4-Phenyl-3-[2-(phenylamino)ethyl]-1H-1,2,4-triazole-5(4H)-thione was used as a starting compound for the synthesis of the corresponding 1,2,4-triazol-3-ylthioacetohydrazide, which reacts with isatins and various aldehydes bearing aromatic and heterocyclic moieties provided target hydrazones. Their cytotoxicity was tested by the MTT assay against human melanoma IGR39, human triple-negative breast cancer (MDA-MB-231), and pancreatic carcinoma (Panc-1) cell lines. The selectivity of compounds towards cancer cells was also studied. In general, the synthesized compounds were more cytotoxic against the melanoma cell line. N′-(2-oxoindolin-3-ylidene)-2-((4-phenyl-5-(2-(phenylamino)ethyl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide, N′-((1H-pyrrol-2-yl)methylene)-2-((4-phenyl-5-(2-(phenylamino)ethyl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide and N′-(2-hydroxy-5-nitrobenzylidene)-2-((4-phenyl-5-(2-(phenylamino)ethyl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide were identified as the most active among all synthesized compounds in 3D cell cultures. N′-(4-(dimethylamino)benzylidene)-2-((4-phenyl-5-(2-(phenylamino)ethyl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide inhibited all cancer cell migration, was characterized as relatively more selective towards cancer cells, and could be further tested as an antimetastatic candidate.

A series of target 4-substituted-5-(2-(pyridine-2-ylamino)ethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thiones and their chloro analogs 7–21 were synthesized in a reaction of the selected aldehydes with the corresponding 4-amino-1,2,4-triazole-3-thiones 5 and 6, which were obtained from 3-(pyridin-2-ylamino)propanoic acid (3) or 3-((5-chloropyridin-2-yl)amino)propanoic acid (4), respectively, with thioacetohydrazide. The antibacterial and antifungal activities of the synthesized hydrazones were screened against the bacteria Escherichia coli, Staphylococcus aureus, and Mycobacterium luteum and the fungi Candida tenuis and Aspergillus niger by agar diffusion and serial dilution methods. 4-Amino-5-(2-((5-chloropyridin-2-yl)amino)ethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (6) and 4-(benzylideneamino)-5-(2-(pyridin-2-ylamino)ethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (7) were identified as exceptionally active (MIC 0.9 µg/mL) against the fungus C. tenuis. 5-Chloropyridine derivatives bearing 4-benzylidene 8, 2-nitrobenzylidene 10, pyridinylmethylene 16, and 4-methylthiobenzylidene 21 moieties showed very high antibacterial activity (MIC 3.9 µg/mL) against the M. luteum strain. The cell viability screening of the synthesized compounds using triple-negative breast cancer MDA-MB-231 and glioblastoma U-87 cell lines by MTT assay identified three active hydrazones, of which 5-(2-(pyridin-2-ylamino)ethyl)-4-((pyridin-3-ylmethylene)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (15) had the highest effect on the viability of cells (IC₅₀ value 39.2 ± 1.7 μM against MDA-MD-231). The in silico molecular modeling results suggested that these three most active hydrazones might have influenced the mitogen-activated protein kinase pathway through the inhibition of BRAF and MEK serine–threonine protein kinases. 5-(2-((5-Chloropyridin-2-yl)amino)ethyl)-4-((4-(methylthio)benzylidene)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (21) demonstrated the highest affinity among them.

A synthesis of 2-((4-phenyl-5-(2-(p-tolylamino)ethyl)-4H-1,2,4-triazol-3-yl)thio)-N’-(1-phenylethylidene)acetohydrazide from 2-[(3-{2-[(4-methylphenyl)amino]ethyl}-4-phenyl-4,5-dihydro-1H-1,2,4-triazol-5-yl)sulfanyl]acetohydrazide and acetophenone is reported. The title compound has been tested to possess 1.5-fold higher antioxidant ability than the control, butylated hydroxytoluene, as determined by a Ferric reducing antioxidant power assay.