Compound 38, a novel potent and selective antagonist of adenosine A_2A receptor, enhances arousal in mice

Adenosine A_2A receptor (A_2AR) plays a pivotal role in the regulation of sleep-wake behaviors. We previously reported an A_2AR selective antagonist compound 38 with an IC₅₀ value of 29.0 nM. In this study, we investigated its effect on sleep-wake regulation in mice. Wild-type (WT) mice were administered compound 38 (3.3, 5.0, 7.5, 15, 30 mg/kg, i.p.) at 9:00, and electroencephalography and electromyography were simultaneously recorded. We showed that administration of compound 38 exhibited a dose-dependent effect on wakefulness promotion. To investigate the impact of compound 38 on sleep rebound, we conducted a 6 h (13:00–19:00) sleep deprivation experiment. We found that administration of compound 38 (30 mg/kg) produced a wakefulness-promoting effect lasting for 1 h. Subsequently, we explored the critical role of A_2AR in the wakefulness-promoting effect of compound 38 using A_2AR knockout (KO) mice and their WT littermates. We found that compound 38 enhanced wakefulness in WT mice, but did not have an arousal-promoting effect in A_2AR KO mice, suggesting that the arousal-promoting effect of compound 38 was mediated by A_2AR. We conducted immunohistochemistry and selectively ablated A_2AR-positive neurons using cell type-specific caspase-3 expression, which revealed an essential role of A_2AR-positive neurons in the nucleus accumbens shell for the arousal-promoting effect of compound 38. In conclusion, as a novel A_2AR antagonist, compound 38 promotes wakefulness in mice via the A_2AR and exhibits promising applications for further advancements in the field of sleep–wake disorders.