Background: A group of substituted benzothiazoles from a research project was found
to have low microsomal clearance. However, these compounds had very high clearance in vivo.
Methods: In the present study, the clearance mechanism of two of the structural analogs, was investigated
in vitro and in vivo.
Results: In vitro studies showed the formation of corresponding non-P450 dependent oxidative
metabolites in S9, cytosol, and hepatocytes. The in vitro formation of these metabolites was observed
in mice, rats, non-human primates, and humans. The dog did not form the corresponding
metabolites in any of the matrices. Inhibition studies with S9 fraction and incubation with human
recombinant aldehyde oxidase (AO) showed that the formation of the corresponding metabolites
was AO dependent. To investigate the role of this pathway in vivo, mice were dosed with compound
A and bile and plasma were analyzed. Most of the metabolites in bile contained the AO-dependent
oxidized benzothiazole moiety, indicating that metabolism involving AO was probably the
main pathway for clearance. The same metabolites were also observed circulating in plasma. Mass
spectrometric analysis of the metabolite showed that the oxidation was on the benzothiazole moiety,
but the exact position could not be identified. Isolation of the metabolite of compound A and
analysis by NMR confirmed the structure of the metabolite as C2 carbon oxidation of the thiazole
ring resulting in carboxamide moiety. Further comparison of both metabolites with corresponding
authentic standards confirmed the structures.
Conclusion: To our knowledge, such an observation of in vitro and in vivo oxidation of substituted
benzothiazole by AO has not been reported before. The results helped the medicinal chemists design
compounds that avoid AO-mediated metabolism and with better ADME property.