Five New Diterpenoids from an Okinawan Soft Coral, Cespitularia sp.

Five new diterpenoids 1–5 were isolated from an Okinawan soft coral, Cespitularia sp., together with the known diterpenoid, alcyonolide (6). New diterpenoid structures were elucidated by spectroscopic methods and by comparison of their NMR data with those of related compounds. Alcyonolide (6) was cytotoxic against HCT 116 cells (IC50 5.85 μM), while these new diterpenoids 1–5 were much less active (IC50 28.2–91.4 μM).


Introduction
Soft corals are rich sources of structurally unique and biologically active metabolites [1,2]. As part of our continuous search for bioactive secondary metabolites from Okinawan marine organisms [3][4][5], we isolated and characterized five new diterpenoids 1-5 as well as the known alcyonolide (6) [6] from a soft coral, Cespitularia sp. (Figure 1). Alcyonolide was the major constituent of ethyl acetate extracts. The carbon skeleton of 1-6 corresponds to a seco-type variety of xenicins, possessing a nine-membered carbocyclic ring trans-fused to a dihydropyran ring [7][8][9]. The biogenesis of compounds 1-6 presumably proceeds after completion of the xenicin-type carbon framework [7][8][9]. Herein, we report the isolation, structure elucidation, and cytotoxicity of the isolates from the soft corals.
The high resolution nanospray-ionization MS ( Among the four methyls, one was associated with the ketonic carbonyl (HMBC correlations of H 3 -18/C-7, -8), another was assigned to the methyl ester (HMBC correlation of H 3 -1′/C-6), and the remaining methyls were part of an isobutenyl group (HMBC correlations of H 3 -16/C-14, -15, -17 and H 3 -17/C-14-C-16). Comparison of NMR data of 1 and 6 revealed similarities. However, there were several significant differences that indicated the presence of new functional groups in 1. The major difference was the presence of a methyl ester and the absence of an acetal group, an acetyl group and an oxygenated methine in 1. Three major spin systems were constructed on the basis of COSY correlations, as shown in Figure 2      The ∆ 4,12 configuration was assigned as Z on the basis of NOEs (H-3/H-13 and H-4a/H-12). Since overlap of the H-4a and H-11a proton signals in CDCl 3 prevented us from determining the configuration of the ring junction at C-4a/C-11a, NMR spectra were recorded in C 6 D 6 and clearly separated signals were observed for the H-4a (δ H 2.96) and H-11a (δ H 3.12) (Experimental Section). Irradiation of these signals did not show any NOEs, suggesting their trans orientation as in alcyonolide (6). This was further supported by a large coupling constant (J H4a,11a = 11.6 Hz) [7][8][9]. Compound 1 could be a precursor of alcyonolide (6) [10], and assuming a common biosynthetic route for them, the absolute configuration could be as depicted in Figure 1.  Tables 1 and 2) of 2 are very similar to those of 1, except for NMR resonances of H-4a, C-4a, H-11a and C-11a, suggesting that 1 and 2 could be cis/trans isomers. Extensive analysis of 1D and 2D NMR data led to a planar structure of 2, which places it in the same diterpene class as 1. Geometric configuration of the ring junction at C-4a/C-11a in 2 was assigned to be cis by NOEDS experiments, in which irradiation of H-11a (δ H 3.38 d, J = 6.4 Hz) caused enhancement of H-4a (δ H 3.12 br q, J = 6.4 Hz). NOEs observed between H-3/H-13 and H-4a/H-12 revealed Z configuration of the ∆ 4,12 , as in 1.
Compound 3 also had the same molecular formula as 1 and 2, as deduced from HRNSIMS [m/z 363.2167 (M + H) + , calcd. for C 21 H 31 O 5 , 363.2166]. IR absorption bands at 1730 and 1709 cm −1 indicated the presence of several carbonyl groups. Its 1 H and 13 C NMR data (Tables 1 and 2) also showed similarities to those of 1 and 2. Extensive analysis of 1D and 2D NMR data and comparison of the 1 H and 13 C NMR data with those of 2 led to the same planar structure as 2, except for geometry of the double bond at C-4. In contrast to 2, the ∆ 4,12 was assigned as E configuration in 3 based on NOEs  Figure 1. A positive iodine-starch test also supported the presence of the hydroperoxy group in 4 [11]. Geometric configuration of the ring junction at C-4a/C-11a in 4 was also assigned to be cis by NOEDS experiments, in which irradiation of H-11a (δ H 3.43 d, J = 6.2 Hz) caused enhancement of H-4a (δ H 3.22 br q, J = 6.2 Hz). NOEs observed between H-3/H-13 and H-4a/H-12 revealed Z configuration of the ∆ 4,12 , as in 1 and 2. Compound 4 could be formed by the ene reaction between 2 and a singlet oxygen.
Compound 5 had the same molecular formula as 4, as deduced from HRNSIMS [m/z 417.1891 (M + Na) + , calcd. for C 21 H 30 O 7 Na, 417.1884]. IR spectrum of 5 was almost identical to that of 4 indicating the presence of several carbonyl groups (1742 and 1729 cm −1 ). 1 H and 13 C NMR spectral data (Tables 1 and 2) of 5 were also similar to those of 4, except for NMR resonances of H-3, C-3, H-4a, C-4a and H-13. Extensive analysis of 1D and 2D NMR data and comparison of the 1 H and 13 C NMR data with those of 4 led to the same planar structure as 4, except for geometry of the double bond at C-4. In contrast to 4, the ∆ 4,12 was assigned as E configuration in 5 on the basis of NOEs (H-3/H-12 and H-4a/H-13). The NOE between H-4a (δ H 3.69 br q, J = 6.0 Hz) and H-11a δ H 3.50 d, J = 6.0 Hz) allowed the ring junction to be assigned as cis, as in 2, 3 and 4. Compound 5 could be also formed by the ene reaction between 3 and a singlet oxygen.

General Experimental Procedures
Optical rotation was measured using a JASCO P-1010 Polarimeter. UV spectra were obtained with a HITACHI U-2001 Spectrophotometer. NMR spectra were recorded on a Bruker AvanceIII 500 spectrometer in CDCl 3 or C 6 D 6 . Chemical shifts and coupling constants were given as δ and Hz, respectively. IR spectra were recorded on a JASCO FT/IR-6100 Fourier Transform Infrared Spectrometer. High resolution mass spectra (HRMS) were obtained on an LTQ Orbitrap hybrid mass spectrometer equipped with a nanospray ionization (NSI) source. Open column chromatography was performed on Kieselgel 60 (70-230 mesh, Merck). HPLC was performed using a COSMOSIL Si60 HPLC column (5SL, 10 × 250 mm). Analytical TLC was performed using Kieselgel 60 F 254 DC-fertigplatten (Merck). All solvents were reagent grade.

Animal Materials
The soft coral was collected during low tide from the coast of Zamami Island, Okinawa, Japan, in April 2012, and identified as Cespitularia sp. A voucher specimen was deposited at the University of the Ryukyus (Specimen No. 110312).