Cespitulones A and B, Cytotoxic Diterpenoids of a New Structure Class from the Soft Coral Cespitularia taeniata

Two novel diterpenoids, cespitulones A (1) and B (2), were isolated from extracts of the soft coral Cespitularia taeniata. Both compounds possess an unprecedented bicyclo [10.3.1] ring system with C-C bond connections between C-10 and C-20, and between C-20 and C-11. Their structures were elucidated on the basis of extensive spectroscopic analyses. Compound 1 exhibited significant cytotoxicity against human medulloblastoma and colon adenocarcinoma cancer cells.


Introduction
Marine soft corals are an excellent source of secondary metabolites with novel structures and interesting biological functions [1][2][3][4][5]. Members of the genus Cespitularia, along with morphologically similar Xenia species, inhabit the coral reefs along the coasts of Taiwan. These interesting cnidarians have brilliant colors and their outer layers are covered with thick mucilage. Previously, several OPEN ACCESS members of the genus Cespitularia were reported to contain a series of complex terpenoids, including cespitularins, nitrogen-containing diterpenoids, cespihypotins, cespitulins and cespitulactones [6][7][8][9][10][11][12]. These diterpenoids are thought to be derived from geranylgeranyl pyrophosphate and 1S-verticillene, involving interesting biogenetic pathways similar to those that generate the taxane diterpenes [13][14][15]. To explore novel bioactive metabolites from these invertebrates, we continued our study on Cespitularia taeniata, and have now isolated two novel diterpenoids, designated as cespitulones A (1) and B (2) (Figure 1). Both compounds possess an unprecedented bicyclo [10.3.1] skeleton. Here we report the isolation, structural elucidation, plausible biogenetic pathway, and the cytotoxicity of 1 and 2.

Results
Cespitulone A (1) was obtained as an amorphous solid that analyzed by HRESIMS for the molecular formula C 20 H 30 O 5 , having six degrees of unsaturation. The presences of hydroxyl and carbonyl functions were indicated by IR absorptions at 3419 and 1703 cm −1 . The 1 H-and 13 C NMR spectra (Table 1), along with DEPT NMR data, confirmed the presence of two carbonyls (δ C 212.4 and 207.4), and illustrated a trisubstituted olefin (δ C 131.7 CH, 133.6 C; δ H 5.58 d, J = 9.1 Hz), a 1,1-disubstituted olefin (δ C 144.9) with an exomethylene group (δ C 115.5 CH 2 ; δ H 4.87s, 4.92s), and one aliphatic quaternary carbon (δ C 45.6, C-15). In addition, two oxygenated methine carbons (δ C 69.8 CH, 77.4 CH), an oxygenated tertiary carbon 34.3 and δ C 24.6), and three methyl groups (δ C 26.9, 27.2, (δ C 89.1 C), six methylene carbons (δ C 31.5, 38.7, 46.9, 47.7, 18.6) with their corresponding proton chemical shifts (δ H 1.54, 1.32, 1.88) were observed. Since 1 contained two carbonyls and two double bonds, the carbon framework of cespitulone A must be bicyclic. Analysis of the COSY NMR data for 1 established the connectivities of H-9/H-9, Me-19/H-7/H-6, H-6/H-5/H-18/H-3/H-2/H-1 and H-13/H-14/H-1. These coupled with the HMBC NMR correlations of H-20/C-10, C-11, C-12, and H-9/C-10, and H-13/C-12, allowed the positions of the carbonyls at C-10 and C-12 and the hydroxyl at C-20 to be assigned. Thus, C-20 could be positioned between the C-10 carbonyl and the tertiary oxygenated C-11 carbon. This suggested that 1 contains an unusual bicyclic system in which the C-20 methyl group (as in cespitularines) was somehow modified and incorporated into the ring system. Analysis of other HMBC correlations, including Me-16/C-11, C-15; Me-17/C-11, C-15; H-9/C-7, C-8 as well as H-5/C-4, C-6, C-18, allowed the proposed bicyclo [10.3.1] ring system to be assigned ( Figure 2). The relative configuration of 1 was determined by analysis of NOESY NMR data based upon the assumption that 1 has the same absolute C-1 (H-1β) configuration as that of the Cespitularia-derived cespitulactams, cespitularines, cespihypotins and toxoids [16,17].   Figure 2). The configuration of the hydroxyl at C-6 was further determined by Mosher's reactions to yield compounds 1a and 1b [18]. The results, illustrated in Figure 3, suggested that the C-6 has the S configuration. A computer-generated perspective structure for 1 is shown in Figure 3 by CS Chem 3D version 9.0 using MM2 force field calculation for energy minimization. The results also suggested that C-6 has S configuration and C-11 hydroxy group is α-oriented.   Figure 4. The results also suggested that C-6 has S configuration and the hydroxyl at C-11 is β-oriented.  Scheme 1 illustrates a plausible biogenetic pathway for 1 and 2 based upon previous publications [7,8,16]. Cespitularin C might be a putative precursor of 1 and 2. Compound 1 is probably produced via intermediates a, b and c involving steps of epoxydation, rearrangement (1,2 methyl shift), oxidation, ring expansion, hydroxylation. Compound 2 may be derived from the same pathway, but through further hydroxylation, dehydration and ketolization of cation c. The Meinwald type rearrangement may be involved to give a ketone directly in the second step [19].

Scheme 1. Plausible biogenetic pathway of 1 and 2.
The isolated compounds 1 and 2 were evaluated for cytotoxicity against human medulloblastoma (Daoy) and colon adenocarcinoma (WiDr) cancer cell lines. As a result, cespitulone A showed significant in vitro cytotoxicity against human medulloblastoma (Daoy) and colon adenocarcinoma (WiDr) cancer cells with IC 50 values of 8.7 and 6.7 µM, respectively [20]. Mitomycin was used as a positive control with IC 50 at 0.3 μM.

Cytotoxicity Assay
The cytotoxic activities of compounds against human medulloblastoma (Daoy) and colon adenocarcinoma (WiDr) cancer cell lines cells were assayed by the MTT (3-(4,5-dimethylthiazole-2yl)-2,5-diphenyltetrazolium bromide) colorimetric assay as previously described [21]. Samples and control standard drugs were prepared at a concentration of 1, 10, 40, and 100 μg/mL. After seeding 2880 cells/well in a 96-well microplate for 3 h, 20 μL of sample or standard agent was placed in each well and incubated at 37 °C for 3 days. After removing the medium from the microplates, the cells were fixed with 10% formaldehyde in 0.9% saline for 30 min, dyed with 1% (w/v) methylene blue in 0.01 M borate-buffer (100 μL/well) for 30 min. The 96-well plate was dipped into a 0.01 M borate-buffer solution four times in order to remove the dye. Then, 100 μL/well of EtOH-0.1 M HCl (1:1) was added as a dye eluting solvent, and the absorbance was measured on a microtiter plate reader (Dynatech, MR 7000) at a wavelength of 650 nm. The ED 50 value was defined by a comparison with the untreated cells as the concentration of test sample resulting in 50% reduction of absorbance. Mitomycin was used as a standard compound.

Conclusions
Our investigation on constituents of Taiwanese soft coral Cespitularia taeniata has resulted in the isolation of two novel diterpenoids (1 and 2), which possess an unprecedented bicyclo [10.3.1] ring system with C-C bond connections between C-10 and C-20, and between C-20 and C-11. Cespitulone A (1) exhibited significant cytotoxicity against human medulloblastoma (Daoy) and colon adenocarcinoma (WiDr) cancer cells.