Antifungal Resveratrol Derivatives Figure 4. Effect of compound 3 on laccase production by B. cinerea. Kinetics of laccase activity in absence (%) or presence (m) of 100 ug ml21 of compound 3, (A). Specific activity of laccase produced by B. cinerea after six days of incubation, (B). Different letters indicate that the means are significantly different at P,0.05. doi:10.1371/journal.pone.0025421.g004 its corresponding demethoxylated stilbene (resveratrol) does not. It was also shown that pterostilbene and 39-hydroxypterostilbene, the natural 3,5-dimethoxy analog of piceatannol, but not resveratrol and piceatannol, induce apoptosis in tumor cells [18] and that both compounds, were able to induce apoptosis, in the two Fas- ligand resistant lymphoma cell lines, HUT78B1 and HUT78B3, and the multi drug-resistant leukemia cell lines HL60-R and K562-ADR (a Bcr-Abl-expressing cell line resistant to imatinib mesylate) [17]. We conclude that compounds 1, 2 and 3 could have additional relevant biological properties, as recently indicated for A549 lung carcinoma [28], and inhibition of lipopolysaccha- ride-induced nitric oxide production [29], where, in contrast with our results, the trimethoxy derivative 1 was the most active. Phenolic compounds such as resveratrol induce laccase pro- duction in B. cinerea, and these enzymes participate in the oxidation of these compounds which aids in their biological activity [6]. Our studies demonstrate that non-phenolic compounds as compound 3 also increase laccase production. In conclusion, antifungal activity and target site on B. cinerea can be modulated through structural modifications of compounds. The two principal features that differ among the title compounds 1, 2 and 3 and resveratrol (and pterostilbene) are: increasing number of methoxy groups and the 2-furyl moiety. The presence of methoxy groups improves the antifungal activity of stilbene derivatives while the presence of furyl or phenyl groups determines the biological target on the fungus. Methoxylation is an important molecular feature for membrane penetration while subsequent demethoxyla- tion may be necessary to make hydroxyls available for increasing antifungal activity. This latter process seems to be more efficient with the dimethoxy species 3 than the trimethoxy derivative 1. The specific role of the 5-membered ring will be investigated synthesizing structurally modified 2-furyl derivatives that will be accompanied by antifungal tests. Materials and Methods Ethics Statement An ethics statement is not required for this work. Test compounds The compounds used in this study (Figure 1) were the natural compounds resveratrol and pterostilbene, (Sigma Chemical Co., St. Louis, MO, USA), and synthetic (E)-3,4,5-trimethoxy-b- (2- furyl)-styrene (1), (E)-4-methoxy-b-(2-furyl)-styrene (2) and (E)-3,5- PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e25421