Design and synthesis of novel spirocyclic carboxylic acids as potent and orally bioavailable DGAT1 inhibitors and their biological evaluation.

A series of novel spirocyclic DGAT1 inhibitors containing the oxadiazole motif were designed and synthesized for biological evaluation. Several compounds exhibited potent diacylglycerol acyltransferase 1 (DGAT1) inhibitory activity. Optimization of the series led to the identification of five lead compounds 8, 9, 10, 11 and 12 that showed excellent in-vitro activity with IC50 values ranging from 7 to 20 nM against human DGAT1. All compounds demonstrated good druggability as well as microsomal stability and safety profiles such as hERG and CYP. Compound 12 significantly reduced plasma triglyceride levels in-vivo in the mouse model of acute lipid challenge. Significant reduction in plasma TG excursion was observed, thus indicating DGAT1 inhibition in-vivo.

Discovery and evaluation of 1H-pyrrolo[2,3-b]pyridine based selective and reversible small molecule BTK inhibitors for the treatment of rheumatoid arthritis.

In a pursuit to identify reversible and selective BTK inhibitors, two series based on 7H-pyrrolo[2,3-d]pyrimidine and 1H-pyrrolo[2,3-b]pyridine as the hinge binding core, have been identified. Structure activity relationship (SAR) exploration led to identification of two advanced lead molecules, 11 and 13, which demonstrated desired BTK inhibitory potency in different cellular assays, excellent selectivity in a panel of 50 diverse kinases, favorable in vivo PK properties in mice and anti-arthritic effect in a mouse model of CIA.

Synthesis and biological evaluation of novel pyrrolidine acid analogs as potent dual PPARα/γ agonists.

The design, synthesis and structure-activity relationships of a novel series of 3,4-disubstituted pyrrolidine acid analogs as PPAR ligands is outlined. In both the 1,3- and 1,4-oxybenzyl pyrrolidine acid series, the preferred stereochemistry was shown to be the cis-3R,4S isomer, as exemplified by the potent dual PPARα/γ agonists 3k and 4i. The N-4-trifluoromethyl-pyrimidinyl pyrrolidine acid analog 4i was efficacious in lowering fasting glucose and triglyceride levels in diabetic db/db mice.

Early ultrasound therapy for rehabilitation after zone II flexor tendon repair.

BACKGROUND: In our Institute, most of the patients treated for hand injuries were industrial workers with poor compliance. For rehabilitation after zone II flexor tendon repair, we had tried various early mobilization protocols. As these protocols demanded a degree of commitment from the patients, our results were suboptimal. Hence, to improve the results, we implemented a new rehabilitation protocol by administering the pulsed ultrasound therapy during the early phase of tendon healing. MATERIALS AND METHODS: This is a prospective study done over a period of five years from January 2008 to January 2013. A total of 100 patients and 139 digits with zone II flexor tendon injuries were studied. After randomization, we administered pulsed ultrasound therapy of different frequencies and intensities for a total of 72 patients and 99 digits and formulated three groups. The results of ultrasound treated cases were compared with each other and with the results of cases treated by immobilization protocol. The results were analyzed using 'Original Strickland' criteria. RESULTS: 72% excellent-good results in ultrasound (Group 1) protocol, 75% excellent-good results in ultrasound (Group 2) protocol, and 77% excellent-good results in ultrasound (Group 3) protocol were achieved. There was no case of rupture in the first two groups. The rupture rate was 7% in ultrasound (Group 3) protocol. Only 25% excellent-good results were obtained in the immobilization protocol. CONCLUSION: After zone II flexor tendon repair, pulsed ultrasound therapy during the early rehabilitation phase is safe and effective. The results are comparable to early mobilization protocols.

Design, synthesis and structure-activity relationships of azole acids as novel, potent dual PPAR alpha/gamma agonists.

The design, synthesis and structure-activity relationships of a novel series of N-phenyl-substituted pyrrole, 1,2-pyrazole and 1,2,3-triazole acid analogs as PPAR ligands are outlined. The triazole acid analogs 3f and 4f were identified as potent dual PPARalpha/gamma agonists both in binding and functional assays in vitro. The 3-oxybenzyl triazole acetic acid analog 3f showed excellent glucose and triglyceride lowering in diabetic db/db mice.

Design, synthesis, and structure-activity relationships of piperidine and dehydropiperidine carboxylic acids as novel, potent dual PPARalpha/gamma agonists.

Several series of substituted dehydropiperidine and piperidine-4-carboxylic acid analogs have been designed and synthesized as novel, potent dual PPARalpha/gamma agonists. The SAR of these series of analogs is discussed. A rare double bond migration occurred during the basic hydrolysis of the alpha,beta-unsaturated dehydropiperidine esters 12, and the structures of the migration products were confirmed through a series of 2D NMR experiments.

Discovery of azetidinone acids as conformationally-constrained dual PPARalpha/gamma agonists.

A novel class of azetidinone acid-derived dual PPARalpha/gamma agonists has been synthesized for the treatment of diabetes and dyslipidemia. The preferred stereochemistry in this series for binding and functional agonist activity against both PPARalpha and PPARgamma receptors was shown to be 3S,4S. Synthesis, in vitro and in vivo activities of compounds in this series are described. A high-yielding method for N-arylation of azetidinone esters is also described.

Muraglitazar, a novel dual (alpha/gamma) peroxisome proliferator-activated receptor activator, improves diabetes and other metabolic abnormalities and preserves beta-cell function in db/db mice.

Muraglitazar, a novel dual (alpha/gamma) peroxisome proliferator-activated receptor (PPAR) activator, was investigated for its antidiabetic properties and its effects on metabolic abnormalities in genetically obese diabetic db/db mice. In db/db mice and normal mice, muraglitazar treatment modulates the expression of PPAR target genes in white adipose tissue and liver. In young hyperglycemic db/db mice, muraglitazar treatment (0.03-50 mg . kg(-1) . day(-1) for 2 weeks) results in dose-dependent reductions of glucose, insulin, triglycerides, free fatty acids, and cholesterol. In older hyperglycemic db/db mice, longer-term muraglitazar treatment (30 mg . kg(-1) . day(-1) for 4 weeks) prevents time-dependent deterioration of glycemic control and development of insulin deficiency. In severely hyperglycemic db/db mice, muraglitazar treatment (10 mg . kg(-1) . day(-1) for 2 weeks) improves oral glucose tolerance and reduces plasma glucose and insulin levels. In addition, treatment increases insulin content in the pancreas. Finally, muraglitazar treatment increases abnormally low plasma adiponectin levels, increases high-molecular weight adiponectin complex levels, reduces elevated plasma corticosterone levels, and lowers elevated liver lipid content in db/db mice. The overall conclusions are that in db/db mice, the novel dual (alpha/gamma) PPAR activator muraglitazar 1) exerts potent and efficacious antidiabetic effects, 2) preserves pancreatic insulin content, and 3) improves metabolic abnormalities such as hyperlipidemia, fatty liver, low adiponectin levels, and elevated corticosterone levels.

Discovery of Potent and Selective A2A Antagonists with Efficacy in Animal Models of Parkinson's Disease and Depression.

Adenosine A2A receptor (A2AAdoR) antagonism is a nondopaminergic approach to Parkinson's disease treatment that is under development. Earlier we had reported the therapeutic potential of 7-methoxy-4-morpholino-benzothiazole derivatives as A2AAdoR antagonists. We herein described a novel series of [1,2,4]triazolo[5,1-f]purin-2-one derivatives that displays functional antagonism of the A2A receptor with a high degree of selectivity over A1, A2B, and A3 receptors. Compounds from this new scaffold resulted in the discovery of highly potent, selective, stable, and moderate brain penetrating compound 33. Compound 33 endowed with satisfactory in vitro and in vivo pharmacokinetics properties. Compound 33 demonstrated robust oral efficacies in two commonly used models of Parkinson's disease (haloperidol-induced catalepsy and 6-OHDA lesioned rat models) and depression (TST and FST mice models).

Design and synthesis of N-[(4-methoxyphenoxy)carbonyl]-N-[[4-[2-(5- methyl-2-phenyl-4-oxazolyl)ethoxy]phenyl]methyl]glycine [Muraglitazar/BMS-298585], a novel peroxisome proliferator-activated receptor alpha/gamma dual agonist with efficacious glucose and lipid-lowering activities.

Muraglitazar/BMS-298585 (2) has been identified as a non-thiazolidinedione PPAR alpha/gamma dual agonist that shows potent activity in vitro at human PPARalpha (EC(50) = 320 nM) and PPARgamma(EC(50) = 110 nM). Compound 2 shows excellent efficacy for lowering glucose, insulin, triglycerides, and free fatty acids in genetically obese, severely diabetic db/db mice and has a favorable ADME profile. Compound 2 is currently in clinical development for the treatment of type 2 diabetes and dyslipidemia.

Fat oxidation, fitness and skeletal muscle expression of oxidative/lipid metabolism genes in South Asians: implications for insulin resistance?

BACKGROUND: South Asians are more insulin resistant than Europeans, which cannot be fully explained by differences in adiposity. We investigated whether differences in oxidative capacity and capacity for fatty acid utilisation in South Asians might contribute, using a range of whole-body and skeletal muscle measures. METHODOLOGY/PRINCIPAL FINDINGS: Twenty men of South Asian ethnic origin and 20 age and BMI-matched men of white European descent underwent exercise and metabolic testing and provided a muscle biopsy to determine expression of oxidative and lipid metabolism genes and of insulin signalling proteins. In analyses adjusted for age, BMI, fat mass and physical activity, South Asians, compared to Europeans, exhibited; reduced insulin sensitivity by 26% (p = 0.010); lower VO2max (40.6±6.6 vs 52.4±5.7 ml x kg(-1) x min(-1), p = 0.001); and reduced fat oxidation during submaximal exercise at the same relative (3.77±2.02 vs 6.55±2.60 mg x kg(-1) x min(-1) at 55% VO2max, p = 0.013), and absolute (3.46±2.20 vs 6.00±1.93 mg x kg(-1) x min(-1) at 25 ml O(2) x kg(-1) x min(-1), p = 0.021), exercise intensities. South Asians exhibited significantly higher skeletal muscle gene expression of CPT1A and FASN and significantly lower skeletal muscle protein expression of PI3K and PKB Ser473 phosphorylation. Fat oxidation during submaximal exercise and VO2max both correlated significantly with insulin sensitivity index and PKB Ser473 phosphorylation, with VO2max or fat oxidation during exercise explaining 10-13% of the variance in insulin sensitivity index, independent of age, body composition and physical activity. CONCLUSIONS/SIGNIFICANCE: These data indicate that reduced oxidative capacity and capacity for fatty acid utilisation at the whole body level are key features of the insulin resistant phenotype observed in South Asians, but that this is not the consequence of reduced skeletal muscle expression of oxidative and lipid metabolism genes.

Genetic and gene expression studies implicate renin and endothelin-1 in edema caused by peroxisome proliferator-activated receptor gamma agonists.

OBJECTIVE: Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists can cause peripheral edema in susceptible individuals. To investigate the mechanistic basis underlying this adverse event, we performed a candidate gene analysis of patients enrolled in clinical trials of muraglitazar, an investigational PPARalpha/gamma dual agonist, and developed a cell culture-based gene expression assay and nonhuman primate model of edema to study the edemagenic properties of PPARgamma agonists. METHODS: A total of 213 single nucleotide polymorphisms (SNPs) in 63 genes were genotyped in 730 participants. Chi-square and logistic regression analyses were used to test for association with edema. Transcriptional responses to PPARgamma agonists were evaluated in Calu-6 cells using quantitative real-time PCR. Male Cynomolgus monkeys were treated with PPAR agonists and were evaluated for edema using MRI. RESULTS: SNPs in renin (rs2368564) and endothelin-1 (rs5370) were associated with reduced risk of edema (P=0.003 and P=0.028, respectively) and an SNP in beta1 adrenergic receptor (rs1801253) was associated with increased susceptibility to edema (P=0.034). Gene expression studies revealed that renin and endothelin-1 were regulated by PPARgamma in Calu-6 cells. A survey of 10 PPARgamma agonists further revealed that a compound's in vitro potency was correlated with its edemagenic potential leading to the prediction that one of three previously uncharacterized PPARgamma agonists would cause less edema. This prediction was validated in a nonhuman primate model of PPARgamma agonist-induced edema. CONCLUSION: Our results implicate a key role for renin and endothelin-1 in the edema caused by PPARgamma agonists and demonstrate how knowledge gained from pharmacogenetic studies can be applied in drug discovery.

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.

The dual peroxisome proliferator-activated receptor alpha/gamma activator muraglitazar prevents the natural progression of diabetes in db/db mice.

There are two major defects in type 2 diabetes: 1) insulin resistance and 2) insulin deficiency due to loss of beta-cell function. Here we demonstrated that treatment with muraglitazar (a dual peroxisome proliferator-activated receptor alpha/gamma activator), when initiated before or after the onset of diabetes in mice, is effective against both defects. In study 1, prediabetic db/db mice were treated for 12 weeks. The control mice developed diabetes, as evidenced by hyperglycemia, hyperinsulinemia, reduced insulin levels in the pancreas, blunted insulin response to glucose, and impaired glucose tolerance. The muraglitazar-treated mice had normal plasma glucose, and insulin levels, equivalent or higher pancreatic insulin content than normal mice, showed a robust insulin response to glucose and exhibited greater glucose tolerance. In study 2, diabetic db/db mice were treated for 4 weeks. The control mice displayed increased glucose levels, severe loss of islets, and their isolated islets secreted reduced amounts of insulin in response to glucose and exendin-4 compared with baseline. In muraglitazar-treated mice, glucose levels were reduced to normal. These mice showed reduced loss of islets, and their isolated islets secreted insulin at levels comparable to baseline. Thus, muraglitazar treatment decreased both insulin resistance and preserved beta-cell function. As a result, muraglitazar treatment, when initiated before the onset of diabetes, prevented development of diabetes and, when initiated after the onset of diabetes, prevented worsening of diabetes in db/db mice.

Comparative metabolism of radiolabeled muraglitazar in animals and humans by quantitative and qualitative metabolite profiling.

Muraglitazar (Pargluva), a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) activator, has both glucose- and lipid-lowering effects in animal models and in patients with diabetes. This study describes the in vivo and in vitro comparative metabolism of [(14)C]muraglitazar in rats, dogs, monkeys, and humans by quantitative and qualitative metabolite profiling. Metabolite identification and quantification methods used in these studies included liquid chromatography/mass spectrometry (LC/MS), LC/tandem MS, LC/radiodetection, LC/UV, and a newly described mass defect filtering technique in conjunction with high resolution MS. After oral administration of [(14)C]muraglitazar, absorption was rapid in all species, reaching a concentration peak for parent and total radioactivity in plasma within 1 h. The most abundant component in plasma at all times in all species was the parent drug, and no metabolite was present in greater than 2.5% of the muraglitazar concentrations at 1 h postdose in rats, dogs, and humans. All metabolites observed in human plasma were also present in rats, dogs, or monkeys. Urinary excretion of radioactivity was low (<5% of the dose) in all intact species, and the primary route of elimination was via biliary excretion in rats, monkeys, and humans. Based on recovered doses in urine and bile, muraglitazar showed a very good absorption in rats, monkeys, and humans. The major drug-related components in bile of rats, monkeys, and humans were glucuronides of muraglitazar and its oxidative metabolites. The parent compound was a minor component in bile, suggesting extensive metabolism of the drug. In contrast, the parent drug and oxidative metabolites were the major components in feces, and no glucuronide conjugates were found, suggesting that glucuronide metabolites were excreted in bile and hydrolyzed in the gastrointestinal tract. The metabolites of muraglitazar resulted from both glucuronidation and oxidation. The metabolites in general had greatly reduced activity as PPARalpha/gamma activators relative to muraglitazar. In conclusion, muraglitazar was rapidly absorbed, extensively metabolized through glucuronidation and oxidation, and mainly eliminated in the feces via biliary excretion of glucuronide metabolites in all species studied. Disposition and metabolic pathways were qualitatively similar in rats, dogs, monkeys, and humans.

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.