In vitro microsomal metabolic studies on secondary aromatic amides

Previous studies showed that amides are metabolites arising from certain secondary aromatic amines. However, some analogue amines did not lead to the formation of the corresponding amides when metabolised under identical conditions. We, therefore, wished to establish the factors preventing detection of amides. In the present study, we thought that amide detection as metabolites from secondary anilines may depend on the hydrolytic rate of the corresponding amide. We studied the in vitro hepatic microsomal metabolism of four aromatic amides i.e. N-(4-nitrobenzoyl)aniline (N4NBZA), N-benzoyl-4-nitroaniline (NBZ4NA), N-benzoylaniline (NBZA) and N-benzoyl-2,4,6-trimethylaniline (NBZTMA) which were (or not) detected following microsomal metabolism of secondary anilines in previous studies. Following the preparation, characterisation and separation of substrates and potential metabolites, incubations were carried out using rabbit microsomal preparations fortified with NADPH. The substrates and potential metabolites were extracted into dichloromethane and analysed by TLC, HPLC and UV. The results indicated that both steric and electronic factors may influence hydrolysis of amides. Three amides i.e. N4NBZA, NBZ4NA and NBZA yielded hydrolytic metabolites, whereas, NBZTMA did not. Para hydroxylated metabolites were also detected from N4NBZA and NBZA. These observations support the concept that one reason for not detecting amides as metabolites from secondary anilines in previous studies could be due to their rapid hydrolysis to the corresponding primary amines.