Synthesis and evaluation of pteroic acid-conjugated nitroheterocyclic phosphoramidates as folate receptor-targeted alkylating agents

A novel nitroheterocyclic bis(haloethyl)phosphoramidate prodrug linked thro ugh lysine to a pteroic acid has been prepared and evaluated as a potential alkylating agent, to target tumor cells that overexpress the folate recept or. The prodrug exhibited IC50 values in the micromolar range and was 10-40 0-fold less cytotoxic in vitro than the phosphoramidate that lacks the lysi ne-pteroyl moiety. The data does not support a contribution of the folate r eceptor to cytotoxicity. In an attempt to determine the basis for the decre ased cytotoxicity in the pteroyl-lysyl analogue, compounds were prepared in which the lysine-pteroyl moiety was replaced with lysine alone or with an n-propyl group. The n-propyl and the lysyl analogues were on average 3.8- a nd 21-fold less potent than the unsubstituted bis(haloethyl)phosphoramidate , respectively. Chemical reduction of the prodrugs followed by P-31 NMR kin etics demonstrated that all of the phosphoramidate anions cyclized to the a ziridinium ion at similar rates and gave comparable product distributions, suggesting that changes in chemical activation did not account for the diff erences in cytotoxicity. It, is likely that folate receptor-mediated transp ort is not sufficient to deliver adequate intracellular concentrations of t he cytotoxic phosphoramide mustard.