Natural Cembrane Diterpenoids From the Soft Coral Sinularia querciformis

Abstract

Chemical examination of Sinularia querciformis afforded one new cembranoid, querciformolide F (1), along with four known cembranoids, sinulariolone (2), granosolide A (3), querciformolide A (4), and sinulariolide (5). The structures of these compounds were determined by extensive spectroscopic (IR, ESIMS, ¹H NMR, and ¹³C NMR) analysis and by comparison with those previously reported in the literature. Compounds 2 to 4 were found to exhibit significant anti-inflammatory activity in lipopolysaccharide (LPS)-induced RAW264.7 mouse macrophages through attenuating the expression of the inducible nitric oxide synthase (iNOS) proteins.

Introduction

Octocorals belonging to the genus Sinularia (phylum Cnidaria, class Anthozoa, order Alcyonacea, family Alcyoniidae) have been found to be a rich source of cembranoid diterpenes^1-7 and steroids^8-9. Compounds of these two types from Sinularia are reported to exhibit anti-inflammatory,^6-7 and cytotoxic^3-6 activities. In order to discover marine natural products with new chemical structures and bioactive activities, we conducted research on the soft coral Sinularia querciformis collected from the waters of Southern Taiwan, leading to the isolation of one new cembranoid, querciformolide F (1), along with four known cembranoids, sinulariolone (2), ¹⁰ granosolide A (3), ¹¹ querciformolide A (4), ¹¹ and sinulariolide (5) ¹² (Figure 1). The anti-inflammatory potential of compounds 1 to 5 were assayed against the expression of the pro-inflammatory iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) proteins in lipopolysaccharide (LPS)–stimulated RAW264.7 macrophagic cells.

Figure 1.

Structures of compounds 1 to 5.

Results and Discussion

Querciformolide F (1) was isolated as a colorless oil that showed a sodiated adduct ion peak in its HRESIMS at m/z 389.19354 (M  +  Na)⁺, accounted for the molecular formula, C₂₀H₃₀O₆ (Calcd for C₂₀H₃₀O₆  +  Na, 389.19346), indicating six degrees of unsaturation in the compound. The IR spectrum revealed absorptions for hydroxyl (ν_max 3391 cm⁻¹) and ε-lactone (ν_max 1710cm⁻¹) groups. The ¹³C-NMR spectrum of 1 (Table 1), showed signals of 20 carbons, which were further identified by the assistance of the distortionless enhancement by polarization transfer (DEPT) spectrum as three methyls, six sp³ methylenes, three sp³ methines (including two oxymethines), one sp² methylene, two sp² methines, three sp³ quaternary carbons and two sp² quaternary carbons (including one ester carbonyl). The NMR signals observed at δ_C 169.4 (C), 144.3 (C), 124.7 (CH₂), 88.7 (C), 35.7 (CH), 33.4 (CH₂), 32.2 (CH₂) and δ_H 6.31 (s), 5.47 (s) showed the presence of an α-methylene-ε-lactone ring by comparing the very similar NMR data of the cembranoids with the same seven-membered lactone ring. Signals resonating at δ_C 61.1 (C), 61.3 (CH) and δ_H 2.89 (1H, dd, J  =  10.4, 3.2 Hz) revealed the presence of a trisubstituted epoxide. The NMR signals at δ_C 83.6 (C) and δ_H 7.34 (1H, brs) showed the presence of a hydroperoxy group at a methine carbon. One 1,2-disubstituted double bond was also identified from NMR signals appearing at δ_C 125.9 (CH), 134.4 (CH), and δ_H 5.44 (1H, d, J  =  16.0 Hz) and 5.68 (1H, ddd, J  =  16.0, 10.0, 4.8 Hz).

Table 1.

¹H and ¹³C NMR Spectroscopic Data for Querciformolide F.

Position	δH (J in Hz) a	δC, b type c
1	2.68 dd (12.0, 7.2)	35.7,	CH
2	1.42 m	32.9,	CH2
3	2.89 dd (10.4, 3.2)	61.3,	CH
4		61.1,	C
5/5´	2.74 dd (12.0, 4.8); 1.71 dd (12.0, 10.0)	42.9,	CH2
6	5.68 ddd (16.0, 10.0, 4.8)	125.9,	CH
7	5.44 d (16.0)	134.4,	CH
8		83.6,	C
9	2.03 m	33.3,	CH
10/10´	1.92 m; 1.61 m	27.9,	CH2
11	3.48 d (10.0)	76.7,	CH
12		88.7,	C
13	1.48 m	33.4,	CH2
14	2.31 m	32.2,	CH2
15		144.3,	C
16		169.4,	C
17/17´	6.31 s; 5.47 s	124.7,	CH2
18	1.29 s	16.4,	CH3
19	1.40 s	24.9,	CH3
20	1.43 s	27.5,	CH3
OOH	7.34 br s

^a

Spectra recorded at 400 MHz in CDCl₃ at 25 °C.

^b

Spectra recorded at 100 MHz in CDCl₃ at 25 °C.

^c

Multiplicity deduced by DEPT spectra.

From the ¹H–¹H COSY spectrum of 1, the separate spin systems of H₂ to 13/H₂ to 14/H-1/H₂ to 2/H-3, H₂ to 5/H-6/H-7 and H₂ to 9/H₂ to 10/H-11 enabled the identification of three different structural units, which were assembled with the assistance of an HMBC experiment. These data, together with the key HMBC correlations of H₃ to 18 to C-3, C-4 and C-5; H₃ to 19 to C-7, C-8 and C-9; H₃ to 20 to C-11, C-12 and C-13 and H₂ to 17 to C-1, C-15 and C-16 permitted the establishment of the carbon skeleton. The relative configurations of the six chiral centers at C-1, C-3, C-4, C-8, C-11, and C-12 in 1 were elucidated by detailed analysis of the nuclear Overhauser effect (NOE) correlations (Figure 2). H-1 (δ_H 2.68, dd, J  =  12.0, 7.2 Hz) showed an NOE interaction with H-11 (δ_H 3.48, d, J  =  10.0 Hz), and H₃ to 18 (δ_H 1.29, s); therefore, assuming the α-orientation of H-1, H-11 and H₃ to 18 this should also be positioned on the α-face. One of the methylene protons at C-10 (δ_H 1.92, m) exhibited NOE correlation with H-11 and was characterized as H-10α, while the other (δ_H 1.61, m) was assigned as H-10β. NOE correlations observed between H₃ to 18 and H-6 (δ_H 5.68, ddd, J  =  16.0, 10.0, 4.8 Hz), H-6 and H-5α (δ_H 2.74, dd, J  =  12.0, 4.8 Hz), reflected the α-orientation of H-1, H-5α, H-6, H-10α, H-11, and H₃ to 18. The J values for both H-6 and H-7 (16.0 Hz) further confirmed the E-configuration of the 6,7-double bond. Also, the NOE correlations observed for H-7 (δ_H 5.44, d, J  =  16.0 Hz) with H₃ to 19 (δ_H 1.40, s), and H₃ to 20 (δ_H 1.43, s), H₃ to 19, and H-10β, reflected the β-orientation of H-7, H-10β, H₃ to 19, and H₃ to 20. From the above observations and further analysis of other NOE interactions, the 1R*, 3S*, 4S*, 8R*, 11R* and 12R* relative configurations of 1 were established (Supplemental Materials, Figures S1–S10).

Figure 2.

Key COSY (Fx1), HMBC (Fx2), and protons with NOESY (Fx3) correlations of 1.

The in vitro anti-inflammatory activities of compounds 1 to 5 were measured by examining the inhibition of LPS (lipopolysaccharide)-induced upregulation of iNOS (inducible nitric oxide synthetase) and COX-2 (cyclooxygenase-2) proteins in macrophages using Western blotting analysis. RAW264.7 cells were obtained from the American Type Culture Collection (ATCC TIB-71, Manassas, VA, USA). ¹³ Compounds 2 to 4 were found to inhibit iNOS release by 82.8, 84.5, and 77.3%, respectively (Figure 3 and Table 2).

Figure 3.

Effect of compounds 1 to 5 (10 μM) on pro-inflammatory iNOS and COX-2 protein expressions in LPS-stimulated murine macrophage cell line RAW264.7 by western blotting analysis. Data were normalized to those of cells treated with LPS alone, and the cells treated with dexamethasone (10 μM) were used as a positive control. Data are expressed as the mean  ±  SEM (n  =  3). Significant difference from the cells treated with LPS (p < .05).

Table 2.

Effects of Compounds 1 to 5 on LPS-Induced iNOS and COX-2 Protein Expressions in Macrophages.

Compounds (10 μM)	Inos	COX-2
	Expression (% of LPS)
Control	0.44  ±  0.07	0.93  ±  0.33
Vehicle	100.0  ±  3.5	100.0  ±  3.0
1	94.2  ±  3.8	100.1  ±  2.0
2	82.8  ±  3.1	95.7  ±  2.9
3	84.5 ±  2.7	98.6  ±  4.3
4	77.3  ±  3.9	97.2  ±  4.9
5	102.1  ±  7.4	108.5  ±  6.4
DEX a	65.6  ±  1.4	22.0  ±  4.3

^a

Dexamethasone (DEX, 10 μM) was used as a positive control.

Materials and Methods

General Experimental Procedures

A digital polarimeter (model P-1010; JASCO Corp., Tokyo, Japan) was used to determine optical rotations of the samples. IR spectra were collected using a Nicolet iS5 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). NMR spectra were taken on a Varian NMR Mercury Plus spectrometer operating at 400 MHz for ¹H and 100 MHz for ¹³C in CDCl₃ using the residual CHCl₃ signal (δ_H 7.26 ppm) and CDCl₃ (δ_C 77.1 ppm) as the internal standard for ¹H and ¹³C NMR, respectively; coupling constants (J) are given in Hz. For ESIMS and HRESIMS, the results were obtained using a SolariX FTMS mass spectrometer (7 Tesla; Bruker, Bremen, Germany). Gravity column chromatography was performed on silica gel (range, 230 to 400 mesh, Merck). TLC was carried out on precoated Kieselgel 60 F₂₅₄ (0.2 mm, Merck) and compounds were visualized by spraying with 10% H₂SO₄ solution followed by heating. High-performance liquid chromatography (HPLC) was performed using a system comprised of a Hitachi L-7100 pump and a Rheodyne 7725 injection port. A semi-preparative normal phase column (Hibar 250 × 10 mm, Supelco, silica gel 60, 5 μm) was used.

Animal Material

Specimens of S. querciformis were manually collected by an underwater diver at a depth of 10 to 15 m from the sea around southern Taiwan on third June 2016. A representative sample of the soft coral (voucher no.: NMMBA-TW-SC-2015-0603) is stored in the National Museum of Marine Biology and Aquarium, Taiwan.

Extraction and Isolation

S. querciformis (wet/dry weight  =  1870/570 g) was sliced and extracted with a mixture of methanol: dichloromethane (1:1). The extract was partitioned between ethyl acetate (EtOAc) and H₂O. The EtOAc layer (7.60 g) was applied to a silica gel column and eluted with a gradient of n-hexane : EtOAc : acetone (from 100% n-hexane to 100% acetone) to yield 13 fractions, A–M. Fraction I was chromatographed using Si gel CC using n-hexane : EtOAc (1:1) to obtain fractions I1-I19. Fraction I11 was separated by NP-HPLC using a mixture of n-hexane : acetone (7:3) to yield querciformolide F (1) (1.7 mg). Fraction L was chromatographed by Si gel CC using n-hexane : EtOAc : acetone (from 100% n-hexane to 100% acetone) to obtain fractions L1-L12. Fraction L7 was separated by NP-HPLC using a mixture of n-hexane : acetone (3:1) to yield sinulariolone (2) (3.9 mg). Fraction I7 was separated by NP-HPLC using a mixture of n-hexane : acetone (4:1) to yield granosolide A (3) (0.7 mg), and sinulariolide (5) (2.7 mg). Fraction G was chromatographed by Si gel CC using n-hexane : EtOAc : acetone : MeOH (from 100% n-hexane to 100% MeOH) to obtain fractions G1-G15. Fraction G8 was separated by NP-HPLC using a mixture of n-hexane : EtOAc (4:3) to yield querciformolide A (4) (2.9 mg).

Querciformolide F (1). colorless oil; [α] −27.7 (c 0.1, CHCl₃); IR ν_max 3391, 2923, 1710, 1260 cm^–1; ¹H (CDCl₃, 400 MHz) and ¹³C (CDCl₃, 100 MHz) NMR data, see Table 1; ESIMS m/z 389 (M  +  Na)⁺; HRESIMS m/z 389.19354 (M  +  Na)⁺ (Calcd for C₂₀H₃₀O₆  +  Na, 389.19346).

Anti-Inflammatory Test

A macrophage (RAW264.7) cell line was purchased from ATCC. We measured the in vitro anti-inflammatory activities of 1 to 5 by examining the inhibition of LPS-simulated upregulation of the iNOS (inducible nitric oxide synthetase) and COX-2 (cyclooxygenase-2) proteins in macrophages using Western blotting analysis. ¹³

Conclusion

One new cembranoid, querciformolide F (1), and four known cembranoids were isolated from the soft coral Sinularia querciformis. These characteristic α-methylene-ε-lactone-containing cembranoids were elucidated using spectroscopic methods. Compounds 2 to 4 displayed inhibitory effects on the production of iNOS at a concentration of 10 μM. These results revealed a series of anti-inflammatory lead compounds which could be referred for future medicinal modifications.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X211059299 - Supplemental material for Natural Cembrane Diterpenoids From the Soft Coral Sinularia querciformis

Supplemental material, sj-docx-1-npx-10.1177_1934578X211059299 for Natural Cembrane Diterpenoids From the Soft Coral Sinularia querciformis by Yi-Ying Wu, Chia-Ching Hsieh, Chia-Ying Li and Wen-Huei Chang, Jih-Jung Chen, Kuei-Hung Lai, Zhi-Hong Wen, Hsu-Ming Chung in Natural Product Communications