This letter describes the construction of conformationally constrained quinazoline analogues. Structure−activity relationship studies led to the identification of the lead compound 9n. Compound 9n exhibits effective in vitro activity against A431 WT,overexpression and H1975 [L858R/T790M] cancer cell lines but is significantly less effective against EGFR negative cancer cell lines (SW620, A549, and K562). Compound 9n was also assessed for potency in enzymatic assays and in vivo antitumor studies. The results indicated that 9n is a potent kinase inhibitor against both wild-type and T790M mutant EGFR kinase. Meanwhile, an oral administration of 9n at a dose of 200 mg/kg produced a considerable antitumor effect in a A431 xenograft model, as compared to gefitinib. A preliminary pharmacokinetic study of 9n also indicates it has good pharmacokinetic properties, and therefore, it is a good starting point for further development. KEYWORDS: Anticancer, kinase inhibitor, EGFR, conformationally constrained T he clinical success of a number of kinase-directed drugs indicates that kinase represent therapeutically relevant targets. The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTKs), which plays an important role in the regulation of cell growth, differentiation, and survival. 1 Because of their multidimensional role in the progression of cancer, EGFR and its family members have emerged as attractive targets for anticancer therapy. 2 Accordingly, targeting EGFR has been intensely pursued, with the approval of gefitinib, erlotinib, and lapatinib for use in the clinic. 3−7 As is common with many therapies, challenges with respect to treatment resistance emerge over time. This situation is certainly true of EGFR inhibitor therapy. Clinical studies have demonstrated the occurrence of resistance to gefitinib with the T790M mutation accounting for 50% of the clinically observed resistant mutations.In order to overcome the T790M mutation related drug resistance, a variety of irreversible inhibitors were developed. These compounds contain a Michael acceptor moiety designed to form a covalent bond with the conserved cysteine residue (Cys797) at the lip of the EGFR ATP binding site. 8,9 So far, canertinib, 10,11 neratinib, 12,13 dacomitinib, 14 and afatinib 15 have been developed as second generation irreversible inhibitors with limited clinical efficacy. These drugs demonstrate the utility of quinazoline derivatives as an attractive scaffold for the development of EGFR inhibitors. To date, many studies have been targeted at finding new structures based on quinazolines that are potent EGFR inhibitors. 16−18 All of these inhibitors share a common feature: upon the binding to EGFR, a hydrogen bond forms between the N1 atom of the compound and the backbone NH of Met769 in the hinge region.To investigate the potential use of a novel scaffold as an EGFR ATP binding site inhibitor, a series of conformationally constrained quinazoline derivatives were synthesized (consisting of...