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Biologically active metabolites from fungi. Part 16:
q
New
preussomerins J, K and L from an endophytic fungus: structure
elucidation, crystal structure analysis and determination of
absolute con®guration by CD calculations
Karsten Krohn,
a,p
Ulrich Flo
Èrke,
a
Markus John,
a
Natalia Root,
a
Klaus Steingro
Èver,
a
Hans-Ju
Èrgen Aust,
b
Siegfried Draeger,
b
Barbara Schulz,
b
Sa
Ândor Antus,
c
Miklos Simonyi
d
and Ferenc Zsila
d
a
Fachbereich Chemie und Chemietechnik, Universita
Èt Paderborn, Warburger Stra
b
e 100, D-33098 Paderborn, Germany
b
Institut fu
Èr Mikrobiologie der Technischen Universita
Èt Braunschweig, Spielmannstra
b
e 7, D-38106 Braunschweig, Germany
c
Department of Organic Chemistry, University of Debrecen, Egyetem ter 1, H-4010 Debrecen, Hungary
d
Department of Molecular Pharmacology, Institute of Chemistry CRC, H-1525 Budapest, P.O.B. 17, Hungary
Received 23 February 2001; accepted 13 March 2001
AbstractÐ Three known preussomerins, G (1), H (2) and I (3), and three new representatives, J (4), K (5) and L (6), were isolated from an
endophytic fungus, a Mycelia sterila, from Atropa belladonna. Their absolute con®guration was determined by comparison of calculated and
experimental CD spectra. q2001 Published by Elsevier Science Ltd.
1. Isolation
In connection with our ongoing search for biologically
active metabolites from fungi, we investigated the con-
stituents of an endophytic fungus, Mycelia sterila, isolated
following surface sterilization from the root of Atropa
belladonna. Since the fungus did not sporulate it could not
be characterized taxonomically. The culture extract of the
fungus was found to have antibacterial and antifungal
properties. Extensive column and preparative thin layer
chromatography of the ethyl ether extract afforded six
pure compounds 1±6(Scheme 1), 1being the least polar
and 6the most polar metabolite. All spots showed a typical
faint blue ¯uorescence on TLC plates and an identical blue-
gray color of the spots on heating, indicating a common
class of natural products. Compounds 1±3are identical
with the known preussomerins G±I,
2
whereas preussomerin
J(4), preussomerin K (5) and preussomerin L (6) are new
representatives of these bisspirobisnaphthalene natural
products.
Preussomerins were ®rst isolated by Gloer et al. from
cultures of the coprophilous (dung-colonizing) fungus
Preussia isomera Cain.
3,4
The six preussomerins A±F
isolated by Gloer et al., showed antifungal activity toward
early-successional coprophilous fungi, thus supporting the
theory of interspecies competition among coprophilous
fungi. Later, preussomerin D was also isolated from the
endophyte Hormonema dematioides
5
and the new preus-
somerins G±I (1±3) from a dung-inhabiting coelomycetous
fungus.
2
Singh et al. also discovered that they inhibit
farnesyl-protein transferase (FPTase). FPTase inhibitors
are potential agents against cancers with mutated ras gene
such as colon or pancreatic carcinomas.
6
Very recently, the
racemic preussomerins G and I
7
and several analogues
8
were prepared by chemical synthesis.
2. Structure determination
The structure elucidation is exempli®ed in more detail on
the most polar metabolite 6, mp 171±1738C, ®rst isolated in
pure crystalline form from methanol. The optically active
compound ([
a
]
D
20
2557) showed comparatively low solu-
bility in nonpolar (e.g. cyclohexane) or very polar (e.g.
methanol) solvents, but could easily be dissolved in
dichloromethane/5% methanol. This indicated the presence
of free hydroxyl groups, con®rmed by a band at 3382 cm
21
in the IR spectrum. Another band at 1722 cm
21
indicated
the presence of a carbonyl group. The molecular mass
of C
20
H
14
O
8
(14 unsaturations) was deduced from the
Tetrahedron 57 (2001) 4343± 4348Pergamon
TETRAHEDRON
0040±4020/01/$ - see front matter q2001 Published by Elsevier Science Ltd.
PII: S0040-4020(01)00320-9
q
For part 15 of series see Ref. 1.
Keywords: fungal metabolites; spiroketal bisnaphthalenes; preussomerins;
antifungal agents.
p
Corresponding author. Tel.: 152-51-602172; fax 152-51-602172;
e-mail: kk@chemie.uni-paderborn.de
K. Krohn et al. / Tetrahedron 57 (2001) 4343± 43484344
high-resolution mass spectrum (HRMS, m/z382.06888) in
agreement with the number of resonances for carbon and
hydrogen atoms, detected in the NMR spectra (see below).
The data of the
1
H and
13
C NMR spectra, together with the
correlations resulting from the H,H-COSY and HMBC
experiments, are listed in Table 1.
The
13
C NMR spectrum indicates a total number of 14 sp
2
-
hybridisized carbon atoms, three of which are directly
connected to a heteroatom while two others are resonances
for carbonyl groups. Very characteristic are signals at
d
95.66 and 95.71, indicating the presence of two ketal
carbon atoms. Signals at
d
70.76 and 70.97 designate
carbon atoms directly connected with heteroatoms and
two further resonances at
d
43.21 and 43.56 are caused
by aliphatic carbon atoms in the neighborhood of hetero-
atoms.
In addition to three singlets for hydroxy groups at
d
3.39,
3.68 and 10.16 (chelated phenolic OH), two groups of multi-
plets at
d
7± 8 for aromatic and
d
3± 5 for aliphatic
protons can be detected in the
1
H NMR spectrum. In the
aromatic region, the presence of two and three vicinal
protons, respectively, can be deduced from the coupling
pattern. The low-®eld signals at
d
4.75 are caused by
two methine protons, directly bound to carbon atoms
bearing oxygen atoms that couple with diastereotopic
methylene protons at
d
3.62 and 2.99, respectively. Due
Scheme 1. Structures of preussomerins 1±6.
Table 1.
1
H and
13
C NMR data and correlations from the H,H-COSY and HMBC of preussomerin L (6)
C number
13
C HMBC
1
H(Jin Hz) H,H COSY
1 202.24 3, 8 10.16, 10-OH, s ±
10195.98 90,3
0,2a
0,2b
07.63, 90-H, dd (1.0, 7.8) 80,7
0
9 158.10 7, 8 7.48, 80-H, t 90,7
0
60152.33 70,8
07.21, 70-H, dd (1.0, 8.2) 90,8
0
6 144.41 7, 8 7.16, 7-H, d (9.2) 8
100(5 0) 132.22 70,8
0,9
06.97, 8-H, d (9.2) 7
50(100) 132.12 2a0,2b
04.75, 3/30-H, 2 t 2a,2b/2a0,2b0
7 126.89 8 3.68, 3-OH, s
70122.63 80,9
03.59, 2a-H, dd (3.2, 18.3) 3, 2b
80(90) 121.65 90,7
0,8
03.49, 2a0-H, dd (3.2, 18.3) 3 0,2b
0
90(80) 121.55 903.39, 3 0-OH, s
8 121.08 7 2.97, 2b-H, dd (2.7, 18.3) 3, 2a
5 119.55 8 2.92, 2b0-H, dd (2.7, 18.3) 30,2a
0
10 114.15 7
4095.71 30,2a
0,2b
0
4 95.66 3
3(3
0) 70.97
30(3) 70.76
2(2
0) 43.56
20(2) 43.21
K. Krohn et al. / Tetrahedron 57 (2001) 4343 ±4348 4345
to the similarity of the top and bottom part of the molecule,
many signals have nearly identical chemical shifts and
represent two protons. The coupling constants of 3- and
30-H with the neighboring methylene protons at 2- and
20-C are J2.7 and J3.2 Hz, excluding 1,2-transdiaxial
positions of the respective protons.
Analysis of the complete set of spin systems in combination
with connectivities deduced from two-dimensional tech-
niques (H,H-COSY, HMQC and HMBC) allowed the
construction of two independent fragments Aand B
(Scheme 2). A combination of the fragments Aand Bin
consideration of the total number of 14 unsaturations ®nally
leads to structure 6for the most polar metabolite (Scheme
1). The new preussomerin L (6) is the bisreduction product
of a hypothetical 2,3- and 2 0,30-diepoxy preussomerin, a
metabolite which has not yet been isolated from natural
sources.
Interestingly, two major independent fragments can also be
seen in the EI mass spectrum for all six compounds 1±6,
suggesting the af®liation to the same class of compounds.
As proposed by Singh et al.,
2
a simpli®ed fragmentation
pattern for preussomerin L (6) is presented in Scheme 3.
The peak of the top fragment I(m/z189 or 191) indicates
a hydroxylated or epoxidized fragment, respectively. In the
bottom fragment II, peaks for both
b
-elimination products
(m/z174) or
b
-substituted fragments occur.
The relative stereochemistry of preussomerin L (6)
9
was
established by X-ray diffraction analysis of a single crystal
obtained from methanol. As shown in Fig. 1, the aromatic
rings are in almost perpendicular positions in the rigid but
relatively strainless skeleton and quasi-boat conformations
are adopted by the hydroaromatic rings in agreement with
the
1
H NMR coupling constants.
The similarity of the spectral data of the other metabolites
1±5, isolated from the endophytic fungus, con®rmed
membership in the preussomerin class of natural products.
Structure elucidation can thus be restricted to data com-
parison and typical differences to those of other known
preussomerins. The three unpolar compounds were identical
with the preussomerins G (1), H (2) and I (3) (Scheme 1) as
identi®ed by Singh et al.,
2
showing desaturation or
saturation at positions 20and 30or a methoxy group at
C-30.PreussomerinI(3), which had previously only been
obtained as an oily compound, could be obtained as a
crystalline material, mp 130±1328C. All spectroscopic data
were in good agreement with those reported in the literature.
2
The next most polar compound, preussomerin J (4), was
isolated in small amounts (1.3 mg). The mass spectrum
and the molecular formula C
22
H
14
O
9
suggested an acetyl-
ation product of preussomerin K. This was con®rmed by
typical changes in the NMR spectra. Additional signals
for carbon atoms appeared at
d
169.4 and 20.94 for an
acetyl group. The triplet for 3 0-H is typically shifted low-
®eld from
d
4.78 for preussomerin K (5) to 5.93 in the
acetylation product 4. The coupling constants for the bottom
aliphatic part of the molecule remain essentially unchanged
(J
2',3'
2.9 Hz), indicating the same conformation for the
acetylation product preussomerin J (4)asin5. It is con-
ceivable that preussomerin J (4) is an artifact, formed during
extraction with ethyl acetate from preussomerin K (5)orby
Michael addition of acetic acid to preussomerin G (1).
The IR and UV data for the oily next polar compound,
preussomerin K (5), are almost identical to those of preus-
somerin L (6). The molecular formula C
20
H
12
O
8
, deter-
mined by HRMS, shows two hydrogen atoms fewer than
the more polar metabolite 6. In the
1
H NMR spectrum, two
low ®eld signals for aliphatic protons at
d
3.84 and 4.26
with a coupling constant of J4.0 Hz can be detected. The
entire pattern is typical for protons attached to an epoxide
Scheme 2. Fragments Aand Bas deduced from the NMR spectra of 6.
Scheme 3. Typical MS fragmentation of preussomerins exempli®ed on
preussomerin L (6).
Figure 1. The molecule of preussomerin L (6) in the crystal.
K. Krohn et al. / Tetrahedron 57 (2001) 4343± 43484346
ring and is almost identical to the corresponding area in
preussomerin G±I.
2
The location of the epoxide ring at
C-2 and C-3 was unambiguously assigned by a long-range
coupling of H-3 to the carbonyl carbon at C± 1. The NMR
spectra of the bottom part of 5corresponded to that of
preussomerin L (6) and thus structure 5could be assigned
to preussomerin K.
3. The absolute con®guration
The absolute con®guration of preussomerin A (7) was
established by Weber et al.
3
by acid-catalyzed degradation
(probably via intermediate (8)to(2)-regiolone (9)in
combination with X-ray analysis for determination of the
relative stereochemistry (Scheme 4). Interestingly, this
experiment established also the stereochemical correlation
to the palmarumycins as shown by the similarity of inter-
mediate 8to palmarumycin C
10
(10).
10
The optical rotations measured for the new compounds 4±6
and those for the preussomerins A or G are all negative.
Unfortunately, the data deviate too much (preussomerin L
(1):
a
20
D2557; preussomerin A:
a
20
D2212
4
; preus-
somerin G:
a
20
D2668
2
) to be a solid basis for the assign-
ment of the absolute con®guration to the new
preussomerins. Thus, we decided to determine their
absolute con®gurations by comparison of calculated and
experimental CD spectra. This method was previously
applied successfully to elucidate the absolute con®gurations
of several palmarumycins.
11± 13
In fact, the preussomerins
are ideal substrates for this kind of determination since
they are conformationally very rigid molecules, ®xed by
their bisspiro structure. Not surprisingly, calculations
using the Spartan package
14
of preussomerin L (6) revealed
one major conformer, 18 kJ/mol higher in energy than the
conformation with next lowest energy. Also, this conforma-
tion was nearly identical to that shown in Fig. 1, obtained by
X-ray structure analysis. Thus, for the calculation of the CD
spectrum of preussomerin L (6), it was suf®cient to consider
only the two enantiomers of this major conformation. The
results of these calculations, employing the BDZDO/
MCDSPD program package
15
and the comparison with the
experimental CD spectrum are shown in Figs. 2 and 3.
In Fig. 2, the experimental spectrum matches that of the
calculated spectrum (dotted line) of structure 6of preus-
somerin L. By contrast, the calculated spectrum for the
enantiomer ent-6is almost of the opposite shape to that of
the experimental spectrum. Consequently, the absolute
con®guration for preussomerin L (6) was assigned beyond
any doubt to be 3R,3
0R,4S,4S0. The CD spectra of all of the
other preussomerins G±K (1±5) were calculated in a similar
manner. Comparison of calculated with the experimental
data con®rmed their absolute con®guration as shown in
Scheme 1 and the CD maxima are listed in Table 2.
16
The
same result was obtained on the basis of the negative sign of
n!
p
p
Cotton effect located at 330 ±334 nm, caused by the
aryl ketone chromophore of these molecules using
Snatzke's rule.
17
4. Biological activity
The crude ethyl acetate extracts showed activity against the
Gram-positive bacterium Bacillus megaterium as well as
against the fungus Microbotryum violaceum. The activity
of the puri®ed individual preussomerins against bacterial,
fungal and algal test organisms is compiled in Table 3. The
activity against the fungi Microbotryum violaceum and
Eurotium repens is relatively moderate as compared to
those of related preussomerins A±F against coprophilous
Scheme 4. Degradation of preussomerin A (7) to regiolone (9) and com-
parison with palmarumycin C
10
(10).
Figure 2. Experimental CD spectrum and calculated spectrum (dotted line)
of structure 6of preussomerin L.
Figure 3. Experimental CD spectrum and calculated spectrum (dotted line)
of structure ent-6: Ð , measured spectrum; £, calculated spectra.
K. Krohn et al. / Tetrahedron 57 (2001) 4343 ±4348 4347
fungi as determined by Weber et al.
3
The remarkable
FPTase inhibitory activity of preussomerins, notably of
preussomerin G, has already been mentioned.
2
5. Experimental
General methods and instrumentation are covered elsewhere.
18
5.1. Cultivation and isolation
The fungus was cultivated for 70 days at ambient tempera-
ture on two different media: biomalt and malt-soya semi-
solid agar as described previously.
18
The combined cultures
(6 l) were homogenized and extracted ®rst with petroleum
ether (0.5 l) and than four times with ethyl acetate (1 l). The
biologically active metabolites were located mainly in the
ethyl acetate extract (3.3 g) as shown by TLC and bio-
autograms. The crude extract was then suspended in a
mixture of cyclohexane and dichloromethane (1:1, 30 ml),
®ltered, and the ®ltrate concentrated at reduced pressure to
yield 3.0 g of soluble crude material. The residue was sepa-
rated into ®ve fractions by column chromatography on silica
gel (400 g), using gradients of dichloromethane/ethyl
acetate (85:15, 50:50, 0:100). The less polar fraction
(2.1 g) contained mainly fatty acids and lipids. The remain-
ing four fractions were each further puri®ed by preparative
TLC chromatography on silica gel (1 mm, Macherey and
Nagel). The least polar fraction was developed twice with
CH
2
Cl
2
/petroleum ether 2:5 to yield 13.1 mg of preus-
somerin G (1) as a yellow solid.
2
The next fraction was
developed with CH
2
Cl
2
to yield 5.4 mg of preussomerin I
(2).
2
Crystallization from cyclohexane/dichloromethane
gave yellow crystals, mp 130±1328C. Preparative TLC
separation of the fraction next in polarity (CH
2
Cl
2
) gave
1.3 mg of preussomerin H (3).
2
The more polar fractions
were separated on TLC plates and developed with increas-
ingly polar eluent (CH
2
Cl
2
/2, 3, and 5% of methanol,
respectively) to yield the preussomerins J (4) (4.0 mg), K
(5) (8.2 mg), and L (6) (7.9 mg). Preussomerin L crystal-
lized from small amounts of methanol (mp 171±1738C). All
preussomerins could be detected on TLC plates by irradia-
tion with 254 nm UV light or by spraying with a solution of
2,4-dinitrophenylhydrazine or molybdenum phosphate.
5.2. X-Ray crystallographic study of 619
Crystal data: C
20
H
14
O
8
,M
r
382.31, hexagonal, space group
P6
1
,a15.274(2), c15.074(7) A
Ê,V3045.5(15) A
Ê
3
,Z6,
D
c
1.251 Mg/cm
3
. It was very arduous obtaining a fairly
suitable crystal for data collection: Bruker AXS P4 diffract-
ometer; Mo-K
a
radiation,
l
0.71073 A
Ê, graphite mono-
chromator, crystal size 0.45£0.30£0.28 mm
3
,T293(2) K,
v
-scan, 2.1#Q#25.08,216#h#16, 216#k#1, 21#l#
17, Lp correction, 3 standard re¯ections were recorded
every 400 re¯ections and showed a decrease of 12%;
4523 re¯ections collected, 2011 independent re¯ections
(R
int
0.061), m0.098 mm
21
. Structure solution by direct
methods, full-matrix least-squares re®nement based on 2010
F
2
values and 256 parameters, hydrogen atoms at calculated
positions were re®ned as riding models; all but hydrogen
atoms re®ned anisotropically. Max (D/
s
),0.001,
G
oof
1.011, R1(I.2(
s
)I)0.081, wR2(all data)0.277. It
was not possible to successfully re®ne various diffuse elec-
tron density residues which must be assigned to solvent
molecules. Program used: SHELXTL NT 5.10.
20
5.2.1. Preussomerin G (1). Yellow solid. R
f
0.62 (dichloro-
methane). C-10 was not correctly assigned in the
13
C-NMR
spectrum of preussomerin G (1).
2
Our data, based on HMBC
correlations, are given below.
13
C-NMR (300 MHz,
CDCl
3
):
d
52.17 (d, C-3), 53.70 (d, C-2), 89.98 (s, C-40),
93.96 (s, C-4), 109.82 (s, C-5), 115.21 (s, C-10), 120.59 (s,
C-50), 120.70 (d, C-90), 120.86 (d, C-7 0), 121.31 (d, C-8),
126.73 (d, C-7), 130.32 (s, C-100), 131.15 (d, C-80), 133.57
(d, C-20), 140.73 (d, C-30), 143.39 (s, C-6), 148.76 (s, C-6 0),
156.11 (s, C-9), 183.73 (s, C-10), 195.45 (s, C-1).
Table 2. CD-Maxima of preussomerins G± L (1±6)
Preussomerin G (1) Preussomerin H (2) Preussomerin I (3)
l
(nm) D
1
(cm
2
mol
21
)
l
(nm) D
1
(cm
2
mol
21
)
l
(nm) D
1
(cm
2
mol
21
)
369 22.75 331 23.44 332 25.62
318 24.62 276 22.87 277 24.92
264 sh 24.79 262 0.43 262 0.11
244 223.38 236 sh 27.7 238 sh 210.22
217 250.15 218 237.95 218 271.63
Preussomerin J (4) Preussomerin K (5) Preussomerin L (6)
l
(nm) D
1
(cm
2
mol
21
)
l
(nm) D
1
(cm
2
mol
21
)
l
(nm) D
1
(cm
2
mol
21
)
331 23.94 331 23.35 334 21.37
275 22.72 276 23.19 269 22.73
237 sh 212.67 240 sh 26.53 237 sh 24.31
218 254.81 218 251.16 219 238.82
Table 3. Biological activity of preussomerins 1±6against microbial test
organisms, agar diffusion assay
Compound Bm. Chl. Eur. Fu. Mm. Mv.
10.6 0 0.9 1.1 0.1 0.6± 1
20.3± 0.5 0.1±0.2 ± ± ± 0.8± 1.3
30.2 0.3 0.5± 0.6 ± 0.1 0.4±0.8
40.4± 0.5 0.1 0.8 ± ± 0.8±1.2
50.5± 0.7 0.5 0.5 ± ± 1.1
60.2± 0.5 0 ± 0 0.2 0.4
Concentration: 50 ml of the given 2 mg/ml solution per plate; test organ-
isms: Bm.Bacillus megaterium, Mv.Microbotryum violaceum,
Mm.Mycotypha microspora, Eur.Eurotium repens, Fu.Fusarium
oxysporum, Chl.Chlorella fusca.
K. Krohn et al. / Tetrahedron 57 (2001) 4343± 43484348
5.2.2. Preussomerin H (2). Oil; R
f
0.5 (dichloromethane).
5.2.3. Preussomerin I (3). Mp 130±1328C (dichloro-
methane/cyclohexane); R
f
0.71 (dichloromethane).
5.2.4. Preussomerin J (4). Oil; R
f
0.43 (SiO
2
,CH
2
Cl
2
);
a
20
D2218 (c 0.08, CH
2
Cl
2
). IR:
n
3389, 2919, 2852,
1734, 1641, 1263, 1098, 1031, 922, 798 cm
21
.UV
(CH
2
Cl
2
):
l
max
(log
1
)360 (2.14), 319 (2.14), 253
(2.70), 228 (2.85) nm.
1
H-NMR (600 MHz, CDCl
3
):
d
1.99 (s, 3H, OCOCH
3
), 3.05 (dd, 1H, J18.8 Hz, J
2.9 Hz, 2a 0-H), 3.47 (dd, 1H, J18.8 Hz, J3.3 Hz, 2b0-
H), 3.82 (d, 1H, J4.0 Hz, 3-H), 4.23 (d, 1H, J4.0, 2-H),
5.93 (t, 1H, 30-H), 6.95 (d, 1H, J
9.1 Hz, 8-H), 7.05 (d, 1H,
J9.14 Hz, 7-H), 7.09 (dd, 1H, J8.2 Hz, J1.0 Hz, 70-H),
7.44 (t, 1H, 80-H), 7.68 (dd, 1H, J7.9, J1.0 Hz, 90-H),
10.12 (s, 1H, 10-OH).
13
C-NMR (600 MHz, CDCl
3
):
d
20.94 (q, OCOCH
3
), 40.26 (t, C-20), 52.07 (d, C-3),
53.48 (d, C-2), 70.62 (d, C-30), 92.20 (s, C-40), 93.08 (s,
C-4), 110.20 (s, C-10), 115.69 (s, C-5), 119.75 (s, C-50),
120.74 (d, C-90), 120.89 (d, C-70), 121.46 (d, C-8), 121.76
(d, C-80), 126.43 (d, C-7), 131.14 (s, C-100), 142.49 (s, C-6),
149.66 (s, C-60), 156.40 (s, C-9), 169.40 (s, OCO), 192.42 (s,
C-10), 195.58 (s, C-1). HRMS (EI) (C
22
H
14
O
9
)calcd
422.06320; found: 422.06378 ^1.4 ppm.
5.2.5. Preussomerin K (5). R
f
0.17 (SiO
2
,CH
2
Cl
2
); oil;
a
20
D2150 (c 0.04, CH
2
Cl
2
). IR
n
3415, 2924, 2846,
1708, 1465, 1419, 1274, 1025, 932 cm
21
. UV (CH
2
Cl
2
):
l
max
(log
1
)363 (1.11), 314 (1.23), 250 (1.64), 260
(1.45), 230 (1.86) nm.
1
H-NMR (300 MHz, CDCl
3
):
d
3.03 (dd, 1H, J2.7 Hz, J18.3 Hz, 2a 0-H), 3.37 (dd,
1H, J3.3 Hz, J18.3 Hz, 2b0-H), 3.84 (d, 1H, J4.0 Hz,
3-H), 3.53 (s, 1H, 30-OH), 3.84 (t, 1H, 30-H), 4.26 (d, 1H,
J4.0 Hz, 2-H), 6.94 (d, 1H, J9.2 Hz, 8-H), 7.02 (d, 1H,
J9.2 Hz, 7-H), 7.05 (dd, 1H, J8.1 Hz, J1.0 Hz, 7 0-H),
7.39 (t, 1H, 80-H), 7.65 (dd, 1H, J7.7, J1.0 Hz, 90-H),
10.12 (s, 1H, 1-H).
13
C-NMR (300 MHz, CDCl
3
):
d
41.30
(t, C-20), 52.17 (d, C-3), 53.67 (d, C-2), 70.28 (d, C-30),
93.58 (s, C-4), 94.38 (s, C-40), 110.26 (s, C-10), 115.04 (s,
C-5), 119.81 (s, C-50), 121.13 (d, C-90), 120.32 (d, C-7),
121.52 (d, C-70), 126.46 (d, C-8), 130.85 (s, C-100),
131.15 (d, C-80), 142.52 (s, C-6), 149.73 (s, C-60), 156.06
(s, C-9), 193.39 (s, C-10), 195.64 (s, C-1). HRMS(EI)
(C
20
H
12
O
8
) calcd. 380.05347; found: 380.05322 ^0.8 ppm.
5.2.6. Preussomerin L (6). Mp 171±1738C; R
f
0.61 (SiO
2
,
CH
2
Cl
2
/MeOH 95:5);
a
20
D2557 (c 0.11, CH
2
Cl
2
). IR
(KBr)
n
3382 cm
21
, 2925, 2851, 1722, 1649, 1434,
1044, 870. UV (CH
2
Cl
2
):
l
max
(log
1
)359 nm (2.96),
315 (2.89), 258 (3.52), 226 (3.61), 194 (2.92).
1
H-NMR
(600 MHz, CDCl
3
):
d
2.92 (dd, J18.3 Hz, J2.7 Hz,
2b0-H), 2.97 (dd, J18.3 Hz, J2.7 Hz, 2b-H), 3.39 (s, 3 0-
OH), 3.49 (dd, J3.2 Hz, J18.3 Hz, 2a 0-H), 3.59 (dd,
J3.2 Hz, J18.3 Hz, 2a-H), 3.68 (s, 1H, 30-OH), 4.75
(dd, 2H, 3, 30-H), 6.97 (d, 1H, J9.2 Hz, 8-H), 7.16 (d,
1H, J9.2 Hz, 7-H), 7.21 (dd, 1H, J8.2 Hz, J1 Hz, 7 0-
H), 7.48 (t, 1H, 8 0-H), 7.63 (dd, 1H, J7.8, J1.0 Hz, 9 0-H),
10.16 (s, 1H, 10-OH).
13
C-NMR (150 MHz, CDCl
3
):
d
43.21 (t, C-2/C-20) 43.56 (t, C-20/C-2), 70.76 (d, C-3/
C-30), 70.97 (d, C-30/C-3), 95.66 (s, C-4), 95.71 (s, C-40),
114.15 (s, C-10), 119.55 (s, C-5), 121.08 (d, C-8), 121.55 (d,
C-90/C-80), 121.65 (d, C-8 0/C-9 0), 122.63 (d, C-70), 126.89
(d, C-7), 132.12 (s, C-50/C-100), 132.22 (s, C-100/C-5 0),
144.41 (s, C-6), 152.33 (s, C-60), 158.10 (s, C-9), 195.98
(s, C-10), 202.24 (s, C-1). HRMS(EI) (C
20
H
14
O
8
) calcd.
382.06795; found: 382.06888 ^2.4 ppm.
Acknowledgements
We thank the BMBF and BASF AG (Germany) and the
Hungarian National Science Foundation (OTKA-T 34250)
for ®nancial support.
References
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kk@chemie.uni-paderborn.de.
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19. Data (excluding structure factors) have been deposited with
the Cambridge Crystallographic Data Centre as supplemen-
tary publications no. CCDC 148953. Copies of the data can be
obtained free of charge on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK (fax: 144-(0)1223-336-033;
e-mail: deposit@ccdc.cam.ac.uk).
20. SHELXTL NT, Ver. 5.10, Bruker Analytical X-ray Systems,
Madison, Wisconsin, USA.
