Tetracyclic terpenoids from Dasyscyphus niveus, dasyscyphins D and E

Article abstract of DOI:10.1021/np800355a

Cultures of the ascomycete Dasyscyphus niveus have yielded two new tetracyclic dasyscyphin-type terpenoids (1 and 2), and their structures were elucidated by NMR spectroscopy and X-ray crystallography. The absolute configuration of dasyscyphin D (1) was d

Full text of DOI:10.1021/np800355a

1654
J. Nat. Prod. 2008, 71, 1654–1656
Tetracyclic Terpenoids from Dasyscyphus niWeus, Dasyscyphins D and E
Johannes C. Liermann,† Heinz Kolshorn,‡ Heidrun Anke,§ Eckhard Thines,§ and Till Opatz*,†
Institut fu¨r Organische Chemie, UniVersita¨t Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany, Institut fu¨r Organische
Chemie, UniVersita¨t Mainz, Duesbergweg 10-14, D-55128 Mainz, Germany, Institut fu¨r Biotechnologie und Wirkstoff-Forschung (IBWF),
Erwin-Schro¨dinger-Strasse 56, D-67663 Kaiserslautern, Germany
ReceiVed June 16, 2008
Cultures of the ascomycete Dasyscyphus niVeus have yielded two new tetracyclic dasyscyphin-type terpenoids (1 and 2), and
their structures were elucidated by NMR spectroscopy and X-ray crystallography. The absolute configuration of
dasyscyphin D (1) was determined by synthesis and NMR spectroscopy of diastereomeric MTPA esters. Both compounds
inhibited the germination of conidia of Magnaporthe grisea at 25 µg/mL.
The tiny, hairy, white, cup-shaped fruiting bodies of the
ascomycete Dasyscyphus niVeus (German: Schneeweisses Haar-
becherchen) can be found growing on decaying oak or beech wood
in European, North American, and New Zealandian forests.1
Various compounds have recently been isolated from fermentation
broths of this fungus. Structure elucidation of these compounds,
the dasyscyphins, has been described along with their strong
cytotoxic activity.2,3 Here, we report on the isolation and structure
elucidation of two novel structurally related terpenoids (1 and 2)
from submerged cultures of D. niVeus collected near Kaiserslautern.
In a screening of extracts obtained from submerged cultures of
ascomycetes, strain A79-88 exhibited antifungal activity, no antibacte-
rial activity, and only very low cytotoxic activity. This selective
biological activity prompted us to isolate the responsible compounds.
The ascomycete was grown in YMG medium until the carbon source
was consumed. The culture filtrate was extracted with ethyl acetate,
and the resulting crude mixture was subjected to silica gel column
chromatography (CC). Further purification by preparative HPLC
yielded two nonpolar compounds, 1 (5.5 mg) as colorless crystals (mp
105-106 °C) and 2 (3.3 mg) as a yellow oil.
Analysis of 13C NMR and DEPT data revealed the presence of
22 carbon atoms and 30 C-H protons in either case, suggesting
that 1 and 2 were closely related structural isomers. The elemental
composition for both compounds was determined to be C22H32O2
1
by HRMS. This is in accordance with H NMR data and proved
the presence of 32 protons and thus two protons bound to
heteroatoms. Furthermore, 13C and DEPT spectra of compound 1
showed a methine carbon at δ 79.5, whereas the spectra of 2 show
a methylene group at δ 65.3. This led to the conclusion that both
be hydroxylated in 2. Only compound 1 possessed two geminal
methyl groups, whereas in both compounds one methyl group
resonated at δ 2.20, characteristic for toluene-like aromatics. The
substitution pattern of the phenol was established to be that of a
5,6-anellated ortho-cresole by HMBC and NOESY experiments.
One of the aromatic protons in 1 showed NOE correlations to
geminal methylene protons at δ 3.01 and 2.66, which was confirmed
by a corresponding HMBC correlation. Both protons had COSY
and NOESY correlations to the multiplet at δ 1.62 (1H), located
on a tertiary center. An HMBC correlation of this proton as well
as of the methyl protons at δ 1.23 with the ortho-position of the
phenol suggested the presence of a five-membered ring with a
methylated ring junction fused to the cresole moiety.
Analysis of further HMBC, COSY, and NOESY correlations
revealed the presence of a fused trans-decalin system carrying an
angular methyl group. In compound 1, the carbinol center is bound
to a quaternary carbon carrying the two remaining methyl groups.
In its isomer 2, the aliphatic OH group is instead located on one of
the geminal methyl groups. The structures of 1 and 2 are based on
a drimane-type sesquiterpene anellated to an indane ring system, a
compounds carry a hydroxy group, which is secondary in 1 and
primary in 2. The UV spectra of methanolic solutions of both
compounds exhibited absorption maxima characteristic of a ben-
zenoid system. In combination with a quaternary carbon atom
resonating at δ 150.3 in both compounds, this was indicative for a
substituted phenol. Tetrasubstitution of the aromatic ring was
concluded from two ortho-coupled aromatic protons (AB spin
system). The elemental composition of 1 and 2 requires seven
double-bond equivalents, four of which are attributed to the benzene
ring. As no further evidence for unsaturation was found, the
molecule must contain three additional rings.
Compound 1 had five methyl groups, all of which appeared as
singlets in the 1H NMR spectrum. In contrast, compound 2 shows
only four methyl singlets; thus, one of the methyl groups in 1 should
* To whom correspondence should be addressed. Tel: +49-(0)40-42838-
4239. E-mail: opatz@chemie.uni-hamburg.de.
† Universita¨t Hamburg.
‡ Universita¨t Mainz.
§ Institut fu¨r Biotechnologie und Wirkstoff-Forschung (IBWF).
10.1021/np800355a CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/05/2008

Notes
Journal of Natural Products, 2008, Vol. 71, No. 9 1655
of COSY and NOESY experiments (Figure 3). By comparison of the
data with those of vitetrifolin A12 and similar compounds reported in
the literature11,13-15 the absolute configuration of 1 was determined
to be S at C-3, being in accordance with that of dasyscyphins A-C.
Since compound 2 does not carry a secondary OH group, the Mosher
method is not suitable in this case. Nevertheless, the close structural
relation between all known dasyscyphins suggests that 2 should have
the same absolute configuration.
Compound 1 completely inhibited conidial germination in Mag-
naporthe grisea 70-15 at 20 µg/mL; compound 2 was slightly less
active with 100% inhibition being observed at 25 µg/mL. Under
minimal conditions, no germ tubes were observed at these concentra-
tions. In the disk diffusion assay with Penicillium chrysogenum (DSM
848), Paecilomyces Variotii (DSM 1961), Rhizomucor miehei (Tu¨ 284),
or Nematospora coylii (ATTC 10647), both compounds showed no
inhibition at 50 µg/disk. Cytotoxic activity toward HeLA S3 cells was
not observed up to concentrations of 50 µg/mL. For dasyscyphins B
and C weak antibacterial and antifungal but strong cytotoxic activities
have been reported, whereas dasyscyphin A was devoid of activity.3
In the conidia germination assay, dasyscyphins B and C exhibited
similar activities to those of D and E. Therefore, we conclude that the
antifungal activities detected during the screening were due to the
presence of dasyscyphins B-E.
Figure 1. Characteristic NOE contacts in 1 and 2.
Experimental Section
General Experimental Procedures. Melting points were determined
with a Dr. Tottoli apparatus and are uncorrected. Optical rotations were
measured with a Perkin-Elmer 241 polarimeter at 546 and 578 nm and
were extrapolated to 589 nm. UV and IR spectra were measured with
a Perkin-Elmer Lambda-16 spectrophotometer and a Bruker IFS48 FTIR
spectrometer, respectively. 1H NMR (400 MHz) and 13C NMR (100.6
MHz) were recorded with a Bruker Avance-II spectrometer. NOESY
spectra (500 MHz) were measured with a Bruker DRX-500 spectro-
meter. The spectra were measured in CDCl3, and the chemical shifts
were referenced to the residual solvent signal (CDCl3: δH ) 7.26 ppm,
δC ) 77.16 ppm).16 APCI-MS spectra were measured with a Hewlett-
Packard MSD1100. ESIMS spectra were measured with a Finnigan
MAT95 spectrometer; HREIMS spectra were measured with a direct
inlet VG70SE spectrometer using PFK as internal reference. Reactions
were monitored on Merck TLC aluminum sheets (silica gel 60 F254).
Diethyl ether was distilled and stored over KOH pellets. CH2Cl2 was
dried by distillation from CaH2. Triethylamine was dried by distillation
from LiAlH4. MTPA chlorides were prepared from the commercially
available acids as described in the literature.17
Figure 2. Crystal structure of 1·CH3OH (ORTEP, ellipsoids drawn
at 50% probability).
Scheme 1. Synthesis of (+)- and (-)-MTPA Esters
Producing Organism. Dasyscyphus niVeus strain A79-88 was
isolated from spore prints of fruiting bodies growing on a dead twig of
beech wood collected near Kaiserslautern, Germany. The specimen
showed all characteristics of the genus and the species.1,18 The strain
was maintained at 4 °C on agar slants on yeast malt glucose medium
(YMG) composed of (g/liter) yeast extract (4, Hartge Ingredients,
Hamburg), malt extract (10, Fra¨nkle & Eck, Fellbach), glucose (4),
and agar (20), pH 5.5. Mycelial cultures of strain A79-88 are deposited
in the culture collection of the Institute of Biotechnology and Drug
Research (IBWF e.V.).
Fermentation and Isolation. Fermentations were carried out in
YMG medium in a 20 L fermentor (Biostat A-20, Braun Melsungen)
at 22 °C with aeration (3.0 L/min) and agitation (120 rpm). A well-
grown culture (200 mL) in the same medium was used as inoculum,
and the antifungal activity was measured with samples withdrawn every
day in a spore germination assay using Magnaporthe grisea as test
organism. After 13 days of fermentation, when the antifungal activity
moiety which is closely related to the skeleton of the dasyscyphins
A-C that were isolated from another strain of Dasyscyphus
niVeus.2,3 These three compounds were also present in crude extracts
of strain A79-88. Dasyscyphin B features a similarly substituted
aromatic ring, which is modified in dasyscyphins A and C.
Therefore, 1 and 2 were given the names dasyscyphins D and E.
The tetracyclic framework of the dasyscyphins is also found in
akaol,4 while pelorol5,6 has an epimeric skeleton. Synthetic
analogues of these marine sesquiterpene derivatives have recently
been reported to be modulator prodrugs for the SHIP-1 enzyme.7,8
The relative configuration of both compounds was determined
by NOESY experiments. Characteristic NOE contacts are depicted
in Figure 1. For compound 1, the structure and relative configuration
were confirmed by X-ray crystallography (Figure 2).
Unfortunately, the collected diffraction data did not allow the
determination of its absolute configuration. Instead, this was achieved
using the well-established Mosher method.9-11 The diastereomeric
MTPA-diesters 3a and 3b were formed by reaction of 1 (1 mg each)
with (+)- and (-)-MTPA chloride, triethylamine, and catalytic amounts
of DMAP in dry CH2Cl2. Although full characterization of the esters
was hampered by the small amounts obtained after chromatographic
purification, characteristic 1H NMR shifts were evaluated by analysis
Figure 3. Characteristic shift differences in 3a and 3b. R ) (+)/
(-)-MTPA, ∆δ ) δ(+) - δ(-).

1656 Journal of Natural Products, 2008, Vol. 71, No. 9
Notes
had reached the maximum and the glucose in the medium was
consumed, the culture fluid (14.5 L) was separated from the mycelia
and extracted with EtOAc (8 L). The mycelium containing no active
compounds was discarded. The organic extract was dried with Na2SO4
and concentrated to yield 4.2 g of crude product, which was further
purified by silica gel chromatography (silica gel 60, Merck Darmstadt,
107 g, column size 17 cm × 4.5 cm). Elution with cyclohexane-EtOAc
(7:3) yielded 98.1 mg of a product containing compounds 1 and 2.
The final purification by preparative HPLC (Merck LiChroSpher RP18,
5 µm, column 25 × 250 mm, flow 20 mL/min; isocratic mode in
MeCN-H2O, 7:3 v/v) resulted in 5.5 mg of pure dasyscyphin D (1)
and 3.3 mg of dasyscyphin E (2).
Biological Assays. Hela S3 (ATCC CCL 2.2 human cervix
carcinoma) cell lines were grown in DMEM medium with 65 µg/mL
penicillin G and 100 µg/mL streptomycin sulfate. The cells (105/mL)
were incubated in microtiter plates with the compounds at 37 °C in a
humidified atmosphere containing 5% CO2. Viable cells were counted
under the microscope after 24 and 48 h.
(3H, s, H3-21); 13C NMR, HSQC, HMBC (100.6 MHz, CDCl3) δ 150.3
(C, C-16), 144.1 (C, C-12), 135.6 (C, C-17), 129.1 (CH, C-14), 120.8
(C, C-15), 116.5 (CH, C-13), 65.3 (CH2, C-20), 62.5 (CH, C-9), 53.4
(CH, C-5), 47.7 (C, C-8), 41.4 (CH2, C-1), 38.6 (C, C-4), 37.3 (C,
C-10), 35.7 (CH2, C-3), 34.8 (CH2, C-7), 32.5 (CH2, C-11), 31.0 (CH3,
C-18), 26.7 (CH3, C-19), 19.7 (CH2, C-6), 18.3 (CH2, C-2), 16.7 (CH3,
C-21), 15.5 (CH3, C-22); APCIMS m/z pos. 311.2 [M - OH]+ (100),
neg. 309.2 [M - H3O]- (6), 327.2 [M - H]- (100); HREIMS m/z
328.2413 (calcd for C22H32O2, 328.2402).
3,16-Bis(r-methoxy-r-(trifluoromethyl)phenylacetyl)dasyscy-
phin D (3). Dasyscyphin D 1 (1.0 mg, 3.0 µmol) was dissolved in dry
CH2Cl2 (1 mL) under argon atmosphere. MTPA chloride (5.0 mg, 19.8
µmol), DMAP (1.0 mg, 8.2 µmol), and dry triethylamine (10 µL, 7.2
mg, 6.9 µmol) were added. The solution was stirred at room temperature
for 1 h. Ether (5 mL) was added, and the resulting solution was washed
with aqueous 1 M HCl (5 mL) and saturated aqueous Na2CO3 (5 mL).
The organic phase was dried over Na2SO4 and the solvent was removed
in vacuo to furnish the diesters as yellow oils.
The antifungal activity was measured in a spore germination assay
with M. grisea 70-15 conidia in water (5 × 104/mL). Germination was
evaluated after 18 h under the microscope and compared to the control
containing no inhibitor. The number of ungerminated conidia or conidia
with significantly shorter germ tubes (germ tube length <30% of
the control) was assessed. A total of 3 × 100 conidia were counted.
The antifungal spectrum of the compounds was evaluated using the
conventional agar diffusion assay.
Compound 3a. Prepared from (+)-MTPA chloride. Yield: 1.0 mg
(44%). Characteristic 1H NMR shifts: COSY, NOESY (500 MHz,
CDCl3) δ 4.72 (H-3), 1.73 (HR-2), 1.65 (Hꢀ-2), 1.11 (H-1), 0.94 (H-5),
0.78 (H3-19), 0.73 (H3-20), 0.43 (H3-21).
Compound 3b. Prepared from (-)-MTPA chloride. Yield: 1.0 mg
(44%). ESIMS: m/z 761 [M + H]+ (46), 783 [M + Na]+ (86), 799 [M
+ K]+ (88). Characteristic 1H NMR shifts: COSY, NOESY (500 MHz,
CDCl3): δ 4.69 (H-3), 1.69 (HR-2), 1.55 (Hꢀ-2), 1.20 (H-5), 1.10 (H-
1), 0.88 (H3-19), 0.75 (H3-20), 0.42 (H3-21).
Dasyscyphin D (1): Colorless crystals (5.5 mg); mp 105-106 °C;
[R]25 +2.92 (c 0.49, CHCl3); UV (MeOH) λmax (log ε) 205 (4.53),
D
Acknowledgment. We thank Dr. S. Franke (University of Hamburg)
for the HREIMS data, Dr. D. Schollmeyer (University of Mainz) for
the X-ray crystallographic analysis of dasyscyphin D (1), and the
Kompetenzzentrum der integrierten Naturstoff-Forschung. The expert
technical assistance of A. Spohn (IBWF) is gratefully acknowledged.
275 (2.91) nm; IR (KBr) νmax 3436, 2934, 1628, 1586, 1473, 1388,
1221, 1106, 1031, 798, 558 cm-1; 1H NMR, COSY, NOESY (400 MHz,
CDCl3) δ 6.87 (1H, d, J ) 7.5 Hz, H-14), 6.62 (1H, d, J ) 7.5 Hz,
H-13), 4.53 (1H, s, OH-16), 3.26 (1H, dd, J ) 11.3, 4.6 Hz, H-3),
3.01 (1H, dd, J ) 16.6, 8.2 Hz, H-11a), 2.68 (1H, dt, J ) 13.6, 6.2
Hz, H-7a), 2.66 (1H, br d, J ) 16.6 Hz, H-11b), 2.20 (3H, s, H3-22),
1.78 (1H, mc, H-7b), 1.73 (1H, dd, J ) 13.2, 3.5 Hz, H-1a), 1.71-1.58
(3H, m, H-2a, H-6a, H-9), 1.54 (1H, ddd, J ) 12.8, 3.5, 1.5 Hz, H-2b),
1.42 (1H, dddd, J ) 13.5, 11.0, 8.8, 4.8 Hz, H-6b), 1.31 (1H, br s,
OH-3), 1.23 (3H, s, H3-18), 1.06 (1H, dd, J ) 13.2, 4.3 Hz, H-1b),
1.01 (3H, s, H3-19), 0.99 (1H, dd, J ) 11.0, 4.9 Hz, H-5), 0.78 (3H, s,
H3-20), 0.52 (3H, s, H3-21); 13C NMR, HSQC, HMBC (100.6 MHz,
CDCl3) δ 150.3 (C, C-16), 143.8 (C, C-12), 136.0 (C, C-17), 129.1
(CH, C-14), 120.8 (C, C-15), 116.5 (CH, C-13), 79.5 (CH, C-3) 62.2
(CH, C-9), 51.2 (CH, C-5), 47.6 (C, C-8), 39.6 (CH2, C-1), 39.2 (C,
C-4), 37.2 (C, C-10), 33.9 (CH2, C-7), 32.4 (CH2, C-11), 30.7 (CH3,
C-18), 28.3 (CH3, C-19), 27.3 (CH2, C-2), 19.5 (CH2, C-6), 15.9 (CH3,
C-21), 15.5 (CH3, C-20), 15.5 (CH3, C-22); APCIMS m/z pos. 311.2
[M - OH]+ (100) neg. 309.2 [M - H3O]- (10), 327.2 [M - H]- (100);
HREIMS m/z 328.2397 (calcd for C22H32O2, 328.2402).
Supporting Information Available: 1H, 13C, and 2D NMR spectra
of compounds 1 and 2 and CIF of 1. This material is available free of
charge via the Internet at http://pubs.acs.org.
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X-ray Crystallographic Data of 1 ·CH3OH. Colorless single
crystals of 1·CH3OH were grown by slow evaporation of a methanolic
solution of 1. Data were obtained at 193 K on a Turbo CAD4
diffractometer with graphite-monochromated Cu KR radiation. Formula
C22H32O2 ·CH3OH, crystal size 0.032 × 0.064 × 0.265 mm3, orthor-
hombic, space group P21212, a ) 16.491(2) Å, b ) 19.995(4) Å, c )
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CHCl3); UV (MeOH) λmax (log ε) ) 204 (4.59), 271 (2.99) nm; IR
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1
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) 7.5 Hz, H-14), 6.62 (1H, d, J ) 7.5 Hz, H-13), 4.50 (1H, s, OH-16),
3.73 (1H, br d, J ) 10.9 Hz, H-20a), 3.42 (1H, d, J ) 10.9 Hz, H-20b),
2.99 (1H, dd, J ) 16.5, 7.8 Hz, H-11a), 2.79 (1H, m, H-7a), 2.63 (1H,
d, J ) 16.5 Hz, H-11b), 2.20 (3H, s, H3-22), 1.83 (1H, ddd, J ) 13.7,
4.7, 3.1 Hz, H-3a), 1.76-1.55 (4H, m, H-1a, H-6a, H-7b, H-9), 1.48
(1H, m, H-2a), 1.41 (1H, m, H-2b), 1.34 (1H, m, H-6b), 1.23 (3H, s,
H3-18), 1.15 (1H, dd, J ) 11.6, 3.7 Hz, H-5), 1.04 (1H, br s, OH-20),
0.99 (3H, s, H3-19), 0.95 (1H, m, H-3b), 0.93 (1H, m, H-1b), 0.45
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NP800355A