Cytotoxic Steroids from the Vietnamese Soft Coral Sinularia conferta

Ninh Thi Ngoc; Pham Thi Mai Huong; Nguyen Van Thanh; Nguyen Thi Phuong Chi; Nguyen Hai Dang; Nguyen Xuan Cuong; Nguyen Hoai Nam; Do Cong Thung; Phan Van Kiem; Chau Van Minh

doi:10.1248/cpb.c16-00881

Abstract

Twelve steroids, including five new compounds 1–5, were isolated and structurally elucidated from a methanol extract of the Vietnamese soft coral Sinularia conferta. Their cytotoxic effects against three human cancer cell lines, lung carcinoma (A-549), cervical adenocarcinoma (HeLa), and pancreatic epithelioid carcinoma (PANC-1), were evaluated using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assays. Among isolated compounds, 10 exhibited potent cytotoxic effects on all three tested cell lines with IC₅₀ values of 3.64±0.18, 19.34±0.42, and 1.78±0.69 µM, respectively.

Steroids are a highly diverse group of metabolically active compounds. 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⁾ Among marine organisms, Sinularia soft corals (phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea) are a rich source of steroids and terpenoids,^1–3) with many hydroxylated steroids having cytotoxic effects.^2,4–9)

To date, the soft coral Sinularia conferta is a little investigated species with several publications have been reported on isolation of some steroid, diterpenoid, sesquiterpene, and ceramide derivatives.^10–15) In continuation of our recent investigations on cytotoxic steroids from Vietnamese Sinularia soft corals,^16,17) the current paper addressed the isolation, structure elucidation, and cytotoxic evaluation of twelve steroids, including five new compounds, from this soft coral.

Results and Discussion

A methanol extract of the S. conferta afforded twelve steroids (Fig. 1) after subjecting it on various chromatographic separations. The known compounds were identified as 7α-methoxyergosta-5,24(28)-diene-3β-ol (6),^18,19) ergosta-5-ene-3β,7α-diol (7),²⁰⁾ 3β,7α-dihydroxyergosta-5,24(28)-diene (8),²¹⁾ 3β-hydroxyergosta-5,24(28)-diene-7-one (9),²²⁾ ergosta-24(28)-ene-3β,5α,6β-triol-6-acetate (10),²³⁾ ergosta-24(28)-ene-3β,5α,6β-triol (11),^20,23) and ergosta-3β,5α,6β-triol (12),²⁴⁾ by spectroscopic experiments, one and two dimensional (1D- and 2D)-NMR and MS, and comparison with reported data.

Fig. 1. Structures of Compounds 1–12

Compound 1 was isolated as a white powder with molecular of C₃₁H₅₂O₂, determined by a quasi-molecular ion peak at m/z 457.4046 [M+H]⁺ on high-resolution electrospray ionization (HR-ESI)-MS. Its NMR features are characteristic for a gorgosterol derivative possessing a cyclopropane ring in side chain with presence of four high-field protons at δ_H 0.18 (1H, m, H-22), 0.24 (1H, m, H-24), −0.13 (1H, t, J=4.5 Hz, H_β-30), and 0.45 (1H, dd, J=4.5, 9.0 Hz, H_α-30).^16,25) In addition, the signals of two oxymethines [δ_C 71.4 (C-3) and 73.9 (C-7)/δ_H 3.62 (1H, m, H-3) and 3.30 (1H, br d, J=4.5 Hz, H-7)], one methoxy group [δ_C 56.8/δ_H 3.36 (3H, s), 7-OMe], and one trisubstituted endocyclic double bond [δ_C 146.1 (C, C-5) and 120.8 (CH, C-6)/δ_H 5.75 (1H, dd, J=1.0, 4.5 Hz, H-6)] were also observed. Detailed analysis of the ¹H, ¹³C-NMR, and heteronuclear single quantum coherence spectroscopy (HSQC) spectra led to assignment of all ¹H-NMR data with the relevant ¹³C-NMR values of 1 as shown in Table 1. The ¹H–¹H correlation spectroscopy (COSY) experiment revealed the proton–proton correlations of H₂-1/H₂-2/H-3/H₂-4 and H-6/H-7/H-8/H-9/H₂-11/H₂-12. These data, together with the heteronuclear multiple bond correlation (HMBC) cross-peaks of H-19 (δ_H 0.98) with C-1 (δ_C 36.8), C-5 (δ_C 146.1), C-9 (δ_C 42.8), and C-10 (δ_C 37.4); H-6 (δ_H 5.75) with C-4 (δ_C 42.3), C-8 (δ_C 37.3), and C-10 (δ_C 37.4); and 7-OMe (δ_H 3.36) with C-7 (δ_C 73.9), confirmed positions of the two oxymethines C-3 and C-7, double bond at C-5/C-6 and attachment of the methoxy group at C-7. Detailed analysis of other COSY and HMBC correlations (Fig. 2) clearly confirmed the planar structure of 1. The proton signal of H-3 at δ_H 3.62 (1H, m) is indicative for an α-orientation of H-3²⁶⁾ (versus t, J=2.5 or 3.0 Hz for β-orientation of H-3).^27,28) This was further confirmed by the ¹³C-NMR chemical shift for C-3 (δ_C 71.4) of 1, which was similar to that of 3β-hydroxycholest-5-en-7-one at δ_C 70.61,²⁶⁾ and quite different from that of aragusterol G (with 3α-OH group) at δ_C 66.4.²⁹⁾ The ¹³C-NMR chemical shifts at C-5 (δ_C 146.1), C-7 (δ_C 73.9), C-9 (δ_C 42.8), and C-14 (δ_C 48.9) of 1 were similar to those of 7α-methoxyergosta-5,24(28)-diene-3β-ol (6) at δ_C 146.2 (C-5), 73.7 (C-7), 42.4 (C-9), and 48.8 (C-14)^18,19) and 3β-hydroxy-7α-methoxy-24β-ethyl-cholest-5-ene (schleicheol 2) at δ_C 146.1 (C-5), 73.9 (C-7), 42.7 (C-9), and 49.1 (C-14),³⁰⁾ but quite different from those of 3β-hydroxy-7β-methoxy-24β-ethyl-cholest-5-ene (schleicheol 1) at δ_C 144.0 (C-5), 81.9 (C-7), 48.5 (C-9), and 56.5 (C-14),³⁰⁾ unambiguously indicated a β-orientation of H-7, which was further confirmed by a nuclear Overhauser spectroscopy (NOESY) correlation (Fig. 3) of H-7 (δ_H 3.30) with H-8 (δ_H 1.48). Moreover, the ¹³C-NMR data for the side-chain of 1 were essentially identical to those of 7-oxogorgosterol,¹⁶⁾ crassumsterol,³¹⁾ and 7β-hydroxygorgosterol,³²⁾ suggesting the same configurations in the side-chain of these compounds, which was also supported by NOESY data (Fig. 3). Thus, the structure of 7α-methoxygorgosterol was determined for compound 1.

Table 1. ¹H- (CDCl₃, 500 MHz) and ¹³C-NMR (CDCl₃, 125 MHz) Spectroscopic Data of 1 and 2

Pos.	1		2
Pos.	δ_C	δ_H (J in Hz)	δ_C	δ_H (J in Hz)
1	36.8	1.16 m/1.83 m	36.8	1.16 m/1.83 m
2	31.5	1.50 m/1.85 m	31.5	1.52 m/1.84 m
3	71.4	3.62 m	71.5	3.61 m
4	42.3	2.30 m/2.33 m	42.3	2.30 m/2.34 m
5	146.1	—	146.1	—
6	120.8	5.75 dd (1.0, 4.5)	120.8	5.73 d (4.0)
7	73.9	3.30 br d (4.5)	73.9	3.29 br d (4.0)
8	37.3	1.48 m	37.2	1.49 m
9	42.8	1.32 m	42.8	1.32 m
10	37.4	—	37.5	—
11	20.9	1.45 m/1.50 m	20.8	1.45 m/1.50 m
12	39.2	1.20 m/1.99 m	39.1	1.15 m/1.95 m
13	42.6	—	42.1	—
14	48.9	1.51 m	49.1	1.50 m
15	24.5	1.08 m/1.65 m	24.3	1.08 m/1.63 m
16	28.3	1.32 m/2.07 m	28.2	1.28 m/1.88 m
17	57.7	1.32 m	55.7	1.30 m
18	11.5	0.64 s	11.5	0.66 s
19	18.3	0.98 s	18.3	0.98 s
20	35.3	1.01 m	36.2	1.37 m
21	21.2	1.01 br s	18.9	0.92 d (6.5)
22	32.2	0.18 m	33.8	0.96 m/1.40 m
23	25.8	—	30.5	0.96 m/1.38 m
24	50.8	0.24 m	39.1	1.20 m
25	32.1	1.57 m	31.5	1.57 m
26	21.3	0.86 d (6.5)	17.6	0.79 d (6.5)
27	22.2	0.96 d (6.5)	20.5	0.86 d (6.5)
28	15.4	0.96 d (6.5)	15.5	0.78 d (6.5)
29	14.3	0.91 s
30	21.5	β −0.13 t (4.5)
		α 0.45 dd (4.5, 9.0)
OMe	56.8	3.36 s	56.8	3.36 s

Assignments were confirmed by HSQC, HMBC, COSY, and NOESY experiments.

Fig. 2. Key COSY (

) and HMBC (

) Correlations of 1, 3, and 5

Fig. 3. Key NOESY Correlations of 1 and 5

The ¹H- and ¹³C-NMR data of 2 were similar to those of 1 (Table 1), except for difference in the signals of the side chain. The presence of four sec-methyl proton signals [δ_H 0.92 (H-21), 0.79 (H-26), 0.86 (H-27), and 0.78 (H-28), each 3H, d, J=6.5 Hz], suggested for an ergostane-like side chain of 2, which was also supported by HR-ESI-MS with a quasi-molecular ion peak at m/z 431.3889 [M+H]⁺. In addition, the structure 7α-methoxy-ergosta-5-ene-3β-ol of 2 was further confirmed by 2D-NMR data, a good agreement of the ¹H- and ¹³C-NMR data for the side chain of 2 with those of ergosta-5-ene-3β,7α-diol (7),²⁰⁾ and the coexistence of these two compounds in S. conferta.

The HR-ESI-MS of 3 revealed a quasi-molecular ion peak at m/z 415.3575 [M+H]⁺, corresponding to a molecular of C₂₈H₄₆O₂. The ¹H- and ¹³C-NMR data of 3 were similar to those of 2 (Tables 1, 2) and 7,²⁰⁾ indicating the presence of an ergostane-like side chain. The ¹H- and ¹³C-NMR signals of the steroid nucleus demonstrated the presence of an oxymethine group [δ_C 67.3 (C-3)/δ_H 4.24 (1H, br dd, J=7.0, 8.0 Hz, H-3)], a trisubstituted double bond [δ_C 132.5 (CH, C-4) and 147.0 (C, C-5)/δ_H 6.15 (1H, s, H-4)], and a ketone group [δ_C 203.1 (C-6)]. The ¹H–¹H COSY experiment revealed the proton–proton correlations of H₂-1/H₂-2/H-3/H-4. This evidence and HMBC cross-peaks of H-19 (δ_H 1.01) with C-1 (δ_C 34.8), C-5 (δ_C 147.0), C-9 (δ_C 51.3), and C-10 (δ_C 38.4) and those of H-4 (δ_H 6.15) with C-6 (δ_C 203.1) and C-10 (δ_C 38.4), confirmed positions of the oxymethine C-3, double bond at C-4/C-5 and ketone C-6. The 3β-OH configuration was determined by circular dichroism (CD) spectrum of 3 with a negative Cotton effect at 253 nm, which was similar to that of 3β-hydroxycholest-4-en-6-one pocessing a negative Cotton effect at 246 nm³³⁾ and opposite to that of 3α-hydroxycholest-4-en-6-one with a positive Cotton effect at 225 nm.³³⁾ Consequently, the structure of 3 was elucidated as 3β-hydroxyergosta-4-ene-6-one.

Table 2. ¹H- (500 MHz) and ¹³C-NMR (125 MHz) Spectroscopic Data of 3–5

Pos.	3^a)		4^a)		5^b)
Pos.	δ_C	δ_H (J in Hz)	δ_C	δ_H (J in Hz)	δ_C	δ_H (J in Hz)
1	34.8	1.45 m/1.78 m	34.8	1.45 m/1.78 m	33.2	1.38 m/1.67 m
2	28.4	1.55 m/2.06 m	28.4	1.54 m/2.05 m	27.9	1.60 m/1.84 m
3	67.3	4.24 br dd (7.0, 8.0)	67.3	4.24 br t (7.5)	73.0	5.19 m
4	132.5	6.15 s	132.5	6.15 s	38.0	1.62 m/2.18 m
5	147.0	—	146.9	—	76.6	—
6	203.1	—	203.1	—	76.4	3.48 br s
7	46.4	1.90 dd (12.0, 16.0)	46.4	1.88 dd (12.5, 16.0)	35.3	1.54 m/1.72 m
		2.55 dd (4.0, 16.0)		2.56 dd (4.0, 16.0)
8	34.2	1.85 m	34.2	1.83 m	31.6	1.75 m
9	51.3	1.22 m	51.3	1.22 m	46.5	1.42 m
10	38.4	—	38.4	—	39.4	—
11	20.8	1.43 m/1.58 m	20.8	1.42 m/1.58 m	22.2	1.37 m/1.40 m
12	39.3	1.23 m/2.06 m	39.3	1.23 m/2.06 m	41.5	1.20 m/2.02 m
13	42.6	—	42.6	—	44.0	—
14	56.7	1.13 m	56.7	1.12 m	57.6	1.17 m
15	24.0	1.10 m/1.65 m	23.9	1.10 m/1.57 m	25.2	1.12 m/1.63 m
16	28.0	1.30 m/1.88 m	28.0	1.30 m/1.88 m	29.3	1.30 m/1.88 m
17	55.9	1.15 m	55.9	1.15 m	57.4	1.12 m
18	11.9	0.69 s	11.9	0.70 s	12.6	0.73 s
19	19.8	1.01 s	19.8	1.01 s	17.1	1.20 s
20	36.1	1.38 m	35.6	1.42 m	36.9	1.45 m
21	18.8	0.93 d (7.0)	18.6	0.95 d (6.5)	19.2	0.99 d (7.0)
22	33.6	0.95 m/2.35 m	34.6	1.15 m/1.54 m	36.0	1.16 m/1.58 m
23	30.6	1.05 m/1.37 m	31.0	1.88 m/2.09 m	32.1	1.93 m/2.13 m
24	39.1	1.20 m	156.7	—	157.9	—
25	31.5	1.58 m	33.8	2.22 m	34.9	2.24 m
26	17.6	0.79 d (7.0)	21.9	1.04 d (6.5)	22.3	1.05 d (7.0)
27	20.5	0.86 d (7.0)	22.0	1.03 d (6.5)	22.4	1.06 d (7.0)
28	15.4	0.78 d (7.0)	106.1	4.66 s/4.72 s	106.8	4.67 s/4.74 s
OAc
1′					172.8	—
2′					21.3	2.02 s

a) Recorded in CDCl₃. b) Recorded in CD₃OD. Assignments were confirmed by HSQC, HMBC, COSY, and NOESY experiments.

The ¹H- and ¹³C-NMR data of 4 were similar to those of 3 (Table 3), except for difference in the signals of the side chain with the presence of a terminal double bond [δ_C 156.7 (C, C-24) and 106.1 (CH₂, C-28)/δ_H 4.66 and 4.72, H₂-28, each 1H, br s] in 4 replaced for a methine and a sec-methyl in 3. This was also confirmed by HR-ESI-MS at m/z 413.3419 [M+H]⁺, corresponding to a molecular of C₂₈H₄₄O₂. The HMBC cross-peaks of H-26 (δ_H 1.04) with C-24 (δ_C 156.7), C-25 (δ_C 33.8), and C-27 (δ_C 22.0); H-27 (δ_H 1.03) with C-24 (δ_C 156.7), C-25 (δ_C 33.8), and C-26 (δ_C 21.9); and those of H-28 (δ_H 4.66 and 4.72) with C-23 (δ_C 31.0), C-24 (δ_C 156.7), and C-25 (δ_C 33.8), clearly determined the position of the terminal double bond at C-24/C-28. Thus, compound 4 was elucidated as 3β-hydroxyergosta-4,24(28)-diene-6-one.

Table 3. Cytotoxic Activity of the Active Compounds 10–12 against A-549, HeLa, and PANC-1 Cell Lines

Compounds	IC₅₀^a) (µM)
Compounds	A-549	HeLa	PANC-1
10	3.64±0.18	19.34±0.42	1.78±0.69
11	78.73±2.11	30.5±0.77	9.35±0.37
12	27.12±1.69	24.64±1.28	20.51±2.72
Camptothecin^b)	12.65±1.01	—	—
Etoposide^b)	—	27.99±2.01	1.17±0.42

a) Data represent the mean±standard deviation (S.D.) of triplicate experiments. b) Positive control; — Not tested.

Compound 5 was also isolated as a white powder with a molecular of C₃₀H₅₀O₄, determined by a quasi-molecular ion peak at m/z 475.3787 [M+H]⁺ on HR-ESI-MS. Its NMR features are characteristic for a sterol with ergosta-24(28)-ene structure determined by good agreement of the ¹H- and ¹³C-NMR data for the side-chain of 5 with those of 4 (Table 2). The ¹H- and ¹³C-NMR spectra for steroid nucleus of 5 exhibited signals of two oxymethine groups [δ_C 73.0 (C-3) and 76.4 (C-6)/δ_H 5.19 (1H, m, H-3) and 3.48 (1H, br s, H-6)] and one oxygenated quaternary carbon [δ_C 76.6 (C-5)]. In addition, an acetate group was identified by signals at δ_C 172.8 (C, C-1′) and 21.3 (CH₃, C-2′)/δ_H 2.02 (3H, s, H-2′). The ¹H–¹H COSY experiment revealed the proton–proton correlations of H₂-1/H₂-2/H-3/H₂-4 and H-6/H-7/H-8/H-9/H₂-11/H₂-12. This evidence and HMBC cross-peaks of H-19 (δ_H 1.20) with C-1 (δ_C 33.2), C-5 (δ_C 76.6), C-9 (δ_C 46.5), and C-10 (δ_C 39.4); H-4 (δ_H 2.18) and H-6 (δ_H 3.48) with C-5 (δ_C 76.6), and H-3 (δ_H 4.24) with C-1′ (δ_C 172.8), confirmed positions of the oxymethines C-3 and C-6, oxygenated quaternary carbon C-5, and attachment of the acetate at C-3. The configurations at C-3, C-5, and C-6 of 5 were assigned to be identical to those of muriflasteroid A,³⁴⁾ based on an agreement of the ¹H- and ¹³C-NMR data for the steroid nucleus of these two compounds, which were further confirmed by NOESY experiment (Fig. 3). Thus, compound 5 was elucidated as ergosta-24(28)-ene-3β,5α,6β-triol-3-acetate.

All isolated compounds were tested for their cytotoxic activity against three human cancer cell lines as lung carcinoma (A-549), cervical adenocarcinoma (HeLa), and pancreatic epithelioid carcinoma (PANC-1), using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assays.^35,36) As the results, compounds 10, 11, and 12 exhibited cytotoxicity against three tested cell lines (Table 3) while the other compounds were not active (IC₅₀>100 µM). Comparison of the activities of compound 10 with those of the positive controls showed that 10 (IC₅₀ values of 3.64±0.18, 19.34±0.42, and 1.78±0.69 µM, respectively) were more potent than camptothecin on A-549 (IC₅₀=12.65±1.01 µM) and etoposide on HeLa cell lines (IC₅₀=27.99±2.01 µM), and as strong as etoposide on PANC-1 cell line (IC₅₀=1.17±0.42 µM). Whereas, compounds 11 and 12 showed significant to moderate cytotoxic activities against three cell lines with the IC₅₀ values ranging from 9.35±0.37 to 78.73±2.11 µM. From the above results, ergosta-24(28)-ene-3β,5α,6β-triol-6-acetate (10) exerts potent cytotoxic effects on all three tested human cancer cell lines and may be a potential candidate for further molecular mechanism-of-action studies. In addition, consideration the structures of isolated compounds suggested that the 3β,5α,6β-triol pattern and location of the acetate substituent might play an important role for cytotoxic activity of these compounds.

Experimental

General Experimental Procedures

Optical rotations were determined on a JASCO P-2000 polarimeter (Hachioji, Tokyo, Japan). CD spectra were recorded with a Chirascan (Applied Photophysics, U.K.) spectropolarimeter. High resolution mass spectra were recorded on an IonSpec 4.7 Tesla spectrometer (Varian, U.S.A.). The ¹H-NMR (500 MHz) and ¹³C-NMR (125 MHz) spectra were recorded on Bruker AM500 and AVANCE III HD 500 (Billerica, MA, U.S.A.) FT-NMR spectrometers with tetramethylsilane (TMS) was used as an internal standard. Medium pressure liquid chromatography (MPLC) was carried out on a Biotage–Isolera One system (SE-751 03 Uppsala, Sweden). Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck, Darmstadt, Germany), YMC*GEL (ODS-A, 12 nm S-150 mm, YMC Co., Ltd., Japan), and Diaion HP-20 (Supelco) resins. TLC used pre-coated silica gel 60 F₂₅₄ (1.05554.0001, Merck) and RP-18 F_254S plates (1.15685.0001, Merck), and compounds were visualized by spraying with aqueous 10% H₂SO₄ and heating for 3–5 min.

Biological Material

The samples of S. conferta (Dana, 1846) were collected near the Con Co island, Quangtri, Vietnam, in May 2015, and identified by Prof. Do Cong Thung, Institute of Marine Environment and Resources, VAST. A voucher specimen (SCO-052015) was deposited at the Institute of Marine Environment and Resources and Institute of Marine Biochemistry, VAST, Vietnam.

Extraction and Isolation

Dried bodies of the soft coral S. conferta (2.5 kg) were extracted three times with methanol (5 L each) under ultrasonic condition. The resulting solutions were filtered, combined, and concentrated under reduced pressure to obtain the methanol residue (SCO.M, 150.0 g), which was suspended in water and extracted in turn with n-hexane and CH₂Cl₂ resulting in extracts of n-hexane (SCO.H, 100.0 g), CH₂Cl₂ (SCO.D, 6.0 g), and water layer. Extract SCO.H (100.0 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 eight fractions, SCO.H1–SCO.H6. Fraction SCO.H5 (10 g) was further separated on RP-18 MPLC using the mobile phase of MeOH–H₂O (gradient 2 : 1→10 : 1, v/v) to give seventeen subfractions, SCO.H5A1–SCO.H5A17. Subfraction SCO.H5A14 (800 mg) was separated into ten smaller fractions, SCO.H5A14A–SCO.H5A14K, by Silica gel CC using CH₂Cl₂–acetone (10 : 1, v/v) as eluent. Fraction SCO.H5A14C (80 mg) was purified by YMC CC eluting with acetone–H₂O (5 : 1, v/v), followed by Silica gel CC with n-hexane–ethyl acetate (4.5 : 1, v/v) to obtain compounds 1 (2.0 mg), 2 (1.2 mg), 5 (1.0 mg), and 6 (3.0 mg). Fraction SCO.H5A14F (100 mg) was further separated by Silica gel CC with n-hexane–acetone (2 : 1, v/v), followed by YMC CC eluted with acetone–H₂O (4 : 1, v/v), to furnish compounds 3 (0.7 mg), 4 (3 mg), and 9 (5.0 mg). Compounds 7 (1.0 mg) and 8 (2.0 mg) were purified from fraction SCO.H5A14H (60 mg) after subjecting it on YMC CC eluted with acetone–H₂O (5 : 1, v/v). Fraction SCO.H5A14K (50 mg) was purified by Silica gel CC eluting with n-hexane–acetone (5 : 1, v/v) to give compound 10 (9.5 mg). Subfraction SCO.H5A12 (700 mg) was separated into five smaller fractions, SCO.H5A12A–SCO.H5A12E, by Silica gel CC with CH₂Cl₂–acetone (10 : 1, v/v). Fraction SCO.H5A12D (34 mg) was purified by Silica gel CC eluting with n-hexane–acetone (3 : 1, v/v), followed by YMC CC with methanol–H₂O (10 : 1, v/v), to obtain compounds 11 (2.4 mg) and 12 (1.0 mg).

7α-Methoxygorgosterol (1)

White powder, [α]_D²⁵ −55.0 (c=0.06, CHCl₃); ¹H-NMR (CDCl₃, 500 MHz) and ¹³C-NMR (CDCl₃, 125 MHz) are given in Table 1; HR-ESI-MS m/z 457.4046 [M+H]⁺ (Calcd for C₃₁H₅₃O₂⁺, 457.4040).

7α-Methoxy-ergosta-5-ene-3β-ol (2)

White powder, [α]_D²⁵ −78.3 (c=0.06, CHCl₃); ¹H-NMR (CDCl₃, 500 MHz) and ¹³C-NMR (CDCl₃, 125 MHz) are given in Table 1; HR-ESI-MS m/z 431.3889 [M+H]⁺ (calcd for C₂₉H₅₁O₂⁺, 431.3883).

3β-Hydroxyergosta-4-ene-6-one (3)

White powder, [α]_D²⁵ −9.5 (c=0.07, CHCl₃); CD (c 2.41×10⁻⁴ M, MeOH) λ_max (mdeg): 323 (−8.85), 253 (−1.37), and 202 (+50.73) nm; ¹H-NMR (CDCl₃, 500 MHz) and ¹³C-NMR (CDCl₃, 125 MHz) are given in Table 2; HR-ESI-MS m/z 415.3575 [M+H]⁺ (Calcd for C₂₈H₄₇O₂⁺, 415.3570).

3β-Hydroxyergosta-4,24(28)-diene-6-one (4)

White powder, [α]_D²⁵ −14.5 (c=0.05, CHCl₃); ¹H-NMR (CDCl₃, 500 MHz) and ¹³C-NMR (CDCl₃, 125 MHz) are given in Table 2; HR-ESI-MS m/z 413.3419 [M+H]⁺ (Calcd for C₂₈H₄₅O₂⁺, 413.3414).

Ergosta-24(28)-ene-3β,5α,6β-triol-3-acetate (5)

White powder, [α]_D²⁵ −15.0 (c=0.07, MeOH); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) are given in Table 2; HR-ESI-MS m/z 475.3787 [M+H]⁺ (Calcd for C₃₀H₅₁O₄⁺, 475.3782).

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 the provision of the spectroscopic instrument 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, Cell Lines and Cell Culture, Cell Proliferation Assay, 1D- and 2D-NMR spectra for the new compounds 1, 3, and 5.

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