(25S)-Cholesten-26-oic acid derivatives from an Indonesian soft coral Minabea sp.

Article abstract of DOI:10.1016/j.steroids.2009.04.002

(25S)-3-Oxocholesta-1,4-dien-26-oic acid (1) and a new (25S)-18-acetoxy-3-oxocholesta-1,4-dien-26-oic acid (2) were isolated from a soft coral Minabea sp. (cf. aldersladei) collected in North Sulawesi, Indonesia, together with two known cholic-acid-type c

Full text of DOI:10.1016/j.steroids.2009.04.002

Steroids 74 (2009) 758–760
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Steroids
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(25S)-Cholesten-26-oic acid derivatives from an Indonesian
soft coral Minabea sp.
Weifang Wanga, Jong-Soo Leea,1, Takahiro Nakazawaa, Kazuyo Ukaia, Remy E.P. Mangindaanb,
Defny S. Wewengkanga,b, Henki Rotinsulub, Hisayoshi Kobayashic,
Sachiko Tsukamotod, Michio Namikoshia,∗
a Department of Natural Product Chemistry, Tohoku Pharmaceutical University, Aoba-ku, Sendai 981-8558, Japan
b Faculty of Fisheries and Marine Science, Sam Ratulangi University, Kampus Bahu, Manado 95115, Indonesia
c Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
d Graduate School of Science, Chiba University, Inage-ku, Chiba 263-8522, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
(25S)-3-Oxocholesta-1,4-dien-26-oic acid (1) and a new (25S)-18-acetoxy-3-oxocholesta-1,4-dien-26-
oic acid (2) were isolated from a soft coral Minabea sp. (cf. aldersladei) collected in North Sulawesi,
Indonesia, together with two known cholic-acid-type compounds, 3-oxochol-1,4-dien-24-oic acid (3)
and 3-oxochol-4-en-24-oic acid (4). The structures of these compounds were determined on the basis of
their spectroscopic data. The absolute stereochemistry at C-25 of 2 was determined by comparative 1H
NMR study using chiral anisotropic reagents [(S)- and (R)-phenylglycine methyl esters]. This is the first
to report compound 1 as a natural product.
Received 26 February 2009
Received in revised form 6 April 2009
Accepted 7 April 2009
Available online 16 April 2009
Keywords:
Soft coral
Minabea sp.
© 2009 Elsevier Inc. All rights reserved.
(25S)-3-Oxocholesten-26-oic acid
3-Oxocholen-24-oic acid
Steroidal carboxylic acids
1. Introduction
2. Experimental
Marine organisms produce steroidal components with variety
of structures [1]. One of the interesting properties of structures is a
high degree of oxidation, and steroidal carboxylic acids and deriva-
tives have been detected in marine organisms more frequently
than terrestrial organisms. Occurrence of steroidal carboxylic acid
derivatives, which were sometimes oxygenated at various sites, has
been shown in starfish, nudibranches, sponges, and, especially, soft
corals. Soft corals are a rich source of biologically active natural
products [1]. In the course of our studies on bioactive metabolites of
marine organisms, we isolated four steroidal carboxylic acids (1–4,
Fig. 1) from a soft coral Minabea sp. collected in Indonesia and found
that 2 was a new compound and that 1 was obtained for the first
time as a natural product. We describe herein the isolation and
structure elucidation of compounds 1 and 2.
2.1. General procedures
NMR spectra were measured on a JEOL JNM-AL-400 or JNM-LA-
600 NMR spectrometer in CDCl3 (ıH 7.24, ıC 77.0). Mass spectra
were obtained by a JEOL JMS-MS 700 mass spectrometer (EI or
FAB mode). UV and IR spectra were recorded on HITACHI U-3310
and on Perkin-Elmer Spectrum One FT-IR spectrometers, respec-
tively. Optical rotations were recorded with a JASCO DIP-370 digital
polarimeter. Fetal bovine serum (FBS) was obtained from GIBCO
after checking the lot, and all other reagents and chemicals for
bioassays were of the highest grade available commercially.
2.2. Organism, extraction, and isolation
Minabea sp. (most likely Minabea cf. aldersladei) was collected
by scuba diving at the Lembeh Strait, Indonesia. The voucher spec-
imen is deposited at the Faculty of Fisheries and Marine Science,
Sam Ratulangi University as 04-09-30 = 2-1a. The soft coral was
immediately cut into small pieces and soaked in ethanol on the
boat. The EtOH extract was evaporated and the residue was parti-
tioned between n-hexane (200 mL) and MeOH–H2O (9:1, 200 mL).
∗
Corresponding author. Tel.: +81 22 727 0219; fax: +81 22 727 0219.
E-mail address: mnami@tohoku-pharm.ac.jp (M. Namikoshi).
On leave from Division of Marine Life Science, Gyeongsang National University,
1
Tongyeong, Kyungnam, Korea 650-160.
0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.04.002

W. Wang et al. / Steroids 74 (2009) 758–760
759
2.2.2. (25S)-18-Acetoxy-3-oxocholesta-1,4-dien-26-oic acid (2)
White powder. [˛D25] + 13.2◦ (c 0.11, CHCl3); UV (MeOH) ꢀmax nm
(log ε) 243 (4.14); IR (KBr) ꢁmax 2945, 1737, 1600, and 1238 cm−1
;
1H and 13C NMR, see Table 1; EIMS m/z 470 [M]+, 410, 397, 289, 267,
173, 147, and 121. HREIMS: m/z 470.3042 (M+, calcd. for C29H42O5,
470.3033).
2.3. Reaction of 2 with (S)- and (R)-phenylglycine methyl esters
(PGMEs) [4,5]
Compound 2 (0.6 mg) and (S)-PGME hydrochloride (5.2 mg)
were dissolved in DMF (0.5 mL), and PyBOP (2.6 mg), HOBT (2.8 mg),
and N-methylmorpholine (100 ␮L) were successively added to the
solution at 0 ◦C. After stirred at room temperature for 4 h, EtOAc
(10 mL) was added to the reaction mixture, and the solution was
washed successively with 7% HCl (10 mL 2×), saturated NaHCO3
(10 mL 2×), and saturated NaCl (10 mL 2×), dried (Na2SO4), and
evaporated. The residue was purified by HPLC (ODS, gradient elu-
tion with 50–100% MeOH in H2O) to give the (S)-PGME amide
(0.2 mg).
Fig. 1. Structures of compounds 1–4.
The aqueous MeOH layer was concentrated, dissolved in water, and
extracted successively with EtOAc and n-BuOH. The EtOAc extract
showed activity against L1210 and V79 cells and was separated on a
SiO2 column with CHCl3–MeOH (gradient elution) into six fractions.
The third fraction (50 mg) was subjected to Sephadex LH-20 column
chromatography with CHCl3–MeOH (1:1) and then with MeOH
followed by reversed-phase HPLC (ODS, 80% MeOH–H2O contain-
ing 0.05% trifluoroacetic acid) to yield compounds 1 (1.5 mg), 2
(1.1 mg), 3 (11 mg), and 4 (1.2 mg). The same fraction also gave two
known cytotoxic briareine-type diterpenes, minabein-4 (1.0 mg)
and minabein-6 (1.4 mg) [2,3].
The (R)-PGME amide (0.2 mg) was prepared from 0.6 mg of 2 and
(R)-PGME hydrochloride in a similar procedure as above.
2.3.1. (S)-PGME amide of 2
1H NMR data (600 MHz, CDCl3): ı 7.01 (1H, d, J = 10.2 Hz, H-1),
6.21 (1H dd, J = 10.2, 1.8 Hz, H-2), 6.05 (1H, s, H-4), 4.22 (1H, d,
J = 11.7 Hz, H-18a), 3.93 (1H, d, J = 11.7 Hz, H-18b), 2.07 (3H, s, -OAc),
1.20(3H, s H3-19), 1.10(3H, d, J = 6.6 Hz, H3-27), 0.96(3H, d, J = 6.2 Hz,
H3-21), 7.25–2.35 (5H, m, Ph of PGME), 6.37 (1H, d, J = 7.3 Hz, NH
of PGME), 5.56 (1H, d, J = 7.3 Hz, CH of PGME), 3.71 (3H, s, OMe of
PGME).
2.2.1. (25S)-3-Oxocholesta-1,4-dien-26-oic acid (1)
White powder. [˛D25] + 5.3◦ (c 0.16, CHCl3); UV (MeOH) ꢀmax nm
(log ε) 244 (4.07); IR (KBr) ꢁmax 2945, 1706, and 1662 cm−1 1H
;
and 13C NMR data, see Table 1; FABMS (glycerol): m/z 413 [M+H]+;
HREIMS: m/z 412.2972 (M+, calcd. for C27H40O3, 412.2977).
2.3.2. (R)-PGME amide of 2
1H NMR data (600 MHz, CDCl3): ı 7.01 (1H, d, J = 10.3 Hz, H-1),
6.21 (1H dd, J = 10.3, 2.2 Hz, H-2), 6.05 (1H, s, H-4), 4.20 (1H, d,
J = 11.7 Hz, H-18a), 3.90 (1H, d, J = 11.7 Hz, H-18b), 2.05 (3H, s, -OAc),
1.20 (3H, s H3-19), 1.13 (3H, d, J = 7.0 Hz, H3-27), 0.89 (3H, d, J = 6.6 Hz,
H3-21), 7.26–2.34 (5H, m, Ph of PGME), 6.40 (1H, d, J = 7.0 Hz, NH
of PGME), 5.57 (1H, d, J = 7.0 Hz, CH of PGME), 3.71 (3H, s, OMe of
PGME).
Table 1
1H (400 M) and 13 C (100 M) NMR dataa for compounds 1 and 2 in CDCl3.
1
2
ıC
ıH (J in Hz)
ıC
ıH (J in Hz)
1
2
3
4
5
6
7
8
156.4
127.3
186.7
123.7
170.0
33.0
33.7
35.5
52.4
43.8
22.9
39.5
42.7
55.4
24.4
28.1
56.0
12.0
18.7
35.6
18.5
35.7
23.7
34.0
39.1
180.8
17.0
–
7.05 d (10.0)
6.22 dd (10.0, 2.0)
–
6.07 brs
–
2.35 m, 2.46 m
1.10 m, 1.93 m
1.60 m
1.02 m
–
1.60 m, 1.79 m
1.14 m, 2.01 m
–
156.2
127.4
186.7
123.8
169.6
32.8
33.7
35.7
52.3
43.7
22.8
34.7
45.4
55.0
24.1
27.7
56.2
62.6
18.8
35.7
18.7
35.7
23.4
34.0
39.0
180.5
17.1
7.04 d (10.0)
6.24 dd (10.0, 2.0)
–
6.08 brs
–
2.34 m, 2.45 m
1.10 m, 1.93 m
1.59 m
1.04 m
–
2.4. Antimicrobial assay
Compounds 1–4 were tested for antimicrobial activity against
Staphylococcus aureus IAM 12544T, Escherichia coli IAM 12119T,
Saccharomyces cerevisiae IAM 14383T, and Mucor hiemalis IAM
6088. Test compounds were dissolved in methanol or ethanol and
40 ␮L of each solution was absorbed on a disk (8 mm in diam-
eter). After incubation, diameters of the inhibition zones were
measured.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
OAc
1.59 m, 1.70 m
1.06 m, 2.40 m
–
2.5. Cytotoxicity testing
0.98 m
1.15 m
1.17 m, 1.58 m
1.23 m, 1.81 m
1.10 m
0.71 s
1.21 s
1.15 m, 1.64 m
1.38 m, 1.86 m
1.09 m
4.22 d (11.7), 3.94 d (11.7)
1.21 s
Cytotoxicity of compounds 1–4 was tested against Chinese ham-
ster V79 and murine leukemia L1210 cell lines. V79 cells were grown
as a monolayer culture in Eagle’s MEM (Nissui Seiyaku Co., Ltd.,
Tokyo, Japan) with 10% heat-inactivated FBS. The relative plating
efficiencies against V79 cells were determined as the ratio of the
number of colonies in various concentrations of samples to that in
the sample-free control, as described in previous papers [6,7]. Two
hundred cells were seeded on a 60/15-mm plastic plate with 4 mL
culture medium and incubated overnight at 37 ◦C. After each sam-
ple in DMSO (4 ␮L) was added to the culture medium, cells were
further cultured for four days. The numbers of colonies in the sam-
ple plates were counted and compared with those in the control
cultures.
1.38 m
1.42 m
0.88 d (6.6)
0.99 m, 1.35 m
1.15 m, 1.33 m
1.37 m, 1.58 m
2.44 m
0.97 d (6.3)
0.99 m, 1.35 m
1.14 m, 1.34 m
1.36 m, 1.58 m
2.45 m
–
–
1.16 d (6.8)
–
–
1.17 d (7.1)
–
2.07 s
171.4
21.1
–
a
Assigned by 1H–1H COSY, HMQC, and HMBC experiments.

760
W. Wang et al. / Steroids 74 (2009) 758–760
The 1H NMR shift differences (ꢂı = ı(S) − ı(R)) between (S)- and
(R)-PGME amide derivatives of 2 were calculated following the
Kusumi’s method [4,5], and the ꢂı value of −0.03 was detected
for the H3-27 signals. On the contrary, signals due to H-18a, H-18b,
OAc, and H3-21 showed ꢂı values of +0.02, +0.03, +0.02, and +0.07,
respectively. Consequently, the absolute configuration at the C-25
position of 2 was determined as (S), and on the usual biosynthetic
precedents, 1 was assigned to have the (25S)-configuration.
Antimicrobial activity against Gram-positive (S. aureus) and
negative bacteria (E. coli), yeast (S. cerevisiae), and a filamentous
fungus (M. hiemalis) and cytotoxicity against V79 and L1210 cells
were examined, and compounds 1–4 showed no apparent activ-
ity at 100 ␮g/disk (antimicrobial), 10 ␮M (V79 cells), and 50 ␮g/mL
(L1210).
Growth inhibitory activity of compounds 1–4 against L1210 cells
was tested in 96-well plastic plates by XTT [2,3-bis(2-methoxy-4-
nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] (cell prolif-
eration kit II®). Compounds were dissolved in MeOH and 10 ␮L of
each sample solution was poured in a well and the solvent evapo-
rated in a clean bench. The suspension of L1210 cells in RPMI 1640
medium (4 × 104 cells/mL, 100 ␮L) was added into each well and the
number of vital cells in the sample wells after 72 h was compared
with those in the control (MeOH) wells.
3. Results and discussion
As a part of our investigation on biologically active metabolites
from marine micro- and macro-organisms, an Indonesian soft coral
Minabea sp. was studied since the EtOAc extract showed cytotox-
icity against L1210 and V79 cells. HPLC separation of the active
fraction afforded two bioactive known compounds, minabein-4 and
minabein-6 [2,3], and four steroidal carboxylic acids 1–4 (Fig. 1).
Structures of two known compounds 3 and 4 were assigned on the
basis of their spectroscopic data and comparison with the reported
values for 3-oxochol-1,4-dien-24-oic acid (3) [8,9] and 3-oxochol-
4-en-24-oic acid (4) [10,11].
Marine organisms will attract our attention as a rich source of
novel steroidal components with interesting biological activities
[1].
Acknowledgements
We thank Dr. T. Oda of Keio University for the cytotoxicity
bioassay against V79 cells. This work was supported in part by
Grant-in-Aid for Scientific Research (No. 18032033) and for Scien-
tific Research on Priority Areas (No. 17035029) from the Ministry
of Education, Culture, Sports, Science and Technology (MEXT) of
Japan. One of us (J.-S. L.) appreciates the Japan Society for the Pro-
motion of Science (JSPS) for financial support to study in Japan (The
Invitation Fellowship for Research in Japan, Long-Term).
Compound 1 showed the [M+H]+ ion at m/z 413 in the FABMS,
and the molecular formula C27H40O3 was determined from HREIMS
and NMR data. The 1H NMR spectrum of 1 revealed four methyl sig-
nals at ı 0.71 (3H, s), 0.88 (3H, d, J = 6.6 Hz), 1.16 (3H, d, J = 6.8 Hz),
and 1.21 (3H, s) ascribable to methyl groups of a steroidal com-
pound. A 1,4-dien-3-one structure at the A ring was deduced from
the analysis of NMR data for 1 (Table 1) and confirmed by compar-
ison of IR, UV, and NMR data for 1 with those for 3. Compound 1
had three more carbons than 3, and the difference was observed
at the side chain. The presence of an ␣-methylcarboxylic acid moi-
ety in 1 was determined from 1H–1H COSY and HMBC spectra of
1. The cholestane structure was assigned from NOE correlations,
which were detected between H-4/H-6, H3-19/H-1, H3-19/H-8, H3-
19/H-11, H3-18/H-8, H3-18/H-11, H3-18/H-20, and H3-21/H-17 in
the NOESY spectrum of 1. The structure of 1 was, therefore, eluci-
dated as 3-oxocholesta-1,4-dien-26-oic acid. The Me ester of 1 has
been isolated from an Antarctic soft coral Anthomastus bathyproc-
tus [12], and the reported 13C NMR data for this compound were
very similar to those for 1 (ꢂı: −0.1 to +0.6 ppm) except for the
signal due to C-26, which was observed at ıC 180.8 (ꢂı: +3.4 ppm)
in the spectrum of 1. Compound 1 was obtained as one of degra-
dation products from cholesterol by Pseudomonas sp. NCIB 10590
under aerobic conditions, but NMR data and the stereochemistry at
C-25 were not reported [8]. This is the first instance to show 1 as a
natural product.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2009.04.002.
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