Xeniaphyllane-Derived Terpenoids from Soft Coral Sinularia nanolobata

Abstract

A novel tetranorditerpenoid, sinubatin A (1) (having an unprecedented carbon skeleton), a new norditerpenoid, sinubatin B (2) (a 4,5-epoxycaryophyllene possessing an unusual methylfuran moiety side chain), and a known diterpenoid, gibberosin J (3) were isolated from soft coral Sinularia nanolobata. The structures of the new compounds were elucidated by extensive analysis of spectroscopic data.

1. Introduction

Soft corals of genus Sinularia (Alcyoniidae) have been reported to be a rich source of novel structures and bioactive terpenoids and steroids [1]. Previous studies on the sample of Sinularia nanolobata Verseveldt have resulted in the isolation of diterpenoids [2,3,4,5] and sesquiterpenoids [3,4], and steroids [5,6]. During the course of our search of bioactive compounds from marine organisms, a chemical investigation on the secondary metabolites of S. nanolobata from Taiwanese waters has afforded a novel tetranorditerpenoid, sinubatin A (1) (possessing an unprecedented carbon skeleton), a new norditerpenoid, sinubatin B (2) (a 4,5-epoxycaryophyllene possessing an unusual methylfuran moiety side chain), and gibberosin J (3) (Figure 1). The structures of 1 and 2 were determined by extensive spectroscopic analysis. The chemical structure of gibberosin J (3) was determined by comparison of its infrared (IR), high resolution electron spray ionization mass spectrum (HR-ESI-MS), and nuclear magnetic resonance (NMR) spectroscopic data with the literature data [7].

2. Results and Discussion

Chromatographic separation on the acetone extract resulted in the isolation of two new terpenoids, sinubatin A and B (1 and 2), as well as a known compound, gibberosin J (3). The HR-ESI-MS, ¹³C NMR, and DEPT spectroscopic data of sinubatin A (1) established its molecular formula as C₁₉H₂₈O₅. ¹³C NMR and DEPT spectrum of 1 showed the presence of four methyl, five sp³ methylene, four sp³ methine, one sp² methylene, two sp³ quaternary, one sp² quaternary, and two carbonyl carbons. The presence of an exomethylene in 1 was shown by the NMR data [δ_H 4.90 (1H, s), 5.01 (1H, s); δ_C 114.0 (CH₂), 150.7 (C)] (

Table 1. NMR spectral data of 1.

Position	δH a	(J in Hz)	δC b	Type	COSY	HMBC	NOESY
1	2.34	m	45.3,	CH	2, 9	11, 12	5
2α	1.45	m	27.7,	CH2	1	-	-
2β	1.57	m			1, 3β	-	9
3α	1.00	td (12.8, 4.8)	38.4,	CH2	2β	1, 4, 16	3β, 5
3β	2.09	m			2β	-	3α
4	-	-	59.6,	C		-	-
5	2.92	dd (10.8, 4.0)	63.8,	CH	6β, 16	-	1, 3α
6α	2.30	m	30.2,	CH2	-	-	-
6β	1.31	m			5	-	6α
7α	2.16	m	29.2,	CH2	6α	6, 9	6β
7β	2.32	m			6α	8, 15	-
8	-	-	150.7,	C		-	-
9	2.71	td (9.6, 9.2)	48.7,	CH	1, 10α, 10β	-	2β, 10β, 14, 15a
10α	1.85	dd (10.4, 9.6)	36.2,	CH2	9	9, 11, 12, 14	-
10β	1.74	dd (10.4, 9.2)			9	8	9, 14
11	-	-	38.3,	C	-	-	-
12	4.74	s	79.4,	CH	-	10, 11, 13, 14, carbonyl (OAc-12)	1, 10α
13	-	-	169.2,	qC	-	-	-
14	1.14	s	15.2,	CH3	-	1, 10, 11, 12	2α, 2β, 9, 10β
15a	5.01	s	114.0,	CH2	-	7, 8, 9	9, 10α, 15b
15b	4.90	s			-	7, 9	7α
16	1.19	s	17.1,	CH3	5	3, 4, 5	3β, 6β, 9
OAc-12	2.15	s	20.6,	CH3	OMe-13	carbonyl (OAc-12)	-
	-	-	170.8,	C	-	-	-
OMe-13	3.71	s	51.9,	CH3	OAc-12	13	-

^a Spectrum recorded at 400 MHz in CDCl₃. ^b Spectrum recorded at 100 MHz in CDCl₃.

). The NMR data [δ_C 59.6 (C), 63.8 (CH), δ_H 2.92 (1H, dd, J = 10.8, 4.0 Hz)] (Table 1) indicated a trisubstituted epoxide in 1. The NMR spectrum contained signals for a secondary acetoxyl [δ_H 4.74 (1H, s), 2.15 (3H, s); δ_C 79.4 (CH), 20.6 (CH₃), and 170.8 (C)] (Table 1). The presence of a methyl ester [δ_H 3.71 (3H, s); δ_C 51.9 (CH₃), 169.2 (C)] was shown in the NMR spectrum. From the data of ¹H–¹H COSY correlations (Figure 2), we established two partial structures of consecutive proton systems extending from H-10 to H-3 through H-9 and from H-16 to H-7 through H-4. HMBC correlations of (a) CH₃-16 to C-3, C-4, and C-5, (b) H₂-15 to C-7, C-8, and C-9, (c) CH₃-14 to C-1, C-10, C-11, and C-12, (d) CH-12 to C-10, C-11,C-13, and C-14 connected four partial structures and concluded the planar structure of 1, as shown in Figure 2. The above functionalities revealed that sinubatin A (1) possesses a novel xeniaphyllane-derived tetranorditerpene skeleton. The relative configuration of 1 was established from a NOESY experiment. NOE correlations of H₃-14/H-9 and H₃-16/H-9 pointed H₃-14, H-9 and H₃-16 to be on the β-side of the molecule. NOE correlation of H-1/H-5 suggested H-1 and H-5 were on the α-side of the molecule. (Figure 3).

HR-ESI-MS of sinubatin B (2) showed a pseudomolecular ion peak at m/z 309.1842 [M + Na]⁺, consistent with the molecular formula C₁₉H₂₆O₂, and seven degrees of unsaturation. The ¹³C NMR spectrum (

Table 2. NMR spectral data of 2.

Position	δH a	(J in Hz)	δC b	Type	COSY	HMBC	NOESY
1	2.47	td (10.0,8.4)	49.3,	CH	2β, 9	3, 8, 9, 11, 17	2α, 3α, 5
2α	1.72	m	27.2,	CH2	3α, 3β	1, 3, 11	1, 3α, 3β
2β	1.54	m			1, 3α, 3β	1	3β, 9
3α	0.98	td (13.2, 5.2)	38.8,	CH2	2α, 2β, 19	2, 4, 5, 19	1, 2α, 5
3β	2.07	dt (13.2,3.6)			2α, 2β	-	2α, 2β, 19
4	-	-	59.7,	C	-	-	-
5	2.92	dd (10.4, 4.0)	63.7,	CH	6α, 6β	3, 6	1, 3α, 6α
6α	2.28	m	30.1,	CH2	5	4, 5, 7	5
6β	1.37	m			5, 7α, 7β	-	7β
7α	2.44	ddd (12.8, 8.0, 4.0)	29.8,	CH2	6β	5, 6, 8, 9	18b
7β	2.17	ddd (12.8, 8.0, 4.4)			6α, 6β	5, 6, 8, 9	9
8	-	-	151.4,	C	-	-	-
9	2.74	td (9.6, 8.4)	47.9,	CH	1, 10α, 10β	1, 7, 8, 10	2β, 7β, 10β, 18a, 19
10α	2.33	dd (10.8, 9.6)	37.8,	CH2	9, 10β	9, 11, 12, 17	18a
10β	1.87	dd (10.8, 8.4)			9, 10α	1, 16	9, 10α, 17
11	-	-	36.9,	C	-	-	-
12	-	-	160.2,	C	-	-	-
13	5.83	d (4.0)	103.7,	CH	-	-	17
14	5.84	dd (4.0, 0.8)	105.8,	CH	-	-	16
15	-	-	150.6,	C	-	-	-
16	2.28	d (0.8)	13.6,	CH3	-	14, 15	14
17	1.35	s	17.9,	CH3	-	1, 10, 11, 12	2α, 2β, 9, 13
18a	5.09	s	113.5,	CH2	7β	7, 8, 9	9, 10α
18b	4.93	s			7β	7, 8, 9	7β
19	1.23	s	17.0,	CH3	3	3, 4, 5	3β, 9

^a Spectrum recorded at 400 MHz in CDCl₃. ^b Spectrum recorded at 100 MHz in CDCl₃.

) of 2 displayed 19 carbon signals, and a DEPT experiments indicated the presence of three methyl, five sp³ methylene, three sp³ methine, two sp² methine, one sp² methylene, two sp³ quaternary, and three sp² quaternary carbons. The ¹³C and ¹H NMR spectra (Table 2) revealed the presence of a trisubstituted epoxides [δ_H 2.92 (dd, J = 10.4, 4.0 Hz); δ_C 63.7 (CH) and 59.7 (C)], a 2,5-disubstituted furan [δ_H 5.83 (d, J = 4.0 Hz), 5.84 (dd, J = 4.0, 0.8 Hz), and 2.28 (d, J = 0.8 Hz); δ_C 103.7 (CH), 105.8 (CH), 150.6 (C), 160.2 (C), 13.6 (CH₃)] [8] and an exomethylene [δ_H 5.09 (s) and 4.93 (s); δ_C 113.5 (CH₂) and 151.4 (C)]. Thus, the tetracyclic structure of 2 was revealed. From the ¹H–¹H COSY spectrum of 2, it was also possible to identify two different structural units (Figure 2), which were assembled with the assistance of an HMBC experiments. Key HMBC correlations (Figure 2) of H₃-19 to C-3, C-4, and C-5; H₃-18 to C-7, C-8, and C-9; H₃-17 to C-1, C-10, C-11, and C-12 indicated that compound 2 was a 4,5-epoxycaryophyllene having a methylfuran on C-11. The relative configuration of 2 was determined from a NOESY experiment. NOE correlations of H₃-19/H-9 and H₃-17/H-9 suggested H₃-19, H-9 and H₃-17 to be on the β-side of the molecule. NOE correlation of H-1/H-5 indicated H-1 and H-5 were on the α-side of the molecule. (Figure 3). Compound 2 was the first caryophyllene possessing a methylfuran on C-11.

Compounds 1–3 were tested for cytotoxicity against mouse lymphocytic leukemia (P-388), human colon adenocarcinoma (HT-29), and human lung epithelial carcinoma (A-549) tumor cell lines. Compound 3 exhibited cytotoxicity against P-388, A549, and HT-29 with ED₅₀ values of of 1.0, 1.2, and 0.5 μg/mL, respectively. However, compounds 1 and 2 were not cytotoxic to P-388, A549 and HT-29 cell lines. Compounds 1–3 were also examined for antiviral activity against human cytomegalovirus (HCMV) and did not show anti-HCMV activity.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were obtained on a JASCO P1020 digital polarimeter (Tokyo, Japan). UV and IR spectra were determined on JASCO V-650 (JASCO, Tokyo, Japan) and JASCO FT/IR-4100 spectrophotometers (JASCO, Tokyo, Japan), respectively. NMR spectra were recorded on a Varian MR 400 NMR spectrometer (Santa Clara, CA, USA) at 400 MHz for ¹H and 100 MHz for ¹³C. ¹H NMR chemical shifts are expressed in δ (ppm) referring to the solvent peak δ_H 7.27 for CHCl₃ and coupling constants are expressed in Hertz (Hz). ¹³C NMR chemical shifts are expressed in δ (ppm) referring to the solvent peak δ_C 77.0 for CDCl₃. MS were obtained by a Bruker APEX II mass spectrometer (Bruker, Bremen, Germany). Precoated silica gel plates (Merck, Kieselgel 60 F₂₅₄, 0.25 mm) and precoated RP-18 F_254s plates (Merck) were used for thin-layer chromatography (TLC) analysis. Silica gel 60 (Merck, Darmstadt, Germany, 230–400 mesh) and LiChroprep RP-18 (Merck, 40–63 µm) were used for column chromatography. High-performance liquid chromatography (HPLC) (Hitachi, Tokyo, Japan) was carried out using a Hitachi L-7100 pump (Hitachi, Tokyo, Japan) equipped with a Hitachi, L-7400 UV detector (Hitachi, Tokyo, Japan) at 220 nm together with a semi-preparative reversed-phased column (Merck, Hibar LiChrospher RP-18e, 5 µm, 250 mm × 25 mm).

3.2. Animal Material

The soft coral S. nanolobata was collected by hand using scuba at San-Shin-Tai, Taitong County, Taiwan, in July 2008, at a depth of 7 m. A voucher specimen (SST-009) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

3.3. Extraction and Separation

The frozen soft coral (3.0 kg) was chopped into small pieces (about 1 cm) and extracted with acetone in a percolator at room temperature. The acetone extract (30.0 g) of S. nanolobata was concentrated under reduced pressure to a brown gum, which was partitioned between EtOAc and H₂O. The EtOAc-soluble fraction (30 g) was applied to Si 60 CC using n-hexane–EtOAc mixtures of increasing polarity for elution. Fraction 12, eluted with n-hexane–EtOAc (6:1), was further purified by reverse-phase HPLC (MeOH–H₂O, 60:40) to obtain 1 (1.5 mg). Fraction 3, eluted with n-hexane–EtOAc (80:1), was further purified by reverse-phase HPLC (MeOH–H₂O, 85:15) to afford 2 (2.6 mg). Fraction 18, eluted with n-hexane–EtOAc (1:4), was further purified by reverse-phase HPLC (MeOH–H₂O, 65:35) to obtain 3 (5.0 mg).

Sinubatin A (1): Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{25}$ −19.2 (c 0.38, CHCl₃); IR (neat) ν_max 2934, 1742, 1442, 1373 and 1420 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; ESI-MS m/z 359 [M + Na]⁺; HR-ESI-MS m/z 359.1837 (calcd. for C₁₉H₂₈O₅Na, 359.1834).

Sinubatin B (2): Colorless oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{25}$ +17.2 (c 0.65, CHCl₃); IR (neat) ν_max 2961, 2925, 1261, 1094, 1020, 799 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; ESIMS m/z 309 [M + Na]⁺; HR-ESI-MS m/z 309.1832 (calcd. for C₁₉H₂₆O₂Na, 309.1830).

3.4. Biological Assay

Cytotoxicity assay and anti-HCMV assay were conducted as previously described [9].

4. Conclusions

The chemical study of soft coral S. nanolobata led to the isolation of a novel tetranorditerpenoid, sinubatin A (1) (having an unprecedented carbon skeleton), a new norditerpenoid, sinubatin B (2) (a 4,5-epoxycaryophyllene possessing an unusual methylfuran moiety side chain), and gibberosin J (3). Compound 3 exhibited cytotoxicity toward P-388, A549, and HT-29 with ED₅₀ values of 1.0, 1.2. and 0.5 μg/mL, respectively. However, compounds 1 and 2 were not cytotoxic to P-388, A549 and HT-29 cell lines. Compounds 1–3 did not show anti-HCMV activity.