title: Epimer interconversion, isomerization, and hydrolysis of tetrahydrouridine: implications for cytidine deaminase inhibition.
abstract: Tetrahydrouridine (THU) is an inhibitor of cytidine deaminase (CDA), the enzyme responsible for the deactivation of ara-C and other cytidine analogues in vivo, and therefore is capable of improving the therapeutic efficacy of these antitumor agents. In aqueous solution formulations, THU exists as a mixture of epimers differing in stereochemistry of the 4-OH substituent. The aims of this study were to investigate the interconversion kinetics of the epimers of THU, the CDA inhibitory effects of these epimers, and the stability and degradation mechanisms of THU epimer mixtures in aqueous solution with the ultimate goal of developing optimal conditions for a parenteral formulation of THU. A stability indicating HPLC assay utilizing a derivatized beta-cyclodextrin column was developed to separate the two epimers of THU and to monitor their reversible isomerization to their beta-ribopyranosyl counterparts and their hydrolysis to form N-glycosidic bond cleavage products. MS and one- and two-dimensional (1)H- and (13)C-NMR measurements were conducted to identify THU epimers and degradation products and to quantitatively model the degradation kinetics. The interconversion reaction between the two THU epimers is acid catalyzed with a first-order rate constant for conversion of epimer 1(1) to epimer 1(2) of (7.4 +/- 0.3) x 10(-3) h(-1) and an equilibrium constant ([1(2)]/[1(1)] of 1.7 +/- 0.1 at pH 7.4 and 25 degrees C. Epimer interconversion was therefore sufficiently slow at pH 7.4 to allow the isolation of each and evaluation of their CDA inhibitory activities utilizing 1% (w/v) mouse kidney homogenates as a source for cytidine deaminase and cytidine as a substrate. Inhibition constants for the two THU epimers (1(1) and 1(2)) were determined to be 8 +/- 1 x 10(-7) M and 6.2 +/- 0.2 x 10(-8) M, respectively. Studies at elevated temperature suggested that THU degradation from epimer mixtures is biphasic with the initial rate of disappearance being acid catalyzed and first order in initial THU concentration, thus ruling out dimerization as a potential reaction mechanism. NMR/MS analyses revealed that the major degradation products included the beta-ribopyranosyl THU isomers (two epimers), the reduced pyrimidinone base (tetrahydrouracil), and various anomers of D-ribose formed through N-glycosidic bond cleavage, and the products of subsequent reactions of the base. Kinetic modeling of the data obtained from both HPLC and NMR measurements indicated that in an acidic solution THU beta-ribofuranosyl --> beta-ribopyranosyl isomerization is a rapid equilibrium reaction, which proceeds through an intermediate observable in 1H-NMR, and is followed by slower N-glycosidic bond hydrolysis. All the reactions between THU, its ribopyranosyl isomers, the intermediate, and the base are acid catalyzed and appear to proceed through the same sugar ring-opened intermediate (carbinolamine), consistent with previous literature.
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007536:Isomerism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D008956:Models, Chemical; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D013767:Tetrahydrouridine / Q000737:chemistry* / Q000494:pharmacology; D013997:Time Factors; D014867:Water / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D012997:Solvents; D014867:Water; D013767:Tetrahydrouridine; D003564:Cytidine Deaminase