Design and synthesis of novel proline based factor XIa selective inhibitors as leads for potential new anticoagulants.

A series of 4, 4-disubstituted proline analogs were designed, synthesized, and tested for selective inhibition of blood coagulation factor XIa in search of new non-vitamin K antagonists based oral anticoagulants for potential prevention and treatment of thrombotic diseases. Starting from a potent thrombin (FIIa) inhibitor chemotype with FIIa IC50 = 1 nM and FXIa IC50 = 160 nM, medicinal chemistry iterations guided by molecular modeling and structure-based drug design led to steady improvement of FXIa potency while dialing down thrombin activity and improving selectivity. Through this exercise, a thousand-fold enhancement of selectivity over thrombin was achieved with some analogs carrying factor XIa inhibition potencies in the 10 nM range. In this communication, we discuss the design principles and structure activity relationship (SAR) of these novel FXIa selective inhibitors.

Discovery of hydroxy pyrimidine Factor IXa inhibitors.

The synthesis and structure activity relationship development of a pyrimidine series of heterocyclic Factor IXa inhibitors is described. Increased selectivity over Factor Xa inhibition was achieved through SAR expansion of the P1 element. Select compounds were evaluated in vivo to assess their plasma levels in rat.

Discovery of the oxazabicyclo[3.3.1]nonane derivatives as potent and orally active GPR119 agonists.

The design and synthesis of two conformationally restricted oxazabicyclo octane derivatives as GRP119 agonists is described. Derivatives of scaffold C, with syn configuration, have the best overall profiles with respect to solubility and in vivo efficacy. Compound 25a was found to have extremely potent agonistic activity and was orally active in lowering blood glucose levels in a mouse oral glucose tolerance test at a dose of 0.1 mg/kg.

Discovery of MK-1462: GLP-1 and Glucagon Receptor Dual Agonist for the Treatment of Obesity and Diabetes.

Peptide-based analogues of the gut-derived incretin hormone, glucagon-like peptide 1 (GLP1), stimulate insulin secretion in a glucose-dependent manner. Currently marketed GLP1 receptor (GLP1R) agonists are safe and effective in the management of Type 2 diabetes but often offer only modest weight loss. This has prompted the search for safe and effective alternatives to enhance the weight loss component of these treatments. We have demonstrated that concomitant activation GLP1R and the glucagon receptor (GCGR) can improve glucose metabolism and provide superior weight loss when compared to selective GLP1R agonism in preclinical species. This paper will highlight chemistry structure-activity relationship optimization and summarize in vivo efficacy studies toward the discovery of a once daily balanced dual agonist 12 (MK-1462), which was advanced into clinical trials.

Discovery of a nortropanol derivative as a potent and orally active GPR119 agonist for type 2 diabetes.

The lead optimization studies of a series of GPR119 agonists incorporating a nortropanol scaffold are described. Extensive structure-activity relationship (SAR) studies of the lead compound 20f led to the identification of compound 36j as a potent, single digit nanomolar GPR119 agonist with high agonist activity. Compound 36j was orally active in lowering blood glucose levels in a mouse oral glucose tolerance test and increased plasma insulin levels in a rat hyperglycemic model. It showed good to excellent pharmacokinetic properties in rats and monkeys and no untoward activities in counter-screen assays. Compound 36j demonstrated an attractive in vitro and in vivo profile for further development.

Discovery of novel chemotypes to a G-protein-coupled receptor through ligand-steered homology modeling and structure-based virtual screening.

Melanin-concentrating hormone receptor 1 (MCH-R1) is a G-protein-coupled receptor (GPCR) and a target for the development of therapeutics for obesity. The structure-based development of MCH-R1 and other GPCR antagonists is hampered by the lack of an available experimentally determined atomic structure. A ligand-steered homology modeling approach has been developed (where information about existing ligands is used explicitly to shape and optimize the binding site) followed by docking-based virtual screening. Top scoring compounds identified virtually were tested experimentally in an MCH-R1 competitive binding assay, and six novel chemotypes as low micromolar affinity antagonist "hits" were identified. This success rate is more than a 10-fold improvement over random high-throughput screening, which supports our ligand-steered method. Clearly, the ligand-steered homology modeling method reduces the uncertainty of structure modeling for difficult targets like GPCRs.

Polypharmacy through Phage Display: Selection of Glucagon and GLP-1 Receptor Co-agonists from a Phage-Displayed Peptide Library.

A promising emerging area for the treatment of obesity and diabetes is combinatorial hormone therapy, where single-molecule peptides are rationally designed to integrate the complementary actions of multiple endogenous metabolically-related hormones. We describe here a proof-of-concept study on developing unimolecular polypharmacy agents through the use of selection methods based on phage-displayed peptide libraries (PDL). Co-agonists of the glucagon (GCG) and GLP-1 receptors were identified from a PDL sequentially selected on GCGR- and GLP1R-overexpressing cells. After two or three rounds of selection, 7.5% of randomly picked clones were GLP1R/GCGR co-agonists, and a further 1.53% were agonists of a single receptor. The phages were sequenced and 35 corresponding peptides were synthesized. 18 peptides were potent co-agonists, 8 of whom showed EC50 ≤ 30 pM on each receptor, comparable to the best rationally designed co-agonists reported in the literature. Based on literature examples, two sequences were engineered to stabilize against dipeptidyl peptidase IV cleavage and prolong the in vivo half-life: the engineered peptides were comparably potent to the parent peptides on both receptors, highlighting the potential use of phage-derived peptides as therapeutic agents. The strategy described here appears of general value for the discovery of optimized polypharmacology paradigms across several metabolically-related hormones.

Discovery of MK-8282 as a Potent G-Protein-Coupled Receptor 119 Agonist for the Treatment of Type 2 Diabetes.

The ever-growing prevalence of type 2 diabetes in the world has necessitated an urgent need for multiple orally effective agents that can regulate glucose homeostasis with a concurrent reduction in body weight. G-Protein coupled receptor 119 (GPR119) is a GPCR target at which agonists have demonstrated glucose-dependent insulin secretion and shows beneficial effects on glycemic control. Herein, we describe our efforts leading to the identification of a potent, oral GPR-119 agonist, MK-8282, which shows improved glucose tolerance in multiple animal models and has excellent off-target profile. The key design elements in the compounds involved a combination of a fluoro-pyrimidine and a conformationally constrained bridged piperidine to impart good potency and efficacy.

The melanin-concentrating hormone receptor 1, a novel target of autoantibody responses in vitiligo.

Vitiligo is a common depigmenting disorder resulting from the loss of melanocytes in the skin. The pathogenesis of the disease remains obscure, although autoimmune mechanisms are thought to be involved. Indeed, autoantibodies and autoreactive T lymphocytes that target melanocytes have been reported in some vitiligo patients. The objective of this study was to identify pigment cell antigens that are recognized by autoantibodies in vitiligo. Using IgG from vitiligo patients to screen a melanocyte cDNA phage-display library, we identified the melanin-concentrating hormone receptor 1 (MCHR1) as a novel autoantigen related to this disorder. Immunoreactivity against the receptor was demonstrated in vitiligo patient sera by using radiobinding assays. Among sera from healthy controls and from patients with autoimmune disease, none exhibited immunoreactivity to MCHR1, indicating a high disease specificity for Ab's against the receptor. Inhibition of MCH binding to its receptor by IgG from vitiligo patients was also shown.

Discovery of orally efficacious melanin-concentrating hormone receptor-1 antagonists as antiobesity agents. Synthesis, SAR, and biological evaluation of bicyclo[3.1.0]hexyl ureas.

Melanin-concentrating hormone (MCH) is a cyclic, nonadecapeptide expressed in the CNS of all vertebrates that regulates feeding behavior and energy homeostasis via interaction with the central melanocortin system. Regulation of this interaction results in modulation of food intake and body weight gain, demonstrating significant therapeutic potential for the treatment of obesity. The MCH-1 receptor (MCH-R1) has been identified as a key target in MCH regulation, as small molecule antagonists of MCH-R1 have demonstrated activity in vivo. Herein, we document our research in a bicyclo[3.1.0]hexyl urea series with particular emphasis on structure-activity relationships and optimization of receptor occupancy, measured both in vitro and via an ex vivo binding assay following an oral dosing regimen. Several compounds have been tested in vivo and exhibit oral efficacy in relevant acute rodent feeding models. In particular, 24u has proven efficacious in chronic rodent models of obesity, showing a statistically significant reduction in food intake and body weight over a 28 day study.

Effects of a selective melanin-concentrating hormone 1 receptor antagonist on food intake and energy homeostasis in diet-induced obese mice.

Melanin concentrating hormone (MCH) is a cyclic neuropeptide expressed in the lateral hypothalamus that plays an important role in energy homeostasis. To investigate the pharmacological consequences of inhibiting MCH signaling in murine obesity models, we examined the effect of acute and chronic administration of a selective MCH1 receptor antagonist (SCH-A) in diet-induced obese (DIO) and Lep(ob/ob) mice. Oral administration of SCH-A for 5 consecutive days (30 mg/kg q.d.) produced hypophagia, a loss of body weight and adiposity, and decreased plasma leptin levels in DIO mice, and hypophagia and reduced weight gain in Lep(ob/ob) mice. Chronic administration of SCH-A to DIO mice decreased food intake, body weight and adiposity, and plasma leptin and free fatty acids. These effects were accompanied by increases in several hypothalamic neuropeptides. Acute administration of SCH-A (30 mg/kg) prevented the decrease in energy expenditure associated with food restriction. These results indicate that MCH1 receptor antagonists may be effective in the treatment of obesity.

Discovery of bicycloalkyl urea melanin concentrating hormone receptor antagonists: orally efficacious antiobesity therapeutics.

Melanin concentrating hormone (MCH) is involved in regulation of food intake and energy homeostasis. Antagonists of the MCH receptor are expected to affect food intake and weight gain, making MCH-R1 an attractive target for obesity treatment. Herein, we report the discovery of a novel, orally active series of MCH-R1 antagonists that exhibit in vivo efficacy in rodent obesity models.

Biaryl ureas as potent and orally efficacious melanin concentrating hormone receptor 1 antagonists for the treatment of obesity.

Herein, we report a small molecule MCH-R1 antagonist which demonstrates oral efficacy in chronic rodent models. Substituted phenyl biaryl urea derivatives were synthesized and evaluated as MCH-R1 antagonists for the treatment of obesity. The structure-activity relationship studies in this series resulted in identification of urea 1 as a potent and selective MCH-R1 antagonist. Compound 1 exhibited oral efficacy in chronic (28 d) rodent models at 3-30 mpk showing significant reduction in food intake and weight gain relative to controls.

In vitro pharmacological characterization of vorapaxar, a novel platelet thrombin receptor antagonist.

Vorapaxar is a novel protease-activated receptor-1 (PAR1) antagonist recently approved for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. The present study provides a comprehensive in vitro pharmacological characterization of vorapaxar interaction with the PAR1 receptor on human platelets. Similar studies were performed with a metabolite of vorapaxar (M20). Vorapaxar and M20 were competitive PAR1 antagonists that demonstrated concentration-dependent, saturable, specific, and slowly reversible binding to the receptor present on intact human platelets. The affinities of vorapaxar and M20 for the PAR1 receptor were in the low nanomolar range, as determined by saturation-, kinetic- and competitive binding studies. The calculated Kd and Ki values for vorapaxar increased in the presence of plasma, indicating a decrease in the free fraction available for binding to the PAR1 receptor on human platelets. Vorapaxar was also evaluated in functional assays using thrombin or a PAR1 agonist peptide (SFLLRN). Vorapaxar and M20 completely blocked thrombin-stimulated PAR1/ß-arrestin association in recombinant cells and abolished thrombin-stimulated calcium influx in washed human platelets and vascular smooth muscle cells. Moreover, vorapaxar and M20 inhibited PAR1 agonist peptide-mediated platelet aggregation in human platelet rich plasma with a steep concentration response relationship. Vorapaxar exhibited high selectivity for inhibition of PAR1 over other platelet GPCRs. In conclusion, vorapaxar is a potent PAR1 antagonist exhibiting saturable, reversible, selective binding with slow off-rate kinetics and effectively inhibits thrombin's PAR1-mediated actions on human platelets.

Improved Stability of Proline-Derived Direct Thrombin Inhibitors through Hydroxyl to Heterocycle Replacement.

Modification of the previously disclosed (S)-N-(2-(aminomethyl)-5-chlorobenzyl)-1-((R)-2-hydroxy-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamide 2 by optimization of the P3 group afforded novel, low molecular weight thrombin inhibitors. Heterocycle replacement of the hydroxyl functional group helped maintain thrombin in vitro potency while improving the chemical stability and pharmacokinetic profile. These modifications led to the identification of compound 10, which showed excellent selectivity over related serine proteases as well as in vivo efficacy in the rat arteriovenous shunt. Compound 10 exhibited significantly improved chemical stability and pharmacokinetic properties over 2 and may be utilized as a structurally differentiated preclinical tool comparator to dabigatran etexilate (Pro-1) to interrogate the on- and off-target effects of oral direct thrombin inhibitors.

Cloning and characterization of rhesus monkey MCH-R1 and MCH-R2.

Rhesus monkey MCH-R1 and MCH-R2 receptors were cloned. Amino acid homology is 98.8% between monkey and human MCH-R1, while monkey and human MCH-R2 are 98% homologous. Binding and intracellular signaling characteristics of the monkey receptors were compared with the human homologues. The results demonstrate that MCH binds to the monkey MCH-R1 receptor with a K(d) of 6.5 nM and monkey MCH-R2 with a K(d) of 2.2 nM similar to K(d) values for human MCH-R1 and MCH-R2. Additionally, monkey MCH-R1 couples through G(i)/G(o) and G(q)-type G proteins similar to human MCH-R1 whereas monkey and human MCH-R2 utilize the G(q) signaling pathway.

Activation of extracellular signal regulated protein kinase by neuropeptide Y and pancreatic polypeptide in CHO cells expressing the NPY Y(1), Y(2), Y(4) and Y(5) receptor subtypes.

Neuropeptide Y (NPY), 36-amino acid amidated peptide expressed in central and peripheral neurons, regulates a variety of physiological activities, including food intake, energy expenditure, vasoconstriction, anxiolysis, nociception and ethanol consumption. NPY binds to a family of G-protein coupled receptors whose activation results in inhibition of adenylyl cyclase activity. To more fully characterize the signal transduction pathways utilized by the NPY receptor subtypes, the pathways leading to phosphorylation of the extracellular signal regulated protein kinases 1 and 2 (ERK) have been compared in CHO cells expressing each of the four cloned human NPY receptor subtypes, Y(1), Y(2), Y(4) and Y(5). NPY Y(1), Y(2), Y(4) and Y(5) receptor-mediated ERK phosphorylation was blocked by pertussis toxin (PTX) exposure, indicating that all four receptors are coupled to inhibitory G(i/o) proteins. Exposure to the protein kinase C (PKC) inhibitor GF109203X diminished Y(1), Y(2) and Y(4) receptor-mediated ERK phosphorylation but completely blocked Y(5) receptor-mediated ERK phosphorylation. Additionally, Y(5) receptor-mediated ERK phosphorylation was inhibited by the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin to a greater extent than was Y(1)-mediated ERK phosphorylation. These results demonstrate that in CHO cells, the Y(5) receptor and the Y(1), Y(2) and Y(4) receptors utilize different pathways to activate ERK.

Inhibition of Factor XI activity as a promising antithrombotic strategy.

Prevention and treatment of thromboembolic disorders with minimal bleeding risk remains a significant unmet medical need. Studies in Factor XI (FXI)-deficient humans and experimental animal models suggest that targeting FXI in humans provides antithrombotic benefits with reduced bleeding liability compared with current standard of care. In this review, we describe an exciting era in the discovery and development of antithrombotic agents as multiple therapeutic modalities for FXI(a) inhibition progress through preclinical and clinical development.

In vivo responsiveness to ezetimibe correlates with niemann-pick C1 like-1 (NPC1L1) binding affinity: Comparison of multiple species NPC1L1 orthologs.

Ezetimibe is the first in class 2-azetidinone that decreases plasma cholesterol by blocking intestinal cholesterol absorption. Ezetimibe effectively reduces plasma cholesterol in several species including human, monkey, dog, hamster, rat, and mouse, but the potency ranges widely. One potential factor responsible for this variation in responsiveness is diversity in ezetimibe metabolism. After oral administration, ezetimibe is glucuronidated. Both ezetimibe and the glucuronide lower plasma cholesterol; however, the glucuronide exhibits greater potency. Recent identification of Niemann-Pick C1 Like-1 (NPC1L1) as the molecular target of ezetimibe enables direct binding studies to be performed. Here, we report the cloning of NPC1L1 derived from multiple species and assess amino acid sequence homology among human, monkey, dog, hamster, rat, and mouse. The rank order of affinity of glucuronidated ezetimibe for NPC1L1 in each species correlates with the rank order of in vivo activity with monkey > dog > hamster and rat >> mouse. Ezetimibe analogs that bind to NPC1L1 exhibit in vivo cholesterol-lowering activity, whereas compounds that do not bind NPC1L1 are inactive. Specific structural components of ezetimibe are identified as critical for binding to NPC1L1. The results demonstrate that small variations in ezetimibe structure or in NPC1L1 amino acid sequence can profoundly influence ezetimibe/NPC1L1 interaction and consequently in vivo activity. The results demonstrate that the ability of compounds to bind to NPC1L1 is the major determinant of in vivo responsiveness.

SAR study of bicyclo[4.1.0]heptanes as melanin-concentrating hormone receptor R1 antagonists: taming hERG.

To improve the ex vivo potency of MCH inhibitor 1a and to address its hERG liability, a structure-activity study was carried out, focusing on three regions of the lead structure. Introduction of new side chains with basic nitrogen improved in vitro and ex vivo bindings. Many potent compounds with K(i)<10nM were discovered (compounds 6a-j) and several compounds (14-17) had excellent ex vivo binding at 6h and 24h. Attenuating the basicity of nitrogen on the side chain, and in particular, introduction of a polar group such as aminomethyl on the distal phenyl ring significantly lowered the hERG activity. Further replacement of the distal phenyl group with heteroaryl groups in the cyclohexene series provided compounds such as 28l with excellent ex vivo activity with much reduced hERG liability.