Renierosides, cerebrosides from a marine sponge Haliclona (Reniera) sp

Article abstract of DOI:10.1021/np070078u

Guided by the brine shrimp lethality assay, eight new cerebrosides (1-8) have been isolated from an extract of the marine sponge Haliclona (Reniera) sp. A novel feature of these cerebrosides was the presence of unprecedented amide-linked long-chain fatty acid moieties. The planar structures of the cerebrosides (1-8) were established by 1D and 2D NMR spectroscopic techniques, mass spectrometric analyses, and chemical degradation methods. The isolated compounds did not display cytotoxicity to a panel of five human solid tumor cell lines.

Full text of DOI:10.1021/np070078u

J. Nat. Prod. 2007, 70, 1481–1486
1481
Notes
Renierosides, Cerebrosides from a Marine Sponge Haliclona (Reniera) sp.
†
,#
†
†
‡
§
⊥
|
Tayyab A. Mansoor, Pramod B. Shinde, Xuan Luo, Jongki Hong, Chong-O. Lee, Chung Ja Sim, Byeng Wha Son, and
Jee H. Jung*
,
†
College of Pharmacy, Pusan National UniVersity, Busan 609-735, Korea, College of Pharmacy, Kyung Hee UniVersity, Seoul 130-701, Korea,
Korea Research Institute of Chemical Technology, Daejon 305-343, Korea, Hannam UniVersity, Daejon 306-791, Korea, Basic Science
Research Institute, Pukyong National UniVersity, Busan 608-737, Korea, and Faculdade de Farmácia, UniVersidade de Lisboa, AV. D. Forças
Armadas, Lisboa 1600-083, Portugal
ReceiVed February 21, 2007
Guided by the brine shrimp lethality assay, eight new cerebrosides (1–8) have been isolated from an extract of the
marine sponge Haliclona (Reniera) sp. A novel feature of these cerebrosides was the presence of unprecedented
amide-linked long-chain fatty acid moieties. The planar structures of the cerebrosides (1–8) were established by
1
D and 2D NMR spectroscopic techniques, mass spectrometric analyses, and chemical degradation methods. The
isolated compounds did not display cytotoxicity to a panel of five human solid tumor cell lines.
Cerebrosides are glycosphingolipids, consisting of a ceramide
and a single sugar residue (glucose or galactose) at C-1. The
hydrophobic ceramide portion involves a sphingoid base and
an amide-linked fatty acyl chain. These amphipathic molecules
1
,2
have been reported to exhibit antitumor/cytotoxic, anti-HIV-
3
4
5
6
1
, neuritogenic, antihepatotoxic, immunosuppressive, immuno-
7 8 2
modulatory, cyclooxygenase-2 inhibitory, antifungal, antimi-
crobial, and antifouling activities.
9
10
Sponges of the genus Haliclona have been studied extensively
and afforded around 200 compounds belonging to different
classes. These compounds include the cytotoxic tertiary alkaloids
1
1
haliclonacyclamines, antifungal pentacyclic alkaloid papua-
1
2
13
mine, antitumor alkaloid manzamine, and anti-inflammatory
1
4
cyclic depsipeptides halipeptins, to name a few. A bioactivity-
guided chemical study of a marine sponge Haliclona (Reniera)
sp. collected from the Korean waters (see the Supporting
Information), has led to the isolation of eight new cerebrosides
(
1–8). The MeOH extract of the sponge, active in the brine
shrimp assay (LD50 126 µg/mL), was partitioned between CH Cl
and H O, and the CH Cl layer was further partitioned between
2
2
2
2
2
aqueous MeOH (LD50 27 µg/mL) and n-hexane (LD50 45 µg/
mL). The n-hexane layer was chromatographed on a normal-
phase column, followed by reverse-phase high-performance
liquid chromatography (RP-HPLC) to yield eight new cerebro-
sides (1–8). Herein, we describe the isolation, structure elucida-
tion, and biological evaluation of these new compounds (1–8).
anomeric proton at δH 4.26), an amide linkage (a nitrogenated
methine proton at δ 3.97 and a carbonyl carbon at δ 177.2), and
H
C
Renieroside A
molecular formula was assigned as C52
resolution fast-atom-bombardment mass spectrometry (HRFABMS;
1
(1) was isolated as a white amorphous solid. Its
a long chain (terminal methyl protons at δ
H
0.89 and methylene
H95NO
9
on the basis of high-
protons at δ
glycosphingolipid. In the C NMR spectrum, the carbon resonances
appeared at δ 62.7 (CH ), 71.6 (CH), 75.0 (CH), 77.9 (CH), 78.0
(CH), and 104.7 (CH), revealing the presence of a ꢀ-glucopyra-
H
1.24–1.38) were observed, indicating its nature as a
13
-
1
13
[
M - H] at m/z 876.6946, ∆ + 1.7 mmu) and H and C NMR
2
spectroscopic analyses. The characteristic signals of a sugar (an
1
5
noside. The coupling constant of the anomeric proton at δ 4.26
d, JHH ) 7.5 Hz; δ 104.7) further confirmed the ꢀ configuration
of the glucose unit (in the case of R-glucopyranoside: JHH ) 3.7
(
C
*
To whom correspondence should be addressed. Tel: 82-51-510-2803.
Fax: 82-51-513-6754. E-mail: jhjung@pusan.ac.kr.
†
16
Pusan National University.
Kyung Hee University.
Korea Research Institute of Chemical Technology.
Hannam University.
Pukyong National University.
Present address: Universidade de Lisboa.
C
Hz; δ 98.5). The absolute configuration of the glucose moiety
‡
1
was deduced to be D (Vide infra). The H NMR spectrum showed
two olefinic proton signals at δ 5.73 (1H, dt, J ) 15.0, 5.5 Hz,
H-5) and 5.48 (1H, dd, J ) 15.0, 7.5 Hz, H-4), attributed to the
§
⊥
|
#
4
1
typical ∆ double bond of a sphingosine. The large vicinal coupling
1
0.1021/np070078u CCC: $37.00

2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/12/2007

1
482 Journal of Natural Products, 2007, Vol. 70, No. 9
Notes
Figure 1. Key COSY and HMBC correlations of compounds 1
and 6.
constants of these olefinic protons (J ) 15.0 Hz) clearly indicated
9
an E geometry for the double bond. The correlation spectroscopy
Figure 2. Key FAB-CID tandem mass fragmentations of the [M
(
COSY) correlations between the H-2 signal (δ 3.97, 1H, m) and
both the H-3 (δ 4.12, 1H, m) and the H-1 (δ 4.13, 1H, m; 3.68,
H, m) signals defined the 2-amino-1,3-dioxygenated fragment.
+
+
Na] ions of the FAMEs derived from 1–3 and 6–8.
1
1
be essentially identical to those of 1, which confirmed that
renieroside A (2) was also a glycosphingolipid and differed only
Furthermore, in the H NMR spectrum, the characteristic signals
for a conjugated diene were observed at δ 5.99 (2H, m, H-9, H-10),
2
1
13
in the length of the lipid base or the lipid amide units. The difference
5
.56 (1H, m, H-8), and 5.54 (1H, m, H-11). The C NMR
was in the fatty acid portion because methyl (2R,17Z)-2-hydroxy-
chemical shifts of allylic carbons C-7 and C-12 at δ 33.0 indicated
1
hexacos-17-enoic acid ([R]23 –4, MeOH) was obtained by the
the E geometry of the diene moiety. The key heteronuclear
D
methanolysis of 2. The location of the double bond was determined
by FAB-CID-MS/MS analysis of the [M + Na]+ ion at m/z 447.
The enhanced peaks of allylic cleavages at m/z 347 and 293
indicated the location of the double bond at C-17 (Figure 2). This
unsaturated fatty acid has been reported in chill-resistant rye, oats,
multiple-bond correlation (HMBC) correlations from H-7 to C-5,
C-6, and C-9 and from H-6 to C-7, and COSY correlations from
H-6 to H-8, were useful to locate the position of double bonds in
the sphingosine base (Figure 1). In addition, the HMBC spectrum
also revealed the correlation from H-1 at δ 4.13 (1H, m) and 3.68
1
9
20
and wheat, and in the pig brain. In reports from the terrestrial
sources, however, the stereochemistry of the hydroxyl group at C-2
of the fatty acid has not been defined. The methanolysis of 2 also
yielded an R- and ꢀ-glucopyranosyl mixture. The optical rotation
(
1H, m) to the anomeric carbon at δ 104.7. The chemical shifts of
C-2 at δ 54.6 and C-3 at δ 73.1 supported the D-erythro
stereochemistry at C-2 and C-3, which was in agreement with those
of synthetic D-erythro-ceramide (δ 55.2, 73.6) and D-glucosyl-D-
17
of this mixture ([R]26 +70.8, MeOH) identified glucose as the D
erythro-ceramide (δ 53.8, 72.6). In case of threo isomers of these
model compounds, C-2/C-3 were reported to appear at δ 55.2/71.2
D
isomer. On the basis of these data, the structure of renieroside A
2
1
7
(2) was established as 1-O-ꢀ-D-glucopyranosyl-(2S,3R,4E,8E,10E)-
-[(2′R,17′Z)-2′-hydroxyhexacos-17′-enoylamino]-octadeca-4,8,10-
and δ 53.7/70.7, respectively. Methanolysis of 1 yielded fatty
2
acid methyl ester (FAME), and its molecular formula was estab-
+
+
triene-1,3-diol.
56 3
lished as C29H O ([M + H] at m/z 453, [M + Na] at m/z 475).
The location of the double bond was determined by collision-
induced-dissociation fast-atom-bombardment tandem mass spec-
3
The molecular formula of renieroside A (3) was found to be
49 9
C H89NO on the basis of high-resolution FAB mass spectroscopy
+
+
1
13
trometry (FAB-CID-MS/MS) analysis of the [M + Na] ion at m/z
([M + Na] at m/z 858.6436, ∆ +0.1 mmu) and H and C NMR
1
13
4
4
75. Allylic cleavages were observed as enhanced peaks at m/z
03 and 349, indicating the location of the double bond at C-21′
spectroscopic results. Again, the H and C NMR spectroscopic
data were found to be nearly identical to those of renierosides A
and A . The methanolysis of 3 gave rise to a different FAME. Its
1
2
1′
(
Figure 2). The geometry of ∆ was assigned as Z on the basis of
2
chemical shifts of allylic carbons C-20′ and C-23′, which appeared
molecular formula was established as C26
H
50
O
3
on the basis of
6
22
+
at δ 27.2. The optical rotation of the ester ([R]
D
–4, MeOH)
FABMS data ([M + Na] at m/z 433). Together with the optical
1
23
identified it as the R isomer. On the basis of these data, the fatty
acid moiety was deduced to be (2R,21Z)-2-hydroxyoctacos-21-enoic
acid. To the best of our knowledge, this appears to be the first report
on the isolation of 2-hydroxyoctacos-21-enoic acid from any natural
or synthetic source. However, (2R,21Z)-2-methoxyoctacos-21-enoic
acid has been reported as a naturally occurring R-methoxy fatty
rotation value ([R]
D
–1, MeOH), the fatty acid portion in
renieroside A was defined as a (2R,16Z)-2-hydroxypentacos-16-
3
enoic acid. The location of the double bond was determined by
+
FAB-CID-MS/MS analysis of the [M + Na] ion at m/z 433. Allylic
cleavages were observed as enhanced peaks at m/z 333 and 279,
indicating the location of the double bond at C-16 (Figure 2).
Similarly, as for the fatty acid of 2, it has also been reported in
1
8
acid from a marine sponge Higginsia tethyoides. On the basis of
the above mentioned data, the structure of renieroside A (1) was
defined as 1-O-ꢀ-D-glucopyranosyl-(2S,3R,4E,8E,10E)-2-[(2′R,21′Z)-
′-hydroxyoctacos-21′-enoylamino]-octadeca-4,8,10-triene-1,3-
diol.
The molecular formula of renieroside A
amorphous solid, was assigned as C50
19
20
chill-resistant rye, oats, and wheat, and in the pig brain, without
1
information about stereochemistry at C-2. All these data were in
agreement with the proposed structure of renieroside A (3) as 1-O-
3
ꢀ-D-glucopyranosyl-(2S,3R,4E,8E,10E)-2-[(2′R,16′Z)-2′-hydroxy-
pentacos-16′-enoylamino]-octadeca-4,8,10-triene-1,3-diol.
2
2
(2), isolated as a white
H
91NO
9
on the basis of high-
4 9
Renieroside A (4) showed a molecular formula of C46H85NO ,
+
resolution FAB mass spectroscopy ([M + Na] at m/z 872.6594,
as deduced from high-resolution FAB mass spectroscopy ([M +
1
13
+
1
13
∆
+0.2 mmu) and the results of H and C NMR spectroscopic
Na] at m/z 818.6123, ∆ +0.1 mmu) as well as H and C NMR
spectroscopic results. The acid hydrolysis of 4 gave rise to a
interpretations (Tables 1 and 2). The NMR results were found to

Journal of Natural Products, 2007, Vol. 70, No. 9 1483
1
a
13
b
c
Table 1. H and C NMR Data of Renieroside A
1
(1) (CD
3
OD)
position
δH
δC
position
δH
δC
lipid base unit
1
N-acyl unit
4.13 (m)
.68 (m)
3.97 (m)
4.12 (m)
5.48 (dd, J ) 15.0, 7.5 Hz)
5.73 (dt, J ) 15.0, 5.5 Hz)
2.14 (m)
2.09 (m)
5.56 (m)
5.99 (m)
5.99 (m)
5.54 (m)
2.06 (m)
1.38–1.24 (m)
1.38–1.24 (m)
0.89 (t, J ) 7.0 Hz)
69.8
1′
2′
3′
177.2
72.8
35.6
3
4.12 (dd, J ) 8.0, 4.8 Hz)
1.71 (m)
1.56 (m)
1.38–1.24 (m)
2.02 (q, J ) 6.0 Hz)
5.34 (m)
1.38–1.24 (m)
0.89 (t, J ) 7.0 Hz)
2
3
4
5
6
7
8
9
54.6
73.1
131.2
134.3
33.1
4′–19′ or 24′–26′
20′ or 23′
21′ or 22′
27′
29–33
27.2
131.1
22.9
33.0
131.7
132.3
132.3
133.5
33.0
29–33
22.9
14.4
28′
14.4
d
glucose unit
d
1
1
1
1
1
1
0
1
2
3–16
7
8
1′′
2′′
3′′
4′′
5′′
6′′
4.26 (d, J ) 7.5 Hz)
3.18 (m)
104.7
75.0
78.0
71.6
77.9
62.7
3.35 (m)
3.28 (m)
3.28 (m)
e
e
3.87 (dd, J ) 12.0, 1.0 Hz)
3
.71 (m)
a
Multiplicities and coupling constants are in parentheses (measured at 500 MHz). Measured at 100 MHz. The H and 13C NMR data of 2–5 were
b
c
1
identical to those of 1 for the lipid base and glucose units (for differences in NMR data of N-acyl units of 2–5, please refer to their respective FAMEs
d
e
in Experimental Section). Values with same superscript within same column may be interchanged. Overlapped with solvent peak (assignments were
secured by HSQC experiment).
1
a
13
b
c
Table 2. H and C NMR Data of Renieroside B
1
(6) (CD
3
OD)
position
δH
δC
position
δH
δC
lipid base unit
1
N-acyl unit
4.13 (m)
.67 (m)
3.98 (m)
4.13 (m)
5.48 (dd, J ) 15.0, 8.0 Hz)
5.73 (dt, J ) 15.0, 6.5 Hz)
2.09 (m)
69.8
1′
2′
3′
177.2
72.8
35.5
3
4.12 (dd, J ) 8.0, 4.8 Hz)
1.69 (m)
1.54 (m)
1.38–1.24 (m)
2.02 (q, J ) 6.0 Hz)
5.34 (m)
1.38–1.24 (m)
0.89 (t, J ) 7.0 Hz)
2
3
4
5
6
7
8
9
54.6
73.1
131.2
134.3
33.1
36.1
130.8
134.8
135.9
131.8
33.2
29–33
22.9
14.6
d
4′–19′ or 24′–26′
20′ or 23′
21′ or 22′
27′
29–33
27.2
131.1
22.9
2.21 (m)
5.33 (m)
28′
14.6
glucose unit
1
1
1
1
1
1
1
0
1
2
3–16
7
8
9
6.02 (d, J ) 15.5 Hz)
5.52 (dt, J ) 15.0, 7.0 Hz)
2.07 (q, J ) 7.0 Hz)
1.38–1.28 (m)
1.38–1.28 (m)
0.89 (t, J ) 7.0 Hz)
1.71 (s)
1′′
2′′
3′′
4′′
5′′
6′′
4.26 (d, J ) 7.5 Hz)
3.18 (m)
104.7
75.0
78.0
71.8
77.9
62.7
3.35 (m)
3.28 (m)
3.28 (m)
e
e
3.87 (dd, J ) 12.0, 1.0 Hz)
3.71 (m)
12.4
a
b
Multiplicities and coupling constants are in parentheses (measured at 500 MHz). Assignments were based on HSQC and HMBC experiments (500
c
1
13
MHz). The H and C NMR data of 7 and 8 were identical to those of 6 for the lipid base and glucose units (for differences in NMR data of N-acyl
units of 7 and 8, please refer to their respective FAMEs in Experimental Section). Values with same superscript within same column may be
interchanged. Overlapped with solvent peak (assignments were secured by HSQC experiment).
d
e
saturated FAME, which was characterized as (2R)-hydroxy-
Renieroside B
its molecular formula was established as C53H97NO
1
(6) was isolated as a white amorphous solid, and
on the basis of
1
docosanoic acid on the basis of its H NMR, FABMS, and optical
9
2
2
13
rotation ([R]
D
–19, MeOH) data. This fatty acid has been reported
high-resolution FABMS and C NMR spectroscopic data. The exact
from several marine sponges including Verongula gigantea and
mass of the [M - H]- ion at m/z 890.7089 matched well with the
2
1
1
13
Aplysina archeri. All these data were in agreement with the
proposed structure for renieroside A (4) as 1-O-ꢀ-D-glucopyrano-
syl-(2S,3R,4E,8E,10E)-2-[(2′R)-hydroxydocosanoylamino]-octadeca-
,8,10-triene-1,3-diol.
Renieroside A (5) was isolated as a white amorphous solid. Its
molecular formula was found to be C48
expected molecular formula (890.7085, ∆ +0.4 mmu). Its H and C
4
NMR spectroscopic data were clearly different from those of reniero-
sides A –A (1–5), indicating the difference was in the sphingosine
base. The H NMR spectrum of 6 revealed the presence of an additional
methyl group (δ 1.71, δ 12.4). Furthermore, a lone downfield doublet
at δ 6.02 (1H, d, J ) 15.5 Hz), rather than a multiplet as in the case
of renierosides A –A , suggested the possible substitution was either
1
5
1
4
5
H
C
H
89NO
9
on the basis of high-
+
resolution FAB mass spectroscopy ([M + Na] at m/z 846.6433,
–0.2 mmu) and the results of H and C NMR spectroscopic
1
5
1
13
∆
at C-9 or C-10 of the conjugated diene. Two different sets of COSY
correlations were useful to solve this issue. The H-6 showed COSY
correlation to H-7, which was in turn correlated with H-8. Another
partial structure was established according to the COSY correlations
from H-10 through H-13, suggesting the presence of a methyl group
at C-9 (Figure 1). In addition, the HMBC correlations from H
C-8, -9, and -10, and a weak long-range COSY correlation between
H -19 and H-8, further confirmed that the methyl group was located
3
1
13
interpretations. The H and C NMR and mass spectroscopic data
of 5 were found to be nearly identical to those of renieroside A
4). Its methanolysis gave rise to a different FAME, which was
identified as (2R)-hydroxytetracosanoic acid on the basis of its low-
resolution FABMS, NMR, and optical rotation ([R]22
–4, MeOH)
data. This fatty acid has been reported in several marine sponges
including Verongula gigantea and Aplysina archeri. All these data
were in agreement with the structure for renieroside A (5) as 1-O-
-D-glucopyranosyl-(2S,3R,4E,8E,10E)-2-[(2′R)-hydroxytetracosanoy-
lamino]-octadeca-4,8,10-triene-1,3-diol.
4
(
D
3
-19 to
21
5
at C-9. These 2D NMR spectroscopic interpretations were also helpful
in defining the location of double bonds in the sphingosine base. The
comparison of spectroscopic data of 6 with those reported supported
ꢀ
1

1
484 Journal of Natural Products, 2007, Vol. 70, No. 9
Notes
the theory that the long-chain base of 6 had the same skeleton and
stereochemistry. The acid hydrolysis of 6 yielded the same FAME as
that of 1, as confirmed by the comparison of NMR, low-resolution
FABMS, FAB-CID-MS/MS, and optical rotation data (see the
Experimental Section). On the basis of all these data, the structure of
Experimental Section
General Experimental Procedures. Optical rotations were mea-
sured using a JASCO P-1020 polarimeter. H and C NMR spectra
were recorded on Varian UNITY 400 and Varian INOVA 500
spectrometers. Chemical shifts are reported with reference to the
1
13
respective residual solvent or deuterated solvent peaks (δ
H
3.30 and
renieroside
B
1
(6) was defined as 1-O-ꢀ-D-glucopyranosyl-
δ
C
49.0 for CD OD). FABMS data were obtained on a JEOL JMS
3
(2S,3R,4E,8E,10E)-2-[(2′R,21′Z)-2′-hydroxyoctacos-21′-enoylamino]-
SX-102A. HRFABMS data were obtained on a JEOL JMS SX-101A.
HPLC was performed with a C18-5E Shodex packed column (250 ×
10 mm, 5 µm, 100 Å) using a Gilson 133-RI detector.
9-methyloctadeca-4,8,10-triene-1,3-diol.
The molecular formula of renieroside B
2
(7), isolated as a white
amorphous solid, was assigned as C51
H
93NO
9
on the basis of high-
Animal Material. The sponge, Haliclona (Reniera), was collected
by hand using scuba gear (20 m depth) in October of 2001, off Ulleung
Island, Korea. The collected sample was frozen immediately. The
specimen (sample No. J01U-6) was identified as Haliclona (Reniera)
species by C.J.S. It was thickly encrusting on the shell surface. The
surface was smooth. The color was purple in life and beige in alcohol.
The texture was soft and fragile. The megascleres were thick oxea
(200–260 µm × 8–10 µm) and thin oxea (100–180 µm × 1–5 µm). A
voucher specimen of the sponge (registry No. Spo. 45) was deposited
at the Natural History Museum, Hannam University, Daejon, Korea.
Extraction and Isolation. The frozen sponge (7 kg) was extracted
with MeOH at room temperature. The MeOH extract (29.2 g) showed
toxicity against brine shrimp larvae (LD50 126 µg/mL). The MeOH
+
resolution FAB mass spectroscopy ([M + Na] at m/z 886.6746, ∆
1
13
–
0.2 mmu) and the results of H and C NMR spectroscopic
interpretations (Tables 1 and 2). The NMR results were found to be
essentially identical to those of 6, which confirmed that renieroside
B (7) was also a C-9 methylated cerebroside and differed only in the
2
length of lipid base or lipid amide units. In addition to the pseudo-
-
molecular ion peak at m/z 862 [M - H] , the FABMS of 7 also
exhibited an intense fragment peak at m/z 700, which was produced
by elimination of the glucosyl unit from the molecular ion. The
difference in the side chain was similarly located in the fatty acid
portion because of the isolation of methyl (2R,17Z)-2-hydroxyhexacos-
2 2 2 2 2
extract was partitioned between CH Cl and H O. The CH Cl layer
1
7-enoic acid in the methanolysis of 7, which was same as the FAME
of 2 (see the Experimental Section). Therefore, the structure of
renieroside B (7) was proposed as 1-O-ꢀ-D-glucopyranosyl-
(
6.6 g, LD50 203 µg/mL) was further partitioned between aqueous
MeOH (1.9 g, LD50 27 µg/mL) and n-hexane (3.9 g, LD50 45 µg/mL).
A portion of the n-hexane fraction (400 mg) was subjected to Si gel
60 (15–40 µm) column chromatography with a solvent system of 100%
2
(2S,3R,4E,8E,10E)-2-[(2′R,17′Z)-2′-hydroxyhexacos-17′-enoylamino]-
2 2 2 2
CH Cl to 80% CH Cl with MeOH, to afford 10 fractions. Fraction 7
9-methyloctadeca-4,8,10-triene-1,3-diol.
(
1
4
70 mg) was subjected to RP-HPLC (C18-5E Shodex packed, 250 ×
3
The molecular formula of renieroside B (8) was found to be
0 mm, 5 µm, 100 Å) eluted with 100% MeOH to afford cerebrosides
(1.1 mg), 3 (2.1 mg), 2 (2.6 mg), 5 (1.2 mg), 7 (2.4 mg), 8 (0.8 mg),
52 9
C H95NO on the basis of high-resolution FAB mass spectroscopy
+
1
13
(
[M + Na] at m/z 900.6903, ∆ –0.2 mmu) as well as H and
C
1 (1.2 mg), and then 6 (0.8 mg).
Renieroside A (1), 1-O-ꢀ-D-Glucopyranosyl-(2S,3R,4E,8E,10E)-
2-[(2′R,21′Z)-2′-hydroxyoctacos-21′-enoylamino]-octadeca-4,8,10-
1
13
NMR spectroscopic results. Again, the H and C NMR spectro-
scopic data were found to be nearly identical to those of renierosides
B and B . The acid hydrolysis of 8 gave rise to (2R,20Z)-2-
1 2
1
2
2
triene-1,3-diol. White amorphous solid. [R]
D
+10 (c 0.1, MeOH).
1
13
H and C NMR data, see Table 1. FABMS (+ ve mode) m/z: 860
hydroxyheptacos-20-enoic acid (see the Experimental Section). The
location of the double bond was determined by FAB-CID-MS/MS
+
+
+
[
(M + H) – H
2
O] , 900 [M + Na] , 698 [(M + H) – H O – 162] ; (-
2
-
-
ve mode) m/z 876 [M - H] , 714 [(M - H) - 162] , 696 [(M - H)
+
analysis of the [M + Na] ion at m/z 461. Allylic cleavages were
-
-
162 - H
, 876.6929).
Methanolysis of 1. Cerebroside (0.70 mg) was dissolved in 5% 1
2
O] . HRFABMS (- ve mode) m/z: 876.6946 (calcd for
observed as enhanced peaks at m/z 389 and 335, indicating the
location of the double bond at C-20 (Figure 2). As far as the authors
are aware, the 2-hydroxyheptacos-20-enoic acid, isolated as a part
of cerebroside, has previously been reported from neither natural
nor synthetic sources. All these data were in agreement with the
52 9
C H94NO
M HCl–MeOH (700 µL), and the mixture was refluxed on a magnetic
stirrer for 18 h at 80 °C. The reaction mixture was then cooled and
extracted with n-hexane (3 mL × 3). The n-hexane layer was evaporated
under N to yield the FAME (methyl 2-hydroxyoctacos-21-enoic acid;
2
3
proposed structure for renieroside B (8) as 1-O-ꢀ-D-glucopyranosyl-
2
2
1
0
.2 mg). [R]
δ 5.34 (2H, m, H-21, H-22), 4.12 (1H, dd, J ) 8.0, 4.8 Hz, H-2), 3.71
3H, s, OCH ), 2.02 (4H, q, J ) 6.0 Hz, H-20, H-23), 1.61 (2H, m,
H-3), 1.28 (40H, m, H-4–H-19, H-24–H-27), 0.89 (3H, t, J ) 7.0 Hz,
D 3
–4 (c 0.1, MeOH). H NMR data (CD OD, 400 MHz):
(2S,3R,4E,8E,10E)-2-[(2′R,20′Z)-2′-hydroxyheptacos-20′-enoylamino]-
9
-methyloctadeca-4,8,10-triene-1,3-diol.
(
3
The prominent feature of six of these cerebrosides (1–3 and 6–8)
1
3
was the presence of very long chain fatty acid moieties. Some of
these fatty acids were unprecedented (fatty acids of 1, 6, and 8),
while others have been reported for the first time from a marine
source (fatty acids of 2, 3, and 7). The very long chain fatty acids
have been reported to be biosynthesized in marine primary
producers by the condensation of precursor fatty acids with malonyl-
CoA, using different enzymes (desaturases and elongases). The
desaturases insert a double bond at a specific carbon atom in the
fatty acid chain, and the elongases elongate the precursors in two-
carbon increments. It might be speculated that similar elongation
and desaturation from precursor fatty acids, followed by hydroxy-
lation, led to the formation of unsaturated hydroxylated fatty acids
in these cerebrosides. Further investigation on the metabolic
pathways and participation of these large molecules in cellular
functions of the lower sessile animals is of profound interest.
H-28). C NMR data (CD
3
OD, assignments based on HMBC and
HSQC experiments, 500 MHz): δ 177.2 (C-1), 131.1 (C-21, C-22),
7
2.8 (C-2), 52.0 (OCH
3
), 35.5 (C-3), 29.0–33.0 (C-4–C-19, C-24–C-
2
6), 27.2 (C-20, C-23), 22.9 (C-27), 14.6 (C-28). FABMS: m/z 453
+
+
+
[M + H] , 475 [M + Na] . FAB-CID-MS/MS: m/z 475 [M + Na]
(100), 459 (0.08), 445 (0.07), 431 (0.06), 417 (0.22), 403 (0.26), 389
(0.07), 375 (0.04), 363 (0.03), 349 (0.24), 335 (0.08), 321 (0.07), 307
2
2
(0.12), 293 (0.11), 279 (0.09), 265 (0.08), 251 (0.07), 237 (0.08), 223
(0.08), 209 (0.06), 195 (0.06), 181 (0.03), 167 (0.05), 153 (0.02), 139
(
0.03), 125 (0.08), 111 (0.13).
Renieroside A (2), 1-O-ꢀ-D-Glucopyranosyl-(2S,3R,4E,8E,10E)-
-[(2′R,17′Z)-2′-hydroxyhexacos-17′-enoylamino]-octadeca-4,8,10-
2
2
2
5
triene-1,3-diol. White amorphous solid. [R]
D
+4 (c 0.18, MeOH).
1
13
+
H and C NMR data, see Table 1. FABMS: m/z 872 [M + Na] .
HRFABMS (+ ve mode): m/z 872.6594 (calcd for C50
72.6592).
Methanolysis of 2. Cerebroside (2.32 mg) was subjected to
methanolysis similarly to 1 to yield a FAME (methyl-2-hydroxyhexa-
9
H91NO Na,
8
The cerebrosides (1–8) were evaluated for cytotoxicity against
a panel of five human solid tumor cell lines including human lung
cancer (A549), human ovarian cancer (SK-OV-3), human skin can-
cer (SK-MEL-2), human CNS cancer (XF498), and human colon
cancer (HCT15) cell line, but were found inactive (ED50 > 10 µg/
mL) to all cell lines in the panel.
2
3
1
cos-17-enoic acid; 1.1 mg). [R]
CD OD, 500 MHz): δ 5.34 (2H, m, H-17, H-18), 4.12 (1H, dd, J )
.0, 4.8 Hz, H-2), 3.71 (3H, s, OCH
D
–4 (c 0.1, MeOH). H NMR data
(
8
3
3
), 2.02 (4H, q, J ) 6.0 Hz, H-16,
H-19), 1.63 (2H, m, H-3), 1.28 (36H, m, H-4–H-15, H-20–H-25), 0.90
(3H, t, J ) 7.0 Hz, H-26). 13C NMR data (CD OD, assignments based
3
on HMBC and HSQC experiments, 500 MHz): δ 177.2 (C-1), 131.1

Journal of Natural Products, 2007, Vol. 70, No. 9 1485
1
(
1
C-17, C-18), 72.8 (C-2), 52.0 (OCH
3
), 35.5 (C-3), 29.0–33.0 (C-4–C-
21-enoic acid; 0.2 mg): [R]22
(CD
8.0, 4.8 Hz, H-2), 3.71 (3H, s, OCH
H-23), 1.61 (2H, m, H-3), 1.28 (40H, m, H-4–H-19, H-24–H-27), 0.90
D
–18 (c 0.02, MeOH). H NMR data
5, C-20–C-24), 27.2 (C-16, C-19), 22.9 (C-25), 14.6 (C-26). FABMS:
3
OD, 500 MHz): δ 5.34 (2H, m, H-21, H-22), 4.12 (1H, dd, J )
+
+
m/z 447 [M + Na] . FAB-CID-MS/MS: m/z 447 [M + Na] (100),
3
), 2.02 (4H, q, J ) 6.0 Hz, H-20,
4
3
2
1
31 (0.13), 417 (0.16), 403 (0.22), 389 (0.24), 375 (0.19), 361 (0.25),
47 (0.24), 333 (0.11), 321 (0.18), 307 (0.15), 293 (0.32), 279 (0.21),
65 (0.20), 251 (0.17), 237 (0.14), 223 (0.15), 209 (0.13), 195 (0.10),
81 (0.07), 167 (0.08), 153 (0.04), 139 (0.07), 125 (0.18), 111 (0.29).
The aqueous MeOH layer of the hydrolysate was evaporated under
1
3
(3H, t, J ) 7.0 Hz, H-28). C NMR data (CD OD, assignments based
3
on HMBC and HSQC experiments, 500 MHz): δ 177.2 (C-1), 131.1
(C-21, C-22), 72.8 (C-2), 52.0 (OCH ), 35.5 (C-3), 29.0-33.0 (C-4–C-
3
19, C-24–C-26), 27.2 (C-20, C-23), 22.9 (C-27), 14.6 (C-28). FABMS:
+
+
a vacuum to remove HCl. The resulting residue was eluted with MeOH
m/z 453 [M + H] , 475 [M + Na] . FAB-CID-MS/MS: m/z 475 [M
+ Na] (100), 459 (0.08), 445 (0.07), 431 (0.06), 417 (0.22), 403 (0.26),
389 (0.07), 375 (0.04), 363 (0.03), 349 (1.45), 335 (0.08), 321 (0.07),
307 (0.12), 293 (0.11), 279 (0.18), 265 (0.08), 251 (0.07), 237 (0.08),
23 (0.08), 209 (0.06), 195 (0.06), 181 (0.03), 167 (0.05), 153 (0.02),
39 (0.03), 125 (0.08), 111 (0.13).
+
on a Sephadex LH-20 column to afford methyl glucopyranoside
mixture of two anomers, 0.4 mg). [R]26
+70.8 (c 0.05, MeOH). H
1
(
D
NMR data (CD
R-anomer), 4.16 (1H, d, J ) 7.8 Hz, H-1, ꢀ-anomer), 3.52 (3H, s, OCH
-anomer), 3.39 (3H, s, OCH , R-anomer).
Renieroside A (3), 1-O-ꢀ-D-glucopyranosyl-(2S,3R,4E,8E,10E)-
-[(2′R,16′Z)-2′-hydroxypentacos-16′-enoylamino]-octadeca-4,8,10-
3
OD, 500 MHz): δ 4.64 (1H, d, J ) 3.8 Hz, H-1,
3
,
2
1
ꢀ
3
3
Renieroside B
(7), 1-O-ꢀ-D-Glucopyranosyl-(2S,3R,4E,8E,10E)-
-[(2′R,17′Z)-2′-hydroxyhexacos-17′-enoylamino]-9-methyloctadeca-
2
2
2
4
2
5
triene-1,3-diol. White amorphous solid. [R]
D
+3 (c 0.14, MeOH).
22
,8,10-triene-1,3-diol. White amorphous solid. [R]
D
+3 (c 0.16,
1
13
+
H and C NMR data, see Table 1. FABMS: m/z 858 [M + Na] .
1
13
MeOH). H and C NMR data, see Table 2. FABMS (+ ve mode):
m/z 886 [M + Na] ; (– ve mode): m/z 862 [M - H] , 700 [(M - H)
HRFABMS (+ ve mode): m/z 858.6436 (calcd for C49
58.6435).
Methanolysis of 3. Cerebroside (1.83 mg) was subjected to
methanolysis similarly to 1 to yield a FAME (methyl 2-hydroxypen-
H89NO
9
Na,
+
-
8
-
–
162] . HRFABMS (+ ve mode): m/z 886.6746 (calcd for
Na, 886.6748).
Methanolysis of 7. Cerebroside (2.08 mg) was subjected to
methanolysis similarly to 1 to yield a FAME (methyl-2-hydroxyhexa-
51 9
C H93NO
2
3
1
tacos-16-enoic acid; 1.2 mg). [R]
CD OD, 500 MHz): δ 5.34 (2H, m, H-16, H-17), 4.12 (1H, dd, J )
8.0, 4.8 Hz, H-2), 3.71 (3H, s, OCH ), 2.02 (4H, q, J ) 6.0 Hz, H-15,
H-18), 1.62 (2H, m, H-3), 1.28 (34H, m, H-4–H-14, H-19–H-24), 0.90
3H, t, J ) 7.0 Hz, H-25). 13C NMR data (CD
OD, assignments based
on HMBC and HSQC experiments, 500 MHz): δ 177.2 (C-1), 131.1
C-16, C-17), 72.8 (C-2), 52.0 (OCH ), 35.5 (C-3), 29.0–33.0 (C-4–C-
4, C-19–C-23), 27.2 (C-15, C-18), 22.9 (C-24), 14.6 (C-25). FABMS:
D
–1 (c 0.1, MeOH). H NMR data
(
–
3
23
1
cos-17-enoic acid; 0.9 mg). [R]
CD OD, 500 MHz): δ 5.34 (2H, m, H-17, H-18), 4.12 (1H, dd, J )
.0, 4.8 Hz, H-2), 3.71 (3H, s, OCH ), 2.02 (4H, q, J ) 6.0 Hz, H-16,
H-19), 1.61 (2H, m, H-3), 1.28 (36H, m, H-4–H15, H-20–H-25), 0.89
D
–1 (c 0.07, MeOH). H NMR data
3
(
3
8
3
(
3
13
(
3H, t, J ) 7.5 Hz, H-26). C NMR data (CD
on HMBC and HSQC experiments, 500 MHz): δ 177.2 (C-1), 131.1
C-17, C-18), 72.8 (C-2), 52.0 (OCH ), 35.5 (C-3), 29.0–33.0 (C-4–C-
5, C-20–C-24), 27.2 (C-16, C-19), 22.9 (C-25), 14.6 (C-26). FABMS:
3
OD, assignments based
(
1
3
(
3
+
m/z 433 [M + Na]. FAB-CID-MS/MS: m/z 433 [M + Na] (100), 417
1
(
(
(
(
0.11), 403 (0.16), 389 (0.09), 375 (0.13), 361 (0.14), 347 (0.11), 333
0.13), 319 (0.05), 307 (0.10), 293 (0.08), 279 (0.11), 265 (0.08), 251
0.07), 237 (0.06), 223 (0.07), 209 (0.05), 195 (0.05), 181 (0.03), 167
0.04), 153 (0.02), 139 (0.03), 125 (0.05), 111 (0.06).
+
+
m/z 447 [M + Na] , FAB-CID-MS/MS: m/z 447 [M + Na] (100),
4
3
2
1
31 (0.08), 417 (0.11), 403 (0.15), 389 (0.14), 375 (0.13), 361 (0.11),
47 (0.13), 333 (0.04), 321 (0.14), 307 (0.11), 293 (0.16), 279 (0.11),
65 (0.09), 251 (0.08), 237 (0.08), 223 (0.07), 209 (0.07), 195 (0.06),
81 (0.05), 167 (0.05), 153 (0.02), 139 (0.03), 125 (0.06), 111 (0.11).
The aqueous MeOH layer of the hydrolysate was evaporated under
4
Renieroside A (4), 1-O-ꢀ-D-Glucopyranosyl-(2S,3R,4E,8E,10E)-
2
-[(2′R)-hydroxydocosanoylamino]-octadeca-4,8,10-triene-1,3-
1
13
diol. White amorphous solid. H and C NMR data, see Table 1.
a vacuum to remove HCl. The resulting residue was eluted with MeOH
on a Sephadex LH-20 column to afford methyl glucopyranoside
+
FABMS: m/z 818 [M + Na] . HRFABMS (+ ve mode): m/z 818.6123
(
9
calcd for C46H85NO Na, 818.6122).
2
7
1
(
mixture of two anomers, 0.4 mg). [R]
D
+62.4 (c 0.04, MeOH). H
OD, 500 MHz): δ 4.64 (1H, d, J ) 3.8 Hz, H-1,
Methanolysis of 4. Cerebroside (0.71 mg) was subjected to
NMR data (CD
3
methanolysis similarly to 1 to yield a FAME (methyl-2-hydroxy-
R-anomer), 4.16 (1H, d, J ) 7.8 Hz, H-1, ꢀ-anomer), 3.52 (3H, s, OCH
-anomer), 3.39 (3H, s, OCH , R-anomer).
Renieroside B (8), 1-O-ꢀ-D-Glucopyranosyl-(2S,3R,4E,8E,10E)-
2-[(2′R,20′Z)-2′-hydroxyheptacos-20′-enoylamino]-9-methyloctadeca-
3
,
2
2
1
docosanoic acid; 0.2 mg). [R]
CD OD, 500 MHz): δ 4.12 (1H, dd, J ) 8.0, 4.8 Hz, H-2), 3.71 (3H,
s, OCH ), 1.61 (2H, m, H-3), 1.28 (36H, m, H-4–H-21), 0.89 (3H, t, J
7.0 Hz, H-22). C NMR data (CD
and HSQC experiments, 500 MHz): δ 177.2 (C-1), 72.8 (C-2), 52.0
OCH ), 35.5 (C-3), 29.0–33.0 (C-4–C-20), 22.9 (C-21), 14.6 (C-22).
FABMS: m/z 393 [M + Na] .
Renieroside A (5), 1-O-ꢀ-D-glucopyranosyl-(2S,3R,4E,8E,10E)-
-[(2′R)-hydroxytetracosanoylamino]-octadeca-4,8,10-triene-1,3-
D
–19 (c 0.02, MeOH). H NMR data
ꢀ
3
(
3
3
3
13
)
3
OD, assignments based on HMBC
1
13
4
,8,10-triene-1,3-diol. White amorphous solid. H and C NMR data,
+
see Table 2. FABMS (+ ve mode): m/z 900 [M + Na] ; (– ve mode):
(
3
-
-
+
m/z 876 [M - H] , 714 [(M - H) – 162] . HRFABMS (+ ve mode):
m/z 900.6903 (calcd for C52 Na, 900.6905).
H95NO
9
5
Methanolysis of 8. Cerebroside (0.37 mg) was subjected to
methanolysis similarly to 1 to yield a FAME (methyl-2-hydroxyhep-
2
1
13
diol. White amorphous solid. H and C NMR data, see Table 1.
FABMS (+ ve mode): m/z 846 [M + Na] ; (– ve mode): m/z 822 [M
2
2
1
+
tacos-20-enoic acid; 0.15 mg). [R]
data (CD OD, 500 MHz): δ 5.34 (2H, m, H-20, H-21), 4.12 (1H, dd,
J ) 8.0, 4.8 Hz, H-2), 3.71 (3H, s, OCH ), 2.02 (4H, q, J ) 6.0 Hz,
H-19, H-22), 1.61 (2H, m, H-3), 1.28 (38H, m, H-4–H-18, H-23–H-
D
–61 (c 0.01, MeOH). H NMR
-
-
3
-
H] , 660 [(M - H) – 162] . HRFABMS (+ ve mode): m/z 846.6433
3
(
calcd for C48
Methanolysis of 5. Cerebroside (0.76 mg) was subjected to
methanolysis similarly to 1 to yield a FAME (methyl-2-hydroxytetra-
9
H89NO Na, 846.6435).
1
3
2
6), 0.89 (3H, t, J ) 7.0 Hz, H-27). C NMR data (CD
3
OD,
2
2
1
assignments based on HMBC and HSQC experiments, 500 MHz): δ
cosanoic acid; 0.4 mg). [R]
CD OD, 500 MHz): δ 4.12 (1H, dd, J ) 8.0, 4.8 Hz, H-2), 3.71 (3H,
s, OCH ), 1.61 (2H, m, H-3), 1.28 (40H, m, H-4–H-23), 0.89 (3H, t, J
7.0 Hz, H-24). C NMR data (CD
and HSQC experiments, 500 MHz): δ 177.2 (C-1), 72.8 (C-2), 52.0
OCH ), 35.5 (C-3), 29.0-33.0 (C-4–C-22), 22.9 (C-23), 14.6 (C-24).
FABMS: m/z 421 [M + Na] .
Renieroside B (6), 1-O-ꢀ-D-Glucopyranosyl-(2S,3R,4E,8E,10E)-
-[(2′R,21′Z)-2′-hydroxyoctacos-21′-enoylamino]-9-methyloctadeca-
D
–4 (c 0.1, MeOH). H NMR data
1
77.2 (C-1), 131.1 (C-20, C-21), 72.8 (C-2), 52.0 (OCH
3
), 35.5 (C-3),
(
3
2
9.0–33.0 (C-4–C-18, C-23–C-25), 27.2 (C-19, C-22), 22.9 (C-26), 14.6
(C-27). FABMS: m/z 461 [M + Na] . FAB-CID-MS/MS: m/z 461 [M
Na] (100), 445 (0.1), 431 (0.08), 417 (0.08), 403 (0.18), 389 (0.17),
75 (0.1), 361 (0.07), 349 (0.4), 335 (0.17), 321 (0.06), 307 (0.12),
293 (0.09), 279 (0.07), 265 (0.08), 251 (0.07), 237 (0.05), 223 (0.06),
09 (0.06), 195 (0.04), 181 (0.03), 167 (0.04), 153 (0.02), 139 (0.01),
25 (0.06), 111 (0.08).
3
+
13
)
3
OD, assignments based on HMBC
+
+
3
(
3
+
2
1
1
2
4
2
2
,8,10-triene-1,3-diol. White amorphous solid. [R]
D
+10 (c 0.1,
1
13
Acknowledgment. This work was supported by the Korea Science
and Engineering Foundation (KOSEF) grant funded by the Korean
government (MOST; No. R01-2004-000-10467-0).
MeOH). H and C NMR data, see Table 2. FABMS (+ ve mode):
+
+
m/z 914 [M + Na] , 874 [(M + H) – H
2
O] , 712 [(M + H) – H
2
O –
+
-
-
1
62] ; (– ve mode): m/z 890 [M - H] , 728 [(M - H) – 162] , 711
-
[
(
(M - H) – H
calcd for C53
Methanolysis of 6. Cerebroside (0.42 mg) was subjected to
methanolysis similarly to 1 to yield a FAME (methyl-2-hydroxyoctacos-
2
O – 162] . HRFABMS (– ve mode): m/z 890.7089
1
H96NO
9
, 890.7085).
Supporting Information Available: H NMR, HSQC, low-resolu-
tion FAB mass, and FAB-CID tandem mass spectra of compounds 1
and 6, FAB-CID tandem mass spectra of compounds 2 and 8, and a

1
486 Journal of Natural Products, 2007, Vol. 70, No. 9
Notes
color photograph of the sponge Haliclona (Reniera) sp. This material
is available free of charge via the Internet at http://pubs.acs.org.
(12) Baker, B. J.; Scheuer, P. J.; Shoolery, J. N. J. Am. Chem. Soc. 1988,
1
10, 965–966.
(
(
13) Sakai, R.; Higa, T. J. Am. Chem. Soc. 1986, 108, 6404–6405.
References and Notes
14) Randazzo, A.; Bifulco, G.; Giannini, C.; Bucci, M.; Debitus, C.; Cirino,
(
1) Jin, W.; Rinehart, K. L.; Jares-Erjiman, A. E. J. Org. Chem. 1994,
9, 144–147.
G.; Gomez-Paloma, L. J. Am. Chem. Soc. 2001, 123, 10870–10876.
5
(15) Bock, K.; Pederson, C. In AdVances in Carbohydrate Chemistry and
Biochemistry, Jipson, R. S., Horfon, D., Eds.; Academic Press: New
York, 1983; Vol. 41, pp 27–46.
(
(
2) Li, H.; Matsunaga, S.; Fusetani, N. Tetrahedron 1995, 51, 2273–2280.
3) Weber, K. T.; Hammache, D.; Fantini, J.; Ganem, B. Biorg. Med.
Chem. Lett. 2000, 10, 1011–1014.
(16) Costantino, V.; Fattorusso, E.; Mangoni, A. Liebigs. Ann. Chem. 1995,
2133–2136.
(17) Sarmientos, F.; Schwarzmann, G.; Sandhoff, K. Eur. J. Biochem. 1985,
146, 59–64.
(
4) Yamada, K.; Hara, E.; Miyamoto, T.; Higuchi, R.; Isobe, R.; Honda,
S. Eur. J. Org. Chem. 1998, 371–378.
(
5) Jung, J. H.; Lee, C.; Kim, Y. C.; Kang, S. S. J. Nat. Prod. 1996, 59,
3
19–322.
(
18) Ayanoglu, E.; Kornprobst, J. M.; Aboud-Bichara, A.; Djerassi, C.
Tetrahedron Lett. 1983, 24, 1111–1114.
19) Imai, H.; Ohnishi, M.; Kinoshita, M.; Kojima, M.; Ito, S. Biosci.,
Biotechnol. Biochem. 1995, 59, 1309–1313.
(
(
6) Seki, M.; Kayo, A.; Mori, K. Tetrahedron Lett. 2001, 42, 2357–2360.
7) Costantino, V.; Fattorusso, E.; Mangoni, A.; Rosa, M. D.; Ianaro, A.;
Maffia, P. Tetrahedron 1996, 52, 1573–1578.
(
(
8) Kang, S. S.; Kim, S. J.; Son, K. H.; Kim, H. P.; Chang, H. W. Chem.
Pharm. Bull. 2001, 49, 321–323.
(
(
20) Kishimoto, Y.; Radin, N. S. J. Lipid Res. 1964, 5, 94–97.
21) Carballeira, N. M.; Shalabi, F.; Nergon, V. Lipids 1989, 24, 229–
(
9) Chen, J. H.; Cui, G. Y.; Liu, J. Y.; Tan, R. X. Phytochemistry 2003,
2
32.
22) Carballeira, N. M.; Shalabi, F.; Maldonado, M. E. Lipids 1990, 25,
35–237.
6
4, 903–906.
(
(
(
10) Hattori, T.; Adachi, K.; Shizuri, Y. J. Nat. Prod. 1998, 61, 823–826.
11) Clark, R. J.; Field, K. L.; Charan, R. D.; Garson, M. J.; Brereton,
I. M.; Willis, A. C. Tetrahedron 1998, 54, 8811–8826.
2
NP070078U