Evaluation of the Anti-Neuroinflammatory Activity of Theophylline Derivatives in Activated Microglia

Abstract ctive: Microglial activation-mediated neuroinflammation is a pivotal pathological hallmark of various neurological disorders. Although theophylline suppresses pro-inflammatory cytokine secretion, and 1,2,3-triazole scaffolds possess intrinsic anti-inflammatory properties, the potential of their hybrid architectures remains largely unexplored. This study evaluates the anti-neuroinflammatory efficacy of a novel series of theophylline-1,2,3-triazole derivatives. Methods: The target theophylline-1,2,3-triazole hybrids were synthesized via copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry. Their inhibitory effects on microglial inflammation and associated cytotoxicity were evaluated using Griess and MTT assays, respectively. Furthermore, real-time quantitative PCR (RT-qPCR) and Western blot analyses were employed to assess the expression of key inflammatory mediators in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Results and Discussion: Among the 16 synthesized derivatives, compound 2f exhibited the most potent inhibitory effect against microglial inflammation. At a concentration of 10 μM, compound 2f suppressed nitric oxide (NO) production to 29.18 ± 6.51% compared to the LPS-treated control, displaying an IC50 value of 2.31 ± 0.029 μM with no detectable cytotoxicity. Mechanistically, compound 2f significantly attenuated the expression of classical M1 phenotypic markers, including NO, IL-1β, IL-6, TNF-α, iNOS, and COX-2, in LPS-challenged microglia. Conclusions: Compound 2f effectively mitigates the inflammatory response in LPS-induced BV-2 microglial cells without affecting cell viability. These findings highlight the therapeutic potential of compound 2f in managing microglial hyperactivation and provide a structural blueprint for designing novel anti-neuroinflammatory agents.