Discovery of Anilino-1,4-naphthoquinones as Potent EGFR Tyrosine Kinase Inhibitors: Synthesis, Biological Evaluation, and Comprehensive Molecular Modeling

Abstract

Epidermal growth factor receptor (EGFR) has been recognized as one of the attractive targets for anticancer drug development. Herein, a set of anilino-1,4-naphthoquinone derivatives (3-18) was synthesized and investigated for their anticancer and EGFR inhibitory potentials. Among all tested compounds, three derivatives (3, 8, and 10) were selected for studying EGFR inhibitory activity (in vitro and in silico) due to their most potent cytotoxic activities against six tested cancer cell lines (i.e., HuCCA-1, HepG2, A549, MOLT-3, MDA-MB-231, and T47D; IC₅₀ values = 1.75-27.91 μM), high selectivity index (>20), and good predicted drug-like properties. The experimental results showed that these three promising compounds are potent EGFR inhibitors with nanomolar IC₅₀ values (3.96-18.64 nM). Interestingly, the most potent compound 3 bearing 4-methyl substituent on the phenyl ring displayed 4-fold higher potency than the known EGFR inhibitor, erlotinib. Molecular docking, molecular dynamics simulation, and MM/GBSA-based free energy calculation revealed that van der Waals force played a major role in the accommodations of compound 3 within the ATP-binding pocket of EGFR. Additionally, the 4-CH₃ moiety of the compound was noted to be a key chemical feature contributing to the highly potent EGFR inhibitory activity via its formations of alkyl interactions with A743, K745, M766, and L788 residues as well as additional interactions with M766 and T790.

Figure 1

Chemical structures of furano-1,2-naphthoquinone I, shikonin, benzoylacrylic acid shikonin II, plumbagin, aminonaphthoquinones, and erlotinib.

Scheme 1. Synthesis of Anilino-1,4-naphthoquinone Derivatives 3–18

Figure 2

EGFR tyrosine kinase inhibitory effects of 3, 8, 10, and erlotinib. Data are shown as means ± SEM of three independent experiments.

Figure 3

Alignment of docked compounds 3 (black), 8 (cyan), and 10 (orange) inside the ATP-binding pocket of the EGFR tyrosine kinase domain. The binding orientation of crystallized erlotinib (gray) was used as the reference.

Figure 4

Two-dimensional ligand–protein interactions of three investigated naphthoquinones in complex with EGFR. (A) Most potent compound 3, (B) compound 8, (C) compound 10, and (D) co-crystallized ligand, erlotinib.

Figure 5

Time evolution of RMSD (top), #contacts (middle), and R_g (bottom) of compound 3 in complex with the EGFR tyrosine kinase domain.

Figure 6

Superimposition between the docked structure and the final MD structure of compound 3 in complex with the EGFR tyrosine kinase domain.

Figure 7

Left panel: (A) ΔG_bind,residue and (B) energy contribution of compound 3 in complex with the EGFR tyrosine kinase domain. Right panel: Representative 3D structures showing the orientation of ligand 3 in the ATP-binding site drawn from the last MD snapshot. The contributing residues and vdW contributions are colored according to their ΔG_bind,residue and energy contribution values, respectively.