In vitro biotransformation of 2,6,9-trisubstituted purine-derived cyclin-dependent kinase inhibitor bohemine by mouse liver microsomes

Abstract

1. Biotransformation pathways of the cyclin-dependent kinase inhibitor 6-benzylamino-2-(3-hydroxypropylamino)-9-isopropylpurine (bohemine) by mouse liver microsomes in vitro were investigated. 2. Metabolite profiles of [8-³H]-labelled bohemine were established by TLC/³H-autoradiography and enzymatic and MS analyses were used to elucidate the chemical structures of the metabolites. The structures of the main primary metabolites were confirmed by synthesis of authentic compounds. 3. A schema of the primary NADPH-dependent pathways has been proposed involving N² - and N9-dealkylation, N⁶-debenzylation, aromatic hydroxylation, and C2 side chain oxidation of bohemine. Three of the primary metabolites detected, 6-(benzylamino)-2-(3-hydroxypropylamino)purine (M4), 6-amino-2-(3-hydroxypropylamino)-9-isopropylpurine (M5) and 6-(4-hydroxybenzylamino)-2-(3-hydroxypropylamino)-9-isopropylpurine (M6), all retaining their parent primary hydroxyl group, were subsequently shown to be converted, by a liver cytosolic NAD⁺-dependent system, into their corresponding carboxylic acids. M6 was subject to microsomal glycosidations requiring UDP-sugar donors. NADPH-dependent conversion of M6 into M5 by microsomes was also demonstrated. 4. Cytochrome P450 (CYP) enzymes-selective inhibitors were used to identify CYPs involved in bohemine biotransformation. The findings suggested that CYP2a and CYP3a substantially contributed to the NADPH-dependent bohemine transformation in vitro. 5. The findings will facilitate experiments designed to dissect enzymatic systems catalysing clearance of C2, C6,N9-trisubstituted purine compounds from mammalian tissues.