New Sinularianin Sesquiterpenes from Soft Coral Sinularia sp.

Four new sesquiterpenes, sinularianins C–F (3–6), together with known sinularianins A (1) and B (2) were identified from a South China Sea soft coral Sinularia sp. Compounds 1–6 were evaluated for inhibition of NF-κB activation using the cell-based HEK293 NF-κB luciferase reporter gene assay. Compounds 1 and 4 were exhibited a potent effect with inhibitory rates of 41.3% and 43.0% at the concentration of 10 µg/mL, respectively.


OPEN ACCESS
China, at a depth of 10 m. Sinularianin A and B have been isolated from the Formosan coral Sinularia sp., but their anti-inflammatory activation were tested for the first time. Similar sesquiterpenes had been isolated mostly from the plant Valeriana officinalis, which was used as an anti-inflammatory remedy in Europe, and were active as inhibitors of NF-κB [19]. In this paper, we describe the isolation, structure elucidation, and the NF-κB inhibitory potential of these compounds.

Results and Discussion
The soft coral Sinularia sp. was dissolved in 85% EtOH, and the extract separated by silica gel column chromatography, Sephadex LH-20, and semi-preparative HPLC to obtain new sesquiterpenes, sinularianins C-F (3-6), and two known compounds (1, 2) ( Figure 1). Sinularianins A (1) and B (2) were previously isolated from the soft coral Sinularia sp., collected off the northeastern Taiwan coast, in May 2004, at a depth of 10 m. Sinularianin A (1) possesses an unprecedented bicyclic skeleton sinulariolane. Sinularinin B (2) was the only example of valerenane-related sesquiterpene with a spiro-butenolide moiety [10]. The valerenane-related sesquiterpenes had been firstly reported from the plant Valeriana officinalis [20,21], and several representatives have been reported from a marine alga [22] and a soft coral [23]. Sinularinin A (1) and B (2), were reisolated and identified by comparison of their 1 H and 13 C NMR data with those reported [10].
Sinularianin C (3) was isolated as a colorless oil. Its molecular formula was established as C 16 Figures S3 and S4). Both the 1 H and 13 C NMR spectra of 3 showed a close similarity to those of 2 [10]. However, the close comparison of the 13 C NMR spectroscopic data of 2 and 3 revealed some differences: one trisubstituted double bond in 2 was changed to the epoxy three-menbered ring (δ C 61.8, 59.5) in 3, and an additional methoxyl (δ C 50.8, δ H 3.17, 3H, s, H-16) was observed in 3. This assumption was supported by the correlation of H-11 to C-4, C-5, and C-6, H-6 to C-5, and C-7, H-7 to C-5 and C-6 in the HMBC spectrum ( Figure 2). Furthermore, the methoxyl substituent was determined to be connected to position C-1 on the basis of the HMBC correlation from 16-OMe to C-1 (Supplementary Figure S5).    Figure S7). Its 1 H and 13 C NMR spectroscopic data were also very similar to those of 2 ( Supplementary Figures S8 and S9). However, a close inspection of their 1 H NMR spectroscopic data revealed some difference: H-4 and H 3 -10 were shifted downfield from 2.57 to 2.90, and from 1.12 to 1.26 respectively, and H-9 was shifted upfield from 1.99 to 1.55. This suggested that the configuration at H-1 and H-4 in 4 should be β orientation compared to α orientation in 2, which was supported by the NOESY experiment ( Figure 3). In the NOESY spectrum, H-9 showed correlation with H 3 -10, H-4, and H-7β, suggesting the β orientations of H-4, H-9, H-7β, and H 3 -10 (Supplementary Figure S10).
Sinularianin E (5) was isolated as a colorless oil, and assigned the molecular formula of C 16 Table 2). The gross structure of 5 was established by the assistance of extensive 2D NMR analysis ( Figure 2). The methoxycarbonyl was confirmed by HMBC correlations from 16-OMe to C-1. The methyl protons resonating at δ H 1.40 and the quaternary carbon resonating at δ C 72.9 indicated that this methyl and a hydroxyl group should be positioned at C-2 by the HMBC correlations from H-15 to C-1, C-2, and C-3 (Supplementary Figure S15). The olefinic methyls (δ H 2.14, s; 1.74, s) attached at C-6 and C-10 were confirmed by the HMBC correlations from H-14 to C-5, C-6, and C-7 and H-13 to C-9, C-10, and C-11. Furthermore, the HMBC correlations from H-9 to C-8, and C-10, H-12 to C-10, and C-11 established the terminal diene unit. Other key informative HMBC correlations from H-3 to C-2, and C-4, H-5 to C-4, H-8 to C-7, C-9, and C-10, established the planar structure of 5. The double bond at C-5 was assigned the Z-geometry on the basis of the downfield chemical shifts of C-14 (δ H 19.7) [24]. The geometry of the disubstituted double bond (C-9) was determined to be E by comparison of the spectral data with those reported in literature [24], whereas the configurations at C-2 remained to be determined. On the basis of above evidences, compound 5 was then identified, and named sinularianin E.  Figure S16). Analysis of 1 H and 13 C NMR data revealed the presence of four methyl groups, three methylene carbons, five methine carbons, and four quaternary carbons (Supplementary Figures S17-S19).  Table 2). The HMBC correlations from H-9 to C-8, and C-10, H-12 to C-10, and C-11, H-13 to C-10, and C-11 established the terminal diene unit (Supplementary Figure S20). The key HMBC correlations of H 3 -16 to C-2, C-3, and C-4 and H-2 to C-1, C-3, C-4, and C-5 indicated the presence of a five-membered carbocycle containing a ketone carbonyl and a trisubstituted double bond (Figure 2), as well as by comparison of the data with that of in agreement with the data of cycloabiesesquine A [25]. The two fragments may be connected via the correlations of H-15 to C-5, C-6, and C-7, H-14 to C-6 and H-7 to C-6, C-7, and C-8 in the HMBC spectrum. Two double bonds in the molecule possessed 2Z and 9E configuration on the basis of the chemical shifts of C-16 and C-13 (δ 15.6 and 11.6, respectively) [24,25].
Although sinularianins E (5) and F (6) formally displayed a quite different skeleton from that of sinularianins A-D (1-4), however, they are actually related to each other. From a biosynthetic aspect, sinularinins A-D (1-4), and F (6) could be generated from sinularinin E (5), via different reaction cascades as illustrated in the hypothetical biosynthetic pathway (Scheme 1). As a precursor, sinularianin E (5) potentially could be transformed into the key intermediate A by dehydration reaction. Intermediate A could be through different intramolecular Diels Alder cyclization reaction to form sinularianin A (1) or valerenolic acid, respectively, and the latter was further modified to produce sinularianin B (2). Intermediate A could be also adapted by Michael addition under the H 2 O attack and then immediately lactonized, followed by a DA cyclization to yield sinularianin B (2), which after epoxidation and dehydration potentially could be produce epoxide sinularianin C (3). Intermediate A might form sinularianin F (6) by an aldol condensation.

Scheme 1. Proposed biosynthetic pathway for 1-6.
Nuclear factor-κ B (NF-κB) plays a key role in regulating the immune response to infection. Incorrect regulation of NF-κB has been linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development [26]. Compounds 1-6 were evaluated for inhibition of NF-κB activation using the cell-based HEK293 NF-κB luciferase reporter gene assay. At concentration of 10 µg/mL, sinularianin A and D exhibits a potent effect with inhibitory rates of 41.3%, and 43.0%, respectively. At the same concentration, other compounds showed moderate effects at the same ( Table 3). The past studies have provided biochemical evidence of valerenane-related sesquiterpenes as anti-inflammatory agents acting via the NF-κB inhibitory potential. The valerenic acid (3) reduced NF-κB activity to 25% at concentration of 100 µg/mL [19].

General Experimental Procedures
The NMR spectra were recorded on a Bruker AC 500 NMR spectrometer with TMS as an internal standard. IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer. UV spectra were recorded on a Shimadzu UV-2600 UV-Vis spectrophotometer. Optical rotations were measured on a PerKin Elmer 341 polarimeter using a 1 dm path length cell. HR-ESI-MS data were measured on AQUITY UPLC/Q-TOF mass spectrometer. ESI-MS data were measured on Bruker's amaZon SL ion trap

Animal Material
The soft coral Sinularia sp. was collected from Dongluo Island, Hainan province of China in July 2009 (7-10 m depth) and identified by Professor Hui Huang, South China Sea Institute of Oceanology, Chinese Academy of Sciences. A voucher specimen (No. 0907010) was deposited in the CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.

Extraction and Isolation
The fresh soft coral (wet, 6 kg) was extracted three times with 95% EtOH (20 L). The extract was concentrated under reduced pressure, and partitioned between H 2 O (4 L) and CHCl 3 (4 L); the CHCl 3 layer (120 g) was further partitioned between 85% EtOH (4 L) and petroleum ether (PE; 4 L) to yield 85% EtOH (34 g) and PE (75.6 g) fractions. The 85% EtOH fraction was separated by silica gel column using CHCl 3 /MeOH to yield 11 portions (Frs. s1-s11). Fr. s3 was purified by silica gel column to yield 12 portions, and portion 10 was further purified with semi-preparative HPLC, eluting with