eliminated in vacuo. The obtained alcohol derivative, 1,2,3- trimethoxy-5-benzyl alcohol, (16.6 mmol) was dissolved in anhydrous CH2Cl2 and stirred at 0uC, then PBr3 (8.3 mmol) was added and the temperature raised to 25uC; after 30 min the alcohol was completely reacted as seen from TLC (n-hexane/ethyl acetate). Water was then added and ethyl ether (3630 ml) used for extraction. The organic phase was washed with a saturated solution of NaCl and dried with anhydrous Na2SO4. Upon ether elimination at low pressure, 15 mmol of the corresponding benzyl bromide were obtained. This was dissolved in toluene (150 ml) and 15 mmol di Ph3P were added with stirring. After refluxing for 12 h and TLC checking (n-hexane/ ethyl acetate 9:1), the obtained suspension was decanted at room temperature and filtered. The product was washed with cold toluene and 13.9 mmol of the phosphonium salt were obtained. This was dissolved in 100 ml of isopropyl alcohol and 28.2 mmol of LiOH?H2O were added. This solution was stirred and refluxed for 15 min. 2-Formyl-furane (13.9 mmol) was then added and refluxing performed until disappearance of the furyl aldehyde (TLC, n-hexane/ethyl acetate 9:1), achieved in 3 h. The solution was brought to 25uC and the solvent eliminated at low pressure. The product was dissolved in CHCl3 (10 ml) and the resulting solution introduced in a silica gel column (h = 10 cm, W = 4 cm) using n-hexane/ethyl acetate 10:1 as eluent. The obtained olefin (13.2 mmol) is a mixture of both diastereoisomers E/Z ca. 1:1. The cis-trans isomerization was performed by dissolving in CHCl3 and adding iodine (1.3 mmol) in 12 h, as followed with TLC. The organic solution was separated and treated first with a saturated solution of sodium thiosulphate and later with a saturated solution of NaCl. After drying with anhydrous Na2SO4, 11.8 mmol of the expected (E)-3,4,5- trimethoxy-b-(2- furyl)-styrene were obtained. Compounds 2 and 3 were synthesized in analogous manner, but using 1-methoxy-4- benzaldehyde (for compound 2) and 1,3-dimethoxy-5-benzalde- hyde (for compound 3), respectively, as starting material, instead of 1,2,3-trimethoxy-5-benzaldehyde for compound 1. The scheme synthesis of compound 1 is shown Figure S1, those of compounds 2 and 3 are in Figure S2. Spectroscopic data (E)-3,4,5-Trimethoxy-b-(2-furyl)-styrene (compound 1). 1H-NMR(CDCl3),d(ppm): 3.86 (s, 3H, OCH3); 3.91 (s, 6H, OCH3); 6.36 (d, 1H, J = 2.93 Hz, H2-furan); 6.45 (dd 1H J1 = J2 = 2.93 Hz, H3-furan); 6.70 (s, 2H, H2-H6-Ar); 6.79 (d, 1H, J = 16.8 Hz, Holefin); 6.97 (d, 1H, J = 16.8 Hz, H-olefin); 7.39 (bd, 1H, J = 2.93 Hz, H4- furan). 13C-NMR(CDCl3), d(ppm): 55.86 (m-CH3); 60.65 (p-CH3); 103.35 (C2-C6-Ar); 108.16 (C2-furan):111.45(C3-furan); 115.85 (Cb- olefin); 126.9 (C1-Ar) 132.5 (Ca-olefin); 137.88 (C4-Ar); 141.85 (C1- furan); 152.96 (C4-furan); 153.20 (C3-C5-Ar). IR (CHCl3, 1%), n(cm21): 1598; 1470; 1459; 1253; 1154. MS m/z: 260 (M+) [29,32– 33]. MP: 78.7–79.8uC. Yield, 75% (3.07 g), colorless. (E)-4-Methoxy-b-(2-furyl)-styrene (compound 2). 1H-NMR (CDCl3), d(ppm): 3.82 (s, 3H, OCH3); 6.29 (d, 1H, J = 2.93 Hz, H2- furan); 6.40 (d, 1H, J = 2.93 Hz, H3-furan); 6.75 (d, 1H, J = 16.8 Hz, H- olefin); 6.87 (bd, 2H, J = 8.8 Hz, H3-H5-Ar); 6.98 (d, 1H, J = 16.8 Hz, H-olefin); 7.39 (bd, 2H, J = 8.8 Hz, H2-H5-Ar). 7.40 (d, 1H, J = 2.93 Hz, H4-furan). 13CNMR(CDCl3), d(ppm): 55.16 (CH3); 107.57 (C2-furan); 111.48 (C3-furan); 114.15 (C3-C5, Ar); 126.84 (Cb- olefin); 127.5 (C2-C6-Ar); 128.12 (C1-Ar); 129.83 (Ca-olefin); 141.67 (C1- furan); 153.6 (C4-furan); 159.33 (C4-Ar). IR (CHCl3, 1%), n(cm21): 1642; 1560. MS m/z: 200 (M+) [34–40]. MP: 73.7–74.3uC [34,37]. Yield, 72% (2.64 g), colorless. (E)-3,5-Dimethoxy-b-(2-furyl)- styrene (compound 3). 1HNMR(CDCl3), d(ppm): 3.82 (s, 6H, OCH3); 6.37 (d, 1H, Antifungal Resveratrol Derivatives J = 2.93 Hz, H2-furan); 6.38 (d, 1H,J = 2.19 Hz, H4-Ar); 6.42 (dd 1H J1 = J2 = 2.93 Hz, H3-furan); 6.61 (d, 2H, J = 2.19 Hz, H2-H6- Ar); 6.84 (d, 1H, J = 16.8 Hz, H-olefin); 6.95 (d, 1H, J = 16.8 Hz, H-olefin); 7.40 (bd, 1H, J = 2.93 Hz, H4-furan) 13C- NMR(CDCl3), d(ppm): 54.8 (CH3); 99.8 (C4-Ar). 104.1 (C2-C6- Ar); 110.1 (C2-furan); 111.4 (C3-furan); 116.7 (Ca-olefin); 126.9 (Cb-olefin); 138.9(C1-Ar). 141.9 (C1-furan); 153.6 (C4-furan); 160.8 (C3-C5-Ar). IR (CHCl3, 1%), n(cm21): 1602; 1470; 1430; 1200; 1150. MS m/z: 230 (M+) [29,32,38,41–43]. MP: 105.9– 107.7uC. Yield, 70% (2.88 g), colorless. The H-NMR of compounds 1, 2 and 3, are depicted in Figures S3, S4 and S5. The C-NMR of compound 2 is shown in Figure S6 and that of compound 3 in Figure S7. Fungal isolate and culture condition In this study, isolate T50 of B. cinerea was used. This was originally isolated from a naturally infected tomato (Solanum lycopersicum cv. Roma) [44] and was maintained on malt-yeast extract agar slants [2% (w/v) malt extract, 0.2% (w/v) yeast extract and 1.5% (w/v) agar] at 4uC. The fungus was grown in the dark on malt-yeast extract agar medium [2% (w/v) malt extract, 0.2% (w/v) yeast extract and 1.5% (w/v) agar) or soft agar (2% (w/v) malt extract, 0.2% (w/v) yeast extract and 0.6% (w/v) agar]. In the mechanism of action analysis, liquid minimum medium [KH2PO4 (1 g l21), K2HPO4 (0.5 g l21), MgSO4?7H2O (0.5 g l21), KCl (0.5 g l21), FeSO4?7H2O (0.01 g l21)] pH 6.5, 25 mol l21 ammonium tartrate as a nitrogen source, and 1% (w/ v) glucose as carbon source were used. Effect of the compounds on the mycelial growth of B. cinerea on solid media The fungitoxicity of the compounds was assessed using the radial growth test on malt-yeast extract agar [45]. Test compounds were dissolved in methanol-DMSO (24:1) and added at final concentrations of 10, 20, 40, 80 or 100 mg ml21. The final methanol-DMSO concentration was identical in control and treatment assays. Mycelial growth diameters were measured daily. After 96 hours of incubation the inhibition percentages relative to the control with methanol-DMSO were calculated. Results were expressed as effective concentration (ED50) that reduced mycelial growth by 50%, determined by regressing the inhibition of radial growth values (percent control) against the values of compound concentration. These experiments were done in triplicate. Effect of the compounds on germination of B. cinerea conidia Conidial germination assays were carried out on microscope slides coated with soft agar medium (2 mm thickness). The added compounds were dissolved in methanol-DMSO (24:1) at a final concentration of 10 or 100 mg ml21. The slides were inoculated with dry conidia obtained from sporulated mycelia (1 week old), placed in a humid chamber (90% relative humidity), and incubated in the dark at 22uC for 7 h. Control contained methanol-DMSO at the same concentration as treatments. Conidial germination was determined directly on the slides at intervals of 1 h. The percentage of germination was estimated by counting the number of germinated conidia in five microscope fields each containing approximately 40 conidia. Conidia were considered germinated when the germ tube length was equal to or greater than conidial diameter. These experiments were done in triplicate. PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e25421