Lobocrassins A–E: New Cembrane-Type Diterpenoids from the Soft Coral Lobophytum crassum

Five new cembrane-type diterpenoids, lobocrassins A–E (1–5), were isolated from the soft coral Lobophytum crassum. The structures of cembranes 1–5 were established by spectroscopic and chemical methods and by comparison of the spectral data with those of known cembrane analogues. Lobocrassin A (1) is the first cembranoid possessing an α-chloromethyl-α-hydroxy-γ-lactone functionality and is the first chlorinated cembranoid from soft corals belonging to the genus Lobophytum. Lobocrassins B (2) and C (3) were found to be the stereoisomers of the known cembranes, 14-deoxycrassin (6) and pseudoplexaurol (7), respectively. Lobocrassin B (2) exhibited modest cytotoxicity toward K562, CCRF-CEM, Molt4, and HepG2 tumor cells and displayed significant inhibitory effects on the generation of superoxide anion and the release of elastase by human neutrophils.


Introduction
Among the diterpenoids isolated from octocorals, the cembrane-type metabolites are the largest group of compounds [1], and the soft coral Lobophytum crassum (family Alcyoniidae) has been proven to be a rich source of cembrane-type compounds [2][3][4][5][6][7][8][9][10][11][12][13]. In our continuing research on novel substances from the octocorals distributed in the waters of Taiwan at the intersection of the Kuroshio current and the South China Sea surface current, the soft coral L. crassum was studied to determine the properties of its organic extract, which displayed cytotoxicity toward MCF-7 (human breast adenocarcinoma) and HeLa (human cervical carcinoma) cells (IC 50 = 10.2 and 8.8 µg/mL, respectively). Five new cembrane derivatives, lobocrassins A-E (1-5) (Figure 1), were isolated. In this paper, we report the isolation, structure determination, and bioactivity of cembranes 1-5.

Results and Discussion
Lobocrassin A (1) was isolated as a colorless oil, and the molecular formula for this compound was determined to be C 20 13 C NMR and DEPT data with the molecular formula indicated that there must be an exchangeable proton, which required the presence of a hydroxy group. This deduction was supported by a broad absorption in the IR spectrum at 3385 cm -1 . The IR spectrum also showed a strong band at 1778 cm -1 , consistent with the presence of a γ-lactone moiety. The 13 C NMR data for 1 confirmed the presence of twenty carbon signals (Table 1), characterized by DEPT as three methyls, seven sp 3 methylenes, two sp 2 methines, three sp 3 methines, three sp 2 quaternary carbons, and two sp 3 quaternary carbons. Based on the 1 H and 13 C NMR spectra (Table 1) OH-15/C-17, permitted the elucidation of the carbon skeleton. The vinyl methyls attached at C-8 and C-12 were confirmed by the HMBC correlations between H-7, H 2 -9/C-19; H 3 -19/C-7, C-8, C-9; and H-11/C-20; H 3 -20/C-11, C-12, C-13 and were further supported by the allylic couplings between H-7/H 3 -19 and H-11/H 3 -20. The C-3/4 epoxide group was confirmed by the HMBC correlations between H 2 -2, H 2 -5/C-3; H 2 -2, H-5a, H-6a/C-4; and H 3 -18/C-3, C-4, C-5. The presence of a hydroxy group at C-15 was deduced from the HMBC correlations between the hydroxy proton (δ H 4.03, br s, OH-15) with C-1, C-15, C-16, and C-17.  The intensity of hydrogenated molecular (M + 2 + H) + isotope peaks observed in the ESIMS and HRESIMS spectra [(M + H) + :(M + 2 + H) + = 3:1] provided strong evidence for the presence of a chlorine atom in 1. The methylene unit at δ C 44.5 (CH 2 -16) was more shielded than expected for an oxygenated C-atom and was correlated to the methylene protons at δ H 3.79 (H-16a) and 3.53 (H-16b) in the HMQC spectrum. These two protons showed a typical geminal coupling pattern with each other (J = 11.6 Hz), and these two proton signals were 2 J-correlated with C-15 and 3 J-correlated with C-1 and C-17 in the HMBC spectrum, demonstrating the attachment of a chlorine atom at C-16. Based on the above findings, the molecular framework of 1 was established unambiguously.
The relative configuration of 1 was elucidated from the interactions observed in a NOESY experiment. Most naturally occurring cembrane-type natural products from soft corals belonging to the order Alcyonacea have the H-1 in the β-orientation [14]. In the NOESY experiment for 1 (Figure 2), correlations observed between H-7 and H 2 -9 and H-11 and H 2 -13, as well as the lack of correlation between H-7/H 3 -19 and H-11/H 3 -20, reflected the E geometry of the double bonds at C-7/8 and C-11/12. Additionally, H-1 correlated with H-13b (δ H 2.52), whereas H-14 showed responses to H-13a (δ H 2.67), and the absence of correlation between H-1 and H-14 suggested a trans-fused γ-lactone in 1. Moreover, it was found that H-14 showed interactions with H-3 and H 3 -20. Thus, assuming the α-orientation of H-14, H-3 should be positioned on the α face. In addition, H 3 -18 was found to interact with H-2a (δ H 2.14), but not with H-3, revealing the trans geometry of the trisubstituted epoxide. H-1 correlated with H-16a/b, indicating that the C-16 methylene was situated on the β face in 1. Based on the above findings, the structure of 1 was elucidated and the chiral centers for 1 were assigned as 1S*, 3S *, 4S*, 14S*, and 15S*.
Lobocrassin D (4) had a molecular formula of C 22  δ C 170.6, acetate carbonyl; 21.0, acetate methyl) in 4. Furthermore, acetylation of 3 gave a less polar product, which was found to be identical with natural product 4 and confirmed as cembranoid 4.

General Experimental Procedures
Optical rotations were measured on a Jasco P-1010 digital polarimeter. Infrared spectra were recorded on a Varian Diglab FTS 1000 FT-IR spectrometer; peaks are reported in cm -1 . The NMR spectra were recorded on Varian Mercury Plus 400 or Varian Inova 500 NMR spectrometers using the residual CHCl 3 signal (δ H 7.26 ppm) as an internal standard for 1 H NMR and CDCl 3 (δ C 77.1 ppm) for 13 C NMR. Coupling constants (J) are given in Hz. 1 H and 13 C NMR assignments were supported by 1 H-1 H COSY, HMQC, HMBC, and NOESY experiments. ESIMS were recorded on a Thermo Finnigan LCQ ion trap or a Bruker APEX II mass spectrometer. HRESIMS data were recorded on Thermo Fischer Scientific LTQ Orbitrap XL or a Bruker APEX II mass spectrometers. 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 Hitahci L-7455 photodiode array detector, and a Rheodyne injection port. A normal phase column (Hibar 250 × 10 mm, Merck, silica gel 60, 5 µm) was used for HPLC.

Animal Material
Specimens of the soft corals L. crassum were collected by hand using scuba equipment off the coast of northeast Taiwan at a depth of 10 m in August 2007 and stored in a freezer until extraction. A voucher specimen (NMMBA-TW-SC-2007-33) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Superoxide Anion Generation and Elastase Release by Human Neutrophils
Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were carried out according to previously described procedures [24,25]. 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.

Conclusions
In previous studies, a series of cembrane-type diterpenoids of potential medical interest were isolated from octocorals belonging to the genus Lobophytum. All corals, including reef-building corals and soft corals, are considered threatened species due to global climate change and habitat destruction. Therefore, the maintenance and culture of these interesting marine invertebrates as sources of new natural products of potential medical relevance is important. In our continuing search for novel substances from marine organisms originally collected from the Indo-Pacific Ocean, the hope is to identify extracts that exhibit interesting bioactivity. As an example, the bioactive cembranoid lobocrassin B (2) was isolated in this study. L. crassum was collected and transplanted back to tanks equipped with a flow-through sea water system. Advanced bioactivity testing for this compound will be carried out if sufficient material can be collected from culture-type species.