Bioactive cembranoids from the soft coral Sinularia crassa

Article abstract of DOI:10.3390/md9101955

Eight new cembranoids, crassarines A-H (1-8) were isolated from the Formosan soft coral Sinularia crassa. Compounds 1-3 represent the rare cembranoids with a 1,12-oxa-bridged tetrahydrofuran ring, while 4 and 5 are the firstly discovered 1,11-oxa-bridged tetrahydropyranocembranoids. The absolute configuration of 6 was determined using the Mosher's method. Compounds 6 and 8 were found to significantly inhibit the expression of both pro-inflammatory iNOS and COX-2 proteins at 10 μM, respectively, while compounds 4-8 were found to be non-cytotoxic toward the selected human liver cancer cells.

Full text of DOI:10.3390/md9101955

Mar. Drugs 2011, 9, 1955-1968; doi:10.3390/md9101955
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Bioactive Cembranoids from the Soft Coral Sinularia crassa
Chih-Hua Chao 1,2, Kuei-Ju Chou 1, Chiung-Yao Huang 1, Zhi-Hong Wen 1,3, Chi-Hsin Hsu 1,3
,
Yang-Chang Wu 4, Chang-Feng Dai 5 and Jyh-Horng Sheu 1,3,
*
1
Department of Marine Biotechnology and Resources, National Sun Yat-sen University,
Kaohsiung 804, Taiwan; E-Mails: chaochihhua@hotmail.com (C.-H.C.);
jzusmile@hotmail.com (K.-J.C.); betty8575@yahoo.com.tw (C.-Y.H.);
wzh@mail.nsysu.edu.tw (Z.-H.W.); hsuch@mail.nsysu.edu.tw (C.-H.H.)
Chinese Medicinal Research and Development Center, China Medical University and Hospital,
Taichung 404, Taiwan
2
3
4
Asian Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan
College of Chinese Medicine, China Medical University, Taichung 404, Taiwan;
E-Mail: yachwu@mail.cmu.edu.tw
5
Institute of Oceanography, National Taiwan University, Taipei, Taiwan;
E-Mail: corallab@ntu.edu.tw
* Author to whom correspondence should be addressed; E-Mail: sheu@mail.nsysu.edu.tw;
Tel.: +886-7-5252000 ext. 5030; Fax: +886-7-5255020.
Received: 18 August 2011; in revised form: 26 September 2011 / Accepted: 9 October 2011 /
Published: 17 October 2011
Abstract: Eight new cembranoids, crassarines A–H (1–8) were isolated from the Formosan
soft coral Sinularia crassa. Compounds 1–3 represent the rare cembranoids with a
1,12-oxa-bridged tetrahydrofuran ring, while 4 and 5 are the firstly discovered
1,11-oxa-bridged tetrahydropyranocembranoids. The absolute configuration of 6 was
determined using the Mosher’s method. Compounds 6 and 8 were found to significantly
inhibit the expression of both pro-inflammatory iNOS and COX-2 proteins at 10 µM,
respectively, while compounds 4–8 were found to be non-cytotoxic toward the selected
human liver cancer cells.
Keywords: Sinularia crassa; crassarines A–H; anti-inflammatory

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1. Introduction
Soft corals were proven to be a rich source of terpenoids [1]. We previously have isolated a series of
bioactive cembrane- [2–4] and norcembrane- [5–8] diterpenoids from the Formosan soft corals of the
genus Sinularia. Although this genus has been well studied regarding bioactive constituents, previous
investigations on an Indian soft coral Sinularia crassa (Tixier-Durivault, 1951) had resulted in the
isolation of only a sphingosine and a steroid possessing anti-inflammatory [9,10] and 5α-reductase
inhibitiory activities [11], respectively. This prompted us to investigate the bioactive compounds from
the Formosan soft coral S. crassa and the present study has led to the isolation of eight new cembranoids,
crassarines A–H (1–8, see Chart 1) from the ethanolic extract of this organism. The structures of these
compounds have been established by extensive spectroscopic analysis and chemical method. The
anti-inflammatory activity of 1–8 to inhibit up-regulation of the pro-inflammatory iNOS (inducible
nitric oxide synthase) and COX-2 (cyclooxygenase-2) proteins in LPS (lipopolysaccharide)-stimulated
RAW264.7 macrophage cells and the cytotoxicity of compounds 4–8 against a panel of cancer cell lines
including human liver carcinoma (HepG2 and HepG3), human breast carcinoma (MCF-7 and
MDA-MB-231), and human lung carcinoma (A-549) were evaluated in order to discover bioactive
natural products.
Chart 1. The structures of crassarines A–H (1–8).
2. Results and Discussion
The HRESIMS of crassarine A (1) exhibited a pseudomolecular ion peak at m/z 361.2353 [M + Na]+,
consistent with a molecular formula of C20H34O4, appropriate for four degrees of unsaturation. The IR
spectrum of 1 showed a broad absorption band at 3461 cm−1 and a strong absorption band at
1698 cm−1, implying the presence of hydroxy and carbonyl groups. The latter was identified as a
ketone functionality from the carbon resonance at δ 211.8 (Table 1). In addition, carbon resonances at
δ 133.3 (CH) and 134.3 (CH) were attributed to the presence of an 1,2-disubstituted double bond. The
above functionalities accounted for two of the four degrees of unsaturation, suggesting a bicyclic
structure in 1. By interpretation of 1H−1H COSY correlations, it was possible to establish three partial
structures from both H-7 and H3-19 to H-8, H-8 to H-11, H2-13 to H2-14, and both H3-16 and H3-17 to
H-15. Subsequently, these partial structures were connected by the HMBC correlations (Figure 1).
According to the downfield-shifted carbon chemical shifts at δ 88.1 (C-1, C), 75.0 (C-11, CH), and

Mar. Drugs 2011, 9
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85.7 (C-12, C) [12] as well as the HMBC correlations from H3-20 to C-11, C-12, and C-13 and H3-16
(or H3-17) to C-17 (or C-16), C-15, and C-1, an ether linkage between C-1 and C-12 forming a
tetrahydrofuran (THF) ring and a hydroxy group at C-11 were assigned for 1. The location of C-6
ketone was suggested from the carbon resonances of the adjacent methylenes at δ 53.3 (C-5) and 51.6
(C-7). This was further confirmed by the HMBC correlations from both H2-7 and H2-5 to C-6. In
addition, the HMBC correlations from H3-18 to C-3, C-4, and C-5 helped to locate the C-2/C-3 double
bond and a hydroxy group at quaternary C-4 (δ 71.4). Hence, the planar structure of 1, a cembranoid
possessing a 1,12-bridged tetrahydrofuran ring, was established as shown in Figure 1.
Table 1. 13C NMR spectroscopic data of compounds 1−8.
#
1 a
1 b
2 c
3 a
4 a
5 a
6 d
7 d
8 d
1
2
3
4
5
6
7
8
88.1
133.3
134.3
71.4
53.3
211.8
51.6
28.9
32.5
29.4
75.0
85.7
35.2
30.9
37.7
18.0
17.7
28.9
22.6
23.4
87.6
133.8
135.1
70.7
56.4
209.5
49.4
25.8
32.7
26.5
71.1
86.4
36.7
30.4
38.0
18.3
17.8
31.1
22.1
20.8
88.6
133.4
136.4
72.4
52.7
212.9
51.1
26.4
32.9
26.8
77.0
84.7
34.6
31.7
38.6
18.2
17.6
29.8
22.3
23.5
170.9
21.0
88.8
133.2
136.5
72.4
52.7
213.0
51.2
26.4
33.0
26.9
77.0
84.7
34.4
31.9
38.5
18.2
17.5
29.7
22.3
24.0
77.5
131.6
139.0
73.4
54.0
215.2
53.1
30.8
32.4
26.0
76.2
70.0
37.1
28.4
40.2
17.3
16.8
28.9
22.0
18.8
77.7
130.8
138.3
71.7
50.8
215.7
54.2
28.5
29.7
24.4
74.7
70.1
36.9
28.8
40.3
17.2
16.8
24.5
20.7
19.5
147.2
119.1
121.7
135.4
38.5
25.2
126.7
136.7
75.3
32.3
57.0
59.5
36.4
24.3
34.4
22.5
22.3
17.3
11.7
18.5
147.7
118.6
122.9
134.8
39.4
25.5
130.1
138.0
33.7
25.5
59.1
60.3
35.4
24.1
33.5
22.3
22.7
16.8
59.4
19.0
146.2
107.7
146.8
117.0
109.6
151.1
35.3
30.4
30.2
24.8
65.4
60.7
40.5
24.2
35.2
21.6
21.1
9.1
9
10
11
12
13
14
15
16
17
18
19
20
OAc
20.0
15.2
CHO
160.9
a Spectra were measured in CDCl3 (100 MHz); b Spectra were measured in pyridine-d5 (100 MHz);
c Spectra were measured in CDCl3 (125 MHz); d Spectra were measured in C6D6 (100 MHz).
The E geometry for the C-2/C-3 double bond was deduced from a 16.0 Hz coupling constant
(Table 1) between H-2 and H-3. The relative configuration of 1 was determined by the interpretation of
NOE correlations (Figure 2). The NOE correlations between H3-20/H3-16 (or H3-17), H-11/H-13a
(δH 2.61), H-11/H-8, and H3-20/H2-13 suggested the 1S*,8S*,11R*,12S* configuration as depicted in
Figure 2. In addition, the NOE correlations observed for H-2 with both H-15 and H3-18 and for H3-18
with H-3 suggested the 4S* configuration. In order to understand the orientation of 4-OH and 11-OH, the
pyridine-induced solvent shifts were measured [13,14]. The significant differences of chemical shifts
(Δδ = δ CDCl3 − δ C5D5N ) due to pyridine-induced deshielding effect of hydroxy group were observed

Mar. Drugs 2011, 9
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for H-7a (Δδ = −0.93 ppm), H3-20 (Δδ = −0.24 ppm), and H-13a (Δδ = −0.63 ppm) (Table 2), suggesting
that 4-OH is close to H-7a, and the 11-OH is not only close to H-13a but also gauche-oriented to H3-20,
as shown in Figure 2. To determine the absolute configuration, we applied the Mosher’s method
on 1. However, we were unable to prepare the corresponding Mosher esters of 1 by usual reaction
conditions [3,4]. This might be due to the steric hindrance of THF ring adjacent to C-11.
Figure 1. Selected 1H−1H COSY (▬) and HMBC (→) correlations of 1−8.
OH
OH
O
R2
O
O
O
5
O
R1
OH
O
O
OR
R = H
R = Ac
R = CHO
1
2
3
R1 = OH, R2 = H
R1 = H, R2 = OH
6
7
and
4
8
Figure 2. Selected NOE correlations for compounds 1, 4, 6, and 8.

Mar. Drugs 2011, 9
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Table 2. 1H NMR Spectroscopic Data of Compounds 1−3 and 8.
1, δH (J in Hz) a 1, δH (J in Hz) b 2, δH (J in Hz) c 3, δH (J in Hz) a 8, δH (J in Hz) d
#
2
3
5
5.73, s
5.73, s
6.28, d (16.0)
6.04, d (16.0)
a: 2.98, d (13.0)
b: 2.87, d (13.0)
a: 3.38, dd
(16.0, 4.0)
b: 2.04, dd
(16.0, 9.6)
5.75, s
5.75, s
5.74, s
5.74, s
5.95, s
a: 2.79, d (15.6)
b: 2.61, d (15.6)
a: 2.45, dd
(15.6, 8.4)
b: 2.23, dd
(15.6, 5.2)
2.02, m
a: 2.89, d (15.0) a: 2.89, d (15.0) 5.73,s
b: 2.48, d (15.0) b: 2.48, d (15.0)
a: 2.52, dd
(18.0, 8.5)
b: 2.16, dd
(18.0, 4.0)
2.29, m
a: 2.49, dd
(18.0, 8.5)
b: 2.18, dd
(18.0, 4.0)
2.29, m
7
a: 2.44, br d (12.4)
b: 2.02, m
8
9
2.41, m
1.96, m
1.46, m
1.30, m
1.37, m
1.38, m
1.30, m
0.97, m
0.99, m
0.93, m
10 a: 1.56, m
b: 1.25, m
a: 2.18, m
b: 1.63, m
a: 1.44, m
b: 1.38, m
a: 1.48, m
b: 1.37, m
a: 1.82, m
b: 1.20, m
2.36, dd
(10.0, 2.0)
11
3.24, br d (9.6)
3.76, d (10.4)
4.80, br d (10.5) 4.90, br d (8.4)
a: 2.61, ddd
(12.4, 8.4, 2.4)
b: 1.75, m
13 a: 1.98, m
b: 1.68, m
a: 1.80, m
b: 1.60, m
a: 1.98, m
a: 1.84, m
b: 1.64, m
a: 2.01, m
a: 2.40, m
b: 1.04, m
a: 3.55, dd
(12.4, 9.2)
b: 2.02, m
2.22, m
14 a: 1.96, m
a: 2.12, m
b: 1.89, m
15 1.76, m
b: 1.88, m
1.81, m
b: 1.87, m
1.75, m
b: 1.86, m
1.75, m
16 0.87, d (6.8)
17 0.86, d (6.8)
0.92, d (6.8)
0.92, d (6.8)
1.61, s
0.86, d (6.8)
0.84, d (6.8)
1.25, s
0.86, d (6.8)
0.84, d (6.8)
1.25, s
1.00, d (6.0)
1.04, d (6.0)
1.88, s
18
1.37, s
19 0.98, d (6.4)
0.94, d (6.8)
1.49, s
0.91, d (6.4)
1.15, s
0.92, d (6.8)
1.18, s
0.82, d (6.4)
1.23, s
20
1.25, s
OAc
CHO
4-OH
2.09, s
8.18,s
4.47, s
4.45, s
a Spectra were measured in CDCl3 (400 MHz); b Spectra were measured in pyridine-d5 (400 MHz);
c Spectra were measured in CDCl3 (500 MHz); d Spectra were measured in C6D6 (400 MHz).
HRESIMS analysis of crassarine B (2) provided a molecular formula of C22H36O5 ([M + Na]+
m/z 403.2463). The 1H and 13C NMR spectroscopic data of 2 were close to those of 1. A comparison of
NMR spectroscopic data of 2 with those of 1 indicated that 2 possesses an acetoxy group [δC 170.9 (C),
δC 21.0 (CH3); δH 2.09], which was suggested to be attached at C-11 due to the downfield-shifted proton
resonance at δH 4.08 (1H, br d, J = 10.5 Hz, H-11) in comparison with the relevant case of 11-OH
analogue 1 (δH 3.24, 1H, br d, J = 9.6 Hz, H-11). The structure elucidation of 2 was accomplished by an
extensive analysis of its 2D NMR correlations, which led to the establishment of its planar structure, as
shown in Figure 1. Except for the C-11 substituent and the THF ring in both compounds 1 and 2, the
differences were observed for the chemical shifts of protons and carbons around the C-4 asymmetric
center, in particular those of H3-18 (δH 1.37 and δC 28.9 for 1; δH 1.25 and δC 29.8 for 2). These

Mar. Drugs 2011, 9
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observations suggested that the configuration at C-4 in 2 should be opposite to that in 1. Moreover, 1 and
2 shared the same NOE correlations around asymmetric centers C-1, C-8, C-11, and C-12. To confirm
the above elucidation, 1 was acetylated to obtain 1a, which displayed different 1H NMR spectrum to that
of 2 (see Experimental). Consequently, 2 was determined to be the 4-epi-11-O-acetyl derivative of 1.
The 13C and 1H NMR spectral data of 3 are very similar to that of 2 (Tables 1 and 2); however, 1H NMR
spectrum of 3 showed a singlet at δ 8.18 which correlates with carbon signal at δ 160.9 in the HSQC
spectrum, indicating the presence of a formyloxy group at C-11 in 3. On the basis of the above data,
3 was identified as the 11-O-formyl derivative of 2. Literature review showed that this is the first
cembranoid with a formyloxy group.
Crassarine D (4) possesses the same molecular formula as that of 1. The 13C NMR data (Table 1) of
4 were mostly similar to those of 1, except for those of sp3 oxygenated carbons, suggesting that they
vary mainly in the heterocyclic ring. The upfield shift for H-11 from δ 3.24 (1H, br d, J = 9.6 Hz) in 1
to δ 3.02 (1H, d, J = 8.8 Hz) in 4 indicates that an ether linkage should be located between C-1 and
C-11 to form a tetrahydropyran (THP) ring. The HMBC correlation from H-11 to C-1 (δ 77.5, C)
confirmed the presence of this THP ring in 4, rather than the THF ring in 1. The detailed analysis of
the correlations observed in the COSY, HMBC, and HSQC spectra further assigned all the
spectroscopic data and established the planar structure of 4 (Figure 1). The E geometry of C-2/C-3
double bond was also deduced from the coupling constant (16.0 Hz) between H-2 and H-3. NOE
correlations between H3-20/H-14a, H3-17/H-14a, H3-20/H-13a, and H-11/H-13b suggested that H-11 is
an axial proton and oriented oppositely to H3-20. Both H-11 and H-8 were suggested to be positioned
on the same face based on the observation of NOE correlations between H-11/H-8, H-8/H-10a, and
H-10a/H-11. In addition, H-3 showed NOE correlations with both H3-18 and H-15 (Figure 2),
revealing that H3-18 should be pointed toward the same orientation as that of the isopropyl group.
Consequently, the 1S*,4R*,8S*,11S*,12R* configuration was suggested for 4. Crassarine E (5) has the
1
13
same molecular formula as that of 4. The H and C NMR spectroscopic data as well as the proton
coupling patterns of 5 are similar to those of 4. A comparison of NMR spectroscopic data of 5 with
those of 4 showed some differences in chemical shifts for protons and carbons neighboring C-4 and
C-8, suggesting that they are epimeric at either C-4 or C-8. The NOE correlation between H3-18 and
H-2 in 5, instead of H3-18 and H-3 in 4 (Figure 2) suggested that compound 5 is a 4-epimer of 4.
Crassarine F (6) was assigned a molecular formula of C20H32O2, according to the HRESIMS and
NMR spectroscopic data (Tables 1 and 3). The IR absorption band at 3300 cm−1 revealed the presence
of hydroxy group. A tetrasubstituted 1,3-butadiene [δH 6.06 (1H, d, J = 10.4 Hz) and 5.90 (1H, dd,
J = 10.4, 1.2 Hz); δC 147.2 (C), 135.4 (C), 121.7 (CH), and 119.1 (CH)], a trisubstituted double bond
[δH 5.50 (1H, dd, J = 7.2, 6.0 Hz); δC 136.7 (C), and 126.7 (CH)], and a trisubstituted epoxide [δH 2.87
(1H, dd, J = 7.6, 6.0 Hz); δC 59.5 (C) and 57.0 (CH)] were also evident. Above NMR signals suggested
6 to be the 1,3-diene cembranoid with an epoxy group [15]. The 11,12-epoxy group was assigned
by the HMBC correlations from H3-20 to C-11, C-12, and C-13 and H2-14 to C-1, C-2, and C-13
(Figure 1). The COSY cross peaks of H2-10/H-11 and H2-10/H-9 as well as the HMBC correlations
from H3-19 to C-7, C-8, and C-9 assigned the hydroxy group at C-9 (δC 75.3, CH). These findings and
the detailed COSY and HMBC correlations established the planar structure of 6, as shown in Figure 1.
The relative configuration of 6 was determined by the interpretation of NOESY spectrum. The crucial
NOE correlations (Figure 2) between H-2/H3-18, H-2/H-15, and H-9/H-7 indicated the E geometry for

Mar. Drugs 2011, 9
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all double bonds and suggested a s-trans geometry for the 1,3-diene. NOE correlations between
H-11/H-3, H-11/H-14a, and H-3/H-14a showed that these protons should be pointed toward the core of
14-membered ring. Furthermore, the absence of NOE correlation between H-11 and H3-20 and the
presence of correlation between H-9 and H3-20 suggested the 9S*,11S*,12S* configuration, as
depicted in Figure 2. The absolute configuration of 6 was determined by the application of Mosher’s
method [16,17]. The (S)- and (R)-MTPA esters of 6 (6a and 6b, respectively) were prepared using the
corresponding (R)- and (S)-MTPA chloride, respectively. The determination of chemical shift differences
for the protons neighboring C-9 led to the assignment of the 9S configuration in 6 (Figure 3). Thus, the
absolute configuration of 6 was determined as 9S, 11S, 12S.
Table 3. 1H NMR Spectroscopic Data of Compounds 4−7.
#
2
3
5
4 a, δH (J in Hz)
5.81, d (16.0)
5.89, d (16.0)
a: 2.80, d (16.0)
b: 2.72, d (16.0)
5 a, δH (J in Hz)
5.58, d (16.0)
6.07, d (16.0)
a: 3.01, d (16.6)
b: 2.41, d (16.6)
6 b, δH (J in Hz)
6.06, d (10.4)
5.90, dd (10.4, 1.2) 6.02, d (10.8)
2.04, m
7 b, δH (J in Hz)
6.08, d (10.8)
2.00, m
7
a: 2.39, dd (13.6, 11.2) a: 2.46, dd (11.6, 2.8) 2.10, m
b: 2.16, dd (13.6, 2.4) b: 2.07, dd (12.0, 11.6)
a: 2.13, m
b: 2.00, m
8
9
1.92, m
a: 1.32, m
b: 1.18, m
1.96, m
5.50, dd (7.2, 6.0)
4.00, dd (8.0, 3.2)
5.26, dd (9.2, 5.2)
a: 2.36, m
b: 2.29, m
a: 1.72, m
b: 1.64, m
3.00, dd (6.8, 5.2)
a: 1.91, m
b: 1.62, m
a: 2.40, m
b: 1.90, m
a: 1.56, m
b: 0.99, m
a: 1.57, m
b: 1.26, m
3.19, d (10.4)
a: 1.72, m
b: 1.51, m
a: 1.65, m
b: 1.59, m
1.80, m
10 a: 1.49, m
b: 1.19, m
11 3.02, d (8.8)
13 a: 1.74, m
b: 1.57, m
14 a: 1.68, m
b: 1.59, m
15 1.77, m
a: 1.99, m
b: 1.67, m
2.87, dd (7.6, 6.0)
a: 1.85, m
b: 1.52, m
a: 2.23, m
b: 1.92, m
2.16, m
2.21, m
16 0.78, d (6.8)
17 0.91, d (6.8)
#
0.80, d (7.0)
0.90, d (7.0)
5 a, δH (J in Hz)
1.38, s
0.99, d (6.8)
0.99, d (6.8)
6 b, δH (J in Hz)
1.65, s
1.00, d (6.8)
0.99, d (6.8)
7 b, δH (J in Hz)
1.63, s
4 a, δH (J in Hz)
18 1.37, s
19 0.98, d (6.4)
1.00, d (6.4)
1.40, s
3.93, d (12.0)
3.89, d (12.0)
1.15, s
20 1.11, s
1.15, s
1.12, s
a Spectra were measured in CDCl3 (400 MHz); b Spectra were measured in C6D6 (400 MHz).
The HRESIMS data of crassarine G (7) revealed a molecular formula of C20H32O2, the same as that
of 6. The IR spectrum of 7 disclosed the presence of hydroxy group (νmax 3434 cm−1). A comparison of
the NMR spectroscopic data of 7 (Tables 1 and 2) with those of 6 revealed that the hydroxy-containing
methine (C-9) in 6 was replaced by a sp3 methylene in 7. It was also found that resonances appropriate
1
for H3-19 in 6 were absent from the H and 13C NMR spectra of 7 and replaced by signals for a
hydroxymethyl group [δH 3.93 and 3.89 (each 1H, d, J = 12.0 Hz); δC 59.4 (CH2)]. Careful inspection
of the 2D NMR spectra of 7 confirmed the above elucidation.

Mar. Drugs 2011, 9
Figure 3. 1H NMR chemical shift differences of MTPA esters of 6.
1962
The HRESIMS and 13C NMR spectroscopic data of crassarine H (8) established a molecular
formula of C20H30O2 and six degrees of unsaturation. The 13C NMR spectrum showed the presence of a
trisubstituted double bond [δC 146.2 (C) and 107.7 (CH)] and a trisubstituted epoxide [δC 65.4 (CH)
and 60.7 (C)]. In addition, the carbon resonances at δC 9.1 (CH3, C-18), 151.1 (C, C-6), 146.8 (C, C-3),
109.6 (CH, C-5), and 117.0 (C, C-4) are attributed to the presence of a 2,5-dialkyl-3-methylfuran [18].
This furan moiety and the trisubstituted double bond were found to be conjugated according to the
downfield-shifted proton resonance of H-2 at δ 5.95 (1H, s) [18]. This was further confirmed by the
HMBC correlations from H-2 to C-1, C-3, C-14, and C-15, H3-18 to C-3, C-4, and C-5, and H-5 to C-3,
C-4, and C-6. The above data together with the detailed inspection of the COSY and HMBC correlations
of 8 established its planar structure (Figure 1). The relative configuration of 8 was determined mainly by
the assistance of the NOESY experiment. The key NOE correlations between H-2 and both H-15 and
H3-18 indicated an E geometry of C-1/C-2 double bond (Figure 2). The trans epoxy group was
deduced by the NOE correlations between H-11/H-13b and H3-20/H-13a. In addition, H-8 showed an
NOE correlation with H3-20, instead of H-11, suggesting the 8S*,11S*,12S* configuration for 8.
The anti-inflammatory activity of diterpenoids 1–8 against the accumulation of pro-inflammatory
iNOS and COX-2 proteins in RAW264.7 macrophage cells stimulated with LPS was evaluated using
immunoblot analysis. At a concentration of 10 µM (Figure 4), 8 was found to significantly reduce the
levels of iNOS protein (35.8 ± 10.7%), compared with the control cells stimulated with LPS only.
At the same concentration, 6 could reduce COX-2 expression (65.6 ± 6.2%) by LPS treatment.
Cytotoxicity of diterpenoids 4–8 against HepG2, HepG3, MCF-7, MDA-MB-231, and A-549 cancer
cell lines was also evaluated. The results showed that the tested compounds were found to be inactive
(IC50 > 20 μM) toward the above cancer cell lines after 72 h exposure.

Mar. Drugs 2011, 9
Figure 4. Effect of compounds 1–8 at 10 μM on the LPS-induced pro-inflammatory iNOS
1963
and on COX-2 protein expression of RAW264.7 macrophage cells by immunoblot analysis.
(A) Immunoblots for iNOS and β-actin, and relative density of iNOS; (B) Immunoblots for
COX-2 and β-actin, and relative density of COX-2. The values are means ± SEM (n = 6).
The relative intensity of the LPS alone stimulated group was taken as 100%. Under the same
experimental conditions, 10 μM CAPE (caffeic acid phenethyl ester; Sigma Chemical
Company, St. Louis, MO, USA) reduced the levels of the iNOS and COX-2 protein to
0.8 ± 4.5% and 75.6 ± 12.2%, respectively, relative to the control cells stimulated with LPS.
* Significantly different from lipopolysaccharide (LPS) alone stimulated group (P < 0.05).
(A)
3. Experimental Section
3.1. General Experimental Procedures
(B)
The melting point was determined using a Fisher-Johns melting point apparatus. Optical rotations
were determined with a JASCO P1020 digital polarimeter. IR spectrum was obtained on a JASCO
FT/IR-4100 spectrophotometer. The NMR spectra were recorded on a Bruker AVANCE 300 FT-NMR (or
Varian 400 MR NMR/Varian Unity INOVA 500 FT-NMR) instrument at 300 MHz (or 400/500 MHz) for
1H (referenced to TMS, δH 0.00 ppm, for both CDCl3 and C5D5N and 7.15 ppm for C6D6) and 75 MHz
(or 100/125 MHz) for 13C (referenced to δC 77.0 for CDCl3, to 128.0 ppm for C6D6, and to internal TMS
at δC 0.0 ppm for C5D5N). ESIMS were recorded by ESI FT-MS on a Bruker APEX II mass
spectrometer. Silica gel 60 (Merck, 230−400 mesh) and LiChroprep RP-18 (Merck, 40–63 μm) were
used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and
precoated RP-18 F254S plates (Merck, 1.05560) were used for TLC analyses. High-performance liquid
chromatography (HPLC) was performed on a Hitachi L-7100 pump equipped with a Hitachi L-7400 UV
detector at 210 nm and a semi-preparative reversed-phase column (Merck, Hibar Purospher RP-18e, 5 μm,
250 × 10 mm).

Mar. Drugs 2011, 9
1964
3.2. Animal Material
The soft coral Sinularia crassa was collected by hand using scuba off the coast of Sansiantai, Taitung
county, Taiwan, in July 2008, at a depth of 10 m, and was stored in a freezer (−20 °C). This soft coral
was identified by one of the authors (C.-F.D.). A voucher specimen (Specimen No. SST-03) was
deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.
3.3. Extraction and Isolation
The frozen bodies of S. crassa (1.1 kg fresh wt) were minced and extracted with EtOH (3 × 2 L, each
for 1 day) at room temperature. The organic extract was concentrated to an aqueous suspension and was
further partitioned between EtOAc and H2O. The EtOAc extract (17.0 g) was fractionated by open
column chromatography on silica gel using n-hexane-EtOAc and EtOAc-MeOH mixtures of increasing
polarity to yield 32 fractions. Fraction 19, eluting with n-hexane–EtOAc (5:1), was further separated by
silica gel column chromatography with gradient elution (n-hexane-EtOAc, 24:1 to 0:1) and followed
by RP-18 open column (MeOH-H2O, 50% to 100%) to yield three subfractions (19A–19C).
Subfraction 19A was subjected to RP-18 HPLC (MeOH-H2O, 90%) to obtain compound 8 (2.2 mg).
Similarly, compounds 2 (1.1 mg) and 3 (1.0 mg) were obtained from subfraction 19C using RP-18
HPLC (MeOH-H2O, 75%). Subfraction 19B was fractionated over silica gel using gradient elution
(n-hexane-EtOAc, 24:1 to 0:1) to yield three subfractions (19B-1–19B-3). Compounds 4 (3.4 mg) and
5 (2.3 mg) were obtained from subfractions 19B-1 and 19B-2, respectively, using RP-18 HPLC
(MeOH-H2O, 66%). Subfraction 19B-3 was subjected to normal phase HPLC (n-hexane-EtOAc, 2:1)
to obtain 1 (2.3 mg). Fractions 22 to 24, eluting with n-hexane–EtOAc (1:1), were combined and
further separated over silica gel column chromatography (n-hexane–EtOAc, gradient elution, 18:1 to 0:1)
to give a residue containing terpenoids. This residue was separated over RP-18 column
chromatography using gradient elution (MeOH-H2O, 50% to 100%) to obtain two subfractions (23A
and 23B). Subfraction 23A was further purified by RP-18 HPLC (MeOH-H2O, 75%) to yield
compound 6 (1.8 mg). In the same manner, compound 7 (8.7 mg) was obtained from subfraction 23B
using RP-18 HPLC (MeOH-H2O, 80%).
Crassarine A (1): colorless oil; [α]24 −93(c 0.20, CHCl3); IR (KBr) νmax 3461, 2963, 2928, 2873,
D
1698, 1455, 1380 cm−1; 1H NMR and 13C NMR data, see Tables 1 and 2; ESIMS m/z 361 [M + Na]+;
HRESIMS m/z 361.2353 [M + Na]+ (calcd for C20H34O4Na, 361.2355).
Crassarine B (2): colorless oil; [α]24 −13 (c 0.11, CHCl3); IR (KBr) νmax 3288, 2957, 2925, 2855,
D
1
13
1732, 1698, 1453, 1372, 1237 cm−1; H NMR and C NMR data, Tables 1 and 2; ESIMS m/z 403
[M + Na]+; HRESIMS m/z 403.2463 [M + Na]+ (calcd for C22H36O5Na, 403.2460).
Crassarine C (3): colorless oil; [α]24 −45 (c 0.10, CHCl3); IR (KBr) νmax 3483, 2955, 2925, 2855,
D
1
13
1725, 1698, 1455, 1375, 1171 cm−1; H NMR and C NMR data, Tables 1 and 2; ESIMS m/z 389
[M + Na]+; HRESIMS m/z 389.2302 [M + Na]+ (calcd for C21H34O5Na, 389.2304).
Crassarine D (4): colorless oil; [α]24 −48 (c 0.34, CHCl3); IR (KBr) νmax 3386, 2955, 2925, 2855,
D
1716, 1458, 1268, 1036 cm−1; 1H NMR and 13C NMR data, Tables 1 and 3; ESIMS m/z 361 [M + Na]+;
HRESIMS m/z 361.2354 [M + Na]+ (calcd for C20H34O4Na, 361.2355).

Mar. Drugs 2011, 9
1965
Crassarine E (5): colorless oil; [α]24 −27 (c 0.23, CHCl3); IR (KBr) νmax 3453, 2957, 2925, 2855,
D
1713, 1458, 1261, 1044 cm−1; 1H NMR and 13C NMR data, Tables 1 and 3; ESIMS m/z 361 [M + Na]+;
HRESIMS m/z 361.2357 [M + Na]+ (calcd for C20H34O4Na, 361.2355).
Crassarine F (6): colorless oil; [α]24 −63 (c 0.18, CHCl3); IR (KBr) νmax 3300, 2960, 2926, 2857,
D
1
13
1668, 1458, 1380, 1255, 1036 cm−1; H NMR and C NMR data, Tables 1 and 3; ESIMS m/z 327
[M + Na]+; HRESIMS m/z 327.2302 [M + Na]+ (calcd for C20H32O2Na, 327.2300).
Crassarine G (7): colorless oil; [α]24 −41 (c 0.73, CHCl3); IR (KBr) νmax 3434, 2959, 2928, 2872,
D
1671, 1459, 1383, 1011 cm−1; 1H NMR and 13C NMR data, Tables 1 and 3; ESIMS m/z 327 [M + Na]+;
HRESIMS m/z 327.2302 [M + Na]+ (calcd for C20H32O2Na, 327.2300).
Crassarine H (8): colorless oil; [α]24 −12 (c 0.22, CHCl3); IR (KBr) νmax 2955, 2922, 2855, 1458,
D
1
1380 cm−1; H NMR and 13C NMR data, Tables 1 and 2; ESIMS m/z 325 [M + Na]+; HRESIMS
m/z 325.2145 [M + Na]+ (calcd for C20H30O2Na, 325.2143).
3.4. Acetylation of 1
To a stirring solution of compound 1 (0.1 mg) in pyridine (1 mL) was successively added excess
acetic acid anhydrous (0.2 mL). After the mixture was stirred over night at room temperature, H2O
(0.3 mL) was added, and this mixture was subsequently extracted with EtOAc (5 × 6 mL). The combined
EtOAc extract was successively washed with saturated aqueous NaHCO3 and brine. The organic layer
was dried over anhydrous Na2SO4 and concentrated to give a residue, which was chromatographed on
silica gel with n-hexane-EtOAc (2:1) as eluent to afford 1a (0.1 mg) which showed a [M + Na]+ peak at
1
m/z 403 in ESIMS spectrum. Selected H NMR (CDCl3, 300 MHz) spectrum of 1a: δ 5.89 (1H, d,
J = 15.9 Hz, H-2 or H-3), 5.77 (1H, d, J = 15.9 Hz, H-2 or H-3), 4.83 (1H, br d, J = 9.9 Hz, H-11), 2.95
(1H, d, J = 15.0 Hz, H-5a), 2.46‒2.56 (2H, m, H-5b, H-7a), 2.08 (3H, s, OCOCH3), 1.37 (3H, s, H3-18),
1.20 (3H, s, H3-18), 0.85‒0.89 (9H, overlapped, H3-19, H3-16, and H3-17).
3.5. Preparation of (S)- and (R)-MTPA Esters of 6
To a solution of 6 (0.5 mg) in pyridine (0.4 mL) was added (R)-MTPA chloride (25 μL), and the
mixture was allowed to stand for 3 h at room temperature. The reaction was quenched by the addition of
1.0 mL of H2O, and the mixture was subsequently extracted with EtOAc (3 × 1.0 mL). The EtOAc layers
were combined, dried over anhydrous MgSO4, and evaporated. The residue was subjected to short silica
gel column chromatography using n-hexane-EtOAc (8:1) to yield the (S)-MTPA ester, 6a (0.3 mg). The
same procedure was used to prepare the (R)-MTPA ester, 6b (0.4 mg from 0.5 mg of 1), with (S)-MTPA
chloride. Selected 1H NMR (CDCl3, 300 MHz) of 6a: δ 7.38−7.50 (5H, m, Ph), 6.14 (1H, d, J = 11.4 Hz,
H-2), 6.00 (1H, d, J = 11.4 Hz, H-3), 5.61–5.71 (2H, overlapped, H-7 and H-9 ), 3.69 (1H, d, J = 12.0 Hz,
H-11), 3.56 (3H, s, OMe), 1.80 (3H, s, H3-18), 1.39 (3H, s, H3-19), 1.10 (3H, s, H3-20), 1.07 (3H, d,
1
J = 6.9 Hz, H3-16 or H3-17), 1.03 (3H, d, J = 6.9 Hz, H3-16 or H3-17); selected H NMR (CDCl3,
300 MHz) of 6b: δ 7.38−7.50 (5H, m, Ph), 6.13 (1H, d, J = 11.4 Hz, H-2), 5.98 (1H, d, J = 11.4 Hz, H-3),
5.67–5.78 (2H, overlapped, H-7 and H-9), 3.70 (1H, d, J = 10.2 Hz, H-11), 3.52 (3H, s, OMe) 1.78
(3H, s, H3-18), 1.22 (3H, s, H3-19), 1.13 (3H, s, H3-20), 1.12 (3H, d, J = 6.9 Hz, H3-16 or H3-17), 1.03
(3H, d, J = 6.7 Hz, H3-16 or H3-17).

Mar. Drugs 2011, 9
1966
3.6. Cytotoxicity Testing
Compounds were assayed for cytotoxicity against human liver carcinoma (HepG2 and HepG3),
human breast carcinoma (MCF-7 and MDA-MB-231), and human lung carcinoma (A-549) cells using
the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method [19]. Freshly
trypsinized cell suspensions were seeded in 96-well microtiter plates at densities of 5000‒10,000 cells
per well with tested compounds added from DMSO-diluted stock. After 3 days in culture, attached cells
were incubated with MTT (0.5 mg/mL, 1 h) and subsequently dissolved in DMSO. The absorbency at
550 nm was then measured using a microplate reader. The IC50 is the concentration of agent that reduced
cell growth by 50% under the experimental conditions.
3.7. In Vitro Anti-Inflammatory Assay
Macrophage (RAW264.7) cell line was purchased from ATCC. In vitro anti-inflammatory activities
of tested compounds were measured by examining the inhibition of LPS induced upregulation of iNOS
and COX-2 proteins in macrophage cells using western blotting analysis [20,21].
4. Conclusions
Cembranoids with a 1,12-oxa-bridged THF ring, such as compounds 1–3, are rare in natural
products. Incensole [22], incensole oxide [23], and incensole acetate [24] are the cembranoids of this
class which were isolated from frankincense, the resin produced by the plant Boswellia carteri. It is
also noteworthy that the formyloxyl cembranoid, such as 3, and the 1,11-oxa-bridged
tetrahydropyranocembranoids, such as 4 and 5, were discovered for the first time.
Acknowledgments
This work was supported by grants from the National Science Council of Taiwan
(NSC98-2113-M-110-002-MY3) and Ministry of Education (98C031702) awarded to J.-H. S.
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Samples Availability: Not available.
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