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

We analyzed the P2X4 receptor structure-activity relationship of a known antagonist 5 , a 1,5-dihydro-2 H -naphtho[1,2- b ][1,4]diazepine-2,4(3 H )-dione. Following extensive modification of the reported synthetic route, 4-pyridyl 21u (MRS4719) and 6-methyl 22c (MRS4596) analogues were most potent at human (h) P2X4R (IC 50 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 N -phenyl ring aza-scan identified 21u with 3-fold higher activity than 5 . Compounds 21u and 22c showed neuroprotective and learning- and memory-enhancing activities in a mouse middle cerebral artery occlusion (MCAO) model of ischemic stroke, with potency of 21u > 22c . 21u dose-dependently reduced infarct volume and reduced brain atrophy at 3 and 35 days post-stroke, respectively. Relevant to clinical implication, 21u also reduced ATP - induced [Ca 2+ ] 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.