Design, Computational, Synthesis, and Anti-Cancer Evaluation of a Multifunctional 2,3-Dihydro-1,3,4-Thiadiazole-Benzenesulfonamide Hybrid as a Triple Inhibitor of CAIX, CAXII, and EGFR

A novel 2,3-dihydro-1,3,4-thiadiazole-benzenesulfonamide hybrid, N-(4-(([Formula: see text]-1-((([Formula: see text]-5-acetyl-3- (4-sulfamoylphenyl)-1,3,4-thiadiazol-2(3H)-ylidene)hydrazono)ethyl)phenyl)-4-chloro benzamide (N-ASTPC), was rationally designed as a triple inhibitor targeting carbonic anhydrase IX (CAIX), carbonic anhydrase XII (CAXII), and the epidermal growth factor receptor (EGFR) — key targets overexpressed in cancer. Molecular docking and 200-ns molecular dynamics (MD) simulations confirmed stable and high-affinity interactions of N-ASTPC with the active sites of all three targets. MM-GBSA binding free energy calculations and ProLIF interaction profiling further supported these findings, while principal component analysis (PCA) revealed limited conformational drift, indicating robust complex formation. Additionally, density functional theory (DFT) assessments affirmed N-ASTPC’s electronic stability and favorable reactivity. Biological assays demonstrated potent inhibitory activity with IC 50 values of 0.039 [Formula: see text]M, 0.04 [Formula: see text]M, and 0.05 [Formula: see text]M for CAIX, CAXII, and EGFR, respectively, comparable or superior to standard inhibitors (acetazolamide and erlotinib). Regarding in vitro cytotoxicity, N-ASTPC displayed selective toxicity toward cancer cells over normal fibroblasts, with notable effects in MDA-MB-231 and MCF-7 breast cancer lines compared to doxorubicin. Flow cytometry showed that N-ASTPC induces significant S-phase arrest and robust early apoptosis, confirmed by a [Formula: see text]5.7-fold increase in Bax, [Formula: see text]4.5-fold increase in caspase-3, and [Formula: see text]2-fold decrease in Bcl-2 expression — consistent with mitochondrial apoptotic pathway activation. Collectively, these findings establish N-ASTPC as a promising multifunctional anti-cancer lead compound capable of simultaneously disrupting tumor cell metabolism and signaling via CAIX, CAXII, and EGFR inhibition. Its multi-modal mechanism and selective apoptotic induction underscore its potential for further preclinical development, particularly in hypoxic and triple-negative breast cancer models.