Calyceramides A-C: Neuraminidase inhibitory sulfated ceramides from the marine sponge Discodermia calyx

Article abstract of DOI:10.1016/S0040-4020(01)00163-6

Three new sulfated ceramides, calyceramides A-C (1-3) were isolated as inhibitors of neuraminidase from the marine sponge Discodermia calyx. Their structures were determined by spectroscopic and chemical methods. The ceramides inhibited neuraminidase with IC₅₀ values of 0.2-0.8 μg mL^-1.

Full text of DOI:10.1016/S0040-4020(01)00163-6

TETRAHEDRON
Pergamon
Tetrahedron 57 *2001) 3013±3017
Calyceramides A±C: neuraminidase inhibitory sulfated
ceramides from the marine sponge Discodermia calyxq
Yoichi Nakao, Kentaro Takada, Shigeki Matsunaga and Nobuhiro Fusetanip
Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan
Received 8 December 2000; accepted 8 February 2001
AbstractÐThree new sulfated ceramides, calyceramides A±C *1±3) were isolated as inhibitors of neuraminidase from the marine sponge
Discodermia calyx. Their structures were determined by spectroscopic and chemical methods. The ceramides inhibited neuraminidase with
IC50 values of 0.2±0.8 mg mL21. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
sponge were partitioned between CH2Cl2 and H2O. The
aqueous layer was further extracted with n-BuOH. The
CH2Cl2 layer was chromatographed on silica gel, while
the n-BuOH layer was separated by ODS ¯ash chromato-
graphy. The active fractions from each chromatography
were combined and puri®ed by repeated reversed-phase
Neuraminidase cleaves an a-linked terminal N-acetyl-
neuraminic acid from glycoproteins, glycolipids, and oligo-
saccharide.2 In several viral and bacterial infections,
neuraminidase is known to play important roles; in¯uenza
virus employs this enzyme to detach itself from the infected
cell in the budding stage, thus indicating requirement of
neuraminidase for replication of the virus. Therefore, selec-
tive inhibitors of neuraminidase are potential therapeutic
agents against in¯uenza.3 In fact, neuraminidase inhibitors,
such as zanamivir *GG167)4 and oseltamirvir *GS4104),5
which were designed on the basis of the crystal structure
of the enzyme±substrate complex, showed a broader anti-
viral spectra, better tolerance, and induce less resistance
than M2 inhibitors such as amantadine and rimantidine.6
HPLC to afford calyceramides A *1, 3.0 mg, 2.8£1027
%
yield based on wet weight), B *2, 1.5 mg, 1.4£1027%),
and C *3, 1.5 mg, 1.4£1027%).
15'
O
2'
14'
1'
HN
4'
OH
2
NaO3SO
18
3
1
7
OH
19
1
14'
O
13'
11'
In our research program to explore potential drug leads from
Japanese marine invertebrates, we found signi®cant
inhibitory activity against neuraminidase in the hydrophilic
extract of the marine sponge Discodermia calyx collected
off Sikine-jima Island. Bioassay-guided separation of the
extract led to the isolation of three active compounds,
calyceramides A±C, which were elucidated as sulfated cera-
mides by spectral and chemical methods. This paper deals
with isolation and structure elucidation of those inhibitors.
HN
20
OH
NaO3SO
NaO3SO
19
OH
O
2
15'
HN
16'
OH
18
OH
3
2. Results and discussion
Calyceramide
A
*1) had a molecular formula of
The combined MeOH and EtOH extracts of the frozen
q Part 106 of the Bioactive Marine Metabolites series.1
C34H66NO7SNa which was determined by high-resolution
FABMS *m/z 632.4590 *D 22.1 mmu)). The IR spectrum
exhibited bands attributable to sulfate *1230 cm21) and
amide *1644 cm21) functionalities. The presence of a sulfate
group was also supported by the ion peaks at m/z 97
Keywords: biologically active compounds; enzyme inhibitors; marine
metabolites; sponges.
*HOSO3)2 and 80 *SO3)2 in FABMS. The H NMR spec-
1
p
Corresponding author. Tel.: 181-3-5841-5299; fax: 181-3-5841-8166;
e-mail: anobu@mail.ecc.u-tokyo.ac.jp
trum revealed the presence of four secondary methyls at d
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020*01)00163-6

3014
Y. Nakao et al. / Tetrahedron 57 22001) 3013±3017
Table 1. NMR data of calyceramide A *1) in CD3OD
C/H No.
dC
d
H *J in Hz)
1H±1H COSY
H-1b,
H-1a, H-2
H-1b
H-4
H-3, H-5
H-4, H-6
H-5, H-7
H-6
HMBC
1a
1b
2
3
4
5
6
7
73.1
4.15, dd *10.4, 3,4)
4.26, dd *10.4, 4.6)
4.01, ddd *8.1, 4.6, 3.4)
4.12, dd *8.1, 7.3)
5.45, dd *15.0, 7.3)
5.72, dt *15.0, 6.9)
2.02, dt *7.3, 6.9)
1.34, m
C-2, C-3
53.9
72.8
130.6
135.4
34.3
C-1, C-3, C-10
C-2
C-3, C-6
C-3, C-6, C-7
C-4, C-5, C-7
C-6, C-8
30.4
8±15
16
17
18
19
10
30±32
29.1
40.2
23.0
23.0
177.1
72.9
35.8
1.2±1.4
1.16, m
1.52, m
0.87, d *6.5)
0.87, d *6.5)
H-15, H-17
H-16, H-18, H-19
H-17
C-15, C-17, C-18, C-19
C-15, C-16, C-18, C-19
C-16, C-17
H-17
C-16, C-17
20
3.97, dd *8.1, 3.9)
1.52, m
1.69, m
1.2±1.4
1.16, m
1.52, m
0.87, d *6.5)
0.87, d *6.5)
H-30a, H-30b
H-20, H-30b
H-20, H-30a
C-10
C-20, C-40
30a
30b
40 ±110
120
130
140
150
30±32
28.5
40.2
23.0
23.0
H-110, H-130
H-120, H-140, H-150
H-130
C-110, C-130, C-140, C-150
C-110, C-120, C-140, C-150
C-120, C-130
H-130
C-120, C-130
0.87 *12H), three heteroatom-bearing methines at d 3.97,
4.01, and 4.12, and an oxygenated methylene protons at d
4.15 and 4.26, two ole®nic protons at d 5.45 and 5.72, and a
huge methylene envelope at d 1.2±1.4 *Table 1). Interpreta-
C-2 and C-3 in 7 was inferred from the coupling constant
between H-2 and H-3 *Jˆ8.6 Hz) as well as from ROESY11
cross peaks between H-3/H3-21 and H-1b/H3-21 *Fig.1).
1
tion of the H±1H COSY and HMQC7 spectra resulted in
Absolute con®guration of calyceramide A *1) was deter-
mined by the application of the modi®ed Mosher's
method.12 Treatment of 1 with *S)-*1)- and *R)-*2)-
MTPACl in CH2Cl2/pyridine *1:1) to yield the correspond-
ing 3,20-bis-MTPA esters. The distribution of positive and
negative Dd *dS2dR) values around the MTPA esters
implied R-stereochemistry for both C-3 and C-20 *Fig. 2).
Therefore, the stereochemistry at C-2 was S.
two partial structures *C-1 to C-7 and C-20 to C-40), both of
which were connected to long aliphatic chains. An HMBC8
correlation between H-2 and C-10 not only con®rmed the
position of this nitrogen atom, but also connected the two
partial structures through an amide bond.
To determine the length of each alkyl chain, calyceramide A
*1) was hydrogenated followed by methanolysis9,10 to afford
sphingosine 4 and a-hydroxy fatty acid 5. The former was
converted to the bis-p-bromobenzoate 6 whose molecular
formula was deduced to be C33H47Br2NO4 on the basis of
FABMS data, thus implying that the sphingosine had 19
carbons, while the a-hydroxy acid had 15 carbons
*Scheme 1).
Calyceramide B *2) had the molecular formula same as that
of 1. Although the low-®eld region of the 1H NMR spectrum
was superimposable on that of 1, their methyl signals were
apparently different; one of the four doublet methyls in 1
was replaced by a triplet one. Further analysis of the 2D
NMR spectra indicated that 2 had an iso- and an anteiso-
terminus. To determine their structures, calyceramide B *2)
was hydrogenated followed by acid hydrolyzed, and the
resulting sphingosine moiety was converted to bis-p-bromo-
benzoate 8. FABMS and 1H NMR data of 8 were consistent
with the C20 sphingosine with an iso-terminus, thereby
The relative stereochemistry at C-2 and C-3 was determined
as follows. Calyceramide A *1) was treated with 2,2-
dimethoxypropane and a catalytic amount of p-TsOH in
CH2Cl2 to afford acetonide 7.9 The anti-relationshipof
Scheme 1.

Y. Nakao et al. / Tetrahedron 57 22001) 3013±3017
3015
O
presence of a C18-sphingosine portion. Accordingly, the
fatty acid portion was a-hydroxy C16 iso acid. The 2S, 3R,
20R-stereochemistry was deduced for 3 from the result of the
modi®ed Mosher's analysis and comparison of H NMR
data with that of 1.
HN
OH
1
2
3
O
O
7
3. Biological activity
Calyceramides A±C inhibited neuraminidase from the
bacterium Clostridium perfrigens with IC50 values of 0.4,
0.2 and 0.8 mg mL21, respectively, while the known
inhibitor 4-acetylneuraminic acid showed only weak
activity in our assay *IC50 1.5 mg mL21).
8.4 Hz
8.6 Hz
H
H
1a
H
O
2
Me
N
O
H
21
1b
3
Me
H
4. Conclusions
Three new ceramide 1-sulfates named calyceramides A±C
were isolated as neuraminidase inhibitors from the marine
sponge D. calyx. Their structures were determined by spec-
troscopic and chemical methods. Our compounds were
closely related to ceramide 1-sulfates isolated from the
bryozoan Watersipora cucullata9 as inhibitors of DNA
topoisomerase I. It is of interest that calyceramides A±C
inhibited neuraminidase signi®cantly.
ROESY
Figure 1. Coupling constants and ROESY correlations of acetonide 7.
OSO3Na
-0.14
-0.12
O
+0.03
+0.07
+0.01
+0.18
2
+0.04
5
1'
4
-0.04
N
+0.08
+0.22
H
OMTPA
OMTPA
5. Experimental
Figure 2. Dd values for the MTPA esters of 1.
5.1. General experimental procedures
disclosing that the fatty acid portion was an a-hydroxyl C14
acid with an anteiso-terminus. The absolute stereochemistry
1H and 13C NMR spectra were recorded on a JEOL A600
1
NMR spectrometer. H and 13C NMR chemical shifts were
1
was determined to be 2S, 3R, 20R on the basis of both H
referenced to the solvent peaks: dH 3.30 and dC 49.0 for
CD3OD, dH 2.04 and dC 29.8 for acetone-d6, dH 7.24 and
dC 77.0 for CDCl3. Optical rotation was measured on a
JASCO DIP-1000 digital polarimeter in MeOH. FAB
mass spectra were obtained on a JEOL SX102/SX102
tandem mass spectrometer using triethanolamine or m-nitro-
benzylalcohol as the matrices. IR spectra were recorded on a
JASCO FT/IR-5300 spectrometer. Fluorescence for inhibi-
tion assay was measured with a Molecular Device Spectra
MAX GEMINI apparatus.
NMR data of the acetonide and the modi®ed Mosher's
method as in the case of 1. The stereochemistry at C-110
remains to be determined.
Br
O
NH
Br
O
O
OH
5.2. Collection and isolation
8
Sponge samples were collected by hand using SCUBA at
depths of 15±20 m off Sikine-jima Island, 200 km south of
Tokyo, immediately frozen, and kept frozen at 2208C until
processed. The frozen sponges *10 kg) were homogenized
and extracted with MeOH and EtOH. The combined
extracts were concentrated and partitioned between
CH2Cl2 and H2O. The aqueous layer was further extracted
with n-BuOH. The CH2Cl2 layer was chromatographed on
silica gel with a CHCl3/MeOH system, while the n-BuOH
extract was chromatographed on an ODS column with an
aq. MeOH system. The active fractions obtained from both
columns were combined and separated by repeated
Br
O
NH
Br
O
O
OH
9
The molecular formula of calyceramide C *3) was also
identical with that of 1. The H NMR spectrum contained
1
two doublet [d 0.86, *6H)] and one triplet [d 0.88, *3H)]
methyl signals, indicating the iso-terminus of either sphin-
gosine or fatty acid portion. Bis-p-bromobenzoate 9 derived
from 3 exhibited a triplet methyl signal at d 0.83 in the H
NMR spectrum, while its FABMS data indicated the
reversed-phase HPLC *Cosmosil 5C18-AR II
20£250 mm). Final puri®cation was done by recycling
reversed-phase HPLC *Develosil C30-VG5,
f
f
1
20£250 mm) with 96% MeOH containing 0.3 M NaClO4
to afford calyceramides A *1, 3.0 mg, 2.8£1027% yield

3016
Y. Nakao et al. / Tetrahedron 57 22001) 3013±3017
based on wet weight), B *2, 1.5 mg, 1.4£1027%), and C *3,
silica gel column with CHCl3/MeOH *199:1) to yield
acetonide 7.
1.5 mg, 1.4£1027%).
5.2.1. Calyceramide A ,1). Colorless solid; [a]D ˆ124.88
1H NMR *acetone-d6) d 5.71 *1H, dt, Jˆ15.0, 6.9 Hz, H-5),
5.34 *1H, dd, 15.0, 6.9, H-4), 4.32 *1H, dd, 8.6, 6.9, H-3),
3.94 *1H, dd, 7.3, 3.8, H-20), 3.79 *1H, m, H-2), 3.77 *1H,
dd, 8.4, 4.6 H-1b), 3.68 *1H, m, H-1a), 1.99 *2H, m, H-6),
1.66 *1H, m, H-30b), 1,51 *1H, m, H-30a), 1.34 *2H, m, H-7);
FABMS m/z 592 *M1H)1 *pos.).
20
*c 0.10, MeOH); IR *®lm) 3850, 3563, 1644, 1540, 1472,
1
1230, 1102 cm21; H and 13C NMR data, see Table 1;
HRFABMS *neg.) m/z 632.4539 *M2Na)2 [C34H66NO7S
*D 22.1 mmu)]
20
5.2.2. Calyceramide B ,2). Colorless solid; [a]D ˆ114.58
*c 0.10, MeOH); IR *®lm) 3600, 3100, 1732, 1651, 1540,
1466, 1377, 1033 cm21; 1H NMR *CD3OD) d 5.72 *1H, dt,
Jˆ15.4, 6.6 Hz, H-5), 5.45 *1H, dd, 15.4, 7.7, H-4), 4.27
*1H, dd, 10.4, 5.0, H-1b), 4.11 *1H, dd, 10.4, 3.8, H-1a),
4.10 *1H, dd, 8.1, 7.7, H-3), 4.01 *1H, ddd, 8.1, 5.0, 3.4,
H-2), 3.97 *1H, dd, 8.1, 3.8, H-20), 2.02 *2H, dt, 7.7, 6.6,
H-6), 1.69 *1H, m, H-30b), 1,52 *1H, m, H-30a), 1.52 *2H, m,
H-18, H-110), 1.38 *2H, m, H-7), 1.2±1.4 *m, H-8-16, H-40-
100), 1.16 *4H, m, H-17, H-120), 0.87 *6H, d, 6.5, H-19,
H-20), 0.86 *3H, t, 7.1, H-130), 0.85 *3H, d, 6.5, H-140);
HRFABMS *neg.) m/z 632.4590 *M2Na)2 [C34H66NO7S
*D 13.0 mmu)]
5.2.7. Bis-p-bromobenzoate 8. Calyceramide B *0.4 mg)
was hydrogenated and converted to bis-p-bromobenzoate
as the same manner as that of 1. FABMS *pos.) m/z 694,
696, and 698 *M1H)1; 1H NMR *CDCl3) d 7.82, 7.78, 7.54,
7.49, 4.59 *H-1b), 4.57 *H-1a), 4.34 *H-2), 4.13 *H-3), 1.33,
1.22, 1.12, 0.83.
5.2.8. Bis-p-bromobenzoate 9. Calyceramide C *0.4 mg)
was acid hydrolyzed without prior hydrogenation to afford
sphingosine. The sphingosine was converted to bis-p-
bromobenzoate as the same manner as that of 1. FABMS
*pos.) m/z 664, 666, and 668 *M1H)1; 1H NMR *CDCl3) d
7.83, 7.78, 7.54, 7.50, 5.75 *H-5), 5.52 *H-4), 4.58 *H-1b),
4.56 *H-1a), 4.35 *H-2), 4.13 *H-3), 1.33, 1.22, 1.12, 0.84.
20
5.2.3. Calyceramide C ,3). Colorless solid; [a]D ˆ116.98
*c 0.10, MeOH); IR *®lm) 3600, 3100, 1732, 1651, 1538,
1
1466, 1377, 1217, 1024 cm21; H NMR *CD3OD) d 5.72
5.2.9. 3,20-Bis-,R)-MTPA ester of calyceramide A ,1). A
mixture of 1 *0.2 mg) and *S)-*1)-MTPACl *10 ml) in
CH2Cl2/pyridine *1:1, 40 ml) was stirred at room tempera-
ture for 30 min. The reaction mixture was diluted with
H2O *3 mL) and extracted with CHCl3 *3£3 mL). The
organic layer was concentrated under reduced pressure to
furnish the 3,20-bis-*R)-MTPA ester, which showed an
*M12MTPA2H)2 peak at m/z 1064 in FABMS *neg.).
1H NMR *CD3OD) d 5.83 *1H, dt, Jˆ15.8, 6.6 Hz, H-5),
5.55 *1H, dd, H-3), 5.26 *1H, dd, 15.8, 8.9, H-4), 5.12 *1H,
dd, H-20), 4.31 *1H, m, H-2), 4.10 *1H, dd, 10.5, 5.0, H-1b),
4.06*1H, dd, 10.5, 3.5, H-1a), 1.78 *1H, m, H-30b), 1.41 *1H,
m, H-30a), 1.18 *1H, m, H-4a0).
*1H, dt, Jˆ15.4, 6.6 Hz, H-5), 5.44 *1H, dd, 15.4, 7.5, H-4),
4.25 *1H, dd, 10.4, 4.8, H-1b), 4.11 *1H, dd, 10.4, 3.4,
H-1a), 4.10 *1H, dd, 8.1, 7.5, H-3), 4.01 *1H, ddd, 8.1,
4.8, 3.4, H-2), 3.97 *1H, dd, 8.1, 3.8, H-20), 2.02 *2H, dt,
7.3, 6.6, H-6), 1.69 *1H, m, H-30b), 1,52 *1H, m, H-30a),
1.52 *1H, m, H-140), 1.38 *2H, m, H-7), 1.2±1.4 *m, H-8-17,
H-40-120), 1.16 *2H, m, H-130), 0.88 *3H, t, 6.9, H-18), 0.86
*6H, d, 6.6, H-150, H-160); HRFABMS *neg.) m/z 632.4558
*M2Na)2 [C34H66NO7S *D 20.2 mmu)]
5.2.4. Hydrogenation and acid hydrolysis of 1. Methanol
solution of calyceramide A *1, 0.4 mg in 0.4 mL) was stirred
under the atmosphere of H2 over 10% Pd±C *catalytic
amounts) for 4 h at room temperature to give a hydro-
genated derivative, which showed an *M2Na)2 peak at
m/z 634 in FABMS *neg.). This was treated with 1N HCl
in 83% MeOH at 708C for 18 h. The reaction mixture was
extracted with n-hexane. To the aqueous alcoholic layer
were added CHCl3 and H2O, and the mixture was shaken.
The organic phase was evaporated to furnish sphingosine 4.
5.2.10. 3,20-Bis-,S)-MTPA ester of calyceramide A ,1). 3,
20-Bis-*S)-MTPA ester of 1 was prepared as described
1
above. H NMR *CD3OD) d 5.91 *1H, dt, Jˆ15.0, 6.9 Hz,
H-5), 5.59 *1H, dd, H-3), 5.44 *1H, dd, 15.0, 8.6, H-4), 5.13
*1H, dd, H-20), 4.27 *1H, m, H-2), 3.98 *1H, dd, 10.5, 3.4,
H-1b), 3.92 *1H, dd, 10.5, 5.8, H-1a), 1.85 *1H, m, H-3b0),
1.44 *1H, m, H-3a0), 1.40 *1H, m, H-4a0); FABMS m/z 1064
*M12MTPA2H)2.
5.2.5. Bis-p-bromobenzoate 6. p-Bromobenzoylchloride
*0.5 mg) and DMAP *catalytic amounts) were added to a
solution of 4 in CH2Cl2/pyridine *1:1, 200 ml), and the
mixture was stirred at room temperature for 12 h. The reac-
tion mixture was diluted with H2O and extracted with
CHCl3. The organic layer was evaporated and puri®ed by
normal phase HPLC *Cosomosil 5SL-2, f 10£250 mm)
with CHCl3 to yield 6, which showed 1:2:1 *M1H)1
peaks at m/z 680, 682, 684 in FABMS *pos.).
1
5.2.11. 3,20-Bis-,R)-MTPA ester of calyceramide B. H
NMR *CD3OD) d 5.83, 5.55, 5.26, 5.12 4.31, 4.10, 4.06,
1.78, 1.41, 1.18.
1
5.2.12. 3,20-Bis-,S)-MTPA ester of calyceramide B. H
NMR *CD3OD) d 5.91, 5.59, 5.44, 5.13, 4.27, 3.98, 3.92,
1.85, 1.44, 1.40.
1
5.2.13. 3,20-Bis-,R)-MTPA ester of calyceramide C. H
NMR *CD3OD) d 5.83, 5.55, 5.26, 5.12 4.31, 4.10, 4.06,
1.78, 1.41, 1.18.
5.2.6. Acetonide 7. A mixture of 1 *0.8 mg), 2,2-
dimethoxypropane *50 ml) and p-TsOH *catalytic amounts)
in CH2Cl2 *100 ml) was stirred at room temperature for 3 h.
After addition of triethylamine *5 ml) and H2O *2 mL), the
reaction mixture was extracted with Et2O *3£2 mL). The
Et2O extract was evaporated and chromatographed on a
1
5.2.14. 3,20-Bis-,S)-MTPA ester of calyceramide C. H
NMR *CD3OD) d 5.91, 5.59, 5.44, 5.13, 4.27, 3.98, 3.92,
1.85, 1.44, 1.40.

Y. Nakao et al. / Tetrahedron 57 22001) 3013±3017
3017
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5.3. Enzyme inhibition assay
Neuraminidase inhibitory activity was examined using
neuraminidase from the bacterium Clostridium perfringens
*Sigma N-2876). The substrate, 4.45 mM 20-*4-methyl-
umbelliferyl)-a-d-N-acetylneuraminic acid *M-8639) in
0.1 M NaOAc buffer *pH 4.2) *90 ml) was added to the
mixture of 2.25£1023 unit of the enzyme in the same buffer
*90 ml) and a sample in DMSO *20 ml); the mixture was
incubated at 378C for 30 min. The amounts of 4-methyl-
umbelliferone released was measured by ¯uorescence at
450 nm by an excitation at 360 nm.13
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Acknowledgements
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This work was partly supported by Grant-in-Aid from the
Ministry of Education, Science, Culture and Sports of Japan
and the JSPS, `Research for the Future Program' *JSPS-
RFTF 96I00301).
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