Secondary Metabolites from the Soft Coral Sinularia arborea

Abstract

Two new 13-hydroxycembrane diterpenoids, arbolides A (1) and B (2), along with a known trihydroxysteroid, crassarosterol A (3), were isolated from the soft coral Sinularia arborea. The structures of new cembranes 1 and 2 were elucidated by spectroscopic methods. Steroid 3 was found to exhibit cytotoxicity toward K562 and MOLT-4 leukemia.

1. Introduction

Previous studies on the chemical constituents of soft corals belonging to the genus Sinularia have led to the isolation of a number of interesting secondary metabolites and some of these were found to possess extensive bioactivities [1,2,3]. Continuation investigation on the chemical constituents of the marine invertebrates collected off the waters of Taiwan, two new cembrane-type diterpenoids, arbolides A (1) and B (2), and a known steroid, crassarosterol A (3) [4], were isolated from the soft coral Sinularia arborea (family Alcyonacea) (Figure 1). In this paper, we describe the isolation, structure determination and cytotoxicity of compounds 1–3.

Figure 1. The soft coral Sinularia arborea and the structures of arbolides A (1), B (2) and crassarosterol A (3).

Figure 1. The soft coral Sinularia arborea and the structures of arbolides A (1), B (2) and crassarosterol A (3).

Sinularia arborea

2. Results and Discussion

Arbolide A (1) was isolated as a colorless oil that gave a pseudomolecular ion [M + Na]⁺ at m/z 359.2195 in the HRESIMS, indicating the molecular formula C₂₀H₃₂O₄ (calcd for C₂₀H₃₂O₄Na, 359.2198) (5° of unsaturation). The IR spectrum of 1 showed a broad band at 3345 cm⁻¹, consistent with the presence of hydroxy group. The ¹³C NMR and DEPT spectra of 1 showed that this compound had 20 carbons (Table 1), including three methyls, six sp³ methylenes, two sp² methylenes, four sp³ methines, an sp² methine, an sp³ quaternary carbon and three sp² quaternary carbons. The presence of a trisubstituted epoxide containing a methyl substituent was established from the NMR signals at δ_C 61.4 (CH), 61.1 (C) and δ_H 2.72 (1H, dd, J = 6.0, 6.0 Hz) and further confirmed by the proton signal of a methyl singlet at δ_H 1.27 (3H, s) (Table 1). A trisubstituted and two 1,1-disubstituted carbon-carbon double bonds were identified from the NMR signals at δ_C 138.4 (C), 125.3 (CH) and δ_H 5.48 (1H, br s); δ_C 147.3 (C), 113.2 (CH₂) and δ_H 5.19 (1H, s), 5.15 (1H, s); δ_C 148.1 (C), 111.2 (CH₂) and δ_H 4.75 (2H, br s), respectively. A hydroperoxy-bearing methine (δ_H 4.38, 1H, dd, J = 7.2, 4.8 Hz, δ_C 88.4, CH) [5,6,7] and a hydroxy-bearing methine (δ_H 3.92, 1H, dd, J = 6.0, 6.0 Hz, δ_C 76.4, CH) were identified from the characteristic NMR signal analysis. These data, combined with the five degrees of unsaturation implied by the molecular formula, suggested a bicyclic structure for 1.

Table 1. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, ¹H–¹H COSY and HMBC correlations for cembrane 1.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	2.02 m	43.1, CH	H2-2, H2-14	C-2, -3, -13, -15, -17
2	1.89 m; 1.23 m	32.9, CH2	H-1, H-3	C-1, -3, -4, -14, -15
3	2.72 dd (6.0, 6.0)	61.4, CH	H2-2	C-2
4		61.1, C
5	1.96 m; 1.30 m	33.7, CH2	H2-6	C-3, -4, -6, -7, -18
6	1.66 m; 1.52 m	28.6, CH2	H2-5, H-7	C-4, -5, -7, -8
7	4.38 dd (7.2, 4.8)	88.4, CH	H2-6	C-5, -6, -19
8		147.3, C
9	2.24 m	31.3, CH2	H2-10, H2-19	C-8, -10, -11
10	2.41 m; 2.27 m	25.9, CH2	H2-9, H-11	C-9
11	5.48 br s	125.3, CH	H2-10, H3-20	n.o. a
12		138.4, C
13	3.92 dd (6.0, 6.0)	76.4, CH	H2-14	C-1, -12, -14, -20
14	1.80 dd (6.8, 6.0)	38.2, CH2	H-1, H-13	C-1, -2, -12, -13, -15
15		148.1, C
16	1.71 br s	18.8, CH3	H2-17	C-1, -15, -17
17	4.75 br s	111.2, CH2	H3-16	C-1, -15, -16
18	1.27 s	17.0, CH3		C-3, -4, -5
19	5.19 s; 5.15 s	113.2, CH2	H2-9	C-7, -8, -9
20	1.69 s	13.6, CH3	H-11	C-11, -12, -13
7-OOH	7.89 br s			n.o.

^a n.o. = not observed.

Table 1. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, ¹H–¹H COSY and HMBC correlations for cembrane 1.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	2.02 m	43.1, CH	H2-2, H2-14	C-2, -3, -13, -15, -17
2	1.89 m; 1.23 m	32.9, CH2	H-1, H-3	C-1, -3, -4, -14, -15
3	2.72 dd (6.0, 6.0)	61.4, CH	H2-2	C-2
4		61.1, C
5	1.96 m; 1.30 m	33.7, CH2	H2-6	C-3, -4, -6, -7, -18
6	1.66 m; 1.52 m	28.6, CH2	H2-5, H-7	C-4, -5, -7, -8
7	4.38 dd (7.2, 4.8)	88.4, CH	H2-6	C-5, -6, -19
8		147.3, C
9	2.24 m	31.3, CH2	H2-10, H2-19	C-8, -10, -11
10	2.41 m; 2.27 m	25.9, CH2	H2-9, H-11	C-9
11	5.48 br s	125.3, CH	H2-10, H3-20	n.o. a
12		138.4, C
13	3.92 dd (6.0, 6.0)	76.4, CH	H2-14	C-1, -12, -14, -20
14	1.80 dd (6.8, 6.0)	38.2, CH2	H-1, H-13	C-1, -2, -12, -13, -15
15		148.1, C
16	1.71 br s	18.8, CH3	H2-17	C-1, -15, -17
17	4.75 br s	111.2, CH2	H3-16	C-1, -15, -16
18	1.27 s	17.0, CH3		C-3, -4, -5
19	5.19 s; 5.15 s	113.2, CH2	H2-9	C-7, -8, -9
20	1.69 s	13.6, CH3	H-11	C-11, -12, -13
7-OOH	7.89 br s			n.o.

^a n.o. = not observed.

From the ¹H–¹H COSY spectrum of 1 (Table 1 and Figure 2), the separate spin systems of H-13/H₂-14/H-1/H₂-2/H-3, H₂-5/H₂-6/H-7 and H₂-9/H₂-10/H-11 were differentiated. These data, together with the key HMBC correlations between protons and quaternary carbons (Table 1 and Figure 2), such as H₂-2, H₂-5, H₂-6, H₃-18/C-4; H₂-6, H₂-9/C-8; H-13, H₂-14/C-12; and H-1, H₂-2, H₂-14, H₂-17/C-15, established the main carbon skeleton of 1. The vinyl methyls at C-12 and C-15 were confirmed by the HMBC correlations between H₃-20/C-11, -12, -13 and H₃-16/C-1, -15, -17; and further supported by the allylic couplings between H-11/H₃-20 and H₂-17/H₃-16 in the ¹H–¹H COSY spectrum of 1. The exocyclic carbon-carbon double bonds at C-8 and C-15 were established by the HMBC correlations between H₂-19/C-7, -8, -9 and H₂-17/C-1, -15, -16; and further confirmed by the allylic couplings between H₂-9/H₂-19 and H₂-17/H₃-16 in the ¹H–¹H COSY spectrum of 1. The hydroperoxy-bearing methine unit at δ_C 88.4 was more shielded than that expected for a hydroxy-bearing methine [5,6], and was correlated to the methine proton appearing at δ_H 4.38 in the HMQC spectrum. Thus, the remaining hydroxy group should be positioned at C-13, as indicated by the key ¹H–¹H COSY correlations and characteristic NMR signals.

Figure 2. ¹H–¹H COSY and selected HMBC correlations (protons→quaternary carbons) for 1.

Figure 2. ¹H–¹H COSY and selected HMBC correlations (protons→quaternary carbons) for 1.

The relative configuration of 1 was elucidated mainly from a NOESY spectrum. In the NOESY experiment for 1 (Figure 3), H-1 correlated with H-13, but not with H-3 and H₂-14, and H-3 showed correlations with H₂-14, revealing the S*-, R*- and R*-configurations of the chiral carbons C-1, C-3 and C-13, respectively, by modeling analysis. H-3 did not exhibit correlation with H₃-18, reflecting the trans stereochemistry of 3,4-epoxide. Additionally, correlations between H-11 and H-13, as well as the lack of correlation between H-11/H₃-20, reflected the E geometry of the double bond at C-11/12. Furthermore, by comparison of the proton chemical shift and coupling pattern of H-7 in 1 (δ_H 4.38 dd, J = 7.2, 4.8 Hz) with those of known cembrane analogues, manaarenolides A (δ_H 4.52, t, J = 3.5 Hz) and B (δ_H 4.40 dd, J = 11.5, 3.5 Hz), which were found to possess 7α- and 7β-hydroperoxy group in their structures, respectively [5], the 7-hydroperoxy group in 1 was proven to be β-oriented and possessing an S*-configuration.

Figure 3. Key NOESY correlations of 1.

Figure 3. Key NOESY correlations of 1.

The HRESIMS spectrum of 2 (arbolide B) exhibited a pseudomolecular ion at m/z 327.2298 [M + Na]⁺, consistent with the molecular formula C₂₀H₃₂O₂ and implying five degrees of unsaturation. The IR spectrum revealed the presence of hydroxy group (ν_max3419 cm⁻¹). The structure of cembrane 2 was deduced from its ¹³C NMR and DEPT spectra (Table 2), which showed that this compound has 20 carbons, including four methyls, seven methylenes (including an sp² CH₂), five methines (including two sp² CH) and four quaternary carbons (including three sp² quaternary carbons). From the ¹H and ¹³C NMR spectra (Table 2), 2 was found to possess three olefinic groups (δ_H 5.05, 1H, ddq, J = 6.4, 6.4, 1.2 Hz; δ_C 123.2, CH; 135.1, C; δ_H 5.34, 1H, dd, J = 6.4, 6.4 Hz; δ_C 127.2, CH; 135.8, C; δ_H 4.71, 1H, dd, J = 2.0, 1.6 Hz; 4.64, 1H, dd, J = 1.6, 0.8 Hz; δ_C 147.6, C; 111.0, CH₂). Signals at δ_C 63.3 (CH), 60.6 (C), 16.7 (CH₃) and δ_H 2.74 (1H, dd, J = 10.0, 2.8 Hz), 1.23 (3H, s) revealed the presence of a methyl-containing trisubstituted epoxide. Detailed analysis of the ¹H–¹H COSY and HMBC correlations (Table 2 and Figure 4) further established the planar structure of 2 as a cembrane-type diterpenoid bearing a hydroxy group at C-13, two trisubstituted carbon-carbon double bonds at C-7/8 and C-11/12, a 1,1-disubstituted carbon-carbon double bond at C-15/17 and a methyl-containing epoxide at C-3/4.

Table 2. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, ¹H–¹H COSY and HMBC correlations for cembrane 2.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	2.01 m	40.7, CH	H2-2, H2-14	C-3, -13, -15
2	1.77 m; 1.31 m	33.5, CH2	H-1, H-3	C-1, -3, -4, -14, -15
3	2.74 dd (10.0, 2.8)	63.3, CH	H2-2	C-2
4		60.6, C
5	2.06 m; 1.27 m	38.1, CH2	H2-6	C-3, -4, -6, -7
6	2.29 m; 2.10 m	24.0, CH2	H2-5, H-7	C-4, -5, -7, -8
7	5.05 ddq (6.4, 6.4, 1.2)	123.2, CH	H2-6, H3-19	C-6, -9, -19
8		135.1, C
9	2.20 m; 2.04 m	38.8, CH2	H2-10	C-7, -8, -10, -11, -19
10	2.16 m	24.1, CH2	H2-9, H-11	C-8, -9, -11, -12
11	5.34 dd (6.4, 6.4)	127.2, CH	H2-10	C-9, -10, -13, -20
12		135.8, C
13	3.87 br d (10.0)	75.3, CH	H2-14	C-11, -20
14	1.82 ddd (13.6, 10.8, 2.8) 1.73 ddd (13.6, 10.0, 3.6)	37.3, CH2	H-1, H-13	C-1, -2, -12, -15
15		147.6, C
16	1.67 br s	18.5, CH3	H2-17	C-1, -15, -17
17	4.71 dd (2.0, 1.6); 4.64 dd (1.6, 0.8)	111.0, CH2	H3-16	C-1, -15, -16
18	1.23 s	16.7, CH3		C-3, -4, -5
19	1.63 br s	16.2, CH3	H-7	C-7, -8, -9
20	1.67 s	13.0, CH3		C-11, -12, -13

Table 2. ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, ¹H–¹H COSY and HMBC correlations for cembrane 2.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	2.01 m	40.7, CH	H2-2, H2-14	C-3, -13, -15
2	1.77 m; 1.31 m	33.5, CH2	H-1, H-3	C-1, -3, -4, -14, -15
3	2.74 dd (10.0, 2.8)	63.3, CH	H2-2	C-2
4		60.6, C
5	2.06 m; 1.27 m	38.1, CH2	H2-6	C-3, -4, -6, -7
6	2.29 m; 2.10 m	24.0, CH2	H2-5, H-7	C-4, -5, -7, -8
7	5.05 ddq (6.4, 6.4, 1.2)	123.2, CH	H2-6, H3-19	C-6, -9, -19
8		135.1, C
9	2.20 m; 2.04 m	38.8, CH2	H2-10	C-7, -8, -10, -11, -19
10	2.16 m	24.1, CH2	H2-9, H-11	C-8, -9, -11, -12
11	5.34 dd (6.4, 6.4)	127.2, CH	H2-10	C-9, -10, -13, -20
12		135.8, C
13	3.87 br d (10.0)	75.3, CH	H2-14	C-11, -20
14	1.82 ddd (13.6, 10.8, 2.8) 1.73 ddd (13.6, 10.0, 3.6)	37.3, CH2	H-1, H-13	C-1, -2, -12, -15
15		147.6, C
16	1.67 br s	18.5, CH3	H2-17	C-1, -15, -17
17	4.71 dd (2.0, 1.6); 4.64 dd (1.6, 0.8)	111.0, CH2	H3-16	C-1, -15, -16
18	1.23 s	16.7, CH3		C-3, -4, -5
19	1.63 br s	16.2, CH3	H-7	C-7, -8, -9
20	1.67 s	13.0, CH3		C-11, -12, -13

Figure 4. ¹H–¹H COSY and selected HMBC correlations (protons→quaternary carbons) for 2.

Figure 4. ¹H–¹H COSY and selected HMBC correlations (protons→quaternary carbons) for 2.

The relative structure of 2 was elucidated by analysis of NOESY correlations, as shown in Figure 5. In the NOESY experiment for 1, H-1 correlated with H-13, but not with H-3, and H-3 showed correlations with H₂-14, but not with H₃-18 revealing the S*-, R*-, R*- and R*-configurations of the chiral carbons C-1, C-3, C-4 and C-13, respectively, by modeling analysis. Correlations observed between H-7/H₂-9 and H-11/H-13, as well as the lack of correlation between H-7/H₃-19 and H-11/H₃-20, reflected the E geometry of the double bonds at C-7/8 and C-11/12.

Figure 5. The NOESY correlations of 2.

Figure 5. The NOESY correlations of 2.

The steroid 3 was found to be identical with the known compound, crassarosterol A, which was first isolated from a Formosan soft coral Sinularia crassa, on the basis of the comparison of its physical and spectroscopic data with those reported previously [4].

Cytotoxicity of compounds 1–3 toward K562 (human erythromyeloblastoid leukemia), MOLT-4 (human acute lymphoblastic leukemia), HTC-116 (human acute promyelocytic leukemia), DLD-1 (human colorectal adenocarcinoma), T-47D (human breast ductal carcinoma), MDA-MB-231 (human breast adenocarcinoma) and MCF-7 (human breast adenocarcinoma) cells was studied, and the results are shown in Table 3. These data showed that crassarosterol A (3) exhibited significant cytotoxicity towards K562 and MOLT-4 leukemia.

Table 3. Cytotoxic data of compounds 1–3.

	Cell lines IC50 (μg/mL)
Compounds	K562	MOLT-4	HTC-116	DLD-1	T-47D	MDA-MB-231	MCF-7
1	NA	NA	NA	NA	NA	NA	NA
2	NA	19.0	NA	NA	NA	NA	NA
3	2.5	0.7	19.0	NA	NA	NA	NA
Doxorubicin a	0.3	0.001	0.06	1.1	0.3	0.4	10.0

^a Doxorubicin was used as a positive control. NA = not active at 20 μg/mL for 72 h.

Table 3. Cytotoxic data of compounds 1–3.

	Cell lines IC50 (μg/mL)
Compounds	K562	MOLT-4	HTC-116	DLD-1	T-47D	MDA-MB-231	MCF-7
1	NA	NA	NA	NA	NA	NA	NA
2	NA	19.0	NA	NA	NA	NA	NA
3	2.5	0.7	19.0	NA	NA	NA	NA
Doxorubicin a	0.3	0.001	0.06	1.1	0.3	0.4	10.0

^a Doxorubicin was used as a positive control. NA = not active at 20 μg/mL for 72 h.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were measured at a Jasco P-1010 digital polarimeter (Japan Spectroscopic Corporation, Tokyo, Japan). Infrared spectra were recorded on a Varian Diglab FTS 1000 FT-IR spectrometer (Varian Inc. Palo Alto, CA, USA); peaks are reported in cm⁻¹. NMR spectra were recorded on a Varian Mercury Plus 400 NMR spectrometer (Varian Inc.) using the residual CHCl₃ signal (δ_H 7.26 ppm) as the internal standard for ¹H NMR and CDCl₃ (δ_C 77.1 ppm) for ¹³C NMR. Coupling constants (J) are given in Hz. ESIMS and HRESIMS were recorded using a Bruker APEX II mass spectrometer (Bruker, Bremen, Germany). Column chromatography was performed on silica gel (230–400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F₂₅₄ (0.25 mm, Merck); spots were visualized by spraying with 10% H₂SO₄ solution followed by heating. The normal phase HPLC (NP-HPLC) was performed using a system comprised of a Hitachi L-7110 pump (Hitachi Ltd. Tokyo, Japan) and a Rheodyne 7725 injection port (Rheodyne LLC, Rohnert Park, CA, USA). Two normal phase columns (Supelco Ascentis^® Si Cat #:581515-U, 25 cm × 21.2 mm, 5 μm; 581514-U, 25 cm × 10 mm, 5 μm, Sigma-Aldrich. Com. St. Louis, MO, USA) were used for NP-HPLC.

3.2. Animal Material

Specimens of the octocoral Sinularia arborea [8] were collected by hand using scuba equipment off the coast of southern Taiwan in October, 2012, and stored in a freezer (−20 °C) until extraction. A voucher specimen (NMMBA-TWSC-1200X) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

3.3. Extraction and Isolation

Specimens of the soft coral Sinularia arborea (wet weight 1.6 kg, dry weight 576 g) were minced and extracted with ethyl acetate (EtOAc). The EtOAc extract left after removal of the solvent (12.5 g) was separated by silica gel and eluted using a mixture of n-hexane/EtOAc in a stepwise fashion from 100:1–pure EtOAc to yield 11 fractions A–K. Fraction G was separated by NP-HPLC, using a mixture of n-hexane and acetone (6:1) to yield 26 subfractions G1–G26. Fraction G13 was repurified by NP-HPLC, using a mixture of n-hexane and EtOAc (4:1) to yield 9 subfractions G13A–G13I. Fraction G13G was repurified by NP-HPLC, using a mixture of n-hexane and acetone (7:2, flow rate: 2.0 mL/min) to yield 2 (3.7 mg, t_R = 8 m). Fraction G13I was repurified by NP-HPLC, using a mixture of dichloromethane and acetone (10:1, flow rate: 1.0 mL/min) to yield 1 (2.9 mg, t_R = 332 m). Fraction H was purified by NP-HPLC, using a mixture of n-hexane and EtOAc (2:1) to obtain 14 subfractions H1–H14. Fraction H12 was repurified by NPLC, using a mixture of n-hexane and acetone (3:1, flow rate: 2.0 mL/min) to yield 3 (1.2 mg, t_R = 149 m).

Arbolide A (1): colorless oil; +12 (c 0.15, CHCl₃); IR (neat) ν_max 3445 cm⁻¹; ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, see Table 1; ESIMS: m/z 359 [M + Na]⁺; HRESIMS: m/z 359.2195 (calcd for C₂₀H₃₂O₄Na, 359.2198).

Arbolide B (2): colorless oil; −3 (c 0.19, CHCl₃); IR (neat) ν_max 3419 cm⁻¹; ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, see Table 2; ESIMS: m/z 327 [M + Na]⁺; HRESIMS: m/z 327.2298 (calcd for C₂₀H₃₂O₂Na, 327.2300).

Crassarosterol A (3): white powder; −16 (c 0.06, CHCl₃) (ref. [4], −45 (c 0.66, CHCl₃)); IR (neat) ν_max 3396 cm⁻¹; ESIMS: m/z 453 [M + Na]⁺. The ¹H and ¹³C NMR data of 3 are in full agreement with those reported previously [4].

3.4. Cytotoxicity Testing

Cytotoxicity of compounds 1–3 was assayed with a modification of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method. Cytotoxicity assays were carried out according to previously described procedures [9,10].

4. Conclusions

In our continuing investigation on the chemical constituents of marine invertebrates collected off the waters of Taiwan, the soft coral Sinularia arborea has resulted in the isolation of two new cembrane-type diterpenoids, arbolides A (1) and B (2) and a known trihydroxysteroid crassarosterol A (3) [4]. Steroid 3 was found to exhibit selective cytotoxicity toward the human leukemia K562 and MOLT-4 cells. To the best of our knowledge, this is the first time that the natural substances from Sinularia arborea have been reported.