Structure–Activity Relationship and Neuroprotective Activity of 1,5-Dihydro-2H-naphtho[1,2-b][1,4]diazepine-2,4(3H)-diones as P2X4 Receptor Antagonists

Abstract

We analyzed the P2X4 receptor structure-activity relationship of a known antagonist <b>5</b>, a 1,5-dihydro-2<i>H</i>-naphtho[1,2-<i>b</i>][1,4]diazepine-2,4(3<i>H</i>)-dione. Following extensive modification of the reported synthetic route, 4-pyridyl <b>21u</b> (MRS4719) and 6-methyl <b>22c</b> (MRS4596) analogues were most potent at human (h) P2X4R (IC₅₀ 0.503 and 1.38 μM, respectively, and selective versus hP2X1R, hP2X2/3R, hP2X3R). Thus, the naphthalene 6-, but not 7-position was amenable to substitution, and an <i>N</i>-phenyl ring aza-scan identified <b>21u</b> with 3-fold higher activity than <b>5</b>. Compounds <b>21u</b> and <b>22c</b> showed neuroprotective and learning- and memory-enhancing activities in a mouse middle cerebral artery occlusion (MCAO) model of ischemic stroke, with potency of <b>21u</b> > <b>22c</b>. <b>21u</b> dose-dependently reduced infarct volume and reduced brain atrophy at 3 and 35 days post-stroke, respectively. Relevant to clinical implication, <b>21u</b> also reduced ATP<b>-</b>induced [Ca²⁺]_i influx in primary human monocyte-derived macrophages. This study indicates the translational potential of P2X4R antagonists for treating ischemic stroke, including in aging populations.