Regioisomeric aromatic dihydroxylation of propranolol. Use of monohydroxylated intermediates for structural assignments of the metabolites formed in vitro.

The formation of dihydroxylated propranolol metabolites [(HO)2-Ps], determined as their trimethylsilyl derivatives, was investigated using GC/MS techniques. Tentative structural assignment for the (HO)2-Ps was achieved by matching retention times of the (HO)2-Ps arising from the incubation of synthetic mono-HO-P regioisomers in the presence of the rat liver 9000g supernatant. Seven compounds were identified as (HO)2-Ps: 2,3-, 3,4-, 3,7-, 4,6-, 4,8-, 5,6-, and 7,8-(HO)2-P. Five of these regioisomers were observed as metabolites when propranolol was the substrate: 4,8-, 3,4-, 5,6-, and 4,6-(HO)2-P. The pathway for the formation of (HO)2-Ps was investigated by incubating propranolol in the presence of rat liver microsomes under an 18O2 atmosphere. 18O2 was the source of both hydroxyl group oxygen atoms indicating that sequential hydroxylation occurs. Mono-HO-Ps, not having hydroxyl groups at C-4 or C-5, proved to be the best substrates for the second hydroxylation. Propranolol is a better substrate than is 4-HO-P for formation of (HO)2-Ps. The regioselectivity of the second hydroxylation is predictable on the basis of expected sites of electrophilic substitution on the mono-HO-P intermediates. Substrate stereoselectivity in the formation of (HO)2-Ps was determined.