Discovery of Potent EGFR Inhibitors With 6-Arylureido-4-anilinoquinazoline Derivatives

Abstract

According to the classical pharmacophore fusion strategy, a series of 6-arylureido-4-anilinoquinazoline derivatives ( Compounds 7a - t ) were designed, synthesized, and biologically evaluated by the standard CCK-8 method and enzyme inhibition assay. Among the title compounds, Compounds 7a , 7c , 7d , 7f , 7i , 7o , 7p , and 7q exhibited promising anti-proliferative bioactivities, especially Compound 7i , which had excellent antitumor activity against the A549, HT-29, and MCF-7 cell lines (IC₅₀ = 2.25, 1.72, and 2.81 μM, respectively) compared with gefitinib, erlotinib, and sorafenib. In addition, the enzyme activity inhibition assay indicated that the synthesized compounds had sub-micromolar inhibitory levels (IC₅₀, 11.66-867.1 nM), which was consistent with the results of the tumor cell line growth inhibition tests. By comparing the binding mechanisms of Compound 7i (17.32 nM), gefitinib (25.42 nM), and erlotinib (33.25 nM) to the EGFR, it was found that Compound 7i could extend into the effective region with a similar action conformation to that of gefitinib and interact with residues L85, D86, and R127, increasing the binding affinity of Compound 7i to the EGFR. Based on the molecular hybridization strategy, 14 compounds with EGFR inhibitory activity were designed and synthesized, and the action mechanism was explored through computational approaches, providing valuable clues for the research of antitumor agents based on EGFR inhibitors.

Keywords: EGFR; anti-proliferative bioactivities; enzyme activity inhibition assay; molecular docking; molecular dynamic simulation.

FIGURE 1

(A) Antitumor drugs with 4-anilinoquinazoline and diaryl urea structural fragments; (B) design scheme of 6-ureido-4-anilinoquinazoline derivatives.

FIGURE 2

Synthetic route of the target Compounds 7a–t.

FIGURE 3

EGFR enzyme activities of the Compound 7 series at 1 μm.

FIGURE 4

Alignment of initial docking poses of the three systems: erlotinib is marked in pale green, gefitinib is in lemon, and Compound 7i is in light pink.

FIGURE 5

RMSD values of the protein receptor (yellow), ligand (deep gray), and binding sites (green) of the constructed systems as the function of time of the second simulation: (A) Compound 7i system; (B) gefitinib system; (C) erlotinib system.

FIGURE 6

Superimposition of the initial and representative conformations of the three studied systems: (A) erlotinib system (representative conformation is colored in green; initial conformation is marked in pale green); (B) gefitinib system (representative conformation is colored in yellow; initial conformation is in limon); (C) Compound 7i system (representative conformation is in magenta; initial conformation is in light-pink).

FIGURE 7

Per-residue binding free energy decomposition of all the studied systems: the residues with energy contribution (absolute value ≥ 0.1) are selected.

FIGURE 8

Schematic diagram of three ligands’ binding modes in the EGFR - gefitinib is in yellow, erlotinib is in green, and Compound 7i is in magenta: (A) Region A, the residues interacting with the common fragment; (B) Region B, the residues interacting with the varied fragment.