Design and optimization of 5-hydroxy-4H-benzo[1,4]oxazin-3-one-derived β2-adrenoceptor agonists with potent anti-asthmatic activity.

β2-Adrenergic receptor (β2-AR) agonists, as classic bronchodilators, serve as the cornerstone for asthma treatment. Herein, we designed and synthesized a series of derivatives based on the 5-hydroxy-4H-benzo[1,4]oxazin-3-one scaffold. Based on a comprehensive evaluation of four core indicators, including cAMP accumulation assays in HEK293 cells overexpressing human β2-AR or β1-AR, guinea pig tracheal strip assays, and in vitro cytotoxicity, compound C3 exhibited the best overall performance (β2-AR EC50 = 7 pM, β2/β1 selectivity > 97-fold; isolated tracheal relaxation Emax = 136.88, pD2 = 8.32; human bronchial epithelial cell BEAS-2B CC50 = 50.07 μM) and was selected as the candidate molecule. In a mouse asthma model, C3 administered at 200 μg/kg/day (458.5 nmol/kg/day) markedly suppressed inflammatory cell activity and improved lung function, showing anti-asthmatic efficacy comparable to that of salmeterol at 300 μg/kg/day (721.7 nmol/kg/day). In conclusion, C3 is a β2-AR agonist with potential application value, holding promise for the treatment of asthma.