Asterosaponins from the Far Eastern starfish Leptasterias ochotensis and their anticancer activity

Article abstract of DOI:10.1016/j.steroids.2014.05.027

Six new asterosaponins, leptasteriosides A-F (3-8), one new and one previously known asterogenins (1, 2) were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis. The structures of 1-8 were elucidated by extensive NMR and ESI-MS techniques. Compounds 2-8 showed slight or moderate cytotoxic activities against cancer cell lines RPMI-7951 and T-47D. The asterosaponins 3-5 demonstrated a significant inhibition of RPMI-7951 and T-47D cell colony formation in soft agar clonogenic assay in nontoxic doses.

Full text of DOI:10.1016/j.steroids.2014.05.027

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Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
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Asterosaponins from the Far Eastern starfish Leptasterias ochotensis
and their anticancer activity
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Timofey V. Malyarenko, Alla A. Kicha, Natalia V. Ivanchina, Anatoly I. Kalinovsky, Roman S. Popov,
Olesya S. Vishchuk, Valentin A. Stonik
7 Q1
⇑
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G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Pr. 100-let Vladivostoku 159, 690022 Vladivostok, Russia
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a r t i c l e i n f o
a b s t r a c t
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Article history:
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Six new asterosaponins, leptasteriosides A–F (3–8), one new and one previously known asterogenins (1,
2) were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis. The struc-
tures of 1–8 were elucidated by extensive NMR and ESI-MS techniques. Compounds 2–8 showed slight
or moderate cytotoxic activities against cancer cell lines RPMI-7951 and T-47D. The asterosaponins
3–5 demonstrated a significant inhibition of RPMI-7951 and T-47D cell colony formation in soft agar
clonogenic assay in nontoxic doses.
Received 20 February 2014
Received in revised form 23 May 2014
Accepted 29 May 2014
Available online xxxx
20 Q2
Keywords:
Asterosaponins
Starfish
Leptasterias ochotensis
Cytotoxicity
Ó 2014 Published by Elsevier Inc.
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Clonogenic assay
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1. Introduction
from the alcoholic extract of animals collected off the Sea of Okhotsk
near Shantar Islands. We describe the structures of new steroid
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The low molecular weight metabolites from the starfish (phylum
Echinodermata, class Asteroidea) are characterized by a remarkable
diversity of polar steroids, including polyhydroxysteroids and
related mono- and biosides as well as steroid oligoglycosides named
as asterosaponins [1–6]. These substances usually represent very
complicated mixtures, whose separations into individual compo-
nents are rather difficult. The majority of asterosaponins contains
compounds 1, 3–8, which were isolated along with known asterog-
enin 2, as well as their cytotoxic properties against human cancer
cell lines RPMI-7951 and T-47D, and the capabilities of compounds
3–5 to inhibit the colony formation of RPMI-7951 and T-47D cells in
a soft agar clonogenic assay in vitro.
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2. Experimental
3b,6a-dihydroxysteroid aglycones with the 9(11)-double bond
and an O-sulfate group at C-3. Their carbohydrate chains include,
as a rule, five or six monosaccharide units attached to C-6 of the
aglycon. Steroid metabolites from starfish, especially glycosides,
have been reported to show a wide spectrum of biological activities,
including cytotoxic, antiviral, antibacterial, antibiofouling, neurito-
genic and antifungal effects [1–6]. Recently, we have shown that
some of the new asterosaponins from the starfish Hippasteria
kurilensis, Asteropsis carinifera and Lethasterias fusca exhibit a
significant inhibition of the human tumor HT-29, HCT-116, RPMI-
7951, and T-47D cells colony formation in soft agar clonogenic assay
[7–9].
2.1. General methods
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Optical rotations were determined on a PerkinElmer 343 polar-
imeter. The 1H and 13C NMR spectra were recorded on a Bruker
Avance III 700 spectrometer at 700.13 and 176.04 MHz, respec-
tively, with tetramethylsilane used as an internal standard. The
HR ESI mass spectra were recorded on an Agilent 6510 Q-TOF
LC/MS mass spectrometer; the samples were dissolved in MeOH
(c 0.001 mg/mL). The CD spectrum was determined on a Jasco
500A spectropolarimeter in MeOH. HPLC separations were carried
out on an Agilent 1100 Series chromatograph that was equipped
So far, polar steroids of the Far Eastern starfish Leptasterias ochot-
ensis (order Forcipulatida, family Asteriidae) have not been studied.
Herein, we report results of our study on the asterosaponin fractions
with a differential refractometer; the Diasfer-110-C18 (10
l
m,
250 mm ꢀ 15 mm) and Discovery C18 (5
l
m, 250 mm ꢀ 10 mm)
columns were used. GC analysis was conducted on an Agilent
6580 Series apparatus, with the carrier gas He (1.7 mL/min) at
100 °C (0.5 min) to 250 °C (5 °C/min, 10 min) and the capillary
column HP-5 MS (30 m ꢀ 0.25 mm), the length of the program
⇑
Corresponding author. Tel.: +7 4232 312360; fax: +7 4232 314050.
E-mail address: stonik@piboc.dvo.ru (V.A. Stonik).
http://dx.doi.org/10.1016/j.steroids.2014.05.027
0039-128X/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: Malyarenko TV et al. Asterosaponins from the Far Eastern starfish Leptasterias ochotensis and their anticancer activity.
Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.05.027

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was 45 min. The temperatures of the injector and the detector
were 150 and 280 °C, respectively. Low-pressure column liquid
chromatography was performed with the Polychrom 1 (powdered
2.4.2. (22E)-6
a
-Hydroxy-5
a
-cholesta-9(11),20(22)-dien-23-one-3b-yl
sulfate, sodium salt (2)
C27H41O6SNa, amorphous powder; ½a D25
ꢁ
ꢂ 12:0ꢃ (c 0.2, MeOH);
Teflon, Biolar, Latvia), Si gel KSK (50–160
lm, Sorbpolimer, Krasno-
1H and 13C NMR data are listed in Tables 1 and 2; (+)HR ESI-MS
m/z 539.2441 [M+Na]+ (calcd. for C27H41O6SNa2, 539.2414);
(ꢂ)HR ESI-MS m/z 493.2625 [MꢂNa]ꢂ (calcd. for C27H41O6S,
493.2629).
dar, Russia). Sorbfil silica gel plates (4.5 ꢀ 6.0 cm, 5–17
l
m, Sorb-
polimer, Krasnodar, Russia) were used for thin-layer
chromatography. The mediums Minimum Essential Medium Eagle
(MEM), Roswell Park Memorial Institute (RPMI-1640), McCoy 5a
(McCoy), fetal bovine serum (FBS), L-glutamine, and gentamicin
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2.4.3. Leptasterioside B (3)
C57H93O26SNa, amorphous powder; ½a D25
ꢁ
þ 3:0ꢃ (c 0.18, MeOH);
were purchased from Biolot (Russia). The CellTiter 96 nonradioac-
tive cell proliferation assay kit was purchased from Promega (Mad-
ison, WI).
1H and 13C NMR data are listed in Tables 1–3; (+)ESI-MS/MS of the
ion [M+Na]+ at m/z 1271: 1151 [(M+Na)–NaHSO4]+, 1005 [(M+Na)–
NaHSO4–C6H10O4]+, 859 [(M+Na)–NaHSO4–2 ꢀ C6H10O4]+, 713
[(M+Na)–NaHSO4–3 ꢀ C6H10O4]+; (+)HR ESI-MS m/z 1271.5481
[M+Na]+ (calcd. for C57H93O26SNa2, 1271.5466); (ꢂ)ESI-MS/MS of
the ion [MꢂNa]ꢂ at m/z 1225: 1079 [(MꢂNa)–C6H10O4]ꢂ, 933
[(MꢂNa)–2 ꢀ C6H10O4]ꢂ, 787 [(MꢂNa)–3 ꢀ C6H10O4]ꢂ, 655
[(MꢂNa)–3 ꢀ C6H10O4–C5H8O4]ꢂ, 509 [(MꢂNa)–4 ꢀ C6H10O4–
C5H8O4]ꢂ, 491 [(MꢂNa)–4 ꢀ C6H10O4–C5H8O4–H2O]ꢂ, 97 [HSO4]ꢂ;
(ꢂ)HR ESI-MS m/z 1225.5678 [MꢂNa]ꢂ (calcd. for C57H93O26S,
1225.5681).
97
2.2. Animal material
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Specimens of L. ochotensis Brandt, 1851 (order Forcipulatida,
family Asteriidae) were collected at a depth of 20–40 m in the
Sea of Okhotsk near the Island of Bolshoy Shantar during the
research vessel Akademik Oparin 29th scientific cruise in August
2003. Species identification was carried out by Dr. A.V. Smirnov
(Zoological Institute of the Russian Academy of Sciences, St. Peters-
burg, Russia). A voucher specimen [no. 029-052] is on deposit at
the marine specimen collection of the G.B. Elyakov Pacific Institute
of Bioorganic Chemistry of the FEB RAS, Vladivostok, Russia.
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2.4.4. Leptasterioside A (4)
C56H91O26SNa, amorphous powder; ½a D25
ꢁ
þ 7:5ꢃ (c 0.17, MeOH);
1H and 13C NMR data are listed in Tables 1–3; (+)ESI-MS/MS of the
ion [M+Na]+ at m/z 1257: 1137 [(M+Na)–NaHSO4]+, 1111 [(M+Na)–
C6H10O4]+, 991 [(M+Na)–NaHSO4–C6H10O4]+, 965 [(M+Na)–2 ꢀ
C6H10O4]+, 845 [(M+Na)–NaHSO4–2 ꢀ C6H10O4]+; (+)HR ESI-MS m/z
1257.5302 [M+Na]+ (calcd. for C56H91O26SNa2, 1257.5309); (ꢂ)ESI-
MS/MS of the ion [MꢂNa]ꢂ at m/z 1211: 1065 [(MꢂNa)–C6H10O4]ꢂ,
919 [(MꢂNa)–2 ꢀ C6H10O4]ꢂ, 773 [(MꢂNa)–3 ꢀ C6H10O4]ꢂ, 641
107
2.3. Extraction and isolation
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The fresh animals (0.35 kg) were chopped and extracted twice
with EtOH at 20 °C. The H2O/EtOH layer was evaporated, and the
residue was dissolved in H2O (0.5 L). The H2O-soluble materials
were passed through a Polychrom 1 column (6.5 cm ꢀ 21 cm),
eluted with distilled H2O until a negative chloride ion reaction
was obtained, and eluted with EtOH. The combined EtOH eluate
was evaporated to give a brownish residue (4.8 g). This material
was chromatographed over a silica gel column (4 cm ꢀ 20 cm) using
CHCl3/EtOH (stepwise gradient, 8:1–1:2) to yield six fractions, 1–6,
that were then analyzed by TLC on silica gel plates in the eluent sys-
tem BuOH/EtOH/H2O (4:1:2). Fractions 1, 2, 4, and 5 mainly con-
tained the free polyhydroxysteroids and related glycosides,
fraction 3 mainly contained the free sulfated asterogenins, and frac-
tion 6 mainly contained the asterosaponins. HPLC separation of
[(MꢂNa)–3 ꢀ C6H10O4–C5H8O4]ꢂ,
495
[(MꢂNa)–4 ꢀ C6H10O4–
C5H8O4]ꢂ, 477 [(MꢂNa)–4 ꢀ C6H10O4–C5H8O4–H2O]ꢂ, 97 [HSO4]ꢂ;
(ꢂ)HR ESI-MS m/z 1211.5519 [MꢂNa]ꢂ (calcd. for C56H91O26S,
1211.5525).
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2.4.5. Leptasterioside C (5)
C58H95O27SNa, amorphous powder; ½a D25
ꢁ
þ 15:7ꢃ (c 0.45, MeOH);
1H and 13C NMR data are listed in Tables 1–3; (+)ESI-MS/MS of the ion
[M+Na]+ at m/z 1301: 1181 [(M+Na)–NaHSO4]+, 1155 [(M+Na)–
C6H10O4]+, 1035 [(M+Na)–NaHSO4–C6H10O4]+, 1009 [(M+Na)–2 ꢀ
C6H10O4]+, 889 [(M+Na)–NaHSO4–2 ꢀ C6H10O4]+, 743 [(M+Na)–
fraction 3 on a Diasfer-110-C18 column (10
2.5 mL/min) with EtOH/H2O (65:35) as an eluent system followed
by the further separation on a Discovery C18 column (5 m,
lm, 250 mm ꢀ 15 mm,
NaHSO4–3 ꢀ C6H10O4]+,
701
[(M+Na)–3 ꢀ C6H10O4–C6H10O5]+;
(+)HR ESI-MS m/z 1301.5548 [M+Na]+ (calcd. for C58H95O27SNa2,
1301.5571); (ꢂ)ESI-MS/MS of the ion [MꢂNa]ꢂ at m/z 1255: 1109
[(MꢂNa)–C6H10O4]ꢂ, 963 [(MꢂNa)–2 ꢀ C6H10O4]ꢂ, 817 [(MꢂNa)–
l
250 mm ꢀ 10 mm, 2.5 mL/min) with MeOH/H2O (70:30) as an
eluent system yielded pure 1 (2.0 mg, tR 16.5 min, Rf 0.77) and 2
(1.3 mg, tR 22.4 min, Rf 0.79). HPLC separation of fraction 6 on a
3 ꢀ C6H10O4]ꢂ,
655
[(MꢂNa)–3 ꢀ C6H10O4–C6H10O5]ꢂ,
509
[(MꢂNa)–4 ꢀ C6H10O4–C6H10O5]ꢂ, 491 [(MꢂNa)–4 ꢀ C6H10O4–
C6H10O5–H2O]ꢂ, 97 [HSO4]ꢂ; (ꢂ)HR ESI-MS m/z 1255.5788 [MꢂNa]ꢂ
(calcd. for C58H95O27S, 1255.5787).
Diasfer-110-C18 column (10
with EtOH/H2O (60:40) as an eluent system and further separation
on a Discovery C18 column (5
lm, 250 mm ꢀ 15 mm, 2.5 mL/min)
l
m, 250 mm ꢀ 10 mm, 2.5 mL/min)
with MeOH/H2O (70:30) as an eluent system gave pure 3 (1.8 mg,
tR 12.3 min, Rf 0.48), 4 (1.7 mg, tR 13.8 min, Rf 0.48), 5 (4.5 mg, tR
11.7 min, Rf 0.47), 6 (1.5 mg, tR 11.3 min, Rf 0.48), 7 (1.8 mg, tR
13.1 min, Rf 0.46), and 8 (1.8 mg, tR 14.2 min, Rf 0.46).
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2.4.6. Leptasterioside D (6)
C57H91O27SNa, amorphous powder; ½a D25
ꢁ
þ 9:2ꢃ (c 0.15, MeOH);
1H and 13C NMR data are listed in Tables 1–3; (+)ESI-MS/MS of the
ion [M+Na]+ at m/z 1285: 1165 [(M+Na)–NaHSO4]+, 1139 [(M+Na)–
C6H10O4]+, 1019 [(M+Na)–NaHSO4–C6H10O4]+, 993 [(M+Na)–2 ꢀ
C6H10O4]+, 873 [(M+Na)–NaHSO4–2 ꢀ C6H10O4]+, 847 [(M+Na)–
3 ꢀ C6H10O4]+, 727 [(M+Na)–NaHSO4–3 ꢀ C6H10O4]+, 685 [(M+Na)–
3 ꢀ C6H10O4–C6H10O5]+; (+)HR ESI-MS m/z 1285.5260 [M+Na]+
(calcd. for C57H91O27SNa2, 1285.5258); (ꢂ)ESI-MS/MS of the ion
[MꢂNa]ꢂ at m/z 1239: 1093 [(MꢂNa)–C6H10O4]ꢂ, 947 [(MꢂNa)–
2 ꢀ C6H10O4]ꢂ, 801 [(MꢂNa)–3 ꢀ C6H10O4]ꢂ, 639 [(MꢂNa)–3 ꢀ
C6H10O4–C6H10O5]ꢂ, 493 [(MꢂNa)–4 ꢀ C6H10O4–C6H10O5]ꢂ, 475
[(MꢂNa)–4 ꢀ C6H10O4–C6H10O5–H2O]ꢂ, 97 [HSO4]ꢂ; (ꢂ)HR ESI-MS
m/z 1239.5470 [MꢂNa]ꢂ (calcd. for C57H91O27S, 1239.5474).
135
2.4. Spectral data of new compounds
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2.4.1. (23S)-6a,23-Dihydroxy-5a-cholesta-9(11),20(21)-dien-3b-yl
sulfate, sodium salt (1)
C27H43O6SNa, amorphous powder; ½a D25
ꢁ
ꢂ 8:5ꢃ (c 0.2, MeOH);
the 1H and 13C NMR data are listed in Tables 1 and 2; (+)HR ESI-
MS m/z 541.2587 [M+Na]+ (calcd. for C27H43O6SNa2, 541.2583);
(ꢂ)HR ESI-MS m/z 495.2771 [MꢂNa]ꢂ (calcd. for C27H43O6S,
495.2780).
Please cite this article in press as: Malyarenko TV et al. Asterosaponins from the Far Eastern starfish Leptasterias ochotensis and their anticancer activity.
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Table 1
1H NMR data of 1–8 (35 °C, C5D5N, J in Hz).a
Position
1
1
2
3, 5
4
6
7
8
1.68 td (13.2, 3.0)
1.43 m
2.54 m
1.91 m
4.95 m
3.40 brd (12.8)
1.77 m
1.42 m
3.84 m
2.44 m
1.67 m
1.42 m
2.50 m
1.88 m
4.92 m
3.36 brd (12.2)
1.73 m
1.41 m
3.83 m
1.68 m
1.42 m
2.83 brd (14.0)
1.90 m
4.91 m
3.50 brd (11.3)
1.69 m
1.51 m
3.79 m
2.73 td (12.6, 4.9)
1.33 m
1.66 m
1.40 m
2.82 m
1.90 m
4.91 m
3.48 brd (8.8)
1.70 m
1.51 m
3.79 m
2.73 m
1.31 m
2.15 m
1.62 m
1.38 m
2.80 m
1.88 m
4.90 m
3.51 brd (11.0)
1.68 m
1.49 m
3.77 m
1.67 m
1.42 m
2.80 m
1.89 m
4.90 m
3.52 brd (13.1)
1.68 m
1.67 m
1.42 m
2.80 m
1.89 m
4.90 m
3.52 m
1.68 m
1.50 m
3.78 m
2.72 m
1.32 m
2.14 m
2
3
4
5
6
7
1.51 m
3.78 td (11.0, 4.5)
2.72 td (12.3, 4.6)
1.33 m
2.44 td (12.0, 4.4)
1.28 m
2.13 m
2.70 td (12.5, 5.7)
1.31 m
2.10 m
1.26 m
2.15 m
8
9
2.15 m
2.13 m
10
11
12
5.28 brd (5.7)
2.14 m
5.30 brd (5.9)
2.09 m
5.27 brd (5.8)
2.32 m
5.21 brs
2.05 brs
5.24 brd (5.8)
1.96 m
5.28 brd (5.9)
2.33 m
5.25 brd (5.9)
2.30 m
2.01 m
2.06 m
1.94 m
2.12 m
2.07 m
13
14
15
1.41 m
1.78 m
1.26 m
1.90 m
1.75 m
2.38 dd (10.4, 8.6)
0.66 s
1.42 m
1.82 m
1.29 m
1.92 m
1.69 m
2.22 m
0.58 s
1.32 m
1.85 m
1.29 m
2.25 m
1.86 m
1.66 m
1.07 s
1.36 m
1.80 m
1.25 m
1.91 m
1.75 m
2.34 t (9.7)
0.64 s
1.35 m
1.84 m
1.30 m
1.92 m
1.68 m
2.18 t (9.8)
0.56 s
1.30 m
1.86 m
1.29 m
2.22 m
1.97 m
1.75 m
1.06 s
1.32 m
1.85 m
1.30 m
2.24 m
1.79 m
1.70 m
1.03 s
16
17
18
19
20
21
0.98 s
0.96 s
0.99 s
0.96 s
0.96 s
0.99 s
0.98 s
5.20 brs
5.03 brs
2.58 dd (13.7, 6.4)
2.44 m
4.20 m
2.30 s
6.24 s
1.50 s
5.20 brs
5.02 brs
2.58 dd (13.7, 6.2)
2.43 dd (13.7, 6.3)
4.21 m
2.29 s
6.24 s
1.45 s
1.59 s
22
23
24
1.86 m
2.93 d (2.3)
3.12 td (5.8, 2.3)
2.90 d (15.6)
2.70 d (15.6)
2.36 m
2.28 m
1.63 m
2.37 d (7.0)
1.65 m
2.38 d (6.9)
1.50 m
2.50 qwin (7.0)
1.50 m
1.51 m
1.46 m
25
26
27
28
2.19 m
1.01 d (6.7)
1.03 d (6.7)
2.24 m
0.94 d (6.4)
0.94 d (6.4)
2.33 m
2.21 m
1.02 d (6.7)
1.04 d (6.6)
2.26 m
0.95 d (7.0)
0.95 d (7.0)
1.78 m
0.95 d (6.4)
0.96 d (6.4)
2.01 sext (7.0)
0.83 d (6.9)
0.93 d (6.9)
1.02 d (6.9)
1.07 d (6.8)
1.08 d (6.8)
4.90 brs
4.87 brs
a
Assignments from 700 MHz 1H–1H COSY-45, HSQC, HMBC (8 Hz), and NOESY (270 ms) data.
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
229
230
231
232
233
234
235
236
2.4.7. Leptasterioside E (7)
m/z 1271: 1157 [(MꢂNa)–C7H14O]ꢂ, 1125 [(MꢂNa)–C6H10O4]ꢂ,
1011 [(MꢂNa)–C7H14O–C6H10O4]ꢂ, 979 [(MꢂNa)–2 ꢀ C6H10O4]ꢂ,
865 [(MꢂNa)–C7H14O–2 ꢀ C6H10O4]ꢂ, 833 [(MꢂNa)–3 ꢀ C6H10-
O4]ꢂ, 719 [(MꢂNa)–C7H14O–3 ꢀ C6H10O4]ꢂ, 557 [(MꢂNa)–
C57H93O28SNa, amorphous powder; ½a D25
ꢁ
þ 10:0ꢃ (c 0.18, MeOH);
1H and 13C NMR data are listed in Tables 1–3; (+)ESI-MS/MS of the
ion [M+Na]+ at m/z 1303: 1183 [(M+Na)–NaHSO4]+, 1083
[(M+Na)–NaHSO4–C6H12O]+, 1037 [(M+Na)–NaHSO4–C6H10O4]+,
937 [(M+Na)–NaHSO4–C6H12O–C6H10O4]+, 891 [(M+Na)–NaHSO4–
2 ꢀ C6H10O4]+, 745 [(M+Na)–NaHSO4–3 ꢀ C6H10O4]+; (+)HR
ESI-MS m/z 1303.5340 [M+Na]+ (calcd. for C57H93O28SNa2,
1303.5364); (ꢂ)ESI-MS/MS of the ion [MꢂNa]ꢂ at m/z 1257: 1157
[(MꢂNa)–C6H12O]ꢂ, 1111 [(MꢂNa)–C6H10O4]ꢂ, 1011 [(MꢂNa)–
C6H12O–C6H10O4]ꢂ, 965 [(MꢂNa)–2 ꢀ C6H10O4]ꢂ, 865 [(MꢂNa)–
C7H14O–3 ꢀ C6H10O4–C6H10O5]ꢂ,
411
[(MꢂNa)–C7H14O–4 ꢀ
C6H10O4–C6H10O5]ꢂ,
393
[(MꢂNa)–C7H14O–4 ꢀ C6H10O4–
C6H10O5–H2O]ꢂ, 97 [HSO4]ꢂ; (ꢂ)HR ESI-MS m/z 1271.5737
[MꢂNa]ꢂ (calcd. for C58H95O28S, 1271.5736).
237
238
2.5. Acid hydrolysis and determination of absolute configurations of
monosaccharides
C6H12O–2 ꢀ C6H10O4]ꢂ,
819
[(MꢂNa)–3 ꢀ C6H10O4]ꢂ,
719
[(MꢂNa)–C6H12O–3 ꢀ C6H10O4]ꢂ, 657 [(MꢂNa)–3 ꢀ C6H10O4–
C6H10O5]ꢂ, 511 [(MꢂNa)–4 ꢀ C6H10O4–C6H10O5]ꢂ, 493 [(MꢂNa)–
4 ꢀ C6H10O4–C6H10O5–H2O]ꢂ, 97 [HSO4]ꢂ; (ꢂ)HR ESI-MS m/z
1257.5584 [MꢂNa]ꢂ (calcd. for C57H93O28S, 1257.5580).
239
240
241
242
243
244
245
246
247
248
249
250
251
252
The acid hydrolysis of 4 (1.7 mg) was carried out in a solution of
2 M trifluoroacetic acid (TFA) (1 mL) in a sealed vial on a H2O bath
at 100 °C for 2 h. The H2O layer was washed with CHCl3
(3 ꢀ 1.0 mL) and concentrated in vacuo. One drop of concentrated
TFA and 0.5 mL of
L-(ꢂ)-2-octanol (Aldrich) were added to the
220
221
222
223
224
225
226
227
228
2.4.8. Leptasterioside F (8)
sugar mixture, and the sealed vial was heated on a glycerol bath
at 130 °C for 6 h. The solution was evaporated in vacuo and treated
with a mixture of pyridine/acetic anhydride (1:1, 0.6 mL) for 24 h
at room temperature. The acetylated 2-octylglycosides were ana-
lyzed by GC using the corresponding authentic samples prepared
by the same procedure. The following peaks of four tautomeric
forms (two pyranoses and two furanoses) for each monosaccharide
C58H95O28SNa, amorphous powder; ½a D25
ꢁ
þ 6:4ꢃ (c 0.18, MeOH);
1H and 13C NMR data are listed in Tables 1–3; (+)ESI-MS/MS of the
ion [M+Na]+ at m/z 1317: 1203 [(M+Na)–C7H14O]+, 1197
[(M+Na)–NaHSO4]+, 1083 [(M+Na)–NaHSO4–C7H14O]+, 937
[(M+Na)–NaHSO4–C7H14O–C6H10O4]+, 791 [(M+Na)–NaHSO4–
C7H14O–2 ꢀ C6H10O4]+,
451
[(M+Na)–NaHSO4–4 ꢀ C6H10O4–
C6H10O5]+; (+)HR ESI-MS m/z 1317.5551 [M+Na]+ (calcd. for
C58H95O28SNa2, 1317.5520); (ꢂ)ESI-MS/MS of the ion [MꢂNa]ꢂ at
were detected in the hydrolysate of 4:
D-quinovose (tR 23.98, 24.10,
24.52, and 24.79 min), -fucose (tR 23.98, 24.22, 24.46, and
D
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Table 2
285
286
287
288
cultured in RPMI-1640 and MEM medium supplemented with 10%
fetal bovine serum (FBS) and penicillin–streptomycin solution. The
cultures were maintained at 37 °C in a 5% CO2 incubator (MCO-
18AIC, ‘‘Sanyo’’, Japan).
13C NMR data of 1–8 (35 °C, C5D5N).
Position
1
2
3, 5
35.9
4
6
7
8
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
28
36.0
29.3
78.6
31.0
50.5
68.2
43.2
36.4
146.5
38.2
116.4
40.9
42.0
53.5
25.3
26.4
56.0
12.8
19.3
147.5
111.6
47.4
68.2
47.1
24.9
24.0
22.1
35.9
29.1
78.6
30.9
50.4
68.1
43.2
36.2
146.4
38.1
116.1
40.6
43.2
53.4
25.3
25.2
59.9
12.6
19.1
157.1
20.7
124.2
200.3
53.6
25.2
22.5
22.5
35.8
29.4
77.3
30.6
49.3
80.3
41.4
36.0
146.0
38.2
116.4
40.7
41.7
53.4
25.1
26.3
55.8
12.6
19.1
147.5
111.6
47.4
68.0
46.9
24.8
23.9
21.9
35.8
29.3
77.3
30.7
49.2
80.6
41.5
35.9
35.9
29.4
77.4
30.6
49.3
80.8
41.5
35.2
145.5
38.2
116.5
42.2
41.7
53.5
25.1
22.9
59.4
13.3
19.1
71.5
23.2
65.6
53.2
41.1
26.6
22.4
22.8
35.9
29.4
77.3
30.5
49.2
80.6
41.4
35.1
29.4
77.4
30.6
49.3
80.5
41.5
35.2
145.4
38.1
116.6
42.4
41.4
54.0
25.1
22.9
58.8
13.5
19.1
73.6
26.0
43.3
29.4
156.7
34.1
21.9
21.9
106.3
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
2.7.2. Cytotoxicity assay
The MTS assay was used as an indicator of cell viability as deter-
mined by the mitochondrial-dependent reduction of formazan
[10]. Briefly, T-47D (5 ꢀ 104/well) and RPMI-7951 (3 ꢀ 104/well)
cells were seeded in 200 lL of complete RPMI-1640/10% FBS and
146.0
38.3
116.1
40.4
43.3
53.4
25.2
25.2
59.8
12.6
19.0
157.1
20.6
124.2
200.3
53.5
25.2
22.5
22.5
145.4
38.2
116.5
42.2
41.5
53.8
24.9
23.1
59.2
13.4
19.1
73.6
26.9
53.4
215.8
53.7
29.8
18.3
21.2
11.8
MEM/10% FBS mediums, respectively, and cultured for 24 h at
37 °C in a 5% CO2 incubator. The cell monolayer was washed with
phosphate buffered saline (PBS) to remove unattached cells. The
attached cells were incubated with fresh medium containing vari-
ous concentrations of asterosaponins (0–200
lM) for 24 h. Subse-
quently, the cells were incubated with 15 L MTS reagent for 3 h,
l
and the absorbance of each well was measured at 490/630 nm
using microplate reader (Power Wave XS, American). The 50%
inhibitory concentration (IC50) was defined as the concentration
of asterosaponins that caused a 50% reduction in cell viability. All
tested samples were assayed in triplicate.
305
306
307
308
309
310
311
312
313
314
315
316
317
318
2.7.3. Soft agar clonogenic assay [11]
T-47D and RPMI-7951 cells (2.4 ꢀ 104/mL) were grown in 1 mL
of 0.3% Basal Medium Eagle (BME) agar containing 10% FBS, 2 mM
L
-glutamine, and 25
ted (control) or treated with compound 2 at concentration of
100 M, compounds 6–8 at concentration of 50 M, and com-
pounds 3, 4, and 5 at concentrations of 7, 1, and 10 M, respec-
tively. RPMI-7951 cells were not treated (control) or treated with
compound 2 at concentration of 100 M, compounds 6–8 at con-
centration of 50 M, and compounds 3–5 at concentration of
10 M, respectively. The cultures cells were maintained at 37 °C
lg/mL gentamicin. T-47D cells were not trea-
l
l
253
254
255
256
257
258
259
24.94 min), and
The retention times of the authentic samples were as follows:
-quinovose (tR 24.02, 24.13, 24.55, and 24.82 min), -fucose (tR
24.01, 24.24, 24.49, and 24.97 min), -glucose (tR 28.27, 28.86,
29.06, and 29.38 min), -quinovose (tR 23.78, 24.30, and
24.87 min), -fucose (tR 24.32, 24.60, and 24.84 min), and -glucose
D-glucose (tR 28.32, 28.82, 29.02, and 29.32 min).
l
D
D
l
D
l
L
l
L
L
in a 5% CO2 incubator for 14 days. The number of cell colonies were
scored using a Motic AE 20 microscope (China) and the Motic
Image Plus computer program.
(tR 28.40, 28.62, 28.72, and 29.38 min).
260
2.6. Preparation of the MTPA esters of compound 1
319
320
321
322
323
2.7.4. Statistical analysis
261
262
263
264
265
266
Aliquots (0.8 mg) of compound 1 were treated with R-(ꢂ)- and
S-(+)- -methoxy- -(trifluoromethyl)-phenylacetyl (MTPA) chlo-
ride (8 L) in dry pyridine (200 L) for 2 h at room temperature,
to obtain the corresponding S-(ꢂ)- and R-(+)-MTPA esters. After
removal of the solvent, the products were purified on a Si gel col-
umn (1 ꢀ 3.5 cm) using CHCl3/EtOH.
All assays were performed at least in triplicate. The results are
expressed as the mean standard deviation (SD). A Student’s t-test
was used to evaluate the data with the following significance lev-
els: ⁄ p < 0.05, ⁄⁄ p < 0.01, ⁄⁄⁄ p < 0.001.
a
a
l
l
324
3. Results and discussion
267
268
269
270
271
272
273
2.6.1. 6,23-Di-(S)-MTPA ester of 1
1H NMR (700 MHz, CD3OD): 0.54 (3H, s, H3-18), 0.82 (3H, d,
J = 6.7 Hz, H3-27), 0.83 (3H, d, J = 6.7 Hz, H3-26), 1.06 (3H, s, H3-
19), 1.36 (1H, m, H0-24), 1.43 (1H, m, H-25), 1.50 (1H, m, H-24),
2.33 (1H, m, H0-22), 2.42 (1H, m, H-22), 4.94 (1H, m, H-3), 4.96
(1H, brs, H0-21), 5.01 (1H, brs, H-21), 5.06 (1H, m, H-6), 5.31 (1H,
m, H-23), 5.40 (1H, brd, J = 5.7 Hz, H-11).
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
The concentrated ethanol extract of L. ochotensis was subjected
to sequential separation by chromatography on columns with Poli-
chrom-1, silica gel followed by HPLC on semipreparative Diasfer-
110-C18, Discovery C18 and analytical Diasfer-110-C18 columns
to yield one new aglycone of asterosaponins–asterogenin 1, six
new asterosaponins, named as leptasteriosides A–F (3–8), and
one known asterogenin 2 (Fig. 1). Compound 2 was identified by
comparison of its 1H and 13C NMR, and MS spectra as rare genuine
274
275
276
277
278
279
280
2.6.2. 6,23-Di-(R)-MTPA ester of 1
1H NMR (700 MHz, CD3OD): 0.53 (3H, s, H3-18), 0.92 (6H, 2 ꢀ d,
J = 6.7 Hz, H3-26, H3-27), 1.06 (3H, s, H3-19), 1.44 (1H, m, H0-24),
1.58 (1H, m, H-24), 1.64 (1H, m, H-25), 2.24 (1H, m, H0-22), 2.33
(1H, m, H-22), 4.87 (1H, brs, H0-21), 4.92 (1H, brs, H-21), 4.93
(1H, m, H-3), 5.08 (1H, m, H-6), 5.29 (1H, m, H-23), 5.42 (1H,
brd, J = 5.7 Hz, H-11).
aglycone,
namely
sodium
(20E)-6a-hydroxy-5a-cholesta-
9(11),20(22)-dien-23-one-3b-yl sulfate, recently found as a steroid
moiety of new asterosaponins from the starfish Leptasterias hylodes
reticulata and A. carinifera [8,12]. The corresponding desulfated ast-
erosapogenin was earlier obtained from hydrolysate of glycosides
from Astropecten aurantiacus and Marthasterias glacialis by Italian
scientists [13]. The assignment of all proton and carbon resonances
by the 2D NMR experiments 1H-1H COSY, HSQC, HMBC, and NOESY
confirmed the structure of 2 (Tables 1 and 2, Fig. 2). The presence
of the cross-peak H-22/Hb-16 in the NOESY spectrum and the
chemical shift of C-17 at dC 59.8 (dC 51.2 and 59.1 in model
(20Z)- and 20(E)-cholesta-5,20(22)-dien-3b-ols, respectively
281
2.7. Bioassay methods
282
283
284
2.7.1. Cell culture
The T-47D (ATCC # HTB-133) human breast cancer and RPMI-
7951 (ATCC # HTB-66) human malignant melanoma cell lines were
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Table 3
1H and 13C NMR data and HMBC and NOESY correlations of the oligosaccharide moieties of compound 3–8 (35 °C, C5D5N, J in Hz).a
Position 3, 4
5, 6, 7, 8
dH
dC
HMBC
NOESY
dH
dC
HMBC
NOESY
Qui I
1
2
3
4
5
6
4.80 d (7.6)
105.0 C6-agl
H3,H5-Qui I, H6-agl
H4-Qui I
H1-Qui I, H1-Xyl
H2,H6-Qui I
H1-Qui I
4.80 d (7.8)
3.98 t (8.5)
3.76 t (8.8)
3.48 t (8.8)
3.66 m
105.2 C6-agl
H3,H5-Qui I, H6-agl
H4-Qui I
H1-Qui I, H1-Glc
H2,H6-Qui I
H1-Qui I
3.98 t (8.1)
3.78 t (9.2)
3.55 t (9.2)
3.67 m
73.9
90.3
74.4
71.8
18.3
C1,C3-Qui I
C2,C5-Qui I, C1-Xyl
C3,C6-Qui I
73.6
91.0
74.5
71.5
18.3
C1,C3-Qui I
C1-Glc, C2-Qui I
C3,C5-Qui I
1.58 d (6.4)
C4,C5-Qui I
H4-Qui I
1.55 d (6.2)
C4,C5-Qui I
H4-Qui I
Xyl or Glc
1
2
3
4
5
4.99 d (7.4)
4.11 brt (8.9)
4.16 m
4.16 m
4.48 dd (12.4, 4.2) 64.4
3.76 m
104.5 C3-Qui I
H3,H5-Xyl, H3-Qui I
H1-Qui II
H1,H5-Xyl
4.98 d (7.7)
4.15 m
4.15 m
4.11 brt (5.8)
3.97 brd (7.9)
103.6 C3-Qui I
H3,H5-Glc, H3,H4-Qui I
H1-Qui II
H1,H5-Glc
H6-Glc, H1-Fuc I
H1,H3-Glc, H1-Fuc I
81.5
75.7
78.8
C3-Xyl
C4-Xyl
C3-Xyl
81.4
75.7
80.5
76.7
C1,C3-Glc
C2-Glc
C1-Fuc I
C1,C3,C4-Xyl
C4-Xyl
H1,H3-Xyl
6
4.62 d (12.7)
4.52 dd (13.0, 6.5)
61.1
H4-Glc
H4-Glc
Qui II
1
2
3
4
5
6
5.33 brd (7.7)
4.09 m
4.09 m
4.07 m
3.71 m
104.7 C2-Xyl
H5-Qui II, H2-Xyl
5.32 brd (6.1)
4.09 m
4.10 m
4.11 m
3.68 m
104.4 C2-Glc
H3,H5-Qui II, H2-Glc
76.1
76.7
75.3
73.4
17.7
C1,C3-Qui II
76.0
76.5
75.3
73.2
17.5
C3-Qui II
H5-Qui II
H6-Qui II
H1,H3-Qui II
H4-Qui II
H1,H5-Qui II
H6-Qui II
H1,H3-Qui II
H4-Qui II
C3-Qui II
C2-Qui II
C6-Qui II
C4,C5-Qui II
1.78 d (6.4)
C4,C5-Qui II
1.77 d (6.4)
Fuc I
1
2
3
4
5
6
4.85 d (7.9)
4.35 dd (9.5, 7.4)
4.10 m
4.00 brd (3.2)
3.78 q (6.5)
1.45 d (6.5)
102.0 C4-Xyl
H3,H5-Fuc I
4.90 d (7.5)
4.42 t (7.5)
4.07 dd (10.0, 2.9) 74.8
3.98 d (3.2)
3.77 m
1.45 d (6.4)
102.7 C4-Glc
81.9 C1-Fuc II, C1,C3-Fuc I H1-Fuc II
H3,H5-Fuc I, H4,H5-Glc
82.9
74.9
71.5
71.4
16.8
C1,C3-Fuc I, C1-Fuc II H1-Fuc II
C2-Fuc I
C2,C3-Fuc I
C1,C6-Fuc I
C4,C5-Fuc I
H1,H5-Fuc I
H5,H6-Fuc I
H1,H3,H4-Fuc I
H4-Fuc I
C2-Fuc I
H1,H5-Fuc I
H6-Fuc I
H1,H3-Fuc I
H4-Fuc I
71.7
71.5
16.9
C2-Fuc I
C4,C6-Fuc I
C4,C5-Fuc I
Fuc II
1
2
3
4
5
6
4.83 d (7.4)
107.0 C2-Fuc I
H3,H5-Fuc II, H2-Fuc I 4.96 d (7.9)
4.43 t (7.5)
106.8 C2-Fuc I
73.6
H3,H5-Fuc II, H2-Fuc I
4.40 dd (9.1, 8.4)
3.97 dd (9.8, 3.3)
3.93 brd (4.0)
3.62 q (6.3)
73.7
74.9
72.4
71.7
17.0
C1,C3-Fuc II
C2-Fuc II
C2-Fuc II
C1,C4,C6-Fuc II
C5-Fuc II
H1,H5-Fuc II
H5,H6-Fuc II
H1,H3,H4-Fuc II
H4-Fuc II
4.00 dd (9.6, 4.1)
3.96 brs
3.65 m
75.0
72.4
71.5
16.6
C2-Fuc II
C2-Fuc II
C1,C4-Fuc II
C5-Fuc II
H1,H5-Fuc II
H6-Fuc II
H1,H3-Fuc II
H4-Fuc II
1.46 d (6.4)
1.47 d (6.5)
a
Assignments from 1H 700.13 MHz, 13C 176.04 MHz, 1H–1H COSY, 1D TOCSY, HSQC, HMBC (8 Hz), and NOESY (270 ms) data.
21
22
24
26
OH
18
20
23
25
27
12
17
OH
11
13
14
16
15
19
H
3 R = A, R1 = H
5 R = A, R1 = CH2OH
9
6
1 R = H
4 R = A, R1 = H
1
10
8
2
3
H
5
7
4
O
NaO3SO
OH
O
H
OR
QuiI
O
H3C
O
Glc or Xyl
R1
2 R = H
6 R = A, R1 = CH2OH
HO
7 R = A, R1 = CH2OH
OH
O
FucI
HO
H3C
O
O
O
HO
OH
O
O
OH
QuiII
HO
H3C
O
O
OH
FucII
H3C
HO
8 R = A, R1 = CH2OH
HO
HO
A
HO
Fig. 1. The structures of compounds 1–8 isolated from L. ochotensis.
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346
347
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349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
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401
402
403
404
405
406
[13,14]) indicated the E-configuration of the 20(22)-double bond in
2. This compound was isolated in the native sulfated form from
starfish for the first time.
configuration of C-23 in 1 was deduced by Mosher’s method. Treat-
ment of compound 1 with R-(ꢂ)- and S-(+)-MTPA chloride gave (S)-
and (R)-MTPA esters, respectively, the proton signals of which
were assigned by 1H-1H COSY experiments (see Section 2). The
The molecular formula of compound 1 was determined to be
C27H43O6SNa from the [M+Na]+ sodiated-molecular ion peak at
m/z 541.2587 (calcd. for C27H43O6SNa2, 541.2570) in the (+)HR
ESI-MS and the [MꢂNa]ꢂ ion peak at m/z 495.2771 (calcd. for
C27H43O6S, 495.2786) in the (ꢂ)HR ESI-MS. The fragment ion peak
at m/z 97 [HSO4]ꢂ in the (ꢂ)ESI-MS/MS of the ion at m/z 495
[MꢂNa]ꢂ exhibited the presence of a sulfate group in 1. The 1H
and 13C NMR spectra of 1 (Tables 1 and 2) showed the presence
of proton and carbon signals of two angular Me groups (dH 0.66,
0.98; dC 12.8, 19.3), the 9(11)-double bond (dH 5.28; dC 116.4,
146.5), one methine group (dH 4.95, dC 78.6), associated with an
O-sulfate group, and one methine group, bearing a hydroxyl group
positive
dH values for H2-24, H-25, H3-26, and H3-27 indicated the
(23S)-configuration in 1 [15]. Thus, the structure of compound 1
was elucidated to be sodium (23S)-6 ,23-dihydroxy-5 -cholesta-
D
dH values (dS ꢂ dR) for H2-21 and H2-22 and negative
D
a
a
9(11),20(21)-dien-3b-yl sulfate. This compound was not so far
found in free or glycosylated forms among starfish polar steroids.
The molecular formula of leptasterioside B (3) was determined
to be of C57H93O26SNa from the [M+Na]+ sodiated-molecular ion
peak at m/z 1271.5481 (calcd. for C57H93O26SNa2, 1271.5466) in
the (+)HR ESI-MS and the [MꢂNa]ꢂ ion peak at m/z 1225.5678
(calcd. for C57H93O26S, 1225.5681) in the (ꢂ)HR ESI-MS. The
fragment ion peaks at m/z 1151 [(M+Na)–NaHSO4]+ in the (+)ESI-
MS/MS of the ion at m/z 1271 [M+Na]+ and at m/z 97 [HSO4]ꢂ in
the (ꢂ)ESI-MS/MS of the ion at m/z 1225 [MꢂNa]ꢂ exhibited the
presence of a sulfate group in 3. The 1H and 13C NMR spectra con-
(dH 3.84, dC 68.2), all characteristic of 3-O-sulfate 3b,6a-dihydroxy
steroid nucleus of asterosaponins [1–6]. The proton and carbon sig-
nals of aglycon side chain demonstrated the presence of two sec-
ondary Me groups (dH 1.01, 1.03; dC 24.0, 22.1), two methylene
groups (dH 2.58, 2.44; 1.63, 1.50; dC 47.4, 47.1), one methine group
(dH 2.19; dC 24.9), a disubstituted double bond (dH 5.20, 5.03, both
brs; dC 111.6, 147.5), and one methine group bearing an hydroxyl
group (dH 4.20; dC 68.2). Analysis of the 2D NMR data (1H–1H COSY,
HSQC, HMBC, and NOESY) led to the assignment of all the proton
and carbon resonances of 1 (Fig. 2). The HMBC and NOESY correla-
tions, such as H2-21/C-17, C-22 and H-17, H3-18, H-22, H-23, H-24;
H2-22/C-17, C-21, C-23 and H-21, H-24; H2-24/C-23 and H-21,
H-22, H3-26, H3-27; H3-26/C-24, C-25, C-27 and H-23, H-24; and
H3-27/C-24, C-25, C-26 and H-23, H-24, supported the presence
firmed the presence of a
D a-dihydroxy steroid nucleus
9(11)-3b,6
with a sulfate group linked at C-3 and carbohydrate chain linked
at C-6 as in the compounds 1 and 2. The proton and carbon signals
of the aglycon side chain indicated the presence of a 24(28)-double
bond (dH 4.90, 4.87; dC 156.7, 106.3), one tertiary Me group (dH
1.50; dC 26.0), two secondary Me groups (dH 1.07, 1.08; dC
2 ꢀ 21.9), two methylene groups (dH 1.86, 2.36, 2.28; dC 43.3,
29.4), one methine group (dH 2.33; dC 34.1), and a tertiary carbon
atom associated with oxygen (dC 73.6). All these chemical shifts
belonging to the aglycon part of 3 were similar to those of astero-
side D from the starfish Asterias amurensis [16]. Analysis of the 2D
NMR data (1H-1H COSY, HSQC, HMBC, and NOESY) led to the
unusual 20(21) double bond and confirmed the
D
20(21)-23-
hydroxycholestane structure of the side chain. The absolute
HO
OH
-O3SO
-O3SO
OR
1, 4
O
OR
3, 5
OH
O
-O3SO
OH
-O3SO
OR
7
2, 6
OR
O
-O3SO
OR
8
Fig. 2. 1H–1H COSY and key HMBC correlations of compounds 1–8.
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assignment of all the proton and carbon resonances of aglycon part
of 3. The (20S)-configuration was assumed on the basis of NOESY
correlations of H3-18/H3-21, H3-21/Hb-12 and H-22/Hb-16 as well
as the chemical shift of H3-21 at dH 1.50 [17]. These evidence
application of extensive 2D NMR analysis of compounds 1, 3
and 4 exhibited that steroid aglycon of 4 is identical to the com-
pound 1, while oligosaccharide moiety of 4 is the same as that of
glycoside 3 (Tables 1–3). Thus, the structure of new asterosaponin
allowed us to propose the sodium (20S)-6a,20-dihydroxy-5a-cho-
– leptasterioside A (4) was defined to be sodium (23S)-6
fucopyranosyl-(1 ? 2)-b- -fucopyranosyl-(1 ? 4)-[b- -quinovopyr-
anosyl-(1 ? 2)]-b- -xylopyranosyl-(1 ? 3)-b- -quinovopyranosyl}-
23-hydroxy-5 -cholesta-9(11),20(21)-dien-3b-yl sulfate [19].
a-O-{b-D-
lesta-9(11),24(28)-dien-3b-yl sulfate as aglycon moiety in 3.
D
D
In addition to the above-mentioned signals, the 1H NMR spec-
trum of 3 exhibited five resonances in the downfield region due
to anomeric protons at dH 4.80, 4.99, 4.85, 4.83, and 5.33 that cor-
related in the HSQC experiment with corresponding carbon signals
at dC 105.0, 104.5, 102.0, 107.0, and 104.7, respectively (Table 3).
Along with mass spectral information, these data showed the pres-
ence of five monosaccharide residues in the oligosaccharide moiety
of 3. The coupling constants (7.4–7.9 Hz) of the anomeric proton
atoms were indicative of a b-configurations of all glycosidic bonds.
The existence of four 6-deoxy-sugar units was supported by four
methyl doublets at dH 1.58, 1.46, 1.45, and 1.78. The irradiation
of anomeric protons in the 1D TOCSY experiments gave the chem-
ical shifts and coupling constants of H-1–H-5 of quinovose and
xylose residues, and of H-1–H-4 of fucose residues; whereas the
irradiation of resonances of the corresponding methyl groups gave
the signals of H-5 of fucose residues. 1H–1H COSY, HSQC, HMBC,
and NOESY experiments led to the assignment of all the proton
and carbon signals of the carbohydrate chain of 3. The spectral data
of the oligosaccharide moiety strictly coincided with those of ter-
D
D
a
The molecular formula of leptasterioside C (5) was determined
to be C58H95O27SNa from the [M+Na]+ sodiated-molecular ion peak
at m/z 1301.5548 (calcd. for C58H95O27SNa2, 1301.5571) in the
(+)HR ESI-MS and the [MꢂNa]ꢂ ion peak at m/z 1255.5788 (calcd.
for C58H95O27S, 1255.5787) in the (ꢂ)HR ESI-MS. The fragment-ion
peaks at m/z 1181 [(M+Na)–NaHSO4]+ in the (+)ESI-MS/MS of the
ion at m/z 1301 [M+Na]+ and at m/z 97 [HSO4]ꢂ in the (ꢂ)ESI-
MS/MS of the ion at m/z 1255 [MꢂNa]ꢂ exhibited the presence of
a sulfate group in 5. Detailed comparison of NMR data of steroid
aglycons of 5 and 3 clearly showed that leptasterioside C (5) have
the same steroid moiety, namely (20S)-6a,20-dihydroxy-5a-cho-
lesta-9(11),24(28)-dien-3b-yl sulfate, bearing an O-olygosaccha-
ride chain at C-6.
The extensive NMR analysis of compounds 5 and 3 indicated that
both glycosides possess the related pentasaccharide moieties, but
differed in the replacement of the 2,4-di-O-substituted b-
anosyl residue for the 2,4-di-O-substituted b- -glucopyranosyl unit
(Table 3). The carbon signals of the last monosaccharide coincided
with those of 2,4-di-O-substituted b- -glucopyranosyl residue in
D-xylopyr-
D
minal b-
internal 3-O-substituted b-
tuted b- -xylopyranosyl, and 2-O-substituted b-
D
-fucopyranosyl and b-
-quinovopyranosyl, 2,4-di-O-substi-
-fucopyranosyl
D-quinovopyranosyl residues, and
D
D
D
D
the reported spectra of some known asterosaponins [12]. The (+)
and (ꢂ)ESI-MS/MS spectra of the molecular ions at m/z 1301
[M+Na]+ and 1255 [MꢂNa]ꢂ showed fragmentary peaks with the
consecutive losses of four 6-deoxyhexoses and one hexose units
(see Section 2). The presence of the NOESY and HMBC cross-peaks
between H-1 of Quip I and H-6 (C-6) of the aglycon, H-1 of Glcp and
H-3 (C-3) of Quip I, H-1 of Quip II and H-2 (C-2) of Glcp, H-1 of Fucp I
and H-4 (C-4) of Glcp, H-1 of Fucp II and H-2 (C-2) of Fucp I, exhibited
interglycosidic linkages and attachment of the oligosaccharide chain
at C-6 in 5. Acid hydrolysis of glycoside 5 with 2 M TFA was carried
out to ascertain its monosaccharide composition. Alcoholysis of the
mixture of sugars by (R)-(ꢂ)-octanol followed by acetylation, GC
analysis, and comparison with corresponding derivatives of standard
monosaccharides according to the procedure of Leontein et al. [20]
residues in the earlier reported spectra of the known regularoside
B [18].
The sequence of monosaccharide units in the carbohydrate chain
of 3 was confirmed by ESI-MS/MS data. In fact, the (ꢂ)ESI-MS/MS
spectrum of the molecular anion peak [MꢂNa]ꢂ at m/z 1225 showed
fragmentary peaks obtained due to the losses of sugar units: m/z
1079 [(MꢂNa) ꢂ 146]ꢂ, loss of 6-deoxyhexose; 933 [(MꢂNa) ꢂ 2
ꢀ 146]ꢂ, losses of two 6-deoxyhexoses; 787 [(MꢂNa) ꢂ 3 ꢀ 146]ꢂ,
losses of three 6-deoxyhexoses; 655 [(MꢂNa) ꢂ 3 ꢀ 146 ꢂ 132]ꢂ,
losses of three 6-deoxyhexoses and pentose; 509 [(MꢂNa) ꢂ 4 ꢀ
146 ꢂ 132]ꢂ, loss of
a carbohydrate chain. Respectively, the
(+)ESI-MS/MS spectrum of the sodiated-molecular ion peak at m/z
1271 showed, besides the ion peak at m/z 1151 [(M+Na)–NaHSO4]+,
a series of fragmentary peaks: m/z 1005 [(M+Na)–NaHSO4–146]+,
losses of sulfate group and 6-deoxyhexoses; 859 [(M+Na)–NaHSO4–
2 ꢀ 146]+, losses of sulfate group and two 6-deoxyhexoses; 713
[(M+Na)–NaHSO4–3 ꢀ 146]+, losses of sulfate group and three
6-deoxyhexoses. The attachment of the oligosaccharide chain to
the steroid aglycon and the position of interglycosidic linkages were
deduced from long-range correlations in the NOESY and HMBC
spectra (Table 3). There were the cross-peaks between H-1 of Quip
I and H-6 (C-6) of aglycon, H-1 of Xylp and H-3 (C-3) of Quip I, H-1
of Quip II and H-2 (C-2) of Xylp, H-1 of Fucp I and H-4 (C-4) of Xylp,
H-1 of Fucp II and H-2 (C-2) of Fucp I. On the basis of all the above
allowed us to establish the D-configuration for all the monosaccha-
ride units comprising the carbohydrate moiety of 5 (quinovose,
glucose and fucose). Thus, the structure of leptasterioside C (5) was
defined as sodium (20S)-6
pyranosyl-(1 ? 4)-[b- -quinovopyranosyl-(1 ? 2)]-b-
osyl-(1 ? 3)-b- -quinovopyranosyl}-20-hydroxy-5 -cholesta-9(11),
a-O-{b-D-fucopyranosyl-(1 ? 2)-b-D-fuco-
D
D-glucopyran-
D
a
24(28)-dien-3b-yl sulfate. Previously this type of carbohydrate
chain was not found in the other asterosaponins.
We assumed the D-configurations of fucose, quinovose, xylose
and glucose units in all co-occurring asterosaponins 3, 4, 6–8 by
analogy with glycoside 5.
mentioned data, the structure of leptasterioside
elucidated to be sodium (20S)-6 -O-{b- -fucopyranosyl-(1 ? 2)-
b- -fucopyranosyl-(1 ? 4)-[b- -quinovopyranosyl-(1 ? 2)]-b-
xylopyranosyl-(1 ? 3)-b- -quinovopyranosyl}-20-hydroxy-5
cholesta-9(11),24(28)-dien-3b-yl sulfate.
B
(3) was
On the basis of extensive 2D NMR analysis of glycosides 5–8, we
suggested that oligosaccharide moiety and steroid nucleus of 5 are
identical to those of glycosides 6–8. The molecular formula of lep-
tasterioside D (6) was determined to be C57H91O27SNa from the
[M+Na]+ sodiated-molecular ion peak at m/z 1285.5260 (calcd.
for C57H91O27SNa2, 1285.5258) in the (+)HR ESI-MS and the
[MꢂNa]ꢂ ion peak at m/z 1239.5470 (calcd. for C57H91O27S,
1239.5474) in the (ꢂ)HR ESI-MS. The fragment-ion peaks at m/z
1165 [(M+Na)–NaHSO4]+ in the (+)ESI-MS/MS of the ion at m/z
1285 [M+Na]+ and at m/z 97 [HSO4]ꢂ in the (ꢂ)ESI-MS/MS of the
ion at m/z 1239 [MꢂNa]ꢂ exhibited the presence of a sulfate group
in 6. Moreover, a detailed NMR analysis the proton and carbon sig-
nals of the steroid aglycon in glycoside 6 showed that it contains
the genuine asterogenin 2. Glycosidation at C-6 was supported
a
D
D
D
D
-
-
D
a
The molecular formula of leptasterioside A (4) was determined
to be C56H91O26SNa from the [M+Na]+ sodiated-molecular ion peak
at m/z 1257.5302 (calcd. for C56H91O26SNa, 1257.5309) in the
(+)HR ESI-MS and the [MꢂNa]ꢂ ion peak at m/z 1211.5519 (calcd.
for C56H91O26S, 1257.5525) in the (ꢂ)HR ESI-MS. The fragment-ion
peaks at m/z 1137 [(M+Na)–NaHSO4]+ in the (+)ESI-MS/MS of the
ion at m/z 1257 [M+Na]+ and at m/z 97 [HSO4]ꢂ in the (ꢂ)ESI-
MS/MS of the ion at m/z 1211 [MꢂNa]ꢂ exhibited the presence of
a sulfate group in 4. The comparison of 1H, 13C NMR spectra and
Please cite this article in press as: Malyarenko TV et al. Asterosaponins from the Far Eastern starfish Leptasterias ochotensis and their anticancer activity.
Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.05.027

STE 7589
No. of Pages 9, Model 5G
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by the values of the C-6 signal at dC 80.6 in the 13C NMR spectrum
of 6 compared to dC 68.1 in the 13C NMR spectrum of 2. From these
evidence we determined that asterosaponin 6 contained 3-O-sul-
of leptasterioside F (8) was established to be sodium (20S,24S)-
539
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6
a
-O-{b-
D
-fucopyranosyl-(1 ? 2)-b-
D
-fucopyranosyl-(1 ? 4)-[b-
-quinovo-
a-cholest-9(11)-en-23-one-3b-yl
D-
quinovopyranosyl-(1 ? 2)]-b-
pyranosyl}-20-hydroxy-24-methyl-5
sulfate.
D-glucopyranosyl-(1 ? 3)-b-
D
fated 6-O-glycosilated (20E)-6a,3b-dihydroxy-5a-cholesta-9(11),
20(22)-dien-23-one aglycon. All proton and carbon resonances of
6 were received from the 1H–1H COSY, HSQC, HMBC, and NOESY
experiments and confirmed the structure of the both aglycon and
the carbohydrate moiety. Therefore, the structure of leptasterioside
Previously, we studied the anticancer activities of starfish ste-
roid glycosides on the T-47D and RPMI-7951 cell lines. In the con-
tinuation of our experiments, we investigated the anticancer
activities of substances 2–8 on the same cell lines. These experi-
ments are necessary for comprehension the structure–activity
relations of starfish steroid compounds.
The in vitro cytotoxicities of compounds 2–8 against human
breast T-47D and melanoma RPMI-7951 cancer cell lines was eval-
uated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) assay. Comp-
ound 2 did not show cytotoxic effect against T-47D cell line within
D (6) was established to be sodium (20E)-6
(1 ? 2)-b- -fucopyranosyl-(1 ? 4)-[b- -quinovopyranosyl-(1 ? 2)]-
b- -glucopyranosyl-(1 ? 3)-b- -quinovopyranosyl}-5 -cholesta
-9(11),20(22)-dien-23-one-3b-yl sulfate.
a-O-{b-D-fucopyranosyl-
D
D
D
D
a
Leptasterioside E (7) has the molecular formula C57H93O28SNa
from the [M+Na]+ sodiated-molecular ion peak at m/z 1303.5340
(calcd. for C57H93O28SNa2, 1303.5364) in the (+)HR ESI-MS and
the [MꢂNa]ꢂ ion peak at m/z 1257.5584 (calcd. for C57H93O28S,
1257.5580) in the (ꢂ)HR ESI-MS. The fragment-ion peaks at m/z
1183 [(M+Na)–NaHSO4]+ in the (+)ESI-MS/MS of the ion at m/z
1303 [M+Na]+ and at m/z 97 [HSO4]ꢂ in the (ꢂ)ESI-MS/MS of the
ion at m/z 1257 [MꢂNa]ꢂ exhibited the presence of a sulfate group
in 7. The presence of the proton and carbon signals of one tertiary
Me group (dH 1.45; dC 23.2), two secondary Me groups (dH 0.95,
0.96; dC 22.4, 22.8), and the 22,23-epoxy group [dH 2.93 (d,
J = 2.3), 3.12 (td, J = 5.8, 2.3); dC 65.6, 53.2] in the NMR spectra
revealed the 20-hydroxy-22,23-epoxy-cholestane side chain in 7,
found for the first time in tenuispinoside A from the starfish Cosc-
inasterias tenuispina [21]. The all proton and carbon resonances
associated with the aglycon moiety in 7 were derived from
1H–1H COSY, HSQC, HMBC, and NOESY spectra (Fig. 2). The carbon
signals of steroid side chain in the 13C NMR spectrum of 7 coin-
cided well to those reported for tenuispinoside A, in which config-
urations of asymmetric centers C-20, C-22, and C-23 were
established by comparison with synthesized model compounds
[21]. So, glycoside 7 was suggested to have the same 20R,22R,23S
stereochemistry. In the NOESY spectrum, the cross-peaks between
H3-21/Hb-12, H-17, H3-18, H-22; H-22/Hb-16, H-17, H3-21; and
H-23/H3-21 confirmed the total structure of the side chain. Thus,
the structure of leptasterioside E (7) was determined to be sodium
a 10–200
against RPMI-7951 cell line with IC50 values of 143
6–8 demonstrated slight cytotoxic effects against T-47D cell line
with IC50 values of 68, 143, and 111 M, and against RPMI-7951
cell lines with IC50 values of 151, 151, and 91 M, respectively.
l
M concentration range, but had weak cytotoxic activity
lM. Glycosides
l
l
However, asterosaponins 3, 5, and, particularly, 4 were shown to
be more cytotoxic against T-47D cell line with IC50 values of 10,
23 and 2
effects against RPMI-7951 cell line were not so strong (IC50 values
of 17, 30, and 27 M, respectively). Therefore, the compound 4 was
lM, respectively. It is of interest that their cytotoxic
l
highly active against T-47D cell line only.
In present study soft agar clonogenic assay was used to evaluate
the effects of the compounds 2–8 on colony formation of cancer
cells. The soft agar assay for tumor cells colony formation are appli-
cable for a broad scope of human tumors and are proved to be use-
ful in the studies on chemosensitivity of human cancers [11]. As
described in Experimental section, the T-47D and RPMI-7951 cell
lines were treated with compounds 2–8 at concentrations that
were lower than IC50 of investigated compounds. The compounds
were incubated with the cells in the soft agar matrix at 37 °C in a
5% CO2 atmosphere for 2 weeks.
Our results provided that investigated compounds at nontoxic
doses inhibited spontaneous colony formation of cancer cells dif-
ferently. Compound 2 did not inhibit colony formation of T-47D
(20R,22R,23S)-6
(1 ? 4)-[b- -quinovopyranosyl-(1 ? 2)]-b-
b- -quinovopyranosyl}-20-hydroxy-22,23-epoxy-5
en-3b-yl sulfate.
a
-O-{b-
D
-fucopyranosyl-(1 ? 2)-b-
-glucopyranosyl-(1 ? 3)-
-cholest-9(11)-
D-fucopyranosyl-
D
D
D
a
cells at concentration up to 100
lM. The treatment of T-47D cells
by 3, 4, and 5 at concentrations 7, 1, and 10
l
M reduced the num-
The molecular formula of leptasterioside F (8) was determined
to be C58H95O28SNa from the [M+Na]+ sodiated-molecular ion peak
at m/z 1317.5551 (calcd. for C58H95O28SNa2, 1317.5520) in the
(+)HR ESI-MS and the [MꢂNa]ꢂ ion peak at m/z 1271.5737 (calcd.
for C58H95O28S, 1271.5736) in the (ꢂ)HR ESI-MS. The fragment-ion
peaks at m/z 1197 [(M+Na)–NaHSO4]+ in the (+)ESI-MS/MS of the
ion at m/z 1317 [M+Na]+ and at m/z 97 [HSO4]ꢂ in the (ꢂ)ESI-
MS/MS of the ion at m/z 1271 [MꢂNa]ꢂ demonstrated the presence
of a sulfate group. The 1H and 13C NMR spectra suggested the pres-
ence of a 20-hydroxy-23-keto-24-methyl-cholestane side chain in
8 with one tertiary Me group (dH 1.59; dC 26.9), three secondary Me
groups (dH 0.83, 0.93, and 1.02; dC 18.3, 21.2, and 11.8), adjacent
with carbonyl methylene group [dH 2.90 (d, J = 15.6), 2.70 (d,
J = 15.6); dC 53.4], and 23-keto group (dC 215.8). The 1H–1H COSY,
HSQC, HMBC, and NOESY experiments confirmed the presence of
the known 3-O-sulfated aglycon thornasterol B in 8 (Fig. 2),
ber of colonies on 51%, 76%, and 34%, respectively, when compared
with untreated cells (control, 0% of inhibition) (Fig. 3). On the other
hand, compounds 6–8 were less effective in this experiment; the
inhibition activity was 40–60% for these compounds at concentra-
tion up to 50 lM (data not shown). It is important to note that
compound 4 possessed the highest inhibitory effect on colony for-
mation of breast cancer T-47D cell lines at low concentration.
It should be noted that the effects of compounds on colony for-
mation also depended on type of cell lines. Melanoma is certain to
be the most aggressive and lethal tumor. That is why melanoma
RPMI-7951 cells are rather resistant to 2–8 compared to breast
cancer T-47D cells. The results obtained for the RPMI-7951 cells
indicated that compounds 2, 6–8 slightly inhibited colony forma-
tion at concentrations up to 50
lM. However, compounds 3, 4,
and 5 at a concentration 10 M effectively reduced the number
l
of colonies on 81%, 93%, and 15%, respectively, in comparison with
nontreated cells (control, 0% of inhibition) (Fig. 3). Cisplatin, an
anticancer drug widely used for treating various types of tumor,
was used as a positive control in our study. It was showed that
compound 4 at concentration was more effective in inhibition of
colony formation of T-47D and RPMI-7951 cells than cisplatin.
Based on these observations, leptasterioside A (4) seems to be
promising for further investigation as cancer preventive and anti-
cancer agent.
i.e.,
6a,20-dihydroxy-24-methyl-5a-cholest-9(11)-en-23-one-
3b-yl sulfate [22]. The negative Cotton maximum ([h]305 = ꢂ272)
in the CD spectrum of 8 was similar to that of acanthaglycoside F
([h]275 = ꢂ685) with (20S,24S)-thornasterol B as an aglycon [22]
in
contrast
with
([h]289 = ꢂ7748,
[h]284 = ꢂ6028
and
[h]284 = ꢂ6884, resp.) in spectra of hippasterioside C, versicosides
B and C with (20S,24R)-thornasterol B as an aglycon [7,22]. As a
result, the (20S,24S)-configuration was assigned, and the structure
Please cite this article in press as: Malyarenko TV et al. Asterosaponins from the Far Eastern starfish Leptasterias ochotensis and their anticancer activity.
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Acknowledgments
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The study was supported by the Program FEB RAS «Far East»,
Grants No. 14-04-00341-a and No. 14-04-31314mol-a from the
RFBR, Grant No. 12-III-A-05-058 from FEB RAS, and Grant of
President of Russia No. 148.2014.4 supporting leading Russian
scientific schools.
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Appendix A. Supplementary material
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2014.05.
027.
Please cite this article in press as: Malyarenko TV et al. Asterosaponins from the Far Eastern starfish Leptasterias ochotensis and their anticancer activity.
Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.05.027