C-4-functionalized cyclogeraniols from saffron: Structural diversity, Enantiomerism, and stereochemistry-dependent anti-melanogenic activity

Crocus sativus L. (saffron) is known for its depigmenting and anti-melanogenic properties, yet the chemical diversity and bioactivity of its cyclogeraniol-derived (CGLs) minor metabolites remain insufficiently explored. In this study, phytochemical investigation of saffron stigmas led to the isolation of a series of C-4-functionalized cyclogeraniols, including six previously undescribed compounds, crosativoids A-F (1-6). Their structures and absolute configurations were elucidated by comprehensive spectroscopic analysis, single-crystal X-ray diffraction, and ECD calculations. These metabolites represent three distinct C-4 linkage patterns, namely CC, CN, and CO linkages. Among them, CGL 1 features a rare C-4C-C bond and an unprecedented 6/5/6/6 polycyclic framework, whereas CGLs (±)-2 and (±)-3 are cyclogeraniol hybrids bearing uracil and hypoxanthine-derived moieties, respectively. Chiral analysis of the CN and CO derivatives revealed intrinsic enantiomerism and suggested preferential formation of the C-4R configuration in saffron-derived cyclogeraniols. A plausible biosynthetic pathway involving C-4-selective nucleophilic addition was proposed. Biological evaluation showed that the 4R (-)-enantiomer of CGL 2 inhibited melanin accumulation in B16F10 cells and downregulated the MITF/TYR axis, with an SPR-derived affinity value of 455 nM toward tyrosinase. These findings expand the structural and stereochemical diversity of saffron cyclogeraniols and identify C-4-functionalized derivatives as promising anti-melanogenic candidates.