Climacostol, a defense toxin of Climacostomum virens (protozoa, ciliata), and its congeners

Article abstract of DOI:10.1016/j.tet.2003.09.105

Climacostol (1), a defense toxin of the heterotrich ciliate Climacostomum virens was established as 5-(Z)-non-2-enyl-benzene-1,3-diol. The structure was rigorously confirmed by the total synthesis. The two congeners of climacostol contained in this ciliat

Full text of DOI:10.1016/j.tet.2003.09.105

Tetrahedron 60 (2004) 7041–7048
Climacostol, a defense toxin of Climacostomum virens
(protozoa, ciliata), and its congeners
Miyuki Eiraku Masaki,a Shouji Hiro,a Yoshinosuke Usuki,a Terue Harumoto,b
Masayo Noda Terazima,b Federico Buonanno,c Akio Miyakec and Hideo Iioa
,
*
aDepartment of Material Science, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan
bGraduate School, Nara Women’s University, Nara 630-8506, Japan
cDepartment of Molecular, Cellular and Animal Biology, University of Camerino, Camerino (MC) 62032, Italy
Received 22 July 2003; revised 17 September 2003; accepted 17 September 2003
Available online 19 June 2004
Abstract—Climacostol (1), a defense toxin of the heterotrich ciliate Climacostomum virens was established as 5-(Z)-non-2-enyl-benzene-
1,3-diol. The structure was rigorously confirmed by the total synthesis. The two congeners of climacostol contained in this ciliate were
determined as 5-(Z,Z)-undeca-2,5-dienyl-benzene-1,3-diol (2) and 5-(Z,Z,Z)-undeca-2,5,8-trienyl-benzene-1,3-diol (3).
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
obtained by preparative TLC on silica gel developed with
MeOH/CH2Cl2 7:93. The biologically active fraction
contained climacostol and compounds A and B. The
mixture were further purified by preparative TLC impreg-
nated with 15% silver nitrate which was prepared in
CH3CN8 (developing solvent MeOH/CH2Cl2 1:9) to give
three fractions A (Rf value¼0.31), B (Rf value¼0.17) and C
(Rf value¼0.05). The fraction A (1.0 mg), the fraction B
(0.1 mg) and the fraction C (0.2 mg) consisted, respectively,
of mostly pure climacostol (1), a 1:1 mixture of 1 and
new compound 2, and mostly pure new compound 3. We
have quitted further purification of the fraction B due to
its scarcity. LD50 of 1 for D. margaritifer (D3-I) was
0.71 mg/ml.6
The heterotrich ciliates Stentor coeruleus and Blepharisma
japonicum, respectively, have a blue pigment stentorin1 and
red pigments blepharismins2 in their extrusive organelles
(pigment granules).3 These pigments have been studied as
photoreceptors,4 but recently we showed that they also have
the function of chemical defense against predators.5 In
another heterotrich ciliate Climacostomum virens, we found
a defense toxin contained in its colorless extrusive
organelles (cortical granules),6 identified it as 5-(Z)-non-2-
enyl-benzene-1,3-diol, and named it climacostol.7 We also
found that this ciliate has two congeners of climacostol. In
this paper, we describe isolation, characterization and
synthesis of climacostol and the structural elucidation of
its congeners in detail.
2.2. Structure of climacostol and its congeners
The molecular formula of climacostol (1) was estimated as
C15H22O2 by high-resolution mass spectroscopy (HRMS):
m/z [M]þ found 234.1630, calcd 234.1620. In the prelimi-
2. Result and discussion
1
nary study, the H and 13C NMR spectra of a mixture of
2.1. Isolation of climacostol and its congeners
climacostol (1), 2 and 3 had been recorded on the machine
for 500 MHz, and the result was published in the
preliminary paper.7 Herein, pure climacostol (1) was
Whole cells (0.8 mL) of Climacostomum virens were dipped
in aqueous 75% EtOH (39 mL). After removal of the cells
by filtration and concentration of the filtrate, the residue was
partitioned between dichloromethane and water. From the
organic layer, a biologically active fraction which is highly
toxic against the raptorial ciliate Dileptus margaritifer was
1
submitted to measure the H and 13C NMR spectra on the
machine for 600 MHz. The 1H and 13C NMR spectra (Table
1) showed the presence of two phenolic OH protons and the
close resemblance of chemical shifts of the aromatic protons
at d 6.25 (2H, d, J¼1.9 Hz, H-4, 6) and 6.18 (1H, t,
J¼1.9 Hz, H-2) and carbons at d 156.8 (2C, C-1, 3), 144.4
(C-5), 108.0 (2C, C-4, 6) and 100.4 (C-2) of 1 to that of
olivetol (5-pentyl-benzene-1,3-diol) suggested that 1 is a
derivative of 5-alkenyl resorcinol. The 1H–1H COSY
Keywords: Biologically active compounds; Toxins; Phenols; Resorcinol;
Cobalt compounds.
*
Corresponding author. Tel.: þ81-6-6605-2562; fax: þ81-6-6605-3152;
e-mail address: iio@sci.osaka-cu.ac.jp
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.09.105

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M. E. Masaki et al. / Tetrahedron 60 (2004) 7041–7048
Table 1. 1H NMR (600 MHz) and 13C NMR (150 MHz) data for climacostol (1) in CDCl3
Position
dH
dC
COSY
HMBC
NOEsy
1,3
2
4,6
—
156.8 (2C)
100.4
108.0 (2C)
144.4
33.2
127.3
131.5
27.3
29.7
29.0
31.8
22.6
6.18 (1H, t, J¼1.9 Hz)
6.25 (2H, d, J¼1.9 Hz)
—
1, 3, 4, 6
1, 2, 3, 10
10
5
10
3.28 (2H, d, J¼6.1 Hz)
4, 6, 20
10
4, 5, 6, 20, 30
10, 40
40
10
20
5.50 (1H, m)
5.51 (1H, m)
30
40
10, 40, 50
20, 30, 50
30, 40, 60, 70
50, 70, 80
50, 60, 80, 90
60, 70, 90
70, 80
40
2.11 (2H, q, J¼6.9 Hz)
1.35–1.41 (2H, m)
1.25–1.34 (2H, m)
1.25–1.34 (2H, m)
1.25–1.34 (2H, m)
0.89 (3H, t, J¼6.9 Hz)
4.70 (2H, s)
30, 50
40
50
60
70
80
90
80
90
1,3 –OH
14.1
spectrum of 1 indicated that the presence of Ar–CH2–
CHvCH–CH2. The olefinic protons at d 5.50 (1H, m, H-20)
and 5.51 (1H, m, H-30) were correlated with the protons at d
3.28 (2H, d, J¼6.1 Hz, H-10) and 2.110(2H, q, J¼6.9 Hz,
H-40), which deduced the presence of a 2 -non-enyl group as
the side chain attached to resorcinol at the 5-position. The
side chain of 1, in especially C-60, 70, 80 was assigned by
the HMQC and HMBC spectra (Table 1). The EI-MS/MS
experiment was carried out in positive mode by choosing
the [M]þ ion (m/z 234) as the precursor ion. The fragment
ion peaks of the EI-MS/MS spectrum were observed at m/z
219 [M2CH3]þ, 205 [M2C2H5]þ, 191 [M2C3H7]þ, 177
[M2C4H9]þ, 163 [M2C5H11]þ, 149 [M2C6H13]þ, 124
[MþH–CHvCHC6H13]þ, which confirmed the presence
benzene-1,3-diol. Previously, the structure of 5-(Z)-hepta-
dec-2-enyl-benzene-1,3-diol, a derivative of 5-(20-alkenyl)-
resorcinol, was reported as a possible dermatitis allergen
from the latex of a mango.9 The position of the olefin was
assumed from the chemical shifts0 of 1H NMR (CCl4,
60 MHz) d 5.33 (2H, br t, H-20, 3 ), 2.25–2.60 (2H, m,
H-10), 1.80–2.20 (2H, m, H-40), and fragment peaks of
MS. The incompatibility of the data to that of climacostol
suggests the necessity of reinvestigating the structure of
the compound from the mango. To our best knowledge,
climacostol is the first example of 5-alkenylresorcinol
having a double bond at the 20-position.
Analysis of 1H NMR for compound 2 was carried out in the
1
of double bonds at D20,3 as shown in Figure 1. The
1:1 mixture of climacostol and 2. The H NMR spectra
0
Z-configuration of D2 ,3 -olefin was determined by the
observation of NOE between the 10- and 40-methylene
protons. Thus, the structure of a new toxic compound
climacostol (1) was determined to be 5-(Z)-non-2-enyl-
(Table 2) of 2 showed the presence of two phenolic OH
protons, and the aromatic protons at d 6.26 (2H, d, J¼
2.3 Hz, H-4, 6) and 6.18 (1H, t, J¼2.3 Hz, H-2) suggested
1
that 2 is also a derivative of 5-alkenyl resorcinol. The H
Figure 1. Climacostol and its congeners from Climacostomum virens.
Table 2. 1H NMR (600 MHz) data of 2 and 3 in CDCl3
2
3
Position
dH
NOEsy
dH
COSY
NOEsy
2
4,6
6.18 (1H, t, J¼2.3 Hz)
6.26 (2H, d, J¼2.3 Hz)
3.31 (2H, d, J¼6.3 Hz)
5.47–5.56 (1H, m)
5.47–5.56 (1H, m)
2.87 (2H, t, J¼6.5 Hz)
5.34–5.44 (1H, m)
5.34–5.44 (1H, m)
2.04–2.08 (2H, m)
ND
6.19 (1H, t, J¼2.3 Hz)
6.25 (2H, d, J¼2.3 Hz)
3.32 (2H, d, J¼6.6 Hz)
5.51–5.55 (1H, m)
5.51–5.55 (1H, m)
2.91 (2H, t, J¼5.6 Hz)
5.38–5.42 (1H, m)
5.38–5.42 (1H, m)
2.82 (2H, t, J¼5.5 Hz)
5.31–5.36 (1H, m)
5.38–5.42 (1H, m)
2.06–2.08 (2H, m)
0.97 (3H, t, J¼7.5 Hz)
4.68 (2H, s)
4,6
2
10
10
40
20
6,40
20
10
40
30
40
10, 70
30, 50
40
10, 70
50
60
70
a
70
40
60, 80
70
40, 100
80
90
ND
ND
ND
4.67 (2H, s)
100
90, 110
100
a
100
70
110
1,3 –OH
ND, not determined.
a
Observed in benzene-d6.

M. E. Masaki et al. / Tetrahedron 60 (2004) 7041–7048
7043
NMR spectrum also showed the presence of the olefinic
protons at d 5.47–5.56 (2H, m, H-20, 30), 5.34–5.44 (2H, m,
H-50, 60), the b0enzylic methylene protons at d 3.31 (2H, d,
J¼6.3 Hz, H-1 ), the double-allylic methylene protons at d
2.87 (2H, t, J¼6.5 Hz, H-40), and the allylic methylene
protons at d 2.04–2.08 (2H, m, H-70), indicating that the
presence of a dienyl group as the side chain attached to
resorcinol. Although the carbon length of 2 could not be
determined from the 1H NMR spectrum, comparison of the
LC/ESI-MS/MS spectrum (vide infra) with that of 3 allowed
us to elucidate the side chain of 2. The LC/ESI-MS
spectrum showed m/z 259 [M2H]2 ion peak, which
suggested the carbon length of the side chain as C11. The
LC/ESI-MS/MS experiment was carried out on negative
ions by choosing the [M2H]2 ion (m/z 259) as the precursor
ion. The fragment ion peaks of the LC/ESI-MS/MS
spectrum were observed at m/z 244 [M2H–CH3]2, 230
[M2H–C2H5]2, 215 [M22H–C3H7]2, 201 [M22H–
C4H9]2, 187 [M22H–C5H11]2, 161 [M22H–CHvCHC5-
H11]2, 147 [M22H–CH2CHvCHC5H11]2, 122 [M2H–
protons at d 2.82 (2H, t, J¼5.5 Hz, H-70), and the allylic
methylene protons at d 2.06–2.08 (2H, m, H-100), which
0
0
suggested the0side chain of 3 is the skipped triene at D2 ,3
,
0
0
0
D5 ,6 and D8 ,9 with the carbon length of C11. The protons at
d 2.06–2.08 (2H, m, H-100) and the triplet protons at d 0.97
(3H, t, J¼7.5 Hz, H-110) showed the terminal ethyl function.
Thus, the undeca-20,50,80-trienyl group is added to resorcinol
at the 5-position 0as the side chain. The Z-configuration of
0
0
0
0
0
D2 ,3 -olefin, D5 ,6 -olefin and D8 ,9 -olefin were determined,
respectively,0by the observation of N0OE between the 10- and
40-protons, 4 - and 70-protons, and 7 - and 100-protons. The
proposed structure was further supported by LC/ESI-MS/
MS spectrum. The LC/ESI-MS/MS experiment was carried
out on negative ions by choosing the [M2H]2 ion (m/z 257)
as the precursor ion. The fragment peaks in the LC/ESI-MS/
MS spectrum of 3 were observed at m/z 227 [M22H–
C2H5]2, 202 [M2H–CHvCHC2H5]2, 187 [M22H–CH2-
CHvCHC2H5]2, 161 [M22H–CHvCHCH2CHvCHC2-
H5]2, 147 [M22H–CH2CHvCHCH2CHvCHC2H5]2,
122 [M2H–CHvCHCH2CHvCHCH2CHvCHC2H5]2,
108 [M2H–CH2CHvCHCH2CHvCHCH2CHvCHC2-
H5]2, which confirmed the position of three olefins at
CHvCHCH2CHvCHC5H11]2,
108
[M2H–CH2
CHvCHCH2CHvCHC5H11]2, which confirmed the pre-
sence of double bonds at D2,3 and D5,6 as shown in Figure 1.
The configuration of the double bonds was determined to
be both cis by the observation of NOE’s between the 10- and
40-methylene protons, and the 40- and 70-methylene protons.
Thus, the structure of 2 was established as 5-(Z,Z)-undeca-
2,5-dienyl-benzene-1,3-diol.
0
0
0
0
0
0
D2 ,3 , D5 ,6 and D8 ,9 . Thus, the structure of 3 was
determined to be 5-(Z,Z,Z)-undeca-2,5,8-trienyl-benzene-
1,3-diol.
2.3. Synthesis of climacostol
The structure and biological activity of climacostol were
confirmed by the synthesis of 1.10 The synthesis of
climacostol (1) was achieved in six steps (Scheme 1).
Phenolic hydroxyl groups of commercially available 3,5-
dihydroxybenzaldehyde was protected with tert-butyldi-
methylsilyl groups, and reacted with 1-octyne using n-BuLi
at 278 8C to give the acetylenic adduct 5 in 91% yield.
Reductive removal of the hydroxyl group at C10 of the
adduct 5 was unsuccessful, because of migration of the
unsaturated bond. The triple bond of 5 was protected by
treatment with dicobalt octacarbonyl to afford the corre-
sponding cobalt complex 6 in 64% yield. The hydroxyl
group at the benzylic position of 6 was activated by
complexation of triple bond with dicobalt octacarbonyl and
the reduction of the hydroxyl group with triethylsilane in the
presence of BF3·OEt211 proceeded to afford 7 in 89% yield.
The molecular formula of compound 3 was estimated as
C17H21O2 by FAB-HRMS (m/z [M2H]2 found 257.1528
calcd 257.1542). The 1H NMR spectra (Table 2) showed the
presence of two phenolic OH protons, and the aromatic
protons at d 6.25 (2H, d, J¼2.3 Hz, H-4, 6) and 6.19 (1H, t,
J¼2.3 Hz, H-2), suggested that 3 is also a derivative of
1
5-alkenyl resorcinol. The H–1H COSY NMR spectrum
of 3 showed that the olefinic proton at d 5.51–5.55 (1H, m,
H-20), the olefinic protons at d 5.51–5.55 (1H, m, H-30) and
5.38–5.42 (1H, m, H-50), the olefinic protons at d 5.38–5.42
(1H, m, H-60) and 5.31–5.36 (1H, m, H-80), and the olefinic
proton at d 5.38–5.42 (1H, m, H-90) were correlated,
respectively, to the benzylic protons at d 3.32 (2H, d,
J¼6.6 Hz, H-10), the double-allylic methylene protons at d
2.91 (2H, t, J¼5.6 Hz, H-40), the double-allylic methylene
Scheme 1. Reagents and conditions. (a) 1-octyne, n-BuLi, THF, 278 8C, 91%. (b) Co2(CO)8, Et2O, 220 8C!25 8C, 64%. (c) Et3SiH, BF3·OEt2, CH2Cl2, 89%.
(d) n-Bu3SnH, benzene, 65 8C, 93%. (e) 70% HF·pyridine, THF, 86%.

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M. E. Masaki et al. / Tetrahedron 60 (2004) 7041–7048
Figure 2. Synthetic analogues of climacostol.
In this reaction, a regio- and stereoselective hydrosilylation
product was also formed as a minor side product, whose
structure was confirmed by leading to 8. The configuration
of the olefin in 8 has not been determined. The hydrosilyl-
ation of an acetylene cobalt complex was also reported by
Isobe et al.12 The Z-olefin 9 was obtained by reductive
decomplexation of the cobalt carbonyl under the Isobe’s
conditions13 in 93%. Climacostol (1) was finally synthe-
sized by deprotection of the silyl group of 9 in HF·pyridine.
Synthetic climacostol (1) exhibited identical properties to
those for the natural product. The toxic activity against D.
margaritifer was as high as that of the natural product.
Recently, Mori et al. reported a simpler synthesis of
climacostol based on the Wittig reaction method starting
from [3,5-bis-(tert-butyl-dimethyl-silanyloxy)-phenyl]-
acetic acid methyl ester.14
quantitative yield. Z-olefin 9 was reduced with 10 wt%
palladium-on-charcoal as a catalyst to give 12 in 80% yield,
and deprotection of the silyl group afforded the alkyl
derivatives 13 in 83% yield (Fig. 2).
2.4. The biosynthesis and the biological activity of
climacostol
We consider that there is a close relation between the
biosynthesis of climacostol and that of stentorin, a toxic
pigment from Stentor coeruleus, though the chemical
structure of them is considerably different to each other. A
hypothetical pathway for the biosynthesis of long-chain
resorcinolic lipids15 would be also true for the biosynthesis
of climacostol and its congeners. Namely, climacostol (C15)
and its congeners (C17) would be synthesized from the C16-
and C18-polyketide, respectively, with the cyclization and
the decarboxylation, as shown in Figure 3. Stentorin is also
likely generated from C16-polyketide accompanied by the
addition reaction of C1 to form isopropyl group resulting
2,4,5,7-tetrahydroxy-3-isopropylanthrone, and followed by
the dimerization reaction of the anthrone.2a,16 The finding of
The alkynyl and alkyl derivatives of climacostol were also
synthesized from 7 and 9, respectively, in order to do the
biological evaluation. Thus, 7 was oxidized with I2 to yield
10 in 90% yield, followed by deprotection of the silyl group
by HF·pyridine to afford the alkynyl derivative 11 in
Figure 3. The putative biosynthesis of climacostol (1) and its congeners, and stentorin.

M. E. Masaki et al. / Tetrahedron 60 (2004) 7041–7048
7045
climacostol as defense toxin of C. virens is consisting with
the suggestion that the primary function of stentorin and
blepharismin is the defense against the predator rather than
photoreception.5b,17
60F254 0.25 mm), and preparative TLC was done on Merck
silica gel plates (Art5744 Kiesel gel 60F254 0.5 mm). Silica
gel column chromatography was carried out on Daisogel
IR-60 (63/210 mm).
Cell–cell interaction by means of extrusomes (extrusive
organelles in protist) in ciliates was reviewed by Miyake.17
Secondary metabolites of ciliates were scarcely studied
except the pigments mentioned above, keronopsin from
Pseudokeronopsis rubra,18 and euplotin,19 raikovenal20 and
epoxyfocardin21 from Euplotes species. The structure,
synthesis and biological activity of resorcinolic lipids
were recently reviewed in detail.15,22 More than 100 of
5-alkyl and 5-alkenylresorcinol homologues have been
found mainly from a variety of higher plants, including
those in the Proteaceae, Anacardiaceae, Ginkogoaceaen
and Graminae families, but rarely found in animal and
microorganism. Resorcinolic lipids are reported to show
significant biological activities,15,22 including antibacterial,
antiparasitic and cytotoxic activity as growth regulator,
inhibition of DNA and RNA synthesis,23 inhibitory effect on
enzymes,24 nematicidal activities,25 interaction with bio-
logical membranes, and as modulator of lipid oxidation. In
recent studies, climacostol was found to cleave DNA in the
presence of CuCl2,26 and also climacostol specifically
inhibits the respiratory chain complex I in mitochondria.27
4.2. Microbial material
Stock W-24 of Climacostomum virens, provided by Dr.
Tavrovskaya (see Acknowledgement), was originally green
due to symbiotic algae. By culturing in the dark, colorless
clones (200–300 mm in length, 150–200 mm in width)
were obtained. Stock D3-I of D. margaritifer (ca. 800 mm in
length, ca. 60 mm in width) formerly D. anser, provided by
Dr. Tavrovskaya, was used as a predator of C. virens. Both
ciliates were grown on Sathrophilus sp., a small ciliate
grown on the boiled lettuce medium, and concentrated by
centrifugation, washed by and suspended in SMB-III,28
a
balanced salt solution (called SMB below).6 Culture,
handling of ciliates and experiments were performed at
24^1 8C.
4.3. Biological assay
Toxicity-test of climacostol: Ten cells of a ciliate were
placed in 250 mL of SMB solution of climacostol of various
concentrations, kept in a dark moist chamber, and the
number of surviving cells were counted after 24 h. The LD50
concentration of climacostol for a ciliate was obtained based
on the concentration–survival curve of the ciliate. At the
late stage of this work, we noticed that the way to dilute the
stock solution of climacostol (5 mg/mL ethanol solution)
influences the result of toxicity measurement of climacostol.
For example, if the ethanol solution was first diluted at 100,
10, and 5 mg/mL and then further diluted, measured LD50
concentrations for D. margaritifer (D3-I) were 1.8, 0.88,
and 0.71 mg/mL, respectively.6
Biological activities including lethal toxicity against the
predatory ciliate D. margaritiefer of the congeners 2 and 3,
and the synthetic alkyl and alkynyl derivatives will be
reported in due course.
3. Conclusion
A potent toxin against D. margaritifer was found in the 75%
ethanol extract of C. virens. A new toxin climacostol of
C. virens, a lethal toxin against the predator, was isolated
and its structure was determined. The two congeners of
climacostol were also isolated and their structures were
determined. Furthermore, the synthesis of climacostol was
carried out and the structure of the natural product was
confirmed.
4.4. Extraction and isolation
Whole cells (0.8 mL) of Climacostomum virens were dipped
in aqueous 75% EtOH (39 mL). After removal of the cells
by filtration, the EtOH solution was concentrated under the
reduced pressure. To the residue was added water and then
extracted with CH2Cl2. The organic layer was washed with
brine, dried over Na2SO4 and evaporated under reduced
pressure. The residue was subjected to the preparative TLC
(developed with MeOH/CH2Cl2, 7:93), and then eluted with
MeOH/CH2Cl2, 1:4. The biologically active fraction was
further purified by the preparative TLC impregnated with
15% silver nitrate which was prepared in CH3CN.8 The
residue was dissolved in CH2Cl2, charged on the preparative
TLC impregnated with 15% silver nitrate in CH3CN. The
plate was developed with MeOH/CH2Cl2, 1:9. Three
fractions were observed by 2.5% FeCl3·6H2O in EtOH.
Each of fractions was scraped off and eluted with MeOH/
CH2Cl2, 1:4 to give climacostol (1) (1.0 mg, Rf value¼
0.31), a 1:1 mixture of 1 and 2 (0.1 mg, Rf value¼0.17), and
3 (0.2 mg, Rf value¼0.05).
4. Experimental
4.1. General
1H and 13C NMR spectra were recorded on a Bruker DRX-
600 for 600 MHz, Varian Unity-500 for 500 MHz, JEOL
JNM-LA400 for 400 MHz or JEOL JNM-LA300 for
300 MHz. Chemical shifts (d) are given in ppm relative to
1
tetramethysilane (d 0.00) or CHCl3 (d 7.26) for H NMR
and d 77.0 for 13C NMR as internal standard. Mass spectra
were obtained on a JEOL JMS-700T. IR spectra were
obtained on a JUSCO A-100. Reaction solvents were
reagent grade. Diethyl ether and tetrahydrofuran (THF)
were distilled from sodium benzophenone ketyl. Dichloro-
methane (CH2Cl2) was distilled from phosphorous oxide.
Dimethyformamide (DMF) was distilled from calcium
hydride. Analytical thin-layer chromatography (TLC) was
performed on Merck silica gel plates (Art5715 Kiesel gel
1
4.4.1. Climacostol (1). H NMR (CDCl3, 600 MHz) and
13C NMR (CDCl3, 150 MHz), see Table 1; EI-MS m/z 234
[M]þ; EI-MS/MS m/z 219 [M2CH3]þ, 205 [M2C2H5]þ,
191 [M2C3H7]þ, 177 [M2C4H9]þ, 163 [M2C5H11]þ, 149

7046
M. E. Masaki et al. / Tetrahedron 60 (2004) 7041–7048
[M2C6H13]þ, 124 [MþH–CHvCHC6H13]þ. HRMS
(FABþ) calcd for C15H22O2 [M]þ 234.1620, found
234.1630.
1 M HCl. The organic layer was dried over Na2SO4,
evaporated under reduced pressure, and the residue was
purified by silica gel column chromatography (n-hexane/
1
AcOEt, 9:1) to give 6 (2.06 g, 64%): H NMR (CDCl3,
4.4.2. 5-(Z,Z)-Undeca-2,5-dienyl-benzene-1,3-diol (2). 1H
NMR (CDCl3, 600 MHz), see Table 2; LC/ESI-MS
(in negative mode) m/z 259 [M2H]2; LC/ESI-MS/MS
(in negative mode) m/z 244 [M2H–CH3]2, 230
[M2H–C2H5]2, 215 [M22H–C3H7]2, 201 [M22H–
C4H9]2, 187 [M22H–C5H11]2, 161 [M22H–CHvCHC5-
H11]2, 147 [M22H–CH2CHvCHC5H11]2, 122 [M2
H–CHvCHCH2CHvCHC5H11]2, 108 [M2H–CH2-
CHvCHCH2CHvCHC5H11]2. HRMS (EIþ) calcd for
C17H24O2 [M]þ 260.1776, found 260.1763.
400 MHz) d 6.54 (2H, d, J¼1.7 Hz, H-2, 6), 6.23 (1H, t,
J¼1.7 Hz, H-40), 5.76 (1H, d, J¼3.2 Hz, H-10), 2.77 (1H, t,
J¼5.6 Hz, H-4 ), 2.74 (1H, t, J¼6.0 Hz, H-40), 2.23 (1H, d,
J¼3.2 Hz, –OH), 1.58–1.69 (2H, m, H-50), 1.42–1.49 (2H,
m), 1.31–1.38 (4H, m), 0.98 (18H, s, Si–C(CH3)3), 0.91
(3H, t, J¼7.1 Hz, H-90), 0.185 (6H, s, Si–CH3), 0.179 (6H,
s, Si–CH3); 13C NMR (CDCl3, 100 MHz) d 199.9 (3C),
199.5 (3C), 156.8 (2C), 146.2, 111.6, 110.3 (2C), 101.5,
98.9, 73.9, 33.7, 31.9, 31.6, 29.3, 25.6 (6C), 22.6, 18.2 (2C),
14.0, 24.4 (4C); IR (nujol) 2975, 2950, 2880, 2120, 2080,
2050, 1603, 1470, 1390, 1350, 1265, 1180, 1040, 1020, 845,
790, 730 cm21; FAB-MS m/z 678 [M23CO]þ 650
[M24CO]þ, 622 [M25CO]þ, 594 [M26CO]þ; HRMS
(FABþ) calcd for C30H48O6Si2Co2 [M23CO]þ 678.1653,
found 678.1664.
4.4.3. 5-(Z,Z,Z)-Undeca-2,5,8-trienyl-benzene-1,3-diol
1
(3). H NMR (CDCl3, 600 MHz), see Table 2; LC/ESI-
MS (in negative mode) m/z 257 [M2H]2; LC/ESI-MS/MS
(in negative mode) m/z 227 [M22H–C2H5]2, 202 [M2H–
CHvCHC2H5]2, 187 [M22H–CH2CHvCHC2H5]2,
161 [M22H–CHvCHCH2CHvCHC2H5]2, 147 [M2
2H–CH2CHvCHCH2CHvCHC2H5]2, 122 [M2H–
CHvCHCH2CHvCHCH2CHvCHC2H5]2, 108 [M2H–
CH2CHvCHCH2CHvCHCH2CHvCHC2H5]2. HRMS
(FAB2) calcd for C17H21O2 [M2H]2 257.1542, found
257.1528.
4.5.3. 1,3-Bis-(tert-butyl-dimethyl-silanyloxy)-5-non-2-
ynyl-benzene cobalt complex (7). To a solution of 6
(1.1 g, 1.4 mmol) and Et3SiH (0.22 mL, 1.4 mmol) in
CH2Cl2 (14 mL) was added BF3·OEt2 (0.16 mL,
1.4 mmol) dropwise under the argon atmosphere. After the
mixture was refluxed for 30 min, the reaction mixture was
added to saturated aqueous NaHCO3, extracted with
CH2Cl2. The organic layer was washed with brine, dried
over Na2SO4, and the solvent was removed under reduced
pressure to give 7 (960 mg, 89%), and the hydrosilylation
product as a minor product whose tert-butyldimethysilyl
groups were removed by HF·pyridine to afford 8; data for 7:
1H NMR (CDCl3, 400 MHz) d 6.36 (2H, d, J¼2.1 Hz,
H-4,6), 6.22 (1H, t, J¼0 2.1 Hz, H-2), 3.95 (2H, s, H-10),
2.78–2.82 (2H, m, H-4 ), 1.59–1.67 (2H, m, H-50), 1.42–
1.48 (2H, m), 1.31–1.37 (4H, m), 0 0.98 (18H, s, Si–
C(CH3)3), 0.91 (3H, t, J¼7.3 Hz, H-9 ), 0.18 (12H, s, Si–
CH3); 13C NMR (CDCl3, 100 MHz) d 199.9 (3C), 199.5
(3C), 156.75, 156.67, 146.1, 111.5, 110.3 (2C), 101.5,
98.9, 73.9, 33.9, 31.9, 31.6, 29.3, 25.6 (6C), 22.6, 18.1 (2C),
14.0, 24.5 (4C); IR (nujol) 2975, 2950, 2880, 2120, 2075,
2050, 1603, 1480, 1470, 1390, 1350, 1265, 1180,
4.5. Synthesis of climacostol and its derivatives
4.5.1. 1-[3,5-Bis-(tert-butyl-dimethyl-silanyloxy)-phe-
nyl]-non-2-yn-1-ol (5). 1-Octyne (720 mg, 6.5 mmol) was
dissolved in THF (13 mL) and cooled to 278 8C under an
argon atmosphere, and n-BuLi (1.6 M solution in hexane,
3.4 mL, 5.5 mmol) was added dropwise. After the mixture
was stirred for 1.5 h at 278 8C, a solution of 3,5-bis-(tert-
butyl-dimethyl-silanyloxy)-benzaldehyde
(4)
(2.0 g,
5.5 mmol) in THF (10 mL) was added at 278 8C. The
reaction temperature was gradually raised to 25 8C and
stirred overnight. To the reaction mixture was added
saturated aqueous NH4Cl and extracted with diethyl ether.
The organic layer was washed with brine, and dried over
Na2SO4. The solvent was removed under reduced pressure
to give 5 (2.36 g, 91%): 1H NMR (CDCl3, 400 MHz) d 6.66
(2H, d, J¼2.20Hz, H-2, 6), 6.28 (1H, t, J¼2.2 Hz, H-4), 5.31
(1H, br s, H-1 ), 2.263 (1H, t, J¼7.1 Hz, H-40), 2.258 (1H, t,
J¼7.1 Hz, H-40), 2.04 (1H, br s, 10-OH), 1.50–1.57 (2H, m,
H-50), 1.36–1.43 (2H, m), 1.26–1.34 (4H, m), 0.98 (18H, s,
Si–C(CH3)3), 0.88 (3H, H-90), 0.20 (12H, s, Si–CH3); 13C
NMR (CDCl3, 100 MHz) d 156.6 (2C), 143.3, 111.70,
111.65 (2C), 87.4, 79.8, 64.6, 31.3, 28.6 (2C), 25.7 (6C),
22.5, 18.8, 18.2 (2C), 14.0, 24.4 (4C); IR (neat) 2980, 2955,
2925, 2880, 1605, 1465, 1405, 1380, 1350, 1270, 1175,
1040, 1015, 990, 950, 840, 790, 750, 690, 675 cm21; EI-MS
m/z 476 [M]þ; HRMS (FABþ) calcd for C27H48O3Si2 [M]þ
476.3142, found 476.3157.
1040, 1020, 840, 790 cm21
;
FAB-MS m/z 662
[M23CO]þ, 635 [M23COþH]þ, 606 [M25CO]þ, 461
[M2Co2(CO)6þH]þ; HRMS (FABþ) calcd for
C30H48O5Si2Co2 [M23CO]þ 662.1704, found 662.1714;
1
data for 8: H NMR (CDCl3, 400 MHz) d 6.20 (2H, d,
J¼1.7 Hz, H-4,6), 6.15 (1H, t, J¼1.7 Hz, H-2), 5.920(2H, t,
J¼6.8 Hz, H-30), 4.66 (2H, s, OH), 3.37 (2H, s, H-1 ), 2.14
(2H, q, J¼6.8 Hz, H-40), 1.37–1.45 (2H, m, H-50),0 1.20–
1.30 (6H, m, H-60, 70, 80), 0.87 (3H, t, J¼7.1 Hz, H-9 ), 0.83
(9H, t, J¼7.9 Hz, Si–CH2CH3), 0.46 (6H, q, J¼7.9 Hz, Si–
CH2CH3).
4.5.4. 1,3-Bis-(tert-butyl-dimethyl-silanyloxy)-5-non-2-
enyl-benzene (9). To a solution of 7 (130 mg, 0.17 mmol)
in benzene (1.7 mL) was added n-Bu3SnH (0.09 mL,
0.34 mmol). After the reaction mixture was stirred for 3 h
at 65 8C, the solvent was evaporated and then the residue
was purified by silica gel column chromatography with
n-hexane to give 9 (73 mg, 93%): 1H NMR (CDCl3,
400 MHz) d 6.30 (2H, d, J¼2.2 Hz, H-4, 6), 6.17 (1H, t,
J¼2.2 Hz, H-2), 5.50 (2H, t, J¼4.8 Hz, H-20, 30), 3.27 (2H,
4.5.2. 1-[3,5-Bis-(tert-butyl-dimethyl-silanyloxy)-phe-
nyl]-non-2-yn-1-ol cobalt complex (6). To a solution of 5
(2 g, 4.2 mmol) in diethyl ether (5 mL) was added a solution
of Co2(CO)8 (2.1 g, 6.1 mmol) in diethyl ether (20 mL) at
220 8C under an argon atmosphere. The cooling bath was
removed and the reaction mixture was stirred for 17.5 h at
room temperature. The reaction mixture was washed with

M. E. Masaki et al. / Tetrahedron 60 (2004) 7041–7048
7047
d, J¼4.8 Hz, H-10), 2.12 (2H, m, H-400), 1.42–1.52 (2H, m,
H-50), 1.21–1.41 (6H, m, H-60, 70, 8 ), 0.98 (18H, s, Si–
C(CH3)3), 0.90 (3H, m, H-90), 0.18 (12H, s, Si–CH3);
13C NMR (CDCl3, 100 MHz) d 156.4 (2C), 143.2, 131.0,
127.8, 113.5 (2C), 109.5, 33.4, 31.8, 29.7, 29.0, 27.3,
25.7 (6C), 22.7, 18.2 (2C), 14.1, 24.5 (4C). IR (neat) 2950,
2925, 2850, 1590, 1450, 1335, 1250, 1160, 1020, 1000,
830, 775 cm21; FAB-MS m/z 463 [MþH]þ, HRMS
(FABþ) calcd for C27H50O2Si2 [M]þ 462.3349, found
462.3342.
4.5.8. 1,3-Bis-(tert-butyl-dimethyl-silanyloxy)-5-nonyl-
benzene (12). To a solution of 9 (334 mg, 0.72 mmol) in
MeOH (14 mL) was added 10% palladium-on-charcoal
catalyst and the mixture was stirred under a hydrogen
atmosphere at 25 8C for 1 h. The reaction mixture was
filtered through the Celite pad, and the filtrate was
evaporated under reduced pressure and then purified by
silica gel column chromatography (n-hexane/diethyl ether,
40:1) to give 12 (270 mg, 80%): 1H NMR (CDCl3,
400 MHz) d 6.28 (2H, d, J¼2.1 Hz, H-4, 6), 6.16 (1H, t,
J¼2.1 Hz, H-2), 2.460 (2H, t, J¼0 7.8 Hz, H-10), 1.24–1.30
(14H, m, H-20, 30, 4 , 50, 60, 7 , 80), 0.97 (18H, s, Si–
C(CH3)3), 0.88 (3H, t, J¼6.8 Hz, H-90), 0.18 (12H, s, Si–
CH3).
4.5.5. 5-(Z)-Non-2-enyl-benzene-1,3-diol (climacostol
(1)). To a solution of 9 (87.7 mg, 0.19 mmol) in THF
(1.1 mL) was added 70% HF·pyridine (0.19 mL) at 0 8C
under an argon atmosphere. After the mixture was stirred for
1 h at 0 8C, the reaction temperature was gradually raised to
25 8C. The reaction mixture was added to saturated aqueous
NaHCO3, and extracted with diethyl ether. The organic
layer was washed with 1 M HCl, dried over Na2SO4, and
evaporated under reduced pressure. The residue was
purified by silica gel column chromatography (n-hexane/
4.5.9. 5-Nonyl-benzene-1,3-diol (13). To a solution of 12
(250 mg, 0.54 mmol) in THF (18 mL) dissolved in the
Teflon vial was added 70% HF·pyridine (1.46 mL) dropwise
using the Teflon syringe at 0 8C under an argon atmosphere.
After the reaction temperature was gradually raised to 25 8C
over 2 h, the reaction mixture was quenched with saturated
aqueous NaHCO3, added 1 M HCl and then extracted with
AcOEt. The organic layer was dried over anhyd. MgSO4,
evaporated under reduced pressure and then purified by
silica gel column chromatography (n-hexane/AcOEt, 5:2)
1
AcOEt, 1:1) to give climacostol (1) (38.3 mg, 86%). H
NMR and 13C NMR spectra were identical with those of
natural 1; IR (nujol) 2980, 2960, 2880, 1615, 1480, 1390,
1350, 1320, 1170, 1020, 980, 850 cm21; LC/ESI-MS/MS
(in negative mode) m/z 217 [M22H–CH3]2, 203 [M22H–
C2H5]2, 189 [M22H–C3H7]2, 175 [M22H–C4H9]2, 161
[M22H–C5H11]2, 147 [M22H–C6H13]2, 122 [M2H–
CHvCHC6H13]2, 108 [M2H–CH2CHvCHC6H13]2.
1
to give 13 (105 mg, 83%): H NMR (CDCl3, 400 MHz) d
6.25 (2H, d, J¼2.2 Hz, H-4, 6), 6.18 (1H, t, J0¼2.2 Hz, H-2),
4.82 (2H, s, OH), 2.48 (2H, t, J0¼7.8 Hz, H-1 )0, 1.56 (2H, m,
H-20), 1.23–1.29 (12H, m, H-3 , 40, 50, 60, 70, 8 ), 0.88 (3H, t,
J¼7.0 Hz, H-90).
4.5.6. 1,3-Bis-(tert-butyl-dimethyl-silanyloxy)-5-non-2-
ynyl-benzene (10). To
a solution of 7 (211 mg,
0.28 mmol) in THF (5 mL) was added I2 (0.50 g,
3.94 mmol) in THF (10 mL). After the mixture was stirred
at 25 8C for 2 h, was added saturated aqueous NaHCO3 and
saturated aqueous NaHSO3 at 0 8C, and extracted with
diethyl ether. The organic layer was washed with brine, and
dried over anhyd. MgSO4. The solvent was removed under
reduced pressure and then purified by silica gel column
chromatography (n-hexane/diethyl ether, 40:1) to give 10
(117 mg, 90%): 1H NMR (CDCl3, 400 MHz) d 6.46 (2H, d,
J¼2.2 Hz, H-4, 6), 6.19 (1H, t, J¼2.2 Hz, H-2), 3.45 (2H, t,
J¼2.4 Hz, H-10), 2.18–2.23 (2H, m, H-40), 1.48–1.54 (2H,
m, H-50), 1.34–1.44 (2H, m, H-60), 1.27–1.34 (4H, m, H-70,
80), 0.97 (18H, s, Si–C(CH3)3), 0.89 (3H, t, J¼7.0 Hz, H-90),
0.19 (12H, s, Si–CH3).
Acknowledgements
Thanks are due to the Analytical Division at Osaka City
University for analyses, mass spectra measurements
and 600 MHz NMR measurements. We thank Dr.
M. Tavrovskaya, Institute of Cytology, Russian Academy
of Sciences, St. Petersburg for the strain of Climacostomum
virens and the strain of D. margaritifer. This work was
partially supported by Grand-in-Aid for Scientific Research
of MEXT Japan, and Italian MURST, which are gratefully
appreciated.
References and notes
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After the reaction temperature was gradually raised to 25 8C
over 2 h, the reaction was quenched with saturated aqueous
NaHCO3, and was added 1 M HCl, and then extracted with
AcOEt. The organic layer was dried over anhyd. MgSO4,
evaporated under reduced pressure and then purified by
silica gel column chromatography (n-hexane/AcOEt, 5:2) to
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