Stereodivergent double Thia-Michael diversification of parthenin enables reduced cytotoxicity with retained anti-inflammatory activity.

Parthenin, a sesquiterpene lactone bearing dual Michael acceptor motifs, was employed as a reactive natural product scaffold for stereodivergent diversification via a mild base-promoted, one-pot double Thia-Michael addition strategy, representing the first systematic report of dual thiol functionalization on this scaffold. Reactions with aliphatic and aromatic thiols afforded fifteen sulfur-functionalized derivatives under mild, room-temperature conditions with distinct substrate-dependent stereochemical outcomes. Aliphatic thiols consistently produced single diastereomers, whereas aromatic thiols yielded separable α/β mixtures arising from stereochemical divergence at C-3. In all cases, the C-7 thiol substituent and adjacent C-6a hydroxyl group adopt a conserved syn orientation (α-face), as confirmed by detailed 1D/2D NMR analysis and single-crystal X-ray diffraction. Biological evaluation in LPS-stimulated RAW 264.7 macrophages revealed that thiol modification significantly reduces the intrinsic cytotoxicity of parthenin under the tested cellular conditions while retaining anti-inflammatory activity. Among the derivatives, compound 2a showed high cell viability (92 ± 5.42% at 10 μM), potent nitric oxide inhibition (63 ± 4.57%, EC₅₀ = 7.35 μM), and low cytotoxicity in RAW 264.7 macrophages (IC₅₀ = 35.36 μM), along with concentration-dependent suppression of IL-6 and TNF-α. Molecular docking suggested favorable binding compatibility of 2a within the iNOS active site, with additional interactions predicted for IKK2. ADMET analysis indicated favorable drug-like properties and elimination of predicted mutagenicity relative to parthenin. Overall, this strategy enables controlled modulation of electrophilicity to generate potentially safer anti-inflammatory analogues.