Anti-fertilization activity of a spirocyclic sesquiterpene isocyanide isolated from the marine sponge Geodia exigua and related compounds

Article abstract of DOI:10.1271/bbb.80071

(-)-10-epi-Axisonitrile-3, a spirocyclic sesquiterpene isocyanide obtained from the marine sponge Geodia exigua, immobilized sperm of sea urchin and starfish to block fertilization at the minimum effective concentration of 0.4 μg/ml. On the other hand, fertilized eggs developed normally to the gastrula stage in the presence of a 250-times higher concentration of the isocyanide. Analysis by ³¹P NMR revealed an accumulation of phosphocreatine and a depletion of inorganic phosphate in the isocyanide-treated sperm, suggesting that (-)-10-epi-axisonitrile-3 inhibited the phosphocreatine shuttle participating in the high-energy phosphate metabolism, thereby immobilizing sperm to block fertilization. No analogs of (-)-10-epi-axisonitrile-3 containing different functionalities or isocyanides with different carbon skeleton exhibited such activity.

Full text of DOI:10.1271/bbb.80071

Biosci. Biotechnol. Biochem., 72 (7), 1764–1771, 2008
Anti-Fertilization Activity of a Spirocyclic Sesquiterpene Isocyanide
Isolated from the Marine Sponge Geodia exigua and Related Compounds
1
3
Emi OHTA,1 Mylene M. UY,2; Shinji OHTA, Mihoko YANAI,
*
y
1;
Toshifumi HIRATA,2 and Susumu IKEGAMI
1Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Japan
2Department of Mathematical and Life Sciences, Graduate School of Science,
Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan
3Natural Science Center for Basic Research and Development, Hiroshima University,
1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan
Received January 30, 2008; Accepted April 1, 2008; Online Publication, July 7, 2008
[doi:10.1271/bbb.80071]
(À)-10-epi-Axisonitrile-3, a spirocyclic sesquiterpene
isocyanide obtained from the marine sponge Geodia
exigua, immobilized sperm of sea urchin and starfish to
block fertilization at the minimum effective concentra-
tion of 0.4 ꢀg/ml. On the other hand, fertilized eggs
developed normally to the gastrula stage in the presence
of a 250-times higher concentration of the isocyanide.
Analysis by 31P NMR revealed an accumulation of
phosphocreatine and a depletion of inorganic phosphate
in the isocyanide-treated sperm, suggesting that (À)-10-
epi-axisonitrile-3 inhibited the phosphocreatine shuttle
participating in the high-energy phosphate metabolism,
thereby immobilizing sperm to block fertilization. No
analogs of (À)-10-epi-axisonitrile-3 containing different
functionalities or isocyanides with different carbon
skeleton exhibited such activity.
biological activity of spirocyclic sesquiterpenes revealed
that 1 potently inhibits fertilization of sea urchin and
starfish gametes by immobilizing sperm without affect-
ing the embryogenesis of the fertilized eggs. In this
paper, we report the selective anti-fertilization activity
of 1 and its mode of action. The effects of 2–4 and
several isocyanides and formamides bearing different
carbon skeletons on echinoderm fertilization and em-
bryogenesis are also described.
1
2
3
4
R = NC
R
R = NHCHO
6
R = NHCO2CH3
10
R = NHCOCH2N(CH3)CH2COOCH3
Materials and Methods
Key words: (ꢀ)-10-epi-axisonitrile-3; fertilization in-
hibition; sperm; 31P NMR; structure-activi-
ty relationship
General procedures. IR spectra were recorded on a
Jasco FT/IR-5300 spectrometer (Jasco, Tokyo). NMR
spectra were recorded on JEOL GSX500 and LA500
spectrometers. Mass spectra were obtained with a
JEOL SX102A spectrometer. Elemental analyses
were performed on a Perkin Elmer 2400II CHNS/O
analyzer (Perkin Elmer, Waltham, MA).
Marine organisms are a rich source of biologically
active terpene isocyanides.1–3) Some of them exhibit
antimalarial,4–7) antifouling,8,9) antimicrobial,10) antifun-
gal,11) and antiparasitic12) activities.
In our continuing search for selective inhibitors of
echinoderm fertilization and of embryonic development
from marine organisms,13–16) we have isolated new
spirocyclic sesquiterpenes, (ꢀ)-10-epi-axisonitrile-3 (1),
exiguamide (2), exicarbamate (3), and exigurin (4),
from the marine sponge Geodia exigua Thiele, and have
reported the inhibitory activity of 2 on the spicule
formation of sea urchin.17,18) Further investigation of the
Bioassay using sea urchin gametes and embryos.
Winter sea urchin (Hemicentrotus pulcherrimus) and
summer sea urchin (Anthocidaris crassispina) were used
in these experiments during the winter (from February to
March) and summer seasons (from July to September)
respectively. Experiments were performed at 20 ꢁC, and
filtered seawater was used throughout. Eggs and sperm
y
To whom correspondence should be addressed. Tel: +81-749-64-8160; Fax: +81-749-64-8140; E-mail: s ikegami@nagahama-i-bio.ac.jp
Current address: Department of Chemistry, College of Sciences and Mathematics, Mindanao State University-Iligan Institute of Technology,
Andres Bonifacio Avenue, 9200 Iligan City, Philippines
*
Abbreviations: EIMS, electron impact mass spectrometry; FABMS, fast-atom bombardment mass spectrometry

Anti-Fertilization Activity of Marine Isocyanide
1765
were collected by injection of 0.5 M KCl solution to
the mature female and male sea urchin respectively,
allowing them to shed into beakers filled with filtered
seawater. Sample solutions were prepared by adding
DMSO solutions of test compounds to seawater to give
final DMSO concentrations of less than 0.2%. DMSO
had no effect on the biological phenomena observed at
the concentrations tested. For the fertilization-inhibition
assay, 1 drop of a diluted sperm suspension was added to
serially diluted sample solutions, and 10 min later, 1
drop of an egg suspension was added. Thirty min later,
the presence of moving sperm and the fertilization
envelope were examined under a light microscope. For
the development-inhibition assay, 2-cell stage embryos
were placed into serially diluted sample solutions. After
they were cultured to the 16-cell stage, the embryos
were transferred to seawater. They were periodically
examined for cytological changes.
described in our previous paper.18) Compound 13 was
isolated from a marine sponge, Acanthella sp., and
identified as kalihinol F on the basis of spectral data, as
described in our previous paper.19)
Synthetic isocyanides. Compound 5 was synthesized
from cyclohexanone and 1,4-dibromobutane via spiro-
[4.5]decan-6-one (5a) and spiro[4.5]dec-6-ylamine (5b).
Compounds 7, 9, and 12 were prepared by coupling of
commercially available amine derivatives with CHCl3.
Compounds 6, 8, 10, and 11 were purchased from
Sigma-Aldrich (St. Louis, MO).
Spiro[4.5]decan-6-one (5a). Following the procedure
described by Mousseron et al.,20) to a solution of
t-BuOK (2.2 g, 20 mmol) in t-BuOH (20 ml) and dry
benzene (15 ml) were added cyclohexanone (1.0 g,
10 mmol) and 1,4-dibromobutane (2.2 g, 10 mmol).
The reaction mixture was refluxed for 5 h at 120 ꢁC.
After cooling and the addition of 5% HCl to the reaction
mixture, the benzene layer was separated and the
aqueous layer was extracted with ether. The combined
organic extracts were dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane/EtOAc = 99:1 as
an eluent) to give 5a (0.80 g, 25%) as a colorless oil. IR
ꢀmax (film) cmꢀ1: 2,942, 2,864, 1,707, 1,447; NMR ꢁH
(500 MHz, CDCl3): 1.25–1.71 (m, 12H), 1.94 (m, 2H),
2.29 (t, 2H, J ¼ 6:5 Hz); NMR ꢁC (125 MHz, CDCl3, a
selected signal): 214.0 (C=O); EIMS m=z (rel. intensity)
152 Mþ (60), 111 (100).
Bioassay using starfish gametes and embryos. Oo-
cytes and sperm of the starfish Asterina pectinifera were
taken from ovarian and testicular fragments respective-
ly. All experiments were carried out at 20 ꢁC using 90%
filtered natural seawater throughout. Sample solutions
were prepared as described above. Maturation of
oocytes was induced by the addition of 1-methyladenine
to give a final concentration of 1 mM. Maturing oocytes
were inseminated 1 h after the start of 1-methyladenine
treatment. In the fertilization-inhibition assay, mature
eggs and sperm suspensions were used. The assay was
performed in the same manner as described for the
sea urchin. In the development-inhibition assay, fertil-
ized eggs were added in serially diluted sample solutions
within 30 min of insemination, after which they were
examined periodically for cytological changes.
Spiro[4.5]dec-6-ylamine (5b). Following the proce-
dure described by Borch et al.,21) a solution of 5a (0.40 g,
2.6 mmol), ammonium acetate (2.5 g, 33 mmol) and
NaBH3CN (0.18 g, 2.8 mmol) in absolute MeOH (10 ml)
was stirred for 48 h at room temperature. Concentrated
HCl was added to acidify to pH 2 or lower, and the
MeOH was removed in vacuo. The residue was taken up
in water and extracted with ether. The aqueous solution
was brought to alkalinity over pH 10 using a KOH
solution, saturated with NaCl, and extracted with ether.
The combined organic extracts were dried over Na2SO4
and concentrated in vacuo to give crude 5b (0.29 g,
73%). IR ꢀmax (film) cmꢀ1: 3,364, 2,928, 2,861, 1,449;
NMR ꢁH (500 MHz, CDCl3): 1.04–1.56 (m, 16H), 2.51
(dd, 1H, J ¼ 8:5, 3.7 Hz, CHNH2), 2.54 (br s, 2H, NH2);
NMR ꢁC (125 MHz, CDCl3, a selected signal): 56.3
(CHNH2); FABMS m=z 154 ½M þ Hꢃþ.
Measurement of 31P NMR spectra of sperm. Testic-
ular fragments of the starfish A. pectinifera were used
as intact quiescent dry sperm (approximately 2 ꢂ
1010 cells/ml). Dry sperm were diluted with 3-fold
Ca2þ-free seawater to prepare a suspension of actively
motile sperm (approximately 5 ꢂ 109 cells/ml). DMSO
solution of 1 was added to the motile sperm suspension
to give a final 1 concentration of 40 mg/ml (approx-
imately 5 ꢂ 109 cells/ml). These sperm suspensions
were prepared at 4–10 ꢁC. In NMR measurements,
3 ml of a sperm suspension with or without 1 was placed
into a 10-mm diameter NMR tube. Measurements were
performed at 10 ꢁC in a JEOL GSX500 spectrometer
operating at 202 MHz for 31P observation using 45ꢁ
pulses and a repetition time of 1.0 s without proton
decoupling for 10 min (600 scans). Chemical shifts were
measured relative to H3PO4 dissolved in D2O (0 ppm)
in a capillary tube, which was added as an external
standard in a 10-mm diameter NMR tube.
6-Isocyanospiro[4.5]decane (5). Following the pro-
cedure described by Sasaki et al.,22) a mixture of
crude 5b (100 mg, 0.58 mmol), benzyltriethylammonium
chloride (10 mg), benzene (5 ml), and 50% aqueous
KOH (8 ml) was vigorously stirred for 30 min at room
temperature. To the resulting emulsion was added
CHCl3 (80 mg, 0.67 mmol) in benzene (3 ml) on ice
for 1 h. Stirring was continued for 2 h at room temper-
Natural products. Compounds 1–4 were obtained
from the marine sponge Geodia exigua Thiele, as

1766
E. OHTA et al.
ature. The benzene layer was separated, washed with
water, dried over Na2SO4, and concentrated in vacuo.
The residue was passed through a silica gel column and
eluted with hexane to give 5 (11 mg) as a colorless
volatile oil from crude 5b in 11% yield. IR ꢀmax (film)
cmꢀ1: 2,940, 2,865, 2,135, 1,449; NMR ꢁH (500 MHz,
CDCl3): 1.20–1.82 (m, 16H), 3.42 (br t, 1H, J ¼ 3:7 Hz,
CHNC); NMR ꢁC (125 MHz, CDCl3, selected signals):
60.7 (t, JN-C ¼ 5:7 Hz, CHNC), 154.4 (t, JN-C ¼ 5:7 Hz,
NC); EIMS m=z (rel. intensity) 162 ½M ꢀ Hꢃþ (38),
134 (100). It has been reported that some isocyanide
compounds show an ½M ꢀ Hꢃþ ion abundantly in their
EIMS.23) HREIMS m=z 162.1279 ½M ꢀ Hꢃþ (calcd. for
C11H16N, 162.1282).
procedure described for 7. IR ꢀmax (film) cmꢀ1: 2,137,
1,514, 1,445; NMR ꢁH (500 MHz, CDCl3): 1.85 (d, 3H,
J ¼ 6:7 Hz), 5.59 (br q, 1H, J ¼ 6:7 Hz), 7.52–7.93 (m,
7H, naphthyl); NMR ꢁC (125 MHz, CDCl3, selected
signals): 51.0 (t, JN-C ¼ 5:7 Hz, CHNC), 156.8 (t,
JN-C ¼ 5:7 Hz, NC); EIMS m=z (rel. intensity) 181 Mþ
(49), 180 ½M ꢀ Hꢃþ (67), 166 (88), 153 (100). Anal.
Found: C, 86.10; H, 6.06; N, 7.63%. Calcd for C13H11N:
C, 86.15; H, 6.12; N, 7.73%.
Formamides. The formamides were generally pre-
pared as follows: to the corresponding isonitrile analog
was added acetic acid, and the solution was stirred at
room temperature for 20 h. After the solvent was
removed, the residue was passed through a short silica
gel column, which was then eluted with varying ratios of
hexane-ethyl acetate mixture to give the corresponding
formamide as a mixture of s-cis and s-trans isomers,
the s-cis isomer being the major product. Compound 15
was purchased from Sigma-Aldrich.
2-Adamantylisocyanide (7). This compound was
prepared as colorless crystals (mp 178–180 ꢁC) from
2-adamantylamine hydrochloride in 63% yield accord-
ing to the procedure described for the conversion from
5b to 5. IR ꢀmax (film) cmꢀ1: 2,909, 2,857, 2,133;
NMR ꢁH (500 MHz, CDCl3): 1.62–2.16 (m, 14H), 3.80
(br s, 1H, CHNC); NMR ꢁC (125 MHz, CDCl3, selected
signals): 58.9 (t, JN-C ¼ 5:7 Hz, CHNC), 154.5 (t,
JN-C ¼ 5:7 Hz, NC); EIMS m=z (rel. intensity) 161 Mþ
(10), 160 ½M ꢀ Hꢃþ (20), 134 (100). Anal. Found: C,
81.97; H, 9.23; N, 8.50%. Calcd. for C11H15N: C, 81.94;
H, 9.38; N, 8.69%.
6-Formylaminospiro[4.5]decane (14). IR ꢀmax (film)
cmꢀ1: 3,263, 1,663; NMR ꢁH (500 MHz, CDCl3): 1.23–
1.75 (m, 16H), 3.17 (dt, 0.3H, J ¼ 3:7, 9.5 Hz), 3.94
(dt, 0.7H, J ¼ 3:7, 9.2 Hz), 5.51 (br, 0.7H, NH), 5.75
(br, 0.3H, NH), 8.08 (d, 0.3H, J ¼ 12:2 Hz, CHO),
8.20 (br s, 0.7H, CHO); NMR ꢁC (125 MHz, CDCl3,
selected signals): 52.4, 57.4 (CHNHCHO), 160.7, 164.0
(NHCHO); FABMS m=z 182 ½M þ Hꢃþ, 137 [M-
NHCHO]þ; HRFABMS m=z 182.1538 ½M þ Hꢃþ
(calcd. for C11H19NO, 182.1545).
Stearylisocyanide (9). Following the procedure de-
scribed by Gokel et al.,24) NaOH pellets (6 g) were
added in portions to H2O (6 ml) under constant vigorous
stirring. A solution of stearylamine (1.4 g, 5.0 mmol),
CHCl3 (2.4 g, 20 mmol), and benzyltriethylammonium
chloride (0.18 g) in CH2Cl2 (6 ml) was added dropwise
to the alkaline solution over a period of 20 min.
After initiation of the addition, the reaction mixture
began to boil spontaneously, and subsided within 1 h.
Stirring was continued for an additional 1 h and the
mixture was diluted with ice-cooled water. The organic
layer was separated and the aqueous layer was extracted
with CH2Cl2. The combined organic extracts were
successively washed with water and 5% NaCl and dried
over K2CO3, and the solvent was removed in vacuo. The
residue was purified by silica gel column chromatog-
raphy (hexane/diethyl ether = 98:2 as an eluent) to
furnish 9 (0.136 g, 10%) as a yellow oil. IR ꢀmax (film)
cmꢀ1: 2,924, 2,853, 2,145, 1,466; NMR ꢁH (500 MHz,
CDCl3): 0.88 (t, 3H, J ¼ 6:7 Hz), 1.26 (m, 32H), 3.37
(br t, 2H, J ¼ 6:1 Hz, CH2NC); NMR ꢁC (125 MHz,
CDCl3, selected signals): 41.5 (t, JN-C ¼ 6:7 Hz,
CH2NC), 155.8 (t, JN-C ¼ 5:7 Hz, NC); EIMS m=z (rel.
intensity) 279 Mþ (24), 278 ½M ꢀ Hꢃþ (49), 96 (100).
Anal. Found: C, 81.52; H, 13.32; N, 4.99%. Calcd. for
C19H37N: C, 81.65; H, 13.34; N, 5.05%.
2-Adamantylformamide (16). IR ꢀmax (film) cmꢀ1
:
3,299, 1,688, 1,657; NMR ꢁH (500 MHz, CDCl3): 1.64–
1.93 (m, 14H), 3.62 (d, 0.3H, J ¼ 9:2 Hz), 4.15 (d, 0.7H,
J ¼ 8:5 Hz), 5.94 (br, 0.7H, NH), 6.23 (br, 0.3H, NH),
8.15 (d, 0.3H, J ¼ 12:2 Hz, CHO), 8.18 (br s, 0.7H,
CHO); NMR ꢁC (125 MHz, CDCl3, selected signals):
52.2, 56.0 (CHNHCHO), 1þ60.3, 163.8 (NHCHO);
FABMS m=z 180 ½M þ Hꢃ , 135 [M-NHCHO]þ;
HRFABMS m=z 180.1387 ½M þ Hꢃþ (calcd for
C11H18NO, 180.1388).
1,1,3,3-Tetramethybutylformamide (17). Compound
17 was identified as 1,1,3,3-tetramethylbutylformamide
1
from its IR, H NMR, 13C NMR, and FABMS spectral
data. IR ꢀmax (film) cmꢀ1: 3,293, 1,682; NMR ꢁH (500
MHz, CDCl3): 0.84 (s, 9H, t-Bu), 1.21 (s, 3H, Me), 1.26
(s, 3H, Me), 1.41 (s, 1H, H-2), 1.61 (s, 1H, H-2), 6.20 (br
s, 0.5H, NH), 7.38 (br d, 0.5H, J ¼ 12:2 Hz, NH), 7.83
(d, 0.5H, J ¼ 1:5 Hz, CHO), 8.04 (d, 0.5H, J ¼ 12:2 Hz,
CHO); NMR ꢁC (125 MHz, CDCl3, selected signals):
54.7, 55.4 (CNHCHO), þ 160.6, 163.2 (NHCHO);
FABMS m=z 158 ½M þ Hꢃ , 113 [M-NHCHO]þ.
(R)-(+)-1-(1-Naphthyl)ethylisocyanide (12). This
compound was prepared as a yellow oil from (R)-(+)-
1-(1-naphthyl)ethylamine in 35% yield according to the
Stearylformamide (18). IR ꢀmax (film) cmꢀ1: 3,295,
1,645; NMR ꢁH (500 MHz, CDCl3): 0.85 (t, 3H, J ¼
7:0 Hz, Me), 1.20–1.35 (m, 30H), 1.45–1.55 (m, 2H),

Anti-Fertilization Activity of Marine Isocyanide
1767
3.18 (dd, 2H, J ¼ 13:4, 6.7 Hz), 3.18 (q, 0.4H, J ¼
6:7 Hz), 3.26 (q, 1.6H, J ¼ 6:7 Hz), 5.89 (br, 0.8H,
NH), 5.95 (br, 0.2H, NH), 8.00 (d, 0.2H, J ¼ 12:2 Hz,
CHO), 8.12 (br s, 0.8H, CHO); NMR ꢁC (125 MHz,
CDCl3, selected signals): 38.1, 41.7 (CH2NHCHO),
161.2, 164.6 (NHCHO); FABMS m=z 298 ½M þ Hꢃþ;
HRFABMS m=z 298.3112 ½M þ Hꢃþ (calcd for
C19H40NO, 298.3109).
A
B
Benzylformamide (19). Compound 19 was identified
1
Fig. 1. Inhibition of Fertilization of Starfish by (ꢀ)-10-epi-Axisoni-
trile-3 (1).
as benzylformamide from its IR, H NMR, 13C NMR,
and FABMS spectral data. IR ꢀmax (film) cmꢀ1: 3,281,
1,651, 1,535, 1,499, 1,454; NMR ꢁH (500 MHz, CDCl3):
4.33 (d, 0.2H, J ¼ 6:1 Hz), 4.44 (d, 1.8H, J ¼ 6:1 Hz),
6.46 (br, 0.1H, NH), 6.79 (br, 0.9H, NH), 7.22–7.38
(m, 5H, phenyl), 8.05 (d, 0.1H, J ¼ 11:6 Hz, CHO),
8.14 (br s, 0.9H, CHO); NMR ꢁC (125 MHz, CDCl3,
selected signals): 41.8, 45.5 (CH2NHCHO), 161.2,
164.7 (NHCHO); FABMS m=z 136 ½M þ Hꢃþ, 91
[M-NHCHO]þ.
Starfish sperm were incubated in the presence (panel A) and
absence (panel B) of 10 mg/ml 1 for 10 min before the addition of
an egg suspension. The photographs were taken 10 min after the
addition of the egg suspension. Bar indicates 50 mm.
Table 1. Inhibitory Effects of 1–4 on the Fertilization and Gastru-
lation of Winter Sea Urchin (Hemicentrotus pulcherrimus) and Starfish
(Asterina pectinifera)
Minimum inhibitory concentration (mg/ml)
Compound
Fertilization
Sea urchin Starfish
0.4 0.4
>100 >100
Gastrulation
ꢂ-Methylbenzylformamide (20). Compound 20 was
identified as ꢂ-methylbenzylformamide from IR,
1H NMR, 13C NMR, and FABMS spectral data. IR
ꢀmax (film) cmꢀ1: 3,275, 1,661, 1,535, 1,495, 1,452;
NMR ꢁH (500 MHz, CDCl3): 1.50 (d, 2.4H, J ¼ 6:7 Hz,
Me), 1.55 (d, 0.6H, J ¼ 6:7 Hz, Me), 4.67 (quin, 0.2H,
J ¼ 6:7 Hz), 5.19 (quin, 0.8H, J ¼ 6:7 Hz), 6.03 (br,
0.8H, NH), 6.23 (br, 0.2H, NH), 7.25–7.37 (m, 5H,
phenyl), 8.12 (d, 0.2H, J ¼ 11:6 Hz, CHO), 8.13 (br s,
0.8H, CHO); NMR ꢁC (125 MHz, CDCl3, selected
signals): 47.6, 51.6 (CHNHCHO), 160.2, 164.1
(NHCHO); FABMS m=z 150 ½M þ Hꢃþ, 105 [M-
NHCHO]þ.
Sea urchin
>100
0.1
Starfish
1
2
3
4
>100
25
>100
>100
>100
>100
>100
>100
>100
6.3
4, only exiguamide (2) inhibited gastrulation, thereby
produced spicule-deficient larvae at the minimum
effective concentration of 0.1 mg/ml, as described
below.
When starfish (Asterina pectinifera) gametes were
treated with 1–4, only 1 exhibited an inhibitory effect on
fertilization at a concentration of 0.4 mg/ml, as shown in
Fig. 1, while 2–4 did not affect fertilization even at
concentrations higher than 100 mg/ml. (ꢀ)-10-epi-Ax-
isonitrile-3 (1) did not affect the embryogenesis of
fertilized eggs, while 2 and 4 inhibited gastrulation at
the minimum effective concentrations of 25 and 6.3 mg/
ml respectively (Table 1). These findings indicate that
(ꢀ)-10-epi-axisonitrile-3 (1) is a selective inhibitor of
echinoderm fertilization.
When starfish or sea urchin unfertilized eggs were
admixed with sperm of the corresponding species in the
presence of 1 at 0.4 mg/ml, the sperm around the eggs
were found to be immotile, indicating that the inhibition
of fertilization by 1 was due to inhibition of sperm
motility.
Upon dilution in seawater, the sperm of the sea urchin
and starfish released from testicular fragments initiated
respiration and became motile. Dynein ATPase initiates
flagellar movement by driving ATP hydrolysis to
produce ADP. Energy utilization by the flagellum of
motile sperm is tightly coupled to the rate of energy
production by the mitochondria. This coupling depends
on the transport of high-energy phosphate compounds
(R)-(+)-1-(1-Naphthyl)ethylformamide (21). IR ꢀmax
(film) cmꢀ1: 3,275, 1,655, 1,532; NMR ꢁH (500 MHz,
CDCl3): 1.69 (d, 2.4H, J ¼ 6:7 Hz), 1.70 (d, 0.6H,
J ¼ 6:7 Hz), 5.47 (quin, 0.2H, J ¼ 6:7 Hz), 5.86 (br,
0.8H, NH), 6.01, (quin, 0.8H, J ¼ 6:7 Hz m), 6.16 (br,
0.2H, NH), 7.44–8.10 (m, 7H, naphthyl), 8.12 (br s,
0.8H, CHO), 8.20 (d, 0.2H, J ¼ 12:2 Hz, CHO); NMR
ꢁC (125 MHz, CDCl3, selected signals): 43.4, 47.9
(CHNHCHO), 160.0, 164.1 (NHCHO); FABMS m=z
200 ½M þ Hꢃþ; HRFABMS m=z 200.1069 ½M þ Hꢃþ
(calcd for C13H14NO, 200.1076).
Results and Discussion
Inhibitory activity of (ꢀ)-10-epi-axisonitrile-3 (1) on
echinoderm fertilization
Sea urchin (Hemicentrotus pulcherrimus) gametes
were treated with 1–4 to investigate their effects on
fertilization. At concentrations of 0.4 mg/ml and greater,
1 prevented fertilization. On the other hand, compounds
2, 3, or 4 did not affect fertilization even at a
concentration of 100 mg/ml (Table 1). Although em-
bryogenesis of fertilized eggs was unaffected by 1, 3, or

1768
E. OHTA et al.
H3PO4 (external standard)
NH2
O
O
NC
a
b
c
Pi
ATP
PCr
γ
α
β
A
B
C
5a
Scheme 1. Synthetic Route for 5.
5b
5
Reagents and conditions: a, 1,4-dibromobutane, t-BuOK, t-BuOH,
benzene, reflux; b, ammonium acetate, NaBH3CN, MeOH, r.t.;
c, CHCl3, benzyltriethylammonium chloride, aq KOH, benzene, r.t.
that the isocyanide group attached at C-6 of 1 plays a
critical role in the inhibition of echinoderm fertilization.
In order to determine the structural requirements for the
inhibitory activity, we prepared some isocyanides, as
follows: Synthesis of a simple spirocyclic isocyanide,
6-isocyanospiro[4.5]decane (5), was achieved by pre-
viously reported methods,20–22) as outlined in Scheme 1.
Spiro[4.5]decan-6-one (5a) was prepared by alkaline-
catalyzed coupling of cyclohexanone with 1,4-dibromo-
butane.20) Compound 5a was reduced to an amine
derivative (5b),21) followed by alkaline-catalyzed cou-
pling of the amino group with chloroform to yield 5.22)
Other isocyanide compounds which have an aliphatic
ring (6 and 7), a linear chain (8 and 9), or an aromatic
moiety (10, 11, and 12) were commercially available
or were derived from the corresponding amines. Com-
pound 13, kalihinol F,28) a diterpene with three iso-
cyanide groups, was isolated from the marine sponge
Acanthella sp. as described previously.19)
5
0
-5
-10
-15
-20
δP
Fig. 2. 31P NMR Spectra of Starfish Sperm Measured at 10 ꢁC.
A, Dry sperm; B, sperm diluted 1:4 in Ca2þ-free sea water
(pH 8.2) (600 scans); C, after the addition of 1 (40 mg/ml) to the
sperm in Ca2þ-free sea water (pH 8.2) (600 scans).
from the mitochondria to a flagellar axoneme, mediated
by the phosphocreatine energy shuttle.25,26) To analyze
phosphate metabolites of the 1-immobilized starfish
sperm, we assigned 31P signals due to inorganic
phosphate (Pi) and high-energy phosphate compounds
in intact starfish sperm in the 31P NMR spectra. As
shown in Fig. 2A, quiescent dry sperm accumulated
phosphocreatine (PCr), but had low levels of Pi.25,27)
When the sperm were diluted to 25% in seawater, a
decrease in PCr and an increase in Pi were observed
(Fig. 2B), accompanied by sperm mobilization. The
ratio of 31P signal areas of Pi to PCr was 1:0.2. The
addition of 1 to motile sperm caused an arrest of sperm
motility, and concomitantly a decrease in Pi and an
increase in PCr were observed in the 31P NMR spectrum
(Pi: PCr = 1:1), as shown in Fig. 2C. On the other hand,
when a simple spirocyclic isocyanide 5, whose synthesis
is described below, was added to the motile sperm, the
ratio of 31P signal areas of Pi to PCr was unchanged (Pi:
PCr = 1:0.2; spectral data not shown). These observa-
tions suggest that 1 inhibits the phosphocreatine energy
shuttle.25,26) To the best of our knowledge, 1 is the first
isocyanide compound found to exhibit selective inhib-
itory activity on sperm motility.
R
R
R
5 R = NC
14 R = NHCHO
7 R = NC
16 R = NHCHO
6 R = NC
15 R = NHCHO
R
R
9 R = NC
18 R = NHCHO
8 R = NC
17 R = NHCHO
R
R
R
12 R = NC
21 R = NHCHO
10 R = NC
19 R = NHCHO
11 R = NC
20 R = NHCHO
CN
HO
NC
O
NC
Inhibitory activity of various isocyanides on echino-
derm fertilization
13
When sea urchin gametes were used as the test
material, compounds 5–13 exhibited weak or no
Among spirocyclic sesquiterpenes 1–4, only 1 ex-
hibited anti-fertilization activity. These findings indicate

Anti-Fertilization Activity of Marine Isocyanide
Table 2. Inhibitory Effects of 5–13 on the Fertilization and Gas-
1769
trulation of Summer Sea Urchin (Anthocidaris crassispina) and
Starfish (Asterina pectinifera)
Minimum inhibitory concentration (mg/ml)
Compound
Fertilization
Sea urchin Starfish
Gastrulation
Sea urchin
Starfish
5
6
50
>100
50
50
>100
25
>100
50
50
13
>100
13
7
8
>100
>100
100
>100
>100
>100
100
>100
>100
>100
>100
25
100
>100
100
100
3.1
9
10
11
12
13
>100
100
>100
13
>100
100
1.6
inhibition on fertilization, as summarized in Table 2.
When starfish gametes were used as the test material, 5,
7, and 12 weakly inhibited fertilization, but 6, 8, 9, 10,
11, and 13 did not. Notably, the inhibition of sea urchin
and starfish fertilization by 5, 7, and 12 was not due to
the blockade of sperm motility as was observed for 1,
suggesting that they act on gamete fusion or later stages
of fertilization.
When fertilized eggs of sea urchin or starfish were
used as the test material, kalihinol F (13), an inhibitor of
topoisomerase I,19) inhibited the gastrulation of starfish.
Compounds 5–12 exhibited relatively weak or no
inhibitory activity on embryogenesis (Table 2).
These findings indicate that 1 is a highly selective
inhibitor of sperm motility, and that the structural
requirements for the activity are stringent. Although
several natural inhibitors of echinoderm fertilization
have been obtained from marine organisms, e.g.,
jaspisin,13,15) callyspongins A and B,16) and halenaquinol
sulfate,29,30) all of them inhibit sperm-egg fusion, but do
not affect sperm motility. There are very few, if any,
selective inhibitors of sperm motility.
Fig. 3. Effect of Exiguamide (2) on the Development of Sea Urchin
Embryos.
Embryos were cultured in the presence (panels A, C, and E) and
absence (panels B, D, and F) of 0.2 mg/ml of 2 from the 2-cell stage.
After they were cultured to the 16-cell stage, the embryos were
transferred to seawater. The photographs were taken 2.5 h (panels A
and B), 24 h (panels C and D), and 48 h (panels E and F) after
insemination. Bar indicates 20 mm.
Table 3. Inhibitory Effects of Formamides 2 and 14–21 on the
Spicule Formation and Fertilization of Winter Sea Urchin (Hemi-
centrotus pulcherrimus)
Minimum inhibitory concentration (mg/ml)
Compound
Spicule formation
Fertilization
Inhibitory activity of various formamides on spicule
formation of sea urchin
2
14
15
16
17
18
19
20
21
0.1
>100
>100
>100
>100
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
Exiguamide (2), a spirocyclic sesquiterpene analogue
with a formylamino group at C-6, inhibited the micro-
mere formation of sea urchin embryos and subsequent
spicule formation at inhibitory concentrations of
0.1–3.0 mg/ml (Fig. 3), as previously reported.5,6) On
the assumption that the formylamino group plays a
critical role in the inhibition of spicule formation of the
sea urchin embryo, we prepared several formamides
(14–21) by hydrolysis of the corresponding isonitriles
(5–12) and examined their effects on spiculogenesis and
fertilization.
As shown in Table 3, only 18 inhibited spicule
formation but did not affect fertilization. The other
synthetic formamides 14–17 and 19–21, affected neither
spicule formation nor fertilization. These findings
suggest that the structural requirements for the inhibitory
>100
>100
>100
activity on spicule formation of sea urchin are stringent.
In conclusion, the spiro[4.5]decene skeleton of (ꢀ)-
10-epi-axisonitrile-3 (1) and exiguamide (2) plays an
important role in exhibiting the selective biological
activity, and the functionality attached to the skeleton
is also crucial to the biological activity. Since the
sesquiterpene isocyanide (ꢀ)-10-epi-axisonitrile-3 (1) is
a major metabolite of the marine sponge Geodia exigua

1770
E. OHTA et al.
membrane fusion in echinoderm gametes with jaspisin,
a novel antihatching substance isolated from a marine
sponge. J. Biol. Chem., 269, 23262–23267 (1994).
14) Ohta, S., Kobayashi, H., and Ikegami, S., Isojaspisin:
a novel styryl sulfate from a marine sponge, Jaspis sp.,
that inhibits hatching of sea urchin embryos. Tetra-
hedron Lett., 35, 4579–4580 (1994).
15) Ohta, S., Kobayashi, H., and Ikegami, S., Jaspisin: a
novel styryl sulfate from the marine sponge, Jaspis
species. Biosci. Biotechnol. Biochem., 58, 1752–1753
(1994).
16) Uno, M., Ohta, S., Ohta, E., and Ikegami, S., Call-
yspongins A and B: novel polyacetylene sulfates from
the marine sponge Callyspongia truncata that inhibit
fertilization of starfish gametes. J. Nat. Prod., 59, 1146–
1148 (1996).
17) Uy, M. M., Ohta, S., Yanai, M., Ohta, E., Hirata, T., and
Ikegami, S., Exiguamide, a new spirocyclic sesquiter-
pene from the marine sponge Geodia exigua that inhibits
cell fate specification during sea urchin embryogenesis.
Biorg. Med. Chem. Lett., 12, 3037–3039 (2002).
18) Uy, M. M., Ohta, S., Yanai, M., Ohta, E., Hirata, T.,
and Ikegami, S., New spirocyclic sesquiterpenes
from the marine sponge Geodia exigua. Tetrahedron,
59, 731–736 (2003).
Thiele, it is suggested that its potent anti-fertilization
property brings about a the sponge-living marine
environment unsuitable for echinoderm reproduction.
Acknowledgments
We thank Mr. Hitoshi Fujitaka of Hiroshima Univer-
sity for NMR measurements. This study was supported
in part by a Grant-in-Aid from the Ministry of
Education, Culture, Sports, Science, and Technology
of Japan.
References
1) Chang, C. W. J., Naturally occurring isocyano/isothio-
cyanato and related compounds. Fortschr. Chem. Org.
Naturst., 80, 1–186 (2000).
2) Garson, M. J., and Simpson, J. S., Marine isocyanides
and related natural products: structure, biosynthesis and
ecology. Nat. Prod. Rep., 21, 164–179 (2004).
3) Blunt, J. W., Copp, B. R., Hu, W., Munro, M. H. G.,
Northcote, P. T., and Prinsep, M. R., Marine natural
products. Nat. Prod. Rep., 24, 31–84 (2007), and
previous reviews in the series.
4) Angerhofer, C. K., Pezzuto, J. M., Konig, G. M., Wright,
A. D., and Sticher, O., Antimalarial activity of sesqui-
terpenes from the marine sponge Acanthella klethra. J.
Nat. Prod., 55, 1787–1789 (1992).
5) Wright, A. D., Konig, G. M., Angerhofer, C. K.,
Greenidge, P., Linden, A., and Desqueyroux-Faundez,
R., Antimalarial activity: the search for marine-derived
natural products with selective antimalarial activity. J.
Nat. Prod., 59, 710–716 (1996).
6) Wright, A. D., Wang, H., Gurrath, M., Konig, G. M.,
Neumann, G., Loria, P., Foley, M., and Tilley, L.,
Inhibition of heme detoxification processes underlies the
antimalarial activity of terpene isonitrile compounds
from marine sponges. J. Med. Chem., 44, 873–885
(2001).
7) Singh, C., Srivastav, N. C., and Puri, S. K., In vivo active
antimalarial isonitriles. Bioorg. Med. Chem. Lett., 12,
2277–2279 (2002).
8) Hirota, H., Tomono, Y., and Fusetani, N., Terpenoids
with antifouling activity against barnacle larvae from the
marine sponge Acanthella cavernosa. Tetrahedron, 52,
2359–2368 (1996).
9) Okino, T., Yoshimura, E., Hirota, H., and Fusetani, N.,
New antifouling sesquiterpenes from four nudibranchs
of the family Phyllidiidae. Tetrahedron, 52, 9447–9454
(1996).
10) Chang, C. W. J., Patra, A., Baker, J. A., and Scheuer,
P. J., Kalihinols, multifunctional diterpenoid antibiotics
from marine sponges Acanthella spp. J. Am. Chem. Soc.,
109, 6119–6123 (1987).
11) Trimurtulu, G., and Faulkner, D. J., Six new diterpene
isonitriles from the sponge Acanthella cavernosa. J. Nat.
Prod., 57, 501–506 (1994).
19) Ohta, E., Ohta, S., Hongo, T., Hamaguchi, Y., Andoh,
T., Shioda, M., and Ikegami, S., Inhibition of chromo-
some separation in fertilized starfish eggs by kalihinol F,
a topoisomerase I inhibitor obtained from a marine
sponge. Biosci. Biotechnol. Biochem., 67, 2365–2372
(2003).
20) Mousseron, M., Jacquier, R., and Christol, H., Rear-
rangements acido-catalyses (1er memoire). Nouvelles
syntheses d0ꢂ-spirocetones. Bull. Soc. Chim. Fr., 346–
356 (1957).
21) Borch, R. F., Bernstein, M. D., and Durst, H. D.,
Cyanohydridoborate anion as a selective reducing agent.
J. Am. Chem. Soc., 93, 2897–2904 (1971).
22) Sasaki, T., Eguchi, S., and Katada, T., Synthesis of
adamantane derivatives. XXV. Synthesis and reactions
of 1- and 2-adamantyl isocyanides. J. Org. Chem., 39,
1239–1242 (1974).
23) Gillis, R. G., and Occolowitz, J. L., Isocyanides. III.
Electron impact study of aliphatic isocyanides. J. Org.
Chem., 28, 2924–2925 (1963).
24) Gokel, G. W., Widera, R. P., and Weber, W. P.,
Phase-transfer Hofmann carbylamine reaction: tert-butyl
isocyanide (2-Methylpropane, 2-isocyano). Org. Syn.,
55, 96–99 (1976).
25) van Dorsten, F. A., Wyss, M., Wallimann, T., and
Nicolay, K., Activation of sea-urchin sperm motility is
accompanied by an increase in the creatine kinase
exchange flux. Biochem. J., 325, 411–416 (1997).
26) Tombes, R. M., and Shapiro, B. M., Metabolite
channeling: a phosphorylcreatine shuttle to mediate high
energy phosphate transport between sperm mitochond-
rion and tail. Cell, 41, 325–334 (1985).
27) Winkler, M. M., Matson, G. B., Hershey, J. W. B.,
and Bradbury, E. M., 31P-NMR study of the activation
of the sea urchin egg. Exp. Cell Res., 139, 217–222
(1982).
12) Alvi, K. A., Tenenbaum, L., and Crews, P., Anthelmintic
polyfunctional nitrogen-containing terpenoids from
marine sponges. J. Nat. Prod., 54, 71–78 (1991).
13) Ikegami, S., Kobayashi, H., Myotoishi, Y., Ohta, S.,
and Kato, K. H., Selective inhibition of exoplasmic
28) Patra, A., Chang, C. W. J., Scheuer, P. J., Duyne, G. D.
V., Matsumoto, G. K., and Clardy, J., An unprecedented

Anti-Fertilization Activity of Marine Isocyanide
1771
triisocyano diterpenoid antibiotic from a sponge. J. Am.
Chem. Soc., 106, 7981–7983 (1984).
lation of CD spectra. Tetrahedron Lett., 26, 3833–3836
(1985).
29) Kobayashi, M., Shimizu, N., Kitagawa, I., Kyogoku, Y.,
Harada, N., and Uda, H., Absolute stereostructures of
halenaquinol and halenaquinol sulfate, pentacyclic
hydroquinones from the Okinawan marine sponge
Xestospongia sapra, as determined by theoretical calcu-
30) Ikegami, S., Kajiyama, N., Ozaki, Y., Myotoishi, Y.,
Miyashiro, S., Takayama, S., Kobayashi, M., and
Kitagawa, I., Selective inhibition of membrane fusion
events in echinoderm gametes and embryos by halena-
quinol sulfate. FEBS Lett., 302, 284–286 (1992).