Bioactive pregnane-type steroids from the soft coral Scleronephthya gracillimum

Article abstract of DOI:10.1016/j.tet.2012.09.060

Nine new steroids, sclerosteroids A-I (1, 5, 6, 8-13), along with 18 known metabolites (2-4, 7, 14-27), were isolated from the soft coral Scleronephthya gracillimum. These structures were elucidated on the basis of detailed spectroscopic analysis. The absolute configurations of sugar moieties in steroidal glycosides 10-13 were determined by HPLC analysis of the o-tolylthiocarbamate derivatives of the liberated sugars from hydrolysis of these steroidal giycosides. Cytotoxic and anti-inflammatory activities of these compounds were measured in vitro.

Full text of DOI:10.1016/j.tet.2012.09.060

Tetrahedron 68 (2012) 9694e9700
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Bioactive pregnane-type steroids from the soft coral Scleronephthya gracillimum
,
,d,
Hui-Yu Fang a y, Chih-Chuang Liawa y, Chih-Hua Chao a, Zhi-Hong Wen a, Yang-Chang Wu b,
Chi-Hsin Hsu a, Chang-Feng Dai c, Jyh-Horng Sheu a
,d,
*
a Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804,Taiwan
b Graduate Institute of Integrated Medicine, China Medical University, Taichung 40402, Taiwan
c Institute of Oceanography, National Taiwan University, Taipei 106, Taiwan
d Asia Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 June 2012
Received in revised form 11 September 2012
Accepted 11 September 2012
Available online 18 September 2012
Nine new steroids, sclerosteroids AeI (1, 5, 6, 8e13), along with 18 known metabolites (2e4, 7, 14e27),
were isolated from the soft coral Scleronephthya gracillimum. These structures were elucidated on the
basis of detailed spectroscopic analysis. The absolute configurations of sugar moieties in steroidal gly-
cosides 10e13 were determined by HPLC analysis of the o-tolylthiocarbamate derivatives of the liberated
sugars from hydrolysis of these steroidal giycosides. Cytotoxic and anti-inflammatory activities of these
compounds were measured in vitro.
Keywords:
Scleronephthya gracillimum
Sclerosteroids
Ó 2012 Elsevier Ltd. All rights reserved.
O-Tolylthiocarbamate
Cytotoxic activity
Anti-inflammatory activity
1. Introduction
analysis and chemical methods. Among them, compound 1 showed
significant cytotoxicity against human liver carcinoma (HepG2),
Soft coral of the genus Scleronephthya have been found to be an
important source of pregnane-type steroids.1e5 Some of the preg-
nanes possess important bioactivities, such as cytotoxic,4e6 anti-in-
flammatory,7 antiplasmodial,8 and antibacterial activities.9 In
continuation of our previous studies of discovering bioactive steroids
from soft corals,10e19 the chemical investigation on Scleronephthya
gracillimum was carried out and led to the isolation of 9 new
pregnane-type steroids, sclerosteroids AeI (1, 5, 6, 8e13), and 18
known compounds, stereonsteroid A (2),4 ceratosteroid C (3),20
ceratosteroid D (4),20 pregna-1,20-dien-3-one (7),21 3-(4-O-acetyl-
human breast carcinoma (MDA-MB-231), meanwhile compounds
10,11, 20, and 26 were found to exhibit moderate cytotoxicityagainst
human liver carcinoma (HepG2 and Hep3B), human breast carci-
noma (MDA-MB-231 and MCF-7), human lung carcinoma (A-549),
and human oral cancer (Ca9-22) cells. Besides, compounds 1, 5, 9,15,
and 23 could significantly inhibit the accumulation of the pro-
inflammatory iNOS (inducible nitric oxide synthase) protein
6-deoxy-
b
-galactopyranosyloxy)-19-norpregna-1,3,5(10),20-tetrae-
ne (14),22 glaucasterol (15),23 pregna-5,20-dien-3
b-ol-3-acetate
(16),24 (5 -ol-3-acetate (17),25 pregna-20-
a,14a,17b)-pregn-20-en-3b
en-3-one (18),21 19-norpregna-1,3,5(10),20-tetraen-3-ol (19),26
stereonsteroid H (20),4 stereonsteroid G (21),4 pregnene glycoside
(22),27 stereosteroid F (23),4 pregn-20-en-3-O-
a-fucopyranoside
(24),28 stereonsteroid E (25),4 stereonsteroid D (26),4 and ximaste-
roid D (27) (Fig.1 and Supplementary data Fig. S1).5 The structures of
new compounds were established by extensive spectroscopic
* Corresponding authors. Tel.: þ886 7 5252000x5030; fax: þ886 7 525 5020;
e-mail address: sheu@mail.nsysu.edu.tw (J.-H. Sheu).
y
These authors have the same contribution to the work.
Fig. 1. Structure of new compounds isolated from S. gracillimum.
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.09.060

H.-Y. Fang et al. / Tetrahedron 68 (2012) 9694e9700
9695
in LPS (lipopolysaccharide)-stimulated RAW264.7 macrophage cells.
Compounds 1, 5, and 9 also showed activity in inhibiting the ex-
pression of COX-2 protein in the same cells.
(J¼12.0 Hz). HMBC correlations from H2-19 to C-10, C-9, C-5, and
C-1 also confirmed this elucidation (Fig. S2). The relative ste-
reochemistry of 1 was determined by the 2D NOE experiment
(Fig. S3). The observed NOESY correlations between H-20 and
H3-18, H-14 and H-17, H2-19 and H-8, H-9 and H-5 and H-3
2. Results and discussion
revealed the
-orientation of H-3, H-5, H-9, H-14, and H-17. On the basis of
the above spectroscopic data, the structure of 1 was established
as 19-acetoxy-5 -pregn-20-en-3-ol.
Compound 1 was identified to be the 19-acetylated derivative of
2 by comparison of their physical (optical rotation) and spectro-
scopic (1H and 13C NMR) data, meanwhile 3-acetylated 1 was also
identical to 4 by comparison of their spectroscopic data. Due to the
amount limitation, the absolute configuration of 1 was indirectly
b-orientation of H3-18, H2-19, and H-8 and the
a
Sclerosteroid A (1) has a molecular formula of C23H36O3 as
determined by HRESIMS, appropriate for six degrees of unsatu-
ration. The 13C NMR and DEPT spectra of 1 showed the presence
of 23 carbon signals, including 2 methyls, 10 sp3 methylenes,
6 sp3 methines, 1 sp2 methine, 1 sp2 methylene, and 1 sp2 and
2 sp3 quaternary carbons (Table 1). The 1H NMR showed the
presence of a tertiary methyl (dH 0.57, 3H, s), an acetoxymethyl
a
(
dH 4.21, 1H, d, J¼12.0 Hz; 4.35, 1H, d, J¼12.0 Hz; dH 2.06, 3H, s),
determined by the (S)- and (R)-
phenylacetic (MTPA) esters of 2 (2a and 2b, respectively).29,30
The values of Dd (R-MTPA ester of 2)] for
a-methoxy-a-(trifluoromethyl)
a methine with an hydroxy group (dH 3.64, 1H, m), and a vinyl
group (dH 4.95, 1H, br d, J¼16.8 Hz; 4.96, 1H, br d, J¼10.8 Hz;
5.74, 1H, ddd, J¼16.8, 10.8, 7.6 Hz) (Table 1). These spectroscopic
data showed that 1 might have a 3-hydroxy pregnane skeleton
with an acetoxymethyl substituent at C-10 on the basis of the
disappearance of a H3-19 singlet around dH 0.80e1.10 and the
presence of an AB doublet at dH 4.21 (J¼12.0 Hz) and 4.35
[
d
(S-MTPA ester of 2)ꢀ
d
H-3, H-4, and H-6 were positive, while the values of Dd for H-1 and
H-2 were negative, revealing the S-configuration at C-3 (Fig. 2).
Sclerosteroid B (5) has a molecular formula of C25H36O4 as de-
termined by HRESIMS, appropriate for six degrees of unsaturation.
Table 1
1H and 13C NMR spectroscopic data for compounds 1, 5, 6, 8, and 9
No.
1
1
5
6
8
9
Mult.c dH
dC
mult.c dH
dC
Mult.c dH
dC
mult.c dH
dC
mult.c dH
a
d
a
d
b
e
a
d
a
d
dC
31.9 CH2
0.92 m
2.21 dt
(13.6, 3.6)
1.30 m
1.85 m
3.64 m
31.5 CH2
1.41 m
2.03 m
152.1 CH
7.01 d (10.5)
33.9 CH2
1.05 m
2.51 dt (13.2, 3.2)
28.6 CH2
1.12 m
2.61 dt
(14.8, 3.2)
1.58 m
1.88 m
4.74 m
2
3
4
31.6 CH2
70.8 CH
38.4 CH2
25.4 CH2
70.0 CH
36.4 CH2
1.72 m
1.94 m
5.22 m
130.7 CH
6.04 d (10.5)
29.3 CH2
72.8 CH
35.8 CH2
1.53 m
1.94 m
4.76 ddd (16.0,
11.2, 4.8)
1.50 m
27.4 CH2
72.6 CH
34.0 CH2
200.0
C
1.37 m
1.67 m
1.55 m
41.5 CH2
2.27 dd
(18.0, 5.0)
2.58 dd
1.60 m
1.53 m
1.82 m
(18.0, 14.5)
2.03 m
1.56 m
5
6
45.1 CH
28.2 CH2
1.29 m
1.22 m
1.29 m
0.91 m
1.74 m
1.48 m
0.76 m
145.0,
122.2 CH
C
44.1 CH
27.3 CH2
44.3 CH
28.5 CH2
1.49 m
1.32 m
44.0 CH
27.7 CH2
1.48 m
1.28 m
1.47 m
0.91 m
1.74 m
1.71 m
0.92 m
5.47 br s
7
32.0 CH2
32.3 CH2
2.09 m
2.20 m
1.80 m
0.88 m
31.4 CH2
1.03 m
1.81 m
1.56 m
1.15 m
31.7 CH2
0.94 m
1.79 m
1.46 m
0.93 m
31.8 CH2
8
9
35.9 CH
54.6 CH
33.1 CH
54.4 CH
36.2 CH
50.3 CH
35.9 CH
52.2 CH
35.6 CH
52.4 CH
10
11
38.0
21.8 CH2
C
40.5
21.1 CH2
C
42.2
21.3 CH2
C
50.6
22.3 CH2
C
81.3 C
1.34 m
1.63 m
0.97 m
1.67 m
1.34 m
1.60 m
0.97 m
1.68 m
1.52 m
1.90 m
1.08 m
1.79 m
1.35 m
1.74 m
1.02 m
1.68 m
22.4 CH2
37.8 CH2
1.89 m
1.71 m
0.95 m
1.69 m
12
37.9 CH2
37.6 CH2
37.4 CH2
37.4 CH2
13
14
15
43.6
55.9 CH
24.7 CH2
C
43.5
55.6 CH
24.6 CH2
C
43.6
56.1 CH
24.6 CH2
C
43.5
55.7 CH
24.7 CH2
C
43.5 C
55.6 CH
24.9 CH2
0.99 m
1.16 m
1.66 m
1.54 m
1.77 m
1.93 m
0.57 s
4.21 d (12.0)
4.35 d (12.0)
0.93 m
1.18 m
1.67 m
1.54 m
1.78 m
1.91 m
0.61 s
4.14 d (11.2)
4.49 d (11.2)
1.03 m
1.69 m
0.97 m
1.23 m
1.73 m
1.55 m
1.72 m
1.96 m
0.54 s
1.01 m
1.70 m
1.21 m
1.58 m
1.79 m
1.94 m
0.66 s
16
27.2 CH2
27.1 CH2
27.3 CH2
1.56 m
1.78 m
1.99 m
0.63 s
4.33 d (12.0)
4.48 d (12.0)
27.2 CH2
27.2 CH2
17
18
19
55.3 CH
13.0 CH3
62.9 CH2
55.2 CH
12.9 CH3
66.9 CH2
55.2 CH
13.1 CH3
62.1 CH2
55.3 CH
13.0 CH3
178.4 C
55.3 CH
13.0 CH3
20 139.8 CH
21 114.5 CH2
5.74 ddd (16.8, 139.6 CH
10.8, 7.6)
4.95 br d (16.8) 114.7 CH2
4.96 br d (10.8)
5.74 ddd (17.4, 139.4 CH
10.4, 7.2)
4.95 br d (17.4) 114.9 CH2
4.96 br d (10.4)
5.75 ddd (17.5, 139.7 CH
10.5 7.5)
4.97 br d (17.5) 114.6 CH2
4.98 br d (10.5)
5.73 ddd (16.8,
10.8, 7.6)
4.96 br d (16.8)
4.97 br d (10.8)
139.9 CH
114.5 CH2
5.76 ddd (16.4,
10.8, 7.6)
4.96 br d (16.4)
4.97 br d (10.8)
OAc 171.2
C
171.0
171.1
C
C
170.7
21.0 CH3
C
170.7
21.4 CH3
C
170.8
21.4 CH3
C
21.2 CH3
2.06 s
1.95 s
2.02 s
2.03 s
21.2 CH3
21.4 CH3
2.05 s
2.06 s
a
b
c
Recorded at 100 MHz in CDCl3 at 25 ꢁC.
Recorded at 125 MHz in CDCl3 at 25 ꢁC.
Multiplicities deduced by DEPT (The chemical shifts referenced to residual signal of CDCl3 at
d 77.0 ppm.).
d
e
Recorded at 400 MHz in CDCl3 at 25 ꢁC.
Recorded at 500 MHz in CDCl3 at 25 ꢁC (The chemical shifts referenced to TMS at
d 0.0 ppm.).

9696
H.-Y. Fang et al. / Tetrahedron 68 (2012) 9694e9700
Sclerosteroid E (9) has a molecular formula of C22H34O4 as de-
termined by the HRESIMS and 13C NMR spectroscopic data, de-
duced six degrees of unsaturation. The 1H NMR spectroscopic data
of 9 showed one methyl signal (dH 0.66, 3H, s), one oxymethine (dH
4.74,1H, m), and a vinyl group (dH 4.96, 1H, br d, J¼16.4 Hz; 4.97, 1H,
br d, J¼10.8 Hz; 5.76, 1H, ddd, J¼16.4, 10.8, 7.6 Hz) (Table 1), re-
vealing that 9 should have 3-acetoxy pregnane skeleton similar to
that of 8. Comparison of the 1H and 13C NMR spectral data of 8 and 9
showed that the C-19 carboxylic carbon (dC 178.4) in 8 was dis-
appeared and C-10 was downfield-shifted from dC 50.6 (in 8) to 81.3
(in 9), while a proton signal was present at dH 6.97 (1H, br s) cor-
responding to a hydroperoxy group in 9. The relative stereochem-
istry of 9 was further established by 2D NMR experiments,
including HMBC and NOESY (Figs. S2 and S3). The NOE correlations
Fig. 2. 1H NMR chemical shift differences Dd
esters of 2.
(
dS dR) in parts per million for the MTPA
ꢀ
The 13C NMR and DEPT spectra of 5 showed the presence of twenty-
five carbon signals, including three methyls, nine sp3 methylenes,
five sp3 methines, two sp2 methine, one sp2 methylene, and three
sp2 and two sp3 quaternary carbons (Table 1). The 1H NMR spectra
of 5 showed the presence of a tertiary methyl (dH 0.61, 3H, s), an
acetoxymethyl (dH 4.14, 1H, d, J¼11.2 Hz; 4.49, 1H, d, J¼11.2 Hz; dH
2.06, 3H, s), a methine with an acetoxy group (dH 5.22, 1H, m; dH
2.05, 3H, s), a vinyl proton (dH 5.47, 1H, brs), and a terminal vinyl
group (dH 4.95, 1H, br d, J¼17.4 Hz; 4.96, 1H, br d, J¼10.4 Hz; 5.74,
1H, ddd, J¼17.4, 10.4, 7.2 Hz) (Table 1). These spectroscopic data
of 9 between H3-18 and H-8 suggested that H3-18 and H-8 were
oriented, also correlations between H-5 and H-9; H-17 and H-14
suggested that H-5, H-9, H-14, and H-17 were all -oriented. The
key NOE correlations of OOH-10 with Hb-4 and H-3 with Ha-2
suggested both the hydroperoxy and acetoxy groups should be
oriented. On the basis of the above analysis, the structure of 9 was
established as 3 -acetoxy-19-nor-10 -hydroperoxypregn-20-ene.
b-
a
b
-
b
b
The molecular formula of sclerosteroid F (10) was found to be
C29H46O7 by HRESIMS, DEPT, and 13C NMR data, indicating seven
degrees of unsaturation. The 1H and 13C NMR spectra of 10 dis-
played the signals for a vinyl group (dH 4.96, 1H, br d, J¼17.2 Hz;
4.97, 1H, br d, J¼10.0 Hz; 5.76, 1H, ddd, J¼17.2, 10.0, 7.6 Hz; dC 114.5,
139.8), an ester carbonyl (dC 170.9), and an acetate methyl group (dH
2.17, 3H, s; dC 21.2). Therefore, 10 possesses five rings. The 1H and
13C NMR spectroscopic data of 10 were similar to those of 3 (Table
2), except for the appearing of six additional carbon signals at dC
97.3 (CH), 66.8 (CH), 73.9 (CH), 70.9 (CH), 65.8 (CH), and 16.0 (CH3),
an anomeric proton signal at dH 5.04 (1H, d, J¼4.0 Hz), as well as
a methyl doublet at dH 1.26, suggesting the presence of a 60-deox-
yhexose unit. This hexose appeared to be the C-30 monoacetate
derivative of fucopyranose by comparison of 1H and 13C NMR data
with those reported previously4 and on the basis of the results of
1He1H COSY, HMBC, and NOESY experiments, in particular the
HMBC correlation from H-30 (dH 5.05) to the acetate carbonyl car-
bon (dC 170.9) (Fig. S2). The sugar was found to be connected to C-3
of the aglycon by HMBC correlation of H-10 and C-3. The anomeric
proton H-10 (dH 5.04) has a small coupling constant, indicating the
equatorial orientation of this proton. The relative configuration of
the aglycon of 10 was further determined by NOESY experiment
(Fig. S3). The absolute configuration of the sugar moiety in 10 was
determined by reversed-phase HPLC analysis of its o-tolylisothio-
carbamate.32,33 The liberated fucose from acid hydrolysis of 10 was
showed that 5 might have a 3-O-acetoxyl-D5,6-pregnane skeleton
with an acetoxymethyl substituent at C-10 on the basis of the
disappearance of a H3-19 singlet around dH 0.80e1.10 and the
presence of an AB doublet at dH 4.14 (J¼11.2 Hz) and 4.49
(J¼11.2 Hz) and a broad-singlet vinyl proton (dH 5.47). The HMBC
correlations from H2-19 to C-10, C-9, C-5, and C-1 also confirmed
this elucidation (Fig. S2). The relative stereochemistry of 5 was
determined by the 2D NOE experiment. The observed NOESY cor-
relations between H3-18 and both H-20 and H-11
H-17 and H-9, H2-19 and H-8, and H-3 and H-1
-orientation of H3-18, H2-19, and H-8 and the -orientation of H-3,
H-9, H-14, and H-17 (Fig. S3). On the basis of the above spectro-
b
, H-14 and both
a
revealed the
b
a
scopic data, the structure of
diacetoxypregna-5,20-diene.
5 was established as 3b,19-
Sclerosteroid C (6) was obtained as an amorphous solid. Its
molecular formula, C23H32O3, was established by HRESIMS, exhib-
iting eight degrees of unsaturation. The 1H and 13C NMR, involving
the DEPT spectra, exhibited the presence of a tertiary methyl (dH
0.63 s; dC 13.1), a primary acetoxymethyl group (dH 4.33, 1H, d,
J¼12.0 Hz; 4.48,1H, d, J¼12.0 Hz; dC 62.1), a vinyl group (dH 4.97, 1H,
br d, J¼17.5 Hz; 4.98, 1H, br d, J¼10.0 Hz; 5.75, 1H, ddd, J¼17.5, 10.5,
7.5 Hz; dC 114.9, 139.4), and an a,b-unsaturated carbonyl group (dH
6.04, 1H, d, J¼10.5 Hz; dH 7.01, 1H, d, J¼10.5 Hz; dC 130.7, 152.1, and
200.0) (Table 1). From above data and extensive 2D NMR (Figs. S2
and S3) and CD data analysis,31 6 should be the 19-acetoxyl de-
rivative of pregna-1,20-dien-3-one (7).21
treated with L-cysteine methyl ester followed by reaction with
o-tolylisothiocyanate to afford the corresponding o-tolylisothio-
carbamate derivative. The retention time of this sugar derivative
determined by HPLC analysis was found to be consistent with that
of o-tolylisothiocarbamate derivative prepared from standard
HRESIMS of sclerosteroid D (8) exhibited a [MþNa]þ peak at m/z
397.2355 (calcd for C23H34O4Na, 397.2357) and established the
molecular formula of C23H34O4, implying seven degrees of unsa-
turation. The 1H NMR spectroscopic data of 8 showed one charac-
teristic methyl signal (dH 0.54, 3H, s), one oxymethine (dH 4.76, 1H,
ddd, J¼16.0, 11.2, 4.8 Hz), and a vinyl group (dH 4.96, 1H, br d,
J¼16.8 Hz; 4.97, 1H, br d, J¼10.8 Hz; 5.73, 1H, ddd, J¼16.8, 10.8,
7.6 Hz) (Table 1), revealing that 8 should have the same 3-acetoxy
pregnane skeleton as 3. Comparison of the 13C NMR spectral data
of 3 and 8 showed that the C-19 oxymethylene carbon (dC 60.8) in 3
was disappeared and replaced by a downfield-shifted carbonyl
carbon (dC 178.4) in 8 and C-10 was downfield-shifted from dC 39.3
(in 3) to 50.6 (in 8), thus the C-19 carboxylic acid functionality of 8
was elucidated (Table 1). NOE correlations of 8 further established
the relative stereochemistry of this steroid (Fig. S3), identical to
those of 3 and 4. From above data and extensive 2D NMR (Figs. S2
L-fucose. The structure of 10 was assigned as 3
b a-L-
-(30-O-acetyl-
fucopyranosyloxy)pregn-20-en-19-ol.
Sclerosteroid G (11) has a molecular formula, C31H48O8, as de-
termined by HRESIMS. The 1H and 13C NMR spectroscopic data of 11
resembled those of 10, except that the presence of a primary hy-
droxy group at C-19 in 10 was replaced by a primary acetoxy group
in 11 (Table 2). Acid hydrolysis of 11 also yielded L-fucose by HPLC
analysis of its o-tolylisothiocarbamate derivative. The relative
configuration and connection of the aglycon and sugar residue of 11
were further determined by 1He1H COSY, HMBC, and NOESY ex-
periments (Figs. S2 and S3). Thus, the structure of 11 was assigned
as 3b a-L-fucopyranosyloxy)pregn-20-en-19-acetate.
-(30-O-acetyl-
Sclerosteroid H (12) had a molecular formula of C29H44O7 as
established by HRESIMS. The 1H and 13C NMR spectroscopic data of
12 were similar to those of 10, except for the replacement of the
and S3), the structure of 8 was assigned as 3b-acetoxypregn-20-en-
19-oic acid.

H.-Y. Fang et al. / Tetrahedron 68 (2012) 9694e9700
9697
Table 2
1H and 13C NMR spectroscopic data for compounds 10e13
No.
10
11
12
13
a
c
a
c
a
c
a
c
dC
31.3
Mult.b
CH2
dH
dC
32.0
Mult.b
CH2
dH
dC
Mult.b
CH2
dH
dC
37.5
Mult.b
CH2
dH
1
2
0.83 m
2.28 dt (13.6, 3.6)
1.50 m
1.90 m
3.63 m
1.37 m
1.68 m
1.20 m
1.21 m
0.90 m
2.24 dt (13.6, 3.2)
1.43 m
1.86 m
3.62 m
1.32 m
1.73 m
1.25 m
1.27 m
31.0
30.4
0.96 m
2.42 dt (13.6, 3.6)
1.39 m
1.94 m
3.61 m
1.29 m
1.82 m
1.39 m
1.52 m
1.08 m
1.68 m
1.55 m
1.79 m
3.50 m
1.27 m
1.53 m
1.09 m
1.27 m
29.8
CH2
29.4
CH2
CH2
29.3
CH2
3
4
77.4
34.9
CH
CH2
76.9
34.6
CH
CH2
76.9
36.0
CH
CH2
77.5
34.3
CH
CH2
5
6
44.9
28.2
CH
CH2
44.9
28.2
CH
CH2
43.3
28.3
CH
CH2
44.8
28.8
CH
CH2
1.92 m
7
32.0
CH2
0.90 m
1.70 m
1.49 m
0.70 m
31.9
CH2
0.90 m
1.73 m
1.48 m
0.73 m
32.0
CH2
1.06 m
1.89 m
1.42 m
0.96 m
32.2
CH2
0.92 m
1.72 m
1.39 m
0.68 m
8
9
10
11
36.1
54.9
39.4
22.6
CH
CH
C
35.9
54.6
38.1
21.8
CH
CH
C
37.0
52.8
51.8
21.4
CH
CH
C
35.6
54.6
35.7
20.8
CH
CH
C
CH2
1.51 m
1.67 m
0.98 m
1.68 m
CH2
1.29 m
1.61 m
0.92 m
1.65 m
CH2
1.26 m
1.73 m
1.00 m
1.67 m
CH2
1.30 m
1.54 m
1.01 m
1.70 m
12
38.0
CH2
37.8
CH2
37.3
CH2
37.1
CH2
13
14
15
43.7
55.8
24.7
C
CH
CH2
43.7
55.9
24.7
C
CH
CH2
43.4
55.7
24.6
C
CH
CH2
43.6
55.6
24.8
C
CH
CH2
1.01 m
1.18 m
1.66 m
1.54 m
1.77 m
1.94 m
0.63 s
3.80 d (12.0)
3.93 d (12.0)
5.76 ddd (17.2,
10.0, 7.6)
4.96 br d (17.2)
4.97 br d (10.0)
5.04 d (4.0)
3.93 m
5.05 dd (10.4, 2.8)
3.83 br s
4.10 q (6.6)
1.26 d (6.6)
0.98 m
1.18 m
1.65 m
1.56 m
1.78 m
1.92 m
0.58 s
4.20 d (12.0)
4.37 d (12.0)
5.75 ddd (17.2,
10.6, 8.0)
4.96 br d (17.2)
4.97 br d (10.6)
5.03 d (4.0)
3.90 dt (10.2, 4.0)
5.04 dd (10.2, 2.8)
3.84 br s
0.94 m
1.20 m
1.70 m
1.55 m
1.78 m
1.94 m
0.52 s
0.99 m
1.17 m
1.69 m
1.58 m
1.80 m
1.94 m
0.61 s
16
27.1
CH2
27.1
CH2
27.1
CH2
27.2
CH2
17
18
19
55.4
13.3
60.8
CH
CH3
CH2
55.3
13.0
62.7
CH
CH3
CH2
55.3
12.8
208.4
CH
CH3
CH
55.4
12.9
12.3
CH
CH3
CH
10.03 s
0.84 s
20
21
139.8
114.5
CH
139.8
114.5
CH
139.5
114.7
CH
5.73 ddd (17.6,
10.2, 7.6)
4.97 br d (17.6)
4.98 br d (10.2)
5.00 d (3.6)
3.90 m
5.02 dd (10.4, 3.6)
3.82 br s
4.06 q (6.4)
1.25 d (6.4)
139.9
114.3
CH
5.76 ddd (16.4,
10.8, 8.0)
4.96 br d (16.4)
4.98 br d (10.8)
5.13 d (3.6)
4.88 dd (10.2, 3.6)
4.02 dd (10.2, 3.2)
3.83 d (3.2)
CH2
CH2
CH2
CH2
10
97.3
66.8
73.9
70.9
65.8
16.0
170.9
21.2
CH
CH
CH
CH
CH
CH3
C
97.1
66.8
73.9
70.9
65.8
16.0
170.7
171.2
21.2
21.2
CH
CH
CH
CH
CH
CH3
C
C
CH3
CH3
97.4
66.8
73.8
71.0
65.9
16.0
170.8
21.2
CH
CH
CH
CH
CH
CH3
C
94.7
72.1
68.6
72.4
65.3
16.1
171.5
21.1
CH
CH
CH
CH
CH
CH3
C
20
30
40
50
4.09 q (6.8)
1.26 d (6.8)
4.13 q (6.4)
1.31 d (6.4)
60
OAc
CH3
2.17 s
CH3
2.16 s
CH3
2.16 s
2.04 s
2.18 s
a
Recorded at 100 MHz in CDCl3 at 25 ꢁC.
Multiplicities deduced by DEPT (The chemical shifts referenced to residual signal of CDCl3 at
Recorded at 400 MHz in CDCl3 at 25 ꢁC (The chemical shifts referenced to TMS at
b
c
d
77.0 ppm.).
d
0.0 ppm.).
C-10 hydroxymethyl group in 10 by an aldehyde (dC 208.4; dH 10.03)
in 12, as also evidenced by the HMBC correlations from H-19 (dH
10.03) to C-10 (dC 51.8), C-9 (dC 52.8), C-5 (dC 43.3), and C-1 (dC 31.0)
experiments (Figs. S2 and S3). Consequently, 13 was determined as
-(20-O-acetyl-
-fucopyranosyloxy)pregna-20-ene.
The cytotoxicity of compounds 1e5, 10e13, 20e23, 25, and 26
3b
a-L
(Table 2). Acid hydrolysis of 12 also gave
L
-fucose as the sugar
against six human cancer cell lines, including hepatoma HepG2 and
Hep3B cells, breast cancer MDA-MB-231 and MCF-7 cells, lung
carcinoma A-549 cells, and gingival cancer Ca9-22 cells, was shown
in Table 3. Among them, compound 1 exhibited stronger cytotox-
icity against HepG2 and MDA-MB-23 with IC50 values of 19.5 and
residue. The relative configuration and connection of the aglycon
and sugar residue of 12 were further determined by 1He1H COSY,
HMBC, and NOESY experiments (Figs. S2 and S3). Thus, the struc-
ture of 12 was assigned as 3b a-L-fucopyranosyloxy)
-(30-O-acetyl-
pregna-20-en-19-al.
15.8 mM, respectively. Compounds 10, 11, 20, and 26 showed
Sclerosteroid I (13) has the molecular formula of C29H46O6, de-
termined by HRESIMS. The 1H and 13C NMR spectroscopic data of 13
resembled those of 12, except for the presence of a methyl sub-
stituent at C-10 in 13 and the small variation in sugar moiety (Table
2). The sugar moiety of 13 was readily assigned to be the 2-O-
moderate cytotoxicity toward all six human cancer lines. Com-
pounds 2, 3, 12, 22, and 23 showed selective moderate cytotoxicity
toward hepatoma cells, HepG2 or Hep3B, and gingival cancer Ca9-
22 cells, whereas these compounds showed inactive toward the
other three cancer cell lines.
The anti-inflammatory activities of 1e5, 7e27 were evaluated
against the accumulation of pro-inflammatory iNOS and COX-2
proteins in RAW264.7 macrophage cells stimulated with LPS,
which were measured by immunoblot analysis (Fig. 3). At a con-
acetyl-
a
-fucose by interpretation of 1He1H COSY correlation to-
gether with an HMBC cross-peak from H-20 to acetate carbonyl
carbon (Fig. S2). The HMBC correlation from H-10 (dH 5.13) to C-3 (dC
77.5) revealed that the sugar residue was attached to C-3 of the
aglycon moiety (Fig. S2). Acid hydrolysis of 13 also liberated
L-fu-
centration of 10 mM, compounds 1, 5, and 9 significantly reduced
cose. The relative configuration and connection of the aglycon and
sugar residue of 13 were further determined by 2D NMR
the levels of iNOS protein to 28.4ꢂ9.4, 27.7ꢂ9.9%, and 25.4ꢂ6.4%,
respectively, relative to control cells stimulated with LPS only,

9698
H.-Y. Fang et al. / Tetrahedron 68 (2012) 9694e9700
Table 3
indicated that compounds 1, 5, and 9 might become the effective
anti-inflammatory agents. None of the other compounds at 10
Cytotoxicity data of compounds isolated from S. gracillimum
m
M
were inhibitory against the expression of pro-inflammatory iNOS
and COX-2 proteins in RAW264.7 macrophage cells.
Compound
Cell lines, IC50
(mM)
HepG2 Hep3B Ca9-22 A-549 MCF-7 MDA-MB-231
The present study demonstrates that the soft coral, S. gracilli-
mum, can produce new pregnane-type metabolites, and some of
these compounds showed useful anti-inflammatory effects. Thus,
the chemical constituents of soft corals of this genus might be
worthy of further investigation of discovering new bioactive agents.
b
b
b
1
2
3
4
19.5
45.7
46.1
36.8
35.0
35.6
29.0
da
26.7
da
15.8
da
44.4
48.8
39.3
44.3
da
da
da
da
35.3
34.7
46.2
da
42.2
da
b
b
b
5
41.3
30.8
28.0
da
10
11
12
13
20
21
22
23
25
26
29.3
28.1
36.3
31.5
28.9
31.2
30.3
32.2
da
28.9
27.2
37.9
30.8
26.9
28.1
28.4
37.3
36.6
24.7
0.3
30.6
28.7
da
38.7
34.1
da
3. Experimental
37.4
28.4
33.2
32.1
da
28.9
29.7
30.8
da
da
da
da
30.1
27.8
32.0
da
da
da
34.1
da
3.1. General experimental procedures
da
da
Melting points were determined using a Fisher-Johns melting
point apparatus. Optical rotation was measured on a JASCO P1020
polarimeter. Ultraviolet spectra were recorded on a JASCO V-650
spectrophotometer. IR spectra were recorded on JASCO FT/IR-4100
infrared spectrophotometer. CD spectrum was measured on
a JASCO J-815 spectrophotometer. The NMR spectra were recorded
on a Bruker Avance 300 NMR spectrometer at 300 MHz for 1H and
75 MHz for 13C, on a Varian MR 400 NMR spectrometer at 400 MHz
for 1H and 100 MHz for 13C, or on a Varian Unity INOVA 500 FT-
NMR spectrometer at 500 MHz for 1H and 125 MHz for 13C. LRMS
and HRMS were obtained by ESI on a Bruker APEX II mass spec-
trometer. Silica gel 60 (Merck, 230e400 mesh) was used for column
chromatography. Precoated silica gel plates (Merck Kieselgel 60
F254 0.2 mm) were used for analytical TLC. High-performance liquid
chromatography was performed on a Hitachi L-7100 HPLC appa-
da
da
23.5
26.8
1.1
28.9
1.9
31.0
2.2
27.0
2.1
Doxorubicin 0.3
a
d, Inactive with IC50>50 mM.
b
Non-tested.
ratus with a C-18 column (250ꢃ10 mm, 5
mm).
3.2. Material
The soft coral S. gracillimum was collected at Green Island, Tai-
wan, in January, 2008, at a depth of 10 m, and was stored in
a freezer until extraction. A voucher specimen (NSYSU-SG001) was
deposited in the Department of Marine Biotechnology and Re-
sources, National Sun Yat-sen University.
3.3. Extraction and isolation
The frozen organisms (1.3 kg fresh wet) were sliced and
extracted exhaustively with EtOH (3ꢃ2 L). The organic extract was
concentrated to an aqueous suspension, which was further parti-
tioned between EtOAc and water. The EtOAc extract (9.6 g) was
fractionated by column chromatography on silica gel using n-hex-
ane/EtOAc and EtOAc/MeOH mixtures of increasing polarity to yield
40 fractions. Fraction 5, eluting with n-hexane/EtOAc (60:1), was
subjected to a Sephadex LH-20 column, using acetone as mobile
phase, to afford two separated subfractions. Subfraction 1 was
further separated by reversed-phase HPLC (MeOH/H2O, 95:5) to
afford 16 (2.5 mg) and 17 (4.1 mg). Fraction 12, eluting with
n-hexane/EtOAc (6:1), was further separated by silica gel column
chromatography (n-hexane/acetone, 10:1) and followed by
reversed-phase HPLC (MeOH/H2O, 85:15) to afford 4 (7.1 mg), 7
(1.4 mg), 18 (6.4 mg), and 19 (3.4 mg). Fraction 13, eluting with n-
hexane/EtOAc (4:1), was further separated by silica gel column
chromatography (n-hexane/EtOAc, 10:1) and followed by reversed-
phase HPLC (MeOH/H2O, 85:15) to afford 1 (8.0 mg), 5 (3.4 mg), 8
(2.7 mg), 9 (2.7 mg), and 15 (5.4 mg). Fraction 14, eluting with
n-hexane/EtOAc (2:1), was further separated by silica gel column
chromatography (n-hexane/EtOAc, 10:1) and followed by reversed-
phase HPLC(MeOH/H2O, 9:1) to afford 3 (13.4 mg) and 6 (0.7 mg).
Fraction 18, eluting with n-hexane/EtOAc (1:6), was rechromato-
graphed over a Sephadex LH-20 column using acetone as the mo-
bile phase to afford six subfractions (A1eA6). Subfractions A2 and
Fig. 3. Effect of isolates (10 mM) from S. gracillimum on the LPS-induced pro-in-
flammatory iNOS and on COX-2 protein expression of RAW264.7 macrophage cells by
immunoblot analysis. (A) Quantification of immunoblots iNOS. (B) Quantification of
immunoblots COX-2. The values are meansꢂSEM (n¼6). The relative intensity of the
LPS alone stimulated group was taken as 100%. (C) Quantification of immunoblots of
b-actin. *Significantly different from LPS alone stimulated group (*P<0.05). Control:
stimulated with LPS.
meanwhile compounds 15 and 23 moderately reduced iNOS level
to 56.7ꢂ4.4% and 61.9ꢂ6.5%, respectively. At this same concentra-
tion, compounds 1, 5, and 9 could reduce COX-2 expression to
5.4ꢂ1.3%, 6.7ꢂ3.5%, and 20.6ꢂ10.0%, respectively. Both results

H.-Y. Fang et al. / Tetrahedron 68 (2012) 9694e9700
9699
A3 were combined and separated by reversed-phase HPLC (CH3CN/
H2O, 9:1 to 3:1) to afford compounds 10 (6.2 mg), 11 (2.3 mg), 12
(8.1 mg), 13 (6.2 mg), 14 (1.7 mg), 2 (15.0 mg), 20 (13.4 mg), 21
(20.8 mg), 22 (8.7 mg), 23 (15.9 mg), 24 (1.5 mg), 25 (8.2 mg), 26
(14.3 mg), and 27 (3.7 mg).
hexane/EtOAc (4:1) to yield the diacetyl derivative 4 (2.5 mg, 91%),
the specific rotation of which was in agreement with that of the
natural product 4.
3.3.12. Acetylation of 2. To a stirring solution of 2 (5.0 mg) in pyr-
idine (0.3 mL) was added Ac2O (250 mL, 16 mg Ac2O diluted in 2 mL
3.3.1. Sclerosteroid A (1). Colorless gum; [
a
]
25 ꢀ28.2 (c 0.4, CHCl3);
pyridine). The mixture was quenched by H2O (0.1 mL) after stirred
over night at room temperature, and subsequently extracted by
ethyl acetate. The organic layer was concentrated to give a residue,
which was chromatographed on silica gel with n-hexane/EtOAc
(8:1) as the eluent to afford 19-acetylated derivative (1, 0.7 mg),
3-acetylated one (3, 1.2 mg), 3,19-diacetylated one (4, 0.3 mg), and
the recovered 2 (3.0 mg).
D
IR (KBr) nmax 3403, 2927, 2866, 1738, 1448, 1240, and 1038 cmꢀ1
;
13C and 1H NMR data, see Table 1 ESIMS m/z 383 [MþNa]þ;
HRESIMS m/z 383.2562 [MþNa]þ (calcd for C23H36O3Na, 383.2560).
3.3.2. Sclerosteroid B (5). Colorless gum; [
a
]
25 þ72.4 (c 0.16, CHCl3);
D
IR (KBr) nmax 2932, 2870, 1739, 1239, and 1025 cmꢀ1
;
13C and 1H
NMR data, see Table 1 ESIMS m/z 423 [MþNa]þ; HRESIMS m/z
423.2511 [MþNa]þ (calcd for C25H36O4Na, 423.2508).
3.3.13. Preparation of (S)-and (R)-MTPA esters of 2. To a solution of
2 (1 mg) in pyridine (0.4 mL) was added R-(ꢀ)-
a
m
-methoxy-
L), and the mix-
a-(tri-
25
3.3.3. Sclerosteroid C (6). Amorphous solid; [
a
]
ꢀ29.2 (c 0.07,
fluoromethyl)phenylacetyl (MTPA) chloride (25
D
CHCl3); IR (KBr) nmax 2923, 2868, 1742, 1682, 1229, and 1036 cmꢀ1
;
ture was allowed to stand over night at room temperature. The
reaction was quenched by the addition of 1.0 mL of H2O, and the
mixture was subsequently extracted with EtOAc (3ꢃ1.0 mL). The
EtOAc-soluble layers were combined, dried over anhydrous MgSO4,
and evaporated. The residue was subjected to short silica gel col-
umn chromatography over using n-hexane/EtOAc (3:1) to yield the
(S)-MTPA ester, 2a (0.8 mg, 87 %). The same procedure was used to
prepare the (R)-MTPA ester, 2b (0.7 mg, 76 %) from the reaction of
(S)-MPTA chloride with 2 in pyridine. Selective 1H NMR (CDCl3,
UV (MeOH) lmax (log ) 226 (7.34) nm; CD (c 0.07, MeOH) lmax (D 3 )
3
204 (ꢀ14.0), 230 (13.0), and 336 (ꢀ2.1); 13C and 1H NMR data, see
Table 1 ESIMS m/z 379 [MþNa]þ; HRESIMS m/z 379.2249 [MþNa]þ
(calcd for C23H32O3Na, 379.2250).
25
3.3.4. Sclerosteroid D (8). White powder; [
a]
ꢀ36.0 (c 0.10,
D
CHCl3); IR (KBr) nmax 2945, 2868, 2842, 1733, 1687, 1242, and
1034 cmꢀ1 13C and 1H NMR data, see Table 1 ESIMS m/z 397
;
[MþNa]þ; HRESIMS m/z 397.2355 [MþNa]þ (calcd for C29H46O6Na,
400 MHz) of 2a:
d
0.845 (1H, m, H-1a), 2.000 (1H, dt, J¼13.6 and
397.2357).
3.6 Hz, H-1b), 1.753 (1H, m, H-2), 4.962 (1H, m, H-3), 1.418 (1H, m,
H-4), 1.347 (1H, m, H-6), 1.154 (1H, m, H-6); Selective 1H NMR
25
3.3.5. Sclerosteroid E (9). Colorless oil; [
a
]
ꢀ13 (c 0.1, CHCl3); IR
(CDCl3, 400 MHz) of 2b:
and 3.2 Hz, H-1b), 1.871 (1H, m, H-2), 4.956 (1H, m, H-3), 1.373 (1H,
m, H-4), 1.290 (1H, m, H-6), 1.122 (1H, m, H-6).
d
0.917 (1H, m, H-1a), 2.149 (1H, dt, J¼14.0
D
(neat) nmax 3390, 2926, 2864, 1718, 1447, 1250 and 1031 cmꢀ1
;
13C
and 1H NMR data, see Table 1 ESIMS m/z 385 [MþNa]þ; HRESIMS
m/z 385.2355 [MþNa]þ (calcd for C22H34O4Na, 385.2357).
3.3.14. Determination of sugar configuration. Authentic samples of
D-fucose and L-cysteine methyl ester hydrochloride (each 0.5 mg)
25
3.3.6. Sclerosteroid F (10). Amorphous solid; [
a
]
ꢀ45.8 (c 0.11,
D
CHCl3); IR (KBr) nmax 3452, 2925, 2868, 1723, 1377, 1249, 1072, and
were dissolved in pyridine (0.1 mL) and heated at 60 ꢁC for 1 h. To
the mixture was added o-tolylisothiocyanate (0.5 mg in 0.1 mL
pyridine) and heated at 60 ꢁC for additional 1 h. The reaction
mixture was directly analyzed by reversed-phase HPLC (Mightysil
RP-18 GP column; 4.6ꢃ250 mm; 25% CH3CN in 50 mM H3PO4;
0.5 mL/min; 35 ꢁC) and detected at 250 nm to give the retention
time of the o-tolylthiocarbamate of sugar. The retention time of
1030 cmꢀ1 13C and 1H NMR data, see Table 2 ESIMS m/z 529
;
[MþNa]þ; HRESIMS m/z 529.3141 [MþNa]þ (calcd for C29H46O7Na,
529.3144).
25
3.3.7. Sclerosteroid G (11). Amorphous solid; [
a]
ꢀ41.0 (c 0.16,
D
CHCl3); IR (KBr) nmax 3453, 2935, 2868, 1736, 1372, 1240, 1073, and
1034 cmꢀ1
;
13C and 1H NMR data, see Table 2 ESIMS m/z 571
the o-tolylthiocarbamate derived from L-fucose, L-cysteine methyl
[MþNa]þ; HRESIMS m/z 571.3247 [MþNa]þ (calcd for C31H48O8Na,
ester, and o-tolylisothiocyanate was obtained by the same manner.
A solution of the glycoside (0.3 mg for each) in 0.6 M HCl/di-
oxane (1:1 v/v, 0.2 mL) was heated at 90 ꢁC for 4 h. After cooling,
the solution was neutralized with Amberlite IRA400 (OHꢀ form),
and the resin was removed by filtration. The filtrate was extracted
with EtOAc. The aqueous layer was dried in vacuo and the afforded
571.3245).
25
3.3.8. Sclerosteroid H (12). Amorphous solid; [
a
]
ꢀ44.2 (c 0.12,
D
CHCl3); IR (KBr) nmax 3440, 2936, 2869, 1720, 1380, 1245, 1073,
1035 cmꢀ1 13C and 1H NMR data, see Table 2 ESIMS m/z 527
;
[MþNa]þ; HRESIMS m/z 527.2985 [MþNa]þ (calcd for C29H44O7Na,
residue was dissolved in pyridine (0.1 mL) containing L-cysteine
527.2988).
methyl ester (0.5 mg), followed by heating at 60 ꢁC for 1 h. A 0.1 mL
solution of o-tolylisothiocyanate (0.5 mg) in pyridine was added to
the mixture, which was again heated at 60 ꢁC for additional 1 h, to
yield the corresponding o-tolylthiocarbamate derivative. Reversed-
phase HPLC analysis of the o-tolylthiocarbamate derivatives de-
rived from the hydrolyte of the glycosides 10e13 showed peaks at
42.3, 42.2, 42.3, and 42.8 min, respectively, while the tR values for
25
3.3.9. Sclerosteroid I (13). Amorphous solid; [
a
]
ꢀ14.2 (c 0.38,
D
CHCl3); IR (KBr) nmax 3436, 2932, 2868, 1739, 1376, 1245, and
1046 cmꢀ1 13C and 1H NMR data, see Table 2 ESIMS m/z 513
;
[MþNa]þ; HRESIMS m/z 513.3192 [MþNa]þ (calcd for C29H46O6Na,
513.3190).
standard L-fucose and D-fucose derivatives were observed at 42.8
and 39.4 min, respectively, suggesting the presence of an L-fucose
residue in 10e13.
25
3.3.10. Pregna-1,20-dien-3-one (7). [
a
]
D
þ31.5 (c 0.23, CHCl3); lit.
[
a]
D þ35.4 (c 0.5, CHCl3);21 CD (c 0.14, MeOH) lmax
(D
3
) 206 (ꢀ3.4),
238 (5.4), and 338 (ꢀ0.2); lit. CD (MeOH) [
q]
237 4770.31
3.3.11. Acetylation of 1. A solution of 1 (1.9 mg) in pyridine (0.2 mL)
was mixed with Ac2O (0.1 mL), and the mixture was stirred at room
temperature for 24 h. After evaporation of excess reagent, the res-
idue was subjected to column chromatography over Si gel using n-
3.4. Cytotoxicity testing
Cell lines were purchased from the American Type Culture
Collection (ATCC). Cytotoxicity assays were performed using the

9700
H.-Y. Fang et al. / Tetrahedron 68 (2012) 9694e9700
10. Lu, Y.; Lin, Y.-C.; Wen, Z.-H.; Su, J.-H.; Sung, P.-J.; Hsu, C.-H.; Kuo, Y.-H.; Chiang,
M. Y.; Dai, C.-F.; Sheu, J.-H. Tetrahedron 2010, 66, 7129e7135.
11. Chao, C.-H.; Wen, Z.-H.; Chen, I. M.; Su, J.-H.; Huang, H.-C.; Chiang, M. Y.; Sheu,
J.-H. Tetrahedron 2008, 64, 3554e3560.
MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bro-
mide] colorimetric method.34,35
3.5. In vitro anti-inflammatory assay
12. Su, J.-H.; Lo, C.-L.; Lu, Y.; Wen, Z.-H.; Huang, C.-Y.; Dai, C.-F.; Sheu, J.-H. Bull.
Chem. Soc. Jpn. 2008, 81, 1616e1620.
13. Chen, B.-W.; Su, J.-H.; Dai, C.-F.; Wu, Y.-C.; Sheu, J.-H. Bull. Chem. Soc. Jpn. 2008,
81, 1304e1307.
14. Su, J.-H.; Lin, F.-Y.; Dai, C.-F.; Wu, Y.-C.; Hsu, C.-H.; Sheu, J.-H. Bull. Chem. Soc. Jpn.
2007, 80, 2208e2212.
15. Su, J.-H.; Lin, F.-Y.; Huang, H.-C.; Dai, C.-F.; Wu, Y.-C.; Hu, W.-P.; Hsu, C.-H.; Sheu,
J.-H. Tetrahedron 2007, 63, 703e707.
16. Ahmed, A. F.; Hsieh, Y.-T.; Wen, Z.-H.; Wu, Y.-C.; Sheu, J.-H. J. Nat. Prod. 2006, 69,
1275e1279.
Macrophage (RAW264.7) cell line was purchased from ATCC.
In vitro anti-inflammatory activities of compounds 1e5, and 7e27
were measured by examining the inhibition of LPS (lipopolysac-
charide)-stimulated upregulation of iNOS (inducible nitric oxide
synthase) and COX-2 (cyclooxygenase-2) proteins in macrophage
cells using Western blot analysis.36
17. Su, J.-H.; Tseng, Y.-J.; Huang, H.-H.; Ahmed, A. F.; Lu, C.-K.; Wu, Y.-C.; Sheu, J.-H.
J. Nat. Prod. 2006, 69, 850e852.
Acknowledgements
18. Chao, C.-H.; Huang, L.-F.; Yang, Y.-L.; Su, J.-H.; Wang, G.-H.; Chiang, M. Y.; Wu,
Y.-C.; Dai, C.-F.; Sheu, J.-H. J. Nat. Prod. 2005, 68, 880e885.
19. Sheu, J.-H.; Chao, C.-H.; Wang, G.-H.; Hung, K.-C.; Duh, C.-Y.; Chiang, M. Y.; Wu,
Y.-C.; Wu, C.-C. Tetrahedron Lett. 2004, 45, 6413e6416.
20. Lu, C.-K.; Wang, S.-K.; Duh, C.-Y. Bull. Chem. Soc. Jpn. 2011, 84, 943e946.
21. Seo, Y.; Jung, J. H.; Rho, J.-R.; Shin, J.; Song, J.-I. Tetrahedron 1995, 51, 2497e2506.
22. Tomono, Y.; Hirota, H.; Imahara, Y.; Fusetani, N. J. Nat. Prod. 1999, 62,
1538e1541.
This work was supported by grants from the National Science
Council of Taiwan NSC-100-2320-B-110-001-MY2 to J.-H.S.
Supplementary data
23. Kobayashi, M.; Shizaka, T.; Mitsuhashi, H. Chem. Pharm. Bull. 1983, 31,
1803e1805.
24. Kingston, J. F.; Gregory, B.; Fallis, A. G. J. Chem. Soc., Perkin Trans. 1 1979,
2064e2068.
Structures of known compounds, 1H and 13C NMR spectra for 1,
5, 6, 8e13 and their 2D NMR correlations. Supplementary data
associated with this article can be found in the online version, at
http://dx.doi.org/10.1016/j.tet.2012.09.060.
25. Higg, M. D.; Faulkner, D. J. Steroids 1977, 30, 379e388.
26. Blackman, A. J. H. A.; Skelton, B. W.; White, A. H. Aust. J. Chem. 1985, 38,
565e573.
ꢀ
27. Cobar, O. M.; Rodríguez, A. D.; Padilla, O. L. J. Nat. Prod. 1997, 60, 1186e1188.
References and notes
28. Huang, X. P.; Deng, Z. W.; Ofwegen, L. V.; Li, J.; Fu, H. Z.; Zhu, X. B.; Lin, W. H. J.
Asian Nat. Prod. Res. 2006, 8, 287e291.
29. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113,
4092e4096.
30. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Org. Chem. 1991, 56,
1296e1298.
31. Schow, S. R.; McMorris, T. C. Steroids 1977, 30, 389e392.
32. Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Chem. Pharm. Bull. 2007,
55, 899e901.
33. Chao, C.-H.; Chou, K.-J.; Huang, C.-Y.; Wen, Z.-H.; Hsu, C.-H.; Wu, Y.-C.; Dai, C.-F.;
Sheu, J.-H. Mar. Drugs 2012, 10, 439e450.
34. Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D.
L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48,
589e601.
35. Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.; Tierney, S.; Nofziger,
T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R. Cancer Res. 1988, 48, 4827e4833.
36. Chao, C.-H.; Wen, Z.-H.; Wu, Y.-C.; Yeh, H.-C.; Sheu, J.-H. J. Nat. Prod. 2008, 71,
1819e1824.
1. Yan, X.-H.; Guo, Y.-W.; Zhu, X.-Z.; Mollo, E.; Cimino, G. Youji Huaxue 2004, 24,
1233e1238.
2. Kittakoop, P.; Suttisri, R.; Chaichantipyuth, C.; Vethchagarun, S.; Suwanborirux,
K. J. Nat. Prod. 1999, 62, 318e320.
3. Han, L.; Wang, C.-Y.; Huang, H.; Shao, C.-L.; Liu, Q.-A.; Qi, J.; Sun, X.-P.; Zhai, P.;
Gu, Y.-C. Biochem. Syst. Ecol. 2010, 38, 243e246.
4. Wang, S.-K.; Dai, C.-F.; Duh, C.-Y. J. Nat. Prod. 2006, 69, 103e106.
5. Yan, X.-H.; Liu, H.-L.; Guo, Y.-W. Steroids 2009, 74, 1061e1065.
6. Ioannou, E.; Abdel-Razik, A. F.; Alexi, X.; Vagias, C.; Alexis, M. N.; Roussis, V.
Tetrahedron 2008, 64, 11797e11801.
7. Chao, C.-H.; Wen, Z.-H.; Su, J.-H.; Chen, I. M.; Huang, H.-C.; Dai, C.-F.; Sheu, J.-H.
Steroids 2008, 73, 1353e1358.
ꢀ
~
ꢀ
ꢀ
8. Gutierrez, M.; Capson, T. L.; Guzman, H. M.; Gonzalez, J.; Ortega-Barría, E.;
ꢀ
Quinoa, E.; Riguera, R. J. Nat. Prod. 2006, 69, 1379e1383.
ꢀ
9. Diaz-Marrero, A. R.; Porras, G.; Aragon, Z.; de la Rosa, J. M.; Dorta, E.; Cueto, M.;
D’Croz, L.; Mate, J.; Darias, J. J. Nat. Prod. 2011, 74, 292e295.
ꢀ