Antibacterial sphingolipid and steroids from the black coral Antipathes dichotoma

Article abstract of DOI:10.1248/cpb.58.1635

From the black coral Antipathies dichotoma, a sphingolipid (2S*,3S*,4E,8E)-2N-[tetradecanoyl]-4(E),8(E)-icosadiene-1, 3-diol (1) and a steroid (22E)-methylcholesta-5,22-diene-1 α,3 β,7 α-triol (2) were isolated. Other known compounds, 3β,7 α-dihydroxy-cholest-5-ene (3) (22E,24S),5α,8α-epidioxy-24- methylcholesta-6,22-dien-3β-ol (4) and (22E,24S),5α,8α- epidioxy-24-methylcholesta-6,9(11),22-trien-3β-ol (5). The structures were established on the basis of NMR spectroscopic analysis and comparison with literature. The antibacterial activity of five compounds was evaluated.

Full text of DOI:10.1248/cpb.58.1635

December 2010
Note
Chem. Pharm. Bull. 58(12) 1635—1638 (2010)
1635
Antibacterial Sphingolipid and Steroids from the Black Coral Antipathes
dichotoma
a
b
a
,a
*
Sultan Samran AL-LIHAIBI, Seif-Eldin Nasr AYYAD, Fekry SHAHER, and Walied Mohamed ALARIF
a Department of Marine Chemistry, Faculty of Marine Sciences, King Abdulaziz University; P. O. Box 80207, Jeddah
21589, Saudi Arabia: and b Department of Chemistry, Faculty of Science, King Abdulaziz University; P. O. Box 80203,
Jeddah 21589, Saudi Arabia. Received May 11, 2010; accepted August 31, 2010; published online September 21, 2010
From the black coral Antipathies dichotoma, a sphingolipid (2S*,3S*,4E,8E)-2N-[tetradecanoyl]-4(E),8(E)-
icosadiene-1, 3-diol (1) and a steroid (22E)-methylcholesta-5,22-diene-1a,3b,7a-triol (2) were isolated. Other
known compounds, 3b,7a-dihydroxy-cholest-5-ene (3) (22E,24S),5a,8a-epidioxy-24-methylcholesta-6,22-dien-
3b-ol (4) and (22E,24S),5a,8a-epidioxy-24-methylcholesta-6,9(11),22-trien-3b-ol (5). The structures were estab-
lished on the basis of NMR spectroscopic analysis and comparison with literature. The antibacterial activity of
five compounds was evaluated.
Key words black coral; Antipathes dichotoma; sphingolipid; trihydroxy steroid; antibacterial
Antipathes dichotoma (PALLAS) belongs to zoanthoid black the positions of double bonds and the absolute configuration
coral. It has some pharmaceutical uses, such as relieving of 1, the acid methanolysis method of Gaver and Sweeley11)
fever and softening hard mass. Few literatures about the which yield a fatty acid methyl ester (FAME) methyl tetrade-
chemical constituents of black corals that reported nine canoate m/z 242 detected by GCMS, the presence of tetrade-
steroids from A. subpinnata,1,2) and four alkaloids from A. canoyl moiety confirmed by the characteristic ion at m/z 211
dichotoma.3) Sphingolipids form a biologically important [CH3(CH2)12CO]ϩ. So the molecular formulas of FAME and
class of compounds,4) some of which have been reported to sphingosine are C15H30O and C20H39NO2, respectively. The
exhibit antihepatotoxic, antitumor and immunostimulatory double bonds and hydroxyl groups should be in sphingosine
activities,5,6) inhibition of atherosclerosis7) and as secondary moiety and their positions could be determined by inspection
messengers.8)
of 1H–1H COSY spectrum, two methylene (C-1) protons at d
3.70 and 3.95 correlated with the methine proton (C-2) at d
3.91 which is correlated with the methine (C-3) proton at d
Results and Discussion
Compound 1 has a molecular formula of C34H65NO3 4.32, the methine (C-3) proton at d 4.32 correlated with the
which was determined from high resolution (HR)-FAB-MS olefinic (C-4) proton at d 5.54 (dt, Jϭ15.0, 6.0 Hz) which is
data (m/z 558.4876 [MϩNa]ϩ). Calcd 558.4862, electron in turn correlated with another olefinic (C-5) proton at d 5.78
ionization-mass spectra (EI-MS) (m/z 535) and 13C-NMR. (dt, Jϭ15.0, 6.6 Hz), the olefinic proton at d 5.78 correlated
The IR spectra showed the hydroxyl and amide NH group with two methylene (C-6) at d 2.15 (q, Jϭ6.6 Hz) that corre-
bands at 3340 and 3320 cmϪ1, the band at 1640 cmϪ1 was lated with another two methylene (C-7) protons at d 2.08 (q,
due to CONH group.
Jϭ6.6 Hz), which correlated with the olefinic (C-8) proton at
1
The H-NMR spectrum (Table 1) revealed the presence of d 5.36 (dt, Jϭ15.0, 6.0 Hz), that proton correlated with the
two primary methyls at d 0.88 (6H, t, Jϭ7.2 Hz), two hetero olefinic (C-9) proton at d 5.43 (dt, Jϭ15.0, 6.0 Hz) that is
bearing-methines at d 3.91 and 4.32 and oxygenated methyl- correlated with the two methylene (C-10) protons at d 1.97
ene protons at d 3.70 and 3.95, four olefinic protons at d (q, Jϭ7.8 Hz). The above discussion implied that the two OH
5.54, 5.78, 5.43 and 5.36, an NH proton at d 6.20 and a huge groups are at C-1 and C-3, and two double bonds one at C-
methylene envelope at d 1.3 (Table 1). The 13C-NMR and 4/C-5 and another between C-8/C-9 (double bonds are trans
distortionless enhancement by polarization transfer (DEPT) oriented owing to the values of chemical shifts of allylic
spectral data of 1 were supportive of the above analysis, methylene dCϾ30 and the J values).12) Consideration of bio-
showing a carbonyl group at dC 174.1, two double bonds at genesis and steric hinderance of sphingolipids, generally
dC 134.3, 131.4, 129.1 and 128.9, three oxygenated or other were acknowledged to determine the absolute stereochem-
hetero atomized carbons at dC 74.6, 62.4 and 54.4, aliphatic istry of the phytosphingosine moiety. On the basis of the 13C-
methylenes at dC 22.7—36.8 and two methyls at dC 14.2. NMR spectral data, the relative stereochemistries at C-2 (d
The downfield doublet at d 6.28 (NH) was deuterium-ex- 54.4) and C-3 (d 74.6) were deduced to be 2S and 3S.7) Thus,
changeable, and there was no any correlation between this the structure of 1 was established as (2S*,3S*,4E,8E)-2N-
signal and any carbon in the heteronuclear multiple quantum [tetradecanoyl]-4(E),8(E)-icosadiene-1,3-diol.
coherence (HMQC) spectrum. On the other hand, a correla-
Compound 2 has a molecular formula of C28H46O3 which
tion from d 6.28 (NH) to d 3.91 (m), and the correlations was determined from HR-FAB-MS data (m/z 453.3357
from d 6.28 (NH) to dC 174.1(C-1Ј), 36.8 (C-2Ј), 62.4 (C-1), [MϩNa]ϩ). Calcd 453.3341, EI-MS (m/z 535) and EI-MS
54.4 (C-2) and 74.6 (C-3) were observed in the 1H–1H corre- (m/z 430) together with 13C-NMR, implying six degrees of
lation spectroscopy (COSY) and heteronuclear multiple bond unsaturation. The presence of hydroxyl and olefinic function-
connectivity (HMBC) spectra, respectively. All the above alities was deduced from IR absorptions at 3355 and
data suggested 1 is a ceramide (sphingolipid).9,10) In order to 1643 cmϪ1. Three hydroxyls in the molecule were estimated
determine the lengths of sphingosine and fatty acid chains, from ion peaks appearing at m/z 412 (MϪH2O)ϩ, and 394
∗ To whom correspondence should be addressed. e-mail: walied1737@yahoo.com
© 2010 Pharmaceutical Society of Japan

1636
Vol. 58, No. 12
Table 1. NMR Spectral Data of Compounds 1 and 2 in CDCl3 (d, ppm, J, Hz).
1
2
No.
No.
1
1
d H (m, J, Hz)
d
13C
d H (m, J, Hz)
d
13C
1a
1b
2
3
4
5
6
7
8
9
10
11—18
19
20
1Ј
2Ј
3Ј
3.70 (dd, 10.8, 3.6)
3.95 (dd, 11.4, 3.6)
3.91 (m)
62.4
1
2a
2b
3
4a
4b
5
6
7
8
9
10
11a
11b
12a
12b
13
14
15a
15b
16a
16b
17
18
19
20
21
22
23
24
25
26
27
28
3.63 (br s)
2.06 (m)
1.77 (m)
4.08 (tt, 10.8, 6, 4.8)
2.17 (m)
73.6
23.9
—
67.7
40.6
—
144.0
117.5
75.9
39.4
37.1
43.1
22.9
—
33.19
—
43.42
55.8
28
—
30.0
—
54.7
12.33
19.7
39.2
22.33
135.6
132.2
43.73
31
54.4
74.6
128.9
131.4
32.4
32.2
134.3
129.1
32.6
29
25.8
14.2
174.1
36.8
32.3
29
4.32 (br s)
5.54 (dt, 15, 6)
5.78 (dt, 15, 6.6)
2.15 (q, 6.6)
2.08 (q, 6.6)
5.36 (dt, 15, 6.6)
5.43 (dt, 15, 6.6)
1.97 (q, 7.8)
1.30 (m)
2.14 (m)
—
5.36 (br d, 2.4)
3.65 (s)
1.45 (m)
1.66 (m)
—
1.30 (m)
0.88 (t, 7.2)
1.50 (m)
1.44 (m)
1.18 (m)
2.01 (m)
2.23 (t, 7.2)
1.64 (m)
1.30 (m)
1.30 (m)
0.88 (t, 7.2)
6.28 (d, 7.2)
—
4Ј—12Ј
13Ј
14Ј
1.20 (m)
1.85 (m)
1.43 (m)
1.90 (m)
1.28 (m)
1.15 (m)
0.60 (s)
1.09 (s)
22.7
14.2
—
NH
1.80 (m)
1.02 (d, 6.6)
5.16 (dd, 15, 7.2)
5.18 (dd, 15, 7.2)
2.02 (m)
1.47 (m)
0.82 (d, 6.6)
0.84 (d, 6.6)
0.92 (d, 6.8)
22.1
20.8
18.9
(MϪ2H2O)ϩ and 376 (MϪ3H2O)ϩ in the EI-MS spectrum, br d, Jϭ2.4 Hz) but no with H-9 (d 1.66, m) or H-14 (d 1.20,
and from the NMR signals (Table 1) resonating at dC 75.9, m). Also from H-NMR data, the orientations of OH groups
73.6 and 67.7 (each CH) and d 3.65 (1H, s), d 3.63 (1H, at C-1, C-3 and C-7 were further confirmed to be a, b and a,
br s), d 4.08 (1H, tt, Jϭ10.8, 6, 4.8 Hz), respectively. Two respectively, on the basis of peak shape and coupling con-
double bonds (dC 144.0, qc, 135.6, CH, 132.2, CH, 117.5, stants where H-1 (d 3.65, br s), H-3 (d 4.08, tt, Jϭ10.8, 6,
CH; d 5.36, 5.18, 5.16 each 1H) were also indicated in com- 4.8 Hz) and H-7 (d 3.63, m). The E geometry of the double
pound 2. Moreover, the 1H-NMR spectrum exhibited two sin- bond at C-22/C-23 supported from NOE interactions of H-22
glets at 1.08 and 0.60 (each 3H, s) and four doublets at d (d 5.16, dd, Jϭ15, 7.2 Hz) with H3-28 (d 0.92, d, Jϭ6.8 Hz)
0.82, 0.84, 0.92 and 1.02 (each 3H, d, Jϭ6.6 Hz). These find- and H-23 (d 5.18, dd, Jϭ15, 7.2 Hz) with H-20 (d 1.80, m)
ings together with the 28 carbon signals in the 13C-NMR sug- and H-23 with H-25 (d 1.47, m). Furthermore the double
gested a trihydroxy methylcholesta skeleton. Analysis of bond at C-22/C-23 is trans oriented owing to the values of
1H–1H COSY and HMBC correlations established structure chemical shifts of allylic methine dCϾ30 and the J values
of compound 2 as illustrated in Fig. 1, where positions of (15 Hz).12) On the basis of the above findings, the structure of
3OH groups and two double bonds were determined to be at compound 2 was established as (22E)-methylcholesta-5,22-
C-1, C-3, C-7, C-5/C-6 and C-22/C-23, respectively. The diene-1a,3b,7a-triol.
stereochemistry of the three hydroxy groups was established
Antibacterial Activity Biological activity of the com-
on the basis of nuclear Overhauser effect spectroscopy pounds 1—5, were screened for their antibacterial activity by
(NOESY) correlation. The hydroxyl group at C-1 and C-3 disk-diffusion technique13) against Gram-positive (Bacillus
were determined to be a and b orientations, respectively, de- subtilis) and Gram-negative bacteria (Pseudomonas aerugi-
pending on strong nuclear Overhauser effect (NOE) interac- nosa), at 1 mg/ml concentration. The inhibited zone diame-
tion of H3-19 (d 1.09, s) with H-1 (d 3.63, br s), H3-19 with ters were measured and recorded in Table 2. Furacin was
H-4b (d 2.14, m), and H-4a (d 2.17, m) with H-3 (d 4.08, tt, used as a standard compound. The minimal inhibitory con-
Jϭ10.8, 6.0, 4.8 Hz) (Fig. 2). The b orientation of the OH centration (MIC) of the strongly active compounds were also
group at C-7 was deduced on the basis of significant correla- measured and listed in Table 2.
tion of H-7 (d 3.65, s) with H-8 (d 1.45) and H-6 (d 5.36,
The data obtained from Table 2 shows that sphingolipid

December 2010
1637
Fig. 1. Selected 1H–1H COSY and HMBC Correlations of 1 and 2
Experimental
General Experimental Procedure Optical rotations were measured on
ATAGO POLAX-L 2 polarimeter. EI/MS analyses were carried out on a Shi-
madzu-QP 2010. 1H-NMR spectra were recorded at 600 MHz, and 13C-NMR
at 75 MHz. Chemical shifts are given in ppm relative to tetramethyl silane
(TMS) as internal standard. Thin layer chromatography was performed on
silica gel (Kieselgel 60, F254) of 0.25 mm layer thickness. Gel filtration was
carried out using Sephadex LH-20. Spots were detected by using
ethanol/sulfuric acid as spray reagent. The black coral (Coelenterata, Hexa-
corallia) A. dichotoma, fam. Antipatharia identified by Prof. Manfred
Grasshoff, and was collected using SCUBA, from Hakel area, Saudi Arabia,
in the Red Sea in Jan. 2009. A voucher specimen was deposited in the Fac-
ulty of Marine Science (B.C. 2009.77).
Fig. 2. Key NOESY Correlations of 2
Table 2. Diameter of Inhibition Zone in mm at Concentration Level of
1 mg/ml and Minimum Inhibitory Concentration (MIC) in mg/ml
Extraction and Isolation The air dried black coral (1.5 kg) was soaked
in a mixture of chloroform/methanol (1 : 1) and allowed to stand at room
temperature for few days. Filtration and evaporation to dryness under re-
duced pressure. Half of the residue (30 g) was fractionated by column chro-
matography over silica gel with gradient hexane–EtOAc and then with gradi-
ent dichloromethane–acetone. Fractions of 50 ml were collected, and those
containing a single compound were combined and further purified to give
the following compounds.
(2S*,3S*,4E,8E)-2N-[Hexadecanoyl]-4(E),8(E)-octadecadiene-1,3-diol
(1) Colorless amorphous powder; mp 123—125 °C (30 mg, 0.002%);
Rfϭ0.52 (Si gel, n-hexane–EtOAc, 1 : 1); [a]D ϩ83 (cϭ0.1, CHCl3). IR
(neat) n cmϪ1: OH and amide NH (3340—3200 cmϪ1), amide carbonyl
(1640 cmϪ1) and olefinic double bond (1620 cmϪ1); HR-FAB-MS data (m/z
558.4876 [MϩNa]ϩ). Calcd 558.4862; EI-MS m/z 535 [M, C34H65NO3]ϩ.
1H- and 13C-NMR: Table 1.
B. subtilis
MIC (mg/ml)
P. aeruginosa
MIC (mg/ml)
Compound
1
2
3
4
5
17.9
0.4
12.7
0.6
6.3
—
17.9
0.3
17.6
0.5
18.2
0.2
9.8
—
9.0
—
10.0
—
11.1
—
Methanolysis of 1 Compound 1 (20.0 mg) was refluxed with 0.9 M HCl
in 82% aq. MeOH (10 ml) for 18 h. The mixture was extracted with n-
hexane and evaporation of the hexane yielded a fatty acid methyl ester
(FAME). GC-MS analysis of FAM gave a single fatty acid, methyl tetrade-
canoate m/z 242 [Mϩ, C15H30O2], 211 [MϪOMe]ϩ, 199 [MϪC3H7]ϩ.
(22E)-Methylcholesta-5,22-diene-1a,3b,7a-triol (2) White powder;
(1) exhibits potent activity against B. subtilis and P. aerugi-
nosa. While the epidioxy steroids (4, 5) and the trihydroxy
steroid (2) show potent activity against B. subtilis.

1638
Vol. 58, No. 12
(1992).
mp 138—140 °C; (36 mg, 0.0024%); Rfϭ0.55 (Si gel, n-hexane–acetone,
6 : 4); [a]D Ϫ30 (cϭ0.1, CHCl3); IR (neat) nmax: 3355 (OH), 2959, 2930,
2870, 1653 (olefinic double bond) cmϪ1; HR-FAB-MS data (m/z 453.3357
[MϩNa]ϩ). Calcd 453.3341; EI-MS m/z 430 (100, [M, C28H46O3]ϩ), 418
(5.7, [MϪH2O]ϩ), 399 (3.5, [MϪH2OϪMe]ϩ), 394 (1.2, [MϪ2H2O]ϩ), 376
(19.5, [MϪ3H2O]ϩ) 314 (11.7), 303 (41.1), 285 (7.3), 249 (11.2),173 (11.3),
145 (13.0), 133 (15.0), 123 (51.2), 105 (40.8):1H- and 13C-NMR: Table 1.
3b,7a-Dihydroxy-cholest-5-ene (3) The fraction eluted with hexane–
EtOAc (6 : 4), was further purified by preparative TLC of silica gel using
same eluant system to give a pure material 130—133 °C (15 mg, 0.0001%);
[a]D Ϫ98 (cϭ0.1, CHCl3). Compound 3 was identified by comparison of its
1H- and 13C-NMR with ref. 14.
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(22E,24S),5a,8a-Epidioxy-24-methylcholesta-6,22-dien-3b-ol (4) and
(22E,24S),5a,8a-Epidioxy-24-methylcholesta-6,9(11),22-trien-3b-ol (5)
The fraction eluted with hexane–EtOAc (7 : 3), was further purified by
preparative TLC of silica gel using same eluant system to give a material
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1
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their NMR spectral data and by comparison with refs. 15 and 16, respec-
tively.
Acknowledgments The authors are thankful to the researcher’s assis-
tants for their help in the laboratories work. Deanship of Scientific Research
(DSR) is also acknowledged for the financial support.
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