Design, synthesis and evaluation of novel nitroxide-gentiopicroside hybrids as anti-inflammatory agents with free radical scavenging activity

Abstract

Inflammatory processes are often accompanied by oxidative stress, with these two phenomena mutually reinforcing each other in a vicious cycle. Therefore, developing anti-inflammatory agents that combine with ROS scavenging is highly meaningful. In this study, 24 novel anti-inflammatory derivatives were designed and synthesized through the hybridization of the COX-2 inhibitor gentiopicroside (GPS) and the ROS scavenging nitroxide radical scaffold. The novel derivatives were subjected to in vitro evaluation of COX-2 inhibitory activity, alongside assessment of free radical scavenging activity using the DPPH assay. The most promising compound GPS-5 exhibited very potent COX-2 inhibitory activity (IC₅₀ = 0.42 μM) and free radical scavenging activity. Furthermore, in RAW 264.7 macrophages, pretreatment with the most promising compound GPS-5 significantly suppressed the LPS-induced production of nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and ROS. In addition, GPS-5 was able to attenuate LPS-induced cell polarization. Importantly, in the TPA-induced ear edema model, compound GPS-5 at 0.5 mg/ear achieved 74.5% inhibition, superior to celecoxib (67.8%). Molecular docking studies further showed that GPS-5 exhibits superior binding affinity to the COX-2 (ΔG_bind = -12.9 kcal/mol) active site compared to celecoxib. These findings establish compound GPS-5 as a promising candidate for the treatment of inflammatory diseases. Furthermore, this work provides a foundation for the development of next-generation anti-inflammatory therapeutics with enhanced potency.