Cytotoxic and anti-inflammatory cembranoids from the Dongsha Atoll soft coral Sarcophyton crassocaule

Article abstract of DOI:10.1016/j.bmc.2010.01.036

Five new cembranoids, sarcocrassocolides A-E (1-5), along with three known cembranoids 6-8, have been isolated from a Formosan soft coral Sarcophyton crassocaule. The structures of the new metabolites were elucidated by extensive spectroscopic analysis and the absolute configuration of 1 was determined by a modified Mosher's method. Compounds 1-4 exhibited significantly cytotoxic activity against a limited panel of cancer cell lines. Compounds 1-4, 6 and 8 were shown to exert significant in vitro anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells. Compound 6 also significantly inhibited the accumulation of pro-inflammatory COX-2 protein.

Full text of DOI:10.1016/j.bmc.2010.01.036

Bioorganic & Medicinal Chemistry 18 (2010) 1936–1941
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cytotoxic and anti-inflammatory cembranoids from the Dongsha Atoll soft coral
Sarcophyton crassocaule
Wan-Yu Lin a, Jui-Hsin Su b, Yi Lu a, Zhi-Hong Wen a, Chang-Feng Dai c, Yao-Haur Kuo d, Jyh-Horng Sheu a,e,
*
a Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan
b Department of Biological Science and Technology, Meiho Institute of Technology, 23 Ping Kuang Road, Neipu Hsiang, Pingtung 912, Taiwan
c Institute of Oceanography, National Taiwan University, Taipei 112, Taiwan
d National Research Institute of Chinese Medicine, Taipei 112, Taiwan
e 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:
Five new cembranoids, sarcocrassocolides A–E (1–5), along with three known cembranoids 6–8, have
been isolated from a Formosan soft coral Sarcophyton crassocaule. The structures of the new metabolites
were elucidated by extensive spectroscopic analysis and the absolute configuration of 1 was determined
by a modified Mosher’s method. Compounds 1–4 exhibited significantly cytotoxic activity against a lim-
ited panel of cancer cell lines. Compounds 1–4, 6 and 8 were shown to exert significant in vitro anti-
inflammatory activity in LPS-stimulated RAW264.7 macrophage cells. Compound 6 also significantly
inhibited the accumulation of pro-inflammatory COX-2 protein.
Received 3 December 2009
Revised 14 January 2010
Accepted 15 January 2010
Available online 22 January 2010
Keywords:
Soft coral
Sarcophyton crassocaule
Cembranoids
Ó 2010 Elsevier Ltd. All rights reserved.
Inflammatory
Sarcocrassocolides
1. Introduction
COX-2 (cyclooxygenase-2) proteins in LPS (lipopolysaccharide)-
stimulated RAW264.7 macrophage cells was also evaluated. The
Many cembrane-type compounds have been shown to exhibit
cytotoxicity1–6 and anti-inflammatory activity.7–11 During the
course of our search for bioactive metabolites from marine inverte-
brates, many bioactive cembrane-derived compounds have been
discovered from soft corals of the genera Sinularia,1–5,7–9 Lobophy-
tum,10,11 and Sarcophyton.12,13 Our investigation on the chemical
constituents of the Dongsha Atoll soft coral Sarcophyton crassocaule
yielded five new cembranoids, sarcocrassocolides A–E (1–5), along
with three known cembranoids, sarcocrassolide (6),14 sinularolide
(7)15 and 13-acetoxysarcocrassolide (8).14 The structures of 1–5
were established by detailed spectroscopic analysis, including
extensive examination of 2D NMR (1H–1H COSY, HMQC, HMBC
and NOESY) correlations. The absolute configuration of 1 was fur-
ther determined using a modified Mosher’s method. The cytotoxic-
ity of compounds 1–4 against human breast carcinoma (MCF-7),
human colon carcinoma (WiDr), human laryngeal carcinoma
(HEp-2) and human medulloblastoma (Daoy) cells was studied,
and the ability of 1–4 and 6–8 to inhibit the up-regulation of
pro-inflammatory iNOS (inducible nitric oxide synthase) and
results revealed that compounds 1–4 are all cytotoxic towards
the above cancer cells, 3 being the most cytotoxic. Compounds
1–4, 6 and 8 were found to effectively inhibit the expression of
iNOS protein, while 6 was the only one seen also to significantly re-
duce the level of COX-2 protein in the above assay.
2. Results and discussion
The HRESIMS (m/z 413.1938 [M+Na]+) of 1 established the
molecular formula C22H30O6, appropriate for eight degrees of
unsaturation, and the IR spectrum revealed the presence of hydro-
xy (3487 cmꢀ1) and lactonic carbonyl (1769 cmꢀ1) groups. The 13
C
NMR (Table 1) and DEPT spectroscopic data showed signals of
three methyls (including one acetate methyl), five sp3 methylenes,
two sp2 methylenes, five sp3 methines (including four oxymeth-
ines), one sp2 methine, one sp3 and five sp2 quaternary carbons
(including two ester carbonyls). The NMR signals (Tables 1 and
2) observed at dC 170.1 (qC), 140.8 (qC), 120.5 (CH2), 81.0 (CH),
and 36.3 (CH), and dH 6.20, 5.65 (each, 1H, d, J = 2.0 Hz), 4.50
(1H, t, J = 2.8 Hz), and 3.64 (1H, dd, J = 6.4, 3.2 Hz) revealed
the presence of an
a-methylene-c-lactonic group by comparing
* Corresponding author.
the very similar NMR data of the five-membered lactone ring in
E-mail address: sheu@mail.nsysu.edu.tw (J.-H. Sheu).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.036

W.-Y. Lin et al. / Bioorg. Med. Chem. 18 (2010) 1936–1941
1937
18
H
H
19
OH
14
OH
O
4
7
17
O
O
8
3
1
15
16
R
R
R
9
O
O
11
13
O
10
12
O
O
O
20
1: R = OAc
3: R = H
2: R = OAc
4: R = H
5: R = OCOEt
6: R = H
7: R = OH
8: R = OAc
Table 1
13C NMR data for compounds 1–5
C#
1a
36.3, CH
39.4, CH2
73.5, CH
84.4, qC
2b
3a
4a
5b
c
1
2
3
4
35.4, CH
41.4, CH2
74.9, CH
86.0, qC
41.8, CH
38.2, CH2
73.3, CH
84.3, qC
41.4, CH
40.9, CH2
74.9, CH
85.7, qC
36.9, CH
34.7, CH2
60.1, CH
60.5, qC
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
37.9, CH2
30.9, CH2
79.4, CH
149.8, qC
33.4, CH2
26.9, CH2
129.1, CH
127.4, qC
77.1, CH
81.0, CH
140.8, qC
170.1, qC
120.5, CH2
21.4, CH3
111.0, CH2
14.5, CH3
20.7, CH3
169.4, qC
37.8, CH2
28.2, CH2
82.6, CH
148.3, qC
28.0, CH2
26.9, CH2
126.4, CH
128.5, qC
77.0, CH
81.8, CH
140.9, qC
170.1, qC
120.9, CH2
18.9, CH3
112.3, CH2
14.8, CH3
20.7, CH3
169.3, qC
38.4, CH2
31.9, CH
79.6, CH
149.1, qC
34.1, CH2
28.5, CH2
129.8, CH
127.6, qC
46.0, CH
80.7, CH
139.1, qC
168.9, qC
121.6, CH2
22.3, CH3
110.2, CH2
17.7, CH3
38.4, CH2
29.1, CH2
82.4, CH
147.2, qC
29.3, CH2
28.1, CH2
127.8, CH
127.7, qC
46.2, CH2
81.4, CH
138.9, qC
168.6, qC
122.0, CH2
20.4, CH3
111.3, CH2
17.9, CH3
37.4, CH2
23.4, CH2
123.0, CH
135.1, qC
37.9, CH2
24.8, CH2
129.0, CH
128.7, qC
77.2, CH
80.9, CH
139.7, qC
169.4, qC
121.0, CH2
17.4, CH3
16.8, CH3
14.7, CH3
13-Acetate
13-Propionate
8.9, CH3
27.5, CH2
172.5, qC
a
Spectra recorded at 100 MHz in CDCl3.
Spectra recorded at 125 MHz in CDCl3.
Attached protons were deduced by DEPT experiments.
b
c
the known compounds 6 and 8.14 Signals appearing at dC 111.0
(CH2), 149.8 (qC), dH 4.90 (1H, s) and 4.92 (1H, s) revealed the pres-
ence of the other 1,1-disubstituted carbon–carbon double bond.
One trisubstituted double bond was also identified from the NMR
signals at dC 127.4 (qC) and 129.1 (CH) and at dH 5.48 (1H, dd,
J = 10.0, 5.6 Hz). In the 1H–1H COSY spectrum, it was possible to
identify three different structural units, which were assembled
with the assistance of an HMBC experiment (Fig. 1). Key HMBC cor-
relations of H3-18 to C-3, C-4 and C-5; H2-19 to C-7, C-8 and C-9;
H3-20 to C-11, C-12 and C-13; H2-17 to C-1, C-15 and C-16; and
H2-5 and H2-6 to C-4 permitted connection of the carbon skeleton.
Furthermore, the acetoxy group positioned at C-13 was confirmed
from the HMBC correlations of H-13 (d 5.36) and protons of an ace-
tate methyl (d 2.01) to the ester carbonyl carbon at d 169.4 (C). A
hydroxy group was positioned at C-3, as both of the (S)- and (R)-
MTPA esters of 1, prepared for the determination of absolute con-
figuration (later discussed), showed significant differences in the
chemical shifts of H-3 and C-3 in comparison with those of 1. In
considering the degrees of unsaturation and molecular formula,
an ether linkage has to be placed between C-4 and C-7. On the basis
of the above analysis, the planar structure of 1 was established
unambiguously.
The relative configuration of 1 was elucidated by the analysis of
NOE correlations, as shown in Figure 2. It was found that H-1 (d
3.64, dd, J = 6.4, 3.0 Hz) showed NOE interaction with H3-18 (d
1.21, s); therefore, assuming a b-orientation of H-1, H3-18 should
also be positioned on the b-face. One of the methylene protons at
C-2 (d 1.94, m) exhibited NOE correlations with both H-1 and H3-
18 and was characterized as H-2b, while the other proton was as-
signed as H-2a (d 1.75, m). NOE correlations observed between H-
2
a
and H-14, and H-14 and H-3, reflected the
a
-orientation of H-
*
14, and hence the S configuration of C-3. Furthermore, H-14
exhibited NOE correlation with H-13 and H-3 exhibited NOE inter-
action with H-7, revealing the
a
-orientations of H-7 and H-13, and
*
hence the S configuration of C-13. The E geometry of the trisubsti-
tuted C-11/C-12 double bond was also assigned from the NOE cor-
relation of H3-20 (d 1.71, s) with H-10 (d 2.15, m), but not with
olefinic proton H-11, and also the upper field chemical shift of C-
20 (d 14.5). On the basis of the above findings and detailed exam-
ination of other NOE correlations (Fig. 2), the relative configuration
of compound 1 was determined. Furthermore, the absolute config-
uration of 1 was finally determined using a modified Mosher’s
esterification method.16 The (S)- and (R)-MTPA esters of 1 (1a
and 1b, respectively) were prepared using the corresponding (R)-

1938
W.-Y. Lin et al. / Bioorg. Med. Chem. 18 (2010) 1936–1941
Table 2
1H NMR data for compounds 1–5
H#
1a
2b
3a
4a
5b
1
2
3.64, dd (6.4, 3.2)
1.94, m
1.75, m
3.49, dd (9.2, 3.2)
1.98, m; 1.90, m
2.05, m; 1.78, m
4.17, dd (10.4, 4.4)
2.37, m
3.66, br d (10.0)
1.88, m
1.69, m
3.37, dd (9.5, 3.0)
1.90, m; 1.75, m
2.15, m; 1.76, m
4.52, dd (9.5, 4.5)
2.33, m
3.43, ddd (5.6, 3.2, 1.6)
1.94, m
1.84, m
3.53, dd (8.0, 4.0)
1.89, m; 1.78, m
2.00, m; 1.77, m
4.14, dd (11.2, 4.0)
2.29, m
3.40, d (8.4)
1.83, m
1.75, m
3.43, t (4.0)
1.86, m; 1.77, m
2.14, m; 1.79, m
4.49, dd (8.8, 4.0)
2.32, d (14.0)
1.83, m
3.06, d (12.0)
1.83, ddd (15.0, 12.5, 3.5)
1.69, ddd (15.0, 7.0, 3.5)
2.64, dd (7.5, 3.5)c
1.34, m
2.14, m; 2.09, m
5.02, t (6.5)
c
3
5
6
7
9
2.22, dd (14.0, 6.5)
2.04, m
1.82, m
10
2.28, m
2.15, m
2.45, dddd (14.0, 10.5, 7.0, 3.5)
2.14, m
2.25, m
2.13, m
2.40, m
2.12, m
2.39, ddd (15.0, 9.5, 9.5)
2.15, m
11
13
5.48, dd (10.0, 5.6)
5.36, s
5.49, t (7.5)
5.27, s
5.29, dd (10.4, 5.2)
2.55, d (14.0)
2.12, m
4.40, dt (10.4, 3.2)
6.25, d (2.0)
5.69, d (2.0)
1.22, s
5.34, t (7.2)
2.48, d (12.8)
2.02, t (12.4)
4.33, d (11.6)
6.25, s
5.30, br t (7.0)
5.39, s
14
17
4.50, t (2.8)
6.20, d (2.0)
5.65, d (2.0)
1.21, s
4.39, br s
6.21, d (2.0)
5.67, d (2.0)
1.20, s
4.60, t (2.5)
6.25, d (2.0)
5.62, d (1.5)
1.29, s
5.70, s
1.20, s
18
19
20
4.92, s; 4.90, s
1.71, s
5.10, s; 4.99, s
1.73, s
4.93, s; 4.89, s
1.67, s
5.08, s; 5.00, s
1.69, s
1.63, s
1.72, s
13-Acetate
13-Propionate
2.01, s
1.97, s
2.26, q (7.5)
1.09, t (7.5)
a
b
c
Spectra recorded at 400 MHz in CDCl3.
Spectra recorded at 500 MHz in CDCl3.
J values (in Hz) in parentheses.
18
4
tion with H3-18 (d 1.20, s), but not with H-3 and H3-20, revealing
the b-orientation of H-7. Further analysis of other NOE interactions
revealed that 2 possessed the same relative configurations at C-1,
C-3, C-4, C-13 and C-14 as those of 1 (Fig. 2). Therefore, 2 was
found to be the C-7 epimer of 1.
OH
19
7
17
15
O
3
8
9
1
11
13
O
14
16
10
12
O
O
Compound 3 was shown by HRESIMS to possess the molecular
formula C20H28O4 (m/z 355.1883 [M+Na]+). The IR spectrum of 3 re-
vealed the presence of hydroxy (3472 cmꢀ1) and lactonic carbonyl
(1760 cmꢀ1) groups. Comparison of the 1H and 13C NMR data (Ta-
bles 1 and 2) of compounds 1 and 3 showed that the structure of 3
should be very close to that of 1, with the exception of signals as-
signed to C-13, where an acetoxymethine (dH 5.36, 1H, s; dC 77.1)
in 1 is replaced by a methylene (dH 2.55, 1H, d, J = 14.0 Hz, dH
2.12, 1H, m; dC 26.6) in 3. The overall planar structure of 3 was fully
established by analyzing the 1H–1H COSY and HMBC correlations
(Fig. 1). The relative stereochemistry of 3 was confirmed to be
20
O
1
OH
O
O
O
3
*
*
*
*
*
1R , 3S , 4R , 7R and 14S from the following NOESY correlations
(Fig. 4): both H-7 (d 4.14) and H-14 (d 4.40) with H-3 (d 3.53),
and H3-18 (d 1.22) with H-1 (d 3.43). These results, together with
other detailed NOE correlations of 3 (Fig. 4), unambiguously estab-
lished the structure of sarcocrassocolide C, as shown in formula 3.
Thus, 3 is the 13-deacetoxy derivative of 1.
O
O
Compound
4 was found to have the molecular formula
O
O
:1H-1H COSY
: HMBC
C22H30O6, as indicated by HRESIMS, suggesting 4 to be an isomer
of 3. By 2D NMR spectroscopic data, including 1H–1H COSY, HMQC
and HMBC, compound 4 was shown to possess the same planar
structure as that of 3. By comparison of the related 1H and 13C
NMR spectroscopic data of 4 (Tables 1 and 2) with those of 3, 4
was found to be the C-7 epimer of 3; this was further confirmed
by analysis of the NOE correlations (Fig. 4) of 4, which revealed
that H-7 was correlated with H3-18.
O
5
Figure 1. 1H–1H COSY and HMBC correlations for 1, 3 and 5.
(ꢀ) and (S)-(+)-MTPA chloride, respectively. The determination of
d values (dS–dR) for protons neighboring C-3 led to the assign-
ment of the S configuration at C-3 in 1 (Fig. 3).
D
Compound 2 possessed the same molecular formula (C22H30O6)
as that of 1, as revealed from HRESIMS. Furthermore, it was found
that the NMR spectroscopic data of 2 were very similar to those of
1. By analysis of the 2D NMR (1H–1H COSY, HMQC, and HMBC) cor-
relations, compound 2 was shown to possess the same planar
structure as that of 1. From the NOESY spectrum (Fig. 2), it was
found that H-7 (d 4.52, dd, J = 9.5, 4.5 Hz) showed an NOE interac-
The HRESIMS spectrum of 5 exhibited a molecular ion peak at
m/z 411.2150 [M+Na]+, consistent with the molecular formula
C23H32O5 and implying eight degrees of unsaturation. Comparison
of the 1H and 13C NMR data of 5 (Tables 1 and 2) with those of 8
showed that the structure of both compounds are very similar.
The 1H and 13C NMR data (Tables 1 and 2) revealed that 5 is the
13-O-deacetyl-13-O-propionyl derivative of 8.

W.-Y. Lin et al. / Bioorg. Med. Chem. 18 (2010) 1936–1941
1939
17
18
4
3
1
3
4
1
14
7
14
7
12
11
19
20
1
3
7
2
4
Figure 2. Key NOESY correlations for 1 and 2.
Figure 4. Key NOESY correlations for 3 and 4.
+0.037
tion of a limited panel of cancer cell lines, including MCF-7, WiDr,
HEp-2 and Daoy carcinoma cells was then evaluated. The results
showed that compound 3, the most potent of compounds 1–4,
exhibited cytotoxicity against MCF-7, WiDr, HEp-2 and Daoy can-
+0.014 +0.031
H
H
OR
4
-0.022
H
-0.016
1
7
O
3
H
+0.017
-0.037
8
9
cer cell lines with ED50s of 2.0, 1.2, 2.6 and 3.2 lg/mL, respectively.
AcO
Furthermore, metabolites 1, 2 and 4 also were found to exhibit sig-
nificant cytotoxicity towards some of the above four cancer cells
(Table 3). The in vitro anti-inflammatory effects of compounds 1–
4 and 6–8 were also tested. The inhibition of LPS-induced up-reg-
ulation of pro-inflammatory proteins, iNOS and COX-2 in macro-
H
-0.023
15
14
13
16
11
12
O
O
20
phage cells was assayed by immunoblot analysis. At
a
1a: R= (S)-MTPA
1b: R= (R)-MTPA
concentration of 10 lM, compounds 1–4, 6 and 8 were found to
Figure 3. 1H NMR chemical shift differences
esters of 1.
D
d (dS ꢀ dR) in ppm for the MTPA
Table 3
Cytotoxicity (ED50
lg/mL) of compounds 1–4
It is noteworthy to mention that metabolites 1–4 are cembra-
noids possessing a tetrahydrofuran moiety with a rarely found
4,7-ether linkage, which has been discovered previously only in
the soft coral Eunicea mammosa.17 As the known compounds 6–8
have been found to exhibit cytotoxicity toward several cancer cell
lines,14,15 the cytotoxicity of compounds 1–4 against the prolifera-
Compound
MCF-7
WiDr
HEp 2
Daoy
1
2
3
4
4.2
3.2
2.0
4.1
0.14
4.2
3.2
1.2
1.8
0.15
6.2
4.5
2.6
4.0
0.07
8.8
5.6
3.2
5.4
0.14
Mitomycin C

1940
W.-Y. Lin et al. / Bioorg. Med. Chem. 18 (2010) 1936–1941
significantly reduce the levels of iNOS protein to 13.7 5.2%,
13.3 5.0%, 4.6 1.3%, 7.0 3.1%, 1.1 0.9% and 6.2 0.5%, respec-
tively, relative to control cells stimulated with LPS only. Further-
more, at the same concentration, metabolite 6 could strongly
reduce COX-2 expression (3.9 2.3%) with LPS treatment. Thus,
compounds 1–4, 6 and 8 might be useful anti-inflammatory agents,
while 6 is the most promising as it showed potent inhibitory activ-
ity against expression of both iNOS and COX-2 proteins (Fig. 5). On
the basis of the above results, we suggest that further investigation
of 6 in terms of its anti-inflammatory activities would be worth-
while for future drug development.
400 mesh) was used for column chromatography. Precoated silica
gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for ana-
lytical TLC. High-performance liquid chromatography was per-
formed on a Hitachi L-7100 HPLC apparatus with a Merck Hibar
Si-60 column (250 ꢁ 21 mm, 7
lm) and on a Hitachi L-2455 HPLC
apparatus with a Inertsil ODS-3 column (250 ꢁ 20 mm, 5
lm).
3.2. Organism
S. crassocaule (specimen no. 20070402) was collected by hand
using scuba off the coast of Dongsha, Taiwan, in April 2007, at a
depth of 5–10 m, and stored in a freezer until extraction. A voucher
sample was deposited at the Department of Marine Biotechnology
and Resources, National Sun Yat-sen University.
3. Experimental
3.1. General experimental procedures
3.3. Extraction and separation
Melting points were determined using a Fisher–Johns melting
point apparatus. Optical rotations were measured on a JASCO P-
1020 polarimeter. Ultraviolet spectra were recorded on a JASCO
V-650 spectrophotometer. IR spectra were recorded on a JASCO
FT/IR-4100 infrared spectrophotometer. The NMR spectra were re-
corded on a Varian 400MR FT NMR (or Varian Unity INOVA500 FT
NMR) instrument at 400 MHz (or 500 MHz) for 1H and 100 MHz (or
125 MHz) for 13C in CDCl3. LRMS and HRMS were obtained by ESI
on a Bruker APEX II mass spectrometer. Silica gel (Merck, 230-
The frozen bodies of S. crassocaule (0.5 kg, wet wt) were minced
and exhaustively extracted with EtOAc (1 L ꢁ 5). The EtOAc extract
(7.3 g) was chromatographed over silica gel by column chromatog-
raphy and eluted with EtOAc in n-hexane (0–100%, stepwise) then
with acetone in EtOAc (50–100%, stepwise) to yield 28 fractions.
Fraction 10 eluted with n-hexane–EtOAc (6:1) and was further
purified over silica gel using n-hexane–EtOAc (7:1) to afford six
subfractions (A1–A6). Subfraction A2 was separated by silica gel
using n-hexane–CH2Cl2 (8:1) to afford 6 (20.0 mg). Fraction 13
eluted with n-hexane–EtOAc (3:1) and was further purified over
silica gel using n-hexane–acetone (5:1) to afford six subfractions
(B1–B6) and 8 (60.0 mg). Subfraction B6 was separated by nor-
mal-phase HPLC using n-hexane–acetone (4:1) and further purified
by reverse-phase HPLC using MeOH–H2O (4:1) to afford 5 (1.0 mg).
Fraction 15 eluted with n-hexane–EtOAc (2:1) and was further
purified over silica gel using n-hexane–acetone (3:1) to afford se-
ven subfractions (C1–C7). Subfraction C3 was separated by nor-
mal-phase HPLC using n-hexane–acetone (7:2) and further
purified by reverse-phase HPLC using MeOH–H2O (2.3:1) to afford
3 (6.2 mg), 4 (5.7 mg) and 7 (1.3 mg). Fraction 18 eluted with n-
hexane–EtOAc (1:1) and was further purified over silica gel using
n-hexane–acetone (3:1) to afford seven subfractions (D1–D7). Sub-
fraction D3 was separated by reverse-phase HPLC using MeOH–
H2O (1.5:1) to afford 1 (10.2 mg) and 2 (3.0 mg).
β-actin
iNOS
A
100
80
60
*
40
*
*
20
0
*
*
*
*
6b
LPSa 1b 2b 3b 4b
7b 8b
3.3.1. Sarcocrassocolide A (1)
White solid; ½a 2D5
ꢂ
+17.7 (c 0.4, CHCl3); UV (MeOH) kmax 204
(log e = 3.5); IR (neat) mmax 3487, 2971, 2940, 1769, 1748, 1436,
β-actin
COX-2
B
1374, 1358,1275,1228 cmꢀ1; 1H NMR and 13C NMR data see Tables
1 and 2; ESIMS m/z 413 [M+Na]+; HRESIMS m/z 413.1938 [M+Na]+
(calcd for C20H30O6Na, 413.1940).
120
100
80
60
40
20
0
3.3.2. Sarcocrassocolide B (2)
White solid; ½a 2D5
ꢀ71.7 (c 0.3, CHCl3); UV (MeOH) kmax 204
ꢂ
(log e = 3.5); IR (neat) mmax 3481, 2965, 2951, 1755, 1456, 1444,
1386, 1370, 1346, 1270, 1227 cmꢀ1 1H NMR and 13C NMR data
;
see Tables 1 and 2; ESIMS m/z 413 [M+Na]+; HRESIMS m/z
413.1943 [M+Na]+ (calcd for C20H30O6Na, 413.1940).
*
6b
3.3.3. Sarcocrassocolide C (3)
White solid; ½a 2D5
ꢂ
+89.4 (c 0.6, CHCl3); UV (MeOH) kmax 207
(log e = 3.6); IR (neat) mmax 3472, 2969, 2930, 1760, 1442, 1405,
LPSa 1b 2b 3b 4b
7b 8b
1269, 1213 cmꢀ1 1H NMR and 13C NMR data see Tables 1 and 2;
;
Figure 5. Effect of compounds 1–4 and 6–8 on iNOS and COX-2 proteins expression
of RAW264.7 macrophage cells by immunoblot analysis. (A) Immunoblots of iNOS
and b-actin; (B) immunoblots of COX-2 and b-actin. The values are mean SEM
(n = 6). Relative intensity of the LPS alone stimulated group was taken as 100%.
Under the same experimental condition CAPE (caffeic acid phenylethyl ester,
ESIMS m/z 355 [M+Na]+; HRESIMS m/z 355.1883 [M+Na]+ (calcd
for C20H28O4Na, 355.1885).
3.3.4. Sarcocrassocolide D (4)
10
respectively. *Significantly different from LPS alone stimulated group (*P <0.05).
aStimulated with LPS, bstimulated with LPS in the presence of 1–4 and 6–8 (10
M).
lM) reduced the levels of the iNOS and COX-2 to 2.5 3.7% and 67.2 13.4%,
White solid; ½a 2D5
ꢂ
+17.2 (c 0.6, CHCl3); UV (MeOH) kmax 215
(log e = 3.8); IR (neat) mmax 3462, 2968, 2949, 1748, 1456, 1268,
l

W.-Y. Lin et al. / Bioorg. Med. Chem. 18 (2010) 1936–1941
1941
1215 cmꢀ1
;
1H NMR and 13C NMR data see Tables 1 and 2; ESIMS
and COX-2 (cyclooxygenase-2) proteins in macrophages cells using
western blotting analysis.20,21
m/z 355 [M+Na]+; HRESIMS m/z 355.1883 [M+Na]+ (calcd for
C20H28O4Na, 355.1885).
Acknowledgments
3.3.5. Sarcocrassocolide E (5)
Colorless oil; ½a D25
ꢀ47.5 (c 0.1, CHCl3); UV (MeOH) kmax 203
ꢂ
This work was supported by grants from the Ministry of Educa-
tion (97C031702) and National Science Council of Taiwan (NSC 98-
2113-M-110-002-MY3) awarded to J.-H. Sheu.
(log e = 3.5); IR (neat) mmax 2923, 2853, 1770, 1749, 1385, 1271,
1223 cmꢀ1 1H NMR and 13C NMR data see Tables 1 and 2; ESIMS
;
m/z 411 [M+Na]+; HRESIMS m/z 411.2150 [M+Na]+ (calcd for
C20H30O6Na, 411.2147).
Supplementary data
3.3.6. Preparation of (S)- and (R)-MTPA esters of 1
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.01.036.
To a solution of 1 (2.0 mg) in pyridine (100
lL) was added (R)-
(ꢀ)- -methoxy- -(trifluoromethyl)phenylacetyl chloride (10
a
a
l
L),
and the solution was then allowed to stand overnight at room tem-
perature. The reaction mixture was added to 1.0 mL of H2O, fol-
lowed by extraction with EtOAc (1.0 mL ꢁ 3). The EtOAc-soluble
layers were combined, dried over anhydrous MgSO4 and evapo-
rated. The residue was purified by a short silica gel column using
acetone–n-hexane (1:2) to yield the (S)-MTPA ester 1a (1.8 mg,
60%). The same procedure was applied to obtain the (R)-MTPA es-
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