2-Methyl-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-5-imine, an edaravone analog, exerts neuroprotective effects against acute ischemic injury via inhibiting oxidative stress

Oxidative stress plays an indispensable role in the pathogenesis of cerebral ischemia. Inhibiting oxidative stress has been considered as an effective approach for stroke treatment. Edaravone, a free radical scavenger, has been shown to prevent cerebral ischemic injury. However, the clinical efficacy of edaravone is limited because it has a low scavenging activity for superoxide anions (O2$–). Here, we report that 2-methyl-5H-benzo[d]pyrazolo[5,1-b][1,3] oxazin-5-imine, a novel small-molecule compound structurally related to edaravone, showed a stronger inhibitory effect on oxidative stress in vitro. In vivo, 2-methyl-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-5-imine reversed transient middle cerebral artery occlusion-induced dysfunctions of superoxide dismutases and malondialdehyde, two proteins crucial for oxidative stress,suggesting a strengthened antioxidant system. Moreover, 2-methyl-5H-benzo[d] pyrazolo[5,1-b][1,3]oxazin-5-imine decreased blood brain barrier permeability. Then, we found that 2-methyl-5Hbenzo[d]pyrazolo[5,1-b][1,3]oxazin-5-imine had a stronger neuroprotective effect than edaravone. More importantly, 2-methyl-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-5-imine decreased not only infarct size and neurological deficits in the acute phase but also modified neurological severity score and escape latency in Morris water maze task in the delayed period, indicating enhanced neuroprotection, sensorimotor function and spatial memory. Together, thesevfindings suggest that 2-methyl-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-5-imine could be a preferable option for stroke treatment.