Design, synthesis and biological evaluation of indane derived GPR40 agoPAMs.

GPR40 (FFAR1 or FFA1) is a G protein-coupled receptor, primarily expressed in pancreatic islet ß-cells and intestinal enteroendocrine cells. When activated by fatty acids, GPR40 elicits increased insulin secretion from islet ß-cells only in the presence of elevated glucose levels. Towards this end, studies were undertaken towards discovering a novel GPR40 Agonist whose mode of action is via Positive Allosteric Modulation of the GPR40 receptor (AgoPAM). Efforts were made to identify a suitable GPR40 AgoPAM tool molecule to investigate mechanism of action and de-risk liver toxicity of GPR40 AgoPAMs due to reactive acyl-glucuronide (AG) metabolites.

Discovery and development of benzo-[1,2,4]-triazolo-[1,4]-oxazepine GPR142 agonists for the treatment of diabetes.

A novel series of benzo-[1,2,4]-triazolo-[1,4]-oxazepine GPR142 agonists are described. The series was designed to address the suboptimal PK (pharmacokinetic) and off-target profile of a class of N-aryl-benzo-[1,4]-oxazepine-4-carboxamides, represented by 1, that were identified from a high-throughput screen of the Merck compound collection for GPR142 agonists. This work led to the discovery of 3-phenoxy-benzo-[1,2,4]-triazolo-[1,4]-oxazepine 47, a potent GPR142 agonist with an off-target and PK profile suitable for in vivo studies. This compound and a related analogue 40 were shown to be active in mouse oral glucose tolerance tests (OGTTs). Furthermore, a GPR142 knock-out mouse OGTT study with compound 40 provides evidence that its glucose-lowering effect is mediated by GPR142.

SAR exploration at the C-3 position of tetrahydro-ß-carboline sstr3 antagonists.

MK-4256, a tetrahydro-ß-carboline sstr3 antagonist, was discontinued due to a cardiovascular (CV) adverse effect observed in dogs. Additional investigations revealed that the CV liability (QTc prolongation) was caused by the hERG off-target activity of MK-4256 and was not due to sstr3 antagonism. In this Letter, we describe our extensive SAR effort at the C3 position of the tetrahydro-ß-carboline structure. This effort resulted in identification of 5-fluoro-pyridin-2-yl as the optimal substituent on the imidazole ring to balance sstr3 activity and the hERG off-target liability.

Discovery of substituted (4-phenyl-1H-imidazol-2-yl)methanamine as potent somatostatin receptor 3 agonists.

We report SAR studies on a novel non-peptidic somatostatin receptor 3 (SSTR3) agonist lead series derived from (4-phenyl-1H-imidazol-2-yl)methanamine. This effort led to the discovery of a highly potent low molecular weight SSTR3 agonist 5c (EC50=5.2 nM, MW=359). The results from molecular overlays of 5c onto the L-129 structure indicate good alignment, and two main differences of the proposed overlays of the antagonist MK-4256 onto the conformation of 5c lead to inversion of antagonism to agonism.

Rapid Evolution of a Fragment-like Molecule to Pan-Metallo-Beta-Lactamase Inhibitors: Initial Leads toward Clinical Candidates.

With the emergence and rapid spreading of NDM-1 and existence of clinically relevant VIM-1 and IMP-1, discovery of pan inhibitors targeting metallo-beta-lactamases (MBLs) became critical in our battle against bacterial infection. Concurrent with our fragment and high-throughput screenings, we performed a knowledge-based search of known metallo-beta-lactamase inhibitors (MBLIs) to identify starting points for early engagement of medicinal chemistry. A class of compounds exemplified by 11, discovered earlier as B. fragilis metallo-beta-lactamase inhibitors, was selected for in silico virtual screening. From these efforts, compound 12 was identified with activity against NDM-1 only. Initial exploration on metal binding design followed by structure-guided optimization led to the discovery of a series of compounds represented by 23 with a pan MBL inhibition profile. In in vivo studies, compound 23 in combination with imipenem (IPM) robustly lowered the bacterial burden in a murine infection model and became the lead for the invention of MBLI clinical candidates.

Synthesis and cannabinoid-1 receptor binding affinity of conformationally constrained analogs of taranabant.

The design, synthesis, and binding activity of ring constrained analogs of the acyclic cannabinoid-1 receptor (CB1R) inverse agonist taranabant 1 are described. The initial inspiration for these taranabant derivatives was its conformation 1a, determined by (1)H NMR, X-ray, and molecular modeling. The constrained analogs were all much less potent than their acyclic parent structure. The results obtained are discussed in the context of a predicted binding of 1 to a homology model of CB1R.

Synthesis and evaluation of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-aminopropanamide as human cannabinoid-1 receptor (CB1R) inverse agonists.

Obesity is a chronic medical condition that is affecting large population throughout the world. CB1 as a target for treatment of obesity has been under intensive studies. Taranabant was discovered and then developed by Merck as the 1st generation CB1R inverse agonist. Reported here is part of our effort on the 2nd generation of CB1R inverse agonist from the acyclic amide scaffold. We replaced the oxygen linker in taranabant with nitrogen and prepared a series of amino heterocyclic analogs through a divergent synthesis. Although in general, the amine linker gave reduced binding affinity, potent and selective CB1R inverse agonist was identified from the amino heterocycle series. Molecular modeling was applied to study the binding of the amino heterocycle series at CB1 binding site. The in vitro metabolism of representative members was studied and only trace glucuronidation was found. Thus, it suggests that the right hand side of the molecule may not be the appropriate site for glucuronidation.

Conformational analysis and receptor docking of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (taranabant, MK-0364), a novel, acyclic cannabinoid-1 receptor inverse agonist.

X-ray crystallographic, NMR spectroscopic, and computational studies of taranabant afforded similar low-energy conformers with a significant degree of rigidity along the C11-N13-C14-C16-C17 backbone but with more flexibility around bonds C8-C11 and C8-O7. Mutagenesis and docking studies suggested that taranabant and rimonabant shared the same general binding area of CB1R but with significant differences in detailed interactions. Similar to rimonabant, taranabant interacted with a cluster of aromatic residues (F(3.36)200, W(5.43)279, W(6.48)356, and Y(5.39)275) through the two phenyl rings and with F(2.57)170 and L(7.42)387 through the CF 3-Pyr ring. The notable distinction between taranabant and rimonabant was that taranabant was hydrogen-bonded with S(7.39)383 but not with K(3.28)192, while rimonabant was hydrogen-bonded with K(3.28)192 but not with S(7.39)383. The strong hydrogen bonding between the amide NH of taranabant and hydroxyl of S(7.39)383 was key to the superior affinity of taranabant to CB1R.

Characterization of a novel and selective cannabinoid CB1 receptor inverse agonist, Imidazole 24b, in rodents.

We document in vitro and in vivo effects of a novel, selective cannabinoid CB(1) receptor inverse agonist, Imidazole 24b (5-(4-chlorophenyl)-N-cyclohexyl-4-(2,4-dichlorophenyl)-1-methyl-imidazole-2-carboxamide). The in vitro binding affinity of Imidazole 24b for recombinant human and rat CB(1) receptor is 4 and 10 nM, respectively. Imidazole 24b binds to human cannabinoid CB(2) receptor with an affinity of 297 nM; in vitro, it is a receptor inverse agonist at both cannabinoid CB(1) and CB(2) receptors as it causes a further increase of forskolin-induced cAMP increase. Oral administration of Imidazole 24b blocked CP-55940-induced hypothermia, demonstrating cannabinoid CB(1) receptor antagonist efficacy in vivo. Using ex vivo autoradiography, Imidazole 24b resulted in dose-dependent increases in brain cannabinoid CB(1) receptor occupancy (RO) at 2h post-dosing in rats, indicating that approximately 50% receptor occupancy is sufficient for attenuation of receptor agonist-induced hypothermia. Imidazole 24b administered to C57Bl/6 mice and to dietary-induced obese (DIO) Sprague-Dawley rats attenuated overnight food intake with a minimal effective dose of 10 mg/kg, p.o. Administration had no effect in cannabinoid CB(1) receptor-deficient mice. DIO rats were dosed orally with vehicle, Imidazole 24b (1, 3 or 10 mg/kg), or dexfenfluramine (3 mg/kg) for 2 weeks. At 3 mg/kg, Imidazole 24b reduced cumulative food intake, leading to a non-significant decrease in weight gain. Imidazole 24b at 10 mg/kg and dexfenfluramine treatment inhibited food intake and attenuated weight gain. These findings suggest that selective cannabinoid CB(1) receptor inverse agonists such as Imidazole 24b have potential for the treatment of obesity.

Constraining the amide bond in N-sulfonylated dipeptide VLA-4 antagonists.

The integrin VLA-4 is implicated in several inflammatory disease states. In search of non-peptidic antagonists of VLA-4, rotational constraints were imposed on the amide bond of prototypical N-sulfonylated dipeptide VLA-4 antagonists. By judicious structural modification of the side chains, trisubstituted imidazoles with moderate binding potencies were obtained, for example, 19, VLA-4 IC(50)=237 nM.

Discovery of N-{(1S,2S)-2-(3-cyanophenyl)- 3-[4-(2-[18F]fluoroethoxy)phenyl]-1-methylpropyl}- 2-methyl-2-[(5-methylpyridin-2-yl)oxy]propanamide, a cannabinoid-1 receptor positron emission tomography tracer suitable for clinical use.

The discovery of a structurally distinct cannabinoid-1 receptor (CB1R) positron emission tomography tracer is described. Starting from an acyclic amide CB1R inverse agonist (1) as the lead compound, an efficient route to introduce 18F to the molecule was developed. Further optimization focused on reducing the lipophilicity and increasing the CB1R affinity. These efforts led to the identification of [18F]-16 that exhibited good brain uptake and an excellent signal-to-noise ratio in rhesus monkeys.

GPR40 partial agonist MK-2305 lower fasting glucose in the Goto Kakizaki rat via suppression of endogenous glucose production.

GPR40 (FFA1) is a fatty acid receptor whose activation results in potent glucose lowering and insulinotropic effects in vivo. Several reports illustrate that GPR40 agonists exert glucose lowering in diabetic humans. To assess the mechanisms by which GPR40 partial agonists improve glucose homeostasis, we evaluated the effects of MK-2305, a potent and selective partial GPR40 agonist, in diabetic Goto Kakizaki rats. MK-2305 decreased fasting glucose after acute and chronic treatment. MK-2305-mediated changes in glucose were coupled with increases in plasma insulin during hyperglycemia and glucose challenges but not during fasting, when glucose was normalized. To determine the mechanism(s) mediating these changes in glucose metabolism, we measured the absolute contribution of precursors to glucose production in the presence or absence of MK-2305. MK-2305 treatment resulted in decreased endogenous glucose production (EGP) driven primarily through changes in gluconeogenesis from substrates entering at the TCA cycle. The decrease in EGP was not likely due to a direct effect on the liver, as isolated perfused liver studies showed no effect of MK-2305 ex vivo and GPR40 is not expressed in the liver. Taken together, our results suggest MK-2305 treatment increases glucose stimulated insulin secretion (GSIS), resulting in changes to hepatic substrate handling that improve glucose homeostasis in the diabetic state. Importantly, these data extend our understanding of the underlying mechanisms by which GPR40 partial agonists reduce hyperglycemia.

Design and Synthesis of Novel, Selective GPR40 AgoPAMs.

GPR40 is a G-protein-coupled receptor expressed primarily in pancreatic islets and intestinal L-cells that has been a target of significant recent therapeutic interest for type II diabetes. Activation of GPR40 by partial agonists elicits insulin secretion only in the presence of elevated blood glucose levels, minimizing the risk of hypoglycemia. GPR40 agoPAMs have shown superior efficacy to partial agonists as assessed in a glucose tolerability test (GTT). Herein, we report the discovery and optimization of a series of potent, selective GPR40 agoPAMs. Compound 24 demonstrated sustained glucose lowering in a chronic study of Goto Kakizaki rats, showing no signs of tachyphylaxis for this mechanism.

Discovery of N-[(1S,2S)-3-(4-Chlorophenyl)-2- (3-cyanophenyl)-1-methylpropyl]-2-methyl-2- {[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), a novel, acyclic cannabinoid-1 receptor inverse agonist for the treatment of obesity.

The discovery of novel acyclic amide cannabinoid-1 receptor inverse agonists is described. They are potent, selective, orally bioavailable, and active in rodent models of food intake and body weight reduction. A major focus of the optimization process was to increase in vivo efficacy and to reduce the potential for formation of reactive metabolites. These efforts led to the identification of compound 48 for development as a clinical candidate for the treatment of obesity.

Discovery and Optimization of a Novel Triazole Series of GPR142 Agonists for the Treatment of Type 2 Diabetes.

GPR142 has been identified as a potential glucose-stimulated insulin secretion (GSIS) target for the treatment of type 2 diabetes mellitus (T2DM). A class of triazole GPR142 agonists was discovered through a high throughput screen. The lead compound 4 suffered from poor metabolic stability and poor solubility. Lead optimization strategies to improve potency, efficacy, metabolic stability, and solubility are described. This optimization led to compound 20e, which showed significant reduction of glucose excursion in wild-type but not in GPR142 deficient mice in an oral glucose tolerance test (oGTT) study. These studies provide strong evidence that reduction of glucose excursion through treatment with 20e is GPR142-mediated, and GPR142 agonists could be used as a potential treatment for type 2 diabetes.

Discovery of MK-1421, a Potent, Selective sstr3 Antagonist, as a Development Candidate for Type 2 Diabetes.

The imidazolyl-tetrahydro-ß-carboline class of sstr3 antagonists have demonstrated efficacy in a murine model of glucose excursion and may have potential as a treatment for type 2 diabetes. The first candidate in this class caused unacceptable QTc interval prolongation in oral, telemetrized cardiovascular (CV) dogs. Herein, we describe our efforts to identify an acceptable candidate without CV effects. These efforts resulted in the identification of (1R,3R)-3-(4-(5-fluoropyridin-2-yl)-1H-imidazol-2-yl)-1-(1-ethyl-pyrazol-4-yl)-1-(3-methyl-1,3,4-oxadiazol-3H-2-one-5-yl)-2,3,4,9-tetrahydro-1H-ß-carboline (17e, MK-1421).

The discovery and potential of N-sulfonylated dipeptide VLA-4 antagonists.

Through a directed screening of a combinatorial library containing carboxylic acids, N-sulfonylated dipeptides were identified as leads in the Merck Research Laboratories VLA-4 antagonist program. Further optimization quickly identified subnanomolar compounds with varying degrees of specificity over the related integrin alpha4beta7. Various metabolic liabilities were identified and addressed. However, the pharmacokinetic properties of nearly all compounds in this class were unacceptable. Other leads were identified with apparent good oral bioavailability, but these were generally associated with very high plasma protein binding and a loss of potency. The mechanism of high plasma clearance was identified in the rat as the organic acid transporter, mrp-2. Compounds were identified that were not substrates of mrp-2, but they still suffered from poor oral bioavailability. Finally, a shift in strategy to identifying VLA-4 antagonists that would be suitable as candidates for inhalation therapy resulted in the preparation of compounds with exception tight binding properties. These compounds were superior to BIO-1211 in the ovalbumin-sensitized mouse model of eosinophil trafficking to the lung. One particular compound had an exceptionally long off-rate with a KD < or = 2 pM. The evolution of the structure activity relationships in our laboratories and strategies for improving potencies and pharmacokinetic profiles are the subject of this review.

Addressing the metabolic activation potential of new leads in drug discovery: a case study using ion trap mass spectrometry and tritium labeling techniques.

Metabolic activation of drug candidates to electrophilic reactive metabolites that can covalently modify cellular macromolecules may result in acute and/or idiosyncratic immune system-mediated toxicities in humans. This presents a significant potential liability for the future development of these compounds as safe therapeutic agents. We present here an example of an approach where sites of metabolic activation within a new drug candidate series were rapidly identified using online liquid chromatography/multi-stage mass spectrometry on an ion trap mass spectrometer. This was accomplished by trapping the reactive intermediates formed upon incubation of compounds with rat and human liver microsomes as their corresponding glutathione conjugates and mass spectral characterization of these thiol adducts. Based on the structures of the GSH adducts identified, potential sites and mechanisms of bioactivation within the chemical structure were proposed. These metabolism studies were interfaced with iterative structural modifications of the chemical series in order to block these bioactivation sites within the molecule. This strategy led to a significant reduction in the propensity of the compounds to undergo metabolic activation as evidenced by reductions in the irreversible binding of radioactivity to liver microsomal material upon incubation of tritium-labeled compounds with this in vitro system. With the efficiency and throughput achievable with such an approach, it appears feasible to identify and address the metabolic activation potential of new drug leads during routine metabolite identification studies in an early drug discovery setting.

Diamine Derivatives as Novel Small-Molecule, Potent, and Subtype-Selective Somatostatin SST3 Receptor Agonists.

A novel class of small-molecule, highly potent, and subtype-selective somatostatin SST3 agonists was discovered through modification of a SST3 antagonist. As an example, (1R,2S)-9 demonstrated not only potent in vitro SST3 agonist activity but also in vivo SST3 agonist activity in a mouse oral glucose tolerance test (OGTT). These agonists may be useful reagents for studying the physiological roles of the SST3 receptor and may potentially be useful as therapeutic agents.

Investigation of Cardiovascular Effects of Tetrahydro-ß-carboline sstr3 antagonists.

Antagonism of somatostatin subtype receptor 3 (sstr3) has emerged as a potential treatment of Type 2 diabetes. Unfortunately, the development of our first preclinical candidate, MK-4256, was discontinued due to a dose-dependent QTc (QT interval corrected for heart rate) prolongation observed in a conscious cardiovascular (CV) dog model. As the fate of the entire program rested on resolving this issue, it was imperative to determine whether the observed QTc prolongation was associated with hERG channel (the protein encoded by the human Ether-à-go-go-Related Gene) binding or was mechanism-based as a result of antagonizing sstr3. We investigated a structural series containing carboxylic acids to reduce the putative hERG off-target activity. A key tool compound, 3A, was identified from this SAR effort. As a potent sstr3 antagonist, 3A was shown to reduce glucose excursion in a mouse oGTT assay. Consistent with its minimal hERG activity from in vitro assays, 3A elicited little to no effect in an anesthetized, vagus-intact CV dog model at high plasma drug levels. These results afforded the critical conclusion that sstr3 antagonism is not responsible for the QTc effects and therefore cleared a path for the program to progress.