The di(4-acetoxybenzyl) ester of methylphosphonate 4 (X = H, R = Me) and the di(4-acyloxybenzyl) esters of methoxycarbonylmethylphosphonate 4 (X = MeO2C, R = Me, Et, Pr, Pr(i), Bu or Bu(t)) were prepared from the appropriate benzyl alcohol and phosphonic dichloride. At pD 8.0 and 37-degrees-C, both series of compounds hydrolyse with half-lives greater than 24 h to the corresponding mono(4-acyloxybenzyl) esters 5 (X = H or MeO2C, R = Me, Et, Pr, Pr(i) Bu or Bu(t)) which were prepared by treatment of the di(4-acyloxybenzyl) esters 4 with sodium or lithium iodide. The mono(4-acyloxybenzyl) esters 5 (X = H, R = Me) and 5 (X = MeO2C, R = Me, Et, Pr, Pr(i) or Bu(t)) undergo chemical hydrolysis to methylphosphonate 6 (X = H), and methoxycarbonylmethylphosphonate 6 (X = MeO2C) respectively, together with 4-hydroxybenzyl alcohol and the appropriate acylate anion. The rates of hydrolysis of the mono(4-acyloxybenzyl) esters decrease as the length and steric bulk of the acyl group increases, with half-lives ranging from approximately 150 h for the acetyl analogues to 2240 h for the pivaloyl derivative. The hydrolyses of the di- and mono-(4-acyloxybenzyl) esters were catalysed by porcine liver carboxyesterase (PLCE), and in all cases the acylate anion was formed. The rate of enzymatic hydrolysis was most rapid for the 4-butanoyloxybenzyl and 4-isobutanoyloxybenzyl analogues. The methoxycarbonyl ester of the phosphonoacetate analogues was not cleaved by PLCE. The methylphosphonate generated from the reaction of 4 (X = H, R = Me) in the presence of esterase and (H2O)-O-18, did not contain O-18 attached directly to phosphorus. These results suggest that both the chemical and enzymatic hydrolyses of the mono(4-acyloxybenzyl) esters and the PLCE-catalysed hydrolyses of the di(4-acyloxybenzyl) esters proceed via hydrolysis of the acyl group to give the acylate anion and the unstable 4-hydroxybenzyl esters. The electron-donating 4-hydroxy group facilitates the cleavage of the benzyl-oxygen bond with the formation of the 4-hydroxybenzyl carbonium ion 9, which readily reacts either with water or the phosphate buffer. The 4-acyloxybenzyl phosphoesters provide the first example of a protecting group which will enable the bioactivation of phosphonate prodrugs at rates appropriate to biological systems.
