Utility of MetaSite in Improving Metabolic Stability of the Neutral Indomethacin Amide Derivative and Selective Cyclooxygenase-2 Inhibitor 2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-phenethyl-acetamide

Abstract

ABSTRACT:Prediction of the metabolic sites for new compounds, synthesized or virtual, is important in the rational design of compounds with increased resistance to metabolism. The aim of the present investigation was to use rational design together with MetaSite, an in silico tool for predicting metabolic soft spots, to synthesize compounds that retain their pharmacological effects but are metabolically more stable in the presence of cytochrome P450 (P450) enzymes. The model compound for these studies was the phenethyl amide (1) derivative of the nonsteroidal anti-inflammatory drug (NSAID) indomethacin. Unlike the parent NSAID, 1 is a potent and selective cyclooxygenase-2 (COX-2) inhibitor and nonulcerogenic anti-inflammatory agent in the rat. This pharmacological benefit is offset by the finding that 1 is very unstable in rat and human microsomes because of extensive P4503 A4/2D6-mediated metabolism on the phenethyl group, experimental observations that were accurately predicted by MetaSite. The information was used to design analogs with polar (glycinyl) and/or electron-deficient (fluorophenyl, fluoropyridinyl) amide substituents to reduce metabolism in 1. MetaSite correctly predicted the metabolic shift from oxidation on the amide substituent to O-demethylation for these compounds, whereas rat and human microsomal stability studies and pharmacokinetic assessments in the rat confirmed that the design tactics for improving pharmacokinetic attributes of 1 had worked in our favor. In addition, the fluorophenyl and pyridinyl amide derivatives retained the potent and selective COX-2 inhibition demonstrated with 1. Overall, the predictions from MetaSite gave useful information leading to the design of new compounds with improved metabolic properties.In the early drug discovery phase, many compounds have high oxidative metabolic instability mediated by cytochrome P450 (P450) enzymes, which often results in poor oral pharmacokinetics in preclinical species. Whereas high-throughput in vitro microsomal assays that monitor metabolic stability provide a convenient means for "rankordering" large numbers of compounds, information pertaining to identification of "soft spots" for unstable compounds cannot be discerned from such analyses and requires separate metabolite identification studies. Unlike microsomal stability assays, metabolite identification experiments are low-throughput and cannot keep pace with high-speed chemistry efforts. In addition, there are scenarios in which metabolism studies will limit the labile site(s) to a certain region within the molecule. To guide chemists in the right direction, information on the exact metabolic site is often preferred. Therefore, in addition to existing in vitro assays, a value added proposition would be the use of in silico tools to precisely predict regiochemistry of metabolism for compounds (synthesized or only virtual), information that could be used for rational design of pharmacologically active compounds with improved metabolic properties.MetaSite is an in silico tool t...

Development of QSAR models for microsomal stability: identification of good and bad structural features for rat, human and mouse microsomal stability

“…The method uses flexible molecular interaction fields generated by GRID to determine the probability of an atom being the site of metabolism. Previous studies indicated that MetaSite's first three ranked predictions were experimentally confirmed for about 84% of the cases [12,[18][19][20]. The method not only predicts the metabolic soft spots of a compound, but also highlights the molecular moieties that help to direct the molecule in the cytochrome cavity such that the site of metabolism is in proximity to the catalytic center.…”

“…The metabolic soft spots of the resulting compounds were predicted with MetaSite [12,22]. Although this method is not designed to predict the relative rate of metabolism of a compound, the identification of the metabolic soft spots could serve as a useful guide for designing [12,[18][19][20] have described success rate of 84% in predicting the correct site of metabolism when its top three predictions are considered. Thus, we visualized the first three ranked predicted soft spots for each of the unstable compounds and identified some substructures that always contained predicted soft spots.…”

(2-Arylethenyl)-1,3,5-triazin-2-amines as a novel histamine H4 receptor ligands

“…The MetaSite computational procedure was used to predict the most likely sites of 6 metabolism and the structures of its metabolites [27,28]. The plot of MetaSite in silico predictions for sites of metabolism for compound 6 by liver model is shown below (Fig.…”

11C-Labeled Analogs of Indomethacin Esters and Amides for Brain Cyclooxygenase-2 Imaging: Radiosynthesis, in Vitro Evaluation and in Vivo Characteristics in Mice

“…This relationship may be due to binding to plasma proteins which is expected to increase with larger log P 7.4 values of the indomethacin analogs. 30) These radiotracers likely bind to the plasma proteins similar to other NSAIDs, including indomethacin binding with high affinity to serum albumin. 31,32) Non-specific binding to albumin and other plasma proteins is known to correlate positively and linearly in vitro with increasing lipophilicity.…”