Design of a chimeric 3-methyl-1,2,3-triazene with mixed receptor tyrosine kinase and DNA damaging properties: A novel tumor targeting strategy

The mixed epidermal growth factor receptor (EGFR)-DNA targeting properties of SMA41, a 6-(3-methyl-1,2,3-triazen-1-yl) 4-anilinoquinazoline designed t o release N-4-m-tolyl-quinazoline-4,6- diamine henceforth referred to as SM A52 [an inhibitor of EGFR tyrosine kinase (TK)] and methyldiazonium (a DNA methylating species) were studied in the O(6-)methylguanine-DNA methyltrans ferase (MGMT)-proficient and high EGFR-expressing epidermoid carcinoma of t he vulva cell line A431. The effects of SMA41 were compared with those of S MA52 alone, and temozolomide (TEM), a clinical prodrug of 5-(3-methyltriaze n- 1-yl) imidazole-4-carboxamide (MTIC) that is inactive in MGMT-proficient cells. The results showed that 1) the chimeric SMA41 could degrade in seru m-containing medium (t(1/2) of similar to 30 min) to generate, as predicted , the free inhibitor SMA52 as the most abundant metabolite (similar to 81% yield); 2) in contrast to SMA52 alone, the chimeric SMA41 and TEM induced s ignificant DNA damage in A431 cells after 30-min or 2-h drug exposures, as confirmed by alkaline single-cell gel microelectrophoresis (comet) assay; 3 ) SMA41 showed 5-fold greater affinity for the ATP binding site of EGFR tha n independently synthesized SMA52 in an enzyme assay and blocked EGF-induce d tyrosine phosphorylation and EGFR autophosphorylation in A431 cells in a dose-dependent manner; 4) these mixed targeting properties of SMA41, combin ed with its ability to be converted to another potent EGFR TK inhibitor (e. g., SMA52) by hydrolytic cleavage, translated into over 8-fold greater anti proliferative activity than TEM, which showed no EGFR targeting properties (IC50 competitive binding >100 muM); 5) under continuous drug exposure (3-6 -day sulforhodamine and clonogenic assays), SMA41 was almost equipotent wit h SMA52; however, in a short 2-h drug exposure followed by incubation in dr ug-free media, SMA52 showed an almost complete loss of antiproliferative ac tivity over the whole dose range. In contrast, SMA41 retained almost 100% o f its activity, indicating a more sustained growth inhibitory activity. The results in toto suggest that the superior antiproliferative activity of SM A41 may be due to a combination of events associated with its binary EGFR T K and DNA targeting properties.