Metabolite identification via LC-SPE-NMR-MS of the in vitro biooxidation products of a lead mGIu5 allosteric antagonist and impact on the improvement of metabolic stability in the series

Detailed information on the metabolic fate of lead compounds con be a powerful tool for on informed approach to the stabilization of metabolically labile compounds in the lead optimization phase. The combination of high performance liquid chromotography (HPLC) with nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) has been used to give comprehensive structural data on metabolites of novel drugs in development. Recently, increased automation and the embedding of online solid-phase extraction (SPE) into a integrated LC-SPE-NMR-MS system have improved enormously the detection limits of this approach. The new technology platform allows the analysis of complex mixtures from microsome incubations, combining low material requirements with relatively high throughput. Such characteristics make it possible to thoroughly characterize metabolites of selected compounds at earlier phases along the path to lead identification and clinical candidate selection, thus providing outstanding guidance in the process of eliminating undesired metabolism and detecting active or potentially toxic metabolites. Such an approach was applied at the lead identification stage of a backup program on metobotropic glutamate receptor 5 (mGlu5) allosteric inhibition. The major metabolites of a lead 5-aminothiazole-4-carboxylic acid amide I were synthesized and screened, revealing significant in vitro activity and possible involvement in the overall pharmacodynamic behavior of 1. The information collected on the metabolism of the highly active compound I was pivotal to the synthesis of related compounds with improved microsomal stability.