Alternethanoxins A and B, polycyclic ethanones produced by Alternaria sonchi, potential mycoherbicides for Sonchus arvensis biocontrol

Article abstract of DOI:10.1021/jf9014944

Alternaria sonchi is a fungai pathogen isolated from Sonchus arvensis and proposed as a biocontrol agent of this noxious perennial weed. Different phytotoxic metabolites were isolated from solid culture of the fungus. Two new polycyclic ethanones, named a

Full text of DOI:10.1021/jf9014944

6656 J. Agric. Food Chem. 2009, 57, 6656–6660
DOI:10.1021/jf9014944
Alternethanoxins A and B, Polycyclic Ethanones Produced by
Alternaria sonchi, Potential Mycoherbicides for Sonchus
arvensis Biocontrol
†
A
NTONIO
E
VIDENTE,*,† BIANCAVALERIA
‡
P
UNZO,† ANNA
NDREA OTTA
A
NDOLFI
§
,
A
LEXANDER
B
ERESTETSKIY
,
AND
A
M
†
ꢀ
Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Universita di
‡
ꢀ
Napoli Federico II, Via Universita 100, 80055 Portici, Italy, All-Russian Institute of Plant Protection,
Russian Academy of Agricultural Science, Pushkin, Saint Petersburg 196608, Russia, and §Istituto di
Chimica Biomolecolare, CNR, Comprensorio Olivetti, Edificio 70, Via Campi Flegrei 34,
80078 Pozzuoli, Italy
Alternaria sonchi is a fungal pathogen isolated from Sonchus arvensis and proposed as a biocontrol
agent of this noxious perennial weed. Different phytotoxic metabolites were isolated from solid
culture of the fungus. Two new polycyclic ethanones, named alternethanoxins A and B, were
characterized using essentially spectroscopic and chemical methods. Tested by leaf disk-puncture
assay on the fungal host plant and a number of nonhost plants, alternethanoxins A and B were
shown to be phytotoxic, whereas they did not possess antimicrobial activity tested at 100 μg/disk.
Hence, alternethanoxins A and B have potential as nonselective natural herbicides. Some
structure-activity relationship observations were also made.
KEYWORDS: Sonchus arvensis; Alternaria sonchi; phytotoxins; polycyclic ethanones; alternethano-
xins A and B; bioherbicides
INTRODUCTION
weed pathogens can be a source of such useful metabolites. For
instance, potent phytotoxins were isolated from culture filtrate or
mycelia of some mycoherbicidal fungi belonging to the genera
Alternaria, Ascochyta, Drechslera, Ophiobolus, Phoma, and many
others (9, 10). Recently, the fungus Alternaria sonchi has been
evaluated as a possible biocontrol agent of sowthistle (11). Species
belonging to the genus Alternaria are known to produce bioactive
metabolites, including nonhost phytotoxins, for example, sola-
napyrones, isolated from cultures of A. solani, the causal agent of
early blight of tomato and potato (12); dextrusins and cyclodep-
sipeptides, isolated from A. brassicae, which causes diseases on
numerous oil-yielding, vegetable, condiment, ornamental, and
wild and some cultivated and wild noncruciferous plants (13);
brefeldin and R,β-dehydrocurvularin isolated from A. zinniae (14)
and several phytotoxins belonging to different groups of natural
compounds including toxic tetramic acids, dibenzo[R]pyrone
moiety containing compounds, and alternatoxins I and II (15, 16).
Phytotoxins produced by A. sonchi have not been studied so far.
Considering the interest in bioactive metabolites produced by
weed pathogens as sources of novel natural herbicides, it seemed
interesting to investigate the production of toxins by this species
of Alternaria.
Perennial weeds are common problems in different crops. They
are especially harmful in agricultural systems with reduced
herbicide usage because of their tolerance to traditional mechan-
ical control methods. Such a typical plant species is Sonchus
arvensis L., commonly called perennial sowthistle (1, 2). This
weed grows vigorously and forms dense patches. It spreads with
underground shoots growing horizontally that give rise to new
aerial shoots. Prolific seed production by S. arvensis serves for the
occupation of new area. This plant species is considered to be an
important weed in Europe and North America as it infests many
habitats such as cultivated fields, roadsides, pastures and range-
lands, railway embankments, and lawns. It is not easy to eradicate
S. arvensis, and all control methods require follow-up: combina-
tions of mechanical, cultural, and chemical methods are more
effective than any single method used alone (3). Herbicides
recommended for chemical control of S. arvensis in nonorganic
cropping systems are restricted to a few active substances
(clopyralid, dicamba, chlorsulfuron, bentazon, phenoxy acids)
(2,4,5). Obviously, new compounds should be actually developed
as herbicides against this weed.
Microbial phytotoxins or their synthetic analogues could be
used for the development of new agrochemicals against
weeds (6, 7). Many plant pathogens, especially necrotrophic
and hemibiotrophic fungi, are capable of producing phytotoxins
responsible for disease development (8). Therefore, appropriate
This study was undertaken to isolate, elucidate the structures
of, and characterize the biological activity of two new phytotoxic
polycyclic ethanones produced in solid culture by A. sonchi,
named alternethanoxins A and B (1 and 2). Their structure was
determined by extensive use of spectroscopic (essentially NMR
and MS techniques) and chemical methods.
*Author to whom correspondence should be addressed (telephone
þ39 0812539178; fax þ39-081-2539186; e-mail evidente@unina.it).
pubs.acs.org/JAFC
Published on Web 07/13/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 15, 2009 6657
Table 1. 1H and 13C NMR Data of Alternethanoxins A (1) and B (2)a,b
1
2
compd position
δC mc
δH
HMBC
MeCO
δC mc
δH
HMBC
1
148.8 s
128.6 s
109.5 d
160.0 s
160.0 s
109.5 d
153.0 s
167.3 s
122.2 d
131.0 d
121.4 d
130.4 s
109.8 s
148.8 s
112.4 s
107.2 d
161.1 s
155.6 s
111.4 d
150.8 s
169.2
MeCO
2
HOC-C(3), H-3, MeCO
HOC-C(3), MeCO
HOC-C(3)
3
6.21 s
6.21 s
MeCO
H-3
6.73 s
6.62 s
4
5
H-3
6
6a
7
H-10, H-9
H-8, OMe
H-10, H-9
H-8
H-8, H-9
H-8, OMe
H-9
8
7.46 d (J = 7.5 Hz)
7.34 dd (J = 7.5 and 7.1 Hz)
7.11 d (J = 7.1 Hz)
125.4 d
122.2 d
152.2 s
155.6 s
118.6 s
7.36 d (J J=9.0 Hz)
7.47 d (J = 9.0 Hz)
9
H-8
10
10a
10b
11
12
13
MeO
MeCO
MeCO
H-8
H-9
H-9
H-6 and/or H-3
52.5 q
198.7 s
22.0 q
3.62 s
2.23 s
53.2 q
180.1 s
22.7 q
4.00 s
2.43 s
H-3
H-3
H-3
H-3
1
1
1
a The chemical shifts are in δ values (ppm) from TMS. b 2D H, H (COSY) 13C, H (HSQC) NMR experiments delineated the correlations of all the protons and the
corresponding carbons. c Multiplicities were assigned by DEPT spectra.
MATERIALS AND METHODS
by preparative TLC on silica gel [eluent CHCl3/i-PrOH (95:5, v/v)], to
yield eight fractions. The residue (51 mg) of the sixth fraction appeared
to be a homogeneous yellow solid, which was named alternethanoxin A (1,
Rf 0.38; 51.0 mg). The residue of the fourth fraction was purified by
preparative TLC on reverse phase [eluent EtOH/H2O (6:4, v/v)] to yield an
amorphous solid, which was named alternethanoxin B (2, Rf 0.47; 2.2 mg).
General Experimental Procedure. Optical rotation was measured in
CHCl3 solution on a JASCO (Tokyo, Japan); IR spectra were recorded as
neat on a Perkin-Elmer (Norwalk, CT) Spectrum One FT-IR spectro-
meter, and UV spectra were taken in MeCN solution on a Perkin-Elmer
spectrophotometer. 1H spectra were recorded at 600 and 400 MHz, in
CDCl3 on Bruker (Kalsrhue, Germany) spectrometers. 13C NMR spectra
were recorded at 150, 100, and 75 MHz, in the same solvent and using the
same instruments. The same solvent was used as internal standard. Carbon
multiplicities were determined by DEPT experiments (17). DEPT, COSY-
45, TOCSY, HSQC, HMBC, and NOESY experiments (17) were per-
formed using Bruker microprograms. ESI and HRESI MS spectra were
recorded on Waters Micromass and Agilent coupled to JEOL AccuTOF
(Milford, MA) instruments. Analytical and preparative TLC was per-
formed on silica gel (Kieselgel 60 F254, 0.25 and 0.50 mm, respectively,
Alternethanoxin A (1). was obtained as an amorphous solid: [R]25
-
D
16° (c 0.2); IR νmax 3341, 1697, 1635, 1583, 1515, 1291 cm-1; UV λmax
(log ε) nm 381 (sh), 299 (3.82), 241 (4.07); 1H and 13C NMR spectra, see
Table 1; HRESIMS (þ), m/z 627 [2M þ Na]þ, 325.0701 [C16H14NaO6
calcd 325.0688, M þ Na]þ, 287 [M - Me]þ.
Alternethanoxin B (2). was obtained as an amorphous solid: [R]25
-
D
32.5° (c 0.1); IR νmax 3232, 1688, 1656, 1608, 1589, 1519, 1291, 1259 cm-1
;
UV λmax (log ε) nm 381 (3.6), 294 (3.8), 262 (4.4), 237 (4.3); 1H and 13
C
NMR spectra, see Table 1; HRESI MS, (þ) m/z 623 [2M þ Na]þ 323.0541
[C16H12NaO6 calcd 323.0532, M þ Na]þ.
Merck, Darmstadt, Germany) or reverse phase (Whatman, KC18 F254
,
Triacetylalternethanoxin A (3). Alternethanoxin A (1, 10.0 mg) was
acetylated with acetic anhydride (70 μL) and pyridine (70 μL) at room
temperature overnight. The reaction was stopped by the addition of
MeOH and evaporated by a N2 stream. The residue (11.0 mg) was purified
by preparative TLC on silica gel [(eluent CHCl3/i-PrOH (98:2, v/v)],
yielding the triacetyl derivative of alternethanoxin A (3) as an amorphous
solid (Rf 0.56, 8.0 mg): [R]25D -15° (c 0.2); IR νmax 1770, 1724, 1670, 1620,
1575, 1433, 1176 cm-1; UV λmax (log ε) nm 287 (sh), 253 (3.93); 1H NMR,
δ 7.82 (1H, d, J=7.7 Hz, H-8), 7.50 (1H, dd, J=8.0, 7.7 Hz, H-9), 7.40 (1H,
d, J=8.0 Hz, H-10), 6.86 (2H, each s, H-3 and H-6) 3.72 (3H, s, OMe), 2.40
(3H, MeCO), 2.02 (3H, MeCOO), 1.95 (6H, 2 ꢀ MeCOO); 13C NMR,
δ 188.4 (MeCO), 168.8 (2 ꢀ MeCOO), 168.5 (MeCOO), 165.9 (C-7), 150.5
(2C, s, C-4 and C-5), 147.6 (C-1), 144.2 (2C, s, C6a and C-10a), 136.2 (C-
10b), 130.6 (C-2), 129.0 (d, C-9), 127.3 and 127.2 (2C, d, C-8 and C-10),
122.0 (2C, d, C-3 and C-6), 52.5 (OMe), 21.55 (MeCOO), 20.5 (3 ꢀ
MeCOO); ESIMS (þ), m/z 879 [2M þ Na]þ, 451[M þ Na]þ.
0.20 mm, Maidstone, U.K.) plates; the spots were visualized by exposure
to UV light or by spraying first with 10% H2SO4 in methanol and then
with 5% phosphomolybdic acid in ethanol, followed by heating at 110 °C
for 10 min. For column chromatography (CC) Kieselgel 60, 0.063-
0.200 mm, was used (Merck, Darmstadt, Germany).
Fungus. The fungus A. sonchi Davis was isolated from diseased leaves
of S. arvensis, and monoconidial isolate (S-102) was deposited in the
culture collection of All-Russian Research Institute of Plant Protection,
Pushkin, Saint Petersburg, Russia. The isolate was maintained in sterile
tubes containing potato dextrose agar.
Production, Extraction, and Purification of Alternethanoxins A
and B (1 and 2). A. sonchi was grown on autoclaved pearl barley in 10
1000-mL Erlenmeyer flasks (pearl barley, 100 g; water, 60 mL) for 21 days
in darkness. Fungal metabolites were extracted from dry mycelium
according to a slightly modified protocol of Evidente et al. (18). The dried
material was extracted with a mixture of acetone/2% NaCl (1:1, 2 L). After
evaporation of acetone, the aqueous residue was extracted with EtOAc
(3 ꢀ 500 mL). The organic extracts were combined, dried (Na2SO4), and
evaporated under reduced pressure, yielding a brown oily residue (975 mg).
The organic extract, showing high phytotoxicity, was purified by silica gel
column chromatography eluted with the CHCl3/i-PrOH (9:1, v/v), to give
11 groups of homogeneous fractions. Fractions were tested for bioactivity
against S. arvense as described below, and those showing phytotoxicity
were further purified. The residue (174.7 mg) of the fourth fraction was
purified by silica gel column, eluted with CHCl3/i-PrOH (95:5, v/v), to
yield five fractions. The residue (88 mg) of the second fraction was purified
Alternethanoxin A Dimethyl Ether (4). To alternethanoxin A (1,
4.0 mg), dissolved in MeOH (0.5 mL), was added an ethereal solution of
diazomethane. The reaction was carried out overnight at room tempera-
ture in the dark. The reaction was stopped by evaporation under N2
stream. The residue (4.2 mg) was purified by preparative TLC on silica gel
[(eluent petroleum/Me2CO (8:2, v/v)], yielding alternethanoxin A dimethyl
ether (4) as an amorphous solid (Rf 0.31, 2.0 mg): [R]25D-17 (c 0.2); IR
ν
max 2923, 1721, 1628, 1600, 1574, 1464, 1277 cm-1; UV λmax (log ε) nm
337 (sh), 285 (3.84); 1H NMR, δ 12.95 (ΟH, s), 7.61 (1H, d, J=7.8 Hz,
H-8), 7.36 (1H, dd, J=7.8, 7.7 Hz, H-9), 7.11 (1H, d, J=7.7 Hz, H-10), 6.46
(1H, s, H-3), 6.05 (1H,s, H-6), 3.74 (3H, s, OMe), 3.72 (3H, s, OMe), 3.31

6658 J. Agric. Food Chem., Vol. 57, No. 15, 2009
Evidente et al.
(3H, s, OMe), 2.29 (3H, s, MeCO); ESIMS (þ), m/z 683 [2MþNa]þ, 353
[M þ Na]þ.
(S)-R-Methoxy-R-trifluorophenylacetate (MTPA) Ester of
Alternethanoxin A (5). (R)-(-)-MPTA-Cl (20 μL) was added to
alternethanoxin A (1, 2.0 mg) and dissolved in dry pyridine (40 μL). The
mixture was kept at room temperature. After 12 h, the reaction was complete,
and MeOH was added. The pyridine was removed by a N2 stream. The
residue was purified by preparative TLC on silica gel [(eluent petroleum/
Me2CO (7:3, v/v)] yielding 5 as a homogeneous solid (Rf 0.39, 2.0 mg): [R]25
D
-12.7 (c 0.15); IR νmax 3374, 1771, 1725, 1637, 1595, 1284, 1214 cm-1; UV
λ
max log (ε) 290 (3.9), 225 (sh) nm; 1H NMR, δ 7.97 (1H, d, J=7.5 Hz, H-8),
7.51 (1H, dd, J=8.0, 7.5 Hz, H-9), 7.46-7.31 (5H, m, Ph), 7.39 (1H, d, J=
8.0 Hz, H-10), 6.37 (1H, s, H-6), 5.93 (1H, s, H-3), 3.76 (3H, s, OMe), 3.44
(3H, s, OMe), 2.23 (3H, s, MeCO); ESIMS (þ), m/z 541 [M þ Na]þ.
(R)-R-Methoxy-R-trifluorophenylacetate (MTPA) Triester
of Alternethanoxin A (6). (S)-(þ)-MPTA-Cl (20 μL) was added to
alternethanoxin A (1, 2.0 mg) and dissolved in dry pyridine (40 μL). The
reaction was carried out under the same conditions used for preparing 5
from 1. Purification of the crude residue by preparative TLC on silica gel
[(eluent petroleum/Me2CO (7:3, v/v)] yielding 6 as homogeneous solid
Figure 1. Structures of alternethanoxin A (1), its derivatives (3-6), and
alternethanoxin B (2).
that of the proton (H-6) of the aldehyde group bonded at C-6 of
the aromatic A ring and hemiacetalized with a phenolic group at
C-5 of the aromatic C ring. These results were in full agreement
with the absorption bands for hydroxy, conjugated carbonyl, and
aromatic groups observed in the IR spectrum (21), as well as with
the absorption maxima exhibited in the UV spectrum at 381, 299,
and 241 nm (20). These partial structures were supported by the
data of the 13C NMR spectrum (Table 1) and the couplings
observed in the HSQC spectrum (17). The aromatic protonated
carbons as well as the methoxy and acetyl groups were observed
at the typical chemical shift values of δ 131.0, 122. 2, 121.4, 109.5,
52.5, and 22.0 for C-9, C-8, C-10, C-3, MeO, and MeCO,
respectively (22). The same spectrum also showed significant
signals for the carbonyl and the hemiacetalic carbon (C-6) at δ
198.7 and 109.5, with the latter overlapped to the C-3 signal. The
signals of the three and five quaternary carbons of the aromatic A
and C rings resonated at very typical chemical shift values of δ
167.3, 153.0, and 130.4 for C-7, C-6a, and C-10a and at δ 148.8,
160.0 (double signals), 128.6, and 109.8 for C-1, C4 and C-5, C-2,
and C-10b and were essentially assigned on the basis of the
couplings observed in the HMBC spectrum (17) (Table 1). The
couplings reported in Table 1 also allowed us to deduce the
presence of a 2,6-pentasubstituted-2H-4-dehydropyran ring (B)
accounting for the remaining unsaturation, which was joined to
the other two rings (A and C) by the bridge-head carbons C-6a
and C-10a and C-5 and C-10b, respectively. These findings
allowed us to assign the chemical shift to all of the carbons and
the corresponding protons (Table 1) as well as to alternethanoxin
A the structure of a 1-(1,4,6-trihydroxy-7-methoxy-6H-benzo-
(d)chromen-2-yl)ethanone (1, Figure 1).
This structure was supported by other couplings observed in
the HMBC spectrum (Table 1) and the data from the HRESIMS
spectrum, recorded in positive modality, which showed sodium
clusters formed by the toxin itself and the corresponding dimer
at m/z 325.0701, [M þ Na]þ, and 627 [2M þ Na]þ and the frag-
mentation peak at m/z 287 [M-Me]þ, which was generated by
the molecular ion by loss of a methyl residue.
The structure of alternethanoxin A was confirmed by prepar-
ing two key derivatives having spectroscopic properties that were
fully consistent with structure 1. By usual acetylation with acetic
anhydride and pyridine, alternethanoxin A was converted into
the corresponding triacetyl derivative 3 (Figure 1), the IR spec-
trum of which showed the significant absence of hydroxy groups
and the presence of bands due to more ester carbonyl groups. Its
1H and 13C NMR spectra differed from those of 1 for the
significant presence of the signals of the three acetoxy groups at
δ 2.02 and 1.95 (two MeCOO) and at δ 168.8 (two MeCOO),
(Rf 0.55, 1.7 mg): [R]25 -33.6 (c 0.13); IR νmax 1769, 1728, 1670, 1621,
D
1452, 1265, 1211, 1168 cm-1; UV λmax (log ε) nm 287 (sh), 256 (4.62); 1H
NMR, δ 7.58-7.25 (15H, m, Ph), 7.49 (1H, d, J=7.6 Hz, H-8), 7.19 (1H,
dd, J=8.0, 7.6 Hz, H-10), 6.92 (1H, d, J=8.0 Hz, H-10), 6.76 (1H, s, H-6),
6.72 (1H, s, H-3), 3.55 (3H, s, OMe), 3.52 (3H, s, OMe), 3.45 (3H, s, OMe),
3.35 (3H, s, OMe), 2.38 (3H, s, MeCO); ESIMS (þ), m/z 973 [MþNa]þ.
Leaf Disk-Puncture Assay. Culture filtrates of A. sonchi, its organic
extract, the chromatographic fractions, and pure compounds 1-4 were
assayed by leaf disk-puncture bioassay on S. arvensis and a number of
nonhost plants. The plants were produced from pieces of underground
shoots or seeds and grown in a greenhouse. The disks (10 mm diameter)
were cut off well-expanded leaves with a cork borer, placed on moistened
filter paper, and punctured by a sharp needle in the center. Crude organic
extract, chromatographic fractions, and pure compounds were dissolved
in a small amount of EtOH and thenbrought up to desirable concentration
with distilled H2O. The final concentration of EtOH in test solutions was
5% v/v, which is nontoxic to leaves of all plants in the control. Droplets
(10 μL) of the test solution were applied on the disks and then incubated in
transparent plastic boxes at 24 °C under a 12 h photoperiod. After 2 days
of incubation, the diameter of the necrotic lesions (mm) was measured.
Antimicrobial Assay. Antifungal activity of the alternethanoxins A
and B was assayed on Saccharomyces cerevisiae, and their antibacterial
activity was tested on Xanthomonas campestris, Escherichia coli, and
Bacillus subtillis at the concentration 100 μg per disk according to the
method previously described (19).
RESULTS AND DISCUSSION
The organic extract obtained from the solid culture of A. sonchi,
showing a high phytotoxic activity on S. arvensis, was purified by a
combination of column chromatography and preparative TLC on
silica gel and reverse phase to obtain two pure phytotoxic meta-
bolites. Their close relationship was shown by 1H and 13C NMR
investigations, and they were named alternethanoxins A (1) and B
and (2) (Figure 1) (51.0 and 2.2 mg/kg, respectively) on the basis of
fungus source and their carbon skeleton.
Alternethanoxin A showed a molecular weight of 302 asso-
ciated with a molecular formula of C16H14O6, consistent with 10
unsaturations, 9 of which were due to a 1,2,3-trisubstituted (A)
and a pentasubstituted (C) aromatic ring and to a carbonyl
1
group. In fact, the H NMR (Table 1) and COSY (17) spectra
showed two doublets (J = 7.5 and J = 7.1 Hz) and a double
doublet (J=7.5 and J=7.1 Hz) at the typical chemical shift values
for a suitable trisubstituted aromatic ring at δ 7.46 (H-8), 7.11
(H-10), and 7.34 (H-9) (20). The same spectrum showed three
singlets due to the proton (H-3) of the pentasubstituted aromatic
ring and another proton, a methoxy, and an acetyl group at δ
6.21, 3.62, and 2.23 (20). The singlet at δ 6.21, which integrated for
two protons, was due to the overlapping of the H-3 signal and

Article
J. Agric. Food Chem., Vol. 57, No. 15, 2009 6659
168.5 (MeCOO), and 20.5 (three MeCOO). In the same spectra
also the downfield shifts (Δδ=0.65) of the overlapped signals of
H-3 and H-6 at δ 6.86 and (Δδ=12.5) of C-3 and C-6 at δ 122.0
were observed. The ESIMS spectrum showed sodium clusters
formed bytriacetylalternethanoxin A itselfand the corresponding
dimer at m/z 451 [MþNa]þ and 879 [2MþNa]þ.
The absolute stereochemistry of the secondary hydroxylated
carbon C-6 of alternethanoxin A (1) was determined by applying
Mosher’s method (23-25). By reaction with the R-(-)-R-meth-
oxy-R-trifluorophenylacetate (MTPA) and S-(þ)MTPA chlor-
ides, alternethanoxin A was converted in the corresponding
diastereomeric S-MTPA ester and R-MTPA triesters (5 and 6,
respectively), the spectroscopic data ofwhich were consistentwith
the structure assigned to 1. The comparison between the 1H
NMR data of the S-MTPA ester (5) and those of the R-MTPA
triester (6) of 1 [Δδ (5 - 6): H-3, -0.79; H-8, þ0.48; H-9, þ0.42;
H-10, þ0.49; MeO, þ0.21; and MeCO, -0.15] allowed us to
assign an R-configuration at C-6. In alternethanoxin B C-6
has the opposite stereochemistry with respect to 1, so that an
S-configuration could be assigned to this chiral carbon in 2.
Compounds 1-4 were tested at a range of concentrations from
0.1 to 4 mg/mL on leaf disks of S. arvense. Only alternethanoxins
A and B (1 and 2) were shown to be phytotoxic. Small necrotic
lesions were seen at concentrations of 1 and 2 mg/mL for 1 and 2,
respectively. At the highest concentration of 1 and 2 (4 mg/mL)
lesions reached 4 and 2 mm in diameter, respectively. When tested
at the concentration of 2 mg/mL on leaf disks of a number plant
species (Cirsium arvense, Calendula officinalis, Taraxacum offici-
nalis, Amaranthus rethroflexus, Convolvulus arvensis, Aegopodium
podagraria, Elytrigia repens, Phleum pratense), alternethanoxins
A and B showed similar nonspecific activities (lesions ∼ 1-2 mm
diameter).
Furthermore, the inactivity of derivatives 3 and 4 demon-
strated that the phenolic hydroxy group at C-4 of C ring is a
structural feature important for the phytotoxicity, whereas the
activity ofalternethanoxin B showedthatthe other one at C-1 and
the hemiacetal hydroxyl group at C-6 are nonessential. The
reduction of both the hemiacetal and the acetyl groups at C-6
and C-2 and the eletrophilic substitution of one or more hydro-
gens of rings A and C with a suitable group could contribute to
demonstrating the importance of the benzo(d)crhomene moiety
and the role of the acetyl group. Neither 1 nor 2 demonstrated
antibiotic or antifungal activity when tested at 100 μg/disk on
Bacillus subtilis, Xanthomonas campestris, Escherichia coli, and
Saccharomyces cerevisiae.
Alternethanoxins A and B are two fungal metabolites in which
an ethanone group was bonded to an original polysubstituted
benzo(d)chromene and dioxacyclopenta[def ]phenanthrene resi-
due, respectively, and occur for the first time as natural com-
pounds with interesting biological activity. In particular, the main
fungal metabolite alternethanoxin A (1) and also alternethanoxin
B (2) showed potential herbicidal properties.
A number of well-known fungal metabolites (alternariol, its
monomethyl ether, altenuene, and altenuisol), which belong to a
class of toxic metabolites containing a dibenzo[R]pyrone moiety,
are structurally close to alternethanoxin A. These compounds are
produced by different Alternaria species isolated from plant
material, and their antibiotic, cytotoxic, and teratogenic activities
are usually stressed (15). Interestingly, alternethanoxins A and B
did not demonstrate antimicrobial activity. Furthermore, the
closest compounds to alternethanoxin A from the group of
ethanones appeared to be the acetophenones, namely, cynan-
diones A-D, cynanchone, and analogues, isolated from the
roots of different Cynanchum plant species and showing poten-
tial pharmacological applications (26). Compounds close to
alternethanoxin B are those belonging to the cylopenta[d,e,f ]-
phenathrene group including the steriols, toxic metabolites pro-
duced by some Fusarium sporotrichiella strains, isolated from
naturally infected grain (27).
By reaction with an ethereal solution of diazomethane over-
night at room temperature 1 was converted into the dimethyl
ether derivative 4 (Figure 1). Its 1H NMR spectrum differed from
that of 1 only for the presence of two more singlets due to the new
methoxy groups at δ 3.74 and 3.31. Probably the phenolic
hydroxy group at C-1 was not methylated as it was hydrogen
bonded with the carbonyl group at C-2, generating a stable six-
membered ring as shown by the singlet observed at a typical
chemical shift value of δ 12.95 (20). The ESI mass spectrum of 5
showed sodium clusters formed by dimethyl alternethanoxin A
itself and the corresponding dimer at m/z 353 [M þ Na]þ and 683
[2MþNa]þ.
Alternethanoxin B showed a molecular weight of 300 asso-
ciated with a molecular formula of C16H12O6 as deduced from its
HRESIMS spectrum and consistent with 11 unsaturations. It
differs from alternethanoxin A in that it lacks two hydrogens and
one unsaturation more. They showed very similar IR and UV
spectra, and comparison of their 1H and 13C NMR spectra
(Table 1) showed very close structures with the only difference
in the substitution of the aromatic A ring. In fact, its 1H NMR
spectrum showed two ortho-coupled aromatic protons resonat-
ing as doublets (J=9.0 Hz) at δ 7.47 and 7.36 and assigned to H-9
and H-8, which coupled in the HSQC spectrum with the aromatic
protonated carbons at δ 122.2 and 125.4, and the absence of
H-10 (20, 22). The 13C NMR spectrum also showed a significant
downfield shift (Δδ=30.8) of C-10 attributable to the presence of
a tetrasubstituted furan ring, accounting for the additional
unsaturation. This new ring probably was generated by the
attachment of the oxygen at C-1 of the C ring to the carbon
C-10 of the A ring. This partial structure was also consistent with
the couplings observed in the COSY and HSQC spectra and
essentially with those of the quaternary carbons recorded in the
HMBC spectrum (Table 1). Furthermore, examination of the 1H
NMR and COSY spectra also showed different chemical shift
values for the protons (H-3) of the pentasubstituted aromatic C
ring and of the hemiacetalized aldehyde group (H-6) resonating at
δ 6.73 and 6.62, respectively, which coupled in the HSQC with the
signals at δ 107.2 and 111.4 (C-3 and C-6), respectively. These
findings suggested an opposite stereochemistry at C-6 in 2 with
respect to 1, which was also supported by the presence in the 1H
NMR spectrum of 2 of a singlet at δ 12.20 due to the hemiacetalic
hydroxy group, which is probably hydrogen bonded to the
methoxy group at C-7 and generating a stable six-membered
cycle. This result was confirmed by the couplings observed in
the NOESY spectra (17) of 1 and 2. In fact, the NOESY spect-
rum of 1, besides the expected effect observed between H-3
and the methyl of the acetyl group at C-2, also showed an
effect between H-6 and the methoxy group at C-7. This
latter effect was significantly absent in the NOESY spectrum
of 2.
These findings allowed us to assign the chemical shift values to
all of the carbons and the corresponding protons (Table 1) and to
alternethanoxin B the structure of 1-(7,9-dihydroxy-1-methoxy-
9H-4,8-dioxacyclopenta[def]phenanthren-5-yl)ethanone (2, Figure 1).
This structure was supported by the other couplings observed in the
HMBC spectrum (Table 1) and by the data of the HRESIMS,
recorded in positive modality, which showed sodium clusters formed
by the toxin itself and the corresponding dimer at m/z 323.0541 [Mþ
Na]þ and 623 [2MþNa]þ.
Because of the structural relationship of alternethanoxins A
and B to some mycotoxins of Alternaria spp., their antimicrobial

6660 J. Agric. Food Chem., Vol. 57, No. 15, 2009
Evidente et al.
activity should be assayed on more species of microorganisms. It
seems to be important also to elucidate the role of these metabo-
lites in the life cycle of their producer. If there is a correlation
between the production of these toxins and the virulence of the
fungus, the toxin production can be used as a marker for selection
of strains of A. sonchi with higher herbicide potential, hence
increasing their commercial value.
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A. Cytochalasins Z1, Z2 and Z3, three 24-oxa[14]cytochalasans
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ACKNOWLEDGMENT
We thank M. Zampa, CNR, Pozzuoli, Italy for mass spectra.
The NMR spectra were recorded in the laboratory of the Istituto
di Chimica Biomolecolare del CNR, Pozzuoli, Italy.
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Received May 5, 2009. Revised manuscript received June 29, 2009.
Accepted July 1, 2009. This work was carried out within the project
“Enhancement and Exploitation of Soil Biocontrol Agents for Bio-
Constraint Management in Crops” (Contract FOOD-CT-2003-
001687), which is financially supported by the European Commission
within the 6th FP of RTD, Thematic Priority 5;Food Quality and
Safety. The research was also in part supported by a grant from Regione
Campania L.R. 5/02 and a grant of the Government of Saint Petersburg.
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