Discovery of a GPR40 Superagonist: The Impact of Aryl Propionic Acid α-Fluorination.

GPR40 is a G-protein-coupled receptor which mediates fatty acid-induced glucose-stimulated insulin secretion from pancreatic beta cells and incretion release from enteroendocrine cells of the small intestine. GPR40 full agonists exhibit superior glucose lowering compared to partial agonists in preclinical species due to increased insulin and GLP-1 secretion, with the added benefit of promoting weight loss. In our search for potent GPR40 full agonists, we discovered a superagonist which displayed excellent in vitro potency and superior efficacy in the Gαs-mediated signaling pathway. Most synthetic GPR40 agonists have a carboxylic acid headgroup, which may cause idiosyncratic toxicities, including drug-induced-liver-injury (DILI). With a methyl group and a fluorine atom substituted at the α-C of the carboxylic acid group, 19 is not only highly efficacious in lowering glucose and body weight in rodent models but also has a low DILI risk due to its stable acylglucuronide metabolite.

GPR40-Mediated Gα12 Activation by Allosteric Full Agonists Highly Efficacious at Potentiating Glucose-Stimulated Insulin Secretion in Human Islets.

GPR40 is a clinically validated molecular target for the treatment of diabetes. Many GPR40 agonists have been identified to date, with the partial agonist fasiglifam (TAK-875) reaching phase III clinical trials before its development was terminated due to off-target liver toxicity. Since then, attention has shifted toward the development of full agonists that exhibit superior efficacy in preclinical models. Full agonists bind to a distinct binding site, suggesting conformational plasticity and a potential for biased agonism. Indeed, it has been suggested that alternative pharmacology may be required for meaningful efficacy. In this study, we described the discovery and characterization of Compound A, a newly identified GPR40 allosteric full agonist highly efficacious in human islets at potentiating glucose-stimulated insulin secretion. We compared Compound A-induced GPR40 activity to that induced by both fasiglifam and AM-1638, another allosteric full agonist previously reported to be highly efficacious in preclinical models, at a panel of G proteins. Compound A was a full agonist at both the Gαq and Gαi2 pathways, and in contrast to fasiglifam Compound A also induced Gα12 coupling. Compound A and AM-1638 displayed similar activity at all pathways tested. The Gα12/Gα13-mediated signaling pathway has been linked to protein kinase D activation as well as actin remodeling, well known to contribute to the release of insulin vesicles. Our data suggest that the pharmacology of GPR40 is complex and that Gα12/Gα13-mediated signaling, which may contribute to GPR40 agonists therapeutic efficacy, is a specific property of GPR40 allosteric full agonists.

Discovery of a novel potent GPR40 full agonist.

Compound 12 is a GPR40 agonist that realizes the full magnitude of efficacy possible via GPR40 receptor agonism. In vitro and in vivo studies demonstrated superior glucose lowering by 12 compared to fasiglifam (TAK-875), in a glucose dependent manner. The enhanced efficacy observed with the full agonist 12 was associated with both direct and indirect stimulation of insulin secretion.

Fasiglifam (TAK-875): Mechanistic Investigation and Retrospective Identification of Hazards for Drug Induced Liver Injury.

TAK-875, a GPR40 agonist, was withdrawn from Phase III clinical trials due to drug-induced liver injury (DILI). Mechanistic studies were conducted to identify potential DILI hazards (covalent binding burden (CVB), hepatic transporter inhibition, mitochondrial toxicity, and liver toxicity in rats) associated with TAK-875. Treatment of hepatocytes with radiolabeled TAK-875 resulted in a CVB of 2.0 mg/day, which is above the threshold of 1 mg/day considered to be a risk for DILI. Covalent binding to hepatocytes was due to formation of a reactive acyl glucuronide (AG) and, possibly, an acyl-CoA thioester intermediate. Formation of TAK-875AG in hepatocytes and/or in vivo was in the order of non-rodents > human (in vitro only) > rat. These data suggest that non-rodents, and presumably humans, form TAK-875AG more efficiently than rats, and that AG-mediated toxicities in rats may only occur at high doses. TAK-875 (1000 mg/kg/day) formed significant amounts of AG metabolite (≤32.7 µM) in rat liver that was associated with increases in ALT (×4), bilirubin (×9), and bile acids (×3.4), and microscopic findings of hepatocellular hypertrophy and single cell necrosis. TAK-875 and TAK-875AG had similar potencies (within 3-fold) for human multi-drug resistant associated protein 2/4 (MRP2/4) and bile salt export pump, but TAK-875AG was exceptionally potent against MRP3 (0.21 µM). Inhibition of MRPs may contribute to liver accumulation of TAK-875AG. TAK-875 also inhibited mitochondrial respiration in HepG2 cells, and mitochondrial Complex 1 and 2 activities in isolated rat mitochondria. In summary, formation of TAK-875AG, and possibly TAK-875CoA in hepatocytes, coupled with inhibition of hepatic transporters and mitochondrial respiration may be key contributors to TAK-875-mediated DILI.

Discovery of novel benzo[b]thiophene tetrazoles as non-carboxylate GPR40 agonists.

GPR40 partial agonism is a promising new mechanism for the treatment of type 2 diabetes mellitus with clinical proof of concept. Most of the GPR40 agonists in the literature have a carboxylic acid functional group, which may pose a risk for idiosyncratic drug toxicity. A novel series of GPR40 agonists containing a tetrazole as a carboxylic acid bioisostere was identified. This series of compounds features a benzo[b]thiophene as the center ring, which is prone to oxidation during phase 1 metabolism. Following SAR optimization targeting GPR40 agonist activity and intrinsic clearance in microsomes (human and rat), potent and metabolically stable compounds were selected for in vivo evaluation. The compounds are efficacious at lowering blood glucose in a SD rat oGTT model.

Indazole-based ligands for estrogen-related receptor α as potential anti-diabetic agents.

Estrogen-related receptor α (ERRα) is an orphan nuclear receptor that has been functionally implicated in the regulation of energy homeostasis. Herein is described the development of indazole-based N-alkylthiazolidenediones, which function in biochemical assays as selective inverse agonists against this receptor. Series optimization provided several potent analogues that inhibited the recruitment of a co-activator peptide fragment in vitro (IC50s < 50 nM) and reduced fasted circulating insulin and triglyceride levels in a sub-chronic pre-diabetic rat model when administered orally (10 mg/kg). A multi-parametric optimization strategy led to the identification of 50 as an advanced lead, which was more extensively evaluated in additional diabetic models. Chronic oral administration of 50 in two murine models of obesity and insulin resistance improved glucose control and reduced circulating triglycerides with efficacies similar to that of rosiglitazone. Importantly, these effects were attained without the concomitant weight gain that is typically observed with the latter agent. Thus, these studies provide additional support for the development of such molecules for the potential treatment of metabolic diseases.

Discovery of novel potent imidazo[1,2-b]pyridazine PDE10a inhibitors.

Design and optimization of a novel series of imidazo[1,2-b]pyridazine PDE10a inhibitors are described. Compound 31 displays excellent pharmacokinetic properties and was also evaluated as an insulin secretagogue in vitro and in vivo.

Benzo[d]imidazole Transient Receptor Potential Vanilloid 1 Antagonists for the Treatment of Pain: Discovery of trans-2-(2-{2-[2-(4-Trifluoromethyl-phenyl)-vinyl]-1H-benzimidazol-5-yl}-phenyl)-propan-2-ol (Mavatrep).

Reported herein is the design, synthesis, and pharmacologic characterization of a class of TRPV1 antagonists constructed on a benzo[d]imidazole platform that evolved from a biaryl amide lead. This design composes three sections: a 2-substituted 5-phenyl headgroup attached to the benzo[d]imidazole platform, which is tethered at the two position to a phenyl tail group. Optimization of this design led to the identification of 4 (mavatrep), comprising a trifluoromethyl-phenyl-vinyl tail. In a TRPV1 functional assay, using cells expressing recombinant human TRPV1 channels, 4 antagonized capsaicin-induced Ca(2+) influx, with an IC50 value of 4.6 nM. In the complete Freund's adjuvant- and carrageenan-induced thermal hypersensitivity models, 4 exhibited full efficacy, with ED80 values of 7.8 and 0.5 mg/kg, respectively, corresponding to plasma levels of 270.8 and 9.2 ng/mL, respectively. On the basis of its superior pharmacologic and safety profile, 4 (mavatrep) was selected for clinical development for the treatment of pain.

Transient receptor potential melastatin 8 channel inhibition potentiates the hypothermic response to transient receptor potential vanilloid 1 activation in the conscious mouse.

OBJECTIVES: Mild decrease in core temperature (therapeutic hypothermia) provides lasting neuroprotection following cardiac arrest or cerebral ischemia. However, current methods for producing therapeutic hypothermia trigger a cold-defense response that must be countered by sedatives, muscle paralytics, and mechanical ventilation. We aimed to determine methods for producing hypothermia in the conscious mouse by targeting two transient receptor potential channels involved in thermoregulation, two transient receptor potential (TRP) channels involved in thermoregulation, TRP vanilloid 1 (TRPV1) and TRP melastatin 8 (TRPM8). DESIGN: Controlled prospective animal study. SETTING: Research laboratory at academic medical center. SUBJECTS: Conscious unrestrained young and aged male mice. INTERVENTIONS: Mice were treated with the TRPV1 agonist dihydrocapsaicin, a TRPM8 inhibitor ("compound 5"), or their combination and the effects on core temperature (Tcore) were measured by implanted thermocouples and wireless transponders. MEASUREMENTS AND MAIN RESULTS: TRPV1 agonist dihydrocapsaicin produced a dose-dependent (2-4 mg/kg s.c.) drop in Tcore. A loading dose followed by continuous infusion of dihydrocapsaicin produced a rapid and prolonged (> 6 hr) drop of Tcore within the therapeutic range (32-34°C). The hypothermic effect of dihydrocapsaicin was augmented in aged mice and was not desensitized with repeated administration. TRPM8 inhibitor "compound 5" (20 mg/kg s.c.) augmented the drop in core temperature during cold exposure (8°C). When "compound 5" (30 mg/kg) was combined with dihydrocapsaicin (1.25-2.5 mg/kg), the drop in Tcore was amplified and prolonged. CONCLUSIONS: Activating warm receptors (TRPV1) produced rapid and lasting hypothermia in young and old mice. Furthermore, hypothermia induced by TRPV1 agonists was potentiated and prolonged by simultaneous inhibition of TRPM8.

Enhancement of kinase selectivity in a potent class of arylamide FMS inhibitors.

Structure-activity relationship (SAR) studies on a highly potent series of arylamide FMS inhibitors were carried out with the aim of improving FMS kinase selectivity, particularly over KIT. Potent compound 17r (FMS IC50 0.7 nM, FMS cell IC50 6.1 nM) was discovered that had good PK properties and a greater than fivefold improvement in selectivity for FMS over KIT kinase in a cellular assay relative to the previously reported clinical candidate 4. This improved selectivity was manifested in vivo by no observed decrease in circulating reticulocytes, a measure of bone safety, at the highest studied dose. Compound 17r was highly active in a mouse pharmacodynamic model and demonstrated disease-modifying effects in a dose-dependent manner in a strep cell wall-induced arthritis model of rheumatoid arthritis in rats.

Shivering and tachycardic responses to external cooling in mice are substantially suppressed by TRPV1 activation but not by TRPM8 inhibition.

Mild decrease of core temperature (32-34°C), also known as therapeutic hypothermia, is a highly effective strategy of neuroprotection from ischemia and holds significant promise in the treatment of stroke. However, induction of hypothermia in conscious stroke patients is complicated by cold-defensive responses, such as shivering and tachycardia. Although multiple thermoregulatory responses may be altered by modulators of thermosensitive ion channels, TRPM8 (transient receptor potential melastatin 8) and TRPV1 (TRP vanilloid 1), it is unknown whether these agents affect cold-induced shivering and tachycardia. The current study aimed to determine the effects of TRPM8 inhibition and TRPV1 activation on the shivering and tachycardic responses to external cooling. Conscious mice were treated with TRPM8 inhibitor compound 5 or TRPV1 agonist dihydrocapsaicin (DHC) and exposed to cooling at 10°C. Shivering was measured by electromyography using implanted electrodes in back muscles, tachycardic response by electrocardiography, and core temperature by wireless transmitters in the abdominal cavity. The role of TRPM8 was further determined using TRPM8 KO mice. TRPM8 ablation had no effect on total electromyographic muscle activity (vehicle: 24.0 ± 1.8; compound 5: 23.8 ± 2.0; TRPM8 KO: 19.7 ± 1.9 V·s/min), tachycardia (ΔHR = 124 ± 31; 121 ± 13; 121 ± 31 beats/min) and drop in core temperature (-3.6 ± 0.1; -3.4 ± 0.4; -3.6 ± 0.5°C) during cold exposure. TRPV1 activation substantially suppressed muscle activity (vehicle: 25.6 ± 3.0 vs. DHC: 5.1 ± 2.0 V·s/min), tachycardia (ΔHR = 204 ± 25 vs. 3 ± 35 beats/min) and produced a profound drop in core temperature (-2.2 ± 0.6 vs. -8.9 ± 0.6°C). In conclusion, external cooling-induced shivering and tachycardia are suppressed by TRPV1 activation, but not by TRPM8 inhibition. This suggests that TRPV1 agonists may be combined with external physical cooling to achieve more rapid and effective hypothermia.

Discovery of vinylcycloalkyl-substituted benzimidazole TRPM8 antagonists effective in the treatment of cold allodynia.

Thermosensitive transient receptor potential melastatin 8 (TRPM8) antagonists are considered to be potential therapeutic agents for the treatment of cold hypersensitivity. The discovery of a new class of TRPM8 antagonists that shows in vivo efficacy in the rat chronic constriction injury (CCI)-induced model of neuropathic pain is described.

Optimization of a potent class of arylamide colony-stimulating factor-1 receptor inhibitors leading to anti-inflammatory clinical candidate 4-cyano-N-[2-(1-cyclohexen-1-yl)-4-[1-[(dimethylamino)acetyl]-4-piperidinyl]phenyl]-1H-imidazole-2-carboxamide (JNJ-28312141).

A class of potent inhibitors of colony-stimulating factor-1 receptor (CSF-1R or FMS), as exemplified by 8 and 21, was optimized to improve pharmacokinetic and pharmacodynamic properties and potential toxicological liabilities. Early stage absorption, distribution, metabolism, and excretion assays were employed to ensure the incorporation of druglike properties resulting in the selection of several compounds with good activity in a pharmacodynamic screening assay in mice. Further investigation, utilizing the type II collagen-induced arthritis model in mice, culminated in the selection of anti-inflammatory development candidate JNJ-28312141 (23, FMS IC(50) = 0.69 nM, cell assay IC(50) = 2.6 nM). Compound 23 also demonstrated efficacy in rat adjuvant and streptococcal cell wall-induced models of arthritis and has entered phase I clinical trials.

Identification of diaryl ether-based ligands for estrogen-related receptor α as potential antidiabetic agents.

Estrogen-related receptor α (ERRα) is an orphan nuclear receptor that has been functionally implicated in the regulation of energy homeostasis. Herein is described the development of diaryl ether based thiazolidenediones, which function as selective ligands against this receptor. Series optimization provided several potent analogues that inhibit the recruitment of a coactivator peptide fragment in in vitro biochemical assays (IC(50) < 150 nM) and cellular two-hybrid reporter assays against the ligand binding domain (IC(50) = 1-5 µM). A cocrystal structure of the ligand-binding domain of ERRα with lead compound 29 revealed the presence of a covalent interaction between the protein and ligand, which has been shown to be reversible. In diet-induced murine models of obesity and in an overt diabetic rat model, oral administration of 29 normalized insulin and circulating triglyceride levels, improved insulin sensitivity, and was body weight neutral. This provides the first demonstration of functional activities of an ERRα ligand in metabolic animal models.

Design and optimization of benzimidazole-containing transient receptor potential melastatin 8 (TRPM8) antagonists.

Transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel that is thermoresponsive to cool to cold temperatures (8-28 °C) and also may be activated by chemical agonists such as menthol and icilin. Antagonism of TRPM8 activation is currently under investigation for the treatment of painful conditions related to cold, such as cold allodynia and cold hyperalgesia. The design, synthesis, and optimization of a class of selective TRPM8 antagonists based on a benzimidazole scaffold is described, leading to the identification of compounds that exhibited potent antagonism of TRPM8 in cell-based functional assays for human, rat, and canine TRPM8 channels. Numerous compounds in the series demonstrated excellent in vivo activity in the TRPM8-selective "wet-dog shakes" (WDS) pharmacodynamic model and in the rat chronic constriction injury (CCI)-induced model of neuropathic pain. Taken together, the present results suggest that the in vivo antagonism of TRPM8 constitutes a viable new strategy for treating a variety of disorders associated with cold hypersensitivity, including certain types of neuropathic pain.

Developments in factor Xa inhibitors for the treatment of thromboembolic disorders.

Thromboembolic diseases are the leading causes of morbidity and mortality in the developed world. Anticoagulants provide effective treatment for venous or arterial thromboembolism. Two coagulation factors, factor Xa (fXa) and thrombin, are the primary targets under active investigation for anticoagulant therapy. fXa, in contrast to the multifunctional roles of thrombin in the coagulation cascade, converts prothrombin to thrombin collectively at the junction of the intrinsic and extrinsic pathway of coagulation. The effectiveness of fXa inhibitors as antithrombotic agents and their potentially reduced bleeding risks may offer superior therapeutic profiles with respect to thrombin inhibitors. After decades of research, many fXa inhibitors are now in the advanced stages of clinical trials. Unlike most reviews, which only provide incremental updates, this review provides an overview of fXa and the medicinal chemistry of its inhibitors. Overviews on coagulation models, antithrombotic therapy, and fXa will be provided, followed by the evolution of the medicinal chemistry of fXa inhibitors over the past few decades.

Reducing ion channel activity in a series of 4-heterocyclic arylamide FMS inhibitors.

During efforts to improve the bioavailability of FMS kinase inhibitors 1 and 2, a series of saturated and aromatic 4-heterocycles of reduced basicity were prepared and evaluated in an attempt to also improve the cardiovascular safety profile over lead arylamide 1, which possessed ion channel activity. The resultant compounds retained excellent potency and exhibited diminished ion channel activity.

Discovery and clinical evaluation of 1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-[2,2-difluoro-2-phenylethylamino]pyrazinone (RWJ-671818), a thrombin inhibitor with an oxyguanidine P1 motif.

We have identified RWJ-671818 (8) as a novel, low molecular weight, orally active inhibitor of human alpha-thrombin (K(i) = 1.3 nM) that is potentially useful for the acute and chronic treatment of venous and arterial thrombosis. In a rat deep venous thrombosis model used to assess antithrombotic efficacy, oral administration of 8 at 30 and 50 mg/kg reduced thrombus weight by 87 and 94%, respectively. In an anesthetized rat antithrombotic model, where electrical stimulation of the carotid artery created a thrombus, 8 prolonged occlusion time 2- and 3-fold at 0.1 and 1.0 mg/kg, i.v., respectively, and more than doubled activated clotting time and activated partial thromboplastin time at the higher dose. This compound had excellent oral bioavailability of 100% in dogs with an estimated half-life of approximately 3 h. On the basis of its noteworthy preclinical data, 8 was advanced into human clinical trials and successfully progressed through phase 1 studies.