Steroidal triglycosides, kurilensosides A, B, and C, and other polar steroids from the Far Eastern starfish Hippasteria kurilensis

Article abstract of DOI:10.1021/np070637x

Three novel steroidal triglycosides, designated as kurilensosides A, B, and C (1-3), were isolated along with a new steroidal diglycoside, kurilensoside D (4), and two new (6, 7) and one known (5) polyhydroxysteroid from the alcoholic extract of the Far Eastern starfish Hippasteria kurilensis. Compounds 1-3 are the first triglycosides containing two carbohydrate chains found from starfish. The structures of 1-7 were elucidated by spectroscopic methods (mainly 2D NMR) and chemical derivatization. Glycosides 1-4 and steroids 6 and 7 inhibited sea urchin egg fertilization by sperm preincubated with these compounds.

Full text of DOI:10.1021/np070637x

J. Nat. Prod. 2008, 71, 793–798
793
Steroidal Triglycosides, Kurilensosides A, B, and C, and Other Polar Steroids from the Far
Eastern Starfish Hippasteria kurilensis
Alla A. Kicha, Natalia V. Ivanchina, Anatoly I. Kalinovsky, Pavel S. Dmitrenok, Irina G. Agafonova, and Valentin A. Stonik*
Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Pr. 100-let VladiVostoku 159, 690022
VladiVostok, Russian Federation
ReceiVed NoVember 11, 2007
Three novel steroidal triglycosides, designated as kurilensosides A, B, and C (1–3), were isolated along with a new
steroidal diglycoside, kurilensoside D (4), and two new (6, 7) and one known (5) polyhydroxysteroid from the alcoholic
extract of the Far Eastern starfish Hippasteria kurilensis. Compounds 1–3 are the first triglycosides containing two
carbohydrate chains found from starfish. The structures of 1–7 were elucidated by spectroscopic methods (mainly 2D
NMR) and chemical derivatization. Glycosides 1–4 and steroids 6 and 7 inhibited sea urchin egg fertilization by sperm
preincubated with these compounds.
Starfish (phylum Echinodermata, class Asteroidea) have
proven to be an especially rich source of structurally unusual
and biologically active polar steroids. Often these steroids occur
as complicated mixtures of highly oxygenated sterols, very
difficult to separate into individual compounds. In a continuation
of our search for new polar steroidal metabolites from Far
Eastern starfish,1 we have examined the alcoholic extract of the
spiny red starfish Hippasteria kurilensis Fisher, 1911 (order
Valvatida, family Goniasteridae). Employing column chroma-
tography on silica gel and Florisil followed by a final separation
using reversed-phase HPLC, we have isolated four new steroidal
glycosides, designated as kurilensosides A, B, C, and D (1–4),
along with two new (6, 7) and one known (5) polyhydroxy-
steroids. Kurilensosides A, B, and C (1–3) are unprecedented
examples of steroidal triglycosides, containing two carbohydrate
chains, in which a monosaccharide unit is attached to C-3 and
a disaccharide unit is located at C-24 of a cholestane aglycon.
Earlier only three triglycosides, monilosides G, H, and I, with a
carbohydrate chain only at C-29 were known from the starfish
Fromia monilis,2 in contrast with more than two hundred
different mono- and diglycosides of polyhydroxysteroids isolated
from starfish so far.
the side chain. The planar structure of 1 was determined on the
basis of the correlations in the HMBC spectrum (Table 1). The
analysis of the NMR spectral data of 1 indicated that kurilensoside
AwasrelatedtolinckosideL1,3containingthesame3ꢀ,4ꢀ,6ꢀ,8,15R,24-
hexahydroxy cholestane aglycon bearing a 2-O-methyl-ꢀ-D-xy-
lopyranosyl residue at C-3. However, 1 also has a disaccharide chain
at C-24. Glycosidation at C-24 was supported by the shifts of the
C-23, C-24, and C-25 signals (δ 28.4, 83.8, and 31.3) in the 13C
NMR spectrum of 1 in comparison with the corresponding values
(δ 31.6, 78.1, and 34.6) for steroids having unsubstituted 24-
hydroxycholestane side chains.3 Acid hydrolysis of 1 with aqueous
2 M CF3COOH gave arabinose and 2-O-methylxylose in a 2:1 ratio
that was confirmed by TLC and GC analysis of the corresponding
aldononitrile peracetates. The (+)-MALDI-TOFMS showed a series
of fragmentations with the following sugar losses: m/z 871 [(M +
Na) - 132]+ loss of arabinose; 857 [(M + Na) - 146]+ loss of
2-O-methylxylose, 769 [(M + Na) - 102 - 132]+ loss of a sulfate
group and arabinose. The (-)-MALDI-TOFMS also indicated a
series of fragmentations with the following sugar losses: m/z 825
[(M - Na) - 132]- loss of arabinose; 811 [(M - Na) - 146]-
loss of 2-O-methylxylose; 679 [(M - Na) - 132 - 146]- loss of
arabinose and 2-O-methylxylose. The common L-configuration for
two arabinose units and D-configuration for the 2-O-methylxylose
unit were preferred according to those most often encountered
among the starfish steroidal glycosides.4 The carbon and proton
signals as well as the corresponding coupling constants of the
terminal monosaccharide in the disaccharide moiety at C-24 in the
NMR spectra of 1 strictly coincided with those of the R-L-
arabinofuranosyl residue.5 The signals of an internal monosaccha-
ride coincided with those of a 2′-substituted 3′-O-sulfate-R-L-
arabinofuranosyl unit reported for the first time for miniatoside B6
and differed from those of a nonsulfated 2′-substituted R-L-
arabinofuranosyl unit.4 So, the signal of C-3′ in the 13C NMR
spectrum of 1 was downfield shifted (δ 77.6 f 83.3) and the signal
of C-2′ was upfield shifted (δ 92.4 f 87.9) in addition to those of
the nonsulfated 2′-substituted R-L-arabinofuranosyl unit, which
confirmed the location of sulfate group at C-3′. By careful
examination of monosaccharide signals with the aid of 1H-1H
COSY and HMBC data, the R-L-Araf-(1 f 2)-3′-O-sulfate-R-L-
Araf disaccharide unit attached to C-24 was determined. The linkage
of terminal R-L-Araf at C-2′ was indicated by the cross-peak H-1′′/
C-2′, the linkage of internal 3′-O-sulfate-R-L-Araf at C-24 was
confirmed by the cross-peak H-1′/C-24, and the attachment of a
2-O-Me-Xylp to C-3 was fixed by the long-range correlations of
H-1′′′/C-3 in the HMBC spectrum. Kurilensoside A was subjected
to partial hydrolysis with aqueous acetic acid to give monoside
Results and Discussion
The molecular formula of kurilensoside A (1) was determined
as C43H73O21SNa from the [M + Na]+ sodiated molecular ion at
m/z 1003.4223 (calcd for C43H73O21SNa2, 1003.4160) in the HR
(+)-MALDI-TOFMS and the [M - Na]- ion at m/z 957 in the
(-)-MALDI-TOFMS. The fragment ion peak at m/z 901 [(M +
Na) - SO3Na + H]+ in the (+)-MALDI-TOFMS exhibited the
presence of a sulfate group in the glycoside. The 1H, 13C, and DEPT
NMR spectra of 1 showed the presence of 43 carbon atoms,
including five methyl groups, 11 methylenes, 23 methines, two
quaternary carbons, one oxygenated quaternary carbon, and one
1
methoxyl group (Tables 1 and 3). The H NMR spectrum of 1
exhibited three resonances in the downfield region due to anomeric
protons (δ 5.12, 5.18, and 4.46) that correlated in the HSQC
experiment with carbon signals at δ 108.0, 109.6, and 102.6,
respectively. Along with mass spectra, these data revealed the
existence of three monosaccharide residues and a hexahydroxy-
1
substituted steroidal moiety in glycoside 1. H-1H COSY cross-
peaks indicated sequences of protons at C-1 to C-7, C-9 to C-12
through C-11, C-14 to C-17, C-20 to C-21, and C-22 to the end of
* To whom correspondence should be addressed. Tel: 7 (4232) 312340.
Fax: 7 (4232) 31450. E-mail: stonik@piboc.dvo.ru.
10.1021/np070637x CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/22/2008

794 Journal of Natural Products, 2008, Vol. 71, No. 5
Kicha et al.
Table 1. 1H NMR and HMBC Data for Compounds 1–3 (500 MHz, CD3OD)a
1
2
3
position
δH (J in Hz)
HMBCb
δH (J in Hz)
HMBCb
δH (J in Hz)
HMBCb
1
2
3
4
5
6
7
1.73 m 1.01 m
1.98 m 1.70 m
3.65 m
4.25 m
1.22 m
4.25 m
2.40 dd (2.9, 15.1) 1.59
dd (3.3, 15.2)
1.70 m 0.99 m
1.91 m 1.62 m
3.58 m
1.70 m 0.99 m
1.91 m 1.63 m
3.58 m
4.40 br s
4.40 br s
10, 19
1.46 dd (2.3, 11.7)
4.26 dd (2.9, 11.6)
3.92 d (2.9)
6, 10, 19
1.46 dd (2.3, 11.7)
4.26 dd (2.9, 11.6)
3.91 d (2.8)
6, 10, 19
5, 6, 8, 9
6, 8
8
9
0.97 m
1.09 dd (3.3, 12.6)
1.09 dd (3.4, 12.7)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1′
1.82 m 1.48 m
1.96 m 1.23 m
1.82 m 1.43 m
1.95 m 1.13 m
1.80 m 1.42 m
1.95 m 1.13 m
1.16 d (9.5)
4.28 dt (3.0, 9.4)
1.91 m 1.76 m
1.32 m
0.95 s
1.43 s
13, 15, 18
1.40 d (5.4)
4.53 ddd (2.1, 5.1, 7.6)
2.40 m 1.38 m
0.99 m
1.26 s
1.16 s
8, 13, 15, 17, 18
13
1.40 d (5.4)
8, 13, 15, 17, 18
4.53 ddd (1.8, 5.5, 7.5)
2.40 m 1.38 m
1.00 m
1.26 s
1.16 s
12, 13, 14, 17
1, 5, 9, 10
17
12, 13 14, 17
1, 5, 9, 10
12, 13, 14, 17
1, 5, 9, 10
1.33 m
1.52 m
1.52 m
0.90 d (6.8)
1.61 m 0.96 m
1.59 m 1.28 m
3.34 m
20, 22
0.93 d (6.5)
1.63 m 0.98 m
1.59 m 1.32 m
3.30 mb
17, 20
0.93 d (6.5)
1.63 m 1.00 m
1.58 m 1.34 m
3.30 mb
17, 20, 22
1.87 m
1.83 m
1.83 m
0.90 d (6.8)
0.88 d (6.8)
5.12 br s
4.32 d (1.9)
4.59 dd (1.9, 5.9)
4.16 dt (3.0, 5.5)
24, 25, 27
24, 25, 26
24, 3′
3′, 1′′
2′
0.90 d (6.8)
0.89 d (6.8)
4.93 d (1.5)
3.97 dd (1.7, 3.5)
3.94 dd (3.5, 6.2)
4.06 dt (4.0, 6.2)
24, 25, 27
24, 25, 26
24, 4′
0.90 d (6.8)
0.89 d (6.8)
4.90 d (2.1)
3.96 dd (2.1, 4.2)
3.85 m
24, 25, 27
24, 25, 26
24, 4′
3′
2′
3′
4′
2′
3′
4.05 ddd (3.4, 4.7, 6.9)
5′
3.88 dd (3.1, 12.1) 3.72 4′
3.96 m 3.73 dd (3.8, 11.3) 3′, 4′, 1′′
3.91 dd (5.5, 11.3) 3.72 1′′ 3′, 4′, 1′′
dd (5.4, 12.1)
dd (3.4, 11.3)
1′′
2′′
3′′
4′′
5′′
5.18 d (1.5)
3.98 dd (1.5, 3.4)
3.86 dd (3.4, 6.3)
3.90 ddd (3.4, 4.7, 6.3) 3′′
3.70 dd (3.4, 12.1) 3.62 3′′, 4′′
dd (4.7, 12.1)
3′′, 4′′, 2′
3′′
2′′
4.28 d (7.5)
3.18 t (9.0)
5′
1′′
4.33 d (7.6)
2.87 dd (7.5, 9.0)
3.32 t (8.7)
3.48 m
3.84 dd (5.5, 12.0) 3.16 1′′, 3′′ 1′′
dd (10.0, 11.7)
5′
1′′, 3′′, 2′′-OMe
3.30 mc
3.48 m
3.86 dd (5.3, 11.3) 3.20
dd (10.8, 11.3)
1′′, 4′′
2′′-OMe
3.58 s
2′′
1′′′
2′′′
3′′′
4′′′
5′′′
4.46 d (7.4)
2.90 dd (7.4, 8.9)
3.35 t (8.9)
3.47 m
3
4.43 d (7.4)
2.91 dd (7.6, 9.0)
3.41 t (8.9)
3.17 m
3
4.45 d (7.6)
2.89 dd (7.5, 9.0)
3.34 t (8.9)
3.47 m
3
1′′′, 3′′′, 2′′′-OMe
2′′′, 4′′′
1′′′, 3′′′, 2′′′-OMe
2′′′, 4′′′
1′′′, 3′′′, 2′′′-OMe
2′′′, 4′′′
3.82 dd (5.1, 11.3) 3.17 1′′′, 3′′′, 4′′′ 1′′′, 3′′′, 4′′′ 4.00 dd (4.7, 11.0) 3.11 m 1′′′, 3′′′, 4′′′ 1′′′, 4′′′ 3.82 dd (5.3, 11.9) 3.15 1′′′, 3′′′ 1′′′
dd (10.1, 11.4) dd (10.2, 11.7)
3.61 s
2′′′-OMe 3.61 s
4′′′-OMe
2′′′
3.61 s
3.45 s
2′′′, 4′′′
4′′′
2′′′
a
Assignments from H-1H COSY and HSQC data. b HMBC correlations, optimized for 8 Hz. c Overlapped with the solvent signal.
1
3
1
1a, which was identified by H NMR data as linckoside L1. The
stereochemistry of C-24 was determined as S by analysis of the 1H
NMR spectrum of (R)-MTPA ester obtained by reaction of 1a with
S-(+)-R-methoxy-R-(trifluoromethyl)phenylacetyl (MTPA) chloride
in dry pyridine. The H3-26 and H3-27 signals were observed at δ
0.80 and 0.82, which coincided with those reported for (R)-MTPA
derivative (24S)-24-hydroxysteroids.3,4 Hence, the structure of
kurilensoside A (1) was elucidated as sodium (24S)-3-O-(2-O-
methyl-ꢀ-D-xylopyranosyl)-24-O-[R-L-arabinofuranosyl-(1 f 2)-
3-O-sulfate-R-L-arabinofuranosyl]-5R-cholestane-3ꢀ,4ꢀ,6ꢀ,8,15R,24-
hexol.
HSQC experiment with the corresponding carbons at δ 109.5, 105.4,
and 102.3, respectively. The 1H, 13C, DEPT NMR, and MS spectra
exhibited the presence three monosaccharide residues and a
1
heptahydroxy-substituted steroidal moiety in glycoside 2. The H
NMR data of the steroidal moiety of 2 were related to those of
compound 5a (derived from co-occurring steroid 5 by solvolysis;
see Experimental Section), and thus 2 was expected to have the
same 3ꢀ,4ꢀ,6R,7R,8,15ꢀ,24-heptahydroxy substitution pattern. The
proton and carbon signals attributable to the aglycon moiety were
assigned by the application of 2D NMR experiments including
1H-1H COSY, HSQC, and HMBC. Glycosidation at C-3 and C-24
was supported by the downfield shifts of the H-3 (δ 3.58) and H-24
(δ 3.30) in the 1H NMR spectrum of 2 with respect to the
corresponding values of δ 3.44 and 3.20 in 5a. The (+)-LSIMS
showed a fragmentation with the following sugar losses: m/z 753
[(M + Na) -178]+ loss of 2,4-di-O-methylxylose; 621 [(M + Na)
- 178 - 132]+ loss of 2,4-di-O-methylxylose and xylose. The (-)-
The molecular formula of kurilensoside B (2) was determined
as C44H76O19 from the [M + Na]+ sodiated molecular ion at m/z
931.4821 (calcd for C44H76O19Na, 931.4879) in the HR (+)-
MALDI-TOFMS and the [M - H]- ion at m/z 907 in the (-)-LSI
MS. The 1H NMR spectrum of 2 showed signals for three anomeric
protons at δ 4.93, 4.28, and 4.43, which were correlated in the

Steroidal Triglycosides from Hippasteria kurilensis
Journal of Natural Products, 2008, Vol. 71, No. 5 795
Table 2. 1H NMR Data for Compounds 4, 6, and 7 (500 MHz,
CD3OD, J in Hz)a
The HR (+)-MALDI-TOFMS of kurilensoside D (4) showed a
sodiated molecular ion peak at m/z 769.4394 [M + Na]+ (calcd
for C38H66O14Na, 769.4351). The fragment ion peak in the (-)-
LSI MS at m/z 599 [(M - H) - 164]- corresponded to the loss of
a methoxylated pentose. Comparison of the NMR data of compound
4 with those of 3 revealed the presence of the same 2-O-Me-ꢀ-D-
Xylp-(1 f 5)-R-L-Araf disaccharide unit attached to C-24 of the
steroid side chain (Tables 2 and 3). The generality of the signals
in the NMR spectra of 4 attributable to the steroid nucleus was
similar to those of 3. However, the signal of H-6 in 4 appeared as
triplet doublets at δ 4.15 instead of doublet doublets at δ 4.26 of
3 due to the lack of a hydroxyl group at C-7. Furthermore, the
signals of C-2 (δ 26.2), C-3 (δ 73.7), and C-4 (δ 69.1) in 4 were
shifted in comparison with the analogous signals in 3 (δ 24.8, 81.2,
66.5, respectively) due to the absence of a monosaccharide residue
at C-3. 2D NMR (1H-1H COSY, HSQC, and HMBC) analysis
indicated that the aglycon moiety contains a 3ꢀ,4ꢀ,6R,8,15ꢀ,24-
hexahydroxyoxidation pattern, and the structure of kurilensoside
D was proposed as 4.
position
4b
6
7
1
2
3
1.70 m 0.96 m
1.82 m 1.54 m
3.42 m
1.71 m 1.01 m
1.83 m 1.57 m
3.47 ddd (3.8, 4.9, 3.49 ddd (3.9, 5.2,
1.72 m 1.00 m
1.88 m 1.62 m
11.9)
4.21 br s
1.72 dd (2.3, 12.2) 1.23 m
5.04 dd (2.8, 12.0) 4.24 m
11.9)
4.05 br s
4
5
6
7
4.25 br s
0.93 m
4.15 td (4.3, 10.8)
2.44 dd (4.3, 12.3) 4.23 d (2.9)
1.30 m
2.40 dd (3.0, 15.0)
1.59 dd (2.9, 14.9)
8
9
0.81 dd (3.2, 12.6) 1.09 m
0.97 m
10
11
12
1.78 m 1.43 m
1.97 dd (3.0, 12.4) 1.95 m 1.15 m
1.15 m
1.82 m 1.43 m
1.81 m 1.47 m
1.96 m 1.23 m
13
14
15
0.98 d (5.7)
1.42 d (5.8)
1.17 d (9.7)
4.41 ddd (2.1, 5.6, 4.48 ddd (2.1, 5.7, 4.27 dt (3.2, 9.5)
7.7)
7.7)
16
17
18
19
20
21
22
23
24
25
26
27
2.38 m 1.39 m
0.97 m
1.25 s
1.15 s
1.51 m
0.92 d (6.6)
1.62 m 0.98 m
1.57 m 1.33 m
3.30 mc
1.82 m
0.90 d (6.8)
0.89 d (6.8)
2.23 m 1.37 m
1.01 m
1.28 s
1.22 s
2.20 m
1.02 d (6.6)
5.42 dd (8.0, 15.2) 1.60 m 0.99 m
5.37 dd (6.8, 15.5) 1.55 m 1.21 m
3.67 t (6.5)
1.65 m
0.90 d (6.7)
0.86 d (6.7)
1.91 m 1.69 m
1.34 m
0.95 s
1.42 s
1.36 m
Kurilensosides A, B, and D (1, 2, and 4) contain rare carbohy-
drate moieties with (1 f 5) bonds between the monosaccharide
units. Earlier this type of asterosaponin carbohydrate chain was
known only for mediasterosides M1-M3 from Mediaster murrayi7
and crossasteroside P4 from Crossaster papposus.8
0.91 d (6.0)
The known compound 5 was identified by comparison of the
MS and NMR data with those reported.9 It was previously isolated
from the Far Eastern starfish Hippasteria phrygiana.9
3.19 m
1.61 m
0.90 d (6.7)
0.88 d (6.7)
The HR (+)-MALDI-TOFMS of compound 6 showed a sodiated
molecular ion peak at m/z 607.2587 [M + Na]+ (calcd for
C27H45O10SNa2, 607.2529). The fragment ion peaks at m/z 505 [(M
+ Na) - 102]+ and 487 [(M + Na) - 120]+ in the (+)-LSIMS
and at 97 [HSO4]- and 80 [SO3]- in the (-)-LSIMS exhibited the
presence of a sulfate group in the steroid. Detailed comparison of
NMR data of 6 with those of 5 clearly indicated that compound 6
contained the identical 3ꢀ,4ꢀ,6R,7R,8,15ꢀ-hexahydroxysteroidal
nucleus with a sulfoxy group at C-6 and differed from 5 only in
the presence of the signals attributable to a 22(23)-double bond
(δH 5.42 and 5.37, δC 139.3 and 130.1) in the 24-hydroxyholestane
side chain. The sequence of H-17 to H-27 correlating with the
corresponding carbon atoms was revealed using 1H-1H COSY and
HSQC experiments. The HMBC correlations supported the total
structure of the side chain: Me-21/C-17, C-20, C-22; H-22/C-23;
H-23/C-17, C-24; Me-26/C-24, C-25, C-27; Me-27/C-24, C-25,
C-26. In addition, the J ) 15.2 Hz suggested a trans-configuration
for a 22(23)-double bond. Thus, steroid 6 was determined to be a
∆22-derivative of 5.
a Assignments from 1H-1H COSY and HSQC data. 1H NMR data
for the disaccharide moiety are identical with those of kurilensoside C
(3); see Table 1. c Overlapped with the solvent signal.
b
LSIMS also indicated a peak at m/z 729 [(M - H) -178]-
coinciding with the loss of 2,4-di-O-methylxylose. By analysis of
1
the H and 13C NMR data, a 2,4-di-O-methyl-ꢀ-D-xylopyranosyl
residue7,8 attached to C-3 and ꢀ-D-xylopyranosyl-(1 f 5)-R-L-
arabinofuranosyl disaccharide unit attached to C-24 were identified.
The signals of the ꢀ-D-Xylp-(1 f 5)-R-L-Araf disaccharide unit in
the 13C NMR spectrum coincided well with those reported for
mediasteroside M2.7 The attachment of the carbohydrate fragments
and the presence of the (1 f 5) interglycosidic linkage was
confirmed by the HMBC experiment, which showed the following
cross-peaks: H-1′(Ara)/C-24, H-1′′(Xyl)/C-5′, and H-1′′′(2,4-di-O-
Me-Xyl)/C-3. The stereochemistry at C-24 was expected to be S
by analogy with co-occurring compound 1. Thus, the structure of
kurilensoside B was defined as 2.
The HR (+)-MALDI-TOFMS of compound 7 exhibited a
sodiated molecular ion peak at m/z 491.3369 [M + Na]+ (calcd
for C27H48O6Na, 491.3349). The NMR data of 7 were very similar
to those of the aglycon moiety of kurilensoside A (1). The signals
of H-3, H-24, C-3, and C-24 of steroid 7 were shifted from δH
3.49 to 3.65 and 3.19 to 3.34, and from δC 73.1 to 80.6 and 78.1
to 83.8, respectively, in comparison with glycoside 1, as a result
of lack of carbohydrate units at the C-3 and C-24 positions. The
total structure of 7 was confirmed by the HMBC correlations H-5/
C-10, C-19; H-6/C-8, C-10; H-7eq/C-5; H-14/C-13, C-18; H-16/
C-17; H-18/C-12, C-13, C-14, C-17; H-19/C-1, C-5, C-9, C-10;
H-21/C-17, C-20, C-22; H-26/C-24, C-25, C-27; H-27/C-24, C-25,
C-26. Thereby, the steroid 7 was proved to be a free aglycon of 1.
Although some glycosides of polyhydroxysteroids with the same
aglycon have been previously reported from starfish,3–5 the native
steroid 7 was encountered for the first time.
The HR (+)-MALDI-TOFMS of kurilensoside C (3) showed a
sodiated molecular ion peak at m/z 931.4934 [M + Na]+ (calcd
for C44H76O19Na, 931.4879). The detailed comparison of its NMR
data with those of 2 clearly indicated that glycoside 3 has the same
steroidal aglycon with the 5′-substituted R-L-arabinofuranosyl unit
attached to C-24 and differs from 2 only in the signals of the
terminal monosaccharide units. By analysis of the 1H-1H COSY,
HSQC and HMBC experiments, two 2-O-methyl-ꢀ-D-xylopyranosyl
residues attached to C-3 of the steroid nucleus and to C-5′ of the
R-L-arabinofuranosyl unit were identified. The fragment ion peak
in the (-)-LSIMS at m/z 743 [(M - H) - 164]- corresponding to
the loss of 2-O-methylxylose was confirmed at its terminal position.
The location of the methoxy groups at C-2′′ and C-2′′′ of the
corresponding xylopyranose units (2 and 3 in the formula) was
supported by the reciprocal HMBC correlations between the
methoxy groups and C-2′′ and C-2′′′, respectively. The signals of
Glycosides 1-4 and steroids 6 and 7 were assayed for inhibition
of the egg fertilization by sperm of the sea urchin Strongylocentrotus
nudus preincubated with these compounds (sperm test). Kurilen-
sosides B, C, and D (2–4) and polyhydroxysteroid 7 exhibited 100%
inhibition at 5.5 × 10-5, 5.5 × 10-5, 6.7 × 10-5, and 1.3 × 10-5
the 2-O-Me-ꢀ-D-Xylp-(1 f 5)-R-L-Araf disaccharide unit in the 13
C
NMR spectrum corresponded well to those reported for medias-
teroside M1.7 Accordingly, the structure of kurilensoside C was
established as 3.

796 Journal of Natural Products, 2008, Vol. 71, No. 5
Kicha et al.
Table 3. 13C NMR Data for Compounds 1–4, 6, and 7 (CD3OD, 125.8 MHz)
position
1
2
3
4
6
7
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1′
41.0
25.9
80.6
74.7
50.5
76.2
45.3
76.8
57.7
36.9
19.3
42.7
45.4
66.4
70.1
41.9
56.0
15.3
18.6
36.5
19.0
33.0
28.4
83.8
31.3
18.5
18.1
108.0
87.9
83.3
84.0
62.8
39.7
24.9
81.3
66.5
47.4
66.6
76.7
79.3
51.2
37.9
18.9
43.0
44.2
56.6
71.2
42.8
58.0
16.5
16.9
36.5
19.1
32.9
28.8
85.0
31.9
18.4
18.3
109.5
83.8a
79.2
83.9a
69.8
39.7
24.8
81.2
66.5
47.4
66.7
76.7
79.3
51.2
37.9
18.9
43.0
44.2
56.6
71.2
42.8
57.9
16.5
16.9
36.5
19.1
32.9
28.7
84.8
31.7
18.4
18.3
109.3
83.9
79.0
83.0
70.1
39.7
26.2
73.7
69.1
57.2
64.8
49.8
77.4
58.4
38.2
19.2
43.4
44.4
62.8
71.1
42.6
58.0
16.5
17.0
36.5
19.0
32.8
28.8
84.9
31.8
18.4
18.3
109.3
83.9
79.0
83.0
70.1
39.6
26.6
73.0
69.4
46.7
76.6
75.0
79.4
51.2
38.5
18.8
42.8
44.1
56.5
71.0
43.4
57.7
16.5
16.8
40.6
20.8
139.3
130.1
78.9
35.3
18.9
18.7
41.0
26.5
73.1
77.5
50.6
76.2
45.2
76.8
57.6
36.7
19.3
42.7
45.5
66.5
70.1
41.7
55.8
15.3
18.7
36.3
19.0
33.3
31.5
78.1
34.6
19.5
17.5
2′
3′
4′
5′
1′′
2′′
3′′
4′′
5′′
2′′-OMe
1′′′
2′′′
3′′′
4′′′
5′′′
2′′′-OMe
4′′′-OMe
109.6
83.7
78.6
85.7
62.7
105.4
74.9
77.7
71.2
67.0
102.3
84.7
77.7
71.1
66.8
61.0
105.4
84.6
77.2
71.3
66.9
61.1
105.3
84.6
77.2
71.2
66.9
61.1
102.6
84.7
77.6
71.3
66.8
61.0
102.3
84.7
76.8
81.0
64.2
61.1
59.0
a Assignment may be reversed.
raphy was performed using Amberlite XAD-2 (20–80 mesh, Sigma,
Chemical Co.), Si gel KSK (50-160 µm, Sorbpolimer, Krasnodar,
Russia), and Florisil (200–300 mesh, Aldrich Chemical Co.). Sorbfil
Si gel plates (4.5 × 6.0 cm, 5–17 µm, Sorbpolimer, Krasnodar, Russia)
in the eluent system BuOH/EtOH/H2O (4:1:2) were used for thin-layer
chromatography.
Animal Material. Specimens of Hippasteria kurilensis Fisher, 1911
(order Valvatida, family Goniasteridae) were collected by dredging at
a depth of 100 m near Matua Island (Kuril Islands) in the Sea of
Okhotsk (research vessel Akademik Oparin, 29th scientific cruise) in
July 2003. Species identification was carried out by Dr. A. V. Smirnov
(Zoological Institute of the Russian Academy of Science, St. Petersburg,
Russia). A voucher specimen [no. 029-26] is on deposit at the marine
specimen collection of the Pacific Institute of Bioorganic Chemistry,
Vladivostok, Russia.
Extraction and Isolation. The fresh animals (770 g) were chopped
and extracted twice with EtOH at 20 °C. The H2O/EtOH layer was
evaporated, and the residue was dissolved in H2O (1 L). The H2O-
soluble fraction was passed through an Amberlite XAD-2 column (7
× 20 cm) and eluted with distilled H2O until a negative chloride ion
reaction was obtained, followed by elution with EtOH. The combined
EtOH eluate was evaporated to give a brownish material (3.4 g). The
resulting total fraction of steroidal compounds was chromatographed
on a Si gel column (4 × 18 cm) using CHCl3/EtOH (stepwise gradient,
4:1 f 1:6), and then obtained fractions were purified on a Florisil
M, respectively. The sulfated substances 1 and 6 were nonactive
in the sperm test below 5 × 10-5 M and showed weaker activities,
with EC100 values of 8.9 × 10-5 and 11 × 10-5 M, respectively.
Experimental Section
General Experimental Procedures. Optical rotations were deter-
mined on a Perkin-Elmer polarimeter model 343. The 1H and 13C NMR
spectra were recorded on a Bruker DPX 300 spectrometer at 300 and
75.5 MHz, respectively, and on a Bruker DRX 500 spectrometer at
500 and 125.8 MHz, respectively, using signals of CD3OD (3.30 ppm
in the 1H NMR spectrum and 49.0 ppm in the 13C NMR spectrum) as
the internal standard. MALDI-TOF mass spectra were recorded on a
Bruker Biflex III laser-desorption mass spectrometer coupled with
delayed extraction using a N2 laser (337 nm). Samples of the tested
compounds were dissolved in MeOH (1 mg/mL), and 1 µL aliquots
were analyzed using a R-cyano-4-hydroxycinnamic acid (CCA) matrix.
LSI mass spectra were recorded on an AMD-604S mass spectrometer
(AMD, Germany) with an accelerating voltage of 8 keV and an energy
of Cs+ ions of 10–12 keV. For recording the mass spectra, a sample
was dissolved in MeOH (10 mg/mL) and an aliquot (1 µL) was analyzed
using glycerol (Sigma) as the matrix.
HPLC separations were carried out on a Agilent 1100 Series
chromatograph equipped with a differential refractometer. Diasfer-110-
C18 (10 µm, 250 × 15 mm) and Kromasil 100A-C18 (5 µm, 250 ×
4.6 mm) columns were used. Low-pressure column liquid chromatog-

Steroidal Triglycosides from Hippasteria kurilensis
Journal of Natural Products, 2008, Vol. 71, No. 5 797
Chart 1
1
column (2.5 × 15 cm) using CHCl3/EtOH (stepwise gradient, 4:1 f
1:2). HPLC separation of collected subfractions, containing nonsulfated
steroids, on a Diasfer-110-C18 column (10 µm, 250 × 15 mm, 2.5
mL/min) with 65% EtOH as the eluent system, followed by purification
on a Kromasil 100A-C18 column (5 µm, 250 × 4.6 mm, 0.5 mL/min)
with 80% MeOH as the eluent system, yielded pure 2 (1.4 mg, Rf 0.65),
3 (2.2 mg, Rf 0.65), 4 (1.7 mg, Rf 0.75), and 7 (1.0 mg, Rf 0.85). HPLC
separation of collected subfractions, containing sulfated steroids, on a
Diasfer-110-C18 column (10 µm, 250 × 15 mm, 2.5 mL/min) with
55% EtOH as the eluent system gave pure 1 (3.7 mg, Rf 0.55), 5 (1.0
mg, Rf 0.78), and 6 (1.1 mg, Rf 0.78).
Compound 6: amorphous powder; [R]25 (0 (c 0.1, MeOH); H
D
NMR data, see Table 2; 13C NMR data, see Table 3; LSIMS(+) m/z
607 [M + Na]+, 505 [(M + Na) - 102]+; 487 [(M + Na) - 120]+,
454 [(M + Na) - 153]+; LSIMS(-) m/z 561 [M - Na]-, 408 [(M -
Na) -153]-, 97 [HSO4]-, 80 [SO3]-; HR MALDI-TOFMS (+) m/z
607.2587 [M + Na]+ (calcd for C27H45O10SNa2, 607.2529).
Compound 7: amorphous powder; [R]25 +14.7 (c 0.1, MeOH);
D
1H NMR data, see Table 2; 13C NMR data, see Table 3; EIMS m/z (%)
468 [M]+ (3), 450 [M - H2O]+ (75), 432 [M - 2H2O]+ (100), 414 [M
- 3H2O]+ (75), 396 [M - 4H2O]+ (30), 378 [M - 5H2O]+ (8), 321
(78); HR MALDI-TOFMS (+) m/z 491.3369 [M + Na]+ (calcd for
C27H48O6Na, 491.3349).
Kurilensoside A (1): amorphous powder; [R]25D -48.0 (c 0.1; H2O/
1
MeOH, 3:1); H NMR data, see Table 1; 13C NMR data, see Table 3;
Acid Hydrolysis of 1. A solution of glycoside 1 (1 mg) in aqueous
2 M CF3COOH (1 mL) was heated at 100 °C for 2 h in a sealed
ampule. The reaction mixture was evaporated in vacuo, and the
residue was dissolved in H2O and extracted with CHCl3 twice. The
monosaccharides 2-O-methylxylose and arabinose were identified
in the aqueous layer by TLC on Si gel in BuOH/EtOH/H2O (4:1:2).
The aqueous layer was evaporated to dryness. Then, pyridine (0.5
mL) and NH2OH · HCl (2 mg) were added to the dried residue, and
the mixture was heated at 90 °C for 40 min. After that time, 0.5
mL of Ac2O was added and the heating at 90 °C was continued for
a further 1 h. The solution was concentrated, and the resulting
aldononitrile peracetates were analyzed by GLC using standard
aldononitrile peracetates as reference samples.
MALDI-TOFMS (+) m/z 1003 [M + Na]+, 901 [(M + Na) - 102]+,
871 [(M + Na) - 132]+, 857 [(M + Na) - 146]+, 769 [(M + Na) -
102 - 132]+, 637 [(M + Na) - 102 - 2 × 132]+, 619 [637 - H2O]+,
601 [637 - 2 × H2O]+; MALDI-TOFMS (-) m/z 957 [M - Na]-,
825 [(M - Na) - 132]-, 811 [(M - Na) - 146]-, 679 [(M - Na) -
132 - 146]-; HR MALDI-TOFMS (+) m/z 1003.4223 [M + Na]+
(calcd for C43H73O21SNa2, 1003.4160).
Kurilensoside B (2): amorphous powder; [R]25D -16.8 (c 0.1; H2O/
1
MeOH, 3:1); H NMR data, see Table 1; 13C NMR data, see Table 3;
LSIMS(+) m/z 931 [M + Na]+, 753 [(M + Na) - 178]+, 621 [(M +
Na) - 178 - 132]+; LSIMS(-) m/z 907 [M - H]-, 729 [(M - H)
-178]-; HR MALDI-TOFMS (+) m/z 931.4821 [M + Na]+ (calcd
for C44H76O19Na, 931.4879).
Partial Acid Hydrolysis of 1. A solution of 1 (2 mg) in 50%
aqueous CH3COOH (1 mL) was stirred at 54 °C for 48 h. The
reaction mixture was evaporated in vacuo, and the residue was
subjected to HPLC separation on a Diasfer-110-C18 column (5 µm,
250 × 4 mm, 0.5 mL/min) with 65% EtOH as the eluent system to
Kurilensoside C (3): amorphous powder; [R]25D -23.2 (c 0.1; H2O/
1
MeOH, 3:1); H NMR data, see Table 1; 13C NMR data, see Table 3;
LSIMS(+) m/z 931 [M + Na]+; LSIMS(-) m/z 907 [M - H]-, 743
[(M - H) - 164]-; HR MALDI-TOFMS (+) m/z 931.4934 [M +
Na]+ (calcd for C44H76O19Na, 931.4879).
1
give 1a (0.5 mg). H NMR data of 1a were identical with those of
Kurilensoside D (4): amorphous powder; [R]25D -4.6 (c 0.1; H2O/
linckoside L1.3
1
MeOH, 1:6); H NMR data, see Table 2; 13C NMR data, see Table 3;
Preparation of (R)-MTPA Ester of Compound 1a. Compound 1a
(0.5 mg) was treated with S-(+)-R-methoxy-R-(trifluoromethyl)phe-
nylacetyl (MTPA) chloride (3 µL) in dry pyridine (100 µL) for 4 h at
room temperature. After removal the solvent, the product was purified
on a Si gel column (0.8 × 9 cm) using hexane/CHCl3 (1:4) to obtain
LSIMS(+) m/z 769 [M + Na]+; LSIMS(-) m/z 745 [M - H]-, 599
[(M - H) -146]-; HR MALDI-TOFMS (+) m/z 769.4394 [M + Na]+
(calcd for C38H66O14Na, 769.4351).
Compound 5: amorphous powder; [R]25D +8.1 (c 0.1, MeOH); 1H
NMR (CD3OD, 500 MHz) δ 0.89 (3H, d, J ) 6.7 Hz, H3-27), 0.91
(3H, d, J ) 6.7 Hz, H3-26), 0.94 (3H, d, J ) 6.6 Hz, H3-21), 1.23 (3H,
s, H3-19), 1.26 (3H, s, H3-18), 1.41 (1H, d, J ) 5.8 Hz, H-14), 1.72
(1H, dd, J ) 2.3, 12.2 Hz, H-5), 2.35 (1H, m, H-16), 3.20 (1H, m,
H-24), 3.48 (1H, m, H-3), 4.21 (1H, br s, H-4), 4.24 (1H, d, J ) 2.7
Hz, H-7), 4.50 (1H, ddd, J ) 2.1, 5.5, 7.6 Hz, H-15), 5.03 (1H, dd, J
) 2.7, 12.1 Hz, H-6); 13C NMR data were identical with those reported
by Levina et al.;9 MALDI-TOFMS (+) m/z 609 [M + Na]+, 507 [(M
+ Na) - 102]+, 489 [(M + Na) - 120]+.
1
the corresponding (R)-MTPA ester of 1a (0.3 mg): selected H NMR
(CD3OD, 500 MHz) δ 0.80 (3H, d, J ) 6.7 Hz, H3-27), 0.82 (3H, d,
J ) 6.7 Hz, H3-26), 0.92 (3H, d, J ) 6.2 Hz, H3-21), 0.98 (3H, s,
H3-18), 1.37 (3H, s, H3-19).
Solvolysis of 5. A solution of 5 (1.0 mg) in a mixture of pyridine
(0.5 mL) and dioxane (0.5 mL) was heated at 120 °C for 5 h. The
reaction mixture was evaporated to dryness and purified on a Florisil
column (0.8 × 2 cm) using CHCl3/EtOH (3:1) to obtain the desulfated
1
derivative 5a (0.5 mg): H NMR (CD3OD, 500 MHz) δ 0.88 (3H, d,

798 Journal of Natural Products, 2008, Vol. 71, No. 5
Kicha et al.
J ) 6.7 Hz, H3-27), 0.90 (3H, d, J ) 6.7 Hz, H3-27), 0.94 (3H, d, J )
6.6 Hz, H3-21), 1.15 (3H, s, H3-19), 1.26 (3H, s, H3-19), 1.40 (1H, d,
J ) 5.5 Hz, H-14), 1.47 (1H, dd, J ) 2.2, 11.6 Hz, H-5), 2.36 (1H, m,
H-16), 3.20 (1H, m, H-24), 3.44 (1H, m, H-3), 3.91 (1H, d, J ) 2.9
Hz, H-7), 4.18 (1H, br s, H-4), 4.26 (1H, dd, J ) 2.8, 11.6 Hz, H-6),
4.52 (1H, ddd, J ) 2.0, 5.5, 7.5 Hz, H-15).
References and Notes
(1) Ivanchina, N. V.; Kicha, A. A.; Kalinovsky, A. I.; Dmitrenok, P. S.;
Chaikina, E. L.; Stonik, V. A.; Gavagnin, M.; Cimino, G. J. Nat. Prod.
2006, 69, 224–228.
(2) Casapullo, A.; Finamore, E.; Minale, L.; Zollo, F.; Carré, J. B.; Debitus,
C.; Laurent, D.; Folgore, A.; Galdiero, F. J. Nat. Prod. 1993, 56, 105–
115.
(3) Kicha, A. A.; Ivanchina, N. V.; Kalinovsky, A. I.; Dmitrenok, P. S.;
Palyanova, N. V.; Pankova, T. M.; Starostina, M. V.; Gavagnin, M.;
Stonik, V. A. Nat. Prod. Commun. 2007, 2, 41–46.
(4) Minale, L.; Riccio, R.; Zollo, F. In Progress in the Chemistry of
Organic Natural Products; Herz, W., Kirby, G. W., Moore, R. E.,
Steglich, W., Tamm, Ch., Eds.; Springer-Verlag: Vienna, 1993; Vol.
62, pp 75–308.
(5) Pizza, C.; Minale, L.; Laurent, D.; Menou, J. L. Gazz. Chim. Ital.
1985, 115, 585–589.
(6) D’Auria, M. V.; Iorizzi, M.; Minale, L.; Riccio, R.; Uriarte, E. J. Nat.
Prod. 1990, 53, 94–101.
(7) Kicha, A. A.; Kalinovsky, A. I.; Ivanchina, N. V.; Stonik, V. A. J.
Nat. Prod. 1999, 62, 279–282.
(8) Kicha, A. A.; Kalinovsky, A. I.; Stonik, V. A. Khim. Prir. Soedin.
1993, 257–260.
Sperm Test. The sea urchin Strongylocentrotus nudus sperm and
eggs were used as test material for bioassays according to the method
of Kobayashi10 with minor modification. Two drops of the sperm
solution was added to 25 mL of sea water and mixed just prior to
each test. Then 0.9 mL of sperm suspension was added to each well
of a 24-well microplate, including 0.1 mL of toxicant solution in
distilled H2O. After 15 min of exposure, approximately 1000
prewashed eggs were added to the sperm-toxicant mixture and
fertilization was allowed to proceed for 15 min. The eggs were then
preserved in 2% formaldehyde, and fertilization was tabulated by
scoring the presence or absence of a complete fertilization membrane
in a subsample of about 100 eggs. Fertilization in seawater controls
was g98%. Data for every compound were analyzed from three
independent measurements. The results were expressed as a percent-
age relative to the controls. Means and standard errors for each
treatment were calculated, using SigmaPlot 3.02 software (Jandel
Corporation).
(9) Levina, E. V.; Kalinovsky, A. I.; Andriyashchenko, P. V.; Dmitrenok,
P. S.; Evtushenko, E. V.; Stonik, V. A. Russ. Chem. Bull. 2004, 53,
2634–2638.
(10) Kobayashi, N. In Ecotoxicological Testing for the Marine EnVironment;
Persoone, G., Jaspers, E., Claus, C., Eds.; State University of Ghent
and Institute of Marine Scientist Research: Bredene: Belgium, 1984;
Vol. 1, pp 798–800.
Acknowledgment. The research described in this publication was
made possible in part by Program of Presidium of RAS “Molecular
and Cell Biology”, Grant No. 08-04-00599 from the RFBR, Grants
No. 06-II-CM-05-001 and 06-III-B-05-128 from FEB RAS, and
Grant No. 2813.2008.4 supporting leading Russian scientific schools.
NP070637X