Ectyoplasides A-b - Unique triterpene oligoglycosides from the Caribbean sponge Ectyoplasia ferox

Article abstract of DOI:10.1002/(SICI)1099-0690(199901)1999:1<231::AID-EJOC231>3.0.CO;2-U

Two novel norlanostane triglycosides, ectyoplasides A (1) and B (2), have been isolated from the methanolic extracts of the Caribbean sponge Ectyoplasia ferox (Raspaliidae, Axinellida). Their structures have been established by extensive application of high-resolution FABMS and two- dimensional NMR techniques, supported by chemical and enzymatic degradation methods. Ectyoplasides A and B have been shown to possess identical sugar chains (composed of two β-galactose units and one α-arabinose unit), but to differ in their aglycone moieties, which possess nortriterpene skeletons with an unprecedented substitution pattern on ring A. Ectyoplasides A and B show moderate cytotoxic activity when tested in vitro towards various cell lines.

Full text of DOI:10.1002/(SICI)1099-0690(199901)1999:1<231::AID-EJOC231>3.0.CO;2-U

FULL PAPER
Ectyoplasides A–B – Unique Triterpene Oligoglycosides from the
Caribbean Sponge Ectyoplasia ferox
Francesco Cafieri, Ernesto Fattorusso,*[a] Orazio Taglialatela-Scafati
Dedicated to the memory of Professor Luigi Minale
Keywords: Saponins / Terpenoids / Sponges / Ectyoplasia ferox
Two novel norlanostane triglycosides, ectyoplasides A (1)
and B (2), have been isolated from the methanolic extracts of
the Caribbean sponge Ectyoplasia ferox (Raspaliidae,
Axinellida). Their structures have been established by
extensive application of high-resolution FABMS and two-
dimensional NMR techniques, supported by chemical and
enzymatic degradation methods. Ectyoplasides A and B have
been shown to possess identical sugar chains (composed of
two β-galactose units and one α-arabinose unit), but to differ
in their aglycone moieties, which possess nortriterpene
skeletons with an unprecedented substitution pattern on ring
A. Ectyoplasides A and B show moderate cytotoxic activity
when tested in vitro towards various cell lines
dition, 1 and 2 possess an invariant triglycosidic chain as a
sugar portion, linked at C-3.
Introduction
Ectyoplasia ferox, Duchassaing and Michelotti (Demos-
pongiae, family Raspaliidae, order Axinellida) is a brownish
orange sponge commonly found as an encrusting species
along the coasts of the Caribbean Sea.[1] In spite of its wide
diffusion, very little is known about the secondary meta-
bolism of E. ferox and, to date, only one report on the phos-
pholipid fatty acid composition has appeared in the litera-
ture.[2] However, during a recent symposium, Pawlik et al.[3]
reported that a mixture of nonseparable polar metabolites
obtained from this sponge has been found to exhibit strong
antifeedant activity.
In the course of our research program aimed at the chem-
istry of Caribbean marine organisms and focused partic-
ularly on the biologically active secondary metabolites, we
have recently begun a chemical investigation of this sponge.
Our interest in this organism started when we became
aware, through preliminary 1H-NMR and chromatographic
analyses, that its polar extracts were surprisingly rich in ter-
penoid saponins, an unusual feature for a marine sponge.
The present paper deals with the isolation and structural
elucidation of two major components of this mixture,
ectyoplasides A (1) and B (2). Their chemical structures,
determined by extensive application of high-resolution
FABMS and 2D-NMR techniques, are characterized by an
unprecedented substitution pattern at ring A of their agly-
cone moieties, which have been identified as nortriterpenes
with a tetracyclic skeleton of the lanostane type. In ad-
Results and Discussion
A specimen of E. ferox, collected by hand along the
coasts of San Salvador Island, Bahamas, was exhaustively
extracted with MeOH at room temperature and the fresh
methanolic extracts were successively partitioned against n-
hexane, CCl4, CHCl3, and n-butanol, following a slightly
modified Kupchan method.[4] The butanol-soluble mate-
rial, which appeared to be the richest in saponins, was in-
itially separated by MPLC on silica gel (230Ϫ400 mesh),
eluting with a solvent gradient system of increasing polarity
from EtOAc to MeOH. Fractions eluted with MeOH/
EtOAc (7:3) were pooled and then further purified by re-
versed-phase HPLC (eluent MeOH/H2O, 8:2) yielding pure
ectyoplasides A (1, 13.5 mg) and B (2, 4.5 mg) as white,
amorphous solids.
Our structural assignment began with the most abundant
triglycoside, ectyoplaside A (1). It exhibits an intense
pseudomolecular peak in the positive-ion HRFAB mass
spectrum at m/z 953.4734 [MϪ ϩ Naϩ ϩ Hϩ], while two
diagnostic peaks were detected at m/z 951.4560 [MϪ ϩ Naϩ
Ϫ Hϩ] and m/z 929.4731 [MϪ] in the negative-ion HRFAB
mass spectrum. Combination of these data allowed us to
assign the molecular formula C46H73NaO19 to 1, which was
further confirmed by NMR evidence.
The presence of a number of signals between δ ϭ 3.2 and
δ ϭ 5.5 in the 1H-NMR spectrum (CD3OD) of 1 (Table 1),
attributable to protons on carbon atoms bearing hetero-
atom substitution, accompanied by typical terpene reson-
ances including six methyl signals, led us to presume a ter-
[a]
Dipartimento di Chimica delle Sostanze Naturali,
via D. Montesano 49, I-80131 Napoli, Italy
Fax: (internat.) ϩ 39-81/7486552
1
E-mail: fattoru@unina.it or scatagli@unina.it
pene-glycosidic nature for 1. Analysis of the 13C- and H-
Eur. J. Org. Chem. 1999, 231Ϫ238
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
1434Ϫ193X/99/0101Ϫ0231 $ 17.50ϩ.50/0
231

F. Cafieri, E. Fattorusso, O. Taglialatela-Scafati
FULL PAPER
NMR spectra (Tables 1 and 2) of 1 was therefore divided oxy group linked at C-14 (as indicated by the resonance of
into two phases, the first dedicated to the elucidation of C-14 at δ ϭ 62.3 in the 13C-NMR spectrum, which matched
the aglycone structure, which was mainly performed on the very well that reported for penasterol,[6] and by the HMBC
native saponin 1, while the second phase focused on the correlation peaks of C-28 with 15-H2) must in this case be
carbohydrate subunit, the structure of which was deduced a carboxylate on the basis of the following evidence: (i)
from data obtained from the peracetyl derivative 1a.
FABMS data; (ii) absorption bands at νmax ϭ 1635, 1573,
The molecular formula C29H45NaO5 was deduced for the and 1454 cmϪ1 in the IR spectrum (KBr) of 1, which are
aglycone moiety of ectyoplaside A (1) on the basis of 13C- typical stretching values of the carboxylate group; (iii) the
NMR data, and was corroborated by a fragmentation peak 13C-NMR signal of C-28 at δ ϭ 186.5, attributable to a
detected at m/z 497 in the positive-ion FABMS of 1. In- carboxylate rather than to a carboxylic group because of its
terpretation of COSY- and HOHAHA-NMR spectra al- relatively low-field chemical shift,[6,7] and (iv) the signal at
1
lowed us to assign all the H signals to five spin systems δ ϭ 1.43, long-range-coupled with 18-H3 (δ ϭ 0.66) and
(essentially one for each ring, and the other for the side assigned to 12ax-H, whose signal appears upfield-shifted
chain) and to elucidate the proton sequence within each relative to the data of other penasterol saponins,[5] because
spin system, aided by comparisons with published data.[5] it resides in spatial proximity to the C-28 carboxylate rather
All the proton resonances were unambiguously associated than a carboxyl group.
1
with relevant carbon atoms on the basis of a 2D 1H-de-
With regard to the structure of ring A, its H- and 13C-
tected HMQC spectrum, whereas an HMBC experiment, NMR resonances appear quite different from those usually
showing 3J cross-peaks, was used to determine the reso- reported for lanostane nuclei.[5Ϫ7] These variations can be
nances of tetrasubstituted carbon atoms (complete assign- explained by considering that in this case only one methyl
ment given in Table 2).
group is linked to C-4, the second being replaced by a
This analysis allowed us to identify the aglycone moiety hydroxy group. This was suggested by the molecular for-
of 1 as a unique nor derivative of penasterol, a lanostane- mula and by the resonance at δ ϭ 72.0 of C-4, inferred
related triterpene with a rare 14-carboxy group, first iso- from its HMBC cross-peaks with 2-H2 (δ ϭ 2.70 and 2.00),
lated from the sponge Penares sp.[6] and previously found 5-H (δ ϭ 1.35) and 6-H2 (δ ϭ 1.92 and 1.63). The presence
as the terpene moiety in other spongal saponins (see below). of this OH group at C-4 leads to dramatic deshielding ef-
In particular, in this case the aglycone actually possesses a fects on the adjacent protons. In particular, 29-H3 resonates
penasterol skeleton with two additional hydroxy groups, at δ ϭ 1.40, with a downfield shift of almost 0.5 ppm; the
linked to C-22 in the side chain and to C-4 (ring A) the 5-H signal is shifted to δ ϭ 1.35; the 19-H3 signal, long-
latter replacing the usual methyl group. Moreover, the carb- range-coupled with 1ax-H, is shifted to δ ϭ 1.10; and 2ax-
232
Eur. J. Org. Chem. 1999, 231Ϫ238

Ectyoplasides AϪB Ϫ Unique Triterpene Oligoglycosides
FULL PAPER
Table 1. 1H-NMR data (500 MHz) of ectyoplasides A (1) and B
(2)[a]
Table 2. 13C-NMR data (125 MHz) of compounds 1 and 2 in
CD3OD
Position
1
2
Position
1
2
δH (int., mult., J in Hz)
δH (int., mult., J in Hz)
δC (mult.)
δC (mult.)
1ax
1eq
2ax
2eq
3
1.84 (1 H, br. dd, 12.5, 6.7)
1.26 (1 H, m)
2.70 (1 H, dt, 12.5, 9.7)
2.00[b]
1.83 (1 H, br. dd, 12.3, 6.7)
1.26 (1 H, m)
2.78 (1 H, dt, 12.3, 9.7)
2.00[b]
1
41.2 (CH2)
33.4 (CH2)
93.2 (CH)
72.0 (C)
56.3 (CH)
24.8 (CH2)
30.2 (CH2)
138.4 (C)
140.0 (C)
36.1 (C)
41.3 (CH2)
33.5 (CH2)
95.0 (CH)
76.5 (C)
50.1 (CH)
27.4 (CH2)
31.1 (CH2)
140.3 (C)
140.0 (C)
36.6 (C)
2
3
4
3.27 (1 H, br. t, 9.7)
3.70 (1 H, br. t, 9.7)
1.57 (1 H, m)
2.05[b]
5
5
1.35[b]
6
6ax
6eq
7ax
7eq
11
1.92 (1 H, m)
1.63 (1 H, m)
2.05[b]
7
2.15[b]
8
2.16[b]
9
2.10[b]
2.16[b]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
1Ј
2.16 (2 H, m)
2.16 (2 H, m)
25.4 (CH2)
30.1 (CH2)
46.4 (C)
25.4 (CH2)
30.2 (CH2)
46.4 (C)
12ax
12eq
15a
15b
16a
16b
17
1.43[b]
1.44[b]
2.00[b]
2.00[b]
2.12[b]
2.13[b]
62.3 (C)
62.1 (C)
1.66[b]
1.66[b]
32.2 (CH2)
28.0 (CH2)
55.2 (CH)
14.5 (CH3)
21.9 (CH3)
44.3 (CH)
15.2 (CH3)
76.1 (CH)
38.4 (CH2)
126.2 (CH)
136.5 (C)
32.2 (CH2)
28.4 (CH2)
55.2 (CH)
14.6 (CH3)
22.1 (CH3)
44.3 (CH)
15.1 (CH3)
76.1 (CH)
38.4 (CH2)
126.2 (CH)
136.4 (C)
2.03[b]
2.03[b]
1.36[b]
1.35[b]
1.56 (1 H, m)
0.66 (3 H, br. s)
1.10 (3 H, br. s)
1.47 (1 H, m)
0.94 (3 H, d, 4.9)
3.66 (1 H, br. t, 7.2)
2.18 (1 H, dt, 12.5, 7.1)
2.08[b]
1.56[b]
18
0.65 (3 H, br. s)
1.13 (3 H, br. s)
1.46 (1 H, m)
0.95 (3 H, d, 4.6)
3.65 (1 H, br. t, 7.2)
2.19 (1 H, dt, 12.2, 7.1)
2.11[b]
19
20
21
22
23a
23b
24
5.15 (1 H, br. t, 7.1)
1.66 (3 H, br. s)
1.73 (3 H, br. s)
1.40 (3 H, br. s)
5.15 (1 H, br. t, 7.1)
1.67 (3 H, br. s)
1.75 (3 H, br. s)
4.03 (1 H, d, 13.2)
3.85 (1 H, d, 13.2)
4.54 (1 H, d, 7.7)
3.81 (1 H, t, 7.7)
3.74[b]
21.3 (CH3)
28.2 (CH3)
186.5 (C)
21.3 (CH3)
28.2 (CH3)
186.5 (C)
26
27
29a
29b
1Ј
29.1 (CH3)
109.3 (CH)
81.1 (CH)
74.7 (CH)
73.7 (CH)
77.9 (CH)
67.0 (CH2)
103.5 (CH)
73.3 (CH)
72.9 (CH)
81.4 (CH)
66.0 (CH2)
107.2 (CH)
79.0 (CH)
78.8 (CH)
73.3 (CH)
77.4 (CH)
67.5 (CH2)
60.2 (CH2)
109.2 (CH)
79.6 (CH)
74.4 (CH)
73.9 (CH)
77.9 (CH)
66.2 (CH2)
103.4 (CH)
71.0 (CH)
72.8 (CH)
81.2 (CH)
66.2 (CH2)
106.6 (CH)
79.0 (CH)
78.9 (CH)
73.1 (CH)
77.4 (CH)
67.5 (CH2)
4.48 (1 H, d, 7.7)
3.81 (1 H, t, 7.7)
3.71[b]
2Ј
2Ј
3Ј
3Ј
4Ј
4Ј
3.78[b]
3.78[b]
5Ј
5Ј
3.75[b]
3.75[b]
6Ј
6Јa,b
1ЈЈ
3.52[b]
3.52[b]
1ЈЈ
2ЈЈ
3ЈЈ
4ЈЈ
5ЈЈ
1ЈЈЈ
2ЈЈЈ
3ЈЈЈ
4ЈЈЈ
5ЈЈЈ
6ЈЈЈ
5.48 (1 H, d, 3.1)
3.80 (1 H, dd, 6.8, 3.1)
3.88 (1 H, br. d, 6.8)
4.11 (1 H, br. s)
3.77[b]
5.48 (1 H, d, 3.1)
3.80 (1 H, dd, 6.8, 3.1)
3.90 (1 H, br. d, 6.8)
4.14 (1 H, br. s)
3.77[b]
2ЈЈ
3ЈЈ
4ЈЈ
5ЈЈa
5ЈЈb
1ЈЈЈ
2ЈЈЈ
3ЈЈЈ
4ЈЈЈ
5ЈЈЈ
6ЈЈЈa,b
3.75[b]
3.76[b]
4.38 (1 H, d, 7.7)
3.61 (1 H, dd, 8.2, 7.7)
3.52[b]
4.39 (1 H, d, 7.5)
3.60 (1 H, dd, 8.2, 7.5)
3.52[b]
3.85[b]
3.85[b]
3.75[b]
3.75[b]
4.14 (2 H, m)
4.14 (2 H, m)
[a] Measured in CD3OD. Ϫ [b] Overlapped with other signals.
The simultaneous presence of methyl and hydroxy groups
at C-4 of ectyoplaside A is a unique feature and, to the
best of our knowledge, has not previously been reported for
H, because of its spatial proximity to the OH group, reso- tetracyclic triterpenoids.[8] The biosynthetic origin of this
nates at δ ϭ 2.70. These are very low-field resonances rela- moiety, as well as of the carboxylate group on C-14, is not
tive to the values usually reported for normal lanostane obvious at first sight. However, they are probably intro-
skeletons.[5] Finally, the sugar unit could be confidently duced by oxidation steps not normally present in the typical
placed at C-3 of ring A by interpretation of the key HMBC pathway from lanosterol to steroids.
correlation peak between 1Ј-H (δ ϭ 4.48) and the signifi-
Information derived from COSY and HOHAHA spectra
cantly downfield-shifted C-3 signal (δ ϭ 93.2). This was was also used to follow the scalar couplings within the side
further confirmed by the strong ROESY peak between 1Ј- chain of 1, in which the presence of a hydroxy group at-
H and 3-H (δ ϭ 3.27). The above data, together with other tached at C-22 was deduced. In particular, we used as start-
diagnostic spatial couplings revealed by the ROESY spec- ing points the two methyl signals of 26-H3 (δ ϭ 1.66) and
trum of 1 (outlined in Figure 1), also allowed us to deduce 27-H3 (δ ϭ 1.73) (the relevant 1H-NMR resonances of
the relative configuration of ring A, in particular the lo- which were assigned based on the ROESY cross-peak be-
cation of the OH group in the β (axial) position, and to tween 27-H3 and 24-H), which show long-range couplings
assign all its proton resonances.
with 24-H (δ ϭ 5.15) and with 23-H2 (δ ϭ 2.18 and 2.08).
Eur. J. Org. Chem. 1999, 231Ϫ238
233

F. Cafieri, E. Fattorusso, O. Taglialatela-Scafati
FULL PAPER
tons through an HMQC experiment. Unfortunately, the re-
maining 1H-NMR sugar signals are in an excessively
crowded region and the superimposition of several key sig-
nals prevented us from measuring coupling constants and
thus from deducing directly the relative stereochemistry of
the monosaccharides. To overcome this difficulty, a derivati-
zation of the saponin 1 appeared indispensable, and, to this
end, 3 mg of 1 was acetylated with acetic anhydride in pyri-
dine under standard conditions, to afford the peracetyl de-
rivative 1a. We repeated the COSY, HOHAHA and ROESY
spectra on this molecule (1H-NMR assignments reported in
the Experimental Section), and detailed analysis of these
data permitted the unambiguous identification of the sugar
portion of the molecule.
The signal at δ ϭ 4.60 in the 1H-NMR spectrum (CDCl3)
of 1a exhibits a ROESY coupling (see Figure 1) with 3-H
(δ ϭ 3.33) of the aglycone moiety, and thus could be attri-
buted to 1Ј-H. Starting from this signal, we could identify,
on the basis of COSY and HOHAHA evidence, the se-
quence of a hexose unit, in which the relatively high-field
Figure 1. Key ROESY correlation peaks detected for compound 1
The latter signals show couplings with 24-H and with a low- resonances of 5Ј-H (δ ϭ 3.85) pointed to the pyranose
field proton resonating at δ ϭ 3.66 (22-H), which correlates form. Within this spin system, the axial-axial 1Ј-H/2Ј-H and
in the HMQC spectrum with a carbon signal at δ ϭ 76.1, 2Ј-H/3Ј-H and the axial-equatorial 3Ј-H/4Ј-H relationships
while it shows no other correlation peaks in the COSY were deduced by measurement of the coupling constants,
spectrum.
whereas the axial position of 5Ј-H is indicated by the strong
The relative configuration of the two chiral centers C-20 ROESY correlation peak with 3Ј-H (δ ϭ 5.05). On the basis
and C-22 of the side chain was deduced by considering a of this evidence, the inner monosaccharide could be iden-
1
specific feature of the H-NMR spectrum, namely the very tified as a β-galactopyranose. Furthermore, the relatively
small (J < 1 Hz) coupling constant between 22-H and 20- high-field chemical shift of 2Ј-H (δ ϭ 4.05) and its ROESY
H. This unusual value points to a preferential conformation correlation peak with 1ЈЈ-H (δ ϭ 5.45) clearly prove the 2Ј-
in which the dihedral angle between these two atoms is near position to be the glycosidic linkage site. Following the
to 90°. To assess this, the two alternative 20R-22R/20S-22S same type of analysis, the second sugar unit was identified
and 20R-22S/20S-22R structures were subjected to a molec- as a pentose in the pyranose form, in which the axial-axial
ular dynamic analysis in the CHARMm force field. The coupling between 2ЈЈ-H and 3ЈЈ-H and the axial-equatorial
conformational behavior of the molecules was explored relationships between 1ЈЈ-H and 2ЈЈ-H and between 3ЈЈ-H
with high-temperature (1000 K) molecular dynamics simu- and 4ЈЈ-H led to an assignment as an α-arabinopyranose.
lation, generating a set of 250 conformations for each mole- Finally, the third sugar must be linked at the 4ЈЈ-position
cule. The energies of these structures were minimized and on the basis of a ROESY cross-peak between 4ЈЈ-H (δ ϭ
analysis of the obtained results indicates that for both iso- 4.35) and 1ЈЈЈ-H (δ ϭ 4.48), and was easily identified as a
mers a set of low-energy conformations of the side chain second β-galactopyranose because it exhibits a pattern of
exists at room temperature. Interestingly, in these preferen- coupling constants identical to that previously measured for
tial conformations, the 20-H/22-H angle ranges from 78 to the first monosaccharide. Thus, we succeeded in completely
90° in the RR/SS isomer, and from 45 to 55° for the RS/SR defining the sugar chain of saponin 1.
isomer. Consequently, we can confidently assign the RR/SS
In order to confirm the proposed structure of ectyopla-
configuration at centers C-20/C-22 from the above NMR side A (1) and to deduce the absolute configuration of the
data. Then, if we assume that the aglycone part of our sap- monosaccharides, 4 mg of the saponin 1 was subjected to
onin possesses the absolute configuration invariably found acidic methanolysis with 1  HCl in 85% MeOH for 2 h at
in all lanostane derivatives isolated to date,[8] we can assign 70°C. The resulting mixture was neutralized with Ag2CO3,
the S configuration to C-20, and hence to C-22 as well. This centrifuged, and the supernatant was concentrated to dry-
configuration has previously been found in C-22-hy- ness under nitrogen. The residue was then partitioned be-
droxylated triterpenes isolated from various sources.[9Ϫ11] tween H2O and CHCl3. The polar layer (fraction A, 2.2
1
Moreover, very small values for the coupling constant of mg) was found by H-NMR analysis to be composed of a
20-H with 22-H were also measured in these cases.
mixture of monosaccharide methyl glycosides. Fraction A
In the sugar portion of the molecule, the existence of a was then divided into two portions and the first of these
triglycosidic chain was initially deduced from the 13C-NMR (0.8 mg) was subjected to a persilylation reaction with TRI-
spectrum of 1, which shows the resonances of three anom- SIL-Z. The persilylated products were analyzed by GC MS,
eric carbon atoms (δ ϭ 109.3, 103.5, and 107.2, respec- giving peaks with the same retention times and the same
tively). These were correlated to the relevant anomeric pro- mass spectra as the persilylated galactopyranose methyl gly-
234
Eur. J. Org. Chem. 1999, 231Ϫ238

Ectyoplasides AϪB Ϫ Unique Triterpene Oligoglycosides
FULL PAPER
coside and arabinopyranose methyl glycoside prepared as
above. A second portion (1.4 mg) of fraction A was per-p-
bromobenzoylated under standard conditions and the re-
sulting mixture, after work-up, was separated by HPLC on
a Whatman Partisil column (eluent n-hexane/diethyl ether,
85:15). Only two major UV-absorbing peaks were collected
and comparison of their 1H-NMR spectra with those re-
ported in the literature[12] allowed their identification as
methyl 2,3,4,6-tetra-O-(p-bromobenzoyl)-α-galactopyrano-
side (3) and methyl 2,3,4-tri-O-(p-bromobenzoyl)-β-arabino-
pyranoside (4). The  configuration of the hexose (3; CD:
∆ε236 ϭ Ϫ30.0, ∆ε254 ϭ ϩ65.2; A ϭ ϩ90.5) and the  con-
figuration of the pentose (4; CD: ∆ε236 ϭ Ϫ33.1, ∆ε254
ϭ
ϩ100.2; A ϭ ϩ135.5) monosaccharide derivatives were de- droxy-30-norlanosta-8(9),24-diene-14-carboxylate.
A
sec-
duced by comparing their exciton split CD curves with ond saponin, also obtained from the butanolic extract of E.
those reported by Liu and Nakanishi.[12]
ferox, was named ectyoplaside B (2). The pseudomolecular
ion peak at m/z 969.4590 [MϪ ϩ Naϩ ϩ Hϩ] in the posi-
tive-ion HRFABMS and peaks at m/z 967.4500 [MϪ ϩ Naϩ
Ϫ Hϩ] and at m/z 945.4691 [MϪ] in the negative-ion
HRFABMS indicated the molecular formula C46H73NaO20
for 2.
The chemical structure of ectyoplaside B (2) was mainly
deduced by comparing its spectroscopic data with those of
1
compound 1. In particular, H- and 13C-NMR spectra of
2 (complete assignment shown in Tables 1 and 2) reveal
significant differences only in some protonic resonances of
the first two rings of the aglycone moiety. Indeed, in 2 also
the second methyl group at C-4 is replaced by a hydroxy-
methyl group [29-H2: δ ϭ 4.03 and 3.85 (d, J ϭ 13.2 Hz);
Next, H-NMR analysis of the apolar layer (fraction B, C-29: δ ϭ 60.2 (3J coupled with 3-H and 5-H in the HMBC
2.0 mg) obtained from the acid methanolysis showed this to spectrum)], in accordance with the molecular formula,
be composed of a complex mixture of degradation products which indicates only one more oxygen atom than 1. This
derived from the aglycone moiety of the saponin 1, prob- CH2OH group causes marked deshielding effects, above all
ably due to facile dehydration at C-22 (giving a conjugated on 3-H (δ ϭ 3.70 in 2, δ ϭ 3.27 in 1), 5-H (δ ϭ 1.57 in 2,
diene), and possibly also at C-4, as well as various products δ ϭ 1.35 in 1) and 6eq-H (δ ϭ 2.15 in 2, δ ϭ 1.63 in 1).
1
resulting from migrations of the double bond ∆8(9). This
A small amount of ectyoplaside B was then subjected to
mixture was analyzed no further.
acetylation, affording the peracetyl derivative 2a. Detailed
1
This extreme instability of the saponin 1 under acidic analysis of the H-NMR spectrum of 2a (reported in the
conditions convinced us that the intact aglycone of 1 could Experimental Section), performed with the aid of 2D-NMR
be obtained only by an enzymatic method. To this end, 5 experiments, revealed that the sugar portion of 2 is identical
mg of the saponin 1 was incubated for 72 h at 38°C in to that of 1, thus allowing a complete description of the
phosphate/citrate buffer (pH ϭ 5) with the glycosidase mix- structure of ectyoplaside B (2) as sodium 3β-O-[β--galac-
ture extracted from Charonia lampas. After neutralization topyranosyl-(1Ǟ4)-α--arabinopyranosyl-(1Ǟ2)-β--galac-
and filtration, the mixture obtained was dried and par- topyranosyl]-4β,22β,29-trihydroxy-30-norlanosta-8(9),24-
titioned between water and CHCl3. Chromatographic and diene-14-carboxylate.
spectroscopic evidence showed that the polar layer con-
Ectyoplasides A (1) and B (2) were tested in vitro for
tained the unreacted saponin and a mixture of partial glyco- cytotoxic activity with J774 (murine monocyte-macro-
sides, while small amounts of the nortriterpene 5 were pre- phage), WEHI164 (murine fibrosarcoma), and P388 (mu-
sent in the apolar fraction. The structure of compound 5, rine leukemia) cell lines. Both compounds were mildly cyto-
1
deduced by HRFABMS and H-NMR spectroscopy, is in toxic, exhibiting IC50 values ranging from 8.5 to 11.0 µg/
full agreement with that previously determined for the agly- mL.
cone moiety of the saponin 1. The 1H-NMR spectrum of 5
Saponins are widely encountered as secondary meta-
has been completely assigned by detailed inspection of bolites among the higher plants. From the marine environ-
COSY and HOHAHA spectra and is reported in the Exper- ment, saponins have mainly been isolated from echino-
imental Section.
derms,[13] such as starfish (steroidal glycosides) and sea cu-
The above data show that the structure of ectyoplaside A cumbers (lanostane glycosides), although some examples of
(1) is sodium 3β-O-[β--galactopyranosyl-(1Ǟ4)-α--ara- pregnane- or cholestane-type steroidal monoglycosides have
binopyranosyl-(1Ǟ2)-β--galactopyranosyl]-4β,22β-dihy- also been reported from gorgonians,[14] fish,[15] and soft co-
Eur. J. Org. Chem. 1999, 231Ϫ238
235

F. Cafieri, E. Fattorusso, O. Taglialatela-Scafati
FULL PAPER
rals.[16] In contrast, spongal saponins are extremely rare,
and although hundreds of Porifera species have been ana-
lyzed with regard to their chemical compositions, only two
or three saponin skeletons have yet been found, namely in
erylosides.[17Ϫ19] formoside[5] (mostly glycosides of penas-
terol derivatives, from Erylus spp.) and sarasinosides[20Ϫ23]
(mostly glycosides of norlanostane derivatives, from As-
teropus spp.).
Italy. The sponge (55 g dry weight after extraction), frozen immedi-
ately after collection and kept frozen until extraction, was first
homogenized and then exhaustively extracted with methanol (4 ϫ
500 mL). The obtained extract was diluted with MeOH/H2O (9:1)
and then partitioned against n-hexane (3 ϫ 500 mL) to yield an
apolar extract weighing 1.4 g. Successively, the water content of the
hydromethanolic phase was adjusted to 20% (v/v) and 40% (v/v)
and the solution was partitioned against CCl4 (3 ϫ 500 mL) and
CHCl3 (3 ϫ 500 mL), respectively, to afford a carbon tetrachloride
(0.8 g) and a chloroform (2.5 g) extract. Finally, all the MeOH was
It should be noted that all these triterpene saponins have
been isolated from sponges of the order Choristida (also evaporated from the methanolic layer, and the aqueous solution
thus obtained was partitioned against nBuOH. The butanol-soluble
material (5.7 g), which was the richest in saponins, was subjected
to MPLC purification on silica gel (230Ϫ400 mesh), eluting with a
solvent gradient system of increasing polarity from EtOAc to
MeOH. Fractions eluted with MeOH/EtOAc (7:3) were combined
and then further purified by reversed-phase HPLC (eluent: MeOH/
H2O, 8:2; flow rate 0.7 mL/min), yielding pure ectyoplasides A (1,
13.5 mg) and B (2, 4.5 mg).
known as Astrophorida) and so the isolation of ectyo-
plasides is especially remarkable because they represent,
along with the very recently reported ulososides,[24] the first
saponins found in a non-Choristida sponge (the Axinellida
order of E. ferox is far from the Choristida order from a
phylogenetic point of view). In addition, this paper must be
considered as the first report on the chemical composition
of an Ectyoplasia sponge.
25
Ectyoplaside A (1): White, amorphous solid. Ϫ [α]D ϭ ϩ3 (c ϭ
0.002 in MeOH). Ϫ IR (KBr): ν˜ ϭ 3406, 2924, 1635, 1573, 1454,
1348, 1261 cmϪ1. Ϫ 1H- and 13C-NMR (CD3OD): See Tables 1
and 2. Ϫ FABMS (positive ions, glycerol/thioglycerol matrix); m/z:
953, 497. Ϫ FABMS (negative ions, thioglycerol matrix); m/z: 951,
Experimental Section
General: Optical rotations (MeOH or CHCl3): PerkinϪElmer 192
polarimeter equipped with a sodium lamp (λ ϭ 589 nm) and a 10-
cm microcell. Ϫ IR (KBr): Bruker IFS-48 spectrophotometer. Ϫ
UV spectra (CH3CN): Beckman DU70 spectrophotometer. Ϫ CD
spectra (CH3CN): JASCO 500A polarimeter. Ϫ MS: Low- and
high-resolution FAB mass spectra (CsI ions, glycerol/thioglycerol
matrix): VG Prospec (Fisons) mass spectrometer equipped with an
FAB source. EI mass spectra (70 eV): VG Prospec (Fisons) mass
929. Ϫ HRFABMS (positive ions); m/z: 953.4734 [MϪ ϩ Naϩ
ϩ
Hϩ]; calcd. for C46H74NaO19: 953.4721. Ϫ HRFABMS (negative
ions); m/z: 951.4560 [MϪ ϩ Naϩ Ϫ Hϩ]; calcd. for C46H72NaO19
951.4573; m/z: 929.4731 [MϪ]; calcd. for C46H73O19: 929.4751.
:
Ectyoplaside A Decaacetate (1a): 3.5 mg of ectyoplaside A (1) was
peracetylated with Ac2O/pyridine at room temperature for 12 h
yielding 4 mg of ectyoplaside A decaacetate (1a) as a colorless,
amorphous oil. Ϫ [α]D25 ϭ ϩ11 (c ϭ 0.002 in CHCl3). Ϫ 1H NMR
(CDCl3, J in Hz): δ ϭ 5.45 (d, J ϭ 3.2, 1ЈЈ-H), 5.40 (d, J ϭ 3.1,
4ЈЈЈ-H), 5.28 (br. d, J ϭ 2.8, 4Ј-H), 5.22 (t, J ϭ 8.1, 2ЈЈЈ-H), 5.07
(dd, J ϭ 7.1, 2.8, 3Ј-H), 5.05 (24-H, overlapped), 4.95 (dd, J ϭ 8.1,
3.1, 3ЈЈЈ-H), 4.93 (22-H, overlapped), 4.85 (dd, J ϭ 9.5, 3.2, 2ЈЈ-H),
4.83 (br. d, J ϭ 9.5, 3ЈЈ-H), 4.59 (d, J ϭ 7.9, 1Ј-H), 4.48 (d, J ϭ
8.1, 1ЈЈЈ-H), 4.33 (br. s, 4ЈЈ-H), 4.20 (6Јa-H and 6Јb-H, overlapped),
4.18 (6ЈЈЈa-H and 6ЈЈЈb-H, overlapped), 4.05 (dd, J ϭ 7.9, 7.1, 2Ј-
H), 3.85 (t, J ϭ 6.6, 5Ј-H and 5ЈЈЈ-H), 3.50 (br. s, 5ЈЈ-H2), 3.33 (br.
d, J ϭ 11.5, 3-H), 2.50 (br. dd, J ϭ 11.5, 11.2, 2ax-H), 2.30 (m,
23a-H), 2.15 (11-H2, overlapped), 2.15Ϫ1.95 (10 CH3CO signals,
each s), 2.14 (23b-H, overlapped), 2.11 (7ax-H, overlapped), 2.05
(7eq-H, overlapped), 1.96 (12eq-H, overlapped), 1.95 (15a-H, over-
lapped), 1.88 (2eq-H, overlapped), 1.85 (1ax-H, overlapped), 1.85
(16a-H, overlapped), 1.83 (6ax-H, overlapped), 1.69 (br. s, 27-H3),
1.62 (br. s, 26-H3), 1.60 (m, 15b-H), 1.55 (dd, J ϭ 7.3, 5.5, 20-H),
1.48 (br. s, 29-H3), 1.37 (6eq-H, overlapped), 1.35 (12ax-H, over-
lapped), 1.35 (5-H, overlapped), 1.32 (16b-H, overlapped), 1.25
(1eq-H, overlapped), 1.25 (17-H, overlapped), 1.02 (br. s, 19-H3),
0.95 (d, J ϭ 7.3, 21-H3), 0.58 (br. s, 18-H3). Ϫ FABMS (positive
ions, CsI ions, glycerol matrix); m/z: 1373 [MϪ ϩ Naϩ ϩ Hϩ].
1
spectrometer equipped with an EI source. Ϫ H- (500.1 MHz) and
13C- (125.0 MHz) NMR spectra: Bruker AMX-500 spectrometer;
chemical shifts are referenced to the residual solvent signal
(CD3OD: δH ϭ 3.34, δC ϭ 49.0; CDCl3: δH ϭ 7.26). Methyl, meth-
ylene, and methine carbon atoms were distinguished by DEPT
experiments. Homonuclear 1H connectivities were determined by
performing COSY experiments. The 2D-HOHAHA experiment
was performed in the phase-sensitive mode (TPPI) with an MLEV-
17 sequence for mixing.[25] One-bond heteronuclear 1H-13C connec-
tivities were determined with the Bax-Subramanian[26] HMQC
pulse sequence on using a BIRD pulse 0.50 s before each scan in
order to suppress the signals originating from protons not directly
bound to 13C (interpulse delay set for JCH ϭ 135 Hz). During the
1
acquisition time, 13C broad-band decoupling was performed with
the GARP sequence. Two- and three-bond 1H-13C connectivities
were determined by HMBC[27] experiments optimized for a 2,3J of
9 Hz. Ϫ Medium-pressure liquid chromatography (MPLC): Büchi
861 apparatus with an SiO2 column (230Ϫ400 mesh). Ϫ HPLC
separations: Waters 501 apparatus equipped with a refractometer
detector and with Hibar RP-18 LiChrospher (250 ϫ 4 mm) col-
umns. Other separations were achieved using a Whatman Partisil
PXS M9 column with a UV detector (λ ϭ 260 nm). Ϫ GC-MS
analyses: Hewlett-Packard 5890 gas chromatograph with a mass-
selective detector MSD HP 5790 MS. A fused-silica column (25 m
ϫ 0.20 mm HP-5; cross-linked 25% Ph-Me silicone, 0.33 mm film
thickness) was used. Carrier gas: hydrogen, 10 mL/min; tempera-
ture 135°C.
Molecular Modeling: Computer modeling studies were carried out
using the Quanta/CHARMm 4.0 program (Molecular Simulations
Inc., 200 Fifth Avenue, Waltham, MA 02154) with a Silicon Graph-
ics Personal Iris 4D-35G computer. No explicit solvent molecules
were included in these calculations, but a distance-dependent di-
electric constant (RDIE) was used to partially compensate for the
absence of solvent. Molecular dynamics (MD) simulations involved
a heating period of 5.0 ps, followed by a 5.0 ps equilibration period
and then 100 ps of dynamics simulation. The time step of inte-
Collection, Extraction and Isolation: A specimen of Ectyoplasia
ferox was collected by hand along the coasts of San Salvador Is-
land, and identified by Prof. M. Pansini (Universita di Genova). A
`
voucher sample (no. SS 1305) has been deposited at the Diparti-
`
mento di Chimica delle Sostanze Naturali, Universita di Napoli, gration was 1 fs. During MD calculations, bond lengths involving
236 Eur. J. Org. Chem. 1999, 231Ϫ238

Ectyoplasides AϪB Ϫ Unique Triterpene Oligoglycosides
hydrogen atoms were kept fixed using the SHAKE algorithm. The 2.12 (7ax-H, overlapped), 2.11 (15a-H, overlapped), 2.05 (7eq-H,
FULL PAPER
coordinates produced by the simulation were saved every 0.5 ps,
giving 250 structures. Each of these was subjected to energy minim-
ization using the conjugated gradient protocol.
overlapped), 2.03 (16a-H, overlapped), 2.00 (12eq-H, overlapped),
1.92 (2eq-H, overlapped), 1.90 (dd, J ϭ 10.5, 8.2, 12ax-H), 1.85
(1ax-H, overlapped), 1.85 (6ax-H, overlapped), 1.79 (d, J ϭ 8.9,
6eq-H), 1.73 (br. s, 27-H3), 1.66 (br. s, 26-H3), 1.66 (15b-H, over-
lapped), 1.56 (dd, J ϭ 7.2, 5.0, 17-H), 1.47 (dd, J ϭ 7.0, 5.0, 20-
H), 1.38 (br. s, 29-H3), 1.34 (5-H, overlapped), 1.33 (16b-H, over-
lapped), 1.26 (1eq-H, overlapped), 1.12 (br. s, 19-H3), 0.93 (d, J ϭ
7.0, 21-H3), 0.67 (br. s, 18-H3).
Methanolysis and Silylation: 4 mg of ectyoplaside A (1) was dis-
solved in 1  HCl in 85% MeOH and the solution was heated at
70°C in a stoppered reaction vial for 2 h. After cooling, the reac-
tion mixture was neutralized with Ag2CO3, centrifuged, and the
supernatant was concentrated to dryness under N2. The residue
25
was then partitioned between water (2 ϫ 5 mL) and CHCl3 (2 ϫ Ectyoplaside B (2): White, amorphous solid. Ϫ [α]D ϭ Ϫ12 (c ϭ
5 mL) yielding a polar layer (fraction A, 2.2 mg) and an apolar
layer (fraction B, 2.0 mg). Fraction B was not analyzed further,
whereas fraction A was divided into two portions. The first of these
(0.8 mg) was dissolved in TRISIL Z (0.15 mL, N-trimethylsilylimi-
0.002 in MeOH). Ϫ IR (KBr): ν˜ ϭ 3500, 2914, 1635, 1573, 1454,
1348, 1261 cmϪ1. Ϫ 1H and 13C NMR (CD3OD): See Tables 1 and
2. Ϫ FABMS (positive ions, glycerol/thioglycerol matrix); m/z: 969.
Ϫ FABMS (negative ions, thioglycerol matrix); m/z: 967, 945. Ϫ
dazole in pyridine, Pierce Chemical Co.), the solution was left at HRFABMS (positive ions); m/z: 969.4590 [MϪ ϩ Naϩ ϩ Hϩ];
35°C for 15 min, and then analyzed by GC MS. Peaks obtained
for this mixture exhibited the same retention times and the same
mass spectra as those obtained from the standard persilylated
methyl glycosides of galactose and arabinose, prepared in the
same manner.
calcd. for C46H74O20Na: 969.4670. Ϫ HRFABMS (negative ions);
m/z: 967.4500 [MϪ ϩ Naϩ Ϫ Hϩ]; calcd. for C46H72O20Na:
967.4512; m/z: 945.4691 [MϪ]; calcd. for C46H73O20: 945.4701.
Ectyoplaside B Undecaacetate (2a): 2.0 mg of ectyoplaside B (2)
was peracetylated with Ac2O/pyridine at room temperature for 12
h, yielding 2.4 mg of ectyoplaside B undecaacetate (2a) as a color-
p-Bromobenzoylation and CD: A second portion of fraction A (1.4
mg) was dissolved in dry pyridine (1.5 mL) and treated with p-
bromobenzoyl chloride (20 mg) and a catalytic amount of 4-(di-
methylamino)pyridine. The mixture was stirred overnight at 60°C,
cold water was then added, and after 30 min, the mixture was ex-
tracted with CHCl3. The obtained extract was washed successively
with saturated aqueous NaHCO3 and water, and then the solvent
was evaporated under reduced pressure. The benzoate mixture thus
obtained was separated by HPLC: Whatman Partisil PXS M9 col-
umn, eluent n-hexane/diethyl ether (85:15), UV detector (λ ϭ 260
nm). Only two major peaks were collected.
25
1
less, amorphous oil. Ϫ [α]D ϭ Ϫ15 (c ϭ 0.002 in CHCl3). Ϫ H
NMR (CDCl3, J in Hz): δ ϭ 5.45 (d, J ϭ 3.2, 1ЈЈ-H), 5.40 (d, J ϭ
3.1, 4ЈЈЈ-H), 5.28 (br. s, 4Ј-H), 5.22 (t, J ϭ 8.1, 2ЈЈЈ-H), 5.05 (24-H,
overlapped), 4.95 (d, J ϭ 7.3, 3Ј-H), 4.94 (dd, J ϭ 8.1, 3.1, 3ЈЈЈ-H),
4.93 (22-H, overlapped), 4.85 (dd, J ϭ 9.8, 3.2, 2ЈЈ-H), 4.82 (d, J ϭ
12.9, 29a-H), 4.71 (br. d, J ϭ 9.8, 3ЈЈ-H), 4.46 (d, J ϭ 8.1, 1Ј-H),
4.46 (d, J ϭ 8.1, 1ЈЈЈ-H), 4.38 (br. s, 4ЈЈ-H), 4.22 (dd, J ϭ 10.7, 6.0,
6Јa-H), 4.16 (d, J ϭ 12.9, 29b-H), 4.08 (d, J ϭ 6.3, 6ЈЈЈa-H and
6ЈЈЈb-H), 4.05 (dd, J ϭ 10.7, 6.0, 6Јb-H), 4.02 (dd, J ϭ 8.1, 7.3, 2Ј-
H), 3.89 (3-H, overlapped), 3.87 (t, J ϭ 6.3, 5ЈЈЈ-H), 3.78 (t, J ϭ
6.0, 5Ј-H), 3.50 (br. s, 5ЈЈ-H2), 2.52 (br. dd, J ϭ 11.4, 11.0, 2ax-H),
2.30 (m, 23a-H), 2.15Ϫ1.95 (11 s, 11 CH3CO signals), 2.15 (23b-
H, overlapped), 2.11 (7ax-H, overlapped), 2.10 (11-H2, overlapped),
2.05 (7eq-H, overlapped), 1.96 (15a-H, overlapped), 1.95 (12eq-H,
overlapped), 1.88 (2eq-H, overlapped), 1.88 (6ax-H, overlapped),
1.85 (16a-H, overlapped), 1.80 (1ax-H, overlapped), 1.69 (br. s, 27-
H3), 1.60 (br. s, 26-H3), 1.58 (6eq-H, overlapped), 1.58 (15b-H,
overlapped), 1.55 (dd, J ϭ 6.6, 5.7, 20-H), 1.53 (5-H, overlapped),
1.38 (dd, J ϭ 10.3, 8.2, 12ax-H), 1.31 (16b-H, overlapped), 1.25
(17-H, overlapped), 1.22 (1eq-H, overlapped), 1.04 (br. s, 19-H3),
0.98 (d, J ϭ 6.6, 21-H3), 0.58 (br. s, 18-H3). Ϫ FABMS (positive
ions, CsI ions, glycerol matrix); m/z: 1431 [MϪ ϩ Naϩ ϩ Hϩ].
Methyl
2,3,4,6-Tetra-O-(p-bromobenzoyl)-α--galactopyranoside
(3): EIMS; m/z: 926. Ϫ 1H NMR (CDCl3): δ ϭ 7.92, 7.85, 7.82,
7.63, 7.61, 7.57, 7.53, 7.39 (8 d, J ϭ 8.8 Hz, each 2 H, ArH), 5.97
(d, J ϭ 3.5 Hz, 1 H, 4-H), 5.90 (dd, J ϭ 10.5 and 3.5 Hz, 1 H, 3-
H), 5.60 (dd, J ϭ 10.5 and 3.5 Hz, 1 H, 2-H), 5.28 (d, J ϭ 3.5 Hz,
1 H, 1-H), 4.58 (m, 2 H, 5-H and 6a-H), 4.37 (m, 1 H, 6b-H). Ϫ
CD (CH3CN): ∆ε236 ϭ Ϫ30.0, ∆ε254 ϭ ϩ65.2; A ϭ ϩ90.5.
Methyl 2,3,4-Tri-O-(p-bromobenzoyl)-β--arabinopyranoside (4):
1
EIMS; m/z: 713. Ϫ H NMR (CDCl3): δ ϭ 8.03, 7.88, 7.82, 7.69,
7.59, 7.57, 7.19 (each 2 H, ArH), 5.93 (dd, J ϭ 10.5 and 3.5 Hz, 1
H, 3-H), 5.77 (d, J ϭ 3.5 Hz, 1 H, 4-H), 5.70 (dd, J ϭ 10.5 and
3.5 Hz, 1 H, 2-H), 5.21 (d, J ϭ 6.5 Hz, 1 H, 1-H), 4.18 (d, J ϭ
12.5 Hz, 1 H, 5a-H), 3.97 (d, J ϭ 12.5 Hz, 1 H, 5b-H). Ϫ CD
(CH3CN): ∆ε236 ϭ Ϫ33.1, ∆ε254 ϭ ϩ100.2; A ϭ ϩ135.5.
Bioassays, Cells and Materials: WEHI164 cells (murine fibrosar-
coma cell line) were maintained in adhesion on Petri dishes with
DulbeccoЈs Modified EagleЈs Medium (DMEM) supplemented
with 10% heat-inactivated fetal bovine serum (FBS), 25 m
HEPES, penicillin (100 U/mL) and streptomycin (100 µg/mL). J774
cells (murine monocyte/macrophage cell line) were grown in sus-
pension culture, in Techne stirrer bottles spun at 25 rpm and incu-
bated at 37°C, in DMEM medium supplemented with 10% FBS,
25 m Hepes, glutamine (2 m), penicillin (100 U/mL) and strepto-
mycin (100 µg/mL). P388 cells (murine leukemia cell line) were
grown in adhesion on Petri dishes with L-15 (Leibovitz) medium
supplemented with 10% FBS, 25 m HEPES, penicillin (100 U/
mL) and streptomycin (100 µg/mL). All reagents for cell culture
were purchased from Cellbio; MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-phenyl-2H-tetrazolium bromide] and 6-mercaptopurine were
obtained from Sigma.
Enzymatic Hydrolysis: A solution of ectyoplaside A (1, 5 mg) in
phosphate/citrate buffer (1.5 mL) at pH ϭ 5.0 was incubated with
10 mg of glycosidase mixture from Charonia lampas (Scikagaku
kogyo) at 38°C for ca. 72 h. The mixture was then neutralized,
filtered, and the filtrate was partitioned between H2O and CHCl3.
The aqueous layer was concentrated to dryness and the residue was
found to contain salts, the unreacted saponin, and a mixture of
partial glycosides. The organic extract was dried with Na2SO4, fil-
tered, and concentrated in vacuo, to afford a fraction which was
shown to contain compound 5 (ca. 0.3 mg).
Compound 5: Colorless, amorphous oil. HRFABMS (positive ions,
glycerol matrix); m/z: 475.3430 [M ϩ Hϩ]; calcd. for C29H47O5:
1
475.3421. Ϫ H NMR (CD3OD): δ ϭ 5.15 (br. t, J ϭ 9.5, 24-H),
3.67 (dd, J ϭ 7.8, 5.6, 22-H), 3.21 (br. d, J ϭ 11.0, 3-H), 2.61 (br.
dd, J ϭ 10.1, 9.2, 2ax-H), 2.20 (11ax-H, overlapped), 2.17 (23a-H,
overlapped), 2.16 (11eq-H, overlapped), 2.12 (23b-H, overlapped),
Cytotoxic Activity: WEHI164, J774, P388 (4 ϫ 103 cells) were
placed in 96-well plates and allowed to adhere at 37°C in 5% CO2/
95% air for 2 h. Thereafter, the medium was replaced with 50 µL
Eur. J. Org. Chem. 1999, 231Ϫ238
237

F. Cafieri, E. Fattorusso, O. Taglialatela-Scafati
FULL PAPER
Marine Natural Products, Athens, 2Ϫ6 Nov. 1997, Book of
Abstracts, p. 40.
of fresh medium and then 75 µL aliquots of 1:2 (v/v) serial dilution
of test compounds 1 and 2 were added and the cells were incubated
for 72 h. The cells viability was assessed through an MTT conver-
sion assay[28]. After 72 h, 25 µL of MTT (5 mg/mL) was added
and the cells were incubated for a further 3 h. Subsequently, the
cells were lyzed and the dark-blue crystals were solubilized with
100 µL of a solution containing 50% (v/v) N,N-dimethylformamide
and 20% (w/v) SDS, adjusted to pH ϭ 4.5. The optical density
(OD) of each well was measured with a microplate spectrophoto-
meter (Titertek Multiskan MCC/340) equipped with a 620-nm fil-
ter. The viability of each cell line in response to treatment with
compounds 1 and 2 was calculated as:% dead cells ϭ 100 Ϫ (OD
treated/OD control) ϫ 100. The cytotoxic activities are expressed
as IC50 values (µg/mL): ectyoplaside A (1): WEHI164: 9.5, J774:
10.5, P388: 8.5; ectyoplaside B (2): WEHI164: 10.2, J774: 10.5,
P388: 9.5. All measurements were performed in triplicate and the
data reported are mean values.
[4]
S. M. Kupchan, R. W. Britton, M. F. Ziegler, C. W. Sigel, J.
Org. Chem. 1973, 38, 178Ϫ179.
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