Highly hydroxylated steroids of the starfish Archaster typicus from the Vietnamese waters

Article abstract of DOI:10.1016/j.steroids.2010.05.012

Five new steroidal compounds, including an unusual glucoside, along with several known steroids were isolated from the starfish Archaster typicus collected in shallow waters of Quang Ninh province (Vietnam). Three new compounds are 27-nor-cholestane deriv

Full text of DOI:10.1016/j.steroids.2010.05.012

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Steroids 75 (2010) 897–904
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Highly hydroxylated steroids of the starfish Archaster typicus from the
Vietnamese waters
Natalia V. Ivanchinaa, Alla A. Kichaa, Trinh T.T. Huongb, Anatoly I. Kalinovskya, Pavel S. Dmitrenoka,
Irina G. Agafonovaa, Pham Q. Longb, Valentin A. Stonika,∗
a Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Pr. 100-let Vladivostoku 159, 690022 Vladivostok, Russia
b Institute of Nature Products Chemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Hanoi, Viet Nam
a r t i c l e i n f o
a b s t r a c t
Article history:
Five new steroidal compounds, including an unusual glucoside, along with several known steroids
were isolated from the starfish Archaster typicus collected in shallow waters of Quang Ninh province
(Vietnam). Three new compounds are 27-nor-cholestane derivatives and the other two are 24,26-
dihydroxycholestane derivatives. A biogenesis pathway for the unusual side chain of 27-nor-cholestane
derivatives is proposed. Isolated compounds presented moderate toxic effects in the sperm- and 8-
blastomere tests on embryonal development of the sea urchin Strongylocentrotus intermedius.
© 2010 Elsevier Inc. All rights reserved.
Received 16 March 2010
Received in revised form 7 May 2010
Accepted 10 May 2010
Available online 21 May 2010
Keywords:
Polyhydroxylated steroids
Glycoside
Starfish
Archaster typicus
1. Introduction
diseases [9]. We decided to study polar steroids from this species
hoping to isolate new compounds of this rare series and extract new
Some echinoderms, especially sea cucumbers and starfish,
are characterized by steroids with unique structural features
that are products of unusual biosyntheses. For example, radiola-
belling experiments have indicated an unusual steroid biosynthetic
pathway in the sea cucumber Eupentacta fraudatrix, involving
transformation of squalene to parkeol instead of lanosterol fol-
lowed by subsequent demethylation from C-4 [1]. As result
unusual sterols such as 4␣,14␣-dimethylcholest-9(11)-en-3␤-ol
and 14␣-methylcholest-9(11)-en-3␤-ol are formed [2]. Unusual
biosynthetic transformations were also detected in the studies of
polar steroids from starfish. In fact, starfish contain a great num-
ber of polar steroids, including polyhydroxylated derivatives, many
of which have no counterpart within other animals [3–5]. Earlier,
studies on the starfish Archaster typicus collected off Nouméa, New
Caledonia by Minale’s group have yielded nine new polar steroids
[6,7], four of which had a 27-nor-cholestane skeleton. Detection
of polyhydroxysteroids with the 27-nor-cholestane side chain was
very noteworthy because similar natural products have not been
found in the animal kingdom except a few rare findings, including
a minor steroid from the sponge Axinella cannabina [8] and 27-
nor-5␤-cholestane-3␣,7␣,12␣,24S,25␰-pentaol, isolated as a major
steroid constituent from the urine and feces of patients with liver
structural information concerning unusual steroid biosynthesis in
this species. The starfish A. typicus was collected near coast-line
of Quang Ninh province (Vietnam). Separation of ethanol extract,
using different chromatographic techniques, led to five new polar
steroids (1–5), including four new highly hydroxylated steroids (1,
3–5) and a glycoside named as typicusoside A (2), along with the
six previously known compounds (6–11).
2. Experimental
2.1. General methods
Optical rotations were determined on a PerkinElmer polarime-
ter Model 343. The 1H and 13C NMR spectra were recorded on
a Bruker DRX 500 spectrometer at 500 and 125.8 MHz, respec-
tively, using tetramethylsilane as an internal standard. HR ESI mass
spectra were recorded on an Agilent 6510 Q-TOF LC/MS mass spec-
trometer; samples were dissolved in MeOH (c 0.001 mg/mL).
HPLC separations were carried out on an Agilent 1100 Series
chromatograph equipped with
a differential refractometer.
Diasfer-110-C18 (10 ␮m, 250 mm × 15 mm) and Diasfer-110-C18
(5 ␮m, 250 mm × 4.6 mm) columns were used. Low pressure
column liquid chromatography was performed using Polychrome
1
(powdered Teflon, Biolar, Latvia), Si gel KSK (50–160 ␮m,
∗
Sorbpolimer, Krasnodar, Russia), and Florisil (200–300 mesh,
Aldrich Chemical Co.). Sorbfil Si gel plates (4.5 × 6.0 cm,
Corresponding author. Tel.: +7 4232 312360; fax: +7 4232 314050.
E-mail address: stonik@piboc.dvo.ru (V.A. Stonik).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.05.012

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N.V. Ivanchina et al. / Steroids 75 (2010) 897–904
Table 1
1H NMR data of 1–5 (500 MHz, J in Hz)a.
Position
1
1b
2c,d
3b
4b
5b
1.72 m
1.01 m
1.81 m
1.53 m
3.42 m
4.23 brs
2.17 m
1.68 m
2.32 m
2.04 m
4.78 m
4.92 d (3.9)
1.59 m
1.33 m
1.75 m
1.56 m
4.02 m
3.92 d (3.9)
1.72 m
1.00 m
1.72 m
1.46 m
3.47 m
2.17 m
1.72 m
0.99 m
1.72 m
1.47 m
3.47 m
2.17 m
2
3
4
1.18 m
1.19 m
5
6
7
0.91 m
1.01 m
1.02 m
4.07 td (4.2, 10.1)
2.15 dd (4.4, 13.1)
1.70 dd (11.0, 13.1)
4.95 d (11.3)
4.97 d (11.4)
4.32 dd (4.9, 11.5)
2.18 dd (11.6, 13.1)
1.82 dd (4.9, 13.1)
3.63 td (4.3, 10.9)
2.29 dd (4.3, 13.2)
1.67 m
3.63 td (4.3, 10.8)
2.28 dd (4.3, 13.3)
1.67 m
8
9
1.55 m
3.15 dd (3.0, 13.0)
2.22 dd (3.6, 13.0)
1.57 m
1.57 m
10
11
1.61 m
1.34 m
1.72 m
1.54 m
2.15 m
1.62 m
2.25 m
1.80 m
1.63 m
1.22 m
1.75 m
1.55 m
1.65 m
1.40 m
1.76 m
1.54 m
1.65 m
1.41 m
1.78 m
1.53 m
12
13
14
15
16
4.41 dd (4.2, 9.5)
1.87 m
1.62 m
1.97 q (9.6)
1.10 s
1.18 s
1.34 m
0.84 d (6.5)
1.54 m
0.97 m
1.53 m
4.81 m
2.11 m
2.01 m
2.32 m
1.44 s
4.42 dd (4.1, 9.4)
1.89 dt (9.5, 13.6)
1.62 m
1.99 q (9.6)
1.10 s
1.29 s
1.36 m
0.84 d (6.5)
1.56 m
0.95 m
4.92 m
2.07 m
1.96 m
2.02 m
1.15 s
1.02 s
1.39 m
0.85 d (6.5)
1.52 m
4.95 dd (4.3, 9.1)
2.10 m
2.00 m
2.06 m
1.18 s
1.02 s
1.63 m
0.87 d (6.5)
2.35 m
1.99 m
17
18
19
20
21
22
2.01 s
1.41 m
0.92 d (6.5)
1.64 m,
1.06 m
1.65 m,
1.29 m
3.76 m
1.69 m,
1.60 m
0.95 t (7.5)
0.98 m
23
1.54 m
1.32 m
3.58 m
1.69 m
1.68 m/1.62 m
1.42 m/1.48 m
4.04 t (6.9)/4.06 m
6.91 ddd (6.3, 8.7, 15.5)
1.25 m
3.36 m
1.48 m
1.37 m
24
25
6.07 d (15.5)
2.23 s
26
27
0.92 t (7.5)
3.57 dd (6.6, 10.6)
3.43 m
0.87 d (7.0)
4.09 dm (14.5)
4.12 td (1.3, 14.5)/4.11 td (1.3, 14.5)
5.05 m/5.06 m
5.11 q (1.5)/5.09 q (1.5)
a
Assignments from 1H-1H COSY, HSQC, and HMBC data.
Measured in CD3OD.
b
c
Measured in C5D5N.
d
The 1H NMR data of the ␣-d-glucopyranosyl unit of compound 2 (500 MHz, J in Hz, C5D5N): 5.40 d (1H, d, J = 3.9, H-1ꢀ), 4.10 (1H, dd, J = 3.9, 9.7 Hz, H-2ꢀ), 4.55 (1H, t, J = 9.1,
H-3ꢀ), 4.19 (1H, t, J = 9.0, H-4ꢀ), 4.43 (1H, m, H-5ꢀ), 4.39 (1H, dd, J = 5.3, 11.2, H-6ꢀ), 4.51 dd (1H, dd, J = 2.3, 11.2, Hꢀ-6ꢀ).
5–17 ␮m, Sorbpolimer, Krasnodar, Russia) in the eluent system
BuOH/EtOH/H2O (4:1:2) were used for thin-layer chromatography.
GLC analysis was carried out on an Agilent 6580 Series apparatus,
carried gas He (1.7 mL/min) at 100 ◦C (0.5 min) → 250 (5 ◦C/min,
10 min), capillary column HP-5 MS (30 m × 0.25 mm). Tempera-
tures of injector and detector were 150 and 280 ◦C, respectively.
material (29.7 g). The resulting total fraction of steroidal com-
pounds was chromatographed on a Si gel column (6.5 cm × 20 cm)
using CHCl3/EtOH (stepwise gradient, 4:1 → 1:6) and the fractions
obtained were purified on a Florisil column (4 cm × 17 cm) using
CHCl3/EtOH (stepwise gradient, 4:1 → 1:2). HPLC separation of
the collected subfractions on a Diasfer-110-C18 column (10 ␮m,
250 mm × 15 mm, 2.5 mL/min) with EtOH/H2O (55:45) as an elu-
ent system yielded pure 6 (1 mg, Rf 0.68), 7 (1.5 mg, Rf 0.71), 8
(1 mg, Rf 0.70), 9 (37 mg, Rf 0.85), 10 (64 mg, Rf 0.75), 11 (20.5 mg,
Rf 0.65), and several additional subfractions of polyhydroxys-
teroidal mixtures. Further HPLC separation of the subfractions on
a Diasfer-110-C18 column (5 ␮m, 250 mm × 4.6 mm, 0.5 mL/min)
with MeOH/H2O/1 M NH4OAc (80:19:1) as the eluent system gave
1 (3.0 mg, Rf 0.75), with MeOH/H2O/1 M NH4OAc (70:29:1) led to
2 (2.0 mg, Rf 0.61) and 5 (2.2 mg, Rf 0.70). MeOH/H2O/1 M NH4OAc
(65:34:1) as the eluent system yielded 3 (1.4 mg, Rf 0.71) and 4
(2.8 mg, Rf 0.63).
2.2. Animal material
Specimens of A. typicus (order Valvatida, family Archasteridae)
were collected from shallow waters of Quang Ninh province (Viet-
nam) in August 2008 and were identified by Dr. Do Cong Thung,
the Institute of Marine Resources and Environment. A voucher
specimen [No. SB 08-2008] is deposited at the Institute of Natural
Products Chemistry, VAST, Vietnam.
2.3. Extraction and isolation
Freshly collected animals (7 kg) were minced and extracted
three times with EtOH at room temperature. The combined extract
was evaporated, and the residue dissolved in H2O (1 L). The
H2O-soluble fraction was passed through a Polychrome-1 column
(7 cm × 26 cm) and eluted with distilled H2O until a negative chlo-
ride ion reaction was obtained. This was followed by elution with
EtOH. The combined EtOH eluate was evaporated to give a brownish
2.4. Spectral data of new compounds
2.4.1. (24R)-27-Nor-5˛-cholestane-3
ˇ,4ˇ,6˛,8,14,15˛,24-heptaol (1)
C
26H46O7, amorphous powder; [˛]25 + 13.3◦ (c 0.1, EtOH); 1H
D
and 13C NMR data, see Tables 1 and 2; (−)ESIMS m/z 469 [M−H]−;
(−)ESIMS/MS of the ion [M−H]− at m/z 469: 451 [(M−H)–H2O]−,

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N.V. Ivanchina et al. / Steroids 75 (2010) 897–904
899
Table 2
13C NMR data of 1–5 (125.8 MHz).
Position
1a
2b,c
3a
4a
5a
39.9
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
40.1
26.2
73.7
69.1
56.8
64.9
45.8
79.5
49.9
37.8
18.5
35.3
48.5
85.1
69.8
40.2
51.7
17.1
17.1
36.3
18.9
33.1
34.5
74.6
30.8
10.2
32.9
26.8
68.2
72.5
78.2
70.7
73.5
79.7
40.6
39.6
17.9
34.9
48.3
83.7
69.0
37.5
50.9
16.8
17.4
35.4
18.4
31.4
29.8
79.4
27.5
9.8
33.1
26.3
69.4
72.7
78.7
66.7
39.3
79.1
41.6
40.2
18.3
35.4
48.6
85.2
69.9
40.1
51.7
17.2
17.3
36.4
18.8
33.7
32.3
73.7
41.5
66.4
10.6
39.9
31.5
72.2
32.4
53.2
67.7
44.7
79.3
48.9
37.6
19.1
35.0
48.1
85.5
77.8
37.2
51.5
17.4
14.3
36.1
18.8
33.1
33.2/33.5
75.0/74.4
152.5
62.9/63.1
110.4/110.0
31.5
72.2
32.4
53.3
67.7
44.7
79.3
48.8
37.6
19.1
34.9
48.2
85.5
77.8
37.3
51.5
17.4
14.3
36.2
19.0
40.4
149.9
133.5
201.3
26.7
a
Measured in CD3OD.
Measured in C5D5N.
b
c
The 13C NMR data of the ␣-d-glucopyranosyl unit of compound 2 (125.8 MHz, C5D5N): 98.9 (C-1ꢀ), 73.8 (C-2ꢀ), 75.2 (C-3ꢀ), 72.3 (C-4ꢀ), 74.2 (C-5ꢀ), 62.8 (C-6ꢀ).
433 [(M−H)–2H2O]−, 415 [(M−H)–3H2O]−, 397 [(M−H)–4H2O]−,
379 [(M−H)–5H2O]−; (+)HRESIMS m/z 493.3138 [M+Na]+ (calcd for
[M−Na]−; (−)ESIMS/MS of the ion [M−Na]− at m/z 529: 511
[(M−Na)–H2O]−, 445 [(M−Na)–C5H7OH]−, 97 [HSO4]−, 80 [SO3]−;
(+)HRESIMS m/z 575.2258 [M+Na]+ (calcd for C26H41O9SNa2,
575.2261).
C26H46O7Na, 493.3136).
2.4.2. (24R)-24-O-˛-d-glucopyranosyl-27-nor-5˛-cholestane-
3ˇ,4ˇ,5,6˛,7ˇ,8,14,15˛,24-nonaol
(2)
2.5. Preparation of the MTPA esters of compound 1
C
32H56O14, amorphous powder; [˛]25 + 11.1◦ (c 0.2, EtOH); 1
H
D
Aliquots (1.0 mg) of compound 1 were treated with S-(+)- and
R-(−)-␣-methoxy-␣-(trifluoromethyl)-phenylacetyl (MTPA) chlo-
ride (15 ␮L) in dry pyridine (200 ␮L) for 2 h at room temperature.
After removal of the solvent, the products obtained were purified
by HPLC on a Diasfer-110-C18 column (5 ␮m, 250 mm × 4.6 mm,
1 mL/min) with 92% MeOH as the eluent system to obtain the cor-
responding (R)- and (S)-MTPA esters of 1.
and 13C NMR data, see Tables 1 and 2; (−)ESIMS m/z 663 [M−H]−;
(+)ESIMS m/z 687 [M+Na]+; (+)ESIMS/MS of the ion at m/z 687: 669
[(M+Na)–H2O]+, 525 [(M+Na)–162]+, 507 [(M+Na)–162–H2O]+, 489
[(M+Na)–162–2H2O]+; (+)HRESIMS m/z 687.3575 [M+Na]+ (calcd
for C32H56O14Na, 687.3562).
2.4.3. 5˛-Cholestane-3ˇ,4ˇ,5,6˛,8,14,15˛,24,26-nonaol (3)
C
27H48O9, amorphous powder; [˛]25 + 9.3◦ (c 0.14, EtOH); 1H
D
and 13C NMR data, see Tables 1 and 2; (−)ESIMS m/z 515 [M−H]−;
(−)ESIMS/MS of the ion [M−H]− at m/z 515: 497 [(M−H)–H2O]−,
479 [(M−H)–2H2O]−, 461 [(M−H)–3H2O]−, 443 [(M−H)–4H2O]−,
425 [(M−H)–5H2O]−, 407 [(M−H)–6H2O]−; (+)HRESIMS m/z
539.3194 [M+Na]+ (calcd for C27H48O9Na, 539.3191).
2.5.1. 3,6,24-tri-(S)-MTPA ester of 1
1H NMR (500 MHz, CD3OD): 0.77 (3H, d, J = 6.5 Hz, H3-21), 0.92
(1H, m, H-22), 0.93 (3H, t, J = 7.5 Hz, H3-26), 1.08 (3H, s, H3-18), 1.27
(1H, m, H-20), 1.28 (3H, s, H3-19), 1.33 (1H, m, Hꢀ-22), 1.39 (1H, m,
H-5), 1.45 (1H, m, H-23), 1.52 (1H, m, H-16), 1.67 (1H, m, Hꢀ-23),
1.69 (2H, m, H-25), 1.72 (1H, m, H-7), 1.76 (1H, m, Hꢀ-16), 1.90 (1H,
m, H-17), 2.41 (1H, dd, J = 4.5, 13.0 Hz, Hꢀ-7), 4.29 (1H, brs, H-4), 4.40
(1H, dd, J = 4.2, 9.5 Hz, H-15), 4.86 (1H, m, H-3), 4.97 (1H, m, H-24),
5.53 (1H, td, J = 4.5, 11.2 Hz, H-6).
2.4.4. 5˛-Cholest-25(27)-ene-3ˇ,6˛,8,14,15˛,24,26-heptaol
15-O-sulfate, sodium salt (4)
C
27H45O10SNa, amorphous powder; [˛]25 + 85◦ (c 0.1, EtOH);
D
1H and 13C NMR data, see Tables
1
and 2; (−)ESIMS m/z
561 [M−Na]−; (−)ESIMS/MS of the ion [M−Na]− at m/z 561:
543 [(M−Na)–H2O]−, 97 [HSO4]−, 80 [SO3]−; (+)ESIMS m/z 607
[M+Na]+, 487 [(M+Na)–NaHSO4]+; (+)HRESIMS m/z 607.2535
[M+Na]+ (calcd for C27H45O10SNa2, 607.2523).
2.5.2. 3,6,24-tri-(R)-MTPA ester of 1
1H NMR (500 MHz, CD3OD): 0.80 (3H, t, J = 7.5 Hz, H3-26), 0.83
(3H, d, J = 6.5 Hz, H3-21), 1.02 (1H, m, H-22), 1.11 (3H, s, H3-18), 1.29
(3H, s, H3-19), 1.34 (1H, m, H-20), 1.38 (1H, m, H-5), 1.46 (1H, m,
Hꢀ-22), 1.52 (1H, m, H-23), 1.60 (1H, m, H-16), 1.62 (2H, m, H-25),
1.72 (1H, m, Hꢀ-23), 1.82 (1H, m, Hꢀ-16), 1.97 (2H, m, H-7, H-17),
2.42 (1H, dd, J = 4.5, 13.0 Hz, Hꢀ-7), 3.86 (1H, brs, H-4), 4.40 (1H, dd,
J = 4.2, 9.5 Hz, H-15), 4.78 (1H, m, H-3), 4.97 (1H, m, H-24), 5.59 (1H,
td, J = 4.5, 11.2 Hz, H-6).
2.4.5.
(23E)-27-Nor-25-oxo-5˛-cholest-23-ene-3ˇ,6˛,8,14,15˛-pentaol
15-O-sulfate, sodium salt (5)
C
26H41O9SNa, amorphous powder; [˛]25 + 43.7◦ (c 0.2, EtOH);
D
1H and 13C NMR data, see Tables 1 and 2; (−)ESIMS m/z 529

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Fig. 1. Structures of compounds 1–8 isolated from A. typicus.
2.6. Acid hydrolysis and determination of the absolute
configuration of glucose in 2
The solution was evaporated in vacuo and treated with a mixture
of pyridine/acetic anhydride (1:1, 0.4 mL) for 24 h at room tem-
perature. The acetylated (−)-2-octylglycosides were analyzed by
GC using the corresponding authentic samples of d-glucose and
l-glucose prepared by the same procedure. The following peaks
were detected in hydrolysate of 2: tR values 28.27, 28.86, 29.06,
and 29.38 min. tR Values of authentic samples were: d-glucose (tR
values 28.27, 28.86, 29.06, and 29.38 min) and l-glucose (tR values:
28.40, 28.62, 28.72, and 29.38 min).
Acid hydrolysis of 2 (1.0 mg) was carried out in 2 M triflu-
oroacetic 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× 0.5 mL)
and concentrated in vacuo. One drop of concentrated TFA and
0.5 mL of (−)-2-octanol (Aldrich) were added to the sugar and
the sealed vial was heated on a glycerol bath at 130 ◦C for 6 h.