Hydrolysis of S-2-(3-aminopropylamino)ethylphosphorothioate (WR-2721)

Abstract

The hydrolysis reaction of S-2-(3-aminopropylamino)ethylphosphorothioate (WR-2721), a radioprotective agent currently undergoing clinical trials, was studied under a variety of experimental conditions in order to provide more complete data and to reconcile significant differences found between two previous studies. 31P NMR spectroscopy was primarily used to follow the reaction, but comparable results were also obtained in parallel studies using a spectrophotometric technique and a technique involving liquid chromatography with electrochemical detection, in which the free sulfhydryl product, 2-(3-aminopropylamino)ethanethiol (WR-1065), was measured. Upon hydrolysis, inorganic phosphate and the free sulfhydryl group were formed by cleavage of the P-S bond. The reaction rate versus pH profile at 30° in 42.5 mM buffer, μ. = 127.5 mM, showed primarily hydrolysis of the monoanion. with an acid-catalyzed reaction below pH 1.5 to 2.0 involving the neutral species of the ester. The energy of activation at pH 4.0 in 42.5 mM acetate buffer was 25.7 kcal/mole (23.1 kcal/mole by liquid chromatography with electrochemical detection). The entropy of activation at pH4.0, 36° was positive, and there was a deuterium isotope effect on the reaction. A small buffer effect on the rate of the reaction at pH 4.0 and pH 5.0 was found to include contributions from both general acid and general base catalysis. These data are consistent with a mechanism for hydrolysis of the monoanion involving a partially rate-determining proton transfer to the sulfur atom and the formation of metaphosphate ion, which is rapidly hydrolyzed to inorganic phosphate.