Reduction of 3'-azido-3'-deoxythymidine (AZT) and AZT nucleotides by thiols. Kinetics and product identification.

3'-Azido-3'-deoxythymidine (AZT), AZT 5'-monophosphate, and AZT 5'-triphosphate (AZTTP) were reduced by dithiothreitol with second-order rate constants of 2.30 x 10(-3), 1.50 x 10(-3), and 7.46 x 10(-4) M-1 s-1, respectively. Handlon and Oppenheimer reported that AZT is quantitatively reduced by thiols to 3'-amino-3'-deoxythymidine (Handlon, A. L., and Oppenheimer, N. J. (1988) Pharm. Res. (N.Y.) 5, 297-299). In the present report, multiple products of this reaction were identified by the techniques of UV spectroscopy, phosphate analysis, coelution with authentic standards from reversed-phase high pressure liquid chromatography, two-dimensional NMR spectroscopy, and mass spectrometry. The product mixture from reduction of AZT 5'-monophosphate at pH 7.1 and 25 degrees C was composed of 2,3'-anhydro-beta-D-threo-thymidine 5'-monophosphate (6.4%), 3'-amino-3'-deoxythymidine 5'-monophosphate (19.6%), beta-D-threo-thymidine 5'-monophosphate (6.8%), thymine and 3-amino-2,3-dideoxyribal 5-monophosphate (8.9%), beta-D-threo-thymidine 3',5'-cyclic monophosphate (9.1%), 3'-deoxy-2',3'-didehydrothymidine 5'-monophosphate (31.5%), and 3',5'-anhydro-beta-D-threo-thymidine (17.8%). Thymine and 3',5'-anhydro-beta-D-threo-thymidine were also products of reduction of AZT and AZTTP. Furthermore, the nucleosides of the above monophosphates were products of reduction of AZT, and the corresponding triphosphates were products of reduction of AZTTP. The product ratios were dependent on the level of phosphorylation of AZT and on the pH of the reaction. Mechanisms for formation of these products are proposed.

The use of low-energy collisionally activated dissociation negative-ion tandem mass spectrometry for the characterization of dog and human urinary metabolites of the drug BW 1370U87.

Liquid chromatography (LC) combined with atmospheric pressure chemical ionization mass spectrometry was used to identify phase I and II metabolites of the drug BW 1370U87 in dog and human urine. Additional analysis of individual high-performance liquid chromatograph fractions collected from dog urine by combined gas chromatography/mass spectrometry identified one metabolite which was not detected by LC/MS methods. Using negative-ion LC/MS, the majority of BW 1370U87 metabolites in human urine were identified as glucuronic acid conjugates of phase I oxidative metabolites. The negative-ion fragmentation produced by low-energy collisionally activated decomposition (CAD), studied by tandem mass spectrometry experiments, confirmed that these compounds were drug-related and allowed metabolite structures to be assigned. Product-ion spectra of the metabolites were dominated by the loss of neutral molecules from even-electron deprotonated [M-H]- ions.