Discovery and optimization of 2-pyridones as dual h-DHFR/EGFRTK inhibitors with immunomodulatory potential; design, synthesis, anti-proliferative activity, and apoptosis inducer

Liver and colorectal cancers present considerable health challenges, underscoring the need to identify innovative targeted therapeutics. Tumor progression can be prevented by targeting EGFR-TK and h-DHFR as essential molecular targets. In this context, we synthesized a new series of 2-pyridones from the reaction of 2-cyanoacrylamide with active methylene or 2-cyanoacetanilide with activated double bonds under basic conditions. The structure of the synthesized 2-pyridones was confirmed through microanalysis and spectroscopic data. In comparison to doxorubicin, the spiro 2-pyridine derivative 9b exhibited the highest anti-proliferative activity, demonstrating IC₅₀ values of 6.89 ± 0.4 μM and 5.68 ± 0.3 μM against HepG-2 and Caco-2 cell lines, respectively, with nearly 2-fold increase in efficacy observed in Caco-2 cells. Additionally, compound 9b demonstrated a significant safety profile concerning normal cells (WI-38), as indicated by selectivity index values of 14.66 and 12.09 against the Caco-2 and HepG-2 cell lines, respectively. Moreover, flow cytometry analysis revealed that compound 9b halted the cell cycle at the G1/S phase in Caco-2 treated cells, demonstrating an increase in the percentage of cells undergoing both early and late apoptosis. The apoptotic potential was corroborated by the up-regulation of BAX and the down-regulation of Bcl-2 levels. Compound 9b exhibited significant inhibitory activity against h-DHFR, with an IC₅₀ value of 0.192 ± 0.011 μM, compared to methotrexate (IC₅₀ = 0.191 ± 0.011 μM). Furthermore, compound 9b demonstrated EGFR inhibitory activity, with IC₅₀ of 0.109 ± 0.005 μM, which is close to the inhibition observed with Lapatinib (IC₅₀ = 0.044 ± 0.002 μM). Compound 9b had better immunomodulatory properties with significant inhibitory efficacy on TNF-α and IL-6, with IC₅₀ values of 0.40 ± 0.03 pg/mL and 0.60 ± 0.02 pg/mL, respectively. These values indicate a greater potency than the positive control drug Lapatinib, which displayed IC₅₀ values of 0.41 ± 0.03 pg/mL and 0.74 ± 0.05 pg/mL for TNF-α and IL-6, respectively. In addition, in silico metabolism prediction using SwissADME and BioTransformer tools revealed that compound 9b is a potential inhibitor of CYP2C9 and CYP3A4, and is predicted to undergo metabolic transformations primarily via aromatic hydroxylation and ketone reduction, while maintaining acceptable stability of its ester moiety. Finally, the molecular docking assessment, together with the direct in vitro enzymatic inhibition results, confirmed that the 2-pyridone derivative 9b can potently bind to and inhibit both EGFR and h-DHFR through favorable binding interactions.