Plasmin-activated prodrugs for cancer chemotherapy. 1. Synthesis and biological activity of peptidylacivicin and peptidylphenylenediamine mustard

Abstract

Many tumors contain elevated levels of plasminogen activator and thus produce elevated levels of the protease plasmin in the milieu of the tumor. It should be possible to prepare peptidyl prodrug derivatives of anticancer drugs that would be locally activated by tumor-associated plasmin. As an initial test of this hypothesis, the peptidyl prodrugs were synthesized of the anticancer drugs (.alpha.S,5S)-.alpha.-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin, AT-125) and N,N-bis(2-chloroethyl)-p-phenylenediamine (phenylenediamine mustard) by mixed anhydride coupling of the parent drug with the protected tripeptide, Boc-D-Val-Leu-Lys(Boc)-OH, followed by deprotection and trifluoroacetic acid. The prodrugs showed an increased selective in vitro cytotoxicity for Rous sarcoma virus transformed chicken embryo fibroblasts (which produce elevated levels of plasminogen activator) compared to nontransformed fibroblasts (which produce low levels of plasminogen activator). In the presence of the plasmin inhibitor, p-nitrophenyl p''-guanidinobenzoate at 2 .mu.g/ml, the selectivity of the phenylenediamine mustard prodrug was reduced, but there was no effect on the cytotoxicity of the free drug. The prodrug analog D-valylleucyl-D-lysylphenylenediamine mustard (in which L-Lys was replaced by D-Lys) was inactive. The prodrug derivative of acivicin did not display selective toxicity for transformed cells when the cells were cultured in plasminogen-free medium. Apparently, plasmin hydrolysis is necessary for the activation of the prodrugs. The prodrugs were tested in vivo for antitumor activity. The prodrug of acivicin, like acivicin itself, was inactive against the B16 melanoma, a murine tumor that produces high levels of plasminogen activator. This prodrug was active against the M5076 [mouse ovarian] carcinoma, a tumor that displays only moderate levels of plasminogen activator; however, despite the fact that the prodrug was 2- to 3-fold less toxic on a molar basis than acivicin, there was no evidence of an increased therapeutic index. The prodrug of phenylenediamine mustard was also slightly less toxic than the parent drug, but again there was no evidence for an improved therapeutic index against the B16 tumor.