New 2-alkoxycyanopyridine derivatives as inhibitors of EGFR, HER2, and DHFR: Synthesis, anticancer evaluation, and molecular modeling studies

New series of substituted 2-alkoxycyanopyridine derivatives were synthesized and evaluated for their in vitro and in vivo Anticancer activities. Comparing the evaluated activities against Cancer cell lines to the broad-spectrum Anticancer doxorubicin, and the kinase inhibitor sorafenib, compounds 3a, 4b, 4c, 7a, and 8d demonstrated superior Anticancer efficacy with elevated safety profiles and selectivity indices, particularly against MCF7 breast Cancer. For exploration of their mechanism of action, assays for inhibition of EGFR, HER2 kinase, and DHFR were performed. The promising synthesized compounds exhibited potent dual kinase EGFR/HER2 inhibitory activity with IC₅₀values of 0.248/0.156 μM for 4b and 0.138/0.092 μM for 4c. Additionally, with IC₅₀ values of 0.138 and 0.193 M, respectively, 4b and 4c had the greatest DHFR inhibitory activity that was comparable to methotrexate. In the MCF7 breast Cancer cell line, they caused arrest at the S phase of the cell cycle and exhibited Apoptosis induction activity. With restored Caspase-3 immunoexpression, the anti-breast Cancer assay performed in vivo of 4b and 4c demonstrated a substantial decrease in tumor volume. Results from molecular modeling were in agreement with biological assays proving the importance of the 3-caynopyridine, two substituted phenyl rings attached to central pyridine ring, and propoxy side chain moieties for binding with the receptors. As 4c works by inhibiting both EGFR/HER2 kinase, DHFR enzymes, in addition to cellular Apoptosis, it could be viewed as a model of compounds possessing a multi-targeting Anticancer activity. Collectively, compounds 4b and 4c might represent prototypes for further development as Anticancer molecules.

Alkoxycyanopyridine; Anti-breast cancer in vivo assay; Cell cycle analysis; DHFR inhibition; EGFR/HER2 kinase inhibition; Pharmacophore prediction; Synthesis.