Cytotoxic ceramides from the Red Sea sponge Spheciospongia vagabunda

Article abstract of DOI:10.1007/s00044-015-1394-9

Extracts of Egyptian marine organisms from the Red Sea were screened for their anticancer activity using sulforhodamine B assay. The extract of the Red Sea sponge Spheciospongia vagabunda possessed promising anticancer activity against HepG2 (liver cancer cell line) and MCF-7 (breast cancer cell line). Isolation of three new ceramides: N-[(2S,3S,4R)-1,3,4-trihydroxytetradecan-2-yl] tridecanamide (1), (R)-2′-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxypentacosan-2-yl] octadecanamide (2) and (R,Z)-2′-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxytricosan-2-yl) nonadec-10-enamide (3) was accomplished via bioassay-guided fractionation. Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR and HRMS. Compounds 2 and 3 displayed high potential cytotoxicity against HepG2 (IC₅₀ 24.7 and 21.3 μM, respectively) and MCF-7 (IC₅₀ 26.8 and 29.8 μM, respectively), compared with doxorubicin as control drug.

Full text of DOI:10.1007/s00044-015-1394-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1394-9
ORIGINAL RESEARCH
Cytotoxic ceramides from the Red Sea sponge Spheciospongia
vagabunda
Enas Elsayed Eltamany1 Amany K. Ibrahim1 Mohamed M. Radwan2,3
•
•
•
Mahmoud A. ElSohly2,4 Hashim A. Hassanean1 Safwat A. Ahmed1
•
•
Received: 31 August 2014 / Accepted: 27 June 2015
Ó Springer Science+Business Media New York 2015
Abstract Extracts of Egyptian marine organisms from
the Red Sea were screened for their anticancer activity
using sulforhodamine B assay. The extract of the Red Sea
sponge Spheciospongia vagabunda possessed promising
anticancer activity against HepG2 (liver cancer cell line)
and MCF-7 (breast cancer cell line). Isolation of three new
ceramides: N-[(2S,3S,4R)-1,3,4-trihydroxytetradecan-2-yl]
tridecanamide (1), (R)-20-hydroxy-N-[(2S,3S,4R)-1,3,4-tri-
hydroxypentacosan-2-yl] octadecanamide (2) and (R,Z)-20-
hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxytricosan-2-yl) non-
adec-10-enamide (3) was accomplished via bioassay-gui-
ded fractionation. Structure elucidation was achieved using
spectroscopic techniques, including 1D and 2D NMR and
HRMS. Compounds 2 and 3 displayed high potential
cytotoxicity against HepG2 (IC50 24.7 and 21.3 lM,
respectively) and MCF-7 (IC50 26.8 and 29.8 lM, respec-
tively), compared with doxorubicin as control drug.
Keywords Spheciospongia vagabunda Á Ceramide Á
Cytotoxic activity
Introduction
Sponges have been considered as a gold mine for the che-
mists. More than 12,000 compounds have been isolated from
marine sources with hundreds of new compounds still being
discovered every year (Joseph and Sujatha, 2011). Marine
sponges produced an enormous array of antitumor, antiviral,
anti-inflammatory, immunosuppressive, antibiotic, and
other bioactive molecules that can affect the pathogenesis of
many human diseases (Sipkema et al., 2005).
Marine sponges were used to produce several potent
cytotoxic bioactive compounds, including alkaloids, ster-
oids, terpenes, peptides, macrolides, polyketides and sph-
ingosine-related compounds like sphingosine-4-sulfates
(Salma et al., 2009).
Many highly bioactive sphingolipids have been discov-
ered from marine organisms and were able to act as bio-
mediators of activation, cell agglutination, intracellular
communication and cell development (Tan and Chen,
2003). Sphingolipids may be particularly important as a
source of multiple signalling molecules, with impact on
cancer development, particularly colon cancer. The sph-
ingolipid-induced anticancer effect is probably derived
from breakdown products such as ceramide and sphin-
gosine (Rui-Dong, 2005). Jaspine B, an anhydrophy-
tosphingosine, inhibited the growth of human and murine
melanoma cells (and also of human cervix epitheloid car-
cinoma) (Safaeian et al., 2009). Moreover, an agelasphin
derivative, a glycosphingolipid isolated from the sea
sponge Agelas mauritiamus, has been in phase I clinical
trials for the treatment of cancer (Salomon et al., 2004).
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-015-1394-9) contains supplementary
material, which is available to authorized users.
& Safwat A. Ahmed
safwat_aa@yahoo.com
1
Department of Pharmacognosy, Faculty of Pharmacy, Suez
Canal University, Ismailia, Egypt
2
National Center for Natural Products Research, School of
Pharmacy, University of Mississippi, University, MS 38677,
USA
3
Pharmacognosy Department, Faculty of Pharmacy,
Alexandria University, Alexandria, Egypt
4
Department of Pharmaceutics, School of Pharmacy,
University of Mississippi, University, MS 38677, USA
123

Med Chem Res
The marine Red Sea environment, being one of the most
biodiverse in the world, offers a potential for producing
novel drugs and prototypes. There are few studies about
chemical constituents of the marine sponge Spheciospon-
gia vagabunda. Steroidal compounds as well as phthalate
derivatives have been isolated from Spheciospongia sp.
(Safaeian et al., 2009; Ma et al., 2004; Whitson et al.,
2008). The increasing threat of cancer has initiated a
renewal of interest in the search for novel anticancer
agents. An encouraging number of marine-organism-
derived anticancer drugs are either already in human cancer
clinical trials or advancing in preclinical development
toward that vitally important objective. Others are in earlier
stages of development (Pettit et al., 2004). As our research
efforts are oriented to discover anticancer drug candidates
based on marine organism constituents, we have investi-
gated a promising lead offered by the marine sponge S.
vagabunda from the Red Sea. We herein report the isola-
tion and identification of three new ceramides (1, 2 and 3)
as well as evaluation of their cytotoxicity against liver and
breast cancer cell lines (Fig. 1).
temperature (-24 °C) until processed. Voucher specimens
were deposited at the Zoological Museum of the University
of Amsterdam under registration numbers ZMAPOR19759
and in the herbarium section of Pharmacognosy Department,
Faculty of Pharmacy, Suez Canal University, Ismailia,
Egypt, under registration number SAA-14.
Extraction, bioassay-guided fractionation
and isolation
Spheciospongia vagabunda was dried (350 g), grounded
and extracted with a mixture of MeOH/CH2Cl2 (1:1)
(3 9 2 L) at room temperature. The extract was evaporated
under vacuum to afford 25 g residue. The residue was
subjected to vacuum liquid chromatography on a flash
silica gel using hexane, EtOAc and MeOH gradient. The
crude extract as well as its fractions was assayed for their
anticancer activity against both HepG2 and MCF-7 cell
lines. The EtOAc-eluted fraction (2 g) showed promising
anticancer activity and then subjected to silica gel column
chromatography eluted with gradients of hexane/EtOAc
(50:50, (v/v)—100 % EtOAc) and EtOAc–MeOH (95:0—
100 % MeOH). Ten subfractions were collected and sub-
mitted to cytotoxic activity. The bioactive subfraction was
rechromatographed on silica gel CC eluted with hexanes/
EtOAc (70:30—100 % EtOAc) and then EtOAc/MeOH
(95:5–75:25) to afford compound 1 (6.3 mg) and a mixture
of 2 and 3, which was further purified by Sephadex LH-20
using CH2Cl2–MeOH (1:1) to give 2 (9.6 mg) and 3
(8.2 mg). The isolated pure compounds were evaluated for
their anticancer activity.
Materials and methods
General
1H NMR (400 MHz), 13C NMR (100 MHz), DEPT-135
and 2D-NMR spectra were recorded using the residual
solvent signal as an internal standard on a Varian AS 400.
IR spectra were measured on a Bruker Tensor 27. High-
resolution mass spectra were measured using a Bruker
BioApex.
Ceramide hydrolysis
Fatty acid methyl esters were identified using Hewlett
Packard (HP) gas liquid chromatography, series model
6890 equipped with flame ionization detector (FID). A
capillary column (HP-INNOWAX, polyethylene glycol,
30 m 9 530 lm, film thickness 1.00 lm) was used in
separation of fatty acids. The injector port temperature was
set at 250 °C (splitless mode) and a pressure of 14.81 psi
and the detector cell at 275 °C. The flow rate of the carrier
gas (N2) was 30 mL/min. The initial column temperature
was 70 °C and increased to 200 °C by the rate of 4 °C/
min., then isothermally for a total run time of 32.5 min.
Pre-coated silica gel G-25 UV254 plates were used for
thin-layer chromatography (20 9 20 cm) (E. Merck). Sil-
ica gel Purasil 60A, 230–400 mesh was used for flash
column chromatography (Whatman).
Three milligram of each isolated compound was heated
separately with 5 mL of 1 M HCL in 15 mL of MeOH for
4 h at 90 °C. The mixture was extracted with hexane, and
the hexane layer was concentrated under vacuum to give the
hydroxyl fatty acid methyl esters of 2 and 3, while in the
case of compounds 1, fatty acid methyl ester was obtained.
The hydroxyl fatty acid methyl esters of 2 and 3 were
separately subjected to Lemieux oxidation (Sun et al., 2006;
Kuksis, 1978; Lemieux and Von Rudlo, 1955). In this
reaction, 0.023 mol/L aqueous KMnO4, 0.09 mol/L NaIO4
(2.0 mL), t-BuOH (1.0 mL) and 0.04 mol/L aqueous
K2CO3 (0.5 mL) were slowly added to the hydroxyl fatty
acid methyl esters of 2 and 3. The mixtures were stirred for
24 h at room temperature, quenched with 2.5 mol/L H2SO4
(0.5 mL) and saturated aqueous Na2SO3, and then extracted
with Et2O (5 9 3 mL). The organic layer was dried over
Na2SO4. Finally, the concentrated, dried residue was
esterified with excess CH2N2 in Et2O overnight. The
resulting esters were analyzed using GC–MS.
Animal organism, collection and identification
The sponge S. vagabunda was collected from Ras Mohamed
at the Egyptian Red Sea, air-dried and stored at low
123

Med Chem Res
Fig. 1 Chemical structure for
compounds 1, 2 and 3
O
13`
HN
2
1`
OH
4
HO
3
1
OH
1
HO
O
18`
2`
NH
OH
HO
OH
2
19`
HO
O
11`
2`
10`
NH
OH
HO
OH
3
1
for C42H84NO5: 682.6349); H NMR and 13C NMR see
Isolates
Table 1.
N-[(2S,3S,4R)-1,3,4-trihydroxytetradecan-2-yl] tridecanamide
(1): white substance; HR–ESI–MS (positive ion mode) m/
z = 456.4018 [M?H]? (calcd. for C27H54NO4: 456.4053); 1H
NMR and 13C NMR see Table 1.
(R)-20-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxypentacosan-
2-yl] octadecanamide (2): white substance; HR–ESI–MS
(positive ion mode) m/z = 698.6694 [M?H]? (calcd.
for C43H88NO5: 698.6662); 1H NMR and 13C NMR see
Table 1.
Cytotoxic activity
The cytotoxicity of the investigated extracts as well as the
isolated compounds was measured by the sulforhodamine
B (SRB) assay as described by (Skehan et al., 1990). This
was performed on three human cell lines: HepG2 (liver
cancer cell line), MCF-7 (breast cancer cell line) and HFB-
4 (normal human melanocytes) which were kindly pro-
vided by the National Cancer Institute (Kasr El Ainy
Street, Cairo, Egypt). The cells were plated into 96-mul-
tiwell plates (104 cells/well) for 24 h before treatment with
(R,Z)-20-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxytricosan-
2-yl) nonadec-10-enamide (3): white substance; HR–ESI–
MS (positive ion mode) m/z = 682.6406 [M?H]? (calcd.
123

Med Chem Res
Table 1 1H-(400 MHz) and 13C-NMR data (100 MHz) of compounds 1, 2 and 3 in C6D5N
Compound 1 Compound 2
Compound 3
Position
dC
dH
Position
dC
dH
Position
dC
dH
1
62.2
52.4
76.0
73.4
33.0
26.9
30.0
28.0
39.6
28.5
23.1
13.0
175.0
36.9
25.8
30.0
32.4
22.5
13.0
–
4.75 (2H, m)
1
62.8
53.0
76.3
74.0
32.9
30.4
30.8
30.7
36.5
30.7
23.7
15.1
175.0
73.2
32.9
30.4
30.8
23.7
15.1
–
4.53 (2H, m)
5.13 (1H, m)
4.41 (1H, m)
4.31 (1H, m)
1.24(2H, m)
1
62.0
53.0
76.3
73.4
33.3
30.0
30.0
26.7
37.7
29.7
23.0
14.4
175.3
72.5
35.7
25.9
30.0
29.7
128.3
132.8
29.7
30.1
35.8
23.0
14.4
–
4.63 (2H, m)
5.14 (1H, m)
4.52 (1H, m)
4.37 (1H, m)
1.27 (2H, m)
1.27(2H, m)
1.27(2H, m)
1.27 (2H, m)
1.27 (2H, m)
1.27 (2H, m)
1.27 (2H, m)
0.89 (3H, t, J = 6.8)
–
2
5.43 (1H, m)
2
2
3
4.46 (1H, m)
3
3
4
4.33 (1H, m)
4
4
5
1.24 (2H, m)
5
5
6
1.24 (2H, m)
6
1.24(2H, m)
6
7–9
10
11
12
13
14
10
1.24 (2H, m)
7–20
21
22
23
24
25
10
20
30
40
50–160
170
180
NH
1.24 (2H, m)
1.24 (2H, m)
1.24 (2H, m)
1.24 (2H, m)
1.24 (2H, m)
0.88 (3H, t, J = 6.8)
–
7–18
19
20
21
22
23
10
20
30
40
50–80
90
100
110
120
120–160
170
180
190
NH
1.24 (2H, m)
1.24 (2H, m)
1.24 (2H, m)
1.24 (2H, m)
0.94 (3H, m, J = 6.8)
–
20
30
2.10 (2H, t, J = 7.6)
1.95 (2H, m, J = 7.6)
1.24 (2H, m)
4.64 (1H, m, J = 7.6)
2.05 (2H, m)
1.24 (2H, m)
1.24 (2H, m)
1.24 (2H, m)
0.88 (3H, t, J = 6.8)
8.61 (1H, d, J = 8.4)
4.63 (1H, m)
2.05 (2H, m)
1.27 (2H, m)
1.27 (2H, m)
2.25 (2H, m)
6.32 (1H, m)
6.70 (1H, m)
2.25 (2H, m)
1.27 (2H, m)
1.27(2H, m)
1.27 (2H, m)
0.89(3H, t, J = 6.8)
8.60 (1H, d, J = 8.4)
40–100
110
120
130
NH
1.24 (2H, m)
1.24 (2H, m)
0.94 (3H, m, J = 6.8)
8.37 (1H, d, J = 8.4)
the extract and the pure compound to allow attachment of
cells to the plate’s wall. Cells were routinely cultured in
Dulbecco’s modified Eagle’s medium (DMEM). Different
concentrations of the tested samples (0, 50, 100, 150 and
200 lg/mL in DMSO) were added to the cell monolayer.
Triplicate wells were prepared for each individual dose.
Monolayer cells were incubated with the extracts under test
for 48 h, at 37 °C and in atmosphere of 5 % CO2. After 48 h,
the cells were fixed, washed and stained with SRB stain.
Excess stain was washed with acetic acid, and the attached
stain was recovered with Tris–EDTA buffer. Color intensity
was measured in an ELISA reader. Moreover, the IC50 (the
dose that reduces survival to 50 %) was calculated.
19.7 (lg/mL), respectively, bioactivity-guided chromato-
graphic processing was carried out. Three pure compounds
were isolated (Fig. 1) and spectroscopically analyzed.
Here, the structure elucidation procedures for the three
compounds are described.
Compound 1 was isolated as white amorphous powder,
and its HRESIMS mass spectrum displayed a pseudo-
molecular ion peak at m/z 456.4018 [M-H]-, which when
combined with detailed 13C NMR spectrum and DEPT
analysis indicated a molecular formula of C27H54NO4,
1
representing one unit of unsaturation. The H NMR spec-
trum in C5D5N showed resonances of an amide proton
doublet at d 8.37 (1H, d, J = 8.4 Hz) and protons of a long
methylene chain at d 1.24, indicating a sphingolipid
skeleton (Ahmed et al., 2008). Characteristic resonances of
a 2-amino-1,3,4-triol unit of the hydrocarbon chain were
observed at dH 5.43 (1H, m), 4.75 (2H, m), 4.46 (1H, m)
Results and discussion
1
Based on the potential antitumor activity of the S. vaga-
bunda extract against HepG2 (liver cancer cell line) and
MCF-7 (breast cancer cell) with IC50 values of 28.1 and
and 4.33 (1H, m) in the H NMR spectrum, corresponding
to carbon resonances at dC 52.4 (CHN), 62.2 (CH2O), 76.0
(CHOH) and 73.4 (CHOH) in the HMQC spectrum,
123

Med Chem Res
1
respectively. In addition, the H NMR spectrum showed
resonances corresponding to aliphatic hydrocarbons at d
0.94 (6H, t, J = 6.8 Hz, H-26 and H-200), 1.24 (overlapped
H, m), 1.95 (2H, m, H-30) and 2.50 (t, J = 7.6 Hz, H-20).
The 13C NMR spectrum showed resonances due to two
terminal methyl groups at dH 0.94 13.0 and an amide
carbonyl at d 175.0. Analysis of the 1H–1H COSY, HMQC
and HMBC spectra led to the assignment of the proton and
carbon signals for 1. The positions of the hydroxyl groups
sphingosine-type cerebroside. The structure of 2 was
assigned by analysis of 2D NMR data, including HMQC
and HMBC experimental methods. Examination of the
HMBC spectrum showed that the amide methine C-2 was
coupled with the NH doublet at d 8.61, the oxymethylene
protons at d 4.53 (H-1) and the oxymethine proton at d 4.41
(H-3). The 13C NMR spectrum showed signals due to two
terminal methyl groups in aliphatic hydrocarbon chains at d
15.1 and an amide carbon at d 175.0. The HMBC corre-
lations between H-3 and the carbons at d 74.0 and d 53.0
also showed that there was another hydroxyl function at
C-4. The HMBC correlation of the carbonyl at 175.0 to
H-20 confirmed the presence of a side chain of an R-hy-
droxyl fatty acid. GC–MS analysis of the fatty acid methyl
ester of 2 was carried out after hydrolysis and oxidation in
which compound 2 was treated with aqueous HCl/MeOH
resulting in methanolysis, giving R-hydroxyacid methyl
ester and phytosphingosine as the products. The R-hy-
droxyacid methyl ester was converted into a fatty acid
methyl ester via Lemieux oxidation using KMnO4 and
NaIO4 (Sun et al., 2006; Kuksis, 1978; Lemieux and Von
Rudlo, 1955).
were confirmed from the H–1H COSY [H2-1/H-2, H-2/H-
1
3, H-3/H-4 and H-4/H2-5] correlations and also from the
HMBC [H2-1/C-2 (2JCH), H2-1/C-3 (3JCH), H-2/C-3 (2JCH),
H-3/C-2 (2JCH), H-3/C-4 (2JCH), H-4/C-2 (3JCH), H-4/C-6
(3JCH)] correlations, which led to the assignment of C-1/C-
2/C-3/C-4. The GC–MS analysis of the fatty acid methyl
ester of 1 was carried out after hydrolysis, which exhibited
a peak at m/z 228 corresponding to a C13 fatty acid methyl
ester, indicating the presence of terminal fatty acid tride-
canoic acid (C13:0).
The configuration of the ceramide moieties of 1 was
1
assigned by comparing the physical data, H NMR and 13C
NMR with analogs reported in the literature, which indicated
the absolute configurations at C-2, C-3 and C-4 to be 2S,
3S and 4R, respectively (Meyer and Guyot, 2002). Detailed
analysis of the COSY and HMBC correlations was found to
be in complete agreement with the proposed structure for 1.
Therefore, 1 was determined to be N-[(2S,3S,4R)-1,3,4-tri-
hydroxytetradecan-2-yl] tridecanamide.
The GC–MS analysis of the fatty acid methyl ester of 2
gave a peak with a molecular ion of m/z 284.2 corre-
sponding to a C17:0 fatty acid methyl ester (heptadecanoic
methyl ester).
The configuration of the ceramide moieties was assigned
1
by comparing its physical data, H NMR and 13C NMR
Compound 2 Its structure elucidation began with anal-
ysis of the HRMS data. The HRESIMS mass spectrum of 2
displayed a pseudo-molecular ion peak at m/z 698.6694
[M?H]?, which when combined with detailed analysis of
the 13CNMR spectrum and DEPT, indicated a molecular
formula of C43H88NO5 representing one unit of unsatura-
(C5D5N) with analogs reported in the literature, in which
the optical rotation ?17.41 (c 1.00, MeOH) and the
chemical shifts of C-2 (d 53.0), C-3 (d 76.3), C-4 (d 74.0)
and C-20 (d 73.2) in C5D5N were in good agreement with
those of the known natural ceramide gracilamide B (Sun
et al., 2006). This evidence indicated the absolute config-
urations of C-2, C-3, C-4 and C-20 to be 2S, 3S, 4R and 20R,
respectively. Detailed analysis of the COSY and HMBC
correlations was found to be in complete agreement with
the proposed structure of 2. The structure of 2 was
1
tion. The H NMR spectrum of 2 displayed typical reso-
nances of an aliphatic chain (d 1.24) and several multiplets
from d 4.31–5.13, together with an amide function at d 8.61
(1H, d, J = 8.4 Hz), which was reminiscent of
a
Table 2 IC50 values of the compounds 1, 2, 3 and doxorubicin on breast cancer cell line (MCF-7), liver cancer cell line (HEPG2) and normal
human melanocytes (HFB-4)
Sample
Cell lines
HepG2 IC50 (lM)
MCF-7 IC50 (lM)
HFB-4
Compound 1
107.1 0.07
24.7 0.18
21.3 0.1
[200 lM
[200 lM
[200 lM
[ 200 lM
14.7
Compound 2
26.8 0.02
29.8 0.13
5.46 0.08
Compound 3
Doxorubicin (positive control)
7.36 0.03
HepG2: liver cancer cell line, MCF-7: breast cancer cell line and HFB-4: human melanocytes (normal cell line). Each data point represents the
mean SD of three independent experiments (significant differences at p \ 0.05). Compounds with IC50 values [200 lM are considered
inactive
123

Med Chem Res
correlation of the carbonyl at dC 175.3 to H-20 confirmed
the presence of a side chain of an R-hydroxyl fatty acid.
GC–MS analysis of the fatty acid methyl ester of 3 was
carried out after hydrolysis and oxidation in which com-
pound 3 was treated with aqueous HCl/MeOH resulting in
methanolysis, giving a R-hydroxyacid methyl ester and
phytosphingosine as the products. The R-hydroxyacid
methyl ester was converted into a fatty acid methyl ester
via oxidation using KMnO4 and NaIO4 (Sun et al., 2006;
Kuksis, 1978; Lemieux and Von Rudlo, 1955).
MCF-7
1.2
1
0.8
0.6
0.4
0.2
0
Compound 1
compound 2
Compound 3
-50
0
50
100
150
Concentration:
M
The GC–MS analysis of the fatty acid methyl ester of 3
gave a peak with a molecular ion of m/z 296 which cor-
responded to a C18:1 fatty acid methyl ester (9-octadecenoic
acid (Z) methyl ester).
Fig. 2 Line graph representing the effect of the compounds 1, 2 and
3 on breast carcinoma cell line (MCF-7)
The configuration of the ceramide moieties was assigned
by comparison of the physical data, 1H NMR and 13C NMR
(C5D5N) with analogs as reported in the literature, in which
the optical rotation ?17.41 (c 1.00, MeOH) and the
chemical shifts of C-2 (d 53.0), C-3 (d 76.3), C-4 (d 73.4)
and C-20 (d 72.5) in C5D5N were in good agreement with
those of the known natural ceramide gracilamide B (Sun
et al., 2006). This evidence indicated the absolute config-
urations of C-2, C-3, C-4 and C-20 to be 2S, 3S, 4R and 20R,
respectively. Detailed analysis of the COSY and HMBC
correlations was found to be in complete agreement with
the proposed structure of 3. The structure of 3 was deter-
mined to be (R,Z)-20-hydroxy-N-[(2S,3S,4R)-1,3,4-trihy-
droxytricosan-2-yl)nonadec-10-enamide.
HepG2
1.2
1
0.8
0.6
Compound 1
0.4
0.2
0
Compound 2
Compound 3
-50
0
50
100
150
Concentration: µM
Fig. 3 Line graph representing the effect of the compounds 1, 2 and
3 on liver carcinoma cell line (HepG2)
Anticancer activity of S. vagabunda extract, fractions as
well as the pure compounds was measured by SRB assay
(Table 2; Figs. 2, 3). Screening of 1, 2 and 3 resulted in
promising anticancer activities against MCF-7 and HEPG2
cell lines. The inhibitory properties of these compounds are
compared with standard doxorubicin. Compound 3 has the
highest activity against HEPG2 with IC50 21.3 lM.
However, compound 2 possessed the highest activity
against MCF-7 with IC50 value of 26.8 lM, while com-
pound 1 has the lowest activity against HepG2 and was
inactive against MCF-7, IC50 value [200 lM. All the
tested compounds showed selective cytotoxicity against
tumor cell lines as they did not possess cytotoxic effect on
HFB-4 normal cell line.
determined to be (R)-20-hydroxy-N-[(2S,3S,4R)-1,3,4-tri-
hydroxypentacosan-2-yl] octadecanamide.
Compound 3 The HRESIMS mass spectrum of 3 dis-
played a pseudo-molecular ion peak at m/z 682.6406
[M?H]?, which when combined with detailed analysis of
the 13CNMR spectrum and DEPT, indicated a molecular
formula of C42H84NO5 representing two units of unsatu-
1
ration. The H NMR spectrum of 3 displayed typical res-
onances of an aliphatic chain (dH 1.27) and characteristic
resonances of a 2-amino-1,3,4,20-tetraol unit of the
hydrocarbon chain were observed at d 5.10 (H-2), d 4.63
(H-1 and H-20), d 4.52 (H-3) and d 4.31 (H-4), together
with an amide function at d 8.60 (1H, d, J = 8.4 Hz) and
two olefinic protons at 6.32 (m, H-100), 6.70 (m, H-110)
which was reminiscent of an unsaturated sphingosine-type
cerebroside. The structure of 3 was assigned by analysis of
2D NMR data, including HMQC and HMBC experimental
methods. Examination of the HMBC spectrum showed that
the amide methine C-2 was coupled with the NH doublet at
d 8.60.
Supporting information
1H and 13C-NMR spectroscopic data for compounds 1, 2
and 3 as well as the effect of the isolated compounds on the
cancer cell lines are available as supporting information.
1H and 13C NMR data for 3 were almost identical to 2.
However, the difference between 3 and 2 was the presence
of unsaturated double bond [dH 6.32 (m, H-100), 6.70 (m,
H-110); dC 128.3 (C-100), 132.8 (C-110)]. The HMBC
Acknowledgments The authors are grateful to R.W.M. van Soest,
Faculty of Science, Zoological Museum Amsterdam, for taxonomic
identification of the sponge samples. Thanks are also due to the
123

Med Chem Res
Egyptian Environmental Affairs Agency (EEAA) for facilitating
sample collection along the coasts of the Red Sea.
Rui-Dong D (2005) Anticancer compounds and sphingolipid
metabolism in the colon. In Vivo 19:293–300
Safaeian S, Hosseini H, Abbas Pour Asadolah A, Farmohamadi S
(2009) Antimicrobial activity of marine sponge extracts of
offshore zone from Nay Band Bay Iran. J Mycol Med 19:11–16
Salma Y, Lafont E, Therville N, Carpentier S, Bonnafe M-J, Levade
Conflict of interest The authors declare no conflict of interest.
´
T, Genisson Y, Andrieu-Abadie N (2009) The natural marine
References
anhydrophytosphingosine, Jaspine B, induces apoptosis in
melanoma cells by interfering with ceramide metabolism.
Biochem Pharm 78:477–510
Ahmed SA, Khalifa SI, Hamann MT (2008) Antiepileptic ceramides
from the Red Sea sponge Negombata corticata. J Nat Prod
71:513–515
Joseph B, Sujatha S (2011) Pharmacologically important natural
products from marine sponges. Indian J Nat Prod 4:5–12
Kuksis A (1978) Hand book of lipid research fatty acids and
glycerides; separation and determination of structure of fatty
acids, vol 1. Springer, New York, pp 1–76
Lemieux RU, Von Rudlo E (1955) Periodate-permanganate oxida-
tions I. Oxidation of olefins. Can J Chem 33:1701–1709
Ma W, Xiao D, Deng S (2004) Studies on chemical constituents of
marine sponge Spheciospongia vagabunda from the South China
Sea. Chin J Mar Drugs 23:15–17
Salomon CE, Magarvey NA, Sherman DH (2004) Merging the
potential of microbial genetics with biological and chemical
diversity: an even brighter future for marine natural products
drug discovery. Nat Prod Rep 21:105–121
Sipkema D, Franssen M, Osinga R, Tramper J, Wijffels R (2005)
Marine sponge as pharmacy. Mar Biotechnol 7:142–162
Skehan P, Storeng R, Scudiero D, Monks A, Mcmahon J, Vistica D,
Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New
colorimetric cytotoxicity assay for anticancer-drug screening.
J Nat Cancer Inst 82:1107–1112
Sun Y, Xu Y, Liu K, Hua H, Zhu H, Pei Y (2006) Gracilarioside and
gracilamides from the Red Alga Gracilaria asiatica. J Nat Prod
69:1488–1491
Tan RX, Chen JH (2003) The cerebrosides. Nat Prod Rep 20:509–534
Whitson E, Bugni TS, Chockalingam PS, Concepcion GP, Harper
MK, He M, Hooper JNA, Mangalindan GC, Ritacco F, Ireland
CM (2008) Spheciosterol sulfates, PKCf inhibitors from a
philippine sponge Spheciospongia sp. J Nat Prod 71:1213–1217
Meyer M, Guyot M (2002) New Spingosines from the marine sponge
Grayella cyatophora. J Nat Prod 65:1722–1723
Pettit GR, Xu J-P, Chapuis JC, Pettit RK, Tackett LP, Doubek DL,
Hooper JNA, Schmidt JM (2004) Antineoplastic agents. 520.
Isolation and structure of irciniastatins A and B from the Indo-
Pacific marine sponge Ircinia ramosa.
47:1149–1152
J
Med Chem
123