Orostanal, a novel abeo-sterol inducing apoptosis in leukemia cell from a marine sponge, Stelletta hiwasaensis

Article abstract of DOI:10.1016/S0040-4039(01)01278-3

A sterol derivative, orostanal, was obtained from a marine sponge of Stelletta hiwasaensis. Spectroscopic analysis and synthetic study revealed its structure as a novel 5(6→7)abeo-sterol. Orostanal and its synthetic analog induce apoptosis in human acute promyelotic leukemia cell.

Full text of DOI:10.1016/S0040-4039(01)01278-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 6349–6351
Orostanal, a novel abeo-sterol inducing apoptosis in leukemia
cell from a marine sponge, Stelletta hiwasaensis
a
a
a
a,
Tomofumi Miyamoto, Kota Kodama, Yuko Aramaki, Ryuichi Higuchi * and
b
Rob W. M. Van Soest
a
Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
b
Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, PO Box 94766,
1090 GT Amsterdam, Netherlands
Received 28 May 2001; accepted 13 July 2001
Abstract—A sterol derivative, orostanal, was obtained from a marine sponge of Stelletta hiwasaensis. Spectroscopic analysis and
synthetic study revealed its structure as a novel 5(6“7)abeo-sterol. Orostanal and its synthetic analog induce apoptosis in human
acute promyelotic leukemia cell. © 2001 Elsevier Science Ltd. All rights reserved.
2
8
Apoptosis is defined as a physiological and pro-
grammed cell death, which is distinguished from necro-
sis. Apoptosis plays an important role not only in the
development but also in the maintenance of homeosta-
sis. Abnormal control of apoptosis may play a role in
the development and survival of cancer as well as other
diseases. It is well known that natural anti-cancer
drugs, etoposide and camptothecin, induce apoptosis in
cancer cells, so that the substance which induces apop2-
tosis must be useful for human cancer chemotherapy.
24
1
29
2
7
H
H 7
H
3
5
Orostanal (1)
HO
CHO
6
OH
5
Orostanal (1) was obtained as an amorphous solid.
The IR spectrum revealed absorption bands due to
hydroxyl (3502 cm ) and carbonyl (1715 cm ). The
H, C NMR and HSQC spectral data suggested the
−
1
−1
While searching for biologically active substances from
marine invertebrates, we isolated a novel sterol named
orostanal (1) as an active constituent inducing apopto-
sis, from a Japanese marine sponge, Stelletta hiwasaen-
sis. In this paper, we will report on the isolation and
structural determination of 1.
1
13
presence of one primary methyl, two secondary
methyls, two tertiary methyls, ten methylenes, seven
methines, two quaternary carbons, one oxygenated
methine, one oxygenated quaternary carbon, one exo-
methylene, and one aldehyde (Table 1). The HR-EI MS
of 1 showed a molecular ion peak at m/z 444.3607 (D
+0.4 mmu) corresponding to the molecular formula of
S. hiwasaensis (wet weight 2.0 kg) was collected by
hand at depths of 10 to 15 m off Oro Island, Fukuoka
3
1
1
Prefecture, Japan, in 1997. The n-hexane soluble frac-
C H O . The H– H COSY, TOCSY, and HSQC–
29 48 3
tion (7.86 g) of the acetone extract obtained from S.
hiwasaensis showed chromatin condensation and DNA
fragmentation at 33 mg/mL against human acute
TOCSY spectra of 1 afforded three partial structures, A
[C1ꢀC4], B[C6ꢀC12, C21, C23] and C[C25ꢀC27, C29].
These three partial structures and three quaternary
carbones, two tertiary methyls, and exo-methylene were
merged by the aid of the HMBC experiment as shown
in Fig. 1.
4
promyelotic leukemia cell (HL-60). Bioassay-guided
separation of the active fraction by silica-gel chro-
matography (CHCl :MeOH/10:0“1:1), followed by
3
reversed-phase column chromatography (95% MeOH/
H O), and reversed-phase HPLC (100% MeOH) gave
an active compound (1) (3.6 mg, 1.8×10 % yield).
The relative stereochemistry except C-25 of 1 was
assigned on the basis of the NOESY correlations, and
the stereochemistry of C-25 was elucidated by compari-
2
−
4
1
*
Corresponding author. Tel.: +81-92-642-6635; fax: +81-92-642-6639;
son of the H NMR data with those of (25S)- and
6
e-mail: rhiguchi@phar.kyushu-u.ac.jp
(25R)-24(28)-dehydroaplysterols as shown in Fig. 2.
0
040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01278-3

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350
T. Miyamoto et al. / Tetrahedron Letters 42 (2001) 6349–6351
1
13
Table 1. H and C NMR spectral data of 1 and 2
1
lC
2
lC
No.
lH
HSQC-TOCSYa
1
2
3
4
5
6
7
8
9
26.8 (t)
28.0 (t)
67.4 (d)
44.3 (t)
84.2 (s)
204.6 (d)
63.9 (d)
40.0 (d)
50.5 (d)
45.5 (s)
21.6 (t)
39.7 (t)
44.8 (s)
56.2 (d)
24.6 (t)
28.3 (t)
55.6 (d)
12.5 (q)
18.4 (q)
35.6 (d)
18.8 (q)
34.6 (t)
30.4 (t)
155.2 (s)
41.7 (d)
28.3 (t)
19.8 (q)
107.2 (t)
12.0 (q)
1.62 (m), 1.31 (m)
1.54 (m), 1.60 (m)
4.05 (quintet, 3.2)
1.66 (dd, 3.1, 14.9), 2.08 (brd)
–
9.63 (d, 2.8)
2.17 (dd, 2.8, 9.0)
2.04 (m)
1.22 (m)
–
1.32 (m), 1.38 (m)
1.06 (m), 2.00 (m)
–
2, 3, 4
1, 3, 4
1, 2, 4
3
26.8 (t)
28.0 (t)
67.4 (d)
44.3 (t)
84.3 (s)
204.6 (d)
64.0 (d)
40.1 (d)
50.5 (d)
45.5 (s)
21.6 (t)
39.7 (t)
44.8 (s)
56.2 (d)
24.6 (t)
28.3 (t)
55.7 (d)
12.5 (q)
18.4 (q)
35.6 (d)
18.7 (q)
36.2 (t)
23.8 (t)
39.5 (t)
28.0 (d)
22.5 (q)
22.8 (q)
–
7
6, 8, 9, 11, 12, 14
7, 9, 11, 12, 14
7, 8, 11, 12
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
7, 9, 11, 12
9, 11
1.12 (m)
7, 8, 15, 16
7, 9, 15, 16, 17
15, 17, 20, 21
15, 21, 22
–
1.04 (m), 1.40 (m)
1.22 (m), 1.80 (m)
1.08 (m)
0.65 (3H, s)
0.86 (3H, s)
–
1.36 (m)
15, 21, 22, 23
16, 17, 20, 22, 23
17, 20, 21, 23
20, 21, 22
–
26, 27, 29
25, 27, 29
25, 26, 29
–
0.87 (3H, d, 6.6)
1.08 (m), 1.47 (m)
1.74 (m), 1.98 (m)
–
1.93 (m)
1.25 (m), 1.37 (m)
0.92 (3H, d, 6.6)
4.61 (2H, brs)
0.76 (3H, t, 7.3)
25, 26, 27
–
a
The mixing time was set to 90 ms.
C
B
2
1
H2
H2
C
CH
CH 17
CH2
CH2
C
H3C 20
C
CH2
23
CH25 CH3
29
H3C
12
CH3
27
CH2
A
C
H2C
HC
H3C
C
H
C
14
1
CH2
8
HMBC COSY & TOCSY
H2C
C
H2
CH
13C
1H
CH
HO
C4 H2
C
CH7
CHO
OH
6
1
1
Figure 1. H– H COSY, TOCSY and HMBC correlations of 1.
The absolute configuration of 1 was determined by
1 and 2 and will be reported at a later date. To our
knowledge, 6-5-6-5 fused rings sterol was only found in
taiwaniasterols, which were isolated from the leaves of
comparison of the CD spectrum with that of KPN-2001
(
2), which is a synthetic analog of 1 prepared from
7
8
cholesterol (Scheme 1). The negative Cotton effects
Taiwania cryptomerioides.
were observed at uext 301 nm (Dm −34.0) in 1 and u
ext
3
02 nm (Dm −20.3) in 2. Since the absolute configuration
is 1, the same as that of natural cholesterol, accord-
ingly, the final structure of 1 was determined.
Acknowledgements
Orostanal (1) and KPN-2001 (2) induced apoptosis in
HL-60 cells at 10 mg/mL, and inhibited 50% cell growth
at 1.7 and 2.2 mM, respectively. Further work is in
progress on the mode of action for apoptosis caused by
This work was supported in part by a Grant-in-Aid for
Scientific Research (C) (No. 12672055) from The Min-
istry of Education, Science, Sports and Culture, Japan.

T. Miyamoto et al. / Tetrahedron Letters 42 (2001) 6349–6351
6351
4
.62(s)
0
.87(d)
H3C
CH2
CH3
0
.87(d)
1
9
11
N
CH3
0.92(d)
1
8
0.76(t)
H C
3
H
4.62(s)
CH2
CH
6
3
1
28
(25S)-24(28)-dehydroaplysterol
H
O
CH
H
H
2
H
HO
H
H8
H C
H
3
29
H
CH3
0.76(t)
H
20
25
27
5
7
H
CH3
H
H
H
14 H15
HO
H
H
0.92(d)
4.89(s)
CH2
3
H
H
0.87(d)
H3C
H
NOE
CH3
N
CH3
.93(d)
0
.76(t)
0
(25R)-24(28)-dehydroaplysterol
Figure 2. Relative stereochemistry of 1.
Scheme 1.
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2
1
972, 26, 239.
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Astrophorida, family Ancorinidae. A voucher specimen
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Compound 2: amorphous solid, [h] =+29.6° (c=0.30,
D
−
1
CHCl ), IR (CHCl , cm ) 3502, 2930, 2871, 1715, EIMS
3
3
4
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119.
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3
5
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D
−
1
CHCl ), IR (CHCl , cm ) 3502, 2930, 2871, 1715, 1638,
3
3
+
1
3
(
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1
13
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600 MHz) and C (150 MHz) NMR in CDCl , see Table
J=3.1, H-7), C NMR (600 MHz), see Table 1.
ext
13
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1998, 48, 1392–1397.
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