760
MALYARENKO et al.
complete structures have been found, including column (5
the absolute stereochemistry, for three new steroid MeOH–H2O–1
polyols.
μ
m, 10
NH4OAc system (75 : 24 : 1). Comꢀ
pound (III) was obtained (1.2 mg, R 45.0 min). The
× 250 mm, 2.5 ml/min) in the
M
t
results of the chromatographic separation of the steꢀ
roid fractions with the isolation of compounds
EXPERIMENTAL
(I)
⎯
(
XIII) are shown in Table 1.
(24R,25 )ꢀ24ꢀmethylꢀ ꢀcholestaneꢀ
,16 ,26ꢀhexaol (I), an amorphous compound,
[α]2D0 + 23
0.1 of MeOH). (+)ꢀHRꢀESI mass specꢀ
trum, : 505.3466 [
+ Na]+ (calculated for
С28Н50О6Na, 505.3499). (–)ꢀHRꢀESI mass specꢀ
The 1Н and 13C NMR spectra (
δ, ppm; J, Hz) were
S
5
α
3β,6α,8,ꢀ
recorded in Bruker DPXꢀ300 (1H, 300, 13C, 75.5 MHz),
Bruker DRXꢀ500 (1H, 500, 13C, 125.7 MHz), and
15
β
β
(
c
13
Bruker Avance III 700 (1H, 700, C, 176 MHz) specꢀ
m/z
M
trometers with the inner standard being CD3OD
δC 49.0 ppm, δH 3.30 ppm). The optical rotation was
(
m/z: 481.3544 [M −
Н]− (calculated for
measured on a Perkin Elmer 343 polarimeter in
MeOH. The mass spectra of ESI were obtained on an
Agilent 6510 QꢀTOF mass spectrometer (United
States). The samples were dissolved in MeOH (with
0.01 mg/ml). HPLC was performed in an Agilent
chromatograph with a refractometric detector. TLC
was performed on Sorbfil plates with a layer of CTXꢀ1A
trum,
С28Н49О6, 481.3535). Spectra of 1Нꢀ and 13C NMR
are shown in Table 2.
(22E,24R,25 )ꢀ24ꢀmethylꢀ
S
5αꢀcholestꢀ22ꢀeneꢀ
3β
, 6 ,8,15 ,16β,26ꢀhexaol (II), an amorphous comꢀ
α
β
pound, [α]2D0 + 6
spectrum, : 503.3340 [
С28Н48О6Na, 503.3343). (–)ꢀHRꢀESIꢀmassꢀspecꢀ
(
c
0.1 of MeOH). (+)ꢀHRꢀESI mass
+ Na]+ (calculated for
silica gel (5–17
foil. The substances were detected by concentrated
H2SO4 with the subsequent heating of the plates at 110
μm, Sorbpolimer, Russia) fixed on
m/z
M
m/z: 479.3385 [M −
Н]− (calculated for
°С
trum,
over the course of 10 min. Preparative separations were
made using column chromatography on Amberlite
XADꢀ2 (20–80 mesh, Sigma Chemical Co.), KSK silica
gel (50–160 μm, Sorbpolimer, Russia), and florisil (100–
200 mesh, Aldrich, United States).
Animals. Samples of the starfish A. carinifera were
collected in January 2005 by diving from a depth of 5–
10 m in the South China Sea (Van Fong Bay, coast of
Vietnam) during the 30th research voyage on board
the RV Akademik Oparin. The determination of the
species of the starfish was made by A.V. Smirnov (Zooꢀ
logical Institute, Russian Academy of Sciences,
St. Petersburg, Russia).
Isolation of compounds (I)–(III). The fresh starꢀ
fishes (500 g) were twice extracted with ethanol while
heating in a water bath. The ethanol extract was conꢀ
centrated in a vacuum, dissolved in H2O (0.5 l), and
С28Н47О6, 479.3378). Spectra of 1Нꢀ and 13C NMR are
shown in Table 2.
(22
,6α,8,15
E
,24
R
,16
,25
S
)ꢀ24ꢀmethylꢀ
5αꢀcholestꢀ22ꢀeneꢀ3β,ꢀ
4β
β
β
,26ꢀheptaol (III), an amorphous
compound, [α]2D0 + 7
mass spectrum, : 519.3283 [
for С28Н48О7Na, 519.3292). (–)ꢀHRꢀESI mass specꢀ
(
c
0.1 of MeOH). (+)ꢀHRꢀESI
+ Na]+ (calculated
m/z
M
trum, m/z: 495.3332 [M− ,
Н]− (calculated for С28Н47О7
495.3327). Spectra of 1Нꢀ and 13C NMR are shown in
Table 2.
Preparation of MTPA esters of compounds (I) and
(II). The substance in an amount of 0.8 mg was disꢀ
solved in 200
μl of dry pyridine, 10 μl of chloroanhyꢀ
dride of (S)ꢀ(+)ꢀМТРА was added (SigmaꢀAldrich,
Germany), it was kept for 2 h at room temperature,
and then the solution was concentrated under a vacꢀ
uum. The dry residue was purified by HPLC on a
passed through a column (7 × 26 cm) with Amberlit
XADꢀ2. The column was washed with H2O until the
absence of Cl⎯ ions in the eluate and then with ethaꢀ
nol. The ethanol eluate was evaporated, and the
obtained total fraction of the steroid compounds was
consecutively chromatographed in columns with silica
Diasferꢀ110ꢀCꢀ18 column (5
1 ml/min) in the МеОН Н2О system (97 : 3). The
output was 0.7 mg of )ꢀ(+)ꢀМТРА ester of comꢀ
pound ( ) and 0.7 mg of )ꢀ(+)ꢀМТРА ester of comꢀ
pound (II).
3,6,26ꢀTriꢀ
The spectrum of 1H NMR (CD3OD), selected signals:
0.83 (3 H, d, 6.8. Me28); 0.85 (3 H, dd, 6.9, Me27);
0.91 (3 H, d, 6.7, Me21); 1.09 (3 H, s, Me19); 1.24
(3 H, s, Me18); 2.59 (1 H, dd, 4.5, 12.0, H7); 4.18
(1 H, t, 7.0,H16); 4.19 (1 H, dd, 6.6, 10.9, Н 26);
4.24 (1 H, dd, 6.6, 10.9,H26); 4.32 (1 H, dd, 6.7, 5.7,
H15); 4.83 (1 H, m, H3); 5.29 (1 H, dt, 4.2, 11.1, H6).
3,6,26ꢀTriꢀ( )ꢀ(+)ꢀMTPAꢀester of compound (II).
The spectrum of 1H NMR (CD3OD), selected signals:
0.92 (3 H, d, 6.8, Me28); 0.92 (3 H, d, 6.9, Me27);
enriched with compound (III). This fraction was addiꢀ 1.02 (3 H, d, 6.7, Me21); 1.10 (3 H, s, Me19); 1.27
tionally purified by HPLC on a Discovery C18 (3 H, s, Me18); 2.58 (1 H, dd, 4.5, 12.0, H7); 4.05
μm, 4 × 250 mm,
−
(
R
(R
I
gel (
gradient, 4 :
the system СНСl3–EtOH (stepwise gradient, 5 :
: 2). Several fractions were obtained containing by
6
×
16 cm) in the СНСl3–EtOH system (stepwise
(R)ꢀ(+)ꢀМТРА ester of compound (I).
1
1
: 4) and with florisil ( 14 cm) in
2 ×
1
J
J
1
J
TLC data polyhydroxysteroids (Rf within 0.42–0.80)
in the toluol–EtOH system (9 : 5). The obtained fracꢀ
tion were divided by HPLC on a Diasfer column
J
J
J
'
J
J
110ꢀC18 (10
EtOH–H2O system (65 : 35); this led to the isolation of
compounds ( ) (1.5 mg, R 47.3 min), (II) (1.3 mg,
R 63.2 min), and (IV)–(XIII) and of the fraction
μm, 15 × 250 mm, 2.5 ml/min) in the
J
R
I
t
t
J
J
J
J
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
No. 6
2010
Malyarenko
