Novel application of proton nuclear magnetic resonance spectroscopy in the identification of 2'-chloronordiazepam metabolites in the dog

Abstract

The only metabolite of 2'-chloronordiazepam, 7-chloro-1,3-dihydro-5-(2'-chlorophenyl)-2H-1,4-benzodiazepin-2-one (1), previously identified in the dog is lorazepam (2), which is a product of 3-hydroxylation. Two phenolic metabolites (3 and 4) in the dog corresponding to 4'-hydroxylation of the 5-phenyl ring and 9-hydroxylation of the fused benzene ring, respectively, have now been identified. The structure of the 9-hydroxy isomer 4 is deduced simply from the observed NMR spectral AB (J meta = 2.5 Hz) pattern of the protons of the fused benzene ring. In contrast, since a 2'-chloro substituent is present on the 5-phenyl ring of the parent drug, the usual method of recognizing 4'-hydroxylation of this ring by observation of AA'BB' multiplets in the proton nuclear magnetic resonance (1H NMR) spectra is inapplicable. Hence, a novel method is introduced to identify the 4'-hydroxy isomer 3, based on attributing different sets of NMR substituent effect parameters to hydroxyl groups, depending on whether these groups are meta or para to the benzodiazepinimine function. The urinary plus fecal excretion of 2-4 by one dog given in a single oral 10 mg/kg dose of 14C-labeled 1 amounted to 20, 5, and 7% of the dose, respectively; the urinary metabolites were excreted predominantly as conjugates of glucuronic acid and/or sulfate.