| We recently developed a novel strategy termed “combi-targeting” that seeks to design “combi-molecules” which are able to not only block EGFR but to also damage DNA. Previous studies that sought to stabilize triazene based combi-molecules were based on masking the 1,2,3-triazene chain with a 2-acetoxymethylene group, leading to the synthesis of RB24 and RB107. The half-lives of these two molecules proved to be only about 5 minutes longer than their parent triazenes. The novel series of molecules presented herein were designed containing hydrolysable groups to modulate the kinetics of their degradation. These include vinyl, acetoxymethyloxyl, and p-nitrophenol carbamates. Kinetic studies determined that the vinyl carbamate ZRL2 was the most stable of the series. However, its vinyl counterpart ZRL1 designed to be cleaved by a basic neighbouring group was the least stable. The half-lives of all of the other molecules were significantly longer than that of RB107. Specifically, ZRL1 had a half-life approximately 20 min longer and ZRS1, ZRL4, ZRL5 40-55 min longer. The molecules were designed to release a fluorescent aminoquinazoline, FD105, upon degradation. This allowed us to observe its intracellular release by fluorescent microscopy. ZRL1 generated the highest level of fluorescence and its more stable counterpart, ZRL2, produced levels that were barely detectable. Studies directed at determining the dual EGFR-DNA targeting abilities of the molecules showed that: (a) all of the compounds were capable of blocking the EGFR tyrosine kinase activity in an isolated enzyme assay and in MDAMB468 cells, (b) all of the molecules, except for the most stable compound ZRL2, were capable of inducing dose-dependent DNA damage, and (c) induction of DNA damage was associated with cell cycle arrest in G2/M. Using a growth inhibition assay, it was determined that all of the molecules could: (a) block the growth of MDAMB468 cells and (b) preferentially inhibit the EGFR transfectant NIH3T3 HER14 over its wild type counterpart. In the growth inhibition assay using the MDAMB468 cells, the most potent drug was ZRL4, while the most stable molecule, ZRL2, remained the least potent drug of the series.;The results in toto suggest that ZRL2, which was the most stable molecule, was not able to hydrolyze fast enough to release the DNA damaging triazene, thus acting primarily as a bulky EGFR inhibitor. This study has allowed us to define a stable compound as one that can decompose under physiological conditions at a rate that is slower than that of an acetoxymethyl triazene but fast enough to generate the cytotoxic DNA damaging agents, while remaining capable of inhibiting EGFR. Based on these criteria, the combi-molecules ZRS1 and ZRL4 were selected for in vivo studies. |
