Diversity of antimicrobial pyrenophorol derivatives from an endophytic fungus, Phoma sp.

Article abstract of DOI:10.1002/ejoc.200800404

Pyrenophorol (1) and (-)-dihydropyrenophorin (3), two known macrodiolides, were isolated together with four new analogues (2, 4-6) and three ring-opened derivatives (7-9) from Phoma sp., an endophytic fungus isolated from Lycium intricatum from Gomera. The structures of the new compounds were elucidated by detailed spectroscopic analysis, comparison with reported data, and chemical interconversion. The absolute configurations were determined by chemical correlations and a modified Mosher's method. The diversity of these seven newly discovered metabolites not only extends the pyrenophorol macrocyclic family, but also gives insight into the biosynthetic interconnections, in particular by isolation of the open-chain precursors. Preliminary studies showed antimicrobial activity of these compounds against the fungus Microbotryum violaceum, the alga Chlorella fusca, and the bacteria Escherichia coli and Bacillus megaterium. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200800404

FULL PAPER
DOI: 10.1002/ejoc.200800404
Diversity of Antimicrobial Pyrenophorol Derivatives from an Endophytic
Fungus, Phoma sp.[‡]
Wen Zhang,[a,b] Karsten Krohn,*[b] Hans Egold,[b] Siegfried Draeger,[c] and Barbara Schulz[c]
Dedicated to Professor Dr. Gerald Henkel on the occasion of his 60th birthday
Keywords: Absolute configuration / Antimicrobial activity / Endophytic fungus / Macrodiolides / Phoma sp. / Pyrenophorol
Pyrenophorol (1) and (–)-dihydropyrenophorin (3), two
known macrodiolides, were isolated together with four new
analogues (2, 4–6) and three ring-opened derivatives (7–9)
from Phoma sp., an endophytic fungus isolated from Lycium
tends the pyrenophorol macrocyclic family, but also gives in-
sight into the biosynthetic interconnections, in particular by
isolation of the open-chain precursors. Preliminary studies
showed antimicrobial activity of these compounds against
intricatum from Gomera. The structures of the new com- the fungus Microbotryum violaceum, the alga Chlorella
pounds were elucidated by detailed spectroscopic analysis,
comparison with reported data, and chemical interconver-
sion. The absolute configurations were determined by chemi-
fusca, and the bacteria Escherichia coli and Bacillus mega-
terium.
cal correlations and a modified Mosher’s method. The diver- (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
sity of these seven newly discovered metabolites not only ex-
Germany, 2008)
hydroxy acid subunits. Many of these diolides show strong
antifungal,[2,7,22] antihelmintic,[4,6,25] or phytotoxic ac-
tivity.[5,23,24] This broad spectrum of bioactivity and the
unique structure of pyrenophorol (1) and its analogues have
also attracted great attention from synthetic chemists.[26–44]
As part of our continuing investigations to elucidate new
bioactive metabolites from fungi, we previously analyzed
the culture extract of an endophytic Phoma sp., isolated
from the plant Fagonia cretica. This work resulted in the
isolation of tetrahydropyrenophorol, a hydrogenated pyren-
ophorol derivative.[7] The structure of the tetrahydropyreno-
phorol was unambiguously confirmed by X-ray single-crys-
tal structure analysis with its absolute configuration solved
by the solid-state CD/TDDFT method.[7] In connection
with our ongoing screening for biologically active second-
ary metabolites from fungi,[45] we have now investigated an-
other endophytic Phoma sp. (internal strain no. 8874) iso-
lated from Lycium intricatum, a Mediterranean plant from
the island of Gomera, Spain. The crude ethyl acetate extract
of the biomalt agar culture showed good antifungal activity
against Microbotryum violaceum and moderate algicidal ac-
tivity against Chlorella fusca. Fractionation of the ethyl ace-
tate extract led to the isolation and structural determination
of known pyrenophorol (1) and (–)-dihydropyrenophorin
(3), together with four new analogues (2, 4–6) and three
novel ring-opened derivatives (7–9) (Figure 1). The four
new analogues are 4-acetylpyrenophorol (2), 4Ј-acetyldihyd-
ropyrenophorin (4), cis-dihydropyrenophorin (5), and tetra-
hydropyrenophorin (6), while the three ring-opended deriv-
Introduction
Naturally occurring macrodiolides, which can be isolated
from various fungi and marine sponges, are an interesting
family of secondary metabolites with pronounced biological
activity. A prominent example from marine sponges is swin-
holide A, possessing a large 44-membered ring structure.[1]
The macrodiolides can roughly be divided into two groups:
They form either homodimers, constructed of two identical
monomeric units, or heterodimers with two different sub-
units. Examples with a symmetrical 16-membered ring
system are pyrenophorol (1),[2–7] vermiculine,[8–10] and
elaiophylin;[11–13] representatives of heterodimeric 14-mem-
bered rings include colletodiol[14–18] and grahamim-
ycin A1.[19] Within the homodimers, pyrenophorol (1) and
analogues such as pyrenophorin (10)[20–23] or (–)-dihy-
dropyrenophorin (3)[24] represent the simplest group within
the cluster of 16-membered macrocyclic dilactones. A typi-
cal arrangement of these naturally occurring diolide 16-
membered rings is the head-to-tail connection of the two C8
[‡] Biologically Active Secondary Metabolites from Fungi, 36. Part
35: Ref.[45]
[a] Research Center for Marine Drugs, School of Pharmacy,
Second Military Medical University,
325 Guo-He Road, Shanghai 200433, P. R. China
[b] Department of Chemistry, Universität Paderborn,
Warburger Straße 100, 33098 Paderborn, Germany
Fax: +49-5251-603245
E-mail: k.krohn@uni-paderborn.de
[c] Institut für Mikrobiologie, Technische Universität Braunschweig,
Spielmannstraße 7, 31806 Braunschweig, Germany
4320
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 4320–4328

Antimicrobial Pyrenophorol Derivatives
Figure 1. Structures 1–9 isolated from Phoma sp.
atives are structurally correlated to (–)-dihydropyrenopho- chemistry of pyrenophorol (1) was confirmed by X-ray dif-
rin (3) and named seco-dihydropyrenophorin (7), 7Ј-acetyl- fraction analysis.[6] Its absolute configuration was deter-
seco-dihydropyrenophorin (8), and seco-dihydropyrenopho- mined by enantioselective syntheses[42,43] and also by the
rin-1,4-lactone (9). We herein report on the isolation, struc- synthesis of ent-1.[44] (–)-Dihydropyrenophorin (3) had been
tural elucidation, and bioactivities of these compounds.
previously isolated from Drechslera avenae.[24] The structure
was elucidated by spectroscopic analysis and oxidation to
the diketone pyrenophorin (10) by using Jones’ reagent. Py-
renophorin (10) is a good antifungal and herbicidal agent
Results and Discussion
The fungus Phoma sp. was cultivated on biomalt agar and has been isolated from Pyrenophora avenae,[20] Stem-
medium for four weeks, and then extracted with ethyl ace- phylium radicinum,[21,22] and Drechslera avenae.[23]
1
tate. The crude extract was fractionated on a silica gel col-
Interestingly, some H NMR signals of pyrenophorol (1)
umn, followed by Sephadex LH-20 column chromatography vary considerably with the concentrations above 20 mg/mL,
to yield pure compounds 1, 4, 7, 8, and mixtures containing whereas the 13C NMR signals remain virtually unchanged.
compounds 2, 3, 5, 6 and 9, respectively. Further silica col- When the concentration is increased from 20 to 60 mg/mL,
umn chromatography gave compounds 2, 3, 5, 6 and 9 in a noticeable upfield shift of the 1H NMR signals is ob-
pure form. The structures were elucidated by spectroscopic served, probably due to intermolecular interactions at
analysis in combination with chemical interconversions.
higher concentration (Table 1).
The structure of pyrenophorol (1), the major metabolite
Compound 2 was isolated as an optically active, colorless
of the title fungus, was determined by detailed spectro- oil. The molecular formula C18H26O7 was established by
scopic analysis and comparison with reported data. Pyreno- HRESIMS. The IR spectrum of 2 indicated the presence of
phorol (1) was previously isolated from the fungus Byssoch- a hydroxy group (3603 cm–1) and an α,β-unsaturated ester
lamys nivea,[2] and subsequently found in Stemphylium group (1717, 1244, 1134 cm–1). The presence of α,β-unsatu-
radicinum,[3] Alternaria alternata,[4] Drechslera avenae,[5] rated esters was also supported by signals for two pairs of
Byssochlamys nivea,[6] and Phoma sp.[7] The relative stereo- olefinic protons (δH = 6.77, 5.93; 6.85, 5.93 ppm) in the
Table 1. NMR spectroscopic data for 1 in different concentrations (c).
No.
a: c = 2 and 20 mg/mL
δH, m, J [Hz]
b: c = 60 mg/mL
δH, m, J [Hz]
∆δ = δa – δb
δC, m[a]
δC, m[a]
∆δH
∆δC
1,1Ј
2,2Ј
3,3Ј
165.0, s
122.2, d
149.4, d
69.8, d
30.5, t
165.3, s
122.0, d
149.1, d
69.9, d
30.5, t
–0.3
0.2
0.3
–0.1
0
5.98, dd, 15.3, 1.5
6.90, dd, 15.3, 5.0
4.30, m
5.98, dd, 15.8, 1.0
6.83, dd, 15.8, 5.0
4.19, dd, 12.3, 6.3
1.79, dd, 13.3, 7.3
1.79, dd, 13.3, 7.3
1.57, m
0
0.07
0.11
0.05
0.12
0.08
0.07
0.09
0.07
4,4Ј
5a,5aЈ
5b,5bЈ
6a,6aЈ
6b,6bЈ
7,7Ј
1.84, m
1.91, m
1.65, m
1.74, m
5.13, m
1.28, d, 6.5
29.0, t
28.8, t
0.2
1.67, m
5.04, m
1.21, d, 6.5
70.4, d
18.3, q
70.3, d
18.4, q
0.1
–0.1
8,8Ј
[a] By DEPT sequence.
Eur. J. Org. Chem. 2008, 4320–4328
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W. Zhang, K. Krohn, H. Egold, S. Draeger, B. Schulz
FULL PAPER
1
1
downfield area of the H NMR spectrum. The H NMR
Compound 4 was obtained as an optically active color-
spectrum of 2 was similar to that of 1, except for the pres- less oil with the molecular formula C18H24O7, established
1
ence of an additional methyl signal at δH = 2.06 (s) ppm by HREIMS. The H and 13C NMR spectra of 4 (Table 2)
(Table 2). This signal, in conjunction with two carbon reso- resembled those of 3, revealing their similar structures. The
nances at δC = 169.8 (s) and 21.1 (q) ppm in its 13C NMR presence of signals at δH = 2.10 ppm and δC = 169.7,
spectrum (Table 2), suggested the presence of an acetyl 21.0 ppm in combination with the remarkable downfield
group in 2. The location of the acetoxy group at C-4 was shift of the methine proton signal (δH = 5.31 ppm in 4 and
shown by the downfield shift of the respective proton signal δH = 4.39 ppm in 3) indicated the presence of an acetoxy
at C-4 at δH = 4.30 ppm in 1 to δH = 5.20 ppm in 2. An group in 4. Thus, compound 4 was established as 4Ј-acetyl-
additional structural proof came from chemical intercon- dihydropyrenophorin. This was further confirmed by acety-
version: Acetylation of both 1 and 2 gave an identical 4,4Ј- lation of 3 to 4, identical with the natural product
diacetylpyrenophorol (11)[4] (Scheme 1). Thus, compound 2 (Scheme 1).
was established as 4-acetylpyrenophorol.
Compound 5 was isolated as an optically active colorless
oil. Its molecular formula C16H22O6, established by HRE-
IMS, was the same as that of 3. H and 13C NMR of 5
1
Table 2. NMR spectroscopic data for compounds 2 and 4.
showed great similarity to those of (–)-dihydropyreno-
phorin (3),[24] suggesting the same macrocyclic skeleton.
However, a difference was observed in the NMR resonances
of the ketone subunit from C-2 to C-4 (Table 3), involving
one carbonyl group and the conjugated double bond. More
importantly, the large coupling constant between the ole-
finic protons in 3 (3J2,3 = 15.5 Hz) markedly decreased in
No.
4-Acetylpyrenophorol (2)
δH, m, J [Hz]
4Ј-Acetyldihydropyrenophorin (4)
δC, m[a]
δH, m, J [Hz]
δC, m[a]
1
2
3
164.8, s
5.93, dd, 15.5, 1.0 124.1, d
6.77, dd, 15.5, 6.7 143.8, d
164.6, s
131.6, d
139.5, d
200.8, s
36.0, t
6.58, d, 16.0
6.95, d, 16.0
4
5.20, m
1.86, m
1.86, m
1.58, m
1.70, m
72.0, d
27.9, t
5a
5b
6a
6b
7
2.58, ddd, 13.5, 8.0, 4.5
2.71, ddd, 13.5, 9.0, 4.5
2.01, m
1
the H NMR spectrum of 5 (3J2,3 = 11.5 Hz), indicating
28.8, t
32.3, t
a (Z) geometry of the double bond. This conclusion was
supported by the diagnostic NOE correlation between 2-H
and 3-H in the NOESY spectra. The presence of a second-
ary alcohol at C-4Ј in 5 allowed the determination of its
absolute stereochemistry by using a modified Mosher’s
method.[46–48] The (S)- and (R)-MTPA esters of cis-dihy-
dropyrenophorin (5) were prepared by treatment at room
temperature with (R)- and (S)-α-methoxy-α-(trifluoro-
methyl)phenylacetyl (MTPA) chloride in dry pyridine,
2.03, m
5.04, m
1.26, d, 6.5
5.07, m
1.23, d, 7.0
69.6, d
18.6, q
165.2, s
70.8, d
19.6, q
165.3, s
122.3, d
145.7, d
70.7, d
27.7, t
8
1Ј
2Ј 5.93, dd, 15.5, 1.0 122.5, d
3Ј 6.85, dd, 15.5, 6.5 148.5, d
4Ј
5aЈ
5bЈ
6aЈ
6bЈ
7Ј
5.94, dd, 15.5, 1.5
6.98, dd, 15.5, 4.0
5.31, m
4.21, m
1.80, m
1.80, m
1.58, m
1.70, m
5.07, m
1.24, d, 7.0
2.06, s
70.7, d
30.7, t
1.93, m
1.93, m
1.67, m
1.73, m
5.13, m
1.26, d, 6.5
2.10, s
28.9, t
28.1, t
respectively. Significant ∆δ values (∆δ = δ(S)-MTPA ester
–
70.4, d
18.6, q
21.1, q
169.8, s
70.8, d
18.2, q
21.0, q
169.7, s
δ(R)-MTPA ester) for the protons near to the chiral center C-3
were observed as shown in Figure 2 (a). According to
Mosher’s rule, the absolute configuration at C-4Ј was deter-
mined as (S), the same as those in its analogues 1–4.
8Ј
OAc
[a] By DEPT sequence.
Scheme 1. Chemical interconversion of pyrenophorin derivatives: (a) Cl3C6H2COCl, iPr2NEt, DMAP, pyridine, PhMe, room temp., 20 h,
60%.
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Eur. J. Org. Chem. 2008, 4320–4328

Antimicrobial Pyrenophorol Derivatives
Table 3. NMR spectroscopic data for compounds 5 and 6.
No.
cis-Dihydropyrenophorin (5)
No.
Tetrahydropyrenophorin (6)
δH, m, J [Hz]
δH, m, J [Hz]
δC, m[a]
δC, m[a]
1
2
164.4, s
124.6, d
1
2a
2b
3a
3b
4
5a
5b
6a
6b
7
172.1, s
28.6, t
6.03, d, 11.5
6.45, d, 11.5
2.39, ddd, 16.8, 6.5, 4.0
2.74, ddd, 16.8, 12.0, 4.5
2.44, ddd, 17.0, 6.5, 4.5
2.86, ddd, 17.0, 12.0, 4.0
3
142.9, d
37.3, t
4
5a
5b
6a
6b
7
8
1Ј
2Ј
3Ј
203.4, s
37.5, t
207.5, s
39.1, t
2.44, dt, 17.5, 7.0
2.92, dt, 17.5, 7.0
1.78, m
2.51, ddd, 18.0, 11.5, 3.5
2.62, ddd, 18.0, 12.0, 3.0
1.77, m
28.9, t
28.8, t
2.18, m
5.09, m
1.31, d, 6.5
2.16, m
4.91, m
1.28, d, 6.0
69.6, d
19.7, q
165.8, s
122.3, d
147.8, d
70.1, d
29.9, t
71.7, d
20.3, q
165.6, s
122.6, d
149.0, d
71.2, d
30.1, t
8
1Ј
2Ј
3Ј
6.01, d, 15.0
6.83, dd, 15.0, 4.5
4.50, m
5.89, dd, 15.5, 0.7
6.77, dd, 15.5, 6.5
4.28, m
4Ј
4Ј
5aЈ
5bЈ
6aЈ
6bЈ
7Ј
1.83, m
1.88, m
1.59, m
1.73, m
4.90, m
1.23, d, 6.5
5aЈ
5bЈ
6aЈ
6bЈ
7Ј
1.77, m
1.82, m
1.59, m
1.68, m
4.91, m
1.22, d, 6.5
28.9, t
29.7, t
71.2, d
18.9, q
70.1, d
18.7, q
8Ј
8Ј
[a] By DEPT sequence.
method[46–48] confirmed the (S) configurtion at C-4Ј [Fig-
ure 2 (b)]. The structure was thus determined as (–)-
(7R,4ЈS,7ЈR)-6.
The polar compound 7 was isolated as an optically active
colorless oil, with the molecular formula of C16H24O7, as
deduced from HRESIMS. The IR spectrum of 7 was remi-
niscent of that of dihydropyrenophorin (3), showing func-
tional absorption bands for hydroxy groups (3396 cm–1)
and an α,β-unsaturated ester group (1722, 1279, 1058 cm–1).
1
Figure 2. ∆δ (δ(S) – δ(R)) values [Hz] for the MTPA esters of 5 (a) The similarity was also observed in the H and 13C NMR
and 6 (b).
spectra of 7 (Table 4) with some differences in the signals
for C-1, C-7Ј and C-8Ј. In particular, the signal for 7Ј-H in
1
Based on the formation of the pyrenophorol family in the H NMR was shifted upfield in 7 (δH = 3.77 ppm) with
the same fungus, the related negative optical rotations of respect to that in 3 (δH = 5.20 ppm), displaying a character-
pyrenophorol ([α]2D0 = –13.3[7] and –14.9[4]) with values istic signal for a secondary alcohol proton instead of an
measured for our derivatives (see Exp. Sect.), and the con- ester proton. In addition, the analysis of the HMBC spec-
sistent chemical correlations, we assume identical absolute trum of 7 revealed a long-range correlation from 7-H to C-
configuration of the derivatives presented in Scheme 1. The 1Ј, but no correlation from 7Ј-H to C-1 in contrast to the
structure of 5 was thus determined as a double-bond isomer situation in 3. These facts, in conjunction with the marked
of 3, and is assigned as (–)-(7R,4ЈS,7ЈR)-5.
increase in the polarity of 7, suggested an open-chain deriv-
Compound 6, an optically active colorless oil, had the ative of 3 by cleavage of the C-1–C-7Ј lactone. Treatment of
molecular formula C16H24O6 as deduced from HRESIMS, 7 with diazomethane gave the methyl ester 12 (Scheme 1),
and thus possessing two mass units more than 3 or 5. which further validated the presence of a carboxylic acid
1
Again, the H and 13C NMR spectra of 6 (Table 3) showed group in 7. Interestingly, a simultaneous 1,3-dipolar cyclo-
similarity to those of 3, except that the signals for the addition was observed,[49,50] and the diazomethane was also
double bond of the unsaturated ketone (δC = 131.0, trapped by the highly activated, conjugated double bond to
139.7 ppm; δH = 6.59, 6.99 ppm) in 3 were replaced by sig- form a pyrazole ring system.[51] The presence of two nitro-
nals for a pair of methylene groups (δC = 28.6, 37.3 ppm; gen atoms was proven by the molecular formula
δH = 2.39, 2.74, and 2.44, 2.86 ppm) in 6. In addition, both C18H28N2O7 as deduced from HREIMS and further sup-
the signals for the ketone and ester carbonyl atoms were ported by a positive reaction with the Dragendoff reagent,
1
shifted downfield (δC = 164.4, 200.2 ppm in 3; δC = 172.1, and a complete H and 13C NMR assignment based on 2D
207.5 ppm in 6) due to the disappearance of the conjugated NMR spectra. In particular, the HMBC correlations from
system. Thus, compound 6 was identified as the 2,3-dihydro NH to C-2, C-3, C-9, from 9-H2 to C-1, C-2, C-3, and from
analogue of dihydropyrenophorin (3). A modified Mosher’s 2-H to C-1 and C-3, suggested the pyrazole ring substruc-
Eur. J. Org. Chem. 2008, 4320–4328
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W. Zhang, K. Krohn, H. Egold, S. Draeger, B. Schulz
FULL PAPER
Table 4. NMR spectroscopic data for compounds 7 and 8.
No.
seco-Dihydropyrenophorin (7)
7Ј-Acetyl-seco-dihydropyrenophorin (8)
δH, m, J [Hz]
δC, m[a]
δH, m, J [Hz]
δC, m[a]
1
2
3
167.5, s
131.5, d
139.4, d
199.5, s
37.1, t
167.7, s
132.0, d
139.2, d
199.6, s
37.1, t
6.59, d, 16.0
6.95, d, 16.0
6.60, d, 16.0
6.95, d, 16.0
4
5a
5b
6a
6b
7
8
1Ј
2Ј
3Ј
4Ј
5aЈ
5bЈ
6aЈ
6bЈ
7Ј
8Ј
OAc
2.65, t, 7.2
2.65, t, 7.2
1.88, m
1.93, m
4.95, m
2.65, t, 7.5
2.65, t, 7.5
1.88, m
1.92, m
4.95, m
29.8, t
29.8, t
70.3, d
19.9, q
166.4, s
119.9, d
150.8, d
70.8, d
32.9, t
70.4, d
19.9, q[b]
166.3, s
120.2, d
150.6, d
70.2, d
1.22, d, 6.0
1.23, d, 6.0
5.93, dd, 16.0, 1.5
6.85, dd, 16.0, 4.7
4.23, m
5.92, dd, 15.5, 1.5
6.83, dd, 15.5, 5.0
4.22, m
1.55, m
1.68, m
1.51, m
1.60, m
3.77, m
1.15, d, 6.0
1.55, m
1.55, m
1.53, m
1.68, m
4.87, m
1.18, d, 6.5
1.98, s
32.0, t
35.0, t
31.4, t
67.7, d
23.2, q
70.8, d
19.8, q[b]
21.2, q
171.3, s
[a] By DEPT sequence. [b] Signals maybe interchangeble.
ture. Not unexpectedly, the diazomethane adduct was a lations from C-1 to 2-H2, 3-H2, and 4-H, as deduced from
mixture of two isomers as evident from the appearance of the HMBC spectrum. This compound was assigned as seco-
signal pairs in the NMR spectra. The structure and abso- dihydropyrenophorin-1,4-lactone (9); the configuration at
lute configuration of the acid 7 was unambiguously C-4 was not determined.
confirmed by cyclization to
3
using
a
modified
Yamaguchi procedure [Scheme 1 (a)].[52,53]
Table 5. NMR spectroscopic data for compound 9.
Compound 8 was isolated as an optically active, colorless
No. δH, m, J [Hz]
δC, m[a] No.
δH, m, J [Hz]
δC, m[a]
oil with the molecular formula C18H26O8, as deduced from
1
177.2, s
28.8, t
1Ј
2Ј
166.4, s
1
HRESIMS. The H and 13C NMR spectra of 8 (Table 4)
2a 2.52, dd, 9.5, 6.5
2b 2.52, dd, 9.5, 6.5
6.60, dd, 15.7, 1.2 120.1, d
were closely related to those of 7, except for the presence of
an additional acetoxy group [δH = 1.98 (s) ppm; δC = 171.3
(s), 21.2 (q) ppm]. The location of the acetoxy group at C-
7 was assured by the downfield shift of the 7Ј-H signal (δH
= 4.87 ppm in 8; δH = 3.77 ppm in 7), and compound 8
was determined as 7Ј-acetyl-seco-dihydropyrenophorin. The
long-range correlation from 7Ј-H to the acetyl carbonyl
atom (δC = 171.3 ppm) in the HMBC spectrum further con-
firmed the proposed structure.
Compound 9 was obtained as an optically active color-
less oil. Its molecular formula C16H26O6 was established by
HRESIMS, indicating four double-bond equivalents. The
IR spectrum of 9 showed the presence of a lactone carbonyl
functionality (1774 cm–1), hydroxy groups (3603 cm–1), and
an α,β-unsaturated ester group (1721, 1282, 1193 cm–1).
Comparison of the 1H and 13C NMR spectra of 9 (Table 5)
3a
1.83, m
27.9, t
3Ј
6.91, dd, 15.7, 4.7 150.5, d
3b 2.32, dt, 13.5, 6.5
4
5a
5b
6a
6b
7
4.49, m
1.64, m
1.78, m
1.64, m
1.78, m
80.4, d
4Ј
4.28, m
1.65, m
1.80, m
1.55, m
1.65, m
70.9, d
33.2, t
31.3, t[b] 5aЈ
5bЈ
31.6, t[b] 6aЈ
6bЈ
35.1, t
5.01, m
70.1, d
20.0, q
7Ј
8Ј
3.81, m
1.19, d, 7.0
67.8, d
23.5, q
8
1.24, d, 6.5
[a] By DEPT sequence. [b] Signals maybe interchangeable.
Bioactivity
The isolated compounds 1–12 were tested in an agar dif-
with those of 7 revealed identical chemical shifts for the fusion assay for their antibacterial, antifungal and algicidal
signals from C-7 to C-7Ј, presenting a characteristic (E) properties and compared with a set of controls (penicillin,
double bond [δH = 6.60 (dd, J = 15.7, 1.2 Hz), 6.91 (dd, J nystatin, actidione, and tetracycline; Table 6). Compounds
= 15.7, 4.7 Hz) ppm; δC = 120.1 (d), 150.5 (d) ppm] and a 2–6, 8, and 11 inhibited all four test organisms, whereas
conjugated ester group (δC = 166.4 ppm), which accounted compounds 1, 10, and 12 inhibited Microbotryum violaceum
for two double-bond equivalents. The remaining two and the alga Chlorella fusca. Compounds 7 and 9 were anti-
double-bond equivalents were attributed to a lactone ring fungal against Microbotryum violaceum and antibacterial
in the structure, confirmed by diagnostic long-range corre- against Escherichia coli and Bacillus megaterium.
4324
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 4320–4328

Antimicrobial Pyrenophorol Derivatives
Table 6. Agar diffusion assays for antibacterial, antifungal, and antialgal activities.[a,b]
Compound
Escherichia coli
Bacillus megaterium
Microbotryum violaceum
Chlorella fusca
1
0
7
9
11
9
10
8
10
12
11
6
7
7
10
7
10
9
11
9
10
12
0
10
0
2
3
4
9
5
10
12
8
10
10
0
10
0
18
18
7
6
10
10
9
7
9
23
7
0
10
0
35
0
7
8
9
6
10
10
11
11
14
18
0
9
11
13
10
0
12
Penicillin
Tetracycline
Nystatin
Actidione
Acetone
0
20
50
0
0
0
[a] 50 µL of substance dissolved in acetone (1 mg/mL) were applied to a filter disc and sprayed with a suspension of the respective test
organism. [b] Radii of the zones of inhibition are given in mm.
0.040–0.063 mm) was used for column chromatography. Precoated
silica gel plates (Merck, G60 F-254 or G50 UV-254) were used
Conclusion
The discovery of an array of pyrenophorol derivatives,
including the minor constituents, demonstrates the produc-
tivity of the fungus and is a beautiful example of chemical
diversity, extending the pyrenophorol family. This chemical
diversity may be due to reduced enzyme specificity or to
non-enzymatic side reactions. In particular, the co-occur-
rence of the open-chain acids as putative biosynthetic pre-
cursors is noteworthy. They have rarely been isolated to-
gether with macrodiolides, perhaps due to their polar na-
ture. The stereochemical investigation and in particular the
chemical interconversion showed that the absolute configu-
ration of all the pyrenophorol members seems to be uni-
form. They have (R) configuration at C-7 and C-7Ј, and (S)
configuration at C-4 and/or C-4Ј, regardless of the oxi-
dation state, double-bond geometry, or cyclization mode.
Obviously, compounds 7–9 are structurally related to pyr-
enophorol (1), (–)-dihydropyrenophorin (3) and the other
new analogues 2 and 4–6. The diversity and interconversion
of the metabolites starting from the same skeleton is created
by just a few simple chemical transformations: Reduction
of carbonyl groups, oxidation of hydroxy groups, hydrogen-
ation of double bonds, lactonization of hydroxy acid to lac-
tone of different ring size, and finally simple acetylation of
hydroxy groups. The acetylation on the secondary alcohol
at C-7Ј in 8 and lactonization between C-1 and C-4 in 9
may have prevented further macrolactoniztion of these
structures. The isolation of twelve different derivatives by
isolation or chemical transformations and comparison of
their data enabled the complete assignment of all signals in
the 1H and 13C NMR spectra of the pyrenophorol
family that have not previously been completely re-
ported.[20–24,36–42]
for analytical and preparative thin-layer chromatography (TLC),
respectively. NMR spectra were recorded at 293 K with a Bruker
Avance 500 spectrometer. Chemical shifts are reported in parts per
million (δ) by using CDCl3 as an internal standard with coupling
constants (J) in Hz. 1H and 13C NMR assignments were supported
by 1H-1H COSY, HMQC, HMBC, NOESY, and NOEDIFF ex-
periments. Optical rotations were measured with a Perkin–Elmer
241 MC polarimeter at the sodium D line. IR spectra were recorded
with a Nicolet-510P spectrophotometer, peaks are reported in cm–1.
UV spectra were recorded with a UV-2101PC spectrophotometer,
peaks are reported in nm. Mass spectra and high-resolution mass
spectra were recorded with MAT 8200 and Micromass LCT mass
spectrometers.
Culture, Extraction and Isolation: The endophytic fungus Phoma
sp., internal strain No. 8874, was isolated following surface sterilis-
ation from the leaves of Lycium intricatum, a succulent woody
shrub from Gomera, and was cultivated on 12 L of 5% w/v biomalt
solid agar media at room temperature for 28 d.[54,55] The culture
media were then extracted with ethyl acetate to afford 7.8 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 dichloromethane in 2-propanol (95:5,
90:10, 80:20, 60:40, 40:60) to give 14 subfractions. Fractions 3, 8,
and 11 gave pure 4 (7.2 mg), 1 (552.0 mg), and 7 (119.0 mg), respec-
tively, by simple Sephadex LH-20 CCs, eluted with CHCl2/MeOH
(30:1 for 4, 10:1 for 1, and 3:1 for 7). Fraction 5 was split by a
Sephadex LH-20 CC (CHCl2/MeOH, 10:1) followed by silica gel
CC (400–600 mesh, petroleum ether/acetone, 5:1) to afford 3
(24.4 mg) and 5 (1.0 mg). Fraction 6 was subjected to a Sephadex
LH-20 CC (CHCl2/MeOH, 10:1) and was then fractionated by sil-
ica gel CC (400–600 mesh, petroleum ether/acetone, 4:1) to yield 2
(10.0 mg) and 6 (2.1 mg). Finally, a Sephadex LH-20 CC (CHCl2/
MeOH, 3:1) on fraction 9 gave pure 8 (16.3 mg) and crude 9, which
was purified by a subsequent silica gel CC (400–600 mesh, petro-
leum ether/acetone, 4:3) to afford the pure product (9, 6.7 mg).
Data for Pyrenophorol (1): Colorless crystals, m.p. 130–133 °C.
[α]2D0 = –10.2 (c = 1.04, CHCl3). 1H and 13C NMR spectroscopic
data: see Table 1. HRESIMS: m/z = 313.16456 (calcd. 313.16511
for C16H25O6).
Experimental Section
General Experimental Procedures: For microbiological methods
and culture conditions, see refs.[54–56] Commercial silica gel (Merck,
Eur. J. Org. Chem. 2008, 4320–4328
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4325

W. Zhang, K. Krohn, H. Egold, S. Draeger, B. Schulz
FULL PAPER
Data for 4-Acetylpyrenophorol (2): Colorless oil, [α]2D0 = –23.9 (c =
3.5 Hz, 2 H, 5b-H, 5Јb-H), 2.54 (ddd, J = 14.0, 8.0, 3.5 Hz, 2 H,
2.44, CHCl ). IR (CHCl ): ν = 3603, 2934, 2874, 1717, 1652, 5a-H, 5Јa-H), 2.13 (m, 2 H, 6b-H, 6Јb-H), 2.08 (m, 2 H, 6a-H, 6Јa-
˜
max
3
3
1466, 1370, 1244, 1134, 1028 cm–1. 1H and 13C NMR spectroscopic
data: see Table 2. HRESIMS: m/z = 377.15757 (calcd. 377.15762
for C18H26O7Na).
H), 1.28 (d, J = 6.5 Hz, 6 H, 8-H3, 8Ј-H3) ppm. 13C NMR
(125 MHz, CDCl3): δ = 199.6 (s, C-4, C-4Ј), 164.9 (s, C-1, C-1Ј),
139.7 (s, C-3, C-3Ј), 131.4 (s, C-2, C-2Ј), 72.2 (d, C-7, C-7Ј), 37.2
(t, C-5, C-5Ј), 32.1 (t, C-6, C-6Ј), 19.6 (q, C-8, C-8Ј) ppm. HRE-
IMS: m/z = 308.12626 (calcd. 308.12598 for C16H20O6).
Data for Dihydropyrenophorin (3): Colorless oil, [α]2D0 = –10.7 (c =
1.00, CHCl ). IR (CHCl ): ν = 3537, 2934, 2853, 1717, 1652,
˜
max
3
3
1455, 1355, 1289, 1174, 1023 cm–1. 1H NMR (500 MHz, CDCl3):
δ = 6.99 (d, J = 15.5 Hz, 1 H, 3-H), 6.98 (dd, J = 15.7, 3.7 Hz, 1
H, 3Ј-H), 6.59 (d, J = 15.5 Hz, 1 H, 2-H), 5.99 (dd, J = 15.5, 2.0 Hz,
1 H, 2Ј-H), 5.20 (m, 1 H, 7Ј-H), 5.00 (m, 1 H, 7-H), 4.39 (m, 1 H,
4Ј-H), 2.70 (ddd, J = 13.0, 8.0, 4.0 Hz, 1 H, 5b-H), 2.56 (ddd, J =
13.0, 8.5, 4.5 Hz, 1 H, 5a-H), 2.07 (m, 2 H, 6-H2), 2.01 (m, 1 H,
5Јb-H), 1.88 (m, 1 H, 5Јa-H), 1.75 (m, 2 H, 6Ј-H2), 1.32 (d, J =
6.5 Hz, 3 H, 8Ј-H3), 1.26 (d, J = 6.5 Hz, 3 H, 8-H3) ppm. 13C NMR
(125 MHz, CDCl3): δ = 200.2 (s, C-4), 165.5 (s, C-1Ј), 164.4 (s, C-
1), 150.3 (s, C-3Ј), 139.7 (s, C-3), 131.0 (s, C-2), 121.4 (s, C-2Ј), 71.1
(d, C-7Ј), 70.7 (d, C-7), 69.7 (d, C-4Ј), 37.2 (t, C-5), 32.4 (t, C-6),
30.3 (t, C-5Ј), 28.4 (t, C-6Ј), 19.6 (q, C-8), 17.7 (q, C-8Ј) ppm.
HREIMS: m/z = 310.14149 (calcd. 310.14164 for C16H22O6).
Synthesis of Pyrenophorin (10) by MnO2 Oxidation of Dihydropyr-
enophorin (3): The same reaction was performed with dihydropyr-
enophorin (3) (5.0 mg, 0.016 mmol) and active MnO2 (3.5 mg,
0.040 mmol) in dry CH2Cl2 (1.0 mL). Stirring for 40 h afforded
identical pyrenophorin (10, 4.1 mg, 83%).[20–23,36–42]
Synthesis of 4,4Ј-Diacetylpyrenophorol (11) by Acetylation of
Pyrenophorol (1): To a solution of 1 (9.0 mg) in dry pyridine
(0.5 mL) were added 2 drops of Ac2O. The mixture was kept at
room temperature for 16 h to afford, after usual workup, 11[4]
quantitatively (11.4 mg) as colorless oil. [α]2D0 = –49.3 (c = 1.20,
CHCl3). 1H NMR (500 MHz, CDCl3): δ = 6.77 (dd, J = 15.7,
6.7 Hz, 2 H, 3-H, 3Ј-H), 5.98 (dd, J = 15.7, 0.7 Hz, 2 H, 2-H, 2Ј-
H), 5.22 (m, 2 H, 4-H, 4Ј-H), 5.09 (m, 2 H, 7-H, 7Ј-H), 2.08 (s, 6
H, Me of OAc), 1.86 (m, 4 H, 5-H2, 5Ј-H2), 1.74 (m, 2 H, 6b-H,
6Јb-H), 1.62 (m, 2 H, 6a-H, 6Јa-H), 1.26 (d, J = 6.5 Hz, 6 H, 8-
H3, 8Ј-H3) ppm. 13C NMR (125 MHz, CDCl3): δ = 169.7 (s, OAc),
164.7 (s, C-1, C-1Ј), 143.7 (s, C-3, C-3Ј), 124.1 (s, C-2, C-2Ј), 72.0
(d, C-4, C-4Ј), 69.8 (d, C-7, C-7Ј), 28.8 (t, C-6, C-6Ј), 27.9 (t, C-5,
C-5Ј), 21.0 (q, OAc), 18.5 (q, C-8, C-8Ј) ppm.
Data for 4Ј-Acetyldihydropyrenophorin (4): Colorless oil, [α]2D0
=
–27.9 (c = 0.72, CHCl ). IR: (CHCl ) ν = 3044, 2939, 2866,
˜
max
3
3
1727, 1682, 1450, 1375, 1289, 1184, 1134 cm–1. H and 13C NMR
spectroscopic data: see Table 2. HREIMS: m/z = 352.15291 (calcd.
352.15218 for C18H24O7).
1
Data for cis-Dihydropyrenophorin (5): Colorless oil, [α]2D0 = –12.8 (c
Synthesis of 4,4Ј-Diacetylpyrenophorol (11) by Acetylation of 4-Ace-
tylpyrenophorol (2): The same reaction was performed with 2
(2.0 mg) to afford an identical sample of 4,4Ј-diacetylpyrenophorol
(11)[4] (2.2 mg, 100%) as a colorless oil.
= 0.10, CHCl ). IR (CHCl ): νmax = 3609, 2925, 2862, 1719, 1606,
˜
3
3
1464, 1380, 1279, 1179, 1025 cm–1. 1H and 13C NMR spectroscopic
data: see Table 3. HREIMS: m/z = 310.14180 (calcd. 310.14164 for
C16H22O6).
Acetylation of Dihydropyrenophorin (3): Treatment of 3 (3.0 mg)
with Ac2O according to the above procedure, afforded an acetate
in quantitative yield (3.4 mg, 100%), identical with the natural
product 4.
Data for Tetrahydropyrenophorin (6): Colorless oil, [α]2D0 = –11.0 (c
= 0.21, CHCl ). IR (CHCl ): νmax = 3632, 2934, 2858, 1723, 1602,
˜
3
3
1461, 1370, 1285, 1189, 1014 cm–1. 1H and 13C NMR spectroscopic
data: see Table 3. HRESIMS: m/z = 335.14650 (calcd. 335.14706
for C16H24O6Na).
Esterification of cis-Dihydropyrenophorin (5) with (R)-MTPA Chlo-
ride: Treatment of 5 (0.3 mg) with (R)-MTPA chloride (5 µL) in
dry pyridine (0.5 mL), stirring at room temperature overnight and
purification by a mini silica gel column chromatography (300 mesh,
petroleum ether/EtOAc, 6:1) afforded the (S)-MTPA ester of 5
(0.37 mg, 72%). 1H NMR (500 MHz, CDCl3): δ = 6.69 (dd, J =
16.2, 7.1 Hz, 1 H, 3Ј-H), 6.44 (d, J = 11.7 Hz, 1 H, 3-H), 6.00 (d,
J = 11.7 Hz, 1 H, 2-H), 5.92 (d, J = 16.2 Hz, 1 H, 2Ј-H), 5.54 (m,
1 H, 4Ј-H), 5.09 (m, 1 H, 7-H), 4.84 (m, 1 H, 7Ј-H), 2.89 (dt, J =
17.0, 7.0 Hz, 1 H, 5b-H), 2.40 (dt, J = 17.0, 6.6 Hz, 1 H, 5a-H),
2.16 (m, 1 H, 6b-H), 1.84 (m, 1 H, 5Јb-H), 1.76 (m, 1 H, 5Јa-H),
1.74 (m, 1 H, 6a-H), 1.62 (m, 1 H, 6Јb-H), 1.57 (m, 1 H, 6Јa-H),
1.30 (d, J = 6.4 Hz, 3 H, 8-H3), 1.17 (d, J = 6.4 Hz, 3 H, 8Ј-H3)
ppm.
Data for seco-Dihydropyrenophorin (7): Colorless oil, [α]2D0 = –12.0
(c = 2.35, MeOH). IR (CHCl ): ν
= 3396, 2979, 2929, 1722,
˜
3
max
1279, 1183, 1058 cm–1. 1H and 13C NMR spectroscopic data: see
Table 4. HRESIMS: m/z
= 351.14142 (calcd. 351.14197 for
C16H24O7Na).
Data for 7Ј-Acetyl-seco-dihydropyrenophorin (8): Colorless oil,
[α]2D0 = –4.4 (c = 0.79, MeOH). IR (CHCl ): ν
= 3507, 2938,
˜
3
max
2863, 1727, 1380, 1264, 1179, 1048 cm–1. 1H and 13C NMR spectro-
scopic data: see Table 4. HRESIMS: m/z = 393.15199 (calcd.
393.15254 for C18H26O8Na).
Data for seco-Dihydropyrenophorin-1,4-lactone (9): Colorless oil,
[α]2D0 = –12.1 (c = 0.67, MeOH). IR (CHCl ): ν
= 3603, 2943,
˜
3
max
2863, 1774, 1721, 1663, 1462, 1282, 1193, 1018 cm–1. 1H and 13C
NMR spectroscopic data: see Table 5. HRESIMS: m/z = 337.16266
(calcd. 337.16271 for C16H26O6Na).
Esterification of cis-Dihydropyrenophorin (5) with (S)-MTPA Chlo-
ride: The same reaction of 5 with (S)-MTPA chloride afforded the
(R)-MTPA ester of 5 (0.37 mg, 72%). 1H NMR (500 MHz,
CDCl3): δ = 6.62 (dd, J = 15.8, 7.2 Hz, 1 H, 3Ј-H), 6.44 (d, J =
11.9 Hz, 1 H, 3-H), 6.02 (d, J = 11.9 Hz, 1 H, 2-H), 5.83 (d, J =
15.8 Hz, 1 H, 2Ј-H), 5.51 (m, 1 H, 4Ј-H), 5.07 (m, 1 H, 7-H), 4.88
(m, 1 H, 7Ј-H), 2.85 (dt, J = 17.0, 7.4 Hz, 1 H, 5b-H), 2.51 (dt, J
= 17.0, 6.7 Hz, 1 H, 5a-H), 2.13 (m, 1 H, 6b-H), 1.92 (m, 1 H, 5Јb-
H), 1.88 (m, 1 H, 5Јa-H), 1.73 (m, 1 H, 6a-H), 1.70 (m, 1 H, 6Јb-
H), 1.64 (m, 1 H, 6Јa-H), 1.29 (d, J = 6.4 Hz, 3 H, 8-H3), 1.21 (d,
J = 6.4 Hz, 3 H, 8Ј-H3) ppm.
Synthesis of Pyrenophorin (10) by MnO2 Oxidation of Pyrenophorol
(1): To a stirred solution of 1 (10.0 mg, 0.032 mmol) in dry CH2Cl2
(2.0 mL) was added active MnO2 (13.9 mg, 0.16 mmol). The re-
sulting suspension was stirred at room temperature for 50 h, the
MnO2 was filtered off and repeatedly washed with Et2O (5 mL) to
afford 10[20–23,36–42] (9.4 mg, 90%) as colorless crystals. M.p. 175–
176 °C (ref.[42] 177–178 °C). [α]2D0 = –40.3 (c = 1.3, CHCl3) {ref.[42]
[α]2D0 = –55.9 (c = 0.44, EtOH)}. H NMR (500 MHz, CDCl3): δ =
1
6.93 (d, J = 16.0 Hz, 2 H, 3-H, 3Ј-H), 6.48 (d, J = 16.0 Hz, 2 H,
2-H, 2Ј-H), 5.03 (m, 2 H, 7-H, 7Ј-H), 2.65 (ddd, J = 14.0, 9.0,
Esterification of Tetrahydropyrenophorin (6) with (R)-MTPA Chlo-
ride: Compound 6 (0.5 mg) was treated with (R)-MTPA chloride
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 4320–4328

Antimicrobial Pyrenophorol Derivatives
according to the above procedure to afford the (S)-MTPA ester of
6 (0.70 mg, 83%). H NMR (500 MHz, CDCl3): δ = 6.68 (dd, J =
15.8, 7.2 Hz, 1 H, 3Ј-H), 5.92 (d, J = 15.8 Hz, 1 H, 2Ј-H), 5.52 (m,
ate agar growth medium for the respective test organism and sub-
sequently sprayed with a suspension of the test organism.[56] The
test organisms were the gram-negative bacterium Escherichia coli,
1
1 H, 4Ј-H), 4.90 (m, 1 H, 7-H), 4.85 (m, 1 H, 7Ј-H), 2.89 (ddd, J the gram positive bacterium Bacillus megaterium (both grown on
= 17.2, 10.9, 2.8 Hz, 1 H, 3b-H), 2.71 (ddd, J = 16.0, 10.9, 2.8 Hz, NB medium), the fungus Microbotryum violaceum, and the alga
1 H, 2b-H), 2.65 (ddd, J = 18.5, 9.5, 2.8 Hz, 1 H, 5b-H), 2.51 (ddd, Chlorella fusca (both grown on MPY medium). Reference sub-
J = 18.5, 8.4, 2.8 Hz, 1 H, 5a-H), 2.39 (ddd, J = 17.2, 5.8, 2.8 Hz, stances were penicillin, nystatin, actidione and tetracycline. The ra-
1 H, 3a-H), 2.33 (ddd, J = 16.0, 5.8, 2.8 Hz, 1 H, 2a-H), 2.13 (m, dius of zone of inhibition was measured in mm. These microor-
1 H, 6b-H), 1.85 (m, 1 H, 5Јb-H), 1.74 (m, 1 H, 5Јa-H), 1.72 (m, 1 gansims were chosen because (a) they are non-pathogenic and (b)
H, 6a-H), 1.59 (m, 1 H, 6Јb-H), 1.56 (m, 1 H, 6Јa-H), 1.28 (d, J = had in the past proved to be accurate initial test organisms for
6.2 Hz, 3 H, 8-H3), 1.17 (d, J = 6.4 Hz, 3 H, 8Ј-H3) ppm.
antibacterial, antifungal and antialgal/herbicidal activities. Com-
mencing at the outer edge of the filter disc, the radius of zone of
inhibition was measured in mm.
Esterification of Tetrahydropyrenophorin (6) with (S)-MTPA Chlo-
ride: Compound 6 (0.5 mg) was treated with (S)-MTPA chloride
according to the above procedure to afford the (R)-MTPA ester of
1
6 (0.70 mg, 83%). H NMR (500 MHz, CDCl3): δ = 6.61 (dd, J =
Acknowledgments
15.7, 7.1 Hz, 1 H, 3Ј-H), 5.83 (d, J = 15.7 Hz, 1 H, 2Ј-H), 5.51 (m,
1 H, 4Ј-H), 4.89 (m, 1 H, 7Ј-H), 4.88 (m, 1 H, 7-H), 2.89 (ddd, J
= 16.0, 10.8, 3.0 Hz, 1 H, 3b-H), 2.72 (ddd, J = 16.0, 10.8, 3.0 Hz,
1 H, 2b-H), 2.64 (ddd, J = 18.6, 11.7, 3.0 Hz, 1 H, 5b-H), 2.50
(ddd, J = 18.6, 8.6, 3.0 Hz, 1 H, 5a-H), 2.38 (ddd, J = 16.0, 6.2,
3.0 Hz, 1 H, 3a-H), 2.34 (ddd, J = 16.0, 6.2, 3.0 Hz, 1 H, 2a-H),
2.12 (m, 1 H, 6b-H), 1.90 (m, 1 H, 5Јb-H), 1.84 (m, 1 H, 5Јa-H),
1.71 (m, 1 H, 6a-H), 1.70 (m, 1 H, 6Јb-H), 1.64 (m, 1 H, 6Јa-H),
1.26 (d, J = 6.2 Hz, 3 H, 8-H3), 1.21 (d, J = 6.4 Hz, 3 H, 8Ј-H3)
ppm.
We thank Tatjana Stolz and Kathrin Meier for excellent technical
assistance.
[1] M. Doi, T. Ishida, M. Kobayashi, I. Kitigawa, J. Org. Chem.
1991, 56, 3629–3632.
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1
with very small variations in the H and 13C NMR spectra for the
1
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3.91 (ddd, J = 17.5, 9.5, 2.0 Hz, 2 H, 9b-H ϫ2), 3.87 (m, 4 H, 9a-
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1.78 (m, 2 H, 5Јb-H ϫ2), 1.66 (m, 2 H, 6Јb-H ϫ2), 1.63 (m, 2 H,
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Yamaguchi Macrolactoniztion of seco-Dihydropyrenophorin (7): To
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Eur. J. Org. Chem. 2008, 4320–4328
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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Received: April 23, 2008
Published Online: July 15, 2008
4328
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 4320–4328