Doravirine is non-nucleoside reverse transcriptase inhibitor (NNRTI) currently in phase III clinical trials for the treatment of HIV infection. Herein we describe a robust kilo-scale synthesis for its manufacture. The structure and origin of major impurities were determined and their downstream fate-and-purge studied. This resulted in a redesign of the route to introduce the key nitrile functionality via a copper mediated cyanation which allowed all impurities to be controlled to an acceptable level. The improved synthesis was scaled to prepare similar to 100 kg batches of doravirine to supply all preclinical and clinical studies up to phase III. The synthesis affords high-quality material in a longest linear sequence of six steps and 37% overall yield.

In our efforts to develop CGRP receptor antagonists as backups to MK-3207, 2, we employed a scaffold hopping approach to identify a series of novel oxazolidinone-based compounds. The development of a structurally diverse, potent (20, cAMP + HS IC50 = 0.67 nM), and selective compound (hERG IC50 = 19 μM) with favorable rodent pharmacokinetics (F = 100%, t1/2 = 7 h) is described. Key to this development was identification of a 3-substituted spirotetrahydropyran ring that afforded a substantial gain in potency (10 to 35-fold).

The optimization of a novel series of non-nucleoside reverse transcriptase inhibitors (NNRTI) led to the identification of pyridone 36. In cell cultures, this new NNRTI shows a superior potency profile against a range of wild type and clinically relevant, resistant mutant HIV viruses. The overall favorable preclinical pharmacokinetic profile of 36 led to the prediction of a once daily low dose regimen in human. NNRTI 36, now known as MK-1439, is currently in clinical development for the treatment of HIV infection.

BACKGROUND:Obesity is considered as a systemic chronic low grade inflammation characterized by increased serum pro-inflammatory proteins and accumulation of macrophages within white adipose tissue (WAT) of obese patients. C5L2, a 7-transmembrane receptor, serves a dual function, binding the lipogenic hormone acylation stimulating protein (ASP), and C5a, involved in innate immunity.AIM:We evaluated the impact of C5L2 on macrophage infiltration in WAT of wildtype (Ctl) and C5L2 knock-out (C5L2(-/-)) mice over 6, 12 and 24 weeks on a chow diet and moderate diet-induced obesity (DIO) conditions.RESULTS:In Ctl mice, WAT C5L2 and C5a receptor mRNA increased (up to 10-fold) both over time and with DIO. By contrast, in C5L2(-/-), there was no change in C5aR in WAT. C5L2(-/-) mice displayed higher macrophage content in WAT, varying by time, fat depot and diet, associated with altered systemic and WAT cytokine patterns compared to Ctl mice. However, in all cases, the M1 (pro-) vs M2 (anti-inflammatory) macrophage proportion was unchanged but C5L2(-/-) adipose tissue secretome appeared to be more chemoattractant. Moreover, C5L2(-/-) mice have increased food intake, increased WAT, and altered WAT lipid gene expression, which is reflected systemically. Furthermore, C5L2(-/-) mice have altered glucose/insulin metabolism, adiponectin and insulin signalling gene expression in WAT, which could contribute to development of insulin resistance.CONCLUSION:Disruption of C5L2 increases macrophage presence in WAT, contributing to obesity-associated pathologies, and further supports a dual role of complement in WAT. Understanding this effect of the complement system pathway could contribute to targeting treatment of obesity and its comorbidities.

AIM:To develop a 3T3-L1 pre-adipocyte model for evaluating the secretion of acylation stimulating protein (ASP) and an ELISA assay for measuring ASP production and investigate the effects and related potential mechanisms of metabolic drugs on the secretion of ASP, and on the complement C3, triglyceride (TG) mass, nonesterified fatty acids (NEFA) release and fatty acid (FA) uptake into adipocytes.METHODS:After differentiated, 3T3-L1 pre-adipocytes were treated with chylomicrons, metformin, rosiglitazone, rimonabant for 48 h. ASP and C3 were measured using a sandwich ELISA. NEFA levels were measured using enzymatic colorimetric kits. FA uptake was measured in a bottom-reading fluorescent microplate reader. TG mass and protein levels were determined using enzymatic colorimetric assay and the Bradford assay, respectively.RESULTS:Chylomicrons increased ASP production (up to 411%±133%, P<0.05). Rosiglitazone and rimonabant decreased ASP production (-53% to -85%, P<0.05), associated with a decrease in the precursor protein C3 (-37% to -65%, P<0.01). By contrast, metformin also decreased ASP (-54% to -100%, P<0.05), but with no change in precursor protein C3. In addition, metformin decreased TG mass (maximum -60%, P<0.05) and real-time FA uptake (maximum -75%, P<0.05).CONCLUSION:3T3-L1 pre-adipocyte model is successfully developed, which can be used for evaluating the effects of metabolic drugs on the secretion of ASP. ELISA assay for measuring ASP production is also developed.

Process research and development of a synthetic route towards a novel renin inhibitor (1) is described. The highly convergent synthetic route provided 1 in 15% yield on multikilogram scale with a longest linear sequence of 11 steps. The use of catalytic hydrogenation features prominently in our design. The proper choice of N-methylpyridone surrogate was also important, and we describe a method for the easy conversion of 2-methoxypyridines to N-methylpyridones using cheap and readily available reagents.

Background: The complement system is well known for its role in innate immunity via the classical, the alternative and the lectin pathways, although recent investigations suggest expanding roles in adipose tissue. Properdin stabilizes C3 convertase following alternative complement activation. Properdin is also present in adipose tissue, localized to adipocyte membranes.Aim: We evaluated the potential role of properdin in energy metabolism using properdin deficient (PKO) mice and cell based assays.Results: PKO mice have a diet-dependent increase in weight gain compared to wild-type (WT) littermates on a high fat diet (P < 0.05), directly related to 51% increase in relative fat mass (PKO: 35.8 +/- 2.2% body fat vs. WT: 23.6 +/- 2.2%, P < 0.01). PKO mice have decreased energy expenditure (P < 0.01), and altered postprandial lipid clearance (P < 0.01). However glucose metabolism was unchanged after a glucose tolerance test vs. WT mice. In murine 3T3-L1 adipocytes, addition of properdin had no effect on C3 or ASP production but almost completely inhibited the insulin-mediated stimulation of fatty acid uptake and incorporation into TG. Properdin had no effect on basal or insulin-stimulated glucose transport in either 3T3-L1 adipocytes or L6 rat skeletal muscle cells.Conclusion: Thus properdin may be added to the growing list of complement proteins (C3, adipsin, factor 8, ASP (C3adesArg), factor H, C1q and C3aR) which influence lipid metabolism, energy storage and insulin resistance, and further support the hypothesis of a dual role of complement in adipose tissue. (C) 2012 Elsevier Ltd. All rights reserved.

Background Complement 3 (C3), a key component of the innate immune system, is involved in early inflammatory responses. Acylation stimulating protein (ASP; aka C3adesArg), a C3 cleavage product, is produced in adipose tissue and stimulates lipid storage. We hypothesized that, depending on the diet, chronic ASP administration in C3−/− mice would affect lipid metabolism and insulin sensitivity via an adaptive adipose tissue inflammatory response. Methodology/Principal Findings C3−/− mice on normal low fat diet (ND) or high fat diet (HFD) were chronically administered recombinant ASP (rASP) for 25 days via an osmotic mini-pump. While there was no effect on food intake, there was a decrease in activity, with a relative increase in adipose tissue weight on ND, and a shift in adipocyte size distribution. While rASP administration to C3−/− mice on a ND increased insulin sensitivity, on a HFD, rASP administration had the opposite effect. Specifically, rASP administration in C3−/− HFD mice resulted in decreased gene expression of IRS1, GLUT4, SREBF1 and NFκB in muscle, and decreased C5L2 but increased JNK, CD36, CD11c, CCR2 and NFκB gene expression in adipose tissue as well as increased secretion of proinflammatory cytokines (Rantes, KC, MCP-1, IL-6 and G-CSF). In adipose tissue, although IRS1 and GLUT4 mRNA were unchanged, insulin response was reduced. Conclusion The effects of chronic rASP administration are tissue and diet specific, rASP administration enhances the HFD induced inflammatory response leading to an insulin-resistant state. These results suggest that, in humans, the increased plasma ASP associated with obesity and cardiovascular disease could be an additional factor directly contributing to development of metabolic syndrome, insulin resistance and diabetes.

In this paper, we report the development of different synthetic routes to MK-7246 (1) designed by the Process Chemistry group. The syntheses were initially designed as an enabling tool for Medicinal Chemistry colleagues in order to rapidly explore structure-activity relationships (SAR) and to procure the first milligrams of diverse target molecules for in vitro evaluation. The initial aziridine opening/cyclodehydration strategy was also directly amenable to the first GMP deliveries of MK-7246 (1), streamlining the transition from milligram to kilogram-scale production needed to support early preclinical and clinical evaluation of this compound. Subsequently a more scalable and cost-effective manufacturing route to MK-7246 (1) was engineered. Highlights of the manufacturing route include an Ir-catalyzed intramolecular N-H insertion of sulfoxonium ylide 41 and conversion of ketone 32 to amine 31 in a single step with excellent enantioselectivity through a transaminase process. Reactions such as these illustrate the enabling impact and efficiency gains that innovative developments in chemo- and biocatalysis can have on the synthesis of pharmaceutically relevant target molecules.

Obesity is characterized by chronic low-grade inflammation with increased adipose tissue pro-inflammatory cytokine production. Acylation stimulating protein (ASP) stimulates triglyceride synthesis and glucose transport via its receptor C5L2. Circulating ASP is increased in obesity, insulin resistance and metabolic syndrome. The present study examines the effects of normal (50 nM), high physiological (200 nM) and pathological (600 nM) levels of ASP on inflammatory changes in 3T3-L1 adipocytes and J774 macrophages and the underlying mechanisms involved. Treatment with ASP for 24h increased monocyte chemoattractant protein-1 (MCP1, 800%, P<0.001) and keratinocyte-derived chemokine (KC, >150%, P<0.01) secretion in adipocytes in a dose-dependent manner, with no effect on IL-6 or adiponectin. In macrophages, ASP had no effect on these cytokines. C5a, a ligand for C5L2 and C5aR receptors, differed from ASP. Macrophage-adipocyte coculture increased MCP-1 and adiponectin secretion, and ASP further enhanced secretion (P<0.001 and P<0.05, respectively) at doses of 50 nM and 200 nM. ASP increased Ser(468) and Ser(536) phosphorylation of p65 NFκB in a time- and concentration-dependent manner (P<0.05) as well as phosphorylation of Akt Ser(473) (p=0.02). ASP and insulin stimulations of Ser(536) p65 NFκB phosphorylation were comparable (both p<0.05) but not additive. Both inhibition of PI3kinase (with wortmannin) and NFκB (with BAY11-7085) prevented ASP stimulation of MCP-1 and KC secretion in adipocytes. These findings suggest that ASP, especially at high physiologic doses, may stimulate specific inflammatory cytokines in adipocytes through PI3kinase- and NFκB-dependant pathways, thus further promoting macrophage infiltration and local inflammation in obese adipose tissue.

Obesity is associated with inflammation characterized by increased infiltration of macrophages into adipose tissue. C5aR-like receptor 2 (C5L2) has been identified as a receptor for acylation-stimulating protein (ASP) and the inflammatory factor C5a, which also binds C5aR. The present study examines the effects of ligands ASP and C5a on interactions between the receptors C5L2 and C5aR in 3T3-L1 adipocytes and J774 macrophages. BRET experiments indicate that C5L2 and C5aR form homo- and heterodimers in transfected HEK 293 cells, which were stable in the presence of ligand. Cell surface receptor levels of C5L2 and C5aR increased during 3T3-L1 adipocyte differentiation; both receptors are also highly expressed in J774 macrophages. Using confocal microscopy to evaluate endogenous receptors in adipocytes following stimulation with ASP or C5a, C5L2 is internalized with increasing perinuclear colocalization with C5aR. There is little C5a-dependent colocalization in macrophages. While adipocyte-conditioned medium (ACM) increased C5L2-C5aR colocalization in macrophages, this was blocked by C5a. ASP stimulation increased Akt (Ser(473)) phosphorylation in both cell types; C5a induced slight Akt phosphorylation in adipocytes with less effect in macrophages. ASP, but not C5a, increased fatty acid uptake/esterification in adipocytes. C5L2-C5aR homodimerization versus heterodimerization may thus contribute to differential responses obtained following ASP vs C5a stimulation of adipocytes and macrophages, providing new insights into the complex interaction between these two cell types within adipose tissue. Studying the mechanisms involved in the differential responses of C5L2-C5aR activation based on cell type will further our understanding of inflammatory processes in obesity.

MK-6096 is an orexin receptor antagonist in clinical trials for the treatment of insomnia. Herein we describe its first kilogram-scale synthesis. Chirality on the a-methylpiperidine core was introduced in a biocatalytic transamination using a three-enzyme system with excellent enantioselectivity (>99% ee). Low diastereoselectivity of the lactam reduction was overcome by development of a camphor sulfonic acid salt formation and dr upgrade. A chemoselective O-alkylation with 5-fluoro-2-hydroxypyridine was optimized and developed. Overall, 1.2 kg of MK-6069 was prepared in nine steps and 13% overall yield.

Process research and development of a synthetic route towards a novel 5-lipoxygenase inhibitor is described. The synthetic route provided 1 in 27% yield in nine steps (seven steps in the longest linear sequence) and was performed on kilogram scale. The synthesis began with the preparation of the coumarin core via an efficient von Pechmann condensation. The triazole fragment was obtained via a regioselective copper-catalyzed [3 + 2] cycloaddition between a chiral alkyne and the coumarin azide.

MK-8141 inhibits renin, an aspartyl protease that is targeted for the regulation of high blood pressure and heart failure. The synthesis of MK-8141 features a diastereoselective Dieckmann cyclization of the diester C to give the 3,9-diazabicyclo-[3.3.1]nonenes D and E (1:8.5).

A convergent approach towards nonsymmetrical 2,5-diester pyrroles is described. The building blocks can be easily assembled in less than four steps allowing for facile construction of diversity. The synthesis uses a rhodium-catalyzed NH insertion, followed by a one-pot deprotection-condensation to yield the desired pyrroles.