New spirocyclic sesquiterpenes from the marine sponge Geodia exigua

Article abstract of DOI:10.1016/S0040-4020(02)01619-8

Three new spirocyclic sesquiterpenes designated exiguamide (1), exicarbamate (2) and exigurin (3), together with (-)-10-epi-axisonitrile-3 (4), have been isolated from the marine sponge Geodia exigua. All four compounds possess the spiro[4.5]decene skeleton and their structures were determined on the basis of spectroscopic data. The structure of 1 was confirmed by X-ray crystallographic analysis and the absolute configuration was determined by applying the modified Mosher's method on its amine derivative. Exiguamide (1) inhibited cell fate specification during sea urchin embryogenesis at a minimum inhibitory concentration of 0.4μM.

Full text of DOI:10.1016/S0040-4020(02)01619-8

Tetrahedron 59 (2003) 731–736
New spirocyclic sesquiterpenes from the marine sponge
Geodia exigua
b
b
a
Mylene M. Uy,a Shinji Ohta,b Mihoko Yanai, Emi Ohta, Toshifumi Hirata
,
*
and Susumu Ikegamib
,
*
aDepartment of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama,
Higashi-Hiroshima 739-8526, Japan
bInstrument Center for Chemical Analysis, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan
Received 25 November 2002; revised 12 December 2002; accepted 16 December 2002
Abstract—Three new spirocyclic sesquiterpenes designated exiguamide (1), exicarbamate (2) and exigurin (3), together with (2)-10-epi-
axisonitrile-3 (4), have been isolated from the marine sponge Geodia exigua. All four compounds possess the spiro[4.5]decene skeleton and
their structures were determined on the basis of spectroscopic data. The structure of 1 was confirmed by X-ray crystallographic analysis and
the absolute configuration was determined by applying the modified Mosher’s method on its amine derivative. Exiguamide (1) inhibited cell
fate specification during sea urchin embryogenesis at a minimum inhibitory concentration of 0.4 mM. q 2003 Elsevier Science Ltd. All rights
reserved.
1. Introduction
The sea urchin embryo provides an excellent model to study
the molecular mechanisms of cell fate specification.1 In the
fourth cleavage during the early development of sea urchins,
eight blastomeres of the same size divide unequally into
four larger macromeres, four smaller micromeres, and eight
mesomeres. Micromeres give rise to spiculogenetic primary
mesenchyme cells at later developmental stages. In the
course of our search for selective inhibitors of echinoderm
embryonic development from marine organisms,2–10 we
found that the methanolic extract of Geodia exigua Thiele
inhibited the formation of micromeres in sea urchin
embryos. A preliminary examination of the bioactive
hexane-soluble extract led to the isolation of a new
spirocyclic sesquiterpene designated exiguamide (1).11
Further investigation of the extract has resulted in the
isolation of two new analogous sesquiterpenes designated
exicarbamate (2) and exigurin (3) in addition to (2)-10-epi-
2. Results and discussion
The MeOH extract of the sponge G. exigua was partitioned
between hexane and H2O. Subjection of the bioactive
hexane-soluble fraction to ODS column chromatography
with MeOH–H2O gradient mixtures afforded several
fractions. Further individual purification of three fractions
on silica gel column employing 0–100% EtOAc in hexane
as eluent furnished compounds 1–4.
axisonitrile-3 (4). We now report on the isolation, structure
elucidation and biological activities of these sesquiterpenes.
Exiguamide (1) was obtained as colorless crystals (mp 139–
1408C) upon crystallization from MeOH–H2O. Its molecu-
lar formula, C16H27NO, was determined by high resolution
FAB mass spectrometry (HRFABMS) (m/z 250.2174
[MþH]þ, D þ0.3 mmu). The IR spectrum showed the
(1657 cm21) functionalities. Analysis of the 1H NMR
Keywords: biologically active compounds; natural products; spiro
compounds; sponges.
presence of NH (3323 cm21) and amide and CvC
*
Corresponding authors. Tel.: þ81-824-24-7487; fax: þ81-824-24-7486;
e-mail: ohta@sci.hiroshima-u.ac.jp
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01619-8

732
M. M. Uy et al. / Tetrahedron 59 (2003) 731–736
Table 1. 1H NMR spectral data of 1–4
Position
1a
2b
3b
4b
1
2
3a
3b
4a
4b
6
6-NH
7
8ax
8eq
9ax
9eq
10
11
12
5.58 br s
5.53 br s
5.67 br s
2.82 m
2.14 ddd (15.6, 8.8, 2.5)
2.23 ddd (12.2, 7.5, 2.5)
1.64 m
4.48 dd (11.1, 3.5)
7.56 d (11.1)
1.36 m
1.02 m
1.66 m
1.84 m
1.43 m
1.71 m
1.55 m
0.98 d (6.6)
1.12 d (6.6)
1.68 br s
5.25 br s
2.34 ddd (15.8, 8.8, 7.5)
1.99 br dd (15.8, 8.8)
1.80 ddd (12.6, 7.5, 2.1)
1.42 ddd (12.6, 8.8, 8.8)
3.82 dd (10.5, 3.5)
7.62 br d (10.5)
1.11 m
1.31 m
1.56 m
1.74 tt (13.5, 5.4)
1.44 m
1.47 m
1.30 m
0.86 d (6.5)
0.68 d (6.5)
1.68 br s
0.95 d (7.5)
8.08 br s
2.74 ddd (15.8, 8.7, 7.6)
2.09 ddd (15.8, 8.7, 2.1)
2.27 ddd (12.5, 7.6, 2.1)
1.60 ddd (12.5, 8.7, 8.7)
4.08 dd (10.5, 3.5)
4.59 d (10.5)
1.26 m
0.79 qd (13.5, 3.5)
1.46 m
1.69 tt (13.5, 3.5)
1.32 m
1.57 m
1.40 m
0.88 d (6.6)
2.11 ddd (16.4, 8.1, 8.1)
1.95 ddd (16.4, 9.0, 2.9)
2.28 ddd (13.0, 8.1, 2.9)
1.82 ddd (13.0, 9.0, 8.1)
3.51 br s
0.96 m
1.49 m
1.49 m
1.56 m
1.42 m
1.55 m
1.72 m
0.86 d (6.6)
0.82 d (6.6)
1.51 br s
1.29 d (7.8)
13
14
15
16
1.05 d (6.6)
1.62 br s
0.87 d (7.6)
1.14 d (7.5)
16-OCH3
17
17-NCH3
18
19-OCH3
3.55 s
3.05 s (2H)
2.09 s
2.85 s (2H)
3.28 s
Coupling constants, JH–H (in Hz), are given in parentheses.
a
Data measured in DMSO-d6 at 500 MHz.
Data measured in C6D6 at 500 MHz.
b
(Table 1), 13C NMR (Table 2), 1H–1H COSY, HMQC and
HMBC spectral data revealed 1 to be a spiro[4.5]decene
having the formylamino, isopropyl and two methyl groups,
thus allowing the gross structure of 1. The relative
stereochemistry of 1 was elucidated on the basis of
difference NOE experiments and confirmed by X-ray
crystallographic analysis (Fig. 1).11
[MþH]þ, D 20.9 mmu). Analyses of the 1H and 13C NMR
spectral data revealed that 2 possessed the same spiro[4.5]-
decene skeleton as 1 differing only on the functional group
attached to C-6 and that the formylamino moiety in 1 was
replaced by the methoxycarbonylamino functionality which
was supported by IR absorption bands at 3366 (N–H) and
1711 cm21 (CvO). Detailed analysis of the 1H–1H COSY,
HMQC and HMBC (Fig. 2) spectral data allowed
determination of the gross structure of 2. The relative
stereochemistry of 2 was elucidated on the basis of
difference NOE experiments. An enhancement of signals
of three protons on C-15 and one of the protons on C-8
(H-8ax) upon irradiation of the NH proton indicated that
they were all in cis 1,3-diaxial positions relative to one
another around the six-membered ring, and defined a chair
conformation for the cyclohexane ring, as shown in Figure
3. Irradiation of the olefinic proton (H-1) enhanced the
signals of H-6, H-7, H-9ax, H-10 and H3-14, placing these
protons on the same face of the cyclohexane ring. These
Exicarbamate (2) had a molecular formula of C17H29NO2,
which was determined by (þ)-HRFABMS (m/z 280.2267
Table 2. 13C NMR spectral data of 1–4
Position
1a
2b
3b
4b
1
2
3
4
5
6
7
8
131.77 d
139.29 s
34.67 t
33.76 t
56.16 s
50.33 d
43.37 d
19.41 t
29.42 t
36.34 d
28.99 d
21.05 q
20.36 q
16.78 q
15.87 q
161.16 d
131.62 d
140.88 s
35.38 t
34.48 t
56.96 s
55.33 d
44.36 d
19.92 t
29.75 t
37.11 d
29.87 d
21.21 q
21.15 q
16.99 q
16.59 q
157.22 s
51.78 q
131.82 d
140.81 s
35.48 t
34.85 t
57.10 s
52.39 d
44.77 d
20.36 t
30.15 t
37.24 d
30.20 d
21.51 q
21.22 q
17.07 q
16.54 q
168.98 s
130.20 d
141.67 s
34.69 t
34.99 t
55.14 s
59.43 d
44.07 d
19.70 t
29.62 t
37.12 d
30.02 d
20.78 q
19.98 q
16.72 q
16.31 q
161.44 s
9
10
11
12
13
14
15
16
16-OCH3
17
17-NCH3
18
19
61.55 t
43.19 q
58.41 t
170.64 s
51.03 q
19-OCH3
a
Data measured in DMSO-d6 at 125 MHz.
Data measured in C6D6 at 125 MHz.
b
Figure 1. Perspective view of the crystal struture of 1.

M. M. Uy et al. / Tetrahedron 59 (2003) 731–736
733
2 indicates that the relative stereochemistry of 3 is the same
as 1 and 2.
Compound 4 {[a]2D5 243.58} was obtained as a colorless oil
that gave a molecular ion at m/z 231.1980 corresponding to
the molecular formula C16H25N (D 20.7 mmu) in the
1
HREIMS. The H and 13C NMR spectral data of 4 were
identical to those of 10-epi-axisonitrile-3,12 indicating that 4
has the same relative structure as 10-epi-axisonitrile-3, the
absolute configuration of which has not been determined.
(2)-10-epi-Axisonitrile-3 (4) was converted into 1 by
hydration. Exiguamide (1) was hydrolyzed to an amine
derivative 5 with 10% HCl. The absolute configuration at
C-6 of 5 was elucidated by the application of the modified
Mosher’s method.13 Two portions of 5 were treated
separately with (2)- and (þ)-a-methoxy-a-(trifluoro-
methyl)phenylacetyl chloride (MTPACl) in pyridine to
afford the (S)- and (R)-a-methoxy-a-(trifluoromethyl)-
phenylacetyl (MTPA) derivatives (6a and 6b), respectively.
The Dd (dS2dR) values are shown in Figure 5. The Dd
values for H-1, H2-3, H2-4, H-10, H3-14 and H3-15 were
negative. The values for H-7, H2-8, H-11, H3-12 and H3-13
were positive. In accordance with the modified Mosher
model, the absolute configuration of C-6 was determined to
be R. Thus, the absolute stereochemistry of exiguamide (1)
and (2)-10-epi-axisonitrile-3 (4) was elucidated to be
5S,6R,7S,10S.
Figure 2. Key 1H–13C HMBC correlations observed for 2.
Due to the paucity of material, the absolute stereochemistry
of 2 and 3 has not been experimentally determined.
However, because 1–4 were derived from the same origin,
it is likely that 2 and 3 share the same absolute
stereochemistry (5S,6R,7S,10S) as that of 1 and 4.
Figure 3. Key NOEs observed for 2.
findings indicate that 2 possesses the same relative
stereochemistry as 1.
Chemical investigation of marine sponges belonging to the
Geodiidae family has led to the isolation of interesting new
bioactive compounds which include a series of sterols14 and
steroids,15 geodiastatins,16 barettin,17 geodiamolides,18,19
geodiatoxins,20geodisterol,21 geodin A magnesium salt,22
stelleferin riboside23 and stellettins A and B.23 Recently,
geoditins A and B were isolated from G. japonica.24
However, to the best of our knowledge, there have been no
reports of sesquiterpenes bearing the spiro[4.5]decene
skeleton from this genus. Such a carbon skeleton has only
been found within the genera Axinella,25 Acanthella26 and
Topsentia.27It has been shown by previous studies,25,28–32
that isocyanides/isonitriles often co-exist with the corre-
sponding thiocyanates and formamides in a single animal
species. In some cases, the corresponding amines or
Exigurin (3) gave a molecular ion at m/z 364.2732
appropriate for the molecular formula C21H36N2O3 (D
þ0.6 mmu) in the HREIMS. Its IR spectrum indicated the
presence of NH (3393 cm21), carbonyl ester (1742 cm21
)
and amide (1645 cm21) functionalities. Extensive analysis
of the NMR data showed that 3 is a C-6 functional group
analog of 1 and 2. The HMBC spectral data (Fig. 4)
indicated that the substituent at C-6 in 3 is a 2-(methoxy-
carbonylmethyl-methyl-amino)-acetylamino group. The
1
1
similarity of the H and 13C chemical shifts and H–1H
coupling constants at C-1 to C-15 between 3 and both 1 and
Figure 4. Key 1H–13C HMBC correlations observed for 3.
Figure 5. Dd values [(dS2dR) in ppm] for MTPA esters 6a and 6b.

734
M. M. Uy et al. / Tetrahedron 59 (2003) 731–736
Table 3. Inhibitory effects of 1–4 on developmental events of sea urchin
embryos
transferred to seawater. They were periodically observed
for any cytological changes.
Event
Minimum inhibitory concentration (mM)
3.3. Sponge sample
1
2
3
4
The marine sponge G. exigua Thiele (Order Astrophorida,
Family Geodiidae) was collected off Oshima, Kagoshima
Prefecture, Japan in July 2001 and was identified by
Professor Patricia R. Bergquist of the University of
Auckland, New Zealand. A voucher specimen is kept in
the laboratory of the authors (S. O.).
Formation of micromeres
Formation of spicule
0.4
0.4
.360
.360
.270
.270
.430
.430
ammonium chloride salt forms were also isolated.33 We
believe this is the first occurrence of an isonitrile co-existing
with 2-(methoxycarbonylmethyl-methyl-amino)-acetyl-
amino, methoxycarbonylamino, and formylamino groups.
3.4. Extraction and isolation
The sponge sample (160 g, wet weight) was cut into small
pieces and soaked in MeOH (500 mL£3) for 3 d in the
refrigerator. The pooled MeOH extract was concentrated in
vacuo to afford a residue which was partitioned between
H2O (200 mL) and hexane (150 mL£3). The hexane soluble
extract (1.1 g; 0.68% wet weight) was then subjected to
medium pressure column chromatography on ODS employ-
ing solvent gradient MeOH–H2O to give nine fractions.
Individual purification of three fractions on a silica gel
column using varying ratios of EtOAc–hexane solvent
system afforded 1 (8.0 mg; 0.005% wet weight), 2 (2.1 mg;
0.001% wet weight), 3 (0.4 mg; 0.0003% wet weight) and 4
(63.8 mg; 0.04% wet weight), respectively.
When fertilized eggs of the sea urchin, Hemicentrotus
pulcherrimus, were cultured in the presence of 0.4–
12.0 mM exiguamide (1), they divided equally to form 16-
cell embryos that were comprised of sixteen cells of the
same size. In a control experiment, normal embryos formed
four macromeres, four micromeres, and eight mesomeres at
the same 16-cell stage. After passing through the blastula
and then gastrula stages, the 1-treated embryos developed to
form spicule-deficient plutei (Table 3). There are very few
substances having the biological activity.34 Compounds
2–4 did not exhibit such activity. Therefore, it is concluded
that the difference of functional groups attached at the C-6
of these compounds reflects the difference of the inhibitory
activities. Exiguamide (1) is considered to be a useful tool
for elucidating the mechanism of cell fate specification
during sea urchin embryogenesis.35
3.4.1. Exiguamide (1). Colorless crystals; mp 139–1408C;
[a]2D5 þ31.78 (c 0.08, CHCl3); IR (KBr) nmax 3323,
1
1657 cm21; H NMR, see Table 1; 13C NMR, see Table
2; (þ)-HRFABMS m/z 250.2174 [MþH]þ (calcd for
C16H28NO, 250.2171); (2)-FABMS m/z 248 [M2H]2.
3. Experimental
3.1. General
3.4.2. Exicarbamate (2). Colorless oil; [a]2D5 þ288 (c 0.02,
1
CHCl3); IR (film) nmax 3366, 1711 cm21; H NMR, see
Table 1; 13C NMR, see Table 2; (þ)-HRFABMS m/z
Melting point was recorded on a Yanagimoto micromelting
point apparatus and is uncorrected. IR spectra were obtained
on a JASCO FT/IR-5300 spectrometer. Optical rotations
were measured on a JASCO DIP-370 digital polarimeter.
NMR spectra were recorded on JEOL GSX500 and LA500
spectrometers at 258C. Mass spectra were obtained from a
JEOL SX102A spectrometer.
280.2267 [MþH]þ (calcd for C17H30NO2, 280.2276).
3.4.3. Exigurin (3). Colorless oil; [a]2D5 2328 (c 0.03,
1
CHCl3); IR (film) nmax 3393, 1742, 1645 cm21; H NMR,
see Table 1; 13C NMR, see Table 2; HREIMS m/z 364.2732
[M]þ (calcd for C21H36N2O3, 364.2726); EIMS m/z (rel.
intensity) 364 [M]þ (19), 305 [M2COOCH3]þ (6), 248
[M2CH2N(CH3)CH2COOCH3]þ (4), 116 (100).
3.2. Bioassay for developmental inhibitors using sea
urchin embryos
3.4.4. (2)-10-epi-Axisonitrile-3 (4). Colorless oil; [a]D25
243.58 (c 0.64, CHCl3); IR (film) nmax 2132, 1653,
Experiments were performed at 208C, and filtered seawater
was used throughout. Eggs and sperm were collected by
electric stimulation of 10 V to the mature female and male
sea urchin, respectively, allowing them to shed into beakers
filled with filtered seawater. After washing three times with
seawater, eggs were fertilized by the addition of the diluted
sperm suspension. Fertilized eggs were washed three times
with seawater, then allowed to develop in seawater until
they reached desired developmental stages. The DMSO
solution of sample to be tested was added to the suspension
of embryos to give final concentrations of DMSO less than
0.1% in seawater. DMSO had no effects on the phenomena
observed at the concentrations tested. To assay for
embryogenesis, 2 or 4-cell stage embryos were placed into
serially diluted sample solutions. After having been cultured
until the 16-cell stage, sample-treated embryos were
1
1456 cm21; H NMR, see Table 1; 13C NMR, see Table
2; HREIMS m/z 231.1980 [M]þ (calcd for C16H25N,
231.1987); EIMS m/z (rel. intensity) 231 [M]þ (32), 216
[M2CH3]þ (77), 204 [M2HCN]þ (38), 188 (50), 161
(100).
3.5. Single crystal X-ray analysis of 1
Crystal data: C16H27NO, M¼249.40, monoclinic, space
group C2 (no. 5), Z¼8, a¼24.4390 (8), b¼8.8390 (4),
3
˚
˚
c¼16.8350 (8) A, b¼115.786 (2)8, V¼3274.5 (2) A ,
F(000)¼1104, m(Mo Ka)¼0.62 cm21, Dc¼1.012 g cm23
,
T¼295 K. The reflection data were collected on a Mac
Science DIP 2030 imaging plate area detector with graphite
˚
monochromated Mo Ka radiation (l¼0.71069 A) from a

M. M. Uy et al. / Tetrahedron 59 (2003) 731–736
735
colorless
crystal
of
approximate
dimensions
2.4 Hz, H-3b), 1.90 (1H, ddd, J¼12.8, 7.9, 2.4 Hz, H-4a),
1.82 (1H, dddd, J¼14.0, 12.8, 4.9, 4.3 Hz, H-9ax), 1.71 (3H,
br s, H-14), 1.68 (1H, m, H-8eq), 1.58 (1H, ddd, J¼12.8,
8.5, 8.5 Hz, H-4b), 1.51 (1H, m, H-9eq), 1.24 (1H, m, H-7),
1.10 (1H, m, H-8ax), 1.05 (1H, m, H-11), 1.00 (3H, d,
J¼7.6 Hz, H-15), 0.83 (3H, d, J¼6.7 Hz, H-12), 0.72 (3H,
d, J¼6.7 Hz, H-13); EIMS m/z 437 [M]þ.
0.7£0.4£0.2 mm. A total of 3076 independent reflections
were collected of which 1568 were considered to be
observed [I.2.90s(I)]. The structure was solved by direct
methods36 and expanded using Fourier techniques.37 The
non-hydrogen atoms were refined anisotropically by full-
matrix least-squares refinement. Hydrogen atoms were
refined isotropically. The structure was finally refined to
R¼0.056 (Rw¼0.076). Crystallographic data for the struc-
ture in this paper has been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication
number CCDC 185123. Copies of the data can be obtained,
free of charge, on application to CCDC, 12 Union Road,
Cambridge, CB2 1EZ, UK (fax: þ44-1223-336033; e-mail:
deposit@ccdc.cam.ac.uk).
Acknowledgements
The authors thank Captain A. Goh and the crew of R/V
Toyoshio-Maru of Hiroshima University for the help in the
collection of the sponge sample, Professor Patricia
R. Bergquist, The University of Auckland, New Zealand,
for the identification of the sponge specimen, and Mr
Hitoshi Fujitaka, Hiroshima University, for the NMR
measurements. This study was supported in part by a
Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
3.6. Conversion of 4 into 1
To a solution of 4 (29 mg) in EtOH was added acetic acid
(2 mL) and the mixture was stirred at room temperature for
18 h. After the solvent was removed, the residue was
purified by silica gel column chromatography (eluted with
20% EtOAc–hexane) to give 28 mg of the product. The IR,
1H NMR and MS spectral data and [a]D of the product were
identical to those of 1.
References
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3.7. Preparation of the (S)- and (R)-MTPA esters
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To a solution of 1 (27 mg) in EtOH was added 10% HCl
(1.5 mL). The mixture was allowed to react at 608C for 4
d.38 After removal of the solvent, the residue was treated
through a short ODS column (eluted with 40% MeOH–H2O
to 100% MeOH) to give a crude fraction (13 mg) of the
amine derivative 5 {(þ)-FABMS m/z 222 [MþH]þ}. The
fraction was used for the preparation of MTPA esters
without further purification. To a solution of the amine
derivative 5 (6 mg) in pyridine (dried over CaH2) was added
4-dimethylaminopyridine and (2)-MTPACl at room tem-
perature and stirred for 2.5 h. After evaporation of the
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(5% EtOAc–hexane) to afford the (S)-MTPA ester (6a).
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1
3.7.1. (S)-MTPA ester (6a). H NMR (CDCl3) d 7.56 and
7.40 (5H, m, MTPA phenyl protons), 6.98 (1H, br d,
J¼10.5 Hz, NH), 5.48 (1H, br s, H-1), 3.98 (1H, dd, J¼10.5,
3.5 Hz, H-6), 3.38 (3H, s, MTPA CH3O2), 2.39 (1H, ddd,
J¼16.5, 8.5, 7.9 Hz, H-3a), 2.02 (1H, ddd, J¼16.5, 8.5,
2.4 Hz, H-3b), 1.82 (1H, tt, J¼13.0, 4.5 Hz, H-9ax), 1.76
(1H, m, H-8eq), 1.73 (1H, ddd, J¼12.5, 7.9, 2.4 Hz, H-4a),
1.69 (3H, br s, H-14), 1.58 (1H, m, H-10), 1.52 (1H, m,
H-9eq), 1.44 (1H, ddd, J¼12.5, 8.5, 8.5 Hz, H-4b), 1.34
(1H, m, H-11), 1.26 (1H, m, H-7), 1.18 (1H, qd, J¼13.0,
3.5 Hz, H-8ax), 0.92 (3H, d, J¼6.5 Hz, H-12), 0.89 (3H, d,
J¼7.5 Hz, H-15), 0.83 (3H, d, J¼6.5 Hz, H-13); EIMS m/z
437 [M]þ.
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