New marine cembrane-type diterpenoids from the Okinawan soft coral Clavularia koellikeri

Article abstract of DOI:10.1021/np000309w

Seven new marine diterpenoids having a cembrane skeleton were isolated from the Okinawan soft coral Clavularia koellikeri. Their structures were determined based on the results of spectroscopic analysis and chemical conversions. Compound 1 showed cytotoxic activity against human colorectal adenocarcinoma cells (DLD-1, IC₅₀ 4.2 μg/mL) and strong growth inhibition against human T lymphocytic leukemia cells (MOLT-4, IC₅₀ 0.9 μg/mL).

Full text of DOI:10.1021/np000309w

J . Nat. Prod. 2000, 63, 1647-1652
1647
New Ma r in e Cem br a n e-typ e Diter p en oid s fr om th e Ok in a w a n Soft Cor a l
Cla vu la r ia koelliker i
Makoto Iwashima, Yuuki Matsumoto, Haruko Takahashi, and Kazuo Iguchi*
Laboratory of Bioorganic Chemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi,
Hachioji, Tokyo 192-0392, J apan
Received J une 19, 2000
Seven new marine diterpenoids having a cembrane skeleton were isolated from the Okinawan soft coral
Clavularia koellikeri. Their structures were determined based on the results of spectroscopic analysis
and chemical conversions. Compound 1 showed cytotoxic activity against human colorectal adenocarcinoma
cells (DLD-1, IC50 4.2 µg/mL) and strong growth inhibition against human T lymphocytic leukemia cells
(MOLT-4, IC50 0.9 µg/mL).
Soft corals of the genus Clavularia, inhabiting the
Okinawan coral reefs, are known to contain secondary
metabolites with unique structures and remarkable bio-
logical activities. During the course of our investigation on
1
biologically active substances from Clavularia spp., seven
new cembrane-type diterpenoids (1-7) having an R-meth-
ylene γ-lactone moiety were isolated from Clavularia
koellikeri (family Clavularidae). These compounds were
found to be congeners of the kericembrenolides isolated
2
previously from C. koellikeri. This paper describes struc-
ture elucidations of diterpenoids 1-7 through spectroscopic
analysis and chemical conversions.
Specimens of C. koellikeri collected on the coral reef of
Ishigaki Island (Okinawa Prefecture, J apan) were im-
mersed in MeOH. The MeOH extract was successively
partitioned between EtOAc and H
soluble portion, and then the aqueous layer was extracted
with n-BuOH to afford n-BuOH- and H O-soluble portions.
2
O to afford an EtOAc-
2
The EtOAc-soluble portion was chromatographed on a Si
gel column, eluting in turn with hexanes, hexanes-EtOAc,
EtOAc, and MeOH, to afford eight fractions. Compound 1
(0.0015% yield) from the fifth and sixth fractions; com-
pounds 2 (0.0004%), 3 (0.0002%), 5 (0.0004%), 6 (0.0002%),
and 7 (0.0003%) from the seventh fraction, and compound
spectrum (Table 2). The 1H NMR spectrum of 1 also
disclosed signals for three olefinic protons at δ 5.03 (H-3),
4
(0.0013%) from the fourth fraction were isolated by
repeated purification using flash column chromatography,
medium-pressure liquid chromatography (MPLC), and
5
.15 (H-11), and 5.16 (H-7) ppm; four oxymethine protons
either bearing three acetates or in the γ-lactone group at
δ 5.30 (H-2), 5.31 (H-14), 5.57 (H-6), and 5.67 (H-10) ppm;
three methyl groups connecting with an olefin at δ 1.68
2
HPLC. Known diterpenoids (kericembrenolides, stolon-
3
2,4
idiol, and neodolabellenol ) were also isolated and identi-
fied.
(H-19), 1.82 (H-20), and 1.88 (H-18) ppm; and three methyl
groups in acetate esters at δ 2.00, 2.02, and 2.04 ppm. The
Resu lts a n d Discu ssion
1
1
H- H COSY spectrum demonstrated correlations from
1
H-1 to H-3, H-5 to H-7, H-9 to H-11, and H-13 to H-1. H-
The molecular formula of compound 1 was found to be
1
H long-range correlations were also observed between
26 34 8
C H O by HREIMS. Signals for all 26 carbons appeared
1
3
H-1 and the H-16 protons, H-3 and H-18, H-7 and H-19,
and H-11 and H-20, by proton-decoupling analysis. These
spectroscopic findings and the degree of unsaturation (10)
showed 1 to have a 14-membered cembrane-type diterpe-
noid skeleton with an R-methylene γ-lactone. After assign-
ments between all the direct C-H bondings were made
based on HMQC, the gross structure was determined by
HMBC analysis. The correlations according to HMBC are
shown in Figure 1.
in the C NMR spectrum of 1 (Table 1). The DEPT
3
spectrum exhibited signals for six methyls, three sp
methylenes, five sp methines, one sp2 methylene, three
3
sp2 methines, and eight sp2 quaternary carbons. The IR
spectrum of 1 showed absorptions due to the functionalities
-
1
of an acetate ester (1731, 1237 cm ) and an R-methylene
-
1
γ-lactone (1763, 1665 cm ). The presence of the R-meth-
ylene γ-lactone system in 1 was also demonstrated by UV
absorption at 208 (ꢀ 12 600) nm and signals at δ 5.66 (H-
1
1
6a) and 6.35 (H-16b) ppm observed in the H NMR
The structure of 1 containing three acetoxyl groups at
C-6, C-10, and C-14 was similar to that of kericembrenolide
2
C with two acetate esters at C-6 and C-9, except for the
*
To whom correspondence should be addressed. Tel.: +81-426-76-7273.
Fax: +81-426-76-7282. E-mail: onocerin@ls.toyaku.ac.jp.
position of one acetate substituent, which was shifted from
1
0.1021/np000309w CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/10/2000

1
648 J ournal of Natural Products, 2000, Vol. 63, No. 12
Iwashima et al.
Ta ble 1. 13C NMR Data for Compounds 1-7 (125 MHz, CDCl3,
δ ppm)
position
1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
46.2
73.4
48.0
72.9
46.3
73.6
45.7
73.9
42.9
78.0
42.8
79.0
43.0
79.2
124.3 124.4 123.5 123.9 126.8 123.9 123.2
139.7 139.5 141.1 141.3 141.6 139.3 140.6
42.3
68.9
127.0 127.1 131.4 129.3 125.1 126.3 131.4
137.3 137.3 134.9 138.5 136.0 138.3 135.2
44.4
66.9
126.7 126.4 126.9 128.6 123.8 129.2 125.1
138.0 138.3 137.9 129.7 131.9 138.9 140.9
41.9
73.6
136.4 138.4 136.5 136.9 140.2 140.0 140.1
123.7 122.8 123.5 123.1 122.2 122.0 121.9
169.3 169.8 169.4 169.7 170.4 169.9 170.0
19.9
16.3
15.8
20.9
21.2
21.3
42.3
68.9
41.8
66.8
45.5
66.7
44.9
67.9
42.3
69.0
45.2
66.4
44.5
67.0
44.5
66.4
38.4
23.4
39.2
23.9
48.2
64.9
44.7
67.3
0
1
2
3
4
5
6
7
8
9
0
44.9
72.6
45.2
73.4
42.1
74.0
36.3
32.6
36.0
32.5
35.8
32.3
F igu r e 2. Key NOE correlations for 1.
spectrum exhibited correlations between H-3 and H-5, H-7,
and H-9, and H-11 and H-13 to disclose the (3E,7E,11E)
stereochemistries. The chemical shift values of the three
methyl carbon signals for C-18, C-19, and C-20 (δ 19.9,
19.8
16.3
16.1
21.2
21.3
20.0
15.8
16.0
20.9
21.2
19.7
15.4
15.8
21.0
16.0
15.5
15.4
21.4
19.8
16.3
15.2
21.3
19.9
15.9
15.6
21.3
1
6.3, and 15.8 ppm, respectively), which appeared in the
1
3
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
high-field area in the C NMR spectrum also supported
the all-E configurations. The relative configurations at C-1,
C-2, and C-14 were determined by the coupling constants
170.0 170.0 170.1 170.2 170.4 170.0 170.3
170.1 170.2 170.2
170.1
1
from the H NMR data (J 1,2 ) 3.4 Hz, J 1,14 ) 3.7 Hz) and
NOE correlations between H-1 and H-3, and H-2 and H-13,
as shown in Figure 2. The relative configurations of the
remaining two chiral centers at C-6 and C-10 were deduced
from the following NOE analysis. NOE correlations be-
tween H-1 and H-20, H-1, and H-3 indicated that these
protons (H-1, H-3, and H-20) were oriented to the same
side, while NOE correlations between H-2 and H-18, H-18
and H-7 and between H-18 and H-11 demonstrated that
these protons (H-2, H-7, H-11, and H-18) were oriented in
the opposite direction. Considering the relationships of
these protons, the relative configurations at C-6 and C-10
were determined by the NOE correlations between H-6 and
H-3, H-6 and H-19, H-19 and H-10, and H-10 and H-20, as
shown in Figure 2.
The absolute configuration of each chiral center in 1 was
determined by the modified Mosher’s method using 2-(2′-
naphthyl)-2-methoxyacetic acid ester (2NMA ester, Scheme
F igu r e 1. HMBC for 1.
5
1
). Compound 1 was treated with potassium carbonate in
2
C-9 to C-10 and the presence of another acetate at C-14.
The stereochemistry for the three trisubstituted olefins of
MeOH at room temperature to obtain triol 8. In this
reaction the Michael-type addition of MeOH occurred
concomitantly with the R-methylene γ-lactone moiety, to
1
was determined by NOESY analysis. The NOESY
Ta ble 2. 1H NMR Data for Compounds 1-3 (500 MHz, CDCl3, δ ppm, J in Hz)
position
1
2
3
1
2
3
5
2.66 (m)
2.57 (m)
2.66 (m)
5.30 (dd, 3.4, 8.8)
5.03 (br d, 8.8)
2.17 (m)
5.33 (dd, 3.6, 8.9)
5.03 (br d, 8.9)
2.17 (m)
5.31 (dd, 3.9, 8.7)
4.93 (br d, 8.7)
2.16 (dd, 10.9, 13.7)
2.53 (br d, 13.7)
4.54 (ddd, 3.9, 9.2, 10.4)
5.24 (br d, 9.2)
2.20 (br d, 12.4)
2.64 (dd, 4.3, 12.4)
5.66 (m)
5.17 (br d, 8.8)
2.11 (dd, 8.7, 13.3)
2.64 (m)
5.31 (dd, 3.9, 8.7)
5.65 (d, 2.1)
2
.62 (m)
2.62 (m)
6
7
9
5.57 (ddd, 5.4, 9.1, 10.0)
5.16 (br d, 9.1)
2.18 (m)
5.58 (ddd, 4.3, 9.0, 10.9)
5.16 (br d, 9.0)
2.18 (m)
2
.61 (m)
2.61 (m)
1
1
1
0
1
3
5.67 (ddd, 5.4, 9.0, 10.1)
5.15 (br d, 9.0)
2.17 (dd, 8.8, 13.5)
5.66 (ddd, 5.3, 8.9, 10.6)
5.13 (br d, 8.9)
2.20 (dd, 8.8, 13.4)
2.50 (dd, 3.8, 13.4)
4.18 (dd, 3.8, 8.8)
5.65 (d, 2.2)
2
.54 (br d, 13.5)
1
1
4
6
5.31 (dd, 3.7, 8.8)
5.66 (d, 1.9)
6
.35 (d, 2.4)
6.42 (d, 2.6)
6.34 (d, 2.6)
1
1
2
AcO
AcO
AcO
8
9
0
1.88 (3H, br s)
1.68 (3H, br s)
1.82 (3H, br s)
2.00 (3H, s)
2.02 (3H, s)
2.04 (3H, s)
1.87 (3H, br d, 0.9)
1.68 (3H, br s)
1.76 (3H, br s)
2.02 (3H, s)
1.89 (3H, br s)
1.65 (3H, br s)
1.81 (3H, br s)
2.01 (3H, s)
2.04 (3H, s)
2.04 (3H, s)

Diterpenoids from Soft Coral Clavularia
J ournal of Natural Products, 2000, Vol. 63, No. 12 1649
Sch em e 2
F igu r e 3. δ∆ Values for 2-NMA ester of 1.
Sch em e 1
tions indicated the presence of an R-methylene γ-lactone
-
1
[νmax 1759, 1667 cm , λmax 211 nm (ꢀ 4500)] moiety and
-
1
-1
hydroxyl (3415 cm ) and acetate (1731, 1260 cm ) func-
tionalities. The 13C and H NMR spectra of 3 (Tables 1 and
1
2
) were quite similar to those of 2, except for the signal at
1
δ 4.54 ppm in H NMR due to a proton assigned as that at
C-6 bearing a secondary hydroxyl group. 2D NMR analysis
determined the gross structure of 3 to be a 6-hydroxy
analogue of 1. The absolute configuration was established
by acetylation of 3 to form the corresponding triacetate.
The spectral data and optical rotation value for the
give a methyl ether at C-16 with an unknown stereochem-
istry at the newly arisen chiral center (C-15). Half of the
amount of 8 was converted into (R)-2NMA triester 9, and
the other half was converted into (S)-2NMA triester 10.
1
After taking H NMR spectra of both diastereomers, the
δ∆ [δ(R)-ester - δ(S)-ester] value of each corresponding proton
was calculated, and they are summarized in Figure 3.
Positive and negative δ∆ values are systematically ar-
ranged at the left side and right side of each chiral center,
to disclose absolute configurations of 6S, 10S, and 14S.
These findings established the absolute configurations of
triacetate derived from 3 ([R]
those of natural 1.
D
-18.8°) were identical with
The molecular formula of compound 4 was determined
to be C22
30 4
H O
from both HREIMS and 13C NMR data
(Table 1). The absorptions observed in the IR and UV
spectra suggested the presence of an R-methylene γ-lactone
-1
1
to be 1S, 2S, 6S, 10S, and 14S.
[νmax 1765, 1667 cm , λmax 229 nm (ꢀ 2500)] and an acetate
-
1
Compound 2 was shown to have the molecular formula
ester (1731, 1241 cm ). Signals for three olefinic methyls
C
24
H
32
O
7
by HREIMS. The IR and UV spectra of 2
at δ 1.61, 1.70, and 1.89 ppm and for one acetate at δ 2.00
1
1
indicated absorptions due to an R-methylene γ-lactone [νmax
ppm were observed in the H NMR spectrum (Table 3). H-
-
1
1
1
759, 1665 cm , λmax 218 nm (ꢀ 3000)]. The IR spectrum
H COSY and HMBC analysis suggested the gross struc-
-1
2
also exhibited acetate ester (1731, 1239 cm ) and hydroxyl
ture to be the same as that of kericembrenolide A (11).
-
1
1
(3452 cm ) functionalities. The H NMR of 2 showed three
However, the spectral data of 4 and 11 were not identical.
NOESY analysis supported the structure of 4 as well as
the same relative configuration of 11, except at C-6. These
observations indicated 4 to be the epimer at C-6 of 11. The
chemical conversion of 11 was thus carried out to determine
the absolute configuration of 4, as shown in Scheme 2.
Methanolysis of the C-6 acetate ester in 11 with potas-
sium carbonate in MeOH accompanied by the addition of
MeOH to the R-methylene γ-lactone moiety gave the
corresponding C-6 alcohol 12 as a single compound after
characteristic signals at δ 1.68, 1.76, and 1.87 ppm due to
olefinic methyl protons, two three-proton singlets at δ 2.02
and 2.04 ppm due to the two acetoxyl moieties, and a signal
at δ 4.18 ppm assigned as the proton on the carbon bearing
the secondary hydroxyl group (Table 2). After assignment
of each direct C-H bonding from HMQC data, the C NMR
and 1H NMR data of 2 were compared with those of 1,
indicating that the gross structure of 2 was a deacetyl
analogue of 1 at C-14. The structure was confirmed by
correlations observed in H- H COSY and HMBC spectra.
The absolute stereochemistry of 2 was established by the
following chemical transformation. Acetylation of 2 pro-
vided the corresponding triacetate 1, the spectral data and
1
3
1
1
6
chromatographic separation. Mitsunobu inversion was
effected for 12 with acetic acid, triphenylphosphine, and
diisopropyl azodicarboxylate at room temperature to afford
13. Compound 4 was similarly converted to the corre-
sponding methyl ether 13 via 14, as also shown in Scheme
2. The spectral data, including the optical rotation for each
compound 13 obtained from 4 and 11, were almost identi-
cal, thus establishing the structure of 4 to be 6-epikericem-
brenolide A (1S,2S,6R).
the optical rotation ([R] -18.0°) of which were identical
D
to those of natural 1. From these results, it was concluded
that the structure for 2 was the 14-hydroxy analogue of 1
with the same absolute configuration.
Compound 3 was found to have the molecular formula,
C
24
H
32
O
7
, the same as that of 2, by the combination of
HREIMS data for compounds 5, 6, and 7, as well as their
C NMR (Table 1) data, indicated the same molecular
1
3
13
HREIMS and C NMR analysis. The IR and UV absorp-

1
650 J ournal of Natural Products, 2000, Vol. 63, No. 12
Iwashima et al.
Ta ble 3. 1H NMR Data for Compounds 4-7 (500 MHz, CDCl3, δ ppm, J in Hz)
position
4
5
6
7
1
2
3
5
2.83 (m)
5.33 (dd, 4.0, 8.8)
4.94 (br d, 8.8)
2.65 (ddd, 2.6, 5.3, 9.9)
4.91 (dd, 2.6, 10.1)
5.15 (br d, 10.1)
2.17 (dd, 10.7, 12.2)
2.59 (dd, 5.2, 12.2)
5.71 (ddd, 5.2, 9.9, 10.7)
5.00 (br d, 9.9)
2.00 (ddd, 4.3, 4.6, 12.5)
2.25 (m)
2.12 (br dd, 4.3, 14.5)
2.30 (ddd, 4.6, 9.9, 14.5)
4.85 (br dd, 4.3, 9.9)
2.08 (m)
2.42 (m)
4.84 (dd, 4.0, 8.9)
5.00 (br d, 8.9)
2.17 (dd, 10.9, 14.9)
2.59 (br dd, 4.3, 14.9)
5.58 (ddd, 4.3, 9.9, 10.9)
5.14 (br d, 9.9)
2.12 (dd, 8.3, 12.6)
2.66 (br dd, 5.2, 12.6)
4.59 (ddd, 5.2, 8.3, 10.6)
2.40 (m)
4.83 (dd, 4.4, 8.7)
4.92 (br d, 8.7)
2.10 (m)
2.16 (dd, 9.1, 15.7)
2
.62 (dd, 4.0, 15.7)
2.63 (m)
6
7
9
4.60 (dt, 4.0, 9.1)
5.18 (br d, 9.1)
2.15 (m)
4.54 (ddd, 4.2, 9.4, 10.9)
5.16 (br d, 9.4)
2.20 (dd, 10.7, 11.7)
2.62 (m)
2
.24 (m)
2.13 (m)
.22 (m)
1
0
5.66 (ddd, 5.2, 8.8, 10.7)
2
1
1
1
3
5.04 (br d, 5.4)
2.13 (m)
5.17 (br d, 10.6)
2.09 (m)
2.32 (m)
1.59 (m)
1.68 (m)
5.57 (d, 2.2)
6.27 (d, 2.6)
1.83 (3H, br s)
1.66 (3H, br s)
1.79 (3H, br s)
2.02 (3H, s)
5.13 (br d, 8.8)
2.11 (br d, 14.7)
2.30 (br d, 14.7)
1.70 (m)
1.88 (m)
5.57 (d, 2.4)
2
.39 (br d, 14.1)
2.23 (m)
1.65 (m)
1
1
4
6
5.25 (ddd, 1.9, 3.6, 12.1)
1
.68 (m)
5.62 (d, 2.3)
5.60 (d, 2.0)
6.26 (d, 2.2)
1.72 (3H, br s)
1.75 (3H, br s)
1.61 (3H, br s)
2.03 (3H, s)
6
.32 (d, 2.7)
6.26 (d, 2.7)
1
1
2
8
9
0
1.89 (3H, br d, 0.6)
1.61 (3H, br s)
1.70 (3H, br s)
2.00 (3H, s)
1.83 (3H, br d, 0.6)
1.67 (3H, br s)
1.73 (3H, br s)
2.04 (3H, s)
AcO
penoids, kericembrenolides D and F, were also examined
in the same cell line, which showed the growth inhibition
for DLD-1 and MOLT-4 to be equivalent to those of 1. Other
new diterpenoids are currently under investigation.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured with a J ASCO DIP-370 automatic polarimeter.
IR spectra were recorded with a Perkin-Elmer FT-IR 1600
spectrophotometer and UV spectra with a J ASCO V-520
spectrophotometer. All NMR spectra were recorded with a
1
13
1
Bruker DRX-500 ( H; 500 MHz, C; 125 MHz), a DPX-400 ( H;
1
4
00 MHz), and a Varian Gemini-300 ( H; 300 MHz) instrument
in CDCl . H- H COSY, NOESY, HMQC, and HMBC NMR
spectra were measured with a Bruker DRX-500 using standard
1
1
3
F igu r e 4. NOE correlations for 6.
formula, C22
Bruker pulse sequences. Chemical shifts are given on a δ (ppm)
scale with CHCl ( H; 7.26 ppm, C; 77.0 ppm) as the internal
3
1
H
30
O
5
, for each compound. H NMR (Table 3),
1
13
IR, and UV spectra suggested these compounds to have
cembrane-type structures, including the functionalities of
one acetate, one alcohol, and three trisubstituted olefins
with E configurations. The 2D NMR analysis for compound
standard (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad). Mass spectra were obtained with a
Micromass Auto Spec spectrometer. Column chromatography
was carried out on Merck Si gel 60 (70-230 mesh), and flash
column chromatography was performed on Merck Si gel 60
(230-400 mesh). MPLC was carried out with a KHLC-201-43
(Kusano) apparatus using a CIG prepack column (Si gel, CPS-
HS-221-05, for normal phase and ODS Si gel, CPO-HS-221-
20, for reversed phase). HPLC was conducted with a YMC-
Pack SIL-06 column (Si gel, SH-043-5-06, normal phase) and
a YMC-Pack ODS-AM column (ODS Si gel, SH-343-5AM,
reversed phase).
5
indicated the gross structure to be 14-acetoxy-6-hydroxy-
cembra-(3E,7E,11E,15)-tetraen-17,2-olide. Acetylation of 5
provided the 6,14-diacetate 15. The spectral data of 15 were
identical to those of kericembrenolide C,2 thus showing
compound 5 to have the absolute configuration (1S,2S,6S,-
1
4S).
The H and 13C NMR spectra of compounds 6 and 7 were
1
quite similar to each other. Based on the 2D NMR analysis,
An im a l a n d Ma ter ia l. The soft coral C. koellikeri (order
Stolonifera, family Clavularidae) was collected from a coral
reef of Ishigaki Island, Okinawa Prefecture, J apan, in No-
vember 1997, at a depth of 1-2 m. A voucher specimen (No.
IR-SC-97-1) is deposited at Tokyo University of Pharmacy and
Life Science, Tokyo, J apan.
Extr a ction a n d Isola tion . Wet specimens (1.87 kg) were
immersed in MeOH (3 × 3 L). After filtration, the combined
extracts were concentrated under reduced pressure. The
MeOH extract (164 g) was partitioned between EtOAc and
6
1
1
and 7 were determined respectively to have 6-acetoxy-
0-hydroxy- and 10-acetoxy-6-hydroxy-cembra-(3E,7E,-
1E,15)-tetraen-17,2-olide gross structures. The NOESY
analysis for compound 6, as shown in Figure 4, indicated
the relative configurations were 1S*, 2S*, 6S*, and 10S*.
Compound 7 was found to have the same relative configu-
rations according to the NOESY analysis. Although the
absolute configurations for 6 and 7 are under investigation,
these compounds are assumed to have the same stereo-
chemistry as those of compounds 1-3.
Compound 1, obtained as the major cembranolide in the
present study, was examined for cytotoxic activities in vitro
for human colorectal adenocarcinoma cells (DLD-1) and
human T-lymphocyte leukemia cells (MOLT-4). Compound
H
2
O, and then the aqueous layer was extracted with n-BuOH.
Each layer was concentrated under reduced pressure to obtain,
in turn, EtOAc- (27.8 g), n-BuOH- (12.5 g), and H O- (72.6 g)
2
soluble portions. An aliquot of the EtOAc-soluble portion (11.0
g) was chromatographed on a Si gel column (500 g). Stepwise
elution with hexanes (300 mL), hexanes-EtOAc (9:1, 4:1, 2:1,
1
showed the proliferation of DLD-1 to inhibit completely
1
:1, and 1:2, each 300 mL), EtOAc (300 mL), and MeOH (600
at 0.6 µM/mL (IC50 4.2 µg/mL) and that of MOLT-4 to
inhibit at 0.6 µM/mL (IC50 0.9 µg/mL). The known diter-
mL) afforded eight fractions. Repeated separation and puri-
fication of fractions 5 and 6 (2.16 g) [eluted with hexanes-

Diterpenoids from Soft Coral Clavularia
J ournal of Natural Products, 2000, Vol. 63, No. 12 1651
EtOAc (1:1 and 1:2)] using Si gel flash column chromatogra-
HPLC [hexanes-i-PrOH (12:1)] to afford (R)-2NMA triester
phy, MPLC (normal and reversed phase), and HPLC gave 1
9 (2.5 mg, 88% yield) as a colorless oil.
2
1
(
11.0 mg) along with kericembrenolides D and E. The seventh
Com p ou n d 9: H NMR (CDCl
3
, 400 MHz) δ ppm 1.43 (3H,
fraction (0.58 g) [eluted with EtOAc] was subjected to Si gel
column chromatography. Further purification by MPLC (nor-
mal and reversed phase) and HPLC afforded compounds 2 (2.9
mg), 3 (1.8 mg), 5 (2.8 mg), 6 (1.2 mg), and 7 (1.9 mg) along
br s, H-20), 1.55 (3H, br s, H-18), 1.59 (3H, br s, H-19), 2.21
(1H, m, H-1), 2.46 (1H, m, H-15), 3.45 (1H, dd, J ) 4.6, 11.7
Hz, H-16a), 3.61 (1H, dd, J ) 6.2, 11.7 Hz, H-16b), 4.90 (1H,
br d, J ) 11.2 Hz, H-3), 4.91 (1H, br d, J ) 6.1 Hz, H-11), 4.96
(1H, br d, J ) 11.1 Hz, H-7), 4.99 (1H, dd, J ) 6.1, 11.2 Hz,
H-2), 5.09 (1H, dd, J ) 3.9, 11.4 Hz, H-14), 5.60 (1H, m, H-10),
5.58 (1H, m, H-6). The H-5, -9, and -13 proton signals are
difficult to discern.
Ester ifica tion of 8 w ith (S)-2NMA. To a mixture of 8 (1.2
mg, 0.003 mmol), (S)-2NMA (2.0 mg, 0.11 mmol), and DMAP
3
(0.4 mg, 0.003 mmol) in CHCl (1 mL) under an argon
atmosphere was added EDC (2.0 mg, 0.010 mmol) at room
temperature, and the mixture was stirred for 1 h. After
removal of urea derivatives by Si gel column chromatography,
the crude product was purified by HPLC [hexanes-i-PrOH
(12:1)] to afford (S)-2NMA triester 10 (3.2 mg, 100% yield) as
3
with stolonidiol and its acetate. The fourth fraction (2.47 g)
[eluted with hexanes-EtOAc (2:1)] was subjected to Si gel
column chromatography to give five fractions. Further puri-
fication of fraction 2 (2.03 g) by MPLC (normal and reversed
phase) and HPLC afforded compound 4 (9.6 mg) along with
2
,4
2
neodolabellenol and kericembrenolides A, B, and C.
2
5
Com p ou n d 1: colorless, viscous oil; [R]
CHCl
max 1763, 1731, 1665, 1237 cm
Tables 1 and 2, respectively; HREIMS m/z 474.2252 [calcd for
D
-19.5° (c 0.13,
3
); UV (EtOH) λmax (log ꢀ) 208 (4.10) nm; IR (dry film)
-
1
13
1
ν
;
C NMR and H NMR, see
+
C
26
H
34
O
8
, 474.2254 (M) ].
Com p ou n d 2: colorless, viscous oil; [R]
CHCl ); UV (EtOH) λmax (log ꢀ) 218 (3.48) nm; IR (dry film)
max 3452, 1759, 1731, 1665, 1239 cm-1
see Tables 1 and 2, respectively; HREIMS m/z 432.2139 [calcd
2
5
D
-19.2° (c 0.19,
3
a colorless oil.
Com p ou n d 10: H NMR (CDCl
13
1
1
ν
;
C NMR and H NMR,
3
, 400 MHz) δ ppm 1.28 (3H,
br s, H-20), 1.57 (3H, br s, H-19), 1.63 (3H, br s, H-18), 1.92
(1H, m, H-15), 2.14 (1H, m, H-1), 3.22 (1H, dd, J ) 4.6, 11.8
Hz, H-16a), 3.39 (1H, dd, J ) 5.7, 11.8 Hz, H-16b), 4.84 (1H,
br d, J ) 9.0 Hz, H-3), 4.90 (1H, br d, J ) 10.1 Hz, H-11), 4.95
(1H, br d, J ) 11.0 Hz, H-7), 4.95 (1H, m, H-14), 5.00 (1H, dd,
J ) 5.6, 9.0 Hz, H-2), 5.53 (1H, ddd, J ) 4.7, 9.1, 10.1 Hz,
H-10), 5.59 (1H, ddd, J ) 3.2, 7.8, 10.4 Hz, H-6). The H-5, -9,
and -13 proton signals are difficult to discern.
+
for C24
Com p ou n d 3: colorless, viscous oil; [R]
CHCl ); UV (EtOH) λmax (log ꢀ) 211 (3.65) nm; IR (dry film)
max 3415, 1759, 1731, 1667, 1260 cm-1
see Tables 1 and 2, respectively; HREIMS m/z 372.1926 [calcd
32 7
H O , 432.2148 (M) ].
2
5
D
-35.0° (c 0.12,
3
13
1
ν
; C NMR and H NMR,
+
for C22
Com p ou n d 4: colorless, viscous oil; [R]
CHCl ); UV (EtOH) λmax (log ꢀ) 229 (3.39) nm; IR (dry film)
max 1765, 1731, 1667, 1241 cm
Tables 1 and 3, respectively; HREIMS m/z 298.1930 [calcd for
H
28
O
5
, 372.1937 (M - CH
3 2
CO H) ].
2
5
D
+102.3° (c 0.13,
3
Acetyla tion of 2. Compound 2 was treated with acetic
anhydride (0.1 mL) in pyridine (1.5 mL) at room temperature
for 1 h. After concentration under reduced pressure, the
-
1
13
1
ν
;
C NMR and H NMR, see
+
C
20
H
26
O
2
, 298.1933 (M - CH
Com p ou n d 5: colorless, viscous oil; [R]
CHCl ); UV (EtOH) λmax (log ꢀ) 215 (3.44) nm; IR (dry film)
max 3461, 1759, 1731, 1665, 1230 cm-1
see Tables 1 and 3, respectively; HREIMS m/z 314.1870 [calcd
3
CO
2
H) ].
residue was purified by HPLC [hexanes-i-PrOH (12:1)] to
2
5
25
D
-35.6° (c 0.18,
obtain triacetate ester 1 [1.2 mg, 83% yield, [R]
D
-18.0° (c
3
0.15, CHCl
3
)] as a colorless oil. The spectral data were identical
13
1
ν
; C NMR and H NMR,
to those of 1.
Acetyla tion of 3. Compound 3 was treated with acetic
anhydride (0.2 mL) in pyridine (0.5 mL) at room temperature
for 1 h. After concentration under reduced pressure, the
residue was purified by HPLC [hexanes-i-PrOH (15:1)] to
obtain the corresponding triacetate ester 1 [1.2 mg, 90% yield,
+
for C20
Com p ou n d 6: colorless, viscous oil; [R]
CHCl ); UV (EtOH) λmax (log ꢀ) 213 (3.52) nm; IR (dry film)
max 3443, 1759, 1731, 1666, 1240 cm-1
see Tables 1 and 3, respectively; HREIMS m/z 314.1873 [calcd
H
26
O
3
, 314.1882 (M - CH
3 2
CO H) ].
2
5
D
-25.8° (c 0.12,
3
13
1
ν
; C NMR and H NMR,
[R]25
D
3
-18.8° (c 0.12, CHCl )] as a colorless oil. The spectral
+
for C20
Com p ou n d 7: colorless, viscous oil; [R]
CHCl ); UV (EtOH) λmax (log ꢀ) 219 (3.48) nm; IR (dry film)
max 3417, 1760, 1731, 1667, 1241 cm-1
see Tables 1 and 3, respectively; HREIMS m/z 314.1926 [calcd
H
26
O
3
, 314.1882 (M - CH
3
CO
2
H) ].
data of this triacetate were identical to those of 1.
2
5
D
-44.0° (c 0.15,
Ch em ica l Con ver sion of 11 to 12. To a mixture of
2
3
kericembrenolide A (11, 5.5 mg, 0.02 mmol) in MeOH (1 mL)
13
1
ν
;
C NMR and H NMR,
was added potassium carbonate (3 mg) at room temperature.
The mixture was vigorously stirred for 1 h at this temperature,
and then EtOAc (2 mL) was added. The mixture was filtered
by passing over a small plug of Si gel column, the plug was
rinsed twice with 2 mL of EtOAc, and the combined filtrates
were concentrated under reduced pressure. The oily residue
was purified by Si gel column chromatography [hexanes-
EtOAc (4:1) as eluent] to obtain 12 (5.2 mg, 100% yield) as a
+
for C20
H
34
O
8
3 2
, 314.1882 (M - CH CO H) ].
Ch em ica l Con ver sion of 1 to 8. To a mixture of 1 (5.0
mg, 0.02 mmol) in MeOH (1 mL) was added potassium
carbonate (5 mg) at room temperature. The reaction mixture
was vigorously stirred for 1 h at this temperature, and then
EtOAc (2 mL) was added. The mixture was filtered by passing
over a small plug of Si gel column, the plug was rinsed twice
with 2 mL of EtOAc, and the combined filtrates were concen-
trated under reduced pressure. The oily residue was purified
by Si gel column chromatography [hexanes-EtOAc (2:1) as
colorless oil.
Com p ou n d 12: H NMR (CDCl
1
3
, 300 MHz) δ ppm 1.56 (3H,
br s, H-20), 1.69 (3H, br s, H-19), 1.80 (3H, br s, H-18), 2.19
(1H, m, H-1), 2.40 (1H, m, H-15), 3.34 (3H, s, OMe), 3.56 (1H,
dd, J ) 4.4, 11.8 Hz, H-16a), 3.61 (1H, dd, J ) 6.6, 11.8 Hz,
H-16b), 4.64 (1H, dd, J ) 4.7, 8.5 Hz, H-2), 4.68 (1H, ddd, J )
4.2, 9.3, 10.8 Hz, H-6), 4.73 (1H, m, H-11), 4.93 (1H, br d, J )
8.5 Hz, H-3), 5.17 (1H, br d, J ) 9.3 Hz, H-7). The H-5, -9, -10,
and -13 proton signals are difficult to discern.
eluent] to obtain triol 8 (3.1 mg, 68% yield) as a colorless oil.
1
Com p ou n d 8: H NMR (CDCl
3
, 400 MHz) δ ppm 1.65 (3H,
br s, H-19), 1.74 (3H, br s, H-20), 1.83 (3H, br s, H-18), 2.63
1H, m, H-1), 2.86 (1H, m, H-15), 3.34 (3H, s, OMe), 3.61 (1H,
(
dd, J ) 3.9, 9.4 Hz, H-16a), 3.65 (1H, dd, J ) 5.8, 9.4 Hz,
H-16b), 4.09 (1H, dd, J ) 3.0, 11.7 Hz, H-14), 4.53 (1H, m,
H-10), 4.59 (1H, ddd, J ) 3.3, 8.1, 10.1 Hz, H-6), 4.93 (1H, br
d, J ) 8.8 Hz, H-3), 5.23 (1H, br d, J ) 9.5 Hz, H-11), 5.28
Mitsu n obu In ver sion 6 of 12. To a mixture of 12 (2 mg,
0.006 mmol), acetic acid (2.0 mg, 0.034 mmol), and triph-
enylphosphine (Ph P, 12.1 mg, 0.046 mmol) in THF (0.5 mL)
3
under an argon atmosphere was added diisopropyl azodicar-
boxylate (0.02 mL, 0.06 mmol) at room temperature, and the
mixture was stirred for 1 h. After removal of urea derivatives,
(
1H, dd, J ) 6.2, 8.8 Hz, H-2). The H-5, -7, -9, -13, and OH
proton signals are difficult to discern.
Ester ifica tion of 8 w ith (R)-2NMA. To a mixture of 8 (1.2
mg, 0.003 mmol), (R)-2NMA (2 mg, 0.011 mmol) and 4-(di-
3
Ph P, and triphenylphosphine oxide by Si gel column chro-
methylamino)pyridine (DMAP, 0.4 mg, 0.003 mmol) in CHCl
1 mL) under an argon atmosphere was added 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC, 2.0
mg, 0.010 mmol) at room temperature, and the mixture was
stirred for 1 h. After removal of urea derivatives by Si gel
column chromatography, the crude product was purified by
3
matography, the crude product was purified by HPLC [hex-
anes-i-PrOH (12:1)] to afford 13 (0.6 mg, 26% yield) as a
colorless oil.
(
Com p ou n d 13: [R]25
(CDCl
H-18), 1.74 (3H, br s, H-19), 2.25 (1H, m, H-1), 2.40 (1H, m,
+85.9° (c 0.12, CHCl
); 1H NMR
D
3
3
, 300 MHz) δ ppm 1.60 (3H, br s, H-20), 1.70 (3H, br s,

1
652 J ournal of Natural Products, 2000, Vol. 63, No. 12
Iwashima et al.
H-15), 3.36 (3H, s, H-OMe), 3.59 (1H, dd, J ) 4.4, 11.1 Hz,
H-16a), 3.73 (1H, dd, J ) 5.9, 11.1 Hz, H-16b), 4.59 (1H, m,
H-11), 4.71 (1H, dd, J ) 2.7, 10.8 Hz, H-2), 4.91 (1H, m, H-6),
Acetyla tion of 5. Compound 5 was treated with acetic
anhydride (0.3 mL) in pyridine (0.5 mL) at room temperature
for 1 h. After concentration under reduced pressure, the
residue was purified by HPLC [hexanes-i-PrOH (15:1)] to
5
.17 (1H, br d, J ) 8.8 Hz, H-7), 5.33 (1H, br d, J ) 10.8 Hz,
H-3), The H-5, -9, -11, and OH proton signals are difficult to
obtain the corresponding diacetate ester 15 [0.8 mg, 68% yield,
+
25
discern; EIMS m/z 330 (M - CH
3
CO
2
H) .
[R]
D
3
-67.5° (c 0.01, CHCl )] as a colorless oil. The spectral
Ch em ica l Con ver sion of 4 to 14. To a mixture of 4 (2.0
mg, 0.006 mmol) in MeOH (1 mL) was added potassium
carbonate (5 mg) at room temperature. The mixture was
vigorously stirred for 1 h at this temperature, and then EtOAc
data of this diacetate were identical to those of kericembre-
nolide C.2
Ack n ow led gm en t. The authors express their appreciation
to Dr. Y. Shida, Tokyo University of Pharmacy and Life
Science, for conducting MS measurements, and Dr. Y. Ichi-
hara, Meiji Institute of Health Science, for conducting the
bioassay.
(2 mL) was added. The mixture was filtered by passing over a
small plug of Si gel column, rinsed twice with 2 mL of EtOAc,
and the combined filtrates were concentrated under reduced
pressure. The oily residue was purified by Si gel column
chromatography [hexanes-EtOAc (2:1) as eluent] to obtain 14
Refer en ces a n d Notes
(
1.9 mg, 100% yield) as a colorless oil.
1
Com p ou n d 14: H NMR (CDCl
br s, H-20), 1.72 (3H, br s, H-19), 1.73 (3H, br s, H-18), 2.28
1H, m, H-1), 2.41 (1H, m, H-15), 3.37 (3H, s, OMe), 3.58 (1H,
3
, 400 MHz) δ ppm 1.59 (3H,
(1) (a) Kikuchi, H.; Tsukitani, Y.; Iguchi, K.; Yamada, Y. Tetrahedron
Lett. 1982, 23, 5171-5174; Kikuchi, H.; Tsukitani, Y.; Iguchi, K.;
Yamada, Y. Tetrahedron Lett. 1983, 24, 1549-1552. (b) Iwashima,
M.; Okamoto, K.; Iguchi, K. Tetrahedron Lett. 1999, 40, 6455-6459;
Iwashima, M.; Nara, K.; Iguchi, K. Steroids 2000, 65, 130-137.
(2) Kobayashi, M.; Son, B. W.; Kyogoku, Y.; Kitagawa, I. Chem. Pharm.
Bull. 1986, 34, 2306-2309.
(
dd, J ) 3.4, 9.4 Hz, H-16a), 3.73 (1H, dd, J ) 4.5, 9.4 Hz,
H-16b), 4.72 (1H, dd, J ) 6.6, 9.4 Hz, H-2), 4.90 (1H, br t, J )
6
.3 Hz, H-11), 5.11 (1H, br d, J ) 9.2 Hz, H-7), 5.34 (1H, br d,
(
3) (a) Mori, K.; Iguchi, K.; Yamada, N.; Yamada, Y.; Inouye, Y.
Tetrahedron Lett. 1987, 28, 5673-5676. (b) Mori, K.; Iguchi, K.;
Yamada, N.; Yamada, Y.; Inouye, Y. Chem. Pharm. Bull. 1988, 36,
2840-2852.
J ) 9.4 Hz, H-3), 5.70 (1H, ddd, J ) 4.5, 9.2, 10.9 Hz, H-6),
The H-5, -9, -10, -13, and -14 proton signals are difficult to
discern; EIMS m/z 330 (M - CH
+
3
CO
2
H) .
(
4) Bowden, B. F.; Braekman, J . C.; Coll, J . C.; Mitchell, S. J . Aust. J .
Chem. 1980, 33, 927-932.
5) (a) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J . Am. Chem.
Soc. 1991, 113, 4092-4096. (b) Kusumi, T.; Takahashi, H.; Xu, P.;
Fukushima, T.; Asakawa, Y.; Hashimoto, T.; Kan, Y.; Inouye, Y.
Tetrahedron Lett. 1994, 35, 4397-4400.
Acetyla tion of 14. Compound 14 was treated with acetic
anhydride (0.2 mL) in pyridine (0.5 mL) at room temperature
for 1 h. After concentration under reduced pressure, the
residue was purified by HPLC [hexanes-i-PrOH (15:1)] to
obtain the corresponding acetate ester [1.3 mg, 61% yield,
(
(
6) Mitsunobu, O. Synthesis 1981, 1-28.
2
5
[R]
D
3
+93.8° (c 0.15, CHCl )] as a colorless oil. The spectral
data of this acetate were identical to those of 13.
NP000309W