Synthesis of sulfanyl derivatives of 1,2,4-triazoles via an acid catalyzed intramolecular cyclization of isothiosemicarbazones: structural characterization, E/Z isomerism, mechanistic insights and in vitro cytotoxicity

Abstract

Sulfanyl derivatives of 1,2,4-triazoles (CL1–CL3) were synthesized via a Lewis or Brønsted acid catalyzed intramolecular cyclization of corresponding isothiosemicarbazones (TL1–TL3). All the synthesized isothiosemicarbazones and their cyclized sulfanyl 1,2,4-triazole derivatives were well characterized by spectroscopic techniques and single crystal XRD analyses. The E/Z isomerism of the isothiosemicarbazones was elucidated using NMR spectroscopy and single crystal XRD analysis. Mechanistic investigations, supported by controlled experiments and spectroscopic evidence, revealed that the cyclization proceeded via an ionic pathway with the evolution of hydrogen. The cytotoxic effect of the cyclized sulfanyl 1,2,4-triazole derivatives (CL1–CL3) was evaluated by MTT assay against MDA-MB-231 (breast), MCF-7 (breast), and HeLa (cervical) cancer cell lines, as well as HEK-293 (kidney) normal cell line, taking 5-fluorouracil (5-FU) as a reference drug. All the substituted sulfanyl-1,2,4-triazoles showed higher cytotoxicity toward MDA-MB-231 and HeLa cells than 5-FU, while exhibiting low toxicity toward HEK-293 cells, indicating good selectivity toward cancer cells. Substituted compounds [CL2 (p-OCH₃) and CL3 (p-NO₂)] displayed enhanced activity compared to the unsubstituted one (CL1), indicating the influence of the substituents on cytotoxicity. Fluorescence staining assays (AO/EB, Hoechst 33342, Rhodamine 123 and DCFH-DA) further supported the observed cytotoxic effects, and suggested that the compounds promoted apoptotic cell death via intracellular reactive oxygen species (ROS) generation, depletion of mitochondrial membrane potential (MMP) and damage to nuclear material.