New sesquiterpenoids from the South China Sea soft corals Clavularia viridis and Lemnalia flava

A detailed chemical investigation of the South China Sea soft corals Clavularia viridis and Lemnalia flava yielded four new halogenated laurane-type sesquiterpenoids, namely, isobromolaurenisol (1), clalaurenol A (2), ent-laurenisol (3), clalaurenol B (4), and the new aromadendrane-type sesquiterpenoid claaromadendrene (6), together with three known sesquiterpenoids (5, 7, and 8). Their structures were determined by extensive spectroscopic analysis and by comparison with the previously reported analogues. In a bioassay, compounds 1, 2 and 4 exhibited interesting inhibitory activities in vitro against PTP1B and NF-κB.


Introduction
Marine soft corals are important sources of biologically active compounds, which made them attractive targets for natural product chemists. Soft corals of the genus Clavularia (class Octocorallia, order Alcyonacea, family Clavulariidea), are prolific sources of numerous biologically active compounds [1][2][3][4]. A variety of structurally unique sesquiterpenes, including aromadendranes [5], maalianes [5], elemanes [6], and trinorguaianes [7][8][9], have been isolated since the early 1980s from several species of Clavularia. Soft corals of the genus Lemnalia are also a rich source of sesquiterpenoids and diterpenoids with various intriguing carbon skeletons, such as nardosinanes, neolemnanes, and ylanganes [10]. Many of these secondary metabolites have attracted a lot of attention for further synthetic and pharmacological studies due to their potent bioactivities ranging from neuroprotective, cytotoxic, to anti-inflammatory properties [10]. In the framework of our ongoing research for the bioactive metabolites from South China Sea soft corals [11,12], we made the collection of the title samples Clavularia viridis and Lemnalia flava off the Xisha Islands, Hainan Province, China. The chemical investigation of two title animals led to the isolation of four new halogenated laurane-type sesquiterpenoids 1-4, one new aromadendrane-type sesquiterpenoid 6) together with three related known compounds 5, 7 and 8 ( Figure 1). Herein, the isolation, structure elucidation and bioactivity evaluation of these compounds are presented.
Isobromolaurenisol (1) was obtained as an optically active colorless oil. Its molecular formula, C 15 H 18 OBr 2 , was deduced by HR-ESIMS with ion peaks at m/z 370.9657, [M − H] -(calcd for C 15 H 17 OBr 2 , 370.9646), indicating six degrees of unsaturation. The 13 C NMR and DEPT spectra contained signals attributable to three methyls, two sp 3 methylenes, one sp 3 methine, one sp 3 quaternary carbon, three sp 2 methines, and five sp 2 quaternary carbons ( Table 1). The typical resonances at δ C 145.6, δ C 113.0, δ H/C 7.30/136.8, δ C 123.4, δ C 153.0, δ H/C 6.71/116.8 revealed the presence of a 1,2,4,5-tetrasubstituted benzene ring, and the signals at δ H/C 6.08/99.1, δ C 154.2 indicated the existence of a trisubstituted double bond. All the above evidence suggested the laurane nature of this molecule, and literature research revealed that 1 should be an isomer of a known laurane-type terpenoid bromolaurenisol (1a) [16,17] due to their extremely similar NMR data and the same molecular weight (Figure 1). In fact, the main difference between 1 and 1a happened only at the tetrasubstituted benzene ring with the substituents exchange between C-7 and C-10 ( Figure 1). The assignment of the planar structure of 1 has been further confirmed by 2D NMR experiments, including 1 H, 1 H COSY, HSQC, and HMBC, with the key correlations shown in Figure 2. In particular, the hydroxy group (δ H 4.68, s) was confirmed to be attached at C-10 by the clear HMBC correlation from OH to C-10 and C-11.
The relative configuration of 1 was established by a NOESY experiment (Figure 3), in which the correlations of H 3 -13 (δ H 1.29, s) with H-2 (δ H 3.56, q, J = 7.2 Hz) and H-5β (δ H 2.34, m) indicated that these protons were on the same side of the molecule and were tentatively assigned to be β-oriented, while the correlation of H-5α (δ H 1.88, m) and H 3 -14 (δ H 0.74, d, J = 7.3) at C-2 indicated CH 3 -14 was α-oriented. Besides, the trisubstituted olefin (Δ 3/15 ) was determined to be in E configuration due to the clear NOE correlations of H-15 with H 3 -13 and H 3 -14. In view of the above evidences, the relative configuration of compound 1 was determined as 1R*,2R*, the same as 1a [16,17].
Compound 2 was isolated as an optically active colorless oil. The molecular formula, C 15 H 19 OBr, was established by the mo-   13 C NMR spectra showed great similarities with those of the co-occurring 1, which indicated the same laurane skeleton. In fact, compound 2 differed from 1 only by the debromonation at the C-15 position, which was in agree with the lack of 78/80 units in its mass compared to that of 1. The planar structure of 2 was further confirmed by its 2D NMR data ( Figure 2). The relative configurations of the chiral centers on the cyclopentane ring were determined to be the same as 1 by inspection of the proton coupling constants (Table 1) and NOESY experiments ( Figure 3). Thus, compound 2 was determined to be the debrominated derivative of 1, namely, clalaurenol A.  [α] D 20 −16.0 (c 0.10, MeOH)} were found to be opposite to that of laurenisol (+85.9) [18]. Thus, compound 3 can be assigned as the enantiomer of 3a, named ent-laurenisol.
Clalaurenol B (4) was obtained as an optically active colorless oil. The molecular formula, C 15 (Figure 2), revealed that compound 6 had the same planar structure as 6a and co-occurring 7 differing only in the stereochemistry. The relative configura-tion of 6 was established by NOESY correlations (Figure 3) in which the correlations of H-6 (δ H 0.62, dd, J = 11.4, 9. suggesting these protons were on the opposite orientation. In view of the above evidences, the relative configuration of compound 6 was determined as 4R*,5S*,6R*,7R*. In fact, the only difference between compounds 6 and 6a was the configuration of the hydroxy group at C-1 with α-orientation for 6 while β-orientation for 6a [19,20]. Further, due to the influence of the configuration inversions of C-1, the 13 C NMR chemical shift of the carbon at C-1 (δ C 85.5, qC), was apparently upfield shifted (Δδ = −3.0) comparing to compound 6a (Table 2), giving the further support of the assigned structure for 6 ( Figure 1). Thus, compound 6 was determined as a C-1 isomer of ent-1-hydroxyalloaromadendrene (6a), namely, claaromadendrene.
In bioassays, all the isolated compounds were tested for protein tyrosine phosphase-1B (PTP1B) and NF-κB inhibitory activity.
In the PTP1B inhibitory assay, the inhibitory effects of compounds 1-8 were evaluated against PTP1B, and the result showed that compounds 1, 2 and 4 had a moderate PTP1B inhibitory activity with IC 50 values of 18.8, 21.8 and 15.6 μM, respectively. The known PTP1B inhibitor oleanolic acid (IC 50 = 3.0 μM) were used as positive control in this assay. In NF-κB inhibitory assay, compounds 2 and 4 showed the most potent NF-κB signaling pathway inhibition with IC 50 values of 6.8 and 7.3 μM, respectively, while compound 1 showed moderate activity with an IC 50 value of 19.9 μM (Table 3).
A # and B # , representing oleanolic acid and bortezomib, respectively, were used as the positive controls.

Conclusion
In summary, eight sesquiterpenoids (1)(2)(3)(4)(5)(6)(7)(8), belonging to four different structural types, were isolated from two South China Sea soft corals (C. viridis and L. flava) for the first time. The discovery of these metabolites extended the structural diversity and complexity of sesquiterpenoids derived from soft corals C. viridis and L. flava. In fact, to our knowledge, naturally occurring laurane-(1-4) and cuparane-derived (5) sesquiterpenoids, are extremely rare in soft corals. Previously, such sesquiterpenoids have only been isolated from the red algae of the genus Laurencia [14,16,17,21] and some sea hares that prey on it [13,22]. In this paper, the chemical investigation of two different soft corals collected off the South China Sea, which belong to two different genera, have resulted in the discovery of two common new halogenated laurane-type sesquiterpenoids (3 and 4). Based on these findings, other than prey-predator relationship, the common symbiotic organisms in the algae and the soft corals might be the source of these metabolites. In fact, many investigations have proved that [23] numerous natural products are actually produced by microbes and/or microbial interactions with the "host from whence it was isolated". Further chemical investigation of these soft corals in the South China Sea as well as their associated microorganisms should be conducted to verify the true origin of these metabolites and to further understand the real biological/ecological roles they played in the life cycle of the title animals in the South China Sea.
The promising PTP1B inhibitory activity of laurane-type sesquiterpenoids [24] in a previously report from our group, inspired us to test the PTP1B inhibitory activity of compounds 1-4. Among them, compound 3 was inactive against PTP1B enzyme, whereas compounds 1, 2 and 4 exhibited considerable PTP1B inhibitory activity with IC 50 values of 18.8, 21.8, and 15.6 μM, respectively. Compounds 1, 2 and 4 also showed strong NF-κB inhibitory activity with IC 50 values of 19.9, 6.8 and 7.3 μM, respectively. With regard to their structure-activity relationship, the bromine atom on the benzene ring may play the key functional role in the inhibitory activity. This study could thus provide a clue for the further biological study and structure modification of marine brominated laurane sesquiterpenoid derivatives towards new effective PTP1B and/or NF-κB inhibitors.

Experimental General experimental procedures
Optical rotations were measured on a Perkin-Elmer 241MC polarimeter. IR spectra were recorded on a Nicolet-Magna FT-IR 750 spectrometer. EIMS and HR-EIMS spectra were recorded on a Finnigan-MAT-95 mass spectrometer. HR-ESIMS spectra were recorded on a Q-TOF Micro LC-MS-MS mass spectrometer. The NMR spectra were measured on a Bruker DRX-500 spectrometer with the residual CHCl 3 (δ H 7.26 ppm, δ C 77.2 ppm) as internal standard. Chemical shifts are expressed in δ (ppm) and coupling constants (J) in Hz. 1

Collection of biological materials
The

Extraction and isolation
The lyophilized bodies of C. viridis (80 g, dry weight) were minced into pieces and exhaustively extracted with acetone at room temperature (4 × 1 L

PTP1B inhibitory activity assay
The recombinant PTP1B catalytic domain was expressed and purified according to a previous report [24]. The enzymatic ac-tivities of the PTP1B catalytic domain were determined at 30 °C by monitoring the hydrolysis of pNPP. Dephosphorylation of pNPP generated the product pNP, which was monitored at an absorbance of 405 nm with an EnVision multilabel plate reader (Perkin-Elmer Life Sciences, Boston, MA). In a typical 100 L assay mixture containing 50 mmol/L 3-morpholinopropanesulfonic acid, pH 6.5, 2 mmol/L pNPP, and 30 nmol/L recombinant PTP1B, activities were continuously monitored and the initial rate of hydrolysis was determined by using the early linear region of the enzymatic reaction kinetic curve. NF-κB signaling pathway inhibitory activity assays NF-κB signaling pathway inhibitory activity was evaluated according to the previously reported protocol [25]. Stable HEK293/NF-κB cells were plated into 384-well plates at a concentration of approximate 2500 cells per well. After culturing overnight, compounds were added to the medium at a final concentration of 0.1 μg/mL. HEK293/NF-κB cells were seeded into 96-well cell culture plates (Corning, NY, USA) and allowed to grow for 24 h. The cells were then treated with compounds, followed by stimulation with TNF-α. 6 h later, the luciferase substrate was added to each well, and the released luciferin signal was detected using an EnVision microplate reader. The IC 50 was calculated with Prism 4 software (Graphpad, San Diego, CA) from the nonlinear curve fitting of the percentage of inhibition (% inhibition) versus the inhibitor concentra-

Supporting Information
Supporting Information File 1 Spectral data of compounds 1-4 and 7.