Discovery of\nthe First BRD4 Second Bromodomain (BD2)-Selective\nInhibitors

Pan-BD2 inhibitors have been shown to retain an antileukemia\neffect\nand display less dose-limiting toxicities than pan-BET inhibitors.\nHowever, it is necessary to consider the potential off-target toxicity\nassociated with the inhibition of four BET BD2 proteins. To date,\nno BRD4 BD2 domain selective inhibitor has been reported. Based on\nour previous pan-BD2 inhibitor <b>12</b> (XY153), we successfully\nidentified <b>16o</b> (XY221) as the first BRD4 BD2-selective\ninhibitor. <b>16o</b> demonstrated potent binding affinity for\nBRD4 BD2 (IC<sub>50</sub> = 5.8 nM), along with high pan-BD2 selectivity\n(667-fold over BRD4 BD1) and BRD4 BD2 domain selectivity (9–32-fold\nover BRD2/3/T BD2). The BRD4 BD2 selectivity of <b>16o</b> was\nfurther confirmed by the BLI assay, showing 66–144-fold selectivity\nover other BET BD2 domains. <b>16o</b> exhibited good liver\nmicrosomal stability (<i>T</i><sub>1/2</sub> > 120 min)\nand pharmacokinetic properties (<i>F</i> = 13.1%). These\ndata indicate that <b>16o</b> may serve as a valuable candidate\nfor BRD4 BD2 advancing epigenetic research.