Chenopodolans A-C: Phytotoxic furopyrans produced by Phoma chenopodiicola, a fungal pathogen of Chenopodium album

Article abstract of DOI:10.1016/j.phytochem.2013.10.007

Three tetrasubstituted furopyrans, named chenopodolans A-C, were isolated together with the well known fungal metabolite (-)-(R)-6-hydroxymellein from the liquid culture of Phoma chenopodiicola, a fungal pathogen proposed for the biological control of Chenopodium album, a common worldwide weed of arable crops. The structures of chenopodolans A-C were established by spectroscopic and chemical methods as 2-(3-methoxy-2,6-dimethyl-7aH-furo[2,3-b]pyran-4-yl)- butane-2,3-diol, 1-(3-methoxy-2,6-dimethyl-7aH-furo[2,3-b]pyran-4-yl)ethanol and 3-methoxy-2,6-dimethyl-4-(1-methylpropenyl)-7aH-furo[2,3-b]pyran, respectively. The absolute configuration R to the hydroxylated secondary carbon (C-11) of the side chain at C-4 of chenopodolan A was determined by applying an advanced Mosher's method. Assayed by leaf puncture on host and non-host weeds chenopodolans A and B, and the 11-O-acetylchenopodolan A showed a strong phytotoxicity. These results showed that the nature of the side chain attached to C-4 is an important feature for the phytotoxicity. A weak zootoxic activity was only showed by chenopodolan B.; 2013 Elsevier Ltd. All rights reserved.

Full text of DOI:10.1016/j.phytochem.2013.10.007

Phytochemistry 96 (2013) 208–213
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Chenopodolans A–C: Phytotoxic furopyrans produced by Phoma
chenopodiicola, a fungal pathogen of Chenopodium album
Alessio Cimmino a, Anna Andolfi a, Maria Chiara Zonno b, Fabiana Avolio a,
Alexander Berestetskiy c, Maurizio Vurro b, Antonio Evidente a,
⇑
a Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy
b Istituto di Scienze delle Produzioni Alimentari, Consiglio Nazionale delle Ricerche, Via Amendola 122/O, 70125 Bari, Italy
c All-Russian Institute of Plant Protection, Russian Academy of Agricultural Sciences, Pushkin, Saint-Petersburg 196608, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2013
Received in revised form 25 September 2013
Available online 6 November 2013
Three tetrasubstituted furopyrans, named chenopodolans A–C, were isolated together with the well
known fungal metabolite (À)-(R)-6-hydroxymellein from the liquid culture of Phoma chenopodiicola, a
fungal pathogen proposed for the biological control of Chenopodium album, a common worldwide weed
of arable crops. The structures of chenopodolans A–C were established by spectroscopic and chemical
methods as 2-(3-methoxy-2,6-dimethyl-7aH-furo[2,3-b]pyran-4-yl)-butane-2,3-diol, 1-(3-methoxy-2,6-
dimethyl-7aH-furo[2,3-b]pyran-4-yl)ethanol and 3-methoxy-2,6-dimethyl-4-(1-methylpropenyl)-7aH-
furo[2,3-b]pyran, respectively. The absolute configuration R to the hydroxylated secondary carbon
(C-11) of the side chain at C-4 of chenopodolan A was determined by applying an advanced Mosher’s
method. Assayed by leaf puncture on host and non-host weeds chenopodolans A and B, and the 11-O-
acetylchenopodolan A showed a strong phytotoxicity. These results showed that the nature of the side
chain attached to C-4 is an important feature for the phytotoxicity. A weak zootoxic activity was only
showed by chenopodolan B.
Keywords:
Chenopodium album
Phoma chenopodiicola
Phytotoxins
Furopyrans
Chenopodolans A–C
(À)-(R)-6-Hydroxymellein
Herbicides
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Considering that the organic extract obtained from the culture
filtrates of the fungus proved to be much more phytotoxic then
Chenopodium album L., also known as common lambsquarters or
fat hen, is a ubiquitous weed of arable crops such as sugar beet and
maize (Holm et al., 1977). The difficulties in managing this weed
have increased the interest in the use of fungal pathogens as a pos-
sible option for its control. Ascochyta caulina (P. Karst.) v.d. Aa and
v. Kest, has been proposed as a fungal pathogen for the control of C.
album (Kempenaar, 1995; Kempenaar et al., 1996). More recently
another pathogenic member of the Sphaeropsidales, Phoma che-
nopodiicola, has been proposed as a potential biocontrol agent. A
new phytotoxic unrearranged ent-pimaradiene diterpene, named
chenopodolin, was isolated from the liquid culture of this pathogen
and characterized as (1S,2S,3S,4S,5S,9R,10S,12S,13S)-1,12-acetoxy-
2,3-hydroxy-6-oxopimara-7(8),15-dien-18-oic acid 2,18-lactone
(Cimmino et al., 2013). At a concentration of 2 mg/mL, the toxin
showed phytotoxic activity, causing necrotic lesions to leaves of
Mercurialis annua, Cirsium arvense and Setaria viridis. The bioassay
of five prepared derivatives indicated that the hydroxy group
the expected by the chenopodolin content, it was hypothesized
that other compounds could be present in the extract, having addi-
tional or synergistic effects.
This manuscript reports on: (a) the isolation and the chemical
characterization of three further novel phytotoxic metabolites pro-
duced by P. chenopodiicola, named chenopodolans A, B and C; (b)
the preliminary studies of their biological activities to evaluate
their potential as natural and safe herbicides; (c) the absolute con-
figuration of the secondary hydroxylated carbon (C-11) of the side
chain at C-4 of chenopodolan A by applying an advanced Mosher’s
method (Othani et al., 1989).
2. Results and discussion
The organic extract obtained from the liquid culture of
P. chenopodiicola, having a high phytotoxic activity described
below, was purified by CC and TLC detailed in the experimental
section. As recently reported, chenopodolin was obtained from
at C-3 and the
a,b-unsaturated ketone at C-6 are important
features for the phytotoxicity, and the acetyl group at C-1 to be
not (Cimmino et al., 2013).
fraction
6 of the first chromatographic column as a new
unrearranged ent-pimaradiene diterpene (Cimmino et al., 2013).
Furthermore, a compound was purified from fraction 4, identified
as (À)-(R)-6-hydroxymellein (7, Fig. 1) by its physic (OR) and spec-
troscopic (1H NMR and ESIMS (+)) data, which were identical to
⇑
Corresponding author. Tel.: +39 081 2539178; fax: +39 081 674330.
E-mail address: evidente@unina.it (A. Evidente).
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.10.007

A. Cimmino et al. / Phytochemistry 96 (2013) 208–213
209
those previously reported (Islam et al., 2007). The structure was
also supported by data from ESIMS (À) and APCI (+) which showed
the pseudomolecular ions at m/z 193 [MÀH]À and 195 [M+H]+,
respectively. The (À)-(R)-6-hydroxymellein (7) was previously iso-
lated from different fungi, e.g. Penicillium verruculosum (Ito et al.,
1992) and Lachnum papyraceum (Stadler et al., 1995) and also from
carrot (Marinelli et al., 1996). The bioassay-guided purification of
fractions 1 and 5 of the original column allowed isolation of three
further metabolites, two of which have phytotoxic activity, thus
confirming the hypothesis that the fungus could produce several
bioactive metabolites.
both as two singlets at d 1.94 and 3.91, respectively, and those of a
2,3-dihydroxybutandiol side chain. Indeed, the proton of the sec-
ondary hydroxylated carbon (C-11) of this latter appeared as a
quartet (J = 6.6 Hz) at d 3.88 being coupled with the adjacent
methyl group (Me-12), which was observed as doublet
(J = 6.6 Hz) ad d 1.20. The methyl group (Me-9) bonded to the qua-
ternary hydroxylated carbon (C-10) of the same residue resonated
as a singlet at d 1.33. The correlations observed in the HSQC spec-
trum (Berger and Braun, 2004) allowed to assign the chemical
shifts of these 7 protonated carbons at d 137.1, 13.4, 8.8, 56.3,
73.7, 17.4 and 23.2 (C-5, C-13, C-8, OMe, C-11, C-12 and C-9,
respectively) (Breitmaier and Voelter, 1987). The other five olefinic
carbons, three of which were oxygenated, were assigned on the ba-
sis of the correlations observed in the HMBC spectrum (Table 2)
(Berger and Braun, 2004). Thus, the signals observed at d 165.9,
165.0, 160.0, 129.5 and 103.1 were assigned to C-3, C-2, C-6, C-4
and C-3a, respectively. Similarly, the signal at d 76.1 was assigned
to C-10. On the basis of the couplings observed in the HMBC spec-
trum, C-3a represents the other bridgehead carbon between the
pyran and the furan rings (Breitmaier and Voelter, 1987). The cou-
plings observed in the same spectrum between Me-9 and C-5, and
between H-5 and C-10 allowed to locate the 2,3-butandiol side
chain on C-4. Considering that all the chemical shifts were assigned
to protons and carbons of 1, as reported in Table 1, chenopodolan A
could be formulated as 2-(3-methoxy-2,6-dimethyl-7aH-furo[2,3-
b]pyran-4-yl)-butane-2,3-diol (1).
The structure assigned to 1 was supported by the data from its
HRESI MS and NOESY spectra. The HRESI MS showed the dimeric
sodiated form, the potassium and sodium clusters at m/z 559
[2M+Na]+, 307 [M+K]+, 291.1217 [M+Na]+, respectively. By H2O
loss the sodium cluster produced the ion at m/z 273. The APCI spec-
trum showed the pseudomolecular ion at m/z 269 [M+H]+ which,
by loss of H2O, produced the ion at m/z 251.
The NOESY spectrum (Table 3) (Berger and Braun, 2004)
showed the correlations between the protons of the side chain
(H-11, Me-9 and Me-12) with H-5, the correlation between H-7a
with both Me-13 and the methoxy group, and that of H-11 with
Me-9 and Me-12. Due to these correlations, in agreement with
the inspection of a Dreiding model of 1 with the exception of the
butandiol side chain at C-4, chenopodolan A appears to have a
quite flatted rigid structure.
The preliminary 1H and 13C NMR investigation showed that the
three metabolites had similar structures, but very different from
that of chenopodolin. Being novel, and belonging to the naturally
occurring furopyran group, they were named chenopodolans A, B
and C.
Chenopodolan
A proved to have the molecular formula
C14H20O5, deduced by its HR ESIMS, with five hydrogen deficien-
cies, and molecular weight of 268. Three of these deficiencies were
associated with one tri- and two tetra-substituted conjugated dou-
ble bonds, as deduced from the preliminary inspection of the 1H
and 13C NMR spectra (Table 1), and in agreement with the typical
bands and absorption maxima observed in the IR (Nakanishi and
Solomon, 1977) and UV (Scott, 1964) spectra. The other two unsat-
urations could be attributed to two rings. Indeed, very significant
signals were observed in the same spectra at d 6.21 (s)/92.8 (CH)
for a carbon bonded to two oxygen atoms and this represents
one of the bridgehead carbons (C-7a) between one pyran and one
furan ring. These findings are in agreement with the presence of
a olefinic proton (H-5) and its adjacent methyl group (Me-13)
belonging to the pyran ring, which coupled (J < 1 Hz) in the COSY
spectrum (Berger and Braun, 2004), and resonated as two singlets
at the typical chemical shift values of d 6.59 and 2.21 (Pretsch et al.,
2000), respectively. The 1H NMR spectrum showed also the signals
of another methyl (Me-8) and that of a methoxy group resonating
11
13
O
O
O
O
7
6
5
1
7a
3a
8
2
3
4
The structure assigned to 1 was confirmed by preparing the 11-
O-acetyl derivative (4) by routine acetylation with pyridine and
acetic anhydride. The IR spectrum of 4 showed the typical band
of the acetoxy group together with that of the tertiary hydroxy
group. Its 1H NMR spectrum differed from that of 1 only for the
10
O
9
O
10
9
OH
OH
OR
11
12
downfield shift of H-11 (Dd 1.12), always appearing as a quartet
(J = 6.4 Hz) at d 5.00, and for the presence of the singlet of the acet-
yl group at d 2.08. Its ESIMS spectrum showed the dimeric sodiated
form [2M+Na]+, the potassium [M+K]+ and sodium [M+Na]+ clus-
ters at m/z 643, 349 and 333, respectively. By loss of H2O the latter
ion generated the peak at m/z 315. The APCI mass spectrum
showed the pseudomolecular ion [M+H]+ at m/z 311; this latter
produced the ions at m/z 293 and 233, by successive losses of
H2O and AcOH, respectively.
Several attempts to crystallize chenopodolans A, as well as B
and C, carried out by changing solvent mixtures and temperatures,
failed probably also due to the limited amounts available of the
metabolites. Thus, X-ray analysis could not be performed to assign
the relative configuration to 1, 2 and 3.
2
1 R=H
4 R=Ac
5 R=S-MPTA
6 R=R-MPTA
O
O
H
4
1
5
3
4a
8a
6
7
2
O
8
O
9
10
12
OH
O
For chenopodolan A, the absolute configuration of the second-
ary hydroxylated carbon (C-11) group of the 2,3-butanediol at-
tached to C-4 was determined by applying an advanced Mosher’s
7
11
3
method (Othani et al., 1989). By reaction with R-(À)-
-trifluoromethylphenylacetyl (MTPA) and S-(+)MTPA chlorides,
chenopodolan A (1) was converted into the corresponding diaste-
a-methoxy-
Fig. 1. Structures of: chenopodolans A–C (1–3, respectively), 11-O-acetyl-, and 11-
O-S-MTPA and 11-O-R-MTPA esters (4–6, respectively) of chenopodolan A; and (À)-
(R)-6-hydroxymellein (7).
a

210
A. Cimmino et al. / Phytochemistry 96 (2013) 208–213
Table 1
1H and 13C NMR data of chenopodolans A–C (1–3, respectively).a,b
Position
1
2
3
dCc
dH (J in Hz)
dCc
dH (J in Hz)
dCc
dH (J in Hz)
2
3
3a
4
5
6
7a
8
165.0 s
165.9 s
103.1 s
129.5 s
137.1 d
160.0 s
92.8 d
8.8 q
23.2 q
76.1 s
73.7 s
17.4 q
13.4 q
56.3 q
164.7 s
165.5 s
103.1 s
126.8 s
136.9 d
159.3 s
92.9 d
8.7 q
164.5 s
165.4 s
101.5 s
123.3 s
136.2 d
160.3 s
91.8 d
8.1 q
132.5 s
128.6 d
16.5 q
13.5 q
8.1 q
6.59 s
6.54 s
7.00 s
6.21 s
1.94 (3H) s
1.33 (3H) s
6.19 s
1.96 (3H) s
4.72 (1H) q (J = 6.0 Hz)
1.35 (3H) d (J = 6.0 Hz)
1.95 (3H) s
6.15 s
1.83 (3H) s
9
64.9 d
23.3 q
12.7 q
10
11
12
13
OMe
5.61 (1H) q (J = 6.9 Hz)
1.76 (3H) d (J = 6.9 Hz)
1.94 (3H) s
2.03 (3H) s
3.91 s
3.88 (1H) q (J = 6.6 Hz)
1.20 (3H) d (J = 6.6 Hz)
2.21 (3H) s
3.91 s
56.1 q
3.91 s
55.5 q
a
b
c
The chemical shifts are in d values (ppm) from TMS.
2D 1H, 1H (COSY) 13C, 1H (HSQC) NMR experiments delineated the correlations of all the protons and the corresponding carbons.
Multiplicities were assigned by DEPT spectrum.
Table 2
Table 4
HMBC data of chenopodolan A–C (1–3, respectively).
1H NMR data of 11’-O-(S)- and 11’-O-(R)-MTPA esters of chenopodolan A (5 and 6,
respectively).
Carbon
1
2
3
5 dH (J in Hz)
6 dH (J in Hz)
2
Me-8
Me-8
3
H-7a, OMe
H-7a, OMe
H-7a, OMe
5
6.419 s
6.522 s
3a
4
5
6
7a
8
H-7a, Me-8
H-7a, Me-8
H-7a, H-9, Me-11
Me-11, H-9
H-5, H-7a, Me-11
H-7a, Me-12
H-5, H-7a, Me-13
H-10, Me-13
7a
11
OMe
13
8
6.162 s
5.238 q (J = 6.4 Hz)
3.905 s
2.135 (3H) s
1.951 (3H) s
1.331 (3H) s
1.381 d (J = 6.4 Hz)
3.574 s
6.211 s
5.241 q (J = 6.4 Hz)
3.917 s
2.198 (3H) s
1.966 (3H) s
1.385 (3H) s
1.323 d (J = 6.4 Hz)
3.525 s
H-7a, H-5, Me-13
Me-13, Me-9
H-5, H-7a, Me-13
H-5, H-7a, Me-13
9
9
Me-10
H-5
Me-11
Me-11
H-10
H-5, Me-12
12
OMe
Ph
10
11
12
H-5, Me-9, Me-12
Me-9, Me-12
7.72-7.35 m
7.72-7.38 m
reomeric S-MTPA and R-MTPA monoesters at C-11 (5 and 6,
respectively), whose spectroscopic data were consistent with the
structure assigned to 1. Subtracting the chemical shift of the pro-
tons (Table 4) of the 11-O-R-MTPA (6) from that of 11-O-S-MTPA
Δδ: −0.049
Δδ: −0.063
H
13
O
Δδ: −0.015
8
O
7
6
5
1
7a
2
(5) esters, the
D
d (5–6) values for all of the protons were deter-
d values were located
3a
Δδ: −0.0108
mined as reported in Fig. 2. The positive
D
3
4
on the right-hand side, and those with negative values on the
left-hand side of the model A as reported in Othani et al. (1989).
This model allowed the assignment of the R configuration at
C-11. Then 1 was formulated (11R) 2-(3-methoxy-2,6-dimethyl-
7aH-furo[2,3-b]pyran-4-yl)-butane-2,3-diol (1).
Chenopodolans B and C differed from chenopodolan A for the
side chain attached to C-4. In chenopodolan B (molecular weight
224 due to its molecular formula of C12H16O4), this chain is a 1-eth-
anol, as results by comparison of its 1H and 13C NMR data with
those of 1 (Table 1). The data of COSY, HSQC and HMBC (Table 2)
spectra of 2, which showed the signals for the tetrasubstituted
furopyran moiety very similar to those of 1, allowed to locate the
side chain at C-4. However, instead of the signals of the 2,
3-butanediol, those typical for the 1-ethanol residue were ob-
Δδ: −0.012
10
11
O
Δδ: −0.054
9
OH
O
12
Ph
Δδ: +0.058
OMe
O
CF3
Fig. 2. Structures of 3-O-S- and 3-O-R-MTPA esters of chenopodolan A (5 and 6,
respectively), reporting the
system.
D
d value obtained by comparison (5-6) of each proton
Table 3
NOESY data of of chenopodolans A–C (1–3).
1
2
3
Irradiated
Observed
Irradiated
Observed
Irradiated
Observed
H-5
H-7a
H-11
Me-9, H-11, Me-12
M-13, OMe
Me-9, Me-12
H-7a
H-5
H-9
OMe, Me-11
Me-10
Me-10, Me-11
H-5
H-7a
H-10
Me-11
OMe, Me-13
H-5, Me-11

A. Cimmino et al. / Phytochemistry 96 (2013) 208–213
211
served at d 4.72 (1H, q, J = 6.0 Hz)/64.9 (CH-9) and 1.35 (3H, d,
J = 6.0 Hz)/23.3 (Me-10). Considering all the chemical shifts as-
signed to the protons and the carbons of 2 (Table 1), chenopodolan
completely inactive (data not shown). These results showed that
the nature of the side chain attached to C-4 is an important feature
for the phytotoxicity. In particular, the presence of the tertiary
hydroxy group in 1 (C-10), which become secondary in 2 (C-9) is
very important for the activity. 3 was completely inactive and this
last result probably is due to the different side chain at C-4 being a
1-methyl propenyl. The activity of the 11-O-acetylchenopodolan A
(4) further supported this result, because in this derivative the sec-
ondary hydroxy group was acetylated only at C-11 whilst it is free
at C-10. Compound 7 proved to have almost the same toxicity of 1
and 2.
In the assay for zootoxicity (Artemia salina), only chenopodolan
B was weakly active, causing around 24% larval mortality after 48 h
of exposure to the metabolite (data not shown). All the other com-
pounds were inactive. Assayed on fungi (Geotrichum candidum) and
gram positive (Bacillus subtilis) and gram negative (Escherichia coli),
none of the metabolites showed antimicrobial activities (data not
shown).
In conclusion, three novel furopyrans, named chenopodolans
A–C, were isolated from P. chenopodiicola, a fungus proposed for
the biocontrol of C. album. Chenopodolans A and B could have addi-
tive phytotoxic activities with chenopodolin and (À)-(R)-6-
hydroxymellein produced by the same fungus. Furopyrans are very
rare as naturally occurring compounds. The only example of this
type is aracemosolone, isolated from root bark of Bauhinia racemo-
sa Lamk (Renuka et al., in press). Some heteroannelated furopyrans
were synthesized as herbicides (Kiyoshi et al., 1997). Thus, consid-
ering the unusual structures associated to the preliminary biolog-
ical activities, it seems the new compounds would deserve
further attentions for better understanding their biological proper-
ties and to evaluate their potential as lead for novel herbicides of
natural origin.
B
could be formulated as 1-(3-methoxy-2,6-dimethyl-7aH-
furo[2,3-b]pyran-4-yl)ethanol.
The structure assigned to 2 was supported by the data from its
HRESI MS and NOESY spectra. The HRESI MS spectrum showed the
dimeric sodiated form [2M+Na]+, the potassium[M+K]+ and sodium
[M+Na]+ clusters at m/z 471, 263, 247.0934, respectively. By loss of
H2O the latter generated the ion at m/z 229. The APCI mass spec-
trum showed the pseudomolecular ion [M+H]+ at m/z 225 which,
by loss of H2O, generated the ion at m/z 207.
The NOESY spectrum (Table 3) showed correlations of the pro-
ton H-5 with Me-10, H-7a with Me-11 and OMe, and H-9 with Me-
10 and Me-11. These results, similarly to 1 and in agreement with
the inspection of a Dreiding model, showed a enough flatted rigid
structure with the exception of the ethanol side chain at C-4.
It was not possible to assign the absolute configuration to the
secondary hydroxylated carbon (C-9) of the ethanol residue of 2
because all the attempts made to convert it into the corresponding
diastereomeric S-MTPA and R-MTPA esters, by reaction with R-(À)-
a-methoxy-a-trifluoromethylphenylacetyl (MTPA) and S-(+)MTPA
chlorides, as above reported for chenopodolan A (1), failed. This
probably could be a consequence of a steric hindrance due to the
bulky of the reagent group and to the proximity of the ethanol res-
idue to the furopyran ring system.
In chenopodolan C (3), which showed a molecular weight of 234
due to its molecular formula of C14H18O3, the side chain was a 1-
methylpropenyl residue. The comparison of its 1H and 13C NMR
data (Table 1), based also on the COSY, HSQC and HMBC (Table 2)
couplings with those of 1 showed that the signals of the same tet-
rasubstituted furopyran moiety were very similar and supported
the location of the side chain at C-4. Furthermore, the signals of
the 2,3-butanediol were absent in the 1H NMR spectrum, while
those for a 1-methylpropenyl residue side chain were observed.
In particular, the olefinic proton (H-10) and its adjacent methyl
group (Me-11) resonated as quartet (J = 6.9 Hz) and a doublet
(J = 6.9 Hz) at d 5.61 and 1.76, while the other methyl group reso-
nated as singlet at d 1.94 (Me-12). The corresponding carbons in
the 13C NMR spectrum were observed at d 128.6, 16.5 and 13.5
(C-10, C-11 and C-12, respectively) while the quaternary olefinic
carbon C-9 resonated at d 132.5.
Considering all the chemical shifts assigned to protons and car-
bons of 3 (Table 1) chenopodolan C could be formulated as 3-meth-
oxy-2,6-dimethyl-4-(1-methylpropenyl)-7aH-furo[2,3-b]pyran.
The structure assigned to 3 was supported by the data from its
HRESI MS and NOESY spectra. The HRESI MS spectrum showed the
trimeric [3M+Na]+ and dimeric [2M+Na]+ sodiated form, the potas-
sium [M+K]+ and sodium [M+Na]+clusters, and the pseudomolecu-
lar ion [M+H]+ at m/z 725, 491, 273, 257.1164 and 235.1343,
respectively.
3. Experimental
3.1. General
Optical rotation was measured in CHCl3 on a Jasco P-1010 digital
polarimeter unless otherwise noted; IR spectra were recorded as
glassy film on a Perkin-Elmer Spectrum One FT-IR Spectrometer
and UV spectra were recorded in MeOH solution on a Perkin-Elmer
Lambda 25 UV/Vis spectrophotometer. 1H and 13C NMR spectra
were recorded at 400/100 MHz in CDCl3 on Bruker spectrometers.
The same solvent was used as internal standard. Carbon multiplic-
ities were determined by DEPT spectra (Berger and Braun, 2004).
DEPT, COSY-45, HSQC, HMBC and NOESY experiments (Berger and
Braun, 2004) were performed using Bruker microprograms. HRESI
and ESI and APCIMS spectra were recorded on Waters Micromass
Q-TOF Micro and Agilent Technologies 6120 Quadrupole LC/MS
instruments, respectively. Analytical and preparative TLC were per-
formed on silica gel plates (Merck, Kieselgel 60 F254, 0.25); the spots
were visualized by exposure to UV light and/or by spraying first
with 10% H2SO4 in MeOH and then with 5% phosphomolybdic acid
in EtOH, followed by heating at 110 °C for 10 min. Column chroma-
tography was performed on silica gel (Merck, Kieselgel 60, 0.063–
0.200 mm). Solvent systems: (A) CHCl3–i-PrOH (95:5); (B) CHCl3;
(C) n-hexane–Me2CO (6:4); (D) CHCl3–iPrOH (97:3).
The NOESY spectrum (Table 3) showed the correlations be-
tween the proton H-5 with those of Me-11, H-7a with both Me-
13 and the methoxy group, and H-10 with H-5 and Me-11. The
lacking of a NOE correlation between H-10 and Me-12 allowed to
assign a E-stereochemistry to the double bond of the side chain
at C-4. Furthermore, as already reported for 1 and 2, and in agree-
ment with the inspection of a Dreiding model, these results con-
firm for 3 a quite flatted rigid structure, with the exception of
the 1-methylpropenyl side chain at C-4.
3.2. Fungal strain
Assayed on punctured detached leaves on Sonchus oleraceus, M.
annua and C. album, chenopodolan B proved to be the most effec-
tive, causing spread necrosis on all the three plant species tested
(level 4; data not shown). Chenopodolan A and 11-O-acetylchenop-
odolan A proved to have almost the same toxicity, being active in
particular against S. oleraceus and M. annua. Chenopodolan C was
The fungus was isolated from diseased leaves of C. album and
identified as P. chenopodiicola (Berestetskiy, unpublished). A mono-
conidial isolate was deposited in the culture collection of both the
All-Russian Research Institute of Plant Protection, Pushkin,
Saint-Petersburg, Russia (I-13.2) and the Institute of Science of

212
A. Cimmino et al. / Phytochemistry 96 (2013) 208–213
Food Production, Bari, Italy (ITEM 12534). The isolate was routinely
grown and maintained in plates and slants containing potato-
dextrose agar (PDA, Sigma–Aldrich, Chemie Gmbh, Buchs,
Switzerland).
(loge
): 328 (3.8), 227 (4.3); 1H NMR spectrum, d 6.51 (s, H-5),
6.21 (s, H-7a), 5.00 (q, J = 6.4 Hz, H-11), 3.91 (s, OMe), 2.20 (s,
Me-13), 2.08 (s, MeCO), 1.94 (s, Me-8), 1.39 (s, Me-9), 1.26 (d,
J = 6.4 Hz, Me-12); ESIMS (+) m/z: 643 [2M+Na]+, 349 [M+K]+,
333 [M+Na]+, 315 [M+NaÀH2O]+; APCIMS (+) m/z: 311 [M+H]+,
293 [M+HÀH2O]+, 233 [M+HÀAcOH]+.
3.3. Production, extraction, and purification of chenopodolans A–C
(1–3)
3.8. 11-O-(S)-
a
-methoxy-
a
-trifluoromethyl-
a
-phenylacetate (MTPA)
The fungus was grown in 1 L Erlenmeyer flasks containing
300 mL of a defined mineral (Pinkerton and Strobel, 1976) as re-
cently described (Cimmino et al., 2013). The material obtained by
lyophilising the culture filtrates (4.6 L) of the fungus was extracted
by EtOAc (4 Â 1 L); the organic extract (1.04 g), having high phyto-
toxic activity, was fractionated by CC eluted with the solvent
system A, as already described (Cimmino et al., 2013). Ten homo-
geneous fraction groups were collected and groups from 1 to 6
proved to be phytotoxic. The purification of the residue of fraction
6 (62 mg) led to obtain chenopodolin. The residue (39 mg) of frac-
tion 5 of the original column was further purified by preparative
TLC, eluted with solvent system A, yielding two homogenous solid
compounds that, being two new furopyrans, were named chenop-
odolan A and B (1, Rf 0.39, 5.6 mg, 1.2 mg/L, 2, Rf 0.53, 3.1 mg,
0.7 mg/L, respectively). The purification by TLC of the residue
(13.7 mg) of fraction 4 (eluent system A) yielded a white amor-
phous solid (7, Rf 0.67, 8.7 mg, 1.9 mg/L) which was identified as
(À)-(R)-6-hydroxymellein, as reported below. The purification of
the residue of fraction 1 (30 mg) by preparative TLC, eluted with
the solvent system B, yielded a yellow homogeneous solid; being
a new furopyran it was named chenopodolan C (3, Rf 0.45,
10.4 mg, 2.3 mg/L).
ester of 1 (5)
(R)-(À)-MPTA-Cl (20
dry pyridine (20
l
L) was added to 1 (1.0 mg) dissolved in
l
L). The mixture was kept at room temperature
for 1 h and then the reaction stopped by adding MeOH. Pyridine
was removed by an N2 stream. The residue (2.5 mg) was purified
by preparative TLC, eluted with solvent system D, yielding 5 as a
homogeneous oil (Rf 0.31, 1.1 mg). It had: IR mmax 3456, 1725,
1679, 1657, 1550, 1275 cmÀ1; UV kmax nm (log
e) 271 (3.34) 224
(sh); 1H NMR spectrum see Table 4; ESIMS (+) m/z 507 [M+Na]+.
3.9. 11-O-(R)-a-methoxy-a-trifluoromethyl-a-phenylacetate (MTPA)
ester of 1 (6)
(S)-(+)-MPTA-Cl (20
dry pyridine (20 L). The reaction was carried out under the same
lL) was added to 1 (1.0 mg) dissolved in
l
conditions used for preparing 5 from 1. The purification of the
crude residue (2.2 mg) by preparative TLC eluted with solvent sys-
tem D, allowed to obtain 6 as a homogeneous oil (Rf 0.31, 1.2 mg).
It had: IR mmax 3472, 1728, 1676, 1657, 1597, 1273 cmÀ1; UV kmax
nm (loge
) 270 (3.31), 225 (sh); 1H NMR spectrum see Table 4;
ESIMS (+) m/z 507 [M+Na]+.
3.4. Chenopodolan A (1)
3.10. (À)-(R)-6-hydroxymellein (7)
25
25
Compound 1: [
a
]
D: +8.5 (c = 0.24); IRmmax 3407, 1679, 1618,
) 307 (3.7), 214 (4.2); 1H and
Compound 7, had: [
a]
D: À40.0 (c = 0.4, MeOH); IRmmax 3544,
18
1555, 1456 cmÀ1; UV kmax nm (log
e
1664, 1625, 1585, 1540, 1251 cmÀ1 [lit, Islam et al., 2007: [
À51 (c = 0.10, MeOH); IR (KBr) = 3600–2800, 1651, 1632, 1587,
a
]
:
D
13C NMR spectra: see Table 1; HRESI MS (+) m/z: 559 [2M+Na]+,
307 [M+K]+, 291.1217 [M+Na]+ (calcd for C14H20NaO5 291.1208),
273 [M+NaÀH2O]+; APCIMS (+) m/z: 269 [M+H]+, 251
[M+HÀH2O]+.
m
1503, 1477, 1386, 1290, 1257, 1221, 1195, 1170, 1120, 1067,
854, 796, 737 cmÀ1]; 1H NMR spectrum is very similar to that pre-
viously reported and recorded (Islam et al., 2007) in the same con-
ditions, except for the following signals, d: 2.88 (1H, dd, J = 16.0
and 10.0 Hz, H-4A), 2.84 (1H, dd, J = 16.0 and 6.0 Hz, H-4B); ESIMS
(+) m/z 217 [M+Na]+; ESIMS (À) m/z 193 [MÀH]À; APCIMS (+) m/z
195 [M+H]+.
3.5. Chenopodolan B (2)
25
Compound 2: [a] D: +8.6 (c = 0.20); IR mmax 3417, 1673, 1610,
1547, 1454 cmÀ1; UV kmax nm (log ) 303 (3.8), 214 (4.3); 1H and
e
13C NMR spectra: see Table 1; HRESI MS (+) m/z: 471 [2M+Na]+,
263 [M+K]+, 247.0934 [M+Na]+ (calcd for C12H16NaO4 247.0946),
229 [M+NaÀH2O]+; APCI m/z: 225 [M+H]+, 207 [M+HÀH2O]+.
3.11. Biological activities
Each metabolite was first dissolved in a minimum amount of
MeOH (10À1 M or not higher than 2% in the final solutions) and
then diluted with distilled water to the desired concentrations.
The following bioassays were performed:
3.6. Chenopodolan C (3)
25
Compound 3: [
a
]
D: +4.6 (c = 0.20); IR mmax 1673, 1626, 1607,
Leaf puncture assay: The compounds were tested by using a leaf
puncture assay on 3 plant species (C. album L., M. annua L., and
S. oleraceus L.) (Evidente et al., 2005). Pure compounds were tested
1547 cmÀ1; UV kmax nm (log ) 336 (3.6), 253 (3.7); 1H and 13C
e
NMR spectra: see Table 1; HRESI MS (+) m/z: 725 [3M+Na]+, 491
[2M+Na]+, 273 [M+K]+, 257.1164 [M+Na]+ (calcd for C14H18NaO3
257.1154), 235.1343 [M+H]+ (calcd for C14H19O3 235.1334).
at 6.85 Â 10À3 M by applying a droplet (20
ll, 2% MeOH) of solu-
tion to detached leaves previously punctured with a needle. Five
replications (droplets) on separate leaves were used for each
metabolite and for each plant species tested. Leaves were kept in
a moistened chamber under continuous fluorescent lights. Symp-
toms were estimated visually between 3 and 5 days after droplet
application, by using a visual scale from 0 (no symptoms) to 4
(necrosis wider than 1 cm). Control treatments were carried out
by applying droplets not containing the metabolites.
3.7. 11-O-Acetylchenopodolan A (4)
Chenopodolan A (1, 2.5 mg) was acetylated with pyridine
(50 lL) and Ac2O (50 lL) at room temperature for 1 h. The reaction
was stopped by addition of MeOH and the azeotrope, obtained by
the addition of benzene, was evaporated by an N2 stream. The oily
residue (5.0 mg) was purified by preparative TLC eluted with the
solvent system C, to give the 11-O-acetyl derivative 4 of chenopod-
olan A as a homogeneous compound (Rf 0.62, 2.4 mg). Derivative 4
had: IR mmax 3423, 1733, 1676, 1615, 1552, cmÀ1; UV kmax nm
Antimicrobial bioassay: The antimicrobial activity was tested
against three microorganisms by using an agar diffusion
assay according to the protocol previously described (Bottalico
et al., 1990). In particular, the antifungal activity was tested on

A. Cimmino et al. / Phytochemistry 96 (2013) 208–213
213
Bottalico, A., Capasso, R., Evidente, A., Randazzo, G., Vurro, M., 1990. Cytochalasins:
structure–activity relationship. Phytochemistry 29, 93–96.
Breitmaier, E., Voelter, W., 1987. Carbon-13 NMR Spectroscopy. VCH, Weinheim, pp.
183–280.
Cimmino, A., Andolfi, A., Zonno, M.C., Avolio, F., Santini, A., Tuzi, A., Berestetskyi, A.,
Vurro, M., Evidente, A., 2013. Chenopodolin: a phytotoxic unrearranged ent-
pimaradiene diterpene produced by Phoma chenopodicola, a fungal pathogen for
Chenopodium album biocontrol. J. Nat. Prod. 76, 1291–1297.
G. candidum (ITEM 781) grown on PDA, whereas the antibiotic
activity was assayed against B. subtilis (strain S106.2) (a gram po-
sitive bacterium) grown on TGYA (tryptic glucose yeast agar – Bio-
life, Bothell, WA) and E. coli (ATCC 29425) (a gram negative
bacterium), grown on LB agar (Sigma, St. Louis, MO). Up to 50 lg
of each metabolite was applied per diskette. Three replications
were performed for each compound. The eventual presence of an
inhibition halo of the microbial growth was visually assessed
1 day after the application.
Zootoxic activity assay: The zootoxic activity was evaluated on A.
salina L. larvae (brine shrimps) by using the protocol already de-
scribed (Bottalico et al., 1990). The metabolites were tested at
1.7 Â 10À4 M, with four replications for each compound. Forty-
eight hours after assay performing, the number of dead larvae
was counted, and toxicity was then expressed as percentage of
dead larvae referred to the total.
Evidente, A., Andolfi, A., Vurro, M., Fracchiolla, M., Zonno, M.C., Motta, A., 2005.
Drazepinone
a
tetrasubstituted tetrahydronaphthofuroazepinone with
herbicidal activity produced by Dreschlera siccans. Phytochemistry 66, 715–721.
Holm, L.G., Pluckett, D.L., Pancho, J.V., Herberger, J.P., 1977. The World’s Worst
Weeds (Distribution and Biology). University Press of Hawaii, Honoloulou, pp.
84–91.
Islam, S.Md., Ishigami, K., Watanabe, H., 2007. Synthesis of (À)-mellein, (+)-
ramulosin, and related natural products. Tetrahedron 63, 1074–1079.
Ito, M., Maruhashi, M., Sakai, N., Mizoue, K., Hanada, K., 1992. NG-011 and NG-012,
novel potentiators of nerv growth factor. I. Taxononomy, isolation, and physico-
chemical and biological properties. J. Antibiot. 45, 1559–1615.
Kempenaar, C., 1995. Studies on the Biological Control of Chenopodium album by
Ascochyta caulina, Doctoral Thesis. University of Wageningen, The Netherlands.
Kempenaar, C., Horsten, P.J.F.M., Scheepens, P.C., 1996. Spore germination and
disease development of Chenopodium album after application of Ascochyta
caulina as a mycoherbicide. Eur. J. Plant Pathol. 102, 143–153.
Statistical analysis: All the bioassays were performed twice with
at least 3 replicates. When appropriate, standard deviation was
determined.
Kiyoshi, A., Fumiaki, K., Hiroyuki, S., Takeshi, K., Sadafumi, K., 1997. Eur. Pat. Appl.,
EP 755935 A1 19970129.
Marinelli, F., Zanelli, U., Ronchi, V.N., 1996. Toxicity of 6-methoxymellein and 6-
hydroxymellein to the producing carrot cells. Phytochemistry 42, 641–643.
Nakanishi, K., Solomon, P.H., 1977. Infrared Absorption Spectroscopy, second ed.
Holden Day, Oakland, pp. 17–44.
Othani, I., Kusumi, T., Ishitsuka, M.O., Kakisawa, H., 1989. An application of an
advanced Mosher’s method. Tetrahedron 30, 3147–3150.
Pinkerton, F., Strobel, G.A., 1976. Serinol as an activator of toxin production in
attenuated cultures of Heliminthosporium sacchari. Proc. Natl. Acad. Sci. U.S.A.
73, 4007–4011.
Acknowledgements
The NMR spectra were recorded by Mrs Dominique Melck in the
laboratory of the ‘‘Istituto di Chimica Biomolecolare del CNR’’,
Pozzuoli, Italy. Prof. A. Evidente is associated to the ‘‘Istituto di
Chimica Biomolecolare del CNR, Pozzuoli, Italy’’.
Pretsch, E., Bühlmann, P., Affolter, C., 2000. Structure Determination of Organic
Compounds – Tables of Spectral Data. Springer-Verlag, Berlin, pp. 161–243.
Renuka, J., Namita, Y., Teena, B., Satish, J., in press. A new pentacyclic phenol and
other constituents from the rot bark of Bauhinia racemosa Lamk Nat. Prod. Res.
http://dx.doi.org/10.1080/14786419.2013.771351.
Scott, A.I., 1964. Interpretation of Ultraviolet Spectra of Natural Products. Pergamon
Press LTD, Oxford, pp. 15–162.
Stadler, M., Anke, H., Sterner, O., 1995. Metabolites with nematicidal and
antimicrobial activities from the ascomycete Lachnum papyraceum (Karst)
Karst. III. Production of novel isocoumarin derivatives isolation and biological
activities. J. Antibiot. 48, 261–266.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2013.
10.007.
References
Berger, S., Braun, S., 2004. 200 and More Basic NMR Experiments: A Practical
Course, first ed. Weinheim, Wiley-VCH.