Discovery of 5-(2',4'-difluorophenyl)-salicylanilides as new inhibitors of receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis.

To improve the inhibitory potency of lead compound NDMC101 on RANKL-induced osteoclastogenesis, a series of new 5-(2',4'-difluorophenyl)-salicylanilide derivatives were synthesized and evaluated for osteoclast inhibition by using TRAP-staining assay. Among them, both of compounds 6d and 6i showed three-fold increase in osteoclast-inhibitory activities compared to NDMC101 at half-inhibitory concentration. Further, the mechanistic study showed that 6d and 6i could suppress RANKL-induced osteoclastogenesis-related genes, such as NFATc1, c-fos, TRAP, and cathepsin K. Their inhibitory activities were further confirmed by including specific inhibition of NF-κB and NFATc1 expression levels in nucleus. In addition, 6d and 6i also could significantly attenuate bone-resorbing activity of osteoclasts by performing pit formation assay. Thus, a new class of 5-(2',4'-difluorophenyl)-salicylanilide derivatives may be considered as essential lead structures for the further development of anti-resorptive agents.