Cytosporin-related compounds from the marine-derived fungus Eutypella scoparia

Article abstract of DOI:10.1016/j.tet.2008.03.016

Chemical investigation of the culture broth of the fungus Eutypella scoparia ICB-OBX, isolated from the marine pulmonate mollusc Onchidium sp., led to the finding of novel compounds 1 and 2, structurally related to angiotensin II binding inhibitors cytosporins, along with unrelated known nitrogen metabolites (compounds 3-5). The structure and the relative stereochemistry of the novel metabolites were assigned mainly by a detailed analysis of two-dimensional NMR techniques whereas the absolute stereochemistry was proposed by modified Mosher's method. Compound 2 contains an unusual cyclic carbonate functionality that is rare among natural products.

Full text of DOI:10.1016/j.tet.2008.03.016

Tetrahedron 64 (2008) 5365–5369
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Cytosporin-related compounds from the marine-derived fungus Eutypella scoparia
,
b
a
c
Maria Letizia Ciavatta a , M. Pilar Lopez-Gresa , Margherita Gavagnin , Rosario Nicoletti ,
*
Emiliano Manzo a, Ernesto Mollo a, Yue-Wei Guo d, Guido Cimino a
a Istituto di Chimica Biomolecolare, C.N.R., Pozzuoli 80078, Italy
b Centro de Ecolog´ıa Qu´ımica Agr´ıcola, Universidad Polite´cnica de Valencia, Campus de Vera, Edificio 6C, 46022 Valencia, Spain
c Istituto Sperimentale per il Tabacco, C.R.A., Scafati 84018, Italy
d State Key Laboratory of Drug Research, Institute of Materia Medica Shanghai, Institutes for Biological Sciences, Chinese Acadmy of Sciences, 201203 Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical investigation of the culture broth of the fungus Eutypella scoparia ICB-OBX, isolated from the
marine pulmonate mollusc Onchidium sp., led to the finding of novel compounds 1 and 2, structurally
related to angiotensin II binding inhibitors cytosporins, along with unrelated known nitrogen metabo-
lites (compounds 3–5). The structure and the relative stereochemistry of the novel metabolites were
assigned mainly by a detailed analysis of two-dimensional NMR techniques whereas the absolute stereo-
chemistry was proposed by modified Mosher’s method. Compound 2 contains an unusual cyclic carbonate
functionality that is rare among natural products.
Received 4 December 2007
Received in revised form 19 February 2008
Accepted 6 March 2008
Available online 18 March 2008
Keywords:
Hexahydrobenzopyrane derivatives
Cytosporins
Ó 2008 Elsevier Ltd. All rights reserved.
Marine fungus
Eutypella scoparia
Carbonate
NMR
Mosher method
Pulmonata
1. Introduction
examined the secondary metabolites produced in the culture broth
of the fungus Eutypella scoparia ICB-OBX, which was isolated from
Marine microorganisms such as bacteria and fungi inhabit every
environment of the sea and are rich sources of pharmacologically
active compounds.1 The interest in marine microorganisms has
grown in the last few years due to the possibility of getting abun-
dant biomass by in vitro culturing and consequently large amounts
of secondary metabolites. Among marine microorganisms, fungi
have shown great potential due to the high structural diversity of
the natural products produced.2 The majority of the chemical
data reported in the literature refer to fungi derived from sponges
and algae whereas only a few papers deal with the secondary
metabolites of mollusc-derived fungi.2,3 In particular, only the gas-
tropod mollusc, the sea hare Aplysia kurodai, has been reported as
a source of fungal isolates producing compounds with antitumor
and cell adhesion inhibition activities.4
the external part of the mollusc. This study led to two novel mole-
cules 1 and 2, along with known nitrogen compounds 3–5, previ-
ously reported from fungi and other natural sources.7,8
Compounds 1 and 2 are structurally related to cytosporin A,
cytosporin B, and cytosporin C (6), isolated from an endophytic
strain of the taxonomically related fungus Cytospora and showed
to be angiotensin II binding inhibitors.9
In this paper, the chemical characterization of the new metabo-
lites that we named cytosporin D (1) and cytosporin E (2) is
described.
2. Results and discussion
In the course of our investigation on marine molluscs5 we ana-
lyzed the pulmonate gastropod Onchidium sp., collected along the
coast of South China Sea that was found to be characterized by a
series of unprecedented polypropionates.6 Subsequently we also
The EtOAc extract of the culture broth was analyzed by TLC
using different eluent systems. The secondary metabolite pattern
was found to be dominated by the presence of a main compound
along with a series of minor components. The extract was purified
by subsequent silica gel column chromatographic steps to give the
novel compounds 1 (5.1 mg) and 2 (9.0 mg), along with the main
metabolite phenochalasin B (3, 28.3 mg), cyclo-(
D)-Pro-(D)-Leu (4,
* Corresponding author. Tel.: þ39 0818675243; fax: þ39 0818041770.
E-mail address: lciavatta@icmib.na.cnr.it (M.L. Ciavatta).
3.9 mg), and cyclo-( )-Pro-( )-Phe (5, 2.0 mg).
D
D
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.03.016

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M.L. Ciavatta et al. / Tetrahedron 64 (2008) 5365–5369
The already known compounds 3–5 were identified by compar-
an oxirane ring as reported in formula 1. To confirm the suggested
gross structure, a sample of compound 1 was acetylated by Ac2O/
DMPA in CH2Cl2 leading to the triacetyl derivative 1a, which was
analyzed by 1H NMR. Diagnostic acylation shifts were observed
for H-3, H-7, and H2-13 (see Section 3) in agreement with the pro-
posed structure. Compound 1 exhibits a carbon skeleton character-
ized by a polyketide moiety and a terpenoid part, reported only
previously for cytosporins A–C [e.g., cytosporin C (6)], which
were isolated some years ago from an endophytic Cytospora sp.9
The full structure characterization of cytosporins A–C has never
been published whereas their pharmacological properties in the
inhibition of angiotensin II binding have been described.9,10 The
structure proposed for compound 69 displayed strong analogies
with compound 1 differing only in the length of the reduced chain
at C-8 and in the presence of the aldehyde function at C-9 replacing
the hydroxymethyl group. However, the unassigned 13C NMR data
reported for 6 match almost completely with those of compound 1.
Our tentative assignment (see Section 3) displayed the main
difference for the C-7 carbon value. This anomaly could be justified
either by a different orientation of the hydroxy group at C-7 that
was not determined for cytosporin C (6) or by the influence of
the double bond in the side chain of cytosporin D (1).
ing their spectral data (1H and 13C NMR, MS, and [a]D) with the
literature.7,8 The new molecules 1 and 2 were characterized as
described below.
Cytosporin D (1) showed the molecular formula C19H30O5 as
deduced by the sodiated-molecular peak at m/z 361.1988 in the
HRESIMS spectrum indicating five degrees of unsaturation. The
13C NMR spectrum disclosed seven sp3 carbons between d 57.4
and 77.7 that were assigned to carbons linked to oxygen (one pri-
mary, four secondary, and two tertiary), eight sp3 high-field reso-
nating signals between d 14.3 and 36.5, and four sp2 carbons at
d 126.7 (d, C-14), 130.7 (s, C-8), 133.5 (s, C-9), and 135.7 (d, C-15)
indicating two double bonds. The remaining unsaturations
required by the formula were thus attributed to three rings. The
1H NMR spectrum showed two olefinic signals at d 6.48 (H-14)
and 6.15 (H-15), which were attributed to the protons of a disubsti-
tuted double bond, four methine signals due to protons linked to
oxygenated carbons at d 4.65 (H-7), 4.53 (H-10), 3.68 (H-3), and
3.29 (H-6), and an AB system at d 4.41 (H-13 part A), 4.08 (H-13
part B), assigned to an hydroxymethyl, two methyl singlets at
d 1.30 (H3-11) and 1.25 (H3-12), and a methyl triplet at d 0.95
(H3-20). A series of multiplet signals due to five additional methyl-
enes completed the spectrum (Table 1). Analysis of 1H–1H COSY
spectrum clearly defined some spin systems: the chain C-14/C-20
[d 6.48 (d, 16, H-14); 6.15 (dt, 7, 16, H-15); 2.23 (td, 7, H2-16);
1.52–1.43 (m, H2-17); 1.42–1.33 (m, H2-18); 1.42–1.33 (m, H2-19);
and 0.95 (t, 7, H3-20)], an isolated oxygenated AB system [d 4.41
(d, 12, H-13A) and d 4.08 (d, 12, H-13B)], two vicinal oxygenated
methines (d 4.65, br s, H-7 and d 3.29, br s, H-6), and an ABX system
constituted by a methylene linked to carbinolic proton [d 2.32 (t, 12,
H-4ax); d 1.70 (dd, 5, 12, H-4eq); and d 3.68 (dd, 5, 12, H-3)]. A
series of HMBC correlations aided us to link these partial struc-
tures. In particular, C-2 had diagnostic correlations with the
methyls H3-11 and H3-12 and with the protons H-3 and H-10.
The carbon C-5 displayed cross-peaks with the protons H-6, H-7,
and the methylene H2-4 whereas C-9 showed correlations with
methines H-10, H-7, and H-14, as well as with the isolated hydroxy-
methyl H2-13 (Table 1). These data were consistent with a tricyclic
structure containing a hexahydrobenzopyrane moiety fused with
The relative stereochemistry of cytosporin D (1) could only par-
tially be defined by NMR analysis. The hydroxyl at C-3 was equato-
rial as inferred by the multiplicity of the carbinolic proton H-3 (dd,
J¼12.0 and 4.8 Hz) consistent with its axial orientation. A diagnostic
NOE effect was observed between H-10 (d 4.53) and the axial
methyl H3-11 (d 1.30; 16.4, C-11) supporting the axial orientation
of H-10. The other steric effects observed in a series of NOE differ-
ence experiments were not useful to establish the orientation of
both the epoxide ring at C-5/C-6 and the hydroxyl group at C-7 of
cytosporin D (1). However, a cis relationship between the epoxide
ring and H-10 was strongly supported by the comparison of 13C
NMR values of compounds 1 and 6 whereas the orientation of
7-OH was suggested by analysis of the spectral data of the co-occur-
ring cytosporin E (2).
The molecular formula of compound 2 was C20H30O7 as deduced
from the sodiated-molecular peak at m/z 405.1883 in the HRESIMS
spectrum, implying six degrees of unsaturation and an additional

M.L. Ciavatta et al. / Tetrahedron 64 (2008) 5365–5369
5367
Table 1
NMR data of cytosporin D (1) and cytosporin E (2) in CD3OD
C
1
2
d
13Ca
mb
d
1Hc
m (J, Hz)
HMBCd
d
13Ca
mb
d
1Hc
m (J, Hz)
HMBCd
2
3
4
77.7
74.0
36.5
s
d
t
d
H3-11, H3-12, H-10, H-3, H-4eq
H3-11, H3-12, H-4eq, H-4ax
77.6
71.4
42.3
s
d
t
d
H3-11, H3-12, H-10, H-3, H-4eq
H3-11, H3-12, H-4eq, H-4ax
3.68
dd (5,12)
t (12)
dd (5,12)
4.05
dd (5,12)
dd (5,12)
t (12)
2.32ax
1.70eq
d
2.30eq
1.90ax
d
5
6
7
8
57.4
62.1
65.2
130.7
133.5
67.6
16.4
27.9
59.7
s
H-4eq, H-4ax, H-10, H-6, H-7
H-4eq, H-4ax, H-10, H-7
H-6, H-14
H-14, H-10, H-7, H2-13
H-15, H-10, H-7, H2-13, H-6
H2-13, H-4eq
67.6
81.0
75.3
130.1
135.5
70.3
16.7
s
H-4eq, H-4ax, H-10, H-6, H-7
H-4eq, H-4ax, H-10, H-7
H-6, H-14
H-14, H-15, H-10, H-7, H2-13
H-15, H-14, H-10, H-7, H2-13, H-6
H2-13, H-4eq
d
d
s
3.29
4.65
d
br s
br s
d
d
s
4.78
5.58
d
dd (8,2)
d (8)
9
s
d
s
d
10
11
12
13
d
q
q
t
4.53
1.30
1.25
4.41A
4.08B
6.48
6.15
2.23
1.52–1.43
1.42–1.33
1.42–1.33
0.95
br s
s
s
d (12)
d (12)
d (16)
dt (7,16)
td (7)
m
d
q
q
t
4.25
1.30
1.25
4.45A
4.18B
6.53
6.08
2.25
1.52–1.43
1.42–1.33
1.42–1.33
0.95
d (2)
s
s
d (12)
d (12)
d (16)
dt (7,16)
td (7)
m
28.2
60.1
14
15
16
17
18
19
20
21
126.7
135.7
34.5
30.1
32.5
23.5
14.3
d
d
d
t
t
t
H2-16, H-7
H2-16, H2-17
H-14, H-15
H-15
125.5
136.8
34.5
30.0
32.5
23.6
14.3
d
d
t
t
t
t
q
s
H2-16, H-7
H2-16, H2-17
H-14, H-15
H-15
m
m
t (7)
m
m
t (7)
t
q
H2-17, H3-20
H2-17, H3-20
H-7, H-6
d
d
156.0
d
a
b
c
Bruker 300 MHz.
Multiplicity deduced by DEPT.
Bruker 600 MHz.
Significant HMBC correlations (J¼8 and 6 Hz).
d
CO2 unit with respect to 1. The 1H NMR spectrum of 2 showed
strong analogies with that of cytosporin D (1) (Table 1) indicating
the presence of the same hexahydrobenzopyrane skeleton with
an alkyl chain at C-8. In particular, the major differences were
observed for H-6 and H-7 protons resonating in compound 2 at
d 4.78 (dd, J¼8 and 2 Hz) and 5.58 (d, J¼8 Hz), respectively, whereas
in compound 1 these signals resonated at d 3.29 (br s) and 4.65 (br
s) (Table 1). This was consistent with a different substitution
pattern in this part of the molecule. The presence of a carbonate
moiety in compound 2 was suggested by a strong IR absorption
at 1650 cmꢀ1 as well as by a diagnostic 13C NMR signal at d 156.0.
The 13C NMR spectrum of 2 showed in addition the expected four
sp2 carbons, seven sp3 carbons linked to oxygen, and eight sp3
high-field resonating signals, similar to those of 1, accounting for
20 carbons as required by mass formula (Table 1).
derivative 2a, which still retained a free tertiary hydroxyl group
(5-OH). A clear cross peak between H-10 (d 4.25) and 5-OH (d
5.60) was observed in the spectrum recorded in DMSO-d6 (see
Section 3) thus inferring the same orientation of the angular sub-
stituents at C-5 and C-10. Finally, in a NOE difference experiment,
the irradiation of H-6 (d 4.78) caused the enhancement of both
H-7 (d 5.58) and H-4eq (d 2.30) signals indicating that the carbonate
cycle was trans-oriented with respect to the angular 5-OH. Accord-
ingly, a strong NOE effect was observed between H-15 and H-7.
Once the relative stereochemistry of cytosporin E (2) was estab-
lished, a possible correlation between the two co-occurring metab-
olites 1 and 2 was evaluated. From a biogenetic point of view,
compound 2 could be considered as derived from compound 1. In
fact, it should be formed by the attack of a carbonate ion at C-6 of
the oxirane ring of compound 1 from the less hindered face. The
esterification of the carbonate unit by the vicinal hydroxyl at C-7
should give the closure of the ring leading to cytosporin E (2).
This implies that the relative stereochemistry at C-5, C-6, and C-7
of cytosporin D (1) should be that reported with the 5,10 cis-junc-
tion and the hydroxyl group at C-7 trans-oriented with respect to
the oxirane ring.
Finally, the absolute stereochemistry of cytosporin D (1) was
determined by applying the modified Mosher’s method.13 After
protection of the primary hydroxyl group at C-13 with DMT (see
Section 3), 1b was reacted with (ꢀ)-R- and (þ)-S-MTPA chlorides
to obtain S-ester (1c) and R-ester (1d), respectively. The Dd values
(DSꢀDR) observed for the signals of protons nearby the hydroxyl
groups at C-3 and C-7 (see Table 2) indicated the S and the R con-
figuration, respectively, as reported in the structure. In particular,
the S configuration at C-3 led to assignment of the absolute stereo-
chemistry at carbons C-5, C-6, and C-10, whereas the observed Dd
values after Mosher esterification at C-7 assigning the R absolute
stereochemistry at the chiral center, confirmed the suggested trans
relationship between the oxirane ring and 7-OH. Even though the
optical rotation of cytosporin D {[a]D ꢀ3.0 (c 0.5, CH3OH)} is oppo-
site to that of cytosporin E {[a]D þ59.9 (c 0.29, CH3OH)}, the same
absolute stereochemistry could be suggested for both compounds
on the basis of biogenetical consideration. The structure 6, closely
The 1H–1H COSY spectrum of 2 revealed the presence of the
same spin systems as 1 that were connected by HMBC analysis to
give the same carbon framework. In particular, diagnostic HMBC
correlations between C-21 and both protons H-6 and H-7 led us
to link the carbonyl to both oxygen atoms at C-6 and C-7 obtaining
a cyclic carbonate moiety, as reported in formula 2. Such function-
ality, which is quite rare in nature, has been reported for metabo-
lites of fungi of genera Phoma11 and Eupenicillium12 [e.g.,
phomoxin C (7)12].
A sample of compound 2 was submitted to acetylation giving
the acetyl derivative 2a, which was characterized by NMR spectros-
copy and mass spectrometry. Diagnostic acylation shifts were
observed for H-3 and H2-13 (see Section 3) in agreement with
the proposed structure.
The relative stereochemistry of cytosporin E (2) was determined
by analysis of both 1H–1H coupling constants and NOE difference
experiments. Analogously with 1, the multiplicity of the carbinolic
proton H-3 (d 4.05, dd, J¼11.8 and 4.8 Hz) indicated the equatorial
orientation of the hydroxyl group at C-3 whereas significant NOE
interactions between H-10 (d 4.25) and the axial methyl H3-11 (d
1.30) supported the axial orientation of H-10. The cis-junction of
the hexahydrobenzopyrane moiety of cytosporin E (2) was deter-
mined by analyzing a NOESY experiment carried out on the acetyl

5368
M.L. Ciavatta et al. / Tetrahedron 64 (2008) 5365–5369
Table 2
3.0 g, per liter), prepared with artificial seawater. This fermentation
was performed at 28 ꢁC, without shaking for 30 days, in a total
volume of 4 L.
NMR selective assignments of 1c (S-ester) and 1d (R-ester) in CD3OD
1c (S-ester)
d H
1d (R-ester)
d H
Dd
m (J, Hz)
m (J, Hz)
H-3
H2-4
4.48
2.26
1.91
3.39
6.27
4.48
0.99
1.20
6.02
5.86
dd (4.8,12.0)
t (12.0)
dd (4.8,12.0)
s
s
s
s
s
4.95
2.52
2.04
3.53
6.18
4.61
1.01
1.19
5.89
5.66
dd (4.8, 12.0)
t (12.0)
dd (4.8, 12.0)
s
s
s
s
s
ꢀ0.47
ꢀ0.26
ꢀ0.13
ꢀ0.14
þ0.09
ꢀ0.13
ꢀ0.022
þ0.01
þ0.13
þ0.20
3.3. Extraction and isolation
H-6
H-7
After incubation the mycelium was removed from the culture
broth by filtration. Then, the broth (4 L) was extracted with ethyl
acetate four times (400 mLꢂ4). The resulting organic extract was
evaporated under vacuum to give a brown oil (400 mg) and parti-
tioned by column chromatography on silica gel using stepwise
gradient elution from CHCl3 and increasing amounts of CH3OH to
give nine fractions, two of which containing compounds 1–5. The
less polar fraction was further purified on silica gel column (n-hex-
H-10
H3-11
H3-12
H-14
H-15
d (16.0)
dt (7.0,16.0)
d (16.0)
dt (7.0, 16.0)
ane/EtOAc gradient) to obtain cyclo-(
D)-Pro-(D)-Leu (4, 3.9 mg) and
related to cytosporin D (1), is that suggested for cytosporin C, but
optical rotation and stereochemical data are not reported in the
paper.
cyclo-( )-Pro-( )-Phe (5, 2.0 mg). The more polar fraction was puri-
D
D
fied on silica gel column (n-hexane/EtOAc gradient) to give pheno-
chalasin B (3, 28.3 mg), cytosporin D (1, 5.1 mg), and cytosporin E
(2, 9.0 mg), in order of polarity.
Cytosporin D (1) and E (2) were tested in antimicrobial assays
against Staphylococcus aureus, Escherichia coli, and Candida albicans.
No inhibitory activity was observed for the two molecules.
In conclusion, two new molecules have been isolated from the
marine-derived fungus E. scoparia. This species, which has been
reported from many and diverse environments ranging from soil
in Antarctica14 to tropical forests of Australia15 and Thailand,16
was isolated here for the first time from a marine source. However,
this is not surprising because other closely related fungi (e.g., Cyto-
spora) mostly known in association with woody plants have been
also reported from marine invertebrates, in accordance with the
suggestion that fungi have the capability of adapting to harsh
environments.
3.3.1. Cytosporin D (1)
Pale yellow oil; [a]D ꢀ3.0 (c 0.5, CH3OH); IR (liquid film) 3426
(br), 2928, 1792, 1507, 1248 cmꢀ1; HRESIMS: found 361.1988
(361.1991 calculated for C19H30O5Na); 1H and 13C NMR in CD3OD
see Table 1.
3.3.2. Cytosporin E (2)
Pale yellow oil; [a]D þ59.9 (c 0.29, CH3OH); IR (liquid film) 3339
(br), 2936, 1792, 1651, 1462, 1375, 1032 cmꢀ1; HRESIMS: found
405.1883 (405.1889 calculated for C20H30O7Na); 1H and 13C NMR
data in CD3OD see Table 1.
1H and 13C NMR data in DMSO-d6 (d in ppm, J in Hz): 3.78–3.70
(H-3, m), 2.10 (H-4eq, dd, 13, 6), 1.74 (H-4ax, t, 13), 4.70 (H-6, d, 8),
5.52 (H-7, d, 8), 4.08 (H-10, s), 1.13 (H3-11, s), 1.06 (H3-12, s), 4.28 (H-
13A, dd, 13, 4), 3.92 (H-13B, dd, 13, 6), 6.46 (H-14, d, 16), 5.92–5.84
(H-15, m), 2.18–2.11 (H2-16, m), 1.44–1.35 (H2-17, m), 1.34–1.25 (H2-
18, m),1.34–1.25 (H2-19, m), 0.88 (H3-20, t, 7), 4.84 (3-OH, d, 5), 4.70
(13-OH, br t, 5), 5.28 (5-OH, br s); 76.0 (C-2, C), 69.2 (C-3, CH), 41.3
(C-4, CH2), 67.0 (C-5, C), 79.3 (C-6, CH), 73.5 (C-7, CH), 136.5 (C-8, C),
127.5 (C-9, C), 68.1 (C-10, CH),16.3 (C-11, CH3), 28.0 (C-12, CH3), 58.5
(C-13, CH2), 125.0 (C-14, CH), 133.0 (C-15, CH), 34.0 (C-16, CH2), 29.5
(C-17, CH2), 29.0 (C-18, CH2), 22.0 (C-19, CH2), 14.8 (C-20, CH3),
155.0 (C-21, C).
3. Experimental
3.1. General experimental procedure
Silica gel chromatography was performed using pre-coated
Merck F254 plates and Merck Kieselgel 60 powder. Optical rotations
were measured on a Jasco DIP 370 digital polarimeter. IR spectra
were recorded on a Biorad FTS 155 FT-IR spectrophotometer.
NMR experiments were recorded at ICB-NMR Service Centre. 1D
and 2D NMR spectra were acquired in CD3OD (shifts are referenced
to the solvent signal) on a Bruker DRX-600 operating at 600 MHz,
using an inverse TCI CryoProbe fitted with a gradient along the
Z-axis. 13C NMR were recorded on a Bruker DPX-300 operating at
300 MHz using a dual probe. High resolution ESIMS were per-
formed on a Micromass Q-TOF MicroÔ coupled with a HPLC Waters
Alliance 2695. The instrument was calibrated using a PEG mixture
from 200 to 1000 MW (resolution specification 5000 FWHM, devi-
ation <5 ppm rms in the presence of a known lock mass).
3.3.3. Cytosporin C (6)
Tentative assignment (d in ppm, CDCl3) of the reported carbon
values:9 77.2 (C-2), 74.3 (C-3), 35.5 (C-4), 57.8 (C-5), 62.4 (C-6),
55.8 (C-7), 157.7 (C-8), 129.9 (C-9), 68.9 (C-10), 16.0 (C-11), 27.8
(C-12), 189.7 (C-13), 31.6 (C-14), 29.9 (C-15), 29.1 (C-16), 22.3 (C-
17), 13.9 (C-18).
3.2. Fungal material and fermentation
3.3.4. Compound 1a
Cytosporin D (1) (1 mg) was dissolved in anhydrous CH2Cl2
(0.5 mL) with a catalytic amount of DMAP and 10 mL of acetic anhy-
dride was added to this solution. The reaction was stirred overnight
at room temperature. CH2Cl2 was removed in vacuo and the residue
was worked up by preparative TLC (SiO2, eluent CHCl3/CH3OH,
98:2) and the resulting UV band was scraped to afford pure 1a
(0.7 mg). Rf 0.9 (CHCl3/CH3OH, 98:2); [a]D 0.0 (c 0.13, CH3OH); IR
(liquid film) 2936, 2855, 1738, 1375, 1223, 1037 cmꢀ1; selected 1H
NMR signals (d in ppm): 6.00 (H-7), 4.85 (H-3), 4.85–4.70 (H2-13),
2.12 (3H, 13-COCH3), 2.06 (6H, 3-COCH3 and 7-COCH3). ESIMS:
m/z 465 [MþH]þ, 405 [Mꢀ60þH]þ, 345 [Mꢀ2ꢂ60þH]þ, 285
[Mꢀ3ꢂ60þH]þ; HRESIMS: found 464.2416 (464.2410 calculated
for C25H34O8).
Isolate ICB-OBX was recovered from the surface of pulmonate
Onchidium sp. collected in the intertidal zone along the coast of
Lingshui County (Hainan province, China), during December
2005. On the basis of ITS sequence data it was identified as
E. scoparia by the Centraalbureau voor Schimmelcultures (CBS,
Utrecht, the Netherlands). The Genbank number for E. scoparia is
AF 373064.
The fungus E. scoparia ICB-OBX was grown on malt extract agar
(MEA; malt extract, mycological peptone, agar) at 28 ꢁC. After
a week of cultivation the mycelial plugs were cut by a cork borer
and inoculated into Erlenmeyer flasks containing 500 mL of malt
extract broth (MEB; malt extract 17.0 g, mycological peptone

M.L. Ciavatta et al. / Tetrahedron 64 (2008) 5365–5369
5369
3.3.5. Compound 1b
1.02 (H3-12, s), 4.82 (H-13A, d, 14), 4.70 (H-13B, d, 14), 6.45 (H-14,
d, 16), 6.05–5.97 (H-15, m), 2.21–2.13 (H2-16, m), 1.44–1.36 (H2-
17, m), 1.24–1.18 (H2-18, m), 1.32–1.26 (H2-19, m), 0.88 (H3-20, t,
7), 5.60 (5-OH, s), 2.03 (3-COCH3, s), 2.01 (13-COCH3, s); 75.0
(C-2, C), 72.3 (C-3, CH), 37.5 (C-4, CH2), 67.0 (C-5, C), 79.0 (C-6,
CH), 74.3 (C-7, CH), 130.0 (C-8, C), 132.0 (C-9, C), 69.2 (C-10, CH),
16.5 (C-11, CH3), 27.7 (C-12, CH3), 61.0 (C-13, CH2), 124.0 (C-14,
CH), 135.5 (C-15, CH), 32.9 (C-16, CH2), 28.2 (C-17, CH2), 30.2 (C-
18, CH2), 21.9 (C-19, CH2), 14.1 (C-20, CH3), 154.2 (C-21, C), 170.0
(3-COCH3- and 13-COCH3, C), 21.5 (3-COCH3 and 13-COCH3, C).
Compound 1 (5.0 mg, 0.015 mM) was stirred in anhydrous
CH2Cl2 (1 mL), catalytic amount of DMAP, and 5.0 mg of 4,4’-dime-
thoxytrytil chloride (0.015 mM, DMT) for 2 h at room temperature.
CH2Cl2 was removed in vacuo and the reaction mixture was puri-
fied by chromatography in a Pasteur pipette (SiO2, CHCl3). Rf 0.5
(CHCl3/CH3OH, 95:5); [a]D þ12.0 (c 0.05, CH3OH); IR (liquid film)
2922, 2847, 1651, 1516, 1462, 1254, 1032 cmꢀ1; EIMS: m/z 640
[M]þ; 1H and 13C NMR data in CD3OD (d in ppm, J in Hz): 3.66
(H-3, dd, 13, 4), 2.24 (H-4ax, t, 13), 1.64 (H-4eq, dd, 13, 4), 3.26
(H-6, br s), 4.63 (H-7, br s), 4.51 (H-10, br s), 1.29 (H3-11, s), 1.30
(H3-12, s), 3.66 (H-13A, d, 11), 3.80 (H-13B, d, 11), 6.06–5.98
(H-14, m), 6.06–5.98 (H-15, m), 2.09–2.00 (H2-16, m), 1.34–1.38
(H2-17, m), 1.43–1.34 (H2-18, m), 1.36–1.25 (H2-19, m), 0.93 (H3-
20, t, 7), 6.84–7.52 (Ar–H, 13H), 3.80 (OCH3, s, 6H); 77.9 (C-2, C),
73.9 (C-3, CH), 36.5 (C-4, CH2), 57.5 (C-5, C), 61.6 (C-6, CH), 65.1
(C-7, CH), 134.4 (C-8, C), 130.0 (C-9, C), 68.6 (C-10, CH), 16.4 (C-11,
CH3), 27.9 (C-12, CH3), 61.2 (C-13, CH2), 127.8 (C-14, CH), 135.3
(C-15, CH), 34.3 (C-16, CH2), 30.7 (C-17, CH2), 32.4 (C-18, CH2),
23.0 (C-19, CH2), 14.4 (C-20, CH3), 55.7 (OCH3), 114.0–158.0 (Ar).
Acknowledgements
The authors thank ICB personnel: Mrs. D. Ricciardi for technical
support, Mr. R. Turco for artwork, and Mrs. D. Melck for biological
assays. Dr. A.W.A.M. de Cock of CBS and Dr. A. Ricelli of ICB (branch
of Rome) are gratefully acknowledged for the taxonomy of E. scopa-
ria and for valuable discussion, respectively. This work was partially
supported by the Italian-Chinese bilateral project CNR-CAS ‘Chem-
ical studies on bioactive molecules with biotechnological interest
from Chinese marine invertebrates’.
3.3.6. Compound 1c (S-MTPA-ester)
Compound 1c was prepared by treating 2 mg of 1b with
0.005 mL of R-(ꢀ)-MTPA chloride in dry CH2Cl2 (0.5 mL) with cata-
lytic amount of DMAP under stirring for 16 h at room temperature.
The ester was purified by chromatography in a Pasteur pipette
(SiO2, CHCl3). Rf 0.9 (CHCl3/CH3OH, 98:2); [a]D þ9.8 (c 0.06,
CH3OH); IR (liquid film) 2928, 2855, 1752, 1507, 1462, 1246, 1173,
1024, 709 cmꢀ1; selected 1H NMR values are in Table 2.
References and notes
1. Jensen, P. R.; Fenical, W. Marine Microorganisms and Drug Discovery: Current
Status and Future Potential. In Drugs from the Sea; Fusetani, N., Ed.; S. Karger
AG: Basel, 2000; pp 6–29.
2. Bugni, T. S.; Ireland, C. M. Nat. Prod. Rep. 2004, 21, 143–163.
3. Saleem, M.; Shaiq Ali, M.; Hussain, S.; Jabbar, A.; Ashraf, M.; Lee, Y. S. Nat. Prod.
Rep. 2007, 24, 1142–1152.
4. Numata, A.; Iritani, M.; Yamada, T.; Minora, K.; Matsumura, E.; Yamori, T.;
Tsuruo, T. Tetrahedron Lett. 1997, 38, 8215–8218.
3.3.7. Compound 1d (R-MTPA-ester)
Compound 1d was prepared by treating 2 mg of 1b with
0.005 mL of S-(ꢀ)-MTPA chloride in dry CH2Cl2 (0.5 mL) with cata-
lytic amount of DMAP under stirring for 16 h at room temperature.
The ester was purified by chromatography in a Pasteur pipette
(SiO2, CHCl3). Rf 0.9 (CHCl3/CH3OH, 98:2); [a]D ꢀ30.5 (c 0.01,
CH3OH); IR (liquid film) 2955, 2847, 1749, 1508, 1462, 1252, 1168,
1026, 702 cmꢀ1; selected 1H NMR values are in Table 2.
5. Wahidullah, S.; Guo, Y.-W.; Fakhr, I. M.; Mollo, E. Molluscs; Cimino, G., Gavagnin,
M., Eds.; Springer: Berlin, 2006; Vol. 43, pp 175–198; Ciavatta, M. L.; Lopez, M.
P.; Gavagnin, M.; Manzo, E.; Mollo, E.; D’Souza, L.; Cimino, G. Molecules 2006, 11,
808–816; Manzo, E.; Gavagnin, M.; Bifulco, G.; Cimino, P.; Di Micco, S.; Ciavatta,
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3.3.8. Compound 2a
Cytosporin E (2) (1 mg) was dissolved in anhydrous CH2Cl2
(0.5 mL) with catalytic amount of DMAP and 10 mL of acetic anhy-
dride was added to this solution. The reaction was stirred overnight
at room temperature. CH2Cl2 was removed in vacuo and the residue
was worked up by preparative TLC (SiO2, eluent CHCl3/CH3OH,
95:5) and the resulting UV band was scraped to afford pure 2a
(0.9 mg). Rf 0.5 (CHCl3/CH3OH, 95:5); [a]D þ5.0 (c 0.03, CH3OH);
IR (liquid film) 2955, 2847, 1749, 1645, 1462, 1352, 1068, 1030,
702 cmꢀ1; selected 1H NMR signals in CD3OD (d in ppm): 5.25 (H-
3), 4.95–4.82 (H2-13), 2.08, 2.10 (6H, 3-COCH3 and 13-COCH3).
ESIMS: m/z 489 [MþNa]þ, 407 [Mꢀ60þH]þ; HRESIMS: found
489.2110 (489.2101 calculated for C24H34O9Na).
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2771–2775.
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1H and 13C NMR assignments in DMSO-d6 (d in ppm, J in Hz):
5.03 (H-3, dd, 12, 6), 2.25 (H-4eq, dd, 12, 6), 1.84 (H-4ax, t, 12),
4.78 (H-6, d, 8), 5.60 (H-7, d, 8), 4.00 (H-10, br s), 1.23 (H3-11, s),
16. Pongcharoen, W.; Rukachaisirikul, V.; Phongpaichit, S.; Rungjindamai, N.;
Sakayaroj, J. J. Nat. Prod. 2006, 69, 856–858.