Design, synthesis and biological evaluation of novel pyrazolo-pyrimidin-amines as potent and selective BTK inhibitors.

To discover the best-in-class Bruton's Tyrosine Kinase (BTK) inhibitors, for th treatment of autoimmune disorders like cancer (B-Cell Lymphoma (BCL)) and rheumatoid arthritis (RA), in the present investigation, novel structural optimizations were carried out. Introduction of novel bicyclic amine linkers and aromatic backbone led to series of compounds 9a-h and 14a-u. Compound 14b was found to be potent, orally bioavailable, selective and irreversible BTK inhibitor. In vitro, 14b showed IC50 of 1.0 nM and 0.8 nM, in BTK and TMD8 assays, respectively. In vivo,14b displayed robust efficacy in collagen-induced arthritis (CIA) and TMD8 xenograft models, which could be correlated with its improved oral bioavailability. In the repeated dose acute toxicity study, 14b showed no adverse changes, indicating that the BTK inhibitor 14b could be viable therapeutic option for the treatment of autoimmune disorders.

Discovery of diaminopyrimidine-carboxamide derivatives as JAK3 inhibitors.

Selective inhibition of janus kinase (JAK) has been identified as an important strategy for the treatment of autoimmune disorders. Optimization at the C2 and C4-positions of pyrimidine ring of Cerdulatinib led to the discovery of a potent and orally bioavailable 2,4-diaminopyrimidine-5-carboxamide based JAK3 selective inhibitor (11i). A cellular selectivity study further confirmed that 11i preferentially inhibits JAK3 over JAK1, in JAK/STAT signaling pathway. Compound 11i showed good anti-arthritic activity, which could be correlated with its improved oral bioavailability. In the repeat dose acute toxicity study, 11i showed no adverse changes related to gross pathology and clinical signs, indicating that the new class JAK3 selective inhibitor could be viable therapeutic option for the treatment of rheumatoid arthritis.

Discovery of 1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-ones based novel, potent and PI3Kδ selective inhibitors.

PI3Kδ is implicated in various inflammatory and autoimmune diseases. For the effective treatment of chronic immunological disorders such as rheumatoid arthritis, it is essential to develop isoform selective PI3Kδ inhibitors. Structure guided optimization of an imidazo-quinolinones based pan-PI3K/m-TOR inhibitor (Dactolisib) led to the discovery of a potent and orally bioavailable PI3Kδ isoform selective inhibitor (10h), with an improved efficacy in the animal models.

Coagonist of glucagon-like peptide-1 and glucagon receptors ameliorates nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is often associated with obesity and type 2 diabetes. Coagonists of glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) are under clinical investigation for the treatment of obesity and type 2 diabetes. In this study, we have demonstrated the effect of a balanced coagonist in the treatment of NAFLD using mouse models. GLP-1R agonist exendin-4, glucagon, and coagonist (Aib2 C24 chimera2) were administered to C57BL6/J mice, in which NAFLD was induced by carbon tetrachloride (CCl4) treatment after high-fat diet (HFD) feeding, and choline-deficient, L-amino-acid-defined HFD (CDAHFD) feeding. Repeated dose administration of coagonist significantly attenuated liver inflammation and steatosis induced by acute and long-term treatment with CCl4 in HFD-fed mice. Coagonist markedly attenuated the CDAHFD-induced expression of TIMP-1, MMP-9, TNF-α, MCP-1, COL1A1, and α-SMA. It also inhibited progression of hepatic steatosis and fibrosis in mice. Exendin-4 was better than glucagon, but coagonist was most effective in reduction of hepatic inflammation as well as steatosis. Coagonist of GLP-1R and GCGR improved NAFLD in C57BL6/J mice. This effect is mediated by reduction in lipotoxicity and inflammation in liver.

Sensitive and specific LC-ESI-MS/MS method for determination of ZYDPLA1, a novel long-acting dipeptidyl peptidase 4 inhibitor in rat plasma: An application for toxicokinetic study in rats.

A rapid and highly specific assay was developed and validated for the estimation of ZYDPLA1 in rat plasma using liquid chromatography coupled to tandem mass spectrometry with positive electrospray ionization. Method validation comprised of parameters such as specificity, matrix effect, precision, accuracy, recovery, stability, etc. The assay procedure involved a simple protein precipitation of ZYDPLA1 and alprazolam (internal standard) from rat plasma using acetonitrile. Chromatographic separation was achieved with a gradient mobile phase comprising: (A) 0.2% ammonia in purified water; (B) 0.1% formic acid in isopropyl alcohol/methanol (1: 1 v/v); and (C) acetonitrile at a flow rate of 1 mL/min on an ACE-5, C18 (4.6 × 50 mm) column with a run time of 5.5 min. The quantitation of ZYDPLA1 was achieved by the summation of four multiple reaction mode transitions (m/z 399.7 → 383.0, 399.7 → 276.10, 399.7 → 153.20 and 399.7 → 127.20), while that of the internal standard was by a single multiple reaction mode transition (m/z 309.10 → 281.00). The lower limit of quantitation achieved was 0.01 µg/mL and the method showed linearity from 0.01 to 25 µg/mL. The intra- and inter-day precision (%CV) of the quality control samples was within 8.81% and accuracy was ±10% of nominal values. This novel method was applied for evaluation of toxicokinetics of ZYDLA1 in rats.

Central GLP-1 receptor activation improves cholesterol metabolism partially independent of its effect on food intake.

Glucagon-like peptide-1 (GLP-1) receptor agonists modulate lipid metabolism, apart from controlling glucose homeostasis. We investigated the role of central GLP-1 receptor (GLP-1R) agonism in regulation of hepatic lipid metabolism in cholesterol-fed hamsters. Cholesterol-fed hamsters were treated by intracerebroventricular (i.c.v.) route with exendin-4, as acute or repeated dose regimen and compared with hamsters pair-fed to the exendin-treated hamsters and with hamsters co-treated with GLP-1 antagonist exendin-9. Effect of acute treatment was observed on food intake, tyloxapol-induced hypertriglyceridemia, and corn oil induced post prandial lipemia. Plasma and hepatic lipids and changes in the expression of hepatic genes involved in lipid metabolism were assessed after chronic administration. Acute, as well as repeated dose, treatment of exendin-4 showed significant changes in hepatic lipids, circulating fatty acids, triglycerides, LDL, and cholesterol. Expression of SREBP-1c was reduced while that of LDLR and CYP7A1 was increased after the repeated dose treatment, and there was no change in HMG CoA reductase. These changes were blocked by co-treatment of exendin-9, and not replicated by pair feeding to the significant extent. Central GLP-1 receptor activation showed profound effects on peripheral lipid metabolism, which were partially independent of its effect on food intake.

Design, synthesis and biological evaluation of novel aminomethyl-piperidones based DPP-IV inhibitors.

A series of novel aminomethyl-piperidones were designed and evaluated as potential DPP-IV inhibitors. Optimized analogue 12v ((4S,5S)-5-(aminomethyl)-1-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-4-(2,5-difluorophenyl)piperidin-2-one) showed excellent in vitro potency and selectivity for DPP-IV over other serine proteases. The lead compound 12v showed potent and long acting antihyperglycemic effects (in vivo), along with improved pharmacokinetic profile.

Cannabinoid receptor 1 antagonist treatment induces glucagon release and shows an additive therapeutic effect with GLP-1 agonist in diet-induced obese mice.

Cannabinoid 1 (CB1) receptor antagonists reduce body weight and improve insulin sensitivity. Preclinical data indicates that an acute dose of CB1 antagonist rimonabant causes an increase in blood glucose. A stable analog of glucagon-like peptide 1 (GLP-1), exendin-4 improves glucose-stimulated insulin secretion in pancreas, and reduces appetite through activation of GLP-1 receptors in the central nervous system and liver. We hypothesized that the insulin secretagogue effect of GLP-1 agonist exendin-4 may synergize with the insulin-sensitizing action of rimonabant. Intraperitoneal as well as intracerebroventricular administration of rimonabant increased serum glucose upon glucose challenge in overnight fasted, diet-induced obese C57 mice, with concomitant rise in serum glucagon levels. Exendin-4 reversed the acute hyperglycemia induced by rimonabant. The combination of exendin-4 and rimonabant showed an additive effect in the food intake, and sustained body weight reduction upon repeated dosing. The acute efficacy of both the compounds was additive for inducing nausea-like symptoms in conditioned aversion test in mice, whereas exendin-4 treatment antagonized the effect of rimonabant on forced swim test upon chronic dosing. Thus, the addition of exendin-4 to rimonabant produces greater reduction in food intake owing to increased aversion, but reduces the other central nervous system side effects of rimonabant. The hyperglucagonemia induced by rimonabant is partially responsible for enhancing the antiobesity effect of exendin-4.

Co-agonist of glucagon and GLP-1 reduces cholesterol and improves insulin sensitivity independent of its effect on appetite and body weight in diet-induced obese C57 mice.

Dual agonism of glucagon and glucagon-like peptide-1 (GLP-1) receptors reduce body weight without inducing hyperglycemia in rodents. However, the effect of a co-agonist on insulin sensitivity and lipid metabolism has not been thoroughly assessed. Diet-induced obese (DIO) mice received 0.5 mg·kg(-1) of co-agonist or 2.5 mg·kg(-1) of glucagon or 8 µg·kg(-1) of exendin-4 by subcutaneous route, twice daily, for 28 days. A separate group of mice was pair-fed to the co-agonist-treated group for 28 days. Co-agonist treatment reduced food intake and reduced body weight up to 28 days. In addition, it reduced leptin levels and increased fibroblast growth factor 21 (FGF21) levels in plasma, when compared with control and pair-fed groups. Co-agonist treatment decreased triglyceride levels in serum and liver and reduced serum cholesterol, mainly due to reduction in low-density lipoprotein (LDL) cholesterol. These changes were not seen with pair-fed controls. Co-agonist treatment improved glucose tolerance and increased insulin sensitivity, as observed during glucose and insulin-tolerance test, hyperinsulinemic clamp, and reduced gluconeogenesis, as observed in pyruvate-tolerance test. The effects on insulin sensitivity and lipid levels are mostly independent of the food intake or body weight lowering effect of the co-agonist.

Long-acting peptidomimetics based DPP-IV inhibitors.

Pyrrolidine based peptidomimetics are reported as potent and selective DPP-IV inhibitors for the treatment of T2DM. Compounds 16c and 16d showed excellent in vitro potency and selectivity towards DPP-IV and the lead compound 16c showed sustained antihyperglycemic effects, along with improved pharmacokinetic profile.

Discovery of liver selective non-steroidal glucocorticoid receptor antagonist as novel antidiabetic agents.

Series of benzyl-phenoxybenzyl amino-phenyl acid derivatives (8a-q) are reported as non-steroidal GR antagonist. Compound 8g showed excellent h-GR binding and potent antagonistic activity (in vitro). The lead compound 8g exhibited significant oral antidiabetic and antihyperlipidemic effects (in vivo), along with liver selectivity. These preliminary results confirm discovery of potent and liver selective passive GR antagonist for the treatment of T2DM.

Discovery of potent, selective and orally bioavailable triaryl-sulfonamide based PTP1B inhibitors.

A novel series of pTyr mimetics containing triaryl-sulfonamide derivatives (5a-r) are reported as potent and selective PTP1B inhibitors. Some of the test compounds (5o and 5p) showed excellent selectivity towards PTP1B over various PTPs, including TCPTP (in vitro). The lead compound 5o showed potent antidiabetic activity (in vivo), along with improved pharmacokinetic profile. These preliminary results confirm discovery of highly potent and selective PTP1B inhibitors for the treatment of T2DM.

ZYKR1, a novel, potent, and peripherally selective kappa opioid receptor agonist reduces visceral pain and pruritus in animal models.

Opioid receptor agonists are effective analgesic agents. Central activation of the mu and/or kappa opioid receptors (KOR) is associated with CNS side effects, which limits their effectiveness. Recent studies indicated that peripherally restricted, selective KOR agonists were potent analgesics and devoid of CNS-related side effects. To confirm this hypothesis, we designed a novel, potent, and peripherally restricted KOR-selective agonist, ZYKR1. The analgesic efficacy, brain penetration and safety of ZYKR1 were assessed in pre-clinical models. ZYKR1 showed KOR agonistic activity in the cAMP assay, with an EC50 of 0.061 nM and more than 105-fold selectivity over the mu and delta opioid receptors (EC50 > 10 µM). ZYKR1 was not found to bind mu, delta opioid, and NOP receptors in radioligand binding assays. ZYKR1 produced concentration-dependent inhibition of electrically evoked contractions in isolated mouse vas deferens with an IC50 of 1.6 nM ZYKR1 showed peripheral restriction and potent analgesic efficacy in various in-vivo animal models (acetic acid induced visceral pain mouse model, ED50: 0.025 mg/kg, IV; ovariohysterectomy induced postoperative pain rat model, ED50: 0.023 mg/kg, IV; and C48/80 induced pruritus mouse model, ED50: 0.063 mg/kg, IV). In addition, ZYKR1 was devoid of motor coordination, physical dependence, dysphoria, and respiratory depression at 30, 400, 10 and 10-fold of efficacy dose, respectively. In conclusion, ZYKR1 has potent antinociceptive action in visceral pain and pruritus with limited CNS side effects in preclinical models owing to its peripheral restriction.

Modulation of PPAR subtype selectivity. Part 2: Transforming PPARα/γ dual agonist into α selective PPAR agonist through bioisosteric modification.

A novel series of oxime containing benzyl-1,3-dioxane-r-2-carboxylic acid derivatives (6a-k) were designed as selective PPARα agonists, through bioisosteric modification in the lipophilic tail region of PPARα/γ dual agonist. Some of the test compounds (6a, 6b, 6c and 6f) showed high selectivity towards PPARα over PPARγ in vitro. Further, highly potent and selective PPARα agonist 6c exhibited significant antihyperglycemic and antihyperlipidemic activity in vivo, along with its improved pharmacokinetic profile. Favorable in-silico interaction of 6c with PPARα binding pocket correlate its in vitro selectivity profile toward PPARα over PPARγ. Together, these results confirm discovery of novel series of oxime based selective PPARα agonists for the safe and effective treatment of various metabolic disorders.

Effect of structurally constrained oxime-ether linker on PPAR subtype selectivity: Discovery of a novel and potent series of PPAR-pan agonists.

A novel series of thaizole and oxazole containing phenoxy acetic acid derivatives is reported as PPAR-pan agonists. Incorporation of structurally constrained oxime-ether based linker in the chemotype of a potent PPARδ selective agonist GW-501516 was adapted as designing strategy. In vitro, selected test compounds 12a, 12c, 17a and 18a showed PPAR-pan agonists activities and among these four compounds tested, 12a emerged as highly potent and efficacious compound, while 17a exhibited moderate and balanced PPAR-pan agonistic activity. In vivo, selected test compounds 12a and 17a exhibited significant anti-hyperglycemic and anti-hyperlipidemic activities in relevant animal models. These results support our hypothesis that the introduction of structurally constrained oxime-ether linker between lipophilic tail and acidic head plays an important role in modulating subtype selectivity and subsequently led to the discovery of potent PPAR-pan agonists.

Combination of Lorcaserin and GLP-1/glucagon Coagonist Improves Metabolic Dysfunction in Diet Induced-obese Mice.

BACKGROUND: Obesity and diabetes are major metabolic disorders that progress to severe morbidity and mortality. Neuroendocrine mechanisms controlling energy balance indicate that combination therapies are needed to sustain weight loss. Lorcaserin was one of the approved therapies for the treatment of obesity, which is recently withdrawn because a safety clinical trial, shows an increased occurrence of cancer. Coagonist of glucagon-like-peptide-1 (GLP-1) and glucagon receptors is a novel investigational therapy demonstrated to have both anti-obesity and anti-diabetic effect. Here, we investigated the effect of combination of lorcaserin and a GLP-1 and glucagon receptors coagonist in diet-induced obese (DIO) mice model. METHODS: The diet-induced obese C57BL/6J mice were used to assess acute and chronic effect of lorcaserin, coagonist of GLP-1and glucagon receptors and their combination on food intake, body weight, and biochemical parameters. RESULTS: In acute study, combination of lorcaserin and coagonist causes synergistic reductions in food intake and body weight. Repeated treatment of combination of lorcaserin and coagonist showed enhanced body weight loss over time, which is due to reduction in fat mass (subcutaneous, retroperitoneal, mesenteric and epididymal fat pad) compared to individual therapy. Also, suppression of locomotor activity seen with lorcaserin was not evident in combination with coagonist. No additive effect was observed in glucose tolerance (intraperitoneal glucose tolerance test or insulin tolerance test), serum lipids, hepatic lipids, and energy expenditure in combination group. CONCLUSION: These data suggest that combination of lorcaserin and coagonist could be a better combination to induce body weight loss.

ZYBT1, a potent, irreversible Bruton's Tyrosine Kinase (BTK) inhibitor that inhibits the C481S BTK with profound efficacy against arthritis and cancer.

Bruton's tyrosine kinase (BTK) plays a central and pivotal role in controlling the pathways involved in the pathobiology of cancer, rheumatoid arthritis (RA), and other autoimmune disorders. ZYBT1 is a potent, irreversible, specific BTK inhibitor that inhibits the ibrutinib-resistant C481S BTK with nanomolar potency. ZYBT1 is found to be a promising molecule to treat both cancer and RA. In the present report we profiled the molecule for in-vitro, in-vivo activity, and pharmacokinetic properties. ZYBT1 inhibits BTK and C481S BTK with an IC50 of 1 nmol/L and 14 nmol/L, respectively, inhibits the growth of various leukemic cell lines with IC50 of 1 nmol/L to 15 µmol/L, blocks the phosphorylation of BTK and PLCγ2, and inhibits secretion of TNF-α, IL-8 and IL-6. It has favorable pharmacokinetic properties suitable for using as an oral anti-cancer and anti-arthritic drug. In accordance with the in-vitro properties, it demonstrated robust efficacy in murine models of collagen-induced arthritis (CIA) and streptococcal cell wall (SCW) induced arthritis. In both models, ZYBT1 alone could suppress the progression of the diseases. It also reduced the growth of TMD8 xenograft tumor. The results suggested that ZYBT1 has high potential for treating RA, and cancer.

Activation of GLP-1 and Glucagon Receptors Regulates Bile Homeostasis Independent of Thyroid Hormone.

BACKGROUND: Balanced coagonists of glucagon-like peptide-1 (GLP-1) and glucagon receptors are emerging therapies for the treatment of obesity and diabetes. Such coagonists also regulate lipid metabolism, independent of their body weight lowering effects. Many actions of the coagonists are partly mediated by fibroblast growth factor 21 (FGF21) signaling, with the major exception of bile homeostasis. Since thyroid hormone is an important regulator of bile homeostasis, we studied the involvement of thyroid hormone in coagonist-induced changes in lipid and bile metabolism. METHODS: We evaluated the effect of a single dose of coagonist Aib2 C24 chimera2 at 150 to 10000 µg/kg on tetraiodothyronine (T4) and triiodothyronine (T3) in high-fat diet-induced obese (DIO) mice and chow-fed mice. Repeated dose treatment of coagonist (150 µg/kg, subcutaneously) was assessed in four mice models namely, on lipid and bile homeostasis in DIO mice, propylthiouracil (PTU)-treated DIO mice, methimazole (MTM)-treated DIO mice and choline-deficient, L-amino acid-defined, highfat diet (CDAHFD)-induced nonalcoholic steatohepatitis (NASH). RESULTS: Single dose treatment of coagonist did not alter serum T3 and T4 in chow-fed mice and DIO mice. Coagonist treatment improved lipid metabolism and biliary cholesterol excretion. Chronic treatment of GLP-1 and glucagon coagonist did not alter serum T3 in hypothyroid DIO mice and CDAHFDinduced NASH. Coagonist increased serum T4 in DIO mice after 4 and 40 weeks of treatment, though no change in T4 levels was observed in hypothyroid mice or mice with NASH. CONCLUSION: Our data demonstrate that coagonist of GLP-1 and glucagon receptors does not modulate bile homeostasis via thyroid signaling.

Coagonist of glucagon-like peptide-1 and glucagon receptors ameliorates kidney injury in murine models of obesity and diabetes mellitus.

AIM: To investigate the role of glucagon-like peptide-1 (GLP-1)/glucagon receptors coagonist on renal dysfunction associated with diabetes and obesity. METHODS: Chronic high-fat diet fed C57BL/6J mice, streptozotocin-treated high-fat diet fed C57BL/6J mice and diabetic C57BLKS/J db/db mice were used as models of diabetes-induced renal dysfunction. The streptozotocin-treated high-fat diet fed mice and db/db mice were treated with the GLP-1 and glucagon receptors coagonist (Aib2 C24 Chimera2, 150 µg/kg, sc) for twelve weeks, while in chronic high-fat diet fed mice, coagonist (Aib2 C24 Chimera2, 150 µg/kg, sc) treatment was continued for forty weeks. Kidney function, histology, fibrosis, inflammation, and plasma biochemistry were assessed at the end of the treatment. RESULTS: Coagonist treatment decreased body weight, plasma lipids, insulin resistance, creatinine, blood urea nitrogen, urinary albumin excretion rate and renal lipids. In kidney, expression of lipogenic genes (SREBP-1C, FAS, and SCD-1) was decreased, and expression of genes involved in ß-oxidation (CPT-1 and PPAR-α) was increased due to coagonist treatment. In plasma, coagonist treatment increased adiponectin and FGF21 and decreased IL-6 and TNF-α. Coagonist treatment reduced expression of inflammatory (TNF-α, MCP-1, and MMP-9) and pro-fibrotic (TGF-ß, COL1A1, and α-SMA) genes and also improved histological derangement in renal tissue. CONCLUSION: Coagonist of GLP-1 and glucagon receptors alleviated diabetes and obesity-induced renal dysfunction by reducing glucose intolerance, obesity, and hyperlipidemia.

Coagonist of GLP-1 and glucagon decreases liver inflammation and atherosclerosis in dyslipidemic condition.

Dyslipidemia enhances progression of atherosclerosis. Coagonist of GLP-1 and glucagon are under clinical investigation for the treatment of obesity and diabetes. Earlier, we have observed that coagonist reduced circulating and hepatic lipids, independent of its anorexic effects. Here, we investigated the role of coagonist of GLP-1 and glucagon receptors in complications of diet-induced dyslipidemia in hamsters and humanized double transgenic mice. Hamsters fed on high fat high cholesterol diet were treated for 8 weeks with coagonist of GLP-1 and glucagon receptors (75 and 150 µg/kg). Pair-fed control was maintained. Cholesterol fed transgenic mice overexpressing hApoB100 and hCETP with coagonist (300 µg/kg) for 4 weeks. After the completion of treatment, biochemical estimations were done. Coagonist treatment reduced triglycerides in plasma, liver and aorta, plasma cholesterol and hepatic triglyceride secretion rate. Expressions of HMG-CoA reductase and SBREBP-1C were reduced and expressions of LDLR, CYP7A1, ABCA1 and ABCB11 were increased in liver, due to coagonist treatment. Coagonist treatment increased bile flow rate and biliary cholesterol excretion. IL-6 and TNF-α were reduced in plasma and expression of TNF-α, MCP-1, MMP-9 and TIMP-1 decreased in liver. Treatment with coagonist reduced oxidative stress in liver and aorta. Energy expenditure was increased and respiratory quotient was reduced by coagonist treatment. These changes were correlated with reduced hepatic inflammation and lipids in liver and aorta in coagonist treated hamsters. Coagonist treatment also reduced lipids in cholesterol-fed transgenic mice. These changes were independent of glycaemia and anorexia observed after coagonist treatment. Long term treatment with coagonist of GLP-1 and glucagon receptor ameliorated diet-induced dyslipidemia and atherosclerosis by regulating bile homeostasis, liver inflammation and energy expenditure.