Eunicellin-Based Diterpenoids, Hirsutalins S–V, from the Formosan Soft Coral Cladiella hirsuta

Four new eunicellin-type hirsutalins S–V (1–4), along with a known compound (–)-6α-hydroxy polyanthellin A (5), were isolated from the soft coral Cladiella hirsuta. The structures of the metabolites were determined by extensive spectroscopic analysis. Cytotoxity of compounds 1–5 against the proliferation of a limited panel of cancer cell lines was measured. Anti-inflammatory activity of compounds 1–5 was evaluated by measuring their ability in suppressing superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils.


Introduction
The chemical investigations on soft corals of the genus Cladiella and Klyxum  have afforded series of eunicellin-based diterpenoids, of which many have been shown to exhibit attracting biological activities [8,. We have previously isolated some bioactive eunicellins and steroids from a Taiwanese soft coral Cladiella hirsuta. Our recent studies of C. hirsuta have led to the discovery of 18 eunicellin-based diterpenoids, hirsutalins A-R [29][30][31], some of which have been found to possess cytotoxic [29,31] and anti-inflammatory activities [29][30][31]. In this paper, we further report the isolation of four new eunicellin-based compounds, hirsutalins S-V along with a known compound (-)-6α-hydroxy polyanthellin A (5) [32] from C. hirsuta (Chart 1). The structures of new compounds were determined by extensive spectroscopic analysis. Cytotoxicity of 1-5 against a limited panel of cancer cell lines and their anti-inflammatory activity, determined by their ability to inhibit the generation of superoxide anion and elastase release in N-formyl-methionyl-leucyl-phenylalanine/ cytochalasin B (fMLP/CB)-induced human neutrophils, were studied in order to discover bioactive compounds from marine environment.

Results and Discussion
Hirsutalin S (1) was isolated as a colorless oil. The HRESIMS (m/z 485.2512) of 1 established a molecular formula of C26H38O7. The IR spectrum of 1 showed the presence of hydroxy and carbonyl groups from absorptions at 3463 and 1740 cm −1 , respectively. The 1 H and 13 C NMR data of 1 (Table 1) were found to be closely resembled to those of known metabolite hirsutalin R [32]. The only difference was the presence of 2-acetoxybutanoate (δC 169.0 (C), 73.9 (CH), 24.5 (CH2), and 9.7 (CH3); 171.0 (C) and 20.6 (CH3)) in 1, instead of 2-butyryloxy butanoate at C-3 of hirsutalin R [32]. This was supported by the HMBC interaction of H-2′′ (δ 2.16) with carbonyl carbon resonating at δ 171.0. Moreover, the 13 C NMR spectroscopic data (Table 1) of 1 showed the presence of two 1,1-disubstituted carbon-carbon double bonds (δC 147.6 (C) and 118.3 (CH2); 145.2 (C) and 111.6 (CH2)). The molecular framework of 1 was established by the complete analysis of its COSY and HMBC correlations ( Figure 1). In the NOESY spectrum of 1, the correlations between H-10 with H-1; H-1 with H3-19, suggested that H-1, H-10 and H3-19 are β-oriented. Besides, correlations of H-2 with H3-15 and H-14; H-9 with H-14, suggested that H-2, H-9, H-14 and H3-15 are α-oriented. Furthermore, the asymmetric center at C-18 was suggested to be R-configured on the basis of NOE correlations between the β-oriented H-1 and H3-19 and between the α-oriented H-2 and H-18. As the absolute configuration of hirsutalin A [29] and that of hirsutalin J except C-2′ configuration [30] have been completely assigned based on Mosher's method, thus, the absolute configuration of 1, except that of C-2′, should be revealed as depicted.  Hirsutalin T (2) was also afforded as a colorless oil. Its molecular formula C30H46O9, was determined by HRESIMS (m/z 573.3036). The 13 C NMR spectrum (Table 1) showed the presence of the 2-acetoxybutanoate (δC 171.2 (C), 74.1 (CH), 24.3 (CH2), and 9.3 (CH3); 171.0 (C) and 20.5 (CH3)) [29] and an n-butyrate (δC173.9 (C), 36.2 (CH2), 18.5 (CH2), and 13.7 (CH3)). Comparison of the NMR data of 2 with those of the known compound hirsutalin A [29], it was found that a 2-hydroxybutyrate at C-3 and a methylene proton at C-4 in hirsutalin A were replaced by a hydroxy group and 2-acetoxybutanoate in 2, respectively. This was confirmed by the downfield shift of C-3 (δC 86.9) of hirsutalin A, relative to that of 2 (δC 74.3), and the HMBC connectivity from H-4 (δ 5.08) to the carbonyl carbon resonating at δ171.2 (C) ( Table 1). The structure of 2 was unambiguously determined by the extensive analysis of 1 H-1 H COSY and HMBC (Figure 1), and NOESY correlations ( Figure 2). Moreover, compound 2 was obtained as a C-2′ epimeric mixture with a ratio of about 1:1 reflected by a pair of signals in the 13 C NMR spectrum. Experiments were tried to separate an individual epimer but they were all unsuccessful.
The new eunicellin, hirsutalin U (3), gave the molecular formula C28H44O8S, on the basis of HRESIMS data (m/z 563.2657). NMR spectroscopic data of 3 (Table 2) showed the presence of the 3-methylsulfoxylpropionate substituent (δC 171.8 (C), 48.92 (CH2), 27.1 (CH2), and 38.6 (CH3)) [13] and an n-butyrate (δC 175.4 (C), 36.5 (CH2), 18.5 (CH2), and 13.7 (CH3)). The spectroscopic data (IR, 1 H NMR, and 13 C NMR) of 3 have similar structural features as those of a known one, hirsutalin J [30], except for the 2-butyryloxybutanoate at C-4 and the hydroxy group at C-20 in hirsutalin J were replaced by a n-butyrate group and 3-methylsulfoxylpropionate substituent in 3, respectively. A paired methyl singlets at δ 2.58/2.59 in an approximate 1:1 ratio in the 1 H NMR spectrum, and the doubling of signals of above methyl group with nearly the equal intensities in 13 C NMR spectrum were observed, suggested the occurrence of nearly equal quantities of R and S-configured sulfoxide moiety (Table 2). Thus, compound 3 is possibly to be an artifact arisen from the oxidation of its sulfide precursor. The analysis of NOE correlations of 3 revealed the same relative configuration at C-1, C-2, C-3, C-4 C-6, C-9, C-10, C-14 and C-18 as that of 2. The similar 1 H NMR, COSY, HMBC correlations ( Figure 1) and the analysis of NOE correlations of 3 further revealed the same relative configuration of both compounds. Thus, the structure of 3 was established.   Hirsutalin V (4) was obtained as a colorless oil with a molecular formula of C28H46O8. IR absorptions of 4 showed the presence of hydroxy and carbonyl groups at 3395 and 1738 cm −1 , respectively. Two ester carbonyl carbons (δC 169.1 and 173.5) were correlated with the methine proton (H-2′, δH 4.77, t, J = 6.4 Hz) of a 2-butyryloxybutanoate unit in the HMBC spectrum. By comparison of the NMR data of 4 with those of hirsutalin C [29], it was found that a C-7/C-16 double bond in hirsutalin C was replaced by an oxymethine bearing a methyl and a hydroxy group in 4, as confirmed by HMBC correlations observed from H3-16 (δ 1.25, 3H, s) to C-6 (δ 80.6, CH), C-7 (δ 77.0, C) and C-8 (δ 45.5, CH2). The planar structure of 4 was confirmed by careful analysis of COSY, HMBC, and NOESY correlations as shown in Figures 1 and 3. Compounds 1-4 are likely in the same enantiomeric series as hirsutalin A and hirsutalin J, based on a shared biosynthetic pathway. Thus, these compounds were suggested to possess the absolute configurations as shown in structures 1-5.

General Experimental Procedures
Silica gel (230-400 mesh, Merck, Darmstadt, Germany) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical TLC. High-performance liquid chromatography was performed on a Hitachi L-2455 HPLC apparatus (Hitachi Ltd., Tokyo, Japan) with a Supelco C18 column (250 × 21.2 mm, 5 μm). NMR spectra were recorded on a Varian UNITY INOVA-500 FT-NMR a Varian 400MR FT-NMR instrument (Varian Inc., Palo Alto, CA, USA) at 400 MHz for 1 H and 100 MHz for 13 C in CDCl3. LRMS and HRMS were obtained by ESI on a Bruker APEX II mass spectrometer (Bruker, Bremen, Germany). Optical rotations were measured on a JASCO P-1020 polarimeter. IR spectra were recorded on a JASCO FT/IR-4100 infrared spectrophotometer (Japan Spectroscopic Corporation, Tokyo, Japan).

Animal Material
The animal Cladiella hirsuta was collected by hand using SCUBA off the coast of Sianglu Islet (23°32′ N, 119°38′ E) in the region of Penghu Islands, in June 2008, at a depth of 10 m, and was stored in a freezer until extraction. A voucher sample (PI-20080610-17) was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

Cytotoxicity Testing
Cell lines were purchased from the American Type Culture Collection (ATCC). Cytotoxicity assays of compounds 1-5 were performed using the Alamar Blue assay [33,34].

In Vitro Anti-Inflammatory Assay
Human neutrophils were obtained using dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were performed according to previously described procedures. [35,36]. LY294002, a phosphatidylinositol-3-kinase inhibitor, was used as a positive control for inhibition of superoxide anion generation and elastase release with percentage inhibitions of 96.1% ± 4.9% in 10 μg/mL and 97.9% ± 7.7% in 10 μg/mL, respectively.

Conclusions
Our investigation demonstrated that the soft coral, C. hirsuta, could be a good source of bioactive substances. It is worthwhile to mention that eunicellin-type metabolite containing a sulfoxide, compound 3, was discovered for the first time from the soft coral C. hirsuta. Compound 1 was shown to display inhibitory activity against elastase release.