Four bioactive new steroids from the soft coral Lobophytum pauciflorum collected in South China Sea

Introduction

The unique and complicated marine environment makes soft corals a treasure-house of secondary metabolites with great variety and bioactivities. Previous chemical studies on soft corals Lobophytum, widely distributed in the world, resulted in the identification of lobane diterpene [1], cembranoids [2], and biscembranoids [3] with different bioactivities. Moreover, structurally specific steroids containing side chains with 23,24-dimethyl groups and (17)20E double bond, have been reported to be frequently isolated from soft corals of this genus, some of them exhibited anti-inflammatory [4], cytotoxic [5,6], and antibacterial activities [7].

To search for bioactive natural products, we have investigated the chemical constituents of the soft coral Lobophytum pauciflorum, collected from Xisha Island in the South China Sea. In the present paper, we describe the isolation of four new polyhydroxylated steroids lobophysterols E–H (1–4), together with three known compounds (5–7) (Figure 1). The structure of the new compounds was established by extensive spectroscopic analysis and comparing with the spectroscopic data of the previously reported structurally-related compounds. Compounds 1 and 2 represented rare examples of steroids with both 23,24-dimethyl groups and 17(20)E double bond. In particular, compound 1 also has a tetracyclic skeleton with a methyl group at C-4. The absolute configuration of 1–3 was determined by X-ray analysis. Herein, we report the isolation, structure elucidation, and bioactivities of these compounds.

Results and Discussion

Compound 1 was isolated as a white powder. Its molecular formula was established as C₃₀H₅₀O₃ by HRESIMS from the molecular ion peak at m/z 481.3648 [M + Na]⁺. The ¹H NMR data (Table 1) showed 5 methyl singlets (δ_H 0.76, CH₃-19; δ_H 0.84, CH₃-18; δ_H 1.19, CH₃-26; δ_H 1.19, CH₃-27; δ_H 1.67, CH₃-21), 3 methyl doublets (δ_H 0.81, CH₃-29; δ_H 0.84, CH₃-28; δ_H 1.00, CH₃-30), an oxymethine (δ_H 3.13, 1H, m) and a series of methylene multiplets located between δ_H 1.25 and δ_H 2.40. The ¹³C NMR and DEPT spectra exhibited the presence of 30 carbon signals, including a carbonyl group (δ_C 210.0), a tetrasubstituted double bond (δ_C 144.4 and 124.2), one oxygenated sp³ secondary carbon (δ_C 76.3) and one oxygenated sp³ quaternary carbon (δ_C 74.2). As two of the six degrees of unsaturation were occupied by the double bond and carbonyl group, the remaining four unsaturations of 1 corresponded to a tetracyclic skeleton.

The ¹H,¹H-COSY experiment (Figure 2) revealed the proton–proton correlations of H-1/H-2/H-3/H-4, H-7/H-8/H-9/H-11/H-12, H-8/H-14/H-15/H-16, and H-22/H-23/H-24/H-28/29. These data, together with the HMBC correlations (Figure 2) from H-19 to C-1/C-5/C-9/C-10, from H₃-30 to C-4/C-5, from H-5/H-7 to C-6, from H₃-18 to C-12/C-13/C-14/C-17, from H₃-21 to C-17/C-20/C-22, and from H₃-26 to C-24/C-25 confirmed the establishment of the carbon skeleton of the 23,24-dimethycholestane with a methyl group at C-4. Thus, the planar structure of 1 was established as shown in Figure 1.

The relative configuration of 1 was deduced by the NOESY spectrum (Figure 3). The NOESY correlations of H-4 with H₃-19, H-8 with H₃-18 and H₃-19 suggested the β-orientation of H-4, H-8, H₃-18, and H₃-19. Moreover, H₃-30 showed NOESY correlations with H-3/H-5, H-5 with H-9, and H-9 with H-14 indicating the α-orientation of H-3, H-5, H-9, H-14, and H₃-30. Furthermore, the NOESY correlations of H₃-18 with H₃-21 suggested the E geometry of Δ¹⁷⁽²⁰⁾. Finally, the absolute configuration of compound 1 was established by single-crystal X-ray diffraction analysis (Figure 4) carried out using Cu Kα radiation with a Flack parameter of 0.0(2).

Compound 2, was isolated as a white powder with molecular formula C₂₉H₄₈O₄, established by HREIMS at m/z 483.3446 [M + Na]⁺. The ¹H and ¹³C NMR data (Table 1) of 2 exhibited very similar typical features of compound 1, the difference between the two compounds occurred in ring A: an OH was located at C-5, but a missing methyl group at C-4 in 2, which was in agreement with the ¹³C NMR spectrum and the molecular mass. The hydroxylation at C-5 was deduced from the HMBC correlations (Figure 2) from H₃-19/H-4 to C-5. Moreover, the HMBC correlations found from H-4 to C-5/C-6, H-7 to C-6, and H₃-18/H₃-21 to C-17 confirmed the location of a ketone carbonyl at C-6 and the double bond at C-17, respectively. Thus, the planar structure of 2 was established as shown in Figure 1. The relative configuration of 2 was deduced by the cross-peaks shown by a 2D NOESY spectrum (Figure 3). The NOE correlations of H-8 with H₃-18 and H₃-19, and H-9 (δ_H 1.94) with H-14 (δ_H 1.50) indicated the β-orientation of H-8/H₃-18/H₃-19, while α-orientation of H-9/H-14. Furthermore, the NOESY correlation of H₃-21 with H₃-18 suggests the E geometry of ∆¹⁷⁽²⁰⁾. The absolute configuration of 2 was established by single-crystal X-ray diffraction analysis (Figure 4) carried out using Cu Kα radiation with a Flack parameter of 0.3(4).

Compound 3 was isolated as a white powder with molecular formula C₂₈H₄₈O₄, established by HRESIMS at m/z 449.3626 [M + H]⁺. The ¹H and ¹³C NMR data (Table 1) of 3 were very similar to those of its analog compound 2, showing the identical signals of the tetracyclic parent nucleus. The difference between the two compounds occurred in the side chain: A single bound was located at C-17/C-20 in 3 instead of the double bound in 2 and the absence of a methyl unit at C-23, in agreement with the ¹³C NMR spectrum and the molecular mass. The HMBC correlations (Figure 2) found from H₃-21 to C-17/C-20/C-22, from H₃-26 to C-25/C-27/C-24, and from H₃-28 to C-24/C-25, together with the ¹H,¹H-COSY correlation (Figure 2) from H-22 to H-24 confirmed the side chain of compound 3. Thus, the planar structure of compound 3 was established, which was the same as the known compound (3β,5α)-25-trihydroxy-24S-methylcholestan-6-one [8]. The difference was the configuration of C-5, which was established as S by single-crystal X-ray diffraction analysis (Figure 4) carried out using Cu Kα radiation with a Flack parameter of −0.11(9). Thus, the absolute configuration of 3 was established.

Compound 4 was obtained as a yellow powder. Based on the HRESIMS data (m/z 427.3569 [M + H]⁺), the molecular formula was determined to be C₂₉H₄₆O₂, 14 mass units less than compound 5 [6]. By comparing the NMR data (Table 1) of 4 and 5, it is obvious that they possess the same parent nucleus. The major difference between them was that the side chain of 4 lacks a methyl group at C-23, which can also be proved by the molecular mass. The concrete structure of the side chain was established by the COSY and HMBC correlations (Figure 2). The ¹H,¹H-COSY experiment revealed that the proton–proton correlation of H-17/H-20/H-21/H-22/H-23/H₂-29. These data, together with the key HMBC correlations from H₃-28 to C-23/C-24/C-25 and from H₃-26/H₃-27 to C-24/C-25, confirmed the structure of the side chain of compound 4. The NOESY correlations (Figure 3) from H-1a (δ_H 1.22) to H-3 and H-9, H-9 to H-14, H-1b (δ_H 1.95) to H₃-19, H-8 to H₃-18 and H₃-19, H₃-18 to H-20 indicated that H-3, H-9, H-14, and H-17 were orientated on α-face, while 3-OH, H-8, H₃-18, and H₃-19 were positioned on the β-face. Further, the NMR data of 4 for the side chain were almost identical to the known compound 8, demethylgorgosterol [9,10]. Thus, the structure of compound 4 was assigned as shown in Figure 1.

Biological activity

The cytotoxic activities of compounds 1–7 were evaluated against three cancer cell lines (HL-60, K562, and Hela), but only compound 1 exhibited weak cytotoxic activity against K562 cells with an IC₅₀ value of 19.03 μM. The investigation of anti-inflammatory activities of lobophysterols E–H with classic transgenic fluorescent zebrafish models (Figure 5) showed that compound 1 exhibited moderate activity, with an inhibition rate of 32% (20 μM).

Conclusion

In conclusion, four new steroids lobophysterols E–H (1–4) and three known analogs were isolated from the soft coral Lobophytum pauciflorum collected at Xisha Island, China. Compounds 1 and 2 represented rare examples of steroids featuring both 23,24-dimethyl groups and 17(20)E double bond. Moreover, compound 1 has a tetracyclic skeleton with a methyl group at C-4. The absolute configuration of 1–3 was determined by X-ray diffraction analyses. Compounds 1–7 were subjected to a cytotoxic activity evaluation against HL-60, K562, and Hela cells, only compound 1 exhibited weak cytotoxic activity against K562 cells with an IC₅₀ value of 19.03 μM. In addition, compound 1 exhibited a moderate anti-inflammatory effect in zebrafish.

Experimental

General experimental procedures

NMR spectra were measured via an Agilent DD2-500 spectrometer (500 MHz for ¹H NMR and 125 MHz for ¹³C NMR). Chemical shifts are reported in parts per million (ppm) with coupling constants (J) in hertz relative to the solvent peaks; δ_H 3.31 and δ_C 49.0 for CD₃OD; δ_H 7.26 and δ_C 77.16 for CDCl₃. HRESIMS data were surveyed on a Thermo LTQ-Orbitrap mass spectrometer. IR spectra were recorded on a Nicolet NEXUS 470 spectrophotometer using KBr pellets. UV spectra were recorded on a Jasco J-815 CD spectropolarimeter. Optical rotations were measured with a Jasco P-1020 polarimeter. Semi-preparative HPLC (Agilent Technologies 1260 Infinity II) equipped with a reversed-phase column ((YMC-packed C18, 5 µm, 250 × 10 mm, 2.0 mL/min) was used to purify samples. Silica gel (300–400 mesh, Qingdao) was used for column chromatography (CC), and precoated silica gel plates (GF254, Qingdao) were used for TLC.

Soft coral material

The soft coral Lobophytum pauciflorum was collected from Yongle Islands of Xisha Islands of the South China Sea in May 2012. The sample was identified by Pingjyun Sung, National Museum of Marine Biology and Aquarium (NMMBA), Checheng, Pingtung 944, Taiwan, China. The voucher specimen (No. XS-2012-27), frozen at −20 °C, was deposited at the School of Medicine and Pharmacy, Ocean University of China, P. R. China.

Extraction and isolation

The frozen bodies of Lobophytum pauciflorum (3.6 kg, wet weight; 1.1 kg, dry weight) were cut into pieces and exhaustively extracted with MeOH five times at room temperature. The solvent was removed under reduced pressure and the combined organic extract was desalted three times by anhydrous methanol. The desalted residue (110 g) was subjected to silica gel column chromatography (CC) eluted with two gradient systems, PE/acetone (1:0 to 1:1) and subsequently CH₂Cl₂/MeOH (15:1 to 1:1) to afford 8 fractions. Fraction 4 (14.6 g) was split (chromatographed on) by silica gel eluting with a gradient of PE/acetone (30:1 to 1:1) to give three subfractions (F41–F43). Subfraction F41 (2.1 g) was chromatographed over silica gel column (PE/acetone, 50:1 to 2:1) to give seven subfractions (F411–F417), F411 (330 mg) was purified by semi-preparative HPLC (ODS, 5 µm, 250 × 10 mm; methanol/water, 95:5, v/v; 2.0 mL/min) to afford compound 4 (2.3 mg), 5 (4.7 mg), 6 (15.5 mg) and 7 (10.2 mg). Fr.412 (210 mg) was chromatographed on semi-preparative HPLC (ODS, 5 µm, 250 × 10 mm; methanol/water, 85:15, v/v; 2.0 mL/min) to give compound 1 (2.5 mg), 2 (5.8 mg) and 3 (13.3 mg).

Identification of new compounds

Compound 1: colorless crystals; [α]_D²⁵ −19.28 (c 0.13, MeOH); IR (KBr) ν_max: 3389, 2944, 2871, 1707, 1603, 1467, 1380 cm⁻¹; ¹H and ¹³C NMR data (CDCl₃, 500 and 125 MHz) see Table 1; HRESIMS (m/z) [M + Na]⁺ calcd for C₃₀H₅₀O₃Na, 481.3652; found, 481.3648.

Compound 2: colorless crystals; [α]_D²⁵ −12.77 (c 0.3, MeOH); IR (KBr) ν_max: 3361, 2926, 2855, 1702, 1651, 1459, 1376 cm⁻¹; ¹H and ¹³C NMR data (CD₃OD, 500 and 125 MHz) see Table 1; HRESIMS (m/z) [M + Na]⁺ calcd for C₂₉H₄₈O₄Na, 483.3445; found, 483.3446.

Compound 3: colorless crystals; [α]_D²⁵ −18.36 (c 0.5, MeOH); IR (KBr) ν_max: 3390, 2938, 1702, 1459, 1376 cm⁻¹; ¹H and ¹³C NMR data (CDCl₃, 500 and 125 MHz) see Table 1; HRESIMS (m/z) [M + H]⁺ calcd for C₂₈H₄₉O₄, 449.3625; found, 449.3626.

Compound 4: yelllow crystals; [α]_D²⁵ −27.83 (c 0.13, MeOH); IR (KBr) ν_max: 3391, 2957, 2872, 1683, 1650, 1558, 1540, 1357 cm⁻¹; ¹H and ¹³C NMR data (CDCl₃, 500 and 125 MHz) see Table 1; HRESIMS (m/z) [M + H]⁺ calcd for C₂₉H₄₇O₂, 427.3571; found, 427.3569.

Cytotoxicity assays

In vitro cytotoxicity was determined by the MTT method against K562 (chronic myeloid leukemia) and HL-60 (human promyelocytic leukemia) cell lines, and by the SRB method against the Hela cell line.

Zebrafish maintenance

Adult zebrafish were cultivated by Qilu University of Technology (Jinan, China). Transgenic zebrafish [Tg: zlyz-EGFP and Tg (vegfr2: GFP)] expressing enhanced green fluorescent protein (EGFP) was used in this article. The conditions of the maintenance complied with guidelines of the Organization for Economic Co-operation and Development (OECD). The zebrafish were maintained under a 14/10 h light/dark cycle at the temperature (28 ± 0.5°C) in a closed flow-through system with charcoal-filtered tap water to ensure normal spawning.

CuSO₄-induced model of zebrafish

In a manner similar to literature reference [11], healthy zebrafish larvae were selected into 24-well plates (n = 10/well) in a 2 mL final volume of embryo medium at 3 dpf and divided into five groups: a control group (fresh fish water), a model group: 40 μM CuSO₄, a positive drug group: 50 μM indomethacin (Solarbio, China) and drug groups: 20 μM CuSO₄ (Sigma-Aldrich, St. Louis, MO, USA) was added to the drug groups and incubated for 1 h after treatment with different compounds for 2 h. All treatments were performed in triplicate. Each zebrafish larva was photographed by a fluorescence microscope (AXIO, Zoom. V16), and the number of macrophages was counted by using the Image-Pro Plus software.