A Homodimer of Withaferin A Formed by Base-Promoted Elimination of Acetic Acid from 27‑O‑Acetylwithaferin A Followed by a Diels–Alder Reaction

Treatment of 27-O-acetylwithaferin A (2) with the non-nucleophilic base, 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), afforded 5β,6β-epoxy-4β-hydroxy-1-oxo-witha-2(3),23(24),25(27)-trienolide (3) and 4, a homodimer of withaferin A resulting from a Diels–Alder [4 + 2] type cycloaddition of the intermediate α,β-dimethylene-δ-lactone (9). Structures of 3 and 4 were elucidated using HRMS and 1D and 2D NMR spectroscopic data. The structure of 4 was also confirmed by single crystal X-ray crystallographic analysis of its bis-4-O-p-nitrobenzoate (8). Formation of withaferin A homodimer (4) as the major product suggests regio- and stereoselectivity of the Diels–Alder [4 + 2] cycloaddition reaction of 9. Acetylation of 2–4 afforded their acetyl derivatives 5–7, respectively. Compounds 2–4 and 6–8 were evaluated for their cytotoxic activities against four prostate cancer (PC) cell lines (LNCaP, 22Rv1, DU-145, and PC-3) and normal human foreskin fibroblast (HFF) cells. Significantly, 4 exhibited improved activity compared to the other compounds for most of the tested cell lines.