Design and Synthesis of 9α-Hydroxyandrost-4-ene-3,17-dione Derivatives as Novel Potent Antiosteoporosis Agents

A series of novel D-ring-fused heterocyclic 9α-hydroxy-4-androstene-3,17-dione (9α-OH-AD) derivatives were synthesized and evaluated for inhibition of RANKL-induced osteoclastogenesis. Among them, the most potent compound 38 (SH494) exhibited an IC₅₀ value of 8.4 nM, representing an approximately 1,110-fold increase in potency compared with the hit compound 9α-OH-AD (IC₅₀ = 9.33 μM). Mechanistic studies revealed that SH494 markedly downregulated the expression of key osteoclastogenic markers at both the transcriptional (Nfatc1, Trap, Ctsk, and Mmp9) and protein (c-Fos, Ctsk, and Mmp9) levels. SH494 markedly inhibited RANKL-induced phosphorylation of p38. Furthermore, SH494 activated the Nrf2 signaling pathway, upregulating the Cat and Gclc genes at the mRNA level and the HO-1 enzyme at the protein level. This reduced intracellular ROS accumulation and normalized mitochondrial membrane potential (ΔΨm). In vivo, SH494 markedly reduced ovariectomy-induced osteoclast activity and alleviated osteoporosis. Therefore, SH494 could serve as a promising lead for the development of novel antiosteoporotic agents.