In vitrobiotransformation 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-(3)H]-labelled bohemine were established by TLC/(3)H-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 N(2)- and N9-dealkylation, N(6)-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.

High Performance Liquid Chromatography‐Electrochemistry‐Electrospray Ionization Mass Spectrometry (HPLC/EC/ESI‐MS) for Detection and Characterization of Roscovitine Oxidation Products

“…Its identity has been confirmed by a comparison of its retention time with that of a synthetic Olomoucine II standard and by the QqTOF accurate mass MS analysis. A similar hydroxylation of the benzene ring was observed in in vitro study of bohemine [45].…”

“…Metabolism of the similar trisubstituted purine, bohemine, has been studied to some degree with the determination of certain primary metabolism routes [44,45]. in vivo metabolism of R-roscovitine has been examined after administration to rats [22,24], mice [23] and its in vitro metabolism was examined by an interaction with selected human and animal microsomes [22].…”

Metabolism of the Trisubstituted Purine Cyclin-Dependent Kinase Inhibitor Seliciclib (R-Roscovitine) in Vitro and in Vivo

“…Our results suggested that CYP2B6-mediated metabolism can occur at low levels of the enzyme, whereas the rate of CYP3A4-mediated metabolism is low until higher levels of the enzyme are present. CYP3A4 is responsible for metabolism of many xenobiotics (Guengerich, 1999) and has been previously shown to be at least partly responsible for metabolism of bohemine, a trisubstituted purine derivative with a close structural relationship to seliciclib (Rypka et al, 2002). We also made the interesting observation that seliciclib inhibits CYP3A4 in vitro at micromolar concentrations that can be achieved in the clinic (de la Motte and Gianella-Borradori, 2004).…”

“…The metabolism of the related trisubstituted purine, bohemine, has been studied in some detail in vitro, and routes of primary metabolism and glucuronidation have been determined (Chmela et al, 2001;Rypka et al, 2002;Cervenková et al, 2003). Metabolism of roscovitine has also been examined after i.v.…”

Hydrolytic cleavage ofN6-substituted adenine derivatives by eukaryotic adenine and adenosine deaminases

“…6-Amino-2-(3-hydroxypropylamino)-9-isopropylpurine and 6-amino-2-[(1-hydroxymethyl)propyl]amino-9-isopropylpurine, which were synthesized following a protocol published previously [30], were provided by Dr Libor Havlíček [Institute of Experimental Botany, Academy of Sciences of the Czech Republic (ASCR), Prague, Czech Republic].…”