Design, synthesis and biological evaluation of novel 4-anlinoquinazoline derivatives as EGFR inhibitors with the potential to inhibit the gefitinib-resistant nonsmall cell lung cancers

Abstract

A series of quinazoline derivatives with benzylidene hydrazine carboxamide were designed and synthesised as EGFR inhibitors. Most compounds exhibited exceptional anti-proliferative activity against A549, HepG2, MCF-7 and H1975 cells. Furthermore, six compounds demonstrated excellent inhibition activity against EGFR^WT with the IC₅₀ value both less than 2 nM. Among the six compounds, 44 exhibited the strongest activity (0.4 nM) and potently inhibited EGFR^L858R/T790M (0.1 μM). Excitingly, the most potent compound 14 showed excellent enzyme inhibitory activity with 6.3 nM and 8.4 nM for both EGFR^WT and EGFR^T790M/L858R. The result of AO single staining and Annexin V/PI staining showed that the compound 14 and 44 could induce remarkable apoptosis of A549 cells. The compound 14 arrested the cell cycle at the S phase and compound 44 arrested the cell cycle at the G0 phase in A549 cells. These preliminary results demonstrate that compound 14 and 44 may be promising lead compound-targeting EGFR.

Keywords: EGFR; NSCLC; Quinazoline derivatives; benzylidene hydrazine; inhibitors.

Figure 1.

The chemical structures of several small molecule EGFR inhibitors.

Figure 2.

Structures and design strategy for target compounds 9–54.

Scheme 1.

Synthetic route of target compounds 9–54.

Scheme 2.

Synthetic route of side chains 57a-l and 60a–f.

Figure 3.

Morphologic changes of A549 cells under inverted microscopy and fluorescence microscopy. 3(a) the control group cell treated with nothing; 3(b) Experimental group treated with 1.33 μM concentration of afatinib. 3(c) Experimental group treated with 1.59 μM concentration of 14. 3(d) Experimental group treated with 0.41 μM concentration of 44.

Figure 4.

Cell apoptosis analysis on A549 cells treated with compounds 14 and 44 at 2.5 μM detected by FCM.

Figure 5.

Cell cycle distribution of A549 cells treated at 2.5 μM compound 14 and 44 for 48 h detected by FCM.

Figure 6.

The protonated state of several important residue were adjusted by using AutoDock 4.5 in favour of forming reasonable hydrogen bond with the ligand and Molecular docking analysis was carried out by the Discover Studio 4.5 visualizer to explore the binding model for the active site of EGFR with its ligand. Docking simulations showed that the four selected compounds (afatinib, BMC201725-9o, 14 and 44) occupied the ATP site of EGFR kinase 6(a). Then we employed 3 D interaction map 6(b), 2 D diagram of the compound 14 6(c) and 2 D diagram of the compound 44 6(d) to display the interaction with the targeted protein (4G5P).