Design, Synthesis, and Biological Evaluation of Heterocycle-Fused Celastrol Derivatives as Potent Antiosteoporosis Agents by Blocking RANKL-Induced Activation of the NF-κB and MAPK Signaling Pathways

Celastrol, an effective component of <i>Tripterygium wilfordii</i>, possesses remarkable antiosteoporosis activity, but its quinone-methyl substructure can induce severe toxicity. Herein, a series of heterocycle-fused Celastrol derivatives were designed and synthesized, and cell-based assays demonstrated that compound <b>19u</b>, containing a pyrazine-fused scaffold, exhibited significantly greater inhibitory activity and selectivity (IC₅₀ = 0.07 μM, SI = 82.57) against RANKL-induced osteoclastogenesis than those of Celastrol (IC₅₀ = 0.27 μM, SI = 3.93) but had no effect on osteoblast differentiation. Notably, mechanistic studies revealed that compound <b>19u</b> bound directly to RANKL and subsequently blocked the RANKL-induced activation of the NF-κB and MAPK pathways. Moreover, studies in zebrafish and ovariectomized mice osteoporosis models confirmed that compound <b>19u</b> significantly alleviated bone loss with a better safety profile compared to Celastrol. Collectively, these findings highlight the potential of the pyrazine-fused Celastrol scaffold in the discovery of small-molecule RANKL inhibitors, and compound <b>19u</b> warrants further investigation.