiatherogenic factors liver X receptor alpha and ATP-binding cassette-transporter A1. Furthermore, both agents enhanced cholesteryl ester hydrolase mRNA expression and inhibited acyl-CoA cholesterol acyltransferase-1 mRNA expression and cholesteryl ester accumulation in macrophages. In this respect, their potencies appeared to correspond to their PPARalpha agonistic activities. These results suggest that pioglitazone has an equally beneficial effect on antiatherogenic events to rosiglitazone, despite being almost 10 times less potent than a PPARgamma agonist.     

AZ 242, a novel PPARalpha/gamma agonist with beneficial effects on insulin resistance and carbohydrate and lipid metabolism in ob/ob mice and obese Zucker rats

Abnormalities in fatty acid (FA) metabolism underlie the development of insulin resistance and alterations in glucose metabolism, features characteristic of the metabolic syndrome and type 2 diabetes that can result in an increased risk of cardiovascular disease. We present pharmacodynamic effects of AZ 242, a novel peroxisome proliferator activated receptor (PPAR)alpha/gamma agonist. AZ 242 dose-dependently reduced the hypertriglyceridemia, hyperinsulinemia, and hyperglycemia of ob/ob diabetic mice. Euglycemic hyperinsulinemic clamp studies showed that treatment with AZ 242 (1 micromol/kg/d) restored insulin sensitivity of obese Zucker rats and decreased insulin secretion. In vitro, in reporter gene assays, AZ 242 activated human PPARalpha and PPARgamma with EC(50) in the micro molar range. It also induced differentiation in 3T3-L1 cells, an established PPARgamma effect, and caused up-regulation of liver fatty acid binding protein in HepG-2 cells, a PPARalpha-mediated effect. PPARalpha-mediated effects of AZ 242 in vivo were documented by induction of hepatic cytochrome P 450-4A in mice. The results indicate that the dual PPARalpha/gamma agonism of AZ 242 reduces insulin resistance and has beneficial effects on FA and glucose metabolism. This effect profile could provide a suitable therapeutic approach to the treatment of type 2 diabetes, metabolic syndrome, and associated vascular risk factors.     

PPARalpha /gamma ragaglitazar eliminates fatty liver and enhances insulin action in fat-fed rats in the absence of hepatomegaly

Peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma agonists lower lipid accumulation in muscle and liver by different mechanisms. We investigated whether benefits could be achieved on insulin sensitivity and lipid metabolism by the dual PPARalpha/gamma agonist ragaglitazar in high fat-fed rats. Ragaglitazar completely eliminated high-fat feeding-induced liver triglyceride accumulation and visceral adiposity, like the PPARalpha agonist Wy-14643 but without causing hepatomegaly. In contrast, the PPARgamma agonist rosiglitazone only slightly lessened liver triglyceride without affecting visceral adiposity. Compared with rosiglitazone or Wy-14643, ragaglitazar showed a much greater effect (79%, P < 0.05) to enhance insulin's suppression of hepatic glucose output. Whereas all three PPAR agonists lowered plasma triglyceride levels and lessened muscle long-chain acyl-CoAs, ragaglitazar and rosiglitazone had greater insulin-sensitizing action in muscle than Wy-14643, associated with a threefold increase in plasma adiponectin levels. There was a significant correlation of lipid content and insulin action in liver and particularly muscle with adiponectin levels (P < 0.01). We conclude that the PPARalpha/gamma agonist ragaglitazar has a therapeutic potential for insulin-resistant states as a PPARgamma ligand, with possible involvement of adiponectin. Additionally, it can counteract fatty liver, hepatic insulin resistance, and visceral adiposity generally associated with PPARalpha activation, but without hepatomegaly.     

Structure-based design of indole propionic acids as novel PPARalpha/gamma co-agonists

In the quest for novel PPARalpha/gamma co-agonists as putative drugs for the treatment of type 2 diabetes and dyslipidemia, we have used a structure-based design approach to identify propionic acids with a 1,5-disubstituted indole scaffold as potent PPARalpha/gamma activators. Compounds 13, 24, and 28 are examples of submicromolar dual agonists with different alpha/gamma EC50 ratios that are selective against the delta-isoform. Analysis of the X-ray complex structure of PPARgamma with the indole propionic acid 13 provides a rationalization for some of the observed SAR.     

Synthesis, in vitro and in silico evaluation of l-tyrosine containing PPARalpha/gamma dual agonists

A novel series of l-tyrosine derivatives have been reported with potential PPARalpha/gamma dual agonistic activity. In vitro cell based PPARalpha/gamma transactivation studies have shown compound 4a and compound 4f to be the most potent PPARgamma and PPARalpha activators, respectively. Molecular docking studies performed on these series of compounds have complemented the experimental results and have led to interesting inferences.     

Discovery of tertiary aminoacids as dual PPARalpha/gamma agonists-I

A novel series of potent dual agonists of PPARalpha and PPARgamma, the alkoxybenzylglycines, was identified and explored using a solution-phase library approach. The synthesis and structure-activity relationships of this series of dual PPARalpha/gamma agonists are described.     

Design, synthesis, and evaluation of novel aryl-tetrahydropyridine PPARalpha/gamma dual agonists

Aryl-tetrahydropyridine derivatives were prepared and their PPARalpha/gamma dual agonistic activities were evaluated. Among them, compound (S)-5b was identified as a potent PPARalpha/gamma dual agonist with an EC(50) of 1.73 and 0.64 microM in hPPARalpha and gamma, respectively. In diabetic (db/db) mice, compound (S)-5b showed good glucose lowering efficacy and favorable pharmacokinetic properties.     

SAR-oriented discovery of peroxisome proliferator-activated receptor pan agonist with a 4-adamantylphenyl group as a hydrophobic tail

3-(4-Alkoxyphenyl)propanoic acid derivatives were prepared as candidate peroxisome proliferator-activated receptor (PPAR) alpha/delta/gamma pan agonists, based on our previous SAR studies directed toward the development of subtype-selective PPAR agonists. Those studies indicated that the steric bulkiness of substituents introduced at the distal benzene ring had an important influence on PPAR activity. The finding that a 4-adamantyl derivative exhibited not only PPARalpha/delta activity but also significant PPARgamma activity prompted us to search for structurally novel phenylpropanoic acid derivatives with more potent adipocyte differentiation activity than the well-known PPARgamma agonist, rosiglitazone, as well as well-balanced PPARalpha and PPARdelta agonistic activities. A representative phenylpropanoic acid derivative (12) bearing a 4-adamantylphenyl substituent proved to be a well-balanced PPAR-pan agonist with activities to regulate the expression of genes involved in lipid and glucose homeostasis, and should be useful as a candidate drug for the treatment of altered PPAR function.     

Synthesis and evaluation of azaindole-alpha-alkyloxyphenylpropionic acid analogues as PPARalpha/gamma agonists

A series of azaindole-alpha-alkyloxyphenylpropionic acid analogues was synthesized and evaluated for PPAR agonist activities. Structure-activity relationship was developed for PPARalpha/gamma dual agonism. One of the synthesized compound 7a was identified as a potent, selective PPARalpha/gamma dual agonist.     

Determination of MK-0767 enantiomers in human plasma by normal phase LC-MS/MS

A sensitive and selective analytical method for the enantioselective determination of MK-0767, a dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist, in human plasma has been developed and validated. The chromatography is based on normal-phase chiral separation on a Kromasil, 5 microm, CHI-DMB 250 mm x 4.6 mm column. The detection involves the direct introduction of the normal phase eluent into MS/MS without the addition of a post-column reagent. Atmospheric pressure chemical ionization (APcI) mode was selected as the ion source in this method. With proper sample handling and processing procedures, ex vivo interconversion of the enantiomers was kept to minimum during sample collection, preparation and short term storage of frozen human plasma samples. The method was successfully utilized to determine the concentrations of MK-0767 enantiomers in human plasma to support pharmacokinetic investigation in man.     

Synthesis and structure-activity relationships of thiadiazole-derivatives as potent and orally active peroxisome proliferator-activated receptors alpha/delta dual agonists

Replacement of the methyl-thiazole moiety of GW501516 (a PPARdelta selective agonist) with [1,2,4]thiadiazole gave compound 21 which unexpectedly displayed submicromolar potency as a partial agonist at PPARalpha in addition to the high potency at PPARdelta. A structure-activity relationships study of 21 resulted in the identification of 40 as a potent and selective PPARalpha/delta dual agonist. Compound 40 and its close analogs represent a new series of PPARalpha/delta dual agonists. The high potency, high selectivity, significant gene induction, excellent PK profiles, low P450 inhibition or induction, and good in vivo efficacy in four animal models support 40 being selected as a pre-clinical study candidate, and may render 40 as a valuable pharmacological tool in elucidating the complex roles of PPARalpha/delta dual agonists, and the potential usage for the treatment of metabolic syndrome.     

Modulation of PPAR receptor subtype selectivity of the ligands: aliphatic chain vs aromatic ring as a spacer between pharmacophore and the lipophilic moiety

Oxazole containing glycine and oximinobutyric acid derivatives were synthesized as PPARalpha agonists by incorporating polymethylene spacer as a replacement of commonly used phenylene group that connects the acidic head with lipophilic tail. Compound 13a was found to be a selective and potent PPARalpha agonist. Further 1,3-dioxane-2-carboxylic acid derivative 20 was synthesized by replacing the tetramethylene spacer of NS-220, a selective PPARalpha agonist with phenylene group and found to exhibit PPARalpha/gamma dual agonism. These results suggest that compounds possessing polymethylene spacer between pharmacophore and lipophilic tail exhibit predominantly PPARalpha agonism whereas those with an aromatic phenylene spacer shows PPARalpha/gamma dual agonism.