Mar. Drugs 2014, 12, 840-850; doi:10.3390/md12020840
OPEN ACCESS
marine drugs
ISSN 1660-3397www.mdpi.com/journal/marinedrugs
Article
Bioactive Cembranoids, Sarcocrassocolides P–R,
from the Dongsha Atoll Soft Coral Sarcophyton
crassocaule
Wan-Yu Lin 1,†, Bo-Wei Chen 1,†, Chiung-Yao Huang 1, Zhi-Hong Wen 1, Ping-Jyun Sung 2,3, Jui-Hsin Su 2,3, Chang-Feng Dai 4 and Jyh-Horng Sheu 1,5,6,7,8,*
1 Department of Marine Biotechnology and Resources, National Sun Yat-sen University,
Kaohsiung 804, Taiwan; E-Mails: [email protected] (W.-Y.L.); [email protected] (B.-W.C.); [email protected] (C.-Y.H.); [email protected] (Z.-H.W.)
2 National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan; E-Mails:
[email protected] (P.-J.S.); [email protected] (J.-H.S.)
3 Graduate Institute of Marine Biotechnology and Department of Life Science and
Institute of Biotechnology, National Dong Hwa University, Pingtung 944, Taiwan
4 Institute of Oceanography, National Taiwan University, Taipei 112, Taiwan; E-Mail:
5 Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807,
Taiwan
6 Department of Medical Research, China Medical University Hospital, China Medical
University, Taichung 404, Taiwan
7 Asia Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804,
Taiwan
8 Frontier Center for Ocean Science and Technology, National Sun Yat-sen University,
Kaohsiung 804, Taiwan
† These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail:
[email protected]; Tel.: +886-7-5252000 (ext. 5030); Fax: +886-7-5255020.
Received: 12 November 2013; in revised form: 13 December 2013 / Accepted: 17 January 2014 / Published: 28 January 2014
Abstract: New cembranoids, sarcocrassocolides P–R (1–3) and four known compounds
(4–7) were isolated from the soft coral Sarcophyton crassocaule. The structures of the metabolites were determined by extensive spectroscopic analysis. Compounds 3–5 and 7 were shown to exhibit cytotoxicity toward a limited panel of cancer cell lines and all compounds 1–7 displayed potent in vitro anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells by inhibiting the expression of inducible
nitric oxide synthase (iNOS) protein. Compound 7 also showed significant activity in reducing the accumulation of cyclooxygenase-2 (COX-2) protein in the same macrophage cells.
Keywords: soft coral; Sarcophyton crassocaule; cytotoxic activity; anti-inflammatory
activity
1. Introduction
Marine terpenoids are of considerable interest due to their unique structures and wide range of biological activities [1]. The macrocyclic cembrane-derived compounds are known to be the major diterpenoidal metabolites in soft corals [2–9]. In previous studies of the secondary metabolites from soft corals of Taiwan waters, a series of bioactive cembranoids was discovered from soft corals belonging to the genera Sinularia [10–17],
Lobophytum [18–21], Sarcophyton [22–28] and Pachyclavularia [29]. Some of these
metabolites have been shown to exhibit cytotoxic activity against the growth of various cancer cell lines [11–13,15,17,19–28], and/or anti-inflammatory activity [10,11,14–19]. Our previous studies on the chemical constituents of a Dongsha Atoll soft coral S.
crassocaule have yielded 15 new cembranoids, sarcocrassocolides A–O, of which several
compounds were shown to exhibit significant cytotoxic and anti-inflammatory activities [30–32]. Our continuing chemical study on the same collection of this organism again led to the isolation of three new cembranoids, sarcocrassocolides P–R (1–3) (Chart 1 and Supplementary Figures S1–S9) along with four known compounds, crassocolides A, B, D, and E (4–7) [23] (Chart 1). The structures of 1–3 were established by extensive spectroscopic analysis, including careful examination of 2D NMR (1H–1H COSY, HSQC,
HMBC and NOESY) correlations. The cytotoxicity of compounds 1–7 against human colon adenocarcinoma (DLD-1), human T-cell acute lymphoblastic leukemia (CCRF-CEM), and human promyelocytic leukemia (HL-60) cell lines was studied, and the ability of 1–7 to inhibit the up-regulation of pro-inflammatory iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) proteins in LPS (lipopolysaccharide)-stimulated RAW264.7 macrophage cells was also examined. Compounds 1–7 were shown to exhibit cytotoxicity towards the above cancer cells, with 5 being the most cytotoxic.
Chart 1. Structures of new metabolites 1–3, and known compounds 4–7.
OAc 1：R1=OH R2=OAc 2：R1=OAc R2=OH HO 3 1 3 7 11 17 18 19 20 14 13 4R1 R2 O O O O O R2 4：R1=OH; R2=OAc 5：R1=OAc; R2=OAc 6：R1=OH; R2=H R1 O O HO O O O 7 Mar. Drugs 2014, 12 842
2. Results and Discussion
The HRESIMS spectrum of sarcrocrassocolide P (1) established the molecular formula C24H34O7, appropriate for eight degrees of unsaturation, and the IR spectrum revealed the presence of a hydroxyl (3445 cm−1) and carbonyl (1767 cm−1) group. The 13C NMR and DEPT (Distortionless Enhancement by Polarization Transfer) (Table 1) spectroscopic data showed signals of five methyls (including two acetate methyls), five sp3 methylenes, one sp2 methylene, four sp3 methines (including three oxymethines), two sp2 methines, one sp3 and six sp2 quaternary carbons (including two ester carbonyls). The NMR signals (Table 1) at δC 170.1 (C), 140.5 (C), 120.9 (CH2), 79.1 (CH), and 38.5 (CH), and δH 6.24, 5.65 (each, 1H, d, J = 2.0 Hz), 5.28 (1H, brs), and 3.11 (1H, d, J = 9.5 Hz) showed the presence of an α-methylene-γ-lactonic group by comparing with the NMR data of known cembranoids with the same five-membered lactone ring [30–32]. Two
trisubstituted double bonds were also identified from NMR signals appearing at δC 135.8 (C), 125.7 (CH) and δH 5.08 (1H, t, J = 7.0 Hz), and at δC 130.3 (C), 127.3 (CH) and δH 5.32 (1H, dd, J = 10.0, 3.5 Hz), respectively. In the COSY spectrum, it was possible to identify three partial structures, which were assembled with the assistance of an HMBC experiment. Key HMBC correlations of H3-18 to C-3, C-4 and C-5; H3-19 to C-7, C-8 and C-9; H3-20 to C-11, C-12 and C-13 and H2-17 to C-1, C-15 and C-16 permitted the establishment of the carbon skeleton (Figure 1). Furthermore, the acetoxy group
positioned at C-13 was confirmed from the HMBC correlations of the methyl protons of an acetate (δH 1.99) to the ester carbonyl carbon at δC 169.3 and the oxymethine signal at 77.5 (C-13, CH). The downfield chemical shift for H3-18 (δ 1.44 s) and the 13C NMR signals at δC 89.9 (C) showed the presence of an acetate group at C-4. The geometries of trisubstituted double bonds at C-7/C-8 and C-11/C-12 are both E, as the chemical shifts for C-19 and C-20 were upfield shifted to 16.0 and 14.5 ppm. On the basis of the above analysis, the planar structure of 1 was established. The relative structure of 1 was
elucidated by the NOE correlations, as shown in Figure 2. The NOE interaction of H-1 (δ 3.11) with H-3 (δ 3.73) and H-11 (δ 5.32) revealed the β-orientation of H-1 and H-3 [23,30–32]. H-3 showed NOE correlation with H3-18 (δ 1.32, s), thus H3-18 should also be positioned on the β-face. The E geometry of the trisubstituted double bonds at C-7/C-8 and C-11/C-12 were confirmed from the NOE correlations of H3-19 (δ 1.67) with one proton of H2-6 (δ 2.26), and H3-20 with H-10. H-14 (δ 5.28) exhibited NOE correlations with both H-13 (δ 5.40) and H3-20, but not with H-1, indicating the α-orientation of both H-13 and H-14. These results, together with other detailed NOE correlations of 1 (Figure 2), unambiguously established the structure of sarcocrassocolide P, as shown in formula 1 (Chart 1). Therefore, the relative stereochemistry of compound 1 was determined. Table 1. NMR spectroscopic
data for Sarcrocrassocolides M–
O (1–3). Sarcrocrassocolide P (1)
position δC, mult. a δH (J in Hz) b δC, mult. a δH (J in Hz) b δC, mult. c δH (J in Hz) d
1 38.5, CH 3.11, brd (9.5) c 37.7, CH 3.06, brs 40.5, CH 3.02, d (11.0) 2 37.3, CH2 1.80, m 35.7, CH2 2.05, t (5.0) 39.3, CH2 2.14, m 1.32, ddd (14.5, 10.5, 9.5) 1.80, m 1.82, ddd (19.0, 5.5, 1.5) 3 73.1, CH 3.73, t (10.0) 75.8, CH 5.04, dd (6.5, 5.0) 71.6, CH 4.25, d (5.0) 4 89.9, C 74.7, C 150.5, C
