Following the discovery of 4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline (4; PD 153035) as an extremely potent (IC(50) 0.025 nM) inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), several fused tricyclic quinazoline analogues have been prepared and evaluated for their ability to inhibit the enzyme. The most potent compound was the linear imidazo[4,5-g]quinazoline (8), which exhibited an IC(50) of 0.008 nM for inhibition of phosphorylation of a fragment of phospholipase C-gamma-1 as substrate. While N-methyl analogues of 8 showed similar potency, analogous N-[2-(dimethylamino)ethyl] derivatives were less effective. The next most potent compounds were the linear pyrazoloquinazolines (19 and 20) (IC(50)s 0.34 and 0.44 nM) and pyrroloquinazoline (21) (IC(50) 0.44nM), while several other linear tricyclic ring systems of similar geometry to 8 (triazolo-, thiazolo-, and pyrazinoquinazolines) were less effective. In the imidazo[4,5-g]quinazoline and pyrroloquinazoline series, the corresponding angular isomers were also much less effective than the linear ones. These results are consistent with structure-activity relationship studies previously developed for the 4-[(3-bromophenyl)amino] quinazolines, which suggested that small electron-donating substituents at the 6- and 7-positions were desirable for high potency. Cellular studies of the linear imidazoloquinazoline 8 show that it can enter cells and rapidly and very selectively shut down EGF-stimulated signal transmission by binding competitively at the ATP site of the EGFR.