Synthesis and anticancer evaluation of novel thioimidazole derivatives bearing a trimethoxyphenyl moiety

Nitrogen-containing heterocycles remain central to drug discovery, with imidazole standing out for its versatility and biological relevance. Sulfur substitution yields thioimidazoles, which offer altered electronic properties and pharmacokinetics. In parallel, the 3,4,5-trimethoxyphenyl (TMP) group is recognized as a privileged pharmacophore in oncology, anchoring ligands within protein binding pockets and contributing to multi-target activity. Here, we combined these motifs to synthesize a series of thioimidazole–TMP conjugates. Their activity was tested across human cancer cell models representing distinct tissue origins, alongside non-malignant lung fibroblasts. Among the library, compound 13b emerged as a lead, showing sub-micromolar potency in the most sensitive cancer cells while remaining inactive in fibroblasts, yielding a selectivity index above 20. Mechanistic studies demonstrated that 13b induces a canonical apoptotic program: Annexin V positivity, caspase-3/7 activation, and γH2AX foci consistent with DNA damage. Crucially, these effects extended beyond two-dimensional assays. In three-dimensional spheroid models, which better mimic tumor architecture, 13b penetrated compact structures, suppressed growth, and induced apoptosis in a concentration-dependent manner. Together, these findings indicate that merging thioimidazole and TMP frameworks yields a hybrid scaffold with strong promise in anticancer research. Compound 13b therefore represents a promising candidate for further mechanistic investigation and optimization toward improved drug-like properties.