L-663,581 [FG 8205; 7-chloro-4,5-dihydro-5-methyl-3-(5-(1-methylethyl)- 1,2,4-oxadiazol-3-yl)6H-imidazo(1,5-A)(1.4)-benzodiazepine-6-one] is an investigational partial agonist of benzodiazepine receptors for possible application in the treatment of anxiety. Previous studies have shown that the drug is eliminated mainly by biotransformation in rats, dogs and monkeys. Two metabolites, mono- and bis-hydroxy analogs were demonstrated to be active in vitro. The potency of benzodiazepine receptor binding (Ki) is 3.7 nM for the parent drug, 3.3 nM for the mono-hydroxylated metabolite and 1.2 nM for the bishydroxylated metabolite, respectively. Although the metabolites are as potent as, or more so than the parent drug in vitro, they are inactive in rats in a conditioned emotional response model. This study was designed to explore the underlying mechanisms responsible for the discrepancy between in vitro and in vivo activity of the metabolites. After i.v. administration of L-663,581 or its preformed metabolites, the metabolites were cleared more slowly than the parent drug. After i.p. administration at the same dose, the areas under the curve of the metabolites were 2 to 5 times that of the parent drug. Thus, the lack of in vivo activity of the metabolites cannot be explained by the absorption and/or elimination kinetics. Brain uptake studies indicated that the permeability of the blood-brain barrier is high for L-663,581 but very poor for the metabolites; the brain extraction ratio was about 0.6 for L-663,581 and less than 0.03 for the metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)