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Mar. Drugs 2014, 12(5), 3060-3071; doi:10.3390/md12053060

Article
Klymollins T–X, Bioactive Eunicellin-Based Diterpenoids from the Soft Coral Klyxum molle
Fang-Yu Chang 1, Fang-Jung Hsu 1, Chi-Jen Tai 1, Wen-Chi Wei 2, Ning-Sun Yang 2,3,4,* 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: tako0225j@gmail.com (F.-Y.C.); fangjungh@gmail.com (F.-J.H.); jean801023@hotmail.com (C.-J.T.)
2
Agricultural Biotechnology Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 115, Taiwan; E-Mail: jackwei@gate.sinica.edu.tw
3
Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan
4
Department of Life Science, National Central University, Taoyuan 320, Taiwan
5
Division of Marine Biotechnology, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan
6
Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung 804, Taiwan
7
Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan
8
Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan
*
Authors to whom correspondence should be addressed; E-Mails: nsyang@gate.sinica.edu.tw (N.-S.Y.); sheu@mail.nsysu.edu.tw (J.-H.S.); Tel.: +886-2-2787-2067 (N.-S.Y.); +886-7-525-2000 ext 5030 (J.-H.S.); Fax: +886-2-2787-2066 (N.-S.Y.); +886-7-525-5020 (J.-H.S.).
Received: 4 January 2014; in revised form: 18 April 2014 / Accepted: 29 April 2014 /
Published: 22 May 2014

Abstract

: Five new eunicellin-based diterpenoids, klymollins T–X (15), along with two known compounds (6 and 7) have been isolated from the soft coral Klyxum molle. The structures of these new metabolites were elucidated by extensive spectroscopic analysis and by comparison with related known compounds. Compound 5 was found to exert significant in vitro anti-inflammatory activity against LPS-stimulated RAW264.7 macrophage cells. Furthermore, compounds 4 and 7 were shown to exhibit cytotoxicity against a limited panel of human cancer cell lines.
Keywords:
soft coral; Klyxum molle; eunicellin-based diterpenoids; klymollins; cytotoxic activity; anti-inflammatory activity

1. Introduction

Soft corals are known to be a rich source of terpenoidal metabolites [1]. Many studies about the discovery of versatile molecular structures and bioactivities of eunicellin-type compounds from soft corals have been reported recently [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18]. Our previous studies on the secondary metabolites of a Formosan soft coral Klyxum molle have resulted in the isolation of a series of new eunicellin-based diterpenoids, klymollins A–S [19,20]. In our continuing investigation effort to discover new metabolites from the soft coral K. molle, we have identified five new eunicellin-type metabolites, klymollins T–X (15) (Chart 1 and Supplementary Figures S1–S15), along with two known eunicellin-based diterpenoids, sclerophytin A (6) and sclerophytin B (7) [21] (Chart 1). The molecular structures of these compounds, including their relative configurations, were established by detailed spectroscopic analysis and by comparison with related physical and spectral data of known compounds. The ability of compounds 17 to inhibit IL-6 (interlukin-6) and TNF-α (tumor necrosis factor α) expression in LPS (lipopolysaccharide)-stimulated murine RAW264.7 macrophage cells and the cytotoxicity of 37 against five human cancer cell lines, human T cell lymphoblast-like cell line (CCRF-CEM), human erythromyeloblastoid leukemia (K562), human acute lymphoblastic leukemia cell line (Molt 4), human ductal breast epithelial tumor cell line (T47D) and human colorectal adenocarcinoma cell line (DLD-1) were evaluated.

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Chart 1. Structures of metabolites 17.

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Chart 1. Structures of metabolites 17.
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2. Results and Discussion

Klymollin T (1) was obtained as a colorless oil. The HRESIMS (m/z 545.2722 [M + Na]+) of 1 provided its molecular formula as C28H42O9, implying the presence of eight degrees of unsaturation. The IR spectrum of 1 revealed the presence of hydroxy and carbonyl groups from absorptions at 3481 and 1746 cm–1, respectively. The 13C NMR spectroscopic data of 1 exhibited 28 carbon signals (Table 1), which were assigned by the aid of DEPT spectrum to six methyls (including two acetate methyl at δC 21.2 and 20.8), seven methylenes (including one oxymethylene at δC 53.0), nine methines (including five oxymethines at δC 91.6, 80.6, 74.2, 73.0 and 72.9), and six quaternary carbons (including three sp2 oxygenated quaternary carbons at δC 172.0, 170.1 and 170.1, two sp3 oxygenated quaternary carbons at δC 84.9 and 55.7, and one sp2 quaternary carbon at δC 150.5). The NMR data of 1 in C6D6 (Table 1) showed the presence of one n-butyrate (δC 172.0, C; 37.8, CH2; 19.2, CH2; and 14.1, CH3; and δH 1.98, 2H, m; 1.53, 2H, m; and 0.83, 3H, t, J = 7.5 Hz), one 1,1-disubstituted double bond (δC 116.9, CH2 and 150.5, C; and δH 5.24, 1H, d, J = 2.0 Hz and 4.86, 1H, brs), one terminal epoxide (δC 53.0, CH2 and 55.7, C; δH 2.31, d and 1.99, d, each 1H, J = 5.0 Hz) and two acetate groups (δC 170.1, C; 170.1, C; 20.8, CH3 and 21.2, CH3; and δH 1.62, s, and 1.82, s, each 3H), respectively. Analysis of HMQC correlations showed that proton signals appearing at δH 2.33 (1H, m), 2.17 (1H, t, J = 8.5 Hz), 3.97 (1H, br s), and 5.00 (1H, d, J = 5.5 Hz) were correlated to two ring juncture methine carbons at δC 42.4 and 43.5 and two oxymethine carbons at δC 91.6 and 80.6, respectively. Therefore, the remaining three degrees of unsaturation identified 1 as a tricyclic diterpenoid. In addition, the COSY correlations of 1 assigned three isolated consecutive proton spin systems (Figure 1). The molecular framework of 1 was further established by HMBC correlations (Figure 1). Furthermore, H-12 (δ 5.24) and an acetate methyl (δ 1.62) exhibited HMBC correlations to the acetate carbonyl carbon (δ 170.1), and H-13 exhibited HMBC correlation to the n-butyrate carbonyl carbon (δ 172.0), revealing the location of an acetate at C-12 and an n-butyrate at C-13. The location of an acetate group at C-3 was then deduced by the chemical shifts of C-3 (δ 84.9) and H3-15 (δ 1.75). From the above results, the structure of compound 1 was shown to be related to that of the known compound, klymollin C [19]. Comparison of the NMR data of them revealed that the replacement of the acetoxy group at C-13 in klymollin C by an n-butyryloxy group in 1.

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Figure 1. Selected COSY (▬) and HMBC (→) correlations of 1, 3 and 4.

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Figure 1. Selected COSY (▬) and HMBC (→) correlations of 1, 3 and 4.
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The relative configuration of 1 was determined by comparison of the chemical shifts with klymollin C and was further confirmed by NOE correlations (Figure 2). The NOE correlations between H-12 and H-13, and between H-13 with H-1, H-10 and H-12 suggested that H-12 and H-13 were β-oriented and the relative configuration of 1 was proposed as 1R*, 2R*, 3R*, 6S*, 9R*, 10S*, 11S*, 12S*, 13S* and 14R*.

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Figure 2. Key NOESY Correlations for 1.

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Figure 2. Key NOESY Correlations for 1.
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The HRESIMS of klymollin U (2) exhibited a [M + Na]+ peak at m/z 545.2723 established the same molecular formula as that of 1. The 1H and 13C NMR data of 2 (Table 1) were similar to those of 1, revealed the presence of two acetoxy groups (δC 170.0, C; 169.4, C; 22.5, CH3 and 21.1, CH3; and δH 2.00, s, 3H and 1.92, s, 3H), one n-butyryloxy group (δC 172.6, C; 36.5, CH2; 18.8, CH2; 13.5, CH3; and δH 2.38, m, 2H; 1.70, m, 2H; 1.00, t, 3H, J = 7.6 Hz), one 1,1-disubstituted double bond (δC 116.8, CH2 and 150.0, C; and δH 5.36, brs and 4.98, brs, each 1H), one terminal epoxide (δC 53.6, CH2 and 55.2, C; δH 2.87, d and 2.64, d, each 1H, J = 4.8 Hz). The positions of one n-butyrate group and one acetate group at C-12 and C-13, respectively, was confirmed by the HMBC correlations of H-12 and the oxymethylene protons (δH 2.38) to the n-butyryloxy carbonyl carbon (δ 172.6), and H-13 and an acetate methyl (δH 2.00) to the acetate carbonyl carbon (δ 170.0). Thus, the remaining one acetate group had to be positioned at C-3, an oxygen-bearing quaternary carbon resonating at δ 84.6 ppm. A more detailed analysis of the 1H and 13C NMR spectroscopic data and correlations in the 1H–1H COSY and HMBC spectra led to the establishment of the gross structure of 2 (Figure 1). The stereochemistry of 2 was finally confirmed by comparison of its NMR data and NOE correlations with 1.

Table Table 1. 13C and 1H NMR data for compounds 13.

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Table 1. 13C and 1H NMR data for compounds 13.
1 a2 b3 b
δHδCδHδCδHδC
12.33 m42.4 (CH) c2.38 t (7.2) d41.8 (CH)2.20 m46.0 (CH)
23.97 brs e91.6 (CH)3.79 brs90.8 (CH)3.58 brs90.9 (CH)
3 84.9 (C) 84.6 (C) 86.7 (C)
41.49 m28.5 (CH2)1.53 m27.8 (CH2)2.10 m37.6 (CH2)
2.35 m 2.26 m 1.80 t (10.8)
51.62 m 34.4 (CH2)1.69 m34.3 (CH2)2.76 m32.8 (CH2)
2.30 m 2.20 m 1.92 t (12.0)
64.24 dd (9.5, 4.0) d73.0 (CH)4.29 d (8.0)72.5 (CH) 113.4 (C)
7 150.5 (C) 150.0 (C)2.16 m38.8 (CH)
82.57 d (13.5)41.4 (CH2)2.48 d (13.6)40.7 (CH2)2.41 ddd (16.0, 6.8, 3.2)36.0 (CH2)
2.71 dd (13.5, 4.0) 2.78 dd (13.6, 4.0) 1.68 m
95.00 d (5.5)80.6 (CH)4.90 m80.1 (CH)4.05 dt (8.8, 3.2)82.1 (CH)
102.17 t (8.5)43.5 (CH)2.26 t (10.4)42.9 (CH)3.92 t (8.0)46.6 (CH)
11 55.7 (C) 55.2 (C) 148.1 (C)
125.24 d (2.0)74.2 (CH)4.92 brs73.6 (CH)2.02 m31.2 (CH2)
2.20 m
135.11 dd (11.0, 2.0)72.9 (CH)4.91 d (10.4)72.2 (CH)0.99 m1.70 m24.9 (CH2)
142.11 m41.8 (CH)2.01 m40.7 (CH)1.26 m43.2 (CH)
151.75 s22.9 (CH3)1.65 s22.1 (CH3)1.31 s23.2 (CH3)
165.24 d (2.0)116.9 (CH2)4.98 brs116.8 (CH2)1.36 d (7.6)19.1 (CH3)
4.86 brs5.36 brs
171.99 d (5.0)53.0 (CH2)2.64 d (4.8)53.6 (CH2)4.77 brs109.8 (CH2)
2.31 d (5.0) 2.87 d (4.8) 4.70 d (1.2)
182.06 m28.3 (CH)2.03 m27.3 (CH)1.70 m29.1 (CH)
190.89 d (7.0)16.3 (CH3)0.84 d (7.2)15.3 (CH3)0.95 d (6.8)21.9 (CH3)
201.12 d (7.0)24.5 (CH3)1.09 d (7.2)24.0 (CH3)0.76 d (6.8)15.4 (CH3)
3-OAc 170.1 (C) 169.4 (C)
1.82 s21.2 (CH3)1.92 s22.5 (CH3)
6-OMe 3.26 brs48.5 (CH3)
12-OAc 170.1 (C)
1.62 s20.8 (CH3)
12-OCOPr 172.6 (C)
2.38 m36.5 (CH2)
1.70 m18.8 (CH2)
1.00 t (7.6)13.5 (CH3)
13-OAc 170.0 (C)
2.00 s21.1 (CH3)
13-OCOPr 172.0 (C)
1.98 m37.8 (CH2)
1.53 m19.2 (CH2)
0.83 t (7.5)14.1 (CH3)

a 13C and 1H spectra recorded at 125 and 500 MHz in C6D6; b 13C and 1H spectra recorded at 100 and 400 MHz in CDCl3; c Deduced from DEPT; d J values (Hz) in parentheses; e Broad signal.

Molecular formula C21H34O3 with five degrees of unsaturation was assigned to klymollin V (3) from its HRESIMS data (m/z 357.2405 [M + Na]+). The NMR spectroscopic data of 3 (Table 1) showed the presence of one 1,1-disubstituted double bond (δC 109.8, CH2 and 148.1, C; δH 4.77, brs and 4.70, d, J = 1.2 Hz, each 1H) and a methoxyl group (δH 3.26, 3H, brs). Analysis of HMQC, COSY and HMBC correlations (Figure 1) showed that proton signals appearing at δH 2.20 (1H, m), 3.92 (1H, t, J = 8.0 Hz), 3.58 (1H, brs), and 4.05 (1H, dt, J = 8.8 and 3.2 Hz) were correlated to two ring-juncture methine carbons at δC 46.0 and 46.6 and two oxymethine carbons at δC 90.9 and 82.1, respectively. Furthermore, one oxygenated quaternary carbon δC 86.7 (C-3) and one deoxygenated quaternary carbon δC 113.4 (C-6), implied that C-3 and C-6 were linked through an oxygen to form a tetrahydrofuran ring. The HMBC correlation of the methoxyl protons (δ 3.26) to C-6 (δ 113.4) suggested the substitution of a methoxyl group at C-6. Thus, the molecular framework of 3 was established. The relative stereochemistry of 3 was deduced by careful interpretation of the NOE correlations (Figure 3). The key NOE correlations of 3 showed interactions between H-1 and H-10, and H3-15; H-10 and H3-16 and H-17a (δ 4.77); and H3-15 and 6-OMe. Thus, all of H-1, H-10, H3-15, H3-16 and 6-OMe should be the β face. NOE correlations were also detected between H-14 and H-2, H-2 and H-4α (δ 2.10), revealing the α-orientation of both H-2 and H-14, as suggested by a molecular model of 3 (Figure 3). On the basis of the above findings, the structure of compound 3, including the relative stereochemistry, was unambiguously established.

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Figure 3. Key NOESY Correlations for 3 and 4.

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Figure 3. Key NOESY Correlations for 3 and 4.
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Klymollin W (4) showed the pseudomolecular ion peak [M + Na]+ at m/z 445.2563 in the HRESIMS and the molecular formula was determined as C24H38O6. NMR spectroscopic data of 4 (Table 2) showed the presence of two acetoxy groups (δC 171.9, 169.6, 22.4 and 21.4; δH 2.11, s and 2.08, s, each 3H). The NMR data of 4 was found to be similar to those of known compound 7 [15], the only difference is the replacement of the hydroxy group at C-3 in 7 by one acetoxy group in 4. The stereochemistry of compound 4 was also determined by the NOESY spectrum (Figure 3), which exhibited NOE correlations of H-10 with H-1 and H-8β (δ 2.04), H-8β with H3-16, establishing the β-orientation of H3-16. On the basis of these results and observed NOE correlations (Figure 3), the structure of metabolite 4 was determined.

Table Table 2. 13C and 1H NMR data for compounds 4 and 5.

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Table 2. 13C and 1H NMR data for compounds 4 and 5.
45
δHδCδHδC
12.18 m45.7 (CH) a2.19 m45.9 (CH)
23.63 brs b92.1 (CH)3.59 brs92.3 (CH)
3 86.7 (C) 86.4 (C)
42.04 m35.7 (CH2)1.94 m35.6 (CH2)
2.60 dd (15.6, 8.8) c 2.63 m
51.26 m29.2 (CH2)1.37 m27.4 (CH2)
1.59 m 1.95 m
65.63 d (6.0)84.7 (CH)5.22 d (6.0)83.4 (CH)
7 75.6 (C) 72.9 (C)
81.86 m45.9 (CH2)1.81 dd (14.0, 3.6)45.2 (CH2)
2.04 m 2.08 m
94.17 q (7.2)78.1 (CH)3.85 ddd (3.6, 7.6, 11.2)78.6 (CH)
102.98 t (7.2)53.8 (CH)3.06 t (7.6)53.8 (CH)
11 147.6 (C) 147.4 (C)
122.04 m31.5 (CH2)2.05 m31.6 (CH2)
2.28 m 2.31 m
131.02 m24.6 (CH2)1.02 m24.6 (CH2)
1.72 m 1.72 m
141.29 m43.9 (CH)1.23 m43.9 (CH)
151.39 s22.9 (CH3)1.38 s23.1 (CH3)
161.20 s23.7 (CH3)1.27 s25.4 (CH3)
174.62 brs109.5 (CH2)4.64 brs109.9 (CH2)
4.68 brs 4.71 brs
181.72 m29.0 (CH)1.74 m29.0 (CH)
190.97 d (7.2)21.9 (CH3)0.98 d (7.2)21.9 (CH3)
200.79 d (7.2)15.4 (CH3)0.79 d (7.2)15.4 (CH3)
3-OAc 169.6 (C) 169.6 (C)
2.11 s22.4 (CH3)2.13 s22.5 (CH3)
6-OAc 171.9 (C) 170.1 (C)
2.08 s21.4 (CH3)2.08 s21.3 (CH3)

13C and 1H spectra recorded at 100 and 400 MHz in CDCl3; a Deduced from DEPT; b Broad signal; c J values (Hz) in parentheses.

The HRESIMS of klymollin X (5) exhibited a [M + Na]+ ion peak at m/z 445.2563, which was consistent with the molecular formula of C24H38O6. Furthermore, it was found that the NMR data of 5 (Table 2) were very similar to those of 4, suggesting that 5 might be a regioisomer of 4. From NOESY spectrum, it was found that the α-oriented H-9 (δ3.85) showed NOE interaction with H-8α (δ 1.81), and the later exhibited further interaction with H3-16. This inferred the α-orientation of the methyl substituent at C-7. Further analysis of other NOE interactions revealed that 5 possessed the same relative configurations at C-1, C-2, C-3, C-9, C-10, C-12 and C-14 as those of 4. Therefore, compound 5 was found to be the C-7 epimer of 4.

Eunicellin-type diterpenoids isolated from Formosan soft corals was reported to have anti-inflammatory activities [22]. Therefore, the in vitro anti-inflammatory effects of compounds 17 were tested by examining the inhibitory activity of these compounds toward the LPS-induced up-regulation of pro-inflammatory proteins, IL-6 and TNF-α in murine RAW264.7 macrophage cells (Figure 4). At a concentration of 25 μM, compound 5 significantly reduce the level of IL-6, relative to the control cells stimulated with LPS only. However, these metabolites did not reduce the expression of TNF-α effectively. The cytotoxicity of the diterpenoids 3–7 against five human carcinoma cell lines, CCRF-CEM, K562, Molt 4, T47D and DLD-1 were also evaluated by the MTT assay. Cytotoxicity of 1 and 2 was not measured due to the paucity of these two compounds. Among the tested compounds, 7 showed stronger activity against the proliferation of four cancer cell lines (ED50 values of CCRF-CEM, K562, Molt 4 and T47D were 4.2, 15.0, 16.5 and 12.4 μg/mL), and 4 exhibited cytotoxicity toward CCRF-CEM, Molt 4 and T47D cancer cell lines with ED50 values of 9.6, 8.5 and 19.9 μg/mL, respectively. These results together with our previous findings [19,20], demonstrated that the soft coral K. molle is a good source of bioactive substances which deserve for further biomedical investigations.

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Figure 4. Effect of compounds 17 on LPS-induced IL-6 and TNF-α expression in RAW264.7 macrophage cells by ELISA analysis. The 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). a stimulated with LPS, b stimulated with LPS in the presence of 17 (25 μM).

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Figure 4. Effect of compounds 17 on LPS-induced IL-6 and TNF-α expression in RAW264.7 macrophage cells by ELISA analysis. The 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). a stimulated with LPS, b stimulated with LPS in the presence of 17 (25 μM).
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3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were measured on a JASCO P-1020 polarimeter. IR spectra were recorded on a JASCO FT/IR-4100 infrared spectrophotometer. ESIMS and HRESIMS were obtained with a Bruker APEX II mass spectrometer. NMR spectra were recorded in C6D6 or CDCl3, either on a Varian UNITY INOVA-500 FT-NMR, a Varian 400MR FT-NMR, or a Bruker AMX-300 FT-NMR. Silica gel (230–400 mesh, Merck, Darmstadt, Germany) was used for column chromatography. Precoated silica gel plates (Kieselgel 60 F-254, 0.2 mm, Merck, Darmstadt, Germany) were used for analytical TLC (Merck, Darmstadt, Germany). High-performance liquid chromatography (HPLC) was performed on a Hitachi L-2130 HPLC apparatus (Hitachi, Tokyo, Japan) with a Supelco C18 column (250 × 21.2 mm, 5 μm, Supelco, Bellefonte, USA) and a Hitachi L-2455 diode array detector (Hitachi, Tokyo, Japan).

3.2. Animal Material

The soft coral Klyxum molle was collected by hand using scuba along the coast of Peng-Hu Islands, Taiwan, in June 2008 at a depth of 10 m, and was stored in a freezer until extraction. A voucher sample (PI-20080610) was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

3.3. Extraction and Separation

The frozen bodies of K. molle (1.3 kg, wet weight) were sliced and exhaustively extracted with EtOAc (3 × 10 L). The organic extract was concentrated to an aqueous suspension and was partitioned between EtOAc and H2O. The EtOAc layer was dried with anhydrous Na2SO4. After removal of solvent in vacuo, the residue (22 g) was subjected to column chromatography on silica gel and eluted with EtOAc in n-hexane (0%–100% of EtOAc, gradient) and further with MeOH in EtOAc of increasing polarity to yield 31 fractions. Fraction 18, eluted with n-hexane–EtOAc (8:1), was further chromatographed over silica gel with a gradient elution using a minture of n-hexane–acetone (7:1) to afford three subfractions (F18B1–F18B3) and compound 7 (39.6 mg). Subfractions F18B1 was subjected to reversed-phase HPLC (CH3CN–H2O, 1.2:1 to 1.6:1) in order to purify compounds 3 (4.0 mg), 4 (7.4 mg), 5 (4.7 mg) and 6 (9.2 mg). Fraction 20, obtained from n-hexane–EtOAc (1:2), was further purified over silica gel using n-hexane–acetone (4:1) to afford four subfractions (F20B1–F20B4). Subfraction F20B4 was separated by reversed-phase HPLC (CH3CN–H2O, 1:1) to afford compounds 1 (1.5 mg) and 2 (1.7 mg).

Klymollin T (1): colorless oil; Marinedrugs 12 03060 i001 −67 (c 0.15, CHCl3); IR (neat) νmax 3481, 2956, 2922, 2874, 2852, 1746, 1456, 1372, 1240, 1098 and 1043 cm−1; 13C and 1H NMR data (500 MHz; C6D6), see Table 1; ESIMS m/z 545 [M + Na]+; HRESIMS m/z 545.2722 [M + Na]+ (calcd for C27H40O11Na, 525.2726).

Klymollin U (2): colorless oil; Marinedrugs 12 03060 i001 −57 (c 0.17, CHCl3); IR (neat) νmax 3481, 2958, 2927, 2875, 2854, 1735, 1456, 1370, 1243, 1098 and 1043 cm−1; 13C and 1H NMR data (400 MHz; CDCl3), see Table 1; ESIMS m/z 545 [M + Na]+; HRESIMS m/z 545.2723 [M + Na]+ (calcd for C27H40O11Na, 545.2726).

Klymollin V (3): colorless oil; Marinedrugs 12 03060 i001 −18 (c 1.14, CHCl3); IR (neat) νmax 2953, 2931, 2877, 1735, 1636, 1467, 1373, 1227, 1183, 1082 and 1038 cm–1; 13C and 1H NMR data (400 MHz; CDCl3), see Table 1; LRESIMS m/z 357 [M + Na]+; HRESIMS m/z 357.2405 [M + Na]+ (calcd for C21H34O3Na, 357.2406).

Klymollin W (4): colorless oil; Marinedrugs 12 03060 i001 +14 (c 2.11, CHCl3); IR (neat) νmax 3466, 2959, 2935, 2872, 1732, 1644, 1448, 1370, 1250, 1103, 1049 and 1023 cm–1; 13C and 1H NMR data (400 MHz; CDCl3), see Table 2; LRESIMS m/z 445 [M + Na]+; HRESIMS m/z 445.2563 [M + Na]+ (calcd for C24H38O6Na, 445.2566).

Klymollin X (5): colorless oil; Marinedrugs 12 03060 i001 +18 (c 1.34, CHCl3); IR (neat) νmax 3478, 2959, 2932, 2871, 1735, 1645, 1431, 1371, 1115 and 1021 cm–1; 13C and 1H NMR data (400 MHz; CDCl3), see Table 2; LRESIMS m/z 445 [M + Na]+; HRESIMS m/z 445.2568 [M + Na]+ (calcd for C24H38O6Na, 445.2566).

3.4. Cytotoxicity Testing

Cell lines were purchased from the American Type Culture Collection (ATCC). Cytotoxicity assays of 37 were performed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] colorimetric method [23,24].

3.5. In Vitro Anti-Inflammatory Assay

Mouse macrophage cell line, RAW264.7, was purchased from ATCC. In vitro anti-inflammatory activities of compounds 17 were measured by examining the inhibition of LPS induced upregulation of IL-6 and TNF-α in macrophages cells [25].

4. Conclusions

New eunicellin-based diterpenoids were isolated together with known compounds from the soft coral Klyxum molle. Compound 5 could significantly inhibit the release of IL-6 in LPS-induced mouse RAW264.7 macrophage cell line. Also, compounds 4 and 7 showed moderate to weak cytotoxicity.

Supplementary Files

  • Supplementary File 1:

    Supplementary Information (PDF, 1357 KB)

  • Acknowledgements

    This research was supported by grants from the National Science Council of Taiwan (NSC 102-2113-M-110-001-MY2), Aim for the Top University Program (02C030205) from Ministry of Education of Taiwan and National Sun Yat-sen University–Kaohsiung Medical University Joint Project (NSYSU–KMU 02C030117), awarded to Jyh-Horng Sheu.

    Author Contributions

    Ning-Sun Yang and Jyh-Horng Sheu designed the experiment and contributed to manuscript preparation. Fang-Yu Chang, Fang-Jung Hsu and Chi-Jen Tai carried out the experiment and wrote the manuscript. Wen-Chi Wei performed and analyzed the bioassay.

    Conflicts of Interest

    The authors declare no conflict of interest.

    References

    1. Blunt, J.W.; Copp, B.R.; Keyzers, R.A.; Munro, M.H.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 2013, 30, 237–323. [Google Scholar] [CrossRef]
    2. Wu, S.-L.; Su, J.-H.; Wen, Z.-H.; Hsu, C.-H.; Chen, B.-W.; Dai, C.-F.; Kuo, Y.-H.; Sheu, J.-H. Simplexins A–I, eunicellin-based diterpenoids from the soft coral Klyxum simplex. J. Nat. Prod. 2009, 72, 994–1000. [Google Scholar] [CrossRef]
    3. Chen, B.-W.; Wu, Y.-C.; Chiang, M.Y.; Su, J.-H.; Wang, W.-H.; Fan, T.-Y.; Sheu, J.-H. Eunicellin-based diterpenoids from the cultured soft coral Klyxum simplex. Tetrahedron 2009, 65, 7016–7022. [Google Scholar] [CrossRef]
    4. Chen, Y.-H.; Tai, C.-Y.; Hwang, T.-L.; Weng, C.-F.; Li, J.-J.; Fang, L.-S.; Wang, W.-H.; Wu, Y.-C.; Sung, P.-J. Cladielloides A and B: New eunicellin-type diterpenoids from an Indonesian octocoral Cladiella sp. Mar. Drugs 2010, 8, 2936–2945. [Google Scholar] [CrossRef]
    5. Chen, B.-W.; Chang, S.-M.; Huang, C.-Y.; Chao, C.-H.; Su, J.-H.; Wen, Z.-H.; Hsu, C.-H.; Dai, C.-F.; Wu, Y.-C.; Sheu, J.-H. Hirsutalins A–H, eunicellin-based diterpenoids from the soft coral Cladiella hirsuta. J. Nat. Prod. 2010, 73, 1785–1791. [Google Scholar] [CrossRef]
    6. Hassan, H.M.; Khanfar, M.A.; Elnagar, A.Y.; Mohammed, R.; Shaala, L.A.; Youssef, D.T.A.; Hifnawy, M.S.; El Sayed, K.A. Pachycladins A–E, prostate cancer invasion and migration inhibitory eunicellin-based diterpenoids from the Red Sea soft coral Cladiella pachyclados. J. Nat. Prod. 2010, 73, 848–853. [Google Scholar] [CrossRef]
    7. Chen, B.-W.; Chao, C.-H.; Su, J.-H.; Wen, Z.-H.; Sung, P.-J.; Sheu, J.-H. Anti-inflammatory eunicellin-based diterpenoids from the cultured soft coral Klyxum simplex. Org. Biomol. Chem. 2010, 8, 2363–2366. [Google Scholar] [CrossRef]
    8. Wu, S.-L.; Su, J.-H.; Lu, Y.; Chen, B.-W.; Huang, C.-Y.; Wen, Z.-H.; Kuo, Y.-H.; Sheu, J.-H. Simplexins J–O, eunicellin-based diterpenoids from a Dongsha Atoll soft coral Klyxum simplex. Bull. Chem. Soc. Jpn. 2011, 84, 626–632. [Google Scholar] [CrossRef]
    9. Chen, Y.-H.; Tai, C.-Y.; Su, Y.-D.; Chang, Y.-C.; Lu, M.-C.; Weng, C.-F.; Su, J.-H.; Hwang, T.-L.; Wu, Y.-C.; Sung, P.-J. Discovery of new eunicellins from an Indonesian octocoral Cladiella sp. Mar. Drugs 2011, 9, 934–943. [Google Scholar] [CrossRef]
    10. Tai, C.-J.; Su, J.-H.; Huang, M.-S.; Wen, Z.-H.; Dai, C.-F.; Sheu, J.-H. Bioactive eunicellin-based diterpenoids from the soft coral Cladiella krempfi. Mar. Drugs 2011, 9, 2036–2045. [Google Scholar] [CrossRef]
    11. Lee, Y.-N.; Tai, C.-J.; Hwang, T.-L.; Sheu, J.-H. Krempfielins J–M, new eunicellin-based diterpenoids from the soft coral Cladiella krempfi. Mar. Drugs 2013, 11, 2741–2750. [Google Scholar] [CrossRef]
    12. Tai, C.-J.; Su, J.-H.; Huang, C.-Y.; Huang, M.-S.; Wen, Z.-H.; Dai, C.-F.; Sheu, J.-H. Cytotoxic and anti-inflammatory eunicellin-based diterpenoids from the soft coral Cladiella krempfi. Mar. Drugs 2013, 11, 788–799. [Google Scholar] [CrossRef]
    13. Cai, Y.-S.; Yao, L.-G.; Di Pascale, A.; Irace, C.; Mollo, E.; Taglialatela-Scafati, O.; Guo, Y.-W. Polyoxygenated diterpenoids of the eunicellin-type from the Chinese soft coral Cladiella krempfi. Tetrahedron 2013, 69, 2214–2219. [Google Scholar] [CrossRef]
    14. Chen, T.-H.; Lu, M.-C.; Chang, Y.-C.; Su, Y.-D.; Chen, Y.-H.; Lin, N.-C.; Fang, L.-S.; Wu, Y.-C.; Sung, P.-J. Discovery of new eunicellin-based diterpenoids from a Formosan soft coral Cladiella sp. Mar. Drugs 2013, 11, 4585–4593. [Google Scholar] [CrossRef]
    15. Shih, F.-Y.; Chen, T.-H.; Lu, M.-C.; Chen, W.-F.; Wen, Z.-H.; Kuo, Y.-H.; Sung, P.-J. Cladieunicellins K and L, new eunicellin-based diterpenoids from an octocoral Cladiella sp. Int. J. Mol. Sci. 2013, 14, 21781–21789. [Google Scholar] [CrossRef]
    16. Chen, B.-W.; Wang, S.-Y.; Huang, C.-Y.; Chen, S.-L.; Wu, Y.-C.; Sheu, J.-H. Hirsutalins I–M, eunicellin-based diterpenoids from the soft coral Cladiella hirsuta. Tetrahedron 2013, 69, 2296–2301. [Google Scholar] [CrossRef]
    17. Li, T.-T.; Tang, X.-L.; Chen, C.-L.; Zhang, X.-W.; Wu, R.-C.; Zhu, H.-Y.; Li, P.-L.; Li, G.-Q. New eunicellin diterpenes and 9,10-secosteroids from the gorgonian Muricella sibogae. Helv. Chim. Acta 2013, 96, 21781–21789. [Google Scholar]
    18. Lai, D.; Liu, D.; Deng, Z.; van Ofwegen, L.; Proksch, P.; Lin, W. Antifouling eunicellin-type diterpenoids from the gorgonian Astrogorgia sp. J. Nat. Prod. 2012, 75, 1595–1602. [Google Scholar] [CrossRef]
    19. Hsu, F.-J.; Chen, B.-W.; Wen, Z.-H.; Huang, C.-Y.; Dai, C.-F.; Su, J.-H.; Wu, Y.-C.; Sheu, J.-H. Klymollins A–H, bioactive eunicellin-based diterpenoids from the Formosan soft coral Klyxum molle. J. Nat. Prod. 2011, 74, 2467–2471. [Google Scholar] [CrossRef]
    20. Lin, M.-C.; Chen, B.-W.; Huang, C.-Y.; Dai, C.-F.; Hwang, T.-L.; Sheu, J.-H. Eunicellin-based diterpenoids from the Formosan soft coral Klyxum molle with inhibitory activity on superoxide generation and elastase release by neutrophils. J. Nat. Prod. 2013, 76, 1661–1667. [Google Scholar] [CrossRef]
    21. Sharma, P.; Alam, M. Sclerophytins A and B. Isolation and structures of novel cytotoxic diterpenes from the marine coral Sclerophytum capitalis. J. Chem. Soc. Perkin Trans. I 1988, 2537–2540. [Google Scholar] [CrossRef]
    22. Wei, W.-C.; Sung, P.-J.; Duh, C.-Y.; Chen, B.-W.; Sheu, J.-H.; Yang, N.-S. Anti-inflammatory activities of natural products isolated from soft corals of Taiwan between 2008 and 2012. Mar. Drugs 2013, 11, 4083–4126. [Google Scholar] [CrossRef]
    23. Alley, M.C.; Scudiero, D.A.; Monks, A.; Hursey, M.L.; Czerwinski, M.J.; Fine, D.L.; Abbott, B.J.; Mayo, J.G.; Shoemaker, R.H.; Boyd, M.R. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 1988, 48, 589–601. [Google Scholar]
    24. Scudiero, D.A.; Shoemaker, R.H.; Paull, K.D.; Monks, A.; Tierney, S.; Nofziger, T.H.; Currens, M.J.; Seniff, D.; Boyd, M.R. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 1988, 48, 4827–4833. [Google Scholar]
    25. Wei, W.-C.; Lin, S.-Y.; Chen, Y.-J.; Wen, C.-C.; Huang, C.-Y.; Palanisamy, A.; Yang, N.-S.; Sheu, J.-H. Topical application of marine briarane-type diterpenes effectively inhibits 12-O-tetradecanoylphorbol-13-acetate-induced inflammation and dermatitis in murine skin. J. Biomed. Sci. 2011, 18, 94. [Google Scholar] [CrossRef]
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