The himanimides, new bioactive compounds from Serpula himantoides (Fr.) Karst.

Abstract

In a screening of basidiomycete cultures from Chile for the production of antibiotics we identified a Serpula himantoides strain as a producer of metabolites inhibiting the growth of bacteria and fungi. Bioactivity guided purification resulted in the isolation of four new antibiotics. Their structures were elucidated by spectroscopic methods. All four compounds are succinimide and maleimide derivatives, of which two are N-hydroxylated.

Full text

Page 1

The Himanimides, New Bioactive Compounds from Serpula himantoides
(Fr.)Karst.
Pedro Aquevequea,b, Timm Ankeb,* and Olov Sternerc,*
aUniversidad de Conception, Casilla 2407, Apartado 10, Conception 3, Chile
bLehrbereich Biotechnologie der Universität, Paul-Ehrlich-Straße 23,
D-67663 Kaiserslautern, Germany. Fax: +49-6312052999. E-mail: anke@rhrk.uni-kl.de
cDepartment of Organic and Bioorganic Chemistry, Lund University, P. O.Box 124,
S-221 00 Lund, Sweden. Fax: +46-462228209. E-mail: Olov.Sterner@orgk2.lth.se
* Authors for correspondence and reprint requests
Z. Naturforsch. 57c, 257Ð262 (2002); received November 7/December 3, 2001
Himanimides, Antibiotics, Basidiomycete
In a screening of basidiomycete cultures from Chile for the production of antibiotics we
identified a Serpula himantoides strain as a producer of metabolites inhibiting the growth of
bacteria and fungi. Bioactivity guided purification resulted in the isolation of four new antibi-
otics. Their structures were elucidated by spectroscopic methods. All four compounds are
succinimide and maleimide derivatives, of which two are N-hydroxylated.
Introduction
Serpula himantoides (Coniophoraceae), a resu-
pinate member of the aphyllophorales is of wide-
spread occurrence in the temperate zones of
Europe and the Americas. It causes an intensive
brown rot on gymnosperms and more rarely on
angiosperms. S. lacrymans, a closely related spe-
cies causes the destructive dry rot of wooden struc-
tures in buildings. Both species contain the pulvi-
nic acid derivatives xerocomic acid, variegatic
acid, and variegatorubin, pigments typically en-
countered within the boletes (Gill and Steglich,
1987). The production of polyacetylenes by S. lac-
rymans has been reported (Jones and Thaller,
1984).
Materials and Methods
General
For analytical HLPC, a Hewlett Packard 1090
series II instrument (column: Merck LiChrocart
125Ð4 filled with LiChrospher 100 RP18) was
used. Preparative HPLC was performed with a
Jasco model PU-980 system with diode array de-
tector. TLC analyses were performed on Mach-
erey-Nagel AlugramSil
plates and visualised with anisaldehyde/sulfuric
acid 1:1 (5% in ethanol) and heating up to 120 ∞C.
1H NMR (500 MHz) and13C NMR (125 MHz)
G/UV254precoated
0939Ð5075/2002/0300Ð0257 $ 06.00
” 2002 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com ·
D
were recorded at room temperature with a Bruker
ARX500 spectrometer with an inverse multinu-
clear 5 mm probehead equipped with a shielded
gradient coil. The spectra were recorded in
CDCl3, and the solvent signals (7.26 and 77.0 ppm,
respectively) were used as reference. The chemical
shifts (δ) are given in ppm, and the coupling con-
stants (J) in Hz. COSY, HMQC and HMBC ex-
periments were recorded with gradient enhance-
ments using sine shaped gradient pulses. For the
2D heteronuclear correlation spectroscopy the re-
focusing delays were optimised for1JCH= 145 Hz
andnJCH= 10 Hz. The raw data were transformed
and the spectra were evaluated with the standard
Bruker XWIN-NMR software (rev. 010101). Mass
spectra were recorded with a Jeol SX102 spec-
trometer, while the UV spectra were obtained
with a Perkin Elmer λ16 and the IR spectra with
a Bruker IFS 48 instrument. The optical rotations
were measured with a Perkin-Elmer 141 polarime-
ter at 22 ∞C.
Producing organism and fermentation
Fruiting bodies of Serpula himantoides were col-
lected in Concepcion, Chile, growing on Eucalyp-
tus globulus in autumm 1995. Mycelial cultures of
strain 95099 were derived from the spore print of
a fruting body. A voucher specimen of the fungus
is deposited in the herbarium of the Department

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258P. Aqueveque et al. · New Antibiotics from Serpula himantoides
Biotechnology, University of Kaiserslautern. The
strain 95099 is kept on YMG agar containing (g/
liter): Glucose 4, malt extract 10, yeast extract 4,
agar 20, pH 5.5. Fermentation was carried out in
YMG medium at 24 ∞C in a 100 liter fermentor
(Braun Biostat U) with aeration (15 liter air/min)
and agitation (120 rpm); 10 liters of a well-growth
culture (10 days) in the same medium were used
as inoculum. During fermentation, aliquots of the
culture fluid (200 ml) were extracted with ethyl ac-
etate. The combined extracts were dried with
Na2SO4and concentrated in vacuo. (40 ∞C). The
extracts were disolved in methanol and this solu-
tion (100 µg/ml) was used to determine the antimi-
crobial activity against bacteria and fungi. The fer-
mentation was stopped after 240 h when the
antibacterial (Bacillus brevis, B. subtilis) activity of
the extract had reached its peak.
Isolation
The compounds were extracted from the culture
filtrate by absortion onto Mitsubishi DIAION HP-
21 resin. The resin was washed with methanol and
the himanimides eluted with acetone yielding
4.13 g of crude product. This was applied onto a
column (25 ¥ 250 mm) containing silica gel
(0.063Ð0.2 mesh, Merck 60). Elution with cyclo-
hexane-ethylacetate 1:1 v/v yielded 615.4 mg of an
enriched product which was applied onto another
silica gel column. (15 ¥ 145 mm, 0.063Ð0.2 mesh,
Merck 60 ). From this column 64,7 mg of 95099Ð
11 were obtained by elution with cyclohexane-
ethylacetate 8:2 v/v. Elution with cyclohexane-
ethylacetate 7:3 v/v yielded 198 mg of a himani-
mide A (1) containing fraction. From this 5.6 mg
of pure 1 was obtained by preparative HPLC (col-
umn: Macherey and Nagel, 250 ¥ 21.2 mm contain-
ing Nucleosil C18 (7 µm) flow rate: 5 ml/min; Gra-
dient 0Ð70% MeOH in 40 min; Rt1: 24 min.
Elution of the silica gel column with cyclohex-
ane-ethylacetate 3:7 v/v yielded 1633 mg of a frac-
tion containing himanimide B (2) and himanimide
D (4). A repetition of silica gel chromatography
as described above resulted in 167.6 mg of pure 2
and 6 mg of pure 4.
Himanimide A (1) (3-[4-(3-methylbut-2-enoxy)-
phenyl-4-phenylmethyl-2,5-dioxo-3-pyrroline) was
obtained as a yellow oil. UV (MeOH), λmax (log
ε): 230 nm (4.1) and 356 nm (3.6). IR (KBr): 3450,
2925, 1735, 1600, 1510, 1465, 1380, 805, 745 and
700 cmÐ1. See Tables I and II for NMR data.
FABHRMS: (348.1598, M+H+, C22H22O3N re-
quires 348.1600).
Himanimide B (2) (3-[4-(2,3-dimethyl-3-methyl-
butoxy)phenyl-4-phenylmethyl-2,5-dioxo-3-
pyrroline) was obtained as a yellow oil. UV
(MeOH), λmax (log ε): 229 nm (4.3) and 356 nm
(3.8). IR (KBr): 3445, 2930, 1715, 1605, 1510, 1455,
1350, 1255, 1180, 1085, 1030, 840, 745 and 700 cmÐ1.
See Tables I and II for NMR data. FABHRMS:
(382.1659, M+H+, C22H24O5N requires 382.1654).
Himanimide C (3) (N-hydroxy-3-[4-(3-methyl-
but-2-enoxy)phenyl-4-phenylmethyl-2,5-dioxo-3-
pyrroline) was obtained as a yellow oil. UV
(MeOH), λmax (log ε): 232 nm (4.3), 290 nm (3.7)
and 374 nm (3.6). IR (KBr): 3470, 2925, 1720,
1605, 1510, 1250, 1180, 1080, 840 and 700 cmÐ1.
See Tables I and II for NMR data. FABHRMS:
(364.1546, M+H+, C22H22O4N requires 364.1549).
Himanimide D (4) (N-hydroxy-3-[4-(3-methyl-
but-2-enoxy)phenyl-4-phenylmethyl-2,5-dioxo-3-
pyrrolidine) was obtained as a colourless oil, [α]D
0∞ (c 0.5 in CHCl3). UV (MeOH), λmax (log ε):
225 sh (4.1), 277 nm (3.4). IR (KBr): 3450, 2925,
1710, 1610, 1510, 1455, 1385, 1230, 1060, 1005, 835,
750 and 700 cmÐ1. See Tables I and II for NMR
data. FABHRMS: (366.1729, M+H+, C22H24O4N
requires 366.1705).
Biological assays
Antimicrobial activitiy was determined in the
serial dilution assay or the plate diffusion assay as
described by Anke et al. (1989). Test for cytotoxic-
ity towards HL-60 cells (human promyelocytic leu-
kemia, ATCC CCL 240), Colo 320 (human colon
adenocarcinoma, DSMZ ACC 144) and L1210
(lymphocytic leukemia, mouse, ATCC CCL219)
was determined as reported by Zapf et al. (1995).
Inhibition of growth of germinated seeds of Setaria
italica and Lepidium sativum was tested as de-
scribed by Anke et al. (1989). Inhibition of ap-
pressorium formation in germinating conidia of
Magnaporthe grisea strain P1 was measured as de-
scribed previously by Thines et al. (1997).
Results and Discussion
Fermentations were harvested 190Ð200 h after
inoculation when the antibiotic content of the cul-

Page 3

P. Aqueveque et al. · New Antibiotics from Serpula himantoides
259
Fig. 1. The structures of himani-
mides A (1), -B (2), -C (3) and -D
(4).
ture broth had reached its maximum. The com-
pounds were isolated from the culture fluid as de-
scribed in the experimental section. The mycelia
did not contain antibiotic activity and were dis-
carded. The structures (see Fig. 1) of the four me-
tabolites were determined by spectroscopic tech-
niques. High resolution MS experiments revealed
the elemental composition of the four metabolites,
and these were in agreement with the1H and13C
NMR data (presented in Tables I and II). 2D
NMR experiments, COSY NOESY, HMQC and
HMBC, were used to establish the structures and
to assign all signals. The composition of himani-
mide A (1) is C22H22O3N, and the unsaturation
index is consequently 13. The presence of two ben-
zene rings, one as a benzyl group and the other as
a 1,4-disubstituted benzene, together accounting
for eight unsaturations, was indicated by the NMR
data, and the ninth is situated in the 3-methylbut-
2-enoxy substituent on C-4? (positioned by the
HMBC correlation between 5?-H2and C-4?). The
remaining part, consisting of C4HO2N and ac-
counting for four unsaturations, is substituted at
C-2 by the para-substituted benzene (as indicated
by the HMBC correlation from 2?-H to C-2) and
at C-3 by the benzyl group (indicated by the
Table I.1H (500 MHz) NMR data (δ; multiplicity; J) for
himanimide A (1), B (2), C (3) and D (4). The spectra
were recorded in CDCl3, and the solvent signal for
CHCl3(7.26 ppm) was used as reference. The coupling
constants J are given in Hz.
1234
H
2
3
2?
3?
Ð
Ð
7.53, d, 8.8
6.97, d, 8.8
Ð
Ð
7.54, d, 8.7
6.99, d, 8.7
Ð
Ð
7.52, d, 8.8
6.95, d, 8.8
6.74, d, 8.4
3.58, d, 3.6
3.11, m
6.80, d, 8.4
5?
4.56, d, 6.9 4.19, dd, 2.9, 9.6 4.55, d, 6.8
4.07, dd, 7.7, 9.6
3.84, dd, 2.9, 7.7 5.49, t, 6.8
1.34, s
1.29, s
3.94, s
4.41, 6.6
6?
8?
9?
1?
5.50, t, 6.9
1.82, s
1.76, s
3.96, s
5.44, t. 6.6
1.79, s
1.71, s
3.18, dd, 4.3, 13.6
3.03, dd. 7.7, 13.6
7.10, d, 7
7.24, t, 7
7.21, t, 7
Ð
1.81, s
1.76, s
3.94, s
3?
4?
5?
NH
7.22, d, 7.2
7.31, t, 7
7.24, t, 7
7.39, brs
7.20, d, 7.5
7.30, t, 7.5
7.23, t, 7
7.62, brs
7.18, d, 7
7.27, t, 7
7.21, t, 7
Ð
HMBC correlations from 1?-H2to C-2, C-3 and C-
4). The remaining proton, which is not attached to
a carbon according to the HMQC spectrum, ap-
pears as a broad singlet at 7.39 ppm in the
NMR spectrum and correlates to C-1, C-2, C-3
1H

Page 4

260P. Aqueveque et al. · New Antibiotics from Serpula himantoides
Fig. 2. Fermentation of Serpula hi-
mantoides and production of hi-
manimides.
Table II.13C (125 MHz) NMR data (δ; multiplicity) for
himanimide A (1), B (2), C (3) and D (4). The spectra
were recorded in CDCl3, and the solvent signal for
CDCl3(77.0 ppm) was used as reference. The multiplici-
ties of the carbon signals were determined indirectly
from HMQC experiments.
1234
C
1
2
3
4
1?
2?
3?
4?
5?
6?
7?
8?
9?
1?
2?
3?
4?
5?
170.8; s
139.2; s
136.9; s
171.3; s
120.8; s
131.1; d
115.0; d
160.5; s
65.0; t
119.2; d
138.7; s
25.8; q
18.2; q
29.8; t
137.2; s
128.4; d
128.9; d
126.8; d
170.8; s
138.9; s
137.4; s
171.3; s
121.6; s
131.2; d
114.9; d
159.9; s
69.3; t
75.7; d
71.7; s
26.6; q
25.0; q
29.8; t
137.1; s
128.3; d
128.9; d
126.9; d
167.8; s
136.2; s
133.5; s
168.4; s
120.5; s
131.2; d
115.0; d
160.6; s
65.0; t
119.1; d
138.7; s
25.8; q
18.2; q
29.9; t
136.8; s
128.3; d
128.9; d
126.9; d
172.8; s
47.5; d
48.2; d
173.8; s
127.9; s
128.9; d
115.2; d
158.5; s
64.8; t
119.5; d
138.2; s
25.8; q
18.2; q
35.3; t
136.4; s
129.4; d
128.8; d
127.2; d
and C-4 in the HMBC spectrum. Taken together,
these data are only compatible with the suggested
3,4-disubstituted maleimide derivative 1. Himani-
mide B (2) is oxidised relative to 1, its elemental
composition is C22H24O5N and comparison of the
NMR data reveals that the oxidation has taken
place in the 3-methylbut-2-enoxy substituent.
Both C-6? and C-7? are saturated but oxygenated
in 2, and the C-4? substituent must therefore be
2,3-dihydroxy-3-methylbutoxy. Also himanimide
C (3), with the composition C22H22O4N, is oxi-
dised compared to 1, but the 1D NMR data of
the two compounds are very similar. The only
differences are observed in the maleimide part,
where the13C signals are slightly upshifted, and
no imide proton could be seen in the1H NMR
spectrum. The conclusion is consequently that
himanimide C (3) is a N-hydroxylated maleimide
derivative, unusual in nature but previously sug-
gested to be produced by the slime mould Arc-
yria denudata (as N-hydroxyarcyroxepin A and
N-hydroxyarcyroxepin B, see Dictionary of Natu-
ral Products on CD, Chapman and Hall 2001).
Finally, himanide D (4) with the composition
C22H22O4N, is reduced compared to 3, and com-
parison of the 1D NMR data reveals that the
change has taken place in the maleimide ring.
COSY correlations from 1?-H2to 3-H and on to
2-H suggest that 4 is a succinimide derivative,
the magnitude of the vicinal coupling constants
are in agreement with this and the HMBC corre-
lations both to and from the succinimide ring
confirm the suggested structure. The configura-
tion of C-2 and C-3 appear to be as shown in
Figure 1, as no NOESY correlations can be ob-
served between 1?-H2and 2-H. Himanide D (4)

Page 5

P. Aqueveque et al. · New Antibiotics from Serpula himantoides
261
is optically inactive, and it is possible that a
racemisation takes place during the isolation pro-
cedure.
The antimicrobial spectrum and cytotoxic activ-
ity of the himanimides are presented in Table III
and Table IV respectively. The himanimide C (3)
exhibited fungicidal effects specially against Al-
ternaria porri, Aspergillus ochraceus and Pythium
irregulare
froma concentration
(69.6 µm) on. Fungistatic effects were observed
against Absidia glauca, Cladosporium cladospori-
odes, Curvularia lunata, Zygorhynchus moelleri,
Nadsonia fulvescens, and Saccharomyces cerevisiae
strain is 1. It also showed moderate or weak anti-
of25 µg/ml
Table III. Antimicrobial activity of compounds isolated from Serpula himantoides.
MIC [µg/ml]
Himanimide Organism
ABCD
Filamentous fungi
Absidia glauca +
A. glauca Ð
Alternaria porri
Ascophyta pisi
Aspergillus ochraceus
Cladosporium clodosporioides
Curvularia lunata
Fusarium fujikuroi
F. oxysporum
Mucor miehei
Paecilomyces variotii
Penicillium islandicum
P. notatum
Pythium irregulare
Zygorhynchus moelleri
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
25†
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
25†
Ð
10s
Ð
25†
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
10s 25†
10s
10s
Ð
Ð
100s
Ð
Ð
Ð
10s 25†
10s
100†
Ð
Ð
Ð
Ð
Ð
Yeast
Nadsonia fulvescens
Nematospora corily
Rhodotula glutinis
Saccharomyces cerevisiae is 1
S. cerevisiae α S 288 c
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
10s
Ð
Ð
10s
Ð
Ð
Ð
25s
Ð
Bacteria
Arthrobacter citreus
Bacillus brevis
B. subtilis
B. licheniformis
Corynebacterium insidiosum
Mycobacterium phlei
Streptomyces sp. ATCC 23836
Escherichia coli K12
Salmonella typhimurium TA 98
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
50†
Ð
Ð
Ð
Ð
Ð
Ð
Ð
10s 100†
25s 50†
25s 100†
50†
Ð
25s
Ð
Ð
25s
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð no effects up 100 µg.
† = bacterio-/-fungicidal.
s = bacterio-/-fungistatic.
bacterial activity against Gram-positive bacteria
and yeast. No activity was observed against Gram-
negative bacteria. 3 also showed strong cytotoxic
activity on HL 60 with IC50-values of 10 µg/ml
(27.5 µm) against HL 60 and moderated against
L1210 (25 µg/ml, 69.6 µm) whereas lower cytotoxic
effects were observed with Colo 320. None of the
other three compounds exhibited considerable an-
timicrobial effects against the bacteria, yeast, fungi
and different cell lines tested, suggesting that the
biological activity of himanide C (3) is linked to
the N-hydroxylated maleimide moiety. On the
other hand, none of the compounds exhibited phy-
totoxic activities up to concentration of 100 µg/disc