Antifouling and fungicidal resorcylic acid lactones from the sea anemone-derived fungus cochliobolus lunatus

Article abstract of DOI:10.1021/jf500248z

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.

Full text of DOI:10.1021/jf500248z

Article
pubs.acs.org/JAFC
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
°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
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 (TA26-
46), 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.
1
at 400 MHz for H and 100 MHz for 13C on a Bruker AVANCE 400
1
spectrometer (Bruker, Kalsruhe, Germany) and at 500 MHz for H
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 high-
resolution 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
Received: January 7, 2014
Revised: March 17, 2014
Accepted: March 17, 2014
Published: March 17, 2014
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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Figure 1. Structures of resorcylic acid lactones 1−11 isolated from C. lunatus (TA26-46).
Co., Ltd., Yantai, China) were used for thin layer chromatography
(TLC).
Cochliomycin D (1). White amorphous powder; [α]25 −125 (c
D
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;
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.
1
IR (KBr) νmax 3747, 3648, 1700, 1650, 1540, 1510 cm−1; H 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).
a
1
Table 1. H NMR Data for 1−4 in DMSO-d6
position
1
2
3
4
3
6.35, d (2.4)
6.48, d (2.4)
6.42, d (15.9)
6.31, brs
6.55, brs
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)
5
1′
6.21, d
(15.9)
2′
3′
6.18, ddd (15.9, 6.33, m
7.8, 6.7)
6.20, ddd (15.7, 6.17, ddd (15.7,
9.0, 5.2) 10.4, 3.1)
2.29, m 2.36, m
2.31, m
2.45, m
2.20, m
2.13, m
2.12, m
3.89, m
2.15, m
4.02, m
4′
5′
3.79, m
3.86, m
4.25, t (5.9)
4.27, t (5.6)
4.30, dd (5.3,
2.6)
4.32, dd (5.1,
4.0)
7′
8′
9′
6.21, d (15.9)
6.13, d
(16.2)
6.46, d (15.7)
6.49, d (15.7)
6.82, ddd (15.9, 6.71, dt
9.8, 5.2)
6.80, ddd (15.7, 6.81, ddd (15.7,
7.9, 5.7)
(16.2, 7.8)
8.7, 6.2)
2.67, ddd (14.4, 2.59, m
5.2, 2.1)
2.65, m
2.58, ddd (14.4,
6.2, 2.8)
2.40, ddd (14.4, 2.45, m
9.8, 7.0)
2.42, m
5.24, m
2.44, ddd (14.4,
8.7, 6.6)
10′
5.33, m
5.02, m
5.13, m
11′
1.32, d (6.3)
10.46, s
1.33, d (5.5) 1.30, d (6.3)
10.04, s 10.08, s
1.35, d (6.0)
10.36, s
2-OH
4′-OH
5′-OH
4.94, d (5.6)
5.18, d (5.9)
4.91, d (6.1) 5.00, d (5.6)
5.02, d (5.6) 5.24, d (5.3)
4.99, d (5.7)
5.17, d (5.1)
3.74, s
4-OCH3 3.75, s
3.73, s
3.72, s
a
400 MHz for 1, 500 MHz for 2−4, δ in ppm, J in Hz.
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Article
1
a
Table 2. 13C NMR Data for 1−4 in DMSO-d6
Compound 1a. White amorphous powder; H 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, H-
5′), 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]+.
position
1
2
3
4
1
110.4, C
114.3, C
112.9, C
111.6, C
2
158.8, C
155.8, C
156.7, C
157.8, C
3
100.3, CH
161.8, C
100.5, CH
160.7, C
100.3, CH
161.0, C
100.1, CH
161.3, C
4
5
103.6, CH
138.9, C
100.9, CH
136.3, C
101.9, CH
137.3, C
102.6, CH
138.3, C
6
1′
2′
3′
4′
5′
6′
129.2, CH
130.7, CH
36.5, CH2
72.7, CH
75.8, CH
200.2 C
129.0, CH
131.3, CH
35.7, CH2
71.5, CH
73.6, CH
199.9, C
129.2, CH
130.5, CH
36.0, CH2
73.0, CH
78.8, CH
200.1, C
128.6, CH
130.5, CH
36.0, CH2
71.9, CH
78.2, CH
200.7, C
1
Compound 3a. White amorphous powder; H 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]+.
7′
8′
9′
131.0, CH
143.9, CH
37.5, CH2
69.9, CH
19.7, CH3
167.9, C
131.3, CH
143.9, CH
37.9, CH2
70.5, CH
20.4, CH3
167.6, C
129.7, CH
142.7, CH
37.5, CH2
69.8, CH
19.9, CH3
167.6, C
130.3, CH
142.4, CH
37.9, CH2
70.2, CH
20.1, CH3
168.2, C
10′
11′
12′
4-OCH3
55.3, CH3
55.3, CH3
55.2, CH3
55.3, CH3
a
100 MHz for 1, 125 MHz for 2−4, δ in ppm.
1
Cochliomycin E (2). White amorphous powder; [α]25D −73 (c 0.10,
Compound 4a. White amorphous powder; H 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, H-
5′), 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, H-
11′), 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 chromatog-
raphy and semipreparative HPLC to obtain 1b (2.9 mg). Tri-p-
bromobenzoyl 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.
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]25 −23.3 (c
D
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]25 −50 (c
D
0.20, MeOH) {literature [α]24 −27.0 (c 0.33, MeOH)};6 CD (0.83
D
mM, MeOH) λmax (Δε) 235 (+12.61), 273 (−6.34), 316 (−1.07), 350
1
(+0.04) nm; H 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]25 −94 (c 0.10,
D
MeOH) {literature [α]24 −75.9 (c 0.41, MeOH)};7 CD (2.21 mM,
D
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]25 +43.3 (c 0.10, MeOH) {literature
Compound 1b. White amorphous powder; CD (0.87 mM,
D
1
[α]24 +25.4 (c 5.4, CH2Cl2)}.8 Zeaenol (7): [α]25 −79 (c 0.15,
CH2Cl2) λmax (Δε) 262 (−11.64), 242 (+11.18) nm; H NMR (500
D
D
MeOH) {literature [α]24 −92 (c 0.54, MeOH)}.9 LL-Z1640-1 (8):
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, p-
bromobenzoyl), 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, p-
bromobenzoyl), 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]+.
D
[α]25 −91.7 (c 0.10, MeOH) {literature [α]24 −80 (c 0.48,
D
D
MeOH)}.7 Paecilomycin F (9): [α]25 −112.5 (c 0.04, MeOH)
D
{literature [α]24 −106.4 (c 0.28, MeOH)}.10 Cochliomycin A (10):
D
[α]25 +32.7 (c 0.10, MeOH) {literature [α]24 +10.5 (c 0.43,
D
D
MeOH)}.5 Aigialomycin B (11): [α]25 −26.7 (c 0.05, MeOH)
D
{literature [α]24 −10 (c 0.27, CHCl3)}.2
PreparationDof 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 semi-
preparative 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.
Compound 3b. White amorphous powder; CD (0.32 mM,
1
CH2Cl2) λmax (Δε) 276 (−0.63), 249 (+5.40) nm; H 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, p-
bromobenzoyl), 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-
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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]+.
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, Thielaviop-
sis 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.
Compound 4b. White amorphous powder; CD (0.87 mM,
1
CH2Cl2) λmax (Δε) 266 (+21.28), 243 (−8.26) nm; H 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, p-
bromobenzoyl), 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, p-
bromobenzoyl), 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, H-
11′); 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).
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 fuckeliana, 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 glass-
house-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
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
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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
1H 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 C-
4′ (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
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
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of the 2D NMR [1H−1H correlation spectroscopy (COSY),
heteronuclear multiple-quantum coherence (HMQC), and
heteronuclear multiple-bond correlation (HMBC)] spectro-
scopic 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
1H−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.
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
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.
determined by their CD spectra (Figure 5). The negative
1
Subsequently, detailed analysis of the H 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 p-
bromobenzoyl groups. The anticlockwise manner in space of
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/6-
31+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 lowest-
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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.
Table 3. Antifouling Activity
compd
EC50 (μg/mL)
17.3
6.67
LC50/EC50
1
>50
>50
>50
>50
>50
>50
20.3
3
4
18.1
22.5
5
6
1.82
6a
3.85
1.23
a
SeaNine 211
The structures of the other known compounds, deoxy-
aigialomycin C (5),8 LL-Z1640-2 (6),7 zeaenol (7),9 LL-
Z1640-1 (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 p-
dimethylaminobenzoyl 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
a
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 para-
doxa, 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
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a
Table 4. Fungicidal Activity of 6 on Whole-Plant Assays
b
c
c
c
d
d
d
pathogen
test method
Pi
Pi
preventative
Pi
Pi
persistence
Pv
Pv
Pv
drench
curative
preventative
curative
persistence
compd
rate (ppm)
eff
eff
eff le
eff
eff
eff le
eff
eff
eff
6
200
60
20
6
−
−
93
86
38
−
98
98
92
17
0
15
30
0
11
11
11
0
81
75
69
63
63
−
−
−
−
−
60
40
15
15
10
−
−
−
−
−
96
96
94
91
50
100
84
63
0
0
100
100
87
93
53
−
−
−
−
−
−
−
−
−
100
100
99
93
0
0
0
0
2
0
0
0
mefenoxam
200
60
20
6
98
98
98
33
−
−
−
−
−
−
−
−
−
98
93
15
0
−
−
100
100
31
13
−
−
−
−
−
−
−
−
−
−
92
89
78
78
−
−
−
−
−
100
100
99
98
−
−
−
−
−
2
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
0.6
0.2
200
60
20
6
−
−
−
−
mandipropamid
98
98
96
91
−
93
88
90
70
−
−
−
−
2
0.6
−
−
−
−
a
Scores are given as percentages of disease control relative to untreated plants (100 indicates complete control of disease symptoms, 0 indicates no
b
c
control); −, not tested at that rate; le, late evaluation. Mefenoxam and mandipropamid as positive controls. P. infestans (tomato). P. infestans
(potato). P. viticola (grapevine).
d
(tomato) at concentrations of 200 and 60 ppm and of U. viciae-
fabae (bean) at 100 ppm, as well as the hyphal growth of P.
dissimile at 2 ppm.
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.
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 rate-
for-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
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.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
1H NMR, 13C NMR, H−1H COSY, and MS spectra of 1−3;
HMQC and HMBC spectra of 1 and 3; H NMR, 13C NMR,
1
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.
AUTHOR INFORMATION
Corresponding Author
*Tel./Fax: 86-532-82031536. E-mail: changyun@ouc.edu.cn.
■
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
dx.doi.org/10.1021/jf500248z | J. Agric. Food Chem. 2014, 62, 3183−3191

Journal of Agricultural and Food Chemistry
Article
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Ittiworapong, P. Hamigeromycins C−G, 14-membered macrolides
from the fungus Hamigera avellanea BCC 17816. Tetrahedron 2010,
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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).
(15) Snatzke, G.; Angeli, C.; Decorte, E.; Moimas, F.; Kojie-
́ ́
Prodie,
Notes
B.; Ruzic-Toros, Z.; Sunjic, V. Preparation via diastereoselective
́
̌
́
̌
hydrogenation, absolute conformation and configuration of exoge-
neous anabolic zeranol ((3S,7R)-3,4,5,6,7,8,9,10,11,12-decahydro-
7,14,16-trihydroxy-3-methy-1H-2-benzoxacyclotetradecin-1-one).
Helv. Chim. Acta 1986, 69, 734−748.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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́
(16) Gelo, M.; Raza, Z.; Sunjic, V.; Guo, J.; Snatzke, G.
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.
Determination of the conformation of E and Z zearalenone and
their 7α- and 7β-hydroxy congeners. Tetrahedron: Asymmetry 1991, 2,
1005−1010.
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