Oxygenated Cembranoids from a Formosan Soft Coral Sinularia gibberosa
Atallah F. Ahmed,†,‡Zhi-Hong Wen,†Jui-Hsin Su,†Ya-Ting Hsieh,†Yang-Chang Wu,§Wan-Ping Hu,|and Jyh-Horng Sheu*,†,⊥
Department of Marine Biotechnology and Resources, National Sun Yat-sen UniVersity, Kaohsiung 804, Taiwan, Republic of China, Department of Pharmacognosy, Faculty of Pharmacy, Mansoura UniVersity, Mansoura 35516, Egypt, Graduate Institute of Natural Products, Kaohsiung Medical UniVersity, Kaohsiung 807, Taiwan, Republic of China, Faculty of Biotechnology, Kaohsiung Medical UniVersity,
Kaohsiung 807, Taiwan, Republic of China, and Asia-Pacific Ocean Research Center, National Sun Yat-sen UniVersity, Kaohsiung 804, Taiwan, Republic of China
ReceiVed July 24, 2007
Chemical investigation of a Formosan soft coral, Sinularia gibberosa, has led to the isolation of eight oxygenated cembranoids, 1-8, including seven new compounds, gibberosenes A-G (2-8). None of these compounds were found to be cytotoxic toward a limited panel of cancer cell lines. Compound 1 significantly inhibited the accumulation of the pro-inflammatory COX-2 protein of the LPS-stimulated RAW264.7 macrophage cells.
Marine terpenoids are considered to be metabolites of great interest due to their unique structures and wide range of biological activities.1Cembranoids1–7and norcembranoids1,8–11were found
to comprise the main terpenoidal content in octocorals. Our previous chemical investigations on soft corals of the genus Sinularia have afforded several cembrane-,12norcembrane-,8–11and
xeniaphyllane-based diterpenoids.13,14Some of these metabolites exhibit cytotoxic
activity against the growth of various cancer cell lines.9–13We have
previously isolated three polyoxygenated sterols from a Formosan soft coral, Sinularia gibberosa, Tixier-Durivault (Alcyoniidea).15
Our further chemical examination of this soft coral has led to the isolation of seven new oxygenated cembranoids, gibberosenes A-G (2-8), along with a known metabolite, (+)-11,12-epoxysarcophytol A (1).16–18The structures of the new metabolites were determined
on the basis of extensive spectroscopic analysis, including 2D NMR (1H–1H COSY, HMQC, HMBC, and NOESY) spectroscopy. The
macrocyclic rings of all structures, except that of 4, were found to possess a conjugated diene. None of the metabolites (1–8) were found to be cytotoxic (IC50’s > 20µg/mL) to human tumor cell
lines, including liver (Hep G2), breast (MCF-7 and MDA-MB-23), and lung (A-549) carcinoma cells. At a concentration of 10
µM, only compound 1 demonstrated an ability to inhibit the
accumulation of the pro-inflammatory proteins inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysac-charide (LPS)-stimulated RAW264.7 macrophage cells.
Results and Discussion
The sliced bodies of the soft coral S. gibberosa were extracted exhaustively with EtOH, and then the concentrated EtOH extract was partitioned between CH2Cl2and H2O. The combined CH2Cl2
-soluble fraction was concentrated under reduced pressure, and the residue was repeatedly chromatographed to yield diterpenoids 1–8. All compounds were obtained as colorless oils. Compound 1 was found to be the known compound (+)-11,12-epoxysarcophytol A.16–18
The new metabolite gibberosene A (2) exhibited a pseudomo-lecular ion peak in the HRESIMS at m/z 383.2196 [M + Na]+, establishing the molecular formula C22H32O4and seven degrees of
unsaturation. An acetoxy group (IR 1748 cm-1;δH2.08, 3H, s;δC
170.5, qC and 20.8, CH3) and a trisubstituted epoxide (δH 3.08,
1H, dd, J ) 9.5, 4.0 Hz;δC60.0, qC and 57.6, CH) were revealed
in 2. Comparison of the NMR data (Tables 1 and 2) of 2 with those of 1 suggested that both compounds are related epoxycem-branoids. One proton (δ 5.91, s) attached to an olefinic carbon (δ
103.6, CH) was found to exhibit HMBC correlations with three sp2quaternary carbons (δ 159.8, 140.5, and 131.0), indicating the * To whom correspondence should be addressed. Tel: 886-7-5252000,
ext. 5030. Fax: 886-7-5255020. E-mail: [email protected]. †Department of Marine Biotechnology and Resources, National Sun Yat-sen University.
‡Mansoura University.
§Graduate Institute of Natural Products, Kaohsiung Medical University. |
Faculty of Biotechnology, Kaohsiung Medical University.
⊥Asia-Pacific Ocean Research Center, National Sun Yat-sen University.
10.1021/np070356p CCC: $40.75 2008 American Chemical Society and American Society of Pharmacognosy Published on Web 01/17/2008
presence of a trisubstituted furan ring in 2. The presence of a trisubstituted double bond was elucidated from the two sp2carbon
signals at δ 132.1 (qC) and 126.6 (CH) and the corresponding
proton signal atδ 4.79. Therefore, 2 was defined as a cembranoid
with an acetoxy group, an epoxide, a trisubstituted olefin, and a trisubstituted furan moiety. From the 1H-1H COSY spectrum
(Figure 1), a partial structure of a proton spin system extending from H-7 (δ 4.79, 1H, m) to H3-18 (δ 1.20, 3H, d, J ) 7.5 Hz)
through H-4 (δ 2.77, 1H, m) could be established, assigning a
secondary methyl group at C-4. Furthermore, it was also found that the chemical shifts of C-7 to C-12 were quite similar to those of 1, implying the location of an epoxide at C-11 and C-12 in 2. Moreover, the HMBC correlations found from the oxymethine proton atδ 5.91 (1H, s) to the ester carbonyl carbon (δ 170.5, qC),
epoxide carbons (δ 57.6, CH, C-11 and 60.0, qC, C-12), and one
of the oxygenated furanoid carbons (δ 140.5, qC), and from H3-18
to the other oxygenated furanoid carbon (δ 159.8, qC), revealed
the C-13 position of the acetoxy group and the presence of the 3,14-oxygen-bound furan. On the basis of the above findings and other detailed correlations in the COSY and HMBC spectra, the planar structure of 2 was established, as illustrated in Figure 1.
The stereochemistry of 2 was determined on the basis of NOE correlations and by comparison of NMR data with 1. The similarity in chemical shifts (Tables 1 and 2) of the epoxy carbons and protons (C-11, C-12, H-11, and H3-20) in 1 and 2, and the biosynthetic
relation of these two metabolites, suggested an R-orientation of the epoxide oxygen in 2. Thus, a β-orientation of H3-20 and an
R-orientation of H-11 were proposed. Due to the overlapping of H-13 and H-2 signals (δ 5.91, 2H, s) on measuring the1H NMR in
CDCl3, we also measured the NOESY spectrum of 2 in C6D6, which
showed signals of both H-13 and H-2 atδ 6.31 (s) and 5.78 (s),
respectively. Therefore, the NOE interaction (measured in C6D6)
Table 1. 1H NMR Data for 1 and Gibberosenes A-E (2-5)
H# 1a 2a 3b 4a 5a 2 5.99 d (10.5)c 5.91 s 6.02 d (11.0) 5.24 d (5.0) 6.08 d (16.5) 3 5.76 d (10.5) 5.81 d (11.0) 3.55 d (5.0) 5.80 d (15.5) 4 2.77 m 5R 2.18 2H, m 1.76 2H, m 2.21 m 2.01 m 1.82 2H, m 5β 2.05 m 1.49 m 6R 2.22 2H, m 2.36 m 2.25 m 2.18 m 2.21 2H, m 6β 1.76 m 2.12 m 1.99 m 7 5.11 br dd (5.5, 5.5) 4.79 m 5.21 dd (8.6, 5.2) 5.27 dd (7.0, 5.5) 5.05 m 9R 2.26 m 2.10 m 2.25 m 2.31 m 2.15 dd (10.0,7.5) 9β 2.09 m 1.89 ddd (14.0, 10.0, 3.5) 2.19 m 2.07 m 2.21 m 10R 1.86 dddd (13.5, 13.5, 7.5, 3.5) 2.17 dd (14.5, 4.0) 1.63 m 2.20 m 1.62 m 10β 1.50 dddd (13.5, 13.5, 6.0, 3.5) 1.36 m 1.52 m 1.39 m 1.91 m 11 3.19 dd (7.0, 7.0) 3.08 dd (9.5, 4.0) 3.01 dd (9.0, 4.2) 3.03 dd (11.0, 3.5) 2.63 d (9.5) 13R 1.98 dd (10.5, 5.0) 5.80 d (9.5) 13β 2.11 dd (10.5, 7.0) 5.91 s 5.29 dd (9.0, 4.2) 3.88 dd (11.0, 3.5) 14R 4.73 dd (7.0, 5.0) 2.70 dd (14.4, 4.2) 2.45 dd (14.0, 11.0) 5.06 d (9.0) 14β 1.95 dd (14.4, 9.0) 2.26 dd (14.0, 3.5)
15 2.67 septet (6.5) 2.92 septet (6.5) 2.45 septet (6.5) 2.37 septet (7.0) 2.42 septet (7.0) 16 1.07 3H, d (6.5) 1.15 3H, d (6.5) 1.03 3H, d (6.5) 1.07 3H, d (7.0) 1.04 3H, d (7.0) 17 1.09 3H, d (6.5) 1.19 3H, d (6.5) 1.05 3H, d (6.5) 1.11 3H, d (7.0) 1.01 3H, d (7.0) 18 1.74 3H, s 1.20 3H, d (7.5) 1.74 3H, s 1.21 3H, s 1.37 3H, s 19 1.59 3H, s 1.62 3H, s 1.48 3H, s 1.65 3H, s 1.59 3H, s 20 1.30 3H, s 1.32 3H, s 1.27 3H, s 1.34 3H, s 1.27 3H, s OAc 2.08 3H, s 2.10 3H, s 2.09 3H, s
aSpectra recorded at 500 MHz.bSpectra recorded at 300 MHz in CDCl
3at 25°C.cThe J values are in Hz in parentheses.
Table 2. 13C NMR Data for 1 and Gibberosenes A-D (2-5)
C# 1a 2b 3a 4b 5a 1 148.5 (qC)c 131.0 (qC) 142.3 (qC) 146.0 (qC) 149.1 (qC) 2 118.4 (CH) 103.6 (CH) 120.4 (CH) 123.1 (CH) 122.6 (CH) 3 119.6 (CH) 159.8 (qC) 121.8 (CH) 60.9 (CH) 141.6 (CH) 4 136.8 (qC) 34.7 (CH) 136.6 (qC) 61.3 (qC) 73.9 (qC) 5 38.4 (CH2) 35.5 (CH2) 39.5 (CH2) 37.2 (CH2) 42.3 (CH2) 6 25.0 (CH2) 26.6 (CH2) 25.9 (CH2) 22.6 (CH2) 23.8 (CH2) 7 127.0 (CH) 126.6 (CH) 126.6 (CH) 126.4 (CH) 130.4 (CH) 8 133.7 (qC) 132.1 (qC) 133.9 (qC) 134.4 (qC) 132.2 (qC) 9 36.5 (CH2) 36.7 (CH2) 36.7 (CH2) 36.7 (CH2) 37.2 (CH2) 10 24.2 (CH2) 25.9 (CH2) 24.4 (CH2) 23.8 (CH2) 24.3 (CH2) 11 58.7 (CH) 57.6 (CH) 57.7 (CH) 57.1 (CH) 60.7 (CH) 12 60.0 (qC) 60.0 (qC) 60.9 (qC) 63.8 (qC) 62.5 (qC) 13 42.2 (CH2) 67.8 (CH) 74.2 (CH) 68.6 (CH) 73.9 (CH) 14 65.8 (CH) 140.5 (qC) 30.4 (CH2) 34.7 (CH2) 117.9 (CH) 15 27.7 (CH) 24.4 (CH) 32.5 (CH) 32.6 (CH) 33.9 (CH) 16 24.3 (CH3) 23.9 (CH3) 23.1 (CH3) 22.8 (CH3) 22.2 (CH3) 17 23.9 (CH3) 23.9 (CH3) 22.2 (CH3) 21.6 (CH3) 21.8 (CH3) 18 17.3 (CH3) 21.3 (CH3) 16.7 (CH3) 18.2 (CH3) 28.2 (CH3) 19 15.1 (CH3) 14.8 (CH3) 14.9 (CH3) 14.7 (CH3) 15.1 (CH3) 20 19.5 (CH3) 15.6 (CH3) 16.0 (CH3) 14.7 (CH3) 15.3 (CH3) OAc 20.8 (CH3) 21.2 (CH3) 21.8 (CH3) 170.5 (qC) 170.7 (qC) 170.5 (qC)
aSpectra recorded at 75 MHz. bSpectra recorded at 125 MHz in
CDCl3at 25°C.cAttached protons were determined by DEPT experi-ments. The values are in ppm downfield from TMS.
displayed by the β-oriented methyl at C-12 (δ 1.28, s) with the
acetoxymethine proton H-13 reflected the R-orientation of the acetoxy group at C-13. Moreover, the consecutive NOE correlations (measured in CDCl3) of H-11 (δH3.08, 1H, dd, J ) 9.5, 4.0 Hz)
with H-10R (δ 2.17, dd, J ) 14.5, 4.0 Hz), H-10R with H-9R (δ
2.10, m) and not with H-9β (δ 1.89, ddd, J ) 14.0, 10.0, 3.5 Hz),
and H-9R with H3-19 (δ 1.62, s), H3-19 with H-6R (δ 2.36, m)
and the correlations (measured in C6D6) of H-4 (δ 2.55, ddq, J )
14.0, 7.0, 3.5 Hz) with H-5β (δ 1.59, m), H-5β with H-7 (δ 4.88,
dd, J ) 7.0, 7.0 Hz), H-7 with H-9β (δ 1.81, ddd, J ) 14.0, 11.0,
3.0 Hz), H-9β with H-10β (δ 1.32, m), and H-10β with H3-20 (δ
1.28, s) indicate the R-orientation of H3-18. The E geometry of the
trisubstituted C-7/C-8 double bond was also assigned from the NOE correlation (measured in C6D6) of H3-19 (δ 1.43, s) with H-6 (δ
2.27, ddd, J ) 14.0, 8.0, 8.0 Hz) and from the higher field chemical shift of C-19 (δ 14.8, in CDCl3). On the basis of the above findings
and other detailed NOE correlations (Figure 2), the structure of gibberosene A (2) was deduced as (4S*,11S*,12R*,13R*,7E)-13-acetoxy-3(14),11(12)-diepoxycembra-2,7,14-triene.
Gibberosene B (3) was found to have the molecular formula C22H34O3, as indicated from the HRESIMS (m/z 369.2405 [M +
Na]+) and NMR data (Tables 1 and 2). The IR (1740 cm-1), EIMS (m/z 286 [M – AcOH]+), and NMR data (δH2.10, 3H, s;δC170.7,
qC and 21.2, CH3) suggested the presence of an acetoxy group. A
tetrasubstituted diene (δH6.02 and 5.81, each 1H, d, J ) 11.0 Hz; δC142.3, qC, 136.6, qC, 121.8, CH, and 120.4, CH), a trisubstituted
double bond (δH5.21, 1H, dd, J ) 8.6, 5.2 Hz;δC133.9, qC and
126.6, CH), and a trisubstituted epoxide (δH3.01, 1H, dd, J ) 9.0,
4.2 Hz;δC60.9, qC and 57.7, CH) were also evident. The HMBC
correlations (Figure 1) between H3-20 (δ 1.27, s) and epoxy carbons
C-11, C-12, and the oxymethine carbon atδ 74.2 (C-13), and from
H-13 (δ 5.29, dd, J ) 9.0, 4.2 Hz) to a carbonyl carbon (δ 170.7),
confirmed the C-13 location of the acetoxy group. These findings and other detailed COSY and HMBC correlations observed for 3 established the gross structure of 3, as shown in Figure 1. The NOE correlations displayed by theβ-oriented methyl on the epoxide (H3
-20) with H-13 disclosed the R-orientation of the 13-OAc, as shown in Figure 2. Moreover, the strong NOE interactions exhibited by H-2 with both methyls at C-15 and C-4, and H3-19 with H-6 (Figure
2), revealed the E geometries of the double bonds at C-1/C-2, C-3/ C-4, and C-7/C-8. Thus, gibberosene B (3) was unambiguously identified as (11S*,12R*,13S*,1E,3E,7E)-13-acetoxy-11,12-ep-oxycembra-1,3,7-triene.
Gibberosene C (4) showed the pseudomolecular ion peak [M + Na]+ at m/z 343.2250 in the HRESIMS, corresponding to the molecular formula C20H32O3and five degrees of unsaturation. The
IR absorption at 3350 cm-1indicated the presence of a hydroxy group in 4. Comparison of the NMR data of 4 with those of 3 revealed the replacement of one double bond (δH5.81, 1H, d, J )
11.0 Hz; δC 136.6, qC and 121.8, CH) in 3 by a trisubstituted
epoxide moiety in 4 (δH3.55, 1H, d, J ) 5.0 Hz;δC61.3, qC and
60.9, CH). The proton of this epoxide was found to exhibit a1H-1H
COSY correlation with an olefinic proton (δ 5.24, d, J ) 5.0 Hz,
H-2), which in turn was found to be HMBC-correlated to the isopropyl methine carbon (δ 32.6, CH, C-15). Therefore, the second
epoxide of 4 should be positioned at C-3/C-4. Moreover, the HMBC correlation found from the epoxide-bound H3-20 to the oxymethine
carbon atδ 68.6 assigned the C-13 location of the hydroxy group.
These and other detailed COSY and HMBC correlations (Figure 1) were employed to establish the planar structure of 4. The E geometries of the two trisubstituted double bonds at C-1/C-2 and C-7/C-8 were indicated by the NOE interactions of H-2 with the methyls at C-15 and H3-19 with H2-6, respectively. The similar
splitting patterns and J values of H-13 in both 3 and 4, and the NOE interaction of H3-20 with H-13, assigned the R-orientation
of the 13-OH. Moreover, the epoxide at C-3/C-4 was established as a trans-oxacyclopropane from the strong NOE correlations displayed by H-2 with the methyls at C-4 and C-15, and those observed for H-3 with H-14R and H-7, but not with H3-18. These
results, together with other detailed analysis of NOE correlations of 4 (Figure 2), unambiguously established the structure of
gibberosene C (4) as (3S*,4S*,11S*,12S*,1E,7E)-3(4),11(12)-diepoxy-13-hydroxycembra-1,7-diene.
Gibberosene D (5) was found to have the molecular formula C22H34O4, as revealed from the HRESIMS (m/z 385.2353 [M +
Na]+) and NMR data (Tables 1 and 2), implying six degrees of unsaturation. The IR absorptions at 1734 and 3350 cm-1and the ion peaks at m/z 303 [M – AcOH + H]+and 285 [M – AcOH – H2O + H]+indicated the presence of one acetoxy and one hydroxy
group. Moreover, the NMR data (Tables 1 and 2) designated one trisubstituted epoxide (δH2.63, 1 H, d, J ) 9.5 Hz;δC62.5, qC
and 60.7, CH) and three olefins, including one trans 1,2-disubsti-tuted double bond (δH 6.08 and 5.80, each 1H, d, J ) 16.5 and
15.5 Hz;δC122.6, CH, 141.6, CH). As in the case of 3, similar
HMBC correlations from H3-20 (δ 1.27, 3H, s) to the oxymethine
carbon (δ 73.9, CH, C-13) and from H-13 (δ 5.80, 1H, d, J ) 9.5
Hz) to the carbonyl carbon (δ 170.5, qC) and C-1 (δ 149.1) assigned
the C-13 position of the acetoxy group. One olefinic double bond was found to be located at C-14/C-1 from the 1H-1H COSY
correlations between H-13 and the olefinic proton atδ 5.06 (1H,
d, J ) 9.0 Hz, H-14), while the other (a 1,2-disubstituted double bond) was found to be positioned at C-2/C-3, as H-3 (δ 5.80)
exhibited HMBC correlations with both C-1 and C-2 (δ 122.6, CH).
Thus, these two double bonds constitute a 1,3-butadiene moiety from C-3 (δ 141.6, CH) to C-2 and then to C-1 and C-14. The
HMBC correlations from methyl protons resonating atδ 1.37 (s)
to an oxygenated carbon C-4 (δ 73.9, qC) and C-3 revealed the
C-4 position of this methyl group. The epoxide should be located at C-11/C-12, as H3-20 showed HMBC correlations not only to
C-13 but also to the epoxy carbons C-11 and C-12 (Figure 1). Thus, the gross structure of 5 was established as 11,12-epoxy-4-hydroxy-cembra-2,7,14-triene.
The Z geometry of the trisubstituted double bond at C-14/C-1 in 5 was established from the NOE interactions of H-14 with the isopropyl proton H-15. As in the cases of 2-4, the NOE interaction observed for H-13 with H3-20 but not with the epoxide H-11
indicated the R-orientation of the C-13 acetoxy group, as shown in Figure 2. Moreover, the NOESY spectrum showed correlations between H-3 and H-7, H-2 and H3-18, H3-18 and H2-6, H2-6 and
H3-19, and H3-19 and H3-20, revealing the β-orientation of the
methyl substitution at C-4. The above observations established the structure of gibberosene D (5) as (4S*,11S*,12R*,13S*,2E,7E,14Z)-13-acetoxy-11,12-epoxy-4-hydroxycembra-2,7,14-triene.
The related metabolite gibberosene E (6) has the molecular formula C24H36O5, as indicated by the HRESIMS (m/z 427.2462,
[M + Na]+) and NMR data (Table 3). It was found that 6 differs from 1-5 by the presence of an additional acetoxy group (IR 1743 cm-1and ESIMS m/z 307 [M – 2 AcOH + Na]+) in 6. Moreover, the NMR data revealed the presence of four trisubstituted double bonds and the absence of an epoxide group. Again, two of these double bonds represented a conjugated diene moiety, as shown in the structure of 6 (δH6.21 and 6.07, each 1H, d, J ) 11.6 Hz;δC
139.2, qC, 137.8, qC, 124.2, CH, and 121.9, CH). The interpretation of the 1H-1H COSY and HMBC correlations (Figure 1) were
successively used to determine the positions of the hydroxy group, double bonds, and the acetoxy groups and resulted in the establish-ment of the planar structure of 6 as 13,14-diacetoxy-9-hydroxy-cembra-1,3,7,11-tetraene.
On the basis of the biogenetic relationship of 6 with 2–5, it was assumed that in 6 the acetoxy group at C-13 has the same orientation as those of 2–5, as shown in Figure 2. It was found that H-13 exhibited NOE correlations with H-15 and H3-20, but not with H-14,
which further correlated with H-11 but not with H3-20. Thus, both
H-13 and H3-20 should be positioned on theβ-face and H-14 should
be situated on the R-face. This was further supported by comparison of the splitting patterns of H-13 and H-14 of 6 (d, J ) 9.2 Hz) with those of the known cembranoid 9 (d, J ) 9.7 Hz, for both H-13 and H-14), isolated from the soft coral Cladiella kashmani (Alcyoniidea).19 Moreover, the NOE correlations (Figure 2)
observed from H3-20 to H-10β, Η-10β to H-9, and the olefinic
H-11 (δ 5.53, 1H, br dd, J ) 6.6, 6.6 Hz) to H-14 (δ 6.14, 1H, d, J ) 9.2 Hz) but not to H-9 indicated the R-orientation of the
hydroxy group at C-9 and the E-configuration of the double bond at C-11/C-12. Therefore, the structure of gibberosene E was unequivocally established as (9S*,13R*,14R*,1Z,3E,7E,11E)-13,14-diacetoxy-9-hydroxycembra-1,3,7,11-tetraene.
Gibberosene F (7) was found to have the molecular formula C24H36O5, as indicated by the HRESIMS (m/z 427.2463, [M +
Na]+), suggesting 7 to be an isomer of 6. The IR, UV, MS, and
Table 3. 1H and13C NMR Data for Gibberosenes E-G (6-8)
6 7 8 C/H 1Ha 13Cb 1Ha 13Cb 1Ha 13Cb 1 139.2 (qC) 139.2 (qC) 146.9 (qC) 2 6.21 d (11.6)c 124.1 (CH)d 6.21 s 124.2 (CH) 6.03 d (11.1) 118.9 (CH) 3 6.07 d (11.6) 121.9 (CH) 6.21 s 123.8 (CH) 5.81 d (11.1) 121.4 (CH) 4 137.8 (qC) 134.7 (qC) 135.0 (qC) 5 2.17 m 39.7 (CH2) 2.26 dd (11.7, 3.0) 49.2 (CH2) 2.09-2.16 2H, m 38.8 (CH2) 2.24 m 2.43 dd (11.7, 11.7) 6 2.22-2.26 2H, m 25.3 (CH2) 4.49 m 67.7 (CH) 2.15-2.22 2H, m 25.0 (CH2) 7 5.34 br dd (6.0, 6.0) 124.2 (CH) 5.12 d (8.5) 128.9 (CH) 5.00 br dd (5.0, 5.0) 127.1 (CH) 8 136.9 (qC) 137.4 (qC) 131.3 (qC) 9 4.17 br s 74.7 (CH) 2.16 m; 2.24 m 39.0 (CH2) 2.13 m; 2.46 br d (12.5) 47.9 (CH2) 10 2.39 m; 2.42 m 32.2 (CH2) 2.10 m; 2.16 m 24.8 (CH2) 4.54 ddd (12.5, 9.0, 3.7) 66.6 (CH) 11 5.53 br dd (6.6, 6.6) 124.2 (CH) 5.44 m 128.7 (CH) 5.15 d (9.0) 128.4 (CH) 12 133.4 (qC) 131.3 (qC) 140.7 (qC) 13 5.39 d (9.2) 77.3 (CH) 5.34 d (10.0) 76.0 (CH) 2.05 m; 2.23 m 40.0 (CH2) 14 6.14 d (9.2) 72.8 (CH) 6.16 d (10.0) 72.7 (CH) 2.23 m; 2.36 m 28.5 (CH2) 15 2.52 septet (6.6) 29.8 (CH) 2.53 septet (6.9) 28.8 (CH) 2.34 m 34.4 (CH) 16 1.04 3H, d (6.6) 25.3 (CH3) 1.01 3H, d (6.9) 25.8 (CH3) 1.05 3H, d (6.3) 22.6 (CH3) 17 1.05 3H, d (6.6) 23.7 (CH3) 1.07 3H, d (6.9) 23.3 (CH3) 1.07 3H, d (6.3) 21.9 (CH3) 18 1.75 3H, s 16.3 (CH3) 1.78 3H, s 17.2 (CH3) 1.74 3H, s 17.3 (CH3) 19 1.44 3H, s 13.4 (CH3) 1.41 3H, s 15.6 (CH3) 1.57 3H, s 16.8 (CH3) 20 1.65 3H, s 16.3 (CH3) 1.63 3H, s 17.5 (CH3) 1.69 3H, s 17.1 (CH3) OAc 2.01 3H, s 21.1 (CH3) 2.01 3H, s 21.1 (CH3) 169.9 (qC) 170.0 (qC) 2.05 3H, s 21.1 (CH3) 2.05 3H, s 21.2 (CH3) 170.5 (qC) 170.5 (qC) aSpectra recorded at 300 MHz in CDCl
3.bSpectra recorded at 75 MHz in CDCl3.cJ values (in Hz) parentheses.dAttached protons were deduced by DEPT experiments.
NMR (Table 3) spectra of 7 also resembled those of 6 and indicated the presence of two acetoxy groups, one hydroxy group, and four trisubstituted double bonds, including a conjugated diene. Owing to the complete overlap of the two olefinic proton signals (δ 6.21,
2H, s, in CDCl3, H-2 and H-3), the1H NMR spectrum of 7 was
also measured in C6D6, where H-2 and H-3 appeared as two
separated doublets (J ) 11.5 Hz) atδ 6.24 and 6.42, respectively.
The chemical shifts of CH2-5 were unveiled primarily by a HMBC
correlation of H3-18 to C-5 (measured in CDCl3). As C-5 (δ 49.2)
of 7 was found to be significantly downfield-shifted relative to that of 6, the hydroxy group should be located at C-6 (δ 67.7) of 7.
This was further supported by the 1H-1H COSY correlations
(measured in CDCl3, Figure 1) observed between the
hydroxyme-thine proton (δ 4.49, m, H-6) and both H2-5 (δ 2.43, dd, J ) 11.7,
11.7 Hz and 2.26, dd, J ) 11.7, 3.0 Hz) and H-7 (δ 5.12, d, J )
8.5 Hz). These findings and other detailed HMBC correlations (Figure 1) confirmed the planar structure of 7. The R configurations at both C-13 and C-14 in 7 might have been the same as those of
6, on the basis of the quite similar chemical shifts, splitting patterns,
and J values for H-13 and H-14 of both compounds. NOE interactions (measured in C6D6) between one of the C-5 methylene
protons (δ 2.14, dd, J ) 11.0, 11.0 Hz) and both H-3 and H-7,
H-7 and H-3, and H-3 and H-14 revealed that this C-5 proton should be assigned as H-5R. Furthermore, H-5β (δ 2.31, d, J ) 11.0 Hz)
showed NOE correlation with H-6, reflecting the R-orientation of the 6-OH. The above findings, together with other detailed correlations (measured in C6D6) in the NOESY spectrum of 7
(Figure 2), established the structure of gibberosene F (7) as (6R*,13R*,14R*,1Z,3E,7E,11E)-13,14-diacetoxy-6-hydroxycembra-1,3,7,11-tetraene.
Gibberosene G (8), [R]25
D) -159 (CHCl3), exhibited a
quasi-molecular ion peak at m/z 311.2348 [M + Na]+in the HRESIMS, appropriate for the molecular formula C20H32O. The oxygen atom
in the molecule was attributable to the presence of one hydroxy group (IR 3373 cm-1; EIMS m/z 270 [M – H2O]+). The
hy-droxymethine proton (δ 4.54, ddd, J ) 12.5, 9.0, 3.7 Hz) showed
COSY correlations with a set of methylene protons (δ 2.13, m and
2.46, br d, J ) 12.5 Hz, H2-9) and an olefinic proton (δ 5.15, d, J
) 9.0 Hz, H-11), while C-9 was HMBC correlated with H3-19 (δ
1.57, 3H, s). Thus, the hydroxy group in 8 should be located at C-10. The E-geometries of the four double bonds at C-1/C-2, C-3/ C-4, C-7/C-8, and C-11/C-12 were determined by the NOE interactions displayed by the methyl protons at C-15 with H-2, H-2 with H3-18, H2-6 with H3-19, and H3-20 with H-10, respectively.
The structure of gibberosene G (8) was established as (-)-(1E,3E,7E,11E)-10-hydroxycembra-1,3,7,11-tetraene.
There have been several reports concerning the chemical constituents of S. gibberosa,7,14,15,20–26and the diterpenoids
identi-fied have shown significant variation among different sampling times and/or locations. A specimen collected from Kenting, the same location where S. gibberosa was obtained for analysis in this study, contained cembranoids with molecular structures very similar to those in our present work.21The results suggest that the chemical
composition of this coral may be affected by geographic and/or time variations.
The cytotoxicity study of diterpenoids 1–8 against the growth of Hep G2, MCF-7, MDA-MB-23, and A-549 cancer cell lines showed that none of these compounds have cytotoxicity against cell growth (IC50’s > 20µg/mL). As 1 and 3 were obtained in
larger quantities than the other metabolites, the in Vitro anti-inflammatory effect of diterpenoids 1 and 3 was tested. In this assay, the accumulation of the pro-inflammatory iNOS and COX-2 proteins of LPS-stimulated RAW 264.7 macrophage cells was evaluated using the immunoblot analysis. It was found that compound 1 at 10µM reduced the levels of the iNOS and COX-2
proteins to 76.2 ( 6.7% and 54.0 ( 6.2%, respectively, relative to the control cells stimulated with LPS alone. However, the same
concentration of the related compound 3 did not produce any inhibition of LPS-induced iNOS and COX-2 expression (Figure 3). The toxicity of 1 and 3 to RAW264.7 cells was also assessed by trypan blue staining and expression of β-actin. The results
indicated that RAW264.7 cell survival andβ-actin expression were
not affected by the presence of 1 and 3 at 10µM.
Experimental Section
General Experimental Procedures. Optical rotations were
mea-sured on a Jasco DIP-1000 digital polarimeter. IR spectra were recorded on a Jasco FT-5300 infrared spectrophotometer. NMR spectra were recorded on a Bruker AVANCE-DPX 300 FT-NMR at 300 MHz for 1H and 75 MHz for13C or on a Varian Unity INOVA 500 FT-NMR at 500 MHz for1H and 125 MHz for13C, respectively, in CDCl
3or C6D6 using TMS as internal standard. Low-resolution MS data were obtained by EI on a VG Quattro GC/MS spectrometer or by ESI on a Bruker APEX II mass spectrometer. HRMS data were recorded by ESI FT-MS on a Bruker APEX II mass spectrometer. Si gel 60 (Merck, 230–400 mesh) and Sephadex LH-20 (Pharmacia) were used for open CC. Precoated Si plates (Merck, Kieselgel 60 F254, 0.2 mm) were used for analytical TLC analyses. Isolation by HPLC was performed by a Shimadzu SPD-10A instrument equipped with a normal-phase column (Hibar Lichrosorb Si-60, 7µm, 250 × 25 mm) or a reversed-phase
column (Hibar Purospher RP-18e, 5µm, 250× 10 mm).
Animal Material. The soft coral S. gibberosa was collected by
hand using scuba off the coast of Kenting, Taiwan, in June 2004, at
Figure 3. Effect of compounds 1 and 3 on iNOS and COX-2 protein
expression of RAW264.7 macrophage cells by immunoblot analysis. (A) Immunoblots of iNOS andβ-actin; (B) immunoblots of COX-2
andβ-actin; values are mean ( SEM (n ) 6). Relative intensity of
the LPS alone stimulated group was taken as 100%. *Significantly different from LPS alone stimulated group (*P < 0.05).aCells not stimulated, bstimulated with LPS, cstimulated with LPS in the presence of 3 (10µM),dstimulated with LPS in the presence of 1 (10µM).
depths of 15 to 20 m and stored in a freezer until extraction. A voucher sample (SC-61) was deposited at the Department of Marine Biotech-nology and Resources, National Sun Yat-sen University.
Extraction and Separation. The sliced bodies of the soft coral S. gibberosa (1.3 kg, wet wt) were exhaustively extracted with EtOH (1
L× 4). The organic layer was filtered and concentrated under vacuum, and the residue of aqueous suspension was partitioned between CH2Cl2 and H2O. The solvent-free CH2Cl2extract (10 g) was subjected to CC on Si gel and eluted with EtOAc in n-hexane (0–100%, gradient) to yield 23 fractions.1H NMR spectroscopy was employed to detect the terpenoid-rich fractions. Fraction 4, eluted with EtOAc–n-hexane (1:9), was further isolated over Si gel using EtOAc–n-hexane (1:20) followed by reversed-phase HPLC, using acetone–H2O (4:1) to yield
3 (5.5 mg). Fraction 6, eluted with EtOAc–n-hexane (1:4), was
rechromatographed over a Sephadex LH-20 column, using acetone as the mobile phase to afford 10 subfractions (F1–F10). Subfraction F7 was separated by normal-phase HPLC, using EtOAc–n-hexane (1:20) to afford 1 (9.7 mg) and a crude mixture of terpenoids. The latter was further isolated by reversed-phase HPLC, using MeOH–H2O (6:1), to afford 4 (1.4 mg) and 8 (2.7 mg), respectively. Fraction 8, eluted with EtOAc–n-hexane (1:2), was rechromatographed over Si gel using EtOAc–n-hexane (1:11) followed by normal-phase HPLC, using EtOAc–n-hexane (1:7), to afford 2 (1.5 mg). Fraction 9, eluted with EtOAc–n-hexane (1:1), was purified on a Sephadex LH-20 column, using acetone as the mobile phase, and then further purified by normal-phase HPLC, using EtOAc–n-hexane (1:5), to give 5 (2.0 mg). Fraction 10, eluted with EtOAc–n-hexane (1:1), was rechromatographed on a column of Sephadex LH-20 column, using acetone as the mobile phase, and then further separated by normal-phase HPLC, using EtOAc–n-hexane (1:3), to afford 6 (3.0 mg) and 7 (2.8 mg).
Gibberosene A (2): colorless oil; [R]25
D-52 (c 0.6, CHCl3); IR (neat)νmax2966, 2926, 2868, 1748, 1653, 1458, 1373, 1233 cm-1; UV
λmaxMeOH nm (log ) 225 (3.89);1H and13C NMR data (CDCl3), see Tables 1 and 2, respectively;1H NMR (C
6D6, 500 MHz)δ 6.31 (1H, s, H-13), 5.78 (1H, s, H-2), 4.88 (1H, dd, J ) 7.0, 7.0 Hz, H-7), 3.34 (1H, dd, J ) 9.0, 4.0 Hz, H-11), 2.88 (1H, septet, J ) 7.0 Hz, H-15), 2.55 (1H, ddq, J ) 14.0, 7.0, 3.5 Hz, H-4), 2.27 (1H, ddd, J ) 14.0, 8.0, 8.0 Hz, H-6R), 2.09 (1H, m, H-10R), 1.93 (1H, br d, J ) 14.0 Hz, H-9R), 1.81 (1H, ddd, J ) 14.0, 11.0, 3.0 Hz, H-9β), 1.75 (3H, s, OAc), 1.69 (2H, m, H-5R and H-6β), 1.59 (1H, m, H-5β), 1.43 (3H, s, H3-19), 1.32 (1H, m, H-10β), 1.28 (3H, s, H3-20), 1.11 (1H, d, J ) 7.0 Hz, H3-18), 1.10 (3H, d, J ) 7.0 Hz, H3-17), 1.09 (3H, d, J ) 7.0 Hz, H3-16); EIMS m/z 360 [8.6, (M)+], 318 (6.6), 300 [2.2, (M – AcOH)+], 179 (96.4); ESIMS m/z 399 [12, (M + K)+], 383 [100, (M + Na)+], 301 [61, (M – AcOH + H)+]; HRESIMS m/z 383.2196 (calcd for C22H32O4Na, 383.2198).
Gibberosene B (3): colorless oil; [R]25
D+50 (c 0.9, CHCl3); IR (neat)νmax2961, 2921, 2860, 1740, 1655, 1456, 1373, 1236 cm-1; UV
λmaxMeOH nm (log ) 241 (3.97);1H and13C NMR data (CDCl3), see Tables 1 and 2, respectively; EIMS m/z 346 [0.8, (M)+], 286 [2.5, (M – AcOH)+], 259 (8.2), 243 (7.3), 191 (35.3), 175 (51.0); ESIMS m/z 385 [25, (M + K)+], 369 [100, (M + Na)+]; HRESIMS m/z 369.2405 (calcd for C22H34O3Na, 369.2406).
Gibberosene C (4): colorless oil; [R]25
D-66 (c 0.6, CHCl3); IR (neat)νmax3350, 2960, 2920, 2858, 1458, 1387 cm-1;1H and13C NMR data (CDCl3), see Tables 1 and 2, respectively; ESIMS m/z 343 [100, (M + Na)+]; HRESIMS m/z 343.2250 (calcd for C20H32O3Na, 343.2249).
Gibberosene D (5): colorless oil; [R]25
D+49 (c 1.0, CHCl3); IR (neat)νmax3350, 2960, 2918, 2860, 1734, 1647, 1456, 1373, 1238 cm-1; UVλmaxMeOH nm (log ) 239 (3.50);1H and13C NMR data (CDCl3), see Tables 1 and 2, respectively; ESIMS m/z 401 [15, (M + K)+], 385 [100, (M + Na)+], 325 [7, (M – AcOH + Na)+], 303 [23, (M – AcOH + H)+
], 285 [23, (M – AcOH – H2O + H)+]; HRESIMS m/z 385.2353 (calcd for C22H34O4Na, 385.2355).
Gibberosene E (6): colorless oil; [R]25
D+79 (c 1.0, CHCl3); IR (neat)νmax3422, 2962, 2926, 2868, 1743, 1647, 1456, 1373, 1244 cm-1; UVλmaxMeOH nm (log ) 248 (4.02);1H and13C NMR data (CDCl3), see Table 3; ESIMS m/z 443 [84, (M + K)+], 427 [100, (M + Na)+], 307 [5, (M – 2 AcOH + Na)+]; HRESIMS m/z 427.2462 (calcd for C24H36O5Na, 427.2460).
Gibberosene F (7): colorless oil; [R]25
D+128 (c 1.0, CHCl3); IR (neat)νmax3422, 2959, 2926, 2857, 1743, 1649, 1456, 1373, 1244 cm-1; UVλmaxMeOH nm (log ) 253 (4.01);1H and13C NMR data (CDCl3), see Table 3;1H NMR (C 6D6, 300 MHz)δ 6.61 (1H, d, J ) 10.0 Hz, H-14), 6.42 (1H, d, J ) 11.5 Hz, H-3), 6.24 (1H, d, J ) 11.5 Hz, H-2), 5.75 (1H, d, J ) 10.0 Hz, H-13), 5.58 (1H, m, H-11), 4.78 (1H, d, J ) 8.1 Hz, H-7), 4.25 (1H, br dd, J ) 11.0, 8.1 Hz, H-6), 2.56 (1H, septet, J ) 6.6 Hz, H-15), 2.31 (1H, d, J ) 11.0 Hz, H-5β), 2.14 (1H, dd, J ) 11.0, 11.0 Hz, H-5R), 1.95 (1H, m, H-9), 1.93 (1H, m, H-10), 1.91 (2H, m, H-9, H-10), 1.79 (3H, s, OAc), 1.72 (3H, s, OAc), 1.69 (1H, d, J ) 7.0 Hz, H3-20), 1.52 (3H, s, H3-18), 1.15 (3H, s, H3-19), 1.09 (3H, d, J ) 6.6 Hz, H3-17), 0.99 (3H, d, J ) 6.6 Hz, H3-16); ESIMS m/z 427 [100, (M + Na)+], 285 [55, (M – 2 AcOH + H)+]; HRESIMS m/z 427.2463 (calcd for C24H36O5Na, 427.2460).
Gibberosene G (8): colorless oil; [R]25
D-159 (c 1.0, CHCl3); IR (neat)νmax3373, 2959, 2926, 2857, 1670, 1456, 1381, 1261 cm-1; UV
λmaxMeOH nm (log ) 250 (3.98);1H and13C NMR data (CDCl3), see Table 3; EIMS m/z 288 [9.9, (M)+], 270 (1.2, [M – H2O]+); ESIMS
m/z 311 [100, (M + Na)+]; HRESIMS m/z 311.2348 (calcd for C20H32ONa, 311.2350).
Cytotoxicity Testing. Cell lines were purchased from the American
type Culture Collection (ATCC). Cytotoxicity assays of compounds
1–8 were performed using the MTT
[3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method.27,28
In Vitro Anti-inflammatory Assay. The anti-inflammatory assay
was modified from Ho et al.29and Park et al.30Murine RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC, No TIB-71) and cultured in Dulbecco’s modified essential medium (DMEM) containing 10% heat-inactivated fetal bovine serum, at 37°C in a humidified 5% CO2-95% air incubator under standard conditions. Inflammation in macrophages was induced by incubating them for 16 h in a medium containing only LPS (0.01µg/mL; Sigma)
without the presence of test compounds. For the anti-inflammatory activity assay, compounds 1 and 3 were added to the cells 5 min before LPS challenge, respectively. Then, cells were washed with ice-cold PBS, lysed in ice cold lysis buffer, and then centrifuged at 20000g for 30 min at 4 °C. The supernatant was decanted from the pellet and retained for Western blot analysis. Protein concentrations were determined by the DC protein Assay kit (Bio-Rad) modified by the method of Lowry et al.31Samples containing equal quantities of proteins were subjected to SDS-polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes (PVDF; Immobilon-P, Millipore, 0.45µM pore
size). The resultant PVDF membranes were incubated with blocking solution and incubated for 180 min at room temperature with antibody against inducible nitric oxide synthase (iNOS; 1:1000 dilution; Trans-duction Laboratories) and cyclooxygenase-2 (COX-2; 1:1000 dilution; Cayman Chemical) protein. The blots were detected using ECL detection reagents (Perkin-Elmer, Western Blot Chemiluminescence Reagent Plus) according to the manufacturer’s instructions and finally exposed to X-ray film (Kodak X-OMAT LS, Kodak). The membranes were reprobed with a monoclonal mouse anti-β-actin antibody (1:2500,
Sigma) as the loading control. After X-ray film scanning, the integrated optical density of the bands was estimated (Image-Pro plus 4.5 software) and normalized to the background values. Relative variations between the bands of the drug-treated samples and the samples treated with LPS alone were calculated in the same image.
Acknowledgment. This work was supported by grants from the
Ministry of Education (95C030313) and National Science Council of Taiwan (NSC 95-2113-M-110-011-MY3) awarded to J.-H.S.
Supporting Information Available:1H and13C NMR spectra of
2–8 are available free of charge via the Internet at http://pubs.acs.org.
Table 4. Growth Inhibition (%) of Cancer Cells by Compounds 1–8 at 20µg/mL
Hep G2 MCF-7 MDA-MB-231 A-549
1 45.5 33.4 27.2 2 -a 41.3 3 24.0 21.5 4 5 6 31.3 21.0 25.8 7 8 23.3 doxorubicinb 66.9 51.6 66.6 73.2
aGrowth inhibition is less than 20%.bDoxorubicin used at 0.5 µg/
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