Article

pubs.acs.org/jnp

Metabolites from the Endophytic Fungus Curvularia sp. M12 Act as

Motility Inhibitors against Phytophthora capsici Zoospores
Muhammad Abdul Mojid Mondol,† Jannatul Farthouse,‡ Mohammad Tofazzal Islam,‡ Anja Schüffler,§
and Hartmut Laatsch*,†
†
Institute for Organic and Biomolecular Chemistry, Georg-August-University Göttingen, Tamannstrasse 2, D-37077 Göttingen,
Germany
‡
Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh
§
Institute of Biotechnology and Drug Research, D-67663 Kaiserslautern, Germany
*
S Supporting Information

ABSTRACT: The endophytic fungus Curvularia sp., strain

M12, was isolated from a leaf of the medicinal plant Murraya
koenigii and cultured on rice medium followed by chemical
screening of the culture extract. Chromatographic analysis led
to the isolation of four new compounds, murranofuran A (1),
murranolide A (2), murranopyrone (3a), and murranoic acid
A (4a), along with six known metabolites, N-(2-hydroxy-6-
methoxyphenyl)acetamide (5), curvularin (6), (S)-dehydro-
curvularin (7), pyrenolide A (8), modiolide A (9), and 8-hydroxy-6-methoxy-3-methylisocoumarin (10). The structures of the
known compounds were confirmed by comparing ESI HR mass spectra, 1H and 13C NMR, and optical rotation data with values
reported in the literature. The planar structures of the new compounds were elucidated by extensive analysis of 1D and 2D NMR
and mass data. The absolute configurations of the new compounds were established by coupling constant analysis, modified
Mosher’s method, and CD data. Compound 8 showed a strong motility impairing activity against Phytophthora capsici zoospores
at a low concentration (100% at 0.5 μg/mL) in a short time (30 min). Compounds 2, 3a, 6, 7, 9, and 10 exhibited zoospore
motility impairment activity at higher concentrations (IC50: 50−100 μg/mL).

P eronosporomycetes, also known as water molds, are a

group of fungus-like eukaryotic microorganisms, phyloge-
netically related to brown algae. They include saprophytes as
well as pathogens of plants, insects, crustaceans, fish, and other
vertebrate animals. Saprophytic peronosporomycetes play key
roles in decomposition and recycling of organic matter;
however plant pathogenic species, especially those of the
genus Phytophthora (the “plant destroyer” in Greek), are
particularly devastating phytopathogens. They cause diseases in
important crop species such as potato, tomato, pepper,
soybean, cucurbits, pumpkin, squash, and eggplant as well as
do environmental damage in natural ecosystems, resulting in
multibillion dollar losses every year.1 Some species of
Phytophthora, notably Phytophthora capsici, are highly dynamic
and destructive pathogens of vegetables, with a wide range of
hosts.2 One of the most distinguishing characteristics of the
peronosporomycete organisms is the production of motile
zoospores in their early life stages. These zoospores play an
important role in the infection of plants.3−5
Peronosporomycetes were traditionally classified as fungi, but
in fact have little in common with them, exhibiting marked
differences in physiology, biochemistry, and genetics. While the
cell walls of fungi are composed mainly of chitin, the
peronosporomycetes contain glucan and cellulose. Fungicides
that target chitin biosynthesis will therefore have no effect on
peronosporomycetes. This lack of effect may cause farmers to
© 2017 American Chemical Society and
American Society of Pharmacognosy
increase the concentration of the fungicides and the frequency
of their usage, factors that may lead to the spread of resistance
in other species. In addition, preparations that in fact
demonstrate activity against peronosporomycetes often contain
copper, tin, or other persistent chemicals, whose toxicity
represent enormous threats to the ecosystem, human health,
and the environment in general.6 Therefore, there is a very
urgent need for natural agents that are biodegradable and will
exclusively control peronosporomycete phytopathogens. We
hope to find such agents in endophytes.
Endophytic fungi are ubiquitous in the plant kingdom. They
produce bioactive secondary metabolites that may help plants
to defend themselves against microorganisms, insects, and
herbivores7−11 and thus may also have agricultural applications.
In our search for natural agents to control peronosporomycetes,
we studied the curry plant, Murraya koenigii. Its leaf extract is
used as an antimicrobial agent in Ayurvedic medicine and also
has insecticidal properties.12 On screening this plant for
endophytic fungi, a Curvularia sp. strain (M12) was isolated
using M2 agar medium. On the basis of prescreening results (1H
NMR and TLC), a culture of the strain was scaled-up on rice
medium. Chromatographic purification of the ethyl acetate
Received: August 28, 2016
Published: February 14, 2017
347 DOI: 10.1021/acs.jnatprod.6b00785
J. Nat. Prod. 2017, 80, 347−355
Journal of Natural Products Article

Chart 1

Table 1. 13C and 1H NMR Data for 1 and 2 in CD3OD

1 2
no. δ Ca type δH, mult. (J in Hz)b δ Ca type δH, mult. (J in Hz)c
1 157.6 CH 7.36, d (1.3) 172.1 C
2 118.7 C 123.9 CH 6.27, d (5.7)
3 51.8 CH 3.20, ddd (3.9, 2.6, 1.3) 156.9 CH 7.37, d (5.7)
4 112.9 CH 5.48, d (2.6) 111.5 C
5 47.3 CH 3.12, qd (7.1, 3.9) 41.6 CH2 2.19, 2.15, ABX (15.2, 8.5, 2.1)
6 212.9 C 76.9 CH 2.97, ddd (8.5, 8.6, 2.2)
7 29.8 CH3 2.15, s 60.9 CH 3.04, dd (8.6, 4.4)
8 13.1 CH3 1.00, d (7.1) 58.2 CH 2.79, dd (8.2, 4.4)
9 194.8 C 67.0 CH 3.55, dq (8.2, 6.4)
10 59.3 C 20.4 CH3 1.24, d (6.4)
11 30.7 CH2 1.89, qd (7.5, 1.6) 51.6 CH3 3.19, s
12 8.7 CH3 0.77, t (7.5) 58.2 CH3 3.31, s
13 20.7 CH3 1.32, s
14 176.3 C
15 56.7 CH3 3.47, s
16 53.0 CH3 3.66, s
a
Measured at 125 MHz. bMeasured at 300 MHz. cMeasured at 600 MHz.

extract led to the isolation of four new (1, 2, 3a, and 4a) and six HRMS, consistent with the molecular formula C16H24O6. This
known compounds (5−10). The isolation, structure elucida- was confirmed by 13C and 1H NMR data (Table 1). In the 13C
tion, and antizoospore activity of these compounds are NMR spectrum, 16 resonances were observed and ascribed to
discussed here. five sp2 carbons (three carbonyl and two olefinic carbons) and

■ RESULTS AND DISCUSSION

Compound 1 was isolated as an optically active, amorphous
solid with a pseudomolecular ion peak [M + H]+ in the ESI
348
11 sp3 carbons with the help of phase-sensitive HSQC.
The planar structure of compound 1 (Figure 1) was
determined via extensive analysis of 2D NMR data. The
olefinic methine H-1 (δC 157.6, δH 7.36) showed HMBC
DOI: 10.1021/acs.jnatprod.6b00785
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Journal of Natural Products
Figure 1. COSY (bold bonds) and HMBC (arrows) correlations for 1−3a, 4a, and 5.
couplings with C-2, C-3, and C-4. H-3 showed a COSY
correlation with H-4 and H-5. Additional HMBC correlations
with C-1, C-2, and C-4 (Figure 1) indicated a dihydrofuran
moiety. The methoxy carbon C-15 showed an HMBC
correlation with the dihydrofuran carbon C-4 (δC 112.9),
forming an acetal moiety. The resonance of the keto carbonyl at
212.9 (C-6), a COSY correlation between H-5 and H3-8, and
HMBC correlations between H3-7/C-5,6 and H-5/C-3,6
identified a butan-2-one fragment that was connected via C-5
with C-3 of the dihydrofuran, as confirmed by the HMBC
correlations of H-3 with C-5 and C-8 and of H-5 and H-8 with
C-3.
The remaining partial formula, C7H11O3, is accounting for a
carbomethoxy group, a carbonyl, a methyl, an ethyl fragment,
and a quaternary carbon, forming a 2-ethyl-2-methyl-3-
ketopropionate (Figure 1). The alternative 2-keto-3-ethyl-3-
methylpropionate was excluded on the basis of the HMBC
correlations and calculated NMR data. In the HMBC spectrum,
the methyl singlet at δH 1.32 (H3-13) correlated with both
carbonyls and with the ethyl residue, so that all fragments must
be connected via the remaining quaternary carbon at δC 59.3
(C-10). Only two natural products with this propionate
fragment seem to be known: specifernin13 and berkelic
acid.14 Taking into account the different type of the ketone
(aliphatic instead of conjugated), their respective NMR data are
in good agreement with the data measured for the 1 fragment.
We did not find examples of the isomeric 3-ethyl-3-
methylpyruvic acid fragment in the natural product literature.15
We observed only a weak HMBC correlation between H-1 of
the dihydrofuran and C-9 of this ketopropionate ester fragment,
but not between C-9 and H-3. There was, however, only one
way to connect both fragments via free valences, resulting in
structure 1, which we named murranofuran A with respect to
the origin of the endophyte.
According to the coupling constant between H-3 and H-4 (J
= 2.6 Hz), the dihydrofuran protons of 1 should be in a trans-
position; DFT calculations predicted correspondingly a
dihedral angle of ∼120° and a coupling constant of 2.6 Hz,
as in the experiment. The syn-facial orientation of H-4 and the
butanone side chain was further confirmed by strong NOE
signals between H-4/H-5 and H-4/H3-8, so that a (3R,4R) or a
(3S,4S) configuration, respectively, resulted.
To further investigate the configuration, the electronic
circular dichroism (ECD) spectra of all eight pairs of
enantiomers were calculated for 1, using ab initio methods on
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Article
a high level of theory (see Experimental Section). The
calculated ECD spectra are dominated by the stereoelectronic
effects of C-3 and C-4. When these are in the S-configuration,
both centers are contributing a positive but different Cotton
effect (3S < 4S) to the CD spectrum (see Supporting
Information, Figures S1 and S2). This is also the reason that,
in the calculations, not only the four trans-(3S,4S,5R*,10R*)
isomers but also the four cis-(3R,4S,5R*,10R*) dihydrofurans
showed a positive Cotton effect and a similar curve shape to
that observed for compound 1. The influences of C-5 and C-10
on the ECD shape are minor and appear at shorter wavelength
(compare Figures S2 and S3, Supporting Information).
Additionally, a positive optical rotation at 589 nm was
predicted for all eight (4S)-isomers, as found experimentally
for 1.
Due to the trans-orientation of the substituents at C-3,4,
murranofuran A must have the absolute (3S,4S) configuration,
as the (3R,4R) isomers would show a negative Cotton effect.
The ECD spectra calculated for the (3S,4S,5R,10R) and
(3S,4S,5R,10S) isomers are a better fit with the experimental
CD spectrum of 1 (Figure 2). However, a further confirmation
Figure 2. Experimental ECD curve of murranofuran A (1) and
calculated curve (thin line) for the (3S,4S,5R,10R) isomer.
of the configuration at C-5 and C-10 based on ECD and NMR
data was not possible, and therefore only the (3S,4S)
configuration is reliable. Further details are discussed in the
Supporting Information. It is worth mentioning that 2,3-
dihydrofurans are very rare in nature, with most of those
reported to date being fungal metabolites related to aflatoxins.16
Murranolide A (2) was obtained as an optically active,
amorphous solid. The molecular formula of C12H18O6 was
assigned on the basis of (+)-ESI HRMS and NMR data (Table
1). The IR absorptions at νmax 3442 and 1768 cm−1 together
DOI: 10.1021/acs.jnatprod.6b00785
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Journal of Natural Products
with resonances in the 13C NMR spectrum at 172.1 and 67.0
were indicative of a butenolide and hydroxy groups,
respectively.
In the COSY data, two spin systems from H-2 to H-3 and
from H2-5 to H3-10 were identified. The carbonyl signal at δC
172.1 (C-1), along with the acetal resonance at δC 111.5 (C-4),
the COSY correlation between H-2 and H-3, and the HMBC
correlations between H-2,3/C-1 and H-2,3/C-4, suggested the
presence of an α,β-unsaturated five-membered lactone ring.
The second spin system was connected to C-4 of the
butenolide, as confirmed by an HMBC correlation of H2-5
with C-3,4 (Figure 1). One of the methoxy groups (δH 3.19, δC
51.6, OMe-11) was attached to C-4, while the other one at δH
3.31 (δC 58.2, OMe-12) was connected to C-6, as determined
by HMBC correlations of H3-11 and H3-12 with C-4 and C-6,
respectively. In addition to the butenolide there must be an
additional ring to account for the remaining double-bond
equivalent. Using the carbon shifts, this was assigned as an
epoxide at C-7,8. The planar structure of 2 was further
established by comprehensive analysis of 2D NMR data.
Compound 2 is a new oxygenated polyketide, assigned as
murranolide A. The related plakilactones are produced by
marine sponges.17
The relative configuration of the 7,8-epoxide in 2 was derived
from the magnitude of the coupling constant between H-7 and
H-8, whose value (J = 4.4 Hz) indicated a cis disubstitution.
This was confirmed by the nuclear Overhauser signals between
H3-10/H2-5 and H3-10/H-6. The absolute configuration of C-9
in 2 was addressed by the modified Mosher’s method. The
positive ΔδH value for H3-10 and negative ΔδH values for the
remaining protons (Figure 3) indicated a (9S)-configuration in
Figure 3. ΔδH (δH = δS − δR) values for MTPA esters 2a and 2b of 2
and 4c and 4d of 4b.
2.18 The configuration of the remaining stereocenters was
determined by computational methods as for 1. Due to the
(9S)-configuration and the cis-configured oxirane, only two
series with four (4R*,6R*,7R,8S,9S)- and four
(4R*,6R*,7S,8R,9S)-configured diastereomers had to be
calculated. It was expected that the butenolide was responsible
for the Cotton effect at ∼250 nm. The calculations predicted a
negative signal for the (4R)-configuration, while the (6S)-
configuration is responsible for the trough at 220 nm, as was
observed experimentally for 2. Finally, the (4R,6S,7R,8S,9S)-
configuration was assigned for 2 based on experimental versus
calculated ECD curves (Figure 4 and Supporting Information,
Figure S4).
Compound 3a was obtained as white crystals with the
molecular formula C10H12O3 determined by (+)-ESI HRMS.
The presence of hydroxy and carbonyl groups was inferred by
IR absorptions at νmax 3411 and 1714 cm−1, respectively. The
signals in the 1H and 13C NMR spectra of 3a were ascribed to
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Article
Figure 4. Experimental and calculated (thin line) ECD curves of
murranolide A (2).
one CH3, eight CH (including six olefinic), and one carbonyl
carbon by analysis of the HSQC spectrum. In the COSY
spectrum, a continuous coupling sequence was found from H-2
to H3-10 (Figure 1). This spin system was connected with a
carbonyl group (δC 163.1), as indicated by an HMBC
correlation of H-2 and H-3 with C-1. The double-bond
geometry at C-6 and C-8 in 3a was determined as E based on
the coupling constants of H-6 (J = 15.5 Hz) and H-9 (J = 14.4
Hz) (Table 2).
The sequence of three double bonds was interrupted by two
oxymethine carbons. As one double-bond equivalent was left,
the carbons C-4 and C-5 could be bridged by the remaining
oxygen atom, forming epoxide 11. This option is, however,
clearly excluded by the experimental 13C NMR shifts, which are
far away from those expected for an epoxide in an aliphatic
chain (δC ∼55−60 ppm). If we take compound 11, however,
into account as a biosynthetic intermediate, 2,3-dihydropyran-
2-one 3a/3b or butenolide 12 could be formed easily by attack
of the carboxylate on C-5 or C-4 in 11, respectively.
As no suitable HMBC correlations were found to differ-
entiate between these alternatives, the 13C NMR data of 3a/3b
and 12 (both diastereomers) were calculated using ab initio
methods. The results (see Supporting Information, Table S1)
verified the cis-dihydropyrone 3a. Corresponding compounds
from the literature are the isomers musacin D19 and
phomalactone;19 the complete agreement with the data of the
latter confirmed structure 3a additionally (see Supporting
Information, Table S1).
A conformation analysis of the diastereomers predicted for
cis-3a a coupling constant between H-4/H-5 of J = 1.5 Hz. For
trans-3b, a biaxial orientation of H-4 and H-5 dominated in the
conformer mixture, and a value of J = 8.2 Hz was calculated.
The close similarity of experimental and calculated shifts and
the coupling constant between H-4 and H-5 (J = 3.0 Hz)
indicated therefore a (S*,S*)-cis-dihydropyrane, 3a. This was
additionally confirmed by NOESY contacts between H-4 and
H-6, while the cross signal between H-4 and H-7 as expected
for 3b was missing.
Very recently diplopyrone B (3c) has been published, a
stereoisomer of 3a with a (Z)-Δ6 double bond.20 The NMR
data of 3a and 3c are clearly different, but also for 3c, we
reproduced the carbon shifts by ab initio calculations within the
experimental error limits (see Supporting Information, Table
S1). For 3c only the configuration at C-5 had been determined,
but on the basis of the shifts and the H-4,5 coupling constant of
J = 3.1 Hz, from our calculations a (4S,5S)-configuration
(corresponding to the cis-orientation) resulted for 3c as well.
As for compounds 1 and 2, the absolute configuration of 3a
was assigned from CD (Figure 5) and ORD calculations (found
DOI: 10.1021/acs.jnatprod.6b00785
J. Nat. Prod. 2017, 80, 347−355
Journal of Natural Products Article

Table 2. 13C and 1H NMR Data for 3a, 4a, and 5

3a 4a 5
no. δ Ca type δH, mult. (J in Hz)b δ Cc type δH, mult. (J in Hz)d δ Cc type δH, mult. (J in Hz)e
1 163.1 C 170.1f C 115.6 C
2 122.9 CH 6.11, d (9.4) 119.5 CH 5.64, d (12.0) 155.8 C
3 144.5 CH 6.97, dd (9.4, 5.3) 145.3 CH 6.63, t (12.0) 111.6 CH 6.51, dd (8.3, 2.0)
4 63.0 CH 4.20, dd (5.3, 3.0)f 127.3 CH 7.46, dd (15.4, 12.0) 128.7 CH 7.06, t (8.3)
5 80.9 CH 4.89, dd (6.4, 3.0) 147.1 CH 6.05, dd (15.4, 6.2) 103.8 CH 6.53, dd (8.3, 2.0)
6 121.8 CH 5.69, dd (15.5, 6.4) 73.1 CH 4.16, m 153.6 C
7 135.9 CH 6.44, dd (15.5, 10.5) 34.5 CH2 1.55, m
1.67, m
8 130.1 CH 6.08, dd (14.4, 10.5)f 36.2 CH2 1.45, m 173.4 C
1.55, m
9 132.9 CH 5.82, dq (14.4, 6.4) 68.7 CH 3.72, m 22.8 CH3 2.18, s
10 18.2 CH3 1.77, d (7.0) 23.7 CH3 1.15, d (6.0) 56.5 CH3 3.81, s
a
Measured in CDCl3 at 125 MHz. bMeasured in CDCl3 at 300 MHz. cMeasured in CD3OD at 125 MHz. dMeasured in CD3OD at 600 MHz.
e
Measured in CD3OD at 300 MHz. fVery weak signal in CD3OD at 300 MHz.

83.3°, calcd 151.2°) as a (4S,5S) isomer, which we named
murranopyrone.
Figure 5. Experimental ECD curve of murranopyrone (3a, bold line)
and calculated curve for the (4S,5S) (thin line) and (4R,5S) isomer
(dotted line).
Murranoic acid A (4a) was obtained as a colorless oil. In the
positive ion mode, the ESI HR mass spectrum showed a
pseudomolecular ion peak at m/z 223.0941 [M + H]+,
indicating the molecular formula C10H16O4. This was supported
by the 13C NMR data (Table 2). The IR spectrum displayed a
broad absorption band centered at 3350 cm−1 and a sharp band
at 1694 cm−1, suggesting the presence of hydroxy and carbonyl
functionalities, respectively. The 1H and 13C NMR resonances
were ascribed by the HSQC and HMBC spectra to one methyl
(sp3-connected), one surprisingly weak carbonyl, two methyl-
ene (sp3), two aliphatic, and four olefinic methine carbons
(Table 2).
In the COSY spectrum, a consecutive proton coupling
sequence was observed from H-2 to H3-10 (Figure 1). The
geometry of the double bonds at C-2 and C-4 was determined
as Z and E, respectively, based on the coupling constants of H-2
(J = 12.0 Hz) and H-5 (J = 15.4 Hz), respectively. The gross
structure of 4a was derived by 2D NMR data analysis (Figure
1). The absolute configuration of 4a was determined by the
modified Mosher’s method.18 All 1H signals of the (S,S)- and
(R,R)-bis-MTPA ester derivatives of the methyl ester 4b were
351
assigned by COSY experiments. The ΔδH values (ΔδH = δS −
δR) are shown in Figure 3. Negative ΔδH values for H-2 to H2-6
and positive values for H2-8 to H3-10 corresponding to the ΔδH
pattern for diesters of anti-1,4-diols (type C system) reported
by Riguera21 indicated the absolute configuration of C-6 and C-
9 in 4a as (R,R). The structure of 4a was therefore conclusively
determined as (2Z,4E,6R,9R)-6,9-dihydroxydeca-2,4-dienoic
acid.
The molecular formula of 5 was determined as C9H11NO3
based on (+)-ESI HRMS and supported by the 13C NMR
spectrum (Table 2). The IR spectrum exhibited an absorption
at 1645 cm−1, indicating the presence of an amide group. The
1
H and 13C NMR spectra revealed methyl, methoxy, and
aromatic signals. The 1H NMR data showed the typical pattern
of a 1,2,3-trisubstituted benzene ring. This was confirmed via
the observed coupling constants and the COSY correlations
(Figure 1 and Table 2). The structure of compound 5 was
conclusively determined as N-(2-hydroxy-6-methoxyphenyl)-
acetamide, based on spectroscopic data analysis. Compound 5
has been synthesized previously,10 but as no spectroscopic data
are available in the literature, they are presented herein. To the
best of our knowledge, this is the first reported isolation of
compound 5 from nature.
The structures of further known compounds were confirmed
by ESI HRMS, 1H and 13C NMR, and optical rotation data,
along with database analysis,16 as curvularin (6),22,23 (S)-
dehydrocurvularin (7),24,25 pyrenolide A (8),26 modiolide A
(9), 26,27 and 8-hydroxy-6-methoxy-3-methylisocoumarin
(10).28
Compounds 2, 3a, and 8−10 are likely derived from
hydroxylated and/or unsaturated decanoic acids such as 4a,
which are also potential precursors of 3-methylisocoumarins,
phthalides, pyrones, and the respective ring-opened acids. Most
of their aliphatic derivatives belong to either the decarestrictines
or cephalosporolides. Although several hundred C10-lactones
were reported, less than 50 of the respective unbranched free
C10-acids (type 4a) seem to be known. The 2,3-unsubstituted
butenolides of type 2 are especially rare in nature. We found
less than 20 of these in the literature.15,16 A certain degree of
similarity is observed only with ramariolides A and B.29
Compounds 1, 2, 3a, 4a, and 5−10 were subjected to
motility inhibitory and zoosporicidal activity tests against
P. capsici. The concentration and time-course activities are
presented in Table 3. Clearly, zoospore motility halting activity
DOI: 10.1021/acs.jnatprod.6b00785
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Journal of Natural Products Article

Table 3. Motility Inhibitory and Zoosporicidal Activities of 1, 2, 3a, 4a, and 5−10 against the Late Blight Phytopathogen
P. capsici
motility inhibitory and zoosporicidal activities over control (% ± SE)a
15 min 30 min 45 min 60 min
no. dose (μg/mL) inhibition lysis inhibition lysis inhibition lysis inhibition lysis
1 100 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
400 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 25 ± 4 0 ± 0
500 0 ± 0 0 ± 0 78 ± 5 0 ± 0 100 ± 0 0 ± 0 100 ± 0 5 ± 1
2 50 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
100 0 ± 0 0 ± 0 0 ± 0 0 ± 0 27 ± 4 0 ± 0 50 ± 8 0 ± 0
300 0 ± 0 0 ± 0 10 ± 0 0 ± 0 35 ± 5 0 ± 0 58 ± 4 0 ± 0
500 0 ± 0 0 ± 0 35 ± 4 0 ± 0 48 ± 6 0 ± 0 73 ± 5 0 ± 0
3a 100 0 ± 0 0 ± 0 14 ± 3 0 ± 0 50 ± 6 0 ± 0 71 ± 6 0 ± 0
200 10 ± 0 0 ± 0 28 ± 4 0 ± 0 62 ± 5 0 ± 0 82 ± 7 0 ± 0
300 43 ± 9 0 ± 0 52 ± 4 0 ± 0 81 ± 9 0 ± 0 87 ± 9 0 ± 0
500 60 ± 4 0 ± 0 100 ± 0 0 ± 0 100 ± 0 5 ± 1 100 ± 0 7 ± 2
4a 100 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 18 ± 3 0 ± 0
400 0 ± 0 0 ± 0 5 ± 1 0 ± 0 11 ± 3 0 ± 0 32 ± 5 0 ± 0
500 5 ± 1 0 ± 0 100 ± 0 0 ± 0 100 ± 0 6 ± 1 100 ± 0 8 ± 2
5 200 0 ± 0 0 ± 0 0 ± 0 0 ± 0 5 ± 2 0 ± 0 7 ± 1 0 ± 0
400 5 ± 1 0 ± 0 45 ± 3 0 ± 0 59 ± 8 0 ± 0 70 ± 5 0 ± 0
6 50 0 ± 0 0 ± 0 5 ± 2 0 ± 0 9 ± 1 0 ± 0 51 ± 6 0 ± 0
100 0 ± 0 0 ± 0 12 ± 1 0 ± 0 22 ± 3 0 ± 0 69 ± 8 0 ± 0
400 0 ± 0 0 ± 0 58 ± 3 0 ± 0 85 ± 7 0 ± 0 100 ± 0 0 ± 0
7 25 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 11 ± 2 0 ± 0
50 0 ± 0 0 ± 0 0 ± 0 0 ± 0 43 ± 6 0 ± 0 83 ± 9 0 ± 0
100 0 ± 0 0 ± 0 89 ± 5 0 ± 0 100 ± 0 0 ± 0 100 ± 0 0 ± 0
200 57 ± 4 0 ± 0 100 ± 0 0 ± 0 100 ± 0 17 ± 2 100 ± 0 40 ± 6
400 100 ± 0 50 ± 4 100 ± 0 52 ± 3 100 ± 0 55 ± 7 100 ± 0 59 ± 6
8 0.5 81 ± 5 0 ± 0 100 ± 0 0 ± 0 100 ± 0 4 ± 1 100 ± 0 5 ± 1
1.0 90 ± 7 0 ± 0 100 ± 0 0 ± 0 100 ± 0 5 ± 1 100 ± 0 7 ± 2
2.5 100 ± 0 0 ± 0 100 ± 0 5 ± 1 100 ± 0 20 ± 3 100 ± 0 22 ± 7
5 100 ± 0 0 ± 0 100 ± 0 33 ± 5 100 ± 0 45 ± 4 100 ± 0 53 ± 8
10 100 ± 0 52 ± 9 100 ± 0 64 ± 6 100 ± 0 82 ± 0 100 ± 0 94 ± 5
25 100 ± 0 85 ± 7 100 ± 0 99 ± 1 100 ± 0 100 ± 0 nt nt
9 50 0 ± 0 0 ± 0 0 ± 0 0 ± 0 5 ± 1 0 ± 0 10 ± 1 0 ± 0
100 55 ± 4 0 ± 0 65 ± 7 0 ± 0 91 ± 5 0 ± 0 95 ± 4 6 ± 1
10 50 0 ± 0 0 ± 0 5 ± 1 0 ± 0 54 ± 6 0 ± 0 68 ± 7 0 ± 0
100 49 ± 4 0 ± 0 65 ± 7 0 ± 0 72 ± 4 0 ± 0 75 ± 6 0 ± 0
200 66 ± 6 0 ± 0 78 ± 6 0 ± 0 81 ± 5 0 ± 0 83 ± 7 0 ± 0
400 81 ± 5 0 ± 0 90 ± 4 0 ± 0 93 ± 6 0 ± 0 95 ± 4 0 ± 0
500 100 ± 0 0 ± 0 100 ± 0 0 ± 0 100 ± 0 34 ± 6 100 ± 0 50 ± 6
a
Data presented here are average values ± SE of at least three replications in each dose of compound. nt, not tested. Fluazinam 500F was used as
positive control, which impaired motility (100%) and caused subsequent lysis of >50% stopped zoospores within 60 min at 0.04 μg/mL.

was increased with increasing dose and time after the treatment.
The most noticeable zoospore motility inhibitory activity was
exhibited by compound 8, where the highest activity (100%)
was achieved at a very low concentration (0.5 μg/mL) within a
short time (30 min) (Table 3). Interestingly, 53% of stopped
zoospores lysed after treatment with compound 8 at 5.0 μg/
mL. Compounds 2, 3a, 6, 7, 9, and 10 also showed more than
50% motility inhibitory activity (IC50) at a concentration of
50−100 μg/mL. The corresponding activity of the remaining
compounds was negligible.
Motility inhibitory activity against phytopathogenic perono-
sporomycete zoospores has been reported for different classes
of compounds isolated from plants and microbes. The
following inhibitory activity against zoospores was reported:
staurosporine (indolocarbazole, 12% at 1 μg/mL),30 dipropyl-
phloroglucinol (phloroglucinol, 90% at 2 μg/mL),31 quebracho
(polyflavonoid tannin, 26% at 0.5 μg/mL),32 cryptosporiopsin
352
A (polyketide, 88% at 10 μg/mL), cyclo-(L-Phe-L-Leu-L-Leu-L-
Leu-L-Leu) (oligopeptide, 88% at 10 μg/mL),33 and gageote-
trin B (lipopeptide, 63% at 10 μg/mL).34 Thus, pyrenolide A
(8) is one of the most potent zoospore motility inhibitory
metabolites reported to date. Its potent activity is probably due
to the presence of the epoxide in the molecule: the
trichothecenes (which are also epoxide-containing cyclic
lactones) showed pronounced inhibitory and zoosporicidal
activities.35
There is evidence to suggest that motile zoospores locate
hosts and aggregate at the sites of infection in response to
specific chemical signals released from the host.36,37 Flagellar
motility is critical for the life cycle and pathogenicity of
zoosporic phytopathogens, and it is likely that protein kinase is
involved in maintaining flagellar motility.30 Any disruption of
the motility of zoospores considerably decreases potential
pathogenesis caused by peronosporomycete phytopatho-
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J. Nat. Prod. 2017, 80, 347−355
Journal of Natural Products
gens.38,39 Thus, the notorious peronosporomycete phytopath-
ogens can be controlled by halting the motility of zoospores,
thereby preventing their aggregation at the host infection
sites.30
Pyrenolide A (8) showed remarkable motility inhibitory
activity against P. capsici zoospores. As this metabolite is small
in size, is biodegradable, and can be easily synthesized in the
laboratory, this secondary metabolite could be used as a
biopesticide or as a lead compound in the design of a novel
environmentally friendly agrochemical agent against P. capsici
and related phytopathogens.
■
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation values were
measured on a PerkinElmer polarimeter, model 241, at the sodium D
line (λ = 589 nm). CD spectra were recorded by a Jasco J-810
spectropolarimeter. Mass spectra were measured with a Bruker
micrOTOF 10237. The NMR spectra were measured on a Mercury-
300 (300.141 MHz), a Varian Inova 500 (125.707 MHz for 13C NMR
spectra), and a Varian Inova 600 (599.740 MHz) spectrometer.
Chemical shifts (δ) were referenced to CH3OH at 3.30 for 1H and
49.0 for 13C and to CHCl3 at 7.24 for 1H and 77.0 for 13C. IR spectra
were taken on a Jasco FT/IR-4100 type A instrument. Mosher’s
reagent was bought from Sigma-Aldrich, Germany. Size exclusion
chromatography was performed on Sephadex LH-20 (Lipophilic
Sephadex, Amersham Biosciences, Ltd.; purchased from Sigma-Aldrich
Chemie, Steinheim, Germany). Column chromatography was done on
silica gel 60 (0.063 to 0.20 nm) and preparative thin-layer
chromatography (PTLC) on MN-silica gel/p UV254 (both obtained
from Macherey-Nagel GmbH & Co. KG, Düren, Germany). All
solvents used were either analytical grade or distilled prior to use.
Fungal Material and Identification. The healthy leaves of the
curry plant (Murraya koenigii) were collected from the garden of
medicinal plants, Department of Pharmacy, Rajshahi University,
Bangladesh. The leaves were kept in a plastic zipper pack at 4 °C
for two months. A leaf was washed with tap water to remove dust and
debris. The surface of the leaf was then sterilized as described
previously.40 The excess NaOCl was removed by washing the leaves
three times with sterilized water. They were then air-dried on sterile
filter paper. The leaf was cut into ca. 1 × 1 cm pieces using sterilized
scissors and placed on M2 agar (in a Petri dish, with agar prepared
from 10 g glucose, 4 g yeast extract, 4 g malt extract, 18 g agar, and 1 L
tap water). The plate was incubated at 25 °C, and the outgrowing
fungal strain M12 was isolated and maintained on M2 agar. The fungal
strain M12 was identified as Curvularia sp. based on the sequence of its
internal transcribed spacer region (ITS) and morphology. Its ITS
sequence shows 100% identity to C. spicifera strain CBS 274.52, as well
as to C. spicifera strain CBS 199.31 (GenBank Accession No.
JN192387 and HF934915, respectively). This strain is currently
deposited in the Microbial Culture Collection at the Institute of
Organic and Biomolecular Chemistry, Georg-August-University
Göttingen, Germany, under the curatorship of Prof. Laatsch.
Fermentation and Isolation. The fungal strain M12 was
subcultured on M2 agar medium and incubated at 25 °C for 3 days.
Rice (200 g) and tap water (130 mL) were added into each of 24 P-
flasks and then sterilized (121 °C for 15 min). The P-flasks were
inoculated with well-grown agar cultures of the strain M12 (pieces 1 ×
1 cm) and incubated at 30 °C for 25 days. The whole culture medium
was extracted with EtOAc and then filtered through Celite
(diatomaceous earth). The filtrate was concentrated to dryness in
vacuo at 40 °C to obtain a reddish gum. This extract was treated with
cyclohexane to remove fats. The remainder (4.0 g) was then divided
into 70 fractions (F1−70, 15 mL for each fraction) by Sephadex LH-
20 column chromatography, eluting with CH2Cl2−MeOH (1:1).
Fractions F32 and F33 were combined and again subfractionated by
silica column chromatography, eluting with 200 mL of each of the
following solvent mixtures: 100% CH2Cl2 (SF1−31), 4% MeOH in
CH2Cl2 (SF32−53), 8% MeOH in CH2Cl2 (SF54−75), 12% MeOH
353
Article
in CH2Cl2 (SF76−91), and 20% MeOH in CH2Cl2 (SF92, 93).
Compounds 2 (1.7 mg) and 5 (1.0 mg) were purified from combined
fractions SF7 + 8 by PTLC, eluting with CHCl3−n-hexane−MeOH
(1:0.3:0.2). Similarly, fractions 28 and 29 were combined and
subfractionated by silica column chromatography, eluting with 100%
CH2Cl2 (SF1−15), 2% MeOH in CH2Cl2 (SF16−23), 4% MeOH in
CH2Cl2 (SF24−36), and 20% MeOH in CH2Cl2 (SF37−50).
Compounds 1 (1.1 mg), 6 (16.0 mg), and 8 (2.5 mg) were purified
from SF14 + 15, SF25, and SF12 + 13 by PTLC, eluting with CHCl3−
MeOH−n-hexane (2:0.4:0.6), EtOAc−n-hexane (2.1:0.9), and ace-
tone−n-hexane (1:2), respectively, whereas 9 (13.3 mg) was obtained
from SF26 by crystallization. Similarly, fractions 30 and 31 were
combined and subfractionated, eluting with 100% CH2Cl2 (SF1−123),
2% MeOH in CH2Cl2 (SF124−152), 8% MeOH in CH2Cl2 (SF153−
162), and 14% MeOH in CH2Cl2 (SF163−172). Compounds 4a (13.0
mg), 3a (3.6 mg), 7 (8.6 mg), and 10 (2.0 mg) were purified from
SF160−164, SF2, SF3, and SF127 + 128 by PTLC, eluting with
CHCl3−n-hexane−MeOH (1:0.3:0.2), EtOAc−n-hexane (0.1:1.4),
CHCl3−n-hexane−MeOH (1:0.4:0.1), and acetone−n-hexane (2:1),
respectively.
Murranofuran A (1): colorless, amorphous solid; [α]20 D +81.8 (c
0.11, MeOH); CD spectrum in MeOH, see Figure 2; IR νmax 2927,
1724, 1647, 1604, 1455, 1377, 1236, 1104 cm−1; 1H and 13C NMR
(CD3OD), see Table 1; (+)-ESI HRMS m/z 313.1646 [M + H]+
(calcd for C16H25O6, 313.1645).
Murranolide A (2): colorless, amorphous solid; [α]20 D −9.4 (c 0.17,
MeOH); CD spectrum in MeOH, see Figure 4; IR νmax 3442, 2930,
1768, 1457, 1104 cm−1; 1H and 13C NMR (CD3OD), see Table 1;
(+)-ESI HRMS m/z 281.0997 [M + Na]+ (calcd for C12H18O6Na,
281.0995).
Murranopyrone (3a): colorless crystals; [α]20 D +83.3 (c 0.36,
MeOH); CD spectrum in MeOH, see Figure 5; IR νmax 3411, 2923,
1714, 1380, 1260, 1082, 990 cm−1; 1H and 13C NMR (CDCl3), see
Table 2; (+)-ESI HRMS m/z 181.0859 [M + H]+ (calcd for C10H13O3,
181.0859).
Murranoic acid A (4a): colorless oil; [α]20D −43.4 (c 1.3, MeOH);
IR νmax 3350, 2924, 1694, 1431, 1205 cm−1; 1H and 13C NMR data
(CD3OD), see Table 2; (+)-ESI HRMS m/z 223.0941 [M + Na]+
(calcd for C10H16O4Na, 223.0940).
N-(2-Hydroxy-6-methoxyphenyl)acetamide (5): colorless, amor-
phous solid; IR νmax 2929, 1645, 1599, 1536, 1475, 1380, 1250, 1091
cm−1; 1H and 13C NMR (CD3OD), see Table 2; (+)-ESI HRMS m/z
204.0631 ([M + Na]+) (calcd for C9H11NO3Na, 204.0631).
Preparation of (S)-MTPA Ester 2a. Compound 2 (0.5 mg) was
dissolved in 300 μL of pyridine, 10 μL of (R)-(−)-MTPA-Cl was
added, and the mixture was stirred at 20 °C for 16 h and dried under
air flow. The reaction mixture was fractionated first by PTLC, eluting
with CHCl3−n-hexane−MeOH (1:0.4:0.6), and then purified on LH-
20, eluting with 20% MeOH in CH2Cl2, to yield the (S)-MTPA ester
(2a, 0.7 mg).
Compound 2a: colorless oil; 1H NMR (CD3OD, 600 MHz) δH
6.32 (1H, d, J = 5.7 Hz, H-2), 7.41 (1H, d, J = 5.7 Hz, H-3), 2.20 (2H,
m, H2-5), 3.14 (1H, m, H-6), 3.15 (1H, dd, J = 6.9, 4.0 Hz, H-7), 2.98
(1H, dd, J = 9.0, 4.0 Hz, H-8), 5.12 (1H, m, H-9), 1.45 (3H, d, J = 6.5
Hz, H3-10), 3.21 (3H, s, H3-11), 3.35 (3H, s, H3-12), 3.54 (3H, s,
OMe), 7.54−7.41 (5H, m); (+)-ESI HRMS m/z 497.1395 [M + Na]+
(calcd for C22H25F3O8Na, 497.1393).
Preparation of (R)-MTPA Ester 2b. The (R)-MTPA ester (2b) of
compound 2 (0.5 mg) was prepared from (S)-(+)-MTPA-Cl and
purified in the same way as 2a to obtain 2b (0.7 mg).
Compound 2b: colorless oil; 1H NMR (CD3OD, 600 MHz) δH
6.32 (1H, d, J = 5.7 Hz, H-2), 7.41 (1H, d, J = 5.7 Hz, H-3), 2.21 (2H,
m, H2-5), 3.17 (1H, m, H-6), 3.20 (1H, dd, J = 8.3, 4.2 Hz, H-7), 3.07
(1H, dd, J = 9.1, 4.2 Hz, H-8), 5.09 (1H, dd, J = 9.1, 6.5 Hz, H-9), 1.32
(3H, d, J = 6.5 Hz, H3-10), 3.21 (3H, s, H3-11), 3.35 (3H, s, H3-12),
3.57 (3H, s, OMe), 7.55−7.40 (5H, m, Phe); (+)-ESI HRMS m/z
497.1400 [M + Na]+ (calcd for C22H25F3O8Na, 497.1393).
Preparation of Methyl Ester 4b. Compound 4a (6.0 mg) was
dissolved in MeOH, and an excess of diazomethane solution was
added. After a few seconds at room temperature, the mixture was
DOI: 10.1021/acs.jnatprod.6b00785
J. Nat. Prod. 2017, 80, 347−355
Journal of Natural Products
evaporated to dryness and then purified by PTLC, eluting with
CHCl3−n-hexane−MeOH (1:0.4:0.1), to deliver the methyl ester 4b
(4.0 mg).
Compound 4b: colorless, amorphous solid; 1H NMR (CD3OD,
300 MHz) δH 5.67 (1H, d, J = 10.0 Hz, H-2), 6.67 (1H, d, J = 10.0 Hz,
H-3), 7.48 (1H, dd, J = 15.0, 10.0 Hz, H-4), 6.09 (1H, dd, J = 15.0, 6.0
Hz, H-5), 4.18 (1H, m, H-6), 1.55 (1H, m, H-7b), 1.66 (1H, m, H-7a),
1.46 (1H, m, H-8b), 1.55 (1H, m, H-8a), 3.72 (1H, m, H-9), 1.15 (3H,
d, J = 6.0 Hz, H-10), 3.69 (3H, s, OMe); (+)-ESI HRMS m/z
237.1097 [M + Na]+ (calcd for C11H18O4Na, 237.1097).
Preparation of (S,S)-Bis-MTPA Ester 4c. The methyl ester 4b
(2.0 mg) was dissolved in 1.0 mL of pyridine; 15 μL of (R)-
(−)-MTPA-Cl was added to the reaction vial, stirred at 20 °C for 16 h,
dried under air flow, redissolved in EtOAc, washed with H2O, and
purified by PTLC, eluting with CHCl3−n-hexane−MeOH (1:0.3:0.4),
to obtain 4c (3.0 mg).
Compound 4c: colorless oil; 1H NMR (CD3OD, 600 MHz) δH
5.74 (1H, d, J = 11.4 Hz, H-2), 6.58 (1H, t, J = 11.4 Hz, H-3), 7.47
(1H, overlapped, H-4), 5.83 (1H, dd, J = 15.5, 6.7 Hz, H-5), 5.48 (1H,
m, H-6), 1.58 (1H, m, H-7b), 1.63 (1H, m, H-7a), 1.60 (2H, m, H-8),
5.12 (1H, m, H-9), 1.32 (3H, d, J = 6.2 Hz, H-10), 7.48−7.33 (10H,
m), 3.70, 3.54, 3.49 (3 × 3H, s, 3 OMe); (+)-ESI HRMS m/z
669.1894 [M + Na]+ (calcd for C31H32F6O8Na, 669.1893).
Preparation of (R,R)-Bis-MTPA Ester 4d. The (R,R)-bis-MTPA
ester 4d was prepared from 4b (2.0 mg) and (S)-(+)-MTPA-Cl in the
same way as for 4c to obtain 4d (3.4 mg).
Compound 4d: colorless oil; 1H NMR (CD3OD, 600 MHz) δH
5.76 (1H, d, J = 11.3 Hz, H-2), 6.66 (1H, t, J = 11.3 Hz, H-3), 7.61
(1H, dd, J = 15.6, 11.3 Hz, H-4), 6.08 (1H, dd, J = 15.6, 6.3 Hz, H-5),
5.60 (1H, m, H-6), 1.70 (1H, m, H-7b), 1.76 (1H, m, H-7a), 1.47 (2H,
m, H-8), 5.03 (1H, m, H-9), 1.13 (3H, d, J = 6.2 Hz, H-10), 7.34
(10H, m), 3.70, 3.57, 3.47 (3 × 3H, s, 3 OMe); (+)-ESI HRMS m/z
669.1894 [M + Na]+ (calcd for C31H32F6O8Na, 669.1893).
Computational Methods. Conformer distributions were searched
using a systematic approach with MMFF, implemented in
Spartan’14.41 The geometries of all resulting conformers within an
energy range of <25 kJ/mol above the global minimum were further
optimized, first with HF/3-21G and then within <15 kJ/mol above the
minimum with DFT using the wB97X-D functional and the 6-31G*
basis set. The resulting geometries with preliminary Boltzmann factors
of >0.001 were used to calculate the ECD spectra with rB3LYP/6-
311G(2d,p), the ORD data with rwB97XD/6-311G(d,p), and the
energies for the final Boltzmann factors (without further geometry
optimization) with wB97XD/6-311+G(2df,2p), using Gaussian 09.42
The contributions of all remaining conformers were averaged with
respect to their final Boltzmann factors.
Bioassay. P. capsici was isolated from chili peppers and kindly
provided by Prof. W. Yuanchao of Nanjing Agricultural University,
China; the strain was routinely maintained on 10% V8 agar media43 at
25 °C in the dark. Samples of mycelia, sporangia, and zoospores were
prepared according to the following steps. Hyphal tip plugs were used
to inoculate 30 mL of sterile clarified 10% V8 agar43 in Petri dishes (90
mm diameter). These were then incubated at 25 °C in the dark for 4
days. To induce the sporangium formation, the agar culture from each
1- to 2-week-old Petri dish was cut into six pieces, covered with sterile
distilled water, and kept in new Petri dishes at 25 °C in the dark. The
water in the dishes was replaced by new sterile distilled water every 12
h. After changing the distilled water three times, plenty of sporangia
had been produced. To stimulate the release of zoospores, the culture
with sporangia was placed at 4 °C for 30 min and then incubated at
room temperature for another 30 min. The sporangia and zoospores
were checked under an Olympus light microscope. These zoospores
remained motile for 10−12 h in sterilized water and were used for the
bioassay. Stock solutions of pure compounds were first prepared in
dimethyl sulfoxide (DMSO) and then diluted with distilled water. The
concentration of DMSO in the zoospore suspension never exceeded
1% (v/v), a condition that does not affect zoospore motility. The
bioassay was carried out as described earlier.34 Briefly, 40 μL of sample
solution was added to 360 μL of zoospore suspension (ca. 1 × 105 per
mL) taken in a dish of plant tissue culture multiwell plate to make a
354
Article
final volume of 400 μL and then quickly mixed with a glass rod; 1%
aqueous DMSO was used as control. The motility of zoospores was
observed under a light microscope at 10-fold magnification.
Quantification of time-course changes of motility and lysis of
zoospores were carried out as described previously.32
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00785.
1
H and 13C NMR spectra for compounds 1, 2, 3a, 4a,
and 5; 1H NMR data for 3a at 300 MHz in CD3OD;
calculated CD spectra for compounds 1 and 2; and NMR
data for 3a (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Tel (H. Laatsch): +49 551 3933211. E-mail: hlaatsc@gwdg.de.
ORCID
Muhammad Abdul Mojid Mondol: 0000-0002-8495-4498
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We thank the Alexander von Humboldt Foundation for a
Georg Forster Fellowship to M.A.M.M. and financial support
for this research. The authors are indebted to Dr. H.
Frauendorf and Dr. M. John for MS and NMR measurements,
respectively; we also thank Dr. S. Hickford for careful language
polishing. J.F. and M.T.I. are thankful to the World Bank for
funding under an HEQEP subproject CP #2071 to UGC in
Bangladesh.
■
REFERENCES
(1) Haverkort, A. J.; Boonekamp, P. M.; Hutten, R.; Jacobsen, E.;
Lotz, L. A. P.; Kessel, G. J. T.; Visser, R. G. F.; Vossen, E. A. G. Potato
Res. 2008, 51, 47−57.
(2) Lamour, K. H.; Stam, R.; Jupe, J.; Huitema, E. Mol. Plant Pathol.
2012, 13, 329−337.
(3) Judelson, H. S.; Blanco, F. A. Nat. Rev. Microbiol. 2005, 3, 47−58.
(4) Walker, C. A.; West, P. Fungal Biol. Rev. 2007, 21, 10−18.
(5) Judelson, H. S.; Roberts, S. Eukaryotic Cell 2002, 1, 687−695.
(6) Damalas, C. A.; Eleftherohorinos, I. G. Int. J. Environ. Res. Public
Health 2011, 8, 1402−1419.
(7) Kim, H. Y.; Choi, G. J.; Lee, H. B.; Lee, S. W.; Lim, H. K.; Jang,
K. S.; Son, S. W.; Lee, S. O.; Cho, K. Y.; Sung, N. D.; Kim, J. C. Lett.
Appl. Microbiol. 2007, 44, 332−337.
(8) Schulz, B.; Boyle, C.; Draeger, S.; Römmert, A.-K.; Krohn, K.
Mycol. Res. 2002, 106, 996−1004.
(9) Zhang, D. X.; Nagabhyru, P.; Schardl, C. L. Plant Physiol. 2009,
150, 1072−1082.
(10) Clay, K.; Cheplick, G. P. J. Chem. Ecol. 1989, 15, 169−182.
(11) Prestidge, R. A.; Ball, O. J. -P. In Multitrophic Interactions in
Terrestrial Systems; Gange, A. C.; Brown, V. K., Eds.; Oxford: Blackwell
Scientific Press: London, 1997; Chapter 2, pp 171−192.
(12) Thodsare, N. H.; Bhatt, P.; Srivastava, R. P. J. Entomol. Zool.
Stud. 2014, 2, 201−205.
(13) Nakajima, H.; Hamasaki, T.; Maeta, S.; Kimura, Y.; Takeuchi, Y.
Phytochemistry 1990, 29, 1739−1743.
(14) Stierle, A. A.; Stierle, D. B.; Kelly, K. J. Org. Chem. 2006, 71,
5357−5360.
(15) The Dictionary of Natural Products on CD-ROM. 2014.
Chapmann & Hall/CRC Press. A detailed description is available on
DOI: 10.1021/acs.jnatprod.6b00785
J. Nat. Prod. 2017, 80, 347−355
Journal of Natural Products Article

the internet via http://dnp.chemnetbase.com-/intro/ M., Ritchie, J.; Rosta, E.; Sherrill, C. D.; Simmonett, A. C.; Subotnik, J.
DNPIntroduction.pdf (August 2014). E.; Woodcock, H., III; Zhang, W.; Bell, A. T.; Chakraborty, A. K.;
(16) Laatsch, H. AntiBase, a database for rapid structural determination Chipman, D. M.; Keil, F. J., Warshel, A.; Hehre, W. J.; Schaefer, H. F.,
of microbial natural products, and annual updates; Wiley-VCH: III; Kong, J.; Krylov, A. I.; Gilla, P. M. W.; Head-Gordon, M. Phys.
Weinheim, Germany, 2013; see http://wwwuser.gwdg.de/~hlaatsc/ Chem. Chem. Phys. 2006, 8, 3172−3191; SPARTAN’14; Wavefunction:
antibase.htm. Irvine, CA, USA, 2014.
(17) Micco, S. D.; Zampella, A.; D’Auria, M. V.; Festa, C.; De (42) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Marino, S.; Riccio, R.; Butts, C. P.; Bifulco, G. Beilstein J. Org. Chem. Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
2013, 9, 2940−2949. B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
(18) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Soc. 1991, 113, 4092−4096. Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
(19) Wu, H.-Y.; Wang, Y.-L.; Tan, J.-L.; Zhu, C.-Y.; Li, D.-X.; Huang, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, A., Jr.;
R.; Zhang, K.-Q.; Niu, X.-M. J. Agric. Food Chem. 2012, 60, 5604− Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,
5608. K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
(20) Masi, M.; Maddau, L.; Linaldeddu, B. T.; Cimmino, A.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega,
D’Amico, W.; Scanu, B.; Evidente, M.; Tuzi, A.; Evidente, A. J. Agric. N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.;
Food Chem. 2016, 64, 217−225. Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
(21) Freire, F.; Seco, J. M.; Emilio, Q.; Riguera, R. J. Org. Chem. Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;
2005, 70, 3778−3790. Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.;
(22) Mohapatra, D. K.; Rahaman, H.; Pal, R.; Gurjar, M. K. Synlett Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .;
2008, 12, 1801−1804. Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09;
(23) Tadross, P. M.; Virgil, S. C.; Stoltz, B. M. Org. Lett. 2010, 12, Gaussian, Inc.: Wallingford, CT, 2009.
1612−1614. (43) Guo, L. Y.; Ko, W. H. Appl. Environ. Microbiol. 1993, 59, 2323−
(24) Vurro, M.; Evidente, A.; Andolfi, A.; Zonno, M. C.; Giordano, 2325.
F.; Motta, A. Plant Sci. 1998, 138, 67−79.
(25) Greve, H.; Schupp, P. J.; Eguereva, E.; Kehraus, S.; Kelter, G.;
Maier, A.; Fiebig, H.-H.; König, G. M. Eur. J. Org. Chem. 2008, 2008,
5085−5092.
(26) Trisuwan, K.; Rukachaisirikul, V.; Phongpaichit, S.; Preedanon,
S.; Sakayaroj, J. Arch. Pharmacal Res. 2011, 34, 709−714.
(27) Evidente, A.; Cimmino, A.; Berestetskiy, A.; Andolfi, A.; Motta,
A. J. Nat. Prod. 2008, 71, 1897−1901.
(28) Gremaud, G.; Tabacchi, R. Nat. Prod. Lett. 1994, 5, 95−103.
(29) Centko, R. M.; Ramon-Garcia, S.; Taylor, T.; Patrick, B. O.;
Thompson, C. J.; Miao, V. P.; Andersen, R. J. J. Nat. Prod. 2012, 75,
2178−2182.
(30) Islam, M. T.; Tiedemann, A.v.; Laatsch, H. Mol. Plant-Microbe
Interact. 2011, 24, 938−947.
(31) Islam, M. T.; Tiedemann, A. v. World J. Microbiol. Biotechnol.
2011, 27, 2071−2079.
(32) Islam, M. T.; Ito, T.; Sakasai, M.; Tahara, S. J. Agric. Food Chem.
2002, 50, 6697−6703.
(33) Talontsi, F. M.; Facey, P.; Tatong, M. D. K.; Islam, M. T.;
Frauendorf, H.; Draeger, S.; Tiedemann, Av.; Laatsch, H. Phytochem-
istry 2012, 83, 87−94.
(34) Tareq, F. S.; Lee, M. A.; Lee, H.-S.; Lee, Y.-J.; Lee, J. S.; Hasan,
C. M.; Islam, M. T.; Shin, H. J. Org. Lett. 2014, 16, 928−931.
(35) Mondol, M. A. M.; Surovy, M. Z.; Islam, M. T.; Schüffler, A.;
Laatsch, H. J. Agric. Food Chem. 2015, 63, 8777−8786.
(36) Horio, T.; Kawabata, Y.; Takayama, T.; Tahara, S.; Kawabata, J.;
Fukushi, Y.; Nishimura, H.; Mizutani, J. Experientia 1992, 48, 410−
414.
(37) Morris, P. F.; Ward, E. W. B. Physiol. Mol. Plant Pathol. 1992, 40,
17−22.
(38) Judelson, H. S.; Blanco, F. A. Nat. Rev. Microbiol. 2005, 3, 47−
58.
(39) Judelson, H. S.; Roberts, S. Eukaryotic Cell 2002, 1, 687−695.
(40) Talontsi, F. M.; Douanla-Meli, C.; Laatsch, H. Z. Naturforsch., B:
J. Chem. Sci. 2013, 68b, 1259−1264.
(41) Shao, Y.; Molnar, L. F; Jung, Y.; Kussmann, J.; Ochsenfeld, C.;
Brown, S. T.; Gilbert, A. T. B.; Slipchenko, L. V.; Levchenko, S. V.;
O’Neill, D. P.; DiStasio, R. A., Jr.; Lochan, R. C.; Wang, T.; Beran, G. J.
O.; Besley, N. A.; Herbert, J. M.; Lin, C. Y.; Voorhis, T. V.; Chien, S.
H.; Sodt, A.; Steele, R. P.; Rassolov, V. A.; Maslen, P. E.; Korambath,
P. P.; Adamson, R. D.; Austin, B.; Baker, J.; Byrd, E. F. C.; Dachsel, H.;
Doerksen, R. J.; Dreuw, A.; Dunietz, B. D.; Dutoi, A. D.; Furlani, T. R.;
Gwaltney, S. R.; Heyden, A.; Hirata, S.; Hsu, C.-P.; Kedziora, G.;
Khalliulin, R. Z.; Klunzinger, P.; Lee, A. M.; Lee, M. S.; Liang, W. Z.;
Lotan, I.; Nair, N.; Peters, B.; Proynov, E. I.; Pieniazek, P. A.; Rhee, Y.

355 DOI: 10.1021/acs.jnatprod.6b00785

J. Nat. Prod. 2017, 80, 347−355