New Cytotoxic Cembranolides from the Soft Coral Lobophytum michaelae

Six new cembranolides, michaolides L–Q (1–6), and a known cembranolide, lobomichaolide (7) were isolated from the CH2Cl2 extract of the soft coral Lobophytum michaelae. Their structures were established by extensive spectral analysis. The anti-HCMV (human cytomegalovirus) activity of 1–7 and their cytotoxicity against selected cell lines were evaluated.

After assignments between all the C-H bondings were made based on an HSQC experiment, the planar structure was determined by HMBC analysis. The correlations according to HMBC are shown in Figure 3. The stereochemistry for the trisubstituted olefins of 1 was determined by NOESY analysis. The NOESY correlations between H-7 and H-9, and H-11 and H-13 disclosed the E configurations for the trisubstituted olefins. The chemical shift values at δ C 15.6 and 15.9 (for C-19 and C-20, respectively) also supported the E configurations [2,3]. The NOESY correlations ( Figure 4       Michaolide M (2) was shown to have the molecular formula of C 24 H 34 O 9 by HRESIMS and from its 13 C NMR data. The 1 H and 13 C NMR spectral data (Table 1)  Michaolide N (3) analyzed for C 26 H 32 O 10 from its HRESIMS and NMR spectroscopic data. The NMR features of compound 3 were analogous to those of 2 with exception that the secondary acetoxyl attached to C-10 was shifted to C-9 and the methyl attached to C-12 was replaced by an aldehyde [δ H 10.14, δ C 190.3] ( Table 1). 1 H-1 H COSY cross peaks ( Figure 6) between H-9 and H-10/H-11 as well as HMBC correlations (Figure 7) between H-20 and C-11/C-12/C-13 as well as between H 3 -19 and C-7/C-8/C-9 helped to ascertain these assignments. The relative stereochemistry of 3 was     Michaolide P (5) was shown to have the molecular formula of C 22 H 30 O 5 by HRESIMS and from its 13 C NMR data. The 1 H and 13 C NMR spectral data ( Table 2) of 5 closely resembled those of 1 except for the replacement of the trisubstituted epoxy by a trisubstituted olefin at Δ 3 . HMBC correlations ( Figure 9) between H 3 -18 and C-3/C-4/C-5 confirmed the presence of a trisubstituted olefin at C-3. The relative stereochemistry was determined by NOESY correlations (Figure 8    The cytotoxicity toward P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), A-549 (human lung epithelial carcinoma) tumor cells, and human embryonic lung (HEL) cells of michaolides L-Q (1-6) and lobomichaolide (7) were shown in Table 3. Non-cytotoxic cembranoid, michaolide O (4) was tested for anti-HCMV activity and showed a negative result (IC 50 > 200 μM/mL). The α-exo-methylene-γ-lactone moiety is important for cytotoxicity by comparing the cytotoxicity of 4 with those of 1-3, 5, and 6 [4]. The absolute stereochemistry of the known cembranolide, lobomichaolide (7) [2,3] should be drawn as in Figure 2 since cembranolides 1 and 7 both exhibited positive optical rotations.

General Experimental Procedures
Optical rotations were determined with a JASCO P1020 digital polarimeter. Ultraviolet (UV) and infrared (IR) spectra were obtained on JASCO V-650 and JASCO FT/IR-4100 spectrophotometers, respectively. NMR spectra were recorded on a Varian MR 400 NMR spectrometer at 400 MHz for 1 H and 100 MHz for 13 C. 1 H NMR chemical shifts are expressed in δ (ppm) referred to the solvent peaks δ H 7.27 for CDCl 3 , and coupling constants are expressed in Hz. 13 C NMR chemical shifts are expressed in δ (ppm) referred to the solvent peaks δ C 77.0 for CDCl 3 . ESI-MS were recorded by ESI FT-MS on a Bruker APEX II mass spectrometer. Silica gel 60 (Merck, Germany, 230-400 mesh) and LiChroprep RP-18 (Merck, 40-63 μm) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F 254 , 0.25 mm) and precoated RP-18 F 254s plates (Merck) were used for thin-layer chromatography (TLC) analysis. High-performance liquid chromatography (HPLC) was carried out using a Hitachi L-7100 pump equipped with a Hitachi L-7400 UV detector at 220 nm together with a semi-preparative reversed-phase column (Merck, Hibar LiChrospher RP-18e, 5 μm, 250 × 25 mm).

Biological Material
The soft coral L. michaelae Tixier-Durivault (Alcyoniidae) was collected at Ken-Ting, Ping-Tong County, Taiwan, in June 2002, at a depth of 3-4 m and was stored for 2 weeks in a freezer until extraction. Identification was kindly verified by Prof. Keryea Soong, Institute of Marine Biology, National Sun Yat-sen, Taiwan. A voucher specimen, MR-004, was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Taiwan.

Cytotoxicity Assay
Cytotoxicity was determined on P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung epithelial carcinoma) tumor cells using a modification of the MTT colorimetric method according to a previously described procedure [25,26]. The provision of the P-388 cell line was supported by J.M. Pezzuto, formerly of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. HT-29 and A-549 cell lines were purchased from the American Type Culture Collection.

Anti-HCMV Assay
To determine the effects of natural products upon HCMV cytopathic effect (CPE), confluent human embryonic lung (HEL) cells grown in 24-well plates were incubated for 1 h in the presence or absence of various concentrations of tested natural products. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of 24-well dish. Antiviral activity was expressed as IC 50 (50% inhibitory concentration), or compound concentration required to reduce virus induced CPE by 50% after 7 days as compared with the untreated control. To monitor the cell growth upon treating with natural products, an MTT-colorimetric assay was employed [27].