Cyclolobatriene, a novel prenylated germacrene diterpene, from the soft coral Lobophytum pauciflorum

Article abstract of DOI:10.1016/j.bmc.2011.12.012

A new 10-membered-ring diterpene, cyclolobatriene (1), along with three other known diterpenes, lobatriene (2), eunicol (3), and fuscol (4), were isolated from the Okinawan soft coral Lobophytum pauciflorum. Their structures were established by extensive

Full text of DOI:10.1016/j.bmc.2011.12.012

Bioorganic & Medicinal Chemistry 20 (2012) 687–692
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Bioorganic & Medicinal Chemistry
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Cyclolobatriene, a novel prenylated germacrene diterpene, from the soft coral
Lobophytum pauciflorum
Sudhakar V.S. Govindam a, Yukio Yoshioka b, Akihiko Kanamoto c, Takeshi Fujiwara c, Tetsuji Okamoto b,
Makoto Ojika a,
⇑
a Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
b Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8553, Japan
c OP Bio Factory Co., Ltd, Naha 901-0152, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new 10-membered-ring diterpene, cyclolobatriene (1), along with three other known diterpenes, lob-
atriene (2), eunicol (3), and fuscol (4), were isolated from the Okinawan soft coral Lobophytum pauciflo-
rum. Their structures were established by extensive NMR spectroscopic analyses. Cyclolobatriene (1) is an
additional example of rare prenylated germacrenes. Although 1, due to a 10-membered-ring structure,
exists as an equilibrium mixture of three conformers, the NMR measurement in CDCl3 at 7 °C enabled
us to assign the NMR signals of the three, which is the first example of the complete NMR assignment
of all the existing conformers of germacrene-type compounds. Cyclolobatriene (1) was thermally unsta-
ble and converted into 2 through Cope rearrangement upon heating at 70 °C. Eunicol (3) also possesses
the same prenylated germacrene structure as 1, showing similar physico-chemical properties to 1. All
Received 7 November 2011
Revised 6 December 2011
Accepted 7 December 2011
Available online 14 December 2011
Keywords:
Soft coral
Cyclolobatriene
Cytotoxic
Lobophytum
Lobane diterpene
Germacrene
four compounds 1–4 showed cytotoxic effect with IC50’s of 0.64, 0.41, 0.35 and 0.52 lM, respectively,
against human epidermoid carcinoma A431 cells.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
example of the rare prenylated germacrenes in nature. In the pres-
ent paper, we describe isolation, cytotoxicity, and structure eluci-
dation of 1–4 (Fig. 1), with complete NMR assignment of three
conformers of 1 and 3.
Soft corals of the subclass Alcyonaria (Octocorallia) are known
as rich sources of lipids, sesquiterpenes, diterpenes and steroids
with wide structural diversity and pronounced bioactivities.1 As a
study on marine metabolites with biological activities, we have
tested the cytotoxicity of an extracts library established from hun-
dreds of marine organisms including a number of soft corals. As a
result, we found a relatively high activity for an extract from the
Okinawan soft coral Lobophytum pauciflorum Ehrenberg (Family
Alcyoniidae), from which we have isolated a novel diterpene,
cyclolobatriene (1), and three related metabolites [lobatriene (2),
eunicol (3) and fuscol (4)] (Fig. 1). These metabolites are structur-
ally interesting, because they possess a ‘prenylated’ germacrene (1
and 3) or a ‘prenylated’ elemene (2 and 4) skeleton which are not
common as natural products. Several prenylated elemenes (or lo-
banes2) have been isolated from a gorgonian Eunicea fusca3,4 and
some soft corals belong to the genus Lobophytum,2,5 Sarcophyton,6
and Sinularia.7–9 But very few prenylated germacrenes like 1 have
been isolated so far; one from brown algae Dilophus ligulatus,10
and three from a soft coral Lobophytum sp.11 and the gorgonian
Eunicea fusca.12 Therefore, cyclolobatriene (1) is an additional
2. Results and discussion
The soft coral Lobophytum pauciflorum was collected off the
Okinawan coast of Japan and extracted with methanol. The ethyl
acetate-soluble fraction of the methanol extract was subjected to
silica gel flash column chromatography followed by recycling HPLC
gave cyclolobatriene (1) and the known related compounds lobatri-
ene (2), eunicol (3), and fuscol (4). All four compounds 1–4 showed
cytotoxic effect with IC50’s of 0.64, 0.41, 0.35 and 0.52 lM, respec-
tively, against human epidermoid carcinoma A431 cells.
Cyclolobatriene (1), ½a D26
ꢀ
+57 (c 0.139, CHCl3), appears as a col-
orless oil. It exhibited IR absorption at 3472 cmꢁ1 due to hydroxyl
group. It showed a molecular ion peak at m/z 327.2292 [M+Na]+ in
the high-resolution ESI-TOF-MS that is compatible with the molec-
ular composition of C20H32O2. Broadened 1H NMR (CDCl3, 27 °C)
signals in the olefin region with <0.5 integral values indicated
the existence of more than one conformer (Fig. 2 and 27 °C). This
phenomenon made the structure elucidation challenging.
Attempts to measure the average NMR spectrum of the
conformers at 70 °C were discouraged by thermal instability of 1
⇑
Corresponding author. Tel./fax: +81 052 789 4284.
E-mail address: ojika@agr.nagoya-u.ac.jp (M. Ojika).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.012

688
S. V. S. Govindam et al. / Bioorg. Med. Chem. 20 (2012) 687–692
17
8
8
5
4
5
12
O
12
O
10
10
17
1
4
1
19
19
15
20
15
20
18
18
16
16
OH
OH
Lobatriene (2)
Cyclolobatriene (1)
OH
OH
Eunicol (3)
Fuscol (4)
6
7
2
3
O
O
OH
OH
2,3-dihydrocyclolobatriene (5)
6,7-dihydrocyclolobatriene (6)
Figure 1. The molecular structures of compounds 1–6.
towards partial isomerisation to 2 (Fig. 2 and 70 °C). Such a rear-
rangement was reported in the conversion of germacrene and its
derivatives toward their 10-membered ring elemene congen-
ers.13–17 However, the broadened NMR signals of 1 decoalesced
and became distinct when the temperature decreased to 7 °C in
CDCl3, therefore the NMR analyses were performed under these
conditions (Fig. 2 and 7 °C).
The decoalesced NMR signals enabled us to assign the NMR data
separately to each conformer (a, b and c). The presence of six dis-
tinct signals [dH 4.46 (br d, H2, a), 4.76 (br d, H6, a), 4.92 (br d, H6,
c), 4.99 (br t, H6, b), 5.06 (m, H2, b), 5.19 (br t, H2, c)] in the olefinic
region due to the two olefinic protons indicated the existence of
three conformational isomers (Table 1). The integral values of
these peaks revealed that the populations of the three conformers
(a, b and c) were in 44:32:24 ratios at 7 °C in CDCl3. The proton-
carbon direct connectivities were established by the HMQC spec-
trum. There were signals for three oxygen-bearing carbons [C14
(dC 80.3), C15 (dC 71.7) and C18 (dC 67.5) for conformer a] in 13C
NMR spectrum (Table 2). The HMBC correlation of H18 methylene
protons (dH 4.19) with two olefinic carbons [C11 (dC 144.7), C12 (dC
115.4)] and an oxygen-bearing carbon atom [C14 (dC 80.3)] and the
DQF-COSY correlation of H12–H13–H14 (Fig. 3) suggested the
presence of an oxene as a partial structure of 1. The occurrence
of a (CH3)2C(OH)– group was inferred by HMBC correlations
(Fig. 3) between the 1H methyl signals at d 1.16 (3H, s, H19), d
1.21 (3H, s, H20) and the quaternary 13C signal at d 71.7 (C15).
The NMR signals of the 10-membered-ring part were distinctive
for each of the three conformers, which was extremely helpful to
assign the chemical shifts of three conformers (a, b and c). The
DQF-COSY correlations of the two olefinic protons (H2 and H6)
with their adjacent methylene groups (H1 and H5) and the HMBC
Figure 2. Olefinic 1H NMR signals of cyclolobatriene (1) at different temperatures.
The asterisks (⁄) indicate signals due to commencement of isomerization to
lobatriene (2). The signals of three conformers are indicated by the letters a, b,
and c.

S. V. S. Govindam et al. / Bioorg. Med. Chem. 20 (2012) 687–692
689
Table 1
1H NMR data of the three conformers of cyclolobatriene (1) at 7 °C (600 MHz, CDCl3)
9
Position
Conformer a
Conformer b
Conformer c
12
17
1
dH (mult., J in Hz)
dH (mult., J in Hz)
dH (mult., J in Hz)
4
10
1
2
2.01 (m), 2.16 (m)
4.46 (br d, 10.2)
1.86 (m), 2.17 (m)
2.08 (m), 2.23 (m)
4.76 (br d, 11.2)
2.02 (m), 2.37 (m)
1.58 (m), 1.73 (m)
1.82 (m)
1.71 (m), 2.19 (m)
5.06 (m)
2.13 (m)
2.08 (m), 2.35 (m)
4.99 (br t, 7.6)
1.78 (m), 2.10 (m)
1.44 (m), 1.60 (m)
1.71 (m)
1.71 (m), 2.13 (m)
5.19 (br t, 7.6)
2.08 (m), 2.17 (m)
2.08 (m), 2.37 (m)
4.92 (br d, 11.2)
2.30 (m), 1.55 (m)
1.58 (m), 1.73 (m)
1.79 (m)
14
19
15
18
16
4
O
1
5
6
8
OH
20
9
10
12
13
14
16
17
18
19
20
OH
5.51 (br s)
1.94 (m), 2.12 (m)
3.26 (m)
1.51 (s)
1.37 (s)
4.19 (m)
1.16 (s)
1.21 (s)
2.51 (s)
5.51 (br s)
1.94 (m), 2.13 (m)
3.26 (m)
1.46 (s)
1.53 (s)
4.19 (m)
1.16 (s)
1.21 (s)
2.51 (s)
5.51 (br s)
1.94 (m), 2.12 (m)
3.26 (m)
1.47 (s)
1.66 (s)
4.19 (m)
1.16 (s)
1.21 (s)
2.51 (s)
O
5
OH
Table 2
13C NMR chemical shifts of the three conformers of cyclolobatriene (1) at 7° C
(150 MHz, CDCl3)
Position
Conformer a
Conformer b
Conformer c
O
6
1
2
3
4
34.8
131.4
129.2
39.3
32.2
124.8
136.2
36.9
30.1
126.4
137.0
38.6
OH
5
6
7
8
26.7
24.1
24.1
HMBC NOESY
COSY
126.4
138.1
41.6
121.7
138.1
41.3
128.8
134.5
34.4
Figure 3. 2D NMR correlations in cyclolobatriene (1), 2,3-dihydrocyclolobatriene
(5) and 6,7-dihydrocyclolobatriene (6).
9
33.9
31.8
33.9
10
11
12
13
14
15
16
17
18
19
20
47.3
44.8
42.5
144.7
115.4
25.2a
80.3b
71.7
16.7
16.2
67.5
23.5
143.3
115.5
25.1a
80.2b
71.7
15.4
16.7
67.5
23.5
142.0
116.0
25.1a
80.2b
71.7
15.4
22.1
67.5
23.5
of H16–H3AH2AH1–H10–H9–H8 and H4–H5–H6, and the HMBC
correlations of H16AC2, C3, C4; H17–C6, C7, C8; H12–C10; and
H10–C11 confirmed the 10-membered-ring structure with a dou-
ble bond between C6–C7 (Fig. 3). In 6, the DQF-COSY correlations
of H1–H2, H4–H5, H6–H7–H–17, H8–H9–H10 and the HMBC cor-
relations of H16–C2, C3, C4; H17–C6, C7, C8; H5–C6 confirmed
the double bond between C2–C3 (Fig. 3). Finally, the NOE correla-
tions shown in Fig. 3 confirmed the E geometry of C6–C7 in 5 and
C2–C3 in 6, confirming the structure of 1. The three dimensional
structures of the conformers a–c are discussed in the last part.
The heat-induced Cope rearrangement of 1 into 2 that occurred
during NMR measurement at 70 °C was beneficial in determining
its absolute configuration. Since the NMR data and optical rotation
26.1
26.1
26.1
a,b
Interchangeable within the same sign.
relationship of methyl groups (H16 and H17) with C2, C3, C4, and
C6, C7, C8, respectively, enabled us to establish the two partial
structures of –CH@C(CH3)–CH2– (Fig. 3). These 2D NMR correla-
tions were similar in all three conformers. The structure 1 for
cyclolobatriene was finally established by connecting these partial
structures to each other by HMBC and HOHAHA spectra.
(½a 2D6
ꢀ
+85) of the isomerised product 2 were in good agreement
with the reported data of lobatriene (½a D26
ꢀ
+86.7)9, we concluded
that the absolute configurations at C-10 and C-14 of 1 are same
as that of 2.
Attempts to identify the double bond geometries at C2 and C6
using NOESY correlations at 7 °C were unsuccessful due to the
overlapping of the correlating signals of the three conformers.
However, the E geometry of the two trisubstituted olefins at C2
and C6 were determined by partial hydrogenation followed by
the NOESY analysis of the two dihydro derivatives 5 and 6
(Fig. 1). The structures of 5 and 6 were confirmed by comparisons
of the 1H NMR data with that of 1 (7 °C) and also by analysing the
2D NMR spectra. The broad olefinic signals of H2 and H6 of 1 were
replaced by a signal at 5.45 (1H, m, H6) in 5, and 5.36 (1H, br s, H2)
in 6. Correspondingly, a vinylogous methyl signal was shifted to
relatively high field of 0.89 (d, J = 6.9 Hz, H16) in 5 and 0.81 (d,
J = 6.2 Hz, H17) in 6 when compared to 1. These facts suggest that
5 and 6 are 2,3-dihydrocyclolobatriene and 6,7-dihydrocyclolob-
atriene, respectively. In compound 5, the DQF-COSY correlations
Eunicol (3) is another 10-membered-ring compound isolated in
the present study. The isolation has been reported previously.11 In
this compound as well, the broadened NMR signals of the three
conformers (a:b:c = 44:32:24) decoalesced when temperature de-
creased to 7 °C as in the case of 1. This facilitated the complete
assignment of the NMR data for the three conformers (see Section
3.2.3), whereas the previous authors reported the NMR data of one
major conformer only.12
Eunicol (3), like 1, may underwent thermal isomerization to an
elemene congener, fuscol (4), which was also isolated in this study
and identified by comparing the 1H NMR data with published data
3,5
(see Section 3.2.4).
The thermal instability of these 10-mem-
bered-ring diterpenes (1 and 3) towards their corresponding elem-
ene congeners (2 and 4) via the Cope rearrangement implies that
most of the elemenes or elemene-type diterpenes are probably

690
S. V. S. Govindam et al. / Bioorg. Med. Chem. 20 (2012) 687–692
artifacts of their precursor germacrenes or germacrene-type 10-
membered-ring diterpenes. To best of our knowledge, the present
study provided the first complete NMR assignment of all the exist-
ing conformers of germacrene-type compounds.
shifts (Table 3) and NOESY correlations (Fig. 4) to those of 1 indi-
cated that the three conformers a, b, and c of 3 possess UU, UD,
and DU conformations, respectively. Despite the existence of the
three conformational isomers for 1 and 3, they exclusively isome-
rise to 2 and 4, respectively, because the Cope rearrangement of
germacrene-type compounds is known to proceed through the
most stable chair–chair transition state, to which the UU con-
former a is structurally closest.19,25
The 10-membered-ring sesquiterpenes have been well studied
for their conformational isomers as well as their geometry in trans-
annular reactions through variable-temperature NMR studies,
molecular mechanics, NOE spectrums and X-ray analysis.18–23
Germacrene A and related germacrene-type sesquiterpenes, for
example, germacrene B, costunolide, hedycaryol, and dehydroger-
macrene A, are known to have the four possible conformers,
namely UU, UD, DU and DD, in reference to the up (U) and down
(D) orientations of C16 and C17 methyl groups to the plane of
the 10-membered-ring (Fig. 4). A high-filed 1H NMR study of ger-
macrene A at variable temperatures was reported and confirmed
the presence of three conformational isomers a, b and c in a ratio
of 52:29:19 at ꢁ20 °C.24 The major conformer a exhibited rela-
tively high-field signals at 4.51 (H2), 4.78 (H6), 1.37 (H17), and
1.52 ppm (H16) (Table 3), as suggested for the UU conformer of he-
dycaryol and dihydropregeijerene.23,24 Similarly, the major con-
former a of cyclolobatriene (1) exhibited relatively high-field
signals at 4.46 (H2), 4.76 (H6), 1.37 (H17), and 1.51 (H16) (Table
3), suggesting that the major conformer a of 1 possesses the UU
conformation (Fig. 4). The other two downfield sets of 1H NMR sig-
nals of 1 correspond to the less populated conformers b (32%) and c
(24%), which were predicted to have UD or DU conformations. In
germacrene A, the second most populated conformer b and the
third populated c exhibit as having UD and DU conformations,
respectively.23 In the present study, the relatively downfield ole-
finic signals [4.99 (H6), 5.06 (H2)] and methyl singlets [1.46
(H16), 1.53 (H17)] of the conformer b of cyclolobtriene (1) are in
agreement with the UD conformer of germacrene A (Table 3).23
The NMR data of the third conformer c of 1 at 4.92 (H6), 5.19
(H2), 1.47 (H16), and 1.66 (H17) are in parallel to those of the
DU conformer of germacrene A (Table 3).23 These conformational
assignments were supported by the NOESY correlations as shown
in Figure 4. Likewise, in eunicol (3), the comparable NMR chemical
3. Experimental section
3.1. General experimental procedures
Thin-layer chromatography (TLC) was performed by using pre-
coated silica gel 60 F254 plates (Art. 5715, Merck) or ODS RP-18 F254
plates (Art. 15389, Merck). Flash column chromatography was per-
formed with an intelligent HPLC pump 880-PU (JASCO Corp.) and a
ternary gradient 880-02 unit (JASCO Corp). Silica gel (40
Flash column (Yamazen Science, Inc) of size
l
m) Hi-
L
(26 mm
i.d. ꢂ 100 mm) was used in flash column chromatography. HPLC
was performed on a high-pressure gradient system equipped with
980-PU pumps and UV-970 detector or an MD-915 photodiode ar-
ray UV detector (JASCO Corp.). Recycling system was generated by
using a LC-8A pump (Shimadzu Corp.). FT–IR spectrum was re-
corded on a FT/IR 400 spectrophotometer (JASCO). Mass spectra
(MS) were recorded on a Mariner Biospectrometry Workstation
(Applied Biosystems, Inc.) in the positive ESI mode. NMR spectra
were recorded on an AMX2 600 (600 MHz for 1H) spectrometer
(Bruker). The NMR chemical shifts (ppm) were referenced to the
solvent peaks of dH 7.26 (residual CHCl3) and dC 77.0 for CDCl3 solu-
tions. The cytotoxicity assays were carried out on human epider-
moid carcinoma A431 cells by
a model Zf Coulter counter
(Coulter Electronics).26
3.2. Isolation of 1–4
The specimens of the soft coral L. pauciflorum (wet weight
210 g) were collected by scuba off Taketomijima Island, Okinawa,
Japan, and were deposited as KTG-070618-006K in OP Bio Factory.
The species was identified by Professor Yehuda Benayahu, Depart-
ment of Zoology, Tel-Aviv University, Israel. The fresh samples
were extracted with MeOH. The MeOH extract was concentrated,
and the residue was partitioned between EtOAc and water. The or-
ganic layer was concentrated under reduced pressure to yield
crude extract (2.259 g). Flash column chromatography of a part
of the crude extract (1.023 g) on Silica gel Hi-Flash column (L-size,
silica gel 30 g), with linear gradient from 10 to 70% EtOAc (30 min)
in hexane gave a total of eight fractions. The fourth fraction
(330.1 mg) showed a cytotoxic activity. A portion (20.0 mg) of
the fourth fraction was separated by recycling HPLC [ODS YMC-
PAK (20 mm i.d. ꢂ 250 mm) column, 85% aq MeOH, detected at
205 nm, 2 recycles] to afford 1 (7.8 mg), 2 (1.2 mg), and a mixture
of 3 and 4 (1.1 mg). To obtain enough of 3 and 4, another portion of
the fourth fraction (53.6 mg) was purified by the same recycling
HPLC (3 recycles) to afford pure 3 (0.5 mg) and 4 (1.8 mg).
CH3
CH3
CH3
17
H
6
R
R
2
10
H
H
CH3
16
H
H
UU (= a)
UD (= b)
H
H
CH3
H
R
R
CH3
CH3
CH3
H
H
DD
DU (= c)
3.2.1. Cyclolobatriene (1)
R=
R=
R=
colorless viscous oil; ½a D26
ꢀ
+57° (c 0.139, CHCl3); IR (film) 3472,
OH
1098, 1077, 844, 756 cmꢁ1; MS (ESI+) m/z 287.2 [MꢁOH]+, 327.1
[M+Na]+; HRMS (ESI) calcd. for C20H32O2Na [M+Na]+ 327.2295,
found 327.2292.
O
OH
cyclolobatriene (1)
germacrene A
eunicol (3)
Figure 4. Possible conformations of cyclolobatriene (1) and eunicol (3) in compar-
ison with those of germacrene A. The conformers are denoted as UU, UD, DU, and
DD in reference to the U (up) and D (down) orientations of C16 and C17 methyl
groups on the 10-membered ring.23 The NOESY correlations observed for 1 and 3
are shown with dotted curved arrows.
3.2.2. Lobatriene (2)
colorless viscous oil; ½a D26
ꢀ
+85.5 (c 0.098, CHCl3) (reported data:
½
a 2D5 +86.7 (c 0.19, CHCl3))9; 1H NMR (600 MHz, CDCl3) d 5.81 (1H,
ꢀ

S. V. S. Govindam et al. / Bioorg. Med. Chem. 20 (2012) 687–692
691
Table 3
Selected 1H NMR data (in CDCl3) of conformers a, b and c of germacrene A, cyclolobatriene (1) and eunicol (3)
Compound
Conformer
H-2
H-6
H-16
H-17
Germacrene A (ꢁ20 °C)23
a (UU)
b (UD)
c (DU)
4.51 (d, 10.0)
5.07 (dd, 9.5, 6.6)
5.21 (t, 7.9)
4.78 (dd, 11.2, 4.0)
5.01 (t, 8.1)
4.94 (d, 12.1)
1.52 (s)
1.46 (s)
1.48 (s)
1.37 (s)
1.54 (s)
1.68 (s)
Cyclolobatriene (1) (7 °C)
Eunicol (3) (7 °C)
a (UU)
b (UD)
c (DU)
4.46 (br d, 10.2)
5.06 (m)
5.19 (br t, 7.6)
4.76 (br d, 11.2)
4.99 (br t, 7.6)
4.92 (br d, 11.2)
1.51 (s)
1.46 (s)
1.47 (s)
1.37 (s)
1.53 (s)
1.66 (s)
a (UU)
b (UD)
c (DU)
4.52 (br d, 7.4)
5.07 (br d, 7.4)
5.19 (br s)
4.78 (br d, 10.8)
5.02 (br t, 6.3)
4.93 (br d, 11.5)
1.52 (s)
1.47 (s)
1.49 (s)
1.39 (s)
1.54 (s)
1.62 (s)
dd, J = 18.0, 10.2 Hz, H-6), 5.57 (1H, br d, J = 4.8 Hz, H-12), 4.90 (1H,
d, J = 18.0 Hz, H-5b), 4.89 (1H, d, J = 10.2 Hz, H-5a), 4.82 (1H, s, H-
4b), 4.58 (1H, s, H-4a), 4.20 (2H, m, H-18), 3.26 (1H, dd, J = 10.8,
3.0 Hz, H-14), 2.43 (1H, s, OH), 2.15 (1H, m, H-13b), 1.99 (1H, m,
H-2), 1.96 (1H, m, H-13a), 1.84 (1H, m, H-10), 1.70 (3H, s, H-16),
1.57 (1H, m, H-9b), 1.55 (2H, m, H-1), 1.50–1.40 (2H, m, H-8),
1.45 (1H, m, H-9a), 1.21 (3H, s, H-19), 1.17 (3H, s, H-20), 0.99
(3H, s, H-17); 13C NMR (150 MHz; CDCl3) d 150.1 (C-6), 147.5 (C-
3), 141.2 (C-11), 116.4 (C-12), 112.2 (C-4), 110.0 (C-5), 80.3 (C-
14), 71.7 (C-15), 68.1 (C-18), 52.7 (C-2), 41.7 (C-10), 39.8 (C-8),
39.7 (C-7), 32.8 (C-1), 27.0 (C-9), 26.1 (C-19), 25.3 (C-13), 24.7
(C-16), 23.7 (C-20), 16.6 (C-17); MS (ESI) m/z 287.2 [MꢁOH]+,
327.1 [M+Na]+.
24.2 (C-5), 22.1 (C-17), 15.5 (C-16), 14.1 (C-18); MS (ESI) m/z
271.2 [MꢁOH]+.
3.2.4. Fuscol (4)
colorless oil; ½a D29
ꢀ
+14 (c 0.099, CHCl3) (reported data: [a]
D
+16.3)3; 1H NMR (400 MHz, CDCl3) d 6.49 (1H, dd, J = 15.2,
11.0 Hz, H-13), 5.87 (1H, d, J = 11.0 Hz, H-12), 5.82 (1H, dd,
J = 16.8, 11.6 Hz, H-6), 5.76 (1H, d, J = 15.2 Hz, H-14), 4.91 (1H, d,
J = 16.8 Hz, H-5b), 4.90 (1H, d, J = 11.6 Hz, H-5a), 4.81 (1H, s, H-
4b), 4.58 (1H, s, H-4a), 2.00 (2H, m, H-2, 10), 1.79 (3H, s, H-18),
1.70 (3H, s, H-16), 1.35 (6H, s, H-19, 20), 1.6–1.2 (6H, m, H-1, 8,
9), 1.00 (3H, s, H-17); MS (ESI) m/z 271.2 [MꢁOH]+.
3.3. Hydrogenation of cyclolobatriene (1)
3.2.3. Eunicol (3)
colorless viscous oil; ½a D29
ꢀ
ꢁ15 (c 0.15, CHCl3) (reported data:
Compound 1 (4.6 mg) was hydrogenated with PtO2 (2 mg) in
EtOH (2 mL) for 2 h. The reaction mixture was filtered through a cot-
ton plug, and the filtrate was concentrated. The crude material was
subjectedto HPLC [Develosil ODS-UG-5 (10 mm i.d. ꢂ 250 mm), 90%
aq MeOH, 4 mL/min, detected at 205 nm] to give two dihydro deriv-
atives 5 (0.7 mg, tR = 15.1 min) and 6 (0.5 mg, tR = 15.9 min).
½
a 2D0
ꢀ
ꢁ22.8 (c 0.00395, CH2Cl2))12
;
1H NMR (600 MHz, CDCl3,
7 °C) conformer a: d 6.44 (1H, dd, J = 15.3, 10.9 Hz, H-13), 5.83
(1H, d, J = 10.9 Hz, H-12), 5.75 or 5.77 (1H, d, J = 15.3 Hz, H-14),
4.78 (1H, br d, J = 10.8 Hz, H-6), 4.52 (1H, br d, J = 7.4 Hz, H-2),
2.36 (1H, m, H-8b), 2.24 (1H, m, H-5b), 2.18 (1H, m, H-4b), 2.12
(2H, m, H-1), 2.08 (1H, m, H-5a), 2.04 (1H, m, H-8a), 1.97 (1H, m,
H-10), 1.87 (1H, m, H-4a), 1.75 or 1.77 (3H, s, H-18), 1.67 (1H, m,
H-9b), 1.59 (1H, m, H-9a), 1.52 (3H, s, H-16), 1.39 (3H, s, H-17),
1.35 (6H, s, H-19, 20), conformer b: d 6.44 (1H, dd, J = 15.3,
10.9 Hz, H-13), 5.83 (1H, d, J = 10.9 Hz, H-12), 5.77 or 5.75 (1H, d,
J = 15.3 Hz, H-14), 5.07 (1H, br d, J = 7.4 Hz, H-2), 5.02 (1H, br t,
J = 6.3 Hz, H-6), 2.34 (1H, m, H-5b), 2.27 (1H, m, H-8b), 2.12 (1H,
m, H-1b), 2.12 (1H, m, H-4b), 2.09 (1H, m, H-5a), 2.06 (1H, m, H-
4a), 1.84 (1H, m, H-1a), 1.77 or 1.75 (3H, s, H-18), 1.70 (1H, m,
H-10), 1.69 (1H, m, H-8a), 1.59 (1H, m, H-9b), 1.54 (3H, s, H-17),
1.47 (3H, s, H-16), 1.43 (1H, m, H-9a), 1.35 (6H, s, H-19, 20), con-
former c: d 6.44 (1H, dd, J = 15.3, 10.9 Hz, H-13), 5.83 (1H, d,
J = 10.9 Hz, H-12), 5.77 or 5.75 (1H, d, J = 15.3, H-14), 5.19 (1H, br
t, H-2), 4.93 (1H, br d, J = 11.5 Hz, H-6), 2.38 (1H, m, H-5b), 2.34
(1H, m, H-8b), 2.17 (1H, m, H-5a), 2.11 (1H, m, H-4b), 2.09 (1H,
m, H-8a), 2.06 (1H, m, H-1b), 2.06 (2H, m, H-9), 1.92 (1H, m, H-
4a), 1.85 (1H, m, H-1a), 1.77 or 1.75 (3H, s, H-18), 1.62 (3H, s, H-
17), 1.49 (3H, s, H-16), 1.35 (6H, s, H-19, 20); 13C NMR (150 MHz,
CDCl3, 7 °C) conformer a: d 147.0 (C-11), 139.1 or 138.9 (C-14),
138.9 (C-7), 131.4 (C-2), 129.2 (C-3), 126.3 (C-6), 122.9 (C-13),
121.2 (C-12), 71.0 (C-15), 53.2 (C-10), 41.6 (C-8), 39.4 (C-4), 34.5
(C-1), 33.3 (C-9), 29.82 (C-19 and 20), 26.6 (C-5), 16.7 (C-16),
16.3 (C-17), 14.5 (C-18), conformer b: d 145.5 (C-11), 138.9 or
139.1 (C-14), 138.9 (C-7), 136.2 (C-3), 124.9 (C-2), 122.9 (C-13),
121.6 (C-12), 121.6 (C-6), 71.0 (C-15), 50.1 (C-10), 41.2 (C-8),
37.0 (C-4), 32.4 (C-1), 31.1 (C-9), 29.8 (C-19 and 20), 24.2 (C-5),
16.7 (C-17), 15.45 (C-16), 14.1 (C-18), conformer c: d 144.4 (C-
11), 138.9 or 139.1 (C-14), 138.1 (C-3), 135.4 (C-7), 128.8 (C-6),
122.9 (C-13), 126.3 (C-2), 121.8 (C-12), 71.0 (C-15), 48.1 (C-10),
38.8 (C-4), 34.2 (C-8), 30.2 (C-1), 29.8 (C-19 and 20) 26.6 (C-9),
3.3.1. 2,3-Dihydrocyclolobatriene (5)
Colorless oil; IR (film) 3469, 1164, 1124, 1098, 1073 cmꢁ1 1H
;
NMR (600 MHz, CDCl3) d 5.49 (1H, d, J = 4.8 Hz, H-12), 5.45 (1H,
m, H-6), 4.18 (1H, d, J = 15.0 Hz, H-18b), 4.13 (1H, d, J = 15.0 Hz,
H-18a) 3.25 (1H, dd, J = 10.8, 3.6 Hz, H-14), 2.42 (1H, br s, OH),
2.20 (1H, m, H-5b), 2.19 (1H, m, H-8b), 2.11 (1H, m, H-13b),
1.97 (1H, m, H-8a), 1.95 (1H, m, H-5a), 1.92 (1H, m, H-13a),
1.86 (1H, br s, H-10), 1.67 (3H, s, H-17), 1.56 (1H, m, H-3),
1.51 (1H, m, H-9b), 1.44 (1H, m, H-9a),1.37 (1H, m, H-4b),
1.31 (1H, br s, H-4a), 1.21 (3H, s, H-20), 1.22 (2H, m, H-1),
1.24 (1H, m, H-2b), 1.16 (3H, s, H-19), 1.02 (1H, br s, H-2a),
0.89 (3H, d, J = 6.9 Hz, H-16); 13C NMR (150 MHz; CDCl3) d
141.5 (C-11), 133.8 (C-7), 126.4 (C-6), 116.9 (C-12), 80.4
(C-14), 71.7 (C-15), 67.9 (C-18), 41.2 (br, C-10), 39.8 (C-8),
34.9(C-2), 34.0 (C-4), 31.2 (C-9), 28.7 (C-1 and 3), 26.2 (C-20),
25.6 (C-5), 25.4 (C-13), 23.8 (C-16), 23.7 (C-19), 16.9 (C-17);
HRMS (ESI) calcd for
329.2480.
C
20H34O2Na [M+Na]+ 329.2451, found
3.3.2. 6,7-Dihydrocyclolobatriene (6)
Colorless oil; IR (film) 3464, 1163, 1129, 1098, 1076 cmꢁ1 1H
;
NMR (600 MHz, CDCl3) d 5.52 (1H, d, J = 4.2 Hz, H-12), 5.36 (1H,
br s, H-2), 4.22 (1H, d, J = 15.6 Hz, H-18b), 4.17 (1H, d, J = 15.6 Hz,
H-18a) 3.27 (1H, dd, J = 11.1, 3.0 Hz, H-14), 2.42 (1H, s, OH), 2.17
(1H, m, H-1b), 2.14 (1H, m, H-13b), 2.13 (1H, m, H-4b), 2.00 (1H,
m, H-4a), 1.94 (1H, m, H-13a), 1.90 (2H, m, H-1a and H-10), 1.64
(3H, s, H-16), 1.61 (1H, m, H-9b), 1.60 (1H, m, H-5b), 1.38 (1H,
m, H-9a), 1.36 (1H, s, H-5a), 1.34 (1H, s, H-6b), 1.26 (1H, m, H-7),
1.21 (3H, s, H-20), 1.19 (1H, m, H-8b), 1.17 (3H, s, H-19), 1.09

692
S. V. S. Govindam et al. / Bioorg. Med. Chem. 20 (2012) 687–692
(1H, m, H-8a), 0.91 (1H, m, H-6a), 0.81 (3H, d, J = 6.2 Hz, H-17); 13
C
References and notes
NMR (150 MHz; CDCl3) d 142.8 (C-11), 135.0 (C-3), 125.0 (C-2),
116.0 (C-12), 80.4 (C-14), 71.7 (C-15), 67.9 (C-18), 42.0 (C-10),
40.0 (C-4), 36.6 (C-8), 32.1 (C-6), 31.5 (C-1), 29.5 (C-7), 28.5
(C-9), 26.1 (C-20), 25.3 (C-13), 23.7 (C-19), 22.6 (C-5), 22.0
(C-17), 16.5 (C-16); HRMS (ESI) calcd for C20H34O2Na [M+Na]+
329.2451, found 329.2453.
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lobatriene (2)
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Compound 1 (4.3 mg) was partially isomerised to 2 while mea-
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(0.7 mL). The resultant mixture was purified by recycling HPLC
[ODS YMC-PAK (20 mm i.d. ꢂ 250 mm) column, 85% aq MeOH, de-
tected at 205 nm, 2 recycles] to afford 1 (0.6 mg) and 2 (1.8 mg).
Lobatriene (2): ½a D26
ꢀ
+85 (c 0.099, CHCl3) (ref. data:9
½
a 2D6
+86.7 (c
ꢀ
0.19, CHCl3).
16. Colby, S. M.; Crock, J.; Dowdle-Rizzo, B.; Lemaux, P. G.; Croteau, R. Proc. Natl.
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We are grateful to Professor Yehuda Benayahu (Tel-Aviv Uni-
versity) for identifying the marine animal and to Professor Jun-ichi
Tanaka (Ryukyu University) for helpful discussion. This work was
supported in part by JSPS KAKENHI (23592962). S. V. S. Govindam
is grateful to the Ministry of Education, Culture, Sports, Science and
Technology, Japan for the award of Japanese Government Foreign
Student Scholarship (MEXT No. 090015). The authors are thankful
to Dr. P. W. L. Lai and Dr. C. Musick of Academic Writing Unit,
School of Liberal Arts & Languages, Nagoya University, for checking
the English language of this manuscript.
26. Onodera, K.; Fukatsu, T.; Kawai, N.; Yoshioka, Y.; Okamoto, T.; Nakamura, H.;
Ojika, M. Biosci. Biotechnol. Biochem. 2004, 68, 848.