DNA interaction of the tyrosine protein kinase inhibitor PD153035 and its N-methyl analogue.

The brominated anilinoquinazoline derivative PD153035 exhibits a very high affinity and selectivity for the epidermal growth factor receptor tyrosine kinase (EGF-R TK) and shows a remarkable cytotoxicity against several types of tumor cell lines. In contrast, its N-methyl derivative, designated EBE-A22, has no effect on EGF-R TK but maintains a high cytotoxic profile. The present study was performed to explore the possibility that PD153035 and its N-methyl analogue might interact with double-stranded DNA, which is a primary target for many conventional antitumor agents. We studied the strength and mode of binding to DNA of PD153035 and EBE-A22 by means of absorption, fluorescence, and circular and linear dichroism as well as by a relaxation assay using human DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to show that both drugs bind to DNA and behave as typical intercalating agents. In particular, EBE-A22 unwinds supercoiled plasmid, stabilizes duplex DNA against heat denaturation, and produces negative CD and ELD signals, as expected for an intercalating agent. Extensive DNase I footprinting experiments performed with a large range of DNA substrates show that EBE-A22, but not PD153035, interacts preferentially with GC-rich sequences and discriminates against homooligomeric runs of A and T which are often cut more readily by the enzyme in the presence of the drug compared to the control. Altogether, the results provide the first experimental evidence that DNA is a target of anilinoquinazoline derivatives and suggest that this N-methylated ring system is a valid candidate for the development of DNA-targeted cytotoxic compounds. The possible relevance of selective DNA binding to activity is considered. The unexpected GC-selective binding properties of EBE-A22 entreat further exploration into the use of N-methylanilinoquinazoline derivatives as tools for designing sequence-specific DNA binding ligands.

Synthesis and antiproliferative properties of 4-aminoquinazoline derivatives as inhibitors of EGF receptor-associated tyrosine kinase activity.

The mitogenic action of EGF is mediated by ligand-induced autophosphorylation of the EGF receptor (EGF-R), which is commonly overexpressed in numerous human cancers. Inhibitors of receptor tyrosine kinase (RTK) activity could therefore be considered as effective potential antitumor agents. For this purpose, 4-aminoquinazoline derivatives were prepared and evaluated for their ability to inhibit RTK activity and the autophosphorylation of EGF-R. In addition, these compounds were tested on A431 cell growth to estimate their antiproliferative effect. The results showed that the substituent at the 4-position of the quinazoline ring must be an aromatic amine carrying small lipophilic electron-withdrawing groups on the 3- (or 2-) position of the phenyl ring. This aromatic moiety might be far from the quinazoline provided that the linking group is conformationally restricted, such as with piperazine. Hydrophilic and non-aromatic substituents such as morpholine gave completely inactive compounds. Introduction of a bulk at the 2-position of the quinazoline ring in 2,4-diaminoquinazolines or tricyclic compounds led to inactive products. This study reports additional structure-activity relationships of a well-characterized series to develop new inhibitors of EGF-R-associated tyrosine kinase activity.