A series of novel C-28 amino acid/amide conjugates and oligo(ethylene glycol)-modified C-2, C-3 pyrazole-fused betulinic acid derivatives were designed, synthesized, and evaluated for their inhibitory effects on RANKL-induced osteoclastogenesis. Among these, compound 18, bearing a C-28 oligo(ethylene glycol) amino amide ester linkage, exhibited the most potent inhibitory activity. It demonstrated an IC50 of 7.96 nM against RANKL-induced osteoclastogenesis in RAW264.7 cells, representing a 10-fold increase in potency compared to the XJ13 (IC50 = 0.08 μM) and achieved >50 % inhibition at 0.01 μM. Importantly, the inhibitory effects of these compounds on RANKL-induced osteoclast differentiation were not attributed to cytotoxicity, as evidenced by the minimal cytotoxicity of compound 18 at 10 μM. Mechanistic studies showed that compound 18 could dose-dependently suppress the expression of osteoclast marker genes (TRAP, CTSK) and proteins (c-Fos, MMP-9). Furthermore, in an ovariectomized (OVX) mouse model, compound 18 (10 and 20 mg/kg, intraperitoneally, i.p.) dose - dependently prevented bone loss by improving key micro-CT parameters and decreasing serum bone resorption markers (CTx). Overall, compound 18 emerged as a highly promising candidate for the treatment of RANKL-driven osteoporosis.
