GNPS-guided isolation of anti-osteoporosis neoclerodane diterpenoids from Ajuga bracteosa.

Neo-Clerodane diterpenoids featuring an α, β-unsaturated-γ-lactone ring exhibit potent anti-osteoclastogenic activity. To find more compounds of this type from Ajuga bracteosa, LC-MS/MS analysis combination with Global Natural Products Social Molecular Networking (GNPS) were used to guide the isolation process. This approach led to the identification of seven previously undescribed neoclerodane diterpenoids (1-7), along with three known analogues (8-10). Their structures were elucidated through NMR spectroscopic analysis, ECD calculations, and X-ray crystallography. Fragmentation pathways of reference compounds 8 and 9 were also elucidated to aid structural interpretation. All isolated compounds were assayed for their anti-osteoporosis. Most compounds significantly inhibited RANKL-induced osteoclast formation, with IC50 values ranging from 1.73 to 27.48 μM. Primary structure-activity relationships (SARs) were subsequently established. These findings confirm neoclerodanes as promising lead compounds for the development of anti-osteoporosis agents and highlight that GNPS-guided isolation based on bioactivity is an efficient strategy for identifying active constituents in Herbal Medicine.