Discovery and SAR of aryl hydroxy pyrimidinones as potent small molecule agonists of the GPCR APJ.

This article describes the discovery of aryl hydroxy pyrimidinones and the medicinal chemistry efforts to optimize this chemotype for potent APJ agonism. APJ is a G-protein coupled receptor whose natural agonist peptide, apelin, displays hemodynamic improvement in the cardiac function of heart failure patients. A high throughput screen was undertaken to identify small molecule hits that could be optimized to mimic the apelin in vitro response. A potent and low molecular weight aryl hydroxy pyrimidinone analog 30 was identified through optimization of an HTS hit and medicinal chemistry efforts to improve its properties.

Characterization of monoacylglycerol acyltransferase 2 inhibitors by a novel probe in binding assays.

Monoacylglycerol acyltransferase 2 (MGAT2) is a membrane-bound lipid acyltransferase that catalyzes the formation of diacylglycerol using monoacylglycerol and fatty acyl CoA as substrates. MGAT2 is important for intestinal lipid absorption and is an emerging target for the treatment of metabolic diseases. In the current study, we identified and characterized four classes of novel MGAT2 inhibitors. We established both steady state and kinetic binding assay protocols using a novel radioligand, [(3)H]compound A. Diverse chemotypes of MGAT2 inhibitors were found to compete binding of [(3)H]compound A to MGAT2, indicating the broad utility of [(3)H]compound A for testing various classes of MGAT2 inhibitors. In the dynamic binding assays, the kinetic values of MGAT2 inhibitors such as Kon, Koff, and T1/2 were systematically defined. Of particular value, the residence times of inhibitors on MGAT2 enzyme were derived. We believe that the identification of novel classes of MGAT2 inhibitors and the detailed kinetic characterization provide valuable information for the identification of superior candidates for in vivo animal and clinical studies. The current work using a chemical probe to define inhibitory kinetics can be broadly applied to other membrane-bound acyltransferases.

Cell-based assay of MGAT2-driven diacylglycerol synthesis for profiling inhibitors: use of a stable isotope-labeled substrate and high-resolution LC/MS.

To demonstrate monoacylglycerol acyltransferase 2 (MGAT2)-mediated enzyme activity in a cellular context, cells of the murine secretin tumor cell-1 line of enteroendocrine origin were used to construct human MGAT2-expressing recombinant cell lines. Low throughput and utilization of radiolabeled substrate in a traditional TLC technique were circumvented by development of a high-resolution LC/MS platform. Monitoring incorporation of stable isotope-labeled D31-palmitate into diacylglycerol (DAG) allowed selective tracing of the cellular DAG synthesis activity. This assay format dramatically reduced background interference and increased the sensitivity and the signal window compared with the TLC method. Using this assay, several MGAT2 inhibitors from different chemotypes were characterized. The described cell-based assay adds a new methodology for the development and evaluation of MGAT2 inhibitors for the treatment of obesity and type 2 diabetes.

Biphenyl Acid Derivatives as APJ Receptor Agonists.

The APJ receptor and its endogenous peptidic ligand apelin have been implicated as important modulators of cardiovascular function, and APJ receptor agonists may be beneficial in the treatment of heart failure. In this article, we describe the discovery of a series of biphenyl acid derivatives as potent APJ receptor agonists. Following the identification of initial high-throughput screen lead 2, successive optimization led to the discovery of lead compound 15a. Compound 15a demonstrated comparable in vitro potency to apelin-13, the endogenous peptidic ligand for the APJ receptor. In vivo, compound 15a demonstrated a dose-dependent improvement in the cardiac output in male Sprague Dawley rats with no significant changes in either mean arterial blood pressure or heart rate, consistent with the hemodynamic profile of apelin-13 in an acute pressure volume loop model.

Discovery of 6-(aminomethyl)-5-(2,4-dichlorophenyl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamides as potent, selective dipeptidyl peptidase-4 (DPP4) inhibitors.

Continued structure-activity relationship (SAR) exploration within our previously disclosed azolopyrimidine containing dipeptidyl peptidase-4 (DPP4) inhibitors led us to focus on an imidazolopyrimidine series in particular. Further study revealed that by replacing the aryl substitution on the imidazole ring with a more polar carboxylic ester or amide, these compounds displayed not only increased DPP4 binding activity but also significantly reduced human ether-a-go-go related gene (hERG) and sodium channel inhibitory activities. Additional incremental adjustment of polarity led to permeable molecules which exhibited favorable pharmacokinetic (PK) profiles in preclinical animal species. The active site binding mode of these compounds was determined by X-ray crystallography as exemplified by amide 24c. A subsequent lead molecule from this series, (+)-6-(aminomethyl)-5-(2,4-dichlorophenyl)-N-(1-ethyl-1H-pyrazol-5-yl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamide (24s), emerged as a potent, selective DPP4 inhibitor that displayed excellent PK profiles and in vivo efficacy in ob/ob mice.