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.

Recombinant irisin induces weight loss in high fat DIO mice through increase in energy consumption and thermogenesis.

OBJECTIVE: Irisin is known to be an important metabolic regulator of glucose and lipid metabolism. The aims of the present study are to assess the role of mouse Irisin in obesity and energy metabolism and its glucose and lipid-lowering effects in a high-fat diet-induced obesity (DIO) mice model. METHODS: DIO mice were treated with recombinant murine Irisin or vehicle, and parameters such as body weight, feed intake, glucose, and lipid levels, obesity, energy consumption, and insulin sensitivity were assessed. mRNA and protein levels of UCP1 and different thermogenesis biomarker were evaluated by quantitative real-time PCR and Western blot, respectively, in tissues and major metabolic organs. RESULTS: Irisin decreased body weight and whole-body fat mass in DIO mice in a dose dependent manner due to marked increases in total energy expenditure. It also lowered blood glucose, insulin, and lipid levels and possibly reversed hepatic steatosis. Irisin improved hepatic and peripheral insulin sensitivity in DIO mice along with body weight reduction and adiposity. Gene expression of UCP1 in different organs (adipose tissue and major organs, i.e., liver, kidney, heart, brain, and spleen) have suggested the role of irisin is global. Gene expression profile of different biomarkers in spleen suggest a profound role of Irisin in inflammation. Liver tissue have also shown significant increase of UCP1 expression in dose dependent manner which suggest a role of irisin in liver.

Discovery and pharmacological evaluation of indole derivatives as potent and selective RORγt inverse agonist for multiple autoimmune conditions.

Targeting nuclear receptor RORγ is recognized to be beneficial in multiple autoimmune disorders. We disclosed new indole analogues as potent RORγ inverse agonists. RO-2 as one of the potent and orally bioavailable compounds was evaluated in various models of autoimmune disorder. It showed potent suppression of downstream markers of RORγt activity in murine and human primary cells, ex vivo PD assay and in multiple animal models of autoimmune diseases. The results indicate the potential of these indole analogues as orally bioavailable small molecule inverse agonists of RORγt, efficacious in various Th17 driven models of autoimmune disorders.

Discovery and evaluation of novel FAAH inhibitors in neuropathic pain model.

Conceptual design and modification of urea moiety in chemotype PF-3845/04457845, the bench marking irreversible inhibitor of fatty acid amide hydrolase (FAAH), led to discovery of a novel nicotinamide-based lead 12a having reversible mechanism of action. Focused SAR around the pyridine heterocycle (Ar) in 12a (Tables 1 and 2) resulted into four shortlisted compounds, (-)-12a, (-)-12i, (-)-12l-m. The required (-)-enantiomers were obtained via diastereomeric resolution of a novel chiral dissymmetric intermediate 15. Based on comparative profile of FAAH potency, metabolic stability in liver microsome, liability of inhibiting major hCYP450 isoforms, rat PK, and brain penetration ability, two SAR optimized compounds, (-)-12l and (-)-12m, were selected for efficacy study in rat model of chemotherapy-induced peripheral neuropathy (CIPN). Both the compounds exhibited dose related antihyperalgesic effects, when treated with 3-30 mg/kg po for 7 days. The effects at 30 mg/kg are comparable to that of PF-04457845 (10 mg/kg) and Tramadol (40 mg/kg).

Evaluation of separate role of intestine and liver in first pass metabolism of budesonide in rat.

1. Budesonide, a potent topical corticosteroid, reported to have low oral bioavailability in mice, rat, dog and human due to rapid first pass metabolism. However, there is insufficient information available in literature regarding the role of intestine and or liver responsible for the first pass metabolism of budesonide. 2. Current study in rats investigates the role of intestine and liver in first pass metabolism of budesonide using two in vivo models. Additionally, budesonide was also evaluated in in vitro assays such as thermodynamic solubility, permeability in Caco-2 cells and stability in simulated gastric (SGF), intestinal fluids (SIF) to understand the underlaying cause for low oral bioavailability. 3. Budesonide showed low oral, intra-duodenal and high intra-portal bioavailability in rat. In a dual vein cannulated rat model, intestinal and hepatic extraction ratios calculated based upon intestinal availability (Fa·Fg) and hepatic availability (Fh), suggests hepatic extraction of budesonide is minimal compared to intestinal. 4. In vitro results suggest, solubility and permeability may not be a barrier for the observed low oral bioavailability in rats. 5. Correlating the in vitro and in vivo data together, it can be concluded that, intestine might be playing major role in first pass metabolism of budesonide.

Modifications of flexible nonyl chain and nucleobase head group of (+)-erythro-9-(2's-hydroxy-3's-nonyl)adenine [(+)-EHNA] as adenosine deaminase inhibitors.

A series of terminal nonyl chain and nucleobase modified analogues of (+)-EHNA (III) were synthesized and evaluated for their ability to inhibit adenosine deaminase (ADA). The constrained carbon analogues of (+)-EHNA, 7a-7h, 10a-c, 12, 13, 14 and 17a-c appeared very potent with Ki values in the low nanomolar range. Thio-analogues of (+)-EHNA 24a-e wherein 5'C of nonyl chain replaced by sulfur atom found to be less potent compared to (+)-EHNA. Docking of the representative compounds into the active site of ADA was performed to understand structure-activity relationships. Compounds 7a (Ki: 1.1nM) 7b (Ki: 5.2nM) and 26a (Ki: 5.9nM) showed suitable balance of potency, microsomal stability and demonstrated better pharmacokinetic properties as compared to (+)-EHNA and therefore may have therapeutic potential for various inflammatory diseases, hypertension and cancer.

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 novel xanthine derivatives as potent and selective A2B adenosine receptor antagonists for the treatment of chronic inflammatory airway diseases.

Adenosine induces bronchial hyperresponsiveness and inflammation in asthmatics through activation of A2B adenosine receptor (A2BAdoR). Selective antagonists have been shown to attenuate airway reactivity and improve inflammatory conditions in pre-clinical studies. Hence, the identification of novel, potent and selective A2BAdoR antagonist may be beneficial for the potential treatment of asthma and Chronic Obstructive Pulmonary Disease (COPD). Towards this effort, we explored several prop-2-ynylated C8-aryl or heteroaryl substitutions on xanthine chemotype and found that 1-prop-2-ynyl-1H-pyrazol-4-yl moiety was better tolerated at the C8 position. Compound 59, exhibited binding affinity (Ki) of 62 nM but was non-selective for A2BAdoR over other AdoRs. Incorporation of substituted phenyl on the terminal acetylene increased the binding affinity (Ki) significantly to <10 nM. Various substitutions on terminal phenyl group and different alkyl substitutions on N-1 and N-3 were explored to improve the potency, selectivity for A2BAdoR and the solubility. In general, compounds with meta-substituted phenyl provided better selectivity for A2BAdoR compared to that of para-substituted analogs. Substitutions such as basic amines like pyrrolidine, piperidine, piperazine or cycloalkyls with polar group were tried on terminal acetylene, keeping in mind the poor solubility of xanthine analogs in general. However, these substitutions led to a decrease in affinity compared to compound 59. Subsequent SAR optimization resulted in identification of compound 46 with high human A2BAdoR affinity (Ki = 13 nM), selectivity against other AdoR subtypes and with good pharmacokinetic properties. It was found to be a potent functional A2BAdoR antagonist with a Ki of 8 nM in cAMP assay in hA2B-HEK293 cells and an IC50 of 107 nM in IL6 assay in NIH-3T3 cells. Docking study was performed to rationalize the observed affinity data. Structure-activity relationship (SAR) studies also led to identification of compound 36 as a potent A2BAdoR antagonist with Ki of 1.8 nM in cAMP assay and good aqueous solubility of 529 µM at neutral pH. Compound 46 was further tested for in vivo efficacy and found to be efficacious in ovalbumin-induced allergic asthma model in mice.

Discovery of liver-directed glucokinase activator having anti-hyperglycemic effect without hypoglycemia.

Glucokinase activators (GKAs) are among the emerging drug candidates for the treatment of type 2 diabetes (T2D). Despite effective blood glucose lowering in clinical trials, many pan-GKAs "acting both in pancreas and liver" have been discontinued from clinical development mainly because of their potential to cause hypoglycemia. Pan-GKAs over sensitize pancreatic GK, resulting in insulin secretion even at sub-normoglycemic level which might be a possible explanation for hypoglycemia. An alternative approach to minimize the risk of hypoglycemia is to use liver-directed GKAs, which are reported to be advancing well in clinical development. Here, we report the discovery and structure-activity relationship (SAR) studies on a novel 2-phenoxy-acetamide series with the aim of identifying a liver-directed GKA. Incorporation of a carboxylic acid moiety as an active hepatocyte uptake recognizing element at appropriate position of 2-phenoxy-acetamide core led to the identification of 26, a potent GKA with predominant liver-directed pharmacokinetics in mice. Compound 26 on oral administration significantly reduced blood glucose levels during an oral glucose tolerance test (oGTT) performed in diet-induced obese (DIO) mice, while showing no sign of hypoglycemia in normal C57 mice over a 10-fold dose range, even when dosed at fasted condition. Together, these data demonstrate a liver-directed GKA has beneficial effect on glucose homeostasis with reduced risk of hypoglycemia.

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.

Design and synthesis of novel, potent and selective hypoxanthine analogs as adenosine A1 receptor antagonists and their biological evaluation.

Multipronged approach was used to synthesize a library of diverse C-8 cyclopentyl hypoxanthine analogs from a common intermediate III. Several potent and selective compounds were identified and evaluated for pharmacokinetic (PK) properties in Wistar rats. One of the compounds 14 with acceptable PK parameters was selected for testing in in vivo primary acute diuresis model. The compound demonstrated significant diuretic activity in this model.

Design, Synthesis of Novel, Potent, Selective, Orally Bioavailable Adenosine A2A Receptor Antagonists and Their Biological Evaluation.

Our initial structure-activity relationship studies on 7-methoxy-4-morpholino-benzothiazole derivatives featured by aryloxy-2-methylpropanamide moieties at the 2-position led to identification of compound 25 as a potent and selective A2A adenosine receptor (A2AAdoR) antagonist with reasonable ADME and pharmacokinetic properties. However, poor intrinsic solubility and low to moderate oral bioavailability made this series unsuitable for further development. Further optimization using structure-based drug design approach resulted in discovery of potent and selective adenosine A2A receptor antagonists bearing substituted 1-methylcyclohexyl-carboxamide groups at position 2 of the benzothiazole scaffold and endowed with better solubility and oral bioavailability. Compounds 41 and 49 demonstrated a number of positive attributes with respect to in vitro ADME properties. Both compounds displayed good pharmacokinetic properties with 63% and 61% oral bioavailability, respectively, in rat. Further, compound 49 displayed oral efficacy in 6-OHDA lesioned rat model of Parkinson diseases.

A2B adenosine receptor antagonists: Design, synthesis and biological evaluation of novel xanthine derivatives.

A2BAdoR is a low affinity adenosine receptor that functions by Gs mediated elevation of cAMP and subsequent downstream signaling. The receptor has been implicated in lung inflammatory disorders like COPD and asthma. Several potent and selective A2BAdoR antagonists have been reported in literature, however most of the compounds suffer from poor pharmacokinetic profile. Therefore, with the aim to identify novel, potent and selective A2BAdoR antagonists with improved pharmacokinetic properties, we first explored more constrained form of MRS-1754 (4). To improve the metabolic stability, several linker modifications were attempted as replacement of amide linker along with different phenyl or other heteroaryls between C8 position of xanthine head group and terminal phenyl ring. SAR optimization resulted in identification of two novel A2BAdoR antagonists, 8-{1-[5-Oxo-1-(4-trifluoromethyl-phenyl)-pyrrolidin-3-ylmethyl]-1H-pyrazol-4-yl}-1,3-dipropyl-xanthine (31) and 8-(1-{2-Oxo-2-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]-ethyl}-1H-pyrazol-4-yl)-1,3-dipropyl-xanthine (65), with high binding affinity (Ki = 1 and 1.5 nM, respectively) and selectivity for A2BAdoR with very good functional potency of 0.9 nM and 4 nM, respectively. Compound 31 and 65 also displayed good pharmacokinetic properties in mice with 27% and 65% oral bioavailability respectively. When evaluated in in vivo mice model of asthma, compound 65 also inhibited airway inflammation and airway reactivity in ovalbumin induced allergic asthma at 3 mpk dose.

Impact of APCI ionization source in liquid chromatography tandem mass spectrometry based tissue distribution studies.

Measurement of test article concentration in tissue samples has been an important part of pharmacokinetic study and has helped to co-relate pharmacokinetic/pharmacodynamic relationships since the 1950s. Bioanalysis of tissue samples using LC-MS/MS comes with unique challenges in terms of sample handling and inconsistent analyte response owing to nonvolatile matrix components. Matrix effect is a phenomenon where the target analyte response is either suppressed or enhanced in the presence of matrix components. Based on previous reports electrospray ionization (ESI) mode of ionization is believed to be more affected by matrix components than atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionization. To explore the impact of ionization source with respect to bioanalysis of tissue samples, five structurally diverse compounds - atenolol, verapamil, diclofenac, propranolol and flufenamic acid - were selected. Quality control standards were spiked into 10 different biological matrices like whole blood, liver, heart, brain, spleen, kidney, skeletal muscle, eye and skin tissue and were quantified against calibration standards prepared in rat plasma. Quantitative bioanalysis was performed utilizing both APCI and ESI mode and results were compared. Quality control standards when analyzed with APCI mode were found to be more consistent in terms of accuracy and precision as compared with ESI mode. Additionally, for some instances, up to 20-fold broader dynamic linearity range was observed with APCI mode as compared with ESI mode. As phospholid interferences have poor response in APCI mode, protein precipitation extraction technique can be used for multimatrix quantitation, which is more amenable to automation. The approach of multiple biological matrix quantitation against a single calibration curve helps bioanalysts to reduce turnaround time. Copyright © 2016 John Wiley & Sons, Ltd.

Discovery of pyrazole carboxylic acids as potent inhibitors of rat long chain L-2-hydroxy acid oxidase.

Long chain L-2-hydroxy acid oxidase 2 (Hao2) is a peroxisomal enzyme expressed in the kidney and the liver. Hao2 was identified as a candidate gene for blood pressure (BP) quantitative trait locus (QTL) but the identity of its physiological substrate and its role in vivo remains largely unknown. To define a pharmacological role of this gene product, we report the development of selective inhibitors of Hao2. We identified pyrazole carboxylic acid hits 1 and 2 from screening of a compound library. Lead optimization of these hits led to the discovery of 15-XV and 15-XXXII as potent and selective inhibitors of rat Hao2. This report details the structure activity relationship of the pyrazole carboxylic acids as specific inhibitors of Hao2.

Discovery of a potent and selective small molecule hGPR91 antagonist.

GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC(50): 0.8 µM)-originally synthesized in Merck for Bradykinin B(1) Receptor (BK(1)R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5 g (IC(50): 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC(50): 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay.

Potent and Selective Inhibitors of Long Chain l-2-Hydroxy Acid Oxidase Reduced Blood Pressure in DOCA Salt-Treated Rats.

l-2-Hydroxy acid oxidase (Hao2) is a peroxisomal enzyme with predominant expression in the liver and kidney. Hao2 was recently identified as a candidate gene for blood pressure quantitative trait locus in rats. To investigate a pharmacological role of Hao2 in the management of blood pressure, selective Hao2 inhibitors were developed. Optimization of screening hits 1 and 2 led to the discovery of compounds 3 and 4 as potent and selective rat Hao2 inhibitors with pharmacokinetic properties suitable for in vivo studies in rats. Treatment with compound 3 or 4 resulted in a significant reduction or attenuation of blood pressure in an established or developing model of hypertension, deoxycorticosterone acetate-treated rats. This is the first report demonstrating a pharmacological benefit of selective Hao2 inhibitors in a relevant model of hypertension.

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.

Pharmacological and x-ray structural characterization of a novel selective androgen receptor modulator: potent hyperanabolic stimulation of skeletal muscle with hypostimulation of prostate in rats.

A novel, highly potent, orally active, nonsteroidal tissue selective androgen receptor (AR) modulator (BMS-564929) has been identified, and this compound has been advanced to clinical trials for the treatment of age-related functional decline. BMS-564929 is a subnanomolar AR agonist in vitro, is highly selective for the AR vs. other steroid hormone receptors, and exhibits no significant interactions with SHBG or aromatase. Dose response studies in castrated male rats show that BMS-564929 is substantially more potent than testosterone (T) in stimulating the growth of the levator ani muscle, and unlike T, highly selective for muscle vs. prostate. Key differences in the binding interactions of BMS-564929 with the AR relative to the native hormones were revealed through x-ray crystallography, including several unique contacts located in specific helices of the ligand binding domain important for coregulatory protein recruitment. Results from additional pharmacological studies effectively exclude alternative mechanistic contributions to the observed tissue selectivity of this unique, orally active androgen. Because concerns regarding the potential hyperstimulatory effects on prostate and an inconvenient route of administration are major drawbacks that limit the clinical use of T, the potent oral activity and tissue selectivity exhibited by BMS-564929 are expected to yield a clinical profile that provides the demonstrated beneficial effects of T in muscle and other tissues with a more favorable safety window.

Molecular cloning, stable expression and cellular localization of human alpha1-adrenergic receptor subtypes: effect of charcoal/dextran treated serum on expression and localization of alpha1D -adrenergic receptor.

The cDNAs encoding for three subtypes of adrenergic receptors, alpha1A-, alpha1B- and alpha1D-ARs, were cloned and expressed in HEK 293 cells. Expression of alpha1A- and alpha1B-AR subtypes in HEK 293 cells was stable even with increased passages but that of alpha1D-AR was not. Cellular localization studies using immunofluorescence and flow cytometry revealed that expression of alpha1A- and alpha1B-ARs was primarily localized on the cell membrane whereas expression of alpha1D-AR was predominantly intracellular. Our studies clearly demonstrated that the culturing of the recombinant cell lines expressing alpha1D-AR in charcoal/dextran treated fetal bovine serum (FBS) resulted in targeting of alpha1D-AR to the cell membrane and thus, significantly improving its stability and availability for ligand binding studies.