In silico and in vitro evaluation of novel carbothioamide-based and heterocyclic derivatives of 4-(tert-butyl)-3-methoxybenzoic acid as EGFR tyrosine kinase allosteric site inhibitors

Abstract

Cancer is an elaborate sequence of disease grades that include uncontrolled cell growth and division, invasion, and metastasis. Overexpression of the epidermal growth factor receptor (EGFR) causes an abnormal signal transduction and is directly linked to cancer development. Most EGFR tyrosine kinase inhibitors (TKIs) are ATP-competitive inhibitors that frequently cause EGFR mutations or chemoresistance. Therefore, targeting the EGFR TK allosteric site has become a highly sought after cancer treatment strategy. Ten new derivatives of 4-(tert-butyl)-3-methoxybenzoic acid containing carbothioamide (compounds 3a-e), triazole (compounds 4a-d) or oxadiazole (compound 5) moieties were designed as EGFR TK allosteric site inhibitors as deduced in silico. The structures of these derivatives were characterized by chemical spectroscopic methods (ATR-FTIR, 1HNMR, 13CNMR and HRESI-MS). According to the molecular docking studies, compounds 3e and 3d showed the highest docking scores (ΔG), which was confirmed by molecular dynamic (MD) simulation studies. The synthesized derivatives, specifically compounds 3d and 3e, exhibited favorable pharmacokinetic profile. In vitro, the newly synthesized derivatives were evaluated for their cytotoxicity against A549 (lung adenocarcinoma), HepG2 (hepatocellular carcinoma), and HCT-116 (colorectal) cancer cell lines via the MTT assay, flow cytometry, RT-PCR, immunoblotting, and kinase inhibition assay. The cytotoxicity results showed that compound 3d was cytotoxic to the three tested cell lines, achieving the lowest IC50 concentration against A549 cancer cells. Compound 3d targeting EGFR tyrosine kinase caused cell cycle arrest at the G2/M phase and induction of the ER-mediated apoptosis pathway. In silico and in vitro antitumor activity findings of compound 3d demonstrated that it is a promising EGFR tyrosine kinase allosteric site inhibitor.