The in Vitro Enzymic Labilities of Chemically Distinct Phosphomonoester Prodrugs

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Abstract
The kinetics of decomposition of phosphomonoesters of hydroxy-methyl-5,5-diphenylhydantoin (1), estrone (2), 17β-testosterone (3), 1-phenylvinyl alcohol (4), and 17α-testosterone (5) were studied in rat whole blood at 25 and/or 37°C. As the acidity of the leaving hydroxyl group of the phosphomonoester increased, there was a tendency for the rate of hydrolysis to increase, except for the anomalous behavior of 4, which was consistent with its relative rate of hydrolysis in aqueous solutions (1). In addition, the kinetics of hydrolysis of 1–5 and p-nitrophenyl phosphate (p-NPP) were studied in the presence of isolated alkaline phosphatases from a variety of sources. The initial rate of production of 17α- and 17β-testosterone from their respective phosphate esters (5 and 3), in the presence of human placental alkaline phosphatase, revealed that 3 was hydrolyzed 5.3-fold more rapidly than 5. This difference in reactivity might have been the result of differences in the stereochemical and/or steric nature of the two isomers. For p-NPP, 1, 2, and 4, the kcat and kcat / Km values determined in the presence of the various alkaline phosphatases showed little variation, whereas for 3, the catalytic constants, kcat and kcat / Km, were found to be dramatically less than those found for p-NPP, 1, 2, and 4. This suggested that the reaction steps, involving the noncovalent binding of the phosphomonoester to the enzyme and/or the nucleophilic displacement of the leaving alcohol of the phosphomonoester by the reactive amino acid residue of the enzyme, might have been less favorable in the case of 3, where the carbon atom of the ester linkage was secondary and was associated with a rigid ring system.