New sphingosines from a gorgonian, Pseudopterogorgia australiensis Ridley, of the Indian Ocean

Article abstract of DOI:10.1021/np040022p

Two new sphingosines, (2S,3R)-2-(docosanoyl amino)nonadecane-1,3-diol (1) and (2S,3S,4R)-2-[(2′R)-2′-hydroxynonadecanoylamino]nonadecane-1,3, 4-triol (3), along with the known (2S,3R,4E)-2-(heptadecanoylamino)octadec-4- ene-1,3-diol, have been isolated from Pseudopterogorgia australiensis, of the Indian Ocean. The structures were deduced from spectral and chemical methods. Compounds 1-3 showed moderate antibacterial activity.

Full text of DOI:10.1021/np040022p

J . Nat. Prod. 2004, 67, 1423-1425
1423
New Sp h in gosin es fr om a Gor gon ia n , P seu d opter ogor gia a u str a lien sis Rid ley, of
th e In d ia n Ocea n †
N. Krishna,‡ P. Muralidhar,‡ M. Muralikrishna Kumar,‡ D. Venkata Rao,*,‡ and Ch. Bheemasankara Rao§
Department of Pharmaceutical Sciences and Department of Organic Chemistry, Andhra University,
Visakhapatnam 530 003, India
Received J anuary 30, 2004
Two new sphingosines, (2S,3R)-2-(docosanoyl amino)nonadecane-1,3-diol (1) and (2S,3S,4R)-2-[(2′R)-2′-
hydroxynonadecanoylamino]nonadecane-1,3,4-triol (3), along with the known (2S,3R,4E)-2-(hepta-
decanoylamino)octadec-4-ene-1,3-diol, have been isolated from Pseudopterogorgia australiensis, of the
Indian Ocean. The structures were deduced from spectral and chemical methods. Compounds 1-3 showed
moderate antibacterial activity.
In continuation of our studies on marine organisms,1-5
we have examined the gorgonian Pseudopterogorgia aus-
traliensis collected from the Tuticorin coast of the Indian
Ocean. A number of terpenoids, sterols,6 and a sphin-
golipid7 were isolated from various Pseudopterogorgia
species. Cytotoxic, ichthyotoxic, neurotoxic, anti-inflamma-
tory, analgesic, and antibacterial activities were reported
for some of the compounds isolated from this genus.6,7
A
derivative of the terpenoid glycoside, pseudopterosin E,
isolated from P. elisabethae, is undergoing phase I clinical
trials as an anti-inflammatory agent.8 Herein we report
the isolation of sphingolipids from the organism.
Sphingolipids exist widely in the membranes of eukary-
otic cells and play important roles in many physiological
processes.9 Sphingolipids have been isolated from a number
of marine organisms including sea stars, sea anemones,
gorgonians, sponges, tunicates, dinoflagellates, and algae.10
Some of the sphingolipids are reported to have cytotoxic,
antitumor, immunostimulatory, antifungal, antimicrobial,
antiviral, and Ca2+-ATPase activities.10 KRN 7000, a
glycosphingolipid isolated from Agelas sp., is under phase
I clinical trials as an anticancer agent.11
The EtOAc fraction of the methanol extract of the shade-
dried organism, upon chromatography over MPLC, yielded
compounds 1-3 as colorless crystals. These compounds had
IR absorptions for the hydroxyl and amide NH (3500-3200
cm-1), amide carbonyl (1640 cm-1), and a long aliphatic
chain (2910-2850 cm-1) and had no significant UV absorp-
tions above 200 nm. The presence of carbons and protons
corresponding to an amide, a long methylene chain, and a
terminal methyl in 13C and 1H NMR spectra and a close
scrutiny of the spectra suggested a sphingolipid frame for
the compounds.5
HMQC spectra. The lengths of the acyl and alkyl chains
were determined by employing methanolysis,12 and the
resultant methyl ester was identified as methyl docosanoate
(GC-MS, m/z 354) and the basic moiety on acetylation gave
1,3-diacetoxy-2-acetaminononadecane (GC-MS, m/z 441).
The relative stereochemistries at C-2 and C-3 were as-
signed as 2S, 3R on the basis of 13C NMR chemical shifts
of C-2 (δ 53.8) and C-3 (δ 72.5), which are consistent with
those reported for glycosyl D(+)-(2S,3R)-sphingosine (δ
53.8, 72.6).13,14 Thus compound 1 could be described as
(2S,3R)-2-(docosanoylamino)nonadecane-1,3-diol.
Compound 3 analyzed for C38H77NO5 by positive ion
FABMS ([M + H]+, m/z 628) and elemental analysis. The
presence of four hydroxyls was suggested by the presence
of three oxymethine carbons at δ 76.7, 73.1, and 72.7 and
an oxymethylene carbon at δ 62.1 and the formation of
tetraacetate. The absence of a triplet at δ 2.2-2.5 and
1
careful study of H-1H COSY and HMQC spectral data
Compound 1, analyzing for C41H83NO3 by positive ion
FABMS ([M + H]+, m/z 638) and elemental analysis,
showed the presence of two oxygenated carbons at δ 62.5
and 72.5 in the 13C NMR spectrum and formed a diacetate
(Ac2O/Py) suggestive of two hydroxyls. A triplet at δ 2.25
suggested a -CH2-CO-NH- skeleton. The hydroxyls on
revealed the positions of hydroxyls on C-1, C-3, C-4, and
C-2′. The fatty acyl and alkyl chains were ascertained by
the method employed earlier vide infra. The relative
stereochemistries at C-2, C-3, and C-4 were assigned as
2S, 3S, 4R based on comparison of optical rotation15 and
13C NMR chemical shifts of C-2 (δ 53.1), C-3 (δ 76.7), and
C-4 (δ 73.1) with literature data of natural15-17 and
synthetic sphingamines.18 Thus compound 3 is described
as (2S,3S,4R)-2-[(2′R)-2′-hydroxynonadecanoylamino]nona-
decane-1,3,4-triol.
1
C-1 and C-3 were ascertained from the H-1H COSY and
† Dedicated to the late Dr. D. J ohn Faulkner (Scripps) and the late Dr.
Paul J . Scheuer (Hawaii) for their pioneering work on bioactive marine
natural products.
Compound 2 analyzed for C35H69NO3 by positive ion
FABMS ([M + H]+, m/z 552) and elemental analysis. The
1H and 13C NMR spectra of the 2 were found to be identical
with those of (2S,3R,4E)-2-(heptadecanoylamino)octadec-
* To whom correspondence should be addressed. Tel: 91-891-2530235.
Fax: 91-891-2755547. E-mail: dvraodod@rediffmail.com.
‡ Department of Pharmaceutical Sciences.
§ Department of Organic Chemistry. Present address: ‘Bhargavi’, 9-40-
7, Pithapuram Colony, Visakhapatnam 03, A.P, India.
10.1021/np040022p CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/23/2004

1424 J ournal of Natural Products, 2004, Vol. 67, No. 8
Notes
CH2CH3); positive ion FABMS m/z 628, 330, 312, 294; anal. C
72.61%, H 12.49%, N 2.19%, calcd for C38H77NO5, C 72.66%,
H 12.36%, N 2.23%.
4-ene-1,3-diol reported earlier from the red algae Amansia
glomerata.19
Compounds 1-3 showed moderate antibacterial activity,
at 1 mg/mL concentration, against Gram-positive bacteria
Bacillus pumilis, B. subtilis, and Staphylococcus aureus
and Gram-negative bacteria Escherichia coli, Proteus vul-
garis, and Pseudomonas aeruginosa, by cup-diffusion
method.20 None of the compounds showed antifungal activ-
ity against Candida albicans and Aspergillus niger.
Acetyla tion . Dry pyridine (0.5 mL) and Ac2O (1.0 mL) were
added to 5 mg of compounds 1 and 3 separately and left
overnight. Usual workup and crystallization in each case
yielded 1a and 3a . Compound 1a : [R]2D5 +13.8° (c 0.1, CHCl3);
1H NMR (CDCl3, 300 MHz) δ 6.62 (1H, d, J ) 8.9 Hz, NH),
1.90-2.01 (6H, s, -COCH3), 1.25 (br s, -CH2-), 0.85 (6H, t,
J ) 8.9 Hz, CH2CH3). Compound 3a : [R]2D5 +11.5° (c 0.1,
CHCl3); 1H NMR (CDCl3, 300 MHz) δ 6.63 (1H, d, J ) 8.9 Hz,
NH), 5.10 (2H, dd, J ) 6.9, 10.8 Hz, H-3, H-2′), 4.93 (1H, m,
H-4), 4.44 (1H, m, H-2), 4.35 (1H, dd, J ) 6.4,10.6 Hz, H-1a),
4.03 (1H, dd, J ) 3.1,11.7 Hz, H-1b), 2.03-2.18 (12H, s,
-COCH3), 1.84 (1H, m, H-3′), 1.66 (4H, m, H-5, H-4′), 1.25
(54H, br s, -CH2-), 0.88 (6H, t, J ) 8.9 Hz, CH2CH3); 13C
NMR (CDCl3, 75 MHz) δ 62.4 (CH2, C-1), 47.8 (CH, C-2), 72.7
(CH, C-3), 72.2 (CH, C-4), 31.8 (CH2, C-5), 171.3 (C, C-1′), 74.0
(CH, C-2′), 31.9 (CH2, C-3′), 31.8 (CH2, C-4′), 29.7-22.7 (CH2,
-CH2-), 14.1 (CH3, CH2CH3), 20.6-21.0 (CH3, -COCH3),
169.9-170.9 (C, COCH3).
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. 1H and 13C NMR
spectra were recorded on a Bruker DRX spectrometer operat-
ing at 300 and 75 MHz, respectively. The chemical shift values
were reported in parts per million units, and the coupling
constants were in Hz. Positive ion FABMS was recorded on a
J EOL-Sx-120/DA-6000 mass spectrometer using a beam of
argon/xenon (2-8 keV) and m-nitrobenzoyl alcohol as the
matrix. Optical rotations were taken on a J ASCO DIP-370
polarimeter. Elemental analysis was carried out on a Carlo
Erba 1108 analyzer. IR spectra were recorded on a Perkin-
Elmer 881 instrument. Melting points were recorded on a
Boitus melting point apparatus and were uncorrected. Silica
gel column chromatography was carried out using silica gel
(finer than 200#, ACME), gel filtration was carried out using
LH20 (Sephadex LH20, Pharmacia Biotech), and MPLC was
performed on a Buchi B-688 MPLC system. GC-MS experi-
ments were carried out on a Shimadzu GCMS-QP 5050A
system.
Meth a n olysis. Compounds 1 and 3 (10 mg) were separately
treated with 3 mL of 1 N HCl in methanol at 90 °C for 15 h
with magnetic stirring. The fatty acid methyl ester so obtained
in each case was extracted with n-hexane and analyzed by
GC-MS. In each case a single compound was obtained. The
MeOH/H2O phase was evaporated, and the residue was
acetylated. Purification by filtration over a Sephadex LH20
column (CH2Cl2/MeOH, 1:1) gave the acetylated sphingamines.
The sphingamines were subjected to GC-MS in each case.
1
Meth yl ester fr om 1: H NMR (CDCl3, 300 MHz) δ 2.22
An im a l Ma ter ia l. The gorgonian Pseudopterogorgia aus-
traliensis was collected from the Tuticorin Coast, India (8°45′
N, 78°12′ E) at a depth of about 10 m. A voucher specimen
was deposited in the Marine Organisms Museum in the
Department of Pharmaceutical Sciences, Andhra University,
Visakhapatnam, India (Voucher No. AU2-183).
(2H, t, J ) 7.2 Hz, H-2), 3.53 (3H, s, OCH3), 1.84 (m, H-3),
1.25 (br s, -CH2-), 0.85 (3H, t, J ) 6.4 Hz, CH2CH3); GC-
MS m/z 354 [M]+.
1
Meth yl ester fr om 3: H NMR (CDCl3, 300 MHz) δ 5.13
(1H, dd, J ) 6.8, 3.8 Hz, H-2), 3.52 (3H, s, OCH3), 2.12 (2H,
m, H-3), 1.84 (2H, m, H-4), 1.50 (2H, m, H-5), 1.25 (26H, br s,
-CH2), 0.88 (3H, t, CH2CH3); GC-MS m/z 328 [M]+.
Extr a ction a n d Isola tion . The organism was shade dried
and powdered (dry wt ca. 1 kg), and the powder was extracted
with MeOH eight times at room temperature. The combined
alcoholic extract was concentrated under reduced pressure,
and the EtOAc-soluble fractions were pooled and concentrated
under reduced pressure after drying on anhydrous MgSO4 to
yield a crude residue (25 g). It was chromatographed over
MPLC (silica gel, finer than 200#, ACME) using eluants with
increasing polarity starting from n-hexane through EtOAc to
MeOH. Fractions eluted with 30% and 50% EtOAc in hexane
and EtOAc yielded, after repeated purification over MPLC,
compounds 1 (50 mg), 2 (30 mg), and 3 (25 mg), respectively.
Com p ou n d 1: colorless crystals (CHCl3); mp 131-133 °C;
[R]2D5 +28.5° (c 0.1, CHCl3); IR (KBr) νmax 3520, 3460, 1640,
2910-2850 cm-1; 1H NMR (CDCl3, 300 MHz) δ 6.36 (1H, d, J
) 7.4 Hz, NH), 3.70 (1H, d, J ) 4.0 Hz, H-1a), 3.44 (1H, br s,
H-1b), 3.91 (1H, m, H-2), 4.31 (1H, br s, H-3), 2.01 (1H, m,
H-4), 2.25 (2H, t, J ) 7.5 Hz, H-2′), 1.52 (2H, m, H- 3′), 1.25
(br s, -CH2-), 0.88 (6H, t, J ) 6.4 Hz, CH2CH3); 13C NMR
(CDCl3, 75 MHz) δ 62.5 (CH2, C-1), 53.8 (CH, C-2), 72.5 (CH,
C-3), 34.5 (CH2, C-4), 173.6 (C, C-1′), 36.8 (CH2, C-2′), 25.8
(CH2, C-3′), 31.9-29.2 (CH2, -CH2-), 14.1 (CH3, CH2CH3);
positive ion FABMS m/z 638 [M +H]+, 620, 314, 286; anal. C
77.19%, H 13.09%, N 2.26%, calcd for C41H83NO3, C 77.16%,
H 13.12%, N 2.20%.
Com p ou n d 3: colorless crystals (MeOH); mp 101-103 °C;
[R]2D5 +8.5° (c 0.1, pyridine); IR (KBr) νmax 3500-3200, 2910-
2850, 1680 cm-1; 1H NMR (pyridine-d5, 300 MHz) δ 8.59 (1H,
d, J ) 7.2 Hz, NH), 4.65 (1H, m, H-1a), 4.51 (1H, m, H-1b),
5.12 (1H, m, H-2), 4.41 (1H, m, H-3), 4.23 (1H, dt, J ) 8.8, 4.4
Hz, H-4), 1.91 (4H, m, H-5, H-4′), 1.72 (4H, m, H-6, H-5′), 4.54
(1H, dd, J ) 4.8, 7.0 Hz, H-2′), 2.21 (2H, m, H-3′), 1.25 (50H,
br s, -CH2-), 0.86 (6H, t, J ) 6.6 Hz, CH2CH3); 13C NMR
(pyridine-d5, 75 MHz) δ 62.1 (CH2, C-1), 53.1 (CH, C-2), 72.7
(CH, C-3), 73.1 (CH, C-4), 34.8 (CH2, C-5), 30.5 (CH2, C-6),
175.8 (C, C-1′), 76.7 (CH, C-2′), 35.8 (CH2, C-3′), 34.2 (CH2,
C-4′), 32.3 (CH2, C-5′), 29.9-26.0 (CH2, -CH2-), 14.0 (CH3,
Acetyl sp h in ga m in e fr om 1: 1H NMR (CDCl3, 300 MHz)
δ 6.60 (1H, d, J ) 9.4 Hz, NH), 4.39 (1H, dd, J ) 9.6, 4.6 Hz,
H-1a), 4.93 (1H, dd, J ) 9.8, 3.7 Hz, H-1b), 5.08 (1H, m, H-2),
4.28 (1H, m, H-3), 1.96 (2H, m, H-4), 1.26 (br s, -CH2-), 0.85
(3H, t, J ) 6.0 Hz, CH2CH3); GC-MS m/z 441 [M]+.
Acetyl sp h in ga m in e fr om 3: 1H NMR (CDCl3, 300 MHz)
δ 8.63 (1H, d, J ) 8.4 Hz, NH), 5.14 (1H, m, H-2), 4.65 (1H,
m, H-1a), 4.52 (1H, m, H-3), 4.20 (1H, dt, J ) 8.8, 4.4, H-4),
3.83 (1H, m, H-1b), 2.18-2.03 (12H, s, COCH3), 1.90 (2H, m,
H-5), 1.46 (2H, m, H-6), 1.25 (24H, br s, -CH2-), 0.86 (3H, t,
J ) 6.4 Hz, CH2CH3); GC-MS m/z 499 [M]+.
Ack n ow led gm en t. The authors thank Dr. P. A. Thomas,
CMFRI, Trivandrum for identification of the organism, and
The University Grants Commission, Department of Ocean
Development, New Delhi, for financial support. Thanks are
due to RSIC, CDRI, and Lucknow for spectral data.
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NP040022P