Innovative Amino-Functionalization of Pyrido[2,3-d]pyrimidine Scaffolds for Broad Therapeutic Applications Supported by Computational Analyses

<b>Background:</b> Derivatives of Pyrido[2,3-<i>d</i>]pyrimidine-6-carboxylate are promising multi-target scaffolds. This study focused on synthesizing 16 amino-functionalized derivatives and evaluating their dual anticancer and antibacterial activities, supported by mechanistic and computational analyses. <b>Objectives:</b> Design and synthesize derivatives, evaluate cytotoxicity against HeLa, HepG-2, and MCF-7 (selectivity against WI-38), investigate EGFR^WT and EGFR^T790M inhibition, assess cell cycle, apoptosis, and migration effects, antibacterial efficacy against <i>E. coli</i> and <i>P. aeruginosa</i>, and perform in silico ADMET, docking, molecular dynamics, DFT, and antiviral predictions. <b>Methods:</b> Synthesized 16 derivatives; tested for cytotoxicity, EGFR inhibition, cell cycle, apoptosis, migration; assessed antibacterial activity; performed ADMET profiling, molecular docking, molecular dynamics, and DFT calculations. <b>Results:</b> Derivatives <b>1</b>, <b>2</b>, and <b>7</b> showed highest cytotoxicity (IC₅₀ = 3.98-17.52 μM; WI-38 IC₅₀ = 64.07-81.65 μM). Compound <b>1</b> potently inhibited EGFRWT (IC₅₀ = 0.093 μM) and EGFRT790M (IC₅₀ = 0.174 μM), induced G0/G1 arrest (74.86%) and apoptosis (26.37%), and reduced MCF-7 migration (69.63%). Moderate antibacterial activity observed (MIC = 50 μg/mL). ADMET indicated favorable pharmacokinetics, low CYP inhibition, negative mutagenicity, and oral toxicity class III. Molecular dynamics confirmed stable binding (EGFRWT RMSD 3 Å; EGFRT790M 3.5-4.6 Å) with persistent hydrogen bonds. In silico antiviral evaluation suggested strong binding to HCV NS5A (-9.36 kcal/mol), SARS-CoV-2 Mpro (-9.82 kcal/mol), and <i>E.coli</i> DNA gyrase (-10.25 kcal/mol). <b>Conclusions:</b> Compound <b>1</b> exhibits dual anticancer and antibacterial activity, supported by mechanistic and computational analyses, highlighting pyrido[2,3-<i>d</i>]pyrimidines as promising multi-target therapeutic scaffolds.