Eunicellin-Based Diterpenoids, Hirsutalins N–R, from the Formosan Soft Coral Cladiella hirsuta

New eunicellin-type hirsutalins N–R (1–5), along with two known eunicellins, (6 and 7) were isolated from the soft coral Cladiella hirsuta. The structures of the metabolites were determined by extensive spectroscopic analysis. Cytotoxic activity of compounds 1–7 against the proliferation of a limited panel of cancer cell lines was measured. The in vitro anti-inflammatory activity of compounds 1–7 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 several eunicellin-based diterpenoids, of which many have been shown to exhibit interesting bioactivities [8,. Our recent chemical study of a Taiwanese soft coral Cladiella hirsuta has led to the discovery of 13 eunicellin-based diterpenoids hirsutalins A-M [29,30] and seven steroids hirsutosterols A-G [31] some of which have been found to possess cytotoxic [29] and anti-inflammatory activities [29,30]. In this paper we further report the isolation of five new eunicellin-based compounds, hirsutalins N-R (Chart 1), along with two known compounds, (1R*,2R*,3R*,6S*,7S*,9R*,10R*,14R*)-3-butanoyloxycladiell-11(17)-en-6,7-diol (6) [6], and hirsutalin E (7) [29] from C. hirsuta (Chart 2). The structures of new compounds were determined by extensive spectroscopic analysis. Cytotoxicity of 1-7 against a limited panel of cancer cell lines and their anti-inflammatory activity, determined by their ability to inhibit the generation of super oxide anion and elastase release in N-formyl-methionyl-leucylphenylalanine/cytochalasin B(fMLP/CB)-induced human neutrophiles, were studied in order to discover bioactive compounds for future new drug development.

Results and Discussion
Hirsutalin N (1) was isolated as a colorless oil. The HRESIMS (m/z 461.2518) of 1 established a molecular formula of C 24 H 38 O 7 . The IR spectrum of 1 showed the presence of hydroxy and carbonyl groups from absorptions at 3451 and 1733 cm −1 , respectively. The 13 C NMR of 1 exhibited 24 carbon signals as expected which were found to be similar to these of a known metabolite hirsutalin I (8, Chart 3) [30], the difference being that the hydroxymethyl group attached at C-18 in hirsutalin I was replaced by a methyl group in 1. This was confirmed by 1 H NMR spectrum of 1 which shows the presence of two isopropyl methyls at δ 0.73 (d, J = 7.2 Hz) and 0.97 (d, J = 7.2 Hz) ( Table 1). Also, NMR data revealed that the n-butanoyloxy group at C-3 in 8 was replaced by an acetoxy group in 1. Key HMBC correlations from H-2 to C-6; H-1, H 2 -8, and H-10 to C-9; H 3 -15 to C-2, C-3 and C-4; H 3 -16 to C-6, C-7 and C-8; H 3 -17 to C-10, C-11 and C-12; and both H 3 -19 and H 3 -20 to C-14 and C-18, permitted the assembly of the carbon skeleton of 1. Based on above results and HMBC correlations (Figure 1), the planar structure of 1 was established. Further, comparison of the NOE correlations of 1 ( Figure 2) with those of hirsutalin I, the relative configuration of 1 was thus determined to be the same.  Hirsutalin O (2) was also afforded as a colorless oil. Compound 2 has a molecular formula C 24 H 38 O 6 , as determined by HRESIMS. In comparing NMR data of 2 with those of the known compound simplexin A (9, Chart 3) [11], it was found that the n-butanoyloxy group at C-3 and the hydroxy group at C-6 in simplexin A (9) were replaced by a hydroxy group and acetoxy group in 2, respectively, as confirmed by the downfield shift of C-3 (δ C 81.3) of 1, relative to that of 2 (δ C 74.0), and the HMBC connectivity from H-6 (δ 5.19) to the carbonyl carbon resonating at δ 170.5 (C) ( Table 1). The relative configuration of 2 was confirmed to be the same as that of 9 by analysis of NOE correlations ( Figure 3). The new eunicellin, hirsutalin P (3), has a molecular formula C 23 H 40 O 6 as determined by HRESIMS. The spectroscopic data (IR, 1 H NMR, and 13 C NMR) of 3 were similar to those of a known one, klysimplex G (10, Chart 3) [12], except that the acetoxy group at C-3 and the hydroxy group at C-6 in 10 were replaced by a hydroxy group and methoxy group, respectively, in 3. The similar 1 H NMR data 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 Q (4) was obtained as a colorless oil and exhibited a molecular formula C 22 H 36 O 5 . IR absorptions of 4 showed the presence of hydroxy and carbonyl groups at 3421 and 1724 cm −1 , respectively. The NMR spectroscopic data revealed the presence of a trisubstituted double bond (δ H 5.28, s, 1H; δ C 128.4, CH and 139.4, C) ( Table 2). One ester carbonyl (δ C 170.2) was assigned from the 13 C NMR spectrum and was HMBC correlated with an acetate methyl (δ H 1.99 s). The chemical shift of H 3 -15 at δ 1.18 indicated the presence of a hydroxy group substitution at C-3, the same as that in compounds 2 and 3. The presence of an acetoxy group at C-11 could be seen from the more downfield shift of H 3 -17 (δ 1.53), in comparison with that of H 3 -15 (δ 1.18). The planar structure of metabolite 1 was elucidated by analysis of COSY and HMBC correlations (Figure 1). The Z geometry of the double bond at C-7 and C-8 was evidenced by the presence of NOE correlation between H-8 and H 3  A structurally-related metabolite, hirsutalin R (5), was also isolated as a colorless oil with a molecular formula of C 28 H 42 O 7 . Two ester carbonyl carbons (δ C 169.0 and 173.5) were correlated in the HMBC spectrum with the methine proton (H-2′, δ H 4.76 t, J = 6.8 Hz) of a 2-butyryloxybutanoate unit. Moreover, the 13 C NMR spectroscopic data ( Table 2) of 5 showed the presence of two 1, 1-disubstituted carbon-carbon double bonds (δ C 147.7 (C) and 118.4 (CH 2 ); 145.2 (C) and 111.6 (CH 2 )). Comparison of the NMR data of 5 with those of hirsutalin C (11, Chart 3) [29] revealed that the only difference between both compounds is the replacement of the hydroxy group in hirsutalin C by a ketone (δ C 206.5) at C-6 in 5. The absolute configuration of hirsutalin A [29] and hirsutalin J [30] have been completely assigned based on NOE correlations and Mosher's method. Compounds 1-5 are likely in the same enantiomeric series as hirsutalin A and hirsutalin J, based on a shared biosynthetic pathway. Thus, these compounds are suggested to possess the absolute configurations as shown in formula 1-5.  Cytotoxicity of compounds 1-7 against the proliferation of a limited panel of cancer cell lines, including P388 (murine leukemia), K562 (human erythro myeloblastoid leukemia), A549 (human lung adenocarcinoma), and HT-29 (human colon adenocarcinoma), was evaluated. Compound 5 was found to exhibit cytotoxicity toward P388 and K562 cell lines with IC 50 values of 13.8 and 36.3 μM (Table 3). Compound 7 displayed cytotoxicity toward A549 cell line with IC 50 value of 37.2 μM. Other metabolites were found to be inactive against the four cancer cells. The neutrophil pro-inflammatory responses to compounds 1-7 were evaluated by suppressing N-formyl-methionyl-leucyl-phenylalanine/ cytochalasin B (fMLP/CB)-induced superoxide anion (O 2 •− ) generation and elastase release in human neutrophils, as shown in Table 4. At a concentration of 10 μg/mL, none of compounds were able to significantly reduce the expression of superoxide anion generation, relative to the control cells stimulated with fMLP/CB only. At the same concentration, compound 1 was found to significantly inhibit the elastase release (31.7% ± 3.2% inhibition) in the same fMLP/CB-stimulated neutrophils.

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-7100 HPLC apparatus with 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 400MR FT-NMR instrument (Varian Inc, Palo Alto, CA, USA) at 400 MHz for 1 H and 100 MHz for 13 C in CDCl 3 . 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 1-7 were performed using the Alamar Blue assay [32,33].

In Vitro Anti-Inflammatory Assay
Human neutrophils were obtained using dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were carried out according to previously described procedures. [34,35]. LY294002, a phosphatidylinositol-3-kinase inhibitor, was used as a positive control for inhibition of superoxide anion generation and elastase release with IC 50 0.6 ± 0.1 and 1.2 ± 0.3 μg/mL [36].

Conclusions
Five new eunicellin-type compounds, hirsutalins N-R (1-5) and two known eunicellin-type compounds (6 and 7), were discovered from the soft coral C. hirsuta. Compound 5 displayed cytotoxicity against the proliferation of P388 and K562 cancer cells possibly due to the presence of the α,β-unsaturated ketone group. Compound 1 was found to effectively inhibit the elastase release in FMLP/CB-induced human neutrophils.