In Vitro and In Silico Evaluation of the Antitumor Potential of New Benzotriazole–1,2,4-Triazole Hybrids

Background/Objectives: One of the major problems facing the medical world is cancer, which causes millions of deaths each year. Nitrogen-containing heterocycles appear to offer a potential solution to this problem. Thus, the present study aimed to synthesize and characterize—both physicochemically and biologically—the new compounds obtained by combining benzotriazole and 1,2,4-triazole scaffolds. Methods: Each of the four cell lines (HaCaT—human keratinocyte, A375—melanoma, A549—non-small cell lung cancer, and HT-29—colorectal adenocarcinoma) was treated for 48 h with concentrations ranging from 10 to 100 µM of the four compounds. After 48 h of treatment, changes in cell viability were assessed using the Alamar Blue assay. These findings were supplemented by microscopic analysis of metabolic changes, as well as alterations in the actin cytoskeleton and nuclei, using staining techniques. Results: The results showed that MG-1 (p-chlorophenyl-) and MG-3 (p-(N,N-dimethylamino)phenyl-) are the most active compounds in our study, with IC50 values for each tumor line as follows: 75.43 ± 2.77, 77.15 ± 1.32, and 96.28 ± 1.72 μM in A375, A549, and HT-29 cells, and 78.97 ± 3.08, 56.78 ± 2.57, and 69.19 ± 3.97 μM, respectively. In HaCaT cells, IC50 values remained above 100 μM for all four hybrids. In silico screening identified MEK1 as the most favorable target for the investigated series, while 100 ns molecular dynamics simulations and MM-GBSA analysis supported stable binding of MG-1 and MG-3, with predicted binding free energies of −54.83 and −40.70 kcal/mol, respectively. Conclusions: Even with a limited set of compounds, we can conclude that the biological effect observed in our study depended on both the cell line and the substituent attached to the aryl moiety. The results obtained for MG-1 and MG-3 suggest that these compounds may serve as suitable starting points for further structural optimization and investigation of the mechanisms of action.