Activated factor XI (FXIa) inhibitors are promising novel anticoagulants with low bleeding risk compared with current anticoagulants. The discovery of potent FXIa inhibitors with good oral bioavailability has been challenging. Herein, we describe our discovery effort, utilizing nonclassical interactions to improve potency, cellular permeability, and oral bioavailability by enhancing the binding while reducing polar atoms. Beginning with literature-inspired pyridine N-oxide-based FXIa inhibitor 1, the imidazole linker was first replaced with a pyrazole moiety to establish a polar C-H···water hydrogen-bonding interaction. Then, structure-based drug design was employed to modify lead molecule 2d in the P1' and P2' regions, with substituents interacting with key residues through various nonclassical interactions. As a result, a potent FXIa inhibitor 3f (Ki = 0.17 nM) was discovered. This compound demonstrated oral bioavailability in preclinical species (rat 36.4%, dog 80.5%, and monkey 43.0%) and displayed a dose-dependent antithrombotic effect in a rabbit arteriovenous shunt model of thrombosis.

G-protein coupled receptor kinase 2 (GRK2), which is upregulated in the failing heart, appears to play a critical role in heart failure (HF) progression in part because enhanced GRK2 activity promotes dysfunction of β-adrenergic signaling and myocyte death. An orally bioavailable GRK2 inhibitor could offer unique therapeutic outcomes that cannot be attained by current heart failure treatments that directly target GPCRs or angiotensin-converting enzyme. Herein, we describe the discovery of a potent, selective, and orally bioavailable GRK2 inhibitor, 8h, through high-throughput screening, hit-to-lead optimization, structure-based design, molecular modelling, synthesis, and biological evaluation. In the cellular target engagement assays, 8h enhances isoproterenol-mediated cyclic adenosine 3′,5′-monophosphate (cAMP) production in HEK293 cells overexpressing GRK2. Compound 8h was further evaluated in a human stem cell-derived cardiomyocyte (HSC-CM) contractility assay and potentiated isoproterenol-induced beating rate in HSC-CMs.

(2S,3R,4R,5S,6R)-2-Aryl-5,5-difluoro-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diols and (2S,3R,4R,5S,6R)-2-aryl-5-fluoro-5-methyl-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diols were discovered as dual inhibitors of sodium glucose co-transporter proteins (e.g. SGLT1 and SGLT2) through rational drug design, efficient synthesis, and in vitro and in vivo evaluation. Compound 6g demonstrated potent dual inhibitory activities (IC50 = 96 nM for SGLT1 and IC50 = 1.3 nM for SGLT2). It showed robust inhibition of blood glucose excursion in an oral glucose tolerance test (OGTT) in Sprague Dawley (SD) rats when dosed at both 1 mg/kg and 10 mg/kg orally. It also demonstrated postprandial glucose control in db/db mice when dosed orally at 10 mg/kg.

A novel series of indazole/indole derivatives were discovered as glucagon receptor (GCGR) antagonists through scaffold hopping based on two literature leads: MK-0893 and LY-2409021. Further structure-activity relationship (SAR) exploration and optimization led to the discovery of multiple potent GCGR antagonists with excellent pharmacokinetic properties in mice and rats, including low systemic clearance, long elimination half-life, and good oral bioavailability. These potent GCGR antagonists could be used for potential treatment of type II diabetes.

Type 2 diabetes mellitus (T2DM) is characterized by chronically elevated plasma glucose levels. The inhibition of glucagon-induced hepatic glucose output via antagonism of the glucagon receptor (GCGR) using a small-molecule antagonist is a promising mechanism for improving glycemic control in the diabetic state. The present work discloses the discovery of indazole-based beta-alanine derivatives as potent GCGR antagonists through an efficient enantioselective synthesis and structure-activity relationship (SAR) exploration and optimization. Compounds within this class exhibited excellent pharmacokinetic properties in multiple preclinical species. In an acute dog glucagon challenge test, compound 13K significantly inhibited glucagon-mediated blood glucose increase when dosed orally at 10 mg/kg.

A new series of (2S, 3R, 4R, 5S, 6R)-5-fluoro-6-(hydroxymethyl)-2-aryltetrahydro-2H-pyran-3,4-diols as dual inhibitors of sodium glucose co-transporter proteins (SGLTs) were disclosed. Two methods were developed to efficiently synthesize C-5-fluoro-lactones 3 and 4, which are key intermediates to the C-5-fluoro-hexose based C-aryl glucosides. Compound 2b demonstrated potent hSGLT1 and hSGLT2 inhibition (IC50 = 43 nM for SGLT1 and IC50 = 9 nM for SGLT2). It showed robust inhibition of blood glucose excursion in oral glucose tolerance test (OGTT) in Sprague Dawley (SD) rats and exerted pronounced antihyperglycemic effects in db/db mice and high-fat diet-fed ZDF rats when dosed orally at 10 mg/kg.

In the small intestine glucose is transported apically across the enterocyte by sodium/glucose cotransporter 1 (SGLT1; slc5a1) and its inhibition lowers the post‐prandial blood glucose excursion; however, questions remain about the effect of altered luminal glucose in the intestine. Using 13 C6‐glucose LC/MS/MS detection we first quantified the inhibition of intestinal glucose uptake in SGLT1 −/− and −/+ mice in vivo, and pharmacologically by cmpd B (IC50 <20 nM for rSGLT1 & rSGLT2) in isolated rat jejunum mounted in Ussing chambers. After Cmpd B was dosed chronically, morphometric intestinal changes were assessed in Zucker Diabetic Fatty (ZDF) rats and cecal/fecal bacterial 16s RNA analysis performed in mice. A high fat diet In C57BL/6mice had variable effects on sglt1 mRNA expression (increased, decreased or not changed) in 3 separate experiments. Thus, there is no consistent impact of diet on SGLT1 mRNA expression. Jejunum mucosal SGLT1 mRNA expression was confirmed absent in −/− mice whereas +/− mice had ~ half the level of expression. After an oral 13 C6‐glucose tolerance test (oGTT) the AUC was similar in WT and SGLT1 +/− mice, and reduced to 25% of AUC in −/− mice (−/−3,851±384, P<0.05 by ANOVA compared to −/+ 18,682±3,277 and WT 14,674±1,616; n=7–8/group). Apically administered 13 C6‐glucose to rat isolated muscle‐stripped jejunum in the presence of cmpd B (1 μM) decreased the amount detected basolaterally (14.2 ± 1.8μM, n=16; versus 8.7 ± 1.3μM, n=12 after 90 mins). In ZDF rats treated daily with 0.3 or 1.0 mg/kg for up to 3 weeks, decreased HbA 1c , and total GLP‐1 during an oGTT increased after the higher dose. Analysis of GI tissue showed no difference in jejunum mucosal SGLT1 or GLUT2 mRNA relative expression, and no gross mucosal adaption (villus height, villus width or mucosal weight). In C57BL/6mice, high sucrose diet or cmpd B in mice fed normal chow (1.0 mg/kg p.o. daily for 5 days) alone did not change cecal glucose concentration but, when combined with high sucrose diet, cecal glucose was increased (30.6 ± 3.4 mg/dL versus 17.1 ± 4.7 mg/dL, n=8/group). Switching to high sucrose diet changed cecal/fecal bacterial diversity by day 2, with some differences in bacterial relative abundance; however, neither microbial diversity nor abundance was correlated with cecal glucose concentrations. In conclusion, SGLT1 inhibition or genetic ablation reduces intestinal glucose uptake (although glucose is still absorbed). but any remaining excess luminal glucose is not associated with intestinal adaptation or bacterial population changes during the time frames studied.

Monoacylglycerol acyltransferase enzymes (MGAT1, MGAT2, and MGAT3) convert monoacylglycerol to diacylglycerol (DAG). MGAT1 and MGAT2 are both implicated in obesity-related metabolic diseases. Conventional MGAT enzyme assays use radioactive substrates, wherein the product of the MGAT-catalyzed reaction is usually resolved by time-consuming thin layer chromatography (TLC) analysis. Furthermore, microsomal membrane preparations typically contain endogenous diacylglycerol acyltransferase (DGAT) from the host cells, and these DGAT activities can further acylate DAG to form triglyceride (TG). Our mass spectrometry (liquid chromatography-tandem mass spectrometry, or LC/MS/MS) MGAT2 assay measures human recombinant MGAT2-catalyzed formation of didecanoyl-glycerol from 1-decanoyl-rac-glycerol and decanoyl-CoA, to produce predominantly 1,3-didecanoyl-glycerol. Unlike 1,2-DAG, 1,3-didecanoyl-glycerol is proved to be not susceptible to further acylation to TG. 1,3-Didecanoyl-glycerol product can be readily solubilized and directly subjected to high-throughput mass spectrometry (HTMS) without further extraction in a 384-well format. We also have established the LC/MS/MS MGAT activity assay in the intestinal microsomes from various species. Our assay is proved to be highly sensitive, and thus it allows measurement of endogenous MGAT activity in cell lysates and tissue preparations. The implementation of the HTMS MGAT activity assay has facilitated the robust screening and evaluation of MGAT inhibitors for the treatment of metabolic diseases.

Activation of cyclic 1,3-diones with PyBroP generates phosphonium salt intermediates which readily undergo Pd-catalyzed Suzuki—Miyaura coupling with boronic acids and pinacol esters as well as Pd/Cu-catalyzed Sonogashira coupling with terminal alkynes.

Monoacylglycerol acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DAG) from free fatty acids (FFA) and sn-monoacylglycerol (MG), the two major hydrolysis products of dietary fat. To demonstrate MGAT2-mediated cellular activity of triglyceride (TG) synthesis, we utilized 1-oleoyl-glycerol-d5 as a substrate to trace MGAT2-driven 1-oleoyl-glycerol-d5 incorporation into TG in HEK293 cells stably expressing human MGAT2. The oleoyl-glycerol-d5 incorporated major TG species were then quantified by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) in a 96-well format. Conventional MGAT2 target-engagement in vivo assays measure the elevation of total plasma TG by orally dosing a bolus of TG oil. We developed a novel LC/ESI/MS/MS-based fat absorption assay to assess the ability of MGAT2 inhibitors to inhibit fat absorption in CD1 mice by a meal tolerance test consisting of a mixture of liquid Boost plus® and 0.59 g/kg U13C-TG oil. The newly resynthesized plasma heavy TGs containing three 13C in the glycerol backbone and two U13C-acyl-chains, which represented the digested, absorbed and resynthesized TGs, were then quantitated by LC/ESI/MS/MS. With this assay, we identified a potent MGAT2 inhibitor that blocked MGAT2-mediated activity in vitro and in vivo. The use of 1-oleoyl-glycerol-d5 and U13C-TG oil followed by LC/ESI/MS/MS detection of stable-isotopic labeled DAG, TG, or glycerol provides a wide range of applications to study pathophysiological regulation of the monoacylglycerol pathway and MGAT2 activity.

Phosphonium salt-activated, Pd-catalyzed Suzuki-Miyaura and Sonogashira cross-coupling reactions of cyclic 1,3-diones in the synthesis of β-substituted cyclic enones are described. These transformations exhibit good isolated yield and high generality with respect to both substrates and coupling partners. Extension of the substrate scope to cyclic 1,3-dione equivalents, such as 2-cyanocyclohexanone (4), is also briefly examined.

A facile synthesis of 2-(3-alkynyl-2,3-dihydrobenzofuran-3-yl)acetate (1a, 1b), with a unique alkynyl-benzylic quaternary center on a dihydrobenzofuran ring, was accomplished. The key step of the synthesis is the aryl radical cyclization to the tethered enyne carboxylate to generate the unique alkynyl-benzylic quaternary center. The synthesis started from simple building blocks with high flexibility and constructed the complex template in a total of four steps. With several functional groups available, compounds of structure 1a and 1b could be easily further derivatized.

Previously disclosed H-4 receptor modulators, the triamino substituted pyridines and pyrimidines, contain a free primary amino (-NH2) group. In this Letter we demonstrate that an exocyclic amine (NH2) is not needed to maintain affinity, and also show a significant divergence in the SAR of the pendant diamine component. These des-NH2 azacycles also show a distinct functional spectrum, that appears to be influenced by the diamine component; in the case of the 1,3-amino pyrimidines, the preferred diamine is the amino pyrrolidine instead of the more common piperazines. Finally, we introduce 3,5-diamino pyridazines as novel histamine H-4 antagonists. (C) 2014 Elsevier Ltd. All rights reserved.

Design, synthesis, and biological evaluation of pyridazine-based, 4-bicyclic heteroaryl-piperidine derivatives as potent stearoyl-CoA desaturase-1 (SCD1) inhibitors are described. In a chronic study of selected analog (3e) in Zucker fa/fa (ZF) rat, dose-dependent decrease of body weight gain and plasma fatty acid desaturation index (DI) in both C16 and C18 are also demonstrated. The results indicate that the plasma fatty acid DI may serve as an indicator for direct target engagement and biomarker for SCD1 inhibition.

Complement C1s protease inhibitors have potential utility in the treatment of diseases associated with activation of the classical complement pathway such as humorally mediated graft rejection, ischemia-reperfusion injury (IRI), vascular leak syndrome, and acute respiratory distress syndrome (ARDS). The utility of biphenylsulfonyl-thiophene-carboxamidine small-molecule C1s inhibitors are limited by their poor in vivo pharmacokinetic properties. Pegylation of a potent analog has provided compounds with good potency and good in vivo pharmacokinetic properties.

With regard to drug discovery, two glycokinases are of particular interest based upon clinical data; ketohexokinase ( EC 2.7.1.3 , 1‐3) and glucokinase/hexokinase 4 ( EC 2.7.1.2 , 4‐8). Yet, modulators for either target have had limited success in pharmaceutical development. Despite, decades of basic and applied research, only a few small molecule activators for glucokinase have made it to phase 1 clinical trial. This could in fact, be due to the basic assay designs used for and the initial steps in drug discovery. Traditionally, glycokinase activities have been monitored by radioactive, enzyme‐linked, fluorescence polarization detection of ATP turnover. Described herein is a novel label‐free means of directly monitoring the bi‐bi reaction for a ketohexokinase where detection of enzyme activity utilizes mass spectroscopy. Here, turnover of ATP and fructose to ADP and fructose 1‐P, respectively, can be monitored directly and simultaneously. The selectivity of analyte detection is maximized by MRM, allowing for detection of each analyte by LCMSMS from both cells and tissues. Basic enzyme characterization is demonstrated using HTMSMS. In addition, the mechanisms of action for two unrelated compounds and the implications of target engagement of pharmaceutically active compounds from “test tube” to tissue samples are discussed.

Two new series of potent and selective dual EGFR/ErbB-2 kinase inhibitors derived from novel thienopyrimidine cores have been identified. Isomeric thienopyrimidine cores were evaluated as isosteres for a 4-anilinoquinazoline core and several analogs containing the thieno[3,2-d]pyrimidine core showed anti-proliferative activity with IC50 values less than 1μM against human tumor cells in vitro.