1-Hydroxy- and 2-hydroxy-3-methylcholanthrene: regioselective and stereoselective formations in the metabolism of 3-methylcholanthrene and enantioselective disposition in rat liver microsomes

Abstract

Absolute configurations of enantiomeric 1-hydroxy-3-methylcholanthrene (1-OH-3MC) and 2-hydroxy-3-methyl-cholanthrene (2-OH-3MC) were determined by the exciton chirality circular dichroism (CD) method as their p-nitrobenzoate derivatives. Enantiomers of 1-OH-3MC were resolved by HPLC using a column packed with chiral stationary phase (CSP) (R)-N-(3,5-dinitrobenzoyl)phenylglycine covalently bonded to gamma-aminopropylsilanized silica. Enantiomers of 2-OH-3MC were resolved as diastereomeric (-)-methoxyacetates by normal-phase HPLC. 1-OH-3MC and 2-OH-3MC, formed in the metabolism of 3MC by liver microsomes from untreated, phenobarbital (PB)-treated and 3MC-treated male Sprague-Dawley rats, were first isolated as a mixture by reversed-phase HPLC and subsequently separated by normal-phase HPLC. Concentration ratios of [1-OH-3MC]:[2-OH-3MC] formed in the metabolism of 3MC by three rat liver microsomal preparations (at 0.5 mg protein per ml of incubation mixture and an incubation time of 10 min) were found to be: 30:70 (control), 21:79 (PB treated) and 10:90 (3MC treated) respectively. R/S enantiomer ratios of 1-OH-3MC formed in the metabolism of 3MC by three rat liver microsomal preparations were determined by CSP HPLC: 35:65 (control), 39:61 (PB treated) and 46:54 (3MC treated) respectively. R/S enantiomer ratios of 2-OH-3MC formed in the metabolism of 3MC by three rat liver microsomal preparations were determined by CD spectral data: 14:86 (control), 6:94 (PB treated) and 6:94 (3MC treated) respectively. Metabolism of racemic 1-OH-3MC and 2-OH-3MC by all three rat liver microsomal preparations was found to be substrate enantioselective; the rate of 1S-OH-3MC metabolism was faster than that of 1R-OH-3MC, whereas the rate of 2R-OH-3MC metabolism was faster than that of 2S-OH-3MC.