6-Substituted 3,4-Benzocoumarins: A New Structural Class of Inducers and Inhibitors of CYP1A1-Dependent Activity

Abstract

A new synthetic route was utilized to prepare 6-substituted 3 ,4-benzocoumarins where the substituents were iodo, fluoro, trifluoromethyl, bromo, chioro, isopropyl, ethyl, t-butyl, methyl, hydrogen, amino, phenyl, or nitro; 3,4-naphthocoumarin was also synthesized. The relative affinities of these congeners for the aryl hydrocarbon (Ah) receptor were determined using rat hepatic cytosol and 2,3,7,8-[ 3H]tetrachlorodibenzo- p-dioxin ([ 3H]TCDD) as the radioligand. In addition, the Ah receptor agonist activity of the 6-substituted 3,4-benzocoumarins was determined from their concentration-dependent induction of ethoxyresorufin O-deethylase (EROD) activity. In contrast with many other structural classes of halogenated aromatics, there was not a correlation between the structure-binding versus structure induction relationships for the 6-substituted 3,4-benzocoumarins. These results suggested that some of these congeners may exhibit partial Ah receptor antagonist activities and this was investigated by determining the inhibitory effects of 6-substituted 3,4-benzocoumarins on TCDD-induced EROD activity in rat hepatoma H4II E cells in culture. Only four compounds (6-isopropyl, 6-phenyl, 6-fluoro, and 6- t-butyl) inhibited the TCDD-induced response (21.7 to 64.4% inhibition) and the mechanism of action of the most active inhibitor, 6- t-butyl-3,4-benzocoumarin, was further investigated. In contrast, with other partial Ah receptor antagonists such as α-naphthoflavone, cotreatment of rat hepatoma H4II E cells with 1 nM TCDD plus 1 and 10 μM 6- t-butyl-3,4-benzocoumanin did not result in decreased levels of the Ah receptor complex (liganded with TCDD). In addition, there was not significant inhibition of TCDD-induced CYPlAl mRNA levels or protein as determined by Northern and Western blot analyses. The results suggest that 6- t-butyl-3,4-benzocoumanin or one of its metabolites is a post-translational inhibitor of CYPlAl-dependent enzyme (EROD) activity in this cell line and thus represents a novel Ah receptor-independent inhibition of CYPlAl.