Halipanasterol,a New Sterol Isolated From the Marine Sponge Halichondria panicea

Abstract

A new sterol (1) and 8 known (2-9) compounds were isolated from the methanol extract of the Vietnamese marine sponge Halichondria panicea (Pallas, 1766) by various chromatographic methods. Their chemical structures were determined as 5α,6α-epoxy-(22E)-ergosta-3β,7α-diol-8,22,25-triene (1), (22E)-ergosta-7,22,25-triene-3β,5α,6β-triol (2), 3β-hydroxycholest-5ene-7-one (3), cholesterol sulfate (4), 3-formamidotheonellin (5), para-hydroxybenzylideneacetone (6), indole-3-carbaldehyde (7), thymidine (8), and adenosine (9) by using a combination of HR-ESI-MS, 1D and 2D NMR spectral data, as well as by comparison with the previous literature. This is the first report of these compounds from H panicea. Compounds 1 and 2 exhibited cytotoxic effects on Hep-G2, SK-Mel-2, LU-1, and MCF-7 cell lines with IC₅₀ values of 9.27 ± 1.25, 11.34 ± 1.18, 15.45 ± 1.47, and 10.85 ± 1.19 µM (for 1), and 6.45 ± 0.77, 9.74 ± 1.03, 7.08 ± 0.64, 5.61 ± 0.39 µM (for 2), compared to the positive control compound, ellipticine, 1.60 ± 0.37, 2.53 ± 0.51, 1.82 ± 0.20, and 2.17 ± 0.66 µM, respectively.

Keywords

demospongiae Halichondriidae halipanasterol sterol

Introduction

The sponge Halichondria panicea (Demospongiae class, Halichondria order, Halichondriidae family) is very widely distributed and difficult to classify. The MeOH and CHCl₃-MeOH extracts of H panicea exhibited hemolytic activity on chicken and human erythrocytes. The MeOH and aqueous extracts produced an immunostimulant effect, and all extracts revealed angiogenic activity; the aqueous extract yielded 9 bands by SDS-PAGE on 12% gel.¹ Previous phytochemical studies showed that H panicea contained sterols,^2‐4 glycosphingolipids,⁵ demospongic acids,⁶ sesquiterpenes,^7,8 lipids,^9‐11 and other constituents.^12‐14 In 2019, 7 new unusual polysulfated steroids were isolated from the marine sponge Halichondria vansoesti collected in the territorial waters of Vietnam, and some of them exhibited cytotoxic activities on human prostate cancer cells PC-3 and 22Rv1.¹⁵ In the course of our ongoing study on marine sponges,¹⁶ we report herein the isolation and structure identification of 1 new and 8 known compounds from the methanol extract of H panicea. The cytotoxic activity of compounds 1-5 on some cancer cell lines was also evaluated.

Results and Discussion

Compound 1 was obtained as a colorless solid. Its molecular formula was determined to be C₂₈H₄₂O₃ based on the quasi-molecular ion peaks at m/z 461.2793 [M+³⁵Cl]⁻ (calcd. for [C₂₈H₄₂O₃³⁵Cl]⁻: 461.2822, Δ = 6.0 ppm) and m/z 463.2753 [M+³⁷Cl]⁻ (calcd. for [C₂₈H₄₂O₃³⁷Cl]⁻: 463.2783, Δ = 6.0 ppm) in the negative mode HR-ESI-MS. The ¹H NMR spectrum of 1 revealed 5 methyls [δ_H 0.59, 1.14, 1.68 (each 3H, s), 1.02 (s) and 1.08 (d, J = 7.0 Hz)], 4 olefinic protons [δ_H 4.69 (s), 4.71 (s), 5.25 (1H, dd, J = 16.0, 6.5 Hz), and 5.29 (1H, dd, J = 16.0, 6.5)], 3 oxygenated methine protons [δ_H 3.95 (m), 3.31 (d, J = 2.5 Hz), and 4.22 (1H, d, J = 2.5 Hz)], and 14 aliphatic protons belonging to 7 methylene groups (δ_H 1.30-2.18). The ¹³C NMR spectrum of 1 exhibited 28 carbon signals due to 6 nonprotonated, 8 methylene, 9 methine, and 5 methyl carbons by the HSQC spectrum. Four oxygen-bearing carbons were identified at δ_C 68.6 (C-3), 65.6 (C-5), 62.6 (C-6), and 67.1 (C-7), 3 double bonds at δ_C 126.9 (C-8), 134.5 (C-9), 135.7 (C-22), 131.6 (C-23), 149.8 (C-25), and 108.8 (C-26). The above observation suggested that 1 should be an ergostane-type sterol having 3 double bonds, 2 hydroxy groups at C-3 and C-7, and an epoxy bridge between C-5 and C-6. Detailed analysis of the ¹H and ¹³C NMR data indicated that the structure of 1 was similar to those of 5α,6α-epoxy-(22E)-ergosta-8,22-diene-3β,7α-diol, except for the addition of a terminal double bond at C-25/C-26 (Table 1).^17–20 These suggestions were further confirmed by the HSQC, COSY, and HMBC spectra. The HMBC correlations from H₃-19 (δ_H 1.14) to C-5 (δ_C 65.6), C-9 (δ_C 134.5) and C-10 (δ_C 38.0), from H-6 (δ_H 3.31) to C-7 (δ_C 67.1) and C-8 (δ_C 126.9), from H₃-21 (δ_H 1.02) to C-17, C-20, C-22 (δ_C 135.7), from H₃-28 (δ_H 1.08) to C-23 (δ_C 131.6), C-24 (δ_C 43.6), and C-25 (δ_C 149.8), and from H₃-27 (δ_H 1.68) to C-24, C-25, and C-26 (δ_C 108.8) indicated 3 double bonds at C-8/C-9, C-22/C-23, and C-25/C-26, and the hydroxy group at C-7. The large proton coupling constant (J_{H−22/H−23} = 16.0 Hz) suggested the trans double bond at C-22/C-23. The very small J value of H-6 and H-7 (J = 2.5 Hz) confirmed that these protons did not have the same axial orientation. The ¹³C NMR data of C-5 (δ_C 65.6), C-6 (δ_C 62.6), and C-9 (δ_C 134.5) of 1 were very similar to the corresponding data of 5α,6α-epoxy-(22E,24R)-ergosta-8,22-diene-3β,7α-diol (1a) (δ_C 65.7, 62.6, and 134.4, respectively),¹⁷ 5α,6α-epoxy-23-demethylgorgost-8-ene-3β,7α-diol (δ_C 65.6, 62.6, and 134.6, respectively),²¹ and (24R)-5α,6α-epoxy-24-ethylcholest-8-ene-3β,7α-diol (δ_C 65.6, 62.5, and 134.5, respectively)²² measured in the same solvent (CDCl₃). In addition, the chemical shifts of H-18 (δ_H 0.59) and H-19 (δ_H 1.14) of 1 were similar to the experimental, as well as the computational data for the structure 3β,7α-dihydroxy-5α,6α-epoxy-Δ⁸.²³ The above observation suggested the 5α,6α-epoxy-ergosta-3β,7α-diol-8-ene structure of 1. Furthermore, one of the 2 H₂-4 protons appeared at δ_H 2.18 as a doublet of doublets with a large coupling constant (J = 11.5 and 12.0 Hz), which completely confirmed the β/axial-configuration of H-3.^22,23 However, the absolute configuration of C-24 was not determined. Based on the above evidence, the structure of 1 was elucidated as 5α,6α-epoxy-(22E)-ergosta-3β,7α-diol-8,22,25-triene, a new compound named halipanasterol (Supplemental Figures S2-S13) (Figure 1).

Figure 1.

Chemical structures of compounds 1-9 and 1a.

Table 1.

¹H and ¹³C NMR Spectroscopic Data for Compound 1.

Pos.	^a,b δ _C	^a,cδ_H (mult, J in Hz)	Pos.	^a,b δ _C	^a,cδ_H (mult, J in Hz)
1	30.2	1.60 (m), 1.79 (m)	15	23.4	1.30 (m), 2.13 (m)
2	30.9	1.61 (m), 1.98 (m)	16	29.0	1.34 (m), 1.80 (m)
3	68.6	3.95 (m)	17	53.7	1.20 (m)
4	39.2	1.45*, 2.18 (dd, 11.5, 12.0)	18	11.3	0.59 (s)
5	65.6	-	19	22.8	1.14 (s)
6	62.6	3.31 (d, 2.5)	20	40.2	2.06 (m)
7	67.1	4.22 (d, 2.5)	21	20.7	1.02 (s)
8	126.9	-	22	135.7	5.29 (dd, 16.0, 6.5)
9	134.5	-	23	131.6	5.25 (dd, 16.0, 6.5)
10	38.0	-	24	43.6	2.70 (m)
11	23.8	2.05 (m)	25	149.8	-
12	35.7	1.41, 1.95	26	108.8	4.69 (s), 4.71 (s)
13	42.1	-	27	20.6	1.68 (s)
14	49.6	2.19 (m)	28	18.9	1.08 (d, 7.0)

(*) Overlapped signals.

The other compounds were identified as (22E)-ergosta-7,22,25-triene-3β,5α,6β-triol (2),^19,22 3β-hydroxycholest-5ene-7-one (3),²⁴ cholesterol sulfate (4),²⁵ 3-formamidotheonellin (5),²⁶ para-hydroxybenzylideneacetone (6),²⁷ indole-3-carbaldehyde (7),²⁸ thymidine (8), and adenosine (9)²⁹ (The experimental data for compounds 2-9 are shown in Supplemental Material). Their NMR and physical data were compared with those reported in the literature and found to match them very well. The structures were further confirmed by the HRESIMS, HSQC, and HMBC spectra (Supplemental Figures S14-S44).

In continuation of our screening program for cytotoxic compounds from marine sponges, compounds 1-5 were evaluated for their cytotoxic effects on 4 human cancer cell lines [lung carcinoma (SK-LU-1), breast carcinoma (MCF-7), hepatocellular carcinoma (Hep-G2), and melanoma (SK-Mel-2)]. First, compounds 1-5 were screened at concentrations of 100 and 20 μM (Supplemental Table S2). The experiments with cell death percentages over 50% obtained from compounds 1 and 2 were further performed as dose-dependent studies to find the corresponding IC₅₀ values. Compounds 1 and 2 exhibited the most cytotoxic effects on Hep-G2, SK-Mel-2, LU-1, and MCF-7 cell lines with IC₅₀ values of 9.27 ± 1.25, 11.34 ± 1.18, 15.45 ± 1.47, and 10.85 ± 1.19 µM (for 1), and 6.45 ± 0.77, 9.74 ± 1.03, 7.08 ± 0.64, and 5.61 ± 0.39 µM (for 2), compared to the positive control compound, ellipticine, 1.60 ± 0.37, 2.53 ± 0.51, 1.82 ± 0.20, and 2.17 ± 0.66 µM, respectively.

Materials and Methods

General Experimental Procedures

The used characterization equipment is the same as described in our previous work.³⁰

Refer to Supplemental Material.

Marine Samples

The marine sponge samples (NCCT-B101) were collected in Vanphong, Nhatrang, Vietnam in May 2020 and identified as H panicea (Pallas, 1766) by one of our authors, Prof. Do Cong Thung. The voucher specimen has been deposited at IMBC, VAST.

Extraction and Isolation

The frozen sponge sample of H panicea (4.0 kg) was defrosted, washed with water, cut into small pieces, and then extracted with methanol in an ultrasonic bath, 3 times (each 10 L MeOH, room temperature, 60 min). After filtration, the filtrate was evaporated under reduced pressure to give the methanol extract (53 g). This was suspended in 1 L of distilled water and extracted with dichloromethane to give a dichloromethane soluble fraction (HP1, 18 g). This was chromatographed on a silica gel column, eluting with a gradient solvent system of n-hexane/acetone (100/1-0/1, vol/vol) to give 5 fractions HP1A-HP1E. Fraction HP1B (3.5 g) was loaded onto a silica gel column and eluted with n-hexane/ethyl acetate (2.5/1, vol/vol) to give 3 subfractions HP1B1-HP1B3. Fraction HP1B1 (210 mg) was purified on a reversed-phase C-18 resin (RP-18) column, eluting with MeOH/water (1.5/1, vol/vol) to give compound 2 (8.0 mg). Fraction HP1B2 (250 mg) was purified on a reversed-phase C-18 resin (RP-18) column, eluting with MeOH/water (2.0/1, vol/vol) to give compounds 1 (6.5 mg) and 2 (7.5 mg). Fraction HP1C (3.2 g) was chromatographed on a reversed-phase C-18 resin (RP-18) column, eluting with acetone/water (2.0/1, vol/vol) to give 2 fractions HP1C1 (1.0 g) and HP1C2 (1.5 g). HP1C1 (900 mg) was chromatographed on a silica gel column eluting with CH₂Cl₂/acetone (6/1, vol/vol) to give compounds 6 (7.8 mg) and 7 (8.2 mg). HP1C2 (1.4 g) was further chromatographed on a YMC column eluting with MeOH/water (1.5/1, vol/vol) to give compounds 4 (9.0 mg) and 8 (8.3 mg). Fraction HP1E (2.9 g) was chromatographed on a silica gel column eluting with n-hexane/acetone (1/1, vol/vol) to give 2 fractions HP1E1 (860 mg) and HP1E2 (1.2 g). HP1E1 was further purified on a YMC column eluting with MeOH/water (1/1, vol/vol) to give compounds 3 (6.7 mg), 5 (10.1 mg), and 9 (12 mg).

Halipanasterol (1)

Colorless solid, mp. 234-236 °C; : +9.0 (c 0.1, MeOH); HRESIMS m/z m/z 461.2793 [M+³⁵Cl]⁻ (calcd. for [C₂₈H₄₂O₃³⁵Cl]⁻: 461.2822, Δ = 6.0 ppm) and m/z 463.2753 [M+³⁷Cl]⁻ (calcd. for [C₂₈H₄₂O₃³⁷Cl]⁻: 463.2783, Δ = 6.0 ppm); ¹H NMR (CDCl₃, 500 MHz) δ (ppm), ¹³C NMR (CDCl₃, 125 MHz) δ (ppm) data (see Table 1).

Biological Assays

Refer to Supplemental Material.

Conclusions

Phytochemical study of the methanol extract of the Vietnamese marine sponge H panicea (Pallas, 1766) led to the isolation of one new, 5α,6α-epoxy-(22E)-ergosta->3β,7α-diol-8,22,25-triene (1), and 8 known compounds, (22E)-ergosta-7,22,25-triene-3β,5α,6β-triol (2), 3β-hydroxycholest-5ene-7-one (3), cholesterol sulfate (4), 3-formamidotheonellin (5), para-hydroxybenzylideneacetone (6), indole-3-carbaldehyde (7), thymidine (8), adenosine (9). Their chemical structures were determined by HR-ESI-MS, ESI-MS, 1D, and 2D NMR spectral data, as well as by comparison with the previous literature. Compounds 1 and 2 exhibited cytotoxic effects on Hep-G2, SK-Mel-2, LU-1, and MCF-7 cell lines with IC₅₀ values ranging from 1.60 ± 0.37 to 10.85 ± 1.19 µM. Furthermore, this is the first report of these compounds from H panicea.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221088061 - Supplemental material for Halipanasterol, a New Sterol Isolated From the Marine Sponge Halichondria panicea

Supplemental material, sj-docx-1-npx-10.1177_1934578X221088061 for Halipanasterol, a New Sterol Isolated From the Marine Sponge Halichondria panicea by Phan Van Kiem, Do Thi Trang, Bui Huu Tai, Nguyen Xuan Nhiem, Dan Thi Thuy Hang, Do Cong Thung, Do Thi Thao, Pham Hai Yen and Phan Thi Thanh Huong in Natural Product Communications

Footnotes

Authors’ Contribution

Research idea, BH Tai, NX Nhiem, PV Kiem; Isolation, DTT Hang, DTH Trang, PH Yen, PTT Huong; Cytotoxic test: DT Thao; Collect sample: DC Thung; Structure elucidation and writing, BH Tai, PV Kiem.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research is funded by Vietnam Academy of Science and Technology under grant number TĐDLB0.01/20-22.

ORCID iDs

Phan Van Kiem

Do Thi Thao

Supplemental Material

Supplemental material for this article is available online.

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