New glycosphingolipids from the fungus Catathelasma ventricosa

Article abstract of DOI:10.1021/np030153a

Three new glycosphingolipids with a cis-Δ¹⁷-fatty acyl moiety, namely, catacerebrosides A-C (1-3), along with two known glycosphingolipids, cerebrosides B and D, six known ergostane-type sterols, and tyrosamine were isolated from the fungus Catathelasma ventricosa. The structures of 1-3 were elucidated on the basis of spectroscopic analysis and chemical methods.

Full text of DOI:10.1021/np030153a

J . Nat. Prod. 2003, 66, 1013-1016
1013
New Glycosp h in golip id s fr om th e F u n gu s Ca ta th ela sm a ven tr icosa
Zha-J un Zhan and J ian-Min Yue*
State Key Laboratory of Drug Research, Institute of Materia Medica, Shanghai Institute for Biological Sciences,
Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park,
Shanghai, 201203, People’s Republic of China
Received April 7, 2003
1
7
Three new glycosphingolipids with a cis-∆ -fatty acyl moiety, namely, catacerebrosides A-C (1-3), along
with two known glycosphingolipids, cerebrosides B and D, six known ergostane-type sterols, and
tyrosamine were isolated from the fungus Catathelasma ventricosa. The structures of 1-3 were elucidated
on the basis of spectroscopic analysis and chemical methods.
Glycosphingolipids and their breakdown products are
important components in the cell membranes of animals
and plants and are emerging as important second mes-
sengers for various cellular processes, such as cell cycle
arrest, differentiation, senescence, apoptosis, and others.1
A series of new glycosphingolipids and sphingolipids has
been isolated recently from natural sources.5-8 Glyco-
sphingolipids and sphingolipids possess a variety of biologi-
Catacerebroside A (1), a white amorphous solid, showed
a protonated molecular ion at m/z 838.6887 [M + H] (calcd
+
for C H NO , 838.6772) in the positive HRESIMS, cor-
4
9
92
9
responding to the molecular formula C H NO . An IR
4
9
91
9
-4
-1
absorption band at 3394 cm indicated the existence of
hydroxyl and amide groups. The typical IR absorptions at
-1
1647 and 1539 cm suggested compound 1 was a second-
ary amide derivative, which was supported by the presence
1
,2
7
cal activities, e.g., antihepatotoxic activity, ionophoric
of a nitrogen-attached carbon signal at δ 55.1 and a
activity for Ca2 ions,9 immunomodulating activity,
+
10,11
13
carbonyl signal at δ 177.7 in the C NMR spectrum (Table
inhibition of the proliferation of lymphocytes stimulated
with concanavalin A,12 and cyclooxygenase-2 inhibition.13
The fungus Catathelasma ventricosa (PK.) Sing. (Tri-
cholomataceae), mainly distributed in the southwestern
region of mainland China, has not been investigated
chemically before. In the current study, three new gly-
1
1
). In the H NMR spectrum (Table 1), one olefinic proton
signal at δ 5.33 (1H, m, H-8), assignable to a trisubstituted
double bond, and four olefinic proton signals at δ 5.68 (1H,
dd, J ) 15.3, 7.4 Hz, H-4), 5.93 (1H, dt, J ) 15.3, 5.8 Hz,
H-5), and 5.53 (2H, m, H-17′ and H-18′), attributable to
the presence of two disubstituted double bonds, were
1
7
cosphingolipids with a unique cis-∆ -fatty acyl moiety,
namely, catacerebrosides A-C (1-3), together with cere-
broside B, cerebroside D, six sterols, and tyrosamine were
isolated from this fungus. The structures of the new
glycosphingolipids (1-3) were elucidated on the basis of
spectroscopic analysis and chemical methods.
1
evident. The H NMR spectrum also showed the presence
of three methyl groups at δ 1.79 (3H, s, H-19) and 1.07
1
(
6H, t, J ) 7.2 Hz, H-18 and H-24′). Comparison of the H
1
3
14
and C NMR data of 1 with those of cerebrosides A-D
indicated that compound 1 is most likely a glycosphingo-
lipid analogue. The amino alcohol moiety could be identified
as a sphingosine unit by the characteristic signals that
1
13
appeared in the H NMR and C NMR spectra, especially
4
8
due to the presence of typical ∆ and ∆ double bonds and
a Me-19 group. A glucopyranosyl moiety could be distin-
1
13
guished in the structure of 1 on the basis of H and
C
NMR spectra. Methanolysis of compound 1 afforded 2-hy-
droxytetracos-17-enoic acid methyl ester, which was identi-
fied by EIMS and ozonolysis. The double bond was located
between C-17′ and C-18′ by ozonolysis of 2-hydroxytetracos-
1
7
7-enoic acid methyl ester to produce heptaldehyde, C H14O,
which was identified by GC-EIMS and co-injection with an
authentic sample.
The linkages of the three parts of the molecule of 1 were
resolved from the HMBC spectrum. The carbon signal at
δ 177.7 (C-1′) correlated with the proton signals at δ 4.19
(
H-2) and 4.18 (H-2′). The proton signal at δ 4.19 (H-2) gave
cross-peaks with carbon signals at δ 73.4 (C-3) and 70.3
C-1), and the latter also correlated with the proton signal
at δ 4.46 (H-1′′). The carbon signal at δ 73.4 (C-3) showed
cross-peaks with the proton signals at δ 5.68 (H-4) and 5.93
(
The ethanolic crude extract of the whole bodies of the
fungus was partitioned with ethyl acetate and water to
obtain an ethyl acetate-soluble fraction, which was then
subjected to extensive column chromatography to afford
three new compounds, catacerebrosides A (55 mg), B (41
mg), and C (62 mg), as well as nine known compounds.
(H-5). In turn, the proton signal at δ 5.93 (H-5) showed
correlations with two methylene carbons at δ 34.9 (C-6)
and 29.2 (C-7), while the proton signal at δ 5.33 (H-8)
correlated with the carbon signals at δ 137.3 (C-9), 41.3
(C-10), 34.9 (C-6), 29.2 (C-7), and 16.7 (C-19). The planar
*
To whom correspondence should be addressed. Tel: 86-21-50806600.
Fax: 86-21-50807088. E-mail: jmyue@mail.shcnc.ac.cn.
structure of 1 was thus formulated. The assignments (Table
1
0.1021/np030153a CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/08/2003

1
014 J ournal of Natural Products, 2003, Vol. 66, No. 7
Notes
Ta ble 1. 1H and 13C NMR Data of Compound 1a
position
1
δH
δC
position
δH
δC
4.30 (1H, dd, 10.5, 3.5)
70.3
3′
1.70, 1.80 (each 1H, m)
36.4
3
.90 (1H, dd, 10.5, 7.0)
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
4.19 (1H, m)
55.1
73.4
131.6
135.1
34.9
4′
5′-15′
16′
17′, 18′
19′
20′-21′
22′
23′
24′
1′′
2′′
3′′
4′′
5′′
6′′
1.52 (2H, m)
1.40-1.50
26.7
30.8-31.4
28.5
131.4
28.5
30.8-31.4
33.6
24.2
15.0
105.2
75.5
78.5
72.0
78.5
4.31(1H, t, 6.9)
5.68 (1H, dd, 15.3, 7.4)
5.93 (1H, dt, 15.3, 5.8)
2.25 (2H, m)
2.23 (2H, m)
5.33 (1H, m)
2.24 (2H, m)
5.53 (2H, m)
2.24 (2H, m)
1.40-1.50
29.2
125.3
137.3
41.3
29.5
30.8-31.4
33.6
24.2
15.0
16.7
1.40-1.50
1.40-1.50
0
1
2.17 (2H, t, 7.6)
1.48 (2H, m)
1.40-1.55
1.40-1.55
1.40-1.55
1.07 (3H, t, 7.2)
1.79 (3H, s)
1.07 (3H, t, 7.2)
4.46 (1H, d, 8.0)
3.38 (1H, t, 7.8)
3.49 (1H,m)
3.50 (1H, m)
3.51 (1H, m)
4.06 (1H, d, 11.7)
2-15
6
7
8
9
63.2
3
.86 (1H, dd, 11.7, 5.2)
1
2
′
′
177.7
73.6
4.18 (1H, m)
a
Spectra were measured in CD3OD; J values are in Hz.
1
13
1
) of the H NMR and C NMR data of 1 were achieved
chain aliphatic chains, one of which possesses a double
bond. Except for the oxygenated carbon signals of the sugar
moiety, there were three other oxygenated methine carbon
1
1
unambiguously from the H- H COSY, HMQC, and HMBC
spectra.
The large vicinal coupling constants of H-4 and H-5
signals (at δ 76.0, 72.7, 72.5) and one secondary carbon
4
13
(
J ) 15.3 Hz) clearly indicated an E-geometry of the ∆
signal (δ 70.6) attached to oxygen appearing in the
C
1
4
8
double bond in 1. The ∆ double bond was also assigned
as E on the basis of the upfield shifted carbon signal of
Me-19.15 The cis-configuration of the ∆ double bond was
NMR spectrum. Accordingly, compound 2 was seen to be
also a glycosphingolipid analogue. Methanolysis of 2 gener-
ated 2-hydroxytetracos-17-enoic acid methyl ester, which
was identical with the methanolysis product found in
compound 1. Apart from this fatty acid moiety and the
sugar unit, the remaining part of the molecule 2 was a
moiety of 1,3,4-trihydroxy-2-aminooctadecane. The con-
nectivity of the three moieties was determined from the
HMBC spectrum, in which the carbonyl signal at δ 175.8
correlated with proton signals at δ 8.55 (NH), 5.26 (H-2),
and 4.60 (H-2′), and the H-1′′ proton signal correlated with
C-1 at δ 70.6. Both the H-1a and H-1b signals at δ 4.50
and 4.75, respectively, showed cross-peaks with C-1′′ at δ
105.7. The full assignments (Experimental Section) of the
17′
evident from the significantly upfield shifted carbon signals
of C-16′ (28.5) and C-19′ (28.5)1
3,15
and the relatively small
coupling constants of H-17′ and H-18′ (W/2 ≈ 3.7 Hz). The
1
3
carbon signals of the sugar moiety in the C NMR
spectrum suggested a â-configuration, which was confirmed
by the large coupling constant of H-1′′ at δ 4.46 (1H, d,
J ) 8.0 Hz) and H-2′′ at δ 3.38 (1H, t, J ) 7.8 Hz). On the
basis of biogenetic reasoning, and the co-isolation from C.
ventricosa of cerebrosides B and D, which have the same
sugar with a D-configuration, the sugar moiety of 1 was
assigned as â-D-glucopyranosyl. By considering biogenetic2
and steric factors, the chemical shift of H-2 and the
chemical shifts of the carbon signals of C-1 to C-3, C-1′,
and C-2′ of glucosphingolipids and sphingolipids may be
utilized to determine their absolute stereochemistry.8,17,18
The proton signal at δ 4.19 (H-2) and the carbon signals
at δ 70.3 (C-1), 55.1 (C-2), 73.6 (C-3), 177.7 (C-1′), and 73.4
1
13
H NMR and C NMR data of 2 were determined from
1
1
the H- H COSY, HMQC, and HMBC spectra.
The ∆1 double bond of 2 was determined to be Z by the
7′
upfield shifted carbon chemical shifts of C-16′ (27.7) and
C-19′ (27.7)1
3,15
and the relatively small coupling constant
of H-17′ and H-18′ (W/2 ≈ 5.3 Hz). The proton signal at δ
5.26 (H-2) and the carbon signals at δ 70.6 (C-1), 51.9 (C-
2), 76.0 (C-3), 72.5 (C-4), 175.8 (C-1′), and 72.7 (C-2′) of 2
were very close to those of model glycosphingolipids, such
as aralia cerebroside8 and pokeweed cerebrosides with
2S,3S,4R-stereochemistry. The structure of 2 was thus
determined as 1-O-â-D-glucopyranosyl-(2S,3S,4R)-2-[(2′R,-
17′Z)-2′-hydroxy-17-tetracosenoylamino]octadecane-1,3,4-
triol.
(
C-2′) of 1 were in good agreement with those reported for
glycosphingonines (as model structures) with a 2S,3R,2′R
configuration.7
fore as 1-O-â-D-glucopyranosyl-(2S,3R,4E,8E)-2-[(2′R,17′Z)-
′-hydroxy-17′-tetracosenoylamino]-9-methyl-4, 8-octadec-
,14,16
The structure of 1 was assigned there-
13
2
adiene-1,3-diol.
Catacerebroside B(2) showed the molecular formula
C
8
48
H
93NO10, as deduced from the positive HRESIMS at m/z
+
44.6862 [M + H] (calcd for C48
IR absorption band at 3405 cm indicated the presence of
hydroxyl and amide groups. The typical IR absorptions at
H
94NO10, 844.6878). The
Catacerebroside C (3) was obtained as a white amor-
phous powder. The positive HRESIMS revealed a pro-
-
1
+
tonated molecular formula of C48
H
94NO11 [M + H] (m/z
-
1
860.6841, calcd 860.6827). The IR and H and 13C NMR
spectra (Experimental Section) of compound 3 were very
similar to those of 2 except for the presence of one more
hydroxyl group, and suggested compound 3 was an ana-
logue of 2. Methanolysis of compound 3 afforded 2,3-
dihydroxytetracos-17-enoic acid methyl ester, which was
1
1
643 and 1535 cm suggested compound 2 was also a
secondary amide, as supported by the presence of a
nitrogen-attached carbon signal at δ 51.9 and a carbonyl
signal at δ 175.8 in the 13C NMR spectrum. The presence
of a â-glucopyranosyl moiety and a double bond was evident
1
13
from the H and C NMR spectra (Experimental Section).
The 1H NMR data showed the presence of two olefinic
proton signals at δ 5.50 (2H, m), multiple proton signals
between δ 1.26-1.38, and a triplet proton signal at δ 0.93
1
identified by EIMS (m/z 412) and H NMR and by analysis
of its ozonolysis products on GC-EIMS. A 1,3,4-trihydroxy-
2-aminooctadecane moiety was identified by spectral analy-
sis in a manner analogous to that for compound 2. The
(
6H, t, J ) 7.6 Hz), suggesting the presence of two long-

Notes
J ournal of Natural Products, 2003, Vol. 66, No. 7 1015
HMBC spectrum showed cross-peaks for the carbonyl
signal at δ 174.3 with the proton signals at δ 8.66 (NH),
3â,5R,6â-triol (190 mg). Fraction 5 (2.5 g) was purified on a
3
silica gel column eluted with CHCl -MeOH-HCOOH (10:1:
0
.1) to afford 22E,24R-ergosta-7,22-dien-3-O-â-D-glucopyrano-
5
.28 (H-2), 4.70 (H-2′), and 4.51 (H-3′). In turn, the H-2
side (21 mg) and tyrosamine (11 mg). Fraction 6 (2.1 g) was
subjected to a column containing MCI gel CHP 20P eluted with
signal at δ 5.28 (H-2) correlated with the carbon signals
at δ 70.3 (C-1), 75.8 (C-3), and 174.3 (C-1′), and the H-1′′
proton signal at δ 4.97 correlated with C-1 at δ 70.3.
The ∆1 double bond in the fatty acid moiety was
determined to be Z, and the stereochemistry of phytosphin-
gosine unit in 3 was assigned as 2S,3S,4R for the same
reasons as in 2. One more hydroxyl was present in the fatty
acid moiety of compound 3, and by comparison with the
fatty acid moiety of compound 2, the chemical shifts of C-1′
and C-2′ were affected. Because there are no suitable
models of glycosphingolipids or sphingolipids with a 2′,3′-
dihydroxy fatty acyl moiety, the absolute configurations of
C-2′ and C-3′ could not readily be established from the
available data. The structure of compound 3 was thus
assigned as 1-O-â-D-glucopyranosyl-(2S,3S,4R)-2-[(17′Z),2′,3′-
dihydroxy-17-tetracosenoylamino]octadecane-1,3,4-triol. This
is the first report of a glycosphingolipid with a 2,3-
dihydroxyl fatty acyl moiety.
9
0% methanol in water to afford 1 (55 mg), 2 (41 mg), 3 (62
mg), and a mixture of cerebrosides B and D (110 mg), which
was further applied to a reversed-phase C18 silica gel column,
eluted with 90% MeOH in water, to yield cerebroside B (41
mg) and cerebroside D (32 mg).
7′
2
0
Ca ta cer ebr osid e A (1): white amorphous powder; [R]
D
-
5.0° (c 1.1, MeOH); IR (KBr) νmax 3394 (OH), 2922, 1647, 1539
-
1
1
13
(
amide), 1467, 1082 (C-O) cm ; H and C NMR (see Table
+
1
8
); positive ESIMS m/z 860 [M + Na] ; positive HRESIMS m/z
+
38.6887 [M + H] (calcd for C49
9
H92NO , 838.6772).
Meth a n olysis of Ca ta cer ebr osid e A (1). An aliquot (10.3
mg) of 1 was dissolved in 5 mL of methanol containing 5%
HCl and refluxed for 18 h. The reaction mixture was neutral-
ized with NaHCO
aqueous solution was extracted with n-hexane three times, and
the organic phase was dried with anhydrous Na SO . After
3
and diluted with 10 mL of water. The
2
4
removal of solvent, 3.8 mg of 2-hydroxytetracos-17-enoic acid
+
methyl ester was afforded: EIMS m/z 396 [M] (48), 365 (31),
+
1
337 [M - COOCH
5 (100).
Ozon olysis of 2-Hyd r oxytetr a cos-17-en oic Acid Meth yl
3
]
(55), 111 (52), 97 (88), 83 (95), 69 (92),
Cerebrosides B and D were identified on the basis of H
5
and 13C NMR spectra and ESIMS. Tyrosamine, 22E,-
14
19
2
7
4R-ergosta-7,22-diene-3â,5R,6â,9R-tetraol, 22E,24R-ergosta-
Ester . Ozone was passed into a stirred solution of 2-hydroxy-
tetracos-17-enoic acid methyl ester (3 mg) in 5 mL of anhy-
drous MeOH-CH Cl (1:1) at -78 °C, and the ozonolysis was
2
0
,22-diene-3â,5R,6â,-triol, ergosterol peroxide, 22E,24R-
ergosta-7,22-dien-3â-ol, 22E,24R-ergosta-7,22-dien-3-O-â-
D-glucopyranoside, and ergosta-4,6,8(14),22-tetraen-3-
one2
2
2
2
1
contained until the solution became blue. The solution was
purged with nitrogen and allowed to come to room tempera-
2,23
1
were identified on the basis of H NMR spectral
ture. Heptaldehyde, C
identified by GC-MS at m/z 114 (C
co-injection with an authentic sample.
Ca ta cer ebr osid e B (2): white amorphous powder; [R]
+5.0° (c 3.0, C H N); IR (KBr) ν 3405 (OH), 2922, 1643, 1535
7
H
14O, produced by ozonolysis was
data and comparison with authentic samples (co-TLC).
7
H
14O, t 6.23 min) and by
R
Exp er im en ta l Section
2
0
D
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were determined on a Perkin-Elmer 341 polarimeter at room
temperature. UV spectra were measured on a Shimadzu UV-
5
5
max
-
1 1
(amide), 1467, 1080 (C-O) cm ; H NMR (C D N, 400 MHz)
5
5
δ 8.55 (1H, d, J ) 9.1 Hz, NH), 5.50 (each 1H, m, H-17′, H-18′),
5.26 (1H, m, H-2), 4.94 (1H, d, J ) 7.7 Hz, H-1′′), 4.75 (1H,
dd, J ) 10.7, 6.6 Hz, H-1a), 4.60 (1H, m, H-2′), 4.52 (1H, d,
J ) 11.6 Hz, H-6a′′), 4.50 (1H, dd, J ) 10.7, 4.5 Hz, H-1b),
2
10A spectrometer. IR spectra were recorded on a Perkin-
Elmer 577 spectrometer using KBr disks. NMR spectra were
measured on a Bruker AM-400 spectrometer with TMS as
internal standard. EIMS (70 eV) was carried out on a Finni-
4
.34 (1H, m, H-3), 4.32 (1H, dd, J ) 11.6, 5.5 Hz, H-6b′′), 4.22
1H, m, H-4), 4.20 (1H, m, H-4′′), 4.19 (1H, m, H-3′′), 4.01 (1H,
t, J ) 8.0 Hz, H-2′′), 3.87 (1H, m, H-5′′), 2.11 (1H, m, H-5a),
.10 (1H, m, H-3a′), 2.07 (each 2H, m, H-16′, H-19′), 2.00 (1H,
(
gan-MAT 95 mass spectrometer, and ESIMS was recorded on
DECA
a Finnigan LCQ
mass spectrometer. HRESIMS were
2
measured on a Thermo Electron Corporation FT-mass spec-
trometer. All solvents used were of analytical grade (Shanghai
Chemical Plant, Shanghai, People’s Republic of China). Silica
gel (200-300 mesh) was used for column chromatography, and
precoated silica gel GF254 plates (Qingdao Marine Chemical
Plant, Qingdao, People’s Republic of China) were used for TLC.
m, H-3b′), 1.97 (1H, m, H-5b), 1.85 (2H, m, H-4′), 1.80 (2H, m,
H-6), 1.26-1.38 (52H, m, H-7-H-17, H-5′-H-15′, H-20′-H-
1
3
2
3′), 0.93 (each 3H, t, J ) 7.6 Hz, H-18, H-24′); C NMR
(C
5
D
5
N, 100 MHz) δ 175.8 (C-1′), 130.4 (C-17′, C-18′), 105.7
C-1′′), 78.6 (C-3′′, C-5′′), 76.0 (C-3), 75.3 (C-2′′), 72.7 (C-2′),
2.5 (C-4), 71.6 (C-4′′), 70.6 (C-1), 62.8 (C-6′′), 51.9 (C-2), 35.7
C-3′), 34.1 (C-5), 32.2 (C-16, C-22′), 29.5-30.5 (C-7-C-15,
C-5′-C-15′, C-20′-C-21′), 27.7 (C-16′, C-19′), 26.7 (C-4′), 26.5
C-6), 23.1 (C-17, C-23′), 14.5 (C-18, C-24′); negative ESIMS
(
7
(
C
18 reversed-phased silica gel (150-200 mesh, Merck) and MCI
gel (CHP20P, 75-150 µm, Mitsubishi Chemical Industries
Ltd.) were also used for column chromatography.
(
-
+
F u n ga l Ma ter ia l. Catathelasma ventricosa was collected
from the Kunming area of Yunnan Province, the People’s
Republic of China, in late J uly of 1996, and authenticated by
Professor Mu Zang of Kunming Institute of Botany, where a
voucher specimen (HKAS30227) was deposited.
Extr a ction a n d Isola tion . A 7.5 kg quantity of the minced
fresh mushroom bodies was extracted with 95% ethanol
extensively at room temperature to give a dark crude extract
m/z 842 [M - H] ; positive HRESIMS m/z 844.6862 [M + H]
(calcd for C H NO , 844.6878).
4
8
94
10
Meth a n olysis of 2. Methanolysis of 2 (5.5 mg) by the
procedure described for compound 1 also afforded 2-hydroxy-
tetracos-17-enoic acid methyl ester (1.6 mg).
2
0
Ca ta cer ebr osid e C (3): white amorphous powder; [R]
+3.0° (c 1.0, C N); IR (KBr) νmax 3363 (OH), 2920, 1628, 1541
5
(amide), 1467, 1078 (C-O) cm ; H NMR (C D N, 400 MHz)
D
5
H
5
-
1 1
5
(304 g), which was dissolved in water (3 L) to form a
δ 8.66 (1H, d, J ) 9.2 Hz, NH), 5.51 (each 1H, m, H-17′, H-18′),
5.28 (1H, m, H-2), 4.97 (1H, d, J ) 7.7 Hz, H-1′′), 4.70 (1H,
dd, J ) 10.6 Hz, 6.0 Hz, H-1a), 4.70 (1H, m, H-2′), 4.52 (1H, d,
J ) 11.7 Hz, H-6a′′), 4.51 (1H, m, H-3′), 4.50 (1H, dd, J ) 10.6,
4.6 Hz, H-1b), 4.31 (1H, dd, J ) 11.7, 5.4 Hz, H-6b′′), 4.29 (1H,
m, H-3), 4.23 (1H, m, H-4), 4.19 (1H, m, H-4′′), 4.17 (1H, m,
H-3′′), 3.98 (1H, m, H-2′′), 3.84 (1H, m, H-5′′), 2.23 (1H, m,
H-5a), 2.13 (each 2H, m, H-4′, H-16′, H-19′), 1.93 (1H, m, H-5b),
1.85 (each 2H, m, H-6, H-5′), 1.25-1.45 (50H, m, H-7-H-17,
H-6′-H-15′, H-20′-H-23′), 0.87 (each 3H, t, J ) 6.8 Hz, H-18,
suspension and then partitioned with ethyl acetate to afford
an ethyl acetate-soluble fraction E (58 g). This fraction was
subjected to column chromatography eluted with petroleum
ether containing increasing amounts of acetone to afford
fractions 1-6. Fraction 2 (25.2 g) was recrystallized from
CHCl to yield 22E,24R-ergosta-7,22-dien-3â-ol (20.5 g). Frac-
3
tion 3 (6.1 g) was applied to a silica gel column eluted with
petroleum ether-ethyl acetate (8:1) to give ergosterol peroxide
(125 mg) and ergosta-4,6,8(14),22-tetraen-3-one (78 mg). Frac-
tion 4 (5.5 g) was separated on a silica gel column eluted with
CHCl -MeOH (15:1) to yield 22E,24R-ergosta-7,22-diene-
â,5R,6â,9R-tetraol (32 mg) and 22E,24R-ergosta-7,22-diene-
H-24′); 13C NMR (C
5 5
D N, 100 MHz) δ 174.3 (C-1′), 130.3 (C-
3
17′, C-18′), 105.5 (C-1′′), 78.5 (C-3′′), 78.4 (C-5′′), 76.2 (C-2′),
75.8 (C-3), 75.1 (C-2′′), 73.6 (C-3′), 72.6 (C-4), 71.5 (C-4′′), 70.3
3

1
016 J ournal of Natural Products, 2003, Vol. 66, No. 7
Notes
(
C-1), 62.7 (C-6′′), 51.8 (C-2), 34.3 (C-5), 32.7 (C-4′), 32.1 (C-
(6) Noda, N.; Tanaka, R.; Miyahara, K.; Sukamoto, T. Chem. Pharm. Bull.
1
996, 44, 895-899.
1
2
1
6, C-22′), 29.6-30.5 (C-7-C-15, C-6′-C-15′, C-20′-C-21′),
7.6 (C-16′, C-19′), 26.6 (C-6, C-5′), 23.0 (C-17, C-23′), 14.3 (C-
(
(
(
7) J ung, J . H.; Lee, C. O.; Kim, Y. C.; Kang, S. S. J . Nat. Prod. 1996,
5
9, 319-322.
+
8, C-24′); positive ESIMS m/z 882 [M + Na] ; positive
8) Kang, S. S.; Kim, J . S.; Xu, Y. N.; Kim, Y. H. J . Nat. Prod. 1999, 62,
1059-1060.
+
HRESIMS m/z 860.6841 [M + H] (calcd for C48
H
94NO11
,
9) Shibuya, H.; Hawasshima, K.; Sakagami, M.; Kawanishi, H.; Shi-
momura, M.; Ohashi, K.; Kitagawa, I. Chem. Pharm. Bull. 1990, 38,
8
60.6827).
Meth a n olysis of 3. Methanolysis of 3 (20.5 mg) by the
procedure described for compound 1 afforded 2,3-dihydroxy-
2
933-2938.
(
10) Natori, T.; Koezuka, Y.; Higa, T. Tetrahedron Lett. 1993, 34, 5591-
1
tetracos-17-enoic acid methyl ester (6.6 mg): H NMR (CDCl
δ 5.34 (2H, t-like, J ) 4.8 Hz, H-17, H-18), 4.23 (1H, d, J )
.5 Hz, H-2), 3.81 (1H, m, H-3), 3.79 (3H, s, -OCH ), 2.00 (4H,
3
)
5592.
(11) Natori, T.; Morita, M.; Akimoto, K.; Koezuka, Y. Tetrahedron 1994,
5
0, 2771-2784.
3
3
(
12) Costantino, V.; Fattorusso, E.; Mangoni, A.; Di Rosa, M.; Ianaro, A.
J . Am. Chem. Soc. 1997, 119, 12465-12470.
m, H-16, H-19), 1.25 (long chain), 0.85 (3H, t, J ) 7.0, H-24);
+
+
+
EIMS m/z 412 [M] (14), 394 [M - H
2
O] (8), 376 [M - 2H
2
O]
(
13) Kang, S. S.; Kim, J . S.; Son, K. H.; Kim, H. P.; Chang, H. W. Chem.
Pharm. Bull. 2001, 49, 321-323.
+
(
2), 317 [M - 2H
2
O - COOCH
3
]
(13), 111 (10), 97 (17), 90
(100), 83 (20), 69 (24), 55 (25). Ozonolysis of 2,3-dihydroxytet-
(14) Sitrin, R. D.; Chan, G.; Dingerdissen, J .; Debrosse, C.; Mehta, R.;
Roberts, G.; Rottschaefer, S.; Staiger, D.; Valenta, J .; Snader, K. M.;
Stedman R. J .; Hoover, J . R. E. J . Antibiot. 1988, 41, 469-480.
racos-17-enoic acid methyl ester also yielded heptaldehyde,
14O, identified by GC-MS at m/z 114 (C 6.23 min)
and by co-injection with an authentic sample.
C
7
H
7 R
H14O, t
(
(
(
(
(
15) Stothers, J . B.Carbon-13 NMR Spectroscopy; Academic Press: New
York, 1972.
16) Chen, X. S.; Wu, Y. L.; Chen, D. H. Tetrahedron Lett. 2002, 43, 3529-
Ack n ow led gm en t. Financial support by the National
3
532.
Scientific Foundation of the People’s Republic of China
(
People’s Republic of China (2002CB512807) is gratefully
acknowledged. We thank Prof. Mu Zang of Kunming Institute
of Botany for the identification of the fungus.
17) Sugiyama, S.; Honda, M.; Komori, T. Liebigs Ann. Chem. 1990, 1069-
30025044) and the Ministry of Science and Technology of the
1078.
18) Sugiyama, S.; Honda, M.; Higuchi, R.; Komori, T. Liebigs Ann. Chem.
1
991, 349-356.
19) Aldrich Library of 13C and 1H FT NMR Spectra, 1992; Vol. 2, p 612B.
(20) Yue, J . M.; Chen, S. N.; Lin, Z. W.; Sun, H. D. Phytochemistry 2001,
6, 801-806.
21) Takaishi, Y.; Ohasih, T.; Tomimatsu, T. Phytochemistry 1989, 28,
45-947.
22) Tanaka, N.; Hosoi, K.; Tanaka, D.; Takahashi, M. Chem. Pharm. Bull.
5
Refer en ces a n d Notes
(
(
(
9
(
(
(
(
1) Hannun, Y. A.; Bell, R. M. Science 1989, 243, 500-507.
2) Kolter, T.; Sandhoff, K. Angew. Chem., Int. Ed. 1999, 38, 1532-1568.
3) Cremeti, E. A.; Fischl, S. A. Lipids 2000, 35, 937-945.
1
996, 44, 843-846.
23) Gonzalez, G. A.; Barrera, B. J .; Marante, J . T. F. Phytochemistry 1983,
22, 1049-1050.
4) Harouse, J . M.; Bhat, S.; Spitalnik, S. L. Science 1991, 253, 320-
3
23.
(
5) Lourenco, A.; Lobo, A. M.; Rodriguez, B.; J imeno, M. L. Phytochem-
NP030153A
istry 1996, 43, 617-619.