Pinnisterols A–C, New 9,11-Secosterols from a Gorgonian Pinnigorgia sp.

Three new 9,11-secosterols, pinnisterols A–C (1–3), were isolated from a gorgonian coral Pinnigorgia sp., collected off the waters of Taiwan. The structures of these compounds were elucidated on the basis of spectroscopic methods. The new sterols 1 and 3 displayed significant inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils, and sterol 1 was found to show moderate cytotoxicity in hepatic stellate cells (HSCs).


Introduction
Studies on the chemical constituents of octocorals collected off the waters of Taiwan, at the intersection of the Kuroshio current and the South China Sea surface current, have led to the isolation of a series of interesting 9,11-secosterols from Cespitularia hypotentaculata [1], Cladiella hirsuta [2], Steroids of this type were found to possess interesting bioactivities, such as cytotoxic [1][2][3][4][5][6], antiinflammatory [3] and antiviral [5,6] activities. In our continuing investigation of bioactive natural products obtained from Formosan soft corals, three new 9,11-secosterols, pinnisterols A-C (1-3), were obtained from a gorgonial coral identified as Pinnigorgia sp. (phylum Cinidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea, family Gorgoniidae) (Figure 1). The structures of secosterols 1-3 were elucidated by spectroscopic methods and by comparison of their NMR features with those of related secosterol analogues. We report herein the isolation, structure determination and bioactivity of secosterols 1-3.

Results and Discussion
The new metabolite pinnisterol A (1) was isolated as a colorless oil, and its molecular formula was established as C 30 H 48 O 6 (seven degrees of unsaturation) from a sodium adduct at m/z 527 in the electrospray ionization mass spectrum (ESIMS) and further supported by a high-resolution electrospray ionization mass spectrum (HRESIMS) at m/z 527.33440 (calcd. for C 30 H 48 O 6 + Na, 527.33431). The 13 C and distortionless enhancement polarization transfer (DEPT) spectroscopic data of 1 showed that this compound has 30 carbons (Table 1), including seven methyls, seven sp 3 methylenes (including an oxymethylene), seven sp 3 methines (including two oxymethines), three sp 3 quaternary carbons (including one oxygenated quaternary carbon), three sp 2 methines and three sp 2 quaternary carbons (including one ketonic carbonyl and one ester carbonyl). The IR spectrum of 1 revealed the presence of hydroxy (ν max 3546 cm´1), ester (ν max 1736 cm´1) and α,β-unsaturated ketone (ν max 1683 cm´1) groups. The latter structural feature was confirmed by the presence of signals at δ C 204.9 (C-9), 139.5 (CH-7) and 136.6 (C-8) in the 13 C NMR spectrum. A disubstituted olefin was identified from the signals of carbons at δ C 134.3 (CH-22) and 133.1 (CH-23), and was confirmed by two olefin proton signals at δ H 5.24 (1H, m, H-22) and 5.22 (1H, m, H-23) ( Table 1). Four doublets at δ H 1.04 (3H, J = 6.8 Hz), 0.81 (3H, J = 6.8 Hz), 0.83 (3H, J = 7.2 Hz) and 0.91 (3H, J = 6.8 Hz) were due to the H 3 -21, H 3 -27, H 3 -26 and H 3 -28 methyl groups, respectively. Two sharp singlets for H 3 -18 and H 3 -19 appeared at δ H 0.74 and 1.31, respectively. In the 1 H NMR spectrum, one acetyl methyl signal (δ H 2.00, 3H, s) was observed. Therefore, metabolite 1 must be a tricyclic compound.  (Table 1). The relative configuration of 1 was elucidated from the correlations observed in a nuclear Overhauser effect spectroscopy (NOESY) experiment and by comparison of NMR data with those of a known secosterol, aplidiasterol B (3β,5α,6β,11-tetrahydroxy-9,11-secocholest-7-en-9-one) (4) (Figure 1), isolated from a Mediterranean ascidian Aplidium conicum [7]. The relative stereochemistries at C-3, C-5, C-6, C-10, C-13, C-14 and C-17 in 1 were found to be the same as those The configuration at C-24 was suggested to be R* on the basis of the 13 C NMR chemical shift of C-28 (δ C 17.6). It was reported that the 13 C NMR value of C-28 resonates at δ C 17.68 ppm in the 24R* epimer of a known sterol, (22E,24R)-24-methylcholesta-5,22-dien-3β-ol, with the same chain, and the 24S* epimer, (22E,24S)-24-methylcholesta-5,22-dien-3β-ol, has a relative 0.4 ppm downfield chemical shift ( Figure 3) [8]. Based on the above findings, the structure, including the relative configuration of 1, was suggested.  Pinnisterol B (2) was isolated as a colorless oil, and its molecular formula was established as C32H52O8 (seven degrees of unsaturation) by HRESIMS at m/z 587.35558 (calcd. for C32H52O8 + Na, 587.35544). The IR spectrum of 2 indicated the presence of hydroxy (3420 cm −1 ), ester (1728 cm −1 ) and α,β-unsaturated ketone (1678 cm −1 ) groups. The whole series of spectroscopic data obtained from one-dimensional (1D) and two-dimensional (2D) NMR experiments ( Table 2) clearly indicated that secosterol 2 had the same core structure as secosterol 1, the differences being limited to the presence in 2 of the addition of an acetoxy group to substitute the alkene at C-23. The 1 H and 13 C NMR data assignments of pinnisterol B (2) were compared with the values of 1. The HMBC correlations observed fully supported the locations of the functional groups, and, hence, pinnisterol B (2) was assigned as structure 2, with the same relative configurations as secosterol 1 in the core rings A-C; the chiral carbons C-3, C-5, C-6, C-10, C-13, C-14 and C-17 of 2 were identical to those of 1, and the 1 H and 13 C NMR chemical shifts and proton coupling constants were also in agreement.   Pinnisterol B (2) was isolated as a colorless oil, and its molecular formula was established as C32H52O8 (seven degrees of unsaturation) by HRESIMS at m/z 587.35558 (calcd. for C32H52O8 + Na, 587.35544). The IR spectrum of 2 indicated the presence of hydroxy (3420 cm −1 ), ester (1728 cm −1 ) and α,β-unsaturated ketone (1678 cm −1 ) groups. The whole series of spectroscopic data obtained from one-dimensional (1D) and two-dimensional (2D) NMR experiments ( Table 2) clearly indicated that secosterol 2 had the same core structure as secosterol 1, the differences being limited to the presence in 2 of the addition of an acetoxy group to substitute the alkene at C-23. The 1 H and 13 C NMR data assignments of pinnisterol B (2) were compared with the values of 1. The HMBC correlations observed fully supported the locations of the functional groups, and, hence, pinnisterol B (2) was assigned as structure 2, with the same relative configurations as secosterol 1 in the core rings A-C; the chiral carbons C-3, C-5, C-6, C-10, C-13, C-14 and C-17 of 2 were identical to those of 1, and the 1 H and 13 C NMR chemical shifts and proton coupling constants were also in agreement.  Pinnisterol B (2) was isolated as a colorless oil, and its molecular formula was established as C 32 H 52 O 8 (seven degrees of unsaturation) by HRESIMS at m/z 587.35558 (calcd. for C 32 H 52 O 8 + Na, 587.35544). The IR spectrum of 2 indicated the presence of hydroxy (3420 cm´1), ester (1728 cm´1) and α,β-unsaturated ketone (1678 cm´1) groups. The whole series of spectroscopic data obtained from one-dimensional (1D) and two-dimensional (2D) NMR experiments ( Table 2) clearly indicated that secosterol 2 had the same core structure as secosterol 1, the differences being limited to the presence in 2 of the addition of an acetoxy group to substitute the alkene at C-23. The 1 H and 13 C NMR data assignments of pinnisterol B (2) were compared with the values of 1. The HMBC correlations observed fully supported the locations of the functional groups, and, hence, pinnisterol B (2) was assigned as structure 2, with the same relative configurations as secosterol 1 in the core rings A-C; the chiral carbons C-3, C-5, C-6, C-10, C-13, C-14 and C-17 of 2 were identical to those of 1, and the 1 H and 13 C NMR chemical shifts and proton coupling constants were also in agreement.   (Table 3). It was found that the NMR signals of 3 were similar to those of 2, except that the signals corresponding to the 3-hydroxy group in 2 were replaced by signals for an acetoxy group in 3. The correlations obtained from a NOESY experiment of 3 also showed that the configurations of chiral centers in the core rings A-C in 3 were identical to those of 2. However, the configurations of chiral carbons C-23 and C-24 of secosterols 2 and 3 were not determined at this stage.  In the biological activity testing, secosterols 1 and 3 displayed significant inhibitory effects on the release of elastase (IC 50 = 3.32 and 2.81 µM) and inhibitory effects on the generation of superoxide anions (IC 50 = 2.33 and 2.50 µM) by human neutrophils (Table 4) [9,10]. Secosterol 2 did not show activity in the anti-inflammatory test, which indicated that an acetoxy substituent at C-3 would enhance the activity by comparison with the structure and anti-inflammatory data of 2 with those of 3. Furthermore, in the cytotoxicity testing, secosterol 1 was found to show moderate cytotoxicity towards the HSC-T6 cells at a concentration of 10 µM (inhibition rate 46.5%) after 24 h testing and secosterols 2 and 3 were not active at the highest concentration tested (Figure 4), implying that the functional groups in the side-chain of secosterols 1-3 would influence the activity.

General Experimental Procedures
Optical rotations were measured on a Jasco P-1010 digital polarimeter (Japan Spectroscopic

Anti-Hepatofibric Assay
The anti-hepatofibric effects of tested secosterols 1-3 were assayed using a WST-1 assay method. Anti-hepatofibric assays were carried out according to the procedures described previously [12].

Generation of Superoxide Anions and Release of Elastase by Human Neutrophils
Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were carried out according to previously described procedures [9,10]. Briefly, superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate.

Conclusions
Our continuing investigations demonstrated that octocorals belonging to the genus Pinnigorgia are good sources of 9,11-secosterols. Pinnisterols A (1) and B (2) are potentially anti-inflammatory, and may become lead compounds in future marine anti-inflammation drug development [13,14]. The results of this study suggested that continuing investigation of new secosterols together with examination of the potentially useful bioactivities of this marine organism is worthwhile for future drug development.