New Sinularianin Sesquiterpenes from Soft Coral Sinularia sp.

Abstract

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.

1. Introduction

The genus Sinularia is the most widely distributed soft coral, consisting of almost 90 species, of which more than 50 have been chemically examined [1,2,3,4,5]. Up to now, Sinularia has yielded many metabolites, including sesquiterpenes, diterpenes, alkaloids, and polyhydroxylated steroids [6,7,8,9,10,11,12]. These metabolites display a wide range of biological activities, such as antimicrobial, anti-inflammatory, and cytotoxic activities [13,14,15,16,17,18]. In our endeavor to explore the bioactive secondary metabolites from marine invertebrates, sinularianins A (1) and B (2) were reisolated along with four new sesquiterpenes, sinularianins C–F (3–6) from soft coral Sinularia sp., collected at Dongluo Island, Hainan province, 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.

2. 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).

Figure 1.

Structures of the compounds 1–6.

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 ¹H and ¹³C NMR data with those reported [10].

Sinularianin C (3) was isolated as a colorless oil. Its molecular formula was established as C₁₆H₂₂O₄ on the basis of the positive HRESIMS at m/z 301.1416 (Calcd for C₁₆H₂₂NaO₄, 301.1416), indicating six degrees of unsaturation (Supplementary Figure S1). The ¹H NMR spectrum (Table 1) revealed the presence of four singlet methyls (δ_H 1.00, 1.41, 1.86, 3.17), three methylene signals (δ_H 1.95, 1H, m; 1.53, 1H, m; 1.96, 1H, m; 1.56, 1H, m; 2.26, 1H, d, J = 16.0 Hz; 1.76, 1H, dd, J = 16.0, 5.0 Hz), three methine signals (2.00, 1H, d, J = 13.0 Hz; 2.43, 1H, m; 3.11, 1H, d, J = 5.0 Hz), and one olefinic proton (δ_H 7.17, 1H, d, J = 1.5 Hz) (Supplementary Figure S2). The ¹³C NMR spectra, together with HSQC, showed 16 signals for four methyls (δ_C 10.3, 20.5, 21.3, 50.8), three sp³ methylenes (δ_C 23.6, 35.7, 38.7), three sp³ methines (δ_C 41.1, 50.4, 59.5), three sp³ oxygenated quaternary carbons (δ_C 61.8, 84.2, 86.5), one sp² methine (δ_C 154.7), one sp² quaternary carbon (δ_C 129.5), and one carbonyl carbon (δ_C 175.6) (Supplementary Figures S3 and S4). Both the ¹H and ¹³C NMR spectra of 3 showed a close similarity to those of 2 [10]. However, the close comparison of the ¹³C 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).

Table 1.

¹H and ¹³CNMR spectroscopic data for compounds 3 (500/125 MHz, in MeOD, δ in ppm, J in Hz) and 4 (in CDCl₃).

Position	3		4
	1H	13C	1H	13C
1		84.2		78.4
2	1.95 m	35.7	1.92 m	43.1
	1.53 m		1.79 m
3	1.96 m	23.6	2.01 m	25.9
	1.56 m
4	2.43 m	41.1	2.90 m	41.8
5		61.8		137.5
6	3.11 d (5.0)	59.5	5.26 s	117.2
7	2.26 d (16.0)	38.7	2.53 m	39.4
	1.76 dd (16.0, 5.0)		1.91 m
8		86.5		85.6
9	2.00 d (13.0)	50.4	1.55 d (12.5)	55.5
10	1.00 s	21.3	1.26 s	28.5
11	1.41 s	20.5	1.74 s	20.5
12	7.17 d (1.5)	154.7	7.03 d (1.5)	150.9
13		129.5		129.7
14		175.6		174
15	1.86 s	10.3	1.94 d (1.5)	10.6
16	3.17 s	50.8

Figure 2.

Selected HMBC correlations (H → C)of compounds 3, 5, and 6.

The relative stereochemistry of 3 was established by the detailed analysis of correlations observed in the NOESY spectrum (Figure 3). In the NOESY spectrum, H-9 showed correlation with H-7β (δ_H 2.26, d, J = 16.0 Hz), which in turn correlated with H-12, suggesting the β orientations of H-9 and H-12. Furthermore, NOE interactions were observed between H₃-10/H-4, H₃-11/H-4, H₃-11/H-6, and H-6/H-7α (δ_H 1.76, dd, J = 16.0, 5.0 Hz), while both H₃-10 and H-4 did not show correlations with H-9, suggesting the α orientation of H₃-10, H₃-11, H-4, and H-6 (Supplementary Figure S6).

Figure 3.

Selected NOE correlations of compounds 3 and 4.

Sinularianin D (4) was isolated as a colorless oil. The ESI-MS showed the [M + Na]⁺ ion at m/z: 271 (Supplementary Figure S7). Its ¹H and ¹³C NMR spectroscopic data were also very similar to those of 2 (Supplementary Figures S8 and S9). However, a close inspection of their ¹H NMR spectroscopic data revealed some difference: H-4 and H₃-10 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 H₃-10, H-4, and H-7β, suggesting the β orientations of H-4, H-9, H-7β, and H₃-10 (Supplementary Figure S10).

Sinularianin E (5) was isolated as a colorless oil, and assigned the molecular formula of C₁₆H₂₄O₄ by the positive HRESIMS at m/z 303.1563 (Calcd for C₁₆H₂₄NaO₄, 303.1572) (Supplementary Figure S11). The ¹H and ¹³C NMR spectroscopic data of 5 indicated sixteen carbon signals: four singlet methyls, four methylenes, three olefinic methines, and five quaternary carbons (Supplementary Figures S12–S14). The ¹H NMR spectrum showed signals of four olefinic protons (δ_H 5.42, 1H, t, J = 7.0 Hz; 6.36, 1H, dd, J = 17.5, 10.5 Hz; 5.13, 1H, d, J = 17.5 Hz; 4.96, 1H, d, J = 10.5 Hz), one methoxy group (δ_H 3.72), two vinyl methyls (δ_H 2.14, s; 1.74, s), and one other methyl (δ_H 1.40, s) (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.

Table 2.

¹H and ¹³C NMR spectroscopic data for compounds 5 and 6 (500/125 MHz, in CDCl₃, δ in ppm, J in Hz).

Position	5		6
	1H	13C	1H	13C
1		176.5		203.2
2		72.9	5.86 s	131.2
3	3.15 d (17.5)	52.9		174.9
	2.80 d (17.5)
4		199.3	4.79 s	75.3
5	6.01 s	122.9	2.65 s	61.5
6		160.3		78
7	2.22 m	40.9	1.82 m	34.1
			2.12 m
8	2.34 m	25.8	2.19 m	22.1
	2.20 m
9	5.42 t (7.0)	130.8	5.52 m	132.3
10		135		134.1
11	6.36 dd (17.5, 10.5)	141	6.36 dd (17.0, 10.5)	141.5
12	5.13 d (17.5)	111.3	5.10 d (17.0)	110.7
	4.96 d (10.5)		4.94 d (10.5)
13	1.74 s	11.7	1.76 s	11.6
14	2.14 s	19.7	1.03 s	22.7
15	1.40 s	26.2	3.21 s	48.5
16	3.72 s	52.7	2.16 s	15.6

Sinularianin F (6) was isolated as a colorless oil. It was assigned a molecular formula of C₁₆H₂₄O₃ by positive HR-ESI-MS at m/z 287.1613 (Calcd for C₁₆H₂₄NaO₃, 287.1623) (Supplementary Figure S16). Analysis of ¹H and ¹³C NMR data revealed the presence of four methyl groups, three methylene carbons, five methine carbons, and four quaternary carbons (Supplementary Figures S17–S19). The ¹H NMR spectrum showed signals of five olefinic protons (δ_H 5.86, s; 5.52, m; 6.36, dd, J = 17.0, 10.5 Hz; 5.10, d, J = 17.0 Hz; 4.94, d, J = 10.5 Hz), one oxygenated methane (δ_H 4.79, s), one methoxy group (δ_H 3.21, s), two vinyl methyls (δ_H 1.76, s; 2.16, s), and one other methyl (δ_H 1.03, s) (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 H₃-16 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 H₂O 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].

Table 3.

Inhibitory rates of NF-κB activation of compounds 1–6.

Concentration	IR (%)
	1	2	3	4	5	6
10 µg/mL	41.3	29.6	24.3	43.0	30.0	36.1

3. Experimental Section

3.1. 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 LC/MS. Materials for column chromatography were silica gel (100–200, 200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China), Sephadex LH20 (40–70 µm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), and YMC Gel ODS-A (12 nm, S-50 µm YMC, MA, USA). The silica gel GF₂₅₄ (0.4–0.5 mm) used for TLC were supplied by the Qingdao Marine Chemical Factory, Qingdao, China. HPLC was carried on shimadzu LC-10ATvp with YMC ODS SERIES (YMC-Pack ODS-A, 250 × 10 mm I.D., S-5 µm, 12 nm).

3.2. 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.

3.3. 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 H₂O (4 L) and CHCl₃ (4 L); the CHCl₃ 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 CHCl₃/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 MeOH/H₂O = 65:35 at a flow rate of 2 mL/min, to afford 1 (6.0 mg) and 2 (7.2 mg). Fr. s5 was purified by Sephadex LH-20 using CHCl₃/MeOH = 1:1 to yield 3 portions, and portion 1 was further purified with semi-preparative HPLC, eluting with MeOH/H₂O = 57:43 at a flow rate of 2 mL/min, to afford 5 (2.2 mg) and 6 (2.6 mg). Fr. s6 was separated by silica gel column using PE/EtOAc to yield 7 portions, and portion 1 was further purified with semi-preparative HPLC, eluting with MeOH/H₂O = 70:30 at a flow rate of 2 mL/min, to afford 3 (2.2 mg) and 4 (3.7 mg).

Sinularianin C (3): Colorless oil; ¹H- and ¹³C-NMR (see Table 1); HR-ESI-MS m/z 301.1416 [M + Na]⁺, (Calcd for C₁₆H₂₂NaO₄, 301.1416).

Sinularianin D (4): Colorless oil; = −6.0 (c = 0.01, MeOH); UV (MeOH): λ_max (log ε) = 204.2 (1.70); IR (KBr) ν_max 3421, 2927, 2854, 1735, 1666 cm^–1 (Supplementary Figure S21); ¹H- and ¹³C-NMR (see Table 1); ESI-MS m/z 271 [M + Na]⁺, 519 [2M + Na]+.

Sinularianin E (5): Colorless oil; ¹H- and ¹³C-NMR (see Table 2); HR-ESI-MS m/z 303.1563 [M + Na]⁺, (Calcd for C₁₆H₂₄NaO₄, 303.1572).

Sinularianin F (6): Colorless oil; ¹H- and ¹³C-NMR (see Table 2); HR-ESI-MS m/z 287.1613 [M + Na]⁺, (Calcd for C₁₆H₂₄NaO₃, 287.1623).

3.4. The Cell-Based HEK293 NF-κB Luciferase Reporter Gene Assay

All compounds were evaluated for inbibition of NF-κB activation using the cell-based HEK 293 NF-κB luciferase reporter gene assay according to the previously reported procedures [19].

4. Conclusions

The investigation of bioactive natural products from a Hainan soft coral, Sinularia sp., has led to the isolation of four new sesquiterpenes, sinularianins C–F (3–6), along with two other analogues, sinularianins A (1) and B (2). Compounds 1 and 4 were exhibited a potent inhibitory effect with inhibitory rates of 41.3% and 43.0% at the concentration of 10 µg/mL, respectively. The discovery of new compounds 3–6 has added to an extremely diverse and complex array of soft coral sesquiterpenes.

Supplementary Files

Supplementary File 1

Supplementary Information (PDF, 1507 KB)

Conflicts of Interest

The authors declare no conflict of interest.