A New Cubitane Diterpenoid from the Soft Coral Sinularia crassa

A new cubitane diterpenoid, crassalone A (1), was isolated from the marine soft coral Sinularia crassa. The structure was determined by extensive spectroscopic analyses. Compound 1 is not cytotoxic (IC50 > 20 μg/mL) toward the four human cancer cell lines tested (HL60, MDA-MB-231, HCT-116 and DLD-1).


Introduction
Marine soft corals of the genus Sinularia have attracted a great deal of attention in light of the structural diversity and wide range of biological activities of their metabolites [1]. Moreover, Formosan soft corals of the genus Sinularia have been shown to be rich sources of structurally unique and bioactive natural products [2][3][4][5][6]. In the investigation of secondary metabolites in marine invertebrates, the diterpenoid cubitane was first reported in 1984 as being obtained from the Caribbean gorgonian octocoral Eunicea calyculata [7]. In other reports, studies of the chemical constituents of octocorals have led to the isolation of various cubitane diterpenoids [7][8][9]. Recently, we have isolated OPEN ACCESS two cubitane diterpenoids from the soft coral Sinularia triangula [10,11]. Our recent study of the chemical constituents on Sinularia crassa ( Figure 1) had led to the isolation of two cembranoids [12]. In this paper, we further report the isolation of a new cubitane diterpenoid, crassalone A (1, Figure 2). The structure of 1 was established by extensive spectroscopic analysis, including careful examination of 2D-NMR ( 1 H-1 H COSY, HMQC, HMBC and NOESY) correlations. The cytotoxicity of compound 1 against human promyelocytic leukemia (HL60), human breast adenocarcinoma (MDA-MB-231), human colon adenocarcinoma (HCT-116) and human colorectal carcinoma (DLD-1) cell lines was also studied.

Results and Discussion
Crassalone A (1) was obtained as a colorless oil. The HR-ESI-MS spectrum of 1 exhibited a molecular ion peak at m/z 327.2298 [M+Na] + , and established a molecular formula C 20 H 32 O 2 , implying five degrees of unsaturation. The IR spectrum of 1 showed a broad absorption band at 3422 cm 1 and a strong absorption band at 1708 cm 1 , implying the presence of hydroxy and carbonyl groups, respectively. Inspection of the 13 C-NMR and DEPT spectral data of 1 (Table 1) in CDCl 3 , indicated the presence of 20 carbon signals of a diterpenoid. These signals were ascribable to carbons of four methyls, five sp 3 methylenes, two sp 2 methylenes, four sp 3 methines (including an oxygenated carbon at δ 75.3) and one sp 2 methine. The remaining four signals appearing in the lower field region of the spectrum are due to the quaternary carbons of thee olefinic carbons (δ 151.7, 147.9 and 142.9) and one ketone carbonyl (δ 211.3). The 1 H-NMR spectral data revealed the presence of four olefinic methylene protons ( 5.03, 4.90, 4.89 and 4.79, each s) and one olefinic proton ( 5.35, d, J = 9.5 Hz). By interpretation of 1 H-1 H COSY correlations, it was possible to establish four partial structures from H-1 to H-4 and H 3 -13, from H 2 -6 to H 2 -7, from H-9 to H-10, and from H-15 to both H 3 -16 and H 3 -17 ( Figure 2). These data, together with the HMBC correlations ( Figure 2) from H 2 -2 to C-12, H 2 -6 to C-4, C-5 and C-8, H-9 to C-7, H-10 and H 2 -12 to C-11 (carbonyl carbon) established the connectivity within the 12-membered ring. A 1,1-disubstituted double bond attached at C-5 was confirmed by the HMBC correlations from H 2 -14 to C-4, C-5 and C-6. One methyl group attached at C-1 was confirmed by the HMBC correlations from H 3 -13 to C-1, C-2 and C-12. Furthermore, two isopropyl moieties attached at C-8 and C-10 were confirmed by the HMBC correlations from both methyl H 3 -16 and H 3 -17 to C-8 and C-15 and H 3 -19 to C-10, C-18 and C-20. Thus, 1 was found to possess one trisubstituted olefin at C-8/C-9, one ketone group at C-11, and two 1,1-disubstituted double bonds at C-5/C-14 and C-18/C-20, respectively. The relative structure of 1 was elucidated by the analysis of NOE correlations, as shown in Figure 3. The NOE correlations observed between H-9 and H-15 reflected the E geometry of double bonds at C-8/C-9. Moreover, the E geometry of double bond at C-8/C-9 further established by comparison of the-NMR data of 1 in CDCl 3 with those of two related compounds, calyculone B (2) and calyculone C (3) (Figure 4), also measured in CDCl 3 . The proton shifts of two protons [H-9 ( H = 5.35) and H-15 ( H = 2.35)] of 1, were found to be the same as those of 2 [H-9 ( H = 5.37) and H-15 ( H = 2.28)] [7]. Also, the carbon shifts of C-9 ( C = 120.0) and C-15 ( C = 33.0) were found to be more closer to that of 2 [C-9 ( C = 119.8) and C-15 ( C = 31.7)] relative to that of 3 [C-9 ( C = 117.1) and C-15 ( C = 27.5)], too [7]. Thus, it was suggested that the double bond of 1 at C-8/C-9 should be E geometry. Due to the overlapping of H-4 and H-10 signals ( 4.024.03 ppm) on measuring the 1 H-NMR in CDCl 3 , we also measured the NOESY spectrum of 1 in pyridine-d 5

General Procedures
Optical rotation values were measured using a Jasco P-1010 digital polarimeter. IR spectra were recorded on a Varian Digilab FTS 1000 Fourier transform infrared spectrophotometer. NMR spectra were recorded on a Varian Unity INOVA 500 Fourier transform-nuclear magnetic resonance (FT-NMR) instrument at 500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR, respectively, in CDCl 3 and pyridine-d 5 . ESIMS and HESIMS data were recorded with a Bruker APEX II mass spectrometer. Gravity column chomatography was performed on silica gel (230-400 mesh, Merck, Darmstadt, Germany). Thin layer chomatography (TLC) was carried out on precoated Kieselgel 60 F254 (0.2 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 (Tokyo, Japan) and a Rheodyne 7725 injection (Cotati, USA) port. A preparative normal phase column (Hibar 250 × 21.2 mm, Supelco, silica gel 60, 5 μm, Bellefonte, USA) was used for HPLC.

Animal Material
The marine soft coral S. crassa (Tixier-Durivault, 1945) was collected by scuba divers at a depth of around 10 m off the coast of Taitung County, Taiwan, in October 2011, and the sample was frozen immediately after collection. A voucher sample was deposited at the National Museum of Marine Biology and Aquarium, Taiwan (specimen No. 2011-1012-7).

Conclusions
In previous studies, a series of the cubitane diterpenoids had been isolated from two gorgonian corals Eunicea calyculata [7,8] and Eunicea laciniata [9], and one soft coral Sinularia triangula [10,11]. The present investigation demonstrated that the metabolite 1 was inactive (IC 50 's >20 μg/mL) towards the growth of HL60, MDA-MB-231, DLD-1 and HCT-116 cancer cells. Among the cubitane diterpenoids discovered from marine organisms, only one compound (sinutriangulin A) has been found to possess weak cytotoxicity toward the two cancer cells (CCRF-CEM and DLD-1). Therefore, compounds of this class have not been found to exhibit significant cytotoxicity from our results and previous reports.