A New 5α,8α-Epidioxysterol from the Soft Coral Sinularia gaweli

Abstract

A new sterol, (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methyl-cholest-6,9(11)-dien-3β-ol (1), and two known sterols, (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methylcholest-6-en-3β-ol (2) and 24-methylenecholestane-1α,3β,5α, 6β,11α-pentol (3), were isolated from the soft coral Sinularia gaweli. The structure of sterol 1 was established by spectroscopic methods and by comparison of the spectral data with those of known analogues. The cytotoxicity of sterols 1–3 towards various tumor cells is reported.

1. Introduction

Soft corals belonging to the genus Sinularia have been well-recognized as marine organisms containing various natural products that show interesting bioactivities [1,2,3]. A series of cytotoxic [4,5,6,7,8,9,10,11,12], anti-inflammatory [7,11,12,13] and antiviral [10] steroids have been isolated from Sinularia sp. octocorals collected off the waters of Taiwan. In continuation with our search for new natural substances, the organic extract of soft coral Sinularia gaweli (Figure 1) was studied, which displayed meaningful signals in NMR studies. Previous investigations of the chemical constituents of S. gaweli yielded two norcembranoidal diterpenes, 5-episinuleptolide acetate and scabrolide D [14]. In further studies of S. gaweli, a new sterol, (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methylcholest-6,9(11)-dien-3β-ol (1), and two known sterols, (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methylcholest-6-en-3β-ol (2) [4] and 24-methylenecholestane-1α,3β,5α,6β,11α-pentol (3) [15,16], were isolated (Figure 1).

Figure 1. The soft coral Sinularia gaweli and the structures of (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methylcholest-6,9(11)-dien-3β-ol (1), (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methylcholest-6-en-3β-ol (2) and 24-methylenecholestane-1α,3β,5α,6β,11α-pentol (3).

Figure 1. The soft coral Sinularia gaweli and the structures of (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methylcholest-6,9(11)-dien-3β-ol (1), (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methylcholest-6-en-3β-ol (2) and 24-methylenecholestane-1α,3β,5α,6β,11α-pentol (3).

2. Results and Discussion

(22R,23R,24R)-5α,8α-Epidioxy-22,23-methylene-24-methylcholest-6,9(11)-dien-3β-ol (1) was isolated as a white powder. The molecular formula of 1 was established as C₂₉H₄₄O₃ (eight degrees of unsaturation) from a [M+Na]⁺ molecule at m/z 463.3192 in HRESIMS (calcd for C₂₉H₄₄O₃Na, 463.3188). The ¹³C-NMR and DEPT spectra of 1 showed this compound to have 29 carbons (Table 1), including six methyls, seven sp³ methylenes, eight sp³ methines, three sp² methines, four sp³ quaternary carbons and an sp² quaternary carbon. From the NMR spectra (Table 1), the presence of three oxygenated C atoms at δ_C 82.7 (C-5), 78.4 (C-8) and 66.3 (CH-3) in the ¹³C-NMR spectrum and an oxymethine proton at δ_H 4.02 (1H, m, H-3) in the ¹H-NMR spectrum was determined. This sterol was further recognized as a 5α,8α-epidioxysterol by the presence of the characteristic signals for H-6 (δ_H 6.60, J = 8.0 Hz) and H-7 (δ_H 6.28, J = 8.0 Hz) in the ¹H-NMR spectrum [4,17]. Four protons appeared at δ_H 0.14 (2H, m, H₂-29), 0.33 (1H, m, H-23) and 0.55 (1H, m, H-22), indicating the presence of a cyclopropyl moiety in 1. Two singlets, which appeared at δ_H 0.68 (3H) and 1.09 (3H), were attributed to Me-18 and Me-19, respectively. Four doublets at δ_H 0.91 (3H, J = 6.4 Hz), 0.86 (3H, J = 6.8 Hz), 0.89 (3H, J = 6.8 Hz) and 0.92 (3H, J = 6.4 Hz) were due to the presence of Me-21, Me-26, Me-27 and Me-28, respectively. The above data suggested that 1 is a peroxysteroid containing a 22,23-methylene-24-methyl moiety in the side chain.

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

Position	δH (J in Hz)	δC, Mult.	1H–1H COSY	HMBC (H→C)
1	2.11 m; 1.70 m	32.6, CH2	H2-2	n.o.
2	1.91 m; 1.55 m	30.6, CH2	H2-1, H-3	C-3
3	4.02 m	66.3, CH	H2-2, H2-4	n.o.
4	2.14 dd (13.6, 2.0); 1.92 dd (13.6, 11.6)	36.1, CH2	H-3	C-2, -3, -5, -10
5		82.7, C
6	6.60 d (8.0)	130.8, CH	H-7	C-4, -5, -8
7	6.28 d (8.0)	135.4, CH	H-6	C-5, -8, -9, -14
8		78.4, C
9		142.5, C
10		37.9, C
11	5.42 dd (6.0, 2.0)	119.8, CH	H2-12	C-8, -10, -12, -13
12	2.28 dd (16.8, 6.0); 2.09 dd (16.8, 2.0)	41.2, CH2	H-11	C-9, -11, -13, -14, -17
13		44.1, C
14	1.83 dd (12.0, 8.0)	47.8, CH	H2-15	C-12, -15
15	1.75 m; 1.61 m	21.2, CH2	H-14, H2-16	C-8, -13, -16
16	2.20 m	28.4, CH2	H2-15, H-17	n.o.
17	1.49 m	57.4, CH	H2-16, H-20	n.o.
18	0.68 s	12.6, CH3		C-12, -13, -14, -17
19	1.09 s	25.5, CH3		C-1, -5, -9, -10
20	0.88 m	39.7, CH	H-17, H3-21, H-22	C-17
21	0.91 d (6.4)	19.0, CH3	H-20	C-20, -22
22	0.56 m	24.2, CH	H-20, H-23, H2-29	n.o.
23	0.33 m	25.1, CH	H-22, H-24, H2-29	n.o.
24	0.55 m	44.9, CH	H-23, H-25, H3-28	n.o.
25	1.64 m	32.8, CH	H-24, H3-26, H3-27	C-24
26	0.86 d (6.8)	18.5, CH3	H-25	C-24, -25, -27
27	0.89 d (6.8)	20.7, CH3	H-25	C-24, -25, -26
28	0.92 d (6.4)	15.8, CH3	H-24	C-24, -25
29	0.14 m	10.5, CH2	H-22, H-23	C-20, -22, -24

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

Position	δH (J in Hz)	δC, Mult.	1H–1H COSY	HMBC (H→C)
1	2.11 m; 1.70 m	32.6, CH2	H2-2	n.o.
2	1.91 m; 1.55 m	30.6, CH2	H2-1, H-3	C-3
3	4.02 m	66.3, CH	H2-2, H2-4	n.o.
4	2.14 dd (13.6, 2.0); 1.92 dd (13.6, 11.6)	36.1, CH2	H-3	C-2, -3, -5, -10
5		82.7, C
6	6.60 d (8.0)	130.8, CH	H-7	C-4, -5, -8
7	6.28 d (8.0)	135.4, CH	H-6	C-5, -8, -9, -14
8		78.4, C
9		142.5, C
10		37.9, C
11	5.42 dd (6.0, 2.0)	119.8, CH	H2-12	C-8, -10, -12, -13
12	2.28 dd (16.8, 6.0); 2.09 dd (16.8, 2.0)	41.2, CH2	H-11	C-9, -11, -13, -14, -17
13		44.1, C
14	1.83 dd (12.0, 8.0)	47.8, CH	H2-15	C-12, -15
15	1.75 m; 1.61 m	21.2, CH2	H-14, H2-16	C-8, -13, -16
16	2.20 m	28.4, CH2	H2-15, H-17	n.o.
17	1.49 m	57.4, CH	H2-16, H-20	n.o.
18	0.68 s	12.6, CH3		C-12, -13, -14, -17
19	1.09 s	25.5, CH3		C-1, -5, -9, -10
20	0.88 m	39.7, CH	H-17, H3-21, H-22	C-17
21	0.91 d (6.4)	19.0, CH3	H-20	C-20, -22
22	0.56 m	24.2, CH	H-20, H-23, H2-29	n.o.
23	0.33 m	25.1, CH	H-22, H-24, H2-29	n.o.
24	0.55 m	44.9, CH	H-23, H-25, H3-28	n.o.
25	1.64 m	32.8, CH	H-24, H3-26, H3-27	C-24
26	0.86 d (6.8)	18.5, CH3	H-25	C-24, -25, -27
27	0.89 d (6.8)	20.7, CH3	H-25	C-24, -25, -26
28	0.92 d (6.4)	15.8, CH3	H-24	C-24, -25
29	0.14 m	10.5, CH2	H-22, H-23	C-20, -22, -24

From the ¹H−¹H COSY spectrum, several structural units, including H₂-1/H₂-2/H-3/H₂-4, H-6/H-7, H-11/H₂-12, H-14/H₂-15/H₂-16/H-17/H-20/H-22/H-23/H-24/H-25/H₃-26(H₃-27), H-20/H₃-21, H-22/ H₂-29, H-23/H₂-29 and H-24/H₃-28, were identified (Table 1 and Figure 2). These data, together with the key HMBC correlations between protons and quaternary carbons, such as H₂-4, H-6, H-7, H₃-19/C-5; H-6, H-7, H-11, H₂-15/C-8; H-7, H₂-12, H₃-19/C-9; H₂-4, H-11, H₃-19/C-10; and H-11, H₂-12, H₂-15, H₃-18/C-13, permitted the elucidation of the main carbon skeleton of 1(Table 1 and Figure 2). The ring junctions C-18 and C-19 methyl groups were positioned at C-13 and C-10 from the HMBC correlations between H₃-18/C-12, -13, -14, -17 and H₃-19/C-1, -5, -9, -10. An oxymethine unit at δ_C 66.3 correlated to the methine proton at δ_H 4.02 in the HMQC spectrum, proving the attachment of a hydroxy group at C-3.

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

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

Because of the signals for protons H-22/H-24 and H-20/H₃-21, H₃-26, H₃-27 are overlapped in the ¹H spectrum of 1, it is difficult to judge the relative configuration of the cyclopropyl moiety by their NOE effect in the NOESY spectrum. However, by comparison of the ¹H- and ¹³C-NMR chemical shifts of Me-21, Me-26, Me-27 and Me-28 with those of a known epidioxysterol, (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methylcholest-6-en-3β-ol (2) [4,18] and four synthetic demethyl-gorgosterol isomers [19] (Figure 3), it was suggested that the stereochemistry of 1 at the side chain should be assigned as 22R, 23R and 24R, as per those of 2. The assignment of the carbon shifts of 1 was based on the comparison of these data with those of the tetracyclic system of 2 [4]. In the HMQC spectrum of 1, the doublet methyls appearing at δ_H 0.86 (J = 6.8 Hz, H₃-26) and 0.92 (J = 6.4 Hz, H₃-28) showed ¹J-correlations with δ_C 18.5 and 15.8, respectively; and the methine protons appearing at δ_H 0.33 (m, H-23) and 0.56 (m, H-22) showed ¹J-correlations with δ_C 25.1 and 24.2, respectively. We suggest that the partial ¹H and ¹³C-NMR chemical shifts for the side chain of steroid 2 that were reported previously should be re-examined [4,20]. Based on the above findings, the structure of 1 was tentatively established as (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methylcholest-6,9 (11)-dien-3β-ol.

In previous studies, the 5α,8α-epidioxy sterols were supposed to have arisen from ∆^5,7-sterols by photooxidization during storage and/or chromatographic separation [21,22,23] with a self-perpetuating mechanism [23]. ∆^5,7-Sterol analogues were not obtained from S. gaweli; at this point it is difficult to infer whether epidioxysterol 1 from S. gaweli is a natural product or an artifact.

Figure 3. The ¹H and ¹³C-NMR chemical shifts of the side-chain methyl groups of epidioxysterols 1 and 2 and synthetic isomers of demethylgorgosterols [4,18,19].

Figure 3. The ¹H and ¹³C-NMR chemical shifts of the side-chain methyl groups of epidioxysterols 1 and 2 and synthetic isomers of demethylgorgosterols [4,18,19].

Sterols 2 and 3 were identified as (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methylcholest-6-en-3β-ol and 24-methylenecholestane-1α,3β,5α,6β,11α-pentol, which have been previously isolated from a Formosan soft coral Sinularia sp. [4] and an Andaman Sea soft coral Sinularia dissecta [15,16], respectively. Their spectral data were in full agreement with those of previously reported.

The cytotoxicity of sterols 1–3 towards K562 (human erythromyeloblastoid leukemia), MOLT-4 (human acute lymphoblastic leukemia) and HL-60 (human promyelocytic leukemia) cells was studied, and the results are shown in Table 2. These data showed that sterol 3 exhibited significant cytotoxicity towards HL-60 cells.

Table 2. Cytotoxic data of sterols 1–3.

Compounds	Cell lines IC50 (μg/mL)
	K562	MOLT-4	HL-60
1	NA	15.70	NA
2	NA	NA	12.14
3	9.71	6.91	3.39
Doxorubicin a	0.20	0.01	0.03

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

Table 2. Cytotoxic data of sterols 1–3.

Compounds	Cell lines IC50 (μg/mL)
	K562	MOLT-4	HL-60
1	NA	15.70	NA
2	NA	NA	12.14
3	9.71	6.91	3.39
Doxorubicin a	0.20	0.01	0.03

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

3. Experimental

3.1. General Procedures

Optical rotation values were measured with a Jasco-P1010 digital polarimeter. Infrared spectra were obtained on a Varian Diglab FTS 1000 FT-IR spectrophotometer. NMR spectra were recorded on a Varian Mercury Plus 400 FT-NMR at 400 MHz for ¹H and 100 MHz for ¹³C in CDCl₃ or C₅D₅N at 25 °C. ESIMS and HRESIMS data were recorded on a Bruker APEX II mass spectrometer. 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) and spots were visualized by spraying with 10% H₂SO₄ solution followed by heating. Normal phase HPLC (NP-HPLC) was performed using a system comprised of a Hitachi L-7110 pump, a Hitachi L-7455 photodiode array detector and a Rheodyne 7725 injection port. A normal phase column (Supelco Ascentis^® Si Cat #:581515-U, 25 cm × 21.2 mm, 5 μm) was used for NP-HPLC. Reverse phase HPLC (RP-HPLC) was performed using a system comprised of a Hitachi L-7100 pump, a Hitachi L-2455 photodiode array detector and a Rheodyne 7725 injection port. A reverse phase column (Varian Polaris C18-A, 250 mm × 10 mm, 5 μm) was used for RP-HPLC.

3.2. Animal Material

Specimens of the soft coral Sinularia gaweli were collected by hand using scuba equipment off the coast of Sansiantai, Taitung County, Taiwan on Oct. 13, 2011, and stored in a freezer (−20 °C) until extraction. This organism was identified by comparison with previous descriptions [24]. A voucher specimen (NMMBA-TWSC-11007) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

3.3. Extraction and Isolation

The freeze-dried and minced material of Sinularia gaweli (wet weight 1.30 kg, dry weight 328 g) was extracted with ethyl acetate (EtOAc) at 25 °C (2 L × 10). The EtOAc extract left after removal of the solvent (11.4 g) was separated by silica gel and eluted using n-hexane/EtOAc/acetone in a stepwise fashion to yield 14 fractions A–N. Fraction F was separated by NP-HPLC using a mixture of n-hexane and acetone (5:1) as the mobile phase to afford the subfractions F1–5. Subfraction F3 was further purified by RP-HPLC using a mixture of methanol (MeOH) and H₂O (97:3, flow rate: 1.0 mL/min) to afford sterols 1 (0.5 mg, t_R = 40 m) and 2 (0.5 mg, t_R = 48 m). Fraction N was separated by NP-HPLC using a mixture of dichloromethane (CH₂Cl₂) and EtOAc as the mobile phase to afford the subfractions N1–10. Subfraction N9 was further purified by RP-HPLC using a mixture of MeOH and H₂O (9:1, flow rate: 1.0 mL/min) to afford sterol 3 (1.2 mg, t_R = 31 m).

(22R,23R,24R)-5α,8α-Epidioxy-22,23-methylene-24-methylcholest-6,9(11)-dien-3β-ol (1): +158 (c 0.03, CHCl₃); m.p. 218−220 °C; IR (neat) υ_max 3445, 1644 cm⁻¹; ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data, see Table 1; ESIMS m/z 463 [M+Na]⁺; HRESIMS: m/z 463.3192 (calcd for C₂₉H₄₄O₃Na, 463.3188).

(22R,23R,24R)-5α,8α-Epidioxy-22,23-methylene-24-methylcholest-6-en-3β-ol (2): +20 (c 0.02, CHCl₃) (Ref. [4], +35 (c 0.1, CHCl₃)); IR (neat) υ_max 3438, 1638 cm⁻¹; ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR data were found to be in full agreement with those reported previously [4,18]; ESIMS m/z 465 [M+Na]⁺; HRESIMS: m/z 465.3347 (calcd for C₂₉H₄₆O₃Na, 465.3344).

24-Methylenecholestane-1α,3β,5α,6β,11α-pentol (3): −3 (c 0.06, CHCl₃) (Ref. [15], −4 (c 1.60, CHCl₃)); IR (neat) υ_max 3380, 1216 cm⁻¹; ¹H (400 MHz, C₅D₅N) and ¹³C (100 MHz, C₅D₅N) NMR data were found to be in full agreement with those reported previously [15]; ESIMS: m/z 487 [M+Na]⁺; HRESIMS: m/z 487.3402 (calcd for C₂₈H₄₈O₅Na, 487.3399).

3.4. Cytotoxicity Testing

The cytotoxicity of sterols 1–3 was assayed using 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 [25,26].

4. Conclusions

Steroid metabolites are major constituents of the extracts of Sinularia spp. octocorals distributed in the waters off Taiwan [4,5,6,7,8,9,10,11,12,13]. Our studies on the chemical constituents of Sinularia gaweli have led to the isolation of a new epidioxysterol, (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methyl- cholest-6,9(11)-dien-3β-ol (1), along with two known sterols, (22R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methylcholest-6-en-3β-ol (2) and 24-methylenecholestane-1α,3β,5α,6β,11α-pentol (3). Sterol 3 was found to exhibit significant cytotoxicity against HL-60 tumor cells, and this result suggested that sterol 3 is worthy of further biomedical investigation. The soft coral S. gaweli has begun to be transplanted to culturing tanks with a flow-through sea water system located in the National Museum of Marine Biology and Aquarium, Taiwan for the extraction of additional natural products in order to establish a stable supply of bioactive material.