In vivo metabolism of 2,6,9-trisubstituted purine-derived cyclin-dependent kinase inhibitor bohemine in mice: glucosidation as the principal metabolic route.
Synthetic cyclin-dependent kinase inhibitors have recently been referred to as effective antiproliferative agents. This study was conducted to characterize clearance of a 3H-labeled, trisubstituted purine-type inhibitor, 8-[3H]bohemine [6-benzylamino-2-(3-hydroxypropylamino)-9-isopropylpurine], in mice. Radioactivity profiles were analyzed by liquid scintillation counting and by thin layer chromatography followed by autoradiography. Metabolite structures were elucidated by mass spectrometry, NMR, and enzymatic analyses. Bohemine was rapidly and completely metabolized in vivo and disappeared from circulation during the first 60 min following intravenous administration. The metabolites were partly eliminated by the hepatobiliary tract and partly by renal excretion. The terminal hydroxyl group located at the C2 side chain of bohemine made the compound susceptible to main metabolic attacks, i.e., distinct types of conjugation reactions with glycosyl donors as well as an oxidative reaction. Other pathways were of relatively minor significance. Bohemine O-beta-D-glucoside was the most abundant metabolite to be excreted. The enzymatic mechanism responsible for bohemine glucosidation in vitro required the presence of a UDP-glucoside donor. Additional glycosidation products were observed after inclusion of UDP-glucuronide, UDP-xylose, UDP-galactose, or UDP-N-acetylglucosamine into microsomal incubates. Glycosidations occurred faster in the kidney incubates than in hepatic ones. The second principal bohemine metabolite was a carboxylic acid, 6-benzylamino-2-(2-carboxyethylamino)-9-isopropylpurine. A cytosolic, 4-methylpyrazole-sensitive alcohol dehydrogenase class I was shown to mediate oxidation of the terminal hydroxyl group of bohemine into this acid, which was the only metabolite found in the blood in significant amounts. However, it displayed only weak cyclin-dependent kinase-1-inhibitory activity (IC(50) > 100 microM) when compared with that of bohemine (IC(50) approximately 1 microM).
Chmela
Z
Z
Department of Pathological Physiology, Medical Faculty, Palacký University, Hnevotínská 3, CZ-775 Olomouc, Czech Republic.
Veselý
J
J
Lemr
K
K
Rypka
M
M
Hanus
J
J
Havlícek
L
L
Krystof
V
V
Michnová
L
L
Fuksová
K
K
Lukes
J
J
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D002264:Carboxylic Acids / Q000097:blood / Q000737:chemistry / Q000494:pharmacology; D002458:Cell Fractionation; D002855:Chromatography, Thin Layer; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D015394:Molecular Structure; D011687:Purines / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D014316:Tritium
D002264:Carboxylic Acids; D004791:Enzyme Inhibitors; D006027:Glycosides; D011687:Purines; C118034:bohemine; D014316:Tritium; D000426:Alcohol Dehydrogenase; D018844:Cyclin-Dependent Kinases