of resveratrol. In this study, we also explore the antifungal activity against B. cinerea isolate T50 of stilbene derivatives having increasing number of methoxy groups since methoxy group substitution has a marked influence on biological properties [16– 18]. Density functional theory (DFT) calculations were used to analyze the fungitoxic structure-activity relationship of these compounds. The effect of the most active furyl resveratrol derivative on laccase production, oxygen consumption and membrane integrity was also evaluated. Results and Discussion Antifungal activity characterization Figure 1 shows the compounds used to determine antifungal activity against B. cinerea. Resveratrol and pterostilbene were used as controls. The effect of these compounds on in vitro mycelia growth in solid media was determined after 96 hours of incubation (Table 1). Compounds 1, 2, and 3 were more active than the control compounds and compound 3 with two methoxy groups had the highest antifungal activity. In addition, the biological effect of these compounds on the conidia germination of B. cinerea was evaluated after 7 h of incubation (Figure 2). At 100 mg ml21, pterostilbene completely inhibited conidia germination and the other compounds showed about 70% inhibition. At a lower concentration, 10 mg ml21, resveratrol stimulated germination, compounds 2 and 3 and pterostilbene showed about 50% inhibition and compound 1 showed about 20% inhibition. The Figure 1. Compounds used in this study. 1: (E)-3,4,5-trimethoxy-b- (2-furyl)-styrene, 2: (E)-4-methoxy-b-(2-furyl)-styrene, 3: (E)-3,5-di- methoxy-b-(2-furyl)-styrene. Resveratrol and pterostilbene were utilized as control. doi:10.1371/journal.pone.0025421.g001 Antifungal Resveratrol Derivatives title compounds did not produce morphological changes in the germ tube (data not shown). Since antifungal activity requires compounds 1, 2 and 3 to cross the fungal membrane it was expected that compound 1 with its three methoxy groups would be the most effective; in contrast, the most active species was the dimethoxy compound 3. However, under fungal attack, demethoxylation (or demethylation) of our compounds could produce hydroxyl formation. One mechanism to account for the lower activity of compound 1 is its incomplete demethoxylation and theoretical studies were performed to explore this hypothesis. Geometry optimizations of compounds 1, 2 and 3, and their population charges were calculated and analyzed (Figure 3). For the trimethoxy derivative 1 the highest positive charge (0.472) is located on position 3 of the aromatic ring (Figure 3A) suggesting this atom might be susceptible to a potential nucleophilic attack by a water molecule to generate an aromatic hydroxyl group [19–20]. Geometry optimization of the corresponding demethoxylated compound 1 derivative, (E)-4,5-dimethoxy,3-hydroxy-b-(2-furyl)- styrene, suggests further demethoxylation is more difficult (Figure 3A, right) since the atomic charge on the C-4 atom is much more negative (20.120), whereas C-5 has a smaller charge (0.315) than a C adjacent to O(furyl) (0.335). In addition, this derivative shows a stabilizing interaction between H(hydroxyl) and the adjacent methoxy group: the H(hydroxyl) points to the O(methoxy) in position 4, with H-bond distance H - - O = 2.079 A˚. Intramolecular hydrogen bonds are common and stabilizing features in polyhydroxy stilbenes; for instance, picea- tannol shows a H-bond distance of interaction slightly longer, 2.139 A˚ (Figure 3D). Moreover, in (E)-4,5-dimethoxy,3-hydroxy- b-(2-furyl)-styrene the hydroxyl O-H bond is 0.981 A˚ (Figure 3A right), slightly longer than the equivalent hydroxyl bond length in piceatannol of 0.974 A˚ (Figure 3D). Repeating this analysis for compound 3, with results shown in Figure 3B left, the C-3 atom of compound 3 has charge 0.655, and upon demethoxylation, the methoxy carbon at position C-5, retains its high charge (0.618) (Figure 3B right). Therefore, contrary to what happens to compound 1, the first demethoxylation step in compound 3 does not affect the second demethoxylation step in its derivative (E)-3- hydroxy-5-methoxy,b-(2-furyl)-styrene. To complete these theo- retical studies, we calculated charges on the monomethoxy compound 2, and noted the C-ipso associated with C(methoxy) charge is 0.491 (Figure 3C left), and similar to that for the C(methoxy) in position 3 of compound 1 (0.472), (Figure 3A, left). In addition, compound 2’s demethoxylated derivative, (E)-4- hydroxy,b-(2-furyl)-styrene has a shorter O-H bond distance, 0.973 A˚ (Figure 3C, right), than the O-H distance in (E)-4,5- dimethoxy,3-hydroxy-b-(2-furyl)-styrene, 0.981 A˚ (Figure 3A right), confirming that compound 1’s hydroxyl group establishes an intramolecular H-bond interaction with the adjacent methoxy group, thereby making further demethoxylation of compound 1 difficult. These studies correlate with and suggest an explanation for the weaker antifungal activity of compound 1 compared to compound 3. Experimentally, we demonstrated that the antifungal activity of resveratrol is markedly lower than that of compounds 1, 2 and 3, and this may be related to the higher polarity that impedes diffusion through fungal membranes. Table 1 confirms this correlation: compound 3 has a LogP value of 4.3, closely related to the other methoxylated compounds, whereas resveratrol has a lower LogP (3.0). Also pterostilbene shows a higher logP than resveratrol, as expected, but not as high as compound 3. PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e25421