Abstract
The structures of four hitherto unidentified metabolites of nitrazepam in the rabbit were elucidated using ultra violet (UV), infrared (IR), nuclear magnetic resonance (NMR) and mass spectrometry. They were 2'-benzoyl-2-hydroxy-4'-nitroacetoanilide (M-I), hydroxylated 2-amino-5-nitrobenzophenone (M-II), 7-amino-8-hydroxynitrazepam (M-III) and 2-formamido-5-nitrobenzophenone (M-IV). These metabolites were excreted primarily in conjugated from except for M-IV. Urinary excretion of nitrazepam and its metabolites were studied by preparative thin-layer chromatography (TLC) and characteristic colorimetric methods before and after enzymic hydrolysis of the conjugates. 2-amino-3-hydroxy-5-nitrobenzophenone (3-OH-ANB) was the major metabolite following oral administration. The excretion of 7-amino-nitrazepam and 7-acetamidonitrazepam increased, but that of 3-OH-ANB decreased following intraperitoneal administration. In both types of administration, M-I to IV were minor metabolites (about 1% of the dose), but M-I and M-IV were considered to be intermediates leading to 2-amino-5-nitrobenzophenone. On the basis of these data, the metabolic pathways of nitrazepam in the rabbit were postulated in Chart 1.
