Bis-Oxadiazole Assemblies as NO-Releasing Anticancer Agents

Background: Malignant pleural mesothelioma (MPM) is an aggressive, asbestos-associated cancer characterized by dysregulated nitric oxide (NO) signaling and increased NO levels that facilitate tumor progression. Paradoxically, this aberrant NO environment creates a therapeutic vulnerability that can be exploited by NO-donor prodrugs, which overwhelm cellular defenses with cytotoxic concentrations of NO, inducing nitrosative stress and apoptosis. Within this framework, oxadiazole-based scaffolds have emerged as a promising platform for prodrug development owing to their versatile chemistry and potential as novel NO donors or synergistic agents. In our previous studies, we developed several series of hybrid architectures incorporating 1,2,5-oxadiazole 2-oxide (furoxan) and 1,2,4-oxadiazole scaffolds, producing compounds with diverse and tunable NO-donor activities. We further observed that the cytotoxicity of these hybrids was significantly influenced by the substituents introduced at position 3 of the furoxan ring. Methods: We designed and synthesized a series of bis(1,2,4-oxadiazolyl)furoxans to systematically investigate their NO-donating capacity, cytotoxicity against MPM cell lines, selectivity over healthy lung fibroblasts, and underlying anticancer mechanisms. Results: The bis(1,2,4-oxadiazolyl)furoxans exhibited lower overall cytotoxicity but significantly higher selectivity compared with previously studied 3-cyano-4-(1,2,4-oxadiazolyl)furoxans. Their NO-releasing properties showed a strong correlation with their ability to induce mitochondrial damage, as evidenced by membrane depolarization. Moreover, the incorporation of specific substituents, such as a furan ring, on the 1,2,4-oxadiazole moiety introduced an additional mechanism of action through the induction of reactive oxygen species. Conclusions: Analysis of cancer cell death confirmed that these compounds acted through a multimodal mechanism dependent on both NO release and the specific substituents on the 1,2,4-oxadiazole moiety.