Antifouling and Fungicidal Resorcylic Acid ... - ACS Publications

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Antifouling and Fungicidal Resorcylic Acid Lactones from the Sea Anemone-Derived Fungus Cochliobolus lunatus Qing-Ai Liu,† Chang-Lun Shao,† Yu-Cheng Gu,‡ Mathias Blum,‡ Li-She Gan,§ Kai-Ling Wang,† Min Chen,† and Chang-Yun Wang*,† †

Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ Jealott’s Hill International Research Centre, Syngenta, Bracknell, Berkshire, RG42 6EY, United Kingdom § Institute of Modern Chinese Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China S Supporting Information *

ABSTRACT: Three new 14-membered resorcylic acid lactones, cochliomycins D−F, 1−3, and eight known analogues, 4−11, were isolated from the sea anemone-derived fungus Cochliobolus lunatus. Compounds 1−4 are diastereomers differing from each other by the absolute configurations of the 4′,5′-diol chiral centers. The absolute configurations of 1−4 were established by the CD exciton chirality method and TDDFT ECD calculations. In antifouling assays, 1, 3−6, and 6a exhibited potent antifouling activities against the larval settlement of the barnacle Balanus amphitrite at nontoxic concentrations, with EC50 values ranging from 1.82 to 22.5 μg/mL. Noticeably, fungicide whole-plant assays indicated that 6 showed excellent activity on the Plasmopara viticola preventative test at 6 ppm and concentration-dependent activity on the Phytophthora infestans preventative application at 200, 60, and 20 ppm. Preliminary structure−activity relationships are also discussed. KEYWORDS: Cochliobolus lunatus, resorcylic acid lactone, antifouling activity, fungicidal activity, whole-plant assay

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°C. UV spectra were recorded on a Unico UV/vis 2802 PCS spectrophotometer (Unico Instrument Co., Ltd., Shanghai, China). CD spectra were recorded on a JASCO J-810 circular dichroism spectrometer (Jasco, Tokyo, Japan). IR spectra were recorded on a Nicolet Nexus 470 spectrometer (Thermo Nicolet Corporation, Madison, WI, USA) using KBr pellets. NMR spectra were recorded at 400 MHz for 1H and 100 MHz for 13C on a Bruker AVANCE 400 spectrometer (Bruker, Kalsruhe, Germany) and at 500 MHz for 1H and 125 MHz for 13C on an Agilent DD2 NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA). Chemical shifts (δ) are reported in parts per million (ppm), using tetramethylsilane (TMS) as the internal standard; coupling constants (J) are in hertz (Hz). Electrospray ionization mass spectrometry (ESIMS) and highresolution electrospray ionization mass spectrometry (HRESIMS) spectra were obtained on a Micromass Q-TOF spectrometer (Waters, Manchester, U.K.). High-performance liquid chromatography (HPLC) separation was performed using a Hitachi L-2000 prep-HPLC system coupled with a Hitachi L-2455 photodiode array detector (Hitachi, Tokyo, Japan). The HPLC column used was a 150 mm × 7.8 mm i.d., 7 μm, Kromasil C18 column (Akzo Nobel, Amsterdam, Holland), with a 10 mm × 4.6 mm i.d. guard column of the same material (Dalian Elite Analytical Instruments Co., Ltd., Dalian, China). Silica gel (100− 200 and 200−300 mesh) (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex LH-20 (Amersham Biosciences Inc., Piscataway, NJ, USA), and octadecylsilyl silica gel (45−60 μm) (Merck KGaA, Darmstadt, Germany) were used for column chromatography. TLC silica gel GF254 plates (Yantai Zi Fu Chemical

INTRODUCTION 14-Membered resorcylic acid lactones, with a variety of biological activities, such as antifungal,1 antimalarial,2,3 and nematicidal4 properties, have raised continuing interest in this family of natural products from researchers. In our previous study, we obtained seven resorcylic acid lactones from the fungus Cochliobolus lunatus (M351) derived from the gorgonian Dichotella gemmacea collected in the South China Sea.5 The potent antifouling activities of these compounds merited further investigation as models for the discovery of new antifouling molecules. However, further research was hindered by strain-specific variations regarding the quantity of metabolite production as well as the seemingly capricious behavior of this fungal strain, especially in altering metabolite profiles when recultured. Consequently, the marine fungi library in our laboratory were further examined with the expectation of identifying another metabolically stable strain with the ability to produce resorcylic acid lactones. Fortunately, C. lunatus (TA2646), another fungal strain isolated from the sea anemone Palythoa haddoni, also collected in the South China Sea, was found to produce resorcylic acid lactones. Herein, we report the isolation and structure elucidation of the resorcylic acid lactones from C. lunatus (TA26-46), as well as the biological evaluation of all of the isolated compounds and their derivatives. In addition, the potential of resorcylic acid lactones as agricultural fungicides is discussed.

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Received: Revised: Accepted: Published:

MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were measured on a WZ-1 polarimeter (Shimadzu, Tokyo, Japan) at 25 © 2014 American Chemical Society

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January 7, 2014 March 17, 2014 March 17, 2014 March 17, 2014 dx.doi.org/10.1021/jf500248z | J. Agric. Food Chem. 2014, 62, 3183−3191

Journal of Agricultural and Food Chemistry

Article

Figure 1. Structures of resorcylic acid lactones 1−11 isolated from C. lunatus (TA26-46). Cochliomycin D (1). White amorphous powder; [α]25D −125 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 198 (3.27), 232 (4.30), 271 (3.88), 312 (3.62) nm; CD (1.38 mM, MeOH) λmax (Δε) 219 (+8.07), 246 (+10.95), 273 (−14.44), 310 (−3.85), 351 (+0.31) nm; IR (KBr) νmax 3747, 3648, 1700, 1650, 1540, 1510 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 385.1 [M + Na]+, 747.2 [2M + Na]+; HRESIMS m/z 385.1266 [M + Na]+ (calcd for C19H22O7Na, 385.1258).

Co., Ltd., Yantai, China) were used for thin layer chromatography (TLC). Fungal Strain. The fungal strain Cochliobolus lunatus (TA26-46) was isolated from a piece of fresh tissue from the inner part of the sea anemone Palythoa haddoni, collected from the Weizhou coral reef (21° 02′ N, 109° 06′ E) in the South China Sea in April 2010. The fungus was identified as C. lunatus according to its morphological traits and a molecular protocol by amplification and sequencing of the DNA sequences of the ITS region of the rRNA gene. The 545 base pair ITS sequence had 100% sequence identity to that of C. lunatus (JN943462). The strain was deposited in the Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, P.R. China, with the GenBank (NCBI) access number JF819163. Fermentation, Extraction, and Isolation. The fungal strain C. lunatus (TA26-46) was fermented on solid media in 100 1-L Erlenmeyer flasks (each containing 80 g of rice, 120 mL of water, and 3.6 g of sea salt) at 28 °C for 4 weeks. The fermented solid medium was extracted three times with 300 mL of EtOAc for each Erlenmeyer flask. The combined EtOAc solution was evaporated to dryness under a vacuum to give an EtOAc extract. The EtOAc extract (80.0 g) was subjected to vacuum liquid chromatography (60.0 cm × 8.0 cm i.d.) on silica gel (100−200 mesh) using step-gradient elution with 5 L of petroleum ether/EtOAc (20:1 to 0:1, v/v) to afford five fractions (fractions 1−5). Fraction 4 was purified repeatedly by column chromatography (60.0 cm × 3.0 cm i.d.) on silica gel (200− 300 mesh) eluted with 3 L of EtOAc/MeOH (v/v, 10:1) to yield 7 (2.0 g). Fraction 3 was isolated by column chromatography (60.0 cm × 3.0 cm i.d.) on silica gel (200−300 mesh) eluted with 3 L of petroleum ether/EtOAc (10:1 to 7:3, v/v), then subjected to Sephadex LH-20 column chromatography (120.0 cm × 3.0 cm i.d.) with 1 L petroleum ether/CH2Cl2/MeOH (2:1:1, v/v/v), and further purified by using semipreparative HPLC on an octadecylsilyl (ODS) column (150 mm × 7.8 mm i.d., 7 μm, Kromasil C18). The dried eluate was dissolved in 5 mL of MeOH and microfiltered. A 100-μL injection volume was used at a flow rate of 2 mL/min and elution of MeOH/ water (11:9, v/v) to give 1 (15.0 mg), 2 (1.3 mg), 3 (2.3 mg), 4 (55.5 mg), 5 (145.5 mg), 6 (250.0 mg), 7 (5.0 mg), 8 (20.3 mg), 9 (1.5 mg), 10 (25.0 mg), and 11 (1.2 mg). The structures of the isolated compounds are depicted in Figure 1.

Table 1. 1H NMRa Data for 1−4 in DMSO-d6 position

6.35, d (2.4) 6.48, d (2.4) 6.42, d (15.9)

2′

4′ 5′

6.18, ddd (15.9, 7.8, 6.7) 2.31, m 2.20, m 3.79, m 4.25, t (5.9)

7′

6.21, d (15.9)

8′

6.82, ddd (15.9, 9.8, 5.2) 2.67, ddd (14.4, 5.2, 2.1) 2.40, ddd (14.4, 9.8, 7.0) 5.33, m 1.32, d (6.3) 10.46, s 4.94, d (5.6) 5.18, d (5.9) 3.75, s

3′

9′

10′ 11′ 2-OH 4′-OH 5′-OH 4-OCH3 a

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1

3 5 1′

2 6.31, brs 6.55, brs 6.21, d (15.9) 6.33, m 2.45, 2.13, 3.86, 4.27,

m m m t (5.6)

6.13, d (16.2) 6.71, dt (16.2, 7.8) 2.59, m

3

4

6.30, d (2.2) 6.48, d (2.2) 6.24, d (15.7)

6.32, d (2.3) 6.45, d (2.3) 6.27, d (15.7)

6.20, ddd (15.7, 9.0, 5.2) 2.29, m 2.12, m 3.89, m 4.30, dd (5.3, 2.6) 6.46, d (15.7)

6.17, ddd (15.7, 10.4, 3.1) 2.36, m 2.15, m 4.02, m 4.32, dd (5.1, 4.0) 6.49, d (15.7)

6.80, ddd (15.7, 7.9, 5.7) 2.65, m

6.81, ddd (15.7, 8.7, 6.2) 2.58, ddd (14.4, 6.2, 2.8) 2.44, ddd (14.4, 8.7, 6.6) 5.13, m 1.35, d (6.0) 10.36, s 4.99, d (5.7) 5.17, d (5.1) 3.74, s

2.45, m

2.42, m

5.02, m 1.33, d (5.5) 10.04, s 4.91, d (6.1) 5.02, d (5.6) 3.73, s

5.24, m 1.30, d (6.3) 10.08, s 5.00, d (5.6) 5.24, d (5.3) 3.72, s

400 MHz for 1, 500 MHz for 2−4, δ in ppm, J in Hz. dx.doi.org/10.1021/jf500248z | J. Agric. Food Chem. 2014, 62, 3183−3191


Article

Compound 1a. White amorphous powder; 1H NMR (500 MHz, acetone-d6) δ 11.37 (1H, s, 2-OH), 7.21 (1H, dt, J = 16.7, 7.1 Hz), 7.17 (1H, dd, J = 15.3, 2.0 Hz), 6.51 (1H, d, J = 2.5 Hz, H-5), 6.44 (1H, d, J = 2.5 Hz, H-3), 6.15 (1H, d, J = 16.7 Hz), 6.07 (1H, ddd, J = 15.3, 10.1, 3.7 Hz), 5.40 (1H, m, H-10′), 5.04 (1H, d, J = 8.8 Hz, H5′), 4.05 (1H, ddd, J = 8.8, 4.1, 2.9 Hz, H-4′), 3.86 (3H, s, 4-OCH3), 2.82−2.91 (2H, m), 2.66 (1H, ddd, J = 15.5, 7.7, 1.2 Hz), 2.54 (1H, ddd, J = 16.0, 10.1, 2.7 Hz), 1.47 (3H, d, J = 6.3 Hz, H-11′), 1.41 (3H, s, H-14′), 1.37 (3H, s, H-15′); 13C NMR (acetone-d6, 125 MHz) δ 196.4 (C), 171.1 (C), 165.3 (C), 165.0 (C), 146.7 (C), 143.6 (CH), 135.8 (CH), 133.8 (CH), 127.0 (CH), 110.0 (C), 108.7 (CH), 105.7 (C), 101.0 (CH), 79.4 (CH), 77.6 (CH), 71.9 (CH), 56.0 (OCH3), 38.0 (CH2), 34.2 (CH2), 27.3 (CH3), 26.4 (CH3), 20.2 (CH3); ESIMS m/z 403.3 [M + H]+, 425.3 [M + Na]+, 827.5 [2M + Na]+. Compound 3a. White amorphous powder; 1H NMR (500 MHz, acetone-d6) δ 11.60 (1H, s, 2-OH), 7.06 (1H, dt, J = 16.1, 6.2 Hz), 6.83 (1H, d, J = 15.6 Hz), 6.60 (1H, d, J = 16.1 Hz), 6.50 (1H, d, J = 2.2 Hz, H-5), 6.42 (1H, d, J = 2.2 Hz, H-3), 6.07 (1H, ddd, J = 15.6, 8.5, 5.9 Hz), 5.53 (1H, m, H-10′), 4.99 (1H, d, J = 6.7 Hz, H-5′), 4.64 (1H, ddd, J = 9.6, 6.7, 2.0 Hz, H-4′), 3.85 (3H, s, 4-OCH3), 2.86 (1H, m), 2.70−2.60 (2H, m), 2.44 (1H, m), 1.56 (3H, s, H-15′), 1.47 (3H, d, J = 6.5 Hz, H-11′), 1.36 (3H, s, H-14′); 13C NMR (acetone-d6, 125 MHz) δ 196.6 (C), 171.3 (C), 165.2 (C), 164.8 (C), 143.2 (C), 142.3 (CH), 134.0 (CH), 130.8 (CH), 128.6 (CH), 109.8 (C), 108.5 (C), 105.9 (CH), 100.9 (CH), 81.9 (CH), 77.9 (CH), 71.7 (CH), 55.9 (OCH3), 37.3 (CH2), 34.4 (CH2), 27.0 (CH3), 25.9 (CH3), 18.7 (CH3); ESIMS m/z 425.0 [M + Na]+. Compound 4a. White amorphous powder; 1H NMR (500 MHz, acetone-d6) δ 11.39 (1H, s, 2-OH), 7.06 (1H, dt, J = 15.5, 7.9 Hz), 6.96 (1H, dd, J = 15.5, 2.2 Hz), 6.55 (1H, d, J = 2.6 Hz, H-5), 6.48 (1H, d, J = 15.5 Hz), 6.42 (1H, d, J = 2.6 Hz, H-3), 6.03 (1H, ddd, J = 15.5, 10.0, 3.9 Hz), 5.58 (1H, m, H-10′), 4.85 (1H, d, J = 6.6 Hz, H5′), 4.65 (1H, ddd, J = 10.9, 6.6, 2.6 Hz, H-4′), 3.85 (3H, s, 4-OCH3), 2.69 (1H, ddd, J = 15.7, 6.4, 2.7 Hz), 2.65−2.60 (2H, m), 2.34 (1H, dt, J = 16.2, 10.5 Hz), 1.59 (3H, s, H-14′), 1.47 (3H, d, J = 6.5 Hz, H11′), 1.35 (3H, s, H-15′); 13C NMR (acetone-d6, 125 MHz) δ 196.7 (C), 171.1 (C), 165.2 (C), 164.8 (C), 143.2 (C), 142.2 (CH), 133.7 (CH), 132.1 (CH), 128.8 (CH), 109.9 (C), 109.9 (C), 108.2 (CH), 101.7 (CH), 82.3 (CH), 78.5 (CH), 72.2 (CH), 55.9 (OCH3), 37.7 (CH2), 35.3 (CH2), 27.0 (CH3), 26.0 (CH3), 19.0 (CH3); ESIMS m/z 425.0 [M + Na]+. Preparation of Tri-p-bromobenzoyl Derivatives, 1b, 3b, and 4b. Cochliomycin D, 1 (2.0 mg), was treated with p-bromobenzoyl chloride (15 mg) and 4-N,N-dimethylaminopyridine (DMAP, 2.5 mg) in 1.5 mL of pyridine/CH2Cl2 (1:2) at 40 °C for 2 h. The mixture was diluted with EtOAc and washed with H2O and 1 M NaHCO3, and the organic layer was concentrated under a vacuum to obtain a colorless solid. It was then purified by Sephadex LH-20 column chromatography and semipreparative HPLC to obtain 1b (2.9 mg). Tri-pbromobenzoyl derivatives 3b (0.5 mg) and 4b (1.8 mg) were prepared similarly from cochliomycin F, 3 (0.3 mg), and (7′E)-6′-oxozeaenol, 4 (1.2 mg), respectively. Compound 1b. White amorphous powder; CD (0.87 mM, CH2Cl2) λmax (Δε) 262 (−11.64), 242 (+11.18) nm; 1H NMR (500 MHz, CDCl3) δ 8.05 (2H, d, J = 8.5 Hz, p-bromobenzoyl), 7.90 (2H, d, J = 8.5 Hz, p-bromobenzoyl), 7.75 (2H, d, J = 8.5 Hz, pbromobenzoyl), 7.66 (2H, d, J = 8.5 Hz, p-bromobenzoyl), 7.57 (2H, d, J = 8.5 Hz, p-bromobenzoyl), 7.49 (2H, d, J = 8.5 Hz, pbromobenzoyl), 7.11 (1H, ddd, J = 15.5, 8.7, 5.4 Hz), 6.93 (1H, d, J = 15.5 Hz), 6.88 (1H, d, J = 2.2 Hz, H-5), 6.73 (1H, d, J = 2.2 Hz, H-3), 6.34 (1H, d, J = 16.1 Hz), 6.12 (1H, m), 5.78 (1H, d, J = 3.4 Hz), 5.77 (1H, m), 5.33 (1H, m), 3.85 (3H, s, 4-OCH3), 2.84 (1H, dt, J = 14.7, 7.4 Hz), 2.64 (2H, m), 2.42 (1H, m), 1.20 (3H, d, J = 6.3 Hz, H-11′); ESIMS m/z 933.0 [M + Na]+. Compound 3b. White amorphous powder; CD (0.32 mM, CH2Cl2) λmax (Δε) 276 (−0.63), 249 (+5.40) nm; 1H NMR (500 MHz, CDCl3) δ 8.04 (2H, d, J = 8.5 Hz, p-bromobenzoyl), 7.95 (2H, d, J = 8.5 Hz, p-bromobenzoyl), 7.85 (2H, d, J = 8.5 Hz, pbromobenzoyl), 7.66 (2H, d, J = 8.5 Hz, p-bromobenzoyl), 7.61 (2H, d, J = 8.5 Hz, p-bromobenzoyl), 7.57 (2H, d, J = 8.5 Hz, p-

Table 2. 13C NMRa Data for 1−4 in DMSO-d6

a

position

1

2

3

4

1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 4-OCH3

110.4, C 158.8, C 100.3, CH 161.8, C 103.6, CH 138.9, C 129.2, CH 130.7, CH 36.5, CH2 72.7, CH 75.8, CH 200.2 C 131.0, CH 143.9, CH 37.5, CH2 69.9, CH 19.7, CH3 167.9, C 55.3, CH3

114.3, C 155.8, C 100.5, CH 160.7, C 100.9, CH 136.3, C 129.0, CH 131.3, CH 35.7, CH2 71.5, CH 73.6, CH 199.9, C 131.3, CH 143.9, CH 37.9, CH2 70.5, CH 20.4, CH3 167.6, C 55.3, CH3



100 MHz for 1, 125 MHz for 2−4, δ in ppm.

Cochliomycin E (2). White amorphous powder; [α]25D −73 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 198 (3.33), 223 (4.10), 271 (3.93), 312 (3.66) nm; CD (0.83 mM, MeOH) λmax (Δε) 220 (+5.53), 245 (+3.86), 278 (−1.99) nm; IR (KBr) νmax 3745, 3646, 1696, 1647, 1556, 1518 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 385.1 [M + Na]+; HRESIMS m/z 385.1269 [M + Na]+ (calcd for C19H22O7Na, 385.1258). Cochliomycin F (3). White amorphous powder; [α]25D −23.3 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 198 (3.09), 231 (4.19), 271 (3.95), 312 (3.67) nm; CD (3.86 mM, MeOH) λmax (Δε) 228 (+2.20), 248 (+2.62), 274 (−1.77), 316 (+0.16) nm; IR (KBr) νmax 3747, 3648, 1700, 1650, 1556, 1521 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 385.1 [M + Na]+, 747.1 [2M + Na]+; HRESIMS m/z 385.1268 [M + Na]+ (calcd for C19H22O7Na, 385.1258). (7′E)-6′-Oxozeaenol (4). White amorphous powder; [α]25D −50 (c 0.20, MeOH) {literature [α]24D −27.0 (c 0.33, MeOH)};6 CD (0.83 mM, MeOH) λmax (Δε) 235 (+12.61), 273 (−6.34), 316 (−1.07), 350 (+0.04) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 385.1 [M + Na]+, 747.2 [2M + Na]+. LL-Z1640-2 (6). White amorphous powder; [α]25D −94 (c 0.10, MeOH) {literature [α]24D −75.9 (c 0.41, MeOH)};7 CD (2.21 mM, MeOH) λmax (Δε) 216 (+3.82), 247 (+7.22), 272 (−13.60), 343 (+0.71) nm. Specific Rotation Data for Known Compounds 5 and 7−11. deoxy-Aigialomycin C (5): [α]25D +43.3 (c 0.10, MeOH) {literature [α]24D +25.4 (c 5.4, CH2Cl2)}.8 Zeaenol (7): [α]25D −79 (c 0.15, MeOH) {literature [α]24D −92 (c 0.54, MeOH)}.9 LL-Z1640-1 (8): [α]25D −91.7 (c 0.10, MeOH) {literature [α]24D −80 (c 0.48, MeOH)}.7 Paecilomycin F (9): [α]25D −112.5 (c 0.04, MeOH) {literature [α]24D −106.4 (c 0.28, MeOH)}.10 Cochliomycin A (10): [α]25D +32.7 (c 0.10, MeOH) {literature [α]24D +10.5 (c 0.43, MeOH)}.5 Aigialomycin B (11): [α]25D −26.7 (c 0.05, MeOH) {literature [α]24D −10 (c 0.27, CHCl3)}.2 Preparation of the Acetonide Derivatives, 1a, 3a, and 4a. A mixture of 1 (3.0 mg), 2,2-dimethoxypropane (1.5 mL), and p-TsOH (0.2 mg) was stirred at room temperature for 1 h. Saturated aqueous NaHCO3 (5 mL) was then added, and the reaction mixture was extracted with EtOAc (5 mL × 3). The organic solvents were removed under a vacuum, and the crude mixture was subjected to semipreparative HPLC to obtain 1a (2.8 mg). By the same procedures, the acetonides 3a (1.0 mg) and 4a (2.5 mg) were prepared from 3 (1.2 mg) and 4 (3.0 mg), respectively. 3185

dx.doi.org/10.1021/jf500248z | J. Agric. Food Chem. 2014, 62, 3183−3191


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Figure 2. HPLC profile of resorcylic acid lactone constituents (fraction 3) of C. lunatus (TA26-46). bromobenzoyl), 7.05 (1H, m), 6.92 (1H, d, J = 15.2 Hz), 6.88 (1H, d, J = 2.2 Hz, H-5), 6.72 (1H, d, J = 2.2 Hz, H-3), 6.52 (1H, d, J = 16.0 Hz), 6.13 (1H, m), 6.03 (1H, dd, J = 2.8, 0.5 Hz), 5.83 (1H, m), 5.32 (1H, m), 3.85 (3H, s, 4-OCH3), 1.99−2.82 (4H, m), 1.11 (3H, d, J = 6.3 Hz, H-11′); ESIMS m/z 933.2 [M + Na]+. Compound 4b. White amorphous powder; CD (0.87 mM, CH2Cl2) λmax (Δε) 266 (+21.28), 243 (−8.26) nm; 1H NMR (500 MHz, CDCl3) δ 8.05 (2H, d, J = 8.5 Hz, p-bromobenzoyl), 7.77 (2H, d, J = 8.5 Hz, p-bromobenzoyl), 7.66 (2H, d, J = 8.5 Hz, pbromobenzoyl), 7.63 (2H, d, J = 8.5 Hz, p-bromobenzoyl), 7.54 (2H, d, J = 8.5 Hz, p-bromobenzoyl), 7.41 (2H, d, J = 8.5 Hz, pbromobenzoyl), 7.04 (1H, ddd, J = 15.5, 10.3, 5.2 Hz), 6.96 (1H, d, J = 2.3 Hz, H-5), 6.78 (1H, d, J = 2.3 Hz, H-3), 6.67 (1H, d, J = 15.5 Hz), 6.41 (1H, d, J = 15.5 Hz), 6.29 (1H, m), 6.12 (1H, d, J = 3.9 Hz), 5.95 (1H, m), 5.30 (1H, m), 3.87 (3H, s, 4-OCH3), 2.76 (2H, m), 2.49 (1H, dd, J = 13.8 Hz, 5.1), 2.32 (1H, m), 1.02 (3H, d, J = 6.3 Hz, H11′); ESIMS m/z 933.2 [M + Na]+. Double-Bond Isomerization of LL-Z1640-2 (6) to Yield 4. Compound 6 (3.0 mg) was dissolved in 2.0 mL of pyridine, and the solution was stirred vigorously at 50 °C for 2 h. H2O (5 mL) was then added, and the reaction mixture was extracted with EtOAc (5 mL × 3). The organic solvents were removed under a vacuum, and the crude mixture was subjected to semipreparative HPLC to give the reaction product, the spectroscopic data of which were consistent with 4 (2.2 mg). Biological Assays. Antifouling Bioassay. The antifouling activities against the larval settlement of the barnacle were determined using cyprids of Balanus amphitrite Darwin according to literature procedures.11 Larval settlement assays were performed using 24-well polystyrene plates (Becton Dickinson, Franklin Lakes, NJ, USA). The tested compounds were first dissolved in a small amount of dimethyl sulfoxide (DMSO) and then diluted with filtered seawater to achieve final concentrations of 50, 20, 10, 5, 2.5, 1.25, and 0.625 μg/mL. About 15−20 competent larvae were gently transferred into each well with 1 mL of testing solution in three replicates, and wells containing larvae in filtered seawater with DMSO only served as a control. SeaNine 211 (Rohm & Haas, Philadelphia, PA, USA), a new type nontoxic antifouling agent developed by Rohm & Haas, was used as a positive control. The plates were incubated for 24−48 h at 23 °C. The effects of the test samples against biofouling were determined by examining the plates under a dissecting microscope to check for (1) settled larvae and (2) nonsettled larvae, as well as (3) any possible toxic effects of the treatments, such as death or paralysis of larvae, which were also recorded. The EC50 (50% inhibition of settlement of cyprids in comparison with the control) and LC50 (50% lethality of cyprids in

comparison with the control) were calculated using the Probit software program with the means of three repeated experiments using different batches of larvae. Antifungal Assays. Antifungal bioassays were conducted following the National Center for Clinical Laboratory Standards (NCCLS) recommendations.12 Three phytopathogenic fungal strains, Thielaviopsis paradoxa, Pestalotia calabae, and Glorosprium musarum, were grown on potato dextrose agar. Targeted microbes (three to four colonies) were prepared from broth culture (28 °C for 48 h), and the final spore suspensions of fungi were 104 mycelial fragments/mL. Test samples (100 μM as stock solution in DMSO and serial dilutions) were transferred to a 96-well clear plate in triplicate, and the suspension of the test organisms was added to each well achieving a final volume of 200 μL. Ketoconazole was used as a positive control. After incubation, the absorbance at 492 nm was measured with a microplate reader. The minimum inhibitory concentration (MIC) was defined as the lowest test concentration that completely inhibited the growth of the test organisms. Fungicide Screening.13 Fungicidal activities were tested by pathogens on leaf-piece assays at a rate of 100 ppm for Septoria tritici on wheat and Uromyces viciae-fabae on bean and at rates of 200 and 60 ppm for Phytophthora infestans on tomato. Fungicidal activities were also evaluated in mycelial growth tests on artificial media against Pythium dissimile, Alternaria solani, Botryotinia f uckeliana, and Gibberella zeae, at rates of 20 and 2 ppm. Rates of tested compounds were adjusted to accommodate intrinsic differences in sensitivities of pathogens in the different test systems. All testing was undertaken on 96-well microtiter plates using azoxystrobin and prochloraz as positive controls. Chemicals were applied to leaf pieces prior to inoculation with spores of the pathogen or in the case of the artificial media assays. The plates were stored in controlled-environment cabinets from 4 to 14 days, depending on the assay, and disease inhibition or mycelial growth was then assessed. Each well was scored using a three-banded system, with complete inhibition of mycelial growth or disease symptoms as 99, partial inhibition as 55, and no inhibition as 0. Fungicide Whole-Plant Assay. Fungicide whole-plant assessment was conducted against P. infestans and Plasmopara viticola on glasshouse-based whole-plant assays. All preventative, curative, and persistence tests against P. infestans on potato and P. viticola on grapevine were applied at rates of 200, 60, 20, 6, and 2 ppm as foliar sprays, whereas the preventative soil drench test against P. infestans on tomato was applied at rates of 20, 6, and 2 ppm/kg of soil. Mefenoxam and mandipropamid were used as positive controls. Chemical applications were made 2 days prior to inoculation for preventative foliar tests, 3 days prior to inoculation for drench tests, 6 days prior to 3186



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Figure 3. Structures and key NOE correlations of acetonide derivatives 1a, 3a, and 4a.

Figure 4. Structures and CD spectra of 1b, 3b, and 4b and Newman projections showing possible local conformations in the 4′R,5′S (1b), 4′R,5′R (3b), and 4′S,5′S (4b) isomers. inoculation for persistence tests, and 1 day after inoculation for curative tests. For the inoculation, sporangial suspensions were made up to the required rate (approximately 70000 sporangia/mL for P. viticola and 50000 sporangia/mL for P. infestans) and applied to the treated plants using a hand-held spray gun. After inoculation, the plants were kept under controlled conditions either in the glass house for trials with P. viticola (14 h of light/day at 100% relative humidity and 20 °C) or in a climate chamber for trials with P. infestans (14 h of light/day at 100% relative humidity and 18 °C). Assessments of disease development were made visually between 5 and 11 days after treatment, depending on the assay. The preventative and persistence tests against P. infestans also included a second evaluation of disease development 2 days after the initial assessment.

semipreparative HPLC guided by the characteristic UV absorption spectra of resorcylates. Cochliomycin D, 1, was isolated as a white powder, and its molecular formula was established to be C19H22O7 (nine degrees of unsaturation) by HRESIMS. The 1H NMR spectrum (Table 1) exhibited an exchangeable proton singlet at δH 10.46 assignable to the chelated phenolic hydrogen. The downfield 1 H NMR spectrum revealed two meta-coupled aromatic proton signals (δH 6.48, d, J = 2.4 Hz; 6.35, d, J = 2.4 Hz), and two trans double bonds (δH 6.42, d, J = 15.9 Hz and 6.18, ddd, J = 15.9, 7.8, 6.7 Hz; 6.21, d, J = 15.9 Hz and 6.82, ddd, J = 15.9, 9.8, 5.2 Hz). Additional resonances attributable to three oxymethines (δH 3.79, m; 4.25, t, J = 5.9 Hz; and 5.33, m), one oxymethyl (δH 3.75, s), and one methyl (δH 1.32, d, J = 6.3 Hz) were observed. The 13C NMR spectrum (Table 2) revealed the presence of one keto carbonyl (δC 200.2), one ester carbonyl (δC 167.9), and one tetra-substituted benzene ring. These spectroscopic features suggest that 1 belongs to the family of resorcylic acid lactones and is very similar to (7′E)-6′oxozeaenol, 4.6,9 The conspicuous differences between the 13C NMR spectrum of 1 and that of 4 are the chemical shifts of C4′ (δC 72.7 in 1 vs 71.9 in 4) and C-5′ (δC 75.8 in 1 vs 78.2 in 4). The planar structure of 1 was further confirmed on the basis

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RESULTS AND DISCUSSION An EtOAc extract from the solid fermentation of the fungus Cochliobolus lunatus (TA26-46) was separated by vacuum liquid chromatography on silica gel using step-gradient elution to yield five fractions (fractions 1−5). Fraction 3 showed a series of peaks in the HPLC profile (Figure 2) with similar UV absorptions at 223, 271, and 312 nm, indicative of the presence of resorcylic acid lactones.2 Fraction 4 showed only a main peak in the HPLC profile with the same UV absorptions as the of peaks in fraction 3. Eleven resorcylic acid lactones, 1−11, were isolated from fractions 3 and 4 by column chromatography and 3187



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of the 2D NMR [1H−1H correlation spectroscopy (COSY), heteronuclear multiple-quantum coherence (HMQC), and heteronuclear multiple-bond correlation (HMBC)] spectroscopic data. The connection from C-1′ to C-5′ and the local structure from C-7′ to C-11′ were addressed on the basis of the 1 H−1H COSY correlations. A trans-olefin (C-1′/C-2′) was attached to the benzene ring at C-6 position, as indicated by the HMBCs from the olefinic proton H-1′ to C-1, C-5, and C-6 and from H-2′ to C-6. The carbonyl (δC 200.2) was placed at the C-6′ position, because the HMBC spectrum exhibited correlations from H-5′, H-7′, and H-8′ to this carbon. Thus, the planar structure of cochliomycin D, 1, was established as the same as that of 4. Cochliomycins E and F, 2 and 3, were also isolated as white powders, with the same molecular formula, C19H22O7, as 1 based on HRESIMS. The 1H and 13C NMR spectra of 2 and 3 (Tables 1 and 2) were strikingly similar to those of 1. Detailed analysis of the 1D and 2D NMR spectra established the same planar structures for 2 and 3 as for 1. Subsequently, detailed analysis of the 1H and 13C NMR spectra of 1−4 (Tables 1 and 2) revealed that these four compounds showed high similarity in NMR spectroscopic data. The significant differences among them were the carbon signals of the oxymethines at C-4′ and C-5′. These NMR features implied that 1−4 are diastereomers differing from each other by the absolute configurations of the 4′,5′-diol chiral centers. The relative configurations of 4′,5′-diol in 1, 3, and 4 were determined by nuclear Overhauser effect (NOE) experiments of their acetonide derivatives. Compounds 1, 3, and 4 were treated with p-TsOH·H2O in 2,2-dimethoxypropane, which gave the respective acetonide derivatives 1a, 3a, and 4a. In the selective NOE experiments of 1a, irradiation of H-4′ (δH 4.05) resulted in the enhancement of the signal for H3-14′ (δH 1.41, s), and irradiation of H-5′ (δH 5.04) resulted in the enhancement of the signal for H3-15′ (δH 1.37, s) (Figure 3). Therefore, H-4′ and H-5′ should be placed on opposite sides of the acetonide ring, suggesting a trans-fused acetonide in 1a. Similarly, the selective NOE experiments indicated that both 3a and 4a were cis-acetonide derivatives (Figure 3). The acetonide formation of 2 was not conducted because of its limited quantity. It should be noted that the cis-fused acetonide derivative of 4 was reported previously,9 but its absolute configuration has not been determined. The absolute configurations of the 4′,5′-diol in 1, 3, and 4 were established by applying CD exciton chirality method.14 To obtain derivatives suitable for CD exciton chirality analysis, additional chromophores had to be introduced into the original structures. The p-bromobenzoyl chromophore was selected to avoid the interference of the enone chromophore, which has an n−π* transition occurring around 335 nm. Acylation of 1, 3, and 4 with excess p-bromobenzoyl chloride in CH2Cl2 and pyridine gave their respective triacyl derivatives, 1b, 3b, and 4b (Figure 4). The CD spectrum of 1b (Figure 4) showed a negative first Cotton effect at 262 nm (Δε = −11.76) and a positive second Cotton effect at 242 nm (Δε = +11.29), suggesting negative chirality for 1b. Furthermore, according to the Karplus formula, the 3JH4′−H5′ value of 3.4 Hz of 1b suggests the Newman projections of four possible conformations for the 4′R,5′S isomer, A1, A2, A3, and A4 (Figure 4). Examinations of the macrocycle conformations with a molecular model strongly suggested that conformations corresponding to the projections A3 and A4 were disfavored due to steric hindrance of the two pbromobenzoyl groups. The anticlockwise manner in space of

two p-bromobenzoyl chromophores in the projections A1 and A2 were consistent with the negative chirality of 1b. Thus, on the basis of the exciton chirality rule, the absolute configuration of 1b was defined as 4′R,5′S. Similarly, 3b exhibited negative chirality in the first Cotton effect at 276 nm (Δε = −0.63) in its CD spectrum, consistent with the anticlockwise manner in the projection B1, suggesting the 4′R,5′R configuration, whereas 4b showed positive chirality in the first Cotton effect at 266 nm (Δε = +21.28), matching the clockwise manner in the projection C1, which revealed the 4′S,5′S configuration (Figure 4). Therefore, the absolute configurations of the 4′,5′-diol in 1, 3, and 4 were designated as 4′R,5′S, 4′R,5′R, and 4′S,5′S, respectively. The absolute configurations at C-10′ in 1−4 were determined by their CD spectra (Figure 5). The negative

Figure 5. CD spectra of 1−4 and 6.

Cotton effects around 275 nm suggested the S configuration at C-10′ for 1−4, by comparison with that of the coisolated known compound 6 and other similar resorcylic acid lactones described in the literature.15,16 Furthermore, 4 could be obtained by a double-bond isomerization reaction from 6, whose absolute configuration was described as 4′S,5′S,10′S in the literature.7 This isomerization experiment further confirmed the absolute configuration of 4. Because 1, 3, and 4 were determined above as having the 4′R,5′S,10′S, 4′R,5′R,10′S, and 4′S,5′S,10′S configurations, respectively, the absolute configuration of 2 was tentatively assigned as 4′S,5′R,10′S. To verify the absolute configuration of 2, time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculations were applied to 2, together with its stereoisomer 1 with the enantiomeric 4′,5′-diol. First, conformational analysis was carried out by Monte Carlo searching using molecular mechanism with the MMFF94 force field in the Spartan program.17 The results showed four lowest-energy conformers for 1 and five for 2 whose relative energies were within 2 kcal/mol. Subsequently, the conformers were reoptimized using DFT at the B3LYP/631+G(d) level in a vacuum with the Gaussian 09 program.18 The B3LYP/6-31+G(d) harmonic vibrational frequencies were further calculated to confirm their stability. The energies, oscillator strengths, and rotational strengths of the first 30 electronic excitations were calculated using the TDDFT methodology at the B3LYP/6-311++G(2d, 2p) level in a vacuum. The ECD spectra were simulated by the overlapping Gaussian function,19 in which the first 15 velocity rotatory strengths for 1 and the first 11 for 2 were employed. To obtain the final ECD spectrum, the simulated spectra of the lowest3188



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Figure 6. B3LYP/6-311++G(2d,2p)//B3LYP/6-31+G(d)-calculated ECD spectra (red) of 4′R,5′S-1 and 4′S,5′R-2 and experimental ECD spectra (black) of 1 (σ = 0.2) and 2 (σ = 0.4).

energy conformations were averaged according to the Boltzmann distribution theory and their relative Gibbs free energies (ΔG). In the 200−400-nm region, the experimental ECD spectrum of 1 showed a small positive first Cotton effect around 350 nm, whereas 2 showed a negative effect around this band. The calculated spectra for the two compounds matched the corresponding experimental spectra (Figure 6). Therefore, qualitative analysis of the above information confirmed the absolute configuration of 2 as 4′S,5′R,10′S. The structures of the other known compounds, deoxyaigialomycin C (5),8 LL-Z1640-2 (6),7 zeaenol (7),9 LLZ1640-1 (8),7 paecilomycin F (9),10 cochliomycin A (10),5 and aigialomycin B (11),2 were determined by comparison of their spectroscopic, MS, and specific rotation data with those in the literature. It is worth pointing out that this is the first report of 5 as a natural product. Its relative and absolute configurations were confirmed using NOE experiments of its acetonide derivative and the CD exciton chirality method with its pdimethylaminobenzoyl derivative. From the chemistry point of view, these resorcylic acid lactones might be unstable because of the existence of enone moieties and many hydroxy groups, especially phenol hydroxy groups, causing chemical conversions under acidic conditions, such as intramolecular hetero Michael addition20 and carbon migration of hemiacetal.3 During acquisition of their NMR spectra, all compounds were found to be quite stable, and no trace of conversions was observed in deuterated solvents (DMSO-d6, acetone-d6, and CDCl3), even with prolonged time of more than 3 months. The stability of these lactones under various conditions is worth further investigation. In our previous study, the resorcylic acid lactones showed potent antifouling activity against the larval settlement of the barnacle Balanus amphitrite.5 In our current research, the isolated resorcylic acid lactones and the acetonide derivatives 1a and 3a−6a were also evaluated for their antifouling activities. Compounds 1, 3−6, and 6a exhibited potent antifouling activity at nontoxic concentrations with the EC50 values of 17.3, 6.67, 18.1, 22.5, 1.82, and 3.85 μg/mL, respectively (Table 3), which were lower than the standard requirement of an EC50 value of 25 μg/mL established by the U.S. Navy program.21 The cis-enone moiety in 6 (1.82 μg/mL) improved the EC50 value approximately 9-fold over that of 4 (18.1 μg/mL) with trans-enone, indicating that the cis-enone functionality might contribute to the antifouling activity. Moreover, the antifouling activity of 6a (EC50 of 3.85 μg/mL), more than 6 times that of 4a (EC50 > 25 μg/mL), also confirmed this possibility. Our

Table 3. Antifouling Activity

a

compd

EC50 (μg/mL)

LC50/EC50

1 3 4 5 6 6a SeaNine 211a

17.3 6.67 18.1 22.5 1.82 3.85 1.23

>50 >50 >50 >50 >50 >50 20.3

Positive control.

previous study indicated that the acetonide functionality might have a positive effect on antifouling activity.5 For the enone resorcylic acid lactones, however, the acetonide functionality (1a, 3a, 4a, and 6a) was found to decrease the antifouling activity significantly (1a, 3a, 4a: EC50 > 25 μg/mL). Comparison of the activities of 1−4 indicated that the configurations of 4′,5′-diol might affect the antifouling activity, with the 4′R,5′R configuration in 3 exhibiting the highest activity. The above findings suggest that small changes in the structures, especially the enone and acetonide functionalities and hydroxy configurations, among these resorcylic acid lactones can result in obvious changes of antifouling activity. The therapeutic ratio (LC50/EC50) is a way of expressing the effectiveness of the compound in relation to its toxicity. A compound with LC50/EC50 > 15 is often considered to be a nontoxic antifouling compound, and a much higher LC50/EC50 ratio is highly recommended when selecting candidate compounds.22 All of the active compounds, 1, 3−6, and 6a, had high therapeutic ratios (LC50/EC50 > 50), suggesting that they might be useful as environmentally benign antifouling agents. Simultaneously, the antifungal activities of all isolated compounds against phytopathogenic fungi Thielaviopsis paradoxa, Pestalotia calabae, and Glorosprium musarum were evaluated. Noticeably, among the tested compounds, only 6 exhibited promising inhibitory activity against P. calabae, with an MIC value of 0.39 μM, which was approximately 25-fold more potent than that of the positive control, ketoconazole (MIC = 10 μM). As a result of its antifungal activity, 6 was provided to Syngenta for agricultural fungicide screening. In primary- and secondary-tier fungicide assays, 6 showed moderate activity against a limited range of pathogens in laboratory-based tests. It totally inhibited the disease development of P. infestans 3189



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Table 4. Fungicidal Activity of 6 on Whole-Plant Assaysa Pib drench

pathogen test method compd 6

mefenoxam

mandipropamid

Pic preventative

Pic curative

Pic persistence

Pvd preventative

Pvd curative

Pvd persistence

rate (ppm)

eff

eff

eff le

eff

eff

eff le

eff

eff

eff

200 60 20 6 2 200 60 20 6 2 0.6 0.2 200 60 20 6 2 0.6

− − 93 86 38 − − − 100 100 99 98 − − − − − −

98 98 92 17 0 98 98 98 33 − − − − − − − − −

15 30 0 0 0 98 93 15 0 − − − − − − − − −

11 11 11 0 0 − 92 89 78 78 − − − − − − − −

81 75 69 63 63 − − − − − − − 98 98 96 91 − −

60 40 15 15 10 − − − − − − − 93 88 90 70 − −

96 96 94 91 50 100 84 63 0 − − − − − − − − −

0 0 0 0 0 − 100 100 31 13 − − − − − − − −

100 100 87 93 53 − − − − − − − − − 100 100 99 93

a

Scores are given as percentages of disease control relative to untreated plants (100 indicates complete control of disease symptoms, 0 indicates no control); −, not tested at that rate; le, late evaluation. Mefenoxam and mandipropamid as positive controls. bP. infestans (tomato). cP. infestans (potato). dP. viticola (grapevine).

with an IC50 value of 3.24 μM. The antibacterial, antiviral, insecticidal, and herbicidal activities of the isolated and synthetic resorcylic acid lactones were also determined, but no significant activity was observed. In summary, 11 resorcylic acid lactones, 1−11, including three new compounds, 1−3, were isolated from the sea anemone-derived fungus C. lunatus (TA26-46). The absolute configurations of 1−4 were established by the CD exciton chirality method and TDDFT ECD calculations. Compounds 1, 3−6, and 6a exhibited potent antifouling activities against the larval settlement of the barnacle B. amphitrite, of which 6 showed the highest activity with low toxicity. More remarkably, 6 showed excellent agricultural fungicidal activity against P. viticola and P. infestans on the whole-plant assay, which would be helpful for the research and development of new natural potential fungicide leads.

(tomato) at concentrations of 200 and 60 ppm and of U. viciaefabae (bean) at 100 ppm, as well as the hyphal growth of P. dissimile at 2 ppm. To further characterize its fungicidal potency, 6 was assessed against P. infestans and Plasmopara viticola on glass-house-based whole-plant assays. Mefenoxam and mandipropamid were used as positive controls. The selection of the standard was based on the properties of candidate active ingredients and the nature of the assays. Mefenoxam is highly systemic; therefore, it was used as the standard in the drench and curative assays. Mandipropamid has an excellent rain-fastness and long-lasting activity because it sticks and distributes quite well in the wax layer of the cuticle, so it was used in persistence test. For the preventive test, both standards can be used, one to ensure that the method is working sufficiently. In this case, the standards serve as quality control for the respective test methods. The results indicated that 6 displayed good activity on both tests, stronger than originally observed on the primary- and secondary-tier assays (Table 4). On the preventative foliar applications against P. infestans (potato), 6 showed potent ratefor-rate activity, with disease control rates (DCRs) of 98%, 98%, and 92% at 200, 60, and 20 ppm, respectively, comparable to the values for the positive control mefenoxam (98%, 98%, and 98%) in the initial assessment (Table 4). Compound 6 also showed promising activity on the P. infestans drench test (DCR of 86% at 6 ppm). More remarkably, the DCR of 6 was 91% at 6 ppm on the preventative foliar applications against P. viticola (grapevine) compared to the positive control mefenoxam (6 ppm, DCR = 0), highlighting the promising activity of 6 against P. viticola (Table 4). In the persistence application test against P. infestans, 6 showed weaker activity than in the preventative application test, and it also produced no significant activity on the curative tests against either P. infestans or P. viticola. In addition, the cytotoxicity of 6 was also determined against tumor cell lines of Hela, A549, HCT-116, HL-60, and K562. It was found that 6 showed cytotoxicity against only HCT-116

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ASSOCIATED CONTENT

* Supporting Information S

1

H NMR, 13C NMR, 1H−1H COSY, and MS spectra of 1−3; HMQC and HMBC spectra of 1 and 3; 1H NMR, 13C NMR, 1D NOE, and MS spectra of 1a, 3a, and 4a; 1H NMR and MS spectra of 1b, 3b, and 4b; TDDFT ECD simulation of 1 and 2; structure and 1D NOE spectrum of compound 5a; structure and CD spectrum of 5b and Newman projections showing possible local conformations in the 4′S,5′S isomer (5b); and NMR and MS data for 5a and 5b. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel./Fax: 86-532-82031536. E-mail: [email protected]. Funding

This work was supported by the Program of the National Natural Science Foundation of China (Nos. 41130858, 41322037, 41376145); the Program for New Century Excellent 3190



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(14) Isaka, M.; Chinthanom, P.; Kongthong, S.; Supothina, S.; Ittiworapong, P. Hamigeromycins C−G, 14-membered macrolides from the fungus Hamigera avellanea BCC 17816. Tetrahedron 2010, 66, 955−961. (15) Snatzke, G.; Angeli, C.; Decorte, E.; Moimas, F.; Kojié-Prodié, B.; Ružić-Toroš, Z.; Sunjić, V. Preparation via diastereoselective hydrogenation, absolute conformation and configuration of exogeneous anabolic zeranol ((3S,7R)-3,4,5,6,7,8,9,10,11,12-decahydro7,14,16-trihydroxy-3-methy-1H-2-benzoxacyclotetradecin-1-one). Helv. Chim. Acta 1986, 69, 734−748. (16) Gelo, M.; Raza, Z.; Š unjić, V.; Guo, J.; Snatzke, G. Determination of the conformation of E and Z zearalenone and their 7α- and 7β-hydroxy congeners. Tetrahedron: Asymmetry 1991, 2, 1005−1010. (17) Spartan 04; Wavefunction Inc.: Irvine, CA, 2004. (18) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr., Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09 Revision C.01; Gaussian, Inc.: Wallingford, CT, 2009. (19) Stephens, P. J.; Harada, N. ECD cotton effect approximated by the Gaussian curve and other methods. Chirality 2010, 22, 229−233. (20) Isaka, M.; Chinthanom, P.; Veeranondha, S.; Supothina, S. Novel cyclopropyl diketones and 14-membered macrolides from the soil fungus Hamigera avellanea BCC 17816. Tetrahedron 2008, 64, 11028−11033. (21) Rittschof, D. Fouling and natural products as antifoulants. In Recent Advances in Marine Biotechnology; Fingerman, M., Nagabhushanam, R., Thompson, M. F., Eds.; Oxford & IBH Publishing Company: New Delhi, India, 1999; pp 245−257. (22) Qian, P.-Y.; Xu, Y.; Fusetania, N. Natural products as antifouling compounds: Recent progress and future perspectives. Biofouling 2010, 26, 223−234.

Talents in University, Ministry of Education of China (No. NCET-11-0472); and the Special Financial Fund of Innovative Development of Marine Economic Demonstration Project (GD2012-D01-001). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Biology Team at Syngenta for the fungicide assay and Dr. Naomi Pain of Syngenta for proofreading of this manuscript. We appreciate Prof. Pei-Yuan Qian and Dr. Ying Xu, Division of Life Science, the Hong Kong University of Science and Technology, Hong Kong, China, for the antifouling bioassay.

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REFERENCES

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Antifouling and Fungicidal Resorcylic Acid ... - ACS Publications

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