Durumolides A-E, anti-inflammatory and antibacterial cembranolides from the soft coral Lobophytum durum

Article abstract of DOI:10.1016/j.tet.2008.07.104

Chemical investigations on the acetone extract of the soft coral Lobophytum durum have afforded five new cembranoids with a trans-fused α-methylene-γ-lactone, durumolides A-E (1, 6, and 8-10), and five previously characterized cembrane-based diterpenoids (2-5 and 7). The structures of the isolated metabolites were elucidated through extensive spectroscopic analyses, while the relative stereochemistry of 4 was confirmed by X-ray diffraction analyses. Moreover, the absolute configurations of 3-5 and 8 were established by application of modified Mosher's method. The anti-inflammatory effects, antibacterial activities, and inhibition assay of HCMV (Human cytomegalovirus) endonuclease activity of these isolated metabolites 1-10 were evaluated in vitro.

Full text of DOI:10.1016/j.tet.2008.07.104

Tetrahedron 64 (2008) 9698–9704
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Durumolides A–E, anti-inflammatory and antibacterial cembranolides
from the soft coral Lobophytum durum
,
Shi-Yie Cheng a, Zhi-Hong Wen a, Shu-Fen Chiou a, Chi-Hsin Hsu a e, Shang-Kewi Wang b,
,e,
Chang-Feng Dai c, Michael Y. Chiang d, Chang-Yih Duh a
*
a Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
b Department of Microbiology, Kaohsiung Medical University, Kaohsiung 807, Taiwan
c Institute of Oceanography, National Taiwan University, Taipei, Taiwan
d Department of Chemistry, National Sun Yat-sen University, Kaohsiung 804, Taiwan
e Center of Asia-Pacific Marine Researches, National Sun Yat-sen University, Kaohsiung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 June 2008
Received in revised form 25 July 2008
Accepted 25 July 2008
Available online 30 July 2008
Chemical investigations on the acetone extract of the soft coral Lobophytum durum have afforded five
new cembranoids with a trans-fused a-methylene-g-lactone, durumolides A–E (1, 6, and 8–10), and five
previously characterized cembrane-based diterpenoids (2–5 and 7). The structures of the isolated
metabolites were elucidated through extensive spectroscopic analyses, while the relative stereochem-
istry of 4 was confirmed by X-ray diffraction analyses. Moreover, the absolute configurations of 3–5 and 8
were established by application of modified Mosher’s method. The anti-inflammatory effects, antibac-
terial activities, and inhibition assay of HCMV (Human cytomegalovirus) endonuclease activity of these
isolated metabolites 1–10 were evaluated in vitro.
Keywords:
Lobophytum durum
Cembranoid
Ó 2008 Elsevier Ltd. All rights reserved.
a
-Methylene-g-lactone
Anti-inflammatory effects
Antibacterial activities
HCMV endonuclease activity
1. Introduction
R1
R1
O
Soft corals belonging to the genus Lobophytum (Alcyoniidae)
have proven to be a rich source of macrocyclic cembrane-type
diterpenes and their cyclized derivatives.1–16 Some of these me-
tabolites have been shown to exhibit cytotoxic properties.2–6 The
ongoing search for bioactive constituents prompted us to in-
vestigate the secondary metabolites of the soft coral Lobophytum
durum (Tixier-Durivault, 1956), chemical constituents and bio-
activity of which were not reported previously. We have succeeded
R2
R2
R3
H
O
O
O
O
1 R1= OAc R2= (R)-OH R3= OH
2 R1= OAc R2= (S)-OH R3= H
3 R1= OAc R2= (R)-OH R3= H
4 R1= OH R2= (R)-OH R3= H
5 R1= OH R2= (S)-OH R3= H
6 R1= OAc R2= OH
7 R1= OAc R2= H
8 R1= OH R2= OH
in the isolation of five new cembranoids possessing a
-lactone ring trans-fused to a 14-membered ring, durumolides
A–E (1, 6, and 8–10), and five previously characterized -methy-
lene-
-lactone-bearing cembranolides (2–5 and 7)10–13,17 from the
a-methylene-
g
O
H
O
OAc
a
HO
H
g
acetone extract of the organism. In the article, we report the
structural elucidation, anti-inflammatory effects, antibacterial
activities, and inhibition assay of HCMV endonuclease activity of
these metabolites.
H
HO
O
O
O
O
O
H
10
9
2. Results and discussion
The acetone extract of L. durum was partitioned between EtOAc
and H2O to afford the EtOAc-soluble fraction, which was then
* Corresponding author. Tel.: þ886 7 5252000x5036; fax: þ886 7 5255020.
E-mail address: yihduh@mail.nsysu.edu.tw (C.-Y. Duh).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.07.104

S.-Y. Cheng et al. / Tetrahedron 64 (2008) 9698–9704
9699
Table 1
subjected to column chromatography on silica gel. The fractions
1H NMR spectral data of compounds 1 and 6
containing terpenoids were selected after the 1H NMR spectrum
revealed the presence of various terpenoids, and these fractions
were further purified by a series of normal and reversed-phase
HPLC to afford 1–10 (see Section 3).
H#
1a
6a
1
2
3
2.78 m
2.73 m
a: 1.87 br d (14.1)b; b: 1.50 m
2.78 m
a: 2.38 m; b: 2.34 m
5.27 t (7.6)b
a: 2.32 m; b: 2.25 m
a: 2.27 m; b: 2.22 m
4.89 br t (7.5)
a: 2.15 m; b: 2.10 m
a: 2.36 m; b: 2.19 m
5.36 br dd (8.4, 4.7)
3.75 d (8.0)
4.22 dd (8.0, 2.3)
a: 6.32 d (1.9); b: 5.73 d (1.5)
4.55 s
1.62 s
1.72 s
2.07 s
Compound 4 has been previously isolated from the soft coral
Lobophytum crassum.12 Definitive support for the proposed struc-
ture of 4 was provided by X-ray crystallographic analysis of color-
less crystals (Fig. 1). The appropriate stereochemistry of 4 was
determined by spectroscopic method according to Mosher’s acyl-
ation for absolute configuration determination of chiral alcohols.18
Analysis of the DdSꢀR values (Fig. 2) according to the Mosher model
pointed to an R configuration for C-13 of 4, because H2-9, H2-10, H-
11, Me-19, and Me-20 of (S)-MTPA ester 4a were less shielded by
the phenyl ring of MTPA products. Therefore, the absolute stereo-
chemistry of 4 was established unambiguously.
5
6
7
9
10
11
13
14
17
18
19
20
OAc
a: 2.46 m; b: 1.18 td (12.3, 2.6)
a: 2.39 m; b: 2.16 m
5.36 br d (6.0)
4.11 dd (14.3, 2.0)
a: 2.78 m; b: 2.35 m
5.47 br d (5.7)
4.08 br s
4.08 br s
a: 6.32 d (2.7); b: 6.05 d (2.2)
a: 4.43 d (12.4); b: 3.84 d (12.4)
1.71 s
1.74 s
2.14 s
Durumolide A (1), appeared as a colorless oil, was isolated as the
most polar metabolite. Its HRESIMS (m/z 429.1890, [MþNa]þ) and
NMR spectroscopic data (Tables 1 and 2) established the molecular
formula C22H30O7, implying the existence of eight degrees of
unsaturation. The IR absorptions of 1 at 1765 and 1663 cmꢀ1
a
Spectra were measured in CDCl3 (300 MHz).
J values (in Hz) are in parentheses.
b
Table 2
13C NMR spectral data of compounds 1, 6, and 8–10
revealed the presence of an
a-methylene-g-lactone moiety. This
was further indicated from the 1H NMR signals at
d
6.32 (1H, d,
C#
1a
42.1 (CH)c
6a
42.0 (CH)c
8a
42.2 (CH)c
9a
38.8 (CH)c
10b
42.8 (CH)c
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
OAc
31.1 (CH2)
62.5 (CH)
60.3 (qC)
32.5 (CH2)
24.1 (CH2)
129.0 (CH)
137.8 (qC)
77.9 (CH)
33.2 (CH2)
126.8 (CH)
133.5 (qC)
80.8 (CH)
81.8 (CH)
138.3 (qC)
168.8 (qC)
124.5 (CH2)
63.6 (CH2)
11.1 (CH3)
12.5 (CH3)
20.8 (CH3)
170.8 (qC)
34.1 (CH2)
126.6 (CH)
135.8 (qC)
34.6 (CH2)
24.3 (CH2)
123.5 (CH)
133.9 (qC)
37.5 (CH2)
24.4 (CH2)
131.3 (CH)
131.9 (qC)
79.1 (CH)
83.7 (CH)
138.4 (qC)
169.6 (qC)
123.3 (CH2)
61.6 (CH2)
17.0 (CH3)
13.2 (CH3)
20.9 (CH3)
170.9 (qC)
33.8 (CH2)
124.5 (CH)
140.3 (qC)
34.6 (CH2)
24.2 (CH2)
123.7 (CH)
133.9 (qC)
37.6 (CH2)
24.4 (CH2)
131.0 (CH)
132.1 (qC)
78.9 (CH)
83.8 (CH)
138.6 (qC)
169.8 (qC)
123.2 (CH2)
60.0 (CH2)
16.9 (CH3)
13.3 (CH3)
36.1 (CH2)
71.0 (CH)
75.4 (qC)
31.2 (CH2)
63.9 (CH)
64.5 (qC)
34.1 (CH2)
22.0 (CH2)
124.3 (CH)
136.3 (qC)
37.5 (CH2)
24.9 (CH2)
131.1 (CH)
132.9 (qC)
79.5 (CH)
84.6 (CH)
138.9 (qC)
169.7 (qC)
124.3 (CH2)
67.6 (CH2)
17.2 (CH3)
14.3 (CH3)
20.9 (CH3)
171.6 (qC)
30.1 (CH2)
24.1 (CH2)
124.3 (CH)
136.2 (qC)
38.5 (CH2)
25.2 (CH2)
132.5 (CH)
132.1 (qC)
81.4 (CH)
82.1 (CH)
138.5 (qC)
168.9 (qC)
124.7 (CH2)
199.3 (CH)
15.8 (CH3)
12.5 (CH3)
a
b
c
Spectra were measured in CDCl3 (75 MHz).
Spectra were measured in CDCl3 (100 MHz).
Multiplicities are deduced by HSQC and DEPT experiments.
Figure 1. X-ray ORTEP diagram of 4.
J¼2.7 Hz) and 6.05 (1H, d, J¼2.2 Hz) and 13C NMR signals at
d 168.8
(qC, C-16), 138.3 (qC, C-15), 124.5 (CH2, C-17), 81.8 (CH, C-14), and
42.1 (CH, C-1). A strong IR spectrum absorption at 1745 cmꢀ1 in-
dicated the presence of an acetoxy group. Two secondary hydroxyls
were recognized as being present in 1 from its 1H NMR signals at
−0.01
+0.01
−0.01
OAc
O
−0.04 OR
+0.05
O
−0.02
−0.04
−0.01
−0.03
−0.03
−0.08
−0.02
+0.07
+0.03 +0.05
−0.01
−0.01
+0.15
RO
4.11 (1H, dd, J¼14.3, 2.0 Hz) and 4.08 (1H, br s) and 13C NMR
RO
H
H
+0.10
d
+0.05
−0.02
+0.08
−0.05
+0.09
signals at
d 77.9 (CH, C-9) and 80.8 (CH, C-13), as well as from
O
O
O
O
+0.08
a broad IR absorption at 3458 cmꢀ1. Moreover, the 13C NMR signals
at 137.8 (qC, C-8), 133.5 (qC, C-12), 129.0 (CH, C-7), 126.8 (CH,
+0.16
+0.08
3
4
d
C-11), 62.5 (CH, C-3), and 60.3 (qC, C-4) assigned two trisubstituted
double bonds and one trisubstituted epoxide in 1, respectively. The
above functionalities also account for six of the eight degrees of
unsaturation, suggesting a bicyclic structure in 1. By interpretation
of 1H–1H COSY correlations, it was possible to establish three partial
structures of consecutive proton systems extending from H-3 to
H2-13 through H2-2, H-1, and H-14, from H2-5 to H-7, and from H-9
to H-11, as well as long-range COSY correlations between H-1/
H2-17, H3-19/H-7, and H3-20/H-11. Moreover, the connectivities of
these partial structures were further established by the HMBC
correlations (Fig. 3). The long-range 1H–13C correlations observed
−0.10
+0.10
−0.02
+0.04
OR
OR
−0.01
+0.01
+0.08
−0.05
−0.08
−0.02
−0.04
+0.01
O
+0.03
+0.01
−0.03
−0.05
−0.14
−0.05
+0.06
−0.05
−0.02
+0.04
−0.02
+0.04
RO
H
RO
−0.39
+0.02
−0.02
−0.04
−0.06
−0.09
−0.08
+0.26
−0.11
+0.16
O
O
O
O
+0.13
+0.09
−0.08
+0.07
5
R= MTPA
8
Figure 2. Selected 1H NMR DdSꢀR values in parts per million for the S- and R-MTPA
esters of 3–5 and 8 in CDCl3.

9700
S.-Y. Cheng et al. / Tetrahedron 64 (2008) 9698–9704
O
O
O
O
O
O
HO
O
HO
O
OHHO
O
O
O
O
O
O
1
6
9
O
H
O
OH
HO
HO
O
O
O
O
8
10
) and key HMBC (/) correlations of 1, 6, and 8–10.
Figure 3. 1H–1H COSY (
from H2-18 to C-3, C-4, C-5, and the carbonyl carbon of 18-OAc
indicated the position of the epoxide at C-3 and C-4. In addition, the
HMBC correlations from H3-19 to C-7, C-8, and C-9 and from H3-20
to C-11, C-12, and C-13 led the assignment of the two secondary
hydroxyls at C-9 and C-13. The locations of the two double bonds at
C-7/C-8 and C-11/C-12 were clarified by analysis of the above HMBC
correlations. Thus, the gross structure of durumolide A was
observations, durumolide A (1) was established as (1R
9S ,13R ,14R ,7E,11E)-18-acetoxy-9,13-dihydroxy-3,4-epoxycembra-
7,11,15(17)-trien-16,14-olide.
*
,3R
*
,4S ,
*
*
*
*
Compound 5 has been previously isolated from the soft coral
Sinularia gibberosa.17 The stereochemistry of 5 assigned by NOESY
spectrum was compatible with those of 5 offered by computer-
generated perspective model using MM2 force field calculations
(Fig. 4), in which the close contacts of atoms calculated in space
were consistent with the NOESY correlations. The NOESY spectrum
indicated that 5 possessed the same configuration as 4 at the C-1,
C-3, C-4, and C-14 stereocenters. The significant NOESY correlation
differences between 4 and 5 demonstrated that the hydroxyl group
at C-13 of 5 possessed the S configuration, which was also identified
by Mosher’s esterification.18 Analysis of the DdSꢀR values (Fig. 2)
according to the Mosher model pointed to an S configuration for
C-13 of 5. Thus, compound 5 was formulated as (1R,3R,4S,13S,
14R,7E,11E)-13,18-dihydroxy-3,4-epoxycembra-7,11,15(17)-trien-16,
14-olide.
assigned as 1, possessing an a-methylene-g-lactone ring fused to
a 14-membered ring at C-1 and C-14. A computer-modeled 3D
structure (Fig. 4) of 1 was generated by using MM2 force field
calculations for energy minimization with the molecular modeling
program Chem 3D Ultra 9.0. The relative stereochemistry of 1
assigned by NOESY spectrum was compatible with those of 1 of-
fered by computer modeling, in which the close contacts of atoms
calculated in space were consistent with the NOESY correlations.
The NOE correlations (Fig. 4) between H-1/H-3, H-1/H-11, H-1/H-
13, H-11/H-13, H-11/H-9, H-3/H-5b (
H-3/H-7, H-7/H-9, H-1/H-2a ( 1.81), H-3/H-2a, H-14/H-2b (
H-14/H3-20, and H-2b/H-17b (
d
1.18), 18-OAc/H-5a (
d
2.46),
d
d 1.50),
d
6.05) indicated the relative con-
Compound 6 was obtained as a colorless oil, which was analyzed
for the molecular formula C22H30O4 by HRESIMS coupled with the
DEPT and 13C NMR spectroscopic data (Table 2). The presence of an
figurations for the 14-membered ring carbons, which were iden-
tical to the X-ray diffraction data of compound 4. From the above
Figure 4. Key NOE correlations and computer-generated perspective model using MM2 force field calculations for 1, 5, 6, and 8–10.

S.-Y. Cheng et al. / Tetrahedron 64 (2008) 9698–9704
9701
a
-methylene-
signals at
NMR signals at
g
-lactone moiety was indicated from the 1H NMR
stereochemistry of 8 as established by the NOESY experiment. In
order to resolve the absolute stereochemistry of 8, we determined
the absolute configuration at C-13 using a modified Mosher’s es-
d
6.32 (1H, d, J¼1.9 Hz) and 5.73 (1H, d, J¼1.5 Hz) and 13
C
d
169.6 (qC, C-16),138.4 (qC, C-15),123.3 (CH2, C-17),
83.7 (CH, C-14), and 42.0 (CH, C-1). The 1H and 13C NMR spectro-
scopic data of 6 contained resonances for three trisubstituted
double bonds at C-3 and C-4 [dH 5.27 (t, J¼7.6 Hz, 1H); dC 135.8 (qC)
and 126.6 (CH)], C-7 and C-8 [dH 4.89 (br t, J¼7.5 Hz, 1H); dC 133.9
(qC) and 123.5 (CH)], and C-11 and C-12 [dH 5.36 (br dd, J¼8.4,
4.7 Hz,1H); dC 131.9 (qC) and 131.3 (CH)]. From the above evidences,
6 was suggested to be a bicyclic cembranolide. The assignments of
proton and carbon signals were achieved by application of COSY,
HSQC, and HMBC experiments (Fig. 1). Metabolite 6 was analogous
to those of 712 except for the resonances in the vicinity of C-13. The
exception was that the resonances for the methylene at C-13 were
replaced by those of a secondary hydroxyl. The COSY correlations
(Fig. 3) from H-13 to H-3 through H2-2, H-1, and H-14, and HMBC
correlations (Fig. 3) from H3-20 to C-11, C-12, and C-13 suggested
these assignments. The stereochemistry of 6 was determined
through inspection of the NOESY NMR spectrum as well as a com-
puter-generated lower energy conformation using MM2 force field
calculations (Fig. 4). From the NOESY spectrum of 6, H-13 was found
to show the NOE correlations with both H-1 and H-11, indicating
the R configuration of the secondary hydroxyl attached at C-13. The
NOE correlations between H2-18/H2-2, H3-19/H2-6, H3-20/H2-10,
H-3/H2-5, H2-9/H-7, and H-11/H-13, respectively, reflected the E
geometry of the three trisubstituted double bonds in the molecule.
On the basis of the aforementioned results and other detailed
NOESY correlations (Fig. 4), the structure of durumolide B (6) was
terification.18 The bis-(S)- and (R)-
a
-methoxy-a-(trifluoromethyl)-
a
-phenylacetic (MTPA) esters for 8 (8a and 8b, respectively) were
prepared by using the corresponding R-(ꢀ)- and S-(þ)-MTPA
chloride, respectively. The determination of DdSꢀR values (Fig. 2) for
the protons neighboring C-13 led to the assignment of the R con-
figuration at C-13 in 8. Thus, durumolide C (8) was established as
(1R,13R,14R,3E,7E,11E)-13,18-dihydroxycembra-3,7,11,15(17)-tet-
raen-16,14-olide.
(1R
*
,3R
*
,4R
*
,13S
*
,14S ,7E,11E)-18-Acetoxy-4-hydroxy-3,13-epoxy-
*
cembra-7,11,15(17)-tetraen-16,14-olide (9) was assigned a molecu-
lar formula of C22H30O6, according to its HRESIMS and NMR
spectroscopic data (Tables 2 and 3). To confirm the number of
primary or secondary OH groups, compound 9 was submitted to
acetylation with Ac2O in pyridine at room temperature overnight.
The obtained negative result showed that compound 9 didn’t
possess any primary or secondary OH groups in the original
structure. The position of the ether linkage at C-3/C-13 was thus
confirmed by the above observations. The acetoxy function posi-
tioned at C-18 was confirmed by the HMBC correlations from H2-18
to C-3, C-4, C-5 and the carbonyl carbon of OAc-18. The 1H–1H COSY
spectrum correlations of 9 were similar to those of 6. These data,
together with the 1H–13C long-range correlations observed in the
HMBC experiments (Fig. 3), established the planar structure of 9.
The relative stereochemistry of 9 was deduced from a 2D NOESY
experiment, which indicated that H-1, H-11, and H-13 are located
on the same side of the ring system, whereas H-3, H-14, and H2-18
are oriented toward the opposite side. The computer-modeled
structure of 9 was generated by CS Chem 3D version 9.0 using MM2
force field calculations for energy minimization, as shown in
Figure 4. The result was consistent with the stereochemistry of 9
as established by NOESY experiments. Accordingly, the structure
of durumolide D (9) was established unambiguously.
unambiguously elucidated and assigned as (1R
*
,13R
*
,14R ,3E,
*
7E,11E)-18-acetoxy-13-hydroxycembra-3,7,11,15(17)-tetraen-16,14-
olide.
Compound
8 gave a formula of C20H28O4, from the in-
terpretation of its HRESIMS and 13C NMR spectroscopic data (Table
2). The NMR features (Tables 2 and 3) of 8 were analogous to those
of 6 with the exception that the resonances for the primary hy-
droxyl at C-18 were replaced by those of an acetoxy group. The
HMBC correlations from H2-18 to C-3, C-4, C-5, and the carbonyl
carbon of OAc-18 suggested these assignments. The computer-
modeled structure of 8 was generated by CS Chem 3D version 9.0
using MM2 force field calculations for energy minimization, as
shown in Figure 4. The result was consistent with the
Durumolide E (10) analyzed for C20H26O5 from HRESIMS and 13
C
NMR spectroscopic data (Table 2), indicating eight degrees of
unsaturation. The IR spectrum of 10 showed the presence of hy-
droxy (3457 cmꢀ1), aldehyde functionality (2846 and 1716 cmꢀ1),
and
a-methylene-g
-lactone (1767 and 1660 cmꢀ1) moieties. The
NMR features (Tables 2 and 3) of 10 were similar to those of 4,
except for the replacement of the oxymethylene by an aldehyde
group [dH 9.34 (1H, d, J¼0.8 Hz) and dC 199.3 (CH)] at C-18. This was
supported by HMBC correlations (Fig. 3) from H-18 to C-3, C-4, and
C-5. The relative stereochemistry and the detailed 1H NMR spec-
troscopic data assignment of 10 were determined mainly by the
assistance of the NOESY experiment (Fig. 4). The NOESY spectrum
indicated that 10 possessed the similar configurations for each ring
junction and chiral center as 4 at C-1, C-3, C-4, C-13, and C-14. On
the basis of the above observations and other detailed NOESY cor-
Table 3
1H NMR spectral data of compounds 8–10
H#
8a
9a
10b
1
2
2.73 m
a: 2.40 m; b: 2.29 m
2.91 m
a: 1.99 m; b: 1.78 m
2.76 m
a: 1.97 dt (16.8, 2.0)c;
b: 1.70 m
3
5
5.19 t (7.5)c
a: 2.26 m; b: 2.17 m
3.57 br d (8.9)c
a: 1.91 m; b: 1.72 m
3.17 dd (8.4, 2.0)
a: 2.51 m;
relations (Fig. 4), compound 10 was fully elucidated as (1R
13S ,14R ,7E,11E)-13-hydroxy-18-oxo-3,4-epoxycembra-7,11,15(17)-
trien-16,14-olide.
Noteworthy is the conclusion concerning the bicyclic ring
junction in these isolated metabolites possessing the -methylene-
-lactone moiety. An X-ray crystallographic analysis of compound 4
* * *
,3R ,4S ,
b: 1.21 td (15.2, 2.4)
a: 2.63 m; b: 2.16 m
5.05 br t (7.2)
a: 2.36 m; b: 2.14 m
a: 2.53 m; b: 2.20 m
5.46 dd (10.0, 2.8)
4.04 br s
4.05 br d (6.4)
a: 6.38 d (3.2);
b: 6.15 d (2.8)
9.34 d (0.8)
*
*
6
7
9
10
11
13
14
17
a: 2.32 m; b: 2.17 m
4.89 br d (7.0)
2.09 m
a: 2.38 m; b: 2.13 m
5.34 br d (5.7)
3.75 d (7.7)
4.23 dd (7.7, 2.3)
a: 6.29 d (1.9);
b: 5.71 d (1.6)
4.10 s
2.24 m
5.19 br t (6.4)
2.23 m
a: 2.39 m; b: 2.25 m
5.46 br t (5.8)
3.83 d (8.8)
4.27 dd (8.8, 3.4)
a: 6.32 d (2.0);
b: 5.76 d (2.0)
a: 4.22 d (11.9);
b: 4.14 d (11.9)
1.67 s
a
g
proved the presence of a trans-fused lactone ring. Biosynthetically,
the tetrahydro-2H-pyran ring of compound 9 may be formed by the
hydroxyl at C-13 attacking to C-3 by acid-catalyzed ring-opening of
the 3,4-epoxide.
Compounds 1–6, 8, and 10 revealed greater antibacterial po-
tential than the positive control (ampicillin) against Salmonella
enteritidis. Compound 3 exhibited significant antibacterial activity
18
19
20
1.61 s
1.71 s
1.58 s
1.71 s
1.78 s
OAc
2.05 s
at a concentration of 50
a concentration of 100 g/disk and compounds 1 and 10 at a con-
centration of 200 g/disk. Compounds 7, 9, and ampicillin exhibited
mg/disk; compounds 2, 4–6, and 8 at
a
b
c
Spectra were measured in CDCl3 (300 MHz).
Spectra were measured in CDCl3 (400 MHz).
J values (in Hz) are in parentheses.
m
m

9702
S.-Y. Cheng et al. / Tetrahedron 64 (2008) 9698–9704
Figure 5. Effect of compounds 1–10 at 10
mM on the LPS-induced pro-inflammatory iNOS and COX-2 protein expression of RAW 264.7 macrophage cells by immunoblot analysis;
(A) immunoblot of iNOS; (B) immunoblot of COX-2; (C) immunoblot of
b-actin. The values are meanꢁSEM (n¼5). Relative intensity of the LPS alone stimulated group was taken as
100%. *Significantly different from LPS-stimulated (control) group (*P<0.05).
significant antibacterial activity at a concentration of 250
The present result suggested that the presence of -methylene-
-lactone moiety is important for significant activity against
m
g/disk.
0.25 mm) and precoated RP-18 F254s plates (Merck, 1.05560) were
used for analytical TLC analyses. High-performance liquid chro-
matography (HPLC) was carried out using a Hitachi L-7100 pump
equipped with a Hitachi L-7400 UV detector at 220 nm and a
semi-preparative reversed-phase column (Merck, Hibar Purospher
a
g
S. enteritidis.19 However, the results for inhibition of HCMV endo-
nuclease activity assay are all negative at a concentration of 1 mg/mL.
In vitro anti-inflammatory activity of compounds 1–10 was
tested using LPS-stimulated cell. Stimulation of RAW 264.7 cell
with LPS resulted in up-regulation of the pro-inflammatory iNOS
and COX-2 protein (Fig. 5). Compounds 1, 4, 5, and 8 at concen-
RP-18e, 5
phenylacetyl and R-(ꢀ)-
m
m, 250ꢂ10 mm). S-(þ)- -Methoxy-
a
a-trifluoromethyl-
a
-methoxy-a-trifluoromethylphenylacetyl
chloride were obtained from ACROS Organics (Geel, Belgium).
tration of 10 mM, significantly reduced the levels of the iNOS
3.2. Animal materials
protein (34.7ꢁ7.9%, 0.9ꢁ0.3%, 1.4ꢁ0.2%, and 0.0ꢁ0.0%, respectively)
and COX-2 protein (62.5ꢁ4.3%, 63.7ꢁ4.0%, 42.9ꢁ10.1%, and
42.5ꢁ8.6%, respectively) compare with the control cells (LPS alone).
Under the same concentration, compounds 3, 6, 9, and 10 did not
inhibit the COX-2 protein expression, but significantly inhibited
iNOS protein expression (0.2ꢁ0.3%, 0.0ꢁ0.1%, 0.5ꢁ0.7%, and
0.1ꢁ0.1, respectively, compare with control cells) by LPS stimula-
The soft coral L. durum was collected by hand using scuba at
Dongsha Islands located in northeastern South China Sea in April
2007, at a depth of 8 m, and was stored in a freezer for 2 months
until extraction. This soft coral was identified by one of the
authors (C.-F.D.). A voucher specimen (TS-13) was deposited in the
Department of Marine Biotechnology and Resources, National Sun
Yat-sen University.
tion. Moreover, the house keeping protein b-actin was not changed
by the presence of compounds 1, 3–6, and 8–10 at the same
concentration. Both compounds 2 and 7 significantly inhibited
pro-inflammatory iNOS and COX-2 protein expression, however, it
3.3. Extraction and isolation
presented cytotoxic activity for significant inhibition of
protein expression. Under the same experimental condition, 10
CAPE (caffeic acid phenylthyl ester; Sigma Chemical. Company, St.
Louis, MO) reduced the levels of the iNOS and COX-2 protein to
1.5ꢁ2.1% and 70.2ꢁ11.5%, respectively, relative to the control cells
stimulated with LPS. The primary anti-inflammatory results
suggested that the existence of a-methylene-g-lactone moiety is
required for the observed activity against LPS-stimulated RAW
264.7 cell.
b-actin
The frozen soft coral (1.2 kg) was chopped into small pieces and
extracted with acetone for 24 h at room temperature. The quantity
of solvent used for each extraction (2.0 L) was at least three times
the amount of the soft coral material used (1.2 kg). The acetone
extracts were evaporated to dryness under reduced pressure. Then,
the acetone extract was partitioned between EtOAc and H2O. The
resulting EtOAc extract (30.0 g) was subjected to column chroma-
tography on silica gel using n-hexane, n-hexane–EtOAc, and EtOAc–
MeOH mixtures of increasing polarity for elution, to furnish 40
fractions. 1H NMR spectroscopy was employed to detect the ter-
penoid-rich fractions. Duplicate samples (20 mg) of fraction 12
(0.73 g) eluted with n-hexane–EtOAc (4:1) was further separated
by preparative TLC using n-hexane–EtOAc (6:1) to yield 7 (12 mg).
Fraction 17 (0.14 g) eluted with n-hexane–EtOAc (1:6) was further
purified by reversed-phase HPLC using MeOH–H2O (75:25) to
afford 2 (15 mg), 3 (24 mg), 6 (4 mg), and 9 (18 mg). Fraction 18
(2.16 g) eluted with n-hexane–EtOAc (1:8) was fractionated over
Sephadex LH-20 (100% MeOH) to produce 14 subfractions. A sub-
fraction 18–11 (202 mg) was subjected to column chromatography
on reversed-phase C-18 gel column eluting with 50% MeOH in H2O
to afford a mixture (90 mg) that was further purified by reversed-
phase HPLC using 70% MeOH in H2O to give 4 (24 mg) and 5
(18 mg). Fraction 19 (1.18 g) eluted with n-hexane–EtOAc (1:10)
was subjected to a silica gel column using n-hexane–EtOAc mix-
tures of increasing polarity for elution, to give 10 subfractions.
Subfraction 19–5 (336 mg) eluted with n-hexane–EtOAc (1:2) was
subjected to a reversed-phase C-18 gel eluting with 70% MeOH in
H2O to afford a mixture (214 mg) that was further purified by
mM
3. Experimental
3.1. General experimental procedures
Optical rotations were determined with a JASCO P1020 digital
polarimeter. IR spectra were recorded on a JASCO FT/IR-4100 in-
frared spectrophotometer. UV spectra were obtained on a JASCO
V-650 spectrophotometer. The NMR spectra were recorded on
a Bruker Advance 300 NMR spectrometer at 300 MHz for 1H and
75 MHz for 13C or on a Varian MR 400 NMR at 400 MHz for 1H and
100 MHz for 13C, respectively, using CDCl3 with TMS as internal
standard. Chemical shifts are given in
d (ppm) and coupling con-
stants in hertz. ESIMS were recorded by ESI FT-MS on a Bruker
APEX II mass spectrometer. The crystallographic data were col-
lected on a Rigaku AFC7S diffractometer using graphite-mono-
chromated Mo K
LiChroprep RP-18 (Merck, 40–63
matography. Precoated Si gel plates (Merck, Kieselgel 60 F254
a
radiation. Si gel 60 (Merck, 230–400 mesh) and
mm) were used for column chro-
,

S.-Y. Cheng et al. / Tetrahedron 64 (2008) 9698–9704
9703
reversed-phase HPLC using 65% MeOH in H2O to give 1 (2 mg).
Similarly, compound 10 (3 mg) was obtained by separation of
a subfraction 19–8 (48 mg) eluted with n-hexane–EtOAc (1:4) on
a reversed-phase HPLC eluting with 75% MeOH in H2O. Subfraction
19–6 (202 mg) eluted with n-hexane–EtOAc (1:3) was subjected to
column chromatography on reversed-phase C-18 gel eluting with
65% MeOH in H2O to afford a mixture (138 mg) that was further
purified by reversed-phase HPLC using 70% MeOH in H2O to give 8
(10 mg).
H-5b). Selected 1H NMR(CDCl3, 300 MHz) of 3b: dH 7.33–7.49 (5H,
m, Ph), 6.27 (1H, d, J¼2.7 Hz, H-17a), 5.96 (1H, d, J¼2.3 Hz, H-17b),
5.50 (1H, br dd, J¼8.9, 3.2 Hz, H-11), 5.26 (1H, d, J¼9.3 Hz, H-13),
4.92 (1H, br t, J¼7.0 Hz, H-7), 4.18 (1H, br dd, J¼8.8, 6.8 Hz, H-14),
4.20 (1H, d, J¼12.1 Hz, H-18a), 3.82 (1H, d, J¼12.1 Hz, H-18b), 3.56
(3H, s, OCH3), 2.76 (1H, m, H-3), 2.76 (1H, m, H-1), 2.37 (1H, m,
H-10a), 2.03 (3H, s, 18-OAc), 1.76 (1H, dt, J¼14.6, 2.8 Hz, H-2a), 1.53
(3H, s, Me-20), 1.42 (3H, s, Me-19), 1.26 (1H, td, J¼12.4, 3.5 Hz,
H-5b).
3.3.1. Durumolide A (1)
3.5. Preparation of bis-(R)- and (S)-MTPA esters of 4, 5, and 8
24
Colorless, viscous oil; [
(log
a
]
þ93.0 (c 0.2, CHCl3); UV (MeOH) lmax
D
3
) 208 (3.76) nm; IR (KBr) nmax 3458, 2952, 1765, 1745, 1663,
In the same manner as shown above, two aliquots of 4 (1.0 mg)
were dissolved in dry pyridine (0.5 mL) and allowed to react
overnight with (R)- and (S)-MTPA chloride (one drop), affording the
bis-(S)- and bis-(R)-MTPA ester 4a (0.4 mg) and 4b (0.3 mg), re-
spectively. For compounds 5 and 8, the same procedure as above
was carried out to obtain the corresponding bis-(S)- and bis-(R)-
MTPA esters (5a, 5b, 8a, and 8b, respectively). Selected 1H
NMR(CDCl3, 300 MHz) of 4a: dH 7.42–7.61 (10H, m, Phꢂ2), 6.32 (1H,
d, J¼3.0 Hz, H-17a), 5.97 (1H, d, J¼2.5 Hz, H-17b), 5.61 (1H, br dd,
J¼8.6, 4.1 Hz, H-11), 5.45 (1H, d, J¼9.0 Hz, H-13), 4.94 (1H, br t, J¼
6.8 Hz, H-7), 4.42 (1H, d, J¼12.2 Hz, H-18a), 4.22 (1H, d, J¼12.2 Hz,
H-18b), 4.10 (1H, br dd, J¼15.7, 8.9 Hz, H-14), 3.56 (3H, s, OCH3),
3.53 (3H, s, OCH3), 2.82 (1H, m, H-3), 2.82 (1H, m, H-1), 1.76 (1H, dt,
J¼14.7, 3.2 Hz, H-2a), 1.67 (3H, s, Me-20), 1.62 (3H, s, Me-19).
Selected 1H NMR(CDCl3, 300 MHz) of 4b: dH 7.42–7.61 (10H, m,
Phꢂ2), 6.33 (1H, d, J¼3.0 Hz, H-17a), 6.00 (1H, d, J¼2.5 Hz, H-17b),
5.53 (1H, br dd, J¼8.6, 4.1 Hz, H-11), 5.36 (1H, d, J¼9.0 Hz, H-13),
4.96 (1H, br t, J¼6.8 Hz, H-7), 4.46 (1H, d, J¼12.2 Hz, H-18a), 4.17
(1H, d, J¼12.2 Hz, H-18b), 4.15 (1H, br dd, J¼15.7, 8.9 Hz, H-14), 3.65
(3H, s, OCH3), 3.55 (3H, s, OCH3), 2.83 (1H, m, H-3), 2.83 (1H, m,
H-1),1.84 (1H, dt, J¼14.7, 3.2 Hz, H-2a),1.59 (3H, s, Me-20),1.45 (3H,
s, Me-19). Selected 1H NMR(CDCl3, 400 MHz) of 5a: dH 7.39–7.52
(10H, m, Phꢂ2), 6.24 (1H, d, J¼3.2 Hz, H-17a), 5.91 (1H, d, J¼2.8 Hz,
H-17b), 5.54 (1H, br s, H-13), 4.90 (1H, br dd, J¼8.4, 4.0 Hz, H-11),
4.83 (1H, br t, J¼7.2 Hz, H-7), 4.40 (1H, d, J¼12.0 Hz, H-18a), 4.23
(1H, d, J¼12.0 Hz, H-18b), 4.18 (1H, dd, J¼6.4, 2.0 Hz, H-14), 3.54
(3H, s, OCH3), 3.51 (3H, s, OCH3), 3.09 (1H, m, H-1), 2.75 (1H, dd,
J¼7.6, 2.8 Hz, H-3), 2.38 (1H, m, H-10a), 2.23 (1H, m, H-10b), 2.11
(1H, m, H-6a), 2.10 (1H, m, H-5a), 2.05 (1H, m, H-6b), 1.98 (1H, m,
H-9a), 1.84 (1H, m, H-9b), 1.75 (1H, dt, J¼14.8, 3.6 Hz, H-2a), 1.69
(3H, s, Me-20), 1.52 (3H, s, Me-19), 1.49 (1H, m, H-2b), 1.23 (1H, td,
J¼14.0, 3.2 Hz, H-5b). Selected 1H NMR(CDCl3, 400 MHz) of 5b: dH
7.40–7.62 (10H, m, Phꢂ2), 6.21 (1H, d, J¼3.2 Hz, H-17a), 5.90 (1H, d,
J¼2.8 Hz, H-17b), 5.65 (1H, br s, H-13), 5.29 (1H, br dd, J¼8.4, 4.0 Hz,
H-11), 4.97 (1H, br t, J¼7.2 Hz, H-7), 4.50 (1H, d, J¼12.0 Hz, H-18a),
4.16 (1H, dd, J¼6.4, 2.0 Hz, H-14), 4.13 (1H, d, J¼12.0 Hz, H-18b),
3.57 (3H, s, OCH3), 3.46 (3H, s, OCH3), 3.11 (1H, m, H-1), 2.80 (1H,
dd, J¼7.6, 2.8 Hz, H-3), 2.47 (1H, m, H-10a), 2.31 (1H, m, H-10b), 2.19
(1H, m, H-6a), 2.12 (1H, m, H-5a), 2.07 (1H, m, H-6b), 2.02 (1H, m,
H-9a), 1.90 (1H, m, H-9b), 1.79 (1H, dt, J¼14.8, 3.6 Hz, H-2a), 1.77
(3H, s, Me-20), 1.57 (3H, s, Me-19), 1.48 (1H, m, H-2b), 1.19 (1H, td,
J¼14.0, 3.2 Hz, H-5b). Selected 1H NMR(CDCl3, 400 MHz) of 8a: dH
7.38–7.54 (10H, m, Phꢂ2), 6.29 (1H, d, J¼2.0 Hz, H-17a), 5.67 (1H, d,
J¼1.2 Hz, H-17b), 5.45 (1H, br t, J¼8.0 Hz, H-11), 5.28 (1H, br t,
J¼7.2 Hz, H-3), 5.12 (1H, d, J¼7.6 Hz, H-13), 4.82 (1H, d, J¼12.0 Hz,
H-18a), 4.79 (1H, br d, J¼6.8 Hz, H-7), 4.69 (1H, d, J¼12.0 Hz, H-
18b), 4.20 (1H, dd, J¼7.6, 2.0 Hz, H-14), 3.53 (3H, s, OCH3), 3.50 (3H,
s, OCH3), 2.68 (1H, m, H-1), 2.36 (1H, m, H-2a), 2.31 (1H, m, H-10a),
2.23 (1H, m, H-2b), 2.20 (1H, m, H-10b), 1.68 (3H, s, Me-20), 1.59
(3H, s, Me-19). Selected 1H NMR (CDCl3, 400 MHz) of 8b: dH 7.36–
7.52 (10H, m, Phꢂ2), 6.32 (1H, d, J¼2.0 Hz, H-17a), 5.72 (1H, d,
J¼1.2 Hz, H-17b), 5.24 (1H, br t, J¼7.2 Hz, H-3), 5.19 (1H, br t,
J¼8.0 Hz, H-11), 4.96 (1H, d, J¼7.6 Hz, H-13), 4.74 (2H, br s, H-18),
4.73 (1H, br d, J¼6.8 Hz, H-7), 4.22 (1H, dd, J¼7.6, 2.0 Hz, H-14), 3.63
1442, 1378, 1267, 1244, 1128, 1049, 952 cmꢀ1; 1H NMR and 13C NMR
data, see Tables 1 and 2; ESIMS m/z 429 [MþNa]þ; HRESIMS m/z
429.1890 [MþNa]þ (calcd for C22H30O7Na 429.1889).
3.3.2. Durumolide B (6)
24
Colorless, viscous oil; [
(log
a
]
þ41.5 (c 0.4, CHCl3); UV (MeOH) lmax
D
3
) 209 (3.85) nm; IR (KBr) nmax 3448, 2952, 1762, 1744, 1660,
1439, 1383, 1234, 1128, 1049, 952 cmꢀ1; 1H NMR and 13C NMR data,
Tables 1 and 2; ESIMS m/z 397 [MþNa]þ; HRESIMS m/z 397.1990
[MþNa]þ (calcd for C22H30O4Na 397.1991).
3.3.3. Durumolide C (8)
24
Colorless, viscous oil; [
(log
1383, 1244, 1128, 1044, 956 cmꢀ1
a
]
þ15.4 (c 1.0, CHCl3); UV (MeOH) lmax
D
3) 208 (3.72) nm; IR (KBr) nmax 3453, 2948, 1767, 1660, 1442,
;
1H NMR and 13C NMR data, see
Tables 2 and 3; ESIMS m/z 355 [MþNa]þ; HRESIMS m/z 355.1887
[MþNa]þ (calcd for C20H28O4Na 355.1885).
3.3.4. Durumolide D (9)
24
Colorless, viscous oil; [
(log
a
]
þ10.2 (c 1.8, CHCl3); UV (MeOH) lmax
D
3
) 210 (3.78) nm; IR (KBr) nmax 3453, 2952, 1767, 1744, 1660,
1442, 1378, 1267, 1244, 1142, 1044, 947 cmꢀ1; 1H NMR and 13C NMR
data, see Tables 2 and 3; ESIMS m/z 413 [MþNa]þ; HRESIMS m/z
413.1942 [MþNa]þ (calcd for C22H30O6Na 413.1940).
3.3.5. Durumolide E (10)
24
Colorless, viscous oil; [
(log
a
]
þ32.0 (c 0.3, CHCl3); UV (MeOH) lmax
D
3
) 208 (3.74) nm; IR (KBr) nmax 3457, 2952, 2846, 1767, 1716,
1660, 1442, 1378, 1244, 1044, 952 cmꢀ1; 1H NMR and 13C NMR data,
see Tables 2 and 3; ESIMS m/z 369 [MþNa]þ; HRESIMS m/z
369.1681 [MþNa]þ (calcd for C20H26O5Na 369.1678).
3.4. Preparation of (R)- and (S)-MTPA esters of 3
Duplicate (1.0 mg) samples of 3 were prepared for both (R)- and
(S)-MTPA chloride acylation reactions. In separate vials, the sam-
ples were dissolved in 0.5 mL of dry pyridine and allowed to react
overnight at room temperature with (R)- and (S)-MTPA chloride
(one drop), respectively. The reaction was quenched by the addition
of 1.0 mL of H2O, followed by extraction with EtOAc (3ꢂ1.0 mL). The
EtOAc-soluble layers were combined, dried over anhydrous MgSO4,
and evaporated. The residue was subjected to a short silica gel
column eluting with n-hexane–EtOAc (5:1) to yield (S)-MTPA ester
(3a) (0.6 mg). The (R)-MTPA ester (3b) (0.4 mg) was prepared with
(S)-MTPA chloride according to the same procedure as described
above. Selected 1H NMR(CDCl3, 300 MHz) of 3a: dH 7.33–7.47 (5H,
m, Ph), 6.25 (1H, d, J¼2.7 Hz, H-17a), 5.92 (1H, d, J¼2.3 Hz, H-17b),
5.55 (1H, br dd, J¼8.9, 3.2 Hz, H-11), 5.36 (1H, d, J¼9.3 Hz, H-13),
4.95 (1H, br t, J¼7.0 Hz, H-7), 4.16 (1H, br dd, J¼8.8, 6.8 Hz, H-14),
4.19 (1H, d, J¼12.1 Hz, H-18a), 3.83 (1H, d, J¼12.1 Hz, H-18b), 3.48
(3H, s, OCH3), 2.78 (1H, m, H-3), 2.78 (1H, m, H-1), 2.45 (1H, m,
H-10a), 2.02 (3H, s, 18-OAc), 1.73 (1H, dt, J¼14.6, 2.8 Hz, H-2a), 1.69
(3H, s, Me-20), 1.57 (3H, s, Me-19), 1.31 (1H, td, J¼12.4, 3.5 Hz,

9704
S.-Y. Cheng et al. / Tetrahedron 64 (2008) 9698–9704
(3H, s, OCH3), 3.50 (3H, s, OCH3), 2.70 (1H, m, H-1), 2.37 (1H, m, H-
2a), 2.22 (1H, m, H-2b), 2.18 (1H, m, H-10a), 2.11 (1H, m, H-10b),
1.61 (3H, s, Me-20), 1.55 (3H, s, Me-19).
detected using ECL detection reagents (Perkin–Elmer, Western Blot
Chemiluminescence Reagent Plus) according to the manufacturer
instructions.
3.6. Crystallographic data and X-ray structure analysis of
compound 420
3.9. Inhibition of endonuclease activity assay
The HCMV UL76 encodes an endonuclease25 was purified from
E. coli BL21-codon DE3-RIL strain, which was transformed with
calmodulin-tagged UL76 plasmid pCBP-UL76. The double-stranded
A
suitable colorless crystal (0.8ꢂ0.8ꢂ0.6 mm3) of
4 was
obtained by slow evaporation from the mixture CH2Cl2–MeOH
(1:5) solution. Crystal data:
C20H28O5$H2O, orthorhombic,
supercoiled DNA
4X174 was purified by CsCl gradient and used as
Mr¼366.44 g/mol; a¼9.2869(19) Å, b¼9.3375(19) Å, c¼21.969(4) Å,
substrate.26 Tested compounds were series diluted and incubated
V¼1905.1(7) Å3, space group P212121, Z¼4, Dcalcd¼1.278 g/cm3,
with 1 mg DNA substrate, reaction buffer, and UL76 endonuclease at
l
¼0.71073 Å,
m
(Mo K
a
)¼0.093 mmꢀ1, F(000)¼792, T¼298(2) K. A
37 ꢃC for 1 h. After cleavage reaction, DNA was resolved in 1%
agarose gel. Concentrations of tested compounds that exert 50% of
inhibition of cleaved substrate (IC50) were recorded.
total of 2170 reflections were collected in the range 1.85<q<26.00,
of which 2170 unique reflections with I>2s(I) were used for the
analysis. The data were solved using the direct method, and the
structure was refined by full-matrix least-squares procedure on F2
values. All non-hydrogen atoms were refined with anisotropic
thermal parameters. The hydrogen atom positions were geo-
metrically idealized and allowed to ride on their parent atoms.
The final indices were R1¼0.0327, wR2¼0.0861 with goodness-
of-fit¼1.032.
Acknowledgements
This work was supported by grants from the National Science
Council (NSC96-2320-B-110-003-MY3) and Ministry of Education
(96C031703) of the Republic of China awarded to C.-Y. D.
3.7. In vitro antimicrobial activity
References and notes
Bacterial strains were grown in LB (Luria-Bertani) broth medium
for 24 h at 37 ꢃC. LB hard agar (17 mL, 1.5% agar) was poured into
sterile Petri dishes (9 cm) and allowed to set. Molten LB soft agar
(2.7 mL, 0.7% agar, 45 ꢃC) was inoculated with 0.3 mL broth culture
of the test organism and poured over the base hard agar plates
forming a homogenous top layer. Sterile paper disc (Advantec,
8 mm) were placed onto the top layer of the LB agar plates. Ten
microliters of the tested compounds (1–10) were applied on to each
filter paper discs. Ampicillin and same solvents were served as
positive and negative control. All plates were incubated at 37 ꢃC,
24 h for antibacterial activity evaluation. The antimicrobial activity
of all isolated metabolites (1–10) was tested from 6.25, 12.5, 25, 50,
1. Blunt, J. W.; Copp, B. R.; Hu, W.-P.; Munro, M. H. G.; Northcote, P. T.; Prinsep,
M. R. Nat. Prod. Rep. 2008, 25, 35–94 and literature cited in previous reviews.
2. Higuchi, R.; Miyamoto, T.; Yamada, K.; Komori, T. Toxicon 1998, 36, 1703–1705.
3. Matthee, G. F.; Konig, G. M.; Wright, A. D. J. Nat. Prod. 1998, 61, 237–240.
4. Wang, S.-K.; Duh, C.-Y.; Wu, Y.-C.; Wang, Y.; Cheng, M.-C.; Soong, K.; Fang, L.-S.
J. Nat. Prod. 1992, 55, 1430–1435.
5. Coval, S. J.; Patton, R. W.; Petrin, J. M.; James, L.; Rothofsky, M. L.; Lin, S. L.; Patel,
M.; Reed, J. K.; McPhil, A. T.; Bishop, W. R. Bioorg. Med. Chem. Lett. 1996, 6,
909–912.
6. Duh, C.-Y.; Wang, S.-K.; Huang, B.-T.; Dai, C.-F. J. Nat. Prod. 2000, 63, 884–885.
7. Yamada, K.; Ryu, K.; Miyamoto, T.; Higuchi, R. J. Nat. Prod. 1997, 60, 798–801.
8. Subrahmanyam, C.; Rao, C. V.; Anjaneyulu, V.; Satyanarayana, P.; Rao, P. V. S.
Tetrahedron 1992, 48, 3111–3120.
9. Uchio, Y.; Eguchi, S.; Kuramoto, J.; Nakayama, M.; Hase, T. Tetrahedron Lett. 1985,
26, 4487–4490.
10. Tursch, B.; Braekman, J. C.; Daloze, D.; Herin, M.; Karlsson, R. Tetrahedron Lett.
1974, 43, 3769–3772.
11. Kashman, Y.; Groweiss, A. Tetrahedron Lett. 1977, 13, 1159–1160.
12. Kinamoni, Z.; Groweiss, A.; Carmely, S.; Kashman, Y. Tetrahedron 1983, 39,
1643–1648.
100, 200, 250, 500 up to 1000 mg/disk on S. enteritidis (ATCC13076).
The bacterial strain was obtained from the American Type Culture
Collection. The antibiotic activity evaluation method was con-
ducted based on previously reports.21
13. Kashman, Y.; Carmely, S.; Groweiss, A. J. Org. Chem. 1981, 46, 3592–3596.
14. Rashid, M. A.; Gustafson, K. R.; Boyd, M. R. J. Nat. Prod. 2000, 63, 531–533.
15. Bowden, B. F.; Coll, J. C.; Tapiolas, D. M. Aust. J. Chem. 1983, 36, 2289–2295.
16. Bowden, B. F.; Coll, J. C.; Decosta, M. S. L.; Desilva, E. D.; Mackay, M. F.;
Mahendran, M.; Willis, R. H. Aust. J. Chem. 1984, 34, 545–552.
17. Li, G.; Zhang, Y.; Deng, Z.; van Ofwegen, L.; Proksch, P.; Lin, W. J. Nat. Prod. 2005,
68, 649–652.
18. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113,
4092–4096.
19. Djeddi, S.; Karioti, A.; Sokovic, M.; Stojkovic, D.; Seridi, R.; Skaltsa, H. J. Nat. Prod.
2007, 70, 1796–1799.
3.8. In vitro anti-inflammatory assay
The anti-inflammatory assay was modified from Ho et al.22 and
Park et al.23 Murine RAW 264.7 macrophages were obtained from
the American Type Culture Collection (ATCC, No. TIB-71). The cells
were activated by incubation in medium containing Escherichia coli
LPS (0.01 mg/mL; Sigma) for 16 h in the presence or absence of
various compounds. Then, cells were washed with ice-cold PBS,
lysed in ice-cold lysis buffer, and then centrifuged at 20,000g for
30 min at 4 ꢃC. The supernatant was decanted from the pellet and
retained for Western blot analysis. Protein concentrations were
determined by the DC protein assay kit (Bio-Rad) modified by the
method of Lowry et al.24 Samples containing equal quantities of
protein were subjected to SDS-polyacrylamide gel electrophoresis,
and the separated proteins were electrophoretically transferred to
polyvinylidene difluoride membranes (PVDF; Immobilon-P, Milli-
20. Crystallographic data for 4 have been deposited with the Cambridge Crystal-
lographic Data Centre (deposition number CCDC 689497). Copies of the data
can be obtained, free of charge, on application to the Director, CCDC, 12 Union
Road, Cambridge CB21EZ, UK (fax: þ44 1223 336033 or e-mail: deposit@ccdc.
cam.ac.uk).
21. (a) Hou, L.; Shi, Y.; Zhai, P.; Le, G. J. Ethnopharmacol. 2007, 111, 227–231; (b)
Arias, M. E.; Gomez, J. D.; Cudmani, N. M.; Vattuone, M. A.; Isla, M. I. Life Sci.
2004, 75, 191–202.
22. Ho, F. M.; Lai, C. C.; Huang, L. J.; Kuo, T. C.; Chao, C. M.; Lin, W. W. Br. J. Phar-
macol. 2004, 141, 1037–1047.
23. Park, E. K.; Shin, Y. W.; Lee, H. U.; Kim, S. S.; Lee, Y. C.; Lee, B. Y.; Kim, D. H. Biol.
Pharm. Bull. 2005, 28, 652–656.
24. Lowry, D. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193,
265–275.
25. Knizewski, L.; Kinch, L.; Grishin, N. V.; Rychlewski, L.; Ginalski, K. J. Virol. 2006,
80, 2575–2577.
26. Tachiwana, H.; Shimura, M.; Nakai-Murakami, C.; Tokunaga, K.; Takizawa, Y.;
Sata, T.; Kurumizaka, H.; Ishizaka, Y. Cancer Res. 2006, 66, 627–631.
pore, 0.45 mm pore size). The resultant PVDF membranes were
incubated with blocking solution and incubated for 180 min with
antibody against inducible nitric oxide synthase (iNOS; 1:1000
dilution; Transduction Laboratories) and cyclooxygenase-2 (COX-2;
1:1000 dilution; Cayman Chemical) protein. The blots were