Microsomal hydroxylation of specifically deuterated monosubstituted benzenes. Evidence for direct aromatic hydroxylation

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Abstract
The aromatic hydroxylation of 6 pairs of selectively deuterated monosubstituted benzenes was investigated with rat liver microsomes of various induction states. The substrates studied included 3,5-D2C6H3X and 2,4,6-D3C6H2X, where X = Br, CN, NO2, OCH3, CH3, or Ph, respectively. The deuterium content of the ortho, meta, and para hydroxylated metabolites, as well as side chain oxidation products, was determined by capillary gas chromatography-mass spectroscopy. These data were analyzed according to a hypothetical model in which a molecule of substrate can undergo either direct aromatic hydroxylation (defined as obligatory and complete loss of deuterium from the site of hydroxylation) or indirect aromatic hydroxylation (defined as the obligatory and complete shift of deuterium to an adjacent position, followed by its partial loss as governed by a kinetic deuterium isotope effect). From this and other analyses of the data the following conclusions were reached. The relative extent of meta hyroxylation increased and the total yield of metabolites decreased as the substituents X became more electron withdrawing. The induction state of the microsomes altered the regioselectivity of hydroxylation (2,3,4, or side chain) noticeably and predictably but had little or no effect on the retention or loss of deuterium during each hydroxylation. With each substrate and at each ring position hydroxylation occurred by a combination of direct and indirect mechanisms. The relative importance of direct vs. indirect mechanisms did not vary in a simple manner with either the position of hydroxylation or the nature of the substituent X. For 20 different hydroxylations the value of the isotope effect (kH/kD [fractional rate constant]) governing retention or loss of deuterium was 4.05 .+-. 0.2. These observations provide strong independent support for the existence of direct pathways for aromatic hydroxlation and for the obligatory intermediacy of a cyclohexadienone in indirect aromatic hydroxylations.