Discovery of Diphenyl Ether Derivatives as Novel BK_Ca Channel Activators: Structure-Activity Relationship, Cryo-EM Complex Structures, and <i>In Vivo</i> Animal Studies

The BK_Ca channel, a large-conductance calcium-activated potassium channel, plays a crucial role in maintaining the homeostasis of the micturition cycle and airway-related functions. In this study, we optimized a novel BK_Ca channel activator, <b>4d</b>, with a diphenyl ether structure identified from library screening. This led to the discovery of potent activators, <b>10b</b> (EC₅₀ = 0.12 μM, cell-based assay) and <b>51b</b>, an orally bioavailable derivative. Compound <b>10b</b> demonstrated potent <i>in vivo</i> efficacy in a spontaneous hypertensive rat (SHR) of urinary incontinence model, while compound <b>51b</b> showed dose-dependent cough suppression efficacy with an ED₅₀ of 11.8 mg/kg in a citric acid-induced cough model. Furthermore, we reported the cryo-electron microscopy (cryo-EM) structures of the BK_Ca channel in complex with <b>10b</b> and <b>51b</b> at resolutions of 2.8 and 3.4 Å. Based on structural analyses, we determined the binding sites and key interaction residues of <b>51b</b>, which were validated via mutation studies.