We created new thiosemicarbazones and their cyclized heterocyclic compounds, examined them, and evaluated their biological activity in silico and in vitro. Two pyrene-based thiosemicarbazone compounds, (E)-N'-(pyren-1-ylmethylene) pyrrolidine-1-carbothiohydrazide (PP1) and (E)-N'-(pyren-1-ylmethylene) morpholine-4-carbothiohydrazide (PM1) were prepared from pyrrolidine-1-carbothiohydrazide and morpholine-4-carbothiohydrazide with pyrene-1-carboxaldehyde. The 2-(pyren-1-yl)-5-(pyrrolidin-1-yl)-1,3,4-thiadiazole (PP2) and 4-(5-(pyren-4-yl)-1,3,4-thiadiazol-2-yl)morpholine (PM2) were obtained unex-pectedly from the cyclization of PP1 and PM1 in the presence of Mn(OCOCH3)2.4H2O. FT-IR, UV-Visible, NMR, and HR-MS were used to accurately portray the structure of the compounds. The molecular com-position of PP1, PP2 and PM2 were ascertained by the single-crystal X-ray diffraction (XRD) method. The compounds are ordered and dispersed in three dimensions according to the density functional the-ory (DFT) findings, which are in good agreement with the XRD-derived crystal structures. The molecular docking analysis revealed the various possible interactions of drugs with the non-small cell lung cancer protein (EGFR with a non-covalent inhibitor). The binding energies for the compounds were found to be in the following order PP2 > PP1 > PM1 > PM2, and molecular dynamic simulation was used to further examine the binding mechanism of the most active compound (PP2). These compounds bound strongly with calf thymus (CT)-DNA, which was well described by absorption and emission studies. This demon-strated that PP2 had a better ability to bind (Kb value of 1.95 x 10 6 and Kq value of 2.43 x 10 4 ). Similarly, the synergy of bovine serum albumin (BSA) with compounds also was examined and showed PP2's sig-nificant binding potential (Kb value of 2.94 x 10 4 and Kq value of 6.87 x 10 4 ). Additionally, using an MTT assay against a few human cancer cells, the substances' in vitro cell viability was evaluated, includ-ing HepG-2 (hepatocellular carcinoma) and T24 (bladder), as well as Vero (normal kidney epithelial cells isolated from an African green monkey). With IC50 values of 13.16 mu M (Cisplatin-IC50 = 49.9 mu M) and 37.67 mu M (Cisplatin-IC50 = > 50 mu M), respectively, the findings demonstrated that PP2 displayed efficient cytotoxicity towards HepG-2 and T24 cells.(c) 2022 Elsevier B.V. All rights reserved.