Hydrolytic Reactions of Thymidine 5′‐O‐Phenyl‐N‐Alkylphosphoramidates, Models of Nucleoside 5′‐Monophosphate Prodrugs

Abstract To obtain detailed data on the kinetics of hydrolytic reactions of triester‐like nucleoside 5′‐ O ‐aryl‐ N ‐alkylphosphoramidates, potential prodrugs of antiviral nucleoside monophosphates, the hydrolysis of diastereomeric ( R P / S P ) thymidine 5′‐{ O ‐phenyl‐ N ‐[(1 S )‐2‐oxo‐2‐methoxy‐1‐methylethyl]phosphoramidate} ( 3 ), a phosphoramidate derived from the methyl ester of L ‐alanine, has been followed by reversed‐phase HPLC over the range from H 0 =0 to pH 8 at 90 °C. According to the time‐dependent product distributions, the hydrolysis of 3 proceeds at pH<4 by two parallel routes, namely by nucleophilic displacement of the alaninyl ester moiety by a water molecule and by hydrolysis of the carboxylic ester linkage that allows intramolecular attack of the carboxy group on the phosphorus atom, thereby resulting in the departure of either thymidine or phenol without marked accumulation of any intermediates. Both routes represent about half of the overall disappearance of 3 . The departure of phenol eventually leads to the formation of thymidine 5′‐phosphate. At pH>5, the predominant reaction is hydrolysis of the carboxylic ester linkage followed by intramolecular displacement of a phenoxide ion by the carboxylate ion and hydrolysis of the resulting cyclic mixed anhydride into an acyclic diester‐like thymidine 5′‐phosphoramidate. The latter product accumulated quantitatively without any indication of further decomposition. Hydroxide‐ion‐catalyzed POPh bond cleavage of the starting material 3 occurred as a side reaction. Comparative measurements with thymidine 5′‐{ N ‐[(1 S )‐2‐oxo‐2‐methoxy‐1‐methylethyl]phosphoramidate} ( 4 ) revealed that, under acidic conditions, this diester‐like compound is hydrolyzed by PN bond cleavage three orders of magnitude more rapidly than the triester‐like 3 . At pH>5, the stability order is reversed, with 3 being hydrolyzed six times as rapidly as 4 . Mechanisms of the partial reactions are discussed.