C
ladielloides C and D: Novel Eunicellin-Based Diterpenoids
from an Indonesian Octocoral Cladiella sp.
Chia-Ying Tai,1,2Yung-Husan Chen,2Tsong-Long Hwang,3Lee-Shing Fang,4 Wei-Hsien Wang,2,5,6 Ming-Chin Liu,2 Jui-Hsin Su,1,2Yang-Chang Wu,7,8and Ping-Jyun Sung*1,2,5,6,9
1Graduate Institute of Marine Biotechnology, National Dong Hwa University, Pingtung 944, Taiwan 2National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan
3Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan 4Department of Sport, Health, and Leisure, Cheng Shiu University, Kaohsiung 833, Taiwan
5Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan 6Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan
7Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University,
Taichung 404, Taiwan
8Natural Medicinal Products Research Center, China Medical University Hospital, Taichung 404, Taiwan
9Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan
Received December 13, 2010; E-mail: [email protected]
Chemical investigation on an Indonesian octocoral identified as Cladiella sp. has led to the isolation of two novel eunicellin-based diterpenoids, cladielloides C (1) and D (2). The structures of 1 and 2 were established by spectroscopic methods. Compound 1 exhibited significant cytotoxicity toward CCRF-CEM tumor cells and metabolites 1 and 2 displayed moderate inhibitory effects on superoxide anion generation by human neutrophils.
Previous investigations on the chemical constituents of octocorals belonging to the genus Cladiella have resulted in a series of interesting eunicellin-based (2,11-cyclized cembra-noid) diterpenoids.112 The compounds of this type were
reported from various octocorals including the genera Acaly-cigorgia,13 Astrogorgia,14 Briareum (=Solenopodium),15,16
Eleutherobia,17 Eunicella,18,19 Klyxum (=Alcyonium),2025
Litophyton,26 Muricella,27 Pachyclavularia,2830
Sclerophy-tum,31 and Sinularia.32 Most eunicellins were found to
possess complex structures and various interesting bioactiv-ities.1,33,34 In continuation of our search for bioactive
substances from the marine invertebrates distributed in the tropical West Pacific Ocean, an Indonesian octocoral identi-fied as Cladiella sp. was studied, and its extract exhibited cytotoxicity toward the tumor cell lines DLD-1 (human colorectal adenocarcinoma), HL-60 (human promyelocytic leukemia), and P388D1 (macrophage-like murine tumor cells) with IC50= 2.7, 8.9, and 7.2 ¯g mL¹1, respectively. In our
previous studies, seven eunicellin-based diterpenoids, includ-ing cladielloides A and B and cladieunicellins AE, were obtained from this organism.11,12 Our further investigation
on the natural products from this soft coral has led to the isolation of two novel eunicellins, cladielloides C (1) and D (2) (Chart 1). In this paper, we report the isolation, structure determination, and bioactivity of the above new diterpenoids 1 and 2.
Results and Discussion
Cladielloide C (1) was isolated as a colorless oil that gave a molecular ion [M + Na]+at m/z 485.2516 in the HR-ESI-MS,
indicating the molecular formula C26H38O7(calcd for C26H38
-O7+ Na, 485.2515) and implying eight degrees of
unsatura-tion. The IR spectrum of 1 showed bands at 3449 and 1745 cm¹1, consistent with the presence of hydroxy and ester groups. From the 1H and 13C NMR spectra (Table 1), 1 was
found to possess a trisubstituted olefin (¤H5.44, 1H, m, H-12;
¤C132.5, s, C-11; 121.5, d, C-12), an exocyclic carboncarbon
double bond (¤H 5.03, 1H, s, H-16a; 5.39, 1H, s, H-16b; ¤C
149.1, s, C-7; 114.9, t, C-16), and a 2-acetoxybutanoate (¤H
2.12, 3H, s;¤C20.5, q; 170.7, s;¤H 1.00, 3H, t, J= 7.6 Hz;
1.90, 2H, m; 4.91, 1H, dd, J= 7.2, 5.2 Hz; ¤C9.4, q; 24.3, t;
73.5, d; 170.1, s) group. In the 1H NMR spectrum of 1, two
doublets at ¤H0.98 and 0.80 (each 3H, d, J= 6.8 Hz, H3-19
and H3-20) were deduced to be from two methyls of an
isopropyl group. A singlet of the tertiary methyl bonded to an oxygenated quaternary carbon was due to the resonance of a signal at ¤H 1.29 (3H, s, H3-15). In addition, a suite of
resonances of proton signals at ¤H2.46 (1H, m, H-1), 2.81 (1H,
br s, H-10), 4.07 (1H, d, J= 2.4 Hz, H-2), 4.04 (1H, m, H-9), and carbon signals at ¤C42.0 (d, C-1), 46.2 (d, C-10), 83.6
(d, C-2), and 81.8 (d, C-9), indicated the presence of a tetrahydrofuran structural unit. Thus, from the above data, four
degrees of unsaturation were accounted for, and the proposed skeleton of 1 was suggested to be a eunicellin-based metabolite with four rings.
From the 1H1H COSY spectrum of 1 (Table 1), it was possible to identify the separate spin systems among H-1/H-2; H-4/H2-5; H2-8/H-9/H-10/H-1; H-12/H2-13/14/1;
H-14/H-18/H3-19 (H3-20), which were assembled with the
assistance of an HMBC experiment (Table 1). The key HMBC correlations between the protons and quaternary carbons of 1, such as H-5, H3-15/C-3; H2-5, H-8, H2-16/C-6; H2-8, 9,
H-16b/C-7; and H-9, H-10, H3-17/C-11, permitted elucidation of
the carbon skeleton. The location of the 2-acetoxybutanoate group in 1 was confirmed by an HMBC correlation between H-4 (¤H5.31) and the 2-acetoxybutanoate carbonyl (¤C170.1,
s, C-1¤) and further supported by the HMBC correlations between H-2¤ (¤H4.91) and the 2-acetoxybutanoate carbonyl at
170.1 (s, C-1¤) and acetate carbonyl at ¤C170.7 (s, C-1¤¤). Thus,
the remaining hydroxy group should be positioned at C-6, an oxygenated quaternary carbon. The C-6 hydroxy group was concluded to be a part of a hemiketal constellation on the basis of a characteristic carbon signal at ¤C 104.4 (s, C-6). The
HMBC correlations between H2-5 (¤H2.98 and 2.49) and each
of the two oxygenated low-field quaternary carbons at ¤C104.4
(s, C-6) and 86.5 (s, C-3) suggested the presence of a C-3/6 etherlinkage. The ether bridge between C-2 and C-9 was also supported by the HMBC correlations between H-2/C-9 and H-9/C-2. The vinyl methyl at C-11 was confirmed by the HMBC correlations between H3-17/C-10, -11, -12 and further
supported by the allylic coupling between the olefin proton H-12 and the vinyl methyl Me-17 in the 1H1H COSY
spectrum.
The relative configuration of 1 elucidated mainly by NOESY spectrum was compatible with those of 1 offered by computer modeling (Figure 1), in which the close contacts of atoms calculated in space were consistent with the NOESY correla-tions. In the NOESY experiment, H-1 correlated with H-10, H3
-15, and H3-20,indicating that H-1, H-10, and H3-15, and the
isopropyl group were situated on the same face; they were assigned as ¢ protons, as H-14 was ¡-oriented. H-2 showed correlations with H-1, H3-15, and H-18; and a small coupling
constant was found between H-1 and H-2 (J = 2.4 Hz), indicating that both the chiral centers C-2 and C-3 should be assigned as R* form by modeling analysis. H-4 correlated with H3-15 and one proton of C-5 methylene (¤H2.49), reflecting the
¡-orientation of 2-acetoxybutanoate at C-4. Furthermore, H-9 correlated with H2-8, H-14, and H3-17. From consideration of
molecular models, H-9 was found to be reasonably close to H2-8, H-14, and H3-17, when H-9 was¡-oriented in 1. Based
on the above findings, the structure, including the relative stereochemistry of 1 was established, and the chiral centers for the carbon skeleton of 1 were assigned as 1R*, 2R*, 3R*, 4S*, 6S*, 9R*, 10R*, and 14R*. By detailed analysis, the partial structurein the ten-membered ring of 1 was found to be similar with those of known eunicellin derivatives, hirsutalins BD (35) (Chart 1), which were isolated from an octocoral, Cladiella hirsuta.10 However, the stereochemistry of the
acetoxy groupin the 2-acetoxybutanoate moiety has not been determined at this stage.
Our present study also has led to the isolation of a new eunicellin 2 (cladielloide D). IR absorptions at 3423, 1715, and 1691 cm¹1, suggested the presence of hydroxy, ketone, and ¡,¢-unsaturated aldehyde groups in 2. The molecular formula
H H O H O H OH 1 2 3 6 7 10 11 12 13 14 4 5 9 16 20 8 17 18 19 15 1' 2' 3' 4' 1'' 2'' O O O O H H O OH H H O O 2 1 H H O H H OH R3 H R1 R2 H H O H H OH HO O O O O H 1R 2R 3R 6S 9R 10R 14R 2'R 18R
3 : R1 = OH, R2 = 2-butyryloxybutanoate, R3 = CH2OAc 4 : R1 = 2-butyryloxybutanoate, R2 = H, R3 = CH2OH 5 : R1 = 2-acetoxybutanoate, R2 = H, R3 = CH2OH
6
C20H30O4 was deduced from HR-ESI-MS at m/z 357.2044
(calcd for C20H30O4+ Na, 357.2042). Inspection of the NMR
data (Table 2) by the assistance of DEPT and HMQC spectra revealed the presence of five methyls, two sp3methylenes, six
sp3methines (including two oxymethines), an sp3oxygenated
quaternary carbon, a trisubstituted olefin, a 1,2-disubstituted double bond, and two carbonyls. The1H NMR spectrum also
showed the presence of five methyls including a methyl attached to an oxygenated quaternary carbon (¤H1.32, 3H, s,
H3-15), a vinyl methyl (¤H1.67, 3H, d, J= 1.2 Hz, H3-17), a
methyl attached to a carbonyl carbon (¤H2.20, 3H, s, H3-16),
and two methyls of an isopropyl group (¤H 0.98, 3H, d,
J= 6.8 Hz; 0.82, 3H, d, J = 6.8 Hz, H3-19 and H3-20). Three
proton signals at ¤H6.35 (1H, dd, J= 15.6, 8.0 Hz, H-5), 6.89
(1H, d, J= 15.6 Hz, H-4), and 9.58 (1H, d, J = 8.0 Hz, H-6) were assigned as the ¡,¢-olefinic protons and the aldehyde proton of the ¡,¢-unsaturated aldehyde group containing a trans-disubstituted carboncarbon double bond. By comparison of the1H and13C NMR data of 2 with those of 1, it was found
that resonances at¤H3.91 (H-2) and 4.00 (H-9) were attributed
to the protons of two oxymethines in the tetrahydrofuran unit. The 13C NMR spectrum showed signals at ¤
C207.8 (s, C-7),
193.7 (d, C-6), 162.4 (d, C-4), and 130.2 (d, C-5) further supporting the presence of a normal ketone and an ¡,¢-unsaturated aldehyde. The chemical shifts of two methine protons, located at two ring-junction carbons of the six-membered ring and an ether ring, ¤H 2.50 (H-10) and 2.38
(H-1), were assigned based on the results of1H1H COSY and
HMQC experiments. Based on the above observations and by analysis of1H1H COSY and HMBC spectral data as shown in
Table 2, the molecular framework of 2 was established.
Table 1. 1H and13C NMR Data,1H1H COSY, and HMBC Correlations for 1
Position ¤Ha) ¤Cb) 1H1H COSY HMBC (H¼ C)
1 2.46 m 42.0 (d)d) H-2, H-10, H-14 n.o.e) 2 4.07 d (2.4)c) 83.6 (d) H-1 C-9, -14 3 86.5 (s) 4 5.31 t (9.2) 79.1 (d) H2-5 C-2, -15, -1¤ 5¡ 2.98 dd (12.8, 9.2) 43.4 (t) H-4, H-5¢ C-4, -6 ¢ 2.49 dd (12.8, 9.2) H-4, H-5¡ C-3, -4, -6 6 104.4 (s) 7 149.1 (s) 8¡ 2.88 dd (14.4, 4.4) 41.9 (t) H-8¢, H-9 C-7, -9, -16 ¢ 2.67 dd (14.4, 5.2) H-8¡, H-9 C-6, -7, -16 9 4.04 m 81.8 (d) H2-8, H-10 C-2, -7, -11 10 2.81 br s 46.2 (d) H-1, H-9 C-1, -8, -9, -11 11 132.5 (s) 12 5.44 m 121.5 (d) H2-13, H3-17 n.o. 13¡ 2.03 m 22.7 (t) H-12, H-13¢, H-14 n.o. ¢ 1.85 m H-12, H-13¡, H-14 n.o. 14 1.34 m 38.8 (d) H-1, H2-13, H-18 n.o. 15 1.29 s 22.9 (q) C-2, -3, -4 16a 5.03 s 114.9 (t) H-16b C-6, -8 b 5.39 s H-16a C-6, -7, -8 17 1.64 br s 22.3 (q) H-12 C-10, -11, -12 18 1.82 m 28.2 (d) H-14, H3-19, H3-20 C-19, -20 19 0.98 d (6.8) 21.7 (q) H-18 C-14, -18, -20 20 0.80 d (6.8) 17.3 (q) H-18 C-14, -18, -19 1¤ 170.1 (s) 2¤ 4.91 dd (7.2, 5.2) 73.5 (d) H2-3¤ C-1¤, -3¤, -4¤, C-1¤¤ 3¤ 1.90 m 24.3 (t) H-2¤, H3-4¤ C-1¤, -2¤, -4¤ 4¤ 1.00 t (7.6) 9.4 (q) H2-3¤ C-2¤, -3¤ 1¤¤ 170.7 (s) 2¤¤ 2.12 s 20.5 (q) C-1¤¤
a) Spectra measured at 400 MHzin CDCl3at 25 °C. b) Spectra measured at 100 MHzin CDCl3at 25 °C. c) J values
(in hertz) in parentheses. d) Attached protons were deduced by DEPT and HMQC experiments. e) n.o.: not observed.
1 2 9 3 4 6 7 16 10 11 12 17 18 19 20 15 5 8 14 13 H/H (Å) H-1/H-2 H-1/H-10 H-1/H3-15 H-1/H3-20 H-2/H3-15 H-2/H-18 H-4/H-5β 2.41 H-4/H3-15 H-8α/H-9 2.26 H-8β/H-9 2.91 H-9/H-14 H-9/H3-17 2.76 2.73 2.35 2.13 2.30 2.71 2.12 2.24 2.55
Figure 1. Key NOESY correlations and computer-gener-ated perspective model using MM2 force field calculations for 1.
The relative stereochemistry of 2 was determined mainly by a NOESY experiment and the results are illustrated in Figure 2. A correlation between H-1 with H-10, suggested that these two protons are on the same side of the molecule and assigned as ¢-oriented. The oxymethine proton H-2 exhibited a correlation with H-14. Thus, H-2 and H-14 should be positioned on the ¡ face. H-9 showed correlations with H2-8 and H-14 and this
proton exhibited coupling with H2-8 (J= 6.8, 3.6 Hz) and H-10
(J= 8.0 Hz), indicating that H-9 was ¡-oriented. Based on the abovefindings, the chiral centers of 2 were assigned as 1R*, 2R*, 9R*, 10R*, and 14R*. However, due to the free rotation of the carboncarbon bond between C-2 and C-3, the stereo-chemistry of C-3 hydroxy group is not determined, although a correlation between H-2 and Me-15 was observed in the
NOESY spectrum of 2. Because cladiellolides AD were isolated from the same animal,11the stereochemistry at C2C3
part of cladiellolide D was deduced to be same as that of cladiellolides AC. Geometric optimization of 2 was per-formed with Chem3D Pro software. The conformation search suggested that the most stable conformation and the calculated minimum energy for 2 are shown in Figure 2. It was found that the calculated distances between those protons having key NOESY correlations of 2 are all shorter than 3 ¡ as shown in Figure 2. It is worth noting that the eunicellin metabolites possessing a cleavage bond between C-6/7 are rarely found. Cladielloide D (2) is the second 6,7-secoeunicellin ever discovered.29
In a previous study, the absolute configuration of a known eunicellin analog, hirsutalin A (6) (Chart 1),10 which was
isolated from the octocoral belonging the same genus Cladiella as that of eunicellins 1 and 2, was determined using a modified Mosher’s method. Thus, the new eunicellins 1 and 2 are assumed to have the same absolute configuration as 6 because these compounds were all isolated from the same genus collected from the tropical West Pacific Ocean.
The cytotoxicity of eunicellins 1 and 2 toward a limited panel of tumor cell lines, including CCRF-CEM (human T cell acute lymphoblastic leukemia), HL-60, DLD-1, and P388D1 cells was evaluated (Table 3). The results showed that cladielloide C (1) exhibited significant cytotoxicity toward CCRF-CEM cells. The in vitro anti-inflammatory effects of metabolites 1 and 2 were tested. Metabolites 1 and 2 displayed moderateinhibitory effects on superoxide anion generation by human neutrophils at 10 ¯g mL¹1, respectively (Table 4).
Table 2. 1H and13C NMR Data,1H1H COSY, and HMBC Correlations for 2
Position ¤Ha) ¤Cb) 1H1H COSY HMBC (H¼ C)
1 2.38 ddd (9.6, 8.0, 4.0)c) 40.4 (d)d) H-2, H-10, H-14 n.o.e) 2 3.91 d (4.0) 87.0 (d) H-1 C-14 3 74.8 (s) 4 6.89 d (15.6) 162.4 (d) H-5 C-3, -6 5 6.35 dd (15.6, 8.0) 130.2 (d) H-4, H-6 C-3 6 9.58 d (8.0) 193.7 (d) H-5 C-5 7 207.8 (s) 8a 2.75 dd (16.0, 6.8) 48.0 (t) H-8b, H-9 C-7, -9 b 2.87 dd (16.0, 3.6) H-8a, H-9 C-7, -9 9 4.00 ddd (8.0, 6.8, 3.6) 79.0 (d) H2-8, H-10 n.o. 10 2.50 br t (8.0) 47.4 (d) H-1, H-9 C-8, -9, -11, -12, -14 11 129.9 (s) 12 5.53 m 123.7 (d) H2-13, H3-17 n.o. 13¡ 2.02 m 23.3 (t) H-12, H-13¢, H-14 n.o. ¢ 1.91 m H-12, H-13¡, H-14 C-14 14 1.46 m 38.9 (d) H-1, H2-13, H-18 C-13 15 1.32 s 23.7 (q) C-2, -3, -4 16 2.20 s 30.9 (q) C-7, -8 17 1.67 d (1.2) 22.7 (q) H-12 C-10, -11, -12 18 1.76 m 27.6 (d) H-14, H3-19, H3-20 C-19, -20 19 0.98 d (6.8) 21.8 (q) H-18 C-14, -18, -20 20 0.82 d (6.8) 17.4 (q) H-18 C-14, -18, -19 3-OH 3.66 s C-15
a) Spectra measured at 400 MHzin CDCl3at 25 °C. b) Spectra measured at 100 MHzin CDCl3at 25 °C. c) J values
(in hertz) in parentheses. d) Attached protons were deduced by DEPT and HMQC experiments. e) n.o.: not observed.
1 2 3 4 5 6 7 16 9 10 11 14 18 20 19 15 17 H/H (Å) H-1/H-10 H-2/H-14 H-2/H3-15 H-8α/H-9 2.43 H-8β/H-9 2.36 H-9/H-14 2.63 2.71 2.35 2.75
Figure 2. Key NOESY correlations and computer-gener-ated perspective model using MM2 force field calculations for 2.
Experimental
General Experimental Procedures. Optical rotation values were measured with a JASCO-P1010 digital polar-imeter. Infrared spectra were obtained on a VARIAN DIGLAB FTS 1000 FT-IR spectrometer. NMR spectra were recorded on a VARIAN MERCURY PLUS 400 FT-NMR at 400 MHzfor
1H and 100 MHzfor 13C in CDCl
3at 25 °C. Proton chemical
shifts were referenced to the residual CHCl3signal (¤H7.26). 13C NMR spectra were referenced to the center peak of CDCl
3
at ¤C77.1. ESI-MS and HR-ESI-MS data were recorded on
BRUKER APEX II mass spectrometer. Column chromatog-raphy was performed on silica gel (230400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F254(0.25 mm, Merck) and spots were visualized
by spraying with 10% H2SO4 solution followed by heating.
HPLC was performed using a system comprised of a HITACHI L-7100 pump, a HITACHI L-7455 photodiode array detector, and a RHEODYNE 7725 injection port. A normal phase column (Hibra 250 © 10 mm, Merck, silica gel 60, 5 ¯m) was usedfor HPLC.
Animal Material. The octocoral Cladiella sp. were collected from Indonesia in 2004 and stored in a freezer until extraction. A voucher specimen was deposited in the National Museum of Marine Biology and Aquarium (NMMBA), Taiwan. This organism was identified by comparison with previous descriptions.36,37
Extraction and Isolation. Sliced bodies of Cladiella sp. (wet weight 402 g, dry weight 144 g) were extracted with a mixture of MeOH and CH2Cl2 (1:1). The extract was
partitioned between EtOAc and H2O. The EtOAc layer was
separated on silica gel and eluted using n-hexane/EtOAc
(stepwise, 25:1pure EtOAc) to yield the 19 fractions AS. Fraction G was repurified by normal-phase HPLC, using the mixtures of n-hexane and EtOAc as a mobile phase to afford compound 2 (4:1). Fraction L was separated by normal-phase HPLC, using the mixtures of CH2Cl2and acetone as a mobile
phase to afford compound 1 (27:1).
Cladielloide C (1): Colorless oil (1.2 mg); ½¡23D +220 (c 0.02, CHCl3); IR (neat):¯max3449, 1745 cm¹1;1H (CDCl3,
400 MHz) and13C (CDCl
3, 100 MHz) NMR data, see Table 1;
ESI-MS: m/z 485 [M + Na]+; HR-ESI-MS: m/z 485.2516
(calcd for C26H38O7+ Na, 485.2515).
Cladielloide D (2): Colorless oil (2.1 mg); ½¡23
D +4
(c 0.11, CHCl3); IR (neat): ¯max 3423, 1715, 1691 cm¹1; 1H
(CDCl3, 400 MHz) and13C (CDCl3, 100 MHz) NMR data, see
Table 2; ESI-MS: m/z 357 [M + Na]+; HR-ESI-MS: m/z
357.2044 (calcd for C20H30O4+ Na, 357.2042).
Cytotoxicity Testing. The cytotoxicity of compounds 1 and 2 was assayed with a modification of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colori-metric method. Cytotoxicity assays were carried out according to procedures described previously.38,39
Molecular Mechanics Calculations. Implementation of the MM2 force filed40 in Chem3D Pro software from
Cam-bridge Soft Corporation, Cambridge, MA, USA (ver 9.0, 2005), was used to calculate molecular models.
Human Neutrophil Superoxide Anion Generation and Elastase Release. Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Superoxide generation and elastase release were carried out according to procedures described previously.41,42 Briefly,
superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase release experiments were performed using MeOSuc AlaAlaProValpnitroanilide as the elastase substrate.
This research work was supported by grants from the National Museum of Marine Biology and Aquarium (Grant Nos. 100100101 and 100200311); National Dong Hwa University; Asia-Pacific Ocean Research Center, National Sun Yat-sen University (No. 98C031702); and the National Science and Technology Program for Biotechnology and Pharmaceuticals, National Science Council (Grant No. NSC 99-2323-B-291-001 and 98-2320-B-291-001-MY3), Taiwan, awarded to P.-J.S.
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