Cladielloides A and B: New Eunicellin-Type Diterpenoids from an Indonesian Octocoral Cladiella sp

Two new eunicellin-type diterpenoids, cladielloides A (1) and B (2), which were found to possess a 2-hydroxybutyroxy group in their structures, were isolated from an Indonesian octocoral identified as Cladiella sp. The structures of eunicellins 1 and 2 were elucidated by spectroscopic methods. Cladielloide B (2) exhibited moderate cytotoxicity toward CCRF-CEM tumor cells and this compound displayed significant inhibitory effects on superoxide anion generation and elastase release by human neutrophils.

The relative configuration of 1 was elucidated from the interactions observed in a NOESY experiment. In the NOESY experiment of 1 (Table 2)  In order to determine the absolute configuration, the eunicellin 1 was treated with (−) or (+)-MTPA chloride to yield the (S)-and (R)-MTPA esters 1a and 1b, respectively [12][13][14]. Comparison of the 1 H NMR chemical shifts for 1a and 1b (Δ values shown in Figure 1) led to the assignment of the S-configuration at C-6. The C-24 hydroxy group in the 2-hydroxybutyrate moiety was also assigned as R-configuration. Therefore, the absolute configurations of all chiral centers of 1 were assigned as 1R, 2R, 3R, 4S, 6S, 9R, 10R, 14R, 24R.  (Table 3), IR, and MS) were similar to those of 1. However, the polarity of 2, which was checked by TLC, was substantially different from that of 1, indicating that these two compounds are isomers. In the 1 H NMR spectrum of 2, an acetate methyl was observed at  H 2.14 (3H, s). The additional acyl group was found to be a 2-hydroxybutyrate group, which showed six contiguous protons ( H 1.02, 3H, t, J = 7.2 Hz; 1.91, 2H, m; 4.87, 1H, dd, J = 6.8, 6.0 Hz). The 13 C NMR signal at  C 170.2 (s) correlated with the signal of an oxymethine proton at  H 4.87 in the HMBC spectrum and was consequently assigned as the carbon atom of the 2-hydroxybutyrate carbonyl. A correlation observed in the HMBC experiment of 2 further revealed the connectivity between H-4 ( H 5.21) and the carbonyl carbon ( C 170.2) of 2-hydroxybutyrate unit and demonstrated the location of the 2-hydroxybutyrate to be at C-4. The position of acetoxy group at C-6 was also confirmed by the connectivity between the oxymethine proton at  H 4.66 (H-6) and the ester carbonyl at  C 171.6 (s) in the HMBC spectrum of 2.
Thus, the remaining hydroxy group should be positioned at C-3. In addition, by comparison of the NOESY correlations of 2 with those of 1, the chiral centers of 2 were confirmed to be the same as those of 1. C-6, -7, -9, -10, -16 C-6, -7, -9, -10, -16 The cytotoxicity of metabolites 1 and 2 toward various tumor cell lines, including DLD-1, HL-60, CCRF-CEM (human T-cell acute lymphoblastic leukemia), and P388D1 cells was evaluated. The results, in Table 4, show that eunicellin 2 exhibited moderate cytotoxicity toward CCRF-CEM cells. The in vitro anti-inflammatory effects of metabolites 1 and 2 were tested. Metabolite 2 displayed significant inhibitory effects on superoxide anion generation and elastase release by human neutrophils at 10g/mL (Table 5). Table 5. Inhibitory effects of diterpenoids 1 and 2 on superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB.

General Experimental Procedures
Optical rotation values were measured with a JASCO P-1010 digital polarimeter at 25 °C. Infrared spectra were obtained on a VARIAN DIGLAB FTS 1000 FT-IR spectrometer. The NMR spectra were recorded on a VARIAN MERCURY PLUS 400 FT-NMR at 400 MHz for 1 H and 100 MHz for 13 C, in CDCl 3 at 25 °C . Proton chemical shifts were referenced to the residual CHCl 3 signal (  7.26 ppm). 13 C NMR spectra were referenced to the center peak of CDCl 3 at  C 77.1 ppm. ESIMS and HRESIMS data were recorded on a BRUKER APEX II mass spectrometer. Column chromatography was performed on silica gel (230-400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F 254 (0.25 mm, Merck) and spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. HPLC was performed using a system comprised of a HITACHI L-7100 pump, a HITACHI photodiode array detector L-7455, and a RHEODYNE 7725 injection port. A normal phase column (Hibar 250 × 10 mm, Merck, silica gel 60, 5 m,) was used for 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, Taiwan (NMMBA). This organism was identified by comparison with previous descriptions [15,16].

Extraction and Isolation
Slices of Cladiella sp. (wet weight 924 g) were extracted with a mixture of MeOH and CH 2 Cl 2 (1:1) and the residue was partitioned between EtOAc and H 2 O. The EtOAc layer was subjected to silica gel column chromatography and eluted using a mixture of n-hexane and EtOAc (stepwise, 100:1 to pure EtOAc) to obtain 19 fractions A-S. Fractions K and N were repurified by normal phase HPLC, using the mixture of n-hexane/ethyl acetate to afford 2 (2.4 mg, 5.5:1) and 1 (7.9 mg, 3:1), respectively.

Preparation of (S)-and (R)-MTPA Esters of Cladielloide A (1)
To a solution of 1 (1 mg) in pyridine (0.4 mL), R-(−)--methoxy--(trifluoromethyl) phenylacetyl (MPTA) chloride (25 L) was added, and the mixture was allowed to stand for 24 h at room temperature. The reaction was quenched by addition of 1.0 mL of water, and the mixture was subsequently extracted with EtOAc (3 × 1.0 mL). The EtOAc-soluble layers were combined, dried over anhydrous MgSO 4 and evaporated. The residue was subjected to column chromatography over silica gel using n-hexane-EtOAc (13:2) to yield the (S)-MTPA ester, 1a (0.7 mg, 44%). The same procedure was used to prepare the (R)-MTPA ester, 1b (1.4 mg, 89%), from the reaction of (S)-MPTA chloride with 1 in pyridine. The key 1 H NMR chemical shift differences Δ ( S −  R ) in ppm for the MTPA esters of 1 are shown in Figure 1.

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 the procedures described previously [19,20]. Briefly, superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate.