1286

Notes

Chem. Pharm. Bull. 50(9) 12861289 (2002)

Vol. 50, No. 9

New Biologically Active Marine Sesquiterpenoid and Steroid from the

Okinawan Sponge of the Genus Axinyssa
Makoto IWASHIMA,a,1) Ikuo TERADA,a Kazuo IGUCHI,*,a and Takao YAMORIb
a
School of Life Science, Tokyo University of Pharmacy and Life Science; Horinouchi, Hachioji, Tokyo 1920392, Japan:
and b Cancer Chemotherapy Center, Japanese Foundation for Cancer Research; Kami-ikebukuro, Toshima-ku, Tokyo
1708455, Japan. Received May 2, 2002; accepted June 3, 2002

A new bisabolane-type sesquiterpenoid, (E )-3-isocyanobisabolane-7,10-diene (1), and a new epidioxyergostane-type steroid, 9(11)-dehydroaxinysterol (2), were isolated from the Okinawan sponge of the genus
Axinyssa. Their structures were elucidated based on the results of spectroscopic analysis and chemical conversion. Epidioxysterol 2 was found to show significant growth inhibitory effects against human cancer cell lines.
Key words marine sponge; genus Axinyssa; bisabolane sesquiterpenoid; 5a ,8a -epidioxysterol

During the course of our investigations of biologically active chemical substances from the Okinawan invertebrates,24)
a bisabolane-type sesquiterpenoid (E)-3-isocyanobisabolane7,10-diene (1) and an ergostane-type steroid 9(11)-dehydroaxinysterol (2) were newly isolated from a sponge of the
genus Axinyssa along with known compounds such as
axinysterol (3).5) This paper describes the structural elucidation of these compounds, the lethal bioassay for brine
shrimp6) of 1, and the growth inhibitory activity against
human cancer cell lines7) of 2.
The isolation and purification were carried out as described in the Experimental section.
The molecular formula of 1 was found to be C16H25N by
high-resolution electron impact MS (HR-EI-MS). All the
carbons in 1 appeared in the 13C-NMR spectrum (Table 1).
The distortionless enhancement by polarization transfer
(DEPT) spectrum indicated four methyls, five sp3 methylenes, one sp3 methine, two sp2 methines, one sp3 quaternary
carbon, and three sp2 quaternary carbons. Two of the quaternary carbon signals at d 56.3 (JNC55.5 Hz) and 156.1
(JNC55.5 Hz) ppm in C6D6 were both observed as a triplet
due to the coupling with 14N.8) These findings together with
the IR absorption at 2130 cm21 strongly suggest the presence
of an isonitrile group (NC) in 1. The 1H-NMR analysis
showed the presence of three olefinic methyls at d 1.45 (s),
1.61 (s), and 1.71 (s) ppm, one methyl group connected to
quaternary carbon at d 1.00 (t, J52.0 Hz) ppm, and two
olefinic protons at d 5.14 (t, J57.1 Hz) and 5.27 (t, J5
7.1 Hz) ppm (Table 1). All the connections for each carbon
proton direct bonding were made from the analysis of 13C1H
correlation spectroscopy (13C1H COSY). Partial structures
NMR Data of 1 in C6D6 (d ppm)a)

Table 1.

No.
1
2
3
4
5
6
7
8
a)

of 1, as shown in Fig. 1, were deduced from 1H1H correlation spectroscopy (1H1H COSY).
The gross structure of 1 was established by the following
heteronuclear multiple bond correlation (HMBC) analysis
(Fig. 1). 1H13C long-range correlations observed from H-2
(CH2) and H-4 (CH2) to C-3, and from H-15 (CH3) to C-2,
C-3 and C-4, indicated the carboncarbon connections
around C-3. Correlations from H-8 (CH) to C-6, from H-9

13

26.7
38.4
56.3 (t, 5.5)b)
38.4
26.7
44.7
137.8
122.6

0.98 (dtd, 4.2, 11.4, 13.9), 1.35 (m)

1.49 (m), 1.54 (m)
1.49 (m), 1.54 (m)
0.98 (dtd, 4.2, 11.4, 13.9), 1.35 (m)
1.57 (m)
5.14 (t, 7.1)

No.
9
10
11
12
13
14
15
16

13

27.4
123.7
131.3
25.8
17.7
14.6
24.8
156.1 (t, 5.5)b)

2.78 (2H, t, 7.1)

5.27 (t, 7.1)
1.71 (3H, s)
1.61 (3H, s)
1.45 (3H, s)
1.00 (3H, t, 2.0)

13

C; 125 MHz. 1H; 500 MHz, J in Hz. Assignments of the 13C and 1H signals were based on HMQC. b) JNC in Hz. Coupled to 14N observed as 1 : 1 : 1.6)

To whom correspondence should be addressed.

e-mail: [email protected]

2002 Pharmaceutical Society of Japan

September 2002

1287
Table 2.

NMR Data of 2 and 3 in CDCl3 (d ppm)

2a)

No.
13

C (125 MHz)

Fig. 1.

Partial Structures and Important HMBC for 1

Fig. 2. Important NOE Correlations and trans-Diaxial Relationship between H-1, H-5, and H-6

(CH2) to C-7, and from H-14 (CH3) to C-7 and C-8 demonstrated the connections around C-7. Finally, correlations from
H-9 (CH2) to C-11, from H-10 (CH) to C-12 and C-13, and
from H-12 (CH3) and H-13 (CH3) to C-10 and C-11, indicated the terminal structure of the side chain in 1. These
spectroscopic findings together with the unsaturation number
(U55) disclosed that compound 1 had the structure of a bisabolane-type sesquiterpenoid with an isonitrile moiety at C3.810) This gross structure of 1 indicated that the molecule
of 1 has a plane of symmetry across the C-3 and C-6 positions on the cyclohexane ring, and thus 1 is achiral and optically inactive; [a ]D25 0.0 (CHCl3). However, the stereochemistry of the olefin moiety and the relative configuration between C-3 and C-6 must be determined.
Stereochemistry of the trisubstituted olefin between C-7
and C-8 was confirmed to be an E configuration by nuclear
Overhauser effect (NOE) analysis, as shown in Fig. 2. The
relative configuration between C-3 and C-6 was revealed by
the combined analysis of NOESY and J values observed in
1
H-NMR spectrum. As shown in Fig. 2, NOE correlations
between the methyl protons (H-15) at C-3 and one of the
methylene protons at both C-1 (H-1a) and C-5 (H-5a) [d 0.98
(dtd, J54.2, 11.4, 13.9 Hz)] indicated that H-15, H-1a, and
H-5a were directed to the same side of the cyclohexane ring.
The 1H1H large coupling constant (J511.4 Hz) of the triplet
signal of H-1a and H-5a demonstrated these protons to be
axial and to couple with the adjacent axial protons (H-2, H-6
for H-1 and H-4, H-6 for H-5). These findings indicated H-6
to be axially directed to the opposite side of H-15, H-1a, and
H-5a, and thus the side chain group at C-6 to be equatorially
directed, as shown in Fig. 2. The structure of 1 was thus concluded to be (E)-3-isocyanobisabolane-7,10-diene.
The biological activity of 1 was examined to estimate the
lethal concentration (LC50) for brine shrimp,6) and compound
1 showed strong activity at LC50 0.1 m g/ml. The other bioactivities for 1 are currently under investigation.
The molecular formula of compound 2, C28H40O3, was determined by HR-EI-MS analysis. IR, 1H-, and 13C-NMR
spectra of 2 were quite similar to those of axinysterol (3),5)
which had the molecular formula C28H42O3, suggesting 2 to

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

32.6
30.6
66.3
36.1
82.7
135.5
130.7
78.3
142.6
38.0
119.7
41.2
43.6
48.2
20.9
28.6
55.8
12.9
25.5
39.7
20.5
135.2
132.0
43.6
149.7
108.9
20.6
18.8

C (100 MHz)

1.68 (m), 2.06 (m)

1.57 (m), 1.92 (m)
4.00 (tt, 5.1, 11.4)
1.92 (dd, 11.4, 13.7), 2.10 (m)
6.28 (d, 8.5)
6.59 (d, 8.5)

5.42 (dd, 1.9, 6.0)

2.08 (m), 2.26 (dd, 6.0, 17.0)
1.83 (dd, 8.2, 12.3)
1.57 (m), 1.68 (m)
1.38 (m), 1.78 (m)
1.37 (m)
0.73 (3H, s)
1.09 (3H, s)
2.05 (m)
1.00 (3H, d, 6.6)
5.23 (dd, 7.8, 15.3)
5.29 (dd, 6.7, 15.3)
2.71 (quint, 6.7)
4.69 (m), 4.70 (m)
1.67 (3H, s)
1.08 (3H, d, 6.7)

a) Assignments of the 13C and 1H signals were based on

Values with the same subscript may be interchanged.

Fig. 3.

13

H (500 MHz, J in Hz)

13

34.8
30.2
66.6
37.1
82.3
135.4b)
130.8
79.5
51.2 (CH)
37.1
20.7 (CH2)c)
39.5
44.7
51.8
23.5c)
28.7c)
56.3
13.0
18.3
39.6
20.69d)
135.5b)
132.0
43.7
149.8
109.0
20.74d)
18.9

C1H COSY.

b, c, d )

Important NOE Correlations and Long-Range Coupling (Bold)

be a dehydro-congener of 3. Differences between 2 and 3

were found in the NMR spectra, due to the presence of a new
trisubstituted olefin at d 5.42 (dd, J51.9, 6.0 Hz) ppm in the
1
H-NMR, and at d 119.7 (CH) and 142.6 (C) ppm in the 13CNMR spectra of 2 (Table 2).
Two-dimensional NMR spectra [1H1H COSY, 1H-detected heteronuclear multiple quantum coherence (HMQC),
and HMBC] indicated that this new double bond is located
between the C-9 and C-11 positions. The relative configuration of 2 was determined based on the following evidence.
NOESY analysis focused on the ring junctures of the
steroidal skeleton confirmed that 2 has the same relative configuration as that of 3 (Fig. 3). The NOE correlation between
H-14 and H-12a , which showed long-range coupling (Wshape) with the angular methyl group (H-18) in the COSY
spectrum, as shown in the bold lines in Fig. 3, exhibited the
trans configuration between H-14 and H-18. The relative
configuration of H-17 was assigned as an a orientation due
to the NOE correlation between H-12a and H-17. The-a ori-

1288

Vol. 50, No. 9

of 2 in the presence of 10% palladium on carbon in ethyl acetate gave 4 {[a ]D25 113.3}. The authentic diol 4 {[a ]D25
110.6} was independently prepared from ergosterol in two
steps with photosensitized oxidation,5,12) in which the desired
epidioxysterol 5 was obtained as a minor product, followed
by catalytic hydrogenation. The spectral data of 4 derived
from 2 were identical to those of authentic 4, including the
sign of its optical rotation value. These findings together with
the spectral analysis confirmed the structure of this new
sterol 2 to be 9(11)-dehydroaxinysterol.
The growth inhibitory properties of 2 against cancer cells
were examined with a disease-oriented panel of 39 human
cancer cell lines (HCC panel) in the Japanese Foundation for
Cancer Research.7) Compound 2 exhibited a strong growth
inhibitory effect against some ovarian cancer cells such as
OVCAR-3 at IC50 0.19 m g/ml (log GI50 26.20) and OVCAR8 at IC50 0.22 m g/ml (log GI50 26.14), as shown in Table 3,
and also indicated significant growth inhibition in 21 human
cancer cell lines at less than 0.60 m g/ml. Estimation of its
bioactivities is now underway, including in vivo bioassay.
Chart 1

Table 3. Growth Inhibitory Activity (IC50) of 2 against Human Cancer

Cell Linesa)
Origin of
cancer
Breast

Lung

Stomach

Kidney

Cell line

IC50
(m g/ml)

HBC-4
BSY-1
HBC-5
MCF-7
MDA-MB-231
NCI-H23
NCI-H226
NCI-H522
NCI-H460
A549
DMS273
DMS114
St-4
MKN1
MKN7
MKN28
MKN45
MKN74
RXF-631L
ACHN

0.85
0.60
0.96
0.36
1.26
0.54
0.63
0.57
0.81
0.96
0.54
0.48
0.69
0.42
0.48
0.84
0.54
0.54
0.72
0.51

Origin of
cancer
Colon

Cell line

HCC2998
KM-12
HT-29
HCT-15
HCT-116
Ovary
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
CNSb)
U251
SF-268
SF-295
SF-539
SNB-75
SNB-78
Prostate
DU-145
PC-3
Melanoma LOX-IMVI

IC50
(m g/ml)
0.57
0.60
0.57
0.75
0.48
0.19
0.60
0.54
0.22
0.81
0.63
1.02
0.75
0.84
2.16
1.17
0.54
0.57
0.60

a) Each IC50 value was estimated from its GI50 value. b) CNS, central nervous
system.

entation of the epidioxy group was determined by the NOE

correlations between H-18 and H-7 (olefinic proton). The relative configuration of the angular methyl group at C-10 was
indicated by the NOE correlation between H-6 (olefinic proton) and H-19, and the stereochemistry at C-3 was deduced
from the J values of H-3 (J55.1, 11.4 Hz). The stereochemistry of two chiral centers in the side chain was assigned by
comparison of the 13C-NMR data from C-20 to C-28 with
those in 3 (Table 2). The chemical shift values of nine carbons in 2 were essentially identical to those of 3.
The following chemical conversions5) from 2 to the known
diol 411) were carried out to establish the absolute configuration of 2, as summarized in Chart 1. Catalytic hydrogenation

Experimental
Optical rotations were measured with a JASCO DIP-370 automatic polarimeter. IR spectra were recorded with a Perkin-Elmer FT-IR 1600 spectrophotometer. NMR spectra were recorded with a Bruker DRX-500 and
DPX-400 spectrometer (1H, 500 and 400 MHz; 13C, 125 and 100 MHz) in
C6D6 for 1 and in CDCl3 for 2, respectively. Two-dimensional NMR spectra
(1H1H COSY, 13C1H COSY, HMQC, HMBC, and NOESY) were measured using standard Bruker pulse sequences. Chemical shifts are given on a
d (ppm) scale with either CHCl3 (1H, 7.26 ppm; 13C, 77.0 ppm) for CDCl3 or
C6H6 (1H, 7.20 ppm; 13C, 128.0 ppm) for C6D6 as the internal standard (s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad). Mass spectra
were recorded with a Micromass Auto Spec spectrometer. Reverse-phase
ODS column chromatography was carried out on YMC S-50 gel (ODSAM120-S50). Normal-phase flash column chromatography was performed
on Merck silica gel 60 (230400 mesh). Medium-pressure liquid chromatography (MPLC) was carried out with a Kusano KHLC-201-43 apparatus using a CIG prepack column (silica gel, CPS-HS-221-05, for normal
phase and ODS silica gel, CPO-HS-221-20, for reverse phase). HPLC with a
recycling loop was performed on a YMC-Pack SIL-06 column (silica gel,
SH-043-5-06, normal phase) and a YMC-Pack ODS-AM column (ODS,
SH-343-5AM, reverse phase).
Extraction and Isolation The sponge of the genus Axinyssa (order
Halichondria, family Desmospongiae) was collected from a coral reef near
Ishigaki Island, Okinawa Prefecture, Japan in June 1999, at a depth of 5
10 m. A voucher specimen (No. S-99-3) was deposited at the Tokyo University of Pharmacy and Life Science, Tokyo, Japan. Wet specimens (706 g)
were immersed in MeOH (332 l). After filtration, MeOH was removed
under reduced pressure. The MeOH extract (47.5 g) was partitioned between
hexane and H2O, and the hexane layer was concentrated under reduced pressure to give a hexane-soluble portion (11.4 g). Most of the hexane-soluble
portion (11.3 g) was chromatographed on an ODS column (200 g). Stepwise
elution with MeOHH2O (9 : 1 for five fractions, 19 : 1 for two fractions,
200 ml of each) and MeOH (400 ml) gave eight fractions. The fifth fraction
(1.23 g) [eluted with MeOHH2O (9 : 1)] containing sesquiterpenoids and
the final fraction (1.20 g) containing sterols were obtained. Each fraction
was subjected to normal-phase silica gel flash column chromatography, followed by MPLC separation to obtain almost pure 1 and 2, respectively. Further purification was carried out with recycled HPLC to afford 1 [11.8 mg,
using a normal-phase column, hexane2-propanol (20 : 1)] and 2 [13.4 mg,
using a reverse-phase column, MeOHH2O (19 : 1)]. Axinysterol (3,
339 mg) and some sesquiterpenoids possessing a bisabolane skeleton were
also obtained in this separation.
(E)-3-Isocyanobisabolane-7,10-diene (1): Colorless oil. [a ]D25 0.0
(c50.75, CHCl3). IR (dry film) n max cm21: 2130. 1H- and 13C-NMR, see
Table 1. HR-EI-MS m/z: 230.1912 (Calcd for C16H25N: 230.1909 [M2H]1).
9(11)-Dehydroaxinysterol (2): White amorphous solid. [a ]D25 178.9
(c50.89, CHCl3). IR (dry film) n max cm21: 3389, 2962, 1644. 1H- and 13CNMR, see Table 2. HR-EI-MS m/z: 424.2971 (Calcd for C28H40O3: 424.2977
[M]1).

September 2002
Catalytic Hydrogenation of 2 to 4 A mixture of 2 (0.6 mg) and 10%
palladium on charcoal (2 mg) in ethyl acetate (0.5 ml) was vigorously stirred
under a hydrogen atmosphere at room temperature for 3.5 h. The mixture
was filtered through a short Celite column, rinsed with ethyl acetate, and the
filtrate was concentrated under reduced pressure. The residue was purified
by silica gel flash column chromatography [hexaneethyl acetate (3 : 1)] and
HPLC [hexane2-propanol (9 : 1)] to obtain diol 4 (0.6 mg, 100% yield) as a
white solid. [a ]D25 113.3 (c50.06, CHCl3).11) IR (dry film) n max cm21:
3406, 3311, 2926. 1H-NMR (400 MHz, CDCl3) d ppm: 0.62 (3H, s, H-18),
0.78 (3H, d, J56.8 Hz, H-27), 0.79 (3H, d, J56.8 Hz, H-26), 0.83 (3H, d,
J56.9 Hz, H-28), 0.94 (3H, d, J56.5 Hz, H-21), 1.13 (3H, s, H-19), 4.12
(1H, tt, J55.2, 10.6 Hz, H-3). 13C-NMR (100 MHz, CDCl3) d ppm: 11.0,
15.5, 17.6, 18.9, 20.5, 23.1, 23.3, 23.4, 23.5, 28.7, 29.5, 29.7, 30.7, 31.0,
31.5, 33.7, 36.7, 36.7, 39.1, 40.8, 41.9, 42.1, 51.6, 54.6, 67.2, 74.3, 129.5,
132.8. HR-EI-MS m/z: 398.3534 (Calcd for C28H46O: 398.3549 [M2
H2O]1).
Preparation of 4 from Ergosterol The oxidation reaction was carried
out according to the procedure described in the literature.5,12) The mixture
of products was separated by silica gel flash column chromatography
[hexaneethyl acetate (4 : 1)], followed by MPLC separation [reverse phase,
MeOHH2O (9 : 1)] to afford 9(11)-dehydro-5a ,8a -epidioxyergosterol (5,
25 mg, 12% yield) as a faint yellow solid. A mixture of 5 (4.9 mg) and 10%
palladium on charcoal (5 mg) in ethyl acetate (3 ml) was vigorously stirred
under a hydrogen atmosphere at room temperature for 3 h. The mixture was
filtered through a short Celite column, and the filtrate was concentrated
under reduced pressure. The residue was purified by silica gel flash column
chromatography [hexaneethyl acetate (1 : 1)], followed by HPLC separation
[normal phase, hexane2-propanol (9 : 1)] to obtain diol 4 (2.8 mg, 59%
yield) as a white solid. [a ]D25 110.6 (c50.11, CHCl3). The spectral data of
4 prepared by this method were identical to those of 4 from natural 2.
Acknowledgments The authors thank Dr. Y. Shida at the Tokyo Univer-

1289
sity of Pharmacy and Life Science for measurement of mass spectra.
References and Notes
1) Present address: Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University,
2630 Sugitani, Toyama 9300194, Japan.
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29772981 (2002).
3) Watanabe K., Tsuda Y., Iwashima M., Iguchi K., J. Nat. Prod., 63,
258260 (2000).
4) Iwashima M., Nara K., Nakamichi Y., Iguchi K., Steroids, 66, 2532
(2001).
5) Iguchi K., Shimura H., Yang Z., Yamada Y., Steroids, 58, 410413
(1993).
6) Meyer B. N., Ferrigni N. R., Putnam J. E., Jacobsen L. B., Nichols D.
E., McLaughlin J. L., Planta Med., 45, 3134 (1982).
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40424049 (1999).
8) Gulavita N. K., de Siva E. D., Hagadone M. R., Karuso P., Scheuer P.
J., J. Org. Chem., 51, 51365139 (1986).
9) Kassuhlka K. E., Potts B. C. M., Faulkner D. J., J. Org. Chem., 56,
37473750 (1991) and references cited therein.
10) Sullivan B. W., Faulkner D. J., Okamoto K. T., Chen M. H. M., Clardy
J., J. Org. Chem., 51, 51345136 (1986).
11) Hallsworth A. S., Henbest H. B., Wrigley T. I., J. Chem. Soc., 1957,
19691981 (1957). This paper described the optical rotation of 4 as
[a ]D 134 without any information about either the concentration or
the solvent.
12) Windaus A., Brunken J., Ann. Chem., 460, 225235 (1928).