N-butylphthalide (NBP) and ligustrazine (TMP) triazole hybrids target the KEAP1-NRF2 pathway to inhibit ferroptosis and exert brain neuroprotectivity.

Ischemic stroke is a life-threatening disease, its pathological progression involves multiple factors, including oxidative stress, apoptosis, and ferroptosis. Our previous study demonstrated that hybridizing N-butylphthalide (NBP) with ligustrazine (TMP) yielded promising anti-ischemic compounds. In this study, we further introduced a triazole structure into NBP-TMP hybrids and synthesized 20 novel compounds. Their neuroprotective activities were evaluated on OGD/R induced SH-SY5Y cells and primary hippocampal neurons, leading to the identification of preferred compounds 8a, 8b and 8d at the concentration of 6.25 μM, which surpassed the neuroprotective activity of the positive control NBP. Among them, 8a exhibited the highest protective activity, with a protection percentage of 75.6 %. Further mechanistic studies revealed that compounds 8a, 8b and 8d maintained intracellular redox homeostasis to resist oxidative stress and inhibit apoptosis in vitro. Specifically, compound 8a exerted neuroprotective effects by modulating the KEAP1-NRF2 pathway: it bound to KEAP1, enhanced NRF2 dissociation and nuclear translocation, facilitated the generation of downstream antioxidant factors, thereby reducing intracellular ‌reactive oxygen species (ROS) levels and effectively protecting neuronal mitochondria. Finally, in vivo experiments demonstrated that compound 8a (20 mg/kg) significantly ameliorated cerebral injury in rats with ischemia-reperfusion injury. Furthermore, it reduced cerebral oxidative stress by modulating the KEAP1-NRF2 pathway and inhibited neuronal apoptosis and ferroptosis in the brain, which is consistent with the results in vitro. In conclusion, our results indicated that 8a serves as a promising candidate for stroke treatment and may facilitate the development of future anti-ischemic drugs.