Design, synthesis, and anticancer evaluation of Biginelli-derived DHPM-amide hybrids bearing clinically inspired kinase-directed pharmacophores: EGFR docking and DFT studies.

A series of twelve novel dihydropyrimidine (DHPM)-amide hybrid conjugates (6a-c, 8a-c, 10a-c, and 12a-c) were rationally designed, synthesized, and biologically evaluated as potential EGFR-targeted anticancer agents against A549 non-small cell lung carcinoma (NSCLC) cells. The target compounds were constructed via a convergent two-step protocol: Biginelli multicomponent condensation to assemble the DHPM-carboxylic acid core (4a-c), followed by EDC·HCl/HOBt-mediated chemoselective amide coupling with four clinically inspired amine fragments derived from erlotinib/gefitinib (series 10), cabozantinib (series 8), imatinib (series 12), and a sorafenib bioisostere (series 6). All compounds were characterized by IR, 1H NMR, 13C NMR, and low-resolution ESI-MS. MTT cytotoxicity screening identified compound 10b as the most potent analogue (IC50 = 16.11 ± 0.11 µg mL-1, equivalent to 23.31 µM), exceeding the molar potency of the reference methotrexate (IC50 = 15.00 ± 0.48 µg mL-1, equivalent to 33.01 µM). It should be noted that methotrexate is a dihydrofolate reductase inhibitor included solely as a general cytotoxicity control and is not a mechanistically matched EGFR comparator; the comparison therefore reflects relative cytotoxic potency only and does not imply EGFR selectivity. Molecular docking against EGFR (PDB 1M17), an erlotinib-bound inactive-conformation structure, returned the most favourable score for 10b (score = -7.819 kcal mol-1), with predicted hydrogen bonds to ASP831 and LYS721, a halogen contact at ILE765, and an extensive 15-residue hydrophobic network; these poses indicate compatibility with the erlotinib-binding pocket rather than a confirmed ATP-competitive binding mode. DFT calculations at B3LYP/6-311G(d,p) characterised the electronic structure of the isolated molecule (ΔE = 4.045 eV; ω = 3.155 eV) and are reported as a physicochemical description only, without implying a causal link to cellular activity. Normal mode analysis indicated conformational stability of the 10b-EGFR complex, collectively identifying 10b as a promising lead for further optimisation and mechanistic evaluation, for which direct EGFR kinase assays remain necessary.