Bioactive isocoumarins isolated from the endophytic fungus Microdochium bolleyi

Article abstract of DOI:10.1021/np800095g

Three new isocoumarin derivatives (2-4) were isolated together with monocerin (1) from Microdochium bolleyi, an endophytic fungus from Fagonia cretica, a herbaceous plant of the semiarid coastal regions of Gomera. Compounds 2 and 3 are both 12-oxo epimers of 1, and 4 is a ring-opened derivative of 1. The structures were elucidated by detailed spectroscopic analysis and comparison with reported data. The absolute configurations were determined by a modified Mosher's method. Compounds 1, 3, and 4 showed good antifungal, antibacterial, and antialgal activities against Microbotryum violaceum, Escherichia coll, Bacillus megaterium, and Chlorella fusca. Compound 2 was moderately antifungal and antialgal.

Full text of DOI:10.1021/np800095g

1078
J. Nat. Prod. 2008, 71, 1078–1081
Bioactive Isocoumarins Isolated from the Endophytic Fungus Microdochium bolleyi
Wen Zhang,†,‡ Karsten Krohn,*,‡ Siegfried Draeger,§ and Barbara Schulz§
Research Center for Marine Drugs, School of Pharmacy, Second Military Medical UniVersity, 325 Guo-He Road, Shanghai 200433, People’s
Republic of China, Department of Chemistry, UniVersity of Paderborn, Warburger Strasse 100, 33098 Paderborn, Germany, and Institut fu¨r
Mikrobiologie, Technische UniVersita¨t Braunschweig, Spielmannstrasse 7, 31806 Braunschweig, Germany
ReceiVed February 11, 2008
Three new isocoumarin derivatives (2-4) were isolated together with monocerin (1) from Microdochium bolleyi, an
endophytic fungus from Fagonia cretica, a herbaceous plant of the semiarid coastal regions of Gomera. Compounds 2
and 3 are both 12-oxo epimers of 1, and 4 is a ring-opened derivative of 1. The structures were elucidated by detailed
spectroscopic analysis and comparison with reported data. The absolute configurations were determined by a modified
Mosher’s method. Compounds 1, 3, and 4 showed good antifungal, antibacterial, and antialgal activities against
Microbotryum Violaceum, Escherichia coli, Bacillus megaterium, and Chlorella fusca. Compound 2 was moderately
antifungal and antialgal.
Monocerin (1) and analogues have been isolated as antifungal,
insecticidal, and phytotoxic secondary metabolites from several
fungal sources including Drechslera monoceras (Exserohilum
monoceras, Helminthosporium monoceras),1 Exserohilum turcum,2–4
and Fusarium larVarum.5,6 It is a nonspecific toxin and also is a
nonspecific inhibitor of seed germination.3 Its biological action
could be caused by interference with selected stages of the cell
division cycle.7 Monocerins have attracted the attention of synthetic
chemists because of their unique structural feature, cis-fused
furobenzopyranones, and their broad spectrum of activity.8–12 Ring-
opened derivatives, such as 5, were isolated from Fusarium
larVarum5,6 and demonstrated toxic activity toward insects.5,6,13
The total synthesis of several of these compounds (fusarentins) was
completed by Simpson and co-workers.9,14
In our ongoing screening for biologically active secondary
metabolites from fungi,15 we investigated metabolites produced by
the endophytic fungus Microdochium bolleyi (internal strain no.
8880), isolated from Fagonia cretica, a herbaceous plant of the
semiarid coastal regions of Gomera. We have continued investigat-
ing endophytes from this biotope, since previous investigations had
shown that a particularly high proportion of endophytes from plants
from Gomera were biologically active.16 The fungus was cultivated
on biomalt agar medium. The crude ethyl acetate extract of the
culture showed very good antifungal activity against Microbotryum
Violaceum and moderate algicidal activity against Chlorella fusca.
Fractionation of the ethyl acetate extract led to the isolation and
structural determination of the known monocerin (1), together with
two new analogues and a ring-opened derivative (2-4). We herein
report on the isolation, structural elucidation including relative and
absolute configuration, and the results of some bioactivity tests on
these compounds.
acetate. The crude extract was fractionated on a silica gel column,
followed by Sephadex LH-20 column chromatography to yield a
crude mixture containing 1-4. Further silica column chromatog-
raphy or preparative TLC gave the pure compounds.
The structure of monocerin (1), the major metabolite, was
determined by detailed spectroscopic analysis and comparison with
reported data.1,5,8,12,17,18 In particular, the distinct NOE effect of
H-4 with H-3 and H-10 in the NOESY and NOEDIFF spectra
indicated that these protons had the same (ꢀ) ꢁrientation. The optical
rotation values ([R]20 ) +46.6 for 1, literature1,9,18 +48.2 to
D
+53.0) confirmed the established structure as 1, namely,
(3R,4R,10S)-1 as shown.
Compound 2 was obtained as an optically active colorless oil.
The molecular formula C16H20O7 was established by HREIMS,
indicating seven double-bond equivalents. The IR spectrum of 2
indicated OH (3603 cm-1) and ester carbonyl (1737 cm-1
)
functionalities and a pentasubstituted aromatic system (3027, 1668,
1600, 1430, 861 cm-1). This evidence was in agreement with the
observation of signals in the 13C NMR and DEPT spectra (see
Experimental Section) for four secondary oxygenated carbons (δC
81.2, d; 75.9, d; 74.7, d; 65.0, d), one ester carbonyl atom (δC 167.7,
s), and six aromatic atoms (δΗ 158.7, s; 156.3, s; 137.4, s; 130.9,
s; 104.5, d; 102.0, s), accounting for five double-bond equivalents.
The remaining double-bond equivalents were due to two additional
rings in the molecule.
The 1H and 13C NMR spectra of compounds 1 and 2 were similar,
with differences observed in the side chain carbons: A methylene
group (δC 19.0, t; δH 1.25, m, 1.33, m) in 1 was replaced by an
oxo-methine (δC 65.0, d; δH 4.01, m) in 2. The signals for the methyl
group were shifted downfield with the proton triplet being replaced
by a doublet (δC 24.0, q, δH 1.19, d, J ) 6.0 in 2; δC 13.9, q, δH
0.82, t, J ) 7.4 in 1). These facts suggested that 2 was a 12-hydroxy
analogue of 1. The proposed structure was confirmed by a proton
1
spin system from H-4 to H3-13, established by a H-1H COSY
experiment and the distinct NOE enhancement of H-4 with H-3,
H-9ꢀ, and H-10 observed in the NOESY and NOEDIFF spectra.
The absolute configuration at C-12, bearing a secondary OH group,
was determined as R by a modified Mosher method,19–21 as shown
in Figure 1. The absolute configuration cannot be based on
comparison of the deviating optical rotations of 1 and 2 (see
Experimental Section). However, it is reasonable to assume a
biosynthetic relationship between monocerin (1) and the co-
occurring isocoumarins 2-4. On the basis of the established
absolute configurations of 1 and 4 (see below), compound 2 was
therefore tentatively determined to be (12R)-12-hydroxymonocerin.
The fungus Microdochium bolleyi was cultivated on biomalt agar
medium for 4 weeks at 21 °C and subsequently extracted with ethyl
* To whom correspondence should be addressed. Tel: (+49) 5251-
602172. Fax: (+49) 5251-603245. E-mail: k.krohn@uni-paderborn.de.
† Second Military Medical University.
‡ University of Paderborn.
§ Technische Universita¨t Braunschweig.
10.1021/np800095g CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/30/2008

Notes
Journal of Natural Products, 2008, Vol. 71, No. 6 1079
Table 1. Agar Diffusion Assays for Antibacterial, Antifungal,
and Antialgal Activities (0.05 mg of substance)a
Escherichia
coli
Bacillus
megaterium
Microbotryum Chlorella
compound
Violaceum
fusca
1
2
10
0
6
0
gi 23
7
8
6
3
4
8
6
7
14
18
0
9
9
0
10
10
0
10
18
18
0
0
0
penicillin
tetracycline
nystatin
actidione
acetone
gi 10
0
35
0
0
20
50
0
0
0
Figure 1. ∆δ (δS - δR) values (in Hz) for the MTPA ester of 2.
a Radii of the zones of inhibition are given in mm. gi ) growth
inhibition; i.e., there was some growth within the zone of inhibition.
of the ring system. Compound 4 was thus elucidated as
(3R,4R,10R)-4 as shown.
Compound 4 is structurally correlated to fusarentin 6,7-dimethyl
ether (5) and analogues, a series of metabolites coisolated with
moncerins from the fungus Fusarium larVarum.5,6 Biosynthetic
studies revealed that both groups of metabolites are of heptaketide
origin,andthefusarentinsmayactasprecursorstothemonocerins.17,18
The same biosynthetic relationship probably exists between the
compounds identified in this study.
The isolated compounds 1-4 were tested in an agar diffusion
assay for their antifungal, antibacterial, and algicidal properties
toward Microbotryum Violaceum, Escherichia coli, Bacillus mega-
terium, and Chlorella fusca (Table 1). Whereas compounds 1, 3,
and 4 inhibited all four test organisms, compound 2 was antialgal
against Chlorella fusca and antifungal against Microbotryum
Violaceum, but was not antibacterial. These activities are consistent
with results of our previous investigations in which we compared
the antifungal activities of endophytic fungi and fungi associated
with algae from different marine habitats.16 Isolates from the marine
biotopes from the island of Gomera were particularly active in our
tests.
Figure 2. ∆δ (δS - δR) values (in Hz) for the MTPA ester of 4.
Compound 3 had the molecular formula C16H20O7, the same as
that of 2. The IR and UV spectra of 3 resembled those of 2, and
the 1H and 13C NMR spectra of 3 were nearly identical to those of
2. A difference was observed in the 13C NMR resonances of C-10
(δ 78.4 in 3, 75.9 in 2) and C-12 (δ 67.2 in 3, 65.0 in 2). The
distinct NOE enhancement of H-4 with H-3, H-9ꢀ, and H-10 in
NOESY and NOEDIFF spectra revealed the same polycyclic
skeleton of 3 and 2. The structural difference was thus attributed
to the opposite configuration at C-12. Due to the probable
biosynthetic relationship with monocerin (1) and isocoumarin 4,
compound 3 was determined to be the C-12 epimer of 2, namely,
(12S)-12-hydroxymonocerin.
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 241 MC polarimeter at the sodium D-line. UV
spectra were recorded on a UV-2101PC spectrophotometer; peaks are
reported in nm. IR spectra were recorded on a Nicolet-510P spectro-
photometer; peaks are reported in cm-1. NMR spectra were recorded
at 293 K on a Bruker Avance-500 spectrometer. Chemical shifts are
reported in parts per million (δ), using CDCl3 as an internal standard
Compound 4 had the molecular formula C16H22O7, indicating
six double-bond equivalents. The IR and UV spectra of 4 were
reminiscent of those of 1, showing absorption bands indicating OH
groups, carbonyl groups, and those typical of a pentasubstituted
phenyl group. The observation of a chelated proton, resonating at
1
with coupling constant (J) in Hz. H and 13C NMR assignments were
1
δ 11.04 in the H NMR spectrum, indicated that rings A and B
supported by 1H-1H COSY, HMQC, HMBC, NOESY, and NOEDIFF
experiments. Mass spectra and high-resolution mass spectra were
performed on a MAT 8200 mass spectrometer. Commercial silica gel
(Merck, 0.040-0.063 mm) was used for column chromatography.
Precoated silica gel plates (Merck, G60 F-254 or G50 UV-254) were
used for analytical and preparative thin-layer chromatography (TLC),
respectively. Microbiological methods and culture conditions are as
described in the literature.24,25
were unchanged. This was confirmed by a comparison of the 13C
NMR signals of 1 and of 4 related to ring A. Signals of one ester
carbonyl (δC 169.0, s) and six aromatic carbons (δΗ 158.8, s; 156.1,
s; 136.9, s; 136.5, s; 102.9, d; 101.8, s) were in agreement with the
corresponding values for 1, accounting for five double-bond
equivalents. The remaining double-bond equivalent was attributed
to the fused pyranone (ring B).
Culture, Extraction, and Isolation. The endophytic fungus Mi-
crodochium bolleyi, internal strain No. 8880, was isolated following
surface sterilization from Fagonia cretica, growing in Gomera, and
was cultivated on 12 L of 5% w/v biomalt solid agar medium at room
temperature for 28 days.24,25 The culture was extracted with ethyl
acetate to afford 5 g of a residue after removal of the solvent under
reduced pressure. The extract was subjected to column chromatography
(CC) on silica gel, eluted with a gradient of petroleum ether in ethyl
acetate (90:10, 50:50, 0:100), to give 14 subfractions. Fraction 8 gave
the main metabolite 1 (166.0 mg) after CC on Sephadex LH-20 (CHCl2/
MeOH, 9:1), followed by silica gel CC (400-600 mesh, CH2Cl2).
Fraction 9 was fractionated using Sephadex LH-20 CC (CHCl2/MeOH,
5:1) and then purified by preparative TLC (CH2Cl2/2-propanol, 50:1)
to yield 4 (8.0 mg). Fraction 12 was first subjected to Sephadex LH-
20 CC (CHCl2/MeOH, 2:1) and then preparative TLC (CH2Cl2/2-
propanol, 30:1) to give compounds 2 (2.5 mg) and 3 (3.4 mg).
Analysis of the 1H-1H COSY spectrum readily established the
proton spin system from H-4 to H3-13 and assigned both secondary
OH groups at C-4 and C-10. The small coupling constant (3J3,4
)
2.0) between H-3 and H-4 suggested a cis configuration of these
protons, which was further confirmed by a NOE difference
experiment. The absolute configuration of both secondary alcohol
groups was determined by comparison of the NMR data of their
MTPA esters. As shown in Figure 2, the negative ∆δ (δS - δR)
values for H-3, H-4, H2-9, and H-10 corresponded to a typical ∆δ
pattern for diesters of R,R-1,4-diol with chiral anisotropic reagents
as reported by Riguera and co-workers.22 Therefore, the configu-
ration of both chiral atoms at C-4 and C-10 was elucidated as R.
As a consequence, the absolute configuration at C-3 was also
determined as R on the basis of the established relative configuration

1080 Journal of Natural Products, 2008, Vol. 71, No. 6
Notes
1
Monocerin (1): colorless oil; [R]20 +46.6 (c 2.43, in MeOH); H
7-OCH3), 2.56 (1H, ddd, J ) 13.9, 7.8, 4.9, H-9ꢀ), 2.53 (1H, ddd, J )
13.6, 7.8, 3.3, H-11b), 2.10 (1H, dd, J ) 13.9, 5.0, H-9R), 1.97 (1H,
ddd, J ) 13.6, 9.3, 4.0, H-11a), 1.26 (3H, d, J ) 6.5, H3-13).
Esterification of 4 with MTPA Chloride. Both (S)- and (R)-MTPA
esters of 4 (4S, 4R) were obtained by treatment of 4 (0.6 mg,
respectively) with (R)- and (S)-MTPA chlorides (5 µL) in dry pyridine
(0.5 mL), stirred at room temperature overnight. The MTPA esters (0.87
mg, 62% yielding) were purified by minicolumn chromatography on
silica gel (300 mesh, petroleum ether/EtOAc, 7:1).
D
NMR (500 MHz, CDCl3) δ 11.21 (1H, s, 8-OH), 6.53 (1H, s, H-5),
4.98 (1H, ddd, J ) 6.1, 3.1, 1.0 Hz, H-3), 4.46 (1H, d, J ) 3.1 Hz,
H-4), 4.02 (1H, m, H-10), 3.86 (3H, s, 6-OCH3), 3.79 (3H, s, 7-OCH3),
2.52 (1H, ddd, J ) 14.5, 8.5, 6.1 Hz, H-9ꢀ), 2.03 (1H, ddd, J ) 14.5,
6.0, 1.0 Hz, H-9R), 1.60 (1H, m, H-11b), 1.49 (1H, m, H-11a), 1.33
(1H, m, H-12b), 1.25 (1H, m, H-12a), 0.82 (3H, t, J ) 7.4 Hz, H3-13);
13C NMR (125 MHz, CDCl3) δ 167.8 (C, C-1), 158.6 (C, C-6), 156.0
(C, C-8), 137.2 (C, C-7), 131.3 (C, C-4a), 104.5 (CH, C-5), 101.9 (C,
C-8a), 81.3 (CH, C-3), 78.5 (CH, C-10), 74.3 (CH, C-4), 60.6 (CH3,
7-OCH3), 56.2 (CH3, 6-OCH3), 39.0 (CH2, C-9), 38.1 (CH2, C-11),
19.0 (CH2, C-12), 13.9 (CH3, C-13); EIMS m/z 308.1 [M]+, 265.0,
247.1, 209.0, 167.0, 148.0, 81.1, 41.1.
1H NMR of 4S-MTPA ester (500 MHz, CDCl3): δ 10.92 (1H, s,
8-OH), 6.72 (1H, s, H-5), 5.81 (1H, d, J ) 1.6, H-4), 5.12 (1H, m,
H-10), 4.25 (1H, br t, J ) 6.9, H-3), 3.95 (3H, s, 6-OCH3), 3.95 (3H,
s, 7-OCH3), 2.02 (1H, ddd, J ) 15.8, 8.6, 6.5, H-9b), 1.83 (1H, ddd,
J ) 15.8, 7.4, 4.0, H-9a), 1.66 (1H, m, H-11b), 1.52 (1H, m, H-11a),
1.26 (2H, m, H2-12), 0.91 (3H, t, J ) 7.3, H3-13).
Compound 2: colorless oil; [R]20D -12.7 (c 0.11 in CH2Cl2/MeOH,
1:1); UV (CH2Cl2/MeOH, 1:1) λmax (log ꢀ) 304 (3.18), 275.2 (3.66),
229.0 (3.99) nm; IR (CHCl3) νmax 3603, 3027, 1737, 1668, 1600, 1430,
1119, 861 cm-1; 1H NMR (500 MHz, CDCl3) δ 11.24 (1H, s, 8-OH),
6.58 (1H, s, H-5), 5.05 (1H, ddd, J ) 6.0, 3.0, 1.0 Hz, H-3), 4.56 (1H,
d, J ) 3.0 Hz, H-4), 4.41 (1H, m, H-10), 4.01 (1H, m, H-12), 3.94
(3H, s, 6-OCH3), 3.90 (3H, s, 7-OCH3), 2.65 (1H, ddd, J ) 14.5, 8.5,
6.0 Hz, H-9ꢀ), 2.20 (1H, ddd, J ) 14.5, 5.5, 1.0 Hz, H-9R), 1.82 (1H,
ddd, J ) 14.0, 8.0, 3.5 Hz, H-11b), 1.73 (1H, ddd, J ) 14.0, 9.0, 4.0
Hz, H-11a), 1.19 (3H, d, J ) 6.0 Hz, H3-13); 13C NMR (125 MHz,
CDCl3) δ 167.7 (C, C-1), 158.7 (C, C-6), 156.3 (C, C-8), 137.4 (C,
C-7), 130.9 (C, C-4a), 104.5 (CH, C-5), 102.0 (C, C-8a), 81.2 (CH,
C-3), 75.9 (CH, C-10), 74.7 (CH, C-4), 65.0 (CH, C-12), 60.7 (CH3,
7-OCH3), 56.3 (CH3, 6-OCH3), 44.6 (CH2, C-11), 39.5 (CH2, C-9),
24.0 (CH3, C-13); HREIMS m/z 324.12094 (calcd for C16H20O7,
324.12094).
1H NMR of 4R-MTPA ester (500 MHz, CDCl3): δ 10.87 (1H, s,
8-OH), 6.67 (1H, s, H-5), 5.94 (1H, d, J ) 1.6, H-4), 5.27 (1H, m,
H-10), 4.50 (1H, br t, J ) 6.9, H-3), 3.94 (3H, s, 6-OCH3), 3.92 (3H,
s, 7-OCH3), 2.24 (1H, ddd, J ) 15.4, 7.8, 6.9, H-9b), 2.01 (1H, ddd,
J ) 15.4, 7.3, 4.6, H-9a), 1.67 (1H, m, H-11b), 1.53 (1H, m, H-11a),
1.26 (2H, m, H2-12), 0.87 (3H, t, J ) 7.3, H3-13).
Agar Diffusion Test for Biological Activity. Compounds 1-4 were
dissolved in acetone at a concentration of 1 mg/mL. Fifty microliters
of the solutions (50 µg) was pipetted onto a sterile filter disk
(Schleicher & Schuell, 9 mm), which was placed onto an appropriate
agar growth medium for the respective test organism and subse-
quently sprayed with a suspension of the test organism.25 The test
organisms were the Gram-negative bacterium Escherichia coli, the
Gram-positive bacterium Bacillus megaterium (both grown on NB
medium), the fungus Microbotryum Violaceum, and the alga Chlo-
rella fusca (both grown on MPY medium). Reference substances
were penicillin, nystatin, actidione, and tetracycline. Commencing
at the outer edge of the filter disk, the radius of the zone of inhibition
was measured in millimeters. These microorgansims were chosen
because (a) they are nonpathogenic and (b) they had in the past
proved to be accurate initial test organisms for antibacterial,
antifungal, and antialgal/herbicidal activities.
Compound 3: colorless oil; [R]20D +4.7 (c 0.17 in CH2Cl2/MeOH,
1:1); UV (CH2Cl2/MeOH, 1:1) λmax (log ꢀ) 275.2 (4.21), 229.4 (4.54)
nm; IR (CHCl3) νmax 3603, 3021, 1731, 1674, 1600, 1457, 1166, 854
cm-1; H NMR (500 MHz, CDCl3) δ 11.23 (1H, s, 8-OH), 6.57 (1H,
1
s, H-5), 5.06 (1H, dd, J ) 6.0, 3.0 Hz, H-3), 4.60 (1H, d, J ) 3.0 Hz,
H-4), 4.35 (1H, m, H-10), 3.98 (1H, m, H-12), 3.95 (3H, s, 6-OCH3),
3.91 (3H, s, 7-OCH3), 2.67 (1H, ddd, J ) 14.5, 8.5, 6.0 Hz, H-9ꢀ),
2.22 (1H, dd, J ) 14.5, 5.5 Hz, H-9R), 1.84 (1H, ddd, J ) 14.0, 9.0,
6.0 Hz, H-11b), 1.75 (1H, dt, J ) 14.0, 4.0 Hz, H-11a), 1.18 (3H, d,
J ) 6.0 Hz, H3-13); 13C NMR (125 MHz, CDCl3) δ 167.6 (C, C-1),
158.7 (C, C-6), 156.3 (C, C-8), 137.5 (C, C-7), 130.6 (C, C-4a), 104.5
(CH, C-5), 102.0 (C, C-8a), 80.8 (CH, C-3), 78.4 (CH, C-10), 75.0
(CH, C-4), 67.2 (CH, C-12), 60.7 (CH3, 7-OCH3), 56.3 (CH3, 6-OCH3),
44.8 (CH2, C-11), 39.6 (CH2, C-9), 23.6 (CH3, C-13); HREIMS m/z
324.12096 (calcd for C16H20O7, 324.12094).
Acknowledgment. We thank BASF AG and the Bundesministerium
fu¨r Bildung and Forschung (BMBF), project no. 03F0360A, for
supporting our research work, and Kathrin Meier and Tatjana Stolz
for excellent technical assistance.
References and Notes
Compound 4: colorless oil; [R]20D -4.3 (c 0.58 in CH2Cl2/MeOH,
1:1); UV (CH2Cl2/MeOH, 1:1) λmax (log ꢀ) 306.0 (3.11), 273.4 (3.66),
229.8 (43.97) nm; IR (CHCl3) Vmax 3603, 3027, 1737, 1674, 1620, 1515,
(1) Aldridge, D. C.; Turner, W. B. J. Chem. Soc., Sect. C 1970, 18, 2598–
2600.
(2) Robeson, D. J.; Strobel, G. A. Agric. Biol. Chem. 1982, 46, 2681–
2683.
1457, 1116, 839 cm-1; H NMR (500 MHz, CDCl3) δ 11.04 (1H, s,
1
8-OH), 6.56 (1H, s, H-5), 4.73 (1H, ddd, J ) 5.5, 5.5, 2.0 Hz, H-3),
4.66 (1H, d, J ) 2.0 Hz, H-4), 4.06 (1H, m, H-10), 3.94 (3H, s,
6-OCH3), 3.89 (3H, s, 7-OCH3), 2.23 (1H, ddd, J ) 14.5, 7.5, 3.0 Hz,
H-9b), 2.06 (1H, ddd, J ) 14.5, 7.5, 5.5 Hz, H-9a), 1.57 (1H, m, H-11b),
1.54 (1H, m, H-11a), 1.49 (1H, m, H-12b), 1.40 (1H, m, H-12a), 0.97
(3H, t, J ) 7.0 Hz, H3-13); 13C NMR (125 MHz, CDCl3) δ 169.0 (C,
C-1), 158.8 (C, C-6), 156.1 (C, C-8), 136.9 (C, C-7), 136.5 (C, C-4a),
102.9 (CH, C-5), 101.8 (C, C-8a), 79.5 (CH, C-3), 68.0 (CH, C-10),
66.5 (CH, C-4), 60.7 (CH3, 7-OCH3), 56.3 (CH3, 6-OCH3), 39.7 (CH2,
C-11), 37.6 (CH2, C-9), 18.8 (CH2, C-12), 13.9 (CH3, C-13); HREIMS
m/z 326.13652 (calcd for C16H22O7, 326.13655).
Esterification of 2 with MTPA Chloride. Both (S)- and (R)-MTPA
esters of 2 (2S, 2R) were obtained by treatment of 2 (0.6 mg,
respectively) with (R)- and (S)-MTPA chlorides (5 µL) in dry pyridine
(0.5 mL) catalyzed with dimethylaminopyridine and stirred at room
temperature overnight. The MTPA esters (1.25 mg, 89% yielding) were
purified by a minicolumn chromatography on silica gel (300 mesh,
petroleum ether/EtOAc, 5:1).
(3) Cuq, F.; Herrmann-Gorline, S.; Klaebe, A.; Rossignol, M.; Petitprez,
M. Phytochemistry 1993, 34, 1265–1270.
(4) Lim, C.-H. Agric. Chem. Biotechnol. (Engl Ed.) 1999, 42, 45–47.
(5) Grove, J. F.; Pople, M. J. Chem. Soc., Perkin Trans. 1 1979, 2048-
2051.
(6) Claydon, N.; Grove, J. F.; Pople, M. J. InVertebr. Pathol. 1979, 33,
364–367.
(7) Cuq, F.; Brown, S. C.; Petitprez, M.; Alibert, G. Plant Cell Rep. 1995,
15, 138–142.
(8) Mori, K.; Takaishi, H. Tetrahedron 1989, 45, 1639–1646.
(9) Dillon, M. P.; Simpson, T. J.; Sweeney, J. B. Tetrahedron Lett. 1992,
33, 7569–7572.
(10) Ko, B.-S. Han’guk Nonghwa Hakhoechi 1994, 37, 402–408.
(11) Mallareddy, K.; Rao, S. P. Tetrahedron 1996, 52, 8535–8544.
(12) Cassidy, J. H.; Farthing, C. N.; Marsden, S. P.; Pedersen, A.; Slater,
M.; Stemp, G. Org. Biomol. Chem. 2006, 4, 4118–4126.
(13) Grove, J. F.; Pople, M. Mycopathologia 1981, 76, 65–67.
(14) McNicholas, C.; Simpson, T. J.; Willett, N. J. Tetrahedron Lett. 1996,
37, 8053–8056.
(15) Zhang, W.; Krohn, K.; Zia-Ullah; Flo¨rke, U.; Pescitelli, G.; Di Bari,
L.; Antus, S.; Kurta´n, T.; Rheinheimer, J.; Draeger, S.; Schulz, B.
Chem. Eur. J. 2008, published online.
(16) Schulz, B.; Draeger, S.; dela Cruz, T.; Rheinheimer, J.; Siems, K.;
Loesgen, S; Bitzer, J.; Schloerke, O.; Zeeck, A.; Ines Kock, I.; Hussain,
H.; Dai, J.; Krohn, K. Bot. Mar. 2008, accepted.
(17) Scott, F. E.; Simpson, T. J.; Trimble, L. A.; Vederas, J. C. J. Chem.
Soc., Chem. Commun. 1984, (12), 756–758.
(18) Axford, L. C.; Simpson, T. J.; Willis, C. L. Angew. Chem, Int. Ed.
2004, 43, 727–730.
(19) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512–519.
1H NMR of 2S-MTPA ester (500 MHz, CDCl3): δ 6.56 (1H, s, H-5),
5.25 (1H, m, H-12), 4.95 (1H, dd, J ) 4.9, 3.1, H-3), 4.44 (1H, d, J )
3.1, H-4), 3.97 (3H, s, 6-OCH3), 3.91 (1H, m, H-10), 3.91 (3H, s,
7-OCH3), 2.35 (1H, ddd, J ) 13.8, 8.7, 4.9, H-9ꢀ), 2.28 (1H, ddd, J )
13.0, 6.8, 2.9, H-11b), 2.04 (1H, dd, J ) 13.8, 4.0, H-9R), 1.90 (1H,
ov, H-11a), 1.33 (3H, d, J ) 6.3, H3-13).
1H NMR of 2R-MTPA ester (500 MHz, CDCl3): δ 6.56 (1H, s, H-5),
5.26 (1H, m, H-12), 5.02 (1H, dd, J ) 4.9, 3.0, H-3), 4.52 (1H, d, J )
3.0, H-4), 4.16 (1H, m, H-10), 3.99 (3H, s, 6-OCH3), 3.91 (s, 3H,

Notes
Journal of Natural Products, 2008, Vol. 71, No. 6 1081
(20) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Org. Chem.
1991, 56, 1296–1298.
(21) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092–4096.
(22) Seco, J. M.; Martino, M.; Quinoa, E.; Riguera, R. Org. Lett. 2000, 2,
3261–3264.
(24) Schulz, B.; Sucker, J.; Aust, H.-J.; Krohn, K.; Ludewig, K.; Jones,
P. G.; Doering, D. Mycol. Res. 1995, 99, 1007–1015.
(25) Ho¨ller, U.; Wright, A. D.; Matthe´e, G. F.; Ko¨nig, G. M.; Draeger, S.;
Aust, H.-J.; Schulz, B. Mycol. Res. 2000, 104, 1354–1365.
(23) Krohn, K.; Florke, U.; Rao, M. S.; Steingrover, K.; Aust, H.-J.;
Draeger, S.; Schulz, B. Nat. Prod. Lett. 2001, 15, 353–361.
NP800095G