Secosteroids and Norcembranoids from the Soft Coral Sinularia nanolobata

Abstract

Two new 9,11-secosteroids, 22α-acetoxy-24-methylene-3β,6α,11-trihydroxy-9,11-seco-cholest-7-en-9-one (1) and 11-acetoxy-24-methylene-1β,3β,6α-trihydroxy-9,11-seco-cholest-7-en-9-one (2), as well as two known norcembranoids, 5-epi-sinuleptolide (3) and sinuleptolide (4), were isolated from the soft coral Sinularia nanolobata. The structures of these metabolites were elucidated on the basis of extensive spectroscopic analysis. The anti-HCMV (human cytomegalovirus) activity of 1–4 and its cytotoxicity against selected cell lines were evaluated.

1. Introduction

Soft corals have been proven to be a rich source of 9,11-secosterols [1,2,3,4,5,6,7,8,9,10,11,12,13,14] and C-4 norcembranoids [15,16,17,18,19,20,21,22,23,24]. 9,11-Secosteroids were found to possess cytotoxic [2,3,5,6,8,10,11,12,13] and anti-inflammatory activities [13]. C-4 Norcembranoids were found to possess cytotoxic [18,19,20,25], anti-inflammatory [23,24], and antiviral activities [23]. As part of the continuing search for bioactive metabolites from marine invertebrates, we explored the secondary metabolites of the soft coral Sinularia nanolobata (Verseveldt, 1977) which was collected from Xiao-Liuqiu Island, Pingtung County, Taiwan. Chromatographic separation of the EtOAc extract of the soft coral resulted in the isolation of new secosteroids 1 and 2, along with two known norcembranoids, 5-epi-sinuleptolide (3) and sinuleptolide (4) [21] (Figure 1). The structures of these metabolites were established by extensive spectroscopic analysis. The anti-HCMV (human cytomegalovirus) activity of 1–4 and cytotoxicity against P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung carcinoma) cancer cell lines were evaluated in vitro.

Figure 1. Structures of Metabolites 1–6.

Figure 1. Structures of Metabolites 1–6.

2. Results and Discussion

Metabolite 1 was isolated as colorless needles. Its molecular formula, C₃₀H₄₈O₆, was established by HRESIMS (m/z 527.3351, [M + Na]⁺), implying seven degrees of unsaturation. The presence of hydroxyl groups was suggested by a strong absorption band at 3459 cm⁻¹ in the IR spectrum. The ¹³C NMR and DEPT (Table 1) spectroscopic data showed signals of six methyls, eight sp³ methylenes (including one oxymethylene), one sp² methylene, eight sp³ methines (including three oxymethines), one sp² methine, two sp³ and four sp² quaternary carbons. The quaternary carbon signal at δ_C 205.4 combined with the chemical shifts of H₃-18 (δ 0.67, s), H₃-19 (δ 1.14, s), H₃-21 (δ 1.02, d, J = 6.8 Hz), H₃-26 (δ 1.03, d, J = 6.8 Hz), H₃-27 (δ 1.03, d, J = 6.8 Hz), H-3 (δ 3.61, m), H-6 (δ 4.30, dd, J = 10.0, 1.6 Hz), H-7 (δ 6.61, d, J = 1.6 Hz), and H₂-11 (each 1H, δ 3.70, m; δ 3.93, m) were found to be similar to those of 3β,6α,11-trihydroxy-9,11-seco-5α-cholest-7-en-9-one (5) [4]. Moreover, the signals appearing at δ_C 170.7 (qC), 152.2 (qC), 108.8 (CH₂), 74.7 (CH), and 21.2 (CH₃) indicated the presence of an acetoxy and an exomethylene in 1. This was further supported by the ¹H NMR signals of acetoxy and exomethylene protons at δ_H 1.99 (3H, s) and 4.71 and 4.80 (each 1H, s), respectively. From the COSY spectrum measured in CDCl₃, it was possible to establish five proton sequences from H-1 to H-7, H₂-11 to H₂-12, H-14 to H-17, H-21 to H₂-23 through H-20, and H-25 to H₃-26 and H₃-27 (Figure 2). Key HMBC correlations of H₂-4 to C-3 and C-5; H-7 to C-5, C-9 and C-14; H₂-12 to C-13 and C-14; H-14 to C-7, C-8, C-9, C-13, C-15 and C-18; H₃-18 to C-12, C-13, C-14, and C-17; H₃-19 to C-1, C-5, C-9, and C-10; H-20 to C-21; H₃-21 to C-17, C-20 and C-22; H-22 to C-24; H₂-23 to C-25; H₃-26 to C-24, C-25, and C-27; H₃-27 to C-24, C-25, and C-26; H₂-28 to C-23, C-24 and C-25; H₃-OAc to OAc-22 permitted the establishment of the secosterol-type skeleton of 1.

Table 1. ¹H and ¹³C NMR Spectroscopic Data for compounds 1 and 2.

Position	1		2
	δH a	δC b	δH a	δC b
1	1.48 m; 1.90 m	31.8, CH2 d	3.87 dd (12.0, 4.4)	70.7, CH
2	1.48 m; 1.94 m	30.5, CH2	1.52 m; 2.14 m	37.8, CH2
3	3.61 m	69.9, CH	3.71 m	67.4, CH
4	1.45 m; 2.31 m	32.8, CH2	1.43 m; 2.29 m	32.2, CH2
5	1.80 m	48.5, CH	1.70 m	46.4, CH
6	4.30 dd (10.0, 1.6) c	69.4, CH	4.38 dd (9.2, 2.0)	69.1, CH
7	6.61 d (1.6)	147.8, CH	6.58 d (1.6)	147.4, CH
8		136.5, qC		136.8, qC
9		205.4, qC		206.5, qC
10		44.9, qC		49.0, qC
11	3.70 m; 3.93 m	59.3, CH2	4.16 td (10.4, 6.4); 4.23 td (10.4, 5.2)	61.2, CH2
12	1.04 m; 1.64 m	40.7, CH2	1.15 m; 1.70 m	36.8, CH2
13		46.3, qC		46.0, qC
14	3.47 dd (9.6, 8.8)	42.2, CH	3.25 dd (10.8, 9.6)	42.2, CH
15	1.62 m	26.4, CH2	1.62 m	26.4, CH2
16	1.65 m; 1.91 m	27.0, CH2	1.45 m; 1.87 m	26.2, CH2
17	1.75 m	45.9, CH	1.67 m	50.1, CH
18	0.67 s	16.9, CH3	0.69 s	17.0, CH3
19	1.14 s	16.2, CH3	1.17 s	9.8, CH3
20	1.78 m	39.8, CH	1.46 m	34.7, CH
21	1.02 d (6.8)	11.8, CH3	1.02 d (6.4)	18.8, CH3
22	5.12 m	74.7, CH	1.18 m; 1.56 m	34.0, CH2
23	2.17 m	32.6, CH2	1.89 m; 2.11 m	31.6, CH2
24		152.2, qC		156.5, qC
25	2.20 m	33.4, CH	2.21 m	33.8, CH
26	1.03 d (6.8)	21.6, CH3	1.02 d (6.8)	21.8, CH3
27	1.03 d (6.8)	22.1, CH3	1.02 d (6.8)	22.0, CH3
28	4.71 s; 4.80 s	108.8, CH2	4.66 s; 4.73 s	106.2, CH2
OAc	1.99 s	21.2, CH3 170.7, qC	2.01 s	21.1 CH3 171.2, qC

^a Spectra recorded at 400 MHz in CDCl₃; ^b Spectra recorded at 100 MHz in CDCl₃; ^c J values (in Hz) are in parentheses; ^d Carbon types are deduced by HSQC and DEPT experiments.

Table 1. ¹H and ¹³C NMR Spectroscopic Data for compounds 1 and 2.

Position	1		2
	δH a	δC b	δH a	δC b
1	1.48 m; 1.90 m	31.8, CH2 d	3.87 dd (12.0, 4.4)	70.7, CH
2	1.48 m; 1.94 m	30.5, CH2	1.52 m; 2.14 m	37.8, CH2
3	3.61 m	69.9, CH	3.71 m	67.4, CH
4	1.45 m; 2.31 m	32.8, CH2	1.43 m; 2.29 m	32.2, CH2
5	1.80 m	48.5, CH	1.70 m	46.4, CH
6	4.30 dd (10.0, 1.6) c	69.4, CH	4.38 dd (9.2, 2.0)	69.1, CH
7	6.61 d (1.6)	147.8, CH	6.58 d (1.6)	147.4, CH
8		136.5, qC		136.8, qC
9		205.4, qC		206.5, qC
10		44.9, qC		49.0, qC
11	3.70 m; 3.93 m	59.3, CH2	4.16 td (10.4, 6.4); 4.23 td (10.4, 5.2)	61.2, CH2
12	1.04 m; 1.64 m	40.7, CH2	1.15 m; 1.70 m	36.8, CH2
13		46.3, qC		46.0, qC
14	3.47 dd (9.6, 8.8)	42.2, CH	3.25 dd (10.8, 9.6)	42.2, CH
15	1.62 m	26.4, CH2	1.62 m	26.4, CH2
16	1.65 m; 1.91 m	27.0, CH2	1.45 m; 1.87 m	26.2, CH2
17	1.75 m	45.9, CH	1.67 m	50.1, CH
18	0.67 s	16.9, CH3	0.69 s	17.0, CH3
19	1.14 s	16.2, CH3	1.17 s	9.8, CH3
20	1.78 m	39.8, CH	1.46 m	34.7, CH
21	1.02 d (6.8)	11.8, CH3	1.02 d (6.4)	18.8, CH3
22	5.12 m	74.7, CH	1.18 m; 1.56 m	34.0, CH2
23	2.17 m	32.6, CH2	1.89 m; 2.11 m	31.6, CH2
24		152.2, qC		156.5, qC
25	2.20 m	33.4, CH	2.21 m	33.8, CH
26	1.03 d (6.8)	21.6, CH3	1.02 d (6.8)	21.8, CH3
27	1.03 d (6.8)	22.1, CH3	1.02 d (6.8)	22.0, CH3
28	4.71 s; 4.80 s	108.8, CH2	4.66 s; 4.73 s	106.2, CH2
OAc	1.99 s	21.2, CH3 170.7, qC	2.01 s	21.1 CH3 171.2, qC

^a Spectra recorded at 400 MHz in CDCl₃; ^b Spectra recorded at 100 MHz in CDCl₃; ^c J values (in Hz) are in parentheses; ^d Carbon types are deduced by HSQC and DEPT experiments.

Figure 2. Selected ¹H−¹H COSY (▬) and HMBC (→) correlations of 1 and 2.

Figure 2. Selected ¹H−¹H COSY (▬) and HMBC (→) correlations of 1 and 2.

The relative configurations of eight chiral centers at C-3, C-5, C-6, C-10, C-13, C-14, C-17, and C-20 in 1 were found to be the same as those of 3β,6α,11-trihydroxy-9,11-seco-5α-cholest-7-ene-9-one [4] (Figure 2). Key NOE correlations for 1 showed interactions between H-20/H-22. Thus, OAc-22 should be α-oriented. The above data established the structure of compound 1 as 22R-acetoxy-3β,6α,11-trihydroxy-24-methylene-9,11-secocholestan-9-one.

Metabolite 2 possessed the same molecular formula (C₃₀H₄₈O₆) as that of 1, as revealed from HRESIMS. The ¹³C NMR spectral data of 2 were found to be close to those of 1, except for the signals due to C-1, C-11, and C-22. COSY correlations from H-1 to H-2, H₂-11 to H₂-12, and H-20 to H₂-22. as well as HMBC correlations from H-1 to C-10, C-9, from H₂-11 to 11-OAc, and from Me-21 to C-17, C-20, C-22 confirmed the presence of a hydroxyl at C-1, an acetoxyl at C-11, and the absence of the acetoxyl at C-22 (Figure 2). Furthermore, the configuration of C-1 was deduced from the NOE correlations of H-1/H-5 and H-3/H-5 (Figure 3). Thus, the structure of compound 2 was established as 11-acetoxy-24-methylene-1β,3β,6α-trihydroxy-9,11-seco-cholest-7en-9-one.

Figure 3. Key NOESY Correlations for 1 and 2.

Figure 3. Key NOESY Correlations for 1 and 2.

Cytotoxicity of compounds 1–4 against the proliferation of a limited panel of cancer cell lines, including P-388, A549, and HT-29, were evaluated (Table 2). Metabolites 1–3 displayed weak cytotoxicity against P-388, with IC₅₀ of 10.2, 27.8, and 15.7 μg/mL, respectively. Compounds 1–4 was also examined for antiviral activity against human cytomegalovirus (HCMV) using a human embryonic lung (HEL) cell line. Compounds 4 showed anti-HCMV activity with ED₅₀ of 1.92 μg/mL (Ganciclovir showed anti-HCMV activity with ED₅₀ of 0.12 μg/mL).

Table 2. Cytotoxicity and anti- human cytomegalovirus (HCMV) activity of 1–4.

Compounds	IC50 (μg/mL)				Anti-HCMV
	A549	HT-29	P-388	HEL
1	>50	>50	10.2	>50	>50
2	>50	>50	27.8	>50	>50
3	>50	>50	15.7	>50	>50
4	>50	>50	>50	>50	1.92

Table 2. Cytotoxicity and anti- human cytomegalovirus (HCMV) activity of 1–4.

Compounds	IC50 (μg/mL)				Anti-HCMV
	A549	HT-29	P-388	HEL
1	>50	>50	10.2	>50	>50
2	>50	>50	27.8	>50	>50
3	>50	>50	15.7	>50	>50
4	>50	>50	>50	>50	1.92

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were measured on a JASCO P1020 digital polarimeter. IR spectra were recorded on a JASCO FT/IR4100 infrared spectrophotometer. The NMR spectra were recorded on a Varian MR 400 FT-NMR at 400 MHz for ¹H and 100 MHz for ¹³C, in CDCl₃ using solvent peak as internal standard. LRMS and HRMS were obtained by ESI on a Bruker APEX ΙΙ mass spectrometer. Silica gel 60 (Merck, Darmstadt, Germany, 230–400 mesh) and LiChroprep RP-18 (Merck, 40–63 μm) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F₂₅₄, 0.25 mm) and precoated RP-18 F_254s plates (Merck) were used for TLC analysis. High-performance liquid chromatography was carried out using a Hitachi L-7100 pump equipped with a Hitachi L-7400 UV detector at 220 nm together with a semi-preparative reversed-phase column (Merck, Hibar LiChrospher RP-18e, 5 μm, 250 × 25 mm).

3.2. Animal Material

The octocoral S. nanolobata was collected by hand using scuba at the Xiao Liuqiu, Pingtung County, Taiwan, in November 2011, at a depth of 6 m. This soft coral was identified by Prof. Chang-Fong Dai, Institute of Oceanography, National Taiwan University. A voucher specimen (SL-10) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

3.3. Extraction and Separation

The frozen soft coral (1.5 kg) was chopped into small pieces and extracted with EtOAc (3L × 3) in a percolator at room temperature. The EtOAc extract of S. nanolobata was concentrated to a dark brown gum (12.3 g). The EtOAc residue was subjected to Si 60 CC using n-hexane-EtOAc-MeOH mixtures of increasing polarity for elution. Fraction 15~21, eluted with n-hexane-EtOAc (1:10) to EtOAc-MeOH (2:1), was further subjected to Si 60 CC EtOAc-MeOH (5:1) to give four subfractions. A subfraction 15~21-1, was purified by reverse-phase HPLC (MeOH-H₂O, 55:45) to afford 3 (50.0 mg) and 4 (35.0 mg). A subfraction 15~21-4, was purified by reverse-phase HPLC (MeOH-H₂O, 85:15) to afford 1 (2.5 mg) and 2 (3.1 mg).

22α-acetoxy-24-methylene-3β,6α,11-trihydroxy-9,11-seco-cholest-7-en-9-one (1): White powder; [α]²⁵_D = +54 (c 0.6, CHCl₃); IR (KBr) ν_max 3459, 2956, 1733, 1031, 1009 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 527 [M + Na]⁺; HRESIMS m/z 527.3351 (calcd for C₃₀H₄₈O₆Na, 527.3348).

11-acetoxy-24-methylene-1β,3β,6α-trihydroxy-9,11-seco-cholest-7-en-9-one (2): White powder; [α]²⁵_D = +16 (c 0.8, CHCl₃); IR (KBr) ν_max 3444, 2925, 1741, 1260, 1036 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 527 [M + Na]⁺; HRESIMS m/z 527.3345 (calcd for C₃₀H₄₈O₆Na, 527.3348).

3.4. Cytotoxicity Testing

Cytotoxicity was determined on P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung epithelial carcinoma) tumor cells using a modification of the MTT colorimetric method according to a previously described procedure [26,27]. The provision of the P-388 cell line was supported by J.M. Pezzuto, formerly of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. HT-29 and A-549 cell lines were purchased from the American Type Culture Collection. To measure the cytotoxic activities of tested compounds, five concentrations with three replications were performed on each cell line. Mithramycin was used as a positive control.

3.5. Anti-HCMV Assay

To determine the effects of natural products upon HCMV cytopathic effect (CPE), confluent human embryonic lung (HEL) cells grown in 24-well plates were incubated for 1 h in the presence or absence of various concentrations of tested natural products with three replications. Ganciclovir was used as a positive control. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of a 24-well dish. Antiviral activity was expressed as IC₅₀ (50% inhibitory concentration), or compound concentration required to reduce virus induced CPE by 50% after 7 days as compared with the untreated control. To monitor the cell growth upon treating with natural products, an MTT-colorimetric assay was employed [28].

4. Conclusions

In the previous studies, 5-epi-sinuleptolide (3) and sinuleptolide (4) both showed inhibition of iNOS protein by LPS stimulation [23], TNF-α production in a dose-dependent manner [24], and nitric oxide (NO) production [24]. 5-Epi-sinuleptolide (3) inhibited human skin cancer cells growth [25]. Sinuleptolide (4) exerted antiviral activity against HCMV (human cytomegalovirus) cells [23]. 9,11-Secosteroid 6 with epoxide at C-5 and C-6 exhibited cytotoxicity against HT-29 cells [29]. This investigation of soft coral S. nanolobata collected at Xiao-Liuqiu Island (Pingtung County, Taiwan) has led to the isolation of two new 9,11-secosteroids (1 and 2) and two known compounds, 5-epi-sinuleptolide (3) and sinuleptolide (4). Metabolites 1–3 displayed weak cytotoxicity and selectivity against P-388, with IC₅₀ of 10.2, 27.8, and 15.7 μg/mL, respectively. Metabolite 4 exhibited antiviral activity against HCMV with ED₅₀ of 1.92 μg/mL.