5-Azacytidine Hydrolysis Kinetics Measured by High-Pressure Liquid Chromatography and 13C-NMR Spectroscopy

Hydrolysis of 5-azacytidine, an experimental anticancer drug, in aqueous buffers was measured using a high-pressure liquid chromatographic (HPLC) procedure and a 13C-NMR method. The former utilized a 17.5-micron Aminex A-6 strong cation-exchanger column eluted with 0.4 M, pH 4.6 ammonium formate buffer at a flow rate of 0.4 ml/min. The hydrolysis sequence as well as the existence of a labile intermediate, N-formylguanylribosylurea, was unequivocally established using 6-13C-5-azacytidine and NMR spectral techniques. A reversible ring opening step to the N-formylguanylribosylurea with an equilibrium constant of 0.58 +/- 0.03 between pH 5.6 and 8.5, followed by an irreversible formation of guanylribosylurea, was found by HPLC. The data confirm previous assumptions on the hydrolytic kinetics. The pH dependency of hydrolysis was examined, and the hydrolysis profile gave a normal V shape with the most stable pH at 7.0. Rather stable intravenous dosage forms can be formulated.