In our pursuit of developing effective anticancer drugs, a dipyridyl thiourea derivative (1) was designed and synthesized via a one-pot reaction at room temperature. The structure of the synthesized compound was confirmed using FT-IR, 1H NMR, mass spectrometry, elemental analysis, and single-crystal X-ray crystallography. Both 1H NMR spectral analysis and single-crystal X-ray analysis revealed the formation of strong intramolecular hydrogen bonds in 1. Hirshfeld surface analysis was also employed to quantify various interactions within the structure. Density Functional Theory (DFT) calculations, performed at the B3LYP/6-31G(d) level with solvent effects in THF, revealed a planar geometry favoring π⋯π stacking. The HOMO–LUMO gap of 4.58 eV indicated moderate reactivity and good charge delocalization. HOMO charge density was localized on the sulfur atom, while LUMO was delocalized across the aromatic core. The cytotoxic potential of compound 1 was assessed using the Trypan Blue exclusion assay against Dalton's Lymphoma (DL) cells, a murine T-cell lymphoma model, revealing a dose-dependent inhibition with an IC50 of 22.5 μM. Furthermore, minimal cytotoxicity was observed in normal peripheral blood mononuclear cells (PBMCs), highlighting the selectivity of 1. Moreover, molecular docking studies further demonstrated favorable binding of compound 1 at the trimeric interface of mouse tumor necrosis factor, stabilized predominantly by hydrophobic interactions and a few hydrogen bonds. These findings collectively support the potential of 1 as a selective anticancer agent, warranting further biological validation.
