Exploration of a Novel Thiadiazole Derivative: Design, Synthesis, Biological Evaluation (In Vitro and in Silico), and DFT Studies.

In this study, we describe the stepwise synthesis and thorough structural elucidation of a newly designed thiadiazole-based hybrid molecule, referred to as compound 5. Its chemical framework was confirmed through an array of spectroscopic analyses, including H and C-NMR, infrared spectroscopy, and HRMS, ensuring a reliable structural validation. By using density functional theory optimized geometry and vibratinoal frequencies were computed. The Natural Bond orbital analysis confirmed the stability of this derivative. Molecular Electro Static potential used to identify electrophilic and nucleophilic area. The visual study of weak interactions NCI and RDG analysis carried out. The biological potential of compound 5 was then explored through in vitro cytotoxicity screening against three distinct human cancer cell lines: MCF-7 (breast carcinoma), A549 (lung carcinoma), and PC3 (prostate carcinoma). The MTT assay, with doxorubicin employed as the reference standard, demonstrated that compound 5 exerted measurable cytotoxicity, particularly against A549 lung cancer cells, yielding an IC of 25.12 µM, which highlights its moderate antitumor efficiency in comparison to the control drug. To complement the experimental findings, a comprehensive in silico investigation was performed. Molecular docking analyses revealed that thiadiazole 5 displayed a notable affinity toward the Tumor Necrosis Factor (TNF) binding site, reflected by a binding energy of -8.69 kcal/mol. Molecular dynamics (MD) simulations further substantiated the robustness of this interaction by confirming the long-term stability of the 5-TNF complex. Binding free energy calculations (MM-GBSA) corroborated these results, as ΔG_bind values consistently remained strongly negative throughout the simulation timeframe. In addition, PCA and RDF evaluations indicated a stable conformational behavior of compound 5, comparable or even superior to the reference molecule. Finally, free energy landscape mapping supported these observations by revealing energetically favorable and well-defined conformational states. Taken together, these experimental and theoretical insights suggest that compound 5 represents a promising scaffold for the development of new anticancer agents, with both structural stability and significant biological relevance.