Steroid Constituents from the Soft Coral Sinularia nanolobata

Abstract

Six steroids (1–6), including the two new compounds 3β,4α-dihydroxyergosta-5,24(28)-diene (1) and 24(S),28-epoxyergost-5-ene-3β,4α-diol (2), were isolated from the methanol extract of the Vietnamese soft coral Sinularia nanolobata. Their structures were elucidated by spectroscopic methods including one and two dimensional (1D- and 2D)-NMR, Fourier transform ion cyclotron resonance (FT-ICR)-MS, and circular dichroism (CD). Compound 2 exhibited moderate cytotoxicity against the acute leukemia (HL-60) cell line with IC₅₀ value of 33.53±4.25 µM and weak effect on the hepatoma cancer (HepG2) and colon adenocarcinoma (SW480) cell lines with IC₅₀ values of 64.35±7.00 and 71.02±4.00 µM, respectively.

Steroids are a highly diverse group of metabolically active compounds. The typical sterols have a 3β-hydroxy-Δ⁵-(or Δ⁰-) cholestane nucleus and a C₈–C₁₀ side chain. Marine organisms are of particular interest for research due to their high content of oxysterols, which are involved in a variety of biological activities.¹⁾ Soft corals are marine invertebrates of the order Alcyonacea, subclass Octocorallia, class Anthozoa, and phylum Cnidaria. Among these marine invertebrates, the genera Cespitularia, Clavularia, Gersemia, Lobophytum, Nephthea, Sarcophyton, and Sinularia are the most prolific.²⁾ The Sinularia species were found to contain many hydroxylated steroids having cytotoxic effects.^3–9)

Recently, we reported five sterols from the soft coral S. microspiculata and evaluation of their cytotoxic effects.¹⁰⁾ In continuation of our ongoing research on the cytotoxic steroids of Vietnamese Sinularia soft corals, we report herein the isolation, structure elucidation, and cytotoxic evaluation of six sterols (Fig. 1), including two new compounds, from the soft coral S. nanolobata.

Fig. 1. Structures of 1–6

Results and Discussion

A methanol extract of the Vietnamese soft coral S. nanolobata revealed six sterols (1–6, Fig. 1) after subjecting it on various chromatographic separations. Detailed analysis of the spectroscopic data one and two dimensional (1D- and 2D)-NMR and MS and comparison with literature values led to elucidation of the known compounds as dissesterol (3),¹¹⁾ 3β-hydroxy-24-methylenecholest-5-en-7-one (4),^12,13) 16α-hydroxysarcosterol (5),¹⁰⁾ and sarcophytosterol (6).¹⁴⁾

Compound 1 was isolated as a colorless powder with the molecular formula C₂₈H₄₆O₂ determined by Fourier transform ion cyclotron resonance (FT-ICR)-MS at m/z 415.35764 [M+H]⁺. The NMR features indicated an ergosterol derivative, one main constituent of Sinularia soft corals.³⁾ The ¹H- and ¹³C-NMR spectra of 1 showed typical signals of two oxymethines [δ_C 76.8 (C-3), 75.2 (C-4)/δ_H 3.26 (1H, ddd, J=11.0, 8.0, 4.5 Hz, H-3) and 4.06 (1H, d, J=8.0 Hz, H-4)], one trisubstituted endocyclic double bond [δ_C 142.1 (C, C-5), 117.8 (CH, C-6)/δ_H 5.74 (1H, t, J=3.0 Hz, H-6)], one 1,1-disubstituted double bond [δ_C 156.9 (C, C-24), 106.0 (CH₂, C-28)/δ_H 4.66, 4.71 (H-28), each 1H, br s], two tert-methyls [δ_C 11.9 (C-18), 20.3 (C-19)/δ_H 0.69 (H-18), 1.03 (H-19), each 3H, s], and three sec-methyls [δ_C 18.7 (C-21), 21.9 (C-26), 22.0 (C-27)/δ_H 0.95 (H-21), 1.02 (H-26), 1.03 (H-27), each 3H, d, J=7.0 Hz]. The NMR data of 1 were similar to those of 4,^12,13) except for the presence of an oxymethine group in 1 instead of a ketone group in 4. The ¹H–¹H correlation spectroscopy (COSY) confirmed a connectivity of H₂-1/H₂-2/H-3/H-4. This evidence and the heteronuclear multiple bond correlation (HMBC) cross-peaks of H₃-19 (δ_H 1.03) with C-1 (δ_C 36.7), C-5 (δ_C 142.1), C-9 (δ_C 50.5), and C-10 (δ_C 38.1) and H-6 (δ_H 5.74) with C-4 (δ_C 75.2) indicated the positions of the two hydroxyl groups at C-3 and C-4 and the trisubstituted double bond C-5/C-6. Detailed analysis of other COSY and HMBC correlations (Fig. 2) clearly confirmed the planar structure of 1. The 3β,4α-dihydroxy configurations were determined by the ¹³C-NMR chemical shifts at C-3 (δ_C 76.8), C-4 (δ_C 75.2), C-5 (δ_C 142.1), and C-6 (δ_C 117.8) of 1, which were essentially identical to those of cholest-5-ene-3β,4α-diol at δ_C 76.5 (C-3), 75.1 (C-4), 142.0 (C-5), and 117.8 (C-6)¹⁵⁾ and quite different from those of cholest-5-ene-3β,4β-diol at δ_C 77.2 (C-3), 72.6 (C-4), 142.9 (C-5), and 128.9 (C-6).¹⁵⁾ The α-orientation of 4-OH group was also supported by a nuclear Overhauser spectroscopy (NOESY) correlation of H-4 (δ_H 4.06) with H-19 (δ_H 1.03). The large coupling constant (J=8.0 Hz) of H-4 indicated trans-relationship between H-3 and H-4 (versus J=4.0 Hz for cis-relationship as in case of cholest-5-ene-3β,4β-diol)¹⁵⁾ and thus β-orientation of 3-OH. The β-orientation of 3-OH was also supported by NOESY correlations of H_a-1 (δ_H 1.14) with H-3 (δ_H 3.26) and H-9 (δ_H 1.00). Moreover, NOESY correlations of H-8 (δ_H 1.47) with H-18 (δ_H 0.69)/H-19 (δ_H 1.03) and H-14 (δ_H 1.02) with H-17 (δ_H 1.14) were also observed (Fig. 2). These NOESY data determined all trans junctions of A to D rings. Thus, the structure of 1 was clearly elucidated as 3β,4α-dihydroxyergosta-5,24(28)-diene.

Fig. 2. Key COSY (), HMBC (), and NOESY () Correlations of 1

The molecular formula of 2 was identified as C₂₈H₄₆O₃, by FT-ICR-MS with a quasi-molecular ion peak at m/z 453.33444 [M+Na]⁺. The ¹H- and ¹³C-NMR data of 2 (Table 1) were similar to those of 1 except for the difference in the data of the side-chain with presence of an oxygenated quaternary carbon [δ_C 62.8 (C-24)] and an oxymethylene group [δ_C 50.5 (C-28)/δ_H 2.53, 2.59 (H-28), each 1H, d, J=5.0 Hz] in 2 instead of the 1,1-disubstituted double bond in 1. These two carbon signals were strongly shifted upfield indicated the presence of an epoxy bridge between C-24/C-28, which was also confirmed by the agreement of the ¹³C-NMR data for the side-chain of 2 with those of 24,28-epoxyergost-5-ene-3β,7α-diol¹⁶⁾ and by HMBC correlations of H-26 (δ_H 0.90) and H-27 (δ_H 0.95) with C-24 (δ_C 62.8) and H-25 (δ_H 1.77) with C-28 (δ_C 50.5). The absolute configuration 24S of 2 was assigned by circular dichroism (CD) spectrum with a negative Cotton effect at 286 nm.¹⁷⁾ Consequently, the structure 24(S),28-epoxyergost-5-ene-3β,4α-diol was elucidated for 2.

Table 1. The ¹H- and ¹³C-NMR Data for 1 and 2

Position	1		2
	δCa,b)	δHa,c) mult. (J in Hz)	δCa,b)	δHa,c) mult. (J in Hz)
1	36.7	1.14 m/1.85 m	36.7	1.14 m/1.85 m
2	28.1	1.60 m/1.90 m	28.1	1.60 m/1.90 m
3	76.8	3.26 ddd (11.0, 8.0, 4.5)	76.6	3.26 ddd (11.0, 7.5, 4.5)
4	75.2	4.06 d (8.0)	75.1	4.05 d (7.5)
5	142.1	—	142.0	—
6	117.8	5.74 t (3.0)	117.8	5.74 t (3.0)
7	31.5	1.57 m/2.11 m	31.5	1.60 m/2.12 m
8	31.4	1.47 m	31.4	1.45 m
9	50.5	1.00 m	50.5	1.00 m
10	38.1	—	38.0	—
11	20.9	1.49 m	20.9	1.49 m
12	39.7	1.18 m/2.03 m	39.7	1.18 m/2.01 m
13	42.3	—	42.3	—
14	56.7	1.02 m	56.7	1.01 m
15	24.2	1.11 m/1.63 m	24.3	1.11 m/1.62 m
16	28.2	1.30 m/1.88 m	28.2	1.30 m/1.90 m
17	56.0	1.14 m	55.8	1.12 m
18	11.9	0.69 s	11.9	0.68 s
19	20.3	1.03 s	20.2	1.02 s
20	35.8	1.44 m	35.8	1.40 m
21	18.7	0.95 d (7.0)	18.7	0.92 d (6.5)
22	34.7	1.17 m/1.56 m	30.3	1.32 m/1.42 m
23	31.0	1.90 m/2.10 m	28.1	1.45 m/1.85 m
24	156.9	—	62.8	—
25	33.8	2.23 m	31.7	1.77 m
26	21.9	1.02 d (7.0)	17.7	0.90 d (6.5)
27	22.0	1.03 d (7.0)	18.4	0.95 d (6.5)
28	106.0	4.66 br s/4.71 br s	50.5	2.53 d (5.0)/2.59 d (5.0)

a) Recorded in CDCl₃. b) 125 MHz. c) 500 MHz. All assignments were done by HSQC, HMBC, ¹H–¹H COSY, and NOESY experiments.

The isolated compounds were evaluated for their cytotoxic activity against a panel of eight human cancer cell lines including HepG2 (hepatoma cancer), HL-60 (acute leukemia), KB (epidermoid carcinoma), LNCaP (prostate cancer), LU-1 (lung cancer), MCF7 (breast cancer), SK-Mel2 (melanoma), and SW480 (colon adenocarcinoma) using sulforhodamine B (SRB) method.¹⁸⁾ The new compound 24(S),28-epoxyergost-5-ene-3β,4α-diol (2) exhibited moderate cytotoxicity against the HL-60 (IC₅₀=33.53±4.25 µM) cell line and weak effect on the HepG2 (IC₅₀=64.35±7.00 µM) and SW480 (IC₅₀=71.02±4.00 µM) cell lines relative to that of the positive control (ellipticine: IC₅₀=1.91±0.36, 1.95±0.28, 2.23±0.16 µM against HL-60, HepG2, SW480, respectively). Sarcophytosterol (6) was previously found to have weak cytotoxic effect against the HL-60 (IC₅₀=89.02±9.93 µM) cell line.¹⁰⁾ No cytotoxicity against any of the tested cancer cell lines (IC₅₀>100 µM) were observed for the other compounds. Consideration the structures of 1 and 2 suggested that the C-24/C-28 epoxy bridge might play an important role for the cytotoxic activity of these compounds.

Experimental

General Experimental Procedures

Optical rotations were determined using a Jasco P-2000 digital polarimeter. CD spectra were recorded with a Chirascan (Applied Photophysics, U.K.) spectropolarimeter. High resolution mass spectra were recorded on a Varian 910 FT-ICR mass spectrometer. The NMR spectra were recorded on a Bruker AVANCE III HD 500 spectrometer with tetramethylsilane (TMS) as an internal standard. Medium pressure liquid chromatography (MPLC) was carried out on a Biotage–Isolera One system. TLC was performed on Kieselgel 60 F₂₅₄ (1.05715; Merck) or RP-18 F_254s plates. Spots were visualized by spraying with 10% aqueous H₂SO₄ solution, followed by heating for 3–5 min. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70–230, 230–400 mesh, Merck) and YMC*GEL resins (ODS-A, 12 nm S-150 µm, YMC Co., Ltd., Japan).

Biological Material

The samples of soft coral S. nanolobata Verseveldt, 1977 were collected in Lang Co., Hue, Vietnam, in April 2015 and identified by Professor Do Cong Thung (Institute of Marine Resources and Environment, VAST, Vietnam). Voucher specimens (No. SN201504) were deposited at the Institute of Marine Biochemistry, VAST.

Extraction and Isolation

Dried bodies of the soft coral S. nanolobata (1.5 kg) was extracted three times with methanol under ultrasonic condition. The resulting solutions were filtered, combined, and concentrated under reduced pressure to obtain the methanol residue (SNM, 140 g), which was suspended in water and extracted in turn with n-hexane and dichloromethane resulting in extracts of n-hexane (SNH, 47 g), dichloromethane (SND, 5 g), and an aqueous layer. Extract SNH (47 g) was crude separated on silica gel MPLC using the mobile phase of n-hexane–acetone (gradient 50 : 1→1 : 1, v/v) to obtain six fractions, SNH1–SNH6. Fraction SNH4 (7.3 g) was further separated on silica gel MPLC using the mobile phase of n-hexane–ethyl acetate (gradient 5 : 1→1 : 1, v/v) to give seven subfractions, SNH4A–SNH4G. Subfraction SNH4B (2.1 g) was further separated into nine smaller fractions, SNH4B1–SNH4B9, by YMC CC eluting with methanol–water (2 : 1, v/v). Compounds 5 (5.0 mg) and 6 (3.5 mg) were obtained from subfraction SNH4B7 (300 mg) after purification with YMC CC eluted with acetone–water (3 : 1, v/v), followed by silica gel CC with n-hexane–acetone (3.5 : 1, v/v). Fraction SNH4B6 (150 mg) was purified by silica gel CC eluting with n-hexane–acetone (4 : 1, v/v) to obtain compounds 1 (3.5 mg) and 2 (2.5 mg). Fraction SNH5 (5.0 g) was further separated on RP-18 MPLC with methanol–water (2 : 1, v/v) to give six subfractions, SNH5A–SNH5G. Finally, purification of subfraction SNH5F (130 mg) by silica gel CC eluting with n-hexane–ethyl acetate (2 : 1, v/v) to obtain compounds 3 (2.6 mg) and 4 (6.0 mg).

3β,4α-Dihydroxyergosta-5,24(28)-diene (1)

Colorless powder; [α]_D²⁵ −25° (c=0.05, CHCl₃); FT-ICR-MS m/z 415.35764 [M+H]⁺ (Calcd for C₂₈H₄₇O₂⁺, 415.35706); ¹H- and ¹³C-NMR data, see Table 1.

24(S),28-Epoxyergost-5-ene-3β,4α-diol (2)

Colorless powder; [α]_D²⁵ −12° (c=0.05, MeOH); FT-ICR-MS m/z 453.33444 [M+Na]⁺ (Calcd for C₂₈H₄₆NaO₃⁺, 453.33392); CD (c=2.30×10⁻³ M, MeCN) λ_max ([θ]): 286 (−574.8) nm; ¹H- and ¹³C-NMR data, see Table 1.

Cytotoxic Assays

Cytotoxic evaluations were performed by following the previously described protocols.^19,20)

Acknowledgments

This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under Grant number 104.01–2013.31. The authors are grateful to the Institute of Chemistry, VAST for measurement of the NMR and mass spectra; Dr. Do Thi Thao, Institute of Biotechnology, VAST for the cytotoxicity evaluation; and Dr. Bui Huu Tai, Institute of Marine Biochemistry, VAST for measurement of the CD spectra.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials, 1D- and 2D-NMR spectra for the new compounds 1 and 2.

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