Four new cembrane diterpenes isolated from an Okinawan soft coral Lobophytum crassum with inhibitory effects on nitric oxide production

Article abstract of DOI:10.1248/cpb.58.1203

Four new cembrane diterpenes (1-4) and fifteen known cembranoids (5-19) were isolated from an Okinawan soft coral Lobophytum crassum. The structures of these four new cembranoids were determined on the basis of spectroscopic evidence. In particular, the a

Full text of DOI:10.1248/cpb.58.1203

September 2010
Regular Article
Chem. Pharm. Bull. 58(9) 1203—1209 (2010)
1203
Four New Cembrane Diterpenes Isolated from an Okinawan Soft Coral
Lobophytum crassum with Inhibitory Effects on Nitric Oxide Production
Mpanzu WANZOLA, Takaaki FURUTA, Yasuhisa KOHNO, Shunichi FUKUMITSU, Shuhei YASUKOCHI,
Kosuke WATARI, Chiaki TANAKA, Ryuichi HIGUCHI, and Tomofumi MIYAMOTO*
Graduate School of Pharmaceutical Sciences, Kyushu University; 3–1–1 Maidashi, Higashi-ku, Fukuoka 812–8582,
Japan. Received June 14, 2010; accepted June 29, 2010; published online July 1, 2010
Four new cembrane diterpenes (1—4) and fifteen known cembranoids (5—19) were isolated from an Oki-
nawan soft coral Lobophytum crassum. The structures of these four new cembranoids were determined on the
basis of spectroscopic evidence. In particular, the absolute stereochemistry of 1, 2, 5 and 6 were elucidated by the
application of the modified Mosher’s method and circular dichroism (CD) spectral data. The inhibitory effects of
some isolates were evaluated on nitric oxide (NO) production against a murine macrophage-like cell line (Raw
264.7). Cembranoids consisting of a-methylene-g-lactone, exhibited the significant effect on NO production.
Key words cembrane diterpene; soft coral; Lobophytum crassum; absolute stereochemistry; nitric oxide production
Soft corals are well known to be a rich source of terpene bicyclic ring system of 1. Interpretation of correlation spec-
metabolites, especially diterpenes of the cembranoid se- troscopy (COSY) and total correlation spectroscopy (TOCSY)
ries,1,2) which are naturally occurring diterpenens containing spectra, in combination with heteronuclear multiple bond
a 14-carbon ring. Many of them are of great interest as they correlation (HMBC) experiments, led to the planar structure
exhibit a wide range of biological activities including ichthy- of 1 (Fig. 2). The COSY and TOCSY spectra of 1 revealed
otoxic, anti-cancer, anti-inflammatory, anti-human immuno- three spin systems from H-3 [dH 5.13 (d, 9.6)] to H-13 [dH
deficiency virus (HIV), and anti-microbial activities.3—9) In 5.18 (t, 6.0)], H-5 [dH 2.22] to H-7 [dH 4.98 (t, 6.1)], and H-9
the continuation of our ongoing research for bioactive marine [dH 2.00, 2.02] to H-11 [dH 5.53 (t, 6.7)]. The HMBC corre-
natural products, we have investigated the n-hexane extract lations from three olefinic methyls [H-18 (dH 1.66)/C-3 (dC
of the Lobophytum crassum (von Marenzeller, 1886) which 121.2), C-5 (dC 39.2)], [H-19 (dH 1.58)/C-7 (dC 123.9), C-9
has exhibited a significantly inhibitory effect on nitric oxide (dC 34.6)], and [H-20 (dH 1.63)/C-11 (dC 72.2), C-13 (dC
(NO) production against lipopolysaccharide (LPS)-induced 124.6)], an oxymethine [H-2 (dH 5.28)/C-16 (dC 170.5) and
Raw 264.7. Chemical investigation revealed that the n- an exomethylene [H-17 (dH 5.66, 6.22)/C-16 (dC 170.5), C-
hexane-soluble material in this soft coral included four new 17 (dC 121.6), C-1 (dC 42.8)] easily merged these three spin
(1—4) and fifteen known (5—19) cembranoids (Fig. 1). In systems and the presence of an a-methylene-l-lactone adja-
this paper, we report on the isolation and structure elucida- cent to the 14-membered ring. Thus, the planar structure of 1
tion of four new cembranoids 1—4 and also the determina- was depicted in Fig. 2. The E-geometry of the D3 and D7 was
tion of absolute stereochemistry of the new 1 and 2 and the determined by the nuclear Overhauser effect (NOE) correla-
known cembranoids 5 and 6. Furthermore, the inhibitory ef- tions between H-3/H-5 and H-7/H-9 (dH 2.00), and the Z-
fect of some isolates was evaluated on NO production geometry of the D12 was determined by the strong NOE be-
against Raw264.7.
tween H-13 and H-20. The cis-fused a-methylene-l-lactone
The frozen L. crassum collected in Okinawa was com- and the 14-membered ring was deduced from the NOE corre-
pletely extracted in n-hexane. The extract was partitioned lation between H-1 and H-2. In addition, NOE correlations
between n-hexane and aqueous methanol. The n-hexane solu- were observed between H-3 and H-2/H-5/H-7, H-7 and H-
ble-material was subjected to silica gel flash column chro- 9/H11, H-13 and H-1/H-17/H-20. These data revealed the
matography and recycling HPLC, followed by the reversed conformation of the 14-membered ring and the relative con-
phase (RP) HPLC to afford four new cembranoids 1—4 and figuration of C-11. Thus, the relative stereochemistry of 1
fifteen known cembranoids 5—19.
was determined to be 1S*, 2S* and 11S* as shown in Fig. 3.
The molecular formula of 1 was determined to be The absolute stereochemistry of 1 was determined using
C22H30O4 by the combination of electrospray ionization modified Mosher’s method.10) Cembranoid 1 was first con-
(ESI)-time-of-flight (TOF)-MS [m/z 381.2039, Calcd 381.2036 verted to the 11-hydroxy derivative 1a and then prepared
(MϩNa)ϩ] and 13C-NMR spectroscopic data revealing eight (R)- or (S)-a-methoxy-a-(trifluoromethyl)phenylacetic acid
degrees of unsaturation. The IR absorptions at 1759 and (MTPA) esters (1b, 1c), respectively. The proton chemical
1666 cmϪ1 and UV absorption at 229 nm were indicative of shifts of each MTPA ester were assigned by 1D- and 2D-
1
the presence of an a-methylene-l-lactone moiety. The H- NMR experiments. The distribution of Dd (dSϪdR) values
and 13C-NMR spectra of 1 showed signals assignable to three indicates that the absolute configuration of C-11 is S configu-
olefinic methyls, one acetylmethyl, three trisubstituted dou- ration. Thus, the absolute configuration at C-1 and C-2 was
ble bonds, one exomethylene, two oxymethines, one aliphatic then deduced to be both S configurations (Chart 1).
methine, five methylenes, and two ester carbonyls. Six of the
Compound 2 displayed a molecular ion peak at m/z
eight degrees of unsaturation in 1 were attributed to four 381.2037 (MϩNa)ϩ and gave the same molecular formula
double bonds and two ester carbonyls (Tables 1, 2). The C22H30NaO4 as for 1. From H- and 13C-NMR data, com-
1
remaining two degrees of unsaturation were attributed to the pound 2 was found to be an isomer of 1. The IR spectrum of
∗ To whom correspondence should be addressed. e-mail: miyamoto@phar.kyushu-u.ac.jp
© 2010 Pharmaceutical Society of Japan

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Vol. 58, No. 9
Fig. 1. Structures of Compounds 1—19
Table 1. 1H-NMR (600 MHz) Data of Compounds 1—4 in CDCl3
Proton
1
2
3
4
1
2
3.03 (m)
5.28 (dd, 7.7, 9.6)
3.02 (m)
4.87a)
2.56 (m)
—
1.97b (m)
6.24 (d, 11.6)
2.10a a)
3
5
5.13 (d, 9.6)
2.22 (2H)a)
5.08 (d, 9.4)
2.17 (2H)a)
5.15 (t, 7.0)
2.11a a)
6.30 (d, 11.6)
2.04 (2H)a)
2.17b a)
6
2.22 (2H)a)
2.18b a)
2.15a a)
2.16 (2H)a)
2.29a a)
4.87a)
2.23b (m)
4.96 (br t, 6.9)
2.04b (m)
2.08a a)
7
9
4.98 (t, 6.1)
2.00b (m)
2.02a (m)
1.61b a)
1.85a (m)
5.53 (t, 6.7)
5.18 (t, 6.0)
4.96 (br t, 6.9)
2.02b a)
2.02b a)
2.17a a)
2.18b a)
2.11a a)
10
2.10 (2H)a)
2.09 (2H)a)
2.29a a)
5.02 (t, 6.1)
2.29b (br)
2.42a (m)
4.97 (m)
11
13
5.05 (dd, 5.4, 6.5)
1.70b a)
5.05 (br s)
2.37 (2H, m)
1.95a a)
14
2.30b (m)
2.41a (m)
—
1.54b a)
5.97 (dd, 3.9, 10.0)
1.76a (m)
—
15
16
17
—
—
2.51 (m)
—
—
1.02 (3H, d, 6.9)
1.06 (3H, d, 6.9)
5.66a (d, 2.4)
6.22b (d, 2.7)
1.66 (3H, s)
5.70 (d, 1.9)
6.34 (d, 2.5)
1.67 (3H, s)
5.49 (s)
6.17 (d, 1.2)
1.56 (3H, s)
18
4.65 (d, 12.4)
4.73 (d, 12.4)
1.46 (3H, s)
1.54 (3H, s)
2.00 (3H, s)
2.05 (3H, s)
19
20
OAc
1.58 (3H, s)
1.63 (3H, s)
2.00 (3H, s)
1.57 (3H, s)
1.64 (3H, s)
2.02 (3H, s)
1.57 (3H, s)
1.52 (3H, s)
OMe
—
—
3.73 (3H, s)
a) Submerged by other signals.

September 2010
1205
Table 2. 13C-NMR (150 MHz) Data of Compounds 1—4 in CDCl3
2, similar to that of 1, also revealed the presence of an a-
methylene-l-lactone (1759, 1663 cmϪ1) and an ester group
Carbon
1
2
3
4
1
(1737 cmϪ1) moieties. The H- and 13C-NMR spectra of 2
also exhibited some similarities to those of 1. The exometh-
ylene in 2 was confirmed by the presence of two characteris-
tic proton signals at dH 5.70 (d, 1.9, 1H) and dH 6.34 (d, 2.5,
1H), both connected to C-17 at dC 124.2. The presence of
two carbonyl esters in 2 was supported by 13C-NMR signals
1
2
3
4
5
6
7
8
9
42.8 (d)
77.7 (d)
121.2 (d)
141.2 (s)
39.2 (t)
24.9 (t)
123.9 (d)
134.3 (s)
34.6 (t)
29.2 (t)
72.2 (d)
135.8 (s)
124.6 (d)
27.3 (t)
138.8 (s)
170.5 (s)
121.6 (t)
15.9 (q)
16.5 (q)
17.7 (q)
21.1 (q)
170.3 (s)
—
46.9 (q)
76.8 (d)
123.2 (d)
141.7 (s)
38.5 (t)
24.3 (t)
124.8 (d)
133.9 (s)
38.6 (t)
24.3 (t)
127.9 (d)
129.4 (s)
39.9 (t)
71.7 (d)
134.8 (s)
170.0 (s)
124.2 (t)
16.2 (q)
15.4 (q)
18.3 (q)
21.0 (q)
170.1 (s)
—
9.6 (d)
32.8 (t)
123.5 (d)
135.4 (s)
38.9 (t)
24.9 (t)
125.8 (d)
133.4 (s)
39.5 (t)
23.8 (t)
122.3 (d)
133.9 (s)
34.2 (t)
29.2 (t)
144.7 (s)
168.0 (s)
123.6 (t)
15.4 (q)
15.3 (q)
17.7 (q)
—
146.0 (s)
120.0 (d)
126.4 (d)
133.0 (s)
35.4 (t)
25.8 (t)
123.9 (d)
134.8 (s)
38.6 (t)
24.6 (t)
126.9 (d)
130.0 (s)
41.9 (t)
72.5 (d)
28.1 (d)
24.8 (q)
23.8 (q)
61.7 (t)
15.7 (q)
18.5 (q)
21.0 (q)
21.3 (q)
170.0 (s)
170.5 (s)
—
1
at dC 170.0 and dC 170.1. H- and 13C-NMR signals at dH
4.87 (m), dC 124.8; dH 5.02 (d, 6.1), dC 127.9; dH 5.08 (d,
9.4), dC 123.2 suggested the presence of three trisubstituted
double bonds in 2 (Tables 1, 2). By the analysis of HSQC,
COSY, TOCSY and HMBC spectral data, the acetoxy group
was located at C-14 [dH 4.97 (m), dC 71.7]. Additionally, the
double bond at C-12/C-13 in 1 shifted to C-11/C-12 in 2.
Thus, the planar structure was established as shown in Fig. 2.
The E-geometry of the three trisubstituted double bonds in 2
was determined by the NOE correlations between H-3 and
H-5, H-7 and H-9 and H-11 and H-14. Furthermore, a cis-
fused ring and the a oriented acetoxy at C-14 were confirmed
by the intense NOE correlations, demonstrating 1R*, 2S*,
and 14S* configurations of 2. The absolute configuration in 2
was determined by following the same procedure subsequent
to 1. Then, the absolute configuration of 2 was determined to
be 1R, 2S, and 14S (Chart 1).
10
11
12
13
14
15
16
17
18
19
20
OAc
OAc
OAc
OAc
OMe
—
—
—
51.7 (q)
—
—
—
—
The molecular formula of C21H32O2 for 3 was determined
Fig. 2. 1H–1H COSY, TOCSY, and Selected HMBC Correlations of Compounds 1—4
Fig. 3. Stereochemistry of Compounds 1—4, and Selected NOE Correlations
Structures were refined by performing an optimized using MM2.

1206
Vol. 58, No. 9
Chart 1. Preparation of MTPA Esters of Compounds 1, 2, and 5, and Their Dd Values
by the ESI-TOF-MS, and therefore, possessed six degrees of HMBC spectrum showed that the two olefinic methyls, H3-19
unsaturation. A signal of a carbonyl ester at 1714 cmϪ1 ap- and H3-20, were coupled to C-7, C-8, C-9 and C-11, C-12,
1
peared in the IR spectrum of 3. The H-NMR spectra of 3 C-13, respectively. In the same way, the oxymethylene pro-
displayed characteristic proton signals for the exomethylene tons at C-18 [(dH 4.65 (d, 12.4) and dH 4.73 (d, 12.4)] exhib-
1
at dH 5.49 (s, 1H) and dH 6.17 (d, 1.2, 1H). In addition, H- ited the HMBC correlations to C-3, C-4, C-5 and also to one
and 13C-NMR data of 3 revealed the presence of three of the carbonyl carbons of the ester group found in 4. The
olefinic protons corresponding to trisubstituted double bonds oxymethine proton [dH 5.97 (dd, 3.9, 10.0)] at C-14 showed
at dH 5.15 (t, 7.0), dH 4.96 (br s) and dH 5.05 (dd, 5.4, 6.5) the HMBC correlations to the second ester group and also to
with olefinic carbon signals at dC 123.5 and 135.4, dC 125.8 the methine C-15. Finally, the COSY and TOCSY correla-
and 133.4, and dC 122.3 and 133.9, respectively. Moreover, tions between H-2 and H-3, H-6 and H-5/H-7, H-10 and H-9/
the proton resonance appearing at dH 3.73 and carbon reso- H-11, H-13 and H-14 achieved the connection of all partial
nances at dC 51.7 and 168.0 unequivocally revealed the pres- structures, and which led to the proposed structure of 4
ence of a methyl ester moiety. It was then concluded that 3 is (Fig. 2).
a monocyclic compound. The cembranoid backbone of 3 was
The stereochemistry of the four double bonds and that of
assembled through the interpretation of COSY, TOCSY and the acetate group in 4 were established by analyzing the
HMBC correlations (Fig. 2). The geometry of all three dou- NOESY spectrum of 4. The NOE correlations from H-2 to
ble bonds in 3 was characterized to be of the E-geometry, in H2-18 and from H-3 to H-5 allowed us to confer both D1 and
accordance with the NOE correlations, because the NOE cor- D3 Z-geometry. The Z, Z nature of the conjugated diene in 4
relations between H-3 and H-5, H-7 and H-9, H-11 and H-13 was confirmed by the coupling constant of H-2/H-3
were observed. Furthermore, the NOE correlations between (Jϭ11.6 Hz). On the other hand, the NOE correlations from
H-1 and H-3/H-11, H-7 and H-3/H-11 were observed, the H-7 to H-9, H-11 to H2-13 and those from H-3 to H-7 and H-
methine proton at C-1 was oriented to the same side of the 14, H-7 to H-11, H-11 to H-14 revealed that the geometry of
three olefinic protons in 3. Finally, the relative configuration D7 and D11 were both E, and that the acetate group at C-14
of the stereogenic center at C-1 in 3 was determined to be was on the opposite side of the olefinic protons H-3, H-7 and
1R* from a perspective of biosynthetic pathway (Fig. 3).
H-11 (Fig. 3).
The molecular ion peak at m/z 411.2505 (MϩNa)ϩ in the
Compound 5 was a known compound isolated previously
ESI-TOF-MS spectrum of 4, in combination with 13C-NMR, from L. denticulatum11) and the Okinawan soft coral
allowed us to establish the molecular formula of 4 as Sinularia mayi.12) The structure of compound 5 was estab-
C24H36O4. The IR spectrum of 4 showed characteristic peaks lished by comparison of 1H-NMR data reported in the litera-
at 1732 cmϪ1 attributable to the ester group. The presence of ture. The relative and absolute stereochemistry of 5 has not
two doublet secondary methyl groups [dH 1.02 (d, 6.9) and been determined yet. Then again, from the NOE correlations
dH 1.06 (d, 6.9)] suggested an isopropyl group (C-15/C- of 5 and by applying Modified Mosher’s method as for 1 and
16/C-17). In fact, the presence of this isopropyl unit was con- 2, we have determined the absolute configuration of 5 to be
firmed by the COSY correlations between the methine proton 1S, 2S, and 13S (Chart 1).
H-15 [dH 2.51 (m)] to both secondary methyl groups (H3-16
Compounds 1, 2, and 5 were confirmed to be isomeric
and H3-17). Proton signals at dH 1.46 and dH 1.54 were cembranoids, which differ from each other in the location of
assigned to olefinic methyl groups (H3-19 and H3-20). The the acetoxy group and the position of one double bond at D11

September 2010
1207
or D12. Furthermore, compound 6, which is the deacetoxy cranolides.18) From the above results, the absolute configura-
cembranoid of 1, 2, and 5, was isolated from L. michaelae,13) tion of 1, 2, 5, and 6 as obtained by Modified Mosher’s
and S. mayi,14) and the spectral data and specific rotation method and the CD analysis, is still the same and can be eas-
were identical with those reported. However, the absolute ily established as shown in Fig. 4.
stereochemistry was reported to be 1R and 2R15) which indi-
The inhibitory effects of compounds 1—3, 5—16 on LPS-
cated the enantiomer of our isolates.
induced NO production against Raw 264.7 cells were evalu-
To clear up any doubts, we examined the absolute stereo- ated. Compounds 1, 2, and 5—12 showed significant in-
chemistry of 1, 2, 5, and 6 using the circular dichroism (CD) hibitory effect of NO production, and their IC50 values were
spectra. The Cotton effect in the 260 nm region corresponds less than 10 mM without any cytotoxic effect (Table 3). These
to an n–p* transition in the a-methylene-g-lactone chro- data indicated that the a-methylene-g-lactone moiety obvi-
mophore. Because the positive Cotton effect at 260 nm was ously plays a role for an inhibitory effect on NO produc-
observed in the CD spectra of 1, 5, and 6, the absolute stere- tion.19) The inhibitory mechanism of these cembranoids was
ochemistry of 6 was identical to those of 1 and 5. On the confirmed by the inhibition of inducible NO synthase (iNOS)
other hand, the CD spectrum of 2 exhibited a negative Cotton expression via suppression of a transcription factor nuclear
effect in the same region contrary to compounds 1, 5 and 6. factor kB (NF-kB).20) These biological properties will be re-
At first glance, the inversion in the sign of the Cotton effect ported in a specialized journal. It is expected these cembra-
between 240 nm and 260 nm observed for 2 indicated the op- noids will contribute to the development of therapies for anti-
posite absolute configuration. However, previous studies on inflammatory diseases.
correlations between the sign of the CD spectrum and the ab-
solute configuration in g-lactones provided evidence of the Experimental
General Experimental Procedures Optical rotations were measured
with a Jasco Dip-370 digital polarimeter at 23 °C. IR spectra were measured
with a JASCO FT/IR-410 spectrophotometer. CD spectra were measured
dependence of the sign of the n–p* Cotton effect upon the
location of Cb relative to the planar lactone system.16,17) The
inversion in the sign of the Cotton effect in 2 can be
explained from the conformational change in the lactone ring
as likewise reported for some sesquiterpenes, such as germa-
with a Jasco J-720W spectropolarimeter at 22 °C. The NMR spectra were
recorded on a Varian INOVA 600 operating at 600 MHz for 1H and 150 MHz
for 13C in CDCl3. 1H- and 13C-NMR chemical shifts are reported in ppm (d)
Fig. 4. CD Spectra of Compounds 1, 2, 5, and 6, and Their Absolute Stereochemistry

1208
Vol. 58, No. 9
ica gel column with n-hexane/EtOAc (25/1), then to the RP-HPLC [MeOH/
H2O (80/20)] to yield 15 (2.0 mg).26) Fr. 7Ј (0.4 g) was chromatographed on
reverse phase column (Cosmosil 140 C18 Prep, Nakalai Tesque) using
MeOH/H2O (9/1) to give four fractions (Fr. 7Ј-1—Fr. 7Ј-4). Fr. 7Ј-1 (114.3
mg) was subjected to Recycling HPLC, and RP-HPLC [MeOH/H2O
(85/15)] to afford 1 (6.4 mg), 2 (10.4 mg) and 5 (65.7 mg).11,12) Finally, Fr. 8Ј
(401.0 mg) was subjected to Recycling HPLC to afford seven fractions (Fr.
8Ј-1—Fr. 8Ј-7). Fr. 8Ј-4 was chromatographed on a reversed phase column
(Cosmosil) using MeOH/H2O (85/15) to give two fractions (Fr. 8Ј-4-1—Fr.
8Ј-4-2). Fr. 8Ј-4-1 (27.2 mg) was then purified by a reverse phase HPLC on a
YMC Pack Pro C18 [250ϫ10 mm, MeOH/H2O (80/20)] to yield 11 (20.0
mg).28)
Table 3. Inhibitory Effect of Compounds 1—3, and 5—16 on LPS-
Induced NO Production (IC50, mM), and Cell Viability at the Concentration
of 10 and 50 mM (% of Control)
a)
a)
a)
Compounds
IC50
10 mM
50 mM
1
2
3
3.8Ϯ0.97
4.0Ϯ0.91
Ͼ50
107.3Ϯ5.5
100.0Ϯ6.6
106.0Ϯ6.3
26.1Ϯ6.7
50.5Ϯ9.1
108.1Ϯ3.1
4
NT
5
6
7
8
9
10
11
12
13
14
15
16
17
4.8Ϯ0.75
2.6Ϯ0.12
3.8Ϯ1.97
5.7Ϯ1.77
7.9Ϯ1.19
2.4Ϯ0.21
4.9Ϯ1.33
6.4Ϯ2.13
Ͼ50
Ͼ50
Ͼ50
16.6Ϯ1.70
NT
105.9Ϯ7.2
103.5Ϯ10.1
99.9Ϯ1.4
28.5Ϯ12.0
31.3Ϯ10.0
78.3Ϯ6.1
Compound 1: Coloress oil; [a]D23 ϩ40.0 (cϭ0.26, CHCl3); IR (CHCl3)
cmϪ1: 2933, 1759, 1727, 1666, 1438, 1372, 1249, 1112, 1018, 982; UV lmax
(n-hexane) nm (log e): 229 (3.74); EI-MS m/z: 358 [M]ϩ; ESI-TOF-MS m/z:
381.2039 [MϩNa]ϩ (Calcd for C22H30O4Na, 381.2036).1H- and 13C-NMR
see Tables 1 and 2.
97.9Ϯ2.4
92.0Ϯ9.2
102.2Ϯ0.53
101.2Ϯ1.7
104.3Ϯ2.1
103.2Ϯ1.9
100.8Ϯ0.20
103.3Ϯ0.70
100.2Ϯ2.4
97.2Ϯ0.05
101.5Ϯ5.8
98.6Ϯ0.19
74.9Ϯ15.2
104.3Ϯ2.8
106.5Ϯ1.9
102.4Ϯ1.5
101.9Ϯ1.3
45.8Ϯ5.5
Compound 2: Coloress oil; [a]D23 Ϫ79.1 (cϭ0.34, CHCl3); IR (CHCl3)
cmϪ1: 2925, 1759, 1737, 1663, 1437, 1374, 1217, 1130, 1027, 978, 942; UV
l
max (n-hexane) nm (log e): 213 (4.21); EI-MS m/z: 358 [M]ϩ; ESI-TOF-MS
m/z: 381.2036 [MϩNa]ϩ (Calcd for C22H30O4Na, 381.2036); 1H- and 13C-
NMR see Tables 1 and 2.
Compound 3: Coloress oil; [a]D23 ϩ22.3 (cϭ0.13, CHCl3); IR (CHCl3)
cmϪ1: 2927, 1714, 1456, 1438, 1141, 948; EI-MS m/z: 316 [M]ϩ; ESI-TOF-
MS m/z: 339.2284 [MϩNa]ϩ (Calcd for C21H32O2Na, 339.2295); 1H- and
13C-NMR see Tables 1 and 2.
18
NT
19
NT
L-NMAA
32.9Ϯ5.90
96.7Ϯ1.0
97.5Ϯ1.4
Compound 4: Coloress oil; [a]D23 ϩ93.3 (cϭ0.09, CHCl3); IR (CHCl3)
cmϪ1: 2928 1732, 1244, 1020, 960; ESI-TOF-MS m/z: 411.2505 [MϩNa]ϩ
(Calcd for C24H36O4Na, 411.2506 ); 1H- and 13C-NMR see Tables 1 and 2.
Compound 5—19: The known compounds were identified by comparison
a) Each value represents the meanϮS.D. (nϭ5) of three experiments.
of their H- and 13C-NMR data and specific rotations with previously pub-
1
and referenced to the solvent signal (CDCl3: dHϭ7.24, dCϭ77.2). EI-MS
were performed on Gas Chromatography Mass Spetrometer-QP5050 (Direct
injection). ESI-TOF-MS were measured with a Bruker microTOF mass
spectrometer. Chromatographic separations were carried out using silica gel
(Merck Silica gel 60 F254), Recycling Preparative HPLC (JAIGEL 1Hϫ2,
600ϫ20 mm i.d., CHCl3, JAI) with a JAI Model LC-9201 system, and
RP HPLC (Cosmosil cholester, 250ϫ4.6 mm i.d., Nakalai Tesque, YMC
Pack Pro C18, 250ϫ10 mm i.d., YMC) with a Jasco PU2089 gradient pump
and PU2075 UV/VIS detector. Thin layer chromatography (TLC) analysis
were carried out using Merck precoated TLC plates Silica gel 60 F254 and
lished data.
Preparation of Secondary Alcohol Derivative (1a) To a solution of
compound 1 (9.9 mg, 27.6 mmol) in dry MeOH (2.5 ml) at room temperature
were added potassium carbonate (20.1 mg, 145.4 mmol) and the resulting
mixture was stirred for 22 h at room temperature. The reaction mixture was
quenched by adding some amount of ion-exchange resine (Dowex 50WX8-
200) and then filtrated. The filtrate was dried with N2, and the resulting
residue was subjected to silica gel column chromatography with n-hexane/
1
EtOAc (8/1—4/1) to give 2.2 mg of 1a. H-NMR (CDCl3) d: 2.61 (1H, m,
H-1), 5.17 (1H, dd, Jϭ7.6, 9.5 Hz, H-2), 5.05 (1H, d, Jϭ9.7 Hz, H-3), 4.93
(1H, br t, H-7), 4.45 (1H, t, Jϭ6.4 Hz, H-11), 2.20 (1H, m, H-15), 3.54 (1H,
dd, Jϭ3.7, 9.5 Hz, H-17), 3.63 (1H, m, H-17Ј), 1.60 (3H, s, H-18), 1.55 (3H,
s, H-19), 1.63 (3H, s, H-20), 3.28 (3H, s, OMe-17).
RP-18F254s
.
Animal Material The soft coral L. crassum (No. OCE 2007-01) was
collected on a coral reef off Sesoko-cho (Okinawa, Japan) by hand in Janu-
ary 2008 at a depth of 2—3 m. A voucher specimen was deposited at the
Graduate School of Pharmaceutical Sciences, Kyushu University.
Preparation of (R)- or (S)-MTPA Esters (1b, 1c) 1.1 mg (3.2 mmol) of
1a was dissolved in 1.5 ml of dry methylene chloride. The solution was
treated with (R)-a-methoxy(trifluoromethyl)phenylacetic acid (MTPA)
(45.7 mg, 195.1 mmol), N,NЈ-dicyclohexyl carbodiimide DCC (45.1 mg,
218.6 mmol) and a catalytic amount of 4-dimethylaminopyridine (DMAP).
The solution was stirred at room temperature for 91 h and filtered. The fil-
trate was dried with N2 and subjected to silica gel column with n-hexane/
EtOAc (12/1) to afford (R)-MTPA ester (1b, 0.8 mg, 1.5 mmol). With (S)-
MTPA acid, 1a (1.1 mg) was esterified in the same manner to yield (S)-
Extraction and Isolation The fresh material of L. crassum was
chopped and exhaustively extracted with n-hexane (10 lϫ3). After removal
of solvent in vacuo, 90.38 g of the n-hexane extract was obtained. Part of
this extract (5.5 g) was chromatographed over silica gel with n-hexane/Et2O
(9/1—0/1, stepwise) to yield 10 fractions, and Fr. 7 was identified as lobo-
phytol acetate (7, 422.9 mg).21) Fr. 3 (235.0 mg) was subjected to recycling
preparative HPLC to yield denticulatolide (12, 28.8 mg),22) and 8 (112.6
mg).23) Fr. 4 (70.0 mg) was also subjected to recycling HPLC to yield 10
(15.2 mg).24) Part of the n-hexane extract (21.1 g) was dissolved in n-hexane
and was further partitioned against 90% of aqueous methanol to yield a n-
hexane soluble part (7.2 g). Flash chromatography of this part (7.0 g) on sil-
ica gel with n-hexane/EtOAc yielded 13 fractions (Fr. 1Ј—Fr. 13Ј). Part of
Fr. 1Ј (149.0 mg) was applied to RP-HPLC [MeOH–H2O (85/15)] to yield
19 (14.1 mg).25) Fr. 2Ј (714.0 mg) was chromatographed over silica gel with
n-hexane/EtOAc (30/1) to afford Fr. 2Ј-1 (15.8 mg) and Fr. 2Ј-2 (490.8 mg).
Part of Fr. 2Ј-2 (250.0 mg) was subjected to recycling HPLC to give eight
fractions (Fr. 2Ј-2-1—Fr. 2Ј-2-8). Purification of Fr. 2Ј-2-4 (8.5 mg) by RP-
HPLC [MeOH/H2O (85/15)] resulted in the isolation of 3 (1.4 mg) and 17
(1.6 mg).26) Fr. 2Ј-2-5 (12.3 mg) was purified RP-HPLC [MeOH/H2O (85/15
and 80/20) to give 16 (1.8 mg),26) 17 (1.0 mg), and 18 (0.7 mg).27) Fr. 3Ј
(101.0 mg) was flash chromatographed over silica gel using n-hexane/EtOAc
(20 : 1) to give five fractions. Fr. 3Ј-5 (47.6 mg) was subjected to recycling
HPLC to afford four fractions (Fr. 3Ј-5-1—Fr. 3Ј-5-4). Fr. 3Ј-5-2 (6.6 mg)
was purified by RP-HPLC [CH3CN/H2O (70/30)] to afford 4 (1.0 mg). Fr. 4Ј
(0.5 g) was subjected to silica gel column with n-hexane/EtOAc (14/1) to
afford six fractions (Fr. 4Ј-1—Fr. 4Ј-6). Fr. 4Ј-5 (277.9 mg) was chro-
matographed over silica gel using n-hexane/EtOAc (14/1) to give 6
(217.1 mg).14) Fr. 4Ј-6 was purified by RP-HPLC [MeOH–H2O (85/15)] to
give 13 (16.3 mg)26) and 14 (4.9 mg).27) Fr. 5Ј (50.1 mg) was subjected to sil-
1
MTPA ester (1c, 0.8 mg). 1b: H-NMR (CDCl3) d: 5.16 (2H, overlap, H-2,
H-3), 2.17 (1H, m, H-6), 2.24 (1H, m, H-6Ј), 5.01 (1H, br t, H-7), 1.63 (1H,
m, H-10), 1.91 (overlap, H-10Ј), 5.83 (1H, br t, H-11), 5.22 (1H, br t, H-13),
1
1.613 (3H, s, H-18), 1.52 (3H, s, H-19), 1.44 (3H, s, H-20). 1c: H-NMR
(CDCl3) d: 5.17 (2H, overlap, H-2, H-3), 2.14 (1H, m, H-6), 2.23 (1H, m, H-
6Ј), 5.00 (1H, br t, H-7), 1.53 (overlap, H-10), 1.86 (1H, br ?, H-10Ј), 5.88
(1H, br t, H-11), 5.25 (1H, br t, H-13), 1.615 (3H, s, H-18), 1.51 (3H, s, H-
19), 1.56 (3H, s, H-20).
Preparation of Secondary Alcohol Derivative (2a), and (R)- , (S)-
MTPA Esters (2b, 2c) from 2 Secondary alcohol derivative (2a), and
(R)- , (S)-MTPA esters (2b, 2c) were prepared as described for 1 using 2
1
(4.7 mg, 13.1 mmol). 2a (2.2 mg): H-NMR (CDCl3) d: 2.39 (1H, m, H-1),
4.88 (1H, dd, Jϭ9.1 Hz, H-2), 5.04 (1H, d, Jϭ9.2 Hz, H-3), 4.84 (1H, br t,
H-7), 4.93 (1H, br t, H-11), 3.68 (1H, br d, H-14), 3.00 (1H, m, H-15), 3.60
(1H, d, Jϭ3.2 Hz, H-17), 3.63 (1H, d, Jϭ5.0 Hz, H-17Ј), 1.64 (3H, s, H-18),
1.51 (3H, s, H-19), 1.56 (3H, s, H-20), 3.29 (3H, s, OMe-17). 13C-NMR
(CDCl3) d: 49.9 (CH, C-1), 79.2 (CH, C-2), 124.7 (CH, C-3), 143.0 (qC, C-
4), 39.9 (CH2, C-5), 25.9 (CH2, C-6), 126.4 (CH, C-7), 135.6 (qC, C-8), 40.0
(CH2, C-9), 25.9 (CH2, C-10), 127.7 (CH, C-11), 132.0 (qC, C-12), 46.3
(CH, C-13), 72.8 (CH2, C-14), 43.1 (CH, C-15), 181.1 (qC, C-16), 60.6
(CH2, C-17), 18.7 (CH3, C-18), 17.6 (CH3, C-19), 20.1 (CH3, C-20). 2b

September 2010
1209
(0.9 mg): 1H-NMR (CDCl3) d: 4.51 (1H, dd, Jϭ8.9 Hz, H-2), 5.04 (2H,
overlap, H-3, H-14), 4.90 (1H, br t, H-7), 5.10 (1H, br t, H-11), 2.40 (1H,
br d, H-13), 3.72 (1H, dd, Jϭ3.62, 9.74 Hz, H-17), 1.64 (3H, s, H-18), 1.57
(3H, s, H-19), 1.63 (3H, s, H-20). 2c (0.8 mg): 1H-NMR (CDCl3) d: 4.59
(1H, dd, Jϭ8.9 Hz, H-2), 5.07 (2H, overlap, H-3, H-11, H-14), 4.90 (1H,
br t, H-7), 5.10 (1H, br t, H-11), 2.28 (1H, br d, H-13), 3.75 (1H, dd, Jϭ3.62,
9.74 Hz, H-17), 1.64 (3H, s, H-18), 1.57 (3H, s, H-19), 1.61 (3H, s, H-20).
Preparation of Secondary Alcohol Derivative (5a), and (R)- , (S)-
MTPA Esters (5b, 5c) from 5 Secondary alcohol derivative (5a), and
(R)- , (S)-MTPA esters (5b, 5c) were prepared as described for 1 using 5
(22.7 mg, 63.0 mmol). 5a (11.1 mg): 1H-NMR (CDCl3) d: 2.98 (1H, m, H-1),
5.36 (1H, dd, Jϭ7.8, 10.8 Hz, H-2), 5.09 (overlap, H-3), 2.19 (overlap, H-5),
2.04 (1H, br d, Jϭ15.9 Hz, H-6), 4.71 (1H, br d, Jϭ8.7 Hz, H-7), 1.81 (1H,
m, H-9), 2.19 (overlap, H-9Ј), 1.96 (1H, m, H-10), 2.19 (overlap, H-10Ј),
5.06 (overlap, H-11), 3.74 (1H, d, Jϭ10.5 Hz, H-13), 1.52 (overlap, H-14),
1.90 (1H, m, H-14Ј), 2.47 (1H, dt, Jϭ4.2, 12.4 Hz, H-15), 3.60 (2H, d, Jϭ
4.2 Hz, H-17), 1.66 (3H, s, H-18), 1.57 (3H, s, H-19), 1.58 (3H, s, H-20),
3.36 (3H, s, OMe-17). 13C-NMR (CDCl3) d: 38.7 (CH, C-1), 77.5 (CH, C-
2), 119.4 (CH, C-3), 143.2 (qC, C-4), 39.6 (CH2, C-5), 24.4 (CH2, C-6),
125.4 (CH, C-7), 133.3 (qC, C-8), 39.7 (CH2, C-9), 22.9 (CH2, C-10), 126.5
(CH, C-11), 136.9 (qC, C-12), 76.9 (CH, C-13), 35.8 (CH2, C-14), 44.7
(CH, C-15), 176.6 (qC, C-16), 69.7 (CH2, C-17), 15.0 (CH3, C-18), 9.6
(CH3, C-19), 15.1 (CH3, C-20). 5b (1.1 mg): 1H-NMR (CDCl3) d: 5.45 (1H,
dd, Jϭ7.7, 10.8 Hz, H-2), 5.08 (1H, d, Jϭ10.8 Hz, H-3), 4.71 (1H, br d, Jϭ
8.7 Hz, H-7), 5.28 (1H, br, H-11), 5.16 (1H, d, Jϭ11.1 Hz, H-13), 3.60 (2H,
d, Jϭ4.0 Hz, H-17), 1.76 (3H, s, H-18), 1.58 (3H, s, H-19), 1.38 (3H, s, H-
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Jϭ6.9, 10.8 Hz, H-2), 5.07 (1H, d, Jϭ10.8 Hz, H-3), 4.71 (1H, br d, Jϭ
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Acknowledgment We wish to thank the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) of Japan for the financial support
to Mr. Mpanzu Wanzola.